EPA 600/R-13/068 | August 2013 | www.epa.gov/ord
United States
Environmental Protection
Agency
Systematic Evaluation of
Aggressive Air Sampling for
Bacillus anthracis Spores
Assessment and Evaluation Report
Office of Research and Development
National Homeland Security Research Center
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EPA /600/R/13/068
August 2013
Systematic Evaluation of Aggressive Air Sampling for
Bacillus anthracis Spores
Assessment and Evaluation Report
National Homeland Security Research Center
Office of Research and Development
U.S. Environmental Protection Agency
Research Triangle Park, NC 27711
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Disclaimer
The United States Environmental Protection Agency (EPA), through its Office of Research and
Development's National Homeland Security Research Center, funded and managed this investigation
through EP-C-09-027 WA 2-28 and 3-28 with ARCADIS U.S., Inc. This report has been peer and
administratively reviewed and has been approved for publication as an EPA document. It does not
necessarily reflect the views of the EPA. 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. EPA
does not endorse the purchase or sale of any commercial products or services.
Questions concerning this document or its application should be addressed to:
Sang Don Lee, 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:919-541-4531
Fax: 919-541-0496
E-mail: lee.sangdon@epa.gov
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Acknowledgments
This effort was managed by the principal investigator from the EPA's Office of Research and
Development's National Homeland Security Research Center (NHSRC), utilizing the support from the
EPA's Chemical, Biological, Radiological, and Nuclear Consequence Management Advisory Team
(CBRN CMAT) within the Office of Solid Waste and Emergency Response (OSWER), Office of
Emergency Management (OEM). The contributions of the entire team are acknowledged.
Project Team:
Sang Don Lee, Ph.D. (Principal Investigator)
National Homeland Security Research Center, Office of Research and Development,
US Environmental Protection Agency
Research Triangle Park, NC 27711
M. Worth Calfee, Ph.D.
National Homeland Security Research Center, Office of Research and Development,
US Environmental Protection Agency
Research Triangle Park, NC 27711
Shawn P. Ryan, Ph.D.
National Homeland Security Research Center, Office of Research and Development,
US Environmental Protection Agency
Research Triangle Park, NC 27711
Leroy Mickelsen
Office of Emergency Management, Office of Solid Waste and Emergency Response,
US Environmental Protection Agency
Research Triangle Park, North Carolina, United States
Stephen Wolfe
United States Environmental Protection Agency, Region 5, West Lake, Ohio, United States
Jayson Griffin
Office of Emergency Management, Office of Solid Waste and Emergency Response,
US Environmental Protection Agency
Research Triangle Park, North Carolina, United States
Rebecca Connell
Office of Emergency Management, Office of Solid Waste and Emergency Response,
US Environmental Protection Agency
Washington, DC, United States
Richard Rupert
Region 3
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US Environmental Protection Agency
Philadelphia, PA, United States
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. The support provided by Tanya
Medley (NHSRC) in acquiring the vast quantities of supplies required for the completion of this project is
also acknowledged.
Additionally, the authors would like to thank the peer reviewers for their significant contributions.
Specifically, the efforts of Erin Silvestri (NHSRC), Tim Dean (EPA), Tony Intrepido (Cubic Applications,
Inc.) and Dino Mattorano (CBRN CMAT) are recognized.
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Table of Contents
Disclaimer 0
Acknowledgments 1
List of Figures 5
List of Tables 5
List of Appendices 5
List of Acronyms and Abbreviations 6
Executive Summary 7
1 Introduction 8
1.1 Process 8
1.2 Project Objectives 9
1.3 Experimental Approach 9
1.3.1 Testing Approach 9
2 Materials and Methods 10
2.1 Testing Chamber 10
2.2 AAS 10
2.3 Test Materials and Deposition 11
2.3.1 Test Coupons Preparation 11
2.3.2 Bacillus Spore Preparation 12
2.3.3 Coupon Inoculation 12
2.4 Test Matrix 13
2.5 Sampling and Analytical Procedures 14
2.5.1 Sponge Wipe Surface Sampling and Extraction 14
2.5.2 Vacuum Sock Surface Sampling and Extraction 15
2.5.3 Hi-Vol Aerosol Sampling 15
2.5.4 Swab Surface Sampling 17
2.6 Sampling Strategy 17
2.6.1 Sampling/Monitoring Points 21
2.7 Sample Handling and Custody 22
2.7.1 Cross-contamination Prevention 22
2.7.2 Sample Quantities 22
2.7.3 Sample Containers 23
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2.7.4 Sample Identification 23
2.7.5 Sample Preservation 24
2.7.6 Sample Holding Times 24
2.7.7 Sample Custody 24
2.7.8 Sample Archiving 25
3 Results and Discussion 26
3.1 Definition of Sampling Efficiency 26
3.2 Inoculation and Spore Recovery 26
3.3 Aggressive Air Sampling Results 27
4 Quality Assurance 33
4.1 Sampling, Monitoring, and Analysis Equipment Calibration 33
4.2 Data Quality 34
4.3 QA/QC Checks 34
4.4 Acceptance Criteria for Critical Measurements 36
4.5 Data Quality Audits 38
4.6 QA/QC Reporting 38
5 Summary and Recommendations 39
6 References 40
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List of Figures
Figure 2-1. COMMANDER setup for AAS 11
Figure 2-2. 3 x3 grid of ADAs on a 107 cm x 107 cm coupon 12
Figure 2-3. Hi-Vol Impactor Plate 15
Figure 2-4. Hi-Vol Air Sampler 16
Figure 2-5. AAS pre- and post-fumigation procedure (The background AAS was conducted
with three air samplers operating concurrently.) 18
Figure 2-6. Phase 1 sampling sequence 19
List of Tables
Table 2-1. Test Matrix 13
Table 2-2. Sample Quantities per Test 23
Table 2-3. Sample Coding 24
Table 3-1. Inoculation of Coupons 26
Table 3-2. Pre-Fumigation AAS Recovery 31
Table 3-3. Post-Fumigation AAS Recovery 32
Table 4-1. Sampling and Monitoring Equipment Calibration Frequency 33
Table 4-2. QA/QC Sample Acceptance Criteria 35
Table 4-3. Critical Measurement Acceptance Criteria 37
List of Appendices
Appendix A Miscellaneous Operating Procedures (MOPs) included as a separate document
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List of Acronyms and Abbreviations
AAS
ADA
APPCD
ATCC
B.
BOTE
CBRN
CPU
CM
CMAT
COC
COMMANDER
DCMD
DHS
DPG
DQI
DQO
ECBC
EPA
cm
cm2
FRM
Hi-Vol
ISO
LPM
MDI
MOP
m
mph
NHSRC
NIST
OEM
ORD
PBST
PM
PPE
ppm
ppmv
QA
QAPP
QC
RH
rpm
slpm
SOP
S&T
VHP
WAM
WARRP
®
Aggressive Air Sampling
Aerosol Deposition Apparatus
Air Pollution Prevention and Control Division
American Type Culture Collection
Bacillus
Bioresponse Operational Testing and Evaluation
Chemical, Biological, Radiological, and Nuclear
Colony Forming Units
critical measurement(s)
Consequence Management Advisory Team
chain of custody
Consequence Management and Decontamination Evaluation Room
Decontamination Consequence and Management Division
Department of Homeland Security
Dugway Proving Grounds
Data Quality Indicator
Data Quality Objective
Edgewood Chemical Biological Center
U. S. Environmental Protection Agency
centimeter
square centimeter
Federal Reference Method
high volume
International Organization for Standardization
liters per minute
Metered Dose Inhaler
Miscellaneous Operating Procedure
meter
miles per hour
National Homeland Security Research Center
National Institute of Standards and Technology
Office of Emergency Management
Office of Research and Development
Phosphate Buffered Saline Tween20
Particulate matter
Personal Protective Equipment
part(s) per million
part(s) per million by volume
Quality Assurance
Quality Assurance Project Plan
Quality Control
Relative Humidity
revolutions per minute
Standard liters per minute
Standard Operating Procedure
Science and Technology (Directorate)
Vaporous Hydrogen Peroxide
Work Assignment Manager
Wide Area Recovery and Resiliency Program
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Executive Summary
The primary objectives of this project were to evaluate the Aggressive Air Sampling (AAS) method for use
in sampling Bacillus anthracis spores. Unlike surface sampling, AAS gives airborne concentrations of
respirable size particles that can be used to assess risk of inhalation exposure and as a result, risk of
inhalation anthrax. Surface sampling does not differentiate particle size nor does it allow for determining
the fraction of particles that could become re-aerosolized. The ability to ensure safe re-entry and
reoccupation of spaces after a decontamination event relies on the ability to make accurate
measurements of any residual contamination. The purpose of this evaluation was to identify the relative
sampling efficacy of the AAS method for spore sampling as a function of surface type, spore surface
concentration, and dissemination method. The AAS test results were compared to other currently-used
surface sampling methods (i.e., vacuum socks or sponge wipes).
This study was designed and conducted to characterize the AAS technique. The first part of this
characterization entailed use of AAS on surfaces with relatively high spore (Bacillus atrophaeus) surface
loadings (i.e., ~1 x 104 colony forming units (CPUs) per cm2), deposited via aerosol deposition apparatus
(ADA) pyramids). Secondly, the efficacy of the AAS for clearance sampling (i.e., lower spore surface
loadings) was determined using the initially loaded coupons that had been subjected to various targeted
Vaporous Hydrogen Peroxide (VHP®) fumigation cycles (250 parts per million [ppm] hydrogen peroxide
for 30, 60, or 90 minutes).
Three Federal Reference Method (FRM) PM-10 high volume samplers (Hi-Vols) were used to sample in
sequence (three tandem 20-minute intervals) following aggressive aerosolization of material coupons that
had been inoculated with spores. Use of the Hi-Vols in sequence allowed fora sampling duration of one
hour at a total volumetric rate that would be desirable or required during an actual large-scale field event.
Most, if not all, recovered spores were collected by the first Hi-Vol sampler (1 air exchange); analysis of
samples collected by the second and third samplers showed near or less than background levels. The
AAS procedure can be further improved by optimizing the air sampling flow rate, the number of air
samplers, and the surface agitation method depending on the sampling site characteristics. Further, a
breakdown of recovery from the impactor plate and the filter of the Hi-Vol sampler (D50 of 10 urn) showed
that most of the recovery was typically from the filter fraction under the tested conditions. This means that
50% of 10 urn particles penetrate through the impactor and the other 50% are collected on the impaction
plate.
The results of this study demonstrated that AAS may be a viable option to sample B. anthracis spores.
The overall test results showed AAS results may vary depending on the surface spore characteristics
(e.g., spore size distribution) and environmental conditions (e.g., relative humidity, fumigation, other
activities that may impact the surface condition). When the AAS results were compared as a function of
surface types, AAS recoveries were lowest from the carpet among three tested materials. The laminate
surface showed the highest spore recoveries. The comparative recoveries ranged 0.37% to 5.84% for
pre-fumigation and 0.004% to 1.032% for post-fumigation AAS.
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1 Introduction
The U.S. Department of Homeland Security (DHS) is committed to using cutting-edge technologies and
scientific talent in its quest to protect human health and the environment. The DHS Science and
Technology Directorate (S&T) is tasked with researching and organizing the scientific, engineering, and
technological resources of the United States and leveraging these existing resources into technological
tools to help protect the homeland. Through this project, the U.S. Environmental Protection Agency's
(EPA's) National Homeland Security Research Center (NHSRC) supports this effort through DHS's Wide
Area Recovery and Resiliency Program (WARRP) S&T program.
Accurate measurements of residual contamination are needed to inform decisions on re-entry and
reoccupation of spaces following site decontamination. For a wide area contamination incident, traditional
surface sampling methods (i.e., wipe, swab and vacuum sock) used for clearance sampling may require
an extensive number of samples, which may be time and labor intensive to achieve a reasonable
confidence. Judgmental sampling is an alternate strategy which can reduce the total number of samples.
However, statistical analyses of results from this sampling strategy are not applicable. Innovative
techniques, such as Aggressive Air Sampling (AAS), may prove useful as an additional method to
currently-used surface sampling methods and, with additional research, may be used as an alternative
method in certain situations (i.e., detection of spore presence from unknown hot spots, wide spread
contamination with concentration close to detection limit for surface sampling methods, etc.), and
effectively shorten the timeline to recovery and/or reduce the sampling burden during a response. AAS
could be used for numerous building interiors for rapid sampling with fewer required personnel, and in
certain scenarios such as covert wide area contamination, could reduce the total number of samples per
unit area compared to the current surface sampling methods. Given that surface sampling is one of the
critical bottlenecks in the remediation process, AAS could ultimately result in a decrease in overall
cleanup time. Unlike surface sampling, AAS indicates airborne concentrations of respirable size particles
that can be used to assess risk of inhalation exposure and as a result, risk of inhalation anthrax. Surface
sampling does not differentiate particle size nor does it allow for determining the fraction of particles that
could become re-aerosolized.
This project evaluated the AAS method to determine whether this technique is appropriate for Bacillus
(B.) anthracis spore sampling. The AAS method was used as a supplement to surface sampling following
building decontamination in response to the 2001 intentional 8. anthracis spore contamination incident in
the U.S.[1-6] However, AAS was not used as a primary method for sampling due to a lack of a systematic
and rigorous evaluation of this technique. The intent of this project is to empirically evaluate the AAS
method for sampling 8. anthracis surrogate spores.
Results from this project may be used by EPA's Office of Emergency Management (OEM) and other
stakeholders to decide upon preferred characterization and clearance sampling methods to be employed
during remediation efforts.
1.1 Process
The general process investigated in this project was AAS of selected surfaces contaminated with 8.
atrophaeus spores (i.e., surrogate for 8. anthracis spores). The AAS method was evaluated for carpet,
laminate, and painted drywall to determine whether this technique may be effective for 8. anthracis spore
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sampling from common surfaces. The AAS Standard Operating Procedure (SOP) used during the
Bioresponse Operational Testing and Evaluation (BOTE, Phase I) [7, 8] project was used as the starting
point for this study. Following deposition of the surrogate spores onto the target surface materials via
aerosolization and settling, spore sampling efficacy of AAS was determined before and after fumigation
with hydrogen peroxide. Similarly, the currently- recommended B. anthracis spore surface sampling
methods (i.e., vacuum socks or sponge wipes) were used to quantify the spore abundance on additional
replicates of the test surfaces. These recoveries were then compared to AAS recoveries. This evaluation
identified the relative sampling efficacy of the AAS method for spore sampling as a function of surface
type and spore surface loading.
1.2 Project Objectives
The primary objectives of this project were to evaluate the AAS method for sampling 8. anthracis
surrogate spores.
1.3 Experimental Approach
The experimental approaches used to meet the objectives of this project were:
• Use of controlled chambers, standardized sections of environmental surfaces, and precise spore
inoculums;
• Contamination of materials via aerosol deposition of bacterial spores;
• Quantitative assessment of spore contamination by sampling representative sections of material
before decontamination;
• Application of a prescribed AAS procedure to the test sections;
• Quantitative assessment of residual contamination by sampling test sections and procedural
background level after AAS;
• Determination of AAS effectiveness (comparison of results from positive control samples and test
sections); and
• Documentation of operational considerations (e.g., cross-contamination, procedural time, impacts
on materials and personnel).
1.3.1 Testing Approach
Tests consisted of three parts that were intended to be conducted in duplicate for a total of six tests. The
test matrix, shown in Table 2.1, shows that each test included one loading level (i.e., inoculum
concentration) for each surface type. The target spore surface loading was 1 x 104 CFU/cm2. The three
surface types selected for this study were carpet, laminate flooring, and painted dry wallboard. In addition
to conducting AAS during each test, the laminate and painted dry wallboard coupons were sampled using
the sponge wipe method (Section 2.5.1), and carpet was sampled using the vacuum sock method
(Section 2.5.2). Culture-based methods were subsequently used to quantify the number of spores
recovered by enumeration of CPUs on microbiological growth media after plating serially-diluted aliquots
of sample extracts. Therefore, in this report "recovery" is defined by the number of CPUs observed
following sample collection, extraction, and analysis (dilution-plating or filter-plating).
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2 Materials and Methods
2.1 Testing Chamber
All testing was done in the Consequence Management and Decontamination Evaluation Room
(COMMANDER). This is a specially constructed enclosed, single-access-point chamber (henceforth,
chamber) within the current Homeland Security Enclosure located within High-Bay Room 130 (H130) at
EPA's Research Triangle Park, NC campus. COMMANDER meets the following criteria:
• Supports 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]);
• Has external dimensions of 2.7 m x3.7 m x3 m high;
• Has nominal internal dimensions of 2.5 m x3.4 m x2.7 m; with about41m2 of floor and walls;
• Contains one air-tight entry/exit port with a window;
• Contains a 1.8 m x 1.8 m x 2.4 m high airlock with single 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; and
• Complies with all relevant local and national codes.
The environmental conditions (relative humidity [RH] and temperature) both during the conditioning
phases and during AAS and surface sampling were monitored by a Vaisala (Model 333, Vaisala, Helsinki,
Finland) probe mounted on the side of the chamber. The COMMANDER was kept at a slightly negative
pressure (~ 2" water) to prevent exfiltration of spores from COMMANDER into the general laboratory
space.
2.2 AAS
During the AAS procedure, three consecutive aerosol samples (every 20 minutes) were collected using
three Hi-Vol samplers (Thermo Fisher Scientific Inc., High Volume Air Sampler VFC-PM10.for FRM
RFPS-1287-063, Pittsburgh, PA, (https://www.thermo.com/eThermo/CMA/PDFs/Various/File 52267.pdf
accessed March, 2013)). Collection of the first 20-minute sample was initiated simultaneously with
aggressive agitation of surfaces and forced resuspension of particles. Particle resuspension was
maintained with turbulent air created by the operation of a stainless steel mixing fan (P/N 1729K11,
McMaster-Carr, Atlanta) (shown in Figure 2-1). The resuspension process was performed using a leaf
blower (Toro Power Sweep Electric Blower, Model # 51585) operated at the highest speed
(approximately 260 km per hour (160 miles per hour)). The operating conditions of the leaf blower,
described in Miscellaneous Operating Procedure (MOP) 3166 (Appendix A), were repeated for all the
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tests and set at an angle of 45 degrees, a distance of less than 30 cm from the target coupon, and with a
forced aerosolization time of 1 min per one 35.56 cm x 35.56 cm area (see Appendix A for copies of all
associated MOPs). The leaf blower operator moved the tip of the blower back and forth across the
coupon(s) at approximately 1 meter per second for the total operation time. The exhaust of the Hi-Vol
samplers was recycled within COMMANDER.
For each test, three stainless steel (reference samples) and 15 material coupons (three sampled using
currently-used methods and 12 sampled using AAS pre-fumigation). Following fumigation, the 12
material coupons were seperated into two groups, 9 for post-fumigation AAS, and 3 sampled with
currently-used methods post-fumigation. Surface samples of coupon materials and stainless steel, used
to characterize the inoculation, were collected on the same day of the AAS test. The coupons sampled
with currently-used methods underwent exactly the same series of conditions, prior to the final sampling
event, as the AAS test samples: inoculated, subjected to AAS, fumigated, and then sampled with the
currently-used surface sampling methods rather than AAS.
Figure 2-1. COMMANDER setup for AAS
2.3 Test Materials and Deposition
2.3.1 Test Coupons Preparation
Various surface material types (carpet, laminate flooring, and painted drywall) were represented by 35.56
cm x 35.56 cm and 107 cm x 107 cm coupons, while the reference stainless steel control coupons were
35.56 cm x 35.56 cm. Coupons were fabricated according to MOP 3150-AII, and were sterilized by a 250
ppmv VHP® cycle for 4 hours inside COMMANDER. After sterilization, coupons were degassed for a
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minimum of three days before testing. Coupons were inoculated in H-130 outside the COMMANDER
facility. To prevent cross-contamination, the test coupons were transported and disassembled
(uncovering the ADAs) in the sterilized COMMANDER airlock. Positive material and stainless steel control
coupons also underwent the same transport to COMMANDER; however, they were disassembled and
sampled in H-130, outside COMMANDER.
2.3.2 Bacillus Spore Preparation
The test organism for this work was a powdered spore preparation of 8. atrophaeus (American Type
Culture Collection, ATCC 9372) and silicon dioxide particles. This bacterial species was formerly known
as 8. subtilis var niger and subsequently 8. globigii. The preparation was obtained from the U.S. Army
Dugway Proving Grounds (DPG) Life Science Division. The preparation procedure is reported in Brown
et al.[9] briefly, after 80 - 90 percent sporulation, the suspension was centrifuged to generate a
preparation of about 20 percent solids. A preparation resulting in a powdered matrix containing
approximately 1 x 1011 viable spores per gram was prepared by dry blending and jet milling the dried
spores with fumed silica particles (Degussa, Frankfurt am Main, Germany). The powdered preparation
was loaded into metered dose inhalers (MDIs) by the U.S. Army Edgewood Chemical Biological Center
(ECBC) or by ARCADIS according to a proprietary protocol. Control checks for each MDI were included in
the batches of coupons contaminated with a single MDI.
2.3.3 Coupon Inoculation
Coupons were inoculated with spores of 8. atrophaeus from a MDI using the procedure detailed in MOP
3161-HD and in the article by Calfee et al[10]. . Briefly, each 107 cm x 107 cm coupon was inoculated
using an array of nine Aerosol Deposition Apparatus (ADA)s (see Figure 2-2), each designed to fit one
35.56 cm x 35.56 cm coupon or area of any thickness.
Figure 2-2. 3x3 grid of ADAs on a 107 cm x 107 cm coupon
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Stainless steel and the other 35.56 cm x35.56 cm material coupons were inoculated using an individual
ADA. In accordance with MOP 3161-HD, the MDI was discharged a single time into each dosing
chamber. The spores were allowed to settle onto the coupon surfaces for a minimum period of 18 hours.
After the minimum 18-hour period, the large (107 cm x 107 cm) coupon was then moved to the
COMMANDER airlock where the ADAs were removed. The uncovered coupons were then transferred to
their positions inside COMMANDER. Care was taken not to touch the inoculated surface. Personnel
changed garb (laboratory coat, P95 mask, hair net, boot covers) after handling the coupons to help
prevent cross-contamination from the moving/handling operation. Three small (35.56 cm x35.56 cm)
coupons were also transported into COMMANDER, while an additional three remained in place until the
following day, when surface samples were collected. These small coupons were then walked to the
airlock in the same manner to simulate the transport undergone by test coupons. ADAs remained on the
coupons until just before sampling.
The MDIs provide 200 discharges (50 uL each) per MDI. The number of discharges per MDI was tracked
so that use did not exceed this value. Additionally, in accordance with MOP 3161-HD, the weight of each
MDI was determined after completion of the contamination of each coupon. If an MDI weighed less than
10.5 g at the start of the contamination procedure described in MOP 3161-HD, this MDI was retired and a
new MDI was used. For quality control of the MDIs, stainless steel contamination control coupons were
inoculated as the first, middle, and last coupons within a single group of coupons inoculated by any one
MDI within a single test. If the results from the contamination controls were outside the acceptance
criteria, the results were immediately reviewed to determine the corrective action.
A log was maintained for each set of coupons that was dosed via the method of MOP 3161-HD. Each
record included 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). The ADA affixed to the coupon was labeled and remained on
the coupon until immediately before sampling.
2.4 Test Matrix
The test matrix for Phase 1 testing is shown in Table 2-1. Each test includes a pre- and a post-fumigation
sampling sequence as shown in Figure 2-5.
Table 2-1. Test Matrix
Test#
4
5
6
Test Material
Carpet
Laminate
Drywall
Spore loading
(CPU / cm2)
3 4
10 -10
3 4
10 -10
3 4
10 -10
Dissemination
Method
ADA
ADA
ADA
Replicate
Tests
2
2
2
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2.5 Sampling and Analytical Procedures
Within a single test, AAS of the material sections was completed for background coupons first before
sampling of any test material sections was performed. Surface sampling was done either by sponge wipe
sampling or vacuum sock sampling according to the protocols documented below. The sampled area by
surface sampling methods was 35.56 cm x35.56 cm (14"x 14") for each coupon.
Prior to the sampling event, all materials needed for sampling were prepared using aseptic techniques.
The materials specific to each protocol are included in the relevant sections below. In addition, general
sampling supplies were also needed. A sampling material bin was stocked for each sampling event. The
bin contained enough sponge wipe sampling and vacuum sock sampling kits (prepared according to MOP
3141 A) to accommodate all required samples for the specific test. Additional kits of each type were
included for back up. Sufficient prepared packages of gloves and bleach wipes were included in the bin. A
sample collection bin was used to transport samples back to Biocontaminant Laboratory at EPA's facility
in Research Triangle Park, NC. The exterior of the transport container was disinfected by wiping all
surfaces with a bleach wipe ortowelette moistened with a 0.5 to 0.8% hypochlorite (HOCI) solution (pH-
adjusted bleach) prior to transport from the sampling location to the Biocontaminant Laboratory.
2.5.1 Sponge Wipe Surface Sampling and Extraction
Sponge wipe sampling is the currently-used method for sampling on nonporous-smooth surfaces such as
ceramics, vinyl, metals, painted surfaces, and plastics. Sponge wipe (3M Sponge-Sticks™ (P/N
SSL10NB), made of cellulose) sampling was used as the comparative method for smooth, nonporous
surfaces, and was conducted according to MOP 3165 (for surface samples) or MOP 3169 (for impactor
samples). The general approach is that a moistened sponge is used to wipe a specified area to recover
surface-associated bacteria, viruses, and biological toxins. The impactor samples (see Figure 2-3) were
representative of particulate matter (PM) greater in size than 10 urn. Due to the size and material
characteristics (hard surface with holes) of the impactor, a separate sampling technique was required.
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Figure 2-3. Hi-Vol Impactor Plate
Sponge wipe samples were extracted by stomaching (1 minute, 260 revolutions per minute [rpm]) in 90
ml of Phosphate Buffered Saline with Tween® 20 (PBST) using a Seward® Model 400 circulator
(Seward® Laboratory Systems, Inc., Port Saint Lucie, FL), then concentrated with a centrifuge. Sponge
wipe extraction is described in detail in MOP 6580. This MOP was developed based on the Laboratory
Response Network (LRN) protocol.
2.5.2 Vacuum Sock Surface Sampling and Extraction
Vacuum sock sampling, the currently recommended sampling method for rough and/or porous surfaces,
was used as the comparative method for carpet coupons. Vacuum sock sampling was conducted
according to MOP 3145.
Vacuum sock samples were extracted by first wetting the collection (white) portion of the filter by dipping
in PBST, then cutting the filter with sterile scissors (vertically, then horizontally) into small pieces
(approximately 1 cm x4 cm). As the filter was fractioned, the resulting pieces were allowed to fall into a
120 mL sterile specimen cup (Starplex Scientific LeakBuster Specimen Containers- Fisher Scientific #14-
375-459, Pittsburgh, PA) containing 20 mL sterile PBST. The cups were then agitated (30 minutes, 300
rpm, ambient temperature) using an orbital platform shaker incubator (Lab-Line, Model 3625). Vacuum
sock extraction is described in MOP 6572.
2.5.3 Hi-Vol Aerosol Sampling
Three Hi-Vol air samplers were used to collect aerosols from a large volume of air during each AAS
sampling event. During each sampling campaign, each sampler was equipped with a new sterile 20.32
cm x 25.40 cm borosilicate (QM-A, Whatman) glass fiber filter. These Hi-Vol samplers use a volumetric
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flow-controlled system for sampling of air (1020 to 1240 slpm) for the collection of participate matter with
diameter less than 10 |jm (D50) onto the filter, and greater than 10 |jm onto the impactor plate (shim).
The three samplers were operated for 20 minutes each, in tandem. For instance, the samplers were not
run concurrently, but sequentially, creating a total sampling duration of 1 hour.
Figure 2-4 shows the Hi-Vol sampler. On the top is an inlet designed to collect particulate matter in wind
speeds of less than 30 miles per hour (mph). The 10 urn impactor plates (or shims) are placed in the
opening seen in Figure 2-4. Below the inlet and impactor plates lies the borosilicate borosilicate glass
fiber filter.
During sample recovery, the filters were folded using aseptic technique inside COMMANDER such that
the side exposed to the spores was inside a double fold. The filter was then transferred to a sterile
stomacher bag for extraction. The impactor plates, meant to capture all particles larger than 10 urn, were
also sampled (using the sponge wipe method in accordance with MOP 3169)..
The filter samples were extracted by stomaching (2 minutes, 230 rpm) in 100 ml of PBST using a
Seward® Model 400 circulator (Seward® Laboratory Systems, Inc, Port Saint Lucie, FL). Extraction of
borosilicate filters is described in MOP 6586. To improve the detection limit, the pulp was removed using
a sieve and the resultant liquid filtered according to MOP 6565.
Figure 2-4. Hi-Vol Air Sampler
16
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2.5.4 Swab Surface Sampling
MOP 3135 was used for collecting swab samples. The general approach was to use a moistened swab to
wipe a specified area to recover bacterial spores. Swab samples were collected from coupons before
inoculation for quality assurance of the sterilization method. Typically, one coupon of each material type
per sterilization batch was sampled.
2.6 Sampling Strategy
The AAS procedure consisted of the sequential events listed in Figure 2-5.
17
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Remove ADAs (if necessary)
Start Air Sampler 1 (1000 Lpm)
¥
Leaf Blower Surface Agitation (12 min) of 107 cm x 107 cm and 35.56 cm x 35.56 cm coupons
Stop Air Sampler 1 after 20 minutes, Start Air Sampler 2 (1000 Lpm)
Stop Air Sampler 2 after 20 minutes, Start Air Sampler 3 (1000 Lpm)
Stoc Air Sampler 3 after 20 minutes. Allow settlina time. Retrieve filters.
JL
VHP COMMANDER for Partial Decontamination
Re-enter COMMANDER
Collect surface samples from three35.56 cm x 35.56 cm coupons
Start Air Sampler 1 (1000 Lpm)
Leaf Blower Surface Agitation (9 min) of 107 cm x 107 cm coupon
Stop Air Sampler 1 after 20 minutes, Start Air Sampler 2 (1000 Lpm)
Stop Air Sampler 2 after 20 minutes, Start Air Sampler 3 (1000 Lpm)
Stop Air Sampler 3 after 20 minutes. Allow settling time. Retrieve filters.
VHP® COMMANDER for Sampler Sterilization
Figure 2-5. AAS pre- and post-fumigation procedure (The background AAS was conducted with
three air samplers operating concurrently.)
Figure 2-6 shows the sampling sequence. The sampling sequence and samples collected are detailed
below:
18
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Step 1. AAS background sampling
Coupon setup
One 107 cm x 107
cm test coupon
Three 35.56 cmx
35.56 cm target
material coupons
Three 35.56 cmx
35.56 cm target
material coupons
Three 35.56 cmx
35.56 cm Stainless
I
Step2a: Stainless
reference sampling
Step 3: Pre-Fumigation AAS
I
I
Step 2b: Surface
sampling
Fumigation (VHP®)
I
Step 4: Post-Fumigation
Surface samolina
Step 5: Post-Fumigation AAS
Chamber reset
Figure 2-6. Phase 1 sampling sequence
1. Background Sampling. This sampling sequence occurred initially and between each of the tests
following VHP® sterilization (250 ppmv H2O2 for four hours). One 107 cm x 107 cm sterilized
material coupon, consisting of nine ADAs assembled side-by-side (3 x3), were placed inside an
empty, decontaminated COMMANDER during AAS. ADAs were removed and one background
(negative) AAS sample was collected using all three Hi-Vol samplers concurrently. A leaf blower
was used according to MOP 3165 to aerosolize spore contaminants, if any, from the sterilized
coupon while the procedural aerosol background samples were collected. All MOPs associated
with this testing can be found in Appendix A. This test sequence helped quantify any cross-
contamination that may occur from test personnel.
19
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2. Pre-Fumiqation Surface Sampling. The pre-fumigation surface sampling involved the collection
of samples from three replicate positive coupons (Figure 2-7 B), using the currently-used surface
sampling methods. These sampling events occurred in H130, outside of COMMANDER.
3. MDI control surface reference samples. These control reference samples consisted of three
35.56 cm x 35.56 cm stainless steel coupons (Figure 2-7 A) that were inoculated at the same
time and with the same spore loading as the test coupons, using the ADA deposition method.
These samples were then subjected to surface reference sampling, performed using the sponge
wipe method on the same day as the AAS test samples, but prior to the AAS phase. These
samples were used to measure the stabilty of the MDI over the duration of the inoculation event.
4. Pre-Fumigation AAS. Three consecutive Hi-Vol samples (three tandem 20-minute intervals) were
collected using three Hi-Vol samplers during and following aggressive resuspension of 12 (one
107 cm x 107 cm material coupon and three 35.56 cm x35.56 cm coupons) test coupons (Figure
2-7 C) that were inoculated with spores. The coupon inoculation was performed using nine ADAs
assembled side-by-side (3 x3) outside of COMMANDER for the 107 cm x 107 cm coupons, and
3 individual ADAs for the 35.56 cm x 35.56 cm coupons, which were placed inside COMMANDER
on the day of testing. Although the inoculation was performed horizontally for all coupons, the
carpet and laminate flooring coupons were placed horizontally and the drywall coupons were
placed vertically along the COMMANDER walls during AAS to mimic their real-world orientations.
The Hi-Vol samplers were each operated for 20 minutes at 1000 Lpm (approximate 1 air
exchange rate). The three consecutive 20-minute samples captured a 1-hour period of time,
including the 12-minute agitation and 50-minute settling time.
5. Post- AAS / Post- VHP® Fumigation Surface Sampling. Three post-fumigation surface samples
were collected from the three 35.56 cm x 35.56 cm target material coupons (Figure 2-7 E) before
post-fumigation AAS on the 107 cm x 107 cm material coupons (Figure 2-7 D). These coupons
were inoculated in the same manner as the 107 cm x 107 cm material coupon and underwent the
same pre-fumigation AAS as the test coupon in Step 3.
6. Post-Fumigation AAS. Three consecutive Hi-Vol samples (every 20 minutes) were collected
using three Hi-Vol samplers following an aggressive aerosolization of the 107 cm x 107 cm
material coupon that was sampled during the pre-fumigation AAS (Step 3) and exposed to the
VHP® fumigation process. The post-fumigation AAS was performed to determine the
effectiveness of the technique at lower spore concentration and spores subjected to fumigation.
7. All materials were subjected to a VHP® cycle (250 ppmv for four hours) to reset for the next test.
20
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A
3 - Reference Coupons
(stainless steel)
3 - Positive Control Coupons
(carpet, laminate, wallboard)
12 - Pre-Fumigation AAS Test Coupons
(carpet, laminate, wallboard)
9 - Post-Fumigation AAS Test Coupons
(carpet, laminate, wallboard)
3- Post-Fumigation Positive Control Coupons
(carpet, laminate, wallboard)
Figure 2-7. Schematic of control and test coupons utilized in each test
2.6.1 Sampling/Monitoring Points
All AAS operations were conducted in the COMMANDER. The COMMANDER environmental conditions
were monitored by a Vaisala probe mounted onto the side of the chamber. The COMMANDER flow
measurement was taken at the air outlet. Because COMMANDER is kept at negative pressure (~2"
water) to prevent out-leakage, this sampling point included any in-leakage air. There were three sampling
points inside COMMANDER during air agitation. These sampling points were in a well-mixed area due to
the presence of a fan, and were considered co-located. Surface sampling took place both outside and
inside COMMANDER. Surface samples of stainless steel used to characterize the inoculation, and the
material positive controls were collected outside of COMMANDER on the day of AAS. Surface samples of
the material coupons post VHP® fumigation were sampled inside COMMANDER. Air sampling during
AAS was performed using three Hi-Vol Air Samplers.
Critical measurements during the sampling phase included duration of agitation with the leaf blower and
duration of Hi-Vol operation. Pressure differential and flow rate data from the Hi-Vol samplers were
collected to verify the correct operation of the blowers.
Environmental measurements included temperature (ranged 19 to 35 °C) and RH (ranged 48 to 66 %) of
COMMANDER during all phases of tesing. The COMMANDER air exchange rate was constantly
monitored.
21
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Because current surface sampling techniques are intrusive, they also removed viable spores from the
surface of the section. For example, the sampling method itself changed the spore concentration on the
surface. Therefore each coupon could be sampled only once. Reference stainless steel control coupons
were inoculated concurrently with test coupons and were conditioned at 40% RH along with test coupons,
but did not undergo AAS. Surface sampling was also performed on coupons following AAS.
2.7 Sample Handling and Custody
2.7.1 Cross-contamination Prevention
Sampling presents a significant opportunity for cross-contamination of samples. In an effort to minimize
the potential for cross-contamination, several management control practices were employed
• In accordance with aseptic technique, a sampling team was utilized, and consisted of a "sampler,"
a "support person," and a "sample handler."
• The sampler handled only the sampling media and the support person handled all other supplies.
The sample handler removed ADAs before sampling and moved coupons after sampling. The
sampler sampled the surface according to the appropriate procedure described in Section 2.5.
• The collection medium (e.g., sponge wipe or vacuum sock) was then placed into a sample
container that was opened, held and closed by the support person.
• The sealed sample was handled only by the support person.
• All of the following actions were performed only by the support person, using aseptic technique:
o The sealed bag with the sample was placed into another sterile plastic bag that was then
sealed; that bag was then decontaminated using a bleach wipe.
o The exterior of the transport container was decontaminated by wiping all surfaces with a
bleach wipe ortowelettes moistened with a solution of 5000 ppm hypochlorite prior to
transport from the sampling location to the DCMD Biocontaminant Laboratory.
The sampling crew then changed their gloves in preparation for working with the next sample.
All sampling inside COMMANDER was done wearing clean garb (laboratory coat, P95 mask, hair net and
boot covers) over the HazMat suits worn during leaf blower operation. Clean garb was changed between
each activity (approximately every 10 minutes).
2.7.2 Sample Quantities
Table 2-2 lists the sample quantities for each test. Each aerosol sample consisted of two portions: the
impactor and the borosilicate glass fiber filter.
22
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Table 2-2.
Sample Quantities per Test
Sample Type
Sample
Quantity
Background samples
Background - Negative aerosol samples
Background - Negative surface sample (test area)
1
3
Test Samples
MDI control (stainless steel reference control) samples
Material positive control (Pre-AAS) samples
P re-fumigation AAS aerosol samples
Post-fumigation surface samples
Post-fumigation AAS aerosol sample
4
3
3
3
3
2.7.3 Sample Containers
For each sponge wipe sample, the primary containment was a Seward Stomacher® bags (P/N
BA6041/CLR, West Sussex, England). Secondary containment was sterile sampling bags. The primary,
secondary, and tertiary containment of each vacuum sock sample consisted of separate-sterile sampling
bags. Swabs were placed in the sterile swab containers (part of the swab sampling kit (BactiSwab®
(Lenexa, Kansas)' as obtained from the supplier) and then bagged in two individual sterile sampling bags
as secondary and tertiary containment. Hi-Vol filters were placed in a stomacher bag to be used for
extraction, and then bagged in two individual sterile sampling bags as secondary and tertiary
containment. All biological samples from a single test were then placed in a sterilized container. After
samples were placed in the container for storage and transport to the Biocontaminant Laboratory, 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 Biocontaminant
Laboratory.
2.7.4 Sample Identification
Each material section was identified by a description of the material and a unique sample number. The
sampling team maintained an explicit laboratory log which included records of each unique sample
number and its associated test number, contamination application, any preconditioning and treatment
specifics, and the date treated. The sample codes eased written identification. Once the samples were
transferred to the Biocontaminant Laboratory for extraction and analysis, each sample was additionally
identified by a replicate plate identifier and dilution factor. Table 2-3 specifies the sample identification.
The Biocontaminant Laboratory also included on each plate the date it was placed in the incubator.
23
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Table 2-3. Sample Coding
Sample Identification: 28-TN-(X)M(AA)-C-S#
Category
TN
(X)M
(Material)
AA
(aerosol samples)
C (fumigation state)
S#
(Sample Type)
Example
Code
1A
X
C
L
W
S
AF
AS
1
2
F#
L#
R#
Test Number , followed by an A or B for
duplicate tests
Procedural Background sample
Carpet
Laminate Flooring
Painted Drywall
Stainless Steel (forQC purposes)
AAS aerosol sample (quartz filter)
AAS aerosol sample (impactor)
P re-fumigation
Post-fumigation
Field Blank
Laboratory blank
Replicate
DCMD Biocontaminant Laboratory Plate Identification: 28-TN-(X)M(AA)-C-S#-R-d
28-TN-(X)M(AA)-C-S#
R (Replicate)
d (Dilution factor)
As above
R A-C
1 Oto4, for10EOto10E4
2.7.5 Sample Preservation
Following transfer to the Biocontaminant Laboratory, 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.
2.7.6 Sample Holding Times
After sample collection for a single test was complete, all biological samples were transported to the
Biocontaminant Laboratory immediately, with appropriate chain of custody (COC) form(s). Liquid samples
were stored no longer than 24 hours prior to analysis. Samples of other matrices were stored no longer
than five days before the primary analysis. Typical hold times, prior to analyses, for most biological
samples was < one day for liquid samples and < three days for all others.
2.7.7 Sample Custody
Careful coordination with the Biocontaminant Laboratory was required to achieve successful transfer of
uncompromised samples in a timely manner for analysis. Test schedules were confirmed with the
Biocontaminant Laboratory 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 chain of custody 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
24
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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 (by authorized personnel)
Laboratory test team members received copies of the test plans prior to each test. Pre-study briefings
were held to apprise all participants of the objectives, test protocols, and COC procedures to be followed.
These protocols were required to be consistent with any protocols established by EPA.
In the transfer of custody, each custodian signed, recorded, and dated the transfer on the COC. 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 High Bay Room H130-Ato the Biocontaminant
Laboratory.
• Samples were carefully packed and hand-carried between on-site laboratories. The COC record
showing the identity of the contents accompanied all packages.
2.7.8 Sample Archiving
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 extraction vessel.
25
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3 Results and Discussion
3.1 Definition of Sampling Efficiency
Percent recovery (Equation 1) was calculated based on the recovery (CPU) from all inoculated coupons
subjected to AAS, and the recovery (CPU) per coupon achieved by surface sampling methods. An
equivalent of twelve coupons were present during pre-fumigation AAS (three individual coupons and one
larger coupon containing a 3x3 grid), and an equivalent of nine coupons were present for the post-
fumigation AAS (one coupon containing a 3 x 3 grid).
% relative recovery =
Total CPU recovered from FRM sample
Averaqe from control couponsx total number of coupons
^ couponJ ^ j t-
* 100%
(1)
3.2 Inoculation and Spore Recovery
To demonstrate recovery from borosilicate filters, filters were spiked with 50 or 500 CPU, extracted
according to MOP 6586, and sieved to remove the filter fibers suspensions generated by the extraction
process. Recovery, based on the inoculum, ranged from 41 to 131%. This range of error is typical of
microbiological recovery methods.
Stainless steel coupons were used to verify the magnitude and repeatability of spore loadings for every
inoculation event, and to compare the recoveries from material coupons. Table 3-1 shows the average
recovery (CPU) and the Relative Standard Deviation (RSD) for all six tests. Several different MDIs were
used for inoculation of different tests, which explains some of the variability between tests. While all
inoculation was utilized with B. atrophaeus spores prepared by Dugway Proving Ground, two different
labs (ECBC and EPA) prepared MDIs with spores. MDIs (EPA1 and EPA2) prepared by EPA were
loaded with spores of two different concentrations. Same inoculation source and method were used for
both stainless steel and material coupons within each test. The results in Table 3-1 show that spore
recoveries on laminate and drywall coupons were similar to those from the stainless steel coupons.
However, recoveries from carpet coupons were lower than those on stainless steel.
Table 3-1. Inoculation of Coupons
Test ID
4a
4b
5a
5b
6a
6b
Material
Carpet
Carpet
Laminate
Laminate
Drywall
Drywall
Spore
preparation
ECBC
EPA1
ECBC
EPA2
EPA1
EPA1
Sample Method
Vacuum sock
Vacuum sock
Sponge wipe
Sponge wipe
Sponge wipe
Sponge wipe
Recovery (CPU) from
Stainless Steel
coupons (929 cm2)
Avg.
8.28 X 10s
1.62 X107
8.80 X 10s
1.96 X 10s
1.23 X107
1.84 X107
RSD
17%
25%
25%
22%
32%
24%
Recovery (CPU) from
Material coupons
(929 cm2)
Avg.
2.97 X 10s
1.77 X 10s
4.61 X 10s
3. 01 X 10s
1.23 X107
1.24 X107
RSD
89%
69%
59%
47%
7%
33%
26
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3.3 Aggressive Air Sampling Results
Three consecutive Hi-Vol samples were collected using three Hi-Vol samplers in sequence (20 minute
intervals) for pre- and post-fumigation AAS. The recoveries from the second and third Hi-Vol samplers
were less than 1 % of the recoveries from the first Hi-Vol sampler for all tests. This implies that one air
exchange rate (1000 Ipm for 20 minutes) was sufficient to collect 99% of the total spores collected using
AAS under the tested conditions. Background AAS was conducted with three Hi-Vol samplers running
concurrently. The test results are shown in Figure 3-1.
l.E+05
l.E+04
u»l.E+03
Carpet (4a)
filter •impactor
l.E+01
l.E+00
background pre-fumigation post-fumigation
l.E+05
l.E+04
Carpet (4b)
filter • impactor
l.E+00
background pre-fumigation post-fumigation
27
-------
l.E+06
l.E+05
l.E+04
Sl.E+03
u
l.E+02
l.E+01
l.E+00
l.E+06
l.E+05
l.E+04
21.E+03
u
l.E+02
l.E+01
l.E+00
Laminate (5a)
filter Bimpactor
background pre-fumigation post-fumigation
Laminate (5b)
filter Bimpactor
background pre-fumigation post-fumigation
28
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l.E+06
l.E+05
l.E+04
EJl.E+03
u
l.E+02
l.E+01
l.E+00
Wallboard (6b)
filter • impactor
background pre-fumigation post-fumigation
Figure 3-1. Recovery results from AAS tests
Figure 3-1 shows spore recoveries from filter (blue bars) and impactor (red bars) of background, pre- and
post-fumigation AAS tests. The data shown in Figure 3-1 are the summation of all three samplers for
background, pre- and post-fumigation AAS. Spores were detected from background AAS in all six tests.
The total number of spores from background AAS tests was less than 100 CFUs (except 6a,
approximately 110 CFUs) and this level is less than 0.1% of the pre-fumigation AAS recoveries.
Therefore, the impact by the background spore levels was minimal to pre-fumigation AAS results. For
29
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post-fumigation AAS, spore recoveries were adjusted by subtracting blank AAS spore recoveries for filter
and impactor plate.
As a hypothesis, spore contamination in background AAS may be caused by the aerosol deposition using
ADAs in an area adjacent to the test chamber. Test coupons were inoculated on a day before each AAS
test and uncontrolled spores during deposition were released to the laboratory (close to COMMANDER)
air. Released spores might be (re-)suspended into the air via activities in the laboratory. The spore-
contaminated air might have been introduced to COMMANDER when the COMMANDER door was open
to start the background AAS. Regardless, this detected background did not interfere with the
interpretation of the results of this study.
Test results from carpet are shown in 4a and 4b of Figure 3-1. Test 4a was conducted with the MDI
prepared by ECBC and EPA1 for Test 4b. The pre-fumigation spore recoveries from Test 4a were mostly
from the filter sample (less than 1% from the impactor sample). However, the Test 4b results show that
the spore recoveries were mostly (~95% of total recoveries) from the filter, but a more significant amount
(~5% of total recoveries) of spores were recovered from the impactor plate. This difference may be due
to the use of different MDIs for the two tests. To confirm this, the spore size distributions from two
different MDIs were measured using a particle size instrument (Aerodynamic Particle Sizer, APS 3321,
TSI Inc, Shorevies, MN). The mass mean diameter and standard deviation were 2.63 urn and 2.05 for
the ECBC MDI and 4.91 urn and 6.22 for the EPA1 MDI. Since single 8. atrophaeus spore size is
approximately 0.8 urn, a larger number of agglomerated spores were inoculated to the coupons in Test 4b
compared to Test 4a. The large cross sectional area of the agglomerated spores make for easier
resuspension compared to the single spores. This is observed from the Test 4a and 4b results. The total
number of spore recoveries from Test 4b was approximately 10 times higher than in Test 4a, despite the
overall initial spore loadings being similar (Table 3-1).
Tests 5a and 5b in Figure 3-1 show the results of AAS on laminate surfaces. ECBC MDI was used for
Test 5a and EPA2 for Test 5b. The AAS pre-fumigation results of Test 5a are similar to those of Test 4a.
Most of spores were recovered from the filter sample, not from the impactor (less than 1% of total spore
recovery). Test 5b pre-fumigation AAS results show that the amount of spores from the impactor is ~30%
of the total spore recovery. As explained from the carpet test results, this impactor recovery difference
between Test 5a and 5b is due to different MDI usage for inoculation (i.e., different particle sizes coming
from the different MDIs). Tests 6a and 6b in Figure 3-1 show the results of AAS on painted wallboard
surfaces. Both tests were conducted with EPA1 MDI. The results from both tests show significant spore
recoveries from the impactor as seen in Tests 4b and 5b.
Table 3-2 shows the breakdown results of recovery from the impactor and the filter. The spore recoveries
from the first Hi-Vol sampler were more than 95% of the total spore recoveries (sum of three Hi-Vol spore
recoveries) for all six tests. The AAS test results were further compared to those of the currently-used
sampling methods. The summary is shown in Table 3-2. The comparative recoveries ranged 0.37 to 5.84
30
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Table 3-2.
Pre-Fumigation AAS Recovery
Test
ID
4a
4b
5a
5b
6a
6b
Material
Carpet
Carpet
Laminate
Laminate
Drywall
Drywall
Blank
AAS
Recov
eries1
Average
Recoveries
(Stainless
Steel)
Average
Recoveries
(Material
Coupons)
Total Filter
Recovery
Total
Impactor
Recovery
CFU/cm2
0.05
0.09
0.05
0.02
0.26
0.06
8.90 x103
1.74x104
9.47 x103
2.11 x105
1.32x104
1.98x104
3.20 x102
1.91 x103
4.96x103
3.30 x105
1.32x104
1.33x104
3.38
19.62
290.01
841.50
134.80
43.64
0.001
1.30
0.01
386.13
15.46
10.40
Relative
Recovery
%
1.06
1.10
5.84
0.37
1.13
0.40
1 Total background spores detected during blank AAS prior to pre-fumigation AAS, value is the sum of three Hi-Vol samplers
operated simultaneously (filter and impactor plate recoveries summed)
To determine the application of AAS at lower concentrations and under varied environmental conditions,
the coupons were sampled by AAS before and after VHP® fumigation. Three control coupons were
selected from the pre-fumigation AAS samples, which were fumigated at the same time as post-
fumigation AAS test samples. These coupons were sampled using currently-used surface sampling
methods consistent with each material type and then removed immediately prior to the initiation of the
AAS operation on the test coupon. Varying cycles of VHP® (ppm-hours) fumigation were used to obtain
differing amounts of post-fumigation surface spore concentrations (as a study parameter). Table 3-3
summarizes the results. Spore recoveries from two tests (4a and 5a) were below background spore
recoveries. Collection signatures (percentage of spores collected on the impactor plate and total
recoveries) were similar for pre- and post-fumigation AAS samples. No spores were recovered in Test 4a
and Test 5a. These tests were initially conducted using high VHP® fumigation conditions (ppm-hours).
Later tests (test 4b, 5b, 6a, and 6b) intentionally utilized less effective fumigation conditions in order to be
able to detect the quantifiable amount of spores from coupon surfaces. The % relative recoveries ranged
between 0.004 to 1.032 %. This % relative recovery values were lower than those from the pre-
fumigation AAS tests. This low % relative recovery for post-fumigation AAS might be related to the
impact from the application of AAS during the pre-fumigation tests and/or due to the VHP fumigation
process. The initial AAS application might have removed a significant amount of loosely attached spores.
This phenomenon would likely have a greater impact on AAS than the currently-used surface sampling
methods. In addition, the surface coupons were exposed to hydrogen peroxide and higher relative
humidity than the pre-fumigation AAS. These factors may increase the spore binding characteristics on
surfaces. Therefore, the combination of these factors may have impacted the post-fumigation AAS.
31
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Table 3-3. Post-Fumigation AAS Recovery
Test ID
4a
4b
5a
5b
6a
6b
Material
Carpet
Carpet
Laminate
Laminate
Drywall
Drywall
Fumigation
(ppm -hours)
495
358
501
444
188
211
Average
Recoveries
(Material
Coupons)
Total Filter
Recovery3
Total
Impactor
Plate
Recovery
CFU/cm2
0.01
356.30
0.00
1679.22
3789.02
6986.01
B.Bb
0.28
B.B
6.10
0.40
0.20
B.B
0.06
B.B
11.23
0.02
0.06
Relative
Recovery
%
N.A.C
0.0960
N.A.
1.032
0.011
0.004
a Filter and Impactor plate spore recoveries were adjusted by subtracting background AAS spore recoveries.
b Below background
cNot Applicable
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4 Quality Assurance
This project was performed under an approved Category III Quality Assurance Project Plan titled
Systematic Evaluation of Aggressive Air Sampling for Bacillus anthracis Spores (January 2012).
4.1 Sampling, Monitoring, and Analysis Equipment Calibration
There were SOPs for the maintenance and calibration of all laboratory and DCMD Biocontaminant
Laboratory equipment. All equipment was verified as being certified calibrated or having the calibration
validated by the on-site (RTP, NC) Metrology Laboratory at the time of use. Standard laboratory
equipment such as balances, pH meters, biological safety cabinets 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.
The Hi-Vol samplers use the pressure drop over an orifice to measure the volumetric flow rate. These
were calibrated prior to testing by the APPCD Metrology Laboratory using a National Institute of
Standards and Technology (NIST)-traceable ROOTS® meter.
Table 4-1. Sampling and Monitoring Equipment Calibration Frequency
Equipment
Pipette
Thermometer
RH sensor
Stopwatch
Clock
Scale
Cal i brati on/Ce rtificati on
Gravimetric calibration twice per year
Compare to independent NIST thermometer
(this is a thermometer that is recertified
annually by either NIST or an International
Organization for Standardization (ISO)-17025
facility) value once per quarter.
Compare to calibration salts once a week.
Compare against NIST Official U.S. time at
http://nist.time. qov/timezone.cq i?Eastern/d/-
5/java once every 30 days.
Compare to office U.S. Time (S> time.qov every
30 days.
Sartorius Bl 310 scale: Check calibration with
Class 2 weights prior to each use.
Expected Tolerance
±0.1% weight
±1 °C
±5%
±1 min/30 days
±1 min/30 days
±0.01 g
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4.2 Data Quality
The primary objective of this project was to evaluate the AAS method to determine whether this technique
is appropriate for 8. anthracis spore sampling. This evaluation was to identify the relative sampling
efficacy of the AAS method for spore sampling as a function of surface type, and spore surface
concentration. The AAS test results were to then be compared to other currently-used surface sampling
methods (i.e., vacuum socks or sponge wipes). This section discusses the Quality Assurance/Quality
Control (QA/QC) checks (Section 4.3) and Acceptance Criteria for Critical Measurements (Section 4.4)
considered critical to accomplishing the project objectives.
The Quality Assurance Project Plan (QAPP) in place for this testing was followed with several deviations,
many of which were documented in the relevant sections of this report. Deviations included:
• Use of sieved filter liquid. When written, the QAPP documented the methods used for filtering a
sample with low CPU counts from dilution plating. During testing it was determined that a maximum
volume of 3 ml could be filter plated from quartz filter sample due to the presence of debris from the
quartz. These samples were thus sieved before filtering to improve the limit of detection.
• Loss of data. Digitally acquired flow rate data were lost from a few tests, due to computer
communication problems. However, flow rates at the beginning and end of each test were verified to
confirm that samplers operated as expected during the sampling duration. The activation switch for
each sampler was also verified to be in the "on" position following sampling initiation. For these
reasons, the lost flow rate data were considered ancillary and only necessary to help define the
operational parameters of Hi-Vol samplers.
• The MDIs in hand when the QAPP was written, manufactured by ECBC, failed to operate reliably.
EPA, therefore, manufactured some replacements. Spores from different MDIs may have different
characteristics; however for this study one MDI type (ECBC or EPA prepared) was used for all
samples within a single test. Results obtained during each test were comparative (AAS compared to
currently used sampling method); therefore results presented herein are not affected by MDI type.
4.3 QA/QC Checks
Uniformity of the test materials was a critical attribute to assuring reliable test results. Uniformity was
maintained by obtaining a large enough quantity of material that multiple material sections and coupons
could be constructed with presumably uniform characteristics. Samples and test chemicals were
maintained to ensure their integrity. Samples were stored away from standards or other samples which
could cross-contaminate them.
Supplies and consumables were acquired from reputable sources and were NIST-traceable when
required. Supplies and consumables were examined for evidence of tampering or damage upon receipt
and prior to use, as appropriate. Supplies and consumables showing evidence of tampering or damage
were not used. All examinations were documented and supplies were appropriately labeled. Project
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personnel checked supplies and consumables prior to use to verify that they met specified task quality
objectives and did not exceed expiration dates.
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, the CPU were
enumerated manually and recorded. Critical QC checks are shown in Table 4-2. The acceptance criteria
were set at the most stringent level that could be routinely achieved and are consistent with the data
quality objectives described in Section 4.4. 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 or tests were
included for each set of test conditions. Qualified, trained and experienced personnel using SOPs/MOPs
ensure 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.
Sterility swabs from Test 6a and 6b coupons indicated that the sterilization procedures were insufficient to
kill all the spores present, however contamination was minimal compared to the amount dosed onto test
coupons and was consistent across all coupon types (test and control).
Table 4-2. QA/QC Sample Acceptance Criteria
Sample Type
Negative
Aerosol
Background
Samples
Negative
coupon control
sample
Field Blank
Purpose
Determine extent
of cross-
contamination in
COMMANDER
and from each
sampling
technique
Determine extent
of cross-
contamination in
COMMANDER
Verify the process
of moving
coupons does not
introduce
contamination
Acceptance Criteria
No detectable spores
No detectable spores
No detectable spores
Corrective Actions
If CPU detected,
discuss potential
impact on results with
EPAWAM. Repeat
test if necessary after
identifying and
removing source of
contamination
Values on test
coupons of the same
order of magnitude will
be considered to have
resulted from cross-
contamination.
Discuss the potential
impact on results with
EPAWAM. Repeat
test if necessary after
identifying and
removing source of
contamination
Determine source of
contamination and
remove
Frequency
1 per sample
per sampling
technique per
test
3 per test
1 per sampling
type
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Sample Type
Laboratory
Materials
Blank Tryptic
Soy Agar
Sterility Control
(plate incubated,
but not
inoculated)
Control
Coupons
(stainless steel
and material)
Inoculation
Reference
Coupons
(stainless steel)
Biological
Samples
Purpose
Verify the sterility
of materials used
to analyze viable
spore count
Controls for
sterility of plates
Used to
determine the
extent of
inoculation on the
coupon.
Used to
determine drift in
the MDI
Controls for
outliers in colony
growth
Acceptance Criteria
No detectable spores
No observed growth
following incubation
1E6CFU, ±0.5 log
The CPU recovered
from the first set of
positive controls must
be within 0.5 log of
the second set of
positive controls
CPU counts between
30-300
Each CPU count
must be within 100%
of the other two
replicates
Corrective Actions
Determine source of
contamination and
remove
All plates are
incubated prior to use,
any contaminated
plates were discarded
Outside target range:
discuss potential
impact with EPA
WAM; correct loading
procedure for next test
and repeat depending
on decided impact
Reject results and
repeat test
Replate or filter plate if
CPU outside criteria
Frequency
1-3 per
material per
test
All plates
8 per test
3 per test
Each sample
4.4 Acceptance Criteria for Critical Measurements
The Data Quality Objectives (DQOs) are used to identify the critical measurements (CM) needed to
address the stated objectives and specify tolerable levels of potential errors associated with simulating
the prescribed fumigation environments. The following measurements were deemed to be critical to
accomplish part or all of the project objectives:
• enumeration of spores recovered from the surface of the coupons and aerosol filters
The Data Quality Indicators (DQIs) listed in Table 4-3 are specific criteria used to quantify how well the
collected data met the DQOs. Failure to provide a measurement method or device that meets these goals
results in the rejection of results derived from the CM. For instance, if the plated volume of a sample is not
known (i.e., is not 100% complete), then that sample is invalid. When originally written, the QAPP
specified the flow rate of the Hi-Vol as critical. However, results in this report are not based on volumetric
terms, but in terms of a 20 minute sample. Therefore, failure to collect flow rate data in real time did not
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invalidate the results as long as the duration of the sample could be determined. All Hi-Vol samplers
passed pre- and post-test calibrations.
Table 4-3. Critical Measurement Acceptance Criteria
Critical
Measurement
Plated Volume
CPU/Plate
Sample time
Measurement
device
Pipette
Manual
counting
Clock
Accuracy
±2%
±1 0% (between
2 counters)
±1 minute per hour
Precision
±1%
±5
NA
Detection
Limit
NA
1 CPU
NA
Completeness
1 00%
1 00%
1 00%
Plated volume critical measurement goals were met. All pipettes are calibrated yearly by an outside
contractor (Calibrate, Inc.) and verified to be within 2% tolerance by gravimetric analysis
Plates were quantitatively analyzed (CPU/plate) using a manual counting method. For each set of results
(per test), a second count was performed (by a second technician) on 25 percent of the plates with
significant data (data found to be between 30-300 CPU). All second counts were found to be within 10
percent of the original count.
There are many QA/QC checks used to validate microbiological measurements. These checks include
samples which demonstrate the ability of the Biocontaminant Laboratory to culture the test organism, as
well as to demonstrate that materials used in this effort do not themselves contain spores. The checks
include:
• Negative control coupons: sterile coupons sampled just as inoculated ones were.
• Field blank samples: sample collection kits taken to the field and transferred as samples were.
• Laboratory material samples: includes all materials, individually, used by the DCMD Biocontaminant
Laboratory in sample analysis
• Positive control coupons: coupons inoculated but not subjected to AAS
• Inoculation control coupons: stainless steel coupons inoculated at beginning, middle, and end of each
inoculation campaign, not subjected to AAS, to assess the stability of the MDI during the inoculation
operation.
The Vaisala RH meters were calibrated weekly and were within the factory specifications during each
AAS operation.
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4.5 Data Quality Audits
This project was assigned QA Category III and did not require technical systems or performance
evaluation audits.
4.6 QA/QC Reporting
QA/QC procedures were performed in accordance with the QAPP for this investigation.
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5 Summary and Recommendations
The results of this study demonstrated that AAS may be a viable option to sample 8. anthracis spores.
The overall test results showed AAS results may vary depending on the contamination characteristics
(e.g., spore size distribution) and environmental conditions (e.g., RH, fumigation, other activities that may
impact the surface condition). When the AAS recovery results were compared as a function of surface
types, AAS recoveries were lowest from the carpet among three tested materials. The laminate surface
showed the highest spore recoveries. The % relative recoveries ranged 0.37% to 5.84% for pre-
fumigation and 0.004% to 1.032% for post-fumigation AAS.
AAS is composed of three major components: surface agitation for particle resuspension, resuspended
particle mixing, and air sampling using filtration. The current test used a commercially available leaf lower
(160 mph) to agitate the test surface. Agitation duration, pattern, and applied pressure should be
improved to increase spore resuspension potential by investigating the surface agitation tool and method.
To fully mix the air within the test chamber, one industrial size fan was used. However, excessive mixing
can result in particle loss to walls while insufficient mixing can increase losses due to gravitational
settling. The number of mixing fans, location, and fan speed should be optimized to reduce the
resuspended particle loss. For air sampling, three Hi-Vol samplers were used in this study to sample in
sequence during pre- and post-fumigation AAS. The first Hi-Vol sampler collected more than 95% of
spores. The second and third samplers showed recoveries from inoculated coupons near or less than that
of background levels. Further, a breakdown of recovery from the impactorand the filter of the high volume
sampler showed that most of the recovery is typically from the filter; however, the recovery of spores on
the impactors suggests some spore clumping or spores traveling on other particles of more than 10 urn in
size (see Table 3-2). Background-adjusted recoveries from the first Hi-Vol sampler (first 20 minutes,
including the occurrence of the leaf blower agitation) were higher than the two subsequent Hi-Vol
samplers (second and third 20 minutes). The recoveries from the subsequent samplers were below
background levels. Post-fumigation comparative recoveries were lower than pre-fumigation comparative
recoveries for three tests (4b, 6a, 6b) by a factor of 10 to 1000. These lower recoveries are the results of
the combination of all treatments on the test samples (e.g., AAS and fumigation). Hi-Vol samplers in the
current study used ~1000 Ipm for 20 min. This sampled volume represented 1 air exchange of
COMMANDER volume. High sampling flow rate (less sampling duration) is recommended to minimize the
loss of spores due to wall impaction and/or gravitational settling. Further the number of air samplers and
location should be optimized by considering the surface and volume of sampling site.
Systematic investigations are needed to assess AAS for targeted sampling approaches. Future studies
should seek to determine the application of AAS to varied dissemination scenarios (e.g., hot-spot, low
levels over wide-area, unconfined spaces, etc.), environmental conditions, and application (e.g.,
characterization or clearance sampling).
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6 References
1. Rogers, J.V., C.L. Sabourin, Y.W. Choi, W.R. Richter, D.C. Rudnicki, K.B. Riggs, M.L. Taylor, and
J. Chang, Decontamination assessment of Bacillus anthracis, Bacillus subtilis, and Geobacillus
stearothermophilus spores on indoor surfaces using a hydrogen peroxide gas generator. J Appl
Microbiol, 2005. 99(4): p. 739-48.
2. Beecher, D.J., Forensic application of microbiological culture analysis to identify mail intentionally
contaminated with Bacillus anthracis spores. Appl Environ Microbiol, 2006. 72(8): p. 5304-10.
3. Weis, C.P., A.J. Intrepido, A.K. Miller, P.G. Cowin, M.A. Durno, J.S. Gebhardt, and R. Bull,
Secondary Aerosolization of Viable Bacillus anthracis Spores in a Contaminated US Senate
Office. JAMA, 2002. 288(22): p. 2853-8.
4. Teshale, E.H., J. Painter, G.A. Burr, P. Mead, S.V. Wright, L.F. Cseh, R. Zabrocki, R. Collins,
K.A. Kelley, J.L. Hadler, D.L. Swerdlow, and T. Connecticut Anthrax Response, Environmental
Sampling for Spores of Bacillus anthracis. Emerging Infectious Diseases, 2002. 8(10): p. 1083-
1087.
5. Raber, E., A. Jin, K. Noonan, R. McGuire, and R.D. Kirvel, Decontamination Issues for Chemical
and Biological Warfare Agents: How Clean is Clean Enough? International Journal of
Environmental Health Research, 2001.11(2): p. 128-148.
6. Schmitt, K. and N.A. Zacchia, Total Decontamination Cost of the anthrax Letter Attacks.
Biosecurity and bioterrorism : biodefense strategy, practice, and science, 2012.10(1): p. 98-107.
7. EPA, Bio-Response Operational Testing and Evaluation (BOTE) Test Plan: Attachment D -
Aggressive Air Sampling Standard Operating Procedure. 2011.
8. 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. Applied and Environmental Microbiology,
2007.73(3): p. 706-710.
9. Brown, G.S., R.G. Betty, J.E. Brockmann, D.A. Lucero, C.A. Souza, K.S. Walsh, R.M. Boucher,
M.S. Tezak, M.C. Wilson, T. Rudolph, H.D.A. Lindquist, and K.F. Martinez, Evaluation of Rayon
Swab Surface Sample Collection Method for Bacillus Spores from Nonporous Surfaces. Journal
of Applied Microbiology, 2007. 103(4): p. 1074-1080.
10. Calfee, M.W., S.D. Lee, and S.P. Ryan, A rapid and repeatable method to deposit bioaerosols on
material surfaces. J Microbiol Methods, 2013.
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Appendix A: Miscellaneous Operating Procedures
MOP 3135 Procedure for Sample Collection using BactiSwab™ Collection and Transport Systems
MOP 3141A Procedure for Assembling Irradiated Vacuum Sock Sampling Kits
MOP 3145 Procedure for HEPA Vacuum Sampling of Large and Small Coupons
MOP3150-AII Procedure for Fabrication of35.56 cm x 35.56 cm, 71 cmx71 cm, and 107 cm x 107 cm
Material Coupons
MOP 3161-HD Aerosol Deposition of Spores onto Material Coupon Surfaces using the Aerosol
Deposition Apparatus (ADA) - High Dosing
MOP 3165 Sponge Sample Collection Protocol
MOP 3166 Aerosolization of Contaminated Coupons Using Toro Power Sweep Electric Blower for
Aggressive Air Sampling (AAS)
MOP 3168 Aggressive Air Sampling (AAS) for WA 3-28: Phase I Sampling Approach
MOP 3169 Sponge Sample Collection Protocol for AAS Impactors
MOP 6535a Serial Dilution: Spread Plate Procedure to Quantify Viable Bacterial Spore
MOP 6555: Petri Dish Media Inoculation Using Beads
MOP 6562 Preparing Pre-Measured Tubes with Aliquots Amounts of Phosphate Buffered Saline
with Tween 20 (PBST)
MOP 6563 Swab Streak Sampling and Analysis
MOP 6565 Filtration and Plating of Bacteria from Liquid Extracts
MOP 6572 Recovery of Spores from Vacuum Sock Samples
MOP 6580 Recovery of Bacillus Spores from 3M Sponge-Stick™ Samples
MOP 6586 Recovery of Bacillus Spores from Quartz Filters
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United States
Environmental Protection
Agency
PRESORTED STANDARD
POSTAGE & FEES PAID
EPA
PERMIT NO. G-35
Office of Research and Development (8101R)
Washington, DC 20460
Official Business
Penalty for Private Use
$300
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