EPA 600/R-13/110 | August 2013 | www.epa.gov/ord

Technology Evaluation Report

Decontamination of Soil 

Contaminated with Bacillus 

anthracis Spores

Office of Research and Development

National Homeland Security Research Center
ii

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 
iii

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 
iv

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
v 

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. 
vi 

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-contaminated 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 μL.) 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. 
vii 

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 B. subtilis ............................................... 44 

9.0 References.... ....................................................................................................................45 

Appendix A Moisture and Organic Content of Soil Sampless.... ............................................46 

Appendix B Neutralization Tests for Liquid Decontaminants.... ............................................49 
viii

Figures

Figure 4-1 Schematic of MeBr Decontamination Test Chamber .............................................. 13

Figure 6-1 Summary of Decontamination Efficacies for pH-Amended Bleach Testing on 

Topsoil and AZTD ................................................................................................... 22

Figure 6-2 Summary of Decontamination Efficacies for KlozurTM 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 (KlozurTM) 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 KlozurTM...................... 23

Table 6-4 Inactivation of Bacillus anthracis Spores on AZTD with KlozurTM ....................... 24

Table 6-5 Inactivation of Bacillus subtilis Spores on Topsoil with KlozurTM ......................... 25

Table 6-6 Inactivation of Bacillus subtilis Spores on AZTD with KlozurTM ............................ 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-1 Soil Sample Analysis ............................................................................................... 43

Table A-2 Soil Moisture Assessments Taken During Metam Sodium Tests ............................ 45

Table B-1 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
ix

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 KlozurTM, Seven-Day 

Contact Time, Six Total Applications ..................................................................... 48

Table B-6 Neutralization Testing with Bacillus subtilis Spores with KlozurTM, Seven-Day 

Contact Time, Three Total Applications ................................................................. 49

Table B-7 Neutralization Testing with Bacillus subtilis Spores with KlozurTM, Seven-Day 

Contact Time, Three Total Applications (Days 0, 2 and 4) ..................................... 49

Table B-8 Neutralization Testing with Bacillus subtilis Spores with KlozurTM, Seven-Day 

Contact Time, One Total Application ...................................................................... 50

Table B-9 Neutralization Testing with Bacillus subtilis Spores with KlozurTM, Seven-Day 

Contact Time, Two Total Applications ................................................................... 50
x 

Abbreviations/Acronyms

ATCC American Type Culture Collection

AZTD Arizona Test Dust

B. anthracis Bacillus anthracis (Ames strain)

B. subtilis Bacillus subtilis (ATCC 19659)

BBRC Battelle Biomedical Research Center

BSC biological safety cabinet

°C degree(s) Celsius

CBR Chemical, biological, and radiological

CFU colony forming unit(s)

CI confidence interval

cm centimeter(s)

DNA deoxyribonucleic acid

EPA U.S. Environmental Protection Agency

ft feet

g gram

HCl Hydrogen chloride

H2O2 hydrogen peroxide

hr hour(s)

HS homeland security

HSRP Homeland Security Research Program

kGy Kilogray(s)

LAL Limulus Amebocyte Lysate

L liter(s)

LED Light emitting diode

LR log reduction

M Molarity

MeBr methyl bromide

mg milligram(s)

MITC methyl isothiocyanate

mL milliliter(s)

μL microliter(s)

NaOCl sodium hypochlorite

NaOH sodium hydroxide

Na2S2O8 sodium persulfate

NHSRC National Homeland Security Research 

Center 

ORD Office of Research and Development

oz ounce

PBS phosphate-buffered saline

PBST phosphate-buffered saline + 0.1% Triton® X100

PCR polymerase chain reaction

ppm part(s) per million

PVC polyvinyl chloride

QA quality assurance

QC quality control
xi

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)
1 

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 

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 Nmethyldithiocarbamate 

(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 

KlozurTM 

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 
3 

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 # Biological 

Agent Soil type

Application Frequency

(total number of 

applications)

Contact Time (hr)

1 B. anthracis 

B. subtilis

Topsoil

AZTD

0.5 mL every 15 minutes

for 2 hr (8) 168

2 B. anthracis 

B. subtilis AZTD 0.5 mL every 15 minutesfor 2 hr (8) 24

3 B. anthracis 

B. subtilis AZTD 0.5 mL every 30 minutesfor 2 hr (4) 2 

4 B. anthracis 

B. subtilis AZTD 0.5 mL every 30 minutesfor 1 hr (2) 1 

Table 2-3 Sodium Persulfate (KlozurTM) Test Matrix

Test # Biological 

Agent Soil Type

Application Frequency*

(total number of 

applications)

Contact Time 

(days) 

1 B. anthracis 

B. subtilis

Topsoil

AZTD Every 60 minutes (6) 7 

2 B. anthracis 

B. subtilis

Topsoil

AZTD Every 60 minutes (3) 7

3 B. anthracis 

B. subtilis

Topsoil

AZTD Days 0, 2 and 4 (3) 7

4 B. anthracis 

B. subtilis

Topsoil

AZTD Time 0 and 1 Hr (2) 7

5 B. anthracis 

B. subtilis

Topsoil

AZTD Day 0 (1) 7

* = Each application consisted of 1 mL KlozurTM 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. 
4 

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 # Biological 

Agent Soil type Target Concentration(mg/L) Contact Time (hr)

1 B. anthracis 

B. subtilis

Topsoil

AZTD 212 ± 21 36

2 B. anthracis 

B. subtilis †

Topsoil

AZTD 212 ± 21 36

3 B. anthracis 

B. subtilis ‡

Topsoil

AZTD 212 ± 21 36

4 B. anthracis 

B. subtilis

Topsoil

AZTD 100 ± 10 36

5 B. anthracis

B. subtilis†

Topsoil

AZTD 100 ± 10 36

6 B. anthracis

B. subtilis

Topsoil

AZTD 180 ± 18 36

7 B. anthracis 

B. subtilis

Topsoil

AZTD 140 ± 14 36

8 B. anthracis 

B. subtilis

Topsoil

AZTD 212 ± 21 24

† 2 mL SFW added prior to sample inoculation. ‡

 Samples dried prior to sample inoculation. 

Table 2-5 Metam Sodium Test Matrix

Test 

# Biological Agent† Soil type

Amount 

SFW Added

Prior to 

Addition of

Metam 

Sodium 

(mL)

Quantity of 

Metam 

Sodium 

Applied

(μL)

Soil Sterilization 

Method

Contact 

Time/ 

Aeration 

Time (days)

1 B. anthracis 

B. subtilis

Topsoil

AZTD -- 80 Gamma Irradiation at 40 kGy 5/0

2 B. anthracis 

B. subtilis

Topsoil

AZTD -- 160 Gamma Irradiation at 40 kGy 5/0

3 B. anthracis 

B. subtilis

Topsoil

AZTD 1 160 Gamma Irradiation at 60 kGy 7/7

4 B. anthracis 

B. subtilis

Topsoil

AZTD 2 160 Gamma Irradiation at 

60 kGy 7/7
5 

5 B. anthracis or

B. subtilis

Topsoil

AZTD 3 160 Gamma Irradiation at 60 kGy 7/7

6 B. anthracis or

B. subtilis

Topsoil

AZTD 1 160 Gamma Irradiation at 60 kGy 14/28

7 B. anthracis or

B. subtilis

Topsoil

AZTD 1 160 Autoclave

(121 °C; 1 hr) 7/7

8 B. anthracis or

B. subtilis

Topsoil

AZTD 1 160 Autoclave

(121 °C; 1 hr) 14/28
6 

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 CFU. 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 (HCl) 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 
7 

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* 

Lot, Batch, or 

ASTM No., or

Observation

Manufacturer/

Supplier Name

Pre-sterilized 

moisture content 

(%)

Pre-sterilized 

organic carbon 

content (%)

Topsoil

Earthgro® Topsoil,

Product #: 

71140180

The Scotts 

Company

Marysville, OH

34 9.3

Arizona Test Dust ISO 12103-1, A3 

Medium

Powder 

Technology, Inc. 

Burnsville, MN

0.23 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 (μL) aliquot of a stock suspension of 

approximately 1 x 109

 CFU/mL was dispensed using a micropipette applied as 10 μL 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 
8 

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 oC. 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. 
9 

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] × 100 (1)

where Mean CFUpc is the mean number of CFU recovered from five replicate positive control 

samples of a single material, and CFUspike is the number of CFU inoculated onto each of those 

samples. The value of CFUspike is known from enumeration of the stock spore suspension. 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 log10 reduction) to which viable spores extracted from test samples after 

decontamination were less numerous than the viable spores extracted from positive control 

samples. The logarithm of the CFU abundance from each sample 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 i

th

sample material was calculated as the difference between those mean log values, i.e.:

) (log - ) (log Efficacy = 10 CFUcij 10 CFUtij (2)

where log10 CFUcij refers to the j individual logarithm values obtained from the positive control 

samples, and log10 CFUtij refers to the j 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., j = 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 CFU abundance of 1 was assigned, resulting in a log10 CFU 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 CFU 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 log10 CFUcij and log10 CFUtij

values were also calculated for both the control and test samples (i.e., S2cij and S2tij), and were 

used to calculate the pooled standard error (SE) for the efficacy value calculated in Equation 2, 

as follows: 

55 

2

ij

2

ij tScS

SE +=

(3)
10 

where the number 5 again represents the number j 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 × 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). 
11 

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 (KlozurTM) 

KlozurTM 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). KlozurTM consists of >99% pure sodium persulfate 

(Na2S2O8) in the form of white odorless crystals. In remediation applications, KlozurTM 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 KlozurTM with H2O2 generates sulfate radicals (SO4•), 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 KlozurTM in SFW and diluting to 100 mL. This solution was 11.9% 

persulfate by weight. Commercially prepared 8% H2O2 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 KlozurTM/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 
12 

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 KlozurTM 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% H2O2 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 H2O2 solutions resulted in a H2O2/persulfate molar ratio of 5 to 1, a 

typical ratio recommended for the use of KlozurTM 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.
13 

The MeBr concentration in the test chamber was measured continuously during the contact 

period using a FumiscopeTM 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. 

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 
14 

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 μL/droplet). Decontamination tests were conducted using 

either 80 μL (Test 1) or 160 μL (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 
15 

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.
16 

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 CFU 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. 
17 

Table 5-1 Performance Evaluation Audits 

Measurement Audit

Procedure

Allowable

Tolerance

Actual

Tolerance

Volume of liquid from 

micropipettes Gravimetric evaluation ± 10% ± 0.57%

Time Compared to independent clock ± 2 sec/hr 0 sec/hr

Temperature Compared to independent calibrated 

thermometer ± 2 °C ± 0.36 °C

Relative Humidity Compare to independent calibrated 

hygrometer ± 10% ± 2% 

FumiscopeTM thermal 

conductivity meter

Instrument was certified as calibrated at 

the time of use ± 10% 0%

Balance Compared to independent calibrated 

weight sets ± 0.5g ± 0.03g

5.3.2 Technical Systems Audit 

Observations and findings from the technical systems audit (TSA) 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 planand 

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 TSA 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-amended 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. 
18 

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 followup 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. 
19 

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 KlozurTM 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/H2O2 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. 
20 

Table 6-1 Inactivation of Bacillus anthracis Spores on Soil using pH-Amended Ultra 

Clorox® Germicidal Bleacha

Contact Time

(Number of Applications) 

– Material

Test #

Inoculum (CFU) Mean of Logs of 

Observed CFU

Mean % 

Recovery

Decontamination 

Efficacy ± CIf

Seven Days (8) 

Topsoila - #1

Positive Controlsb 9.87 x 107 7.54 ± 0.04 35.60 ± 3.48 g

Test Samplesc 9.87 x 107 7.19 ± 0.06 15.74 ± 2.29 0.36 ± 0.07

Laboratory Blankd 0 0 - -

Procedural Blanke 0 0 - -

Seven Days (8) –

AZTD - #1

Positive Controls 9.87 x 107 7.88 ± 0.05 77.08 ± 9.72 -

Test Samples 9.87 x 107 0 0 ≥7.88 ± 0.05

Laboratory Blank 0 0 - -

Procedural Blank 0 0 - -

24 Hours (8) -

AZTD - #2

Positive Controls 9.43 x 107 7.87 ± 0.03 79.30 ± 5.07 -

Test Samples 9.43 x 107 0 0 ≥7.87 ± 0.02 

Laboratory Blank 0 0 - -

Procedural Blank 0 0 - - 

Two Hours (4) –

AZTD - #3

Positive Controls 1.05 x 108 7.91 ± 0.04 77.35 ± 6.99 -

Test Samples 1.05 x 108 0 0 ≥7.91 ± 0.03

Laboratory Blank 0 0 - -

Procedural Blank 0 0 - - 

One Hour (2) –

AZTD - #4

Positive Controls 1.04 x 108 7.82 ± 0.06 63.75 ± 9.00 -

Test Samples 1.04 x 108 2.66 ± 1.79 0.0066 ± 0.0088 5.16 ± 1.57

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 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 × SE). g

 “-” Not Applicable.
21 

Table 6-2 Inactivation of Bacillus subtilis Spores on Soil using pH-Amended Ultra 

Clorox® Germicidal Bleacha

Contact Time

(Number of Applications) 

– Material

Test #

Inoculum (CFU) Mean of Logs of 

Observed CFU

Mean % 

Recovery

Decontamination 

Efficacy ± CIf

Seven Days (8) –

Topsoila - #1

Positive Controlsb 1.25 x 108 7.41 ± 0.15 21.58 ± 6.96 g

Test Samplesc 1.25 x 108 7.31 ± 0.03 16.29 ± 1.02 0.10 ± 0.14

Laboratory Blankd 0 0 - -

Procedural Blanke 0 0 - -

Seven Days (8) –

AZTD - #1

Positive Controls 1.25 x 108 7.81 ± 0.16 54.77 ± 17.15 -

Test Samples 1.25 x 108 0 0 ≥7.81 ± 0.14

Laboratory Blank 0 0 - -

Procedural Blank 0 0 - - 

24 Hours (8) -

AZTD - #2

Positive Controls 1.19 x 108 7.93 ± 0.02 71.14 ± 3.40 - 

Test Samples 1.19 x 108 0 0 ≥7.93 ± 0.02

Laboratory Blank 0 0 - -

Procedural Blank 0 0 - - 

Two Hours (4) –

AZTD - #3

Positive Controls 1.14 x 108 7.95 ± 0.03 77.98 ± 5.33 - 

Test Samples 1.14 x 108 0.74 ± 1.66 0.00090 ± 0.0021 7.21 ± 1.46

Laboratory Blank 0 0 - -

Procedural Blank 0 0 - -

One Hour (2) –

AZTD - #4

Positive Controls 8.80 x 107 7.69 ± 0.05 56.40 ± 6.60 - 

Test Samples 8.80 x 107 4.42 ± 1.67 0.23 ± 0.36 3.28 ± 1.47

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 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 × 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. g

 “-” Not Applicable.
22 

Figure 6-1 Summary of Decontamination Efficacies (with 95% confidence 

intervals) for pH-Amended Bleach Testing on Topsoil and AZTD
23 

Table 6-3 Inactivation of Bacillus anthracis Spores on Topsoil with KlozurTM, a

Contact Time

(Number of Applications)

Test #

Inoculum (CFU) Mean of Logs of 

Observed CFU

Mean % 

Recovery

Decontamination 

Efficacy ± CIf

Seven Days (6)† - #1

Positive Controlsb 8.97 x 107 7.82 ± 0.04 74.35 ± 7.50 -

g

Test Samplesc 8.97 x 107 0 0 ≥7.82 ± 0.04

Laboratory Blankd 0 0 - -

Procedural Blanke 0 0 - -

Seven Days (3) † - #2

Positive Controls 8.97 x 107 7.72 ± 0.04 58.51 ± 5.65 -

Test Samples 8.97 x 107 0.65 ± 1.46 0.00041 ± 0.00090 7.07 ± 1.28

Laboratory Blank 0 0 - -

Procedural Blank 0 0 - -

Seven Days (3) ‡ - #3

Positive Controls 1.12 x 108 7.62 ± 0.09 37.98 ± 8.74 -

Test Samples 1.12 x 108 2.09 ± 1.40 0.00093 ± 0.0014 5.53 ± 1.23

Laboratory Blank 0 0 - -

Procedural Blank 0 0 - -

Seven Days (2) † - #4

Positive Controls 1.23 x 108 7.83 ± 0.08 55.45 ± 9.91 -

Test Samples 1.23 x 108 4.03 ± 1.13 0.00089 ± 0.0017 3.80 ± 1.00

Laboratory Blank 0 0 - -

Procedural Blank 0 0 - -

Seven Days (1) - #5

Positive Controls 8.83 x 107 7.68 ± 0.08 54.90 ± 10.81 -

Test Samples 8.83 x 107 6.75 ± 0.31 0.076 ± 0.042 0.93 ± 0.28

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 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 × SE). g

 “-” 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.
24 

Table 6-4 Inactivation of Bacillus anthracis Spores on AZTD with KlozurTM, a

Contact Time

(Number of Applications)

Test #

Inoculum 

(CFU)

Mean of Logs of 

Observed CFU Mean % Recovery Decontamination Efficacy ± CIf

Seven Days (6)† - #1

Positive Controlsb 8.97 x 107 7.87 ± 0.10 83.94 ± 19.52 -

g

Test Samplesc 8.97 x 107 0 0 ≥7.87 ± 0.09

Laboratory Blankd 0 0 - -

Procedural Blanke 0 0 - -

Seven Days (3) † - #2

Positive Controls 8.97 x 107 7.69 ± 0.04 55.21 ± 5.04 -

Test Samples 8.97 x 107 0.31 ± 0.70 0.0000091 ± 0.000018 7.38 ± 0.61

Laboratory Blank 0 0 - -

Procedural Blank 0 0 - -

Seven Days (3) ‡ - #3

Positive Controls 1.12 x 108 7.79 ± 0.10 56.46 ± 12.98 -

Test Samples 1.12 x 108 2.55 ± 0.59 0.00054 ± 0.00051 5.24 ± 0.53

Laboratory Blank 0 0 - -

Procedural Blank 0 0 - -

Seven Days (2) † - #4

Positive Controls 1.23 x 108 7.48 ± 0.31 30.32 ± 25.09 -

Test Samples 1.23 x 108 3.31 ± 1.33 0.025 ± 0.050 4.17 ± 1.19

Laboratory Blank 0 0 - -

Procedural Blank 0 0 - -

Seven Days (1) - #5

Positive Controls 8.83 x 107 7.95 ± 0.13 104.24 ± 32.81 -

Test Samples 8.83 x 107 3.58 ± 0.26 0.0049 ± 0.0026 4.37 ± 0.26

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 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 × SE). g

 “-” 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 6-5 Inactivation of Bacillus subtilis Spores on Topsoil with KlozurTM, a

Contact Time

(Number of Applications)

Test #

Inoculum 

(CFU)

Mean of Logs of 

Observed CFU Mean % Recovery Decontamination Efficacy ± CIf

Seven Days (6)† - #1

Positive Controlsb 1.12 x 108 7.86 ± 0.18 68.71 ± 25.25 -

g

Test Samplesc 1.12 x 108 0 0 ≥7.86 ± 0.16

Laboratory Blankd 0 0 - -

Procedural Blanke 0 0 - -

Seven Days (3) † - #2

Positive Controls 9.30 x 107 7.58 ± 0.05 41.57 ± 5.19 -

Test Samples 9.30 x 107 4.57 ± 2.61 0.71 ± 0.71 3.01 ± 2.28

Laboratory Blank 0 0 - -

Procedural Blank 0 0 - -

Seven Days (3) ‡ - #3

Positive Controls 1.31 x 108 7.72 ± 0.09 40.41 ± 8.02 -

Test Samples 1.31 x 108 4.71 ± 0.53 0.064 ± 0.057 3.01 ± 0.48

Laboratory Blank 0 0 - -

Procedural Blank 0 0 - -

Seven Days (2) † - #4

Positive Controls 1.18 x 108 7.68 ± 0.40 57.02 ± 50.50 -

Test Samples 1.18 x 108 7.20 ± 0.05 13.37 ± 1.59 0.49 ± 0.35

Laboratory Blank 0 0 - -

Procedural Blank 0 0 - -

Seven Days (1) - #5

Positive Controls 9.53 x 107 7.74 ± 0.08 58.91 ± 10.70 -

Test Samples 9.53 x 107 7.54 ± 0.09 37.00 ± 8.84 0.20 ± 0.11

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 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 × SE). g

 “-” 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 

Table 6-6 Inactivation of Bacillus subtilis Spores on AZTD with KlozurTMa

Contact Time

(Number of Applications)

Test #

Inoculum 

(CFU)

Mean of Logs of 

Observed CFU Mean % Recovery Decontamination Efficacy ± CIf

Seven Days (6)† - #1

Positive Controlsb 1.12 x 108 8.01 ± 0.11 94.99 ± 25.64 -

g

Test Samplesc 1.12 x 108 6.91 ± 0.13 7.53 ± 2.07 1.10 ± 0.15

Laboratory Blankd 0 0 - -

Procedural Blanke 0 0 - -

Seven Days (3) † - #2

Positive Controls 9.30 x 107 7.64 ± 0.09 47.89 ± 9.94 -

Test Samples 9.30 x 107 7.69 ± 0.05 52.71 ± 5.42 0.00 ± 0.00

Laboratory Blank 0 0 - -

Procedural Blank 0 0 - -

Seven Days (3) ‡ - #3

Positive Controls 1.31 x 108 8.00 ± 0.03 75.86 ± 5.57 -

Test Samples 1.31 x 108 7.37 ± 0.27 20.88 ± 12.67 0.62 ± 0.24

Laboratory Blank 0 0 - -

Procedural Blank 0 0 - -

Seven Days (2) † - #4

Positive Controls 1.18 x 108 7.89 ± 0.31 76.26 ± 33.36 -

Test Samples 1.18 x 108 7.84 ± 0.04 59.15 ± 5.40 0.05 ± 0.28

Laboratory Blank 0 0 - -

Procedural Blank 0 0 - -

Seven Days (1) - #5

Positive Controls 9.53 x 107 7.96 ± 0.04 95.55 ± 9.06 -

Test Samples 9.53 x 107 7.72 ± 0.12 56.69 ± 14.75 0.24 ± 0.11

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 on five individual samples, the mean percent 

recovery on those five samples, and decontamination efficacy (log reduction). b

 Positive Controls = sample 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 × SE). g

 “-” 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.
27 

† = 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 KlozurTM Liquid Testing on Topsoil and AZTD 
28 

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 #

Actual Mean MeBr 

Concentration ± SD

(mg/L)

Soil Moisture 

Condition

Actual mean 

temperature ± SD (° C)

Actual 

Mean RH ± 

SD

Contact 

Time (hr)

1 213 ± 0.74 No change 25.3 ± 0.41 79.3 ± 3.53 36

2 213 ± 0.73 2 mL SFW 

added 25.2 ± 0.16 82.8 ± 2.16 36

3 213 ± 0.68 Samples dried 25.4 ± 0.53 54.5 ± 2.26 36

4 102 ± 1.36 No change 25.5 ± 0.62 76.9 ± 3.96 36

5 101 ± 0.51 2 mL SFW 

added 25.3 ± 0.53 85.1 ± 1.91 36

6 181 ± 0.68 No change 25.2 ± 0.22 82.0 ± 2.38 36

7 141 ± 0.53 No change 25.2 ± 0.18 82.9 ± 2.08 36

8 213 ± 0.81 No change 25.2 ± 0.30 76.2 ± 4.01 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 
29 

more resistant to MeBr than B. anthracis. However, this result is consistent with a previous 

study.1
30 

Table 7-2 Inactivation of Bacillus anthracis Spores on Topsoil with MeBra

Contact Time

 (Actual Concentration 

[mean mg/L ± SD]) Test #

Inoculum 

(CFU)

Mean of Logs of 

Observed CFU Mean % Recovery Decontamination Efficacy ± CIf

36 Hours (213 ± 0.74) #1

Positive Controlsb 8.97 x 107 7.77 ± 0.037 66.38 ± 5.47 -

g

Test Samplesc 8.97 x 107 0.36 ± 0.82 0.000016 ± 0.000033 7.41 ± 0.72

Laboratory Blankd 0 0 - -

Procedural Blanke 0 0 - -

36 Hours (213 ± 0.73)* #2

Positive Controls 9.97 x 107 7.92 ± 0.065 83.61 ± 12.71 -

Test Samples 9.97 x 107 0.51 ± 1.15 0.000074 ± 0.00016 7.40 ± 1.01

Laboratory Blank 0 0 - -

Procedural Blank 0 0 - -

36 Hours (213 ± 0.68)

† #3

Positive Controls 7.57 x 107 7.65 ± 0.080 60.21 ± 11.30 -

Test Samples 7.57 x 107 1.78 ± 1.75 0.0028 ± 0.0058 5.87 ± 1.54

Laboratory Blank 0 0 - -

Procedural Blank 0 0 - -

36 Hours (102 ± 1.36) #4

Positive Controls 8.73 x 107 7.72 ± 0.080 61.49 ± 11.27 -

Test Samples 8.73 x 107 6.28 ± 0.037 2.18 ± 0.19 1.45 ± 0.08

Laboratory Blank 0 0 - -

Procedural Blank 0 0 - -

36 Hours (101 ± 0.51)* #5

Positive Controls 8.40 x 107 7.81 ± 0.11 79.43 ± 21.13 -

Test Samples 8.40 x 107 6.81 ± 0.075 7.84 ± 1.29 1.00 ± 0.12

Laboratory Blank 0 0 - -

Procedural Blank 0 0 - -

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 6.68 ± 0.79

Laboratory Blank 0 0 - -

Procedural Blank 0 0 - -

36 Hours (141 ± 0.53) #7

Positive Controls 1.43 x 108 8.05 ± 0.020 78.18 ± 3.58 -

Test Samples 1.43 x 108 4.59 ± 0.13 0.028 ± 0.0075 3.46 ± 0.11

Laboratory Blank 0 0 - -

Procedural Blank 0 0 - -

24 Hours (213 ± 0.81) #8

Positive Controls 1.25 x 108 7.79 ± 0.045 49.01 ± 4.82 -

Test Samples 1.25 x 108 4.54 ± 0.38 0.037 ± 0.033 3.25 ± 0.34

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 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 × SE). g

 “-” Not Applicable.

* 2 mL SFW added to samples prior to inoculation. † = Samples dried in oven prior to inoculation. 
31 

Table 7-3 Inactivation of Bacillus anthracis Spores on AZTD with MeBra

Contact Time

(Actual Concentration 

[mean mg/L ± SD]) Test #

Inoculum 

(CFU)

Mean of Logs of 

Observed CFU Mean % Recovery Decontamination Efficacy ± CIf

36 Hours (213 ± 0.74) #1

Positive Controlsb 8.97 x 107 7.74 ± 0.80 62.81 ± 12.48 -

g

Test Samplesc 8.97 x 107 0 0 ≥7.74 ± 0.07

Laboratory Blankd 0 0 - -

Procedural Blanke 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 ≥7.90 ± 0.05

Laboratory Blank 0 0 - -

Procedural Blank 0 0 - -

36 Hours (213 ± 0.68)† #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 7.46 ± 0.60

Laboratory Blank 0 0 - -

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 2.34 ± 0.39

Laboratory Blank 0 0 - -

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 1.40 ± 0.25

Laboratory Blank 0 0 - -

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 6.26 ± 1.29

Laboratory Blank 0 0 - -

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 6.19 ± 1.56

Laboratory Blank 0 0 - -

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 7.17 ± 0.86

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 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 × SE). g

 “-” Not Applicable.

* 2 mL SFW added to samples prior to inoculation. †

 Samples dried in oven prior to inoculation. 
32 

Table 7-4 Inactivation of Bacillus subtilis Spores on Topsoil with MeBra

Contact Time

(Actual Concentration 

[mean mg/L ± SD]) Test #

Inoculum 

(CFU)

Mean of Logs of 

Observed CFU Mean % Recovery Decontamination Efficacy ± CIf

36 Hours (213 ± 0.74) #1

Positive Controlsb 6.10 x 107 7.75 ± 0.047 92.59 ± 9.37 -

g

Test Samplesc 6.10 x 107 5.62 ± 0.045 0.69 ± 0.07 2.13 ± 0.06

Laboratory Blankd 0 0 - -

Procedural Blanke 0 0 - -

36 Hours (213 ± 0.73)* #2

Positive Controls 1.36 x 108 8.00 ± 0.051 73.59 ± 8.68 -

Test Samples 1.36 x 108 6.54 ± 0.063 2.55 ± 0.37 1.46 ± 0.07

Laboratory Blank 0 0 - -

Procedural Blank 0 0 - -

36 Hours (213 ± 0.68)† #3

Positive Controls 1.03 x 108 7.52 ± 0.024 32.37 ± 1.78 -

Test Samples 1.03 x 108 6.74 ± 0.061 5.35 ± 0.68 0.78 ± 0.06

Laboratory Blank 0 0 - -

Procedural Blank 0 0 - -

36 Hours (102 ± 1.36) #4

Positive Controls 9.23 x 107 7.77 ± 0.035 64.53 ± 5.15 -

Test Samples 9.23 x 107 7.45 ± 0.17 32.46 ± 9.93 0.32 ± 0.15

Laboratory Blank 0 0 - -

Procedural Blank 0 0 - -

36 Hours (101 ± 0.51)* #5

Positive Controls 8.50 x 107 7.77 ± 0.014 69.32 ± 2.15 -

Test Samples 8.50 x 107 7.79 ± 0.025 71.93 ± 4.17 0.00 ± 0.00

Laboratory Blank 0 0 - -

Procedural Blank 0 0 - -

36 Hours (181 ± 0.68) #6

Positive Controls 9.53 x 107 7.66 ± 0.083 48.69 ± 9.30 -

Test Samples 9.53 x 107 6.78 ± 0.061 6.30 ± 0.91 0.88 ± 0.09

Laboratory Blank 0 0 - -

Procedural Blank 0 0 - -

36 Hours (141 ± 0.53) #7

Positive Controls 1.42 x 108 8.11 ± 0.076 91.83 ± 15.87 -

Test Samples 1.42 x 108 6.42 ± 0.15 1.91 ± 0.61 1.69 ± 0.15

Laboratory Blank 0 0 - -

Procedural Blank 0 0 - -

24 Hours (213 ± 0.81) #8

Positive Controls 1.36 x 108 8.03 ± 0.0066 79.26 ± 1.21 -

Test Samples 1.36 x 108 7.36 ± 0.22 18.33 ± 6.76 0.67 ± 0.19

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 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 × SE). g

 “-” Not Applicable.

* 2 mL SFW added to samples prior to inoculation. †

 Samples dried in oven prior to inoculation. 
33 

Table 7-5 Inactivation of Bacillus subtilis Spores on AZTD with MeBra

Contact Time

(Actual Concentration 

[mean mg/L ± SD]) Test #

Inoculum

(CFU)

Mean of Logs of 

Observed CFU Mean % Recovery Decontamination Efficacy ± CIf

36 Hours (213 ± 0.74) #1

Positive Controlsb 6.10 x 107 7.77 ± 0.038 96.82 ± 8.23 -

g

Test Samplesc 6.10 x 107 6.30 ± 0.059 3.32 ± 0.45 1.47 ± 0.06

Laboratory Blankd 0 0 - -

Procedural Blanke 0 0 - -

36 Hours (213 ± 0.73)* #2

Positive Controls 1.36 x 108 8.03 ± 0.031 79.01 ± 5.67 -

Test Samples 1.36 x 108 7.49 ± 0.061 23.03 ± 3.47 0.54 ± 0.06

Laboratory Blank 0 0 - -

Procedural Blank 0 0 - -

36 Hours (213 ± 0.68)† #3

Positive Controls 1.03 x 108 7.58 ± 0.071 37.36 ± 5.94 -

Test Samples 1.03 x 108 7.07 ± 0.047 11.57 ± 1.24 0.51 ± 0.07

Laboratory Blank 0 0 - -

Procedural Blank 0 0 - -

36 Hours (102 ± 1.36) #4

Positive Controls 9.23 x 107 7.78 ± 0.063 65.35 ± 8.88 -

Test Samples 9.23 x 107 7.72 ± 0.067 56.86 ± 8.52 0.06 ± 0.08

Laboratory Blank 0 0 - -

Procedural Blank 0 0 - -

36 Hours (101 ± 0.51)* #5

Positive Controls 8.50 x 107 7.77 ± 0.012 68.61 ± 1.97 -

Test Samples 8.50 x 107 7.50 ± 0.22 40.59 ± 19.15 0.27 ± 0.19

Laboratory Blank 0 0 - -

Procedural Blank 0 0 - -

36 Hours (181 ± 0.68) #6

Positive Controls 9.53 x 107 7.77 ± 0.12 64.16 ± 14.69 -

Test Samples 9.53 x 107 7.64 ± 0.052 45.67 ± 5.37 0.14 ± 0.12

Laboratory Blank 0 0 - -

Procedural Blank 0 0 - -

36 Hours (141 ± 0.53) #7

Positive Controls 1.42 x 108 8.04 ± 0.041 76.86 ± 7.25 -

Test Samples 1.42 x 108 8.00 ± 0.026 71.27 ± 4.38 0.03 ± 0.04

Laboratory Blank 0 0 - -

Procedural Blank 0 0 - -

24 Hours (213 ± 0.81) #8

Positive Controls 1.36 x 108 7.92 ± 0.090 62.16 ± 13.14 -

Test Samples 1.36 x 108 7.97 ± 0.024 68.09 ± 3.77 0.00 ± 0.00

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 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 × SE). g

 “-” Not Applicable.

* 2 mL SFW added to samples prior to inoculation. †

 Samples dried in oven prior to inoculation. 
34 

* 2 mL SFW added to each sample prior to inoculation.

†

 samples dried in oven prior to inoculation.

Figure 7-1. Summary of Decontamination Efficacies for MeBr Fumigant Testing 

on Topsoil and AZTD 
35 

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%. 
36 

Table 7-6 Inactivation of Bacillus anthracis Spores on Topsoil with Metam Sodiuma

Contact Time

(Aeration Time)

Test # 

Inoculum 

(CFU)

Mean Logs 

Observed

(CFU ± SD)

Mean % Recovery 

(±SD)

Decontamination 

Efficacy ± CI f

Five Days (0 Days) - #1

Positive Controlsb 1.37 x 108 7.94 ± 0.064 64.82 ± 9.68 -

g

Test Samplesc 1.37 x 108 7.11 ± 0.23 10.69 ± 6.58 0.83 ± 0.21

Laboratory Blankd 0 0 - -

Procedural Blanke 0 0 - -

Five Days (0 Days) - #2

Positive Controls 1.11 x 108 8.00 ± 0.060 90.63 ± 12.64 -

Test Samples 1.11 x 108 3.41 ± 3.11 0.27 ± 0.26 4.59 ± 2.73

Laboratory Blank 0 0 - -

Procedural Blank 0 0 - -

Seven Days (7 Days) † - #3

Positive Controls 1.03 x 108 7.83 ± 0.52 66.08 ± 8.04 -

Test Samples 1.03 x 108 1.82 ± 2.50 0.014 ± 0.020 6.01 ± 2.19

Laboratory Blank 0 0 - -

Procedural Blank 0 0 - -

Seven Days (7 Days) ‡ - #4

Positive Controls 1.04 x 108 7.74 ± 0.062 52.75 ± 7.56 -

Test Samples 1.04 x 108 3.96 ± 0.48 0.014 ± 0.013 3.77 ± 0.42

Laboratory Blank 0 0 - -

Procedural Blank 0 0 - -

Seven Days (7 Days) § - #5

Positive Controls 9.97 x 107 7.89 ± 0.082 79.78 ± 14.46 -

Test Samples 9.97 x 107 1.37 ± 2.01 0.0054 ± 0.12 6.52 ± 1.76

Laboratory Blank 0 0 - -

Procedural Blank 0 0 - -

14 Days (28 Days) † - #6

Positive Controls 9.67 x 107 7.84 ± 0.046 71.09 ± 7.58 -

Test Samples 9.67 x 107 0 0 ≥7.84 ± 0.04

Laboratory Blank 0 0 - -

Procedural Blank 0 0 - -

Seven Days (7 Days) † - #7

Positive Controls 1.11 x 108 8.04 ± 0.0099 98.56 ± 2.26 -

Test Samples 1.11 x 108 6.15 ± 0.22 1.39 ± 0.61 1.89 ± 0.19

Laboratory Blank 0 0 - -

Procedural Blank 0 0 - -

14 Days (28 Days) †- #8

Positive Controls 1.00 x 108 7.94 ± 0.042 87.18 ± 8.42 -

Test Samples 1.00 x 108 4.96 ± 0.40 0.12 ± 0.071 2.98 ± 0.35

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 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 × SE). g

 “-” 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. 
37 

Table 7-7 Inactivation of Bacillus anthracis Spores on AZTD with Metam Sodiuma 

Contact Time

(Aeration Time)

Test # 

Inoculum 

(CFU)

Mean Logs 

Observed

(CFU ± SD)

Mean % Recovery 

(±SD)

Decontamination 

Efficacy ± CI f

Five Days (0 Days) - #1

Positive Controlsb 1.37 x 108 7.84 ± 0.054 51.18 ± 6.26 -

g

Test Samplesc 1.37 x 108 0.63 ± 1.40 0.00020 ± 0.00045 7.22 ± 1.23

Laboratory Blankd 0 0 - -

Procedural Blanke 0 0 - -

Five Days (0 Days) - #2

Positive Controls 1.11 x 108 7.77 ± 0.10 54.47 ± 14.03 -

Test Samples 1.11 x 108 0 0 ≥7.77 ± 0.09

Laboratory Blank 0 0 - -

Procedural Blank 0 0 - -

Seven Days (7 Days)

† - #3

Positive Controls 1.03 x 108 7.81 ± 0.040 63.36 ± 5.83 -

Test Samples 1.03 x 108 0 0 ≥7.81 ± 0.03

Laboratory Blank 0 0 - -

Procedural Blank 0 0 - -

Seven Days (7 Days) ‡- #4

Positive Controls 1.04 x 108 7.68 ± 0.061 46.65 ± 6.47 -

Test Samples 1.04 x 108 0 0 ≥7.68 ± 0.054

Laboratory Blank 0 0 - -

Procedural Blank 0 0 - -

Seven Days (7 Days) § - #5

Positive Controls 9.97 x 107 7.91 ± 0.053 82.57 ± 10.22 -

Test Samples 9.97 x 107 0 0 ≥7.91 ± 0.47

Laboratory Blank 0 0 - -

Procedural Blank 0 0 - -

14 Days (28 Days) †- #6

Positive Controls 9.67 x 107 7.79 ± 0.025 64.14 ± 3.55 -

Test Samples 9.67 x 107 0 0 ≥7.79 ± 0.02

Laboratory Blank 0 0 - -

Procedural Blank 0 0 - -

Seven Days (7 Days) † - #7

Positive Controls 1.11 x 108 7.81 ± 0.045 58.49 ± 5.96 -

Test Samples 1.11 x 108 0 0 ≥7.81 ± 0.04

Laboratory Blank 0 0 - -

Procedural Blank 0 0 - -

14 Days (28 Days) † - #8

Positive Controls 1.00 x 108 7.74 ± 0.062 55.86 ± 7.92 -

Test Samples 1.00 x 108 0 0 ≥7.74 ± 0.05

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 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 × SE). g

 “-” 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. 
38 

Table 7-8 Inactivation of Bacillus subtilis Spores on Topsoil with Metam Sodiuma

Contact Time

(Aeration Time)

Test # 

Inoculum 

(CFU)

Mean Logs 

Observed

(CFU ± SD)

Mean % Recovery 

(±SD)

Decontamination 

Efficacy ± CI f

Five Days (0 Days) - #1

Positive Controlsb 1.07 x 108 7.99 ± 0.010 91.53 ± 2.05 -

g

Test Samplesc 1.07 x 108 7.77 ± 0.094 56.45 ± 11.96 0.22 ± 0.08

Laboratory Blankd 0 0 - -

Procedural Blanke 0 0 - -

Five Days (0 Days) - #2

Positive Controls 1.35 x 108 8.06 ± 0.010 84.15 ± 2.00 -

Test Samples 1.35 x 108 6.97 ± 0.19 7.53 ± 3.51 1.08 ± 0.17

Laboratory Blank 0 0 - -

Procedural Blank 0 0 - -

Seven Days (7 Days)

† - #3

Positive Controls 1.04 x 108 7.99 ± 0.055 95.52 ± 11.83 -

Test Samples 1.04 x 108 6.86 ± 0.21 7.76 ± 3.74 1.13 ± 0.19

Laboratory Blank 0 0 - -

Procedural Blank 0 0 - -

Seven Days (7 Days) ‡- #4

Positive Controls 9.73 x 107 7.72 ± 0.048 54.12 ± 6.10 -

Test Samples 9.73 x 107 7.06 ± 0.19 12.72 ± 5.32 0.66 ± 0.18

Laboratory Blank 0 0 - -

Procedural Blank 0 0 - -

Seven Days (7 Days) § - #5

Positive Controls 9.07 x 107 7.81 ± 0.058 71.51 ± 9.24 -

Test Samples 9.07 x 107 7.18 ± 0.11 16.89 ± 3.61 0.63 ± 0.11

Laboratory Blank 0 0 - -

Procedural Blank 0 0 - -

14 Days (28 Days) †- #6

Positive Controls 8.97 x 107 7.85 ± 0.072 80.13 ± 14.57 -

Test Samples 8.97 x 107 6.75 ± 0.51 8.94 ± 5.63 1.10 ± 0.45

Laboratory Blank 0 0 - -

Procedural Blank 0 0 - -

Seven Days (7 Days) † - #7

Positive Controls 1.13 x 108 7.87 ± 0.035 65.86 ± 5.21 -

Test Samples 1.13 x 108 7.19 ± 0.10 14.09 ± 2.90 0.68 ± 0.09

Laboratory Blank 0 0 - -

Procedural Blank 0 0 - -

14 Days (28 Days) † - #8

Positive Controls 1.06 x 108 7.82 ± 0.070 63.15 ± 10.07 -

Test Samples 1.06 x 108 6.94 ± 0.27 9.23 ± 4.52 0.89 ± 0.24

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 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 × SE). g

 “-” 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. 
39 

Table 7-9 Inactivation of Bacillus subtilis Spores on AZTD with Metam Sodiuma

Contact Time

(Aeration Time)

Test # 

Inoculum 

(CFU)

Mean Logs 

Observed

(CFU ± SD)

Mean % Recovery 

(±SD)

Decontamination 

Efficacy ± CI f

Five Days (0 Days) - #1

Positive Controlsb 1.07 x 108 7.83 ± 0.46 83.14 ± 42.82 -

g

Test Samplesc 1.07 x 108 7.06 ± 0.37 13.99 ± 11.29 0.77 ± 0.52

Laboratory Blankd 0 0 - -

Procedural Blanke 0 0 - -

Five Days (0 Days) - #2

Positive Controls 1.35 x 108 7.87 ± 0.049 55.72 ± 6.17 -

Test Samples 1.35 x 108 4.15 ± 2.59 0.56 ± 0.76 3.72 ± 2.28

Laboratory Blank 0 0 - -

Procedural Blank 0 0 - -

Seven Days (7 Days)

† - #3

Positive Controls 1.04 x 108 8.02 ± 0.030 101.87 ± 7.14 -

Test Samples 1.04 x 108 1.86 ± 2.56 0.019 ± 0.027 6.16 ± 2.24

Laboratory Blank 0 0 - -

Procedural Blank 0 0 - -

Seven Days (7 Days) ‡- #4

Positive Controls 9.73 x 107 7.76 ± 0.033 58.64 ± 4.33 -

Test Samples 9.73 x 107 0 0 ≥7.76 ± 0.029

Laboratory Blank 0 0 - -

Procedural Blank 0 0 - -

Seven Days (7 Days) § - #5

Positive Controls 9.07 x 107 7.85 ± 0.045 78.88 ± 8.00 -

Test Samples 9.07 x 107 0 0 ≥7.85 ± 0.039

Laboratory Blank 0 0 - -

Procedural Blank 0 0 - -

14 Days (28 Days) †- #6

Positive Controls 8.97 x 107 7.80 ± 0.044 70.14 ± 7.45 -

Test Samples 8.97 x 107 6.06 ± 0.13 1.32 ± 0.41 1.74 ± 0.12

Laboratory Blank 0 0 - -

Procedural Blank 0 0 - -

Seven Days (7 Days) † - #7

Positive Controls 1.13 x 108 7.78 ± 0.048 53.27 ± 5.79 -

Test Samples 1.13 x 108 2.97 ± 2.82 0.27 ± 0.59 4.80 ± 2.47

Laboratory Blank 0 0 - -

Procedural Blank 0 0 - -

14 Days (28 Days) † - #8

Positive Controls 1.06 x 108 7.75 ± 0.054 53.34 ± 6.61 -

Test Samples 1.06 x 108 0 0 ≥7.75 ± 0.05

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 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 × SE). g “-” 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. 
40 

* 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.

Test results are presented in numerical order

Figure 7-2. Summary of Decontamination Efficacies for Metam Sodium Fumigant Testing

on Topsoil and AZTD
41 

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. 
42 

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).
43 

Table 8-1 Minimum Treatment Required for Effective Decontamination 

Decontaminant Soil 

type

Microor

-ganism Minimum Treatment for ≥ 6LR

pH-amended 

bleach TS B.a. Not found 

pH-amended 

bleach TS B.s. Not found 

pH-amended 

bleach AZTD B.a. 2 hour contact time, 4 applications 

pH-amended 

bleach AZTD B.s. 2 hour contact time, 4 applications 

Sodium 

persulfate TS B.a. 3 applications every 60 minutes 

Sodium 

persulfate TS B.s. 6 applications every 60 minutes 

Sodium 

persulfate AZTD B.a. 3 applications every 60 minutes 

Sodium 

persulfate AZTD B.s. Not found 

Methyl bromide TS B.a. 180 mg/L MeBr, 24 hour contact time 

Methyl bromide TS B.s. Not found

Methyl bromide AZTD B.a. 140 mg/L MeBr, 24 hour contact time

Methyl bromide AZTD B.s. Not found

Metam sodium TS B.a. 160 μL, 7 day contact time, 7 day aeration time, 1 mL water 

added to soil

Metam sodium TS B.s. Not found

Metam sodium AZTD B.a. 80 μL, 5 day contact time, no aeration period, no moisture added 

to soil

Metam sodium AZTD B.s. 160 μL, 7 day contact time, 7 day aeration time, 1 mL water 

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 a 7-day 

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.

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 pHamended 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. 
44 

8.3 Comparing efficacy 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 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. 
45 

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-11/076, August 

2011. 

2. The Phaseout of methyl bromide. http://www.epa.gov/ozone/mbr/index.html. Accessed 

May 8, 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 

on topsoil. EPA 600/R-10/080, 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 KlozurTM 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. VAPAM HL Restricted Use Pesticide Label. AMVAC Chemicals. 2010. 
46 

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-1. 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-1 Soil Sample Analysis‡

Soil Type

PreSterilizationPost-Gamma 

Irradiation†

PostAutoclave*PreSterilizationPost-Gamma 

Irradiation†

PostAutoclave*

Water Content (%) Fraction Organic Carbon (%, 440 °C)

Topsoil 33.6 32.5 25.4 9.27 9.21 7.29

AZTD 0.233 0.238 0.582 0.399 0.385 0.264

pH Recalcitrant Organic Carbon (%, 750 °C)

Topsoil 6.91 7.23 7.11 1.35 1.39 2.26

AZTD 8.58 8.69 9.09 1.20 1.22 1.15

‡ Data provided by CTL Engineering, Columbus, Ohio. † 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]/A

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 
47 

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. 
48 

Table A-2 Soil Moisture Assessments Taken During Metam Sodium Tests

Test #

Volume of Metam 

Sodium Applied, 

Contact Time

(Days)/ Aeration 

Time (Days)

Soil Sterilization 

Method Soil Type 

Amount SFW 

Added Prior to 

Addition of 

Metam 

Sodium (mL)‡

Estimated Moisture 

Content (%)† of Soil 

Undergoing 

Decontamination

Overnight Time 

Prior to Decon 

Test (hr:min) 

Actual 

Moisture of 

Tested Soil 

(%)* 

Overnight 

Time for 

MoistureTested Soil 

(hr:min)

1 80 μL,

5/0

Gamma Irradiation 

@ 40 kGy

Topsoil

0 

≥ 43.56

18:35

43.56

20:04

AZTD ≥ 15.64 15.64

2 160 μL,

5/0

Gamma Irradiation 

@ 40 kGy

Topsoil

0 

≤ 43.56

21:30

43.56

20:04

AZTD ≤ 15.64 15.64

3 160 μL,

7/7

Gamma Irradiation 

@ 60 kGy

Topsoil

1 

≤ 46.38

22:26

46.38

20:42

AZTD ≤ 18.04 18.04

4 160 μL,

7/7

Gamma Irradiation 

@ 60 kGy

Topsoil

2 

≤ 39.59

26:21

39.59

24:17

AZTD ≤ 18.31 18.31

5 160 μL,

7/7

Gamma Irradiation 

@ 60 kGy

Topsoil

3 

47.62

19:58

47.62

19:58

AZTD 27.06 27.06

6 160 μL,

14/28

Gamma Irradiation 

@ 60 kGy

Topsoil

1 

≤ 46.38

22:54

46.38

20:42

AZTD ≤ 18.04 18.04

7 160 μL,

7/7

Autoclave

(121 °C; 1 hour)

Topsoil 1 38.39

20:43

38.39

20:42

AZTD 18.25 18.25

8 160 μL, 

14/28

Autoclave 

(121 °C; 1 hour)

Topsoil 1 ≤38.39

24:04

38.39 20:42

AZTD ≤18.25 18.25

†

 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
49 

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 CFU 

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 CFU). 

(4) Assess neutralizer effectiveness at terminating decontamination (add spores to 

neutralized decontamination solution; determine CFU 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 μL 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-1 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 KlozurTM for a seven-day contact time with six applications over five 

hours. (Test 1)
50 

• 2.0% STS with KlozurTM for a seven-day contact time with three applications over two 

hours. (Test 2)

• 1.0% STS with KlozurTM for a seven-day contact time with three applications over four 

days. (Test 3)

• 1.0% STS (AZTD) and 2.0% STS (topsoil) KlozurTM for a seven-day contact time with 

one application at time 0. (Test 5)

• 1.0% STS KlozurTM for a seven-day contact time with two applications over one hour. 

(Test 4)

Table B-1 Neutralization Testing with Bacillus subtilis spores with pH-Amended 

Bleach, Seven-Day Contact Time, Eight Total Applications 

Treatment Inoculum

(CFU)

Total Observed 

(CFU)

% of 

Control

Topsoil

pH-amended bleach + spores 1.08 x 108 0 0

pH-amended bleach + PBS + Triton® X-100 + spores 1.08 x 108 6.87 x 107 65.0

PBS + Triton® X-100 + spores (Control) 1.08 x 108 1.06 x 108 -

pH-amended bleach + PBS + Triton® X-100 + 1.0% STS + spores 1.08 x 108 9.96 x 107 94.2

pH-amended bleach + PBS + Triton® X-100 + 1.5% STS + spores 1.08 x 108 1.03 x 108 97.7

pH-amended bleach + PBS + Triton® X-100 + 2.0% STS + spores 1.08 x 108 1.11 x 108 105

AZTD

pH-amended bleach + spores 1.08 x 108 0 0

pH-amended bleach + PBS + Triton® X-100 + spores 1.08 x 108 4.85 x 107 58.7

PBS + Triton® X-100 + spores (Control) 1.08 x 108 8.25 x 108 -

pH-amended bleach + PBS + Triton® X-100 + 1.0% STS + spores 1.08 x 108 7.23 x 107 87.6

pH-amended bleach + PBS + Triton® X-100 + 1.5% STS + spores 1.08 x 108 8.05 x 108 97.6

pH-amended bleach + PBS + Triton® X-100 + 2.0% STS + spores 1.08 x 108 9.18 x 107 111

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 + spores 1.16 x 108 0 0

pH-amended bleach + PBS + Triton® X-100 + Spores 1.16 x 108 0 0

PBS + Triton® X-100 + spores (Control) 1.16 x 108 9.15 x 107 -

pH-amended bleach + PBS + Triton® X-100 + 1.0% STS + spores 1.16 x 108 8.25 x 107 90.2

pH-amended bleach + PBS + Triton® X-100 + 1.5% STS + spores 1.16 x 108 8.73 x 107 95.5

pH-amended bleach + PBS + Triton® X-100 + 2.0% STS + spores 1.16 x 108 8.23 x 107 90.0
51 

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 1.24 x 108 0 0

pH-amended bleach + PBS + Triton® X-100 + spores 1.24 x 108 0 0

PBS + Triton® X-100 + spores (Control) 1.24 x 108 1.06 x 108 -

pH-amended bleach + PBS + Triton® X-100 + 1.0% STS + spores 1.24 x 108 9.77 x 107 92.0

pH-amended bleach + PBS + Triton® X-100 + 1.5% STS + spores 1.24 x 108 1.03 x 108 97.5

pH-amended bleach + PBS + Triton® X-100 + 2.0% STS + spores 1.24 x 108 1.04 x 108 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 bleach + spores 1.07 x 108 0 0

pH-amended bleach + PBS + Triton® X-100 + spores 1.07 x 108 1.44 x 105 0.268

PBS + Triton® X-100 + spores (Control) 1.07 x 108 5.38 x 107 -

pH-amended bleach + PBS + Triton® X-100 + 1.0% STS + spores 1.07 x 108 6.39 x 107 119

pH-amended bleach + PBS + Triton® X-100 + 1.5% STS + spores 1.07 x 108 6.88 x 107 128

pH-amended bleach + PBS + Triton® X-100 + 2.0% STS + spores 1.07 x 108 7.94 x 107 148

Table B-5 Neutralization Testing with Bacillus subtilis Spores with KlozurTM, Seven-Day

Contact Time, Six Total Applications 

Treatment Inoculum

(CFU)

Total Observed 

(CFU)

% of 

Control

Topsoil

KlozurTM + spores 1.11 x 108 0 0

KlozurTM + PBS + Triton® X-100 + spores 1.11 x 108 0 0

PBS + Triton® X-100 + spores (Control) 1.11 x 108 1.20 x 108 -

KlozurTM + PBS + Triton® X-100 + 1.0% STS + spores 1.11 x 108 1.22 x 108 102

KlozurTM + PBS + Triton® X-100 + 2.0% STS + spores 1.11 x 108 1.17 x 108 97.9

KlozurTM + PBS + Triton® X-100 + 5.0% STS + spores 1.11 x 108 1.13 x 108 94.6

AZTD

KlozurTM + spores 1.11 x 108 0 0

KlozurTM + PBS + Triton® X-100 + spores 1.11 x 108 0 0

PBS + Triton® X-100 + spores (Control) 1.11 x 108 1.34 x 108 -

KlozurTM + PBS + Triton® X-100 + 1.0% STS + spores 1.11 x 108 1.43 x 108 107

KlozurTM + PBS + Triton® X-100 + 1.5% STS + spores 1.11 x 108 1.39 x 108 104

KlozurTM + PBS + Triton® X-100 + 2.0% STS + spores 1.11 x 108 1.06 x 108 79.3
52 

Table B-6 Neutralization Testing with Bacillus subtilis Spores with KlozurTM, Seven-Day

Contact Time, Three Total Applications 

Treatment Inoculum

(CFU)

Total Observed 

(CFU)

% of 

Control

Topsoil

KlozurTM + spores 1.24 x 108 0 0

KlozurTM + PBS + Triton® X-100 + spores 1.24 x 108 0 0

PBS + Triton® X-100 + spores (Control) 1.24 x 108 1.15 x 108 -

KlozurTM + PBS + Triton® X-100 + 1.0% STS + spores 1.24 x 108 1.05 x 108 90.9

KlozurTM + PBS + Triton® X-100 + 2.0% STS + spores 1.24 x 108 1.06 x 108 91.9

KlozurTM + PBS + Triton® X-100 + 5.0% STS + spores 1.24 x 108 9.88 x 107 85.7

AZTD

KlozurTM + spores 1.24 x 108 0 0

KlozurTM + PBS + Triton® X-100 + Spores 1.24 x 108 0 0

PBS + Triton® X-100 + spores (Control) 1.24 x 108 1.06 x 108 -

KlozurTM + PBS + Triton® X-100 + 1.0% STS + spores 1.24 x 108 1.12 x 108 106

KlozurTM + PBS + Triton® X-100 + 1.5% STS + spores 1.24 x 108 1.13 x 108 106

KlozurTM + PBS + Triton® X-100 + 2.0% STS + spores 1.24 x 108 1.01 x 108 95.5

Table B-7 Neutralization Testing with Bacillus subtilis Spores with KlozurTM, Seven-Day

Contact Time, Three Total Applications (Days 0, 2 and 4) 

Treatment Inoculum

(CFU)

Total Observed 

(CFU)

% of 

Control

Topsoil

KlozurTM + spores 1.16 x 108 0 0

KlozurTM + PBS + Triton® X-100 + Spores 1.16 x 108 0 0

PBS + Triton® X-100 + spores (Control) 1.16 x 108 1.01 x 108 -

KlozurTM + PBS + Triton® X-100 + 1.0% STS + spores 1.16 x 108 1.04 x 108 103

KlozurTM + PBS + Triton® X-100 + 2.0% STS + spores 1.16 x 108 1.02 x 108 101

KlozurTM + PBS + Triton® X-100 + 5.0% STS + spores 1.16 x 108 9.84 x 107 97.2

AZTD

KlozurTM + spores 1.16 x 108 0 0

KlozurTM + PBS + Triton® X-100 + spores 1.16 x 108 0 0

PBS + Triton® X-100 + spores (Control) 1.16 x 108 9.36 x 107 -

KlozurTM + PBS + Triton® X-100 + 1.0% STS + spores 1.16 x 108 1.21 x 108 130

KlozurTM + PBS + Triton® X-100 + 1.5% STS + spores 1.16 x 108 1.06 x 108 113

KlozurTM + PBS + Triton® X-100 + 2.0% STS + spores 1.16 x 108 9.15 x 107 97.8
53 

Table B-8 Neutralization Testing with Bacillus subtilis Spores with KlozurTM, Seven-Day

Contact Time, One Total Application 

Treatment Inoculum

(CFU)

Total Observed 

(CFU)

% of 

Control

Topsoil

KlozurTM + spores 1.34 x 108 0 0

KlozurTM + PBS + Triton® X-100 + spores 1.34 x 108 0 0

PBS + Triton X-100 + spores (Control) 1.34 x 108 1.25 x 108 -

KlozurTM+ PBS + Triton® X-100 + 1.0% STS + spores 1.34 x 108 1.18 x 108 94.4

KlozurTM + PBS + Triton® X-100 + 2.0% STS + spores 1.34 x 108 1.21 x 108 96.8

KlozurTM + PBS + Triton® X-100 + 5.0% STS + spores 1.34 x 108 1.14 x 108 90.7

AZTD

KlozurTM + spores 1.34 x 108 0 0

KlozurTM + PBS + Triton® X-100 + spores 1.34 x 108 0 0

PBS + Triton X-100 + spores (Control) 1.34 x 108 1.10 x 108 -

KlozurTM + PBS + Triton® X-100 + 1.0% STS + spores 1.34 x 108 1.05 x 108 96.2

KlozurTM+ PBS + Triton® X-100 + 1.5% STS + spores 1.34 x 108 9.00 x 107 82.2

KlozurTM + PBS + Triton® X-100 + 2.0% STS + spores 1.34 x 108 7.81 x 107 71.3

Table B-9 Neutralization Testing with Bacillus subtilis spores with KlozurTM, 7 Day 

Contact Time, 2 Total Applications

Treatment Inoculum

(CFU)

Total Observed 

(CFU)

% of 

Control

Topsoil

KlozurTM + spores 1.17 x 108 0 0

KlozurTM + PBS + Triton® X-100 + spores 1.17 x 108 0 0

PBS + Triton® X-100 + spores (Control) 1.17 x 108 1.27 x 108 -

KlozurTM + PBS + Triton® X-100 + 1.0% STS + spores 1.17 x 108 1.06 x 108 83.3

KlozurTM + PBS + Triton® X-100 + 2.0% STS + spores 1.17 x 108 1.02 x 108 80.5

KlozurTM + PBS + Triton® X-100 + 5.0% STS + spores 1.17 x 108 9.89 x 107 77.7

AZTD

KlozurTM + spores 1.17 x 108 0 0

KlozurTM + PBS + Triton® X-100 + spores 1.17 x 108 0 0

PBS + Triton® X-100 + spores (Control) 1.17 x 108 1.15 x 108 -

KlozurTM + PBS + Triton® X-100 + 1.0% STS + spores 1.17 x 108 1.15 x 108 99.9

KlozurTM + PBS + Triton® X-100 + 1.5% STS + spores 1.17 x 108 1.07 x 108 93.1

KlozurTM + PBS + Triton® X-100 + 2.0% STS + spores 1.17 x 108 1.01 x 108 87.6
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