United States
Environmental Protection
Agency
.ugust
www.epa.gov/or
Technology Evaluation Report
Decontamination of Soil
Contaminated with Bacillus
anthracis Spores
Office of Research and Development
National Homeland Security Research Center
-------
EPA/600/R-13/110
August 2013
Decontamination of Soil Contaminated
with Bacillus anthracis Spores
U.S. Environmental Protection Agency
Research Triangle Park, NC 27711
11
-------
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 and
directed this work through Contract Number EP-C-10-001 with Battelle Memorial Institute. 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
in
-------
Acknowledgments
Contributions of the following individuals and organization to this report are gratefully
acknowledged:
United States Environmental Protection Agency (EPA)
Leroy Mickelson, Consequence Management Advisory Team
Peer reviewers
Dino Mattorano, EPA Consequence Management Advisory Team
Gene Rice EPA National Homeland Security Research Center
Craig Ramsey, US Department of Agriculture, Animal and Plant Health Inspection
Service
Battelle
IV
-------
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 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 four
chemical decontaminants for inactivating Bacillus anthracis (causative agent for anthrax) spores
in soil. The decontaminants that were evaluated included two liquid biocides (pH-amended
bleach and sodium persulfate) and two fumigants (methyl bromide and metam sodium). The
objective of this study was to provide an understanding of the performance (i.e., efficacy) of
these decontamination technologies to guide their use and implementation in HS applications for
hard-to-decontaminate materials such as soil. In the assessment of options for decontamination
following an intentional release of B. anthracis spores, it is important to know what operational
factors can impact the decontamination efficacy.
This investigation focused on decontamination of two types of soil material: topsoil and Arizona
Test Dust (AZTD). These two soil types were selected for testing in an attempt to span the range
in expected organic content of soils. Decontamination efficacy tests were conducted with spores
of B. anthracis or B. subtilis, the latter microorganism included to assess its potential as a
surrogate for future studies related to B. anthracis. Decontamination efficacy was quantified in
terms of log reduction (LR), based on the difference in the number of bacterial spores (as colony
forming units) recovered from the positive controls (soil samples not exposed to decontaminant)
and test samples. Tests were conducted with varying operational parameters (e.g., contact time,
number of applications of the decontaminant, decontaminant concentration) to assess the effect
of these parameters on decontamination efficacy.
Summary of Results
pH-Amended Bleach
At the most robust treatment with pH-amended bleach (seven-day contact time, eight
applications), the decontamination efficacy for topsoil was minimal: less than 0.5 LR for both
microorganisms. In contrast, pH-amended bleach was successful in decontaminating AZTD
with greater than 7.0 LR obtained for both B. anthracis and B. subtilis with four applications and
a two- hour contact time. For AZTD, efficacy generally decreased with decreasing number of
applications and contact time.
Sodium Persulfate
All five tests conducted with sodium persulfate used a contact time of seven days while varying
the number of times the decontaminant was applied to the soil samples. The most efficacious
treatment, in which sodium persulfate was applied to the samples six times, resulted in complete
inactivation (no spores detected) of B. anthracis on both soil materials. The next robust
treatment (three applications of the decontaminant, all applied within the first two hours)
provided greater than a 7 LR for B. anthracis on both soils. Efficacy generally decreased with
decreasing number of persulfate applications. The decontamination efficacy results for topsoil
and AZTD were not significantly different for the majority of tests.
-------
Methyl Bromide
Eight tests were conducted with this fumigant, with concentrations ranging from 100 to 212
milligrams per liter (mg/L). All tests were conducted at 25 °C with a contact time of 36 hours,
except for the last test at 24 hours. The relative humidity (RH) levels in the test chamber were
measured but uncontrolled, although for a few tests, attempts were made to manipulate the RH
by adding or removing moisture from the soil prior to testing. The two most efficacious
treatments evaluated (utilizing 212 mg/L MeBr, 36 hour contact time, no drying of soil) resulted
in complete inactivation of B. anthracis spores on AZTD and greater than 7.0 LR on topsoil.
Overall, MeBr was effective (greater than 6 LR achieved) against B. anthracis on topsoil and
AZTD at 25 °C when using a concentration of at least 180 mg/L and contact time of 36 hours.
(One minor exception is the test in which the soil samples were dried beforehand, which resulted
in a 5.9 LR on topsoil.) As expected, decontamination efficacy generally decreased with
decreasing concentration and contact time. With respect to the effect of soil type, the
decontamination efficacies obtained for B. anthracis were slightly higher on AZTD compared to
topsoil.
Metam Sodium
This decontaminant was significantly more effective on the AZTD compared to the topsoil for
the majority of the tests. For all but one of the eight tests with AZTD, B. anthracis was
completely inactivated, whereas just one test with B. anthracis-contammated topsoil resulted in
completely inactivation. Metam sodium was effective (greater than 6 LR) against B. anthracis
on topsoil in three of the tests conducted.
Operational factors such as doubling the amount of metam sodium applied to the soil materials
improved efficacy significantly for both microorganisms on topsoil and for B. subtilis on AZTD.
(Tests were conducted with either 80 or 160 jiL.) Increasing contact time (up to 14 days)
generally improved efficacy for the inactivation of B. anthracis on topsoil but not significantly.
Decontamination efficacy for B. anthracis on topsoil increased with increasing soil moisture
content, with efficacy greater than 6 LR when the soil moisture was at its highest levels (-46%).
Comparing efficacy results for B. anthracis and B. subtilis
There were no tests in which B. subtilis was inactivated to a significantly higher degree than B.
anthracis, and, for pH-amended bleach, there were no significant differences in decontamination
efficacy for the two microorganisms. For the other three decontaminants, the inactivation
efficacy for B. subtilis was significantly less than the efficacy for B. anthracis for the majority of
the tests conducted. For MeBr in particular, the differences in efficacy for the two
microorganisms were greater than 5-6 LR for more than half of the tests.
VI
-------
Contents
Disclaimer iii
Acknowledgments iiv
Executive Summary v
Abbreviations/Acronyms x
1.0 Introduction 1
2.0 Technology Descriptions and Test Matrices 2
2.1 Technology Descriptions 2
2.2 Test Matrices for Liquid Decontamination 3
2.3 Test Matrices for Fumigant Decontamination 3
3.0 Summary of Test Procedures 6
3.1 Biological Agent 6
3.2 Soil Materials 6
3.3 Preparation of Soil Samples 7
3.4 Sample Extraction and Biological Agent Quantification 8
3.5 Decontamination Efficacy 9
3.6 Discoloration of Soil Materials 10
4.0 Decontamination Procedures 11
4.1 Liquid Decontaminant Preparation 11
4.2 Liquid Decontamination Test and Control Chambers and Procedures 11
4.3 MeBr Fumigation Test and Control Chambers and Procedures 12
4.4 Metam Sodium Fumigation Test and Control Chambers and Procedures 14
5.0 Quality Assurance/Quality Control 16
5.1 Equipment Calibration 16
5.2 QC Results 16
5.3 Audits 16
5.4 Test/Quality Assurance Plan Deviations 17
5.5 QA/QC Reporting 18
5.6 Data Review 18
6.0 Results and Performance Summary for Liquid Biocides 19
6.1 pH-Amended Bleach Results 19
6.2 Sodium Persulfate Results 19
7.0 Results and Performance Summary for Fumigant Biocides 19
7.1 MeBr Results 28
7.2 Metam Sodium Results 35
7.3 Discoloration of Soils 41
8.0 Summary of Results 42
8.1 Decontamination Efficacy 42
8.2 Effect of Soil Type 43
8.3 Comparing Efficacy for B. anthracis and.8. subtilis 44
9.0 References 45
Appendix A Moisture and Organic Content of Soil Sampless 46
Appendix B Neutralization Tests for Liquid Decontaminants 49
vn
-------
Figures
Figure 4-1 Schematic of MeBr Decontamination Test Chamber 13
Figure 6-1 Summary of Decontamination Efficacies for pH-Am ended Bleach Testing on
Topsoil and AZTD 22
Figure 6-2 Summary of Decontamination Efficacies for Klozur Liquid Testing on Topsoil
and AZTD 27
Figure 7-1 Summary of Decontamination Efficacies for MeBr Fumigant Testing on Topsoil
and AZTD 34
Figure 7-2 Summary of Decontamination Efficacies for Metam Sodium Fumigant Testing
on Topsoil and AZTD 40
Figure 7-3 Effect of Topsoil Moisture Content on Decontamination Efficacy for
B. anthracis 41
Figure 8-1 Minimum Treatment Required for Effective Decontamination 43
Tables
Table 2-1 Decontamination Technology Descriptions 2
Table 2-2 pH-Amended Test Matrix 3
Table 2-3 Sodium Persulfate (Klozur™) Test Matrix 3
Table 2-4 MeBr Test Matrix 4
Table 2-5 Metam Sodium Test Matrix 4
Table 3-1 Soil Materials 7
Table 5-1 Performance Evaluation Audits 17
Table 6-1 Inactivation of Bacillus anthracis Spores on Soil using pH-Amended Ultra
Clorox Germicidal Bleach 20
Table 6-2 Inactivation of Bacillus subtilis Spores on Soil using pH-Amended Ultra Clorox®
Germicidal Bleach 21
Table 6-3 Inactivation of Bacillus anthracis Spores on Topsoil with Klozur™ 23
Table 6-4 Inactivation of Bacillus anthracis Spores on AZTD with Klozur™ 24
Table 6-5 Inactivation of Bacillus subtilis Spores on Topsoil with Klozur™ 25
Table 6-6 Inactivation of Bacillus subtilis Spores on AZTD with Klozur 26
Table 7-1 Actual Fumigation Conditions for Tests with MeBr 28
Table 7-2 Inactivation of Bacillus anthracis Spores on Topsoil with MeBr 30
Table 7-3 Inactivation of Bacillus anthracis Spores on AZTD with MeBr 31
Table 7-4 Inactivation of Bacillus subtilis Spores on Topsoil with MeBr 32
Table 7-5 Inactivation of Bacillus subtilis Spores on AZTD with MeBr 33
Table 7-6 Inactivation of Bacillus anthracis Spores on Topsoil with Metam Sodium 36
Table 7-7 Inactivation of Bacillus anthracis Spores on AZTD with Metam Sodium 37
Table 7-8 Inactivation of Bacillus subtilis Spores on Topsoil with Metam Sodium 38
Table 7-9 Inactivation of Bacillus subtilis Spores on AZTD with Metam Sodium 39
Table A-l Soil Sample Analysis 43
Table A-2 Soil Moisture Assessments Taken During Metam Sodium Tests 45
Table B-l Neutralization Testing with Bacillus subtilis Spores with pH-Amended Bleach,
Seven-Day Contact Time, Eight Total Applications 47
Table B-2 Neutralization Testing with Bacillus subtilis Spores with pH-Amended Bleach,
24 Hour Contact Time, Eight Total Applications 47
viii
-------
Table B-3 Neutralization Testing with Bacillus subtilis Spores with pH-Amended Bleach,
120 Minute Contact Time, Four Total Applications 48
Table B-4 Neutralization Testing with Bacillus subtilis Spores with pH-Amended Bleach,
60 Minute Contact Time, Two Total Applications 48
Table B-5 Neutralization Testing with Bacillus subtilis Spores with Klozur™, Seven-Day
Contact Time, Six Total Applications 48
Table B-6 Neutralization Testing with Bacillus subtilis Spores with Klozur™, Seven-Day
Contact Time, Three Total Applications 49
Table B-7 Neutralization Testing with Bacillus subtilis Spores with Klozur™, Seven-Day
Contact Time, Three Total Applications (Days 0, 2 and 4) 49
Table B-8 Neutralization Testing with Bacillus subtilis Spores with Klozur™, Seven-Day
Contact Time, One Total Application 50
Table B-9 Neutralization Testing with Bacillus subtilis Spores with Klozur™, Seven-Day
Contact Time, Two Total Applications 50
IX
-------
Abbreviations/Acronyms
ATCC
AZTD
B. anthracis
B. subtilis
BBRC
BSC
°C
CBR
CPU
CI
cm
DNA
EPA
ft
g
HC1
H2O2
hr
HS
HSRP
kGy
LAL
L
LED
LR
M
MeBr
mg
MITC
mL
HL
NaOCl
NaOH
Na2S2O8
NHSRC
ORD
oz
PBS
PBST
PCR
ppm
PVC
QA
QC
American Type Culture Collection
Arizona Test Dust
Bacillus anthracis (Ames strain)
Bacillus subtilis (ATCC 19659)
Battelle Biomedical Research Center
biological safety cabinet
degree(s) Celsius
Chemical, biological, and radiological
colony forming unit(s)
confidence interval
centimeter(s)
deoxyribonucleic acid
U.S. Environmental Protection Agency
feet
gram
Hydrogen chloride
hydrogen peroxide
hour(s)
homeland security
Homeland Security Research Program
Kilogray(s)
Limulus Amebocyte Lysate
liter(s)
Light emitting diode
log reduction
Molarity
methyl bromide
milligram(s)
methyl isothiocyanate
milliliter(s)
microliter(s)
sodium hypochlorite
sodium hydroxide
sodium persulfate
National Homeland Security Research
Center
Office of Research and Development
ounce
phosphate-buffered saline
phosphate-buffered saline + 0.1% Triton® X-
100
polymerase chain reaction
part(s) per million
polyvinyl chloride
quality assurance
quality control
-------
QMP Quality management plan
RH relative humidity
rpm revolution(s) per minute
SD standard deviation
SE standard error
SFW sterile filtered water (cell-culture grade)
STS sodium thiosulfate
TSA technical systems audit(s)
XI
-------
1.0 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 investigation was to develop an understanding of the effectiveness of four
different chemical biocides or technologies to decontaminate two types of soil materials.
Residual biological agent (such as B. anthracis) on surfaces following decontamination after an
intentional release could present a potential health risk. This report documents the impact of
operational factors on the efficacy of two liquid decontaminants (pH-amended bleach, sodium
persulfate) and two fumigants (methyl bromide, metam sodium) against spores of B. anthracis
and B. subtilis using one cm deep topsoil and AZTD. B. subtilis was included in the tests to
assess its potential use as a benign surrogate microorganism (so that future decontamination tests
could be conducted without the use B. anthracis). Decontamination efficacy was determined
based on the log reduction (LR) in viable spores recovered from the inoculated samples, with and
without exposure to the decontaminants.
-------
2.0 Technology Descriptions and Test Matrices
2.1 Technology Descriptions
Table 2-1 describes the four decontamination technologies (or biocides) evaluated in this
investigation. Information is provided on the manufacturer, product name (where applicable),
chemical components and active ingredients. Some of the decontaminants are mixtures that react
to produce other chemicals that are responsible for the sporicidal activity. Note that Ultra
Clorox® Germicidal Bleach is registered as a disinfectant, but the pH-amended solution is not.
Further details on the chemical composition, preparation, and decontamination application
procedures are provided in Section 4.
Table 2-1 Decontamination Technology Descriptions
Decontaminant
Product Name
and Vendor
Active Ingredients
and Sporicidal
Chemical
Components
EPA
Registration
Methyl Bromide
Methyl Bromide
Matheson Tri-Gas
Basking Ridge, NJ
Methyl bromide
99.5% methyl bromide gas with
0.5% chloropicrin added as a
warning irritant
None
Metam Sodium
Metam
concentrate,
Buckman
Laboratories, Inc.
Memphis, TN
Sodium N-
methyldithiocarbamate
(metam sodium),
methyl isothiocyanate
(MITC)
42.5% metam sodium; aqueous
solution
1448-107
pH-Amended
Bleach
Ultra
Clorox®Germicidal
Bleach,
Clorox®
Professional
Products Co.
Oakland, CA
Sodium hypochlorite,
hypochlorous acid
Sodium hypochlorite 6.15%,
sodium hydroxide <1%; diluted
with sterile filtered water
(SFW); with 5% acetic acid
added to reduce pH to 6.5 - 7.0.
67619-8
(disinfectant)
Sodium Persulfate
Klozur™
FMC Corporation
Philadelphia, PA
Sodium persulfate,
activated with
hydrogen peroxide;
sulfate radicals
Sodium persulfate (Na2S2O8)
>99% purity (used as a 12%
(0.5M) aqueous solution,
activated with an 8% hydrogen
peroxide solution
None
Methyl bromide was selected for testing because it has been demonstrated to be effective against
B. anthracis on building materials1, but has not been tested against B. anthracis on soils.
Furthermore, although MeBr use is being phased out under the Montreal Protocol, MeBr is still
currently and widely used via critical use exemptions as a soil and commodity (quarantine)
fumigant2. Metam sodium was selected for testing because it is the most widely used soil
fumigant in the US3. If proven to be effective against B. anthracis, it would greatly improve
preparedness in the event of an outdoor release of B. anthracis to have a decontaminant that is
widely available and commonly used. Bleach (with its pH lowered) was selected for testing
because this decontaminant has been demonstrated to be effective against B. anthracis on some
materials, is easily made using off-the-shelf chemicals, and is often the decontaminant of choice
for remediation officials4. Lastly, sodium persulfate was included in this evaluation because this
chemical is used to remediate soil contaminated with organic chemicals. In addition, sodium
-------
persulfate was shown to be moderately effective against B. anthracis in soil in screening tests5,
so more robust test conditions were planned for this evaluation.
2.2 Test Matrices for Liquid Decontamination
In general, the conditions selected for testing (e.g., contact time, concentration) were based on
previous, similar B. anthracis efficacy tests (as described above), as well as how the
decontaminants are currently used in practice.
The test matrices for the pH-amended bleach and sodium persulfate (activated with hydrogen
peroxide (H2O2)) liquid sporicide tests are shown in Tables 2-2 and 2-3, respectively. For the
bleach tests, topsoil was tested only once under the most robust test condition (Test 1; it was
completely ineffective). For the sodium persulfate tests, a contact time of seven days was used
for each test. For each test listed below, separate subtests were conducted for each combination
of microorganism and soil type.
Table 2-2 pH-Amended Bleach Test Matrix
Test#
1
2
3
4
Table
Test#
1
2
3
4
5
Biological
Agent
B. anthracis
B. subtilis
B. anthracis
B. subtilis
B. anthracis
B. subtilis
B. anthracis
B. subtilis
2-3 Sodium
Biological
Agent
B. anthracis
B. subtilis
B. anthracis
B. subtilis
B. anthracis
B. subtilis
B. anthracis
B. subtilis
B. anthracis
B. subtilis
Soil type
Topsoil
AZTD
AZTD
AZTD
AZTD
Persulfate (Klozur™)
Soil Type
Topsoil
AZTD
Topsoil
AZTD
Topsoil
AZTD
Topsoil
AZTD
Topsoil
AZTD
Application Frequency
(total number of
applications)
0.5 mL every 15 minutes
for 2 hr (8)
0.5 mL every 15 minutes
for 2 hr (8)
0.5 mL every 30 minutes
for 2 hr (4)
0.5 mL every 30 minutes
for 1 hr (2)
Test Matrix
Application Frequency*
(total number of
applications)
Every 60 minutes (6)
Every 60 minutes (3)
Days 0, 2 and 4 (3)
Time 0 and 1 Hr (2)
Day 0 (1)
Contact Time (hr)
168
24
2
1
Contact Time
(days)
7
7
7
7
7
1 = Each application consisted of 1 mL Klozur followed by 1 mL 8% H2O2.
2.3 Test Matrices for Fumigant Decontamination
The test matrices for the MeBr and metam sodium fumigation tests are shown in Tables 2-4 and
2-5.
-------
For MeBr, all tests were conducted at 25 °C and with RH measured but not controlled.
However, soil moisture did affect the RH level, so soil moisture was adjusted in a few
experiments to assess its impact on RH and subsequent decontamination efficacy.
Metam sodium requires some moisture in the soil to produce MITC gas, the chemical
responsible for biocidal activity6. Two mL of water were therefore added to all soil samples
prior to testing, and additional amounts of water were added to the soils as an experimental
variable. The two methods used to pre-sterilize the soil (irradiation and autoclaving) also
affected soil moisture, so soil sterilization method was also used as a test parameter. Due to the
potential for having residual metam sodium in the soil following the contact time, the soil
samples were allowed to aerate for varying amounts of time (mimicking how the product is used
in the field). Further details on the metam sodium related test procedures may be found in
Section 4.4 and Appendix A.
Table 2-4 MeBr Test Matrix
Test#
1
2
3
4
5
6
7
8
Biological
Agent
B. anthracis
B. subtilis
B. anthracis
B. subtilis^
B. anthracis
B. subtilis^
B. anthracis
B. subtilis
B. anthracis
B. subtilis^
B. anthracis
B. subtilis
B. anthracis
B. subtilis
B. anthracis
B. subtilis
Soil type
Topsoil
AZTD
Topsoil
AZTD
Topsoil
AZTD
Topsoil
AZTD
Topsoil
AZTD
Topsoil
AZTD
Topsoil
AZTD
Topsoil
AZTD
Target Concentration
(mg/L)
212 ±21
212 ±21
212 ±21
100 ±10
100 ±10
180 ±18
140 ±14
212 ±21
Contact Time (hr)
36
36
36
36
36
36
36
24
' 2 mL SFW added prior to sample inoculation.
* Samples dried prior to sample inoculation.
Table 2-5 Metam Sodium Test Matrix
Test
#
1
2
3
4
Biological Agentt
B. anthracis
B. subtilis
B. anthracis
B. subtilis
B. anthracis
B. subtilis
B. anthracis
B. subtilis
Soil type
Topsoil
AZTD
Topsoil
AZTD
Topsoil
AZTD
Topsoil
AZTD
Amount
SFW Added
Prior to
Addition of
Metam
Sodium
(mL)
1
2
Quantity of
Metam
Sodium
Applied
(HL)
80
160
160
160
Soil Sterilization
Method
Gamma Irradiation at
40kGy
Gamma Irradiation at
40kGy
Gamma Irradiation at
60kGy
Gamma Irradiation at
60kGy
Time/
Aeration
Time (days)
5/0
5/0
111
7/7
-------
B. anthracis or
B. subtilis
Topsoil
AZTD
160
Gamma Irradiation at
60kGy
7/7
B. anthracis or
B. subtilis
Topsoil
AZTD
160
Gamma Irradiation at
60kGv
14/28
B. anthracis or
B. subtilis
Topsoil
AZTD
160
Autoclave
(121 °C; 1 hr)
7/7
B. anthracis or
B. subtilis
Topsoil
AZTD
160
Autoclave
(121 °C; 1 hr)
14/28
-------
3.0 Summary of Test Procedures
Test procedures were performed in accordance with a pre-approved Quality Assurance Project
Plan (QAPP) (available upon request) and are summarized in this chapter.
3.1 Biological Agent
The B. anthracis spores used for this testing were prepared from a qualified stock of the Ames
strain at the Battelle Biomedical Research Center (BBRC, West Jefferson, OH). All spore lots
were subject to a stringent characterization and qualification process required by Battelle's
standard operating procedure for spore production. Specifically, all spore lots were characterized
prior to use by observation of colony morphology, direct microscopic observation of spore
morphology and size and determination of percent refractivity and percent encapsulation (of the
vegetative bacterial colonies). In addition, the number of viable spores was determined by colony
count and expressed as colony forming units per milliliter (CFU/mL). Theoretically, once plated
onto bacterial growth media, each viable spore germinates and yields one CPU. Variations in the
expected colony phenotypes were recorded. Endotoxin concentration of each spore preparation
was determined by the Limulus Amebocyte Lysate (LAL) assay to assess whether contamination
from gram-negative bacteria occurred during the propagation and purification process of the
spores. Genomic deoxyribonucleic acid (DNA) was extracted from the spores and DNA
fingerprinting by polymerase chain reaction (PCR) was performed to confirm the genotype. The
virulence of the spore lot was measured by challenging guinea pigs intradermally with a dilution
series of spore suspensions, and virulence was expressed as the intradermal median lethal dose.
To ensure spores are used in testing (and not vegetative cells), various steps are taken, described
as follows. The spore stock is stored in purified water and characterized via visual purity. The
stock is viewed under the microscope, viable spores are then counted and any cell debris is
noted. The spore preparation must have a minimum 95% purity vs. debris and non-viable
spores. The spore prep is also heat shocked prior to removing from our fermenter. In addition,
testing was conducted for robustness of the spores via hydrochloric acid (HC1) resistance.
The B. subtilis spores (BBRC stock culture; American Type Culture Collection [ATCC] 19659)
underwent the same characterization tests as described above for B. anthracis, except that the
LAL assay, DNA fingerprinting, and virulence testing were excluded. Qualitative PCR was
performed using a custom PCR assay to confirm B. subtilis. Primers were designed that targeted
a conserved region of B. subtilis chromosomal DNA because multiple strains of this bacterium
exist.
The stock spore suspensions were prepared in SFW at an approximate concentration of 1 x 109
CFU/mL and stored under refrigeration at 2 to 8 degrees Celsius (°C).
3.2 Soil Materials
Information on the soil types used for testing is presented in Table 3-1. Soil samples were placed
unpacked in one ounce (oz), 1.5 inch diameter glass jars (Qorpak®, #GLC-01596, Bridgeville,
PA) at a depth of one cm for testing. The commercial topsoil used for this evaluation was a
proprietary mixture of soil, composted cow manure, sand, and other ingredients (also
-------
proprietary). Topsoil was selected for testing since it represents a difficult soil to treat in terms
of its organic content. The AZTD was selected for testing since it represents a soil with minimal
organic burden.
Soils used in tests with pH-amended bleach, persulfate, and MeBr were prepared for testing by
sterilization via gamma irradiation at -40 kilogray (kGy; STERIS Isomedix Services,
Libertyville, IL). Soils were pre-sterilized to minimize contamination that could interfere with
colony counting. However, when testing with metam sodium (the last technology to be tested),
endogenous bacteria were observed in the topsoil samples, so additional soil sterilization
methods were evaluated and used for the tests with metam sodium. (It is unclear why this
contamination occurred, since topsoil samples were all from the same lot.) In addition to gamma
irradiation at -40 kGy, samples were gamma irradiated at -60 kGy or autoclaved at 121 °C for
one hr. (Refer to Appendix A for additional details.) Gamma-irradiated soils were sealed in
Lock & Lock containers (Farmers Branch, TX) and autoclaved soils were sealed in sterilization
pouches (Cat # 01-812-51, Fisher Scientific, Pittsburgh, PA) to preserve sterility until the
samples were ready for use.
Table 3-1 Soil Materials
Material*
Topsoil
Arizona Test Dust
Lot, Batch, or
ASTM No., or
Observation
Earthgro® Topsoil,
Product #:
71140180
ISO 12103-1, A3
Medium
Manufacturer/
Supplier Name
The Scotts
Company
Marysville, OH
Powder
Technology, Inc.
Burnsville, MN
Pre-sterilized
moisture content
(%)
34
0.23
Pre-sterilized
organic carbon
content (%)
9.3
0.40
* A soil sample consisted of a 1.5 in diameter glass jar filled with uncompacted soil to a height of 1 cm.
Prior to decontamination testing, samples (pre- and post-sterilization) were analyzed in triplicate
using ASTM D Method 2974-87 for Moisture, Ash and Organic Matter of Peat and Other
Organic Soils7. The results of these tests are shown in Table 3-1 and in more detail in Appendix
A. Note the topsoil has a much higher moisture and organic content compared to the AZTD.
The moisture and organic content did not change significantly after the gamma irradiation of the
samples. However, slight changes were observed in autoclaved samples.
Because the moisture content of soils could impact the decontamination efficacy of metam
sodium, the moisture content of samples used in the tests with metam sodium was also
determined using ASTM Method D 2974-87. Further details and results are found in Appendix
A.
3.3 Preparation of Soil Samples
Test and positive control soil samples (in their jars) were placed on a flat surface within a Class
II biological safety cabinet (BSC) and inoculated with approximately 1 x 108 CFU of viable B.
anthracis or B. subtilis spores per sample. A 100 microliter (jiL) aliquot of a stock suspension of
approximately 1 x 109 CFU/mL was dispensed using a micropipette applied as 10 jiL droplets
across the soil surface. This approach provided a more uniform distribution of spores across the
sample surface than would be obtained through a single drop of the suspension. Further details
on the inoculation methods can be found elsewhere8'9. After inoculation, the samples were left
undisturbed overnight in a Class III BSC to dry under ambient conditions, approximately 22 °C
and 40% relative humidity (RH). A heat shock test was conducted to confirm that no
7
-------
germination of cells occurred (only spores present) while spores were left in soil samples
overnight.
The number and type of replicate samples used for each combination of material, decontaminant,
concentration, and environmental condition included were:
• five test samples (inoculated with B. anthracis or B. subtilis spores and exposed to
decontaminant)
• five positive controls (inoculated with B. anthracis or B. subtilis spores but not exposed
to decontaminant)
• one laboratory blank (inoculated with sterile water only and not exposed to the
decontaminant)
• one procedural blank (inoculated with sterile water only and exposed to the
decontaminant)
On the day following spore inoculation, the jars of soil samples intended for decontamination
(including blanks) were transferred into a test chamber where the decontamination technology
was applied using the apparatus and application conditions specified in Section 4 of this report.
3.4 Sample Extraction and Biological Agent Quantification
At the appropriate decontaminant contact time, spores were extracted from the soil samples by
adding 10 mL of sterile phosphate-buffered saline extraction buffer containing 0.1% Triton®
X-100 surfactant (PBST; Sigma, St. Louis, MO) and neutralizer (to stop sporicidal activity when
liquid decontaminant was used; refer to subsection 4.2.2 and Appendix B) to each sample jar.
The jars were capped and agitated on an orbital shaker for 15 minutes at approximately 200
revolutions per minute (rpm) at room temperature. Further details on these methods can be
found elsewhere8'9.
Residual viable spores were quantified using a dilution plating approach. Following extraction,
the extract was removed and a series of 10-fold dilutions was prepared in sterile water. An
aliquot (0.1 mL) of either the undiluted extract and/or each serial dilution was plated onto tryptic
soy agar in triplicate and incubated for 18-24 hours (hr) at 35-37 °C. Colonies were counted
manually and CFU/mL was determined by multiplying the average number of colonies per plate
by the reciprocal of the dilution. Dilution data representing the greatest number of individually
definable colonies were expressed as arithmetic mean ± standard deviation of the numbers of
CFU observed.
Laboratory blanks controlled for sterility and procedural blanks controlled for viable spores
inadvertently introduced to test samples. The blanks were inoculated with an equivalent amount
of 0.1 mL SFW. The target acceptance criterion was that extracts of laboratory or procedural
blanks were to contain zero CFU of target organism.
After each decontamination test, the BSC III was cleaned thoroughly (using separate steps
involving bleach, ethanol, water, then drying) following procedures established under the BBRC
Facility Safety Plan.
-------
3.5 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/CFUspike] x 100 (1)
where Mean CFUpc is the mean number of CPU recovered from five replicate positive control
samples of a single material, and CFUspike is the number of CPU inoculated onto each of those
samples. The value of CFUspike is known from enumeration of the stock spore suspension. Spore
recovery was calculated for B. anthracis or B. subtilis on each soil sample, and the results are
included in Section 6.
The performance or efficacy of the decontaminants was assessed by determining the number of
viable organisms remaining on each soil test sample after decontamination. Those numbers were
compared to the number of viable organisms extracted from the positive control samples.
The number of viable spores of B. anthracis or B. subtilis in extracts of test and positive control
samples was determined to calculate efficacy of the decontaminant. Efficacy is defined as the
extent (as logio reduction) to which viable spores extracted from test samples after
decontamination were less numerous than the viable spores extracted from positive control
samples. The logarithm of the CPU abundance from each sample extract was determined, and the
mean of those logarithm values was then determined for each set of control and associated test
samples, respectively. Efficacy of a decontaminant for a test organism/test condition on the /'th
sample material was calculated as the difference between those mean log values, i.e.:
Efficacy = (Iog10 CFUcl}) - (Iog10 CFUttj) (2)
where logio CFUcy refers to they individual logarithm values obtained from the positive control
samples, and logio CFUty refers to they individual logarithm values obtained from the
corresponding test samples, and the overbar designates a mean value. In tests conducted under
this plan, there were five positive controls and five corresponding test samples (i.e.,7 = 5) for
each soil sample. A decontaminant that achieves a 6 LR or greater is considered effective10.
In the case where no viable spores were detected in any of the five test sample extracts after
decontamination, a CPU abundance of 1 was assigned, resulting in a logio CPU of zero for that
material. When this occurs, the spore population on the soil sample is considered to be
completely inactivated within the detection limit of 33 CPU per soil sample. With complete
spore inactivation, the decontaminant achieves the maximum efficacy possible or quantifiable.
That is, the final efficacy on that material is reported as greater than or equal to (>) the value
calculated by Equation 2. With complete inactivation, the reported LR value is dependent on the
positive control recovery, and in most cases, the LR > 7.5.
The variances (i.e., the square of the standard deviation) of the logio CFUcy and logio CFUty
values were also calculated for both the control and test samples (i.e., S2Cy and <$%•), and were
used to calculate the pooled standard error (SE) for the efficacy value calculated in Equation 2,
as follows:
(3)
-------
where the number 5 again represents the number y of samples in both the control and test data
sets. Each efficacy result is reported as an LR value with an associated 95% confidence interval
(CI), calculated as follows:
95% CI = Efficacy ± (1.96 x SE) (4)
The significance of differences in efficacy across different test conditions and spore types was
assessed based on the 95% confidence interval of each efficacy result. Differences in efficacy
were judged to be significant if the 95% CIs of the two efficacy results did not overlap. Any
results based on this formula are hereafter noted as significantly different. Note this comparison
is not applicable when the two efficacy results being compared are both reported with LRs as >
some value.
3.6 Discoloration of Soils
The physical effect of the decontaminants on the soil materials was also monitored qualitatively
during the evaluation. This approach provided a gross visual assessment of whether the
decontaminants altered the appearance of the soil, e.g., discoloration. The procedural control
(sample that is decontaminated, but has no spores applied) was visually compared to a laboratory
blank sample (a sample not exposed to the decontaminant and that has no spores applied).
10
-------
4.0 Decontamination Procedures
4.1 Liquid Decontaminant Preparation
4.1.1 pH-Amended Ultra Clorox® Germicidal Bleach
The pH-amended bleach consisted of bleach diluted in water with its pH adjusted by addition of
acetic acid. Specifically, Ultra Clorox® Germicidal Bleach was used, which contains 6.15% by
weight sodium hypochlorite (NaOCl) and <1.0% sodium hydroxide (NaOH) in aqueous solution.
This product has a pH between 11 and 12, and a density of 1.08 to 1.11 grams (g)/mL. The pH
adjustment to 6.5 - 7.0 is achieved by the addition of 5% acetic acid. The primary active
decontaminating agent in this final solution is hypochlorous acid. The recipe for preparation of
pH-amended bleach for use as a decontaminant was as follows:
• Prepare 5% acetic acid solution by diluting 50 mL of glacial acetic acid up to 1 L with SFW
in a volumetric flask.
. Mix 9.4 parts SFW, 1 part Ultra Clorox® Germicidal Bleach, and 1 part 5% acetic acid. The
resulting solution will have a mean total chlorine content (estimated based on dilution) of
about 5,400 parts per million (ppm) (or mg/L). The pH is verified before every test to be 6.5
-6.6.
4.1.2 Sodium Persulfate (Klozur™)
Klozur™ was used as the source of sodium persulfate and is a solid reagent made by FMC
Corporation used for in situ and ex situ chemical oxidation of contaminants in environmental
remediation applications (e.g., soil). Klozur™ consists of >99% pure sodium persulfate
(Na2S2Og) in the form of white odorless crystals. In remediation applications, Klozur™ is
injected into contaminated soil or groundwater and activated by mixing in appropriate
proportions of up to 8% H2O2 by weight, according to instructions published by FMC
Corporation11. Activation of Klozur™ with F^C^ generates sulfate radicals (SCV), which are
capable of destroying a wide range of organic contaminants while maintaining oxidative ability
in a soil (organic) environment. For testing, a 0.5 M solution of sodium persulfate was prepared
by dissolving 12 g of Klozur™ in SFW and diluting to 100 mL. This solution was 11.9%
persulfate by weight. Commercially prepared 8% F^C^ solution was purchased for use in testing.
4.2 Liquid Decontamination Test and Control Chambers and Procedures
4.2.1 Test and Control Chambers
All liquid decontaminant tests were conducted at ambient conditions inside a climate-controlled
laboratory. The temperature inside the testing chamber was equilibrated to the ambient
laboratory temperature of approximately 20 °C. The temperature and RH were both monitored
and recorded with a HOBO® data logger (Onset Computer Corporation, Cape Cod, MA), but no
attempt was made to control either. All experiments took place in a Class III BSC.
4.2.2 Neutralization Determination
Sodium thiosulfate (STS) was used to neutralize Klozur™/H2O2 and pH-amended bleach
decontaminants after the desired contact times were achieved. The optimum concentration of
STS in the extraction buffer was determined in trial runs for each liquid decontaminant and
application regimen (number of applications of the decontaminant and contact time) that was
11
-------
tested. In each of those trials, a range of STS concentrations was assessed to determine the STS
concentration that most effectively stopped the action of the decontaminant (indicated by the
maximum recovery of viable spores in the sample extracts). Further details of the methods and
results of the neutralization trials to determine the optimum amount of STS to use are
summarized in Appendix B.
4.2.3 pH-Amended Bleach Decontamination Procedure
The number of applications and contact times were selected for testing was based on previous
tests with pH-amended bleach on soil materials5. Each application consisted of injecting 0.5 mL
of pH-amended bleach into each sample (or SFW into the positive control samples) using a
laboratory pipette, and mixing the soil and pH- amended bleach solution thoroughly in the
sample jar using a glass stirring rod. The solution was re-applied at intervals specified in Table
2-2, mixing between each application with the stirring rod. After the last indicated application,
all samples were left at ambient temperature and RH (with the cap removed) in the Class III BSC
for the required contact time. At the end of the required time, all samples were extracted as
described in Section 3.4.
4.2.4 Sodium Persulfate Decontamination Procedure
For the Klozur™ tests, a 1 mL volume of the 0.5 M persulfate solution was added to each sample
jar and mixed with a glass stirring rod. A 1 mL volume of the 8% H^C^ activating solution was
immediately applied and mixed in the same manner. SFW was applied to the control samples at
all application times in the same manner. This process was repeated for multiple applications as
shown in Table 2-3. After the last application, all samples were left uncapped in the Class III
BSC until the end of the specified contact time (all tests used a seven-day contact time). At the
end of the contact time, all samples were dry, and extracted as described in Section 3.4. Equal
volumes of the persulfate and F^C^ solutions resulted in a F^CVpersulfate molar ratio of 5 to 1, a
typical ratio recommended for the use of Klozur™ in soil remediation11. A contact time of one
week was selected, based on information indicating this oxidant can persist in subsurface
environments for hours to weeks12.
4.3 MeBr Fumigation Test and Control Chambers and Procedures
Methyl bromide is a colorless and odorless volatile gas. Chloropicrin was added to the MeBr
source gas (0.5% chloropicrin, 99.5% MeBr) as a warning irritant (lacrimator) for the safety of
laboratory staff. The gas mixture was used at full strength and injected into the test chamber at
the indicated concentrations.
Figure 4-1 shows a schematic drawing of the MeBr test chamber and containment system. The
primary test chamber was glass with a 23 L volume (approximately 29 x 29 x 29 cm3). The
chamber was insulated to prevent condensation on the inside chamber walls. The glass chamber
was needed for MeBr as the gas exhibits high penetration through many materials. The high
toxicity and penetrability of MeBr also required a secondary containment chamber for protection
of laboratory personnel. A Class III BSC (SG603, Baker, Sanford, ME) provided secondary
containment. Temperature was controlled using a heated water bath and the RH was uncontrolled
in the MeBr test chamber.
12
-------
The MeBr concentration in the test chamber was measured continuously during the contact
period using a Fumiscope™ Version 5.0 (Key Chemical and Equipment Company, Clearwater,
FL). MeBr was added to the chamber, as necessary, to maintain the specified concentration
within ±10%. The Fumiscope meter was calibrated by the manufacturer for MeBr, displaying
the concentration on a digital light-emitting diode (LED) display in ounces of MeBr per 1000
cubic feet (ft3). One oz per 1000 ft3 is approximately 257 ppm at 25 °C and is approximately 1
mg/L (independent of temperature). The Fumiscope meter included an air pump that pulled a
gas sample from the test chamber through the thermal conductivity meter at a controlled rate and
exhausted the gas back into the test chamber. Moisture was removed from the gas sample before
it was measured in the Fumiscope to eliminate interference from water. At the end of a given
trial, the test chamber was flushed with ambient air to <250 ppm and opened.
Gas concentration
controller
23 L Test Chamber
Gas bottte
Pressure regulator
Automated valve
Nafion tube
Power inlet
Temp/RH probe
Fumiscope
t
KOH or Carbon trap
Temp/RH controller
Pass III Biological Safety Cabinet
Figure 4-1 Schematic of MeBr Decontamination Test Chamber.
A 9 L Lock & Lock® airtight container served as the positive control chamber. After the
addition of control and laboratory blank samples, this chamber was kept at ambient laboratory
temperature with no attempts made to control temperature. The RH in the chamber was also left
uncontrolled to mimic the RH in the test chamber as closely as possible. However, both
temperature and RH were measured with an iTHP-2 temperature and humidity probe (Omega
Engineering, Stamford, CT).
As in previous studies with MeBr1, multiple samples of each soil type were inoculated with the
biological agent and placed on a wire rack inside the test chamber. Blank (i.e., uninoculated) and
positive control (i.e., inoculated but not decontaminated) samples were also prepared for each
soil material and were utilized with data from the test samples (inoculated and decontaminated)
to determine decontamination efficacy.
The eight MeBr tests were conducted at concentrations ranging from 100 to 212 mg/L, as shown
in Table 2-4. All tests were conducted using a 36 hr contact time, except the last test that was
conducted for 24 hr. All tests were conducted at 25 °C and with RH measured but not
controlled. The initial RH levels in the chamber just prior to injection of MeBr were at ambient
13
-------
levels and ranged from approximately 35-65% RH. The RH levels in the chamber increased
over time, presumably due to the release of moisture from the soil samples into the chamber air.
Two of the tests (Tests 2 and 5) were conducted with soils in which 2 mL SFW was added, to
assess the impact on RH levels and decontamination efficacy. One test (Test 3) was conducted
with the soil materials dried prior to testing, also to assess impact on RH level (presumably
would be relatively lower) and decontamination efficacy.
4.4 Metam Sodium Fumigation Test and Control Chambers and Procedures
Metam sodium is a clear or yellow to yellow-green liquid with a slight sulfide odor; the source
we used was an aqueous solution of metam sodium, 42.5% by weight13.
Upon exposure to the environment, metam sodium decomposes to MITC (the primary
biologically active ingredient) and eventually decomposes to hydrogen sulfide and other
degradation products containing hydrogen, sulfur, and nitrogen. The conversion rate of metam
sodium to MITC depends on soil moisture, pH, soil type, temperature, organic content, and other
factors. For optimum performance, it is recommended that the soil be free of clods and soil
moisture be between 50-80% of field capacity. Metam sodium can be applied with tillers,
sprinklers, or other means of distribution to mix into the soil. Once the metam sodium is added to
the soil, it is common practice to place a tarp or cap over the soil to prevent the loss of MITC.
The time between application of the metam sodium and planting depends on whether a tarp is
used and can vary between 2-4 weeks6. After removal of the tarp, the soil may need to be
aerated prior to planting14.
All metam sodium tests were completed under ambient laboratory conditions with no attempt
made to control the temperature or RH. Each sample jar served as its own primary container.
Immediately prior to inoculation of spores to the soil sample, 2 mL of SFW was added to each
test, control, and blank sample and mixed with a glass stirring rod. Once mixed, all test and
control samples were inoculated with biological agent and allowed to dry overnight as described
in Section 3.3. Because of the potential importance of soil moisture in the formation of MITC,
following the overnight drying period, additional amounts of water were added to the soil
samples (varied from 0-3 mL of SFW; refer to Table 2-5), and the mixture was stirred.
During the course of testing with metam sodium, endogenous bacteria (morphologically distinct
from our target organisms) were observed in the topsoil samples. This observation of
endogenous bacteria occurred in the soils that were treated with gamma irradiation at -40 kGy
and also in subsequent soil samples irradiated at -60 kGy or autoclaved at 121 °C for one hr.
Because the sterilization method could impact soil moisture, the moisture content of samples
used in the tests with metam sodium was determined for each test. Refer to Appendix A for
additional details.
Following the addition of any water, the 42.5 % metam sodium solution was applied with a
pipette using small droplets (i.e., 10 jiL/droplet). Decontamination tests were conducted using
either 80 jiL (Test 1) or 160 jiL (Tests 2-8) metam sodium applied to each soil sample. Based on
the surface area of the sample jars used, these amounts correspond to approximately 75 gallons
per acre or 150 gallons per acre, respectively. After application of the metam sodium, the sample
jars were capped. These airtight containers allowed the liquid metam sodium to off-gas to MITC,
and keep the gas in close contact with each soil sample (mimicking a tarped application in
agriculture). Following the conclusion of the contact time, samples were either extracted and
plated (refer to Section 3.4) immediately, or the caps were removed, samples stirred, and then the
14
-------
samples were allowed to aerate with their lids off for a specified amount of time prior to
extraction.
Starting with the fourth metam sodium test, we assessed the presence of MITC (potentially
indicating the presence of residual metam sodium in the soil) after the contact time to determine
the need for aeration of soil samples. Air samples were drawn from the headspace of one B.
anthracis topsoil test sample and one B. anthracis AZTD test sample using specialized MITC
detection tubes (Cat # 800-03485, SKC Gulf Coast Inc., Houston, TX; detection limit of 0.1
ppm). These air samples were taken immediately following the removal of the sample caps and
stirring of each sample. Air samples were then taken again at the end of the aeration period. This
procedure was utilized to ensure that no MITC remained in the headspace of the samples at the
end of the contact time and aeration period. These tests did show the presence of MITC after
each contact time (indicating the potential presence of residual metam sodium), but no MITC
was detected after the same samples were aerated.
15
-------
5.0 Quality Assurance/Quality Control
Quality assurance/quality control (QC) procedures were performed in accordance with the
Quality Management Plan (QMP) and the test/QA Plan. The QA/QC procedures and results are
summarized below.
5.1 Equipment Calibration
All equipment (e.g., pipettes, incubators, biological safety cabinets) and monitoring devices (e.g.,
thermometer, hygrometer) used at the time of evaluation were verified as being certified,
calibrated, or validated.
5.2 QC Results
Quality control efforts conducted during decontaminant testing included positive control samples
(inoculated, not decontaminated), procedural blanks (not inoculated, decontaminated), laboratory
blank (not inoculated, not decontaminated), and inoculation control samples (analysis of the
stock spore suspension).
All positive control results were within the target recovery range of 1 to 150% of the inoculated
spores, and all procedural and laboratory blanks met the criterion of no observed CPU for both
organisms.
Inoculation control samples were taken from the spore suspension on the day of testing and
serially diluted, nutrient plated, and counted to establish the spore density used to inoculate the
samples. The spore density levels met the QA target criterion of 1 x 109 CFU/mL (±1 log) for all
tests.
5.3 Audits
5.3.1 Performance Evaluation Audit
Performance evaluation audits were conducted to assess the quality of the results obtained during
these experiments. Table 5-1 summarizes the performance evaluation audits that were
performed.
No performance evaluation audits were performed to confirm the concentration of B. anthracis
or B. subtilis spores. Unlike chemical analytes, commercially available quantitative standards do
not exist for these organisms. The control samples and blanks support the spore measurements.
16
-------
Table 5-1 Performance
Measurement
Volume of liquid from
micropipettes
Time
Temperature
Relative Humidity
Evaluation Audits
Audit
Procedure
Gravimetric evaluation
Compared to independent clock
Compared to independent calibrated
thermometer
Compare to independent calibrated
hygrometer
Fumiscope™ thermal Instrument was certified as calibrated at
conductivity meter the time of use
Balance
Compared to independent calibrated
weight sets
Allowable
Tolerance
± 10%
± 2 sec/hr
±2°C
± 10%
± 10%
±0.5g
Actual
Tolerance
± 0.57%
0 sec/hr
±0.36°C
±2%
0%
±0.03g
5.3.2 Technical Systems Audit
Observations and findings from the technical systems audit (ISA) were documented and
submitted to the laboratory staff lead. TSAs were conducted on December 6 and December 13,
2011, to ensure that the tests were being conducted in accordance with the test/QA plan and
Quality Management Plan (QMP). As part of the audit, test procedures were compared to those
specified in the test/QA plan and data acquisition and handling procedures were reviewed. None
of the findings of the ISA required corrective action.
5.3.3 Data Quality Audit
At least 10% of the data acquired during the evaluation were audited. The data was traced from
the initial acquisition, through reduction and statistical analysis, to final reporting to ensure the
integrity of the reported results. All calculations performed on the data undergoing the audit were
checked.
5.4 Test/Quality Assurance Plan Deviations
5.4.1 pH-Amended Ultra Clorox® Germicidal Bleach Test Matrix
Table 5 of the test/QA plan shows that four tests would be completed using pH-amended Ultra
Clorox Germicidal Bleach using both topsoil and AZTD. After showing minimal efficacy (0.36
LR against B. anthracis and 0.10 with B. subtilis) under the most robust test conditions planned,
there appeared to be no need to investigate the efficacy of pH-am ended bleach for topsoil further.
The remaining tests were conducted using AZTD only.
5.4.2 Extraction buffer
For the seven-day test using pH-amended Ultra Clorox® Germicidal Bleach, the neutralization
extraction buffer (1.5% sodium thiosulfate [STS]) was made with phosphate-buffered saline
(PBS) instead of the required PBST. This buffer was used to extract all test samples (both topsoil
and AZTD) and associated blanks. Minimal effect on results was expected, as exemplified in the
positive control recovery results.
17
-------
5.5 QA/QC Reporting
Each assessment and audit was documented in accordance with the test/QA plan and QMP. For
these tests, findings were noted (none significant) in the TSA or data quality audit, but no follow-
up corrective action was necessary. The findings for the TSA were minor with one item noted
(see Section 5.4.2, above). The findings for the data quality audit were mostly minor data
transcription errors requiring some recalculation of efficacy results, but none were gross errors in
recording.
5.6 Data Review
Records and data generated in the evaluation received a QC/technical review before they were
utilized in calculating or evaluating results and prior to incorporation in reports.
18
-------
6.0 Results and Performance Summary for Liquid Biocides
6.1 pH-Amended Bleach Results
The quantitative decontamination efficacy results (in terms of LR) for pH-amended bleach
against spores of B. anthracis and B. subtilis are presented in detail in Tables 6-1 and 6-2 and
summarized in Figure 6-1.
For the seven-day contact time (Test 1), the decontamination efficacy on topsoil was minimal
(less than 0.5 LR for both organisms), and because of this lack of efficacy, additional tests under
the less robust conditions were discontinued for topsoil. The poor efficacy is most likely due to
the organic content of the topsoil; refer to Table 3-1.
At this same decontamination treatment for AZTD, both microorganisms were completely
inactivated. In general, the pH-amended bleach was more successful in decontaminating the
AZTD, with greater than 7 LR obtained for both B. anthracis and B. subtilis with only a 2 hr
contact time (Test 3). There were no significant differences in efficacy between the two
microorganisms tested.
6.2 Sodium Persulfate Results
The quantitative efficacy results for Klozur™ are presented in detail in Tables 6-3 through 6-6
and summarized graphically in Figure 6-2. All tests were conducted with a contact time of seven
days, with the number of applications of the sodium persulfate/F^C^ decontaminant ranging
from 1 to 6. The most robust treatment, the six application regimen (Test 1), resulted in
complete inactivation of B. anthracis on both soil materials. The next robust treatment (three
applications of the decontaminant, all applied within the first 2 hours; Test 2) provided greater
than a 7 LR for B. anthracis on both soils. Efficacy generally decreased with decreasing number
of persulfate applications; none of the other sodium persulfate application conditions resulted in
greater than a 6 LR.
When comparing the results for the topsoil and AZTD, the decontamination effectiveness of the
sodium persulfate against B. anthracis was not significantly different for the two soil types for
the majority of tests. Against B. subtilis, however, the sodium persulfate technology was
generally more effective on the topsoil than the AZTD. (The highest LR obtained for B. subtilis
on AZTD was 1.1.) These results generally indicate that the organic content of the topsoil did
not diminish efficacy, which is consistent with its persistent oxidative ability and commercial use
as a soil remediation technology. When comparing results for the two microorganisms, B.
subtilis was significantly more difficult to inactivate than B. anthracis in all but one of the tests.
Two tests were conducted to assess whether the frequency of the application of sodium
persulfate affected decontamination efficacy. In these two tests, we used a contact time of seven
days and three applications of the sodium persulfate, but in one test it was applied once every
hour (Test 2), and in the other, it was applied every 48 hours (Test 3). When applied every hour,
the efficacy was significantly greater than when it was applied every 48 hours (on Days 0, 2 and
4) against B. anthracis: 7.07 vs. 5.53 LR on topsoil and 7.38 vs. 5.24 LR on AZTD. However,
with respect to B. subtilis, the frequency of the application resulted in no significant difference in
efficacy.
19
-------
Table 6-1 Inactivation of Bacillus anthracis Spores on Soil using pH-Amended Ultra
Clorox® Germicidal Bleach"
Contact Time
(Number of Applications)
- Material
Test#
Seven Days (8)
Topsoil3 - #1
Positive Controls'3
Test Samples0
Laboratory Blankd
Procedural Blank6
Seven Days (8) -
AZTD - #1
Positive Controls
Test Samples
Laboratory Blank
Procedural Blank
24 Hours (8) -
AZTD - #2
Positive Controls
Test Samples
Laboratory Blank
Procedural Blank
Two Hours (4) -
AZTD - #3
Positive Controls
Test Samples
Laboratory Blank
Procedural Blank
One Hour (2) -
AZTD - #4
Positive Controls
Test Samples
Laboratory Blank
Procedural Blank
Inoculum (CFU)
9.87 x 107
9.87 x 107
0
0
9.87 x 107
9.87 x 107
0
0
9.43 x 107
9.43 x 107
0
0
1.05 x 108
1.05 x 108
0
0
1.04 x 108
1.04 x 108
0
0
Mean of Logs of
Observed CFU
7.54 ±0.04
7. 19 ±0.06
0
0
7.88 ±0.05
0
0
0
7.87 ±0.03
0
0
0
7.91 ±0.04
0
0
0
7.82 ±0.06
2.66 ± 1.79
0
0
Mean % Decontamination
Recovery Efficacy ± CIf
35.60 ±3.48 z
15.74 ±2.29 0.36 ±0.07
-
-
77.08 ± 9.72
0 >7.88±0.05
-
-
79.30 ±5.07
0 >7.87 ± 0.02
-
-
77.35 ±6.99
0 >7.91±0.03
-
-
63.75 ±9.00
0.0066 ± 0.0088 5. 16 ±1.57
-
-
a Data are expressed as the mean (± SD) of the logs of the number of spores (CFU) observed on five individual samples, the mean percent
recovery on those five samples, and decontamination efficacy (log reduction).
b Positive Controls = samples inoculated, not decontaminated.
c Test Samples = samples inoculated, decontaminated.
d Laboratory Blank = samples not inoculated, not decontaminated.
e Procedural Blank = samples not inoculated, decontaminated.
f CI = confidence interval (± 1.96 x SE).
* "-" Not Applicable.
20
-------
Table 6-2 Inactivation of Bacillus subtilis Spores on Soil using pH-Amended Ultra
Clorox® Germicidal Bleach"
Contact Time
(Number of Applications)
- Material
Test#
Seven Days (8) -
Topsoil3 - #1
Positive Controls'3
Test Samples'
Laboratory Blankd
Procedural Blank6
Seven Days (8) -
AZTD - #1
Positive Controls
Test Samples
Laboratory Blank
Procedural Blank
24 Hours (8) -
AZTD - #2
Positive Controls
Test Samples
Laboratory Blank
Procedural Blank
Two Hours (4) -
AZTD - #3
Positive Controls
Test Samples
Laboratory Blank
Procedural Blank
One Hour (2) -
AZTD - #4
Positive Controls
Test Samples
Laboratory Blank
Procedural Blank
Inoculum (CFU)
1.25 x 108
1.25 x 108
0
0
1.25 x 108
1.25 x 108
0
0
1.19xl08
1.19xl08
0
0
1.14xl08
1.14xl08
0
0
8.80 x 107
8.80 x 107
0
0
Mean of Logs of
Observed CFU
7.41 ±0.15
7.31 ±0.03
0
0
7.81 ±0.16
0
0
0
7.93 ± 0.02
0
0
0
7.95 ±0.03
0.74 ± 1.66
0
0
7.69 ±0.05
4.42 ± 1.67
0
0
Mean % Decontamination
Recovery Efficacy ± CIf
21.58 ±6.96 g
16.29 ±1.02 0.10 ±0.14
-
-
54.77 ±17.15
0 >7.81±0.14
-
-
71. 14 ±3.40
0 >7.93 ± 0.02
-
-
77.98 ±5.33
0.00090 ± 0.0021 7.21 ±1.46
-
-
56.40 ± 6.60
0.23 ±0.36 3.28 ±1.47
-
-
Data are expressed as the mean (± SD) of the logs of the number of spores (CFU) observed on five individual samples, the mean percent
recovery on those five samples, and decontamination efficacy (log reduction).
b Positive Controls = samples inoculated, not decontaminated.
c Test Samples = samples inoculated, decontaminated.
d Laboratory Blank = samples not inoculated, not decontaminated.
e Procedural Blank = samples not inoculated, decontaminated.
f CI = confidence interval (± 1.96 x SE). Differences in efficacy may be significant if the 95% CIs of the two efficacy results do not overlap;
however, this comparison is not applicable when the two efficacy results being compared are both reported with log reductions as > some
value.
8 "-" Not Applicable.
21
-------
I
I
5
pH-Amended Ultra Clorox* Germicidal Bleach
7 Day (8)
Topsoil
7 Day (8)
AZTD
24 Hour (8)
AZTD
Hour (4)
AZTD
1 Hour (2)
AZTD
Contact Time (= of applications)
Soil Type
Figure 6-1 Summary of Decontamination Efficacies (with 95% confidence
intervals) for pH-Amended Bleach Testing on Topsoil and AZTD
22
-------
Table 6-3 Inactivation of Bacillus anthracis Spores on Topsoil with Klozur™'3
Contact Time
(Number of Applications)
Test#
Seven Days (6)1 - #1
Positive Controls'3
Test Samples0
Laboratory Blankd
Procedural Blank6
Seven Days (3) t- #2
Positive Controls
Test Samples
Laboratory Blank
Procedural Blank
Seven Days (3)*- #3
Positive Controls
Test Samples
Laboratory Blank
Procedural Blank
Seven Days (2) t- #4
Positive Controls
Test Samples
Laboratory Blank
Procedural Blank
Seven Days (1) - #5
Positive Controls
Test Samples
Laboratory Blank
Procedural Blank
Inoculum (CFU)
8.97 x 107
8.97 x 107
0
0
8.97 x 107
8.97 x 107
0
0
1.12xl08
1.12xl08
0
0
1.23 x 108
1.23 x 108
0
0
8.83 x 107
8.83 x 107
0
0
Mean of Logs of
Observed CFU
7.82 ±0.04
0
0
0
7.72 ± 0.04
0.65 ±1.46
0
0
7.62 ±0.09
2.09 ± 1.40
0
0
7.83 ±0.08
4.03 ± 1.13
0
0
7.68 ±0.08
6.75 ±0.31
0
0
Mean %
Recovery
74.35 ±7.50
0
-
-
58.51 ±5.65
0.00041 ±0.00090
-
-
37.98 ±8.74
0.00093 ±0.0014
-
-
55.45 ±9.91
0.00089 ±0.0017
-
-
54.90 ± 10.81
0.076 ± 0.042
-
-
Decontamination
Efficacy ± CIf
j.
>7.82 ± 0.04
-
-
-
7.07 ±1.28
-
-
-
5.53 ± 1.23
-
-
-
3. 80 ±1.00
-
-
-
0.93 ±0.28
-
-
Data are expressed as the mean (± SD) of the logs of the number of spores (CFU) observed on five individual samples, the mean percent
recovery on those five samples, and decontamination efficacy (log reduction).
b Positive Controls = samples inoculated, not decontaminated.
c Test Samples = samples inoculated, decontaminated
d Laboratory Blank = samples not inoculated, not decontaminated.
e Procedural Blank = samples not inoculated, decontaminated.
f CI = confidence interval (± 1.96 x SE).
g "-" Not Applicable.
T The decontaminant was applied every 60 minutes until the total number of applications was reached.
* The decontaminant was applied on days 0, 2 and 4.
23
-------
Table 6-4 Inactivation of Bacillus anthracis Spores on AZTD with Klozur™'a
Contact Time T ,
~T , f A .. ,. -. Inoculum
(Number 01 Applications) ,^-^-m
Test# (CFU)
Seven Days (6)f - #1
Positive Controls'3
Test Samples'
Laboratory Blankd
Procedural Blank6
Seven Days (3) t- #2
Positive Controls
Test Samples
Laboratory Blank
Procedural Blank
Seven Days (3)*- #3
Positive Controls
Test Samples
Laboratory Blank
Procedural Blank
Seven Days (2) t- #4
Positive Controls
Test Samples
Laboratory Blank
Procedural Blank
Seven Days (1) - #5
Positive Controls
Test Samples
Laboratory Blank
Procedural Blank
8.97 x 107
8.97 x 107
0
0
8.97 x 107
8.97 x 107
0
0
1.12xl08
1.12xl08
0
0
1.23 x 108
1.23 x 108
0
0
8.83 x 107
8.83 x 107
0
0
Mean of Logs of ,.„ n/ „ Decontamination
™. j A^TT Mean % Recovery _„„. , ~Tf
Observed CFU J Efficacy ± CI
7.87 ±0.10
0
0
0
7.69 ±0.04
0.31 ±0.70
0
0
7.79 ±0.10
2.55 ±0.59
0
0
7.48 ±0.31
3.31 ±1.33
0
0
7.95 ±0.13
3.58 ±0.26
0
0
83.94 ±19.52 -&
0 >7.87 ± 0.09
-
55.21 ±5.04
0.0000091 ±0.000018 7.38 ±0.61
56.46 ±12.98
0.00054 ±0.00051 5.24 ±0.53
30.32 ±25.09
0.025 ±0.050 4.17 ±1.19
-
104.24 ±32.81
0.0049 ± 0.0026 4.37 ± 0.26
-
Data are expressed as the mean (± SD) of the logs of the number of spores (CFU) observed on five individual samples, the mean percent
recovery on those five samples, and decontamination efficacy (log reduction).
b Positive Controls = samples inoculated, not decontaminated.
c Test Samples = samples inoculated, decontaminated.
d Laboratory Blank = samples not inoculated, not decontaminated.
e Procedural Blank = samples not inoculated, decontaminated.
f CI = confidence interval (± 1.96 x SE).
g "-" Not Applicable.
T The decontaminant was applied every 60 minutes until the total number of applications was reached.
* The decontaminant was applied on days 0, 2 and 4.
24
-------
Table 6-5 Inactivation of Bacillus subtilis Spores on Topsoil with Klozur
TM,a
Contact Time
(Number of Applications)
Test#
Seven Days (6)t - #1
Positive Controls'3
Test Samples0
Laboratory Blankd
Procedural Blank6
Seven Days (3) t- #2
Positive Controls
Test Samples
Laboratory Blank
Procedural Blank
Seven Days (3)*- #3
Positive Controls
Test Samples
Laboratory Blank
Procedural Blank
Seven Days (2) t- #4
Positive Controls
Test Samples
Laboratory Blank
Procedural Blank
Seven Days (1) - #5
Positive Controls
Test Samples
Laboratory Blank
Procedural Blank
Inoculum
(CFU)
1.12xl08
1.12xl08
0
0
9.30 x 107
9.30 x 107
0
0
1.31 xlO8
1.31 xlO8
0
0
l.lSxlO8
l.lSxlO8
0
0
9.53 x 107
9.53 x 107
0
0
Mean of Logs of
Observed CFU
7.86 ±0.18
0
0
0
7.58 ±0.05
4.57 ±2.61
0
0
7.72 ±0.09
4.71 ±0.53
0
0
7.68 ±0.40
7.20 ±0.05
0
0
7.74 ±0.08
7.54 ±0.09
0
0
Mean % Recovery
68.71 ±25.25
0
-
41.57±5.19
0.71 ±0.71
-
40.41 ± 8.02
0.064 ± 0.057
-
57.02 ±50.50
13.37 ±1.59
-
58.91 ±10.70
37.00 ±8.84
Decontamination
Efficacy ± CIf
_s
>7.86±0.16
-
3.01 ±2.28
-
3.01 ±0.48
-
0.49 ±0.35
-
.
0.20 ±0.11
Data are expressed as the mean (± SD) of the logs of the number of spores (CFU) observed on five individual samples, the mean percent
recovery on those five samples, and decontamination efficacy (log reduction).
Positive Controls = samples inoculated, not decontaminated.
Test Samples = samples inoculated, decontaminated.
Laboratory Blank = samples not inoculated, not decontaminated.
Procedural Blank = samples not inoculated, decontaminated.
CI = confidence interval (± 1.96 x SE).
"-" Not Applicable.
The decontaminant was applied every 60 minutes until the total number of applications was reached.
The decontaminant was applied on days 0, 2 and 4.
25
-------
Table 6-6 Inactivation of Bacillus subtilis Spores on AZTD with Klozur
™a
Contact Time
(Number of Applications)
Test#
Seven Days (6)t - #1
Positive Controls'3
Test Samples0
Laboratory Blankd
Procedural Blank6
Seven Days (3) t- #2
Positive Controls
Test Samples
Laboratory Blank
Procedural Blank
Seven Days (3)*- #3
Positive Controls
Test Samples
Laboratory Blank
Procedural Blank
Seven Days (2) t- #4
Positive Controls
Test Samples
Laboratory Blank
Procedural Blank
Seven Days (1) - #5
Positive Controls
Test Samples
Laboratory Blank
Procedural Blank
Inoculum
(CFU)
1.12xl08
1.12xl08
0
0
9.30 x 107
9.30 x 107
0
0
1.31 xlO8
1.31 xlO8
0
0
l.lSxlO8
l.lSxlO8
0
0
9.53 x 107
9.53 x 107
0
0
Mean of Logs of
Observed CFU
8.01±0.11
6.91 ±0.13
0
0
7.64 ±0.09
7.69 ±0.05
0
0
8.00 ±0.03
7.37 ±0.27
0
0
7.89 ±0.31
7.84 ±0.04
0
0
7.96 ±0.04
7.72 ±0.12
0
0
Mean % Recovery
94.99 ±25.64
7.53 ±2.07
-
47.89 ±9.94
52.71 ±5.42
-
75.86 ±5.57
20.88 ±12.67
-
76.26 ±33. 36
59.15 ±5.40
-
95.55 ±9.06
56.69 ± 14.75
Decontamination
Efficacy ± CIf
_s
1.10±0.15
-
0.00 ± 0.00
-
0.62 ± 0.24
-
0.05 ±0.28
-
.
0.24 ±0.11
Data are expressed as the mean (± SD) of the logs of the number of spores (CFU) observed on five individual samples, the mean percent
recovery on those five samples, and decontamination efficacy (log reduction).
Positive Controls = sample inoculated, not decontaminated.
Test Samples = samples inoculated, decontaminated.
Laboratory Blank = samples not inoculated, not decontaminated.
Procedural Blank = samples not inoculated, decontaminated .
CI = confidence interval (± 1.96 x SE).
"-" Not Applicable.
The decontaminant was applied every 60 minutes until the total number of applications was reached.
The decontaminant was applied on days 0, 2 and 4.
26
-------
Klozur™ on Topsoil
IB. antlimcis
iB. snbti/is
7Day(6)T 7Day(3)t 7 Day (3); 7Day(2)t
ContnctTime (= of applications)
7Day(l)
Klozur™ on Arizona Test Dust
iB.mithrads
iB.siibtilis
' Day (6)t
7Day(3)t
Day (2)T
'Day (1)
Contact Time (# of applications)
t = The decontaminant was applied every 60 minutes until the total number of applications was reached.
* =The decontaminant was applied at the start of the test and on day 2 and 4.
Figure 6-2 Summary of Decontamination Efficacies (with 95% confidence
intervals) for Klozur Liquid Testing on Topsoil and AZTD
27
-------
7.0 Results and Performance Summary for Fumigant Biocides
7.1 MeBr Results
The decontamination efficacy of MeBr against B. anthracis and B. subtilis was evaluated at
target concentrations ranging from 100 to 212 mg/L at a target temperature of 25 °C, using a
contact time of 36 hr for all tests except for one test performed at 24 hr. The RH levels in the test
chamber were uncontrolled, but for a few tests, attempts were made to manipulate RH by adding
or removing moisture from the soil prior to testing. The actual fumigation conditions for each
test, including RH, are shown in Table 7-1. The detailed decontamination efficacy results are
shown in Tables 7-2 through 7-5 and summarized graphically in Figure 7-1.
Table 7-1 Actual Fumigation Conditions for Tests with MeBr
Test#
1
2
3
4
5
6
7
8
Actual Mean MeBr
Concentration ± SD
(mg/L)
213 ±0.74
213 ±0.73
213 ±0.68
102 ±1.36
101 ±0.51
181 ±0.68
141 ±0.53
213 ±0.81
Soil Moisture Actual mean Contact
Condition temperature ± SD (° C) lvlean^ai Time (hr)
No change
2mLSFW
added
Samples dried
No change
2mLSFW
added
No change
No change
No change
25. 3 ±0.41
25.2 ±0.16
25.4 ±0.53
25. 5 ±0.62
25. 3 ±0.53
25.2 ±0.22
25.2 ±0.18
25.2 ±0.30
79.3 ±3.53
82. 8 ±2.16
54.5 ±2.26
76.9 ±3. 96
85.1 ±1.91
82.0 ±2.38
82.9 ±2.08
76.2 ±4.01
36
36
36
36
36
36
36
24
The two most robust treatment conditions in terms of concentration, contact time, and soil
moisture (212 mg/L MeBr, 36 hour, no drying of soil; Tests 1 and 2) resulted in complete
inactivation of B. anthracis spores on AZTD, and > 7.0 LR on topsoil. Overall, MeBr was
effective (greater than 6 LR achieved) against B. anthracis on topsoil and AZTD at 25 °C when
using a concentration of at least 180 mg/L and contact time of 36 hours. One minor exception is
the test in which the soil samples were dried beforehand (Test 3), which resulted in a
comparatively lower RH (55%), and a lower decontamination efficacy (LR of 5.9) on topsoil.
The two tests in which water was added to the soil beforehand (Tests 2 and 5) resulted in slightly
higher RH levels (compared to tests under the same conditions without water added), but the
added water had no significant effect or resulted only in slightly decreased decontamination
efficacy for B. anthracis. As expected, decontamination efficacy generally decreased with
decreasing concentration and contact time.
With respect to the effect of soil type, the decontamination efficacies obtained for B. anthracis
were generally slightly higher on AZTD compared to topsoil, although half of the test results for
the two soil types were not significantly different. B. subtilis was significantly more difficult to
inactivate compared to B. anthracis for all tests conducted. The highest LR value obtained for B.
subtilis was 2.1, with the majority of the LR values for B. subtilis below 1.0. The scope of this
study did not allow for us to examine the mechanisms to explain why B. subtilis is significantly
28
-------
more resistant to MeBr than B. anthracis. However, this result is consistent with a previous
study.1
29
-------
Table 7-2 Inactivation of Bacillus anthracis Spores on Topsoil with MeBra
Contact Time T , ,.„ ,.T ,.
,. , ,„ ... Inoculum Mean 01 Logs 01 ,.„ „. „
(Actual Concentration ,^-^-m m. ., ^-n Mean % Recovery
r /T _•_ CT»I\ T * u (CFU) Observed CFU J
[mean mg/L ± SD]) Test #
36 Hours (213 ± 0.74) #1
Positive Controls'3 8.97 x 107 7.77 ± 0.037 66.38 ± 5.47
Test Samples' 8.97 x 107 0.36 ± 0.82 0.000016 ± 0.000033
Laboratory Blankd 0 0
Procedural Blank6 0 0
36 Hours (213 ± 0.73)* #2
Positive Controls 9.97 x 107 7.92 ± 0.065
Test Samples 9.97 x 107 0.51 ± 1.15 0.
Laboratory Blank 0 0
Procedural Blank 0 0
36Hours(213±0.68)t#3
Positive Controls 7.57 x 107 7.65 ± 0.080
Test Samples 7.57 x 107 1.78 ± 1.75
Laboratory Blank 0 0
Procedural Blank 0 0
36 Hours (102 ± 1.36) #4
Positive Controls 8.73 x 107 7.72 ± 0.080
Test Samples 8.73 x 107 6.28 ± 0.037
Laboratory Blank 0 0
Procedural Blank 0 0
36 Hours (101 ± 0.51)* #5
Positive Controls 8.40 x 107 7.81 ±0.11
Test Samples 8.40 x 107 6.81 ± 0.075
Laboratory Blank 0 0
Procedural Blank 0 0
83.61 ±12.71
.000074 ± 0.00016
60.21 ±11.30
0.0028 ± 0.0058
61.49 ±11.27
2.18 ±0.19
79.43 ±21. 13
7.84 ± 1.29
36 Hours (181 ± 0.68) #6
Positive Controls 8.83 x 107 7.66 ± 0.036 51.55 ±4.31
Test Samples 8.83 x 107 0.97 ± 0.90 0.000031 ± 0.000031
Laboratory Blank 00
Procedural Blank 0 0
36 Hours (141 ± 0.53) #7
Positive Controls 1.43 x 108 8.05 ± 0.020
Test Samples 1.43 x 108 4.59 ±0.13
Laboratory Blank 0 0
Procedural Blank 0 0
24 Hours (213 ± 0.81) #8
Positive Controls 1.25 x 108 7.79 ± 0.045
Test Samples 1.25 x 108 4.54 ± 0.38
Laboratory Blank 0 0
Procedural Blank 0 0
a Data are expressed as the mean (± SD) of the logs of the number of spores (CFU) observed
recovery on those five samples, and decontamination efficacy (log reduction).
b Positive Controls = samples inoculated, not decontaminated.
c Test Samples = samples inoculated, decontaminated.
11 Laboratory Blank = samples not inoculated, not decontaminated.
e Procedural Blank = samples not inoculated, decontaminated .
f CI = confidence interval (± 1.96 x SE).
8 "-" Not Applicable.
* 2 mL SFW added to samples prior to inoculation.
T = Samples dried in oven prior to inoculation.
78.18 ±3.58
0.028 ± 0.0075
49.01 ±4.82
0.037 ±0.033
on five individual samples,
Decontamination
Efficacy ± CIf
7.41 ±0.72
7.40 ± 1.01
5.87 ± 1.54
1.45 ±0.08
1.00 ±0.12
6.68 ± 0.79
3.46 ±0.11
3.25 ±0.34
the mean percent
30
-------
Table 7-3 Inactivation of Bacillus anthracis Spores on AZTD with MeBra
Contact Time T , ,.„ ,.T ,.
,. , ,„ ... Inoculum Mean 01 Logs 01 ,.„ „. „
(Actual Concentration ,^-^-m /-*. ., ^TT Mean % Recovery
r /T _•_ CT»I\ T * u (CFU) Observed CFU J
[mean mg/L ± SD]) Test #
36 Hours (213 ± 0.74) #1
Positive Controls'3 8.97 x 107 7.74 ±0.80 62.81 ± 12.48
Test Samples' 8.97 x 107 0 0
Laboratory Blankd 0 0
Procedural Blank6 0 0
36 Hours (213 ± 0.73)* #2
Positive Controls 9.97 x 107 7.90 ± 0.062 80.52 ± 11.32
Test Samples 9.97 x 107 0 0
Laboratory Blank 00
Procedural Blank 0 0
36Hours(213±0.68)t#3
Positive Controls 7.57 x 107 7.77 ±0.050 77.75 ± 9.08
Test Samples 7.57 x 107 0.30 ±0.68 0.000010 ± 0.000019
Laboratory Blank 00
Procedural Blank 0 0
36 Hours (102 ± 1.36) #4
Positive Controls 8.73 x 107 7.75 ± 0.056 64.83 ± 8.69
Test Samples 8.73 x 107 5.41 ± 0.44 0.38 ± 0.20
Laboratory Blank 00
Procedural Blank 0 0
36 Hours (101 ± 0.51)* #5
Positive Controls 8.40 x 107 7.72 ±0.061 63.33 ± 8.79
Test Samples 8.40 x 107 6. 32 ±0.28 2. 89 ±1.49
Laboratory Blank 00
Procedural Blank 0 0
36 Hours (181 ± 0.68) #6
Positive Controls 8.83 x 107 7.75 ± 0.052 63.87 ± 7.55
Test Samples 8.83 x 107 1.49 ± 1.47 0.00056 ± 0.0011
Laboratory Blank 00
Procedural Blank 0 0
36 Hours (141 ± 0.53) #7
Positive Controls 1.43 x 108 8.13 ± 0.031 94.69 ± 6.79
Test Samples 1.43 x 108 1.94 ± 1.78 0.00074 ± 0.00069
Laboratory Blank 00
Procedural Blank 0 0
24 Hours (213 ± 0.81) #8
Positive Controls 1.25 x 108 7.87 ±0.084 60.85 ±11. 51
Test Samples 1.25 x 108 0.70 ±0.98 0.000022 ± 0.000034
Laboratory Blank 00
Procedural Blank 0 0
a Data are expressed as the mean (± SD) of the logs of the number of spores (CFU) observed on five individual samples,
recovery on those five samples, and decontamination efficacy (log reduction).
b Positive Controls = samples inoculated, not decontaminated.
c Test Samples = samples inoculated, decontaminated.
11 Laboratory Blank = samples not inoculated, not decontaminated.
e Procedural Blank = samples not inoculated, decontaminated.
f CI = confidence interval (± 1.96 x SE).
8 "-" Not Applicable.
* 2 mL SFW added to samples prior to inoculation.
T Samples dried in oven prior to inoculation.
Decontamination
Efficacy ± CIf
_s
>7.74 ± 0.07
>7.90 ± 0.05
7.46 ± 0.60
2.34 ±0.39
1.40 ±0.25
6.26 ±1.29
6.19 ±1.56
7.17 ±0.86
the mean percent
-------
Table 7-4 Inactivation of Bacillus subtilis Spores on Topsoil with MeBra
Contact Time
(Actual Concentration
[mean mg/L ± SD]) Test #
36 Hours (213 ± 0.74) #1
Positive Controls'3
Test Samples0
Laboratory Blankd
Procedural Blank6
36 Hours (213 ± 0.73)* #2
Positive Controls
Test Samples
Laboratory Blank
Procedural Blank
36Hours(213±0.68)t#3
Positive Controls
Test Samples
Laboratory Blank
Procedural Blank
36 Hours (102 ± 1.36) #4
Positive Controls
Test Samples
Laboratory Blank
Procedural Blank
36 Hours (101 ± 0.51)* #5
Positive Controls
Test Samples
Laboratory Blank
Procedural Blank
36 Hours (181 ± 0.68) #6
Positive Controls
Test Samples
Laboratory Blank
Procedural Blank
36 Hours (141 ± 0.53) #7
Positive Controls
Test Samples
Laboratory Blank
Procedural Blank
24 Hours (213 ± 0.81) #8
Positive Controls
Test Samples
Laboratory Blank
Procedural Blank
Inoculum
(CFU)
6.10 xlO7
6.10 xlO7
0
0
1.36 xlO8
1.36 xlO8
0
0
1.03 x 108
1.03 x 108
0
0
9.23 x 107
9.23 x 107
0
0
8.50 x 107
8.50 x 107
0
0
9.53 x 107
9.53 x 107
0
0
1.42 x 108
1.42 x 108
0
0
1.36 xlO8
1.36 xlO8
0
0
Mean of Logs of
Observed CFU
7.75 ± 0.047
5.62 ± 0.045
0
0
8.00 ±0.051
6.54 ± 0.063
0
0
7.52 ±0.024
6.74 ±0.061
0
0
7.77 ±0.035
7.45 ±0.17
0
0
7.77 ±0.014
7.79 ± 0.025
0
0
7.66 ± 0.083
6.78 ±0.061
0
0
8. 11 ±0.076
6.42 ±0.15
0
0
8.03 ± 0.0066
7.36 ±0.22
0
0
Mean % Recovery
92.59 ±9.37
0.69 ± 0.07
-
-
73.59 ±8.68
2.55 ±0.37
-
-
32.37 ± 1.78
5.35 ±0.68
-
-
64.53 ±5. 15
32.46 ± 9.93
-
-
69.32 ±2.15
71.93 ±4.17
-
-
48.69 ± 9.30
6.30 ±0.91
-
-
91. 83 ±15.87
1.91 ±0.61
-
-
79.26 ± 1.21
18.33 ±6.76
-
-
Decontamination
Efficacy ± CIf
_s
2.13 ±0.06
-
-
-
1.46 ±0.07
-
-
-
0.78 ± 0.06
-
-
-
0.32 ±0.15
-
-
-
0.00 ±0.00
-
-
-
0.88 ±0.09
-
-
-
1.69 ±0.15
-
-
-
0.67 ±0.19
-
-
a Data are expressed as the mean (± SD) of the logs of the number of spores (CFU) observed on five individual samples, the mean percent
recovery on those five samples, and decontamination efficacy (log reduction).
b Positive Controls = samples inoculated, not decontaminated.
c Test Samples = samples inoculated, decontaminated.
11 Laboratory Blank = samples not inoculated, not decontaminated.
e Procedural Blank = samples not inoculated, decontaminated.
f CI = confidence interval (± 1.96 x SE).
8 "-" Not Applicable.
* 2 mL SFW added to samples prior to inoculation.
T Samples dried in oven prior to inoculation.
32
-------
Table 7-5 Inactivation of Bacillus subtilis Spores on AZTD with MeBra
Contact Time
(Actual Concentration
[mean mg/L ± SD]) Test #
36 Hours (213 ± 0.74) #1
Positive Controls'3
Test Samples0
Laboratory Blankd
Procedural Blank6
36 Hours (213 ± 0.73)* #2
Positive Controls
Test Samples
Laboratory Blank
Procedural Blank
36Hours(213±0.68)t#3
Positive Controls
Test Samples
Laboratory Blank
Procedural Blank
36 Hours (102 ± 1.36) #4
Positive Controls
Test Samples
Laboratory Blank
Procedural Blank
36 Hours (101 ± 0.51)* #5
Positive Controls
Test Samples
Laboratory Blank
Procedural Blank
36 Hours (181 ± 0.68) #6
Positive Controls
Test Samples
Laboratory Blank
Procedural Blank
36 Hours (141 ± 0.53) #7
Positive Controls
Test Samples
Laboratory Blank
Procedural Blank
24 Hours (213 ± 0.81) #8
Positive Controls
Test Samples
Laboratory Blank
Procedural Blank
Inoculum
(CFU)
6.10 xlO7
6.10 xlO7
0
0
1.36 xlO8
1.36 xlO8
0
0
1.03 x 108
1.03 x 108
0
0
9.23 x 107
9.23 x 107
0
0
8.50 x 107
8.50 x 107
0
0
9.53 x 107
9.53 x 107
0
0
1.42 x 108
1.42 x 108
0
0
1.36 xlO8
1.36 xlO8
0
0
Mean of Logs of
Observed CFU
7.77 ±0.038
6.30 ±0.059
0
0
8.03 ±0.031
7.49 ±0.061
0
0
7.58 ±0.071
7.07 ± 0.047
0
0
7.78 ±0.063
7.72 ± 0.067
0
0
7.77 ±0.012
7.50 ±0.22
0
0
7.77 ±0.12
7.64 ±0.052
0
0
8.04 ±0.041
8.00 ± 0.026
0
0
7.92 ±0.090
7.97 ± 0.024
0
0
Mean % Recovery
96.82 ±8.23
3.32 ±0.45
79.01 ±5.67
23.03 ±3.47
37.36 ±5.94
11.57 ±1.24
65.35 ±8.88
56.86 ±8.52
-
68.61 ±1.97
40.59 ±19.15
-
64. 16 ±14.69
45.67 ±5.37
-
76.86 ±7.25
71.27 ±4.38
62.16 ±13. 14
68.09 ±3.77
Decontamination
Efficacy ± CIf
_s
1.47 ±0.06
0.54 ±0.06
0.51 ±0.07
0.06 ±0.08
-
.
0.27 ±0.19
-
0.14 ±0.12
-
0.03 ±0.04
0.00 ±0.00
a Data are expressed as the mean (± SD) of the logs of the number of spores (CFU) observed on five individual samples, the mean percent
recovery on those five samples, and decontamination efficacy (log reduction).
b Positive Controls = samples inoculated, not decontaminated.
c Test Samples = samples inoculated, decontaminated.
11 Laboratory Blank = samples not inoculated, not decontaminated.
e Procedural Blank = samples not inoculated, decontaminated.
f CI = confidence interval (± 1.96 x SE).
8 "-" Not Applicable.
* 2 mL SFW added to samples prior to inoculation.
T Samples dried in oven prior to inoculation.
33
-------
MeBron Top soil
15. anthracis
•B. subtilis
Contact Time (ing L)
MeBron Arizona Test Dust
15. fiutlnncis
IB. stibtilis
Contact Time (ing L)
* 2 mL SFW added to each sample prior to inoculation.
T samples dried in oven prior to inoculation.
Figure 7-1. Summary of Decontamination Efficacies for MeBr Fumigant Testing
on Topsoil and AZTD
34
-------
7.2 Metam Sodium Results
The detailed decontamination efficacy results for metam sodium against B. anthracis and B.
subtilis on topsoil and AZTD are summarized in Tables 7-6 through 7-9 and summarized
graphically in Figure 7-2.
In terms of the number of test conditions in which the soil samples were completely inactivated,
the metam sodium was significantly more effective against both microorganisms on the AZTD
compared to the topsoil for the majority of the tests. For example, in all but one of the eight tests
on AZTD, B. anthracis was completely inactivated, whereas there was just one test (Test 6) on
topsoil in which B. anthracis was completely inactivated.
In all the tests on topsoil, B. subtilis was significantly more difficult to inactivate compared to B.
anthracis. On AZTD, B. subtilis was significantly more difficult to inactivate compared to B.
anthracis in half the tests.
The effect of doubling the amount of metam sodium applied to the soil materials can be seen in
reviewing results for Tests 1 and 2. Efficacy improved significantly for both microorganisms on
topsoil and for B. subtilis on AZTD.
Increasing contact time and aeration time generally improved efficacy for the inactivation of B.
anthracis on topsoil but not significantly. This effect can be seen by comparing the results
between Test 2 and 3 (contact time/aeration time increased from 5/0 to 7/7 days); Tests 5 and 6
(contact time/aeration time increased from 7/7 to 14/28 days using 60 kGy irradiated soils); and
Tests 7 and 8 (contact time/aeration time increased from 7/7 to 14/28 days for autoclaved soils).
Similar improvements in efficacy were seen with B. subtilis on AZTD.
The moisture content of the soil samples was affected by the amount of water added prior to
decontamination testing, the soil sterilization method, and the overnight dry time; refer to
Appendix A for further details. The effect of moisture content on decontamination efficacy of
the metam sodium is readily apparent in the results for B. anthracis on topsoil (refer to Figure 7-
3). In Figure 7-3, results are aggregated by contact time/aeration time (results for Tests 1 and 2
are excluded because different amounts of metam sodium were used), which shows that efficacy
increases with increasing levels of moisture. For Tests 3, 5, and 6, in which efficacy was greater
than 6.0, the soil moisture was notably at its highest levels. The effect of moisture on efficacy
was not readily apparent for the results with B. subtilis on topsoil (all results less than a 1.13 LR)
or for both microbes on AZTD (all results greater than 7 LR for B. anthracis). For AZTD, the
lack of apparent effect of moisture may be because the moisture content for Tests 3-8 was
generally uniform at approximately 18%, with one exception for Test 5, which had a moisture
content at 27%.
35
-------
Table 7-6 Inactivation of Bacillus anthracis Spores on Topsoil with Metam Sodium3
Contact Time
(Aeration Time)
Test#
Five Days (0 Days) - #1
Positive Controls
Test Samples0
Laboratory Blankd
Procedural Blank6
Five Days (0 Days) - #2
Positive Controls
Test Samples
Laboratory Blank
Procedural Blank
Seven Days (7 Days) T - #3
Positive Controls
Test Samples
Laboratory Blank
Procedural Blank
Seven Days (7 Days) * - #4
Positive Controls
Test Samples
Laboratory Blank
Procedural Blank
Seven Days (7 Days) § - #5
Positive Controls
Test Samples
Laboratory Blank
Procedural Blank
14 Days (28 Days) T- #6
Positive Controls
Test Samples
Laboratory Blank
Procedural Blank
Seven Days (7 Days) T - #7
Positive Controls
Test Samples
Laboratory Blank
Procedural Blank
14 Days (28 Days) T - #8
Positive Controls
Test Samples
Laboratory Blank
Procedural Blank
Inoculum
(CFU)
1.37xl08
1.37xl08
0
0
l.llxlO8
l.llxlO8
0
0
1.03 xlO8
1.03 xlO8
0
0
1.04 xlO8
1.04 xlO8
0
0
9.97 xlO7
9.97 xlO7
0
0
9.67 xlO7
9.67 xlO7
0
0
l.llxlO8
l.llxlO8
0
0
1.00 xlO8
1.00 xlO8
0
0
Mean Logs
Observed
(CFU ± SD)
7.94 ±0.064
7.11 ±0.23
0
0
8.00 ±0.060
3.41±3.11
0
0
7.83 ±0.52
1.82 ±2.50
0
0
7.74 ±0.062
3. 96 ±0.48
0
0
7.89 ±0.082
1.37±2.01
0
0
7.84 ±0.046
0
0
0
8.04 ±0.0099
6.15 ±0.22
0
0
7.94 ±0.042
4.96 ±0.40
0
0
Mean % Recovery Decontamination
(±SD) Efficacy ±CIf
64.82 ± 9.68 -£
10.69 ±6.58 0.83 ±0.21
-
-
90.63 ± 12.64
0.27 ±0.26 4.59 ±2.73
-
-
66.08 ± 8.04
0.014 ±0.020 6.01 ±2.19
-
-
52.75 ±7.56
0.014 ±0.013 3.77 ±0.42
-
-
79.78 ±14.46
0.0054 ±0.12 6.52 ±1.76
-
-
71.09 ±7.58
0 >7.84±0.04
-
-
98.56 ±2.26
1.39±0.61 1.89±0.19
-
-
87. 18 ±8.42
0.12±0.071 2.98±0.35
-
-
Data are expressed as the mean (± SD) of the logs of the number of spores (CFU) observed on five individual samples, the mean percent
recovery on those five samples, and decontamination efficacy (log reduction).
Positive Controls = samples inoculated, not decontaminated.
Test Samples = samples inoculated, decontaminated.
Laboratory Blank = samples not inoculated, not decontaminated.
Procedural Blank = samples not inoculated, decontaminated .
CI = confidence interval (± 1.96 x SE).
"-" Not Applicable.
2 mL SFW added to all samples prior to inoculation.
1 mL SFW added prior to addition of metam sodium.
2 mL SFW added prior to addition of metam sodium.
3 mL SFW added prior to addition of metam sodium.
36
-------
Table 7-7 Inactivation of Bacillus anthracis Spores on AZTD with Metam Sodium3
Contact Time
(Aeration Time)
Test#
Five Days (0 Days) - #1
Positive Controls
Test Samples0
Laboratory Blankd
Procedural Blank6
Five Days (0 Days) - #2
Positive Controls
Test Samples
Laboratory Blank
Procedural Blank
Seven Days (7 Days) T - #3
Positive Controls
Test Samples
Laboratory Blank
Procedural Blank
Seven Days (7 Days) f - #4
Positive Controls
Test Samples
Laboratory Blank
Procedural Blank
Seven Days (7 Days) § - #5
Positive Controls
Test Samples
Laboratory Blank
Procedural Blank
14 Days (28 Days) T- #6
Positive Controls
Test Samples
Laboratory Blank
Procedural Blank
Seven Days (7 Days) T - #7
Positive Controls
Test Samples
Laboratory Blank
Procedural Blank
14 Days (28 Days) T - #8
Positive Controls
Test Samples
Laboratory Blank
Procedural Blank
Inoculum
(CFU)
1.37xl08
1.37xl08
0
0
l.llxlO8
l.llxlO8
0
0
1.03 xlO8
1.03 xlO8
0
0
1.04 xlO8
1.04 xlO8
0
0
9.97 xlO7
9.97 xlO7
0
0
9.67 xlO7
9.67 xlO7
0
0
l.llxlO8
l.llxlO8
0
0
1.00 xlO8
1.00 xlO8
0
0
Mean Logs
Observed
(CFU ± SD)
7.84 ±0.054
0.63 ±1.40
0
0
7.77 ±0.10
0
0
0
7.81 ±0.040
0
0
0
7.68 ±0.061
0
0
0
7.91 ±0.053
0
0
0
7.79 ±0.025
0
0
0
7.81 ±0.045
0
0
0
7.74 ±0.062
0
0
0
Mean % Recovery Decontamination
(±SD) Efficacy ±CIf
51. 18 ±6.26 -g
0.00020 ± 0.00045 7.22 ±1.23
-
-
54.47 ±14.03
0 >7.77±0.09
-
-
63. 36 ±5.83
0 >7.81±0.03
-
-
46. 65 ±6.47
0 >7.68± 0.054
-
-
82.57 ±10.22
0 >7.91±0.47
-
-
64.14 ±3.55
0 >7.79±0.02
-
-
58.49 ±5.96
0 >7.81±0.04
-
-
55. 86 ±7.92
0 >7.74±0.05
-
-
a Data are expressed as the mean (± SD) of the logs of the number of spores (CFU) observed on five individual samples, the mean percent
recovery on those five samples, and decontamination efficacy (log reduction).
b Positive Controls = samples inoculated, not decontaminated.
c Test Samples = samples inoculated, decontaminated.
d Laboratory Blank = samples not inoculated, not decontaminated.
e Procedural Blank = samples not inoculated, decontaminated.
f CI = confidence interval (± 1.96 x SE).
8 "-" Not Applicable.
* 2 mL SFW added to all samples prior to inoculation.
t 1 mL SFW added prior to addition of metam sodium.
* 2 mL SFW added prior to addition of metam sodium.
§ 3 mL SFW added prior to addition of metam sodium.
37
-------
Table 7-8 Inactivation of Bacillus subtitis Spores on Topsoil with Metam Sodium3
Contact Time
(Aeration Time)
Test#
Five Days (0 Days) - #1
Positive Controls
Test Samples0
Laboratory Blankd
Procedural Blank6
Five Days (0 Days) - #2
Positive Controls
Test Samples
Laboratory Blank
Procedural Blank
Seven Days (7 Days) T - #3
Positive Controls
Test Samples
Laboratory Blank
Procedural Blank
Seven Days (7 Days) f - #4
Positive Controls
Test Samples
Laboratory Blank
Procedural Blank
Seven Days (7 Days) § - #5
Positive Controls
Test Samples
Laboratory Blank
Procedural Blank
14 Days (28 Days) T- #6
Positive Controls
Test Samples
Laboratory Blank
Procedural Blank
Seven Days (7 Days) T - #7
Positive Controls
Test Samples
Laboratory Blank
Procedural Blank
14 Days (28 Days) T - #8
Positive Controls
Test Samples
Laboratory Blank
Procedural Blank
Inoculum
(CFU)
1.07 xlO8
1.07 xlO8
0
0
1.35xl08
1.35xl08
0
0
1.04 xlO8
1.04 xlO8
0
0
9.73 x 107
9.73 x 107
0
0
9.07 xlO7
9.07 xlO7
0
0
8.97 xlO7
8.97 xlO7
0
0
1.13xl08
1.13xl08
0
0
1.06 xlO8
1.06 xlO8
0
0
Mean Logs
Observed
(CFU ± SD)
7.99 ±0.010
7.77 ±0.094
0
0
8.06 ±0.010
6.97±0.19
0
0
7.99 ±0.055
6.86 ±0.21
0
0
7.72 ±0.048
7.06 ±0.19
0
0
7.81 ±0.058
7.18±0.11
0
0
7.85 ±0.072
6.75 ±0.51
0
0
7.87 ±0.035
7.19±0.10
0
0
7.82 ±0.070
6.94 ±0.27
0
0
Mean % Recovery Decontamination
(±SD) Efficacy ±CIf
91. 53 ±2.05 -g
56.45 ±11. 96 0.22 ±0.08
84. 15 ±2.00
7.53±3.51 1.08±0.17
-
95. 52 ±11. 83
7.76±3.74 1.13 ±0.19
54.12±6.10
12.72 ±5.32 0.66 ±0.18
-
71. 51 ±9.24
16.89 ±3.61 0.63 ±0.11
-
80.13 ±14.57
8.94±5.63 1.10±0.45
65. 86 ±5.21
14.09 ±2.90 0.68 ±0.09
-
63. 15 ±10.07
9.23 ±4.52 0.89 ±0.24
-
Data are expressed as the mean (± SD) of the logs of the number of spores (CFU) observed on five individual samples, the mean percent
recovery on those five samples, and decontamination efficacy (log reduction).
b Positive Controls = samples inoculated, not decontaminated.
c Test Samples = samples inoculated, decontaminated.
d Laboratory Blank = samples not inoculated, not decontaminated.
e Procedural Blank = samples not inoculated, decontaminated.
f CI = confidence interval (± 1.96 x SE).
8 "-" Not Applicable.
* 2 mL SFW added to all samples prior to inoculation.
t 1 mL SFW added prior to addition of metam sodium.
* 2 mL SFW added prior to addition of metam sodium.
§ 3 mL SFW added prior to addition of metam sodium.
38
-------
Table 7-9 Inactivation of Bacillus subtitis Spores on AZTD with Metam Sodium3
Contact Time
(Aeration Time)
Test#
Five Days (0 Days) - #1
Positive Controls
Test Samples0
Laboratory Blankd
Procedural Blank6
Five Days (0 Days) - #2
Positive Controls
Test Samples
Laboratory Blank
Procedural Blank
Seven Days (7 Days) T - #3
Positive Controls
Test Samples
Laboratory Blank
Procedural Blank
Seven Days (7 Days) f - #4
Positive Controls
Test Samples
Laboratory Blank
Procedural Blank
Seven Days (7 Days) § - #5
Positive Controls
Test Samples
Laboratory Blank
Procedural Blank
14 Days (28 Days) T- #6
Positive Controls
Test Samples
Laboratory Blank
Procedural Blank
Seven Days (7 Days) T - #7
Positive Controls
Test Samples
Laboratory Blank
Procedural Blank
14 Days (28 Days) T - #8
Positive Controls
Test Samples
Laboratory Blank
Procedural Blank
Inoculum
(CFU)
1.07 xlO8
1.07 xlO8
0
0
1.35xl08
1.35xl08
0
0
1.04 xlO8
1.04 xlO8
0
0
9.73 x 107
9.73 x 107
0
0
9.07 xlO7
9.07 xlO7
0
0
8.97 xlO7
8.97 xlO7
0
0
1.13xl08
1.13xl08
0
0
1.06 xlO8
1.06 xlO8
0
0
Mean Logs
Observed
(CFU ± SD)
7.83 ±0.46
7.06 ±0.37
0
0
7.87 ±0.049
4.15±2.59
0
0
8.02 ±0.030
1.86 ±2.56
0
0
7.76 ±0.033
0
0
0
7.85 ±0.045
0
0
0
7.80 ±0.044
6.06 ±0.13
0
0
7.78 ±0.048
2.97 ±2.82
0
0
7.75 ±0.054
0
0
0
Mean % Recovery Decontamination
(±SD) Efficacy ±CIf
83. 14 ±42.82 -g
13. 99 ±11.29 0.77 ±0.52
-
-
55.72 ±6.17
0.56 ±0.76 3.72 ±2.28
-
-
101.87±7.14
0.019 ±0.027 6.16 ±2.24
-
-
58.64 ±4.33
0 >7.76± 0.029
-
-
78.88 ±8.00
0 >7.85± 0.039
-
-
70. 14 ±7.45
1.32±0.41 1.74±0.12
-
-
53.27 ±5.79
0.27 ±0.59 4.80 ±2.47
-
-
53. 34 ±6.61
0 >7.75±0.05
-
-
a Data are expressed as the mean (± SD) of the logs of the number of spores (CFU) observed on five individual samples, the mean percent
recovery on those five samples, and decontamination efficacy (log reduction).
b Positive Controls = samples inoculated, not decontaminated.
c Test Samples = samples inoculated, decontaminated.
d Laboratory Blank = samples not inoculated, not decontaminated.
e Procedural Blank = samples not inoculated, decontaminated.
f CI = confidence interval (± 1.96 x SE).
8 "-" Not Applicable.
* 2 mL SFW added to all samples prior to inoculation.
t 1 mL SFW added prior to addition of metam sodium.
* 2 mL SFW added prior to addition of metam sodium.
§ 3 mL SFW added prior to addition of metam sodium.
39
-------
Metam Sodium on Top soil"
IS. fnitlnncis
iS. siibtilis
5 Day 5 Day
(ODay) (ODay)
7 Day 7 Day 7 Day
(7 Day)t (7 Day); (7 Day)§
14 Day 7 Day 14 Day
(28Day)t (7Day)| (28Day)t
Contact Time (Aeration Time)
N
Metam Sodium on Arizona Test Dust5"
iS. anthrctcis
IS. siibtilis
5 Day 5 Day
(ODay) (ODay)
7 Day 7 Day 7 Day 14 Day 7 Day 14Day
(7Day)t ("Day); (7 Day)§ (28Da>')t (7Day)t (28Day)t
Contact Time (Aeration Time)
* 1 mL SFW added to all samples prior to inoculation.
t 1 mL SFW added prior to addition of metam sodium.
* 1 mL SFW added prior to addition of metam sodium.
^ 3 mL SFW added prior to addition of metam sodium.
Test results are presented in numerical order
Figure 7-2. Summary of Decontamination Efficacies for Metam Sodium Fumigant Testing
on Topsoil and AZTD
40
-------
38.39 <39.59 <46.38 47.62 <38.39 <46.38
Topsoil Moisture Content
Tests 7, 4, 3, and 5 had 7 d contact time, 7 d aeration; Tests 8 and 6 had 14 d contact time, 28 day aeration
Figure 7-3. Effect of Topsoil Moisture Content on Decontamination Efficacy for B.
anthracis
7.3 Discoloration of Soils
At the end of each decontamination test, the procedural blanks were compared visually to the
laboratory blanks, and test samples were compared visually to positive controls, to assess any
impact (i.e., discoloration) the decontaminants may have had on each material type. Based on the
visual appearance of the decontaminated samples, there were no apparent changes in the color of
the two soil types after being exposed to MeBr, metam sodium, pH-amended bleach, or sodium
persulfate.
41
-------
8.0 Summary of Results
8.1 Decontamination Efficacy
The principle goal of this study was to find the necessary decontamination treatment conditions
(e.g., concentration of active ingredient, contact time, number of applications, etc.) to effectively
decontaminate (> 6 LR) topsoil and AZTD using four different biocidal chemistries. The four
decontaminants tested were pH-amended bleach, sodium persulfate, methyl bromide, and metam
sodium. With the exception of pH-amended bleach in topsoil, greater than 6 LR against B.
anthracis was obtained with all four decontaminants for both soil types.
Table 8-1 shows the minimum conditions required to obtain at least a 6 LR for each combination
of decontamination technology, soil type, and microorganism. More stringent conditions, such as
higher concentration, more applications, or longer contact time typically resulted in higher
efficacy, and in some cases, complete inactivation. Conversely, there were a few tests in which
the most stringent treatment evaluated resulted in a LR less than 6 (indicated in Table 8-1 as
"Not found"). Examples of this included the use of pH-amended bleach on topsoil against both
biological agents, and methyl bromide against B. subtilis (on both soil types).
42
-------
Table 8-1 Minimum Treatment Required for Effective Decontamination
Decontaminant
pH-amended
bleach
pH-amended
bleach
pH-amended
bleach
pH-amended
bleach
Sodium
persulfate
Sodium
persulfate
Sodium
persulfate
Sodium
persulfate
Methyl bromide
Methyl bromide
Methyl bromide
Methyl bromide
Metam sodium
Metam sodium
Metam sodium
Metam sodium
Soil
type
TS
TS
AZTD
AZTD
TS
TS
AZTD
AZTD
TS
TS
AZTD
AZTD
TS
TS
AZTD
AZTD
Microor
-ganism
B.a.
B.s.
B.a.
B.s.
B.a.
B.s.
B.a.
B.s.
B.a.
B.s.
B.a.
B.s.
B.a.
B.s.
B.a.
B.s.
Minimum Treatment for > 6LR
Not found
Not found
2 hour contact time, 4 applications
2 hour contact time, 4 applications
3 applications every 60 minutes
6 applications every 60 minutes
3 applications every 60 minutes
Not found
180 mg/L MeBr, 24 hour contact time
Not found
140 mg/L MeBr, 24 hour contact time
Not found
160 (iL, 7 day contact time, 7 day aeration time, 1 mL
added to soil
Not found
water
80 (iL, 5 day contact time, no aeration period, no moisture added
to soil
160 (iL, 7 day contact time, 7 day aeration time, 1 mL
added to soil
One bleach application consisted of 0.5 mL acidified beach, with a mean FAC level of approximately 5,400 ppm and pH 6.5.
One sodium persulfate application consisted of 1 mL 0.5 M sodium persulfate followed by 1 mL 8% H2O2. All tests conducted using
contact time.
All MeBr tests were conducted at 25 °C and RH uncontrolled (all but one test had RH > 75%).
B.a. = B. anthracis; B. s. = B. subtilis.
water
; a 7-day
8.2 Effect of Soil type
The effect of soil type on decontamination efficacy depended on the chemical decontaminant and
to some extent, the microorganism. For example, the decontamination efficacy results for pH-
amended bleach and metam sodium were significantly higher on AZTD (compared to topsoil)
for nearly every test. For the sodium persulfate, however, the decontamination efficacy results
were very similar for the two soil types when testing against B. anthracis. But interestingly, in
the majority of the persulfate tests against B. subtilis, higher efficacy was achieved on topsoil.
For MeBr, the decontamination efficacies obtained for B. anthracis were generally slightly
higher on AZTD compared to topsoil, although half of the test results for the two soil types were
not significantly different.
43
-------
8.3 Comparing efficacy for B. anthracis and B. subtitis
There were no tests in which B. subtilis was inactivated to a significantly higher degree than B.
anthracis, and, for pH-amended bleach, there were no significant differences in decontamination
efficacy for the two microorganisms. For the other three decontaminants, the efficacy for the
inactivation of B. subtilis was significantly less than the efficacy for B. anthracis for the majority
of the tests conducted. For MeBr in particular, the differences in efficacy for the two
microorganisms were greater than 5-6 LR for more than half of the tests.
44
-------
9.0 References
1. Systematic investigation of liquid and fumigant decontamination efficacy against biological
agents deposited on test coupons of common indoor materials. EPA 600/R-l 1/076, August
2011.
2. The Phaseout of methyl bromide, http://www.epa.gov/ozone/mbr/index.html. Accessed
May8, 2013.
3. Metam sodium fact sheet. Journal of Pesticide Reform, 2006: Vol. 26, No. 1.
4. Wood, J.P., Calfee, M.W., Clayton, M., Griffin-Gatchalian, N., and Touati A. Optimizing
acidified bleach solutions to improve sporicidal efficacy on building materials. Letters in
Applied Microbiology, Volume 53, Issue 6, 668-672, December 2011.
5. Evaluation of liquid and foam technologies for the inactivation of Bacillus anthracis spores
ontopsoil. EPA 600/R-l0/080, September 2010.
6. VAPAM HL A soil fumigant solution for all crops. AMVAC Chemicals. 2005.
7. ASTM D 2974-87, Standard Test Methods for Moisture, Ash, and Organic Matter of Peat
and Other Organic Soils, ASTM, March 1993.
http://turf.lib.msu.edu/1990s/1993/930331.pdf Accessed May 8, 2013.
8. Wood, J.P., Choi, Y., and Rogers, J.V. Efficacy of liquid spray decontaminants for
inactivation of Bacillus anthracis spores on building and outdoor materials. J. Appl
Microbiol. 2011, 110, 1262-1273.
9. Rogers, J.V., Sabourin, C.L.K., Choi, Y.W., Richter, W.R., Rudnicki, D.C., Riggs, K.B.,
Taylor, M.L. and Chang, J. (2005), Decontamination assessment of Bacillus anthracis,
Bacillus subtilis, and Geobacillus stearothermophilus spores on indoor surfaces using a
hydrogen peroxide gas generator. Journal of Applied Microbiology, 99: 739-748.
10. Determining the Efficacy of Liquids and Fumigants in Systematic Decontamination Studies
for Bacillus anthracis Using Multiple Test Methods. US EPA Report 600/R-10/088,
December 2010.
11. FMC Environmental Solutions. Procedure for activation of Klozur™ persulfate with an 8%
hydrogen peroxide solution, Document 02-01-EIT-DH, FMC Corporation, Philadelphia, PA,
Copyright 2008 FMC Corporation.
12. Huling, S.G. and Pivetz, B.E. US EPA Engineering Issue, In-situ chemical oxidation.
EPA/600/R-06/072, August 2006. http://www.epa.gov/ada/gw/isco.html. Accessed May 8,
2013.
13. Buckman Laboratories, Inc. Material Safety Data Sheet for Metam Concentrate. 12/4/2009.
14. VAP AM HL Restricted Use Pesticide Label. AM VAC Chemicals. 2010.
45
-------
Appendix A
Moisture and Organic Content of Soil Samples
Initial Soil Analysis
Soil samples (pre- and post-sterilization via gamma irradiation at -40 kGy or autoclaving at 121
°C for one hour) were sent to an independent laboratory for analysis of moisture, organic content
and other characteristics. The samples were analyzed in triplicate using ASTM D 2974-87,
Moisture, Ash and Organic Matter of Peat and Other Organic Soils7. (Moisture and organic
contents are determined based on loss of sample mass at a given temperature.) The results are
shown in Table A-l. The moisture and organic content did not change significantly after the
gamma irradiation of the samples. However, autoclaving of the samples did have more of an
effect on the soil characteristics. Samples of each soil type were confirmed to be sterile following
gamma irradiation and autoclaving by dilution plating samples on tryptic soy agar.
Table A-l Soil Sample Analysis^
Soil Type
Topsoil
AZTD
Topsoil
AZTD
Pre-
Sterilization
Post-Gamma
Irradiation1
Post-
Autoclave*
Water Content (%)
33.6
0.233
32.5
0.238
25.4
0.582
pH
6.91
8.58
7.23
8.69
7.11
9.09
Pre- Post-Gamma
Sterilization Irradiation1
Fraction Organic Carbon
9.27 9.21
0.399 0.385
Post-
Autoclave*
(%, 440 °C)
7.29
0.264
Recalcitrant Organic Carbon (%, 750 °C)
1.35 1.39
1.20 1.22
2.26
1.15
* Data provided by CTL Engineering, Columbus, Ohio.
t Data post-gamma irradiation at ~40 kGy.
* Data post-autoclave at 121 °C for one hour.
Moisture Analysis Related to Metam Sodium Tests
Due to issues with incomplete sterilization of topsoil samples during the metam sodium tests
(indicated by the presence of endogenous bacteria), different methods (higher dose of gamma
radiation, or autoclaving) were tested to mitigate the presence of the non-target bacteria.
Because the sterilization method could affect moisture content, the moisture of the soils was
assessed for each sterilization and metam sodium application method used.
Soil moisture tests were completed following method ASTM D 2974-87. Samples were weighed,
dried in an oven for >16 hours at 105 ± 5 °C, weighed again and the moisture content calculated
as:
Moisture Content, % = [(A-B) x 100]/4
where:
A = mass of the as-received specimen, g
B = mass of the oven-dried specimen, g
The results of the moisture assessments for the metam sodium decontamination testing are
presented in Table A-2. As testing proceeded, we determined that soil moisture wasn't just a
function of sterilization method (e.g., autoclaved soils were generally lower in moisture
compared to irradiated soils) or the amount of water added to the soil, but that overnight drying
time seemed to affect soil moisture results as well. For example, the topsoil used for Test 4, in
which 2 mL water was added, had lower moisture content than the soil for Test 3, which only
46
-------
had 1 mL water added. This discrepancy is most likely due to the longer overnight dry time for
Test 4. Overnight dry times are therefore listed in Table A-2. Further, actual overnight dry
times prior to the application of metam sodium didn't always coincide with the overnight dry
times for conducting the soil moisture test. The soil moisture levels for the decontaminated soil
samples were estimated to be greater than or less than the soil samples that underwent moisture
tests depending on how the overnight dry times compared for the decontaminated soil samples
vs. the soil samples which were tested for moisture.
47
-------
Table A-2 Soil Moisture Assessments Taken During Metam Sodium Tests
Test#
1
2
3
4
5
6
7
8
Volume of Metam
Sodium Applied,
Contact Time
(Days)/ Aeration
Time (Days)
80 nL,
5/0
160 nL,
5/0
160 nL,
7/7
160 nL,
7/7
160 nL,
7/7
160 nL,
14/28
160 nL,
7/7
160 nL,
14/28
Soil Sterilization
Method
Gamma Irradiation
@ 40 kGy
Gamma Irradiation
@ 40 kGy
Gamma Irradiation
@ 60 kGy
Gamma Irradiation
@ 60 kGy
Gamma Irradiation
@ 60 kGy
Gamma Irradiation
@ 60 kGy
Autoclave
(121 °C; 1 hour)
Autoclave
(121 °C; 1 hour)
Soil Type
Topsoil
AZTD
Topsoil
AZTD
Topsoil
AZTD
Topsoil
AZTD
Topsoil
AZTD
Topsoil
AZTD
Topsoil
AZTD
Topsoil
AZTD
Amount SFW
Added Prior to
Addition of
Metam
Sodium (mL)*
0
0
1
2
3
1
1
1
Estimated Moisture
Content (%)* of Soil
Undergoing
Decontamination
>43.56
> 15.64
<43.56
< 15.64
< 46.38
< 18.04
<39.59
< 18.31
47.62
27.06
<46.38
< 18.04
38.39
18.25
<38.39
<18.25
Overnight Time
Prior to Decon
Test(hr:min)
18:35
21:30
22:26
26:21
19:58
22:54
20:43
24:04
Actual
Moisture of
Tested Soil
(%)•
43.56
15.64
43.56
15.64
46.38
18.04
39.59
18.31
47.62
27.06
46.38
18.04
38.39
18.25
38.39
18.25
Overnight
Time for
Moisture-
Tested Soil
(hr:min)
20:04
20:04
20:42
24:17
19:58
20:42
20:42
20:42
Estimated moisture based on the moisture assessment in comparison to the overnight dry times.
* Actual moisture measured during moisture assessment.
* All soil samples had 2 mL SFW added the night before decontamination tests and prior to sample inoculation with spores
48
-------
Appendix B
Neutralization Tests for Liquid Decontaminants
Neutralization Methodology
Neutralization for the pH-amended bleach and sodium persulfate was achieved with STS. The
concentration of STS tested in the neutralization panels was 1.0, 1.5, and 2.0 and 5.0% in the
extraction solution. These STS concentrations were selected based on historical data.
The following evaluations were made in each neutralization panel:
(1) Decontamination effectiveness (add spores to decontaminant solution; determine CPU
without neutralization). No spores should be recovered.
(2) Assess effectiveness of extraction buffer only (PBST without any STS) to neutralize or
dilute sufficiently active ingredient of decontaminant.
(3) Positive control recovery (add spores to extraction buffer without neutralizer, and without
decontaminant; determine CPU).
(4) Assess neutralizer effectiveness at terminating decontamination (add spores to
neutralized decontamination solution; determine CPU with neutralization).
Based on these results, a specific concentration of neutralizer in the extraction buffer was
selected to be used during the liquid decontamination tests.
To specifically assess the neutralizer effectiveness at terminating sporicidal activity (Item 4
above), each decontaminant was applied in the same manner as required for testing and allowed
to stay in contact with the soil for the appropriate contact time. At the end of the contact time, the
extraction buffer containing the tested level of STS was added and the soil was extracted on an
orbital shaker for 15 minutes at 200 rpm at room temperature. A 100 uL aliquot of spores was
then added to each sample, with each sample slowly mixed by hand ten times and dilution plated.
This level was compared to the positive control recovery.
The results of the neutralization panels are shown in Tables B-l through B-9. From these trials,
the following STS concentrations were determined to be sufficient for neutralization in both soil
types (except where indicated) in the following tests:
• 1.5% STS with pH-amended Ultra Clorox® for a seven-day contact time with eight
applications over two hours (Test 1).
• 1.5% STS with pH-amended Ultra Clorox® for a 24 hour contact time with eight
applications over two hours (Test 2).
• 2.0% STS with pH-amended Ultra Clorox for a 120 minute contact time with four
applications over two hours (Test 3).
• 2.0% STS with pH-amended Ultra Clorox for a 60 minute contact time with two
applications over one hour (Test 4).
• 1.0% STS with Klozur™ for a seven-day contact time with six applications over five
hours. (Test 1)
49
-------
• 2.0% STS with Klozur™ for a seven-day contact time with three applications over two
hours. (Test 2)
• 1.0% STS with Klozur™ for a seven-day contact time with three applications over four
days. (Test 3)
• 1.0% STS (AZTD) and 2.0% STS (topsoil) Klozur™ for a seven-day contact time with
one application at time 0. (Test 5)
• 1.0% STS Klozur™ for a seven-day contact time with two applications over one hour.
(Test 4)
Table B-l Neutralization Testing with Bacillus subtilis spores with pH-Amended
Bleach, Seven-Day Contact Time, Eight Total Applications
Treatment
Topsoil
pH -amended bleach + spores
pH-amended bleach + PBS + Triton® X-100 + spores
PBS + Triton® X-100 + spores (Control)
pH -amended bleach + PBS + Triton® X-100 + 1.0% STS + spores
pH-amended bleach + PBS + Triton® X-100 + 1.5% STS + spores
pH -amended bleach + PBS + Triton® X-100 + 2.0% STS + spores
AZTD
pH -amended bleach + spores
pH-amended bleach + PBS + Triton® X-100 + spores
PBS + Triton® X-100 + spores (Control)
pH -amended bleach + PBS + Triton® X-100 + 1.0% STS + spores
pH-amended bleach + PBS + Triton® X-100 + 1.5% STS + spores
pH-amended bleach + PBS + Triton® X-100 + 2.0% STS + spores
noculum
(CFU)
.08 x 10*
.08 x 108
.08 x 108
.08 x 108
.08 x 108
.08 x 108
.08 x 108
.08 x 108
.08 x 108
.08 x 108
.08 x 108
.08 x 108
Total Observed
(CFU)
0
6.87 xlO7
1.06 xlO8
9.96 xlO7
1.03 x 108
l.llxlO8
0
4.85 x 107
8.25 x 108
7.23 x 107
8.05 x 108
9.18 xlO7
%of
Control
0
65.0
-
94.2
97.7
105
0
58.7
-
87.6
97.6
111
Table B-2 Neutralization Testing with Bacillus subtilis Spores with pH-Amended Bleach,
24 Hour Contact Time, 8 Total Applications
Treatment
Inoculum
(CFU)
Total Observed
(CFU)
%of
Control
AZTD
pH -amended bleach +
pH -amended bleach +
PBS + Triton® X-100
pH -amended bleach +
pH -amended bleach +
pH -amended bleach +
spores
PBS + Triton® X-100 H
+ spores (Control)
PBS + Triton® X-100 H
PBS + Triton® X-100 H
PBS + Triton® X-100 H
i- Spores
h 1.0% STS
:r 1.5% STS
:r 2.0% STS
+ spores
+ spores
+ spores
.16
.16
.16
.16
.16
.16
x
x
X
X
X
X
108
108
108
108
108
108
9
8
8
8
0
0
15 x
25 x
73 x
23 x
107
107
107
107
0
0
90.2
95.5
90.0
50
-------
Table B-3 Neutralization Testing with Bacillus subtilis Spores with pH-Amended Bleach,
120 Minute Contact Time, Four Total Applications
Treatment
Inoculum
(CFU)
Total Observed
(CFU)
%of
Control
AZTD
pH -amended bleach + spores
pH -amended bleach + PBS + Triton8
X-100
+ spores
PBS + Triton® X-100 + spores (Control)
pH -amended bleach + PBS + Triton8
pH -amended bleach + PBS + Triton8
pH-amended bleach + PBS + Triton8
X-100
X-100
X-100
+ 1.0% STS
+ 1.5% STS
+ 2.0% STS
+ spores
+ spores
+ spores
.24
.24
.24
.24
.24
.24
X
X
X
X
X
X
10"
108
108
108
108
108
1
9
1
1
06
77
03
04
0
0
X
X
X
X
108
10'
108
108
0
0
-
92.0
97.5
98.2
Table B-4 Neutralization Testing with Bacillus subtilis Spores with pH-Amended
Bleach, 60 Minute Contact Time, Two Total Applications
Treatment
Inoculum
(CFU)
Total Observed
(CFU)
%of
Control
AZTD
pH-amended
pH-amended
PBS + Triton
pH-amended
pH-amended
pH-amended
bleach +
bleach +
81 X-100
bleach +
bleach +
bleach +
spores
PBS + Triton® X-100 -
+ spores (Control)
PBS + Triton® X-100 -
PBS + Triton® X-100 -
PBS + Triton® X-100 -
i- spores
I- 1.0% STS -
I- 1.5% STS -
h 2.0% STS -
i- spores
i- spores
i- spores
.07 x 10"
.07 x 108
.07 x 108
.07 x 108
.07 x 108
.07 x 108
1
5
6
6
7
0
44 x 105
38 x 107
39 x 107
88 x 107
94 x 107
0
0.268
119
128
148
Table B-5
TM
Neutralization Testing with Bacillus subtilis Spores with Klozurllvl, Seven-Day
Contact Time, Six Total Applications
Treatment
Inoculum
(CFU)
Total Observed
(CFU)
%of
Control
Topsoil
Klozur™ +
Klozur™ +
spores
PBS+ Triton®
'X- 100 + spores
PBS + Triton® X-100 + spores (Control)
Klozur™ +
Klozur™ +
Klozur™ +
PBS+ Triton8
PBS+ Triton8
PBS+ Triton8
'X-100 + 1.0% STS
'X-100 + 2.0% STS
'X-100 + 5.0% STS
+ spores
+ spores
+ spores
1.
1.
1.
1.
1.
1.
11
11
11
11
11
11
x
X
X
X
X
X
108
108
108
108
108
108
1.20
1.22
1.17
1.13
0
0
x
x
X
X
108
108
108
108
0
0
-
102
97.9
94.6
AZTD
Klozur™ +
Klozur™ +
spores
PBS+ Triton8
'X- 100 + spores
PBS + Triton® X-100 + spores (Control)
Klozur™ +
Klozur™ +
Klozur™ +
PBS + Triton® X-100 + 1.0% STS
PBS+ Triton®
PBS+ Triton8
'X-100 + 1.5% STS
'X-100 +2.0% STS
+ spores
+ spores
+ spores
1.
1.
1.
1.
1.
1.
11
11
11
11
11
11
X
X
X
X
X
X
108
108
108
108
108
108
1.34
1.43
1.39
1.06
0
0
X
X
X
X
108
108
108
108
0
0
-
107
104
79.3
51
-------
TM
Table B-6 Neutralization Testing with Bacillus subtilis Spores with Klozur11V1, Seven-Day
Contact Time, Three Total Applications
i reatment
Inoculum
(CFU)
Total Observed
(CFU)
%of
Control
Topsoil
Klozur™ +
Klozur™ +
spores
PBS+ Triton8
X-100 +
spores
PBS + Triton18 X-100 + spores (Control)
Klozur™ +
Klozur™ +
Klozur™ +
PBS+ Triton6
PBS+ Triton8
PBS+ Triton8
X-100 +
X-100 +
X-100 +
1.0% STS
2.0% STS
5.0% STS
+ spores
+ spores
+ spores
AZTD
Klozur™ +
Klozur™ +
spores
PBS+ Triton8
X-100 +
Spores
PBS + Triton® X-100 + spores (Control)
Klozur™ +
Klozur™ +
Klozur™ +
PBS+ Triton8
PBS+ Triton8
PBS+ Triton8
X-100 +
X-100 +
X-100 +
1.0% STS
1.5% STS
2.0% STS
+ spores
+ spores
+ spores
.24
.24
.24
.24
.24
.24
X
X
X
X
X
X
10"
108
108
108
108
108
1.15
1.05
1.06
9.88
0
0
X
X
X
X
108
108
108
107
0
0
-
90.9
91.9
85.7
.24
.24
.24
.24
.24
.24
X
X
X
X
X
X
108
108
108
108
108
108
1.06
1.12
1.13
1.01
0
0
X
X
X
X
108
108
108
108
0
0
-
106
106
95.5
Table B-7 Neutralization Testing with Bacillus subtilis Spores with Klozur
Contact Time, Three Total Applications (Days 0, 2 and 4)
TM
, Seven-Day
i reatment
Inoculum
(CFU)
Total Observed
(CFU)
%of
Control
Topsoil
Klozur™
Klozur™
+ spores
+ PBS+ Triton8
X-100
+ Spores
PBS + Triton18 X-100 + spores (Control)
Klozur™
Klozur™
Klozur™
+ PBS+Tritone
+ PBS+ Triton8
+ PBS+ Triton8
X-100
X-100
X-100
+ 1.0% STS
+ 2.0% STS
+ 5.0% STS
+ spores
+ spores
+ spores
AZTD
Klozur™
Klozur™
+ spores
+ PBS+ Triton8
X-100
+ spores
PBS + Triton81 X-100 + spores (Control)
Klozur™
Klozur™
Klozur™
+ PBS+ Triton8
+ PBS+ Triton8
+ PBS+ Triton8
X-100
X-100
X-100
+ 1.0% STS
+ 1.5% STS
+ 2.0% STS
+ spores
+ spores
+ spores
.16
.16
.16
.16
.16
.16
X
X
X
X
X
X
10"
108
108
108
108
108
1.01
1.04
1.02
9.84
0
0
X
X
X
X
108
108
108
107
0
0
-
103
101
97.2
.16
.16
.16
.16
.16
.16
X
X
X
X
X
X
10"
108
108
108
108
108
9.36
1.21
1.06
9.15
0
0
X
X
X
X
10'
108
108
10'
0
0
-
130
113
97.8
52
-------
Table B-8 Neutralization Testing with Bacillus subtilis Spores with Klozur
Contact Time, One Total Application
TM
, Seven-Day
rr Inoculum
Treatment /-rn?™
(C*U)
Total Observed
(CFU)
%of
Control
Topsoil
Klozur™ + spores 1.34 x 108
Klozur™ + PBS + Triton® X-100 + spores 1.34 x 108
PBS + Triton X-100 + spores (Control) 1.34 x 108
Klozur™+ PBS + Triton® X-100 + 1.0% STS + spores 1.34 x 108
Klozur™ + PBS + Triton® X-100 + 2.0% STS + spores 1.34 x 108
Klozur™ + PBS + Triton® X-100 + 5.0% STS + spores 1.34 x 108
0
0
1.25 x 108
LlSxlO8
1.21 xlO8
1.14xl08
0
0
-
94.4
96.8
90.7
AZTD
Klozur™ + spores 1.34 x 108
Klozur™ + PBS + Triton® X-100 + spores 1.34 x 108
PBS + Triton X-100 + spores (Control) 1.34 x 108
Klozur™ + PBS + Triton® X-100 + 1.0% STS + spores 1.34 x 108
Klozur™+ PBS + Triton® X-100 + 1.5% STS + spores 1.34 x 108
Klozur™ + PBS + Triton® X-100 + 2.0% STS + spores 1.34 x 108
Table B-9 Neutralization Testing with Bacillus subtilis spores with
Contact Time, 2 Total Applications
rr Inoculum
Treatment /-rn?™
(C*U)
0
0
LlOxlO8
1.05 x 108
9.00 x 107
7.81 x 107
Klozur™, 7 Day
Total Observed
(CFU)
0
0
-
96.2
82.2
71.3
%of
Control
Topsoil
Klozur™ + spores
Klozur™ + PBS + Triton® X-100 + spores
PBS + Triton® X-100 + spores (Control)
Klozur™ + PBS + Triton® X-100 + 1.0% STS + spores
Klozur™ + PBS + Triton® X-100 + 2.0% STS + spores
Klozur™ + PBS + Triton® X-100 + 5.0% STS + spores
AZTD
Klozur™ + spores
Klozur™ + PBS + Triton® X-100 + spores
PBS + Triton® X-100 + spores (Control)
Klozur™ + PBS + Triton® X-100 + 1.0% STS + spores
Klozur™ + PBS + Triton® X-100 + 1.5% STS + spores
Klozur™ + PBS + Triton® X-100 + 2.0% STS + spores
.17x10"
.17xl08
.17xl08
.17xl08
.17xl08
.17xl08
0
0
1.27 x 108
1.06 x 108
1.02 x 108
9.89 x 107
0
0
-
83.3
80.5
77.7
.17x10"
.17xl08
.17xl08
.17xl08
.17xl08
.17xl08
0
0
1.15xl08
1.15xl08
1.07 x 108
1.01 xlO8
0
0
-
99.9
93.1
87.6
53
-------
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
------- |