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                     technical   BRIEF
                     BUILDING A SCIENTIFIC FOUNDATION FOR SOUND ENVIRONMENTAL DECISIONS
         Decontamination of Soil  Contaminated with
                 Bacillus anthracis Ames Spores
  INTRODUCTION
  Spores of Bacillus anthracis (the causative agent for anthrax) are highly resistant to severe
  environmental conditions and remain viable after decades in soil. Contamination following a
  wide-area release could overwhelm the nation's remediation capacity, drag cleanup out over
  many years and result in significant economic impact.

  Effective *, widely available, and economical decontamination methods that can be employed
  on soils in the event of a wide-area release are needed. Table 1 summarizes the results from
  initial screenings conducted in the laboratory to find efficacious decontaminants for soil. Tests
  were conducted with 1 cm uncompacted topsoil placed in Petri dishes or glass jars of similar
  size.

  Table 1. Efficacy of Decontaminants on Topsoil Contaminated with Bacillus anthracis
  Ames [3]
Decontaminants Tested (Primary
Sporicidal Active Ingredient)
pH-amended Ultra Clorox®
Germicidal Bleach 1 (Hypochlorous
acid)
CASCAD™ SDF (Hypochlorous
acid)
Oxonia Active ® (Peracetic acid)
Klozur™ (Sodium persulfate)
Activated With H2022
Total Contact Time
60 min
120 min
60 min
120 min
60 min
120 min

48 h
Number of
Applications
During Total
Contact Time
4
8
2
4
6
12
6
6
Range of Mean Logio
Reduction Achieved
With Treatment
Oto1
Oto1
Oto1
1to2
Oto1
1to2
1to2
3 to 4
   Sodium hypochlorite at 6.15%, sodium hydroxide at <1 %, and diluted with sterile filtered water, and pH-amended with acetic
  acid at 5% to achieve a pH between 6.5 and 7.
  2 Klozur™ is sodium persulfate and activated with hydrogen peroxide- It was tested at relatively long contact times, typical of
  field-use conditions for remediation of soil contaminated with organic chemicals.
    A minimum log™ reduction of 6.0 is generally considered effective decontamination for 6. anthracis.
                                      1

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SUMMARY OF DECONTAMINATION OF ARIZONA TEST DUST AND TOPSOIL
CONTAMINATED WITH SPORES OF BACILLUS ANTHRACIS AMES

Following the screening tests, pH-amended bleach and sodium persulfate/hydrogen peroxide
(hbCb) were selected for further testing. Additional tests were conducted with pH-amended
bleach (at longer contact times) because of its wide availability. Additional tests were
conducted with persulfate/hbCb because it showed some moderate effectiveness against
anthrax in screening tests. Some other decontaminants shown to be effective against anthrax
on building materials were also included in tests for soil.
In order to identify effective decontamination methods on soils, studies using fumigants and
liquids on Arizona Test Dust (AZTD) and a garden topsoil (represents a difficult soil to treat in
terms of its organic content) were conducted using varying operational parameters.
The fumigants tested were chlorine dioxide (CICb) gas; methyl bromide (MeBr); and metam
sodium [sodium N-methyldithiocarbamate]. The liquids tested were CIO2 aqueous solution;  pH-
amended bleach; and sodium persulfate (Klozur™) activated with hydrogen peroxide
Tests were conducted with varying operational parameters that included contact time; number
of spray applications (liquids); decontaminant concentration; temperature; soil depth; and soil
moisture.
Table 2 summarizes the minimum treatments for each decontaminant that achieved > 6 log™
reduction on at least one soil type [1, 3, 4]. Results for gaseous chlorine dioxide are
summarized separately in Table 3.
Table 2. Minimum Treatment Required for Effective Decontamination of Soil Inoculated
With Bacillus anthracis Ames Spores [1, 2]
Decontaminant
pH-amended Ultra Clorox®
Germicidal Bleach1
Aqueous CI02 3
Sodium persulfate activated
with H202 4
Soil Type
(1 cm depth)
Topsoil
AZTD
Topsoil
AZTD
Topsoil
AZTD
Minimum Treatment to Achieve > 6 Log™
Reduction
Not found 2
4 applications, 2 h contact time
Not found
Not found
3 applications every 30 min
3 applications every 60 min

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 Table 2. Minimum Treatment Required for Effective Decontamination of Soil Inoculated
 With Bacillus anthracis Ames Spores [1,2] continued
Decontaminant
Methyl bromide 5
Metam sodium
Soil Type
(1 cm depth)
Topsoil
AZTD
Topsoil
AZTD
Minimum Treatment to Achieve > 6 Log™ Reduction
180 mg/L, 24 h contact time
140 mg/L, 24 h contact time
160 \iL, 7 day contact time, 7 day aeration time, 1mL water added to
soil
80 |j,L, 5 day contact time, no aeration time, no moisture added to soil
1 One bleach application consisted of 0.5 ml acidified bleach, with mean free available chlorine level of approximately 5,400 ppm
and pH 6.5
2 After 8 spray applications, 7 day contact time < 1 logio reduction was achieved.
3 Aqueous CI02 was tested at most robust condition of 4,000 ppm CI02,2 hr contact time, 4 spray applications
4 One sodium persulfate application consisted of 1 ml 0.5 M sodium persulfate followed by 1mL 8% H202; all tests used 7 day
contact time.
5 All methyl bromide tests conducted at 25 °C and RH uncontrolled (all but one test had > 75% RH).

Table 3 summarizes the effect soil depth has on the efficacy of CIO2 gas and the contact time
needed to achieve at least a 6 logio reduction under two different RH  conditions. Relative
humidity was varied based on observations made in earlier work that RH can affect the efficacy
of chlorine dioxide gas (RH  was not an experimental variable in the other studies) .

Table 3. Minimum Contact Time Required to Achieve > 6 Logio Reduction Using  Chlorine
Dioxide Gas 1 on Soil Contaminated With Bacillus anthracis Ames Spores [4]
Soil Type
Topsoil
AZTD
Topsoil
AZTD
Topsoil
AZTD
Topsoil
AZTD
Percent
Relative
Humidity
75
75
85
85
75
75
85
85
Soil Depth
(cm)
1
1
1
1
2
2
2
2
Minimum Contact Time (h) to Achieve
> 6 Logio Reduction
2
2
2
2
Not found after 4 hours
2
Not found after 4 hours
2
1
 Concentration 3,000 ppm by volume

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DESCRIPTION OF DECONTAMINANTS TESTED

Fumigants
   •  Chlorine Dioxide Gas [4]
      Chlorine dioxide has been shown to be an effective decontaminant on a variety of
      porous and non-porous indoor materials contaminated with B. anthracis spores.
   •  Methyl Bromide [2]
      Methyl bromide has been shown to be an effective decontaminant on a variety of porous
      and non-porous indoor materials contaminated with B. anthracis spores.  It is also used
      as a soil and agricultural commodity fumigant, but is being phased out under the
      Montreal Protocol for Substances that Deplete the Ozone Layer.
   •  Metam Sodium (Sodium N-methyldithiocarbamate) [2]
      (EPA Reg. No. 11448-107) Metam sodium is the most widely used soil fumigant in the
      U.S.

Liquids
   •  CASCAD™ SDF (Hypochlorous acid) [3]
      CASCAD is produced by mixing together two solutions that form hypochlorous acid,
      hydrogen peroxide, along with foaming  and buffering agents.
   •  Oxonia Active ® (Peracetic acid) [3]
      Contains 5.8% peracetic acid. Registered with EPA as a sterilant (1677-129).
   •  Aqueous Chlorine Dioxide [1]
      CIO2 was produced using water, hydrochloric acid, sodium hypochlorite, and sodium
      chlorite. The concentration of CIO2 was adjusted by the amount of sodium hypochlorite.

   •  pH-amended Bleach (acidified) [2, 3]
      Ultra Clorox ® Germicidal Bleach (EPA  Reg. No. 67619-8) was used to make pH-
      amended bleach, which is not registered with EPA. The amended bleach was made by
      mixing 9.4 parts water, 1 part Ultra Clorox® Germicidal Bleach,  and 1 part 5% acetic
      acid (for example, vinegar). The resulting solution should have a pH of about 6.8 and a
      mean total chlorine content of about 6,200 ppm.

   •  Sodium persulfate (Klozur™) Activated with Hydrogen Peroxide [2, 3]
      In the initial screenings of soil decontaminants, Klozur ™ was shown to  be moderately
      effective against B. anthracis Ames on soil (see Table 1). It is typically used to remediate
      soil contaminated with organic chemicals, and was tested against B. anthracis spores at
      the conditions used for organic chemical remediation.

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        LESSONS LEARNED FROM SOIL DECONTAMINATION INVESTIGATIONS
        Minimum treatment conditions to achieve a 6 log™ or greater reduction for B. anthracis Ames
        spores in both tested soil types were found for sodium persulfate activated with hydrogen
        peroxide, methyl bromide, and metam sodium, but were not found for pH-amended bleach or
        aqueous CIO2.

        The Arizona Test Dust was generally easier to decontaminate then the proprietary garden
        topsoil, but this depends on the decontaminant.

        Tests using CIO2 gas showed decontaminant efficacy decreased when soil depth increased.
        CONTACT INFORMATION

        For more information, visit the EPA Web site at http://www.epa.gov/nhsrc/

        Technical Contact: Joseph Wood (wood.joe@epa.gov)

        General Feedback/Questions: Kathy Nickel (nickel.kathy@epa.gov)


        REFERENCES

        [1] U.S. EPA. 2012. Decontamination of Indoor and Outdoor Materials with Aqueous Chlorine
        Dioxide Solutions. Washington, D.C.: U.S. Environmental Protection Agency. EPA/600/R-
        12/516.
        [2] U.S. EPA. 2013. Decontamination of Soil Contaminated with Bacillus anthracis Spores-
        report. Washington, D.C.: U.S. Environmental Protection Agency. EPA/600/R-13/110.
        [3] U.S. EPA. 2010. Evaluation of Liquid and Foam Technologies for the Inactivation of Bacillus
        anthracis Spores in Topsoil. Investigation Report. Washington, D.C.:  U.S. Environmental
        Protection Agency. EPA/600/R-10/080.
        [4] U.S. EPA. 2012. Inactivation of Bacillus anthracis Spores in Soil Matrices with Chlorine
        Dioxide Gas. Washington, D.C.: U.S. Environmental Protection Agency.  EPA/600/R-12/517.
           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.
           This document does not constitute nor should be construed as an EPA endorsement of any
                                 particular product, service, or technology.
August 2014
EPA/600/R-14/189

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