technical BRIEF
INNOVATIVE RESEARCH FOR A SUSTAINABLE FUTURE
w ww. e p a. gov/resea rc h
Research Results to Enhance Management of
Bacillus anthracisContaminated Wash Water
Purpose
This technical brief provides decision makers with practical information
that could be useful for managing and treating decontamination wash
water generated during remediation activities following a Bacillus
anthracis (6. anthracis, anthrax) contamination incident. Research
results related to sampling and analysis methods for challenging water
matrices and various treatment methods are summarized.
Introduction
Following a B, anthracis contamination incident, wash water will likely
be generated during site remediation activities, potentially through
direct use of liquids in decontamination methods, from equipment
decontamination, or through washing personal protective equipment
(PRE) onsite (Figure 1). For example, following the intentional mailing of
letters containing B. anthracis spores in 2001, decontamination of
personnel in PPE working to clean up the U.S. Capitol buildings
generated approximately 14,000 gallons of wash water which required
steam sterilization prior to disposal because of the potential presence
of B. anthracis spores [1], Of recent concern is an urban wide-area
biothreat agent contamination event, which has the potential to
generate much larger volumes of liquid waste from decontamination
activities. This wash water will likely be collected and stored onsite, and
because it poses difficult and unique challenges to the waste stream, it
will potentially require onsite treatment prior to disposal.
Previous work has focused on improving detection and treatment of
highly pathogenic microorganisms in drinking water distribution
systems. These methods have generally involved concentrating
pathogens from a larger volume of tap water to increase the probability
of detection when target organisms are present at low levels.
Traditional drinking water sampling and treatment methods may be
inadequate when applied to decontamination wash water, which poses
unique challenges. Decontamination wash water is generally more
turbid than tap water and can contain high levels of particulate and
U.S. Environmental Protection Agency's
Homeland Security Research Program
(HSRP) develops scientific products based
on 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. The HSRP provides these
products, technical assistance, and
expertise to support EPA's roles and
responsibilities under the National
Response Framework, statutory
requirements, and Presidential Directives.
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organic matter (Figure 2), causing a higher decontaminant demand [2,3], These challenging matrices can also be
problematic when employing traditional methods for concentrating pathogens in drinking water (i.e., particulate
matter causes filter clogging). The U.S. Environmental Protection Agency (EPA) has been developing and testing
sampling and treatment methods designed to overcome these operational challenges for bio-contaminated
wash water.
Sampling and Analysis Methods
Ultrafiltration Concentration
Biothreat agents (e.g., B. anthracis spores) can be present
in low concentrations in decontamination wash water.
Collecting and concentrating particulates in larger water
samples (40-100 liters) would increase the likelihood of
agent detection when sampling to determine the best
treatment and disposal options, but traditional
ultrafiltration methods have proved problematic due to
filter fouling issues arising from high levels of particulate
matter [4].
Axial flow hollow fiber ultrafiltration (HFUF), in which the
filtrate and retentate flow paths of single-use dialysis filters
(Figure 3) are switched, has proved to be an effective concentration method for microorganisms in water with
high levels of particulate matter [5], The axial flow method has a much higher capacity for particulate matter
since the space for particle accumulation is about 500 times larger compared to conventional HFUF. Operating
with recirculation, axial flow HFUF of water with different particulate matter concentrations (0 to 150 mg
solids/L) yielded a recovery of 35 to 53% of B. globigii spores (a nonpathogenic surrogate for B. anthracis).
Recovery of MS2 (a surrogate for pathogenic viruses) was comparable (45% organism recovery at 150 mg
solids/L). Operating without recirculation (dead end method) also proved effective for concentrating spores,
even with a very high solids concentration of
750 mg/L. Although axial flow ultrafiltration
has been shown to be an effective
concentration method in turbid water
matrices, one disadvantage is that
microorganism recovery is more cumbersome
because it involves additional steps not
required as part of traditional HFUF (e.g., filter
dissection is required for maximum biothreat
agent recovery, Figure 3) [5].
Analytical Detection Methods
Real-time polymerase chain reaction (PCR),
rapid viability-polymerase chain reaction (RV-
PCR), and culture methods have all been
identified as suitable methods for detection
of B. anthracis spores in decontamination
wash water samples. When spore
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concentrations are low, ultrafiltration prior to analysis will concentrate the biothreat agent and therefore may
increase the probability of detecting target organisms. Amendments to traditional methods may be necessary to
minimize the likelihood of false negatives with dirty water matrices (e.g., additional washes to remove PCR
inhibitors, or extending incubation times) and to avoid clogging the filter during filter plating due to high
suspended particle loads [6],
Wash Water Treatment Methods
Chlorine
The challenges of disposing of bio-contaminated wash water following cleanup of the B, anthracis-contaminated
U.S. Capitol buildings led the development of the U.S. National Response Team (U.S. NRT) quick reference guide
for the on-site treatment of PRE wash water containing B. anthracis spores [7]. The method described in this
guide calls for a slightly acidic 10% bleach solution (1-part chlorine bleach and 1-part vinegar to 8 parts wash
water, by volume) and a treatment time of 1-2 hours. The addition of vinegar, a dilute acid, decreases the pH of
the resulting solution to ~7, which makes the chlorine species much more germicidal Although this treatment is
efficacious (> 5 logio or 99.999% inactivation in < 1 minute), it also requires a relatively large volume of bleach (a
hazardous material) and has the potential to form chlorine gas, a toxic gas, if too much acid (or the wrong type
of acid) is added. At a pH of 4, chlorine gas levels begin to increase exponentially with decreasing pH.
Therefore, it is better suited for treating relatively small volumes (< 30 gallons) of wash water.
EPA has developed and tested chlorine inactivation
methods that are safer for treating larger volumes
of wash water. Approximately 5% (v/v) bleach (with
no vinegar addition) was sufficient for > 7 logio
inactivation of B. globigii spores in simulated
diluted wash water after a 10 minute exposure at
room temperature, and inactivation occurred more
rapidly when a detergent with buffering agents (i.e.,
1% Alconox®) was added [8]. Chlorine inactivation
efficacy generally decreases with increasing pH and
decreasing temperatures, so contact times should
be adjusted accordingly. Based on the results from
an EPA study [9] in which a wide variety of wash
waters were tested, it is estimated that a 6 logio
inactivation of viable B. anthracis spores in wash
water can be achieved with contact time of 100
minutes and 400 minutes for wash water
temperatures of ~20°C and ~4°C, respectively,
when a 5 % bleach solution is used. This assumes a
safety factor of 2 and is based on results from the
wash waters that were most difficult to treat.
Adjusting the pH of the wash water (following
bleach addition) to ensure that it is below pH 9
(rather than below 7) can still decrease the contact
time necessary for inactivation. A phosphate buffer
Figure 1: Wash water used in bench-scale inactivation
testing [10],
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is an effective way to lower pH without introducing the hazard of chlorine gas formation, which could result
from adding acid in excess of the U.S. NRT published guidelines [9].
Because wash water generated at an actual cleanup of B. anthracis spores will likely have unique characteristics,
EPA has developed a procedure for testing the efficacy of chlorine bleach inactivation of Bacillus spores in actual
wash water generated during site decontamination [10]. In this bench-scale procedure, which should be
conducted in a biosafety level 3 laboratory (because virulent spores could be present in the wash water), a
known amount of B. globigii is added to a known volume of the site-specific wash water (Figure 4). B. globigii
concentrations are measured before and after chlorine bleach addition to the wash water (with initial,
intermediate, and final sampling points), allowing measurement of 99.9999% (or 6 logio removal) inactivation of
the B. globigii spores. The method does not rely on measuring treatment of B. anthracis spores since levels in
the wash water would likely be too low for detection, and, because B. globigii is more resistant to chlorine than
B. anthracis [11], 6 logio inactivation of B. globigii implies an even greater inactivation of B. anthracis spores.
Results from this bench-scale testing can be used to estimate conservative contact times for full-scale wash
water treatment (assuming chlorine concentrations do not decrease substantially) [10].
Acidified Nitrite
Disinfecting large volumes of wash water onsite with traditional drinking water treatment methods or with
other published guidelines (e.g., U.S. NRT [7]) could necessitate transporting large quantities of chemicals (e.g.,
bleach) or placement and installation of large machinery (e.g., ozone or chlorine dioxide generators). Nitrate
salts are relatively inexpensive, available in large quantities, and do not require neutralization with a reducing
agent before discharge to the municipal sewer (although transporting large quantities of salts may provide
alternative challenges, and pH adjustment may be necessary prior to sewer discharge). EPA [12] tested the
efficacy of acidified nitrite for spore disinfection with varying pH, temperature, nitrite concentration, and buffer
types (Butterfield's or phosphate buffered saline, PBS). Spore inactivation occurred more rapidly at lower pH
and was slower in waters at colder temperatures or with a higher ionic strength. At optimal inactivation
conditions (room temperature, pH 2 and low ionic strength), acidified nitrite is an adequate substitute for
chlorine dioxide or free chlorine, and it is more effective than monochloramine at both optimal and suboptimal
conditions. However, at low temperatures and sub-optimal pH conditions, free chlorine is more effective [12].
Water Treatment Units
The EPA HSRP has developed the Water
Security Test Bed (WSTB) in conjunction
with Idaho National Laboratory for
testing decontamination technologies
in previously-serviceable drinking water
pipes. Water flowing through the WSTB
exits into a lagoon, which contains
sediment and algae (Figure 5).
Experiments conducted in 2015 tested
the ability of four different mobile
treatment systems (e.g., EPA's
Advanced Oxidation Process [AOP] UV-
Ozone trailer unit, Figure 6) to treat
biologically-contaminated (with B.	during treatment
Figure 2: EPA's Water Security Test Bed lagoon. Howard Saline ClM 6.0
Chlorination System setup on table (left) with effluent entering lagoon
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globigii) "dirty" water, which has an increased disinfectant demand resulting from the dirt and algae. Treatment
volumes ranged from 1,250 to 5,000 gallons, with experiments running from 5.5 hours to one day. All treatment
units achieved at least a 4 logio removal (99.99%) of B. globigii spores in the lagoon water over the course of the
experiments. See Table 1 for a performance summary of the different mobile water treatment devices tested.
All of the tested treatment devices can be scaled up (or multiple units could be put into place) to treat larger
volumes of water [2].
Table 1. Mobile Water Treatment Device Performance Summary (adapted from U.S. EPA, 2016 [2])
Water
Treatment
Technology
Tested
Capital
Cost
Average Log
Reduction
(B. globigii)
Volume
Treated
(gallons)
Flow
(gallons per
minute)
Performance Summary
EPA Advanced
Oxidative
Process Trailer
(UV and Ozone)
$40,000
4.0
2,000
5
Immediate disinfection, log reduction
was unstable during this study due to
experimental challenges.
Solstreme (UV)
$15,000
3.5 to 4.0
2,000
5
Stable, immediate disinfection, easy
to transport and set up.
Water Step
(Chlorinator)
$8,000
7.0
1,250
not
applicable
6-log reduction in 300 min, lowest
total treated volume.
Hay ward
(Chlorinator)
$4,000
4.3
5,000
40
4-log reduction in 1,350 min, under
most difficult disinfection conditions.
Additional Challenges and
Concerns
Additional considerations may be necessary during
decontamination wash water treatment and
disposal, depending on the inactivation method
employed. For example, disinfectants may react
with constituents in the water matrix other than
the contaminant of concern, and/or disinfectant
concentrations could degrade as a result of
environmental exposure (e.g., sunlight).
Monitoring disinfectant levels throughout the
treatment process may be necessary to ensure
that target concentrations are maintained for the
duration of the contact time. Moreover, reactions
between decontaminants (e.g., chlorine) and
organic or inorganic substances in the wash water
matrices can form disinfection byproducts that
may create a secondary health concern [13], Air quality monitoring (for spores and other contaminants) can be
used to determine if pathogenic bioaerosols are generated during wash water treatment [14].

Figure 3: EPA Advanced Oxidative Process (AOP) mobile
ozone/UV treatment system [2],
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Following biothreat agent inactivation in decontamination wash water, additional water treatment may be
necessary prior to disposal (e.g., before discharging to the publicly-owned water treatment facilities or natural
waterways). For instance, the disinfectant may need to be neutralized with a quenching agent (e.g., sodium
thiosulfate following disinfection with bleach), or the pH of the treated wash water may need to be adjusted. If
present, gloves and other PPE debris may also need to be removed from wash water and disposed of properly.
Communication with local water and sewer authorities is necessary to determine acceptability plans before
releasing any treated water or runoff into sewers or natural waterways [15].
Contact Information
For more information, visit the EPA website at https://www.epa.gov/homeland-securitv-research.
Technical Contact: Vincente Gallardo (gallardo.vincente@epa.gov)
Technical Brief Author: Katherine Ratliff (ratliff.katherine@epa.gov)
General Feedback/Questions: Amelia McCall (mccall.amelia@epa.gov)
Disclaimer
This technical brief was subject to administrative review but does not necessarily reflect the view of the U.S.
Environmental Protection Agency (EPA). No official endorsement should be inferred, as the EPA does not
endorse the purchase or sale of any commercial products or services. Katherine Ratliff was supported by an
appointment to the EPA Research Participation Program administered by the Oak Ridge Institute for Science and
Education (ORISE) through an interagency agreement between the U.S. Department of Energy (DOE) and the
EPA. ORISE is managed by ORAU under DOE contract number DE-SC0014664.
References
1.	U.S. Environmental Protection Agency (U.S. EPA), Federal On-scene Coordinator's Report for the Capitol
Hill Site; Washington, D.C. Philadelphia, Pennsylvania: U.S. Environmental Protection Agency Region 3,
2002. https://response.epa.gov/sites/DCN000305703/files/osc%20report.pdf Accessed 5/14/2019.
2.	U.S. EPA, Testing Large-Volume Water Treatment and Crude-Oil Decontamination Using the EPA Water
Security Test Bed. Cincinnati, Ohio: U.S. Environmental Protection Agency. EPA/600/R-161/126, 2016.
3.	Rose, L.J. and E.W. Rice, Inactivation of bacterial biothreat agents in water, a review. Journal of water
and health, 2014. 12(4): p. 618-633.
4.	U.S. EPA, Bio-response Operational Testing and Evaluation (BOTE) Project Phase 1: Decontamination
Assessment. Washington D.C.: U.S. Environmental Protection Agency. EPA/600/R-13/168, 2013.
5.	Gallardo, V.J., B.J. Morris, and E.R. Rhodes, The use of hollow fiber dialysis filters operated in axial flow
mode for recovery of microorganisms in large volume water samples with high loadings of particulate
matter. Journal of Microbiological Methods, 2019. 160: p. 143-153.
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6.	Shah, S.R., Protocol for Detection of Bacillus anthracis in Environmental Samples During the Remediation
Phase of an Anthrax Incident (Second Edition). Cincinnati, Ohio: U.S. Environmental Protection Agency.
EPA/600/R-17/213, 2017.
7.	U.S. National Response Team, NRT Quick Reference Guide: Bacillus anthracis PPE Wash Water
Decontamination. 2012.
8.	Muhammad, N., V.J. Gallardo, D.A. Schupp, E.R. Krishnan, K.S. Minamyer, and E.W. Rice, Inactivation of
Bacillus spores in decontamination wash down wastewater using chlorine bleach solution. Canadian
Journal of Civil Engineering, 2014. 41(1): p. 40-47.
9.	Gallardo, V.J., D.A. Schupp, J.L. Heckman, E.R. Krishnan, E.W. Rice, Inactivation of Bacillus Spores in
Wash Waters Using Dilute Chlorine Bleach Solutions at Different Temperatures and pH Levels. Water
Environment Research, 2018. 90(2): p. 110-121.
10.	U.S. EPA, A Bench-Scale Procedure for Evaluating Chlorine Bleach Inactivation of Bacillus Spores in Wash
Water from a Cleanup of a Site with Biothreat Agents. Cincinnati, Ohio: U.S. Environmental Protection
Agency. EPA/600/R-18/296, 2019.
11.	Sivaganesan, M., N. Adcock, and E. Rice, Inactivation of Bacillus globigii by chlorination: A hierarchical
Bayesian model. Journal of Water Supply: Research and Technology-AQUA, 2006. 55(1): p. 33-43.
12.	Szabo, J.G., N.J. Adcock, and E.W. Rice, Disinfection of Bacillus spores with acidified nitrite.
Chemosphere, 2014. 113: p. 171-174.
13.	Silva, R.G., J. Szabo, V. Namboodiri, E.R. Krishnan, J. Rodriguez, and A. Zeigler, Evaluation of an
environmentally sustainable UV-assisted water treatment system for the removal of Bacillus globigii
spores in water. Water Supply, 2017. 18(3): p. 968-975.
14.	Chattopadhyay, S., and S. Taft, Exposure Pathways to High-Consequence Pathogens in the Wastewater
Collection and Treatment Systems. Cincinnati, Ohio: U.S. Environmental Protection Agency. EPA/600/R-
18/221, 2018.
15.	U.S. EPA and Water Environment Research Foundation, Collaborative Workshop on Handling,
Management, and Treatment of High-Consequence Biocontaminated Wastewater by Water Resource
Recovery Facilities. Washington D.C.: U.S. Environmental Protection Agency. EPA/600/R-16/054, 2016.
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