United States
Environmental Protection
Agency
EPA 600/R-15/100 I June 2015 I www.epa.gov/research
Expedient Approach for
Decontamination of Biologicals
Indoor Environment
Determination of Operationally
Effective Liquid Decontaminant
Application Methods for Indoor
Decontamination
ASSESSMENT AND EVALUATION REPORT
Office of Research and Development
National Homeland Security Research Center
-------
EPA600-R-15-100
Expedient Approach for Decontamination of Biologicals
Indoor Environment
Determination of Operationally Effective
Liquid Decontaminant Application Methods
for Indoor Decontamination
Assessment and Evaluation Report
National Homeland Security Research Center
Office of Research and Development
U.S. Environmental Protection Agency
Research Triangle Park, NC 27711
-------
Disclaimer
The United States Environmental Protection Agency, through its Office of Research and Development's
National Homeland Security Research Center, funded and directed this investigation through EP-C-09-
027 with ARCADIS U.S., Inc. 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. This report
includes photographs of commercially available products. The photographs are included for purposes of
illustration only and are not intended to imply that EPA approves or endorses the product or its
manufacturer. Environmental Protection Agency does not endorse the purchase or sale of any
commercial products or services.
Questions concerning this document or its application should be addressed to:
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@epa.gov
-------
Acknowledgments
This effort was directed by the principal investigator (PI) from the Office of Research and Development's
(ORD's) National Homeland Research Center (NHSRC), to address critical capability or knowledge gaps
identified by the Program to Align Research and Technology with the Needs of Environmental Response
(PARTNER). A project team consisting of the PI, one additional member from ORD's NHSRC, and two
members from the U.S. Environmental Protection Agency's (EPA's) Office of Solid Waste and Emergency
Response's (OSWER's) Consequence Management and Advisory Division (CMAD) guided this research.
Project Team:
M. Worth Calfee, Ph.D. (PI)
NHSRC, ORD, US EPA
Research Triangle Park, NC 27711
R. Leroy Mickelsen, M.S., P.E.
US EPA, OSWER, Office of Environmental Management (OEM), Chemical, Biological, Radiological, and
Nuclear (CBRN) CMAD
Research Triangle Park, NC 27711
Mike Nalipinski
US EPA, OSWER, OEM, CBRN CMAD
Boston, MA 02203
Sang Don Lee, Ph.D.
NHSRC, ORD, US EPA
Research Triangle Park, NC 27711
The peer-, quality assurance-, and editorial-reviewers of this report are also acknowledged for their input
to this product:
Peer Reviewers;
Shannon Serre (EPA, OEM, CMAD),
Dave Rees (EPA, Region 10),
Randy Schademann (EPA Region 7)
Quality Assurance and Editorial Reviewers:
Ramona Sherman (EPA, ORD, NHSRC)
Eletha Brady-Roberts (EPA, ORD, NHSRC)
Joan Bursey (EPA, ORD, NHSRC)
This effort was completed under U.S. EPA contract #EP-C-09-027 with ARCADIS-US, Inc. The support
and efforts provided by ARCADIS-US, Inc. are gratefully acknowledged.
IV
-------
Table of Contents
Disclaimer iii
Acknowledgments iv
List of Tables ix
List of Acronyms and Abbreviations xi
Executive Summary xiii
1 Introduction 1
1.1 Project Objectives and Process 1
1.2 Experimental Approach 2
1.2.1 Taskl 2
1.2.2 Task 2 3
1.2.3 Definitions of Effectiveness 4
2 Materials and Methods 7
2.1 Task 1 - Evaluation of Material Limits to Spray-Based Wetting Procedures 7
2.1.1 Facility Design 7
2.1.2 Coupon Preparation 8
2.1.3 Taskl Test Matrix 8
2.1.4 Spray Procedure 9
2.1.5 Physical Testing and Analytical Procedures 12
2.1.6 Sample Identification and Tracking 14
2.1.7 Sample Preservation 14
2.1.8 Long-Term Monitoring Schedule 15
2.2 Task 2 - Decontamination Procedures Effectiveness Evaluation 16
2.2.1 Test Chamber 16
2.3 Test Coupon Preparation 17
2.3.1 Ceiling Tile Coupons 18
2.3.2 Wallboard Coupons 20
2.3.3 Carpet Coupons 20
2.3.4 Upholstery Coupons 21
2.3.5 Paper Samples 22
2.3.6 Simple Coupons 22
-------
2.4 Spore Preparation 22
2.5 Coupon Inoculation 23
2.5.1 Inoculation of 14-in by 14-in Coupons 23
2.5.2 Inoculation of 24-in by 24-in Ceiling Tiles 23
2.5.3 MDI Use and Inoculation Recordation 24
2.6 Task 2 Test Matrix 25
2.6.1 Phase One - Method Optimization Testing 25
2.6.2 Phase Two - Decontamination Procedure Testing 25
2.6.3 Post-Decontamination Conditioning 28
2.7 Sampling and Analytical Procedures 29
2.7.1 Sampling Procedures 29
2.7.2 Decontamination Solution Sampling 30
2.7.3 Office Chamber Sampling Procedures 30
2.7.4 Microbiological Analysis 36
2.7.5 Cross Contamination Prevention 38
2.7.6 Sample Containers for Collection, Transport, and Storage 40
2.7.7 Sample Holding Times 40
2.7.8 Sample Handling and Custody 41
2.7.9 Sample Archiving 41
2.7.10 Information Recorded by Field Personnel 41
3 Results and Discussion 43
3.1 Task 1 - Drying Cabinet Conditions 43
3.2 Task 1 - Physical Testing 44
3.2.1 Temporal Evaluation of Wallboard Hardness 44
3.2.2 Evaluation of Moisture Content on Wallboard 46
3.2.3 Temporal Evaluation ofWallboard Roughness 50
3.3 Task 2- Effectiveness Evaluation of Decontamination Procedures 54
3.3.1 Phase 1: Optimization Testing Method Development 54
3.3.2 Phase 2: Mock Office Environment Testing 55
4 Quality Assurance/Quality Control 63
4.1 Sampling, Monitoring, and Analysis Equipment Calibration 63
4.2 Quality Assurance and Quality Control Checks 64
VI
-------
4.3 Acceptance Criteria for Critical Measurements 64
4.3.1 Quality Control Checks 66
4.4 QA/QC Reporting 67
4.4.1 Deviations from the QAPP 68
4.5 Data Quality Audits 69
5 Summary and Recommendations 70
References 72
VII
-------
List of Figures
Figure 2-1. Spray Chamber 7
Figure 2-2. Electric Backpack Sprayer 10
Figure 2-3. Spray Pattern Characterization 11
Figure 2-4. Measurement Location Template for each Set of Replicate Test Coupons 12
Figure 2-5. Extech FHT200 Penetrometer (Hardness Meter) 13
Figure 2-6. James Instruments, Inc., NDTT-M-70 Moisture Meter 13
Figure 2-7. Phase II Plus SRG-4000 Profilometer (Surface Roughness Meter) 14
Figure 2-8. Coupons Stored Vertically Inside the Drying Cabinet 15
Figure 2-9. 0.866-in (22-mm) Ceiling Tile Coupons 19
Figure 2-10. Ceiling with Coupon Sockets for Later Installation of Inoculated Ceiling Tile Test
Coupons 19
Figure 2-11. Wallboard Section with Open Socket Intended for Inoculated Wallboard Coupon 20
Figure 2-12. Carpet Flooring with Coupon Sockets for Inoculated Coupons 21
Figure 2-13. 12-in by 12-in Carpet Tile Coupon Sectioned into Six4-in by6-in Pieces in
Preparation for Extraction Sampling 21
Figure 2-14. Front of Assembled Upholstered Cushion 22
Figure 2-15. Round ADA-2R Schematic (ID = inner diameter) 23
Figure 2-16. Top View of Round ADA-2R 24
Figure 2-17. Round ADA-2R with O-ring Gasket 24
Figure 2-18. Material Coupons in Position Priorto Decontamination 26
Figure 2-19. Carpet Flooring Coupon Sockets for Inoculated Coupon (4-in by 6-in and 12-in by
12-in) Placement 27
Figure 2-20. Office Environment Void of Porous Materials 28
Figure 2-21. Office Material Sample Coupon Layout 31
Figure 2-22. Office Environment Floor Sampling Layout 34
Figure 2-23. Vacuuming AFSD on Subflooring 35
Figure 2-24. Aseptic Transfer of PBST onto Mopping Pad of AFSD for Tile Floor 35
Figure 2-25. Mopping AFSD on Laminate Tile Flooring 36
Figure 3-1. Average Daily RH and Temperature inside the Drying Cabinet 43
Figure 3-2. Material Hardness Evaluation of Unfinished Wallboard 44
Figure 3-3. Material Hardness Evaluation of Matte Latex Finish 45
Figure 3-4. Material Hardness Evaluation of Semi-gloss Finished Wallboard 46
VIM
-------
Figure 3-5. Absorption Capacity by Surface Finish for Varying Liquid Applications 47
Figure 3-6. Surface Moisture Content on Unpainted Wallboard over Time 48
Figure 3-7. Surface Moisture Content of Matte Latex-Finished Wallboard over Time 49
Figure 3-8. Surface Moisture Content of Semi-gloss-Finished Wallboard over Time 50
Figure 3-9. Surface Roughness of Unpainted Wallboard over Time 51
Figure 3-10. Surface Roughness of Matte Latex-Finished Wallboard over Time 52
Figure 3-11. Surface Roughness of Semi-Gloss-Finished Wallboard over Time 52
Figure 3-12. Carpet Recovery Optimization Tests 54
Figure 3-13. Ceiling Tile Extraction Optimization Results 55
Figure 3-14. Procedure 1 Office Environment Floor Sampling Layout 56
Figure 3-15. Pre-decontamination Recoveries - Procedure 1 57
Figure 3-16. Post-Decontamination Recoveries - Procedure 1 57
Figure 3-17. Pre-Decontamination Recoveries - Procedure 2 58
Figure 3-18. Post-Decontamination Recoveries - Procedure 2 59
Figure 3-19. Decontamination Efficacies of Decontamination Procedure 2 on Non-porous and
Porous Decontaminated Materials, by Surface Sampling Method 60
Figure 3-20. Warm Spot Sampling Layout 61
Figure 3-21. Post-Decontamination Recoveries of Spores using Sponge Sticks in Areas Adjacent
to Hotspots - Laminate Flooring 61
Figure 3-22. Sampling Recoveries for both Conventional and Robotic Sampling Approaches 62
List of Tables
Table 1-1. Decontamination Procedure Type Material Handling 3
Figure 2-1. Spray Chamber 7
Table 2-1. Task 1 Test Matrix 9
Figure 2-2. Electric Backpack Sprayer 10
Figure 2-3. Spray Pattern Characterization 11
Table 2-2. Measured Test Parameters - Task 1 11
Figure 2-4. Measurement Location Template for each Set of Replicate Test Coupons 12
Figure 2-5. Extech FHT200 Penetrometer (Hardness Meter) 13
Figure 2-6. James Instruments, Inc., NDTT-M-70 Moisture Meter 13
Figure 2-7. Phase II Plus SRG-4000 Profilometer (Surface Roughness Meter) 14
IX
-------
Figure 2-8. Coupons Stored Vertically Inside the Drying Cabinet 15
Table 2-3. Long-Term Monitoring Schedule 15
Table 2-4. Physical Monitoring Timeline 16
Table 2-5. Coupon Types 17
Table 2-6. Task 2, Phase Two Test Matrix 25
Table 2-7. Sampling and Analytical Procedures 30
Table 2-8. Sampling Methods used for Material Surfaces in the Office Configuration 31
Table 2-9. Frequency of Sampling Monitoring Events 32
Table 2-10. Critical and Non-Critical Measurements 33
Table 3-1. One-Way RM-ANOVA 53
Table 3-1: Sampling Recoveries for both Conventional and Robotic Sampling Approaches 62
Table 4-1. Instrument Calibration Requirements 63
Table 4-2. Critical Measurement Acceptance Criteria 65
Table 4-3. QA/QC Sample Acceptance Criteria 66
Table 4-4. Task 1 Data Quality Criteria for pAB 67
Table 4-5. Task 1 pAB Monitored Points 67
Table 4-6. Task 2 Data Quality Criteria for pAB 67
-------
List of Acronyms and Abbreviations
ABS
AD
AD-2R
AFSD
APPCD
ASTM
ATCC
B.
Bg
BOTE
BSC
CBRN
CPU
CMAD
COC
COMMANDER
Dl
DPG
DQI
DQO
EPA
EtO
FAC
FIFRA
ft
H202
HSRP
ID
in
ISO
LPM
LR
MDI
min
ml
Acrylonitrile butadiene styrene
Aerosol deposition apparatus
Round aerosol deposition apparatus
Automated floor sampling device
Air Pollution Prevention and Control Division
ASTM International
American Type Culture Collection
Bacillus
Bacillus atrophaeus
Bio-response Operational Testing and Evaluation
Biological Safety Cabinet
Chemical, Biological, Radiological, and Nuclear
Colony forming unit(s)
Consequence Management Advisory Division
Chain of custody
Consequence Management and Decontamination Evaluation Room
Deionized
Dugway Proving Ground
Data Quality Indicator
Data Quality Objective
U.S. Environmental Protection Agency
Ethylene oxide
Free available chlorine
Federal Insecticide, Fungicide, and Rodenticide Act
Foot/feet
Hydrogen peroxide
Homeland Security Research Program
Identification, inner diameter
Inch(es)
International Organization for Standardization
Liter(s) per minute
Log reduction
Metered dose inhaler
Minute(s)
Milliliter(s)
XI
-------
MOP Miscellaneous Operating Procedure
NA Not applicable
NHSRC National Homeland Security Research Center
NIST National Institute of Standards and Technology
OEM Office of Emergency Management
ORD Office of Research and Development
OSWER Office of Solid Waste and Emergency Response
P/N Part Number
pAB pH-adjusted bleach
PARTNER Program to Align Research and Technology with the Needs of Environmental
Response
PBST Phosphate Buffered Saline with Tween®20
PFU Plaque Forming Unit
PI Principal Investigator
PPE Personal protective equipment
ppm Part(s) per million
ppmv Parts per million by volume
psi Pounds per square inch
QA Quality Assurance
QAPP Quality Assurance Project Plan
QC Quality Control
RH Relative humidity
RM-ANOVA Repeated Measured Analysis of Variance
s Second(s)
SCADA Supervisory Control and Data Acquisition
SEM Scanning electron microscopy
TSA Tryptic Soy Agar
VHP® Vaporized hydrogen peroxide
WACOR Work Assignment Contracting Officer Representative
XII
-------
Executive Summary
This project supports the mission of the U.S. Environmental Protection Agency's (EPA) Office of
Research and Development's (ORD) Homeland Security Research Program (HSRP) by providing
information relevant to the remediation of areas contaminated with biological agents.
The primary objective of this investigation was to determine the effectiveness of an expedient approach
for indoor decontamination. Such approaches were utilized in previous anthrax remediation efforts, and
their effectiveness has been determined experimentally at the bench-, pilot-, and field-scale. While this
approach has been demonstrated to be effective under certain conditions, the material impact to common
indoor surface types when this approach was used under efficacious conditions has yet to be determined
comprehensively. To close this knowledge gap, the current study evaluated the impacts of spray-based
decontamination procedures on wallboard, one of the most common and abundant indoor surface types,
and a surface type with high likelihood of wetting impact due to its adsorptive properties.
Task 1: Evaluation of Material Limits to Spray-Based Wetting Procedures
In Task 1 of this study, coupons consisting of wallboard coated with one of three surface finishes
(unpainted, matte latex, and semi-gloss) were subjected to spray-based decontamination procedures to
determine the physical impact. Coupons were sprayed with pH-adjusted bleach (pAB) or deionized (Dl)
water to maintain visible wetness for 10, 30 and 60 minutes. Each test included three replicate material
coupons of each finish type. A set of unexposed coupons was used as controls. Physical assessments
(hardness, moisture, surface roughness) were performed on the coupons after application of the spray
procedures over an extended period of time (34 days), to determine if the spray procedures caused
lasting changes (damage) to the material.
In general, none of the spray procedures explored in Task 1 showed evidence of causing lasting effects
on the physical integrity of wallboard, regardless of paint finish. The volume of decontaminant required to
reach the physical limits of the wallboard would be far greater than the volume that would reasonably be
applied during an actual decontamination event. With this information, the focus of the spray-based
decontamination procedures used in Task 2 shifted from the physical limits of the materials to the overall
effectiveness of the spraying procedures. It should be noted that damages to a building structure, other
than to surface/wall materials, is possible during extensive spray-based decontamination operations (i.e.,
damage to sub-floor, or damage to ceilings below spray operations within multi-story buildings); however,
was outside the scope of this investigation.
Task 2: Decontamination Procedures Effectiveness Evaluation
In Task 2 of this study, the decontamination efficacy of non-destructive spray procedures was then
determined in laboratory trials using a non-pathogenic surrogate for Bacillus (B.) anthracis. An indoor
chamber was retrofitted with a mock office environment. Common office materials were inoculated with
known concentrations of surrogate spores, then the space was decontaminated using one of two spray-
based procedures developed from the findings in Task 1. One procedure prescribed the removal
(simulated off-site treatment) of all porous items before spray-based decontamination commenced; the
second procedure evaluated the decontamination efficacy when all items (porous and non-porous) were
decontaminated in-place. Overall effectiveness - a combination of physical and chemical methods to
xiii
-------
reduce and/or inactivate spores of the 8. anthracis surrogate (8. atrophaeus (Bg)) from a contaminated
surface - was determined for both procedures. A secondary objective was to determine the sampling
efficacy of an automated floor sampling device (AFSD) robots following these expedient spraying
decontamination methods.
The surface sampling methods on previously-wetted ceiling tile and carpet materials proved to be
ineffective compared to extractive sampling methods. For upholstery, the antimicrobial properties of this
indoor/outdoor material apparently prevented a conclusive assessment of the decontamination efficacy of
the spraying procedure.
Spraying procedures proved less effective on porous materials than on non-porous materials. These
results suggest that an alternative decontamination method should be considered for porous items in lieu
of a spray-based method. These findings suggest that, if a low-tech approach is to be utilized, porous
materials should be spritzed with decontaminant (i.e., wetted to reduce risk of spore resuspension from
items) and removed from an indoor environment before executing a more efficacious decontamination
procedure of the remaining non-porous items.
Post-decontamination sampling of areas directly adjacent to inoculated areas indicated that viable spores
relocated to areas in which the decontaminant solution pooled. During an actual remediation event, the
transportation of spores to areas where pooling may occur should be anticipated. Additionally, such
pooling areas may provide ideal locations for sampling.
The AFSD room scale post-decontamination collection proved to be a robust method for non-porous
surface materials. The AFSD spore surface recoveries were found to be comparable to the sponge stick
sampling technique. The same results were also found to be consistent for the carpet materials when the
AFSD was compared to the vacuum sock technique for surface sampling. There is an advantage to using
the robot cumulative sampling approach, compared to the conventional sampling techniques that collect a
certain number of discrete samples that can result in some false negatives. These data suggest that
traditional approaches to post-liquid-based-decontamination sampling likely underrepresent the
magnitude of the number of viable spores remaining on surfaces following the treatment.
XIV
-------
1 Introduction
This project supports the mission of the U.S. Environmental Protection Agency's (EPA's) Office of
Research and Development's (ORD's) Homeland Security Research Program (HSRP). EPA's National
Homeland Security Research Center (NHSRC) strives to accomplish the HSRP mission by providing
information, expertise and products that can be widely used to prevent, prepare for, and recover from
public health and environmental emergencies involving chemicals, biologicals, and/or radiologicals.
In 2001, the introduction of a few letters containing Bacillus (B.) anthracis (anthrax) spores into the U.S.
Postal Service system resulted in the contamination of several 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 vaporized hydrogen
peroxide (VHP®). Additional quick, effective and economical decontamination methods having the
capacity to be employed over wide areas (outdoor and indoor) are required to increase preparedness for
repetition of such a release.
In addition to fumigation, which was used primarily in large heavily contaminated facilities, other
decontamination methods were used. These methods included combinations of disposal of contaminated
items, vacuuming, and the use of liquid sporicides such as a pH-adjusted bleach solution. If proven
effective, "lower-tech" decontamination methods could reduce overall remediation costs through
increased decontamination capacity and reduced reliance on specialized contractors. Decontamination
can be accomplished by physical removal of surface-bound contaminants or via inactivation of the spores
with antimicrobial chemicals. Physical removal could be accomplished via on-site removal of the
contamination from the material or physical removal of the material itself (i.e., disposal). Similarly,
inactivation of the contaminant can be done on-site or after removal of the material prior to ultimate
disposal. During the 2001 remediation, the balance between removal and decontamination in-place was
facility-dependent and factored in 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.
Since 2001, a primary focus for facility remediation has been improving confidence in decontamination
methods. Developing and demonstrating lower cost and more readily available/deployable
decontamination options could significantly increase EPA's readiness to respond to a wide area release.
1.1 Project Objectives and Process
The objective of Task 1 (Evaluation of Material Limits to Spray-Based Wetting Procedures) was to
determine the upper limits (without irreversible damage) of liquid application to wallboard building
materials with varying finishes. Representative sections of wallboard (referred to as "coupons") were
wetted with pH-adjusted bleach (pAB) solution at varying time intervals, then were observed over an
extended period of time (34 days), to determine if the spray procedures caused permanent damage to the
materials. Each spray procedure was repeated using deionized (Dl) water for the purpose of performing a
comparative analysis of the damaging effects of the two liquids. Measurements of material moisture,
hardness, and roughness as well as observations of appearance were used to evaluate damage. The
information gathered from this test was to be used for method development in Task 2 of this effort.
-------
The objective of Task 2 (Decontamination Procedures Effectiveness Evaluation) was to determine the
effectiveness of a set of two low-tech decontamination approaches. These approaches were based upon
prior experience from EPA's Bio-response Operational Testing and Evaluation (BOTE) [1] project. One
approach involved entry into the contaminated space, "spritzing" (lightly spraying all surfaces with pAB to
reduce resuspension of spores) and bagging of contaminated porous materials (paper, soft furniture,
ceiling tiles, etc.). The waste bags were removed, and decontamination of the remaining surfaces was
accomplished by treatment (spraying) with sporicidal liquids (in accordance with a previously published
study [2]). The space was allowed to dry, then surface areas were sampled. The second approach was
identical to the first, with the exception that there was no removal of porous items prior to treatment (i.e.,
all items in the space were decontaminated in-place). Air samples were collected during both procedures
to monitor the re-aerosolization of spores.
In an effort to maximize the benefit of the room-scale mock office environment, automated floor sampling
devices (AFSDs) were evaluated for their utility in post-decontamination sampling. Spore recovieries by
two AFSDs were compared with recoveries obtained using conventional sampling methods.
1.2 Experimental Approach
1.2.1 Taskl
Wallboard coupons were prepared with one of three surfaces (unpainted, indoor matte latex, and semi-
gloss). During testing, the surface of the coupons was sprayed to wetness with pAB at To (initial time) and
maintained wetted by reapplying pAB to achieve exposure durations (wet contact time) of 10, 30, and 60
minutes. Each spray procedure was also performed using Dl water to differentiate between wetting
impacts on materials and pAB-specific impacts. Each test included three replicate material coupons of
each finish type. A set of unexposed coupons served as controls. Physical assessments (hardness,
moisture, surface roughness) were performed on the coupons after application of the spray procedures
for a period of 34 days immediately following the decontamination treatment.
The procedural steps for this task are summarized below:
1. Wallboard coupons (14 inches (in) by 14 in) were cut from larger pieces of wallboard.
2. Preliminary physical characterization (hardness, moisture, and surface roughness measurements) of
coupons materials were performed.
3. Prescribed spray procedures were applied.
4. Temporal assessments (daily for the first five days, then weekly for an additional month) on physical
characteristics of wetted coupons were performed.
A One-Way Repeated Measured Analysis of Variance (RM-ANOVA) was used to analyze the large sets
of data gathered over the observation period. RM-ANOVA was used to assess the significance of the
effect the wetting spray treatments (10-, 30-, and 60-minute (min) spray) had on moisture content,
hardness, and surface roughness on wallboards with different paint finishes.
-------
1.2.2 Task 2
An indoor chamber retrofitted with a mock office setup containing office materials inoculated with known
concentrations of surrogate spores was decontaminated using two spray-based procedures (Table 1-1).
In one procedure (Procedure 1), all porous items were removed prior to the initiation of decontaminant
spraying (simulated off-site or ex situ treatment of porous items). The second procedure (Procedure 2)
evaluated the decontamination efficacy when all items (porous and non-porous) were decontaminated in-
place.
Table 1-1. Decontamination Procedure Type Material Handling
Procedure Type
Procedure 1
Procedure 2
Material
Ceiling Tile
Painted Wallboard
Carpet
Upholstery
Paper
ABS* Plastic
Formica Laminate
Vinyl-coated paper
Ceiling Tile
Painted Wallboard
Carpet
Upholstery
Paper
ABS Plastic
Formica Laminate
Vinyl-coated paper
Porous or Non-
porous
Porous
Non-porous
Porous
Porous
Porous
Non-Porous
Non-porous
Non-porous
Porous
Non-porous
Porous
Porous
Porous
Non-Porous
Non-porous
Non-porous
Designated for
Removal
Yes
No
Yes
Yes
Yes
Yes
No
Yes
No
*Acrylonitrile butadiene styrene
Overall effectiveness - a combination of physical and chemical methods to reduce and/or inactivate 8.
atrophaeus (Bg) spores (formerly, 8. globigii; a surrogate for 8. anthracis) from a contaminated surface,
was determined for both procedures.
The procedural steps for this task are summarized below:
1. Inoculation of the materials (coupons) with Bg spores via aerosol inoculation.
2. Placement of the coupons within the office environment.
-------
3. Application of prescribed decontamination methods or procedures.
4. Assessment of residual viable spores (via post-decontamination sampling), starting inoculation (via
sampling of positive controls), and potential cross-contamination (via sampling of negative controls
(blanks)).
5. Deployment of the AFSD for post-decontamination sampling.
6. Determination of decontamination effectiveness as measured by log reduction (e.g., mean of the logs
of test sample recoveries subtracted from mean of the logs of positive control sample recoveries).
7. Documentation of operational considerations (e.g., cross-contamination, procedural time, impacts on
materials and personnel).
In addition to the steps outlined above, all test activities were documented via narratives in laboratory
notebooks, real-time data acquisition, and the use of digital photography. The documentation included,
but was not limited to, any deviations from the test plans and physical impacts on the materials.
All tests were conducted in accordance with internal miscellaneous operating procedures (MOPs), to
ensure repeatability and adherence to the data quality validation criteria set for this project.
1.2.3 Definitions of Effectiveness
The surface decontamination efficacy for each decontamination technique and surface material
combination was evaluated by measuring the difference in the logarithm of the measured colony forming
units (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 (LR) on the specific material surface as defined in Equation 1-1.
where:
rj = ^J H (1-1)
' Nc NS
Surface decontamination effectiveness; the average LR of
TJ. = spores on a specific material surface (surface material
designated by;)
The average of the logarithm (or geometric mean) of the
iog(CFUc^) ^ number of viable spores (determined by CPU) recovered on
— the control coupons (C indicates control and Nc is the number
1\
c of control coupons)
-------
loeCCFC/ )
N
The average of the logarithm (or geometric mean) of the
number of viable spores (determined by CPU) remaining on
the surface of a decontaminated coupon (S indicates a
decontaminated coupon, and Ns is the number of coupons
tested).
When no viable spores were detected, a value of 0.5 CPU was assigned for CFUs.k, and the efficacy was
reported as greater than or equal to the value calculated by Equation 1 -1 .
The standard deviation of the average LR of spores on a specific material (l"|i ) is calculated by Equation
1-2:
where:
C*r)
„ _
' '
Standard deviation of r|i, the average LR of spores on a
specific material surface
The average LR of spores on a specific material surface
(surface material designated by ;)
The average of the LR from the surface of a decontaminated
coupon (Equation 1-3)
Ns = Number of test coupons of a material surface type.
X,. =•
.
where:
_
\og(CFUc) = —
N
c
Represents the "mean of the logs" (geometric mean),
the average of the logarithm-transformed number of
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)
-------
r PI _ Number of CPU on the surface of the kth
decontaminated coupon
Total number (1 ,k) of decontaminated coupons of a
material type.
In this report, decontamination efficacy is generally reported in terms of LR for a particular material.
Results may include whether the average LR fora particular test is > 6.0, since a decontaminantthat
achieves > 6 LR (against a 6-7 log challenge) is generally considered effective during laboratory efficacy
tests for the purposes of registration under the Federal Insecticide, Fungicide, and Rodenticide Act
(FIFRA) [3]. Demonstrated effectiveness in the laboratory can help predict a decontamination
procedure's/product's performance in the field, but the 6 LR benchmark is in no way translatable to
clearance goals.
-------
2 Materials and Methods
2.1 Task 1 - Evaluation of Material Limits to Spray-Based Wetting Procedures
2.1.1 Facility Design
All spray activities involving pAB were performed inside a spray chamber (Figure 2-1) designed
specifically for "low-tech" decontamination of medium-sized material coupons. The spray chamber
promoted operator safety by removing liberated chlorine gas (generated from pAB) through the filtered
exhaust via a readily accessible connection to the facility's air handling system. The spray chamber
dimensions were 4 feet (ft) high by 4 ft wide by 4 ft deep, and the chamber was designed to
accommodate three 14 in by 14 in coupons at a time in horizontal or vertical orientation. In this study, only
the vertical assembly was utilized. The spray chamber was constructed of stainless steel with the
exception of the front face and top, which were clear acrylic plastic. The acrylic door was fitted with ports
to allow the insertion of the sprayer-type delivery system in the direct middle of each vertical coupon.
Figure 2-1. Spray Chamber
To expose all coupons on the same day, spray testing with Dl water was performed inside the
Consequence Management and Decontamination Evaluation Room (COMMANDER), which is described
in detail in Section 2.2.1.
-------
2.1.2 Coupon Preparation
Wallboard coupons were prepared by cutting 14 in by 14 in by 0.5-in sections from a larger 4 ft by 8 ft by
0.5 in wallboard section (Goldbond Model GB00090800, National Gypsum Company, Charlotte, NC).
Coupons then received one of three surface finishes representing different levels of protection that typical
wallboard surfaces may have: minimal protection with no primer or paint (i.e., unpainted wallboard);
moderate protection provided by primer and interior flat (matte) latex paint (Behr® Premium Plus® Interior
Flat White Latex Paint); and the highest level of protection provided by primer and industrial grade semi-
gloss epoxy paint (Sherwin Williams® Pro Industrial™ Pre-Catalyzed Water-based Epoxy, K46-150
Series, Semi-Gloss). Following fabrication of the wallboard coupons (described in Section 2.3),
preliminary weight measurements were collected for all coupons. White water-based latex interior/exterior
multi-surface primer (KILZ 2, Model #20941, Home Depot, Durham, NC) and paints were applied using
roller brushes. Adhesive tape (FT210, Reflectix, Inc., Markleville, IN) was applied to the edges after the
finish. This positioning allowed the tape to remain visible and easily avoided during physical testing.
Preliminary tests conducted on a subset of coupons constructed with joint tape and joint compound (both
applied after the primer and paint) revealed that during the spray application, the joint compound
dissolved and the joint tape separated from the coupon surface. As a result, the spray solution absorbed
more readily into the wallboard through the exposed, unfinished edges. Accordingly, all subsequent tests
utilized adhesive tape (in place of the joint tape) to seal the outer edges of the coupons, thereby
preventing liquid absorption through the edges and ensuring that post-exposure physical
characterizations remained representative of the wallboard material.
It should be noted that wallboard installed within buildings has vulnerabilities to liquid damage not
represented accurately in these coupon-based tests. Extensive spray-based decontamination operations
may facilitate significant liquid absorption and wicking at floor/wallboard unions. Such wicking effects as
well as other impacts likely to result during indoor spray procedures (i.e., damage to sub-floor, or
damage to ceilings below spray operations within multi-story buildings) were outside the scope of this
investigation.
2.1.3 Task 1 Test Matrix
Eighteen tests were performed, as shown in Table 2-1. Paint finish (unpainted, matte latex, or semi-
gloss), visible wetness time (10, 30, or 60 min) and spray solution (pAB or Dl water) were varied across
the tests. For each test, three replicate coupons were exposed to the prescribed conditions; in addition,
three unexposed (not sprayed) coupons of each type were evaluated and served as material controls.
The surface roughness, moisture content, and hardness of each coupon were measured to evaluate
impact of the spray solution. A total of 63 coupons were utilized for this effort.
-------
Table 2-1. Task 1 Test Matrix
Wallboard Finish
Unpainted
Indoor Matte Latex Paint
Semi-Gloss Epoxy Paint
Spray Solution
Dl Water
pAB
Dl Water
pAB
Dl Water
pAB
Wetness Time (min)
10
30
60
10
30
60
10
30
60
10
30
60
10
30
60
10
30
60
Dependent Variables
Surface Roughness,
Moisture Content,
Hardness
2.1.4 Spray Procedure
The pAB was prepared inside a four-gallon electric backpack sprayer (Figure 2-2). Prior to test initiation,
free available chlorine (FAC) was verified to be between 6000 and 6700 parts per million (ppm) and pH
between 6.5 and 7. The target flow rate of the pAB solution was set to 1.2 liters per minute (LPM), and
the flow was checked at the start and at the conclusion of each set of spray applications. The spray
pattern was measured at the onset of each day's testing and was verified to be between 12 in and 14 in in
diameter, as shown in Figure 2-3. The temperature of the solution was recorded but was not considered
to be a critical measurement.
Tests using pAB were performed in the ventilated spray chamber. A set of three replicate wallboard
coupons was installed vertically (most common real-world orientation of drywall) in the spray chamber.
Once all QC requirements were confirmed to be within specification, the material sections were sprayed
through the center port of the chamber door. The chamber door remained in the closed position during
spay application to allow proper venting of fumes generated from the pAB. Coupons were re-sprayed to
achieve the 0 (control), 10, 30, or 60 min of total "visibly wetted" contact time.
The large capacity of the COMMANDER chamber allowed simultaneous initiation of all tests using Dl
water by staggering the spray applications. All coupons designated for 10, 30, and 60 min of visible
wetness were sprayed. After 10 min, those coupons designated for the 30- and 60-min tests were re-
sprayed. Then finally, after 30 min, only those coupons designated for 60 min of visible wetness were re-
sprayed. Each set of coupons was positioned vertically on tables inside the COMMANDER. A guide line
was placed on the table surface, at a measured distance of 1 ft from the front of the coupons, to serve as
a visual aid for proper nozzle positioning during spray application.
-------
For pAB and Dl water spray procedures, the initial spray application was for 15 seconds (s), with each
subsequent spray application for 10 s. The spray application was initiated at the top left corner of the
coupon positioned on the far left. Spraying continued across all three coupon replicates to the top right
corner of the coupon positioned on the far right. Spraying continued across all three coupons in a "Z"
pattern. The frequency of reapplication depended upon liquid type (pAB or Dl water) and surface finish.
Table 2-2 details the test parameters for each test. At the lowest solution level, the sprayer maintained a
pressure of 35 pounds per square inch (psi) and a flow rate of approximately 1.2 LPM.
Figure 2-2. Electric Backpack Sprayer
10
-------
Figure 2-3. Spray Pattern Characterization
Table 2-2. Measured Test Parameters - Task 1
Test Description
Unpainted, Dl Water
Unpainted, pAB
Matte Latex, Dl Water
Matte Latex, pAB
Semi-Gloss, Dl Water
Semi-Gloss, pAB
Contact
Time
10 min
30 min
60 min
10 min
30 min
60 min
10 min
30 min
60 min
10 min
30 min
60 min
10 min
30 min
60 min
10 min
30 min
60 min
Total Spray
Applications
2
8
17
3
10
16
3
8
17
5
11
19
3
8
17
5
14
27
Measured
flow rate
(mL/min)
1140
1140
1140
1200
1224
1164
1140
1140
1140
1164
1188
1200
1140
1140
1140
1188
1200
1140
Total Applied
Volume per Coupon
(mL/coupon)
158
538
1108
233
714
1067
221
538
1108
355
759
1300
221
538
1108
363
966
1741
11
-------
2.7.5 Physical Testing and Analytical Procedures
2.1.5.1 Measurement Quantities and Collection Points
Physical monitoring tests were conducted to determine the physical impact of each liquid type and
wetness duration on the various coupon types. These tests were performed according to the schedule
detailed in Table 2-2. To ensure that reported measurements for each coupon were representative of the
entire test set, three measurements were taken per test, per region; corner, edge, and center. Figure 2-4
shows a test template for a set of three coupons and the test locations on each coupon. Purple indicates
the edge regions, green indicates the corner regions, and blue indicates the center regions. Locations
marked with an "M" were reserved for moisture testing.
Figure 2-4. Measurement Location Template for each Set of Replicate Test Coupons
One measurement (average of three points) was taken from each of the three replicate coupons; an edge
position (purple), center position (blue), and corner position (green), for a total of nine points per test. To
ensure that each measurement was representative, a region sampled within the same test set would be
in a different location relative to the previous coupon(s). For example, if corner testing for the first
replicate was from the upper left corner, corner testing from the second and third replicates would come
from one of the other corners.
2.1.5.2 Hardness Testing
The hardness sampling procedure for wallboard was a modified version of ASTM International (ASTM)
C473-10, "Standard Test Methods for Physical Testing of Gypsum Panel Products". Hardness testing was
performed using the Extech FHT200 penetrometer (Extech Instruments Corporation, Nashua, NH) (Figure
2-5) equipped with a 0.118 in (3 mm) foot. The measurement taken was the maximum force (N) exerted
just before penetrating the surface of the coupon. Due to the destructive nature of hardness testing,
locations designated for moisture testing were avoided.
12
-------
Figure 2-5. Extech FHT200 Penetrometer (Hardness Meter)
2.1.5.3 Moisture Testing
A James Instruments, Inc., NOT T-M-70 (James Instruments, Inc., Chicago, IL) (Figure 2-6) was used to
measure moisture content. The T-M-70 is a pinless meter allowing for repeated sampling of the same
areas without destruction of the coupon surface. Moisture content was measured from one corner, one
edge, and one center of each test set (as shown in Figure 2-4). For each coupon set, one individual
measurement was collected from each replicate coupon; one measurement for the corner, edge, and
center locations, each from a different coupon. For example, if the corner moisture measurement for a set
was made from the first replicate, the edge and center measurements were collected from the second
and third replicates.
Figure 2-6. James Instruments, Inc., NOT T-M-70 Moisture Meter
13
-------
2.1.5.4 Roughness Testing
The Phase II+, SRG-4000 (Figure 2-7) Profilometer (Phase II+, Upper Saddle River, NJ) was used to
measure surface roughness. When the SRG-4000 takes a measurement, a diamond stylus traverses
across the material surface and a piezoelectric pickup records all vertical movement. Peaks and valleys
are recorded and converted into micrometers (urn). Similar to moisture and hardness testing, surface
roughness was measured from one corner, one edge and one center of each replicate set. Due to the
destructive nature of hardness testing, coupon locations designated for moisture content were used for
surface roughness testing.
Figure 2-7. Phase II Plus SRG - 4000 Profilometer (Surface Roughness Meter)
2.7.6 Sample Identification and Tracking
Each coupon was marked on the back with a unique code using a permanent marker prior to the initial
weighing. This identification (ID) also served as a way to verify coupon orientation.
2.1.7 Sample Preservation
Following spraying, the coupons were removed from the test chamber and immediately weighed, then
placed vertically inside positive pressure drying cabinets under a flow of dry compressed air at 5 LPM
(Figure 2-8). Coupons were also stored in these drying cabinets between physical testing events.
14
-------
Figure 2-8. Coupons Stored Vertically Inside the Drying Cabinet
The relative humidity (RH) and temperature inside the cabinet were recorded throughout the duration of
physical monitoring using a Onset® HOBO® U12 temperature /RH data logger (Grainger, Raleigh, NC).
2.1.8 Long-Term Monitoring Schedule
Following the wetting procedure, each coupon set was relocated to the positive-pressure drying cabinets
for 18 hours, prior to the initial physical tests. Table 2-3 shows the long-term monitoring schedule. Table
2-4 provides the elapsed time (days) from the application of the spray procedure for each physical test
event.
Surface roughness tests were not performed during Test 1.
Table 2-3. Long-Term Monitoring Schedule
First Week
Hardness and Moisture testing was
performed every weekday for the first
week (Days 1 to 5).
5 tests (Tests 1-5)
First Month
Hardness, Moisture, and Roughness
testing was performed every week for
four weeks after the conclusion of daily
testing (up to 34 days after exposure).
four tests (Tests 6-9)
15
-------
Table 2-4. Physical Monitoring Timeline
Physical Test Event
1
2
3
4
5
6
7
8
9
Elapsed Time From Spray
Application (Days)
1
2
3
4
5
15
22
28
34
2.2 Task 2 - Decontamination Procedures Effectiveness Evaluation
The objective of Task 2 was to determine the decontamination effectiveness of non-destructive spray
procedures (identified under Task 1 of this effort) in a mock office setting contaminated with Bacillus
spores. In addition, evaluations of two strategies (with and without removal of porous items prior to
application of spray-based procedures) were conducted under Task 2.
2.2.1 Test Chamber
All mock office decontamination activities were conducted inside the COMMANDER chamber.
COMMANDER has the following attributes:
• Allows repeated fabrication of an environment (e.g., furnished office room; outdoor setting)
contained within the chamber.
• Allows for release of biological organisms or chemicals into the chamber.
• Allows for application of a decontamination technology (including fumigation with toxic corrosive
gases).
• Supports entry into the chamber during all of the above mentioned activities (in appropriate
personal protective equipment (PPE)).
• External dimensions of 9 ft by 12 ft by 10 ft high.
• Nominal internal dimensions of 8.3 ft by 11.3 ft by by 8.8 ft high.
• Contains one air-tight entry/exit port with a window.
• Contains a6ftby6ftby8ft high airlock with single air-tight entry/exit port with a window.
• Contains entry/exit ports in line with the enclosure double door to allow for large materials to be
brought into or out of the chamber.
• Complies with all relevant local and national codes.
16
-------
For the current tests, the stainless steel surfaces of the chamber were covered with materials typical of an
indoor office setting. The floor was covered with plywood and then carpet tiles (Shaw Industries, Dalton,
GA). The rear and side walls were framed with two (2-in by 4-in) studs positoned every 18 in on center
and faced with 1/2-in thick wallboard (GoldBond, Part Number (P/N) GB4080-0800, Lowe's, Durham, NC).
These facings spanned the floor to the bottom of an angle iron bracket welded onto the walls
approximately one ft from the ceiling. The wallboard was patched with joint compound (USG Sheetrock,
P/N 380119048, Lowe's, Durham, NC) and joint tape (USG Sheetrock, P/N 382199010) according to
typical building practices, then painted with primer (Kilz, P/N 20005, Home Depot, Durham, NC) and finish
painted (Behr, P/N 105001, Home Depot, Durham, NC). At the top of the walls, a drop ceiling was
installed. The drop ceiling consisted of 1/2-in thick, 2-ft by 2-ft acoustic panels (Armstrong, P/N 949,
Lowe's, Durham, NC) and two plenum grilles to enable conditioning of the chamber using RH and
temperature controls. The chamber contained three 6-ft collapsible tables to hold coupon materials. Open
coupon sockets were left in the ceiling, wallboard, and carpet where inoculated coupons could be placed.
The office environment was then sterilized with a six-hour exposure to 400 ppm hydrogen peroxide
(H2O2) (VHP®) and allowed to off-gas for seven days.
2.3 Test Coupon Preparation
Porous and non-porous materials common to office settings were selected for this investigation. Coupon
descriptions for each material type are listed in Table 2-5.
Table 2-5. Coupon Types
Material
Ceiling Tile
Painted
Wallboard
Carpet
Upholstery
Paper
Porous or
Non-porous
Porous
Non-porous
Porous
Porous
Porous
Description
Building
material
Building
material
Building
material
Office
equipment
Office contents
Designated
for Removal
in
Procedure
1?
Yes
No
Yes
Yes
Yes
Source
Armstrong, P/N SC1135c, Lowe's,
Durham, NC
GoldBond, P/N GB4080-0800,
Lowe's, Durham, NC
See Section 2.3.3 (P/N Multiplicity
Tile 54594, Shaw Industries, Dalton,
GA
See Section 2.3.4
(OnlineFabricStore, P/N 124331-
2645,
https://www. onlinefabricstore.net
Tiger Supplies, P/N 19BX15502,
Irvington NJ
17
-------
ABS
Plastic
Formica
Laminate
Vinyl-
coated
paper
Non-Porous
Non-porous
Non-porous
Office contents
- phone and
printer casing
(simple
coupon)
Office
equipment
(simple
coupon)
Office Contents
(simple
coupon)
Yes
No
Yes
McMaster Carr, P/N 8586K551 ,
Atlanta, GA
Formica Corporation, P/N 949-58-
48X96-900, Lowe's, Durham, NC
Wall Planner, P/N 129647, Office
Depot, Durham, NC
2.3.1 Ceiling Tile Coupons
Ceiling tile coupons used for the Task 2-Phase One tests (Method Optimization Testing described in
Section 2.6.1) consisted of a 12-in by 12-in tile and a 0.866 in (22-mm) tile (Figure 2-9). The 12-in by 12-
in coupons (P/N 87261, Armstrong World Industries, Inc., Lancaster, PA) were prefabricated tiles made of
mineral fiber. The 0.866 in (22-mm) coupons were fabricated on site by excising a 0.866 in (22-mm)
diameter plug from the 12-in by 12-in ceiling tile. The plugs were fastened to 0.709 in (18-mm) aluminum
Scanning Electron Microscopy (SEM) stubs (P/N 16119, Ted Pella, Inc., Redding, CA), then the edges
were wrapped in masking tape (P/N 2020CG, 3M, St. Paul, MN) to prevent deterioration. All coupons
used for Task 2-Phase One tests were placed in ethylene oxide (EtO) pouches and sterilized in an 18-
hour EtO sterilization cycle, then allowed to rest for 48 hours prior to inoculation.
Ceiling tile coupons used in the mock office were prefabricated 24-in by 24-in textured contractor ceiling
tile panel (Armstrong World Industries, Inc., Lancaster, PA). The tiles were installed in a 15-/16-in grid
suspension system, approximately 12 in from the COMMANDER ceiling. In the ceiling, four 24-in by 24-in
coupon sockets (Figure 2-10) were left open for later placement of inoculated and blank coupons. All
ceiling tile test coupons were placed in sterilization bags, sterilized with a four-hour exposure to 250 ppm
H2O2, allowed to degas for at least one week, and stored in a sterilization bag until needed for inoculation.
Ceiling tiles surrounding the open coupon sockets were sampled as negative controls and procedural
blanks and were sterilized during the COMMANDER reset cycle (a four-hour exposure to 250 ppm H2O2)
prior to testing.
18
-------
Figure 2-9. 0.866-in (22-mm) Ceiling Tile Coupons
Figure 2-10. Ceiling with Coupon Sockets for Later Installation of Inoculated
Ceiling Tile Test Coupons
19
-------
2.3.2 Wall board Coupons
Wallboard coupons consisted of painted (interior matte latex) coupons as described in Section 2.1.2.
Three wallboard coupons were cut from the wall retrofitted in COMMANDER to create coupon sockets.
To prevent the absorption of liquid into the exposed wallboard around the socket (Figure 2-11), the edges
were covered with the same adhesive tape used to seal the wallboard coupons in Task 1. Each coupon
was identified so that it would be returned to the original socket after inoculation. Wallboard coupons were
placed in sterilization bags, sterilized with a four-hour exposure to 250 ppm hhCb, allowed to degas for at
least one week, and stored in a sterilization bag until needed for inoculation. The 14-in by 14-in wallboard
sections surrounding the coupon sockets were marked and sampled as negative controls and procedural
blanks and were sterilized during the COMMANDER reset cycle (a four-hour exposure to 250 ppm H2O2)
prior to testing.
Figure 2-11. Wallboard Section with Open Socket Intended for
Inoculated Wallboard Coupon
2.3.3 Carpet Coupons
The carpet coupons were prefabricated 24-in by 24-in 100 % nylon tile (P/N Multiplicity Tile 54594, Shaw
Industries, Dalton, GA). These tiles were placed on the plywood subfloorof the chamber without adhesive
for easy removal. The entire floor was covered by carpet, with the exception of three 12-in by 12-in and
six 14-in by 14-in coupon sockets shown in Figure 2-12, where inoculated coupons would be placed after
inoculation. The 12-in by 12-in and 14-in by 14-in coupons were cut from the 24-in by 24-in tiles. The 12-
in by 12-in coupons were prepared for extraction sampling by cutting them into six 4-in by 6-in sections
(Figure 2-13). All carpet coupons were placed in sterilization bags, sterilized with a four-hour exposure to
250 ppm H2O2, allowed to degas for at least one week, and stored in a sterilization bag until needed for
inoculation.
20
-------
Figure 2-12. Carpet Flooring with Coupon Sockets for Inoculated Coupons
Figure 2-13.12-in by 12-in Carpet Tile Coupon Sectioned into Six 4-in
by 6-in Pieces in Preparation for Extraction Sampling
2.3.4 Upholstery Coupons
For each upholstery coupon (Figure 2-14), a 12-in by 12-in piece of 1-in thick foam padding
(OnlineFabricStore, P/N 124331-2645) was placed in the center of a 14-in by 14-in by %-in thick piece of
plywood. A 24-in by 24-in piece of indoor/outdoor fabric (Bryant Indoor/Outdoor Pine, Bryant Industries
Corp, NY, NY, product discontinued) was used to cover the foam. Excess fabric was folded underneath
and stapled to the back side of the plywood backing.
21
-------
Coupons were placed in sterilization bags, sterilized with a four-hour exposure to 250 ppm hhCb, allowed
to degas for at least one week, and stored in a sterilization bag until needed for inoculation.
Figure 2-14. Front of Assembled Upholstered Cushion
2.3.5 Paper Samples
Paper coupons consisted of stacks of 25 pages of 14-in by 14-in paper (P/N 019BX15502, Tiger
Supplies, Irvington, NJ), stapled together. Coupons were placed in sterilization bags, sterilized with a
four-hour exposure to 250 ppm hhCb, allowed to degas for at least one week, and stored in a sterilization
bag until needed for inoculation.
2.3.6 Simple Coupons
Simple coupons were 14-in x 14-in coupons cut to size from bulk stock. These simple coupons included
ABS plastic (P/N 8586K551, McMaster-Carr, Atlanta, GA), laminate Formica (P/N 949-58-48X96-900,
Formica Corporation, Cincinnati, OH), and vinyl-coated paper (P/N 129647, Office Depot Inc., Boca
Raton, FL,).
Coupons were placed in sterilization bags, sterilized with a four-hour exposure to 250 ppm H2O2, allowed
to degas for at least one week, and stored in a sterilization bag until needed for inoculation.
2.4 Spore Preparation
The test organism for this work was a powdered spore preparation of Bg (American Type Culture
Collection (ATCC) 9372; Manassas, VA) and silicon dioxide particles. This bacterial species was formerly
known as B. subtilis var. niger and subsequently B. globigii, or B. atrophaeus. 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. [4]. Briefly, after 80-90 percent sporulation, the suspension was
centrifuged to generate a preparation of approximately 20 percent solids. A preparation resulting in a
powdered matrix containing approximately 1011 viable spores per gram was prepared by dry blending and
jet milling the dried spores with fumed silica particles (Degussa, Frankfurt am Main, Germany). The
powdered preparation was loaded into metered dose inhalers (MDIs) according to a proprietary protocol.
Control checks for each MDI were included in the batches of coupons contaminated with a single MDI.
22
-------
2.5 Coupon Inoculation
All materials used for inoculation were sterilized with vaporous hydrogen peroxide or steam autoclave
before use.
2.5.1 Inoculation of 14-in by 14-in Coupons
Coupons were inoculated with a target concentration of 2 x 107 spores of Bg from an MDI using the
procedure described by Calfee et al. [5]. Briefly, each coupon was inoculated independently by being
placed into a separate dosing chamber (aerosol deposition apparatus, ADA) designed to fit one 14-in by
14-in coupon of any thickness. The MDI was discharged a single time into the ADA. The spores were
allowed to 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 ADA and placed into service, either to be set in place in
the office chamber, or sampled as positive control coupons. The ADAs were removed only from those
coupons required for a single sample at a time.
2.5.2 Inoculation of24-in by 24-in Ceiling Tiles
A small 3-in by 3-in area of the 24-in by 24-in ceiling tiles designated for extraction sampling was
inoculated using an in-house method developed for subway concrete testing. In brief, the inoculation
procedure involves placing a round ADA (ADA-2R) on the surface of the coupon for inoculation (Figures
2-15, 2-16, and 2-17). The ADA-2R was placed on the coupon, and the MDI was attached to the top of
the ADA-2R. A slide was opened, and the MDI was activated. Following inoculation, the slide was closed
and the MDI was removed. To mark the sampling area, a sterile 3-in by 3-in stainless steel template was
placed around the ADA-2R and traced with a permanent marker. The spores were allowed to settle for at
least 18 hours. This procedure was repeated for each coupon.
O-ring
/
Hose Barb for Vent
Figure 2-15. Round ADA-2R Schematic (ID = inner diameter)
23
-------
Figure 2-16. Top View of Round ADA-2R
Figure 2-17. Round ADA-2R with O-ring Gasket
2.5.3 MDI Use and Inoculation Recordation
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, the weight of each MDI was determined after
completion of the inoculation of each coupon. For quality control of the MDIs, stainless steel inoculation
control coupons (14 in by 14 in) were inoculated as the first, middle, and last coupons within a single
group of coupons inoculated by any one MDI within a single test.
A log was maintained for each set of coupons that was dosed. Each record contained the unique coupon
identifier, 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 inoculation
(dosing).
24
-------
2.6 Task 2 Test Matrix
This work was conducted in two phases. The first phase consisted of a number of small optimization and
proof-of-concept tests needed to inform test procedures in the second phase. The second phase was
conducted to assess the impact of removing porous materials in an office environment on the overall
(room-scale) efficacy of pAB spray-based decontamination procedures.
2.6.1 Phase One - Method Optimization Testing
2.6.1.1 Carpet Extraction-based Sampling Optimization
In preparation for carpet extraction sampling, carpet extraction optimization tests were performed. Two
types of coupons were inoculated with 1 x 107 Bg: 14-in by 14-in stainless steel coupons designated for
sponge stick sampling, and 12-in by 12-in carpet coupons made of six 4-in by 6-in subsections
designated for extraction method recovery. Each of the six 4-in by 6-in subsections was processed
separately, then recoveries were summed during data analysis to estimate recovery from the entire 12-in
by 12-in coupon. Three stainless steel coupons and three 12-in by 12-in carpet coupons per extraction
procedure were analyzed. Three carpet extraction procedures (stomaching and two methods of orbital
shaking) were evaluated for recovery and ease of use.
2.6.1.2 Ceiling Tile Extraction-based Sampling Verification Test
Prior to testing, the ceiling tile extraction method was tested to determine recovery efficiency. Additionally,
in an effort to explore all options for pre- and post-decontamination ceiling tile sampling, the efficiency of
sponge stick samplers was determined for dry ceiling tile and wetted ceiling tile. Briefly, at least 12 ceiling
tile coupons were aseptically transferred to 16-in by 16-in re-sealable plastic bags (P/N 20085T21,
McMaster Carr, Atlanta, GA) and sterilized with EtO. A set of five ceiling tile coupons was extracted
directly. A set of three ceiling tile coupons was sampled with sponge sticks as detailed in Section 2.7.3.2.
A set of four ceiling tiles was spritzed with approximately 10 ml of sterilized Dl water, allowed to absorb
the water (approximately 1 min), then also sampled with a pre-moistened sponge stick.
2.6.2 Phase Two - Decontamination Procedure Testing
Three tests were included in Task 2, Phase Two, as detailed in Table 2-6. The test matrix shows whether
porous materials were removed prior to application of the decontamination procedure and the flooring
type (i.e., porous or non-porous) that was in place for AFSD testing.
Table 2-6. Task 2, Phase Two Test Matrix
Test Number
1
2
3
Decontamination
Procedure
1
2
1
Porous Items
Removed
Yes
No
Yes*
Flooring for
AFSD Testing
Subflooring
Carpet
Tile Flooring
"Only floor tile was inoculated for this test.
25
-------
2.6.2.1 Coupon Transfer into COMMANDER
After coupon inoculation, the dosed coupons were moved into the sterilized chamber that was outfitted as
an office, including the plastic tables that would be used to stage the test coupons (with the exception of
the ceiling tile, the wallboard, and the carpet) in the typical real-world horizontal orientation. These staging
tables would later be sampled after applying the decontamination procedure to characterize the relocation
of spores from the test coupons to the table.
Following inoculation and the prescribed 18-hour deposition, the clamps were removed from the ADAs
and coupons. Each coupon was carried with ADA in place to the airlock where the ADA was removed
outside the COMMANDER airlock door, then passed into the airlock where the coupons were temporarily
staged before placement inside the inner COMMANDER chamber. Once all the test coupons were
transferred inside the airlock, the external door was sealed, and the door to the inner COMMANDER
chamber was opened. Coupons were strategically moved inside and placed in position from the top of the
chamber to the bottom, in their real-world orientations on representative surfaces. Ceiling tile coupons
were inverted and placed in the coupon sockets with the inoculated side oriented downward (see Figure
2-10). Next, the wallboard coupons were affixed to empty coupon sockets in the vertical orientation,
inoculum side oriented outward toward the inside of the room (see Figure 2-11). The remaining materials
(except carpet) were placed horizontally on tables (Figure 2-18).
Figure 2-18. Material Coupons in Position Prior to Decontamination
Finally, carpet coupons were placed in their corresponding sockets, starting with those located near the
rear of the inner COMMANDER chamber and working toward the front (exit) (Figure 2-19). To
characterize AFSD collection efficiency, room scale tests were conducted as part of each test. The
COMMANDER was divided into two sections, one designated for sampling by a traditional human-based
sampling team (sponge stick, vacuum sock, or extraction) and the other intended for sampling by AFSD.
Both sections contained at least four inoculated locations or hot spots, one of which was in a location
difficult to reach for the decontamination team. The flooring areas represented by hot spots included
26
-------
corners, edges and centers. The hot spots were 12-in by 12-in coupons comprised of six 4-in by 6-in
coupons designated for extraction method recovery. The decontamination team was not privy to the
location of the hot spots to prevent bias of treatments.
Figure 2-19. Carpet Flooring Coupon Sockets for Inoculated Coupon
(4-in by 6-in and 12-in by 12-in) Placement
The method for placing floor tile coupons for Test 3 was identical with the method used for placing the
carpet coupons (with the exception of the 4-in by 6-in extraction coupons) for Test 1 and Test 2.
Test coupons and procedural blank coupons were placed in the chamber. Positive control and blank
coupons remained outside. Testing was conducted under ambient conditions.
2.6.2.2 Application of Decontamination Procedures
Once the inoculated coupons were installed, the sampling team spritzed (sprayed until soaked) each
porous item, as well as the exterior of the bags, and all non-porous material surfaces with pAB using a
four-gallon backpack sprayer at a flow rate of approximately 1.2 LPM.
2.6.2.2.1 Decontamination Procedure 1
The decontamination Procedure 1 was conducted as follows:
• Following the placement of inoculated coupons in position from the top to the bottom of the
chamber, all surfaces were spritzed (slightly sprayed to reduce re-suspension of spores) with
pAB solution.
• After the initial spritzing with pAB, all porous and some designated non-porous materials were
removed and spritzed individually with pAB in the following order:
o Upholstery
o Paper
o Vinyl-coated paper
o ABS Plastic
27
-------
o Carpet
o Ceiling Tiles
• The materials were segregated and bagged by material type.
• Formica and wallboard were not removed.
• All items contained in each bag were sprayed again until soaked. The exterior of the inner waste
bags was spritzed prior to being placed in secondary 6 mil contractor bags. When full, the waste
bag was transferred to the airlock.
• After the mock office was completely void of porous materials as shown in Figure 2-20, the
remainining surfaces were sprayed for about three minutes (covering them completely) with
pAB using a UDOR chemical sprayer (Model UAG-1003HU, Pro-Chlorine Gas Powered Chemical
Sprayer, Ultimate Washer Inc., Jupiter, FL), chosen for this test because it was made specifically
for use with chlorine-based solutions.
Figure 2-20. Office Environment Void of Porous Materials
2.6.2.2.2 Decontamination Procedure 2
The decontamination for Procedure 2 was similar to Procedure 1, except that the porous materials were
not removed, they were decontaminated in-place and remained in the mock office until they were
sampled following at least 18 hours of drying (COMMANDER blowers were initiated to promote air
circulation).
2.6.3 Post-Decontamination Conditioning
After decontamination, the indoor environment was dried for six days using approximately two air
exchanges per hour at ambient RH (40-60 % RH) and temperature (17.5-19.0 °C).
28
-------
2.7 Sampling and Analytical Procedures
2.7.1 Sampling Procedures
Within a single test, surface sampling of the materials was completed for all procedural blank coupons
first before sampling of any test material was performed. Surface sampling was done either by wipe
sampling, sponge stick sampling, swab sampling, or vacuum sock sampling in accordance with the
protocol documented below. 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. The general sampling supplies were sterile or sterilized/disinfected for each sampling event.
2.7.1.1 Sponge Stick
3M™ Sponge-Sticks (SSL10 NB, 3M, St. Paul, MN) pre-wetted with neutralizing buffer were used on
non-porous smooth surfaces such as ceramics, vinyl, metals, painted surfaces, and plastics. The general
approach was that a moistened sterile non-cotton pad was used to wipe a specified area to recover
bacteria, viruses, and biological toxins.
2.7.1.2 Swab Sampling
Sterility (growth/no growth) was verified by collecting swab (BactiSwab™ Collection and Transport System,
Cat.# R12100, Remel, Lenexa, KS) samples from example materials (ADAs, ADA gaskets, coupons, etc.)
before use. The general approach was to use a moistened swab to wipe a specified area to recover
bacterial spores.
2.7.1.3 Vacuum Sock Sampling
Vacuum sock (Midwest Filtration, Cincinnati, OH) sampling was used on carpet, upholstery, and paper
material coupons. Vacuum sock sampling was done following a strict protocol that would ensure consistent
and representative sampling of such material types.
Table 2-7 lists the sampling and analytical procedures used in this study. Details are described in the
following sections.
29
-------
Table 2-7. Sampling and Analytical Procedures
Matrix
Air
Air
Ceramics,
Vinyl,
Metals,
Paited
Surfaces,
Plastics
Carpet,
Upholstery,
Paper
Carpet,
Ceiling Tile
All Surfaces
(prior to
inoculation)
Measurement
CPU/volume
RH
CPU/area
CPU/area
CPU/area
Growth/No
growth
Sampling/
Measurement
Method
Aerosol sample
Vaisala Model
HMT 3332
3M Sponge
stick™ 3 sampling
Vacuum sock4
sampling
Extraction
Techniques
Swab5 sampling
Sample Container
Via-Cell®
Bioaerosol
Cassette1
NA
Triple-bagged
Triple-bagged
Triple-bagged
Swab container
Preservation/
Storage
Refrigeration
None
Refrigeration
Refrigeration
Refrigeration
Refrigeration
Maximum
Holding
Times
72 hours
None
72 hours
72 hours
72 hours
72 hours
1 Via-Cell® Bioaerosol Cassette (Zefon International, Ocala, Fl)
2 Vaisala (HMT 333, Vaisala Online Store, https://store.vaisala.com/us/)
33M Sponge-Stick™ (P/N, SSL10NB, 3M, Saint Paul, MN)
4 Vacuum Sock (FAB 20-01-004S, Midwest Filtration, Cincinnati, OH)
5 BactiSwab® (Cat. # R12100, Remel, Lenexa, KS)
2.7.2 Decontamination Solution Sampling
The FAC concentration of the pAB formulations was measured using analytical procedures based on
ASTM Method D2022-89. In short, a 5-mL aliquot was mixed with a buffered potassium iodide (Cat. #
S25493B, Fisher Science Education, Nazareth, PA) solution and iodometrically titrated with sodium
thiosulfate (P/N 7975-32, Ricca Chemical Co., Arlington, TX) to a colorless endpoint. The aliquot was
taken and analyzed immediately after formulation (including mixing). The pH of the solution was
measured with an Oakton pH5 pH meter (Oakton Instruments, Vernon Hills, IL) that was calibrated daily.
The pAB was used within three hours of its preparation.
2.7.3 Office Chamber Sampling Procedures
2.7.3.1 Office Material Sampling Points
Table 2-8 shows a list of the sampling techniques used for each office material. Each material type had
three test (decontaminated) replicate samples, three positive control replicate samples, one
decontaminated procedural blank, and one non-inoculated (negative) sample. The coupon materials
listed in Table 2-8 were placed randomly using three tables as shown in Figure 2-21.
30
-------
Table 2-8. Sampling Methods used for Material Surfaces in the Office Configuration
Material Coupons
Formica
Painted Wallboard
Carpet
Carpet
Ceiling Tile
Ceiling Tile
Office Chair Seat (upholstery coupon)
Office Chair Seat (upholstery coupon)
Paper
Vinyl-coated paper
ABS Plastic
Sample Technique
Sponge stick
Sponge stick
Vacuum sock
Extraction
Vacuum sock
Extraction
Vacuum sock
Extraction
Sponge stick
Sponge stick
Sponge stick
Sample ID
TW
DW
CH(-V)
CH(-E)
TH(-V)
TH (-E)
UH(-V)
UH (-E)
PA
VP
AB
Total Number of Samples
per Event
8
8
8
8
8
8
8
8
8
8
8
Paper
(VS #2)
Vinyl Coated
Paper (SS#1)
ABS Plastic
(SS#1)
Upholstery
(E#1)
Formica
(SS#1)
Upholstery
(VS #2)
Vinyl Coated
Paper (SS #3)
Upholstery
(VS #3)
Table 1
Formica
(SS #3)
Open
ABS Plastic
Blank (SS)
Paper Blank
(VS)
Upholstery
Blank (E)
Open
Formica Blank
(SS)
Formica
(SS #2)
Table 2
ABS Plastic
(SS #3)
Upholstery
(E#3)
Upholstery
(VS#1)
Paper
(VS#1)
Paper
(VS #3)
Vinyl Coated
Paper (SS #2)
ABS Plastic
(SS #2)
Upholstery
(E#2)
Table 3
VS = Vacuum sock sample. SS = Sponge stick sample. E = Extraction sample.
Figure 2-21. Office Material Sample Coupon Layout
Sampling points in the mock office included the inoculated coupon "hotspot" and "warm" areas
surrounding the hotspot that may have been contaminated secondarily. Warm spots surrounding each
wallboard hotspot included one square ft to the left, to the right, and beneath the coupon. Each paper and
simple coupon (ABS plastic, paper, and vinyl-coated paper) was put within a 16-in by 16-in square on a
table, and the area under the coupon was sampled as the corresponding warm area. Each carpet tile not
inoculated (or the area under said carpet tile) was considered a warm carpet sample. For Test 1, carpet
31
-------
was removed during decontamination, leaving subfloor as potential warm zones. The subfloor sampling
was possible as the outline of each carpet tile had been marked on the subfloor prior to laying the carpet.
The sampling team moved strategically through the sampling area, in order to reduce cross
contamination. For example, areas targeted for surface sampling were not touched or disturbed prior to
sample collection in that area. The frequency of sampling and monitoring events is presented in Table 2-
9. Table 2-10 lists the critical and non-critical measurements for each sample.
Table 2-9. Frequency of Sampling Monitoring Events
Sample Type
Test coupon
Negative control
coupon
Positive control
coupon
Procedural blank
coupons
Microbiology
material blanks
pAB
Aerosol sample
RH/temperature
Sample
Number
3 per material
type per
sampling type
1 per material
type
3 per material
type per
sampling type
3 coupons
collocated with
control positive
coupons
1 per material
1 per batch
1 with each
entry
3
Sample/Monitoring
Frequency
1 set per
decontamination
1 set per location
per fumigation
1 set per inoculation
1 set per inoculation
One peruse of
material
1 prior to use
Once during
negative control
sampling inside the
office, application of
the decontamination
procedure, and
post-
decontamination
sampling
Logged every 10
seconds
Sample Location
Office
H130A
H130A
Office
NA
Containment Vessel
Office
Office
Purpose
To determine the number
of viable spores after
decontamination
To determine extent of
cross-contamination
To determine the number
of viable spores deposited
onto the coupons
To determine extent of
cross-contamination
To demonstrate sterility of
extraction and plating
materials
To demonstrate that the
FAC is within QC
specifications
To assess re-
aerosolization of spores
To determine
environmental conditions
inside the office
32
-------
Table 2-10. Critical and Non-Critical Measurements
Sample Type
Test coupon
Negative control coupon
Positive control coupon
Procedural blank coupons
Microbiology material blanks
pAB
Aerosol sample
RH/temperature
Critical Measurements
Plated volume, incubation temperature,
extracted volume, CPU
Plated volume, incubation temperature,
extracted volume, CPU
Plated volume, incubation temperature,
extracted volume, CPU
Plated volume, incubation temperature,
extracted volume, CPU
Plated volume, incubation temperature,
extracted volume, CPU
FAC concentration, pH
Sampled air volume, plated volume, incubation
temperature, extracted volume, CPU
NA
Non-critical
Measurement
Storage time, storage
temperature
Storage time, storage
temperature
Storage time, storage
temperature
Storage time, storage
temperature
Storage time, storage
temperature
Temperature
NA
Temperature and RH of
Office
NA = Not applicable
2.7.3.2 AFSD Collection Efficiency Sampling
To characterize AFSD collection efficiency, room scale tests were conducted: Decontamination
Procedure 1 with ceramic tile flooring, and Decontamination Procedure 2 with carpet flooring. Sampling
was conducted as detailed in Section 2.6.2.1, with the following exception. Both sections had at least
three (rather than four) inoculated locations or hot spots, one of which was in a difficult to reach location
for the decontamination team. Figure 2-22 provides an example of the location of the inoculated areas in
the extraction sampling area and the AFSD sampling area.
33
-------
AFSD Sampling Area
Sampling Area
Figure 2-22. Office Environment Floor Sampling Layout
The sampling team entered COMMANDER using clean entry protocols. All precautions were taken to
avoid the AFSD sampling area. The sampling team collected samples from the right side (designated for
surface (vacuum) and extraction sampling), and then the AFSD was activated in the AFSD sampling area
(left side), shown in Figure 2-22. The sampling team exited the office. The AFSD was retrieved within 18
hours of activation. Recovery and quantification of spores collected by the AFSD was described
previously by Lee et al. [6, 7]. The porous floor surfaces (wood subfloor and carpet) were sampled using
a vacuuming AFSD (NEATO XV-11 ™, Neato Robotics, Inc., Newark, CA), as described previously. The
non-porous floor surface (laminate tile) was sampled with a mopping AFSD (Braava™ 320, iRobot,
Bedford, MA) by aseptically transferring 50 mL of Phosphate Buffered Saline with Tween®20 (PBST) (Lot
# SLBF5740V, Sigma, St Louis, MO) to the mopping cloth (Figure 2-24) prior to initiating the sampling
sequence (Figure 2-25).
34
-------
Figure 2-23. Vacuuming AFSD on Subflooring
Figure 2-24. Aseptic Transfer of PBST onto Mopping Pad of AFSD for
Tile Floor
35
-------
Figure 2-25. Mopping AFSD on Laminate Tile Flooring
2.7.4 Microbiological Analysis
The NHSRC Biocontaminant Laboratory (Biolaboratory) located at the EPA facility in Research Triangle
Park, NC, analyzed samples either qualitatively for spore presence (quality control, swab samples) or
quantitatively for the number of viable spores recovered per sample (CPU). Details of the sampling
procedures are provided below. A laboratory notebook was used to document the details of each
sampling event (or test).
For all sample types, liquid extracts were serially diluted 10-fold (in PBST) and 0.1 mL spread-plated onto
TSA plates in triplicate. Plates were incubated at 35 ± 2 °C for 18 to 24 h and colony-forming units (CFU)
were enumerated visually. Only plates containing between 30 and 300 CFU were utilized for recovery
estimates. Extracts were diluted and replated if none of the 10-fold dilutions resulted in all three plates
containing colony counts within the acceptable range. All extracts were stored at 4 ± 2 °C. Total spore
recovery was calculated by multiplying the mean CFU counts from triplicate plates by the inverse of the
volume plated, by the dilution factor, and finally by the total volume of the extract. Any samples below
countable criteria (30-300 CFU) on the primary dilution plates were subsequently filter-plated to reduce
the detection limit. The filters were placed onto Tryptic Soy Agar (TSA) plates and incubated at 35 ± 2 °C
for 18-24 hours prior to manual enumeration.
Details of the extraction procedures are provided below.
2.7.4.1 Coupon Extraction
During Phase One, a variety of extraction procedures were evaluated for efficacy of spore recovery.
These extraction procedures are described in the sections below.
36
-------
2.7.4.1.1 Stomaching carpet coupons
The stomaching-based carpet extraction procedure used a paddle blender (Stomacher® 400Circulator,
Seward, Ltd., West Sussex, UK) to process the coupons with 120 ml of PBST. The six 4-in by 6-in
inoculated coupon parts were individually transferred from the inoculated site to a stomacher bag
(BA6141/CLR, Seward, Ltd., West Sussex, UK) and processed in the stomacher machine for 2 minutes at
230 RPM. The 120-mL extract was then split into three 50-mL conical tubes, and centrifuged at 3500 xg
for 15 minutes. Following centrifugation, the supernatant was removed aseptically and carefully so as not
to disturb the pellet. The pellets were resuspended in the remaining liquid using the vortex mixer and
recombined into one tube prior to plating.
2.7.4.1.2 Orbital shaker with carpet coupons
This extraction procedure used an orbital shaker to provide physical agitation during spore extraction. The
six 4-in by 6-in inoculated coupon parts were individually transferred aseptically from the inoculated site to
a 10-in by 15-in sterile sampling bag with flat wire closure (P/N 01-002-53, Thermo Fisher Scientific, Inc.,
Waltham, MA). Sterile PBST (180 ml) was aseptically transferred to each sample bag. Secondarily
contained sample bags were placed into the incubated shaker (Model 3626, Lab-Line Instruments Inc.,
Melrose Park, IL). Samples were shaken at room temperature for 30 min at 300 RPM. The extract was
then split into four 50-mL conical tubes and centrifuged at 3500 xg for 15 minutes. Following
centrifugation, the supernatant was removed aseptically and carefully so as not to disturb the pellet. The
pellets were resuspended in the remaining liquid using the vortex mixer and recombined into one tube
prior to plating.
2.7.4.1.3 Stomaching 3-in by 3-in ceiling tiles
To extract ceiling tile, 3-in by 3-in ceiling tile sections were aseptically placed into stomacher bags,
followed by 150 mL of sterile PBST. The samples were allowed to soak in the extraction solution (PBST)
fora minimum of five minutes. Following soaking, the ceiling tiles were manually broken into smaller,
more manageable pieces while still inside the bag. The sample bag with both the sample and PBST was
placed into another stomacher bag (non-closure) for secondary containment and stomached for two
minutes at 230 RPM. Due to the density of the sample, very little foam was produced during the
stomaching process. Therefore, immediately after removing the sample, 50 mL of liquid was removed
from the sample and transferred into a pre-labeled sterile 50-milliliter (mL) conical tube. The extracted
liquid was obtained by squeezing the sample, keeping all debris at the bottom of the bag, while the
debris-free liquid flowed to the top of the bag where it was captured by a sterile serological pipette for
transfer to a pre-labeled sterile conical tube.
2.7.4.1.4 Extraction of 0.866-in (22-mm) diameter ceiling tile
Coupons (0.866-in (22-mm) diameter) were aseptically transferred to sterile pre-labeled conical tubes
(P/N 352098, Falcon, Reynosa, Mexico) followed by 20 mL of PBST prior to extraction. The tubes,
containing coupon and PBST, were placed in an ultrasonic bath for 10 min and subsequently agitated
using a vortex mixer at the highest setting for 2 continuous min. The samples were then homogenized
again using a vortex mixer immediately prior to analysis.
37
-------
2.7.4.1.5 Extraction of 12-in by 12-in ceiling tile
To extract each of the 12-in by 12-in ceiling tiles, a total of two liters of PBST was used. The first liter of
PBST was added to the primary bag containing the ceiling tile. After five min, the coupon was manually
broken into smaller pieces, and the second liter of PBST was added. The sample bags were secured with
a twist tie to prevent leakage, then placed into the incubator shaker retrofitted with a rack with rectangular
cut-outs. The shaker operated for 30 min at 300 RPM. The sample bags were then transferred from the
shaker to a BSC where 100 ml of the liquid extract was aseptically removed by pressing the liquid from
the debris, and the liberated liquid was captured with a sterile serological pipette. The liquid extract was
aseptically transferred to a sterile pre-labeled specimen cup (Starplex, Cleveland, TN) and plated using
wide orifice pipette tips.
2.7.4.2 Method Verification
Prior to testing, preliminary tests were conducted to verify the performance of the extraction-based
sampling methods (Section 2.6.1) intended for use for each porous material (carpet, ceiling tile, and
upholstery). Extraction-based sampling methods for ceiling tile and carpet proved effective and were
implemented for processing coupons generated during testing. The upholstery extraction procedure was
determined to be ineffective and therefore was not used as a sampling method for this effort.
Additionally, a preliminary test was performed to characterize the sampling efficacy of sponge sticks on
previously wetted ceiling tile. To simulate the process of wetting and drying ceiling tile during testing,
representative sections of inoculated ceiling tile were sprayed with sterile Dl water and allowed to dry until
no longer visibly wet. The coupons were then sampled with sponge sticks, along with ceiling tiles that had
never been wetted and stainless steel coupons, for comparative analysis. The sponge-stick technique
results for the ceiling tiles were within the same order of magnitude as the stainless steel coupon
recoveries for similar size coupons (Section 3.3.1) but orders of magnitude lower than the extraction
sampling approach. The use of positive control samples provided verification of the deposition method.
2.7.5 Cross Contamination Prevention
Several management controls were instituted to prevent cross-contamination. This effort was labor-
intensive and required many activities be performed on coupons that were intentionally contaminated
(test coupons and positive controls). Specific procedures were put in place in the effort to prevent cross-
contamination among the groups during sample handling.
There were four primary activities for each test in the Task 2 experimental matrix. These activities were
preparation of the coupons, execution of the decontamination process (including sample recovery),
sampling, and analysis. Specific management controls for each of the four following activities are
described below.
2.7.5.1 Preventing Cross-Contamination during Coupon Preparation
Coupon preparation includes the activities performed on each material coupon prior to conducting the
decontamination procedure. All coupons were sterilized prior to use to reduce background spores on the
test materials; the simple coupons were fumigated with EtO using an Andersen EOGas 333 sterilization
system (Andersen Products, Haw River, NC), while the carpet, upholstery, ceiling tile, and drywall
coupons were fumigated with 400 ppmvhhCb vapor for four hours. On the day of testing, procedural
38
-------
blank coupons were moved into the spray chamber. Test decontamination procedures were conducted on
the procedural blank coupons before any inoculated coupons.
2.7.5.2 Preventing Cross-Contamination during Execution of the Decontamination Process
The COMMANDER airlock was fumigated with 250 parts per million by volume (ppmv) hhCb for four
hours after each chamber entry/exit to help prevent contamination of the chamber by re-entraining spores
from the airlock.
In addition, several techniques were employed throughout the testing schedule to limit cross-
contamination migrating from the laboratory into the airlock and chamber:
• The enclosure area was mopped prior to each entry into the airlock or chamber with Clorox® Clean
Up (Lowe's, Durham, NC). Once the area was mopped, the enclosure doors were closed and access
was restricted except to test personnel.
• When entering the enclosure after Step 1, all personnel stepped onto a disinfectant mat soaked with
Clorox® bleach before donning new clean booties prior to stepping into the enclosure.
• All "clean garb" (i.e., disposable lab coats, nitrile gloves, disposable booties, and disposable bouffant
caps) was kept outside COMMANDER in clean bins prior to use, where contaminating spore
concentrations were expected to be much lower.
• All materials brought into the chamber were pre-sterilized when possible and placed in bins that were
then wiped down with Dispatch® wipes (P/N 69150, Caltech, Middland, Ml) when transported across
the threshold into the enclosure.
• Clean garb was worn by all personnel once they entered the enclosure or airlock, at a minimum.
During application of the decontamination procedures, Level C suits were worn instead.
2.7.5.3 Preventing Cross-Contamination during Sampling
Sampling poses an additional significant opportunity for cross-contamination of samples. In an effort to
minimize the potential for cross-contamination, the following management controls were followed:
• Sampling teams made up of a "sampler," a "support person," and a "sample handler" were utilized for
sampling outside the mock office (positive controls and negative controls), and a two person team
made up of a sampler and a support person was employed inside the mock office. Inside, the support
person also assumed the responsibilities of the sample handler.
• The sampler changed gloves each time after handling items during the sampling event.
• The support person was responsible for handing sterile templates to the sampler.
• The sampler handled only the sampling media and the support person handled all other supplies. The
sampler sampled the surface according to the appropriate procedure.
• The collection medium was placed into a sample container that was opened, held and closed by the
support person.
39
-------
• The resulting sealed sample was handled only by the support person.
All of the following actions were performed only by the support person, using aseptic technique:
• The sealed bag with the sample was placed into another sterile plastic bag and sealed; the external
bag was then decontaminated using a bleach wipe.
• The double-bagged sample was placed into a disinfected sample container for transport.
• 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
NHSRC Biolaboratory.
Additionally, and equally important, the order of sampling was as follows: (1) all blank coupons; (2) all
decontaminated coupons; (3) all positive control coupons. This order ensured that coupons were handled
in an order from lowest level of contamination to the highest level of contamination.
After use, the positive control coupons were placed in a bin of soapy water before disposal. The handling
of the contaminated coupons was done in a way to minimize spore dispersal. One person was tasked
with removing the clamps holding the ADA to the coupon and the removal of the ADA and gasket from
the coupon. A second person, wearing new gloves for each coupon, was then tasked with placing the
coupon into service.
2.7.5.4 Preventing Cross-Contamination during Analysis
General aseptic laboratory technique was followed and was embedded in all procedures used by the
NHSRC Biolaboratory. Additionally, the order of analysis (consistent with the above) was as follows: (1)
all blank coupons; (2) all decontaminated coupons; (3) all positive control coupons.
2.7.6 Sample Containers for Collection, Transport, and Storage
For each sample intended for extraction-based sampling, the primary containment was an individual
sterile sampling bag. The secondary containment was a larger sterile sampling bag. The primary and
secondary containments of each sponge stick sample were separate sterile sampling bags. All biological
samples from a single test were then placed in a disinfected container which served as tertiary
containment. After samples were placed in the container for storage and transport to the NHSRC
Biolaboratory, the container was wiped with a towelette saturated with a hypochlorite solution. A single
container was used for storage in the decontamination laboratory during sampling and for transport to the
NHSRC Biolaboratory. Aliquots of decontamination solutions were analyzed immediately and were thus
not subjected to multilayered containment. Sample Preservation
Following transfer to the NHSRC Biolaboratory, all samples were stored at 4 ± 2 °C until analyzed. All
samples were allowed to equilibrate at room temperature for one hour prior to analysis.
2.7.7 Sample Holding Times
After sample collection for a single test was complete, all biological samples were transported to the
NHSRC Biolaboratory immediately, with appropriate Chain of Custody (COC) form(s). Samples were
stored no longer than five days before the primary analysis. Typical hold time prior to analyses for most
40
-------
biological samples was < two days. Because decontamination solutions are not shelf-stable, they were
analyzed immediately upon collection.
2.7.8 Sample Handling and Custody
Careful coordination with the NHSRC Biolaboratory was required to achieve successful transfer of
uncompromised samples in a timely manner for analysis. Test schedules were confirmed with the
Biolaboratory prior to the start of each test. To ensure the integrity of samples and to maintain a timely
and traceable transfer of samples, an established and proven COC or possession is mandatory. Accurate
records were maintained whenever samples were created, transferred, stored, analyzed, or destroyed.
The primary objective of these procedures was to create an accurate written record that could be used to
trace the possession of the sample from the moment of its creation through the reporting of the results. A
sample was in custody in any one of the following states:
• In actual physical possession;
• In view, after being in physical possession;
• In physical possession and locked up so that no one could tamper with it;
• In a secured area, restricted except to authorized personnel; or
• In transit.
Laboratory test team members received copies of the test plans prior to each test.
In the transfer of custody, each custodian signed, recorded, and dated the transfer on the COC form.
Sample transfer could be on a sample-by-sample basis or on a bulk basis. The following protocol was
followed for all samples as they were collected and prepared for distribution:
• A COC record accompanied the samples. When turning over possession of samples, the transferor
and recipient signed, dated, and noted the time on the record sheet. This record sheet allowed
transfer of custody of a group of samples from Highbay room H130A to the NHSRC Biolaboratory.
• If a sample custodian had not been assigned, the laboratory operator had the responsibility of
packaging the samples for transport. Samples were carefully packed and hand-carried between on-
site laboratories. The COC record showing the identity of the contents accompanied all packages.
2.7.9 Sample A re hiving
All samples and diluted samples were archived for a minimum of two weeks following completion of
analysis. This time allowed for review of the data to be performed to determine if any re-plating of
selected samples was required. Samples were archived by maintaining the primary extract at 4 ± 2 °C in
a sealed 50-mL conical tube.
2.7.10 Information Recorded by Field Personnel
A sampling event log sheet called "Material Tracker" was maintained for each sampling event (or test).
The sampling team members' names, date, run number, and all sample codes with corresponding
coupon codes were recorded on each sheet. Any deviations from sampling protocols were documented in
41
-------
the laboratory notebook. Sample volumes, time of day, results from pH, temperature, and active
ingredient measurements, and observations were also written down in the laboratory notebook.
Digital photographs of selected material coupons with a noticeable change due to the decontamination
procedure were taken after the completion of the sampling of all sections in a test.
42
-------
3 Results and Discussion
3.1 Task 1 - Drying Cabinet Conditions
Figure 3-1 shows the average daily RH and temperature data within coupon drying cabinets, respectively,
for all 34 days of physical monitoring. During the coupon drying phase, the mean RH inside the drying
cabinet ranged between 21 % and 40 % while mean temperatures were between 24 °C and 27 °C.
01
CO
CD
45-
40-
35-
30-
1
Q. 25-
20 -I
—•—Temperature
— RH
0 5 10 15 20 25 30 35
Elapsed Time Since Spray Application (Days)
40
Figure 3-1. Average Daily RH and Temperature inside the Drying Cabinet
43
-------
3.2 Task 1 - Physical Testing
3.2.1 Temporal Evaluation of Wallboard Hardness
Material hardness was measured by the minimum force required to puncture the material surface, using a
penetrometer equipped with a 3 mm foot. As shown in Figure 3-2, the control coupons represent both the
lowest and some of the highest hardness values. The hardness of the control coupons demonstrated high
variability, which was likely due to inherent variability in the wallboard itself or in the measurement
thereof. This variability prevents any firm conclusions from this testing; however, spraying with Dl water or
pAB did not seem to induce significant changes in the hardness of wallboard.
^
I
£
160-
140-
120-
100-
80-
60-
40-
20-
• Control Coi ipons
T 10 min Water r\\ \Afa+cir 1
20 min Water L" VVdlCl I
B
•*I ! i
•it, : x A *
160-
140-
120-
100-
80-
60-
40-
20-
• Control Coupons
T 10 min pAB . .
C Xmln^ PABJ
•
••!f * . i
TT^ *» *
" *
5 10 15 20 25 30
Elapsed Time Since Spray Application (Days)
35
Figure 3-2. Material Hardness Evaluation of Unfinished Wallboard
From Figure 3-2, Day 1 results seem to suggest that the wetting procedure affected the material hardness
compared to the control coupons (sprayed coupons showed a slight reduction in minimum force required
for puncture, compared to controls). However, for all subsequent tests, the data suggest no correlation
between applying the wetting procedure and the material hardness. Any effects the wetting procedures
had on hardness were no longer apparent after a drying time of five days.
44
-------
Figure 3-3 shows that in the case of matte latex-finished wallboard, the average force required to
puncture the coupons varied for each testing event. Similar to unfinished wallboard, the variance in the
control coupon data between test observations was high. Any subtle effects from applying the wetting
procedure may have been obscured by such high variability.
140-
120- m
100J "*.
80- .« *
60^ *A f.
40-
20-
Dl Water j
•
•
•
^
• Control Coupons
T 10 min Water
A 30 min Water
• 60 min Water
•
140-
120-
100-
80-
60-
40-
20-
P^
•• ^^"J
•
|f T i »
T w
T *
T
• Control Coupons
• lOminpAB
A 30 min pAB
T 60 min pAB
t
•
T
A
0 5 10 15 20 25 30
Elapsed time Since Spray Application (Days)
35
Figure 3-3. Material Hardness Evaluation of Matte Latex Finish
Not unlike unfinished and matte latex finished wallboard, the results shown in Figure 3-4 for semi-gloss
finished wallboard suggest that after Day 1, no change was detected in surface hardness for this material.
The type of applied finish appears to have little or no effect on the hardness of the wallboard materials.
45
-------
•\AT\
\*\
-------
20-
15-
10-
5-
0
Bare
MatteLatex
HighGloss
10min 30min
Visible Wetness Time
60min
Figure 3-5. Absorption Capacity by Surface Finish for Varying Liquid Applications
3.2.2.2 Surface Moisture Content
The various materials were evaluated overtime to determine the surface moisture content for the different
liquid application times. Illustrated below is the moisture content for unfinished (unpainted) wallboard
(Figure 3-6). Coupons sprayed with pAB consistently have a moisture content higher than the control
(non-sprayed) coupons at anytime during treatment. This effect is minimal or nonexistent with water
spraying. As expected, the magnitude of the wetting effect on test coupons was greater during
observations performed closer to the wetting procedure (first five days) than for those made during
subsequent testing days. While Dl water may absorb more readily into wallboard material (Figure 3-5),
pAB has a lower capacity for desorption, resulting in a prolonged effect on moisture content.
47
-------
£
c 0.5-
8 •
g> 0.4-1
f^ t • T
i • | • | • | • | • | •
-^ 1 0
^ 0.9-
0.8-
0.7-
0.6-
0.5-
04-
PAB| • Conlrc
L£^J . lOmir
T_ A 30 mir
T 60 mir
•
Viv
V- *
i« * •
^^ • •
•
t
)l Coupons
ipAB
ipAB
ipAB
I
0 5 10 15 20 25 30 35
Elapsed Time since Spray Applications (Days)
Figure 3-6. Surface Moisture Content on Unpainted Wai I board over Time
Illustrated below is the moisture content for matte latex finished wallboard and semi-gloss wallboard
(Figures 3-7 and 3-8, respectively). Coupons sprayed with pAB seem to have a moisture content higher
than the control (non-sprayed) coupons during the first few days of the treatment. However, this effect is
less pronounced with these two materials than with unpainted wallboard. The moisture loss seems to be
influenced more by environmental conditions than by history of surface spraying.
48
-------
Moisture Content (%)
0.75-
0.70-
0.65-
0.60-
0.55-
0.50-
0.45-
Dl WatPr 1 " Control Coupons
30 mn Water
VT 60 rrin Water
. .
I f 1
080
0.75-
0.70-
0.65-
0.60-
0.55-
0.50-
0.45-
ndn_
nAB 1 • Control Coupons
'-•^•J • 10rrinpAB
VA 30 rrin pAB
T 60 rrin pAB
* Y •
T
0 5 10 15 20 25 30 35
Elapsed Time since Spray Application (Days)
Figure 3-7. Surface Moisture Content of Matte Latex-Finished Wai I board over Time
49
-------
=sT
I
8
p
^
(/)
g
080
0.75-
0.70-
0.65-
0.60-
0.55-
0.50-
0.45-
040-
Dl \l\hter b • Control Coupons
^^|^J . mminWatPr
* 30 mn Water
VT 60 rrin Water
n
|Ty
T
080
075-
.
0.70-
0.65-
0.60-
0.55-
0.50-
0.45-
nan_
nABl • Control Coupons
, L|H^J • 10rrinpAB
V« * 30 rrin pAB
VT 60 rrin pAB
• LJ ^ ::
t
0 5 10 15 20 25 30 35
Elapsed Time since Spray Application (Days)
Figure 3-8. Surface Moisture Content of Semi-gloss-Finished Wai I board over Time
3.2.3 Temporal Evaluation of Wai I board Roughness
Roughness data are expressed in "Ra" which is defined as the arithmetic average height of roughness
irregularities measured from the mean within the evaluation length (0.492-in (12.5 mm)).
Shown in Figure 3-9 are the surface roughness values for unpainted wallboard. Each spray procedure
appeared to have an effect on the surface roughness of unpainted wallboard when compared to the
control coupons. This effect was most significant for measurements made closer to the time of wetting
procedure application (five days). In general, exposing unpainted wallboard to pAB for 60 min appears to
have a greater effect on surface roughness than a 60-min exposure to Dl water. During the first few days
following application of the wetting procedures, there appears to be no correlation between surface
roughness of unpainted wallboard and the wetting solution.
50
-------
5.50 -H
5.25-
5.00-
4.75-
4.50-
4.25-
4.00-
3.75-
ni Water b ' Contral COUP0"5
Ul Water • lOninWMer
^ A 60rrinWtter
•A- ^ A
V" I • A
• • c
•
CD
o:
5.25-
5.00-
4.75-
4.50-
4.25-
4.00-
3.75-
350-
1 1 • Centre
pABl * 1°rri
L^^J T Xni
Vy A SOmi
A •
• • I 1 "
•
H
D| Coupons
ipAB
ipAB
ipAB
V
A
•
0 5 10 15 20 25 30 35
Elapsed time since Spray Application (Days)
Figure 3-9. Surface Roughness of Unpainted Wallboard over Time
Figures 3-10 and 3-11 show surface roughness for matte late-x and semi-gloss-finished wallboard,
respectively. As shown in Figure 3-10, high variability in surface roughness measurements obscured any
effects or trends following liquid exposure on matte latex finished wallboard.
Roughness (Ra) values are considerably lower for the semi-gloss-finished wallboard than for other
finishes as seen in Figure 3-11. In general, surface roughness was affected by the spray procedures
during the first few days (five days) following the spraying procedures; however, these effects appeared
to diminish overtime. The type of liquid solution (pAB or water) seem to have no effect on all the
wallboards tested.
51
-------
1
CD
o:
65
6.0-
.
5.5-
5.0-
4.5-
4.0-
3.5-
65-,
6.0-
.
5.5-
.
5.0-
4.5-
4.0-
35-
ni Watpr k * Control Coupons
T Ul Water | 10minWjter
• 60 rrin Water
* t T
• ll A •
A • '
1 .-.AD b • Control Coupons
^^^™ T JU nin pAB
T • 60 rrin pAB
T
^T x • x
/•• • . *
• •
0 5 10 15 20 25 30 35
Elapsed time Since Spray Application (Days)
Figure 3-10. Surface Roughness of Matte Latex-Finished Wallboard over Time
300
2.75-
2.50-
2.25-
2.00-
1.75-
^ 1 50-
^-^ 300-,
CD
^ 2.75-
2.50-
2.25-
2.00-
1.75-
1 50-
r-\\ \ A / j. • • Control Coupons
Ul Water • T 10min Water
v • 60ninWfeter
1 :
1 . p L ^ • Control Coupons
A ^"^™ • A XrrinpAB
^ T 60 rrin pAB
T. V "
^ T •
T T
0 5 10 15 20 25 30 35
Elapsed Time since Spray Applications (Days)
Figure 3-11. Surface Roughness of Semi-Gloss-Finished Wallboard over Time
52
-------
An RM-AN OVA was used to compare the moisture content, hardness, and surface roughness means of a
material with a specific finish for the three independent treatments (10-, 30-, and 60-min spray) over time
(repeated measures). If the difference in the mean values among the treatment groups is greater than
would be expected by chance, there is a statistically significant difference (p<0.05). In this case, multiple
comparisons of the type of treatments (10-, 30-, and 60-min spray) versus the control group (control
coupons) were performed using the Holm-Sidak method via statistical software (Sigmaplot 12.0, San
Jose, CA). The results for the effect of the spray treatment on these measurements are presented in
Table 3-1.
Table 3-1. One-Way RM-ANOVA
Parameter
Moisture
Hardness
Roughness
Wallboard
Finish
Unpainted
Matte Latex
Semi-Gloss
Unpainted
Matte Latex
Semi-Gloss
Unpainted
Matte Latex
Semi-Gloss
RM-ANOVA
<0.001
<0.001
0.001
0.451
0.110
0.914
0.015
<0.001*
<0.001
Significance (p) of Individual Contrasts with Control
10min
pAB
0.001
<0.001
0.021
ns
ns
ns
ns
ns
ns
SOmin
pAB
<0.001
<0.001
ns
ns
ns
ns
0.030
ns
ns
60min
pAB
<0.001
0.001
ns
ns
ns
ns
0.009
ns
0.004
10min
H20
ns
ns
ns
ns
ns
ns
0.030
ns
ns
SOmin
H20
ns
ns
ns
ns
ns
ns
0.008
ns
ns
60min
H20
ns
ns
ns
ns
ns
ns
ns
ns
ns
*ANOVA on Ranks rather than RM ANOVA performed due to Failed Normality Test; none of contrasts between experimental groups
and control group were significantly different.
ns = Not Significant.
The One-Way RM-ANOVA for equal moisture content shows significant statistical differences between
the three pAB treatments overtime when compared with control coupons for both unpainted and matte
latex wallboards (p< 0.001), and to a lesser extent for 10-min pAB treatment for semi-gloss finished
wallboard (p = 0.021). No statistical differences in the moisture content with water treatments overtime
were observed.
The One-Way RM-ANOVA for equal hardness showed no statistical differences on the effect of the
spraying treatment, or time, for all three types of wallboards tested. When this analysis was applied to
determine the effect of water treatment on the wallboard roughness, a statistically significant difference
for both the unpainted and semi-gloss wallboards was observed for both solutions, but no difference for
the matte latex finish wallboard. Subtle differences in the surface texture of individual coupons resulting
from the painting process (roller texturing) resulted in difficulty detecting differences caused specifically by
the wetting process. The surfaces of unpainted wallboard coupons were also visibly inconsistent, with
some areas smoother than others.
53
-------
3.3 Task 2 - Effectiveness Evaluation of Decontamination Procedures
The results presented in this section (Phase 1) consist of a number of small, optimization and proof-of-
concept tests needed to inform test procedures in the second phase. Phase 2 was conducted to assess
the impact of removing porous materials from an office environment on the efficacy of a pAB spray-based
decontamination procedure.
3.3.1 Phase 1: Optimization Testing Method Development
Optimization tests were performed to determine the most efficient extraction method for carpet and ceiling
tile materials.
Carpet Material. Data for both extraction methods (stomacher and orbital shaker) for carpet coupons
were compared to stainless steel controls (sampled by sponge stick) to assess recovery efficiency. The
results, shown in Figure 3-12, seem to indicate slightly better recoveries for the stomacher than the orbital
shaker extraction technique. However, a Two Sample t-test shows that at a 95 % confidence interval, the
difference in the population mean recoveries is not significantly different between the sampling
approaches (p-value of 0.387). Further, the low standard deviations observed for these extraction
techniques demonstrate a high precision within a set of samples.
.
•
107,
a :
g •
I 105-
^H Stainless Steel Coupons
I I Carpet Coupons
— I —
T
1
Sponge Stick Stomacher Orbital Shaker
Extraction Methods
Figure 3-12. Carpet Recovery Optimization Tests
54
-------
Ceiling Tiles. Results from the optimization recovery test results for ceiling tiles are shown in Figure 3-
13. Recoveries using the sponge-stick technique on ceiling tiles were within the same order of magnitude
as the recoveries from stainless steel coupons of similar size.
10 i
IH SpongeSticks
I I Extraction
Material and Sampling Technique
Figure 3-13. Ceiling Tile Extraction Optimization Results
3.3.2 Phase 2: Mock Office Environment Testing
3.3.2.1 Procedure 1 - Porous Material Removal
Procedure 1 was evaluated in the mock office by sampling the pre-decontaminated areas, warm spot
areas such as the areas surrounding each wallboard hotspot, areas on the desk corresponding to areas
beneath removed coupons (ABS plastic, paper, and vinyl-coated paper), and under flooring areas. The
under floor sampling was possible as the outline of each carpet tile had been marked on the subfloor prior
to laying the carpet as illustrated in Figure 3-14.
55
-------
AFSD Sampling Area
Sampling Area
Figure 3-14. Procedure 1 Office Environment Floor Sampling Layout
Figure 3-15 shows the recovery from positive control coupons, representative of the test coupons placed
in the mock office before decontamination. Figure 3-16 shows recovery following removal of porous
materials and decontamination with a spray of pAB. Note that post-decontamination recoveries by AFSDs
(Figure 3-22 and Table 3-1).
56
-------
Material Type
Figure 3-15. Pre-decontamination Recoveries - Procedure 1
LL
O,
40-
30-
10-
0
DL
SpongeStick
Vacuum Sock
DL
Detection Limits
Material Type
Figure 3-16. Post-Decontamination Recoveries - Procedure 1
57
-------
All the warm spots (areas adjacent to the inoculated wallboard coupons, subflooring under the carpet,
and underneath the simple coupons (ABS plastic, paper, table)) were non-detect. Some transfer of
spores from the coupons to the surrounding area (warm zones) did occur in pooled areas on the table.
Two of the three inoculated drywall coupons were non-detect, but 30 viable spores were recovered from
one coupon. As a whole, removing porous materials and spraying with pAB was an effective
decontamination technique, with an average LR of 6.2 on the two materials (Formica and hot spot
drywall) that were not removed. Transport of spores from the carpet to the subfloor during
decontamination was not detected by AFSD or sponge stick sampling.
3.3.2.2 Procedure 2 - Porous Materials Decontaminated In-Place
Figure 3-17 details the average pre-decontamination recoveries from each material for the various
sampling methods employed. The sampling methods used for porous materials included extraction,
vacuum sock, or sponge stick.
Pre-decontamination sampling of all materials (excluding upholstery) showed a starting concentration of
at least 1 x 106 CFU per unit of sampled area. Recoveries were lower than the targeted 1 x 106 CFU for
upholstery. This material has anti-microbial properties that may have negatively biased spore recovery
assays.
LJ_
o
CD
:
.
•
10%
.
106:
:
105-
•
i
i
i
i
I I SpongeStick
I I VaccumSock
^H Extraction
I
i
Material Type
Figure 3-17. Pre-Decontamination Recoveries - Procedure 2
58
-------
During the extraction process, the ceiling tile was observed to form a thick pulpy consistency that may
have prevented a homogeneous distribution of spores within the extraction liquid. The ceiling tile
extraction technique seems to be superior to the sponge stick sampling approach. No viable spores were
recovered from the upholstery extraction sample.
Figure 3-18 shows the average spore recoveries for each material after the application of
Decontamination Procedure 2. The vacuum sock sampling method for carpet returned an average of 9.04
x 101 CFU/ft2, while the extraction sampling method returned an average of 4.86 x 104CFU/ft2. Similarly
for ceiling tile, sponge stick sampling returned an average of less than 1 CFU/ft2, while extraction
sampling returned an average of 5.34 x 105 CFU/ft2. While the difficulty of performing extraction-based
sampling in the field makes it a non-ideal sampling method for both carpet and ceiling tile, it is apparent
that surface sampling severely underrepresents the magnitude of surface contaminants when used on
previously wetted porous materials.
108
107i
8 1°31
I 162!
1 1 SpongeStick
1 1 VaccumSock
1 1 Extraction
fi
n n n
n n
Material Type
Figure 3-18. Post-Decontamination Recoveries - Procedure 2
Decontamination efficacies of 6 LR or greater were achieved on all non-porous materials (Formica, vinyl
coated paper, drywall, and ABS plastic), while efficacy on carpet was 2.04 LR and 1.54 LR for ceiling tile
(Figure 3-19). Comparison of recoveries from surface sampling and extraction sampling again showed
that extraction sampling was the more efficient technique. Consistent with numerous previous studies,
porous materials proved more difficult to decontaminate than their non-porous counterparts.
59
-------
8
g 7-1
01
6-
5-
4-
c 3J
o
I 2-1
1-
0
n n
I I SpongeStick
I I VaccumSock
^H Extraction
Material Type
Figure 3-19. Decontamination Efficacies of Decontamination Procedure 2 on Non-porous and
Porous Decontaminated Materials, by Surface Sampling Method
3.3.2.3 Spore Transport
During the efficiency testing with non-porous (tile) flooring, the sampling team sampled the areas
surrounding "hot" zones, labeled "warm zones" using sponge sticks (Figure 3-20). Figure 3-21 shows the
recovery of spores from these areas adjacent to hotspots. The significant recoveries from Warm Zone 1
and Warm Zone 3 demonstrate that viable spores were transported during application of the
decontamination procedures and concentrated in areas where the decontamination solution collected.
The highest recovery was from Warm Zone 1, which corresponds spatially to the lowest area, closest to
the COMMANDER drain (located underneath the mock office setup). The high recovery of spores where
pooling occurred makes these areas ideal for post-decontamination sampling.
60
-------
AFSD Sampling Area
Sampling Area
Desk
Warm Zone
Warm Zone 6
Warm Zone 5
Warm Zone 4
Warm Zone 2
Warm Zone 3
Warm Zone 1
Drain
Figure 3-20. Warm Spot Sampling Layout
80-
70-
60-
iZ 50-
§
40-
20-
10-
345
Warm Zone ID
Figure 3-21. Post-Decontamination Recoveries of Spores using Sponge
Sticks in Areas Adjacent to Hotspots - Laminate Flooring
61
-------
3.3.2.4 Assessment of AFSD Sampling for Decontaminated Porous and Non-Porous Surfaces
The AFSD room scale post-decontamination collection efficiency evaluation was conducted for both
carpet material and ceramic tiles. The AFSD results were compared to conventional surface sampling
techniques for carpet (vacuum socks) and laminate tiles (sponge sticks). The AFSD recoveries shown in
Figure 3-22 and summarized in Table 3-1 were comparable to the surface sampling techniques for both
materials/sampling techniques. The advantage of using the robots is that robotic sampling is a
cumulative sampling approach, compared to the conventional sampling techniques that collect a certain
number of discrete samples. The probability of sampling in non-contaminated areas is high as shown for
laminate samples where six (6) out of nine (9) samples that covered the entire room were non-detects.
104
10%
b
a:
§
10%
Vacuum Socks
NeatoXV-11
Sponge Sticks
IRobot
carpet laminate Tile
Material Type
Figure 3-22. Sampling Recoveries for both Conventional and Robotic Sampling Approaches
Table 3-1: Sampling Recoveries for both Conventional and Robotic Sampling Approaches
Surface Type
Carpet
Laminate
Recovery (CFU/Room (Non-Detect Samples/Total Samples))
Currently-Used Sampling Methods
Vacuum Sock
7.97 103/ (0/3)
Sponge Stick
92 (6/9)
Robot Sampling
NeatoXV-11
2.36103(0/1)
IRobot
578(0/1)
62
-------
4 Quality Assurance/Quality Control
This project was performed under approved Category III Quality Assurance Project Plans (QAPP):
Decontamination of Bundled/Bagged Waste with pH-Adjusted Bleach. Task 1: Evaluation of Waste
Decontamination Procedures (June 2013), and Expedient Approaches for Decontamination of Biologicals
- Indoor Environment. Task 2: Decontamination Procedures Effectiveness Evaluation (November 2013).
4.1 Sampling, Monitoring, and Analysis Equipment Calibration
There were operating procedures for the maintenance and calibration of all laboratory equipment utilized
for this project. Calibration was validated by EPA's Air Pollution Prevention and Control Division (APPCD)
on-site (Research Triangle Park, NC) Metrology Laboratory at the time of use. Standard laboratory
equipment such as balances, pH meters, 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 Requirements
Equipment
Thermometer
pH meter
HOBO RH sensor
Stopwatch
Micropipettes
Clock
Scale
Calibration/Certification
Compare to independent NIST thermometer (a
thermometer that is recertified annually by either
NIST or an ISO-1 7025 facility value once per quarter)
Perform a two-point calibration with standard buffers
that bracket the target pH before each use
Compare to calibrated RH sensor prior to use
Compare against NIST Official U.S. time at
http://nist.time. qov/timezone.cqi?Eastern/d/-5/iava
(last accessed 4/27/2015) once every 30 days
All micropipettes will be certified as calibrated at least
once per year. Pipettes are recalibrated by
gravimetric evaluation of pipette performance to
manufacturer's specifications every six months by
supplier (Rainin Instruments, Mettler Toledo,
Greifensee, Switzerland)
Compare to office U.S. Time @ time.gov every 30
days
Check calibration with Class 2 weights
Expected Tolerance
±1 °C
±0.1 pH units
±5%
± 1 min/30 days
±2%
± 1 min/30 days
± 0.1 % of weight
NIST = National Institute of Standards and Technology
ISO = International Organization for Standardization
63
-------
4.2 Quality Assurance and Quality Control Checks
Uniformity of the test materials was a critical attribute for assuring reliable test results. Samples and test
chemicals were maintained to ensure their integrity. Samples were stored away from standards or other
samples that could cause cross-contamination.
Supplies and consumables were acquired from reputable sources and were NIST-traceable when
available. Supplies and consumables were examined for evidence of tampering, damage or expiration
dates upon receipt and prior to use, as appropriate. Supplies and consumables showing evidence of
tampering or damage or past expiration dates were not used. All examinations were documented and
supplies were appropriately labeled. Project personnel checked supplies and consumables prior to use to
verify that they met specified task quality objectives and did not exceed expiration dates.
4.3 Acceptance Criteria for Critical Measurements
The Data Quality Objectives (DQOs) define the critical measurements needed to address the stated
objectives and specify tolerable levels of potential errors associated with simulating the prescribed
decontamination environments. The following measurements were deemed to be critical to accomplish
part or all of the project objectives:
• Hardness
• Moisture content
• Surface roughness
• Chlorine concentration, determined by measuring the FAC in the decontaminant solutions
• pH of decontaminant solutions
• Enumeration of CPU
• Flow rate of decontamination solutions
• Plated volume.
Data Quality Indicators (DQIs) for the critical measurements were used to determine whether the
collected data met the Quality Assurance Project Plan (QAPP) quality assurance (QA) objectives. The
critical measurement acceptance criteria are shown in Table 4-2.
When one of the test parameters did not meet the test target value, the test method was repeated or
modified to reach test target values, so that completeness for the task was 100 percent. For example, if
the target FAC concentration in the bleach (decontaminant) solution was not met, the solution was either
prepared again or adjusted according to the MOP. Test RH values were adjusted with data from
calibrated RH sensors. Similarly, if the CFU abundance did not fall within the acceptable range, the
sample was either filtered or re-plated.
Plates were analyzed quantitatively (CFU/plate) using a manual counting method. For each set of results
(per test), a second count was performed on 25 percent of the plates within the quantification range
64
-------
(spread-plates with 30 - 300 CPU, filter-plates with 1-150 CPU). All second counts were found to be
within 10 percent of the original count.
In this study, temperature of decontamination solutions was monitored carefully to determine pot-life of
decontamination solution batches. A temperature increase greater than 5 °C was a threshold designating
that preparation of a new batch of the decontamination solution was needed.
Table 4-2. Critical Measurement Acceptance Criteria
Critical
Measurement
Hardness
Moisture
Content
Surface
Roughness
FAC in pAB
Solution
pH of pAB
Solution
Flowrate of
pAB Sprayer
Plated Volume
CPU per plate
Temperature
of incubation
chamber
Measurement Device
Extech FHT 200 (Extech
Instruments, Nashua, NH)
M-70 Aquameter (James
Instruments, Inc., Chicago, IL)
Phase II SRG-4000 (Phase II+,
Upper Saddle River, NJ)
Hach High Range Bleach Test Kit,
Method 10100 (Hach, Loveland,
CO)
Oakton pH probe (OKPH502; pH5)
(Oakton Instruments, Vernon Hills,
IL)
Volume collected in a graduated
cylinder per time
Micropipette
Manual counting
NIST-traceable thermometer (daily)
Accuracy
Target
±0.5 %
0.1 %
±10%
± 0.06 g/L
± 0.01 pH
± 10%
2%
±10% CPUs/
plate between 1st
and 2nd count
+ 2°C
Actual
NA
NA
±1.2%
N/A
±0.01 pH
NA
<± 2 %8
<10%CFUs/
plate between 1st
and 2nd count
<+ 2°C
Detection Limit
0.3- 196.10 N
0-80 %
0.005 urn -16
urn
4- 12 g/L
(4,000-12,000
ppm) FAC
(quantitation
range)
0.00- 14.00 pH
1 mL/min
NA
1CFU per plate
NA
NA = Not applicable; standard evaluations were not performed for these instruments.
There are many QA/quality control (QC) checks used to validate microbiological measurements. These
checks include samples that demonstrate the ability of the NHSRC Biolaboratory to culture the test
organism, as well as to demonstrate that materials used in this effort did not themselves contain spores.
The checks included:
• Negative control coupons: sterile coupons that underwent the decontamination process;
• Field blank coupons: sterile coupons carried to the decontamination location but not decontaminated;
• Laboratory blank coupons: sterile coupons not removed from the NHSRC Biolaboratory;
• Positive control coupons: coupons inoculated but not fumigated.
65
-------
4.3.1 Quality Control Checks
Quantitative standards do not exist for biological agents. Quantitative determinations of organisms in this
investigation did not involve the use of analytical measurement devices. Rather, CPU were enumerated
manually and recorded. Critical QA/QC checks are shown in Table 4-3. The acceptance criteria were set
at the most stringent level that could be achieved routinely. Positive controls and procedural blanks were
included along with the test samples in the experiments so that well-controlled quantitative values were
obtained. Background checks were also included as part of the standard protocol. Replicate coupons
were included for each set of test conditions. Qualified, trained, and experienced personnel ensured data
collection consistency. When necessary, training sessions were conducted by knowledgeable parties,
and in-house practice runs were used to gain expertise and proficiency prior to initiating the research.
Table 4-3. QA/QC Sample Acceptance Criteria
QC Sample
Procedural
Blank (coupon
without
biological agent)
Positive Control
(Sample from
material coupon
contaminated
with biological
agent but not
subjected to the
test conditions)
Blank plating of
microbiological
supplies
Blank ISA
Sterility Control
(plate incubated,
but not
inoculated)
Chlorine
concentration
PH
Field blank
samples
Information
Provided
Controls for sterility of
materials and methods
used in the procedure
Initial contamination
level on the coupons;
allows for determination
of LR; controls for
confounds arising from
history impacting
bioactivity; controls for
special causes.
Shows plate's ability to
support growth
Controls for sterility of
supplies used in dilution
plating
Controls for sterility of
plates
Concentration of FAC in
the fresh pAB or diluted
bleach solution
Effective concentration
of hydrogen ions in
solution
The level of
contamination present
during sampling
Frequency
1 per test
3 or more
replicates per
test
3 of each supply
per plating event
Each plate
1 peruse
1 per use
1 per sampling
event
Acceptance
Criteria
No observed CFU
For high inoculation,
target loading of 1 x 107
CFU per sample with a
standard deviation of <
0.5 log. (5x106-5x107
CFU/sample). For low
inoculation, target loading
of1x102CFUper
sample with a standard
deviation of < 0.25 log (56
-177 CFU/sample)
No observed growth
following incubation
No observed growth
following incubation
6000-6700 ppm for fresh
pAB or diluted bleach
>6.5and <7.0 for fresh
pAB
Non-detect
Corrective
Action
Reject results of test
coupons on the same
order of magnitude.
Identify and remove
source of contamination
Outside target range:
discuss potential impact
on results with EPA
WACOR*; correct
loading procedure for
next test and repeat
depending on decided
impact.
Sterilize or dispose of
source of contamination.
Re-plate samples
All plates are incubated
prior to use, so any
contaminated plates will
be discarded
Reject solution, replace
reagents and prepare a
new solution
Reject solution, replace
reagents and prepare a
new solution
Clean up environment.
Sterilize sampling
materials before use
*WACOR = Work Assignment Contracting Officer Representative
66
-------
4.4 QA/QC Reporting
QA/QC procedures were performed in accordance with the QAPP for this investigation. Tables 4-4
through 4-6 show the measured quality indicators, measured values for critical test parameters, for Task 1
and Task 2.
Table 4-4. Task 1 Data Quality Criteria for pAB
pAB
Batch
ID
Batch 1
Batch 2
Batch 3
FAC Concentration - per 5 mL pAB Titrated
Target Value
(ppm)
6000 - 6700
6000 - 6700
6000 - 6700
Test
Value
(ppm)
6530
6710
6349
Frequency
Once before testing
Once before testing
Once before testing
PH
Target
Value
6.5-7.0
6.5-7.0
6.5-7.0
Test
Value
6.8
7.0
6.8
Frequency
Once before testing
Once before testing
Once before testing
Table 4-5. Task 1 pAB Monitored Points
Test ID
Test 4
Tests
Tests
Test 12
Test 18
Test 1 1
Test 17
Test 10
Test 16
FAC Concentration - per 5 mL pAB Titrated
pAB Batch and Monitored
Point
Batch 1 , Check 1
Batch 1 , Check 2
Batch 1 , Check 3
Batch 2, Check 1
Batch 2, Check 2
Batch 2, Check 3
Batch 2, Check 4
Batch 3, Check 1
Batch 3, Check 2
Test Value
(ppm)
6530
6049
6009
6710
5348
5027
4727
6349
6089
PH
Test Value
6.8
6.7
6.5
7.0
6.5
6.3
6.2
6.8
6.8
Table 4-6. Task 2 Data Quality Criteria for pAB
67
-------
Test ID
Task 2 -
Test 1
Task 2 -
Test 2
Task 2 -
Tests
FAC Concentration - per 5 mL
pAB Titrated
Target
Value
(ppm)
6000-
6700
6000-
6700
6000-
6700
Test
Value
(ppm)
6069
6570
6049
Frequency
Once before
testing
Once before
testing
Once before
testing
PH
Target
Value
6.5-7.0
6.5-7.0
6.5-7.0
Test
Value
6.8
6.9
6.9
Frequency
Once before
testing
Once before
testing
Once before
testing
Chamber Parameters (SCADA*)
RH
51.3
25.0
63.4
Temp
17.3
18.4
19.9
Frequency
Data recorded at
1 min intervals
for the duration
of the test
Data recorded at
1 min intervals
for the duration
of the test
Data recorded at
1 min intervals
for the duration
of the test
*SCADA = supervisory control and data acquisition
With the exception of hardness and FAC, all critical measurements met the accuracy requirements for this
effort (Table 4-2). Accuracy data for hardness is not available. The hardness instrument, Extech FHT 200,
was not verified prior to each use with a standard material of known hardness. Instead, the hardness of
the wallboard material was compared to that of the control materials. The accuracy of the HACH High
Range Bleach Test Kit was not verified with a chlorite standard prior to use and therefore is not available
for reporting. The flowrate of the backpack sprayer was verified before and after each spray application;
however, the factory setting for the flowrate was unavailable for accuracy calculations.
With the exception of pH and FAC, all critical measurements were 100 % complete. In 91.6 % of the tests
that required use of pAB (Task 1 and Task 2 combined), the acceptance criteria (Table 4-2) were met.
The pAB solution was required to meet the acceptance criteria for FAC (6000 - 6700 ppm) and pH (6.5 -
7.0) at the time of preparation and it was required to be used within three hours of preparation. In Task 1,
after the initial pAB solution quality analyses were performed on each batch, FAC and pH monitoring
continued prior to use for each test (Table 4-5). All batches of pAB solution used in Task 1 met all quality
requirements.
4.4.1 Deviations from the QAPP
As shown in Table 4-4, Task 1 Data Quality Criteria for pAB, the initial FAC concentration test value for
Batch 2 was 10 ppm above the maximum taget value of 6700 ppm. The estimated impact of the slightly
higher FAC concentration on the overall results is negligible.
Table 4-5 shows monitored points of each batch of pAB solution prepared. These points were collected
prior to use for test. While each batch was required to meet the FAC and pH quality criteria upon
preparation, subsequent points collected for monitoring purposes were not subject to the same
acceptance criteria.
68
-------
For consistency, the requirement that each batch of pAB be discarded three hours after preparation was
adopted. Test records indicate that Check 4 for Batch 2 was performed three hours and 42 minutes after
the batch was prepared. This batch was incorrectly used for Test 17 (30 minute pAB application on the
semi-gloss finished wallboard). For this effort, ideally, a fresh batch of pAB would have been prepared
prior to each application. While in some instances the FAC and pH of the pAB may not have been ideal
for laboratory use, during non-ideal field situations, it is unlikely that a new batch of pAB would be made
prior to every application.
4.5 Data Quality Audits
This project was assigned QA Category III and did not require technical systems or performance
evaluation audits. However, all data were subject to peer QC review before being finalized.
69
-------
5 Summary and Recommendations
During Task 1 of this study, the physical limits of wallboard using spray-based decontamination
techniques were evaluated. Wallboard material sections were prepared with three types of finishes
(unfinished, interior matte latex paint, and industrial grade epoxy semi-gloss paint), subjected to pAB or
Dl water spraying, and were then maintained wetted for 0, 10, 30, or 60 minutes. Physical impact was
then monitored over the 34 days that followed the day of spray application.
Lasting material impact was not detected for any of the spray test treatments. Significant impact to
surface moisture content and roughness was detected for all wallboard finish types when pAB was
sprayed initially. These effects were quickly dissipated, as sprayed coupons quickly returned to pre-
sprayed or control coupon condition. Data from some tests show a trend in moisture content with respect
to application duration and type of wallboard finish paint; however, the impact was short-lived and
inconsistent. For example, results from Task 1, Test 1 (first day of testing) indicate the wetting procedure
using both pAB and Dl water affected the hardness of the material when compared to the unexposed
control coupons prior to drying. However, after drying, the hardness of previously wetted coupons was not
significantly different from the hardness of control coupons. Any effect the wetting procedures had on the
material hardness for each finish type diminished so that sprayed coupons were not significantly different
from non-sprayed control coupons 24 hours after application. Surface roughness increased following the
wetting procedures; however, this increase diminished overtime. During the drying process, the surface
texture among coupons within the same replicate set appeared to exhibit more variation than among
different treatment types.
In general, regardless of the liquid type or application duration, the wetting procedures did not appear to
have lasting effects on the physical integrity of the wallboard (other than some observed impact to joint
tape and joint compound). These test results suggest that the volume of decontaminant required to reach
the physical limit of wallboard material would be far greater than what could reasonably be applied during
an actual decontamination event. However, impact to material integrity due to pooling near the base of
floor/wallboard unions may result in significant wicking and thus deleterious effects. This type of effect
would not be detected by the current test design. Nonetheless, since Task 1 suggested that limited
effects on wallboard were observed using spray conditions frequently utilized during surface
decontamination procedures, the focus of Task 2 shifted from the physical limits of the materials to
determining the overall effectiveness of the spraying procedure.
Surface sampling methods of certain porous materials following decontamination were expected to be
inadequate. Therefore, to ensure the objective was met, prior to the onset of Task 2 testing, preliminary
tests aimed at optimizing extraction-based sampling methods for carpet and ceiling tile were performed.
The recoveries for extraction methods utilizing stomaching and orbital shaking were comparable to or
much higher than recoveries for the stainless steel controls. Stomacher-based extraction methods
required less labor and were more cost effective.
Extraction-based sampling of previously-wetted carpet proved superior to the more conventional
vacuum-based sampling technique. Spore recoveries using the sponge-stick technique on ceiling tile
were within the same order of magnitude as the stainless steel control coupons; however, post-
decontamination recoveries for ceiling tiles showed better recoveries with the extraction technique than
with the sponge-stick technique. Alternate sampling methods should be developed and verified for
70
-------
upholstery prior to future testing as neither of the methods tested during this effort (vacuum sock and
extraction) proved effective.
During Task 2 of this study, two spray-based decontamination approaches were evaluated in a mock
office setting. Procedure 1 required removal of porous items prior to spray-based decontamination of the
remaining surfaces. Procedure 2 attempted spray-based decontamination of all items in-place, with no
removal of porous items before the spray-based decontamination commenced.
Results from Task 2 suggest that decontamination efficacies on Formica laminate, vinyl coated paper,
painted wallboard, and ABS plastic were all at least a 6 LR (Procedures 1 and 2), while less than 2 LR
was achieved on carpet and ceiling tile when not removed from the mock office (Procedure 2). Viable
spores were detected following both decontamination procedures (Figures 3-16 and 3-18). The greatest
amount of viable spores detected after decontamination were found on carpet and ceiling tile, when
Procedure 2 (no removal) was rendered. These results suggest that a more stringent decontamination
procedure should be considered for these materials, or a decontamination approach that involves pre-
decontamination removal of these items (followed by more stringent ex situ treatment) should be utilized.
The results suggest that the advantages of Procedure 2 (ease of deployment and reduced ex situ
treatment required, as result of no pre-decontamination removal step) are negated by the low efficacy on
the items slated for removal using Procedure 1.
Following decontamination procedures in the mock office environment, the sampling team sampled the
warm zones (the areas surrounding the inoculated "hot" zones) in addition to hot zones to determine if
contaminants were spread by the decontamination treatment. A significant number of spores were
recovered in two of the six sampled laminate flooring warm zones, demonstrating that viable spores were
transported during application of the decontamination procedures and concentrated in areas where the
decontamination solution collected. The highest recovery was from the area closest to the COMMANDER
drain (located underneath the mock office setup). Transportation of spores with the decontamination
solution should be expected during application of spray-based decontamination procedures. Areas where
decontamination solution is observed pooling may be ideal for post-decontamination sampling.
The AFSD room scale post-decontamination collection efficiency evaluation proved to be a robust method
for non-porous surface sampling. The AFSD spore recoveries were found to be comparable to the
sponge stick sampling technique. The same results were also found to be consistent for the carpet
materials when the AFSD was compared to the vacuum sock technique for surface sampling. The AFSD
results were compared to conventional surface sampling techniques for carpet (vacuum socks) and
laminate tiles (sponge sticks). The advantage of using the robots is that robotic vacuum sampling is a
cumulative sampling approach, compared to the conventional sampling techniques that collect a certain
number of discrete samples. The probability of sampling in non-contaminated areas is high as shown for
laminate samples where six (6) of nine (9) samples that covered the entire room were non-detects.
These data suggest that traditional approaches to post-liquid-based-decontamination sampling likely
underrepresent the magnitude of viable spores remaining on surfaces following the treatment.
71
-------
References
1. U.S. Environmental Protection Agency, Bio-Response Operational Testing and Evaluation
(BOTE) Project. 2013, Report No. EPA/600/R-13/168, Washington D.C., USA.
2. U.S. Environmental Protection Agency, Expedient Approaches for the Management of Wastes
Generated from Biological Decontamination Operations in an Indoor Environment - Evaluation of
Waste Sampling and Decontamination Procedures. 2014, Report No. EPA/600/R-14/262,
Washington D.C.
3. U.S. Environmental Protection Agency, Product Performance Test Guidelines OCSPP 810.2100:
Sterilants—Efficacy Data Recommendations. 2012, Report No. EPA 712-C-07-056, Washington
D.C.
4. Brown, G.S., R.G. Betty, J.E. Brockmann, D.A. Lucero, C.A. Souza, K.S. Walsh, R.M. Boucher,
M. Tezak, M.C. Wilson, and T. Rudolph, Evaluation of a Wipe Surface Sample Method for
Collection of Bacillus Spores from Nonporous Surfaces. Appl. Environ. Microbiol., 2007. 73(3): p.
706-710.
5. Calfee, M.W., S.D. Lee, and S.P. Ryan, A rapid and repeatable method to deposit bioaerosols on
material surfaces. Journal of Microbiological Methods, 2013. 92(3): p. 375-380.
6. Sang Don Lee, M. Worth Calfee, Leroy Mickelsen, Stephen Wolfe, Jayson Griffin, Matt Clayton,
Nicole Griffin-Gatchalian, and A. Touati, Evaluation of Surface Sampling for Bacillus Spores
Using Commercially Available Cleaning Robots. Environ. Sci. Technol, 2013. 47(6): p. 2595-
2601.
7. Sang Don Lee, M. Worth Calfee, Leroy Mickelsen, Matt Clayton, and A. Touati, Scenario-Based
Evaluation of Commercially Available Cleaning Robots for Collection of Bacillus Spores from
Environmental Surfaces. Remediation 2014. 24(2).
72
-------
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
------- |