EPA/600/R-17/343 I September 2017
www.epa.gov/homeland-security-research
United States
Environmental Protection
Agency
Assessment of the Decontamination of Soil
Contaminated with Bacillus anthracis Spores
Using Chlorine Dioxide Gas, Methyl Bromide,
or Activated Sodium Persulfate
.t.
It!
1
I I
Office of Research and Development
Homeland Security Research Program
-------
This page left intentionally blank
-------
Disclaimer
The U.S. Environmental Protection Agency (EPA), through its Office of Research and
Development's (ORD's) National Homeland Security Research Center (NHSRC), funded,
directed, and managed this work through Contract Number EP-C-15-003, Task Order 005, with
MRIGlobal. This report has been peer and administratively reviewed and has been approved for
publication as an EPA document. The views expressed in this report are those of the authors and
do not necessarily reflect the views or policies of the Agency. Mention of trade names or
commercial products does not constitute endorsement or recommendation for use of a specific
product.
Questions concerning this document or its application should be addressed to:
Joseph 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
-------
Acknowledgements
Contributions of the following individuals and organizations to this report are gratefully
acknowledged.
United States Environmental Protection Agency (EPA)
Joseph Wood, Principal Investigator; National Homeland Security Research Center
(NHSRC)
Leroy Mickelsen and Shannon Serre; Office of Land and Emergency Management
(OLEM)
Worth Calfee, Lukas Oudejans, and Erin Silvestri; NHSRC
Peer reviewers
John Archer; NHSRC
Elise Jakabhazy; OLEM
MRIGlobal
-------
Executive Summary
The U.S. Environmental Protection Agency (EPA) Office of Research and Development is
striving to protect human health and the environment from adverse impacts resulting from
environmental contamination (such as from acts of terror) by investigating the effectiveness and
applicability of technologies for homeland security (HS)-related applications. The purpose of this
investigation was to determine the efficacy of three chemical decontaminants for inactivating the
causative agent for anthrax, Bacillus anthracis spores, in soil. The decontaminants that were
evaluated included chlorine dioxide gas, a liquid biocide (sodium persulfate activated with
aqueous hydrogen peroxide), and methyl bromide. The objective of this study was to provide an
understanding of the performance (i.e., efficacy) of these decontamination technologies under a
range of environmental conditions, thus guiding their use and implementation in HS applications
for hard-to-decontaminate materials such as soil.
This investigation focused on decontaminating three types of soil materials - topsoil, sand, and a
clayey soil - at soil depths of up to 5 inches. The three soil types were selected in an attempt to
span the range of soil types (and associated properties, such as organic content, porosity, particle
size). Decontamination efficacy tests were conducted using B. anthracis (Ames strain) spores.
Decontamination efficacy was quantified in terms of log reduction (LR), based on the difference
in the number of bacterial spores (as CFU) recovered from the positive controls (soil samples not
exposed to decontaminant) and test samples. (A decontaminant is considered to be an effective
sporicide if a 6 LR or greater is achieved based upon appropriate laboratory testing.) Tests were
conducted with varying operational parameters (e.g., environmental conditions, contact time,
decontaminant concentration) to assess the effect of these parameters on decontamination
efficacy at each of the soil depths.
Method Development
Prior to conducting the main decontamination studies just described, method development was
required to determine techniques for preparing, placing, and quantitatively recovering B.
anthracis-spiked soil samples from within a larger soil mass. In the end, a 1-gram soil mass was
spiked with 0.1 mL of B. anthracis stock and was then sealed within custom-made Tyvek or
polyvinylidene fluoride envelopes. This packet was referred to as a carrier soil packet, or CSP.
To conduct an experiment, CSPs could then be placed at various depths within a larger soil
column of the same soil type without contaminating the entire mass, were easily recovered as
individual samples, and provided a quantitative amount of B. anthracis spores at each location.
Design of experiments using test columns was also evaluated prior to conducting the main
decontamination studies. Small laboratory-sized test columns typically suffer from wall effects
(e.g., channeling), making it difficult to simulate large outdoor soil characteristics accurately. To
address this problem, we performed a series of method development tests using 4-, 6-, 8-, and
10-in diameter columns, commercially-available biological indicators (Bis) impregnated with
Bacillus atrophaeus spores, and chlorine dioxide (CIO2) gas or sodium persulfate liquid solution.
This approach provided a cost-effective way to evaluate the expected column performance for
treating B. anthracis with either gas or liquid decontaminants. Results showed that a 10-in
diameter column was the preferred design, and that no wall effects were anticipated when Bis or
CSPs were placed along the central axis of the column, down to five inches. Successful
iii
-------
conclusion of the method development phase allowed the project to then proceed to the main
decontamination studies using the 10-inch diameter test columns with the three decontaminants.
Summary of Decontamination Test Results
Chlorine Dioxide
A total of seven ambient-temperature tests were performed using gaseous CIO2 at target
concentrations of 8.4 to 14 mg/L (3,000 to 5,000 ppm), exposure times of 3 to 27.5 hours, and
relative humidity (RH) levels of > 75%. In general, CIO2 was ineffective at decontaminating
topsoil at depths greater than 1". For depths up to 1", results ranged from ineffective to complete
kill, depending on the exact test conditions. The most aggressive test was performed at an
average CIO2 concentration of 14.6 mg/L, 82.5% RH, and a 27.5-hr exposure. These conditions
produced complete kill (> 7 LR) in topsoil at the 1" depth, but was ineffective at greater depths.
Sand was more easily treated by CIO2 than the topsoil, and showed full decontamination down to
2" for most of the test conditions. Furthermore, two of the three 24-hr tests showed complete kill
(>7 LR) at all depths.
Clay was also more easily treated by CIChthan topsoil, and had full decontamination down to 3"
for most of the test conditions. Two of the three 24-hr tests (-3,000 ppm) showed complete kill
(>7 LR) at all depths.
Data from one of the 24-hr tests showed no recovery from any of the clay test samples or
positive controls, prompting additional tests that attempted to evaluate the decay of positive
controls over a 1-week time period. Neither of these two additional tests showed complete decay
in the clay material as had been observed in the previous test.
One test was performed using compacted soils for all three types. This condition showed that
CIO2 was unable to penetrate compacted topsoil to a 1" depth, but did not show a noticeable
effect on either sand or clay.
Sodium Persulfate
Three tests were performed using sodium persulfate (tradename Klozur™ SP) at concentrations
of 0.5 to 1.0 M, activated immediately prior to use by mixing 50/50 (volume basis) with
8%> aqueous hydrogen peroxide. The decontamination liquid was applied to the top of the test
columns in up to six separate applications at either one or two day intervals. Total liquid volumes
varied by experiment and soil type, with a maximum up to 0.54 mL per gram of soil based on a
wetting depth of 6". Contact times were six to seven days, and environmental conditions were
ambient temperature and RH.
The activated sodium persulfate decontamination solution proved to be extremely reactive with
topsoil, and produced a vigorous foaming reaction upon application. Actual decontamination
efficacy of topsoil, however, was poor, and was effective only down to 0.5" for the 0.5M
solutions and to 1" for the 1.0 M solution. Sand showed less reactivity and formed more of a
slurry with the liquid decontamination solution. Decontamination results were also poor for sand,
and was effective to only a 1" depth. Clay, on the other hand, showed complete decontamination
iv
-------
down to 5" with only two applications of activated sodium persulfate, applied at a loading of
0.09 mL/g.
Methyl Bromide
A total of four tests were performed using gaseous methyl bromide (MeBr) at concentrations
ranging from 224 to 325 mg/L (56,000 to 81,250 ppm), exposure times of 48 to 65.5 hrs, ambient
temperature of 20 °C, and RH levels of > 75%. Test 1, conducted at 236 mg/1 MeBr for 48 hr,
was not effective for any soil type, typically having a 1-2 LR. Maintaining similar test conditions
except for increasing contact time (Test 2) or increasing concentration (Test 3) resulted in
generally moderate improvement in efficacy. The pre-wetting of soils (Test 4) did not provide
any improvement in MeBr decontamination efficacy. Efficacy was observed to be generally
higher for topsoil and sand, and lowest for the clay soil; in fact, there were no test conditions in
which any of the clay samples were decontaminated effectively, including the CSPs at the
surface of the clay columns. Lastly, although there were a few exceptions, decontamination
efficacy was generally similar across all depths for a particular soil and test condition, suggesting
that penetration of the MeBr gas through the soil matrices was not a limiting factor.
v
-------
Contents
Executive Summary iii
Acronyms and Abbreviations ix
Section 1. Introduction 1
Section 2. Technology Descriptions and Test Matrices 3
2.1 Technology Descriptions 3
2.2 Test Matrix 4
2.3 Test Matrices for Chlorine Dioxide Decontamination 4
2.4 Test Matrices for Sodium Persulfate Decontamination 5
2.5 Test Matrices for Methyl Bromide Decontamination 5
2.6 Test Matrices for Clay Positive Controls Decay Tests 6
Section 3. Summary of Test Procedures 7
3.1 Biological Agent 7
3.2 Soils 8
3.3 Carrier Soil Packet (CSP) Development and Preparation 10
3.4 Test Column Preparation 10
3.5 Sample Extraction and Biological Agent Quantification 11
3.6 Decontamination Efficacy 11
3.7 Biological Indicator (BI) Handling and Analysis 12
Section 4. Decontamination Procedures 13
4.1 Decontaminant Preparation 13
4.2 Test and Control Chambers and Procedures 14
Section 5. Quality Assurance and Quality Control 17
5.1 Equipment Calibration 17
5.2 Quality Control Results 17
5.3 Audits 19
5.4 Test/Quality Assurance Plan Deviations 20
5.5 QA/QC Reporting 21
5.6 Data Review 22
Section 6. Results and Performance Summary for Chlorine Dioxide 23
6.1 Chlorine Dioxide Test Conditions 23
6.2 Chlorine Dioxide Decontamination Results 25
6.3 Chlorine Dioxide Log Reduction Charts 34
Section 7. Results and Performance Summary for Sodium Persulfate 37
7.1 Sodium Persulfate Test Conditions 37
7.2 Sodium Persulfate Decontamination Results 40
7.3 Sodium Persulfate Log Reduction Charts 44
Section 8. Results and Performance Summary for Methyl Bromide 46
8.1 Methyl Bromide Test Conditions 46
8.2 Methyl Bromide Decontamination Results 50
8.3 Methyl Bromide Log Reduction Charts 55
vi
-------
Section 9. Clay Positive Controls Decay Tests 57
9.1 Clay Positive Controls Decay Test Conditions 57
9.2 Clay Positive Controls Decay Test Results 57
9.3 Clay Positive Controls Decay Test Log Reduction Charts 60
Section 10. Summary of Results and Conclusions 61
Section 11 .References 63
Appendix A. Bacillus Anthracis Source Information and Plasmids Analysis A-l
Appendix B. Soil Properties B-l
Appendix C. CSP Development C-l
Appendix D. Preliminary Tests D-l
Appendix E. ORP Measurements E-1
Figures
Figure 1. Soil Textural Triangle 8
Figure 2. Test Chamber 13
Figure 3. Test 1, CIO:: 8.7 mg/L, 3 hrs, 77% RI1 33
Figure 4. Test 2, CIO:: 10.3 mg/L, 6 hrs, 80% RI I 33
Figure 5. Test 3, CIO2: 8.9 mg/L, 3 hrs, 74% RH, [saturated soil] 34
Figure 6. l est 4. CIO:: 10.1 mg/L, 24 hrs, 81% RH 34
Figure 7. Test 5, CIO2: 9.3 mg/L, 24 hrs, 80% RH, [saturated soil] 35
Figure 8. Test 6, CIO:: 14.6 mg/L, 24 hrs, 83% RH 35
Figure 9. Test 7, CIO2: 9.4 mg/L, 7.75 hrs, 86% RH, [compacted soil] 35
Figure 10. Test 1, SP: 0.5 M, 0.16-0.18 mL/g, 2 applications 43
Figure 11. Test 2, SP: 0.5 M, 0.09 mL/g, 6 applications (topsoil, sand), 2 applications (clay)....43
Figure 12. Test 3, SP: 1.0 M, 0.09 mL/g, 6 applications (topsoil, sand), 1 application (clay) 44
Figure 13. BA Morphology Changes after Exposure to MeBr (left) vs Unexposed (right) 47
Figure 14. Test 1, MeBr: 236 mg/L, 48 hrs, 78% RH 53
Figure 15. Test 2, MeBr: 224 mg/L, 65.5 hrs, 77% RH 53
Figure 16. Test 3, MeBr: 325 mg/L, 48 hrs, 76% RH 54
Figure 17. Test 4, MeBr: 230 mg/L, 48 hrs, 79% RH, saturated soil 54
Figure 18. Test 4a: Clay Positive Control Decay Test, 30% RH 58
Figure 19. Test 7a: Clay Positive Control Decay Test, 92% RH 58
vii
-------
..2
...3
...3
...4
...4
...5
...7
16
.18
19
20
22
.26
.27
.28
.29
.30
.31
.32
.37
.40
.41
.42
.46
49
50
.51
52
55
.56
.57
Decontamination Technology Description
Overall Test Matrix for Decontamination of B. anthracis in Soil
Chlorine Dioxide Test Design
Sodium Persulfate Test Design
Methyl Bromide Test Design
Clay Positive Controls Decay Test Design
Soil Suppliers and Primary Physical Properties
Sample Performance Criteria
Quality Control Results Summary
Performance Evaluation Audits
Comparison of Unburied and Buried Positive Controls
Chlorine Dioxide Test Conditions
Test 1, CIO2 Decontamination Results
Test 2, CIO2 Decontamination Results
Test 3, CIO2 Decontamination Results
Test 4, CIO2 Decontamination Results
Test 5, CIO2 Decontamination Results
Test 6, CIO2 Decontamination Results
Test 7, CIO2 Decontamination Results
Sodium Persulfate Test Conditions
Test 1, SP Decontamination Results
Test 2, SP Decontamination Results
Test 3, SP Decontamination Results
Methyl Bromide Test Conditions
Test 1, MeBr Decontamination Results
Test 2, MeBr Decontamination Results
Test 3, MeBr Decontamination Results
Test 4, MeBr Decontamination Results
Clay Positive Controls Decay Test Conditions
Test 4a, Clay Positive Controls Decay Results
Test 7a, Clay Positive Controls Decay Results
viii
-------
Acronyms and Abbreviations
BA
Bacillus anthracis, or B. anthracis
BI
Biological Indicator
BSC
Biological Safety Cabinet
BSL
Biological Safety Level
CC
Control Column
CFU
Colony Forming Units
COA
Certificate of Analysis
CSP
Carrier Soil Packet
EPA
U.S. Environmental Protection Agency
HS
Homeland Security
HSRP
Homeland Security Research Program
LED
Light Emitting Diode
LR
Log Reduction
MeBr
Methyl bromide
NCCC
Negative Control Control Column
NCTC
Negative Control Test Column
NHSRC
National Homeland Security Research Center
ORP
Oxidation Reduction Potential
PBS
Phosphate-Buffered Saline
PC
Polycarbonate or Positive Control
PCR
Polymerase Chain Reaction
PVC
Polyvinyl chloride
PVDF
Polyvinylidene Fluoride
QA
Quality Assurance
QAPP
Quality Assurance Project Plan
QC
Quality Control
QMP
Quality Management Plan
RH
Relative Humidity
SA
Select Agent
SBA
Sheep Blood Agar
TC
Test Column
-------
Section 1.
Introduction
The U.S. Environmental Protection Agency's (EPA's) Homeland Security Research Program
(HSRP) 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 HSRP is working to develop tools and information that will help
detect the intentional introduction of chemical or biological contaminants in buildings, water
systems, or the outdoor environment; contain these contaminants; decontaminate buildings,
water systems or the outdoor environment; and facilitate the treatment and disposal of materials
resulting from remediation activities.
As part of the above effort, EPA investigates the effectiveness and applicability of technologies
for homeland security (HS)-related applications by developing test plans that are responsive to
the needs of the HSRP's EPA Program Office partners, conducting tests, collecting and
analyzing data, and preparing peer-reviewed reports. All evaluations are conducted in
accordance with rigorous quality assurance (QA) protocols to ensure that data of known and high
quality are generated and that the results are defensible. EPA provides high-quality information
that is useful to decision makers in purchasing or applying the tested technologies.
The purpose of this study was to evaluate technologies for decontaminating soil contaminated
with Bacillus anthracis (Ames) spores. (Note, in the rest of this report, we use the abbreviation
"BA" to refer to spores of the B. anthracis Ames strain.) Decontamination of soil may be
needed in the event of a wide area release of BA. Following such a contamination incident,
spores may migrate below the soil surface by various transport mechanisms such as rainfall.
However, there remains some uncertainty with regard to the depths in which spores may
penetrate soil, and this may depend on a number of factors. For this reason, decontamination
efficacy was evaluated as a function of soil depth in this study.
The decontaminants evaluated were chlorine dioxide (CIO2) gas, activated sodium persulfate
(SP), and methyl bromide (MeBr). This work built on previous laboratory studies involving soil
decontamination [1-4], but was performed at a significantly larger scale. In contrast to prior
studies, this study also investigated the effect of several operating parameters on
decontamination efficacy, with the primary objective of finding the required conditions (e.g.,
concentration, contact time, mass quantity) needed for effective decontamination of soil as a
function of parameters such as soil depth, soil type, moisture, and chemical composition (e.g.,
organic content, levels of selected cations).
Prior to embarking on the testing just described, however, an intensive series of method
development tests was performed to address two primary concerns. First, a means of preparing,
placing, and quantitatively recovering BA-spiked soil samples from within a larger soil mass was
required, leading to development of the carrier soil packet (CSP). Second, a soil test column
design was needed to simulate a large, outdoor soil mass while avoiding wall effects
(e.g. channeling) that are typical with small lab oratory-scale columns. The main body of this
report, however, is focused on the actual decontamination test results. Details regarding the CSP
development and wall-effects tests for the columns are found in the appendices.
1
-------
The project was conducted at MRIGlobal's facilities in Kansas City (425 Volker, Kansas City,
MO) and in Florida (1470 Treeland Blvd, S.E., Palm Bay, FL). Preliminary method development
tests, such as the extraction and recovery of Bacillus atrophaeus (B. atrophaeus) biological
indicators (Bis) from soil, were performed at the Florida location. Wall-effects tests using Bis,
test chamber construction, and all work with Biosafety Level (BSL)-3 Select Agents (SAs),
including spore preparation and testing with BA, were performed at the Kansas City location.
2
-------
Section 2.
Technology Descriptions and Test Matrices
2.1 Technology Descriptions
Table 1 describes the three decontamination technologies evaluated in this study. Information is
provided on the manufacturer, product name (where applicable), chemical components, and
active ingredients. Further details on the chemical composition, preparation, and
decontamination application procedures are provided in Section 4.
Table 1. Decontamination Technology Description
Decontaminant
Product Name and
Vendor
Active Ingredients
Components
EPA
Pesticide
Registration
C102
Minidox-M CIO2
Generator;
ClorDiSys Solutions, Inc.,
Lebanon, NJ
Chlorine dioxide
gas
Chlorine dioxide gas
(CIO2); programmable
from 0 to 30 mg/L.
80802-1
Sodium
Persulfate
Klozur® SP
PeroxyChem
Philadelphia, PA
SP, activated with
8% hydrogen
peroxide (H2O2)
SP (Na2S208) >99%
purity, used as either a
0.5 M or 1.0 M aqueous
solution, activated with
8% hydrogen peroxide.
None
MeBr
Meth-O-Gas® 100
Commodity Fumigant;
Cardinal Professional
Products;
Anaheim, CA
Methyl bromide
100% methyl bromide
5785-11 for
pests
associated
with
agricultural
commodities
Chlorine dioxide was selected for testing because an earlier screening study had shown good
efficacy for inactivating BA in Arizona Test Dust [1], This earlier study had also evaluated the
performance with topsoil under limited laboratory conditions, so this was explored further across
a wider range of test conditions. Sodium persulfate was included in this evaluation because it is
used to remediate soil contaminated with organic chemicals. In addition, SP was shown to be
effective in certain conditions against BA in soil in screening tests [2, 3] and on other outdoor
materials [4], so more detailed testing was planned for this evaluation. Methyl bromide was
selected for testing because it has been demonstrated to be effective against BA on building
materials [5], and was shown to be effective against BA in small-scale soil tests [3],
Furthermore, although MeBr use is being phased out under the Montreal Protocol on Substances
that Deplete the Ozone Layer, MeBr is still currently and widely used via critical use exemptions
as a soil and commodity (quarantine) fumigant [6],
3
-------
2.2 Test Matrix
A total of fourteen decontamination efficacy tests were performed, with an additional two tests
performed to evaluate the decay of positive controls in clay. All of the tests were performed in a
custom-built test chamber using BA at soil depths from zero (on top of surface) up to five inches
within a 10-in diameter column. The exception to this procedure was for the clay positive
controls decay tests, which did not require a soil column. Each soil column had six CSP samples
of the appropriate soil type, with one set on the soil surface (0 inch depth) and the others buried
at depths up to five inches. Five positive CSP controls of each soil type were set up in a separate
Control Chamber and not exposed to the decontaminant. One negative control of each soil type
was also set up in both the Test Chamber and the Control Chamber. The general test conditions
are summarized in Table 2. Initial test conditions (e.g., concentration, relative humidity [RH],
contact time) were selected based on previous test conditions shown to be effective for each
decontaminant.
Table 2. Overall Test Matrix for Decontamination of 6. anthracis in Soil
Type of Test
No. of
Tests
Soil
Types
Decontaminant
Concentration
Exposure
Time
(hrs)
Temperature/RH
Samples3
Chlorine Dioxide
7
Topsoil
Sand
Clay
8.7 -14.6 mg/L
3-27.5
22.2 - 25.3°C
73.5 - 85.8 % RH
TC = 6
CC = 5
NCTC = 1
NCCC = 1
Sodium Persulfate
3
Topsoil
Sand
Clay
0.5-1.0 M
50/50 W/H2O2
0.09-0.18 mL/g
1 - 6 doses
144 -168
(6-7 days)
21.5 - 22.6°C
91.3-92.9 %RH
TC = 6
CC = 5
NCTC = 1
NCCC = 1
Methyl Bromide
4
Topsoil
Sand
Clay
224 - 325 mg/L
48-65.5
19.7-20.1oC
76.3 - 78.7 % RH
TC = 6
CC = 5
NCTC = 1
NCCC = 1
Positive Controls
Decay
2
Clay
NA
0-168 hrs
(7 days)
23.0 - 24.6°C
29.5 - 92.0 % RH
CC = 3b
a Per soil type.
b Per time period.
TC = Test Column.
CC = Positive Control Column.
NCTC - Negative Control, Test Column.
NCCC - Negative Control, Control Column.
2.3 Test Matrices for Chlorine Dioxide Decontamination
The test matrix for CIO2 is presented in Table 3, and shows the nominal target values for each of
the test parameters. Actual measured values are presented later in the results section for CIO2.
Initial target test conditions for Test 1 were 8.4 mg/L (-3000 ppm, with 1 ppm = 2.81 mg/m3, or
0.00281 mg/L for CIO2) and > 75% RH for three hrs. Conditions for each of the subsequent tests
were determined based on results of previous tests. Fumigation conditions for each test were the
same for each soil type.
4
-------
Table 3. Chlorine Dioxide Test Design
Test
No.
Depth Increments
Tested (inches)
Target CIO2
Cone. (mg/L)
Target T
(°C)
Target RH
(%)
Target
Contact Time
(hrs)
Other
Conditions
1
1
8.4
>21
>75
3
2
1
8.4
>21
>75
6
3
0.5
8.4
>21
>75
3
Saturated soil.
4
1
8.4
>21
>75
24
5
1
8.4
>21
>75
24
Saturated soil.
6
1
14.0
>21
>75
24
7
1
8.4
>21
>80
6
Compacted soil.
2.4 Test Matrices for Sodium Persulfate Decontamination
The general approach for testing SP was similar to that of CIO2, and again used three (3) soil
types simultaneously in a 10" diameter column, inside the Test Chamber. Negative Controls and
Positive Controls were identical to the CIO2 testing.
The test matrix for SP is presented in Table 4, and shows the target values for each of the test
parameters. Note that for Tests 2 and 3, decontaminant conditions varied by soil type.
Table 4. Sodium Persulfate Test Design
Test
No.
Depth(s)
Tested
Target SP
Cone.
(mg/L)
Target
T
(°C)
Target
RH
(%)
Target
Contact
Time
(days)
Target Application Conditions
Soil
Quantity
No. of
Doses
Interval
1
1 -in depths
0.5 M,
50/50 with
8% H2O2
Ambient
T
Ambient
RH
7
All
types
0.18 mL/g
2
48-hr
2
0.5-in depths
(topsoil)
1 -in depths
(sand, clay)
0.5 M,
50/50 with
8% H2O2
Ambient
T
Ambient
RH
7
Topsoil
Sand
Clay
0.09 mL/g
0.09 mL/g
0.09 mL/g
CD CD CM
24-hr
24-hr
24-hr
3
0.5-in depths
(topsoil)
1 -in depths
(sand, clay)
1.0 M,
50/50 with
8% H2O2
Ambient
T
Ambient
RH
7
Topsoil
Sand
Clay
0.09 mL/g
0.09 mL/g
0.09 mL/g
6
6
1
24-hr
24-hr
NA
2.5 Test Matrices for Methyl Bromide Decontamination
The test design for MeBr is presented in Table 5, and shows the nominal target values for each of
the test parameters. Actual measured values are presented later in the results section for MeBr.
Initial target test conditions for Test 1 were 212 mg/L (-53,000 ppm, where 1 oz/1000 ft3 ~ 1
mg/L ~ 250 ppm for MeBr) and > 75% RH for 48 hrs. Conditions for each of the subsequent
tests were determined based on results of previous tests. MeBr fumigation conditions for each
test were the same for each soil type.
5
-------
Table 5. Methyl Bromide Test Design
Test
No.
Depth(s) Tested
Target MeBr
Cone. (mg/L)
Target T
(°C)
Target RH
(%)
Target
Contact Time
(hrs)
Other
Conditions
1
1 -in depths
212
>21
>75
48
2
1 -in depths
212
>21
>75
72 (48)a
3
1 -in depths
300
>21
>75
48
4
1 -in depths
212
>21
>75
48
Saturated soil.
a One set of 0" depth samples removed at 48-hrs. All other samples to be removed at 72 hrs.
2.6 Test Matrices for Clay Positive Controls Decay Tests
When no viable spores were recovered from the clay positive controls during the first 24-hour
CIChtest (Test 4, CIO2), we thought this may have been due to interaction between clay and the
spores. In addition, inadvertently, the temperature and RH for the positive controls was higher
than typical (37 °C and 94% RH). In an effort to quantify this potential interaction over a 168-hr
time period, a pair (ambient RH and high RH) of Clay Positive Controls Decay tests were
conducted. These tests used three samples per time point, making a total of 18 spiked samples
overall. One non-soil extraction control was also performed (to assess inoculation level). Refer
to Table 6.
Table 6. Clay Positive Controls Decay Test Design
Test
No.
Depth(s) Tested
Target CIO2
Cone. (mg/L)
Target T
(°C)
Target RH
(%)
Target
Contact Time
(hrs)
Other
Conditions
4a
0 (Clay Positive
Controls Decay)
None
Ambient T
Ambient RH
0, 24, 48, 72,
96, 168
Control Chamber
only, for testing
decay of clay
positive controls.
3 samples each
time period.
7a
0 (Clay Positive
Controls Decay
None
Ambient T
High RH
0, 24, 48, 72,
96, 168
Control Chamber
only, for testing
decay of clay
positive controls.
3 samples each
time period.
6
-------
Section 3.
Summary of Test Procedures
Test procedures were performed according to the approved Quality Assurance Project Plan
(QAPP) and are summarized below.
This project was designed to have two distinct phases. First, a set of preparations and soil column
design tests was performed to determine the best technical approach for performing the actual
decontamination tests. Once the preliminary tests were performed, the actual decontaminant tests
were executed. The main experimental variables for the decontaminant tests included the
decontaminant type (i.e., SP, CIO2, or MeBr), soil type, and the specific physical parameters for
each test (depth of soil, decontaminant concentration and/or quantity, contact time, and RH).
3.1 Biological Agent
B. anthracis (BA) Ames spores were prepared at the Kansas City location using the A0462 strain
from the MRIGlobal Repository (originally from catalog number NR-411 from Biodefense and
Emerging Infections Research Resources Repository, www.beiresources.org). The BA was
prepared according to MRIGlobal procedures, including growth on liquid or solid sporulation
media, microscopic examination to verify spore production, and a heat shock of the preparation
(65°C for 30 minutes in a stationary water bath) to kill any remaining vegetative B. anthracis
Ames cells. Spores were resuspended in 10% glycerol. The concentration of viable spores in the
preparation was determined by viable plate counting methods using sheep blood agar (SBA).
An aliquot of the spore preparation was serially diluted with 100 |iL aliquots plated onto each of
three SBA plates. Plates were incubated for approximately 16-20 hours at 35°C. After
incubation, colony forming units (CFU) were counted to determine spore concentration. Spore
preparation volumes were adjusted to achieve at least 1 x 109 CFU/mL. BA spore preparations
were mixed thoroughly and then aliquoted into 1-mL cryovials and stored at -80°C until use. All
work with BA was conducted within a Biosafety Cabinet (BSC) under BSL-3 containment.
Project requirements stated that the efficacy of the three selected decontaminants would be
evaluated against fully virulent BA. To verify that the Ames stock designated for use on this
project contains pXOl and pX02 virulence plasmids, polymerase chain reaction (PCR) analysis
was performed on the spore preparation using MRIGlobal in-house assays BA2 (targeting capB
on pX02) and BA3 (targeting LF on pXOl).
Appendix A contains a product information sheet for the Ames strain used in this study (A0462,
BEI Resources catalog number NR-411). Appendix A also contains PCR reports for detecting
the virulence plasmids pXOl and pX02 for the determination that was performed two times
during the program. An initial plasmids analysis was performed in February 2016 prior to any
BSL-3 work. A reanalysis was performed in June 2017 during the test series with MeBr. The
presence of pXOl and pX02 plasmids was confirmed in both cases, indicating virulence of the B.
anthracis strain. Assays BA2 and BA3 were used to evaluate serial dilutions of DNA prepared
from the spore preparation. Quantification data indicate detection of both plasmids at the highest
DNA dilution tested. Positive controls for both assays were positive; negative controls for both
assays were negative.
7
-------
3.2 Soils
Three soil types were used for this study: a topsoil, a sandy soil, and a clayey soil. These soils
were purchased from commercial sources as outlined in Table 7. Analyses for moisture and
density were performed at MRIGlobal. All other physical property tests were performed by
Agvise Laboratories (North Dakota). A complete summary of physical properties of the soils is
found in Appendix B.
Soils were purchased in 50-lb bags from a local supplier, and variations could not be avoided due
to the large amounts of soil required for this project. To minimize changes in moisture content
after purchase, soils were stored in a sealed plastic trash bag within a sealed plastic tub.
However, as shown in Appendix B, soil moisture did diminish over time as the study proceeded.
Table 7. Soil Suppliers and Primary Physical Properties
Soil
Type
Lot, Batch, or
Other Description
Manufacturer/
Supplier Name
Density
Range
(g/mL)a
Moisture
Range
(%)b
Organic
Matter
(%)c
Sand/Silt/Clay
(%)d
Topsoil
(loamy)
Timberline
(Oldcastle)
Oldcastle Lawn &
Garden (Home
Depot, local supply)
0.97-1.02
19.6-41.9
5.2
42/33/25
Sand
Play Sand
(Pavestone)
Pavestone (Home
Depot, local supply)
1.14-1.49
CO
0
1
CO
4^
0.0
100/0/0
Clay
Crimson Clay
(Better Baseball)
Better Baseball
(online supply)
1.06-1.28
10.7-28.4
0.2
44/11/45
a Bulk density of undried, uncompacted soil as measured by weight in a beaker.
b Measured by ASTM D2974-87.
c By Walkley-Black method [7],
d Hydrometer method.
Soil density was measured prior to each test by weighing a 200-mL volume of each soil type in a
beaker. Soil moisture was measured according to ASTM D 2974-87 prior to each test. Samples
were weighed, dried in an oven for >16 hrs at 105 ± 5 °C, and weighed again, with the moisture
calculated as:
Moisture Content (%) = [(A-B) x 100]/A
A = mass of the as-received sample, g
B = mass of the oven-dried sample, g
Sand/Silt/Clay moisture content was measured by Agvise Laboratories using the hydrometer
method. The soil textural triangle below in Figure 1 [8] shows results for the three types of soils
tested. The topsoil sample fell into the "loam" category.
8
-------
100
80
SILTY
CLAY
40 / SANm
A CLAY
SILTY CLAY
LOAM
CLAY LOAM
SANDY CLAY
LOAM
20
80
LOAM / SILT LOAM
SANDY LOAM
go
SILT
' \ LOAMY
iANDV SAND
100
Percent Sand
Figure 1. Soil Textural Triangle [8]
Detailed physical characteristics of the soils were obtained from a Series II analysis through
Agvise Laboratories. Full results are contained in Appendix B. A small amount (-0.5 lb, or
-250 g) of each soil type was shipped to the analytical lab in a sealed plastic bag. Series II
analysis consists of the following parameters, as per standard methods on file with Agvise
Laboratories.
• pH (electrode)
• % organic matter (Walkley-Black procedure)
• Cation Exchange Capacity
• Ca, Mg, Na, K, and H (in conjunction with the above Cation Exchange Capacity)
• % moisture (gravimetric loss upon drying)
• % moisture at 1/3 bar (water holding capacity)
9
-------
• % sand, silt, and clay (hydrometer method)
• USDA Textural Class (profiled according to USDA/NRCS System)
• Bulk Density (gm/cm3), (gravimetric weight)
Test soils for general column filling were not sterilized prior to use, and were used "as-is" from
the suppliers. The smaller amounts of soil used for creating carrier soil packets spiked with BA
Ames, however, were sterilized prior to use. Sterilization was performed by autoclaving at 121
°C for two hours.
As a final note, we acknowledge that clayey soils left undisturbed in the environment may
become denser and less permeable to fluids due to their platelet/particle orientation. However,
the procedures we used in the laboratory for the handling of soils and packing into test columns
did not allow for us to use undisturbed clay soils. Thus most likely properties such as density and
transmissivity of the clayey soils used in testing in this study may not be representative of actual
field conditions. Additional tests are recommended to assess this issue.
3.3 Carrier Soil Packet (CSP) Development and Preparation
Carrier Soil Packets (CSPs) were created to provide primary containment of the BA Ames
spores. CSPs also provided a convenient method for spiked soils to be placed at various depths
within a soil column, to be easily recovered post-test, and allowed repeatable sample recovery
and extraction procedures to be used.
The outer "pillow" material of each CSP was made from either Tyvek or polyvinylidine fluoride
(PVDF), depending on the type of test. The initial plan was to use Tyvek (Mesa Labs, medical
grade 1073B, 0.22-|im) throughout the study. Preliminary screening studies, however, showed
that the liquid decontaminant (SP) was not able to penetrate Tyvek as quickly as expected.
Further method development showed that PVDF filter media (EMD Millipore, Billerica, MA;
0.22-|im, hydrophilic) provided the necessary performance for use with SP. Consequently, CSPs
for testing the gas-phase decontaminants (CIO2, MeBr) were made from Tyvek, while CSPs for
the liquid decontaminant (SP) were made from PVDF. Appendix C contains further details on
the CSP method development.
Sterilized, 1-gram quantities of soil were placed into each CSP, and then spiked with ten, 10-|iL
droplets (100-|iL total) of BA Ames spore prep, for a target inoculation level of 108 CFU. Each
CSP was then heat sealed using a lab heat sealer (Uline, Pleasant Prairie, WI). Dimensions of the
CSPs were -1.5" x 2" for the Tyvek and ~3" x 3" for the PVDF.
3.4 Test Column Preparation
Based on results from the preliminary wall effects testing (Appendix D), we fabricated 12" high
test columns from 10" diameter polyvinyl chloride (PVC) pipe. This design allowed CSP
placement at depths up to 5" without a wall effect (i.e. depth = radius).
Columns were fabricated with an open top to allow gas exposure or liquid application to the soil
surface. The bottom of each column was closed with a PVC cap to prevent soil from falling out
and to eliminate the possibility of decontaminant in-leakage from below.
10
-------
Prior to each test, soil density was determined by weighing a 200-mL volume of soil in a glass
beaker. The mass per unit depth (g/in) was then calculated for a 10-in diameter column.
Depending on the test conditions, soil layers of either 1" or V2" depths were weighed using an
A&D (Elk Grove, IL) FX-6000 electronic balance. Soil layers were stored in sealed ZipLoc®
bags inside sealed plastic tubs to maintain moisture content at the original level prior to building
a column for testing. The mass of soil for each 1-inch depth increment varied somewhat from
test to test and for each type of soil; refer to Appendix B for soil density data.
To build each column, a 5" base of soil backfill was first placed on the bottom. Soil layers were
then added one at a time, using the weighed ZipLoc bags and spiked CSPs as needed to meet the
conditions for each test. This technique allowed five depths of CSPs to be tested simultaneously,
along with a 0" depth sample placed on the top surface of the soil. CSPs were always placed in
the center of each soil layer, thus maintaining a minimum clearance of 5" from the side walls of
the column to eliminate any potential wall effects (gas leakage, channeling).
3.5 Sample Extraction and Biological Agent Quantification
Soil samples were extracted by removing soil from the CSP with a sterile spatula and placing it
into 10 mL of sterile phosphate buffered saline (PBS) with 0.1% Triton-X 100. Samples were
agitated at room temperature for 15 minutes at 200 rpm.
Bacillus spores were quantified using viable plate counting methodology with a dilution plating
approach. 100-|iL aliquots were removed from the extraction liquid after agitation, although if
there was too much settling of soil particles, the extraction liquid was gently mixed again to
restore turbidity. From that point, a series of 1:10 dilutions was performed and triplicate 100-|iL
aliquots of each dilution were plated onto sheep blood agar (SB A) culturing media. Plates were
incubated for 16-20 hours at approximately 35 °C. Following incubation, samples were either
immediately counted or were maintained in storage at 4-8 °C until enumeration. After counting
was complete, laboratory staff destroyed all samples by autoclaving. Refer to Appendix C for
further details about CSP development and CFU extraction and quantification method
evaluations.
3.6 Decontamination Efficacy
The mean percent spore recovery from each soil sample was calculated using results from
positive control samples (inoculated, not decontaminated), by means of the following equation:
Mean % Recovery = [Mean CFUpc/CFUtarget] x 100 (1)
where Mean CFUpc is the mean number of CFU counted from five replicate positive control
samples from a single material, and CFUtarget is the number of CFU counted from the non-soil
extraction control sample (i.e., 100-|iL spiked into extraction buffer and recovered, not spiked
onto soil). Mean % recovery was also calculated for each individual soil sample. Results are
included in Sections 6 through 8.
Efficacy was defined as the comparison (as a logio reduction) of viable counts between
individual test samples after decontamination versus the average of the positive controls, as
shown below:
11
-------
Efficacy (as log reduction, or "LR") = (Avg. logio CFUpc) - logio CFUsampie (2)
At any given soil depth, a decontamination that achieved an efficacy of > 6 LR was considered to
be effective [9], We note, however, that while a decontamination efficacy > 6 LR may be
considered "effective" when reporting test results, in an actual BA release event, the goal would
be to minimize the number of recoverable viable spores, regardless of LR.
In cases where no viable spores were detected, a CFU value of 1 was assigned, producing a logio
value of zero for that sample. These samples were considered to be completely inactivated and
therefore, achieved the maximum efficacy possible or quantifiable. That is, the final efficacy was
reported as greater than or equal to (>) the value calculated by Equation 2.
3.7 Biological Indicator (Bl) Handling and Analysis
Prior to performing tests using BA within the custom-made CSPs, a series of preliminary method
development and wall-effects tests was performed using commercially-available biological
indicators, or Bis, from Mesa Laboratories, Inc. Bis consisted of a small stainless steel disc
impregnated with B. atrophaeus spores to a level of 2.8 x 106 CFU per disc, and sealed within a
Tyvek type 1073 (0.22-|im pore size) pouch to maintain sterility.
Method development tests were performed with the Bis as a cost-effective way to determine
fundamental data on the expected performance of the CSPs and potential wall effects for the
column designs. Details and results from these method development tests are included in
Appendices C and D. The standard procedure for extraction and recovery of the Bis was as
follows:
1. Cut Tyvek and aseptically dump BI disc into 15 mL tube containing 10 mL sterile water.
2. Sonicate tube for 15 min.
3. Heat shock (optional) by placing tube in water bath at 80-85 °C for 10 minutes.
4. Analyze by plate counting/serial dilution, using 0.1-mL aliquots.
12
-------
Section 4.
Decontamination Procedures
4.1 Decontaminant Preparation
4.1.1 Chlorine Dioxide
A Minidox-M CIO2 generator (ClorDiSys Solutions, Inc., Lebanon, NJ) was used to produce all
of the CIO2 required for the project. Prior to any laboratory use, the system was inspected and
overhauled by a ClorDiSys field service engineer. The photometer was calibrated and fresh CIO2
cartridges were installed. A complete checklist of the field maintenance performed is in the
project file and is also found in the ClorDiSys Minidox-M Systems Operations Guide [10],
4.1.2 Sodium Persulfate
Klozur® SP (Peroxychem, Philadelphia, PA) was used as the source of SP, and is used for in situ
and ex situ chemical oxidation of contaminants in environmental remediation applications (e.g.,
soil). Klozur® SP is a >99% pure SP (Na2S20x) in the form of white odorless crystals. In
remediation applications, Klozur® SP is injected into contaminated soil or groundwater and is
activated by mixing in appropriate proportions of 8% hydrogen peroxide (H2O2) (or other
activators) by weight. Peroxychem recommends performing a bench-scale study to determine the
optimum hydrogen peroxide to SP molar ratio for the remediation.
A full optimization was beyond the scope of this study. Guidance provided by Peroxychem,
however, states that molar ratios of 1:1 to 10:1 (hydrogen peroxide to persulfate) are generally
used, with a molar ratio of 5:1 typically being sufficient to treat most contaminants under a wide
range of site conditions [11], This information, along with previous EPA studies [2-4], led to an
initial target condition of 0.5 M SP mixed in equal volumes with 8% hydrogen peroxide. This
50/50 mixture provides an actual molar ratio of 4.7 to 1. Later in the test series, the persulfate
concentration was adjusted to 1.0 M, while still maintaining the 50/50 volume ratio with
hydrogen peroxide. This mixture provides an actual molar ratio of 2.35 to 1.
Food-grade 8% hydrogen peroxide (Family Health, Miami FL) was purchased on-line, and fresh,
unopened, 1-pint bottles were used as needed for each test. The hydrogen peroxide/persulfate
solution was always mixed fresh immediately prior to application.
4.1.3 Methyl Bromide
Methyl bromide is a colorless and odorless volatile gas. Due to the toxicity of MeBr, previous
EPA studies have used a commercial blend of 99.5% MeBr with 0.5% chloropicrin added as a
warning irritant [12], Laboratory staff were unable to locate a commercial supplier for MeBr
containing 0.5% chloropicrin, and used 100% MeBr available through Cardinal Professional
Products (Anaheim, CA) as Meth-O-Gas® 100 commodity fumigant. The Cardinal sales
representative said that custom chloropicrin blends were no longer available due to market
conditions (MeBr is now banned except for specific exempt industries).
13
-------
4.2 Test and Control Chambers and Procedures
4.2.1 Test and Control Chambers
All testing was conducted using a custom-built Test Chamber to contain the soil columns
exposed to decontaminant and a Control Chamber to contain positive control samples. The Test
Chamber was a rigid structure with dimensions of 14" x 44" x 20" height, built from aluminum
framing and chemical-resistant PVC walls. Temperature and RH were monitored with a
HMD40/50 probe from Vaisala (Boston, MA). Other chamber conditions were modified slightly
for each decontaminant type, and are described in the subsections below. A picture of the Test
Chamber is shown below in Figure 2.
Figure 2. Test Chamber
The Control Chamber was a small plastic Life-Latch bucket for primary containment, placed into
an incubator within the BSL-3 facility. The incubator temperature and RH were monitored and
logged using an AmegaView centralized monitoring system (Mesa Monitoring, sensor model
#3006-20; Lakewood, CO).
4.2.2 ChlorDiSys Minidox-M
The Test Chamber was connected to the Minidox-M using several W* Teflon tubes, as per the
manufacturer's guidance, for inlet/outlet CIO2 to the photometer, feed CIO2 to the Test Chamber,
purge air to the Test Chamber, and a manual exhaust valve for purge air. Controls on the
Minidox-M also allowed operation of a small humidifier (PureGuardian H1010; Euclid, OH)
through a relay switch linked to a 120-VAC power supply. Dehumidification controls were not
part of the design, thus allowing RH levels to be maintained at or above the set-point.
Temperature controls were also not part of the design, although the Minidox-M did log
14
-------
temperature throughout each test. Ambient operating conditions in the BSL-3 were targeted to be
70°F or greater.
Laboratory staff followed the manufacturer's procedures for operating the Minidox-M. First, the
system performed a "conditioning" step where RH within the Test Chamber was raised to the
target level. Chlorine dioxide gas "exposure" was then introduced to the target concentration and
was held for the target time period. Note that the factory-installed program on the Minidox-M
limited exposure time to a 10-hr time period, requiring lab staff re-entry and manually restarting
the sequence for those tests that ran beyond 10 hours. After completing a test, the gas was
removed and vented to external exhaust.
Chlorine dioxide concentration, temperature, and RH were logged at 1-min intervals throughout
each test.
Vendor-supplied literature has the following information on the system controls:
• Humidifier auto on (RH is >2% below set point); auto off (RH is at the set point).
• CIO2 auto on (CIO2 is 0.3 mg/L below set point); auto off (0.3 mg/L above set point).
• Chlorine dioxide set point range 0 to 30 mg/L.
• Exposure time is 0 to 10 hours (600 minutes).
• Chamber volume is 1 to 28,000 cubic feet.
• Pressure set point, above atmospheric, 0 to 600 Pascals.
4.2.3 Sodium Persulfate Decontamination Procedure
Sodium persulfate was prepared at concentrations of either 0.5 M or 1.0 M and activated by
mixing it 50/50 (volume basis) with 8% hydrogen peroxide. SP dry powder was weighed and
mixed with deionized water to prepare the stock SP solution. (119.1 g dry SP into 1 L of water
yields a 0.5 M solution.) Liquid hydrogen peroxide was mixed with the SP solution in 50/50
proportions immediately prior to soil application. Volumes and contact times were dependent on
conditions determined for each individual test.
Liquid volume of each application was based on the approximate soil saturation point (in units of
mL liquid per gram of soil) that was determined during preliminary testing. The liquid volume to
be applied was then calculated for a wetted depth of 6 inches in a 10-in diameter column. Actual
application of the liquid was performed by pouring the calculated volume through a hand-held
garden sprinkler head onto the top of each column.
For the first test with SP, plastic end caps were in place on the bottom of each column as had
been done with the previous CIO2 tests. Although this design allowed liquid seepage (drainage)
from the bottom, it was found to be more restrictive than desired. Consequently, several small
Vi" holes were drilled into each end cap, improving the liquid drainage for the remaining tests.
Liquid drainage was collected in drip pans located beneath each column. Excess liquid was
removed as needed to prevent the drip pans from overflowing. For the final test in this series, the
oxidation reduction potential (ORP) was measured using a hand-held probe (Hach Pocket Pro™;
Loveland, CO). ORP measurements were taken of the initial (fresh) activated SP and final
15
-------
(partially spent) liquid emerging from the column bottom. Further details of the ORP
measurements can be found in Appendix E.
Temperature and RH were not controlled for the SP testing, but were logged using a Hygroflex
HF53W XMTR T/RH probe (Rotronic Instrument Corp., Hauppauge, NY).
4,2.4 Methyl Bromide Decontamination Procedure
The MeBr concentration in the test chamber was measured continuously using a Fumiscope™
Version 5.0 (Key Chemical and Equipment Company, Clearwater, FL). MeBr was added to the
chamber as necessary to reach the specified concentration, and was maintained at or above the
target set point by using an Omega Engineering CN7523 controller (Norwalk, CT).
The Fumiscope™ is calibrated annually by the manufacturer for MeBr, displaying the
concentration on a digital LED display in ounces of MeBr per 1000 cubic feet. One oz per
1000 ft3 is approximately 257 ppm at 25 °C, and is approximately 1 milligram (mg) per liter
(independent of temperature). MeBr tests were expected to be run at -212 to 300 mg/L
(53,000 to 75,000 ppm). Calibration of the Fumiscope was conducted at the factory by the
supplier. In addition, prior to being transferred to the BSL-3 facility, the Fumiscope™ calibration
was verified with a 75,000 ppm calibration gas from Scott-Marrin, Inc (Riverside, CA).
The Fumiscope™ included an air pump that drew gas from the test chamber through the thermal
conductivity detector at a controlled rate of ~1 LPM, then exhausted the gases back into the test
chamber. Moisture was removed from the gas sample by using a small glass condenser trap just
upstream of the Fumiscope™ At the end of a given trial, the test chamber was flushed with
compressed air and then opened to flush with ambient laboratory air. Worker safety at the 5-ppm
level was confirmed by testing the hood with a hand-held miniRAE 3000 PID sensor (RAE
Systems, Inc., San Jose, CA). The miniRAE was used during Test 1 MeBr before, during, and
after the test to spot check for safety/leaks (whenever staff were in the room taking care of
something. There was not a set schedule.) After successfully demonstrating good engineering
controls during Test 1, the safety office waived the need for using it, provided proper lab
procedure was used, such as hood airflows and equipment setbacks.
Temperature and RH were monitored with the same Rotronic T/RH probe used for the previous
SP tests. RH was controlled at or above the set point by using an Omega CN7523 controller with
a small humidifier identical to the CIO2 tests (i.e., the Pureguardian H1010 ultrasonic
humidifier).
16
-------
Section 5.
Quality Assurance and Quality Control
Quality assurance and quality control (QC) procedures were performed according to the program
Quality Management Plan (QMP) and the QAPP. A summary of QA/QC procedures and results
is below.
5.1 Equipment Calibration
All equipment (e.g., pipettes, incubators, balances) and monitoring devices (e.g., thermometer,
hygrometer, Minidox-M controls) used at the time of testing were verified as being within
calibration or vendor certification.
5.2 Quality Control Results
Quality controls during the program included several types of sample performance controls, as
summarized in Table 8. Specifically, there were two types of inoculation controls: a back titer
(100-|iL of the stock spore suspension on the test day was analyzed by direct plating) and the
non-soil extraction controls (100-|iL of stock spore suspension, spiked into buffer, recovered and
plated identically to samples but without exposure to soil). Recovery of positive controls was
then calculated versus the non-soil extraction control results for each test. Other QC samples
included positive controls for each soil type (inoculated, not decontaminated), negative controls,
and zero-depth controls (inoculated, unburied, placed on the top surface of each soil column,
decontaminated).
Table 8. Quality Control Sample Performance Criteria
Sample
Number of
Samples
Performance
Criteria
Corrective Action if Performance
Criteria are not Attained
Inoculation Control - stock
suspension directly analyzed by
plating (back titer)
One per test at
time-zero.
±1 log from target
value of 1 x 109
CFU/mL
Identify and correct the cause of
incorrect bacteria levels in the stock
suspension.
Inoculation Control - stock
suspension spiked into buffer w/o
soil contact (non-soil extraction
control)
One per test at
time-zero.
±1 log from target
value of 1 x 109
CFU/mL
Identify and correct the cause of
incorrect bacteria levels in the
result.
Positive Control - inoculated, not
exposed to decontaminant, kept in
separate Control Chamber
Five per soil
type
Mean CFU >5% and
<120% of inoculation
control
Discuss results with team lead.
Negative Controls (laboratory
blank) - not inoculated, not
exposed to decontaminant
One per soil
type
No observed CFU
Identify and remove source of
contamination.
Negative Controls (procedural
blank) - not inoculated, exposed to
decontaminant
One per soil
type.
No observed CFU
Identify and remove source of
contamination.
0" Control - inoculated, exposed to
decontaminant, unburied, placed
on top surface of soil
One per soil
type
None; driven by the
effectiveness of the
decontaminant
Compare data with CSPs at
depths; with highly-variable results
attempt to identify source of the
variability.
17
-------
Table 9 contains a summary of the quality control results for each of the tests. A "yes" indicates
that the QC criteria from Table 8 were met for that test.
All back titers met the performance criteria of ±1 log from the target value of 1 x 109 CFU/mL,
except Test 7, CIO2, which had a value higher than 1 x 1010 CFU/mL. In addition, all of the back
titers showed a stock suspension value of at least 1 x 109 CFU/mL, though there were no specific
QC criteria for this. Non-soil extraction controls also met the QC criteria of ±1 log, with one
exception, Test 3, CIO2, that was low (6.6 x 107 CFU). These few anomalies were not expected
to impact results significantly.
For positive controls, a few samples fell beyond the 5% - 120% target criteria. Results shown in
Table 9 show both yes/no and % recovery range for positive controls. (For each soil type, there
were a total of 70 positive controls used in the study: 14 experiments X 5 replicates.) For
topsoil, there were 6 of 70, or 8.6%, positive controls that fell outside this criterion. For sand: 7
of 70, or 10.0%; and for clay: 7 of 70, or 10.0%. Overall, 20 of 210, or 9.5%, of positive controls
fell outside this recovery range.
As per Table 8, performance criteria that were not met were discussed with the principal
investigator on a case-by-case basis, and corrective actions were taken where possible.
Variations in % recovery are believed to be due to variations in the soil content. Since the testing
required large volumes of soil, it was somewhat problematic to maintain consistency across the
entire length of program. Poor recovery of clay positive controls from Test 4, CIO2, led to an
effort to characterize the decay of clay-based positive controls in Tests 4a and 7a.
For the CIO2 and SP tests, all of the 0" controls (spiked CSP placed on the top surface of each
soil column) showed complete kill (i.e., fully inactivated), with no observed CFU, with the
exception of Test 3, SP. This sample did not show complete inactivation, presumably because
the large amount of foaming partially blocked SP penetration into the PVDF packet.
For the MeBr tests, however, several of the CSPs on the soil surfaces (0" controls) unexpectedly
did not show complete inactivation. Only one test (Test 2 MeBr, sand) of the fifteen total 0"
samples showed complete kill. Three of the fifteen total 0" samples clearly showed B. anthracis
colonies, although results fell below the quantitation range of 30 - 300 when undiluted. For
project reporting purposes, these samples were counted as is, with a footnote of "below
quantitation limit", i.e., > 0 but < 30 CFU, as per the project QAPP. Finally, the remaining
eleven 0" samples for the MeBr testing showed a decontamination efficacy that was typically
approximately the same as the buried samples, and frequently in the range of only 0-2 LR.
All negative controls (blanks) met the criteria of no observed CFU.
18
-------
Table 9. Quality Control Results Summary
Test ID
Back
Titer
Non-Soil
Extraction
Control
Positive Controls % Recovery Range
Negative
Controls
0"
Controls
Topsoil
Sand
Clay
1. CIO2
Yes
Yes
Yes (18-40)
No (4-51)
Yes (10-57)
Yes
Yes
2. CIO2
Yes
Yes
Yes (25 - 96)
Yes (37 - 87)
Yes (8 - 69)
Yes
Yes
3. CIO2
Yes
No
No (3-236)
No (1 - 114)
No (4-138)
Yes
Yes
4. CIO2
Yes
Yes
No (17-315)
Yes (25 - 58)
No (0)
Yes
Yes
5. CIO2
Yes
Yes
Yes (27 - 56)
No (116-165)
Yes (46-107)
Yes
Yes
6. CIO2
Yes
Yes
Yes (47-81)
Yes (18-35)
Yes (10-23)
Yes
Yes
7. CIO2
Noa
Yes
Yes (19-47)
Yes (54 - 66)
Yes (7-16)
Yes
Yes
4a. Decay
Yes
Yes
-
-
-
-
-
7a. Decay
Yes
Yes
-
-
-
-
-
1. SP
Yes
Yes
Yes (6-21)
Yes (18-36)
Yes (5 - 8)
Yes
Yes
2. SP
Yes
Yes
Yes (27 - 37)
Yes (46 - 65)
Yes (7 - 22)
Yes
Yes
3. SP
Yes
Yes
Yes (20-47)
Yes (21 - 37)
Yes (18-61)
Yes
Nob
1. MeBr
Yes
Yes
Yes (35 - 53)
Yes (87-113)
Yes (19-42)
Yes
Noc
2. MeBr
Yes
Yes
Yes (19-23)
Yes (84 - 97)
Yes (13-19)
Yes
Nod
3. MeBr
Yes
Yes
Yes (13-27)
Yes (70 - 83)
Yes (9-16)
Yes
Noe
4. MeBr
Yes
Yes
Yes (38 - 50)
Yes (74 - 94)
Yes (20 - 23)
Yes
Noc
a Results were high. No corrective action taken.
b Results did not show complete inactivation, believed due to excessive foaming during decontaminant application.
c Results did not show complete inactivation (48-hr exposure) for any of the three soil types.
d 48-hr 0" controls were not completely inactivated. At 65.5 hrs, sand 0" was completely inactivated, topsoil was >0
but <30 counts (below detection limit), while clay was not completely inactivated.
e 0" controls were >0 but <30 counts (below detection limit) for topsoil and sand, while clay was not completely
inactivated.
5.3 Audits
5.3.1 Performance Evaluation Audit
Performance evaluation audits were conducted to assess the quality of the results obtained during
the experiments. Table 10 summarizes the performance evaluation audits that were performed.
19
-------
Table 10. Performance Evaluation Audits
Measurement
Audit Procedure
Allowable Tolerance
Actual Measurement or
Calibration Check
Volume of liquid from
micropipettes
Annual calibration to meet
manufacturers
specifications, pass or
replace pipette.
±10%
10 |jL size: 1.0%
100 |jL size: 0.8%
1000 |jL size: 0.8%
Minidox-M CIO2
concentration
Photometer calibrated by
manufacturer prior to
testing.
±10%
1%
Fumiscope thermal
conductivity meter
Vendor certified and
calibrated prior to test.
Checked in laboratory
using independent
calibration gas.
±10%
1.4%
Balance
Compared to independent
calibrated weight sets
±0.5 g
0.1 g
Temperature
(lab incubator for controls)
Calibrated once annually
by Amega, pass or
replace sensor.
±2°C
0.6°C
RH
(lab incubator for controls)
Calibrated once annually
by Amega, pass or
replace sensor.
±5%
1.7%
Temperature (refrigerator
for storing extractions)
Calibrated once annually
by Amega and
continuously monitored.
±2-8 °C
1.2°C
Temperature (incubator
used for plates)
Calibrated once annually
by Amega, pass or
replace sensor.
±2°C
0.5°C
Freezer (stock storage)
Calibrated once annually
by Amega, pass or
replace sensor.
±2°C
1.2°C
5.3.2 Technical Systems Audit
As per the QAPP, a technical systems audit was not conducted as part of this program.
5.3.3 Data Quality Audit
As per the QAPP, 10% of the viable plate count data was audited by the QA representative
assigned to this task. The data were traced from the initial acquisition through reduction and final
reporting. Calculations and spreadsheets set up with the data were checked as part of the effort.
The findings of the data quality audit showed that results are of known and acceptable quality.
A copy of the data quality audit report is on file at MRIGlobal.
5.4 Test/Quality Assurance Plan Deviations
5.4. Cu ntc3bl Rsnqg
A countable range of 30 to 300 colonies for viable plating analysis was established in the QAPP
for this project. Practical experience, however, required adjustment of this range on a case-by-
case basis. The BA colonies proved to be quite large, making accurate counting above -150
20
-------
impossible. As a consequence, the analyst was sometimes forced to evaluate the next lowest
dilution and use this value (e.g., 15 counts) even though it fell out of the target quantitation
range. Any results outside the 30 to 300 range were flagged in the raw data given to the principal
investigator for each individual test.
During the MeBr tests, several samples fell outside the quantitation range of 30 to 300 when
undiluted sample aliquot was plated. For reporting purposes, these samples were counted as is,
but footnoted "below quantitation limit, > 0 but <30 counts".
5.4.2 Unburied versus Buried Positive Controls
When the program began, the initial plan was for positive control samples to be buried in a
separate Control Column using the appropriate soil types. However, laboratory tests were
conducted to demonstrate that a simpler method of using unburied positive controls would
produce similar overall results. For Test 1, CIO2, a set of five buried and five unburied positive
controls were evaluated for each soil type. Table 11 compares the two sets of results, which had
averages that agreed to within 15% for all three soil types. Consequently, at the direction of the
principal investigator, all of the remaining tests used only unburied positive controls.
Table 11. Comparison of Unburied and Buried Positive Controls
Unburied
Buried
Difference
(CFU)
(CFU)
(%)
Topsoil
8.67 x107
5.83 x107
1.65 x10s
1.30 x10s
1.02 x10s
9.63 x107
1.44 x10s
1.66 x10s
7.37 x107
2.03 x10s
Topsoil Average
1.14x10s
1.31 x 10s
13.3
Clay
4.20 x107
1.62 x 10s
1.44 x10s
1.15 x 10s
9.30 x107
9.83 x 107
5.73 x107
1.82 x 10s
2.36 x10s
9.27 x 107
Clay Average
1.15x10s
1.30 x 10s
12.7
Sand
1.98 x10s
2.31 x 10s
1.07 x10s
1.18 x 10s
1.10x10s
1.15 x 10s
NAa
1.59 x 10s
2.10x10s
2.20 x 10s
Sand Average
1.56 x10s
1.69 x 10s
7.6
a Sample did not meet criteria of 30-300 counts at any dilution and was not included in the average.
5.5 QA/QC Reporting
Each assessment and audit was documented in accordance with the QAPP and QMP. For this
program, any findings were noted as not significant and no follow-up corrective actions were
necessary.
21
-------
5.6 Data Review
Records and data generated in the testing received a QC/technical review before they were used
in calculations or determination of results, and prior to being incorporated into data reports to the
principal investigator.
22
-------
Section 6.
Results and Performance Summary for Chlorine Dioxide
6.1 Chlorine Dioxide Test Conditions
The actual fumigation conditions measured for the CIO2 test series are presented in Table 12.
Highlights of each test are given in the subsections below. Table 12 also includes summary
information on decontamination efficacy, in terms of the maximum depth at which > 6 LR was
achieved for each soil type for each test.
23
-------
Table 12. Chlorine Dioxide Actual Test Conditions
Test
No.
Depth(s) Tested
Avg. CIO2
Cone.
(mg/L)
Avg. T
(°C)
Avg. RH
(%)
Contact
Time
(hrs)
Other
Conditions
Max depth
(inches)
achieving >
6 LR
topsoil
Max depth
(inches)
achieving >
6 LR
clayey
Max depth
(inches)
achieving >
6 LR
sandy
1
0, 1, 2, 3, 4, 5"
8.7 ± 0.30
25.3 ± 0.44
76.7 ± 3.65
3
0
0
0
Positive Controls
NA
26.5 ± 0.07
44.4 ± 0.33
a
2
0, 1, 2, 3, 4, 5"
10.3 ± 2.98
24.4 ±0.19
79.6 ± 6.49
6
0
2
1
Positive Controls
NA
25.7 ± 0.07
39.5 ± 0.19
a
3
0, 0.5, 1, 1.5, 2, 2.5"
8.9 ± 0.85
24.3 ± 0.33
73.5 ± 3.62
3
Saturated
soil
0
2.5
2
Positive Controls
NA
25.2 ± 0.07
43.8 ± 0.59
a
4
0, 1, 2, 3, 4, 5
10.1 ± 2.16
23.3 ± 0.48
80.6 ± 3.02
24
1
5*
5
Positive Controls
NA
36.7 ±0.10
93.8 ± 1.34
a
5
0, 1, 2, 3, 4, 5"
9.3 ± 1.34
23.2 ± 0.47
80.1 ± 2.76
24
Saturated
soil
0
5
2
Positive Controls
NA
24.6 ± 0.12
27.7 ± 4.37
a
6
0, 1, 2, 3, 4, 5"
14.6 ± 2.13
22.2 ± 0.20
82.5 ± 1.24
27.5
1
5
5
Positive Controls
NA
23.5 ± 0.41
14.7 ± 3.98
a
7
0, 1, 2, 3, 4, 5"
9.4 ± 0.89
22.3 ± 0.27
85.8 ± 0.43
7.75
Compacted
soil
0
3
2
Positive Controls
NA
23.4 ± 0.20
20.3 ± 4.23
a
a Positive controls had no decontaminant applied, but were collected and extracted at the end of the contact time for the test samples.
* No spores recovered from clay positive controls for Test 4, thus test results are questionable
24
-------
6.2 Chlorine Dioxide Decontamination Results
6.2.1 Test 1 CIO2
Target CIO2 concentration was 8.4 mg/L and target RH was >75% for a 3-hr contact time. This
test used both buried and unburied positive control samples as described earlier in Section 5
(QC), while all subsequent tests used only unburied positive control CSPs. Results showed that
the decontamination had virtually no effect for the topsoil and sand columns. The clay column
showed limited effect (up to 2 LR) at depths up to 2".
6.2.2 Test 2 CIO2
Target CIO2 concentration was 8.4 mg/L and target RH was >75% for a 6-hr contact time. This
experiment was meant to determine if efficacy could be improved by increasing contact time,
and in fact, efficacy generally did improve compared to Test 1. Results showed that the
decontamination had a 3.5 LR at 1" and tapered off to no effect by 4" for the topsoil column. The
sand column showed complete kill (8 LR) at 1" and tapered off to no effect by 4". The clay
column showed complete kill (8 LR) at up to 2" and tapered off to no effect by 4".
6.2.3 Test 3 CIO2
Target CIO2 concentration was 8.4 mg/L and target RH was >75% for a 3-hr contact time. The
top 5" of soils were wetted pre-test to near the saturation point, which had been determined
experimentally in preliminary tests to be 0.08 mL/g (topsoil), 0.10 mL/g (sand), and 0.04 mL/g
(clay). CSPs were placed in 0.5-in increments, rather than the 1-in increments used previously,
based on previous efficacy results.
Pre-wetting the soils did improve efficacy somewhat (compared to Test 1), although results were
somewhat mixed. Results showed that for the topsoil, the CIO2 provided a 1 LR at the 2" depth
to the column, but was ineffective at shallower depths. The sand column showed complete kill at
most depths. The clay column showed complete kill at most depths as well. For all three
columns, it is believed that clumping or channeling due to the wetted soil was the cause of
inconsistency across the range of depths.
6.2.4 Test 4 CIO2
Target CIO2 concentration was 8.4 mg/L and target RH was >75% for an extended 24-hr contact
time. Inadvertently, the actual mean temperature for the positive controls was 37 °C and the RH
was elevated to 94%. Results showed that the extended contact time did continue to improve
efficacy somewhat; decontamination had complete kill (8 LR) at 1" and tapered off to no effect
by 3" for the topsoil column. The sand column showed complete kill (7 LR) at all depths. The
clay column appeared to show complete kill at all depths, but positive controls for clay also
showed no recovery, thus making the test results for clay questionable. Refer to Section 9 of this
report for test results for the clay decay tests, conducted to explore reasons for no recovery of
spores from clay positive control CSPs.
25
-------
6.2.5 Test 5 CIO2
Target CIO2 concentration was 8.4 mg/L and target RH was >75% for a 24-hr contact time. The
top 5" of soils were wetted pre-test to near the saturation point, using the same experimentally-
determined levels as in Test 3 CIO2 described earlier. This experiment was conducted to elicit the
effect of pre-wetting of soil, by comparing to Test 4. In this case, the effect of added soil
moisture seems to have diminished efficacy somewhat. (The CIO2 concentration for Test 5 was
somewhat lower than for Test 4, and this may have also contributed to the somewhat lower
efficacy.) Results showed that the decontamination had 1 LR at 1" for the topsoil column. The
sand column showed complete kill (8 LR) at up to 2", dropping off to only a minor effect at 5".
The clay column had complete kill (8 LR) at all depths.
6.2.6 Test 6 CIO2
Target CIO2 concentration was 14.0 mg/L and target RH was >75% for a target 24-hr contact
time. This test was conducted to evaluate whether improved efficacy would occur for the topsoil
with an increased CIO2 concentration. Results showed that the decontamination had complete
kill (8 LR) at 1" for the topsoil column, then tapered off to minor or no effect. This result is
essentially the same for topsoil for Test 4. The sand column showed complete kill (8 LR) at all
depths. The clay column had complete kill (8 LR) at all depths.
6.2.7 Test 7 CIO2
Target CIO2 concentration was 8.4 mg/L and target RH was >80% for a target 6-hr contact time.
This test attempted to duplicate the conditions of Test 2 CIO2, but with compressed soils. Once
soil columns were built, soils were gently compacted by compression with a small hand tool.
Results showed that the decontamination had no effect at any depth for the topsoil column. The
sand column showed complete kill (8 LR) at up to 2". The clay column had complete kill (8 LR)
at up to 3".
Tables 13 through Table 19 show the detailed results for the decontamination of BA spores in
soil using CIO2.
26
-------
Table 13. Test 1, CIO2 Decontamination Results
Test ID, summary
Non-Soil Ext.
Control (CFU)
Log of
Recovered CFU
from the CSP
Mean % Recovery
(vs. Non-Soil
Extraction
Control)
Decontamination
Efficacy
Test 1
8.7 mg/L, 3-hr
Soil Type: Topsoil
Test Sample, 0"
4.13x10s
0
0
8.04
Test Sample, 1"
8.10
31
-0.07
Test Sample, 2"
8.16
35
-0.12
Test Sample, 3"
8.22
40
-0.18
Test Sample, 4"
8.26
44
-0.22
Test Sample, 5"
8.33
52
-0.29
Positive Controls (mean)
4.13x10s
8.04a
00
I
4^
O
O"
—
Negative Controls (TC)
0
0
0
-
Negative Controls (CC)
0
0
0
-
Soil Type: Sand
Test Sample, 0"
4.13x10s
0
0
7.98
Test Sample, 1"
7.82
16
0.17
Test Sample, 2"
8.00
24
-0.02
Test Sample, 3"
8.32
50
-0.33
Test Sample, 4"
8.39
59
-0.40
Test Sample, 5"
8.30
48
-0.31
Positive Controls (mean)
4.13x10s
7.98a
4 - 51b
—
Negative Controls (TC)
0
0
0
-
Negative Controls (CC)
0
0
0
-
Soil Type: Clay
Test Sample, 0"
4.13x10s
0
0
7.98
Test Sample, 1"
5.92
<1
2.06
Test Sample, 2"
6.06
<1
1.92
Test Sample, 3"
7.95
21
0.03
Test Sample, 4"
7.96
22
0.02
Test Sample, 5"
8.03
26
-0.06
Positive Controls (mean)
4.13x10s
7.98a
O
I
cn
O"
—
Negative Controls (TC)
0
0
0
-
Negative Controls (CC)
0
0
0
-
a Average for five positive controls is reported.
b Recovery range for five positive controls is reported.
27
-------
Table 14. Test 2, CIO2 Decontamination Results
Test ID, summary
Non-Soil Ext.
Control (CFU)
Log of
Recovered CFU
from the CSP
Mean % Recovery
(vs. Non-Soil
Extraction
Control)
Decontamination
Efficacy
Test 2
10.3 mg/L, 6-hr
Soil Type: Topsoil
Test Sample, 0"
2.14x10s
0
0
8.11
Test Sample, 1"
4.60
<1
3.51
Test Sample, 2"
7.00
5
1.11
Test Sample, 3"
7.32
10
0.79
Test Sample, 4"
7.33
10
0.77
Test Sample, 5"
7.85
33
0.26
Positive Controls (mean)
2.14x10s
8.11a
25 - 96b
—
Negative Controls (TC)
0
0
0
-
Negative Controls (CC)
0
0
0
-
Soil Type: Sand
Test Sample, 0"
2.14x10s
0
0
8.13
Test Sample, 1"
0
0
8.13
Test Sample, 2"
6.75
3
1.38
Test Sample, 3"
7.03
5
1.10
Test Sample, 4"
7.73
25
0.40
Test Sample, 5"
7.75
26
0.38
Positive Controls (mean)
2.14x10s
8.13a
CO
-vl
I
00
-vl
O"
—
Negative Controls (TC)
0
0
0
-
Negative Controls (CC)
0
0
0
-
Soil Type: Clay
Test Sample, 0"
2.14x10s
0
0
7.87
Test Sample, 1"
0
0
7.87
Test Sample, 2"
0
0
7.87
Test Sample, 3"
5.82
<1
2.05
Test Sample, 4"
8.16
68
-0.29
Test Sample, 5"
8.27
88
-0.40
Positive Controls (mean)
2.14x10s
7.87a
00
I
O)
CD
O"
—
Negative Controls (TC)
0
0
0
-
Negative Controls (CC)
0
0
0
-
a Average for five positive controls is reported.
b Recovery range for five positive controls is reported.
28
-------
Table 15. Test 3, CIO2 Decontamination Results
Test ID, summary
Non-Soil Ext.
Control (CFU)
Log of
Recovered CFU
from the CSP
Mean % Recovery
(vs. Non-Soil
Extraction
Control)
Decontamination
Efficacy
Test 3, saturated soils
8.9 mg/L, 3-hr
Soil Type: Topsoil
Test Sample, 0"
6.67 x107
0
0
7.55
Test Sample, 0.5"
7.07
18
0.48
Test Sample, 1.0"
7.24
26
0.30
Test Sample, 1.5"
7.53
51
0.02
Test Sample, 2.0"
6.20
2
1.35
Test Sample, 2.5"
7.70
76
-0.15
Positive Controls (mean)
6.67 x107
7.55a
3
- 236b
—
Negative Controls (TC)
0
0
0
-
Negative Controls (CC)
0
0
0
-
Soil Type: Sand
Test Sample, 0"
6.67 x107
0
0
6.59
Test Sample, 0.5"
0
0
6.59
Test Sample, 1.0"
3.79
<1
2.79
Test Sample, 1.5"
0
0
6.59
Test Sample, 2.0"
0
0
6.59
Test Sample, 2.5"
2.85
<1
3.74
Positive Controls (mean)
6.67 x107
6.59a
1
- 114b
—
Negative Controls (TC)
0
0
0
-
Negative Controls (CC)
0
0
0
-
Soil Type: Clay
Test Sample, 0"
6.67 x107
0
0
7.18
Test Sample, 0.5"
1.52
<1
5.66
Test Sample, 1.0"
0
0
7.18
Test Sample, 1.5"
2.92
<1
4.26
Test Sample, 2.0"
0
0
7.18
Test Sample, 2.5"
0
0
7.18
Positive Controls (mean)
6.67 x107
7.18a
4
- 138b
—
Negative Controls (TC)
0
0
0
-
Negative Controls (CC)
0
0
0
-
a Average for five positive controls is reported.
b Recovery range for five positive controls is reported. Low recovery possibly due to spore germination in freshly-
autoclaved (moist) soil. Possible lab error also occurred during dilution of some positive controls, and some
samples may be off by 1 log.
29
-------
Table 16. Test 4, CIO2 Decontamination Results
Test ID, summary
Non-Soil Ext.
Control (CFU)
Log of
Recovered CFU
from the CSP
Mean % Recovery
(vs. Non-Soil
Extraction
Control)
Decontamination
Efficacy
Test 4, saturated soils
10.1 mg/L, 24-hr
Soil Type: Topsoil
Test Sample, 0"
1.20 x10s
0
0
8.14
Test Sample, 1"
0
0
8.14
Test Sample, 2"
6.62
3
1.52
Test Sample, 3"
8.95
749
-0.81
Test Sample, 4"
8.89
652
-0.75
Test Sample, 5"
8.14
115
0.00
Positive Controls (mean)
1.20 x10s
8.14a
17 — 315b
—
Negative Controls (TC)
0
0
0
-
Negative Controls (CC)
0
0
0
-
Soil Type: Sand
Test Sample, 0"
1.20 x10s
0
0
7.62
Test Sample, 1"
0
0
7.62
Test Sample, 2"
0
0
7.62
Test Sample, 3"
0
0
7.62
Test Sample, 4"
0
0
7.62
Test Sample, 5"
0
0
7.62
Positive Controls (mean)
1.20 x10s
7.62a
K)
cn
I
cn
00
O"
—
Negative Controls (TC)
0
0
0
-
Negative Controls (CC)
0
0
0
-
Soil Type: Clay
Test Sample, 0"
1.20 x10s
0
0
0C
Test Sample, 1"
0
0
0C
Test Sample, 2"
0
0
oc
Test Sample, 3"
0
0
oc
Test Sample, 4"
0
0
oc
Test Sample, 5"
0
0
oc
Positive Controls (mean)
1.20 x10s
0a
ob
—
Negative Controls (TC)
0
0
0
-
Negative Controls (CC)
0
0
0
-
a Average for five positive controls is reported.
b Recovery range for five positive controls is reported.
c Calculated as "0", with 0% recovery for all positive controls and 0 seen for all test samples.
30
-------
Table 17. Test 5, CIO2 Decontamination Results
Test ID, summary
Non-Soil Ext.
Control (CFU)
Log of
Recovered CFU
from the CSP
Mean % Recovery
(vs. Non-Soil
Extraction
Control)
Decontamination
Efficacy
Test 5, saturated soils
9.3 mg/L, 24-hr
Soil Type: Topsoil
Test Sample, 0"
1.90 x10s
0
0
7.87
Test Sample, 1"
6.78
3
1.09
Test Sample, 2"
7.99
52
-0.12
Test Sample, 3"
7.99
52
-0.12
Test Sample, 4"
8.04
58
-0.17
Test Sample, 5"
7.85
37
0.02
Positive Controls (mean)
1.90 x10s
7.87a
27 - 56b
—
Negative Controls (TC)
0
0
0
-
Negative Controls (CC)
0
0
0
-
Soil Type: Sand
Test Sample, 0"
1.90 x10s
0
0
8.41
Test Sample, 1"
0
0
8.41
Test Sample, 2"
0
0
8.41
Test Sample, 3"
3.52
<1
4.88
Test Sample, 4"
4.22
<1
4.19
Test Sample, 5"
7.76
30
0.65
Positive Controls (mean)
1.90 x10s
8.41a
116 - 165b
—
Negative Controls (TC)
0
0
0
-
Negative Controls (CC)
0
0
0
-
Soil Type: Clay
Test Sample, 0"
1.90 x10s
0
0
8.17
Test Sample, 1"
0
0
8.17
Test Sample, 2"
0
0
8.17
Test Sample, 3"
0
0
8.17
Test Sample, 4"
0
0
8.17
Test Sample, 5"
0
0
8.17
Positive Controls (mean)
1.90 x10s
8.17a
46 - 107b
—
Negative Controls (TC)
0
0
0
-
Negative Controls (CC)
0
0
0
-
a Average for five positive controls is reported.
b Recovery range for five positive controls is reported.
31
-------
Table 18. Test 6, CIO2 Decontamination Results
Test ID, summary
Non-Soil Ext.
Control (CFU)
Log of
Recovered CFU
from the CSP
Mean % Recovery
(vs. Non-Soil
Extraction
Control)
Decontamination
Efficacy
Test 6, saturated soils
14.6 mg/L, 27.5-hr
Soil Type: Topsoil
Test Sample, 0"
4.40 x10s
0
0
8.42
Test Sample, 1"
0
0
8.42
Test Sample, 2"
7.77
13
0.65
Test Sample, 3"
8.01
23
0.41
Test Sample, 4"
8.02
24
0.40
Test Sample, 5"
7.97
21
0.45
Positive Controls (mean)
4.40 x10s
8.42a
4^
I
00
O"
—
Negative Controls (TC)
0
0
0
-
Negative Controls (CC)
0
0
0
-
Soil Type: Sand
Test Sample, 0"
4.40 x10s
0
0
8.05
Test Sample, 1"
0
0
8.05
Test Sample, 2"
0
0
8.05
Test Sample, 3"
0
0
8.05
Test Sample, 4"
0
0
8.05
Test Sample, 5"
0
0
8.05
Positive Controls (mean)
4.40 x10s
8.05a
00
I
CO
cn
O"
—
Negative Controls (TC)
0
0
0
-
Negative Controls (CC)
0
0
0
-
Soil Type: Clay
Test Sample, 0"
4.40 x10s
0
0
7.82
Test Sample, 1"
0
0
7.82
Test Sample, 2"
0
0
7.82
Test Sample, 3"
0
0
7.82
Test Sample, 4"
0
0
7.82
Test Sample, 5"
0
0
7.82
Positive Controls (mean)
4.40 x10s
7.82a
10 - 23b
—
Negative Controls (TC)
0
0
0
-
Negative Controls (CC)
0
0
0
-
a Average for five positive controls is reported.
b Recovery range for five positive controls is reported.
32
-------
Table 19. Test 7, CIO2 Decontamination Results
Test ID, summary
Non-Soil
Extract. Control
(CFU)
Log of
Recovered CFU
from the CSP
Mean % Recovery
(vs. Non-Soil Ext.
Control)
Decontamination
Efficacy
Test 7, compacted soils
9.4 mg/L, 7.75-hr
Soil Type: Topsoil
Test Sample, 0"
1.93 x10s
0
0
7.77
Test Sample, 1"
8.09
63
-0.32
Test Sample, 2"
8.03
56
-0.26
Test Sample, 3"
8.02
54
-0.25
Test Sample, 4"
7.96
47
-0.19
Test Sample, 5"
7.93
44
-0.16
Positive Controls (mean)
1.93 x10s
7.77a
CD
I
O"
—
Negative Controls (TC)
0
0
0
—
Negative Controls (CC)
0
0
0
-
Soil Type: Sand
Test Sample, 0"
1.93 x10s
0
0
8.06
Test Sample, 1"
0
0
8.06
Test Sample, 2"
0
0
8.06
Test Sample, 3"
3.82
0
4.24
Test Sample, 4"
6.08
1
1.98
Test Sample, 5"
7.67
24
0.40
Positive Controls (mean)
1.93 x10s
8.06a
54 - 66b
—
Negative Controls (TC)
0
0
0
-
Negative Controls (CC)
0
0
0
-
Soil Type: Clay
Test Sample, 0"
1.93 x10s
0
0
7.26
Test Sample, 1"
0
0
7.26
Test Sample, 2"
0
0
7.26
Test Sample, 3"
0
0
7.26
Test Sample, 4"
6.10
1
1.16
Test Sample, 5"
7.64
23
-0.39
Positive Controls (mean)
1.93 x10s
7.26a
7 - 16b
—
Negative Controls (TC)
0
0
0
-
Negative Controls (CC)
0
0
0
-
a Average for five positive controls is reported.
b Recovery range for five positive controls is reported.
33
-------
6.3 Chlorine Dioxide Log Reduction Charts
Figures 3 through Figure 9 show results for the CIO2 tests in graphical form. These LR charts
show the same "decontamination efficacy" data that were presented earlier in Section 6.2, but in
a visual format.
Test 1, CI02, 8.7 mg/L, 3 hrs
q
§ 7 4
I
V
-O ^
0)
CC q
>\ # Topsoil
00
° 1
y —• Clay
X
1
0 1 2 3 4 5
Depth (in)
Figure 3. Test 1, CIO2: 8.7 mg/L, 3 hrs, 77% RH.
Topsoil
Figure 4. Test 2, CIO2: 10.3 mg/L, 6 hrs, 80% RH
34
-------
Test 3, CI02, 8.9 mg/L, 3 hrs
Topsoil
Figure 5. Test 3, CIO2: 8.9 mg/L, 3 hrs, 74% RH, [saturated soil]
Test 4, CI02, 10.1 mg/L, 24 hrs
c 7
o
¦»—
u S
¦o
S 3
o
-1 1
-1
~
¦Topsoil
•Clay
•Sand
12 3 4
Depth (in)
Figure 6. Test 4, CIO2: 10.1 mg/L, 24 hrs, 81% RH
35
-------
Topsoil
Figure 7. Test 5, CIO2: 9.3 mg/L, 24 hrs, 80% RH, [saturated soil]
Test 6, CI02,14.6 mg/L, 27.5 hrs, 85% RH
10
c
8
0
—1
0
fi
"O
O)
en
4
SD
0
2
0
t==|
V 1
\
* 1
p 1
» V
\
\
\
2 3
Depth (in)
-Topsoil
-Clay
-Sand
Figure 8. Test 6, CIO2: 14.6 mg/L, 24 hrs, 83% RH
1 2 3 4 5
Depth (in)
Test 1, CI02, 9.4 mg/L, 7.75 hrs
Topsoil
Figure 9. Test 7, CIO2: 9.4 mg/L, 7.75 hrs, 86% RH, [compacted soil]
36
-------
Section 7.
Results and Performance Summary for Sodium Persulfate
7.1 Sodium Persulfate Test Conditions
The actual conditions measured for the SP test series (three tests) are presented in Table 20.
Highlights of each test are given in the subsections below. Table 20 also includes summary
information on decontamination efficacy, in terms of the maximum depth at which > 6 LR was
achieved for each soil type for each test.
37
-------
Table 20. Sodium Persulfate Test Conditions
Other Application Conditions
Max depth
Test
No.
Depth(s)
Tested
SP Cone.
(mg/L)
Avg. T
(°C)
Avg.
RH
(%)
Contact
Time
(hrs)
Soil
Vol/Qty per
dose
No. of
Doses
Interval
(inches)
achieving >
6 LR
Topsoil
1390 mL;
0.177 mL/g
2
48-hr
0
1
0, 1, 2, 3, 4, 5"
0.5 M,
50/50 with
8% H2O2
21.5±
0.70
92.9 ±
7.23
168
(7 days)
Sand
1390 mL;
0.155 mL/g
2
48-hr
1
Clay
1390 mL;
0.159 mL/g
2
48-hr
5
Positive
Controls
NA
23.7 ±
0.49
26.0 ±
13.68
a
NA
NA
NA
NA
0.5, 1.0, 1.5,
2.0, 2.5"
(topsoil)
0, 1, 2, 3, 4, 5"
(sand, clay)
Topsoil
702 mL;
0.09 mL/g
6
24-hr
0.5
2
0.5 M,
50/50 with
8% H2O2
22.0 ±
0.62
91.3 ±
9.04
144
(6 days)
Sand
820 mL;
0.09 mL/g
6
24-hr
1
Clay
792 mL;
0.09 mL/g
2
24-hr
5
Positive
NA
23.8 ±
91.5 ±
NA
NA
NA
NA
Controls
0.35
2.22
0.5, 1.0, 1.5,
2.0, 2.5"
(topsoil)
0, 1, 2, 3, 4, 5"
(sand, clay)
Topsoil
681 mL;
0.09 mL/g
6
24-hr
1
3
1.0 M,
50/50 with
8% H2O2
22.6 ±
0.74
91.9 ±
8.70
168
(7 days)
Sand
876 mL;
0.09 mL/g
6
24-hr
0
Clay
820 mL;
0.09 mL/g
1
NA
4
Positive
Controls
NA
24.5 ±
0.47
94.3 ±
0.16
a
NA
NA
NA
NA
a Positive controls had no decontaminant applied, but were collected and extracted at the end of the contact time for the test samples.
38
-------
7,1.1 Test 1 SP
Target test conditions were two applications of 0.5 M SP (activated by 50/50 mixture with 8%
hydrogen peroxide) separated by 48-hrs, with an overall contact time of 168 hrs (seven days).
Each application was intended to provide wetting down to a 6" depth. A 1390 mL liquid volume
was used for all soil types, making actual wetting levels of 0.177 mL/g (topsoil), 0.155 mL/g
(sand), and 0.159 mL/g (clay) per application.
The SP solution reacted vigorously with the topsoil and created large volumes of foam. The
foaming reaction with sand was minimal but still present. The sand also tended to become more
of a slurry as the liquid was applied, causing buried CSPs to rise to nearer the surface, rather than
remaining at their intended depths. Clay soils did not show any foaming reaction when the SP
was applied.
Results showed that the decontamination had a 1 LR at 1" for the topsoil column, then tapered
off to no effect. The sand column showed complete kill (7 LR) at 1". The clay column had
complete kill (7 LR) at all depths.
7.1.2 Test 2 SP
Based on the results from Test 1 SP, this test was designed to increase the amount of activated
SP for topsoil and sandy soil, but reduce the amount for clay. In addition, we hoped that smaller
(but more and frequent) doses of SP for sand and topsoil would reduce the amount of foaming or
reactivity. Target test conditions were increased to six applications of 0.5 M SP (activated by
50/50 mixture with 8% hydrogen peroxide) separated by 24-hrs, with an overall contact time of
168 hrs (seven days) for topsoil and sand. Given the success with clay in Test 1 SP, only two
applications were performed. The dose or application volumes were reduced to 0.09 mL/g. For
topsoil, CSPs were placed at 0.5-in depths.
The SP solution reactions with topsoil, sand, and clay were as described above for Test 1 SP.
Due to the smaller application volumes, however, foaming was better controlled. The increased
total liquid volumes for sand and topsoil for this test caused spent SP solution to begin emerging
from the column bottoms during or after the third application.
Results showed that the decontamination had improved somewhat for the topsoil, which had
complete kill (7 LR) at 0.5", then tapered off to no effect. The sand column showed complete kill
(7 LR) at 1", then tapered off to no effect. Even with less SP applied, the clay column had
complete kill (8 LR) at all depths.
7.1.3 Test 3 SP
This test was designed to improve efficacy by increasing the concentration of the SP. Target test
conditions were six applications of 1.0 M SP (activated by 50/50 mixture with 8% hydrogen
peroxide) separated by 24-hrs, with an overall contact time of 168 hrs (seven days) for topsoil
and sand. Given the success with clay in Test 2 SP, only one application was performed. As with
Test 2 SP, to reduce the problems with foaming, the application volumes were reduced to
39
-------
0.09 mL/g, with liquid volumes being calculated from the soil density measured for the test. For
topsoil, CSPs were placed at 0.5-in depths, rather than the 1-in depth used previously.
The SP solution reactions with topsoil, sand, and clay were as described above for Test 1 and 2
SP. Due to the smaller application volumes, foaming was again better controlled, but believed
contributed to the recovery of spores from the 0" CSP for topsoil.
Spent SP solution that emerged from the column bottoms was evaluated in a more subjective
manner during this test, with volumes being estimated and oxidation reduction potential (ORP)
measurements being taken. See Appendix E for further details.
Results showed that the decontamination had complete kill (7 LR) down to 1" for the topsoil
column, then dropped off to no effect. The sand column showed a 4 LR at 1", then tapered off to
no effect. The clay column had complete kill (7 LR) at all depths, except at 5", which had a
1 LR.
7.2 Sodium Persulfate Decontamination Results
Table 21 through Table 23 show the detailed results for the decontamination of BA spores in soil
using activated SP.
40
-------
Table 21. Test 1, SP Decontamination Results
Test ID, summary
Non-Soil Ext.
Control (CFU)
Log of
Recovered CFU
from the CSP
Mean % Recovery
(vs. Non-Soil Ext.
Control)
Decontamination
Efficacy
Test 1
Soil Type: Topsoil
Test Sample, 0"
2.23 x10s
0
0
7.29
Test Sample, 1"
5.97
< 1
1.32
Test Sample, 2"
7.64
20
-0.35
Test Sample, 3"
7.53
15
-0.24
Test Sample, 4"
7.61
18
-0.31
Test Sample, 5"
7.33
24
-0.44
Positive Controls (mean)
2.23 x10s
7.29a
6 - 21b
—
Negative Controls (TC)
0
0
0
-
Negative Controls (CC)
0
0
0
-
Soil Type: Sand
Test Sample, 0"
2.23 x10s
0
0
7.75
Test Sample, 1"
0
0
7.75
Test Sample, 2"
5.95
< 1
1.80
Test Sample, 3"
6.98
4
0.77
Test Sample, 4"
7.05
5
0.70
Test Sample, 5"
7.68
21
0.07
Positive Controls (mean)
2.23 x10s
7.75a
00
I
CO
O)
O"
—
Negative Controls (TC)
0
0
0
-
Negative Controls (CC)
0
0
0
-
Soil Type: Clay
Test Sample, 0"
2.23 x10s
0
0
7.14
Test Sample, 1"
0
0
7.14
Test Sample, 2"
0
0
7.14
Test Sample, 3"
0
0
7.14
Test Sample, 4"
0
0
7.14
Test Sample, 5"
0
0
7.14
Positive Controls (mean)
2.23 x10s
7.14a
cn
I
00
O"
—
Negative Controls (TC)
0
0
0
-
Negative Controls (CC)
0
0
0
-
a Average for five positive controls is reported.
b Recovery range for five positive controls is reported.
41
-------
Table 22. Test 2, SP Decontamination Results
Test ID, summary
Non-Soil Ext.
Control (CFU)
Log of
Recovered CFU
from the CSP
Mean % Recovery
(vs. Non-Soil Ext.
Control)
Decontamination
Efficacy
Test 2
Soil Type: Topsoil
Test Sample, 0"
2.63 x10s
0
0
7.91
Test Sample, 0.5"
0
0
7.91
Test Sample, 1.0"
6.59
1
1.31
Test Sample, 1.5"
7.50
12
0.40
Test Sample, 2.0"
8.08
46
-0.18
Test Sample, 2.5"
8.27
70
-0.36
Positive Controls (mean)
2.63 x10s
7.91a
27 - 37b
—
Negative Controls (TC)
0
0
0
-
Negative Controls (CC)
0
0
0
-
Soil Type: Sand
Test Sample, 0"
2.63 x10s
0
0
7.61
Test Sample, 1"
0
0
7.61
Test Sample, 2"
6.66
2
0.96
Test Sample, 3"
4.00
0
3.61
Test Sample, 4"
7.71
20
-0.10
Test Sample, 5"
5.98
0
1.63
Positive Controls (mean)
2.63 x10s
7.61a
7 - 22b
—
Negative Controls (TC)
0
0
0
-
Negative Controls (CC)
0
0
0
-
Soil Type: Clay
Test Sample, 0"
2.63 x10s
0
0
8.16
Test Sample, 1"
0
0
8.16
Test Sample, 2"
0
0
8.16
Test Sample, 3"
0
0
8.16
Test Sample, 4"
0
0
8.16
Test Sample, 5"
0
0
8.16
Positive Controls (mean)
2.63 x10s
8.16a
46 - 65b
—
Negative Controls (TC)
0
0
0
-
Negative Controls (CC)
0
0
0
-
a Average for five positive controls is reported.
b Recovery range for five positive controls is reported.
42
-------
Table 23. Test 3, SP Decontamination Results
Test ID, summary
Non-Soil Ext.
Control (CFU)
Log of
Recovered CFU
from the CSP
Mean % Recovery
(vs. Non-Soil Ext.
Control)
Decontamination
Efficacy
Test 3
Soil Type: Topsoil
Test Sample, 0"
3.87 x10s
4.29
< 1
3.51
Test Sample, 0.5"
0
0
7.80
Test Sample, 1.0"
0
0
7.80
Test Sample, 1.5"
7.91
39
-0.11
Test Sample, 2.0"
7.98
45
-0.18
Test Sample, 2.5"
8.15
67
-0.35
Positive Controls (mean)
3.87 x10s
7.80a
20 - 47b
—
Negative Controls (TC)
0
0
0
-
Negative Controls (CC)
0
0
0
-
Soil Type: Sand
Test Sample, 0"
3.87 x10s
0
0
7.78
Test Sample, 1"
2.82
< 1
4.96
Test Sample, 2"
3.98
< 1
3.80
Test Sample, 3"
4.82
< 1
2.97
Test Sample, 4"
7.10
6
0.69
Test Sample, 5"
7.97
44
-0.19
Positive Controls (mean)
3.87 x10s
7.78a
21 - 37b
—
Negative Controls (TC)
0
0
0
-
Negative Controls (CC)
0
0
0
-
Soil Type: Clay
Test Sample, 0"
3.87 x10s
0
0
7.88
Test Sample, 1"
0
0
7.88
Test Sample, 2"
0
0
7.88
Test Sample, 3"
0
0
7.88
Test Sample, 4"
0
0
7.88
Test Sample, 5"
6.55
2
1.33
Positive Controls (mean)
3.87 x10s
7.88a
00
I
O)
O"
—
Negative Controls (TC)
0
0
0
-
Negative Controls (CC)
0
0
0
-
a Average for five positive controls is reported.
b Recovery range for five positive controls is reported.
43
-------
7.3 Sodium Persulfate Log Reduction Charts
Figure 10 through Figure 12 show results for the SP tests in graphical form. This
"decontamination efficacy" is the same (as log reduction, or LR) data that were presented earlier
in Section 7.2, but in a visual format.
Topsoil
Figure 10. Test 1, SP: 0.5 M, 0.16-0.18 mL/g, 2 applications
Topsoil
Figure 11. Test 2, SP: 0.5 M, 0.09 mL/g, 6 applications (topsoil, sand), 2 applications (clay)
44
-------
Topsoil
Figure 12. Test 3, SP: 1.0 M, 0.09 mL/g, 6 applications (topsoil, sand), 1 application (clay)
45
-------
Section 8.
Results and Performance Summary for Methyl Bromide
8.1 Methyl Bromide Test Conditions
The actual conditions measured for the MeBr test series are presented in Table 24. Highlights of
each test are given in the subsections below. Table 24 also includes summary information on
decontamination efficacy, in terms of the maximum depth at which > 6 LR was achieved for
each soil type for each test.
46
-------
Table 24. Methyl Bromide Test Conditions
Test
No.
Depth(s)
Tested
Methyl
Bromide
Cone. (mg/L)
Avg. T
(°C)
Avg. RH
(%)
Contact
Time
(hrs)
No. of
Samples3
Other
Conditions
Max depth
(inches)
achieving > 6
LR
topsoil
Max depth
(inches)
achieving > 6
LR
clayey
Max depth
(inches)
achieving > 6
LR
sandy
1
0, 1, 2, 3, 4, 5"
236 ± 14.9
19.7 ± 0.11
78.0 ± 3.26
48
TC=5
CC=5
NCTC=1
NCCC=1
None
None
None
None
Positive
Controls
NA
25.3 ± 0.17
93.3 ± 1.93
c
2
0, 1, 2, 3, 4, 5"
224 ±11.5
20.0 ± 0.21
77.3 ±2.68
65.5 (48)b
TC=5
CC=5
NCTC=1
NCCC=1
None
None
None
0
Positive
Controls
NA
24.8 ±0.15
93.6 ± 1.21
c
3
0, 1, 2, 3, 4, 5"
325 ± 33.2
19.9 ± 0.08
76.3 ± 3.27
48
TC=5
CC=5
NCTC=1
NCCC=1
None
0
None
0
Positive
Controls
NA
26.4 ± 1.80
94.3 ± 0.05
c
4
0, 1, 2, 3, 4, 5"
230 ± 16.8
20.1 ± 0.14
78.7 ± 3.43
48
TC=5
CC=5
NCTC=1
NCCC=1
Saturated
soil
None
None
None
Positive
Controls
NA
26.4 ± 0.58
94.3 ± 0.31
c
a Per soil type.
b One 0" sample of each soil type was also removed at 48 hrs.
c Positive controls had no decontaminant applied, but were collected and extracted at the end of the contact time for the test samples.
TC = Test Column.
CC = Positive Control Column.
NCTC - Negative Control, Test Column.
NCCC - Negative Control, Control Column. "None" implies that no CSPs inactivated > 6 LR, including CSPs on top of column (0 inch)
47
-------
8.1.1 Test 1 MeBr
Target MeBr concentration was 212 mg/L and target RH was >75% for a 48-hr contact time.
Results from the first MeBr test showed that the decontamination had virtually no effect for the
topsoil and clay columns, and the sand column had only a 1-2 LR. Furthermore, the 0" samples
were not killed by direct exposure to the MeBr, but instead had decontamination levels nearly
matching those of the buried samples. We were expecting that the samples on top of the soil
surfaces would be effectively decontaminated, based on previous tests with MeBr [3, 5],
(Although we acknowledge that while the CSPs on the soil surfaces did not have soil depth to
impact results, the CSPs were comprised of 1-gram of soil and this may have affected results.)
Since we achieved only minimal inactivation of spores using MeBr at fumigation conditions
which were effective in previous tests, this prompted us to investigate the cause of this
discrepancy.
A visual examination of the agar plates showed that the morphology of the BA CFU was slightly
different from previous tests, in that the colonies were clearly smaller in size. Figure 13 shows an
example of the agar plates for this experiment. Because of this, initially we considered the
possibility that our poor results with MeBr were due to inadvertently using an organism other
than BA. The photo on the left is the MeBr exposed plate at the 1 x 10"2 dilution, and the one on
the right is the unexposed control sample plate at the 1 x 10"7 dilution. Follow-up plating of a
second generation cultured from the colonies on the agar plate pictured on the left side of Figure
13 revealed that the morphology returned to normal, indicating that the spores were being
affected by the MeBr, but had not been actually killed at the time of the test ending (i.e. 48-hrs).
Another explanation is that the plate on the left has ~50X as many CFU, thus competitive
inhibition could explain the smaller colony size initially.
As an added check to provide further evidence to confirm we were in fact using our target
organism, a second PCR analysis again showed the presence of the pXOl and pX02 plasmids in
the affected morphology samples, identical to the results seen at the beginning of the study
(Appendix A). The presence of both plasmids confirms a virulent strain of BA, although not
necessarily the Ames strain. Refer to Section 3.1 and Appendix A for further documentation
regarding the BA used in this study. The BA (Ames) used in this study originated from a stock
obtained from the Biodefense and Emerging Infections (BEI) Research Resources Repository.
Additional discussions between the PI and members of the project team confirmed that the
equipment used for taking measurements of temperature, RH, and MeBr concentration was
functioning properly, and that there was no reason to suspect faulty data for these parameters.
There are several other possibilities that may explain the difference in efficacy results between
the present study and previous studies, but due to scheduling and budgetary constraints, we were
unable to further investigate these. The differences in results may be due (but not limited to) to
differences in microbiological methods, different methods to measure temperature, RH, and
MeBr concentration; different laboratory personnel; or different BA Ames strain.
48
-------
Figure 13. BA Morphology Changes after Exposure to MeBr (left) vs Unexposed (right).
8.1.2 Test 2 MeBr
Target MeBr concentration was 212 mg/L and target RH was >75%, with a target exposure time
increased to 72 hours. The actual exposure time for this test was 65.5, due to miscommunication
between lab staff. In an effort to better characterize the unexpected results from Test 1 MeBr, an
additional set of 0" samples were generated for this test. One set of 0" samples was removed at
the 48-hr contact time (as was done in Test 1), while the second set of 0" samples remained in
place until the test end.
Results showed a measurable increase in efficacy between the 48-hr and 65.5-hr results. As had
occurred in Test 1 MeBr, none of the 0" samples were completely inactivated at the 48-hr time.
For the 65.5-hr time, sand 0" samples were completely inactivated, topsoil 0" depth samples still
contained BA colonies, but were below quantitation limits (< 30 counts at the lowest dilution),
and clay 0" samples were reduced by only a 3.5 LR. As with Test 1 MeBr results, efficacy
seemed to be generally independent of depth of soil, suggesting that penetration of the MeBr gas
through the soils was not a limiting factor.
While the 0" depth was the only set of paired samples allowing direct comparison for the 48-hr
and 65.5-hr points, a general comparison can be made to the 48-hr results from Test 1 MeBr. The
Test 1 MeBr results showed only a 1-2 LR for all soils types at all depths, while the Test 2 MeBr
results were typi cally a 3-4 LR at all depths. Again note that several of the topsoil samples and
one of the sand samples were below CFU quantitation limits (< 30 counts at the highest dilution),
but were not completely inactivated. A visual examination of the agar plates showed that the
morphology of the colonies for Test 2 MeBr essentially matched the morphology of Test 1 MeBr
at both the 48-hr and 65.5-hr time periods.
8.1.3 Test 3 MeBr
In Test 3 MeBr, the target MeBr concentration was increased to 300 mg/L and target RH was
>75% for a 48-hr contact time. The increased MeBr concentration improved efficacy (compared
to Test 1 MeBr), but very few samples were effectively decontaminated.
49
-------
The topsoil sample at a 1" depth was below quantitation limits. Other depths showed a 3-4 LR.
Sand samples were reduced by 4 LR at all depths. Clay samples were reduced by 2-3 LR at all
depths. Morphology of the colonies essentially matched the morphology of the previous MeBr
tests.
8.1.4 Test 4 MeBr
Target MeBr concentration was 212 mg/L and target RH was >75% for a 48-hr contact time.
After constructing the test columns, soils were wetted to their saturation point as had been done
for Test 3 CIO2 and Test 5 CIO2 described earlier, to elicit this effect.
Similar to Test 1 MeBr, results showed that the 0" samples (topsoil, sand, and clay) still
contained BA colonies, and were reduced by < 1 LR. Water-saturated soil did not increase the
decontamination efficacy for the buried samples, as results were essentially the same as, or even
slightly worse than, the 0" samples. Results for this test were similar to results for Test 1 MeBr,
further suggesting that pre-wetting of soils does not improve efficacy.
8.2 Methyl Bromide Decontamination Results
Table 25 through Table 28 show detailed results for the decontamination of BA spores in soil
using MeBr.
50
-------
Table 25. Test 1, MeBr Decontamination Results
Test ID, summary
Non-Soil Ext.
Log of
Mean % Recovery
Decontamination
Control (CFU)
Recovered CFU
from the CSP
(vs. Non-Soil Ext.
Control)
Efficacy
Test 1
Soil Type: Topsoil
Test Sample, 0"
1.16x10s
6.49
3
1.22
Test Sample, 1"
6.44
2
1.26
Test Sample, 2"
6.81
6
0.90
Test Sample, 3"
7.00
9
0.70
Test Sample, 4"
6.82
6
0.88
Test Sample, 5"
6.67
4
1.03
Positive Controls (mean)
1.16x10s
7.70a
CO
cn
I
cn
CO
O"
—
Negative Controls (TC)
0
0
0
—
Negative Controls (CC)
0
0
0
-
Soil Type: Sand
Test Sample, 0"
1.16x10s
6.72
4
1.35
Test Sample, 1"
6.28
2
1.78
Test Sample, 2"
5.66
< 1
2.40
Test Sample, 3"
6.86
6
1.21
Test Sample, 4"
6.72
4
1.34
Test Sample, 5"
6.66
4
1.40
Positive Controls (mean)
1.16x10s
8.06a
87 — 113b
—
Negative Controls (TC)
0
0
0
—
Negative Controls (CC)
0
0
0
-
Soil Type: Clay
Test Sample, 0"
1.16x10s
6.91
7
0.61
Test Sample, 1"
6.90
7
0.62
Test Sample, 2"
6.83
6
0.69
Test Sample, 3"
6.92
7
0.60
Test Sample, 4"
6.68
4
0.84
Test Sample, 5"
6.87
6
0.65
Positive Controls (mean)
1.16x10s
7.52a
CD
I
4^
K)
O"
—
Negative Controls (TC)
0
0
0
-
Negative Controls (CC)
0
0
0
-
a Average for five positive controls is reported.
b Recovery range for five positive controls is reported.
51
-------
Table 26. Test 2, MeBr Decontamination Results
Test ID, summary
Non-Soil Ext.
Control (CFU)
Log of
Recovered CFU
from the CSP
Mean % Recovery
(vs. Non-Soil Ext.
Control)
Decontamination
Efficacy
Test 2: 224 mg/L, 65.5-hr
Soil Type: Topsoil
Test Sample, 0" (48-hr)
1.01 x 108
6.23
2
1.09
Test Sample, 0" (65.5-hr)
2.52c
<1 c
4.80c
Test Sample, 1"
3.52c
< 1c
3.80c
Test Sample, 2"
3.82
< 1
3.51
Test Sample, 3"
2.82c
< 1c
4.51c
Test Sample, 4"
5.52°
< 1c
1.80°
Test Sample, 5"
4.08
< 1
3.24
Positive Controls (mean)
1.01 x 108
7.32a
19 - 23b
—
Negative Controls (TC)
0
0
0
—
Negative Controls (CC)
0
0
0
-
Soil Type: Sand
Test Sample, 0" (48-hr)
1.01 x 108
6.72
6
1.22
Test Sample, 0" (65.5-hr)
0
0
7.94
Test Sample, 1"
3.63
< 1
4.30
Test Sample, 2"
3.66
< 1
4.28
Test Sample, 3"
3.63
< 1
4.31
Test Sample, 4"
3.82c
< 1c
4.11°
Test Sample, 5"
3.61
< 1
4.33
Positive Controls (mean)
1.01 x 108
7.94a
00
4^
I
CD
O"
—
Negative Controls (TC)
0
0
0
-
Negative Controls (CC)
0
0
0
-
Soil Type: Clay
Test Sample, 0" (48-hr)
1.01 x 108
6.87
7
0.36
Test Sample, 0" (65.5-hr)
3.71
< 1
3.52
Test Sample, 1"
4.10
< 1
3.13
Test Sample, 2"
4.10
< 1
3.13
Test Sample, 3"
3.59
< 1
3.64
Test Sample, 4"
4.20
< 1
3.03
Test Sample, 5"
4.14
< 1
3.09
Positive Controls (mean)
1.01 x 108
7.23a
CO
I
CD
O"
—
Negative Controls (TC)
0
0
0
-
Negative Controls (CC)
0
0
0
-
a Average for five positive controls is reported.
b Recovery range for five positive controls is reported.
c One or more plates of sample was below quantitation limit (< 30 CFU) at no dilution.
52
-------
Table 27. Test 3, MeBr Decontamination Results
Test ID, summary
Non-Soil Ext.
Control (CFU)
Log of
Recovered CFU
from the CSP
Mean % Recovery
(vs. Non-Soil Ext.
Control)
Decontamination
Efficacy
Test 1: 325 mg/L, 48-hr
Soil Type: Topsoil
Test Sample, 0"
1.27 x10s
0
0
7.36
Test Sample, 1"
3.52c
0C
3.84c
Test Sample, 2"
3.52
< 1
3.84
Test Sample, 3"
3.78
< 1
3.58
Test Sample, 4"
3.54
< 1
3.82
Test Sample, 5"
3.72
< 1
3.64
Positive Controls (mean)
1.27 x10s
7.36a
13 - 27b
—
Negative Controls (TC)
0
0
0
—
Negative Controls (CC)
0
0
0
-
Soil Type: Sand
Test Sample, 0"
1.27 x10s
0
0
7.98
Test Sample, 1"
3.64
< 1
4.35
Test Sample, 2"
3.83
< 1
4.15
Test Sample, 3"
3.97
< 1
4.01
Test Sample, 4"
3.98
< 1
4.00
Test Sample, 5"
3.94
< 1
4.04
Positive Controls (mean)
1.27 x10s
7.98a
0
1
00
CO
O"
—
Negative Controls (TC)
0
0
0
—
Negative Controls (CC)
0
0
0
-
Soil Type: Clay
Test Sample, 0"
1.27 x10s
4.54
< 1
2.60
Test Sample, 1"
4.39
< 1
2.75
Test Sample, 2"
4.72
< 1
2.42
Test Sample, 3"
4.62
< 1
2.52
Test Sample, 4"
4.18
< 1
2.96
Test Sample, 5"
3.91
< 1
3.23
Positive Controls (mean)
1.27 x10s
7.14a
CD
I
O)
O"
—
Negative Controls (TC)
0
0
0
-
Negative Controls (CC)
0
0
0
-
a Average for five positive controls is reported.
b Recovery range for five positive controls is reported.
c One or more plates of sample was below quantitation limit (< 30 CFU) at no dilution.
53
-------
Table 28. Test 4, MeBr Decontamination Results
Test ID, summary
Non-Soil Ext.
Control (CFU)
Log of
Recovered CFU
from the CSP
Mean % Recovery
(vs. Non-Soil Ext.
Control)
Decontamination
Efficacy
Test 4: 230 mg/L, 48-hr,
saturated soil
Soil Type: Topsoil
Test Sample, 0"
1.02 x10s
6.69
5
0.95
Test Sample, 1"
6.82
6
0.82
Test Sample, 2"
6.95
9
0.69
Test Sample, 3"
7.12
13
0.52
Test Sample, 4"
6.97
9
0.67
Test Sample, 5"
7.08
12
0.55
Positive Controls (mean)
1.02 x10s
7.64a
CO
00
I
cn
o
O"
—
Negative Controls (TC)
0
0
0
—
Negative Controls (CC)
0
0
0
-
Soil Type: Sand
Test Sample, 0"
1.02 x10s
6.96
9
0.97
Test Sample, 1"
7.02
10
0.91
Test Sample, 2"
7.08
12
0.85
Test Sample, 3"
7.08
12
0.85
Test Sample, 4"
7.13
13
0.80
Test Sample, 5"
7.26
18
0.67
Positive Controls (mean)
1.02 x10s
7.93a
I
CD
O"
—
Negative Controls (TC)
0
0
0
-
Negative Controls (CC)
0
0
0
-
Soil Type: Clay
Test Sample, 0"
1.02 x10s
6.74
5
0.60
Test Sample, 1"
6.96
9
0.39
Test Sample, 2"
6.92
8
0.42
Test Sample, 3"
6.96
9
0.38
Test Sample, 4"
7.12
13
0.22
Test Sample, 5"
7.18
15
0.16
Positive Controls (mean)
1.02 x10s
7.34a
20 - 23b
—
Negative Controls (TC)
0
0
0
-
Negative Controls (CC)
0
0
0
-
a Average for five positive controls is reported.
b Recovery range for five positive controls is reported.
54
-------
8.3 Methyl Bromide Log Reduction Charts
Figure 13 through Figure 16 show results for the MeBr tests in graphical form. This
"decontamination efficacy" (as log reduction, or LR) is the same data that were presented earlier
in Section 8.2, but in a visual format.
Test 1, MeBr
9
c 7
o
° 5
3 D
Topsoil
-1 '—
0 1 2 3 4 5
Depth (in)
Figure 14. Test 1, MeBr: 236 mg/L, 48 hrs, 78% RH
Test 2, MeBr
c
o
3
¦a
(D
cc
w
o
2 3
Depth (in)
-Topsoil
¦Clay
-Sand
Figure 15. Test 2, MeBr: 224 mg/L, 65.5 hrs, 77% RH
55
-------
Test 3, MeBr
Q
i r
V
{J D
~s
» 1 '
| < Topsoil
< Sand
^ 3 d
no '
o
1
1
1
1
1
0 1 2 3 4
Depth (in)
Figure 16. Test 3, MeBr: 325 mg/L, 48 hrs, 76% RH
Test 4, MeBr
C
o
u
13
"O
¦ =4
0 1 2 3 4 5
-Topsoil
-Clay
-Sand
Depth (in)
Figure 17. Test 4, MeBr: 230 mg/L, 48 hrs, 79% RH, [saturated soil]
56
-------
Section 9.
Clay Positive Controls Decay Tests
9.1 Clay Positive Controls Decay Test Conditions
Two clay positive controls decay tests were performed during the same time period as the CIO2
testing. Specifically, Test 4a was performed immediately following Test 4, CIO2, and Test 7a
was performed immediately following Test 7, CIO2. The actual conditions measured for these
two tests are presented in Table 29. Test 4a provided data for clay positive controls decay at low
RH, while Test 7a was conducted at high RH.
Table 29. Clay Positive Controls Decay Test Conditions
Test
No.
Depth(s) Tested
Avg. CIO2
Cone. (mg/L)
Avg. T
(°C)
Avg. RH
(%)
Time to
Analysis
(hrs)
Other
Conditions
4a
0 (Clay Positive
Controls Decay)
None
24.6 ± 0.24
29.5 ± 5.8
0, 24, 48, 72,
96, 168
Control Chamber
only, for testing
decay of clay
positive controls.
7a
0 (Clay Positive
Controls Decay
None
23.0 ± 0.26
92.0 ± 1.68
0, 24, 48, 72,
96, 168
Control Chamber
only, for testing
decay of clay
positive controls.
9.2 Clay Positive Controls Decay Test Results
Table 30 and Table 31 show results for the clay positive controls decay tests. Neither test showed
complete decay (no spores recovered, or a LR > 6) as had been observed during Test 4, CIO2.
With this in mind, it is clear that recovery of viable spores from clay is potentially inconsistent
from test-to-test, and may be dependent on variables beyond the scope of this project to fully
explore.
57
-------
Table 30. Test 4a, Clay Positive Controls Decay Results
Log of
Mean % Recovery
Log Reduction
Test ID, summary
Recovered CFUa
Recovered CFU
(vs. Non-Soil
(vs. Non-Soil
from the CSP
Extract. Control)
Extract. Control)
No CIO2 decon, 0-hr
Control Sample 1
1.13x10s
8.05
71
0.15
Control Sample 2
1.07 "
8.03
68
0.17
Control Sample 3
1.12 "
8.05
71
0.15
Positive Control15
5.67 x10s
8.75
358
-0.55
Non-Soil Extract. Control
1.58 x10s
8.20
-
-
No CIO2 decon, 24-hr
Control Sample 1
7.83 x107
7.89
50
0.30
Control Sample 2
8.23 "
7.92
53
0.27
Control Sample 3
6.87 "
7.84
44
0.35
Positive Control15
2.32 x10s
8.37
149
-0.18
Non-Soil Extract. Control
1.56 x10s
8.19
-
-
No CIO2 decon, 48-hr
Control Sample 1
5.33 x107
7.73
28
0.54
Control Sample 2
5.90 "
7.77
31
0.50
Control Sample 3
6.07 "
7.78
32
0.49
Positive Control15
1.79 x10s
8.25
95
0.02
Non-Soil Extract. Control
1.88 x10s
8.27
-
-
No CIO2 decon, 72-hr
Control Sample 1
4.07 x107
7.61
23
0.63
Control Sample 2
7.93 "
7.90
46
0.34
Control Sample 3
4.93 "
7.69
28
0.55
Positive Control15
1.80 x10s
8.26
104
-0.02
Non-Soil Extract. Control
1.74 x10s
8.24
-
-
No CIO2 decon, 96-hr
Control Sample 1
7.60 x107
7.88
45
0.35
Control Sample 2
9.10 "
7.96
54
0.27
Control Sample 3
8.50 "
7.93
50
0.30
Positive Control15
1.49 x10s
8.17
88
0.06
Non-Soil Extract. Control
1.69 x10s
8.23
-
-
No CIO2 decon, 168-hr
Control Sample 1
4.63 x10s
6.67
2.7
1.57
Control Sample 2
1.89 "
6.28
1.1
1.96
Control Sample 3
1.43 "
6.16
<1
2.08
Positive Control15
2.26 x10s
8.35
130
-0.11
Non-Soil Extract. Control
1.74 x10s
8.24
-
a All samples were inoculated with 1.58 x 108 CFU (100-|jL of 1.58 x 109 CFU/mL).
b No extraction, no soil.
58
-------
Table 31. Test 7a, Clay Positive Controls Decay Results
Log of
Mean % Recovery
Log Reduction
Test ID, summary
Recovered CFUa
Recovered CFU
(vs. Non-Soil
(vs. Non-Soil
from the CSP
Extract. Control)
Extract. Control)
No CIO2 decon, 0-hr
Control Sample 1
1.15x10s
8.06
71
0.15
Control Sample 2
9.93 x107
8.00
61
0.21
Control Sample 3
8.70 x107
7.94
54
0.27
Positive Control15
1.72 x10s
8.24
106
-0.03
Non-Soil Extract. Control
1.62 x10s
8.21
-
-
No CI02 decon, 24-hr
Control Sample 1
7.60 x107
7.88
26
0.59
Control Sample 2
1.11 x 10s
8.05
38
0.42
Control Sample 3
9.17 x 107
7.96
31
0.51
Positive Control15
3.77 x10s
8.58
128
-0.11
Non-Soil Extract. Control
2.93 x10s
8.47
-
-
No CIO2 decon, 48-hr
Control Sample 1
5.07 x107
7.70
32
0.50
Control Sample 2
6.50 "
7.81
41
0.39
Control Sample 3
4.43 "
7.65
28
0.55
Positive Control15
1.49 x10s
8.17
94
0.03
Non-Soil Extract. Control
1.58 x10s
8.20
-
-
No CIO2 decon, 72-hr
Control Sample 1
1.07 x10s
8.03
62
0.21
Control Sample 2
7.03 x107
7.85
41
0.39
Control Sample 3
9.40 x107
7.97
55
0.27
Positive Control15
1.31 x 10s
8.12
76
0.12
Non-Soil Extract. Control
1.72 x10s
8.24
-
-
No CIO2 decon, 96-hr
Control Sample 1
2.93 x107
7.47
19
0.72
Control Sample 2
2.73 "
7.44
18
0.75
Control Sample 3
3.83 "
7.58
25
0.61
Positive Control15
1.19x10s
8.07
76
0.12
Non-Soil Extract. Control
1.56 x10s
8.19
-
-
No CIO2 decon, 168-hr
Control Sample 1
5.83 x107
7.77
42
0.38
Control Sample 2
8.20 "
7.91
59
0.24
Control Sample 3
6.87 "
7.84
49
0.31
Positive Control15
4.00 x10s
8.60
286
-0.45
Non-Soil Extract. Control
1.40 x10s
8.15
-
a All samples were inoculated with 1.60 x 108 CFU (0.1 mL of 1.60 x 109 CFU/mL).
b No extraction, no soil.
59
-------
9.3 Clay Positive Controls Decay Test Log Reduction Charts
Figure 17 and Figure 18 show results for the clay positive controls decay tests in graphical form.
While these figures show some decay over the course of the 168-hr time period, a sharp decay to
at least LR of > 6 did not occur. The figures also show a rise and fall over the test period,
illustrating the variability encountered.
Test 4a: Clay Positive Control Decay Test
0.80
0.70
0.60
00
0.50
0.40
P 0.30
° 0.20
0.10
0.00
0
20
40
60
80
100
120
140
160
180
Time (hrs)
0 Sample 1 0 Sample 2 0 Sample 3
Figure 18. Test 4a: Clay Positive Control Decay Test, 30% RH
Test 7a: Clay Positive Control Decay Test
0.80
0.70
0.60
OO
0.50
0.40
o
&_
2 0.30
o
0.20
0.10
0.00
o
0
20
40
60
80
100
120
140
160
180
(O
£ Time (hrs)
< Sample 1 0 Sample 2 < Sample 3
Figure 19. Test 7a: Clay Positive Control Decay Test, 92% RH
60
-------
Section 10.Summary of Results and Conclusions
The decontaminants evaluated in this study (CIO2 gas, activated SP, and MeBr) were selected
based on their efficacious results from previous bench-scale soil decontamination tests in which
only 1-2 cm of topsoil were used in Petri dishes. In this study the scale of testing was enlarged,
vis-a-vis using 10" diameter columns filled with soil to a depth of six inches, and using three
types of soil materials: a topsoil, a sandy soil, and a clayey soil.
Due to the enlarged scale of testing, a method development program was needed to establish
experimental procedures related to preparing, placing, and quantitatively recovering BA spores
from within the large soil mass in the test columns. We settled on an approach in which BA
spores would be contained in CSPs and placed in the center of the soil columns at various depths,
ranging from 0 inches (on soil surface) down to five inches.
CIO2 gas
With CIO2 gas, topsoil was the most difficult of the soil materials to decontaminate. Of the
seven tests conducted, the maximum depth in which topsoil was effectively decontaminated was
just one inch, and this occurred in only two tests (with either extended contact time and/or
increased concentration). For the clay and sandy soils, effective decontamination was achieved in
a few of the tests down to a depth of 5 inches. Sand showed full decontamination down to 2
inches for most of the test conditions, while clay had full decontamination down to 3 inches for
most of the tests. The depth of the soil in which effective decontamination was achieved
generally increased with an increase in contact time or CIO2 concentration.
Sodium Persulfate
Three tests were performed using activated SP, with variations in the concentration, application
rate, the number of applications of this liquid decontaminant, or the contact time. The SP
solution proved to be highly reactive with topsoil, and produced a vigorous foaming reaction
upon application. Decontamination efficacy of topsoil was effective to 0.5 inch for the 0.5 M
solutions and to 1 inch for the 1.0 M solution. Sand showed less reactivity with the SP, but the
activated SP was effective to only a maximum of a 1-inch depth. Clay, on the other hand,
showed complete decontamination down to either 4 or 5 inches in all three tests.
MeBr
Four tests were conducted using MeBr at concentrations ranging from 224 to 325 mg/L, contact
times of 48 to 65.5 hrs, ambient temperature of 20 °C, and RH levels of > 75%. The
decontamination efficacy of MeBr was less than expected based on previous studies using this
fumigant. In the majority of the experiments, BA was not effectively inactivated even at the
surface of the soil columns. This prompted us to confirm again via PCR that the microorganism
we were working with was in fact BA. We are uncertain why the MeBr results in this study are
inconsistent with previous EPA decontamination studies using MeBr, although there are several
possibilities. Further research may be needed to clarify this discrepancy.
In the first test, conducted at 236 mg/1 MeBr for 48 hr, MeBr was not effective for any soil type,
and resulted only in 1-2 LR, including the CSPs on the surface. Maintaining similar test
conditions except for increasing contact time or increasing concentration resulted in only
61
-------
moderate improvement in decontamination efficacy, primarily in the range of 3-4 LR. The pre-
wetting of soils did not provide any improvement in MeBr decontamination efficacy either,
producing efficacy results similar to Test 1.
Overall, efficacy was observed to be generally higher for topsoil and sand, and lowest for the
clay soil. Lastly, although there were a few exceptions, decontamination efficacy was generally
similar across all depths for a particular soil and test condition, suggesting that penetration of the
MeBr gas through the soil matrices was not a limiting factor.
Implications
This study demonstrated that CIChgas and activated SP may be suitable candidates for
decontamination of soil contaminated with BA, depending on a number of factors, but primarily
the soil type and depth of spores. Inactivation of spores beyond 1-inch depth may require more
aggressive decontamination conditions as demonstrated in this study, depending on soil type.
For the activated SP in particular, relatively large amounts of this decontaminant were needed to
be effective for the topsoil and sandy soils. This suggests this decontamination technique may be
more suitable if implemented ex-situ (e.g., soil removed and placed in a tank without drainage),
to allow for longer and improved contact between the BA spores and the decontaminant.
Further research is needed to understand the poor performance of MeBr in the present study, in
light of its demonstrated effectiveness in previous research.
Lastly, future field-scale soil decontamination studies are recommended, in particular to assess
issues related to encountering soils (such as those with clay) that may be denser and less
permeable to decontaminants (as compared to this lab study).
62
-------
Section 11.
References
1. U. S. EPA. Inactivation of Bacillus anthracis Spores in Soil Matrices with Chlorine
Dioxide Gas. United States Environmental Protection Agency, EPA 600/R-12/517,
May 2012.
2. U.S. EPA. Evaluation of Liquid and Foam Technologies for the Inactivation of Bacillus
anthracis spores on topsoil. EPA 600/R-10/080, September 2010.
3. U.S. EPA. Decontamination of Soil Contaminated with Bacillus anthracis Spores.
Technology Evaluation Report. United States Environmental Protection Agency, EPA
600/R-13/110, August 2013.
4. U. S. EPA. Decontamination of Outdoor Materials Contaminated with Anthrax Using
Sodium Persulfate or Chloropicrin. United States Environmental Protection Agency,
EPA 600/R-15/101, July 2015.
5. Wood, J.P., Wendling, M., Richter, W., Lastivka, A., Mickelsen, L. (2016) Evaluation of
the Efficacy of Methyl Bromide in the Decontamination of Building and Interior
Materials Contaminated with B. anthracis spores. Applied and Environmental
Microbiology. April 2016; 82:7 2003-2011; doi: 10.1 128/AEM.03445-15
6. The Phase-out of Methyl Bromide, https://www.epa.gov/ods-phaseout/methyl-bromide
Accessed 8/24/17.
7. Walkley, A. and I.A. Black. 1934. An examination of the Degtjareff method for
determining soil organic matter, and a proposed modification of the chromic acid titration
method. Soil Sci. 37:29-38.)
8. U.S. Department of Agriculture. Soil Survey Manual. Issued March 2017.
9. U.S. EPA. Determining the Efficacy of Liquids and Fumigants in Systematic
Decontamination Studies for Bacillus anthracis Using Multiple Test Methods. United
States Environmental Protection Agency, EPA/600/R-10/088, December 2010.
10. Minidox-M Decontamination System, System Operations Guide, V2.02, ClorDiSys
Solutions, Inc., www.clordisvs.com. (accessed August 2017) Lebanon, NJ (2007).
11. Peroxychem Environmental Solutions. Klozur™ Persulfate Activation Guide. Activating
Klozur™ Persulfate with an 8% Hydrogen Peroxide Solution. Document 03-01-ESD-15.
Copyright 2015 PeroxyChem. Peroxychem.com/remediation.
12. U.S. EPA. Methyl Bromide Decontamination of Indoor and Outdoor Materials
Contaminated with Bacillus anthracis spores. United States Environmental Protection
Agency, EPA/600/R-14/170, August 2014.
63
-------
Appendix A.
Bacillus Anthracis Source Information and Plasmids
Analysis
A-1
-------
The source sheet (catalog number NR-411) from Biodefense and Emerging Infections (BEI)
Research Resources Repository from which all BA (Ames strain) for this study originated is
below. Laboratory staff confirmed the presence of the capB gene (testing for the presence of
plasmid pX02) and the LF gene (testing for the presence of plasmid pXOl) by PCR assay.
Presence of both plasmids, which were found, indicates a virulent strain of the B. anthracis stock
was received from BEI, as expected.
Plasmid confirmation of pXOl and pX02 in the BA employed for this program was done at the
start of the program (February 16, 2016) and again near the end of the program (June 21, 2017).
The instrument employed for these assays was a real-time PCR instrument (Bio-Rad CFX 96
Hercules, CA) using a custom-developed assay that has been validated and confirmed using
numerous strains of BA.
The initial plasmids analysis instrument print-out for pXOl and pX02, February 16, 2016 is
below. The second analysis (June, 2017) instrument print-out is listed second.
A-2
-------
fee/resources
lijODtftNSF & fcMFRGiNG INHrCTIONS
RKSi-.AR! SOURChS Ki S'OSl i'ORY
Product Information Sheet for NR-411
Bacillus anthracis, Strain Ames (A0462)
Catalog No. NR-411
For research use only. Not for human use.
Contributor:
Professor Paul Keim, Keim Genetics Laboratory, Director of
Department of Biological Sciences, College of Arts and
Sciences, Northern Arizona University, Flagstaff, Arizona
Product Description:
Bacteria Classification: Bacillaceae, Bacillus,
Bacillus cereus group
Species: Bacillus anthracis
Strain: Ames (A0462, Asc 159)
Original Source: Bacillus anthracis (B. anthracis), strain
Ames (A0462, Asc 159) was isolated from a dead cow in
Jim Hogg County, Texas in 1981.1,2 The organism was
sent to USAMRIID where it was passaged in a guinea pig
and mistakenly given the name "Ames". In the mid-1980s
B. anthracis, strain Ames was provided to the Chemical
and Biological Defence Sector and the Centre for Applied
Microbiology and Research (CAMR) at Porton Down,
Salisbury, UK. Professor Paul Keim received the bacteria
from CAMR.
B. anthracis is an aerobic, Gram-positive, spore-forming, rod-
shaped bacillus that causes the acute infectious disease
anthrax. Herbivores are the natural hosts and become
infected by consuming soil. Humans are incidentally infected
by coming into contact with infected animals or their
products. B. anthracis virulence is dependent on the
expression of a polysaccharide capsule and an extracellular
toxin that is composed of three proteins: lethal factor, edema
factor, and protective antigen.3
The presence of pX01 and pX02 in NR-411 has been
confirmed by PCR amplification of plasmid-specific
sequences from extracted DNA.
Material Provided:
Each vial contains approximately 0.5 mL of bacterial culture
in 0.5X Tryptic Soy Broth supplemented with 10% glycerol.
Note: If homogeneity is required for your intended use,
please purify prior to initiating work.
Packaging/Storage:
NR-411 was packaged aseptically in screw-capped plastic
cryovials. The product is provided frozen and should be
stored at -60°C or colder immediately upon arrival. For
long-term storage, the vapor phase of a liquid nitrogen
freezer is recommended. Freeze-thaw cycles should be
avoided.
Growth Conditions:
Media:
Tryptic Soy Broth
Tryptic Soy Agar with 5% defibrinated sheep blood
Incubation:
Temperature: 37°C
Atmosphere: Aerobic
Propagation:
4.
Keep vial frozen until ready for use; thaw slowly.
Transfer the entire thawed aliquot into a single tube of
broth.
Use several drops of the suspension to inoculate an
agar slant and/or plate.
Incubate the tubes and plate at 37°C for 24 hours.
Citation:
Acknowledgment for publications should read "The following
reagent was obtained through the NIH Biodefense and
Emerging Infections Research Resources Repository, NIAID,
NIH: Bacillus anthracis, Strain Ames (A0462), NR-411."
Biosafety Level: 3
Appropriate safety procedures should always be used with
this material. Laboratory safety is discussed in the following
publication: U.S. Department of Health and Human Services,
Public Health Service, Centers for Disease Control and
Prevention, and National Institutes of Health. Biosafety in
Microbiological and Biomedical Laboratories. 5th ed.
Washington, DC: U.S. Government Printing Office, 2007; see
www.cdc.aov/od/ohs/biosftv/bmbl5/bmbl5toc.htm. This
publication recommends that all persons working in or
entering laboratory or animal care areas where frequent
activities with clinical specimens or diagnostic cultures of
Bacillus anthracis are being conducted should have
documented evidence of satisfactory vaccination.
Disclaimers:
You are authorized to use this product for research use only.
It is not intended for human use.
Use of this product is subject to the terms and conditions of
the BEI Resources Material Transfer Agreement (MTA). The
MTA is available on our Web site at www.beiresources.ora.
While BEI Resources uses reasonable efforts to include
accurate and up-to-date information on this product sheet,
neither ATCC nor the U.S. Government make any
warranties or representations as to its accuracy. Citations
from scientific literature and patents are provided for
informational purposes only. Neither ATCC® nor the U.S.
Government warrants that such information has been
confirmed to be accurate.
This product is sent with the condition that you are
responsible for its safe storage, handling, use and disposal.
ATCC® and the U.S. Government are not liable for any
Biodefense and Emerging Infections Research Resources Repository E-mail: contact@beiresources.org
www.beiresources.ora Tel: 800-359-7370
Fax: 703-365-2898
© 2004/2005/2007/2009/2010 American Type Culture Collection (ATCC). All rights reserved. :«¦;.-_i2ajo;cio
Page 1 of 2
A-3
-------
te/resources
Brrsrarcm rtsoi¦ rcr'' V J ,-«y's Product Information Sheet for NR-411
damages or injuries arising from receipt and/or use of this
product. While reasonable effort is made to ensure
authenticity and reliability of materials on deposit, the U.S.
Government, ATCC®, their suppliers and contributors to BEI
Resources are not liable for damages arising from the
misidentification or misrepresentation of products.
Use Restrictions:
This material is distributed for internal research, non-
commercial purposes only. This material, its product or its
derivatives may not be distributed to third parties. Except as
performed under a U.S. Government contract, individuals
contemplating commercial use of the material, its products or
its derivatives must contact the contributor to determine if a
license is required. U.S. Government contractors may need
a license before first commercial sale.
References:
1. Kenefic, L. J., et al. "Texas Isolates Closely Related to
Bacillus anthracis Ames." Emera. Infect. Pis. 14 (2008):
1494-1496. PubMed: 18760033.
2. Read, T. D., et al. The Genome Sequence of Bacillus
anthracis Ames and Comparison to Closely Related
Bacteria." Nature 423 (2003): 81-86. PubMed:
12721629. GenBank: AE016879.
3. Oncil, S., S. Oncii, and S. Sakarya. "Anthrax - An
Overview." Med. Sci. Monit. 9 (2003): RA276-RA283.
PubMed: 14586293.
4. Van Ert, M. N., et al. "Strain-Specific Single-Nucleotide
Polymorphism Assays for the Bacillus anthracis Ames
Strain." J. Clin. Microbiol. 45 (2007): 47-53. PubMed:
17093023.
5. Keim, P., et al. "Multiple-Locus Variable-Number
Tandem Repeat Analysis Reveals Genetic Relationships
within Bacillus anthracis." J. Bacteriol. 182 (2000): 2928-
2936. PubMed: 10781564.
ATCC® is a trademark of the American Type Culture
Collection.
Biodefense and Emerging Infections Research Resources Repository E-mail: contact@beiresources.org
www.beiresources.org Tel: 800 359-7370
Fax: 703-365-2898
©2004/2005/2007/2009/2010 American Type Culture Collection (ATCC). All rights reserved.
Page 2 of 2
A-4
-------
Initial plasmids analysis for pXOl and pX02, February 16, 2016.
CD
XC0620160216 1_B A2_B A3_Quant_DK.pcrd
2/16/2016 2:37 PM
Report Information
User: BioRad/lgreene
Data File Name: XC0620160216 1 _BA2_B A3_Quant_DK.pcrd
Data File Path: C:\Users\Public\DocumentsVBio-Rad\CFX\Users\data
Well Group Name: All Wells
Report Differs from Last Save: Yes
Run Setup
Run Information
Run Date: 2/16/2016 12:20 PM
Run User: lgreene
Run Type: User-defined
Plate File: Florida Ba Test.pltd
ID:
Notes:
Sample Volume: 25
Temperature Control Mode: Calculated
Lid Temperature: 105
Base Serial Number: CT013497
Optical Head Serial Number: 785BR11154
Protocol
1: 50.0°C for 2:00
2: 95.0°C for 11:00
3: 95.0°C for 0:15
Plate Read
4: 56.0°C for 1:00
5: GOTO 3, 39 more times
Plate Display
2
3
4
5
6
7
8
9
10
11
12
A
Unk
BA2
BA2 1:10
01
Unk
BA2
BA2 1:10
02
Unk
BA2
BA2 1:10
03
Unk
BA2
BA2 1:100
_01
Unk
BA2
BA2 1:100
_02
Unk
BA2
BA2 1:100
_03
Unk
BA2
BA2 1:100
0_01
Unk
BA2
BA2 1:100
0_02
Unk
BA2
BA2 1:100
0 03
Unk
BA2
BA2 1:100
00 001
Unk
BA2
BA2 1:100
00_002
Unk
BA2
BA2 1:100
00 003
B
Unk
BA2
BA2 1:100
Q00_001
Unk
BA2
BA2 1:100
Q00_002
Unk
BA2
BA2 1:100
000_003
Unk
BA2
BA2 PC 0
Unk
BA2
BA2 PC 0
2
Unk
BA2
BA2 NTC
01
Unk
BA2
BA2 NTC
02
C
Unk
BA3
BA3 1:10
01
Unk
BA3
BA3 1:10
02
Unk
BA3
BA3 1:10
03
Unk
BA3
BA3 1:100
_01
Unk
BA3
BA3 1:100
_02
Unk
BA3
BA3 1:100
_03
Unk
BA3
BA3 1:100
0_01
Unk
BA3
BA3 1:100
0 02
Unk
BA3
BA3 1:100
0 03
Unk
BA3
BA3 1:100
00 001
Unk
BA3
BA3 1:100
00 002
Unk
BA3
BA3 1:100
00 003
D
Unk
BA3
BA3 1:100
000 001
Unk
BA3
BA3 1:100
000 002
Unk
BA3
BA3 1:100
000 003
Unk
BA3
BA3 PC 0
1
Unk
BA3
BA3 PC 0
2
Unk
BA3
BA3 NTC
01
Unk
BA3
BA3 NTC
02
E
A-5
-------
Plate Display
2
3
4
5
6
7
8
9
10
' 11 I 12
F
G
H
I
Quantification
Step #: 3
Analysis Mode: Target
Cq Determination: Single Threshold
Baseline Method:
BA2: Auto Calculated
BA3: Auto Calculated
Threshold Setting:
BA2: 294.70, Auto Calculated
BA3: 306.43, Auto Calculated
Amplification
4000
2000
0
10
20
Quantification Data
Well
Fluor
Target
Content
Sample
Cq
Cq
Mean
Cq
Sid.
Dev
A01
FAM
BA2
Unkii
BA21:10 01
23.70
23.70
0.000
A02
FAM
BA2
Unkn
BA2_1:10_02
23.67
23.67
0.000
AO 3
FAM
BA2
Unkn
BA2J:10_03
23.57
23.57
0.000
A 04
FAM
BA2
Unkn
BA2 1:100 01
27.20
27.20
0.000
A05
FAM
BA2
Unkn
BA2_1:100_02
27.17
27.17
0.000
A06
~fam1
R A?
Unkn
BA2_1;100_03
27.17
27.17
0.000
AO 7
FAM
BA2
Unkn
BA2_1:1000_01
30.85
30.85
0.000
A08
FAM
BA2
Unkn
BA2 1:1000 02
31.06
31.06
0.000
A09
FAM
BA2
Unkn
BA2_i:1000_03
30.86
30.86
0.000
A10
FAM
BA2
Unkn
BA2 1:10000 001
34.53
34.53
0.000
All
FAM
BA2
Unkn
BA2_1:10000_002
34.53
34.53
0.000
A12
FAM
BA2
Unkn
BA2J:10000_003
34.53
34.53
0.000
A-6
-------
Quantification Data
Well
Fluor
X arget
Content
Sample
Cq
Cq
Mean
Cq
Std.
Dev
BOl
FAM
BA2
IJ ilkil
B A2_ 1:1OOOOOOO1
37.58
37.58
O.OOO
B02
FAM
BA2
Unkn
B A2 1:100000_002
38.99
38.99
O.OOO
B03
FAM
BA2
Unkn
BA2 1:1OOOOO 003
37.67
37.67
O.OOO
B04
FAM
BA2
Unkn
BA2_PC_01
38.93
38.93
O.OOO
B05
FAM
BA2
Unkn
BA2 PC 02
38.72
38.72
O.OOO
B06
FAM
BA2
Unkn
BA2 NTC Ol
N/A
O.OO
O.OOO
B07
FAM
BA2
Unkn
BA2 NTC 02
N/A
O.OO
O.OOO
C'Ol
FAM
BA3
Unkn
B A3_ 1:10_01
22.78
22.78
O.OOO
C02
FAM
BA3
Unkn
B A3_ 1:10_02
22.75
22.75
O.OOO
C03
FAM
BA3
Unkn
BA3_1:10_03
22.70
22.70
O.OOO
C04
FAM
BA3
Unkn
BA31:1OOO1
26.33
26.33
O.OOO
COS
FAM
BA3
Unkn
B A3 1:100 02
26.37
26.37
O.OOO
C06
FAM
BA3
Unkn
BA3 1:1OO 03
26.39
26.39
O.OOO
C07
FAM
BA3
Unkn
BA3 1: lOOO Ol
30.03
30.03
O.OOO
COS
FAM
BA3
Unkn
BA3 1:1000 02
30.03
30.03
O.OOO
C09
FAM
BA3
Unkn
BA3 1:1000_03
30.07
30.07
O.OOO
CIO
FAM
BA3
Unkn
BA3_ 1:10000 OO 1
33.92
33.92
O.OOO
CI 1
FAM
BA3
Unkn
BA3 1:10000 002
33.89
33.89
O.OOO
C12
FAM
BA3
Unkn
BA3_1:10000 003
34.05
34.05
O.OOO
DO 1
FAM
BA3
Unkn
BA3_1:100000_001
37.14
37.14
O.OOO
D02
FAM
B A3
Unkn
BA3 1:1 OOOOO 002
37.48
37.48
O.OOO
D03
FAM
B A3
Unkn
BA3 1:1 OOOOO 003
37.1 1
37.11
O.OOO
D04
FAM
BA3
Unkn
BA3 PC Ol
36.08
36.08
O.OOO
D05
FAM
BA3 [
Unkn
B A3_PC_02
35.24
35.24
O.OOO
D06
FAM
BA3
Unkn
BA3NTCO1
N/A
O.OO
O.OOO
DO 7
FAM
BA3
Unkn
BA3NTC02
N/A
O.OO
O.OOO
QC Parameters
Data
Description
Value
Use
Results
Exclude
Wells
All
excluded
wells
Negative
control with
a Cq less
than
38
True
False
NTC with a
Cq less than
38
True
False
NRT with a
Cq less than
38
True
False
Positive
control with
a Cq greater
than
30
True
False
A-7
-------
Data
Description
Value
Use
Results
Exclude
Wells
All
excluded
wells
Unknown
without a
Cq
N/A
True
BA2.B6,
B7.
BA3:D6,
D7.
False
Standard
without a
Cq
N/A
True
False
Efficiency
greater than
110.0
True
Efficiency
less than
90.0
True
Std Curve
R "2 less
than
0.980
True
Replicate
group Cq
Std Dev
greater than
0.20
True
False
A-8
-------
Plasmids confirmation reanalysis of pXOl and pX02, June 21, 2017.
XC0520170621^2 JB A2_B A3__DK.pcrd
7/7/2017 1 1:06 AM
Report Information
User: BioRad/dkiekel
Data File Name: XC0520170621 2_BA2_BA3JDK.pcrd
Data File Path: C:\Uscrs\mducote\AppData\Local\Microsofl\Windows\Temporary Internet
Files\C ontent. Outlook\GKJT 4 Y FC
Well Group Name: All Wells
Report Differs from Last Save: No
Notes
Run Setup
Run Information
Run Date: 6/21/2017 10:46 AM
Run User: dkiekel
Run Type: User-defined
ID:
Notes:
Sample Volume: 25
Temperature Control Mode: Calculated
Lid Temperature: 105
Base Serial Number: CT013504
Optical Head Serial Number: 785BR11128
Protocol
1: 50.0°C for 15:00
2: 95.0°C for 2:00
3: 95.0°C for 0:15
4: 60.0°C for 1:00
Plate Read
5: GOTO 3, 39 more times
Plate Display
2
3
4
5
6
7
8
9
10
11
12
A
Unk
Unk
Unk
Unk
Unk
Unk
Unk
Unk
Unk
Unk
Unk
Unk
BA2
BA2
BA2
BA2
BA2
BA2
BA2
BA2
BA2
BA2
BA2
BA2
BA3
BA3
BA3
BA3
BA3
BA3
BA3
BA3
BA3
BA3
BA3
BA3
S1-1
S1-2
S1-3
S2-1
S2-2
S2-3
PC-1
PC-2
PC-3
NTC-1
NTC-2
NTC-3
B
C
D
E
F
G
H
A-9
-------
Quantification
Step #: 4
Analysis Mode: Fluorophore
Cq Determination: Single Threshold
Baseline Method:
Cy5: Auto Calculated
Tex 615: Auto Calculated
Threshold Setting:
Cy5: 88.16, Auto Calculated
Tex 615: 52.57, Auto Calculated
300
200
100
0
15
JD
25
30
35
Quantification Data
Well
Fluor
T arget
Content
Sample
Cq
Cq
Mean
Cq
Std.
Dev
A01
Cy5
BA3
Unkn
Sl-1
21.93
21.93
0.000
A02
Cy5
BA3
Unkn
Sl-2
21.93
21.93
0.000
AO 3
Cy5
BA3
Unkn
SI-3
21.72
21.72
0.000
A04
Cy5
BA3
Unkn
S2-1
21.70
21.70
0.000
A05
Cy5
BA3
Unkn
S2-2
21.81
21.81
0.000
A06
Cy5
BA3
Unkn
S2-3
21.76
21.76
0.000
A07
Cy5
BA3
Unkn
PC-1
36.69
36.69
0.000
AO 8
Cy5
BA3
Unkn
PC-2
36.26
36.26
0.000
A09
Cy5
BA3
Unkn
PC-3
36.61
36.61
0.000
A10
Cy5
BA3
Unkn
NTC-1
N/A
0.00
0.000
A1 1
Cy5
BA3
Unkn
NTC-2
N/A
0.00
0.000
A12
Cy5
BA3
Unkn
NTC-3
N/A
0.00
0.000
A01
Tex
615
BA2
Unkn
Sl-1
34.54
34.54
0.000
A02
Tex
615
BA2
Unkn
Sl-2
34.81
34.81
0.000
A03
Tex
615
BA2
Unkn
SI-3
35.18
35.18
0.000
A-10
-------
Quantification Data
Well
Fluor
T arget
Content
Sample
Cq
Cq
Mean
Cq
Std.
Dev
A04
Tex
615
BA2
Unkn
S2-1
34.70
34.70
0.000
AOS
Tex
615
BA2
Unkn
S2-2
33.72
33.72
0.000
A06
Tex
615
BA2
Unkn
S2-3
34.76
34.76
0.000
A07
Tex
615
BA2
Unkn
PC-1
34.72
34.72
0.000
AOS
Tex
615
BA2
Unkn
PC-2
34.43
34.43
0.000
A09
Tex
615
BA2
Unkn
PC-3
34.59
34.59
0.000
A10
Tex
615
BA2
Unkn
NTC-1
N/A
0.00
0.000
A11
Tex
615
BA2
Unkn
NTC-2
N/A
0.00
0.000
A12
Tex
615
BA2
Unkn
NTC-3
N/A
0.00
0.000
A-11
-------
Appendix B.
Soil Properties
B-1
-------
Soil density was measured by weighing a 200-mL volume of each soil type in a beaker. Soil
moisture was measured according to ASTM D 2974-87 prior to each test. Samples were
weighed, dried in an oven for >16 hrs at 105 ± 5°C, and weighed again, with the moisture
calculated as:
Moisture Content (%) = [(A-B) x 100]/A
A = mass of the as-received sample, g
B = mass of the oven-dried sample, g
B-2
-------
Table B-1. Ongoing Soil Properties Checks (density, moisture)
Density
(g/mL)
Moisture
(%)
Test/Date
Topsoil
Sand
Clay
Topsoil
Sand
Clay
Notes
Initial (preliminary tests)
1.00
1.39
1.06
-
-
-
Test 1, CI02 (08/05/16)
-
-
-
41.5
5.5
22.3
Density not measured; assumed to be unchanging from Test 1
Test 2, CI02 (08/24/16)
-
-
-
41.9
3.0
22.9
Density not measured; assumed to be unchanging from Test 1
Test 3, CI02 (09/06/16)
1.01
1.49
1.11
27.5
8.4
26.3
Values are initial, unwetted. For testing, soils were wetted to near
saturation by adding 0.08 mL/g (topsoil), 0.04 mL/g (clay), 0.10
mL/g (sand).
Test 4, CI02 (09/26/16)
0.97
1.25
1.27
21.5
5.2
28.4
Test 5, CI02 (11/28/16)
1.02
1.14
1.28
21.6
4.7
15.1
Values are initial, unwetted. For testing, soils were wetted to near
saturation by adding 0.08 mL/g (topsoil), 0.04 mL/g (clay), 0.10
mL/g (sand).
Test 6, CI02 (12/06/16)
0.93
1.14
1.01
19.9
4.4
14.9
Test 7, CI02 (12/21/16)
1.03
1.39
1.16
20.4
3.5
15.6
Uncompressed.
Test 7, CI02 (12/21/16)
1.37
1.59
1.55
20.4
3.5
15.6
Compressed. Same soil as uncompressed, measured after
compression.
Test 1, SP (02/06/17)
1.02
1.16
1.13
20.0
4.2
10.8
Test 2, SP (03/02/17)
1.01
1.18
1.14
20.3
4.2
10.7
Test 3, SP (04/11/17)
0.98
1.26
1.18
19.6
5.1
10.7
Test 1, MeBr (06/06/2017)
0.97
1.18
1.13
21.6
3.4
10.8
Test 2, MeBr (06/19/2017)
0.96
1.19
1.04
24.1
2.1
8.0
Test 3, MeBr (07/05/2017)
1.00
1.18
1.06
21.9
2.7
8.4
Test 4, MeBr (07/19/2017)
1.02
1.22
1.13
25.7
4.5
8.0
Values are initial, unwetted. For testing, soils were wetted to near
saturation by adding 0.08 mL/g (topsoil), 0.04 mL/g (clay), 0.10
mL/g (sand).
B-3
-------
08/18/2016 Agvise Laboratories Series II Soil Characterization Test:
Type
Density3
(g/mL)
Moisture13
(%)
% OMc
% Sand/Silt/Clay
USDA Textural
Class
Cation Exchange
Capacity
(meq/100 g)
pH
(Water)
Buffer pH
(Adams-Evans)
Topsoil (Oldcastle)
0.81
54.1
33.9
64/18/18
Sandy Loam
57.0
8.1
7.9
Sand
(Play Sand)
1.64
2.7
0.1
100/0/0
Sand
4.3
8.4
8.0
Clay
(Crimson Clay)
1.11
31.9
0.2
40/16/44d
Clay
8.3
4.7
7.2
a Disturbed Bulk Density.
b Field capacity at 1/3 bar.
c Organic matter by Walkley-Black method.
d Supplier (Better Baseball) claims mix is 25/40/35 but cites no source or method.
08/18/2016 Agvise Laboratories Series II Soil Characterization Test, Base Saturation Data:
Type
K
(%, ppm)
Ca
(%, ppm)
Mg
(%, ppm)
Na
(%, ppm)
H
(%, ppm)
Topsoil (Oldcastle)
27.8
6,178
42.5
4,839
14.5
989
14.6
1,909
0.7
4
Sand
(Play Sand)
2.5
42
80.7
688
12.7
65
2.1
21
1.9
1
Clay
(Crimson Clay)
0.9
29
16.5
274
4.5
45
0.9
17
77.2
64
B-4
-------
Appendix C.
CSP Development
C-1
-------
Carrier soil packet (CSP) development was performed through a multi-step process that
evaluated the types of materials and techniques that would give good recovery, yet allow
permeation of the decontaminant. Some of the method development steps used a commercially-
available product containing a small stainless steel disc impregnated with B. atrophaeus spores
and sealed within a Tyvek packet. This commercial product, produced by Mesa Labs, is known
as the Apex biological indicator, or BI, while the custom-made packets containing soil spiked
with BA were referred to as CSPs. Using the commercial Bis as the basis for developing BA-
spiked CSPs, six steps were performed for CSP development:
1. Develop a method for removing the Tyvek BI envelopes from the soil columns.
2. Perform extractions of B. atrophaeus spores from the stainless steel carriers within the
Bis (after sitting in soil).
3. Develop a method for aseptic removal of discs from BI Tyvek envelopes.
4. Determine penetration of SP liquid into Tyvek and PVDF envelopes.
5. Develop Tyvek and PVDF envelopes for carrier soil packets.
6. Perform extractions of B. atrophaeus spores from spiked autoclaved soils placed within
the soil contained in a column.
A summary of all the CSP development tests is presented in Table C-l. The resulting data from
these tests are described in the sections below.
Table C-1. Summary of CSP Development Tests
Date
Soil Type
Columns
Description
Location of
Additional
Details
Extraction of 6. atrophaeus from the BI Stainless Steel Carriers
01/06/2016
Topsoil
NA
BI recovery from Tyvek in soil with and
w/o heat shock.
Table C-2
02/12/2016
NA
NA
BI recovery test.
Table C-3
SP Soak (no soil)
02/24/2016
NA
Glass Jars
Bis in Tyvek in SP for 7 days.
Table C-4
05/11/2016
NA
Glass Jars
Bis in Tyvek in SP for 5, 10, 30, 60
min.
Table C-5
06/04/2016
NA
Glass Jars
Bis in PVDF in SP for 5, 10, 30, 60
min.
On file.
06/22/2016
NA
Glass Jars
Bis in PVDF in SP for 5, 10, 30, 60
min.
Table C-6
06/27/2016
NA
Glass Jars
Bis in PVDF in SP for 4 to 48 hrs.
Table C-7
PVDF Development Tests
05/18/2016
NA
NA
PVDF and PC filters immersed in SP.
Visual exam
05/20/2016
NA
NA
PVDF, PC, and Tyvek materials wetted
with a drop of SP.
Visual exam
Extraction of B. atrophaeus from Spiked Autoclaved Soil
05/16/2016
All types
NA
B. atrophaeus-sp\ked soil recovery test
in Tyvek.
Table C-8
06/04/2016
All types
NA
B. atrophaeus -spiked soil recovery
test in PVDF.
Table C-9
C-2
-------
Tyvek Envelope Removal Method Development
Tests were conducted to assess procedures to remove the Tyvek envelopes from soil columns.
Preliminary tests included attaching a wire or dental floss to the envelope and pulling it up
through the soil column. After using this method for the first wall effects test, however, it was
abandoned in favor of simply tipping the soil column on its side, carefully dumping the soil into
a second bucket, and removing each BI envelope by hand as it reached the top surface.
Extraction of B. atrophaeus from the BI carriers
Using the Mesa Labs protocol, the extraction efficiency of B. atrophaeus from the BI stainless
steel carriers within the Tyvek envelopes was evaluated. One experiment was performed on
01/06/2016 using four Bis that sat in soil for 5 days, two of which were heat-shocked for 10 min
at 80-85°C, while two were not (Table C-2).
Table C-2. BI Extraction Efficacy (5 days in soil, with and without heat shock)
Sample
Type
Method
Observed
(CFU)
Recovery
(%)a
1
Apex BI in Tyvek
Standard
2.67 x 10®
95.4
2
Apex BI in Tyvek
Standard
1.64 x 10s
58.6
3
Apex BI in Tyvek
Standard + Heat Shock
1.86 x 10s
66.4
4
Apex BI in Tyvek
Standard + Heat Shock
1.02 x 10s
36.4
a BI standard is 2.8 x 10s CFU, obtained from certificate of analysis (COA).
The standard extraction procedure using the Mesa Labs protocol is as follows:
1. Cut Tyvek, and aseptically dump BI disc into 50 mL tube containing 10 mL sterile water.
2. Sonicate tube for 15 min.
3. Heat shock (optional) by placing tube in water bath at 80-85°C for 10 minutes.
4. Analyze by plate counting/serial dilution.
A second experiment was performed on 02/12/2016 using four Bis. Two Bis were removed from
the Tyvek envelopes and directly recovered using the procedure above. One BI envelope was
stirred in sterile water for 10 minutes prior to recovery. One BI was soaked in bleach for
10 minutes prior to recovery. Table C-3 presents the results.
Table C-3. BI Extraction Efficacy
Sample
Type
T reatment
Observed
(CFU)
Recovery
(%)a
1
Apex BI in Tyvek
Simple recovery
1.50 x 10s
53.6
2
Apex BI in Tyvek
Simple recovery
1.53 x 10s
54.6
3
Apex BI in Tyvek
Stir in sterile water 10 min,
then recover.
1.42 x 10s
50.8
4
Apex BI in Tyvek
Soak in bleach for 10 min,
then recover.
0
0
a BI standard is 2.8 x 10s CFU, obtained from COA.
C-3
-------
Aseptic Removal of BI Discs from Tyvek Envelopes
Procedures were developed for aseptic removal of the stainless steel carriers from the BI Tyvek
and (later) PVDF envelopes. These procedures were then adapted for use with BA inoculated
soil later in the project.
The envelopes were opened by peeling apart the two papers near the top of the envelope or by
cutting with sterile scissors. The stainless steel BI was removed by tipping the opened Tyvek
envelope and allowing the BI disc to fall out, or by grasping the disc by sterile forceps. The BI
discs were placed in 1 mL of sterile phosphate buffered saline extraction buffer containing 0.1%
Triton X-100 surfactant and agitated at -200 rpm at room temperature for 15 minutes. Samples
were diluted and plated as described in the main body of the report and incubated at ~35°C for
16-20 hours. B. atrophaeus was present as orange colonies and enabled discrimination from
contaminants.
Determining Penetration of Sodium Persulfate into Tyvek and PVDF
Envelopes
It was anticipated that SP decontamination would require extended exposure times (1 to 7 days).
Hence, a 5-day test was performed on 02/24/2016 to determine minimum exposure time in which
persulfate permeated the commercially-available Tyvek envelope Bis. Glass jars were set up and
filled with ~1" depth of activated SP (0.5 M, 1:1 with 8% hydrogen peroxide) to allow for
complete immersion of the BI. One BI was placed in each jar and was briefly swirled. Due to the
hydrophobic nature of Tyvek, the Bis tended to float but eventually were permeated. For
comparison, a set of Bis soaked in sterile water was also included. The permeation data from
these experiments are summarized below.
Table C-4. Bis Soaked in Activated Sodium Persulfate (Tyvek Envelope)
Sample
Envelope
T reatment
Observed
(CFU)
Recovery
(%)a
Decon.
Efficacy
(LR)a
15-sec exposure
Tyvek
Activated SP
5.93 x 105
21
0.68
1-day exposure
Tyvek
Activated SP
0
0
6.45
4-day exposure
Tyvek
Activated SP
0
0
6.45
5-day exposure
Tyvek
Activated SP
0
0
6.45
15-sec exposure
Tyvek
Sterile water
1.72 x 10s
62
0.21
1-day exposure
Tyvek
Sterile water
5.43 x 105
19
0.72
4-day exposure
Tyvek
Sterile water
5.23 x 105
19
0.73
5-day exposure
Tyvek
Sterile water
4.53 x 105
16
0.79
a Based on BI standard of 2.80 x 10s CFU (from supplier's COA).
Results showed complete kill (6.45 LR) at the 1-day mark, while the 15-sec sample only showed
partial kill of 0.68 LR. The data from these tests does not unambiguously determine if slow
permeation or slow decontamination rates are the cause of complete kill on or before 24 hours of
exposure. In an attempt to determine the source of the time to kill, a test was performed on
05/11/2016 using Bis contained within the Tyvek and also removed from the Tyvek, in which
100 |iL of SP was directly applied onto the spore-laden concave side of the disc. This application
allowed the surface area of the disc to be entirely covered with liquid, but not overflow. Discs
kept in Tyvek were gently swirled in liquid as had been done during the previous soak test.
C-4
-------
Results show that the persulfate is, in fact, fast-acting upon direct contact with the B. atrophaeus
contained on the BI disc. This test demonstrated that the rate of persulfate permeation through
the Tyvek envelope is significant and affects the actual decontamination rate, and thus an
alternative material was investigated.
Table C-5. Bis in Activated Sodium Persulfate (Direct to Disk vs. Tyvek Envelope)
Sample
Envelope
T reatment
Observed
(CFU)
Recovery
(%)a
Decon.
Efficacy
(LR)a
5-min exposure
none
Activated SP
0
0
6.45
10-min exposure
none
Activated SP
0
0
6.45
30-min exposure
none
Activated SP
0
0
6.45
60-min exposure
none
Activated SP
0
0
6.45
30-min exposure
Tyvek
Activated SP
6.00 x105
21
0.67
60-min exposure
Tyvek
Activated SP
6.47 x104
2
1.64
15-sec exposure
Tyvek
Sterile water
1.10x10s
39
0.41
60-min exposure
Tyvek
Sterile water
1.23 x105
4
1.36
a Based on BI standard of 2.80 x 10s CFU (from supplier's COA).
Porous polyvinylidene fluoride (PVDF) was chosen as a potential candidate based on its known
aqueous permeation rate identified by the manufacturer. Envelopes were custom made by using
90-mm, 0.22-|im, hydrophilic, filters from Millipore (GVWP 00010). Envelopes were made by
folding the filter in half, heat sealing two sides, aseptically transferring a BI disk by pouring it
from a cut Tyvek envelope, and heat-sealing the third side.
A decontamination efficacy test was performed on 06/22/2016 using BI disks in PVDF
envelopes prepared as described. Results showed increasing decontamination efficacy with time
for the PVDF envelopes over the time period of 5 minutes to 60 minutes (Table C-6). Since a
complete kill (LR -6.5) was not seen within the hour, however, a follow-up test was performed
on 06/27/2016 where time periods of 4 to 48 hours were tested. These results are shown in
Table C-7.
Table C-6. Bis in Activated Sodium Persulfate, 5 to 60 min (PVDF vs. Tyvek Envelope)
Sample
Envelope
T reatment
Observed
(CFU)
Recovery
(%)a
Decon.
Efficacy
(LR)b
5-min exposure
PVDF
Activated SP
3.73 x10s
133
0.17
10-min exposure
PVDF
Activated SP
1.97x10®
70
0.45
30-min exposure
PVDF
Activated SP
3.70 x105
13
1.17
60-min exposure
PVDF
Activated SP
7.33x103
< 1
2.88
30-min exposure
Tyvek
Activated SP
2.63x10®
94
0.32
60-min exposure
Tyvek
Activated SP
3.07x10®
110
0.25
60-min exposure
PVDF
Sterile water
5.90x10®
211
-0.03
Control sample
none
Direct to sterile water
5.50x10®
196
NA
a Based on BI standard of 2.80 x 10s CFU (from supplier's COA).
b Based on recovery of control sample and not the COA from the BI supplier.
C-5
-------
Table C-7. Bis in Activated Sodium Persulfate, 4 to 48 hr (PVDF only)
Sample
Envelope
Treatment
Observed
(CFU)
Recovery
(%r
Decon.
Efficacy
(LR)b
4-hr exposure
PVDF
Activated SP
0
0
6.47
18-hr exposure
PVDF
Activated SP
0
0
6.47
24-hr exposure
PVDF
Activated SP
0
0
6.47
48-hr exposure
PVDF
Activated SP
0
0
6.47
Control sample
none
Direct to sterile water
2.93x10®
105
NA
a Based on Bl standard of 2.80 x 106 CFU (from supplier's COA).
b Based on recovery of control sample and not the COA from the Bl supplier.
Developing Tyvek and PVDF Envelopes for Carrier Soil Packets
Physical evaluations (size, shape, material strength) were performed to characterize the Tyvek
envelopes used for making CSPs for the spiked soils. A 1-gram quantity of soil was found to be
appropriate. Heat-sealing the envelopes with a Uline lab heat sealer was found to be sufficient.
Upon determining that Tyvek was not the best choice for SP testing, an alternative material was
sought. A brief review of suppliers showed that polyvinylidene fluoride (PVDF) and
polycarbonate (PC) were available as 0.2-jim, hydrophilic filter media from Millipore. Samples
of both PVDF and PC were obtained and tested for physical strength and ability to heat seal.
Both materials were immersed in activated SP and showed no deterioration after 12 hours.
Finally, both materials were tested for penetration of activated SP by placing a single drop of
liquid on each, and observing the rate of liquid flow through the filter. While both materials were
hydrophilic and allowed penetration, PVDF was clearly superior. Figure C-l is a picture of the
PC (left), PVDF (upper and middle right), and Tyvek (lower right) five minutes after the drop
was placed. Note that complete disbursement of the water drop occurs on the PVDF, while for
the other materials, a portion of the drop was still intact.
Because of the data in this and the previous section, all decontamination tests described in this
report using SP employed PVDF envelopes. The commercially-available Tyvek Bis were simply
cut open and the disc was poured into a PVDF envelope, then heat sealed in place.
C-6
-------
Figure C-1. Persulfate Penetration into PC, PVDF, and Tyvek
Extracting B. atrophaeus from Spiked Autoclaved Soil
Two sets of tests were done to evaluate recovery of B. atrophaeus-spiked soil. One set of tests
was performed using 1-gram quantities of autoclaved soil in Tyvek envelopes. After preliminary
wall-effects tests showed problems with using Tyvek for SP testing, another identical test was
performed using PVDF envelopes.
For the Tyvek envelope tests, soils were spiked ten times with 10-|iL aliquots of B. atrophaeus
(100 |iL total from a master cell bank containing 6.7e8 CFU/mL, resulting in a 6.7e7 CFU spike)
per 1-gram sample. Three samples of each soil type were prepared, along with one negative
control (unspiked) for each soil type. Tyvek and PVDF envelopes were then heat sealed and
placed in a biosafety cabinet (BSC) for 30 minutes. Samples were recovered by cutting
envelopes open with sterile scissors, placing soil into 50-mL conical tubes, adding 10 mL of
extraction buffer, agitating at 200 rpm for 15 minutes, removing 100 |iL of extract, and plating
serial dilutions. Table C-8 shows the results.
Table C-8. B. atrophaeus-Spiked Soil Extraction Efficacy (Tyvek Envelopes)
Sample
Envelope
T reatment
Observed
(CFU)
Recovery
(%)a
Topsoil
Tyvek
Sit 30 min
1.77 x107
40
Topsoil
Tyvek
Sit 30 min
2.03 x107
46
Topsoil
Tyvek
Sit 30 min
1.97 x107
45
Neg. Control (Topsoil)
Tyvek
Sit 30 min
0
0
Sand
Tyvek
Sit 30 min
2.84 x107
64
Sand
Tyvek
Sit 30 min
3.03 x107
69
Sand
Tyvek
Sit 30 min
3.03 x107
69
Neg. Control (Sand)
Tyvek
Sit 30 min
0
0
Clay
Tyvek
Sit 30 min
2.01 x 107
46
Clay
Tyvek
Sit 30 min
2.22 x107
51
Clay
Tyvek
Sit 30 min
2.28 x107
52
Neg. Control (Clay)
Tyvek
Sit 30 min
0
0
No Soil Spike (control)
none
Direct to buffer
4.40 x107
NA
a Based on control sample (direct to buffer) recovery of 4.4 x 107 CFU.
For the PVDF envelope tests, soils were spiked as described above, but ten 10-|iL aliquots of B.
atrophaeus diluted 1:1 with sterile water were used (100 |iL total from a master cell bank of
3.35e8 CFU/mL, resulting in a 3.35e7 CFU spike) per 1-gram sample. One sample of each soil
type was prepared, and one control sample was used. Table C-9 shows the results.
Table C-9. B. atrophaeus-Spiked Soil Extraction Efficacy (PVDF Envelopes)
Sample
Envelope
T reatment
Observed
(CFU)
Recovery
(%)a
Topsoil
PVDF
Sit 30 min
2.00 x107
716
Sand
PVDF
Sit 30 min
7.47 x10s
268
Clay
PVDF
Sit 30 min
9.50 x105
34
No Soil Spike (control)
none
Direct to buffer
2.79 x10s
NA
a Based on control sample (direct to buffer) recovery of 2.79 x 106 CFU.
C-7
-------
Appendix D.
Preliminary Tests
D-1
-------
Several preliminary tests were conducted prior to executing the main study. These tests included
preliminary wall-effects and soil saturation tests, and are summarized in the sections that follow
and also in Table D-l.
Table D-1. Summary of Wall-Effects Tests
Date
Soil Type
Columns
Description
Data Location
CIO2 Wall-Effects Tests
02/18/2016
Topsoil
10", 8", 6" buckets
8.4 mg/L, 3 hr, 75% RH
Table D-2
03/04/2016
Sand
10", 8", 6" columns
8.4 mg/L, 3 hr, 75% RH
Table D-3
03/16/2016
Topsoil
8", 6" columns
8.4 mg/L, 6 hr, 75% RH
Table D-4
03/30/2016
Clay
8", 6" columns
8.4 mg/L, 6 hr, 75% RH
Table D-5
Sodium Persulfate Wall-Effects Tests
03/28/2016
Topsoil
10", 8", 6" columns
-0.1 mL/g, 1 day
Table D-6
04/20/2016
Topsoil
10", 8", 6" columns
0.14 mL/g total, 7 day
Table D-7
06/06/2016
Topsoil
6" column
0.14 mL/g total, 1 day
Table D-8
Sodium Persulfate Wettability Tests
04/11/2016
Topsoil
10" bucket
Multiple applications to match previous
water test and observe similarities.
Visual exam
04/13/2016
Topsoil
8" column
Fresh applications every 20 min until
saturation point is reached at 0.14
mL/g.
Visual exam
Preliminary Wall Effects Tests
Four wall effects tests were performed using CIO2, and three were performed using SP, as
described below.
Chlorine Dioxide with Topsoil, 8.4 mg/L, 3 hr
A wall effects test was performed on 02/18/2016 using CIO2, Bis, commercial topsoil and a
small containment box. Chlorine dioxide was generated by the Minidox-M with a concentration
set point of 8.4 mg/L and an exposure time of 3 hrs. A containment box was made from a 35-
gallon plastic tub (Sterilite™; Townsend, MA) with the top sealed by duct tape. Connections to
the Minidox-M were made by using tubing and standard fittings obtained from ClorDiSys. Three
test columns were built as follows: 10" diameter x 10" depth (5-gallon plastic bucket), 8"
diameter x 10" depth (PVC pipe with an end cap on the bottom); 6" diameter x 10" depth (PVC
pipe with an end cap on the bottom).
Timberline™ topsoil (Oldcastle Lawn & Garden, Inc., available at Home Depot) was used for the
test soil. Soil density was established to be 1.0 g/cm3 based on previous EPA work [4], For test
purposes, this density was obtained by weighing the appropriate mass of soil per unit depth for
the three column diameters tested (6", 8", and 10").
Bis were placed in duplicate in each column at depths of 2" and 5" and were located side by side
in the center of the column (i.e., at least three inches from the wall). A single BI was placed on
the top surface of the soil in each column. Two additional Bis were not placed in the
containment, but were analyzed as controls, making a total of 17 Bis for testing the three
configurations.
D-2
-------
Results showed that the CIO2 gas did not penetrate soil at >2" depth in any of the columns, while
the Bis on the top surface had complete kill. Control samples showed normal recovery in the
range of approximately 50%. Average air temperature was 20.2°C (68.4°F) and average RH was
84.6% during the test. Table D-2 presents the results.
Table D-2. Wall Effects Test (CIO2 and Topsoil, 02/18/2016)
Column
Diameter
(in)
Bl Depth
(in)
Sample
ID
Observed
(CFU)
%
Recovery3
Log of
Recovered
CFU
Decon.
Efficacy
(LR)b
10
0" (top surface)
A
0
0
-
6.22
10
2"
A
1.51 x 10®
54
6.18
0.04
10
2"
B
1.51 x 10®
54
6.18
0.04
10
5"
A
1.55 x10s
55
6.19
0.03
10
5"
B
1.58 x10s
56
6.20
0.02
8
0" (top surface)
A
0
0
-
6.22
8
2"
A
1.36x10®
49
6.13
0.09
8
2"
B
1.43x10®
51
6.15
0.07
8
5"
A
1.53x10®
55
6.18
0.04
8
5"
B
1.53x10®
55
6.18
0.04
6
0" (top surface)
A
0
0
-
6.22
6
2"
A
1.43x10®
51
6.16
0.07
6
2"
B
1.38x10®
49
6.14
0.08
6
5"
A
1.39x10®
50
6.14
0.08
6
5"
B
1.41 x 10®
50
6.14
0.07
Control
Not exposed
A
1.77x10®
63
6.25
NA
Control
Not exposed
B
1.57x10®
56
6.19
NA
a Bl standard is 2.8 x 10s CFU, obtained from certificate of analysis.
b Compared against the average of control samples (last two entries in the table).
Chlorine Dioxide with Sand, 8.4 mg/L, 3 hr
A wall effects test was performed on 03/04/2016 using CIO2, Bis, and commercial sand
(Pavestone™ play sand from Home Depot). The containment box, columns, test depths, CIO2
concentration, and exposure time were identical to the topsoil test described above. Sand density
was determined to be 1.4 g/cm3 by weighing a measured volume of sand in a beaker.
Results showed that the CIO2 gas penetrated sand at the 2" depth, producing a log reduction of
approximately 2, but did not penetrate at the 5" depth. Bis on the top surface had complete kill.
Control samples showed normal recovery in the range of approximately 50%. Average air
temperature was 19.8°C (67.6°F) and ranged from 19.0 to 20.6°C during the test. Average RH
was 81.4%) and ranged from 76.8 to 84.1% during the test. Table D-3 presents the results.
D-3
-------
Table D-3. Wall Effects Test (CIO2 and Sand, 03/04/2016)
Column
Diameter
(in)
Bl Depth
(in)
Sample ID
Observed
(CFU)
Log of
Recovered CFU
Decon. Efficacy
(LR)b
10
0" (top surface)
A
0
-
6.12
10
2"
A
0
-
6.12
10
2"
B
0
-
6.12
10
5"
A
1.42 x10s
6.15
-0.03
10
5"
B
1.46 x10s
6.16
-0.04
8
0" (top surface)
A
0
-
6.12
8
2"
A
0
-
6.12
8
2"
B
0
-
6.12
8
5"
A
1.39x10®
6.14
-0.03
8
5"
B
1.43x10®
6.16
-0.04
6
0" (top surface)
A
0
-
6.12
6
2"
A
6.00 x103
-
6.12
6
2"
B
1.00 x104
-
6.12
6
5"
A
1.25x10®
6.10
0.03
6
5"
B
1.32x10®
6.12
0.00
Control
Not exposed
A
1.33x10®
6.12
NA
Control
Not exposed
B
1.33x10®
6.12
NA
a Bl standard is 2.8 x 10s CFU, obtained from certificate of analysis.
b Compared against the average of control samples (last two entries in the table).
Chlorine Dioxide with Topsoil, 8.4 mg/L, 6 hr
A wall effects test was performed on 03/16/2016 using CIO2, Bis, and commercial topsoil as
described earlier. The containment box was identical to previous tests. Columns were 6" and 8"
PVC pipe. Test depths were from 0 to 6" in 1-in intervals. Chlorine dioxide concentration was
8.4 mg/L, with a total exposure time of 6 hrs.
Table D-4 presents the results for these 6-hr topsoil exposure tests. Results showed that the CIO2
gas did not penetrate the topsoil. All samples except one had a log reduction of <1, indicating a
possible wall effect for that sample. Bis on the top surface had complete kill. Control samples
showed normal recovery in the range of approximately 50%. Average air temperature was
23.2°C (73.8°F) ranging from 21.8°C to 24.5°C during the test. Average RH was 78.8% and
ranged from 73.0 to 86.3% during the test.
D-4
-------
Table D-4. Wall Effects Test (CIO2 and Topsoil, 03/16/2016)
Column
Diameter
(in)
Bl Depth
(in)
Sample ID
Observed
(CFU)
Log of
Recovered CFU
Decon. Efficacy
(LR)b
8
0" (top surface)
A
0
-
6.06
8
1"
A
4.27 x10s
6.63
-0.58
8
2"
A
1.24 x10s
6.09
-0.04
8
3"
A
5.60 x104
4.75
1.31
8
4"
A
5.93 x105
5.77
0.28
8
5"
A
4.83 x105
5.68
0.37
8
6"
A
6.00 x105
5.78
0.28
6
0" (top surface)
A
0
-
6.06
6
1"
A
1.35x10®
6.13
-0.08
6
2"
A
1.35x10®
6.13
-0.07
6
3"
A
1.32x10®
6.12
-0.07
6
4"
A
1.64x10®
6.22
-0.16
6
5"
A
1.10x10®
6.04
0.01
6
6"
A
9.30 x105
5.97
0.09
Control
Not exposed
A
8.27 x105
5.92
NA
Control
Not exposed
B
1.56x10®
6.19
NA
a Bl standard is 2.8 x 10s CFU, obtained from certificate of analysis.
b Compared against the average of controls samples (last two entries in the table).
Chlorine Dioxide with Clay, 8.4 mg/L, 6 hr
A wall effects test was performed on 03/30/2016 using CIO2, Bis, and commercial clay soil as
described earlier. The containment box was identical to previous tests. Columns were 6" and 8"
PVC pipe. Test depths were from 0 to 6" in 1-in intervals. Chlorine dioxide concentration was
8.4 mg/L for an exposure time of 6 hrs. Manual homogenization of the clay was performed
wherein clumps of > -1/4" were broken up by hand during column filling. The clay was not
mechanically sieved.
Table D-5 presents the results for these 6-hr clay tests. Results showed that the CIChgas
penetrated up to 3 to 4" with complete kill, then dropped off to <1 log reduction at greater
depths. Bis on the top surface had complete kill. Control samples showed normal recovery in the
range of approximately 50%. Average air temperature was 23.2°C (73.8°F) and ranged from
21.8 to 24.5°C during the test. Average RH was 78.8% and ranged from 73.0 to 86.3% during
the test.
D-5
-------
Table D-5. Wall Effects Test (CIO2 and Clay, 03/30/2016)
Column
Diameter
(in)
Bl Depth
(in)
Sample ID
Observed
(CFU)
Log of
Recovered CFU
Decon. Efficacy
(LR)b
8
0" (top surface)
A
0
-
6.31
8
1"
A
0
-
6.31
8
2"
A
0
-
6.31
8
3"
A
0
-
6.31
8
4"
A
0
-
6.31
8
5"
A
4.70 x105
5.67
0.64
8
6"
A
9.00 x104
4.95
1.36
6
0" (top surface)
A
0
-
6.31
6
1"
A
0
-
6.31
6
2"
A
0
-
6.31
6
3"
A
0
-
6.31
6
4"
A
1.30 x105
5.14
1.17
6
5"
A
5.70 x105
5.76
0.55
6
6"
A
6.63 x105
5.82
0.49
Control
Not exposed
A
2.04 x10s
6.31
NA
Control
Not exposed
B
2.05 x10s
6.31
NA
a Bl standard is 2.8 x 10s CFU, obtained from certificate of analysis.
b Compared against the average of control samples (last two entries in the table).
Sodium Persulfate with Topsoil, 0.5 M, 0.10 mL/g, 1 day
A wall effects test was performed on 03/28/2016 using activated SP, Bis, and commercial topsoil
as described earlier. No containment box was used for these tests. Three column configurations
were tested with 6", 8", and 10" diameter PVC pipe placed in an open-topped plastic tub (to
contain any drips) in a fume hood. Test depths were at 0, 2", and 5". Sodium persulfate was
mixed at 0.5 M concentration, and was activated by mixing with fresh 8% hydrogen peroxide
immediately prior to application. The liquid was applied at a total volume of 0.09 to 0.10 mL/g
of topsoil calculated for a depth of 5" of soil. Soak time after application was 1 day. One
additional 10" column was also dosed with deionized water as a control. Liquid was applied in
~100-mL increments every 2 minutes as follows:
• 10" column: 6x 97-mL applications, 582 mL total
• 8" column: 4x 100-mL applications, 400 mL total
• 6" column: 2x 100-mL applications, 200 mL total
Table D-6 presents the results of the persulfate/topsoil exposures. Results showed little, if any,
kill versus the controls. Bis on the top surface did not all show complete kill. During later tests,
as previously described, problems were traced to hydrophobic qualities of Tyvek, which
essentially slows down or even repels the persulfate liquid.
Average air temperature was 24.1°C (75.4°F) and ranged from 22.5 to 25.5°C during the test.
Average RH was 19.8% and ranged from 17.0 to 25.0% during the test.
D-6
-------
Table D-6. Wall Effects Test (Sodium Persulfate and Topsoil, 03/28/2016)
Column
Diameter
(in)
Bl Depth
(in)
Sample ID
Observed
(CFU)
Log of
Recovered CFU
Decon. Efficacy
(LR)b
10 control
0" (top surface)
A
2.65 x105
5.42
0.32
10 control
2"
A
3.30 x105
5.52
0.22
10 control
2"
B
3.33 x105
5.52
0.22
10 control
5"
A
3.23 x105
5.51
0.23
10 control
5"
B
contaminated
-
-
10
0" (top surface)
A
3.33 x102
2.52
3.22
10
2"
A
3.27 x105
5.51
0.23
10
2"
B
3.53 x105
5.55
0.19
10
5"
A
2.02 x105
5.30
0.44
10
5"
B
3.83 x105
5.58
0.16
8
0" (top surface)
A
0
—
5.75
8
2"
A
1.79 x105
5.25
0.49
8
2"
B
1.73 x105
5.24
0.50
8
5"
A
2.59 x105
4.41
1.33
8
5"
B
2.88 x104
4.46
1.28
6
0" (top surface)
A
1.03 x103
3.01
2.73
6
2"
A
2.02 x105
5.31
0.44
6
2"
B
1.88 x105
5.27
0.47
6
5"
A
1.82 x105
5.26
0.48
6
5"
B
1.78 x105
5.25
0.49
Control
Not exposed
A
1.04 x10s
6.02
NA
Control
Not exposed
B
2.94 x105
5.47
NA
a Bl standard is 2.8 x 10s CFU, obtained from certificate of analysis.
b Compared against the average of control samples (last two entries in the table).
Sodium Persulfate with Topsoil, 0.5 M, 0.14 mL/g, 7 day
A wall effects test was performed on 04/20/2016 using activated SP, Bis, and commercial topsoil
as described earlier. No containment box was used for these tests. Three columns configurations
were tested with 6", 8", and 10" diameter PVC pipe placed in an open-topped plastic tub (to
contain any drips) in a fume hood. Test depths were from 0 to 6" in 1-inch increments. SP was
mixed at 0.5 M concentration, and was activated by mixing with fresh 8% hydrogen peroxide
immediately prior to application. The liquid was applied at a total volume of 0.14 mL/g of
topsoil calculated for a depth of 6" of soil, the amount determined to be the saturation point of
soil in earlier lab tests. Soak time after application was 7 days. One additional sample was dosed
with deionized water in topsoil as a wet control. Liquid was applied in 2 equal increments, 30
minutes apart, as follows:
• 10" column: two (2) 550-mL applications; 1,100 mL total
• 8" column: two (2) 350-mL applications; 700 mL total
• 6" column: two (2) 195-mL applications; 390 mL total
Table D-7 presents the results of these 7-day persulfate exposures. Results showed little, if any,
kill versus the controls. Unlike the previous test, all Bis on the top surface did show complete
kill, indicating that the 7-day exposure time is more than adequate for the persulfate to permeate
the Tyvek CSP (as was shown earlier in this report).
D-7
-------
Average air temperature was 22.7°C (72.9°F) and ranged from 22.5 to 24.0°C during the test.
Average RH was 48.7% and ranged from 40.0 to 58.0% during the test. Table D-7 presents the
results.
Table D-7. Wall Effects Test (Sodium Persulfate and Topsoil, 04/20/2016)
Column
Diameter
(in)
BI Depth
(in)
Sample ID
Observed
(CFU)
Log of
Recovered CFU
Decon. Efficacy
(LR)b
10
0" (top surface)
A
0
-
5.53
10
1"
A
5.67 x105
5.75
-0.22
10
2"
A
5.97 x105
5.78
-0.25
10
3"
A
7.57 x105
5.88
-0.35
10
4"
A
3.93 x105
5.59
-0.07
10
5"
A
2.56 x105
5.41
0.12
10
6"
A
5.27 x105
5.72
-0.19
8
0" (top surface)
A
0
-
5.53
8
1"
A
3.13 x 105
5.50
0.03
8
2"
A
4.93 x105
5.69
-0.16
8
3"
A
4.97 x105
5.70
-0.17
8
4"
A
3.87 x105
5.59
-0.06
8
5"
A
4.20 x105
5.62
-0.09
8
6"
A
4.97 x105
5.70
-0.17
6
0" (top surface)
A
0
-
5.53
6
1"
A
4.30 x105
5.63
-0.10
6
2"
A
5.70 x105
5.76
-0.23
6
3"
A
3.20 x105
5.51
0.02
6
4"
A
5.77 x105
5.76
-0.23
6
5"
A
5.13 x 105
5.71
-0.18
6
6"
A
3.07 x105
5.49
0.04
Wet Control
Not exposed
A
4.63 x105
5.67
-0.14
Control
Not exposed
A
3.33 x105
5.52
NA
Control
Not exposed
B
3.43 x105
5.54
NA
a Bl standard is 2.8 x 10s CFU, obtained from certificate of analysis.
b Compared against the average of control samples (last two entries in the table).
Sodium Persulfate with Topsoil, 0.5 M, 0.14 mL/g, 1 day, PVDF packets
A wall effects test was performed on 06/06/2016 using activated SP, Bis repackaged in PVDF,
and commercial topsoil as described earlier. PVDF Bis were prepared by cutting open a standard
Tyvek BI packet, pouring the actual BI disc into a PVDF packet, then heat sealing the PVDF.
A single column configuration was used for this test. The column was a 6" PVC pipe placed in
an open-topped plastic tub (to contain any drips) in a fume hood. Test depths were at 0, 1, and
2". SP was mixed at 0.5 M concentration, and was activated by mixing with fresh 8% hydrogen
peroxide immediately prior to application. The liquid was applied at a total loading of 0.14 mL/g
of topsoil calculated for a depth of 6" of soil, the amount determined to be the saturation point of
soil in earlier lab tests. Soak time after application was 1 day. Liquid was applied in 2 equal
increments, 30 minutes apart, as follows:
• 6" column: two (2) 195-mL applications; 390 mL total
D-8
-------
Table D-8 presents the results, which showed -0.4 log reduction versus the controls.
Average air temperature was 22.5°C (72.5°F) and ranged from 22.5 to 23.0°C during the test.
Average RH was 48.4% and ranged from 41.0 to 52.0% during the test.
Table D-8. Wall Effects Test (Sodium Persulfate and Topsoil, 06/06/2016)
Column
Diameter
(in)
Bl Depth
(in)
Sample ID
Observed
(CFU)
Log of
Recovered CFU
Decon. Efficacy
(LR)b
6
0" (top surface)
A
0
—
5.05
6
1"
A
4.37 x104
4.64
0.41
6
2"
A
4.53 x104
4.66
0.39
Control
Not exposed
A
9.50 x104
4.98
NA
Control
Not exposed
B
1.30 x105
5.11
NA
a Bl standard is 2.8 x 10s CFU, obtained from certificate of analysis.
b Compared against the average of control samples (last two entries in the table).
Sodium Persulfate Soil Saturation Tests
Due to the large masses of soil being tested in this project, we were not able to rely on liquid
loading volumes used in previous EPA studies. Previous work (EPA, 2015), for example, used a
liquid volume of 3 applications of 0.18 mL activated SP per gram of soil (three (3) 0.18-mL
applications, or 0.54 mL/g total, which worked well on a petri-dish scale. For the current project,
large test columns containing >25 lbs of soil were required, so it was necessary to experimentally
determine a more appropriate soil saturation point.
An initial test using a 6" depth of topsoil in a 5-gal bucket was performed on 02/11/2016.
Topsoil (density -1.0 g/mL) was weighed and measured. For the 5-gal bucket, this amounts to
3.4 lb/inch of soil depth, or 20.4 lbs for a 6" depth. The bucket was assembled so that the original
bottom was replaced with a metal screen, and elevated onto a platform for observation.
100 mL of deionized water was evenly distributed across the surface of the soil. This was
repeated every 2 minutes, and visual observations were made for leakage of water through the
metal screen at the bottom of the bucket. After 800 mL had been poured onto the soil
(-16 minutes after beginning the test), water began dripping through the metal screen. A beaker
was placed under the dripping water, and approximately 1 mL was collected before the dripping
stopped. Based on these data, a saturation point of 0.09 mL/g was estimated.
The first SP wall-effects test was performed on 03/28/2016 (see previous section) using a volume
of 0.09 mL/g, and a target penetration depth of 5". When no decontamination was observed at
this depth, it was decided to determine the soil saturation point using activated SP, rather than
water. When large volumes of soil are wetted by large volumes of SP, a vigorous oxidizing
reaction occurs that immediately liberates extensive volumes of gas, causing foaming. This, in
turn, causes the liquid to puddle up on the soil surface in a foamy layer that ultimately penetrates
over the next 30 minutes or so. This is a phenomenon not seen during previous EPA studies
because of the relatively small volumes of soil and decontaminant volumes used during petri-
dish sized experiments.
D-9
-------
A SP saturation test was performed on 04/13/2016 that used the same general approach as the
previous water tests. An 8" column was filled to a 6" depth with topsoil (4,952 g), and 100-mL
volumes of activated SP were applied. Applications were initially in 20-min intervals. By the 3rd
application, the reaction had created enough foam to slow penetration of the persulfate, so that
there was standing liquid on the top surface that becomes a sticky, porous area as it seeps in
(Figure D-l). By the 5th application, a penetration rate was achieved such that fresh applications
were slowed to once per hour. Shortly after the 7th application, liquid began dripping from the
bottom surface, indicating that the saturation point had been reached (Figure D-2). Dripping
liquid was captured in a glass beaker and was poured over fresh topsoil in a nearby bucket. No
reaction was observed with the fresh topsoil, suggesting that the captured persulfate was now-
inert, or at least its reactive components reduced.
Figure D-1. Topsoil Persulfate Saturation Test (just prior to 5th application of sodium persulfate)
Figure D-2. Sodium Persulfate Dripping From Bottom of Topsoil Column
The persulfate saturation level for the topsoil was calculated to be 0.14 mL/g. Again, this level is
less than the previous 0.54 mL/g used in earlier EPA work. It is also important to note that the
applications must be spaced well apart, or else surface reactions and gas release will inhibit the
timely flow of sodium persulfate into the soil.
D-10
-------
Appendix E.
ORP Measurements
E-1
-------
During the SP test series, any liquid that permeated through the column was collected in drip
pans located beneath each column. Excess liquid was removed as needed to prevent the drip pans
from overflowing. For the final test in this series (Test 3 SP), the oxidation reduction potential
(ORP) was measured using a hand-held probe (Hach Pocket Pro™). ORP measurements were
taken of the initial (fresh) activated SP and final (partially spent) liquid emerging from the
column bottom. Liquid volumes were also estimated by the laboratory staff.
As shown in Table E-l, liquid emerging from the sand column was nearly the same as the
application volume (876 mL) after the 3rd application, and also showed a clearly different ORP
value from the freshly-mixed solution. For topsoil, ORP from the emerging liquid was only
slightly different from the freshly-mixed solution, and only limited drainage occurred. Clay was
not included in the ORP evaluation because only one liquid application was used, and no liquid
was found to emerge from the bottom of the clay column.
Results show that the ORP changes during permeation through the clay and topsoil.
Interestingly, the ORP, in general, for the topsoil permeate is very close to the activity (i.e., ORP
reading) as freshly-mixed persulfate. In the clay experiments, the ORP increased significantly
and in several cases by almost a factor of two. The ORP device was checked repeatedly between
readings using a certified standard and was always shown to be within specifications. The source
of this increase was not investigated further, but could be due to extraction from the clay and
soil, increasing the ORP reading.
Table E-1. ORP Results for Test 3 SP
ORP Reading of Permeated Liquid3
Volume of Collected Permeate
Day
Application
Topsoil
(mV)
Sand
(mV)
Topsoil
(mL)
Sand
(mL)
0
1
-
-
-
-
1
2
-
-
-
-
2
3
-
638
-
339
3
4
-
661
-
794
4
5
432
653
180
825
5
6
512
559
261
860
a Freshly-mixed SP, activated with 8% hydrogen peroxide, had an average ORP response of 437 mV. The response
to a certified calibration solution was stable at 225 mV (Zobell's solution from Hach).
E-2
-------
vvEPA
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
------- |