United States

Environmental Protection

Agency

Sabre Technical Services Chlorine Dioxide

Gas Generator

Office of Research and Development

National Homeland Security

Research Center

test/qa plan for

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EPA/Battelle Approval of 

Test/QA Plan 

For 

EVALUATION OF SPORICIDAL 

DECONTAMINATION TECHNOLOGIES 

February 2005 

(SIGNATURES ON FILE) 

Original signed by: Original signed by: 

John C.S. Chang 04/04/05 Eletha Brady-Roberts 04/01/05 

John C.S. Chang, Ph.D. Date Eletha Brady-Roberts Date 

Task Order Project Officer Quality Manager 

U.S. EPA U.S. EPA 

Original signed by: Original signed by: 

Thomas Kelly for 03/30/05 Zachary Willenberg 03/30/05 

Karen Riggs Date Zachary Willenberg Date 

Program Manager Quality Manager 

Battelle Battelle 
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Test/QA Plan 

for 

EVALUATION OF SPORICIDAL 

DECONTAMINATION TECHNOLOGIES 

 

February 2005 

(SIGNATURES ON FILE) 

Name David W. Skodack Signature David W. Skodack 

Company Sabre Technical Services L.L.C. 

Date 03/29/05 
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TABLE OF CONTENTS 

 Page 

List of Acronyms ................................................................................................................... v 

Distribution List..................................................................................................................... vi 

A PROJECT MANAGEMENT..................................................................................... 1 

A1 Technology Evaluation Organization ........................................................................ 1 

 A1.1 Battelle ........................................................................................................... 2 

 A1.2 Vendors.......................................................................................................... 5 

 A1.3 EPA................................................................................................................ 5 

 A1.4 Test Facility ................................................................................................... 6 

A2 Problem Definition/Background................................................................................ 7 

A3 Technology Evaluation Description and Schedule.................................................... 8 

 A3.1 Applicability .................................................................................................. 8 

 A3.2 Scope.............................................................................................................. 9 

 A3.3 Schedule......................................................................................................... 10 

A4 Quality Objectives ..................................................................................................... 10 

A5 Special Training/Certification.................................................................................... 12 

 A5.1 General Site Description................................................................................ 12 

 A5.2 Site Operations............................................................................................... 13 

 A5.3 Training.......................................................................................................... 14 

A6 Documentation and Records...................................................................................... 14 

B MEASUREMENT AND DATA AQUISITION ....................................................... 16 

B1 Experimental Design.................................................................................................. 16 

 B1.1 General Test Design....................................................................................... 16 

 B1.2 Scale of Testing and Testing Apparatus ........................................................ 17 

 B1.3 Test Surfaces.................................................................................................. 18 

 B1.4 Biological Agents and Surrogates.................................................................. 19 

 B1.5 Temperature and Relative Humidity Conditions ........................................... 20 

 B1.6 Surface Damage............................................................................................. 20 

B2 Methods Requirements and Procedures..................................................................... 21 

 B2.1 Agents ............................................................................................................ 21 

 B2.2 Coupon-Scale Testing.................................................................................... 21 

 B2.2.1 Preparation of Test Materials.......................................................... 21 

 B2.2.2 Application of Biological Agents to Test Coupons ........................ 21 

 B2.2.3 Confirmation of Surface Application Density ................................ 22 

 B2.2.4 Application of the Decontamination Technology, Monitoring of 

 Test Procedures............................................................................... 22 

 B2.2.5 Determination of Decontamination Efficacy.................................. 22 

 B2.2.6 Observation of Surface Damage ..................................................... 24 

B3 Quality Control Requirements ................................................................................... 25 
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B4 Instrument Calibration and Frequency....................................................................... 25 

B5 Data Acquisition Requirements ................................................................................. 25 

B6 Data Management ...................................................................................................... 26 

 B6.1 Efficacy Calculations..................................................................................... 26 

 B6.2 Statistical Analysis......................................................................................... 26 

C ASSESSMENT/OVESIGHT..................................................................................... 28 

C1 Assessments and Response Actions........................................................................... 28 

 C1.1 Technical Systems Audit ............................................................................... 28 

 C1.2 Performance Evaluation Audits ..................................................................... 28 

C2 Reports to Management ............................................................................................. 28 

D DATA VALIDATION AND USABILITY............................................................... 30 

 

E REFERENCES .......................................................................................................... 31 

APPENDIX 

Detailed Descriptions of Decontamination Technologies and Operating Parameters for Efficacy 

Evaluation 

LIST OF FIGURES 

Figure 1. Organization Chart for the Sporicidal Decontamination Technology 

Evaluation ...................................................................................................... 1 

Figure 2. Compact Glove Box for the Sporicidal Decontamination Technology 

Evaluation ...................................................................................................... 18 
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LIST OF ACRONYMS 

ANOVA two-way analysis of variance 

ATCC American Type Culture Collection 

BSC biosafety cabinet 

BSL biosafety level 

C Celsius 

CDC Centers for Disease Control and Prevention 

CFR Code of Federal Regulations 

CFU colony forming unit 

ClO2 chlorine dioxide 

cm centimeter 

d deep 

EPA U.S. Environmental Protection Agency 

FDA Food and Drug Administration 

h high 

ISO International Organization for Standardization 

L liter 

mL milliliter 

μL microliter 

MREF Medical Research and Evaluation Facility 

NHSRC National Homeland Security Research Center 

ppm parts per million 

QA quality assurance 

QC quality control 

QMP Quality Management Plan 

rpm revolutions per minute 

SD standard deviation 

SOP standard operating procedure 

TOPO Task Order Project Officer 

TSA technical systems audit 

TTEP Technology Testing and Evaluation Program 

UV ultraviolet 

w wide 
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DISTRIBUTION LIST 

Ms. Karen Riggs 

Mr. Zachary Willenberg 

Dr. James Rogers 

Battelle 

505 King Avenue 

Columbus, OH 43201-2693 

Dr. Michael Taylor 

Dr. Harry Stone 

Battelle 

10300 Alliance Rd, Ste 155 

Cincinnati, OH 45242 

Mr. Eric Koglin 

USEPA National Homeland Security 

 Research Center 

944 East Harmon Avenue 

Las Vegas, NV 89119 

Ms. Eletha Brady-Roberts 

USEPA National Homeland Security 

 Research Center 

26 W. Martin Luther King Drive 

Cincinnati, OH 45268 

Dr. John C.S. Chang 

U.S. Environmental Protection Agency 

U.S. EPA Mailroom, E305-03 

Research Triangle Park, NC 27711 

Technology Vendor(s) 

Mr. John Mason 

Sabre Technical Services 

17 Computer Drive East 

Albany, NY 12205
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A PROJECT MANAGMENT

A1 TECHNOLOGY EVALUATION ORGANIZATION 

 The technology evaluation will be performed by Battelle under the direction of the U.S. 

Environmental Protection Agency’s (EPA) National Homeland Security Research Center 

(NHSRC) through the Technology Testing and Evaluation Program (TTEP). This test/quality 

assurance (QA) plan is based on a previously approved test/QA plan per the directive of the Task 

Order Project Officer (TOPO). The organization chart in Figure 1 shows the individuals from 

Battelle, the vendor, and EPA who will have responsibilities in the technology evaluation. The 

responsibilities of these organizations and individuals are summarized in the following 

subsections. 

Figure 1. Organization Chart for the Sporicidal Decontamination Technology Evaluation 

John Chang 

EPA Task Order 

Project Officer 

Karen Riggs 

Battelle TTEP 

Manager 

Michael Taylor 

Building Decontamination 

Technology Area Leader/ 

Task Order Leader 

Zachary Willenberg 

Battelle Quality 

Assurance Manager 

Battelle Management 

Eletha Brady-Roberts 

NHSRC Quality 

Assurance Manager 

Eric Koglin 

EPA/NHSRC 

Project Officer 

Elisha Morrison 

Biological Testing 

QA Coordinator 

Decontamination 

Technology 

Vendor 

Representatives 

James Rogers 

Laboratory Test Coordinator 

Medical Research and 

Evaluation Facility (MREF) 
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A1.1 Battelle 

 Dr. Michael Taylor is Battelle’s Building Decontamination Technology Area Leader and 

Task Order Leader for this technology evaluation. He will have overall responsibility for 

ensuring that the technical, schedule, and cost goals established for testing and evaluation are 

met, and that the procedures employed for testing are consistent with TTEP guidelines. Dr. 

Taylor will serve as the primary interface for the TOPO. Dr. Taylor’s responsibilities are to: 

• Ensure that TTEP procedures are being followed. 

• Select the appropriate laboratory or location for the evaluation. 

• Prepare the draft test/QA plan and evaluation reports. 

• Establish a test schedule. 

• Revise this test/QA plan and evaluation reports in response to reviewers’ comments. 

• Keep the Battelle Program Manager informed of the progress and difficulties in 

planning and conducting the evaluation. 

• Coordinate with the Battelle Quality Assurance Manager for the performance of 

technical and performance audits as required by Battelle or EPA Quality Management 

staff. 

• Have overall responsibility for ensuring that this test/QA plan is followed. 

• Respond to any issues raised in assessment reports and audits, including instituting 

corrective action as necessary. 

• Establish a budget and schedule for the technology evaluation and direct the effort to 

ensure that budget and schedule are met. 

• Coordinate distribution of final test/QA plan and evaluation reports. 

 Ms. Karen Riggs is Battelle’s TTEP Manager. As such, Ms. Riggs will: 

• Maintain communication with EPA’s NHSRC Project Officer on all aspects of the 

program. 

• Monitor adherence to budgets and schedules in this work. 

• Provide the TOPO with monthly technical and financial progress reports. 
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• Review the draft test/QA plan. 

• Review the draft evaluation reports. 

• Ensure that necessary Battelle resources, including staff and facilities, are committed 

to the technology evaluation. 

• Ensure that vendor confidentiality is maintained. 

• Support Dr. Taylor in responding to any issues raised in assessment reports and 

audits. 

 Mr. Zachary Willenberg is Battelle’s Quality Assurance Manager for TTEP. As such, 

Mr. Willenberg will: 

• Review the draft test/QA plan. 

• Maintain communication with EPA Quality Management staff for this program. 

• Conduct a technical systems audit (TSA) at least once during the technology 

evaluation. 

• Audit at least 10% of the evaluation data. 

• Prepare and distribute an assessment report for each audit. 

• Verify implementation of any necessary corrective action. 

• Notify Battelle’s TTEP Manager to issue a stop work order if internal audits indicate 

that data quality is being compromised. Notify the Task Order Leader if such an 

order is issued. 

• Provide a summary of the QA/quality control (QC) activities and results for the 

evaluation reports. 

• Review the draft evaluation reports. 

• Ensure that all quality procedures specified in this test/QA plan and in the Quality 

Management Plan(1) (QMP) are followed. 

 Ms. Elisha Morrison will serve as Battelle’s Biological Testing QA Coordinator and 

assist Mr. Willenberg as necessary. 
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 Dr. James Rogers is Battelle’s Laboratory Test Coordinator for this evaluation. His 

responsibilities are to: 

• Coordinate with vendor representatives to facilitate the performance of the 

evaluation.

• Assist in preparation of the draft test/QA plan. 

• Arrange for use of the test facility and establishment of evaluation schedules.

• Arrange for the availability of qualified staff to conduct the evaluation.

• Assure that the evaluation is conducted in accordance with this test/QA plan.

• Provide input into revision of this test/QA plan, evaluation report, and evaluation 

statement in response to reviewers’ comments.

• Update the Battelle TTEP Manager and Task Order Leader on progress and 

difficulties in planning and conducting the evaluation.

• Coordinate with the Battelle Quality Assurance Manager for the performance of 

technical and performance audits as required by Battelle or EPA Quality Management 

staff.

 A Medical Research and Evaluation Facility (MREF) Laboratory Facilities Coordinator 

will review and approve data and records related to facility operation. This Facilities 

Coordinator will: 

• Review and approve all data and records related to facility operation.

• Provide input on facility procedures for the evaluation report. 

• Provide requisite technical staff during the technology evaluation. 

• Provide any safety training needed by Battelle, vendor, or EPA staff. 
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 Battelle technical staff will support Dr. Taylor in planning and conducting the technology 

evaluation. These staff will: 

• Ensure that the facility is fully functional prior to the times/dates needed in the 

technology evaluation. 

• Adhere to the requirements of the test/QA plan and the program WMP in carrying out 

the technology evaluation. 

• Support Dr. Taylor in responding to any issues raised in assessment reports and audits 

related to facility operation. 

A1.2 Vendors 

 Vendors of the sporicidal decontamination technologies will: 

• Provide input for preparation of the draft test/QA plan. 

• Review this test/QA plan and approve the current version prior to the evaluation of 

their technology. 

• Sign a Vendor Agreement specifying the respective responsibilities of the vendor and 

of Battelle in the evaluation. 

• Provide information on the quantitative response of their sporicidal decontamination 

technology to aid in the planning of the evaluation. 

• Provide the necessary equipment used for their sporicidal decontamination 

technology for use in the technology evaluation. 

• Train Battelle and/or test facility staff in the operation of their sporicidal 

decontamination technology. 

• Provide support, if needed, in use of their sporicidal decontamination technology 

during testing. 

• Review their respective draft evaluation report. 

A1.3 EPA 

 Mr. Eric Koglin is the EPA/NHSRC Project Officer for the EPA contract with Battelle, 

“Testing and Evaluation of Homeland Security-Related Technologies for the Measurement, 
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Sampling, Removal, and Decontamination of Chemical and Biological Agents” under which 

TTEP has been established. 

 Dr. John Chang is the EPA TOPO for Task Order 1113. As such, Dr. Chang will: 

• Have overall responsibility for directing the evaluation process. 

• Review the draft test/QA plan. 

• Approve the final test/QA plan and any subsequent versions. 

• Review the draft evaluation reports. 

• Oversee the EPA review process on the draft test/QA plan and evaluation reports. 

• Coordinate submission of evaluation reports for final EPA approval. 

 Ms. Eletha Brady-Roberts is the NHSRC Quality Assurance Manager for the TTEP. As 

such, Ms. Brady-Roberts will: 

• Review the draft test/QA plan and any subsequent versions. 

• Perform, at her option, one external TSA during the technology evaluation. 

• Notify the EPA TOPO to issue a stop work order if an external audit indicates that 

data quality is being compromised. 

• Prepare and distribute an assessment report summarizing the results of the external 

audit, if one is performed. 

• Review the draft evaluation reports. 

A1.4 Test Facility 

 The location for the technology evaluation described here will be Battelle’s laboratories 

in Columbus and West Jefferson, Ohio. The Columbus facilities to be used are chemical 

laboratories equipped for safe handling of a wide variety of chemicals. The MREF, located in 

West Jefferson, has chemical and biological surety agent laboratories certified for use of 

chemical and biological warfare agents. Other test facilities could be used depending on the 

availability and capability of the facilities. 
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A2 PROBLEM DEFINITION/BACKGROUND 

 Among its responsibilities related to Homeland Security, the EPA has the goal of 

identifying methods and equipment that can be used for decontaminating indoor environments 

following a terrorist attack on a building using chemical or biological agents. In January 2003, 

EPA established the NHSRC to manage, coordinate, and support a wide variety of homeland 

security research and technical assistance efforts. The NHSRC is conducting tests to evaluate 

the performance of commercially-available products, methods, and equipment for 

decontamination of porous and non-porous indoor surfaces contaminated with biological or 

chemical agents. 

 The purpose of this testing is to generate objective performance data that can be used by 

building and facility managers, first responders, groups responsible for building 

decontamination, and other technology buyers and users to make informed purchase and 

application decisions. All potential users need unbiased, high-quality, objective third-party data 

and information in order to assess how well the available decontamination tools will meet their 

performance objectives while protecting human health and the environment. All testing and 

evaluation conducted through the TTEP is under the direction of EPA and is subject to the TTEP 

QMP. In performing each test, Battelle will follow the general procedures described in the 

QMP, and develop a separate test/QA plan that is specific for the type of decontamination 

technology being tested. This particular test/QA plan has been prepared for testing and 

evaluation of decontamination technologies that use gaseous or sporicidal decontamination 

agents [e.g. chlorine dioxide (ClO2), hydrogen peroxide vapor, formaldehyde]. 

 The objective of this test/QA plan is to describe laboratory test procedures that will be 

implemented to determine the efficacy of sporicidal technologies for removing or inactivating 

biological agents or surrogates on a range of representative indoor surfaces. This test/QA plan is 

specifically focused on decontamination of indoor surfaces typical of those found in a public 

building with the ultimate goal of providing technologies for restoring the building to a usable 

state following a terrorist attack. Decontamination of personnel or large equipment items (e.g. 

manufacturing equipment) is not covered in this test/QA plan. Decontamination technology 

testing and evaluation are being performed to generate data indicative of the technology 
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performance or efficacy. For the evaluation conducted under this test/QA plan, quantitative 

assessment of performance is accomplished by sampling and analysis of contaminants before and 

after the implementation of the decontamination technology. The performance parameters to be 

evaluated in the evaluation under this test/QA plan are discussed in the Appendix. 

 One or more biological agents (e.g., spores; vegetative cells, biotoxins) may be released 

inside a building during a terrorist attack. The highly persistent biological warfare agent, 

Bacillus anthracis Ames spores, was selected for this evaluation. 

 Indoor surfaces (e.g., carpet, laminate, wood) representing those found in a typical office 

building have been selected for use in evaluating the decontamination technology. The indoor 

surfaces selected include both porous and non-porous materials (see Section B1.3). 

A3 TECHNOLOGY EVALUATION DESCRIPTION AND SCHEDULE 

 The overall objective of the evaluation called for under this test/QA plan is to determine 

the efficacy of the sporicidal decontamination technologies for removing/inactivating biological 

agents in or on typical indoor surfaces. Evaluation of each technology will be accompanied by 

careful monitoring of dwell time, decontamination agent concentration, temperature, relative 

humidity and other parameters that may impact decontamination efficacy. 

A3.1 Applicability 

 This test/QA plan focuses on the evaluation of commercially available technologies for 

decontaminating indoor surfaces found in a typical office building or subway. This plan 

specifically focuses on building decontamination in the context of use by personnel responsible 

for decontamination after a terrorist attack. Toxic industrial chemicals, chemical warfare agents 

and/or biological warfare agents (including toxins) may pose a threat in the building 

contaminated by a terrorist attack. This plan focuses on the evaluation of technologies that are 

potentially applicable for decontaminating indoor surfaces contaminated with biological warfare 

agents (or toxins). 
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 Technology evaluation testing requires a quantitative basis for establishing the 

performance of the tested technologies. For this evaluation, the performance of each of the 

decontamination technologies will be evaluated by comparing the amount of biological agent 

remaining on the indoor surface (carpet, wallboard, etc.) after decontamination with the amount 

of biological agent that was added to the indoor surface prior to decontamination. 

A3.2 Scope 

 The overall objective of the technology evaluation described in this test/QA plan is to 

evaluate the performance of decontamination technologies using biological agents (including 

toxins) and surrogates under a range of realistic conditions. Testing may be conducted over 

ranges of temperature and relative humidity representing those that might be encountered in a 

decontamination situation in a building environment. 

 The performance parameters on which the decontamination technologies will be 

evaluated under this plan include: 

• Log kill or efficacy [the logarithm (base 10) of the number of colony-forming units 

(CFUs) removed/destroyed by applying the decontamination technology]. 

• Surface damage caused by the decontamination technology. 

 The evaluation to be conducted under this plan is limited to detection of biological 

warfare agents (or surrogates) or toxins in or on individual samples (test coupons) of indoor 

materials. Testing will be conducted in two phases. The initial phase will take place in a naïve 

area (not a biological safety laboratory) and the second phase will take place in a biosafety level 

(BSL) 3 area. 

 In the initial phase, the decontamination technology will be coupled with the test 

chamber, associated monitoring devices will be installed in the test chamber and the vendor staff 

will train the MREF staff regarding use of the decontamination technology. In addition, the 

MREF staff will verify that all sub-systems comprising the testing apparatus are in working 

order. 
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 The second phase of the testing entails evaluating the performance of the 

decontamination technology using live agent. This evaluation will be performed in a BSL-3 in 

the MREF. 

A3.3 Schedule 

The evaluation described in this test/QA plan is expected to commence within two weeks 

after this test/QA plan has been approved. It is anticipated that four weeks will be required to 

complete all testing for a single sporicidal technology. This schedule is predicated upon the 

vendor shipping the decontamination technology to Battelle and training Battelle personnel in the 

use of the equipment in accordance with the overall testing schedule. 

A4 QUALITY OBJECTIVES 

 The performance parameters to be evaluated under this test/QA plan include: 

• Quantitative assessment of decontamination efficacy of sporicidal technologies. 

CFUs in extracts of control test coupons are determined using standard plating 

techniques. Plate counts for three controls (inoculated with 108

 spores per coupon but 

not decontaminated) is determined and each of these values is used to calculate the 

value N/N′ where N is the number of CFUs found on the control coupon and N′ is the 

number of CFUs found on the decontaminated coupon. The log [10] of each of these 

is calculated and the three logs are averaged to obtain a mean log kill ± standard 

deviation (SD). The log kill value is also efficacy discussed in Section B1.1. The log 

kill or efficacy value is used as an indication of the overall effectiveness of the 

decontamination technology. The closer the efficacy value is to 108

 (the number of 

spores applied to the test coupon) the more effective or efficacious the treatment.

• Qualitative assessment of residual biological agent and surrogate spores on test 

surfaces following decontamination and extraction. Following the extraction to 

determine the efficacy value quantitatively, test coupons will be immersed in liquid 

growth medium contained in individual vials, cultured at the respective temperatures, 
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and bacterial growth in the vials will be observed visually at one and seven days after 

immersion. If the growth medium appears cloudy, then the conclusion is drawn that 

viable spores remained on the decontaminated samples following extraction. Growth 

could arise from spores added to the test coupon or reflect growth of indigenous 

organisms. Spores or other organisms giving rise to growth in the nutrient medium 

will be identified by standard culture techniques. A small sample of each positive 

culture will be streak plated onto tryptic soy agar plates and cultured overnight. The 

streak plates will provide information as to whether the organisms present are the 

same as those inoculated on the coupons, or other distinct microorganisms

• Qualitative assessment of spore strips and biological indicators. Following 

implementation of the decontamination technology the spore strips and indicators are 

placed in growth medium. The growth medium is examined at one and seven days 

post-decontamination for evidence of viable spores (medium becomes cloudy). No 

cloudiness indicates no viable spores which indicates 100% kill or a log kill of 106. 

The biological indicators and spore strips have been used in large-scale 

decontaminations to assess the completeness of decontamination. However, the 

spores on the biological indicators and spore strips are not B. anthracis spores but 

surrogates. In addition, the spore strips and biological indicators are more amenable 

to decontamination (biological indicators are a metal disc, spore strips are filter 

paper). The results for biological indicators and spore strips provide a means of 

comparing the effectiveness of the decontamination technology with other results 

obtained using the same or other decontamination technologies.

• Changes in appearance of representative indoor surfaces. Each test coupon is 

visually examined before and after application of the decontamination technology to 

assess whether or not the test coupon material exhibits physical changes (loss or 

change of color etc.). This information provides a general indication of how 

“caustic” the decontamination technology is and whether or not the contents of, for 

example, a subway station would be damaged or destroyed during decontamination.

 
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 Quantitative determinations in this study do not involve the use of analytical 

measurement devices. Rather, bacterial colonies will be enumerated manually and recorded. All 

other determinations will be qualitative. Specific information regarding the individual 

decontamination technologies is included in the Appendix. 

A5 SPECIAL TRAINING/CERTIFICATION 

 These tests are expected to be conducted at the Battelle facility in West Jefferson, Ohio. 

That facility is described below. Alternative facilities could also be used, provided those 

facilities meet all the requirements for safety, security, and testing capability established by this 

test/QA plan. 

A5.1 General Site Description 

 Evaluation of sporicidal decontamination technologies will be conducted at Battelle’s 

MREF located in West Jefferson, Ohio, near Battelle’s headquarters in Columbus, Ohio. The 

following section describes the MREF biofacility. The evaluation will be performed in 

accordance with Battelle’s facility-specific methods and standard operating procedures (SOPs) 

that are cited where appropriate throughout this test/QA plan. 

 The MREF specializes in research, development, testing, and evaluation of medical 

countermeasures against highly pathogenic biological and highly toxic chemical materials. This 

facility is one of a very limited number of U.S. laboratories capable of studying aerosolized 

etiological agents in animal models under BSL-3 containment. This facility maintains state-ofthe-art equipment, and professional and technical staffing expertise to safely conduct testing and 

evaluation of hazardous biological materials under the Food and Drug Administration’s (FDA) 

Good Laboratory Practices Guidelines (21 CFR Part 58). The MREF operates in compliance 

with all applicable Federal, state, and local laws and regulations, including U.S. Army 

regulations and is routinely inspected by personnel from the appropriate government agency. 

Battelle operates the MREF in compliance with requirements contained in 32 CFR 626 and 627, 

Biological Defense Research Programs. The MREF facilities are ISO 9001 certified, accredited 
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by the American Association for the Accreditation of Laboratory Animal Care, and inspected 

and compliant with the U.S. Department of Agriculture, FDA, Drug Enforcement Agency, Ohio 

EPA, U.S. Army Safety Team, U.S. Army Inspector General, U.S. Army Medical Research 

Institute of Chemical Defense Safety and Chemical Operations Branch, U.S. Army Medical 

Research and Materiel Command Office of Animal Care and Use Review, Madison County 

Health Department, and Battelle’s Institutional Animal Care and Use Committee. The MREF 

fully complies with all applicable U.S. Army Regulations, and Federal Government and State of 

Ohio regulations to conduct and support research, development, testing and evaluation studies 

using highly toxic chemical and pathogenic biological materials. The MREF is licensed to ship, 

receive, and handle select agents, as defined by the Centers for Disease Control and Prevention 

(CDC). 

 Testing outlined in this test/QA plan will be performed in the MREF BSL-3 facility, 

which was completed in 1995 and expanded to 31,000 square feet in 2002. The containment 

area within the facility is designed to meet or exceed the BSL-3 facility guidelines published by 

the CDC and National Institute of Health entitled Biosafety in Microbiological and Biomedical 

Laboratories (4th edition, 1999). Included are seven BSL-3 microbiology laboratories that 

contain multiple Class III biosafety cabinets (BSCs) and two autoclaves. Additional laboratories 

within this area include multiple microbiology laboratories equipped with Class II BSCs. Test 

procedures at the MREF are governed by established SOPs that are specified by facility, number, 

and title. 

A5.2 Site Operations 

 Battelle operates the MREF in compliance with all applicable Federal, state, and local 

laws and regulations, including U.S. Army Regulations and the CDC. Battelle’s facilities are 

certified through inspection by personnel from the appropriate government agency. Battelle’s 

MREF is certified to work with both live and surrogate agents including bacterial endospores 

(e.g. B. anthracis), vegetative bacteria, and viruses. Additionally, the MREF is ISO 9001 

certified, performs work under this ISO standard, and is monitored by regular outside ISO 

quality inspections. 
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A5.3 Training 

Because of the hazardous materials involved in this technology evaluation, 

documentation of proper training and certification of the test personnel is mandatory before 

testing takes place. The Battelle Quality Assurance Manager, or a designee, must assure that 

documentation of such training is in place for all evaluation personnel before allowing evaluation 

to proceed. 

All participants in this evaluation (i.e., Battelle, EPA, and vendor staff) will adhere to the 

security, health, and safety requirements of the Battelle facility in which testing will be 

performed. Vendor staff will train Battelle evaluation staff in the use of their decontamination 

technology, but will not be the technology users during the evaluation. To the extent allowed by 

the test facility, vendor staff may observe, but may not conduct, any of the technology evaluation 

activities identified in this test/QA plan. 

Access to restricted areas of the test facility will be limited to staff who have met all the 

necessary training and security requirements. The existing access restrictions of the test facility 

will be followed, i.e., no departure from SOPs will be needed for this evaluation. All visiting 

staff at the test facility will be given a site-specific safety briefing prior to the start of any 

technology evaluation activities. This briefing will include a description of emergency operating 

procedures and the identification, location, and operation of safety equipment (e.g., fire alarms, 

fire extinguishers, eye washes, exits). Evaluation procedures must follow all safety practices of 

the test facility at all times. Any report of unsafe practices in this evaluation, by those involved 

in the evaluation or by other observers, shall be grounds for stopping the evaluation until the 

Quality Assurance Manager and testing personnel are satisfied that unsafe practices have been 

corrected. 

A6 DOCUMENTATION AND RECORDS 

Documentation of training related to technology testing, field testing, data analysis, and 

reporting is maintained for all Battelle technical staff in training files at their respective 

locations. The Battelle Quality Assurance Manager may verify the presence of appropriate 
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training records prior to the start of testing. Battelle will document training by the vendor with a 

form signed by the vendor. Battelle technical staff will have a minimum of a bachelor’s degree 

in science/engineering or have equivalent work experience. 
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B MEASUREMENT AND DATA ACQUISITION 

B1 EXPERIMENTAL DESIGN 

B1.1 General Test Design 

 This test/QA plan specifies procedures for bench-scale testing to evaluate the 

performance of sporicidal decontamination technologies under specified operating conditions 

and ambient conditions for decontaminating small pieces (i.e. test coupons) of indoor materials 

to which biological agents and surrogates have been added. 

 Evaluation of the efficacy of a particular sporicidal technology for decontaminating a 

particular material will be accomplished by determining the differential mean reduction of viable 

agent or surrogate under an experimental treatment compared to mean reduction in the absence 

of the treatment (control). Treatments will be defined in terms of viable agent or surrogate, 

decontamination technology (or lack thereof) and operational use of the technology (e.g., 

concentrations, dwell times, flow/vent rates), and ambient conditions (e.g., temperature, 

humidity). Decontamination efficacy will be assessed by comparing the number of viable 

organisms remaining after decontamination with the number initially applied to the test surface. 

Efficacy will be expressed as the log (base 10) of result of dividing the mean number of viable 

organisms found on the control by the number of viable organisms found on a treated sample. 

Triplicate samples (e.g., 3 carpet, 3 wood, etc.) are subjected to the decontamination technology 

and an efficacy value for each sample is calculated and these three values are used to calculate a 

mean and SD. 

 In advance of each efficacy evaluation, a null hypothesis (HO) will be formulated for the 

mean differential performance of the experimental treatment and control data: 

HO : RTreatment – RControl > c 

HA : RTreatment – RControl ≤ c 

Where: 

RTreatment is the mean removal for the treatment group. 

RControl is the mean removal for the control group. 

c is a constant that could be zero if desirable to identify any significant removal or some 

threshold value for demonstrating minimum efficacy (e.g., 99.9999% removal). 
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The experimental treatment and controls will be defined according to desired planned 

comparisons. For any particular material, the planned comparisons may include: 

• Efficacy of a decontamination technology under selected environmental conditions for a 

biological agent compared to a surrogate(s). 

• Efficacy of a particular decontamination technology under selected environmental 

conditions and a particular biological agent for each of several different configurations of 

the technology (e.g., different concentrations, dwell times) or between use and absence of 

the technology. 

After obtaining the experimental treatment and control data, a statistical comparison will be 

conducted. If sufficient evidence is found (at 95% confidence) that the experimental treatment 

mean removal exceeds the control mean removal above the applicable threshold “c”, the 

treatment will be concluded to be efficacious. 

 A parallel sensitivity analysis will be conducted to determine, based on the variability of 

the data collected, how large a true difference in efficacy would be highly likely (90%) to have 

been identified as statistically significant by this hypothesis test. 

 Throughout the evaluation, the test coupons will be randomly assigned to control and 

experimental groups. Each decontamination technology will be applied in a manner consistent 

with the manufacturer’s recommendations. The technology vendor will provide the equipment 

and training regarding application of their technology. 

 The statistical approaches for these analyses are discussed in Section B6.2. 

B1.2 Scale of Testing and Testing Apparatus 

 The parameters listed above will be evaluated during bench-scale testing in the 

laboratory. A decontamination test chamber, a Compact Glove Box 830-ABC (Plas Labs, Inc., 

Lansing, MI; see Figure 2) will be used for exposing the test coupons to the decontamination 

technology. This test chamber has dimensions of 71 cm w x 59 cm d x 74 cm h (28” x 23” x 

29”) and outer dimensions of 110 cm w x 61 cm d x 79 cm h (43” x 24” x 31”). The test 

chamber has a total volume of 317L (11.2 cubic feet). The test chamber also has a top opening 

of 43 cm x 58 cm (17” x 23”) and an attached transfer chamber that is 30 cm (12”) long and an 
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inner diameter of 28 cm (11”). Glove ports are available for working in the test chamber. The 

test chamber may need to be modified, in accordance with the vendor, to accommodate the 

decontamination technology. The decontaminant (fumigant, foam, liquid, gel) will be directed 

from the vendor’s system into the test chamber under the conditions (dwell time, decontaminant 

concentration, temperature, and humidity) specified by the vendor. The performance tests will 

use test coupons that are approximately 1.9 cm w x 7.5 cm long (3⁄4” x 3”); multiple coupons of 

each indoor material will be contaminated with the agent/surrogate, placed into the test chamber 

and then treated with the decontamination technology. Blank (i.e., uncontaminated) and control 

(i.e., contaminated but not decontaminated) coupons will also be prepared for each test material, 

and results obtained for these coupons will be utilized along with the data resulting from the 

analyses of post-treatment samples to calculate decontamination efficacy. This evaluation 

methodology comprises a highly controlled, reproducible approach to assess decontamination 

efficacy, while simulating a realistic, small-scale application of the decontamination technology. 

B1.3 Test Surfaces 

 A subset of the various structural, decorative, and functional surfaces that may be found 

inside an office building will be used for evaluating sporicidal decontamination technologies. 

The surface materials to be used include both non-porous and porous surfaces representing a 

variety of materials. Test coupons (typically measuring 1.9 cm x 7.5 cm) will be prepared from 

Figure 2. Compact Glove Box for the Sporicidal Decontamination Technology Evaluation 
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larger pieces of stock material. The representativeness and uniformity of the test materials are 

critical attributes to assure reliable evaluation results. Representativeness means that the 

materials used are typical of such materials used in buildings in terms of quality, surface 

characteristics, structural integrity, etc. Uniformity means that all test pieces are essentially 

equivalent for evaluation purposes. Representativeness will be assured by selection of test 

materials that meet industry standards or specifications for indoor use, and by obtaining those 

materials from appropriate suppliers. Uniformity will be maintained by obtaining a large enough 

quantity of material that multiple test samples can be obtained with presumably uniform 

characteristics (e.g., test coupons will be cut from the interior rather than the edge of a large 

piece of material), or by using standardized coupons where available. The test surfaces that will 

be used are listed below with their corresponding sample identification codes in parentheses: 

• Painted (latex, semi-gloss) concrete cinder block (PC) 

• Painted (latex, flat) wallboard paper (PW) 

• Decorative laminate (DL) 

• Galvanized metal ductwork (GM) 

• Glass (GS) 

• Bare pine wood (BWD) 

• Industrial carpet (IC) 

B1.4 Biological Agents and Surrogates 

 The biological agent to be used in the evaluation under this test/QA plan has been 

selected based on an evaluation of potential threats to buildings. (3) The evaluation considered 

availability, lethality, potential delivery pathways and persistence of potential agents. 

 The biological agent used in evaluating the sporicidal decontamination technology will 

be B. anthracis Ames strain spores. The biological surrogates will be used to establish 

correlations between the decontamination efficacy of surrogates and agents. To provide 

correlations with the B. anthracis results, the surrogates Bacillus subtilis (ATCC 19659) and 

Geobacillus stearothermophilus (ATCC 12980) will be used. B. anthracis Ames strain spores 

and surrogate spores will be prepared and characterized according to MREF SOPs. 
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Both spores (B. subtilis and G. stearothermophilus) have commonly been used as surrogates for 

B. anthracis in decontamination technology testing. The G. stearothermophilus surrogate 

exhibits comparatively high resistance to various sporicidal decontaminants. The B. subtilis 

(ATCC 19659) surrogate is the most commonly used surrogate for B. anthracis. 

 Two types of biological indicators will be used under this test/QA plan. A commercial 

spore strip of the same spore type [B. subtilis var niger (B. atrophaeus ATCC 9372) on paper 

backing manufactured by Raven Biological Laboratories] as those used during decontamination 

of U.S. Postal Service facilities contaminated with B. anthracis will be used. In addition, 

biological indicators containing B. stearothermophilus and B. subtilis (Apex Laboratories, Inc.) 

will be included. Each of these biological indicators typically contains a spore population of 

approximately 106

 spores on a stainless steel disc packaged in a Tyvek® envelope. 

B1.5 Temperature and Relative Humidity Conditions 

 Each sporicidal decontamination technology may require different temperature and 

humidity conditions in the space to be decontaminated. The temperature and the relative 

humidity will be controlled and monitored during the decontamination process. Specific 

operating parameters and conditions for each sporicidal decontamination technology are 

specified in the Appendix. 

B1.6 Surface Damage 

 The effect of the decontamination technology on the test coupons will be evaluated 

during the efficacy evaluation procedure. Before and after decontamination of the test coupons, 

the appearance of the decontaminated coupons will be visually inspected, and any obvious 

changes in the color, reflectivity, and apparent roughness of the coupon surfaces will be recorded 

in the evaluation. This comparison will be performed for each of the test materials, before 

extraction or sampling of the decontaminated test coupons. 
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B2 METHODS REQUIREMENTS AND PROCEDURES 

B2.1 Agents 

 B. anthracis (Ames), B. subtilis (ATCC 19659) and G. stearothermophilus (ATCC 

12980) spores will be prepared according to established MREF procedures. (4, 5) Working stock 

suspensions of each spore type will be prepared at a target concentration of approximately 1x109 

CFU/mL. 

B2.2 Coupon-Scale Testing 

B2.2.1 Preparation of Test Materials 

 Each of the test coupons will be cut to 1.9 cm x 7.5 cm size from the interior of a large 

piece of test material. Edges and damaged areas will be avoided in cutting test coupons. The 

test coupons will be visually inspected prior to inoculation with the biological agent or surrogates 

and any surface anomalies will be recorded. Specifications of each test coupon will be recorded. 

On each evaluation day, each coupon will be assigned a unique identifier code by the evaluation 

staff for traceability. Prior to the application of the biological agent or surrogate, the surface of 

each test coupon will be wiped with 70% isopropanol or ethanol to kill endogenous 

microorganisms. This is intended to minimize contamination by microorganisms other than 

those being evaluated. To ensure further cleanliness and prevent contamination of test surfaces, 

sterile technique will be exercised during all phases of handling the test coupons. 

B2.2.2 Application of Biological Agents to Test Coupons 

 Application of biological agent/surrogates to test coupons will be performed in a BSC III 

according to established MREF procedures. (6) Test coupons will be placed lying flat in the 

cabinet and contaminated at challenge levels of approximately 1 x 108 CFU per coupon. A 100 

μL aliquot of a stock suspension (approximately 1 x 109

 CFU/mL) of spores will be dispensed 

using a micropipette as small droplets across the surface of the test coupon. After contamination 

with biological agent or surrogate suspension, the test coupons will remain overnight and 

undisturbed in the BSC III to dry. 
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B2.2.3 Confirmation of Surface Application Density 

 To confirm the application density of biological agents and surrogates, the respective 

spore suspensions used to contaminate the coupons will be re-enumerated on each day of use. 

This enumeration will be carried out as described in Section B2.2.5. 

B2.2.4 Application of the Decontamination Technology, Monitoring of Test Procedures 

 One day following application of biological agent or surrogate, inoculated test coupons 

intended for decontamination (including one blank) will be transferred into the test chamber that 

has been coupled with the decontamination technology. The inoculated control coupons (not 

decontaminated) and one blank will be left undisturbed in the BSC. The decontamination 

technology will be applied in accordance with the vendor’s instructions. The concentration of 

the gaseous decontaminant, dwell time, relative humidity, and temperature will be controlled and 

monitored as described in the Appendix. Following decontamination, the test chamber will be 

cleared using the vendor-supplied method for neutralization of decontamination chemicals. 

B2.2.5 Determination of Decontamination Efficacy 

 The efficacy of a sporicidal technology for neutralizing/inactivating biological agents on 

indoor surface materials will be determined. For building decontamination, the amount of 

biological agent that remains following decontamination needs to be ascertained since residual 

agent could present a potential health risk for building occupants. The performance or efficacy 

of the sporicidal decontamination technology will be assessed by comparing the number of 

viable organisms remaining after decontamination with the number actually added to test 

coupons. For biological agents, there is currently no determined safe level for remaining residual 

organisms; (2) however, a traditional approach for qualifying decontamination performance is the 

assessment of growth of biological indicators pre-positioned inside the space being fumigated. 

Typically, biological indicators contain spore loads of approximately 1 x 106

; therefore, complete 

neutralization of these biological indicators results in a 6-log kill. As stated above there is no 

officially recognized clean level for building decontamination; however, biological indicators 
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(used in other decontamination efforts) will also be used for this evaluation as a means for 

evaluating the efficacy of the sporicidal decontamination technology. 

 The decontaminated, control, and blank coupons will be placed individually in a sterile 

50 mL conical vial to which 10 mL of sterile phosphate-buffered saline containing 0.1% Triton 

X-100 has been added. The purpose of the Triton X-100 is to minimize clumping of spores. For 

spore extraction, the tubes will be agitated on an orbital shaker for 15 minutes at approximately 

200 rpm at room temperature. Each tube will then be heat-shocked for 1 hour at 60o C to 65o C 

(for B. anthracis and B. subtilis) or 90o C to 95o C (for G. stearothermophilus) to kill vegetative 

bacteria. Following the heat-shock, 1.0 mL of the extract will be removed and a series of 

dilutions through 10-7 will be prepared in sterile water. An aliquot (100 μL) of the undiluted 

extract and each serial dilution will be plated onto tryptic soy agar plates in triplicate, allowed to 

dry, and incubated overnight at 35o C to 37o C for B. anthracis and B. subtilis and at 55o C to 60o 

C for G. stearothermophilus. Plates will be enumerated within 18 to 24 hours of plating 

as described in References 7 and 8. The number of CFU/mL will be determined by multiplying 

the average number of colonies per plate by the reciprocal of the dilution. Data will be expressed 

as mean + SD of the numbers of CFUs observed. To calculate the percent recovery of spores 

following treatment, the calculated number of spores remaining on the decontaminated coupons 

will be divided by the calculated number of spores on the control coupons that were not subject 

to decontamination. 

 Decontamination efficacy will be calculated as the log reduction (mean ± SD) in viable 

organisms achieved by the decontamination technology. Efficacy (E) for biological agents or 

surrogates will be calculated as 

E = log (N/N′) 

Where N is the number of viable organisms recovered from the control coupons (i.e., those not 

subjected to decontamination), and N′ is the number of viable organisms recovered from the test 

coupons after decontamination. 

 A separate efficacy calculation will be made for each of the surface materials for the 

biological agent and surrogates. Percent recovery (mean ± SD) will be calculated for each type 

of test material inoculated with each biological agent/surrogate by dividing the number of 
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biological organisms in the treated sample by the number of biological organisms in the control 

(non-decontaminated). For each material and agent/surrogate combination, a mean and range of 

the efficacy values will be reported. Thus, the primary efficacy results from the coupon testing 

will be a matrix table in which each entry shows the mean and range of efficacy results for one 

of the agents/surrogates on one of the surface materials. 

 Based on previous decontamination studies, it is assumed that 100% recovery of spores 

from the inoculated test coupons will not be achieved; therefore, viable spores may remain on the 

test coupons. A qualitative assessment of these spores will be performed to determine whether 

viable spores remain on the decontaminated test coupons. Following the extraction process 

described above, each coupon will be transferred into a sterile 50 mL conical tube containing 20 

mL of tryptic soy broth culture medium. These vials will be cultured at the appropriate 

temperature for B. anthracis or surrogates to encourage viable spore germination and subsequent 

proliferation of vegetative bacteria. At one and seven days post-decontamination the tubes will 

be visually assessed qualitatively for viability. A cloudy culture medium will indicate “growth” 

of viable spores, while clear culture medium will indicate “no growth.” In the seven day samples 

where growth is observed, a loop of the culture medium will be cultured on tryptic soy agar 

plates using a standard streak plate technique. This culturing on tryptic soy agar plates will 

provide a means to determine whether the observed growth in the liquid culture medium is due to 

the B. anthracis or surrogate applied to the coupon, a contaminant, or both. 

 The biological indicators and spore strips will be cultured in tryptic soy broth and 

assessed qualitatively at one and seven days post-decontamination for growth or no growth. 

However, further culturing of any positive samples on tryptic soy agar plates will not be 

performed. 

B2.2.6 Observation of Surface Damage 

Following application of the decontamination technology, each test surface will be 

examined visually to establish whether use of the decontamination approach caused any obvious 

damage to the surface. Observation of surface damage will be performed immediately after 

completion of the decontamination process but before post-decontamination sampling to assess 
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efficacy. If wetted by the decontamination process, the test surface will be allowed to dry before 

any inspection for damage. Visual inspection of the surface will then take place through side-byside comparison of the decontaminated test surface and the control coupons of the same test 

material. Differences in color, reflectivity, and roughness will be assessed qualitatively and 

observations will be made by the evaluation staff and recorded.

B3 QUALITY CONTROL REQUIREMENTS 

 Quantitative standards do not exist for biological agents and surrogates. The 

confirmation procedure, controls, blanks, and method validation efforts will be the basis of 

support for biological evaluation results. 

B4 INSTRUMENT CALIBRATION AND FREQUENCY 

 The equipment needed for the evaluation will be maintained and operated according to 

the quality requirements and documentation of the evaluation facility. Relative humidity and 

temperature in the test chamber will be monitored using a NIST-traceable digital thermometer – 

hygrometer. The accuracy of the instrumentation used to monitor the decontamination reagent 

(e.g. fumigant) will be verified as indicated in the Appendix. 

B5 DATA ACQUISITION REQUIREMENTS 

 There are no data needed for this project implementation that are obtained from nonmeasurement sources such as computer databases, programs, literature files or historical 

databases. 
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B6 DATA MANAGEMENT 

 Data acquisition during the evaluation includes proper recording of the procedures used 

in the evaluation to assure consistency in the evaluation and adherence to this test/QA plan; 

documentation of sampling/evaluation conditions; recording observations regarding the 

condition of the surface of each coupon before and after the decontamination process; and 

recording of efficacy results and evaluation conditions. Data acquisition will be carried out by 

the Battelle testing staff manually and recorded immediately in a consistent format throughout all 

evaluations. All written records will be in ink and any corrections to recorded data will be made 

with a single line through the original entry. The correction will then be entered, initialed, and 

dated by the person making the correction. Any non-obvious correction will include a reason for 

the correction. Strict confidentiality of evaluation data will be maintained. 

B6.1 Efficacy Calculations 

 For biological agents and surrogates, decontamination efficacy will be calculated as 

(described in Section B2.2.5) the reduction in viable organisms achieved by the technology. 

B6.2 Statistical Analysis 

 For each material and species combination, calculating log reduction values will result in 

a total of 63 efficacy values (that is three coupons for each of seven materials analyzed in 

triplicate). In cases where no viable colonies remain after decontamination, one colony will be 

assumed to be present for the purpose of this calculation. A two-way analysis of variance 

(ANOVA) model with main effects for each test organism and test material and interactions will 

be fitted to the efficacy data. This model will be used to compare each mean to zero, compare 

each surrogate to a selected organism, for example B. anthracis (for a specific material), and 

compare each surrogate to a selected organism for porous and non-porous materials. T-tests or 

statistical contrasts will be used for the comparisons, with no adjustment for multiple 

comparisons. The ANOVA model is fitted using the SAS® (Version 8.2) GLM procedure. 

 The evaluation results will be compiled in a report. The report will briefly describe 

TTEP and evaluation procedures as well as all evaluation data and observations. The preparation 
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of the draft report, review of the draft report, the revision of the draft report, final approval, and 

the distribution of the final report, will be conducted as stated in the QMP. 
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C ASSESSMENT/OVERSIGHT 

C1 ASSESSMENTS AND RESPONSE ACTIONS 

 Battelle’s Quality Assurance Manager will audit at least 10% of the evaluation data. The 

Quality Assurance Manager will trace the data from initial acquisition, through reduction and 

statistical comparisons, to final reporting. All data calculations will be checked. 

C1.1 Technical Systems Audit 

 Battelle’s Quality Assurance Manager or designee will perform one TSA during the 

evaluation. The TSA is to ensure the evaluation is performed in accordance with the TTEP QMP 

and the test/QA plan and that QA/QC procedures are implemented. The Quality Assurance 

Manager may review evaluation methods, compare test procedures to those specified in this 

test/QA plan, and review data acquisition and handling procedures. The Quality Assurance 

Manager will prepare a TSA report and the findings must be addressed either by modifications of 

test procedures or by documentation in the evaluation records and final report. At EPA’s 

discretion, EPA QA staff may also conduct an independent on-site TSA during the evaluation. 

The EPA TSA findings will be communicated to evaluation staff at the time of the audit, and 

documented in a TSA report. These findings must be addressed as stated above. 

C1.2 Performance Evaluation Audits 

 No performance evaluation audit will be performed for biological agents and surrogates, 

as quantitative standards for these biological materials do not exist. The confirmation procedure, 

controls, blanks, and method validation efforts will be the basis of support for biological 

evaluation results. 

C2 REPORTS TO MANAGEMENT 

 Each assessment and audit will be documented in accordance with the QMP. Assessment 

reports will include the following: 

• Identification of any adverse findings or potential problems. 
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• Space for response to adverse findings or potential problems. 

• Possible recommendations for resolving problems. 

• Citation of any noteworthy practices that may be of use to others. 

• Confirmation that solutions have been implemented and are effective. 

During the course of any assessment or audit, the Quality Assurance Manager will identify to the 

technical staff performing experimental activities any immediate corrective action that should be 

taken. If serious quality problems exist, the Quality Assurance Manager is authorized to stop 

work. Once the assessment report has been prepared, the Building Decontamination Technology 

Area Leader or Task Order Leader will ensure that a response is provided for each adverse 

finding or potential problem, and will implement any necessary follow-up corrective action. The 

Quality Assurance Manager will ensure that follow-up corrective action has been taken. 
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D DATA VALIDATION AND USABILITY 

 Records generated during the evaluation will receive a QC/technical review before these 

records are used to calculate, evaluate, or report results. This review will be performed by a 

Battelle technical staff member other than the person who originally generated the record. 

Evaluation staff will be consulted as needed to clarify any issues about the data records. The 

review will be documented by the person performing the review by adding his/her initials and 

date to a hard copy of the record being reviewed. This hard copy will then be returned to the 

Battelle staff member who generated or who will be storing the record. 
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E REFERENCES 

1. Quality Management Plan (QMP) for the Technology Testing and Evaluation Program 

(TTEP), Version 1, prepared by Battelle, Columbus, Ohio, January 2005. 

2. Raber, E., Jin, A., Noonan, K., McGuire, R., and Kirvel, R.D. Decontamination Issues for 

Chemical and Biological Warfare Agents: How Clean is Clean Enough? Int. J. Environ. 

Health Res., 11, 128-148 (2001). 

3. Decontamination Technology Testing and Evaluation: Task 1: Technology Identification 

and Selection, prepared for the U.S. EPA under Task Order 1113 by Battelle, Columbus, 

Ohio, December 17, 2004. 

4. Battelle MREF SOP Number: MREF. X-074, “Standard Operating Procedure (SOP) for the 

Production of Bacillus anthracis Spores.” 

5. Battelle MREF SOP Number: MREF. X-093, “Standard Operating Procedure (SOP) for the 

Production of Bacillus anthracis Spores in a Small Fermentor.” 

6. Battelle MREF Facility Safety Plan Annex 12 to Appendix B, “Guidelines for the Use of 

Class II and Class III Biological Safety Cabinets in the MREF Biofacility.” 

7. Battelle MREF SOP Number: MREF. X-054, “Standard Operating Procedure (SOP) for 

Enumeration of BL-2 and BL-3 Bacterial Samples via the Spread Plate Technique.” 

8. Battelle MREF SOP Number: MREF. X-112, “Standard Operating Procedure (SOP) for 

Interpreting and Calculating Enumeration Data for Biological Decontamination Testing.” 
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APPENDIX 

DETAILED DESCRIPTIONS OF DECONTAMINATION TECHNOLOGIES AND 

OPERATING PARAMETERS FOR EFFICACY EVALUATION 

Sabre Technical Services Chlorine Dioxide Fumigant Technology 

General Description: 

ClO2 is not stable as a compressed gas and, therefore, vaporous ClO2 must be produced on-site. 

Decontamination technologies that utilize vaporous ClO2 typically include the equipment and 

chemicals for on-site generation, delivery, removal and neutralization of ClO2. In addition, ClO2 

generation technologies may require concurrent use of equipment for establishing and 

maintaining specific temperature and humidity conditions that are required for safe and effective 

operation of the decontamination process. 

Sabre Equipment Description and Operating Parameters 

The Sabre equipment includes a 20.3 cm x 20.3 cm (8” x 8”) base onto which is mounted a 15.2 

cm x 15.2 cm (6” x 6”), 91.4 cm (36”) high sparging column. A 19 L (5 gallon) container 

(vented through a sodium thiosulfate trap, container placed in an over pack for safety) containing 

15 L of an aqueous solution consisting of 3g/L of ClO2 plus 1000 ppm of chlorite is prepared onsite. The ClO2 solution is pumped (using a peristaltic pump) into the sparging column and air 

from the test chamber is pumped into and through the column to sparge the ClO2 from the liquid 

into the air stream; the air stream re-enters the glove box to establish the desired gaseous ClO2

concentration. Liquid introduction from the reservoir of ClO2/chlorite solution to the sparging 

column is initially at the rate of 60 mL per minute; when the desired ClO2 concentration in the 

test chamber is achieved, the liquid introduction into the sparging column is decreased to 3 mL 

per minute. The spent liquid exiting the sparging column is collected in a reservoir. The air 

from the chamber is recirculated into and out of the sparging column. Temperature for the 

decontamination is held in the range of 23.9-35° C and the relative humidity should be held in 
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the range of 23.9-35° C. A nebulizer (supplied by Battelle for this test) is used to establish the 

desired humidity level in the test chamber. A constant temperature bath is employed to maintain 

the temperature of the liquid entering the sparging tower. The parameters of temperature and 

humidity will not be varied in this evaluation. Total treatment time is 3 hours for 3,000 ppm 

ClO2 in order to achieve a CT of 9,000. 

At the end of the decontamination test the ClO2 in the system is removed by pumping through a 

carbon adsorption column. 

Description of the Method for Real-Time Monitoring of ClO2

The concentration of ClO2 inside the glove box will be monitored using a portable ultraviolet 

(UV) spectrometer (developed by DARPA) for real-time monitoring of the concentration of ClO2 

in air. The UV spectrometer measures the strong 360 nm UV absorption of ClO2 gas, generates 

a 4-20 mA signal and, based on the absorption data, the ClO2 gas concentration is calculated. 

The UV optical beam is produced by a low power light emitting diode and is detected by a 

photodiode. A drawing of the monitor assembly is shown in the Figure below (lower panel), and 

a photograph of the as-built units is shown (upper picture). 

UV LED 

source

UV filter + 

photodiode + 

integrated amlifier

Oakton 3 channel datalogger

Temp sensor

RH sensor

A/D, ClO2 sensor to logger

Battery powered, >15 hours

Datalogger has flash memory

UV LED 

source

UV filter + 

photodiode + 

integrated amlifier

Oakton 3 channel datalogger

Temp sensor

RH sensor

A/D, ClO2 sensor to logger

Battery powered, >15 hours

Datalogger has flash memory

UV LED 

source

UV filter + 

photodiode + 

integrated amlifier

Oakton 3 channel datalogger

Temp sensor

RH sensor

A/D, ClO2 sensor to logger

Battery powered, >15 hours

Datalogger has flash memory

UV LED 

source

UV filter + 

photodiode + 

integrated amlifier

Oakton 3 channel datalogger

Temp sensor

RH sensor

A/D, ClO2 sensor to logger

Battery powered, >15 hours

Datalogger has flash memory
Sporicidal Decontamination Technologies Test/QA Plan 

Date: 3/22/05 

 Version 1 

The calibration of the UV spectrometer will be checked at the start of the study by comparing the 

concentrations indicated by the UV spectrometer (concentrations in the range of 1000 ppmv to 

3000 ppmv) by sampling the air in the glove box and comparing the ClO2 concentrations found 

using OSHA Method ID 202 [see www.osha.gov/dts/sltc/methods/inorganic/id202/id202.html

(Note: This method was modified by reducing the volume of air captured to only 0.5 mL and 

increasing the potassium iodide capture solution volumes to 50 mL)] with concentrations 

determined using the DARPA UV spectrometer. The concentrations of ClO2 as determined 

using the DARPA UV spectrometer will be deemed acceptable if the concentration determined 

using the DARPA UV spectrometer (over the ClO2 concentrations of 1000 ppm to 3200 ppm) 

agrees within ± 30% of the corresponding concentration determined using OSHA Method ID 

202. [Note: Data indicative of the performance of OSHA Method ID 202 will be included in the 

section of the report that describes the effort to calibrate the DARPA UV spectrometer.]