EPA/600/R-18/251 | August 2018
www.epa.gov/homeland-security-research
United States
Environmental Protection
Agency
oEPA
Bleach-Based Biodecontamination
of Subway Materials
Office of Research and Development

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EPA 600/R-18/251
August 2018
Bleach-Based Biodecontamination of Subway Materials
Assessment and Evaluation Report
Lukas Oudejans, Ph.D.
National Homeland Security Research Center
Office of Research and Development
U.S. Environmental Protection Agency
Research Triangle Park, NC 27711
Abderrahmane Touati, Ph.D., Denise Aslett, Ph.D., Ahmed Abdel-Hady, Francis
Delafield, Jason Colon, and Alex Merrell
Jacobs Technology, Inc.

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Disclaimer
The U.S. Environmental Protection Agency (EPA), through its Office of Research and
Development's National Homeland Security Research Center, funded and managed this
investigation through Contract No. EP-C-15-008 with Jacobs Technology, Inc. (Jacobs). Jacobs's
role did not include establishing EPA policy. This report has been peer- and administratively
reviewed and has been approved for publication as an EPA document. This report does not
necessarily reflect EPA's views. No official endorsement should be inferred. This report includes
photographs of commercially available products included for purposes of illustration only and
are not intended to imply that EPA approves or endorses the products shown or their
manufacturers. EPA does not endorse the purchase or sale of any commercial products or
services.
Questions concerning this document or its application should be addressed to the
following individual:
Lukas Oudejans, Ph.D.
Decontamination and Consequence Management Division
National Homeland Security Research Center
U.S. Environmental Protection Agency (MD-E343-06)
Office of Research and Development
109 T.W. Alexander Drive
Research Triangle Park, NC 27711
Telephone No.: (919) 541-2973
Fax No.: (919) 541-0496
E-mail Address: Oudeians.lukas@epa.gov

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Acknowledgments
The principal investigator (PI) from the U.S. Environmental Protection Agency (EPA),
through its Office of Research and Development's National Homeland Security Research Center
(NHSRC), directed this effort with the support of a project team from across EPA. The
contributions of the individuals listed below have been a valued asset throughout this effort.
Project Team
Lukas Oudejans (PI), EPA, NHSRC/DCMD
M. Worth Calfee, EPA, NHSRC/DCMD
Leroy Mickelsen, EPA, OLEM/CMAD
Shannon Serre, EPA, OLEM/CMAD
Joseph Wood, EPA, NHSRC/DCMD
EPA Peer Reviewers
Erin Silvestri, EPA, NHSRC/TCAD
Benjamin Franco, EPA, Region 4
EPA Quality Assurance
Eletha Brady-Roberts, NHSRC
EPA Technical Editing
Joan Bursey
Jacobs Technology, Inc.
Abderrahmane Touati
Denise Aslett
Ahmed Abdel-Hady
Zora Drake-Richman
Francis Delafield
Jason Colon
Alex Merrell

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Executive Summary
The U.S. Environmental Protection Agency's (EPA) Homeland Security Research
Program (HSRP) mission is to advance EPA's capabilities to protect human health and the
environment from adverse impacts arising from terrorist threats and other contamination
incidents. The National Homeland Security Research Center (NHSRC) conducts research under
the HSRP to address gaps in EPA's response to terrorist attacks involving chemical, biological,
radiological, and nuclear contamination.
The response following a chemical or biological incident in a transportation hub like a
subway system requires fast and effective remediation approaches to mitigate cost and reduce the
time for the underground system to return to normal operation. This study is designed to evaluate
"low-tech" decontamination approaches that use readily available off-the shelf equipment for
dispensing decontamination solutions. This effort builds on previous HSRP studies [1,2] which
demonstrated that pH-adjusted bleach (pAB) can be efficacious for inactivating Bacillus
atrophaeus var. globigii (Bg), a surrogate for Bacillus anthracis (Ba), on selected cleaned and
grimed surfaces (at 10 grams per square meter [g/m2]). It was unclear whether excessive grime as
found in e.g., a subway environment would impact the decontamination efficacy. The study,
discussed here, specifically evaluated the potential impact of a significantly higher surface grime
loading (> 100 g/m2) on Bg spore inactivation efficacy for pAB as well as various diluted bleach
(DB) ratios. Sections of the subway material coupons, and granite rocks from track ballast were
inoculated using a metered dose inhaler (MDI). Track ballast decontamination experiments
included tests with a dry track ballast and a post-inoculation wetted ballast mimicking the
possible wetting of contaminated ballast by rain and associated redistribution of spores into the
track ballast bed.
Recovery of spores from bleach-sprayed surfaces (test samples) was compared to
recovery from surfaces that were inoculated but not sprayed (positive control samples).
Decontamination efficacy was expressed as a log reduction (LR) and was determined by
comparing spore recoveries for test samples to those of positive controls. The transfer of viable
organisms to post-decontamination liquid waste was evaluated through quantitative analysis of
decontamination procedure runoff.
Results of this evaluation for these subway materials are summarized below:
•	The pAB and DB (at 20,000 parts per million (ppm) diluted Clorox® Concentrated
Germicidal Bleach) solutions resulted in no recoverable viable Bg spores on concrete,
ceramic, and painted steel surfaces and in all liquid runoff, with no difference in
performance between grimed and cleaned materials.
•	The DB solution with a target free available chlorine (FAC) concentration of 6,000
ppm showed greater than 6 LR on clean material surfaces (concrete, ceramic, and
painted steel), regardless of material type, and only a few remnant spores on some of
the surfaces tested. Less than 6 LR was observed on grimed surfaces, regardless of
material type. The results suggest that grime affects the sporicidal inactivation of
spores on surfaces by one to three orders of magnitude. Cleaning prior to this DB
application of these heavily grimed and contaminated surfaces would be necessary to
achieve full decontamination of grimed materials. Such cleaning step prior to DB

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application would likely also reduce the number of spores on surfaces (not
investigated here).
•	No spores were detected in runoff for all three bleach-based solutions (pAB, 6,000-
ppm FAC DB, and 20,000 ppm DB), regardless of material type (concrete, ceramic,
and painted steel) or surface treatment (cleaned or grimed). The 30-minute contact in
the collected runoff was sufficient to decontaminate all spores displaced during the
spraying process.
•	Decontamination of the top layer of dry granite rocks (track ballast) (pAB and DB at
6,000 and 5,000 ppm, respectively) resulted in no recoverable viable Bg spores in all
three 3" depth layers and effluent.
•	The post-inoculation uniform wetting of the ballast (simulating wetting from a rain
event) resulted in a stratification of spores over the 9-in depth of the ballast track,
depending on the applied water volume per area. Increasing the wetting level resulted
in even larger stratification of the spores in the top, middle, and bottom layer as well
as the runoff.
•	The pAB solution was very effective inactivating all spores (none detected) in all
three ballast layers and in runoff at a volume sprayed per area of 3,740 mL/m2 At a
lower volume of 1,770 mL/m2 sprayed per unit area, pAB was still very effective (no
viable spores detected) in the top 6-in track ballast layer but less effective (LR < 6) in
the bottom layer (greater than 6-in depth).
•	The DB solution, with a target FAC concentration of 4,000 and 5,000 ppm
(corresponding to a dilution ratio of 1 to 20 and 1 to 15 of a Clorox® Concentrated
Germicidal Bleach stock solution with DI water, respectively), achieved greater than
6 LR over the 9-in height of the ballast at a volume sprayed of 2,210 mL/m2 and was
fully effective (no spores detected) at a volume of 3,700 mL/m2. Colony-forming
units (CFU) of spores were observed in the runoff collected during the spraying
process. However, no recoverable CFU were observed following 24 hours of contact
time.

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Contents
Disclaimer	i
Acknowledgments	ii
Executive Summary	iii
Contents	v
Figures	vii
Tables viii
Acronyms and Abbreviations	ix
1	Introduction	1
1.1	Background	1
1.2	Project Description and Objectives	2
2	Experimental Approach	2
3	Experimental Methods and Materials	5
3.1	Test Materials	5
3.1.1 Phase I: Subway Materials	5
3.1.1.1	Coupon Cleaning	5
3.1.1.2	Coupon Griming	6
3.1.1.3	Material Sterilization	 7
3 .1.2 Phase II: Track Ballast	8
3.1.2.1 Track Ballast Decontamination and Sampling Assembly	8
3.2	Test Organism and Inoculation	9
3.2.1	B. atrophaeus var. globigii (Bg), a Surrogate for B. anthracis	9
3.2.2	Bg Spore Inoculation	10
3.3	Decontamination Approach	12
3.3.1	Decontaminants	12
3.3.1.1	pAB Solution	12
3.3.1.2	DB Solution	13
3.3.2	Decontamination Testing Approach	13
3.3.2.1	Phase I: Subway Materials	13
3.3.2.2	Phase II: Track Ballast	15
3.4	Neutralizing Agents for Extracted Samples	17
4	Sampling and Analysis	19
4.1	Sampling Approach	19
4.1.1	Phase I: Subway Materials	19
4.1.1.1	Wipe Samples	19
4.1.1.2	Liquid Runoff Samples	20
4.1.2	Phase II: Track Ballast	20
4.1.2.1	Ballast Rock Samples	20
4.1.2.2	Liquid Runoff Samples	20
4.2	Sampling Frequency and Monitoring Events	20
4.2.1	Phase I: Subway Materials	20
4.2.2	Phase II: Track Ballast	21
4.3	Sample Handling	23
4.3.1	Sample Containers	23
4.3.2	Sample Preservation	23

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4.3.3 Sample Custody	23
4.4	Analytical Procedures	23
4.4.1	Track Ballast Extraction Method	23
4.4.2	Microbiological Analysis	25
4.5	Characterization of Decontamination Solutions	25
4.5.1	Determination of FAC by Titration	25
4.5.2	pH and Temperature Measurements	25
4.6	Determination of Decontamination Efficacy	25
5	Results and Discussion	27
5.1	Phase I: Subway Materials	27
5.1.1	Characterization of Decontamination Solutions	27
5.1.2	Physical Spore Removal	28
5.1.3	Decontamination Efficacy	29
5.1.3.1	pAB Results	29
5.3.1.2	DB Results (Target FAC Concentration: 6,000ppm)	30
5.3.1.3	DB Results (Target FAC Concentration: 20,000ppm)	31
5.2	Phase II: Track Ballast	31
5.2.1	Characterization o/Decontamination Solutions	32
5.2.2	Fate and Transport of Spores	32
5.2.3	Decontamination Efficacy	33
5.2.3.1	Bleach-Based Decontamination Solution following Wetting
of Ballast Material	33
5.2.3.2	Bleach-Based Decontamination Solution with Dry Ballast
Material.	35
6	Summary	37
6.1	Subway Materi al s	37
6.2	Track Ballast	37
7	Quality Assurance and Quality Control	39
7.1	Criteria for Critical Measurements and Parameters	39
7.2	Data QA/QC	39
7.3	QA/QC Checks	40
7.3.1	Check of Integrity of Samples and Supplies	40
7.3.2	NHSRC BioLab Control Checks	41
7.3.3	Data Quality Audit	42
7.3.4	QA Assessments and Corrective Actions	42
8	References	44

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Figures
Figure 3-1. Representative grimed concrete coupon	7
Figure 3-2. Representative track ballast rocks (3/4-in to 2-in sizes)	8
Figure 3-3. Track ballast decontamination and sampling assembly	9
Figure 3-4. MDI canister (A) and actuator (B)	10
Figure 3-5. Round ADA	11
Figure 3-6. 14- by 14-in ADA	11
Figure 3-7. Environmental test chamber	13
Figure 3-8. Spray system setup inside the ETC	14
Figure 3-9. Electric backpack sprayer	16
Figure 3-10. DE broth neutralizer effectiveness	18
Figure 4-1. Track ballast extraction method recovery	24
Figure 5-1. Subway material recoveries after wetting of top layer	28
Figure 5-2. Transport of spores through track ballast after wetting of the top layer	33
Figure 5-3. Decontamination efficacy of bleach-based solutions on track ballast after
wetting of the top layer	35
Figure 5-4. Decontamination efficacy of bleach-based solutions on dry track ballast	36

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Tables
Table 3-1. SNL Grime Recipe	6
Table 3-2. SNL Grime Element Analysis	6
Table 3-3. Subway Materials Decontamination Test Matrix	15
Table 3-4. Track Ballast Decontamination Test Matrix	17
Table 4-1. Sampling Frequency for Subway Material Coupon Tests	21
Table 4-2. Sampling Frequency for Track Ballast Tests	22
Table 5-1. FAC and pH of Bleach-based Decontamination Solutions for Subway
Materials	27
Table 5-2. Spore Removal After Water Spray of Subway Materials	29
Table 5-3. Decontamination Efficacy of pAB Solution on Subway Materials	30
Table 5-4. Decontamination Efficacy of DB Solution on Subway Materials	30
Table 5-5. Decontamination Efficacy of DB Solution on Concrete	31
Table 5-6. FAC and pH of Bleach-Based Decontamination Solutions for Track Ballast
Decontamination Testing	32
Table 5-7. Decontamination Efficacy of Bleach-Based Solutions on Track Ballast
following Wetting	34
Table 7-1. DQIs for Critical Measurements and Parameters	40
Table 7-2. Additional QC Checks for Biological Measurements	42
Table 7-3. Cross-Contamination Assessment of Subway Material Testing	43
Table 7-4. Cross-Contamination Assessment of Track Ballast Testing	43

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Acronyms and Abbreviations
|ig	microgram(s)
|iL	microliter(s)
ADA	aerosol deposition apparatus
AREMA	American Railway Engineering and Maintenance-of-Way Association
ATCC	American Type Culture Collection
Ba	Bacillus anthracis
Bg	Bacillus atrophaeus var. globigii
BioLab	NHSRC Research Triangle Park Microbiology Laboratory
BOTE	Bio-Response Operational Testing and Evaluation Project
CFU	colony-forming unit(s)
Cb	chlorine
COC	chain of custody
cm	centimeter(s)
DB	diluted bleach
DCMD	Decontamination and Consequence Management Division
DE	Dey Engley
DHS	U.S. Department of Homeland Security
DI	deionized
DQI	data quality indicator
DQO	data quality objective
DTRL	Decontamination Technologies Research Laboratory
EC	elemental carbon
EPA	U. S. Environmental Protection Agency
ETC	environmental test chamber
EtO	ethylene oxide
FAC	free available chlorine
FIFRA	Federal Insecticide, Fungicide, and Rodenticide Act
ft	foot
g	gram(s)
g/m2	gram(s) per square meter
HHS	U.S. Department of Health and Human Services
HOC1	hypochlorous acid
HSRP	Homeland Security Research Program
in	inch(es)
Jacobs	Jacobs Technology, Inc.
L	liter(s)

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L/min
liter(s) per minute
LR
log reduction
MDI
metered dose inhaler
m
meter
mg
milligram(s)
mL
milliliter(s)
mL/m2
milliliter(s) per square meter
mL/min
milliliter(s) per minute
mm
millimeter(s)
MTA
New York Metropolitan Transportation Authority
NHSRC
National Homeland Security Research Center
NIOSH
National Institute for Occupational Safety and Health
NIST
National Institute of Standards and Technology
OC
organic carbon
ocr
hypochlorite anion
pAB
pH-adjusted bleach
PBST
phosphate-buffered saline with 0.05% Tween® 20
PI
principal investigator
ppm
part(s) per million
PRB
polyester-rayon blend
PTI
Powder Technology, Inc.
QA
quality assurance
QC
quality control
RH
relative humidity
rpm
revolution(s) per minute
RTP
Research Triangle Park
sec
second(s)
SNL
Sandia National Laboratories
STD
standard deviation
STS
sodium thiosulfate
TCAD
Threat and Consequence Assessment Division
TSA
tryptic soy agar
UTR
Underground Transportation Restoration
XRF
X-ray fluorescence

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1 Introduction
The U.S. Environmental Protection Agency's (EPA) Homeland Security Research
Program (HSRP) provides credible information to protect human health and the environment
from adverse impacts arising from terrorist threats and other contamination incidents. The
National Homeland Security Research Center (NHSRC) conducts research under the HSRP to
provide expertise and guidance on the selection and implementation of decontamination methods
that may significantly reduce the time and cost of wide-area remediation. This project addresses
HSRP strategic goals as described in detail in the "Homeland Security, Strategic Research
Action Plan 2016-2019" [3],
This report discusses a project that evaluates the effectiveness of bleach-based
decontamination methods to remediate contaminated underground transportation infrastructure
such as subway tunnels and platforms after a biological release. The following sections discuss
the project background and the project description and objectives.
1.1 Background
Response to a chemical or biological release in a subway system would require fast and
effective remediation to mitigate cost and reduce the time required for the underground system to
return to normal operation. "Low-tech" decontamination approaches readily available at local
hardware stores (such as a backpack or chemical sprayer in conjunction with bleach-containing
solutions) are easy to use for dispensing a decontamination solution during remediation of large
indoor and outdoor contaminated surfaces.
Previous studies [1,2] conducted by the EPA demonstrated that pH adjusted bleach
(pAB) was effective in inactivating Bacillus atrophaeus var. globigii (Bg), a surrogate for B.
anthracis (Ba) on selected surfaces. In one study [2], a 30-minute pAB contact time resulted in a
greater than or equal to a 6-log reduction (LR) in spores on grime-free drywall and concrete
coupons which were inoculated with spores at a level of 1 x 107 colony-forming units (CFU).
The effect of grime (at 10 grams per square meter [g/m2]) on concrete was found to be minimal,
if any effect was observed at all.
Another study conducted through the interagency Underground Transport Restoration
(UTR) Project [4] with the U.S. Department of Homeland Security (DHS) identified equipment
that could be used or rapidly modified for use in dispensing liquid chemicals to decontaminate
surfaces after a biological contamination incident. The selected equipment, an Air-O-Fan®
sprayer (Air-O-Fan D-40R 3800 L (Air-O-Fan Products Corp, Reedley, CA), was used to
evaluate the sporicidal efficacy of pAB to decontaminate unpainted concrete and ceramic tile
(both materials found in subway tunnels or stations). Each (clean) surface type was inoculated
with 1 x 107 CFU Bg spores and then decontaminated using prescribed Air-O-Fan applications
and selected contact times. Decontamination of ceramic tile resulted in greater than 6 LR, while
no condition was found to be effective for unpainted concrete, even with multiple pAB
applications and 30-minute contact times between applications.
This current study builds on the previous studies discussed above by investigating the
decontamination efficacy of a spray-based "low-tech" decontamination approach using bleach-
based sporicidal solutions to decontaminate grimed and grime-free subway material surfaces.
The novel approach in this study was the use of diluted bleach (DB), in addition to more

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commonly used pAB, to decontaminate surfaces with a higher grime loading (tenfold greater
than in the previous study [2]) and track ballast sections consisting of crushed rocks with voids to
facilitate drainage of water away from the rails and ties. The impact of grime on decontamination
efficacy was assessed by measuring and quantifying residual spores present on surfaces and in
effluents after each step of the defined bleach spraying process. The study provided valuable
information regarding the need for more conventional surface cleaning before the use of pAB
and DB using a "low-tech" spraying approach as an efficacious decontamination strategy in a
subway environment.
1.2 Project Description and Objectives
The general process investigated during this study was the decontamination of surfaces
contaminated with Bg spores. Decontamination can be defined as the process of inactivating or
reducing/removing a contaminant in or on humans, animals, plants, food, water, soil, air, areas,
or items through physical, chemical, or other methods to meet cleanup goals. The primary
objective of this study was to evaluate the impact of grime applied to representative subway
surface materials on surface decontamination efficacy via sprayed bleach application. The impact
of the presence of grime was determined by comparing the surface decontamination efficacies
between grimed subway material coupons that were not cleaned prior to decontamination to
those that were cleaned using a method prescribed by the New York Metropolitan Transportation
Authority (MTA).
Cleaned and grimed concrete, glazed ceramic tile, and painted stainless-steel coupons and
the surface (top) layer of track ballast were inoculated with Bg spores using an aerosol deposition
apparatus (ADA). The inoculated coupon surfaces underwent decontamination by spraying.
Results for spores recovered from sprayed surfaces (test samples) were compared to results for
spore recovery from surfaces that were inoculated but not sprayed (control samples). Surface
decontamination efficacy was calculated as the difference between the average positive control
recoveries and the post-decontamination recoveries for each treated surface. Additional sampling
was conducted to determine the fate of spores when track ballast was wetted with water followed
by bleach decontamination. During these laboratory evaluations, a decontaminant was
considered effective when LR > 6 for a 1 x 106 CFU or greater challenge [5], When no viable
spores were recovered after decontamination, the method was considered highly effective.
Quality control (QC) samples such as procedural blanks (clean and grimed surfaces that
underwent spraying but were not inoculated) and negative controls (clean and grimed surfaces
that were not inoculated and did not undergo spraying) were included to monitor for cross-
contamination. Procedural positive controls (inoculated surfaces that underwent a water-spraying
process) were included in one test to determine the physical removal of spores solely due to
spraying of the liquid onto the surface. The NHSRC Research Triangle Park (RTP) Microbiology
Laboratory (BioLab) quantified the number of viable spores.
2 Experimental Approach
Decontamination testing consisted of two phases. During Phase I, the efficacy of bleach-
based decontamination solutions was evaluated on the surface of cleaned and grimed subway
building materials. Phase II examined the efficacy of bleach-based decontamination solutions on
a three-level assembly of track ballast rocks. The general experimental approach used to meet the

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project objectives is described below.
1.	Material preparation: Test materials for each phase were prepared as discussed below.
•	Phase I test materials: The target materials consisted of subway materials, including
concrete, glazed ceramic tile, and painted stainless steel. The test coupons were
prepared as described in Section 3.1.1.
•	Phase II test materials: The target material consisted of track ballast (crushed stones)
consistent with the American Railway Engineering and Maintenance-of-Way
Association (AREMA) ballast gradation for main running tracks, size 4A. Track
ballast sterilization and the decontamination and sampling assembly are described in
Section 3.1.2.
2.	Inoculation of test coupons with the target organism: The test materials were
inoculated using an aerosol deposition method that delivered a known concentration of
spores in a repeatable fashion. Approximately 1 x 107 spores of Bg, a surrogate organism
for Ba, were deposited onto each test material as discussed in Section 3.2.2.
3.	Preparation of decontamination solutions: The decontamination solutions consisted of
pAB and DB at two different FAC levels), freshly prepared on each test day as discussed
in Section 3.3.1.
4.	Application of decontamination solutions to test materials: Test coupons (three
coupons per test material) prepared from selected subway materials were decontaminated
using the environmental test chamber (ETC) as described in Section 3.3.2.1. The track
ballast material was decontaminated using the electric backpack sprayer described in
Section 3.3.2.2.
5.	Preparation of neutralizing agents: The neutralizing agents included sodium thiosulfate
(STS) and Dey Engley (DE) broth as discussed in Section 3.4. The neutralizing agents
were applied to stop decontamination activity after a prescribed exposure time.
6.	Sample collection: Subway material and ballast samples were collected as summarized
below.
•	Subway Materials: For each material, a wipe sample and a liquid runoff sample were
collected. The runoff samples were collected in conical tubes containing a pre-
determined volume of neutralizing agent as described in Section 4.1.1.
• Track Ballast: Rocks from three 3- inch [in] in layers of a 9-in ballast assembly
were collected separately in 1 L Nalgene bottles ((ThermoFisher Scientific,
Waltham, MA; Item No. 2187-0032). Each bottle was filled no more than half full
to allow room for addition of extraction liquid. Therefore, multiple bottles were
required to collect a single sample. During the spray process, liquid runoff from
each ballast material test was collected in Nalgene bottles containing a pre-
determined volume of neutralizing agent. The ballast sample collections are
described in Section 4.1.2. The ballast material spore extraction procedure,
developed by the BioLab, is described in Section 4.5.1.

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7.	Sample analysis: Viable Bg spores were extracted from the wipe, ballast rock, and liquid
runoff samples, and aliquots were analyzed using the microbiological analysis procedure
described in Section 4.5.2. Viable spore recovery was quantified in terms of CFU present
in each sample.
8.	Determination of decontamination efficacy: Data reduction was performed on
measurements of the total spores (CFU) recovered from each replicate test sample,
reporting; average recovered CFU and standard deviation for each group of test samples.
Section 5.1.3 discusses the determination of decontamination efficacy.

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3 Experimental Methods and Materials
This section describes the experimental methods and materials, including the test
materials, grime, test organism and inoculation, decontamination approach, and neutralizing
agents for extracted samples.
3.1 Test Materials
This study consisted of two phases. During Phase I, the efficacy of bleach-based
decontamination solutions was evaluated on the surface of subway materials. Phase II examined
the efficacy of bleach-based decontamination solutions on a three-level assembly of track ballast
rocks. Each test material is discussed below.
3.1.1 Phase I: Subway Materials
The subway concrete chunks and the ceramic tiles were sourced from a previous research
effort [6] and using dry mechanical methods, were divided into as many coupons as possible
having a minimum 1.5 in x 1.5 in (3.8 centimeters [cm] x 3.8 cm) flat square surface. Painted
construction steel was represented by coupons constructed of unpolished low-carbon steel that
was approximately 1/8-in thick (P/N 8910K401, McMaster Carr, Atlanta, Georgia) and painted
with both primer (P/N 249058, Rust-Oleum Corporation, Vernon Hills, Illinois) and acrylic
enamel (P/N 248647, Rust-Oleum Corporation, Vernon Hills, Illinois). The concrete, glazed
ceramic tile, and painted steel coupons underwent the following cleaning preparation before use.
3.1.1.1	Coupon Cleaning
All subway material coupons were cleaned using a dilute solution of Tide® Institutional
Formula Floor & All-Purpose Cleaner (P&G Professional, Cincinnati, OH). This solution
consisted of 0.28 percent (%) Tide® Institutional Formula Floor & All-Purpose Cleaner by
weight (4.2 grams of cleaner in 1.5 liters [L] of hot water). The coupons were cleaned using the
procedure summarized below, which was adapted from a method used by the MTA.
•	Place coupon flat in a sink, with the top surface facing up.
•	Spray the top surface with 0.28% Tide® solution, using a foaming spray applicator
(Trigger Sprayer, Grainger Item # 3U603 on 32-ounce spray bottle, Grainger Item
# 3U593, W.W. Grainger, Inc., Lake Forest, IL).
•	Scrub the top surface lightly with a 1,5-in soft natural bristle paint brush.
•	Rinse each coupon well under flowing tap water.
•	Stand the coupons on edge on a paper towel.
•	Blow the coupons dry with dry nitrogen to remove surface water.
•	Let the coupons air-dry for 24 hours.
All coupons were sterilized with ethylene oxide (EtO) before use and before the griming
process discussed below.

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Griine Application
Grime was applied to the sampling area of a coupon using an applicator brush (part
#71955T55, McMaster-Carr, Elmhurst, IL). The application process was repeated until a
minimum of 150 milligrams (mg) of grime had been transferred to the surface of the coupon.
The weight of the coupon was determined before and after griming and was noted in a laboratory
notebook. Figure 3-1 shows a representative grimed concrete coupon.
Figure 3-1. Representative grimed concrete coupon.
3.1.1.3 Material Sterilization
All sterilization procedures were performed using an Andersen ethylene oxide (EtO)
sterilizer system that consists of a cartridge, a Humidichip®, a dosimeter, and a bag (EOGas®,
Part No. 333, ANPRO, Haw River, NC) and a sterilization kit (Kit #6, Part No. AN1006,
ANPRO). EtO sterilization was preferred over more conventional autoclave sterilization
considering the potential impact of high temperature on, e.g., the painted steel. The sterilization
procedure is summarized below.
1. All the items to be sterilized were packed in appropriate 12- by 15-in EtO envelopes (Part
No. AN2350), and the bags were sealed. Like materials were sterilized together in EtO
envelopes as follows:
•	Concrete coupons
•	Glazed ceramic tile coupons
•	Painted stainless steel coupons
•	Inoculation control coupons (2.5- by 2.5-in stainless steel)
•	Plastic trays (bottoms and lids)
•	Actuators
•	ADA
•	Deionized (DI) water tank

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2.	Sealed EtO envelopes, dosimeters, Humidichips®, and EtO dispensers were placed in
appropriate sterilization bags.
3.	The sterilization bags were vacuum-sealed and loaded into the EtO sterilizer for an 18-
hour sterilization cycle.
3.1.2 Phase II: Track Ballast
Track ballast, depicted in Figure 3-2, is the foundation of a railway track. The tested track
ballast (Wake Stone Corporation, Cary, NC) followed the American Railway Engineering and
Maintenance-of-Way Association (AREMA) ballast gradation size 4A for main running tracks
with nominal sizes of %-in to 2-in. (approximately 2 to 7 cm) across. AREMA 4A is the main
component of a track ballast gradation [9],
Figure 3-2. Representative track ballast rocks (3A-in to 2-in sizes).
Track ballast rock sampling assembly is discussed below.
3.1.2.1	Track Ballast Decontamination and Sampling Assembly
Figure 3-3 shows the track ballast decontamination and sampling assembly. Three layers
of ballast were assembled in 12- by 12-in arrays, with an overall depth of 9-in, which is greater
than the minimal depth required by AREMA gradation. The layers were separated by removable
metal sieves and were assembled on top of a funneled effluent collection manifold. The layers
were held together by 14- by 14-in metal plates with 12- by 12-in openings to expose the ballast.

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Figure 3-3. Track ballast decontamination and sampling assembly.
3.2 Test Organism and Inoculation
The test organism and inoculation process described in the following sections includes
excerpts from laboratory operating procedures used for these tests.
3.2.1 B. atrophaeus far. globigii (Bg), a Surrogate for B. anthracis
Bg, a surrogate for the spore-forming bacterial agent Ba, was used for this project. Like
Ba, Bg is a soil-dwelling, Gram-positive, aerobic microorganism, but unlike Ba, Bg is non-
pathogenic. Bg forms an orange pigment when grown on nutrient agar, a desirable characteristic
for detecting viable spores in complex environmental samples. Bg has a long history of use in the
biodefense community as a simulant for anthrax [10]

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3.2.2 Bg Spore Inoculation
Each test material was inoculated with approximately 1 « 107 spores of Bg (cultured,
processed, and lyophilized at Dugway Proving Ground, Dugway, UT; annotated as American
Type Culture Collection [ATCC] 9372) using a metered dose inhaler (MDI) (canister Part No.
BK0339783, Bespak, Hertfordshire, England). The MDI canister contained Bg spores suspended
in an ethanol solution and HFA-134A propellant (1,1,1,2-tetrafluoroethane). The MDI canister
was situated inside an actuator as shown in Figure 3-4 so that each time the actuator was
depressed, a repeatable number of spores was deposited [11], The MDI actuator is a small plastic
tube into which the MDI canister is inserted.
Figure 3-4. MDI canister (A) and actuator (B).
Each MDI was charged with a volume of spore preparation plus propellant sufficient to
deliver 200 discharges of 50 microliters (jiL) per discharge. MDI use was tracked so that the
number of discharges of each MDI did not exceed 200.
The inoculation procedure using an MDI involves first placing an Aerosol Deposition
Apparatus [11,12] on the surface of the test material. The ADA is clamped to the test coupon or
the ballast assembly, and the MDI is attached to the top of the ADA. A slide below the MDI is
opened, and the MDI is activated. Following inoculation, the slide is closed, and the MDI is
removed. The assembly remains closed while the spores are allowed to settle for 18 hours prior
to testing. This process is repeated for each test material. A round ADA, shown in Figure 3-5,
was designed for deposition of spores onto a 1.5- by 1.5-in (3.8- by 3.8-cm) square area of a
shard of subway concrete.

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Figure 3-5. Round ADA.
The top layer of ballast was inoculated with Bg spores from an MDI using a 14- by 14-in
(35.6- by 35.6-cm) ADA (Figure 3-6), clamped to the top of the ballast assembly. An MDI was
used to inoculate the top layer through the ADA lid.
ADA vntb lid in
cbsed position
syrioge niter
Figure 3-6. 14- by 14-in ADA.
To ensure the MDIs were functioning properly, stainless-steel control coupons (14- by
14-in) were inoculated prior to, during, and after each test inoculation.

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3.3 Decontamination Approach
This section discusses the decontamination agents and decontamination testing approach.
3.3.1 Decontaminants
The bleach-based decontamination solutions were selected based on published
information indicating that pAB can reduce bacterial spore populations (> 6 LR) under specific
conditions related to concentration, pH, contact time, and material [13], In 2001/2002, EPA
issued crisis exemptions [14] permitting the limited sale, distribution, and use of EPA-registered
bleach products against Ba spores at some facilities and locations, including Capitol Hill, the
U.S. Postal Service Processing and Distribution Centers at Brentwood (Washington, DC) and
Hamilton (Trenton, NJ), the Department of State, the General Services Administration, and
Broken Sound Boulevard (Boca Raton, FL). Each decontamination solution is discussed in detail
below.
3.3.1.1	pAB Solution
Adjusting the pH of bleach from alkaline to acidic ranges enhances the sporicidal activity
of bleach-based decontaminants due to the decreased disassociation of hypochlorous acid
(HOC1) and hypochlorite anions (OC1"). At pH ranges above ~9, most of the solution remains in
the OC1" form, but at pH ranges below ~7, the equilibrium changes so that most of the solution is
in the HOC1 form [15], The sporicidal effectiveness of HOC1 is much greater than the sporicidal
effectiveness of OC1", with a relative effectiveness at killing spores on the order of 100 to 1 by
comparison [16], Additionally, the combination of HOC1, OC1", and Cb in pAB yields an optimal
sporicidal mixture of highly oxidizing chlorine species, together referred to as free available
chlorine (FAC) (adopted from [15]).
The specific conditions for preparing the pAB under the EPA crisis exemptions are
summarized as follows: A bleach solution (5,000 to 6,000 ppm FAC concentration) was prepared
at a pH close to but not above 7 (tested by a paper test strip) by mixing one part bleach (5.25 to
6% sodium hypochlorite concentration) with one part vinegar and eight parts water as follows:
¦	One part bleach (with a 5.25 to 6% sodium hypochlorite concentration)
¦	One part white vinegar
¦	Eight parts water
¦	Bleach and vinegar were not combined directly. Water was first added to the bleach
(two cups water to one cup of bleach), then vinegar (one cup), and then the remaining
water (six cups)
Contact time for all treated surfaces was 60 minutes with repeated applications as
necessary to keep the surfaces wet.
The pAB solution prepared for this project deviated from EPA crisis exemption
conditions by using a more concentrated Clorox® Concentrated Germicidal bleach (8.3%
sodium hypochlorite concentration) while the pH was maintained at ~7. A previous study [17],
suggested that an increase in FAC may increase the sporicidal efficacy of the pAB for some
materials. Further, the contact time for the subway material decontamination testing was reduced
to 30 minutes.

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The listed working volume for this chamber is 16 ft3 (442 L), with internal dimensions in
terms of height by width by depth of 30-in (76 cm) by 30-in (76 cm) by 30-in (76 cm). The test
chamber has temperature and humidity ranges of -73 °F (-58 °C) to 175 °F (79 °C) and 10% to
95% relative humidity (RH), respectively. The chamber was modified to include a custom spray
system that was controlled from outside the unit so that environmental conditions within the test
chamber were not altered during decontamination testing.
Figure 3-8. Spray system setup inside the ETC.
The controller box consists of the spray controller button, a pressure regulator that adjusts
the pressure at which the decontaminant is delivered, the timers that adjust the spray duration,
the controller power supply, and the air lines that power the spray system. To eliminate coupon
overspray and to ensure there is enough space for coupons underneath the nozzles, only three
coupons were sprayed at a time. Three nozzles were deployed during testing: the center nozzle
and both outside nozzles.
The ETC timer was adjusted prior to testing to ensure that the proper volume per square
inch was delivered onto the horizontal coupon surface. Each test coupon was placed flat in a high
quality, thick-grade 83A- by 6- by l3/4-in polypropylene tray (Paksh Novelty, Model No. 26443,
Amazon.com). The trays were sufficiently sized to ensure that the entirety of the spray from each
nozzle was collected. The spray nozzle flow rate was adjusted to deliver a total flow of 11
mL/spray and cover an area of 5 inches in diameter. This spray delivery ensured that the 1.5- by
1.5-in sample area within each coupon received a decontamination solution volume that was
equivalent to the BOTE project application volume per surface area (i.e., 580 mL/m2). Once the
application method was established, decontamination testing was conducted as outlined in Table
3-3.

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Application of the bleach-based decontamination solutions to ballast materials was
conducted with a ShurFlo 4 ProPack Rechargeable Electric Back Pack Sprayer SRS-600
(Pentair-ShurFlo, Costa Mesa, California), shown in Figure 3-9. After rinsing with DI water, the
decontamination solution was prepared directly in the four-gallon tank of the sprayer. The flow
rate of each sprayer was verified to be approximately 1 liter/minute (L/min) before and after each
test using a 250-mL graduated cylinder and a stopwatch. The liquid was collected and volume
recorded based on a 10-second (sec) spray time. The spray height was adjusted so that the spray
cone encompassed the entire 14- by 14-in ballast layer surface.
1
Figure 3-9. Electric backpack sprayer.
Although the amount of pAB (~ 5,400 mL/nr) used was based on findings from the
BOTE project [18], the amount of pAB was increased by a factor of two or more, to compensate
for the large ballast surface area. For initial tests, pAB was sprayed onto the ballast at a flow rate
of 1 L/min for a duration of 5 sec, then re-sprayed 15 min later for the another 5-sec duration.
For later tests, the spray duration was changed to 10 sec to determine if increasing the spray time
would lead to full decontamination
The test matrix summarized in Table 3-4 was designed to examine the decontamination
efficacy of the bleach-based solutions on the surface of the top layer of crushed rock ballast as
well as in the same material middle and bottom layers of the three-layer assembly.

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4 Sampling and Analysis
Decontamination efficacy is determined from the recovered CFU per sample expressed
on a log-10 scale. Sampling results for positive controls of the same material are compared to
post-decontamination results for the test samples. Positive control samples are inoculated on the
same day and analyzed on the same day as test samples but are not decontaminated.
Additional measurements before and during the decontamination procedure also are
required to ensure QC for the testing. These measurements include QC checks on the reagents
and equipment used in the decontamination procedure.
A sampling data log sheet was maintained for each sampling event (or test) that included
each sampling event, the date, test name, sample identification numbers, and other test details
(such as test temperature, final runoff volume, and sample extraction time).
The following sections discuss the sampling approach, sampling frequency and
monitoring events, quality assurance (QA)/QC sampling, sample handling, analytical procedures,
characterization of the decontamination solutions, and determination of the decontamination
efficacy.
4.1 Sampling Approach
The sampling approaches for the subway materials and track ballast are discussed below.
4.1.1 Phase I: Subway Materials
Before the sampling event, all materials needed for sampling were prepared using aseptic
techniques. Nonpowdered surgical gloves were used during sampling. Dispatch® bleach wipes
(The Clorox Company, Oakland, CA) were used to sterilize each sample secondary container. A
sampling material bin was stocked for each sampling event based on the sample quantity. The
bin contained enough wipe sampling kits to accommodate all required samples for the specific
test. Five additional kits were also on hand for backup. Enough prepared packages of gloves and
bleach wipes were included in the bin as well as extra gloves and wipes.
Subway material samples consisted of wipe samples and liquid runoff samples as
discussed below.
4.1.1.1	Wipe Samples
Surface wipe samples from each material coupon were collected using polyester-rayon
blend (PRB) wipes (Curity all-purpose sponges #8042, 2- by 2-in, four-ply, Covidien PLC,
Dublin, Ireland). The protocol used in this project was adopted from the protocol provided by
Busher et al. [21] and Brown et al. [22], In short, the surface of the coupon was wiped, applying
a consistent amount of pressure, and using S-strokes horizontally and vertically to cover the
sample area. The wipe was folded and rolled to fit in a conical tube containing 10 mL of
phosphate-buffered saline with 0.05% Tween® 20 (PBST), and a pre-determined amount of STS
as a neutralizer. For a typical 6,000 ppm FAC, 11 mL volume bleach solution, the STS amount
was approximately 2 mL of a 2.26N stabilized STS titrant solution (HACH, P/N 26869-01,
Ames, Iowa). STS volumes were adjusted depending on expected bleach volume and FAC

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concentration.
4.1.1.2	Liquid Runoff Samples
Liquid runoff samples were collected from each tray that contained a test coupon. After
the wipe sampling of the surface of the coupon, the coupon was removed and the liquid runoff
was transferred to a 50-mL conical tube containing 10 mL of PBST and a pre-determined
amount of STS as a neutralizer. The transfer of the runoff was completed using a 10-mL
serological pipette (Costar® STRIPETTE®, Part No. 4488, Corning Inc., NY). A PRB wipe was
used to wipe the collection tray and remove remaining decontamination liquid. This wipe then
was added to the 50-mL liquid runoff conical tube to constitute one runoff sample.
4.1.2 Phase II: Track Ballast
The Phase II track ballast samples consisted of ballast rock and liquid runoff samples.
4.1.2.1	Ballast Rock Samples
After the decontamination procedure, ballast rocks from each layer were collected in
separate sterile 1 LNalgene containers (# 2187-0032, Thermo Fisher Scientific, Waltham,
Massachusetts). Using sterile gloves, samplers filled the Nalgene bottles to a maximum of half
full (~ 500 mL) to allow room for addition of extraction liquid.
4.1.2.2	Liquid Runoff Samples
Liquid runoff samples of the decontamination solution and water rinse that seeped
through all three layers of the ballast assembly. The samples were collected in 1-L Nalgene
bottles though a collection port located at the bottom of the three-layer ballast assembly. The
total mass of runoff collected was determined by first measuring the weight of the empty
collection bottle (containing only the neutralizer: DE broth) and then measuring the weight of the
bottle with the collected liquid to determine the net volume of the runoff liquid.
4.2 Sampling Frequency and Monitoring Events
The sampling frequency and monitoring events for the subway materials and track ballast
are discussed below.
4.2.1 Phase I: Subway Materials
The front face of each coupon was the only surface sampled in this study. All coupons
were sampled using wipes. The liquid runoff from each coupon was collected during application
of the decontamination solution. For each test, the following samples were collected:
•	Three test coupons and one procedural blank coupon per material type (grimed
concrete, grimed painted stainless steel, grimed glazed ceramic, and cleaned
versions thereof)
•	Three positive control coupons (inoculated but not in contact with
decontamination solution)

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• One negative control (coupon not inoculated or in contact with decontamination
solution) for each material.
Procedural blank coupons served as indicators of procedural contamination between
coupons. The positive control coupons were used to determine recovery and LR of test coupons,
and the negative control coupon aided as an indicator of contamination intrinsic to the laboratory
space.
Table 4-1 lists the sampling frequency for each subway material coupon test.
Table 4-1. Sampling Frequency for Subway Material Coupon Tests
Sum pic Type
Sninplo Description
Uepliciites
I'Vequencv
Purpose
Test coupon
A surface sample collected from
inoculated and decontaminated
subway materials
3 per coupon type
1 set per
material type
Determine the number
of viable spores after
decontamination
Procedural
positive control
coupons
A surface sample collected from
inoculated subway materials and
non-decontaminated subway
materials
3 per coupon type
1 set per
material type
Determine the number
of viable spores
physically removed
from the coupon
Negative control
coupon
A surface sample collected from
non-inoculated and non-
decontaminated subway
materials
1 per coupon type
1 set per
material type
Determine extent of
cross-contamination
and/or the sterility of
coupons
Procedural blank
coupon
A surface sample collected from
non-inoculated and
decontaminated subway
materials
1 per coupon type
1 set per
material type
Determine extent of
cross-contamination
Positive control
coupon
A surface sample collected from
inoculated and non-
decontaminated subway
materials
3 per coupon type,
inoculated as the
first, middle, and
last coupons
1 per
inoculation
event
Determine the number
of viable spores
recoverable from the
coupons
Inoculation control
coupons (stainless
steel)
A surface sample collected from
inoculated and non-
decontaminated stainless steel
3 per inoculation
event, inoculated
immediately
before each
positive control
coupon
1 per
inoculation
Determine the number
of viable spores
deposited onto the
coupons and to assess
the stability of the MDI
Laboratory
material blanks
Plating of aliquots or
representative portions of all
materials used
3 per material
Once per use
of material
Demonstrate sterility of
extraction and plating
materials
4.2.2 Phase II: Track Ballast
After a decontamination event, the sample liquid was collected in a 1 L sterile Nalgene
bottle. The spray frequency varied depending on the testing approach as outlined in Table 4-2.

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Table 4-2. Sampling Frequency for Track Ballast Tests
Siimplo Tjpe Siimpk* Description Qu;m(i(\ I-'iv(|ik*iio Purpose
Post-
decontamination
ballast
Inoculated and
decontaminated ballast
3 samples (top,
middle, and bottom
layers)
3 sets per
test
condition
Determine number of viable
spores in each ballast layer
after decontamination
Positive control
sample
Inoculated and non-
decontaminated ballast
1 sample (top layer)
3 sets per
test
condition
Determine number of viable
spores recoverable from first
ballast layer
Post-procedural
positive control
ballast sample
Inoculated ballast sprayed
with water only
3 samples (top,
middle, and bottom
layers)
3 sets per
test
condition
Determine number of viable
spores physically removed
using DI water only
Negative control
Non-inoculated and non-
decontaminated ballast
1 layer
1 sample
per test
condition
Determine extent of cross-
contamination and/or the
sterility of coupons
Procedural blank
coupon
Non-inoculated and
decontaminated ballast
3 samples (top,
middle, and bottom
layers)
1 set for
both test
conditions
Determine extent of cross-
contamination
Inoculation
control coupons
(stainless steel)
Surface sample from
inoculated and non-
decontaminated stainless
steel
3 per inoculation
event, inoculated at
the beginning,
middle, and end of a
test set
1 per
inoculation
Determine the number of
viable spores deposited onto
and to assess the stability of
the MDI
Runoff (liquid)
Effluent from sprayed pAB
containing STS neutralizer
2 per spray event
1 per spray
event
Determine if residual spores
are transported downstream in
runoff from decontaminating
ballast
Runoff (liquid)
Effluent from DI water
spray
2 per spray event
1 per spray
event
Determine if residual spores
are transported downstream
from spraying decontaminated
ballast
Laboratory
material blanks
Plating of aliquots or
representative portions of all
materials used
3 per material
Once per
use of
material
Demonstrate sterility of
extraction and plating
materials
After an overnight drying process, the ballast material was collected for analysis. A
single ballast assembly was considered one test sample, and each test sample was divided into
three layers (top, middle, and bottom). Because of the sheer amount and volume of rocks
contained in each layer, only a 3- by 3-in center area of each layer was sampled and analyzed
after an overnight drying period. Samples from each layer were collected separately in Nalgene
bottles.
For each test, one positive control sample that consisted of the top layer of the ballast
assembly was collected separately in the same way as the test samples, but (in this case) the
ballast system did undergo decontamination. Likewise, a separate single layer of ballast was
collected and used as a negative control sample (a layer neither inoculated nor in contact with
decontamination solution).
Additionally, three 14- by 14-in stainless-steel coupons per test set were inoculated at the
beginning, middle, and end of each test set inoculation process to verify the consistency of spore
application associated with each MDI as described in Section 3.2.2. These coupons served as
inoculation control coupons.

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4.3	Sample Handling
This section discusses the sample containers and sample preservation.
4.3.1	Sample Containers
For each wipe sample and grime sample, the primary containment container was an
individual, sterile, 50-mL conical tube. Secondary and tertiary containment consisted of sterile
sampling bags.
Liquid effluent samples were collected in individual sterile specimen caps or Nalgene
bottles placed inside pre-labeled sterile bags for secondary containment.
A single container was used for storage in the decontamination laboratory during
sampling and for transport to the BioLab.
4.3.2	Sample Preservation
All sample tubes and bottles were stored in secondary containment and kept together
until processing. All individual sample containers remained sealed while in the decontamination
laboratory, during transport, and until processing in the BioLab.
4.3.3	Sample Custody
After sample collection for a single test was complete, all biological samples were immediately
transported to the NHSRC BioLab accompanied by a Chain of Custody (COC) form.
4.4	Analytical Procedures
This section discusses the method used to determine the best extraction procedure for the
track ballast and the microbiological analysis.
4.4.1 Track Ballast Extraction Method
The purpose of this test was to evaluate several spore extraction methods and select one
that provided the best spore recovery from ballast material used in stackable assemblies. Using
an MDI, a single 12- by 12-in rock layer was uniformly inoculated with 1 x 107 spores of Bg.
Inoculated rocks were aseptically recovered in sterile 1 L Nalgene bottles such that each bottle
was filled to a maximum of half full to leave room for the addition of extraction liquid. Each
sampled layer resulted in the use of eight 1 L Nalgene bottles. The following protocols were used
to evaluate five different extraction techniques:
1.	Extraction Method 1: Aseptically add 400 mL of PBST to each of the eight Nalgene
bottles to immerse the ballast, and sonicate 10 minutes. Aseptically combine all eight
volumes into one sterile container, and then plate a sample at the zero dilution.
2.	Extraction Method 2: Aseptically add 400 mL of PBST to each of the eight Nalgene
bottles to immerse the ballast, sonicate 10 minutes, shake at 250 revolutions per minute
(rpm) for 10 minutes, and then sonicate for another 5 minutes. Aseptically combine all
eight volumes into one sterile container, and then plate a sample at the zero dilution.

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4.4.2 Microbiological Analysis
The BioLab analyzed all samples to check for presence of spores (sterility check
samples) and to quantify the CFU per sample (wipe samples, ballast rocks, and liquid runoff).
For all sample types, PBST was used as the extraction buffer. After the extraction procedure,
each sample was aliquoted and plated in triplicate using a spiral plater (Autoplate 5000,
Advanced Instruments Inc., Norwood, MA), which deposits the sample in exponentially
decreasing amounts across a rotating agar plate in concentric lines to achieve three 10-fold serial
dilutions on each plate. Plates were incubated at 35 ± 2 °C for 16 to 19 hours. The colonies on
each plate were enumerated using a QCount® colony counter (Advanced Instruments Inc.,
Norwood, MA).
Positive control samples were diluted 100-fold (10"2) in PBST before spiral plating, and
samples of unknown concentration were plated undiluted and after a 100-fold dilution. Samples
with known low concentrations were plated undiluted. The QCount® colony counter
automatically calculates the CFU/mL in a sample based on the dilution plated and the number of
colonies that develop on the plate. The QCount® records the data in an MS Excel spreadsheet.
Only plates meeting the threshold of at least 30 CFU were used for spore recovery
estimates. Samples below the 30-CFU threshold were either spiral plated again with a more
concentrated sample aliquot or spread-plated in triplicate on tryptic soy agar (TSA) plates using
1-mL aliquots per plate to achieve a lower detection limit. The plates were incubated at 35 ± 2
°C for 20 to 24 hours before manual enumeration.
4.5	Characterization of Decontamination Solutions
This section discusses the characterization of the two bleach decontamination solutions,
pAB and DB, which involved the determination of FAC by titration and pH and temperature
measurements.
4.5.1	Determination of FAC by Titration
FAC was measured with an iodometric method that uses a HACH digital titrator (Model
#16900, HACH, Loveland, CO) and a HACH reagent titration kit. The HACH digital titrator
manual [23] discusses the titration procedure and FAC concentration.
4.5.2	pH and Temperature Measurements
The pH and temperature of the bleach solutions were measured daily using a calibrated
pH meter (Oakton® Acorn™ pH 5, OAKTON Instruments, Vernon Hills, IL, USA). The
temperature sensor included with the pH meter was factory-calibrated and checked monthly by
comparison of the displayed value to a NIST-certified thermometer or other thermometer known
to be accurate.
4.6	Determination of Decontamination Efficacy
The overall effectiveness of a decontamination technique is a measure of the ability of the
method to inactivate or remove spores from material surfaces while considering viable spores
that might be relocated to runoff and aerosol fractions. Such fugitive biological emissions could

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result in secondary contamination that would necessitate additional remediation.
Data reduction was performed on measurements of the total spores (CFU) recovered from
each replicate sample, average recovered CFU, and standard deviation (STD) for each group of
samples. The groups of samples included the following for each combination of material type
and extracted sample type:
•	Positive control areas (replicates, average, STD);
•	Test areas (replicates, average, STD);
•	Procedural blank coupons.
Efficacy is defined as the LR of the viable Bg spores recovered from the surface of the
test material after the decontamination procedure has been reduced from the positive control
areas (not exposed to the decontamination procedure). LR is calculated as follows:
LR = Mean (Log CFU positive control sample) - Mean (Log CFU test sample) (1)

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6 Summary
The sporicidal efficacy of bleach-based decontamination solutions (pAB and DB) was
evaluated on clean and grimed subway concrete, glazed ceramic tile, and painted stainless steel
coupons and on ballast rock, inoculated with 1 x 101 Bg spores on each respective surface. The
findings and results of this study are summarized below for the subway materials and track
ballast.
6.1	Subway Materials
•	The pAB and DB (20,000 ppm diluted Clorox® Concentrated Germicidal Bleach)
solutions achieved full inactivation of Bg spores on all material surfaces and in all
liquid runoff, with no difference in performance between grimed and clean
materials.
•	The DB solution with a target FAC concentration of 6,000 parts per million (ppm)
showed greater than 6 LR on cleaned material surfaces, regardless of material
type, and only a few recovered CFU on some surfaces tested. Alternately, less
than 6 LR was observed on grimed surfaces, regardless of material type. These
results suggest that grime affects the sporicidal inactivation of spores on surfaces
by one to three orders of magnitude. Therefore, DB at the 6,000-ppm target FAC
concentration apparently was not an effective decontamination solution for
subway materials subject to grime deposits over time. Cleaning of surfaces prior
to application of DB would be necessary to achieve full decontamination of
grimed materials using the DB solution with a 6,000-ppm FAC.
•	No spores were detected in runoff for all three bleach-based solutions (pAB, 6,000-
ppm FAC DB, and 20,000-ppm FAC DB), regardless of material type or surface
treatment (cleaned or grimed). The 30-minute contact in the collected runoff was
long enough to decontaminate all spores displaced during the spraying process.
Hence, the generated runoff during the remediation following a real incident would
not contain spores
6.2	Track Ballast
•	A pre-decontamination wetting results in dissemination and stratification of the
spores over the 9-in depth of the track ballast, depending on the volume per area
sprayed. For example, a 1,760- mL/m2 spray resulted in 83% of the inoculated
spores in the top ballast layer (3-in depth), 15% in the middle 3-in layer, and 2%
in the bottom 3-in layer. No runoff was recovered for this volume of spraying.
Increasing the spray level to 5,680 mL/m2 resulted in even larger stratification of
the spores (28, 14, 7, and 51% in the top, middle, and bottom layers and runoff,
respectively).
•	The pAB solution was very effective, with no spores detected in all three ballast
layers and in runoff at a volume sprayed per area of 3,740 mL/m2. At a lower
volume sprayed per unit area of 1,770 mL/m2, pAB still was very effective (none
detected) in top 6-in track ballast layer but less effective (LR < 6) in the bottom
layer (greater than 6-in depth).

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• The DB solution (4,000 ppm < FAC <5,000 ppm) achieved greater than 6 LR
over the 9-in height of the ballast at a volume sprayed of 2,210 mL/m2 and was
fully effective (none detected) at a volume of 3,700 mL/m2. CFU spores were
observed in the runoff collected during the spraying process. However, full kill
(no spores detected) was observed over 24 hours of contact time. As such,
collected runoff would be free of viable spores. However, if not captures, runoff
would end up in the soil below the ballast bed where the bleach containing runoff
would reduce its reactivity due to the organic loading of the soil leaving residual
spores in the soil.

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7 Quality Assurance and Quality Control
All test activities were documented in laboratory notebooks and digital photographs. The
documentation included, but was not limited to, a record for each decontamination procedure,
any deviations from the quality assurance project plan, and physical impacts on materials. All
tests were conducted in accordance with established EPA Decontamination Technologies
Research Laboratory (DTRL) and NHSRC RTP Microbiology Laboratory procedures to ensure
repeatability and adherence to the data quality validation criteria set for this project. Standard
operating procedures were available for the maintenance and calibration of all laboratory
equipment.
The following sections discuss the criteria for the critical measurements and parameters,
data QA/QC, and QA/QC checks for the project.
7.1	Criteria for Critical Measurements and Parameters
Data quality objectives (DQOs) are used to determine the critical measurements needed
to address the stated project objectives and specify tolerable levels of potential errors associated
with simulating the prescribed decontamination environments. The following measurements
were deemed critical to accomplish part or all the project objectives:
•	Sprayer flow rate,
•	Runoff volume collected,
•	pH of decontamination solutions,
•	FAC concentration of decontamination solutions,
•	Spray time,
•	Exposure time,
•	Temperature of incubation chamber,
•	CFU counts,
•	Plated volume,
•	Neutralizer volume,
•	Total volume per unit area sprayed, and
•	Sample volume collected.
7.2	Data QA/QC
Table 7-1 lists the DQIs and criteria for the critical measurements used to determine if the
collected data met the QA objectives. Volumes of components were measured as accurately as
possible using appropriate measuring equipment (such as volumetric flasks, graduated cylinders,
or scales). Commercial products such as Clorox® bleach and glacial acetic acid (99%) were used
to prepare the decontamination solutions.

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7.3.2 NHSRC BioLab Control Checks
Quantitative standards do not exist for biological agents. Viable spores were counted
using an Advanced Instruments QCount® colony counter. Counts greater than 300 or less than 30
CFUs were considered outside the quantitation range. If the CFU count did not fall within the
acceptable quantitation range, the sample was re-plated at a different volume or dilution and then
re-counted.
Before each batch of plates was enumerated, a QC plate was analyzed, and the result was
verified to be within the range indicated on the back of the QC plate. As the plates were counted,
a visual inspection of colony counts made by the QCount® colony counter was performed.
Obvious count errors made by the software were corrected by adjusting the settings (such as
colony size, light, and field of view) and by re-counting using an edit feature of the QCount®
software that allows manual removal of erroneously identified spots or shadows on the plate or
by adding colonies that the QCount® software may have missed.
The acceptance criteria for the critical CFU counts were set at the most stringent level
routinely achievable. Positive controls were included along with the test samples so that spore
recovery from the different surface types could be assessed. Background checks also were
included as part of the standard protocol to check for unanticipated contamination. Replicate
coupons were included for each set of test conditions to characterize the variability of the test
procedures.
Further QC samples were collected and analyzed to check the ability of the NHSRC
BioLab to culture the test organism as well as to demonstrate that the test materials used did not
contain pre-existing spores. The checks included the following:
•	Positive control coupons: Coupons inoculated in tandem with the test coupons to
demonstrate the highest level of contamination recoverable from a specific
inoculation event.
•	Procedural blank coupons: Material coupons sampled in the same fashion as
test coupons but not inoculated with the surrogate organism before sampling.
•	Sterility checks: Swab samples for biological data quality indicators (DQIs) were
used for sterility checks on coupons, materials, and equipment before use in the
testing. A single, pre-moistened swab sample was collected from each item and
coupon. Grime samples were collected in 50-mL conical tubes for each batch of
grimed coupons as a sterility check.
•	Blank TSA sterility controls: Plates incubated but not inoculated.
•	Replicate plates of diluted microbiological samples: Replicate plates for each
sample.
•	Unexposed field blanks: Material coupons sampled in the same fashion as test
coupons but not inoculated with the surrogate organism before sampling or
exposed to the decontamination process.
Table 7-2 lists the additional QC checks built into the NHSRC BioLab procedures
designed to provide assurance against cross-contamination and other biases in the

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8 References
[1]	EPA. Assessment of liquid and physical decontamination methods for environmental
surfaces contaminated with bacterial spores: Development and evaluation of the
decontamination procedural steps. U.S. Environmental Protection Agency, Office of
Research and Development, Washington, DC, EPA/600/R-12/025, 2012.
[2]	EPA. Assessment of liquid and physical decontamination methods for environmental
surfaces contaminated with bacterial spores: Evaluation of spray method parameters and
impact of surface grime. U.S. Environmental Protection Agency, Office of Research and
Development, Washington, DC, EPA/600/R-12/591, 2012.
[3]	EPA Homeland Security Strategic Research Action Plan 2016-2019. U.S. Environmental
Protection Agency, Office of Research and Development, Washington, DC, EPA/601/K-
15/001, 2015.
[4]	Serre, S. and L. Oudejans. Underground Transport Restoration (UTR) Operational
Technology Demonstration (OTD). U.S. Environmental Protection Agency, Office of
Research and Development, Washington, DC, EPA/600/R-17/272, 2018.
[5]	EPA. Guidance on Test Methods for Demonstrating the Efficacy of Antimicrobial
Products for Inactivating Bacillus Anthracis Spores on Environmental Surfaces. Federal
Insecticide, Fungicide, and Rodenticide Act (FIFRA) Scientific Advisory Panel (SAP)
Meeting Minutes No. 2007-05. Arlington, VA, 2007.
[6]	EPA. Interactions of CIO2 and H2O2 fumigants with dirt and grime on subway concrete.
U.S. Environmental Protection Agency, Office of Research and Development,
Washington, DC, EPA/600/R-14/226, 2014.
[7]	Einfeld, W., Boucher, R.M., Tezak, M.S., Wilson, M.C., and Brown, G.S. 2011.
Evaluation of surface sampling method performance for Bacillus spores on clean and
dirty outdoor surfaces. Sandia National Laboratories Report. SAND2011-4085.
[8]	NIOSH (National Institute for Occupational Safety and Health). 2003. Diesel Particulate
Matter (as Elemental Carbon): Method 5040: Issue 3. Available at
https://www.cdc.gov/niosh/docs/2003-154/pdfs/5040.pdf. Last accessed August 7, 2018.
[9]	CSX. Standard specifications for the design and construction of private sidetracks. Issued
by: Office of the Vice President—Engineering CSX Transportation, Inc. Available at:
https://vvvvvv.csx.com/index.cfm/librarv/files/customers/industrial-development/site-
design-guidelines-and-specifications/. Last accessed August 7, 2018.
[10]	Gibbons, H.S., Broomall, S.M., McNew, L.A., Daligault, H., Chapman, C., Bruce, D.,
Karavis, M., Krepps, M., McGregor, P.A., Hong, C., Park, K.H., Akmal, A., Feldmann,
A., Lin, J.S., Chang, W.E., Higgs, B.W., Demirev, P., Lindquist, J., Liem, A., Fichler, E.,

-------
Read, T.D., Tapia, R., Johnson, S., Bishop-Lilly, K.A., Detter, C., Han, C.,
Sozhamannan, S., Rosenzweig, C.N., and Skowronski, E.W. 2011. Genomic signatures
of strain selection and enhancement in Bacillus atrophaeus var. globigii, a historical
biowarfare simulant. PLoS ONE. 6(3): el7836.
[11]	Lee, S.D., Ryan, S.P., and Snyder, E.G. 2011. Development of an aerosol surface
inoculation method for Bacillus spores. Applied and Environmental Microbiology. 77(5):
1638-1645.
[12]	Calfee, M.W., Lee, S.D., and Ryan, S.P. 2013, A rapid and repeatable method to deposit
bioaerosols on material surfaces. Journal of Microbiological Methods. 92(3): 375-380.
[13]	Calfee, M.W., Wood, J., Mickelsen, L., Kempter, C., Miller, L., Colby, M., Touati, A.,
Clayton, M., Griffin-Gatchalian, N., Payne, S., and Delafield, R. 2012. Laboratory
evaluation of large scale decontamination approaches. Journal of Applied Microbiology.
112(5): 874-882.
[14]	Canter, D.A., Gunning, D., Rodgers, P., O'Connor, L., Traunero, C., and Kempter, C.J.
2005. Remediation of Bacillus anthracis contamination in the U.S. Department of Justice
mail facility. Biosecurity andBioterrorism. 3(2): 119-127.
[15]	Frazer, A.C., Smyth, J.S., and Bhupathiraju, V.K. 2013. Sporicidal efficacy of pH-
adjusted bleach for control of bioburden on production facility surfaces. Journal for
Industrial Microbiology Biotechnology. 40(6): 601-611.
[16]	Brazis, A.R, Leslie, J.E., Kabler, P.W., and Woodward, R.L. 1958. The inactivation of
spores of Bacillus globigii and Bacillus anthracis by free available chlorine. Applied
Microbiology 6(5): 338-342.
[17]	Wood, J.P., Calfee, M.W., Clayton, M., Griffin-Gatchalian, N., and Touati, A. 2011.
Optimizing acidified bleach solutions to improve sporicidal efficacy on building
materials. Letters in Applied Microbiology 53(6): 668-72.
[18]	EPA. Bio-Response Operational Testing and Evaluation (BOTE) Project - Phase 1:
Decontamination assessment. U.S. Environmental Protection Agency, Office of Research
and Development, Washington, DC, EPA/600/R-13/168, 2013.
[19]	Calfee, M.W., Choi, Y., Rogers, J., Kelly, T., Willenberg, Z., and Riggs, K. 2011. Lab-
scale assessment to support remediation of outdoor surfaces contaminated with Bacillus
anthracis spores. Journal of Bioterrorism and Biodefense. 2(3): 1-9.
[20]	Dey, B.P., and Engley, Jr., F.B. 1994. Neutralization of antimicrobial chemicals by
recovery media. Journal of Microbiological Methods. 19(1): 51-58.

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[21]	Busher A., Noble-Wang, J., and Rose, L. 2008. Surface Sampling. Pages 95-131. In
Sampling for Biological Agents in the Environment. Emanuel, P., Roos, J., and Niyogi,
K., Editors. ASM Press, Washington, DC.
[22]	Brown, G.S., Betty, R.G., Brockmann, J.E., Lucero, D.A., Souza, C.A., Walsh, K.S.,
Boucher, R.M., Tezak, M., Wilson, M.C., and Rudolph, T. 2007. Evaluation of a wipe
surface sample method for collection of Bacillus spores from nonporous surfaces.
Applied and Environmental Microbiology 73(3): 706-710.
[23]	See (https://pim-resources.coleparmer.eom/instruction-manual/24Q08-00.pdf ). Last
accessed August 7, 2018.
[24]	EPA. Decontamination of Soil Contaminated with B. anthracis Spores. U.S.
Environmental Protection Agency, Office of Research and Development, Washington,
D C., EPA/600/R-13/110, 2013.

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