EPA 600/R-15/101 I July 2015 I www.epa.gov/research
United States
Environmental Protection
Agency
Decontamination of Outdoor
Materials Contaminated with
Anthrax Using Sodium Persulfate
or Chloropicrin
r :•-
Office of Research and Development
National Homeland Security Research Center
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EPA/600/R-15/101
July 2015
Decontamination of Outdoor Materials
Contaminated with Anthrax Using
Sodium Persulfate or Chloropicrin
U.S. Environmental Protection Agency
Research Triangle Park, NC 27711
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Disclaimer
The U.S. Environmental Protection Agency through its Office of Research and Development
funded, directed, and managed the research described here under contract EP-C-10-001 to
Battelle. It has been subjected to the Agency's review and has been approved for publication.
Note that approval does not signify that the contents necessarily reflect the views of the Agency.
Mention of trade names, products, or services does not convey official EPA approval,
endorsement, or recommendation.
Questions concerning this document or its application should be addressed to:
Joseph P. Wood
National Homeland Security Research Center
Office of Research and Development
U.S. Environmental Protection Agency
Mail Code E343-06
Research Triangle Park, NC 27711
919-541-5029
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Acknowledgments
Contributions of the following individuals and organization to this report are gratefully
acknowledged:
Project team members (all from U.S. Environmental Protection Agency (EPA))
Lawrence Kaelin, Office of Emergency Management, Chemical, Biological, Radiological, and
Nuclear (CBRN) Consequence Management Advisory Division
Leroy Mickelsen, Office of Emergency Management, CBRN Consequence Management
Advisory Division
Carter Williamson, On-Scene Coordinator, Region 4
Peer Reviewers
Worth Calfee, EPA, Office of Research and Development
Michael Nalipinski, EPA Office of Emergency Management, CBRN Consequence Management
Advisory Division
Richard Rupert, On-Scene Coordinator, Region 3
Battelle
11
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Executive Summary
The U.S. Environmental Protection Agency (EPA), Office of Research and Development (ORD),
is striving to protect human health and the environment from adverse impacts resulting from the
release of threat agents. As part of this mission, EPA's National Homeland Security Research
Program is investigating the effectiveness and applicability of technologies for homeland
security related applications. This report provides the results of an assessment to determine the
decontamination efficacy of one soil fumigant (chloropicrin) and one liquid decontaminant
(sodium persulfate activated with hydrogen peroxide) in inactivating Bacillus anthracis
(causative agent for anthrax) spores on outdoor materials. Another objective of the study was to
compare these results with another spore-forming microorganism (Bacillus subtilis) to assess its
potential as a representative surrogate for B. anthracis, for use in field studies and additional lab-
based investigations.
This investigation focused on decontamination of two types of topsoil (one procured from a
garden center and the other obtained from a soil testing laboratory), as well as four porous
outdoor building materials (wood, concrete, asphalt, and brick). Decontamination efficacy tests
were conducted with spores of virulent B. anthracis or non-virulent B. subtilis. Decontamination
efficacy was quantified in terms of log reduction (LR), based on the difference in the number of
bacterial spores recovered from positive controls and test samples. A decontaminant that
achieves an LR value >6 (i.e., at least a 99.9999% reduction in spores from test coupons
compared to positive controls [coupons not exposed to decontaminant]) is considered effective.
Tests were conducted at ambient temperature and relative humidity with various contact times
and application methods to assess the effect of these operational parameters on decontamination
efficacy.
Summary of Results
Chloropicrin
Because chloropicrin is widely used in agriculture as a soil fumigant (an alternative pesticide for
methyl bromide), it was tested for decontamination efficacy against bacterial spores in the two
types of soil mentioned above. The chloropicrin was tested using an application rate equivalent
to the maximum EPA pesticide label amount, and then tested again with twice this amount. The
soil moisture was also increased to determine if this improved efficacy. Unfortunately,
chloropicrin was only minimally effective (the maximum LR achieved was 1.3 LR) in the three
experiments conducted. Because of these low efficacy results when using a relatively large
amount of chloropicrin, decontamination efficacy testing was discontinued after the third test.
Sodium persulfate activated with hydrogen peroxide
In the two tests in which only the soil materials were used, complete inactivation of spores of
both Bacillus species was obtained on both types of soil. There was no difference in results
when the sodium persulfate and the hydrogen peroxide were mixed together prior to application
to the soils, or if the two chemicals were applied to the soils sequentially.
After testing with the soil materials, ten additional tests were conducted with the four other
outdoor materials. Out of these 10 tests, there were only two tests in which > 6.0 LR was
achieved for B. anthracis on all four materials. In terms of the number of tests in which the
average LR of B. anthracis spores was > 6.0 (to provide some perspective on the relative ease of
iii
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decontaminating the different materials), this occurred in eight of the tests for asphalt, five tests
for both wood and brick, and four tests for concrete.
In comparing the inactivation results for the two microorganisms, there was no significant
difference in efficacy results between the two species except in one test in which B. anthracis
was inactivated to a somewhat higher degree.
Impact of Study
This laboratory study provides information on the efficacy of chloropicrin and sodium persulfate
activated with hydrogen peroxide to decontaminate soils and other outdoor materials that have
been contaminated with B. anthracis spores. These results may be useful in the development of
guidance to aid in deployment of such decontaminants after a wide-area release of B. anthracis
spores. This report also provides justification for use of B. subtilis as a surrogate for B. anthracis
for soil and outdoor decon testing with these decontaminants, for use in future field studies and
additional lab-based investigations.
IV
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Contents
Disclaimer i
Acknowledgments ii
Executive Summary iii
Abbreviations/Acronyms viii
1.0 Introduction 1
2.0 Technology Descriptions and Test Matrices 2
2.1 Technology Descriptions 2
2.2 Test Matrices 2
3.0 Test Procedures 5
3.1 Biological Agents 5
3.2 Test Materials 5
3.3 Preparation of Test Materials 7
3.4 Coupon Extraction and Biological Agent Quantification 7
3.5 Decontamination Efficacy 8
3.6 Surface Damage 10
4.0 Decontamination Description and Procedures 11
4.1 Decontaminant Preparation 11
4.2 Decontamination Procedures 11
5.0 Quality Assurance/Quality Control 14
5.1 Equipment Calibration 14
5.2 QC Results 14
5.3 Audits 16
5.4 Test/Quality Assurance Plan Deviations 16
5.5 QA/QC Reporting 17
5.6 Data Review 17
6.0 Summary of Results and Discussion 18
6.1 Chloropicrin 18
6.2 Klozur™ Sodium Persulfate Results 19
6.3 Surface Damage to Materials 29
6.4 Summary and Conclusions 29
7.0 References 31
Figures
Figure 3-1. Coupon types from left to right: wood, concrete, asphalt, brick, and an example
topsoil sample 7
Figure 3-2. Liquid inoculation of coupon using a micropipette 7
Figure 6-1. Summary of decontamination efficacies for chloropicrin testing in Earthgro®
and Agvise® topsoil. Results shown as average log reduction ± 95 confidence
interval 18
Figure 6-2. Summary of decontamination efficacies for Klozur® sodium persulfate testing
against Bacillus spp. in Earthgro® and Agvise® topsoil 21
Figure 6-3. Decontamination efficacy for Klozur® sodium persulfate testing against Bacillus
spp. on wood 22
Figure 6-4. Summary of decontamination efficacy for Klozur® sodium persulfate testing
against Bacillus spp. on concrete 22
Figure 6-5. Summary of decontamination efficacy for Klozur® sodium persulfate testing
against Bacillus spp. on asphalt 23
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Figure 6-6. Summary of decontamination efficacy for Klozur® sodium persulfate testing
against Bacillus spp. on brick 23
Figure 6-7. Summary of Klozur® sodium persulfate efficacy (Tests 3-12) results, by test,
against Bacillus anthracis 24
Table 6-4. Effect of Contact Time (CT) on Klozur® Sodium Persulfate Decontamination
Efficacy against Bacillus anthracis 26
Figure E-l. Summary of Klozur® sodium persulfate efficacy against Bacillus anthracis on
wood, concrete, asphalt, and brick. Results shown in average log reduction ± CL. E2
Tables
Table 2-1. Decontamination Technology Descriptions 2
Table 2-3. Test Matrix for Klozur® Sodium Persulfate Applied to Soils 3
Table 2-4. Test Matrix for Klozur® Sodium Persulfate Applied to Outdoor Materials*^ 3
Table 3-1. Test Materials 6
Table 5-1. Positive Control Spore Recovery Deviations for Bacillus spp 14
Table 5-3. Actual Conditions for Klozur® Sodium Persulfate Tests* 15
Table 5-4 Performance Evaluation Audits 16
Table 6-1. Summary of Average Differences in Klozur® Sodium Persulfate Efficacy
between B. anthracis and B. subtilis 20
Table 6-2. Summary of Bacillus anthracis Average Log Reductions by Material Type 25
Table 6-3. Effect of Contact Time (CT) on Klozur® Sodium Persulfate Efficacy against
Bacillus subtilis in Suspension 25
Table 6.5 Effect of Increasing Klozur® Sodium Persulfate Concentration on Efficacy against
Bacillus anthracis* 26
Table 6-6. Effect of Increasing the Number of Applications of Klozur® Sodium Persulfate
on Efficacy against Bacillus anthracis* 28
Table 6-7. Effect of Application Time Interval on the Efficacy of Klozur® Sodium
Persulfate against B. anthracis*^ 29
Table A-l. Soil Sample Analysis A2
Table A-2. Soil Moisture Assessments for Chloropicrin Tests A3
Table B-l. Neutralization Testing with Bacillus subtilis spores with Klozur® sodium
persulfate, Seven Day Contact Time, Three Total Applications* (Conducted
with Test 3) B2
Table B-2. Neutralization Testing with Bacillus subtilis spores with Klozur® sodium
persulfate, Three Day Contact Time, Four Total Applications* (Conducted with
Test 6) B2
Table B-3. Neutralization Testing with Bacillus subtilis spores with Klozur® sodium
persulfate, One Day Contact Time, Four Total Applications (TEST 9) B3
Table B-4. Neutralization Testing with Bacillus subtilis spores with Klozur® sodium
persulfate, One Day Contact Time, One Application of 16 sprays* (TEST 10) B3
Table B-5. Suspension Testing with Klozur® sodium persulfate on Bacillus spp B4
Table C-l Inactivation of B. anthracis Spores in Earthgro® Topsoil with Chloropicrina Cl
Table C-2 Inactivation of B. subtilis Spores in Earthgro® Topsoil with Chloropicrina C2
Table C-3 Inactivation of B. anthracis Spores in Agvise Topsoil with Chloropicrina C2
Table C-4 Inactivation of B. subtilis Spores in Agvise Topsoil with Chloropicrina C3
Table D-l Inactivation of B. anthracis and B. subtilis Spores in Earthgro® Topsoil with 0.5
M Klozur®'a Sodium Persulfate Dl
Table D-2 Inactivation of B. anthracis and B. subtilis Spores in Agvise® Topsoil with 0.5
M Klozur®'a Sodium Persulfate D2
vi
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Table D-3
Table D-4
Table D-5
Table D-6
Table D-7
Table D-8
Table D-9
Table D-10
Inactivation of B.
Inactivation of B.
Persulfate
Inactivation of B.
Persulfate
Inactivation of B.
Inactivation of B.
Inactivation of B.
Inactivation of B.
Inactivation of B.
anthracis Spores on Wood with Klozur®'a Sodium Persulfate...D3
anthracis Spores on Concrete with Klozur®'a Sodium
D4
anthracis Spores on Asphalt with Klozur®'a Sodium
D5
anthracis Spores on Brick with Klozur®'a Sodium Persulfate....D6
subtilis Spores on Wood with Klozur®'a Sodium Persulfate D8
subtilis Spores on Concrete with Klozur®'a Sodium Persulfate..D9
subtilis Spores on Asphalt with Klozur®'a Sodium Persulfate..Dl 1
subtilis Spores on Brick with Klozur®'a Sodium Persulfate D12
List of Appendices
Appendix A Moisture and Organic Content of Soil Samples Al
Appendix B Neutralization and Suspension Tests with Klozur® Sodium Persulfate Bl
Appendix C Chioropicrin Detailed Test Results Cl
Appendix D Klozur® Sodium Persulfate Detailed Test Results Dl
Appendix E Effects of Materials and Application on Sodium Persulfate Efficacy El
vn
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Abbreviations/Acronyms
ATCC® American Type Culture Collection
B. anthracis Bacillus anthracis (Ames strain)
B. subtilis Bacillus subtilis (ATCC 19659)
BSC biological safety cabinet
CPU colony-forming unit(s)
CI confidence interval
CT contact time
E-beam electron beam
EPA U.S. Environmental Protection Agency
kGy kilogray
LAL Limulus Amebocyte Lysate
Ib Pound
LR log reduction
M molarity
min Minute
mL milliliter(s)
|iL microliter(s)
PBS phosphate-buffered saline
PBST phosphate-buffered saline + 0.1% Triton X-100
PCR polymerase chain reaction
QA quality assurance
QC quality control
QMP Quality Management Plan
RH relative humidity
rpm revolutions per minute
SD standard deviation
SE standard error
SFW sterile filtered water (cell-culture grade)
STS sodium thiosulfate
T&E Testing and Evaluation
TSA technical systems audit(s)
Vlll
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1.0 Introduction
The U.S. Environmental Protection Agency's (EPA's) Homeland Security Research Program is
helping protect human health and the environment from adverse impacts resulting from the
release of chemical, biological, or radiological agents. With an emphasis on decontamination and
consequence management, water infrastructure protection, and threat and consequence
assessment, the research program is working to develop tools and information that will help
detect the intentional introduction of chemical or biological contaminants into buildings or water
systems; contain these contaminants; decontaminate buildings, outdoor environments, or water
systems; and facilitate the disposal of material resulting from restoration activities.
In the event of a large urban release of B. anthracis spores, extensive resources would be
required in the remediation effort. Chemical technologies that are already commercially
available and relatively widely used, such as chemical oxidants used in soil remediation or
agricultural pesticides, could potentially transition to decontaminants for B. anthracis
remediation if the need arises.
In this laboratory study, the efficacy of two decontaminants (chloropicrin [a soil pesticide] and
sodium persulfate activated with hydrogen peroxide [used for remediation of soils contaminated
with organic chemicals) was investigated. Spores of either Bacillus anthracis or Bacillus
subtilis, applied to soil samples and other outdoor building materials (wood, concrete, asphalt,
and brick), were used in the testing. This study builds on previous laboratory research conducted
by EPA to assess decontamination efficacy of sodium persulfate for inactivating B. anthracis
spores in soil ^'2\
An additional objective of this work was to obtain side-by-side efficacy data for B. anthracis
Ames and B. subtilis, to assess the suitability of using B. subtilis as a surrogate for B. anthracis
Ames in future decontamination studies with chloropicrin or sodium persulfate. The Ames strain
of B. anthracis was chosen for testing because this strain was identified in the Amerithrax
incident in 2001(3).
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2.0 Technology Descriptions and Test Matrices
2.1 Technology Descriptions
Table 2-1 describes the two decontaminants tested in this investigation. The information on
product composition is based on vendor-provided information and was not confirmed in this
evaluation. Greater detail on the product preparation and application procedures is provided in
Sections 3 and 4.
Chloropicrin is a volatile liquid used as the active ingredient in tear gas, as it is a powerful
lachrymator. It is also widely used in agriculture as a soil fumigant and pesticide (an alternative
to methyl bromide), and for this reason, it was tested against bacterial spores in soil.
Sodium persulfate activated with hydrogen peroxide produces sulfate radicals, and is used to
remediate soil contaminated with organic chemicals. In recent evaluations, sodium persulfate has
been shown to be effective against B. anthracis in soil*-2-*; therefore, it was decided to test its
decontamination efficacy with additional outdoor materials. Unless otherwise indicated, any
mention of "sodium persulfate" or the product name "Klozur®" in this report implies sodium
persulfate activated with an 8% hydrogen peroxide solution.
Table 2-1. Decontamination Technology Descriptions
Technology
Information
Chloropicrin-100
Fumigant
-omponems
Trinity Manufacturing
Hamlet, NC
99.4% chloropicrin
EPA Registration
62341-15*
Klozur
.®
FMC Corporation
Philadelphia, PA
Sodium persulfate (Na2S2O8) >99%
purity (used as a 12 or 24% [0.5 or 1 M]
aqueous solution, activated with an 8%
hydrogen peroxide solution
None
'registered as a pre-plant fumigant for the control of soil-borne pests, such as wireworms and nematodes
2.2 Test Matrices
The test matrices for chloropicrin and Klozur® sodium persulfate are shown in Tables 2-2
through 2-4. For the chloropicrin test matrix, the variables assessed included the quantity of
chloropicrin added to the soil, as well as the amount of water added to soil (e.g., to achieve the
recommended soil moisture per the EPA pesticide label). Each biological agent was tested
separately with each soil type or material at each test condition (designated by test number).
Further information on the moisture and organic content of the soils is presented in Appendix A.
Table 2-2. Test Matrix for Chloropicrin Applied to Soils*
Biological
Agent
Material
Added Water
Test Number
Chloropicrin
Volume Volume *
B. anthracis;
B. subtilis
Earthgro
Topsoil;
®
®,
27
27
Agvise Topsoil
54
*A11 tests completed using a 14-day contact time (CT) and 7-day aeration time.
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,®,
Table 2-3. Test Matrix for Klozur Sodium Persulfate Applied to Soils
Biological
Agent
B. anthracis
B. subtilis
Material
Earthgro®
Topsoil
Agvise
Topsoil
*Decontaminant applied every 60
lest
Number
1
2
Decontaminant
Details
1 mL of 0.5 Molar (M)
Klozur followed by 1
mL 8% H2O2
2 mL 1 : 1 mixture of 0.5
M Klozur® and 8%
H2O2
Neutralization
Buffer
2% sodium
thiosulfate (STS)
2% SLS
Total
Number of
Applications*
3
3
Contact
Time
(Days)
7
7
minutes until the total number of applications was achieved.
Table 2-4. Test Matrix for Klozur R Sodium Persulfate Applied to Outdoor Materials*^"
Bi°£fj' Material Test Number
3
4
5
Wood 6
B. anthracis Concrete 7
B. subtilis Asphalt 8
Brick 9
10
11
12
Conation
0.5M
0.5 M
1M
1M
1M
1M
1M
1M
1M
0.5 M
of Applications *
3
4
4
4
2
3
4
4§
2#
4
Contact Time
(days)
7
7
7
3
3
3
1
1
1
3
*Decontaminant applied every 60 minutes until the total number of applications was achieved, unless indicated otherwise.
tAll tests conducted with a 1:1 volume mixture of Klozur sodium persulfate and 8% H2O2.
§Lest conducted using four spray applications of decontaminant, but applied (16 sprayer trigger pulls) all at once.
Lests conducted using two spray applications of decontaminant, but applied (8 spray trigger pulls) all at once.
With the persulfate test matrices, a number of operational parameters or variables were assessed
to determine any effect or improvement in decontamination efficacy, and included the following:
• Concentration of Klozur
• Applying the persulfate and hydrogen peroxide separately (Test 1), or pre-mixing the
persulfate and hydrogen peroxide prior to applying to material (Tests 2-12). In previous
tests with persulfate4, the hydrogen peroxide activator was applied separately for safety
reasons, as per vendor recommendations; Test 1 was conducted to confirm these previous
results, using the same number of applications and contact time. However, mixing the
two reagents beforehand and applying as one solution did not present any issues during
testing and would simplify application procedures.
• Number of spray applications. A generic household hand sprayer (Qorpak PLC-03431,
Bridgeville, PA) was used for tests.
• Frequency of the spray applications (e.g., every hour, or all at once)
• Contact time
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In addition, prior to conducting tests on the outdoor materials, a few preliminary tests (not shown
in Table 2-4) were conducted with sodium persulfate in liquid suspension (no materials). These
tests were conducted in conjunction with the persulfate neutralization tests, and were performed
to determine whether sodium persulfate on its own (without activation by hydrogen peroxide)
would provide any biocidal activity against spores. These detailed results are reported in
Appendix B.
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3.0 Test Procedures
This section provides an overview of the procedures that were used for the bench-scale
evaluation of chloropicrin and Klozur sodium persulfate to inactivate 5. anthracis and B. subtilis
spores on two different soil types and four outdoor materials (wood, concrete, asphalt, and
brick).
3.1 Biological Agents
The virulent B. anthracis spores used for this testing were prepared from a qualified stock of the
Ames strain at the Battelle Biomedical Research Center (BBRC, Lot B21, West Jefferson, OH).
The spore lot was subjected to a stringent characterization and qualification process. Specifically,
the spore lot was characterized prior to use by observation of colony morphology, direct
microscopic observation of spore morphology, and size and determination of percent refractivity
and percent encapsulation. In addition, the number of viable spores was determined by colony
count and expressed as colony forming units (CFU)/milliliter (mL). Variations in the expected
colony phenotypes were recorded. Endotoxin concentration of each spore preparation was
determined by the limulus amebocyte lysate (LAL) assay to assess whether contamination from
gram-negative bacteria occurred during the propagation and purification process of the spores.
Genomic deoxyribonucleic acid (DNA) was extracted from the spores and DNA fingerprinting
by polymerase chain reaction (PCR) was done to confirm the genotype. The virulence of the
spore lot was measured by challenging guinea pigs intradermally with a dilution series of spore
suspensions, and virulence was expressed as the intradermal median lethal dose. In addition,
testing was conducted for robustness of the spores via hydrochloric acid (HC1) resistance.
The B. subtilis spores (Battelle Biomedical Research Center stock culture; American Type
Culture Collection [ATCC®] 19659) did not undergo the level of stringency for characterization
(LAL assay, DNA fingerprinting, and virulence testing excluded), but qualitative PCR was done
using a custom PCR assay to confirm B. subtilis. Primers were designed that targeted a
conserved region of B. subtilis chromosomal DNA, since multiple strains of this bacterium exist.
Both stock spore suspensions were prepared in sterile filtered water (SFW; Sigma Aldrich
W3500, St. Louis, MO) at an approximate concentration of 1 x 109 CFU/mL and stored under
refrigeration at 2 to 8 degrees Celsius (°C).
3.2 Test Materials
Two soil types (Earthgro® topsoil and Agvise® Mollisol topsoil) and four outdoor materials
(wood, concrete, asphalt, and brick) were used in the study. Information on these materials is
presented in Table 3-1. The Earthgro® product was purchased from a garden supply store, and
the Mollisol topsoil was obtained from Agvise Laboratories, a soil testing laboratory. The soil
samples were placed uncompacted in 1 ounce, 1.5 inch diameter glass jars (29 mL volume, 3.8
cm; Cat #GLC-01596, Qorpak, Bridgeville, PA) at a depth of approximately 1 centimeter (cm)
for testing (Figure 3-1). (This equates to a soil volume of 11.4 cm3, and a mass of approximately
9-11 grams, depending on moisture content.) Materials were prepared for testing by sterilization
via electron beam (E-beam) irradiation with a dose of approximately 200 kilogray (kGy; Texas
A&M University, College Station, TX) or autoclaving at 121 °C for 1 hour as indicated in Table
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3-1. Small amounts of endogenous bacteria were noted in the topsoil samples following E-beam
irradiation. Because of this, the irradiated topsoil samples were also autoclaved at 121 °C for 2
hours. E-beam irradiated materials were sealed in Ziploc® bags and autoclaved materials were
sealed in sterilization pouches (Cat #01-812-51, Fisher Scientific, Pittsburgh, PA) to preserve
sterility until the samples were ready for use.
Table 3-1. Test Materials
Material"
Lot, Batch, or ASTM No., or
Observation
Manufacturer/
Supplier Name
Approximate
Coupon Size,
width x length x
thickness
Material
Preparation
Earthgro®
Topsoil
Agvise® Topsoil
Wood
Concrete
Asphalt
Brick
Product*: 7 1140 180
Mollisol, HCB-SL-PF,
collected from Eastern, ND
Bare Pine Wood;
Generic Molding
Unpainted; ASTM C90
cinder block
Fine aggregate, salvaged
material from urban parking lot
Belden red shale, chemical
resistant brick (ASTM C-279)
The Scotts Company,
Marysville, OH
Agvise Laboratories,
Northwood, ND
Lowes,
Columbus, OH
Wellnitz
Columbus, OH
Shelly Aggregate and
Asphalt,
Columbus, OH
Hamilton Parker Co.,
Columbus, OH
11.4cm3*
11.4 cm3*
1.9 cm x 7.5 cm x
0.3 cm
1.9 cm x 7.5 cm x
0.6 cm
1.9 cm x 7.5 cm x
0.6 cm
1.9 cm x 7.5 cm x
0.6 cm
E-beam and
Autoclave
E-beam and
Autoclave
E-beam
Autoclave
Autoclave
Autoclave
* Soil samples consisted of 1.5 inch (3.8 cm) diameter glass jars filled with uncompacted soil to height of 1 cm
Prior to decontamination testing, soil samples (pre- and post-sterilization) were characterized by
Agvise Laboratories. The details of these test results are shown in Appendix A. The moisture
content of the soils following E-beam irradiation combined with autoclaved sterilization was
assessed using method ASTM D 2974-87^ and is further explained in Appendix A. Soil
moisture analyses tests were conducted because the moisture content of the soil could impact the
efficacy of chloropicrin; refer to Section 4.2.3 for further details. The most significant changes in
soil samples occurred in the Earthgro® soil grain size (i.e., % sand, % silt, and % clay
composition) as a result of sterilization procedures.
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Figure 3-1. Coupon types from left to right: wood, concrete, asphalt, brick, and an
example topsoil sample.
3.3 Preparation of Test Materials
Test and positive control samples were placed on a flat surface within a Class II or III biological
safety cabinet (BSC) and inoculated with approximately 1 x 108 CPU of viable B. anthracis or B.
subtilis spores per coupon. A 100 microliter (jiL) aliquot of a stock suspension with a target titer
of 1 x 109 CFU/mL was dispensed using a micropipette applied as 10 jiL droplets across the
coupon or soil surface (Figure 3-2). This approach provided a more uniform distribution of
spores across the sample surface than would be obtained through a single drop of the suspension.
After inoculation, the samples were left undisturbed overnight in a Class III BSC to dry under
ambient conditions, approximately 22 °C and 40% relative humidity (RH).
Figure 3-2. Liquid inoculation of coupon using a micropipette.
The number and type of replicate samples used for each combination of material, decontaminant,
concentration, and environmental condition included were:
• five test coupons (inoculated with B. anthracis or surrogate spores and exposed to
decontaminant)
• five positive controls (inoculated with B. anthracis or surrogate spores but not exposed
to decontaminant)
• one laboratory blank (not inoculated and not exposed to the decontaminant)
• one procedural blank (not inoculated and exposed to the decontaminant).
On the day following spore inoculation, samples intended for decontamination (including
procedural blanks) were transferred into a test chamber where the decontaminant was applied
using the application conditions specified in Section 4 of this report.
3.4 Coupon Extraction and Biological Agent Quantification
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For sample extraction, 10 mL of sterile phosphate-buffered saline (PBS) extraction buffer
containing 0.1% Triton X-100 surfactant (PBST; Sigma, St. Louis, MO) or 10 mL of PBST with
the appropriate percentage of neutralizer (based on results shown in Appendix B), was added to
each soil sample jar. For the other outdoor materials, test coupons, positive controls, and blanks
were placed in 50 mL polypropylene conical tubes containing 10 mL of sterile PBST. The jars
and vials were capped, placed on their sides and agitated on an orbital shaker for 15 minutes
(min) at approximately 200 revolutions per minutes (rpm) at room temperature.
The number of spores surviving the treatment, and recovered from the test material was indicated
using a viable plating approach. Following extraction, the extract was removed and a series of
10-fold dilutions was prepared in SFW. An aliquot (0.1 mL) of either the undiluted extract
and/or each serial dilution were plated onto tryptic soy agar in triplicate and were incubated for
18 to 24 hours at 35 to 37 °C. Colonies were counted manually and CFU/mL were determined by
multiplying the average number of colonies per plate by the reciprocal of the dilution. Dilution
data representing the greatest number of individually definable colonies were expressed as
arithmetic mean ± standard deviation (SD) of the numbers of CFU observed. Laboratory blanks
determined sterility, and procedural blanks controlled for viable spores inadvertently introduced
to test coupons. The target acceptance criterion was that extracts of laboratory or procedural
blanks were to contain no CFU.
After each decontamination test, the BSC III was thoroughly cleaned (using separate steps
involving bleach, ethanol, water, then drying).
3.5 Decontamination Efficacy
The mean percent spore recovery from each coupon was calculated using results from positive
control coupons (inoculated, not decontaminated), by means of the following equation:
Mean % Recovery = [Mean CFUpc/CFUSpike] x 100 (1)
Where, Mean CFUpc is the mean number of CFU recovered from five replicate positive control
samples of a single material, and CFUspike is the number of CFU inoculated onto each of those
samples. The value of CFUspike is known from enumeration of the stock spore suspension. One
aliquot of the stock suspension is plated and enumerated on each day of testing to confirm the
CFUsPike concentration. Spore recovery was calculated for B. anthracis or B. subtilis on each
sample, and the results are included in Section 6 and Appendices C and D.
The performance or efficacy of the decontaminants was assessed by determining the number of
viable organisms remaining on each test sample after decontamination. Those numbers were
compared to the number of viable organisms extracted from the positive control samples.
The number of viable spores of B. anthracis or B. subtilis in extracts of test and positive control
samples was determined to calculate efficacy of the decontaminants. Efficacy is defined as the
extent (as logio reduction) to which viable spores extracted from test samples after
decontamination were less numerous than the viable spores extracted from positive control
samples. The logarithm of the CFU abundance from each sample was determined, and the mean
of those logarithm values was then determined for each set of control and associated test
samples, respectively. Efficacy of a decontaminant for a test organism/test condition on the /'th
sample material was calculated as the difference between those mean log values, i.e.:
-------
Efficacy = (Iog10 CFUct]) - (Iog10 CFUtv) (2)
where logic CFUcy refers to they individual logarithm values obtained from the positive control
coupons, logio CFUty refers to they individual logarithm values obtained from the corresponding
test coupons, and the overbar designates a mean value. In tests conducted under this plan, there
were five positive controls and five corresponding test coupons (i.e.,7 = 5) for each coupon. A
decontaminant that achieves a 6 log reduction (LR) or greater is considered effective^.
In the case where no viable spores were found in any of the five test sample extracts after
decontamination, a CPU abundance of 1 was assigned, resulting in a logic CPU of 0 for that
material. This situation occurred when the decontaminant was highly effective, and no viable
spores were found on the decontaminated test samples. In such cases, the final efficacy on that
material was reported as greater than or equal to (>) the value calculated by Equation 2.
The variances (i.e., the square of the SD) of the logio CFUcy and logio CFUty values were also
calculated for both the control and test coupons (i.e., S2cy and -5%), and were used to calculate
the pooled standard error (SE) for the efficacy value calculated in Equation 3, as follows:
(3)
where the number 5 again represents the number y of samples in both the control and test data
sets. Each efficacy result is reported as an average LR value with an associated 95% confidence
interval (CI), calculated as follows:
95% CI = Efficacy ± (1.96 x SE) (4)
The significance of differences in efficacy across different test conditions and spore species was
assessed based on the 95% CI of each efficacy result. Differences in efficacy were judged to be
significant if the 95% CIs of the two efficacy results did not overlap. Any results based on this
formula are hereafter noted as significantly different. Note this comparison is not applicable
when the two efficacy results being compared are both reported with LRs as > some value.
The average difference in efficacy between B. anthracis and B. subtilis was determined by
comparing the results of each test. This difference in efficacy was calculated as follows:
LRa,2 - LRa,l
Avg Difference in Efficacy (LR) = — (5)
n
where the letters a through n represent the material types, the number 1 represents B. anthracis,
and the number 2 represents B. subtilis. The letter n represents the number of materials tested,
with n = 4 for Tests 3 through 12. When both values were > LR (indicating complete
inactivation), these were not included in the formula. A positive value indicates the avirulent B.
subtilis organism was inactivated on average to a higher degree (i.e., it was less resistant) across
the materials tested compared to B. anthracis. Similar comparisons were made between tests to
assess the effect of decontamination parameters such as contact time, concentration, etc. For
some of these comparisons, significant differences in average efficacy between tests were
assessed with a t-test using Microsoft® Excel, according to the formula below:
-------
t = (6)
b*l-*2
where X^ and X2 are the means of Tests 1 and 2, respectively. S^_^ is the standard error of the
difference between Tests 1 and 2. Excel produces a p-value, a statistic calculated from the t-test,
used to assess whether the averages of the two tests are reliably different from each other. Using
this formula, a p-value was assigned where indicated. If the calculated p-value was <0.05, then
the two sets of data were considered to be significantly different.
3.6 Surface Damage
The physical effect of the decontaminants on the soil and materials was also qualitatively
monitored during the evaluation. This approach provided a gross visual assessment of whether
the decontaminants altered the appearance of the test materials. The procedural control (sample
that is decontaminated, but has no spores applied) was visually compared to a laboratory blank
sample (a sample not exposed to the decontaminant and that has no spores applied). Obvious
visible damage might include structural damage, surface degradation, discoloration, or other
aesthetic impacts.
10
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4.0 Decontamination Description and Procedures
4.1 Decontaminant Preparation
4.1.1 Chloropicrin
Chloropicrin-100 was used as the source of chloropicrin and is used as a soil fumigant. The
product was supplied at 99.4% and used at full strength.
4.1.2 Klozur sodium persulfate
Klozur sodium persulfate was used as the source of sodium persulfate. It is a solid reagent
manufactured by FMC Corporation which is used for in situ and ex situ chemical oxidation of
contaminants in environmental remediation applications (e.g., soil/6). Klozur sodium persulfate
consists of >99% pure sodium persulfate (Na2S2Og) in the form of white, odorless crystals. In
remediation applications, Klozur sodium persulfate is injected as an aqueous solution into
contaminated soil or groundwater, and activated by mixing in appropriate proportions of up to
8% H2O2 by weight, according to instructions published by FMC Corporation^. Activation of
Klozur sodium persulfate with FkCh generates sulfate radicals (SO/f*), which are capable of
destroying a wide range of organic contaminants in the presence of soil. For testing, a 0.5 or 1
molar (M) solution of sodium persulfate was prepared by dissolving 12 or 24 g, respectively, of
Klozur sodium persulfate in SFW and diluting to 100 mL. (In terms of mass, a 1 mL application
of 0.5 M or 1.0 M persulfate contains 0.12 g or 0.24 g, respectively.) The 8% FbCh solution was
prepared by diluting a 30% solution (EMD Millipore, Billerica, MA) in SFW.
We note that a few preliminary tests were conducted with sodium persulfate in suspension, with
and without the addition of hydrogen peroxide, to assess any biocidal activity that persulfate may
have on its own. These few suspension tests were conducted in conjunction with the
neutralization tests, and are described in Chapter 6 and in more detail in Appendix B.
4.2 Decontamination Procedures
4.2.1 Test and Control Chambers
All tests were conducted at ambient conditions inside a climate-controlled laboratory. The
temperature and RH inside the testing chamber were equilibrated to the ambient laboratory
temperature of approximately 20 °C and 40% RH. The temperature and RH were both monitored
and recorded with a HOBO® data logger (Onset Computer Corporation, Bourne, MA), but no
attempt was made to control either. All tests took place in a Class III BSC.
4.2.2 Neutralization Determination
Neutralization of Klozur sodium persulfate was achieved using sodium thiosulfate where
indicated. The results of the neutralization tests (performed with B. subtilis only) are summarized
in Appendix B.
Neutralization tests were previously conducted in soils using the same parameters described
here*-2-*. Neutralization of Klozur sodium persulfate was achieved with 2% sodium thiosulfate in
these instances. However, no neutralization tests had previously been completed using the four
outdoor materials (wood, concrete, asphalt, and brick). The optimum concentration of sodium
thiosulfate in the extraction buffer was determined in test runs for multiple Klozur sodium
persulfate application regimens (number of applications and contact times [CTs]) that were
11
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tested. It was determined that no neutralizer was necessary in any tests using the four outdoor
materials. Further details of the methods and results of the neutralization tests are summarized in
Appendix B.
4.2.3 Chloropicrin Decontamination Procedure
All chloropicrin tests were completed under ambient laboratory conditions with no attempt made
to control the temperature or RH. Each sample jar served as its own primary container. For Tests
2 and 3, immediately prior to inoculation of spores to the soil sample (volume of 11.4 cm3, and a
mass of approximately 9-11 grams, depending on moisture content) SFW was added to each test,
control, and blank sample and mixed with a glass stirring rod. (Test 1 was completed without the
addition of SFW.) Water was added in Tests 2 and 3 to assess soil moisture impact on
decontamination efficacy. As indicated on the Chloropicrin 100 Fumigant pesticide label(8), a
50-75% soil moisture based on field capacity is recommended. The Agvise soil required 3 mL
of additional water to reach 50-75% field capacity, whereas the Earthgro® topsoil required only 2
mL to reach 50-75% field capacity. Refer to Appendix A for additional details.
Once mixing in any added water, all test and control samples were inoculated with the biological
agent as described in Section 3.3. Following the addition of the agent, chloropicrin was applied
with a pipette using small droplets (i.e., 10 jiL/droplet), then mixed into the soil with a glass
stirring rod. Decontamination tests were conducted using either 27 jiL or 54 jiL chloropicrin
applied to each soil sample. Based on the surface area of the sample jars used, these amounts
correspond to approximately 350 pounds (Ib) per acre or 700 Ib/acre, respectively. The
maximum application rate specified by the pesticide label for chloropicrin (8536-2) is 350
Ib/acre*-8-*. After application of the chloropicrin, the sample jars were sealed. These airtight
containers allowed the chloropicrin vapor to stay in contact with each soil sample (mimicking a
tarped agricultural application). Following the conclusion of the contact time (CT), the caps were
removed, samples stirred, and then the samples were allowed to aerate with their lids removed
for 7 days prior to extraction.
Air samples were drawn from the headspace of one B. anthracis Earthgro® topsoil test sample
and one B. anthracis Agvise topsoil sample using specialized chloropicrin detection tubes (Cat
#8103421, Draeger Safety Inc., Pittsburgh, PA) for each test. Air samples were taken
immediately following the removal of the sample caps and stirring of each sample. Air samples
were then taken again at the end of the aeration period. This procedure was utilized to ensure that
no chloropicrin remained in the headspace of the soil samples at the end of the aeration period.
These tests did confirm the presence of chloropicrin after the 14 day CT, but no chloropicrin was
detected after the samples were aerated (7 days).
4.2.4 Klozur sodiumpersulfate Decontamination Procedure
For Klozur sodium persulfate Test 1 with soil, an application consisted of the following: 1 mL
volume of the 0.5 M persulfate solution was added to each soil sample (soil sample volume of
11.4 cm3, and a mass of approximately 9-11 grams, depending on moisture content) and mixed
with a glass stirring rod. A 1 mL volume of the 8% FfeCh activation solution was immediately
applied and mixed in the same manner. For Test 2, an application consisted of the following: the
two reagents were mixed in a 1:1 volume ratio prior to the addition of 2 mL of the mixture to the
soil samples. For both Tests 1 and 2, three applications of Klozur sodium persulfate/H202 were
used, and resulted in saturating the soil samples.
12
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For the remainder of the Klozur sodium persulfate tests with the outdoor materials (Tests 3-12),
the sodium persulfate and hydrogen peroxide were mixed together prior to application to the test
samples. The test samples were sprayed until fully wetted (typically about 4 trigger pulls in one
spray application) with a 1:1 volume mixture of either 0.5 M or 1 M persulfate solution and 8%
H2O2 using a handheld sprayer from a distance of approximately 12 inches (30.5 cm). SFW was
applied to the positive control samples at all application times in the same manner. This process
was repeated when multiple spray applications were used. After the last application, all soil
samples were left uncapped and all other materials left undisturbed in the Class III BSC until the
end of the specified CT. At the end of the CT, all samples were extracted as described in Section
3.4. Equal volumes of the 0.5 M persulfate and EbCh solutions resulted in a H2O2/persulfate
molar ratio of 5 to 1, a typical ratio recommended for the use of Klozur sodium persulfate in soil
remediation^. An initial CT of 1 week was selected, based on information indicating the
persulfate can maintain its oxidative ability in subsurface environments for weeks'-6-*. CTs were
scaled down to 1 and 3 days as efficacy data were collected.
13
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5.0 Quality Assurance/Quality Control
QA/quality control (QC) procedures were performed in accordance with the Testing and
Evaluation (TandE) Quality Management Plan (QMP) and the pre-approved test/QA Plan. The
QA/QC procedures and results are summarized below.
5.1 Equipment Calibration
All equipment (e.g., pipettes, incubators, biological safety cabinets) and monitoring devices (e.g.,
thermometer, hygrometer) used at the time of evaluation were verified as being certified,
calibrated, or validated.
5.2 QC Results
QC efforts conducted during decontaminant testing included positive control samples
(inoculated, not decontaminated), procedural blanks (not inoculated, decontaminated), laboratory
blank (not inoculated, not decontaminated), and inoculation control samples (analysis of the
stock spore suspension).
Results for all positive control samples for the non-soil materials were within the target recovery
range of 1 to 150% of the inoculated spores, except for the samples indicated in Table 5-1. If
recovery of CPU for a particular species and material was less than 1%, the test was repeated.
There were three cases in which repeat of the test for a particular species/material still did not
result in positive control recovery over 1%. The implication is that for these three cases, we are
unable to use the 6 LR benchmark for efficacy. Notwithstanding, all results presented in Chapter
6 are based on the original recovery data; all data, including those from repeated tests, are
included in Appendix D.
Table 5-1. Positive Control Spore Recovery Deviations for Bacillus spp.
Test ,, . . .
„ , Material
Number
Wood
Wood
Wood
Brick
5 Brick
Brick
Brick
Brick
Concrete
Organism
B. subtilis
B. subtilis
B. anthracis
B. anthracis
B. anthracis
B. subtilis
B. subtilis
B. anthracis
B. subtilis
Mean % Recovery
0.97
0.96*
0.18
0.02
0.00*
0.99
0.09*
0.43
0.78
Mean Log CFU
Recovered
5.84
5.73*
4.49
2.46
0.00*
5.87
1.77*
5.75
5.96
CFU, colony forming units
*Positive control recovery test repeated again with no increase in percent recovery.
Several of the positive control samples for B. subtilis from soil exceeded the 150% recovery
criterion. Refer to Tables C-2, C-4, D-l, and D-2 in Appendix. Separate soil recovery tests were
conducted, using both species, to troubleshoot the problem, and included the use of heat
shocking samples to inactivate any vegetative cells. (Vegetative cells could cause increased
CFU recovery.) The tests showed no significant difference in results for the heat and non-heat
shock tests, indicating no presence of vegetative organisms. We do not believe these abnormally
14
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high recoveries affect our results, since for the chloropicrin tests, minimal inactivation occurred
regardless of species and recovery. And for the soil tests with persulfate, complete inactivation
occurred, regardless of species or positive control recovery.
All procedural and laboratory blanks met the criterion of no observed CPU for both organisms.
Inoculation control samples were taken from the spore suspension on the day of testing and
serially-diluted, nutrient plated, and counted to establish the spore density used to inoculate the
samples. The spore density levels met the QA target criterion of 1 * 109 CFU/mL (acceptable
range of 1 x 108 to 1 x 1010 CFU/mL) for all tests.
5.2.1 Operational Parameters
The temperature and RH during each test were recorded but not controlled, as described in
Section 4.1. Readings were taken once every minute for the duration of the CT. The actual
operational parameters for each test are shown in Tables 5-2 and 5-3 and reported as the average
value ± SD.
Table 5-2. Actual Conditions for Chloropicrin Tests*
1 19.81 ±0.22 32.01 ± 10.24
2 21.55 ±0.38 45.90 ±4.17
3 21.78 ±0.68 49.85 ±6.38
RH, relative humidity.
*Data reported as average ± SD.
Table 5-3. Actual Conditions for Klozur" Sodium Persulfate Tests*
1
2
3
4
5
6
7
8
9
10
11
12
18.91 ±0.62
18.91 ±0.62
20.31 ±0.32
18.63 ±0.24
18.95 ±0.45
19.28 ±0.30
19.91 ±0.31
20. 19 ±0.26
19.93 ±0.64
20.37 ±0.39
t
20.29 ±0.38
51.16±4.53
51.16±4.53
40.84 ±1.28
63.57 ±3.71
63.60 ±2.90
60.79 ±5.92
54.72 ±0.97
55.83 ±0.67
51.95 ±3.74
50.98 ±4.21
t
44.34 ±3.49
RH, relative humidity.
*Data reported as average ± SD.
^Temperature and RH not recorded for Test # 11. Refer to Section 5.4.
15
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5.3 Audits
5.3.1 Performance Evaluation Audit
Performance evaluation audits were conducted to assess the quality of the results obtained during
these experiments. Table 5-4 summarizes the performance evaluation audits that were
performed.
No performance evaluation audits were performed to confirm the concentration and purity of B.
anthracis or B. subtilis spores because quantitative standards do not exist for these organisms.
The control samples and blanks support the spore measurements.
Table 5-4 Performance Evaluation Audits
Volume of liquid
from micropipettes
Gravimetric evaluation
± 10%
±0.01% to 3.17%
Time
Compared to independent clock
± 2 seconds/hour 0 seconds/hour
Temperature
Compared to independent calibrated thermometer
±2°C
±0.2 to 1.6 °C
Relative Humidity Compare to independent calibrated hygrometer
± 20%
± 0 to 4 %
Balance
Compared to independent calibrated weight sets
±0.5g
± 0 to 0.3 g
5.3.2 Technical Systems Audit
Observations and findings from the technical systems audit (TSA) were documented and
submitted to the laboratory staff lead for response. A TSA was conducted on April 22, 2014, to
ensure the tests were being conducted in accordance with the appropriate test/QA plan and QMP.
As part of the audit, test procedures were compared to those specified in the test/QA plan and
data acquisition and handling procedures were reviewed. None of the findings of the TSA
required corrective action.
5.3.3 Data Quality Audit
At least 10% of the data acquired during the evaluation were audited. A QA auditor traced the
data from the initial acquisition, through reduction and statistical analysis, to final reporting to
ensure the integrity of the reported results. All calculations performed on the data undergoing the
audit were checked.
5.4 Test/Quality Assurance Plan Deviations
The test/QA plan specified that five test coupons, five positive control coupons, one laboratory
blank, and one procedural blank would be included in each test for each organism. For all tests
with Klozur sodium persulfate, only one laboratory blank and one procedural blank were
included for each test and "shared" between the two microorganisms. Blank coupons were not
inoculated and were treated in the same manner as the test or positive control samples (sprayed
with either decontaminant or SFW). There was no differentiation between a B. anthracis blank or
B. subtilis blank as they were not inoculated. The same information could be gathered while
16
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reducing the number of materials used on a single test. Additionally, all blank samples were
negative for the target organisms.
Section 3.2 of the test/QA plan states "The temperature and RH of the control and test chambers
will be measured with a thermometer/hygrometer (Fisher Scientific Cat. No. S66283, Pittsburgh,
PA), and the data will be recorded using a data logger (Onset Part No. U12-001, Bourne, MA)".
For Test 11, the HOBO® was inadvertently not launched inside the test chamber, resulting in no
temperature or RH data at the end of the 1-day contact period. The data for the decontamination
samples were not affected. Data were successfully captured from all other tests, and
demonstrated temperatures and RH were maintained (see Tables 5-2 and 5-3).
5.5 QA/QC Reporting
Each assessment and audit was documented in accordance with the test/QA plan and QMP. For
these tests, findings were noted (none significant) in the data quality audit, but no follow-up
corrective action was necessary. The findings were mostly minor data transcription errors
requiring some recalculation of efficacy results, but none were gross errors in recording. Copies
of the assessment reports were distributed to the EPA QA Manager and laboratory staff. QA/QC
procedures were performed in accordance with the test/QA plan.
5.6 Data Review
Records and data generated in the evaluation received a QC/technical review before they were
utilized in calculating or evaluating results and prior to incorporation in reports.
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6.0 Summary of Results and Discussion
6.1 Chloropicrin
The decontamination efficacy results (average ± 95% CI) for chloropicrin are summarized
graphically in Figure 6-1. Refer to Appendix C for the detailed efficacy test results. Either 27 jiL
or 54 jiL was applied to each soil sample (equivalent to 350 or 750 Ib/acre, respectively).
Chloropicrin in Earthgro® Topsoil
IB. anthracis
3B. subtilis
1 (27) 2 (27)* 3 (54)*
Test Number (Volume Chloropicrin in u,L)
Chloropicrin in Agvise Topsoil
o •
.2 4
CJ
S
1 1
I
d 7
>•-.
CJ
C5
CJ
S1
1
UJ
n -
T
T
T T.^
I1 1
tB. anthracis
%B. subtilis
1 (27) 2 (27)f 3 (54)f
Test Number (Volume Chloropicrin in u,L)
* = 2 mL sterile filtered water added prior to addition of chloropicrin.
t = 3 mL sterile filtered water added prior to addition of chloropicrin.
Figure 6-1. Summary of decontamination efficacies for chloropicrin testing in Earthgro®
and Agvise® topsoil. Results shown as average log reduction ± 95 confidence
interval.
The results indicate that chloropicrin exhibits low inactivation against B. anthracis and B.
subtilis spores in soil. The highest LR of 1.27 was achieved on B. anthracis in Earthgro® topsoil
with double the recommended amount of chloropicrin (54 jiL) and a 14-day CT. Because of
these low efficacy results when using a relatively large amount of chloropicrin, decontamination
efficacy testing was discontinued after the third test.
18
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In Test 3 for the Earthgro® topsoil, a significant difference in efficacy was observed between the
B. anthracis and B. subtilis samples with LRs of 1.27 and 0.09, respectively. In all other tests, no
significant difference between organisms was observed.
The effect of doubling the amount of chloropicrin applied to the soil samples was assessed. No
significant difference in efficacy was observed in either topsoil type or organism type.
Increasing the moisture content of the soil samples also did not show significant improvement in
efficacy (compare the results for Tests 1 and 2, for each soil type).
6.2 Klozur™ Sodium Persulfate Results
A summary of the efficacy results for Klozur sodium persulfate is presented in Figures 6-2
through 6-7 and the detailed qualitative results are provided in Appendices D and E. Tests were
conducted using CTs of one, three, or seven days; with one, two, three, or four applications every
60 minutes. Soil tests were completed using a 0.5 M concentration of Klozur sodium persulfate
followed by an 8% FfeCh activator (Test 1), or by mixing the two reagents together beforehand
and then applying the solution at once (Test 2). The four other outdoor materials were sprayed
with a 1:1 mixture of either 0.5 M or 1 M Klozur sodium persulfate and 8% FfeCh activator as
described in Section 4.2. The effects of the different test parameters and variables are
summarized and discussed below.
6.2.1 Efficacy Comparisons between B. anthracis andB. subtilis
A summary of the results comparing the average difference in decontamination efficacy for the
two microorganisms (B. anthracis and B. subtilis) is shown in Table 6-1. Refer to Appendices D
and E for detailed efficacy results. Comparison of the results for the two species can also be
visualized for each material in Figures 6-2 through 6-6.
The B. subtilis was more resistant than B. anthracis in all tests except for Tests 1 and 2
conducted with the soil materials. (Although for Tests 1 and 2, both microorganisms were
completely inactivated, so this comparison may be inconsequential.) However, except for Test 7,
the average differences in efficacy between the two organisms were not significant. There is no
specific reason to explain why the results for the inactivation of two species in Test 7 were
significantly different.
For concrete (Figure 6-4), the decontamination efficacy was significantly greater for B. anthracis
in every test. This is in contrast to the other three materials, where there were only a few tests
that resulted in decontamination efficacy being significantly different for the two species.
Coincidentally, the average percent positive control recoveries for the two species was
significantly lower for B. subtilis on concrete (t-test), while there were no significant differences
in recovery of the two species from the other three materials.
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Table 6-1.
Test
Number
Summary of Average Differences in Klozur® Sodium Persulfate Efficacy
between B. anthracis and B. subtitis
Klozur®
Concentration
(M)
Application
Application Frequency Contact Average
(total number of Time Difference
applications)! (Days) in Efficacy
1
2
3
4
5
6
7
8
9
10
11
12
0.5
0.5
0.5
0.5
1
1
1
1
1
1
1
0.5
1 mL Klozur/ 1 mL 8% H2O2
2 mL 1 : 1 mixture Klozur and 8% H2O2
1 : 1 mixture Klozur/ 8% H2O2
1 : 1 mixture Klozur/ 8% H2O2
1 : 1 mixture Klozur/ 8% H2O2
1 : 1 mixture Klozur / 8% H2O2
1 : 1 mixture Klozur / 8% H2O2
1 : 1 mixture Klozur/ 8% H2O2
1 : 1 mixture Klozur / 8% H2O2
1 : 1 mixture Klozur / 8% H2O2
1 : 1 mixture Klozur / 8% H2O2
1 : 1 mixture Klozur / 8% H2O2
3
3
3
4
4
4
2
3
4
1 (16 trigger pulls)
1 (8 trigger pulls)
4
7
7
7
7
7
3
3
3
1
1
1
3
0.38
0.52
-1.59
-1.25
-0.24
-1.69
-2.84*
-1.45
-1.16
-2.50
-1.69
-1.86
Results shown in average difference in efficacy (log reduction). A positive result indicates that!?, subtilis was inactivated to a higher
degree (less resistant) thanB. anthracis Ames. *An asterisk denotes a significant difference in efficacy.
f All applications were made at intervals of 60 minutes.
6.2.2 Efficacy of Klozur Sodium Persulfate in Topsoil
The decontamination efficacy was evaluated in topsoil in Tests 1 and 2. In Test 1, 1 mL of 0.5 M
Klozur sodium persulfate was added to the soil samples followed by 1 mL of 8% EbCh. This test
mimicked tests previously conducted^. To assess the difference in efficacy when pre-mixing
equal parts of both reagents, Test 2 was conducted by adding 2 mL of the pre-mixed solution to
each sample. In both tests, the decontaminant was reapplied two times (a total of three
applications) and was left in contact with the soil for 7 days. The results of this comparison are
shown in Figure 6-2. Complete inactivation of B. anthracis and B. subtilis spores was achieved
in both types of soils using both application approaches, confirming previous test results. These
results also provide evidence that mixing Klozur sodium persulfate and EbCh together and
applied as one reagent is as effective as adding the two reagents separately.
20
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0.5 M Klozur™ in Earthgro® and Agvise Topsoil
Figure
• Earthgro® B. anthracis
MAgvise B. anthracis
E Earthgro® B. sub tills
\BAgvise B. subtilis
1 mL Klozur™ followed 1:1 mixture of Klozur™
by 1 ml H2O2 (#1) and H2O2 (#2)
Application (Test #)
6-2. Summary of decontamination efficacies for Klozur® sodium persulfate testing
against Bacillus spp. in Earthgro® and Agvise® topsoil.
6.2.3 Effects of Test Materials on Efficacy for B. anthracis
The LR results for each of the other four materials tested (wood, concrete, asphalt, and brick) are
summarized in the bar graphs in Figures 6-3 through 6-6 by material and in Figure 6-7 by test.
The average LRs achieved across all tests are also shown in Table 6-2.
In general, decontamination efficacy results for concrete, wood, and brick were similar. For the
overall study, there were no significant differences in efficacy among the three materials.
Asphalt was somewhat easier to decontaminate than the other materials, but because of the
variability in results, the average efficacy for asphalt was only significantly higher than concrete.
In terms of the number of tests in which the average LR of B. anthracis spores was > 6.0 (a
benchmark for efficacy to gauge relative ease of decontaminating these materials), this occurred
in eight of the tests for asphalt, five tests for both wood and brick, and four tests for concrete.
The decontamination results for the two soils in Test 2 can be compared to the results for the
other four materials from Test 3, since the decontamination parameters were the same (three
applications, 7 day contact time, 0.5 M Klozur) for the two tests. The soil materials were all
completely decontaminated (no detectable spores), while the other materials were generally more
difficult to decontaminate. That the soils were easier to decontaminate may be attributed to the
mineral content in the soil (e.g., iron) that may assist in activating persulfate to the sulfate
radical(6).
Further details on the decontamination efficacy results are found in Appendices D and E.
21
-------
Klozur™ on Wood
a
o
i
9
8
7
6
5
4
3
2
1
0
fl
3
7 Day
(3)§
4
7 Day
(4)§
5
7 Day
(4)f
6
3 Day
(4)f
7 8
3 Day 3 Day
(2)f (3)f
Test#
Contact Time (# of applications)
9
IDay
(4)f
10
IDay
UB. anthracis
MB. subtilis
Figure 6-3. Decontamination efficacy for Klozur® sodium persulfate testing against
Bacillus spp. on wood.
(Results shown in average log reduction ± confidence interval).
*Indicates complete inactivation
§Test conducted using 0.5 M Klozur®
f Test conducted using 1 M Klozur®
Klozur™ on Concrete
o
•a
1
n
3
7 Day
(3)§
4
7 Day
(4)§
5
7 Day
(4)f
678
)ay 3 Day 3 Day
l)t (2)t (3)t
Test#
Contact Time (# of applications)
J,
UB. anthracis
MB. subtilis
12
3 Day
(4)§
Figure 6-4. Summary of decontamination efficacy for Klozur® sodium persulfate testing
against Bacillus spp. on concrete.
(Results shown in average log reduction ± confidence interval).
§Test conducted using 0.5 M Klozur®
f Test conducted using 1 M Klozur®
22
-------
Klozur™ on Asphalt
a
o
sS
u
. anthracis
. subtilis
Test#
Contact Time (# of applications)
Figure 6-5. Summary of decontamination efficacy for Klozur® sodium persulfate testing
against Bacillus spp. on asphalt.
(Results shown in average log reduction ± confidence interval).
*Indicates complete inactivation
§Test conducted using 0.5 M Klozur®
f Test conducted using 1 M Klozur®
Klozur™ on Brick
y
i
i «
I 5
§• 4
It 3
1? 2
u
IS i
w
n
o u-
1
^^B
¥1
3 4
7 Day 7 Day
(3)§ (4)§
A
T
~~
A
A
-
A
* T T^ il
1
•
A
JL
-
I
'
--
i UB. anthracis
| ^B. subtilis
1
S
5 6 7 8 9 10 11 12
7 Day 3 Day 3 Day 3 Day 1 Day 1 Day 1 Day 3 Day
(4)f (4)f (2)f (3)f (4)f (l)f (l)t (4)§
Test#
Contact Time (# of applications)
Figure 6-6. Summary of decontamination efficacy for Klozur® sodium persulfate testing
against Bacillus spp. on brick.
(Results shown in average log reduction ± confidence interval).
*Indicates complete inactivation
§Test conducted using 0.5 M Klozur®
f Test conducted using 1 M Klozur®
23
-------
Icsl #3
Test #4
i6
I s
Test #5
T<-sl #6
iJJLi
Test #7
Icsl #8
Test #9
Tesi W10
Tesl #11
Test #12
*Complete inactivation achieved
Figure 6-7. Summary of Klozur® sodium persulfate efficacy (Tests 3-12) results, by test,
against Bacillus anthracis.
(Results shown in average log reduction ± confidence interval).
24
-------
Table 6-2. Summary of Bacillus anthracis Average Log Reductions by Material Type
Material Type
Wood
Concrete
Asphalt
Brick
Average (±SD)LR for
5. 84 ±0.62
5.62 ± 0.47
6.54 ±0.76
5.72 ±0.50
Tests 3 12
LR, log reduction
6.2.4 Effect of Contact Time on the Efficacy ofKlozur sodium persulfate
The effect of increasing CT on the effectiveness ofKlozur sodium persulfate as a decontaminant
was first evaluated in suspension (see Appendix B, Table B-5, for details on suspension test
results). Briefly, approximately 108 B. subtilis spores were added to 10 mL of 0.5 M Klozur
sodium persulfate or 10 mL of a 1:1 mixture of 0.5 M Klozur sodium persulfate and 8% IHbCh.
These samples were mixed by hand and plated immediately (within 10 minutes), or left for a CT
of 7 days with no neutralizer (n=3 for each treatment). The results for the colony forming units
recovered after the CT are shown in Table 6-3.
Table 6-3. Effect of Contact Time (CT) on Klozur® Sodium Persulfate Efficacy against
Bacillus subtilis in Suspension
Treatment"
Inoculum
(CFU)
Average
CFU
recovered b
Average % Recovery
<10 Minute CT
10 mL Klozur + B. subtilis spores
5 mL Klozur/5 mL H2O2 + B. subtilis spores
7.20 x 107
6.32 xlO7
6.02 x 107
87.78%
83.61%
7 Day CT
10 mL Klozur + B. subtilis spores
5 mL Klozur/5 mL H2O2 + B. subtilis spores
1.08 xlO8
3.06 xlO4
0.00
0.028%
0.00%
CFU, colony forming units
aAll treatments completed using a 0.5 M concentration ofKlozur®.
bAverage of three samples.
These results show that recovery of spores does decrease (i.e., improved decontamination
efficacy) with increased CT under the conditions being compared. With the 1 week CT, efficacy
improved by approximately three orders of magnitude when in contact with just Klozur sodium
persulfate. The effect of CT was more pronounced when using the mixture for Klozur sodium
persulfate and IHbCh, as there was minimal inactivation of spores after 10 minutes but complete
inactivation of spores after 7 days.
These suspension test results also confirm that the Klozur is more effective when activated with
H2O2. At the one week CT, Klozur by itself provided a 3.5 LR, while the activated Klozur
provided complete inactivation of the spores (an 8 LR).
The effect of CT on LR was also evaluated by keeping all other decontamination application
parameters constant (i.e., Klozur sodium persulfate concentration, application procedures) using
25
-------
the four outdoor materials (wood, concrete, asphalt, brick). These comparisons are summarized
in Table 6-4 via Tests 5, 6, and 9.
Table 6-4. Effect of Contact Time (CT) on Klozur® Sodium Persulfate Decontamination
Efficacy against Bacillus anthracis
Material Type
Wood
Concrete
Asphalt
Brick
lest y
1 Day CT
> 6.68
5.40
7.00
6.71
±C»l V
3 Day CT
>
>
6.54
6.69
6.69
6.80
,
Average
Increase in
Efficacy
0 36*
iest o
3 Day CT
> 6.54
6.69
6.69
> 6.80
iest j
7 Day CT
> 5.98
5.24
> 6.59
> 5.65
,
Average
Increase in
Efficacy
-0 78*
tAll tests completed using 1:1 mixture of 1M Klozur®) and 8% EhCh, applied 4 times every 60 minutes
*Not a significant change in efficacy
With the exception of concrete (when comparing 1 and 3 day CT), no significant effect of
increasing CT on decontamination efficacy was observed when using a 1:1 mixture of 1 M
Klozur sodium persulfate and 8% IHbCh, with four spray applications. Efficacy was relatively
high at these test conditions (> 5 LR, with a number of materials having complete
decontamination), possibly masking the effect of CT. The LR decreased for concrete when
increasing CT from 3 to? days, illustrating the variability and difficulty in working with this
complex and porous material. Overall, when averaging the results for all materials, no significant
differences in efficacy were noted in either comparison.
6.2.5 Effect of Klozur sodium persulfate Concentration on Decontamination Efficacy against
B. anthracis
The effect of increasing the concentration of Klozur sodium persulfate (from 0.5 M to 1.0 M) on
efficacy was also assessed. These results are summarized in Table 6-5.
Table 6.5 Effect of Increasing Klozur® Sodium Persulfate Concentration on Efficacy
against Bacillus anthracis *
Material Type
Wood
Concrete
Asphalt
Brick
Test 4t
0.5M
Klozur
5.35
6.55
5. 91
5.96
Test 5t Average
-, ™ T^I Increase in
I M Klozur Efficacy
> 5.98
5.24 #
> 6.59
> 5.65
Test 6§
0.5M
Klozur
> 6.54
6.69
6.69
> 6.80
Test 12§
IM
Klozur
> 6.71
6.36
7 09
4.83
Average
Increase in
Efficacy
#
*Data are expressed as average decontamination efficacy (log reduction)
t Tests completed using 4 applications every 60 minutes with a 7 Day CT
§Tests completed using 4 applications every 60 minutes with a 3 Day CT
#not a significant change in efficacy
The difference in efficacy was assessed between Tests 4 and 5 (four applications; 7 day CT) and
Tests 6 and 12 (four applications; 3 day CT). There were just two instances in which increasing
the concentration of Klozur significantly improved efficacy. Overall, when averaging the results
for all materials, no significant differences in efficacy were noted in either condition. Although
these results generally do not show significant improvement in efficacy when increasing
26
-------
persulfate concentration, this may be because efficacy was already relatively high at the 0.5 M
concentration. Further testing at lower levels of persulfate may make this effect more readily
apparent.
6.2.6 Effect of Spray Application Approach on Decontamination Efficacy against B. anthracis
Lastly, the effect of the number of applications and the time interval between spray applications
of Klozur sodium persulfate was evaluated. These results are summarized in Tables 6-6 and 6-7
below.
In general, results were mixed in terms of the impact of increasing the number of spray
applications on decontamination efficacy. We would expect improvement in decontamination
efficacy with an increase in the number spray applications, or perhaps only minor improvement
if decontamination efficacies are already relatively high. However, in comparing Tests 7 and 8,
increasing the number of sprays led to an apparent average decrease in efficacy. (In actuality,
there were no significant differences in efficacy for wood and concrete when comparing results
for Tests 7 and 8; and for brick in both Tests 7 and 8, the coupons were completely
decontaminated.)
When increasing the number of applications from three to four using 0.5 M Klozur sodium
persulfate and a 7 day CT (compare Tests 3 and 4), no significant increase in efficacy was
observed. Again, efficacy was relatively high at the Test 3 conditions (> 5 LR), possibly masking
the effect of increasing the number of applications. However, when increasing the number of
applications from three to four using 1 M Klozur sodium persulfate and a 3 day CT (compare
Tests 8 and 6), a significant increase in average efficacy (1.97) was observed.
An average increase (but not significant) in efficacy of 1.01 was observed when the number of
spray applications was increased from 2 to 4. In this comparison between Test 11 and Test 10, a
1:1 mixture of 1 M and 8% H2O2 with a 1 Day CT were used, but the spray applications occurred
all at once.
27
-------
Table 6-6.
Effect of Increasing the Number of Applications of Klozur® Sodium Persulfate
on Efficacy against Bacillus anthracis *
Material Type
Wood
Concrete
Asphalt
Brick
Material Type
Wood
Concrete
Asphalt
Brick
Material Type
Wood
Concrete
Asphalt
Brick
Test 3t
3
Applications
5.63
5.62
7.26
5.40
Test 7§
2
Applications
6.05
4.09
> 7.61
> 7.00
Test 11*
2
Applications
5.92
4.85
6.56
5.03
Test 4t
4
Applications
5.35
6.55
5.91
5.96
Test 8§
3
Applications
4.23
4.77
5.00
> 5.75
Test 10*
4
Applications
> 6.75
6.67
5.67
> 7.30
Average
Increase in
Efficacy
-0.04®
Average
Increase in
Efficacy
-1.25®
Average
Increase in
Efficacy
1.01®
Test 8§ Test 6§
3 4
Applications Applications
4.23 > 6.54
4.77 6.69
5.00 6.69
> 5.75 > 6.80
Average
Increase in
Efficacy
1.97
*Data are expressed as decontamination efficacy (log reduction)
tTests completed using 1:1 mixture of 0.5 M Klozur® and 8% EhCh with a 7 Day contact time (CT)
§Tests completed using 1:1 mixture of 1 M Klozur® and 8% EhCh with a 3 Day CT
#Tests completed using 1:1 mixture of 1 M and 8% EhCh with a 1 Day CT, sprayed all at once (no 1 hr intervals)
<®Not a significant change in efficacy
By comparing Tests 9 and 10, we can assess whether adding the decontaminant at various
intervals (i.e., every 60 min) or adding the same approximate volume of decontaminant in one
application has any effect. Refer to Table 6-7. Both Tests 9 and 10 were conducted using a 1 M
concentration of Klozur sodium persulfate and a 1 day CT. In Test 9, the Klozur sodium
persulfate was applied four times, with 60 min between each application. In Test 10, a similar
quantity of decontaminant used in Test 9 was spray applied all at once, using a "super-soak"
approach. No significant increase in efficacy was observed when applying the decontaminant
over four applications or applying in one super soak application. Again, this lack of significant
effect may be because efficacy was already relatively high at the Test 9 conditions.
28
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Table 6-7.
Effect of Application Time Interval on the Efficacy of Klozur® Sodium
Persulfate against B. anthracis*^
Material Type
Test 9 Test 10
4 4 Average
Applications Applications Increase in
at 1 hr time sprayed all at Efficacy
intervals once
Wood
Concrete
Asphalt
Brick
> 6.68
5.40
7.00
6.71
> 6.75
6.67
5.67
> 7.30
0 18^
*Data are expressed as decontamination efficacy (log reduction)
tAll tests completed using 1:1 mixture of 1 M Klozur® and 8% EhCh with a 1 Day contact time.
§Not a significant increase in effiacy
6.3 Surface Damage to Materials
At the end of each decontamination test, the procedural blanks were visually compared to the
laboratory blanks, and test samples were visually compared to positive controls to assess any
impact the decontaminants may have had on the appearance of each material type. Based on the
visual appearance of the decontaminated samples, no apparent changes in the color of the two
soil types were observed after being exposed to chloropicrin or Klozur sodium persulfate.
Additionally, there were no apparent changes in color or roughness of the four outdoor materials
after exposure to Klozur sodium persulfate.
6.4 Summary and Conclusions
Several tests were conducted in this study to evaluate the decontamination efficacy of two
decontaminants: chloropicrin and sodium persulfate activated with hydrogen peroxide. Spores of
B. anthracis Ames and B. subtilis were used as the test microorganisms on two types of soil and
four other outdoor materials. Decontamination parameters such as chemical concentration,
quantity applied, and contact time were varied in order to find efficacious conditions with the
minimum application requirements.
Chloropicrin
Because chloropicrin is widely used in agriculture as a soil fumigant (an alternative pesticide for
methyl bromide), it was tested for decontamination efficacy against bacterial spores in garden
topsoil and the Agvise soil. The chloropicrin was tested using an application rate equivalent to
the maximum pesticide label amount, and then tested again with twice this amount. The soil
moisture was also increased to determine if this improved efficacy. Unfortunately, chloropicrin
was only minimally effective (the maximum LR achieved was 1.3 LR) in the three experiments
conducted. Because of these low efficacy results when using a relatively large amount of
chloropicrin, decontamination efficacy testing was discontinued after the third test.
29
-------
Sodium persulfate activated with hydrogen peroxide
In a previous study, sodium persulfate activated with hydrogen peroxide (the commercially
available source of sodium persulfate was Klozur™) was shown to be effective in inactivating B.
anthracis spores in garden topsoil when using three applications and a one week contact time(2).
Based on those results, it was decided to conduct further decontamination efficacy tests with
another type of topsoil (a Mollisol soil from Agvise Laboratory) and four other outdoor materials
(wood, concrete, brick, asphalt, and brick).
In the preliminary suspension tests (no materials used), we confirmed that sodium persulfate
activated with hydrogen peroxide was more effective in inactivating spores compared to using
the persulfate by itself (no hydrogen peroxide activation). All subsequent decontamination tests
used sodium persulfate with hydrogen peroxide.
In the two initial tests with only the two soil materials, complete inactivation of both spore
species was obtained on both types of soil, confirming previous test results. Further, there was
no difference in results when the sodium persulfate and the hydrogen peroxide were mixed
together prior to application to the soils, or if the two chemicals were applied to the soils
sequentially.
Ten additional tests were conducted with the four other outdoor materials. Out of these 10 tests,
there were only two tests in which > 6.0 LR was achieved for B. anthracis on all four materials.
This occurred in Test 6, in which 1.0 M Klozur sodium persulfate, four spray applications
(applied atl hour intervals), and a 3 day contact time were used. Greater than 6 LR also occurred
on all four materials in Test 10, when using 1.0 M Klozur sodium persulfate, four spray
applications (applied all at once), and a 1 day contact time.
For the overall study, there were no significant differences in efficacy for wood, concrete, and
brick. Average results for asphalt showed that it was marginally easier to decontaminate than the
other materials, but its average LR was only significantly higher than concrete. In terms of the
number of tests in which the average LR of B. anthracis spores was > 6.0 (a benchmark for
efficacy to gauge relative ease of decontaminating these materials), this occurred in eight of the
tests for asphalt, five tests for both wood and brick, and four tests for concrete.
The effect of the different decontamination application parameters was sometimes difficult to
demonstrate due to the relatively variable results obtained when working with these porous
materials. In most cases, increasing contact time, persulfate concentration, or the number of
spray applications improved decontamination efficacy. But the effect of increasing these
parameters was not always significant (in some cases due to already achieving relatively high
efficacy), and occasionally resulted in decreased efficacy.
With regard to comparing the inactivation results for the two microorganisms: For the sodium
persulfate work encompassing 12 tests, there was no significant difference in efficacy results
between the two species except for one of the tests (Test 7), in which B. anthracis was
inactivated to a somewhat higher degree. These results provide evidence for the use of B.
subtilis as a surrogate for B. anthracis for soil and outdoor decon testing with sodium persulfate,
for use in future field studies and additional lab-based investigations.
30
-------
7.0 References
1. US Environmental Protection Agency. Evaluation of Liquid and Foam Technologies for
the Decontamination of Bacillus anthracis Spores on Topsoil. US Environmental
Protection Agency: Washington, DC. EPA 600/R-10/080, September 2010.
2. Decontamination of Soil Contaminated with Bacillus anthracis Spores: Technology
Evaluation Report. US Environmental Protection Agency: Research Triangle Park, NC.
EPA600/R-13/110, August 2013.
3. US Department of Justice. Amerithrax Investigative Summary. United States Department
of Justice: Washington, DC. (http://www.iustice.gov/archive/amerithrax/docs/amx-
investigative-summary.pdf) Accessed November 6, 2014.
4. ASTM D 2974-87, Standard Test Methods for Moisture, Ash, and Organic Matter of
Peat and Other Organic Soils. ASTM: Philadelphia, PA. March 1993.
http://turf.lib.msu.edu/1990s/1993/930331.pdf
5. US Environmental Protection Agency. Determining the Efficacy of Liquids and
Fumigants in Systematic Decontamination Studies for Bacillus anthracis using Multiple
Test Methods. US EPA: Research Triangle Park, NC. EPA 600/R-10/088, December
2010. http://cfpub.epa.gov/si/si_public record report.cfm?dirEntryId=227175
6. Huling, S.G. and Pivetz, B.E. US EPA Engineering Issue, In-situ chemical oxidation. US
Environmental Protection Agency: Ada, OK. EPA/600/R-06/072. August 2006.
http://www.epa.gov/ada/gw/isco.html. Accessed November 6, 2014.
7. Procedure for Activation of Klozur® Persulfate with an 8% Hydrogen Peroxide Solution,
Document 02-01-EIT-DH, FMC Corporation: Philadelphia, PA, Copyright 2008 FMC
Corporation.
8. Chloropicrin 100 Fumigant Product Label. EPA Reg. No. 8536-2, September 20, 2012
9. Agvise Laboratories. Interpreting a Soil Test Report, Agvise Laboratories. Agvise
Laboratories: Northwood, ND. (Accessed April 16, 2015)
http://www.agvise.com/wp-content/uploads/2012/07/Interrpreting-a-Soil-Test-Report-
high-res.pdf
10. U.S. Department of Agriculture. Guidelines for Estimating Soil Moisture Conditions.
(Accessed April 16, 2015)
http://www.nrcs.usda.gov/wps/portal/nrcs/detail/mt/newsroom/?cid=nrcsl44p2 056491
-------
Appendix A
Moisture and Organic Content of Soil Samples
Initial Soil Analysis
Soil samples (pre- and post-sterilization via electron beam at -200 kGy or autoclaving at 121 °C
for 1 hour) were sent to an independent laboratory (Agvise Laboratories) for analysis of
moisture, organic content and other characteristics.
Additionally, moisture content was determined by analyzing samples in triplicate using ASTM D
2974-87, Moisture, Ash and Organic Matter of Peat and Other Organic Soils*-4-*. These tests were
performed for samples that had been sterilized via electron beam and subsequently autoclaved at
121 °C for 2 hours in their respective sample jars.
The results are shown in Table A-l.
A-l
-------
Table A-l. Soil Sample Analysis
Pre1 A2
HCB-SL-PF
(Agvise®)
71140180
(Earthgro*)
24.0
24.0
26.0
74.0
E3 Pre A E Pre
23.0
43.0
49.0
39.0
53.0
21.0
47.0
35.0
27.0
37.0
% Clay
A
21.0
5.00
USDA Textural Class
E Pre
30.0
22.0
Loam
Clay
Loam
A
Silt
Loam
Sandy
Loam
Clay
Loam
Loam
Bulk Density (gm/cc)
Pre
0.87
1.02
A E
0.79
1.44
0.83
0.98
71140180
(Earthgro)
ation Excha
Capacity
(meq/100 g
Moisture at If
Field Capaci
% Moisture
21.7
13.9
25.9
15.5
44.5
29.9
36.5
25.2
42.9
27.1
6.37
26.7
b Organic M
10.4
26.4
9.50
6.11
13.5
9.89
6.51
9.24
6.38
7.00
71140180
(Earthgro
Topsoil)
'Pre = Pre-Sterilization; 2A = Autoclave @ 121 °C for 2 hours
3E = Electron beam irradiation @ 200 kGy
4AE = Electron beamed @ 200 kGy and subsequently autoclaved @ 121 °C for 2 hours in the glass sample jars. Moisture analyzed by Battelle.
The % listed under the cations Ca, Mg, K, Na, and H represents the percentage of the total amount of cations measured in the sample, and so all will add up to 100%
A-2
-------
Subsequent Moisture Analysis Related to Chloropicrin Tests
After Test 1 with chloropicrin, it was determined that the moisture content of the soil samples
may need to be elevated to increase the efficacy of chloropicrin. Tests 2 and 3 involved the
addition of either 2 or 3 mL of SFW to increase the moisture content (target of 50-75% field
capacity)(10) of the Earthgro® and Agvise soils, respectively. Moisture assessments were
completed following method ASTM D 2974-87. Samples were weighed, dried in an oven for
>16 hours at 105 ± 5 °C, weighed again and the moisture content calculated as:
Moisture Content, % = [(A-B) x 100]/A
where:
A = mass of the as-received specimen, g
B = mass of the oven-dried specimen, g
Additionally, the moisture field capacity was estimated using guidelines established by the U.S.
Department of Agriculture(10).
The results of these moisture assessments for the chloropicrin testing are presented in Table A-2.
Table A-2. Soil Moisture Assessments for Chloropicrin Tests
.hloropicnn
Applied
27
Earthgro® Topsoil
Agvise Topsoil
mnt SFW Added
Prior to Addition of
Chloropicrin (mL)
0
0
Moisture Conte
(%)ofSoil
Undergoing
Decontamination
13.48
6.11
timated
Field Capacity
NA
NA
27
Earthgro Topsoil
Agvise Topsoil
22.71
32.76
50-75
50-75
54
Earthgro Topsoil
Agvise Topsoil
22.71
32.76
50-75
50-75
A-3
-------
Appendix B
Neutralization and Suspension Tests with Klozur® Sodium Persulfate
Neutralization Methodology
Neutralization of the Klozur sodium persulfate was achieved with 2% sodium thiosulfate (STS),
for Tests 1 and 2 with the soil materials; the percent STS was selected based on previous similar
testing and historical data*-2-*.
For Tests 3 through 12, four neutralization panels were conducted using 1% STS. The
decontamination tests for which accompanying neutralization tests were selected in order to span
the range of contact times and application regimes. See Tables B-l through B-4.
Neutralization panels were completed to assess the need for neutralizer when Klozur sodium
persulfate was applied and left in contact with the materials, depending on the spray application
regime and contact time. The following evaluations were made in each neutralization panel:
(1) Decontamination effectiveness (add spores directly to decontaminant solution; determine
CPU without neutralization). No spores should be recovered
(2) Assess effectiveness of extraction buffer only (phoshate-buffered saline with 0.1% Triton
X-100 [PBST] without any STS) to neutralize or dilute sufficiently active ingredient of
decontaminant
(3) Positive control recovery (add spores to extraction buffer without neutralizer, and without
decontaminant; determine colony forming units [CPU])
(4) Assess neutralizer effectiveness at terminating decontaminant (add spores to extraction
buffer containing 1% STS; determine CPU with neutralization).
To assess the neutralizer effectiveness at terminating sporicidal activity (item 4 above), each
decontaminant was applied in the same manner as required for testing and allowed to stay in
contact with the materials for either 1, 3, or 7 days. At the end of the contact time [CT], the
samples were placed in 50 mL conical tubes containing 10 mL PBST + 1% STS and placed on
an orbital shaker for 15 minutes at 200 rpm at room temperature. A 100 jiL aliquot of spores
Bacillus subtilis spores was applied to each sample, with each sample slowly mixed by hand and
dilution plated. This level was compared to the positive control recovery.
The results of the neutralization panels are shown in Tables B-l through B-4. From these tests, it
was determined that no neutralizer would be needed for any Klozur sodium persulfate tests using
the outdoor materials (Tests 3 through 12). In these tests the percent spore recoveries from
samples containing neutralizer were very similar to the samples containing no neutralizer.
Suspension Tests
These tests were conducted using the neutralization methodology, in particular item (1) above.
Table B-5 summarizes the test conditions and results of these tests. Variables used in these tests
included the microorganism, contact time, and the use of the hydrogen peroxide activator. The
results show that sodium persulfate (without hydrogen peroxide) will inactivate spores to some
limited degree (3.54 log reduction [LR]), provided there is sufficient contact time. With the
activator, a small LR is achieved with B. anthracis after only 15 minutes, and complete
inactivation of spores is achieved after one week.
B-l
-------
Table B-l. Neutralization Testing with Bacillus subtilis spores with Klozur® sodium
)ersulfate, Seven Day Contact Time, Three Total Applications* (Conducted with Test 3)
\vg. lotai "/o oi
>served (CFU) Control
0.5 M Klozur + Spores
Wood
PBS + Triton® X-100 + spores (Control)
Klozur + PBS + Triton X-100 + spores
Klozur + PBS + Triton X-100 + 1.0% STS + spores
Concrete
PBS + Triton X-100 + spores (Control)
Klozur + PBS + Triton® X-100 + spores
Klozur + PBS + Triton® X-100 + 1.0% STS + spores
Asphalt
PBS + Triton X-100 + spores (Control)
Klozur + PBS + Triton X-100 + spores
Klozur + PBS + Triton X-100 + 1.0% STS + spores
Brick
PBS + Triton X-100 + spores (Control)
Klozur + PBS + Triton X-100 + spores
Klozur + PBS + Triton X-100 + 1.0% STS + spores
1.08 x 108 :
1.08 x 108 {
1.08 x 108
1.08 x 108
1.08 x 108
1.08 x 108
1.08 x 108
1.08 x 108
1.08 x 108
1.08 x 108
1.08 x 108 {
1.08 x 108 {
1.08 x 108 <
5.06 x 104
5.65 x 107
.02 x 108
.19xl08
.ISxlO8
.01 x 108
.03 x 108
.06 x 108
.04 x 108
.03 x 108
5.60 x 107
5.94 x 107
).82 x 107
0.035
118.23
137.24
85.68
87.20
98.64
97.26
103.99
114.19
PBS, phosphate-buffered saline; STS, sodium thiosulfate.
8% H2O2
*Klozur® denotes 1:1 ratio of 0.5 M Klozur sodium persulfate and
Table B-2. Neutralization Testing with Bacillus subtilis spores with Klozur® sodium
persulfate, Three Day Contact Time, Four Total Applications* (Conducted
with Test 6)
1 Treatment
l.OM Klozur + Spores
(Inoculum
(CFU)
9.13 xlO7
Avg. Total % of
Observed (CFU) Control
0
0
Wood
PBS + Triton® X-100 + spores (Control)
Klozur + PBS + Triton X-100 + spores
Klozur® + PBS + Triton® X-100 + 1.0% STS + spores
9.13 xlO7
9.13 xlO7
9.13 xlO7
1.04 x 108
1.01 xlO8
9.83 x 107
96.80
94.46
Concrete
PBS + Triton X-100 + spores (Control)
Klozur + PBS + Triton X-100 + spores
Klozur + PBS + Triton X-100 + 1.0% STS + spores
9.13 xlO7
9.13 xlO7
9.13 xlO7
1.03 x 108
1.12xl08
1.13xl08
109.27
110.25
Asphalt
PBS + Triton X-100 + spores (Control)
Klozur + PBS + Triton X-100 + spores
Klozur + PBS + Triton X-100 + 1.0% STS + spores
Brick
PBS + Triton X-100 + spores (Control)
Klozur + PBS + Triton X-100 + spores
Klozur + PBS + Triton X-100 + 1.0% STS + spores
PBS, phosphate-buffered saline; STS, sodium thiosulfate; *Klozur®
9.13 xlO7
9.13 xlO7
9.13 xlO7
9.13 xlO7
9.13 xlO7
9.13 xlO7
denotes 1 : 1 ratio of 1
9.95 x 107
1.01 xlO8
9.65 x 107
9.40 x 107
8.79 x 107
8.53 x 107
M Klozur® and 8% H2O2,
101.91
96.92
93.48
90.78
B-2
-------
Table B-3. Neutralization Testing with Bacillus subtilis spores with Klozur® sodium
persulfate, One Day Contact Time, Four Total Applications (TEST 9)
Treatment
Klozur® + Spores
Inoculum
1.12xl08
Avg. Total
Observed (CFU)
0
%of
Control
0
Wood
PBS + Triton® X-100 + spores (Control)
Klozur + PBS + Triton X-100 + spores
Klozur + PBS + Triton X-100 + 1.0% STS + spores
1.12xl08
1.12xl08
1.12xl08
LlSxlO8
1.03 x 108
1.16xl08
86.96
97.75
Concrete
PBS + Triton X-100 + spores (Control)
Klozur + PBS + Triton X-100 + spores
Klozur + PBS + Triton X-100 + 1.0% STS + spores
1.12xl08
1.12xl08
1.12xl08
1.19xl08
1.15xl08
LlSxlO8
96.63
99.72
Asphalt
PBS + Triton X-100 + spores (Control)
Klozur + PBS + Triton X-100 + spores
Klozur + PBS + Triton X-100 + 1.0% STS + spores
1.12xl08
1.12xl08
1.12xl08
1.04 x 108
1.12xl08
1.07 x 108
107.12
102.78
Brick
PBS + Triton X-100 + spores (Control)
Klozur + PBS + Triton X-100 + spores
Klozur + PBS + Triton X-100 + 1.0% STS + spores
1.12xl08
1.12xl08
1.12xl08
9.41 x 107
1.08 x 107
9.78 x 107
114.42
103.90
PBS, phosphate-buffered saline; STS, sodium thiosulfate.
H202.
*Klozur® denotes 1:1 ratio of 1 M Klozur sodium persulfate and 8%
Table B-4. Neutralization Testing with Bacillus subtilis spores with Klozur® sodium
persulfate, One Day Contact Time, One Application of 16 sprays* (TEST 10)
1 Treatment
Klozur® + Spores
Inoculum
l.llxlO8
Avg. Total
Observed (CFU)
0
%of
Control
0
Wood
PBS + Triton® X-100 + spores (Control)
Klozur + PBS + Triton X-100 + spores
Klozur + PBS + Triton X-100 + 1.0% STS + spores
l.llxlO8
l.llxlO8
l.llxlO8
1.87 x 108
8.39 xlO7
1.01 xlO8
44.93
54.11
Concrete
PBS + Triton X-100 + spores (Control)
Klozur + PBS + Triton X-100 + spores
Klozur + PBS + Triton X-100 + 1.0% STS + spores
l.llxlO8
l.llxlO8
l.llxlO8
9.03 x 107
8.18 xlO7
1.14xl08
90.52
126.31
Asphalt
PBS + Triton X-100 + spores (Control)
Klozur + PBS + Triton X-100 + spores
Klozur + PBS + Triton X-100 + 1.0% STS + spores
l.llxlO8
l.llxlO8
l.llxlO8
9.21 x 107
8.90 x 107
9.11 xlO7
96.63
98.95
Brick
PBS + Triton X-100 + spores (Control)
Klozur + PBS + Triton X-100 + spores
Klozur + PBS + Triton X-100 + 1.0% STS + spores
l.llxlO8
l.llxlO8
l.llxlO8
1.12xl08
1.19xl08
9.27 x 107
106.25
82.74
PBS, phosphate-buffered saline; STS, sodium thiosulfate.
H2O2.
*Klozur® denotes 1:1 ratio of 1 M Klozur sodium persulfate and 8%
B-3
-------
Table B-5. Suspension Testing with Klozur® sodium persulfate on Bacillus spp.
Treatme»,
10 mL of 0.5 M Klozur® with no
H2O2 activator
Mixture of 5 mL 0.5 M Klozur with
5 mL 8% H2O2 activator
10 mL of 0.5 M Klozur with no H2O2
activator
Mixture of 5 mL 0.5 M Klozur with
5 mL 8% H2O2 activator
10 mL of 0.5 M Klozur with no H2O2
activator
Mixture of 5 mL 0.5 M Klozur with
5 mL 8% H2O2 activator
Contact time
< 15 minutes
< 15 minutes
< 15 minutes
< 15 minutes
1 week
1 week
Inoculum (CFU) and
Species
8.13xl075.a.
8.13xl075.a.
7.20 x 107 B.s.
7.20 x 107 B.s.
1.08xl085.5.
1.08 xlO8 5.5.
Spores
Recovered
(CFU)
8.02 xlO7
6.76 xlO6
6.32 x 107
6.02 x 107
3.06 x 104
0
Log
reduction
0.01
1.08
0.06
0.08
3.55
>8.03
B.a., Bacillus anthracis; B.s., Bacillus subtilis; CFU, colony forming units
B-4
-------
Appendix C
Chloropicrin Detailed Test Results
Chloropicrin Efficacy Results
The detailed spore recovery and decontamination efficacy results for Chloropicrin against
B. anthracis and B. subtilis on two materials types (Earthgro® and Agvise topsoil) are
shown in Tables C-l through C-4. Zero CPU were observed on all laboratory and
procedural blanks.
Table C-l
Inactivation of B. anthracis Spores in Earthgro® Topsoil with
Chloropicrin"
Test Number
(Chloropicrin Volume)
l(27uL)
Positive Controls'5
Test Samples0
Laboratory"1 and Procedural Blank
2(27uL)*
Positive Controls
Test Samples
Laboratory and Procedural Blank
3 (54 uL)*
Positive Controls
Test Samples
Laboratory and Procedural Blank
r Inoculum
(CFU)
1.23 xlO8
1.23 xlO8
0
1.00 xlO8
1.00 xlO8
0
1.01 xlO8
1.01 xlO8
0
Mean of Logs of
Observed CFU
7.83 ±0.15
6.77 ±0.37
0
8.17±0.10
7.53 ±0.34
0
7.81 ±0.08
6.55 ±0.38
0
Mean %
Recovery
58.16±20.19
5. 88 ±2.80
-
149.80 ±30.18
41.60 ±24.02
-
65.49 ±11. 86
4.49 ±3.22
-
Decontamination
Efficacy ±CIf
g
1.06 ±0.35
-
-
0.63 ±0.31
-
-
1.27 ±0.34
-
a = Data are expressed as the mean (± SD) of the logs of the colony forming units (CFU) observed on five individual
samples, the mean (± SD) percent recovery on those five samples, and decontamination efficacy (log reduction).
b = Positive Controls = inoculated, not decontaminated samples.
c = Test Samples = inoculated, decontaminated samples.
d = Laboratory Blank = not inoculated, not decontaminated sample.
e = Procedural Blank = not inoculated, decontaminated sample.
f = CI = 95% confidence interval (± 1.96 x SE).
g = "-" Not Applicable.
* = 2 mL SFW added to all samples prior to inoculation
C-l
-------
Table C-2 Inactivation of B. subtitts Spores in Earthgro Topsoil with Chloropicrin"
Test Number
(Chloropicrin Volume)
1 (27 uL)
Positive Controls
Test Samples
Laboratory4 and Procedural Blank
2(27uL)*
Positive Controls
Test Samples
Laboratory and Procedural Blank
3 (54 uL)*
Positive Controls
Test Samples
Laboratory and Procedural Blank
1 Inoculum
(CFU)
9.27 xlO7
9.27 xlO7
0
6.40 xlO7
6.40 xlO7
0
7.40 xlO7
7.40 xlO7
0
Mean of Logs of
Observed CFU
8.32±0.13
7.83 ±0.06
0
8.20 ±0.05
7.94 ±0.03
0
8.00 ±0.04
7.91 ±0.04
0
Mean %
Recovery
23 1.72 ±67.63
73. 57 ±9.77
250.94 ±29.88
136.16±8.54
134.97 ±13.21
110.27 ±10.60
Decontamination
Efficacy ±CIf
0.49 ±0.12
.
0.26 ±0.05
.
0.09 ±0.05
1 = Data are expressed as the mean (± SD) of the logs of the colony forming units (CFU) observed on five individual samples, the mean
(± SD) percent recovery on those five samples, and decontamination efficacy (log reduction).
b = Positive Controls = inoculated, not decontaminated samples.
c = Test Samples = inoculated, decontaminated samples.
d = Laboratory Blank = not inoculated, not decontaminated sample.
e = Procedural Blank = not inoculated, decontaminated sample.
f = CI = 95% confidence interval (± 1.96 x SE).
g = "-" Not Applicable.
* = 2 mL SFW added to all samples prior to inoculation
Table C-3 Inactivation of B. anthracis Spores in Agvise Topsoil with Chloropicrin"
Test Number
(Chloropicrin Volume)
l(27uL)
Positive Controls15
Test Samples0
Laboratory"1 and Procedural6 Blank
2(27uL)*
Positive Controls
Test Samples
Laboratory and Procedural Blank
3 (54 uL)*
Positive Controls
Test Samples
Laboratory and Procedural Blank
Inoculum
(CFU)
1.23 xlO8
1.23 xlO8
0
1.00 xlO8
1.00 xlO8
0
1.01 xlO8
1.01 xlO8
0
Mean Logs of
Observed CFU
7.80 ±0.07
7.42 ±0.16
0
8.04 ±0.13
7.21 ±0.63
0
7.70 ±0.05
6.82 ±0.49
0
Mean %
Recovery
51.76±8.58
22.34 ±8.00
114.46±33.99
30. 17 ±27.26
50.44 ±6.30
11.02 ±13.66
Decontamination
Efficacy ±CIf
£
0.38±0.15
0.83 ±0.56
0.89 ±0.43
1 = Data are expressed as the mean (± SD) of the logs of the number of spores (CFU) observed on five individual samples, the mean (±
SD) percent recovery on those five samples, and decontamination efficacy (log reduction).
b = Positive Controls = inoculated, not decontaminated samples.
c = Test Samples = inoculated, decontaminated samples.
d = Laboratory Blank = not inoculated, not decontaminated sample.
e = Procedural Blank = not inoculated, decontaminated sample.
f = CI = 95% confidence interval (± 1.96 x SE).
« = "." Not Applicable.
* = 3 mL SFW added to all samples prior to inoculation
C-2
-------
Table C-4 Inactivation of B. subtilis Spores in Agvise Topsoil with Chloropicrin"
Test Number
(Chloropicrin Volume)
1 (27 uL)
Positive Controls
Test Samples
Laboratory4 and Procedural Blank
2 (27 uL)*
Positive Controls
Test Samples
Laboratory and Procedural Blank
3 (54 uL)*
Positive Controls
Test Samples
Laboratory and Procedural Blank
1 Inoculum
(CFU)
9.27 xlO7
9.27 xlO7
0
6.40 xlO7
6.40 xlO7
0
7.40 xlO7
7.40 xlO7
0
Mean Logs of
Observed CFU
7.74 ±0.08
7.44 ±0.20
0
8.71 ±0.08
7.79 ±0.04
0
8.51 ±0.21
7.41 ±0.20
0
Mean %
Recovery
60.32 ±12.36
32.84 ±19.05
-
804.38 ±131.43
97.50 ±8.85
-
473.24 ±182.49
37.86 ±15.75
-
Decontamination
Efficacy ±CIf
-
0.30±0.19
-
-
0.91 ±0.07
-
-
1.10±0.26
-
1 = Data are expressed as the mean (± SD) of the logs of the colony forming units (CFU) observed on five individual samples, the mean
(± SD) percent recovery on those five samples, and decontamination efficacy (log reduction).
b = Positive Controls = inoculated, not decontaminated samples.
c = Test Samples = inoculated, decontaminated samples.
d = Laboratory Blank = not inoculated, not decontaminated sample.
e = Procedural Blank = not inoculated, decontaminated sample.
f = CI = 95% confidence interval (± 1.96 x SE).
* = 3 mL SFW added to all samples prior to inoculation
C-2
-------
Appendix D
Klozur® Sodium Persulfate Detailed Test Results
The results from all tests (Tests 1 through 12) with Klozur sodium persulfate are detailed in
Tables D-l through D-10.
Table D-l
Inactivation of B. anthracis and B. subtitis Spores in Earthgro® Topsoil with
0.5 M Klozur®'a Sodium Persulfate
Mean L
Observe
anthracis Ames
Seven Days (3) - #1
Positive Controls15 8.13 x 107 7.89 ± 0.04
TestSamplesc 8.13 x 107 0.00±0.00
Laboratory*1 and Procedural6 Blank 0 0
95.03 ±8.87
0.00 ±0.00
>7.89±0.03
Seven Days (3) - #2
Positive Controls 8.13 x 107 7.92 ± 0.03
Test Samples 8.13xl07 0.00 ±0.00
Laboratory and Procedural Blank 0 0
103.54 ±7.71
0.00 ±0.00
B. subtilis
>7.92±0.03
_
Seven Days (3) - #1
Positive Controls
Test Samples
Laboratory and Procedural Blank
Seven Days (3) - #2
Positive Controls
Test Samples
Laboratory and Procedural Blank
8.87 xlO7
8.87 xlO7
0
8.87 xlO7
8.87 xlO7
0
8.18 ±0.07
0.00 ±0.00
0
8.37±0.11
0.00 ±0.00
0
173.39 ±27.81
0.00 ±0.00
268.32 ±66.70
0.00 ±0.00
>8. 18 ±0.06
.
>8.37±0.10
= Data are expressed as the mean (± SD) of the logs of the colony forming units (CFU) observed on five individual samples, the mean (± SD)
percent recovery on those five samples, and decontamination efficacy (log reduction).
= Positive Controls = inoculated, not decontaminated samples.
= Test Samples = inoculated, decontaminated samples.
= Laboratory Blank = not inoculated, not decontaminated sample.
= Procedural Blank = not inoculated, decontaminated sample.
= CI = confidence interval (± 1.96 x SE).
= "-" Not Applicable.
D-l
-------
Table D-2 Inactivation of B. anthracis and B. subtilis Spores in Agvise® Topsoil with 0.5
M Klozur®'a Sodium Persulfate
Contact Time
mber of Application
Test#
Inoculum
Mean Logs of
>served CF
Mean %
Decontamination
fficacv±CIf
B. anthracis Ames
Seven Days (3) - #1
Positive Controls'5
Test Samples0
Laboratory"1 and Procedural6 Blank
Seven Days (3) - #2
Positive Controls
Test Samples
Laboratory and Procedural Blank
8.13 xlO7
8.13 xlO7
0
8.13 xlO7
8.13 xlO7
0
7.65 ±0.10
0.00 ±0.00
0
7.74 ±0.06
0.00 ±0.00
0
56.26 ±13.04
0.00 ±0.00
67.48 ±9.97
0.00 ±0.00
>7.65±0.09
>7.74±0.05
B. subtilis
Seven Days (3) - #1
Positive Controls
Test Samples
Procedural Blank
Seven Days (3) - #2
Positive Controls
Test Samples
Laboratory and Procedural Blank
8.87 xlO7
8.87 xlO7
0
8.87 xlO7
8.87 xlO7
0
8.12±0.19
0.00 ±0.00
0
8.32 ±0.20
0.00 ±0.00
0
158.67 ±56.41
0.00 ±0.00
258.40 ±115.07
0.00 ±0.00
>8.12±0.16
>8.32±0.18
a = Data are expressed as the mean (± SD) of the logs of the colony forming units (CFU) observed on five individual samples, the mean (± SD)
percent recovery on those five samples, and decontamination efficacy (log reduction).
b = Positive Controls = inoculated, not decontaminated samples.
c = Test Samples = inoculated, decontaminated samples.
d = Laboratory Blank = not inoculated, not decontaminated sample.
e = Procedural Blank = not inoculated, decontaminated sample.
f = CI = confidence interval (± 1.96 x SE).
g = "-" Not Applicable.
D-2
-------
Table D-3 Inactivation of B. anthracis Spores on Wood with Klozur®'a Sodium Persulfate
Klozur Concentration -
Contact Time
Number of Applications) -
Test#
lean Logs <
1 served CT
0.5 M Klozur - Seven Days (3) - #3
Positive Controls15 1.22 x 108
Test Samples0 1.22xl08
Laboratoryd and Procedural6 Blank 0
6.47 ±0.26
0.84 ±1.23
0
Mean %
Recove—
2.77 ±1.46
0.000088 ±0.00018
5.63 ±1.10
0.5 M Klozur - Seven Days (4) - #4
Positive Controls 8.73 x 107 6.06 ± 0.25
TestSamples 8.73 x 107 0.70 ±1.57
Laboratory and Procedural Blank 0 0
1.51 ±1.00
0.00074 ±0.0017
5.35 ±1.39
1 M Klozur - Seven Days (4) - #5
Positive Controls 1.03 x 108 4.49 ±1.70
Test Samples 1.03xl08 0.00 ±0.00
Laboratory and Procedural Blank 0 0
0.18±0.18
0.00 ±0.00
>4.49±1.49
1 M Klozur - Seven Days (4) - #5 - Repeat
Positive Controls S.SOxlO7 5.98±0.16 1.50±1.33
Test Samples 8.80x 107 0.00 ±0.00 0.00 ±0.00 >5.98 ±0.35
1 M Klozur - Three Days (4) - #6
Positive Controls 9.90xl07 6.54 ±0.04
TestSamples 9.90xl07 0.00 ±0.00
Laboratory and Procedural Blank 0 0
3.49 ±0.28
0.00 ±0.00
>6.54±0.03
1 M Klozur - Three Days (2) - #7
Positive Controls 1.13xl08 6.78 ±0.19
Test Samples 1.13xl08 0.73 ±1.02
Laboratory and Procedural Blank 0 0
5.68 ±2.30
0.000030 ±0.000051
6.05 ±0.91
1 M Klozur - Three Days (3) - #8
Positive Controls 1.43 x 108 6.62 ± 0.06
Test Samples 1.43xl08 2.39 ±1.74
Laboratory and Procedural Blank 0 0
2.92 ±0.41
0.0033 ±0.0045
4.23 ±1.52
1 M Klozur - One Day (4) - #9
Positive Controls 7.33 x 107 6.68 ± 0.09
Test Samples 7.33 x 107 0.00 ± 0.00
Laboratory and Procedural Blank 0 0
6.60 ±1.37
0.00 ±0.00
>6.68±0.08
1 M Klozur - One Day (1) - #10*
Positive Controls 1.56xl08 6.75 ±0.32
TestSamples 1.56xl08 0.00±0.00
Laboratory and Procedural Blank 0 0
4.61±4.11
0.00 ±0.00
>6.75±0.28
1 M Klozur - One Day (1) - #11*
Positive Controls 9.87 x 107 7.00 ±0.16
TestSamples 9.87xl07 1.08 ±1.48
Laboratory and Procedural Blank 0 0
10.69 ±3.48
0.00022 ±0.00032 5.92 ±1.31
0.5 M Klozur - Three Days (4) - #12
Positive Controls 1.02xl08 6.71 ±0.17
TestSamples 1.02xl08 0.00±0.00
Laboratory and Procedural Blank 0 0
5.35 ±2.27
0.00 ±0.00
>6.71±0.15
" = Data are expressed as the mean (± SD) of the logs of the colony forming units (CFU) observed on five individual samples, the mean (± SD) percent
recovery on those five samples, and decontamination efficacy (log reduction).
b = Positive Controls = inoculated, not decontaminated samples.
c = Test Samples = inoculated, decontaminated samples.
11 = Laboratory Blank = not inoculated, not decontaminated sample.
e = Procedural Blank = not inoculated, decontaminated sample.
f = CI = confidence interval (± 1.96 x SE).
8 = "-" Not Applicable.
*One application (16 trigger pulls)
TOne application (8 trigger pulls)
D-3
-------
Table D-4 Inactivation of B. anthracis Spores on Concrete with Klozur®'a Sodium Persulfate
Klozur® Concentration
Contact Time
(Number of Applications^
Test#
Inoculum
Mean Logs of
Observed
.-contamination
"fficacv±CIf
0.5 M Klozur - Seven Days (3) - #3
Positive Controls'5
Test Samples0
Laboratory4 and Procedural6 Blank
0.5 M Klozur - Seven Days (4) - #4
Positive Controls
Test Samples
Laboratory and Procedural Blank
1 M Klozur - Seven Days (4) - #5
Positive Controls
Test Samples
Laboratory and Procedural Blank
1 M Klozur - Three Days (4) - #6
Positive Controls
Test Samples
Laboratory and Procedural Blank
1 M Klozur - Three Days (2) - #7
Positive Controls
Test Samples
Laboratory and Procedural Blank
1 M Klozur - Three Days (3) - #8
Positive Controls
Test Samples
Laboratory and Procedural Blank
1 M Klozur - One Day (4) - #9
Positive Controls
Test Samples
Laboratory and Procedural Blank
1 M Klozur - One Day (1) - #10*
Positive Controls
Test Samples
Laboratory and Procedural Blank
1 M Klozur - One Day (1) - #11*
Positive Controls
Test Samples
Laboratory and Procedural Blank
0.5 M Klozur - Three Days (4) - #12
Positive Controls
Test Samples
Laboratory and Procedural Blank
1.22 xlO8
1.22 xlO8
0
8.73 x 107
8.73 x 107
0
1.03 xlO8
1.03 xlO8
0
9.90 xlO7
9.90 xlO7
0
1.13xl08
1.13xl08
0
1.43 xlO8
1.43 xlO8
0
7.33 xlO7
7.33 xlO7
0
1.56xl08
1.56xl08
0
9.87 xlO7
9.87 xlO7
0
1.02 xlO8
1.02 xlO8
0
7.16 ±0.26
1.54 ±0.90
0
6.85±0.15
0.30 ±0.68
0
6.34 ±0.44
1.10±1.55
0
6.99 ±0.08
0.30 ±0.68
0
7.27 ±0.25
3.18±1.15
0
6.95 ±0.27
2.17±1.58
0
7.10±0.35
1.70 ±1.85
0
7.03 ±0.08
0.36 ±0.82
0
7.23 ±0.27
2.38 ±2.24
0
6.90 ±0.32
0.54 ±1.21
0
13. 84 ±9.64
0.000066 ±0.000056
8.48 ±2.41
0.0000085 ±0.000017
3.26 ±3.41
0.00036 ±0.00071
10. 12 ±2.08
0.0000075 ±0.00001 5
18.61 ±9.82
0.015 ±0.030
7.15 ±4.07
0.0021 ±0.0040
22.56 ±19.56
0.0084 ±0.019
6.96±1.14
0.0000091 ±0.000019
19.93 ±11.46
0.013 ±0.025
9.57 ±6.32
0.00010 ±0.00022
g
5.62 ±0.82
6.55 ±0.61
5. 24 ±1.41
6.69 ±0.60
4.09 ±1.03
4.77 ±1.41
5.40 ±1.65
6.67 ±0.72
4.85 ±1.98
6.36 ±1.09
= Data are expressed as the mean (± SD) of the logs of the colony forming units (CFU) observed on five individual samples, the mean (± SD) percent
recovery on those five samples, and decontamination efficacy (log reduction).
= Positive Controls = inoculated, not decontaminated samples.
= Test Samples = inoculated, decontaminated samples.
= Laboratory Blank = not inoculated, not decontaminated sample.
= Procedural Blank = not inoculated, decontaminated sample.
= CI = confidence interval (± 1.96 x SE).
= "-" Not Applicable.
;One application (16 trigger pulls)
One application (8 trigger pulls)
D-4
-------
Table D-5 Inactivation of B. anthracis Spores on Asphalt with Klozur®'a Sodium Persulfate
Klozur Concentration
Contact Time
(Number of Applications)
Test#
Inoculum
(CFU)
Mean Logs of
Observed CFU
Mean %
Recovery
Decontamination
Efficacy ±CIf
0.5 M Klozur - Seven Days (3) - #3
Positive Controls'5
Test Samples0
Laboratory"1 and Procedural6 Blank
0.5 M Klozur - Seven Days (4) - #4
Positive Controls
Test Samples
Laboratory and Procedural Blank
1 M Klozur - Seven Days (4) - #5
Positive Controls
Test Samples
Laboratory and Procedural Blank
1 M Klozur - Three Days (4) - #6
Positive Controls
Test Samples
Laboratory and Procedural Blank
1 M Klozur - Three Days (2) - #7
Positive Controls
Test Samples
Laboratory and Procedural Blank
1 M Klozur - Three Days (3) - #8
Positive Controls
Test Samples
Laboratory and Procedural Blank
1 M Klozur - One Day (4) - #9
Positive Controls
Test Samples
Laboratory and Procedural Blank
1 M Klozur - One Day (1) - #10*
Positive Controls
Test Samples
Laboratory and Procedural Blank
1 M Klozur - One Day (1) - #11*
Positive Controls
Test Samples
Laboratory and Procedural Blank
0.5 M Klozur - Three Days (4) - #12
Positive Controls
Test Samples
Laboratory and Procedural Blank
1.22 xlO8
1.22 xlO8
0
8.73 xlO7
8.73 xlO7
0
1.03 xlO8
1.03 xlO8
0
9.90 xlO7
9.90 xlO7
0
1.13xl08
1.13xl08
0
1.43 xlO8
1.43 xlO8
0
7.33 xlO7
7.33 xlO7
0
1.56xl08
1.56xl08
0
9.87 xlO7
9.87 xlO7
0
1.02 xlO8
1.02 xlO8
0
7.26 ±0.14
0.00 ±0.00
0
7.12±0.08
1.21 ±1.14
0
6.59 ±0.37
0.00 ±0.00
0
7.44 ±0.11
0.75 ±1.68
0
7.61 ±0.06
0.00 ±0.00
0
7.44 ±0.21
2.43 ±1.78
0
7.42±0.19
0.42 ±0.95
0
7.40 ±0.12
1.73 ±2.39
0
7.71 ±0.10
1.15±2.57
0
7.40±0.13
0.30 ±0.68
0
15. 56 ±5.65
0.00 ±0.00
15.14±2.80
0.000092 ±0.00011
4.75 ±2.94
0.00 ±0.00
28.61 ±6.61
0.0012 ±0.0026
36.46 ±4.85
0.00 ±0.00
20.74 ±8.58
0.011 ±0.024
39.05 ±18.78
0.000037 ±0.000081
16.50 ±4.01
0.0093 ±0.019
52.81 ± 10.77
0.11 ±0.26
25.41 ±7.39
0.0000073 ±000014
g
>7.26±0.12
5.91 ±1.01
>6.59±0.32
6.69 ±1.48
>7.61±0.05
5.00 ±1.57
7.00 ±0.85
5.67±2.10
6.56 ±2.26
7.09 ±0.61
= Data are expressed as the mean (± SD) of the logs of the colony forming units (CFU) observed on five individual samples, the mean (± SD) percent
recovery on those five samples, and decontamination efficacy (log reduction).
= Positive Controls = inoculated, not decontaminated samples.
= Test Samples = inoculated, decontaminated samples.
= Laboratory Blank = not inoculated, not decontaminated sample.
= Procedural Blank = not inoculated, decontaminated sample.
= CI = confidence interval (± 1.96 x SE).
= "-" Not Applicable.
;One application (16 trigger pulls)
One application (8 trigger pulls)
D-5
-------
Table D-6 Inactivation of B. anthracis Spores on Brick with Klozur®'a Sodium Persulfate
Klozur® Concentration Contact
Time
(Number of Applications)
0.5 M Klozur - Seven Days (3) - #3
Positive Controls'5
Test Samples0
Laboratory4 and Procedural6 Blank
0.5 M Klozur - Seven Days (4) - #4
Positive Controls
Test Samples
Laboratory and Procedural Blank
0.5 M Klozur - Seven Days (4) - #4 - Repeat
Positive Controls
Test Samples
0.5 M Klozur - Seven Days (4) - #4 - Repeat
Positive Controls
Test Samples
1 M Klozur- Seven Days (4) - #5
Positive Controls
Test Samples
Laboratory and Procedural Blank
1 M Klozur - Seven Days (4) - #5 - Repeat
Positive Controls
Test Samples
1 M Klozur- Seven Days (4) - #5 - Repeat
Positive Controls
Test Samples
1 M Klozur - Three Days (4) - #6
Positive Controls
Test Samples
Laboratory and Procedural Blank
1 M Klozur - Three Days (2) - #7
Positive Controls
Test Samples
Laboratory and Procedural Blank
1 M Klozur - Three Days (3) - #8
Positive Controls
Test Samples
Laboratory and Procedural Blank
1 M Klozur - One Day (4) - #9
Positive Controls
Test Samples
Laboratory and Procedural Blank
1 M Klozur - One Day (1) - #10*
Positive Controls
Test Samples
Laboratory and Procedural Blank
1 M Klozur - One Day (1) - #llf
Positive Controls
Test Samples
Laboratory and Procedural Blank
Inoculum
(CFU)
1.22 xlO8
1.22 xlO8
0
8.73 xlO7
8.73 xlO7
0
8.80 xlO7
8.80 xlO7
1.17xl08
1.17xl08
1.03 xlO8
1.03 xlO8
0
8.80 xlO7
8.80 xlO7
1.17xl08
1.17xl08
9.90 xlO7
9.90 xlO7
0
1.13xl08
1.13xl08
0
1.43 xlO8
1.43 xlO8
0
7.33 xlO7
7.33 xlO7
0
1.56xl08
1.56xl08
0
9.87 xlO7
9.87 xlO7
0
Mean Logs of
Observed CFU
6.59 ±0.23
1.20±1.17
0
5.96 ±0.25
0.00 ±0.00
0
0.00 ±0.00
0.00 ±0.00
5.65 ±0.22
0.00 ±0.00
2.46 ±1.45
0.00 ±0.00
0
0.00 ±0.00
0.00 ±0.00
5.65 ±0.22
0.00 ±0.00
6.80±0.17
0.00 ±0.00
0
7.00 ±0.12
0.00 ±0.00
0
5.75 ±0.20
0.00 ±0.00
0
7.01 ±0.16
0.30 ±0.68
0
7.30±0.15
0.00 ±0.00
0
7.21 ±0.13
2.18±1.37
0
Mean % Decontamination
Recovery Efficacy ±CIf
3.60 ±2.00 £
0.000088 ±0.00015 5.40 ±1.04
1.18±0.62
0.00 ±0.00 >5. 96 ±0.22
0.00 ±0.00
0.00 ±0.00 0.00 ±0.00
1
0.56 ±0.46
0.00 ±0.00 >5.65±0.41
0.02 ±0.04
0.00 ±0.00 >2.46±1.27
0.00 ±0.00
0.00 ±0.00 0.00 ±0.00
1
0.56 ±0.46
0.00 ±0.00 >5.65±0.41
6.66 ±2.22
0.00 ±0.00 >6.80±0.14
9.00 ±2.33
0.00 ±0.00 >7.00±0.10
0.43 ±0.22
0.00 ±0.00 >5.75±0.18
14.75 ±5.47
0.000010 ± 0.000020 6.71 ± 0.61
13.46 ±4.56
0.00 ±0.00 >7.30±0.13
17.12 ±5.87
0.0010 ±0.0015 5.03 ±1.20
D-6
-------
0.5 M Klozur - Three Days (4) - #12
Positive Controls 1.02 x 108 6.80 ±0.20 6.76 ±3.06
Test Samples 1.02xl08 1.97 ±1.46 0.0022 ±0.0048 4.83 ±1.29
Laboratory and Procedural Blank 00--
a = Data are expressed as the mean (± SD) of the logs of the colony forming units (CFU) observed on five individual samples, the mean (± SD)
percent recovery on those five samples, and decontamination efficacy (log reduction).
b = Positive Controls = inoculated, not decontaminated samples.
c = Test Samples = inoculated, decontaminated samples.
d = Laboratory Blank = not inoculated, not decontaminated sample.
e = Procedural Blank = not inoculated, decontaminated sample.
f = CI = confidence interval (± 1.96 x SE).
§ = "." Not Applicable.
*One application (16 trigger pulls)
^One application (8 trigger pulls)
D-7
-------
Table D-7 Inactivation of B. subtilis Spores on Wood with Klozur®'a Sodium Persulfate
Klozur® Concentration
Contact Time
(Number of Applications^
Test#
0.5 M Klozur - Seven Days (3) - #3
Positive Controls15 9.43 x 107
Test Samples0 9.43 x 107
Laboratory4 and Procedural6 Blank 0
Mean Logs of
Observed CFU
6.31 ±0.21
0.67 ±1.51
0
Mean %
Recover
contamination
fficacv±CIf
2.38±1.15 £
0.00050 ±0.0011 5.63 ±1.34
0.5 M Klozur - Seven Days (4) - #4
Positive Controls 7.17 x 107 5.84 ± 0.09
Test Samples 7.17xl07 0.30 ±0.68
Laboratory and Procedural Blank 0 0
0.97 ±0.20
0.000010 ±0.000020 5.53 ±0.60
0.5 M Klozur - Seven Days (4) - #4 - Repeat
Positive Controls
Test Samples
7.00 xlO7
7.00 xlO7
5.73 ±0.09
0.00 ±0.00
0.96 ±0.80
0.00 ±0.00
>5.73±0.26
1 M Klozur - Seven Days (4) - #5
Positive Controls 8.47 x 107 6.04 ± 0.15
Test Samples 8.47 x 107 1.89 ±1.78
Laboratory and Procedural Blank 0 0
1.36 ±0.44
0.0019 ±0.0035 4.15 ±1.56
1 M Klozur - Three Days (4) - #6
Positive Controls 7.73 x 107 6.40 ± 0.25
Test Samples 7.73 x 107 0.00 ± 0.00
Laboratory and Procedural Blank 0 0
3.69 ±1.79
0.00 ±0.00
>6.40±0.22
1 M Klozur - Three Days (2) - #7
Positive Controls 8.07 x 107 6.36 ±0.19
Test Samples 8.07 x 107 3.61 ± 0.58
Laboratory and Procedural Blank 0 0
3.04±1.18
0.010±0.011
2.75 ±0.54
1 M Klozur - Three Days (3) - #8
Positive Controls 1.22 x 108 6.26 ± 0.26
Test Samples 1.22 x 108 2.73 ±1.88
Laboratory and Procedural Blank 0 0
1.72 ±0.97
0.0054 ±0.0049
3.53 ±1.66
1 M Klozur - One Day (4) - #9
Positive Controls 7.90 x 107 6.23 ± 0.13
Test Samples 7.90 x 107 1.71 ±1.59
Laboratory and Procedural Blank 0 0
2.24 ±0.73
0.00067 ±0.00079
4.52 ±1.39
1 M Klozur - One Day (1) - #10*
Positive Controls 9.57 x 107 6.04 ± 0.28
Test Samples 9.57 x 107 1.63 ±1.74
Laboratory and Procedural Blank 0 0
1.39 ±1.00
0.0024 ±0.0050 4.41 ±1.54
1 M Klozur - One Day (1) - #11*
Positive Controls 8.10 x 107 6.52 ± 0.23
Test Samples 8.10 x 107 2.84 ± 2.65
Laboratory and Procedural Blank 0 0
4.60 ±2.44
0.12±0.25
3.68 ±2.33
0.5 M Klozur - Three Days (4) - #12
Positive Controls 9.70 x 107 6.12 ± 0.28
Test Samples 9.70 x 107 1.09 ±1.50
Laboratory and Procedural Blank 0 0
1.62 ±1.27
0.00023 ±0.00032 5.02 ±1.34
a = Data are expressed as the mean (± SD) of the logs of the colony forming units (CFU) observed on five individual samples, the mean (± SD) percent
recovery on those five samples, and decontamination efficacy (log reduction).
b = Positive Controls = inoculated, not decontaminated samples.
c = Test Samples = inoculated, decontaminated samples.
d = Laboratory Blank = not inoculated, not decontaminated sample.
e = Procedural Blank = not inoculated, decontaminated sample.
f = CI = confidence interval (± 1.96 x SE).
g = "-" Not Applicable.
*One application (16 trigger pulls)
*One application (8 trigger pulls)
D-8
-------
Table D-8 Inactivation of B. subtilis Spores on Concrete with Klozur8
Persulfate
•a Sodium
Klozur® Concentration
Contact Time
(Number of Applications)
0.5 M Klozur - Seven Days (3) - #3
Positive Controls'5
Test Samples0
Laboratory4 and Procedural6 Blank
0.5 M Klozur - Seven Days (4) - #4
Positive Controls
Test Samples
Laboratory and Procedural Blank
0.5 M Klozur - Seven Days (4) - #4 -
Positive Controls
Test Samples
1 M Klozur - Seven Days (4) - #5
Positive Controls
Test Samples
Laboratory and Procedural Blank
1 M Klozur - Three Days (4) - #6
Positive Controls
Test Samples
Laboratory and Procedural Blank
1 M Klozur - Three Days (2) - #7
Positive Controls
Test Samples
Laboratory and Procedural Blank
1 M Klozur - Three Days (3) - #8
Positive Controls
Test Samples
Laboratory and Procedural Blank
1 M Klozur - One Day (4) - #9
Positive Controls
Test Samples
Laboratory and Procedural Blank
1 M Klozur - One Day (1) - #10*
Positive Controls
Test Samples
Laboratory and Procedural Blank
1 M Klozur - One Day (1) - #llt
Positive Controls
Test Samples
Laboratory and Procedural Blank
0.5 M Klozur - Three Days (4) - #12
Positive Controls
Test Samples
Laboratory and Procedural Blank
Inoculum
(CFU)
9.43 x 107
9.43 x 107
0
7.17 xlO7
7.17 xlO7
0
Repeat
7.00 xlO7
7.00 xlO7
8.47 xlO7
8.47 xlO7
0
7.73 x 107
7.73 x 107
0
8.07 xlO7
8.07 xlO7
0
1.22 xlO8
1.22 xlO8
0
7.90 xlO7
7.90 xlO7
0
9.57 xlO7
9.57 xlO7
0
8.10 xlO7
8.10 xlO7
0
9.70 xlO7
9.70 xlO7
0
a = Data are expressed as the mean (± SD) of the logs of the colony forminj
Mean Logs of
Observed CFU
6.37 ±0.21
5.09 ±0.37
0
5.90±0.18
4.66 ±0.31
0
5.85 ±0.36
3. 91 ±0.07
6.18±0.15
3.27 ±0.88 0
0
6.34 ±0.22
4.26 ±0.46
0
6.10 ±0.24
5.37±0.31
0
5.96±0.13
5.03 ±0.23
0
Mean %
Recovery
2.76 ±1.43
0.17±0.12
1.19±0.49
0.076 ±0.046
1.77 ±1.90
0.013 ±0.006
1.87 ±0.74
.0051 ±0.0042
3.17±1.63
0.033 ±0.023
1.79 ±1.23
0.34±0.19
0.78 ±0.21
0.10 ±0.044
6.63 ±0.31 6.56 ±4.59
3.72 ±0.24 0.0075 ±0.0038
0
6.29 ±0.19
5.52 ±0.32
0
6.46 ±0.17
5.71 ±0.47
0
6.51 ±0.27
4.39 ±0.49
0
I units (CFU) observed on
2.23 ±1.02
0.42 ±0.28
3.74±1.19
0.90 ±0.67
3.84±2.11
0.037 ±0.026
Decontamination
Efficacy ±CIf
g
1.29±0.38
1.25 ±0.32
1.93 ±0.57
2.91 ±0.78
2.08 ±0.45
0.73 ±0.34
0.94 ±0.23
2.91 ±0.34
0.78 ±0.33
0.74 ±0.44
2.11 ±0.49
five individual samples, the mean (± SD)
percent recovery on those five samples, and decontamination efficacy (log reduction).
b = Positive Controls = inoculated, not decontaminated samples.
c = Test Samples = inoculated, decontaminated samples.
D-9
-------
d = Laboratory Blank = not inoculated, not decontaminated sample.
e = Procedural Blank = not inoculated, decontaminated sample.
f = CI = confidence interval (± 1.96 x SE).
i = "." Not Applicable.
*One application (16 trigger pulls)
^One application (8 trigger pulls)
D-10
-------
Table D-9 Inactivation of B. subtilis Spores on Asphalt with Klozur®'a Sodium Persulfate
Klozur® Concentration
Contact Time
(Number of Applications)
Test#
0.5 M Klozur - Seven Days (3) - #3
Positive Controls'5
Test Samples0
Laboratory"1 and Procedural6 Blank
0.5 M Klozur - Seven Days (4) - #4
Positive Controls
Test Samples
Laboratory and Procedural Blank
1 M Klozur - Seven Days (4) - #5
Positive Controls
Test Samples
Laboratory and Procedural Blank
1 M Klozur - Three Days (4) - #6
Positive Controls
Test Samples
Laboratory and Procedural Blank
1 M Klozur - Three Days (2) - #7
Positive Controls
Test Samples
Laboratory and Procedural Blank
1 M Klozur - Three Days (3) - #8
Positive Controls
Test Samples
Laboratory and Procedural Blank
1 M Klozur - One Day (4) - #9
Positive Controls
Test Samples
Laboratory and Procedural Blank
1 M Klozur - One Day (1) - #10*
Positive Controls
Test Samples
Laboratory and Procedural Blank
1 M Klozur - One Day (1) - #llt
Positive Controls
Test Samples
Laboratory and Procedural Blank
0.5 M Klozur - Three Days (4) - #12
Positive Controls
Test Samples
Laboratory and Procedural Blank
Inoculum
(CFU)
9.43 x 107
9.43 x 107
0
7.17 xlO7
7.17 xlO7
0
8.47 xlO7
8.47 xlO7
0
7.73 x 107
7.73 x 107
0
8.07 xlO7
8.07 xlO7
0
1.22 xlO8
1.22 xlO8
0
7.90 xlO7
7.90 xlO7
0
9.57 xlO7
9.57 xlO7
0
8.10 xlO7
8.10 xlO7
0
9.70 xlO7
9.70 xlO7
0
" = Data are expressed as the mean (± SD) of the logs of the colony forminj
Mean Logs of
Observed CFU
7.09 ±0.06
1.58±1.01
0
6.93 ±0.16
2.05 ±1.62
0
6.80 ±0.11
1.68 ±1.09
0
7.20 ±0.21
2.66 ±1.31
0
7.19±0.23
3.71 ±0.96
0
6.43 ±0.25
3.07±1.14
0
7.27±0.18
0.52±1.16
0
7.26 ±0.17
1.79 ±1.78
0
7.17±0.18
2.82 ±1.73
0
7.22 ±0.17
2.93 ±1.68
0
Mean %
Recovery
13. 11 ±2.02
0.00016 ±0.00023
-
12.63 ±4.36
0.0057 ±0.012
-
7.55 ±1.75
0.00020 ±0.00029
-
22.54 ±12.83
0.012 ±0.026
-
21.47±11.19
0.046 ±0.094
-
2.49 ±1.35
0.0058 ±0.0091
-
25.75 ±13.26
0.0010 ±0.00023
-
20. 10 ±7.64
0.0015 ±0.0020
-
19.73 ±9.91
0.0099 ±0.017
-
17.96 ±6.94
0.0049 ±0.0041
-
Decontamination
Efficacy ±CIf
g
5.51 ±0.89
-
-
4.88 ±1.43
-
-
5.11 ±0.92
-
-
4.53 ±1.16
-
-
3.48 ±0.86
-
-
3. 36 ±1.02
-
-
6.75 ±1.03
-
-
5.47 ±1.56
-
-
4.35 ±1.52
-
-
4.28 ±1.48
-
I units (CFU) observed on five individual samples, the mean (± SD)
percent recovery on those five samples, and decontamination efficacy (log reduction).
= Positive Controls = inoculated, not decontaminated samples.
= Test Samples = inoculated, decontaminated samples.
= Laboratory Blank = not inoculated, not decontaminated sample.
= Procedural Blank = not inoculated, decontaminated sample.
= CI = confidence interval (± 1.96 x SE).
= "-" Not Applicable.
;One application (16 trigger pulls)
One application (8 trigger pulls)
D-ll
-------
Table D-10 Inactivation of B. subtilis Spores on Brick with Klozur®'a Sodium Persulfate
Klozur® Concentration
Contact Time
(Number of Applications)
0.5 M Klozur - Seven Days (3) - #3
Positive Controls'5
Test Samples0
Laboratory4 and Procedural6 Blank
0.5 M Klozur - Seven Days (4) - #4
Positive Controls
Test Samples
Laboratory and Procedural Blank
1 M Klozur - Seven Days (4) - #5
Positive Controls
Test Samples
Laboratory and Procedural Blank
1 M Klozur - Seven Days (4) - #5 - Repeat
Positive Controls
Test Samples
1 M Klozur - Seven Days (4) - #5 - Repeat
Positive Controls
Test Samples
1 M Klozur - Three Days (4) - #6
Positive Controls
Test Samples
Laboratory and Procedural Blank
1 M Klozur - Three Days (2) - #7
Positive Controls
Test Samples
Laboratory and Procedural Blank
1 M Klozur - Three Days (3) - #8
Positive Controls
Test Samples
Laboratory and Procedural Blank
1 M Klozur - One Day (4) - #9
Positive Controls
Test Samples
Laboratory and Procedural Blank
1 M Klozur - One Day (1) - #10*
Positive Controls
Test Samples
Laboratory and Procedural Blank
1 M Klozur - One Day (1) - #11*
Positive Controls
Test Samples
Laboratory and Procedural Blank
0.5 M Klozur - Three Days (4) - #12
Positive Controls
Test Samples
Laboratory and Procedural Blank
(CFU)
9.43 xlO7
9.43 xlO7
0
7.17xl07
7.17xl07
0
8.47 xlO7
8.47 xlO7
0
7.00 xlO7
7.00 xlO7
8.63 xlO7
8.63 xlO7
7.73 xlO7
7.73 xlO7
0
8.07 xlO7
8.07 xlO7
0
1.22 xlO8
1.22 xlO8
0
7.90 xlO7
7.90 xlO7
0
9.57 xlO7
9.57 xlO7
0
S.lOxlO7
S.lOxlO7
0
9.70 xlO7
9.70 xlO7
0
a = Data are expressed as the mean (± SD) of the logs of the colony forminj
Mean Logs of
Observed
CFU
6.81 ±0.18
1.88 ±1.07
0
6.64 ±0.10
0.00 ±0.00
0
5. 87 ±0.26
0.00 ±0.00
0
1.10±6..08
0.00 ±0.00
7.07 ±0.02
0.30 ±0.46
6.94±0.15
0.00 ±0.00
0
6.71 ±0.16
0.30 ±0.68
0
6. 13 ±0.44
0.00 ±0.00
0
6.99 ±0.08
0.00 ±0.00
0
7.23 ±0.09
0.40 ±0.89
0
6.83±0.18
0.00 ±0.00
0
7.00 ±0.05
0.62 ±1.38
0
££ '
7.26±3.18
0.00021 ±0.00015
6.19±1.55
0.00 ±0.00
0.99 ±0.51
0.00 ±0.00
0.09 ±0.21
0.00 ±0.00
14.16±4.89
0.0000086 ±0.000017
11. 89 ±4.04
0.00 ±0.00
6.62 ±2.13
0.0000092 ±0.00001 8
1.58±1.33
0.00 ±0.00
12.41 ±2.30
0.00 ±0.00
17.87 ±3.39
0.000022 ± 0.000046
8.96 ±3. 59
0.00 ±0.00
10.47 ±1.22
0.00025 ±0.00055
decontamination
Efficacy ±CIf
£
4.92 ±0.95
>6.64±0.09
>5.87±0.23
>1.10±2.16
1
6.76 ±0.61
>6.94±0.13
6. 40 ±0.61
>6.13±0.39
>6.99±0.07
6.83 ±0.79
>6.83±0.16
6.39±1.21
I units (CFU) observed on five individual samples, the mean (± SD)
percent recovery on those five samples, and decontamination efficacy (log reduction).
D-12
-------
b = Positive Controls = inoculated, not decontaminated samples.
c = Test Samples = inoculated, decontaminated samples.
d = Laboratory Blank = not inoculated, not decontaminated sample.
e = Procedural Blank = not inoculated, decontaminated sample.
f = CI = confidence interval (± 1.96 x SE).
s = "." Not Applicable.
*One application (16 trigger pulls)
TOne application (8 trigger pulls)
D-13
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Appendix E
Effects of Materials and Application on Sodium Persulfate Efficacy
Effect of Test Materials on Klozur® Sodium Persulfate Efficacy
Klozur sodium persulfate tests were conducted using 6 materials: Earthgro® and Agvise® Topsoil
for Tests 1 and 2 (completed as a confirmation of previous decontamination data with Klozur
sodium persulfate(2)); wood, concrete, asphalt, and brick for Tests 3 through 12. A summary of
the results in terms of LR of B. anthracis for Tests 3 through 12 are organized by operational
parameters (application, contact time, etc.) in Figure E-l
E-l
-------
Bare Pine Wood
Unpainted Concrete
3 Day 7 Day 7 Day 1 Day 1 Day 1 Day 3 Day 3 Day 3 Day 7 Day
(4) (3) (4) (l)t (1)§ (4) (2) (3) (4) (4)
0.5 M Klozur™ 1 M Klozur™
Contact Time (Number of Applications)
3 Day 7 Day
(4) (3)
7 Day
(4)
1 Day 1 Day 1 Day 3 Day 3 Day 3 Day 7 Day
(l)t (1)§ (4) (2) (3) (4) (4)
0.5 M Klozur™ 1M Klozur™
Contact Time (Number of Applications)
Asphalt
Brick
0.5 M Klozur111 1 M Klozur™
Contact Time (Number of Applications)
0.5 M Klozur™ 1M Klozur™
Contact Time (Number of Applications)
Figure E-l. Summary of Klozur® sodium persulfate efficacy against Bacillus anthracis on wood, concrete, asphalt, and brick.
Results shown in average log reduction ± CI
*Complete inactivation achieved
tKlozur® applied in 1 application (8 trigger pulls)
§Klozur® applied in 1 application (16 trigger pulls)
E-2
-------
United States
Environmental Protection
Agency
PRESORTED STANDARD
POSTAGE & FEES PAID
EPA
PERMIT NO. G-35
Office of Research and Development (8101R)
Washington, DC 20460
Official Business
Penalty for Private Use
$300
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