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technical BR
Assessment of Liquid and Physical Methods for Decontamination of
Surfaces Contaminated with Bacterial Spores: Evaluation and
Refinement of the Procedural Steps
Introduction
The availability of decontamination methods for use in
the recovery from the release of a biological agent is a
critical need. A release over a wide area could result in
the contamination of many residences, businesses,
public facilities and outdoor areas. In 2001, following
the mailing of letters containing Bacillus anthracis
(anthrax) spores, contaminated facilities were
fumigated with chlorine dioxide or hydrogen peroxide.1
Contamination following a wide-area release could
overwhelm the nation's remediation capacity, dragging
clean up out over many years and resulting in
enormous economic impact. Quick, effective and
economical decontamination methods that can be
employed over wide areas are needed to increase
preparedness for such a release.
U.S. EPA's Homeland Security Research Program
(HSRP) develops products based on scientific
research and technology evaluations. Our products
and expertise are widely used in preventing,
preparing for, and recovering from public health and
environmental emergencies that arise from terrorist
attacks or natural disasters. Our research and
products address biological, radiological, or chemical
contaminants that could affect indoor areas, outdoor
areas, or water infrastructure. HSRP provides these
products, technical assistance, and expertise to
support EPA's roles and responsibilities under the
National Response Framework, statutory
requirements, and Homeland Security Presidential
Directives.
In the document Assessment of Liquid and Physical Decontamination Methods for Environmental
Surfaces Contaminated with Bacterial Spores: Development and Evaluation of the
Decontamination Procedural Steps2, the U.S. Environmental Protection Agency's (EPA)
Homeland Security Research Program reports on its evaluation of an 8-step, low technology
approach to decontaminating anthrax-contaminated surfaces. This approach had been used
previously by EPA in response to contamination incidents involving naturally occurring anthrax
spores.3 The primary objective of this study was to determine through laboratory testing the
effectiveness of individual and various combinations of steps of the procedure. A subsequent
study, Assessment of Liquid and Physical Decontamination Methods for Environmental Surfaces
Contaminated with Bacterial Spores: Evaluation of Spray Method Parameters and Impact of
Surface Grime4, was conducted to further evaluate the decontamination approach. The results
from these studies indicate that shorter, simpler decontamination procedures, can be almost as
effective as the previously employed 8-step procedure.
U.S. Environmental Protection Agency
Office of Research and Development, Homeland Security Research Program
EPA/600/S-13/064
April, 2013
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Overview
In the first study, tests using various combinations of steps from the 8-step decontamination
procedure were performed. The tests used 929 cm2 (196 in2) pieces (coupons) of common
building materials. Since significant differences in decontamination efficacy have been reported
for porous and non-porous materials,
both types of materials were tested5'6.
Porous building materials tested were
carpet, pressure treated deck wood,
and rough cut pine wood; non-porous
building materials tested were painted
8-step Procedure
Step 1: Vacuum Step 5: Wet vacuum
Step 2: pAB mist Step 6: pAB spray
(10 min contact) (30-60 min contact)
Step 3: Detergent Scrub Step 7: Rinse
Step 4: Rinse Step 8: Wet vacuum
wallboard and concrete. The coupons
were inoculated via aerosol deposition with Bacillus atrophaeus spores (a surrogate for anthrax
spores) at approximately 1 x 107 colony forming units (CPU) per coupon. Testing included the
various building materials oriented both vertically and horizontally, depending upon how they may
be encountered in the field.
Attempts were made to adapt the laboratory methods in a manner that more closely mimic field
conditions than previous laboratory tests. This was accomplished by using larger coupons
inoculated by an aerosol method and sampled using wipe and/or vacuum sock sampling methods.
Tests were conducted to determine the comparability of results from this study to previous
laboratory studies that had used smaller coupons, a liquid inoculation and extraction for spore
recovery (i.e., sampling) from the coupons. A thorough description of the test methods, their
development, and decontamination efficacy results is provided in the report.
Surface decontamination effectiveness is a measure of viable spores remaining on a surface after
decontamination. Decontamination efficacy was determined by comparing recoveries from
positive controls to that of coupons subjected to the treatment. Surface decontamination
effectiveness reflects both the inactivation of spores remaining on the surface and the physical
removal of spores from the material surface. Surface decontamination can be achieved by
physically transferring viable spores from the material surface to another media (e.g., rinse water).
Therefore, overall decontamination effectiveness in this study was measured by analyzing
material surfaces, rinsate, wet/dry vacuum filters and exhaust for the presence of viable spores
following administration of the decontamination procedure. Transfer of viable spore to other such
media may require additional treatment and handling.
Figure 1 presents surface decontamination effectiveness (log reduction in viable spores) achieved
using various combinations of steps from the 8-step procedure. The study demonstrated that the
full procedure, with two chemical decontamination steps employing pH-adjusted bleach (pAB),
achieved a surface decontamination effectiveness of greater than 6 log reduction (>99.9999%) for
all material type and orientations (horizontal and vertical). For the application of pAB, surfaces
were sprayed and allowed to remain wetted with the solution for the desired contact time (i.e., 10
min in Step 2 and 30-60 min in Step 6). Applying just the first five decontamination steps (four
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steps for vertical surfaces) achieved a similar (> 6 log) reduction of spores on all surfaces, except
concrete in the vertical position. Vacuuming alone, with a wet/dry shop vacuum, resulted in a less
than one log reduction of spores on all surfaces. This step was determined to not be useful for this
procedure on surfaces that are relatively free of loose debris and dust.
Viable spores were found in the wet/dry vacuum exhaust (past the HEPA filter), wet/dry vacuum
HEPA filters, and rinsate (Figure 2) from all surfaces in both the full 8-step procedure and in the 5-
step procedure, indicating that surface decontamination was achieved by a combination of
chemical inactivation and physical removal. Hence, the full or modified procedure was successful
at surface decontamination, but both procedures resulted in contaminated rinsate and some
breakthrough/bypass of spores through wet/dry vacuum HEPA filters.
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Step 1: Vacuum
Step 2: pAB mist (10 min
contact)
Step 3: Detergent Scrub
Step 4: Rinse
Step 5: Wet Vacuum
Step 6: pAB spray (30-60
minute contact)
Step 7: Rinse
Step 8: Wet vacuum
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Steps 1-8 Steps 1-5 step 1 Steps 3, 4, 5 Steps 4 &5 Steps 1, 3 & 6-8
Figure 1. Surface decontamination shown as the average surface log reduction on material surfaces
per decontamination step or procedure (v = vertical, h = horizontal)2
Figure 2 illustrates the recovery of viable spores within rinse water collected following Steps 3-5.
The presence of viable spores in the rinsate shows that, although the procedure is capable of
achieving a greater than six log reduction of spores on the material surfaces, a significant number
of viable spores are transferred to the rinsate. Overall spore inactivation using either the 5- or 8-
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step procedure was in the order of two to four log reductions. Additional treatment of the rinsate
would be required to achieve overall (complete) inactivation.
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5-Step
• Concrete-v
• Concrete-h
• Painted Wallboard-v
• Painted Wallboard-h
• Rough-cut Wood-v
DCarpet-h
• Deck Wood-h
Step 1: Vacuum
Step 2:10 min bleach mist
Step 3: Detergent Scrub
Step 4: Rinse
Step 5: Wet Vacuum
Step 6: 30-60 minute bleach
Step 7: Rinse
Step 8: Wet vacuum
Figure 2: Viable spores (CPU, colony forming units) contained in rinsate from 8-step and 5-step
surface decontamination procedures2
Tests on the effectiveness of both the original 8-step procedure and the abbreviated 5-step (4-
step on vertical surfaces) procedures involved a pAB mist that maintained a wetted surface for 10
minutes. Tests T1 though T3, shown in Figure 3 below, show the effectiveness of various pAB
spray application regimens. Each pAB spray regimen was followed (10 minutes later) by a de-
ionized water rinse. Test T1 consisted of a single 4-sec pAB spray. T2 consisted of three sprays
at t = 0, 5, and 10 minutes. T3 consisted of one 12-sec spray. All three spray regimens yielded
similar results with greater than 6 log reductions on deck wood surfaces suggesting a short
duration pAB spray can be as effective as the multiple spray regimens, and would generated less
runoff.
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T2
T3
Figure 3: Log reductions in spores on deck wood following spray and rinse2
A subsequent study, Assessment of Liquid and Physical Decontamination Methods for
Environmental Surfaces Contaminated with Bacterial Spores: Evaluation of Spray Method
Parameters and Impact of Surface Grime, was conducted to further refine the decontamination
approach. The study sought to determine if variations in the spraying approach could reduce the
time required to conduct the decontamination procedure and minimize runoff while still effectively
decontaminating the test materials. The second study continued the evaluation of spray
parameters by varying duration and flow rate and evaluating decontamination efficacy, but without
a rinse step that generates most of the runoff. Rough-cut pine wood, painted wallboard and
concrete were chosen as test materials as they represent commonly occurring, yet challenging to
decontaminate, surfaces likely to be encountered during an urban remediation. Consistent with
the first study, the second study also used 929 cm2 coupons inoculated with approximately 1 x 107
CPU Bacillus atrophaeus spores.
The procedures with no rinse step (Table 1) achieved comparable efficacies on non-porous
material surfaces to those with a rinse step (Figure 1). With no rinse step, reductions of about 6
log were observed on non-porous surfaces. A 15 second spray, two 15 second sprays, and a 30
second spray, each without a rinse step, equally achieved the 6 log reduction. This result
suggests that a single spray application of pAB with no vacuum, scrub or rinse steps may be
sufficient for nonporous surfaces. Eliminating the rinse step would minimize runoff,
reaerosolization and procedure duration.
On a porous surface (rough cut pine) neither the 15 second spray, two 15 second sprays, nor
single 30 second spray, without a rinse step, achieved a 6 log reduction. Approximately 3 log
reductions were observed. Spray with pAB followed by rinse achieved a 6 log reduction on the
rough pine surface suggesting that a combination of physical removal and inactivation accounted
for the reduction.
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The tests included in these studies, as well as previous field applications of the full 8-step
procedure had been conducted with freshly prepared pAB. The procedure to date has required
use of the pAB within 15 minutes of preparation. Experiments were conducted to evaluate the
effectiveness of pAB over time (up to 32 hours after preparation). The study showed that aging of
the pAB solution decreases its effectiveness on surfaces that are typically difficult to
decontaminate, such as wood. In contrast, aging seems to have little or no effect (within the first
four hours after preparation) on the ability of pAB to decontaminate the nonporous drywall
coupons. Solutions of pAB older than 4 hours show a marked decline in efficacy for both types of
material (i.e., porous and non-porous) tested. The results indicate that when decontaminating
non porous surfaces, the preparation of pAB solution could possibly be done less frequently and
perhaps in larger batches without compromising decontamination efficacy.
Tests were also conducted to resolve the effect of contamination level on decontamination
efficacy. With the lower inoculum, efficacies for nonporous surfaces ranged from 5 - 6 log
reduction. Tests of the decontamination procedures conducted at a medium level contamination
(1 x 104 to 1 x 10s CPU) on drywall and concrete coupon materials confirmed that full
decontamination (meaning no viable spores detected following decontamination) can be obtained
with a single application of pAB (5 sec/0.09 m2 or 5 sec/ft2). However, no single pAB application
was found to be effective in inactivating/removing spores from low level (1 x 102 CPU) inoculated
pine-wood coupons. Porous surfaces yielded only 0.13 - 3.5 log reduction (Table 2).
Runoff from the spray parameter testing was collected and analyzed for the presence of viable
spores. Runoff from all tests performed on non-porous surfaces contained viable spores
indicating that log reduction on the material surface resulted from a combination of inactivation
and physical removal. No viable spores were detected in the runoff from a 30-sec pAB spray on
the pine wood coupons; whereas, viable spores were present in the runoff from tests utilizing 15-
sec pAB applications.
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Table 1: Surface Decontamination Parametric (high inoculation) Test Results3
Material
Drywall
Concrete
Pine
Wood
Drywall
Concrete
Pine
Wood
Pine
Wood
Pine
Wood
Pine
Wood
Positive Controls (n=3)
Avg. CPU/
Sample
1.72 x 107
1.02 x 107
1.95 x 106
2.05 x 107
8.16 x 106
3.93 x 106
2.31 xlO6
2.31 xlO6
2.31 xlO6
Mean
of
Logs
7.23
7.00
6.27
7.30
6.89
6.57
6.35
6.35
6.35
RSD
(%)
22%
22%
41%
25%
38%
47%
30%
30%
30%
Test Coupons (n=3)
Avg. CPU/
Sample
351
7
3520
6
12
2236
1600
1984
1188
Mean of
Logs
1.54
0.60
3.39
0.26
0.41
2.86
3.03
3.26
3.06
RSD (%)
170%
83%
95%
155%
163%
154%
105%
46%
27%
LR
5.69
6.40
2.87
7.04
6.48
3.71
3.32
3.09
3.28
RSD
(%)
23%
11%
16%
12%
15%
22%
14%
8%
4%
pAB Decontamination Conditions Achieved
Decontamination Steps
1 x 15 second spray, low flow, no
reapplication, no rinse
1 x 15 second spray, high flow, no
reapplication, no rinse
1 x 30 second spray, low flow, no
reapplication, no rinse
2 x 15 second spray, low flow,
sprayed at 0 and 5 min, no rinse
1 x 15 second spray, low flow, no
reapplication, no rinse
Flow Rate
(ml/min)
1030
1030
1040
1340
1340
1360
1030
1060
1030
CPU, colony forming units; LR, log reduction; n, number of replicates; pAB, pH-adjusted bleach; RDS, relative standard deviation
U.S. Environmental Protection Agency
Office of Research and Development, Homeland Security Research Program
EPA/600/S-13/064
April, 2013
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Table 2 Surface Decontamination Parametric (low inoculation) Test Results3
Material
Drywall
Concrete
Pine
Wood
Pine
Wood
Pine
Wood
Pine
Wood
Pine
Wood
Positive Controls (n=3)
Avg. CPU/
Sample
4.36 x105
6.42 x104
4.33 x104
3.23 x102
1.73x102
1.73x102
1.73x102
Mean
of
Logs
5.64
4.78
4.62
2.44
2.12
2.12
2.12
RSD
(%)
16%
42%
34%
55%
93%
93%
93%
Test Coupons (n=3)
Avg.
CPU/
Sample
0.5
0.5
45
47
107
160
40
Mean
of
Logs
-0.20
-0.15
1.11
1.36
1.99
1.84
1.50
RSD
(%)
3%
1%
143
%
136
%
47%
141
%
87%
LR
5.84
4.93
3.51
1.09
0.13
0.28
0.62
RS
D
(%)
0.0
0
0.0
0
0.2
9
0.0
0
0.0
0
0.0
0
pAB Decontamination Conditions
Achieved
Decontamination Steps
1x15 second spray, no
reapplication, no rinse
1 x 30 second spray, no
reapplication, no rinse
1 x 30 second spray, no
reapplication, no rinse
2x15 second spray,
sprayed at 0 and 5 min, no
rinse
2x15 second spray,
sprayed at 0 and 15 min,
no rinse
Flow Rate
(ml/min)
1000
1000
1000
1100
1100
1100
1000
CPU, colony forming units; LR, log reduction; pAB, pH-adjusted bleach; RDS, relative standard deviation
Laboratory testing to date utilized clean coupons, whereas, real-world building urban materials will
likely contain a layer of grime that could impact decontamination. Therefore, tests were conducted
to evaluate the effect of grime (dirty surfaces) on decontamination effectiveness. The effects of
grime on decontamination effectiveness were tested using rough cut pine wood and concrete
coupons. Neat (no grime) and grimed coupons were subjected to spray-based decontamination
procedures that evaluated the additive effects of physical cleaning procedures (scrubbing and
vacuuming) on surface decontamination efficacy. The results of these studies indicate that the
presence of grime does not significantly impact decontamination of these surfaces (Figure 4).
Additionally, neither the use of a surfactant with the pAB solution, nor vacuum and scrub steps on
grime covered surfaces resulted in an observable improvement to the surface decontamination
efficacy. It is important to note that the level of grime used here (1 gram per coupon) may not
represent heavily-soiled surfaces. Larger amounts of grime may indeed affect decontamination
efficacy.
U.S. Environmental Protection Agency
Office of Research and Development, Homeland Security Research Program
EPA/600/S-13/064
April, 2013
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I Concrete-Clean
I Concrete-Grime
Wood-Clean
I Wood-Grime
pAB spray pAB/TSP spray
pAB/TSP spray, vacuum pAB/TSP
scrub spray, scrub
Figure 4: Effect of Grime on Surface Decontamination3
Summary
The 8-step decontamination procedure using a pAB solution was tested to determine the
effectiveness of individual and combinations of steps.
The results from the study entitled Assessment of Liquid and Physical Decontamination Methods
for Environmental Surfaces Contaminated with Bacterial Spores indicate that a shorter, simpler
decontamination procedure can be as effective as the previously employed 8-step
decontamination procedure.
Results suggest that an abbreviated 4-step procedure, comprised of pAB mist (10 min contact
time), detergent scrub, rinse, and wet vacuum steps, was almost as effective as the 8-step
procedure in decontaminating all tested material surfaces (porous and non-porous). Further,
longer (12-sec pAB spray) or repeated pAB misting (three 4-sec
pAB sprays) as the initial step did not result in an increase in
surface decontamination efficacy (total wetted tie kept to 10
min). Rinse water collected during testing with both the 8-step
and abbreviated procedure showed that viable spores were
transferred from the material surfaces to the rinsate. Hence,
Four-Step Procedure
Step 1:10 min bleach mist
Step 2: Detergent Scrub
Step 3: Rinse
Step 4: Wet vacuum
additional treatment of the rinsate is required to increase overall decontamination (spore
inactivation).
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Non-porous Surface
One-Step Procedure
For painted drywall, concrete, non-porous surfaces results
pointed to the potential for even greater simplification of the
decontamination procedure. Results from the second study,
Step 1: 15 mm bleach mist durjng whjch sprgy method parameters were evaluated and no
rinse steps were employed, achieved higher levels of spore inactivation on material surfaces than
did the test involving a rinse step. Testing of decontamination effectiveness using a 15 second
pAB spray alone, with no vacuum, scrubbing or rinse steps was effective for non-porous surfaces,
achieving a 6 log reduction on these surfaces. Hence, for non-porous surfaces, a pAB spray
approach could be used so as to minimize the duration of the procedure, worker exposure,
potential for contaminated runoff, and the potential for reaerosolization of spores by vacuuming
and scrubbing. Porous surfaces were more difficult to decontaminate, as approximately 3 log
reductions was achieved for these materials. These results suggest that porous surfaces are not
adequately decontaminated using the spray alone approach as tested.
Finally, pAB solution was shown to retain its effectiveness up to four hours after its preparation,
particularly for use on non-porous surfaces. Decontamination of porous surfaces with pAB may
require smaller batches prepared more frequently, in order to retain effectiveness of the solution.
Overall, this finding should reduce the frequency, and related personnel time, required to maintain
fresh batches of the decontamination solution during a decontamination effort.
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Contact Information
For more information, visit the EPA Web site at www.epa.gov/nhsrc.
Technical Contacts: Shawn Ryan (ryan.shawn@epa.gov)
Worth Calfee (Calfee.worth@epa.gov)
General Feedback/Questions: Kathy Nickel (nickel.kathy@epa.gov)
References:
1. Sharp RJ, Roberts AG. Anthrax: the challenges for decontamination. Journal of Chemical
Technology and Biotechnology 2006;81:1612-1625.
2. U.S. EPA. Assessment of Liquid and Physical Decontamination Methods for
Environmental Surfaces Contaminated with Bacterial Spores: Development and
Evaluation of the Decontamination Procedural Steps . U.S. Environmental Protection
Agency, Washington, DC, EPA/600/R-12/025, 2012.
3. Snook C, Cardarelli J, Mickelsen R, et al. Medical Toxicology and Public Health: National
Decontamination Team, U.S. Environmental Protection Agency (EPA). Journal of Medical
Toxicology 2008;4:289-291.
4. Assessment of Liquid and Physical Decontamination Methods for Environmental Surfaces
Contaminated with Bacterial Spores: Evaluation of Spray Method Parameters and Impact
of Surface Grime, U.S. Environmental Protection Agency, Washington, DC,
EPA/600/R/12/591,2013.
5. Calfee, M.W., Choi, Y., Rogers, J., Kelly, T., Willenberg, Z., Riggs, K., 2011. Lab-
Scale Assessment to Support Remediation of Outdoor Surfaces Contaminated
with Bacillus anthracis Spores. Journal of Bioterrorism and Biodefense. 2, 1-8.
6. Wood, J.P., Choi, Y.W., Rogers, J.V., Kelly, T.J., Riggs, K.B., Willenberg, Z.J.,
2011. Efficacy of liquid spray decontaminants for inactivation of Bacillus anthracis
spores on building and outdoor materials. J. Appl. Microbiol. 110, 1262-1273.
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