EPA/600/R-18/271 | August 2018
www.epa.gov/homeland-security-research
United States
Environmental Protection
Agency
oEPA
Evaluation of the Removal and
Inactivation of Bacterial Spores
on Outdoor Surfaces Using a
Small-Scale Street Sweeper
Office of Research and Development
Homeland Security Research Program
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EPA 600/R-18/271
August 2018
Evaluation of the Removal and Inactivation
of Bacterial Spores on Outdoor Surfaces Using a
Small-Scale Street Sweeper
Authors:
Joseph Wood
National Homeland Security Research Center
Office of Research and Development
U.S. Environmental Protection Agency
Research Triangle Park, NC 27711
Abderrahmane Touati, Ph. D, Brian Sechrest, Dennis Aslett, and Ahmed
Abdel-Hady
Jacobs Technology, Inc.
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Disclaimer
The U.S. Environmental Protection Agency (EPA), through its Office of Research and
Development's National Homeland Security Research Center, directed and managed this
investigation through Contract No. EP-C-15-008 with Jacobs Technology, Inc. (Jacobs). Jacobs'
role did not include establishing Agency policy. This report has been peer and administratively
reviewed and has been approved for publication as an Environmental Protection Agency
document. It does not necessarily reflect the views of the Environmental Protection Agency. No
official endorsement should be inferred. This report includes photographs of commercially
available products. The photographs are included for purposes of illustration only and are not
intended to imply that EPA approves or endorses the product or its manufacturer. EPA does not
endorse the purchase or sale of any commercial products or services.
Questions concerning this document, or its application should be addressed to the
following individual:
Joseph Wood
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
E-mail Address: wood.ioe@epa.gov
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Acknowledgments
The principal investigator from the U.S. Environmental Protection Agency (EPA), through its
Office of Research and Development's National Homeland Security Research Center (NHSRC),
directed this effort with intellectual support of a project team from across EPA. The input and
contributions of the individuals listed below have been a valued asset throughout this effort.
EPA Project Team
Joseph Wood, NHSRC/DCMD (Principal Investigator)
Leroy Mickelsen, OLEM/CMAD
Shannon Serre, OLEM/CMAD
Worth Calfee, NHSRC/DCMD
Anne Mikelonis, NHSRC/DCMD
Matthew Magnuson, NHSRC/DCMD
Shawn Ryan, NHSRC/DCMD
EPA Quality Assurance
Eletha Brady-Roberts, NHSRC
Jacobs Technology, Inc.
Abderrahmane Touati
Brian Sechrest
Lee Brush
Steve Terll
Denise Aslett
Ahmed Abdel-Hady
Zora Drake-Richman
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Executive Summary
In a wide area release of Bacillus cmthrcicis spores, street sweepers may offer a relatively
quick method of removing and/or inactivating spores from contaminated roadways, parking lots,
and other similar surfaces. Street sweepers typically use water for dust suppression and
scrubbing prior to vacuuming, and this is the type of sweeper evaluated in the present study,
albeit at a smaller scale. In the present study, a commercially available walk-behind floor
scrubber was used to represent a small-scale street sweeper, and was evaluated in several tests, to
determine its ability to remove or inactivate bacterial spores from concrete and asphalt surfaces.
The effectiveness of the walk-behind floor scrubber to decontaminate concrete and
asphalt was tested using the non-pathogenic Bacillus atrophaeus var. globigii (Bg) spores as a
surrogate for contamination with B. anthracis spores (the causative organism of anthrax).
The effectiveness of the floor scrubber was determined by sampling the concrete and
asphalt using a wet vacuum procedure. Air sampling was used to indicate if Bg spores were
aerosolized from the surface during the decontamination procedure. Samples from the floor
scrubber liquid were also taken to determine the disposition of the Bg spores.
Testing was conducted with the floor scrubber using water, water/surfactant solution, pH-
amended bleach (pAB) or sodium dichloro-s-triazinetrione (Dichlor). Removal of Bg spores
from the asphalt surface (74-81%) with water in the floor scrubber was lower than that observed
for the concrete surface (99%). This may have been due to the inhomogeneous characteristics of
the asphalt when compared to the concrete surface.
Additional passes of the floor scrubber over the concrete surface may have led to a -0.5
log reduction increase in removal efficiency, while no increase was determined for asphalt. (We
caution that the statistical significance of these results is lacking and that additional testing is
warranted to be able to draw more definitive conclusions.) The addition of a surfactant to the
water did not improve Bg removal for either surface while the use of the pAB and Dichlor
sporicidal solutions were found to greatly increase decontamination efficacy.
The sporicidal solutions used with the scrubber resulted in > 7 log reduction (99.99999%
removal), with no Bg spores recovered in the post-decontamination samples.
Spread of contamination from the initial "hot spot" was observed during the testing
activities, with alO-fold increase in spread occurring with the use of the scrubber. Bg (1.6 - 4.6
log colony forming units (CFU)) was observed outside of the scrubber test area in all tests except
for with the use of Dichlor on asphalt. Air samples taken during different phases of the testing
were generally highest during the inoculation and overnight settling period, and lowest during
the decontamination phase. The aerosolized Bg collected during the inoculation and overnight
settling period tended to be higher for the asphalt surface compared to the concrete surface.
This study demonstrated that the walk-behind floor scrubber used with a sporicidal
solution, such as pAB or Dichlor, may be useful for the decontamination of concrete and asphalt
surfaces.
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Contents
Disclaimer
Acknowledgments i
Executive Summary ii
Figures v:
Tables viii
Acronyms and Abbreviations ix
1. Introduction 1
2. Experimental Approach 2
2.1 Testing and Sampling Approaches 2
2.2 Test Matrix 3
3. Testing Facility, Equipment, and Materials 5
3.1 Test Site Preparation 5
3.1.1 Storage Building with Concrete Slab Floor 5
3.1.2 Tent Erected on an Asphalt Parking Lot
3.1.3 Test Surface
3.1.3.1 Test Facility Surface Layout
3.2 Equipment Preparation
3.2.1 Street Sweeper
3.2.2 Wet Vacuum Samplers
3.2.3 Back-Pack Sprayer 1
3.2.4 Test Organism and Inoculation 1
3.2.4.1 Bacillus atrophaeus var. globigii (Bg) 1
3.2.4.2 Metered Dose Inhaler and Aerosol Deposition Apparatus (ADA) 1
3.2.5 ADA Inoculation of Surfaces 1
3.2.6 Dry Filter Unit 1
3.2.7 Via-Cell Bioaerosol Sampling Cassette 1
3.2.8 Inoculation Control Coupons 1
3.3 Test Facility, Equipment and Material Sterilization 1
3.3.1 Test Surface Sterilization 1
3.4 Liquid Spraying Agents 1
3.4.1 DI Water 1
iv
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3.4.2 DI Water/0.05% Tween® 20 16
3.4.3 pAB Solution 16
3.4.4 Dichlor (chlorinated granules) 17
3.4.5 Dishwashing Liquid Solution 17
3.4.6 SSDX-12™ Surfactant Formulation 17
3.5 Neutralizing Agents for Extracted Samples 17
4. SAMPLING AND ANALYSIS APPROACH 19
4.1 Sampling Protocol 19
4.1.1 Testing and Sampling Flow Timeline 19
4.1.2 Sampling Operation 24
4.2 Sample Handling 30
4.2.1 Sample Containers 30
4.2.2 Sample Preservation 30
4.3 Microbiological Analyses 30
4.3.1 Sample Extraction 30
4.3.1.1 MDI Inoculum Wipes 30
4.3.1.2 Wet Vacuum Sampler and Sweeper Liquid Samples 31
4.3.1.3 Dry Filter Units 31
4.3.1.4 Via-cell Cassettes 31
4.3.1.5 Swabs 31
4.3.2 Spiral Plating and Filter plating 31
4.4 Decontamination Solutions Characterizations 33
4.4.1 Determination of FA C by Titration 33
4.4.2 pH and Temperature Measurements 33
4.5 Decontamination Efficacy 33
5. Results and Discussion 35
5.1 Dry Aerosol Deposition Evaluation 35
5.2 Spore Recovery from MDI and Positive Controls 36
5.3 Cross-Contamination Evaluation 36
5.3.1 Cross-Contamination Assessment on Concrete Surface 3 7
5.3.2 Cross-Contamination Assessment on Concrete using an Improved ADA 38
5.3.3 Cross-Contamination Assessment on Asphalt using the Improved ADA 41
5.4 Street Sweeper Decontamination/Removal Efficacy 44
5.4.1 Removal Efficacy Using DI Water 44
5.4.1.1 Spore Recovery on Concrete using DI Water and a 1-Pass Sweep 44
5.4.1.2 Spore Recovery on Concrete using DI Water and a 3-Pass Sweep 47
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5.4.1.3 Spore Recovery on Asphalt using DI Water and a 1-Pass Sweep 48
5.4.1.4 Spore Recovery on Asphalt using DI Water and a 3-Pass Sweep 51
5.4.2 Spore Removal Efficacy Using Surfactant-Based Aqueous Solutions 53
5.4.2.1 Spore Removal Efficacy Using Dishwashing Solution on Concrete 53
5.4.2.2 Spore Removal Efficacy Using Dishwashing Solution on Asphalt 55
5.4.2.3 Spore Removal Efficacy Using S SDX-12™ on Concrete 57
5.4.3 Decontamination Efficacy Using Chlorine-Based Aqueous Solutions 59
5.4.3.1 Decontamination Efficacy Using pAB on Concrete 59
5.4.3.2 Decontamination Efficacy Using pAB on Asphalt 61
5.4.3.3 Decontamination Efficacy Using Dichlor on Concrete 63
5.4.3.4 Decontamination Efficacy Using Dichlor on Asphalt 65
5.5 Summary of Results 67
6. Quality Assurance and Quality Control 71
6.1 Measurement Equipment Calibration 71
6.2 Criteria for Critical Measurements and Parameters 72
6.3 Integrity of Samples and Supplies 73
6.4 NHSRC Bio-laboratory Control Checks 73
References 75
Appendix A: Operational Summary Logs for Each Test A-l
Appendix B: Market Summary of Walk-Behind Street Sweeper Specifications B-l
Appendix C. Equipment Sterilization C-l
Figures
Figure 3-1: Pre-engineered Building with a Concrete Slab Floor 6
Figure 3-2: Heavy duty Fire-rated PVC Tent erected on an Asphalt Surface 7
Figure 3-3: Test Areas Schematic 8
Figure 3-4: Tennant T5 Machine and Parts 9
Figure 3-5: Wet Vacuuming with Residential Vacuum sampler (Hoover) 10
Figure 3-6. Electric Backpack Sprayer 11
Figure 3-7. MDI canister (A) and Actuator (B) 12
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Figure 3-8. 14 in x 14 in ADA 13
Figure 3-9. Large (38- by 38-in) ADA Schematic 13
Figure 3-10. Large (38- by 38-in) ADA with a Sliding Base 14
Figure 3-11. Dry Filter Unit 100 15
Figure 4-1: Floor Schematic 20
Figure 4-2: Swab Collection from the Wet Vacuum Nozzle 21
Figure 4-3: Street Sweeper Sweeping Pattern 23
Figure 4-4: Wet Vacuum Sampling Pattern for the "Hot Spot" Area "e" 25
Figure 4-5. Bg Bacterial colonies (CFUs) on a spiral-plated agar plate 32
Figure 4-6. Bg bacterial colonies (CFUs) on a filter plate 33
Figure 5-1: Dry Aerosol Discrete Spatial Distribution using an ADA on a Glass Coupon 35
Figure 5-2: Dry Aerosol Concentric Distribution using an ADA on Glass Coupon 36
Figure 5-3: Assessment of Cross-Contamination Post-Inoculation on Concrete Surface using an ADA 37
Figure 5-4: Assessment of Cross-Contamination Post-Inoculation on Concrete Surface using an Improved
AD 39
Figure 5-5: Assessment of Cross-Contamination Post-Inoculation on Asphalt Surface using an Improved
AD 42
Figure 5-6: Concrete and Asphalt Surface Characteristics 44
Figure 5-7: Log of Spore Recoveries on Concrete using DI Water and a 1-Pass Sweep 45
Figure 5-8: Log of Spore Recoveries on Concrete using DI Water and a 3-Pass Sweep 47
Figure 5-9: Log of Spore Recoveries on Asphalt using DI Water and a 1-Pass Sweep 49
Figure 5-10: Log of Spore Recoveries on Asphalt using DI Water and a 3-Pass Sweep 51
Figure 5-11: Log of Spore Recoveries on Concrete using Dawn® Dishwashing Solution 53
Figure 5-12: Log of Spore Recoveries on Asphalt using Dawn® Dishwashing Solution 55
Figure 5-13: Log of Spore Recoveries on Concrete using SSDX-12™ Solution 57
Figure 5-14: Log of Spore Recoveries on Concrete using pAB Solution 59
Figure 5-15: Log of Spore Recoveries on Asphalt using pAB Solution 61
Figure 5-16: Log of Spore Recoveries on Concrete using Dichlor Solution 63
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Figure 5-17: Log of Spore Recoveries on Asphalt using Dichlor Solution
Tables
Table 2-1: Test matrix
Table 4-1. Test Inoculation Sequence 2
Table 4-2. Typical Samples List for each testing Event 2
Table 4-3. Example of an Operational Summary Log for each testing Event 2
Table 5-1. Cross-Contamination Assessment on Concrete Surface Using an ADA 3
Table 5-2. Cross-Contamination Assessment on Concrete Using an Improved ADA 4
Table 5-3. Cross-Contamination Assessment on Asphalt Surface Using an Improved ADA 4
Table 5-4. Spore Recoveries on Concrete using DI Water and a 1-Pass Sweep 4
Table 5-5. Spore Recoveries on Concrete using DI Water and a 3-Pass Sweep 4
Table 5-6. Spore Recoveries on Asphalt using DI Water and a 1-Pass Sweep 5
Table 5-7. Spore Recoveries on Asphalt using DI Water and a 3-Pass Sweep 5
Table 5-8. Spore Recoveries on Concrete using Dawn® Dishwashing Solution 5
Table 5-9. Spore Recoveries on Asphalt using Dawn® Dishwashing Solution 5
Table 5-10. Spore Recoveries on Concrete using SSDX-12™ Solution 5
Table 5-11. Spore Recoveries on Concrete using pAB Solution 6
Table 5-12. Spore Recoveries on Asphalt using pAB Solution 6
Table 5-13. Spore Recoveries on Concrete using Dichlor Solution 6
Table 5-14. Spore Recoveries on Asphalt using Dichlor Solution 6
Table 6-1. Instrument Calibration Frequency 7
Table 6-2. Critical Measurement Acceptance Criteria 7
Table 6-3. Additional Quality Checks for Biological Measurements 74
viii
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Acronyms and Abbreviations
% percent
ADA aerosol deposition apparatus
ATCC American Type Culture Collection
Ba Bacillus cmthracis
Bg Bacillus atrophaeus var. globigii
BioLab NHSRC RTP Microbiology Laboratory
BSC Biosafety cabinet
CFU colony forming unit(s)
cm centimeter
DCMD EPA Decontamination and Consequence Management Division
DHS Department of Homeland Security
DFU Dry Filter Unit
DI Deionized
DQI data quality indicator
DQO data quality objective
DTRL EPA Decontamination Technologies Research Laboratory
EPA U. S. Environmental Protection Agency
EtO ethylene oxide
FAC free available chlorine
H2O2 hydrogen peroxide
HASP Health and Safety Plan
HSPD Homeland Security Presidential Directive
in inch
ISO International Organization for Standardization
LR Logio reduction
MDI metered dose inhaler
mL Milliliter
MOP Miscellaneous Operating Procedure
NHSRC EPA National Homeland Security Research Center
NIST National Institute of Standards and Technology
ORD Office of Research and Development
pAB pH-adjusted bleach
PBS phosphate-buffered saline
PBST phosphate-buffered saline with 0.05% Tween® 20
PPE personal protective equipment
ppm part per million
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ppmv part per million by volume
PRB polyester-rayon blend
QA Quality Assurance
QAO QA Officer
QC Quality Control
RH relative humidity
RLS Research Laboratory Support
RSD relative standard deviation
RTP Research Triangle Park
SHEM Safety, Health and Environmental Management
STD standard deviation
STS sodium thiosulfate
TLP technical lead person
TSA tryptic soy agar
TSB tryptic soy broth
VHP vaporized hydrogen peroxide
WA Work Assignment
WACOR Work Assignment Contracting Officer Representative
WAL Work Assignment Leader
x
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1. Introduction
The U.S. Environmental Protection Agency's (EPA's) Homeland Security Research
Program (HSRP) provides credible scientific and technical information to protect human health
and the environment from adverse impacts arising from terrorist threats and other contamination
incidents. The HSRP conducts research to provide expertise and inform decisions on the
selection and implementation of decontamination methods that may lead to significant reductions
in the time and cost of wide area remediation efforts.
In a wide area release of Bacillus cmthrcicis spores, street sweepers may offer a relatively
quick method of removing and/or inactivating spores from contaminated roadways, parking lots,
and other similar surfaces. Historically, street sweeping primarily has been conducted to remove
the regular buildup of litter and trash along urban streets. Recent developments in sweeper
technology, such as regenerative sweepers and "dustless sweepers", enable municipalities and
transportation departments to collect smaller particles in a cost-effective manner. For example,
one vendor equips a few of their street sweeper models with highly efficient fabric filters with a
Minimum Efficiency Reporting Value (MERV) rating of 16 (< 95% collection efficiency for
particles with an aerodynamic diameter ranging from 0.3 to 10 microns (Tymco®, 2018). This
type of sweeper technology designed for highly efficient dust control may also be suitable for
collection of bacterial spores.
Street sweepers may also use water for dust suppression and scrubbing prior to
vacuuming (Amato et al., 2010). This is the type of sweeper technology evaluated in the present
study, albeit at a smaller scale. In the present study, a commercially available walk-behind floor
scrubber was evaluated in several tests, to determine its ability to remove and/or inactivate
bacterial spores from concrete and asphalt surfaces. The walk behind floor sweeper we selected
for testing utilizes both water for dust suppression/cleaning as well as a vacuum. These pilot-
scale tests were conducted using a smaller-scale sweeper for proof of concept purposes, with an
eye toward the possibility of future full-scale street sweeper tests, depending on the outcome.
Initial tests were conducted with the floor sweeper using either water or a
water/surfactant solution, to evaluate removal efficiency of Bacillus spores from the test surface.
After the first test on concrete, we noticed that spores had spread to the areas adjacent to the hot
spot (where spores were inoculated). Because of this, additional tests were conducted without the
sweeper, to determine whether the spread of spores was due to the sweeping activity or other
activities such as sampling and inoculation. Additional tests with the sweeper were then
conducted with decontaminant solutions of either pH-adjusted (pAB) or sodium dichloro-s-
triazinetrione (Dichlor) to assess spore inactivation. The tests on concrete were conducted in a
shed, and the tests on asphalt were conducted in a parking lot with a tent enclosure. In the study,
B. atrophaeus var. globigii (Bg) spores were used as a surrogate for B. anthracis. The study also
generated operational information such as duration of the decontamination process, water usage
and collection, re-aerosolization of spores, contamination of adjacent clean areas, and other
associated issues.
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2. Experimental Approach
2.1 Testing and Sampling Approaches
Sections of concrete and asphalt surfaces were inoculated using an aerosol deposition
method that delivered approximately 5 x 107 spores of Bg. The inoculated area (the hot spot) as
well as adjacent areas underwent decontamination using the small-scale street sweeper. These
areas were then sampled, and the spore recoveries from treated surfaces (test samples) were
compared to spore recoveries from surfaces that were inoculated but not treated (positive control
samples). Surface decontamination efficacy or removal efficiency was calculated as the
difference between the average positive control recovery and the post-decontamination recovery
of spores from the treated surface.
QC samples such as procedural blanks (material surfaces that underwent the
sweeping/decontamination process but were not inoculated) and negative controls (surfaces that
were not inoculated and did not undergo the surface treatment process) were included to monitor
for cross-contamination. The National Homeland Security Research Center Research Triangle
Park (NHSRC RTP) Microbiology Lab (BioLab) quantified the number of viable spores.
The general experimental approach used to meet the project objectives is described
below. Prior to the experimental program, market research was conducted to determine the
availability of a walk-behind street sweeper, that could be used to represent full-scale
technology. A battery-powered Walk-Behind floor scrubber, (Model #T5, Tennant Company,
Minneapolis, MN) was selected because of its relatively low-cost, and its use of a water spray for
dust suppression followed by vacuuming. The market research conducted on the walk-behind
sweepers is described in Section 3.2.1
• Site preparation: Since the testing required relatively large surface areas (12-fft x 12-ft
area) to be swept/scrubbed and then sampled, two tests facilities were used: 1) a 28-ft
wide x 22-fft deep x 10-ft high pre-engineered metal storage building with a 4-in concrete
slab, and 2) a 20-fft x 20-ft outdoor tent, erected on an asphalt parking lot. The two
facilities and pre-test preparation of surfaces are described in Section 3.1.
• Reset of Test Surface: Prior to each test, the entire concrete or asphalt surface
underwent a sterilization/decontamination process to help prevent cross-contamination
between tests (referred to as a "reset"). Reset of test surfaces was accomplished using
pAB applied with a backpack sprayer. The test chamber "Reset" is further described in
Section 3.3.1.
• Inoculation of the hot spot test surface with the target organism: The hot spot,
located in the center of the test area, was inoculated each test using an aerosol deposition
method that delivered a known quantity of spores in a repeatable fashion onto a 3-ft x 3-ft
area. A positive control area of the same size was also similarly inoculated but was
located adjacent to the test area. Three inoculation control samples were inoculated with
the same MDIs, but on smaller (14-ft x 14-ft) stainless-steel plates. Approximately 5 x
107 Bg spores were deposited during each inoculation. This is discussed in Section 3.2.4.
• Preparation of the decontamination solutions: Tests were conducted using either
deionized (DI) water, DI water with surfactants, or decontamination solutions in the
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scrubber solution tank. If decontamination solutions of pAB or Dichlor were used, they
were freshly prepared on each test day; Section 3.3 discusses preparation of the test
solutions.
• Sweeping procedure: The decontamination procedure followed a protocol which
consisted of operating the sweeper over the test surface by beginning at one corner of the
test surface area, continuing in a straight path to the opposite side of the test area, then
operating the sweeper back to the starting side while the sweeper was overlapping the
previous path by half its width. This pattern was repeated until the entire test area was
covered. In two of the tests, three passes of the sweeper over the test area were used. The
sweeping protocol is discussed in Section 3-5.
• Sampling Procedures: Each experiment included the following samples. Refer to section
4.12.
• Individual wet vacuums were used for the following samples:
i. one (1) positive control surface area
ii. nine (9) swept surfaces
iii. two (2) pre- and post-sampling non-inoculated test surfaces (negative
controls)
• Sampling with gauze wipes for the three MDI controls
• one (1) sweeper "dirty" tank liquid sampling and a rinse of tank
• aerosol sampling:
i. via Dry Filter Unit (DFU); up to four separate DFU samples were taken in
each experiment: (1) taken during negative sampling; (2) inoculation and
overnight settling; (3) during the Aerosol Deposition Apparatus (ADA)
removal and sweeping; and (4) during surface sampling
ii. (1) Via-Cell cassette sample.
• Sample Analysis. Bg spores were extracted or filtered from the test samples, and aliquots
were analyzed using the procedures for microbiological analysis described in Section 4.3.
Viable spore recovery was quantified in terms of colony forming units (CFU) present in
each sample.
• Fate of the spores: Total viable spores recovered from each replicate test sample were
quantified in terms of CFU, which was used to calculate the average CFU and standard
deviation for each group of test samples. Determination of the decontamination
efficacy/transport of the spores is discussed in Section 4.5.
2.2 Test Matrix
The test matrix for this project is listed in Table 2-1. After the initial test of the sweeper
using DI water on concrete, we found there was a substantial number of spores in the test area
adjacent to the center hot spot. To determine how much of the spread of spores was the result of
operating the sweeper vs. all other activities taking place in the test area (e.g., inoculation,
surface sampling), tests were conducted without the sweeper. Two of these tests were conducted
on concrete, and the third on asphalt. After the initial test on concrete without the sweeper (Test
2), it was clear that cross-contamination occurred before any sweeping took place. Because of
this, changes to some of the equipment and sampling procedures were implemented to reduce the
spread of spores, and another test (Test 3) was conducted on concrete without the sweeper. This
is further discussed in the Results section of the report.
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The main variables tested were the type of surface, sweeper liquid, and number of
sweeper passes. Tests were initially conducted on the concrete surface during the spring and
summer seasons. After those tests were complete, we moved to the asphalt surface. The asphalt
tests took place during the winter. Tests with the decontaminants (pAB and Dichlor) were
conducted after the tests with water/surfactants.
Table 2-1: Test matrix
Test Number
Sweeper Liquid
Test Area
2
None (spore cross-contamination
check; no sweeper used)
Concrete
3
None (spore cross-contamination
check; no sweeper used.) Conducted
after making changes to procedures
and equipment, to mitigate cross-
contamination
Concrete
9
Asphalt
1
DI Water (Single Pass)
Concrete
10
Asphalt
6
DI Water (3 Passes)
Concrete
12
Asphalt
4
SSDX-12™
Concrete
5
Dishwashing Soap
Concrete
11
Asphalt
7
pAB
Concrete
14
Asphalt
8
Dichlor
Concrete
13
Asphalt
4
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3. Testing Facility, Equipment, and Materials
This section describes the experimental testing and materials, including site preparation,
sampling and aerosol deposition equipment, sterilization (reset) of the equipment and materials,
target organism, and type of decontaminant and neutralization agents used.
3.1 Test Site Preparation
Two test facilities were used to evaluate the effectiveness of the sweeper to remove or
inactivate spores. The two test surfaces were concrete and asphalt. Since the testing required
relatively large surface areas (12-ft x 12-ft sampling area) to be swept and then sampled, two test
facilities were used: 1) a metal storage building with a new concrete floor, and 2) an outdoor tent
erected on an asphalt parking lot. The two facilities and preparation of the test surfaces are
described in the following sections.
3.1.1 Storage Building with Concrete Slab Floor
A pre-engineered steel building, placed on a concrete slab, was installed in 2017 at the
EPA/RTP Outdoor Storage Area (OSA). The building is 28-ft wide x 22-fft deep x 10-ft high.
The building is also equipped with an 8- x 8-ft roll-up door, which has sufficient clearance to
accommodate a forklift with a 7-ft mast. The steel building was erected on a 4-in thick concrete
slab, reinforced with welded wire fabric (6x6 W2.9/W2.9). The concrete strength was set to a
minimum of 2500 PSI at 28 days. The sweeper tests were conducted in the new shed/floor prior
to it being used, and thus the concrete floor was clean with minimal cracks. The concrete floor
was also confirmed to be level. A photograph of the outside of the steel building is shown in
Figure 3-1.
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Figure 3-1: Pre-engineered Building with a Concrete Slab Floor
3.1.2 Tent Erected on an Asphalt Parking Lot
A 20-ft x 20-ft. heavy duty fire-rated fabric tent (ShelterLogic Model # 25920, Home
Depot, Durham, NC), complete with walls and door panels, was erected on an asphalt surface
(the parking lot) at the EPA Fluid Modeling Facility (FMF) located on Old Page Road, Durham,
North Carolina. The tent includes six (6) individual clear PVC windows, and two (2) double
zipper doors allowing for multiple entry options. The tent was anchored to the parking lot using
several large concrete blocks. Figure 3-2 is a photograph of the tent test facility used for this
project. In contrast to the new concrete surface, the asphalt surface was weathered, contained
small cracks, and was on a slight incline.
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Figure 3-2: Heavy duty Fire-rated PVC Tent erected on an Asphalt Surface
3.1.3 Test Surface
3.1.3.1 Test Facility Surface Layout
Each test facility (the steel building for the concrete and the heavy-duty tent for the
asphalt) consisted of a 400-ft2 (20- x 20-ft) area, which accommodated one 12 ft by 12 ft sweeper
test area, one 3 - x 3-ft positive control area (located near the sweeper test area), one 3- x 3-ft
negative control area (also located adjacent to the sweeper test area), and other ancillary
equipment needed for testing. The ancillary equipment included Dry Filter Units (DFU) for air
sampling of spores, and temperature and RH data loggers (referred to as HOBO, by Onset
Corp.). The center of the sweeper test area was inoculated with Bg spores (hot spot). Three 1 -ft
square stainless-steel coupons were also inoculated with the MDI as an inoculation control. The
test facility surface layout is shown in Figure 3-3.
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20 ft.
l-ft
lift #
MDIs HOBO
12 ft.
Post-Decon Floor Sampling Areas
3-ft
3-ft
12 ft.
3-ft
Positive
Control
3-ft
20 ft.
Figure 3-3: Test Areas Schematic
3.2 Equipment Preparation
3.2.1 Street Sweeper
Historically, street sweeping has been used to improve aesthetics in urban environments.
Its primary purpose has been to abate the regular buildup of litter and trash along city streets.
Recent developments in sweeper technology such as regenerative sweepers or vacuum sweepers
enable municipalities and transportation departments to collect small particles in a cost-effective
manner. Additionally, some street sweepers utilize water for dust suppression or scrubbing prior
to vacuuming. A summary of some representative walk-behind sweepers/scrubbers is listed in
Appendix B.
The battery-powered Walk-Behind Floor Scrubber (Tennant 5, Tenant Company,
Minneapolis, MN), hereafter called "street sweeper", was selected for this study because it
features a water spray for dust suppression, and it provides a cylindrical brush head for
particle/debris dislodging and capture. (A new brush head was used for each test.) The street
sweeper is also equipped with a squeegee to help achieve high levels of solution recovery. It
comes with both a cleaning solution (clean) tank and a recovery (dirty) tank that can be accessed
easily. A debris tray eliminates operator contact with recovery (dirty) tank solutions. The brushes
and squeegees can be replaced easily, without tools, to minimize operator contact as well as
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cross-contamination. A photo of the Tennant T5 and its parts, as provided by the manufacturer,
are shown in Figure 3-4.
There were three operational conditions the sweeper could be operated. For every test,
the sweeper was operated as follows:
a. Lowest brush pressure
b. Highest solution flow rate (approximately 0.45 gallons per minute)
c. Lowest forward speed (approximately 1.25 ft. per second)
In a typical test with one pass over the 144 ft2 test area, Approximately 2 gallons (7-8 liters) of
solution were dispensed over a period of about 5-6 minutes, and approximately two-thirds of the
solution was recovered.
Figure 3-4: Tennant T5 Machine and Parts
3.2.2 Wet Vacuum Samplers
The Ftoover Dual V Steam Vac All Terrain with Spin Scrub brushes (Model F7452900,
The Hoover Company, North Canton, Ohio), shown in Figure 3-5, was selected for sampling of
the test surface, and the negative and positive control surfaces. (The test surface was gridded into
nine separate sampling areas.) The Hoover F7452900 has Dual V suction technology, multiple
brush speeds, and a 12-amp motor, all designed to help deliver uniform suction across the full
13-in width of the nozzle. It also has separate cleaning solution (clean) and recovery (dirty)
liquid tanks (each with 1-gallon capacity) as well as hand tools for cleaning hard-to-reach areas.
The vacuum sampler was set to "Wash Auto Rinse" mode during vacuum sampling.
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Figure 3-5: Wet vacuum sampling concrete floor
3.2.3 Back-Pack Sprayer
Prior to the initiation of each test, the floors of the test facilities were sterilized
(decontaminated) in their entirety using a ShurFlo 4 ProPack Rechargeable Electric Back Pack
Sprayer SRS-600 (Pentair-ShurFlo, Costa Mesa, California), shown in Figure 3-6. The pAB
solution used for reset of the floor was prepared directly inside the four-gallon container of the
electric backpack sprayer. The sprayer was rinsed with DI water and filled with the pAB solution
prior to use. A second ShurFlo 4 ProPack sprayer was used to rinse the street sweeper's
"recovery tank" with deionized water after emptying it. Appropriate personal protective
equipment, including the use of an air purifying respirator, was used during the pAB spraying
operation.
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Figure 3-6. Electric Backpack Sprayer
3.2.4 Test Organism and Inoculation
3.2.4.1 Bacillus atrophaeus var. glohigii (Bg)
Bacillus atrophaeus var. Niger, formerly known as Bacillus globigii (Bg), a surrogate for
the spore-forming bacterial agent Bacillus anthracis, was used for this project. Like Bacillus
anthracis, Bg is a soil dwelling, gram positive, spore forming, aerobic microorganism, but unlike
B. anthracis, it is non-pathogenic. Bg forms an orange pigment when grown on nutrient agar, a
desirable characteristic when there is a need to detect 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)
3.2.4.2 Metered Dose Inhaler and Aerosol Deposition Apparatus (ADA)
Each test surface was inoculated with approximately 5 « 107 Bg spores, (cultured,
processed, and lyophilized at Dugway Proving Ground, Dugway, UT: American Type Culture
Collection ATCC 9372) using an MDI (Canister Part No. BK0339783, Bespak, Hertfordshire,
England) and an aerosol deposition apparatus (ADA). The MDI canister contained spores
suspended in ethanol and HFA-134A propellant (1,1,1,2-tetrafluoroethane). Each MDI was
charged with a volume of spore preparation plus propellant sufficient to deliver 200 discharges
of 50 microliters (|iL) per discharge. MDI use was tracked so that the number of discharges did
not exceed 200. To inoculate the test surface, the MDI canister was situated inside an actuator
(Figure 3-7) so that each time the actuator was depressed, a repeatable number of spores was
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deposited on the coupon (Calfee et al.)
Figure 3-7. MDI canister (A) and Actuator (B)
3.2.5 ADA Inoculation of Surfaces
All surface inoculations used during this project employed an ADA like the one shown in
Figure 3-8. Each ADA is equipped with an actuator fitting, which is covered by a sliding lid. The
lid remained in the closed position except when opened for inoculations. ADAs were also
equipped with air exhausts located on each corner of the ADA. The exhausts were each covered
with a 0.22 um syringe filter to prevent spore escape and cross contamination of test surfaces. To
further guard against cross contamination, the bottom perimeter of each ADA was fitted with a
1-in wide rubber gasket. The ADA size varied according to the surface area requiring
inoculation, but the inoculation procedure was the same for all test surfaces. A 14-in xl4-in
.ADA was used for MDI-inoculation of the stainless steel controls; a 38-in x 38-in ADA was used
to inoculate the hot spot of the test area as well as the positive control surface area.
Prior to inoculation, an ADA of appropriate size was situated directly on top of a flat test
surface. The MDI + actuator assembly was placed in the actuator fitting on top of the ADA, then
the lid was opened, and the MDI was activated one time to deliver approximately 5 x 101 Bg
spores to the test surface under the ADA. Following inoculation, the lid was closed, and the MDI
assembly was removed. The ADA was left in place for at least 18 hours to allow time for the
spores to settle. This process was repeated for each inoculated surface (inoculation controls,
positive control area).
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Figure 3-8. 14 in x 14 in ADA
To inoculate the 36-in * 36-in "hot spot" on the floor of the test facility, as well as the
positive control, a large 38-in * 38-in ADA was designed and constructed. A schematic is shown
in Figure 3-9.
38 inches 38 inches
Figure 3-9. Large (38— by 38-in) ADA Schematic
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The large (38- by 38-in) ADA had all the features (actuator fitting, air exhausts capped
with 0.22 |im syringe filters, and a perimeter gasket) of the smaller AD As, but a special sliding
base was added. The sliding base was assembled between two perimeter gasket layers, and was
designed to prevent cross contamination of the test areas immediately adjacent to the hot spot
during ADA removal. During inoculation, the sliding base was opened as shown in Figure 3-10.
Just prior to ADA removal, the base was closed. Prior to the sweeping tests, two tests were
conducted using the MDI and the large ADA to assess spore loading and distribution on a glass 3
ft by 3 ft glass coupon.
ADA
closed position
ADA
open position
Figure 3-10. Large (38- by 38-in) ADA with a Sliding Base
3.2.6 Dry Filter Unit
A Dry Filter Unit (DFU 1000, Fact Sheet provided by the Chemical, Biological,
Radiological, & Nuclear Information Resource Center [CBRN IRC]) was used to collect target
spores from ambient air during inoculation, sweeping, and sampling events. The sampling flow
rate of each DFU was checked before each test using a thermo-anemometer probe placed at the
air inlet. Subsequent microbiological analysis was used to determine the presence of aerosolized
Bg spores on the filters. The DFU, shown in Figure 3-11, was configured in a portable,
weathertight carrying case and operated with AC power. The DFU accommodates two (2) filters
(7/8 polyester felt filter disk, 1-micron rating), which fit inside the unit's filter assembly. Each
DFU filter was analyzed separately for Bg spores, and the sum of these two filters' CFU is what
is listed in this report. DFU sampling times were typically less than an hour during negative
surface sampling, ADA removal/sweeping, and test surface sampling following sweeping; the
DFU sampling time for the inoculation and overnight settling period was typically over 18 hours.
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Figure 3-11. Dry Filter Unit 100
3.2.7 Via-Cell Bioaerosol Sampling Cassette
Bioaerosol samples were also collected using Via-Cell cassettes (Product # VIA010, Zefon,
Ocala, FL). The cassette assembly was attached to the top cover of the sweeper, where it
collected an air sample during the sweeping event. The Via-cell sample time was concurrent with
sweeper operation and varied between 5-10 minutes (depending if 1-3 passes of the sweeper
were used), with air sample volumes of 60-150 liters. Results for these samples were used to
estimate the occurrence and magnitude of aerosolized Bg spores caused by sweeping. The air
flow rate of this sampler was much lower than the DFUs and was expected to be 15 L/min
according to the manufacturer.
3.2.8 Inoculation Control Coupons
Stainless steel coupons (14- by 14-in) were used to assess MDI inoculation levels. The
number of spores recovered from these coupons was used to represent the maximum number of
recoverable spores deposited by the MDI (using the 14 by 14-inch ADA) on a surface of the
same size. These coupons were prepared using heavy-duty power hydraulic shears to cut the
metal from larger sheets.
3.3 Test Facility, Equipment and Material Sterilization
3.3.1 Test Surface Sterilization
Prior to each test, the entire floor of the test facility was reset with pAB to help prevent cross-
contamination between tests. The pAB decontamination solution (2.5 gallons) was applied with a
ShurFlo 4 ProPack Rechargeable Electric Back Pack Sprayer. Starting in a back corner (away
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from the test facility entrance), the entire length of the floor was sprayed in a straight back and
forth pattern until reaching the opposite corner. The spray process was repeated twice, each time
with another 2.5 gallons of pAB. The floor was left to air dry at least 72 hours prior to testing.
Other equipment was reset/sterilized as well, and these procedures are described in Appendix
C.
3.4 Liquid Spraying Agents
Chlorine-based decontamination solutions, surfactant-based aqueous cleaning solutions,
and DI water were used in the assessment of the effectiveness of the sweeper in removing or
inactivating spores from the concrete and asphalt test surfaces. In addition, DI water with a
surfactant was used in the wet vacuum samplers. These solutions are described below.
3.4.1 DI Water
DI water was one of the liquids used in the sweeper to evaluate the ability of the sweeper
to remove spores from the concrete and asphalt surfaces. A DI water system (Purologix™, 10
gpm, Holly Springs, NC) was used to obtain the laboratory grade DI water for each test in this
study.
3.4.2 DI Water/0.05% Tween® 20
A sterilized surfactant-based aqueous solution, DI Water/0.05% Tween® 20, was used in
the wet vacuum samplers. The final sampling solution, 0.05% Tween 20 solution, was prepared
by adding 3mL of Tween® 20 (Cat No. BP337-100, Fisher Scientific, Hampton, NH) to 6L of DI
water. The DI/Water/0,05% Tween® 20 was sterilized using a 1-hour gravity cycle sterilization
process at 121 °C before use.
3.4.3 pAB Solution
pAB has been demonstrated to be an effective decontaminant agent in inactivating
bacterial spores, under specific conditions related to concentration, pH, contact time, and
material (Calfee et al.. 2012) In 2001/2002, EPA issued crisis exemptions permitting the limited
sale, distribution, and use of EPA-registered bleach products against Bacillus cmthrcicis spores at
some facilities and locations, including Capitol Hill, the U.S. Postal Service Processing and
Distribution Centers at Brentwood (Washington, DC) and Hamilton (Trenton, NJ), the
Department of State, the General Services Administration, and Broken Sound Boulevard (Boca
Raton, FL).
The pAB solution, prepared for this project, deviated from the specific conditions under
the EPA crisis exemptions, by using a more concentrated Clorox® Concentrated Germicidal
bleach (8% sodium hypochlorite concentration), while maintaining a pH around 7. A previous
study by Wood et.al., 2011, suggested that an increase in free available chlorine (FAC) may
increase the sporicidal efficacy of the pAB for some materials.
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The concentration of household bleach and the strength of white vinegar can vary by
batch and storage time. Therefore, the formulation listed above can vary in pH and chlorine
concentration depending on the starting reagents, and storage time. Before use, the pH and FAC
contents were measured at the start and monitored throughout each test. The pAB solution was
prepared using the following mixture. Appropriate respiratory protection was worn during the
preparation of the pAB to minimize exposure to chlorine gas.
¦ One-part germicidal bleach (with a listed 8% sodium hypochlorite concentration)
¦ One-part white vinegar
¦ Eight parts water
¦ Bleach and vinegar were not combined directly. Water was first added to the bleach
(two cups water to one cup of bleach), then vinegar (one cup), and then the remaining
water (six cups).
3.4.4 Dichlor (chlorinated granules)
Dichlor (Pool Solutions, Pool Supply World, PSW-CSC158-5, B & G Builders Pools &
Spas, Durham, NC) is an effective sanitation agent for swimming pools that is fast dissolving
and prevents harmful bacterial growth. This product is pH neutral, and the listed manufactured
product has 56% chlorine available. Dichlor was prepared by dissolving stabilized chlorinating
granules in deionized water to reach the target concentration of 0.33 lb./gallon of DI water
3.4.5 Dishwashing Liquid Solution
A pre-rinse detergent-water solution was prepared by adding -3.1 part of Dawn® Ultra
Dishwashing Liquid Original Scent (Procter & Gamble, Cincinnati, OH, USA) to 50 parts of
deionized (DI) water followed by thorough mixing. No evaluation of the detergent solution was
required.
3.4.6 SSDX-12™ Surfactant Formulation
SSDX-12™ (AeroSafe Products, Inc., Norcross, GA) is a concentrated detergent, that is
certified for general cleaning of U.S. military aircraft. This cleaning agent was originally
developed as a non-reactive, non-corrosive decontaminant to remove chemical and biological
warfare agents from sensitive military equipment. The product label recommended dilutions for
chemical and biological decontamination is 4:1 to 15:1 water to SSDX-12™ (20% to 6.25% soap
solution. For this project, the latter dilution was applied.
3.5 Neutralizing Agents for Extracted Samples
The presence of bleach-based decontamination solution on the sample surface, or in the
liquid effluent following a decontamination event, could negatively bias CFU quantification
results. Based on previous studies, sodium thiosulfate (STS) was proven to be an effective
neutralizer of bleach on porous and non-porous surfaces (Calfee et al„ 2011), and was used
during the post-decontamination recovery of the sampling liquid from the "dirty" tanks of the
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street sweeper, and the wet vacuum samplers. The volume of STS that was added to the
collection media was based on the FAC level in the sample, which was determined using an
iodometric method for the determination of chlorine dioxide and chlorite using a HACH®
Hypochlorite Test Kit (Model CN-HRDT, Fisher Scientific, Waltham, MA).
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4. SAMPLING AND ANALYSIS APPROACH
The objective of the study was to assess the effectiveness of a small-scale street sweeper
to remove and/or inactivate spores deposited on concrete and asphalt surfaces, and to determine
the fate of the spores that did go through the full sweeping process. The effectiveness was
determined from the recovered CFUs per sample expressed on a log-10 scale. A positive control
sample of the same material was compared to post-sweep test samples. Positive control samples
were inoculated on the same day and analyzed on the same day as test samples but did not go
through the sweeping process.
Additional measurements prior to or during the sweep procedure were also required to
ensure quality control in the testing. These measurements included quality control checks on the
reagents and equipment being used in the decontamination procedure.
A sampling data log sheet was maintained for each sampling event (or test) that included
each sampling event, the date, test name, sample IDs, and other test details such as test
temperature, sample volumes, and sample extraction time.
The following sections discuss the sampling protocol, sample types, and frequency of
sampling and monitoring events.
4.1 Sampling Protocol
Before each test, all materials and equipment needed for sampling were sterilized as
discussed in Section 3-3. and handled using aseptic techniques. Non-powdered, surgical gloves
were used during sampling. Individually wrapped, pre-moistened bleach wipes were placed in
sterile sampling bags. Dispatch® bleach wipes were also used. A sampling material bin was
stocked for each sampling event based on the sample quantity. The bin contained enough wipe
sampling kits to accommodate all required samples for each specific test. The following test
protocol was implemented for each test.
4.1.1 Testing and Sampling Flow Timeline
All tests were conducted in accordance with the following testing and sampling timeline:
Day 1: Test Facility Preparation
The test facility and the floor were prepared ahead of the sampling process as follows:
1. Set up a Relative Humidity(RH)/Temperature logging device (HOBO) in the test
chamber.
2. Line the edges of the test facility floor with absorbent pads or booms to prevent any
possible liquid runoff.
3. Cover the floor seams with duct tape (2").
4. Divide test chamber floor as per Figure 4-1, shown below. Mark the edges of each area
using duct tape. Ensure the inoculated sweep area isn't over floor seams or tape.
5. Divide the sweep area into 9 sections ("a" through "i"), as illustrated in Figure 4-1 for
post-decon sampling.
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6. Sterilize the test facility floor using pAB, as described in Section 3.3.1.
7. Allow at least 72 hours for the floor to dry before testing.
l-ft
l-ft
MDIs
o
HOBO
DFU
a
b
c
d
e
Hot Spot
f
g
h
4.5-ft 3_ft
Negative
Control
3-ft
3-ft
3-ft
4.5-ft
Positive
Control
3-ft
4.5-ft 3-ft 4.5-ft
Post-Decon Floor Sampling Areas
Figure 4-1: Floor Schematic
Day 4: Test Chamber and Sampling Equipment Sterility Checks
The floor and the wet vacuum samplers were checked for sterility, 72 hours after the "Reset" of
the test facility floor, following these sampling procedures:
1. Visual inspection of the floor that it is dry.
2. Don sterile boot covers before entering the shed sweep area
3. Aseptically collect a swab sample from each wet vacuum cleaner nozzle (See Figure 4-2).
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Figure 4-2: Swab Collection from the Wet Vacuum Nozzle
4. Sample the 3-ft x 3-ft negative control area, using one (1) wet vacuum sampler. The
purpose of this sample was to check the adequacy of the reset. Follow the sampling
procedures as outlined Section 4.12.
5. Sample the 3-ft x 3-ft to-be-inoculated (Hot Spot) sampling area "e" (Figure 4-1) using a
second wet vacuum sampler. This second negative control sample was taken from the hot
spot area (prior to area inoculated) to ensure the reset was adequate, and served as an
additional check where the maximum number of spores were in the previous test. Note
we did not start taking this second negative sample until after measures were taken to
improve the inoculation step and mitigate spread of spores.
Day 5: Inoculation
1. Set up ambient RH and Temperature logging device inside the shed/tent to measure
environmental conditions. This device should log continuously until the sampling is
complete.
2. Ensure that section "e" is dry following Day 4 negative sampling
3. Aseptically unwrap four (4) stainless steel coupons. Three (3) to be used as MDI controls,
and one as (1) as a negative control.
4. Obtain one swab sample from one (1) of the stainless steel coupons (MDI control
sample), one (1) from the ADA surface, and one from the ADA's gasket, using the swab
sampling outlined in Section 4.12. Include a blank swab for comparison.
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5. Aseptically place four (4) sterile 14-in x 14-in AD As (VHP sterilized) over the four (4)
stainless steel coupons. Secure the AD As to the coupons using binder clips.
6. Aseptically place two (2) sterile 38-in x 38-in AD As (VHP sterilized); one (1) over the 3-
ft x 3-ft positive control area, and one (1) over the "area "e" or "Hot Spot".
7. Inoculate the three (3) MDI controls, and the two (2) surfaces (positive control, and area
"e"), using a single MDI. Table 4-1 summarizes the inoculation control sequence for each
test.
Table 4-1. Test Inoculation Sequence
Order of Inoculation
Inoculation Location
1
Stainless Steel MDI Control Coupon 1
2
Positive Control Area
3
Stainless Steel MDI Control Coupon 2
4
Inoculated Sweep Area
5
Stainless Steel MDI Control Coupon 3
Day 6: Sweeper Operation and Sampling Sequence
Sweeper Operation
2. Ensure that the sweeper battery is fully charged before use.
3. Verify that the positive control area and MDI control coupons (to be sampled after
sweeping) are completely covered with AD As.
4. Don a fresh pair of sterile boot covers and PPE (laboratory coat, nitrile gloves, P-95
respirator, and safety glasses. A positive air pressure respirator was worn when applying
sporicidal solutions.)
5. Bring the sweeper into the shed. Unwrap the VHP bags off the sweeper. Aseptically
obtain a swab sample from inside the solution recovery tank.
6. Using clean sterile gloves, attach a clean, sterile squeegee to the sweeper. Refer to the
Tennant T5 Operator's manual for detailed instructions. The manual can be accessed
using the following web link: http://www.caliberequipment.com/manuals/WST5-
manual.pdf
7. Connect the vacuum hose to the squeegee assembly.
8. Install a clean, sterile brush to the sweeper.
9. Fill the sweeper solution tank with the target solution in 1 L increments and mark the
solution tube to the desired volume (50 L in general)
10. Make sure the recovery tank is empty and the float shut-off screen is installed and clean.
11. Start the DFU in the test chamber as summarized in Section 4.12 for DFU operation
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12. Aseptically slide the ADA base plate (Figure 3-10) onto the ADA and remove the ADA
from the inoculated section and move it outside the shed (away from the door).
13. Move the sweeper towards test area section a. The following sweeper parameters were
used to operate the sweeper:
a. Lowest Brush Pressure
b. Highest Solution Flow Rate (approximately 0.45 gallons per minute)
c. Lowest Speed (approximately 1.25 ft. per second)
14. Start a timer, and the sweeper, simultaneously and record all the times in the lab
notebook. Start at one corner of the sweep area (Area "a"; Figure 4-1) with a path straight
toward the top of the 12- by 12-foot area (Area "g").
15. Overlap the previous path approximately 6 inches and sweep down the opposite direction
until the bottom of the test area is reached.
16. Repeat this pattern until the entire test area has been swept by the sweeper. Then stop the
test duration timer. The sweeping pattern is illustrated in Figure 4-3.
Figure 4-3: Street Sweeper Sweeping Pattern
17. Turn off the DFU pump. Record the times in the lab notebook.
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18. Remove the sweeper to an area outside the sweep area but still within the test chamber.
Turn the key switch off. Cover the sweeper with Tyvek bags to prevent any possible
cross-contamination.
4.1.2 Sampling Operation
The sampling program involved surface sampling (positive control, swept surfaces "a"
through "i", and the post sampling negative control) using individual wet vacuum samplers as
described in Section 4.12. coupon sampling (three (3) MDI controls, and one (1) negative
control), sweeper "dirty" tank liquid sampling, and aerosol sampling via DFU and Via-Cell
cassettes. The sampling procedures, and sampling flow timeline are summarized below:
Sweeper Sampling
1. Sweeper Solution Recovery Tank Sample
a. While holding the drain hose upward, remove the cap and carefully lower hose to
drain the "dirty" liquid to sterile, pre-labeled Nalgene bottles.
b. Weigh each Nalgene bottle before and after sample collection to determine the
amount of sample collected. Record the values in the lab notebook.
2. Sweeper Dirty Tank DI water Sample
a. Using a backpack sprayer, rinse the recovery tank and debris tray with sterile DI
water. Aseptically transfer the rinsate into different sterile Nalgene bottles (pre-
labeled).
b. Weigh the bottles before and after sample collection to determine the amount of
sample collected. Record the values in the lab notebook.
3. Sterilize the outside of the Nalgene bottles using Dispatch® bleach wipes and place each
bottle in a secondary containment such as a Twirl 'Em bag (sterile bags with round wire
closure, Model No. 14-9555-181, Fisher Scientifics, Hampton, NH).
4. Sterilize the outside of the secondary containment using bleach wipes.
5. Place all samples in the collection bin.
6. Turn off the Via-cell pump and the DFU pump. Record the times in the lab notebook.
7. Aseptically retrieve the Via-cell from the pump and place it in the Via-cell pouch.
8. Retrieve the DFU filters with the filter holders and place them in sterile Twirl 'Em bags.
9. Sterilize the outside of the Via-cell pouch and DFU Twirl 'Em bags using bleach wipes.
10. Place the samples in the collection bin.
Wet Vacuum Sampling
1. Don a fresh pair of sterile boot covers before stepping back into the sweep area.
2. Fill each vacuum sampler with approximately 1 Liter of 0.05% Tween 20. Record all
volumes in the lab notebook.
3. Sample section "a" by moving the wet vacuum sampler, back and forth, in a specified
pattern on the designated sampling area. The sampling procedure is as follows:
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a. Place the vacuum sampler nozzle on the sampling area so that the front edge of
the vacuum sampler nozzle lip coincides with the line defining the beginning of
the sampling area and the side of the nozzle coincides with the one side boundary
of the second strip, as shown in Figure 4-4.
b. Stroke 1: Backward stroke starting at the end of the sampling area
c. Stroke 2: Forward stroke.
d. Move the vacuum sampler horizontally by half of the nozzle width area.
e. Restart the process (steps 2-5) for the next sampling strip to the end of the
sampling area.
6-in
width location Vacuuming
Figure 4-4: Wet Vacuum Sampling Pattern for the "Hot Spot" Area "e"
f. Immediately post-sampling, aseptically transfer the liquid from the wet vacuum
"dirty" tank to a pre-labeled clean Nalgene bottle. Record the volume collected in
the lab notebook. If. a bleach solution is used for the decontamination process, use
pre-labeled clean Nalgene bottle pre-loaded with an appropriate amount of the
neutralizing agent.
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g. Sterilize the outside of the Nalgene bottle using Dispatch® bleach wipes and
place the bottle in a secondary containment such as a Twirl 'Em bag. Sterilize the
outside of the secondary containment using bleach wipes.
h. Place the whole sample in the collection bin.
4. Repeat wet vacuum sampling for each section "b" through "i" of the swept area. Each
section will be sampled separately with a new a new "sterilized" wet vacuum sampler.
The post-sweeping surface sampling was performed as follows: Sampling surface area
"a", followed by areas "b", "c", "d", "f"g", "h", "i", and then the inoculated area "e".
5. Repeat the wet vacuum sampling of the "Negative control" sample, and then the
"Positive control" Sample area.
Wipe Sampling
Wipe sampling was performed for the four (4) MDI control coupons (three (3) positive
controls, and one (1) negative control). The general approach is that a moistened sterile non-
cotton pad (Curity all-purpose sponges #8042, 2-in x 2-in, 4-ply, Covidien PLC, Dublin, Ireland)
is used to wipe a specified area to recover bacteria, viruses, and biological toxins. Sampling was
conducted one coupon at a time. All coupons were placed horizontally for sampling. The wipes
were prepared in the BioLab by aseptically removing the sponges from their packing and placing
them in an unlabeled sterile 50 mL conical tube (Cat. No. 14-959-49A, Fisher Scientific,
Waltham, MA) using sterile forceps. Each transferred sponge was then moistened by adding 2.5
mL of sterile PBST and capped.
The surface area for all samples was 1 -ft2. A template was used to cover the exterior (1-in) of
each coupon leaving a square (12-in x 12-in) exposed for sampling for all coupons. The outer 1-
in of each 14-in x 14-in coupon was not sampled to avoid edge effects (The ADA inoculated a
12-in x 12-in area). The MDI controls were sampled as follows:
1. Don a fresh pair of sterile boot covers and PPE (laboratory coat, nitrile gloves, P-95
respirator, and safety glasses).
2. Remove the clamps from the Blank MDI control coupon, and then remove the ADA from
the coupon.
3. Wipe the surface of the coupon horizontally, using a consistent amount of pressure and
using S-strokes to cover the designated sample area of the coupon.
4. Fold the wipe in half, concealing the exposed side and then wiped the same surface
vertically using the same S-stroke technique.
5. Fold the wipe again and roll it so that it would fit into a conical tube.
6. Place the folded wipe into a conical tube
7. Place the conical tube in a sterile Twirl 'Em bag.
8. Sterilize the outside of the Twirl 'Em bags using bleach wipes
9. Place the sample in the collection bin.
10. Repeat this process for the 3 MDI control coupons and the 1 MDI negative control.
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Aerosol Sampling
Aerosol samples were collected using a Via-Cell cassette attached to the top of the
sweeper and operated only during the sweeping, and DFUs. Initially (the first two tests), only
one DFU sample was taken during each experiment: in Test 1, a DFU sample was taken during
the sweeping operation only, and in Test 2, it sampled only during the floor sampling with the
wet vacuums. After changes were made to the inoculation process and other operations to reduce
the spread of spores, beginning with Test 3, there were four separate DFU air samples taken
during each test, for the following operations: 1) During the negative control surface sampling
prior to inoculation; 2) during inoculation and overnight settling; 3) during the ADA removal and
sweeping process; and 4) during the wet vacuuming sampling sequence. These DFU air samples
were taken separately to help to identify which operations were causing aerosolization of spores.
At the end of each DFU sampling event, the sample retrieval consisted of the following steps:
1. Aseptically retrieve the Via-cell cartridge from the pump and place it in the Via-cell
pouch, and the DFU filters (front and back) from the DFU filters holder and place them
in sterile Twirl 'Em bags.
2. Sterilize the outside of the Via-cell pouch and DFU Twirl 'Em bags using bleach wipes.
3. Place the samples in the collection bin.
Swab Sampling
The general approach for swab sampling was to use a moistened swab (BactiSwab®
Collection and Transport System, R12100, Remel, Thermo Fisher Scientific, Waltham, MA) to
wipe a specified area to recover bacterial spores. Swab samples were collected from all
equipment prior to use to serve as sterility checks. MOPs 3135 and 6563 were followed to
sample and analyze materials for swab sampling. Five (5) sample swabs were taken to check for
the sterility of the test equipment (ADA, ADA gasket, Sweeper, Wet vacuum sampler, and MDI
Control Coupon). The swab sampling procedure is described below:
1. Through the sleeve, crush the BactiSwab ampule at midpoint.
2. Hold BactiSwab tip end up for at least five seconds to allow the medium to wet the swab.
3. Open the package and remove the BactiSwab.
4. Label the plastic tube.
5. Remove the cap-swab from the plastic tube.
6. Swab the surface while spinning the cap-swab between the thumb and index fingers.
7. Return cap-swab to tube.
8. Date and initial each sample tube.
A summary of all the samples taking during a testing event is presented in Table 4-2.
27
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Table 4-2. Typical Sample List for each Test
Sample
Number
Description
Sample Type
Test Result
Pre-Inoculation Sterility Check
Growth or No Growth
1
ADA Sterility check
Swab
2
Gasket Sterility check
Swab
3
Hoover Sterility check
Swab
4
MDI Control Sterility check
Swab
5
Backpack Sprayer
Swab
CFU
6
DI Water Sterility Check
DI Water
7
Negative Control
Wet-Vacuum Liquid
8
Negative Control Area-e (hot spot)
Wet-Vacuum Liquid
9
DFU Filter- During negative control
Filters 1&2
Re-Aerosolization Checks During Inoculation
CFU
10
DFU - During inoculation and settling
Filters 1&2
Post-Inoculation Samples (In Order of Sampling)
CFU
11
Sweeper Recovery (includes a sample direct
from tank and a sample from rinsate of tank)
"Dirty" tank Liquid
12
DFU - During ADA Removal and Sweeping
Filters 1&2
13
Viacell Cassette - Sweeper Operation
Cassette
14
Test Area - a
Wet-Vacuum Liquid
15
Test Area - b
Wet-Vacuum Liquid
16
Test Area - c
Wet-Vacuum Liquid
17
Test Area - d
Wet-Vacuum Liquid
18
Test Area - f
Wet-Vacuum Liquid
19
Test Area - g
Wet-Vacuum Liquid
20
Test Area - h
Wet-Vacuum Liquid
21
Test Area - i
Wet-Vacuum Liquid
22
Test Area - e (hot spot)
Wet-Vacuum Liquid
23
Negative Control - Post-Sampling
Wet-Vacuum Liquid
24
Positive Control
Wet-Vacuum Liquid
25
DFU - During Wet-Vacuum surface Sampling
Filters 1&2
26
Blank MDI Control
Gauze Wipe
27
MDI Inoculation Control-1
Gauze Wipe
28
MDI Inoculation Control-2
Gauze Wipe
29
MDI Inoculation Control-3
Gauze Wipe
28
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The operational summary of the sweeper, environmental conditions, and sampling
equipment are summarized in Table 4-3 for a typical sampling event.
Table 4-3. Example of an Operational Summary Log for each testing Event
Street Sweeper
Volume dispensed (L)
Volume Recovered (L)
Percent Liquid
Liquid/decontaminant
Clean DI water
-
Rinsate
Via-cell Operation
Start Time (H:M)
End Time (H:M)
RunTime
(min)
Flow Rate
(1pm)
Collected
Volume (m3)
DFU Operation
Sample Type
RunTime (HH:MM)
Velocity
(m/s)
Air Flow
Rate (LPM)
Collected
Volume (m3)
DFU Negative Sampling
DFU Inoculation (and overnight) sampling
DFU ADA Removal and sweeping
DFU Wet Vacuum sampling
HOBO Operation
Start Time (HH:MM)
Start Time (HH:MM)
RunTime
(H:M)
Temperature
RH
Wet Vacuum Sampling Operations
Sample location
Volume dispensed (L)
Volume
Recovered
(L)
Areas
Sampled (ft2)
Recovery
(mL/ft2)
Negative Control (Pre-inoculation)
Negative Control (Post-inoculation)
Negative Control Area-e (Pre-inoculation)
Test Area - a
Test Area - b
Test Area - c
Test Area - d
Test Area - e
Test Area - f
Test Area - g
Test Area - h
Test Area - i
Positive Control
29
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4.2 Sample Handling
This section discusses the sample containers and sample preservation.
4.2.1 Sample Containers
For each wipe sample, the primary containment container was an individual, sterile, 50-
mL conical tube. Secondary and tertiary containment consisted of sterile sampling bags. Liquid
samples, from the sweeper and wet vacuum samplers, were collected in sterile Nalgene bottles
placed inside pre-labeled sterile bags for secondary and tertiary containment. Similarly, Via-cell
cartridges, and DFU filters (front and back) were placed in sterile 5.5- x 15-in Twirl 'Em bags.
A single container, serving as quaternary containment, was used for storage in the
decontamination areas during sampling and for transport to the BioLab.
4.2.2 Sample Preservation
After sample collection, sample integrity was maintained by storing the samples in four
containers (one sample collection container, one sterile inner bag, one sterile outer bag with an
exterior sterilized during the sample packaging process, and one sterile container holding all
samples from a test). All individual sample containers remained sealed while in the
decontamination laboratory and during transport.
4.3Microbiological Analyses
The BioLab analyzed samples either qualitatively for spore presence (quality control,
swab samples) or quantitatively for the number of viable spores recovered per sample. Results
were reported in CFU per unit volume. Details of the extraction and analysis procedures are
provided in Sections 4.3.1 and 4.3.2. A laboratory notebook was used to document the details of
each sampling event (or test).
4.3.1 Sample Extraction
Spores were extracted from the sampling medium using the appropriate method for each
sample type, as described below. After extraction, aliquots were removed for either dilution
plating or filter plating, as appropriate for expected spore recoveries.
4.3.1.1 MDI Inoculum Wipes
Wipe samples were received in 50-ml conical tubes. Spores were extracted from the
wipes by adding 20 ml PBST to each sample, then agitating the tubes using a vortex mixer (set to
maximum rotation) for two minutes in 10-second intervals. Aliquots were then removed for
plating.
30
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4.3.1.2 Wet Vacuum Sampler and Sweeper Liquid Samples
Depending on sample levels of turbidity and debris, aliquots were either plated directly or
processed using a method (referred to as the "high debris" method) designed to minimize the
presence of non-target organisms and separate sample particulates from liquid. For the high
debris method, a sample aliquot (volume is dependent on the expected spore concentration) was
removed and placed in a 50-ml conical tube. Sample tubes were heat treated in an 80 °C water
bath for 30 minutes to inactivate non-spore forming microbes. After cooling to room
temperature, tubes were centrifuged at 3,500 x g to pelletize the debris. The resulting supernatant
(all but ~3 ml) was filter plated with a vacuum system as described in Section 4.3.2. The filter-
plated supernatant volume was recorded. The pellet was re-suspended in the remaining
supernatant liquid and spread on tryptic soy agar (TSA) plates using a 1 ml volume per plate
until the entire pellet was plated. Plates for the supernatant and the pellet were manually
enumerated. CFU totals for each were added together to report CFU/sample volume.
4.3.1.3 Dry Filter Units
To extract spores from the DFU filters contained in sterile 50-ml conical tubes, tubes
were placed in a Biosafety Cabinet (BSC) and a 20-ml volume of sterile PBST was added to
each tube. Tubes were vortexed for 2 minutes in 10 second intervals. Aliquots were then
removed for either spiral plating or filter plating, as described in Section 4.3.2.
4.3.1.4 Via-cell Cassettes
Cassettes were aseptically opened in a BSC, and the collection slide was aseptically
placed in a 50-ml conical tube. A volume of 20-ml sterile PBST was added to each tube, then
tubes were vortexed for 2 minutes in 10 second intervals. Aliquots were removed for either spiral
plating or filter plating, as described in Section 4.3.2
4.3.1.5 Swabs
Each swab was removed from the packaging in a BSC, then rotated across the TSA
media surface in a zig-zag pattern until the entire circumference of the swab contacted the media
surface. Plates were incubated at 35 ± 2 °C for 18 to 20 hours. Swab plates were manually
observed to determine "growth" or "no growth" of the target organism.
4.3.2 Spiral Plating and Filter plating
Sample extracts requiring dilution were plated in triplicate using a Spiral Plater
(Autoplate 5000, Advanced Instruments Inc., Norwood, MA). The automated spiral plater
deposits the sample in exponentially decreasing amounts across a rotating agar plate in
concentric lines to achieve three 10-fold serial dilutions on each plate. Plates with Bg samples
were incubated at 35 ± 2 °C for 18 to 20 hours. During incubation, the colonies develop along
the lines where the sample was deposited (see Figure 4-5). Colonies on each plate were
enumerated using a QCount® colony counter (Advanced Instruments Inc., Norwood, MA).
31
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Figure 4-5. Bg Bacterial colonies (CFUs) 011 a spiral-plated agar plate
Positive control samples were diluted 100-fold in PBST before spiral plating, while
samples of unknown concentration were plated with no dilution and with a 100-fold dilution.
Samples with known low concentrations were plated with no dilution. The QCount® colony
counter automatically calculates the CFU/mL in a sample based on the dilution plated and the
number of colonies that develop on the plate. The QCount® records the data in an MS Excel
spreadsheet.
Only spiral plates meeting the threshold of at least 30 CFUs were used for spore recovery
estimates. After quantitation with the QCount® colony counter, samples with plate results below
the 30-CFU threshold were either re-spiral plated with a more concentrated sample aliquot or
filter-plated to achieve a lower detection limit. The filter plate volume was based on the CFU
data from the QCount® result. Filter plating was performed using 100-ml capacity MicroFunnel
units with 0.45 jam GN-6 Metricel membranes (Pall Corporation, Laboratory, Port Washington,
NY) and a vacuum manifold (Pall Corporation, Laboratory, Port Washington, NY). The filters
were placed onto tryptic soy agar (TSA) plates and incubated at 35 ± 2 °C for 20 to 24 hours
before manual enumeration. Figure 4-6 shows filter plates with Bg colonies.
32
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Figure 4-6. Bg bacterial colonies (CFUs) on a filter plate
4.4 Decontamination Solutions Characterizations
4.4.1 Determination of FA C by Titration
Measurements of FAC were performed using an iodometric method that uses a HACH
Digital titrator and a HACH® reagent titration kit (Model #16900, HACH® Loveland, CO). The
titration procedure can be found in the HACH® digital titrator manual (https://pim-
resources.coleparmer.com/instruction-manual/24908-0Q.pdf).
4.4.2 pH and Temperature Measurements
Measurements of pH and temperature of the bleach solutions were perform ed daily using
a calibrated pH meter (Oakton® Acorn™ pH 5, OAKTGN Instruments, USA). The temperature
sensor included with the pH meter was factory-calibrated and checked monthly by comparison of
the displayed value to a NIST-certified thermometer.
4.5 Decontamination Efficacy
Data reduction was performed on measurements of the total spores (CFU) recovered from
each sampled surface or material. The following groups of samples included for each
combination of sweeping/liquid decontaminant scenario are summarized below:
• Positive control areas (Surface positive control area, and MDI inoculation controls)
• Test areas (Inoculated hot spot area, and adjacent areas)
• Negative control areas (Surface negative control area [two], and MDI negative control)
33
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Decontamination or removal efficacy was evaluated as a function of decontamination
solution, surface material, and other parameters. When using sporicidal solutions,
decontamination efficacy was expressed as a logio reduction (LR) of the viable Bg spores
recovered from the test surface following sweeper usage. When using the sweeper with just
water or surfactant solutions, removal efficiency was determined. Typically, for laboratory
assessments of decontamination efficacy, for a 1 x 106 colony forming units (CFU) challenge or
greater, a LR > 6 is considered effective (U.S. EPA, 2007); when no viable spores are recovered
after decontamination treatment, the method may also be considered effective.
Decontamination efficacy (for each test that used sporicidal solutions) was calculated as
follows:
Log CFU positive control sample -Log (Sum of CFU of all nine of the test areas samples: a-i)
Removal efficiency (when not using sporicides) was calculated as follows:
CFU positive control sample - Sum of CFU of all nine of the test areas samples X 100
CFU positive control sample
34
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5. Results and Discussion
Section 5.1 discusses the spatial distribution of spores using a specially designed ADA
Section 5.2 discusses the overall study results for the inoculation levels and positive controls.
Section 5.3 presents the results for the evaluation of spore transfer from the inoculated area "hot
spot" to the adjacent areas. Section 5.4 discusses the removal efficacy and/or inactivation of the
street sweeper for each of the tests using DI water, surfactant-based aqueous solutions, and
chlorine-based sporicidal solutions (Dichlor and pAB). Section 5.5 summarizes the overall study
results and includes summary tables for the surface decontamination as well as air sampling data.
Unless noted otherwise, all results shown in this chapter in the figures are in units of log CFU.
5.1 Dry Aerosol Deposition Evaluation
Two tests were conducted to determine the spatial distribution of Bg spores using a large
(3- x 3-ft) ADA, over a 40- x 40-inch glass coupon. The first test consisted of sampling 29
3-in x 3-in. discretely marked surfaces on the glass coupon. Each surface was sampled using a
separate moistened sterile non-cotton pad wipe. The heat map ([Log CFU]), representing the
spatial distribution of theBg spores, is shown in Figure 5-1. The number of spores was highest in
the center 3- x 3-in. area with a recovery of approximately 6.8 log CFU. The number of spores
gradually decreases from the center to the outer edge of the coupon surface, however, the
outermost corners of the coupon still had greater than 4.8 log CFU recovered.
8
36 in
Figure 5-1: Dry Aerosol Discrete Spatial Distribution using an ADA on a Glass Coupon
(Log CFU)
A second test was conducted to evaluate the spatial distribution of Bg spores using the
same dry deposition method, but with a different sampling plan. The inoculated area was divided
35
-------
into nine (9) concentric sections of equal areas (~ 144 in2). The results for this test, shown in
Fi gure 5-2, confirm the results of the first test with m ore than 60% of the recovered spores were
concentrated in the 12- x 12-in center square. Furthermore, more than 6 log CFU were observed
on the outermost concentric band. These data confirm that the inoculation procedure (MDI
discharge of - 5 x 107 spores of Bg, in combination with a 3-ft x 3-ft ADA) results in a spore
loading distribution greater than 6 Log (CFU/ft2). This inoculation procedure was applied
throughout this project.
6.0-6.25
6.25-6.5
6.5 - 6.75
36 in
Figure 5-2: Dry Aerosol Concentric Distribution using an ADA on Glass Coupon (Log
CFU)
5.2 Spore Recovery from MDI and Positive Controls
Overall, the recovery of Bg spores from the stainless-steel MDI controls (spores
recovered directly via extraction) n= 42) ranged from 6.9-7.8 log CFU, with an average ± SD of
7.6 ± 0.2 log CFU. The average recovery of spores from the concrete positive controls (n=8) was
7.3 ± 0.2 log CFU, and for asphalt (n=6), the average recovery was 7.2 ± 0.1 log CFU. (CFU
were recovered from coupons via wet vacuum sampling.)
5.3 Cross-Contamination Evaluation
Three tests were conducted to determine whether the sweeper or other experimental
activities (such as inoculation or sampling) caused spreading of spores to adjacent areas in the
12- x 12-ft sweep area. These tests were performed according to the sampling protocol discussed
36
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in Section 4.1. but without the operation of the sweeper. The spreading of Bg spores from the hot
spot area "e" to adjacent areas within the test area, as well as to the negative control area, did
occur without the use of the sweeper. The individual test results are discussed below.
5.3.1 Cross-Contamination Assessment on Concrete Surface
The results for the test on concrete to assess spread of spores without the sweeper (i.e.,
spread of spores due to other activities such as reset, inoculation, sampling, etc.; Test 2 from
matrix) are shown in Figure 5-3 in log CFU, and summarized in Table 5-1. The operation logs
for all the sampling equipment and materials are summarized in Appendix A.
The recovered spores from the "Hot Spot", i .e., the inoculated area "e", was the same
order of magnitude as the positive control. The areas adjacent to the hot spot were cumulatively
cross-contaminated with up to 5.7 x 104 CFU, or 0.24% of the total recovered spores from the
tested area. The DFU air sampler was run during the wet vacuum sampling and collected 3.2 log
CFU
6.9
E
.6
B
Inoculation Controls
©
DFU
< X X ~
A 5-ft 3-fl 4.5-ft
Sampling Floor Area
* Denotes Belov/ Detection Limit
Figure 5-3: Transport of spores in concrete test area without use of sweeper (log CFU)
i
i
A 5-ft
3.6
4.4
3.0
A
B
C
t
i
\
1.5*
7.4
3.5
3-fl
D
F
t
i
I
.4
3.7
4.2
3.5
A 5-ft
1
Positive Control
G
H
I
>
t
37
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Table 5-1. Cross-Contamination Assessment on Concrete Surface Using an ADA
Sample Number
Description
Sample Type
Test Result
Pre-Inoculation Sterility Check
Growth/No Growth
1
ADA Sterility check
Swab
NG
2
Gasket Sterility check
Swab
NG
3
Hoover Sterility check
Swab
NG
4
MDI Control Sterility check
Swab
NG
5
Backpack Sprayer
Swab
NG
CFU
6
DI Water Sterility Check
DI Water
<5*
7
Negative Control
Wet-Vacuum Liquid
<28*
Post-Inoculation Samples (In Order of Sampling)
CFU
8
Test Area - a
Wet-Vacuum Liquid
3.9 x 103
9
Test Area - b
Wet-Vacuum Liquid
2.6 x 104
10
Test Area - c
Wet-Vacuum Liquid
1.1 x 103
11
Test Area - d
Wet-Vacuum Liquid
<31*
12
Test Area - f
Wet-Vacuum Liquid
3.2 x 103
13
Test Area - g
Wet-Vacuum Liquid
4.9 x 103
14
Test Area - h
Wet-Vacuum Liquid
1.5 x 104
15
Test Area - i
Wet-Vacuum Liquid
3.1 x 103
16
Test Area - e
Wet-Vacuum Liquid
2.4 x 107
17
DFU during Wet-Vacuum
Sampling
Filters 1&2
1.4 x 103
18
Positive Control
Wet-Vacuum Liquid
2.7 x 107
19
Blank MDI Control
Gauze Wipe
<2*
20
MDI Inoculation Control-1
Gauze Wipe
8.2 x 106
21
MDI Inoculation Control-2
Gauze Wipe
4.0 x 107
22
MDI Inoculation Control-3
Gauze Wipe
4.7 x 107
* Denotes Below Detection Limit
5.3.2 Cross-Contamination Assessment on Concrete using an
Improved ADA
Another test was conducted (Test 3) on concrete without the use of the sweeper, to assess
measures taken to reduce cross-contamination. To reduce spore spread that may occur during the
removal of the ADA, a sliding base (Figure 3-10) was added to the bottom of the ADA.
Additionally, the sampling area for each section was reduced to avoid further cross-
contamination during the wet vacuum sampling process. The adjacent sampling areas were
reduced by 2-in inward, and the sampling area for the inoculated area "e" was reduced from 3-ft
x 3-ft to 2.5- x 2.5-ft. The results of this test are illustrated in Figure 5-4 and summarized in
Table 5-2.
38
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In comparing the results of Test 2 and 3, it may provide some insight as to whether
measures taken helped to mitigate spread of spores. However, we caution that this is essentially
only a comparison of two data points, and further testing would be required to draw more
definitive conclusions. The cross-contamination from the "hot spot" to the adjacent areas from
the initial spreading (Test 2) was 5.7 x 104 CFU, which compares to 4.8 x 103 CFU from Test 3
(a 92% difference). Surface sampling of the negative control area, conducted after sampling the
sweep area, also showed the presence of Bg spores, and was of a level like the grids adjacent to
the hot spot area e. After the reset, but prior to inoculation, the negative control and area "e"
were found to have no detectable spores.
Inoculation Controls
3.0
2.6
2.3
A
B
C
3.1
D
DFU
2.8
2.8
2.6
G
H
I
+ Post-Sampling
Negative Control
3-ft
4 5-ft
2.0
A 5-ft
Positive Control
4.5 -ft 3-ft 4.5-ft
Sampling Floor Area
Figure 5-4: Assessment of Cross-Contamination Post-Inoculation on Concrete Surface
using an Improved ADA (log CFU)
39
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Table 5-2. Cross-Contamination Assessment on Concrete Using an Improved ADA
Sample
Number
Description
Sample Type
Test Result
Pre-Inoculation Sterility Check
Growth/No Growth
1
ADA Sterility check
Swab
NG
2
Gasket Sterility check
Swab
NG
3
Hoover Sterility check
Swab
NG
4
MDI Control Sterility check
Swab
NG
5
Backpack Sprayer
Swab
NG
CFU
6
DI Water Sterility Check
DI Water
<8*
7
Negative Control
Wet-Vacuum Liquid
<4*
8
Negative Control Area-e
Wet-Vacuum Liquid
<10*
9
DFU Filter- during negative sampling
Filters 1&2
<3*
Re-Aerosolization Checks During Inoculation
CFU
10
DFU - during Inoculation of ADA's and
overnight
Filters 1&2
6.2 x 102
Post-Inoculation Samples (In Order of Sampling)
CFU
11
DFU - during ADA Removal
Filters 1&2
<7
14
Test Area - a
Wet-Vacuum Liquid
1.1 X 103
15
Test Area - b
Wet-Vacuum Liquid
3.9 x 102
14
Test Area - c
Wet-Vacuum Liquid
2.0 x 102
15
Test Area - d
Wet-Vacuum Liquid
1.3 x 103
16
Test Area - f
Wet-Vacuum Liquid
7.8 x 101
17
Test Area - g
Wet-Vacuum Liquid
6.6 x 102
18
Test Area - h
Wet-Vacuum Liquid
6.9 x 102
19
Test Area - i
Wet-Vacuum Liquid
3.8 x 102
20
Test Area - e
Wet-Vacuum Liquid
2.5 x 107
21
DFU - during Wet-Vacuum Sampling
Filters 1&2
9.6 x 101
22
Negative Control - Post-Sampling
Wet-Vacuum Liquid
9.1 x 101
23
Positive Control
Wet-Vacuum Liquid
2.5 x 107
24
Blank MDI Control
Gauze Wipe
<2*
25
MDI Inoculation Control-1
Gauze Wipe
4.5 x 107
26
MDI Inoculation Control-2
Gauze Wipe
5.1 x 107
27
MDI Inoculation Control-3
Gauze Wipe
5.1 x 107
* Denotes Below Detection Limit Shown
40
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5.3.3 Cross-Contamination Assessment on Asphalt using the
Improved ADA
The results of this test (Test 9) to assess transport of spores within the test/sweep area and
to the negative control area, without the operation of the sweeper, are illustrated in Figure 5-5
and summarized in Table 5-3.
The transport of spores from the "hot spot" to the adjacent areas (without the use of the
sweeper) was found to be at least one (1) order of magnitude greater than for the concrete surface
(Test 3). However, we caution that this is essentially only a comparison of two data points, and
further testing would be required to draw more definitive conclusions. The cumulated cross-
contamination to the adjacent asphalt surfaces amounted to about 9.1 x 104, or more than 0.46 %
of the total post-sampling recoverable spores. Spores spread outside the 12- x 12-ft test area to
the post-sampling negative control area where the recovery (3.2 x 103 CFU) was the same order
of magnitude as the recovery adjacent to the "hot spot" area. This cross-contamination most
likely occurred during the inoculation process. The aerosolization during the inoculation process
(and overnight settling) was confirmed via the DFU sampler, which recovered 5.7 x 105 CFU.
This was three log CFU greater than what was captured by the DFU during the
inoculation/overnight period for the same test but on the concrete surface (Test 3). (Again, we
caution that this is essentially only a comparison of two data points, and further testing would be
required to draw more definitive conclusions.) The differences in aerosolization rates may be
due to the different characteristics of the two surfaces. The asphalt surface was non-uniform
(cracks, crevices, gaps, etc.) which caused inadequate sealing of the ADA apparatus to the
surface compared to the concrete which was much smoother and allowed a more optimal
adherence of the ADA. Photographs of the two surfaces are shown in Figure 5-6.
41
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mmm
Inoculation Controls
2.9
DFU
3.8
A
3.6
B
3.3
C
3.4
D
.3
3.5
F
3.9
G
4.7
H
3.8
1
1h * "h M k
A 5-ft 3-ft 4.5-ft
Sampling Floor Area
y Post-Sampling
Negative Control
4.5-ft
3-f!
3.5
A 5-ft
Positive Control
Figure 5-5: Assessment of Cross-Contamination Post-Inoculation on Asphalt Surface using
an Improved ADA (log CFU)
42
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Table 5-3. Cross-Contamination Assessment on Asphalt Surface Using an Improved ADA
Sample
Number
Description
Sample Type
Test Result
Pre-Inoculation Sterility Check
Growth/No Growth
1
ADA Sterility check
Swab
NG
2
Gasket Sterility check
Swab
NG
3
Hoover Sterility check
Swab
NG
4
MDI Control Sterility check
Swab
NG
CFU
5
DI Water Sterility Check
DI Water
<3*
6
Negative Control
Wet-Vacuum Liquid
<7*
7
Negative Control Area-e
Wet-Vacuum Liquid
<6*
8
DFU Filter- Negative control
Filters 1&2
<3*
Re-Aerosolization Checks During Inoculation
CFU
9
DFU - Inoculation of ADA's
Filters 1&2
5.7 x 105
Post-Inoculation Samples (In Order of Sampling)
CFU
10
Test Area - a
Wet-Vacuum Liquid
6.2 x 103
11
Test Area - b
Wet-Vacuum Liquid
3.8 x 103
12
Test Area - c
Wet-Vacuum Liquid
6.1 x 103
13
Test Area - d
Wet-Vacuum Liquid
2.8 x 103
14
Test Area - f
Wet-Vacuum Liquid
3.3 x 103
15
Test Area - g
Wet-Vacuum Liquid
7.6 x 103
16
Test Area - h
Wet-Vacuum Liquid
5.6 x 104
17
Test Area - i
Wet-Vacuum Liquid
5.8 x 103
18
Test Area - e
Wet-Vacuum Liquid
2.0 x 107
19
DFU - Wet-Vacuum Sampling
Filters 1&2
8.1 x 102
20
Negative Control - Post-Sampling
Wet-Vacuum Liquid
3.4 x 103
21
Positive Control
Wet-Vacuum Liquid
1.7 x 107
22
Blank MDI Control
Gauze Wipe
<1*
23
MDI Inoculation Control-1
Gauze Wipe
3.0 x 107
24
MDI Inoculation Control-2
Gauze Wipe
3.2 x 107
25
MDI Inoculation Control-3
Gauze Wipe
3.5 x 107
* Denotes Below Detection Limit Shown
43
-------
Figure 5-6: Concrete and Asphalt Surface Characteristics
5.4 Street Sweeper Decontamination/Removal Efficacy
The street sweeper was evaluated for its ability to remove or inactivate spores of Bg with
the use of various liquid decontaminants employed in the sweeper. These included DI water,
surfactant-based aqueous cleaning solutions, and chlorine-based decontamination solutions.
5.4.1 Removal Efficacy Using DI Water
5.4.1.1 Spore Recovery on Concrete using DI Water and a 1-Pass Sweep
The results of this test (Test 1) are illustrated in Figure 5-7 and summarized in Table 5-4.
The spore removal efficiency for the test area was 99.73%, or 2.6 LR. The quantity of spores
recovered from the recovery tank was 7.5 Log CFU. Some of the swept surfaces had little (area
"i") to no contamination (area "f') following the sweeping process.
44
-------
8 ¦ 7.9 ¦ 7.8
Inoculation Controls
Street Sweeper Recovery
2.7
DFU
*
4.5-ft
3.3
3.2
3.0
A
B
C
>
'
*
ii
3.0
4.7
¦
r
3-ft
D
E
\
i
t
i.
7.4
3.4
3.9
1.8
4.5-ft
H
Positive Control
G
I
r
X >¦*-
4.5-ft 3-ft 4.5-ft
Sampling Floor Area
Denotes Below Detection Limit
Figure 5-7: Spore Recovery on Concrete using DX Water and a 1-Pass Sweep (log CFU)
45
-------
Table 5-4. Spore Recoveries on Concrete using DI Water and a 1-Pass Sweep
Sample
Number
Description
Sample Type
Test Result
Pre-Inoculation Sterility Check
Growth/No Growth
1
ADA Sterility check
Swab
NG
2
Gasket Sterility check
Swab
NG
3
Hoover Sterility check
Swab
NG
4
MDI Control Sterility check
Swab
NG
5
Backpack Sprayer
Swab
NG
CFU
6
DI Water Sterility Check
DI Water
<2*
7
Negative Control
Wet-Vacuum Liquid
6.1 x 101
Post-Inoculation Samples (In Order of Sampling)
CFU
8
Sweeper Recovery
"Dirty" tank Liquid
3.3 x 107
9
DFU - Sweeper Operation
Filters 1&2
5.9 x 102
10
Via-cell Cassette - Sweeper Operation
Cassette
<1*
11
Test Area - a
Wet-Vacuum Liquid
2.1 x 103
12
Test Area - b
Wet-Vacuum Liquid
1.6 x 103
13
Test Area - c
Wet-Vacuum Liquid
1.0 x 103
14
Test Area - d
Wet-Vacuum Liquid
9.7 x 102
15
Test Area - f
Wet-Vacuum Liquid
<6*
16
Test Area - g
Wet-Vacuum Liquid
2.3 x 103
17
Test Area - h
Wet-Vacuum Liquid
7.7 x 103
18
Test Area - i
Wet-Vacuum Liquid
5.9 x 101
19
Test Area - e
Wet-Vacuum Liquid
5.1 x 104
20
Positive Control
Wet-Vacuum Liquid
2.5 x 107
21
Blank MDI Control
Gauze Wipe
6.1 x 101
22
MDI Inoculation Control-1
Gauze Wipe
6.2 x 107
23
MDI Inoculation Control-2
Gauze Wipe
8.2 x 107
24
MDI Inoculation Control-3
Gauze Wipe
6.4 x 107
* Denotes Below Detection Limit Shown
*Detection Limit based on 50% probability of 1 spore detection in a sample
46
-------
5.4.1.2 Spore Recovery on Concrete using DI Water and a 3-Pass Sweep
This test (Test 6) was designed to determine if increasing the number of sweeps of the
area would result in an increase of the number of spores removed from the concrete surface. The
results of this test are illustrated in Figure 5-8 and summarized in Table 5-5. The removal
efficiency for the test area was 99.94%, or a LR of 3.2. This removal efficiency may be
compared to Test 1 (LR of 2.6), in which one pass with DI was used. However, we caution that
this is essentially only a comparison of two data points, and further testing would be required to
draw more definitive conclusions about whether additional passes of the sweeper increase
collection efficiency.
Inoculation Controls
Street Sweeper Recovery
3.0
2.9
2.5
A
B
C
(IT)
2.7
3.9
2.8
D
E
F
DFU
2.9
3.0
2.8
G
H
1
| Post-Sampling
Negative Control
3.4
3-fi
4.5-ft
Positive Control
M-
4.5-ft 3-ft 4.5-ft
Sampling Floor Area
Figure 5-8: Spore Recoveries on Concrete using DI Water and a 3-Pass Sweep (log CFU)
47
-------
Table 5-5. Spore Recoveries on Concrete using DI Water and a 3-Pass Sweep
Sample
Number
Description
Sample Type
Test Result
Pre-Inoculation Sterility Check
Growth/No Growth
1
ADA Sterility check
Swab
NG
2
Gasket Sterility check
Swab
NG
3
Hoover Sterility check
Swab
NG
4
MDI Control Sterility check
Swab
NG
5
Backpack Sprayer
Swab
NG
CFU
6
DI Water Sterility Check
DI Water
<2*
7
Negative Control
Wet-Vacuum Liquid
<5*
9
DFU Filter- Negative control
Filters 1&2
<2*
Re-Aerosolization Checks During Inoculation
CFU
10
DFU - Inoculation of ADA's
Filters 1&2
5.8 x 103
Post-Inoculation Samples (In Order of Sampling)
CFU
11
Sweeper Recovery
"Dirty" tank Liquid
4.6 x 107
12
DFU - ADA Removal & Sweeper Operation
Filters 1&2
1.1 x 103
13
Via-cell Cassette - Sweeper Operation
Cassette
<1*
14
Test Area - a
Wet-Vacuum Liquid
1.0 x 103
15
Test Area - b
Wet-Vacuum Liquid
7.8 x 102
16
Test Area - c
Wet-Vacuum Liquid
2.9 x 102
17
Test Area - d
Wet-Vacuum Liquid
4.7 x 102
18
Test Area - f
Wet-Vacuum Liquid
6.8 x 102
19
Test Area - g
Wet-Vacuum Liquid
7.2 x 102
20
Test Area - h
Wet-Vacuum Liquid
9.1 x 102
21
Test Area - i
Wet-Vacuum Liquid
5.8 x 102
22
Test Area - e
Wet-Vacuum Liquid
8.0 x 103
23
DFU - Wet-Vacuum Sampling
Filters 1&2
1.4 x 103
24
Negative Control - Post-Sampling
Wet-Vacuum Liquid
2.4 x 103
25
Positive Control
Wet-Vacuum Liquid
2.1 x 107
26
Blank MDI Control
Gauze Wipe
<3*
27
MDI Inoculation Control-1
Gauze Wipe
2.8 x 107
28
MDI Inoculation Control-2
Gauze Wipe
5.5 x 107
29
MDI Inoculation Control-3
Gauze Wipe
5.0 x 107
*Detection Limit
5.4.1.3 Spore Recovery on Asphalt using DI Water and a 1-Pass Sweep
The results of the asphalt test with DI water (Test 10) are illustrated in Figure 5-9 and
summarized in Table 5-6. The removal efficiency of the sweeper for the test area was 77.40%, or
a LR 0.6.
48
-------
In this test, the spread of spores to test areas adjacent to the hot spot was 8.0 x ID5 CFU,
which is approximately 1 log CFU greater than the spread that occurred on asphalt without the
use of the sweeper (Test 9; 9.1 x 104 CFU). We caution that this is essentially only a comparison
of two data points, and further testing would be required to draw more definiti ve conclusions
regarding the effect of the sweeper operation on the spreading of spores.
This result, in which the operation of the sweeper caused ~ 1 log CFU greater spread to
adjacent areas than without the sweeper, occurred as well on the concrete surface.
5 ¦ 7.6 ¦ 7.6
Inoculation Controls
Street Sweeper Recover/
2.8
DFU
5.0
5.3
4.6
C
B
4.2
D
6.5
4.3
F
5.5
4.6
H
4.5
1
* Post-Sampling
Negative Control
A 5-ft
3-ft
3.1
A 5-f:
Positive Control
4.5-ft 3-fl 4.5-ft
Sampling Floor Area
Figure 5-9: Spore Recovery on Asphalt using DI Water and a 1-Pass Sweep (log CFU)
49
-------
Table 5-6. Spore Recoveries on Asphalt using DI Water and a 1-Pass Sweep
Sample
Number
Description
Sample Type
Test Result
Pre-Inoculation Sterility Check
Growth/No Growth
1
ADA Sterility check
Swab
NG
2
Gasket Sterility check
Swab
NG
3
Hoover Sterility check
Swab
NG
4
MDI Control Sterility check
Swab
NG
5
Backpack Sprayer
Swab
NG
CFU
6
DI Water Sterility Check
DI Water
<1*
7
Negative Control
Wet-Vacuum Liquid
<5*
8
Negative Control Area-e
Wet-Vacuum Liquid
<5*
9
DFU Filter- Negative control
Filters 1&2
<3*
Re-Aerosolization Checks During Inoculation
CFU
10
DFU - Inoculation of ADA's
Filters 1&2
1.4 x 105
Post-Inoculation Samples (In Order of Sampling)
CFU
11
Sweeper Recovery
"Dirty" tank Liquid
2.0 x 107
12
DFU - ADA Removal & Sweeper Operation
Filters 1&2
6.5 x 102
13
Via-cell Cassette - Sweeper Operation
Cassette
<1*
14
Test Area - a
Wet-Vacuum Liquid
9.8 x 104
15
Test Area - b
Wet-Vacuum Liquid
2.2 x 105
16
Test Area - c
Wet-Vacuum Liquid
3.6 x 104
17
Test Area - d
Wet-Vacuum Liquid
1.7 x 104
18
Test Area - f
Wet-Vacuum Liquid
1.8 x 104
19
Test Area - g
Wet-Vacuum Liquid
3.3 x 105
20
Test Area - h
Wet-Vacuum Liquid
4.5 x 104
21
Test Area - i
Wet-Vacuum Liquid
2.8 x 104
22
Test Area - e
Wet-Vacuum Liquid
3.1 x 106
23
DFU - Wet-Vacuum Sampling
Filters 1&2
4.6 x 103
24
Negative Control - Post-Sampling
Wet-Vacuum Liquid
1.2 x 103
25
Positive Control
Wet-Vacuum Liquid
1.7 x 107
26
Blank MDI Control
Gauze Wipe
<1*
27
MDI Inoculation Control-1
Gauze Wipe
3.3 x 107
28
MDI Inoculation Control-2
Gauze Wipe
3.7 x 107
29
MDI Inoculation Control-3
Gauze Wipe
4.3 x 107
*Detection Limit
50
-------
5.4.1.4 Spore Recovery on Asphalt using DI Water and a 3-Pass Sweep
The results of the 3-pass sweep on asphalt (Test 12) are illustrated in Figure 5-10 and
summarized in Table 5-7. Increasing the number of sweeps had no improvement on the total
reduction for the area treated by the street sweeper. The removal efficiency of this test was 74%,
compared to a 77% removal for using just one pass of the sweeper (Test 10).
6 ¦ 7.6 ¦ 7.6
Inoculation Controls
Street Sweeper Recovery
3.8
A
4.1
B
3.8
C
("IT)
3.2
6.7
3.6
D
F
DFU
3.6
G
4.5
H
3.9
I
4.5-ft
3-fl
1.8
Post-Sampling
Negative Control
A 5-ft
Positive Control
M ><
4.5-ft 3-ft 4 5-ft
Sampling Floor Area
Figure 5-10: Spore Recovery on Asphalt using DI Water and a 3-Pass Sweep (log CFU)
51
-------
Table 5-7. Spore Recoveries on Asphalt using DI Water and a 3-Pass Sweep
Sample
Number
Description
Sample Type
Test Result
Pre-Inoculation Sterility Check
Growth/No Growth
1
ADA Sterility check
Swab
NG
2
Gasket Sterility check
Swab
NG
3
Hoover Sterility check
Swab
NG
4
MDI Control Sterility check
Swab
NG
5
Backpack Sprayer
Swab
NG
CFU
6
DI Water Sterility Check
DI Water
<1*
7
Negative Control
Wet-Vacuum Liquid
<4*
8
Negative Control Area-e
Wet-Vacuum Liquid
<5*
9
DFU Filter- Negative control
Filters 1&2
<3*
Re-Aerosolization Checks During Inoculation
CFU
10
DFU - Inoculation of ADA's
Filters 1&2
1.1 X 104
Post-Inoculation Samples (In Order of Sampling)
CFU
11
Sweeper Recovery
"Dirty" tank Liquid
3.7 x 107
12
DFU - ADA Removal & Sweeper Operation
Filters 1&2
2.4 x 102
13
Via-cell Cassette - Sweeper Operation
Cassette
3
14
Test Area - a
Wet-Vacuum Liquid
6.4 x 103
15
Test Area - b
Wet-Vacuum Liquid
1.2 x 104
16
Test Area - c
Wet-Vacuum Liquid
5.7 x 103
17
Test Area - d
Wet-Vacuum Liquid
1.7 x 103
18
Test Area - f
Wet-Vacuum Liquid
4.1 x 103
19
Test Area - g
Wet-Vacuum Liquid
3.7 x 103
20
Test Area - h
Wet-Vacuum Liquid
3.1 x 104
21
Test Area - i
Wet-Vacuum Liquid
7.8 x 103
22
Test Area - e
Wet-Vacuum Liquid
5.5 x 106
23
DFU - Wet-Vacuum Sampling
Filters 1&2
4.0 x 102
24
Negative Control - Post-Sampling
Wet-Vacuum Liquid
6.6 x 101
25
Positive Control
Wet-Vacuum Liquid
2.2 x 107
26
Blank MDI Control
Gauze Wipe
<1*
27
MDI Inoculation Control-1
Gauze Wipe
4.3 x 107
28
MDI Inoculation Control-2
Gauze Wipe
4.3 x 107
29
MDI Inoculation Control-3
Gauze Wipe
3.8 x 107
* Denotes Below Detection Limit Shown
52
-------
5.4.2 Spore Removal Efficacy Using Surfactant-Based Aqueous
Solutions
The testing results using surfactant-based aqueous cleaning solutions on concrete and
asphalt are presented in this section. The two solutions used during these tests were made with a
commercial dishwashing liquid and SSDX-12™, a concentrated detergent that is certified for
general cleaning of U.S. military aircraft.
5.4.2.1 Spore Removal Efficacy Using Dishwashing Solution on Concrete
The results of this test (Test 5) are illustrated in Figure 5-11 and summarized in Table 5-
8. No improvement in removal efficiency was observed with this the use of dishwashing
solution. That is, the overall removal efficiency for this test was 99.7%, which was the same
removal efficiency obtained for the test with DI water only (Test 1).
7.8 ¦ 7.8
Inoculation Controls
Street Sweeper Recovery
2.5
4.1
2.8
A
B
C
( 2.3 )
3.3
4.6
2.6
D
E
F
DFJ
3.2
3.6
2.8
G
H
I
Post-Sampling
Negative Control
3-ft
A 5-ft
1.6
A 5-ft
Positive Control
4.5-ft 3-ft 4.5-ft
Sampling Floor Area
Figure 5-11: Spore Recovery on Concrete using Dawn® Dishwashing Solution (log CFU)
53
-------
Table 5-8. Spore Recoveries on Concrete using Dawn® Dishwashing Solution
Sample
Number
Description
Sample Type
Test Result
Pre-Inoculation Sterility Check
Growth/No Growth
1
ADA Sterility check
Swab
NG
2
Gasket Sterility check
Swab
NG
3
Hoover Sterility check
Swab
NG
4
MDI Control Sterility check
Swab
NG
5
Backpack Sprayer
Swab
NG
CFU
6
DI Water Sterility Check
DI Water
<2*
7
Negative Control
Wet-Vacuum Liquid
<6*
8
Negative Control Area-e
Wet-Vacuum Liquid
<4*
9
DFU Filter- Negative control
Filters 1&2
<3*
Re-Aerosolization Checks During Inoculation
CFU
10
DFU - Inoculation of ADA's
Filters 1&2
3.0 x 104
Post-Inoculation Samples (In Order of Sampling)
CFU
11
Sweeper Recovery
"Dirty" tank Liquid
4.1 x 107
12
DFU - ADA Removal & Sweeper Operation
Filters 1&2
6.5 x 102
13
Via-cell Cassette - Sweeper Operation
Cassette
<1*
14
Test Area - a
Wet-Vacuum Liquid
3.4 x 102
15
Test Area - b
Wet-Vacuum Liquid
1.2 x 104
16
Test Area - c
Wet-Vacuum Liquid
6.7 x 102
17
Test Area - d
Wet-Vacuum Liquid
1.8 x 103
18
Test Area - f
Wet-Vacuum Liquid
4.2 x 102
19
Test Area - g
Wet-Vacuum Liquid
1.7 x 103
20
Test Area - h
Wet-Vacuum Liquid
4.0 x 103
21
Test Area - i
Wet-Vacuum Liquid
6.3 x 102
22
Test Area - e
Wet-Vacuum Liquid
3.8 x 104
23
DFU - Wet-Vacuum Sampling
Filters 1&2
1.2 x 102
24
Negative Control - Post-Sampling
Wet-Vacuum Liquid
3.8 x 101
25
Positive Control
Wet-Vacuum Liquid
2.0 x 107
26
Blank MDI Control
Gauze Wipe
<2*
27
MDI Inoculation Control-1
Gauze Wipe
2.6 x 107
28
MDI Inoculation Control-2
Gauze Wipe
6.9 x 107
29
MDI Inoculation Control-3
Gauze Wipe
5.7 x 107
*Detection Limit
54
-------
5.4.2.2 Spore Removal Efficacy Using Dishwashing Solution on Asphalt
The results of this test are illustrated in Figure 5-12 and summarized in Table 5-9. The
removal efficiency for this test with the use of dish soap (80.7%) was similar to the removal
efficiency on asphalt with just DI water (Test 10; 77.4%). We caution that this is essentially only
a comparison of two data points, and further testing would be required to draw more definitive
conclusions regarding the effect of using soap on removal efficiency.
QBE
Inoculation Controls
Street Sweeper Recovery
DFU
3.3
A
6.0
3.8
C
3.8
D
6.2
3.4
F
4.0
G
5.4
3.5
1
"
^ Post-Sampling
Negative Control
A 5-ft
3-ft
2.8
4.5-ft
Positive Control
->< M-
4.5-ft 3-ft 4.5-ft
Sampling Floor Area
Figure 5-12: Spore Recovery on Asphalt using Dawn® Dishwashing Solution (log CFU)
55
-------
Table 5-9. Spore Recoveries on Asphalt using Dawn® Dishwashing Solution
Sample
Number
Description
Sample Type
Test Result
Pre-Inoculation Sterility Check
Growth/No Growth
1
ADA Sterility check
Swab
NG
2
Gasket Sterility check
Swab
NG
3
Hoover Sterility check
Swab
NG
4
MDI Control Sterility check
Swab
NG
5
Backpack Sprayer
Swab
NG
CFU
6
DI Water Sterility Check
DI Water
<1*
7
Negative Control
Wet-Vacuum Liquid
<4*
8
Negative Control Area-e
Wet-Vacuum Liquid
<6*
9
DFU Filter- Negative control
Filters 1&2
<3*
Re-Aerosolization Checks During Inoculation
CFU
10
DFU - Inoculation of ADA's
Filters 1&2
1.6 x 105
Post-Inoculation Samples (In Order of Sampling)
CFU
11
Sweeper Recovery
"Dirty" tank Liquid
1.6 x 107
12
DFU - ADA Removal & Sweeper Operation
Filters 1&2
3.0 x 102
13
Via-cell Cassette - Sweeper Operation
Cassette
<2*
14
Test Area - a
Wet-Vacuum Liquid
1.8 x 103
15
Test Area - b
Wet-Vacuum Liquid
1.1 x 106
16
Test Area - c
Wet-Vacuum Liquid
5.8 x 103
17
Test Area - d
Wet-Vacuum Liquid
5.9 x 103
18
Test Area - f
Wet-Vacuum Liquid
2.3 x 103
19
Test Area - g
Wet-Vacuum Liquid
1.1 x 104
20
Test Area - h
Wet-Vacuum Liquid
2.5 x 105
21
Test Area - i
Wet-Vacuum Liquid
3.0 x 103
22
Test Area - e
Wet-Vacuum Liquid
1.7 x 106
23
DFU - Wet-Vacuum Sampling
Filters 1&2
1.1 x 104
24
Negative Control - Post-Sampling
Wet-Vacuum Liquid
6.1 x 102
25
Positive Control
Wet-Vacuum Liquid
1.6 x 107
26
Blank MDI Control
Gauze Wipe
7.5 x 102
27
MDI Inoculation Control-1
Gauze Wipe
3.1 x 107
28
MDI Inoculation Control-2
Gauze Wipe
3.3 x 107
29
MDI Inoculation Control-3
Gauze Wipe
2.2 x 107
* Denotes Below Detection Limit Shown
56
-------
5.4.2.3 Spore Removal Efficacy Using SSDX-12™ on Concrete
The results of this test (Test 4) are illustrated in Figure 5-13 and summarized in Table 5-
10. Like the dishwashing solution, no improvement was observed with this formulation, when
compared to DI water only (Test 1), in terms of removal/decontamination efficacy on the treated
surfaces, and the extent of the cross-contamination to the areas adjacent to the 'hot spot" area.
The removal efficiency obtained in this test was 99.38%.
7.6 ¦ 7.7
Inoculation Controls
Street Sweeper Recovery
2.6
DFU
4.6
A
CD
Lo
3.1
C
3.0
D
2.7
F
4.7
G
3.8
H
3.7
I
| Post-Sampling
Negative Control
A 5-ft
3-ft
1.9
A 5-ft
Positive Control
4.5-ft 3-ft 4.5-ft
Sampling Floor Area
Figure 5-13: Spore Recovery on Concrete using SSDX-12™ Solution (log CFU)
57
-------
Table 5-10. Spore Recoveries on Concrete using SSDX-12™ Solution
Sample
Number
Description
Sample Type
Test Result
Pre-Inoculation Sterility Check
Growth/No Growth
1
ADA Sterility check
Swab
NG
2
Gasket Sterility check
Swab
NG
3
Hoover Sterility check
Swab
NG
4
MDI Control Sterility check
Swab
NG
5
Backpack Sprayer
Swab
NG
CFU
6
DI Water Sterility Check
DI Water
<8*
7
Negative Control
Wet-Vacuum Liquid
<4*
8
Negative Control Area-e
Wet-Vacuum Liquid
<5*
9
DFU Filter- Negative control
Filters 1&2
<4*
Re-Aerosolization Checks During Inoculation
CFU
10
DFU - Inoculation of ADA's
Filters 1&2
8.2 x 102
Post-Inoculation Samples (In Order of Sampling)
CFU
11
Sweeper Recovery
"Dirty" tank Liquid
6.1 x 106
12
DFU - ADA Removal & Sweeper Operation
Filters 1&2
3.6 x 102
13
Test Area - a
Wet-Vacuum Liquid
3.6 x 104
14
Test Area - b
Wet-Vacuum Liquid
1.8 x 103
15
Test Area - c
Wet-Vacuum Liquid
1.3 x 103
16
Test Area - d
Wet-Vacuum Liquid
9.9 x 102
17
Test Area - f
Wet-Vacuum Liquid
5.1 x 102
18
Test Area - g
Wet-Vacuum Liquid
4.6 x 104
19
Test Area - h
Wet-Vacuum Liquid
6.6 x 103
20
Test Area - i
Wet-Vacuum Liquid
5.3 x 103
21
Test Area - e
Wet-Vacuum Liquid
1.0 x 105
22
DFU - Wet-Vacuum Sampling
Filters 1&2
1.9 x 102
23
Negative Control - Post-Sampling
Wet-Vacuum Liquid
8.0 x 101
24
Positive Control
Wet-Vacuum Liquid
3.2 x 107
25
Blank MDI Control
Gauze Wipe
<2*
26
MDI Inoculation Control-1
Gauze Wipe
4.4 x 107
27
MDI Inoculation Control-2
Gauze Wipe
4.4 x 107
28
MDI Inoculation Control-3
Gauze Wipe
4.4 x 107
* Denotes Below Detection Limit Shown
58
-------
5.4.3 Decontamination Efficacy Using Chlorine-Based Aqueous
Solutions
5.4.3.1 Decontamination Efficacy Using pAB on Concrete
The results of this test are illustrated in Figure 5-14 and summarized in Table 5-11. As
expected, p.AB was found to be very efficacious in decontaminating the treated surfaces (no
spores were recovered from the sampled areas). Thus, the LR for this test was > 7.33. Note that
no spores were detected in the sweeper recovery tank as well; however, there was transfer of
spores to outside of the test area (4.6 log CFU).
Inoculation Controls
Street Sweeper Recovery
0.6*
0,3*
0.6*
OiD
0.4*
0.4*
0.3*
DFU
0.5*
0.5*
0.7*
4.5-ft
4.6
Post-Sampling
Negative Control
A 5-f:
Positive Control
-*>< >*-
4-5-ft 3-ft 4.5-ft
Sampling Floor Area
Denotes Below Detection Limit
Figure 5-14: Spore Recovery on Concrete using pAB Solution (log CFU)
59
-------
Table 5-11. Spore Recoveries on Concrete using pAB Solution
Sample
Number
Description
Sample Type
Test Result
Pre-Inoculation Sterility Check
Growth/No Growth
1
ADA Sterility check
Swab
NG
2
Gasket Sterility check
Swab
NG
3
Hoover Sterility check
Swab
NG
4
MDI Control Sterility check
Swab
NG
5
Backpack Sprayer
Swab
NG
CFU
6
DI Water Sterility Check
DI Water
<3*
7
Negative Control
Wet-Vacuum Liquid
<4*
8
Negative Control Area-e
Wet-Vacuum Liquid
<4*
9
DFU Filter- Negative control
Filters 1&2
<4*
Re-Aerosolization Checks During Inoculation
CFU
10
DFU - Inoculation of ADA's
Filters 1&2
5.8 x 103
Post-Inoculation Samples (In Order of Sampling)
CFU
11
Sweeper Recovery
"Dirty" tank Liquid
<66*
12
DFU - ADA Removal & Sweeper Operation
Filters 1&2
1.4 x 101
13
Via-cell Cassette - Sweeper Operation
Cassette
<1*
14
Test Area - a
Wet-Vacuum Liquid
<5*
15
Test Area - b
Wet-Vacuum Liquid
<3*
16
Test Area - c
Wet-Vacuum Liquid
<5*
17
Test Area - d
Wet-Vacuum Liquid
<3*
18
Test Area - f
Wet-Vacuum Liquid
<3*
19
Test Area - g
Wet-Vacuum Liquid
<4*
20
Test Area - h
Wet-Vacuum Liquid
<4*
21
Test Area - i
Wet-Vacuum Liquid
<6*
22
Test Area - e
Wet-Vacuum Liquid
<3*
23
DFU - Wet-Vacuum Sampling
Filters 1&2
5.7 x 102
24
Negative Control - Post-Sampling
Wet-Vacuum Liquid
3.8 x 104
25
Positive Control
Wet-Vacuum Liquid
2.2 x 107
26
Blank MDI Control
Gauze Wipe
<1*
27
MDI Inoculation Control-1
Gauze Wipe
2.9 x 107
28
MDI Inoculation Control-2
Gauze Wipe
4.6 x 107
29
MDI Inoculation Control-3
Gauze Wipe
3.5 x 107
* Denotes Below Detection Limit Shown
60
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5.4.3.2 Decontamination Efficacy Using pAB on Asphalt
The results of this test are illustrated in Figure 5-15 and summarized in Table 5-12. pAB
was found to be very efficacious in decontaminating the asphalt surface surfaces (no spores were
detected in the sampled areas), providing a LR of > 7.2. This high efficacy was somewhat
unexpected, since pAB is known to be less effective on organic-based materials compared to
inorganic based materials. In a bench-scale test spray applying pAB to asphalt coupons, only a
3.6 LR was achieved (Calfee et al. 2011). We caution that this is essentially only a comparison of
two data points, and further testing would be required to draw more definitive conclusions.
6 ¦ 7.6 ¦ 7.6
Inoculation Controls
Street Sweeper Recover/
0.8*
o.r
0.6*
0.6*
0.6*
o.r
DFU
0.6*
0.6*
o.r
3-fi
A 5-ft
1.8
Post-Sampling
Negative Control
4.5-ft
Positive Control
-M ~<-
4.5-ft 3-fl 4.5-ft
Sampling Floor Area
Denotes Below Detection Limit
Figure 5-15: Spore Recovery on Asphalt using pAB Solution (log CFU)
61
-------
Table 5-12. Spore Recoveries on Asphalt using pAB Solution
Sample
Number
Description
Sample Type
Test Result
Pre-Inoculation Sterility Check
Growth/No Growth
1
ADA Sterility check
Swab
NG
2
Gasket Sterility check
Swab
NG
3
Hoover Sterility check
Swab
NG
4
MDI Control Sterility check
Swab
NG
5
Backpack Sprayer
Swab
NG
CFU
6
DI Water Sterility Check
DI Water
<3*
7
Negative Control
Wet-Vacuum Liquid
<3*
8
Negative Control Area-e
Wet-Vacuum Liquid
<4*
9
DFU Filter- Negative control
Filters 1&2
<3*
Re-Aerosolization Checks During Inoculation
CFU
10
DFU - Inoculation of ADA's
Filters 1&2
3.7 x 104
Post-Inoculation Samples (In Order of Sampling)
CFU
11
Sweeper Recovery
"Dirty" tank Liquid
<680*
12
DFU - ADA Removal & Sweeper Operation
Filters 1&2
<3*
13
Via-cell Cassette - Sweeper Operation
Cassette
<5
14
Test Area - a
Wet-Vacuum Liquid
<6*
15
Test Area - b
Wet-Vacuum Liquid
<5*
16
Test Area - c
Wet-Vacuum Liquid
<5*
17
Test Area - d
Wet-Vacuum Liquid
<4*
18
Test Area - f
Wet-Vacuum Liquid
<5*
19
Test Area - g
Wet-Vacuum Liquid
<5*
20
Test Area - h
Wet-Vacuum Liquid
<4*
21
Test Area - i
Wet-Vacuum Liquid
<6*
22
Test Area - e
Wet-Vacuum Liquid
<4*
23
DFU - Wet-Vacuum Sampling
Filters 1&2
<3
24
Negative Control - Post-Sampling
Wet-Vacuum Liquid
6.5 x 101
25
Positive Control
Wet-Vacuum Liquid
1.6 x 107
26
Blank MDI Control
Gauze Wipe
<1*
27
MDI Inoculation Control-1
Gauze Wipe
4.1 x 107
28
MDI Inoculation Control-2
Gauze Wipe
3.7 x 107
29
MDI Inoculation Control-3
Gauze Wipe
3.6 x 107
* Denotes Below Detection Limit Shown
62
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5.4.3.3 Decontamination Efficacy Using Dichlor on Concrete
The results of this test are illustrated in Figure 5-16 and summarized in Table 5-13. The
Dichlor solution, like pAB, was found to be very efficacious in decontaminating the treated
surface (no spores were detected in the sampled areas, resulting in a LR of > 6.9). No spores
were detected in the sweeper recovery tank, but the higher detection limit (3.5 log CFU) was due
to the chlorine granules crystalizing during analytical procedures.
Figure 5-16: Spore Recovery on Concrete using Dichlor Solution (log CFU)
63
-------
Table 5-13. Spore Recoveries on Concrete using Dichlor Solution
Sample
Number
Description
Sample Type
Test Result
Pre-Inoculation Sterility Check
Growth/No Growth
1
ADA Sterility check
Swab
NG
2
Gasket Sterility check
Swab
NG
3
Hoover Sterility check
Swab
NG
4
MDI Control Sterility check
Swab
NG
5
Backpack Sprayer
Swab
NG
CFU
6
DI Water Sterility Check
DI Water
<3*
7
Negative Control
Wet-Vacuum Liquid
<4*
8
Negative Control Area-e
Wet-Vacuum Liquid
<5*
9
DFU Filter- Negative control
Filters 1&2
<3*
Re-Aerosolization Checks During Inoculation
CFU
10
DFU - Inoculation of ADA's
Filters 1&2
5.7 x 103
Post-Inoculation Samples (In Order of Sampling)
CFU
11
Sweeper Recovery
"Dirty" tank Liquid
<3,300*
12
DFU - ADA Removal & Sweeper Operation
Filters 1&2
1.9 x 102
13
Via-cell Cassette - Sweeper Operation
Cassette
<1*
14
Test Area - a
Wet-Vacuum Liquid
<3*
15
Test Area - b
Wet-Vacuum Liquid
<2*
16
Test Area - c
Wet-Vacuum Liquid
<3*
17
Test Area - d
Wet-Vacuum Liquid
<3*
18
Test Area - f
Wet-Vacuum Liquid
<2*
19
Test Area - g
Wet-Vacuum Liquid
<3*
20
Test Area - h
Wet-Vacuum Liquid
<3*
21
Test Area - i
Wet-Vacuum Liquid
<4*
22
Test Area - e
Wet-Vacuum Liquid
<3*
23
DFU - Wet-Vacuum Sampling
Filters 1&2
5.7 x 102
24
Negative Control - Post-Sampling
Wet-Vacuum Liquid
1.3 x 104
25
Positive Control
Wet-Vacuum Liquid
8.7 x 106
26
Blank MDI Control
Gauze Wipe
6.5 x 101
27
MDI Inoculation Control-1
Gauze Wipe
3.8 x 107
28
MDI Inoculation Control-2
Gauze Wipe
5.6 x 107
29
MDI Inoculation Control-3
Gauze Wipe
5.3 x 107
* Denotes Below Detection Limit Shown
64
-------
5.4.3.4 Decontamination Efficacy Using Dichlor on Asphalt
The results of this test (Test 13) are illustrated in Figure 5-17 and summarized in Table 5-
14. Dichlor solution was found to be very efficacious in decontaminating the asphalt surfaces (no
spores were recovered in the sampled areas, resulting in a LR of > 7.1). Note that in contrast to
the three other tests utilizing pAB or Dichlor on concrete and asphalt, no spores were detected
outside the test surface area (negative control area), and no spores were detected in the DFU air
sample run during sweeping.
Inoculation Controls
Street Sweeper Recovery
0.7*
0.6*
0.7*
0.6*
0.4*
0.6*
DFU
0.6*
0.5*
0.8*
ijr Post-Sampling
Negative Control
A 5-ft
A 5-ft
Positive Control
>+-
4.5-ft 3-ft 4.5-ft
Sampling Floor Area
Denotes Below Detection Limit
Figure 5-17: Spore Recovery on Asphalt using Dichlor Solution (log CFU)
65
-------
Table 5-14. Spore Recoveries on Asphalt using Dichlor Solution
Sample
Number
Description
Sample Type
Test Result
Pre-Inoculation Sterility Check
Growth/No Growth
1
ADA Sterility check
Swab
NG
2
Gasket Sterility check
Swab
NG
3
Hoover Sterility check
Swab
NG
4
MDI Control Sterility check
Swab
NG
5
Backpack Sprayer
Swab
NG
CFU
6
DI Water Sterility Check
DI Water
<3*
7
Negative Control
Wet-Vacuum Liquid
<3*
8
Negative Control Area-e
Wet-Vacuum Liquid
<4*
9
DFU Filter- Negative control
Filters 1&2
<3*
Re-Aerosolization Checks During Inoculation
CFU
10
DFU - Inoculation of ADA's
Filters 1&2
1.2 x 105
Post-Inoculation Samples (In Order of Sampling)
CFU
11
Sweeper Recovery
"Dirty" tank Liquid
<600*
12
DFU - ADA Removal & Sweeper Operation
Filters 1&2
<3*
13
Via-cell Cassette - Sweeper Operation
Cassette
<1*
14
Test Area - a
Wet-Vacuum Liquid
<5*
15
Test Area - b
Wet-Vacuum Liquid
<5*
16
Test Area - c
Wet-Vacuum Liquid
<5*
17
Test Area - d
Wet-Vacuum Liquid
<5*
18
Test Area - f
Wet-Vacuum Liquid
<4*
19
Test Area - g
Wet-Vacuum Liquid
<5*
20
Test Area - h
Wet-Vacuum Liquid
<4*
21
Test Area - i
Wet-Vacuum Liquid
<7*
22
Test Area - e
Wet-Vacuum Liquid
<3*
23
DFU - Wet-Vacuum Sampling
Filters 1&2
2.2 x 103
24
Negative Control - Post-Sampling
Wet-Vacuum Liquid
<4*
25
Positive Control
Wet-Vacuum Liquid
1.3 x 107
26
Blank MDI Control
Gauze Wipe
<1*
27
MDI Inoculation Control-1
Gauze Wipe
2.7 x 107
28
MDI Inoculation Control-2
Gauze Wipe
2.8 x 107
29
MDI Inoculation Control-3
Gauze Wipe
3.6 x 107
* Denotes Below Detection Limit Shown
66
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5.5 Summary of Results
A summary of the study results related to surface decontamination is shown in Table 5-15.
Aerosol sample results for the study are summarized in Table 5-16.
The key findings from this study can be highlighted as follows:
• The Bg inoculum levels on the stainless-steel control surfaces were consistent throughout
the study, with an average ± SD of 7.6 ± 0.2 log CFU. The average recovery of spores
from the concrete positive controls was 7.3 ± 0.2 log CFU, and for asphalt positive
controls, the average recovery was 7.2 ±0.1 log CFU.
• The spore removal efficacy of the street sweeper on asphalt, for the three tests using only
water (with and without surfactants), ranged from 74-81% (with average ± SD = 77±3.4).
The minimum removal efficiency was 99.4% for the four similar tests on concrete (with
an average ± SD of 99.7±0.2). A simple Microsoft Excel t-test showed these results to be
significantly different, although additional tests and analyses may be warranted to allow
for a more robust determination. The differences in results for the two materials may be
due to the non-uniform characteristics of the asphalt surface, making the dislodging and
capture of the spores more difficult compared to the smooth concrete surface. Most likely
the asphalt results are more representative of what could be expected in a real scenario
with B. anthracis contamination, since the asphalt surface was aged/weathered, whereas
the concrete surface was new and had a smooth surface.
• In the tests using DI water, the addition of surfactants (including the "high tech" military
grade surfactant) did not seem to improve spore removal on either surface. The removal
of spores using three passes of the sweeper on concrete was slightly greater (~ 0.5 LR)
compared to using just one pass, but this improvement was not realized on asphalt. We
caution that determining whether these changes in operation significantly affected
removal efficiency would require additional test data and statistical analyses.
• Transfer of spores from the inoculated 'hot spot" area (grid "e") to the adjacent grids of
the test area occurred with and without the use of the sweeper, and ranged from
approximately 4-6 log CFU. The level of cross-contamination to the adjacent areas
resulting from the operation of the sweeper was approximately 10 times higher in CFU
than without operation of the sweeper. This phenomenon occurred on both surface types.
Additionally, with or without the use of the sweeper, the level of cross-contamination to
adjacent areas outside the hot spot area "e" was approximately 10 times higher on the
asphalt surface compared to the concrete surface.
• The use of pAB and Dichlor formulations were found to be very effective in
decontaminating both the concrete and asphalt surfaces (no spores detected in the sweep
area). In addition, no spores were detected in the sweeper recovery tank following these
tests. However, spores did aerosolize and transfer to outside the sweep area, to the
negative control area.
67
-------
• Of the seven tests in which the sweeper was operated with only aqueous solutions (i.e.,
no sporicidal solutions), the average ± SD for the log CFU recovered from the sweeper
recovery tank was 7.4 ± 0.3. There was no difference in the average recoveries of spores
from the sweeper recovery tank for the two surfaces. The levels of spore recovery from
the sweeper recovery tank was generally the same order of magnitude as the positive
controls. Of the four tests utilizing sporicidal solutions, no spores were detected in the
sweeper recovery tank in any test.
• Following the reset of the test surface, the results for the negative samples from area "e"
and the negative control area outside the sweep area were non-detect for Bg spores
throughout the entire study, except for the first test. These negative samples were
typically collected on Monday following the reset of the test surface (on previous Friday)
using pAB applied with a backpack sprayer, but prior to the inoculation of the test area
"e".
• The results of the negative control area, when sampled after sweeping had taken place,
were positive for Bg spores in every test except Test 13 (Dichlor on asphalt). Log CFU
levels from these samples ranged from 1.6 - 4.6 and are indicative of the reaerosolization
of spores from the test area.
• All DFU air samples taken during surface sampling of the negative control areas were
negative.
• The results for the other DFU air samples were somewhat mixed but allowed for some
observation of trends (although it is not clear if these are significant effects). In terms of
both total CFU collected and CFU/m3, CFU results were generally highest during the
inoculation and overnight settling period, and lowest for the DFU samples taken during
the ADA removal and sweeping event. Also, the CFU collected during the inoculation
and overnight setting period were generally higher for the asphalt surface. Refer to Table
5-16.
68
-------
Table 5-15. Summary of Test Results
Test #
Surface
Decontaminant
Avg MDI
control (log
CFU)
Positive
control (log
CFU)
Log CFU
remaining
in sweep
area*
Removal
Efficiency
(%)
LR
Log CFU -
sweeper
recovery
tank
Log CFU - negative
control area after
sweeping
1
C
DI
7.84
7.39
4.82
99.73
2.57
7.52
NA
2
C
none
7.40
7.42
7.38
NA
NA
NA
NA
3
C
none, improved ADA
7.69
7.40
7.40
NA
NA
NA
1.96
4
C
military soap
7.65
7.50
5.30
99.38
2.21
6.78
1.90
5
C
dish soap
7.67
7.30
4.77
99.70
2.53
7.62
1.58
6
C
DI, 3 passes
7.63
7.33
4.12
99.94
3.21
7.66
3.4
7
C
pAB
7.56
7.33
ND
-
>7.33
ND
4.58
8
C
Dichlor
7.68
6.94
ND
-
>6.94
ND
4.10
9
A
none
7.51
7.23
7.29
NA
NA
NA
3.53
10
A
DI
7.57
7.23
6.59
77.40
0.64
7.31
3.08
11
A
dish soap
7.45
7.20
6.45
80.7
0.72
7.21
2.8
12
A
DI, 3 passes
7.61
7.33
6.75
74.0
0.59
7.57
1.8
13
A
Dichlor
7.48
7.10
ND
-
>7.1
ND
ND
14
A
pAB
7.58
7.19
ND
-
>7.19
ND
ND
Notes: all spore loadings from surfaces and sweeper reported in log CFU. ND = below detection limit. NA = not available, not applicable. DI = deionized water.
C = concrete. A = asphalt. *- Total log CFU from entire sweep/test area after sweeping
69
-------
Table 5-16. Air Sample Data Summary
Test
Surfac
e
Decontaminant
Via-
cell
CFU/
m3
DFU during
inoculation and
overnight settling (log
CFU)
DFU during
inoculation
and overnight
settling
(CFU/m3)
DFU during
ADA removal
and sweeping
(log CFU)
DFU during
ADA removal
and sweeping
(CFU/m3)
DFU during
floor sampling
(log CFU)
DFU during
floor sampling
(CFU/m3)
1
C
DI
ND
NA
NA
2.7
316
NA
NA
2
C
None
NA
NA
NA
NA
NA
3.2
98
3
C
None, improved ADA
NA
2.8
1.9
ND
6.7
2.0
11
4
C
military soap
NA
2.8
2.1
2.6
34
2.3
18
5
C
Dish soap
ND
5.2
79
2.5
80
4.0
8
6
C
DI, 3 passes
ND
3.8
14
3.0
107
3.1
121
7
C
pAB
ND
3.8
13
1.1
2
2.8
4.5
8
C
Dichlor
ND
3.8
13
2.3
25
2.8
60
9
A
None, improved ADA
NA
5.8
1450
NA
NA
2.9
64
10
A
DI
ND
5.1
335
2.8
114
3.7
390
11
A
Dish soap
12
5.2
306
2.5
35
4.0
904
12
A
DI, 3 passes
26
4.0
24
2.4
24
2.6
34
13
A
Dichlor
ND
5.1
250
ND
ND
3.3
249
14
A
pAB
ND
4.6
78
ND
ND
ND
ND
70
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6. Quality Assurance and Quality Control
All test activities were documented in laboratory notebooks and digital photographs. The
documentation included, but was not limited to, a record for each experiment, any deviations
from the quality assurance project plan, and physical impacts on materials. All tests were
conducted in accordance with established EPA Decontamination Technologies Research
Laboratory (DTRL) and BioLab procedures to ensure repeatability and adherence to the data
quality validation criteria set for this project.
The following sections discuss the measurement equipment calibration, the criteria for
the critical measurements and parameters, DQIs, and the quality assurance (QA) and quality
control (QC) checks for the project.
6.1 Measurement Equipment Calibration
The DTRL and Biolab have standard operating procedures for the maintenance and
calibration of all laboratory equipment. All equipment was verified as being certified, calibrated,
or by having the calibration validated by the EPA Air and Energy Management Division's
Metrology Laboratory at the time of use. Table 6-1 summarizes the instrument calibration
frequency.
Table 6-1. Instrument Calibration Frequency
Equipment
Calibration/Certification
Expected Tolerance
Thermometer
Compare to independent NIST thermometer (a thermometer recertified
annually by either NIST or an ISO-17025 facility) value once per quarter
±1 °C
Stop watch
Compare to official U.S. time at time.gov every 30 days
f 1 minute every 30 days
Micropipettes
Certified as calibrated at time of use; recalibrated by gravimetric
evaluation of performance to manufacturer's specifications every year
±5%
Scale
Compare reading to Class S weights
±1%
Serological Pipettes
Certified as calibrated at time of use; recalibrated by gravimetric
evaluation of performance to manufacturer's specifications every year
±5%
Air flow rate for Via-
Cell sampler
Via-Cell cassette will be connected to the Zefon Bio-pump and was
calibrated using an Air-o-cell flow meter (Zefon International, Ocala, FL)
± 20% of 15 L/min
Air flow rate for DFU
sampler
DFU velocity checked using a thermal anemometer; flow rate will be
calculated by multiplying velocity by the cross-sectional area of the
sampling tube
±20% of 800 L/min*
DFU = Dry Filter Unit ISO = International Organization for Standardization L/min = Liter per minute
NIST = National Institute of Standards and Technology
*800 LPM was the expected flow rate without the 1-micron filters installed, and was verified with the anemometer at the beginning
and end of study; flow rates with the filters installed and included in this report were typically < 300 LPM
Any deficiencies were noted in the laboratory notebook. The instrument was adjusted to
meet calibration tolerances and recalibrated within 24 hours. If tolerances were not met after
recalibration, additional corrective action were taken, including the replacement of the
equipment.
71
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6.2 Criteria for Critical Measurements and Parameters
The Data Quality Objectives (DQOs) were used to determine the critical measurements
needed to address the stated objectives and specify tolerable levels of potential error associated
with simulating the prescribed decontamination environments. The following measurements
were deemed to be critical to accomplish part or all project objectives:
• Liquid sample volume collected, from both the sweeper and wet-vacuum dirty tanks
• Aliquot volume (from liquid samples) to be analyzed by the Biolab
• Plated volume
• CFU counts
• Incubation temperature
• Area of inoculation (inoculated sweep area and positive control area)
• Area swept
• Areas that are wet-vacuum sampled (test area, negative control area, and positive
control area)
• Volume of air sampled and time for air sampling
Data quality indicator (DQI) goals for the critical measurements were used to determine
if the collected data met the research objectives. All the acceptance criteria for the critical
parameters listed in Table 6-2 were met for this project.
Table 6-2. Critical Measurement Acceptance Criteria
Critical Measurement
Measurement Device
Accuracy/ Precision
Acceptance Criteria
Volume
Serological pipette
+ 1 mL
+ 10% of target value
Temperature of Incubation
Chamber
NIST-traceable thermometer
+ 2 °C
Not applicable; standard
evaluations not performed for
this instrument
CFU Counts
QCount
50% RSD among the triplicate
plating
50% RSD among the triplicate
plating
Mass or volume of liquid
collected in both street sweeper
and wet vacuum
Weigh Scale
+ 0.1 pound
50% RSD between
test sets
Sampled Air Volume
Meter Box/Pump
5 % bias
Not applicable; standard
evaluations not performed for
this instrument
CFU = Colony forming unit L/min = Liter per minute L/min = Liter per minute mL= Milliliter
min/hour = Minute per hour NIST = National Institute of Standards and Technology
RSD = Relative standard deviation
If the CFU count for bacterial growth did not fall within the target range, the sample was
either filtered or replated. Filter plates were quantitatively analyzed (CFUs per plate) using a
manual counting method. For each set of results per test, a second count will be performed on
25% of the plates within the quantification range (plates with 30 to 300 CFUs).
72
-------
6.3 Integrity of Samples and Supplies
Samples were carefully maintained and preserved to ensure their integrity. Samples were
stored away from standards and other samples that could possibly cross-contaminate them.
Supplies and consumables were acquired from reputable sources and were National Institute of
Standards and Technology traceable whenever possible. Supplies and consumables were
examined for evidence of tampering or damage upon receipt and prior to use, as appropriate.
Supplies and consumables that showed evidence of tampering or damage were discarded. All
examinations were documented, and all supplies were appropriately labeled.
6.4 NHSRC Bio-laboratory Control Checks
Quantitative standards do not exist for biological agents. Viable spores were counted
using a QCount® colony counter. Counts generated that were either greater than 300 or less than
30 were considered outside of the targeted range. If the count of CFU for bacterial growth did
not fall within the target range, the sample was either filtered or re-plated. Re-plates and filter
plates were enumerated manually.
Before each batch of plates were enumerated on the QCount®, a QC plate was analyzed,
and the result was verified to be within the range indicated on the back of the QC plate. As the
plates were being counted, a visual inspection of colony counts made by the QCount® software
was performed. Obvious count errors made by the software were corrected by adjusting the
settings (e.g., colony size, light, and field of view) and recounting or by manually removing or
adding colonies as needed.
The acceptance criteria for the critical CFU measurements were set at the most stringent
level that could be achieved routinely. Inoculum and positive controls were included along with
the test samples in the experiments so that spore recovery from the different surface types could
be assessed. Background checks were also included as part of the standard protocol to check for
unanticipated contamination. Replicate coupons were included for each set of test conditions to
characterize the variability of the test procedures.
Additional QC samples were collected and analyzed to check the ability of the BioLab to
culture the test organism, as well as to demonstrate that materials used in this effort did not
contain spores. The checks included the following:
• Procedural blank coupons: Material coupons sampled in the same fashion as test
coupons but not contaminated with surrogate organism prior to sampling.
Additional QC checks for BioLab procedures are listed in Table 6-3. These provide
assurances against cross-contamination and other biases in microbiological samples.
73
-------
Table 6-3. Additional Quality Checks for Biological Measurements
Sample Type
Frequency
Acceptance Criteria
Information Provided
Corrective Action
Positive control coupon -
sample from material
coupon contaminated with
biological agent and
sampled using the existing
sampling methods
One per test
105 - 107 fori?, globigii
50% RSD between coupons in
each test set
Used to determine the extent
of inoculation on the target
coupon type
If outside range, Identify
and remove source of
variability if possible
Inoculum Control coupon -
stainless steel coupon
contaminated with
biological agent and
sampled using PRB wipes.
Minimum of
three per test
105 - 107 fori?, globigii
50% RSD between coupons in
each test set
Used to determine the extent
of inoculation on the target
coupon type
If outside range, Identify
and remove source of
variability if possible
Procedural blank
coupon without biological
agent that underwent the
sampling procedure
One per test
Non-detect
Controls for sterility of
materials and methods used
in the procedure
Analyze extracts from
procedural blank without
dilution; identify and
remove source of
contamination if possible
Blank tryptic soy agar
sterility control
Plate incubated but not
inoculated
Each plate
No observed growth after
incubation
Controls for sterility of
plates
All plates incubated before
use, so contaminated plates
discarded before use
Replicate plating of diluted
Microbiological Samples
Each sample
Reportable CFUs of triplicate
plates must be within 100 %.
Reportable CFUs are between
30 and 300 per plate
Used to determine the
precision of the replicate
plating
Re-plate sample
The QA/QC control test results (positive and negative controls) were listed for each test
in the Results section. Sterility checks were conducted for all the equipment and materials and
listed for each test in the result section.
In most of the procedural blank and the inoculum control blank samples, no Bg spores
were detected. Some control blanks were found to be contaminated, but they had little or no
effect on the final results. For negative controls, the contamination may have occurred by
incomplete inactivation of spores during VHP cycle.
With respect to the wet vacuum control blanks (negative controls, sampled after the reset
but prior to inoculation), all the negative controls taken outside of the test/sweep area were non-
detect except for the first test. All the wet vacuum negative controls taken of the hot spot area
were non-detect for Bg. These results demonstrate the effectiveness of the reset/sterilization
process.
The negative control area outside the sweep area was sampled twice in each test: after the
reset of the floor, prior to inoculation (allowing to dry over the weekend); and after the sweeper
was operated. Every negative sample prior to inoculation, except for testing DI water on concrete
(Test 1), was non-detect for spores. For the negative samples taken after the sweeper was
operated, in every test this sample was positive for Bg spores, except when Dichlor and pAB
were applied on asphalt. These results were expected and demonstrate the reaerosolization of
spores from the test area and their migration to an area outside the test area. Following Test 2,
the hot spot area was also sampled prior to inoculation, as an additional negative control (to
74
-------
check for the reset effectiveness); in all cases, this sample was non-detect for Bg spores.
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U.S. Environmental Protection Agency (EPA). 2007. "Guidance on Test Methods for
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Stormwater Management in the United States." The National Academies Press,
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Environmental Surfaces Contaminated with Bacterial Spores: Evaluation of Spray
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Acidified Bleach Solutions to Improve Sporicidal Efficacy on Building Materials. Letters
in Applied Microbiology. 2011, 53(6): 668-72
77
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Appendix A: Operational Summary Logs for Each Test
Test 2: Cross-Contamination Assessment on Concrete Surface using a Standard
ADA
HOBO Operation (During Sampling)
Start Time (mm/dd/yyyy; hh:mm)
Start Time
(hh: mm)
Run Time
(hh:mm: ss)
Average
Temperature
Average RH (%)
03/15/2017
03/15/2017
00:65:00
46
26
14:52
13:45
Wet Vacuum Sampling Operat
ions
Sample location
Volume
dispensed (1)
Volume
Recovered (1)
Areas Sampled
(ft2)
Volume sprayed per
unit area (ml/ft2)
Negative Control (Pre-inoculation)
500
275
9.00
55.6
Test Area - a
825
520
20.25
40.7
Test Area - b
530
270
13.50
39.3
Test Area - c
675
430
20.25
33.3
Test Area - d
540
315
13.50
40
Test Area - e
585
420
9.00
65
Test Area - f
600
345
13.50
44.4
Test Area - g
710
485
20.25
35.1
Test Area - h
470
285
13.50
34.8
Test Area - i
765
505
20.25
37.8
Positive Control
715
445
9.00
79.4
Average
629
391
14.7
45.9
Stdev
111
91.9
4.54
14.0
A-1
-------
Test 3: Cross-Contamination Assessment on Concrete Surface using an Improved
ADA
DFUO
jeration
Sample Type
Run Time (min)
Velocity (m/s)
Flow Rate
(1/min)
Collected Volume
(m3)
DFU Negative Sampling
10
1.76
296
2.96
DFU Inoculation and Overnight
1152
1.68
282
325
DFU during ADA Removal
3
1.89
317
0.952
DFU during Wet Vacuuming
27
1.97
331
8.93
HOBO Operation
Start Time (mm/dd/yyyy; hh:mm)
Start Time (hh:
mm)
Run Time
(hh:mm: ss)
Average
Temperature
Average RH (%)
05/08/2017
5/10/2017
43:58:00
60.1
68.3
14:52
10:40
Wet Vacuum Sampling Operations
Sample location
Volume
dispensed (mL)
Volume
Recovered (mL)
Areas
Sampled (ft2)
Volume sprayed per
unit area (mL/ft2)
Negative Control (Pre-inoculation)
490
185
9.00
23.8
Negative Control (Post-inoculation)
535
390
9.00
12.3
Negative Control Area-e (Pre-
inoculation
870
575
9.00
13.6
Test Area - a
950
630
20.3
30.5
Test Area - b
1000
465
13.5
29.0
Test Area - c
920
510
20.2
36.5
Test Area - d
740
375
13.5
26.6
Test Area - e
680
515
6.25
8.3
Test Area - f
895
470
13.5
25.7
Test Area - g
945
795
795
24.1
Test Area - h
930
715
13.5
17.6
Test Area - i
930
670
20.3
28.1
Positive Control
685
485
9.00
12.7
Average
813
522
13.6
22.2
Stdev
162
154
4.93
8.19
A-2
-------
Test 9: Cross-Contamination Assessment on Asphalt Surface using an Improved
ADA
DFU Operation
Sample Type
Run Time (min)
Velocity (m/s)
Flow Rate
(1/min)
Collected Volume (m3)
DFU Negative Sampling
85
2.12
356
30.3
DFU during inoculation/and settling of
the spores overnight Sampling
1225
1.92
322
394
DFU during Wet Vacuuming
40
1.90
319
12.8
HOBO Operation
Start Time (mm/dd/yyyy; hh:mm)
Start Time (hh:
mm)
Run Time
(hh:mm: ss)
Average
Temperature
(°F)
Average RH (%)
11/06/2017
11/08/2017
46:40:00
60.1
68.3
12:30
11:10
Wet Vacuum Sampling Operations
Sample location
Volume
dispensed (mL)
Volume
Recovered (mL)
Areas Sampled
(ft2)
Volume sprayed per
unit area (ml/ft2)
Negative Control (Pre-inoculation)
520
590
9.00
57.8
Negative Control (Post-inoculation)
685
475
9.00
76.1
Negative Control Area-e (Pre-
inoculation)
580
515
9.00
64.4
Test Area - a
895
725
20.25
44.2
Test Area -b
905
640
13.50
67.0
Test Area - c
830
460
20.25
41.0
Test Area - d
770
540
13.50
57.0
Test Area - e
840
545
6.25
134.4
Test Area - f
855
665
13.50
63.3
Test Area - g
955
730
20.25
47.2
Test Area - h
825
575
13.50
61.1
Test Area - i
945
645
20.25
46.7
Positive Control
635
425
9.00
70.6
Average
788
579
13.6
63.9
Stdev
141
98.0
5.1
23.7
A-3
-------
Test 1: Removal Efficacy Determination for DI Water on Concrete (1-Pass Sweep)
Street Sweeper
Initial Volume in the Clean Tank
Liquid Sprayed
Volume
Dispensed (1)
Volume
Recovered (1)
Volume Recovered
(%)
50
Clean DI water
8
5.265
66
Rinsate
1.945
DFU Operation
Sample Type
Run Time (min)
Velocity (m/s)
Flow Rate
(1/min)
Collected Volume
(m3)
DFU during the Sweeping Process
6
1.84
308.93
1.85
Via-cell Operation
Strat Time (hh:mm)
End Time
(hh:mm)
Run Time (min)
Flow Rate
(lpm)
Collected Volume
(m3)
10:51
10:57
6
15
0.09
HOBO Operation
Start Time (mm/dd/yyyy; hh:mm)
Start Time (hh:
mm)
Run Time
(hh:mm)
Average
Temperature
Average RH
(%)
11/06/2017
11/08/2017
46:40
60
68
12:30
11:10
Wet Vacuum Sampling Operations
Sample location
Volume
dispensed (mL)
Volume
Recovered (mL)
Areas
Sampled (ft2)
Volume sprayed per
unit area (ml/ft2)
Negative Control (Pre-inoculation)
535
320
9.00
59
Test Area - a
565
285
20.3
28
Test Area -b
540
310
13.50
40
Test Area - c
475
300
20.25
23
Test Area - d
590
360
13.50
44
Test Area - e
435
260
9.00
48
Test Area - f
530
350
13.50
39
Test Area - g
475
280
20.25
23
Test Area - h
480
320
13.50
36
Test Area - i
615
370
20.25
30
Positive Control
395
215
9.00
44
Average
511
306
14.72
38
Stdev
64.4
43.6
4.54
11
A-4
-------
Test 6: Removal Efficacy Determination for DI Water on Concrete (3-Passes Sweep)
Street Sweeper
Initial Volume in the Clean Tank
Liquid Sprayed
Volume
Dispensed (1)
Volume
Recovered (1)
Volume Recovered
(%)
50
Clean DI water
14.5
11.01
69
Rinsate
2.74
DFU Operation
Sample Type
RunTime (min)
Velocity (m/s)
Flow Rate
(1/min)
Collected Volume
(m3)
DFU during pre-inoculation sampling
14
2.05
344.19
4.819
DFU during inoculation/and settling of
the spores overnight
1152
2.06
345.87
398.441
DFU during the ADA removal and
sweeping process
30
2.05
344.19
10.326
DFU during Wet Vacuuming
34
2.00
335.96
11.423
Via-cell Operation
Strat Time (hh:mm)
End Time
(hh:mm)
RunTime (min)
Flow Rate
(1pm)
Collected Volume
(m3)
09:10
09:20
10
15
0.09
HOBO Operation
Start Time (mm/dd/yyyy; hh:mm)
Start Time (hh:
mm)
Run Time
(hh:mm)
Average
Temperature
Average RH
(%)
06/26/2017
06/29/2017
44:00
79
60
14:00
10:00
Wet Vacuum Sampling Operations
Sample location
Volume
dispensed (mL)
Volume
Recovered (mL)
Areas
Sampled (ft2)
Volume sprayed per
unit area (ml/ft2)
Negative Control (Pre-inoculation)
650
385
9
42.8
Test Area - e (Pre-inoculation)
360
255
9
28.3
Negative Control (Post treatment)
600
325
9
36.1
Test Area - a
895
665
20.3
31.6
Test Area - b
600
355
13.50
49.6
Test Area - c
720
46
20.25
35.1
Test Area - d
615
395
13.50
46.7
Test Area - e
500
235
6.25
67.2
Test Area - f
755
475
13.50
51.9
Test Area - g
680
400
20.25
37.3
Test Area - h
615
320
13.50
57.4
Test Area - i
620
350
20.25
43.0
Positive Control
525
200
9.00
71.7
Average
626
339
13.85
26.3
Stdev
129
147
4.85
10.1
A-5
-------
Test 10: Removal Efficacy Determination for DI Water on Asphalt (1-Pass Sweep)
Street Sweeper
Initial Volume in the Clean Tank
Liquid Sprayed
Volume
Dispensed (1)
Volume
Recovered (1)
Volume Recovered
(%)
50
Clean DI water
7.5
3.535
47
Rinsate
4.096
DFU Operation
Sample Type
RunTime (min)
Velocity (m/s)
Flow Rate
(1/min)
Collected Volume
(m3)
DFU during pre-inoculation sampling
35
2.073
348.05
12.182
DFU during inoculation/and settling of
the spores overnight
1255
2.032
341.17
428.165
DFU during the ADA removal and
sweeping process
17
2.006
336.80
5.726
DFU during Wet Vacuuming
33
2.098
352.25
11.624
Via-cell Operation
Strat Time (hh:mm)
End Time
(hh:mm)
RunTime (min)
Flow Rate
(1pm)
Collected Volume
(m3)
10:52
10:57
5
15
0.075
HOBO Operation
Start Time (mm/dd/yyyy; hh:mm)
Start Time (hh:
mm)
Run Time
(hh:mm)
Average
Temperature
Average RH
(%)
11/27/2017
11/29/2017
46:40
51
53
13:00
11:40
Wet Vacuum Sampling Operations
Sample location
Volume
dispensed (mL)
Volume
Recovered (mL)
Areas
Sampled (ft2)
Volume sprayed per
unit area (ml/ft2)
Negative Control (Pre-inoculation)
620
395
9
68.9
Test Area - e (Pre-inoculation)
730
415
9
77.8
Negative Control (Post treatment)
700
520
9
81.1
Test Area - a
640
465
20.3
31.6
Test Area - b
670
480
13.50
49.6
Test Area - c
710
555
20.25
35.1
Test Area - d
630
425
13.50
46.7
Test Area - e
605
460
6.25
67.2
Test Area - f
700
545
13.50
51.9
Test Area - g
755
620
20.25
37.3
Test Area - h
775
580
13.50
57.4
Test Area - i
870
705
20.25
43.0
Positive Control
645
405
9.00
71.7
Average
696
498
13.85
55.3
Stdev
81
94
4.85
16.0
A-6
-------
Test 12: Removal Efficacy Determination for DI Water on Asphalt (3-Pass Sweep)
Street Sweeper
Initial Volume in the Clean Tank
Liquid Sprayed
Volume
Dispensed (1)
Volume
Recovered (1)
Volume Recovered
(%)
50
Clean DI water
15.1
11.82
85
Rinsate
1.02
DFU Operation
Sample Type
RunTime (min)
Velocity (m/s)
Flow Rate
(1/min)
Collected Volume
(m3)
DFU during pre-inoculation sampling
30
2.04
342.51
10.275
DFU during inoculation/and settling of
the spores overnight
1290
2.01
337.41
435.341
DFU during the ADA removal and
sweeping process
29
2.04
342.51
9.933
DFU during Wet Vacuuming
35
1.97
330.76
11.577
Via-cell Operation
Strat Time (hh:mm)
End Time
(hh:mm)
RunTime (min)
Flow Rate
(1pm)
Collected Volume
(m3)
10:13
10:24
9
15
0.09
HOBO Operation
Start Time (mm/dd/yyyy; hh:mm)
Start Time (hh:
mm)
Run Time
(hh:mm)
Average
Temperature
Average RH
(%)
0/14/2018
2/16/2018
48:20
58
62
11:05
11:25
Wet Vacuum Sampling Operations
Sample location
Volume
dispensed (mL)
Volume
Recovered (mL)
Areas
Sampled (ft2)
Volume sprayed per
unit area (ml/ft2)
Negative Control (Pre-inoculation)
395
290
9
32.2
Test Area - e (Pre-inoculation)
560
360
9
40.0
Negative Control (Post treatment)
575
296
9
32.9
Test Area - a
710
595
20.3
29.4
Test Area - b
640
520
13.50
38.5
Test Area - c
510
365
20.25
18.0
Test Area - d
600
265
13.50
19.6
Test Area - e
610
415
6.25
46.1
Test Area - f
515
415
13.50
30.7
Test Area - g
430
330
20.25
16.3
Test Area - h
705
480
13.50
35.6
Test Area - i
745
605
20.25
29.9
Positive Control
580
338
9.00
37.2
Average
583
405
13.85
31.3
Stdev
104
113
4.85
8.88
A-7
-------
Test 5: Removal Efficacy Determination For Dawn® Dishwashing Solution
Formulation On Concrete
Street Sweeper
Initial Volume in the Clean Tank (L)
Liquid Sprayed
Volume
Dispensed (1)
Volume
Recovered (1)
Volume Recovered
(%)
12
Dishwashing
Solution
8.5
5.87
69
Rinsate
3.135
DFU Operation
Sample Type
Run Time (min)
Velocity (m/s)
Flow Rate
(1/min)
Collected Volume
(m3)
DFU during pre-inoculation sampling
10
2.002
336.05
3.361
DFU during inoculation/and settling of
the spores overnight
1152
1.9609
329.23
379.269
DFU during the ADA removal and
sweeping process
24
2.002
336.05
8.065
DFU during Wet Vacuuming
47
2.002
336.05
15.794
HOBO C
Operation
Start Time (mm/dd/yyyy; hh:mm)
Start Time (hh:
mm)
Run Time
(hh:mm)
Average
Temperature
Average RH
(%)
6/12/2017
6/14/2017
41:18
91
83
14:52
10:10
Wet Vacuum Sampling Operations
Sample location
Volume
dispensed (mL)
Volume
Recovered (mL)
Areas Sampled
(ft2)
Volume sprayed
per unit area
(ml/ft2)
Negative Control (Pre-inoculation)
775
470
9
63.9
Test Area - e (Pre-inoculation)
510
340
9
75.6
Negative Control (Post treatment)
365
170
9
48.9
Test Area - a
720
395
20.3
45.4
Test Area - b
680
365
13.50
47.0
Test Area - c
665
370
20.25
39.0
Test Area - d
875
445
13.50
46.3
Test Area - e
475
265
6.25
80
Test Area - f
625
420
13.50
47.4
Test Area - g
760
455
20.25
45.2
Test Area - h
560
355
13.50
53.7
Test Area - i
618
395
20.25
45.9
Positive Control
505
255
9.00
69.4
Average
626
362
13.85
28.0
Stdev
141
87.1
4.85
9.46
A-8
-------
Test 11: Removal Efficacy Determination for Dawn® Dishwashing Solution
Formulation on Asphalt
Street Sweeper
Initial Volume in the Clean Tank (L)
Liquid Sprayed
Volume
Dispensed (1)
Volume
Recovered (1)
Volume
Recovered (%)
12
Dishwashing
Solution
7.2
4.055
64
Rinsate
4.902
DFU Operation
Sample Type
Run Time (min)
Velocity (m/s)
Flow Rate
(1/min)
Collected Volume
(m3)
DFU during pre-inoculation sampling
85
2.12
355.94
30.255
DFU during inoculation/and settling of
the spores overnight
1459
2.14
359.30
524.219
DFU during the ADA removal and
sweeping process
23
2.18
366.02
8.418
DFU during Wet Vacuuming
33
2.14
359.30
11.857
HOBO C
Operation
Start Time (mm/dd/yyyy; hh:mm)
Start Time (hh:
mm)
Run Time
(hh:mm)
Average
Temperature (°F)
Average RH
(%)
12/26/2017
12/28/2017
48:20
38
51
11:05
11:25
Wet Vacuum Sampling Operations
Sample location
Volume
dispensed (mL)
Volume
Recovered (mL)
Areas Sampled
(ft2)
Volume sprayed
per unit area
(ml/ft2)
Negative Control (Pre-inoculation)
868
358
9
39.8
Test Area - e (Pre-inoculation)
845
462
9
51.3
Negative Control (Post treatment)
604
430
9
47.8
Test Area - a
877
594
20.3
29.3
Test Area - b
723
462
13.50
34.2
Test Area - c
736
467
20.25
23.1
Test Area - d
677
498
13.50
36.9
Test Area - e
609
362
6.25
40.2
Test Area - f
591
384
13.50
28.4
Test Area - g
868
584
20.25
28.8
Test Area - h
650
430
13.50
31.9
Test Area - i
922
652
20.25
32.2
Positive Control
704
462
9.00
51.3
Average
744
473
13.85
36.6
Stdev
118
90.0
4.85
9.10
A-9
-------
Test 4: Removal Efficacy Determination for SSDX-12™ Solution on Concrete
Wet Vacuum Sampling Operations
Sample location
Volume
dispensed (mL)
Volume
Recovered (mL)
Areas Sampled
(ft2)
Volume sprayed
per unit area
(ml/ft2)
Negative Control (Pre-inoculation)
575
270
9
30.0
Test Area - e (Pre-inoculation)
680
435
9
48.3
Negative Control (Post treatment)
440
360
9
40.0
Test Area - a
920
635
20.3
31.4
Test Area - b
635
420
13.50
31.1
Test Area - c
790
480
20.25
23.7
Test Area - d
625
380
13.50
28.1
Test Area - e
720
465
6.25
51.7
Test Area - f
640
400
13.50
29.6
Test Area - g
915
635
20.25
31.4
Test Area - h
725
495
13.50
36.7
Test Area - i
930
605
20.25
29.9
Positive Control
625
355
9.00
39.4
Average
709
457
13.85
34.7
Stdev
147
113
4.85
8.16
A-10
-------
Test 7: Log-Reduction Efficacy Determination for pAB Formulation on Concrete
Street Sweeper
Initial Volume in the Clean Tank (L)
Liquid Sprayed
Volume
Dispensed (1)
Volume
Recovered (1)
Volume
Recovered (%)
12
pAB Solution
7.5
4.65
62
DFU Operation
Sample Type
Run Time (min)
Velocity (m/s)
Flow Rate
(1/min)
Collected Volume
(m3)
DFU during pre-inoculation sampling
14
2.16
362.49
5.075
DFU during inoculation/and settling of
the spores overnight
1375
1.92
322.36
443.249
DFU during the ADA removal and
sweeping process
22
1.91
320.68
7.055
DFU during Wet Vacuuming
39
1.93
324.04
12.678
HOBO Operation
Start Time (mm/dd/yyyy; hh:mm)
Start Time (hh:
mm)
Run Time
(hh:mm)
Average
Temperature (°F)
Average RH
(%)
7/18/2017
7/20/2017
48:24
91
74.5
10:36
11:00
Wet Vacuum Sampling Operations
Sample location
Volume
dispensed (mL)
Volume
Recovered (mL)
Areas Sampled
(ft2)
Volume sprayed
per unit area
(ml/ft2)
Negative Control (Pre-inoculation)
500
325
9
36.1
Test Area - e (Pre-inoculation)
555
305
9
33.9
Negative Control (Post treatment)
485
255
9
28.3
Test Area - a
755
435
20.3
36.11
Test Area - b
405
200
13.50
28.33
Test Area - c
690
430
20.25
33.89
Test Area - d
390
235
13.50
21.48
Test Area - e
530
275
6.25
14.81
Test Area - f
510
225
13.50
21.23
Test Area - g
665
315
20.25
17.41
Test Area - h
550
345
13.50
30.56
Test Area - i
810
560
20.25
16.67
Positive Control
595
325
9.00
15.56
Average
572
473
13.85
25
Stdev
127
90.0
4.85
7.76
A-11
-------
Test 14: Log-Reduction Efficacy Determination for pAB Formulation on Asphalt
Street Sweeper
Initial Volume in the Clean Tank (L)
Liquid Sprayed
Volume
Dispensed (1)
Volume
Recovered (1)
Volume
Recovered (%)
12
pAB Solution
7.5
4.93
66
DFU Operation
Sample Type
Run Time (min)
Velocity (m/s)
Flow Rate
(1/min)
Collected Volume
(m3)
DFU during pre-inoculation sampling
13
2.09
351.49
4.568
DFU during inoculation/and settling of
the spores overnight
1337
2.13
358.29
479.038
DFU during the ADA removal and
sweeping process
27
2.17
365.01
9.855
DFU during Wet Vacuuming
21
2.12
355.61
7.468
HOBO Operation
Start Time (mm/dd/yyyy; hh:mm)
Start Time (hh:
mm)
Run Time
(hh:mm)
Average
Temperature (°F)
Average RH
(%)
3/27/2018
3/28/2017
27:50
53
64
8:45
12:35
Wet Vacuum Sampling Operations
Sample location
Volume
dispensed (mL)
Volume
Recovered (mL)
Areas Sampled
(ft2)
Volume sprayed
per unit area
(ml/ft2)
Negative Control (Pre-inoculation)
675
205
9
22.8
Test Area - e (Pre-inoculation)
600
295
9
32.8
Negative Control (Post treatment)
550
305
9
33.9
Test Area - a
685
420
20.3
20.74
Test Area - b
715
335
13.50
24.81
Test Area - c
550
300
20.25
14.81
Test Area - d
535
240
13.50
17.78
Test Area - e
640
260
6.25
28.89
Test Area - f
490
350
13.50
25.93
Test Area - g
535
290
20.25
14.32
Test Area - h
555
250
13.50
18.52
Test Area - i
525
320
20.25
15.80
Positive Control
515
320
9.00
35.56
Average
582
299
13.85
23.6
Stdev
73.0
54.6
4.85
7.42
A-12
-------
Test 8: Log-Reduction Efficacy Determination for Dichlor Solution on Concrete
Street Sweeper
Initial Volume in the Clean Tank (L)
Liquid Sprayed
Volume
Dispensed (1)
Volume
Recovered (1)
Volume
Recovered (%)
12
Dichlor Solution
7
5.24
75
DFU Operation
Sample Type
Run Time (min)
Velocity (m/s)
Flow Rate
(1/min)
Collected Volume
(m3)
DFU during pre-inoculation sampling
15
2.15
360.81
5.412
DFU during inoculation/and settling of
the spores overnight
1423
1.90
318.84
453.705
DFU during the ADA removal and
sweeping process
22
2.05
344.53
7.5280
DFU during Wet Vacuuming
28
2.02
338.65
9.482
HOBO Operation
Start Time (mm/dd/yyyy; hh:mm)
Start Time (hh:
mm)
Run Time
(hh:mm)
Average
Temperature (°F)
Average RH
(%)
8/01/2017
8/3/2017
49:25
84
68
9:45
11:10
Wet Vacuum Sampling Operations
Sample location
Volume
dispensed (mL)
Volume
Recovered (mL)
Areas Sampled
(ft2)
Volume sprayed
per unit area
(ml/ft2)
Negative Control (Pre-inoculation)
435
315
9
35.0
Test Area - e (Pre-inoculation)
555
365
9
40.6
Negative Control (Post treatment)
895
365
9
33.3
Test Area - a
510
250
20.3
12.35
Test Area - b
365
135
13.50
10.00
Test Area - c
515
245
20.25
12.10
Test Area - d
460
225
13.50
16.67
Test Area - e
565
280
6.25
31.11
Test Area - f
415
180
13.50
13.33
Test Area - g
525
295
20.25
14.57
Test Area - h
520
260
13.50
19.26
Test Area - i
500
315
20.25
15.56
Positive Control
945
505
9.00
56.11
Average
554
282
13.85
23.8
Stdev
172
90.1
4.85
14.1
A-13
-------
Test 13: Log-Reduction Efficacy Determination for Dichlor Solution on Asphalt
Street Sweeper
Initial Volume in the Clean Tank (L)
Liquid Sprayed
Volume
Dispensed (1)
Volume
Recovered (1)
Volume
Recovered (%)
12
Dichlor Solution
7
4.815
69
DFU Operation
Sample Type
Run Time (min)
Velocity (m/s)
Flow Rate
(1/min)
Collected Volume
(m3)
DFU during pre-inoculation sampling
17
2.08
349.73
5.945
DFU during inoculation/and settling of
the spores overnight
1300
2.11
353.93
460.106
DFU during the ADA removal and
sweeping process
31
2.17
365.01
11.315
DFU during Wet Vacuuming
24
2.23
374.41
8.986
HOBO Operation
Start Time (mm/dd/yyyy; hh:mm)
Start Time (hh:
mm)
Run Time
(hh:mm)
Average
Temperature (°F)
Average RH
(%)
2/27/2018
2/28/2018
47:00
47
57
7:45
12:45
Wet Vacuum Sampling Operations
Sample location
Volume
dispensed (mL)
Volume
Recovered (mL)
Areas Sampled
(ft2)
Volume sprayed
per unit area
(ml/ft2)
Negative Control (Pre-inoculation)
525
200
9
22.2
Test Area - e (Pre-inoculation)
360
280
9
31.1
Negative Control (Post treatment)
495
270
9
30.0
Test Area - a
550
250
430
21.2
Test Area - b
630
135
395
29.3
Test Area - c
565
245
410
20.2
Test Area - d
470
225
375
27.8
Test Area - e
505
280
210
23.3
Test Area - f
425
180
340
25.2
Test Area - g
650
295
400
19.8
Test Area - h
540
260
300
22.2
Test Area - i
815
315
600
29.6
Positive Control
520
505
330
36.7
Average
542
349
13.85
26.0
Stdev
112
107
4.85
5.12
A-14
-------
Appendix B: Market Summary of Walk-Behind Street Sweeper Specifications
Brand and
Model Number
Manufacturer
Location
Cleaning Path
Weight (lb)
Filtration
Efficiencv
Volume
Canacitv
Battery Type
and Power
Tennant S10
32 in Single
Side Brash
599
99% at 3
urn
79
24
Tennant S9
Tennant
Minneapolis,
MN
35 in Dual
Side Brash
300
99.97% at
0.3 urn
60
12
Tennant S5
Company
24 in Single
Side Brash
121
99.97% at
0.3 urn
37
12
Tennant T5
26"
Cylindrical
621
N/A - Wet
Vacuum
85
24
Tennant 3640
32 in Single
Side Brash
385
92% at 0.3
um
45
24
Dulevo 52EH
Dulevo
International
Fontanellato,
Italy
35 in Dual
Side Brash
199
90% at 2.5
um
40
12
Minuteinan
Minuteinan
Pingree
28 in Single
269
99.97% at
35
12
KS28
International
Grove, IL
Side Brash
3 um
Minuteman
Minuteinan
Pingree
25 in Single
269
99.97% at
30
24
KS25W
International
Grove, IL
Side Brash
3 um
PowerBoss
HM34BQP
PowerBoss
Pingree
Grove, IL
35 in Single
Side Brash
364
99.97% at
3 um
50
12
Dust Max
DM2800
Industrial Air
Raleigh, NC
28 in Single
165
0.2 um
HEPA
50
12
Dust Max
DM3600
Solutions
Side Brash
187
0.2 um
HEPA
65
12
B-1
-------
Appendix C. Equipment Sterilization
Prior to use, test equipment (street sweeper, wet vacuum samplers, ADAs, and backpack
sprayers) was sterilized using Vaporized Hydrogen Peroxide® (VHP). The hydrogen peroxide
(H2O2) vapor was generated using a STERIS VHP 1000ED system loaded with a 35% H2O2
Vaprox® cartridge. Each sterilization cycle lasted 4 hours at 250 ppm VHP. Cycle efficacy was
verified through the routine use of Biological Indicators (Bis) charged with a minimum of 1 x 106
spores of Geobacillus stearothermophilus (Apex Biological Indicators, Mesa Labs, Bozeman,
MT).
Following each test, a strict and specific reset process was employed for each piece of test
equipment, as described in the next sections:
Street Sweeper Sterilization
• Detach the street sweeper spent brush and squeegee
• Flush 100L of sterile DI water (autoclave sterilized) through the sweeper at least two (2)
times to clean the internal plumbing and remove residual spores. Collect the rinse from this
process and dispose through Chemical Services.
• Remove dirt and debris from the street sweeper wheels by wiping them with a wipe
(Kimberly-Clark wipes, Catalog No. 06-666-1 ID, Fisher scientific, Hampton, NH) wetted
with 70% ethyl alcohol. One (1) liter of 70 % ethyl alcohol was prepared by mixing 700-
ml of Ethyl Alcohol 200 Proof (Pharmco-Aaper, Brookfield, CT, CAS No. 64-17-5) with
300-ml ofDI water.
• Transfer the street sweeper, along with a new brush and a new squeegee, into the airlock
of the Consequence Management and Decontamination Evaluation Room
(COMMANDER), located in the EPA/RTP High Bay Area. The COMMANDER consists
of a stainless steel-lined inner chamber built specifically for decontamination testing, with
internal dimensions of approximately 3.4 m wide by 2.5 m deep by 2.8 m high. The airlock
compartment was used for the VHP sterilization process occurs.
• Wrap the sweeper and ancillary equipment in sterile VHP bags and transport them to the
test site for decontamination testing.
Wet Vacuum Sampler Sterilization
• Fill the "Clean" tank of each wet vacuum sampler with 1 L of pAB.
• Run the wet vacuum sampler to flush the pAB into the "dirty" tank. Dispose of the pAB
collected in the "dirty" tank through Chemical Services.
• Repeat this process two (2) times, using DI water instead of pAB.
• Wrap each vacuum sampler in sterile VHP bags, with the nozzle flap open to allow VHP
inside the equipment and place them into the COMMANDER airlock for VHP sterilization.
• Run the VHP sterilization cycle.
• Keep the sterilized wet vacuum samplers outside the testing area, and do not open the VHP
bags until sampling is started.
C-1
-------
Back pack sprayer Sterilization
• Remove the batteries from the backpack sprayer. Set these aside as they will not undergo
sterilization.
• Wrap each backpack sprayer, with the tank lid open, in a sterile VHP bag. Transfer the
wrapped sprayers into the COMMANDER airlock for VHP sterilization.
• Run the VHP sterilization. Fill the tank with pAB or DI water, as appropriate for the
intended use.
DFU Sterilization
• Wipe the DFU exterior with Dispatch® Bleach Wipes (Clorox Co., Oakland, CA),
followed by a wipe (Kimberly-Clark wipes, Catalog No. 06-666-1 ID, Fisher scientific,
Hampton, NH) wetted with 70% ethyl alcohol.
Stainless Steel MDI Control Coupon Sterilization
• Wrap each stainless- steel coupon (0.020-in thickness (McMaster-Carr) with aluminum foil
and sterilize at 121 °C on a 1-hr gravity cycle in a Steris Amsco Century SV 120 Scientific
Pre-Vacuum Sterilizer (STERIS Corporation, Mentor, USA). The 1-hour cycle is
recommended for inactivation of gram positive spore-forming bacteria. Stainless steel
coupon sterility was evaluated by swab sampling and PRB wipe sampling.
DI-water/Tween Sterilization
• The DI-water/Tween, used in the Hoover wet vacuum samplers, was sterilized in 20-liter
heavy-duty autoclavable Nalgene plastic polypropylene carboy containers, (U.S.
Pharmacopeia Convention (USP) class VI, vacuum-rated for intermittent vacuum use only,
83B closure size, Fisher Catalog No. 02-690-23). A sterility check for the DI water/Tween
was evaluated using an aliquot sample.
C-2
-------
vvEPA
United States
Environmental Protection
Agency
PRESORTED STANDARD
POSTAGE & FEES PAID
EPA
PERMIT NO. G-35
Office of Research and Development (8101R)
Washington, DC 20460
Official Business
Penalty for Private Use
$300
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