EPA/600/R-13/265 | December 2013 | www.epa.gov/ord
United States
Environmental Protection
Agency
Determination of the Difference in
Reaerosolization of Spores off
Outdoor Materials
Interim Report
Ml
Office of Research and Development
National Homeland Security Research Center
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EPA /600-R13-265
December 2013
Determination of the Difference in
Reaerosolization of Spores off Outdoor Materials
Interim Report
National Homeland Security Research Center
Office of Research and Development
U.S. Environmental Protection Agency
Research Triangle Park, NC 27711
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Disclaimer
The United States Environmental Protection Agency, through its Office of Research and Development's
National Homeland Security Research Center, funded and managed this investigation through contract
EP-D-10-070 WA 3-19 with Alion Science and Technology. Funding was received from the Interagency
Agreement HSHQPM-12-X-00118 P00001 between the Environmental Protection Agency and the
Department of Homeland Security. 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 the Environmental Protection Agency
approves or endorses the product or its manufacturer. The Environmental Protection Agency does not
endorse the purchase or sale of any commercial products or services.
Questions concerning this document or its application should be addressed to:
Russell W. Wiener, Ph.D.
Decontamination and Consequence Management Division
National Homeland Security Research Center
U.S. Environmental Protection Agency (MD-D205-03)
Office of Research and Development
109 T.W. Alexander Drive
Research Triangle Park, NC 27709
Phone:919-541-1910
Fax:919-541-0496
E-mail: wiener.russell@epa.gov
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Acknowledgments
The authors would like to acknowledge the support of Alion Science and Technology, funded under EPA
contract EP-D-10-070 and from Interagency Agreement HSHQPM-12-X-00118 P00001 between EPA
and the Department of Homeland Security. In addition the authors would like to acknowledge the
contributions of Arcadis U.S., Inc. for microbiological support and Booz Allen Hamilton Inc. for program
support. The quality assurance review by Joan Bursey is also appreciated.
The contributions of the interagency Scientific Program on Reaerosolization and Exposure (SPORE)
team members listed below are also acknowledged:
DHS:
Donald Bansleben
Matthew Moe
Department of Defense:
K. Wing Tsang
Jeffrey Hogan
Angelo Madonna
EPA:
Shawn Ryan
Marshall Gray
Worth Calfee
Joseph Wood
Sangdon Lee
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Table of Contents
Disclaimer ii
Acknowledgments iii
List of Figures v
List of Tables vi
List of Acronyms and Abbreviations vii
Executive Summary ix
1 Introduction 1
2 Experimental Materials and Methods 3
2.1 Reaerosolization Wind Tunnel 3
2.2 Deposition Chambers 7
2.2.1 Wet Deposition Chamber 7
2.2.2 Dry Deposition Chamber 8
2.3 Test Surrogates 10
2.3.1 Bacillus thuringiensis var. kurstaki 10
2.3.2 Bacillus atrophaeus subspecies globigii 11
2.3.3 Fluorescent Polystyrene Latex Spheres 12
2.4 Test Surface Characterization 13
2.4.1 Hygroscopicity 14
2.4.2 Surface Tension 14
2.4.3 Surface Roughness 14
2.4.4 Surface Area 15
2.5 Test Protocol 16
3 Results and Discussion 18
3.1 Scouting Tests 18
3.2 Current EPA Reaerosolization Experiments 21
3.3 Planned Reaerosolization Experiments 23
4 Quality Assurance 24
4.1 Equipment Calibration 24
4.2 Data Quality Objectives 24
4.3 QA/QC Checks 25
4.4 Acceptance Criteria 26
5 Summary 29
6 References 30
IV
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List of Figures
Figure 2-1: T/RH controlled environmental test chamber 3
Figure 2-2: EPA RWT positioned inside the T/RH chamber 4
Figure 2-3: Open front section of the EPA RWT showing a coupon, lower part of the internal flow
transition unit, and the slotted jet attached to the linear traversing device 4
Figure 2-4: Slotted jet driven by the linear traverse actuator 4
Figure 2-5: Coupon and flow transition unit 4
Figure 2-6: Slotted jet above the coupon at the end of its forward traverse and the air speed
monitoring device (anemometer) 5
Figure 2-7: Last open door showing filter holders 5
Figure 2-8: Four filter holders designed to be serviced using heavy-duty gloves 5
Figure 2-9: Average air velocity from the slotted nozzle as a function of nozzle pressure 6
Figure 2-10: Vertical velocity profiles in RWT #1 (EPA) and RWT #2 (DPG) 7
Figure 2-11: Wet deposition chamber 8
Figure 2-12: Top view of chamber 8
Figure 2-13: Deposition chamber coupon platform 8
Figure 2-14: Dry deposition chamber 9
Figure 2-15: SEM image of bar-coded Btk spores deposited with wet deposition chamber 11
Figure 2-16: SEM image of Bg spores deposited with wet deposition chamber 12
Figure 2-17: SEM image of PSL spheres 1 urn in diameter deposited with wet deposition chamber 12
Figure 2-18: Flow chart describing the test protocol for wet deposition experiments in the RWT 17
Figure 3-1: Scouting test average percent wet-deposited Btk reaerosolized by surface type and
jet velocity 19
Figure 3-2: Calculated percent wet-deposited Btk reaerosolized results from scouting tests with
varying jet velocity for smooth surfaces 20
Figure 3-3: Calculated percent wet-deposited Btk reaerosolized results from scouting tests with
varying jet velocity for rough surfaces 20
Figure 3-4: Calculated percent wet-deposited Btk reaerosolized results from scouting tests with
varying jet velocity for very rough surfaces 21
Figure 3-5: Percent wet-deposited Btk reaerosolized results from five replicates of final experiment 22
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List of Tables
Table 2-1. Dry Deposition Chamber Surface Loading Uniformity Data 10
Table 2-2. Materials/Surfaces Used for Testing 13
Table 3-1. Number of Scouting Tests Completed by Coupon Type 18
Table 3-2. Slotted Jet Settings for Wet Deposition Experiments by Coupon Type 21
Table 4-1. Data Quality Objectives for Experimental Conditions 24
Table 4-2. QA/QC Checks 26
Table 4-3. Spore Acceptance Criteria 26
Table 4-4. Deposition Acceptance Criteria 27
Table 4-5. Reaerosolization Acceptance Criteria 28
Table 4-6. Acceptable Variation among Replicate Experiments 28
VI
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List of Acronyms and Abbreviations
ATF Aerosol Test Facility
atm atmosphere(s)
Ba Bacillus anthracis
Ba-Ames Bacillus anthracis (Ames strain)
Bg Bacillus atrophaeus subspecies globigii
BSL biosafety level
Btk Bacillus thuringiensis var. kurstaki
CPU colony-forming unit(s)
cm centimeter(s)
CV coefficient of variation
DHHS Department of Health and Human Services
DHS Department of Homeland Security
DOD United States Department of Defense
DPG Dugway Proving Ground
DQO data quality objective
ECBC Edgewood Chemical Biological Center
EPA United States Environmental Protection Agency
ft foot/feet
HEPA high efficiency particulate air
in inch(es)
L liter(s)
m meter(s)
mg milligram(s)
ml milliliter(s)
MOP miscellaneous operating procedure
mph mile(s) per hour
NHSRC National Homeland Security Research Center
NIH National Institutes of Health
NIST National Institute of Standards and Technology
nm nanometer(s)
PBS phosphate buffered saline
PBST phosphate buffered saline with 0.05% Tween® 20
PCR polymerase chain reaction
PSL polystyrene latex
QA/QC quality assurance/quality control
QAPP quality assurance project plan
Ra measured distance from the median plane of the surface
RH relative humidity
RTP Research Triangle Park
RWT reaerosolization wind tunnel
VII
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s second(s)
SEM scanning electron microscope/microscopy
SOP standard operating procedure
SPORE Scientific Program on Reaerosolization and Exposure
SS stainless steel
T temperature
TGA thermal gravimetric analyzer
uL microliter(s)
urn micrometer(s)
VIM
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Executive Summary
The United States Department of Defense (DOD), Department of Health and Human Services (DHHS),
Department of Homeland Security (DHS), and Environmental Protection Agency (EPA) execute related
missions in biodefense response and recovery. There are mutual interests in understanding more about
Bacillus anthracis (Ba) reaerosolization and any associated public health risks. These organizations are
partnering through the Scientific Program on Reaerosolization and Exposure (SPORE). The primary
objective of SPORE is to develop an understanding of reaerosolization to make informed decisions
regarding response and mitigation activities that will reduce the risks associated with a Ba release.
Two small reaerosolization wind tunnels (RWTs) have been designed and constructed specifically for
conducting these spore reaerosolization studies. The first RWT is being used at EPA to conduct
reaerosolization experiments using two surrogate bacterial spore species and one inert particulate as the
test subjects. The second RWT has been installed in the biocontainment chamber at Dugway Proving
Ground (DPG) and will be used to conduct reaerosolization experiments using Ba-Ames. The end point
for the experimentation is to compare two sets of data, one acquired from EPA using surrogates and the
second from DPG using Ba-Ames.
Validation tests have been completed at DPG using the same study protocols and one surrogate used in
the EPA facility, Bacillus thuringiensis var. kurstaki (Btk). Reaerosolization results for Bf/
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1 Introduction
This interim project report describes the progress and current status of the research effort to characterize
the reaerosolization of bacterial spores, in particular biological anthrax simulants Bacillus thuringiensis
var. kurstaki (Btk) and atrophaeus subspecies globigii (formerly Bacillus globigii [Bg]), from outdoor
surfaces under different environmental conditions and compare the data with data from identical
experiments carried out using B. anthracis (Ames strain), hereafter referred to as Ba-Ames.
The purpose of this research effort is twofold: Our first and primary focus is to determine the suitability of
the biological simulants Btk and Bg and inert polystyrene latex (PSL) spheres for Ba-Ames. Our second
aim is to provide a quantitative understanding of the reaerosolization of anthrax spores from
environmentally available surfaces.
Over the past decade, the intentional release of the "Category A" biological agent Bacillus anthracis (Ba)
has been deemed a serious public health risk. Such a release has the potential to produce mass
casualties as Ba spores are highly persistent in the environment, thus creating challenges for
decontamination and restoration of infrastructure. Over the past 60 years, numerous studies have
identified the potential for Ba to reaerosolize (become a particulate suspended in air after initial settling).
Unfortunately, there has been a dearth of quantitative information related to the prediction of public health
impacts from reaerosolized Ba spores. Without such information, prediction of the reaerosolization hazard
accompanying an anthrax attack in an outdoor urban environment is problematic. The mechanisms
governing reaerosolization are complex, and developing a quantitative database is scientifically
challenging, time consuming, and expensive.
The United States Department of Defense (DOD), Department of Health and Human Services (DHHS),
Department of Homeland Security (DHS), and Environmental Protection Agency (EPA) execute related
missions in biodefense response and recovery. There are mutual interests in understanding more about
Ba reaerosolization and any associated public health risks. In turn, these organizations are partnering
through the Scientific Program on Reaerosolization and Exposure (SPORE). The primary objective of
SPORE is to develop an understanding of reaerosolization to make informed decisions regarding
response and mitigation activities that will reduce the risks associated with an outdoor Ba release.
The overall SPORE effort will provide guidance to senior leadership, public health officials, and response
personnel so that they can be prepared to respond to and mitigate economic, environmental, and human
health impacts resulting from reaerosolization after an anthrax incident. In such an event, B. anthracis
spores released into an outdoor urban area would disperse and settle on exposed surfaces. The physical
and chemical requirements for decontamination would depend on the initial dispersion and the later
reaerosolization of the biological material as well as the types of surfaces affected, such as concrete,
brick, asphalt, soil, metal piping, plants, plastics, and cloth—the typical materials that make up the
infrastructure of a complex urban environment.
This study is investigating the reaerosolization of Btk and Bg surrogate spores and inert particles in EPA's
National Homeland Security Research Center (NHSRC) Aerosol Test Facility (ATF) located in Research
Triangle Park (RTP), NC. Work completed at EPA will be compared with work conducted at Dugway
Proving Ground (DPG) in Utah. The work at DPG will be carried out in a manner identical to the work
conducted in EPA's ATF but will use Ba-Ames. The testing is being conducted by a project team
consisting of Alion Science and Technology (Durham, NC), RTI International (RTP, NC), ARCADIS US
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(Durham, NC), and DPG, with project oversight from EPA, DHS, and DPG principal investigators and the
Interagency SPORE Workgroup.
Two small reaerosolization wind tunnels (RWTs) have been designed and constructed specifically for
conducting spore reaerosolization studies. The first RWT is being used at EPA to conduct
reaerosolization experiments using two surrogate bacterial spore species and one inert particulate as the
test subjects. The second RWT has been installed in the biocontainment chamber at DPG and will be
used to conduct reaerosolization experiments using Ba-Ames. The end point for the experimentation is to
compare two sets of data, one acquired from EPA using surrogates and the second from DPG using Ba-
Ames. Validation tests are underway at DPG using the same study protocols and one surrogate (Btk)
used in the EPA facility. Reaerosolization results for Btk from EPA and DPG will be compared statistically
to verify that the data produced by both reaerosolization wind tunnels describe the same population.
Once the data have been demonstrated to be equivalent, DPG will conduct experiments using Ba-Ames.
The two data sets will then be evaluated statistically and the behavior of the Ba-Ames and surrogates
compared quantitatively to determine the efficacy of the surrogate in representing Ba-Ames in terms of
environmental reaerosolization.
The wind tunnel experiments are concerned primarily with the combination of two processes, namely,
detachment of spores from a surface followed by their reaerosolization in the tunnel airflow. Numerous
experimental variables affect reaerosolization of particles. Particle variables include size, morphology,
chemistry, hygroscopicity, electrostatic properties, and others. Important surface characteristics also
include morphology, chemistry, hygroscopicity, and electrostatic properties, as well as roughness and
asperity. In addition, fluid variables impact reaerosolization; these key parameters include velocity,
turbulence, boundary layer effects, and humidity.
The experimental variables that have been selected for this study are particulate type (Btk, Bg, PSL, and
Ba-Ames), particulate preparation (liquid slurry, dry powder, and refined powder), jet velocity (low,
medium, and high), surface type (glass, roofing shingles, and concrete), and roughness level of each
surface type (smooth, rough, and very rough). Although innumerable potential biological surrogates exist,
the two chosen for these experiments were selected based on programmatic interests of SPORE, as well
as safety, size similarity, and other key particle properties. At this point, the relative importance of various
spore properties, such as the type of endospore and degree of hygroscopicity, has not been established.
The properties of the test surfaces have been analyzed quantitatively. Experimental variables were
prioritized per the input from the SPORE Workgroup. It is important at this stage of testing that both EPA
and DPG operate using identical protocols, so any parameter that cannot be controlled adequately at
both facilities is deemed unsuitable for evaluation in this set of experiments.
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2 Experimental Materials and Methods
The laboratory reaerosolization studies using surrogates are being conducted at EPA's ATF in RTP, NC.
Processing and analysis of microbiological samples is being conducted in EPA's microbiology laboratory
in RTP. Reaerosolization studies using Ba-Ames will be conducted in DPG's biosafety level (BSL) 3
laboratories at Dugway Proving Ground, UT. The key materials and pieces of equipment used for the
project are described here. These materials and equipment include a small-scale reaerosolization wind
tunnel and deposition chambers for wet and dry deposition of particulate materials. Two identical versions
of each piece of equipment have been designed and constructed by Alion for the project. One set is being
used for tests with surrogates in EPA's ATF, and the other set is being installed at DPG in the BSL 3
facility to be used for tests with Ba-Ames.
2.1 Reaerosolization Wind Tunnel
The RWT at EPA is contained in a temperature (T) and relative humidity (RH) controlled environmental
test chamber (Figure 2-1) within a BSL 1 laboratory. The BSL 1 facility is a standard laboratory module in
EPA's RTP facility.
Figure 2-1: T/RH controlled environmental test chamber.
The RWT (Figures 2-2 through 2-8) is 6 feet (ft) long and has a 9-inch (in) by 9-in cross section. The RWT
is open-ended by design and can thus take advantage of the T/RH environmental settings available
inside the chamber. It operates on the principle of total collection of reaerosolized material from the test
surface on four polyester felt filters. The RWT is fitted with two high efficiency particulate air (HEPA) filters
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to provide clean air and prevent contamination of the chamber and the blower. Its walls are laminated
with aluminum sheeting used in building construction. The top of the RWT is divided into independent
doors that provide access to various areas of the tunnel. One door allows for installing and retrieving the
test coupon, and another door provides access for installing and retrieving test filters. The RWT that was
constructed for use in the BSL 3 facility at DPG is identical to the EPA version except that the doors are
located on the side of the RWT to provide easier access through glove ports.
Figure 2-2: EPA RWT positioned inside the T/RH
chamber. The HEPA filter can be seen at the entry
end and the blower at the far end.
Figure 2-3: Open front section of the EPA
RWT showing a coupon, lower part of the
internal flow transition unit, and the slotted jet
attached to the linear traversing device.
Figure 2-4: Slotted jet driven by the linear
traverse actuator.
Figure 2-5: Coupon and flow transition unit.
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Figure 2-6: Slotted jet above the coupon at the
end of its forward traverse and the air speed
monitoring device (anemometer).
Figure 2-7: Last open door showing filter
holders.
Figure 2-8: Four filter holders designed to be serviced using heavy-duty gloves.
The reaerosolization force applied to the deposited spores is generated by an air jet that traverses over
the surface of the test material (coupon). The air jet is produced by a slotted nozzle, shown in Figure 2-4,
that is connected to a compressed air supply. The air jet applies the reaerosolization force evenly across
the width of the test coupon (Figure 2-5). The actuator allows moving the air jet over the coupon at a
steady speed. The height of the jet, angle of the air impinging on the coupon, and the speed of the
actuator can all be changed as desired, but will be held constant for all experiments. At this time, the air
jet is quantified in terms of the average air velocity exiting the slotted nozzle, calculated by dividing the
total volumetric flow rate of air through the nozzle at each pressure setting by the cross sectional area of
the nozzle opening (Figure 2-9). This air velocity will be equated to a reaerosolization force that can be
translated to activities in the environment that could cause spore reaerosolization (wind, foot traffic,
vehicle traffic, etc.). Scouting trials were conducted to determine appropriate jet velocities to reaerosolize
wet-deposited spores. The trials illustrated that wet deposition of spores results in strong surface
attachment, requiring high jet velocities to dislodge significant quantities of spores. Additional scouting
trials will be conducted for dry-deposited spores to determine appropriate jet velocities after completion of
all wet deposition experiments. These jet velocities are expected to be much lower than those used for
the wet deposition experiments.
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300
250
— 200
150
100
OJ
50
0.5
1.5 2 2.5
Nozzle presure(atm)
3.5
Figure 2-9: Average air velocity from the slotted nozzle as a function of nozzle pressure.
Downwind of the air jet and test coupon in the RWT is the flow transition unit, shown in Figure 2-5. This
unit was designed to minimize loss of particles and at the same time accommodate four filter holders
(Figure 2-8) with round polyester felt filters. The biological extraction requirements impose a need to have
filters no larger than 10 centimeters (cm) in diameter. Therefore, multiple filters have been installed to
obtain a reasonable air velocity and directionality in the tunnel.
Vertical profiles of velocity were measured in both RWTs to verify that they have the same overall flow
characteristics. Results are presented in Figure 2-10. For these measurements, a clean smooth concrete
coupon was placed in the RWT, the air jet was positioned in the center of the coupon, and a hot-wire
anemometer (Series 471 digital thermo-anemometer, Dwyer Instruments, Inc., Michigan City, IN, USA)
was used to measure the velocity 2 cm downwind of the downwind edge of the coupon. Velocity was
measured at eight heights using five different nozzle pressures ranging from 0 to 2 atmospheres (atm).
Statistical analyses were conducted to compare the velocity measurements between the two different
RWTs. In addition to examining whether the measured velocities were significantly different statistically,
the magnitude of the differences was also estimated. The analyses performed here answered the
question of whether velocities in the two RWTs differed from an overall perspective. The EPA RWT (#1)
generally yielded lower measured velocities than the DPG RWT (#2). This difference was established with
a Wilcoxon signed rank test (Hollander and Wolfe, 1973), which gave a p-value of 0.0021. Of the 45
height-pressure combinations, RWT#1 was lower on 34, higher on 10, and one combination had equal
velocities. The magnitude of the RWT #1 minus RWT #2 difference was estimated to be -0.17 meters
(m)/second (s), determined from the Hodges-Lehmann estimator based on Walsh averages (Hollander
and Wolfe, 1973). To summarize, RWT #1 consistently yielded lower velocity measurements with the
overall difference estimated as -0.17 m/s. This difference is acceptable at 7% of the average measured
velocity (2.3 m/s).
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-RWTSlP=O.Oatm —I
RWT«2P=O.Oatm -I
-RWTSlP-0.2Satm —*— RWTK1 P=O.S aim —I
RWT*2P=0.2Satm RWTK2 P=O.Satm --
-RWT«lP=1.0atm —I
RWT«2P=1.0atm -»- RWT*2 P=2.0atm
10 15
Wind velocity |mph)
Figure 2-10: Vertical velocity profiles in RWT #1 (EPA) and RWT #2 (DPG).
2.2 Deposition Chambers
Two separate deposition chambers, a wet chamber and a dry chamber, are being used for deposition of
liquid slurry, dry powder, and refined powder surrogate preparations. Chambers identical to the chambers
being used in EPA's ATF were constructed by Alion and shipped to the DPG facility.
2.2.1 Wet Deposition Chamber
The wet deposition chamber consists of several key components: a vessel, an ultrasonic nozzle, and two
small mixing fans (Figures 2-11 and 2-12). It is designed to fit over the coupon for aerosol generation and
deposition (Figure 2-13). Two identical chambers have been fabricated. The first is in use at EPA, and the
second is in use at DPG.
Several design features have been included in the wet deposition chamber to achieve consistency in
surface loading. An ultrasonic nozzle (model Q060-2-26-17-303-030, Sono-tek Corp., Milton, NY, USA) is
used to atomize the spore solution in the deposition chamber. A microbore was fitted inside the ultrasonic
nozzle to minimize the amount of "dead" liquid trapped inside the nozzle that can drip onto the coupon at
the end of the atomization. The needle inner diameter is sized to produce a median droplet size of 31
micrometers (urn). The needle is directly connected to the dispensing syringe pump via hypodermic
(20 gauge) tubing, providing a well-controlled and steady dispensing rate. As shown in Figure 2-12, the
two small mixing fans (model DF124010BM-3G, Dynatron Corp., Fremont, CA, USA) are positioned close
to the coupon surface at the edges of the coupon at opposite walls of the chamber. This arrangement
produces swirling air that rises along the walls, descends in the center, and then disperses along the
coupon, producing a droplet-laden vortex that swirls downward and ultimately produces a uniform coating
on the coupon.
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Figure 2-11: Wet deposition
chamber.
Figure 2-12: Top view of
chamber.
Figure 2-13: Deposition
chamber coupon platform.
Deposition efficiency was measured for the wet deposition chamber by depositing 0.2 ml of Btk spore
solution onto an array of six stainless steel (SS) coupons that completely covered the test coupon area.
For each deposition test a reference sample was collected by dispensing 0.2 ml of Btk spore solution
directly into a conical tube containing 10 ml of phosphate buffered saline with 0.05% Tween® 20 (PBST).
Spores were extracted from the SS coupons according to MOP 6601 (Arcadis, 2013a) then plated and
incubated, and colony-forming units (CFUs) were enumerated according to MOP 6535a (Arcadis, 2009).
The reference samples were also plated and incubated, and CFUs were enumerated. The average
measured deposition efficiency for the wet deposition chamber from eight separate tests was 50%,
calculated as the ratio of CFUs recovered from the SS coupons divided by the experimentally determined
extraction efficiency of the SS coupons (93%) to CFUs contained in the reference sample. The
repeatability of deposition is estimated by the coefficient of variation (CV) of reference samples dispensed
from the same spore solution. This analysis of the reference sample data from the deposition efficiency
tests shows that the CV is 10%.
2.2.2 Dry Deposition Chamber
A dry powder deposition chamber, shown in Figure 2-14, was designed and constructed to allow uniform
deposition of dry powders onto test material coupons. The dry deposition chamber is suitable for
deposition of any powder aerosol. The chamber is approximately one square foot and is made mostly of
aluminum with a transparent plastic viewing window. The chamber was built to house the 7.75-in by
7.75-in test coupons. A penetration in the top of the lid contains the powder reservoir/aerosol nozzle. The
powder reservoir is designed to contain approximately 100 milligrams (mg) of powder at a time. Once the
test coupon and powder reservoir are loaded, the lid is placed over the coupon, flush on the base plate to
create a seal. The chamber is evacuated to a vacuum of-59 in of water. The vacuum pump is stopped,
and the system is depressurized by admitting air through a switch valve. The rush of air aerosolizes the
powder and transports the particles through the reservoir into the deposition chamber where they begin to
settle onto the test coupon.
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Figure 2-14: Dry deposition chamber.
The amount of vacuum generated inside the chamber controls the amount of aerosol that is deposited
onto the surfaces. A small vacuum will allow only a small rush of air into the chamber, thus producing a
small amount of powder dispersion. Likewise, a higher vacuum will result in a larger amount of powder
movement. When the desired amount of aerosol has been dispersed into the chamber, determined by the
desired pressure difference, the vacuum pump is stopped and the aerosol is allowed to settle by gravity.
The gravitational settling time depends largely on the average radius and mass density of the aerosol
particles and can be calculated as distance from the nozzle to the coupon divided by the settling velocity,
determined by Stokes' law.
After the aerosol has had time to settle within the chamber (10 minutes in this case), the pressure release
valve is opened, allowing HEPA-filtered air to enter the vessel. This process slowly brings the deposition
chamber back to atmospheric pressure. Generally, an additional 10 to 15 minutes is required for the
chamber to depressurize completely before the test coupon can be retrieved.
The chamber design and loading procedure were validated using uranine-tagged amorphous silica and
lyophilized Btk spores to determine if the process resulted in consistent and repeatable surface loading.
Syloid® (W.R. Grace & Co., Baltimore, MD, USA) is the basis of the synthetic aerosol, providing the
micron particle size. The powder is tagged with uranine tracer using an RTI proprietary method (RTI
International, RTP, NC, USA), and the amount of fluorescence per particle is a known quantity. The Btk
spores were provided by Dugway Life Sciences (Dugway, UT, USA). Filters were placed at each corner of
the chamber and in the center. Eight total trials were performed: four fluorescently tagged amorphous
silica trials and four Btk trials. For the Btk trials, the filters were recovered and analyzed for total CFUs per
square centimeter using standard microbiological methods. For the fluorescent silica trials, the filters were
recovered and analyzed using fluorometry. The number of particles present in the sample was calculated
by knowing the amount of fluorescence per particle. Results are shown in Table 2-1. Btk trials 3 and 4
were invalid due to failure to follow the appropriate procedure. The average variance across the coupon
surface for fluorescently tagged amorphous silica was 16 %, and the average repeatability was 13 %.
These values meet the acceptability criteria of 20 % variance. This deposition mechanism should work
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well for the refined spore preparation. Much greater variability was encountered with lyophilized Btk
spores than with the amorphous silica, most likely due to the properties of the Btk spore preparation. The
spore preparation tends to clump and hold an electrostatic charge, increasing the variability in the surface
concentration.
To address this problem, additional work is underway to develop a method to create a dry aerosol with
minimal spore agglomerates from the lyophilized cake. This method will involve suspending the spores in
pure isopropyl alcohol, spraying them with the wet deposition chamber ultrasonic nozzle into a small
chamber to allow the alcohol to evaporate, and introducing the dry spores into the dry deposition chamber
to allow the spores to settle onto the test coupon. This process should provide acceptable variance in
spore surface concentration across the test coupon and between trials.
Table 2-1. Dry Deposition Chamber Surface Loading Uniformity Data.
Results for uranine-tagged amorphous silica and Btk spores presented.
Uranine-Tagged Amorphous Silica (particles/cm^)
Position
Top left
Top right
Bottom right
Bottom left
Center
Average
Std. dev.
CV
Trial 1
50864
30902
48896
51965
36279
43781
9558
22%
Trial 2
49442
39791
41242
59032
41754
46252
8071
17%
Trial 3
38704
32278
44102
47206
48139
42086
6606
16%
Trial 4
43990
43967
49190
53398
53603
48830
4766
10%
Average
45750
36735
45858
52900
44944
Std Dev
5554
6206
3861
4870
7538
CV
12%
17%
8%
9%
17%
Btk Spores (CFU/cm2)
Position
Top left
Top right
Bottom right
Bottom left
Center
Average
Std. dev.
CV
Trial 1
35583
27298
63424
60844
18199
41070
20209
49%
Trial 2
8461
21051
33002
44139
17791
24889
13887
56%
Trial 3
Invalid
-
-
-
Trial 4
Invalid
-
-
-
Average
22022
24175
48213
52491
17995
Std Dev
19178
4418
21512
11812
288
CV
87%
18%
45%
23%
2%
2.3 Test Surrogates
Several species of Bacillus spores are commonly used as surrogates for B. anthracis in laboratories and
field studies, including B. atrophaeus, B. thuringiensis, B. cereus, and B. subtilis, among others
(Greenberg et al., 2010). This project aims to compare reaerosolization of two of these types of bacterial
spores and one inert particle to reaerosolization of Ba-Ames.
2.3.1 Bacillus thuringiensis var. kurstaki
The Ba-Ames genotype and phenotype are similar to B. thuringiensis (EPA, 2012). Btk is a gram-positive
bacterium commonly found in soil. The endotoxin protein produced during sporulation is commonly used
as a pesticide. The average hydrated spore size is 0.8 x 1.4 urn, which is the same as the average
hydrated spore size for B. anthracis (Carrera et al., 2007). Bar-coded Btk is a genetically modified strain
10
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developed by Edgewood Chemical Biological Center (ECBC). The genetic modification alters the DMA
sequence such that polymerase chain reaction (PCR) analysis clearly distinguishes these spores from the
common naturally occurring strain. The applicability of bar-coded Btk as a surrogate for Ba-Ames is under
evaluation in this research.
The bar-coded Btk cells used in this study were cultured by 10-liter (L) batch fermentation. Following
sporulation, the spores were concentrated into a wet pellet, washed three times, and lyophilized. The
lyophilized spores were a dry but very clumpy cake rather than a loose dry powder. The lyophilized spores
were delivered to EPA and mixed with sterile deionized water to make a stock solution that was diluted
with PBST to the desired titer of 108 spores/milliliter (ml). Figure 2-15 shows a scanning electron
microscopy (SEM) image of bar-coded Btk spores deposited using the wet deposition chamber on
aluminum foil. The Btk spores were deposited as 74 % singlets and 21 % doublets with very few large
agglomerates.
Figure 2-15: SEM image of bar-coded Btk spores deposited with wet deposition chamber.
2.3.2 Bacillus atrophaeus subspecies globigii
Bacillus atrophaeus subspecies globigii (Bg) is a gram-positive rod capable of producing endospores.
The endospores are elliptically shaped with dimensions of 0.7-0.8 x 1-1.5 urn and a mean aerodynamic
diameter of approximately 1.1 urn (Carrera et al., 2007). Bg has been used as a surrogate for B. anthracis
in more than 40 studies (Greenberg et al., 2010). Bg cells were fermented, harvested, and lyophilized by
DPG in the same manner as the bar-coded Btk. Figure 2-16 shows a SEM image of Bg spores deposited
with the wet deposition chamber onto aluminum foil. This preparation of Bg was determined to be
unsuitable for reaerosolization testing due to the presence of many vegetative cells (approximately 30 %)
and large agglomerates. DPG is preparing a new batch of Bgto address these problems.
11
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Figure 2-16: SEM image of Bg spores deposited with wet deposition chamber.
2.3.3 Fluorescent Polystyrene Latex Spheres
Internally dyed fluorescent polystyrene latex (PSL) spheres are commercially available and easy to detect
using fluorometry. PSL spheres are commonly used in aerosol sampler and filter evaluation studies. The
PSL spheres currently being used in this project have a diameter of 1 urn and a density of 1.05 g/cm3
(Part #G0100B, Thermo Scientific, Waltham, MA). This diameter and density are comparable with dry 8.
anthracis spore density of 1.17 g/cm3 (Carrera et al., 2007). The spheres are dyed internally with a
fluorescent green dye with excitation and emission wavelengths of 468 and 508 nanometers (nm),
respectively. Figure 2-17 shows a SEM image of fluorescent PSL spheres 1 urn in diameter deposited
with the wet deposition chamber onto aluminum foil.
Figure 2-17: SEM image of PSL spheres 1 urn in diameter deposited with wet deposition chamber.
12
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2.4 Test Surface Characterization
Three types of surfaces—glass, concrete pavers, and roofing shingles—were investigated using
techniques intended to describe the surface features and material characteristics. These materials are
described in Table 2-2. Three subtypes within each category were investigated to provide a range of
roughness within each material type. The three glass types are smooth glass, frosted glass, and bubbled
glass. Glass coupons were purchased from Dan's Glass, Inc. (Raleigh, NC, USA).The three textures of
concrete pavers are smooth, rough, and very rough, based on surface texture produced by acid-etching.
Concrete coupons were purchased from Sonoma Cast Stone Corp. (Petaluma, CA, USA). Roofing felt
(#30W, Warrior Roofing Manufacturing, Tuscaloosa, AL, USA), economy shingles (#241008006, Tarco
Roofing, Little Rock, AR, USA), and architectural shingles (#0601070, GAP Corp., Wayne, NJ, USA) were
chosen for roofing materials. Roofing materials were purchased from a Home Depot home improvement
store in Raleigh, NC. The nine materials were subjected to five material characterization methods. All
data from these tests are with the project statistician, and statistical analyses are pending.
Table 2-2. Materials/Surfaces Used for Testing
Material
Glass
Asphalt roofing
shingles3
Cast concrete
Roughness
Class
Smooth
Rough
Very rough
Smooth
Rough
Very rough
Smooth
Rough
Very rough
Details
Window glass ("float glass")
"Frosted" or "etched" glass
Architectural textured glass
Asphalt-impregnated "roofing felt"b, same
asphalt chemistry as roofing shingles, no
grit
"Economy" shingles, relatively thin and flat
"Architectural" shingles, relatively thicker
with more texture
Earth Crete mold release: cement simple
mold release with no sanding, finishing,
paint, wax, or sealants
Earth Crete heavy acid etch: cement mold
release acid etched using 8 % muriatic acid
with no sanding, finishing, paint, wax, or
sealants (equivalent to typical sidewalk
finish)
Earth Crete extra-heavy acid etch #1 :
cement cast mold aggressive acid etched
using 8 % muriatic acid with no sanding,
finishing, paint, wax, or sealants (equivalent
to skid-resistant finish of parking deck floor)
7-3/4 in x 7-3/4 in x 1/8 in
coupons cut from "single-
weight" glass at glass company
7-3/4 in x 7-3/4 in x 1/8 in
coupons cut from "single-
weight" glass at glass company
7-3/4 in x 7-3/4 in x 1/8 in
coupons cut from "single-
weight" glass at glass company
7-3/4 in x 7-3/4 in squares cut
with utility knife in laboratory
7-3/4 in x 7-3/4 in squares cut
with utility knife in laboratory
7-3/4 in x 7-3/4 in squares cut
with utility knife in laboratory
7-3/4 in x 7-3/4 in x 3/4 in thick
coupons prepared by Sonoma
Stone
7-3/4 in x 7-3/4 in x 3/4 in thick
coupons prepared by Sonoma
Stone
7-3/4 in x 7-3/4 in x 3/4 in thick
coupons prepared by Sonoma
Stone
aAsphalt roofing shingles have been selected as a proxy for asphalt paving. The shingles are readily available, are
of consistent formulation, and are easily handled in the laboratory. The base material of shingles is asphalt-
impregnated fiberglass. The surface grit is usually boiler slag, similar to the stone aggregate in asphalt paving.
bAsphalt-impregnated roofing felt (also called "felt roof deck protection") is felt saturated with asphalt to form a
composite that is essentially the same as the base layer of the shingles.
13
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2.4.1 Hygroscopicity
The hygroscopicity measurement was used to provide an indication of the ability of the materials to
absorb water molecules. A thermal gravimetric analyzer (TGA) (model Q50, TA Instruments, New Castle,
DE, USA) determined changes in weight of samples relative to change in temperature. A small sample
was immersed in water and then wiped dry. The sample was then placed in the TGA, and the reduction in
mass was recorded throughout the programmed heating cycle. Assuming water loss was the only factor
affecting mass change, the hygroscopicity of the sample was calculated. Three samples of each material
were measured for hygroscopicity.
2.4.2 Surface Tension
The surface tension of water droplets on the test material was used as an indicator of the hydrophobic or
hydrophilic nature of the surface. The angle formed between the solid/liquid interface and the liquid/vapor
interface is referred to as the contact angle. A droplet with an angle greater than 90° indicates a
hydrophobic surface. Likewise, a droplet with a contact angle less than 90° indicates a hydrophilic
surface. Contact angles approaching 0° are possible if the droplet has a strong chemical attraction to the
solid surface, resulting in the liquid spreading thinly across the surface. Contact angle measurements
were obtained with a Rame-Hart 200-F1 goniometer (Rame-Hart Instrument Co., Succasunna, NJ, USA).
A distilled water droplet of 10 microliters (uL) was deposited on each surface and an image was captured
of the profile. Imaged, a free image analysis tool developed by the National Institutes of Health (NIH), was
used to measure the incident angles. At least two angle measurements were taken on either side of the
droplet for each material.
2.4.3 Surface Roughness
Surface roughness of the materials was measured on two different scales. A micrometer scale roughness
measurement determines the contact area between the spore and surface. This scale is a factor in the
spore adhesion to the surface. A millimeter scale roughness determines the airflow boundary layer
characteristics across the surface. This scale is a factor in the lift force acting on the spore and
determines detachment. Surface roughness for both scales is reported as the arithmetic mean of the
absolute values of measured distance from the median plane of the surface (Ra).
Micrometer Scale
Two methods quantified the micrometer scale surface roughness of the materials. A Micro Photonics
TR200 surface roughness tester (Micro Photonics Inc., Allentown, PA, USA) was employed first. The
contact profilometer relies on a piezoelectric-operated diamond stylus to measure the contour of the
surface and provides the arithmetic mean profile (in urn) of the surface of interest. All materials except for
the smooth and frosted glass had surface features with scales that exceeded the profilometer range of
motion. Consequently, SEM combined with digital image analysis was employed as an alternative method
of determining roughness on the micrometer scale. Five images were taken from various locations on a
single sample. Three samples of each material were imaged, thus providing 15 images for each material
at a single magnification. Images of the materials were collected at SOOxand 10,000x magnifications.
The SEM images were individually analyzed using Imaged software. All images were preprocessed by
converting them to 8-bit images, enhancing the contrast, applying a topographical threshold value based
on the frequency and intensity of shading in the image, and converting the image to binary. After
preprocessing, the Roughness Calculation plug-in (Chinga and Dougherty, 2006) was used to determine
14
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the surface roughness, Ra. The image-generated roughness value for smooth glass was scaled to the
value measured with the profilometer, thus providing a factor to convert all materials from image-
generated roughness values to actual micrometer scale roughness values.
Millimeter Scale
To quantify the millimeter scale roughness of the test materials, optical images were taken at 2x
magnification. The images were processed in the same manner as the SEM images. However, a z-axis
scale proved to be unattainable for optical images at low magnification. Micro-scale SEM images were
able to provide a greater range of depth and clarity at specific locations on the sampling coupons that
could be compared directly to the function of a profilometer. Optical images could not provide this same
basis of comparison. Instead, ImageJ-derived roughness measurement outputs for optical images were
normalized to the lowest measured sample (smooth glass), thus providing a unitless range for roughness
relative to smooth glass.
2.4.4 Surface Area
Actual Surface Area
The surface area of a given material can vary greatly based on the size of the material as well as the
contour of the surface. A flat surface like smooth glass has very few, if any, ridges or valleys. In contrast,
a rough surface like roofing shingles has many ridges and valleys. Thus the surface area of a smooth
surface will be much less than the surface area of a very rough material of the same size. The amount of
surface area can greatly influence the number of attachment points for a contaminant.
The SEM and optical images used for roughness measurements were also used to measure surface area
for all test materials. All images were preprocessed in Imaged by first using the Set Scale function to
measure a known distance on the image. This known distance was either the scale provided on the SEM
image or, for the optical images, the length of the entire coupon. After the scale was set, the images were
converted to 32-bit images and contrast-enhanced. The SurfCharJ plug-in (Imaged, 2011) was used to
calculate the surface area of the image. The surface area output was normalized with the projected area
of the chosen image and was therefore unitless; however, with knowledge of the pixel length and scale,
the actual surface area was calculated.
Specific Surface Area
The specific surface area measurement gave an indication of the microstructure of the surface of interest.
The specific surface area, in m2/g, of small samples of the test coupons were determined by measuring
the physical adsorption of nitrogen gas molecules on the solid surface with a Micromeritics ASAP 2020
accelerated surface area and porosimetry system (Micromeritics, Norcross, GA, USA). Using the
Brunauer-Emmett-Teller algorithm to interpret the data from the instrument, the internal surface area of
porous materials was measured. The system made multiple measurements per sample, and the standard
deviation was calculated automatically. For all types of glass, four measurements were collected per
coupon, and the majority of these measurements were below the detectable limit for the instrument. For
the pavers, four measurements were taken on three different coupons for each type of paver. Four
measurements were taken on a single coupon for each type of roofing material. Half of the
measurements for roofing felt and rolled roofing were below detectable limits.
15
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2.5 Test Protocol
Figure 2-18 contains a flow chart that provides a brief description of the test protocol for wet deposition
experiments in the RWT. The same procedure is used for experiments at EPA and DPG. However,
implementing the procedure in the laboratory at DPG is complicated by the restrictions imposed on
working with BSL-3 agent, and additional steps are necessary to provide the required levels of
containment at all times.
The samples generated in each experiment include the following:
• Positive control: an approximately 3 to 5 ml sample of the spore solution transferred into a sterile
conical tube as a positive control.
• Reference tube: a reference sample collected for each deposition test by dispensing 0.2 ml of Btk
spore solution directly into a conical tube containing 10 ml of phosphate buffered saline with 0.05%
Tween®20 (PBST).
• Filters: four polyester felt filters used for each experiment to collect spores reaerosolized from the test
coupon and transferred to sterile Stomacher bags for extraction according to MOP 6600 (Arcadis,
2013b).
• Field blank filter: a field blank filter collected for each day of testing in a Stomacher bag as a negative
control.
• Coupon wipes: a wipe sample of the test coupon surface extracted according to MOP 6567 (Arcadis,
2010) and done as a qualitative check that spores were deposited on the coupon surface.
16
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J Dust coupon
t Glue coupon to disposable riser, if needed
J Equilibrate coupon at room T and RH
Wet Deposition Chamber
t Collect positive control sample
J Collect reference tube sample
> Deposit 02 mL of surrogate solution on coupon
> Place coupon in T/RH chamber to equilibrate
j Load autoclaved filters into clean, dry filter holders
J Place filter holders in labeled plastic bags
Reaerosolization Wind Tunnel Test
Place loaded coupon in RWT
Install four filter holders in RWT
Close RWT
Turn on RWT fan with damper closed
Run experiment and simultaneously
>• Open RWT fan damper
Open vatve to air jet
>• Turn traverse switch to forward
When traverse completes motion (- 1 min), simultaneously
Close RWT fan damper
>• Close valve to air jet
>* Turn traverse switch to return
Remove filter holders and place in correct, labeled bags
Remove coupon
Run sweeps to clean RWT
Repeat procedure for all scheduled experiments
>
J
*
Sample Collection
Wipe surface of test coupon after experiment
and place wipe in labeled tube
In clean biosafety cabinet, remove filters from filter
holders and place in labeled Stomacher bags
Place field blank filter in labeled Stomacher bag
Cleanup
Disinfect and dispose of coupon
Clean biosafety cabinet
Wash disassembled filter holders with amended bleach,
rinse with deionized water, and rinse with isopropyl alcohol
Leave clean filter holders to dry in clean biosafety cabinet
Figure 2-18: Flow chart describing the test protocol for wet deposition experiments in the RWT.
17
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3 Results and Discussion
3.1 Scouting Tests
Many scouting tests, listed in Table 3-1, were conducted with wet-deposited Btkto determine the final
experimental procedure and experimental details such as surface loading levels and jet velocities. Fifty-
four individual reaerosolization experiments were run with various loaded coupons at fixed jet velocity
with the purpose of fine-tuning the experimental procedure, assessing suitability of surface loading levels,
and estimating reproducibility and precision of results. Surface loading levels were varied over three
orders of magnitude by changing the spore concentration of the inoculant and the amount of inoculant
applied to the test coupon. The goal was to have the majority of spores deposited as single spores. For
this reason, the spore concentration of the inoculants could not be of a higher order than 108 CFU/mL, or
many spores would be contained in each droplet from the ultrasonic nozzle. The amount of inoculant
deposited on the surface varied between 0.1 ml and 0.6 ml. The volume chosen was 0.2 ml to avoid
any puddles forming on the coupon surface. Therefore, the surface loading level chosen for the wet
deposition experiments was 0.2 ml of 108 CFU/mL.
An additional 18 reaerosolization experiments were run with loaded coupons at multiple jet velocities with
the primary purpose of determining the low, medium, and high jet velocity settings for each surface type.
The jet velocity is set by adjusting the pressure in the compressed air line supplying the slotted nozzle.
Six of the varying jet velocity experiments were conducted at 11 nozzle pressures, from 0 to 2.5 atm, and
12 later experiments were conducted at eight nozzle pressures, ranging from 0 to 3.5 atm. When the
nozzle pressure is set to 0 atm, there is no air flowing through the nozzle, and the reaerosolization force
is the wind velocity of 5.8 mph generated by the RWT fan.
Table 3-1. Number of Scouting Tests Completed by Coupon Type
Material
Glass
Roofing shingles
Concrete
Roughness Class
Smooth
Rough
Very rough
Smooth
Rough
Very rough
Smooth
Rough
Very rough
Tests with Single
Jet Velocity
3
17
0
6
6
0
11
11
0
Tests with Multiple
Jet Velocities
3
3
3
1
1
1
2
2
2
Figure 3-1 presents average percent reaerosolized results from wet-deposited Btk experiments with
single jet velocities. The bars represent the average percent reaerosolized from three separate tests for
each surface type and jet velocity combination, and the error bars represent the CV. The overall trend in
the results was that very little was reaerosolized from the glass surfaces, while significant numbers of
spores were reaerosolized and collected from both concrete and roofing shingle surfaces. In general the
18
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number of spores reaerosolized from concrete and roofing shingle coupons were one to two orders of
magnitude greater than the number of spores reaerosolized from glass coupons.
2.5%
Surface type and jet velocity
Figure 3-1: Scouting test average percent wet-deposited Btk reaerosolized by surface type and jet
velocity. Each bar represents the average of three repetitions, and the error bars represent the CV.
Figures 3-2 through 3-4 present percent reaerosolized with varying jet velocity for each surface type. The
error bars represent the CV calculated from the replicate tests presented in Figure 3-1. There are no error
bars in Figure 3-4 because replicate experiments were not conducted for very rough surfaces in the
scouting tests. For the varying jet velocity experiments, only Filter #1 (the filter on the bottom right in
Figure 2-5) was collected and analyzed for each jet velocity to minimize the number of microbiological
samples for processing and plating to conserve time and resources while still providing valuable data for
determining the low, medium, and high jet velocities to be used for each test surface in the final wet
deposition experiments. The percent reaerosolized shown in Figures 3-2 through 3-4 was calculated as
% reaerosolized =
amount collected on Filter #1 / 0.42
amount deposited on coupon
where 0.42 is the average fraction collected on Filter #1 calculated from results of 33 individual
reaerosolization experiments. A great deal of variability was encountered in the fraction collected on each
of the four filters, and this variability was likely a major contributor to the variability in the results in Figures
3-2 through 3-4.
19
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Wet-deposited Btk, smooth surfaces only
1.4%
1.2%
1.0%
•Smooth shingle
Smooth concrete
-Smooth glass
0.0%
100 150 200
Jet velocity (mph)
250
300
Figure 3-2: Calculated percent wet-deposited Btk reaerosolized results from scouting tests with
varying jet velocity for smooth surfaces.
Wet-deposited Btk, rough surfaces only
0.6%
0.5% --
•Rough shingle
Rough concrete
•Rough glass
0.0%
50
100 150 200
Jet velocity (mph)
250
300
Figure 3-3: Calculated percent wet-deposited Btk reaerosolized results from scouting tests with
varying jet velocity for rough surfaces.
20
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Wet-deposited Btk, very rough surfaces only
1.6%
1 .4%
1.2%
1.0%
0.8%
0.6%
0.4%
0.2%
0.0%
-Very rough shingle
Very rough concrete
Very rough glass
50
100
250
300
150 200
Jet velocity (mph)
Figure 3-4: Calculated percent wet-deposited Btk reaerosolized results from scouting tests with
varying jet velocity for very rough surfaces.
3.2 Current EPA Reaerosolization Experiments
The chosen jet velocities for the final reaerosolization experiments are presented in Table 3-2. The goal in
choosing the jet velocities separately for each surface type was to avoid collecting zero spores and to
obtain results from the three different levels that cover as wide a range as possible for each spore
preparation. This air velocity will be equated to a reaerosolization force that can be translated to activities
in the environment that could cause spore reaerosolization (wind, foot traffic, vehicle traffic, etc.). These
jet velocities were determined in the scouting trials to provide measurable numbers of reaerosolized wet-
deposited spores. Additional scouting trials will be conducted with dry-deposited spores to determine
appropriate jet velocities after completion of all wet deposition experiments. Jet velocities for the dry
deposition experiments are expected to be much lower than those used for the wet deposition
experiments.
Table 3-2. Slotted Jet Settings for Wet Deposition Experiments by Coupon Type
Material
Glass
Roofing shingles
Concrete
Jet Velocity Level
Low
Medium
High
Low
Medium
High
Low
Medium
High
Nozzle Pressure
(atm)
0.98
1.92
3.38
0
0.98
2.97
0.5
1.35
2.97
Jet Velocity
(mph)
103
163
252
5.8a
103
226
67
127
226
aWhen the nozzle pressure is 0, the velocity is that produced by the RWT fan.
21
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For the final reaerosolization tests, a replicate of the complete experiment comprises 27 individual tests
(3 surface materials x 3 roughness levels x 3 jet velocities) executed in a random order. The test order for
each replicate is generated by the project statistician and delivered to the scientists conducting the
experiments. Five replicates of the complete experiment with wet-deposited Btk have been executed in
the EPA RWT. All samples from these tests were extracted, plated, enumerated, and verified by the
microbiology laboratory. Microbiological data were entered in a spreadsheet for analysis. Calculations
from the raw microbiological data include total measured CFU for each sample (filter, wipe, reference
sample tube, or positive control),
CFU
sample
plate counts x extraction volume
plated volume xdilution factor
total CFU collected on the four filters for each test,
CFUtotal =CFUfilter1 +CFUfilter2 +CFUfilter3 + CFUfilter4
and percent reaerosolized for each test,
% reaerosolized = -
CFU
total
CFUref x deposition efficiency
CFUref is the total number of CFUs in the reference tube sample, and the deposition efficiency was
determined by the SS coupon tests described in Section 2.2.1. The five results expressed as percent
reaerosolized for each material/roughness/jet velocity combination were averaged, and the CV was
calculated. Figure 3-5 presents wet-deposited Btk reaerosolization results as a function of the average jet
velocity. The error bars represent the CV. These results have been delivered to the project statistician for
further analysis.
1.4%
1.0% --
0.8% --
0 6%
0.4%
0.0%
-Smooth shingle
• Rough shingle
•Very rough shingle
-Smooth concrete
• Rough concrete
•Very rough concrete
Smooth glass
Rough glass
Very rough glass
50
100 150 200
Average velocity of applied reaerosolization force (mph)
Figure 3-5: Percent wet-deposited Btk reaerosolized results from five replicates of the final
experiment. The error bars represent the CV.
22
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Reaerosolization testing with wet-deposited fluorescent PSL beads is currently underway. As of July 31,
2013, 45 tests of the 81 planned tests (3 complete replicates) have been completed. Extraction of PSL
filter samples is being conducted in the same manner as extraction of biological samples with deionized
water instead of PBST. The solution containing PSL recovered from each filter is split equally between
two 40-mL conical tubes and centrifuged to separate the solids. Most of the supernatant is pipetted off,
and samples are sealed and stored in the dark to prevent degradation of the fluorescent dye. Samples
will be analyzed by fluorometry to quantify the number of PSL beads collected from each test. All
calculations will be the same as above for microbiological data, substituting PSL counts for CPUs.
3.3 Planned Reaerosolization Experiments
Reaerosolization testing with wet-deposited Bg spores will commence when wet-deposition PSL
experiments are complete and a suitable batch of Bg spores is obtained. The most important criterion for
acceptability is that the Bg preparation be > 95 % spores. At this time, three replicates of the complete
experiment are planned for wet deposition Bg.
After wet deposition testing is complete at EPA, the laboratory will be cleaned and decontaminated and
equipment and procedures for dry deposition experiments will be implemented. A limited number of
scouting tests will be conducted with dry bar-coded Btk to determine the jet velocities to be used for dry
deposition reaerosolization experiments. The current plan is to execute three replicates of the complete
experiment for dry deposition bar-coded Btk followed by three replicates of the complete experiment for
dry deposition Bg.
Reaerosolization testing is scheduled to begin at DPG the week of August 5, 2013. The first tests to be
conducted at DPG will be with wet-deposited Btk. Results from these experiments will be delivered to the
project statistician for comparison to results from tests conducted at EPA. When it has been determined
that the system at DPG is producing results that are not statistically significantly different from the results
produced at EPA, testing with wet deposition of Ba-Ames will commence.
23
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4 Quality Assurance
This project was performed under approved Category III quality assurance project plan (QAPP) titled
Determination of the Difference in Resuspension of Spores off of Outdoor Materials, QAPP-ZED-12-02
(Alion, 2012). This QAPP has recently been revised and is in the EPA review and approval process.
4.1 Equipment Calibration
Before beginning experiments, all of the following monitoring equipment was checked against either a
primary or a secondary standard to ensure that the equipment is operating within acceptance criteria:
• A Rotronic model HT205 probe (Rotronic Instrument Corp., Hauppauge, NY, USA) is used to monitor
and control temperature and relative humidity inside the test chamber (last calibration 12/30/11).
• A model DA 410 vane anemometer (Pacer Instruments, Keene, NH, USA) is used to measure the
wind tunnel velocity. The anemometer is returned to the factory for calibration (last calibration
1/30/12).
• A National Institute of Standards and Technology (NIST)-traceable temperature probe is used to
record temperatures in the incubators.
• Class A volumetric glassware is used where possible.
All equipment used in the project is maintained and calibrated according to operation manual
specifications and/or previous investigations.
4.2 Data Quality Objectives
The precision and accuracy goals have been established for each measurement parameter based on
(1) scientific requirements needed to achieve the primary objectives of the study, (2) knowledge of the
measurement system, (3) in-house experience with the sampling and measurement methods, and
(4) other similar research studies. These values are considered provisional, and acceptable target ranges
might be refined after the first measurements are taken and verified in each facility. Data quality
objectives (DQOs) for each major measurement parameter are listed in Table 4-1.
Table 4-1. Data Quality Objectives for Experimental Conditions
Parameter (Method)
Velocity uniformity (vane anemometer)
Analytical measurements (plate cultures)
Temperature/RH (capacitance)
Completeness
(%)
90
50
90
Accuracy
(%)
±10
±10
± 5 (temp)
±10(RH)
Precision
(%)
±20
±15
±2
±5
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Substantial effort will be expended to ensure that samples and measured parameters are representative
of the media and conditions being measured. All data will be calculated and reported in units that are
consistent with similar measurements from partnering organizations to allow for comparability of data
among organizations. DQOs for precision and accuracy are based on prior knowledge of the
measurement system employed and method verification studies, which include the use of replicate
samples and duplicate analyses. Definitions of DQOs are given below.
Completeness: a measure of the amount of verified data obtained from a measurement system compared
to the amount of data that was expected to be obtained under normal conditions. Completeness will be
assessed by reviewing laboratory data logs and laboratory logbooks to ensure that all data are verified
within established objectives.
Accuracy: the degree of agreement of measurements (or an average of measurements) with an accepted
reference or true value. Accuracy is a measure of the bias or systematic error in a system.
Precision: a measure of mutual agreement among individual measurements of the same property, usually
under prescribed similar conditions. Precision is best expressed in terms of the standard deviation.
Various measurements of precision exist depending on the prescribed similar condition.
Representativeness: the degree to which data accurately and precisely represent the characteristics of a
population, process, or environmental condition, or parameter variations at a sampling point.
Representativeness will be assessed by the collection of appropriate numbers of samples and the use of
a verified sampling design.
Comparability: the confidence with which one data set can be compared to another. Comparability of
experimental and numerical data will be ensured by using standard comparison and reporting methods.
All data will be presented in specified and documented units. Comparability will be ensured by the use of
approved SOPs for all instrumentation.
4.3 QA/QC Checks
The quality assurance/quality control (QA/QC) checks that are performed are provided in Table 4-2. Each
time spores are deposited onto a batch of test coupons, two positive controls and one negative control
are performed. The sample for the first positive control is pulled directly from the spore stock solution. The
second positive control sample (called the reference tube) is collected directly from the deposition
chamber ultrasonic nozzle and plated to ensure that the ultrasonic nozzle is not damaging the spores.
Each test coupon was wipe sampled after the experiment was complete as a qualitative check that
spores were deposited on the coupon surface. A negative control (blank) of the phosphate buffer that is
being used is prepared by plating onto an agar plate and spreading evenly using a sterile spreader. In
addition, a wind tunnel blank or background test is run after every third or fourth test coupon experiment
to provide a measurement of the contamination level of the RWT. The background sample is run and
collected onto filters just as the test runs with a clean smooth glass coupon in place. A field blank filter is
also transferred to a Stomacher bag in the laboratory where filters are loaded into the filter holders as a
negative control.
The acceptance criteria of ±5% for temperature and RH at DPG might not be feasible given the limitations
of the system. Temperature and RH data from the tests with Btk will be reviewed and discussed among
project scientists, and the acceptance criteria for these measurements might be revised.
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Table 4-2. QA/QC Checks
Measurement Matrix
QA/QC Check
Acceptance
Frequency Criteria
Corrective Action
Temperature/ RWT
RH
Pressure drop RWT
Chamber Continuous ± 5%
temperature probe
Pressure gauge Continuous ± 5%
Positive control
Microbiological Spore
inoculant
Microbiological PBS without
microbes
Temperature Incubator air NIST-traceable
thermometer
Negative control
Laboratory Filter
contamination
Field blank
2/run
Growth
1/run No growth
Continuous ± 5%
1/day
No growth
Stop sampling and
correct as necessary.
Check filter holders,
RWT seal.
Investigate viability of
microbes.
Clean and disinfect
systems.
Repair incubator.
Dispose of questionable
samples. Repeat
sampler test.
Clean and disinfect
laboratory.
4.4 Acceptance Criteria
The following acceptance criteria tables detail the experimental goals, sources of error, and means of
minimizing error. Tables 4-3 and 4-4 list the established acceptance criteria for the surrogate spores and
their deposition, respectively. The acceptance criteria for the reaerosolization tests are given in Table 4-5.
The data quality objective is less than a 100% standard error in the measurement of reaerosolization
efficiency (amount reaerosolized/amount deposited on surface). Table 4-6 presents the acceptable
variation among replicate experiments.
Table 4-3. Spore Acceptance Criteria
Goal: Btkand Bg have physical and chemical characteristics similar to Ba-Ames.
Controllable Error
Uncontrollable Error
Error Source
Spore size
Spore shape
Spore purity
Spore coating chemistry
Spore surface morphology
Minimization Strategy
Compare SEM images with reference
images.
Compare SEM images with reference
images.
Confirm endotoxin is absent from Btk
and Bg.
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Table 4-4. Deposition Acceptance Criteria
Goal: Minimize loading variability across the test coupon.
Controllable
Error
Uncontrollable
Error
Error Source
Deposition chamber operation (e.g.,
duration, flow, pressure, etc.)
Spore concentration (CFU/mL,
CPU/mass)
Extraction efficiency from filter or
wipe
Spore plating efficiency
Spore loss between deposition and
reaerosolization experiment
Spore agglomeration as liquid drops
or puddles dry, or dry powder is
dispersed
Spore agglomeration due to random
deposition pattern
Spore migration into surface pores
Minimization Strategy
Make sure operation is consistent between
deposition events.
Make sure sample preparation and extraction
from bulk material are consistent.
Make sure efficiency is consistent (most
important). (High extraction efficiency is
desired.)
Make sure plating procedure is consistent.
Handle and transport coupons in a covered
tray (particularly an issue for refined spores).
Limit liquid application to avoid puddle
formation. Minimize drop size to yield one
spore per drop. Make sure powder dispersal
breaks dry agglomerates into discrete
particles.
Goal: Generate a monolayer of individual spores or consistently sized aggregates across the
test surface.
Controllable
Error
Uncontrollable
Error
Error Source
Deposition chamber operation
(e.g., duration, flow, pressure)
Spore concentration (CFU/mL,
CPU/mass)
Spore agglomeration as liquid drops
or puddles dry, or powder is
dispersed
Particle agglomeration due to
random deposition pattern
Particle migration into surface pores
Agglomeration of particles in bulk
material/container
Minimization Strategy
Make sure operation is consistent between
deposition events.
Make sure sample preparation and extraction
from bulk material are consistent.
Limit liquid application to avoid puddle
formation. Minimize drop size to yield one
particle per drop. Make sure powder dispersal
breaks dry agglomerates into discrete
particles.
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Table 4-5. Reaerosolization Acceptance Criteria
Goal: Minimal variability among test surface coupon characteristics.
Controllable
Error
Uncontrollable
Error
Error Source
Surface chemistry analytical error and
surface variability
Surface porosity analytical error and
surface variability
Surface hygroscopicity analytical error
and surface variability
Contact angle analytical error and
surface variability
Surface roughness analytical error and
surface variability
None but variability could be large and
unable to be minimized
Minimization Strategy
Characterize chemistry of a representative
sample of all surfaces by XRF.
Characterize porosity of a representative
sample of all surfaces with a BET.
Characterize water absorption capacity of
the surface by TGA.
Characterize a representative number of all
surfaces with a goniometer.
Characterize roughness height and asperity
frequency of a representative sample of all
surfaces by profilometry.
Table 4-6. Acceptable Variation among Replicate Experiments
Goal: Minimal variance in reaerosolization among replicate experiments at same conditions.
Error Source
Minimization Strategy
Controllable
Error
Wind tunnel operation (carrier air
speed, jet speed, test duration, etc.)
Particle losses within wind tunnel
Filter collection efficiency
Particle extraction efficiency from filter
media
Spore plating variability
Particle loss between reaerosolization
experiment and sample analysis
Make sure operation is consistent within
and among tests.
Ensure proper design to maximize fraction
transported to sample filters.
Make sure filtration efficiency is greater
than 99% for particles larger than 1 urn.
Make sure efficiency (most important) is
consistent. (High extraction efficiency is
desired.)
Use a consistent plating procedure.
Handle and transport coupons in a covered
tray (particular issue for refined surrogates).
Uncontrollable
Error
Random variation in test surface
characteristics
Particle migration into surface pores
Particle and surface chemistry at point
of contact
Evaporation or formation of water
meniscus between particle and
surface
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5 Summary
This interim project report describes the progress and current status of the research effort to characterize
the reaerosolization of Ba surrogate spores and particles from outdoor surfaces under different
environmental conditions and compare the data with data from identical experiments carried out using
Ba-Ames. The greatest part of the effort has been expended in design, construction, and validation of
equipment and development and validation of methods. Following is a list of major tasks completed,
underway, and planned through the end of November 2013.
Tasks Completed
• Designed, constructed, and validated reaerosolization wind tunnels for use at EPA and DPG.
• Designed, constructed, and validated wet deposition chambers for use at EPA and DPG.
• Designed and constructed dry deposition chambers for use at EPA and DPG.
• Conducted scouting trials for wet deposition Btk reaerosolization experiments.
• Completed five replicates of wet deposition Btk reaerosolization experiments at EPA.
• Completed three replicates of wet deposition polystyrene latex (PSL) reaerosolization experiments at
EPA.
• Installed all systems at DPG.
• Wet deposition Bacillus atrophaeus subspecies globigii (Bg) reaerosolization experiments at EPA.
• Wet deposition Btk reaerosolization experiments at DPG for RWT validation.
Tasks Currently Underway
• Dry deposition method development (est. completion 10/4/13).
• Additional deposition efficiency validation tests in EPA and DPG wet deposition chambers (est.
completion 10/4/13).
• Dry deposition efficiency validation tests with EPA and DPG chambers (est. completion 10/11/13).
• Scouting trials for dry deposition Btk reaerosolization experiments at EPA (est. completion 10/11/13).
Tasks Planned for Completion before November 30, 2013
• Wet deposition Ba-Ames reaerosolization experiments at DPG (mid-October through November
2013).
• Dry deposition Btk reaerosolization experiments at EPA (10/7/13-10/18/13).
• Dry deposition Bg reaerosolization experiments at EPA (10/21/13 - 11/8/13).
• Dry deposition PSL reaerosolization experiments at EPA (11/11/13 - 11/22/13).
• Dry deposition Ba-Ames reaerosolization experiments at DPG (November 2013).
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6 References
Alion Science and Technology (Alion). 2012. Determination of the Difference in Resuspension of Spores
off of Outdoor Materials, QAPP-ZED-12-02. Durham, NC.
Arcadis U.S. 2009. Serial Dilution: Spread Plate Procedure To Quantify Viable Bacterial Spores, MOP
6535a, Revision 3.0. Durham, NC.
Arcadis U.S. 2010. Recovery Of Bacillus Spores From Wipe Samples, MOP 6567, Revision 0. Durham,
NC.
Arcadis U.S. 2013a. Recovery of Bacillus Spores from Stainless Steel Coupon Samples, MOP 6601,
Revision 0. Durham, NC.
Arcadis U.S. 2013b. Recovery of Bacillus Spores from Polyester Felt Filters, MOP 6600, Revision 0.
Durham, NC.
Carrera, M.; Zandomeni, R.O.; Fitzgibbon, J., and Sagripanti, J.-L. 2007. Difference between the spore
sizes of Bacillus anthracis and other Bacillus species. Journal of Applied Microbiology 102(2): 303-312.
Chinga, G.; Dougherty, R. 2006. Roughness Calculation, http://rsb.info.nih.gov/ij/plugins/roughness.html,
last accessed September 26, 2013.
Environmental Protection Agency (EPA). 2012. On the Use of Bacillus thuringiensis as a Surrogate for
Bacillus anthracis in Aerosol Research. Technical Report, EPA/600/R-12/596. Research Triangle Park,
NC.
Greenberg, D.L.; Busch, J.D.; Keim, P.; and Wagner, D.M. 2010. Identifying experimental surrogates for
Bacillus anthracis spores: a review. Investigative Genetics 1(1): 4.
Hollander, M. and Wolfe, D.A. 1973. Nonparametric Statistical Methods. John Wiley and Sons, New York,
NY, 503 pages.
Imaged. 2011. SurfCharJ plug-in, Version 1c. http://www.gcsca.net/IJ/SurfCharJ.html, last accessed
September 26, 2013.
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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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