EPA 600/R-14/226 | August 2014 | www.epa.gov/research
United States
Environmental Protection
Agency
Interactions of CI02 and H202
Fumigants with Dirt and Grime on
Subway Concrete
ASSESSMENT AND EVALUATION REPORT
Office of Research and Development
National Homeland Security Research Center
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August 2014
EPA/600/R-14/226
Interactions of CIO2 and H2O2 Fumigants
with Dirt and Grime on Subway Concrete
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, through its Office of Research and Development's
National Homeland Security Research Center, funded and managed this investigation under EPA
Contract Number EP-C-09-027, Work Assignment 4-12 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:
Lukas Oudejans, 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:919-541-2973
Fax:919-541-0496
E-mail: Oudeians.Lukas@epa.gov
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Acknowledgments
The following individuals and organizations are acknowledged for review of this document:
United States Environmental Protection Agency:
Office of Solid Waste and Emergency Response, Office of Emergency
Management
Larry Kaelin
Shannon Serre (on detail from ORD-NHSRC)
Office of Research and Development, National Homeland Security Research
Center
Joe Wood
Ramona Sherman (QA review)
The support and efforts provided by ARCADIS U.S., Inc., are gratefully acknowledged.
IV
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Table of Contents
Disclaimer iii
Acknowledgments iv
Table of Contents v
List of Figures vii
List of Tables viii
List of Acronyms and Abbreviations ix
Executive Summary xi
1 Introduction 1
1.1 Project Objectives 1
1.2 General Approach 2
1.2.1 Definitions of Effectiveness 2
2 Materials and Methods 5
2.1 Fumigation Chamber 5
2.2 Kinetics Chamber 7
2.2.1 Air Exchange System 8
2.3 Coupon Preparation 8
2.3.1 Coupon Cleaning 8
2.4 Method Development I - Material Inoculation 9
2.4.1 Aerosol Deposition Method Modification 9
2.5 Method Demonstration II - Surface Sampling 11
2.6 Experimental Approach 11
2.6.1 Fumigation Tests 11
2.6.2 Material Demand Tests 12
2.7 Sampling and Analytical Procedures 16
2.7.1 Sampling Strategy 16
2.7.2 Sampling Points 17
2.7.3 Sampling Frequency 17
2.7.4 Statistical Approach 19
2.7.5 Sampling Procedures 20
2.8 Sample Handling and Custody 21
2.8.1 Preventing Cross-Contamination during Sampling 21
2.8.2 Preventing Cross-Contamination during Analysis 22
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2.8.3 Representative Samples 22
2.8.4 Sample Quantities 22
2.8.5 Sample Containers for Collection, Transport, and Storage 22
2.8.6 Sample Preservation 22
2.8.7 Sample Archiving 23
3 Results and Discussion 24
3.1 Method Development I 24
3.2 Method Development II 24
3.3 CIO2 Fumigations 25
3.4 H2O2 Fumigations 28
3.5 Material Demand of CIO2 31
4 Quality Assurance 34
4.1 Sampling, Monitoring, and Analysis Equipment Calibration 34
4.2 Data Quality 35
4.3 QA/QC Checks 35
4.4 Acceptance Criteria for Critical Measurements 37
4.5 Data Quality Audits 39
4.6 QA/QC Reporting 39
5 Summary and Recommendations 40
6 References 41
VI
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List of Figures
Figure 2-1. Block Diagram of the CIO2 Fumigation System 6
Figure 2-2. Schematic of the Kinetic Facility 7
Figure 2-3. Cutaway View of Kinetics Chamber 8
Figure 2-4. Round ADA schematic 10
Figure 2-5. Round ADA with O-ring gasket 10
Figure 2-6. Illustration of CIO2 Breakdown during a Decontamination Event 13
Figure 3-1. Recoveries of Various Concrete Sampling Methods 25
Figure 3-2. Average CFU Recovered from Concrete following Exposure to 1500 ppmvCIO2 26
Figure 3-3. Log Reduction of Spores Recovered from Concrete following 1500 ppmvCIO2 27
Figure 3-4. Average CFU Recovered from Concrete following Exposure to 500 ppmvCIO2 28
Figure 3-5. Average CFU recovered from Concrete following Exposure to 250 ppmv VPHP 29
Figure 3-6. Average Recovery from Coupons after Exposure to 150 ppmv VPHP 30
Figure 3-7. Average LR from Coupons after Exposure to 150 ppmv VPHP 31
Figure 3-8. Material Demand Test at 250 ppm CIO2 32
Figure 3-9. Steady-state Reaction Rates of Kinetics Chamber with and without Concrete
Samples 32
Figure 3-10. Concrete Adsorption as a Function of Concentration 33
VII
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List of Tables
Table 2-1. Fumigation Test Matrix 12
Table 2-2. Kinetics Test Matrix 14
Table 2-3. Sample Frequency 17
Table 2-4. Critical and Non-Critical Measurements 18
Table 2-5. Sample Frequency for Material Demand Tests 19
Table 2-6. Critical and Non-Critical Measurements for Material Demand Task 19
Table 3-1. Recovery from Stainless Steel Coupons following Prototype Deposition Method 24
Table 3-2. Recovery of Various Concrete Sampling Methods 24
Table 3-3. Fumigation Test Matrix 26
Table 3-4. H2O2 Fumigation Test Matrix 29
Table 3-5. Average LR of Washed and Unwashed Concrete 30
Table 4-1. Sampling and Monitoring Equipment Calibration Requirements 34
Table 4-2. Analysis Equipment Calibration Frequency 35
Table 4-3. QA/QC Sample Acceptance Criteria 36
Table 4-4. Critical Measurement Acceptance Criteria 38
VIM
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List of Acronyms and Abbreviations
ADA
APPCD
ATCC
ATI
B.
CBR
CBRN
CPU
CI02
CMAD
COC
CT
DAS
DHS
DQO
EMS
EPA
EtO
H202
HSPD
HSRP
ID
LR
MDI
MIT
mSM
MTA
NA
NHSRC
NIST
OPP
ORD
OSWER
pAB
PBST
ppm
ppmv
PRB
aerosol deposition apparatus
Air Pollution Prevention and Control Division
American Type Culture Collection
Analytical Technologies, Inc.
Bacillus
chemical, biological, or radiological
Chemical, Biological, Radiological, and Nuclear
Colony Forming Unit(s)
chlorine dioxide
Consequence Management Advisory Division
chain of custody
concentration x time
data acquisition system
Department of Homeland Security
Data Quality Objective
Environmental Monitoring System
U. S. Environmental Protection Agency
ethylene oxide
hydrogen peroxide
Homeland Security Presidential Directive
Homeland Security Research Program
identification
log reduction
metered dose inhaler
Massachusetts Institute of Technology
modified Standard Method
Metropolitan Transportation Authority
not applicable
National Homeland Security Research Center
National Institute of Standards and Technology
Office of Pesticide Programs
Office of Research and Development
Office of Solid Waste and Emergency Response
pH-adjusted bleach
Phosphate Buffered Saline with 0.05 % TWEEN®20
part(s) per million
part(s) per million volume
Polyester-Rayon Blend
IX
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PTFE Polytetrafluoroethylene
QA Quality Assurance
QAPP Quality Assurance Project Plan
QC Quality Control
RH relative humidity
RSD relative standard deviation
RTP Research Triangle Park
SD standard deviation
SOP standard operating procedure
VHP® (STERIS-registered) Vaporized Hydrogen Peroxide
VPHP Vapor-phase Hydrogen Peroxide
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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) to conduct research
and develop scientific products that improve the capability of EPA to carry out its homeland security
responsibilities. Improving the capability for transit systems to rapidly recover from a biological event has
been identified as a high priority need. The remediation of a transportation hub, like a subway system,
may require the use of volumetric decontamination approaches, such as fumigation with chlorine dioxide
(CIO2) or hydrogen peroxide (H2O2).While previous National Homeland Security Research Center
(NHSRC) studies have shown these fumigants to be highly efficacious if applied under specific
environmental conditions (temperature and relative humidity (RH)), it is unclear what the impact of dirt
and grime is on the efficacy of these fumigants on realistic (subway) building materials. The presence of
dirt and grime may result in a change in sporicidal activity of the fumigant and may therefore require
changes in operational fumigation conditions to reach remediation goals. The primary objective of this
research was to evaluate the impact that dirt and grime, as present on unpainted subway concrete, may
have on the fumigation efficacy.
The impact of dirt and grime on concrete surfaces was also investigated as part of a material demand
study. Building materials like concrete may impact the fumigant concentration by either sorption or
decomposition of the decontaminant. Consequently, higher input fumigant concentrations would be
required to achieve and maintain the targeted effective concentration within an enclosed interior space.
Previous NHSRC material demand efforts for CIO2 and vaporized hydrogen peroxide (VHP®) used clean
(concrete) surfaces. Dirt and grime may increase material demand when present on a material associated
with low or no demand while material demand may possibly decrease if dirt and grime forms a protective
layer on a material associated with higher demand.
Other objectives include determining which sampling procedure provides better recovery from grimed and
cleaned concrete using a prescribed method from the New York City Metropolitan Transportation
Authority (MTA). A method was developed using metered dose inhalers (MDIs) to inoculate 1.5" coupons
of subway concrete. Three surface sampling methods (sponge wipe, cloth wipe, and vacuum sock) were
tested. All three methods showed a recovery comparable to the recovery from stainless steel coupons,
but the sponge wipe method had higher and more repeatable recovery.
Fumigation results for subway concrete using CIO2 were found to be in agreement with fumigation data
available for unpainted cinder block. Here, greater than 6-log reductions in Bacillus spores were observed
for 1500 ppmv CIO2, 75% RH and > 4 hours (h) contact time or 500 ppmv CIO2, 75% RH and > 6 h
contact time (shortest contact time tested). Though this investigation suggests that fumigation of washed
subway concrete can result in different efficacy values than fumigation of the unwashed subway concrete,
the differences are statistically not significant and do not suggest that the presence of grime on concrete
would affect fumigation efficacy.
Fumigation results for subway concrete using VPHP cannot be immediately compared to clean concrete
data as available VPHP fumigation data for concrete are limited to painted concrete. A greater than 6-log
reduction in Bacillus spores on subway concrete was observed for 250 ppm H2O2 (as generated using
Steris VHP® technology), 20% RH and a > 4 h contact time. The 6-log reduction in spores was not
reached at the longest contact time (10 h) at the 150 ppmv H2O2 concentration. Observed differences in
XI
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log reduction between washed and unwashed subway concrete following VHP fumigation were not
statistically significant.
The grimed subway concrete had no detectable material demand when using the CIO2 fumigant. This is
consistent with previously obtained data for clean unpainted concrete cinder block. Material demand
studies using the subway concrete were not conducted for VHP® based on limited availability of subway
concrete material. In addition, previous VHP® material demand tests showed that the presence of
concrete cinder block coupons had a large impact on maintaining the VHP® concentration due to
decomposition of VHP®. In the presence of concrete cinder block, a high (2x) increase in generator output
was required to maintain the target concentration due to degradation of the hydrogen peroxide at the
surface.
Reported results presented here were obtained from a small scale study with a limited amount of subway
concrete (surface) available. Extrapolation of these results to a full scale subway station fumigation
process should be made with caution. Additional fumigation testing on an intermediate or large scale
would assist in such extrapolation of results. Levels and composition of dirt and grime may also vary
significantly throughout a subway (tunnel) system.
XII
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1 Introduction
This project supports the mission of the U.S. Environmental Protection Agency's (EPA) Homeland
Security Research Program (HSRP) by providing relevant information pertinent to the decontamination of
contaminated areas resulting from an act of terrorism. Under Homeland Security Presidential Directive
(HSPD)-10, the U.S. Department of Homeland Security (DHS) is tasked to coordinate with other
appropriate Federal departments and agencies to develop comprehensive plans that "provide for
seamless, coordinated Federal, state, local, and international responses to a biological attack." As part of
these plans, EPA, in a coordinated effort with DHS, is responsible for "developing strategies, guidelines,
and plans for decontamination of persons, equipment, and facilities" to mitigate the risks of contamination
following a biological weapons attack.
EPA's National Homeland Security Research Center (NHSRC) provides expertise and products under the
HSRP that can be used widely to prepare for, respond to, and recover from public health and
environmental emergencies arising from terrorist threats and incidents. The HSRP's research on
biological agent decontamination supports EPA's Office of Solid Waste and Emergency Response
(OSWER) and the Office of Pesticide Programs (OPP). OSWER and its Special Teams, which include the
Chemical, Biological, Radiological, and Nuclear (CBRN) Consequence Management Advisory Division
(CMAD), support the emergency response functions carried out by the Regional Offices. The OPP
supports the decontamination effort by providing expertise on (biological) agent inactivation and ensuring
that the use of pesticides in such efforts is done in accordance with applicable laws. Close collaboration
between the different program offices having homeland security responsibilities is sought to rapidly
increase EPA's capabilities to help the Nation recover from a terrorist event involving the intentional
release of chemical, biological, or radiological (CBR) materials.
In 2001, the introduction of a few letters containing Bacillus anthracis spores into the U.S. Postal Service
system resulted in the contamination of several facilities. In the event of a biological incident in a
transportation hub like a subway system, remediation may require the use of various remediation options
including volumetric decontamination approaches such as fumigation as an effective decontamination
method. Rapid decontamination of subways and other transportation infrastructure is not only critical for
the reoccupancy of the contaminated area but also for the surrounding areas that use the transit system.
Previous NHSRC studies have shown that fumigants like chlorine dioxide (CIO2) and hydrogen peroxide
(H2O2) vapors can be highly efficacious if applied under specific application (temperature and relative
humidity (RH)) conditions. It is, however, unclear what the impact is of dirt and grime on realistic building
materials on the fumigation efficacy. Such presence may result in change in sporicidal activity of the
fumigant and may also require changes in operational fumigation conditions to reach remediation goals
due to changes in material demand.
1.1 Project Objectives
The primary objective was to evaluate the impact that dirt and grime, as present on unpainted subway
concrete, has on CIO2 and VPHP fumigation efficacy. Secondary objectives were (1) to identify which
sampling procedure provided a better recovery of 8. anthracis (surrogate) spores from grimed and
cleaned concrete, and (2) to measure any gross material demand presented by the presence of dirt and
grime on concrete.
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To meet the project objectives, this study was comprised of the following four tasks:
1. Modification of the aerosol deposition method. An aerosol deposition method described by Lee et
al1. and Calfee et al.2 was designed for inoculation of a 12" x 12"
method was modified to deposit on 1.5" x 1.5" concrete coupons.
al1. and Calfee et al.2 was designed for inoculation of a 12" x 12" square. This aerosol deposition
2. Determination of a surface sampling method using the modified aerosol deposition procedure.
Coupons were sampled using three techniques (sponge wipe, wetted wipe, and 37-mm vacuum
filter) to determine one suitable sampling method for use in decontamination tests.
3. Decontamination tests of subway concrete coupons by fumigation followed by sampling to
determine fumigation efficacy.
4. Material demand tests for CIO2 fumigant (only).
1.2 General Approach
The general process investigated in this project was the decontamination of unpainted concrete surfaces
contaminated with Bacillus spores (i.e., surrogates of 8. anthracis). Decontamination can be defined as
the process of inactivating or reducing a contaminant in or on humans, animals, plants, food, water, soil,
air, areas, or items through physical, chemical, or other methods to meet a cleanup goal.
For this effort, decontamination methods included fumigation with CIO2and Vapor-phase Hydrogen
Peroxide (VPHP). Concrete from the floor of the New York Metropolitan Transit Authority (MTA) Old
South Ferry Station was made available for this research. Sections (coupons) of this subway concrete
were inoculated via aerosol deposition. Some coupons underwent fumigation, and recovery of Bacillus
spores from fumigated coupons was compared to recovery from coupons that were inoculated but not
fumigated (positive control coupons). Quality Control (QC) samples such as procedural blank coupons
(coupons that undergo the fumigation process but that are not inoculated) and negative controls (coupons
that do not undergo the fumigation process and are not inoculated) were also included to monitor for
cross-contamination. All samples were analyzed for the quantitative determination of viable spores.
Each of the described 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.
1.2.1 Definitions of Effectiveness
Quantification of Colony Forming Unit (CFU) counts per coupon occurred as a calculated product of the
average counted number of CFU and extraction volume (ml) and divided by the product of plated volume
(ml) and tube dilution factor. Efficacy is defined as the extent (by log reduction) to which the agent
extracted from the coupons after the treatment with the decontamination procedure is reduced below the
agent extracted from positive control areas (not exposed to the decontamination procedure). Efficacy (as
the log reduction [LR]) was calculated using Equation 1-1 for each material within each combination of
decontamination procedure (i) and test material (j) as:
c=l k=l
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where:
L/J\i
= the average log reduction of spores on a specific material surface
the average of the logarithm of the number of viable spores
°&\^r (J Cj' l^lc = (determined by CPU) recovered on the control coupons [c = control,
j = coupon number, and Nc is the number of coupons (1 , /)]
the average of the logarithm of the number of viable spores
V \og((^p7Tf ) / A/" _ (determined by CPU) recovered from the surface of a
k decontaminated coupon [S= decontaminated coupon, k = coupon
number, and Nt is the number of coupons tested (1 , k)]
When no viable spores were detected, the detection limit of the sample was used, and the efficacy
reported as greater than or equal to the value calculated by Eqn. 1-1. The detection limit of a sample
depends on the analysis method and so may vary. The detection limit of a plate is assigned a value of 1
CPU, but the fraction of the sample plated varies. For instance, the detection limit of a 0.1 ml plating of a
20 ml sample suspension is 200 CPU (1 CFU/0.1 ml * 20 ml), but if all 20 ml of the sample were filter
plated, the detection limit would be 1 CPU.
The standard deviation of LRj is calculated by Eqn. 1-2:
where:
= Standard deviation of LRj
the average log reduction of spores on a specific material
surface
The average of the log reduction of each from the surface of a
decontaminated coupon (Equation 1-3)
^ = -J—^ T7 (1 -3)
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where:
Represents the "mean of the logs", the average of the
)/ A/" - l°9arithm transformed number of viable spores (determined by
1JC 'JC CPU) recovered on the control coupons [C= control, j =
coupon number, and Nc is the number of coupons (1, j)]
Number of CPU on the surface of the kth decontaminated
=
coupon
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2 Materials and Methods
2.1 Fumigation Chamber
An opaque chamber (Plas-Labs 830 series glove box, Plas-Labs, Inc., Lansing, Ml, USA) maintained and
controlled a leak-free fumigation atmosphere and allowed for the periodic addition and removal of
coupons during fumigation. Chlorine dioxide was provided by a ClorDiSys "GMP" CIO2 generator
(ClorDiSys Solutions, Inc., Lebanon, NJ, USA). The generator includes real-time feedback control of
concentration via an internal Environmental Monitoring System (EMS) photometric monitor. VPHP was
provided by a STERIS Vaporized Hydrogen Peroxide (VHP)® 1000 ED (STERIS Corp., Mentor, OH,
USA). The VHP® 1000 ED was connected through a custom-designed control system using a feedback
loop from a data acquisition system (DAS).
Humidity of the chamber was controlled by the DAS. A model HMD53 Vaisala RH/temperature sensor
(Vaisala, Inc., Helsinki, Finland) provided a signal used in a feedback loop. When the Vaisala RH sensor
read lower than the RH setpoint, solenoid valves opened to inject humid air from a gas humidity bottle
(model LF-HBA with Nafion® tubing (Fuel Cell Technologies, Inc., Albuquerque, NM, USA). The gas
humidity bottle, heated to 140 °F, passed compressed air through Nafion® tubes surrounded by de-
ionized water, creating a warm air stream saturated with water vapor. Temperature was controlled by
circulation of water through radiators. Figure 2-1 shows the schematic of the CIO2 fumigation system that
was used for these efficacy tests. A similar system was used for the VPHP fumigations by replacing CIO2
systems with VPHP analogs.
Modified Standard Method (mSM) 4500-CIO2-B samples were taken every 60 minutes to confirm the
concentration of CIO2 in the test chamber. VPHP concentrations were also verified using wet chemistry
methods. Multiple fans were used inside the chamber to provide internal mixing. Pressure relief valves
and check valves prevented over-pressurization of the chamber. A room monitor alarmed if there was an
accidental release of fumigant. All fumigation gas was directed through a sorbent trap before release into
a fume hood.
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EMS CIO2
concentration
T
CIO? Generator
Digital signal line
Digital control line
Heated tubing for gas flow
Cooling water line
Figure 2-1. Block Diagram of the CIO2 Fumigation System
The kinetics facility consisted of two chambers connected in series with all the ancillary equipment
required for successful estimation of the material demand. The first chamber was used as a constant-
concentration feed reservoir to the second kinetic chamber in which all the experiments were performed.
A schematic of the kinetic facility is shown in Figure 2-2. The main components added to the existing
laboratory setup are described in the following sections.
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Steam from Gas
Humidity Bottle
CIO2 Flow - heated sample lines
Unheated tubing
Control lines
RH/Temperature probe
Temp.
1
ClorDiSys CIO2
generator (GMP)
CIO2 Monitor, RH
and Temp.,
Pressure
I
Figure 2-2. Schematic of the Kinetic Facility
2.2 Kinetics Chamber
The kinetics chamber (Figure 2-3) is a stainless steel enclosure with an internal volume of 119 liters,
designed for RH and temperature control, and including various ports as shown in Figure 2-2. A fan was
installed to aid with internal mixing.
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Figure 2-3. Cutaway View of Kinetics Chamber
2.2.1 A ir Exchange System
The air exchange was attained by removing air at a set rate from the kinetics chamber. The makeup air
came from either the constant concentration glove box (in the case of the exposure and steady-state
phases) or from ambient laboratory air (for all other phases). The air exchange rate was constant
throughout the entire experiment, set at one exchange per hour to mimic regular air exchanges in an
occupied facilities.
2.3 Coupon Preparation
Chunks of subway concrete from the floor of the New York Metropolitan Transit Authority (MTA) Old
South Ferry Station were provided to EPA by Department of Homeland Security (DHS) Science and
Technology Division via Massachusetts Institute of Technology Lincoln Laboratories (MIT-LL). These
chunks were covered in grit, probably from the deconstruction process. The chunks were divided, using
dry mechanical methods, into as many coupons as possible that contained a minimum 1.5" x 1.5" square.
No information was available on the type of concrete and its age other than that this subway station was
established in the early 1900's.
2.3.1 Coupon Cleaning
A subset of the subway concrete coupons was cleaned using a method adapted from the New York MTA.
This was done to assess whether fumigation of a cleaned concrete surface would result in differences in
log reduction of spores when compared to fumigation results obtained with subway concrete "as
received". The cleaning solution consisted of Tide® Institutional Formula Floor & All-Purpose Cleaner (4.2
g) mixed in 1.5 liters of hot water to create a 0.28 % solution by weight.
The cleaning procedure consisted of the following seven steps:
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1. Place concrete pieces flat in a sink with top surface facing up.
2. Spray with 0.28 % Tide® solution using foaming spray applicator (Trigger Sprayer, Grainger Item
# 3U603 on 32 Ounce Spray Bottle, Grainger Item # 3U593).
3. Scrub lightly with 1.5" soft pure-bristle paintbrush.
4. Rinse each piece well under flowing tap water.
5. Stand pieces on edge on a paper towel.
6. Blow dry with dry nitrogen to remove surface water.
7. Let air dry for 24 hours.
A 1.5" square was outlined using a permanent marker (e.g., black or silver Sharpie®) on each coupon
before inoculation. This square was used to align the aerosol deposition apparatus (ADA) (see Section
2.4) and served to frame the sampling area.
Additionally, after a coupon was dosed via the procedure detailed in Section 2.4.1.2, the coupon was
labeled with the unique identifier. The identification (ID) was written on the side of the coupon using a
permanent marker. The stainless steel coupons were pre-labeled on the underside (non-contaminated)
side using a black Sharpie®.
Concrete coupons were sterilized with EtO before use by placing coupons in an Andersen EOGas 333
Cabinet (Andersen Products Inc., Haw River, NC, USA) which was set at 50 °C. Typically, an 11 g
cartridge of EtO was released into a 22" x 36" diffusion bag containing the items for sterilization. The
contents underwent an 18-hour exposure and degassing cycle.
2.4 Method Development I - Material Inoculation
Coupons were inoculated (loaded) with spores of B. atrophaeus Cformerly B. globigii) (American Type
Culture Collection (ATCC) 9372) from a metered dose inhaler (MDI).1 Method development was required
to reach the targeted deposition of 1 x 106 CPU on a 1.5-inch diameter portion of the concrete surface.
Stainless steel coupons were used to quantify CPU for this effort. Coupons were sterilized with ethylene
oxide (EtO) before use.
2.4.1 Aerosol Deposition Method Modification
The aerosol deposition method described by Lee et al1. and Calfee et al.2 was modified for targeting a
deposition of 1 x 106 CPU on a 1.5-inch diameter portion of the concrete surface. Stainless steel coupons
were used to quantify CPU for this effort. Stainless steel coupons were autoclaved before use and five
coupons were used for each effort. A successful method was required to deliver an average of at least 1 x
106 CPU and have a precision of ±0.5 log to have the ability to demonstrate a 6-log reduction in viable
spores. These coupons were sampled with sponge wipes according to internal operating procedures.
2.4.1.1 Bacillus Spore Preparation
The test organism for this work was a powdered spore preparation of Bacillus atrophaeus (formerly 8.
globigii) (American Type Culture Collection (ATCC) 9372) and silicon dioxide particles. A preparation
resulting in a powdered matrix containing approximately 1 x 1011 viable spores per gram was prepared by
dry blending and jet milling the dried spores with fumed silica particles (Deguss, Frankfurt am Main,
Germany). The powdered preparation was loaded into MDIs (Cirrus, Morrisville, NC, USA) and sealed
after addition of propellant. Control checks for each MDI as described in Section 2.4.1.2 were included in
the batches of coupons contaminated with a single MDI.
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2.4.1.2 Coupon Inoculation
Coupons were inoculated (loaded) with spores of 8. atrophaeus (formerly 8. globigii) from an MDI. In
brief, the inoculation procedure involved placing a round ADA on the top surface of the coupon facing
upwards for inoculation (Figures 2-4 and 2-5). The ADA was clamped to the coupon and the MDI was
attached to the top of the ADA. A slide was opened, and the MDI was activated. Following inoculation, the
slide was closed and the MDI was removed. The spores were allowed to settle for at least 18 hours. This
procedure was repeated for each coupon. Inoculation was done on a laboratory bench with coupons
placed in a bed of sand to keep the irregular shaped coupons upright.
Hose Barb for Vent
Figure 2-4. Round ADA schematic
Figure 2-5. Round ADA with O-ring gasket
10
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Each MDI provides 150 discharges before degradation of concentration. The number of discharges per
MDI was tracked to ensure that use did not exceed this value. Additionally, in accordance with internal
operating procedures, 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, the MDI
was retired and a new MDI was used. For quality control of the MDIs, pairs of positive control coupons
and stainless MDI reference 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.
A log was maintained for each set of coupons that were dosed via this method. Each record in this log
recorded 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).
After inoculation, the coupons remained undisturbed for 18 hours to allow the spores to settle, and then
the coupons were aseptically transferred to sterilized coupon holders or bins for storage before use.
2.5 Method Demonstration II - Surface Sampling
The modified aerosol deposition method described in Section 2.4 was used to inoculate concrete
coupons. Initial sampling methods included sponge wipe,34 37-mm vacuum filter,5 and wetted wipe
sampling.6 Nine replicate coupons were inoculated. A successful sampling method would deliver at least
1 x 106 CPU and have a precision of ±0.5 log. Inoculation at this level allowed to observe a 6-log
reduction in viable spores. Sampling methods were all conducted as per internal operating procedures.
Ease of use and cost were factors in determining the method used for sampling. The NHSRC Research
Triangle Park (RTP) Biocontaminant Laboratory (hereafter referred to as the NHSRC Biocontaminant
Laboratory) quantified the number of viable spores per sample. Following extraction, the resulting
samples were plated in triplicate and CPU were enumerated. One method, sponge wipe, was selected for
further use as part of the actual decontamination testing.
2.6 Experimental Approach
2.6.1 Fumigation Tests
The modified deposition and sampling methods described in Sections 2.4 and 2.5, respectively, were
used to inoculate and sample concrete coupons. Four fumigation scenarios using two fumigation
techniques were used to decontaminate triplicate coupons of two types, cleaned and not cleaned. Log
reduction was calculated by comparing recovery from fumigated coupons to recovery from coupons that
were inoculated but not fumigated (positive controls). The goal was to provide a 6-log reduction in CPU -
a benchmark for determining efficacy of a decontamination procedure - under fumigation conditions
obtainable in the field. The test matrix is shown in Table 2-1.
11
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Table 2-1. Fumigation Test Matrix
Test ID
1
2
3
4
Fumigant
CI02
CIO2
VPHP
VPHP
Concentration
1 500 ppmv*
500 ppmv
250 ppmv
1 50 ppmv
Exposure
Times (hours)
0, 2, 4, and 6
0,6, 12, and 18
0,1,2, and 4
0,4, 7, and 10
Other
Conditions
75 % RH, 24 °C
75 % RH, 24 °C
< 80 % RH
< 80 % RH
* parts per million by volume
Testing was conducted in a glove box and proceeded as follows:
1. Sterilization of all coupons for the test. Coupons were sterilized using EtO. The sterility of the
coupons was verified through the use and sampling of laboratory blank control samples as part of
each test condition (not fumigated).
2. Inoculation of test and positive control coupons with the procedure developed in Section 2.4.
3. Three (3) test coupons per time point and coupon type and one blank coupon per coupon type
(negative controls) were loaded into the glove box.
4. The fumigation with CIO2 was performed using the ClorDiSys GMP and according to the parameters
shown in Table 2-1. The fumigation with H2O2 vapor (VPHP) was performed using the STERIS VHP®
1000ED and according to the parameters shown in Table 2-1.
5. After the exposure time was reached, the coupons were transferred to the airlock, where they were
aerated for 10 min before removal.
6. Coupons were sampled immediately after removal from the airlock. Samples were transferred to the
on-site NHSRC Biocontaminant Laboratory in sterile primary independent packaging within sterile
secondary containment containing logical groups of samples. All samples were accompanied by a
completed chain of custody (COC) form.
In addition to the steps outlined above, all test activities were fully documented during the activity via
narratives in laboratory notebooks, the use of digital photography, and video. The documentation could
also include items such as a record of time required for each decontamination step or procedure, any
deviations from the test plans, and physical impacts on the materials, among others.
2.6.2 Material Demand Tests
Material demand studies were expected to be conducted for both fumigants. The limited availability of
subway concrete coupons did not allow for material demand tests with H2O2 fumigant. The decision to
only study the CIO2 material demand was based on previous studies 7 8 that identified the significant
material demand of VHP® in the presence of unpainted cinder concrete material. The additional dirt and
grime on the concrete surface was not expected to change this high material demand.
The impact of fumigant target concentration on homogeneous and heterogeneous decomposition rates of
CIO2 was assessed during the exposure and aeration phases of a decontamination event. The CIO2
uptake by the subway concrete was quantified and compared to the homogeneous decomposition of CIO2
under the same operating conditions. The kinetics chamber was carefully leak checked to avoid
12
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misinterpreting CIO2 leaks as degradation. If the kinetics chamber did not maintain a vacuum pressure of
2" of water for one minute, corrective actions were taken.
The expected demand of CIO2 is represented schematically in Figure 2-6 for homogeneous and
heterogeneous exposures.
3500
3000
Constant Injection
Homogeneous Mixing
Heterogeneous Mixing
Adsorption and reaction phase |
Pre-conditioning phase
0 60 120 180 240 300 360 420 480 540 600
Run Time (min)
Figure 2-6. Illustration of CIO2 Breakdown during a Decontamination Event
The overall experimental approach consists of different phases defined as follows:
• Pre-conditioning phase: During this phase, the kinetics chamber was ramped from ambient
temperature and RH conditions to the pre-determined temperature and RH conditions for the
test.
• Conditioning phase: The kinetics chamber and its contents were maintained at constant
temperature and RH before exposure to CIO2.The conditioning phase did not last more than
one minute for this effort.
• Adsorption and reaction phase (Exposure phase): The introduction of CIO2 into the kinetic
chamber began. The concentration of CIO2 climbed from zero to a steady-state value.
• Reaction Only Phase (Steady-state): The concentration of CIO2 reached a steady-state
maximum. This phase was defined by a change of less than 5 % in the CIO2 concentration over
a period of one hour.
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• Aeration Phase: This phase began when the Reaction Only Phase was completed. Ambient air
began entering the glove box, reducing the CIO2 concentration from its state-state value. The
aeration phase ended when the concentration in the glove box first fell to below 15 ppm.
• Late Aeration Phase: This phase begins at the end of the Aeration phase and lasts for 12
hours.
The run times and demand rates for each phase shown in Figure 2-6 are presented just for illustration
purposes.
During all phases, the air exchange rate was maintained at a constant value. The only change was in the
source of the makeup air, either ambient air or the constant concentration of CIO2 at constant temperature
and RH from the glove box. No microbiological samples were collected during this task.
2.6.2.1 CIO2 Demand in an Homogeneous Environment
The homogeneous test matrix (Tests 1 - 3 in Table 2-2) was designed to determine the extent of the
kinetics chamber on the decomposition of CIO2.
Table 2-2. Kinetics Test Matrix
Test
Number
1
2
3
4
5
6
Inlet
Concentration
(ppm)
250
500
1500
250
500
1500
RH (%)
75
75
75
75
75
75
Temperature
(°F)
75
75
75
75
75
75
Minimum
Exposure
Time
(hours)
36
18
6
36
18
6
Test
Material
None
Concrete
'As Is'
Surface area of
coupons (in2)
0
170
Each of the above phases, from pre-conditioning to late aeration, was followed in order. Continuous
emission monitoring of CIO2 concentration, RH, and temperature was performed during all phases of the
simulated decontamination event. Extractive samples were also taken during conditioning of the chamber,
charging of the chamber, and aeration of the chamber. These samples were analyzed using mSM 4500-
CIO2-B.
2.6.2.2 CIO2 Demand in the Heterogeneous Environment
The heterogeneous test matrix (Tests 4-6 in Table 2-2) was designed to determine reaction rates of CIO2
in the presence of grimed subway concrete. Operation of the glove box proceeded as in homogeneous
tests; however, the kinetics chamber contained subway concrete of known surface area. Surface area of
the coupons was estimated by wrapping the coupons in a single layer of foil and gravimetrically
determining the area of the foil used.
14
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Modified SM 4500-CIO2-B samples were taken every 60 minutes to confirm the concentration of CIO2 in
the test chamber. Pressure relief valves and check valves prevented over-pressurization of the chamber.
The approach to determination of reaction and absorbance values for heterogeneous kinetic tests is
described below.
1. For each experiment, the total demand per unit time was calculated by multiplying the difference
between the blank concentration and the actual concentration by the flow rate.
Total demand (mg/min) = (C(expenmentai)-C(biank)) x F (2-1)
where: C is concentration (mg/liter) and
F is flow (L/min).
2. Determine the total demand at steady-state concentration, i.e., the highest concentration
reached. If the absorbance stage has been completed, the total demand at this stage should
equal the heterogeneous reaction rate.
3. Plot the total demand per unit surface area at steady state for all available steady-state
concentrations. Use a least-squares model to determine a relationship between concentration
and reaction demand:
Reaction Demand (mg/min/m2) = f(C,t) (2-2)
4. For each experiment, calculate the reaction demand for each time point based on the relationship
determined in Step 3.
5. The adsorption demand may be calculated for each experiment by summing the difference
between the actual total demand and the reaction demand for each time point. Because of mixing
delays, some individual time points may have negative adsorption demand values. However, the
adsorption demand values should average out to a non-negative value.
Adsorption demand (mg/run) = Z(Total demandj - Reaction demandj) (2-3)
for each time interval i.
6. Plot the adsorption demand per unit surface area as a function of steady-state concentrations.
Use a least-squares model to determine a relationship between adsorption and concentration:
Adsorption (mg/m2) = f(C) (2-4)
7. The total demand may be calculated from the following equations:
Total Demand (mg) = Reaction Demand (mg) + Adsorbance Demand (mg) (2-5)
Reaction Demand (mg) = A x f(C,t) (2-6)
15
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where: A = total exposed surface area (m2)
The Reaction Demand component includes a relationship between concentration and time which will
be dependent on generation technique. For instance, if the target fumigation was 9000 ppm*hours,
the total reaction demand would be a function of the duration of the fumigation.
Adsorbance Demand (mg) = A x f(C) (2-7)
where: C = target concentration (mg/L).
2.7 Sampling and Analytical Procedures
Within a single test, extraction or surface sampling was completed for all blank coupons before sampling
of any inoculated coupon was performed. Surface sampling was done either by wipe sampling or vacuum
sampling in accordance with the protocols 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. In addition, general
sampling supplies were also needed. A sampling material bin was stocked for each sampling event. The
bin contained enough wipe sampling and vacuum sampling kits to accommodate all required samples for
the specific test. Additional kits of each type were also included for backup. Sufficiently prepared
packages of gloves and bleach wipes were included in the bin. Extra gloves and wipes were also
included. A sample collection bin was used to transport samples back to the on-site NHSRC
Biocontaminant Laboratory. The exterior of the transport container was decontaminated by wiping all
surfaces with a bleach wipe ortowelette moistened with a solution of pH-adjusted bleach (pAB) prior to
transport from the sampling location to the NHSRC Biocontaminant Laboratory.
Fumigation conditions were reached before coupons were exposed to the fumigant. Fumigation
generation equipment was enclosed inside a spray booth, which necessitated long fumigant injection
tubes. However, the tube length did not seem to affect fumigation conditions. Room alarms were present
for both fumigants.
The glove box used for fumigation was insulated from light and heat. Following fumigation, coupons were
aerated for at least ten minutes before sampling. ADAs were sterilized using EtO before each use.
2.7.1 Sampling Strategy
The primary objective was to study the impact of grime on fumigation effectiveness as to decontaminate
concrete subway surfaces. The effectiveness is measured by the determination of the LR calculated per
Section 1.2.1. Sampling of positive controls was compared to post-decontamination sampling of test
sections for this study. Since current surface sampling techniques are intrusive, they will also remove
viable spores from the surface of the coupon. Positive control coupons were inoculated on the same day
and analyzed on the same day as test coupons but were not decontaminated.
For the material demand task, mass balances of CIO2 fumigant were performed in real time on the kinetic
chamber by constantly monitoring the inlet and outlet concentration of CIO2 as well as the flow rate. To
16
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confirm the readings of the photometers (which also respond to CI2 gas), wet chemistry samples based
on mSM 4500-CIO2-B were collected every hour.
2.7.2 Sampling Points
Each sampling method was used on the surface of coupons inoculated with approximately 1 x 106 spores.
For each inoculation event, additional samples were collected from stainless steel surfaces as MDI
control samples. Wipe samples or vacuum samples were collected by sampling within a 1.5" x 1.5"
sampling template pre-printed on the coupons.
CIO2 and VPHP concentration measurements were collected from fumigation and kinetics chambers
(CIO2 only for kinetics study). Fans in the chambers provided mixing, ensuring that the measurements
were representative.
2.7.3 Sampling Frequency
Table 2-3 lists the frequency of all samples for the fumigation tests.
Table 2-3. Sample Frequency
Sample Type
Test Coupon
Negative Control
Coupon
Procedural Blank
Coupon
Positive Control Coupon
MDI Control Coupons
(stainless steel)
NHSRC Biocontaminant
Laboratory Material
Blanks
H202 Monitor
H202 Wet Chemistry
Samples
H202 Wet Chemistry
Sample Blank
(laboratory air)
CI02 Monitor
mSM 4500-CI02-B Wet
Chemistry Samples
Quantity
3 per coupon type
and fumigation
condition
1 per coupon type
1 per coupon type
3 per coupon type,
inoculated as the
first, middle, and
last coupons
3 per inoculation
event, inoculated
immediately before
each positive
control coupon
3 per material
1
Duration dependent
1
1
Duration-
dependent
Frequency
1 set per fumigation
1 per fumigation
1 per fumigation
(earliest time point)
1 per inoculation
1 per inoculation
Once per use of
material
Real time during
H202 fumigations
1 every 2 hours
during fumigation
1 per H202
fumigation
Real-time during
CI02 fumigations
Once every 60
minutes
Process Type or
Location
Fumigated
Not fumigated
Fumigated
Not fumigated
Not fumigated
NA
Glove box
Glove box
NA
Glove box
Glove box
Purpose
To determine the number of
viable spores after fumigation
To determine extent of cross-
contamination and/or the
sterility of coupons
To determine extent of cross-
contamination during
fumigation
To determine the number of
viable spores recoverable
from the coupons
To determine the number of
viable spores deposited onto
the coupons and to assess the
stability of the MDI
To demonstrate sterility of
extraction and plating
materials
To determine exposure
experienced by the coupons
To validate operation of H202
monitor
Procedure for sample
collection and titration
To determine exposure
experienced by the coupons
To validate operation of CI02
real-time monitors
17
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Sample Type
CI02 Wet Chemistry
Sample Blank
(laboratory air)
RH/Temperature
Quantity
1
1
Frequency
lperCI02
fumigation
Logged every 10
seconds
Process Type or
Location
NA
Glove box
Purpose
To demonstrate blank value
of mSM 4500-CI02-B
To determine environmental
conditions during fumigations
NA = not applicable.
Table 2-4 lists critical and non-critical measurements for each sample.
Table 2-4. Critical and Non-Critical Measurements
Sample Type
Test Coupon
Negative Control
Coupon
Positive Control
Coupon
Field Blank
Coupons
Lab Blank Coupons
NHSRC
Biocontaminant
Laboratory Material
Blanks
hbCb Monitors
H2O2Wet
Chemistry Samples
H2O2Wet
Chemistry Sample
Blank
CIO2 Monitors
mSM 4500-CIO2-B
Wet Chemistry
Samples
CIO2Wet
Chemistry Sample
Blank
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
Plated volume, incubation temperature, extracted
volume, CPU
H2O2 concentration
Volume collected, volume of titrant used
Volume collected, volume of titrant used
CIO2 concentration
Volume collected, volume of titrant used
Volume collected, volume of titrant used
RH and temperature during fumigation
Non-critical
Measurement
Storage time, storage
temperature
Storage time, storage
temperature
Storage time, storage
temperature
Storage time, storage
temperature
Storage time, storage
temperature
Storage time, storage
temperature
NA
Temperature of meter
box, time for collection
Temperature of meter
box, time for collection
NA
Temperature of meter
box, time for collection
Temperature of meter
box, time for collection
NA
NA = not applicable.
Table 2-5 lists the frequency of all samples for the material demand tests.
18
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Table 2-5. Sample Frequency for Material Demand Tests
Sample Type
Coupon Surface Area
CI02 Monitor
mSM 4500-CI02-B Wet
Chemistry Samples
CI02 Wet Chemistry
Sample Blank
(laboratory air)
RH/Temp
Background Demand
Test
Material Demand Tests
Quantity
1
1 per chamber
Duration dependent
1
1
1 per constant
concentration
1 per constant
concentration
Frequency
Each coupon
Real-time during
CI02 fumigations
Once every 60
minutes
1 perCI02
fumigation
Logged every 10
seconds
1
1 per coupon type
Location
Laboratory
Constant source
chamber and
kinetics chamber
Constant Source
chamber and
kinetics chamber
Laboratory air
Kinetics chamber
Laboratory
Laboratory
Purpose
To determine the total
surface area exposed to allow
calculations of demand per
surface area
To determine exposure
experienced by the coupons
To validate operation of CI02
real-time monitors
To demonstrate blank value
of mSM 4500-CI02-B
To determine environmental
conditions during fumigations
To determine material
demand of empty kinetics
chamber
To determine material
demand of grimed concrete
Table 2-6 lists critical and non-critical measurements for each sample collected during the material
demand task.
Table 2-6. Critical and Non-Critical Measurements for Material Demand Task
Sample Type
CIO2 Monitors
mSM 4500-CIO2-B Wet Chemistry
Samples
CIO2 Wet Chemistry Sample Blank
RH/Temp
Background Demand Test
Material Demand Tests
Critical Measurements
CIO2 concentration
Volume collected, volume of titrant used
Volume collected, volume of titrant used
RH and temperature during fumigation
Flow rate, real time concentration, exposure time
Flow rate, real time concentration, surface area
present, mass of concrete present, exposure time
Non-critical
Measurement
Temperature of meter
box, time for collection
Temperature of meter
box, time for collection
Digital video was collected during representative events (inoculation, fumigation, and sampling).
Photographs of selected material coupons with any visible change due to the sampling procedure were
taken after the completion of the sampling.
2.7.4 Statistical Approach
Section 1.2.1 details the methods for determining the efficacy or LR of a fumigation technique for each
coupon location. The Student's t-test was used to evaluate whether a variable such as fumigation
19
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duration had an effect on efficacy. The LR was also plotted against variables such as measured fumigant
concentration to determine any relationship.
2.7.5 Sampling Procedures
2.7.5.1 Polyester-Rayon Blend (PRB) Wipe Sampling
PRB wipe sampling is typically used for small sample areas and is effective on nonporous, smooth
surfaces such as ceramics, vinyl, metals, painted surfaces, and plastics.3 PRB wipe sampling was used
for concrete samples during method devlopment as per internal operating procedures. The general
approach is that a moistened sterile nonwoven PRB pad is used to wipe a specified area to recover
bacteria, viruses, and biological toxins. The protocol that was used in this project has been adopted from
that provided by Busher et al.3 and Brown et al.4 Wipe samples were extracted in 20 ml Phosphate
Buffered Saline with 0.05 % TWEEN®20 (PBST) and subjected to serial tenfold dilution and spread-
plating as per internal operating procedures.
2.7.5.2 37 mm Vacuum Sampling
For concrete samples during method devlopment, 37-mm vacuum sampling using
polytetrafluoroethylene (PTFE) filters (0.3 micron pore size) was used.5 A 1.5" x 1.5" square was
vacuumed on each coupon. The 37-mm samples were extracted as per internal operating procedures
and subjected to tenfold serial dilution and spread-plating.
2.7.5.3 Sponge Wipe Sampling
Sponge wipe sampling was used for concrete samples during method development and as part of the
fumigation testing. Sponge wipe samples were collected using the following two patterns: (1) using the
flat side of the sponge wipe, the surface was sampled using horizontal S-strokes, covering the entire
template area; and (2) the sponge wipe was then flipped over to the opposite side to sample the surface
in a vertical pattern, covering the entire template area. This is an abbreviation of the sampling method
described in detail in the study Rose et al,6 which was designed to sample a larger area than the one
used for this study. Sponge wipe samples were extracted in 90 ml PBST and subjected to tenfold serial
dilution and spread-plating.
2.7.5.4 Wet Chemistry Samples
The CIO2 extractive samples were collected hourly according to internal operating procedures during CIO2
fumigations. The H2O2 extractive samples were collected every two hours during VPHP fumigations.
2.7.5.5 Coupon Spore Enumeration
The NHSRC Biocontaminant Laboratory quantified the number of viable spores per sample (vacuum and
wipe samples). PBST was used as the extraction buffer for all sample types. After the appropriate
extraction procedure, as described in the sections to follow, the buffer was subjected to a five-stage serial
dilution (10~1 to 10~5). The resulting samples were plated in triplicate and incubated overnight at 35 °C ±
2°C. Following incubation, CFU were enumerated as per internal operating procedures. The PBST was
prepared according to internal operating procedures.
20
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The extraction procedure used to recover spores was varied depending upon the different matrices (PRB
wipes, sponge sticks, or vacuum filters) according to internal operating procedures. Other procedures are
described in the following subsections. Samples that have fewer than the reportable limit of 30 CFU/plate
in the undiluted sample underwent filter plating and re-plating. While there are no EPA-approved methods
for spore enumeration from surfaces, the use of positive control samples as the baseline for log reduction
calculations includes a built-in verification of the deposition and enumeration methods.
2.7.5.6 Extractive CIO2 Analysis
CIO2 concentration in an air sample was determined by titration. Briefly, sodium thiosulfate is used to
reduce iodine oxidized by the CIO2. The titration goes from a yellow solution to a colorless endpoint.
2.7.5.7 Extractive H2O2 Analysis
H2O2 concentration in an air sample was measured by titration. Briefly, potassium permanganate is used
to oxidize H2O2 dissolved under acidic conditions. The titration goes from a colorless solution to the first
pink tinge.
2.8 Sample Handling and Custody
2.8.1 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, several management controls were followed.
• In accordance with aseptic technique, a sampling team was utilized, made up of a "sampler," a
"support person," and a "sample handler."
• The sample handler was the only person to handle ADAs or material coupons during the
sampling event.
• The sampler handled only the sampling media and the support person handled all other supplies.
The sampler sampled the surface according to internal operating procedures.
• The collection medium (e.g., PRB wipe or 37-mm cassette) was placed into a sample container
that was opened, held and closed by the support person.
• The sampler placed the 37-mm nozzle directly into the small conical tube with sterile gloves. The
tube 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 double-bagged sample was then placed into a sample container for transport.
o The exterior of the transport container was decontaminated by wiping all surfaces with a
bleach wipe or towelette moistened with a solution of hypochlorite prior to transport from
the sampling location to the NHSRC Biocontaminant Laboratory.
• After the sample was placed into the container for transport, the sample handling team placed the
21
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sampled material in pAB for decontamination and eventual disposal.
• The sampling crew then changed their gloves in preparation for working with the next sample.
Additionally, and equally important, the order of sampling was as follows: (1) all blank coupons; (2)
decontaminated coupons; and (3) positive control coupons. This order ensured that coupons were
handled in an order from lowest level of contamination to the most.
2.8.2 Preventing Cross-Contamination during Analysis
General aseptic laboratory technique was followed and is embedded in the standard operating
procedures (SOPs) and MOPs used by the NHSRC Biocontaminant Laboratory to recover and plate
samples. The SOPs and MOPs document the aseptic technique employed to prevent cross-
contamination.
Additionally, the order of analysis (consistent with the above) was as follows: (1) all blank coupons; (2) all
decontaminated coupons; and (3) all positive control coupons.
2.8.3 Representative Samples
This work was meant to explore the efficacy of fumigants within subway systems. The concrete coupon
materials were taken from an actual subway system. The fumigation conditions are considered
representative of conditions that could be met in the field.
2.8.4 Sample Quantities
The sample quantities are outlined in Table 2-2. Concrete coupon quantities were limited by the finite
amount of New York subway concrete available for testing.
2.8.5 Sample Containers for Collection, Transport, and Storage
For each PRB wipe sample, the primary containment was an individual sterile 50 ml conical tube.
Secondary containment was sterile sampling bags. The primary containment of the 37-mm sample was a
sterile 10" x 5.5" sample bag. The inlet tube for the 37-mm sample was primarily contained in a separate
sterile 15 ml conical tube. The secondary containment of the inlet tube and 37-mm cassette was
separate sterile sampling bags. The primary containment of the sponge wipe was a Seward stomacher
bag (Seward Limited, Worthing, West Sussex, UK), secondarily contained in an individual sterile sampling
bag. 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 NHSRC Biocontaminant Laboratory, the
container was wiped with a towelette saturated with at least 5000 parts per million (ppm) hypochlorite
solution by weight. A single container was used for storage in the decontamination laboratory during
sampling and for transport to the NHSRC Biocontaminant Laboratory.
2.8.6 Sample Preservation
Following transfer to the NHSRC Biocontaminant Laboratory, all samples were stored at 4 °C ± 2 °C until
they were analyzed. Samples were stored no longer than five days before the primary analysis. A typical
holding time, prior to analyses, for most biological samples was two days. All samples were allowed to
equilibrate to room temperature for one hour prior to analysis.
22
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2.8.7 Sample A rchiving
All samples and diluted samples were archived for at least two weeks following completion of analysis.
This time allowed for review of the data to determine if any re-plating of selected samples was required.
Samples were archived by maintaining the primary extract at 4 °C ± 2°C in a sealed extraction tube.
23
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3 Results and Discussion
3.1 Method Development I
The 1.5" round ADA deposition method demonstrated the ability to deliver the desired concentration of at
least 1xio6 CPU per coupon with a better than 0.5 log precision. This ensures that a 6-log reduction in
viable spores can be demonstrated. Recovery from sponge wipes of the four replicate stainless steel
samples is shown in Table 3-1. The table shows repeatable recovery of 1.3 x 107 CPU per sample with a
relative standard deviation (RSD) of 30 %.
Table 3-1. Recovery from Stainless Steel Coupons following Prototype Deposition Method
Replicate
1
2
3
4
CPU/sample
9.56E+06
1.52E+07
1.05E+07
1.82E+07
Log
CPU
7.0
7.2
7.0
7.3
Average
CPU/sample
1.34E+07
SD
4.06E+06
RSD
30%
SD = Standard deviation.
Based on these results, the method describing the 1.5" ADA deposition method, was adopted for all
subsequent inoculations.
3.2 Method Development II
Three sampling methods were evaluated to repeatedly recover CPU from concrete samples inoculated
using the 1.5" round ADA deposition method. The results are displayed in Table 3-2 and Figure 3-1.
Table 3-2. Recovery of Various Concrete Sampling Methods
Sample
Stainless Controls
Sponge Wipe
Wetted Wipe
37-mm Vacuum
Average
1.18E+07
9.27E+06
1.08E+07
2.35E+06
Maximum
1.55E+07
1.05E+07
1.77E+07
3.27E+06
Minimum
8.78E+06
6.83E+06
4.31E+06
1.31E+06
RSD
29%
23%
62%
42%
24
-------
Recovery from Various Concrete Sampling Methods
I Average
IRSD
Stainless Sponge Wetted 37 mm
Controls Wipe Wipe Vacuum
Figure 3-1. Recoveries of Various Concrete Sampling Methods
Results from the sponge wipe of stainless control coupons is shown for reference. Wetted wipe samples
had a large variability, with a relative standard deviation (RSD) of 62 %. Wetted wipe samples are also
not recommended because the sampler has direct contact with the sample. The 37-mm vacuum samples
recovered only 20 % of the spores compared to the stainless steel control samples. For these reasons,
the sponge wipe was chosen as the preferred concrete sampling method for subsequent fumigation tests.
3.3 CIO2 Fumigations
As discussed in Section 2.1, two measurement methods were used for determining CIO2 concentration: a
real-time photometer and the periodic wet chemistry method based on mSM 4500-CIO2-B. For the
duration of the wet chemistry samples, photometer samples were also collected. Wet chemistry values
were 9% higher than average photometer reading at the time of sampling. CIO2 concentrations reported
for the remainder of this report, when based on real-time photometer data, are standardized to the wet
chemistry value using this correction.
Conditions during the two CIO2 fumigations are shown in Table 3-3. Average and standard deviation
values were calculated for the duration of each exposure.
25
-------
Table 3-3. Fumigation Test Matrix
1500 ppmv Test
Target Conditions:
1500 ppmv, 75%RH,24°C
Exposure
Time (h)
2.5
4
6
CI02
(ppmv)
1570 ±40
1570 ±40
1570 ±40
Temp
(°C)
23.3 ±0.02
23. 3 ±0.02
23. 3 ±0.03
RH
(%)
75.0 ±0.1
75.0 ±0.1
75.0 ±0.1
500 ppmv Test
Target Conditions:
500 ppmv, 75 % RH, 24 °C
Exposure
Time (h)
6
12
18
CI02
(ppmv)
570 ± 40
550 ± 40
550 ± 40
Temp
(°C)
23.4 ±0.1
23.4 ±0.1
23.4 ±0.2
RH
(%)
76 ±1
75 ±0.3
76 ±1
Microbiological results from the 1500 ppmv CIO2 test are shown in Figure 3-2.
1 nvin8-,
I .UX IU
1 nvin7-
I .UX IU
CO*
•I 1 nvin6
C I .UX IU
"8
yj -i nvin5-
o5 T-0*10
§1 nvin4-
I .UX IU
s_
n 1 nvin3
I I 1 .UX IU
o
™ 1 DYin2-
D) I .UX IU
&
CD
> 1 rvi n1 -
^ I .UX IU
1 nvin0-
1 .UX 1 U
1500 ppmv CIO2 Fumigation Test
i
• Concrete As Is
• V\feshed Concrete
-•-
• i
i 1
0123456-
Hours Exposure
7
Figure 3-2. Average CFU Recovered from Concrete following Exposure to 1500 ppmv CIO2.
Some spores (1 CFU each on one of the three unwashed concrete coupons and 1 CFU on one of the
three washed concrete coupons) were recovered after 6 hours of exposure. These values are very close
26
-------
to the detection limit of the method. Washed and unwashed concrete behaved similarly, with no statistical
difference between spores recovered from the two types of coupon preparations (t = 0.95). Figure 3-3
shows the same results in terms of log reduction.
Log Reduction (1500 ppm CIO2)
7
6-
II
c 5_
5
'fj 4-
v_) t-
•§
*- Q
I]
0)
> ^ _
0-
• •
• Concrete As Is 1
• Washed Concrete |
01 234567
Hours Exposure
Figure 3-3. Log Reduction of Spores Recovered from Concrete following 1500 ppmv CIO2
Both the 4- and 6-hour fumigation durations indicate a greater than 6-log reduction.
The second test (Figure 3-4) was conducted to look at the efficacy when using a lower CIO2
concentration. This test extended to longer exposure times to duplicate concentration x time (CT)
exposure levels.
27
-------
1 nvin8-,
I .UX I U
1 nvin7-
I .UX I U
CO
r- 1 DY1D6-
S_ 1 .UX 1 VJ
(D A rv,-m5
ŁJT 1.0X10 -
o 1 nvin4-
ffi \ .UX I U
rr 1 nvin3-
LJ_ I .UX I U
O
Pr> 1 nvin2-
D3 1 .UX 1 U
&
CD
> -i rvi n1 -
^ I .UX I U
1 nvin°-
I .UX I U
500 ppmv CIO2 Fumigation Test
• Concrete As Is
• V\^shed Concrete
I 5
20 2 4 6 8 10 12 14 16 18 2
Hours Exposure
0
Figure 3-4. Average CPU Recovered from Concrete following Exposure to 500 ppmv CIO2
Very few spores were recovered from all three of the non-zero time points of the 500 ppmv CIO2
fumigation. No spores were recovered from any of the washed concrete coupons. Of the unwashed
concrete, one spore was recovered from one of the three replicate coupons for the first two time points. A
total of two spores were recovered from all 18 fumigated coupons. Based on this limited dataset, the
results show no significant difference in efficacy for the cleaned and uncleaned concrete subway
surfaces. All three time points provided a 6-log reduction. However, only the longest exposure (18 hours)
provided complete decontamination (no detection).
3.4 H2O2 Fumigations
Conditions during the H2O2 fumigations are shown in Table 3-4. Average and standard deviation values
were calculated for the duration of each exposure.
28
-------
Table 3-4. H2O2 Fumigation Test Matrix
250 ppmv test
Target Conditions:
250 ppmv H202, < 80 % RH
Exposure
Time (h)
1
2
4
H202
(ppmv)
250 ±4
250 ±4
250 ± 1 1
Temp
(°C)
17.0 ±0.1
17.1 ±0.1
17.2 ±0.2
RH
(%)
21 ±1
21 ±1
21 ±1
150 ppmv test
Target Conditions:
1 50 ppmv, < 80 % RH
Exposure
Time (h)
4
7
10
H202
(ppmv)
151 ±13
151 ±12
151 ±11
Temp
(°C)
20.0 ±0.5
20.0 ±0.3
20.0 ±0.5
RH
(%)
11 ±2
11 ±2
11 ±2
Microbiological recovery for the 250 ppmv H2O2 is shown in Figure 3-5.
250 ppmv H2O2 Fumigation Test
1 nnFj-DR
1 nnfj.n~7 •
PO
U. 1 nnF-ufifi
•D
oi 1 nnF-i-nc;
01
>
81 nnfj-nA
01
_ 1 nnF-i.n3
LL.
cj
1 nnfj-m
M
i- 1 nnF-i.ni
^ 1 nnF-i-nn
1 nnr m -
4 Washed Concrete
T • Concrete As Is
;
I
±— — ±— — i— — a— — i
512345
Hours Exposure
Figure 3-5. Average CFU recovered from Concrete following Exposure to 250 ppmv VPHP
While there are differences in the response of washed and unwashed concrete coupons, these
differences are not statistically significant based on the observed variation in the average CFU recovered
for each set of coupons. One- and two-hour fumigation of both concrete types produced some non-detect
coupons. All coupons from the four-hour exposure at 250 ppm were non-detect. Only the 4-hour
fumigation at 250 ppmv VPHP produced an average LR over 6.
These results are in contrast to the fumigation at 150 ppmv VPHP. No tested exposure time at the lower
VPHP concentration provided a 6-log reduction. Average recovery is shown in Figure 3-6.
29
-------
150 ppmv H2O2 Fumigation Test
1 nnFj-DR
1 nnFj-D7 ••
PO
C1 nnFj-HA
•D
oi 1 nnF-i-nc;
01
>
81 nnfj-nA
01
_ 1 nnFj-n^
LL.
cj
1 nnFj-n9
no
(0
i_ -i nnFj.ni
"^ 1 nnF-i-nn
1 nnp m -
~~i
1
;
; T
1
T
1 ^Washed Concrete
1 • Concrete As Is
9
52468
Hours Exposure
10 12
Figure 3-6. Average Recovery from Coupons after Exposure to 150 ppmv VPHP
As in the 250 ppm test, there does not seem to be an effect of grime on concrete before VPHP
fumigation. The apparent benefit at the 4-hour exposure is contradicted by the results of the 10-hour
exposure.
Table 3-5 shows the average LR for both tests in terms of CT. Even at much higher CT values, LR from
the 250 ppmv test was lower. This investigation suggests there is no clear relationship between CT and
efficacy for VPHP fumigation of concrete. For concrete, high concentrations of H2O2 may be needed,
unless very long exposure times are used. An extrapolation of log reduction over exposure time at 150
ppm (see Figure 3-7) suggests that a 6 LR may be reached after 13 hours, compared to 6 hours at 250
ppm.
Table 3-5. Average LR of Washed and Unwashed Concrete
250 ppmv Test
ppm*hours
246
497
989
Washed
Concrete
6.38
4.89
7.12
Concrete
'As Is'
5.41
5.68
7.18
150 ppmv Test
ppm*hours
605
1062
1511
Washed
Concrete
3.65
2.69
2.79
Concrete
'As Is'
2.13
2.91
4.85
30
-------
Average LR from 150 ppmv Test
8nn
7 nn
6nn
DC R nn
01
Sf 400
-------
250 ppm Concrete
1 **
-0.8-
en
•— ' n R -
(N
0
^04-
OT ,
0 -
(
*"W ^^V ^^%
^->V t X
~?
4 m
4
4 •
1 *
« *%
, *S _ r_ r^
*[CIO2]wilh concrete (mg/L)
Modeled [CIO2](no demand)
• Act jal aeration [CIO 2]
Model Aeration [C1O2] (no
demand or ofTgassing)
) 100 200 300 400 500 600 700
Fumigation time (minutes)
Figure 3-8. Material Demand Test at 250 ppm CIO2
No absorbance is seen, as demonstrated by no lag time in the rise of the concentration in the presence of
the grimed concrete. Reaction rates are also very low, as seen in the relative equivalence in the steady-
state concentration. Figure 3-9 shows the reaction rates of the empty (blank) kinetics chamber and of the
kinetics chamber including concrete with a surface area of approximately 170 in2 as a function of CIO2
concentration.
[c*
"55
reaction rate (
03^
00 _
OTC _
09 _
01 R -
01 .
On1; -
•
+ Concrete
* • Blank
* +
012345
CIO2 concentration (mg/L)
Figure 3-9. Steady-state Reaction Rates of Kinetics Chamber with and without Concrete
Samples
32
-------
There is no apparent difference between the reaction rate with the concrete and without, so the reaction
rate was set to zero for the absorbance calculations (see Section 2.6.2.2). These adsorption rates are
shown in Figure 3-10 and again suggest no adsorption.
Concrete Adsorption
_ (
N*
"55
c Ann
o ^uu
'•g.
"D
<
1 nnn
CIO2 concentration (mg/L)
) 1 2 3 4 5
Figure 3-10. Concrete Adsorption as a Function of Concentration.
Negative adsorption rate values are indicative of the limited ability to measure the small difference
between adsorption characteristics of the empty chamber and the chamber with concrete coupons. It is
possible that some material demand could have been detected with larger amounts of concrete in the
chamber, but tests were done with 10 pieces with a surface area of 170 in2 due to the limited availability
of the subway concrete. Neither reaction nor absorbance components of CIO2 demand were detected for
grimed concrete.
33
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4 Quality Assurance
This project was performed under an approved Category III Quality Assurance Project Plan (QAPP) titled
Interaction of Fumigation with Realistic Surfaces from Subway System (June 2013) and an addendum for
Task 4 - Material Demand (February, 2014), both available upon request.
4.1 Sampling, Monitoring, and Analysis Equipment Calibration
There were standard operating procedures for the maintenance and calibration of all laboratory and
NHSRC Biocontaminant Laboratory equipment. All equipment was verified as being certified calibrated or
having the calibration validated by EPA's Air Pollution Prevention and Control Division (APPCD) on-site
(Research Triangle Park, 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 Tables 4-1 and 4-2.
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. Sampling and Monitoring Equipment Calibration Requirements
Equipment
Meter Box
RH Sensor
Stopwatch
Clock
Temperature Sensor
Buret
Calibration/Certification
Volume of gas is compared to NIST-traceable dry
gas meter annually
Compare to 3 calibration salts once a week
Compare against NIST* Official U.S. time at
http://nist.time.qov/timezone.cqi?Eastern/d/-5/iava
once every 30 days.
Compare to office U.S. Time @ time.gov every 30
days
Compare to independent NIST thermocouple
annually
Gravimetric verification of volume performed
annually
Expected Tolerance
±2%
±5%
± 1 min/30 days
± 1 min/30 days
± 2 % full scale
± 1 %
* National Institute of Standards and Technology
All titrants have a certification of analysis with NIST-traceable concentration values.
34
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Table 4-2. Analysis Equipment Calibration Frequency
Equipment
Pipettes
Burets
Pressure
Manometer
Incubator
thermometers
Scale
Calibration
Frequency
Annually
Annually
Annually
Annually
Before each
use
Calibration Method
Gravimetric
Gravimetric
Compared to NIST-
traceable Heiss gauge
Compared to NIST-
traceable thermometer
Compared to Class S
weights
Acceptance
Criteria
± 1 % target
value
± 1 % target
value
± 3 % reading
± 0.2 °C
± 0.01 % target
4.2 Data Quality
The primary objective of this research was to evaluate the impact that dirt and grime, as present on
unpainted subway concrete, have on fumigation efficacy. Secondary objectives included determining
which sampling procedure provides better recovery from grimed and cleaned concrete, and
characterizing subway material before and after cleaning using a prescribed method from the New York
City MTA. 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 QAPP in place for this project was followed with deviations noted as follows:
• Subway concrete coupons were sterilized with EtO rather than being autoclaved as specified in
the QAPP to remove any effect of the autoclave heat on any grime present on the concrete.
• Fumigation conditions listed in the test matrix (Table 2-1) were determined after the QAPP was
approved.
• Concrete availability prevented testing of washed concrete as part of the material demand testing.
4.3 QA/QC Checks
Samples were maintained to ensure their integrity. Samples were stored away from standards or other
samples that could cross-contaminate them. While the size and shape of the concrete coupons varied,
the size of the inoculation and sampling area did not.
Supplies and consumables were acquired from reputable sources and were NIST-traceable when
possible. 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
35
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personnel checked supplies and consumables prior to use to verify that they meet 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, CPU were enumerated
manually and recorded. Critical QC checks are shown in Table 4-3. The acceptance criteria were set at
the most stringent level that could be achieved routinely 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 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
Sample Type
Negative Control
Coupons
Field Blank Coupons
Laboratory Blank
Coupons
Laboratory Material
Coupons
Blank Tryptic Soy Agar
Sterility Control
(plate incubated, but
not inoculated)
Positive Control
Coupons
mSM 4500-CIO2-B Wet
Chemistry
H2O2 Wet Chemistry
Purpose
Determine extent of
cross-contamination
Verify the process of
moving coupons
does not introduce
contamination
Verify the sterility of
coupons following
autoclaving
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
coupons
Validate CIO2
concentration
measurements
Validate H2O2
concentration
measurements
Acceptance Criteria
No detectable spores
No detectable spores
No detectable spores
No detectable spores
No observed growth following
incubation
1 x 10s CPU ±0.5 log
1 5 % of photometric reading
65 % of electrochemical reading
Corrective Actions
Values on test coupons of
the same order of
magnitude will be
considered to have resulted
from cross-contamination
Determine source of
contamination and remove
Determine source of
contamination and remove
Determine source of
contamination and remove
All plates are incubated prior
to use, so any contaminated
plates will be discarded
Outside target range:
discuss potential impact on
results; correct loading
procedure for next test and
repeat depending on
decided impact
Repeat
Check calibration of
electrochemical sensor
Frequency
1 per sample
type
1 per test
3 per test
3 per
material per
test
Each plate
3 per coupon
type per test
1 per hour
2 per hour
36
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Sample Type
Fumigation Extraction
Blank Samples
Puffing Control
Coupons
Replicate Plating of
Diluted Microbiological
Samples
Post-test Calibration of
RH Sensors (Vaisala,
Helsinki, Finland)
Purpose
Validated baseline of
extractive techniques
Used to determine
drift in the MDI
Used to determine
variability in CFU
counts
Used to validate
sensor operation
Acceptance Criteria
Non-detect
The CFU recovered from the
first set of positive controls must
be within 0.5 log of the second
set of positive controls
The reportable CFU of triplicate
plates must be within 100 %.
Reportable CFU are between
30 and 300 CFU per plate
The post-test calibration check
readings must be within 5 % of
target reading
Corrective Actions
Obtain new reagents
Reject results and repeat
test
Re-plate sample
Reject results. Repeat test
as deemed appropriate
Frequency
1 per test
3 per
inoculation
Each sample
1 per test
4.4 Acceptance Criteria for Critical Measurements
The Data Quality Objectives (DQOs) are used to determine 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:
• enumeration of spores on the surface of the concrete coupons;
• concentration measurements to characterize the fumigation conditions.
Table 4-4 lists the quantitative acceptance criteria for critical measurements. Failure to provide a
measurement method or device that met these goals would result in a rejection of results derived from the
critical measurement. For instance, if the plated volume of a sample is not known (i.e., is not 100 %
complete), then that sample is invalid. If a mSM 4500-B sample for CIO2 is lost or does not meet the
criteria for other reasons, then another should be collected to take its place. In contrast, for the real-time
H2O2 measurements, some missing data would not invalidate a test.
37
-------
Table 4-4. Critical Measurement Acceptance Criteria
Critical
Measurement
Plated volume
CFU/plate
CIO2 concentration
H2O2 concentration
Fumigation Time
RH/Temp of
Fumigation
Surface Area
Flow Rate
Surface Area of
Concrete
Measurement
Device
Pipette
Hand counting
mSM 4500-B
ATI sensor
Timer
Vaisala
HMD40Y
Milligram
Balance
Dry Gas Meter
Balance
Accuracy
±2%
±10% (between 2
counters)
± 15 % of photometric
value
±10% range
± 1 second
±5%
+ 0.001 g
±1 %
+ 0.01 g
Precision
±1 %
±10%
±5%
±5%
± 1 second
±3%
+ 0.001 g
±1 %
+ 0.01 g
Detection Limit
NA
1 CFU
10 ppm
10 ppm
1 second
NA
+ 0.001 g
1 % full scale
0.01 g
Completeness
100%
100%
90%
90%
100%
90%
100%
90%
100%
Plated volume critical measurement goals were met. All pipettes are calibrated yearly by an outside
contractor (Calibrate, Inc.).
Plates were quantitatively analyzed (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 with significant data (data found to
be between 30-300 CFU). All second counts were found to be within 10 percent of the original count.
There are many additional QA/QC checks used to validate microbiological measurements. These checks
include samples that demonstrate the ability of the NHSRC 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 placed in glove box and fumigated;
• Field blank coupons: sterile coupons carried to fumigation location but not fumigated;
• Laboratory blank coupons: sterile coupons not removed from NHSRC Biocontaminant
Laboratory;
• Laboratory material coupons: includes all materials, individually, used by the NHSRC
Biocontaminant Laboratory in sample analysis;
• Positive control coupons: coupons inoculated but not fumigated; and
• MDI inoculation control coupons: stainless steel coupons puffed at beginning, middle, and end of
each inoculation campaign, not fumigated, to assess the stability of the MDI during the inoculation
operation.
38
-------
The CIO2 photometer calibrations were checked prior to each test and were within the factory
specifications during each fumigation. The primary CIO2 measurements were the mSM 4500-B extractive
samples. The accuracy and precision of the titration equipment were checked using a single-point NIST-
traceable standard solution. The Analytical Technologies, Inc. (ATI) H2O2 sensor was the primary
measurement device for H2O2 fumigations. The accuracy and precision of this instrument was assessed
by placing the unit in the headspace of a known concentration of H2O2. All DQI goals were met for all
measurements in Table 4-4.
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.
39
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5 Summary and Recommendations
The primary objective of this investigation was to determine the effect of real-world grime on the efficacy
of CIO2 and VPHP fumigations of subway concrete. A method was developed using MDIs to inoculate
small 1.5" coupons of concrete. Three surface sampling methods were tested (sponge wipe, wetted wipe,
and 37- mm vacuum filter); all three methods were comparable for recovery from stainless steel coupons,
but the sponge wipe method had higher and more repeatable recovery.
Fumigation of subway concrete using CIO2 resulted in a greater than 6-log reductions in Bacillus spores
for 1500 ppmv CIO2, 75% RH and > 4 h contact time or 500 ppmv CIO2, 75% RH and > 6 h contact time
(shortest contact time tested). Results suggest that fumigation of washed subway concrete does not
result in different efficacy values than fumigation of the unwashed subway concrete. The observed
differences are minimal and statistically not significant.
No differences were observed in feed concentration and the time required to reach the target CIO2
fumigation conditions with and without grimed subway concrete present. Hence, no CIO2 demand was
observed for this grimed subway concrete (approximately 170 in2 surface area of concrete material).
Fumigation with H2O2 as generated using Steris VHP® technology resulted in a greater than 6-log
reduction in Bacillus spores on subway concrete for 250 ppm H2O2, 20% RH and a > 4 h contact time.
The 6-log reduction in spores could not be reached, even at the longest tested contact time (10 h) at 150
ppmv H2O2 concentration. Observed differences in log reduction between washed and unwashed subway
concrete following VHP® fumigation were not statistically significant.
No material demand studies were conducted for the VHP® fumigant. Previous research efforts indicated a
high demand due to decomposition of hydrogen peroxide at the concrete surface. A recent full scale
VHP® fumigation demonstration, as conducted as part of the Bio-response Operational Testing and
Evaluation (BOTE) project,9 also indicated that material demand is significant. In that study, VHP®
fumigation did not achieve and maintain a 250 ppmv H2O2 concentration for 1.5 h in a 8000 ft2 facility.
This study showed that three of the four fumigation conditions using two different fumigants provided
conditions which demonstrated greater than 6-log reduction of Bacillus spores. No impact was observed
in the log reduction of spores in the presence of dirt and grime on these subway concrete surfaces.
Nevertheless, the high demand observed for VPHP in the presence of substantial amounts of concrete
may make achieving the necessary VPHP fumigation conditions challenging. Other fumigants may need
to be considered as alternatives to chlorine dioxide fumigation of concrete. Fumigants such as methyl
bromide and formaldehyde have, however, so far not been evaluated for fumigation of grimed concrete.
40
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6 References
1. Lee, S. D.; Ryan, S. P.; Snyder, E. G., Development of an aerosol surface
inoculation method for Bacillus spores. Applied and Environmental Microbiology
2011, 77(5), 1638-1645.
2. Calfee, M. W.; Lee, S. D.; Ryan, S. P., A rapid and repeatablemethod to deposit
bioaerosols on material surfaces. Journal of Microbiological Methods 2013, 92, 375-
380.
3. Busher, A. Noble-Wang, J.;Rose L., Surface sampling, in Sampling for Biological
Agents in the Environment. Emanuel, P.; Roos, J.W.; Niyogi, K., Eds. ASM Press:
Washington, DC, 2008; p 95-131.
4. Brown, G. S.; Betty, R. G.; Brockmann, J. E.; Lucero, D. A.; Souza, C. A.; Walsh, K.
S.; Boucher, R. M.; Tezak, M.; Wilson, M. C.; Rudolph, T., Evaluation of a wipe
surface sample method for collection of Bacillus spores from nonporous surfaces.
Applied and Environmental Microbiology 2007, 73 (3), 706-710.
5. Calfee, M. W.; Rose, L. J.; Morse, S.; Mattorano, D.; Clayton, M.; Touati, A.; Griffin-
Gatchalian, N.; Slone, C.; McSweeney, N., Comparative evaluation of vacuum-
based surface sampling methods for collection of Bacillus spores. Journal of
Microbiological Methods 2013, 95 (3), 389-396.
6. Rose, L. J.; Hodges, L.; O'Connell, H.; Noble-Wang, J., National validation study of a
cellulose sponge wipe-processing method for use after sampling Bacillus anthracis
spores from surfaces. Applied and Environmental Microbiology 2011, 77 (23), 8355-
8359.
7. U.S. EPA. Material Demand Studies: Interaction of Chlorine Dioxide Gas with
Building Materials. U.S. Environmental Protection Agency, Washington, DC,
EPA/600/R-08/091,2008
8. U.S. EPA. Material Demand Studies: Materials Sorption of Vaporized Hydrogen
Peroxide. U.S. Environmental Protection Agency, Washington, DC, EPA/600/R-
10/002,2010
9. U.S. EPA. Bio-response Operational Testing and Evaluation (BOTE) Project. U.S.
Environmental Protection Agency, Washington, DC, EPA/600/R-13/168, 2013.
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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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