EPA/600/R-18/283 | September 2018
www.epa.gov/homeland-security-research
United States
Environmental Protection
Agency
oEPA
Evaluation of Electrostatic
Sprayers for Use in a Personnel
Decontamination Line Protocol for
Biological Contamination Incident
Response Operations
Office of Research and Development
Homeland Security Research Program
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&EPA
EPA/600/R-18/283
Evaluation of Electrostatic Sprayers for Use in a
Personnel Decontamination Line Protocol for
Biological Contamination Incident Response
Operations
Assessment and Evaluation Report
National Homeland Security Research Center
Office of Research and Development
U.S. Environmental Protection Agency
Research Triangle Park, NC 27711
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Disclaimer
The U.S. Environmental Protection Agency (EPA), through its Office of Research and
Development's National Homeland Security Research Center, funded and managed this
investigation through Contract No. EP-C-15-008 with Jacobs Technology, Inc. (Jacobs). This
report has been peer and administratively reviewed and approved for publication as an EPA
document. This report does not necessarily reflect the views of the EPA. No official
endorsement should be inferred. This report includes photographs of commercially available
products. The photographs are included for the purpose of illustration only and are not intended
to imply that the EPA approves of or endorses the products or their manufacturers. The EPA
does not endorse the purchase or sale of any commercial products or services.
Questions concerning this report or its application should be addressed to the following
individual:
John Archer, MS, CIH
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) 541-1151
Fax No.: (919) 541-0496
E-mail Address: archer.iohn@epa.gov
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Acknowledgments
The principal investigator from the Office of Research and Development's National Homeland
Security Research Center (NHSRC) directed this effort with support of a project team of staff
from across the U.S. Environmental Protection Agency (EPA). The contributions of the following
individuals are a valued asset throughout this effort:
U.S. EPA Principal Investigator
John Archer, NHSRC/ Decontamination and Consequence Management Division
(DCMD)
U.S. EPA Technical Reviewers
Joseph Wood, NHSRC/DCMD
Elise Jakabhazy, Office of Land and Emergency Management (OLEM),
CBRN Consequence Management Advisory Division (CMAD)
U.S. EPA Product Team
Lukas Oudejans, NHSRC/DCMD
M. Worth Calfee, NHSRC/DCMD
Sang Don Lee, NHSRC/DCMD
Leroy Mickelsen, OLEM/CBRN/CMAD
U.S. EPA Quality Assurance Reviewer
Ramona Sherman, NHSRC
Jacobs Technology, Inc.
Madhura Karnik
Abderrahmane Touati
Denise Aslett
Ahmed Abdel-Hady
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Contents
Disclaimer ii
Acknowledgments iii
Executive Summary 1
1.0 Introduction 1
1.1 Background 1
1.2 Objectives 3
2.0 Experimental Approach 4
3.0 Experimental Materials and Methods 6
3.1 Test Materials 6
3.1.1 Coupon Fabrication 6
3.1.2 Sterilization Process 8
3.2 Test Chamber 9
3.3 Test Organism and Inoculation Procedure 10
3.3.1 Bg Surrogate for Ba 10
3.3.2 Bg Spore Inoculation 10
3.4 Decontamination Equipment, Solution, and Neutralizer 11
3.4.1 Sprayers 11
3.4.1.1 Electric Backpack Sprayer 12
3.4.1.2 Electrostatic Sprayer 12
3.4.2 Decontamination Solution 13
3.4.3 Neutralizing Agent 14
4.0 Decontamination Testing 15
4.1 Test Matrix 15
4.2 Testing Approach 16
5.0 Sampling and Analytical Procedures 18
5.1 Sample Types 18
5.1.1 Wipe Samples 18
5.1.2 Liquid Runoff Samples 19
5.1.3 Aerosol (Air) Samples 19
5.1.4 Sterility Check Swab Samples 19
5.2 Sample Quantities 19
5.3 Sample Handling 20
5.3.1 Sample Containers 20
5.3.2 Sample Preservation 20
5.3.3 Sample Custody 21
5.4 Microbiological Analysis 21
5.5 Decontamination Solution Characterization 22
5.5.1 pH 22
5.5.2 FAC by Titration 22
5.6 Determination of Efficacy 22
6.0 Results and Discussion 24
6.1 Decontamination Efficacy 24
6.2 Spore Disposition (Fate and Transport of Spores) 26
6.3 Liquid Waste Generation 28
6.4 Results Summary and Discussion 30
7.0 Quality Assurance and Quality Control 33
7.1 Criteria for Critical Measurements and Parameters 33
7.2 DQIs 33
7.3 QA/QC Checks 34
7.3.1 Integrity of Samples and Supplies 34
7.3.2 NHSRC BioLab Control Checks 34
7.3.3 Decontamination Solution Verification 36
7.3.4 QA Assessments and Response Actions 37
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References 38
Appendices 40
Figures
Figure 1-1. USEPA Standard Operating Safety Guidelines, Site Control Work Zones 2
Figure 3-1. Test material, Plywood (A) and Coupon Preparation (B) 7
Figure 3-2. Front (A) and Back (B) of Finished Test Coupon on Plywood 7
Figure 3-3. PPE Test Coupons 8
Figure 3-4. Decontamination Test Chamber with Coupon 9
Figure 3-5. MDI Actuator (A) and Canister (B) 10
Figure 3-6. 14- by 14-in ADA with Syringe Filter 11
Figure 3-7. Inoculation Setup 11
Figure 3-8. Electric Backpack Sprayer 12
Figure 3-9. SC-ET HD Air-Assisted Electrostatic Sprayer 13
Figure 4-1. Liquid Runoff Collection Assembly 16
Figure 5-1. Wipe Sampling of Test Coupon 18
Figure 5-2. Via-Cell® Bioaerosol Sampling Cassette 19
Figure 5-3. Bacterial Colonies on Spiral-plated Agar Plate 21
Figure 5-4. Bacterial Colonies on Filter Plate 22
Figure 6-1. Surface Decontamination Efficacy 24
Figure 6-2. Representation of Contact Angle of Liquid Droplets on Coupon Surfaces 25
Figure 6-3. Typical Beading of droplets seen on Butyl, Neoprene, Nitrile, Chemtape®,
Tychem® and Tyvek®* (A) and coalescence of droplets on Latex (B) 25
Figure 6-4. Log CFU Bg Spores in Liquid Runoff Samples 26
Figure 6-5. Percentage of Bg Spores Recovered from Procedural Positive Coupons 27
Figure 6-6. Average Volume of Liquid Waste Generated during Spraying 28
Tables
Table ES-1. Summary of findings by sprayer type 3
Table 3-1. Material Specifications 6
Table 3-2. Sterilization Processes Used 8
Table 3-2. Decontamination Sprayers Tested 12
Table 4-1. Test Matrix 15
Table 4-2. Test Coupon Configuration 15
Table 5-1. Sample Quantities 20
Table 6-1. Sprayer Comparison 31
Table 7-1. DQIs for Critical Measurements 33
Table 7-2. Additional QC Checks for Biological Measurements 36
Table 7-3. Cross-Contamination Assessment of Blank and Negative Control Samples 37
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Acronyms and Abbreviations
|jL microliter(s)
|jm micrometer(s)
ADA Aerosol deposition apparatus
Ba Bacillus anthracis
Bg Bacillus atrophaeus var. globigii
BioLab EPA Microbiology Laboratory
CFU Colony-forming unit(s)
CMAD Consequence Management Advisory Division
CRZ Contamination Reduction Zone
DB Diluted bleach
DCMD Decontamination and Consequence Management Division
DFU Dry Filter Unit
DHS U.S. Department of Homeland Security
Dl Deionized
DQI Data quality indicator
EPA U.S. Environmental Protection Agency
EtO Ethylene oxide
EZ Exclusion Zone
FAC Free available chlorine
ft feet
ID Identification
in inch(es)
H2O2 Hydrogen peroxide
HSRP Homeland Security Research Program
kJ kiloJoule
L liter(s)
LR Log reduction
MDI Metered dose inhaler
min minute
mL milliliter(s)
mL/min milliliter(s) per minute
N Normal
ND Non-detect
NHSRC National Homeland Security Research Center
NIST National Institute of Standards and Technology
pAB pH-adjusted bleach
PBST Phosphate-buffered saline with 0.05% Tween® 20
PPE Personal protective equipment
psi pound(s) per square inch
ppm part(s) per million
QA Quality assurance
QC Quality control
RH Relative humidity
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RSD
Relative standard deviation
RTP
Research Triangle Park, North Carolina
SOP
Standard Operating Protocol
STS
Sodium thiosulfate
SZ
Support Zone
TSA
tryptic soy agar
VHP
vaporized hydrogen peroxide
VMD
Volume mean diameter
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Executive Summary
This project supports the mission of the U.S. Environmental Protection Agency's (EPA's)
Homeland Security Research Program (HSRP) of the Office of Research and Development's
National Homeland Security Research Center (NHSRC) by providing vital scientific data that
can inform decisions for EPA emergency responders. The focus of this study was to provide
information relevant to the decontamination of personnel and personal protective equipment
(PPE) after responding to an act of bioterrorism. To minimize worker exposure and to prevent
the spread of potentially hazardous materials beyond the original areas of contamination, work
zones will be established to allow workers to move between the non-contaminated Support
Zone (SZ), the Contamination Reduction Zone (CRZ) where personnel decontamination takes
place, and the Exclusion Zone (EZ) or area of contamination. A well-established
decontamination line is essential for ensuring that potentially hazardous residues (chemical,
biological or radiological) on worker PPE do not transfer into the SZ. Traditional electric
backpack sprayers or handheld manual sprayers are often used to distribute a liquid
decontaminant over the surfaces of worker PPE, but this process can generate a large volume
of waste and may not always provide decontamination efficacy. Therefore, improved
decontamination line strategies must be investigated to minimize the spread of contamination
and reduce waste disposal costs.
A previous EPA study shows that compared to traditional sprayer systems, an electrostatic
spray technology is more efficient, reduces waste, and delivers a more uniform distribution of
liquids over uneven surfaces (USEPA 2015b). The current study explores the use of
electrostatic sprayers as an alternative to the sprayers currently used in a decontamination line
setting. Specifically, this study compares the performance of an electrostatic sprayer with a
traditional electric backpack sprayer by evaluating the efficacy of each sprayer in removing or
inactivating spores of Bacillus atrophaeus var. globigii (Bg), a surrogate for Bacillus anthracis,
from different types of PPE materials.
A decontamination test chamber was used to evaluate the sprayers. The following seven PPE
materials commonly found in PPE gloves, suits, boots, and related accessories were tested:
nitrile, butyl, latex, Tyvek®, Tychem®, neoprene, and ChemTape®. Coupons measuring 14- by
14-inches were prepared from each PPE material and inoculated with 1 x 107 Bg spores. Test
coupons were then placed in a vertical orientation in the decontamination test chamber and
sprayed with a 10% diluted bleach (DB) decontamination solution until completely wet using
either the backpack or electrostatic sprayer. Spray times for each type of sprayer were
evaluated based on the flow rates as indicated in Table ES-1.
After a 5-minute contact time, the coupons were removed from the test chamber and sampled
using a wipe sampling method. Wipe samples were collected in specimen cups containing a
pre-determined volume of sodium thiosulfate (STS) neutralizing agent used to quench the
decontamination reaction and preserve viable spores present in each sample. Wipe samples
were then analyzed for the presence of viable spores. Overspray liquid runoff and air samples
were also collected and analyzed for the presence of viable spores. The liquid runoff sample
collection bottles also contained STS.
The sprayer decontamination efficacy was determined by comparing the mean Log 10 number of
colony forming units (CFU) observed for the inoculum controls (stainless-steel coupons
ES-1
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inoculated but not exposed to decontamination treatment) to the mean Log 10 number of CFU
observed for the decontaminated test samples.
Overall, both sprayers achieved a surface log reduction (LR) of greater than or equal to 6, with
no statistically significant difference between the two sprayers (p-value = 0.49) (Table ES-1) For
three of the seven test materials, no surface CFU were detected when the electrostatic sprayer
was used. In contrast, there were CFU detected on coupons for all of the traditional backpack
sprayer tests.
An effective personnel decontamination line spray technology will apply decontaminant
solutions to the intended materials with: (1) high efficacy for the contaminant; (2) little to no
cross-contamination among field personnel and equipment; (3) little or no spreading of
contamination beyond the Exclusion Zone; and (4) minimal liquid waste generation. To assess
the transport or migration of viable spores off the test surfaces that could lead to cross
contamination, liquid runoff samples were collected and quantitatively analyzed. Each sprayer
also was evaluated when deionized (Dl) water was substituted for DB, and test coupons were
sprayed under the decontamination spray test conditions to understand how sprayer application
affects the physical removal of spores from a material surface. One runoff sample was collected
per test and analyzed for the number of viable spores (CFU). All of the runoff samples collected
from the backpack sprayer contained a large number of viable spores, whereas all of those
collected from the electrostatic sprayer contained very few to no detectable viable spores.
Runoff samples collected from the backpack sprayer ranged from 5.3 x 104 CFU to 5.0 x 106
CFU with a standard deviation of ± 1.6 x 106. Runoff samples from the electrostatic sprayer
ranged from no CFU detected to 1 spore detected.
The field applicability of a spray technology also depends on its ability to minimize cross-
contamination among field personnel and equipment, to limit the spread of contamination
beyond the area of initial contamination, and to minimize additional risks to personnel. The
number of spores physically removed via liquid runoff from test coupons indicates a potential
cross-contamination risk that could impact the extent of contamination at the site. The
application of decontamination solution using a backpack sprayer was observed to physically
remove almost twice as many spores compared to the electrostatic sprayer, due to the liquid
volume used and the tendency for runoff from the PPE materials. Therefore, use of the
backpack sprayer, as tested in this study, physically removes biological contamination from the
PPE surface and could result in environmental cross-contamination of PPE and other
equipment in a biological decontamination line.
To evaluate a suitable spray technology for a decontamination line, liquid waste generation
assessment is another important parameter to be considered, so quantifying and comparing the
amount of potentially hazardous waste generated by each sprayer type was also an overarching
project objective. Traditional electric backpack sprayers tend to have higher flow rates, resulting
in the application of larger volumes of decontamination liquid, thus generating more liquid
hazardous waste. Additionally, an electrostatic sprayer provides a more uniform distribution
using a minimal amount of decontamination solution over the surface area sprayed, thereby
significantly reducing waste streams and costs associated with liquid hazardous waste disposal.
During decontamination testing, runoff liquid volumes were collected and measured
gravimetrically. The quantity of liquid waste generated by the electrostatic sprayer was almost
75 times less than the amount generated by the backpack sprayer (Table ES-1).
ES-2
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Table ES-1. Summary of findings by sprayer type
Characteristic
Electrostatic Sprayer
Backpack Sprayer
Flow rate (actual)
62 mL/minute
996 mL/minute
Time required to cover a surface
area of 14 in by 14 in (actual)
30 seconds
10 seconds
Sprayer efficacy across all seven
test materials
> 6 LR (except latex
material)
>6 LR
Waste generation (average)
6 mi-
450 mL
ES-3
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1.0 Introduction
The project was conducted to support jointly held missions of the U.S. Department of Homeland
Security (DHS) and the U.S. Environmental Protection Agency (EPA). The EPA's 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.
Within the EPA, the project supports the mission of EPA's HSRP by providing relevant information
pertinent to the decontamination of contaminated zones resulting from a biological incident.
This report discusses a decontamination project that evaluated the decontamination efficacy and
physical migration (transport) of Bacillus spores and operational efficiency of two types of sprayer
technologies: electrostatic and traditional electric backpack sprayers. These sprayers were used to
apply a decontamination solution to materials that are common constituents of emergency responder
personal protective equipment (PPE) under operationally relevant exposure conditions and contact
times. The following sections discuss the project background and objectives.
1.1 Background
Under Homeland Security Presidential Directive 10, the DHS is tasked with coordinating with other
appropriate federal departments and agencies to develop comprehensive plans that "provide for
seamless, coordinated Federal, state, local, and international responses to a biological attack." As
part of these plans, the EPA, in a coordinated effort with DHS, is responsible for "developing
strategies, guidelines, and plans for decontamination of persons, equipment, and facilities" to mitigate
the risks of contamination after a biological weapons attack. EPA's National Homeland Security
Research Center (NHSRC) provides expertise and products that can be widely used to prevent,
prepare for, and recover from public health and environmental emergencies arising from terrorist
threats and incidents. Within the NHSRC, the Decontamination and Consequence Management
Division (DCMD) conducts research to provide expertise and guidance on the selection and
implementation of decontamination methods that may ultimately provide the scientific basis for a
significant reduction in the time and cost of decontamination events. The NHSRC DCMD
decontamination research program goals are to provide: (1) expertise and guidance on the selection
and implementation of decontamination methods; and (2) the scientific basis for a significant reduction
in the time and cost of decontamination events. The NHSRC works with EPA's Office of Emergency
Management, who have revised the biological personnel decontamination line protocol based on a
previous NHSRC PPE decontamination study (USEPA 2015a, USEPA 2015c).
In previous studies, some of the most promising methods for applying decontaminants such as the
electrostatic sprayer were found to be more efficient than the traditional electric backpack sprayer in
uniform distribution for the decontamination of flat surfaces of large building materials (USEPA
2015b). However, these technologies have not been assessed for time-limited (a few minutes)
applications such as the decontamination of personnel PPE and equipment in a biological
decontamination line.
After the release of a hazardous biological substance, the impacted site is characterized and mapped
into controlled work zones to mitigate the spread of further contamination and prepare for cleanup as
shown in Figure 1-1 (USEPA 1992).
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Figure 1-1. USEPA Standard Operating Safety Guides, Site Control Work Zones (USEPA 1992)
The Exclusion Zone (EZ, or Hot Zone), set up downwind of the Support Zone (SZ), is the
contaminated zone and has the highest potential for exposure. The Contamination Reduction Zone
(CRZ) is the transition area between the EZ and the SZ. The decontamination line is located just
inside the CRZ, typically near the exit of the EZ. The purpose of the decontamination line is twofold:
(1) to ensure that potentially harmful or dangerous residues on persons, samples, and equipment are
confined within the CRZ; and (2) to extract personnel from their PPE safely while also protecting
decontamination line personnel and minimizing liquid waste. Personnel who have been performing
decontamination activities exit the EZ and move through the decontamination line in the CRZ, where
traditional electric backpack sprayers or decontamination showers are often used to distribute a
decontamination solution over entry personnel to decontaminate the PPE and remove potentially
harmful surface residues. This process has the potential to generate a significant quantity of liquid
hazardous waste. However, if an electrostatic sprayer technology could be used to achieve the same
purpose but instead deliver a more uniform distribution of decontamination solution over the PPE
surface while using less liquid decontaminant, decontamination efficacy may be improved and waste
streams and their associated costs may be reduced.
This project addresses the direct need to evaluate alternative sprayer technologies and techniques by
assessing the decontamination efficacy and consequences of using an electrostatic sprayer. The
results of this study will be included as an addendum to the EPA Technical Support Working Group
Task CB-CM-3499 final report, "Test Method for Standardized Evaluation of Decontamination
Solutions." The study results will also provide quantitative information relevant to technical and
operational aspects of personnel decontamination, which can assist emergency responders in
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mitigating health hazards to personnel operating in a chemically- or biologically-contaminated
environment and in minimizing cross-contamination
1.2 Objectives
One main objective of this study was to evaluate the decontamination efficacy of electrostatic sprayer
technology for use in a decontamination line. Another objective was to compare sprayer technologies
currently used in decontamination lines for personnel decontamination (i.e., handheld "garden-type"
sprayers) to the electrostatic sprayer technology.
To compare the two technologies, both were tested by applying a diluted bleach decontamination
solution to a variety of constituents commonly found in emergency responder PPE Levels B or C. The
study used operationally relevant exposure conditions and field-appropriate decontamination solution
contact times to evaluate not only the surface log reduction (LR) of Bacillus spores but also the
physical removal and migration of the spores. This study provided quantitative efficacy information
relevant to sprayer decontamination methods. These results identified a useful means to: (1) assist
decision makers and first responders in mitigating health hazards to personnel in the decontamination
line by minimizing reaerosolization; (2) minimize the potential for contaminant migration from the
incident scene; and (3) reduce liquid waste from the personnel decontamination process. Additional
goals were to assess electrostatic sprayer operational efficiency and evaluate any potential safety
hazards involved with its use.
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2.0 Experimental Approach
The testing was conducted at EPA's Research Triangle Park (RTP) facility in North Carolina. The
general experimental approach used to meet the project objectives is described below.
1. Preparation of representative samples of test materials: The following seven PPE
materials used in suits, boots, gloves, and related accessories were selected for testing: nitrile,
butyl, latex, Tyvek®, Tychem®, neoprene, and ChemTape®. Materials were categorized as
plastic (Tychem®, Tyvek®, and ChemTape®) or rubber (nitrile, butyl, latex, and neoprene) for
surface sampling purposes. Test coupons of each material were prepared as described in
Section 3.1.1.
2. Contamination of PPE coupons with a standardized inoculum of the target organism:
The test material coupons were contaminated using an aerosol deposition method that
delivered a known quantity of spores in a repeatable fashion. Approximately 1 x 107 spores of
Bacillus atrophaeus var. globigii (Bg), a surrogate organism for Bacillus anthracis (Ba), were
deposited onto each test material coupon as discussed in Section 3.3.2.
3. Preparation of decontamination solution: The decontamination solution consisted of 10%
diluted bleach (DB), freshly prepared on each test day as discussed in Section 3.4.2.
4. Preparation of neutralizing agent: STS was used as a neutralizing agent as discussed in
Section 3.4.3. STS was applied to stop the decontamination activity after a prescribed
exposure time. STS also was added to procedural blanks, test coupons, and runoff samples.
5. Application of decontamination procedure on test material coupons: Procedural blanks
(non-inoculated coupon) and test coupons (inoculated) were arranged in the test chamber in a
vertical position, then sprayed using either the electric backpack or the electrostatic sprayer in
accordance with the pre-determined test conditions as discussed in Section 3.4. Deionized
(Dl) water was used for the procedural positive coupons, as a control to decouple the physical
spore removal from the surface against the sporicidal activity of the decontamination solution.
After the prescribed five-minute exposure time, coupons were collected and transferred to a
sampling table for wipe sampling as discussed in Section 5.1.1.
6. Coupon sampling: Coupons were sampled using the wipe sampling method described in
Section 5.1.1. Based on the material category (plastic or rubber), either three or two wipe
samples were collected from each coupon. All coupon wipe samples were extracted in
Phosphate Buffered Saline (135 mM NaCI, 2.7 mM KCI, 4.3 mM Na2HP04, 1.4 mM KH2P04)
with 0.05% Tween® 20 (PBST).
7. Collection of runoff: Liquid runoff from the coupons was collected through the chamber drain
outlet in sterile Nalgene® bottles containing pre-determined volumes of STS neutralizer.
8. Sample extraction and analysis: Wipe samples were extracted in PBST, and aliquots of the
wipe extracts and liquid runoff samples were analyzed using an automated system for plating
assays or filter plating to determine the number of colony forming units (CFU) present in each
sample.
9. Determination of decontamination efficacy: Decontamination efficacy, as a function of the
sprayer technology and material type, was measured as LR in viable spores recovered
following treatment, as compared to controls. Typically, for laboratory assessments of
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decontamination efficacy, an LR of 6 or greater is considered effective. Decontamination
efficacy for each coupon was determined by comparing test coupon results to stainless-steel
inoculum control coupon results. Quantitative assessment of residual (background)
contamination was performed by sampling procedural blanks (non-inoculated coupons
exposed to the same decontamination process as the test coupons). The transfer of viable
organisms to decontamination liquid waste was evaluated through quantitative analysis of
spraying procedure residue samples (such as liquid runoff samples). The physical
removal/transfer of spores was evaluated by sampling procedural positives (sprayed with Dl
water instead of DB).
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3.0 Experimental Materials and Methods
This section describes the test materials, test chamber, test organism and inoculation, and
decontamination equipment (sprayers), solution, and neutralizer used to achieve the project
objectives.
3.1 Test Materials
The representativeness and uniformity of test materials are essential in achieving adequate evaluation
results. Materials are considered representative if they are typical of materials currently used in the
field in terms of quality, surface characteristics, and structural integrity. For this project,
representativeness was ensured by: (1) selecting test materials typically representative of PPE, and
(2) obtaining these materials from appropriate suppliers. Uniformity was maintained by obtaining and
preparing a quantity of material sufficient to allow the preparation of multiple test samples with
presumably uniform characteristics (that is, test coupons for each test were prepared using the same
batch of material).
Coupons of the following seven PPE materials were prepared on site: nitrile, butyl, latex, Tyvek®,
Tychem®, neoprene, and ChemTape®. Table 3-1 summarizes the coupon materials, including their
characteristics and sources.
Table 3-1. Material Specifications
Material
PPE Type
Category
Thickness
(inch)
Manufacturer/Supplier Name
Stainless Steel
NA
Metal
0.02
-
Nitrile (Buna-N)
Rubber
0.01 to 0.02
McMaster-Carr Elmhurst, IL
Butyl
Gloves
Rubber
0.06 to 0.07
MSC Industrial Supply Co.
Latex
Rubber
0.01 to 0.02
Melville, NY
Tyvek® 400
Suits
Plastic
0.0059
DuPont
Tychem® QC/2000
Plastic
0.01
Wilmington, DE
Neoprene (chemical-resistant
rubber)
Boots
Rubber
0.120 to 0.130
MSC Industrial Supply Co.
Melville, NY
ChemTape®
Accessory
Plastic
0.0125
Kappler
Guntersville, AL
Coupon fabrication and test material sterilization are discussed below.
3.1.1 Coupon Fabrication
All coupon dimensions were 14- by 14-inches (in). Material coupons were prepared on a plywood
base using the PPE materials listed in Table 3-1. The following materials and equipment were used to
prepare the coupons:
• 0.438-in Plywood (Plytanium 15/32 CAT PS1-09 Pine Plywood Sheathing, from Lowes, Item #
12192)
• PPE materials (Table 3-1)
• 1/4-in staples
• Staple gun
• Safety razor utility knife
• Table saw
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• Tape measure
• Spray adhesive (Product ID 74, 3M Foam Fast Spray Adhesive Clear, Fort Worth, TX)
• Appropriate PRE (including safety glasses, cut-resistant gloves, and safety footwear)
The procedure summarized below was used to prepare all the test coupons.
1. Personnel preparing the coupons donned appropriate PPE, including safety glasses, cut-
resistant gloves, and safety footwear.
2. Using a table saw, a 14- by 14-in square of Plywood was cut.
Figure 3-1. Test material, Plywood (A) and Coupon Preparation (B)
3. Using a safety razor utility knife, a 16- by 16-in square of PPE material was cut. For frail
materials that tend to tear when only a single layer was wrapped around the Plywood (such as
latex), a double layer of material was used to prepare the coupon.
The material square was placed with the backing side up on a table, and the Plywood was
placed over it.
4. The test material was then folded onto the Plywood and stapled in place using a staple gun
(Figure 3-1 B). Thick materials such as butyl and neoprene were stuck to the Plywood using a
spray adhesive. Figure 3-2 shows a finished coupon.
Figure 3-2. Front (A) and Back (B) of Finished Test Coupon on Plywood
5. For ChemTape®, which is 2 in wide, the tape was wrapped on the 14- by 14-in Plywood in
single layers, leaving no gap between adjacent strips.
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Figure 3-3. shows finished coupons of each test material.
Latex Chemtape Tyvek Neoprerie
Butyl Nitrile(Buna-N) Tychem
Figure 3-3. PRE Test Coupons
3.1.2 Sterilization Process
Materials and supplies were sterilized prior to testing using a method suitable for each item.
Sterilization procedures included vaporized hydrogen peroxide (VHP) sterilization, autoclaving, filter
sterilization, ethylene oxide (EtO) sterilization, and pH-adjusted bleach (pAB) sterilization, as
discussed in the below table (Table 3-2.)
Table 3-2. Sterilization Processes Used
Sterilization
Process
Description
Materials/Supplies
Vaporized
Hydrogen
Peroxide®
(VHP)
Sterilization
Before the sterilization process, coupons and sprayers (with the lid
open) were wrapped in bags, and the ADAs were placed in large
plastic bins. Hydrogen peroxide vapor was produced using a
STERIS VHP 1000ED generator loaded with a 35% hydrogen
peroxide (H2O2) Vaprox® cartridge. Each sterilization cycle
generated a maximum concentration of 250 parts per million (ppm)
VHP and lasted four hours. Negative control coupons were used to
verify coupon sterility.
Test material
coupons, Aerosol
deposition
apparatuses
(ADAs), and
Sprayers
Autoclaving
Sterilized using a 30 minute gravity cycle at 121°C in a STERIS
Amsco Century SV 120 Scientific Pre-Vacuum Sterilizer (STERIS
Corporation, Mentor, OH). The stainless-steel coupons measured
14- by 14-in and were carefully wrapped in aluminum foil to
maintain sterility when removed from the autoclave. A sterility
check for the stainless-steel coupons was performed using swabs
(BactiSwab® Collection and Transport System, Remel, Thermo
Fisher Scientific, Waltham, MA).
Stainless-steel
inoculum control
coupons (0.02 inch
thick), Nalgene®
bottles, and
carboys
Filter
sterilization
Sterilized using a vacuum filter (Corning 430513, Bottle Top
Vacuum Filter, 0.22 micrometer (|jm) pore size, 33.2 centimeter CA
membrane, Tewksbury, MA) and a sterile 1-liter (L) Pyrex bottle.
Sterilized Dl water was transferred into a sterile 5 L carboy. A 50-
milliliter (ml.) sample from each 5 L batch was sent to the NHSRC
RTP Microbiology Laboratory (BioLab) for sterility analysis.
Dl water
8
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Sterilization
Process
Description
Materials/Supplies
Ethylene
Oxide (EtO)
sterilization
Sterilized using an Andersen EtO sterilizer system (FN 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, humidichip, and
EtO dispenser.
3. The sterilization bags were vacuum-sealed and loaded
into the EtO sterilizer for an 18 hour sterilization cycle.
Sampling templates
and inoculation
equipment
Sterilization
using pAB
solution
To avoid cross contamination between tests, the interior of the test
chamber was sterilized using pAB immediately before testing. This
process commonly is referred to as "reset" of the test chamber.
The pAB solution was prepared using Dl water, 5% acetic acid,
and bleach in an 8:1:1 ratio, then loaded into the pre-sterilized (with
pAB) tank of a SHURflo 4 ProPack Rechargeable Electric
Backpack Sprayer SRS-600 (Pentair-SHURFlo, Costa Mesa,
CA). The sprayer was used to coat the interior of the test chamber
with pAB. After a 1Q-minute (min) contact time, the chamber was
rinsed with sterile Dl water to remove residual pAB from the
chamber. A swab (BactiSwab® Collection and Transport System,
Remel, Thermo Fisher Scientific, Waltham, MA) sample of the test
chamber was collected for a sterility check.
Interior of the test
chamber
3.2 Test Chamber
The sprayer test chamber is located at EPA's RTF facility in North Carolina. The test chamber
measures 4- by 4- by 4-feet (ft) and was designed to accommodate three 14- by 14-in coupons at a
time in a horizontal or vertical position. For this project, a single PPE coupon was placed in the test
chamber at a time and sprayed in a vertical position as shown in Figure 3-4.
Material Coupon
Figure 3-4. Decontamination Test Chamber with Coupon
9
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Except for the clear acrylic front and top pieces, the test chamber is constructed of solid stainless
steel. The reverse-pyramid design of the chamber bottom allows the collection of coupon runoff
through a central drain with a 3-in diameter. The chamber air is exhausted to the facility's air handling
system through a connection also fitted with a sampling port. The port was used to collect samples
during each test so that the quantity of aerosolized spores could be estimated.
Two HOBO Relative Humidity/Temperature sensors (Model U12, Onset Computer Corporation,
Bourne, MA) were placed around the spraying and inoculation areas. Temperature and humidity were
measured to generate qualitative information in anticipation of helping to explain variations in project
data, if any.
3.3 Test Organism and Inoculation Procedure
Details on the test organism and inoculation process are provided in the following sections.
3.3.1 Bg Surrogate for Ba
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-pigmented colony when grown on nutrient agar, a desirable characteristic for detecting viable
spores in environmental samples. Bg has a long history of use in the biodefense community as a
simulant for anthrax-associated biowarfare and bioterrorism events (Gibbons et al. 2011).
3.3.2 Bg Spore Inoculation
The test coupons were inoculated with Bg spores using a metered-dose inhaler (MDI). The MDI
canister contained Bg spores suspended in ethanol solution, HFA-134A propellant (1,1,1,2-
tetrafluoroethane) gas, and Tween®. The MDI actuator is a small plastic tube in which the MDI
canister is inserted (Figure 3-5(A)).
Figure 3-5. MDI Actuator (A) and Canister (B)
Each time the actuator is depressed, a repeatable number of spores are deposited on the coupon (Lee
et al. 2011). MDIs selected for testing must weigh more than 10.5 grams. MDIs weighing less than 10.5
grams are retired and no longer used. Each test coupon was inoculated independently using the MDI
canister and actuator. The MDIs were weighed before and after inoculation to ensure proper discharge.
10
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For quality control (QC) purposes for the MDIs, a stainless-steel inoculation control coupon was
included as the first, middle, and last coupon inoculated using a single MDI in a single test.
For the MDI inoculation procedure (Lee et al. 2011; Caifee et al. 2013), an ADA measuring 1- by 14-in
was placed on the surface of the test coupon (Figure 3-6).
Figure 3-6. 14- by 14-in ADA with Syringe Filter
The ADA was clamped to the test coupon, and the MDI was attached to the top of the ADA. A slide
below the MDI was opened, and the MDI was activated. After inoculation, the slide was closed and the
MDI was removed. The assembly was kept closed while the spores were allowed to settle for 18 hours
before testing. This process was repeated for each test. (Figure 3-7).
Figure 3-7. Inoculation Setup
3.4 Decontamination Equipment, Solution, and Neutralizer
This section discusses decontamination equipment (sprayers), decontamination solution, and
neutralizer.
3.4.1 Sprayers
The sprayers summarized in Table 3-2 were tested.
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Table 3-2. Decontamination Sprayers Tested
Sprayer Type
Description
Flow Rate
Electric
backpack
SHURFlo 4 ProPack Rechargeable Electric Back Pack
Sprayer SRS-600 (Pentair-SHURFlo, Costa Mesa, CA)
996 mL/min ute
Electrostatic
SC-ET HD electrostatic sprayer (Electrostatic Spraying
Systems ESS, Watkinsville, GA)
62 mL/minute
Each type of sprayer is discussed in more detail below.
3.4.1.1 Electric Backpack Sprayer
The SHURflo 4 SRS 600 ProPack rechargeable electric backpack sprayer used for this project
measures approximately 36 in high by 24 in wide by 6 in long (Figure 3-8). This backpack sprayer has
a variable speed pump, an adjustable spray cone nozzle, and the hose is made of reinforced/braided
PVC. This sprayer has been used in previous EPA decontamination studies and provides a good
representation of the type of handheld sprayer nozzle that is typically used in personnel
decontamination lines.
After sterilization, the 4-gallon tank of the sprayer was filled with 10% DB. The sprayer knob was
tightened on each test day to ensure a consistent cone spray (several inches in diameter) on all
coupons. The consistency of spray was verified by performing a spray pattern test using a
construction paper. Before each test, a stop watch and a 500 ml_ graduated cylinder were used to
verify (in triplicate) that the approximate flow rate of each sprayer was 1,020 milliliters per minute
(mL/min). The liquid was collected and volume recorded based on a 10-second spray time. Readings
were expected to be within 10% of the average. If they were not, the nozzle was tightened or the
sprayer wand was changed, and the flow rate was re-tested until the desired flow rate was achieved.
3.4.1.2 Electrostatic Sprayer
The air-assisted SC-ET HD electrostatic sprayer shown in Figure 3-9 was used in this study.
12
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Figure 3-9. SC-ET HD Air-Assisted Electrostatic Sprayer
This sprayer measures approximately 22 in high by 16 in wide by 10 in long and produces electrically
charged spray droplets that are carried to the target in a gentle low-pressure air stream. The sprayer
tank has a capacity of 4.7 L and a spray gun with hose length of 15 ft. The SC-ET HD ESS system is
intended for light-duty, quick disinfection and sanitization applications and is compatible with most
conventional chemicals. The sprayer also is equipped with a patented MaxCharge™ technology
electrostatic spray gun that delivers droplets with a volume median diameter (VMD) of 40 pm. The
electrostatic charge induced by the MaxCharge™ nozzle is strong enough to allow the droplets to
move in any direction to cover surfaces homogeneously, according to the manufacturer.
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 ft to cover the whole 14- by 14-in test coupon area. A stop watch and a
250-mL graduated cylinder were used to verify (in triplicate) that the approximate flow rate of the sprayer
was 240 miliiliters/minute (mL/min). The liquid was collected and volume recorded based on a 30-
second spray time. Readings were expected to be within 10% of the average. If they were not, the spray
gun was checked for bleach corrosion and re-cleaned if necessary. The flow rate was re-tested until
the desired flow rate was achieved. During operation of the electrostatic backpack sprayer, personnel
wore anti-static gloves (Part No. AS9674S, MCR Safety, Collierville, TN) for safety.
3.4.2 Decontamination Solution
DB (10%) was used as the decontamination agent for this study as referenced in the EPA
Consequence Management Advisory Division's (CMAD's) "BioResponse Decontamination Line
Standard Operating Protocol" (SOP) (USEPA 2015c). The solution was prepared in fresh 1-L batches
on each test day using the procedure summarized below.
1. In a sterile container, 900 mL of D! water was added to 100 mL of Clorox® Concentrated
Germicidal Bleach.
2. The solution was manually mixed for 1 min, resulting in a 10% DB solution.
3. The pH and free available chlorine (FAC) of the solution were measured before use.
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3.4.3 Neutralizing Agent
Neutralizing agents are used to stop the decontamination reaction to achieve a prescribed contact
time. STS has been demonstrated to be effective for bleach on both porous and nonporous surfaces
(Calfee et al. 2011). so it was selected for use during this test. The volume of STS added to the
sample containers (wipe and liquid runoff) was determined by measuring the FAC of the DB solution
using a HACH® Hypochlorite Test Kit (Model CN-HRDT, Fisher Scientific, Waltham, MA). The HACH
test kit uses an iodometric method to determine FAC and chlorite concentrations. Method
development tests were conducted to ensure the effectiveness of STS before its use in this study.
A 2 normal (N) solution of STS was prepared as summarized below.
1. STS pentahydrate (Na2S2C>3 5H2O, 496.4 grams) crystals were added to 1 L of Dl water.
2. The solution was stirred until all the crystals dissolved completely.
3. The 2 N STS solution then was sterilized using a bottle-top filter (150 ml_ Corning Bottle Top
Filter, 0.22 |jm cellulose acetate, 33 millimeter neck, sterile, Catalog No. EK-680516, Corning,
NY) and a vacuum filtration system.
4. Each batch of STS was dated, stored at 4°C, and used within six months of preparation.
14
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4.0 Decontamination Testing
This section discusses the test matrix and approach for the decontamination coupon testing.
4.1 Test Matrix
Table 4-1 summarizes the test matrix characteristics including test material and number of coupons
tested.
Table 4-1. Test Matrix
Test ID
Test Material
Category for
wipe
sampling
Decontamination
Technology
Total No. of
Material
Coupons
1
Nitrile (Buna-N)
Rubber
Backpack Sprayer
12
2
Electrostatic Sprayer
12
3
Butyl
Rubber
Backpack Sprayer
12
4
Electrostatic Sprayer
12
5
Latex
Rubber
Backpack Sprayer
12
6
Electrostatic Sprayer
12
7
Tyvek®
Plastic
Backpack Sprayer
12
8
Electrostatic Sprayer
12
9
Tychem®
Plastic
Backpack Sprayer
12
10
Electrostatic Sprayer
12
11
Neoprene (chemical-
Rubber
Backpack Sprayer
12
12
resistant rubber)
Electrostatic Sprayer
12
13
ChemTape®
Plastic
Backpack Sprayer
12
14
Electrostatic Sprayer
12
Each test used the coupon configuration summarized in Table 4-2.
Table 4-2. Test Coupon Configuration
Type of Coupon
No. per
Test
Contaminated with 107
Bg Spores
Decontaminated
Negative control
1
No
No
Procedural blank
1
No
Yes, 10% DB
Test
3
Yes
Yes, 10% DB
Procedural positive control (blank for
procedural positive coupons)
1
No
Yes, sterile Dl water
Procedural positive
3
Yes
Yes, sterile Dl water
Positive control
3
Yes
No
Stainless-steel inoculation control
(used in calculation of
decontamination efficacy, i.e., LR)
3
Yes
No
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4.2 Testing Approach
The decontamination approach consisted of applying the 10% DB solution to the surface of each 14-
by 14-in coupon until the coupon was completely wet (visually). This process required 10 and 30
seconds for the electric backpack and electrostatic sprayers, respectively.
The migration and physical removal of spores were evaluated as functions of the following:
• Type of sprayer (electric backpack or electrostatic)
• Type of PRE test material
The approach below was used for the testing.
1. Test Chamber Sterilization and Cleaning: Freshly prepared pAB was used to sterilize the
test chamber as discussed in Section 3.1.2.5 before each procedural blank test. In addition, to
avoid biased results in the liquid runoff samples caused by residual bleach, the test chamber
also was cleaned with pAB and sterile D! water before processing the procedural positive
coupons.
2. Coupon Setup: For testing, a single coupon was placed in a vertical orientation in the center
of the test chamber (as shown in Figure 3-4). Procedural blank coupons were always tested
first, followed by test coupons.
3. Liquid Runoff: A clean, sterile Nalgene® bottle (500 mL or 1 L) preloaded with a pre-determined
volume of STS was used to collect liquid runoff by placing the bottle under the drain of the test
chamber (Figure 4-1). The bottles were weighed before and after each test to determine the
volume of liquid runoff generated by each type of sprayer and test material.
Figure 4-1. Liquid Runoff Collection Assembly
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4. Decontaminant application: The 10% DB solution was applied using either the electric
backpack or electrostatic sprayer as summarized below.
a. A spray test was initiated by checking the flow rate of the sprayer as described in Section
3.4.1.1 and Section 3.4.1.2. Later in the test procedure, a spray pattern test was conducted
by spraying from one foot away onto a piece of construction paper measuring 14- by 14-in
mounted in the test chamber in the vertical orientation. The spray pattern was visually
assessed to ensure that the spray was being discharged into the center of the paper.
b. The coupons were sprayed using multiple side-to-side strokes (starting from the top left
side of the coupon and ending at the bottom right, moving downward, in a "Z" pattern) to
completely wet the coupon surface. This step was repeated as often as necessary to
satisfy the required spray duration. Table A-1 in Appendix A presents the spray duration
log. A contact time of five minutes, determined from CMAD's "BioResponse
Decontamination Line SOP" (EPA 2015c) was allowed before sampling. Procedural blanks
(coupons of each test material not contaminated with Bg spores) were processed first,
followed by the test coupons. The physical transfer of spores using both types of sprayers
was evaluated by spraying a set of coupons (Procedural positive control and material
coupons) with sterile Dl water. These coupons were processed using the same procedure
as the test coupons.
After decontamination spraying, residual spores were recovered from the coupons using the wipe
sampling technique discussed in Section 5.1.1 and assessed for viability. Liquid waste (runoff)
samples were also collected and analyzed for viable spores. Together, results from these samples
were used to determine the decontamination efficacy of each type of sprayer under the test conditions
discussed above using 10% DB.
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5.0 Sampling and Analytical Procedures
A sampling data log sheet was maintained for each sampling event (or test) that included each
sample's identification (ID) number, the date, test name, sample description, and sampling start and
end times. Appendix A presents a sample of that the data log. The sample ID numbers and
descriptions were pre-printed on the sampling data log sheet before sampling began. Digital
photographs were taken to document activities throughout the test cycle.
The following sections discuss the sample types, sample quantities, sample handling, microbiological
analysis, decontamination solution characterization, and determination of efficacy.
5.1 Sample Types
The types of samples collected for this study include wipe, liquid runoff, aerosol(air), and sterility
check swab samples, as discussed below.
5.1.1 Wipe Samples
The test materials were categorized as plastic (Tyvek®, Tychem®, and ChemTape®) and rubber
(nitrile, butyl, latex, and neoprene). To minimize cross-contamination of decontaminated coupons,
each coupon surface was being wiped completely to collect surface wipe samples, leaving no
contaminated liquid residue behind. Surface wipe samples were collected using polyester-rayon blend
wipes (Curity all-purpose sponges #8042, 2- by 2-in, four-ply, Covidien PLC, Dublin, Ireland). Three
wipes were used on each plastic material coupon and two wipes were used on each rubber material
coupon. The number of wipes required to effectively remove all liquid from the surface of each
material type was determined as a part of a method development process.
The BioLab prepared the wipes for each test. Using sterile forceps, each four-ply wipe was aseptically
removed from the packing and placed in an unlabeled, sterile, 120-mL specimen cup (Catalog No. 14-
375-462, Fisher Scientific, Waltham, MA). Each wipe was moistened by adding 2.5 ml_ of sterile PBST,
and the cup was capped. The wiping protocol used in this project was adopted from the protocol
described by Busher et al. (2008'; and Brown et al. (2007). The coupon surface was wiped by applying
consistent pressure. An S-stroke motion was used both horizontally and vertically to cover the sample
area as shown in Figure 5-1.
I
Figure 5-1. Wipe Sampling of Test Coupon
After wiping, each wipe was loosely folded and placed in a sterile specimen cup containing PBST (60
mL for plastic materials and 40 m!_ for rubber materials) and a pre-determined amount of STS
neutralizer. Wipe start and end times were recorded using a wipe sampling log (Table A-2 in Appendix
A).
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5.1.2 Liquid Runoff Samples
Decontamination solutions that accumulated through the test chamber collection port (drain) were
collected as liquid runoff samples. Each sample was collected in a 500 ml_ Nalgene® bottle pre-loaded
with a pre-determined volume of STS neutralizer. Runoff collection sample volumes were determined
by subtracting the weight of the collection bottle (containing only the STS neutralizer) from the weight
of the bottle with the runoff sample in it. The weights were recorded using a liquid runoff collection log
(Table A-3 in Appendix A).
5.1.3 A erosol (A ir) Samples
Aerosol samples were collected using Via-Cell® bioaerosol cassettes (Part No. VIA010, Bioaerosol
Sampling Cassette, Zefon International, Ocala, FL) as shown in Figure 5-2.
Figure 5-2. Via-Cell® Bioaerosol Sampling Cassette
During each test, aerosol samples were collected from inside the test chamber interior and from the
test chamber exhaust duct. The initial and final temperature, gas meter volume, and sample flow
change in enthalpy (AH) was recorded for each sample using the Via-Cell® cassette log (Table A-4 in
Appendix A). At the end of each sampling event, the Via-Cell® cartridge was aseptically retrieved from
the pump and placed in the Via-cell® pouch. The outside of the pouch was sterilized using bleach
wipes before transport to the BioLab for analysis.
5.1.4 Sterility Check Swab Samples
Pre-moistened swabs (BactiSwab® Collection and Transport System, Remel, Thermo Fisher
Scientific, Waltham, MA) were used to wipe specified areas to test for the presence of spores. A
single swab sample was collected for each of the following types of equipment for each test:
• ADA and ADA gasket;
• Sprayer (electric backpack or electrostatic);
• Test chamber; and
• Coupons (test material and stainless-steel coupons).
An unused sterile swab sample was used as a laboratory blank.
5.2 Sample Quantities
Table 5-1 summarizes the sample quantities and the number of samples collected during each testing
event.
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Table 5-1. Sample Quantities
Sample Name
Sample Description
Replicates
Samples
Collected
Test coupon (2-3
wipes per coupon)
14 - by 14-in material coupon
inoculated and decontaminated
with DB
3 per material and sprayer type
3 specimen
cups, 1 per
replicate
Procedural positive
coupon (2-3 wipes per
coupon)
14- by 14-in material coupon
inoculated and sprayed with Dl
water
3 per material and sprayer type
3 specimen
cups, 1 per
replicate
Negative control
coupon (2-3 wipes per
coupon)
14- by 14-in material coupon not
contaminated or decontaminated
1 per material and sprayer type
1 specimen cup
per test
Procedural blank
coupon (2-3 wipes per
coupon)
14- by 14-in material coupon not
contaminated but decontaminated
with DB
1 per material and sprayer type
1 specimen cup
per test
Procedural positive
control coupon (2-3
wipes per coupon)
14- by 14-in material coupon not
contaminated but decontaminated
with sterile Dl water
1 per material and sprayer type
1 specimen cup
per test
Positive control
coupon (2-3 wipes per
coupon)
14- by 14-in material coupon
contaminated but not
decontaminated
3 per material and sprayer type
3 specimen
cups, 1 per
replicate
Stainless-steel
inoculation control
coupon (2-3 wipes per
coupon)
14- by 14-in stainless-steel
coupon contaminated but not
decontaminated
3 per inoculation event,
inoculated immediately before
each positive control coupon
3 specimen
cups, 1 per
replicate
Liquid runoff
Effluent from sprayed diluted
bleach containing STS neutralizer
1 per sample type and material
4 per test
Via-cell® cassette
Air sample - chamber and
exhaust duct
Not applicable
2 per test
Sterility check sample
Swab sample and Dl water
sample
Not applicable
7 swabs per test
and 1 Dl water
sample per test
5.3 Sample Handling
After the collection of coupon surface wipe and liquid runoff samples, the samples were sealed in
secondary containment and transported to the BioLab for analysis. This section discusses the sample
containers, preservation, and custody.
5.3.1 Sample Containers
For each wipe sample, the primary container was an individual sterile specimen cup. Secondary and
tertiary containment consisted of sterile sampling bags. Liquid runoff samples were collected in
individual sterile and labeled Nalgene® bottles. A single plastic container was used to store the
samples in the decontamination laboratory during sampling and for transport to the BioLab.
5.3.2 Sample Preservation
All sample specimen cups 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. Upon arrival at the Biolab, samples were
20
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unpackaged immediately and stored at 4 °C until processed. Hold times in the laboratory did not
exceed one week.
5.3.3 Sample Custody
After sample collection for a single test was completed, all biological samples were immediately
transported to the BioLab accompanied by a completed Chain of Custody form.
5.4 Microbiological Analysis
The NHSRC Bio-contaminant Laboratory analyzed all samples for presence (sterility check samples)
and to quantify the CFU per sample (wipe samples, liquid samples, and filter samples). Multiple
wipes used per test coupon were combined into one sample container and extracted together.
Samples were processed using a variety of methods including spiral plating, spread plating, filter
plating and or the high debris method, developed by the BioLab.
For all sample types, the BioLab analyzed samples to quantify the number of viable spores (CFU) per
sample. For all sample types, PBST was used as the extraction buffer. Each sample was aliquoted
and plated in triplicate using a spiral plater (Autoplate 5000, Advanced Instruments Inc., Norwood,
MA), which deposits the extracted sample in exponentially decreasing amounts across a rotating agar
plate in concentric lines to achieve three tenfold serial dilutions on each plate. Plates were incubated
at 35 ± 2 °C for 16 to 19 hours. During incubation, colonies develop along the lines where the sample
was deposited (see Figure 5-3). The colonies on each plate were enumerated using a QCount®
colony counter (Advanced Instruments Inc., Norwood, MA).
Figure 5-3. Bacterial Colonies on Spiral-plated Agar Plate
Positive control samples were diluted 100-fold (10'2) 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® 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 3Q-CFU threshold were either spiral plated again using a less diluted sample
aliquot, spread plated in triplicate, or filter plated. The follow-up plating method and volumes used
were based on the CFU data from the initial QCount® results. All plating was performed on tryptic soy
21
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agar (TSA) plates, and the plates were incubated at 35 ± 2 °C for 20 to 24 hours before manual
enumeration. Figure 5-4 shows a filter plate with colonies of Bg.
Figure 5-4. Bacterial Colonies on Filter Plate
5.5 Decontamination Solution Characterization
This section discusses the characterization of the 10% DB solution, which involved the determination
of pH and temperature and FAC by titration, as discussed below.
5.5.1 pH
The pH of the decontamination solution was measured daily or after each new solution was prepared,
using a calibrated pH meter (Model No. 35614-30, Oakton® pH 150, Oakton Instruments, Vernon Hills,
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 or other thermometer known to be accurate.
5.5.2 FAC by Titration
The FAC of the DB solution was measured immediately after preparation using 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 discusses the titration procedure and FAC concentration (https://pim-
resources.coleparmer.com/instruction-manual/24908-0Q.pdf , accessed August 21. 2018).
5.6 Determination of Efficacy
The overall effectiveness of a decontamination technique is a measure of the ability of the technique
to inactivate or remove spores from material surfaces. Data reduction was performed on
measurements of the total viable spores (CFU) recovered from each sampled surface or material.
Decontamination efficacy for a particular material was calculated in terms of the LR. The number of
spores (CFU) recovered from each test coupon (CFUt) and positive-control coupon (CFUpc) was
transformed to its log™ value. Then, the mean of the log™ values for each test coupon (three
replicates) was subtracted from the mean of the log™ values for each positive control (three
replicates), as follows:
Efficacy (LR) = (log CFUpc)| - |(log CFU,)
22
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where CFUpc is the number of CFU recovered from the inoculum positive control coupons (stainless
steel coupons not decontaminated), and CFUt is the number of CFU recovered from the test coupons.
When filter plates had no CFU detected, a value of 1 CFU was input, resulting in a log value of 0.
Many of the decontamination efficacy results are presented or discussed in terms of whether a 6 LR
of the micro-organism population was obtained for a particular material and test condition. The 6 LR
benchmark is used, since a decontaminant that achieves an LR of 6 or greater (when a 6-7 log
challenge is used) for a particular material is considered an effective sporicidal decontaminant
(USE 37). We caution, however, that effective decontamination in the laboratory setting may not
always transfer to similar efficacy in a field- or full-scale, more realistic setting. Further, a 6 LR still
might not be safe for a highly contaminated area. For example, a 6 LR of spores against a spore
loading of 8 or 9 log CFU would leave significant remaining viable spores and could potentially pose a
health hazard.
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6.0 Results and Discussion
This type of laboratory study was conducted to evaluate actual PPE materials and spray technologies
that may be used in a biological personnel decontamination line. The wet decontamination step may
be conducted after gross decontamination procedures to ensure the biological agent is inactivated
prior to doffing of PPE. This study examined the decontamination efficacy of the two types of sprayers
tested, spore disposition (the transport or migration of spores to the air or as liquid runoff), and the
operational efficiency of each type of sprayer tested as discussed below. A results summary is
provided at the end of this section.
6.1 Decontamination Efficacy
In this section, the decontamination efficacy of the two sprayers (traditional backpack and
electrostatic) is discussed. Decontamination is considered effective when there is an LR of greater
than or equal to 6 or 1 x 106 CFUs (USEPA 2007).
Figure 6-1 summarizes the surface decontamination efficacies for the two sprayers on each tested
material type.
Surface Log Reduction:
Backpack Sprayer vs. Electrostatic Sprayer
9.0 -i
Nitrile Butyl Latex Tyvek Tychem Neoprene Chemtape
¦ Backpack Sprayer (7.2 ± 0.4) ¦ Electrostatic Sprayer [7.0 ± 0.7)
'Denotes no CFU detected above detection limit
Figure 6-1. Surface Decontamination Efficacy
Overall, both sprayers achieved a surface LR > 7 for at least five of the seven PPE material types,
with no statistically significant difference between the two sprayers when all LR values were pooled
and compared (p-value = 0.49). Spore CFU quantities for the inoculum controls were on the order of
107 CFU. For three of the seven test materials, no CFU were detected on the material surfaces when
the electrostatic sprayer was used. In contrast, non-detects were not observed for any of the
backpack sprayer tests. Because residual spores were quantified on the PPE material in many cases,
24
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full decontamination had not occurred on these materials. The slightly lower electrostatic sprayer
efficacy (LR = 5.7) observed for latex may be a result of its observed hydrophilicity but why not see
same effect for other sprayer? The decontamination solution immediately ran off the latex material
upon spraying with the electrostatic sprayer, perhaps preventing the contact time needed to fully
inactivate the Bg spores. Hydrophilicity of the latex material could have resulted in a flat
decontamination solution droplet formation on its surface, causing a lower contact angle as shown in
Figure 6-2).
Contact angle {between
coupon surface and liquid
droplet)
Liquid droplet
Contact angle (between
coupon surface and liquid
droplet)
Liquid droplet
Latex material coupon
Other matenal coupons
Figure 6-2. Representation of Contact Angle of Liquid Droplets on Coupon Surfaces
Hydrophilic surfaces have contact angles of less than 90° (American Chemical Society 2014.)
Hydrophilic surface droplet formation would have resulted in the coalescing of droplets and
subsequent immediate runoff of the decontamination solution. During testing, the electrostatic sprayer
solution did not form proper droplets on the latex material. Instead, the liquid spray was observed to
coalesce and run off the material immediately, preventing the contact time necessary to fully
decontaminate the material. Figure 6-3 shows: A) the beading of solution typically seen on all test
PPE materials except latex as well as B) the coalescence of the beads on latex for the electrostatic
sprayer.
Figure 6-3. Typical Beading of droplets seen on Butyl, Neoprene, Nitrile, Chemtape®, Tychem® and
Tyvek®* (A) and coalescence of droplets on Latex (B)
"Image created using ImageJ software
Finally, the latex material was less robust than the other materials, so the latex material was applied
to the coupons in a double layer to prevent tearing. This variation in coupon preparation may have
contributed to the large standard deviation observed for the electrostatic sprayer and the reduced
surface LR results.
25
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6.2 Spore Disposition (Fate and Transport of Spores)
The field applicability of a spray technology depends not only on its surface decontamination
performance but also its likelihood of transferring spores from a material surface to its surrounding
environment (i.e., cross-contamination). To assess the potential of viable spores to be physically
washed off the test coupon surfaces, all liquids used in the decontamination process were collected
and quantitatively analyzed. To provide a conservative estimate of spore fate and transport, runoff
samples were neutralized immediately upon collection by pre-loading collection tubes with the STS
neutralizing agent.
During each decontamination spray test, coupons of each material type were spray tested in triplicate.
One combined runoff sample was collected per material test and includes runoff from triplicate
coupons into one container, and analyzed for the number of viable spores. Figure 6-4 summarizes the
log number of viable spores (CFU) collected in the runoff samples for each material type.
Viable Spores Recovered in Post-Spray Runoff Samples
8.0
7.0 -
Nitrile Butyl Latex Tyvek Tychem Neoprene ChemTape
¦ Backpack Sprayer ¦ Electrostatic Sprayer
'Denotes no CFU detected exceeding detection limit
Figure 6-4. Log CFU Bg Spores in Liquid Runoff Samples
As the figure shows, all the runoff samples collected from the electric backpack sprayer contained a
large number of viable spores, whereas those collected from the electrostatic sprayer contained very
few to no detectable viable spores. This significant difference in spores collected in runoff between the
two sprayers is due to the considerable less decontaminant used to cover the PPE coupon surface
using the electrostatic sprayer. The application flow rate is higher for the electric backpack sprayer,
which results in more runoff as compared to the electrostatic sprayer. More liquid applied leads to
more physical transport of spores off the PPE material. Table B-1 in Appendix B presents the
decontamination efficacy results for each material in more detail.
The field applicability for a spray technology used for personnel decontamination also depends on its
potential to: (1) minimize cross-contamination among field personnel and equipment; (2) limit the
spread of contamination beyond the site originally impacted; and (3) minimize additional exposure
risks requiring further remediation action. Assessment of these factors requires an understanding of
how a sprayer effects the physical removal of spores from a material surface. Each sprayer also was
26
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evaluated when Dl water was substituted for DB, and the test coupons were sprayed under the
decontamination test conditions. The number of viable spores (CFU) physically removed from test
coupons indicates a potential cross-contamination risk from migration of spores off PPE, which could
be tracked outside the decontamination line area. Figure 6-5 summarizes the recovery of spores for
the procedural positive coupons sprayed with Dl water for each sprayer type and test material.
C
o
Q_
3
O
U
E
o
cN
CD
>
O
o
(D
csl
¦*-»
c
(D
(J
s_
(D
CL
Percent Recovery of Spores from Coupon Materials
When Spraying with Dl Water Only
100%
90%
80%
70%
60%
50%
40%
30%
20%
10%
0%
il .1
Nitrile
Butyl
Latex
Tyvek
Tychem Neoprene Chemtape
I Backpack Sprayer ¦ Electrostatic Sprayer
Figure 6-5. Percentage of Bg Spores Recovered from Procedural Positive Coupons
As implied in the above figure, the backpack sprayer physically removed more spores during the liquid
application for all material types than the electrostatic sprayer, which led to lower percent recovery of
spores from coupon surfaces. Percent recovery was calculated as amount recovered on procedural
positive (CFU)/inoculated controls (CFU) X 100. Percent recoveries from the runoff solution are not
shown in the figure but were consistently higher for the backpack sprayer as compared to the
electrostatic sprayer, indicating that use of the backpack sprayer, as tested in this study, physically
removes biological contamination from the PPE surface and could result in environmental cross-
contamination of PPE and other equipment in a biological decontamination line. Table B-2 in
Appendix B presents results for percent recovery achieved during the Dl water wash-down for each
material and each sprayer in detail. Much greater recovery of spores from the PPE surfaces was
observed with the electrostatic sprayer, with the exception of Tyvek®. We believe that the low
recovery from Tyvek® may have been due to an inoculation malfunction or residual decontaminant in
the test chamber.
Via-Cell® bioaerosol cassette samples were also collected to study the fate of the spores further. Two
cassettes were used to evaluate re-aerosolization during each spray test. One cassette was placed
inside the test chamber, and the other cassette was connected to the exhaust duct of the test
chamber. The sampling was conducted eight diameters downstream and two diameters upstream of
any flow disruptions. The Via-Cell® bioaerosol cassettes were installed after sterilizing the test
chamber. The cassettes were operated only during the spraying of test coupons. During most tests,
27
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no spores were detected in the air samples. Table B-3 in Appendix B presents results for the fate of
spores during aerosol sampling for each material and each sprayer in more detail.
Controlled reaerosolization experiments should be conducted during PPE decontamination spray
tests using other bioaerosol sampling techniques like Dry Filter Units (DFUs) that sample a much
greater volume of air, to validate the results obtained using the above method.
6.3 Liquid Waste Generation
In a previous EPA study evaluating the decontamination line protocol (USEPA 2015a), liquid waste
generated during decontamination was found to be a key carrier of contamination. EPA recommends
avoiding large volumes of liquid waste generation unless a completely effective decontamination
technique (with immediate efficacy) is used. Otherwise, biological contaminants may be transported
outside the decontamination line area. Additionally, liquid waste generated from a biological
decotamination line may be costly to dispose of and will likely cause difficulty in finding a disposal
facility willing to accept the liquid waste.
To evaluate decontamination line suitability for a spray technology, waste assessment must be
considered, so quantifying and comparing the amount of potentially hazardous liquid waste generated
by each sprayer type was a project objective. Traditional backpack sprayers have the potential to
generate a significant quantity of liquid hazardous waste due to the volume sprayed and runoff from
PPE. Additionally, these types of sprayers typically cause overspray (excess liquid that spreads
beyond an area being sprayed) when spraying PPE surfaces, which could lead to cross-
contamination outside the decontamination setup. The electrostatic sprayer could be used to achieve
more uniform distribution of decontamination solution over the surface area sprayed, as well as
forming a "liquid film" that adheres to the material, thereby significantly reducing waste streams and
costs for liquid hazardous waste disposal. During decontamination testing, runoff liquid volumes were
collected and measured gravimetrically. Figure 6-6 summarizes the average amount of liquid waste
produced by each sprayer type over the range of test materials.
Average Volume (mL) of Liquid Waste Collected
During Test Coupon Spraying Procedure
o
in
o
-------
cost savings can be achieved through the use of an electrostatic sprayer for personnel
decontamination.
29
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6.4 Results Summary and Discussion
Average surface decontamination results for both sprayer types indicated an LR of greater than or
equal to 6 for most materials (except latex), suggesting that both sprayer types provide the same level
of decontamination efficacy (p-value = 0.49). However, liquid runoff sample results for the regular
backpack sprayer show a significant number of viable spores in the runoff, indicating that the spores
were washed off the test coupons during the decontamination process. Conversely, for the
electrostatic sprayer, few to no viable spores were observed in the liquid runoff samples for all
material types, suggesting that the spores were not washed off the coupons and were inactivated
during the five-minute contact time using the DB solution.
Overall, the electrostatic sprayer demonstrated the ability to contain spores on the coupon surfaces,
which resulted in a significant reduction in the number of spores that migrated in the pre-neutralized
decontamination runoff compared to the backpack sprayer. In tests using Dl water only, the backpack
sprayer physically removed (through migration) significantly more spores from the PPE coupons than
the electrostatic sprayer, demonstrating the negative consequence of potential contamination to be
transferred from the PPE to the decontamination area, which may lead to cross contamination outside
the CRZ if the spores are not fully inactivated. Additionally, liquid hazardous waste disposal costs
could be increased.
Table 6-1 demonstrates the overall comparison of the two sprayer technologies and highlights the
pros and cons for electrostatic sprayers and traditional backpack sprayers.
30
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Table 6-1. Sprayer Comparison
Traditional Backpack Sprayer
Electrostatic Sprayer (ESS)
Pros
Cons
Pros
Cons
Efficacy
X
>6 log
reduction
X
>6 log reduction
Liquid Spray
Volume
X
X
16X less
Waste
Generated
X
X
75X less
Coupon
Coverage spray
time
X
3X less
X
Droplet particle
size
X
X
Smaller droplet size (40 |jm) leads
to more surface area and better
coverage
Electrostatic
Attraction
X
X
Wraparound effect leads to
multisurface coverage
Electric shock
X
No risk of
electrical
shock
X
Wear anti-static gloves and use
bonding strap to prevent
electrostatic buildup
Cross
contamination
X
Runoff introduces
potential for cross
contamination
X
Very little runoff minimizes cross
contamination
Cost
X
10X less
than ESS
X
Based on the study results, use of the electrostatic sprayer technology in the decontamination line
could reduce the risk for cross-contamination of personnel and equipment compared to the regular
backpack sprayer. Additionally, the electrostatic sprayer generated 75 times less liquid runoff than
the backpack sprayer, suggesting that the electrostatic sprayer could reduce waste volumes and
associated disposal costs.
Although the spray duration of the electrostatic sprayer was three times longer than the traditional
backpack sprayer, the liquid waste from the electrostatic sprayer rarely contained viable spores, and
the waste stream volume was significantly reduced. Therefore, the disadvantage of increased
decontamination line spraying time may be outweighed by the significant advantages in waste
reduction and the decreased risk of personnel cross-contamination and spread of contamination
beyond the impacted site. It is not certain how much longer it will take to fully cover a person with the
31
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electrostatic sprayer once scaled up to a real-world scenario. Therefore, additional experiments are
underway to address the difference in spray duration between the two technologies when
decontaminating a mannequin outfitted with a full Level C PPE ensemble.
Additional pilot-scale studies utilizing more elaborate field-deployable decontamination systems and
full Levels of B or C PPE ensembles are suggested as next steps to confirm these results and clarify
the time and cost impacts of electrostatic sprayer use in a mock decontamination line setting.
Specifically, the time to fully spray and decontaminate a PPE ensemble with the electrostatic sprayer
needs to be evaluated as it will help determine whether the technique is operationally feasible.
32
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7.0 Quality Assurance and Quality Control
All test activities were documented in narratives in laboratory notebooks through digital photographs.
The documentation included, but was not limited to, a record for each spray test procedure, deviations
from the quality assurance project plan, and physical impacts on materials and equipment. All tests
were conducted in accordance with established EPA Decontamination Technologies Research
Laboratory and BioLab procedures to ensure repeatability and adherence to the data quality validation
criteria set for this project.
The following sections discuss the criteria for the critical measurements and parameters, data quality
indicators (DQIs), and quality assurance (QA)/ QC checks for the project.
7.1 Criteria for Critical Measurements and Parameters
Data quality objectives 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. Digital photographs were taken throughout the testing and
sampling phases. The following measurements were deemed critical to accomplish part or all of the
project objectives:
• pH of 10% DB solution;
• FAC of 10% DB solution;
• Volume of liquid needed to wet the coupon surface using sprayers;
• Backpack sprayer spray diameter at 1 foot;
• Electrostatic sprayer diameter at 1 foot;
• Flow rate of backpack sprayer;
• Flow rate of electrostatic sprayer; and
• Temperature and RH (relative humidity).
7.2 DQIs
Critical measurements were used to determine if the collected data met the QA objectives. If a
measurement method or device resulted in data that did not meet the DQIs for the critical
measurements, data derived from the critical measurements 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.
Table 7-1 lists the DQIs for the critical measurements. As the table shows, all the DQIs were within
the target acceptance criteria set for this project.
Table 7-1. DQIs for Critical Measurements
Critical Measurement
Analysis Method
Accuracy/Precision
Acceptance Criteria
CFU per plate
Spiral plater/QCount
50% RSD amongst the
triplicate plating
50% RSD amongst the
triplicate plating
Incubation chamber
temperature
NIST-traceable thermometer
(daily)
O
o
CM
+l
Not applicable
33
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Critical Measurement
Analysis Method
Accuracy/Precision
Acceptance Criteria
Spray application time
NIST-calibrated stopwatch
± 1 minute/hour
± 2 minutes (2*±1 min)
Spray application volume
NIST-calibrated stopwatch
± 1 second/hour
14- by 14-inch coupon
surface wetted with
liquid
PH
pH meter/NIST-traceable
buffer solutions
± 0.01 pH unit
pH > 7
Collection of effluent at
specified time
Graduated cylinder
± 1 mL
± 10% of target value
Sprayer pressure
Class B pressure gauge
± 2 psi
± 20% of target value
Notes: psi = Pounds per square inch; RSD = Relative standard deviation
7.3 QA/QC Checks
Many QA/QC checks were used during this project to ensure that the data collected met all the critical
measurement requirements listed in Table 7-1. The measurement and parameter criteria were set at
the most stringent level that can routinely be achieved. The integrity of each sample during collection
and analysis was evaluated. Control samples and procedural blanks were included along with the test
samples so that well-controlled quantitative values were obtained. Replicate coupons of all materials
were included for each sprayer test.
The integrity of samples and supplies, BioLab control checks, decontamination solution verification,
and QA assessments and response actions are discussed below.
7.3.1 Integrity of Samples and Supplies
Samples were carefully maintained and preserved to ensure their integrity. Samples were stored
away from standards or other samples that could possibly cross-contaminate them.
Project personnel carefully checked supplies and consumables prior to use to verify that they met
specified project quality objectives. Incubation temperature was monitored using NIST-traceable
thermometers. Balances and pipettes are calibrated yearly by the EPA Metrology Laboratory.
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. CFU counts greater than 300 or less than 30 were
considered outside the targeted range. If the CFU count for bacterial growth did not fall within the
targeted range, the sample was re-plated 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 being 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: (1) recounting using an edit feature of the QCount® software that allows manual
34
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removal of erroneously identified spots or shadows on the plate; or (2) adding colonies that the
QCount® software may have missed.
The acceptance criteria for the critical CFU counts were set at the most stringent level that can
routinely be achieved. 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 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 particular inoculation event.
• Unexposed field blank (negative control): Coupons sampled in the same fashion as test
coupons but not inoculated with the surrogate organism before sampling or exposed 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.
• Sample container sterilization: The exterior of the wipe sample container (specimen cup)
and the sterile sampling bags were decontaminated by wiping all surfaces with a bleach wipe
before transport from sampling location to BioLab in a secondary container.
• Sterility checks: Pre-moistened swabs used to wipe specified areas to test for the presence
of spores for sterility checks on coupons (PPE materials and stainless steel), the test chamber,
and sprayers before use in testing as discussed in Section 5.1.4; additionally, Dl water
samples were collected in 50 ml_ conical tubes (Catalog No. 14-959-49A, Fisher Scientific,
Waltham, MA) for each batch of sterilized Dl water used for spray test as a sterility check.
• Blank TSA sterility controls: Plates incubated but not inoculated.
• Replicate plates of diluted microbiological samples: Replicate plates for each sample.
Table 7-2 lists the additional QC checks built into the BioLab procedures designed to provide
assurances against cross-contamination and other biases in the microbiological samples.
35
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Table 7-2. Additional QC 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% RSD between
coupons in each test set
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
Analyze extracts from
procedural blank without
dilution. Identify and
remove source of
contamination if possible.
Unexposed field
blank (negative
control) coupon
One per test
Non-detect
Level of
contamination
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.
Replicate plating of
diluted
microbiological
samples
Each sample
Reportable CFU count of
triplicate plates within
100%; reportable CFU
counts between 30 and
300 CFU per plate
Precision of
replicate plating
Re-plate sample
7.3.3 Decontamination Solution Verification
Volumes of components were measured as accurately as possible using appropriate measuring
equipment such as volumetric flasks, serological pipette tips, and graduated cylinders. Commercial
products such as Clorox® were used as a 10% DB solution source. The concentration of each new
batch of DB was evaluated. Dl water was used to prepare the decontamination solution.
The following parameters of the 10% DB solution were measured prior to each use:
• pH;
• FAC (in ppm);
• Temperature;
• RH.
These readings were recorded as measured. FAC was measured using a HACH® high-range bleach
test kit (Method 10100, Model CN-HRDT), and pH was measured using an Oakton Acorn® Series pH
5 meter (Oakton Instruments, Vernon Hills, IL). Two HOBO Relative Humidity/Temperature sensors
(Model U12, Onset Computer Corporation, Bourne, MA) were used to measure temperature and
humidity around the testing area. Appendix B includes a discussion of the characterization of the
decontamination solution and Table B-4, which summarizes the measurement results.
36
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7.3.4 QA Assessments and Response Actions
The QA assessment and corrective action procedures for this project are intended to provide rapid
detection of data quality problems. Project personnel were intimately involved with the data on a daily
basis so that any data quality issue became apparent soon after it occurred. Some contamination in
QC procedural blank samples and negative control samples was observed in some tests. However,
the contamination was very minimal and had little to no effect on the project results. Table 7-3
summarizes the QA/QC assessment of spore recoveries for the various sample types. As the table
shows, blank and negative sample results were present were at or near the detection limit. Only one
blank sample had a recovery above the acceptable reportable quantification limit of 30 CFU per filter.
With spore recoveries on the order of logs of CFU, this contamination is inconsequential.
Table 7-3. Cross-Contamination Assessment of Blank and Negative Control Samples
Test ID
Material type
Procedural Blank Spore
Recovery (CFU)
Negative Control Spore
Recovery (CFU)
Surface
Runoff
Surface
1
Nitrile (Buna-N)
ND
ND
3
2
ND
ND
ND
3
Butyl
ND
7
ND
4
ND
ND
15
5
Latex
ND
32
1
6
ND
ND
ND
7
Tyvek®
ND
ND
ND
8
ND
ND
ND
9
Tychem®
1
ND
ND
10
ND
ND
ND
11
Neoprene
ND
ND
1
12
ND
ND
ND
13
ChemTape®
ND
ND
ND
14
ND
ND
1
Note: ND = None detected
37
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References
American Chemical Society. 2014. Definitions for Hydrophilicity, Hydrophobicity, and
Superhydrophobicity: Getting the Basics Right. The Journal of Physical Chemistry Letters 5(4):
686-688. On-line address: http://pubs.acs.org/doi/pdf/10.1021/iz402762h , accessed August
21. 2018.
Brown, G.S., R.G. Betty, J.E. Brockmann, D.A. Lucero, C.A. Souza, K.S. Walsh, R.M. Boucher, M. Tezak,
M.C. Wilson, and T. Rudolph. 2007. Evaluation of a Wipe Surface Sample Method for Collection of
Bacillus Spores from Nonporous Surfaces. Applied and Environmental Microbiology 73(3): 706-
710.
Busher, A., J. Noble-Wang, and L. Rose. 2008. Surface Sampling. Sampling for Biological Agents in
the Environment. Emanuel P. Roos and K. Niyogi, Editors. Chapter 5, Pages 95-131. ASM
Press, Washington, DC. doi: 10.1128/9781555817473.
Calfee, M.W., Y. Choi, J. Rogers, T. Kelly, Z. Willenberg, and K. Riggs. 2011. Lab-Scale Assessment
to Support Remediation of Outdoor Surfaces Contaminated with Bacillus anthracis Spores."
Journal of Bioterrorism and Biodefense. 2(3): 1-8.
Calfee, M.W., S.D. Lee, and S.P. Ryan. 2013. A Rapid and Repeatable Method to Deposit Bioaerosols
on Material Surfaces. Journal of Microbiological Methods, 92(3): 375-380.
Gibbons, H.S., S.M. Broomall, L.A. McNew, H. Daligault, C. Chapman, D. Bruce, M. Karavis, M. Krepps,
P.A. McGregor, C. Hong, K.H. Park, A. Akmal, A Feldmann, J.S. Lin, W.E. Chang, B.W. Higgs,
P. Demirev, J. Lindquist, A. Liem, E. Fochler, T.D. Read, R. Tapia, S. Johnson, K.A. Bishop-
Lilly, C. Detter, C. Han, S. Sozhamannan, C.N. Rosenzweig, and E.W. Skowronski. 2011.
Genomic Signatures of Strain Selection and Enhancement in Bacillus atrophaeus var. globigii,
a Historical Biowarfare Simulant. PLoS ONE. (6)3: e17836. doi: 10.1371/journal.pone.0017836.
Kabashima, John, D. K. Giles, and M. P. Parrella. 1995. Electrostatic Sprayers Improve Pesticide
Efficacy in Greenhouses. California Agriculture. 49(4): 31-35.
Lee, S.D., S.P. Ryan, and E.G. Snyder. 2011. "Development of an Aerosol Surface Inoculation Method for
Bacillus Spores." Applied and Environmental Microbiology. 77(5): 1638-1645.
USEPA (U.S. Environmental Protection Agency). 1992. Standard Operating Safety Guides. Office of
Emergency and Remedial Response, Washington, D.C., 9285.1-03, PB92-963414.
USEPA (U.S. Environmental Protection Agency). 2007. Guidance on Test Methods for Demonstrating
the Efficacy of Antimicrobial Products for Inactivating Bacillus anthracis Spores on
Environmental Surfaces. FIFRA Scientific Advisory Panel Meeting. Arlington, VA. SAP Minutes
No. 2007-05.
USEPA (U.S. Environmental Protection Agency). 2015a. Decontamination Line Protocol Evaluation for
Biological Contamination Incidents Assessment and Evaluation Report. National Homeland
Security Research Center, Office of Research and Development, Washington, DC. EPA/600/R-
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USEPA (Environmental Protection Agency). 2015b. Application of Electrostatic and Backpack Sprayer
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USEPA (Environmental Protection Agency). 2015c. "BioResponse Decontamination Line SOP."
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Advisory Division (CBRN CMAD).
39
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Appendices
40
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Appendix A: Data Logs
This appendix includes examples of data logs for the spray duration, wipe sampling, liquid runoff
collection, and Via-Cell® cassettes.
Table A-1. Example of Spray Duration Log
Coupon ID
Description
Spraying Start
Time
Spraying End Time
Comments
DB Spray
91-8-K-BPS-PB-01
Procedural Blank
91-8-K-BPS-TC-01
Test Coupon 1
91-8-K-BPS-TC-02
Test Coupon 2
91-8-K-BPS-TC-03
Test Coupon 3
Dl
Water Spray
91-8-K-BPS-FB-01
Field Positive Blank
91-8-K-BPS-FP-01
Field Positive Control 1
91-8-K-BPS-FP-02
Field Positive Control 2
91-8-K-BPS-FP-03
Field Positive Control 3
Table A-2. Example of Wipe Sampling Log
Coupon ID
Description
Sampling
Start Time
Sampling
End Time
Comments
91-1-N-BPS-X-01
Field Blank
91-1-N-BPS-NC-01
Negative Control
91-1-N-BPS-PB-01
Procedural Blank Wipe
91-1-N-BPS-TC-01
Test Coupon 1
91-1-N-BPS-TC-02
Test Coupon 2
91-1-N-BPS-TC-03
Test Coupon 3
91-1-N-BPS-FB-01
Field Positive Blank Wipe
91-1-N-BPS-FP-01
Field Positive Control 1
91-1 -N-BPS-FP-02
Field Positive Control 2
91-1-N-BPS-FP-03
Field Positive Control 3
91-1-N-BPS-PC-01
Positive Control 1
91-1 -N-BPS-PC-02
Positive Control 2
91-1 -N-BPS-PC-03
Positive Control 3
91-1-N-BPS-IC-01
Inoculum Control 1
91-1-N-BPS-IC-02
Inoculum Control 2
91-1-N-BPS-IC-03
Inoculum Control 3
A-1
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Table A-3. Example of Liquid Runoff Collection Log
Sample ID
Description
Initial Weight
(grams)
Final Weight
(grams)
Comments
91-8-K-BPS-PR-01
Procedural blank runoff
sample
Start Via-Cell® Cassettes
91-8-K-BPS-RF-01
Test coupon runoff sample
After test coupon 1 spray
After test coupon 2 spray
After test coupon 3 spray
Stop Via-Cell® Cassettes
91-8-K-BPS-BR-01
Field positive blank runoff
sample
91-8-K-BPS-FR-01
Test coupon runoff sample
After test coupon 1 spray
After test coupon 2 spray
After test coupon 3 spray
Table A-4. Example of Via-Cell® Cassette Log
Sample ID
Description
Temperature (°C)
Gas Meter Volume
(L)
Sample Flow A H
(kJ)
Initial
Final
Initial
Final
Initial
Final
91-8-K-BPS-VC-01
Inside test chamber
91 -8-K-B P S-VC-02
Test chamber exhaust
duct
Notes:AH = Change in enthalpy; kJ = Kilojoule; L = Liter
A-2
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Appendix B: Data Summary
This appendix presents the data summary tables for decontamination efficacy, log recovery
achieved during the Dl water wash-down, and fate of spores based on aerosol sampling, followed
by a discussion of the characterization of the decontamination solution and a table that includes
the pH, FAC, temperature, and RH results for each testing event.
Table B-1. Decontamination Efficacy
Sprayer Type
Stainless-Steel Inoculum
Control Coupons (CFU)
Test Coupons(CFU)
Surface LR (CFU)
Spores in
Runoff
Average
STD
Average
STD
Average
STD
(Log CFU)
Nitrile (Buna-N)
Backpack
3.44E+07
1.15E+07
1.17E+00
2.90E-01
7.48
0.10
6.50
Electrostatic
4.63E+07
8.04E+06
1.00E+00
ND
7.67
ND
0.10
Butyl
Backpack
1.79E+07
1.53E+07
5.73E+00
6.72E+00
6.73
0.57
6.27
Electrostatic
1.67E+07
1.70E+07
1.54E+00
9.41 E-01
7.08
0.24
0.37
Latex
Backpack
2.59E+07
2.63E+06
2.09E+00
8.31E-01
7.11
0.16
4.72
Electrostatic
9.48E+06
2.20E+06
4.30E+01
4.52E+01
5.64
0.74
ND
Tyvek®
Backpack
3.23E+07
1.74E+07
2.56E+00
2.69E+00
7.26
0.43
6.41
Electrostatic
2.07E+07
1.24E+07
1.00E+00
ND
7.32
ND
ND
Tychem®
Backpack
1.68E+07
8.51 E+06
1.15E+00
2.60E-01
7.17
0.09
6.37
Electrostatic
3.25E+07
3.15E+06
2.91 E+00
2.63E+00
7.16
0.37
0.41
Neoprene
Backpack
2.21 E+07
1.45E+07
3.70E+00
2.11 E+00
6.84
0.31
6.70
Electrostatic
7.71 E+06
4.31 E+06
1.00E+00
ND
6.89
ND
ND
ChemTape®
Backpack
4.04E+07
4.74E+06
1.13E+00
2.24E-01
7.56
0.08
6.65
Electrostatic
2.89E+07
8.47E+06
1.77E+00
7.15E-01
7.23
0.16
0.41
Notes: CFU = Colony-forming unit; ND = None detected; STD =
: Standard deviation
Table B-2. Percent Recovery Achieved during Dl Water Wash-down
Sprayer Type
Average
Recovery (%)
Nitrile
Butyl
Latex
Tyvek®
Tychem®
Neoprene
ChemTape®
Backpack
11.4
3.2
5.8
4.9
1.7
13.6
6.6
Electrostatic
42.3
100.5
62.4
0.0
49.4
66.4
24.6
B-1
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Table B-3. Reaerosolization of Spores Based on Air Sampling
Backpack Sprayer
Electrostatic Sprayer
Material
type
Inside
Chamber
Chamber Duct
Inside
Chamber
Chamber Duct
(CFU)
Nitrile
ND
ND
ND
32.8
Butyl
ND
ND
ND
ND
Latex
ND
ND
ND
ND
Tyvek®
42.4
3.08
86.7
ND
Tychem®
ND
ND
ND
ND
Neoprene
9.38
ND
ND
ND
ChemTape®
1.54
ND
ND
3.08
Notes: CFU = Colony-forming unit ND = None detected
Characterization of Decontamination Solution
For this study, the decontamination solution was 10% DB at a pH ranging from 10 to 12 units
and FAC concentrations ranging from 6,000 to 20,000ppm. The decontamination solution of
10% DB was chosen because it is commonly used actual decontamination lines. Additionally,
two HOBO Relative Humidity/Temperature sensors (Model U12, Onset Computer Corporation,
Bourne, MA) were used to measure temperature and humidity around the testing area. These
sensors were launched before contamination of the coupons (inoculation) and recorded
temperature and humidity data points throughout spraying and sampling events. The average
temperature and RH readings around the test location throughout the testing events were 23 °C
and 46%, respectively. Table B-4 lists the pH and FAC data for the decontamination solution
prepared for each test as well as the temperature and RH results for each testing event.
Table B-4. pH, FAC, Temperature, and RH per Test
Test
Material
Sprayer Type
PH
FAC
(ppm)
Temperature (°C)
RH (%)
ID
DB Solution
HOBO 1
HOBO
2
HOBO 1
HOBO 2
1
Nitrile
Backpack
11.0
8,633
22.73
23.17
47.8
35.5
2
(Buna-N)
Electrostatic
10.8
8,253
22.97
23.20
46.4
46.6
3
Butyl
Backpack
10.6
8,193
22.69
22.81
45.7
37.8
4
Electrostatic
10.9
8,673
23.07
22.92
45.2
48.7
5
Latex
Backpack
11.3
8,133
22.45
22.72
49.7
55.0
6
Electrostatic
11.0
7,952
23.07
22.92
45.2
48.7
7
Tyvek®
Backpack
10.3
8,633
22.47
24.80
50.4
53.5
8
Electrostatic
10.8
6,390
24.67
22.73
52.8
48.2
9
Tychem®
Backpack
11.0
6,450
22.77
22.97
56.47
46.7
10
Electrostatic
11.0
7,812
22.81
26.12
45.7
40.09
11
Backpack
10.8
8,954
22.83
23.76
44.2
47.6
12
Neoprene®
Electrostatic
10.8
8,012
22.70
*No
data
39.1
*No data
13
Backpack
10.2
8,713
22.97
23.20
46.4
46.6
14
ChemTape®
Electrostatic
11.0
7,752
22.70
*No
data
39.1
*No data
Note : For Test#12 and Test#14, HOBO 2 was observed to record to no data.
B-2
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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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