Decontamination of Indoor and Outdoor Materials with Aqueous Chlorine Dioxide Solutions

Home , Chlorine dioxide, Potassium oxide

EPA 600/R-12/516 | May 2012 | www.epa.gov/ord

Office of Research and Development National Homeland Security Research Center

EPA/600/R/12/516 May 2012

Decontamination of Indoor and Outdoor Materials with Aqueous Chlorine Dioxide Solutions

U.S. Environmental Protection Agency

Research Triangle Park, NC 27711

Disclaimer

The U.S. Environmental Protection Agency (EPA), through its Office of Research and Development’s (ORD) National Homeland Security Research Center (NHSRC), funded, directed and managed this work through Contract Number EP-C-10-001 with Battelle. This report has been peer and administratively reviewed and has been approved for publication as an EPA document. 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

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Foreword

Following the events of September 11, 2001, addressing the critical needs related to homeland security became a clear requirement with respect to EPA’s mission to protect human health and the environment. Presidential Directives further emphasized EPA as the primary federal agency responsible for the country’s water supplies and for decontamination following a chemical, biological, and/or radiological (CBR) attack. To support EPA’s mission to assist in and lead response and recovery activities associated with CBR incidents of national significance, the National Homeland Security Research Center (NHSRC) was established to conduct research and deliver products that improve the capability of the Agency and other federal, state, and local agencies to carry out their homeland security responsibilities.

One goal of NHSRC’s research is to provide information on decontamination methods and technologies that can be used in the response and recovery efforts resulting from a CBR release over a wide area. The complexity and heterogeneity of the wide-area decontamination challenge necessitates the understanding of the effectiveness of a range of decontamination options. In addition to effective fumigation approaches, rapidly deployable or readily available surface decontamination approaches have also been recognized as a tool to enhance the capability to respond to and recover from such an intentional CBR dispersion.

Through working with ORD’s program office partners (EPA’s Office of Emergency Management and Office of Chemical Safety and Pollution Prevention) and Regional on-scene coordinators, NHSRC is attempting to understand and develop useful decontamination procedures for wide-area remediation. This report documents the results of a comprehensive laboratory study designed to better understand and maximize the effectiveness of aqueous solutions of chlorine dioxide (ClO2) to decontaminate materials contaminated with Bacillus anthracis spores.

These results, coupled with additional information in separate NHSRC publications (available at www.epa.gov/nhsrc) can be used to determine whether a particular decontamination technology can be effective in a given scenario. NHSRC has made this publication available to the response community to prepare for and recover from disasters involving biological contamination. This research is intended to move EPA one step closer to achieving its homeland security goals and its overall mission of protecting human health and the environment while providing sustainable solutions to our environmental problems.

Jonathan Herrmann, Director National Homeland Security Research Center

Acknowledgments

Contributions of the following individuals and organization to this report are gratefully acknowledged:

United States Environmental Protection Agency (EPA) Office of Research and Development, National Homeland Security Research Center Eletha Brady-Roberts (Quality Assurance) Lukas Oudejans (peer review)

United States Environmental Protection Agency (EPA) Office of Emergency Management, National Decontamination Team Michael Ottlinger (peer review)

United States Environmental Protection Agency (EPA) Office of Research and Development, National Risk Management Research Laboratory Timothy Dean (peer review)

Battelle

Executive Summary

The U.S. Environmental Protection Agency quantified in terms of log reduction, based (EPA), Office of Research and Development on the difference in the number of bacterial is striving to protect human health and the spores recovered from the positive controls environment from adverse impacts resulting and test coupons. Efficacy tests were from acts of terror by investigating the conducted with increasing levels of ClO2 effectiveness and applicability of (1,500 to 4,000 parts per million (ppm)), technologies for homeland security (HS)- contact times (one or two hours) and/or related applications. The purpose of this number of spray applications (two to four investigation was to determine the total spray applications), in order to decontamination efficacy of aqueous maximize the decontamination efficacy for chlorine dioxide (ClO2) solutions in each material (i.e., to achieve a target log inactivating Bacillus anthracis (causative reduction of at least 6.0). In addition to agent for anthrax) spores, as a function of efficacy, the impact of the decontamination material and decontamination parameters process on the materials was visually (concentration, contact time, number of observed and reported. spray applications). The objective of this study was to provide an understanding of the Summary of Results performance of the aqueous ClO2 The aqueous ClO2 decontamination decontamination technology to guide its use technology provided complete inactivation and implementation in HS applications. In of B. anthracis spores on galvanized metal, the assessment of options for decorative laminate, and glass using a 3,000 decontamination following intentional ppm, three-spray, one-hour contact time release of B. anthracis, it is important to treatment. Complete decontamination was know whether and to what extent such not achieved for any of the other materials factors can impact the decontamination for any of the ClO2 decontamination efficacy. conditions tested. The highest average log reductions achieved for B. anthracis on This investigation focused on treated wood, industrial carpet, and decontamination of both indoor and outdoor unpainted concrete were 2.64, 3.40, and materials, including industrial carpet, treated 2.50, respectively. The aqueous ClO2 wood, unpainted concrete, two types of soil decontaminant was the least effective on the material (topsoil and Arizona Test Dust soil materials (topsoil and AZTD), with [AZTD]), decorative laminate, galvanized nearly all log reduction results less than 1.0 metal, and glass. Decontamination efficacy for the six ClO2 conditions tested. Overall, tests were conducted with spores of Bacillus the results show that with robust enough anthracis or Bacillus subtilis, the latter conditions (i.e., 3,000 – 4,000 ppm ClO2, organism included to assess its potential as a one-two hour contact time, and two – four surrogate for future studies related to B. spray applications), the aqueous ClO2 spray anthracis. Decontamination efficacy was technology may be an effective

decontaminant for some nonporous materials such as glass, galvanized metal, and laminate. However, aqueous ClO2 was found to be largely ineffective on porous materials (e.g., wood, carpet, soils, and concrete), even under the most robust conditions tested.

With regard to comparing the decontamination efficacies of B. anthracis and B. subtilis, there were no test results in which B. subtilis was inactivated to a significantly greater degree than B. anthracis. Additionally, no visible damage was observed on any test materials for any of the decontamination conditions tested.

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Contents Foreword ...... iv Acknowledgments...... v Executive Summary ...... vi Tables ...... x Figures...... xiii Abbreviations/Acronyms ...... xiv

1.0 Introduction ...... 1

2.0 Preparation of ClO2 Solutions and Test Matrix ...... 2 2.1 Preparation of Aqueous ClO2 Solutions ...... 2 2.2 Test Matrix ...... 2

3.0 Summary of Test Procedures...... 6 3.1 Preparation of Test Coupons...... 6 3.2 Decontaminant Testing ...... 9 3.2.1 Chlorine Dioxide Liquid Spray Neutralization ...... 9 3.2.2 Chlorine Dioxide Liquid Spray Tests ...... 10 3.3 Decontamination Efficacy ...... 11 3.4 Qualitative Assessment of Surface Damage ...... 12 3.5 Chlorine Dioxide Solution Characterization...... 12

4.0 Quality Assurance/Quality Control ...... 13 4.1 Equipment Calibration ...... 13 4.2 QC Results ...... 13 4.3 Audits ...... 13 4.3.1 Performance Evaluation Audit...... 13 4.3.2 Technical Systems Audit ...... 14 4.3.3 Data Quality Audit ...... 14 4.4 QAPP Amendments and Deviations ...... 15 4.5 QA/QC Reporting ...... 15 4.6 Data Review ...... 15

5.0 Initial Chlorine Dioxide Spray Scoping Tests ...... 16 5.1 Initial Scoping Decontamination Tests Using Galvanized Metal Coupons...... 16 5.2 Scoping Decontamination Tests Using Other Materials ...... 19

6.0 Chlorine Dioxide Spray Efficacy Maximization Tests ...... 24

7.0 Liquid Spray Results for Two vs. Four Total Spray Applications ...... 40 7.1 Description ...... 40 7.1.1 Test Material Composition ...... 40 7.1.2 Subjectivity of Spraying Method ...... 40

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7.1.3 Chemistry of ClO2 Solution ...... 41

7.2 Decontamination Efficacy ...... 43

8.0 Summary of Results ...... 47

9.0 References ...... 51

Appendix A. Spray Deposition and Neutralization Tests ...... 52

Appendix B. Effect of Pre-sterilization of Soil Materials on the Decontamination Efficacy for Aqueous ClO2 Spray ...... 66

Tables

Table 2-1. Initial Scoping Test Matrix for ClO2 Liquid Spray ...... 3 Table 2-2. Efficacy Maximization Test Matrix for ClO2 Liquid Spray ...... 4 Table 2-3. Matrix of Repeat Liquid Spray Tests to Assess Effect of Increasing Number of Spray Applications ...... 5 Table 3-1. Summary of Materials used for Decontaminant Testing ...... 9 Table 4-1. Performance Standards for Amperometric Titration ...... 14 Table 4-2. Performance Evaluation Audits ...... 14 Table 5-1. Inactivation of Bacillus anthracis Spores—Initial Scoping Tests on Galvanized Metal ...... 17 Table 5-2. Inactivation of Bacillus subtilis Spores—Initial Scoping Tests on Galvanized Metal ...... 18 Table 5-3. Summary of Efficacy Values with 95% Confidence Intervals for Initial Scoping Maximization of ClO2 Liquid Sprayed on Galvanized Metal ...... 19 Table 5-4. Inactivation of Bacillus anthracis Spores—3,000 ppm ClO2 Liquid on Building and Outdoor Materials (One Hour Contact, Three Total Spray Applications)...... 21 Table 5-5. Inactivation of Bacillus subtilis Spores—3,000 ppm ClO2 Liquid on Building and Outdoor Materials (One Hour Contact, Three Total Spray Applications)...... 22 Table 5-6. Summary of Efficacy Values with 95% Confidence Intervals for 3,000 ppm ClO2 Liquid on Building and Outdoor Materials (One Hour Contact, Three Total Spray Applications) ...... 23

Table 6-1. Inactivation of Bacillus anthracis Spores—3,000 ppm ClO2 Liquid Sprayed on Building and Outdoor Materials (Four Total Spray Applications, One Hour Contact Time) ...... 26 Table 6-2. Inactivation of Bacillus subtilis Spores—3,000 ppm ClO2 Liquid Sprayed on Building and Outdoor Materials (Four Total Spray Applications, One Hour Contact Time) ...... 27 Table 6-3. Inactivation of Bacillus anthracis Spores—4,000 ppm ClO2 Liquid Sprayed on Building and Outdoor Materials (Two Total Spray Applications, One Hour Contact Time) ...... 28 Table 6-4. Inactivation of Bacillus subtilis Spores—4,000 ppm ClO2 Liquid Sprayed on Building and Outdoor Materials (Two Total Spray Applications, One Hour Contact Time) ...... 29 Table 6-5. Inactivation of Bacillus anthracis Spores—4,000 ppm ClO2 Liquid Sprayed on Building and Outdoor Materials (Four Total Spray Applications, One Hour Contact Time) ...... 30 Table 6-6. Inactivation of Bacillus subtilis Spores—4,000 ppm ClO2 Liquid Sprayed on Building and Outdoor Materials (Four Total Spray Applications, One Hour Contact Time) ...... 31 Table 6-7. Inactivation of Bacillus anthracis Spores—3,000 ppm ClO2 Liquid Sprayed on Building and Outdoor Materials (Four Total Spray Applications, Two Hour Contact Time) ...... 32 Table 6-8. Inactivation of Bacillus subtilis Spores—3,000 ppm ClO2 Liquid Sprayed on Building and Outdoor Materials (Four Total Spray Applications, Two Hour Contact Time) ...... 33 Table 6-9. Inactivation of Bacillus anthracis Spores—4,000 ppm ClO2 Liquid Sprayed on

Building and Outdoor Materials (Four Total Spray Applications, Two Hour Contact Time) ...... 34 Table 6-10. Inactivation of Bacillus subtilis Spores—4,000 ppm ClO2 Liquid Sprayed on Building and Outdoor Materials (Four Total Spray Applications, Two Hour Contact Time) ...... 35 Table 6-11. Summary of Mean Quantitative Efficacy with 95% Confidence Intervals for ClO2 Liquid Spray Efficacy Maximization Results for B. anthracis...... 36 Table 6-12. Summary of Mean Quantitative Efficacy with 95% Confidence Intervals for ClO2 Liquid Spray Efficacy Maximization Results for B. subtilis ...... 37 Table 7-1. Chlorine Dioxide Liquid Batch Chemical Comparison ...... 42 Table 7-2. Inactivation of Bacillus anthracis Spores—Two Applications vs. Four Applications 3,000 ppm ClO2 Liquid Sprayed on Materials (One Hour Contact Time) ...... 44 Table 7-3. Inactivation of Bacillus anthracis Spores—Two Applications vs. Four Applications 4,000 ppm ClO2 Liquid Sprayed on Materials (One Hour Contact Time) ...... 45 Table 7-4. Summary of Decontamination Efficacy Values for Two vs. Four Applications ...... 46 Table 7-5. Comparing Decontamination Efficacy Values for Tests that Were Repeated ...... 46 Table 8-1. Summary of Liquid ClO2 Decontamination Efficacy for Initial Scoping Tests ...... 49 Table 8-2. Summary of Liquid ClO2 Liquid Decontamination Efficacy for Maximization Tests ...... 50 Table A-1. Deposition/Runoff Weight of ClO2 Liquid with 60 Minute Contact Time ...... 52 Table A-2. Neutralization Testing with Bacillus anthracis Spores with 3,000 ppm ClO2 Liquid, One Hour Contact, Two Total Spray Applications ...... 54 Table A-3. Neutralization Testing with Bacillus anthracis Spores with 3,000 ppm ClO2 Liquid, One Hour Contact, Three Total Spray Applications ...... 54 Table A-4. Neutralization Testing with Bacillus subtilis Spores with 3,000 ppm ClO2 Liquid, One Hour Contact, Three Total Spray Applications ...... 55 Table A-5. Neutralization Testing with Bacillus anthracis Spores with 1,500 ppm ClO2 Liquid, One Hour Contact, Four Total Spray Applications ...... 56 Table A-6. Neutralization Testing with Bacillus subtilis Spores with 1,500 ppm ClO2 Liquid, One Hour Contact, Four Total Spray Applications ...... 56 Table A-7. Neutralization Testing with Bacillus anthracis Spores with 2,000 ppm ClO2 Liquid, One Hour Contact, Four Total Spray Applications ...... 57 Table A-8. Neutralization Testing with Bacillus subtilis Spores with 2,000 ppm ClO2 Liquid, One Hour Contact, Four Total Spray Applications ...... 57 Table A-9. Deposition/Runoff Weight of ClO2 Liquid Spray per Maximization Testing Condition and Material ...... 59

Table A-10. Neutralization Testing with Bacillus anthracis Spores with 3,000 ppm ClO2 Liquid, One Hour Contact, Four Total Spray Applications ...... 61 Table A-11. Neutralization Testing with Bacillus subtilis Spores with 3,000 ppm ClO2 Liquid, One Hour Contact, Four Total Spray Applications ...... 61 Table A-12. Neutralization Testing with Bacillus anthracis Spores with 4,000 ppm ClO2 Liquid, One Hour Contact, Two Total Spray Applications ...... 62 Table A-13. Neutralization Testing with Bacillus subtilis Spores with 4,000 ppm ClO2 Liquid, One Hour Contact, Two Total Spray Applications ...... 62 Table A-14. Neutralization Testing with Bacillus anthracis Spores with 4,000 ppm ClO2 Liquid, One Hour Contact, Four Total Spray Applications ...... 63

Table A-15. Neutralization Testing with Bacillus subtilis Spores with 4,000 ppm ClO2 Liquid, One Hour Contact, Four Total Spray Applications ...... 63 Table A-16. Neutralization Testing with Bacillus anthracis Spores with 3,000 ppm ClO2 Liquid, Two Hour Contact, Four Total Spray Applications ...... 64 Table A-17. Neutralization Testing with Bacillus subtilis Spores with 3,000 ppm ClO2 Liquid, Two Hour Contact, Four Total Spray Applications ...... 64 Table A-18. Neutralization Testing with Bacillus anthracis Spores with 4,000 ppm ClO2 Liquid, Two Hour Contact, Four Total Spray Applications ...... 65 Table A-19. Neutralization Testing with Bacillus subtilis Spores with 4,000 ppm ClO2 Liquid, Two Hour Contact, Four Total Spray Applications ...... 65 Table B-1. Comparison of Decontamination Efficacy with 95% Confidence Intervals using Unsterile vs. Sterile Soils for ClO2 Liquid Spray Testing ...... 67

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Figures

Figure 3-1. Inoculation of coupon using a multi-channel micropipette ...... 7 Figure 3-2. Topsoil “coupons” made with Parafilm®-lined Petri dishes ...... 8 Figure 5-1. Summary of efficacies (log reduction) and confidence intervals for initial scoping tests with ClO2 liquid spray on galvanized metal ...... 19 Figure 5-2. Summary of efficacies (log reduction) and confidence intervals for initial scoping tests on building and outdoor materials ...... 23 Figure 6-1a. Summary of efficacies (log reduction) and confidence intervals for liquid spray testing conditions ...... 38 Figure 6-1b. Summary of efficacies (log reduction) and confidence intervals for liquid spray testing conditions ...... 39

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Abbreviations/Acronyms

ATCC American Type Culture Collection AZTD Arizona Test Dust AZ Arizona B. anthracis Bacillus anthracis (Ames strain) B. subtilis Bacillus subtilis (ATCC 19659) BBRC Battelle Biomedical Research Center BSC III biological safety cabinet, Class III C Celsius CGB compact glovebox CFU colony-forming unit(s) CI confidence interval ClO2 chlorine dioxide cm centimeter(s) DNA deoxyribonucleic acid EPA U.S. Environmental Protection Agency g gram(s) HDPE high density polyethylene hr hour(s) HS homeland security L liter(s) LAL Limulus Amebocyte Lysate min minute(s) mg milligram(s) mL milliliter(s) µL microliter(s) N Normal NA not applicable NHSRC National Homeland Security Research Center NIST National Institute of Standards and Technology ORD EPA Office of Research and Development PBS phosphate-buffered saline PCR polymerase chain reaction ppm parts per million ppmv parts per million by volume psi pounds per square inch QA quality assurance QAPP Quality Assurance Project Plan QC quality control RH relative humidity rpm revolutions per minute SD standard deviation SE standard error

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SFW sterile filtered water (cell-culture grade) STS sodium thiosulfate TSA technical systems audit(s)

1.0 Introduction

The U.S. Environmental Protection Agency’s applying the tested technologies. EPA (EPA’s) National Homeland Security provides potential users with unbiased, Research Center (NHSRC) is helping protect third-party information that can supplement human health and the environment from vendor-provided information. Stakeholder adverse impacts resulting from the release of involvement ensures that user needs and chemical, biological, or radiological agents. perspectives are incorporated into the test With an emphasis on decontamination and design so that useful performance consequence management, water information is produced for each of the infrastructure protection, and threat and tested technologies. consequence assessment, NHSRC is working to develop tools and information that will help The purpose of this investigation was to detect the intentional introduction of chemical develop an understanding of the or biological contaminants in buildings, effectiveness of liquid solutions of ClO2 to outdoor environments, or water systems; decontaminate a range of materials. This contain these contaminants; decontaminate investigation focused on decontamination of buildings, outdoor environments, or water indoor and outdoor surfaces typical of those systems; and facilitate the disposal of material found in a public building or outdoors that resulting from remediation efforts. could be contaminated by a biological agent (such as B. anthracis) following an As part of the above effort, EPA investigates intentional release. Residual biological agent the effectiveness and applicability of on surfaces following decontamination after technologies for homeland security (HS)- an intentional release could present a related applications by developing test plans potential health risk. This report documents that are responsive to the needs of the impact of various factors on the efficacy stakeholders, conducting tests, collecting of aqueous ClO2 against spores of B. and analyzing data, and preparing peer- anthracis and B. subtilis on surfaces. reviewed reports. All evaluations are Specifically, tests were conducted to conducted in accordance with rigorous determine the ClO2 liquid spray application quality assurance (QA) protocols to ensure requirements (e.g., in terms of that data of known and high quality are concentration, number of sprays, contact generated and that the results are defensible. time) needed to maximize the inactivation EPA provides high-quality information that efficacy of B. anthracis (or B. subtilis) is useful to decision makers in purchasing or spores as a function of material.

2.0 Preparation of ClO2 Solutions and Test Matrix

2.1 Preparation of Aqueous ClO2 metal coupons with aqueous solutions of Solutions ClO2 at concentrations ranging from 1,500 to 3,000 ppm, as shown in Table 2-1. To prepare the aqueous solutions of ClO2, 6 (Galvanized metal was selected for initial N HCl was mixed into sterile filtered water tests based on results of previous testing.1) (SFW) in a glass bottle. Commercially- In these initial tests, galvanized metal was available Clorox® bleach was added to the completely decontaminated only with the HCl + SFW mixture and mixed carefully. A 3,000 ppm, three-spray treatment. Based on formulation of 25% sodium chlorite solution these results with galvanized metal, (SabreChlor 25, Sabre™) was mixed into additional scoping tests were performed the Clorox® + HCl + SFW solution which using this same 3,000 ppm, three-spray yielded the ClO2 solution. Target ClO2 treatment, with industrial carpet, treated concentrations were achieved by adjusting wood, laminate, topsoil, and glass. Refer to the amount of Clorox®. (Target Chapter 5 for further details. concentrations of ClO2 were between 3,000 to 4,000 ppm depending on the test). Target Since glass and galvanized metal were the chlorite ranges were achieved by adjusting only materials completely decontaminated the amount of sodium chlorite solution, to for both B. anthracis and B. subtilis spores achieve a 50% minimum ratio of chlorite to in the initial scoping tests, glass and ClO2 in the solution. Target pH levels (4 – galvanized metal were eliminated from 7) were achieved by adjusting the amount of further testing, and in their place, AZTD and HCl. (See Section 3.5 for further details on unpainted concrete were included in the test the methods used to measure chlorite and matrix. Thus tests with AZTD and ClO2 concentrations and pH.) The solution unpainted concrete, along with the materials was then transferred into a commercially- from the initial scoping tests that were not available, 480 mL (16 ounce) sprayer with a completely decontaminated, were then cylinder style high density polyethylene conducted at increasing levels of ClO2, (HDPE) bottle (Qorpak® Item No. 7331X, contact time, and/or number of spray Bridgeville, PA). The HDPE bottle was applications with the goal of improving or covered with aluminum foil since ClO2 is maximizing decontamination efficacy. photosensitive. Refer to the decontamination “maximization” test matrix summarized in Table 2-2 and the details presented in 2.2 Test Matrix Chapter 6. An adaptive management approach was used to incorporate new To help target subsequent testing conditions knowledge into the testing as with additional materials, initial scoping decontamination efficacy results became tests were conducted using only galvanized available.

Table 2-1. Initial Scoping Test Matrix for ClO2 Liquid Spray ClO Biological Material 2 # Spray Applications Contact Concentration Agenta (application times) Time (hr) (ppm) B. anthracis 4 or Galvanized metal 1,500 ± 150 1 (Time 0, +15, +30, +45 min) B. subtilis B. anthracis 4 or Galvanized metal 2,000 ± 200 1 (Time 0, +15, +30, +45 min) B. subtilis B. anthracis 2 or Galvanized metal 3,000 ± 300 1 (Time 0, +30 min) B. subtilis B. anthracis 3 or Galvanized metal 3,000 ± 300 1 (Time 0, +20, +40 min) B. subtilis Industrial carpet Treated wood B. anthracis Decorative 3 or 3,000 ± 300 1 laminate (Time 0, +20, +40 min) B. subtilis Topsoil Glass a Tests for B. anthracis and B. subtilis were conducted separately

Table 2-2. Efficacy Maximization Test Matrix for ClO2 Liquid Spray ClO Biological Materials 2 # Spray Applications Contact Concentration Agenta (application times) Time (hr) (ppm) Industrial carpet Treated wood B. anthracis Decorative laminate 4 or 3,000 ± 300 1 Unpainted concrete (Time 0, +15, +30, +45 min) B. subtilis AZ test dust Topsoil Industrial carpet Treated wood B. anthracis Decorative laminate 2 or 4,000 ± 400 1 Unpainted concrete (Time 0, +30 min) B. subtilis AZ test dust Topsoil Industrial carpet Treated wood B. anthracis Decorative laminate 4 or 4,000 ± 400 1 Unpainted concrete (Time 0, +15, +30, +45 min) B. subtilis AZ test dust Topsoil Industrial carpet Treated wood B. anthracis Decorative laminate 4 or 3,000 ± 300 2 Unpainted concrete (Time 0, +15, +30, +45 min) B. subtilis AZ test dust Topsoil Industrial carpet Treated wood B. anthracis Decorative laminate 4 or 4,000 ± 400 2 Unpainted concrete (Time 0, +15, +30, +45 min) B. subtilis AZ test dust Topsoil a Tests for B. anthracis and B. subtilis were conducted separately

Lastly, due to some variability in results with carpet, wood, and unpainted concrete, a few tests were repeated with these materials using B. anthracis spores; this test matrix is shown in Table 2-3. These repeat tests were conducted to assess the effect of increasing the number of spray applications at a given ClO2 concentration while eliminating the possibility that slight differences in the batch preparation of the aqueous ClO2 solutions were affecting decontamination efficacy (i.e., causing variability in results). The results of these repeat tests and further details are discussed in Chapter 7.

Table 2-3. Matrix of Repeat Liquid Spray Tests to Assess Effect of Increasing Number of Spray Applications ClO Biological Materials 2 # Spray Applications Contact Concentration Agent (application times) Time (hr) (ppm) Industrial carpet 2 B. anthracis Treated wood 3,000 ± 300a 1 (Time 0, +30 min) Unpainted concrete Industrial carpet 4 B. anthracis Treated wood 3,000 ± 300a 1 (Time 0, +15, +30, +45 min) Unpainted concrete Industrial carpet 2 B. anthracis Treated wood 4,000 ± 300b 1 (Time 0, +30 min) Unpainted concrete Industrial carpet b 4 B. anthracis Treated wood 4,000 ± 300 1 (Time 0, +15, +30, +45 min) Unpainted concrete a Tested using the same batch of 3,000 ppm ClO2. b Tested using the same batch of 4,000 ppm ClO2.

3.0 Summary of Test Procedures

Test procedures were performed in intradermally with a dilution series of spore accordance with a pre-approved Quality suspensions, and virulence was expressed as Assurance Project Plan (QAPP) and are the intradermal median lethal dose. In summarized in this chapter. addition, testing was conducted for robustness of the spores via HCl resistance. 3.1 Preparation of Test Coupons The stock spore suspension was prepared in SFW at an approximate concentration of 1 x The B. anthracis spores used for this testing 109 CFU/mL and stored under refrigeration were prepared from a qualified stock of the at 2 to 8 °C. The B. subtilis spores did not Ames strain at the Battelle Biomedical undergo the level of stringency for Research Center (BBRC). All spore lots characterization (LAL assay, DNA were subject to a stringent characterization fingerprinting, and virulence testing and qualification process required by excluded), but qualitative PCR was done Battelle’s standard operating procedure for using a custom PCR assay to confirm B. spore production. Specifically, all spore lots subtilis. Primers were designed that targeted were characterized prior to use by a conserved region of B. subtilis observation of colony morphology, direct chromosomal DNA, since multiple strains of microscopic observation of spore this bacterium exist. morphology and size and determination of percent refractivity and percent B. anthracis or B. subtilis spores were encapsulation. In addition, the number of inoculated onto test coupons in an viable spores was determined by colony appropriate biosafety cabinet Level III (BSC count and expressed as colony forming units III) according to established BBRC per milliliter (CFU/mL). Theoretically, once procedures. Inoculated coupons and soils plated onto bacterial growth media, each (topsoil and AZTD) were prepared prior to viable spore germinates and yields one CFU. each day of experimental work. Coupons Variations in the expected colony were placed flat in the BSC III and phenotypes were recorded. Endotoxin inoculated at approximately 1 x 108 total concentration of each spore preparation was spores per coupon. This inoculation on determined by the Limulus Amebocyte coupons was accomplished by dispensing a Lysate (LAL)2 assay to assess whether 100 microliter (µL) aliquot of the spore contamination from gram-negative bacteria stock suspension (approximately 1 x 109 occurred during the propagation and spores/mL) using a multi-channel purification process of the spores. Genomic micropipette as 10 droplets (each of 10 µL DNA was extracted from the spores and volume, Figure 3-1) across the surface of the DNA fingerprinting by polymerase chain test coupon. This approach provided more reaction (PCR) was done to confirm the uniform distribution of spores across the genotype. The virulence of the spore lot was coupon surface than would be obtained measured by challenging guinea pigs through a single drop of the suspension.

Figure 3-1. Inoculation of coupon using a multi-channel micropipette.

The inoculations for the soils were done by environmental laboratory) showed that it dispensing a 100 µL aliquot of the spore had an average water content of 16.2%, an stock suspension (approximately 1 x 109 average fraction organic carbon value of spores/mL) using a single-channel 5.20%, an average recalcitrant organic micropipette as 10 droplets across the carbon value of 2.05%, and an average soil surface of the soils. The reason a single- pH of 7.28. channel micropipette was used to inoculate the soils was that the topsoil and AZTD test The AZTD was also commercially available, “coupons” consisted of these materials and its chemical composition was supplied placed in a Parafilm®-lined, 3.5 cm diameter by the vendor as percent of weight: Silicon x 1.0 cm tall Petri dish to an unpacked depth dioxide (68 to 76%), aluminum oxide (10 to of 1.0 cm (Figure 3-2), so the multichannel 15%), iron (III) oxide (2 to 5%), sodium micropipette dimensions did not match the oxide (2 to 4%), magnesium oxide (1 to Petri dish. The reason the Petri dishes were 2%), titanium dioxide (0.5 to 1%), and lined with Parafilm® was that this inert film potassium oxide (2 to 5%). Analysis of this enabled the easy removal of the soils from AZTD showed that it had an average water the dishes into the extraction tubes in a content of 1.19%, an average fraction single motion without having to scoop the organic carbon value of 0.569%, an average inoculated decontaminated soils which recalcitrant organic carbon value of 1.07%, presented handling and safety issues. and an average soil pH of 8.30.

The commercial topsoil used for this After inoculation, the test coupons remained evaluation was a proprietary mixture of soil, undisturbed overnight in the BSC III to dry composted cow manure, sand, and other thoroughly. Test coupons were then exposed ingredients (also proprietary). Analysis of to the decontaminant the next day (i.e., this topsoil (conducted by a third party within 24 hours after inoculation).

Figure 3-2. Topsoil “coupons” made with Parafilm®-lined Petri dishes.

The origin and specifications of the The porous and nonporous coupons were materials used for test coupons are shown in sterilized before use by gamma-(γ )- Table 3-1. All materials were selected as irradiation (treated wood, industrial carpet, representative types of building and outdoor and decorative laminate) or autoclaving materials. All test coupons were made from (galvanized metal and glass). In the initial new materials. The porous and nonporous scoping test with topsoil, the topsoil was not test coupons were 1.9 by 7.5 cm in size, and sterilized prior to use in order to maintain the soils were 3.5 cm diameter by 1.0 cm tall chemical and physical integrity of the in size. material. But due to concerns that the high background levels of the endogenous The industrial carpet was comprised of organisms could potentially interfere with recycled nylon fiber (90%) that had quantitative results, all subsequent tests with undergone the solution dye process, which the soil materials were sterilized by γ - made the carpet inherently stain-resistant irradiation. Refer to Appendix B for further (particularly to acid-based substances), so no details. The γ -irradiation sterilization additional stain treatment was applied to the method was chosen for the porous materials material during its production. The carpet since the pressure (15 pounds per square was treated, however, with an antimicrobial inch [psi]) and heat (121 °C) from an chemical (zinc omadine). The carpet autoclave could physically alter or damage backing consisted entirely of thermoplastic these coupons. Therefore, the nonporous polyolefin compound (also recycled coupons were sent to be γ -irradiated at content). approximately 40 kilogray by a vendor that specializes in this type of processing

(STERIS Isomedix Services, Libertyville, autoclaved at Battelle by following an IL). The nonporous materials were internal standard operating procedure.

Table 3-1. Summary of Materials used for Decontaminant Testing

Lot/Batch/ Manufacturer/ Coupon Size, Material Material Observation Supplier Name Width x Length Preparation NONPOROUS Brooks Brothers Glass; Glass C1036 1.9 cm x 7.5 cm Autoclave Columbus, OH Galvanized metal Adept Products; NAa 1.9 cm x 7.5 cm Autoclave ductwork West Jefferson, OH A’Jack Inc.; Decorative laminate NA 1.9 cm x 7.5 cm γ - irradiation Columbus, OH

POROUS Grossmans Bargain Carpet Shaw EcoTek 6 1.9 cm x 7.5 cm γ - irradiation Outlet; Columbus, OH Painted-wallboard 05-16-03; Set-E-493; United States Gypsum 1.9 cm x 7.5 cm γ - irradiation paper Roll-3 Company; Chicago, IL 30906, ACQ, 2 x 6 x 8 Lowe’s Top Choice; Treated wood 1.9 cm x 7.5 cm γ - irradiation Prime Springfield, OH 5 parts sand and Wysong Concrete; Unpainted concrete 1.0 cm x 3.0 cm γ - irradiation 2 parts cement Fairborn, OH SOILS

GardenScape, Inc.; 3.5 cm diameter x 1.0 nonsterile, Topsoil PY1A0597 Eau Claire, PA (unpacked) γ - irradiation Powder Technology, Inc.; 3.5 cm diameter x 1.0 Arizona Test Dust ISO 121030-1 γ - irradiation Burnsville, MN (unpacked) aNot applicable.

3.2 Decontaminant Testing that remained on, or ran off from, each material (i.e., “spray-and-weigh”). These 3.2.1 Chlorine Dioxide Liquid Spray ClO2 liquid deposition data were used in Neutralization trial runs to determine the amount of neutralizing agent needed to stop the action Neutralization panels were conducted before of the decontaminant after the prescribed any testing with each aqueous ClO2 liquid contact time. Refer to Appendix A for the spray batch, using coupons that had not been results of these tests. inoculated with spores. In these neutralization panels, the decontaminant was 3.2.2 Chlorine Dioxide Liquid Spray Tests applied and measurements were made with multiple coupons of each material type to Five replicate test coupons (inoculated with determine the amount of the decontaminant B. anthracis or B. subtilis spores and

decontaminated), five replicate positive to stop the decontamination activity of the control coupons (inoculated and not ClO2 liquid. The required concentration of decontaminated, i.e., sprayed only with STS was determined in the neutralization SFW), one procedural blank (not inoculated, panels for each ClO2 test solution or number decontaminated), and one laboratory blank of applications tested. In each of the (not inoculated, not decontaminated) of each neutralization panels, a range of STS coupon material were used in testing under concentrations was tested to determine the each set of testing conditions. All test concentration that most effectively stopped coupons were oriented horizontally (i.e., the action of the ClO2 (as indicated by the lying flat). After the contact time, the ClO2 maximum recovery of viable spores in solution that had pooled on top of each test simulated coupon extracts). The results of coupon was captured, neutralized, and those neutralization panels are shown in subjected to spore extraction along with the Appendix A. associated test coupon. The coupons were extracted by agitation on On the day following inoculation, test an orbital shaker for 15 minutes at coupons intended for decontamination approximately 200 revolutions per minute (including blanks) were separated from the (rpm) at room temperature. (Unsterilized positive controls or coupons not exposed to topsoil was incubated in a water bath for one decontaminant (including blanks) since both hour at 55 to 60 °C to inactivate the sets were inoculated and dried overnight in endogenous flora that were susceptible to the same BSC III. The ClO2 liquid spray heat shock). The unpainted concrete distance (30.5 cm), humidity (≤ 70% RH), coupons were sonicated for 1 hour to and temperature (22 °C ± 2 °C) were the increase extraction efficiency. The soils same for all applications, including the were transferred from the Petri dishes to the positive controls. The positive controls extraction tubes. Following extraction, a (including blanks) were transferred into a 1 mL aliquot of the coupon extract was separate compact glove box, or CGB removed, and a series of dilutions up (Compact Glove Box, Plas-Labs Model No. through 10-7 was prepared in SFW. An 830-ABC, Lansing, MI), where SFW was aliquot (0.1 mL) of the undiluted extract and sprayed using the same type of sprayer. For each serial dilution were then spread plated porous materials and soils, more sprays (i.e., onto tryptic soy agar plates (in triplicate) trigger pulls) were required to wet the and incubated overnight at 35 to 37 ºC. surfaces of the materials since they absorbed Resulting colonies were enumerated within the liquid. 18 to 24 hours of plating. The number of CFU/mL was determined by multiplying the Following the appropriate number of spray average number of colonies for the triplicate applications and contact time, each coupon plates by the reciprocal of the dilution, and (along with any associated pooled accounting for the 0.1 mL volume of the decontaminant) was aseptically transferred extract or dilution that was plated. to a sterile 50 mL conical tube containing 10 mL of sterile phosphate-buffered saline Before further decontamination tests with (PBS) solution with 0.1% Triton X-100 the next ClO2 solution, the BSC III and the surfactant (i.e., 99.9% PBS, 0.1% Triton X- compact glove box (CGB) were thoroughly 100) and the appropriate concentration of cleaned (using separate steps involving sodium thiosulfate (STS) neutralizer needed bleach, ethanol, water, then drying)

following procedures established under the coupons. The blanks were spiked with an BBRC Facility Safety Plan. equivalent amount of 0.1 mL of “stock suspension” that did not contain the Laboratory blanks controlled for sterility biological agent. The target acceptance and procedural blanks controlled for viable criterion was that extracts of laboratory or spores inadvertently introduced to test procedural blanks were to contain no CFU.

The mean percent spore recovery from each coupon material was calculated using results from positive control coupons (spiked, not decontaminated (sprayed with SFW instead of the decontaminant)), 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 coupons of a single material, and CFUspike is the number of CFU spiked onto each of those coupons. 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 coupon material, and the results are included in Chapters 5 through 12.

3.3 Decontamination Efficacy

The performance or efficacy of the ClO2 liquid spray was assessed by determining the number of viable organisms remaining on each test coupon and in any decontaminant run-off from the coupon (for liquid spray testing only) after decontamination. Those numbers were compared to the number of viable organisms extracted from the positive control coupons. The number of viable spores of B. anthracis or B. subtilis in extracts of test and positive control coupons was determined in order to calculate efficacy of the decontaminant. Efficacy is defined as the extent (as log10 reduction) to which viable spores extracted from test coupons after decontamination were less numerous than the viable spores extracted from positive control coupons. The logarithm of the CFU abundance from each coupon extract was determined, and the mean of those logarithm values was then determined for each set of control and associated test coupons, respectively. Efficacy of a decontaminant for a test organism/test condition on the ith coupon material was calculated as the difference between those mean log values, i.e.:

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

where log10 CFUcij refers to the j individual logarithm values obtained from the positive control coupons and log10 CFUtij refers to the j individual logarithm values obtained from the corresponding test coupons, and the overbar designates a mean value. In tests conducted under this plan, there were five control and five corresponding test coupons (i.e., j = 5) for each coupon material. In the case where no viable spores were found in any of the five test coupon extracts after decontamination, a CFU abundance of 1 was assigned, resulting in a log10 CFU of zero for that material. This situation occurred when the decontaminant was highly effective, and no viable spores were found on the decontaminated test coupons. In such cases, the final efficacy on that material was reported as greater than or equal to (≥) the value calculated by Equation 2.

The variances (i.e., the square of the standard deviation) of the log10 CFUcij and log10 CFUtij 2 2 values were also calculated for both the control and test coupons (i.e., S cij and S tij), and were used to calculate the pooled standard error (SE) for the efficacy value calculated in Equation 2, as follows:

2 2tScS SE ij += ij (3) 55

where the number 5 again represents the number j of coupons in both the control and test data sets. Each efficacy result is thus reported as a log reduction value with an associated SE value. The significance of differences in efficacy across different coupon materials and spore types was assessed based on the 95% confidence interval of each efficacy result. The 95% confidence interval (CI) is:

95% CI = Efficacy ± (1.96 × SE) (4)

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 log reductions as ≥ some value.

3.4 Qualitative Assessment of Surface The total resulting iodine is reduced back to Damage iodide when titrated with standard 0.1 Normal (N) [equal to 0.1 molar] sodium Visual inspection of each coupon surface thiosulfate (STS). After this initial reaction took place after the prescribed ClO2 liquid with ClO2, the solution is acidified using 6 and fumigant contact time and application N hydrochloric acid (HCl), which forms rates, through side-by-side comparison of additional chlorite, and is titrated further the decontaminated test surface and control with STS. The total volume (mL) of STS coupons of the same test material. solution titrated is proportional to the Differences in color, reflectivity, and amount of iodine generated, which is roughness were assessed qualitatively, and proportional to the chlorine dioxide observations were documented. concentration.

3.5 Chlorine Dioxide Solution Certified National Institute of Standards and Characterization Technology (NIST)-traceable chlorite standards, appropriately diluted in solution The concentrations of ClO2 solutions were comparable to the sampling solution, were measured using a modified titration method titrated each day of chlorine dioxide testing based on the Standard Method 4500-ClO2 E to verify accuracy. Amperometric Method II3. For this titration method, 5 mL of the ClO2 solution is added The pH of the ClO2 solutions was measured to a 150 mL solution of 5% potassium with a calibrated pH meter (Thermo iodide in phosphate buffer (pH 7.0). Under Scientific, Waltham, MA). these conditions, ClO2 oxidizes the iodide to iodine (and ClO2 is converted to chlorite). 12

4.0 Quality Assurance/Quality Control

Quality assurance/quality control (QA/QC) the coupons. The spore density levels met procedures were performed in accordance the QA target criterion of 1 × 109 CFU/mL with the QAPP (available upon request). (± 25%) for all tests, with the exception of The QA/QC procedures are summarized the following three test conditions: below. • B. anthracis and B. subtilis for the 4.1 Equipment Calibration 3,000 ppm, 1 hour, 2 total spray applications. In these tests, the All equipment (e.g., pipettes, incubators, inoculum spore density was 6.37 x biological safety cabinets, pH meter) and 108 CFU/mL and 6.47 x 108 monitoring devices (e.g., thermometer, CFU/mL, respectively. hygrometer) used at the time of evaluation • B. subtilis for the 3,000 ppm, 1 hour, were verified as being certified, calibrated, 3 total spray applications. In this or validated. test, the inoculum spore density was 6.53 x 108 CFU/mL. 4.2 QC Results Although these few tests did not meet the Quality control efforts conducted during QA target criterion, the results are not decontaminant testing included positive expected to be adversely affected. control coupons (inoculated, not decontaminated), procedural blanks (not 4.3 Audits inoculated, decontaminated), laboratory blanks (not inoculated, not decontaminated), 4.3.1 Performance Evaluation Audit and spike control samples (analysis of the stock spore suspension). Performance evaluation audits were conducted to assess the quality of the results All positive control results were within the obtained during evaluation. target recovery range of 1 to 150% of the spiked spores, and all procedural and Performance standards for amperometric laboratory blanks met the criterion of no titration for 1000 mg/L, 3000 mg/L, and observed CFU for both organisms. 4000 mg/L chlorite using sodium chlorite stock solutions to verify the titration method Spike control samples were taken from the were made and tested. The results for these spore suspension on the day of testing and performance standards are listed in Table 4- serially diluted, nutrient plated and counted 1. to establish the spore density used to spike

Table 4-1. Performance Standards for Amperometric Titration

Sodium Chlorite Measured Chlorite Stock (mg/L) (mg/L) 1,000 ± 100 1,045 3,000 ± 300 3,203 4,000 ± 400 4,113

Temperatures were monitored but no efforts were undertaken to control any of the test temperatures.

No performance evaluation audits were performed to confirm the concentration and purity of B. anthracis or B. subtilis spores because quantitative standards do not exist for these organisms. The control coupons and blanks support the spore measurements.

Table 4-2 summarizes the performance evaluation audits that were performed.

Table 4-2. Performance Evaluation Audits

Audit Allowable Actual Measurement Procedure Tolerance Tolerance Volume of liquid Gravimetric evaluation ± 10% ± 5% from micropipettes Chlorite Amperometric titration ± 10% ± 10% Compared to independently Temperature ± 2 °C ± 2 °C calibrated thermometer Compared to independently Relative Humidity ± 10% < 10% calibrated hygrometer Compare time to independent Time ± 2 sec/hr 0 sec/hr clock or watch value

4.3.2 Technical Systems Audit findings of the TSA required corrective action. TSA records were permanently Contractor QA staff conducted technical stored with the Contractor QA Manager. systems audits (TSAs) on June 17, July 20, August 26 and 27, and November 4, 2010, 4.3.3 Data Quality Audit to ensure that the tests were being conducted in accordance with the appropriate test At least 10% of the data acquired during the plan/QAPP. As part of the audit, test evaluation were audited. The contract QA procedures were compared to those auditor traced the data from the initial specified in the test/QAPP and data acquisition through reduction and statistical acquisition and handling procedures were analysis to final reporting to ensure the reviewed. Observations and findings from integrity of the reported results. All the TSA were documented and submitted to calculations performed on the data the test leader for response. None of the undergoing the audit were checked. 14

findings were mostly minor data 4.4 QAPP Amendments and Deviations transcription errors requiring some recalculation of efficacy results, but none Two deviations were prepared, approved, were gross errors in recording. Copies of the and retained in the test files for this assessment reports were distributed to the evaluation. The first deviation related to the EPA and contractor staff. QA/QC initiation of testing as described in the two procedures were performed in accordance QAPP amendments without fully- with the QAPP. signed/approved amendments in place. A second deviation was for the cases during 4.6 Data Review ClO2 liquid spray testing when the positive controls exceeded the ± 25% criterion from Records and data generated in the evaluation the target inoculum titer. These deviations received a QC/technical review before they are not expected to adversely impact results; were utilized in calculating or evaluating refer to Section 4.2 for more information. results and prior to incorporation in reports. All data were recorded by contractor staff. 4.5 QA/QC Reporting The staff member performing the QC/technical review was involved in the Each assessment and audit was documented experiments and added his/her initials and in accordance with the QAPP. For these the date to a hard copy of the record being tests, findings were noted (none significant) reviewed. This hard copy was returned to in the data quality audit, but no followup the staff member who stored the record. corrective action was necessary. The

5.0 Initial Chlorine Dioxide Spray Scoping Tests

To help target subsequent testing conditions • 3,000 ppm ClO2, 1 hour contact with additional materials, initial scoping time, three total spray applications. tests were performed first using galvanized (Actual ClO2 liquid concentration metal coupons to assist in focusing the ClO2 measured at 3,103 ppm for both B. concentration, contact time, and number of anthracis and B. subtilis testing.) spray applications that would potentially be • 1,500 ppm ClO2, 1 hour contact needed to effectively decontaminate other time, four total spray applications. materials. Galvanized metal and the initial (Actual ClO2 liquid concentration decontamination conditions were selected measured at 1,484 ppm for both B. 1 based on results of previous testing . anthracis and B. subtilis testing.) Specifically, ClO2 liquid spray testing with • 2,000 ppm ClO2, 1 hour contact target concentrations of 1,500; 2,000; and time, four total spray applications. 3,000 ppm; contact time of 1 hour, and (Actual ClO2 liquid concentration varying number of spray applications was measured at 2,024 ppm for both B. conducted on galvanized metal inoculated anthracis and B. subtilis testing.) with either spores of B. anthracis or B. subtilis. As expected, increasing the concentration and number of spray applications improved decontamination efficacy; refer to Tables 5- 5.1 Initial Scoping Decontamination Tests 1 through 5-3and Figure 5-1. Complete Using Galvanized Metal Coupons inactivation within the detection limit was seen only when testing at the condition of The decontamination efficacies were 3,000 ppm ClO2, 1 hour contact time, and evaluated for B. anthracis and B. subtilis on three total spray applications. Both B. galvanized metal for the following anthracis (≥ 7.89) and B. subtilis (≥ 7.70) conditions: were completely inactivated under these conditions. For three of the four B. • 3,000 ppm ClO2, 1 hour contact anthracis tests, a 6 log reduction or greater time, two total spray applications. was achieved, and for the other test with B. (Actual ClO2 liquid concentration anthracis, a log reduction of nearly 6 (5.99) measured at 3,103 ppm for both B. was obtained. (A decontaminant which anthracis and B. subtilis testing.) achieves a 6 log reduction or greater is considered effective.4)

Table 5-1. Inactivation of Bacillus anthracis Spores—Initial Scoping Tests on Galvanized Metal Contact Time, Inoculum Mean of Logs of Mean % Decontamination Applications a (CFU) Observed CFU Recovery Efficacy ± CIf 3000 ppm (1 hr contact, two total sprays) Positive Controlsb 6.37 x 107 7.76 ± 0.070 91.0 ± 15.3 -g Test Couponsc 6.37 x 107 0.680 ± 0.93 < 0.0100 7.08 ± 0.82 Laboratory Blankd 0 0 - - Procedural Blanke 0 0 - - 3000 ppm (1 hr contact , three total sprays) Positive Controls 9.43 x 107 7.89 ± 0.030 83.3 ± 5.90 - Test Coupons 9.43 x 107 0 0 ≥ 7.89 ± 0.030 Laboratory Blank 0 0 - - Procedural Blank 0 0 - - 1500 ppm (1 hr contact, four total sprays) Positive Controls 9.90 x 107 7.84 ± 0.030 69.7 ± 4.10 - Test Coupons 9.90 x 107 1.85 ± 1.1 < 0.01 5.99 ± 0.99 Laboratory Blank 0 0 - - Procedural Blank 0 0 - - 2000 ppm (1 hr contact, four total sprays) Positive Controls 9.43 x 107 7.87 ± 0.080 79.0 ± 15.0 - Test Coupons 9.43 x 107 0.740 ± 1.7 < 0.01 7.12 ± 1.5 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 coupons, the mean percent recovery on those five coupons, and decontamination efficacy (log reduction). b Positive Controls = inoculated, not decontaminated coupons (sprayed with SFW). c Test Coupons = inoculated, decontaminated coupons. d Laboratory Blank = not inoculated, not decontaminated coupon. e Procedural Blank = not inoculated, decontaminated coupon. 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.

Table 5-2. Inactivation of Bacillus subtilis Spores—Initial Scoping Tests on Galvanized Metal Contact Time, Inoculum Mean of Logs of Mean % Decontamination Applications a (CFU) Observed CFU Recovery Efficacy ± CIf 3000 ppm (1 hr contact, two total sprays) Positive Controlsb 6.47 x 107 7.74 ± 0.030 85.4 ± 5.80 -g Test Couponsc 6.47 x 107 0.610 ± 0.84 < 0.01 7.13 ± 0.74 Laboratory Blankd 0 0 - - Procedural Blanke 0 0 - - 3000 ppm (1 hr contact, three total sprays) Positive Controls 6.53 x 107 7.70 ± 0.080 77.4 ± 14.0 - Test Coupons 6.53 x 107 0 0 ≥ 7.70 ± 0.070 Laboratory Blank 0 0 - - Procedural Blank 0 0 - - 1500 ppm (1 hr contact, four total sprays) Positive Controls 1.12 x 108 7.98 ± 0.040 84.6 ± 8.0 - Test Coupons 1.12 x 108 7.21 ± 0.15 15.4 ± 6.0 0.760 ± 0.14 Laboratory Blank 0 0 - - Procedural Blank 0 0 - - 2000 ppm (1 hr contact, four total sprays) Positive Controls 1.05 x 108 7.98 ± 0.040 92.3 ± 9.10 - Test Coupons 1.05 x 108 6.07 ± 0.45 1.75 ± 1.80 1.91 ± 0.40 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 coupons, the mean percent recovery on those five coupons, and decontamination efficacy (log reduction). b Positive Controls = inoculated, not decontaminated coupons (sprayed with SFW). c Test Coupons = inoculated, decontaminated coupons. d Laboratory Blank = not inoculated, not decontaminated coupon. e Procedural Blank = not inoculated, decontaminated coupon. 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.

Table 5-3. Summary of Efficacy Values with 95% Confidence Intervals for Initial Scoping Tests of ClO2 Liquid Sprayed on Galvanized Metal

Efficacy for ClO Liquid, Contact Time, Efficacy for 2 B. anthracis (Ames) Applications B. subtilis

3000 ppm, 1 hr, 2 applications 7.08 ± 0.82 7.13 ± 0.74 3000 ppm, 1 hr, 3 applications ≥ 7.89 ± 0.030a ≥ 7.70 ± 0.070a 1500 ppm, 1 hr, 4 applications 5.99 ± 0.99 0.760 ± 0.14 2000 ppm, 1 hr, 4 applications 7.12 ± 1.50 1.91 ± 0.40

aResult represents complete inactivation within the detection limit of 33.33 CFU/material. 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.

* *

Figure 5-1. Summary of efficacies (log reduction) and confidence intervals for initial scoping tests with ClO2 liquid spray on galvanized metal (asterisk indicates complete inactivation within the detection limit).

5.2 Scoping Decontamination Tests Using hour contact time, this same Other Materials decontamination treatment was used for the additional materials. The actual ClO2 liquid Based on the initial scoping test results with concentration was measured at 3,103 ppm galvanized metal, a second scoping test was for both B. anthracis and B. subtilis testing. conducted using additional materials, i.e., The results are shown in Tables 5-4 and 5-5 unsterilized topsoil, glass, treated wood, and summarized in Table 5-6 and Figure 5- industrial carpet, and decorative laminate. 2. Overall, log reductions varied by material, Since galvanized metal was completely with topsoil decontaminated at the worst decontaminated for both B. anthracis and B. efficacy (less than 0.24 log reduction). subtilis only at the test condition of 3,000 Complete inactivation within the detection ppm ClO2, three spray applications, and 1 limit was observed only on glass and

decorative laminate for B. anthracis (≥ 7.86 efficacy for wood and carpet ranged from and ≥ 7.84, respectively) and on glass for B. approximately 1.5 – 4.0 log reduction. subtilis (≥ 7.88). The decontamination

Table 5-4. Inactivation of Bacillus anthracis Spores—3,000 ppm ClO2 Liquid on Building and Outdoor Materials (1 Hour Contact, Three Total Spray Applications) Inoculum Mean of Logs of Mean % Decontamination Test Material (CFU) Observed CFU Recovery Efficacy ± CIg Topsoila,f Positive Controlsb 1.26 x 108 7.97 ± 0.060 74.6 ± 11.0 -h Test Couponsc 1.26 x 108 7.72 ± 0.060 42.4 ± 5.80 0.240 ± 0.080 Laboratory Blankd 0 0 - - Procedural Blanke 0 0 - - Glass Positive Controls 1.26 x 108 7.86 ± 0.080 58.7 ± 11.0 - Test Coupons 1.26 x 108 0 0 ≥ 7.86 ± 0.070 Laboratory Blank 0 0 - - Procedural Blank 0 0 - - Treated Wood Positive Controls 1.26 x 108 6.92 ± 0.050 6.61 ± 0.76 - Test Coupons 1.26 x 108 4.51 ± 0.18 0.0300 ± 0.01 2.41 ± 0.17 Laboratory Blank 0 0 - - Procedural Blank 0 0 - - Industrial Carpet Positive Controls 1.26 x 108 8.01 ± 0.060 81.8 ± 11 - Test Coupons 1.26 x 108 4.61 ± 0.84 0.150 ± 0.28 3.40 ± 0.73 Laboratory Blank 0 0 - - Procedural Blank 0 0 - - Decorative Laminate Positive Controls 1.26 x 108 7.84 ± 0.050 55.4 ± 6.10 - Test Coupons 1.26 x 108 0 0 ≥ 7.84 ± 0.040 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 coupons, the mean percent recovery on those five coupons, and decontamination efficacy (log reduction). b Positive Controls = inoculated, not decontaminated coupons (sprayed with SFW). c Test Coupons = inoculated, decontaminated coupons. d Laboratory Blank = not inoculated, not decontaminated coupon. e Procedural Blank = not inoculated, decontaminated coupon. f Unsterilized topsoil used for testing. g 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. h “-” Not Applicable.

Table 5-5. Inactivation of Bacillus subtilis Spores—3,000 ppm ClO2 Liquid on Building and Outdoor Materials (1 Hour Contact, Three Total Spray Applications) Inoculum Mean of Logs of Mean % Decontamination Test Material (CFU) Observed CFU Recovery Efficacy ± CIf Topsoila, f Positive Controlsb 1.16 x 108 7.92 ± 0.040 72.0 ± 6.90 -g Test Couponsc 1.16 x 108 7.83 ± 0.030 57.9 ± 4.20 0.0900 ± 0.050 Laboratory Blankd 0 0 - - Procedural Blanke 0 0 - - Glass Positive Controls 1.16 x 108 7.88 ± 0.11 67.4 ± 18.0 - Test Coupons 1.16 x 108 0 0 ≥ 7.88 ± 0.10 Laboratory Blank 0 0 - - Procedural Blank 0 0 - - Treated Wood Positive Controls 1.16 x 108 7.02 ± 0.74 24.9 ± 31.0 - Test Coupons 1.16 x 108 5.59 ± 0.37 0.420 ± 0.23 1.43 ± 0.72 Laboratory Blank 0 0 - - Procedural Blank 0 0 - - Industrial Carpet Positive Controls 1.16 x 108 7.84 ± 0.010 59.5 ± 2.00 - Test Coupons 1.16 x 108 3.76 ± 0.41 0.01 ± 0.01 4.08 ± 0.36 Laboratory Blank 0 0 - - Procedural Blank 0 0 - - Decorative Laminate Positive Controls 1.16 x 108 7.90 ± 0.050 68.4 ± 8.0 - Test Coupons 1.16 x 108 2.77 ± 1.72 0.010 ± 0.02 5.13 ± 1.5 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 coupons, the mean percent recovery on those five coupons, and decontamination efficacy (log reduction). b Positive Controls = inoculated, not decontaminated coupons (sprayed with SFW). c Test Coupons = inoculated, decontaminated coupons. d Laboratory Blank = not inoculated, not decontaminated coupon. e Procedural Blank = not inoculated, decontaminated coupon. f Unsterilized topsoil used for testing. g 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. h “-” Not Applicable.

Table 5-6. Summary of Efficacy Values with 95% Confidence Intervals for 3,000 ppm ClO2 Liquid on Building and Outdoor Materials (1 Hour Contact, Three Total Spray Applications) Efficacy for Efficacy for Test Material B. anthracis (Ames) B. subtilis

Topsoil 0.24 ± 0.08 0.09 ± 0.05 Glass ≥ 7.86 ± 0.07 a ≥ 7.88 ± 0.10 a Treated Wood 2.41 ± 0.17 1.43 ± 0.72 Industrial Carpet 3.40 ± 0.73 4.08 ± 0.36 Decorative Laminate ≥ 7.84 ± 0.04 a 5.13 ± 1.50 aResult represents complete inactivation within the detectable limit of 3.33 CFU/mL. 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.

* * *

Figure 5-2. Summary of efficacies (log reduction) and confidence intervals for initial scoping tests on building and outdoor materials (asterisk indicates complete inactivation within the detection limit).

6.0 Chlorine Dioxide Spray Efficacy Maximization Tests

Additional ClO2 liquid spray testing on (Actual ClO2 liquid concentration building and outdoor materials was measured at an average of 3,980 ppm conducted to determine if increasing the for B. anthracis testing and an ClO2 concentration, number of spray average of 4,047 ppm for B. subtilis applications, and/or contact time would testing). improve decontamination efficacy for the • 3,000 ppm ClO2, two hour contact materials not completely decontaminated time, four total spray applications. under the test conditions described in (Actual ClO2 liquid concentration Chapter 5. Tests for galvanized metal and measured at 3,170 ppm for B. glass were discontinued from further testing anthracis testing and 3,103 ppm for because these materials were completely B. subtilis testing.) decontaminated for the two microorganisms • 4,000 ppm ClO2, two hour contact using 3000 ppm ClO2, three sprays, and one time, four total spray applications. hour contact time. In place of these (Actual ClO2 liquid concentration materials, unpainted concrete and AZTD measured at 3,912 ppm for both B. were added to the efficacy maximization test anthracis and B. subtilis testing.) matrix.

An adaptive management approach was The detailed decontamination efficacy used to incorporate new knowledge into the results are shown in Tables 6-1 through 6- testing as decontamination efficacy results 10, and summarized in Tables 6-11 and 6- became available. Test matrices are 12and in Figure 6-1. Decorative laminate presented in the order in which the tests was the only material to be completely were conducted: decontaminated in some of the tests, and a log reduction ≥ 6.0 was achieved on • 3,000 ppm ClO2, one hour contact laminate for nine of the ten tests. The soil time, four total spray applications. materials (topsoil and AZTD) were the most (Actual ClO2 liquid concentration difficult materials to decontaminate, with all measured at 3,238 ppm for B. average log reduction results for AZTD less anthracis testing and an average of than 1.5 and all log reduction results for 3,137 ppm for B. subtilis testing). topsoil less than 0.31. Except for a few tests, • 4,000 ppm ClO2, one hour contact all of the log reduction results for wood, time, two total spray applications. concrete, and carpet were less than 3.0. The (Actual ClO2 liquid concentration most robust liquid spray treatment of 4,000 measured at 4,047 ppm for both B. ppm ClO2, four spray applications, and two- anthracis and B. subtilis testing.) hour contact time did offer improved • 4,000 ppm ClO2, one hour contact decontamination efficacy compared to some time, four total spray applications. of the other test conditions (all other

variables being equal), but did not with unpainted concrete. A number of significantly improve efficacy compared to factors could be the cause of these outlier the 4,000 ppm ClO2, four spray applications, results, including random experimental one hour contact time except for a few tests. error, but we think these results may also be associated with using different batches for Decontamination efficacy results did not the testing of the ClO2 solutions. A few always improve with increasing ClO2 tests were repeated with these materials to concentration, contact time, or number of assess the effect of increasing the number of spray applications. This effect was more spray applications at a given ClO2 pronounced on carpet, wood, and unpainted concentration while eliminating the concrete, where some test results were much possibility that differences in the batch higher than expected. For example, preparation of the aqueous ClO2 solutions industrial carpet was decontaminated at a were affecting decontamination efficacy log reduction of greater than 7.0 at the 3,000 (i.e., causing variability in results). See ppm, one hour, four-spray test condition, but Chapter 7 for further discussion of potential the log reduction results for carpet were all causes of experimental variability and test less than 3.0 for all the other maximization results. tests. A somewhat similar result occurred

Table 6-1. Inactivation of Bacillus anthracis Spores—3,000 ppm ClO2 Liquid Sprayed on Building and Outdoor Materials (Four Total Spray Applications, One Hour Contact Time) Inoculum Mean of Logs of Mean % Decontamination Test Material f (CFU) Observed CFU Recovery Efficacy ± CI Industrial Carpeta Positive Controlsb 1.12 x 108 7.94 ± 0.05 77.4 ± 8.80 -g Test Couponsc 1.12 x 108 0.82 ± 1.8 < 0.01 7.12 ± 1.6 Laboratory Blankd 0 0 - - Procedural Blanke 0 0 - - Treated Wood Positive Controls 1.12 x 108 7.66 ± 0.43 57.0 ± 39 - Test Coupons 1.12 x 108 4.82 ± 0.04 0.06 ± 0.01 2.85 ± 0.38 Laboratory Blank 0 0 - - Procedural Blank 0 0 - - Decorative Laminate Positive Controls 1.12 x 108 7.84 ± 0.09 62.8 ± 13 - Test Coupons 1.12 x 108 0.68 ± 1.5 < 0.01 7.16 ± 1.30 Laboratory Blank 0 0 - - Procedural Blank 0 0 - - Unpainted Concrete Positive Controls 1.02 x 108 7.32 ± 0.19 21.9 ± 8.1 - Test Coupons 1.02 x 108 1.19 ± 2.70 0.180 ± 0.39 6.13 ± 2.30 Laboratory Blank 0 0 - - Procedural Blank 0 0 - - Arizona Test Dust Positive Controls 1.14 x 108 7.97 ± 0.05 82.2 ± 8.8 - Test Coupons 1.14 x 108 7.85 ± 0.07 62.5 ± 10 0.12 ± 0.08 Laboratory Blank 0 0 - - Procedural Blank 0 0 - - Topsoil Positive Controls 1.14 x 108 7.98 ± 0.06 84.1 ± 10 - Test Coupons 1.14 x 108 7.93 ± 0.06 75.0 ± 11 0.05 ± 0.07 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 coupons, the mean percent recovery on those five coupons, and decontamination efficacy (log reduction).b Positive Controls = inoculated, not decontaminated coupons (sprayed with SFW). c Test Coupons = inoculated, decontaminated coupons. d Laboratory Blank = not inoculated, not decontaminated coupon. e Procedural Blank = not inoculated, decontaminated coupon. 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.

Table 6-2. Inactivation of Bacillus subtilis Spores—3,000 ppm ClO2 Liquid Sprayed on Building and Outdoor Materials (Four Total Spray Applications, One Hour Contact Time) Inoculum Mean of Logs of Mean % Decontamination Test Material (CFU) Observed CFU Recovery Efficacy ± CIf Industrial Carpeta Positive Controlsb 1.20 x 108 7.86 ± 0.07 61.2 ± 9.0 -g Test Couponsc 1.20 x 108 6.59 ± 0.16 3.44 ± 1.1 1.27 ± 0.16 Laboratory Blankd 0 0 - - Procedural Blanke 0 0 - - Treated Wood Positive Controls 1.20 x 108 6.88 ± 0.08 6.43 ± 1.10 - Test Coupons 1.20 x 108 4.84 ± 2.70 0.80 ± 0.60 2.04 ± 2.40 Laboratory Blank 0 0 - - Procedural Blank 0 0 - - Decorative Laminate Positive Controls 1.20 x 108 7.82 ± 0.04 55.3 ± 5.70 - Test Coupons 1.20 x 108 0 0 ≥ 7.82 ± 0.04 Laboratory Blank 0 0 - - Procedural Blank 0 0 - - Unpainted Concrete Positive Controls 9.33 x 107 7.49 ± 0.12 34.3 ± 8.50 - Test Coupons 9.33 x 107 2.10 ± 2.0 < 0.01 5.39 ± 1.80 Laboratory Blank 0 0 - - Procedural Blank 0 0 - - Arizona Test Dust Positive Controls 1.02 x 108 7.94 ± 0.03 85.4 ± 5.60 - Test Coupons 1.02 x 108 7.86 ± 0.06 71.4 ± 10.0 0.08 ± 0.06 Laboratory Blank 0 0 - - Procedural Blank 0 0 - - Topsoil Positive Controls 1.02 x 108 7.89 ± 0.05 75.8 ± 9.60 - Test Coupons 1.02 x 108 7.90 ± 0.04 78.8 ± 7.80 0.005 ± 0.06 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 coupons, the mean percent recovery on those five coupons, and decontamination efficacy (log reduction).b Positive Controls = inoculated, not decontaminated coupons (sprayed with SFW). c Test Coupons = inoculated, decontaminated coupons. d Laboratory Blank = not inoculated, not decontaminated coupon. e Procedural Blank = not inoculated, decontaminated coupon. 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.

Table 6-3. Inactivation of Bacillus anthracis Spores—4,000 ppm ClO2 Liquid Sprayed on Building and Outdoor Materials (Two Total Spray Applications, One Hour Contact Time) Inoculum Mean of Logs of Mean % Decontamination Test Material (CFU) Observed CFU Recovery Efficacy ± CIf Industrial Carpeta Positive Controlsb 1.05 x 108 8.04 ± 0.05 106 ± 11.0 -g Test Couponsc 1.05 x 108 5.22 ± 0.60 0.31 ± 0.34 2.82 ± 0.52 Laboratory Blankd 0 0 - - Procedural Blanke 0 0 - - Treated Wood Positive Controls 1.05 x 108 7.16 ± 0.26 16.3 ± 12.0 - Test Coupons 1.05 x 108 5.21 ± 0.50 0.24 ± 0.21 1.95 ± 0.49 Laboratory Blank 0 0 - - Procedural Blank 0 0 - - Decorative Laminate Positive Controls 1.05 x 108 7.76 ± 0.07 55.8 ± 9.60 - Test Coupons 1.05 x 108 0 0 ≥ 7.76 ± 0.06 Laboratory Blank 0 0 - - Procedural Blank 0 0 - - Unpainted Concrete Positive Controls 1.22 x 108 7.27 ± 0.24 17.3 ± 9.30 - Test Coupons 1.22 x 108 0.68 ± 0.93 < 0.01 6.60 ± 0.84 Laboratory Blank 0 0 - - Procedural Blank 0 0 - - Arizona Test Dust Positive Controls 1.21 x 108 7.99 ± 0.01 81.6 ± 2.40 - Test Coupons 1.21 x 108 7.05 ± 0.12 9.59 ± 2.50 0.94 ± 0.11 Laboratory Blank 0 0 - - Procedural Blank 0 0 - - Topsoil Positive Controls 1.21 x 108 7.98 ± 0.01 78.9 ± 1.10 - Test Coupons 1.21 x 108 7.93 ± 0.01 70.1 ± 1.60 0.05 ± 0.01 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 coupons, the mean percent recovery on those five coupons, and decontamination efficacy (log reduction).b Positive Controls = inoculated, not decontaminated coupons (sprayed with SFW). c Test Coupons = inoculated, decontaminated coupons. d Laboratory Blank = not inoculated, not decontaminated coupon. e Procedural Blank = not inoculated, decontaminated coupon. 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.

Table 6-4. Inactivation of Bacillus subtilis Spores—4,000 ppm ClO2 Liquid Sprayed on Building and Outdoor Materials (Two Total Spray Applications, One Hour Contact Time) Inoculum Mean of Logs of Mean % Decontamination Test Material (CFU) Observed CFU Recovery Efficacy ± CIf Industrial Carpeta Positive Controlsb 8.93 x 107 7.68 ± 0.06 53.7 ± 7.30 -g Test Couponsc 8.93 x 107 5.17 ± 0.19 0.18 ± 0.10 2.51 ± 0.17 Laboratory Blankd 0 0 - - Procedural Blanke 0 0 - - Treated Wood Positive Controls 8.93 x 107 6.59 ± 0.12 4.50 ± 1.40 - Test Coupons 8.93 x 107 5.00 ± 0.22 0.12 ± 0.06 1.59 ± 0.22 Laboratory Blank 0 0 - - Procedural Blank 0 0 - - Decorative Laminate Positive Controls 8.93 x 107 7.63 ± 0.08 48.8 ± 9.10 - Test Coupons 8.93 x 107 1.16 ± 1.1 < 0.01 6.47 ± 1.40 Laboratory Blank 0 0 - - Procedural Blank 0 0 - - Unpainted Concrete Positive Controls 8.93 x 107 7.06 ± 0.16 13.7 ± 5.60 - Test Coupons 8.93 x 107 3.42 ± 0.16 < 0.01 3.64 ± 0.20 Laboratory Blank 0 0 - - Procedural Blank 0 0 - - Arizona Test Dust Positive Controls 1.33 x 108 7.96 ± 0.04 69.5 ± 6.20 - Test Coupons 1.33 x 108 7.83 ± 0.06 51.5 ± 6.90 0.13 ± 0.06 Laboratory Blank 0 0 - - Procedural Blank 0 0 - - Topsoil Positive Controls 1.33 x 108 7.96 ± 0.03 69.5 ± 5.20 - Test Coupons 1.33 x 108 7.92 ± 0.05 63.4 ± 6.80 0.04 ± 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 coupons, the mean percent recovery on those five coupons, and decontamination efficacy (log reduction).b Positive Controls = inoculated, not decontaminated coupons (sprayed with SFW). c Test Coupons = inoculated, decontaminated coupons. d Laboratory Blank = not inoculated, not decontaminated coupon. e Procedural Blank = not inoculated, decontaminated coupon. 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.

Table 6-5. Inactivation of Bacillus anthracis Spores—4,000 ppm ClO2 Liquid Sprayed on Building and Outdoor Materials (Four Total Spray Applications, One Hour Contact Time) Inoculum Mean of Logs of Mean % Decontamination Test Material (CFU) Observed CFU Recovery Efficacy ± CIf Industrial Carpeta Positive Controlsb 1.21 x 108 7.97 ± 0.02 76.5 ± 3.60 -g Test Couponsc 1.21 x 108 6.69 ± 0.10 4.18 ± 1.00 1.27 ± 0.09 Laboratory Blankd 0 0 - - Procedural Blanke 0 0 - - Treated Wood Positive Controls 1.21 x 108 7.01 ± 0.10 8.62 ± 1.90 - Test Coupons 1.21 x 108 5.80 ± 0.09 0.530 ± 0.11 1.21 ± 0.12 Laboratory Blank 0 0 - - Procedural Blank 0 0 - - Decorative Laminate Positive Controls 1.21 x 108 7.90 ± 0.02 66.1 ± 3.30 - Test Coupons 1.21 x 108 0 0 ≥ 7.90 ± 0.02 Laboratory Blank 0 0 - - Procedural Blank 0 0 - - Unpainted Concrete Positive Controls 1.16 x 108 7.74 ± 0.21 52.4 ± 26.0 - Test Coupons 1.16 x 108 5.14 ± 0.69 0.310 ± 0.48 2.61 ± 0.63 Laboratory Blank 0 0 - - Procedural Blank 0 0 - - Arizona Test Dust Positive Controls 1.16 x 108 7.96 ± 0.02 78.8 ± 3.40 - Test Coupons 1.16 x 108 6.50 ± 0.09 2.79 ± 0.66 1.46 ± 0.08 Laboratory Blank 0 0 - - Procedural Blank 0 0 - - Topsoil Positive Controls 1.16 x 108 7.98 ± 0.04 82.2 ± 7.30 - Test Coupons 1.16 x 108 7.81 ± 0.04 55.7 ± 5.30 0.17 ± 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 coupons, the mean percent recovery on those five coupons, and decontamination efficacy (log reduction).b Positive Controls = inoculated, not decontaminated coupons (sprayed with SFW). c Test Coupons = inoculated, decontaminated coupons. d Laboratory Blank = not inoculated, not decontaminated coupon. e Procedural Blank = not inoculated, decontaminated coupon. 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.

Table 6-6. Inactivation of Bacillus subtilis Spores—4,000 ppm ClO2 Liquid Sprayed on Building and Outdoor Materials (Four Total Spray Applications, One Hour Contact Time) Inoculum Mean of Logs of Mean % Decontamination Test Material (CFU) Observed CFU Recovery Efficacy ± CIf Industrial Carpeta Positive Controlsb 1.14 x 108 7.92 ± 0.03 73.5 ± 5.50 -g Test Couponsc 1.14 x 108 7.19 ± 0.04 13.8 ± 1.40 0.73 ± 0.05 Laboratory Blankd 0 0 - - Procedural Blanke 0 0 - - Treated Wood Positive Controls 1.14 x 108 6.94 ± 0.05 7.70 ± 0.93 - Test Coupons 1.14 x 108 6.01 ± 0.32 1.13 ± 0.94 0.930 ± 0.29 Laboratory Blank 0 0 - - Procedural Blank 0 0 - - Decorative Laminate Positive Controls 1.14 x 108 7.86 ± 0.06 64.4 ± 9.10 - Test Coupons 1.14 x 108 0 0 ≥ 7.86 ± 0.06 Laboratory Blank 0 0 - - Procedural Blank 0 0 - - Unpainted Concrete Positive Controls 7.67 x 107 7.29 ± 0.14 26.4 ± 9.30 - Test Coupons 7.67 x 107 5.76 ± 0.39 0.940 ± 0.48 1.52 ± 0.36 Laboratory Blank 0 0 - - Procedural Blank 0 0 - - Arizona Test Dust Positive Controls 7.67 x 107 7.79 ± 0.04 80.6 ± 7.10 - Test Coupons 7.67 x 107 7.57 ± 0.16 51.0 ± 18.0 0.22 ± 0.14 Laboratory Blank 0 0 - - Procedural Blank 0 0 - - Topsoil Positive Controls 7.67 x 107 7.86 ± 0.09 96.2 ± 20.0 - Test Coupons 7.67 x 107 7.74 ± 0.08 73.3 ± 13.0 0.12 ± 0.10 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 coupons, the mean percent recovery on those five coupons, and decontamination efficacy (log reduction).b Positive Controls = inoculated, not decontaminated coupons (sprayed with SFW). c Test Coupons = inoculated, decontaminated coupons. d Laboratory Blank = not inoculated, not decontaminated coupon. e Procedural Blank = not inoculated, decontaminated coupon. 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.

Table 6-7. Inactivation of Bacillus anthracis Spores—3,000 ppm ClO2 Liquid Sprayed on Building and Outdoor Materials (Four Total Spray Applications, Two Hour Contact Time) Inoculum Mean of Logs of Mean % Decontamination Test Material (CFU) Observed CFU Recovery Efficacy ± CIf Industrial Carpeta Positive Controlsb 1.15 x 108 7.96 ± 0.07 80.4 ± 13.0 -g Test Couponsc 1.15 x 108 5.45 ± 0.22 0.270 ± 0.12 2.51 ± 0.20 Laboratory Blankd 0 0 - - Procedural Blanke 0 0 - - Treated Wood Positive Controls 1.15 x 108 7.11 ± 0.09 11.4 ± 2.00 - Test Coupons 1.15 x 108 5.05 ± 0.44 0.150 ± 0.19 2.06 ± 0.39 Laboratory Blank 0 0 - - Procedural Blank 0 0 - - Decorative Laminate Positive Controls 1.15 x 108 7.88 ± 0.07 67.0 ± 11.0 - Test Coupons 1.15 x 108 1.24 ± 1.2 < 0.01 6.64 ± 1.00 Laboratory Blank 0 0 - - Procedural Blank 0 0 - - Unpainted Concrete Positive Controls 1.15 x 108 7.94 ± 0.03 75.6 ± 5.60 - Test Coupons 1.15 x 108 5.72 ± 0.31 0.55 ± 0.33 2.22 ± 0.28 Laboratory Blank 0 0 - - Procedural Blank 0 0 - - Arizona Test Dust Positive Controls 1.15 x 108 7.87 ± 0.03 65.1 ± 4.50 - Test Coupons 1.15 x 108 7.21 ± 0.27 16.3 ± 8.50 0.66 ± 0.24 Laboratory Blank 0 0 - - Procedural Blank 0 0 - - Topsoil Positive Controls 1.15 x 108 7.98 ± 0.02 82.9 ± 4.30 - Test Coupons 1.15 x 108 7.88 ± 0.05 67.0 ± 8.60 0.09 ± 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 coupons, the mean percent recovery on those five coupons, and decontamination efficacy (log reduction).b Positive Controls = inoculated, not decontaminated coupons (sprayed with SFW). c Test Coupons = inoculated, decontaminated coupons. d Laboratory Blank = not inoculated, not decontaminated coupon. e Procedural Blank = not inoculated, decontaminated coupon. 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.

Table 6-8. Inactivation of Bacillus subtilis Spores—3,000 ppm ClO2 Liquid Sprayed on Building and Outdoor Materials (Four Total Spray Applications, 2 Hour Contact Time) Inoculum Mean of Logs of Mean % Decontamination Test Material (CFU) Observed CFU Recovery Efficacy ± CIf Industrial Carpeta Positive Controlsb 9.50 x 107 7.85 ± 0.09 76.5 ± 15.0 -g Test Couponsc 9.50 x 107 7.05 ± 0.07 12.0 ± 1.90 0.80 ± 0.10 Laboratory Blankd 0 0 - - Procedural Blanke 0 0 - - Treated Wood Positive Controls 9.50 x 107 6.98 ± 0.09 10.3 ± 2.50 - Test Coupons 9.50 x 107 6.01 ± 0.44 1.56 ± 1.40 0.97 ± 0.39 Laboratory Blank 0 0 - - Procedural Blank 0 0 - - Decorative Laminate Positive Controls 9.50 x 107 7.94 ± 0.04 91.9 ± 8.50 - Test Coupons 9.50 x 107 5.06 ± 0.86 0.390 ± 0.48 2.88 ± 0.75 Laboratory Blank 0 0 - - Procedural Blank 0 0 - - Unpainted Concrete Positive Controls 9.50 x 107 7.47 ± 0.20 33.7 ± 15.0 - Test Coupons 9.50 x 107 6.47 ± 0.39 4.37 ± 4.20 1.00 ± 0.38 Laboratory Blank 0 0 - - Procedural Blank 0 0 - - Arizona Test Dust Positive Controls 9.50 x 107 7.98 ± 0.04 99.9 ± 9.10 - Test Coupons 9.50 x 107 7.80 ± 0.09 67.5 ± 13.0 0.18 ± 0.08 Laboratory Blank 0 0 - - Procedural Blank 0 0 - - Topsoil Positive Controls 9.50 x 107 7.98 ± 0.04 100± 9.80 - Test Coupons 9.50 x 107 7.93 ± 0.02 89.5 ± 3.90 0.05 ± 0.04 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 coupons, the mean percent recovery on those five coupons, and decontamination efficacy (log reduction).b Positive Controls = inoculated, not decontaminated coupons (sprayed with SFW). c Test Coupons = inoculated, decontaminated coupons. d Laboratory Blank = not inoculated, not decontaminated coupon. e Procedural Blank = not inoculated, decontaminated coupon. 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.

Table 6-9. Inactivation of Bacillus anthracis Spores—4,000 ppm ClO2 Liquid Sprayed on Building and Outdoor Materials (Four Total Spray Applications, Two Hour Contact Time) Inoculum Mean of Logs of Mean % Decontamination Test Material (CFU) Observed CFU Recovery Efficacy ± CIf Industrial Carpeta Positive Controlsb 1.33 x 108 7.90 ± 0.09 60.79 ± 13 -g Test Couponsc 1.33 x 108 5.06 ± 0.17 0.09 ± 0.04 2.84 ± 0.17 Laboratory Blankd 0 0 - - Procedural Blanke 0 0 - - Treated Wood Positive Controls 1.33 x 108 7.22 ± 0.20 13.8 ± 7.60 - Test Coupons 1.33 x 108 4.59 ± 0.71 0.05 ± 0.03 2.64 ± 0.65 Laboratory Blank 0 0 - - Procedural Blank 0 0 - - Decorative Laminate Positive Controls 1.33 x 108 7.89 ± 0.05 58.7 ± 7.20 - Test Coupons 1.33 x 108 0.310 ± 0.69 < 0.01 7.58 ± 0.61 Laboratory Blank 0 0 - - Procedural Blank 0 0 - - Unpainted Concrete Positive Controls 1.15 x 108 7.65 ± 0.16 40.6 ± 16.0 - Test Coupons 1.15 x 108 5.15 ± 0.11 0.12 ± 0.03 2.50 ± 0.16 Laboratory Blank 0 0 - - Procedural Blank 0 0 - - Arizona Test Dust Positive Controls 7.87 x 107 7.90 ± 0.03 100 ± 5.90 - Test Coupons 7.87 x 107 6.62 ± 0.24 5.97 ± 3.70 1.28 ± 0.21 Laboratory Blank 0 0 - - Procedural Blank 0 0 - - Topsoil Positive Controls 1.33 x 108 7.97 ± 0.04 70.6 ± 6.80 - Test Coupons 1.33 x 108 7.66 ± 0.03 34.2 ± 2.70 0.31 ± 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 coupons, the mean percent recovery on those five coupons, and decontamination efficacy (log reduction).b Positive Controls = inoculated, not decontaminated coupons (sprayed with SFW). c Test Coupons = inoculated, decontaminated coupons. d Laboratory Blank = not inoculated, not decontaminated coupon. e Procedural Blank = not inoculated, decontaminated coupon. 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

Table 6-10. Inactivation of Bacillus subtilis Spores—4,000 ppm ClO2 Liquid Sprayed on Building and Outdoor Materials (Four Total Spray Applications, Two Hour Contact Time) Inoculum Mean of Logs of Mean % Decontamination Test Material (CFU) Observed CFU Recovery Efficacy ± CIf Industrial Carpeta Positive Controlsb 1.30 x 108 7.89 ± 0.06 60.5 ± 8.00 -g Test Couponsc 1.30 x 108 5.58 ± 0.24 0.33 ± 0.13 2.31 ± 0.22 Laboratory Blankd 0 0 - - Procedural Blanke 0 0 - - Treated Wood Positive Controls 1.30 x 108 6.95 ± 0.12 7.11 ± 2.20 - Test Coupons 1.30 x 108 4.95 ± 0.77 0.18 ± 0.26 2.00 ± 0.69 Laboratory Blank 0 0 - - Procedural Blank 0 0 - - Decorative Laminate Positive Controls 1.30 x 108 7.98 ± 0.02 73.4 ± 3.10 - Test Coupons 1.30 x 108 0 0 ≥ 7.98 ± 0.02 Laboratory Blank 0 0 - - Procedural Blank 0 0 - - Unpainted Concrete Positive Controls 1.30 x 108 6.65 ± 0.18 3.72 ± 1.80 - Test Coupons 1.30 x 108 5.60 ± 0.37 0.38 ± 0.22 1.05 ± 0.36 Laboratory Blank 0 0 - - Procedural Blank 0 0 - - Arizona Test Dust Positive Controls 1.30 x 108 7.96 ± 0.02 70.4 ± 3.60 - Test Coupons 1.30 x 108 7.27 ± 0.25 16.3 ± 9.70 0.69 ± 0.22 Laboratory Blank 0 0 - - Procedural Blank 0 0 - - Topsoil Positive Controls 1.30 x 108 8.03 ± 0.02 83.3 ± 3.80 - Test Coupons 1.30 x 108 7.96 ± 0.03 69.5 ± 4.70 0.08 ± 0.03 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 coupons, the mean percent recovery on those five coupons, and decontamination efficacy (log reduction).b Positive Controls = inoculated, not decontaminated coupons (sprayed with SFW). c Test Coupons = inoculated, decontaminated coupons. d Laboratory Blank = not inoculated, not decontaminated coupon. e Procedural Blank = not inoculated, decontaminated coupon. 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

Table 6-11. Summary of Mean Quantitative Efficacy with 95% Confidence Intervals for ClO2 Liquid Spray Efficacy Maximization Results for B. anthracis Quantitative Efficacy (mean log reduction) 3000 ppm, 4000 ppm, 4000 ppm, 3000 ppm, 4000 ppm, One hr contact, One hr contact, One hr contact, Two hr contact, Two hr contact, four total spray two total spray four total spray four total spray four total spray Test Material applications applications applications applications applications Industrial Carpet 7.12 ± 1.60 2.82 ± 0.52 1.27 ± 0.09 2.51 ± 0.20 2.84 ± 0.17

Treated Wood 2.85 ± 0.38 1.95 ± 0.49 1.21 ± 0.12 2.06 ± 0.39 2.64 ± 2.6 Decorative 7.16 ± 1.34 ≥ 7.76a ± 0.06 ≥ 7.90a ± 0.02 6.64 ± 1.0 7.58 ± 0.61 Laminate Unpainted 6.13 ± 2.34 6.60 ± 0.84 2.61 ± 0.63 2.22 ± 0.28 2.50 ± 0.16 Concrete Arizona Test Dust 0.12 ± 0.08 0.940 ± 0.11 1.46 ± 0.08 0.66 ± 0.24 1.28 ± 0.21

Topsoil 0.05 ± 0.07 0.05 ± 0.01 0.17 ± 0.05 0.09 ± 0.05 0.31 ± 0.05 aResult represents complete inactivation within the detectable limit of 33.33 CFU/material. 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.

Table 6-12. Summary of Mean Quantitative Efficacy with 95% Confidence Intervals for ClO2 Liquid Spray Efficacy Maximization Results for B. subtilis Quantitative Efficacy (mean log reduction) 3000 ppm, 4000 ppm, 4000 ppm, 3000 ppm, 4000 ppm, One hr contact, One hr contact, One hr contact, Two hr contact, Two hr contact, four total spray two total spray four total spray four total spray four total spray Test Material applications applications applications applications applications Industrial Carpet 1.27 ± 0.16 2.51 ± 0.17 0.73 ± 0.05 0.80 ± 0.10 2.31 ± 0.22

Treated Wood 2.04 ± 2.40 1.59 ± 0.22 0.93 ± 0.29 0.97 ± 0.39 2.00 ± 0.69

Decorative Laminate ≥ 7.82a ± 0.04 6.47 ± 1.40 ≥ 7.86a ± 0.06 2.88 ± 0.75 ≥ 7.98a ± 0.02

Unpainted Concrete 5.39 ± 1.80 3.64 ± 0.20 1.52 ± 0.36 1.00 ± 0.38 1.05 ± 0.36

Arizona Test Dust 0.08 ± 0.06 0.13 ± 0.06 0.22 ± 0.14 0.18 ± 0.08 0.69 ± 0.22

Topsoil 0.005 ± 0.06 0.04 ± 0.05 0.12 ± 0.10 0.05 ± 0.04 0.08 ± 0.03 aResult represents complete inactivation within the detectable limit of 33.33 CFU/material. 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.

* * * * *

Figure 6-1a. Summary of efficacies (log reduction) and confidence intervals for liquid spray testing conditions (asterisk indicates complete inactivation within the detection limit).

Topsoil

Figure 6-1b. Summary of efficacies (log reduction) and confidence intervals for liquid spray testing conditions.

7.0 Liquid Spray Results for Two vs. Four Total Spray Applications

7.1 Description wood, and unpainted concrete) have, historically, been variable in terms of During the course of the ClO2 liquid spray efficacy (log reduction) once decontaminant efficacy maximization testing (refer to has been applied. The reason may be Chapter 6), the decontamination efficacy attributed to the porous nature of the results obtained for some of the porous materials themselves. Not only are the spore materials (industrial carpet, treated wood, suspensions being absorbed, but so is the and unpainted concrete) were variable, i.e., sprayed ClO2 liquid. No evaluations have did not follow expected trends and in some been made to determine what happens to cases, were much higher than expected. For decontaminants or how the materials example, as more spray applications were themselves affect the fate (e.g., activity) of involved (e.g., from two total spray the decontaminants once applied and applications to four total spray applications, absorbed into the coupons. Nonporous but at the same contact time and materials (e.g., laminate) allow the spore concentration), a significantly lower efficacy suspensions to dry as droplets in the place (log reduction) would not be expected. where they were inoculated. These materials Similarly, as testing progressed from a lower also allow the liquid decontaminants to pool ClO2 concentration to a higher concentration and stay visibly wetted for the duration of (but with same contact time and number of the contact times. spray applications), a significantly lower efficacy (log reduction) would not be 7.1.2 Subjectivity of Spraying Method expected. But for some of these materials and tests, lower decontamination efficacies The ClO2 solution is sprayed using a were indeed observed. Several factors may commercially-available trigger sprayer as have contributed to this variability and are described in Chapter 2. Prior to spraying, the discussed below. We do note that some of sprayer was tipped so that it was horizontal these factors may have had minimal impact (i.e., facing the inoculated material coupons on efficacy for the materials already easily in the horizontal configuration) at a distance decontaminated at most or all of the test of 30.5 cm. When sprayed, the trigger was conditions (e.g., decorative laminate), or for pulled while simultaneously sweeping the materials that were minimally sprayer from side-to-side to cover the five decontaminated at all of the test conditions inoculated replicates of one material. The (soil materials). number of trigger pulls was counted to render the materials “fully wetted” for that 7.1.1 Test Material Composition spray application at that time point. The same procedure was followed for each The porous materials that exhibited highly subsequent material until all materials were variable results (industrial carpet, treated sprayed with the ClO2 solution for that time

point (e.g., time 0). This same procedure chlorite to ClO2 were measured or was repeated for additional time points (e.g., calculated. According to the manufacturer, +30 minutes). The subjectivity involved the pH of the ClO2 solution must be between with this method is apparent, but this 4 and 7. Also, the chlorite concentration method was the most reasonable method (ppm) must be within a minimum of 50% of when previous tasks that involved the target ClO2 concentration (e.g., if a specialized sprayers/applicators (depending 3,000 ppm ClO2 liquid is needed, the on the vendor) were taken into chlorite can be no less than 1,500 ppm). consideration. Any development or Every effort, however, was taken during implementation of an automated system was ClO2 liquid spray testing to bring the pH of precluded due to the number of variables the solution as close to 7 as possible because (e.g., nozzle type, pressure requirements, the measured pH was near 7, so, for number of decontaminant component consistency’s sake, every batch of ClO2 reservoirs) associated with each solution was adjusted to be pH 7. Of the 22 decontaminant formulation. times that the ClO2 solution was made for liquid spray testing, the average pH was Since a commercially-available trigger 6.73 ± 0.56, and the average chlorite to ClO2 sprayer was used to dispense and apply the ratio was 84.42 ± 13.42%. See Table 12-1 ClO2 liquid, there was no form of precise for a summary of the chemical control of where the sprayed liquid characteristics and their variability for each deposited. Due to space limitations batch of aqueous ClO2 used in testing. associated with working in a Class III BSC glove box, two material types were placed To eliminate the possibility that some of the per elevated grill. Despite efforts taken to variability in efficacy results for the wood, control the conical spray pattern from the concrete and carpet materials was due to trigger sprayer, a “shadowing effect” may batch-to-batch variability of the aqueous have resulted in over-spraying one material solutions, tests were conducted to see type that landed on the other material on the whether two total spray applications versus same elevated grill , possibly yielding one four total spray applications using the same material actually getting more sprayed ClO2 ClO2 liquid batch (either 3000 ppm or 4000 liquid than intended. ppm ClO2) and contact time would yield the expected results (i.e., higher efficacy with 7.1.3 Chemistry of ClO2 Solution increased number of sprays) on industrial carpet, treated wood, and unpainted concrete Batch-to-batch differences in the pH and inoculated with B. anthracis spores. With other chemical characteristics of the aqueous these additional tests, we would be better ClO2 solutions may also have been a factor able to determine whether increasing the in the variable decontamination efficacy number of spray applications improved results. When a ClO2 solution batch was decontamination efficacy. made, the pH, chlorite, ClO2, and ratio of

Table 7-1. Chlorine Dioxide Liquid Batch Chemical Comparison

Ratio of Test Condition Measured pH Titrated Chlorite Titrated ClO (ppm) [Chlorite] vs. [ClO ] (ClO Liquid Prep #) (4-7) 2 (ppm) 2 2 (50% minimum) 3000 ppm, 60', three apps 6.86 3102.7 3169.7 102% (1) (2) 6.97 3102.7 3203.4 103% (3) 6.57 3102.7 3540.6 114% (4) 6.82 3237.6 3270.8 101% Test Condition Average 6.81 3136.4 3296.1 105% Test Condition Standard 0.170 67.450 168.32 6.05% Deviation 3000 ppm, 60', four apps 6.92 3237.6 1719.7 53.1% (1) (2) 6.93 3237.6 2529.0 78.1% (3) 6.91 3170.2 2562.7 80.8% (4) 6.60 3102.7 2663.9 85.9% (5) 6.98 3237.6 2158.1 66.7% (6) 6.88 3102.7 2630.2 84.8% (7) 6.95 3102.7 2427.8 78.2% Test Condition Average 6.89 3170.2 2384.5 75.4% Test Condition Standard 0.130 67.450 338.16 11.7% Deviation 4000 ppm, 60', two apps (1) 6.90 4181.9 3473.2 83.0% (2) 4.35 4181.9 3102.2 74.2% (3) 6.84 4047.0 3237.1 80.0% (4) 6.77 4047.0 3169.7 78.3% (5) 6.96 4047.0 3102.2 76.7% Test Condition Average 6.36 4101.0 3216.9 78.4% Test Condition Standard 1.13 73.890 153.79 3.35% Deviation 4000 ppm, 60', four apps 6.30 3777.2 3608.0 95.5% (1) (2) 6.98 4115.5 3304.6 80.3% (3) 6.93 4181.9 3203.4 76.6% (4) 6.89 3912.1 3709.2 94.8% (5) 6.81 4047.0 3270.8 80.8% (6) 6.85 3912.1 3473.2 88.8% Test Condition Average 6.79 3991.0 3428.2 86.1% Test Condition Standard 0.250 150.48 201.94 8.05% Deviation Total AVG 6.73 N/A N/A 84.4%

Total STDEV 0.560 N/A N/A 13.4% N/A = Not applicable

7.2 Decontamination Efficacy A comparison of the “maximization” test For the 3,000 ppm ClO2 repeat test (actual results (refer to Chapter 6) and those that ClO2 liquid concentration was 3,238 ppm), were repeated is summarized in Table 7-5. the decontamination efficacies for all At the 3,000 ppm (four sprays) test materials improved when increasing the condition and the 4,000 ppm (two sprays) number of sprays from two to four, as test condition, the repeat test log reduction shown in Table 7-2 and summarized in results are lower than the initial test results Table 7-4. Although increasing the number reported in Chapter 6. At the 4,000 ppm of sprays did improve efficacy at this test (four sprays) test condition, the repeat test condition, the log reduction results were still results were slightly higher or equivalent. In very poor and were all below 2.27. general, the repeat test results are consistent with the other test results for each material An increase in decontamination efficacy (see Chapter 6). Thus the outlying results between the two spray applications versus for industrial carpet and unpainted concrete the four spray applications for 4,000 ppm may have been due to experimental error or ClO2 batch was seen only for industrial some uncontrolled parameter, possibly carpet, as shown in Table 7-3 and related to the ClO2 solution chemistry. summarized in Table 7-4. (Actual ClO2 liquid concentration measured at 4,182 ppm.) Regardless, the highest log reduction achieved in this repeat test condition was 2.31.

Table 7-2. Inactivation of Bacillus anthracis Spores—Two Applications vs. Four Applications 3,000 ppm ClO2 Liquid Sprayed on Materials (One Hour Contact Time) Inoculum Mean of Logs of Mean % Decontamination Test Material (CFU) Observed CFU Recovery Efficacy ± CIf Two Applications Industrial Carpeta Positive Controlsb 1.16 x 108 7.78 ± 0.04 52.5 ± 4.70 -g Test Couponsc 1.16 x 108 7.15 ± 0.20 13.2 ± 6.90 0.64 ± 0.18 Laboratory Blankd 0 0 - - Procedural Blanke 0 0 - - Treated Wood Positive Controls 1.16 x 108 7.08 ± 0.03 10.4 ± 0.66 - Test Coupons 1.16 x 108 5.58 ± 0.39 0.440 ± 0.35 1.50 ± 0.35 Laboratory Blank 0 0 - - Procedural Blank 0 0 - - Unpainted Concrete Positive Controls 1.16 x 108 7.62 ± 0.05 36.2 ± 4.10 - Test Coupons 1.16 x 108 6.45 ± 0.15 2.55 ± 0.71 1.17 ± 0.14 Laboratory Blank 0 0 - - Procedural Blank 0 0 - - Four Applications Industrial Carpet Positive Controls 1.16 x 108 7.75 ± 0.06 49.3 ± 6.50 - Test Coupons 1.16 x 108 5.78 ± 0.32 0.650 ± 0.47 1.97 ± 0.29 Laboratory Blank 0 0 - - Procedural Blank 0 0 - - Treated Wood Positive Controls 1.16 x 108 7.37 ± 0.24 22.0 ± 8.40 - Test Coupons 1.16 x 108 5.10 ± 0.09 0.110 ± 0.02 2.27 ± 0.22 Laboratory Blank 0 0 - - Procedural Blank 0 0 - - Unpainted Concrete Positive Controls 1.16 x 108 7.83 ± 0.06 58.7 ± 8.3 - Test Coupons 1.16 x 108 5.72 ± 0.32 0.55 ± 0.30 2.11 ± 0.29 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 coupons, the mean percent recovery on those five coupons, and decontamination efficacy (log reduction).b Positive Controls = inoculated, not decontaminated coupons (sprayed with SFW). c Test Coupons = inoculated, decontaminated coupons. d Laboratory Blank = not inoculated, not decontaminated coupon. e Procedural Blank = not inoculated, decontaminated coupon. 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.

Table 7-3. Inactivation of Bacillus anthracis Spores—Two Applications vs. Four Applications 4,000 ppm ClO2 Liquid Sprayed on Materials (One Hour Contact Time) Inoculum Mean of Logs of Mean % Decontamination Test Material (CFU) Observed CFU Recovery Efficacy ± CIf Two Applications Industrial Carpeta Positive Controlsb 1.19 x 108 8.07 ± 0.03 100 ± 7.30 -g Test Couponsc 1.19 x 108 6.95 ± 0.15 7.87± 2.50 1.12 ± 0.13 Laboratory Blankd 0 0 - - Procedural Blanke 0 0 - - Treated Wood Positive Controls 1.19 x 108 7.47 ± 0.31 29.6 ± 16.0 - Test Coupons 1.19 x 108 5.80 ± 0.48 0.780 ± 0.57 1.67 ± 0.50 Laboratory Blank 0 0 - - Procedural Blank 0 0 - - Unpainted Concrete Positive Controls 1.19 x 108 8.03 ± 0.15 93.8 ± 26.0 - Test Coupons 1.19 x 108 6.41 ± 0.16 2.29 ± 0.74 1.62 ± 0.19 Laboratory Blank 0 0 - - Procedural Blank 0 0 - - Four Applications Industrial Carpet Positive Controls 1.19 x 108 8.02 ± 0.04 88.6 ± 7.70 - Test Coupons 1.19 x 108 6.25 ± 0.43 2.04 ± 1.30 1.77 ± 0.38 Laboratory Blank 0 0 - - Procedural Blank 0 0 - - Treated Wood Positive Controls 1.19 x 108 7.33 ± 0.40 27.0 ± 31.0 - Test Coupons 1.19 x 108 5.02 ± 0.51 70.2 ± 0.15 2.31 ± 0.57 Laboratory Blank 0 0 - - Procedural Blank 0 0 - - Unpainted Concrete Positive Controls 1.19 x 108 7.91 ± 0.12 70.4 ± 22.0 - Test Coupons 1.19 x 108 6.14 ± 0.43 1.59 ± 1.20 1.77 ± 0.39 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 coupons, the mean percent recovery on those five coupons, and decontamination efficacy (log reduction).b Positive Controls = inoculated, not decontaminated coupons (sprayed with SFW). c Test Coupons = inoculated, decontaminated coupons. d Laboratory Blank = not inoculated, not decontaminated coupon. e Procedural Blank = not inoculated, decontaminated coupon. 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.

Table 7-4. Summary of Decontamination Efficacy Values for Two vs. Four Applications Efficacy for Efficacy for Test Material B. anthracis B. anthracis Two sprays Four sprays 3,000 ppm ClO2 Industrial Carpet 0.64 ± 0.18 1.97 ± 0.29 Treated Wood 1.50 ± 0.35 2.27 ± 0.22 Unpainted Concrete 1.17 ± 0.14 2.11 ± 0.29

4,000 ppm ClO2 Industrial Carpet 1.12 ± 0.13 1.77 ± 0.38 Treated Wood 1.67 ± 0.50 2.31 ± 0.57 Unpainted Concrete 1.62 ± 0.19 1.77 ± 0.39

All efficacy results reported in terms of mean log reduction ± 95% confidence interval

Table 7-5. Comparing Decontamination Efficacy Values for Tests that Were Repeated Efficacy for Efficacy for Test Condition B. anthracis B. anthracis Initial Test (Ch. 6) Repeat Test

3,000 ppm ClO2 – Four sprays, One hr Industrial Carpet 7.12 ± 1.60 1.97 ± 0.29 Treated Wood 2.85 ± 0.38 2.27 ± 0.22 Unpainted Concrete 5.42 ± 2.40 2.11 ± 0.29

4,000 ppm ClO2 – Two sprays, One hr Industrial Carpet 2.82 ± 0.52 1.12 ± 0.13 Treated Wood 1.95 ± 0.49 1.67 ± 0.50 Unpainted Concrete 6.60 ± 0.84 1.62 ± 0.19

4,000 ppm ClO2 – Four sprays, One hr Industrial Carpet 1.27 ± 0.09 1.77 ± 0.38 Treated Wood 1.21 ± 0.12 2.31 ± 0.57 Unpainted Concrete 2.61 ± 0.63 1.77 ± 0.39 All efficacy results reported in terms of mean log reduction ± 95% confidence interval

8.0 Summary of Results

A summary of the decontamination efficacy difficult to decontaminate materials. The results for the initial ClO2 scoping tests is results for the tests to increase shown in Table 8-1. Galvanized metal was decontamination efficacy with aqueous ClO2 effectively decontaminated (having a 6 log are summarized in Table 8-2. In these tests, reduction or greater) in three of the four B. the highest decontamination efficacies were anthracis tests. For the other test with B. observed on the nonporous decorative anthracis, a log reduction of nearly 6 (5.99) laminate; laminate was the only other was obtained. Complete inactivation of material to be completely decontaminated spores for both organisms on galvanized under some of the test conditions. The soil metal was observed only under the test materials (topsoil and AZTD) were the most condition utilizing 3,000 ppm ClO2, one difficult materials to decontaminate, with all hour contact time and three spray log reduction results for AZTD less than 1.5 applications. With this same and all log reduction results for topsoil less decontamination treatment on the additional than 0.31. Except for a few tests (including test materials, glass was the only other some outlying results; discussed below), all material completely decontaminated for both of the log reduction results for treated wood, B. anthracis and B. subtilis spores. The concrete, and industrial carpet were less than decorative laminate material was completely 3.0. decontaminated under this same test condition, but only with regard to B. Unexpectedly, the decontamination efficacy anthracis. Topsoil, treated wood, and results did not always improve with industrial carpet were all ineffectively increasing concentration, contact time, or decontaminated (i.e., having a log reduction number of spray applications. This effect < 6.0), with the highest log reduction was more pronounced on carpet, wood, and achieved on industrial carpet (4.08) with B. unpainted concrete. A few tests were subtilis. repeated (noted in Table 8-2 as “Repeat tests”) with these materials to assess the Based on these initial scoping results, glass effect of increasing the number of spray as well as galvanized metal was eliminated applications at a given ClO2 concentration from further testing, and in their place, while eliminating the possibility that AZTD and unpainted concrete were differences in the batch included in the test matrix. Further preparation/chemistry of the aqueous ClO2 “maximization” testing continued with solutions were affecting decontamination increasing ClO2 levels, number of spray efficacy (i.e., causing variability in results). applications, and contact times, to determine At the 3,000 ppm level, the decontamination the conditions necessary to maximize efficacies for all materials improved decontamination efficacy on the more significantly when increasing the number of

sprays from two to four. At the 4,000 ppm level, efficacy improved significantly only on industrial carpet when increasing the number of sprays. In general, the results for the tests that were repeated were more consistent with the other test results for each material. The outlying results for industrial carpet and unpainted concrete may therefore have been due to unknown experimental error or some uncontrolled parameter, with some possible association with the ClO2 solution chemistry.

The most robust liquid spray treatment of 4,000 ppm ClO2, four spray applications, and two hour contact time did offer improved decontamination efficacy compared to some of the other test conditions (all other variables being equal), but did not significantly improve efficacy compared to the 4,000 ppm ClO2, four spray applications, one hour contact time except for a few tests.

Comparing Log Reductions for B. anthracis and B. subtilis

Of the 39 liquid spray tests in which decontamination efficacies could be compared between B. anthracis and B. subtilis (see Tables 8-1 and 8-2), 21 of the test results were significantly different. For all of these 21 tests, B. subtilis was inactivated to a lesser degree.

Material Compatibility

No visible damage was observed on any test materials for any of the tests conducted.

Table 8-1. Summary of Liquid ClO2 Decontamination Efficacy for Initial Scoping Tests Efficacy for Efficacy for Test Material Test Condition B. anthracis B. subtilis

Galvanized Metal 1500 ppm, 1 hr, 4 apps 5.99 ± 0.99 0.76 ± 0.14b Galvanized Metal 2000 ppm, 1 hr, 4 apps 7.12 ± 1.50 1.91 ± 0.40b Galvanized Metal 3000 ppm, 1 hr, 2 apps 7.08 ± 0.82 7.13 ± 0.74 Galvanized Metal 3000 ppm, 1 hr, 3 apps ≥ 7.89 ± 0.03a ≥ 7.70 ± 0.07a

Topsoilc 3000 ppm, 1 hr, 3 apps 0.24 ± 0.08 0.09 ± 0.05b Glass 3000 ppm, 1 hr, 3 apps ≥ 7.86 ± 0.07a ≥ 7.88 ± 0.10a Treated Wood 3000 ppm, 1 hr, 3 apps 2.41 ± 0.17 1.43 ± 0.72b Industrial Carpet 3000 ppm, 1 hr, 3 apps 3.40 ± 0.73 4.08 ± 0.36 Decorative Laminate 3000 ppm, 1 hr, 3 apps ≥ 7.84 ± 0.04a 5.13 ± 1.50b

All efficacy results reported in terms of mean log reduction ± 95% confidence interval 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. aResult represents complete inactivation within the detection limit of 33.33 CFU/material. b Values in bold for B. subtilis by testing condition are significantly different from corresponding values for B. anthracis. c Tested with unsterilized soil.

Table 8-2. Summary of Liquid ClO2 Decontamination Efficacy for Maximization Tests 3000 ppm, 3000 ppm, 4000 ppm, 4000 ppm, 3000 ppm, 4000 ppm, one hr contact, one hr contact, one hr contact, one hr contact, two hr contact, two hr contact, Test two total spray four total spray two total spray four total spray four total spray four total spray Material applications applications applications applications applications applications B.a. B.a. B.s. B.a. B.s. B.a. B.s. B.a. B.s. B.a. B.s. Industrial 7.12 1.27 2.82 2.51 1.27 0.730 2.51 0.80 2.84 2.31 - Carpet (±1.60) (±0.16)b (±0.52) (±0.17) (±0.09) (±0.05)b (±0.20) (±0.10)b (±0.17) (±0.22)b Industrial 0.64 1.97 1.12 1.77 Carpet (±0.18) ------(±0.29) (±0.13) (±0.38) (Repeat) 2.85 0.67 1.95 2.55 1.21 0.93 2.06 0.97 2.64 2.00 Treated Wood - b b (±0.38) (±0.35) (±0.49) (±2.0) (±0.12) (±0.29) (±0.39) (±0.39) (±0.65) (±0.69) Treated Wood 1.50 2.27 1.67 2.31 ------(Repeat) (±0.35) (±0.22) (±0.50) (±0.57) Decorative 7.16 ≥ 7.82 ≥ 7.76 6.47 ≥ 7.90 ≥ 7.86 6.64 2.88 7.58 ≥ 7.98 - Laminate (±1.30) (±0.04)a (±0.06)a (±0.94)b (±0.02)a (±0.06)a (±1.0) (±0.75)b (±0.61) (±0.02)a Unpainted 5.42 5.39 6.60 3.64 2.61 1.52 2.22 1.00 2.50 1.05 - Concrete (±2.40) (±1.80) (±0.84) (±0.20)b (±0.63) (±0.36)b (±0.28) (±0.38)b (±1.1) (±0.36) Unpainted 1.17 2.11 1.62 1.77 Concrete (±0.14) ------(±0.29) (±0.19) (±0.39) (Repeat) 0.12 0.08 0.94 0.13 1.46 0.22 0.66 0.18 1.28 0.69 AZ Test Dust - b b b b (±0.08) (±0.06) (±0.11) (±0.06) (±0.08) (±0.14) (±0.24) (±0.08) (±0.03) (±0.22) 0.05 < 0.01 0.05 0.04 0.170 0.12 0.09 0.05 0.310 0.08 Top Soil - b (±0.070) (±0.06) (±0.01) (±0.05) (±0.05) (±0.10) (±0.05) (±0.04) (±0.05) (±0.03) All efficacy results reported in terms of mean log reduction ± 95% confidence interval. 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. a Result represents complete inactivation within the detection limit of 33.33 CFU/material. b Values in bold for B. subtilis (B.s.) by testing condition are significantly different from corresponding values for B. anthracis (B.a.). “-” Not tested.

9.0 References

1. Evaluation of Liquid and Foam Standard Methods for the Technologies for the Examination of Water and Waste Decontamination of B. anthracis and Water, 20th Edition; Clesceri, L.S; B. subtilis Spores on Building and Greenberg, A.E.; Eaton, A.D., Eds.; Outdoor Materials. US EPA Report American Public Health Association, 600/R-09/150, November 2009. American Water Works Association, 2. Associates of Cape Cod, Inc., Water Environment Federation: Limulus Amebocyte Lysate Baltimore, MD, 1995; 4-77 to 4-79. CHROMO-LAL Method, Part No. 4. Determining the Efficacy of Liquids PN001087, Rev000, East Falmouth, and Fumigants in Systematic Massachusetts, November 2007 Decontamination Studies for http://www.acciusa.com/pdfs/accPro Bacillus anthracis Using Multiple duct/pisheets/Chromo- Test Methods. US EPA Report LAL%20Insert%20English.pdf 600/R-10/088, December 2010. Accessed February 22, 2012. 3. 4500-ClO2 Chlorine Dioxide, E. Amperometric Method II. In

Appendix A: Spray Deposition and Neutralization Tests

Spray Deposition Results for Initial Scoping Tests (Refer to Chapter 5)

The ClO2 solution was applied from a distance of 30.5 cm (12 inches) to the horizontally- oriented galvanized metal until the materials were fully wetted. Re-application of the ClO2 was made on all coupon surfaces at 30 minutes after the initial application, when the coupon surfaces were tested with two spray applications; at 20 minutes after the initial application, when the coupon surfaces were tested with three spray applications; or at 15 minutes after the initial application, when the coupon surfaces were tested with four spray applications. At 60 minutes after the initial application, each coupon was placed in the 50 mL conical tube that also served to collect excess ClO2 runoff. The test coupons stayed in their horizontal orientation throughout the 60-minute contact time.

To assess ClO2 spray deposition, triplicate coupons of the galvanized metal were weighed prior to application of the ClO2 in the trial runs, and these values were recorded. The triplicate coupons were then sprayed with ClO2 until fully wetted in their horizontal orientations, re- application(s) were made at the appropriate time(s), and, after the 60 minute contact time, each coupon was weighed again. The pre-application weights were then subtracted from the post- application weights, and that difference was added to the weight of decontaminant runoff captured separately from each coupon. The average deposition/runoff weight for each testing condition is shown in Table A1-1.

Table A-1. Deposition/runoff Weight of ClO2 Liquid with 60 Minute Contact Time Average Deposition/Runoff Test Condition Weight (g)

3,000 ppm ClO2, 0.38 ± 0.10 two applications 3,000 ppm ClO2, 0.52 ± 0.066 three applications 1,500 ppm ClO2, 0.61 ± 0.13 four applications 2,000 ppm ClO2, 0.61 ± 0.13 four applications

Neutralization Methodology for Initial Scoping Tests (Refer to Chapter 5)

Neutralizations for the 3,000 ppm (actual concentrations measured at 2,833 ppm for two applications and 3,238 ppm for three applications), 1,500 ppm (actual concentration measured at 1,484 ppm), and 2,000 ppm (actual concentration measured at 2,023 ppm) ClO2 solutions for the

initial scoping tests were achieved with STS. The concentration range of STS used during the neutralization panels was 0.5, 1.0, 1.5, and 2.0% (for 3,000 ppm and two total spray applications only) in the extraction solution. These STS concentrations were based on historical data. The results of the neutralization panels are shown in Tables A-2 to A-8. From these trials, the following STS concentrations were determined to be sufficient for neutralization in the following scoping tests:

• 1.0% STS for Bacillus anthracis spores with 3,000 ppm ClO2 liquid, one hour contact, two total spray applications. The same STS concentration was used for B. subtilis for these conditions. Subsequent neutralization panels included B. subtilis.

• 1.0% STS for Bacillus anthracis spores with 3,000 ppm ClO2 liquid, one hour contact, three total spray applications.

• 0.5% STS for Bacillus subtilis spores with 3,000 ppm ClO2 liquid, one hour contact, three total spray applications.

• 1.0% STS for Bacillus anthracis spores with 3,000 ppm ClO2 liquid, one hour contact, three total spray applications.

• 0.5% STS for Bacillus anthracis spores with 1,500 ppm ClO2 liquid, one hour contact, four total spray applications.

• 1.0% STS for Bacillus subtilis spores with 1,500 ppm ClO2 liquid, one hour contact, four total spray applications.

• 1.0% STS for Bacillus anthracis and B. subtilis spores with 2,000 ppm ClO2 liquid, one hour contact, four total spray applications.

Table A-2. Neutralization Testing with Bacillus anthracis Spores with 3,000 ppm ClO2 Liquid, One Hour Contact, Two Total Spray Applications Total Inoculum % of Treatment Observed (CFU) Control (CFU) a 7 ClO2 + Spores 6.43 x 10 0 0 a,b 6.43 x 107 0 0 ClO2+ PBS + Triton X-100 + Spores PBS + Triton X-100 + Spores (Control)b 6.43 x 107 6.50 x 107 100 a,b 6.43 x 107 6.98 x 107 107 ClO2+ PBS + Triton X-100 + 0.5% STS + Spores

a,b 7 7 ClO2+ PBS + Triton X-100 + 1.0% STS + Spores 6.43 x 10 7.31 x 10 113

a,b 7 7 ClO2+ PBS + Triton X-100 + 1.5% STS + Spores 6.43 x 10 7.29 x 10 112

a,b 7 7 ClO2+ PBS + Triton X-100 + 2.0% STS + Spores 6.43 x 10 6.49 x 10 99.9

a ClO2 volume of 0.38 mL corresponds to mean gravimetric deposition on test materials and density of approximately 1.0 g/mL. b Volume of PBS (10 mL) includes 0.1% of Triton X-100 surfactant and indicated % of STS; total volume for all samples with ClO2 = 10.38 mL (10 mL PBS/Triton X-100/STS + 0.38 mL ClO2).

Table A-3. Neutralization Testing with Bacillus anthracis Spores with 3,000 ppm ClO2 Liquid, One Hour Contact, Three Total Spray Applications Total Inoculum % of Treatment Observed (CFU) Control (CFU) a 7 ClO2 + Spores 9.63 x 10 0 0

a,b 7 ClO2+ PBS + Triton X-100 + Spores 9.63 x 10 0 0 PBS + Triton X-100 + Spores (Control)b 9.63 x 107 9.05 x 107 100

a,b 7 7 ClO2+ PBS + Triton X-100 + 0.5% STS + Spores 9.63 x 10 8.66 x 10 95.8

a,b 9.63 x 107 8.89 x 107 98.3 ClO2+ PBS + Triton X-100 + 1.0% STS + Spores

a,b 9.63 x 107 8.77 x 107 96.9 ClO2+ PBS + Triton X-100 + 1.5% STS + Spores

a ClO2 volume of 0.52 mL corresponds to mean gravimetric deposition on test materials and density of approximately 1.0 g/mL. b Volume of PBS (10 mL ) includes 0.1% of Triton X-100 surfactant and indicated % of STS; total volume for all samples with ClO2 = 10.52 mL (10 mL PBS/Triton X-100/STS + 0.52 mL ClO2).

Table A-4. Neutralization Testing with Bacillus subtilis Spores with 3,000 ppm ClO2 Liquid, One Hour Contact, Three Total Spray Applications Total Inoculum % of Treatment Observed (CFU) Control (CFU) a 7 ClO2 + Spores 4.07 x 10 0 0 a,b 4.07 x 107 0 0 ClO2+ PBS + Triton X-100 + Spores PBS + Triton X-100 + Spores (Control)b 4.07 x 107 8.56 x 107 100 a,b 4.07 x 107 8.54 x 107 99.8 ClO2+ PBS + Triton X-100 + 0.5% STS + Spores

a,b 7 7 ClO2+ PBS + Triton X-100 + 1.0% STS + Spores 4.07 x 10 8.20 x 10 95.8

a,b 7 7 ClO2+ PBS + Triton X-100 + 1.5% STS + Spores 4.07 x 10 7.91 x 10 92.5 a ClO2 volume of 0.52 mL corresponds to mean gravimetric deposition on test materials and density of approximately 1.0 g/mL. b Volume of PBS (10 mL) includes 0.1% of Triton X-100 surfactant and indicated % of STS; total volume for all samples with ClO2 = 10.52 mL (10 mL PBS/Triton X-100/STS + 0.52 mL ClO2).

Table A-5. Neutralization Testing with Bacillus anthracis Spores with 1,500 ppm ClO2 Liquid, One Hour Contact, Four Total Spray Applications Total Inoculum % of Treatment Observed (CFU) Control (CFU) a 8 ClO2 + Spores 1.08 x 10 0 0 a,b 1.08 x 108 0 0 ClO2+ PBS + Triton X-100 + Spores PBS + Triton X-100 + Spores (Control)b 1.08 x 108 9.88 x 107 100 a,b 1.08 x 108 9.87 x 107 99.9 ClO2+ PBS + Triton X-100 + 0.5% STS + Spores

a,b 8 7 ClO2+ PBS + Triton X-100 + 1.0% STS + Spores 1.08 x 10 9.37 x 10 94.9

a,b 8 7 ClO2+ PBS + Triton X-100 + 1.5% STS + Spores 1.08 x 10 9.60 x 10 97.2

a ClO2 volume of 0.61 mL corresponds to mean gravimetric deposition on test materials and density of approximately 1.0 g/mL. b Volume of PBS (10 mL) includes 0.1% of Triton X-100 surfactant and indicated % of STS; total volume for all samples with ClO2 = 10.61 mL (10 mL PBS/Triton X-100/STS + 0.61 mL ClO2).

Table A-6. Neutralization Testing with Bacillus subtilis Spores with 1,500 ppm ClO2 Liquid, One Hour Contact, Four Total Spray Applications Total Inoculum % of Treatment Observed (CFU) Control (CFU) a 7 ClO2 + Spores 8.60 x 10 0 0

a,b 7 ClO2+ PBS + Triton X-100 + Spores 8.60 x 10 0 0 PBS + Triton X-100 + Spores (Control)b 8.60 x 107 7.15 x 107 100

a,b 7 7 ClO2+ PBS + Triton X-100 + 0.5% STS + Spores 8.60 x 10 7.73 x 10 108

a,b 7 7 ClO2+ PBS + Triton X-100 + 1.0% STS + Spores 8.60 x 10 8.48 x 10 119

a,b 8.60 x 107 8.17 x 107 114 ClO2+ PBS + Triton X-100 + 1.5% STS + Spores

Table A-7. Neutralization Testing with Bacillus anthracis Spores with 2,000 ppm ClO2 Liquid, One Hour Contact, Four Total Spray Applications Total Inoculum % of Treatment Observed (CFU) Control (CFU) a 8 ClO2 + Spores 1.16 x 10 0 0 a,b 1.16 x 108 0 0 ClO2+ PBS + Triton X-100 + Spores PBS + Triton X-100 + Spores (Control)b 1.16 x 108 1.05 x 108 100 a,b 1.16 x 108 1.07 x 108 102 ClO2+ PBS + Triton X-100 + 0.5% STS + Spores

a,b 8 8 ClO2+ PBS + Triton X-100 + 1.0% STS + Spores 1.16 x 10 1.14 x 10 108

a,b 8 8 ClO2+ PBS + Triton X-100 + 1.5% STS + Spores 1.16 x 10 1.02 x 10 97.5

Table A-8. Neutralization Testing with Bacillus subtilis Spores with 2,000 ppm ClO2 Liquid, One Hour Contact, Four Total Spray Applications Total Inoculum % of Treatment Observed (CFU) Control (CFU) a 8 ClO2 + Spores 1.10 x 10 0 0

a,b 8 ClO2+ PBS + Triton X-100 + Spores 1.10 x 10 0 0 PBS + Triton X-100 + Spores (Control)b 1.10 x 108 1.16 x 108 100

a,b 8 8 ClO2+ PBS + Triton X-100 + 0.5% STS + Spores 1.10 x 10 1.12 x 10 96.2

a,b 8 8 ClO2+ PBS + Triton X-100 + 1.0% STS + Spores 1.10 x 10 1.12 x 10 96.7

a,b 1.10 x 108 1.12 x 108 96.4 ClO2+ PBS + Triton X-100 + 1.5% STS + Spores

Liquid Spray Deposition Results for Efficacy Maximization Tests (Refer to Chapter 6)

The ClO2 solution was applied from a distance of 30.5 cm (12 inches) to the horizontally oriented test material coupons until they appeared fully wetted. The ClO2 solution was re-applied on all coupon surfaces at the following times for each corresponding testing condition:

• 3,000 ppm ClO2 liquid, one hour contact time, four total spray applications at time 0, +15, +30, and +45 minutes (contact time ends 15 minutes after the +45 minute application).

• 4,000 ppm ClO2 liquid, one hour contact time, two total spray applications at time 0 and +30 minutes (contact time ends 30 minutes after the +30 minute application).

• 4,000 ppm ClO2 liquid, one hour contact time, four total spray applications at time 0, +15, +30, and +45 minutes (contact time ends 15 minutes after the +45 minute application).

• 3,000 ppm ClO2 liquid, two hour contact time, four total spray applications at time 0, +30, +60, and +90 minutes (contact time ends 30 minutes after the +90 minute application).

• 4,000 ppm ClO2 liquid, two hour contact time, four total spray applications at time 0, +30, +60, and +90 minutes(contact time ends 30 minutes after the +90 minute application).

After the required total contact time, each coupon was placed in the 50 mL conical tube that also served to collect the pooled ClO2 solution. The test coupons stayed in their horizontal orientation throughout the respective contact times.

To assess ClO2 spray deposition, triplicate coupons of each test material were weighed prior to application of the ClO2 in the trial runs, and these values were recorded. Then the triplicate coupons were sprayed with ClO2 solution until fully wetted in their horizontal orientations, re- application(s) was made at the appropriate time(s), and, after the respective contact times (60 or 120 minutes), each coupon was weighed again. The pre-application weights were then subtracted from the post-application weights, and that difference was added to the weight of decontaminant runoff captured separately from each coupon. The average deposition/runoff weight of the ClO2 solution from each of the test materials is shown in Table A-9. The total averaged value (0.57 g) over all six materials was then used to estimate the amount of STS needed to neutralize the ClO2 effectively under this testing condition.

Table A-9. Deposition/runoff Weight of ClO2 Liquid Spray per Maximization Testing Condition and Material Average Deposition/Runoff Weight (g) 3000 ppm, 4000 ppm, 4000 ppm, 3000 ppm, 4000 ppm, one hr contact, one hr contact, one hr contact, two hr contact, two hr contact, four total spray two total spray four total spray four total spray four total spray Test Material applications applications applications applications applicationsa Industrial Carpet 0.92 ± 0.32 0.43 ± 0.83 0.56 ± 0.18 1.0 ± 0.17 1.0 ± 0.17 Treated Wood 0.46 ± 0.082 0.29 ± 0.062 0.56 ± 0.070 0.46 ± 0.076 0.46 ± 0.076 Decorative Laminate 0.77 ± 0.18 0.12 ± 0.035 0.21 ± 0.035 0.39 ± 0.042 0.39 ± 0.042 Unpainted Concrete 0.41 ± 0.089 0.28 ± 0.051 0.52 ± 0.14 0.68 ± 0.15 0.68 ± 0.15 Topsoil 0.19 ± 0.090 0.73 ± 0.18 0.85 ± 0.28 0.42 ± 0.13 0.42 ± 0.13 Arizona Test Dust 0.65 ± 0.067 0.43 ± 0.040 0.84 ± 0.28 0.53 ± 0.10 0.53 ± 0.10 Total Average 0.57 ± 0.26 0.38 ± 0.21 0.59 ± 0.24 0.58 ± 0.23 0.58 ± 0.23

aThe same deposition/runoff determined from 3,000 ppm, two hr contact time, four total spray applications used.

Neutralization Methodology for Scoping Tests (Refer to Chapter 6)

Neutralization for the 3,000 ppm and 4,000 ppm ClO2 solutions for the liquid spray maximization tests was achieved with STS. The concentration range of STS used during the neutralization panels was 0.5, 1.0, and 1.5% in the extraction solution. These STS concentration ranges were based on historical data. The results of the neutralization panels are shown in Tables A10 to A19. The results of these trials demonstrated that the following STS concentrations were sufficient for neutralization for the following maximization tests:

• 1.5% and 0.5% STS for B. anthracis and B. subtilis spores, respectively, with 3,000 ppm ClO2 liquid, one hour contact, four total spray applications. (Actual ClO2 liquid concentration measured at 3,238 ppm for both B. anthracis and B. subtilis testing.)

• 0.5% STS for both B. anthracis and B. subtilis spores with 4,000 ppm ClO2 liquid, one hour contact, two total spray applications. (Actual ClO2 liquid concentration measured at 4,182 ppm for both B. anthracis and B. subtilis testing.)

• 1.5% STS for both B. subtilis and B. subtilis spores with 4,000 ppm ClO2 liquid, one hour contact, four total spray applications. (Actual ClO2 liquid concentration measured at 4,182 ppm for both B. anthracis and B. subtilis testing.)

• 1.5% and 1.0% STS for B. anthracis and B. subtilis spores, respectively, with 3,000 ppm ClO2 liquid, two hour contact, four total spray applications. (Actual ClO2 liquid concentration measured at 3,238 ppm for both B. anthracis and B. subtilis testing.)

• 1.5% and 0.5% STS for B. anthracis and B. subtilis spores, respectively, with 4,000 ppm ClO2 liquid, two hour contact, four total spray applications. (Actual ClO2 liquid concentration measured at 4,114 ppm for both B. anthracis and B. subtilis testing.)

Table A-10. Neutralization Testing with Bacillus anthracis Spores with 3,000 ppm ClO2 Liquid, One Hour Contact Time, Four Total Spray Applications Total Inoculum % of Treatment Observed (CFU) Control (CFU) a 8 ClO2 + Spores 1.03 x 10 0 0 a,b 1.03 x 108 0 0 ClO2+ PBS + Triton X-100 + Spores PBS + Triton X-100 + Spores (Control)b 1.03 x 108 8.88 x 107 100 a,b 1.03 x 108 9.36 x 107 105 ClO2+ PBS + Triton X-100 + 0.5% STS + Spores

a,b 8 7 ClO2+ PBS + Triton X-100 + 1.0% STS + Spores 1.03 x 10 9.13 x 10 103

a,b 8 7 ClO2+ PBS + Triton X-100 + 1.5% STS + Spores 1.03 x 10 8.93 x 10 101 a ClO2 volume of 0.57 mL corresponds to mean gravimetric deposition on test materials and density of approximately 1.0 g/mL. b Volume of PBS (10 mL) includes 0.1% of Triton X-100 surfactant and indicated % of STS; total volume for all samples with ClO2 = 10.57 mL (10 mL PBS/Triton X-100/STS + 0.57 mL ClO2).

Table A-11. Neutralization Testing with Bacillus subtilis Spores with 3,000 ppm ClO2 Liquid, One Hour Contact Time, Four Total Spray Applications Total Inoculum % of Treatment Observed (CFU) Control (CFU) a 8 ClO2 + Spores 1.11 x 10 0 0 a,b 1.11 x 108 0 0 ClO2+ PBS + Triton X-100 + Spores PBS + Triton X-100 + Spores (Control)b 1.11 x 108 7.78 x 107 100

a,b 8 7 ClO2+ PBS + Triton X-100 + 0.5% STS + Spores 1.11 x 10 7.68 x 10 98.7

a,b 8 7 ClO2+ PBS + Triton X-100 + 1.0% STS + Spores 1.11 x 10 7.40 x 10 95.2

a,b 8 7 ClO2+ PBS + Triton X-100 + 1.5% STS + Spores 1.11 x 10 8.07 x 10 104 a ClO2 volume of 0.57 mL corresponds to mean gravimetric deposition on test materials and density of approximately 1.0 g/mL. b Volume of PBS (10 mL) includes 0.1% of Triton X-100 surfactant and indicated % of STS; total volume for all samples with ClO2 = 10.57 mL (10 mL PBS/Triton X-100/STS + 0.57 mL ClO2).

Table A-12. Neutralization Testing with Bacillus anthracis Spores with 4,000 ppm ClO2 Liquid, One Hour Contact Time, Two Total Spray Applications Total Inoculum % of Treatment Observed (CFU) Control (CFU) a 8 ClO2 + Spores 1.03 x 10 0 0 a,b 1.03 x 108 0 0 ClO2+ PBS + Triton X-100 + Spores PBS + Triton X-100 + Spores (Control)b 1.03 x 108 1.04 x 108 100 a,b 1.03 x 108 1.15 x 108 110 ClO2+ PBS + Triton X-100 + 0.5% STS + Spores

a,b 8 8 ClO2+ PBS + Triton X-100 + 1.0% STS + Spores 1.03 x 10 1.09 x 10 105

a,b 8 7 ClO2+ PBS + Triton X-100 + 1.5% STS + Spores 1.03 x 10 9.87 x 10 95.1 a ClO2 volume of 0.38 mL corresponds to mean gravimetric deposition on test materials and density of approximately 1.0 g/mL. b Volume of PBS (10 mL) includes 0.1% of Triton X-100 surfactant and indicated % of STS; total volume for all samples with ClO2 = 10.38 mL (10 mL PBS/Triton X-100/STS + 0.38 mL ClO2).

Table A-13. Neutralization Testing with Bacillus subtilis Spores with 4,000 ppm ClO2 Liquid, One Hour Contact Time, Two Total Spray Applications Total Inoculum % of Treatment Observed (CFU) Control (CFU) a 7 ClO2 + Spores 9.43 x 10 0 0 a,b 9.43 x 107 0 0 ClO2+ PBS + Triton X-100 + Spores PBS + Triton X-100 + Spores (Control)b 9.43 x 107 1.05 x 108 100

a,b 7 7 ClO2+ PBS + Triton X-100 + 0.5% STS + Spores 9.43 x 10 9.41 x 10 90.0

a,b 7 7 ClO2+ PBS + Triton X-100 + 1.0% STS + Spores 9.43 x 10 9.13 x 10 87.4

a,b 7 7 ClO2+ PBS + Triton X-100 + 1.5% STS + Spores 9.43 x 10 9.39 x 10 89.8 a ClO2 volume of 0.38 mL corresponds to mean gravimetric deposition on test materials and density of approximately 1.0 g/mL. b Volume of PBS (10 mL) includes 0.1% of Triton X-100 surfactant and indicated % of STS; total volume for all samples with ClO2 = 10.38 mL (10 mL PBS/Triton X-100/STS + 0.38 mL ClO2).

Table A-14. Neutralization Testing with Bacillus anthracis Spores with 4,000 ppm ClO2 Liquid, One Hour Contact Time, Four Total Spray Applications Total Inoculum % of Treatment Observed (CFU) Control (CFU) a 8 ClO2 + Spores 1.32 x 10 0 0 a,b 1.32 x 108 0 0 ClO2+ PBS + Triton X-100 + Spores PBS + Triton X-100 + Spores (Control)b 1.32 x 108 1.21 x 108 100 a,b 1.32 x 108 1.13 x 108 93.1 ClO2+ PBS + Triton X-100 + 0.5% STS + Spores

a,b 8 8 ClO2+ PBS + Triton X-100 + 1.0% STS + Spores 1.32 x 10 1.23 x 10 102

a,b 8 8 ClO2+ PBS + Triton X-100 + 1.5% STS + Spores 1.32 x 10 1.25 x 10 103 a ClO2 volume of 0.59 mL corresponds to mean gravimetric deposition on test materials and density of approximately 1.0 g/mL. b Volume of PBS (10 mL) includes 0.1% of Triton X-100 surfactant and indicated % of STS; total volume for all samples with ClO2 = 10.59 mL (10 mL PBS/Triton X-100/STS + 0.59 mL ClO2).

Table A-15. Neutralization Testing with Bacillus subtilis Spores with 4,000 ppm ClO2 Liquid, One Hour Contact Time, Four Total Spray Applications Total Inoculum % of Treatment Observed (CFU) Control (CFU) a 8 ClO2 + Spores 1.25 x 10 0 0 a,b 1.25 x 108 0 0 ClO2+ PBS + Triton X-100 + Spores PBS + Triton X-100 + Spores (Control)b 1.25 x 108 1.19 x 108 100

a,b 8 8 ClO2+ PBS + Triton X-100 + 0.5% STS + Spores 1.25 x 10 1.15 x 10 96.7

a,b 8 8 ClO2+ PBS + Triton X-100 + 1.0% STS + Spores 1.25 x 10 1.05 x 10 88.3

a,b 8 8 ClO2+ PBS + Triton X-100 + 1.5% STS + Spores 1.25 x 10 1.18 x 10 98.5 a ClO2 volume of 0.59 mL corresponds to mean gravimetric deposition on test materials and density of approximately 1.0 g/mL. b Volume of PBS (10 mL) includes 0.1% of Triton X-100 surfactant and indicated % of STS; total volume for all samples with ClO2 = 10.59 mL (10 mL PBS/Triton X-100/STS + 0.59 mL ClO2).

Table A-16. Neutralization Testing with Bacillus anthracis Spores with 3,000 ppm ClO2 Liquid, Two Hour Contact Time, Four Total Spray Applications Total Inoculum % of Treatment Observed (CFU) Control (CFU) a 8 ClO2 + Spores 1.54 x 10 0 0 a,b 1.54 x 108 0 0 ClO2+ PBS + Triton X-100 + Spores PBS + Triton X-100 + Spores (Control)b 1.54 x 108 1.16 x 108 100 a,b 1.54 x 108 1.10 x 108 95.1 ClO2+ PBS + Triton X-100 + 0.5% STS + Spores

a,b 8 8 ClO2+ PBS + Triton X-100 + 1.0% STS + Spores 1.54 x 10 1.14 x 10 98.6

a,b 8 8 ClO2+ PBS + Triton X-100 + 1.5% STS + Spores 1.54 x 10 1.21 x 10 105 a ClO2 volume of 0.59 mL corresponds to mean gravimetric deposition on test materials and density of approximately 1.0 g/mL. b Volume of PBS (10 mL) includes 0.1% of Triton X-100 surfactant and indicated % of STS; total volume for all samples with ClO2 = 10.59 mL (10 mL PBS/Triton X-100/STS + 0.59 mL ClO2).

Table A-17. Neutralization Testing with Bacillus subtilis Spores with 3,000 ppm ClO2 Liquid, Two Hour Contact Time, Four Total Spray Applications Total Inoculum % of Treatment Observed (CFU) Control (CFU) a 8 ClO2 + Spores 1.55 x 10 0 0 a,b 1.55 x 108 0 0 ClO2+ PBS + Triton X-100 + Spores PBS + Triton X-100 + Spores (Control)b 1.55 x 108 1.16 x 108 100

a,b 8 8 ClO2+ PBS + Triton X-100 + 0.5% STS + Spores 1.55 x 10 1.12 x 10 96.8

a,b 8 8 ClO2+ PBS + Triton X-100 + 1.0% STS + Spores 1.55 x 10 1.19 x 10 103

a,b 8 8 ClO2+ PBS + Triton X-100 + 1.5% STS + Spores 1.55 x 10 1.15 x 10 99.5 a ClO2 volume of 0.59 mL corresponds to mean gravimetric deposition on test materials and density of approximately 1.0 g/mL. b Volume of PBS (10 mL) includes 0.1% of Triton X-100 surfactant and indicated % of STS; total volume for all samples with ClO2 = 10.59 mL (10 mL PBS/Triton X-100/STS + 0.59 mL ClO2).

Table A-18. Neutralization Testing with Bacillus anthracis Spores with 4,000 ppm ClO2 Liquid, Two Hour Contact Time, Four Total Spray Applications Total Inoculum % of Treatment Observed (CFU) Control (CFU) a 8 ClO2 + Spores 1.50 x 10 0 0 ab 1.50 x 108 0 0 ClO2+ PBS + Triton X-100 + Spores PBS + Triton X-100 + Spores (Control)b 1.50 x 108 1.12 x 108 100 ab 1.50 x 108 1.09 x 108 97.1 ClO2+ PBS + Triton X-100 + 0.5% STS + Spores

ab 8 8 ClO2+ PBS + Triton X-100 + 1.0% STS + Spores 1.50 x 10 1.09 x 10 97.1

ab 8 8 ClO2+ PBS + Triton X-100 + 1.5% STS + Spores 1.50 x 10 1.16 x 10 103 a ClO2 volume of 0.59 mL corresponds to mean gravimetric deposition on test materials and density of approximately 1.0 g/mL. b Volume of PBS (10 mL) includes 0.1% of Triton X-100 surfactant and indicated % of STS; total volume for all samples with ClO2 = 10.59 mL (10 mL PBS/Triton X-100/STS + 0.59 mL ClO2).

Table A-19. Neutralization Testing with Bacillus subtilis Spores with 4,000 ppm ClO2 Liquid, Two Hour Contact Time, Four Total Spray Applications Total Inoculum % of Treatment Observed (CFU) Control (CFU) a 8 ClO2 + Spores 1.56 x 10 0 0 a,b 1.56 x 108 0 0 ClO2+ PBS + Triton X-100 + Spores PBS + Triton X-100 + Spores (Control)b 1.56 x 108 1.25 x 108 100

a,b 8 8 ClO2+ PBS + Triton X-100 + 0.5% STS + Spores 1.56 x 10 1.30 x 10 104

a,b 8 8 ClO2+ PBS + Triton X-100 + 1.0% STS + Spores 1.56 x 10 1.26 x 10 101

a,b 8 8 ClO2+ PBS + Triton X-100 + 1.5% STS + Spores 1.56 x 10 1.23 x 10 98.6 a ClO2 volume of 0.59 mL corresponds to mean gravimetric deposition on test materials and density of approximately 1.0 g/mL. b Volume of PBS (10 mL) includes 0.1% of Triton X-100 surfactant and indicated % of STS; total volume for all samples with ClO2 = 10.59 mL (10 mL PBS/Triton X-100/STS + 0.59 mL ClO2).

Appendix B: Effect of Pre-Sterilization of Soil Materials on the Decontamination Efficacy for Aqueous ClO2 Spray

We planned initially to test the topsoil and AZTD in an unsterilized state, to maintain the physical and chemical integrity of the soil materials. However, due to concerns that endogenous flora in the unsterilized topsoil and AZTD could affect our test results, we decided to sterilize the soil materials using gamma irradiation. We did find that sterilizing these soils via gamma irradiation did not change their physical appearance or integrity from an observational standpoint (e.g., no change in color).

To examine the effect that sterilizing the soil materials would have on decontamination efficacy, the following tests were conducted using sterilized and unsterilized topsoil and AZTD materials:

• 3,000 ppm ClO2 liquid spray, 60 minute contact time, four total spray applications, B. anthracis and B. subtilis. (Actual ClO2 liquid concentration measured at 3,103 ppm for both B. anthracis and B. subtilis testing using unsterilized soils. Actual ClO2 liquid concentration measured at 3,238 ppm for both B. anthracis and B. subtilis testing using sterilized soils.)

• 4,000 ppm ClO2 liquid spray, 60 minute contact time, two total spray applications, B. anthracis and B. subtilis. (Actual ClO2 liquid concentration measured at 4,047 ppm for both B. anthracis and B. subtilis testing using unsterilized soils. Actual ClO2 liquid concentration measured at 4,181 ppm for both B. anthracis and B. subtilis testing using sterilized soils.)

The results between the sterilized and unsterilized soil are compared in Table B-1. Although sterilization of soil materials does make a significant difference in the majority of results (based on whether the 95% confidence intervals for the log reduction results overlapped), the decontamination efficacy for these materials remains dismal. Only one out of the 16 tests showed an average log reduction greater than 1.0.

Table B-1. Comparison of Decontamination Efficacy with 95% Confidence Intervals using Unsterile vs. Sterile Soils for ClO2 Liquid Spray Testing

Testing Condition, B. anthracis Efficacy B. subtilis Efficacy Soil Type (Log Reduction ± CI) (Log Reduction ± CI)

3,000 ppm ClO2, one hr, four total spray applications Sterilized AZTD 0.120 ± 0.080a 0.0800 ± 0.060b Non-Sterile AZTD 1.22 ± 0.040a 0.680 ± 0.11b Sterilized Topsoil 0.0500 ± 0.070 < 0.0100c Non-Sterile Topsoil 0.160 ± 0.17 0.150 ± 0.13c

4,000 ppm ClO2, one hr, two total spray applications Sterilized AZTD 0.940 ± 0.11a 0.130 ± 0.060b Non-Sterile AZTD 0.300 ± 0.11a 0.960 ± 0.21b Sterilized Topsoil 0.0500 ± 0.010 0.0400 ± 0.050 Non-Sterile Topsoil 0.0800 ± 0.15 0.130 ± 0.10 aResults significantly different for B. anthracis between sterilized and unsterilized AZTD. bResults significantly different for B. subtilis between sterilized and unsterilized AZTD. cResults significantly different for B. subtilis between sterilized and unsterilized Topsoil

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