EPA/6Q0/R-21/0Q4 | May 2021
www.epa.gov/emergency-response-research
United States
Environmental Protection
Agency
&EPA
Inactivation of a Bacillus Spore
Surrogate on Outdoor Materials Via the
Spray Application of Sodium Dichloro-s-
triazinetrione and Other Chlorine-Based
Decontaminant Solutions
Office of Research and Development
Homeland Security Research Program
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AEPA
EPA 600/R21/004
Inactivation of a Bacillu Spore
Surrogate on Outdoor Materials Via the
Spray Application of Sodium Dichloro-s-
triazinetrione and Other Chlorine-Based
Decontaminant Solutions
Ep.LlSren.G.'ytn <">
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Disclaimer
The U.S. Environmental Protection Agency (EPA), through its Office of Research and
Development's (ORD) Homeland Security Research Program, funded and directed this
investigation through contract EP-C-15-008 with Jacobs Technology Inc. This report has been
peer and administratively reviewed and has been approved for publication as an EPA
document. It does not necessarily reflect the views of the Agency. No official endorsement
should be inferred. EPA does not endorse the purchase or sale of any commercial products or
services.
Questions concerning this document, or its application should be addressed to:
Joseph Wood
Office of Research and Development
U.S. Environmental Protection Agency (MD-E343-06)
109. T.W. Alexander Drive
Research Triangle Park, NC 27711
Phone: 919-541-5029
E-mail: wood.ioe@epa.gov
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The U.S. Environmental Protection Agency (EPA) is charged by Congress with protecting the
Nation's land, air, and water resources. Under a mandate of national environmental laws, the
Agency strives to formulate and implement actions leading to a compatible balance between
human activities and the ability of natural systems to support and nurture life. To meet this
mandate, EPA's research program is providing data and technical support for solving
environmental problems today and building a science knowledge base necessary to manage
our ecological resources wisely, understand how pollutants affect our health, and prevent or
reduce environmental risks in the future.
The EPA's Center for Environmental Solutions and Emergency Response (CESER) within the
Office of Research and Development (ORD) conducts applied, stakeholder-driven research and
provides responsive technical support to help solve the Nation's environmental challenges. The
Center's research focuses on innovative approaches to address environmental challenges
associated with the built environment. We develop technologies and decision-support tools to
help safeguard public water systems and groundwater, guide sustainable materials
management, remediate sites from traditional contamination sources and emerging
environmental stressors, and address potential threats from terrorism and natural
disasters. CESER collaborates with both public and private sector partners to foster
technologies that improve the effectiveness and reduce the cost of compliance, while
anticipating emerging problems. We provide technical support to EPA regions and programs,
states, tribal nations, and federal partners, and serve as the interagency liaison for EPA in
homeland security research and technology. The Center is a leader in providing scientific
solutions to protect human health and the environment.
Gregory Sayles, Director
Center for Environmental Solutions and Emergency Response
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Acknowledgments
The principal investigator from the U.S. Environmental Protection Agency (EPA) 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.
EPA Project Team
Joseph Wood, Principal Investigator, Center for Environmental Solutions and Emergency
Response, Homeland Security and Materials Management Division (CESER/HSMMD)
Leroy Mickelsen, Office of Land and Emergency Management/Chemical, Biological,
Radiological and Nuclear Consequence Management Advisory Division (OLEM/CMAD)
Shannon Serre, OLEM/CMAD
Anne Mikelonis, CESER/HSMMD
Anna Tschursin, OLEM/Office of Resource Conservation and Recovery
Richard Rupert, On-Scene Coordinator Region 3
EPA Quality Assurance
Ramona Sherman, HSMMD QA Director
Jacobs Technology. Inc.
Denise Popeo
Abderrahmane Touati
Denise Aslett
Ahmed Abdel-Hady
Jonathan Sawyer
Wendy Coss
EPA Technical Reviewers
Worth Calfee
Katherine Ratliff
External Technical Reviewers
Robert Miknis, US Department of Agriculture
Vipin Rastogi, US Department of the Army
iv
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Table of Contents
Disclaimer ii
Acknowledgments iv
Appendices vi
List of Figures vi
List of Tables vii
Acronyms and Abbreviations ix
EXECUTIVE SUMMARY x
1 INTRODUCTION 1
1.1 Background 1
1.2 Project Description and Objectives 1
2 EXPERIMENTAL APPROACH 3
2.1 Bench-Scale Experimental Approach 3
2.1.1 General Testing Process 3
2.1.2 Neutralizing Agents for 18-mm Extracted Samples 4
2.2 Pilot-Scale Experimental Approach 5
3 EXPERIMENTAL MATERIALS AND METHODS 8
3.1 Test Materials 8
3.2 Testing Facility 9
3.2.1 Bench-Scale Testing Facility 9
3.2.2 Pilot-Scale Testing Facility 10
3.2.3 Sprayers 11
3.2.3.1 Electrostatic Sprayer 11
3.2.3.2 Electric Backpack Sprayer 12
3.3 Decontamination Solutions 13
3.3.1 Dichlor (chlorinated granules) 13
3.3.2 Diluted Bleach 14
3.3.3 pH adjusted bleach (pAB) 14
4 Test Facility, Equipment and Material Sterilization 15
4.1 Test Surface Reset (Sterilization) 15
4.1.1 Equipment Sterilization - Vaporized Hydrogen Peroxide 15
4.1.2 Stainless-Steel MDI Control Coupon Sterilization - Autoclaving 15
4.1.3 Templates and Inoculation Equipment Sterilization - Ethylene Oxide (EtO) 15
5 Decontamination, Sampling, and Analysis Approach 16
5.1 Decontamination and Sampling Protocol 16
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5.1.1 Testing and Sampling Flow Timeline 16
5.2 Surface Sampling 20
5.2.1 Vacuum Sampling 20
5.2.2 Wipe Sampling 21
5.2.3 Swab Sampling 22
5.3 Sample Handling 23
5.3.1 Sample Containers 23
5.3.2 Sample Preservation 23
5.4 Microbiological Analyses 23
5.4.1 Wipe, Vacuum, and 18 mm Coupon Samples 24
5.4.2 Swabs 24
5.4.3 Spiral Plating and Filter plating 24
5.4.4 pH, Temperature, and FAC Measurements 25
5.5 Decontamination Efficacy 25
6 Results and Discussion 26
6.1 Bench-Scale Results 26
6.1.1 Test conditions 26
6.1.2 Decontamination Efficacy Results 26
6.2 Pilot-Scale Testing 28
6.2.1 Test Conditions 28
6.2.2 Decontamination Efficacy Results 30
7 Quality Assurance and Quality Control 34
7.1 Criteria for Critical Measurements/Parameters 34
7.2 Data Quality Indicators 34
7.3 Integrity of Samples and Supplies 35
7.4 BioLab Control Checks 36
8 References 39
Appendices
Appendix A: Material Coupon Fabrication 41
Appendix B: Test Chamber and Equipment Cleaning and Sterilization Procedures 43
List of Figures
Figure 2-1. 18-mm coupons 3
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Figure 2-2. 18-mm coupon spray stand (a) and coupon spray collectors (b) 4
Figure 2-3. Asphalt, concrete, brick, wood surface characteristics 5
Figure 3-1. Bench-scale spray apparatus for 18-mm coupons 10
Figure 3-2. Decontamination chamber for pilot-scale tests 11
Figure 3-3. Electrostatic sprayer 12
Figure 3-4. SHURflo SRS-600 electric backpack sprayer 13
Figure 5-1. Example alternating concrete and wood coupons in spray chamber 18
Figure 5-2. Spray pattern for electrostatic sprayer 19
Figure 5-3. Filter assembly and Sensidyne Gilian sampling pump 20
Figure 6-1. Bench-scale decontamination results 27
Figure 6-2. Pilot-scale decontamination results 31
List of Tables
Table 2-1. Bench-Scale Spray Test Matrix 7
Table 2-2. Pilot-Scale Spray Test Matrix 7
Table 3-1. Test Coupon Material Specifications 9
Table 3-2. Decontamination Equipment (Sprayers) 11
Table 5-1. Test Inoculation Sequence 17
Table 5-2. Sample Duration 12-in x 12-in Surface Area and 6-in Diameter Coupons 21
Table 5-4. Typical Sterility Check Sample Types for Each Testing Event 23
Table 6-1. Test Conditions for Bench-Scale Decontamination Testing on 18-mm Coupon
Materials 26
Table 6-2. Decontamination Efficacy of Diluted Bleach Solution on 18-mm Coupon Materials 27
Table 6-3. Decontamination Efficacy of Dichlor Solution on 18-mm Coupon Materials 28
Table 6-4. Decontamination Efficacy of pAB Solution on 18-mm Coupon Materials 28
Table 6-5. Test Conditions for Diluted Bleach Decontamination of 14-in x 14-in Coupon Materials 29
Table 6-6. Test Conditions for pAB (High FAC) Decontamination of 14-in x 14-in Coupon
Materials 29
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Table 6-7. Test Conditions for Dichlor Decontamination of 14-in x 14-in Coupon Materials 29
Table 6-8. Test Conditions for pAB (Low FAC) Decontamination of 14-in x 14-in Coupon Materials 30
Table 6-9. Decontamination Efficacy of Diluted Bleach Solution on Large Coupons 31
Table 6-10. Decontamination Efficacy of Dichlor Solution on Large Coupons 32
Table 6-11. Decontamination Efficacy of pAB (6500 ppm FAC) Solution on Large Coupons 32
Table 6-12. Decontamination Efficacy of pAB (20000 ppm FAC) Solution on Large Coupons 32
Table 7-1. DQIs for Critical Measurement 35
Table 7-2. Additional Quality Checks for Biological Measurements 38
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Acronyms and Abbreviations
2N
2 Normal
ADA
aerosol deposition apparatus
Bg
Bacillus atrophaeus var. globigii
BioLab
HSMMD microbiology laboratory
CFU
colony forming unit(s)
Dl
deionized
dichlor
dichloro-s-triazinetrione
DQI
data quality indicator
EPA
U. S. Environmental Protection Agency
ESS
electrostatic sprayer
EtO
ethylene oxide
FAC
free available chlorine
HSMMD
Homeland Security Materials Management Division
in
inch
LR
Logio reduction
MDI
metered-dose inhaler
NIST
National Institute of Standards and Technology
ORD
Office of Research and Development
pAB
pH-adjusted bleach
PBST
phosphate-buffered saline with 0.05% Tween® 20
ppm
part per million
QA
quality assurance
QC
quality control
RH
relative humidity
RSD
relative standard deviation
RTP
Research Triangle Park
SD
standard deviation
STS
sodium thiosulfate
TSA
tryptic soy agar
ix
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EXECUTIVE SUMMARY
The U.S. Environmental Protection Agency's 2020 Homeland Security Strategic Research
Action Plan was established to advance EPA's capabilities to recover from a wide-area
contamination incident, and the study described in this report supports that objective. This
small study evaluated the effectiveness of readily available and inexpensive chlorine-based
decontaminant solutions for inactivating spores of Bacillus atrophaeus var. globigii (Bg); a
widely used surrogate for Bacillus anthracis on outdoor surfaces.
More specifically, the primary purpose of this study was to evaluate the decontamination
efficacy of sodium dichloro-s-triazinetrione (also known as dichlor, a common pool
disinfection chemical), and compare the results to more well-known decontaminants such
as pH-adjusted bleach (pAB) and diluted bleach. The outdoor materials evaluated in this
study included concrete, wood, asphalt, and brick. Initial scoping tests were conducted at
bench-scale using 18-mm diameter coupons of the four materials, with test chemical
application to the coupons via an automated spray apparatus. Following these scoping
tests, the chlorine-based solutions were evaluated at a larger scale, whereby an
electrostatic sprayer was used to apply test chemicals onto 14-inch by 14-inch coupons. In
these pilot-scale tests, the pAB decontaminant was tested at two different free available
chlorine concentrations: one level consistent with a typical pAB formulation (~ 6,000 parts
per million free available chlorine), and one at a higher concentration, typical of the dichlor
and diluted bleach solutions' free available chlorine concentrations (~ 20,000 ppm). In both
scales of tests, decontamination efficacy was determined in terms of logio reduction (LR) of
colony forming units, based on the difference in the number of Bg spores recovered from
surfaces of positive controls and test materials.
For the bench-scale tests, the average decontamination efficacy for dichlor for the four
materials was a LR of 5.7, which was similar to the average LR achieved for the diluted
bleach (an average LR of 5.5 for the four materials). In contrast, the average LR for pAB
was 4.7 for the four materials. For the materials comprised of organic compounds (wood
and asphalt), dichlor achieved the highest decontamination efficacy (> 6.0 LR for both
materials) of the three decontaminant solutions evaluated. This result supports the
hypothesis that the dichlor chemical may be better able to withstand reduction of its biocidal
activity when in contact with organic materials. Although we caveat that this effect was less
pronounced in the pilot-scale tests.
The pilot-scale tests produced somewhat similar decontamination efficacy results. On
average, the decontamination efficacy was highest with the diluted bleach (an average LR
of 6.0 for the four materials), followed by dichlor (an average LR of 5.4 for the four
materials). Decontamination efficacy results for the two pAB solutions were the lowest of
the four decontaminant solutions studied. Additionally, the average efficacy across the four
materials was similar for both pAB solutions (average LR of 4.7-4.8), indicating that the
increased free available chlorine concentration of the pAB had minimal effect. In terms of
having the lowest number of spores recovered after decontamination, the results for diluted
bleach and dichlor were similar; both decontaminants were able to achieve at least one
sample with no spores detected, for every material.
With respect to impact of material type, decontamination efficacy was the highest for the
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brick material in every pilot-scale test, with > 6.0 average LR observed for each of the four
decontaminant solutions tested. Average decontamination efficacy ranged from 4.2-5.7 LR
for the concrete; 3.2-5.9 for the wood; and 4.2-5.6 for the asphalt. In general, the range in
decontamination efficacies observed for the 14-in x 14-in coupons was consistent with
efficacies observed for the smaller 18-mm coupons.
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1 INTRODUCTION
The U.S. Environmental Protection Agency's (EPA's) Homeland Security Strategic
Research Action Plan (U.S. EPA, 2020) was designed to advance EPA's capabilities to
recover from a wide-area contamination incident. The study discussed in this report helps
to fulfill that objective. Specifically, this study evaluated the effectiveness of commercially
available chlorine-based solutions for inactivating Bacillus atrophaeus var. globigii (Bg)
spores (surrogate for Bacillus anthracis) on outdoor surfaces. The chlorine-based
decontaminants that were evaluated included a common pool disinfection chemical,
acidified bleach, also known as pH-adjusted bleach (pAB), and diluted bleach.
1.1 Background
Following a large, intentional release of B. anthracis spores in a metropolitan area, outdoor
materials would likely become contaminated with the biological agent. Materials used in
outside environments are typically porous and may be comprised of organic matter,
making their decontamination difficult. While pAB and dilute bleach have been shown to be
efficacious in inactivating B. anthracis spores on many materials, little information or data
have been reported on the efficacy of using commercial, off-the-shelf pool chemicals
(mixed with water) for inactivation of B. anthracis spores on building and outdoor materials.
In this study, we evaluated the decontamination efficacy for solutions of the commercial,
off-the-shelf pool chemical "dichlor" (i.e., sodium dichloro-s-triazinetrione dihydrate [CAS
Number 51580-86-0], also known as sodium dichloroisocyanurate dihydrate; chemical
formula C3Cl2N3Na03-2H20). We also evaluated solutions of pAB and dilute bleach applied
under the same conditions, to compare with dichlor. The use of a widely available
granular chemical such as dichlor, that could simply be mixed with water at its point of use,
would be an advantageous decontamination tool to have in the event of a wide area
release of B. anthracis spores.
Dichlor is used for chlorination of swimming pools and spas and, when added to water, it
produces in solution sodium cyanurate and hypochlorous acid (Akamatsu et al.,1995). The
equilibrium between chloroisocyanurates and free available chlorine (FAC) may be the
reason for the potential enhanced microbiocidal efficacy of dichlor under organic burden,
as compared to bleach (Coates, 1988).
While a few decontaminants for B. anthracis or sterilants have incorporated the chemical
dichlor in their formulations (e.g., CASCAD™ surface decontamination foam) and have
been evaluated for their efficacy against B. anthracis or surrogate spores (Wood et al.,
2011a; Calfee et al., 2011), few if any decontamination studies have directly evaluated the
commercial, off-the-shelf pool chemicals themselves. In one study, dichlor solutions were
disseminated as a fog and found to be generally more effective on organic materials (e.g.,
carpet, wood) compared to pAB (US EPA, 2017a).
1.2 Project Description and Objectives
The purpose of this small study was to evaluate the decontamination efficacy of dichlor,
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pAB, and dilute bleach solutions when used to decontaminate different outdoor material
surfaces contaminated with Bg (surrogate for B. anthracis) spores. The chlorine-based
solutions were applied with an electrostatic sprayer onto the outdoor materials used in the
study, which included concrete, wood, asphalt, and brick. The asphalt and wood materials
are organic and would provide a glimpse into whether the dichlor solution is more effective
than the dilute bleach or pAB on materials that are challenging to decontaminate.
Decontamination efficacy was assessed through measurement of spores on positive
controls (not exposed to the decontaminant) compared to the number of spores recovered
from test surfaces. Sprayers were used to apply the chlorine-based decontaminant
solutions for both the bench- and pilot-scale tests.
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2 EXPERIMENTAL APPROACH
The general process investigated in this study was the decontamination of surfaces
contaminated with Bg spores using chlorine-based decontaminant spray solutions.
Coupons were inoculated with Bg spores, followed by spraying with selected
decontamination solutions. The recovery of Bg spores from the decontaminated coupons
was compared to recovery from the positive control coupons, which were inoculated but
not sprayed with the decontaminant. The surface decontamination efficacy was calculated
by the difference in mean Iog10 recoveries of positive control samples and those of post-
treated surface samples.
The study was divided into two phases. Initially, a bench-scale decontamination approach
using 18-mm diameter coupons (excised sample materials) was evaluated, followed by
pilot-scale tests using an electrostatic sprayer on 14-inch (in) x 14-in inoculated coupons.
2.1 Bench-Scale Experimental Approach
Decontamination tests were initially executed using small 18-mm brick, wood, asphalt, and
concrete coupons, shown in Figure 2-1. The coupons were assembled by punching 18-
mm diameter circular disks from the bulk material then affixing the punched discs to
aluminum 18-mm scanning electron microscope pin mounts (Ted Pella, p/n 16199; Ted
Pella, Redding, CA) with adhesive tabs (Ted Pella, p/n 16082).
Figure 2-1.18-mm coupons.
2.1.1 General Testing Process
The inoculations and spray procedures took place in a walk-in chemical hood located at
the EPA's Research Triangle Park (RTP) High Bay laboratory (North Carolina). The tasks
were performed on a solid stainless-steel bench placed inside the hood. A custom-made
spray apparatus was used for bench-scale spray applications, as described in detail and in
an approach similar to a previous study (U.S. EPA, 2017b).
Contact time, number of spray applications, and spray time were maintained constant for
each testing sequence. Replicates of three, 18-mm diameter coupons fabricated from
unpainted concrete, asphalt, treated pine-based plywood, brick, and stainless-steel were
inoculated with a nominal, target level of 1-5 x 107 Bg spores using an aerosol deposition
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apparatus (ADA) fitted with an actuator arid a metered-dose inhaler (MDI) described
elsewhere (Lee et al., 2011). For this investigation, stainless-steel coupons were used for
inoculum controls. Material coupons were extracted, plated, and enumerated in the
microbiology laboratory.
The inoculation of the test coupons and controls was performed the day prior to spraying
with the decontaminant solutions. After the chlorine-based solutions' free available chlorine
(FAC) concentration and pH were measured (within 1 hour of use), the prepared
decontaminant solution was transferred to the spray apparatus before use.
The test and procedural blank coupons were placed horizontally in the 18-mm coupon
spray collectors, and the collectors placed inside the spray stand. Figure 2-2(a) shows the
spray apparatus and Figure 2-2(b) shows a close-up photograph of the 18-mm coupons
positioned horizontally in the coupon spray collectors. The decontaminant spray was
applied with the coupon holder directly under the spray nozzle (McMaster-Carr, 32885K52,
Douglasville, GA) with the sprayer setting at 20 psi (flow rated at 0.3 gallons per minute at
this pressure). The sprayer nozzles were maintained approximately 6 inches from the
coupon surface and directed downward onto the top of the coupons. The automated
system provided a 3-second spray at time 0, 5, and 10 minutes, for a total of three sprays
and a total contact time of 15 minutes. (With the three, 3-second sprays, a nominal volume
of 170 ml_ was sprayed over each coupon.) The spray procedure was applied to each of
the four coupon materials simultaneously in a uniform manner.
(a) (b)
Figure 2-2.18-mm coupon spray stand (a) and coupon spray collectors (b).
2.1.2 Neutralizing Agents for 18-mm Extracted Samples
The presence of residual chlorine-based decontaminant solution remaining on the sample
surface after reaching the contact time could negatively bias colony forming unit (CFU)
quantification results. Based on previous studies, sodium thiosulfate (STS) was proven to
be an effective neutralizer for bleach on porous and non-porous surfaces (Calfee et al.,
2011), and was used during the post-decontamination recovery of the 18-mm coupons
retrieved from the spray apparatus. (The coupons were placed in the recovery media/STS
solution immediately following the 15-minute contact time.) The volume of STS that was
added to the collection media was determined by measuring FAC in the sample using an
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iodometrlc method for the determination of chlorine dioxide and chlorite using a HACH®
hypochlorite test kit (Model CN-HRDT, Fisher Scientific, Waltham, MA).
2.2 Pilot-Scale Experimental Approach
The 14-in x 14-in material coupons (brick, wood, and concrete) and 6-in diameter asphalt cores
were inoculated with Bg spores using an ADA inoculation approach (Calfee et al., 2013a).
(Note we had attempted to construct 14-in x 14-in asphalt coupons, but this was a labor-
intensive process and occasionally resulted in coupons that crumbled apart. We decided to use
the smaller, round asphalt coupons/cores that were taken as highway samples by the
Department of Transportation and gifted to us.) The targeted deposition was 107 spores on a
12-in x 12-in center portion of the 14-in x14-in coupon surface. The spores were deposited on
the top 6-in diameter surface of the asphalt cores by the same process as the 14-in x 14-in
coupons but using a different ADA sized for the smaller, round coupons. The total surface of
the top of the asphalt core was sampled. All sample surfaces were analyzed for the quantitative
determination of viable spores. Up-close photographs of the coupon surfaces are shown in
Figure 2-3.
Figure 2-3. Asphalt, concrete, brick, wood surface characteristics.
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The pilot-scale experimental approach is described below.
1. Preparation of representative samples of test materials: Test coupons were
prepared from building materials (asphalt, brick, concrete, and pressure-treated
plywood made from pine), as noted in Section 3.1.
2. Site preparation: Testing was performed on 14-in x 14-in coupons and 6-in diameter
coupons a laboratory decontamination chamber. Pre-test preparation of surfaces are
described in Section 4.
3. Surface Sterilization: Prior to each test, the test chamber underwent a sterilization
reset process to help prevent cross-contamination between tests (commonly referred to
as a reset of the test chamber). Sterilization of the chamber test surfaces was
accomplished using pAB applied with a backpack sprayer. If dichlor or diluted bleach
was used as the test decontaminant, then the selected test decontaminant was used to
reset the chamber instead of pAB. Decontaminant spray was followed by a deionized
(Dl) water rinse. The test chamber reset is described in Section 4.
4. Inoculation of test surfaces with the target organism: The test surfaces were
inoculated using an aerosol deposition method that delivered a known concentration of
spores in a repeatable fashion. A target, nominal inoculation level of 1-5 x 107 Bg
spores were deposited onto each test material, as discussed in Section 5.1.
5. Preparation of the decontamination solutions: Decontamination solutions—pAB,
dichlor, and diluted bleach—were freshly prepared on each test day, and Section 3.3
discusses preparation of the test solutions.
6. Application of decontaminant on test materials: Procedural blank and test coupons
were arranged in the vertical position, with materials alternating, within the test
chamber. The decontamination procedure for 14-in x 14-in coupons followed a spray
test protocol that consisted of spraying the test surface for a 20-second spray per
coupon. A 30-minute waiting period occurred between spray applications. Two
applications of decontaminant were used followed by an application of a Dl water rinse.
The decontamination spray protocol is discussed in Section 5.1. Although it is possible
that a small amount of spores may be transferred to the water rinsate in this process,
the rinse water was not assayed for spores.
7. Coupon Sampling Procedures: The sampling process involved wipe or vacuum
surface sampling dependent upon the coupon material. The sampling consisted of three
(3) positive controls, three (3) test surfaces, and two (2) non-inoculated test surfaces
(field blank and procedural blank) for each material.
8. Sample Extraction and Analysis. Viable Bg spores were extracted from the sampling
media, and aliquots of the extraction liquid were analyzed using the sample procedures
for microbiological analysis described in Section 5.4. Viable spore recovery was
quantified in terms of colony-forming units (CFUs) present in each sample.
The bench-scale and pilot-scale test matrices are listed in Table 2-1 and Table 2-2,
respectively. In the bench-scale tests, pAB was evaluated at its typical FAC concentration
of approximately 6,000-7,000 ppm. But in the pilot-scale tests, we decided to evaluate it at
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the typical concentration and at an FAC concentration comparable with the diluted bleach.
Table 2-1. Bench-Scale Spray Test Matrix
Decontamination
Solution
Test Area
Material
FAC Concentration
Target (PPM)
Concrete
20,000
Dichlor
Asphalt
Wood
Brick
Concrete
Diluted Bleach
Asphalt
20,000
Wood
Brick
Concrete
pAB
Asphalt
6,000
Wood
Brick
FAC = free available chlorine; pAB= pH-adjusted bleach
Table 2-2. Pilot-Scale Spray Test Matrix
Decontamination
Solution
Test Area Material
FAC Concentration
Target (PPM)
Concrete
Dichlor
Asphalt
20,000
Wood
Brick
Concrete
Diluted Bleach
Asphalt
20,000
Wood
Brick
Concrete
Asphalt
6,000
Wood
pAB
Brick
Concrete
Asphalt
20,000
Wood
Brick
FAC=free available chlorine; pAB = pH-adjusted bleach
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3 EXPERIMENTAL MATERIALS AND METHODS
This section describes the test materials, test chamber, spray apparatus, sprayers,
decontamination solution, test organism, inoculation procedures, and neutralizing agents
that were used during the study.
3.1 Test Materials
The representativeness and uniformity of test materials are essential for achieving
adequate evaluation results. Materials are considered representative if they are consistent
in content, quality, surface characteristics, and structural integrity. For this study,
representativeness was ensured by: (1) selecting test materials (2) obtaining these
materials from appropriate suppliers and (3) fabricating coupons as briefly discussed in
this report. Uniformity was maintained by obtaining and preparing a quantity of material
sufficient to allow the preparation of multiple test samples with uniform characteristics (that
is, test coupons per test were prepared using the same batch of material).
Concrete, wood, and brick coupons were prepared on site. Note that for the larger brick
coupons, mortar was used in their construction, but no mortar was used for the smaller 18
mm brick coupons, due to their small size. Asphalt sample cores were obtained from the
North Carolina Department of Transportation (DOT) and received with documentation
stating the material specifications. Table 3-1 lists the test coupon materials including
details such as thickness, category and suppliers or manufacturers. Appendix A describes
construction of coupons made at EPA RTP.
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Table 3-1. Test Coupon Material Specifications
Material
Description
Manufacturer/ Supplier
Name/Location
Coupon
Size
Press ure-
T reated
Plywood
3/4-in thick, 4' by 8' ACQ-D
pressure treated plywood
(made from pine)
Georgia-Pacific/Lowe's
Item #471096, model #34CC (or
equivalent) / Lowe's, Mooresville,
NC. Cut onsite.
14-in x 14-in
Concrete
coupon
14-in x 14-in, by 1-inch
thick
Constructed onsite at the EPA RTP.
(R)
Quikrete concrete mix (Atlanta,
GA). Plywood backing.
14-in x 14-in
Stainless
Steel
Multipurpose Stainless
Steel (48-in x48-in), type
304, #2B mill (unpolished),
0.036-in thick
McMaster-Carr
Atlanta, GA 30374-0100. Cut onsite.
14-in x 14-in
Brick Coupon
with mortar
Z-Brick original
manufactured brick veneer
2.3-in x 8-in each brick
Constructed on site at the EPA RTP
using Lowes Item #54658 Model
#ZC004205 brick veneer and
Quikrete Mortar Mix No.:1102.
Plywood backing.
14-in x 14-in
Asphalt
6-in diameter asphalt cores
with uniform compaction
and composition
Cut to size onsite from asphalt cores
received from North Carolina DOT
6-in diameter,
2 1/2-in
thickness
18-mm
coupon
Wood, asphalt, brick, and
concrete mounted on 18-
mm scanning electron
microscope pin mounts.
Constructed onsite at the EPA RTP.
Wood coupons were constructed
using treated plywood (listed above).
Brick coupons were constructed from
Lowes Item #54658 Model
#ZC004205 brick veneer. Asphalt
coupons were constructed from
melted DOT asphalt cores. Concrete
coupons were constructed from
Quikrete concrete mix
18-mm
3.2 Testing Facility
3.2.1 Bench-Scale Testing Facility
The bench-scale tests with the 18-mm coupon stubs were performed under a fume hood
utilizing a custom-made spray apparatus shown in Figures 3-1. The apparatus used 30-
degree full-cone nozzles to provide precise amounts of decontaminant spray at a known
flow rate and pressure, to all four coupons simultaneously.
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Figure 3-1. Bench-scale spray apparatus for 18-mm coupons.
3.2.2 Pilot-Scale Testing Facility
A designated decontamination spray test chamber was utilized to evaluate the
effectiveness of various decontamination solutions to remove or inactivate spores shown
in Figure 3-2.
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Figure 3-2. Decontamination chamber for pilot-scale tests.
3.2.3 Sprayers
This section describes the types of sprayers used this study. The sprayers, summarized in
Table 3-2, were used during testing for application of the decontaminant or rinse water.
Table 3-2. Decontamination Equipment (Sprayers)
Type
Description
Electric Backpack
(R)
SHURflo 4 ProPack rechargeable electric backpack sprayer SRS-600 (Pentair-
SHURflo, Costa Mesa, CA). This was used only for spraying Dl rinse water.
Electrostatic
MaxCharge™ SC-HD electrostatic sprayer, Electrostatic Spraying Systems,
Watkinsville, GA
Bench-scale
Spray Apparatus
Custom-made spray apparatus
3.2.3.1 Electrostatic Sprayer
The following sections discuss decontamination equipment (sprayers), decontamination
agents, and neutralizer used for extracted samples.
The air-assisted SC-ET HD electrostatic sprayer (ESS; Electrostatic Spraying Systems,
Inc., Watkinsville, GA), as shown in Figure 3-3, was used in this study to apply the
decontaminants at pilot-scale. We chose to use this sprayer for the pilot-scale tests based
on its performance in a previous study, in which it was shown that the ESS provided
similar decontamination efficacy as the backpack sprayer, but with much less waste
decontaminant. (U.S. EPA, 2018a) This ESS (approximately 22-in height x 16-in width x
10-in length) produces electrically charged spray droplets that are carried to the target in a
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low pressure, gentle, air stream. It contains a 4.7 L capacity liquid tank and spray gun with
hose length of 15 feet. From the manufacturer, the SC-ET HD ESS system is intended for
light-duty, quick disinfection and sanitization applications and is compatible with most
conventional chemicals. The ESS is equipped with a patented MaxCharge™ technology
electrostatic spray gun that delivers droplets with a volume median diameter of 40
micrometers (Mm). Air-assisted electrostatic spray technology gives more than twice the
deposition efficiency of hydraulic sprayers and non-electrostatic types of air-assisted
sprayers (Kabashima et al., 1995). Prior to testing, the spray distance was set to 1 foot to
cover the whole 14- by 14-in test area or 6-in diameter of asphalt.
Figure 3-3. Electrostatic sprayer.
A stopwatch and a 250 ml_ graduated cylinder were used to verify the flow rate of the
electrostatic sprayer. The liquid was collected and volume recorded based on a 30-sec
spray time. The approximate average flow rate of the electrostatic sprayer was 124
mL/min. Readings were expected to be within 10% of the average. If not, the spray gun
was checked for bleach corrosion and re-cleaned if necessary. During operation of the
ESS, personnel wore anti-static gloves (Part No. AS9674S, MCR Safety, Collierville, TN)
for safety.
3,2.3.2 Electric Backpack Sprayer
A SHURflo SRS-600 ProPack rechargeable electric backpack sprayer (SHURflo, Cypress,
CA; see Figure 3-4) was used only for application of a Dl water rinse following
decontamination, due to its high flow rate. This backpack sprayer can spray 120 gallons on
12
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one battery charge and is designed with variable speed and an adjustable nozzle for either
light or heavy-duty spraying. The sprayer may be maintained at a pressure of 35 pounds
per square inch (psi) and a flow rate of approximately 1 liter per minute during the spray
application sequence. A stopwatch and a 250 mL graduated cylinder were used to verify
the flow rate of the backpack sprayer. The approximate average flow rate of the backpack
sprayer was 1136 mL/min. The liquid was collected and volume recorded based on a 30-
sec spray time. Readings were expected to be within 10% of the average.
The backpack sprayer was only used for Dl water applications. The sprayer was
decontaminated using a VHP™ cycle in the airlock chamber.
3.3 Decontamination Solutions
3,3.1 Dichlor (chlorinated granules)
The focus of this study was to assess the decontamination efficacy of a pool chemical
solution (dichlor) and to compare it to other chlorine-based decontaminant solutions, for
four outdoor materials contaminated with B. anthracis surrogate spores. The dichlor
solutions were prepared by dissolving granules of sodium dichloro-s-triazinetrione
dihydrate (Pool Solutions, Pool Supply World, P/N PSW-CSC158-5; Brilliance for Spas, B
& G Builders Pools & Spas, Durham, NC) in deionized water, using 0.33 lb per gallon of Dl
water, to achieve a target concentration of 20,000 parts per million (ppm) (20,000 ppm =
2%) FAC. According to the manufacturer's label, the product is pH neutral and has 56%
Figure 3-4. SHURflo SRS-600 electric backpack sprayer.
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available chlorine.
3.3.2 Diluted Bleach
The diluted bleach solution was used with a target FAC concentration of 20,000 ppm (2%)
and prepared by using Clorox® concentrated germicidal bleach stock solution and diluted
with Dl water.
3.3.3 pH adjusted bleach (pAB)
pAB has been demonstrated to be an effective decontaminant in inactivating bacterial
spores, under specific conditions related to concentration, pH, contact time, and material
(Wood etal., 2011a; Calfee etal., 2012). A previous study by Wood et.al., 2011b,
suggested that an increase in FAC may increase the sporicidal efficacy of the pAB for
some materials.
The pAB solution was prepared by using a concentrated Clorox® concentrated germicidal
bleach (6% sodium hypochlorite concentration), while maintaining pH around 7.
The concentration of household bleach and the strength of white vinegar can vary by batch
and storage time. Therefore, the formulation listed above can vary in pH and chlorine
concentration depending on the starting reagents, and storage time. Before use, the pH
and FAC levels were measured at the start and monitored throughout each test. The
formulation for pAB is as follows:
• One-part bleach (with a listed 8% sodium hypochlorite concentration)
• One-part vinegar
• Eight parts water
• Bleach and vinegar are not to be combined together directly. Water was first added to
the bleach (two cups water to one cup of bleach), then vinegar (one cup), followed by
the remaining water (six cups).
The pAB was prepared using germicidal bleach (Clorox® bleach, Clorox Corp., Oakland,
CA), water, and 5% acetic acid (prepared from certified glacial acetic acid, in lieu of
vinegar; vinegar typically contains ~ 5% acetic acid). The final pH of the pAB solution was
6.5-7.0 with a target FAC concentration of 6000-6800 ppm. The pAB with an FAC
concentration between 19,630 and 20,431 ppm was also evaluated in the pilot-scale
portion of the study.
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4 Test Facility, Equipment and Material Sterilization
4.1 Test Surface Reset (Sterilization)
The following is a discussion of the techniques used to "reset" (sterilize) test equipment
after an experiment so that it can be reused in subsequent experiments without causing
contamination from the target spore organism. Different techniques are used depending
on material compatibility with the sterilant.
Prior to each test, the coupon chamber was sterilized (or reset) with pAB, diluted bleach or
dichlor, depending on the decontaminant being evaluated in each particular test, to help
prevent cross-contamination between tests. The sterilization solution (2.5 gallons) was
applied with an electrostatic sprayer. The spray process was repeated twice, each time
with another 2.5 gallons of solution. Dl water was utilized for the final rinse for each reset.
Metal cabinets for storing coupons before and after testing, were sterilized with Dispatch®
wipes followed by wiping with isopropyl alcohol. The cabinets were sealed with tape and
left to air dry at least 24 hours prior to testing. Cabinet A housed test coupons, Cabinet B
housed positive coupons, and Cabinet C housed negative coupons.
4.1.1 Equipment Sterilization - Vaporized Hydrogen Peroxide
Prior to use, test equipment was sterilized using a proprietary vaporized hydrogen
peroxide, VHP® system. The hydrogen peroxide (H2O2) vapor was generated using a
STERIS VHP 1000ED system loaded with a 35% H2O2 Vaprox® cartridge (Steris, Mentor,
OH). Each sterilization cycle lasted 4 hours at 250 ppm VHP vaporized hydrogen peroxide.
Cycle efficacy was verified through the routine use of biological indicators charged with a
minimum of 1 x 106 spores of Geobacillus stearothermophilus (Apex® biological indicators,
Mesa Labs, Bozeman, MT). This process was used to sterilize test material coupons,
ADAs, and backpack sprayers. Prior to the process, coupons and the backpack sprayer
(with lid open) were wrapped in VHP bags, while ADAs were placed in large plastic bins.
Negative control coupons were used to verify coupon sterility.
4.1.2 Stainless-Steel MDI Control Coupon Sterilization - Autoclaving
Stainless-steel inoculum control coupons (0.020-in thickness, McMaster-Carr), concrete
coupons, and carboys were sterilized using a 30-min gravity cycle at 121 °C in a Steris
Amsco Century SV 120 Scientific Pre-Vacuum Sterilizer (STERIS Corporation, Mentor,
OH). The coupons were carefully wrapped with aluminum foil prior to autoclaving, in order
to maintain sterility when removed from the autoclave. The 1-hour cycle is recommended
for inactivation of gram-positive spore-forming bacteria. Sterility check for the stainless-
steel coupons was evaluated by using a swab (BactiSwab® Collection and Transport
System, Remel, Thermo Fisher Scientific, Waltham, MA).
4.1.3 Templates and Inoculation Equipment Sterilization - Ethylene Oxide (EtO)
Sampling templates and inoculation equipment were sterilized using an Andersen ethylene
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oxide (EtO) sterilizer system (PN: 333 EOGas®, Haw River, NC). The sterilization
procedure is summarized below.
1. All the items to be sterilized were packed in appropriate EtO envelopes and sealed.
2. Sealed EtO envelopes were placed in appropriate sterilization bags along with a
dosimeter, humid-chip, and EtO dispenser.
3. The sterilization bags were vacuum-sealed and loaded into the EtO sterilizer for an 18-
hour cycle.
5 Decontamination, Sampling, and Analysis Approach
The following sections discuss the decontamination test procedures, sampling protocol,
sample types, and frequency of sampling and monitoring events for the pilot-scale tests.
5.1 Decontamination and Sampling Protocol
Before each test, all materials and equipment needed for sampling were sterilized as
discussed in Appendix B and handled using aseptic techniques. Non-powdered, surgical
gloves were used during sampling. Individually wrapped, pre-moistened bleach wipes
(Clorox Dispatch® wipes; Clorox Corp., Oakland, CA) were placed in sterile sampling
bags. 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 each specific test. The following test protocol was implemented for each test.
5.1.1 Testing and Sampling Flow Timeline
All the tests were conducted in accordance with the following testing and sampling flow
timeline:
Day 1: Sterilization of Coupons and Equipment
1. Coupons were wrapped in Tyvek® bags (Dupont, Wilmington, DE) and sealed with tape.
Coupon type and date of sterilization were recorded on the tape.
2. Concrete and stainless-steel control coupons were placed in a large autoclave using a
250 °F gravity cycle autoclave.
3. Steris vaporous hydrogen peroxide (VHP®) ED 1000 system was used to sterilize
wrapped wood, brick, and asphalt coupons.
4. The ADA was placed in a designated bin within the chamber. A VHP cycle was run for 4
hours.
5. 12-in x 12-in paper templates were placed in ethylene oxide (EtO) bags (10 per bag). MDI
adaptors were placed in individual small bags. EtO cycle was run.
Day 2: Decontaminate Coupon Cabinets
1. Dispatch wipes were used to clean all surfaces of the coupon storage cabinets
including exterior surfaces.
2. Surfaces were sprayed with Dl water and dried with disposable dry wipes
3. Surfaces were sprayed with isopropyl alcohol.
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4. Tape was placed across the opening of the cabinets stating that the cabinet has been
decontaminated. The date and initials of person who performed the cleaning were
recorded on the tape.
Day 2: Set Up Coupons with ADAs
5. A swab of one sterilized ADA from each batch was collected for a sterility check prior to
set up.
6. Coupons were aseptically unwrapped and ADA was placed on top. The apparatus was
secured with clips.
Day 3: Inoculate Coupons
1. The MDI was removed from the lab refrigerator and let stand to room temperature for a
minimum of 1 hour. The MDI was uncapped and placed in a desiccant bag.
2. The mass of the MDI was determined by analytical balance.
3. An E7 MDI (nominal inoculation in each puff of 7 log CFU) of was used to inoculate
coupons.
4. Coupons and 3 stainless-steel coupons (inoculation controls) were dosed with E7 Bg
MDI. The MDI and actuator were positioned in the ADA top opening. Inoculation was
done per the test sequence listed below in Table 5-1.
Table 5-1. Test Inoculation Sequence
Order of Inoculation
Material
1
Stainless Steel MDI Control Coupon 1
2
Positive Control Coupons
3
Test Coupons
MDI, metered-dose inhaler
5. Inoculated 3 test coupons and 3 positive controls.
6. Positive control coupons and material field blank were not sprayed and remained in the
Clean Positive Coupon Cabinet until sampling.
7. MDI was returned to refrigerated storage.
8. Coupons, procedural blanks, and inoculation controls covered with ADAs were left for
18 hours.
Day 4: Reset Decontamination Chamber
1. Inside walls of chamber (prior to each trial with each material) were sterilized/reset.
2. Using a sprayer solely for resetting equipment, interior surfaces were kept wet with pAB
solution, dichlor, or diluted bleach for 10 minutes. pAB solution was discarded after 3
hours from time of preparation. Dichlor was discarded after 4 hours from preparation
time.
3. Using Dispatch wipes, the inside of chamber door was cleaned.
4. Surfaces were rinsed with Dl water with drain open.
5. After ensuring rinsate was removed from chamber, valve was closed in preparation for
the test.
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Day 4: Perform Decontamination Spray Test
For the pilot-scale tests, two spray applications were made with the electrostatic
sprayer to the coupons: an initial spray, and then another 30 minutes later. Thirty
minutes after the second spray application, the coupons were sprayed with Dl water to
rinse off the decontaminant, thus providing a total contact time of one hour. The
average spray time for each coupon for each spray application was nominally 20
seconds. The spray test sequence was as follows:
1, Flow rate of electrostatic sprayer was determined and recorded.
2. Only three coupons at a time could be placed in the spray chamber (refer to Figure 5-
1); therefore, three coupons were set up in the chamber alternating the material type
(e.g., concrete - wood- concrete for the first round, and then wood-concrete-wood for
the second round). Similarly, asphalt and brick were paired together in a third and fourth
round. (Note, we kept the same spray time for consistency, even though the asphalt
coupons were smaller in area compared to the 14-in x 14-in coupons. This may have
resulted in potentially more decontaminant per surface area applied to the brick
materials.)
Figure 5-1. Example alternating concrete and wood coupons in spray chamber.
3. The coupons were sprayed using multiple side-to-side strokes, moving in a Z pattern to
completely wet the coupons (see for example Figure 5-2.). The total spray time for the
three coupons combined was 1 minute. After the first spray application, the three
coupons dwelled for a contact time of 30 minutes, and then a second spray was
applied.
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Spray 1 - 12" , Top left corner to top
right corner
Spray 2 - 12* , Middle right side to
middJe left side
Spray 3 - 12" , Bottom Left Corner to
Bottom Right Corner
Figure 5-2. Spray pattern for electrostatic sprayer.
4. After the second spray application and allowing another 30-minute contact time, the
coupons were rinsed using Dl water in a backpack sprayer specifically used for Dl water
only. Each coupon was rinsed for 20 seconds.
5. After being sprayed with the Dl water and allowed to drip dry, the coupons were placed in
the sterilized cabinets after each test and allowed to further dry overnight.
Day 5: Perform Coupon Surface Sampling
1. After overnight drying, surface sampling of coupons was conducted using wetted wipes
for smooth surfaces (wood, stainless-steel) or by a vacuum method utilizing 37-mm
cassettes for concrete, brick, and asphalt coupons.
2. Samples were transferred to the EPA's Homeland Security Materials Management
Division (HSMMD) microbiology laboratory (BioLab) for microbiological analysis in sterile,
primary, independent packaging within sterile, secondary containment that holds logical
groups of samples for analysis. All samples were accompanied by a completed chain-of-
custody form.
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5.2 Surface Sampling
5.2.1 Vacuum Sampling
Surface sampling with 37-mm vacuum cassettes was conducted as described below, as
well as by Calfee et al. (2013b) and Mikelonis et al. (2020). Concrete, brick, and asphalt
materials were sampled by this method. The coupons were placed horizontally for
sampling.
The coupons were sampled as follows:
1. Nozzles were sterilized with ethylene oxide. Each nozzle was accompanied by a 1 cm long
piece of Tygon® tubing (United States Plastic Corp., Lima, OH).
2. The cassette kit was pre-assembled as follows:
a. Each 37-mm cassette was labeled with a unique sample ID.
b. The Whirl-Pak® bag (Whirl-Pak, Madison, Wl) was labeled with the same unique
sample ID.
c. The cassette plugs were aseptically removed and a PVC adaptor was placed onto
each end of the cassette. The removed plugs were saved for step (f) below.
d. A 20 cm long piece of tubing was cut with scissors.
e. The 20 cm tubing was placed onto the outlet end of the cassette.
f. The sampling nozzle was placed onto the inlet end of the cassette. This was done
by inserting the 1 cm piece of Tygon tubing into the port at the exit of the nozzle
(see Figure 5-3), and then placing the adapter into the tubing.
Figure 5-3. Filter assembly and Sensidyne Gilian sampling pump.
3. A two-person team was used, employing aseptic technique. The team consisted of a
sampler and a support person,
4. The Vac-U-Go pump power cord was plugged in and the calibrated rotameter was attached
to the Sensidyne1" Gilian''" 12 pump (Sensidyne, St. Petersburg, FL). The pump was
adjusted to the flow rate of 5 LPM. The sampler then donned sterile gloves.
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5. The support person aseptically unwrapped a template from the bag and handed it to the
sampler, taking care to not touch the template.
6. The sampler placed the template onto the coupon surface.
7. The support person opened the sampling supply bin and removed the 37-mm cassette
sample kit from the bin.
8. The support person recorded the sample collection bag ID number on the sampling log
sheet or in the laboratory notebook.
9. The support person:
a. Opened the 37-mm cassette sample kit outer bag and removed the unlabeled 37-
mm cassette assembly bag.
b. Opened the small unlabeled bag containing the 37-mm cassette assembly.
c. Held the bag so that the sampler could remove the kit.
d. The support person held the tubing for the sampler to place the 37-mm cassette
assembly onto the tubing.
10. The sample handler removed the 37-mm cassette assembly from the bag and attached to
the Tygon vacuum tube held by the support person.
11. The support person or assistant recorded the duration of sampling. Table 5-2 displays the
values for total and single pass durations as specified in the test plan.
12. Sampling was performed horizontally using S-strokes to cover the entire area of the
material surface not covered by the template. The nozzle inlet was gently pressed against
the coupon surface while the nozzle is pulled across the coupon.
Table 5-2. Sample Duration 12-in x 12-in Surface Area and 6-in Diameter Coupons
Material
Total Sampling
Duration
Single Pass
Duration
Number of Passes
per Direction
Concrete - 12-in x 12-in
5 minutes
2 1/2 minutes
1
Brick - 12-in x 12-in
5 minutes
2 1/2 minutes
1
Asphalt - 6-in diameter
1 minute
30 seconds
1
5.2.2 Wipe Sampling
Wipe sampling was performed for the materials with smooth surfaces and not likely to
release particles (MDI stainless steel control coupons and the wood coupons) using a
moistened, sterile non-cotton gauze wipe (Curity™ all-purpose sponges #8042, 2-in x 2-in,
4-ply, Covidien PLC, Dublin, Ireland). All coupons were placed horizontally for sampling.
The wipes were prepared by aseptically removing them from their packing and placing
them in an unlabeled sterile 50 ml_ conical tube (Cat. No. 14-959-49A, Fisher Scientific,
Waltham, MA) using sterile forceps. Each transferred wipe was then moistened by adding
2.5 ml_ of sterile phosphate-buffered saline with 0.05% Tween® 20 (PBST) and capped.
A template was used to cover the exterior (1-in) of each coupon leaving a square (12-in x
12-in) exposed for sampling for all coupons. The outer 1-in of each coupon was not
sampled in order to avoid edge effects. The wipe sampling procedure was performed
consistent with previous studies (Brown et al., 2007; US Environmental Protection Agency,
2015). Briefly, the coupons were sampled as follows:
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1. Remove the clamps from the Blank MDI control coupon, and then remove the ADA from
the coupon.
2. Wipe the surface of the coupon horizontally, using a consistent amount of pressure and
using S-strokes to cover the designated sample area of the coupon.
3. Fold the wipe in half, concealing the exposed side and then wipe the same surface
vertically using the same S-stroke technique.
4. Fold the wipe again and roll it so that it would fit into a conical tube.
5. Place the folded wipe into a conical tube.
6. Place the conical tube in a sterile Twirl'Em® bag (Labplas, Quebec, Canada).
7. Sterilize the outside of the Twirl'Em bags using bleach wipes.
8. Place the sample in the collection bin for transport to the BioLab.
5.2.3 Swab Sampling
Swab samples (BactiSwab™ collection and transport system, R12100, Remel, Thermo
Fisher Scientific, Waltham, MA) were collected from cabinets, test chambers, and ADAs to
serve as sterility checks. Swab samples were taken to check for the sterility of the test
equipment. The swab sampling procedure is described below:
1. Open the package and remove the BactiSwab™ swab.
2. Label the plastic tube holding the swab.
3. Remove the cap-swab from the plastic tube.
4. Swab the surface while spinning the cap-swab between the thumb and index fingers.
5. Return cap-swab to tube.
6. Through the Bactiswab ™ sleeve, crush the BactiSwab™ ampule inside the sleeve at
midpoint.
7. Date and initial each sample tube.
A typical summary of sterility check samples taken during a testing event is presented in
Table 5-4.
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Table 5-4. Typical Sterility Check Sample Types for Each Testing Event
Sample Number
Description
Sample Type
Inoculation Sterility Checks
1
ADA/Gasket Sterility check
Swab
2
Cabinet A
Swab
3
Cabinet B
Swab
4
Cabinet C
Swab
5
Chamber prior to testing
Swab
6
Chamber following 1st reset
Swab
7
Chamber following 2nd reset
Swab
8
Blank metered-dose inhaler (MDI) Control
Wipe
9
MDI Inoculation Control-1
Wipe
10
MDI Inoculation Control-2
Wpe
11
MDI Inoculation Control-3
Wipe
5.3 Sample Handling
This section discusses the sample containers used and sample preservation.
5.3.1 Sample Containers
For each wipe sample and 18-mm sample, the primary containment was an individual,
sterile, 50-mL conical tube. Secondary and tertiary containment consisted of sterile
sampling bags. Twirl'Em bags (sterile bags with round wire closure, Model No. 14-9555-
181, Fisher Scientifics, Hampton, NH) were used for 37-mm cassette vacuum samples.
5.3.2 Sample Preservation
After sample collection, sample integrity was maintained by storing samples in four
containers (one sample collection container, one sterile inner bag, one sterile outer bag
with the exterior sterilized during the sample packaging process, and one sterile container
holding all samples from a test). All individual sample containers remained sealed while in
the decontamination laboratory and during transport.
5.4 Microbiological Analyses
Samples were analyzed either qualitatively for spore presence (quality control, swab and
wipe samples) or quantitatively for the number of viable spores recovered per sample.
Results were reported in colony forming units (CFUs) per unit volume.
Spores were extracted using the appropriate method for each sample type, as described
below. After extraction, aliquots were removed for either dilution plating or filter plating, as
appropriate for expected spore recoveries.
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5.4.1 Wipe, Vacuum, and 18 mm Coupon Samples
All samples were received in 50-mL conical tubes. Spores were extracted from the wipes
by adding 20 ml_ PBST to each sample, then agitating the tubes using a vortex mixer (set
to maximum rotation) for two minutes in 10-second intervals. Aliquots were then removed
for plating. Spores were extracted from the vacuum and coupons samples typically using
10 mL PBST.
5.4.2 Swabs
Each swab was removed from the packaging in a biological safety cabinet, then rotated
across the tryptic soy agar (TSA) media surface in a zig-zag pattern until the entire
circumference of the swab contacted the media surface. Plates were incubated at the
appropriate temperature and time based on the Bacillus strain used in the test. Swab
plates were manually observed to determine growth or no growth of the target organism.
5.4.3 Spiral Plating and Filter plating
Sample extracts requiring dilution were plated in triplicate using a spiral plater (Autoplate®
spiral plater, Autoplate 5000, Advanced Instruments Inc., Norwood, MA). The automated
spiral plater 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
with Bg samples were incubated at 35 ± 2 °C for 18 to 20 hours. During incubation, the
colonies develop along the lines where the sample was deposited. Colonies on each plate
were enumerated using a QCount® colony counter (Advanced Instruments Inc., Norwood,
MA).
Positive control samples were diluted 100-fold in PBST before spiral plating, while samples
of unknown concentration were plated with no dilution and with a 100-fold dilution.
Samples with known low concentrations were plated with no dilution. 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® counter records
the data in an MS Excel spreadsheet.
Only spiral plates meeting the threshold of at least 30 CFUs were used for spore recovery
estimates. After quantitation with the QCount® colony counter, samples with plate results
below the 30-CFU threshold were either re-spiral plated with a more concentrated sample
aliquot or filter-plated to achieve a lower detection limit. The filter plate volume was based
on the CFU data from the QCount® result. Filter plating was performed using 100-ml
capacity MicroFunnel™ units (Tredegar Industries, Inc., Richmond, VA) with 0.45 |jm GN-
6 Metricel® membranes (Pall Corporation, Laboratory, Port Washington, NY) and a
vacuum manifold (Pall Corporation, Laboratory, Port Washington, NY). The filters were
placed onto TSA plates and incubated at 35 ± 2 °C for 20 to 24 hours before manual
enumeration.
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5.4.4 pH, Temperature, and FAC Measurements
Measurements of pH and temperature of the decontaminant solutions were performed
each day of testing using a calibrated pH meter (Oakton® Acorn™ pH 5, OAKTON
Instruments, Vernon Hill, IL). The temperature sensor included with the pH meter was
factory-calibrated and checked monthly by comparison of the displayed value to a National
Institute of Standards and Technology (NIST) certified thermometer.
Measurements of FAC were performed using an iodometric method that uses a HACH®
digital titrator and a HACH® reagent titration kit (Model #16900, HACH®, Loveland, CO).
The titration procedure can be found in the HACH® digital titrator manual (https://pim-
resources.coleparmer.com/instruction-manual/24908-0Q.pdf), which is based on Modified
ASTM Method D2202-08.
5.5 Decontamination Efficacy
Decontamination efficacy is reported in terms of log™ reduction (LR) of the viable Bg
spores recovered from the treated test material surface relative to the positive controls.
That is, LR was calculated for each material/decontaminant as follows:
LR = Average of the three log™ CFU positive controls values - average of the three log™
CFU test samples
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6 Results and Discussion
6.1 Bench-Scale Results
6.1.1 Test conditions
Table 6-1 lists the actual FAC concentrations and pH levels for the prepared
decontamination solutions. For the dichlor and diluted bleach tests, two separate
experiments were conducted (one experiment for concrete, the other experiment for the
wood, brick, and asphalt materials). For each test, a contact time of 15 minutes was used.
Table 6-1. Test Conditions for Bench-Scale Decontamination Testing on 18-mm Coupon
Materials
Matprial
FAC (ppm)
PH
Type
Dichlor
Diluted
Bleach
pAB
Dichlor
Diluted
Bleach
pAB
Concrete
21,300
20,300
6,610
NA*
11.3
11.5
6.8
Wood
19,900
20,400
Brick
Asphalt
FAC, free available chlorine; pAB, pH adjusted bleach
inadvertently not measured, but expected to be ~ 6.5, based on pilot-scale tests
6.1.2 Decontamination Efficacy Results
The bench-scale results are summarized in Figure 6-1 and detailed in Tables 6-2, 6-3, and
6-4, for diluted bleach, dichlor, and pAB decontamination solutions, respectively.
Overall, average decontamination efficacy for the three decontaminants was generally > 4
LR, except for a few tests (brick/dichlor and wood/pAB). Full decontamination (no CFUs
detected in the post-decontamination samples), and/or decontamination efficacies greater
than 6 LR were observed for brick decontaminated with diluted bleach; and for concrete,
wood, and asphalt material coupons decontaminated with dichlor. For the organic
materials wood and asphalt, dichlor achieved the highest decontamination efficacy (> 6.0
LR for both materials) of the three decontaminant solutions evaluated. This result
supports the hypothesis that the swimming pool chemical may be better able to maintain
its biocidal activity when in contact with organic materials.
With respect to the decontaminants, the average decontamination efficacies for dichlor (an
average LR of 5.7 for the four materials) and diluted bleach (an average LR of 5.5 for the
four materials) were similar. The average LR for pAB (4.7) for the four materials was the
lowest among the three decontaminant solutions tested. We are unclear why the pAB
was not as effective as the DB, since in a previous study both decontaminants were found
to be similarly effective with DB at a concentration of 20,000 ppm. (U.S. EPA, 2018b)
26
-------
T
concrete wood brick asphalt
Figure 6-1. Bench-scale decontamination results
Table 6-2. Decontamination Efficacy of Diluted Bleach Solution on 18-mm Coupon
Materials
Coupon
Material
Log Positive Controls
(CFU)
Log Test Coupons
(CFU)
Log Reduction
Mean
SD
Mean
SD
Mean
SD
Concrete
7.30
0.09
1.77*
0.79
5.53
0.56
Wood
6.82
0.18
2.14*
1.73
4.69
1.23
Brick
7.39
0.03
0.12*
0.03
7.26
0.03
Asphalt
7.29
0.12
2.67*
1.66
4.63
1.17
CFU, colony forming units; SD, standard deviation
*At least one of the three test samples had no spores detected (DL ~20 CFU for Concrete, and ~2 CFU
for Wood, Brick and Asphalt.) n=3 for each coupon material.
27
8-i
7-
6-
C
o
t>
z>
TD
-------
Table 6-3. Decontamination Efficacy of Dichlor Solution on 18-mm Coupon Materials
Coupon
Positive Controls (log
CFU)
Test Coupons (log
CFU)
Log Reduction
Mean
SD
Mean
SD
Mean
SD
Concrete
7.07
0.43
1.30**
0
5.77
0.21
Wood
7.09
0.28
0.14*
0.03
6.95
0.20
Brick
7.24
0.11
3.66
1.26
3.59
0.89
Asphalt
7.28
0.03
0.86*
0.70
6.42
0.50
CFU, colony forming units; SD, standard deviation
*At least one sample at or below limit of detection (~20 CFU for Concrete, and ~2 for Wood, Brick and
Asphalt.) **all samples ND. n=3 for each coupon material.
Table 6-4. Decontamination Efficacy of pAB Solution on 18-mm Coupon Materials
Coupon
Positive Controls (log
CFU)
Test Coupons (log
CFU)
Log Reduction
Mean
SD
Mean
SD
Mean
SD
Concrete
7.05
0.19
2.16*
1.36
4.90
0.97
Wood
7.19
0.05
3.54
0.56
3.64
0.40
Brick
7.21
0.08
1.29*
2.07
5.92
1.46
Asphalt
7.22
0.03
2.81
2.0
4.42
1.41
CFU, colony forming units; SD, standard deviation
*At least one sample at or below limit of detection (~20 CFU for Concrete, and ~2 for Wood, Brick and
Asphalt). n=3 for each coupon material.
6.2 Pilot-Scale Testing
The following sections discuss the test conditions for the pilot-scale testing and the
decontamination results from these tests.
6.2.1 Test Conditions
The decontamination solutions and concentrations for this study were similar to the ones
used for the bench-scale study, with the exception that pAB was tested at two FAC
concentrations: a typical, relatively low FAC for pAB, and at a higher concentration of FAC,
equivalent to the concentrations used for dichlor and diluted bleach. Tables 6-5 through 6-
8 list the test conditions for the prepared decontamination solutions. For the dichlor, diluted
bleach, and the high FAC pAB, the FAC levels were all approximately 20,000 ppm, while
for the more typical pAB, its FAC level was approximately 6,400 ppm. The pH level for the
dilute bleach was 11.3-11.4, while the pH for the other three decontaminants ranged from
6.4-7.0. Each coupon was sprayed twice for 20 seconds with the electrostatic sprayer,
which had an average flow rate of approximately 124 mL/minute. The total sprayed
volumes of the decontaminant for the two sprays combined are shown in Tables 6-5
28
-------
through 6-8 and ranged from 63-85 mL/coupon. To clarify, this was the total amount
sprayed per coupon, on average, but does not imply this amount was deposited onto each
coupon, due to overspray and runoff.
Table 6-5. Test Conditions for Diluted Bleach Decontamination of 14-in x 14-in Coupon
Materials
Diluted Bleach
Material
Type
FAC
(PPm)
PH
Temp
(°C)
Total Volume
Sprayed
per coupon (ml_)
Concrete
20,600
11.4
22.0
85
Wood
20,600
11.4
22.0
85
Brick
20,800
11.3
20.8
85
Asphalt
20,800
11.3
20.8
85
FAC, free available chlorine
Table 6-6. Test Conditions for pAB (High FAC) Decontamination of 14-in x 14-in
Coupon Materials
pAB
Material
Type
FAC
(PPm)
PH
Temp
(°C)
Total Volume
Sprayed per
coupon (ml_)
Concrete
20,400
7.05
19.0
80
Wood
20,400
7.05
19.0
80
Brick
19,600
6.93
21.9
83
Asphalt
19,600
6.93
21.9
83
FAC, free available chlorine; pH adjusted bleach
Table 6-7. Test Conditions for Dichlor Decontamination of 14-in x 14-in Coupon
Materials
Dichlor
Material
Type
FAC
(PPm)
PH
Temp
(°C)
Total Volume
Sprayed per
coupon (ml_)
Concrete
20,800
6.72
20.7
84
Wood
20,800
6.72
20.7
84
Brick
20,400
6.40
22.6
78
Asphalt
20,400
6.40
22.6
78
FAC, free available chlorine
29
-------
Table 6-8. Test Conditions for pAB (Low FAC) Decontamination of 14-in x 14-in
Coupon Materials
pAB
Material
Type
FAC
(PPm)
PH
Temp
(°C)
Total Volume
Sprayed per
coupon (mL)
Concrete
6,410
6.88
19.2
63
Wood
6,410
6.88
19.2
63
Brick
6,410
6.99
21.5
67
Asphalt
6,410
6.99
21.5
67
6.2.2 Decontamination Efficacy Results
The pilot-scale test results for decontamination efficacy, for each decontamination solution
on 14-in x 14-in material coupons, inoculated with 107Bg spores, are summarized in
Figure 6-2 and detailed in Tables 6-9 through 6-12.
With respect to material effects, decontamination efficacy was the highest for the brick
material in every test, with > 6.0 average LR observed for the four decontamination
solutions tested. Average decontamination efficacy ranged from 4.2-5.7 LR for the
concrete; 3.2-5.9 for the wood; and 4.2-5.6 for the asphalt.
In general, the range in decontamination efficacies observed for the 14-in x 14-in coupons
was similar to what was observed for the smaller 18-mm coupons. The one exception to
this that stood out was the decontamination efficacy for dichlor on brick. In the 18-mm
coupon tests, the efficacy for dichlor on brick was 3.6, while for the larger coupons, the
efficacy was 6.6. We are uncertain how to explain this difference in efficacy, other than
potentially unidentified experimental error, and/or attribute it to the small sample size (only
three replicates were used for each test condition). Another potential factor that may have
contributed to the difference in results was how the coupons were constructed. The 18-
mm coupons were constructed with only the brick material, while the larger coupons
included mortar as well. Additionally, the pilot-scale asphalt coupons (6-inch diameter)
were smaller in surface area compared to the 14-in x 14-in coupons, allowing for
potentially more decontaminant to be applied per unit area. Repetition of these test
conditions would be helpful in elucidating any potential issues.
Wth respect to the decontaminants, on average the decontamination efficacy was highest
with the diluted bleach (an average LR of 6.0 for the four materials), followed by dichlor (an
average LR of 5.4 for the four materials). Decontamination efficacy results for the two pAB
solutions were the lowest among the four decontaminant solutions studied. Additionally,
the average efficacy across the four materials was similar for both pAB solutions (average
LR of 4.7-4.8), indicating that the increased FAC concentration had minimal effect. (We do
acknowledge that decontamination efficacy did increase for concrete at the higher pAB
FAC level, although this difference may not be statistically significant. We are uncertain
30
-------
why the increased concentration of FAC for the pAB solution did not improve efficacy,
other than that efficacy may have been limited by other factors, such as the presence of
organic material and/or that spores were protected by microscopic barriers in the
materials.) In terms of having the lowest number of CFU recovered after decontamination,
the results for diluted bleach and dichlor were similar; both decontaminants were able to
achieve at least one sample with no spores detected, for every material.
8
7
6
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o
03
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-------
Table 6-10. Decontamination Efficacy of Dichlor Solution on Large Coupons
Coupon
Positive Controls (log
CFU)
Test Coupons (log
CFU)
Log Reduction
Mean
SD
Mean
SD
Mean
SD
Concrete
5.93
0.21
1.52*
0.32
4.42
0.27
Wood
7.08
0.20
2.17*
1.78
4.91
1.26
Brick
6.65
0.15
0.06*
0.01
6.59
0.11
Asphalt
6.02
0.35
0.42*
0.32
5.60
0.33
CFU, colony forming units; SD, standard deviation
*At least one sample at or below limit of detection (~20 CFU for Concrete, and ~2 for Wood, Brick and
Asphalt) n=3 for each coupon material.
Table 6-11. Decontamination Efficacy of pAB (6500 ppm FAC) Solution on Large
Coupons
Coupon
Positive Controls (log
CFU)
Test Coupons (log
CFU)
Log Reduction
Mean
SD
Mean
SD
Mean
SD
Concrete
6.52
0.09
2.28
1.12
4.24
0.80
Wood
7.09
0.33
3.87
0.78
3.21
0.59
Brick
7.48
0.09
0.89*
1.25
6.59
0.88
Asphalt
6.39
0.11
1.04
0.46
5.36
0.35
CFU, colony forming units; FAC, free available chlorine; pAB, pH adjusted bleach; SD, standard
deviation
*At least one sample at or below limit of detection (~20 CFU for Concrete, and ~2 for Wood, Brick and
Asphalt) n=3 for each coupon material.
Table 6-12. Decontamination Efficacy of pAB (20000 ppm FAC) Solution on Large
Coupons
Coupon
Positive Controls (log
CFU)
Test Coupons (log
CFU)
Log Reduction
Mean
SD
Mean
SD
Mean
SD
Concrete
7.13
0.28
1.94
0.16
5.19
0.23
Wood
7.25
0.14
3.72
0.89
3.53
0.63
Brick
7.08
0.22
1.05*
0.87
6.03
0.64
Asphalt
6.00
0.45
1.80
0.64
4.21
0.55
CFU, colony forming units; FAC, free available chlorine; pAB, pH adjusted bleach; SD, standard
deviation
*At least one sample at or below limit of detection (~20 CFU for Concrete, and ~2 for Wood, Brick and
Asphalt) n=3 for each coupon material.
32
-------
In summary, the dichlor decontaminant solution was shown to be nearly as effective as the
diluted bleach solution in inactivating the B. anthracis spore surrogate on outdoor
materials, and in the bench-scale tests, was shown to be more effective on the organic
materials. With a more neutral pH than diluted bleach, dichlor solutions could potentially
be more compatible with materials, although this is a research gap that would require
further study. Additionally, dichlor has the advantage that it is sold as solid granules that
could simply be mixed with water at its point of use, thus reducing cost and
environmental/safety issues associated with transport of a hazardous chemical. Thus,
dichlor should be considered an advantageous decontamination tool to have in the event
of a wide area release of B. anthracis spores. Lastly, these chlorine-based decontaminants
were prepared using Dl water; further research is recommended to evaluate whether the
use of tap water in preparing these decontaminant solutions affects decontamination
efficacy. Further research into the decontamination efficacy of other commercial, off-the-
shelf swimming pool granular chemicals, such as calcium hypochlorite, is also
recommended.
-------
7 Quality Assurance and Quality Control
All test activities were documented in laboratory notebooks and via 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 and HSMMD Microbiology Laboratory procedures (as
described in this report) to ensure repeatability and adherence to the data quality
validation criteria set for this study.
The following sections discuss the measurement equipment calibration, the criteria for the
critical measurements and parameters, data quality indicator (DQIs), and the quality
assurance (QA) and quality control (QC) checks for the study.
7.1 Criteria for Critical Measurements/Parameters
The data quality objectives are used to determine the critical measurements needed to
address the stated 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 of the study objectives:
• pH of diluted bleach, dichlor, pAB solutions, and Dl water
• FAC of diluted bleach, dichlor, and pAB solutions
• Flow rate of electrostatic sprayer
• Flow rate of the spray apparatus system
• Plated volume
• CFU counts
• Contact time of decontamination solution
7.2 Data Quality Indicators
The data quality indicators (DQIs) for the critical measurements (listed in Table 7-1) were
used to determine if the collected data met the data quality objectives. If a measurement
method or device resulted in data that did not meet these goals, the data derived from the
critical measurement were rejected. Decisions to accept or reject test results were based
on engineering judgment used to assess the likely impact of the failed criterion on the
conclusions drawn from the data. The acceptance criteria were set at the most stringent
levels that can routinely be achieved. All the DQIs were within the target acceptance
criteria set for this study as shown in Table 7-1.
-------
Table 7-1. DQIs for Critical Measurement
Critical Measurement
Analysis
Method
Accuracy/Precision
Acceptance
Criteria
CFU per plate
Spiral
plater/QCount
50% RSD among
the triplicate plating
50% RSD
among the
triplicate
plating
Temperature of incubation chamber
NIST-traceable
thermometer
(daily)
O
o
CN
+i
Not
applicable;
standard
evaluations
not
performed for
this
instrument
Spray application time
NIST-calibrated
stopwatch
± 1 minute/hour
± 2 minutes
(2 x ± 1
min)
PH
pH meter/
NIST-traceable
buffer solutions
± 0.01 pH unit
Not
applicable;
standard
evaluations
not
performed for
this
instrument
Titration/extraction/sample/neutralizer
volumes volume and extraction
volumes
Burette
± 1 ml_
± 10% of
target value
FAC
Na2S203/KI
titration
± 0.06 gram per liter
± 10% of
target value
CFU, colony forming units; DQI, data quality indicator; FAC, free available chlorine; NIST, National
Institute of Standards and Technology; RSD, relative standard deviation; SD, standard deviation
If the CFU count for bacterial growth did not fall within the target range (acceptance
criteria), the sample was either filtered or replated. All results were recorded, plates were
quantitatively analyzed (CFUs per plate) using a manual counting method. For each set of
results per test, a second count was performed on 25% of the plates within the
quantification range (plates with 30 to 300 CFUs).
7.3 Integrity of Samples and Supplies
Samples were carefully maintained and preserved to ensure their integrity. Samples were
stored away from standards and other samples that could possibly cross-contaminate
them.
Supplies and consumables were acquired from reputable suppliers. Project personnel
35
-------
carefully checked supplies and consumables prior to use to verify that they met specified
study quality objectives. Balances and micropipettes were calibrated yearly by the EPA
Metrology Laboratory.
7.4 BioLab Control Checks
Quantitative standards do not exist for biological agents. Viable spores were counted using
a QCount® colony counter. Counts generated that were either greater than 300 or less
than 30 CFUs per plate were considered outside of the targeted range. If the count of
colony-forming units for bacterial growth did not fall within the target range, the sample
was replated and recounted. Replates and filter plates were enumerated manually.
Before each batch of plates were enumerated on the QCount®, 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 being counted, a visual inspection of colony counts made by the QCount®
software was performed. Obvious count errors made by the software were corrected by
adjusting the settings (e.g., colony size, light, and field of view) and recounting 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 measurements were set at the most stringent
level that could be achieved routinely. Positive controls were included along with the test
samples in the experiments so that spore recovery from the different surface types could
be assessed. Background checks were also included as part of the standard protocol to
check for unanticipated contamination. Three replicate positive control coupons and three
replicate test coupons were included for each set of test conditions to characterize the
variability of the test procedures.
Additional QC samples were collected and analyzed to check the ability of the BioLab to
culture the test organism, as well as to demonstrate that materials used in this effort did
not contain spores. The checks included the following:
• Positive control coupons: Coupons inoculated in tandem with the test coupons to
demonstrate the highest level of inoculation recoverable from a particular inoculation
event.
• Unexposed field blank (negative control): Material coupons sampled in the same
fashion as test coupons but not inoculated with the surrogate organism before sampling
or exposure to the decontamination process.
• Procedural blank coupons: Material coupons handled and sampled in the same
fashion as test coupons but not inoculated with the surrogate organism before
sampling.
• Wipe sample containers: The exterior of the wipe sample container (conical tube) and
the sterile sampling bags were decontaminated by wiping all surfaces with a bleach
wipe before transporting from sampling location to BioLab in a secondary container.
36
-------
• Sterility checks: Swab samples were used for sterility checks on stainless steel
coupons, coupon storage cabinets, spray test chamber, and ADAs before use in
testing, as discussed in Section 5-1
• Blank TSA sterility controls: Plates incubated but not inoculated.
• Replicate plates of diluted microbiological samples: Replicate plates for each
diluted sample.
QC checks for BioLab procedures are listed in Table 7-2. These provide assurances
against cross-contamination and other biases in microbiological samples.
-------
Table 7-2. Additional Quality Checks for Biological Measurements
Sample Type
Frequency
Acceptance Criteria
Information
Provided
Corrective Action
Positive control
coupon
Minimum of
three per
test
1 x 107 for Bg,
50% relative standard
deviation between
coupons in each test set
Used to determine
extent of recovery
of inoculum on
target coupon type
If outside range, discuss
in the results section of
this report
Procedural blank
coupon
One per test
Non-detect
Controls for sterility
of materials and
methods used in
the procedure
Analyze extracts from
procedural blank without
dilution. Identify and
remove source of
contamination, if possible
Unexposed field
blank (negative
control) sample
One per test
Non-detect
Level of
contamination
present during
sampling
Clean up environment;
sterilize sampling
materials before use
Blank TSA sterility
control
Each plate
No observed growth after
incubation
Controls for sterility
of plates
All plates incubated
before use; contaminated
plates discarded before
use
Replicate plating of
diluted
microbiological
samples
Each
sample
Reportable CFU count of
triplicate plates within
100%; reportable CFU
counts between 30 and
300 CFUs per plate
Used to determine
precision of
replicate plating
Re-plate sample
Bg, Bacillus atrophaeus var. globigii; CFU, colony forming units; TSA, tryptic soy agar
The QA/QC control test results for the entire sampling campaign are listed for each test.
Sterility checks were conducted for all the equipment and materials and listed for each test
in the results section. Most of the coupon control blanks (negative control), the EPA
accepted sampling procedure blank (procedural blank), and the inoculum control blanks
(stainless-steel control blank) had no spores detected. A few control blanks were found to
be contaminated, but they had little or no effect on the final results. For negative controls,
the contamination may have occurred by incomplete inactivation of spores from the
materials during the VHP cycle.
-------
8 References
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stability of sodium dichloroisocyanurate. Jpn J Toxicol Environ Health. 41 (2): 134-141.
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M, Wilson MC, and Rudolph T. 2007. Evaluation of a wipe surface sample method for
collection of Bacillus spores from nonporous surfaces. Appl Environ Microbiol. 73(3):
706-710.
Calfee MW, Choi Y, Rogers J, Kelly T, Wllenberg Z, and Riggs K. 2011. Lab-scale assessment
to support remediation of outdoor surfaces contaminated with Bacillus anthracis spores.
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Calfee MW, Ryan SP, Wood JP, Mickelsen L, Kempter C, Miller L, Colby M, Touati A, Clayton
M, Griffin-Gatchalian N, McDonald S, and Delafield R. 2012. Laboratory evaluation of
large-scale decontamination approaches. J Appl Microbiol. 112(5): 874-882.
Calfee MW, Lee SD, and Ryan SP. 2013a. A rapid and repeatable method to deposit
bioaerosols on material surfaces. J Microbiol Methods. 92(3): 375-380.
Calfee MW, Rose LJ, Morse S, Mattorano D, Clayton M, Touati A, Griffin-Gatchalian N, Slone
C, and McSweeney N. 2013b. Comparative evaluation of vacuum-based surface
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Coates D. 1988. Comparison of sodium hypochlorite and sodium dichloroisocyanurate
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efficacy in greenhouses. California Agriculture, July - August, 1995. 49(4): 31-35.
Lee SD, Ryan SP, and Snyder EG. 2011. Development of an aerosol surface inoculation
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U.S. Environmental Protection Agency. 2017b. Evaluation of Spray-Based, Low-Tech
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40
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Appendix A: Material Coupon Fabrication
Coupon preparation is shown below.
Concrete and brick coupons dimensions were 14- by 14-in and were prepared
plywood base.
41
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Asphalt Coupon Cut Asphalt Coupon Asphalt Coupon Holder
18-mm brick, asphalt, and wood coupons attached to stainless-steel with double-sided
tape
Specialized molds for 18-mm concrete coupon
42
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Appendix B: Test Chamber and Equipment Cleaning and Sterilization
Procedures
The pH-adjusted bleach (pAB) solution used to clean the test chamber and equipment
surfaces in both the decontamination lab and BioLab (EPA's Homeland Security Materials
Management Division's microbiology laboratory) will be prepared as a 1:10 dilution of
bleach in deionized (Dl) water pH-adjusted to about 6.8 using glacial acetic acid. The
steps summarized below will be used for cleaning and sterilization.
Test chamber: between each material type and before and after each test
1. Using the backpack sprayer, keep the interior surfaces wet with pAB solution for 10
minutes.
2. With the drain open, rinse the surfaces with Dl water. Collect the rinsate in a carboy for
ultimate disposal.
3. After ensuring that all rinsate has been removed from the chamber, close the chamber
valve in preparation for the next test.
4. Use a mop assembly with a disposable pad to wipe down the interior of the chamber with
isopropyl alcohol or ethanol.
5. Remove to disposable mop pad and place it in a bucket of pAB solution for decontamination
before disposal.
Buckets: after use in a test
1. Fill the buckets with pAB and leave them covered for at least 60 minutes.
2. Rinse all buckets five times with Dl water.
3. Air dry the buckets before use.
Work surfaces: before and after use
1. Wet all surfaces with pAB or using Dispatch® bleach wipes.
2. Rinse with Dl water.
3. Wet and wipe surfaces with isopropyl alcohol or ethanol.
4. Air dry surfaces before use.
5. Alternatively, cover paper can be used and replaced before and after each use.
Sampling templates: before and after use
Autoclave the sample templates using a 250 °C, 30-min gravity cycle autoclave (SV 120
scientific pre-vacuum sterilizer; STERIS Amsco, Mentor, OH).
Coupon cabinets: before and after each test wet and wipe all surfaces with pAB or using
Dispatch® bleach wipes.
1. Rinse with DI water.
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Wet and wipe surfaces with isopropyl alcohol or ethanol.
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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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