EPA/600/R-12/591 | August 2012 | www.epa.gov/ord
United States
Environmental Protection
Agency
Assessment of Liquid and
Physical Decontamination
Methods for Environmental
Surfaces Contaminated with
Bacterial Spores: Evaluation
of Spray Method Parameters
and Impact of Surface Grime
Office of Research and Development
National Homeland Security Research Center
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EPA/600/R/12/591
August 2012
Disclaimer
The United States Environmental Protection Agency, through its Office of Research and Development's
National Homeland Security Research Center, funded and managed this investigation through Contract
#EP-C-09-027 WA 2-25 with ARCADIS, U.S. This report has been peer and administratively reviewed and
has been approved for publication as an Environmental Protection Agency document. It does not
necessarily reflect the views of the Environmental Protection Agency. No official endorsement should be
inferred. The Environmental Protection Agency does not endorse the purchase or sale of any commercial
products or services. This report includes photographs of commercially available products. The photographs
are included for purposes of illustration only and are not intended to imply that Environmental Protection
Agency approves or endorses the product or its manufacturer.
Questions concerning this document or its application should be addressed to the principal investigator on
this effort.
M. Worth Calfee, Ph.D.
Decontamination and Consequence Management Division
National Homeland Security Research Center
U.S. Environmental Protection Agency (MD-E343-06)
Office of Research and Development
109. T.W. Alexander Drive
Research Triangle Park, NC 27711
Phone:919-541-7600
Fax:919-541-0496
E-mail: Calfee.Worth@epamail.epa.gov
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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 capabilities to respond to and recover from such an intentional CBR release.
Through working with Office of Research and Development's (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 surface decontamination
procedures for wide-area remediation. This report documents the results of a laboratory study designed
to better understand the effectiveness of surface cleaning and decontamination methods in an attempt to
develop a readily-deployable treatment procedure for surfaces contaminated with, for example, 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
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Acknowledgments
This effort was initiated following discussions with the U.S. EPA's Office of Solid Waste and Emergency
Response's Office of Emergency Management (OEM) on high-priority research needs to support response
and recovery following incidents involving chemical, biological, or radiological (CBR) agents or materials.
Due to their regulatory responsibilities under the Federal Insecticide, Fungicide, and Rodenticide Act
(FIFRA), the U.S. EPA's Office of Chemical Substances and Pollution Prevention (OCSPP) was also
interested in this effort. The management support from both program offices for the U.S. EPA's Office of
Research and Development (ORD) regarding the contribution that research and development makes
towards the U.S. EPA's preparedness in the homeland security area is greatly appreciated
This effort was managed by the principal investigator from ORD's National Homeland Research Center
(NHSRC), and EPA's Office of Emergency Management, Chemical, Biological, Radiological and Nuclear
(CBRN) Consequence Management Advisory Team.
Project Team:
M. Worth Calfee, Ph.D. (Principal Investigator)
Office of Research and Development, U.S. EPA
Research Triangle Park, NC 27711
R. Leroy Mickelsen, M.S., P.E.
Office of Solid Waste and Emergency Response, U.S. EPA
Research Triangle Park, NC 27711
Shawn P. Ryan, Ph.D.
Office of Research and Development, U.S. EPA
Research Triangle Park, NC 27711
This effort was completed under U.S. EPA contract #EP-C-09-027 with ARCADIS-US, Inc. The support
provided by Tanya Medley (U.S. EPA/ORD/NHSRC) in acquiring the vast quantities of supplies required for
the completion of this project is also acknowledged.
Additionally, the authors would like to thank the peer reviewers for their significant contributions. Specifically,
the efforts of Joe Wood, Marissa Mullins, and Rebecca Connell are recognized.
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Table of Contents
Acknowledgments iii
Appendices vi
List of Tables vi
List of Figures vii
List of Abbreviations & Acronyms ix
Executive Summary xii
1. Introduction 1
1.1 Objectives 2
1.2 General Approach 2
1.2.1 Taskl 4
1.2.2 Tasks 2 and 3 4
1.2.3 TaskS 5
1.2.4 Tasks 2 and 3 Procedure Overview 5
1.3 Definitions of Effectiveness 8
1.3.1 Surface Decontamination Efficacy 8
1.3.1.1 Detection Limits 11
1.3.2 Overall Decontamination Effectiveness (Ultimate Fate of Spores) 11
2. Materials and Methods 12
2.1 Coupon Preparation 12
2.1.1 Effect of Grime on Surface Recovery. 16
2.2 Material Inoculation Procedure 16
2.2.1 Bacillus Spore Preparation 17
2.2.2 Coupon Inoculation Procedure 17
2.3 Experimental Approach 18
2.4 Decontamination Procedure 19
2.5 Test Matrix 21
2.6 Sampling Points 25
2.7 Sampling and Analytical Procedures 27
2.7.1 Extraction Sampling 28
IV
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2.7.1.1 Extraction Method Development 28
2.7.2 Factors Affecting Sampling/Monitoring Procedures 29
2.7.3 Preparation for Sampling/Monitoring 29
2.7.4 Wipe Sampling 29
2.7.5 Swab Sampling 31
2.7.6 Run-off Collection and Sampling 31
2.7.7 Aerosol Sampling 32
2.7.8 pAB Sampling 32
2.7.9 Additional Samples Collected 33
2.7.10 Split Samples 33
2.7.11 Sample Analyses 34
2.7.12 Coupon, Material, and Equipment Cleaning and Sterilization 34
3. Results and Discussion 37
3.1 Task 1: Impact on Efficacy of the Degradation of the pAB Solution over Time 37
3.1.1 Extraction Method Development 37
3.1.2 Efficacy Testing 39
3.2 Task2: Parametric Evaluation of the Decontaminant Application Procedures 41
3.2.1 High Inoculation Tests 41
3.2.1.1 Surface Decontamination 42
3.2.2 Fate of Spores 43
3.2.3 Low Inoculation Decontaminations 47
3.2.4 Wetted Wipe Decontamination 50
3.3 Task 3: Impact of Soiled Surfaces (Grime) on Decon Efficacy 52
3.3.1 Preliminary Tests on Effect of Grime on Recovery 52
3.3.2 Surface Sampling Results 53
3.3.3 Fate of Spores 56
3.4 Assessment of Operational Parameters 57
3.4.1 Time 57
3.4.2 Physical Impacts on Materials 57
3.4.3 Impact on Decontamination Workers 58
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4. Quality Assurance and Quality Control
4.1 Calibration of Sampling/Monitoring Equipment
4.2 Data Quality Indicator (DQI) Goals
4.2.1 Temperature Measurements
4.2.2 pH Measurements
4.2.3 Pressure Measurements
4.2.4 FAC Measurements
4.2.5 Flow Measurements
4.2.6 Positive Control CFU
4.2.7 CFU Counts
4.3 Data Quality Audit
4.4 QA/QC Reporting
4.5 Amendments to and Deviations from the Original QAPP
4.5.1 Formal Amendments
4.5.2 Other QAPP Deviations
4.5.3 Data Quality Indicator Assessment
5. References
59
59
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60
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64
Appendices
Appendix A: Coupon, Test Chamber and Equipment Cleaning and Sterilization Procedures A-l
Appendix B: Miscellaneous Operating Procedures (MOPs) B-1
Appendix C: Spore Deposition and Handling Procedures C-l
Appendix D: Decontamination Procedures D-1
Appendix E: Sampling Procedures E-l
Appendix F: Sample Analyses F-l
Appendix G: QAPP Amendments G-l
List of Tables
Table 2-1. Test Conditions for High Inoculation Parametric Tests (Task 2)
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Table 2-2. Test Conditions for Low Inoculation Parametric Tests (Task 2) 23
Table 2-3. Test Conditions for Wetted Wipe Decontamination Tests (Task 2) 23
Table 2-4. Task 3 Test Matrix, Impact of Grime on Wood and Concrete Surface
Decontaminations 24
Table 2-4. Sample Types 27
Table 2-5. Cleaning Methods and Frequency for Common Test
Materials/Equipment 34
Table 3-1. Recovery from Neutralized (Sprayed) and Control (Not Sprayed)
Test Samples 38
Table 3-2. FAC in Extracts of Wallboard Paper and Wood Coupons 38
Table 3-3. FAC and pH of pAB in the Bulk Container or Following Spraying,
over Time 39
Table 3-4. Task 1 Results - Effect of pAB Age on Wood Surface
Decontamination 40
Table 3-5. Task 1 Results - Effect of pAB Age on Drywall Surface
Decontamination 41
Table 3-6. Test Conditions for High Inoculation Parametric Tests (Task 2) 42
Table 3-7. Task 2 - Surface Decontamination Parametric (high inoculation)
Test Results 45
Table 3-8. Results from 35 ppm FAC Extraction Efficacy Test 47
Table 3-9. Task 2 - Surface Decontamination Parametric (low inoculation)
Test Results 48
Table 3-10. Wetted-Wipe Test Procedure Results (Drywall Coupons) 51
Table 3-11. Task 3-Recovery from Wipe Samples of Grimed Coupons 52
Table 3-12. Task 3 - Results for Grimed and Non-Grimed Decontamination Tests
using pAB and pAB/TSP 55
Table 4-1. Instrument Calibration Frequency 59
Table 4-2. Acceptance Criteria and Test Values for Critical Measurements 60
List of Figures
Figure 1-1. Typical Timeline and Flow Diagram for Each Test
Figure 2-1. 18 mm Rough-Cut Barn Wood Coupon (Task 1)
Figure 2-2. Wallboard Coupon Front (Left) and Back (Right)
Figure 2-3. Rough-Cut Barn Wood Coupon Front (Left) and Back (Right)
Figure 2-4. Curing Concrete (Left) and Final Concrete Coupon (Right)
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Figure 2-5. Application of Grime to Coupons 16
Figure 2-6. Decontamination Chamber 19
Figure 2-7. Spraying Through Center-Aligned Port in the Small Chamber Door 20
Figure 2-8. Sampling Template Centered on a Representative Concrete Coupon 25
Figure 2-9. Spray Chamber Exhaust Duct with Arrow pointing to Sample Port 26
Figure 3-1. FAC and pH of pAB over Time (Task 1) 40
Figure 3-2. Effect of Age on pAB Surface Decontamination Efficacy (Task 1). 41
Figure 3-3. Surface Decontamination Efficacy (Log Reductions, LR) by Material
Type for the Five Parametric Tests (Task 2). 44
Figure 3-4. Recovery of Viable Spores in Rinsates (Task 2). 46
Figure 3-5. Decontamination Efficacy (LR) of Five Decontamination Procedures on
Low-Level Contamination (Task 2) 49
Figure 3-6. Photograph of Wood Fibers on a Filter-plate 50
Figure 3-7. Recovery from Coupons after Three Methods of Wipe Decontamination 52
Figure 3-8. Task 3 Results - The Effect of Grime on Surface Decontamination 53
Figure 3-9. Task 3 Results - Recovery from of Grimed vs. Clean Decontaminated
Surface Samples 54
VIM
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List of Abbreviations & Acronyms
ADA
ANOVA
APPCD
ATCC
BSC
CBR
CBRN
CPU
CI2
DCMD
DHS
Dl
DPG
DQI
DQO
ECBC
EPA
FAC
FIFRA
g
ft
ft2
HEPA
H202
HSRP
HVLP
in
in2
INL
ISO
kGy
Aerosol Deposition Apparatus
Analysis of Variance
Air Pollution Prevention and Control Division
American Type Culture Collection
Biological Safety Cabinet
Chemical, Biological, and Radiological
Chemical, Biological, Radiological, and Nuclear
Colony Forming Unit(s)
Chlorine
Decontamination Consequence and Management Division
Department of Homeland Security
Deionized
Dugway Proving Ground
Data Quality Indicator
Data Quality Objective
Edgewood Chemical Biological Center
U. S. Environmental Protection Agency
Free Available Chlorine
Federal Insecticide, Fungicide, and Rodenticide Act
gram
Foot (Feet)
Square Feet/Foot
High Efficiency Particulate Air
Hydrogen Peroxide
Homeland Security Research Program
High Volume Low Pressure
Inch(es)
square inch
Idaho National Laboratory
International Organization for Standardization
kiloGray
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kPa
Lpm
LR
m2
MDI
min
ml
MOP
NFSA
NHSRC
NIST
NRMRL
OCSPP
OEM
OSHA
ORD
pAB
PBS
PBST
PI
PPE
ppm
ppmv
psi
QA
QAPP
QC
RH
RSD
sec
STS
ISA
TSP
UV
kiloPascal
Liter(s) per Minute
Log Reduction
square meter
Metered Dose Inhaler
minute
milliliter
Miscellaneous Operating Procedure
National Fire Sprinkler Association
National Homeland Security Research Center
National Institute of Standards and Technology
National Risk Management Research Laboratory
Office of Chemical Safety and Pollution Prevention
Office of Emergency Management
Occupational Safety and Health Administration
Office of Research and Development
pH-Adjusted Bleach
Phosphate Buffered Saline (solution)
Phosphate Buffered Saline 0.05% TWEEN®-20
Principal Investigator
Personal Protective Equipment
parts per million
parts per million by volume
pounds per square inch
Quality Assurance
Quality Assurance Project Plan
Quality Control
Relative Humidity
Relative Standard Deviation
second
Sodium Thiosulfate
Trypticase Soy Agar
Trisodium Phosphate
Ultraviolet (light)
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VHP Vaporous Hydrogen Peroxide
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Executive Summary
This project supports the U.S. Environmental Protection Agency (EPA), through its Homeland Security
Research Program (HSRP), to provide relevant information pertinent to the decontamination of
contaminated facilities resulting from a bioterrorism-related incident. The primary focus of this project is to
evaluate and improve the effectiveness and practical application of in situ expedient decontamination
methods to remediate and restore areas contaminated by biological threat agents. These decontamination
techniques would rely on equipment (e.g., garden hoses, portable chemical sprayers, power washers) and
application of liquid decontaminant solutions that can be cost-effective and readily available on site.
The aim of this research was to optimize low-tech decontamination approaches using equipment and
chemicals that should be readily available at local hardware stores, namely, the use of portable battery-
powered chemical sprayers to dispense solutions of pH-adjusted bleach (pAB) onto contaminated surfaces.
First, the temporal impact or the age of the pAB solution was investigated by evaluating the sporicidal
efficacy on a small-scale. Nonvirulent Bacillus spores were used as surrogates for biological agents
because they are easily used in laboratory tests without the risk of laboratory worker infection, and
represent a conservative estimate of decontamination effectiveness. Rough-cut barnwood and primed and
painted wallboard paper were chosen as test materials as they represent commonly occurring, yet
challenging to decontaminate, surfaces likely encountered during an urban (indoor or outdoor) remediation.
The decontamination solutions were pAB prepared fresh (used within 15 minutes after preparation) and pAB
also used at 2, 4, 8, 24, and 32 hours after preparation. Potential negative bias of residual bleach was also
evaluated for these two test materials at this inoculation level (7 ± 0.5 log CPU) and at lower inoculation
levels (2-3 log ±0.5 log CPU).
Second, the performance of pAB spray-based decontamination procedures was evaluated parametrically
with respect to the physical removal, inactivation, and overall fate of spores on "medium-sized" (35.6 cm x
35.6 cm or 14 in by 14 in) drywall, pressure-treated wood, and concrete pieces (coupons). These materials
were utilized neat (no added grime) and chosen because of their common occurrence in indoor and outdoor
facilities. The decontamination procedures involved pAB sprayed onto the surface at two different flow rates
and various reapplication rates. A wetted wipe decontamination approach was also tested on the medium
drywall coupons.
Third, the impact of soiled surfaces (grime) on the sporicidal effectiveness of the low-tech decontamination
procedure was assessed for the same "medium sized" coupons. The "medium-sized" coupons were
evaluated in a custom-built test chamber.
Operational parameters such as processing time and physical impact on materials or decontamination crew
were also determined. Further, to assess the potential for viable spores to be washed off the surfaces, all
liquids used in the decontamination process were collected and analyzed quantitatively.
The results from this study indicate that aging of the pAB decontamination solution decreases its sporicidal
effectiveness on surfaces that are typically difficult to decontaminate, such as those like wood with a higher
organic content. In contrast, aging seems to have little or no effect (within the first four hours after
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preparation) on the ability of pAB to decontaminate the nonporous drywall coupons. Solutions of pAB older
than 4 hours show a marked decline in efficacy for either type of material tested.
The results from the decontamination procedure parametric tests indicated that greater than 6 log reduction
in spores recovered from surfaces - a benchmark for determining efficacy of a decontamination procedure -
can be obtained on grime-free drywall and concrete coupons with a single application of pAB (5 seconds
(sec)/0.09 square meters (m2) or 5 sec/square foot (ft2)), when using the highest flow rate (1344 milliliters
(ml_)/minute (min)) tested. Further testing focused on developing decontamination procedures for the most
challenging material, wood, where 6 log reductions could not be obtained with a single pAB spray
application. For wood, increasing the pAB spray duration from 5 sec/ft2 to 10 sec/ft2 or increasing the
frequency of spraying (two applications rather than a single application) did not provide any statistically
significant improved efficacy in surface decontamination. Longer spray duration and/or additional
applications were outside the scope of this study, in part because longer decontamination procedures
prohibitively increase the cost and complexity of a full-scale response in a real-world scenario. However,
based on these results, significant improvement in decontamination efficacy is not anticipated with longer
spray duration and/or additional applications.
Tests of the decontamination procedures conducted at a medium level contamination challenge (1 x 104 to 1
x 105 CFU) on drywall and concrete coupon materials confirmed that a full decontamination (meaning no
viable spores detected following decontamination) can be obtained with a single application of pAB (5
sec/0.09 m2 or 5 sec/ft2). However, no single decontamination procedure investigated within the scope of
this study was found to be effective in inactivating/removing spores from low level (1 x 102 CFU) inoculated
wood coupons.
The impact of grime (1 gram (g) per coupon) on decontamination efficacy was minimal for rough-cut barn
wood and concrete coupons inoculated with 1 x 10 5 to 1 x 106 spores, when using either a pAB solution or
a pAB/trisodium phosphate (TSP) solution as the decontaminant. The results with this grime loading
suggest no particular advantage to the use of pAB with surfactant (TSP) versus pAB. Further studies should
investigate the efficacy of pAB and pAB with surfactant when used to decontaminate heavily grimed
surfaces (i.e., >1 g/0.09 m2). Additional cleaning activities such as scrubbing or vacuuming did not increase
decontamination efficacy, but may increase the chances that spores will be re-aerosolized. The wetted wipe
decontamination procedures tested provided minimal to moderate decontamination efficacy (0.6 to 3.3 log
reduction).
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1. Introduction
The National Homeland Security Research Center (NHSRC) conducted this study in support of the EPA's
Office of Research and Development's Homeland Security Research Program. The NHSRC aims to provide
expertise and guidance on the selection and implementation of effective and efficient decontamination
methods following biological contamination incidents. The Department of Homeland Security (DHS) is
tasked to coordinate with other appropriate Federal departments and agencies to develop comprehensive
plans which provide for seamless, coordinated Federal, state, local, and international responses to a
biological attack. As part of these plans, EPA, in a coordinated effort with DHS, is responsible for developing
strategies, guidelines, and plans for decontamination of persons, equipment, and facilities to mitigate the
risks of contamination following a biological weapons attack. NHSRC provides expertise and products that
can be widely used to prevent, prepare for, and recover from public health and environmental emergencies
arising from terrorist threats and incidents.
In 2001, the introduction of a few letters containing anthrax spores into the U.S. Postal Service system
resulted in the contamination of numerous government and private facilities. Although most of the facilities in
which these letters were processed or received in 2001 were heavily contaminated, they were successfully
remediated with approaches such as fumigation with chlorine dioxide or vaporous hydrogen peroxide
(VHP®).1 In addition to fumigation used primarily in heavily contaminated facilities, other cleaning methods
were used in secondarily contaminated (e.g., cross-contaminated letters potentially in contact with the
Bacillus anthracis spore-containing letters or tracked from primarily contaminated sites) areas or primarily
contaminated facilities showing a minimal presence of spores.1 During the remediation in 2001, these
methods included combinations of removal and disposal of contaminated items, vacuuming, and the use of
liquid sporicides such as pH-adjusted bleach (pAB) solution to treat surfaces. Additionally, a set of combined
mechanical and chemical procedures (vacuum, scrub/wash and bleach) was used successfully in the
decontamination of a small wooden shed contaminated with Bacillus anthracis spores originating from
animal hides during a drum-making process.2 When effective, such a "lower-tech" approach involving
washing and cleaning with readily available equipment and reagents would significantly increase EPA's
readiness to respond to a wide area release. Accordingly, additional quick, effective, and economical
decontamination methods having the capability to be employed over wide areas (outdoor and indoor) are
needed to increase preparedness fora biological release on a larger spatial scale.
During the decontamination activities following the 2001 anthrax incidents, a combination of removal and in
situ decontamination techniques was used. The balance between the two approaches was facility-specific
and dependent upon many issues (e.g., physical state of the facility). One factor was that such remediation
was unprecedented for the United States Government and no technologies had been proven for such use at
the time (e.g., registered under the Federal Insecticide, Fungicide, Rodenticide Act [FIFRA]). The cost of
disposal proved to be very significant and complicated by the nature of the waste (e.g., finding an ultimate
disposal site). Since 2001, a primary focus for facility remediation has been improving the effectiveness and
practical application of in situ decontamination methods and evaluating waste treatment options to optimize
the decontamination/disposal paradigm. This optimization, recognized as being site-specific, has a
significant impact on reducing the cost and time for the remediation effort.
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In previous studies conducted by NHSRC, data were generated to understand the effectiveness of different
decontamination steps for surfaces inoculated with Bacillus spores via aerosol deposition3. This
understanding was used to develop a recommended method for decontamination of a facility in Durham,
NH, contaminated with natural Bacillus anthracis spores. These studies generated data to develop and test
surface decontamination protocols based upon understanding the effectiveness of individual process steps.
The current study was conducted to build upon the previously developed methods in order to (i) improve the
ease of application (e.g., decrease reapplication time), (ii) improve overall effectiveness under application
conditions of easier use, and (iii) to develop methods effective on grimed surfaces
1.1 Objectives
The primary objective of this study was to improve a series of low-tech decontamination remediation
procedures applied to various surfaces contaminated with viable bacterial spores. The remediation
approach used for this study focused on equipment (i.e., backpack sprayers, etc.) and chemicals (i.e.,
bleach, acetic acid (vinegar), etc.) that should be readily available at local hardware stores. Remediation
activity parameters were chosen with the aim of improving the decontamination parameters by speeding up
slow steps without reducing efficacy and by eliminating ineffective or counterproductive steps. The efficacy
was evaluated by recovery of spores from the surfaces of materials while also considering the potential for
cross-contamination. Operational parameters such as processing time, physical impact on materials or
decontamination crew, and fate of the viable spores (e.g., re-aerosolization, contamination of equipment,
wash water, filters) were also determined. This study was comprised of the following three tasks:
• Task 1 - the impact on efficacy of the degradation of the pAB solution overtime (up to 32
hours) was determined.
• Task 2 - variations on the spray parameters of the decontaminant solution application
procedure were investigated.
• Task 3 - the impact of soiled surfaces (grime) on efficacy was determined.
1.2 General Approach
The general process investigated in this project was decontamination of surfaces contaminated with Bacillus
spores (i.e., surrogates of Bacillus anthracis). Decontamination can be defined as the process of inactivating
or reducing the amount of contamination in or on humans, animals, plants, food, water, soil, air, areas, or
items through physical, chemical, or other methods to meet a cleanup goal. In terms of the surface of a
material, decontamination can be accomplished by physical removal of the contamination or via inactivation
of the contaminant with antimicrobial chemicals, heat, ultraviolet (UV) light, etc. Physical removal could be
accomplished via in situ removal of the contamination from the material or physical removal of the material
itself (i.e., disposal). Similarly, inactivation of the contaminant can be conducted in situ or after removal of
the material for ultimate disposal.
In this research, the basis for the specific decontamination procedure was a process similar to that used by
EPA in Region 1 to decontaminate a wooden shed in Danbury, CT. The process employed for the shed is
documented in the "After Action Report- Danbury Anthrax Incident (U.S. EPA Region 1, September 19,
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2008)"2 and discussed by Snook et al.4 The resulting process that was the baseline for this project can be
summarized in the following sequential procedural steps:
1. Vacuum surfaces with a wet/dry vacuum containing a High Efficiency Particulate Air (HEPA) filter.
2. Mist the surface with the pAB solution until it remains wetted; reapply as necessary to keep the
surface wetted for a contact time of 10 minutes.
3. Scrub the surface using a brush with soap and water.
4. Rinse the surface with water.
5. Vacuum standing water from horizontal surfaces with the wet/dry vacuum.
6. Completely cover the surface with the pAB solution for the desired contact time (i.e., 30-60 min).
7. Rinse the entire surface with water.
8. Vacuum standing water from horizontal surfaces with the wet/dry vacuum.
Results from the previous study3, were used to develop refined decontamination procedures. Refinements
were limited to eliminating steps that have no or negative efficacy and choosing the most effective duration
for steps of variable times. Step 5 of the process, for instance, did not contribute to the efficacy of the
procedure and could be a source for unintended re-aerosolization. Also, the 30 min of pAB exposure was
sufficient to achieve a 6 log reduction of spores, so 30 min was the maximum exposure, instead of the 60
min exposure listed above. The current study attempted to improve the decontaminant application
procedure further by investigating effects of varying pAB spray rates (1000 mL/min and 1300 mL/min) and
re-applications (none, re-application after 5 minutes, and re-application after 15 minutes). The efficacy of
these refined procedures, both with respect to the physical removal and the inactivation of spores, are
evaluated on an operational-scale, with relevant test materials.
The general approach used to meet the objectives of this project was:
• use of controlled chambers, standardized coupons and spore inocula;
• inoculation of small or medium-sized uniform pieces of materials (coupons) with bacterial spores via
aerosol deposition;
• quantitative assessment of initial viable spore surface concentration by sampling positive control
coupons (coupons inoculated with the bacterial spores in the same manner as test coupons but not
subjected to the decontamination treatment being tested);
• application of a prescribed decontamination procedure to the test coupons and procedural blanks;
• quantitative assessment of residual viable spore loading on each material type after application of
combinations of decontamination procedures by sampling test coupons and procedural blanks;
• quantitative and qualitative analysis of viable spores that survive the various decontamination
procedures through transfer to air (via aerosolization) and runoff or rinsate,
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• qualitative analysis of viable spores that survive the various decontamination procedures through
transfer to wet/dry vacuum filters and vacuum exhaust samples;
• determination of surface decontamination efficacy (comparison of concentrations of viable spores from
positive controls and test coupons);
• determination of overall decontamination efficacy (accounting for viable spores transferred to other
media [e.g., rinsate or air] during the decontamination process); and
• documentation of operational considerations (e.g., cross-contamination, procedural time, impacts on
materials and personnel).
Small (18 mm [0.71 in.] diameter, area of 2.6 cm2 [0.4 square inches (in2)]) and medium-sized coupons
(35.6 cm x35.6 cm [14 in by 14 in]; area of 1265 cm2 [196 in2]) of relevant building materials were fabricated
(see Section 2) and sterilized (see Appendix A) in groups identified by sterilization batch number. The small
coupon size was chosen to maximize replicates and minimize sampling effects during the execution of the
Task 1 test matrix (see Section 3.1). The medium coupon size was chosen for application of the physical
decontamination methods (i.e., vacuuming, spraying, and brushing) and field sampling methods (i.e., wipe
and vacuum sock) used in this current study (Tasks 2 and 3). The materials used in this study included
wallboard (primed and painted; vertical orientation to represent a wall), rough-cut barn wood (vertical
orientation with wood slats oriented vertically), and concrete (vertical orientation). All materials used are
considered porous with the exception of the painted wallboard (a sealed surface).
1.2.1 Taskl
In Task 1, two materials (rough-cut pine wood; primed and painted wallboard paper) were used to test the
effectiveness of a pAB solution over time. The materials were inoculated with spores of B. atrophaeus
(formerly, 8. globigii) at 7 log CPU (± 0.5 log CPU). The decontamination solutions were freshly prepared
pAB (used within 15 minutes after preparation), then used at 2, 4, 8, 24, and 32 hours after preparation. The
coupons were sprayed at 0 and 15 minutes with pAB from a backpack sprayer at a flow rate of 1000 mL/min
for a rate consistent with 5 sec/ft2, as determined effective from previous studiesS24. The samples collected
and analyzed for viable bacterial spores during this task included whole-coupon extractions from five (5)
replicate test coupons of each material type and from two sets of positive controls (one per day of testing)
and aliquots of rinsates. Samples were collected directly from the pAB solution for determination of pH and
free available chlorine (FAC). In addition, aliquots of pAB were collected during spray application to
determine post-spray FAC.
1.2.2 Tasks 2 and 3
In Task 2, the efficacy of spray decontamination approaches with various pAB application flow rates and
reapplication scenarios were determined on three materials (rough-cut pine wood; primed and painted
drywall, and concrete). These materials were utilized neat (no added grime) and chosen because of their
common occurrence in indoor and outdoor facilities. Additionally, they represent a broad range of
expected challenge for decontamination. The test materials were inoculated with spores of 8. atrophaeus at
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7 log CPU (± 0.5 log CPU). Recoveries from decontaminated coupons and positive control coupons were
determined by wipe sampling, and used to evaluate decontamination efficacy. The main variables
examined were the duration and frequency of application of decontamination solutions, which were either
pAB or pAB with surfactant (TSP). Scrubbing and vacuuming steps were also investigated for their
contribution to reduction of surface contamination.
1.2.3 TaskS
In Task 3, the impact of surface grime on decontamination efficacy was determined for rough-cut pine wood
and concrete coupons. These materials were chosen due to their commonality and they represent
materials that have been shown to present a challenge for decontamination. Neat (no grime) and grimed
coupons were subjected to spray-based decontamination procedures that determined the additive effects of
physical cleaning procedures (scrubbing and vacuuming) on surface decontamination efficacy. Similar to
Task 2, coupons were inoculated with 7 log CPU (± 0.5 log CPU) spores of B. atrophaeus. Grime was
applied to coupons at 1 g per ft2. Decontamination efficacy was determined by comparing recoveries from
positive controls to that of coupons subjected to the treatment.
1.2.4 Tasks 2 and 3 Procedure Overview
Although a preliminary test matrix was designed for Tasks 2 and 3, these matrices evolved greatly during
this project as testing progressed and results were obtained (i.e., an adaptive management approach).
Because of this evolution, the details of the testing conducted for these tasks are discussed in Section 3
immediately prior to reporting of the results. In this way, the reasoning behind the test progression will be
presented to provide the logic used during the parametric tests.
The projected timeline and flow diagram for the testing approach for Tasks 2 and 3 is shown in Figure 1-1.
Details of the types and numbers of materials tested, as well as the procedures used for inoculation,
decontamination, sampling, and testing are described in Section 2 and in the attached appendices.
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Day1
Projected Timeline
Day 2
Day 3 Day 4
r >
Load
Coupons
into their
Respective
Cabinets
\
Decontamination
Procedure
Application
i
X
Coupon
Drying
Recovery of
Wet/Dry Vacuum
Samples
and
Liquid Samples
Figure 1-1. Typical Timeline and Flow Diagram for Each Test
Day 1 of testing involved coupon inoculation and preparation for testing on Day 2. The required number of
test and positive control coupons were loaded with the target spores at least one day, but no more than four
days, prior to the decontamination procedure. The coupons remained isolated in independent deposition
devices throughout this time.
On Day 2, inoculated coupons were removed from the deposition devices and loaded into their respective
cabinets (positive controls and test coupons into the Test Coupon Cabinets and the procedural blanks into
the Procedural Blank Cabinet). As indicated above, positive control and test coupons were both inoculated
with the target number of viable spores; the test coupons were subjected to the decontamination procedure
being tested while the positive control coupons were not (and were maintained under ambient laboratory
conditions). The purpose of the positive controls is to determine the starting viable spore load on each
coupon type for comparison to the viable spore load on the test coupons after decontamination. Since the
sampling process removes spores from the material surfaces, the procedure of using positive controls and
test coupons to determine effectiveness is common.11'13'14 Procedural blank coupons (negative controls)
were the same materials as the test and positive controls. However, the procedural blanks were intentionally
not inoculated with spores. The blanks were put through the same decontamination procedure as the test
coupons for the purpose of elucidating any potential cross-contamination introduced during the testing
procedure.
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After the coupons were appropriately stored, sets of three coupons were then positioned in the
decontamination chamber and the prescribed decontamination procedure was conducted. Coupons were
placed in a vertical orientation. Procedural blank coupons were subjected to the decontamination procedure
first. The blank coupons were then transferred to a dedicated storage/drying cabinet. Test coupons were
then placed in the chamber for application of the decontamination procedure. The decontamination
procedure was completed on all test coupons of one material type before moving on to the next material.
After the decontamination procedure was applied to a coupon, the coupon was moved to the appropriate
cabinet for drying (test coupons to the Decontaminated Coupon Cabinet and procedural blanks to the
Procedural Blank Cabinet). To be consistent with field procedures, the decontaminated coupons were
completely dry (allowed to dry for at least 24 hours) prior to sampling. After the completion of each set of
coupons, the test chamber was cleaned in accordance with the procedure described in Appendix A. A
coupon set included all blank coupons or all replicates of one material type.
The temperature and pH of the pAB solution and deionized (Dl) water were measured at the initiation of a
test and prior to the start of each test set (i.e., material type). The FAC of the pAB solution was also
measured (see Section 2 for method). The flow rate from the backpack sprayer (SRS-540 Propack,
SHURflo, Cypress, CA) was measured at the start and end of testing of each set of three coupons on which
the sprayer was being used. The spray pattern for the backpack sprayer was confirmed (and adjusted as
needed) prior to the start of a test. These measurements were made to ensure that such parameters were
in accordance with the data quality objectives (DQOs) defined for the project (see Section 4) and did not
confound test results and conclusions. Adjustments were made as necessary to achieve the desired set
points within the acceptable tolerances.
Although surface sampling of the coupons did not occur until Day 3, several other samples were collected to
obtain additional information on the fate of the spores. To assess the potential for viable spores to be
washed off the surfaces, all liquid runoff (rinsate) generated in the decontamination process was collected
and analyzed quantitatively for viable spores. Rinsate samples were a composite of the rinsate from all
replicate coupons of a particular material type per test. Residual decontaminant within rinsates was
neutralized in situ with sodium thiosulfate (STS) to prevent continued action of the pAB to reduce spore
recovery (Section 2.6.7). Quantitative analysis was conducted on rinsate samples so that the magnitude of
spore relocation could be determined. The volume of runoff liquid collected for each coupon set was
measured after collection.
On Day 3, after at least 24 hours of drying, surface sampling of the coupons was performed. A sampled
area of 0.12 m2 (1.27 ft2) per coupon for this study was created by sampling the interior section of each
coupon; a template was used to cover the exterior 0.6 cm (0.25 in) of each coupon leaving a square 34.3
cm by 34.3 cm (13.5 in by 13.5 in) exposed for sampling. Surface sampling of each test coupon was
conducted only once.
The analysis of the samples collected (coupon, filter, rinsate, and exhaust) occurred over a three-day
period. In general, the NHSRC Biocontaminant Laboratory extracted the samples on the day of receipt,
plated on the following day, and then counted colonies on the third day. However, instances occurred when
it was possible to apply the decontamination procedure to only half of the coupons on the first day, with the
remaining half decontaminated on the following day. For these tests, the later samples were analyzed over
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a two-day period. Sample extraction and plating would occur on the day of receipt, or a day later, with
colony counting the following day.
Appendix A describes the procedure for coupon, test chamber, and equipment cleaning and sterilization.
Appendix B contains MOPs, including the aerosol deposition of spores. Appendices C, D, E and F contain
additional details of the inoculation, decontamination, sampling and analysis procedures, respectively.
1.3 Definitions of Effectiveness
The "overall effectiveness (efficacy)" of a decontamination technique is a measure of the ability of the
method to inactivate and/or remove the spores from contaminated building material surfaces (i.e.,
represented by coupons in this study) while taking into account viable spores that may be relocated to
rinsate and/or aerosol fractions. Such fugitive biological emissions could result in secondary contamination
that would necessitate additional remediation strategies.
1.3.1 Surface Decontamination Efficacy
The surface decontamination efficacy for each decontamination technique and surface material combination
was evaluated by measuring the difference in the logarithm of the measured CPU before decontamination
(determined from sampling the positive control coupons) and after decontamination (determined from
sampling the test coupons) for that material. This value is reported as a log reduction on the specific
material surface as defined in Equation 1-1.
77 =— — (1-1)
ft AT AT
N,
where:
Surface decontamination effectiveness: the average log
T! . = reduction of spores on a specific material surface (surface
material designated by;)
The base 10 logarithm of the geometric mean, or the average of
ck) _ the base 10 logarithm of the number of viable spores
— (determined by CPU) recovered on the control coupons (C
c indicates control and Nc is the number of control coupons)
The base 10 logarithm of the geometric mean, or the average of
Nt
"Vloe (CPU ) the base 10 logarithm of the number of viable spores
_t=i = (determined by CPU) remaining on the surface of a
Ns decontaminated coupon (S indicates a decontaminated coupon
and Ns is the number of coupons tested).
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When no viable spores were detected, a value of 0.5 CPU was assigned for CFUs,k (see Section 1.3.1.1)
and the efficacy was reported as greater than or equal to the value calculated by Equation 1-1.
The standard deviation of the average log reduction of spores on a specific material (T\\) is calculated by
Equation 1-2:
SD.
rf,
I(«)2
Ns-l
(1-2)
where:
or) _ Standard deviation of Y\\, the average log reduction of spores on
Vi a specific material surface
„ _ The average log reduction of spores on a specific material
'' surface (surface material designated by;)
The average of the log reduction from the surface of a
A|< —
decontaminated coupon (Equation 1-3)
Ns = Number of test coupons of a material surface type.
XL = '
NS
(1-3)
where:
Iog10 (CFUC ) = —
N
c
Represents the "mean of the logs" (geometric mean),
the average of the logarithm-transformed number of
c k) ^ viable spores (determined by CPU) recovered on the
control coupons (C = control coupons, Nc = number of
control coupons, k = test coupon number and Ns is
the number of test coupons)
CFUs,k = Number of CPU on the surface of the kt
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decontaminated coupon
-.. _ Total number (1 ,k) of decontaminated coupons of a
material type.
The average surface decontamination effectiveness of the decontamination technique for spores recovered
on the surface of building materials, independent of the type of material, was evaluated by comparing the
difference in the logarithm of the CPU before decontamination (from sampling of the positive control
coupons) and after decontamination (from sampling of the test coupons) for all the tested materials. These
data are calculated by determining the arithmetic mean of r\ for all material types according to Equation 1-4
and reported as log reductions of spores for each decontamination technique.
where ji is the overall surface log reduction efficacy for the technique, and N, is the total number of
coupon material types tested with that technique (;' indicates coupon material type).
The standard deviation of Y\rts calculated by Equation 1-5:
where:
_ Standard deviation of T|7; the overall surface log reduction
^T efficacy for the technique
TjT = Overall surface log reduction efficacy for the technique
The average log reduction of spores on a specific material
'* surface (surface material designated by ;)
NJ = Number of coupon material types.
While this method of calculating surface decontamination efficacy is useful for comparing decontamination
methods, the indoor clearance criterion for a facility following actual bioterrorism events has generally been
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no growth of the biocontaminant via culture for all environmental samples.5 Thus, clearance sampling after
use of a particular decontamination method in which CPU of the biocontaminant were detected would
indicate that the decontamination was not adequately effective.
1.3.1.1 Detection Limits
Quantification of viable spores collected by surface sampling techniques was determined according to MOP
6535a (Appendix B). Quantification was accomplished by physical extraction of spores from sampling
media, followed by plating 0.1 ml of serial dilutions of the extraction fluid in triplicate onto trypticase soy
agar (ISA) (Difco,Franklin Lakes, NJ). Following plate incubation, CFU (30 to 300 colonies) were
enumerated from the appropriate plate dilutions. The CFU per sample were calculated according to
Equation 1-6. When fewer than 30 CFU (the quantitation limit) were present on the primary (no dilution)
plates, the extracts were filter-plated as described in Section 2.7.12. When no detectable spores were found
from the filter-plating, a value of 0.5 CFU was assigned as the detection limit for efficacy determinations
(calculation of log reduction). The use of this detection limit value for samples with less than 30 CFU on the
primary plates is consistent with other published methods.6 789 For the current effort, this detection limit was
considered for the plating and, hence, the multiplier of 200 (20 ml extraction fluid for wipe samples divided
by 0.1 ml) was applied for all non-filter sample results. This procedure yielded an overall detection limit of
100 CFU/sample. The addition of the filter-plating method lowered the overall detection limit to the stated
0.5 CFU/sample due to analysis of the entire sample extract.
average CFU from
= ^Plicate allutlonplates or on f liter x 1 x(extract VOlume.mL} (1-6)
sample volume plated or filtered,mL (tube dilution factor)
The number of viable spores in the rinsate was calculated in a similar fashion when all of the rinsate was
filtered. For aliquot sampling, a portion (typically 1,10, and 89 ml) of the rinsate was filtered and the filter
was directly plated. The enumerated CFU from these plates were then multiplied by the inverse fraction of
the rinsate that was filtered. For example, if 100 ml of the 20,000 ml rinsate (the total volume collected
after one coupon set of a test) were plated, then the CFU counts on the filter would be multiplied by
20000/100 (or 200) to represent the total number of spores in the rinsate. If no CFU were present on any
filter plate, then a value of 0.5 CFU was assigned as the detection limit (CFUs,k in Equation 1.1). This
detection limit was still subject to the multiplier, resulting in a detection limit of 100 CFU for the above
example.
1.3.2 Overall Decontamination Effectiveness (Ultimate Fate of Spores)
The surface decontamination efficacy, as calculated in accordance with Equation 1-4, is a measure of the
effectiveness of the procedure to mitigate the contamination on the surface of the materials. The measure of
effectiveness is an aggregate value due to inactivation of the spores on the materials (i.e., due to the
application of a sporicide) and/or physical removal of the spores from the material (e.g., washed/rinsed off or
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removed by the vacuum process). When the spores are physically removed from the surface, viable spores
may remain in the rinsate, become aerosolized by the physical contact of the decontamination procedure, or
be collected on or in any equipment used in the decontamination procedure. Understanding the ultimate fate
of the spores (or overall decontamination effectiveness) is critical to recognizing the utility or appropriate
implementation of the specific decontamination process.
2. Materials and Methods
Coupon materials were chosen to represent common indoor and outdoor surfaces. Equipment and
chemicals utilized for the decontamination methods were selected based upon their high availability at local
hardware stores. Prior to use, all test equipment intended to come in contact with coupons or samples was
sterilized via autoclave sterilization at 121 °C and 103 kiloPascals (kPa) (15 pounds per square inch (psi)),
or by using a STERIS VHP® 1000ED (STERIS Corporation, Mentor, OH) hydrogen peroxide (H2O2)
generator cycle at 250 parts per million by volume (ppmv) H2O2 for four hours. All laboratory work surfaces
were covered with bench liner (Fisherbrand, P/N 14-127-47, Waltham, MA).
2.1 Coupon Preparation
For Task 1, test materials were 18 mm (0.71 in) diameter coupons prepared from primed and painted
wallboard paper and rough-cut barn wood (Figure 2-1). Forthe wallboard coupons, the interior kraft facing
was removed from 1/2-in thick gypsum wallboard (GOLD BOND, Home Depot SKU# 258-350). The paper
was primed with Kilz2 Latex primer and painted with Behr Interior Enamel paint. Circles of 18 mm (0.71 in)
diameter were punched from this primed and painted wallboard paper. Only the exterior surface of the
wallboard was used because the presence of gypsum interferes with spore recovery during extraction. For
the wood coupons, a hole saw (18 mm/0.71 in ID) was used to remove 0.3 cm (1/8 in)-thick circles from the
surface of exterior rough-cut barn wood (2.5 cm x 15.2 cm [1 in x6 in] pressure-treated Brazilian Pine Dog
Ear Picket Fence lumber [Product Number 884-831, Home Depot, Durham, NC]). These circles (wood and
wallboard paper) were then fastened to 18 mm (0.71 in) aluminum stubs (P/N 16119, Ted Pella, Inc.,
Redding, CA) using double sided tape (P/N 16073-2, Ted Pella, Inc., Redding, CA), resulting in a coupon.
These coupons were sterilized prior to use by steam autoclave utilizing a gravity cycle program consistent
with a NHSRC Biocontaminant Laboratory MOP 6570. Appendix B lists all associated MOPs for this project,
which can be found in the project QAPP or associated Amendments (listed in Section 4.5). All 18 mm (0.71
in) diameter (2.6 cm2 or 0.4 in2) coupons were utilized in the vertical orientation during testing.
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Figure 2-1.18 mm Rough-Cut Barn Wood Coupon (Task 1)
For Task 2, test materials were 35.6 cm x 35.6 cm (14 in by 14 in) coupons of rough-cut barn wood, painted
wallboard, and unsealed concrete. For Task 3, test materials were 35.6 cm x 35.6 cm (14 in by 14 in)
coupons of rough-cut barn wood and unsealed concrete. Coupons were sterilized according to procedures
found in Appendix A. All coupons for Tasks 2 and 3 were tested in the vertical orientation. Coupon
fabrication for both tasks is described below and outlined in MOP 3150:
• Painted Wallboard (Figure 2-2): A 35.6 cm x35.6 cm (14 in by 14 in, 1267 cm2 or 196 in2) piece of 1.3
cm (0.5 in)-thick wallboard was cut from a 1.2 m by 2.4 m (4 feet (ft) by 8 ft) sheet (Product Number
258-350, Home Depot, Durham, NC). The cut edges were sealed by applying a skim coat of joint
compound (Product Number 258-725, Home Depot, Durham, NC) to about 3.8 cm (1.5 in) of the
backside edge of the coupon. Using joint tape (5.1 cm or 2 in) (Product Number 430-684, Home Depot,
Durham, NC), one half of the tape (utilizing the factory fold) was applied to the back of the coupon. A
second skim coat of joint compound was applied over the first coat. After the joint compound was dry,
the coupon was turned over and a skim coat of joint compound was applied to the cut edge and about
2.5 cm (1 in) of the front edge. The tape was folded over the edge extending 1.3 cm (0.5 in) over the
front side of the coupon. A second skim coat was applied and allowed to dry. Using a sanding block
(Product Number 733-336, Home Depot, Durham, NC), any rough spots of the joint compound were
removed. One coat of KILZ Latex Primer (Product Number 317-390, Home Depot, Durham, NC) was
applied to the front side of the coupon and allowed to dry. This coat of primer was covered with one coat
of Behr Premium Plus interior flat white latex paint (Product Number 135-992, Home Depot, Durham,
NC). The back side of the coupon received one coat of Behr interior enamel paint (no primer was used
on the back) (Product Number 374-776, Home Depot, Durham, NC).
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Figure 2-2. Wallboard Coupon Front (Left) and Back (Right)
Rough-Cut Barn Wood (Figure 2-3): The material used to fabricate the 35.6 cm x 35.6 cm (14 in by 14
in) (1267 cm2 or 196 in2) coupons of rough-cut barn wood was 2.5 cm by 15.2 cm (1 in by 6 in)(nominal
lumber size, 1.3 cm by 13.7 cm [0.5 in by 5.375 in], actual) pressure-treated Brazilian Pine Dog Ear
Picket Fence lumber (Product Number 884-831, Home Depot, Durham, NC). The coupons were
assembled using two 35.6 cm by 13.7 cm (14 in by 5.375 in) pieces of lumber, plus one 35.6 cm (14 in)
long board ripped to 8.5 cm (3.35 in) wide, resulting in a 1267 cm2 (196 in2) coupon. The three pieces
were assembled with no spaces between boards, attached with 2.5 cm (1 in) staples from the backside,
using the same fence lumber material (perpendicular to the surface boards) as support.
Figure 2-3. Rough-Cut Barn Wood Coupon Front (Left) and Back (Right) (Note Offset of Wood Orientation)
• Concrete (Figure 2-4): Quikrete Sand/Topping (Product Number 10389, Home Depot, Durham, NC)
mix was used to fabricate 2.5 cm (1 in) thick, 35.6 cm x 35.6 cm (14 in by 14 in) coupons. The mix was
prepared according to the manufacturer's instructions and poured into forms. Surfaces were smoothed
with a hand trowel and the coupons were allowed to dry overnight. Once set, the coupons were
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removed and stacked on a pallet where they were wetted and covered with plastic to cure for at least 30
days.
Figure 2-4. Curing Concrete (Left) and Final Concrete Coupon (Right)
For Task 3, several tests required grimed coupons. Recently, a study was conducted to determine the
effects of surface grime on biological sampling efficiency10. The constituents of the grime used in that study
were reportedly similar to grime found on urban surfaces. No other documented recipes of urban grime
were found in the literature, so this grime formulation and concentration (1 g/ft2) was utilized for the current
study.
The constituents of the standardized grime included:
• 94% Arizona fine dust - National Institute of Standards and Technology (NIST)-traceable
• 3% Soot mixture:
10 g carbon black
1 g diesel particulate matter- NIST-traceable
0.5 g new 10W30 motor oil
0.5 g a-pinene (neat)
• 3% Mixture of biological materials:
4 g Lycopodium powder
4 g Ragweed pollen
4 g Paper mulberry mixture
This standardized grime was dissolved in ethanol. As discussed in Section 3.3, the grime was irradiated (40
kilogray, kGy) to sterilize it before use. A High Volume Low Pressure (HVLP) sprayer (CX-10, Croix Air
Products) was charged with an amount of the solution to deliver 1 g of grime to each coupon (See Figure 2-
5). All of the solution was sprayed onto the coupon, and the ethanol was allowed to evaporate from the
coupons under a hood for 15 minutes. This process was repeated for each coupon.
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Figure 2-5. Application of Grime to Coupons
2.1.1 Effect of Grime on Surface Recovery.
To determine the effect of grime on recovery of the target organism, preliminary tests were performed on
stainless steel coupons. Three sterilized stainless steel coupons were placed in the grime deposition hood
(three at a time). Coupons were handled with sterile gloves to minimize contamination. Grime was applied to
the coupons according to MOP 3163 and allowed to dry. Upon drying, coupons were removed from the
hood and placed under sterile aerosol deposition apparatus (ADA) pyramids as if being prepared for
inoculation. The pyramids were then removed and the coupons were sampled according to MOP 3144. The
wipe samples were transferred to the NHSRC Biocontaminant Laboratory, along with a field blank sample
(handled but unused wipe) and a coupon blank (a wipe sample from a sterile coupon).
The NHSRC Biocontaminant Laboratory extracted the samples according to MOP 6567, but then split the
samples into two 10 mL aliquots. One aliquot of each sample was then spiked with 1 x 104 Bacillus
atrophaeus spores (total). Three 10 mL aliquots of phosphate-buffered saline with Tween®20 (PBST) were
also spiked with the same quantity of spores. Recovery was then determined according to MOP 6535a.
2.2 Material Inoculation Procedure
The investigation of the effectiveness of the decontamination procedures requires that a target organism be
applied to a "sterile" material surface (i.e., material inoculation) at a precise target loading (e.g., spores per
piece of material [or coupon]). This section provides details on the target organism and material inoculation
procedures used for this investigation.
Having materials void of all living organisms (i.e., sterile) other than the test organism is necessary in
laboratory testing, albeit not realistic of real-world scenarios, because background contamination can
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confound the ability to collect and detect the target organism. This condition provides for a laboratory test
design which limits potentially confounding, uncontrolled variables, in order to provide a more reliable
understand if the impact of the intended parameters.
The inoculation of the coupons using aerosol deposition of viable spores, as opposed to the more traditional
use of dispensing precise amounts of liquid spore suspensions onto the material surface (liquid inoculation),
was used in this study to more closely represent the nature of the contamination experienced in past
"anthrax" incidents. Liquid inoculation has been the more commonly used method of inoculation for studies
of decontamination efficacy due to the ease and acceptable precision of the application of the spore
suspension.11'12'13'14 Recently, a highly repeatable (i.e., CV<50%) aerosol-based method of coupon
inoculation has been developed.15 This aerosol-based method of inoculation was utilized in this study,
consistent with its predecessor studies324, as it more accurately reflects contamination mechanisms
experienced during the 2001 letter attacks16.
2.2.1 Bacillus Spore Preparation
The test organism for this work was a powdered spore preparation of Bacillus atrophaeus (American Typr
Culture Collection [ATCC 9372]) and silicon dioxide particles. This bacterial species was formerly known as
8. subtilis var. niger and subsequently 8. globigii and is a commonly used surrogate for Bacillus
anthracis.14'23 The preparation was obtained from the U.S. Army Dugway Proving Ground (DPG) Life
Science Division. The preparation procedure is reported in Brown et al.17 Briefly, after 80 - 90 percent
sporulation, the suspension was centrifuged to generate a preparation of about 20 percent solids. A
preparation resulting in a powdered matrix containing approximately 1 x 1011 viable spores per gram was
prepared by dry blending and jet milling the dried spores with fumed silica particles (Deguss, Frankfurt am
Main, Germany). The powdered preparation was loaded into MDIs by the U.S. Army ECBC according to a
proprietary protocol. Quality assurance (QA) documentation is provided by ECBC with each batch of MDIs.
Control checks for each MDI were included in the batches of coupons contaminated with a single MDI. The
low and high inoculation targets used MDIs of differing inoculation doses as described in Section 2.2.2.
2.2.2 Coupon Inoculation Procedure
For Task 1,18 mm diameter (0.71 in) coupons were inoculated (loaded) with spores of 8. atrophaeus from
an MDI using the procedure detailed in MOP 3113.
For Tasks 2 and 3, 35.6 cm x 35.6 cm coupons (14 in by 14 in) were inoculated with spores of 8.
atrophaeus from an MDI using the procedure detailed in MOP 6561 and MOP 3161-LD for the high and low
inoculation levels, respectively. Briefly, each coupon was contaminated independently by being placed into
a separate dosing chamber designed to accommodate one 35.6 cm x 35.6 cm (14 in by 14 in) coupon of
any thickness. In accordance with MOP 6561 or MOP 3161-LD, the MDI was discharged a single time into
the dosing chamber. The spores were allowed to gravitationally settle onto the coupon surfaces for a
minimum period of 18 hours. After the minimum 18-hour period, the coupons were then removed from the
dosing chamber and moved to an isolated cabinet (Test Coupon Cabinet) which contained all loaded
coupons for a single test. Inoculated coupons were handled with care to minimize spore dispersal. One
person was tasked with removing the clamps holding the dosing chamber to the coupon and the removal of
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the dosing chamber and gasket from the coupon. A second person was then tasked with moving the coupon
to the proper location (e.g., test and positive control coupons to the Test Coupon Cabinet and blank
coupons to the Blank Coupon Cabinet). The Test Coupon Cabinet is a steel cabinet (1.2 m wide by 0.6 m
deep by 2.0 m high) (48 in wide by 24 in deep by 78 in high) with twelve shelves each 15.2 cm (6 inches)
apart. Each cabinet could store 36 coupons. Test and positive control coupons were arranged in each
cabinet according to material types. Procedural blank coupons of each material/orientation to be used in a
single test were contained in a separate isolated cabinet (Blank Coupon Cabinet) of similar construction but
with dimensions of 1.2 m wide by 0.6 m deep by 0.9 m high (48 in wide by 24 in deep by 36 in high) with 3
shelves.
There were originally two target contamination ranges: 1 x107 CPU (3 x 106 to 3 x 107 CPU) and 1 x 102
CPU (1.7 x 102 to 5.6 x 101 CPU). MOP 6561 outlines the higher target range and MOP 3161-LD outlines
the lower target range. The lower target proved to be problematic: a new lot of MDIs was not as reliable as
previous lots, and the 1 x 102 CPU target is at the low end of detection. An MDI with a listed concentration of
2 x 109 CPU was used for the 1 x 107 target recoveries, and an MDI with a listed concentration of 4.5 x 106
CPU was used for the low dose target.
The MDIs are claimed to provide 200 discharges per MDI. The number of discharges per MDI was tracked
so that use did not exceed this value. Additionally, in accordance with MOP 6561 and MOP 3161-LD, the
weight of each MDI was determined after completion of the contamination of each coupon. If an MDI
weighed less than 10.5 g at the start of the contamination procedure described in these MOPs, the MDI was
retired and a new MDI was used. For quality control of the MDIs, stainless steel control coupons were
inoculated as the first, middle, and last coupons within a single group of coupons inoculated by any one MDI
within a single test. These inoculation control coupons (35.6 cm x 35.6 cm or 14 in by 14 in) were
contaminated, sampled, and analyzed in the same manner as test coupons.
A log was maintained for each set of coupons that was dosed via the method of MOP 6561 or MOP 3161 -
LD. Each record in this log contained the unique MDI identifier, the date, the operator, the weight of the MDI
before dissemination into the coupon dosing device, the weight of the MDI after dissemination, and the
difference between these two weights. The coupon codes were pre-printed on the log sheet prior to the start
of coupon inoculation (dosing).
2.3 Experimental Approach
All three of the tasks being reported here were conducted in the custom-built test chamber (Figure 2-6) used
in predecessors of this study.32324. The chamber, located in High Bay Room 130 (H130) at EPA's Research
Triangle Park facility, has dimensions of 1.2 m high by 1.2 m wide by 1.2 m deep (4 ft high by 4 ft wide by 4
ft deep) and is designed to accommodate three 0.37 m wide by 0.37 m long (1.2 ft wide by 1.2 ft long)
coupons at a time in either orientation (horizontal or vertical, see below). The chamber is of solid stainless
steel construction with the exception of the front face and top which are fabricated from clear acrylic plastic.
The front face acrylic section is a door allowing full access to the inside of the chamber while standing
outside. The back stainless steel wall contains an assembly to hold the vertically oriented coupons
(maximum three 0.37 m by 0.37 m [1.2 ft by 1.2 ft] coupons at one time). To minimize cross-contamination,
experimentation began with procedural blanks (coupons of each material not contaminated with the target
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spore), followed by the test coupons of each material type. Only one material type occupied the test
chamber at a time with the exception of the blanks, in which all material types were processed concurrently.
The chamber is fitted with connections allowing HEPA-filtered air to enter and filtered exhaust to exit via a
readily accessible connection to the facility's air handling system. The chamber is also designed to be
decontaminated easily between runs using either liquids orfumigants, as needed. Decontamination of the
chamber is discussed in Appendix A.
The bottom of the chamber is pyramidal in shape with a 7.6 cm (3 in) drain in the center. The drain can be
closed or opened either to collect or release the runoff from the coupons during the decontamination
procedure. The bottom of the chamber has a 189 L (50 gallon) collection capacity.
Figure 2-6. Decontamination Chamber
2.4 Decontamination Procedure
It was critical for this project that each step in the decontamination procedure be implemented as uniformly
as possible for all sections and tests. Changes in technique during the study could lead to highly variable
data and bias the data leading to the drawing of erroneous conclusions. Therefore, the methods for each
step were documented in detail in order to provide as much standardization as possible. Staff performing
the decontamination procedures practiced each step in advance. Additional details can be found in
Appendix D.
For spray-based decontamination procedures, the spray wand was inserted into the center port (see Figure
2-7) and moved in and out as necessary to maintain the correct distance from the three coupons while
accomplishing the spray pattern described in Appendix D (decontamination application methods and rinsing
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with water). Every effort was made to perform this step consistently and maintain the correct distance from
all materials. The port also allowed the chamber door to remain closed during application of the
decontamination solutions. During the spraying of the decontamination solutions with the backpack sprayer,
the front face door was closed and sealed. The seal was designed to contain any splashed liquid.
Maintaining the door in a closed position also prevented exposure of the worker to the toxic fumes from
decontamination solutions during application.
The general approach for all spray-based methods was as follows:
1) wet the surface with decontaminant (pAB) using a precise duration and flow rate
2) reapply the decontaminant if prescribed
3) allow the coupons to dry overnight
4) sample surfaces for surviving spores
Figure 2-7. Spraying Through Center-Aligned Port in the Small Chamber Door
Application of the pAB spray on drywall surfaces, though effective against spores, could cause damage to
the drywall. Hence, a decontamination method was evaluated using pAB-wetted wipes and/or spritzing (light
spraying) of decontaminant. Three tests were conducted to determine the optimal wetted wipe
decontamination technique on 35.6 cm x 35.6 cm (14 in by 14 in) drywall coupons in accordance with MOP-
3156:
• Test 1: Spritzing with pAB, then wiping with pAB-wetted wipe
• Test 2: Wiping with SimWipe (SimChem, Product #: 67F020-01-50, Sarasota, FL)
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• Test 3: Combination of wiping with SimWipe, then spritzing with pAB and wiping with pAB-wetted wipe,
consecutively
• Test 4: Spritzing with pAB, then wiping with pAB-wetted wipe on coupon with lower inoculum.
The coupons were spritzed with pAB before the pAB wetted wipe procedure only (Test 1 and Test 3). The
coupons were allowed to dry overnight, and wipe samples were collected from them one day following the
decontamination. Recovery was compared to drywall coupons inoculated the same day as the test coupons
that did not undergo any decontamination procedure (positive controls).
2.5 Test Matrix
In Task 1, the impact of the age of pAB on sporicidal efficacy was evaluated on small scale (18 mm [0.71 in]
diameter; 2.6 cm2 [0.4 in2]) coupons of rough-cut barn wood and primed and painted wallboard paper. The
materials were inoculated with Bacillus atrophaeus (formerly Bacillus globigii) spores at 7 log CPU (± 0.5 log
CPU). The decontamination solutions were freshly prepared pAB (used within 15 minutes after preparation),
and also used at 2, 4, 8, 24, and 32 hours after preparation. The samples collected and analyzed fo viable
bacterial spores during this task included whole-coupon extractions from five (5) replicate test coupons of
each material type and from two sets of positive controls (one per day of testing) and aliquots of rinsates.
Samples were collected directly from the pAB solution for determination of pH and FAC. In addition, aliquots
of pAB were collected during spray application, to determine post-spray FAC.
A simplified decontamination procedure was used for Task 1. The backpack sprayer was used to spray pAB
at 1000 mL/min for 15 seconds at 0 and 15 minutes. The spray pattern, movement, and duration were the
same as for the medium-sized coupons (see Appendix D), and based upon results from predecessor
tests.3'24 Coupons were then collected and placed into extraction buffer to quench the sporicidal activity
promptly following a 30-minute contact time.
The objective of Task 2 was to refine application rates and frequency so that a maximum surface area could
be fully decontaminated (high efficacy), if achievable, in the least amount of time (low effort). For this effort,
Task 2 the overall effectiveness of decontamination methods were evaluated as a function of application
parameters and spore load (surface concentration). Coupons (35.6 cm x 35.6 cm or 14 in x 14 in) of rough-
cut barn wood, painted wallboard, and concrete were positioned vertically during testing, but were
inoculated horizontally with 1 x 107 Bacillus atrophaeus spores via aerosol deposition. Decontamination
procedures were evaluated for each material type with the decontaminant application process occurring in
the spray chamber as described previously.32324. Overall decontamination effectiveness was determined as
a function of the procedures and material types. Samples included wipes from two coupon materials
(painted wallboard and concrete) and from stainless steel inoculation control coupons, vacuum sock
samples from rough-cut barn wood coupons, aliquots of runoffs, samples of aerosolized spores, and
aliquots of the pAB for pH and FAC measurements. Each test included three replicate test coupons, three
replicate positive control coupons, and one procedural blank of each material type, resulting in a total of
seven coupons for each material type during each test condition. The backpack sprayer was used for
application of the pAB solution to all three test materials.
The decontamination procedure used in the test matrix was based upon results reported previously. 32324
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Results from these studies suggest:
• Unless significant amounts of debris are present on the surface to be decontaminated, no vacuuming
step is required before the pAB/TSP solution application;
• Completely cover the surface with pAB solution for the desired 30-minute contact time using the back-
pack sprayer for application to all materials (rough-cut barn wood, painted wallboard, and concrete); and
• For comparison of the efficacy of the spray-based method to the efficacy of wetted wipes during drywall
surface decontaminations, wipes should be wetted with pAB solution.
The sampling strategy included enumeration of viable spores remaining on coupons from decontaminated
and non-decontaminated (control) coupons, enumeration of spores in runoffs and aerosols as a result of
relocation during decontamination, and evaluation of pAB (pH and FAC). Sampling methods are fully
described in Appendix E.
The temperature and pH measurements of the pAB solution and Dl water were conducted at the initiation of
a test and prior to the start of each test set (i.e., material type) throughout a test. The flow rate from the
backpack sprayer was measured at the start and end of each set of three coupons on which the sprayer
was used. The spray pattern for the backpack sprayer was confirmed (and adjusted as needed) prior to the
start of a test. These methods and the quality control criteria are outlined in the project QAPP entitled,
"Quality Assurance Project Plan for the Assessment of Liquid and Physical Decontamination Methods for
Environmental Surfaces Contaminated with Bacterial Spores: Part 4 - Optimization of Method Parameters
and Impact of Surface Grime.
,,18
For Task 2, the test matrix evolved based on the results from previous tests. The matrix as performed is
shown in Tables 2-1 and 2-2.
Table 2-1. Test Conditions for High Inoculation Parametric Tests (Task 2)
Test ID
O1
O2
O3
O4
Materials
Drywall, Concrete,
Wood
Drywall, Concrete,
Wood
Wood
Wood
Decontamination Steps
1 application, 15 seconds spray per 3 coupons,
no reapplication, no rinse
1 application, 15 seconds spray per 3 coupons,
no reapplication, no rinse
1 application, 30 seconds spray per 3 coupons,
no reapplication, no rinse
2 applications (at 0 and 5 min), 15 seconds
spray per 3 coupons, no reapplication, no rinse
Prescribed
Decontaminant
Application Flow Rate
Low(1 L/min)
High(1.5L/min)
Low(1 L/min)
Low(1 L/min)
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O5
Wood
2 applications (at 0 and 15 min), 15 seconds
spray per 3 coupons, no reapplication, no rinse
Low(1 L/min)
Table 2-2. Test Conditions for Low Inoculation Parametric Tests (Task 2)
Test ID
O6
O7
O8
O9
O10
Materials
Drywall, Concrete,
Wood
Drywall, Concrete,
Wood
Wood
Wood
Wood
Decontamination Steps
1 application, 15 seconds spray per 3 coupons,
no reapplication, no rinse
1 application, 30 seconds spray per 3 coupons,
no reapplication, no rinse
1 application, 30 seconds spray per 3 coupons,
no reapplication, no rinse
2 applications (at 0 and 5 min), 15 seconds
spray per 3 coupons, no reapplication, no rinse
2 applications (at 0 and 15 min), 15 seconds
spray per 3 coupons, no reapplication, no rinse
Prescribed
Decontaminant
Application Flow Rate
Low(1 L/min)
Low(1 L/min)
Low(1 L/min)
Low(1 L/min)
Low(1 L/min)
In addition, another set of tests were incorporated into Task 2 to determine the decontamination
effectiveness of wetted wipes at reducing contamination on painted wallboard coupons (Table 2-3). In these
tests, pAB solution was spritzed (lightly sprayed) onto the coupon surface with a spray bottle before
decontamination was attempted with a wetted wipe.
Table 2-3. Test Conditions for Wetted Wipe Decontamination Tests (Task 2)
Test ID
W1
W2
W3
W4
Materials
Drywall
Drywall
Drywall
Drywall
Decontamination Steps
Spritzing with pAB, then wiping with pAB-
wetted wipe
Wiping with SimWipe
Combination of wiping with SimWipe, then
spritzing with pAB and wiping with pAB-wetted
Spritzing with pAB, then wiping with pAB-
wetted wipe
Inoculation Level
High
High
High
Low
In Task 3, the impact of grime on surface decontamination efficacy of rough-cut barn wood and concrete
was determined. The coupons were prepared, inoculated, and sampled in accordance with the procedures
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described in Task 2. In addition, standardized grime (see Section 2.1) was applied to coupons prior to
inoculation, as noted in the test matrix (Table 2-4).
Table 2-4. Task 3 Test Matrix, Impact of Grime on Wood and Concrete Surface Decontaminations
Test
G1
G2
G3
G4
G5
G6
G7
G8
Decontaminant
pAB solution
pAB solution
pAB solution/TSP solution
pAB solution/TSP solution
pAB solution/TSP solution
pAB solution/TSP solution
pAB solution/TSP solution
pAB solution/TSP solution
Material
Wood
Concrete
Wood
Concrete
Wood
Concrete
Wood
Concrete
Wood
Concrete
Wood
Concrete
Wood
Concrete
Wood
Concrete
Inoculation
High
High
High
High
High
High
High
High
Grime
No
Yes
No
Yes
No
Yes
No
Yes
Scrubbing
No
No
No
No
Yes
Yes
Yes
Yes
Vacuuming1
No
No
No
No
No
No
Yes1
Yes1
1 Vacuum step is the first step performed
Samples included wipes from concrete coupons and from stainless steel inoculation control coupons,
vacuum sock samples from rough-cut barn wood coupons, aliquots of runoffs, samples of aerosolized
spores, swab samples of vacuum cleaner parts, and aliquots of the pAB for pH and FAC measurements.
Three replicate test coupons, three replicate positive control coupons, and one procedural blank of each
material type were included in each test during Task 3.
The decontamination procedure (see Appendix D) used for Task 3 was a combination of up to four
decontamination steps:
1. Vacuum the surface using the squeegee attachment of a wet/dry vacuum cleaner.
2. Spray the coupon with decontamination solution for 10 seconds at lowest flow rate (1000 mL/min
for pAB, 1300 mL/min for pAB/TSP solution).
3. Scrub the coupon with a brush.
4. Allow a 30-minute contact time.
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In tests G1 through G6, no vacuum step (step 1) was utilized. In tests G1 through G4, no scrubbing (step 3)
was utilized.
Two decontamination solutions were used: pAB and pAB with TSP substitute surfactant (pAB solution/TSP
solution). Preparation of the two decontamination solutions is described in MOP 3128-A and 3128-B,
respectively.
2.6 Sampling Points
Wipe sample or vacuum sock (Midwest Filtration, Cincinnati OH) samples from test (decontaminated)
coupons were collected after a minimum of 18 hours of drying, when coupon surfaces appeared visibly dry.
Positive control coupons were sampled at the same time as test coupons. Wipe samples and vacuum sock
samples were collected by sampling within a 34.3 cm by 34.3 cm (13.5 in by 13.5 in) sampling template
centered on the coupons (Figure 2-8).
Figure 2-8. Sampling Template Centered on a Representative Concrete Coupon
Extractive samples of Task 1 coupons were taken immediately after decontamination. The coupons were
aseptically transferred to sterile vials while in High Bay Room H130 (the location of the spray chamber).
Positive control extractions were conducted after the last test coupons had been extracted.
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Runoff was neutralized in situ with enough STS to neutralize all bleach sprayed onto the coupons. Aliquots
of runoff were collected immediately after all of the runoff from a decontamination procedure had been
generated. More details of run-off sample collection and neutralization are provided in Section 2.7.
Aliquots of pAB for FAC and pH were collected and analyzed immediately (within 10 minutes) before use.
Aerosol samples were isokinetically collected during active decontamination activities, from the chamber
exhaust duct (Figure 2-9) using a SKC BioSampler® (Model No. 225-9595). The sampling point was eight
diameters downstream of and two diameters upstream of any flow disruptions.
Figure 2-9. Spray Chamber Exhaust Duct with Arrow pointing to Sample Port
Swab samples were collected using MOP 3135 from the vacuum cleaner nozzle and HEPA filter after use.
• For each coupon set, a swab sample was collected from the HEPA-rated filter within the wet/dry
vacuum (if used in the test) and qualitatively analyzed to confirm contamination by the target organism.
Such information is relevant to the treatment of the vacuum after use and the potential spread of
contamination.
• Two or more exhaust samples were collected, one from the blank wet/dry vacuum (when used) and one
or more from the test coupon wet/dry vacuums (when used). These samples were collected because of
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the potential for the wet/dry vacuum to spread contamination via the presence of viable spores in the
exhaust. This potential was assessed by collecting a composite sample from all vacuuming in a test.
2.7 Sampling and Analytical Procedures
Five types of biological samples were included in this project. These samples are described below and in
Table 2-4.
• Extractive samples for quantitative determination of viable spores on coupons were used for Task 1.
• Surface sampling procedures were used in Tasks 2 and 3 to collect samples from the coupon
materials. The sampling procedures included wipe sampling or vacuum sampling for quantitative
determination of viable spores on coupon surfaces. Additionally, wet swab sampling was done to
qualitatively determine the presence of the target organism on wet/dry vacuum filters and for sterility
checks of materials and equipment prior to testing.
• The rinsate generated during the decontamination procedure was collected for each material type
(Tasks 2 and 3).
• HEPA filters from the wet/dry vacuums used for each coupon set were removed and sampled by
swabbing (Task 3).
• Aerosol samples were collected from the exhaust air flow exiting the decontamination chamber during
decontamination operations to quantify spores aerosolized during spray applications (Tasks 2 and 3).
Table 2-4. Sample Types
Sample Type
Extractive
sample
Wipe Sample
Vacuum Sock
Sample
Sample Medium/ Source
18 mm coupons
Stainless steel, drywall, and
concrete coupons
Rough-cut wood coupons
Task
Taskl
Task 2 and 3
Task 2 and 3
Purpose
Quantify CPU recovery
Quantify CPU recovery from
material surface
Quantify CPU recovery from
material surface
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Swab samples
Swab samples
Rinsate Filter
Aerosol
Representative Coupons
Wet/Dry Vacuum filter
Runoff from coupons during
decontamination procedure
Air from chamber during
decontamination procedure
Task 2 and 3
TaskS
Task 2 and 3
Task 2 and 3
Qualitatively indicate sterility of
coupon surface before
inoculation
Qualitatively indicate spore
presence
Quantify CPU in rinsate/runoff
Quantify re-aerosolized CPU
during decontamination
procedure
A sampling event log sheet was maintained for each sampling event (or test). The names of the sampling
team members, date, run number, and all sample codes with corresponding coupon codes were recorded
on each sheet. The coupon codes were pre-printed on the sampling event log sheet prior to the start of
sampling. The materials and equipment used as well as the sampling protocols for sampling are detailed in
Table D-2 in Appendix D.
2.7.1 Extraction Sampling
The 18 mm (0.71 in) coupons used in Task 1 were analyzed by whole-coupon extraction and plating.
Following treatment, coupons were transferred aseptically into 50 ml sterile vials containing 10 ml
Phosphate Buffered Saline (PBS) + 0.05% Tween®20 (PBST) (P3563, Sigma-Aldrich). This operation was
performed in the decontamination chamber in H130. The vials containing the extraction solution with
neutralizing STS and coupon were transferred to the NHSRC Biocontaminant Laboratory, where they were
sonicated for 20 minutes at 42 kHz and 135 Watts using a Branson 8510 ultrasonic water bath to extract
spores from the coupon surface. The solution and coupon were then vortexed 2 minutes to further dislodge
any viable spores. Each vial was briefly re-vortexed immediately before any solution was withdrawn. The
solution was subjected to five sequential 10-fold serial dilutions (when necessary) following MOP 6535a.
Each dilution (0.1 ml) was spread-plated in triplicate onto TSA using sterile beads according to MOP 6555
and incubated at 35°± 2 °C for 18-24 hours. CPU were manually counted.
2.7 A A Extraction Method Development
The 18 mm (0.71 in) stubs were placed directly into extraction solution (PBST) using aseptic techniques
immediately following decontamination. These stubs were still wet from the pAB application and may have
contained enough decontamination liquid to confound enumeration analysis. For this reason, prior to testing
described in Section 3.1.2, the following extraction method development test was performed to determine
the effects of residual decontaminant on recovery estimates.
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Sterilized 18 mm (0.71 in) coupons of rough-cut wood and primed and painted wallboard paper were
attached to the center of a 35.6 cm x 35.6 cm (14" x 14") stainless steel mock medium-sized coupon. The
coupons were sprayed with fresh pAB in accordance with Appendix D, as if they were a subsection of a
medium sized coupon. The backpack sprayer was used to apply pAB at 1000 mL/min for 5 seconds per
stainless steel mock medium-sized coupon, with two applications 15 minutes apart, with an additional 15
minute contact time before extraction. The coupons were then placed in extraction solution. A second set of
sterilized but unsprayed coupons were placed directly in the extraction solution. Both sets were then spiked
with a liquid spore inoculum and allowed to sit for one hour before analysis. Based on the results, discussed
in Section 3.1.1, a molar equivalent amount of STS to neutralize 28 ppm FAC was added to the extraction
liquid fordrywall coupons but not for wood coupons.
2.7.2 Factors Affecting Sampling/Monitoring Procedures
Sampling of coupon surfaces was conducted after coupons that were wetted during the decontamination
procedure had become visibly dry. Drying was allowed to occur in the Decontaminated Coupon Cabinet or
Procedural Blank Cabinet, facilitated by a slight air flow provided by 5 liters per min (Lpm) of filtered and
dried compressed air. All coupons were allowed to dry for at least 18 hours. The actual time that each
coupon was allowed to dry was recorded in the laboratory notebook. The biases associated with sampling
previously wetted surfaces are unclear, but others have suggested reduced recoveries of viable agent on
1Q
such surfaces.
2.7.3 Preparation for Sampling/Monitoring
Within a single test, surface sampling of the material sections was completed for all procedural blank
coupons first before sampling of any test material sections was performed. This order of operations was
followed to minimize the potential for cross-contamination (e.g., sampling progressed from the least
contaminated to the most contaminated samples). Surface sampling was conducted either by wipe sampling
or vacuum sock sampling in accordance with the protocols included in Appendix E. The surface area for all
samples was 0.12 m2 (1.27 ft2).
A sampling material bin was stocked with all appropriate items (consistent with the protocols in Appendix E)
for each sampling event. The bin contained enough wipe sampling kits to accommodate all required
samples for the specific test. An additional kit was also included for backup. Enough gloves and bleach
wipes needed to complete the test were available. A sample collection bin was used to transport samples
back to the NHSRC Biocontaminant Laboratory for analysis following collection. The exterior of the transport
container was decontaminated by wiping all surfaces with a Dispatch® (Clorox) bleach wipe prior to
transport from the sampling location to the NHSRC Biocontaminant Laboratory. To ensure the integrity of
samples and to maintain a timely and traceable transfer of samples, a proven documented chain of custody
(CoC) procedure was followed for each test.
2.7.4 Wipe Sampling
To determine the amount of spores residing on the coupon surface after treatment and assess the
effectiveness of the decontamination procedure, wipe sampling was performed for each painted wallboard
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and concrete coupon. Wipe sampling is typically used for small sample areas and is effective on nonporous
smooth surfaces such as ceramics, vinyl, metals, painted surfaces, and plastics.17 Wipe samples were also
chosen as the preferred surface sampling method for concrete coupons to allow comparisons with results
from predecessor testsS, in which wipe sampling was found to perform better with respect to higher number
of spores recovered for this type of material and lower standard deviations among repeat tests. Wipe
sampling was conducted according to MOP 3144. The general approach is that a moistened sterile
noncotton pad is used to wipe a specified area to recover bacteria, viruses, and biological toxins.17 The
protocol used in this project is described in MOP 3144 and has been adapted from that provided by Busher
etal.,2
6567.
et al.,20 and Brown et al.17 Microbiological analysis of the wipe sample was conducted according to MOP
Wipe samples were generally processed on the same day on which they were collected. The concrete wipe
samples collected a great deal of debris and fine particles. It is speculated that this confounded filter-plate
analyses (conducted in accordance with MOP 6565), as some of the samples yielded no viable spores
recovered, when spread-plate analyses (conducted in accordance with MOP 6535a) indicated viable spores
were recovered. Vacuum Sock Sampling
Vacuum sock sampling was conducted on 35.6 cm x 35.6 cm (14 in by 14 in) rough-cut barn wood coupons
because of the difficulty implementing the wipe sampling procedure on the rough surface, which snags the
wipe fabric. Vacuum sock sampling was conducted according to MOP 3145. Microbiological analysis of the
vacuum sock sample was conducted according to MOP 6572.
Vacuum socks samples were generally processed on the same day on which they were collected. The
rough-cut barn wood samples collected a great deal of debris and large wood fragments. This grade of
wood is neither planed nor weathered and does have a high capacity to splinter. Vacuum sock extracts
were filter-plated in accordance with MOP 6565. Some of these samples resulted in no viable spores
recovered, which may be due in part to the co-collected debris. Figure 2-10 shows a picture of a 1 ml filter
plate (left) and a 14.5 ml filter plate (right). Some samples had to be split among several filters due to the
abundant debris clogging the filter.
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Figure 2-10: Filter Plates of Samples from Rough-Cut Wood.
2.7.5 Swab Sampling
MOP 3135 was followed for collection of swab samples. The general approach is to use a moistened swab
to wipe a specified area to recover bacterial spores. Swab samples were collected from the vacuum cleaner
HEPA filter during Task 3. Swab samples were also used to confirm sterility of materials before use in
experimentation. Swab samples were analyzed according to MOP 6563.
2.7.6 Run-off Collection and Sampling
In a field application, decontaminant runoff could pool in a location with a high oxidation demand (such as
soil). This demand would easily quench remaining sporicidal activity of the decontaminant, resulting in
spores evading decontamination and potentially being relocated to uncontaminated areas. To simulate this
immediate neutralization, STS, which reacts with and neutralizes chlorine and hypochlorite ions, was added
to the runoff collection vessel before use. Prior to decontamination, the runoff collection vessel was charged
with enough STS to neutralize all bleach sprayed onto the coupons. Thus, the hypochlorite component of
the pAB was neutralized immediately upon collection. The runoff from the coupons throughout the entire
decontamination procedure being tested was collected for a given coupon set (material type or all blanks).
After all coupons from a single set were moved to the Decontaminated Coupon Cabinet or Procedural Blank
Cabinet, the chamber was rinsed with Dl water. A pre-weighed, sterile runoff collection carboy collected the
runoff. The total mass of liquid collected was recorded by comparison to the tare value. After collection,
triplicate 100 ml aliquots were taken using aseptic technique. The aliquot collection procedure was
performed as follows:
1. Sampler donned a face mask, pair of examination gloves, disposable lab coat, and bouffant cap.
2. The contents of the carboy were agitated to ensure homogeneity.
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3. The carboy cap was removed.
4. Using a new 100 ml sterile serological pipette, sampler aseptically pipetted 100 ml of sample
into a sterile 4 oz. container.
Step 4 was repeated until triplicate samples were obtained.
The runoff aliquot containers were triple-contained in sterile bags and transported to the NHSRC
Biocontaminant Laboratory for submission and analysis at the conclusion of the entire test. The runoff was
stored at 4 ± 2 °C until processed. Processing occurred within 24 hours.
This method of neutralizing the runoff simulates the worst-case scenario for spore survivability in the run-off.
2.7.7 Aerosol Sampling
A 10 cm (4 in) diameter galvanized duct which was 112 cm (44 in) in length was attached to the chamber to
allow for precise flow measurements and isokinetic sampling. The duct was attached to the chamber using
a coupling and a 90-degree elbow. The sampling port was located 81 cm (32 in)(8 diameters) downstream
from the 90-degree elbow which was connected to the chamber and 30.5 cm (12 in)(3 diameters) from the
bend in the flexible duct that connects to the main exhaust. This 10.2 cm (4 in) galvanized duct was
isokinetically sampled using a standard Method 5-type21 meter box coupled with a SKC BioSampler®
(Model No. 225-9595). Integral to the BioSampler® are critical sonic orifices that require a minimum vacuum
of 38 cm (15 in) Hg across the orifices from the sample pump. This vacuum, in turn, provides a constant
flow of 12.5 Lpm. The inlet of the BioSampler® was connected to a 0.5 cm (0.185 in) buttonhook nozzle so
that the sample gas velocity was isokinetic to the duct velocity. Isokinetic variation was calculated per EPA
Method 521, Section 12.11. The BioSampler® was filled with 15 ml PBST during operation. As discussed in
Section 3, the STS was added to the collection liquid for tests following Test O5. Operation time was limited
to a maximum of 1 hour to prevent evaporation of the liquid, but no sample required operation for more than
30 minutes. The aerosol sample was analyzed in two parts per MOP 6568. To collect particulate that
impinges on or settles in the nozzle and inlet tubing prior to the collection liquid, the nozzle and inlet are
rinsed with 50 ml PBST. This rinse is analyzed independently of the collection liquid itself.
2.7.8 pAB Sampling
The pAB was sampled in two ways for two different analyses. During production of the pAB, a 5 ml aliquot
from the bulk mixing vessel was collected for FAC analysis. To determine the pH, a probe (Acorn pH5,
Oakton, Vernon Hills, IL) was placed in the bulk mixing vessel. During use for decontamination procedures,
a 50 ml aliquot was removed from the backpack sprayer tank prior to use, and a 50 ml aliquot was taken
from the graduated cylinder following the flow measurement taken immediately before use in a
decontamination procedure. The FAC and pH of these aliquots were also determined.
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2.7.9 Additional Samples Collected
Samples were collected to describe both the decontamination process itself and the efficacy of its
application. An overview of these tasks is described below to provide context for the results described in
Section 3.
The objective of the study was to assess the effectiveness of decontamination procedures for reducing
surface contamination and to refine the procedures for maximum benefit (low effort and high effectiveness).
The effectiveness is measured by the determination of the log reduction calculated per Section 1.3. Hence,
surface sampling of the test areas before and after decontamination was required to determine the log
reduction after application of the procedure. Since current surface sampling techniques are intrusive (i.e.,
they remove viable spores from the surface of the section), separate positive control and test coupons had
to be used to compare pre- and post-decontamination recoveries. Positive control coupons were inoculated
on the same day and analyzed on the same day as test coupons, but were not decontaminated.
The effectiveness of removing contamination from the surface of the sections provides critical information
regarding the utility of the procedure. However, field applicability is also dependent upon several other
factors including the ultimate disposition (or fate) of the spores. This latter information is required to provide
information pertinent to the development of a comprehensive site-specific remediation strategy. For
example, if viable spores are washed off materials (e.g., transferred unharmed from surfaces to runoff
water), remediation field strategies might require runoff collection and treatment. Hence, it is important to
gain a holistic understanding of the fate of the spores during decontamination procedures.
To assess the fate of spores during decontamination, several samples in addition to the surface samples
were collected. To assess the maximum potential for viable spores to be washed off the surfaces, all liquids
used in the decontamination process were collected in a vessel with enough STS to neutralize all bleach
sprayed onto the coupons and quantitatively analyzed. These were composite samples for a set of replicate
coupons during a decontamination procedure. Runoff samples were analyzed quantitatively to determine
the disposition of viable spores in this medium. The volume of liquid collected in each section set was
measured after collection. Aerosol samples were also isokinetically sampled from the chamber duct to
assess the potential for re-aerosolization of spores due to the decontamination process itself.
The procedures tested herein were based upon the results from previous studiesS2324, andwere originally
developed based upon use for the remediation of the wooden shed in Danbury, CT, and via an interagency
workgroup on foreign animal disease threats. All materials and equipment utilized during testing, as well as
the sampling protocols, are detailed in Appendices D and E.
2.7.10 Split Samples
While no samples were split for separate analyses, replicate aliquots were taken of some samples (replicate
rinsate sample aliquots collected to analyze a larger portion of the sample yet contain samples in small
vials). In the cases where replicate samples produced results within the acceptable range, these data were
averaged. The BioSampler® includes two portions: a rinse of the nozzle and all glassware exposed to the
aerosol between the duct and the collection liquid, and the collection liquid itself. These two portions of the
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sampler were analyzed independently, yet the data were combined for one estimate of bioaerosol recovery
for each sample.
2.7.11 Sample Analyses
Analyses of all biological samples were conducted in the on-site NHSRC Biocontaminant Laboratory. PBST
was used as the extraction buffer. After the appropriate extraction procedure, as described in Appendix F,
the samples were plated, incubated, and analyzed (CPU enumerated) in accordance with MOP 6535a.
Appropriate dilutions of the extracted sample (i.e., the initial undiluted sample extraction dilution, and up to a
five-stage serial dilution [10~1 to 10~5]) would be plated depending on expected CPU concentration. For
example, the last three dilutions (10~3,10~4 and 10~5) might not be plated fora decontaminated sample if a
low CPU concentration (high decontamination efficacy) was expected.
In addition to the analysis in MOP 6535a, supplementary analysis procedures were used for samples
resulting in less than 30 CPU/sample in the undiluted sample extract (e.g., wipe in the extraction buffer).
These analyses were used to lower the current detection limit associated with MOP 6535a. In accordance
with MOP 6565, Revision 2, samples were filter-plated.
The PBST was prepared according to the manufacturer's directions and in accordance with MOP 6562,
dissolving one packet in one liter of sterile water. The solution was then vacuum-filtered through a sterile
0.22 urn filter unit to sterilize.
The extraction procedure used to recover spores varied depending upon the different matrices (e.g., wipes,
vacuum socks). The procedures are described in Appendix F.
2.7.12 Coupon, Material, and Equipment Cleaning and Sterilization
Several management controls were administered in order to prevent cross-contamination. This project was
labor intensive and required that many activities be performed on coupons that were intentionally
contaminated (test coupons and positive controls) and not contaminated (procedural blanks). The treatment
of these three groups of coupons (positive control, test, and procedural blank) varied for each group. Hence,
specific procedures were put in place in an effort to prevent cross-contamination among the groups.
Each test in the experimental matrix included four primary activities. These activities were preparation of the
coupons, execution of the decontamination process (including sample recovery), sampling, and analysis.
Specific management controls for each of these activities, as well as cleaning methods put in place to
prevent cross-contamination, are shown in Table 2-5. Appendix A details the coupon, test chamber and
equipment cleaning and sterilization procedures.
Table 2-5. Cleaning Methods and Frequency for Common Test Materials/Equipment
Material/Equipment
Use
Cleaning Method
Frequency
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Material/Equipment
Decontamination Procedure
Chamber
Coupon Cabinets
Backpack Sprayer
Distilled Water Tanks
(Reservoir)
Wet/Dry Vacuums
Heads of Wet/Dry Vacuums
Other Bulk Equipment
(Deposition Housing and
Gaskets, Templates, etc.)
All Work Surfaces
Use
Contain coupons during
the application of the
decontamination
procedure being tested
Store coupons prior to
testing and/or sampling
Used to apply
decontamination solution
Utilized during chamber
decontamination (reset)
procedure
Part of the
decontamination
procedure
Part of the
decontamination
procedure
Various
Throughout each test
Cleaning Method
Washing with pH-adjusted
bleach solution, or wiping
with Dispatch® Bleach
Wipes, rinse with Dl water
pH-Adjusted bleach solution
or wiping with Dispatch®
Bleach Wipes, rinse with Dl
water followed by EtOH
Purge with decontamination
solution before use
Bleach solution, soak
overnight
Fumigation with hydrogen
peroxide
Fumigation with hydrogen
peroxide
Fumigation with hydrogen
peroxide or washing with
pAB solution in accordance
with Appendix A.
Cover with new bench liner
Frequency
Before/after each test and
between test materials
Before/after each test
Before each test
Treated before each test
(within 48 hours of the test
start)
Before each use
Before each use
Varied
Before/after each use
(cleaning of surfaces
between handling of
replicate coupons during
sampling)
Due to the amount of waste and reusable items (requiring decontamination after use) generated during this
testing (e.g., sterilization bags, sampling templates, etc.), creation of a rigid plan to segregate such items
was imperative. Reusable items were clearly distinguished and separated from waste items after use and
put in distinct segregated locations within the testing area.
During the decontamination procedure for Task 2, one person (sample handler) was tasked with moving the
coupons to the decontamination chamber. A different person was tasked with moving the treated coupon to
the drying cabinets. Disposable laboratory coats were worn by the sample handler (tasked with moving the
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coupons) to further minimize the potential of cross-contamination. The sample handler donned new gloves
and a new disposable laboratory coat after moving a complete set (i.e., three of test samples) from the test
coupon cabinet to the decontamination chamber.
All bins, buckets, and containers remained closed or covered unless in use (e.g., material being placed into
or extracted from the bin, bucket, or container). Adequate cleaning of all common materials and equipment
was critical in preventing cross-contamination.
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3. Results and Discussion
The test matrix (modification of remediation activity parameters) evolved (i.e., adaptive management
approach) as results were obtained during the testing campaign. Tests were progressive in that details of
one test were used to inform and design subsequent tests in attempts to optimize the decontamination
process (speeding up slow steps without reducing efficacy and eliminating ineffective or counterproductive
steps). Therefore, the ultimate goal was to achieve maximum benefit (low effort and high effectiveness) with
off-the-shelf products.
In addition to reduction of contamination from material surfaces, determination of the ultimate fate of the
spores was also a critical measurement objective. Combined, this information can inform selection or further
development of appropriate, situation-specific decontamination procedures. Following discussion of the
individual decontamination procedure results, the ultimate fate of the spores and decontamination worker
exposure due to the procedures was explored, when possible.
All p-values reported are based on single factor, two-sided analysis of variance (ANOVA) of log-transformed
CPU recovered or log reductions with an alpha value of 0.05.
3.1 Task 1: Impact on Efficacy of the Degradation of the pAB Solution over Time
3.1.1 Extraction Method Development
The results of the extraction method testing described in Section 2.7.1 are shown in Table 3-1. To test the
effect of residual pAB on CPU recovery, extractions of sprayed and unsprayed coupons were spiked with a
liquid spore inoculum as shown in Table 3-1. Five replicate coupons of each type (sprayed, and unsprayed)
were tested.
The residual hypochlorite concentration (FAC) from sprayed coupons may interfere with spore recovery;
thus the neutralization of FAC by STS may be important. A series of tests was completed to determine the
amount of STS solution needed to neutralize the FAC in the buffer solution. Table 3-2 shows the data for the
two sprayed materials tested (six 18-mm wallboard paper coupons and six 18-mm wood coupons) titrated
with 0.000375N STS solution. The coupons were subjected to two 15-second pAB applications using a
starting 6630 ppm FAC bleach solution. When pAB-sprayed drywall coupons were added to the extraction
fluid, the FAC in the buffer rose to between 4 and 28 ppm. A molar equivalent amount of STS was added to
the extraction fluid to neutralize 28 ppm FAC during Tests STS 2 and STS 3, which were conducted with
drywall only, because wood coupons did not show any rise of the FAC in the extraction buffer.
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Table 3-1. Recovery from Neutralized (Sprayed) and Control (Not Sprayed) Test Samples (n = 5).
Test ID
STS1
STS 2
STS3
STS4
Description
NoSTS
Extraction
fluid with
STS
Extraction
fluid with
STS
NoSTS
Material
Drywall
Wood
Drywall
Drywall
Drywall
Wood
Inoculation
Titer
3x107
6x107
4x102
5x102
Sprayed
Avg CPU/
Sample
1.2x107
4.7 x107
1.2x108
5.9 x102
1.3x102
9.9 x102
Mean of
Logs
6.13
7.66
8.06
2.76
1.56
2.90
RSD1
28%
1%
0.4%
4%
57%
11%
Not sprayed
Avg CPU/
Sample
5.2 x107
6.1 x107
8.5 x107
8.1 x102
1.6x103
5.9 x102
Mean of
Logs
7.71
7.78
7.93
2.91
3.06
2.77
RSD
1%
1%
0.3%
2%
12%
2%
T-test
(P-
value)
0.095
0.243
0.0015
0.068
0.033
0.43
1 RSD = Relative standard deviation of Log CPU.
Table 3-2. FAC in Extracts of Wallboard Paper and Wood Coupons
Sample Number
FAC2-D-1
FAC2-D-2
FAC2-D-3
FAC2-D-4
FAC2-D-5
FAC2-D-6
FAC2-W-1
FAC2-W-2
FAC2-W-3
FAC2-W-4
FAC2-W-5
FAC2-W-6
Coupon Material
wallboard paper
T3
o
Volume of Titrant
5.2
20.8
10
10
3.8
3.4
0
0
0
0
0
0
FAC ppm
6.9
27.7
13.3
13.3
5.1
4.5
0
0
0
0
0
0
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The Student's t-test was used to compare recoveries between the two treatments (sprayed and nonsprayed
coupons) of the same material type. The first test (STS 1) was conducted to determine the FAC in the
extract buffer of pAB-sprayed coupons following extraction and to determine recovery if no neutralizer (STS)
was added. These data suggest that residual bleach on sprayed drywall coupons likely reduced recovery.
For instance, recovery from pAB-sprayed drywall coupons was less than recovery from control coupons (p =
0.095), however this difference was not significantly different.
Analysis of the data with Student's t-test suggested that the sprayed and unsprayed results were
significantly different (p < 0.05) for Test STS 2; however, recovery from the pAB-sprayed dry wall coupons
was higher than recovery from unsprayed samples. These data suggest that the presence of neutralized
bleach does not have a negative bias on sample recovery.
Tests STS 3 and STS 4 were subsequently conducted with lower inocula to demonstrate the method would
be reliable at lower concentrations. Extraction of drywall coupons with STS was demonstrated to introduce
no negative bias in recovery due to residual pAB. Samples extracted without STS during STS 4 (low
inoculum) had 10-fold lower recoveries when sprayed with pAB versus the control samples (not sprayed
with pAB). This difference was not apparent in STS 3, when STS was included in the extraction buffer.
These data support the notion that STS is necessary in extraction buffers to reduce downstream effects of
the decontaminant during efficacy testing. STS was not used for extraction of wood coupons, as there was
no FAC from residual pAB detected in STS 1, likely due to beach demand by the wood. Recoveries from
wood were also unaffected by bleach residual in the low inoculum test (STS 4).
3.1.2 Efficacy Testing
FAC was measured from aliquots collected from the bulk container and from the sprayer nozzle. The
sprayed pAB was expected to have lost FAC during the act of spraying. Surprisingly, the opposite was
observed with the FAC of the sprayed fresh pAB nearly twice that of the bulk container. The increase in FAC
of the sprayed pAB was a function of age (or FAC of the original solution). The cause of the increase has
not yet been determined. The results are shown in Tables 3-3 and Figure 3-1.
Table 3-3. FAC and pH of pAB in the Bulk Container or Following Spraying, over Time (n=1)
Time
0
2
4
8
24
32
Bulk FAC ppm
6530
5348
4287
4487
3866
3405
Sprayed FAC ppm
12459
5789
5168
4647
3686
3485
BulkpH
6.8
6.37
6.06
5.72
5.32
5.24
Sprayed pAB pH
7.83
7.1
6.75
6.13
5.48
5.37
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August 2012
•Bulk FAC ppm
•Sprayed FAC ppm
BulkpH
•Sprayed pH
4 8 24
Age of pAB (hours)
32
Figure 3-1. FAC and pH of pAB over Time (Task 1)(n=1)
Table 3-4 shows the results for the pAB decontamination on rough-cut barn wood, and Table 3-5 shows the
results for the decontamination on drywall coupons. Figure 3-2 graphically shows the effect of age on pAB
efficacy. Age of the pAB had little to no effect on the ability of pAB to decontaminate the nonporous drywall
coupons. Wood coupons did offer more protection to the spores, and older pAB did not perform as well as
fresh pAB. These results suggest that it may be more critical to use freshly prepared pAB on difficult to
decontaminate materials, such as those with a higher organic content like wood or grimed materials. One
option is to use backpack sprayers modified with a "soap bottle attachment" filled with an acid of suitable
strength, so that bleach is acidified just prior to spraying. Acidifying bleach as it is sprayed, rather than by
the entire batch, eliminates the problems associated with decreasing FAC overtime.
Table 3-4. Task 1 Results - Effect of pAB Age on Wood Surface Decontamination (n=5)
Wood
Time Elapsed Since pAB Solution
Preparation (hours)
Positive
Fresh (15 min)
2 hours
4 hours
8 hours
24 Hours
32 Hours
Recovery (Mean
CFU/Sample)
9.83x1 0s
4.47 x102
3.97 x103
2.26 x103
9.09 x103
4.67 x104
3.84 x104
Mean of Logs
6.99
2.50
2.98
2.97
3.79
4.34
4.33
RSD
21%
90%
178%
168%
104%
117%
69%
LR
n/a
4.48
4.00
4.02
3.19
2.65
2.65
n/a = not applicable; LR = log reduction
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EPA/600/R/12/591
August 2012
Table 3-5. Task 1 Results - Effect of pAB Age on Drywall Surface Decontamination (n=5)
Drywall
Time Elapsed Since pAB Solution
Preparation (Hours)
Positive
Fresh (15 min)
2 hours
4 hours
8 hours
24 Hours
32 Hours
Recovery (Mean
CPU/Sample)
9.22x1 0s
1.42 x102
8.02 x101
7.34 x101
1.27x103
3.93 x102
3.59 x102
Mean of Logs
6.96
1.95
1.83
1.77
2.55
2.26
2.28
RSD
4%
130%
67%
79%
182%
128%
110%
LR
n/a
5.02
5.14
5.22
4.43
4.72
4.71
7.00
0.00
Effect of Age on pAB Efficacy (n=5)
•Drywall LR
•Wood LR
10 15 20
Age of pAB (hours)
25
30
35
Figure 3-2. Effect of Age on pAB Surface Decontamination Efficacy (Task 1). Data are presented as mean Log
Reduction (surfaces) from five replicate samples, error bars indicate standard deviation.
3.2 Task 2: Parametric Evaluation of the Decontaminant Application Procedures
3.2.1 High Inoculation Tests
Tests O1 through O5 were conducted with a spore load (inoculum) that resulted in 1 x 107 CPU recovered
from positive control samples (i.e., high inoculation tests). These tests were targeted at demonstrating a
greater than 6 log reduction in recovered spores, a benchmark for determining efficacy of a decontamination
procedure.22
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3.2.1.1 Surface Decontamination
Table 3-6 gives a summary of test conditions for the high inoculation optimization tests (1 x 107
CFU/sample). During Test O1, a shortened application procedure was conducted as a starting point forthe
parametric tests. The conditions (1 application, 15 seconds spray per 3 coupons, no reapplication, no rinse)
were chosen as a low efficacy starting point based upon results from previous testing.3'23'24 Results from
Test O1 indicate efficacy was near 6 LR for concrete and drywall, but low (<3 LR) for wood.
Test O2 was performed in an attempt to improve the efficacy with no additional application time. The flow
rate used was the maximum flow of the backpack sprayer (1350 mL/min), using the same spray pattern
used in Test O1. This simple one-step decontamination procedure provided an increased efficacy over Test
O1 procedure and greater than 6 LR for two of the three tested materials (drywall and concrete). However,
the sample size was not large enough to demonstrate that the higher flow rate provided a statistically higher
efficacy.
Table 3-6. Test Conditions for High Inoculation Parametric Tests (Task 2)
Test ID
O1
O2
O3,
O4
O5
Materials
Drywall, Concrete,
Wood
Drywall, Concrete,
Wood
Wood
Wood
Wood
Decontamination Steps
1 application, 15 seconds spray per 3 coupons,
no reapplication, no rinse
1 application, 15 seconds spray per 3 coupons,
no reapplication, no rinse
1 application, 30 seconds spray per 3 coupons,
no reapplication, no rinse
2 applications (at 0 and 5 min), 15 seconds
spray per 3 coupons, no reapplication, no rinse
2 applications (at 0 and 15 min), 15 seconds
spray per 3 coupons, no reapplication, no rinse
Decontaminant
Application Flow Rate
Prescribed
Low(1 L/min)
High(1.5L/min)
Low(1 L/min)
Low(1 L/min)
Low(1 L/min)
Further improvements on the efficacy of decontamination could not be detected on drywall or concrete
surfaces (e.g., near complete inactivation of these surfaces was achieved with one application), so these
materials were not included in Tests O3, O4, and O5, which focused on the most challenging material (i.e.,
wood).
Tests O3, O4, and O5 were performed to determine whether variations in application times could produce a
measured effect in decontamination efficacy. Each test had a total spray time of 30 seconds, but Test O4
and Test O5 had two 15-second applications, each at different times following the first application, while
Test O3 used a single 30-second spray. None of the application methods provided a statistically improved
efficacy in surface decontamination, nor were they significantly different in their effectiveness at wood
surface decontamination (Figure 3-3).
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These data suggest that application methods should be designed with the surface material in mind. Less
rigorous application procedures were highly effective for concrete and drywall. The higher flow rate in Test
O2 may have slightly improved efficacy on wood; however, the marginal benefit in efficacy should be
weighed against the higher volume of solution required. These data are consistent with previous studies,
and support the notion that rough wood surfaces are difficult to decontaminate.
3.2.2 Fate of Spores
Reduction of the number of spores from the surface of decontaminated materials could be due to three
effects:
1. Deactivation of the spores due to the decontamination method;
2. Removal of active spores due to the decontamination method; or
3. Reduction of recovery of active spores due to the decontamination method.
The reduction in recovery (Effect 3) has been suggested by other researchers,19 but was not investigated in
this study.
In order to quantify Effect 2, removal of active spores, samples were collected from the runoff/rinsate and
aerosol generated during spraying. As previously mentioned, the runoff collection vessel was charged with
enough STS to neutralize all bleach sprayed onto the coupons, which provides conditions for the maximum
possible spore survival. This fate of spores in the rinsate, thus, serves as the worst case scenario. In a field
application where there was no oxidative demand in the rinsate, continued exposure to pAB in the rinsate is
expected to reduce spore survivability.
The results are shown in Figure 3-4 and Table 3-7. Rinsate was not collected from Test O1. Figure 3-4
shows the recovery of viable spores in rinsate liquids. The CFU counts /ml derived from 100 ml aliquots
were multiplied by the total volume of rinsate collected. The striking result is the absence of spores in the
rinsate of Test O3. Without further investigation, we are unsure whether the presence of spores in rinsates
during Tests O4 and O5 was due to the two 15-second pAB applications being less effective at killing
spores in the rinsate than the 30-second pAB application, although no more effective at surface
decontamination; or if the spores were present due to cross-contamination. Alternately, the longer
spraying time (30 sec) certainly generated more runoff, as more of the surfaces were saturated for longer
periods of the spray, even though the total spray duration was equal for O3, O4, and O5. More pAB was
expected to "runoff coupon surfaces during Test O3, resulting in more pAB collected in the rinsate sample
and therefore a higher potential for insufficient neutralization. Active pAB in the rinsate sample could have
lowered the number of viable spores recovered and could explain the unexpectedly low abundance of
rinsate spores observed during this test (O3).
43
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Final Report
Project No. RN990273.0025
Revision 0.0
July 2012
9.00
0.00
Efficacies During Parametric Tests
(high inocula, n=3)
I Log reduction
I Inoculum log
CPU
7.04
6.48
Drywall
Concrete
Wood
1 x 15 second spray, low flow, no
reapplication, no rinse
Test Ol
1 x 15 second spray, high flow, no
reapplication, no rinse
Test O2
Wood
1 x 30 second 2 x 15 second 2 x 15 second
spray, low spray, low spray, low
flow, no flow, sprayed flow, sprayed
reapplication, at 0 and 5 at 0 and 15
no rinse min, no rinse min, no rinse
Test O3
Test O4
Test O5
Figure 3-3. Surface Decontamination Efficacy (Log Reductions, LR) by Material Type for the Five Parametric Tests (Task 2).
Error bars indicate standard deviation.
44
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Table 3-7. Task 2 - Surface Decontamination Parametric (high inoculation) Test Results
Test
ID
Test 01
Test 02
Test 03
Test 04
Test 05
Material
Drywall
Concrete
Wood
Drywall
Concrete
Wood
Wood
Wood
Wood
Positive Controls (n=3)
Avg.
CPU/
Sample
1.72 x 107
1.02 x 107
1.95 x 106
2.05 x 107
8.16 xlO6
3.93 x 106
2.31 x 106
2.31 x 106
2.31 x 106
Mean
of
Logs
7.23
7.00
6.27
7.30
6.89
6.57
6.35
6.35
6.35
RSD
(%)
22%
22%
41%
25%
38%
47%
30%
30%
30%
Test Coupons (n=3)
Avg.
CPU/
Sample
351
7
3520
6
12
2236
1600
1984
1188
Mean of
Logs
1.54
0.60
3.39
0.26
0.41
2.86
3.03
3.26
3.06
RSD
(%)
170%
83%
95%
155%
163%
154%
105%
46%
27%
LR
5.69
6.40
2.87
7.04
6.48
3.71
3.32
3.09
3.28
RSD
(%)
23%
11%
16%
12%
15%
22%
14%
8%
4%
pAB Decontamination Conditions
Achieved
Decontamination Steps
1 x 15 second spray, low
flow, no reapplication,
no rinse
1 x 15 second spray, high
flow, no reapplication,
no rinse
1 x 30 second spray, low
flow, no reapplication,
2 x 15 second spray, low
flow, sprayed at 0 and 5
1 x 15 second spray, low
flow, no reapplication,
Flow Rate
(ml/min)
1030
1030
1040
1340
1340
1360
1030
1060
1030
RSD = Relative Standard Deviation of CFU/sample
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Viable Spores Collected in Rinsates during Parametric Tests
1,784
1,315
1,889
<22.48
Drywall Concrete Wood
Wood
Wood
Wood
1 x 15 second spray, high flow, no reapplication, 1 x 30 second 2 x 15 second 2 x 15 second
no rinse
Test O2
spray, low flow,
no
reapplication,
no rinse
Test O3
spray, low flow, spray, low flow,
sprayed at 0 sprayed at 0
and 5 min, no and 15 min, no
rinse
Test O4
rinse
Test O5
Figure 3-4. Recovery of Viable Spores in Rinsates (Task 2). Data are reported as the total CFU recovered from
rinsate samples; this value is printed above the bars in the figure.
During the first round of testing (high inoculation - Test O1 through O5), aerosolized spores were detected.
As described in Section 2.6.8, the aerosol sampling equipment consists of a nozzle and the inlet to the
collection liquid, and the collection liquid itself. Spores were detected only in the rinse of the SKC
BioSampler® nozzle, not in the collection liquid. This observation would be consistent with two scenarios:
1. Spores were in very large clumps and were settling or impacting on the nozzle.
2. Residual droplets of aerosolized bleach were decontaminating or preventing spore recovery from
the SKC collection liquid.
The FAC of the collection liquid in the SKC BioSampler® was measured following Tests O3, O4, and O5 and
was found to be 36.7 ppm, 38.2 ppm, and 32.3 ppm, respectively (mean of 35 ppm).
To determine if this low concentration could account for the absence of spores in the liquid, a test was
designed to measure recovery from spiked extraction fluids. 10 ml of PBST (simulating the BioSampler®
collection liquid) was brought to 35 ppm FAC with pAB, and then spiked with spores. The results are shown
in Table 3-8 .
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Table 3-8. Results from 35 ppm FAC Extraction Efficacy Test (n = 5)
Collection
Liquid
PBST
PBST with
STS
Positive Controls
Avg. CFU/
Sample
3.57E+02
3.49E+02
Mean of
Logs
2.55
2.53
RSD (%)
18%
32%
Samples Spiked with 35 ppm FAC pAB
Avg. CFU/
Sample
5.70E-01
3.57E+02
Mean of
Logs
-0.24
2.55
RSD (%)
1%
18%
p-value
(t-test)
0.0003
0.68
RSD = Relative Standard Deviation of CPU/sample
No spores were collected from the 35 ppm FAC liquid, while the recovery from PBST with STS was the
same as the positive controls. Residual FAC in the BioSampler® collection liquid for Tests O3, O4, and O5
(PBST only) could have prevented detection of the spores in the aerosol phase. The BioSampler® collection
liquid for subsequent tests (Tests O6 through O10, and all Task 3 tests) was PBST spiked with STS to
neutralize entrained pAB.
3.2.3 Low Inoculation Decontaminations
Tests O6 through Test O10 were designed to evaluate the efficacy of the spray-based procedure on
coupons with lower spore inoculations. Log reductions are typically non-linear reactions, with resilient
spores resisting decontamination. The results are tabulated in Table 3-8 and displayed graphically in Figure
3-5.
Low-level inoculations proved difficult to perform in a repeatable manner, so inocula for Tests O6 through
O10 ranged from ~1 x 104to 1 x 106 per coupon (0.09 m2 or 1 ft2). Coupons for Tests O8, O9, and O10 -
including positive control coupons common to all tests - were all inoculated on the same day to reduce test
variation. The MDIs used forthese inoculations were manufactured with a propellant of untested shelf-life,
and many were unable to perform reliably. While this factor makes it difficult to compare log reductions
across tests, the low level inoculations do provide valuable information on the difficulty of full
decontamination.
As shown in Table 3-9, no spores were detected on Test O6 decontaminated concrete or drywall coupons
(detection limit values of 0.5 CFU are listed). Spores were detected on Test O6 decontaminated wood
coupons, however. For Test O7 (wood only), the pAB application time was doubled, and, again, the
decontamination procedure failed to remove or inactivate all spores. For Tests O8, O9, and O10, the
inoculation level was relatively low. During these tests, some surface samples from decontaminated
coupons had more recovered spores than from the positive controls. These results were surprising and
somewhat unexplainable yet do illustrate the notion that low levels of contamination should not be assumed
to be decontaminated easily. None of the decontamination methods tested proved effective on deactivating
spores on wood coupons, even at low contamination levels.
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Table 3-9. Task 2 - Surface Decontamination Parametric (low inoculation) Test Results
Test ID
Test O6
Test O7
Test O8
Test O9
Test O1 0
Material
Drywall
Concrete
Wood
Wood
Wood
Wood
Wood
Positive Controls (n=3)
Avg. CPU/
Sample
4.36 x105
6.42 x104
4.33 x104
3.23 x102
1.73x102
1.73x102
1.73x102
Mean
of
Logs
5.64
4.78
4.62
2.44
2.12
2.12
2.12
RSD
(%)
16%
42%
34%
55%
93%
93%
93%
Test Coupons (n=3)
Avg. CPU/
Sample
0.5
0.5
45
47
107
160
40
Mean
of
Logs
-0.20
-0.15
1.11
1.36
1.99
1.84
1.50
RSD
(%)
3%
1%
143%
136%
47%
141%
87%
LR
5.84
4.93
3.51
1.09
0.13
0.28
0.62
RSD
(%)
0.00
0.00
0.29
0.00
0.00
0.00
pAB Decontamination Conditions Achieved
Decontamination Steps
1x15 second spray, no
reapplication, no rinse
1 x 30 second spray, no
reapplication, no rinse
1 x 30 second spray, no
reapplication, no rinse
2x15 second spray, sprayed at 0
and 5 min, no rinse
2x15 second spray, sprayed at 0
and 15 min, no rinse
Flow
Rate
(ml/min)
1000
1000
1000
1100
1100
1100
1000
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Efficacies During Para met ricTests
(lowinocula) (n=3)
Log reduction
Log CPU Inoculation
1 x 15 second
spray, no
reapplication, no rinse
Test O6
Wood
1x 30 secondlx 30 second2 x15 second2 x15 second
spray, no spray, no spray, at 0 spray, at 0
reapplicationjeapplication, and 5 min, and 15 min,
no rinse no rinse no rinse no rinse
Test O7
Test O8
Test O9 Test O10
Figure 3-5. Decontamination Efficacy (LR) of Five Decontamination Procedures on Low-Level
Contamination (Task 2). Average LR value listed above bar in Figure. Error bars indicate standard
deviation.
The positive control samples often required filter-plate analysis, as undiluted spread-plating of these
samples often resulted in less than 30 CFU. The low-level inoculation tests were complicated by the
presence of wood fibers on the filter following filter-plating, as copious amounts of wood fiber debris covered
the filter and prevented enumeration of CFU (Figure 3-6). For example, 1 ml of a sample may contain 18
CFU on the filter-plate, but no CFU were detected on a filter in which16 ml of the same sample was
analyzed. The higher volume contained more wood debris and prevented discrimination of CFU. This lack of
discrimination resulted in surface samples from wood having a higher detection limit (20 CFU/sample) than
samples from some other materials, which had detection limits closer to 0.5 CFU/sample.
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Figure 3-6. Photograph of Wood Fibers on a Filter-plate. The high debris in these samples confounded the
filter-plate assay and resulted in higher detection limits for surface samples collected from wood.
For future tests with heavy debris, the extraction and plating procedures should be modified to take into
consideration the obscuring effect of the debris. One suggestion is to let the sample sit for two minutes after
it is removed from the orbital shaker to let foam subside and the debris settle, then remove the liquid sample
near the surface. This procedure may help eliminate interference by debris, but may also result in low
recovery estimates as an unknown number of spores may be bound to the settled wood fibers.
The difficulty with recovery, combined with the difficulty in decontamination, point to a major weakness in
confirming adequate decontamination of challenging surfaces (i.e., wood) following a Bacillus spore release.
Spores that are difficult to destroy and detect could pose a significant threat. Further research is warranted to
improve both detection and destruction of spores on such materials.
No spores were detected in either the aerosol phase or rinsate of these low-level inoculation tests. However,
the detection limit for the aerosol samples was very high (400 CFU) due to the small portion of the flow
sampled. The detection limit of the rinsate sample was 5 CFU.
3.2.4 Wetted Wipe Decontamination
The results of the wetted wipe decontamination tests are presented in Table 3-10.
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Table 3-10. Wetted-Wipe Test Procedure Results (Drywall Coupons) (n=3)
Test
W1
W2
W3
W4
Decon
Method
pAB Wetted
Wipe
SimWipe
SimWipe +
pAB Wetted
Wipe
pAB Wetted
Wipe (3
wipes/coupon)
Positive Controls (n=3)
Avg. CPU/
Sample
1.70x107
1.70x107
1.70x107
4.36 x105
Mean of
Logs
7.23
7.23
7.23
5.64
RSD
(%)
6.7%
6.7%
6.7%
16%
Test Coupons (n=3)
Avg. CPU/
Sample
3.58 x105
4.62x1 0s
2.08 x104
1.72x103
Mean of
Logs
5.38
6.66
3.92
3.15
RSD
(%)
95%
3%
109%
67%
LR
1.86
0.57
3.31
2.49
RSD
(%)
28%
2%
28%
15%
Figure 3-7 shows the range of CFU recovered from the decontaminated 35.6 cm x 35.6 cm (14 in by 14 in)
drywall surface. ANOVA reveals that the SimWipe-only decontamination represents a statistically distinct
decontamination method (lowest efficacy). While the combination of the SimWipe and the pAB wetted wipe
seems to be more efficacious than the pAB wetted wipe alone, the Student's t-test two-tailed homoscedastic
value (p-value) is 0.075, suggesting that the variance is not statistically significant. A fourth test (shown in
Table 3-10) was evaluated against a lower spore inoculation on drywall coupons, using three wetted wipes,
one for each direction of wiping as described in MOP 3156. Recovery cannot be compared directly because
of the difference in spore loading before decontamination. However, a comparison of the efficacies suggests
no statistically significant difference between the efficacies of any wipe decontamination procedure which
incorporates pAB (i.e., all instances except the SimWipes alone test). When comparing these results to
those of the efficacy of the two spray-based procedures on drywall it is apparent that even the shortest
spray procedure (one, 15-second decontaminant application, Test O1) yielded much higher efficacies (5.69
LR) than any of the wetted-wipe procedures (Table 3-7 and 3-10). For a more thorough understanding of
the efficacy of wetted wipe decontaminations, these tests should be repeated with larger sample sizes.
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LL.
U
Recovery
1 nnnnnnn
1 nnnnnn
1 nnnnn
mnnn
mnn
inn -
m -
1
•
i
pAB wetted wipe DrySimwipe Dry Simwipe, then pAB
wetted wipe
Figure 3-7. Recovery from Coupons after Three Methods of Wipe Decontamination (n=3). Error bars represent
maximum and minimum values.
3.3 Task 3: Impact of Soiled Surfaces (Grime) on Decon Efficacy
3.3.1 Preliminary Tests on Effect of Grime on Recovery
Initial results showed that the grime contained substantial contamination. To reduce the background
contamination co-collected during sampling, the grime was sterilized by exposure to 40 KGy of gamma
irradiation. Verification tests were then conducted to determine if background contamination was eliminated
by the sterilization procedure and to determine if recovery was affected by the addition of the grime.
No contamination was recovered from wipe samples of sterilized grime (Table 3-11). The value listed for
the unspiked wipe aliquot indicates the detection limit. Recoveries were nearly identical for spiked grime
wipes and spiked PBST extraction buffer alone (p-value = 0.87). These results demonstrated that there was
no recovery bias, either positive or negative, from the presence of irradiated grime.
Table 3-11. Task 3 - Recovery from Wipe Samples of Grimed Coupons (n = 3)
Spiked Grime Wipe
Spiked PBST
Unspiked Wipe
PBST
Avg. CFU/
Sample
9.82 x104
9.73 x104
<1.00x101
<1.05
Mean of
Logs
4.99
4.99
<1.00
<0.02
RSD
(%)
8.8%
1.8%
0.0%
0.0%
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3.3.2 Surface Sampling Results
Figure 3-8 shows the efficacy of the four decontamination procedures on grimed and clean coupons. Note
that some of the differences in LR are due to differences in recovery from the positive control coupons.
Figure 3-9 and Table 3-12 present spore recovery from the surface samples, suggesting no particular
advantage to the use of pAB vs. pAB with surfactant (i.e., no difference in observed efficacy). Results from
these tests are in agreement with results of Task 2, and suggest a single 30-second spray application is
effective for concrete, yet yields approximate a 3 LR for wood coupons (with a 7 Log inoculum), regardless
of the presence of grime. All four decontamination methods yielded equivalent recoveries from coupons,
grimed or clean. This observation suggests no impact of grime at this loading. Tests with higher loadings are
required to extend this finding to more heavily soiled surfaces.
The Effect of Grime on Surface
Decontamination
I Concrete-Clean
I Concrete-Grime
Wood-Clean
I Wood-Grime
pAB spray pAB/TSP spray pAB/TSP spray, vacuum pAB/TSP
scrub spray, scrub
Figure 3-8. Task 3 Results - The Effect of Grime on Surface Decontamination (n = 3). Error bars indicate standard
deviation.
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Recovery from Grimed and Clean Coupons
10000
_OJ
Q.
E
as
in
v
u
•e
3
in
E
g
o
Sri
QC
1000
100
10
I Concrete-Clean
I Concrete-Grime
Wood-Clean
I Wood-Grime
pAB spray pAB/TSP spray pAB/TSP spray, vacuum pAB/TSP
scrub spray, scrub
Figure 3-9. Task 3 Results - Recovery from of Grimed vs. Clean Decontaminated Surface Samples (n=3). Error
bars indicate standard deviation.
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Table 3-12. Task 3 - Results for Grimed and Non-Grimed Decontamination Tests using pAB and pAB/TSP (n = 3)
Test
G1
G2
G3
G3b
G4
G4b
G5
G5
G6
G6
G7
G7
G8
G8
Material
Concrete
Wood
Concrete
Wood
Wood
Concrete
Wood
Concrete
Wood
Concrete
Wood
Concrete
Wood
Concrete
Wood
Concrete
Grime
No
No
Yes
Yes
No
No
Yes
Yes
No
No
Yes
Yes
No
No
Yes
Yes
Positive Controls
Avg. CPU/
Sample
8.80 x105
2.77x1 0s
9.89 x105
1.42 x 10s
6.51 x105
2.38 x105
5.40 x105
2.98 x105
2.78 x105
2.02x1 0s
5.70 x105
3.50x1 0s
2.78 x105
2.02x1 0s
5.70 x105
3.50x1 0s
Mean
of
Logs
5.88
6.40
5.98
6.14
5.81
5.37
5.71
5.45
5.44
6.29
5.75
6.49
5.44
6.29
5.75
6.49
RSD
(%)
57%
46%
31%
26%
14%
20%
37%
42%
5%
30%
16%
59%
5%
30%
16%
59%
Test Coupons
Avg. CPU/
Sample
2
1450
1
353
197
1
70
13
140
1
83
1
147
7
23
1
Mean of
Logs
0.23
3.06
-0.05
2.49
1.97
-0.01
1.52
1.10
2.12
-0.05
1.55
-0.16
2.15
0.32
1.30
-0.17
RSD(%)
15.6%
63.5%
50.6%
60.0%
89.6%
49.7%
136.3%
43.3%
42.9%
48.0%
142.2%
0.7%
28.4%
156.9%
65.5%
1 .6%
LR
5.65
3.34
6.03
3.65
3.84
5.38
4.19
4.35
3.33
6.34
4.20
6.66
3.29
5.97
4.45
6.66
RSD
(%)
1.2%
12.0%
3.4%
7.7%
21.9%
3.7%
15.6%
4.0%
6.0%
3.0%
16.8%
0.0%
4.1%
14.3%
6.8%
0.1%
Decontamination
Solution
pAB
pAB
pAB
pAB
pAB/TSP
pAB/TSP
pAB/TSP
pAB/TSP
pAB/TSP
pAB/TSP
pAB/TSP
pAB/TSP
pAB/TSP
pAB/TSP
pAB/TSP
pAB/TSP
Decontamination
Conditions
(Decontamination
Steps)
1 x 30 second spray, no
reapplication, no rinse
1 x 30 second spray, no
reapplication, no rinse
1 x 30 second spray, no
reapplication, no rinse
1 x 30 second spray, no
reapplication, no rinse
Scrub, then Spray
Scrub, then Spray
Scrub, then Spray
Scrub, then Spray
Vacuum, scrub, and
Spray
Vacuum, scrub, and
Spray
Vacuum, scrub, and
Spray
Vacuum, scrub, and
Spray
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3.3.3 Fate of Spores
During Task 3, rinsate and aerosol samples were collected and analyzed as they were during Task 2. No
spores were detected in any rinsate sample except for the concrete rinsate in Test G1. Only one of the
triplicate samples tested positive for Bacillus spores, suggesting that the presence of these spores may
have been due to cross-contamination during collection. The rinsates were homogenized before aliquots
were collected, so all three replicate samples should have produced similar results.
Figure 3-10 shows the CFU recovered from aerosol samples during decontamination. Aerosol sampling was
conducted during all portions of each procedure including spraying, scrubbing, and vacuuming. Viable
spores were detected in all aerosol samples collected during testing with inoculated (test) samples. Aerosol
samples collected during decontamination of blank coupons did not yield viable spores. Figure 3-10 shows
the combination of both halves of the aerosol sample; the rinse of the nozzle and the collection liquid, which
were analyzed separately according to MOP 6578.
Aerosol-collected Spores during Decontamination of
Grimed and Non-Grimed Coupons
100000
10000
_
Q.
E
S
.
(J
01
O
U
01
ce.
1000
100
10
I Concrete-Clean
I Concrete-Grime
Wood-Clean
I Wood-Grime
pAB spray pAB/TSP spray pAB/TSP spray, vacuum pAB/TSP
scrub spray, scrub
Figure 3-10. Recovery from Aerosol Samples during Task 3 Testing (n = 1)
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The values shown in Figure 3-10 are pooled from all three coupons during a single decontamination. While
some are relatively low, these data represent the re-aerosolized spores from only approximately 0.37 m2 (4
ft2). Furthermore, these data have not been normalized to the total volume of air sampled, for the simple
reason that it is unknown during what portion of the decontamination cycle most of the spores are
aerosolized.
Figure 3-10 suggests that the decontamination procedures with more steps are likely to result in a greater
magnitude of spore re-aerosolization. Figure 3-10 also suggests that laboratory studies using clean
materials may overestimate the actual aerosolization from grimed materials. The concrete coupons used for
this study were not polished concrete, so the surface was sometimes powdery. This powdery surface may
help explain the higher aerosolization from concrete.
The results from these tests suggest that spray or mist should be the first decontamination step, rather than
the use of a vacuum cleaner, as the amount of aerosol-collected spores was highest in tests where
vacuuming was conducted before wetting of surfaces.
3.4 Assessment of Operational Parameters
3.4.1 Time
The time required to decontaminate a batch of coupons depended on the decontamination procedure being
applied. The methods used in Test O1 and O2 were very effective on drywall and concrete and relatively
rapid at 5 seconds/ per 0.09 m2 (ft2). A single worker on a four-hour shift may therefore be able to
decontaminate 260 m2 (2800 ft2). Such an application would require between 61 to 80 L (16 and 21 gallons)
of sporicide per hour, depending on the chosen flow rate. This volume would require the backpack sprayer
(5 gallon capacity) be refilled every 15 to 20 minutes. Due to safety concerns with fatigue while wearing a
National Fire Sprinkler Association (NFSA) Class C suit, cooling vests may be necessary to sustain a 4-hour
shift.
The four-step procedure used in Task 3 would likely require three responders rather than the one responder
per area the spray alone procedure required: one to vacuum the area, one to spray the area, and one to
scrub the area. As a result, this four-step procedure would triple the required personnel.
Since special care was taken to prevent cross-contamination and produce repeatable and documented
decontamination steps, the procedures used in this study may provide an underestimate of field-scale
productivity per person. Field-scale personnel, though, may be hampered by Level C suits and supplied air
respirators (see Section 3.4.3). The Personal Protective Equipment (PPE) used in the field may perhaps be
more restrictive than the PPE used in this study.
3.4.2 Physical Impacts on Materials
The materials used in this study were rugged, and, as expected, did not show any physical changes
following decontamination.
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3.4.3 Impact on Decontamination Workers
For this study, the decontamination steps were performed by personnel outside the chamber housing the
coupons because of the high concentration of chlorine (CI2) gas generated by the pAB. Based upon CI2
concentrations during a field-scale event, respiratory protection may be required. The measured CI2
concentrations in the space may determine whether Occupational Safety and Health Administration (OSHA)
Level C air purifying respirators or supplied air (e.g., Self Contained Breathing Apparatus) might be required.
These refined procedures were much less onerous than procedures used under previous studiesS.
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4. Quality Assurance and Quality Control
This project was performed under an approved Category III QAPP entitled, "Assessment of Liquid and
Physical Decontamination Methods for Environmental Surfaces Contaminated with Bacterial Spores: Part 4
- Optimization of Method Parameters and Impact of Surface Grime (June 2011)".
18
4.1 Calibration of Sampling/Monitoring Equipment
There were standard operating procedures for the maintenance and calibration of all laboratory and
NRMRL/NHSRC Biocontaminant Laboratory equipment. All equipment was verified as being certified
calibrated or having the calibration validated by EPA's Air Pollution Prevention and Control Division's
(APPCD) on-site (RTP, NC) Metrology Laboratory at the time of use. Standard laboratory equipment such
as balances, pH meters, biological safety cabinets (BSCs) and incubators were routinely monitored for
proper performance. Calibration of instruments was done at the frequency shown in Table 4-1. Any
deficiencies were noted. The instrument was adjusted to meet calibration tolerances and recalibrated within
24 hours. If tolerances were not met after recalibration, additional corrective action was taken, possibly
including recalibration or/and replacement of the equipment.
Table 4-1. Instrument Calibration Frequency
Equipment
Thermometer
pH meter
Relative humidity (RH)
sensor
Stopwatch
Clock
Pressure Guage
Scale
Calibration/Certification
Compare to independent NIST thermometer (this is a
thermometer that is recertified annually by either NIST or an
International Organization for Standardization (ISO)-17025
facility) value once per quarter
Perform a 2-point calibration with standard buffers that
bracket the target pH before each use.
Compare to calibration salts once a week.
Compare against NIST Official U.S. time at
http://nist.time.gOV/timezone.cgi7Eastern/d/-5/java once every
30 days.
Compare to office U.S. Time @ time.gov every 30 days.
Compare to independent NIST Pressure gauge annually.
Check calibration with Class 2 weights
Expected Tolerance
±1°C
± 0.1 pH units
±5%
± 1 min/30 days
± 1 min/30 days
±2 psi
± 0.1% weight
59
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4.2 Data Quality Indicator (DQI) Goals
Target acceptance criteria for the critical measurements are shown in Table 4-2 with precision goals.
Table 4-2. Acceptance Criteria and Test Values for Critical Measurements
Measurement
Parameter
FACin
Decontamination
Solution
pHof
Decontamination
Solution
Temperature of
Liquids
Pressure of
Backpack
Sprayer used for
Decontamination
Solution
Spraying
Flow Rate of
Decontamination
Spray
Positive Control
CFU (high
inoculation only)
Test Coupon
CFU
Target
Value
6000-6700
parts per
million
(ppm)
6.5
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Final Report
Project No. RN990273.0025
water is expected to have minimal effect on project results and was therefore allowed to remain outside
specification without corrective action.
The temperature of the pAB was measured only when prepared, not when applied.
4.2.2 pH Measurements
The pH measurements listed in Table 4-2 are of the solution as prepared, not as applied to the coupons
during decontamination. The pAB degrades over its 3-hour lifetime. The Oakton pH probe was calibrated
with pH 7.0 buffer solution per manufacturer's instructions at the start of each test day. All the results were
within the specified target range.
4.2.3 Pressure Measurements
Significant variation in the pressure reading of the backpack sprayer was observed. Some of the variation in
Task 2 was due to the targeted higher flow rate from the sprayer. The higher pressure at this flow rate was
intentional (though the exact value was not known at the writing of the QAPP amendment). The low values
during Task 3 testing were due to a malfunctioning gauge. The flow rates for these tests were within
specification.
4.2.4 FAC Measurements
The HACH High Range Bleach Test Kit was used to titrate a standard solution of 1000 ppm NaCIO2. The
HACH test kit returned a value within 10 percent of the standard. The FAC measurements listed in Table
4-2 are of the solution as prepared, not as applied to the coupons during decontamination. The pAB
degrades over its 3-hour lifetime. The FAC of the bulk solution was measured periodically during testing and
was expected to be lower than the FAC of the fresh, as-prepared solution.
4.2.5 Flow Measurements
The target flow rates listed in the QAPP for the backpack sprayer were based on pAB; this study also used
a pAB/TSP solution, which had higher flow rates at the same sprayer setting. The sprayer was set to its
lowest (or highest for Test O2) setting to provide a spray pattern of 16-in diameter from a distance of 3 ft.
4.2.6 Positive Control CFU
The target concentration was not always met, and, as expected, there was much variation between material
types and material condition (grimed vs. non-grimed). Most inoculation levels did provide sufficient loading
to allow for a 6 log dynamic range.
4.2.7 CFU Counts
Twenty-five percent (25%) of all plates containing CFU within the acceptable range (30-300) were counted
by a second technician. The second enumeration was required to fall within 10 percent of the initial count.
61
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The positive control enumerations (i.e., inocula) were occasionally above or below the target value due to
malfunctioning MDIs, but the inoculations were deemed acceptable by the EPA Principal Investigator (PI)..
4.3 Data Quality Audit
At least 10 percent of the data acquired during the investigation were audited. The QA Manager traced the
data from the initial acquisition through reduction to final reporting to ensure the integrity of the reported
results. All data treatment calculations were checked before inclusion in this report.
4.4 QA/QC Reporting
QA/QC procedures were performed in accordance with the QAPP for this investigation.
4.5 Amendments to and Deviations from the Original QAPP
4.5.1 Formal Amendments
During the course of the projects, six amendments were added to the QAPP by the EPA PI in response to
data results or equipment failures. These amendments, listed in Appendix G, were submitted by e-mail to
the EPA QA officer for formal approval.
4.5.2 Other QAPP Deviations
The QAPP states that, following coupon inoculation, a second person, wearing new gloves for each coupon,
is tasked with moving the coupon to the proper location (e.g., test and positive control coupons to the Test
Coupon Cabinet and blank coupons to the Blank Coupon Cabinet). This person did not wear new gloves for
each sample. Any cross-contamination that occurred between similarly inoculated coupons is not expected
to have biased the results.
Rather than transfer the 18 mm coupons in Task 1 in the BSC as listed in the QAPP, the operation was
performed inside the chamber where the decontamination took place. Transfer of the holding plate from the
chamber to the BSC would expose the coupons to much more risk of contamination.
The QAPP states that the time required for the grimed coupons to appear dry would be recorded in a
laboratory notebook. However the coupons never appeared wet. Instead, a drying time of 15 minutes
(based on application of the grime to paper) was chosen, as listed in the MOP 3163.
Amendment 3 states that all coupons will be spritzed with pAB before wiping. Spritzing was not done with
the SimWipe due to a misinterpretation of the wiping MOP 3156 at the time of testing. The SimWipe method
is expected to have been more effective with the addition of pAB.
The BioSamplers® were not filled with peptone-buffered water as stated in the QAPP due to the
unavailability of peptone-buffered water for the first test. Instead, PBST was used as the collection liquid.
There was no excessive foaming due to the TWEEN®-20 in the PBST.
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The flow rate of 1000 mL/min is listed for Task 3 decontamination. However, the flow rate at the lowest
setting of the sprayer is 1300 mL/min when the pAB/TSP solution is used.
Although rinsate samples were to be processed on the same day as they were taken, due to the length of
time required to collect the samples, they were placed in the refrigerator at 4 ± 2 °C overnight and
processed the next day.
The glassware accompanying the BioSamplers® was to be rinsed with 100 ml of sterile PBST. However,
because the glassware was shorter in length than originally anticipated, only 50 ml of PBST was used to
rinse the glass.
4.5.3 Data Quality Indicator Assessment
Most of the data quality indicators for the critical measurements were within their specified target ranges as
indicated in Table 4-2. However, in some instances, some small deviations were noted such as deionized
water temperatures, sprayers flow rates, or CPU counts. These small deviations in the measurements,
although critical, were consistent throughout the tests and did not affect the affect the intra-test
comparisons.
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5. References
1 Canter, D. A. (2005). "Remediating anthrax-contaminated sites: Learning from the past to protect the
future." Chemical Health and Safety 12(4): 13-19.
2 After Action Report- Danbury Anthrax Incident, U.S. EPA Region 1, September 19, 2008.
3 U.S. EPA. Assessment of Liquid and Physical Decontamination Methods for Environmental Surfaces
Contaminated with Bacterial Spores: Development and Evaluation of the Decontamination Procedural
Steps . U.S. Environmental Protection Agency, Washington, DC, EPA/600/R-12/025, 2012.
4 Snook, C. P., J. Cardarelli, et al. (2008). "Medical toxicology and public health-update on research and
activities at the centers for disease control and prevention and the agency for toxic substances and
disease registry." J Med Toxicol 4(4): 289-291.
5 Canter, D. A. (2005). "Addressing residual risk issues at anthrax cleanups: How clean is safe?" Journal
of Toxicology and Environmental Health 68(11-12): 1017-1032.
6 ASTM International. Standard Test Method E-2414-05. Quantitative sporicidal three-step method (TSM) to
determine sporicidal efficacy of liquids, liquid sprays, and vapor or gases on contaminated carrier
surfaces. West Conshohocken, PA: American Society for Testing and Materials; 2005.
7 AOAC International. (2008). Method 2008.05 - Determining efficacy of liquid sporicides against spores of
Bacillus subtilis on a hard nonporous surface using the quantitative three step method (TSM).
8 Tomasino, S. F., R. M. Pines, et al. (2008). "Determining the efficacy of liquid sporicides against spores
of Bacillus subtilis on a hard nonporous surface using the quantitative three step method: Collaborative
study." Journal of AOAC International 91(4): 833-852.
9 Wood JP. Evaluation of liquid and foam technologies for the decontamination of B. anthracis and B.
subtilis spores on building and outdoor materials. Washington, D.C.: U.S. Environmental Protection
Agency; 2009 November. Report No.: EPA/600/R-09/150.
10 Einfeld, W., R. M. Boucher, et al. (2011). Evaluation of Surface Sampling Method Performance for
Bacillus Spores on Clean and Dirty Outdoor Surfaces. Albuquerque, NM, Sandia National Laboratories:
SAND2011-4085.
11 Rastogi, V. K., L. Wallace, et al. (2009). "Quantitative Method To Determine Sporicidal
Decontamination of Building Surfaces by Gaseous Fumigants, and Issues Related to Laboratory-Scale
Studies." Appl. Environ. Microbiol. 75(11): 3688-3694.
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12 Rogers, J. V., C. L. K. Sabourin, et al. (2005). "Decontamination assessment of Bacillus anthracis,
Bacillus subtilis, and Gee-bacillus stearothermophilus spores on indoor surfaces using a hydrogen
peroxide gas generator." Journal of Applied Microbiology 99(4): 739-748.
13 Tomasino, S. F., V. K. Rastogi, et al. (2010). "Use of Alternative Carrier Materials in AOAC Official
Method(SM) 2008.05, Efficacy of Liquid Sporicides Against Spores of Bacillus subtilis on a Hard,
Nonporous Surface, Quantitative Three-Step Method." Journal of Aoac International 93(1): 259-276.
14 Sagripanti, J. L., M. Carrera, et al. (2007). "Virulent spores of Bacillus anthracis and other Bacillus
species deposited on solid surfaces have similar sensitivity to chemical decontaminants." Journal of
Applied Microbiology 102(1): 11-21.
15 Lee, S. D., S. P. Ryan, et al. (2011). "Development of an Aerosol Surface Inoculation Method for
Bacillus Spores." Appl. Environ. Microbiol. 77(5): 1638-1645.
16 Edmonds, J., P. Clark, et al. (2010). "Multigeneration Cross Contamination of Mail with Bacillus
Species Spores by Tumbling." Appl. Environ. Microbiol. 76(14): 4797-4804.
17 Brown, G. S., R. G. Betty, et al. (2007). Evaluation of rayon swab surface sample collection method for
Bacillus spores from nonporous surfaces." Journal of Applied Microbiology 103(4): 1074-1080.
18
ARCADIS U.S., Inc. Quality Assurance Project Plan for the Assessment of Liquid and Physical
Decontamination Methods for Environmental Surfaces Contaminated with Bacterial Spores: Part 4 -
Optimization of Method Parameters and Impact of Surface Grime. Prepared under Contract No. EP-C-09-
027, Work Assignment No. 2-25. U.S. Environmental Protection Agency, National Homeland Security
Research Center, Research Triangle Park, NC. June 2011.
19 Grand, I., M.-N. Bellon-Fontaine, et al. (2011). "Possible Overestimation of Surface Disinfection
Efficiency by Assessment Methods Based on Liquid Sampling Procedures as Demonstrated by In Situ
Quantification of Spore Viability." Appl. Environ. Microbiol. 77(17): 6208-6214.
20 Busher, A.; Noble-Wang; J.; Rose, L. Surface Sampling. In Sampling for Biological Agents in the
Environment; Emanual, P., Roos, J.W., Niyogi, K., Eds.; ASM Press: Washington, DC, 2008; pp 95 - 131.
21 EPA Method 5 - Determination of Particulate Matter Emissions from Stationary Sources. CFR 40, Part
60.
22 U.S. Environmental Protection Agency (2007) Guidance on test methods for demonstrating the efficacy
of antimicrobial products for inactivating Bacillus anthracis spores on environmental surfaces. In FIFRA
Scientific Advisory Panel Meeting. Arlington, VA: US Environmental Protection Agency (SAP Minutes
No. 2007-05).
23 Calfee, M. W., S. P. Ryan, et al. (2012). "Laboratory evaluation of large-scale decontamination
approaches." Journal of Applied Microbiology 112(5): 874-882.
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24 US Environmental Protection Agency (2011) Effectiveness of Physical and Chemical Cleaning and
Disinfection Methods for Removing, Reducing or Inactivating Agricultural Biological Threat Agents. US
Environmental Protection Agency, Office of Research and Development, National Homeland Security
Research Center. EPA 600/R-11/092, September 2011.
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Appendix A: Coupon, Test Chamber and Equipment Cleaning and Sterilization
Procedures
The pAB solution used for cleaning surfaces in equipment in both the decontamination and NRMRL/NHSRC
Biocontaminant Laboratory was prepared as a 1:10 dilution of bleach in Dl water, pH-adjusted to ~6.8 using
glacial acetic acid.
The following steps were followed for cleaning the decontamination chamber between each material type
and before/after each test:
• Using the back sprayer, the interior surfaces are kept wet with pAB solution for 10 minutes.
• With the drain open, the surfaces are then rinsed with Dl water. The run-off is collected in a carboy and
ultimately discarded.
• After ensuring all runoff is removed from the chamber, the valve is closed in preparation for the next
test.
• A mop assembly with a disposable pad is used to wipe down the interior of the chamber with isopropyl
alcohol orethanol.
• The pad is then removed and placed in a bucket of pAB solution for decontamination prior to disposal.
The following steps are followed for cleaning the wet/dry vacuum (including head assembly) after use in a
test:
• Soak the head assembly in pAB for at least 60 minutes.
• Spray the wet/dry vacuum drum with pAB and maintain wetted for at least 60 minutes.
• Soak the hoses in pAB for at least 60 minutes.
• Rinse all parts with Dl water.
• Air dry prior to re-use.
• Alternatively, the wet/dry vacuums may be fumigated with a STERIS VHP® sterilization cycle. This
cycle entails the use of a STERIS VHP® ARD hydrogen peroxide (H2O2) generator and exposure of all
components of the wet/dry vacuum to H2O2 at 250 ppmvfor4 hours by maintaining this constant
concentration in a decontamination chamber.
• Replace HEPA filters.
The following steps are followed for cleaning the buckets after use in a test:
A-1
-------
• Fill the buckets with pAB and leave them covered for at least 60 minutes.
• Rinse all buckets five times with Dl water.
• Air dry prior to re-use.
The following steps are followed for cleaning the brushes after use in a test:
• Soak the brushes in pAB for at least 60 minutes.
• Rinse with Dl water.
• Air dry prior to re-use.
The following steps are followed for cleaning the work surfaces before and after use:
• Wet all surfaces with pAB solution or using Dispatch® bleach wipes.
• Rinse with Dl water.
• Wet and wipe surfaces with isopropyl alcohol or ethanol.
• Air dry prior to re-use.
• Alternatively, cover paper can be used and replaced before/after each use.
The sampling templates are autoclaved before/after each use.
The following steps are followed for cleaning the coupon cabinets before and after use:
• Wet and wipe all surfaces with pAB solution or using Dispatch® bleach wipes.
• Rinse with Dl water.
• Wet and wipe surfaces with isopropyl alcohol or ethanol.
•
Air dry prior to re-use.
The gaskets used in MOP 6561 during the contamination procedure were cleaned via fumigation with the
STERIS VHP® sterilization cycle. This cycle entails the use of a STERIS VHP® ARD hydrogen peroxide
(H2O2) generator at 250 ppmvfor4 hours by maintaining this constant concentration in a decontamination
chamber.
The carboys were autoclaved according to MOP 6570.
The BioSampler and front end of the BioSampler train were autoclaved according to MOP 6570.
A-2
-------
Concrete coupons were autoclaved according to MOP 6570. Other types of coupons were cleaned via
fumigation with the STERIS VHP® sterilization cycle. This cycle entails the use of a STERIS VHP® ARD
H2O2 generator at 250 ppmv for 4 hours by maintaining this constant concentration in a decontamination
chamber.
A-3
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Appendix B: Miscellaneous Operating Procedures (MOPs)
MOP 3113: Procedure for Deposition of Bacillus Subtilis Spores using a Metered Dose Inhaler
MOP 3128-A Procedure for Preparing pH-Adjusted Bleach Solution
MOP 3128-B Procedure for Preparing pH-Adjusted Bleach Solution with Tri-sodium Phosphate
Substitute
MOP 3135: Procedure for Sample Collection using BactiSwab™ Collection and Transport Systems
MOP 3144: Procedure for Wipe Sampling of Coupons
MOP 3145: Procedure for HEPA Vacuum Sampling of Large and Small Coupons
MOP 3150: Procedure for Fabrication of 14" x 14" Material Coupons
MOP 3156: Procedure for Wetted Wipe Decontamination
MOP 3161-LD: Aerosol Deposition of Spores onto Material Coupon Surfaces Using the Aerosol Deposition
Apparatus (ADA) - Low Dosing
MOP 3163: Aerosol Application of Grime on Material Coupons
MOP 6535a: Serial Dilution: Spread Plate Procedure to Quantify Viable Bacterial Spores
MOP 6555: Petri Dish Media Inoculation Using Beads
MOP 6561: Aerosol Deposition of Spores onto Material Coupon Surfaces Using the Aerosol Deposition
Apparatus
MOP 6562: Preparing Pre-Measured Tubes with Aliquoted Amounts of Phosphate Buffered Saline with
Tween 20 (PBST)
MOP 6563: Swab Streak Sampling and Analysis
MOP 6565: Filtration and Plating of Bacteria from Liquid Extracts
MOP 6567: Recovery of Bacillus Spores from Wipe Samples
MOP 6568: Aseptic Assembly of Wipe Kits
MOP 6570: Use of STERIS Amsco Century SV 120 Scientific Prevacuum Sterilizer
MOP 6571: Recovery of Bacillus Spores from Via-cell Aerosol Sampling Cassettes
B-1
-------
MOP 6572: Recovery of Spores from HEPA Sock (Vacuum Sock) Samples
MOP 6578: Preparation and Analysis for SKC BioSamplers®
B-2
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Appendix C: Spore Deposition and Handling Procedures
The handling of the contaminated coupons for Task 1, including movement to minimize or control spore
dispersal, was done in accordance with MOP 6561. One person was tasked with removing the clamps
holding the dosing chamber to the coupon and the removal of the dosing chamber and gasket from the
coupon. A second person was then tasked with moving the coupon to the proper location (e.g., test and
positive control coupons to the Test Coupon Cabinet and blank coupons to the Blank Coupon Cabinet).
For Task 2, two personnel were used to move the 40 in by 40 in coupons into their vertical positions in
COMMANDER following removal of the deposition devices. This was the only time the coupons were
handled, and this occurred a minimum of two days prior to sampling.
For Task 1, the Test Coupon Cabinet was a steel cabinet (48 in wide by 24 in deep by 78 in high) with
twelve shelves each 6 in apart. Each cabinet held a total of 36 coupons, hence, two Test Coupon Cabinets
were needed for a test. These were labeled as Test Coupon Cabinet 1 and Test Coupon Cabinet 2. Test
and positive control coupons were arranged in each cabinet according to material types. A single material
type was not split among cabinets. Procedural blank coupons of each material/orientation to be used in a
single test were contained in a separate isolated cabinet (Blank Coupon Cabinet) of similar construction,
however, with dimensions of 48 in wide by 24 in deep by 36 in high with 3 shelves.
Each MDI was claimed to provide 200 discharges. The number of discharges per MDI was tracked so that
use did not exceed this value. Additionally, in accordance with MOP 6561, the weight of each MDI was
recorded after completion of the contamination of each coupon. If an MDI weighed less than 10.5 g at the
start of the contamination procedure described in MOP 6561, the MDI was retired and a new MDI used. For
quality control of the MDIs, a contamination control coupon was run as the first, middle, and last coupon
contaminated with a single MDI in a single test. The contamination control coupon was a stainless steel
coupon (1.2 feet by 1.2 feet) that was contaminated in accordance with MOP 6561, sampled, and analyzed.
A log was maintained for each set of coupons that were dosed via the method of MOP 6561. Each record in
this log recorded a unique coupon identifier (see Table C-1), the MDI unique identifier, the date, the
operator, the weight of the MDI before dissemination into the coupon dosing device, the weight of the MDI
after dissemination, and the difference between these two weights. The coupon codes were pre-printed on
the log sheet prior to the start of coupon contamination (dosing).
Additionally, after a coupon was dosed via the above procedure, the coupon was labeled with the unique
identifier using the coding outlined in Table C-1. The label was printed on the side of the coupon using a
permanent marker (e.g., black or silver Sharpie®). The sampling team maintained an explicit laboratory log
which included records of each unique sample number and its associated test number, contamination
application, any preconditioning and treatment specifics, and the date treated. Each coupon was marked
with only the material descriptor and unique code number. Once the coupons were transferred to the
NRMRL/NHSRC Biocontaminant Laboratory for plate counts, each sample was additionally identified by
replicate number and dilution.
C-1
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Table C-1. Coupon Sample Coding
Coupon Identification: 25-TN-(X)M-D-(TT)-SS-NN
Category
TN
(X)M
(Material)
D
(Descriptor)
TT
SS
(Sample Type)
NN
(Sample Number)
Example
Code
A1
X
K
W
D
S
0
P
T
C
X
FB
TT
DE(F/B)
VE (F/B)
FN
FB
R
VN
VF
W
HS
E
NN
Test Number (from Table 2-1 , Table 2-2, Table 2-3,
Table 2-4, or Table 2-5)
Procedural Blank
Concrete
Rough-cut barn wood
Painted wallboard
Stainless Steel (for QC purposes)
Blank coupon
Positive control sample
Test sample
The sample has had contact with the coupon
The sample has had no contact with the coupon
Field Blank
Time point: 00,02,04,08,24,32 (A tests only - Table 2-1 )
Ambient (duct) exhaust (Front or Back half of sample)
Vacuum cleaner exhaust(Front or Back half of sample)
FAC and pH of pAB sample from nozzle
FAC and pH of pAB sample from bulk
Run-off
Swab sample of vacuum nozzle
Swab sample of vacuum filter
Wipe Sample
Vacuum sock sample
Direct extraction
Sequential numbers
NRMRL/NHSRC Biocontaminant Laboratory Plate Identification: 25-TN-(X)M-D-(TT)-SS-NN-R-
d
25-TN-(X)M-D-SS-(T)-NN
R
(Replicate)
As above
R A-C
C-2
-------
Coupon Identification: 25-TN-(X)M-D-(TT)-SS-NN
Category
d(Dilution)
Example
Code
1
Oto4, forlOEOto
10E4
Each material section will be identified by a description of the material and a unique sample number. The
sampling team will maintain an explicit laboratory log which will include records of each unique sample
number and its associated test number, contamination application, any preconditioning and treatment
specifics, and the date treated. Each material section test area sample will be marked with only the material
descriptor and unique code number. The wet/dry vacuum samples and exhaust sample from each test will
be identified with an associated test number and material section type. The sample codes will ease written
identification. Once the coupons are transferred to the NRMRL/NHSRC Biocontaminant Laboratory for plate
counts, each sample will additionally be identified by replicate number and dilution. Table 2-7 specifies the
sample identification. The NRMRL/NHSRC Biocontaminant Laboratory will also include on each plate the
date it was placed in the incubator.
The procedural blank coupons will have a two letter material code, with the first letter being "X". For runoff
samples, however, no material code will be used for the procedural blanks, since both materials will be
present. For the STS tests in Table 2-5, the decontaminated blanks are defined as the test coupons, and the
blanks that do not undergo decontamination are the controls. Not all the categories may be used for all
samples. For instance, a field blank for wipe coupons will be taken every day, but will not be affiliated with
any material. Hence, the first wipe field blank for Test G1 would be 25-G1-FB-W-1. The "C" and "X"
descriptor codes are used for samples such as the vacuum nozzle swab. A code of 25-G1-C-X-VN-1
designates a swab sample from the vacuum nozzle to be used on concrete coupons of Test G1, but before
such use occurred. As such, these "X" descriptors act as background blanks.
C-3
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Appendix D: Decontamination Procedures
Coupon Storage Cabinets
On the decontamination procedure test day, the procedural blank, test, and positive control coupons were
placed into the appropriate cabinets. A total of three cabinets were used to contain the coupons prior to
decontamination (one for the procedural blanks, and two containing the contaminated [positive controls and
test] coupons). One additional cabinet was used to store test coupons for drying after the decontamination
procedure has been applied. The cabinets with their intended purpose are listed in Table D-1.
Table D-1. Coupon Storage Cabinets
Cabinet Name
Test Coupon Cabinet #1
Test Coupon Cabinet #2
Procedural Blank Cabinet
Decontaminated Coupon Cabinet
Description
For storage of contaminated coupons (both positive control and
test coupons); each cabinet can hold 36 coupons, thus, two
cabinets will be needed for all tests
For storage of procedural blank coupons; the cabinet will be
under slight positive pressure in order to prevent contamination
from the test environment (i.e., laboratory) and allow passive air
flow to promote drying.
For storage of all test coupons after application of the
decontamination procedure; the cabinet will be under slight
positive pressure in order to prevent contamination from the
test environment (i.e., laboratory) and allow passive air flow to
promote drying.
Materials and Equipment
The materials and equipment used for the decontamination procedures were standardized as much as
possible and are listed in Table D-2. Decontamination steps are described in the subsequent sections of this
Appendix.
D-1
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Table D-2. Material and Equipment Used in the Decontamination Procedural Steps
Material/Equipment
Wet/dry vacuum
Sprayer
Bleach
Vinegar
Bucket of cleaning
solution
Detergent
Brush
Sponge
Nozzle
Garden hose
Pressure regulator
Description
RIDGID 14 Gallon Pro Vac WD1450
(http://vwwv.homedepot.com/webapp/wcs/stores/servlet/ProductDisplay?storeld=10051&la
ngld=-1&catalogld=10053&productld=100081216&N=10000003+90401+524502+1600)
Head attachment: RIDGID 2-1/2 In. Wet Nozzle (Squeegee) Accessory
(http://vwwv.homedepot.com/webapp/wcs/stores/servlet/ProductDisplay?storeld=10051&la
ngld=-1&catalogld=10053&productld=100046467&N=10000003+90401+524502+1600)
Filter: RIDGID 5-Layer Vacuum HEPA
Filter(http://vwwv.homedepot.com/webapp/wcs/stores/servlet/ProductDisplay?storeld=1005
1&langld=-1&catalogld=10053&productld=100022800)
12 Volt battery-operated 4 gallon backpack sprayer
(http://vwwv.agrisupply.com/product.asp?pn=59540&c2p=ppv&bhcd2=1 237402084)
Ultra Clorox® Regular Bleach (EPA Reg. No. 67619-8)
http://vwwv.clorox.com/products/overview.php7prod id=clb) 6.15% sodium hvpochlorite;
<1% sodium hydroxide
(http://vwvw.thecloroxcompany.com/products/msds/bleach/cloroxregularbleach0505_.pdf)
5% v/v technical grade acetic acid
3 gallons in a 5-gallon plastic pail
Klean-Strip TSP Substitute
(http://vwvw.homedepot.com/webapp/wcs/stores/servlet/ProductDisplay?storeld=10051&la
ngld=-1&cataloged=10053&productld=1 00259541)
Rubbermaid Floor Scrubber
(http://vwvw.homedepot.com/webapp/wcs/stores/servlet/ProductDisplay?storeld=10051&la
ngld=-1&catalogld=10053&productld=1 00644166)
QEP Extra Large Grouting Sponge, 7-1/2 x 5-1/2 x 2 In., Rectangle with Rounded Corners
(http://www.homedepot.com/webapp/wcs/stores/servlet/ProductDisplay?storeld=10051&la
ngld=-1&catalogld=10053&productld=1 00173109)
Standard Adjustable-Flow Garden Hose Nozzle Standard Brass, 4" Length
(McMaster-Carr, P/N 7484T1)
100ft; 5/8 inch diameter
(McMaster-Carr, P/N 7453T2)
Bronze Pressure Regulator-Plumbing-Code Rated Standard, 3/4" NPT Female, 25-75 PS'
(McMaster-Carr, P/N 8138K14)
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Material/Equipment
Bucket of Dl water
Carboy container
Pump
Description
3 gallons in a 5-gallon plastic pail
Carboys; Nalgene; Heavy Duty; polypropylene; Autoclavable; Leakproof; For full vacuum
applications up to 8 Hours; USP class VI, vacuum rated for intermittent vacuum use only;
83B Closure size; capacity: 5.25 gal. (20L)
NSF-Certified Rotary Vane Pump for Water with Motor, Brass, 4.3 Max GPM, 3/4
Horsepower
pH-adjusted Bleach (pAB)Solution
To reduce the impact of "natural" variations in the bleach solution in this study, the pH and chlorine content
was measured at the start and monitored throughout each test. The solution had to have a mean pH close
to, but not above neutral (>6.5 and <7.0) and a mean total chlorine content of 6,000-6,700 ppm. The
temperature of the solution had to be between 18 - 24 °C (64 - 75 °F). Any solution having a pH, chlorine
content or temperature falling outside of this range at anytime was discarded and a fresh pAB solution
prepared. The chlorine content was measured using a Hach high range bleach test kit (Method 10100). The
pH and temperature were measured using an Oakton pH probe (OKPH502; pH5). Dl water was used as the
base for all solutions.
The pAB solution was prepared just prior to the initiation of testing on a particular day and was used within a
window of three hours from the time of preparation (for Tasks 2 and 3). After three, hours, the bleach
solution was tested to see if it still met the QA criteria; if not, it was discarded and a fresh pAB solution
prepared. Additionally, technical grade acetic acid (5% v/v) was used instead of off-the-shelf white vinegar.
This change was expected to reduce the variability to in the pAB solution for the purpose of this study.
The pAB solution was applied to each coupon using a backpack sprayer (see Tables 2-2, 2-3 and 2-4).
Backpack Sprayer Application of Decontaminant
Prior to the start of testing, the spray pattern from the backpack sprayer was tested by spraying at the
appropriate distance (1 ft) onto a piece of 1.2 ft by 1.2 ft blue construction paper mounted in the position of
the material section. The spray was discharged into the center of the paper and the pattern was visually
assessed for consistency with that shown in Figure D-1. The diameter of the spray was checked to ensure
that was within the acceptable limits (12" to 16" diameter at 1 foot for the bleach sprayer, and at 12" to 16"
diameter at 3 feet for Dl water).
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Figure D-1. Bleach Spray Pattern from Nozzle 1 Foot Away
pAB Solution and Application
The pAB solution was applied to each material section using a 4-gallon rechargeable backpack sprayer
(ShurFlo ProPack SRS-600), see Figure D-2). The bleach was applied starting at the upper left or far left
corner and spraying across the section from left to right, then across the section from right to left, back and
forth 5 times for vertical surfaces. A constant spray having a diameter of about 12 inches at the section
surface was used; the surface was completely flooded to wet the surface. The spray nozzle was held a
constant distance of approx. 1 ft from the section surface. The battery-operated sprayer automatically
maintained a constant pressure of 35 psi throughout the application. The pressure was indicated by the
pressure gauge that is located on the sprayer. The flow rate of the pAB at 35 psi is 1000 mL/min. The flow
rate was measured before and after each application to a section. The pAB solution was reapplied
throughout the specified contact time, as outlined by the test plan. The sections were sprayed to completely
wet (or flood) the surface of the materials. During the bleach application, the duration of each spray
application was recorded. This measurement and the recording of flow rate allowed fora determination of
the amount of bleach applied to each material.
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Figure D-2. Backpack sprayer
pAB with TSP Substitute
The pAB with TSP substitute (surfactant) solution was made according to MOP 3128-B. This MOP
describes production of a TSP solution (DAP, SKU 7079860001, Baltimore, MD) made according to the
manufacturer's recommendations (1/4 cup per gallon solution). The solution, instead of being water, also
includes 6000 ppm bleach, neutralized with acetic acid. The general solution is made of 1/4 cup TSP, 12.5
oz (370 ml) germicidal bleach, and 30 oz (900 ml) 5% acetic acid in 1 gallon solution (3.785 liters).
The pAB + TSP solution was applied to each material section using a backpack sprayer (see Figure E-2).
The liquid was applied starting at the upper left or far left corner and spraying across the section from left to
right, then across the section from right to left, back and forth 5 times for vertical surfaces and four times for
horizontal surfaces. A constant spray having a diameter of about 12 inches at the section surface was used;
the surface was completely flooded to wet the surface. The spray nozzle was held a constant distance of
approx. 1 ft from the section surface. The battery-operated sprayer automatically maintained a constant
pressure of 35 psi throughout the application. The pressure was indicated by the pressure gauge that is
located on the sprayer. The flow rate of the pAB with TSP at 35 psi is 1000 mL/min. The flow rate was
measured before and after each application to a section. The pAB with TSP solution was reapplied
throughout the specified contact time and frequency. During the bleach application, the duration of each
spray application was recorded. This measurement and the recording of flow rate allowed for a
determination of the amount of bleach applied to each material.
One exception to this application method was for Tests O1, O2, O3, O6, and O7, a separate set of
wallboard coupons that had the pAB with TSP applied using a sponge. The sponge was squeezed to
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remove excess liquid prior to application to the coupon surface. Sponges were sterilized using VHP® and
were not reused.
Wet/Dry Vacuum
For each material type, a single wet/dry vacuum was used and clearly identified with a label indicating the
test number and material set (control or material type). The wet/dry vacuum exhaust was routed directly to
the air handling system to prevent re-entrainment of particles during this phase. A 6-inch duct was used to
exhaust filtered gas to the facility air handling system. The wet/dry vacuum used was a 6.0 HP unit with a
14-gallon capacity. The make and model are shown in Table E-2. The head attachment that was used in
this study was the 16-inch wet nozzle (squeegee) (Figure D-3).
Figure D-3.16 In. Wet Nozzle (Squeegee) Accessory
For vertical surfaces, the vacuuming action started with the center of the squeegee at the upper right corner
of the material. Nine downward strokes were used, each overlapping the last by 50 percent.
The filter inside the wet/dry vacuum was a 5-layer HEPA-rated filter (Figure D-4) that can be purchased as
an accessory replacement for the standard pleated filter (non-HEPA-rated) provided with the vacuum as
received by the consumer. Proper seating of the HEPA filter was confirmed for each assembly. The details
of the filter can be found in the link provided in Table D-2.
Figure D-4. 5-Layer HEPA Filter
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During application of the procedure, the time required for each material set was recorded. Before and after
each use of the vacuum, the vacuum cleaner was disassembled and sterilized using VHP®.
Brushing
The brush type used in this project was a synthetic fiber brush designed for heavy-duty rough floor
scrubbing and adsorbing cleaning solution (see Figure D-5). The dimensions of the brush are 10 inches
wide by 3 inches deep. The scrubbing action started at the upper right corner of the section. Using a zigzag
stroke, the brush was firmly moved down the surface. The time required for the brushing application to each
section was recorded.
Figure D-5. Brush (handle not shown)
Used brushes were placed into a bucket filled with the pAB solution. Brushes were left in the solution for a
minimum of one hour before being removed, rinsed with Dl water, and left to dry. After the brushes were
dry, a swab sample of the brushes was taken and plated to ensure sterility of the brushes. If viable target
organism was found on any brush, the entire batch was re-treated in the pAB solution, rinsed with Dl water,
dried and then re-sampled. If a positive sample was returned a second time for a batch of brushes, all
brushes were discarded and replaced.
Rinsing with a Garden Hose
Rinsing the decontamination chamber was done using a standard garden hose nozzle as listed in Table D-
2. The water was supplied to the nozzle through a 75 ft garden hose of 5/8 inch diameter. The head
pressure was maintained constant at approx. 60 psi using a pressure regulator listed in Table D-2. The
water was supplied via a closed loop system having a carboy container as the reservoir and a pump to
provide a pressurized stream and continual recirculation. An Oakton pH probe was used to monitor the
temperature and pH of the water throughout the test. The water used in this study was Dl water. Aliquots
(100 ml) were collected for filter plating as sterility checks the day each decontamination procedure was
run. Via adjustment of the nozzle, the spray pattern was controlled to be 1 ft in diameter measured 3 ft from
the nozzle.
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Figure D-7: Dl water supply system
Quality Control Measures
Additional measurements prior to or during the decontamination procedure application are also required in
order to ensure quality control in the testing. These measurements include quality control checks on the
reagents and equipment being used in the decontamination procedure. The pH and chlorine concentration
of the pAB solution has been shown to have a significant impact on the inactivation of Bacillus species
spores. After preparation of the pAB solution, the pH was measured using an Oakton pH probe. Additionally,
the pH was measured during the decontamination testing after each material was run within a test. The CI2
concentration was measured after preparation of the pAB solution by Hach High Range Bleach Test Kit,
Method 10100 (Model CN-HRDT). The temperature was also measured after the mixture was prepared and
prior to use on each material section using a NIST-traceable thermometer.
The water pressure at the head of the garden hose (i.e., faucet) was controlled with a pressure regulator.
The pressure was confirmed prior to each use of the hose. The flow rate and spray pattern from the hose
were checked prior to the start of the decontamination test. The flow rate was measured using an inline flow
meter. The spray pattern was visually verified to be nominally a 1 ft. diameter (10 - 14 in) at the coupon
surface from a distance of 3 ft. between the nozzle and coupon face.
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The time for application of each procedural step and time between procedural steps on each coupon was
measured using a NIST-traceable stopwatch and recorded in the laboratory notebook.
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Appendix E: Sampling Procedures
E.1 Sampling Material and Equipment
The materials and equipment used for sampling are listed in Table E-1.
Table E-1. Material and Equipment Used in Sampling
Material/Equipment
Nonpowdered, sterile surgical gloves
Nonpowdered, non-sterile surgical
gloves
Dust Masks
Disposable lab coats
Disposable Bench Liner
Phosphate Buffered Saline
50 ml_ conical tubes
Sterile sampling bags
Bleach wipes
Wipes
Swabs
Sampling Vacuum
Vacuum socks (sampling)
Carboys (2)
Analytical Filter Units
Vacuum pump
Description
KIMTECH PURE* G3 Sterile Nitrile Gloves, Kimberly-
Clark (VWR P/N HC61 1 10 for extra-large; VWR P/N
HC61 1 90 for large; VWR P/N HC61 1 80 for medium)
Exam gloves (Fisherbrand Powder-Free Nitrile Exam
Gloves, Fisher P/N 19-1 30-1 597D (for large); 19-1 30-
1597C (for medium))
3M Particulate Respirator 8271 , P95
Kimberly-Clark Kleenguard A10 Light Duty Apparel, P/N
40105
No source provided
Phosphate Buffered Saline with TWEEN®20 (Sigma
Aldrich, P/N: P3563-10PAK)
BD Falcon® BlueMax Graduated Tubes, 15mL (Fisher
Scientific P/N 14-959-70C)
Fisherbrand Sterile Sampling Bags (TWIRL'EM) Overpack
Size : 10" by 14", P/N 01-002-53
Inner bag size: 5.5" by 9" (wipe);
Sample Bag Size: 5.5" by 9 "
Dispatch® Bleach Wipes, P/N 69260
Kendall Curity Versalon absorbent gauze sponge 2" by 2"
sterile packed (rayon/polyester blend)
(http://www. mfasco.com/)
Bacti Swab®
(http://www.remelinc.com/lndustrial/
CollectionTransport/BactiSwab.aspx)
Omega Vac, Atrix International
Midwest Filtration, Cincinnati OH. x-cell 100
(http://www.midwestfiltration.com/dust-samplinq.php)
Nalgene autoclavable carboys with tabulation
(20 L) (Fisher Cat# 02-690-23)
150 mL Nalgene Analytical Filter Units (0.2 urn Cellulose
Acetate) (Fisher Cat# 130-4020)
Cast oil-free vacuum Pump with adjustable suction (Fisher
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Material/Equipment
Tubing
Filter cassettes
Sampling pump
Description
Cat#0 1-092-25)
Fisher PVC clear tubing (1/2" i.d., 1/16" thickness) (Fisher
Cat#14-169-7J)
Fisher PVC clear tubing (3/8" i.d., 1/16" thickness) (Fisher
Cat#14-169-7G)
Fisher PVC clear tubing (vacuum tubing)
(3/8" i.d., 1/8" thickness)
(Fisher Cat* 1 4-1 69-7H)
Via-Cell® Bioaerosol Sampling Cassette P/N VIA010
http://www.zefon.com/store/via-cell-bioaerosol-sampling-
cassette.html
Isokinetic Method 5 Source Sampling Console
Model 51 1E
http://www.apexinst.com/products/consoles.htm
E.2 Surface Sampling Procedures
Within a single Task 2 and 3 test, surface sampling of the coupons was completed for all procedural blank
coupons first, followed by all test coupons, and then followed by all positive control coupons.. Surface
sampling was done by wipe sampling in accordance with the protocol documented below. The surface area
for all samples was 1.3 sq. ft. A template was used to cover the exterior 0.25 in. of each Task 2 and 3
coupon leaving a 13.5 in. by 13.5 in. square exposed for sampling. The outer 0.25 in. around each coupon
was not sampled in order to avoid unrepresentative edge effects. A large stainless steel template covering
the entire coupon was used for Task 2 and 3 sampling. This template also prevented the outer edges from
being sampled, and provided a 0.5 in border between samples
Prior to the sampling event, all materials needed for sampling were prepared using aseptic techniques. The
materials specific to each protocol are included in the relevant sections below. In addition, general sampling
supplies were needed. A sampling material bin was stocked for each sampling event, using the information
included in these sampling protocols. The bin contained enough wipe sampling and vacuum sock sampling
kits to accommodate all required samples for the specific test. Additional kits of each type were also
included for backup. Enough prepared packages of gloves and bleach wipes were included in the bin. Extra
gloves and wipes were also included. A sample collection bin was used to transport samples back to the
NRMRL/NHSRC Biocontaminant Laboratory. The exterior of the transport container was decontaminated by
wiping all surfaces with a bleach wipe ortowelette moistened with a solution of pAB prior to transport from
the sampling location to the NRMRL/NHSRC Biocontaminant Laboratory.
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E.2.1 Coupon Wipe Sampling
Wipe sampling is typically used for small sample areas and is effective on nonporous, smooth surfaces such
as ceramics, vinyl, metals, painted surfaces, and plastics.11 The general approach is that a moistened sterile
non-cotton pad is used to wipe a specified area to recover bacteria, viruses, and biological toxins.11 The
protocol that was used in this project is described below and has been adopted from that provided by
Busher et al.11 Brown et al.12, and documented in the INL 2008 Evaluation Protocols.1314 It should be noted
that none of these references provide a validated wipe procedure tor Bacillus spores, as a validated
sampling procedure does not currently exist.
The following procedure was used in this study for Task 2 and 3 wipe sampling of each coupon surface:
1. A three person team was used, employing aseptic technique throughout. The team consisted of a
sampler, coupon handler, and support person.
2. All materials needed for collection of each sample was prepared in advance using aseptic technique. A
sample kit for a single wipe sample was prepared as follows:
a. Two sterile sampling bags (10" by 14", 5.5" by 9 ") and a 50 ml conical tube, capped, were labeled
in accordance with Appendix D. These bags and conical tube had the same label. The 5.5" by 9"
labeled sterile sampling bag was referred to as the sample collection sterile sampling bag.
b. A dry sterile wipe was placed in an unlabeled sterile 50 ml conical tube using sterile forceps and
aseptic technique. The wipe was moistened by adding 5 ml of sterile phosphate buffered saline
with 0.005% TWEEN®-20. The tube was then sealed.
c. The labeled 50 ml conical tube, capped, the unlabeled conical tube containing the pre-moistened
wipe, and the 5.5" by 9" labeled sampling bag were placed into the 10" by 14" labeled sterile
sampling bag. Hence, each labeled sterile sampling bag contained a labeled 50 ml conical tube
(capped), an unlabeled capped conical tube containing a pre-moistened wipe, and an empty
labeled sterile sampling bag.
d. Each prepared bag was one sampling kit.
3. All members of the sampling team each donned a pair of sampling gloves (a new pair per sample); the
sampler's gloves were sterile sampling gloves. All members wore dust masks to further minimize
potential contamination of the samples.
4. The coupon handler removed the coupon from the appropriate cabinet and placed it on the sampling
area. The sampling area was covered with a new piece of lab bench cover for each coupon.
5. The support person recorded the coupon code on the sampling log sheet.
6. The support person removed a template from the bag and handed it to the sampler.
7. The sampler placed the template onto the coupon surface.
8. The support person removed a sample kit from the sampling bin and records the sample tube number
on the sampling log sheet next to the corresponding coupon code just recorded.
9. The support person:
a. Opened the outer sterile sampling bag touching the outside of the bag.
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b. Touching only the outside of the overpack bag, removed and opened the unlabelled conical tube
and poured the pre-moistened wipe onto the sample.
c. Discarded the unlabelled conical tube.
d. Maneuvered the labeled 50 ml conical tube to the end of the outer sterile sampling bag and
loosened the cap.
e. Removed the cap from 50 ml conical tube immediately preceding the introduction of the sample
into the tube.
10. The sampler:
a. Wiped the surface of the sample horizontally using S-strokes to cover the entire sample area of the
coupon using a consistent amount of pressure.
b. Folded the wipe concealing the exposed side and then wiped the same surface vertically using the
same technique.
c. Folded over again and rolled up the folded wipe to fit into the conical tube.
d. Carefully placed the wipe into the 50 ml conical tube that the support person was holding being
careful not to touch the surface of the 50 ml conical tube or plastic sterile sampling bag.
11. The support person then immediately closed and tightened the cap to the 50 ml conical tube and slid
the tube back into the sample collection sterile sampling bag.
12. The support person then put the 50 ml conical tube into the empty labeled 5.5" by 9" sampling bag and
sealed the bag.
13. The support person then sealed the outer sample collection bag now containing the capped 50 ml
conical tube (containing the sample wipe) inside a sealed 5.5" by 9" sample collection bag.
14. The support person then decontaminated the outer sample bag by wiping it with a Dispatch® bleach
wipe.
15. The support person then placed the triple contained sample into the sample collection bin.
16. If sampling from the coupon was completed, the coupon handler moved the coupon and template to the
appropriate location for archival or discarding.
17. All members of the sampling team removed and discarded their gloves.
18. Steps 3 - 17 were repeated for each sample to be collected.
E.2.2 Vacuum Sock Sampling
Vacuum sock sampling is typically used for large porous areas.2 The general approach is that a collection
sock is used to trap dust material.2 The protocol that will be used in this project is depicted below and has
been adopted from that provided by Busher et al.2, Brown et al.8, and documented in the INL 2008
Evaluation Protocols12. None of these references provide a validated vacuuming procedure tor Bacillus
spores.
E-4
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The following procedure will be used in this study for Vacuum Sock sampling of each coupon surfaces:
1. A three-person team will be used, employing aseptic technique. The team will consist of a
sampler, sample handler, and support person.
2. All materials needed for each sample to be collected will be prepared in advance using aseptic
technique A sample kit for a single HEPA vacuum sample will contain the following:
a. Two sampling bags (10" x 14", 5.5" x 9") will be labeled in accordance with Section 4.9.
These bags will have the same label. An additional unlabeled bag contains the HEPA
sock collection assembly listed in Table 4-1. The label will be clearly distinguishable
through the unlabeled bag.
b. The two sterile, labeled sampling bags and the vacuum sock assembly bag will be placed
inside a second 10" x 14" unlabeled bag.
c. Each prepared bag is one Vacuum Sock sampling kit.
3. All members of the sampling team will each don a pair of sampling gloves (a new pair per sample);
the sampler's gloves shall be sterile sampling gloves. All members shall wear dust masks to
further minimize potential contamination of the samples.
4. The sampler will plug in the HEPA vacuum power cord and then don his/her sterile gloves.
5. The HEPA vacuum will be maintained on a rolling cart for easy movement into place.
6. The sampler will hold the vacuum nozzle for the support person to place the vacuum sock assembly
onto the nozzle.
7. The support person will open the sampling supply bin and remove one vacuum sock sample kit
from the bin.
8. The support person will record the sample collection bag number on the sampling log sheet.
9. The sample handler will remove the coupon from the appropriate cabinet and place it on the
sampling area.
10. The support person will remove a template from the bag and hand it to the sampler.
11. The sampler will place the template onto the coupon surface.
12. The support person will record the coupon code on the sampling log sheet next to the
corresponding vacuum sock collection bag number that was just recorded.
13. The support person will:
a. Open the vacuum sock sample kit outer bag and remove the unlabelled vacuum sock
assembly bag.
b. Open the small unlabelled sampling bag containing the vacuum sock assembly and push
the assembly from the bottom to expose the cardboard applicator tube opening.
c. Place the vacuum sock assembly onto the nozzle of the vacuum tube, using the bag to
handle the sock assembly, while the sampler holds the vacuum nozzle.
14. The sampler will:
a. Securely hold the outer edge of the sock onto the tube.
b. Turn on the vacuum with her foot.
c. Not let go of the filter sock while the vacuum is turned on in order to prevent the sock from
being sucked into the vacuum
d. Vacuum "horizontally" using S-strokes to cover the entire area of the material surface not
cover by the template, while keeping the vacuum nozzle perpendicular to the sample
surface.
e. Vacuum the same area "vertically" using the same technique.
f. Turn off the vacuum when sampling is completed.
15. The support person will remove the sock assembly from the nozzle, using the inner sterile sampling
bag.
16. The support person will then seal the inner sterile sampling bag and place it into the outer sterile
sampling bag.
17. The support person will then seal the outer sterile sampling bag.
18. The support person will then seal the 10" x 14" overpack sample bag now containing the outer and
inner bags, the inner containing the vacuum sock assembly. The 3rd size bag will then be wiped
with a bleach wipe and then dispose of the bleach wipe.
19. The sampler will wipe down the nozzle (inside and out) and end of the tubing with bleach wipe;
dispose of the bleach wipe.
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20. The support person will then place the triple contained sample into the sample collection bin.
21. If sampling from the coupon is completed, the sample handler will move the coupon and template to
the appropriate location for archiving or disposal.
22. All members of the sampling team will remove and discard their gloves.
Steps 3-21 will be repeated for each sample to be collected.
E.2.3 Swab Sampling
Swab sampling was used for sterility checks on coupons and equipment prior to use in the testing. A single
swab sample was collected from each item and coupon. MOP 3135 was followed (see Appendix C), which
employs the use of a pre-moistened swab.
E.3 Run-off Collection and Sampling Procedures
During application of the decontamination procedure for each set of Task I coupons, the drain in the
decontamination test chamber remained open. The runoff from the coupons throughout the entire
decontamination procedure being tested was collected for a given coupon set (material type or all blanks)
into a vessel which was pre-dosed with sodium thiosulfate (STS). The volume of sodium thiosulfate needed
to neutralize the total volume of decontamination liquid to be applied was determined by titration, and was
set to 150% in excess. After all coupons from a single set had been moved to the Decontaminated Coupon
Cabinet or Procedural Blank Cabinet, the chamber was rinsed with Dl water. For Task II, a run-off collection
vessel (trough) was placed under the coupon, and curtains arranged such that splashing run-off drained into
the trough. The trough was also dosed with enough STS to neutralize the decontamination liquid.
Analysis of the liquid was accomplished by filter-plating triplicate 100 ml aliquots of each run-off sample.
The collection procedure for the 100 ml aliquots was performed as follows:
1. Sampler donned a face mask, pair of examination gloves, disposable lab coat, and bouffant cap.
2. The contents of the carboy were agitated to ensure homogeneous mixing.
3. The carboy cap was removed.
4. Using a new 50 ml sterile pipette tip, 100 ml of sample was aseptically pipetted into a sterile 100 ml
container.
Step 4 was repeated until triplicate samples were obtained.
The runoff aliquots are triple-contained and transported to the NRMRL/NHSRC Biocontaminant Laboratory
for submission and analysis at the conclusion of the entire test according to MOP 6565 (see Appendix C).
Briefly, spores in the runoff sample were collected onto 0.2 urn pore-size analytical filters by vacuum
filtration (Figure E-1). The filter was then placed (particulate side up) onto bacterial growth media and
incubated 18 ± 2 hours at the optimal growth temperature. After incubation, colonies were enumerated on
the filter surface by visual inspection as shown in Figure E-2.
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Figure E-1. Nalgene Analytical Filter Unit connected to a Filter Unit.
Figure E-2. B. atrophaeus CFU on a Filter Unit.
E.4 Aerosol Sampling Procedures
The use of high-pressure hoses and pressure washers is expected to generate aerosols. There is potential
for generated aerosols to contain viable spores removed from the coupon surfaces. Bioaerosol samples
were collected from the decontamination chamber during all spraying activities. Zefon Via-Cell® Bioaerosol
Sampling Cassettes (Figure E-3) were used to collect aerosol samples. During Via-Cell® sample collection,
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the air concentration of chlorine gas (during pH-amended bleach application) or hydrogen peroxide vapor
(during Spor-Klenz® application) was also monitored.
The Via-Cell® sampler was operated and analyzed according to the manufacturer's recommendations.
(http://www.zefon.com/analytical/downloadA/ia-Cell Lab Manual Booklet.pdf). During Task I, separate Via-
Cell® samples were collected during the liquid decontamination application and the Dl water rinse
application. During Task II, separate Via-Cell® samples were collected before each decontamination step,
two samples during the decontamination step, and after the decontamination procedureto provide some
baseline data similar to the procedural blank during Task I. The Via-Cell® samples were analyzed according
to MOP 6571 (see Appendix C).
Figure E-3 Via-Cell BioAerosol Cassette
Filters are analyzed to determine viable CFUs collected per volume of air sampled.
The following sampling procedure was used to collect the Via-Cell® samples:
1. With a clean pair of gloves, the Via-Cell® was removed from the foil pouch. The cassette and the pouch
were labeled with the sample ID.
2. The small blue plug was removed and the cassette connected to the dry gas meter pump.
3. A leak-check was performed by turning on the pump with the inlet to the Via-Cell® closed capped off.
The flow of air should have stopped. If not, all connections were checked.
4. The cap of the Via-Cell® was removed and affixed in the ambient air around the coupon to be
decontaminated.
5. The starting volume on the dry gas meter (DGM) was recorded and the timer reset.
6. When time to collect a sample, the two switches on the meter box for the pump and the timer were
simultaneously turned on. The sample ID, the time of day and the meter temperature were recorded.
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7. The valve settings on the meter box were adjusted so that the AH pressure reading was 1.1" water.
8. At the end of sample collection, the two switches on the meter box for the pump and the timer were
simultaneously turned off. The final reading on the DGM, the meter temperature, and the elapsed time
were recorded.
9. The cap of the Via-Cell® was replaced and the pump disconnected. The outlet plug was reinserted.
10. The Via-Cell® was placed in the foil pouch. The exterior of the pouch was wiped with a Dispatch® wipe
and placed in secondary containment.
E.5 Sample Preservation
After sample collection, sample integrity was maintained by storage of samples in quadruple containers
(1 - sample collection container, 2 - sterile bag, 3 - sterile bag with exterior sterilized during sample
packaging process, 4 - sterile container holding all samples from a test). All individual sample containers
remained sealed while in the decontamination laboratory or in transport after the introduction of the sample.
The locking lid on the container holding all samples remained closed except for the brief period it is opened
for sample introduction by the support person of the sampling team. The sampling person did not handle
any samples after they were relinquished to the support person during placement into the primary sample
container.
After sample collection for a single test was complete, all samples were transported to the NRMRL/NHSRC
Biocontaminant Laboratory immediately, with appropriate chain of custody form(s).
In the NRMRL/NHSRC Biocontaminant Laboratory, all samples were stored in the refrigerator at 4 ± 2 °C
until they were analyzed. All samples were allowed to stabilize at room temperature prior to analysis.
E.6 Sample Holding Times
All samples were stored in accordance with Section F.5. Liquid samples were stored no longer than 24
hours prior to analysis. Samples of other matrices were stored no longer than five days before the primary
analysis. A typical holding time, prior to analyses, for most biological samples was two days.
During the analysis procedure, samples could be stored in the refrigerator overnight after extraction and
prior to the dilution plating. All samples were allowed to equilibrate to room temperature and were vortexed
for 10 seconds prior to plating.
Because pAB is not shelf-stable, it was analyzed immediately upon collection.
E-9
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Appendix F: Sample Analyses
F.1 Sample Analyses
The APPCD Biocontaminant Laboratory located in E-288 of the RTP, NC, campus facility analyzed all
samples to quantify the number of viable spores per sample. For all sample types except those from the
BioSampler, phosphate buffered saline with 0.05% TWEEN®-20 (PBST) was used as the extraction buffer.
After the appropriate extraction procedure, as described in the sections to follow, the buffer was subjected to
a five stage serial dilution (10~1 to 10"5), plated in triplicate and incubated overnight at 35 ± 2 °C in
accordance with MOP 6535a (see Appendix B). Following incubation, CFUs were enumerated according to
MOP 6567.
The PBST was prepared according to the manufacturer's directions and in accordance with MOP 6562 (see
Appendix C), dissolving one packet in one liter of sterile water. The solution was then vacuum-filtered
through a sterile 0.22 urn filter unit to sterilize.9
The extraction procedure used to recover spores was varied depending upon the different matrices (wipes,
filter socks, wet/dry vacuum filter, liquid, filter cassette) and can be found in MOP 6572 for extraction of
HEPA socks (vacuum socks), MOP 6567 for extraction of wipe samples, MOP 6563 for analyses of swab
samples, MOP 6571 for air sample cassettes. The procedures are described in the following subsections.
F.1.1 Recovery from Wipe Samples
The procedure for the recovery of spores from wipe samples (MOP 6567, Appendix B) was adapted from
the INL 2008 Evaluation Protocols8, and was performed as follows:
1. The analyst donned a fresh pair of gloves. Gloves were changed periodically (at least between batches)
or after direct contact with a sample to reduce contamination.
2. The 50 ml conical tube containing the sample wipe was removed from the double sterile bag and wiped
with a bleach wipe. The analysts changed gloves after the wipe step.
3. PBST (20 ml) was added to each 50 ml conical tube by aseptically pouring a pre-measured volume.
4. The sample was then vortexed for 2 minutes in 10 second bursts, leaving the wipe in the same tube.
5. If the sample sat for more than one minute after Step 4, the sample was re-vortexed individually to
homogenize prior to dilution plating. To complete dilution plating, the conical tube was uncapped and
the cap placed underside up on the Bio Safety Cabinet surface while the aliquot was removed from the
tube. Immediately after the aliquot was removed, the cap was aseptically replaced.
6. Each sample was processed individually. Steps 1-5 were repeated for each sample in the batch.
Dilution plating occurred as described in Section G.1.
F-1
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F.1.2 Recovery from HEPA Socks (Vacuum socks)
The extraction from HEPA sock (vacuum sock) samples is described in MOP 6572.
F.1.3 Recovery from Swabs
Swab sampling procedures were used to confirm sterility of coupons and equipment prior to their use in
testing. The protocol that was used in this project is described in MOP 6563. Each item to be used in the
decontamination process was sampled before use. The randomly selected procedural blank coupon (from
the same sterilization batch as the rest of the coupons of the same type) was also swab sampled before the
inoculation operations began.
F.1.4 Recovery from Liquid
The abundance of viable spores in the runoff samples was determined by filtration of runoff aliquots (MOP
6565). Filter samples were cultured on bacterial growth media, and recovery was determined by
enumerating colony forming units (CFU). The abundance of spores in the original runoff water was
determined by multiplying the calculated abundance of spores per milliliter of aliquot by the total runoff
volume.
F.1.5 Recovery from Wet/Dry Vacuums
The purpose of sampling the wet/dry vacuum after use was to confirm contamination of the unit with the
target organism. The most logical place to sample to confirm contamination was the HEPA filter. The filter
was sampled using the swab protocol described in MOP 3135. Each pleat of the filter was sampled with a
single swab. The vacuum nozzle (squeegee) was also sampled before and after use.
F.1.6 Recovery from 18 mm Coupons
The extraction of the spores from 18 mm coupons was accomplished as follows: (1) the coupons were
transferred into 50 ml sterile vials containing 10 ml PBST; (2) the vials containing the solution and coupon
were sonicated for 20 minutes at 42 kHz and 135 Watts (Branson 8510 ultrasonic waterbath), then vortexed
2 minutes to further dislodge any viable spores; and (3) each vial was briefly re-vortexed immediately before
any solution was withdrawn and subjected to a five stage serial dilution following MOP 6535a.
F.1.7 Recovery from Aerosol Samplers
Aerosol samples collected in the BioSampler were processed according to MOP 6575. The total flow in the
duct was measured using an S-type pitot tube as described in EPA Method 2. This measurement was done
at the beginning and end of the day of decontaminations.
F-2
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Appendix G: QAPP Amendments
1.1.1.1 Amendment 1 (6/16/2011)
The conditions of this first test "O1" were based upon preliminary testing conducted by the WAM and Leroy
Mickelsen with the testing equipment. Conditions for the remaining tests were to be decided once data were
collected and analyzed for test O1.
Table 2-2. Task 2 (Optimization) Test Matrix (Table and numbering directly from original amendment.)
Test
O1
Decontaminant
pAB solution
Inoculation
High1
Application Rate
850 - 900 mL/min flow rate, 1
second/ft2, (SHURflo sprayer set to
low pressure setting with nozzle at
the most restricting position,, i.e.,
mist)
Application frequency
At 0 minutes (no reapplication)
No Rinse,
Coupons dried overnight,
sampled the next day.
G-1
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1.1.1.2 Amendment 2 (6/17/2011)
Amendment 2 detailed tests that were outside the scope of this report.
1.1.1.3 Amendment 3 (9/23/2011)
Amendment to Task 2
The overall objective of Task 2 is to determine the effectiveness of the decontamination methods on larger
coupons (14 in. x 14 in) as a function of application parameters and spore loading. These parameters
include, but are not limited to: initial contamination level, decontamination procedure (including
decontaminant), decontaminant application rate, decontaminant reapplication frequency, and time.
This amendment includes a specific decontamination procedure applied to drywall coupons using wipes
wetted with sporicide or disinfectant.
MOP-3156: Procedure for Wetted Wipe Decontamination
The testing procedures will be conducted according to MOP-3156 included in Appendix A. The purpose of
this MOP is to ensure a consistent and representative procedure for using wetted wipe decontamination.
Test Matrix
Three tests will be conducted to determine the optimal wetted wipe decontamination technique on 14 in. x
14 in drywall coupons in accordance with MOP-3156. All three tests will include moistening of the coupon
surface with a freshly-made pAB solution as stated in MOP-3156, before applying the wiping procedure:
Test 1: pH-Adjusted Bleach (pAB) Wetted Wipe
Test 2: SimWipe or ready-made "tack cloth"
Task 3: Combination of SimWipe and pAB wetted wipe procedures consecutively
Wipe samples from each test will be collected after 18 hours of coupons drying, and appearing visibly dry.
Positive control coupons will be sampled at the same time. Wipe samples are collected by sampling within a
13.5" x 13.5" sampling template centered on the coupons. Each test will require three test, one procedural
blank, and three positive control coupons of drywall coupons, with a total of 7 coupons per test.
Sample ID, Tracking, and Chain of Custody
Each material section will be identified by a description of the material and a unique sample number. The
sampling team will maintain a detailed laboratory log which will include records of each unique sample
number and its associated test number, contamination application, any preconditioning and treatment
specifics, and the date treated. Each material section test area sample will be marked with only the material
descriptor and unique code number. The wet/dry vacuum samples and exhaust sample from each test will
be identified with an associated test number and material section type. The sample codes will ease written
G-2
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identification. Once the coupons are transferred to the NRMRL/NHSRC Biocontaminant Laboratory for plate
counts, each sample will additionally be identified by replicate number and dilution. Table 4-1 specifies the
sample identification. The NRMRL/NHSRC Biocontaminant Laboratory will also include on each plate the
date it was placed in the incubator.
Table 4-1. Sample Coding (Table and numbering directly from original amendment.)
Coupon Identification: 25-WA-(X)M-DD-NN
Category
WA
(X)M
(Material)
DD
(Descriptor)
NN
(Sample Number)
Example
Code
W1
X
D
S
B
P
T
FB
NN
Wipe Procedure test A(1=pAB, 2=SimWipe, 3=
Combined)
Procedural Blank
Painted wallboard
Stainless Steel (for QC purposes)
Blank coupon
Positive control sample
Test sample
Field Blank
Sequential numbers
NRMRL/NHSRC Biocontaminant Laboratory Plate Identification: 25-WA-(X)M-DD-NN-R-d
25-WA-(X)M-DD-NN
R
(Replicate)
d(Dilution)
As above
R A-C
1 0 to 4, for 10 to 10 x104
G-3
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1.1.1.4 Amendment 4 (10/3/2011)
Amendment to Task 2
The overall objective of Task 2 is to determine the decontamination method effectiveness on larger coupons
(14 in. x 14 in) as a function of application parameters and spore loading. These parameters include, but are
not limited to: initial contamination level, decontamination procedure (including decontaminant),
decontaminant application rate, decontaminant re-application frequency, and time.
This amendment includes a test matrix that will determine the efficacy of specific decontamination
procedures for coupon materials loaded with a relatively low inoculum of spore.
1. Coupons Material List
The coupons material list is shown in Table 1.
Table 1. Material Coupon List (Table and numbering directly from original amendment.)
Coupon Material Type
Coupon loading (CPU)
Drywall
Concrete
Wood
Number of Coupons
1x102
9
6
6
Blank
4
3
3
Sample ID, Tracking, and Chain of Custody
Each material section will be identified by a description of the material and a unique sample number. The
sampling team will maintain a detailed laboratory log which will include records of each unique sample
number and its associated test number, contamination application, any preconditioning and treatment
specifics, and the date treated. Each material section test area sample will be marked with only the material
descriptor and unique code number. The sample codes will ease written identification. Once the coupons
are transferred to the NRMRL/NHSRC Biocontaminant Laboratory for plate counts, each sample will
additionally be identified by replicate number and dilution. Table 2 specifies the sample identification. The
NRMRL/NHSRC Biocontaminant Laboratory will also include on each plate the date it was placed in the
incubator.
G-4
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Table 2. Sample Coding (Table and numbering directly from original amendment.)
Coupon Identification: 25-O7-(X)M-D-BB-SS-NN
Category
(X)M
(Material)
D
(Descriptor)
BB (decontamination procedure)
SS
(Sample Type)
NN
(Sample Number)
Example Code
X
K
W
D
0
P
T
FB
O1
WS
HS
NN
Procedural Blank
Concrete
Rough-cut barn wood
Painted wallboard
Blank coupon
Positive control sample
Test sample
Field Blank
BB=O1 For Test procedure , and W1 for Wetted wipe
with Amended Bleach (for Painted Wall board only)
Wipe Sample
Vacuum sock sample
Sequential numbers (1 to 3)
NRMRL/NHSRC Biocontaminant Laboratory Plate Identification: 25-O7-(X)M-D-BB-SS-NN-R-d
25-O7-(X)M-A-D-B-SS-NN
R
(Replicate)
d(Dilution)
As above
R
1
A-C
0 to 4, for 10 to 10 x104
2. Decontamination Testing Protocol
Decontamination Methods
Repeat of Test O1
Spray once with pAB for 15 seconds, no reapplication, and no rinse (identical to Test O1). Allow about 18
hours of spore drying before the sampling phase.
G-5
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Repeat of Test W1 for Painted Dry Wall Only
One test will be conducted to determine the efficacy of the wetted wipe decontamination technique on 1 x
102 contaminated 14 in. x 14 in drywall coupons in accordance with MOP-3156. This test will be similar to
Test W1 that involved moistening of the coupon surface with a freshly-made pAB solution as stated in MOP-
3156, before applying the wetted wiping procedure:
3. Testing Protocol
Testing schedule
Day 1:
• Inoculate 1 x 102 coupons based on earlier results. Leave 1 coupon of each concrete and wood and 2
coupons of drywall as procedural blanks, and 1 coupon of each material as field blanks. An additional
set of sterilized coupons will be required as blanks on Day 3.
Day 2:
• Perform Test O1 decontamination procedure on 1 blank coupon of each type and place them in the
Blank cabinet. Perform W1 procedure on an additional drywall coupon.
Clean chamber.
• Perform decontamination procedure(s) for coupons of each type, and place them Test cabinet 2.
Day 3:
Sample coupons in the following order:
• procedural blanks
• sample coupons
• 1 x 102 positive control,
Analysis
Due to the various levels of spores in the coupons, the following analysis procedure will be followed:
• 1 x 102 positive controls zero dilution plate, 1 ml filter plates
• All other samples 1 ml and remainder filter plates
G-6
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1.1.1.5 Amendment 5 (10/12/2011)
Recovery from Grimed Coupons
To determine the effect of grime on recovery of our target organism, an additional test is necessary. This
test will be performed on stainless steel coupons. Three sterilized stainless steel coupons will be placed in
the grime deposition hood (three at a time). Coupons will be handled with sterile gloves to minimize
contamination. The coupons will undergo the grime application procedure (details to follow) and be allowed
to dry. As soon as the coupons are dry, they will be removed from the hood and placed under sterile ADA
pyramids as if being prepared for inoculation, but no inoculation will take place. Once all three coupons have
been through this procedure, the pyramids will be removed and the coupons will be sampled according to
MOP 3144. The wipe samples will then be sent to the NRMRL/NHSRC Biocontaminant Laboratory, along
with a field blank sample (a handled but unused wipe) and a coupon blank (a wipe sample from a sterile
coupon).
The NRMRL/NHSRC Biocontaminant Laboratory will extract the samples according to MOP 6567, but will
then split the samples into two 10 mL aliquots. One aliquot of each sample will then be spiked with 1 x 104
Bacillus atrophaeus spores (total). Three 10 mL aliquots of PBST will also be spiked with the same quantity
of spores. All samples will be plated at the 0 and -1 dilution, with subsequent plating at higher dilutions
pending results.
Sample IDs will be as follows:
25-GR1 -WN-{1, 2, and 3}: Replicate samples of unspiked portion of wipe sample from grimy coupon
25-GR1-WS-{1, 2, and 3}: Replicate samples of spiked portion of wipe sample from grimy coupon
25-GR1 -FB-1: Field blank wipe
25-GR1 -WX-1: Sterile coupon wipe
25-GR1-P-{1,2, and 3}: Spiked PBST used to verify inoculum
The N and S portions of the first two sample IDs will be appended by the NRMRL/NHSRC Biocontaminant
Laboratory following extraction and splitting.
1.1.1.6 Amendment 6 (1/11/2012}
The overall objective of Task 2 is to determine the effectiveness of the decontamination method on larger
coupons (14 in. x 14 in) as a function of application parameters and spore loading. These parameters
include, but are not limited to: initial contamination level, decontamination, decontaminant application rate,
decontaminant re-application frequency, and time. The test matrix for Task 2 was changed in response to
Test O1 Test results; the decontaminant for this testing sequence is pAB solution.
G-7
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(Table directly from original amendment.)
Test
01
02
O3
O4
O5
O6
071
O8
O9
O10
Inoculation
High
High
High
Low
Very low
Low
Material
Concrete
Dry Wall
Rough-cut wood
Concrete
Dry Wall
Rough-cut wood
Rough-cut wood
Concrete
Dry Wall
Rough-cut wood
Rough-cut wood
Rough-cut wood
Spraying Time
(sec)
15 sec
15 sec
30 sec
15 sec
15 sec
15 sec
30 sec
30 sec
15 sec
15 sec
Sprayed at Time (min)
0
0
0
At 0 and 5 min
At 0 and 15 min
0
0
At 0 and 5 min
At 0 and 15 min
Spray Flow Rate
(mL/min)
1000
1500
1000
1000
1000
1000
1Test O7 was added for a very low inoculation level
In Task 3, sections (14 in x 14 in) of grimed or neat materials positioned as walls will be inoculated with
Bacillus atrophaeus (formerly Bacillus globigii) via aerosol deposition for a chosen decontamination
procedure developed in Task 3 as a function of the procedures and material types used. The test matrix for
Task 3 is presented in this amendment:
Test Procedures: The test procedure will be the same as the one applied for Test O3 (1 x 30 second spray
(1000 ml/min, mist).
G-8
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The test matrix for Task 3 shall be completed as follows: (Table directly from original amendment.)
Test
G1
G2
G3
G4
G5
G6
G7
G8
G92
G102
Decontaminant
pAB solution
pAB solution
pAB solution/TSP solution
pAB solution/TSP solution
pAB solution/TSP solution
pAB solution/TSP solution
pAB solution/TSP solution
pAB solution/TSP solution
pAB solution/TSP solution
pAB solution/TSP solution
Inoculation
High
High
High
High
High
High
High
High
Low
Low
Grime (yes/no)
No
Yes
No
Yes
No
Yes
No
Yes
No
Yes
Scrubbing
No
No
No
No
Yes
Yes
Yes
Yes
No
No
Vacuuming
No
No
No
No
No
No
Yes1
Yes1
No
No
1This step will occur first; ^these tests will be optional upon approval by the WAM and if funding and time
permit.
G-9
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United States
Environmental Protection
Agency
PRESORTED STANDARD
POSTAGE & FEES PAID
EPA
PERMIT NO. G-35
Office of Research and Development (8101R)
Washington, DC 20460
Official Business
Penalty for Private Use
$300
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