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technical BR
Bio-Response Operational Testing
and Evaluation (BOTE) Project
Phase 1 - Decontamination Assessment
INTRODUCTION
The Bio-Response Operational Testing and
Evaluation (BOTE) Project was a multi-
agency effort designed to test and evaluate a
complete response to a biological incident -
from the initial public health and law
enforcement reaction through environmental
remediation. The scenario involved the
intentional release of Bacillus anthracis (Ba)
spores, the causative agent for anthrax,
inside a building. In this study, Bacillus
atrophaeus spp. globigii (Bg) spores were
used as a non-pathogenic surrogate for Ba
spores.
The BOTE Project was conducted in two
distinct phases. Phase 1 was a field-level
decontamination assessment. Phase 2 was
an operational exercise involving key federal
agencies that are responsible for the forensic
investigation, public health assessment, and
remediation following a biological incident.
This summary is focused on Phase 1 of the
project. Phase 1 was designed to assess
three approaches to site remediation after
the release of Bg spores within a building
(Figure 1). The assessment incorporated
recent advances in biological sampling and
decontamination that had previously been
tested in small-scale applications.
BOTE Project Phase 1 Objectives
• Conduct and evaluate field-level application of
three decontamination technologies/protocols
for the cleanup of a building contaminated with
Bacillus anthracis (Ba) spores, the causative
agent for anthrax. Simulants of Ba spores were
used.
• Utilize newly developed biological sampling and
analysis methods for characterization of the
anthrax simulant contamination (concentration
and location) and determination of
decontamination efficacy.
• Collect and analyze results and operational
information from the decontamination
operation.
• Perform a cost analysis of the complete
remediation process.
Determine the exposure to spores associated
with reentry into the building following cleanup.
Figure 1. Two story building, without and with secondary
containment (tenting), at Idaho National Laboratory.
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METHODS
The testing was conducted in a two story office building (4,025 ft2/floor) that was tented to provide
secondary containment of the spores in the building. The building was set up such that each floor
included three rooms furnished with residential materials (e.g., sofa, bed), three rooms furnished
with commercial materials (e.g., desk, file cabinet), one mailroom and one industrial-style workshop
(Figure 2). Each floor had an independent heating, ventilation, and air conditioning (HVAC) system.
Phase 1 testing was conducted in three rounds, each utilizing a different decontamination method
with all other test conditions being consistent across the rounds. Each round (Figure 3) consisted
of preparing the facility, dissemination of Bg spores to achieve target surface loadings,
characterization sampling, decontamination and waste management, post-decontamination
sampling, and facility assessment. After each round, the facility was re-set to its initial configuration
for the start of the next round.
Concentration of approximately 104-106 and 102 viable spores/ft2 were released on the first and
second floors, respectively, for each round in order to test the efficacy of each decontamination
approach under two contamination challenge amounts.
Figure 3. Example rooms (top left to bottom right): mailroom, workshop, residential and
commercial setting.
•-.
ampling •
Reaerosolization
h Sampling —|
Sand Sample Colleciu
Figure 3. Timeline of major activities in each round.
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Round 1: Fumigation with STERIS Corp. Vaporized Hydrogen Peroxide (VHP ) Technology
Figure 4 VHP generation system.
• Full-facility in situ decontamination, including contents (i.e., no materials
were removed prior to fumigation)
• Fumigation of both floors and HVAC system utilizing two VHP® generation
systems (Figure 4), one connected to each floor's air handling unit
• Target fumigation conditions: 250 ppmv of hydrogen peroxide vapor (H2O2)
for 90 min and a cumulative concentration-time product (CT) of 400 ppmv-
hrsat>65°F(~18°C)
• Portable fans were operating inside the facility to aid H2O2 distribution
• Temperature, relative humidity (RH) and H2O2 concentration were
measured continuously at numerous locations
• Spor-Klenz Ready to Use sterilant was sprayed on surfaces on which equipment was to be placed during
fumigation
• The target H2O2 concentration was not achieved at all monitored locations
• Decontamination process was completed in 3 days from set-up through aeration
Round 2: Decontamination process incorporating pH-adjusted bleach spraying
» Preparation: Source reduction was conducted by teams in
Level C PPE; all porous and difficult to decontaminate
materials were removed from the facility (e.g., ceiling tiles,
furniture, carpet, HVAC supply line). Materials were
bagged, sprayed with pH-adjusted bleach (Figure 5), and
removed for sampling and management as waste
• Facility was maintained under negative pressure and
ambient temperature throughout decontamination
» Decontamination: Teams in Level B PPE (due to the
decontaminant) sprayed all interior surfaces including
HVAC return duct with pH-adjusted bleach using a gas-
powered sprayer situated outside the facility; surfaces
were maintained wetted for >10 min.
Figure 5. Bagging porous materials (R) and
spraying with pH-adjusted bleach (L).
• Drying : Portable fans and heaters were run to the facility during drying phase
• The process was planned and implemented by EPA Region 10, based upon field experience from EPA Region 1
and recent EPA lab studies
• Decontamination was completed in 5 days from preparation through drying
Round 3: Fumigation with chlorine dioxide gas (CIO2) (Sabre Technical Services, LLC.)
• Full-facility in situ decontamination; only materials removed were mattresses
and cushions due to the time required to aerate these materials following
decontamination (i.e., preventing sampling due to the toxicity of CIO2)
• A truck-mounted CIO2 gas-generation system was used (Figure 6)
• Target fumigation conditions: 3000 ppmv of CIO2 for 3 hrs and a cumulative
CT of 9000 ppmv-hrs at > 65 °F (-18 °C) and RH > 65%
• Fans were added inside the facility to aid CIO2 distribution, activated carbon
was used to scrub CIO2 during maintenance of negative pressure and
during aeration
• Temperature, RH and CIO2 concentrations were continuously measured at
numerous locations
Figure 6. Truck-mounted gas-
generation system.
floor mean RH was below the target 65%
• Target CIO2 concentration was achieved at all monitored locations; 2"
(63.7±5.9%)
• Decontamination process was completed in 3 days, from set-up (~2 days) through aeration, with the exception
of time required for staging the material used for tenting the facility (on the facility inside the secondary
enclosure)
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SAMPLE COLLECTION
Surface Sampling
The effectiveness of the three decontamination
technologies was determined by measuring the
surface concentrations of viable Bg spores in
colony forming units ((CPU) per ft2), before and
after decontamination. Wipe sampling (Figure 7)
using cellulose sponge-stick wipes and swabs, and
vacuum sampling (using vacuum socks) were the
primary collection methods. These sampling
methods were consistent with current validated or
Centers for Disease Control (CDC) recommended
sampling for Ba spores. Additional surface samples
using Versalon® wipes (gauze wipes) were also
collected for use in an operational assessment of
EPA's rapid-viability polymerase chain reaction
(RV-PCR) analytical method. All sampling
metadata (e.g. time, location, sample type) was
collected using hand-held personal data acquisition
(PDAs) devices and the Sandia National
Laboratories' Building Restoration Operations
Optimization Model (BROOM) software system.
Figure 7. Above: Wipe sampling: sponge-stick and swab.
Below: Vacuum sampling (left) and PDA with BROOM
software (right).
Air Sampling
Aggressive air sampling offers the potential
to reduce the post-decontamination
sampling burden by collecting bulk air
samples that could be used to determine if
contamination remains. Following post-
decontamination surface sampling,
aggressive air sampling was conducted in
two rooms as a secondary evaluation of
decontamination effectiveness and to
compare these results to surface sampling
results. Air samples were collected during
and after the agitation of potential surface
contamination using a leaf blower (Figure 8);
samples were collected using high volume
samplers; and collection media were
analyzed via culture methods. Aggressive air
sampling was conducted successfully after all three decontamination rounds, and results were
comparable to surface sample results. The air sampling results after Round 1 (fumigation with
VHP®) showed the highest concentrations of spores detected in the air; the lowest spore
concentrations were detected for Round 3 (fumigation with
Figure 8. Use of a leaf lower to agitate surface contamination.
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Figure 9. Personnel
decontamination line.
Wastewater Treatment and Sampling
Wash water was collected from the personnel decontamination line
(Figure 9) in 55-gal drums and used to assess the effectiveness of
an on-site bleach treatment procedure. An ultrafiltration
concentrator was used to sample Bg spores in the wash water. The
ultrafiltration device was intended to concentrate spores contained in
a high volume of wastewater into a much smaller volume of water;
thereby, increasing detection sensitivity. Unfortunately, the high
turbidity of the wash water presented operational challenges for the
ultrafiltration method and only a small number of viable spores were
able to be detected in the wash water. The bleach treatment
procedure was, therefore, alternatively assessed by spiking wash
water with additional Bg spores prior to the addition of bleach to
raise the concentration of spores to levels detectable without
concentration. The bleach process was determined to provide
greater than a 3-log reduction of viable spores (the upper limit that
could be determined in this study). Results from the spiked wash
water test were similar to those obtained from laboratory experiments using artificially generated
wash water possessing similar water quality characteristics as field generated wash water. These
findings suggest that the proposed inactivation procedure would be applicable for wash water
derived from similar personnel decontamination activities.
Spore Transport and Reaerosolization
To examine the potential transportation of Bg spores from the initial area of dissemination inside
the building to outside the building, Petri dishes containing sterilized sand (Figure 10) were placed
directly outside the test facility, but within the secondary containment enclosure and around
building entrances, exits and high traffic areas. The detection of Bg in some of these previously
uncontaminated sand samples suggested that spores
have the potential to migrate out of a contaminated
building and settle into the surrounding environment. The
study did not attempt to differentiate when exfiltration
occurred from the facility (i.e., during dissemination or
during subsequent remediation activities).
Figure 10. Tray of Petri dishes containing
sterilized sand.
Reaerosolization was studied by measuring the
concentration of Bg spores in the air within two rooms at
five phases (background, after spore dissemination, prior
to surface sampling, pre-decontamination and post-
decontamination) throughout each round of
decontamination. Air samples were collected using SKC
BioSamplers®. Post-decontamination Bg spores were
detected in the air following Round 1 (Fumigation with Vaporized Hydrogen Peroxide (VHP®)), but
not Rounds 2 (process incorporating removal and spraying of pH-adjusted bleach) or 3 (chlorine
dioxide fumigation). All samples collected after spore dissemination, before surface sampling and
pre-decontamination contained measureable concentrations of spores indicating that airborne
concentrations of the spores persist after dissemination and that spores may, potentially, be
reaerosolized by typical remediation activities under certain conditions.
Exposure Assessment
The surface and air samples collected, as well as the monitoring and assessment of
decontamination operational parameters, provided measurements for the assessment of pre- and
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post-decontamination exposure potential. However, limitations in the data and site-specific
variables currently do not allow for accurate exposure predictions that can be extrapolated to other
sites. Results and lessons learned from the BOTE Project will be used to develop a methodology
for site-specific exposure assessment.
COST ANALYSIS
The BOTE Project cost analysis
estimated the overall cost of the
application of various decontamination
technologies as a function of materials,
time (including labor hours), waste
disposal, and other resources.
• Sampling and analysis costs were
roughly equivalent in all three rounds
of decontamination due to the study
design. As can be seen in Figure 11
(top), sampling and analysis costs
were the largest contributors to the
overall cost. However, since this was
an operational assessment,
considerably more samples were
taken than anticipated for an actual
incident in a building of this size.
Regardless, sampling and analysis
costs are anticipated to be a major
cost factor, which should be
considered in any cleanup of a
biological incident.
• The Incident Command (1C) costs
were also relatively independent of
the decontamination method used in
this project.
• The cumulative costs of the
decontamination processes (e.g.
materials, contracts, labor) were
roughly equivalent for all three
decontamination methods tested
(Figure 11 bottom).
• Waste management costs were
shown to be a significant cost
component particularly for the pH-
adjusted bleach decontamination
IC Cost
Restoration Cost
Decontamination Cost
Average Sampling and Analysis Cost
1.000,000-
800,000 -
- 600,000 -
400,000 -
200,000 -
VHP
pH-Adjjsted
Bleach Process
Waste Management Costs
Cost of Decontamination Materials
Decontamination Contractor Fixed Costs
Cost of Removal Teams Entering
Costs of Decontamination Teams Entering
Cost of Decontamination Line Operations
300,000 -
5? 250.000-
200,000-
VHP'
pH-Adjusted
Bleach Process
Figure 11. Overall all cost components (top) and waste
management cost details (bottom)
process as used in this exercise. Waste characterization sampling was the largest single
component of waste management costs. These costs are specific to the decontamination
processes as they were employed in the BOTE Project and based-upon documented
assumptions made about waste management procedures and costs.
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CONCLUSIONS
Each decontamination method was performed a single time in the BOTE Project; the results and
conclusions should be considered based upon the implementation as described above.
Decontamination costs alone, not considering sampling and analysis or waste management, were
roughly equivalent. Notable differences in waste generation and anticipated associated cost were
documented. The decontamination efficacy findings for Rounds 1, 2, and 3 are summarized in
Figure 12, below.
20
40
80
100
Round 1 Pre-
iii = 291)
Round 1 Post-
in = 276)
Round 2 Prc-
(n =268)
Round 2 Post-
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FOR MORE INFORMATION
The complete report for Phase 1 of the BOTE Project can be found at: www.epa.gov/nhsrc
A video documentary is available at: http://www.epa.gov/nhsrc/video/bote.html
Technical Contacts:
Shawn Ryan (919) 541-0699
ryan.shawn(S)epa.gov
Shannon Serre (919) 541-3817
serre.shannon(S)epa.gov
Communications Contact:
Kathy Nickel (513) 569-7955
nickel.kathy(S)epa.gov
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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
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statutory requirements, and Homeland Security Presidential Directives.
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