United States
Environmental Protection
Agency
EPA/600/R-18/322 | October 2018
www.epa.gov/homeland-security-research
Development of Laboratory
Equipment and protocols for the
Assessment of Rain, Water Wash'
Down, and Channelized Flow for
Removal of Spores on
Urban Surfaces


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mnn
m m
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Office of Research and Development

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EPA/600/R-18/322
September 2018
Development of
Laboratory Equipment and Protocols
for the Assessment of Rain,
Water Wash-Down, and Channelized Flow
for Removal of Spores on Urban Surfaces
Anne Mikelonis and Worth Calfee
U.S. Environmental Protection Agency
Decontamination and Consequence Management Division
National Homeland Security Research Center

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Disclaimer
The U.S. Environmental Protection Agency (EPA) through its Office of Research and
Development funded and managed the research described herein under EP-C-15-008 to Jacobs
Technology Inc. It has been subjected to the Agency's review and has been approved for
publication. Note that approval does not signify that the contents necessarily reflect the views of
the Agency. Any mention of trade names, products, or services does not imply an endorsement
by the U.S. Government or EPA. The EPA does not endorse any commercial products, services,
or enterprises. The contractor role did not include establishing Agency policy.
Questions concerning this document or its application should be addressed to:
Anne Mikelonis, Ph.D., P.E.
U.S. Environmental Protection Agency
Office of Research and Development
National Homeland Security Research Center
Decontamination and Consequence Management Division
109 T.W. Alexander Dr. (MD-E-343-06)
Research Triangle Park, NC 27711
Phone: 919-541-0579

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Table of Contents
1.0 Introduction	8
2.0 Background	8
2.1	Rainfall	8
2.2	Channelized Flow	10
3.0 Rainfall	11
3.1	Characterization Techniques	11
3.2	10-ft. Tall Rainfall Simulator	14
3.3	26-ft. Tall Rainfall Simulator	18
4.0 Channelized Flow	21
5.0 Spray-Down	23
6.0 Quality Assurance	25
7.0 Future Work	28
8.0 References	29
Appendix A. 10-ft. Intensity Heat Maps	31
Appendix B. Spore Washoff Procedure-Rain	36
Appendix C. 10-ft. Rainfall Spore Washoff Results	37
Appendix D. 26-ft. Intensity Heat Maps	45
Appendix E. 26-ft. Rainfall Spore Washoff Results	52
Appendix F. Spore Washoff Procedure-Channel	63
Appendix G. Spore Washoff Data - Channel	65
Appendix H. Channel Velocity Measurements	68
Appendix I. Spore Washoff Procedure-Spray	69
Appendix J. Spore Washoff Data - Spray	72

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List of Figures
Figure 2.1 Continental US precipitation frequency estimate for a two-year recurrence interval
and a one-hour duration rain event	9
Figure 2.2 Continental US precipitation frequency estimate for a 100-year recurrence interval
and a one-hour duration rain event	9
Figure 3.1 10-second Durham, North Carolina, USA rain event data recorded by the Parsivel2. 11
Figure 3.2 Examples of simulated rainfall that did not follow the Gunn-Kinzer curve	12
Figure 3.3 Parsivel2	12
Figure 3.4 Heat map experimental setup	12
Figure 3.5 Example heat map of a nozzle with a non-uniform spray pattern	13
Figure 3.6 Example heat map of a uniform spray pattern	13
Figure 3.7 10-ft. tall rainfall simulator	14
Figure 3.8 10-ft. tall rainfall simulator with mesh screen	14
Figure 3.9 10-ft simulator rainfall removal results (for Bg) at different intensity rain events	17
Figure 3.10 26-ft tall rainfall simulator	18
Figure 3.11 Washoff coupon holders	18
Figure 4.1 Custom Channelized Flow Simulator	21
Figure 4.2 Channelized flow Bg washoff from concrete coupon. Error bars are standard error.. 22
Figure 5.1 Spray Chamber	23
Figure 5.2 Load cell response to different garden hose nozzle tips	24
Figure 5.3 Load cell response to different pressure washer nozzle tips	24
List of Tables
Table 3.1 Rainfall Simulator Design Criteria	11
Table 3.2 10-ft. tall rainfall simulator nozzle summary of intensity and droplet size	15
Table 3.3 26-ft. tall rainfall simulator nozzle summary of intensity and droplet size	19
Table 4.1 Velocities during channelized flow experiments	22
Table 5.1 Pressure washer nozzle spray conditions	23
Table 6.1 Instrument Calibration Frequency	25
Table 6.2 Critical Measurement Acceptance Criteria	26
Table 6.3 QA/QC Sample Acceptance Criteria	27

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Acknowledgments
Contributions of the following individuals and organizations to this report are gratefully
acknowledged:
EPA Project Team
John Archer, ORD/NHSRC/DCMD
Worth Calfee, Ph.D., ORD/NHSRC/DCMD
Sang Don Lee, Ph.D., ORD/NHSRC/DCMD
Anne Mikelonis, Ph.D., P.E., ORD/NHSRC/DCMD
Katherine Ratliff, Ph.D., ORISE Postdoctoral Fellow in ORD/NHSRC/DCMD
Jacobs Technology, Inc. Project Team
Ahmed Abdel-Hady, Jacobs Technology, Inc.
Denise Aslett, Ph.D., Jacobs Technology, Inc.
Lee Brush, Jacobs Technology, Inc.
Jason Colon, Science Systems Applications, Inc.
Kathleen May, Jacobs Technology, Inc.
Joshua Nardin, Jacobs Technology, Inc.
Brian Sechrest, CSS-Dynamac
Ryan Stokes, Jacobs Technology, Inc.
Steve Terll, Jacobs Technology, Inc.
Abderrahmane Touati, Ph.D., Jacobs Technology, Inc.
EPA Technical Reviewers
Leroy Mickelsen, OLEM/OEM/CMAD
Michael Pirhalla, ORD/NHSRC/DCMD
EPA Technical Edit
Joan Bursey, Ph.D., Grantee, The National Caucus and Center on Black Aged, Inc.
EPA Quality Assurance
Eletha Brady Roberts, ORD/NHSRC/IO
Ramona Sherman, ORD/NHSRC/IO

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Acronyms and Abbreviations
ADA	aerosol dose apparatus
Ba	Bacillus anthracis
Bg	Bacillus globigii
Btk	Bacillus thuringiensis var. kurstaki
CONUS	Continental United States
EPA	U.S. Environmental Protection Agency
ft.	foot/feet
h	hour(s)
HSRP	Homeland Security Research Program
ID	identification
in.	inch(es)
ISO	International Organization for Standardization
L	liter(s)
m	meter(s)
|im	micrometer(s)
MDI	metered dose inhaler
mL	milliliter(s)
mm	millimeter(s)
MOP	miscellaneous operating procedure
NHSRC	National Homeland Security Research Center
NIST	National Institute of Standards and Technology
NOAA	National Oceanic and Atmospheric Administration
ORD	EPA Office of Research and Development
Parsivel	PARticle Size and VELocity
PBST	Phosphate buffered saline with 0.05% Tween® 20
PDAQ	portable data acquisition
PVC	polyvinyl chloride
QAPP	Quality Assurance Project Plan
RTP	Research Triangle Park
s	second(s)
SCS	U.S. Soil Conservation Service
TSA	tryptic soy agar
WACOR	Work Assignment Contracting Officer's Representative

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Executive Summary
A large-scale outdoor biological contamination incident requires a better understanding of how
rainfall and water-based decontamination measures would transport contaminants so that
responders can effectively select sampling locations, stage waste, and strategize other recovery
decisions. Over the last few years, the United States Environmental Protection Agency's (EPA's)
Homeland Security Research Program (HSRP), in the Office of Research and Development
(ORD), has built and characterized a laboratory-scale rainfall simulator and a channelized flow
simulator. HSRP has also developed bench-scale power washing testing protocols to study the
movement of Bacillus anthracis (Ba) simulants. The primary focus of this report is to document
the characterization of these apparatuses and discuss the future direction of this work.
Few commercial rainfall simulators are on the market, therefore a custom 10-ft. tall rainfall
simulator was constructed and tested with ten different nozzle configurations over a range of
operating pressures. Various-sized collection bins and a PARticle Size and VELocity (Parsivel)
laser disdrometer (version 2) were used to characterize the simulator for rainfall intensity,
particle size, and velocity. The simulator produced rain events with an intensity range of 0.9 -
6.5 inches (in.)/hour(h). Preliminary washoff experiments were conducted by inoculating
concrete coupons (using a meter dosed inhaler and aerosol deposition device) with Bacillus
globigii (Bg), a simulant for Ba often used in disinfection studies. These spore washoff
experiments removed approximately 5-45% of spores from the coupons within the hour-long
testing window. However, there was no predictable pattern of spore removal by rainfall intensity.
A 26-ft. tall rainfall simulator was therefore constructed to improve the critical shortcomings
(i.e., droplets failing to reach terminal velocity) of the 10-ft. simulator. The 26-ft. tall simulator
was capable of producing rain events that were 0.66 - 4 in./h in intensity. This rainfall range is in
line with being able to simulate up to 100-year, one-hour storm events experienced across the
majority of the continental United States. Currently, 32 coupons (concrete and asphalt) have
been inoculated with Bg or Bacillus thurengiensis kurstaki (Btk), subjected to simulated rain
events, and had spore concentrations quantified in the resulting runoff water. The results of these
tests were provided in the Appendices of this report and will be elaborated upon in forthcoming
publications. Future planned experiments will include rain patterns that involve periods of drying
and variations in rain intensity values.
A custom channelized flow simulator was constructed, and spore washoff experiments were
performed using concrete coupons inoculated with Bg for flows ranging from 25-150
milliliters) (mL)/second (s). Over the course of an hour, approximately 18-35% of spores were
removed from the coupons. However, there were large variations in removal that did not strictly
follow a pattern according to flowrate. A specialized, hydraulically more flexible, benchtop
sediment transport channel was acquired to continue this work using velocities at, above, and
below the predicted threshold of movement.
Highly controlled bench scale spray washing procedures were also evaluated using a load cell to
measure the force of the spray applied. A conventional power washer and garden hose were
used, and brick, asphalt, glass, and concrete coupons inoculated with Bg were tested. At most,
15%) of the spores were removed from the coupon during testing as measured in the runoff water.
For concrete, the total removal was like lower intensity rain events but in a much shorter amount
of time. Experiments examining Btk removal are currently underway. Future work will involve
outdoor testing and explore the use of surfactants as a wash aid.

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1.0 Introduction
The best manner for emergency responders to efficiently and effectively decontaminate a large
outdoor area after a biological release of Bacillus anthracis (Ba) spores is uncertain, both in
terms of technology and process. A large-scale remediation effort will likely take a considerable
length of time to plan and execute. During this time, there is an opportunity for rainfall events to
redistribute contamination. Limited national laboratory capacity and high costs will require
decision makers to target sample locations so maps that highlight potential hotspots this over
time and weather event are desirable. Additional examples of decisions that would be aided by
fate and transport predictions are waste staging locations (i.e., it would be undesirable if waste
areas flooded and washed contamination into clean areas) and evacuation zone delineation.
Contaminate spread due to water may also occur because decision makers choose to pursue
interventions that use water-based washing methods to decontaminate critical outdoor spaces.
Since 2016, the United States Environmental Protection Agency's (EPA's) National Homeland
Security Research Center (NHSRC) has built laboratory capabilities to better understand and
compare the removal of spores from urban surfaces by rain, channelized flow, and washing
(using spray generated from a garden hose and a pressure washer). During this process, several
iterations of custom laboratory equipment and processes were developed. The custom equipment
described in this report was designed and built for these experiments, due to few available off-
the-shelf options. The purpose of this report is to document the characterization of this custom-
built equipment and related processes. As such, this report is separated into three main sections:
1) Rainfall, 2) Overland Flow, and 3) Spray-Down. While biological washoff data have been
collected as part of each stage of this work, this topic is not the focus of this report. Washoff data
are provided in the main body of text only when necessary to illustrate a point relevant to
equipment redesign. However, all washoff data recorded to-date are provided in full detail in the
appendices. These data are still actively being collected. Once complete, several additional
publications thoroughly analyzing these data and providing conclusions and recommendations
regarding the efficacy of different water-based methods at removing spores are planned in
upcoming years. Experiments are also planned to study the removal of spores by different
patterns of rain and dry periods and more complex water matrices.
2.0	Background
2.1	Rainfall
While the current scientific literature lacks research specifically studying the removal of spores
by various water sources (i.e., rain droplets, channelized flow, or spray), there is a wealth of
studies examining the erosivity of soil by rainfall and the washoff of solids from impervious
surfaces by stormwater (e.g., Egodawatta et al., 2007; Shaw et al., 2009; Charbeneau and Barrett,
2016; Gong et al., 2016). Soil erosivity and stormwater washoff have been shown to depend on
rainfall intensity, drop size distribution, and kinetic energy (van Dijk et al., 2002, Panagos et al.,
2017, Wischmeier et al., 1958). Other parameters such as pH, temperature, and organic and
inorganic composition of stormwater may also influence washoff but have not been studied in
detail. Rainfall parameters vary by climatic region across the United States. In any two-year
period, most locations in the continental United States (CONUS) experience less than two in. of
rainfall within any one-hour storm event (Figure 2.1). The largest rain event that most places in
the CONUS will experience within a one-hundred-year period is approximately 4 in./h,

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excluding coastal regions in the southeast and south-central U.S./great plains, likely due to
thunderstorm activity or mesoscale convective systems that last for longer than one hour (Figure
2.2). Very high intensity rainfall values are, however, possible over very short periods of time.
Kilometers
Inches
High : 6
I 5.5
5
4.5
4
3.5
3
2.5
¦ 2
{ 1.5
1
0.5
Low : 0
Data Source: NOAA Atlas 14, Precipitation Frequency Data Server (PFDS):
https://hdsc.nws.noaa.gov/hdsc/pfds/index.html
Figure 2.1 Continental US precipitation frequency estimate for a two-year recurrence interval and a one-hour
duration rain event. (States in white are not yet covered by National Oceanic and Atmospheric
Administration's (NOAA's) Atlas 14 program, but there are active plans to include them in the future.)
Inches
High : 6
5.5
5
4.5
4
3.5
3
2.5
2
1.5
1
0.5
Low : 0
Data Source: NOAA Atlas 14, Precipitation Frequency Data Server (PFDS):
https://hdsc.nws.noaa.gov/hdsc/pfds/index.html
Figure 2.2 Continental US precipitation frequency estimate for a 100-year recurrence interv al and a one-hour
duration rain event. (States in white are not yet covered by NOAA's Atlas 14 program, but there are active
plans to include them in the future.)
Kilometers

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It is important to recognize that intensity is inherently a bulk parameter for rainfall. To
characterize rainfall in more detail (and understand particle detachment and washoff), droplet
size and velocity measurements are necessary. These fundamental parameters also vary by
geography and meteorological event, but the energy relationships and size frequency
distributions that dictate natural rainfall have been studied in detail since the 1940s (Laws, 1941;
Laws and Parsons, 1943). In general, raindrops have median drop size values of less than 4
millimeters (mm) and have corresponding terminal velocities of less than 9 meters (m)/s (van
Dijk et al., 2002). Both values were considered key design criteria for this work.
Over time, rainfall simulators have been constructed in a variety of different styles. Bowyer-
Bower and Burt (1989) and Hall (1970) provide comprehensive reviews of rainfall simulator
designs. In brief, early standard devices were introduced by the United States Soil Conservation
Service (SCS) during the 1930s and 1940s and called the "type F" and "type FA." These
standard devices consisted of two parallel lines of nozzles on each side of an agricultural study
plot that were directed upward to reach a 10-foot maximum spray height before falling to the
ground surface. Type FA worked at reduced pressure compared to the type F, so Type FA was
ultimately favored because it was less expensive to operate. While these devices achieved
intensity values that mimicked naturally-occurring rain events, the drop sizes generated from the
nozzles corresponded only to droplets experienced during lower intensity rain events. Also, their
velocities were lower than the terminal velocities achieved in the natural environment, indicating
that the overall kinetic energy delivered during simulated rain events was not representative of
natural rainstorms and potentially underestimated erosivity resulting from the storm events. To
overcome these shortfalls, artificial rain simulators have evolved creatively. One device used
muslin fabric draped over a horizontal screen of chicken wire with lengths of yarn attached to the
fabric to form larger droplet sizes (Ellison and Pomerene, 1944). Others have used spray nozzles
on a rotating disk (Pall et al., 1983) and telescopic stainless-steel tubing with redistribution
screens (Regmi and Thompson, 2000).
2.2 Channelized Flow
Overland flow of water, also commonly referred to as sheet flow or surface runoff, results when
water cannot infiltrate into the ground and instead moves along the land surface. Hydraulically,
surface runoff is defined as unsteady, shallow, open-channel flow and is represented
mathematically by the Saint-Venant equations (Neelz and Pender, 2009). The flow of surface
runoff is also considered turbulent. The fundamental mechanisms of solids removal during urban
area channelized overland flow (e.g., in roadway gutters) is not a mature area of study. However,
it is reasonable to consider that this area of solids removal shares some of the mechanistic
underpinnings established in the study of sediment transport in river channels, a more robust
field of study. In river sediment transport, the Shields diagram (and variations thereof, including
the Hjulstrom-Sundbog diagram) (Cao et al., 2006) graphically displays the threshold for the
start of sediment movement. Depending on the version of the diagram, the y-axis is provided as
either a dimensionless constant such as a Reynolds number, shear stress, or velocity. The x-axis
represents the diameter of the particle and shading of the diagram indicates how properties of the
flow or sediment affect the threshold of particle movement. If Bacillus anthracis (Ba) spores
(approximate spherical equivalent diameter of 1 micrometer ([j,m) [Chung et al., 2009]) are
assumed to behave like unconsolidated sediment, their movement would start at approximately
0.18 m/s in water (Southard, 2006).


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Diameter i
Figure 3.2 Examples of simulated rainfall that did not follow the Gunn-Kinzer curve.
The collection bin consisted of a plastic receptacle of
size like the coupons used during washoff
experiments (12 in. by 12 in.). Rainfall simulator
water (deionized water) was collected in the bin for a
recorded duration and used to calculate the rain
intensity produced by each nozzle-operating pressure
configuration. The Parsivel2 optical disdrometer was
used to measure drop size distribution and velocity.
The Parsivel2 is a laser-based optical system that
consists of two sensor heads with splash protectors
and measures approximately two feet from the sensor
base to the top of the sensor heads (Figure 3.3). One
side of the sensor is a laser emitter and the other side
is the receiver. The Parsivel2laser-based optical
system produces a horizontal strip of light
approximately 1 in. wide and 7 in. long. If no water
droplets are detected between the emitter and
receiver, then a maximum voltage output is detected.
As precipitation particles pass through the laser
beam, a portion of the beam is blocked, which
reduces the voltage. This voltage drop is correlated
with particle size. The duration of the signal
disturbance is also measured and is used to calculate
the particle velocity. Velocity and particle size are
then used to calculate kinetic energy for each rain
event. Recently, the Parsivel2's performance was
compared to a collocated two-dimensional video
disdrometer (Park et a/., 2017). The two instalments
were in good agreement with respect to droplet size,
intensity, kinetic energy and velocity for rainfall
rates below approximately 0.4 in./h and drop
diameters of 0.02 to 0.16 in. Above 0.8 in./h, the
Figure 3.3 Parsivel2
Figure 3.4 Heat map experimental setup


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3.2 10-ft. Tall Rainfall Simulator
From approximately January 2016
to May 2017, a 10-ft tall rainfall
simulator was used for washoff
experiments (Figure 3.7). The
simulator consisted of a manifold
with locations for up to five
exchangeable misting nozzles
mounted at the top of a metal
frame enclosed by Plexiglass. The
manifold consisted of a 0.25-in.
diameter pipe preceded by a
pressure reducing valve (Watts®
% in. part number LFN45BM1-U,
Water Inlet
Nozzle Manifold & Pressure Gauge
Coupon
Figure 3.7 10-ft. tall rainfall simulator
50 psi). The misting nozzles used
throughout this testing period
were obtained from McMaster-
Carr and Tee-Jet. Although the
manifold was designed to
accommodate up to five nozzles,
only one nozzle (in the center
position) was used for most of the
characterization testing. Table 3.2
summarizes the average droplet size
and intensity parameters for each
nozzle at different operating pressures. The "diffuser", as
referred to in the table, is an optional insert in the nozzle head
and comes in different screen sizes. In general, the operating
pressure did not consistently vary the intensity produced by
each specific nozzle. Also, different nozzles did not display an
appreciable difference in droplet size. More variation in
intensity values was achievable by using different
combinations of nozzles (ranging from a low of 0.9 in/h to the
highest achievable value of 6.5 in/h). However, only the TG-1
nozzle operating at 15 psi produced droplet size and velocity
distributions that followed the Gunn-Kinzer reference curve.
Obtaining spatial uniformity of intensity over the area of the
coupon was also challenging when using the 10-ft. tall
simulator. Appendix A contains all the heat maps that were
collected. In an effort to produce larger droplet sizes, several
simulator characterization tests were conducted using a large mesh screen located several in.
below the nozzle outlets (Figure 3.8 and see Table 3.2 notes). In addition to larger visible
droplets, the presence of the mesh also resulted in much higher intensity values towards the
center of the coupon as compared to the edges (see heat maps marked "mesh" in Appendix A).
Figure 3.8 10-ft. tall rainfall
simulator with mesh screen

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Table 3.2. 10-ft. tall rainfall simulator nozzle summary of intensity and droplet size
12 in. x 12 in. . , t Average
Parsivel Average
Heat Map Pressure Bin _ ^ Kinetic
Nozzle Diiiuser Intensity Droplet „ Notes
Available (psi) Intensity „ / ... Energy
(in./h) size (mm) ,
(in./h) (J/m -h)
TG-1
50
yes
15
1.1
6.2 ± 0.28
1.32 ± 0.01
1782 ± 120
Spray not sufficient to cover 12
in. xl2 in. area
yes
40
1.4
3.6 ±0.28
0.79 ± 0.01
543 ± 60
-
yes
90
5.9
3.1 ±0.59
0.81 ± 0.02
269 ± 63
-
___
15
40
90
0.5
1.0
3.7
7.7 ±0.85
5.7 ±0.24
3.9 ±0.47
1.43 ± 0.02
1.18 ± 0.02
0.82 ± 0.02
2312 ± 316
1622 ± 95
426 ± 53
Parsivel2 spectograph follows
Gunn-Kinzer curve

15
1.7
3.0 ±0.58
0.98 ± 0.03
1069 ± 287
Mesh under nozzle manifold.
Parsivel2 spectograph follows
Gunn-Kinzer curve
yes
yes
40
90
2.6
5.4
6.0 ± 0.74
7.0 ± 1.38
0.98 ± 0.06
0.86 ± 0.01
1783 ± 405
1648 ± 982
Mesh under nozzle manifold.
Generates both mist and large
droplets
Mesh under nozzle manifold.
Generates both mist and large
droplets
100
yes
yes
15
40
1.8
3.3
4.9 ±0.31
3.6 ±0.26
1.25 ± 0.01
0.78 ± 0.01
1339 ± 116
553 ± 86
Spray not sufficient to cover
12Mxl2M area
yes
90
4.8
4.1 ±0.63
0.86 ± 0.02
366 ± 70
-
FL5-VS
50

15


40
produced an uneven droplet spray that cannot be accurately quantified

90
100

15

40


yes
90
SS4.3W
50

15


40 produced an uneven droplet spray that cannot be accurately quantified
90

100

40
1.9
1.4 ±0.11 | 0.81 ± 0.01 | 140 ± 28
-
yes i 40
1.7
0.8 ±0.08 0.62 ± 0.00 47 ± 6
yes | 90
3.7
1.8 ±0.29
0.95 ± 0.01 1 209 ± 59
I 90
5.5
1.4 ±0.25
0.68 ± 0.02 | 72 ± 15



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fall Simulator
•
K
N
During the Summer of 2017, a 26-ft. tall rainfall simulator
was designed and constructed at the EPA's Fluid Modeling
Facility in Research Triangle Park (RTP), North Carolina
(Figure 3.10). 26-ft was the tallest height that could be
accommodated in an indoor facility on campus (a higher
height, in theory, helped more droplets reach terminal
velocity). The staicture was composed of a polyvinyl
chloride (PVC) pipe frame with an approximately 5-ft. by 5-
ft. footprint. The sides of the structure were encased with
thick plastic sheeting to prevent water from spraying outside
a water containment basin. The 26-ft tall simulator was built
with direct plumbing to a deionized water source and a 400-
gallon and a 35-gallon deionized water reservoir next to the
structure. A 3A- horsepower general purpose motorized
pump with pressure gauge was used to supply water via a
hose to the top of the simulator. The nozzle manifold
located at the top-center of the structure was repurposed
from the 10-ft. simulator. The manifold has a capacity to
hold up to five nozzles and has an inlet pressure gauge with
an operating pressure range of 5-100 psi. Additional
accessories for the work area included a computer work
station for operation of the Parsivel2, an area dedicated to
personal protective equipment (harnesses, hardhats, gloves,
and laboratory coats) and a scissor lift for maintenance and
changing nozzles. Coupon holders were also redesigned to
minimize collection of water droplets that did not hit the
coupon. The coupon holders consisted of stainless-steel
exteriors with plastic molds on the interiors to snugly fit
concrete and asphalt coupons (Figure 3.11). In the same
manner as the 10-ft. tall simulator, the 26-ft. simulator was
characterized for uniformity of the spray distribution using
heat maps (Appendix D). Table 3.3 summarizes the average
droplet size and intensity parameters for each nozzle at
different operating pressures.
rainfall simulator
Figure 3.11 Washoff coupon holders

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Table 3.3 26-ft. tall rainfall simulator nozzle summary of intensity and droplet size
12in.xl2in. Parsivel Average _ _
Heat Map Pressure Kinetic Energy
Nozzle Diffuser 	 ... Bin Intensity Intensity Droplet . , Notes
Available (psi) v p J/m2-h
(in./h) (in./h) Size (mm)
TG-1
50
yes
20
0.935
0.73 ±0.16
0.93 ±0.11
207.6 ±54.77
Follows Gunn-Kinzer curve. One hour
heatmap is hotter towards the center, not
an even distribution.
TG-1 (x2)
50
yes
(1 hour
and 22
min.)
20
3.354
2.38 ±0.57
0.73 ±0.04
159 ±40.03
Does not follow Gunn-Kinzer curve. One
hour heatmap is hotter towards bottom
right.
FL5-VS
50
50
yes
yes
40
50
1.829
0.772
0.545 ±0.083
0.55 ± 0.03
0.61 ±0.01
0.61 ±0.01
20.50 ±4.55
54.52 ±6.33
Does not follow the Gunn-Kinzer curve.
Droplets are small. Heat map was collected
over one hour.
Follows Gunn-Kinzer curve but most
droplets are small with few large ones
falling under the curve. Heat map was
collected over one hour.
FL5-VS (x2)
50
yes
50
1.911
0.74 ± 0.16
0.63 ±0.01
37.68 ±24.66
Gunn-Kinzer curve looks very blocky. Heat
map was collected over one hour.
HH-6SQ
NA
yes
(1 hour
and 20
min.)
5
4.512
2.14 ± 0.29
0.69 ±0.07
320.76 ±49.91
Follows Gunn-Kinzer curve. The heat map
showed rainfall not as evenly distributed
during the 20 minute duration test
HH-14WSQ
NA
yes
5
0.671
0.45 ±0.04
0.63 ±0.01
82.73 ±17.21
Follows Gunn-Kinzer curve and heat map
shows even distribution of the droplets.
NA
yes
5
0.640
0.418 ±0.04
0.63 ±0.01
80.18 ±17.05
Follows Gunn-Kinzer curve and heat map
shows even distribution of the droplets.
HH-30WSQ
NA
yes
5
2.859
1.769 ±0.16
0.71 ±0.01
553.28 ±118.28
Follow Gunn-Kinzer curve and heat map
shows even distribution of the droplets
HH-50WSQ
NA
yes
5
4.024
2.53 ±0.57
0.77 ±0.02
1475.43 ±825
Follows Gunn-Kinzer curve and heat map
shows even distribution of the droplets.
Larger particles than previously observed
produced as well.

-------
Table 3.3., continued
12 in. xl2 in. Parsivel Average _
Heat Map Pressure . ^ ^ Kinetic Energy
Nozzle Diffuser Bin Intensity Intensity Droplet , Notes
Available psi ,. ... ,. ... c. , , (J/m2-h)
(in./h) (in./h) Size (mm)
HH-1
NA
yes
iteration 1
5
3.598
3.39 ±0.59
0.76 ±0.24
1475.43 ±825
Follows Gunn-Kinzer curve. Heat map off
center.
NA
yes
iteration 2
5
1.585
1.58 ±0.26
0.67 ±0.01
192.70 ±37.16
Follows Gunn-Kinzer curve, but blocky.
NA
yes
iteration 3
5
1.460
1.26 ±0.162
0.63 ±0.01
132.99 ±18.21
Follows Gunn-Kinzer curve. Heat map
slightly off center.
NA
yes
iteration 4
5
1.280
1.29 ±0.16
0.64 ±0.01
142.23 ± 14.69
Follows Gunn-Kinzer curve. Heat map
slightly off center.
NA
yes
20
3.170
1.60 ±0.25
0.66 ±0.02
27.74 ±18.80
Does not follow Gunn-Kinzer curve. Heat
map off center.
HH-1 (x2)
NA
yes
5
1.950
1.73 ±0.15
0.67 ±0.01
240.27 ±20.73
Follows Gunn-Kinzer curve and has higher
droplets counts. Flowrate drops after first
nozzle, second nozzle not receiving the
same flowrate.
GG-1.5W
NA
yes
5
1.646
1.72 ±0.27
0.67 ±0.02
220.95 ±33.75
Follows Gunn-Kinzer curve, slightly blocky.
Heat map slightly off from center.
GG-2.8W
NA
yes
5
0.915
0.58 ±0.04
0.58 ±0.01
52.05 ±6.52
Follows Gunn-Kinzer curve. Heat map very
evenly distributed
Nozzle Suppliers: Tee-Jet (TG-1, FL5-VS); Spraying Systems Co., iSpray (HH-6SQ, HH-14WSQ, HH-30WSQ, HH-
50WSQ, HH-1, GG-1.5W, GG-2.8W)
The notes in Table 3.3 indicate that the redesign of the simulator made a marked difference in
achieving the design goals of the apparatus. With the additional height, all nozzles, excluding
TG-1 (x2) and FL5-VS at 40 psi, achieved droplet size-velocity combinations that followed the
Gunn-Kinzer curve for natural rain. Ultimately, the final nozzles were selected based upon
having heat maps that demonstrated a uniform spray pattern (see Figure 3.6 for example of a
uniform spray pattern). The nozzles that fit this criterion were HH-1, HH-14WSQ, HH-30WSQ,
HH-50WSQ, and GG-2.8W. These nozzles allowed for an intensity range for the 26-ft. simulator
of approximately 0.66 in/h to 4 in/h, which were also in line with the original design goals.
As of the date of publication, a total of 32 washoff experiments have been conducted using the
26-ft. rainfall simulator. Bg and Btk aerosol spores were deposited onto 12-in. by 12-in. concrete
and asphalt coupons and measured in the runoff water using standard microbiological plating
methods. Bg is traditionally used as a Ba simulant for disinfection research and Btk for aerosol
fate and transport research. Both microorganisms are being tested in this stormwater research for
applicability to historical results from both microorganisms. Appendix E contains the raw data
and rainfall conditions from these runoff experiments. Their analysis will be the subject of future
publications.

-------
4.0 Channelized Flow
A channelized flow simulator was designed to study how spores are removed by sheet flow that
occurs on roads and curbs. The simulator was designed and built from half-inch Plexiglass and
with adjustable feet. The simulator contained a recessed trough for a 14-in. by 14-in. coupon (1-
in.-thick) to be mounted at a 5% slope (Figure 4.1}. During experiments, concrete coupons were
sealed in place to prevent short circuiting. Water was first pumped into a reservoir using an
adjustable pressure gauge, which then flowed over an aluminum foil-covered spillway before
flowing over the concrete surface. The aluminum foil helped to maintain sheet flow. Sheet flow
was achieved for flowrates of 25 rnL/s to 150 mL/s. The simulator contained a removal
collection basin at the outlet orifice to allow for collection of runoff water contaminated with
spores. Appendix F contains a detailed procedure for runoff experiments.
A total of 9 experiments were conducted using the channelized flow simulator (one at 25 mL/s,
uminum Foil Location
Collection basin
ouPon Loi
Pressure gage
Figure 4.1 Custom Channelized Flow Simulator
and two each at 50 mL/s, 75 mL/s, 100 mL/s, and 150 mL/s). Appendix G contains the raw data
(spore counts in collected runoff water) from the experiments shown in Figure 4.2. At the one-
hour time point, more spores were not necessarily removed by higher flow rates (e.g., 75 mL/s
removed more spores than 100 mL/s or 150 mL/s). As indicated by the error bars in Figure 4.2,
there was also variability in the spore removal between tests conducted at the same flow rate.
Since water velocity is a key determinant for inducing movement in river sediment, the
channelized flow simulator was also carefully analyzed for velocity at each of the flow rates used
during the experiments. For the velocity assessment, height of water was measured above each
coupon using a point gauge. The slope of the coupon was accounted for in the calculations.
Velocity was calculated using both volumetric and Manning's Equation approaches (Table 4.1)
(Mays, 2010). Appendix PI contains the raw depth of water data and the equations used to make
the calculations in Table 4.1. The result of the analysis indicates that all experiments were
conducted at similar velocities (i.e., as flow rate increased, so did the water depth, but not water


-------
5.0 Spray-Down
A chamber was used to evaluate the effect of sprayed water
on the removal of spores from concrete, brick, asphalt, and
glass. Coupons were inoculated with spores using an
aerosolized deposition approach and spores were measured in
the spray runoff water. A stainless-steel chamber (40 in. (L) x
38 in. (W) x 36 in. (H), with an acrylic hinged front door was
used for a controlled environment (Figure 5.1). The top of the
chamber included ports through which the test materials were
sprayed. The chamber also contained a sloped basin that led
to an outlet port for sample collection. A horizontal load cell
was used to record the mass (pounds) applied to the
material. Coupons were cut to 5.5-in. * 5.5-in. to match the
load cell dimensions and allowed the force reading to be representative of the water hitting the
coupon. The water delivery system consisted of a conventional garden hose-type bore nozzle (40
psi, with dial that could switch among seven different spray patterns) and a pressure washer rated
at 1600 psi with adjustable slit nozzles. All garden hose nozzles produced a spray that would
cover the entire test coupon, but the pressure washer nozzle tips did not necessarily cover the
coupon. Each pressure washer nozzle type was tested at different distances from the coupon
surface to determine when the entire coupon was covered by the spray (Table 5.1). In Table 5.1,
different pressure washer nozzles are identified according to the spray angle (in degrees). These
designations come predetermined by the manufacturer. "Height" in Table 5.1 refers to the
distance the nozzle was held from the surface of the horizontal surface. "Length" and "width"
refer to the measured spray area that was produced on the horizontal surface at the height and
nozzle condition.
Table 5.1 Pressure washer nozzle spray conditions
Nozzle (°)
Height (ft)
Length (in)
Width (in)
Approximate
Area (in2)
Notes
0
1
3.25
3.25
8.30
All sprays are approximately radial since it is formed
through the perpendicular impact of the water jet
against the concrete surface.
2
4.6
4.6
16.62
3
5.5
5.5
23.76
4
6
6
28.27
5
7.125
7.125
39.87
6
7.5
7.5
44.18
15
1
7
7.25
41 28
All of the widths are generated through splashing,
and therefore approximations. Variance in the width
is affected by unnacounted variables including wind,
slant of the surface, and measurement technique. In
some cases, the center did not seem to get sprayed
with water as much. In instances where the center
did not have the largest width, then the centermost
largest area width was taken.
2
8.25
9
63.62
3
12
9
169.65
4
14.25
11
246.22
5
13.625
9
192.62
6
16
7
175.93
25
1
9
7.5
106.03
2
10.5
6.5
107.21
3
13
7.125
145.49
4
19
8
238.76
5
21.125
8.5
282.06
6
22.5
8.5
300.41
40
1
11.75
4.875
89.98
2
18.375
5.875
169.57
3
21.5
6
202.63
4
25.25
6
237.98
5
33.5
6
315.73
6
43
7
472.81
23
4
Load Cell ar
M
TTjfc fii


/
Jyr ^loped floor for water coilectkajT^^/yJ


-------
During spray down experiments, first uninoculated coupons (procedural blanks) and then
inoculated coupons (test coupons) were mounted in the chamber and sprayed with deionized
water with the runoff water collected and analyzed for spores. Appendix I contains a detailed
procedure and Appendix J contains results of the tests conducted. As of publication, Bg spore
removal data have been collected for both the garden hose and pressure washer from concrete,
brick, asphalt, and glass. At most 15% of spores were removed from the coupons (Appendix J)
The garden hose was just as effective as the pressure washer. Testing to determine the removal
of Btk spores is still underway.
6.0 Quality Assurance
This project used thermometers, stopwatches, micropipettes, scales, pressure gauges, a pH meter,
and a graduated cylinder, calibrated per specifications in Table 6.1.
Table 6.1 Instrument Calibration Frequency
Equipment
Calibration/Certification
Expected Tolerance

Compare to independent NIST thermometer (a thermometer

Thermometer
that is recertified annually by either NIST or an ISO-17025
facility) value once per quarter.
±1 °c
Stopwatch
Compare to official U.S. time (a), time.gov every 30 davs.
Certified as calibrated at time of use. Recalibrated by
± 1 min/30 days
Micropipettes
gravimetric evaluation of performance to manufacturer's
specifications every year.
±5%
Scale
Compare reading to Class S weights every day.
± 1 %
Meter box (pressure
Volume of gas is compared to NIST-traceable dry gas meter
±2%
gauge)
annually.
pH meter
2-point calibration using NIST-traceable buffer solutions
immediately prior to testing
± 0.1 pH units
Graduated cylinder
Collection of effluent at specified time.
+ 1 mL
NIST = National Institute of Standards and Technology; ISO = International Organization for Standardization
The following measurements were deemed critical to accomplishing part or all of the project
objectives: sampling time, sample volume, incubation temperature, count of CFUs, plated volume,
rain rate, and overland flow rate. Data quality indicators (DQIs) for the critical measurements were
used to determine if the collected data met the project objectives. The critical measurement
acceptance criteria are shown in Table 6.2. If the CFU count for bacterial growth does not fall
within the target range, the sample will either be filtered or re-plated. Data shown in the appendices
of this report met the acceptance criteria in Table 6.2.

-------
Table 6.2 Critical Measurement Acceptance Criteria
Critical Measurement
Measurement Device
Accuracy/Precision
Target
Detection
Limit
Time
± 1 min/30 days
± 1 min/30 days
NA
Temperature of
incubation chamber
NIST-traceable thermometer (daily)
± 2 °C
N/A
Counts of CFU
QCount
Check of spiral plater
template that is within
1.82 x 104-2.30 x 104
1 CFU per plate
Plated volume
Collection of effluent at
specified time
Spiral plater
Graduated cylinder
50 % relative standard
deviation among the
triplicate plating
± 1 mL
1 CFU
NA
Collection of effluent
over time
Graduated cylinder and NIST-
calibrated stopwatch
± 5 % of target set point
NA
Rain and overland flow
rate
Graduated cylinder and NIST-
calibrated stopwatch
± 5 % of target set point
NA
Quantitative standards do not exist for biological agents. Quantitative determinations of organisms
in this investigation did not involve the use of analytical measurement devices. Rather, the CFU
were enumerated using a QCount (Advanced Instruments, Norwood, MA, USA) and recorded.
Critical QA/QC checks for the biological results are shown in Table 6-3. Controls and blanks were
included along with the test samples in the experiments so that well-controlled quantitative values
were obtained. Verifying the sterility of samples prior to inoculation and other background checks
were also included as part of the standard protocol of each experiment. Replicate coupons were
also included for each set of test conditions and the following additional types of quality control
samples:
•	Metered dose inhaler (MDI) control coupons: stainless steel coupons inoculated at the
same time as material coupons and sampled by sponge wipe. These coupons are inoculated at
the beginning, middle, and end of each inoculation campaign to assess the stability of the
MDI during the inoculation operation.
•	Procedural blank coupons: sterile coupons that undergo the same sampling process as the
test coupons.
•	Positive control coupons: representative material coupons that are inoculated and sampled
using a wipe sampling technique.
Additionally, a chain of custody form was used when transferring samples from the simulators to
the onsite microbiology laboratory, and a laboratory notebook and an electronic file repository
were maintained. During 2017, this project underwent a laboratory control audit by an external
reviewer and a technical systems audit by NHSRC's quality assurance officers. No substantial
issues were detected during these audits.

-------
Table 6.3 QA/QC Sample Acceptance Criteria
QC Sample
Information
Provided
Frequency
Acceptance Criteria
Corrective Action
Procedural blank
(coupon without
biological agent).
Controls for sterility
of materials and
methods used in the
procedure.
1 per test.
No observed CFU.
Reject results of test
coupons on the same order
of magnitude. Identify and
remove contamination
source.
Positive control
(sample from
material coupon
contaminated with
biological agent
but not subjected
to test conditions).
Initial contamination
level on the coupons;
allows for
determination of log
reduction; controls for
confounds arising
from history impacting
bioactivity; controls
for special causes;
shows plate's ability
to support growth.
3 or more
replicates per test.
For high inoculation,
target loading of 1 x 107
CFU per sample with a
standard deviation of <
0.5 log (5 x 106 - 5 x
107 CFU/sample).
Outside target range:
discuss potential impact on
results with WACOR;
correct loading procedure
for next test and repeat
depending on decided
impact.
Outlier: evaluate/exclude
value.
Blank plating of
microbiological
supplies.
Controls for sterility
of supplies used in
dilution plating.
3 of each supply
per plating event.
No observed growth
following incubation.
Sterilize or dispose of
contamination source. Re-
plate samples.
Blank TSA
sterility control
(plate incubated
but not
inoculated).
Controls for sterility
of plates.
Each plate is
incubated at least
18 but fewer than
24 hours.
No observed growth
following incubation.
All plates are incubated
prior to use. All
contaminated plates will be
discarded.
Procedural blank
samples.
Contamination level
present during
sampling.
1 per sampling
event.
Non-detect.
Clean up environment.
Sterilize sampling materials
before use.
Replicates of
microbiological
dilution plates.
Repeatability of
results.
3 per dilution.
Counts greater than 20
are reportable. Standard
deviation must be
<100 %. Grubbs outlier
test or equivalent.
Sample will be re-plated.
MDI control (wipe
sample from
stainless steel
coupon
contaminated with
biological agent).
Initial contamination
level on coupons.
Shows plate's ability
to support growth.
3 replicates per
MDI use.
Target loading CFU per
sample with a standard
deviation of < 0.5 log.
No evidence of MDI
decay during
inoculation event.
Grubbs outlier test (or
equivalent).
Outside target range:
discuss potential impact on
results with WACOR;
correct loading procedure
for next test and repeat
depending on decided
impact.
Outlier: evaluate stability
ofMDIs.
Pressure of hose
Pressure impacts flow
rate and spray pattern
Per use
4M6 psi
Correct pressure according
to manufacturer's
directions; replace if
problem cannot be resolved

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7.0 Future Work
Decision makers need data and maps to help determine if evacuation zones need to be shifted,
what portions of their infrastructure should be cleaned, where they should situation waste and
how this could change after different sized rain events. Washoff data collection provides not only
a rough understanding of how "sticky" a certain contaminant is, but can also be used to develop
mathematical models that power simulations. The laboratory capabilities described in this report
are tools that can be used on most contaminants of concern and expanded upon to answer
specific questions about certain hydraulic conditions.
The 26-ft. tall rainfall simulator will continue to be used to study spore washoff by different
simulated rain events. Now that its nozzles are well-characterized and produce realistic rain
events for a constant duration intensity, we plan to investigate washoff from rain events with
different patterns of intensity and drying. We also plan to explore the influence of stormwater
quality on the adhesion of both types of simulants. These experiments will have additions of
natural organic matter and metals (e.g. copper and zinc) to the water. The rainfall simulator is
available for washoff studies of chemical and radiological (with safety enhancements)
particulates in addition to biological. A related effort within NHSRC has involved building a
physical model of a portion of a city. The rainfall simulator may be used to simulate storm events
to verify hydraulic models. Ultimately, the washoff curves produced with this simulator will be
used to inform parameterization of stormwater models so that the movement of spores and other
contaminants during rain events may be translated into maps used by emergency responders and
decision makers for sampling, public health decisions, and remediation. These mapping activities
can be combined with other mapping products (e.g., transportation logistics, affected
infrastructure) to allow decision makers to make their decisions based on a situational awareness
of the complex system of systems that are in play during a wide-area response.
The channelized flow experiments will be repeated using Bg and Btk in the Armfield sediment
channel to better understand the velocity conditions that induce movement of spores and to
understand the interplay between material type and movement of spores. Velocity will become a
critical measurement instead of flow for these experiments. Eventually this channel may also be
used to study the fate and transport of spores and other contaminants in river systems.
Finally, the pressure washing laboratory experiments will be completed using Btk spores, and
outdoor tests are being planned to compare to the laboratory results for removal efficacy. Initial
outdoor scoping tests using a pressure washer indicate that it is difficult to generate large enough
volumes of runoff water to collect the spores that have been dislodged. Outdoor tests will seek to
operationalize a rinse procedure that generates collectable volumes of water. Surfactants will
also be considered as wash aids.

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8.0 References
Angulo-Martinez, M., & Barros, A. P. (2015). Measurement uncertainty in rainfall kinetic
energy and intensity relationships for soil erosion studies: An evaluation using PARSIVEL
disdrometers in the Southern Appalachian Mountains. Geomorphology, 228, 28-40.
Bowyer-Bower, T. A. S., & Burt, T. P. (1989). Rainfall simulators for investigating soil response
to rainfall. Soil Technology, 2(1), 1-16.
Cao, Z., Pender, G., & Meng, J. (2006). Explicit formulation of the Shields diagram for incipient
motion of sediment. Journal of Hydraulic Engineering, 732(10), 1097-1099.
Charbeneau, R. J., & Barrett, M. E. (1998). Evaluation of methods for estimating stormwater
pollutant loads. Water Environment Research, 70(7), 1295-1302.
Chung, E., Kweon, H., Yiacoumi, S., Lee, I., Joy, D. C., Palumbo, A. V., & Tsouris, C. (2009).
Adhesion of spores of Bacillus thuringiensis on a planar surface. Environmental Science &
Technology, 44( 1), 290-296.
Egodawatta, P., Thomas, E., & Goonetilleke, A. (2007). Mathematical interpretation of pollutant
wash-off from urban road surfaces using simulated rainfall. Water Research, 41(13), 3025-3031.
Ellison, W. D., & Pomerene, W. H. (1944). A rainfall applicator. Agr. Eng, 25, 220.
Gong, Y., Liang, X., Li, X., Li, J., Fang, X., & Song, R. (2016). Influence of rainfall
characteristics on total suspended solids in urban runoff: A case study in Beijing, China. Water,
8(1), 278.
Gunn, R., & Kinzer, G. D. (1949). The terminal velocity of fall for water droplets in stagnant air.
Journal of Meteorology, 6(4), 243-248.
Hall, M. J. (1970). A critique of methods of simulating rainfall. Water Resources Research, 6(4),
1104-1114.
Kathiravelu, G., Lucke, T., & Nichols, P. (2016). Rain drop measurement techniques: a review.
Water, 5(1), 29.
Laws, J. O. (1941). Measurements of the fall-velocity of water-drops and raindrops. Eos,
Transactions American Geophysical Union, 22(3), 709-721
Laws, J. O., & Parsons, D. A. (1943). The relation of raindrop-size to intensity. Eos,
Transactions American Geophysical Union, 24(2), 452-460.
Mays, L. W. (2010). Water Resources Engineering. John Wiley & Sons.
Neelz, S., & Pender, G. (2009). Desktop review of 2D hydraulic modelling packages, Science
Report SC080035, Joint UK Defra/Environment Agency Flood and Coastal Erosion. Risk
Management R&D Program.
Pall, R., Dickinson, W. T., Beals, D., & McGirr, R. (1983). Development and calibration of a
rainfall simulator. Canadian Agricultural Engineering, 25(2), 181-187.

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Panagos, P., Borrelli, P., Meusburger, K., Yu, B., Klik, A., Lim, K.J., Yang, J.E., Ni, J., Miao,
C., Chattopadhyay, N., Sadeghi, S.H., (2017). "Global rainfall erosivity assessment based on
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Park, S. G., Kim, H. L., Ham, Y. W., & Jung, S. H. (2017). Comparative evaluation of the OTT
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Raupach, T. H., & Berne, A. (2015). Correction of raindrop size distributions measured by
Parsivel disdrometers, using a two-dimensional video disdrometer as a reference. Atmospheric
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Regmi, T. P., & Thompson, A. L. (2000). Rainfall simulator for laboratory studies. Applied
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Van Dijk, A. I. J. M., Bruijnzeel, L. A., & Rosewell, C. J. (2002). Rainfall intensity-kinetic
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Appendix B. Spore Washoff Procedure-Rain
Preparation
Sterilize the following materials in the airlock (4 hours, 200 ppm vaporous hydrogen peroxide):
•	Aerosol dose apparatuses (ADAs) (6)
•	Collection bin with coupon holder (1)
•	Mesh droplet dispersion screen (1)
Sterilize the following materials in the autoclave (250°C gravity cycle):
•	Stainless steel coupons (4)
•	Concrete coupons (3, used coupons re-sterilized in autoclave)
Sterilize the deionized water tank as follows:
•	Pour 50 mL of germicidal bleach into the tank and fill the tank with deionized water. (Let
sit for at least one hour.)
•	Run the tank water through the pump bypass; empty the tank into the sink
•	Fill and empty the tank two times with deionized water; run rinse water through hose to
outside storm drain
•	Fill the tank with deionized water. Test that the free available chlorine is non-detectable
at outlet end. Take a 50-mL sample from both the tank and from the nozzle head to test
sterility.
Day 1
•	Set up tables with bench liner. Remove a metered dose inhaler (E7) from refrigerator.
•	Assemble concrete coupons on table with AD As.
•	Assemble three stainless steel coupons on table with AD As.
•	Perform sterility swabs on a concrete coupon, a stainless-steel coupon, an ADA, mesh,
and manifold
•	Label the coupons with sample ID per QAPP
Day 2
•	Center the nozzle apparatus and set to achieve desired rainfall intensity. Take several
flow checks using a collection bin to verify the desired rate is being achieved, and adjust
settings if necessary.
•	Aseptically place the first concrete coupon into the sterilized bin/coupon holder. Ensure
the coupon is centered underneath the apparatus.
•	Turn rainmaker on and collect the first 5 seconds of runoff in a falcon tube. After this
initial collection periodically collect a 5-second sample after 6, 10, 18, 32, and 60
minutes.
•	Stop the rainmaker and open the outlet valve.
•	Remove the concrete coupon from the coupon holder and set aside. Wipe down the
collection bin with dispatch wipes, sodium thiosulfate wipes, and ethyl alcohol. Run
sterile water through the bin and collection valve; take a free available chlorine
measurement of a rinse sample to verify non-detect for chlorine. Take a sterility swab
sample of the interior of the bin.
•	Wipe sample the inoculation control coupons.
•	Deliver samples to onsite microbiology laboratory with chain of custody for analysis.

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Appendix C. 10-ft. Rainfall Spore Washoff Results
These data were generated using the nozzles outlined in the "description" column of the table.
The colony forming unit (CFU) values were obtained using the NHSRC onsite microbiology lab
at EPA's research facility in Research Triangle Park (RTP)'s miscellaneous operating procedure
(MOP) 6608 for spiral plating, incubating, and enumerating target organisms. The CFU values
reported in this Appendix represent the number of spores collected in the runoff water sample at
the notated timestamp.
Description
Sample ID
CFU
Timestamp
(s from start)
Log CFU
Rain Test with mesh
(2 Ml nozzles)
Bg spores-dry deposition
0.5 in/h
Replicate 1
59-AR0.5-Dl-C-L-la
2.94E+04
0
4.47
59-AR0.5-Dl-C-L-la
4.03E+04
5
4.61
59-AR0.5-Dl-C-L-la
2.51E+04
18
4.40
59-AR0.5-Dl-C-L-la
4.33E+04
40
4.64
59-AR0.5-Dl-C-L-la
2.98E+04
88
4.47
59-AR0.5-Dl-C-L-la
3.43E+04
188
4.54
59-AR0.5-Dl-C-L-la
1.32E+04
397
4.12
59-AR0.5-Dl-C-L-la
1.04E+04
829
4.01
59-AR0.5-Dl-C-L-la
8.00E+03
1727
3.90
59-AR0.5-Dl-C-L-la
3.05E+03
3592
3.48
Rain Test with mesh
(2 Ml nozzles)
Bg spores-dry deposition
0.5 in/h
Inoculation Control
AR0.5/AR6-D1-SS-W-1
2.02E+07
NA
7.31
AR0.5/AR6-D1-SS-W-2
3.70E+07
NA
7.57
AR0.5/AR6-D1-SS-W-3
2.80E+07
NA
7.45
Rain Test with mesh
(2 Ml nozzles)
Bg spores-dry deposition
0.5 in/h
Replicate 2
59-AR0.5-D2-C-L-la
1.57E+05
0
5.20
59-AR0.5-D2-C-L-lb
6.30E+04
5
4.80
59-AR0.5-D2-C-L-lc
3.78E+04
17
4.58
59-AR0.5-D2-C-L-ld
7.73E+04
40
4.89
59-AR0.5-D2-C-L-le
2.29E+04
88
4.36
59-AR0.5-D2-C-L-lf
1.52E+04
188
4.18
59-AR0.5-D2-C-L-lg
3.12E+04
397
4.49
59-AR0.5-D2-C-L-lh
6.95E+03
829
3.84
59-AR0.5-D2-C-L-li
2.55E+03
1727
3.41
59-AR0.5-D2-C-L-lj
4.21E+03
3592
3.62
Rain Test with mesh
(2 Ml nozzles)
Bg spores-dry deposition
0.5 in/h
Inoculation Control
59-AR0.5/AR6-D2-SS-W-
1
4.96E+07
inoculation
control
7.70
59-AR0.5/AR6-D2-SS-W-
2
3.84E+07
inoculation
control
7.58
59-AR0.5/AR6-D2-SS-W-
3
4.34E+07
inoculation
control
7.64

-------
Description
Sample ID
CFU
Timestamp
(s from start)
Log CFU
Rain Test with mesh
(1 M3 nozzle)
Bg spores-dry deposition
1 in/h
Replicate 1
59-ARl-Dl-C-L-la
4.79E+04
0
4.68
59-ARl-Dl-C-L-lb
1.88E+04
5
4.27
59-ARl-Dl-C-L-lc
1.43E+04
17
4.15
59-ARl-Dl-C-L-ld
5.16E+03
91
3.71
59-ARl-Dl-C-L-le
9.54E+03
113
3.98
59-ARl-Dl-C-L-lf
6.30E+03
230
3.80
59-ARl-Dl-C-L-lg
1.11E+04
397
4.05
59-ARl-Dl-C-L-lh
1.79E+04
829
4.25
59-ARl-Dl-C-L-li
1.65E+04
1727
4.22
59-ARl-Dl-C-L-lj
4.24E+03
3592
3.63
Rain Test with mesh
(1 M3 nozzle)
Bg spores-dry deposition
1 in/h
Inoculation Control
59-AR1-D1/D2-SS-W-1
2.98E+07
NA
7.47
59-AR1-D1/D2-SS-W-2
5.42E+07
NA
7.73
59-AR1-D1/D2-SS-W-3
4.86E+07
NA
7.69
Rain Test with mesh
(1 M3 nozzle)
Bg spores-dry deposition
1 in/h
Replicate 2
59-ARl-D2-C-L-la
1.99E+04
0
4.30
59-ARl-D2-C-L-lb
8.40E+03
5
3.92
59-ARl-D2-C-L-lc
2.58E+03
17
3.41
59-ARl-D2-C-L-ld
9.47E+03
40
3.98
59-ARl-D2-C-L-le
1.14E+04
83
4.06
59-ARl-D2-C-L-lf
5.48E+03
188
3.74
59-ARl-D2-C-L-lg
2.53E+04
397
4.40
59-ARl-D2-C-L-lh
2.35E+04
829
4.37
59-ARl-D2-C-L-li
6.80E+03
1727
3.83
59-ARl-D2-C-L-lj
3.60E+03
3592
3.56
Rain Test with mesh
(1 M3 nozzle)
Bg spores-dry deposition
1 in/h
Inoculation Control
59-AR1-D1/D2-SS-W-1
2.98E+07
NA
7.47
59-AR1-D1/D2-SS-W-2
5.42E+07
NA
7.73
59-AR1-D1/D2-SS-W-3
4.86E+07
NA
7.69

-------
Description
Sample ID
CFU
Timestamp
(s from start)
Log CFU
Rain Test with mesh
(M1 + M3 + M5)
Bg spores-dry deposition
3 in/h
Replicate 1
59-AR3-D2-C-L-la
4.37E+05
0
5.64
59-AR3-D2-C-L-lb
3.73E+04
5
4.57
59-AR3-D2-C-L-lc
3.17E+04
17
4.50
59-AR3-D2-C-L-ld
4.11E+04
40
4.61
59-AR3-D2-C-L-le
6.91E+04
88
4.84
59-AR3-D2-C-L-lf
4.24E+04
188
4.63
59-AR3-D2-C-L-lg
2.00E+04
397
4.30
59-AR3-D2-C-L-lh
4.03E+03
829
3.61
59-AR3-D2-C-L-li
7.82E+03
1729
3.89
59-AR3-D2-C-L-lj
2.54E+03
3592
3.41
Rain Test with mesh
(Ml + M3 + M5)
Bg spores-dry deposition
3 in/h
Inoculation Control
59-AR3-D2/D3-SS-W-1
4.78E+07
NA
7.68
59-AR3-D2/D3-SS-W-2
3.76E+07
NA
7.58
59-AR3-D2/D3-SS-W-3
4.50E+07
NA
7.65
Rain Test with mesh
(M1 + M3 + M5)
Bg spores-dry deposition
3 in/h
Replicate 2
59-AR3-D3-C-L-la
9.20E+04
0
4.96
59-AR3-D3-C-L-lb
1.45E+05
6
5.16
59-AR3-D3-C-L-lc
1.33E+05
17
5.12
59-AR3-D3-C-L-ld
7.26E+04
45
4.86
59-AR3-D3-C-L-le
6.09E+04
88
4.78
59-AR3-D3-C-L-lf
4.13E+04
188
4.62
59-AR3-D3-C-L-lg
2.14E+04
397
4.33
59-AR3-D3-C-L-lh
2.96E+04
829
4.47
59-AR3-D3-C-L-li
6.93E+03
1727
3.84
59-AR3-D3-C-L-lj
1.84E+03
3592
3.26
Rain Test with mesh
(Ml + M3 + M5)
Bg spores-dry deposition
3 in/h
Inoculation Control
59-AR3-D2/D3-SS-W-1
4.78E+07
NA
7.68
59-AR3-D2/D3-SS-W-2
3.76E+07
NA
7.58
59-AR3-D2/D3-SS-W-3
4.50E+07
NA
7.65

-------
Description
Sample ID
CFU
Timestamp
(s from start)
Log CFU

59-AR6-Dl-C-L-la
2.78E+04
0
4.44

59-AR6-Dl-C-L-lb
4.12E+04
5
4.61

59-AR6-Dl-C-L-lc
2.62E+04
17
4.42
Rain Test with mesh
59-AR6-Dl-C-L-ld
2.30E+04
40
4.36
(2 M15 + 1 M5)
Bg spores-dry deposition
6 in/h
Replicate 1
59-AR6-Dl-C-L-le
1.21E+05
88
5.08
59-AR6-Dl-C-L-lf
6.10E+04
188
4.79
59-AR6-Dl-C-L-lg
1.94E+04
397
4.29
59-AR6-Dl-C-L-lh
1.52E+04
829
4.18

59-AR6-Dl-C-L-li
6.64E+03
1727
3.82

59-AR6-Dl-C-L-lj
8.31E+03
3592
3.92
Rain Test with mesh
AR0.5/AR6-D1-SS-W-1
2.02E+07
NA
7.31
(2 M15 + 1 M5)
AR0.5/AR6-D1-SS-W-2
3.70E+07
NA
7.57
Bg spores-dry deposition




6 in/h
Inoculation Control
AR0.5/AR6-D1-SS-W-3
2.80E+07
NA
7.45

59-AR6-D2-C-L-la
9.02E+04
0
4.96

59-AR6-D2-C-L-lb
1.47E+05
5
5.17

59-AR6-D2-C-L-lc
2.42E+05
17
5.38
Rain Test with mesh
59-AR6-D2-C-L-ld
1.16E+05
61
5.07
(2 M15 + 1 M5)
Bg spores-dry deposition
6 in/h
Replicate 1
59-AR6-D2-C-L-le
1.18E+05
88
5.07
59-AR6-D2-C-L-lf
4.22E+04
188
4.63
59-AR6-D2-C-L-lg
1.74E+04
397
4.24
59-AR6-D2-C-L-lh
1.66E+04
829
4.22

59-AR6-D2-C-L-li
1.08E+04
1727
4.03

59-AR6-D2-C-L-lj
2.67E+03
3592
3.43
Rain Test with mesh
59-AR0.5/AR6-D2-SS-W-
1
4.96E+07
NA
7.70
(2 M15 + 1 M5)
Bg spores-dry deposition




59-AR0.5/AR6-D2-SS-W-
9
3.84E+07
NA
7.58
6 in/h
Inoculation Control




59-AR0.5/AR6-D2-SS-W-
3
4.34E+07
NA
7.64

-------
Description
Sample ID
CFU
Time stamp
(s from start)
Log CFU
Rain Test no mesh
(2 Ml nozzles)
Bg spores-dry deposition
0.5 in/h
Replicate 1
59-AR0.5N-Dl-C-L-la
8.31E+03
0
3.92
59-AR0.5N-Dl-C-L-lb
1.56E+04
5
4.19
59-AR0.5N-Dl-C-L-lc
2.52E+04
17
4.40
59-AR0.5N-Dl-C-L-ld
3.60E+03
40
3.56
59-AR0.5N-Dl-C-L-le
6.35E+03
88
3.80
59-AR0.5N-Dl-C-L-lf
9.42E+03
188
3.97
59-AR0.5N-Dl-C-L-lg
6.47E+03
397
3.81
59-AR0.5N-Dl-C-L-lh
2.88E+03
829
3.46
59-AR0.5N-Dl-C-L-li
3.09E+03
1727
3.49
59-AR0.5N-Dl-C-L-lj
2.79E+03
3592
3.45
Rain Test no mesh
(2 Ml nozzles)
Bg spores-dry deposition
0.5 in/h
Inoculation Control
59-AR0.5N-D1/D2-SS-W-
1
3.24E+07
NA
7.51
59-AR0.5N-D1/D2-SS-W-
2
4.42E+07
NA
7.65
59-AR0.5N-D1/D2-SS-W-
3
3.26E+07
NA
7.51
Rain Test no mesh
(2 Ml nozzles)
Bg spores-dry deposition
0.5 in/h
Replicate 2
59-AR0.5N-D2-C-L-la
4.13E+04
0
4.62
59-AR0.5N-D2-C-L-lb
4.20E+04
5
4.62
59-AR0.5N-D2-C-L-lc
1.94E+04
17
4.29
59-AR0.5N-D2-C-L-ld
3.40E+03
40
3.53
59-AR0.5N-D2-C-L-le
3.15E+03
88
3.50
59-AR0.5N-D2-C-L-lf
3.12E+03
188
3.49
59-AR0.5N-D2-C-L-lg
1.24E+04
397
4.09
59-AR0.5N-D2-C-L-lh
9.59E+02
829
2.98
59-AR0.5N-D2-C-L-li
3.75E+03
1727
3.57
59-AR0.5N-D2-C-L-lj
5.83E+02
3592
2.77
Rain Test no mesh
(2 Ml nozzles)
Bg spores-dry deposition
0.5 in/h
Inoculation Control
59-AR0.5N-D1/D2-SS-W-
1
3.24E+07
NA
7.51
59-AR0.5N-D1/D2-SS-W-
2
4.42E+07
NA
7.65
59-AR0.5N-D1/D2-SS-W-
3
3.26E+07
NA
7.51

-------
Description
Sample ID
CFU
Timestamp
(s from start)
Log CFU
Rain Test no mesh
(1 M3 nozzle)
Bg spores-dry deposition
1 in/h
Replicate 1
59-ARlN-Dl-C-L-la
1.73E+05
0
5.24
59-ARlN-Dl-C-L-lb
4.58E+04
5
4.66
59-ARlN-Dl-C-L-lc
1.74E+04
35
4.24
59-ARlN-Dl-C-L-ld
1.11E+04
60
4.05
59-ARlN-Dl-C-L-le
1.39E+04
210
4.14
59-ARlN-Dl-C-L-lf
5.04E+03
219
3.70
59-ARlN-Dl-C-L-lg
1.40E+04
397
4.15
59-ARlN-Dl-C-L-lh
3.25E+03
829
3.51
59-ARlN-Dl-C-L-li
3.38E+03
1727
3.53
59-ARlN-Dl-C-L-lj
1.44E+03
3592
3.16
Rain Test no mesh
(1 M3 nozzle)
Bg spores-dry deposition
1 in/h
Inoculation Control
59-AR1N-D1/D2-SS-W-1
6.18E+07
NA
7.79
59-AR1N-D1/D2-SS-W-2
1.09E+08
NA
8.04
59-AR1N-D1/D2-SS-W-3
9.66E+07
NA
7.98
Rain Test no mesh
(1 M3 nozzle)
Bg spores-dry deposition
1 in/h
Replicate 2
59-ARlN-D2-C-L-la
7.58E+04
0
4.88
59-ARlN-D2-C-L-lb
3.44E+04
5
4.54
59-AR1N-D2-C-L-1C
3.67E+04
17
4.56
59-ARlN-D2-C-L-ld
2.51E+04
42
4.40
59-ARlN-D2-C-L-le
7.05E+04
88
4.85
59-ARlN-D2-C-L-lf
2.70E+04
188
4.43
59-ARlN-D2-C-L-lg
1.07E+04
397
4.03
59-ARlN-D2-C-L-lh
6.56E+03
829
3.82
59-ARlN-D2-C-L-li
2.72E+03
1727
3.44
59-ARlN-D2-C-L-lj
3.00E+03
3592
3.48
Rain Test no mesh
(1 M3 nozzle)
Bg spores-dry deposition
1 in/h
Inoculation Control
59-AR1N-D1/D2-SS-W-1
6.18E+07
NA
7.79
59-AR1N-D1/D2-SS-W-2
1.09E+08
NA
8.04
59-AR1N-D1/D2-SS-W-3
9.66E+07
NA
7.98
Rain Test no mesh
(M1 + M3 + M5)
Bg spores-dry deposition
3 in/h
Replicate 1
59-AR3N-Dl-C-L-la
8.06E+03
0
3.91
59-AR3N-Dl-C-L-lb
4.91E+03
5
3.69
59-AR3N-Dl-C-L-lc
1.05E+04
17
4.02
59-AR3N-Dl-C-L-ld
8.66E+03
40
3.94
59-AR3N-Dl-C-L-le
7.56E+03
88
3.88
59-AR3N-Dl-C-L-lf
4.62E+03
188
3.66
59-AR3N-Dl-C-L-lg
1.93E+03
397
3.28
59-AR3N-Dl-C-L-lh
6.91E+03
829
3.84
59-AR3N-Dl-C-L-li
1.79E+03
1727
3.25
59-AR3N-Dl-C-L-lj
1.09E+03
3592
3.04

-------
Description
Sample ID
CFU
Timestamp
(s from start)
Log CFU
Rain Test no mesh
(Ml + M3 + M5)
Bg spores-dry deposition
3 in/h
Inoculation Control
59-AR3N-D1/D2-SS-W-1
4.58E+07
NA
7.66
59-AR3N-D1/D2-SS-W-2
3.66E+07
NA
7.56
59-AR3N-D1/D2-SS-W-3
4.70E+07
NA
7.67
Rain Test no mesh
(M1 + M3 + M5)
Bg spores-dry deposition
3 in/h
Replicate 2
59-AR3N-D2-C-L-la
0
0
0
59-AR3N-D2-C-L-lb
7.34E+03
5
3.87
59-AR3N-D2-C-L-lc
7.03E+03
17
3.85
59-AR3N-D2-C-L-ld
4.49E+03
40
3.65
59-AR3N-D2-C-L-le
2.06E+04
88
4.31
59-AR3N-D2-C-L-lf
1.27E+04
188
4.10
59-AR3N-D2-C-L-lg
4.14E+01
397
1.62
59-AR3N-D2-C-L-lh
5.95E+03
829
3.77
59-AR3N-D2-C-L-li
2.43E+03
1727
3.38
59-AR3N-D2-C-L-lj
7.20E+02
3592
2.86
Rain Test no mesh
(M1 + M3 + M5)
Bg spores-dry deposition
3 in/h
Inoculation Control
59-AR3N-D1/D2-SS-W-1
4.58E+07
NA
7.66
59-AR3N-D1/D2-SS-W-2
3.66E+07
NA
7.56
59-AR3N-D1/D2-SS-W-3
4.70E+07
NA
7.67
Rain Test no mesh
(2 M15 + 1 M5)
Bg spores-dry deposition
6 in/h
Replicate 1
59-AR6N-Dl-C-L-la
2.78E+03
0
3.44
59-AR6N-Dl-C-L-lb
1.07E+04
5
4.03
59-AR6N-Dl-C-L-lc
1.70E+04
17
4.23
59-AR6N-Dl-C-L-ld
1.87E+04
40
4.27
59-AR6N-Dl-C-L-le
9.20E+03
88
3.96
59-AR6N-Dl-C-L-lf
7.26E+03
188
3.86
59-AR6N-Dl-C-L-lg
1.54E+04
397
4.19
59-AR6N-Dl-C-L-lh
1.15E+04
829
4.06
59-AR6N-Dl-C-L-li
5.46E+03
1727
3.74
59-AR6N-Dl-C-L-lj
2.20E+03
3592
3.34
Rain Test with mesh
(2 M15 + 1 M5)
Bg spores-dry deposition
6 in/h
Inoculation Control
59-AR6N-D1/D2-SS-W-1
2.68E+07
NA
7.43
59-AR6N-D1/D2-SS-W-2
4.72E+07
NA
7.67
59-AR6N-D1/D2-SS-W-3
5.66E+07
NA
7.75

-------
Description
Sample ID
CFU
Timestamp
(s from start)
Log CFU
Rain Test with mesh
(2 M15 + 1 M5)
Bg spores-dry deposition
6 in/h
Replicate 2
59-AR6N-D2-C-L-la
8.45E+04
0
4.93
59-AR6N-D2-C-L-lb
2.86E+04
5
4.46
59-AR6N-D2-C-L-lc
8.14E+04
17
4.91
59-AR6N-D2-C-L-ld
1.01E+05
40
5.00
59-AR6N-D2-C-L-le
6.52E+04
88
4.81
59-AR6N-D2-C-L-lf
7.35E+04
188
4.87
59-AR6N-D2-C-L-lg
3.36E+04
397
4.53
59-AR6N-D2-C-L-lh
1.14E+04
829
4.06
59-AR6N-D2-C-L-li
1.68E+03
1727
3.23
59-AR6N-D2-C-L-lj
8.79E+02
3592
2.94
Rain Test with mesh
(2 M15 + 1 M5)
Bg spores-dry deposition
6 in/h
Inoculation Control
59-AR6N-D1/D2-SS-W-1
2.68E+07
NA
7.43
59-AR6N-D1/D2-SS-W-2
4.72E+07
NA
7.67
59-AR6N-D1/D2-SS-W-3
5.66E+07
NA
7.75








-------
Appendix E. 26-ft. Rainfall Spore Washoff Results
These data were generated using the conditions specified in the top portion of the table. The
colony forming unit (CFU) values reported for each test coupon were obtained using the NHSRC
onsite microbiology lab at EPA's research facility in Research Triangle Park (RTP)'s
miscellaneous operating procedure (MOP) 6608 for spiral plating, incubating, and enumerating
target organisms. The CFU values reported in this Appendix represent the number of spores
collected in the runoff water sample at the notated timestamp.
0.6 in./h


Deposition
Dry








Test ID
59-AR0.6








Material Type
Concrete








Spore Type
Bg







Nozzle Part Number
HH-14WSQ






Droplet Volume Mean Diameter
0.627 mm







Kinetic Energy
727 J/(m2h)





Number of Nozzles Used during Test
1







Sample Collection Time
5 s






Intensity (20 min bin measurement)
0.64 in/h






Intensity (heat map)
0.56 in/h






Intensity (Parsivel2)
0.41 in/h






Positive Control
2.17E+07 CFU






Positive Control Stdev
5.99E+06 CFU







Coupon 1


Coupon 2


Coupon 3

Time
Average

Collected
Average
Sample
Collected
Average
Sample
Collected
Stamp
Liquid
Analyzed
Volume
Liquid
Volume
Volume
Liquid
Volume
Volume
(s)
CFU
Volume (mL)
(mL)
CFU
(mL)
(mL)
CFU
(mL)
(mL)
5
5.06E+03
22.20
2.20
4.77E+03
22.10
2.10
5.78E+03
23.10
3.10
10
2.43E+03
21.90
1.90
4.62E+03
22.00
2.00
6.04E+03
22.80
2.80
25
5.18E+02
20.70
0.70
2.82E+03
21.70
1.70
2.05E+02
20.50
0.50
35
5.49E+02
21.10
1.10
1.70E+03
21.30
1.30
1.68E+03
21.50
1.50
95
2.14E+03
22.30
2.30
5.61E+02
20.40
0.40
1.91E+03
22.00
2.00
185
3.21E+03
22.00
2.00
7.79E+02
20.50
0.50
1.77E+03
21.60
1.60
455
1.89E+03
22.80
2.80
6.27E+02
20.90
0.90
6.06E+02
20.90
0.90
845
3.00E+02
21.40
1.40
1.03E+03
21.00
1.00
1.29E+03
21.90
1.90
1805
5.86E+02
21.70
1.70
1.29E+02
21.50
1.50
7.37E+02
24.30
4.30
3600
3.28E+02
22.80
2.80
1.10E+02
21.20
1.20
6.99E+02
22.30
2.30

-------
1 in./h


Deposition
Dry








Test ID
59-AR0.6








Material Type
Concrete








Spore Type
Bg







Nozzle Part Number
GG-2.8W






Droplet Volume Mean Diameter
0.60 mm







Kinetic Energy
46 J/(m2h)





Number of Nozzles Used during Test
1







Sample Collection Time
5 s






Intensity (20 min bin measurement)
0.98 in/h






Intensity (heat map)
0.92 in/h






Intensity (Parsivel2)
0.54 in/h






Positive Control
2.63E+07 CFU






Positive Control Stdev
4.13E+06 CFU







Coupon 1


Coupon 2


Coupon 3


Average

Collected
Average
Sample
Collected
Average
Sample
Collected
Time
Liquid
Analyzed
Volume
Liquid
Volume
Volume
Liquid
Volume
Volume
Stamp (s)
CFU
Volume (mL)
(mL)
CFU
(mL)
(mL)
CFU
(mL)
(mL)
5
3.73E+05
27.80
7.80
1.51E+03
20.70
0.70
7.90E+03
28.00
8.00
10
6.92E+04
23.70
3.70
1.28E+03
20.60
0.60
5.62E+03
25.80
5.80
25
2.21E+04
21.50
1.50
1.70E+03
20.70
0.70
2.34E+03
23.20
3.20
35
2.03E+04
21.90
1.90
1.74E+03
21.00
1.00
2.31E+03
22.40
2.40
95
1.16E+04
21.20
1.20
1.29E+03
21.80
1.80
1.04E+03
21.90
1.90
185
4.47E+03
21.30
1.30
5.85E+02
20.90
0.90
1.14E+03
22.00
2.00
455
1.18E+03
21.50
1.50
4.43E+02
21.10
1.10
1.20E+03
22.20
2.20
845
1.50E+03
21.10
1.10
2.97E+02
21.60
1.60
7.18E+02
22.10
2.10
1805
1.64E+03
23.00
3.00
7.02E+02
24.20
4.20
7.01E+02
21.90
1.90
3600
9.31E+02
25.50
5.50
5.45E+02
26.60
6.60
2.97E+02
22.00
2.00

-------
3.2 in./h


Deposition
Dry









Test ID
59-AR3.2









Material Type
Concrete









Spore Type
Bg








Nozzle Part Number
HH-l







Droplet Volume Mean Diameter
0.83
mm







Kinetic Energy
766.65
J/(m2h)





Number of Nozzles Used during Test
1








Sample Collection Time
5
s






Intensity (20 min bin measurement)
3.2
in/h






Intensity (heat map)
3.26
in/h






Intensity (Parsivel2)
4.09
in/h






Positive Control
3.15E+07
CFU






Positive Control Stdev
5.11E+06
CFU







Coupon 1



Coupon 2


Coupon 3


Average

Collected

Average
Sample
Collected
Average
Sample
Collected
Time
Liquid
Analyzed
Volume

Liquid
Volume
Volume
Liquid
Volume
Volume
Stamp (s)
CFU
Volume (mL)
(mL)

CFU
(mL)
(mL)
CFU
(mL)
(mL)
5
4.84E+03
21.40
1.40

5.18E+03
22.90
2.90
1.78E+04
29.70
9.70
10
3.03E+03
21.20
1.20

2.89E+03
23.10
3.10
5.99E+03
26.50
6.50
25
1.89E+03
22.80
2.80

3.21E+03
23.60
3.60
5.77E+03
26.00
6.00
35
1.92E+03
24.00
4.00

3.47E+03
23.60
3.60
6.44E+03
26.40
6.40
95
1.41E+03
25.10
5.10

1.98E+03
23.00
3.00
1.68E+03
23.70
3.70
185
1.74E+03
24.80
4.80

2.32E+03
25.80
5.80
4.40E+03
29.50
9.50
455
1.44E+03
26.60
6.60

2.73E+03
25.80
5.80
9.72E+02
24.30
4.30
845
1.10E+03
29.00
9.00

2.30E+03
29.50
9.50
2.44E+03
31.70
1.70
1805
6.96E+02
29.00
9.00

1.52E+03
31.70
11.70
6.10E+02
25.40
5.40
3600
3.69E+02
29.90
9.90

3.60E+02
30.00
10.00
1.64E+02
24.60
4.60

-------
4.0 in./h


Deposition
Dry









Test ID
59-AR4.0









Material Type
Concrete









Spore Type
Bg








Nozzle Part Number
HH-50WSQ







Droplet Volume Mean Diameter
0.85
mm







Kinetic Energy
2156
J/(m2h)





Number of Nozzles Used during Test
1








Sample Collection Time
5
s






Intensity (20 min bin measurement)
3.60
in/h






Intensity (heat map)
3.66
in/h






Intensity (Parsivel2)
3.13
in/h






Positive Control
2.51 E+07
CFU






Positive Control Stdev
2.60E+06
CFU







Coupon 1



Coupon 2


Coupon 3


Average

Collected

Average
Sample
Collected
Average
Sample
Collected
Time
Liquid
Analyzed
Volume

Liquid
Volume
Volume
Liquid
Volume
Volume
Stamp (s)
CFU
Volume (mL)
(mL)

CFU
(mL)
(mL)
CFU
(mL)
(mL)
5
1.55E+04
20.68
0.68

3.25E+03
21.14
1.14
1.65E+04
21.21
1.21
10
4.57E+03
20.66
0.66

6.61E+03
21.85
1.85
1.32E+04
21.32
1.32
25
2.24E+03
20.62
0.62

2.13E+03
21.59
1.59
4.18E+03
21.16
1.16
35
3.21E+03
21.06
1.06

1.79E+03
21.39
1.39
1.48E+04
22.43
2.43
95
5.38E+03
23.40
3.40

1.45E+03
22.95
2.95
2.40E+04
25.07
5.07
185
3.38E+03
25.24
5.24

6.80E+02
24.96
4.96
3.32E+03
31.22
11.22
455
2.16E+03
28.19
8.19

3.89E+02
25.71
5.71
1.49E+03
29.79
9.79
845
1.38E+03
30.42
10.42

1.52E+02
25.95
5.95
6.61E+02
26.99
6.99
1805
1.50E+03
33.11
13.11

4.73E+01
25.93
5.93
2.34E+02
31.78
11.78
3600
3.31E+02
38.34
18.34

2.59E+01
28.55
8.55
7.35E+01
26.72
6.72

-------
0.6 in./h


Deposition
Dry









Test ID
59-AR0.6









Material Type
Concrete









Spore Type
Btk








Nozzle Part Number
HH-14WSQ







Droplet Volume Mean Diameter
0.62
mm







Kinetic Energy
76.53
J/(m2h)





Number of Nozzles Used during Test
1








Sample Collection Time
5
s






Intensity (20 min bin measurement)
0.64
in/h






Intensity (heat map)
0.57
in/h






Intensity (Parsivel2)
0.39
in/h






Positive Control
2.84E+07
CFU






Positive Control Stdev
1.28E+07
CFU







Coupon 1



Coupon 2


Coupon 3


Average

Collected

Average
Sample
Collected
Average
Sample
Collected
Time
Liquid
Analyzed
Volume

Liquid
Volume
Volume
Liquid
Volume
Volume
Stamp (s)
CFU
Volume (mL)
(mL)

CFU
(mL)
(mL)
CFU
(mL)
(mL)
5
9.44E+04
24.70
4.70

1.62E+03
21.30
1.30
1.93E+04
26.20
6.20
10
1.96E+04
21.50
1.50

6.76E+02
20.80
0.80
8.59E+03
23.60
3.60
25
2.16E+04
22.20
2.20

8.88E+02
21.40
1.40
3.66E+03
21.80
1.80
35
1.72E+04
21.80
1.80

5.69E+02
20.70
0.70
2.31E+03
21.40
1.40
95
1.44E+04
21.80
1.80

7.95E+02
21.20
1.20
1.36E+03
21.30
1.30
185
1.46E+04
21.80
1.80

7.21E+02
21.20
1.20
4.62E+03
22.00
2.00
455
6.46E+02
20.20
0.20

7.23E+02
21.90
1.90
1.36E+03
21.30
1.30
845
1.53E+03
21.90
1.90

6.73E+02
21.70
1.70
1.38E+03
20.80
0.80
1805
7.92E+02
22.00
2.00

4.22E+02
21.10
1.10
1.01E+03
22.00
2.00
3600
2.76E+02
21.20
1.20

4.79E+02
22.80
2.80
1.96E+02
21.20
1.20

-------
1 in./h


Deposition
Dry









Test ID
59-AR1









Material Type
Concrete









Spore Type
Btk








Nozzle Part Number
GG-2.8W







Droplet Volume Mean Diameter
0.61
mm







Kinetic Energy
43.60
J/(m2h)





Number of Nozzles Used during Test
1








Sample Collection Time
5
s






Intensity (20 min bin measurement)
0.98
in/h






Intensity (heat map)
0.88
in/h






Intensity (Parsivel2)
0.48
in/h






Positive Control
3.67E+07
CFU






Positive Control Stdev
1.28E+07
CFU







Coupon 1



Coupon 2


Coupon 3


Average

Collected

Average
Sample
Collected
Average
Sample
Collected
Time
Liquid
Analyzed
Volume

Liquid
Volume
Volume
Liquid
Volume
Volume
Stamp (s)
CFU
Volume (mL)
(mL)

CFU
(mL)
(mL)
CFU
(mL)
(mL)
5
9.50E+03
22.20
2.20

3.42E+00
22.20
2.20
5.79E+03
26.30
6.30
10
5.98E+03
21.20
1.20

1.89E+00
22.70
2.70
2.43E+03
23.80
3.80
25
5.76E+03
24.40
4.40

1.93E+00
22.00
2.00
1.92E+03
22.90
2.90
35
5.92E+03
23.50
3.50

3.98E+00
21.50
1.50
1.96E+03
22.50
2.50
95
2.39E+03
22.50
2.50

4.04E+00
21.00
1.00
1.56E+03
23.00
3.00
185
3.05E+03
22.10
2.10

1.74E+04
23.70
3.70
1.22E+03
23.10
3.10
455
1.11E+03
22.20
2.20

2.35E+04
22.80
2.80
8.25E+02
22.30
2.30
845
1.23E+03
22.90
2.90

1.08E+03
22.20
2.20
5.13E+02
22.30
2.30
1805
7.42E+02
23.20
3.20

1.08E+03
23.50
3.50
2.88E+04
23.80
3.80
3600
5.76E+02
24.00
4.00

1.20E+02
21.80
1.80
8.16E+02
24.00
4.00

-------
3.2 in./h


Deposition
Dry









Test ID
59-AR3.2









Material Type
Concrete









Spore Type
Btk








Nozzle Part Number
HH-1







Droplet Volume Mean Diameter
0.82
mm







Kinetic Energy
797.03
J/(m2h)





Number of Nozzles Used during Test
1








Sample Collection Time
5
s






Intensity (20 min bin measurement)
3.60
in/h






Intensity (heat map)
3.26
in/h






Intensity (Parsivel2)
4.09
in/h






Positive Control
3.15E+07
CFU






Positive Control Stdev
5.11E+06
CFU







Coupon 1



Coupon 2


Coupon 3


Average

Collected

Average
Sample
Collected
Average
Sample
Collected
Time
Liquid
Analyzed
Volume

Liquid
Volume
Volume
Liquid
Volume
Volume
Stamp (s)
CFU
Volume (mL)
(mL)

CFU
(mL)
(mL)
CFU
(mL)
(mL)
5
2.63E+04
27.60
7.60

1.88E+05
27.30
7.30
2.09E+04
22.20
2.20
10
2.00E+04
28.80
8.80

4.22E+04
23.30
3.30
2.18E+04
25.30
5.30
25
1.37E+04
31.40
11.40

1.39E+05
25.10
5.10
1.62E+05
30.50
10.50
35
1.16E+04
30.90
10.90

1.35E+05
26.10
6.10
1.12E+05
30.20
10.20
95
7.90E+03
30.20
10.20

1.18E+04
27.60
7.60
2.94E+03
24.00
4.00
185
6.97E+03
30.70
10.70

8.31E+03
27.10
7.10
4.37E+03
24.10
4.10
455
3.15E+03
28.90
8.90

8.22E+03
26.70
6.70
2.73E+03
23.70
3.70
845
1.49E+03
26.60
6.60

3.23E+03
30.30
10.30
1.73E+03
23.80
3.80
1805
1.53E+03
29.40
9.40

2.79E+03
29.40
9.40
3.70E+02
28.00
8.00
3600
3.70E+02
29.80
9.80

1.71E+02
29.00
9.00
2.39E+02
28.80
8.80

-------
4.0 in./h


Deposition
Dry









Test ID
59-AR4.0









Material Type
Concrete









Spore Type
Btk








Nozzle Part Number
HH-50WSQ







Droplet Volume Mean Diameter
0.82
mm







Kinetic Energy
797.03
J/(m2h)





Number of Nozzles Used during Test
1








Sample Collection Time
5
s






Intensity (20 min bin measurement)
0.98
in/h






Intensity (heat map)
-
in/h






Intensity (Parsivel2)
0.48
in/h






Positive Control
3.35E+07
CFU






Positive Control Stdev
2.90E+06
CFU







Coupon 1



Coupon 2


Coupon 3


Average

Collected

Average
Sample
Collected
Average
Sample
Collected
Time
Liquid
Analyzed
Volume

Liquid
Volume
Volume
Liquid
Volume
Volume
Stamp (s)
CFU
Volume (mL)
(mL)

CFU
(mL)
(mL)
CFU
(mL)
(mL)
5
1.48E+03
20.80
0.80

1.63E+03
20.80
0.80
1.53E+03
21.10
1.10
10
3.35E+02
20.30
0.30

1.56E+03
21.50
1.50
1.92E+03
21.60
1.60
25
6.45E+02
20.80
0.80

1.85E+03
21.80
1.80
2.46E+03
21.90
1.90
35
8.90E+02
20.70
0.70

7.88E+02
21.00
1.00
1.40E+03
22.30
2.30
95
8.44E+02
21.10
1.10

1.91E+03
29.40
9.40
4.73E+03
25.40
5.40
185
4.39E+02
21.30
1.30

5.01E+03
42.20
22.20
1.14E+03
32.00
12.00
455
6.76E+02
31.40
11.40

1.37E+03
35.10
15.10
1.05E+03
37.00
17.00
845
1.39E+03
34.50
14.50

9.64E+02
37.80
17.80
1.03E+03
38.20
18.20
1805
2.65E+02
30.70
10.70

3.46E+02
40.40
20.40
2.76E+02
38.00
18.00
3600
1.26E+02
34.80
14.80

2.25E+02
41.40
21.40
1.84E+02
39.30
19.30

-------
1.0 in./h


Deposition
Dry









Test ID
59-AR1-A









Material Type
Asphalt









Spore Type
Bg








Nozzle Part Number
GG-2.8W







Droplet Volume Mean Diameter
0.85
mm







Kinetic Energy
2156
J/(m2h)





Number of Nozzles Used during Test
1








Sample Collection Time
5
s






Intensity (20 min bin measurement)
3.60
in/h






Intensity (heat map)
0.92
in/h






Intensity (Parsivel2)
3.12
in/h






Positive Control
1.91E+07
CFU






Positive Control Stdev
2.83E+06
CFU







Coupon 1



Coupon 2


Coupon 3


Average

Collected

Average
Sample
Collected
Average
Sample
Collected
Time
Liquid
Analyzed
Volume

Liquid
Volume
Volume
Liquid
Volume
Volume
Stamp (s)
CFU
Volume (mL)
(mL)

CFU
(mL)
(mL)
CFU
(mL)
(mL)
5
4.29E+03
26.50
6.50

1.73E+05
33.60
13.60
2.70E+03
29.20
9.20
10
2.55E+03
24.80
4.80

3.36E+04
24.50
4.50
1.30E+03
25.30
5.30
25
2.34E+03
24.50
4.50

1.24E+04
21.60
1.60
1.48E+03
25.30
5.30
35
1.39E+03
23.30
3.30

1.06E+03
20.70
0.70
8.53E+02
23.70
3.70
95
2.04E+03
24.00
4.00

2.64E+03
24.24
4.24
3.99E+02
22.80
2.80
185
2.32E+03
22.70
2.70

1.75E+03
23.00
3.00
5.92E+02
23.20
3.20
455
1.76E+03
27.30
7.30

4.52E+01
20.80
0.80
9.10E+02
23.80
3.80
845
5.34E+02
28.10
8.10

1.36E+02
23.60
3.60
4.21E+02
23.20
3.20
1805
5.33E+02
27.00
7.00

1.10E+02
23.90
3.90
4.04E+02
24.10
4.10
3600
7.81E+01
24.80
4.80

3.66E+01
22.50
2.50
7.72E+01
25.30
5.30

-------
1.0 in./h


Deposition
Dry









Test ID
59-AR1-A









Material Type
Asphalt









Spore Type
Btk








Nozzle Part Number
GG-2.8W







Droplet Volume Mean Diameter
0.60
mm







Kinetic Energy
39
J/(m2h)





Number of Nozzles Used during Test
1








Sample Collection Time
5
s






Intensity (20 min bin measurement)
1.10
in/h






Intensity (heat map)
0.92
in/h






Intensity (Parsivel2)
0.52
in/h






Positive Control
1.26E+06
CFU






Positive Control Stdev
2.86E+05
CFU







Coupon 1



Coupon 2


Coupon 3


Average

Collected

Average
Sample
Collected
Average
Sample
Collected
Time
Liquid
Analyzed
Volume

Liquid
Volume
Volume
Liquid
Volume
Volume
Stamp (s)
CFU
Volume (mL)
(mL)

CFU
(mL)
(mL)
CFU
(mL)
(mL)
5
6.72E+03
32.60
12.60

2.51E+03
23.10
3.10
3.72E+03
26.80
6.80
10
2.28E+03
23.50
3.50

3.75E+03
21.40
1.40
5.61E+03
30.20
10.20
25
5.31E+03
34.90
14.90

4.60E+03
27.70
7.70
2.53E+03
25.20
5.20
35
3.30E+03
29.10
9.10

5.35E+03
23.90
3.90
6.70E+03
223.30
203.30
95
4.52E+02
23.50
3.50

4.03E+02
22.10
2.10
7.35E+01
20.70
0.70
185
4.20E+02
23.50
3.50

3.30E+02
22.00
2.00
3.02E+02
21.20
1.20
455
2.89E+02
23.10
3.10

2.73E+02
24.50
4.50
6.17E+01
20.90
0.90
845
8.89E+01
24.10
4.10

7.98E-01
20.10
0.10
1.04E+02
22.30
2.30
1805
1.30E+02
23.90
3.90

1.23E+02
23.80
3.80
5.18E+01
21.60
1.60
3600
4.74E+01
25.60
5.60

2.62E+01
22.80
2.80
3.75E+01
23.80
3.80

-------
4.0 in./h
Deposition
Dry

Test ID
59-AR4.0

Material Type
Asphalt

Spore Type
Btk

Nozzle Part Number
HH-50WSQ

Droplet Volume Mean Diameter
0.82
mm
Kinetic Energy
894
J/(m2h)
Number of Nozzles Used during Test
1

Sample Collection Time
5
s
Intensity (20 min bin measurement)
2.45
in/h
Intensity (heat map)
2.40
in/h
Intensity (Parsivel2)
1.98
in/h
Positive Control
1.26E+07
CFU
Positive Control Stdev
2.06E+06
CFU


Coupon 1


Coupon 2


Average

Collected
Average
Sample
Collected
Time
Liquid
Analyzed
Volume
Liquid
Volume
Volume
Stamp (s)
CFU
Volume (mL)
(mL)
CFU
(mL)
(mL)
5
8.33E+04
31.90
11.90
5.05E+04
29.90
9.90
10
9.67E+04
31.70
11.70
2.80E+04
28.10
8.10
25
5.31E+04
24.80
4.80
6.08E+04
24.80
4.80
35
3.40E+04
28.10
8.10
8.91E+03
22.00
2.00
95
2.47E+02
21.70
1.70
1.21E+04
26.90
6.90
185
4.81E+03
24.50
4.50
5.06E+04
24.00
4.00
455
7.75E+02
24.60
4.60
5.73E+03
25.30
5.30
845
2.16E+03
26.50
6.50
1.04E+03
26.10
6.10
1805
5.61E+02
26.70
6.70
9.84E+02
28.10
8.10
3600
8.00E+02
32.00
12.00
4.53E+02
30.20
10.20

-------
Appendix F. Spore Washoff Procedure-Channel
For these experiments, spores were dry-inoculated onto the surface of a concrete coupon and
positioned at a 5% slope in the custom channelizedflow apparatus. Runoff samples that
correlate with various time points of interest and positive control coupons (concrete coupons
that have been dry-inoculated and sampled with wipes) were collected.
Preparation
Sterilize the following materials in the airlock (4 hours, 200 ppm vaporous hydrogen peroxide):
•	Overflow tank
•	Aerosol dose apparatuses (4)
Sterilize the following materials in the autoclave (250°C gravity cycle):
•	Stainless steel coupons (4)
•	500 mL Nalgene bottles with caps (20)
•	14x14" concrete coupons (4)
Sterilize the deionized water tank as follows:
•	Pour one gallon of germicidal bleach into the tank and fill the tank with DI water.
•	Empty the tank into the sump.
•	Fill and empty the tank two times with DI water.
•	Fill the tank with deionized water.
Day 1
•	Set up tables with bench liner. Remove a metered dose inhaler (E7) from the refrigerator.
•	Assemble three stainless steel coupons with aerosol dose apparatuses.
•	Assemble one concrete coupon with apparatus.
•	Perform sterility swabs on the concrete coupon, one stainless steel coupon and one
aerosol dose apparatus.
•	Inoculate the test set of coupons.
Day 2
•	Transfer the channel outside, level, and cover the weir and area before the coupon holder
with sterilized aluminum foil.
•	Establish a flow rate without the coupon. Record a few measurements to ensure that this
setting achieves the desired flow rate.
•	Take a 50-mL blank sample from the established flow in a specimen cup to test sterility.
•	Stop the flow and dry the test tank with a clean cloth.
•	Set the concrete coupon in the channel and seal the surrounding area with door caulk.
•	Start the flow at the desired flowrate
•	Fill the tank to the top of the weir with water and ensure it does not overflow.
•	Start a timer when the first drop of sample flows down the basin. Simultaneously begin
sample collection as follows:
o For the first 45 seconds, a four-person team will be positioned at the collection
end of the overflow tank. Two samplers collect liquid samples into Nalgene

-------
bottles by opening their respective outlet valves, then closing the valves after a
collection time of 5 seconds (per sample). The third and fourth person will assist
the samplers by receiving and capping collected samples, and supplying empty
Nalgene bottles to the samplers.
o
Collect one
sample between 0 and 5 seconds (sampler 1)
o
Collect one
sample between 5 and 10 seconds (sampler 2)
o
Collect one
sample between 17 and 22 seconds (sampler 1)
o
Collect one
sample between 40 and 45 seconds (sampler 2)
o
Discard the
runoff between 45 seconds and 88 seconds
o
Collect one
sample at 88 seconds
o
Discard the
runoff between 93 seconds and 188 seconds
o
Collect one
sample at 188 seconds
o
Discard the
runoff
o
Collect one
sample at 397 seconds
o
Discard the
runoff
o
Collect one
sample at 829 seconds
o
Discard the
runoff
o
Collect one
sample at 1727 seconds
o
Discard the
runoff
o
Collect one
sample at 3592 seconds
Stop the deionized water pump and discard the remaining runoff.
Record the volumes of each liquid sample.
Wipe sample the three inoculation control coupons.
Deliver all samples to the onsite microbiology laboratory with a chain of custody form.

-------
Appendix G. Spore Washoff Data - Channel


25 mL/s


Deposition
Dry




Test ID
59-A025-D1



Sample Collection Time
5
s



Flow Rate
25
mL/s



Positive Control
2.52E+07
CFU




Average





Liquid
Sample



Time Stamp (s)
CFU
Volume



5
1.59E+05
16.04



10
7.62E+04
21.46



22
5.35E+04
22.84



45
3.56E+05
29.58



93
4.27E+04
26.30



193
6.04E+04
25.62



402
5.03E+03
25.60



834
1.95E+03
25.96



1732
3.81 E+02
27.20



3597
7.51 E+02
25.04



50 m L/s
Deposition
Dry

Deposition
Dry

Test ID
59-A050-D2
Test ID
59-AO50-D3

Sample Collection Time
5
s
Sample Collection Time
5
s
Flow Rate
50
mL/s
Flow Rate
50
mL/s
Positive Control
4.68E+07
CFU
Positive Control
3.84E+07
CFU
Positive Control Stdev
4.41 E+06
CFU
Positive Control Stdev
1.16E+07
CFU

Average
Sample

Average
Sample

Liquid
Volume

Liquid
Volume
Time Stamp (s)
CFU
(mL)
Time Stamp (s)
CFU
(mL)
5
1.50E+06
39.02
5
1.76E+06
14.78
10
1.27E+06
43.88
10
8.49E+05
33.26
22
2.96E+05
46.04
22
2.53E+05
47.56
45
8.93E+04
49.06
45
5.50E+04
51.54
93
1.98E+04
48.06
93
7.98E+04
48.26
193
1.40E+04
52.16
193
1.00E+04
51.28
402
1.38E+04
47.10
402
6.94E+03
52.90
834
3.62E+03
49.58
834
1.29E+03
54.90
1732
5.57E+02
49.06
1732
3.54E+02
52.66
3597
1.73E+02
49.12
3597
1.15E+02
55.64

-------
75 mL/s
Deposition
Dry

Deposition
Dry

Test Date
59-A075-D1
Test ID
59-A075-D2

Sample Collection Time
5
s
Sample Collection Time
5
s
Flow Rate
75
mL/s
Flow Rate
75
mL/s
Positive Control
2.65E+07
CFU
Inoculation Control
4.20E+07
CFU
Positive Control Stdev
3.99E+06
CFU
Positive Control Stdev
1.06E+07
CFU

Average
Sample

Average
Sample

Liquid
Volume

Liquid
Volume
Time Stamp (s)
CFU
(mL)
Time Stamp (s)
CFU
(mL)
5
5.67E+06
62.98
5
3.75E+06
45.72
10
8.22E+05
63.22
10
2.65E+06
58.40
22
3.91 E+05
68.58
22
7.16E+05
69.86
45
1.35E+05
67.72
45
1.37E+05
62.66
93
2.10E+04
70.88
93
3.98E+04
75.72
193
8.50E+03
70.82
193
2.51E+04
72.74
402
4.07E+03
73.58
402
2.78E+03
85.78
834
8.21 E+02
68.42
834
3.95E+03
75.30
1732
1.90E+02
68.92
1732
1.36E+03
67.82
3597
2.83E+02
73.16
3597
6.65E+02
81.86
100 mL/s
Deposition
Dry

Deposition
Dry

Test ID
59-AOlOO-Dl
Test ID
59-A0100-D2

Sample Collection Time
5
s
Sample Collection Time
5
s
Flow Rate
100
mL/s
Flow Rate
100
mL/s
Positive Control
3.55E+07
CFU
Positive Control
2.52E+07
CFU
Positive Control Stdev

CFU
Positive Control Stdev

CFU

Average
Sample

Average
Sample

Liquid
Volume

Liquid
Volume
Time Stamp (s)
CFU
(mL)
Time Stamp (s)
CFU
(mL)
5
1.69E+05
26.96
5
3.05E+06
50.02
10
6.79E+05
62.58
10
4.54E+05
63.46
22
2.96E+05
64.90
22
4.64E+05
68.22
45
9.54E+04
78.56
45
8.31 E+04
80.92
93
1.55E+04
83.92
93
2.71 E+04
99.50
193
2.86E+04
96.94
193
5.18E+03
97.34
402
3.88E+03
89.48
402
1.63E+03
96.76
834
1.59E+03
105.30
834
1.35E+03
94.14
1732
1.10E+04
95.54
1732
2.02E+03
101.12
3597
7.59E+02
94.88
3597
1.10E+03
86.88

-------
150 mL/s
Deposition
Dry

Deposition
Dry

Test ID
59-AO150-D1
Test ID
59-AO150-D2

Sample Collection Time
5
s
Sample Collection Time
5
s
Flow Rate
150
mL/s
Flow Rate
150
mL/s
Positive Control
4.96E+07
CFU
Positive Control
3.55E+07
CFU
Positive Control Stdev
1.39E+07
CFU
Positive Control Stdev
9.64E+06
CFU

Average
Sample

Average
Sample

Liquid
Volume

Liquid
Volume
Time Stamp (s)
CFU
(mL)
Time Stamp (s)
CFU
(mL)
5
5.90E+06
81.92
5
4.35E+05
35.26
10
1.77E+06
96.82
10
1.04E+06
65.98
22
1.61E+05
91.94
22
6.99E+05
101.34
45
1.16E+05
116.18
45
5.19E+05
120.70
93
1.38E+05
138.08
93
1.13E+05
144.36
193
3.18E+04
157.24
193
2.31E+04
154.16
402
9.72E+03
138.90
402
5.46E+03
148.82
834
7.65E+03
152.94
834
7.57E+02
141.96
1732
1.32E+03
140.60
1732
5.37E+02
148.16
3597
4.32E+02
143.94
3597
6.57E+02
145.94


-------
Appendix I. Spore Washoff Procedure-Spray
Purpose
Coupons were inoculated with bacillus spores (Bg or Blk) and decontaminated using a garden
hose with a nozzle on the shower setting or a pressure washer with the 15-degree spray angle tip.
The coupons will have an inoculated area of approximately 4.25 in. x 4.25 in. and will be
sprayed at approximately 34 in. away from the surface of the coupon. The inoculated area
corresponds to the theoretical coverage achieved by the pressure washer at the specified spray
height.
Day 1
•	Prior to coupon inoculation, swab samples of a representative coupon and inoculation
control coupon will be collected.
•	Coupons will be inoculated (loaded) with spores from a metered dose inhaler using the
procedure detailed in Miscellaneous Operating procedure (MOP) 3161M that uses an
aerosol dosing apparatus. The inoculation procedure will involve raising the apparatus to
the same level as the coupon, clamping the coupon onto a 14" x 14" stainless steel piece
with a 5.5-in. x 5.5-in. cutout, and inoculating the coupon so that an even distribution of
spores meets the coupon to ensure that a 14-in. x 14-in. area gets completely inoculated.
If needed, cover any gaps between the 14-in. x 14-in. stainless steel and the 5.5-in. x 5.5-
in. coupon to prevent cross contamination and escaping spores.
•	Once the aerosol dosing apparatus has been secured to the coupon, place the metered
dose inhaler on the top, open the slide, and activate the inhaler.
•	Following inoculation, close the slide and remove the inhaler.
•	Repeat for each coupon.
•	Record the initial and final inhaler mass (verify scale calibration) in the inoculation log.
•	Allow at least 18 hours after inoculating the coupons before testing.
Day 2
Pre-Test Activities:
•	Calibrate a pH meter using pH buffer solutions: 4.0, 7.0, and 10.0
•	Determine the free available chlorine of the bleach feed stock. If the hypochlorite
concentration is less than 7%, DO NOT USE.
•	Prepare 4 L of pH adjusted bleach with a free available chlorine of approximately 7,900
mg/L. The pH adjusted bleach will be prepared using a volumetric ratio of 1:1:8 Clorox®
Concentrated Germicidal bleach: 5% acetic acid: deionized water
o Measure the free available chlorine using a HACH® kit. Record measurement in
laboratory notebook,
o Measure the pH and temperature of the pH adjusted bleach with calibrated pH
meter. Record measurements in laboratory notebook,
o Record the time the pH adjusted bleach was prepared
•	Transfer pH adjusted bleach into a sterile SHURflo backpack sprayer.

-------
•	Spray the wash-down chamber with pH adjusted bleach and allow a 15-minute contact
time with collection port closed.
•	Rinse the chamber with deionized water.
•	As water drains from the chamber, collect a 100-mL sample.
•	Determine the free available chlorine of the sample. If sample is clear (or approximately
the same free available chlorine of tap water), then the sample is free of chlorine; if not,
then re-rinse the chamber until no free available chlorine is present.
Coupon Wash-down and Sampling:
1.	Set up a table with bench liner for sample handling.
2.	Connect the hose to the pressure washer and power on the pressure washer or garden
spray nozzle. If using the pressure washer, allow time to pressurize the water (when
powered on, the washer makes plenty of noise; once it quiets down, the water is
pressurized).
3.	Collect -100 mL of water from the pressure washer outlet. *NOTE: Remove the spray tip
before doing this.
4.	Place the desired spray tip on the pressure washer and spray water so that any air that
may be stuck is removed when the line is purged to ensure that the coupon is sprayed
with the correct angle and force.
5.	Aseptically place the procedural blank coupon on the load cell. Ensure all ports except
the spraying port are covered.
6.	Open and place a sterile Nalgene bottle directly underneath the collection port.
7.	Start the Portable Data Acquisition (PDAQ) (force sensor) to record load cell
measurements.
8.	Go up to the exact height of the wash-down chamber port opening and spray the coupon
straight down vertically for 5 seconds.
9.	Collect, cap, and adequately label the Nalgene bottle in preparation to deliver sample to
the onsite microbiology laboratory.
10.	Aseptically remove the procedural blank coupon. Ensure all ports except the spraying
port are covered.
11.	Rinse the wash-down basin with copious amounts of water.
12.	Open and place a second sterile Nalgene bottle directly underneath the collection port.
13.	Collect a rinse sample of the basin to check for any potential residual spores.
14.	Collect, cap, and adequately label the Nalgene bottle in preparation for delivery of
sample to the biolab.
15.	Remove the aerosol dose apparatus from a test coupon and aseptically place on the load
cell. Ensure all ports except the spraying port are covered.
16.	Open and place a sterile Nalgene bottle directly underneath the collection port.
17.	Start the PDAQ. Properly name the file and write the file name down in the notebook.
18.	Go up to the exact height of the wash-down chamber port opening and spray the coupon
straight down vertically for 5 seconds.

-------
19.	Record the coupon identification (ID), duration of spray, and time in the notebook.
20.	Collect and cap the Nalgene bottle, adequately label in preparation to deliver sample to
the biolab.
21.	Aseptically remove the test coupon from the load cell. Ensure all ports except the
spraying port are covered.
22.	Rinse the wash-down basin with copious amounts of water.
23.	Open and place a second sterile Nalgene bottle directly underneath the collection port.
24.	Collect a rinse sample of the basin to check for any potential residual spores.
25.	Collect, cap, and adequately label the Nalgene bottle in preparation to deliver sample to
the biolab.
26.	Repeat steps 15 through 25 for n number of test coupons.
27.	Perform wipe samples of the coupons listed below following the listed instructions.
The coupons will be removed, and wipe sampled with polyester rayon blend wipes using MOP
3199 as a guide. The sample will be placed in a conical tube containing 10 mL of phosphate
buffered saline with 0.05% TWEEN®20 (PBST).

-------
Appendix J. Spore Washoff Data - Spray
Average of Replicate Coupo
ns

Test IDs
1BCH, lBCHb


Material Type
Concrete


Spore Type
Bg


Spray Method
Garden Hose


Spray Orientation
Vertical


Spray Distance
34
in.

Spray Time
5
s

Number of Replicates
5
per test

Average Positive Control Log CFU
7.18


Stdev Positive Control Log CFU
6.56


Total Average Applied Energy
0.44
J

Applied Energy Standard Error
1.48E-03
J

Energy Flux
16110
J/m2-h

Coupon
Average Log CFU
Standard Error
Avg % Removal
TCI
6.11

93%
TC2
6.08

94%
TC3
6.09

94%
TC4
6.11

93%
TC5
6.03

94%
Average
6.08
0.011
94%
Average of Replicate Coupons

Test IDs
1BBH


Material Type
Brick


Spore Type
Bg


Spray Method
Garden Hose


Spray Orientation
Vertical


Spray Distance
34
in.

Spray Time
5
s

Number of Replicates
5
per test

Average Positive Control Log CFU
7.21


Stdev Positive Control Log CFU
6.20


Total Average Applied Energy
0.20
J

Applied Energy Standard Error
1.48E-03
J

Energy Flux
7233
J/m2-h

Coupon
Average Log CFU
Standard Error
Avg % Removal
TCI
6.06

93%
TC2
6.14

91%
TC3
6.17

90%
TC4
6.08

92%
TC5
6.14

91%
Average
6.12
0.02
91%

-------
Average of Replicate Coupo
ns

Test IDs
1BAH


Material Type
Asphalt


Spore Type
Bg


Spray Method
Garden Hose


Spray Orientation
Vertical


Spray Distance
34
in.

Spray Time
5
s

Number of Replicates
5
per test

Average Positive Control Log CFU
7.08


Stdev Positive Control Log CFU
6.77


Total Average Applied Energy
0.09
J

Applied Energy Standard Error
1.48E-03
J

Energy Flux
3353
J/m2-h

Coupon
Average Log CFU
Standard Error
Avg % Removal
TCI
5.90

93%
TC2
5.67

96%
TC3
5.91

93%
TC4
5.70

95%
TC5
5.79

94%
Average
5.79
0.049
94%
Average of Replicate Coupo
ns

Test IDs
1BGH


Material Type
Glass


Spore Type
Bg


Spray Method
Garden Hose


Spray Orientation
Vertical


Spray Distance
34
in.

Spray Time
5
s

Number of Replicates
5
per test

Average Positive Control Log CFU
7.15


Stdev Positive Control Log CFU
6.54


Total Average Applied Energy
0.14
J

Applied Energy Standard Error
1.48E-03
J

Energy Flux
5034
J/m2-h

Coupon
Average Log CFU
Standard Error
Avg % Removal
TCI
5.14

99%
TC2
4.81

99%
TC3
5.05

99%
TC4
5.18

99%
TC5
5.13

99%
Average
5.06
0.067
99%

-------
Average of Replicate Coupons


Test IDs
3BCH


Material Type
Concrete


Spore Type
Bg


Spray Method
Pressure Washer


Spray Orientation
Vertical


Spray Distance
34
in.

Spray Time
5
s

Number of Replicates
5
per test

Average Positive Control Log CFU
7.44


Stdev Positive Control Log CFU
6.92


Total Average Applied Energy
0.57
J

Applied Energy Standard Error
1.48E-03
J

Energy Flux
21146
J/m2-h

Coupon
Average Log CFU
Standard
Error
Avg % Removal
TCI
6.14

93%
TC2
6.45

85%
TC3
5.90

96%
TC4
6.18

92%
TC5
6.32

89%
Average
6.20
0.093
91%

Average of Replicate Coupons


Test IDs
3BBH


Material Type
Brick


Spore Type
Bg


Spray Method
Pressure Washer


Spray Orientation
Vertical


Spray Distance
34
in.

Spray Time
5
s

Number of Replicates
5
per test

Average Positive Control Log CFU
7.17


Stdev Positive Control Log CFU
6.50


Total Average Applied Energy
0.79
J

Applied Energy Standard Error
1.48E-03
J

Energy Flux
29218
J/m2-h

Coupon
Average Log CFU
Standard
Error
Avg % Removal
TCI
5.88

95%
TC2
6.02

93%
TC3
5.98

94%
TC4
6.00

93%
TC5
5.75

96%
Average
5.93
0.050
94%

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Average of Replicate Coupons


Test IDs
3 BAH


Material Type
Asphalt


Spore Type
Bg


Spray Method
Pressure Washer


Spray Orientation
Vertical


Spray Distance
34
in.

Spray Time
5
s

Number of Replicates
5
per test

Average Positive Control Log CFU
7.00


Stdev Positive Control Log CFU
6.35


Total Average Applied Energy
0.44
J

Applied Energy Standard Error
1.48E-03
J

Energy Flux
16385
J/m2-h

Coupon
Average Log CFU
Standard
Error
Avg % Removal
TCI
5.55

97%
TC2
5.46

97%
TC3
5.64

96%
TC4
5.53

97%
TC5
5.70

95%
Average
5.58
0.042
97%

Average of Replicate Coupons


Test IDs
3BGH


Material Type
Glass


Spore Type
Bg


Spray Method
Pressure Washer


Spray Orientation
Vertical


Spray Distance
34
in.

Spray Time
5
s

Number of Replicates
5
per test

Average Positive Control Log CFU
7.05


Stdev Positive Control Log CFU
6.03


Total Average Applied Energy
0.61
J

Applied Energy Standard Error
1.48E-03
J

Energy Flux
22632
J/m2-h

Coupon
Average Log CFU
Standard
Error
Avg % Removal
TCI
5.60

97%
TC2
5.64

97%
TC3
5.69

96%
TC4
5.86

94%
TC5
5.41

98%
Average
5.64
0.073
96%


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