&EPA
www. e pa. gov/researc h
technical BRIEF
INNOVATIVE RESEARCH FOR A SUSTAINABLE FUTURE
Effectiveness of Outdoor Environment
Decontamination for Biological Agents
Contamination of outdoor environments could result from intentional or accidental releases of
biological materials or human or animal disease outbreaks. Outdoor contamination incidents pose
significant challenges in determining the extent of contamination (sampling and analysis), containing the
contaminant spread (mitigation), and remediating the areas so that re-occupancy or reuse can occur.
Currently, few methods are well-characterized, efficacious, and readily-available for outdoor
decontamination; especially for application over large areas. Further, the range of possible
environmental conditions (e.g., temperature, humidity, precipitation, wind) in the outdoor environment
is typically much greater than that indoors. Extreme temperatures and the presence of natural, organic-
rich materials in outdoor environments are examples that are known to challenge typical (physical and
chemical) decontamination processes. For example, low temperatures can cause liquid-based
decontaminants to freeze and become ineffective. The presence of organic-rich grime can neutralize the
oxidative potential of many chemical-based decontaminants. Understanding the potential challenges to
outdoor decontamination and developing effective solutions to overcome those challenges is critical for
development of wide-area response capabilities. Two recent research studies have begun to address the
knowledge and capability gaps associated with conducting decontamination for biological agents in
outdoor environments and challenging settings [1,2].
The first study aimed to assess the effectiveness of spray-based decontamination methods for
inactivating Bacillus atrophaeus (surrogate for B. cmthracis) spores and bacteriophage MS2 (surrogate
for foot and mouth disease virus) on neat or heavily soiled concrete and treated plywood) (Figure 1) [2],
Decontamination efficacy was assessed for three different decontamination solutions; pH-amended
Bleach (pAB) and Spor-Klenz® Ready-to Use (RTU) were evaluated against B. atrophaeus spores, and 2
percent (%) weight/volume (w/v) citric acid in sterilized deionized (DI) water and pAB were evaluated
against MS2. Three application methods (handheld sprayer, backpack sprayer, and a chemical sprayer)
were utilized to deliver decontaminants to the test surfaces. The evaluation was conducted on two test
material surfaces (concrete and treated plywood), with and without agricultural grime. The handheld
application method was conducted using a bench-scale test spray apparatus to evaluate the pAB and
citric acid spray-based decontamination methods for 18-millimeter (mm) coupons (both grimed and
neat) contaminated with MS2. The backpack and the chemical sprayer application methods were
conducted on a larger scale (14-inch by 14-inch coupons) to better simulate field operations and were
evaluated for both MS2 and B. atrophaeus. For all tests, a wetted surface contact time of 30 minutes was
administered, followed by a surface rinse with water. The fate of the microorganisms in the runoff
generated during the decontamination procedure and in the subsequent rinse step, as well as their
potential re-aerosolization in the air, were also investigated.
1
U.S. Environmental Protection Agency
Office of Research and Development
EPA/600/S-18/223
September 2018

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Figure 1. Photo of Model Agricultural Grime Application to Concrete Test Coupons.
Decontamination tests with B. airophaeus spores indicated that higher efficacies were achieved
on neat materials than on grimed materials, independent of the type of material or application method
(Table 1). pAB was found to be more effective than Spor-Klenz1' RTU for decontaminating neat
concrete materials, while the latter decontaminant was more efficacious for neat plywood materials
independent of application method (backpack sprayer versus chemical sprayer). Viable spore levels
found in rinsate samples were higher for the backpack sprayer tests than for the chemical sprayer tests,
potentially because the chemical sprayer was more effective at physically removing spores before the
rinse step. Relatively high re-aerosolization of spores (greater than 1 st 1Q3 colony forming units [CFU]
per test) was observed during some tests with both the backpack and chemical sprayers.
Decontamination tests with MS2 indicated that 2% citric acid was not efficacious on concrete
and plywood (Tables 2 and 3). However, pAB was found to be efficacious against MS2, with full
decontamination on neat or grimed concrete and limited efficacy for neat or grimed plywood. Further,
few viable viruses were detected in the runoff from pAB tests, unlike for the 2% citric acid formulation,
which had almost complete wash-off (and recovery) of viable viruses from all coupon types. Finally, no
viable MS2 re-aerosolization was observed in any of the conducted tests, independent of the type of
decontamination solution used. However, it should be noted that the Via-CellK bio-aerosol cassette
sampling method, used in this study, was not validated for MS2 sampling or recovery. A summary of the
decontamination results is shown in Tables 1-3.
U.S. Environmental Protection Agency
Office of Research and Development

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Table 1. Decontamination Results for Large Coupon (Lab-Scale) Tests with Bacillus atrophaeus

Decontamination
Application
Method
Material
Decontamination
Coupon
Positive Controls (CFU)
Test Coupons
LR
(CFU)

Type
Liquid
Condition
Average
STD
Average
STD
Average
STD
1
Backpack sprayer
Concrete
pAB
Neat
1.63E+07
1.67E+06
ND
-
7.3
0.02
2
Grimed
1.02E+06
1.77E+05
1.24E+03
8.78E+02
3.0
0.36
3
Backpack sprayer
T reated
pAB
Neat
2.92E+06
1.08E+06
1.99E+02
3.65E+01
6.6
0.90
4
plywood
Grimed
6.46E+051
3.01E+05
6.36E+02
5.99E+02
3.3
0.64
5
Backpack sprayer
Concrete
Spor-Klenz" RTU
Neat
7.21E+06
3.72E+06
2.67E+02
2.03E+02
4.6
0.62
6
Grimed
1.24E+04
1.51E+03
1.01E+02
9.22E+01
2.4
0.66
7
Backpack sprayer
T reated
Spor-Klenz" RTU
Neat
1.59E+07
7.09E+06
ND
-
7.4
0.01
8
plywood
Grimed
1.27E+06
5.26E+05
1.88E+03
2.20E+03
3.1
0.53
9
Chemical sprayer
Concrete
pAB
Neat
2.01E+06
1.46E+06
ND
ND
6.4
0.01
10
Grimed
1.66E+0512
1.44E+05
4.65E+02
4.03E+02
3.5
0.52
11
Chemical sprayer
T reated
pAB
Neat
6.73E+06
2.72E+06
1.27E+00
9.33E-01
6.8
0.27
12
plywood
Grimed
4.29E+051
2.05E+05
1.96E+02
3.40E+02
3.9
0.79
13
Chemical sprayer
Concrete
Spor-Klenz* RTU
Neat
4.94E+041
2.39E+04
5.10E+02
3.33E+02
2.5
1.31
14
Grimed
1.51E+06
2.80E+05
3.60E+01
3.78E+01
4.8
0.43
15

T reated

Neat
9.58E+06
3.09E+05
ND
-
7.1
0.14
16
Chemical sprayer
plywood
Spor-Klenz* RTU
Grimed
Samples were exposed to exccess heat during heat shock process
CFU - colony forming unit; LR - log reduction; STD - standard deviation
Positive control recoveries below 6 logs, prevent achievement of 6 LR
2Some replicates were too contaminated to enumerate.
3
U.S. Environmental Protection Agency
Office of Research and Development

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Table 2. Decontamination Results for Small Coupon (Bench-Scale) Tests with MS2
Decon
Agent
Material
Positive Control
PFU
Test Coupon
PFU
Surface Decontamination Efficacy
(LR)


Average
STD
Average
STD
Average
Cumulative STD

Neat concrete
6.77E+06
2.68E+06
ND
-
7.1
0.12
pAB
Grimed concrete
2.99E+07
2.59E+07
2.83E+05
6.34E+05
6.4
1.3
Neat plywood
1.37E+08
7.97E+07
4.54E+05
1.46E+05
2.4
0.19

Grimed plywood
4.91E+07
7.36E+07
8.57E+05
9.86E+05
3.7
1.7

Neat concrete
3.68E+07
1.24E+07
1.39E+07
7.93E+06
0.46
0.15
2%
Citric
acid
Grimed concrete
6.17E+07
1.03E+08
4.99E+06
4.21E+06
1.1
1.1
Neat plywood
6.21E+07
1.12E+07
3.52E+04
3.83E+04
3.5
0.25

Grimed plywood
6.35E+07
8.05E+07
7.88E+07
6.96E+07
0.08
0.56
LR - log reduction; PFU - plaque forming unit; STD - standard deviation
Table 3. Decontamination Results for Large Coupon (Lab-Scale) Tests with MS2
Decon Agent
Material
Positive Coupon
(PFU)
Test Coupon
(PFU)
Surface Decontamination Efficacy
(LR)


Average
STD
Average
STD
Average
Cumulative STD

Neat Concrete
2.46E+04
6.61E+03
ND
-
4.7
0.06
pAB
Grimed Concrete
1.54E+06
2.65E+05
ND
-
6.2
0.04
Neat Plywood
3.64E+06
-
9.78E+01
4.44E+01
4.8
0.33

Grimed Plywood
4.70E+06
4.71E+04
ND
-
7.0
0.00
2% Citric Acid
Neat Concrete
6.20E+03
6.74E+03
2.89E+03
1.98E+03
0.20
0.36
Grimed Concrete
8.36E+05
3.26E+05
1.15E+02
1.06E+02
4.3
0.35
LR - log reduction; PFU - plaque forming unit; STD - standard deviation
The second study sought to determine the efficacy of spray-applied bleach decontamination
formulations, specifically formulated to remain liquid at low temperatures (i.e., below the freezing point
for water) [1], These non-freezing bleach formulations (NFB) could be beneficial when conducting
remediation activities during cold weather conditions. The materials utilized during testing were glass
and concrete, surface types common to building exteriors in outdoor environments.
The tests were conducted in an environmental test chamber (ETC) so that temperature conditions
ranging from -25 °C to 25 °C could be precisely achieved (Figure 2). An automated spray system,
completely contained within the environmental chamber was developed. The use of this setup allowed
easy control of test parameters (i.e., spray duration, spray pressure, volume of spray, temperature and
relative humidity), and allowed a more realistic challenge to the decontamination method as all
components (spray nozzles, spray reservoir, hoses, etc.) were located inside the chamber and at the test
temperature.
U.S. Environmental Protection Agency
Office of Research and Development

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Eight non-freezing bleach-based formulations were prepared from recipes provided by the EPA-
Environment and Climate Change Canada (ECCC) working group. Each solution was evaluated for its
ability to inactivate Bacillus atrophaeus spores on building material (concrete and glass) surfaces. The
solutions contained de-icing agents that depressed the freezing point of the solutions below the target
test temperatures so that the solutions could be spray-applied. In addition to the NFB solutions,
traditional pH-amended bleach (pAB) solution was included in the evaluations (when temperatures
permitted) as a reference decontamination agent.
Solenoid val\
Figure 2. Photographs of the Spray Apparatus Inside the Temperature-Controlled Environmental Test
Chamber.
Kyrtar tubing
Coupon holding
funnel
Rinsate collection
vials
Oecont a mination
liquid tank
Coupon holding
stand
Figure 3 summ arizes the surface decontamination efficacy results for pAB and three of the most
efficacious NFB formulations. As the figure shows, pAB achieved a surface LR greater than 6 at
temperatures greater than 0 °C on both materials tested. None of the NFB formulations were as effective
as pAB. As the testing temperature was lowered, decontamination efficacy also tended to decrease.
However, at temperatures greater than 0 °C, no test solutions were as effective as pAB, and none were
observed to achieve a 6 LR (Figure 3). Decontamination efficacy data for the pAB solution were
U.S. Environmental Protection Agency
Office of Research and Development

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gathered only to 0 °C because the freezing point of pAB was determined to be -8 °C. Despite the NFB
solutions demonstrating lower decontamination efficacies compared to pAB, these solutions currently
are the only NFB decontaminants evaluated against Bacillus spores. At conditions below -8 °C, these
solutions may be useful in reducing surface-bound spore concentrations during remediation efforts. The
results from this project provide an important baseline that further work can build upon to develop and
characterize new decontamination options under environmentally-challenging conditions such as
freezing temperatures.
pAB
Glass Concrete
L> 7.0
>* 3 0
o 2-0
10DC
Temperature (°C)
25 Bc
S6
*o
¦25aC -10°C 0°C	10°C
Temperature |°C)
25"C
S5
0.0
S 7* °
0
3 6.0
1	8.0
O)
4.0
©

-1
3.0
&
2.0
re

u

it=
1.0
ULJ
0.0
A
B- 8 B"
3.2
•25°C
10"C	0"C	10°C
Temperature (°C)
S7
8.0
7.0
6.0
5.0
4.0
— 3,0
o 2.0
ro
fi
1.0
0.0
"C
o
cc
£
LU
• Glass Con er*t*
2.5
3 3
12
.2StlC
H i
" ¦" I"
-10°C	0°C	10°C	25DC
Temperature (°C)
^Denotes Full Surface Decontamination Based on Detection Limit
Figure 3. Surface Decontamination Efficacy (Log Reduction) for pH-Amended Bleach and three non-
freezing Decontamination Solutions (S5, S6, and S7).
In summary, many factors can influence decontamination efficacy in outdoor environments.
Currently, our grasp of decontamination capabilities is lacking for outdoor areas. Further work is needed
to determine impacts of weather (rain, wind, snow, humidity, extreme temperatures) and surface/matrix
types on our ability to select viable options for remediation. Also, application of decontamination
methods over large areas with readily-available devices, chemicals, supplies, and workers will be
challenging and should be addressed prior to an incident. While the current two studies have begun to
address questions regarding outdoor decontamination, many more need to be answered in order to
develop robust and comprehensive remediation strategies for large, outdoor areas.
6
U.S. Environmental Protection Agency
Office of Research and Development

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Contact Information
For more information, visit the EPA Web site at http://www2.epa.gov/homeland-security-research.
Technical Contact: Worth Calfee (Calfee.Worth@epa.gov)
General Feedback/Questions: Amelia McCall (mccall.amelia@epa.gov)
Disclaimer
The U.S. Environmental Protection Agency through its Office of Research and Development
funded and managed the research described herein under contract EP-C-15-008. This summary has been
subjected to the Agency's review and has been approved for publication. Note that approval does not
signify that the contents reflect the views of the Agency. Mention of trade names, products, or services
does not convey official EPA approval, endorsement, or recommendation.
References
1.	Calfee, W., L. Mickelsen, S. Serre, R. Rupert, AND M. Nalipinski. Evaluation of Spray-Based,
Low-Tech Decontamination Methods under Operationally Challenging Environments: Cold
Temperatures. U.S. Environmental Protection Agency, Washington, DC, EPA/600/R-17/211,
2017.
2.	U.S. EPA. Effectiveness of Spray-Based Decontamination Methods for Spores and Viruses on
Heavily Soiled Surfaces. U.S. Environmental Protection Agency, Washington, DC, EPA/600/R-
16/162, 2016.
U.S. Environmental Protection Agency
Office of Research and Development

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