EPA/600/R-12/586 | August 2012 | www.epa.gov/ord
United States
Environmental Protection
Agency
Evaluation of Hydrogen
Peroxide Fumigation for
HVAC Decontamination
Office of Research and Development
National Homeland Security Research Center
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EPA 600-R-12-586
Evaluation of Hydrogen Peroxide Fumigation for HVAC
Decontamination
Assessment and Evaluation Report
National Homeland Security Research Center
Office of Research and Development
U.S. Environmental Protection Agency
Research Triangle Park, NC 27711
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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 through EP-C-09-
027 WA 2-65 and 3-61 with ARCADIS U.S., Inc. This report has been peer and administratively reviewed
and has been approved for publication as an Environmental Protection Agency document. It does not
necessarily reflect the views of the Environmental Protection Agency. No official endorsement should be
inferred. This report includes photographs of commercially available products. The photographs are
included for purposes of illustration only and are not intended to imply that EPA approves or endorses the
product or its manufacturer. Environmental Protection Agency does not endorse the purchase or sale of
any commercial products or services.
Questions concerning this document or its application should be addressed to:
M. Worth Calfee, Ph.D.
Decontamination and Consequence Management Division
National Homeland Security Research Center
U.S. Environmental Protection Agency (MD-E343-06)
Office of Research and Development
109. T.W. Alexander Drive
Research Triangle Park, NC 27711
Phone:919-541-7600
Fax:919-541-0496
E-mail: Calfee.Worth@epamail.epa.gov
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Acknowledgments
This effort was managed by the principal investigator from ORD's National Homeland Research Center
(NHSRC), utilizing the support from the EPA's CBRN Consequence Management Advisory Team (CMAT)
within the Office of Emergency Management (OEM). The contributions of the entire team are
acknowledged.
Project Team:
M. Worth Calfee, Ph.D. (Principal Investigator)
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
R. Leroy Mickelsen
CBRN CMAT, Office of Emergency Management, Office of Solid Waste and Emergency Response, US
EPA
Research Triangle Park, NC 27711
This effort was completed under U.S. EPA contract #EP-C-09-027 with ARCADIS-US, Inc. The support
and efforts provided by ARCADIS-US, Inc. are gratefully acknowledged. The support provided by Tanya
Medley (U.S. EPA/ORD/NHSRC) in acquiring the vast quantities of supplies required for the completion of
this project is also acknowledged.
Additionally, the authors would like to thank the peer reviewers for their significant contributions.
Specifically, the efforts of Tim Dean (NRMRL, APPCD), Shannon Serre (NHSRC, DCMD), Mike Nalipinski
(Region 1) and Elsbeth Hearn (Region 1) are recognized.
IV
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Table of Contents
Disclaimer iii
Acknowledgments iv
Table of Contents v
List of Tables vii
List of Appendices viii
List of Acronyms and Abbreviations ix
Executive Summary xi
1 Introduction 1
1.1 Process 1
1.2 Project Objectives 2
1.3 Experimental Approach 2
1.3.1 Testing Sequence 2
1.3.2 Definitions of Effectiveness 4
2 Materials and Methods 6
2.1 Facility Design 6
2.2 Hydrogen Peroxide Cycle 7
2.3 Coupon Preparation 9
2.3.1 Test Coupons 9
2.3.2 Positive Control Coupons 10
2.3.3 Spore Preparation 11
2.3.4 Coupon Inoculation and Test Preparation 11
2.4 Test Matrix 11
2.5 Sampling and Analytical Procedures 12
2.5.1 Test Facility Sampling Procedures 12
2.5.1.1 Sampling/Monitoring Points 12
2.5.1.2 Electrochemical Sensor for H2O2 Concentration Measurement 15
2.5.1.3 Duct Flow Rate 15
2.5.2 Microbiological Analysis 15
2.5.2.1 Coupon Spore Enumeration 16
2.5.2.2 Swab Samples 16
2.5.2.3 Method Verification 16
2.6 Sampling Handling and Custody 16
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2.6.1 Prevention of Cross-contamination of Sampling/Monitoring Equipment 16
2.6.2 Preventing Cross-Contamination during Execution of the Decontamination Process 17
2.6.3 Preventing Cross-Contamination during Sampling 17
2.6.4 Preventing Cross-Contamination during Analysis 17
2.6.5 Sample Quantities 17
2.6.6 Sample Containers for Collection, Transport, and Storage 17
2.6.7 Sample Identification 18
2.6.8 Sample Custody 19
2.6.9 Sample Archiving 19
3 Results and Discussion 20
3.1 HVAC Duct Flow Characterization 20
3.1.1 Flow Velocity versus Blower Speed 20
3.1.2 Flow velocity Profile near an Elbow 21
3.2 H2O2 Fumigations - Unlined Duct Results 22
3.3 H2O2 Fumigations-Lined Duct 27
3.3.1 Exposure Phase 27
3.3.2 Desorption from Lined Duct 28
3.3.3 Comparison of Lined and Unlined Duct 30
4 Quality Assurance 32
4.1 Sampling, Monitoring, and Analysis Equipment Calibration 32
4.2 Data Quality 33
4.3 QA/QC Checks 33
4.4 Acceptance Criteria for Critical Measurements 34
4.5 Data Quality Audits 37
4.6 QA/QC Reporting 37
4.7 Amendment to Original QAPP 37
5 Summary and Recommendations 39
References 40
VI
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List of Figures
Figure 2-1. Front (top left), Side (top right), and Top views (bottom) of Duct Design (motor and
round duct connections not shown in this diagram, see next figure) 6
Figure 2-2. Photo of Actual Testing Facility 7
Figure 2-3. Control Loop Schematic 8
Figure 2-4. Test Coupons Holder Setup 10
Figure 2-5. Positive Control Coupon Holder 10
Figure 2-6. Duct Testing Facility with Sampling and Monitoring Locations Indicated by Letters
A-H 13
Figure 3-1. Pre- and Post-Blower Velocity Traverses Inside the Duct 21
Figure 3-2. Velocity Traverse Inside the Duct at Locations D and E 22
Figure 3-3. Sample Response Time forthe ATI Sensors 23
Figure 3-4. Spatial Efficacy Results for Unlined Duct (250 ppm x4 hours) 25
Figure 3-5. VHP Concentration during Exposure and Aeration Phases forthe Lined and
Unlined Duct 28
Figure 3-6. VHP Concentration during the Two Exposure Periods (initial exposure and
subsequent desorption) forthe Lined Duct (Test 01) 29
Figure 3-7. Average LR as a Function of CT 30
List of Tables
Table 2-1. Test Matrix 12
Table 2-2. Frequency of Sampling Monitoring Events 14
Table 2-3. Critical and Non-Critical Measurements 15
Table 2-4. Coupon Sample Coding 18
Table 3-1. Average H2O2 Concentrations during Fumigation 23
Table 3-2. Positive Controls Inoculation Results (n = 4) 24
Table 3-3. Average CFU Recovered from Test Coupons from Unlined Duct Sample Points
(n = 4) 24
Table 3-4. Average Log Reduction During Testing of the Unlined Duct by Sample Location
(n = 4) 26
Table 3-5. Average Log Reduction in Duct (n = 32) 28
Table 3-6. Conditions and Efficacy during Desorption Tests 29
Table 3-7. Comparison of Lined and Unlined Duct 31
VII
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Table 4-1. Sampling and Monitoring Equipment Calibration Frequency 32
Table 4-2. Analysis Equipment Calibration Frequency 33
Table 4-3. QA/QC Sample Acceptance Criteria 35
Table 4-4. Critical Measurement Acceptance Criteria 36
Table 4-5. Proposed Test Matrix 38
List of Appendices
Appendix A Miscellaneous Operating Procedures (MOPs)
VIM
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List of Acronyms and Abbreviations
APPCD
ATCC
B.
BSC
CIO2
CBR
CBRN
CPU
CM
CMAT
COC
CT
DCMD
DHS
Dl
DF
DQI
DQO
ECBC
EPA
FIFRA
GMP
HEPA
H202
hp
HSRP
HVAC
LR
MDI
MOP
NOT
NHSRC
NIST
OCSPP
ORLS
ORD
OSWER
PBST
Air Pollution Prevention and Control Division
American Type Culture Collection
Bacillus
Biological Safety Cabinet
Chlorine dioxide
Chemical, Biological, or Radiological
Chemical, Biological, Radiological, and Nuclear
Colony Forming Units(s)
Critical Measurements
Consequence Management Advisory Team
Chain of custody
Concentration x Time
Decontamination and Consequence Management Division
Department of Homeland Security
Deionized
Decimal Factor
Data Quality Indicator
Data Quality Objective
Edgewood Chemical Biological Center
U. S. Environmental Protection Agency
Federal Insecticide, Fungicide, and Rodenticide Act
A product name, rather than an acronym
High-Efficiency Particulate Air
Hydrogen peroxide
horse power
Homeland Security Research Program
Heating, Ventilation, and Air Conditioning
Log reduction
Metered Dose Inhaler
Miscellaneous Operating Procedure
National Decontamination Team
National Homeland Security Research Center
National Institute of Standards and Technology
Office of Chemical Safety and Pollution Prevention
On-Site Research Laboratory Support
Office of Research and Development
Office of Solid Waste and Emergency Response
Phosphate Buffered Saline Tween20
IX
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PPE Person Protective Equipment
ppm parts per million
QA Quality Assurance
QAPP Quality Assurance Project Plan
QC Quality Control
RH Relative Humidity
SEM Scanning Electron Microscopy
SOP Standard Operating Procedure
TBD To Be Determined
TRIO Taskforce on Research to Inform and Optimize CBR (chemical, biological, and
radiological) Response/Readiness
TSA Tryptic Soy Agar
VHP Vaporized Hydrogen Peroxide
WAM Work Assignment Manager
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Executive Summary
This project supports the mission of the U.S. Environmental Protection Agency's Office of Research and
Development's Homeland Security Research Program (HSRP) by providing information relevant to the
decontamination of areas contaminated as a result of an act of terrorism. The primary objective of this
project was to determine the efficacy of fumigation with hydrogen peroxide (H2O2) vapor on deactivating
spores inside a Heating, Ventilation and Air Conditioning (HVAC) duct. For these tests a STERIS 1000ED
VHP® mobile biodecontamination system was used to generate and inject H2O2 vapor. Secondary
objectives were to determine the effect that flow rate, distance from injection point, flow and pressure
points at turns such as elbows, inlet concentration of fumigant, and fumigant residual effects may have on
the decontamination efficacy. Two types of duct were tested: galvanized metal and galvanized metal lined
internally with fiberglass duct insulation.
The efficacy of H2O2 for the decontamination of an unlined duct varied based on the location in the duct.
For a single fumigation condition, the average log reduction (LR) per location ranged from 0.6 LR to full
decontamination (>7.4 LR, no recoverable viable spores). These results suggest that flow patterns can be
very complex in ductwork, and those complexities can make gaseous decontamination more difficult in
certain locations within the ductwork. Flow separation, eddying, and flow reversal occurred at certain
locations in the duct immediately following elbows. These locations were very difficult to decontaminate in
an unlined, metal duct. Increasing the flow rate through the duct seemed to exacerbate these effects,
though additional research is needed to confirm this result.
Lined duct proved easier to decontaminate than unlined metal duct. The lining absorbed H2O2, and
desorbed it over a period of over 48 hours. This desorption contributed significantly to VHP levels within
the duct following the initial fumigation, and resulted in higher efficacies than observed in unlined
ductwork. The results demonstrate that fumigation with H2O2can be an effective decontaminant on lined
duct even at low concentrations for a prolonged period of time (24 hours). Fumigations with a
concentration-time product (CT) of 550 ppm-hours exposure to H2O2 provided more than a 6 log
reduction.
XI
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1 Introduction
This project supports the mission of the U.S. Environmental Protection Agency's Office of Research and
Development's HSRP by providing information relevant to the decontamination of areas contaminated as
a result of an act of terrorism. Under Homeland Security Presidential Directive -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, the 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. The
EPA's National Homeland Security Research Center (NHSRC) provides expertise and products, through
implementation of the HSRP, that can be widely used to prevent, prepare for, and recover from public
health and environmental emergencies arising from terrorist threats and incidents. The goal of NHSRC"s
decontamination research is to provide products and expertise that guide the selection and
implementation of decontamination methods and provide the scientific basis fora significant reduction in
the time and cost of decontamination events. This research supports the Office of Solid Waste and
Emergency Response (OSWER) and the Office of Chemical Safety and Pollution Prevention (OCSPP).
OSWER, through its Special Teams that includes the CBRN Consequence Management Advisory Team
(CMAT), supports the emergency response functions carried out by the Regional Offices. OCSPP
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 the Federal Insecticide, Fungicide
and Rodenticide Act (FIFRA).
Close collaboration among 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 anthrax spores into the U.S. Postal Service system
resulted in the contamination of several facilities. Although most of the facilities where these letters were
processed or received in 2001 were heavily-contaminated, they were successfully remediated with
approaches such as fumigation with vaporized hydrogen peroxide (VHP®) or chlorine dioxide (CIO2),
including the HVAC ducts.12 While these decontamination methods have been studied extensively for
decontamination of surfaces found in the open spaces of a building (walls, floors, windows, etc.), this
research will help to determine the efficacy of the decontamination method within the confined spaces of
an HVAC system and on the materials found within these systems.
1.1 Process
The general process being investigated in this project is decontamination of 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. In terms of the surface of a
material, decontamination can be accomplished by physical removal of the contamination or via
inactivation of the contaminant with antimicrobial chemicals, heat, ultraviolet light, etc. Physical removal
could be accomplished via in situ removal of the contamination from the material or physical removal of
the material itself (i.e., disposal). Similarly, inactivation of the contaminant can be conducted in situ or
after removal of the material for ultimate disposal. During the decontamination activities following the
1
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results of the 2001 anthrax incidents, a combination of removal and in situ decontamination was used.3
The balance between the two was facility-dependent and factored in many issues (e.g., physical state of
the facility). One factor was that such remediation was unprecedented for the United States Government
and no technologies had been proven for such use at the time. The cost of disposal proved to be very
significant and was complicated by the nature of the waste (e.g., finding an ultimate disposal site).34
Since 2001, a primary focus for facility remediation has been improving the effectiveness and practical
application of in situ decontamination methods and evaluating waste treatment options to be able to
provide the information necessary to optimize the decontamination/disposal paradigm. This optimization
has a significant impact on reducing the cost of and time for the remediation effort.
In this study, the decontamination efficacy was evaluated for H2O2 vapor when used to inactivate Bacillus
spores inside a lab-scale HVAC system. Coupons of HVAC duct material were loaded with spores using a
deposition device. Test materials were 18 mm diameter coupons prepared from the same materials as
the duct. Test and procedural blank coupons were placed in the test duct and decontaminated as
described using H2O2 as the fumigant of choice for this project. After fumigation, the test coupon holders
were removed from each testing section of the duct, and the coupons were then removed from the
coupon holders for spore extraction and quantification. Positive control coupons (i.e., contaminated with
spores but untreated) were used to determine the pre-treatment (i.e., inoculum) loading on each coupon
type. Spores were extracted and quantified from the test coupons, positive control coupons, and QC
samples. Quality control (QC) samples included procedural blank coupons (coupons that underwent the
fumigation process but which were not inoculated) and negative controls (which did not undergo the
fumigation process)
1.2 Project Objectives
The primary objective of this project was to determine the efficacy of the H2O2 fumigation method on
deactivating spores inside an HVAC duct. For this project a STERIS 1000ED VHP® mobile
biodecontamination system was used to generate H2O2 vapor and inject it into the HVAC duct.
Secondary objectives were to determine the effect that flow rate, distance from injection point, flow and
pressure points at turns such as elbows, inlet concentration of fumigant, and fumigant residual effects
may have on the decontamination efficacy. The latter was determined based upon the comparison of the
number of spores (measured as colony forming units (CPUs)) recovered from positive control coupons
versus the recovery from test coupons. The static pressure inside the duct and the concentration of the
fumigant were measured at several locations along the duct. These parameters were used to
characterize the behavior of the fumigant inside the duct.
1.3 Experimental Approach
A closed loop duct was constructed and subjected to fumigation with H2O2 vapor under different operating
conditions. Inoculated coupons of the duct material, whether lined or unlined, were placed at different
points along the duct, flush with the duct surface, and exposed to the decontamination technique. The
efficacy of the decontamination method was measured by comparing the number of colony-forming
Bacillus spores recovered from these test coupons as compared to positive control coupons.
1.3.1 Testing Sequence
Testing was conducted in test ductwork fabricated in High-Bay Room 122-A at EPA's Research Triangle
Park facility. A test matrix was developed at the start of the testing campaign, and this matrix was
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sequentially modified as the results of completed tests were analyzed. In general, the testing sequence
was conducted as follows:
1. Sterilization of all coupons and materials needed for the test. The sterility of the coupons was verified
through the use of laboratory blank control samples.
2. Inoculation of test and positive control coupons with spores of 8. subtilis using a metered dose inhaler
(MDI).
3. Insertion of the test coupon holders loaded with a set of five coupons each (four test coupons and
one negative coupon) at eight defined testing locations along the length of the ductwork. These
locations were chosen specifically to determine a) the potential effects of temporal degradation of
fumigant in the duct, and b) the effect on efficacy due to differing flow patterns within the duct
including low pressure points at either angular or curved turns.
4. Application of a prescribed fumigation sequence with H2O2 vapor using a STERIS VHP® 1000ED
generator. The target test condition (fumigation concentration, duct flow rate, and exposure time) was
set and controlled at the inlet of the ductwork. Relative Humidity (RH) and temperature during testing
were monitored, but not controlled. The fumigant concentration was monitored continuously at three
locations (inlet, mid-, and at the end of the duct closed loop) to determine the concentration profile as
a function of length and time in the duct. After the exposure time was reached, the ductwork was
immediately aerated until fumigant concentrations were low enough to allow safe removal of the test
coupons for analysis.
5. Transfer of the test coupons, procedural blanks, and positive controls to the NHSRC Biocontaminant
Laboratory in sterile primary independent packaging within sterile secondary containment containing
logical groups of samples for analysis. All samples were accompanied by a completed chain of
custody (COC) form.
6. Quantitative assessment of initial viable spore loading by sampling and analysis of positive control
coupons.
7. Quantitative assessment of remaining viable spores on test coupons following treatment, and
quantitative assessment of spores on negative control coupons.
8. Determination of surface decontamination efficacy (comparison of viable spore concentrations from
positive controls and test coupons).
For the lined duct, a series of tests was added to the above testing sequence to determine sporicidal
efficacy of off-gassing H2O2 following the decontamination phase (which is defined in Section 2.2). After
removing the first series of test coupons subjected to the prescribed fumigation conditions, a second
series of inoculated coupons and blank coupons was loaded in the duct at one or two locations for a
quantitative assessment of residual decontaminant off-gassing (low decontaminant concentration) to
remove/inactivate the viable spores during an extended aeration phase.
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In addition to the steps outlined above, all test activities were documented during the activity via
narratives in laboratory journals, real-time data acquisition, and the use of digital photography. The
documentation included, but was not limited to, a record of time required for each decontamination step
or procedure, any deviations from the test plans, and physical impacts on the materials.
All tests were conducted in accordance with developed miscellaneous operating procedures (MOPs),
listed in Appendix A, to ensure repeatability and adherence to the data quality validation criteria set for
this project.
1.3.2 Definitions of Effectiveness
The sporicidal effectiveness (efficacy) of the decontamination technique is a measure of the ability of the
method to inactivate the spores on a contaminated material surface (i.e., represented by coupons in this
study). Efficacy is evaluated by measuring the difference in the logarithm (Log10) of the measured CPU
before decontamination (determined from sampling the positive control coupons) and after
decontamination (determined from sampling the test coupons) for the same type of material. The number
of viable spores was measured as CPU. This value is reported as a Iog10 reduction on the specific
material surface as defined in Equation 1-1.
(1-1)
It N N
JVc JV«
where:
Surface decontamination effectiveness; the average log
reduction of spores on a specific material surface (surface
material designated by;)
The average of the logarithm (or geometric mean) of the
number of viable spores (determined by CPU) recovered on
the positive control coupons (C indicates control and Nc is the
number of control coupons)
The average of the logarithm (or geometric mean) of the
number of viable spores (determined by CPU) remaining on
the surface of a decontaminated coupon (S indicates a test
coupon and Ns is the number of coupons tested).
When no viable spores were detected, a value of 0.5 CPU was assigned to the maximum plated volume
to determine the detection limit for CFUs,k and the efficacy was reported as greater than or equal to the
value calculated by Eqn. 1-1. The choice of 0.5 CPU as the detection limit allowed differentiation between
detect (1 CPU) and non-detect, a vital distinction in a field event.
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The standard deviation of the average log reduction of spores on a specific material (T\\) is calculated by
Eqn. 1-2:
Ns-l
d-2)
where:
_ Standard deviation of T|j, the average log reduction of spores
^ on a specific material surface
~j _ The average log reduction of spores on a specific material
'' surface (surface material designated by;)
The average of the log reduction from the surface of a test
-^k —
coupon (Equation 1-3)
Ns = Number of test coupons of a material surface type.
and
N,
(1-3)
where:
\og(CFUc) =
c
Represents the "mean of the logs" (geometric mean),
the average of the logarithm-transformed number of
viable spores (determined by CPU) recovered on the
control coupons (C = positive control coupons, Nc =
number of positive control coupons, k = test coupon
number and Ns is the number of test coupons)
CFUs,k = Number of CPU on the surface of the kth test coupon
Ns = Total number (1 ,k) of test coupons of a material type.
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2 Materials and Methods
2.1 Facility Design
Testing was conducted in a test ductwork that was fabricated in High-Bay Room 122-A at EPA's
Research Triangle Park facility. Figures 2-1 and 2-2 show a diagram of the test duct, and the actual
testing facility, respectively. The test duct consisted of 16-inch high by 8-inch wide, 18 gauge galvanized
steel duct work within secondary containment (a spray booth, containing an exhaust ventilation system
independent of the High Bay Building). The design was chosen to maximize duct length, provide complex
flow regions including elbows, and fit inside the spray booth chamber (secondary containment). The test
duct included both the square ell 90° turns typical of many HVAC systems and radial ells included to
reduce the total pressure drop. A blower (Model 7C651. modified with 1/4 horsepower (HP) inverter duty
motor, Dayton, Electric Manufacturing, Miles, IL) provided recirculation of fumigant within the ductwork,
when desirable. Due to the higher than normal pressure drop of this duct design, a larger 1/4 HP motor
was required on the blower to provide a full dynamic range of flow rates. The ductwork was made to
disassemble easily and be fabricated in both lined and un-lined forms. Sample ports were fashioned at
various points along the length of the duct to allow coupons to be inserted into the duct flush with the
inside surface of the duct. For the lined test condition, the duct was internally coated with Knauf Sonic XP
1.5# 1" fiberglass duct liner (Knauf Insulation, Shelbyville, IN).
Figure 2-1. Front (top left), Side (top right), and Top views (bottom) of Duct Design (motor and
round duct connections not shown in this diagram, see next figure)
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Figure 2-2. Photo of Actual Testing Facility
2.2 Hydrogen Peroxide Cycle
The H2O2 vapor in this study was generated using a STERIS VHP® 10OOED generator (referred to as
Vaporized Hydrogen Peroxide, or VHP®) loaded with a 35% H2O2 Vaprox®, cartridge. The STERIS
hydrogen peroxide product has been registered by EPA under FIFRA (Reg.# 58779-4). The STERIS
generator was operated with a closed control loop in-line with the duct testing facility (See Figure 2-3). To
control and monitor the concentration of H2O2 in the duct, three Analytical Technology Corp. (Collegeville,
PA) H2O2 electrochemical sensors (model B12-34-6-1000-1) were used to provide real-time concentration
readings. The H2O2 was injected at the duct blower outlet, and the first sensor (located downstream) was
used to control a solenoid valves (V1 and V2 in Figure 2-3) on the control loop. When the sensors
indicated the concentration was above the setpoint, V1 and V2 were switched to the bypass loop.
Sensors located at the duct mid- and end-points were used for monitoring purposes only.
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STERIS
1000ED
Figure 2-3. Control Loop Schematic
Two controllers of the STERIS VHP® 1000ED store the target operating conditions including the desired
time for each fumigation phase, operating pressure, H2O2 injection rate, airflow rates, and target RH. The
controllers also monitor the amount of hydrogen peroxide available in the reservoir and the dryer
capacity.
After the hydrogen peroxide solution reservoir is filled, a VHP® fumigation cycle was programmed to
include three operational phases: Conditioning, Decontamination, and Aeration. To initiate the cycle,
hydrogen peroxide is first pumped from the cartridge to a reservoir.
• Conditioning Phase: The STERIS VHP® 10OOED pulls 17 acfm of air from the duct, pushes it through
a desiccator and a High-Efficiency Particulate Air (HEPA) filter. This dry filtered air is then returned to
the duct, with H2O2 vapor injected into the air stream just before it leaves the STERIS VHP® 1000ED
with a controllable (1-12 g/min) injection rate. The condition phase facilitates reaching the desired
decontamination concentration more quickly in larger sealed enclosures. The condition time is
affected by sterilant injection rate and enclosure volume. The conditions were selected for the
purpose of reducing the total cycle time. Use of the condition phase does not reduce the time of
exposure during the Decontamination Phase.
• Decontamination Phase. A constant flow of the H2O2 vapor/HEPA-filtered air mixture is maintained at
the selected H2O2 injection rate, within the controllable range. The Decontamination time was set for
the length of the test (90 or 240 minutes) with the injection rate adequate to maintain the H2O2
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concentration. The control loop helped improve precision and prevent overshoot with regard to H2O2
vapor concentration.
• Aeration Phase. There are two stages of the Aeration Phase, one provided by the STERIS VHP®
1000ED, and one provided by the PDAQ control system. For the STERIS stage, H2O2 vapor injection
is stopped and the recirculation flow of dry HEPA-filtered air through a catalyst at 17 acfm continues
for 4 hours to reduce the H2O2 concentration within the enclosure. In addition to the STERIS aeration,
a pressure relief blower was used to remove air from the duct and pass it through activated carbon
before release. Laboratory air was used to replace air removed from the duct.
2.3 Coupon Preparation
2.3.1 Test Coupons
Test materials were 18 mm diameter coupons prepared from the same materials as the duct: 18 gauge
galvanized steel (P/N 01170, Eastcoast Metal Distributors, Durham, NC) and liner (Knauf 1.5# 1"
fiberglass. Shelbyville, IN). The liner coupon consisted of a 1 mm-thick slice of the liner (including the
inner, intended surface of exposure) affixed to a galvanized stub using double-sided adhesive tape (P/N
16073-2, Ted Pella, Inc., Redding, CA). The coupons were fastened to 18 mm aluminum stubs (P/N
16119, Ted Pella, Inc., Redding, CA) using an adhesive-backed magnet (P/N 5775K8, McMasterCarr.,
Atlanta, GA). The galvanized coupons were sterilized prior to use by steam autoclave consistent with
NHSRC Biocontaminant Laboratory MOP 6570 (Appendix A). Liner coupons were sterilized using
ethylene oxide (Anderson EOGas Sterilizer, Haw River, NC). Appendix A lists all of the associated MOPs,
which can be found in the project Quality Assurance Project Plan (QAPP) for the Evaluation of Medium
and High Tech Methods for HVAC Decontamination.5
A set of five coupons (four test coupons, and one negative coupon) was collocated on a test coupon
holder (Figure 2-4) and inserted at each testing location immediately before the start of the test. Magnetic
seals were used to ensure that the coupons were aligned with the corresponding holes in the duct. The
test and procedural blank coupon holders were designed so that the surface of the coupon would be
planar with the inner surface of the duct, thereby minimizing flow disruptions.
-------
Magnet
Figure 2-4. Test Coupons Holder Setup
2.3.2 Positive Control Coupons
The positive control coupon holders are slightly different from the test coupon holders, as shown in Figure
2-5. Two to three holders were utilized for each test. Positive controls were inoculated at the beginning,
middle, and end of the test coupon inoculation sequence to ensure that inoculations were equal across all
test coupons.
.
Figure 2-5. Positive Control Coupon Holder
10
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2.3.3 Spore Preparation
The test organism for this work was a powdered spore preparation of 8. subtilis (American Type Culture
Collection (ATCC) 19659; Manassa, VA) 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 metered dose inhalers (MDIs)6 by the U.S. Army Edgewood
Chemical Biological Center (ECBC) according to a proprietary protocol.7 8 Quality assurance
documentation is provided by ECBC with each batch of MDIs. Control checks for each MDI were included
in the batches of coupons contaminated with a single MDI.
2.3.4 Coupon Inoculation and Test Preparation
Coupons of different types of HVAC materials were inoculated (loaded) with spores of 8. subtilis using an
MDI. The deposition of spores onto the coupons is conducted in accordance with a procedure detailed in
MOP 3157 included in Appendix A. In brief, the inoculation procedure involves placing the coupon (18
mm-diameter galvanized HVAC material with or without duct liner attached) on a sterile stub (1 8 mm-
diameterSEM pin stub, Ted Pella, Redding, CA) used for inoculation, and placing it at a precise distance
from an MDI during actuation. Following inoculation, the coupon is transferred to a new sterile stub, and
the original inoculated stub is discarded. This process is repeated for each coupon. To avoid biases
among the positive controls and the test coupons, the following spore loading sequence was adopted:
1 . Inoculate the first set of four positive control coupons (4 total)
2. Inoculate the first four sets of four test coupons (1 6 total)
3. Inoculate the second set of four positive control coupons (when present, 4 total)
4. Inoculate the second four sets of four test coupons (1 6 total)
5. Inoculate the last set of four positive control coupons (4 total)
The MDIs are set to provide up to 200 discharges before degradation of spore concentration. The number
of discharges per MDI was tracked so that use did not exceed this value. Additionally, in accordance with
MOP 3157, 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
3157, it was retired and a new MDI was used.
A log was maintained for each set of coupons that were dosed via the method of MOP 31 57. Each record
in this log contained the unique coupon identifier, the MDI unique identifier, the date, the operator, the
weight of the MDI before dissemination into the coupon dosing device, the weight of the MDI after
dissemination, and the difference between these two weights. After inoculation, the coupons were
aseptically transferred to sterilized coupon holders. Each test required the inoculation of 32 test coupons
and 12 positive control coupons.
2.4 Test Matrix
This work was accomplished in several tests for lined and unlined HVAC ducts, using H2O2 generated by
the STERIS VHP® 1 0OOED as the fumigant of choice. The test matrix shown in Table 2-1 represents the
overall work performed under this project and reflects the modifications to the operating parameters such
11
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as fumigation concentration, exposure time, and flow rate being necessitated as each test's results were
reviewed and evaluated. Note that the numbering for this series of tests starts at 13 since it is part of a
larger matrix outlined in the QAPP entitled "Quality Assurance Project Plan for the Evaluation of Medium
and High Tech Methods for HVAC Decontamination"5, and includes testing of other volumetric
decontaminants such as chlorine dioxide gas and fogging technologies. The numbering scheme for these
tests was kept consistent with the QAPP in order to avoid confusion upon completion of the other phases
of the test plan.
Table 2-1. Test Matrix
Test*
13
14
15
13b
16xa
14b
01
01p
02
02p
03
04xa
04p
Fumigant
H202
H202
H202
H202
H202
H202
H202
H202
H202
H202
None
H202
H202
Concentration (ppm)
250
250
250
250
250
250
250
residual
250
residual
0
50
residual
Exposure time (min)
240
90
240
240
240
90
240
1440
90
1440
90
90
1440
Blower speed
15 Hz
15 Hz
15 Hz
15 Hz
60 Hz
15 Hz
15 Hz
OHz
15 Hz
OHz
15 Hz
15 Hz
OHz
Lined
No
No
No
No
No
No
Yes
Yes
Yes
Yes
Yes
Yes
Yes
a. Test added during the course of the testing program
2.5 Sampling and Analytical Procedures
2.5.1 Test Facility Sampling Procedures
2.5.1.1 Sampling/Monitoring Points
Coupon locations along the test duct were chosen to capture a wide range of in-duct variability in the
dynamic HVAC duct environment. The parameters of interest included the following:
• Distance from the injection/monitoring point. This measurement potentially provided information about
the degradation of the fumigant as it traveled through the duct.
• Height inside the duct. The flow of the air through the duct was expected to be turbulent; however the
highly convoluted flow pattern could produce a stratified flow. Efficacy at each location was measured
in quadruplicate (i.e., four replicate coupons per sample location, each at spatially distinct positions
with regards to height within the duct). A stratified flow was expected to manifest itself as a trend in
efficacy as a function of height inside the duct.
12
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• Pressure points at turns. The flow pattern was expected to have high pressure points on the outside
of 90° turns and low pressure points at the inside of the turns. Sampling locations were chosen at
both points at the same turn (hence the same distance from injection point). Boundary layers could be
thicker at the low pressure points, with lower fumigant concentration reaching the spores.
Other measurements include fumigant concentration, differential pressure (related to flow), RH, and
temperature. Figure 2-6 shows all sampling and monitoring locations in the duct. The frequency of
sampling and monitoring events is presented in Table 2-2. Table 2-3 lists the critical and non-critical
measurements for each sample.
VHP return
Blower
VHP injection —'
RH sensor
VHP sensor
Figure 2-6. Duct Testing Facility with Sampling and Monitoring Locations Indicated by
Letters A-H
13
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Table 2-2. Frequency of Sampling Monitoring Events
Sample Type
Test coupon
Negative control
coupon
Positive control
coupon
Field blank coupons
Laboratory blank
coupons
Biocontaminant
Laboratory material
blanks
H2O2 monitors
H2O2 wet chemistry
samples
H2O2 wet chemistry
sample blank
Flow rate
Pressure of Duct
RH/Temp
Sample
Number
4 per sampling
location, each
at a spatially
distinct height
within the duct
1 per sampling
location
8 to 12 -a set
of 4 inoculated
at the
beginning,
middle, and
end of test
coupon
inoculations
3 coupons
which are co-
located with
test coupons
3 sterile
coupons
3 per material
3 real-time
instruments
3 every 2 hours
1
1
4
4
Sample/Monitoring
Frequency
1 set per location
per fumigation
1 set per location
per fumigation
1 set per inoculation
1 set per inoculation
1 set per fumigation
One set per use of
material
Realtime during
H2O2 fumigations
Once per port every
2 hours
1 perH2O2
fumigations
Logged every 10
seconds
Logged every 10
seconds
Logged every 10
seconds
Sample Location
Shown in Figure 2-6 as
letters A-H
Shown in Figure 2-6 as
letters A-H
NA
NA
NA
NA
Shown in Figure 2-6 at
three locations
Shown in Figure 2-6 at
three locations
NA
Collocated with RH
sensors shown in
Figure 2-6 at 4
locations
Co-located with RH
sensors shown in
Figure2-5 at 4 locations
Shown in Figure 2-6
Purpose
To determine the number
of viable spores after
fumigation
To determine extent of
cross-contamination
To determine the number
of viable spores deposited
onto the coupons
To determine extent of
cross-contamination
To demonstrate sterility of
coupons and extraction
materials.
To demonstrate sterility of
extraction and plating
materials
To determine exposure
experienced by the
coupons and to determine
and degradation within the
duct
To verify proper operation
of H2O2 monitors
To demonstrate correct
operation of MOP 31 43
To determine the flow rate
within the duct.
To help determine the leak
rate of the duct
To determine
environmental conditions
inside the duct
14
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Table 2-3. Critical and Non-Critical Measurements
Sample Type
Test coupon
Negative control coupon
Positive control coupon
Field blank coupons
Laboratory blank coupons
Biocontaminant Laboratory material
blanks
VHP monitors
Flow rate
Pressure in duct
RH/Temperature
Critical Measurements
Plated volume, incubation temperature, extracted
volume, CFU
Plated volume, incubation temperature, extracted
volume, CFU
Plated volume, incubation temperature, extracted
volume, CFU
Plated volume, incubation temperature, extracted
volume, CFU
Plated volume, incubation temperature, extracted
volume, CFU
Plated volume, incubation temperature, extracted
volume, CFU
hbCb concentration
Velocity pressure across duct
NA
RH and temperature of duct
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 and RH of
duct
Pressure in duct, relative
to atmospheric pressure
NA
NA= Not applicable
2.5.1.2 Electrochemical Sensor for H2O2 Concentration Measurement
H2O2 concentration within the duct was monitored using Analytical Technology Corp. (Collegeville, PA)
electrochemical sensors (model B12-34-6-1000-1). The sensors are factory preset to measure from 0 to
1000 ppm H2O2 within an accuracy of 5% of the measured value.
The sensors were also calibrated before each test by exposing the transmitter to the head space of a
known concentration and temperature of hydrogen peroxide solution. MOP 3136 describes the details of
the general procedure for calibration of ATI H2O2 transmitters using wells.
2.5.1.3 Duct Flow Rate
,TM
Pressure differential traverses were performed on the straight line duct using the AIRDATA
MULTIMETER ADM-860 electronic micro manometer from Shortridge Instruments, Inc. (Scottsdale, AZ).
This meter measures air velocities and differential pressures when used with a pitot tube and
automatically corrects for density variation due to local temperatures and barometric pressures. A
sampling grid of 24 points was created (3 horizontal lines at 6, 8, and 10 inches from the vertical direction
of the duct and eight equally spaced sampling points along the three horizontal lines.
2.5.2 Microbiological A nalysis
The NHSRC Biocontaminant Laboratory analyzed all samples qualitatively for spore presence (swab
samples) or quantitatively for the number of viable spores per coupon sample.
15
-------
Details of the sampling and analysis procedures are provided below. A laboratory notebook was used to
document the details of each sampling event (or test).
2.5.2.1 Coupon Spore Enumeration
The day after duct fumigation, 18 mm test, procedural blanks, and positive control coupons were
transferred aseptically into empty 50 ml sterile vials. This operation was performed in H122 at the site of
the duct, so that no spores would be lost in the transfer. The sample vials were then transported to the
NHSRC Biocontaminant Laboratory, where 10 ml of sterile Phosphate Buffered Saline plus Tween®20
(PBST) was aseptically added. The sample vials were then sonicated for 10 minutes using an 8510
Branson (Danbury, CT) ultrasonic cleaner at 44 kHz and 250 Watts. The sonication step was immediately
followed by two continuous minutes of vortexing to further dislodge any viable spores. Each vial was
briefly re-vortexed immediately before any solution was withdrawn for analysis. The solution was
subjected to a five-stage serial dilution following MOP 6535a. Each dilution (0.1 ml) was inoculated onto
trypic soy agar (TSA) plates, spread with sterile beads according to MOP 6555, and incubated at 35 ± 2
°C for 18-24 hours. Plates with 30-300 CPU were counted manually. Any samples below countable
criteria (30 CPU) on the primary dilution plates were filtered following MOP 6565. The filters were
incubated at 35 ± 2 °C for 18-24 hours prior to manual enumeration.
2.5.2.2 Swab Samples
Swab sampling was used for sterility checks of the ductwork prior to each use in the testing. A swab
equipped with a long handle was used to sample each of the eight test points (A through H) in Figure 2-6.
MOP 6563 was followed for collection of swab samples. Swabs were streaked onto TSA and incubated at
35 ± 2 °C for 18-24 hours prior to qualitative growth analysis (presence / absence determination).
2.5.2.3 Method Verification
While there are no approved methods for spore enumeration, the use of positive control samples as the
baseline for log reduction calculations includes a built-in verification of the deposition and enumeration
methods.
2.6 Sampling Handling and Custody
2.6.1 Prevention of Cross-contamination of Sampling/Monitoring Equipment
Several management controls were instituted to prevent cross-contamination. This project was labor
intensive and required that many activities be performed on coupons that were intentionally contaminated
(test coupons and positive controls). Specific procedures were put in place in the effort to prevent cross-
contamination among the groups. Adequate cleaning of all common materials and equipment was critical
in preventing cross-contamination.
There were four primary activities for each test in the experimental matrix. These activities were
preparation of the coupons, execution of the decontamination process (including sample recovery),
sampling, and analysis. The unlined duct coupons were sterilized prior to use by steam autoclave utilizing
a gravity cycle program consistent with NHSRC Biocontaminant Laboratory MOP 6570. Lined coupons
were fumigated with ethylene oxide using an Andersen (Haw River, NC) EOGas 333 sterilization system
to prevent the heat of the autoclave cycle from melting the liner. Specific management controls for each of
the three following activities are described below.
16
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2.6.2 Preventing Cross-Contamination during Execution of the Decontamination
Process
The following management controls were followed in an effort to minimize the potential for cross-
contamination:
• Negative control coupons were present for each test location. Growth on these coupons would
indicate contamination during fumigation or handling.
• Swab samples were taken from inside the duct following the sterilization (reset) fumigation. Growth of
these swab samples would indicate the failure of the sterilization fumigation, and new conditions
would be assigned to the sterilization fumigation. Nearly all initial swabs indicated that the duct was
sterile following reset. In a few instances swab samples indicated the presence of residual
background contamination and sterilization conditions were revised and conducted to reset the duct.
2.6.3 Preventing Cross-Contamination during Sampling
Sampling poses an additional significant opportunity for cross-contamination of samples. In an effort to
minimize the potential for cross-contamination, several management controls were followed.
• Only one coupon holder was handled at a time. Only the outside surfaces of the holders were
touched.
• The coupons were placed in the sterile 50 ml conical tube immediately following post-
decontamination, at the site of the duct.
• The coupons were constructed as separate removable discs, so that the stub did not transfer any
cross-contaminants.
• Cross-contamination was tracked by the negative in situ coupons.
2.6.4 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 was always as follows: (1) all blank coupons; (2) all test
coupons; and (3) all positive control coupons.
2.6.5 Sample Quantities
The sample quantities were outlined previously in Table 2-2. In brief, for each test in Table 2-1, there
were eight coupon sample locations, which yielded 32 test coupons, 8 negative controls for the test
coupons, 8 to 12 positive control coupons, 3 field blank coupons, and 3 laboratory blank coupons.
2.6.6 Sample Containers for Collection, Transport, and Storage
Samples were initially held in the sample holders designed to attach to the duct. These holders were
removed from the duct, and sterile forceps were used to transfer samples to individual, sterile 50 ml
conical tubes. Swabs of the duct interior were placed in the sterile swab containers and then bagged in
17
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two individual sterile sampling bags as secondary and tertiary containment, prior to transfer to the
NHSRC Biocontaminant Laboratory.
After sample collection for a single test was complete, all biological samples were transported to the
NHSRC Biocontaminant Laboratory immediately, with appropriate COC form(s). Samples were stored (4
± 2 °C) no longer than five days before the primary analysis. Typical hold times, prior to analyses, for
most biological samples was < 2 days. All samples were allowed to equilibrate at room temperature for
one hour prior to analysis.
2.6.7 Sample Identification
Each coupon was identified by 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 coupons were transferred to the APPCD Biocontaminant
Laboratory for microbiological analysis, each sample was additionally identified by replicate plate (Petri
dish) number and dilution. Table 2-4 specifies the sample identification. The NHSRC Biocontaminant
Laboratory also included the date each plate was placed in the incubator.
Table 2-4. Coupon Sample Coding
Coupon Identification: 65-TN-LC-RS
Category
65
TN
LC(p)
Location Code
RS
Replicate Sample
Example
Code
65
01
A(P)
PA, Pp(1-2)
PM
PZ, Pp (3-4)
FB
BN
1
Work Assignment designation
Test Number (from Table 2-2)
A through H as shown in Figure 2-1 .(p) denotes post test off-gassing sample
First set of positive controls (at beginning of puffing)
Middle set (if applicable) of positive controls
Last set of positive controls(at end of puffing)
Field Blank
Negative stub sample
The replicate sample ID is dictated by the placement in the holder or stage. The
positive control RS is shown in Figure 3-2, while the sample RS will be similarly
stamped with the numbers 1 through 5. Field and laboratory blank samples are
interchangeable, and are simply assigned a value of 1 through 3 in the order of
processing.
Biocontaminant Lab Plate Identification: 65-TN-LC-RS -R-D
65-TN-LC-RS
R
(Replicate)
D
(Dilution)
As above
R
1
A-C
0 to 4, corresponding to 1 x 10" to 1 x 10"4
18
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Swabs collected as sterility checks were identified by the code 65-TN-SW-LC. The swabs were collected
from each sample location shown in Figure 2-6 according to MOP-3135.
2.6.8 Sample Custody
Careful coordination with the NHSRC 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
accurate written record that can 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 can tamper with it
• In a secured area, restricted except to authorized personnel
• In transit
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.
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 Highbay room H130-A to the NHSRC Biocontaminant
Laboratory.
• If the custodian had not been assigned, the laboratory operator had the responsibility of packaging
the samples for transport. Samples were carefully packed and hand-carried between on-site
laboratories. The COC record showing the identity of the contents accompanied all packages.
2.6.9 Sample A re hiving
All samples and diluted samples were archived for 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 ± 2 °C in a sealed 50 mL conical tube.
19
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3 Results and Discussion
This section presents the results of each test, with details on how and why the concentration, exposure
time, and flow rate parameters were modified for subsequent tests. The investigation of the effectiveness
of H2O2 fumigation required some initial characterization of the duct flow rate, flow pattern, and low
pressure at turns at angular and curved elbows, before commencement of the biological testing. The
results of the duct flow characterization are discussed in Section 3.1. The results of the decontamination
testing for unlined HVAC duct and lined HVAC duct are reported and discussed in Sections 3.2 and 3.3,
respectively. Note that some additional tests were incorporated in the lined duct test matrix to investigate
the effect of out-gassing on the decontamination effectiveness.
3.1 HVAC Duct Flow Characterization
The air velocity inside the unlined duct was characterized as a function of the blower speed and sampling
location. A variable frequency inverter was used to operate the blower at three speeds (15 Hz, 30 Hz, and
60 Hz) that resulted in calculated Reynolds (Re) numbers for the unlined duct all above 105. This value
suggests that the overall bulk flow inside the duct is highly turbulent at all tested flow rates. The flow rate
in the lined duct was not measured, but is still expected to be in the turbulent flow region.
3.1.1 Flow Velocity versus Blower Speed
The flow velocities inside the duct were characterized at the three blower speeds by performing pitot tube
traverses on the straight line of the duct (Location H before the inlet of the blower and location A
downstream of the outlet of the blower).
The velocity profiles at locations A and H inside the duct are shown in Figure 3-1 for each blower speed.
The results show that the flow velocities vary linearly with the speed of the blower, and minimal losses are
registered between the two locations, A and H.
20
-------
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-^^^ — ^ — * rs^^^
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-<-(60Hz, H)
r~^^$ i ^ i T f ]
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Transversal locations inside the duct
Figure 3-1. Pre- and Post-Blower Velocity Traverses Inside the Duct
Note that the geometry of the duct did not provide a position with straightened flow, thus the standard
U.S. EPA Method 29 procedure for measuring flow could not be followed.
3.1.2 Flow velocity Profile near an Elbow
The flow pattern near a round elbow (Location D and E) was characterized at three blower speed ratings
(15 Hz, 30 Hz, and 60 Hz), using a sampling number of 48 points (a 3 x 16 grid).
These measurements were made in the plane of the duct that includes Sample Location E (zero inches
inside the duct) and Sample Location D (16 inches inside the duct). The results shown in Figure 3-2
demonstrate that the flow is affected by the elbow upstream of Location E, with higher flow outside the
bend of the elbow (1 to 8 inches) and decreasing on the inside of the bend (9 to 16 inches) causing flow
reversal and flow separation. While the total flux of fumigant across this plane of the duct is equal to the
total flow rate of the system, the flux at any one point is unknown due to the flow separation. It is unknown
whether there was any flux at sample location D, or whether the flow was simply recirculation. The
calculated bulk Re was greater than 4000, a benchmark for the transition from intermediary to turbulent
flow.
The design of the duct system did not allow similar measurements to be performed at other locations with
preceding elbows, due to the limited space at these locations.
21
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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
Transversal locations inside the duct (in)
Figure 3-2. Velocity Traverse Inside the Duct at Locations D and E
3.2 H2O2 Fumigations - Unlined Duct Results
The first series of tests was completed on the unlined HVAC duct at a H2O2 concentration time of 250
ppmvfor4 hrs per the crisis exemption under Section 18 of FIFRA that authorizes EPA to allow an
unregistered use of a pesticide for a limited time if EPA determines that an emergency condition exists
(http://www.epa.gov/opp00001/factsheets/chemicals/vhp_factsheet.htm). The STERIS registration claim
is that Vaprox® hydrogen peroxide is effective as a Sterilant, Sporicide, Bactericide, Virucide, and
Fungicide at 250 ppm for 90 minutes in sealed enclosures up to 4,000 ft3.
http://www.epa.gov/pesticides/chem search/cleared reviews/csr PC-000595 3-Apr-06 a.pdf.10 The
second parameter investigated was the blower speed (15 Hz and 60 Hz) to determine the effects of the
flow velocity, if any, on the fumigant sporicidal effectiveness.
As discussed in Section 2.4.1, there were three locations for H2O2 and RH sensors. Table 3-1 shows the
average H2O2 concentration during fumigations. Location A was nearest the point of injection, and
Location H was farthest from injection. Spikes in H2O2 concentration were typically short-lived.
22
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Table 3-1. Average H2O2 Concentrations and RH during Fumigation
Test 13
Test13b
Test 14
Test 14b
Test 15
Test 16
H2O2 Location
A (ppm)
Average/
(± Standard
Deviation)
247(±37)
249 (±11)
250(±9)
255 (±10)
243 (±27)
242(±21 )
H202
Location D-E
(ppm)
Average/
(± Standard
Deviation)
232 (±34)
246 (±13)
232(±9)
231 (±10)
244(±27)
230 (±20)
H202
Location H
(ppm)
Average/
(± Standard
Deviation)
225 (±34)
215(±11)
219(±10)
221 (±12)
228(±26)
208 (±19)
RH
Location
A(%)
58.2
48.0
47.3
75.1
51.8
45.1
RH
Location
D-E(%)
59.0
47.7
49.8
72.7
52.4
44.4
RH
Location H
(%)
59.8
51.0
49.1
79.0
56.7
47.3
The measured concentration at Location H was consistently lower than the other two locations. The
sensor may or may not have been in a position of high flux, but the response time was quick for all
sensors, as shown in Figure 3.3. This observation suggests that there was some degradation of H2O2 in
the duct. The products of H2O2 decay include water, so, if there was decay of the H2O2, a rise in RH
throughout the duct may be expected. The generally rising RH values in Table 3-1 during some tests may
further indicate H2O2 decay as the vapor traverses the duct (from A to H).
400
o J
12:20:10 12:20:53 12:21:36 12:22:19 12:23:02 12:23:46 12:24:29 12:25:12 12:25:55
Figure 3-3. Sample Response Time for the ATI Sensors
23
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Two or three sets of positive control coupons were inoculated alongside test coupon sets. These positive
control coupons were done at the beginning and the end, and for later tests, also in the middle of
inoculations. The CPU recovered from these sets of coupons are shown in Table 3-2.
Table 3-2. Positive Controls Inoculation Results (n = 4)
Test 13
Test13b
Test 14
Test 14b
Test 15
Test 16
First set (CPU/sample)
2.95E+07 + 2.09+07
1.36E+07 + 6.57+06
1.46E+07 + 3.81+06
1.53E+07 + 1.02+07
2.52E+07 + 1.26+07
5.98E+06 + 2.10+06
Middle set (CPU/sample)
2.16E+07 + 2.04+07
8.95E+06 + 4.04+06
End set (CPU/sample)
2.39E+07 + 1.47+07
1.39E+07 + 8.36+06
6.03E+06 + 1.75+06
1.47E+07 + 6.90+06
1. 71 E+07 + 2.69+06
7. 15E+06 + 6.00+06
While all of these values met the target dose QA requirements and allow for a 6 log reduction, care must
be taken when interpreting the data not to compare LR values without considering the initial loading.
There was high variability in the post-decontamination recovery (efficacy) data between tests. Tests 13,
13b, and 15 were all replicate tests. The average CPU recovered from these replicate tests are shown in
Table 3-3 and Figure 3-4.
Table 3-3. Average CPU Recovered from Test Coupons from Unlined Duct Sample Points (n = 4)
Test ID
13
13b
15
Location
A
1
29
1
Location
B
1
11300
Samples
lost*
Location
C
1
1
1
Location
D
3500
2
1.88x106
Location
E
1
3
463
Location
F
5
7
1200
Location
G
1
4.51 x105
5.08 x 10s
Location
H
1
1
1910
These samples were mistakenly absent from the duct during exposure.
24
-------
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5 °
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A
i^
B
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c
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7-
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4-
3-
2 -
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n
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i
F
T
1
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F
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i
G
T
J_
1
G
i
H
T
1
1
H
Sample Location
Figure 3-4. Recovery of Positive Controls (green bars) and Spatial Efficacy Results (gray bars)
for Unlined Duct (250 ppm x 4 hours). Efficacy data are reported as mean log reduction
(gray bars) from four replicate coupons per location. Green bars represent mean Log10
positive control recoveries from one replicate coupon collected from each of the eight
sampling locations. Sample locations (A through H) from Figure 2-6.
These data show the difficulty in replicating fumigations for localized efficiency measurements, as there
was much variation both within the duct for a particular fumigation and within a single location between
fumigations. Reasons for the high variability include the following:
25
-------
• Unknown, non-linear kill kinetics: Small variations in RH or spikes in H2O2 concentration could be
much more effective against spores than the average condition.
• Leaks from the coupon holders may have offered protection to some coupons, (i.e., in areas of low
local pressure, a curtain of fresh air entering the duct near the coupon could have protected it from
the fumigant)
• Flow patterns in the duct may depend on (variable) initial conditions.
Several conclusions can be drawn from these data. For sets of coupons that were collocated a distance
from the blower near a flow disturbance, one set, or side, experiences higher fumigant flux than the other
side. The high flow at Location E and low re-circulating flow at Location D (discussed in Section 3.1),
seems to have influenced the efficacy of the fumigant. Location G was also much more difficult to
decontaminate than Location F, located across the duct. Location G would seem to be on the high
pressure side of the curve, but the duct was intentionally designed to create complex flow patterns that
were not easily predicted. Small perturbations in the inside of the duct may have directed flow downward
at that location.
Table 3-4 shows log reduction values for all tests as a function of coupon location. Cells with values
based on detection limit values have been colored blue. Cells with values based on a very small number
(<10) of spores are indicated in red. The lack of contamination observed on any of the negative control
coupons suggests these values were not caused by cross-contamination. A comparison between 90-
minute exposures and 240-minute exposures suggests that, while longer exposure times may provide
higher efficacy, there is no guarantee that higher efficacy will occur, suggesting a non-linear kill curve.
Rather than higher flow rates inside the duct improving contact of the fumigant to the coupons, increasing
blower speed seems to have offered some protection to the spores (T-test comparing 15 Hz LR after 240
minute exposure to 60 Hz LR gives a p-value of 0.0003). Further investigation is needed to explain this
outcome.
Table 3-4. Average Log Reduction During Testing of the Unlined Duct by Sample Location (n = 4).
Blower
Setting
(Hz)
15
60
Exposure
(min)
240
90
240
A
>7.50
5.86
>7.55
>7.22
B
>7.50
5.83
6.57
>7.32 | >7.33
3.92 | 6.28
C
>7.57
>7.38
>7.54
6.50
>7.32
5.40
D
6.47
7.01
1.71
2.15
6.73
4.62
E
>7.57
7.05
6.66
5.16
>7.32
4.29
F
7.02
6.49
5.83
3.88
5.46
4.98
G
>7.57
3.47
2.95
2.51
2.44
3.25
H
>7.50
>7.38
6.30
3.61
>7.33
>6.99
Avg
7.34
6.31
5.50
4.70
6.41
4.97
SD
0.39
1.30
2.29
1.94
1.73
1.23
NOTE: Data in blue cells are based upon detection limit values (no CPUs detected), LR data in red cells are based upon
low post-decon recoveries (<10 CPU).
26
-------
3.3 H2O2 Fumigations - Lined Duct
Internally lined HVAC duct presented a much different fumigation scenario. There are two main
differences in the behavior of airflow in the lined duct versus the unlined duct:
• There are fewer leaks in the lined duct because the presence of the liner covers gaps in the duct
joints. Note that the presence of the liner did not change the possibility of leaks near the coupon
holders.
Note: While leaks were anticipated in both the lined and unlined ducts, these leaks were very minimal
compared to the total amount of bulk airflow inside the ductwork. In addition, the ducts were
constructed using materials and methods typical of residential and commercial ductwork, and
thus any leaks experienced are expected to mimic real-world conditions.
• The liner adsorbs and desorbs fumigant leading to longer aeration times and longer exposures. This
phenomenon is not specific to our facility, but will vary as a function of the material and liner
manufacturer.
The second difference means that the aeration phase of the lined duct is fundamentally different from
unlined duct, even with the exact same fumigation conditions. Due to the desorption of the fumigant over
a long period of time, a series of tests was completed to determine the sporicidal effectiveness of the
fumigant at low fumigant concentration exposure (i.e., low concentrations resulting from fumigant
desorption following a fumigation). A test blank run (no fumigant added) was added to the test matrix to
evaluate any non-fumigant related sporicidal effect on the test coupons. The blank test was conducted
and sampled the same way as the other test runs.
3.3.1 Exposure Phase
The test and procedural blank coupons were present in the duct during the conditioning phase, the
decontamination phase, and for the aeration phase, for a total of approximately 24 hours. It is important to
understand the difference in the fumigation minutes and the exposure minutes for the lined duct. For the
unlined duct, there was no measurable material absorbance and, at the end of the fumigation, the
fumigant concentration declined rapidly. The lined duct, however, exhibited significant desorption during
the aeration period. Figure 3-5 shows a trace of the control sensor during all phases of exposure for a
lined and unlined test at 250 ppm for 4 hours, as well as the hysteresis response of the sensor when
being removed from exposure directly to ambient air.
The trailing concentration during aeration phase of the lined duct (about 30 ppm) contributed a significant
portion of the total exposure in terms of Concentration x Time (CT) or ppm*hours. For instance, the CT
target fumigation was 1000 ppm*hours (250 ppm x 4 hours) for Test 1, but the overall exposure was 75
percent higher due to the contribution of the aeration phase. The contribution of the aeration phase was
an even greater portion of the overall exposure for Tests 02 and 04.
The mean log reduction values for each test are shown in Table 3-5.
27
-------
20
Figure 3-5. H2O2 Concentration during Exposure and Aeration Phases for the Lined and Unlined
Duct
Table 3-5. Average Log Reduction in Duct (n = 32)
Test
Exposure
Test 01
Test 02
Test 04
Exposure
ppm
250
250
50
Fumigation
Minutes
240
90
90
Total
pprrfhours
1760
1400
280
AvgLR
>7.4
>7.3
4.6
RSD
0.1%
1.3%
18.9%
Both fumigation conditions used in Test 01 and Test 02 were very effective, with recovery of fewer than
10 CPU for all samples. Though the exposure time seemed different, the exposure in terms of ppm*hours
was quite similar because of desorption during the aeration phase.
Test 4 was performed with the aim of determining the minimum exposure needed for decontamination.
Ideally, the CT for Test 4 would have been similar to the target CT for Test 02 (375 ppm*hours), but the
kinetics of adsorption/desorption were not well enough understood to predict accurately. The Test 04
conditions were deemed moderately effective, providing only a 4.6 log reduction.
3.3.2 Desorption from Lined Duct
Figure 3-6 shows the sensor responses during Test 01, showing the H2O2 concentration in the lined duct
over a period of 48 hours. The test and procedural blank coupons experienced the CT represented as the
integration of the concentration curve to the left of the first vertical line. The concentration inside the duct
seems to have increased while those coupons were removed (the bump between the two vertical lines in
Figure 3-6), perhaps because of the physical disturbance of the duct or changes in the movement of air
28
-------
around the sensors during this operation. Coupons for the desorption test were placed inside the duct
after the exposure coupons were removed (the CT to the right of the second vertical line).
—ATI 4
—ATI 5
Aflfr
Exposure Coupons -
Desorption Coupons
Figure 3-6. H2O2 Concentration during the Two Exposure Periods (initial exposure and
subsequent desorption) for the Lined Duct (Test 01).
Table 3-6 shows the average concentration, the CT, and the LR of the coupons during the desorption
test. Test 3 (discussed below) was conducted identically to Tests 01, 02, and 04, but with no exposure to
H202.
Table 3-6. Conditions and Efficacy during Desorption Tests
Test ID
Test 01 p
Test 02p
Test 03
Test 04p
Average H2O2
ppm
(Position A)
26.3
23.6
5.8*
9.6*
Average H2O2
ppm
(Position E)
34.7
22.5
-3.9*
6.7*
Average H2O2
ppm
(Position H)
31.6
56.0
5.5*
7.2*
CT
(pprrfhours)
631
570
9
233
LR
>7.3
>7.4
0
0
*These values are below the reported detection limit of the sensor
"p" at the end of each Test ID indicates the test was conducted following (i.e., post-test) the test with similar Test ID
(without the "p")
29
-------
Given the very high efficacy observed for coupons placed in the lined duct following H2O2exposure, Test
3 (no H2O2) was added to the test matrix to verify that residual H2O2, and not some other component of
the duct lining, was causing the inactivation. This control test verified that simple exposure to the duct did
not reduce recovery compared to positive control samples, indicating that exposure to even very low
concentrations of H2O2 over long times can be effective at inactivating spores in lined ducts.
Figure 3-7 shows the LR as a function of CT for all lined tests. These results suggest a critical CT value
around 550 ppm*hours provided very effective decontamination of the lined duct, and that this CT may be
provided with low concentrations of H2O2 vapor. Much higher CTs were required to decontaminate the
unlined duct.
7 _
fi -
QC
g 4
<
3 .
9 _
1
4
0 "
Test 02p Test 02 Test 01
% •
Test 04 Test Olp
Test 03
^ Test 04p
0.0 500.0 1000.0 1500.0 2000.0
ppm*hours
Figure 3-7. Average LR as a Function of CT (lined duct tests)
3.3.3 Comparison of Lined and Unlined Duct
Table 3-7 shows a comparison of Lined and Unlined duct for test conditions common to both duct types.
30
-------
Table 3-7. Comparison of Lined and Unlined Duct. For each set of conditions (250 ppmv H2O2 for 4
hours, or 250 ppmv H2O2 for 90 minutes) decontamination efficacy (LR) from the lined test
was compared by T-test to efficacy of unlined tests, p-values are reported in the last row of
each unlined test column. Exposure (ppmv*hours) is reported as the cumulative CT over 24
hours, as this is the amount of time coupons were inside the duct.
H2O2
ppmv*hours
LR
Student's
T-test
p-value
250 ppmv H2O2for4 hours
Lined test
01
1748
7.4
Unlined Test
13
NA
7.3
0.88
Unlined Test
13b
1250
6.3
8.3x1 0"4
Unlined Test
15
1100
5.5
2.7x10'4
250 ppmv H2O2for90 minutes
Lined test
02
1084
7.2
Unlined
Test 14
482
4.7
4.9x1 0'8
Unlined
Test14b
486
6.4
0.013
A Student's T-test comparing the log reductions for lined and unlined ducts was performed for each
replicate test on the unlined duct (i.e., lined duct versus each of the unlined test replicates for each test
condition). With the exception of Test 13, which had unknown fumigation conditions because of a data
acquisition failure, the p-values of the T-test indicate that lined and unlined duct are systems with
statistically significant differences. Lined duct were more easily decontaminated than unlined duct at the
same target fumigation conditions.
31
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4 Quality Assurance
This project was performed under an approved Category III Quality Assurance Project Plan titled
Evaluation of Medium and High Tech Methods for HVAC Decontamination (July 2011).1
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 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 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 Frequency
Equipment
Meter box
Flow meter
RH sensor
Stopwatch
Clock
Pressure gauges
Calibration/Certification
Volume of gas is compared to NIST-traceable dry gas
meter annually
Calibration using a flow hood and a Shortridge
manometer
Compare to 3 calibration salts once a week.
Compare against NIST Official U.S. time at
http://nist.time.goV/timezone.cgi7Eastern/d/-5/java
once every 30 days.
Compare to office U.S. Time @ time.gov every 30
days.
Compare to independent NIST Pressure gauge
annually.
Expected Tolerance
±2%
±5%
±5%
±1 min/30 days
±1 min/30 days
± 2 %full scale
32
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Table 4-2. Analysis Equipment Calibration Frequency
Equipment
Pipettes
Pressure
Manometer
Incubator
thermometers
Scale
Calibration
Frequency
Annually
Annually
Annually
Before each
use
Calibration Method
Gravimetric
Compared to NIST-
traceable Heiss gauge
Compared to NIST-
traceable thermometer
Compared to Class S
weights
Responsible
Party
External
Contractor
ARCADIS
ARCADIS
Metrology
Laboratory
ARCADIS
Acceptance
Criteria
±1% target
value
±3% reading
± 0.2 °C
±0.01% target
4.2 Data Quality
The primary objective of this project was to determine the efficacy of various fumigation methods on
deactivating spores inside an HVAC duct. Secondary objectives were to determine the effect that flow
rate, distance from injection point, flow and pressure points at turns such as elbows, and inlet
concentration of fumigant may have on the efficacy. 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)1 in place for this testing was followed with several deviations,
many of which were documented in the text above. Deviations included the flow rate in the duct and the
H2O2 wet chemistry. The original test matrix listed the air flows at 450, 900, and 1350 CFM. Due to
excessive air turbulence and lack of an accurate method to measure the velocity, the variable frequency
inverter setting was used to vary flow rate to ensure repeatability. The H2O2 wet chemistry method proved
to be very unreliable and provided no correlation with the actual set point. The ATI sensors used to
monitor the H2O2 concentration were calibrated before each test and were relied on instead. These
deviations did not substantially affect data quality and were necessitated by the test results themselves.
Lined coupons and coupon holders were sterilized using ethylene oxide rather than autoclave due to the
potential incompatibility of the lining material with high temperatures.
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
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
33
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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 4-3. 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 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.
4.4 Acceptance Criteria for Critical Measurements
The Data Quality Objectives (DQOs) define the critical measurements (CM) 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 duct coupons
• concentration measurements to characterize the fumigation conditions
The Data Quality Indicators (DQIs) listed in Table 4-4 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. In contrast, for the real-time H2O2
measurements, some missing data would not invalidate a test.
34
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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
Puffing control
coupons
Fumigation
extraction blank
samples
Post-test
calibration of ATI
H2O2 and Vaissala
RH sensors
Purpose
Determine extent of
cross-contamination
within duct
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
Used to determine
drift and variance in
the MDI
Validated baseline
of extractive
techniques
Used to validate
sensor operation
Acceptance Criteria
None
No detectable spores
No detectable spores
No detectable spores
No observed growth
following incubation.
1e6CFU, ±0.5 log
The CFU recovered from
the first set of positive
controls must be within
0.5 log of the second set
of positive controls
Non-detect
The post-test calibration
check readings must be
within 5% of target
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;
correct loading procedure
for next test and repeat
depending on decided
impact.
Reject results and repeat
test.
Obtain new reagents
Reject results. Repeat
test.
Frequency
1 per sample
location
3 per test
3 per test
3 per material
per test
Each plate
8 per test
2 sets of 4
coupons
1 per test
1 per test
35
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Table 4-4. Critical Measurement Acceptance Criteria
Critical
Measurement
Plated volume
CFU/plate
Fumigation Time
H2C>2 concentration
RH of fumigation
Measurement
device
Pipette
Enumeration by
sight
Timer
ATI sensor
Vaissala
HMT40Y
Accuracy
±2%
±10% (between 2
counters)
±1 second
±10% range
±5%
Precision
±1 %
±10%
± 1 second
±5%
±3%
Detection Limit
NA
1 CPU
1 second
10 ppm
NA
Completeness
100%
100%
100%
90%
90%
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 QA/QC checks used to validate microbiological measurements. These checks include
samples which 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 duct 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
• Inoculation control coupons: aluminum coupons puffed at beginning, middle, and end of each
inoculation campaign, not fumigated, to assess the stability of the puffer during the inoculation
operation.
The ATI H2O2 sensors and Vaissala RH meters were zeroed and spanned prior to each test and were
within the factory specifications during each fumigation.
36
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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.
4.7 Amendment to Original QAPP
The following amendment was added to the QAPP in response to changes necessitated by project
results.
Amendment 1 (11/09/2011)
Table 4-5 (below) was submitted as Amendment #1, and was to replace this table in the original QAPP.
The results from Test 13 showed that H2O2 fumigation was very effective at decontaminating the coupons
at the lowest flowrate. Higher flowrates were expected to further improve efficacy by improving mixing.
Even at the lowest flow rate, concentrations in the duct were similar at all test points. Due to the lack of
H2O2 demand presented by the galvanized duct and to the efficacy of the first tested H2O2 condition (Test
13), this amendment was needed to modify the test matrix. For Test 14, the exposure time rather than the
duct flow rate was changed from conditions in Test 13. New conditions for Test 14 were 250 ppm for 90
minutes (compared to 250 ppm for 240 minutes in Test 13). The results of Test 14 were to be used to
determine the conditions for Test 15, and perhaps Tests 2 and 3.
37
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Table 4-5. Proposed Test Matrix
Test
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
Fumigant
VHP
VHP
VHP
CIO2
CIO2
CIO2
CIO2
CIO2
CIO2
Fog
Fog
Fog
VHP
VHP
VHP
CIO2
CIO2
CIO2
CIO2
CIO2
CIO2
Fog
Fog
Fog
Concentration
(ppm)
250
250
250
3000
3000
3000
200
200
200
TBD
TBD
TBD
250
250
TBD
3000
3000
3000
200
200
200
TBD
TBD
TBD
Exposure Time
(min)
240
240
240
180
180
180
480
480
480
TBD
TBD
TBD
240
90
TBD
180
180
180
480
480
480
TBD
TBD
TBD
Flow Rate in Duct
(acfm)
450
900
1350
450
900
1350
450
900
1350
450
900
1350
450
450
450
450
900
1350
450
900
1350
450
900
1350
Duct Work Lined?
Lined
Lined
Lined
Lined
Lined
Lined
Lined
Lined
Lined
Lined
Lined
Lined
Un-lined
Un-lined
Un-lined
Un-lined
Un-lined
Un-lined
Un-lined
Un-lined
Un-lined
Un-lined
Un-lined
Un-lined
38
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5 Summary and Recommendations
®
The efficacy of fumigation with H2O2, using the STERIS VHP 1000ED, in the unlined duct varied based
on the location in the duct. For a single fumigation condition, the average LR per location ranged from 0.6
LR to full decontamination (7.4 LR). The results suggest that flow patterns can be very complex in
ductwork, and those complexities can make gaseous decontamination more difficult in certain locations
within the ductwork. Flow separation, eddying, and flow reversal occurred at certain locations in the duct
immediately following elbows. These locations were very difficult to decontaminate in the unlined, metal
duct. Increasing the flow rate through the duct seemed to exacerbate these effects, though more studies
are needed to confirm this result.
Lined duct proved easier to decontaminate than metal duct. The lining absorbed H2O2, and desorbed it
over a period of more than 48 hours. This desorption contributed a significant portion of the overall
exposure. The results demonstrate that fumigation with H2O2, per the VHP® process, can be an effective
decontaminant on lined duct even at low concentrations for a prolonged period of time (24 hours).
Exposures with a CT of 550 ppm-hours provided more than a 6 log reduction.
Based on these results, the following recommendations can be made:
• Fumigation with H2O2 shows promise for decontaminating internally-insulated ductwork. Its efficacy
on other types of insulation should be investigated.
• Lower concentrations of H2O2for longer exposure times can be used as an effective decontaminant in
lined duct.
• Desorbing materials could be investigated as a method for H2O2 delivery.
• Given the surprising effect of flow rate in unlined metal duct, efficacy of H2O2 in metal ducts should be
studied under very low flow conditions.
Note: This study utilized the STERIS VHP 1000ED to generate H2O2 vapor. Results obtained using
other methods of H2O2 vapor generation may differ from those of the current study.
39
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References
1 Rupert, R. "Federal On-Scene Coordinator's Report for the Capitol Hill Site, Washington, DC" US EPA,
Region 3, Philadelphia, PA, 125 pages, GS-10F-0076K.
2 Schaudies, R.P. "Analysis of chlorine dioxide remediation of Washington, DC: Bacillus anthracis
contamination," Appendix 15, Hart Senate Office Building HVAC Fumigation Final Report, U.S.
Environmental Protection Agency, Region III, EPA 903-R-03-013.
3 Canter, D.A., D. Gunning, P. Rodgers, L. O'Connor, C. Traunero, C.J. Kempter. "Remediation of
Bacillus anthracis contamination in the U.S. Department of Justice mail facility," Biosecurity and
Bioterrorism. 2005;3(2):119-127.
4 Canter, D.A., T.J. Sgroi, L. O'Connor, C.J. Kempter, "Source Reduction in an Anthrax-Contaminated
Mail Facility," Biosecurity and Bioterrorism: Biodefense Strategy, Practice, and Science. Dec 2009, Vol. 7,
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5ARCADIS U.S., Inc., "Quality Assurance Project Plan for the Evaluation of Medium and High Tech
Methods for HVAC Decontamination," Prepared under Contract No. EP-C-09-027, Work Assignment No.
2-65. U.S. Environmental Protection Agency, National Homeland Security Research Center, Research
Triangle Park, NC. July 2011. (available upon request)
6 Lee, S.D., S.P. Ryan, E.G. Snyder. "Development of an Aerosol Surface Inoculation Method for Bacillus
Spores," Appl. Environ. Microbiol., 77(5):1638, 2011.
7 Rastogi, V.K., L. Wallace, L.S. Smith, S.P. Ryan, B. Martin. "Quantitative method to determine sporicidal
decontamination of building surfaces by gaseous fumigants, and issues related to laboratory-scale
studies," Appl. Environ. Microbiol., 75:3688-3694, 2009.
8 Rogers, J.V., C.L. Sabourin, Y.W. Choi, W.R. Richter, D.C. Rudnicki, K.B. Riggs, M.L. Taylor, J. Chang,
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spores on indoor surfaces using a hydrogen peroxide gas generator," J. Appl. Microbiol. 99: 739- 748,
2005.
9 U.S. Environmental Protection Agency. "Official Method 2 - Measurement of gas volumetric flow rates in
small pipes and ducts". 40 CFR 20.
10 U.S. Environmental Protection Agency. "Memorandum: Efficacy Review for EPA Reg. No. 58779-4,
Vaprox® Hydrogen Peroxide Sterilant; DP Barcode: 321541", USEPA, Office of Prevention, Pesticides,
and Toxic Substances, 2006.
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