EPA/600/R-14/014 | March 2014 | www.epa.gov/ord
United States
Environmental Protection
Agency
Evaluation of Chlorine Dioxide
Gas and Peracetic Acid Fog for
the Decontamination of a Mock
Heating, Ventilation, and Air
Conditioning Duct System
Assessment and Evaluation Report
Office of Research and Development
National Homeland Security Research Center
-------
EPA600-R-14-014
Evaluation of Chlorine Dioxide Gas and Peracetic Acid Fog
for the Decontamination of a Mock Heating, Ventilation, and
Air Conditioning Duct System
Assessment and Evaluation Report
National Homeland Security Research Center
Office of Research and Development
U.S. Environmental Protection Agency
Research Triangle Park, NC 27711
-------
Disclaimer
The United States Environmental Protection Agency, through its Office of Research and Development's
National Homeland Security Research Center, funded and directed this investigation through EP-C-09-
027 WA 3-65 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:
Joseph Wood
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:919-541-5029
Fax:919-541-0496
E-mail: wood.ioe@epa.gov
-------
Acknowledgments
This effort was directed by the principal investigator from the Office of Research and Development's
(ORD) National Homeland Research Center (NHSRC), utilizing the support from the US Environmental
Protection Agency's (EPA's) Chemical, Biological, Radiological, and Nuclear (CBRN) Consequence
Management Advisory Team (CMAT) within the Office of Emergency Management (OEM). The
contributions of the entire team are acknowledged.
Project Team:
Joseph Wood (Principal Investigator)
NHSRC, ORD, US EPA
Research Triangle Park, NC 27711
Worth Calfee, Ph.D.
NHSRC, ORD, US EPA
Research Triangle Park, NC 27711
R. Leroy Mickelsen
CBRN CMAT, OEM, 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.
Ramona Sherman (Quality Assurance)
NHSRC, ORD, US EPA
Cincinnati, OH 45220
Joan Bursey (Editorial)
NHSRC, ORD, US EPA
Research Triangle Park, NC 27711
The peer reviewers of this report are also acknowledged for their input to this product:
Rebecca Connell
CBRN CMAT, OEM, Office of Solid Waste and Emergency Response, US EPA, Washington, D.C.
Lukas Oudejans
NHSRC, ORD, US EPA
Research Triangle Park, NC 27711
Staci Kane
Lawrence Livermore National Laboratory
Livermore, CA
IV
-------
Table of Contents
Disclaimer iii
Acknowledgments iv
Table of Contents v
List of Figures vii
List of Tables vii
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 Definitions of Effectiveness 4
2 Materials and Methods 6
2.1 Facility Design 6
2.2 Decontamination Cycle 9
2.2.1 Chlorine Dioxide 9
2.2.2 Peracetic Acid 10
2.3 Coupon Preparation 11
2.3.1 Test Coupons 11
2.3.2 Positive Control Coupons 11
2.3.3 Spore Preparation 12
2.3.4 Coupon Inoculation and Test Preparation 12
2.4 Test Matrix 13
2.5 Sampling and Analytical Procedures 14
2.5.1 Test Facility Sampling Procedures 14
2.5.2 Microbiological Analysis 18
2.6 Sampling Handling and Custody 19
2.6.1 Prevention of Cross-Contamination of Sampling/Monitoring Equipment 19
2.6.2 Preventing Cross-Contamination during Execution of the Decontamination Process 19
2.6.3 Preventing Cross-Contamination during Sampling 19
2.6.4 Preventing Cross-Contamination during Analysis 20
2.6.5 Sample Quantities 20
-------
2.6.6 Sample Containers for Collection, Transport, and Storage 20
2.6.7 Sample Identification 20
2.6.8 Sample Preservation 21
2.6.9 Sample Holding Times 21
2.6.10 Sample Custody 21
2.6.11 Sample Archiving 22
3 Results and Discussion 23
3.1 HVAC Duct Flow Characterization 23
3.1.1 Flow Velocity versus Blower Speed 23
3.1.2 Flow Velocity Profile Near an Elbow 24
3.2 CIO2 Fumigations- Unlined Duct Results 26
3.3 CIO2 Fumigations- Lined Duct Results 28
3.4 Test Blank Runs- Lined and Unlined Duct 301
3.5 PAA Fog Screening Tests 31
4 Quality Assurance 35
4.1 Sampling, Monitoring, and Analysis Equipment Calibration 35
4.2 Data Quality 36
4.3 QA/QC Checks 36
4.4 Acceptance Criteria for Critical Measurements 37
4.5 Data Quality Audits 40
4.6 QA/QC Reporting 40
5 Summary and Recommendations 41
References 42
VI
-------
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. HVAC Duct Modification for Fogging Tests 8
Figure 2-4. Control Loop Schematic 9
Figure 2-5. Test Coupon Holder Setup 11
Figure 2-6. Positive Control Coupon Holder 12
Figure 2-7. Duct Testing Facility (CIO2) with Sampling and Monitoring Locations Indicated by
Letters A-H 15
Figure 3-1. Post-Blower Velocity Traverses Inside Unlined Duct at Point A 24
Figure 3-2. Velocity Traverse Inside the Duct from Location E to D for Unlined Duct 25
Figure 3-3. H2O2 Levels During Fogging Test 9 33
List of Tables
Table 2-1. Test Matrix 13
Table 2-2. Frequency of Sampling Monitoring Events 16
Table 2-3. Critical and Non-Critical Measurements 17
Table 2-4. Coupon Sample Coding 21
Table 3-1. Average CIO2 Concentrations and RH during Unlined Duct Fumigation 26
Table 3-2. Positive Controls Inoculation Results for Unlined Fumigations (n = 4) 26
Table 3-3. Average CFU Recovered from Test Coupons from Unlined Duct Sample Points
(n = 4) 27
Table 3-4. Log Reduction during Testing of the Unlined Duct by Sample Location (n=4) 28
Table 3-5. Average CIO2 Concentrations and RH during Lined Fumigations 28
Table 3-6. Positive Controls Inoculation Results for Lined Fumigations (n = 4) 29
Table 3-7. Average CFU Recovered from Test Coupons from Lined Duct Sample Points
(n = 4) 30
Table 3-8. Log Reduction during Testing of the Lined Duct by Sample Location (n=4) 30
Table 3-9 Log Reduction of the Blank Tests for Unlined and Lined Duct 31
Table 3-10. Results oft-Tests on Blank Tests 31
Table 3-11. Conditions for PAA Screening Tests 32
VII
-------
Table 3-12. Average Log Reduction and Positive Control Levels for Testing of PAA on Unlined
Duct 34
Table 4-1. Sampling and Monitoring Equipment Calibration Frequency 35
Table 4-2. Analysis Equipment Calibration Frequency 36
Table 4-3. QA/QC Sample Acceptance Criteria 38
Table 4-4. Critical Measurement Acceptance Criteria 39
VIM
-------
List of Acronyms and Abbreviations
APPCD
ATCC
B.
CIO2
CBR
CBRN
CFM
CPU
CM
CMAT
COC
CT
DCMD
DHS
Dl
DQI
DQO
ECBC
EMS
EPA
GMP
H202
HP
HSPD
HSRP
HVAC
KIPB
LR
MDI
MOP
NHSRC
NIST
OEM
OPP
ORD
OSWER
PAA
PBST
Air Pollution Prevention and Control Division
American Type Culture Collection
Bacillus
Chlorine dioxide
Chemical, Biological, or Radiological
Chemical, Biological, Radiological, and Nuclear
Cubic feet per minute
Colony Forming Units(s)
Critical Measurement
Consequence Management Advisory Team
Chain of custody
Concentration xTime
Decontamination and Consequence Management Division
Department of Homeland Security
Deionized
Data Quality Indicator
Data Quality Objective
Edgewood Chemical Biological Center
Environmental Monitoring System
U. S. Environmental Protection Agency
A product name, rather than an acronym
Hydrogen peroxide
horse power
Homeland Security Presidential Directive
Homeland Security Research Program
Heating, Ventilation, and Air Conditioning
Potassium iodide phosphate buffer
Log reduction
Metered Dose Inhaler
Miscellaneous Operating Procedure
National Homeland Security Research Center
National Institute of Standards and Technology
Office of Emergency Management
Office of Pesticide Programs
Office of Research and Development
Office of Solid Waste and Emergency Response
Peracetic acid
Phosphate Buffered Saline with Tween 20
IX
-------
ppm part(s) per million
QA Quality Assurance
QAPP Quality Assurance Project Plan
QC Quality Control
Re Reynolds
RH Relative Humidity
SD Standard deviation
SEM Scanning Electron Microscope/Microscopy
SOP Standard Operating Procedure
ISA Tryptic Soy Agar
VHP Vaporized H2O2
WAM Work Assignment Manager
-------
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
investigation was to determine the efficacy of chlorine dioxide (CIO2) fumigation and fogging with
peracetic acid (PAA) for inactivating bacterial spores (using a surrogate for Bacillus anthracis) inside a
pilot-scale heating, ventilation and air conditioning (HVAC) ductwork system. Tests were conducted at
both high (3,000 parts per million [ppm]) and low (200 ppm) levels of CIO2 with varying contact times or
fogging with PAA at for two different PAA quantities in the fogger. The overall goal of the study was to
provide an understanding of the performance of these decontamination technologies to guide their use
and implementation in homeland security applications for hard-to-decontaminate environments such as
HVAC duct systems. In the assessment of options for decontamination following an intentional release of
8. anthracis spores, it is important to know what products or methods may be used successfully and how
operational factors can impact the decontamination efficacy.
This investigation focused on decontamination of two types of HVAC duct: galvanized metal and
galvanized metal lined internally with fiberglass duct insulation. Decontamination efficacy tests were
conducted with spores of 8. subtilis, a surrogate for 8. anthracis. Decontamination efficacy was quantified
in terms of log reduction (LR), based on the difference in the number of bacterial spores (quantified as
colony forming units) recovered from the positive controls (duct samples not exposed to the
decontaminant) and test samples (placed at eight locations along the length of the duct). Tests were
conducted with varying operational parameters (e.g., contact time, decontaminant concentration, relative
humidity) to assess the effect of these parameters on decontamination efficacy. For example, some tests
were conducted at relatively low concentrations of CIO2gas (200 ppm) but longer contact times, because
this approach could potentially allow for lower capacity CIO2 generation technologies to be used.
Summary of Results
Chlorine Dioxide - Unlined Duct
With the unlined duct, three tests were conducted at 3,000 ppm CIO2 and three tests were conducted at
200 ppm CIO2. For the three tests conducted at 3,000 ppm CIO2, all achieved a >6 LR, and all but two
sampling locations (out of a total of 24) had no viable spores recovered. (Sporicides achieving a LR > 6
are considered effective under efficacy testing requirements developed under the Federal Insecticide,
Fungicide, and Rodenticide Act.) For the three tests conducted at the 200 ppm level, CIO2 was somewhat
less effective than at 3,000 ppm. At 200 ppm, the average LR ranged from 5.7 ± 0.26 (at a four-hour
contact time) to 7.2 ± 0.19 (with an eight-hour contact time), with 15 out of 24 sampling locations having
no viable spores recovered (from the three tests conducted at 200 ppm).
Chlorine Dioxide - Lined Duct
With the fibreglass-lined duct, two tests were conducted at 3,000 ppm CIO2, and two tests were
conducted at 200 ppm CIO2. The average LR for the two tests conducted at 3,000 ppm was 6.4 ± 0.13,
while the average LR for the two tests conducted at 200 ppm was 5.9 ± 0.55. Of the two tests conducted
at 200 ppm CIO2, the one test conducted with a contact time of four hours resulted in all eight sampling
xi
-------
locations having viable spores recovered. While in the other test with 200 ppm, an eight-hour contact
time was used and resulted in only three sampling locations (out of eight) at which viable spores were
recovered.
Peracetic Acid Fog - Unlined Duct
Two tests were conducted with PAA fog, using varying initial amounts of PAA solution in the fogger.
Each test resulted in an average LR of 6.7 ± 0.67. Each test also resulted in having only one sampling
location (out of eight) at which no viable spores were recovered.
Efficacy as a Function of Location in Duct
While there was some variability in the efficacy results by location within the duct system - for each
particular test, there was no specific location within the duct system that tended to be easier or more
difficult to decontaminate in the overall study.
XII
-------
1 Introduction
This project supports the mission of the U.S. Environmental Protection Agency's (EPA) Homeland
Security Research Program (HSRP) by providing relevant information pertinent to the decontamination of
contaminated areas resulting from an act of terrorism. Under Homeland Security Presidential Directive
(HSPD)-10, the U.S. Department of Homeland Security (DHS) is tasked to coordinate with other
appropriate Federal departments and agencies to develop comprehensive plans that "provide for
seamless, coordinated Federal, state, local, and international responses to a biological attack." As part of
these plans, EPA, in a coordinated effort with DHS, is responsible for "developing strategies, guidelines,
and plans for decontamination of persons, equipment, and facilities" to mitigate the risks of contamination
following a biological weapons attack.
EPA's HSRP provides expertise and products that can be used widely to prepare for, respond to, and
recover from public health and environmental emergencies arising from terrorist threats and incidents.
The HSRP's research on biological agent decontamination supports EPA's Office of Solid Waste and
Emergency Response (OSWER) and the Office of Pesticide Programs (OPP). OSWER and its Special
Teams, which include the Chemical, Biological, Radiological, and Nuclear (CBRN) Consequence
Management Advisory Team (CMAT), support the emergency response functions carried out by the
Regional Offices. The OPP supports the decontamination effort by providing expertise on biological agent
inactivation and ensuring that the use of pesticides in such efforts is done in accordance with applicable
laws. Close collaboration between the different program offices having homeland security responsibilities
is sought to rapidly increase EPA's capabilities to help the Nation recover from a terrorist event involving
the intentional release of chemical, biological, or radiological (CBR) materials.
In 2001, the introduction of a few letters containing Bacillus anthracis spores into the U.S. Postal Service
system resulted in the contamination of several facilities. Some of the facilities where these letters were
processed or received in 2001 were heavily contaminated. While the overall facilities were successfully
remediated with approaches such as fumigation with vaporized hydrogen peroxide (VHP®) or chlorine
dioxide (CIO2), including treatment of the heating, ventilation and air conditioning (HVAC) ducts 12, data
specific to the decontamination efficacy of the HVAC system were lacking. Furthermore, while these
decontamination methods have been studied extensively for decontamination of surfaces found in the
open spaces of a building (walls, floors, windows, etc.), the present research helps to determine the
efficacy of the decontamination method within the confined spaces of an HVAC system and on the
materials found within these systems. This study builds on earlier work conducted with VHP® and
published by EPA.3
1.1 Process
The general process being investigated in this project is decontamination of HVAC system 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
1
-------
the results of the 2001 anthrax incidents, a combination of removal and in situ decontamination was
used.4 The balance between the two approaches 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., difficulty in
finding an ultimate disposal site).45 Since 2001, a primary focus for facility remediation has been
improvement of the effectiveness and practical application of in situ decontamination methods and
evaluation of 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, coupons of HVAC duct material were loaded with spores using an aerosol deposition
device. The 18 mm-diameter coupons were prepared from the same materials as the duct. Test and
procedural blank coupons were placed in the test duct and decontaminated using CIO2 fumigation or
using a fog of peracetic acid solution, or PAA. After decontamination (fumigation or fogging), the coupons
were removed for spore extraction and quantification. Positive control coupons (i.e., contaminated with
spores but not subjected to the decontamination process) were used to determine the pre-treatment (i.e.,
inoculum) loading on each coupon type. Quality control (QC) samples included procedural blank coupons
(coupons that underwent the decontamination process, but which were not inoculated) and negative
controls (which did not undergo the decontamination process).
1.2 Project Objectives
The primary objective of this project was to determine the efficacy of the CIO2 fumigation method and
fogging with PAA on inactivating spores inside an HVAC duct. Tests were conducted with varying
operational parameters (e.g., contact time, decontaminant concentration, relative humidity) to assess the
effect of these parameters on decontamination efficacy. For CIO2, some tests were conducted at 3,000
ppm CIO2, the typical or standard level used for anthrax spore decontamination. Other tests were
conducted at relatively low concentrations of CIO2 gas (200 ppm), but for longer contact times, because
this approach could potentially allow for lower capacity chlorine dioxide generation technology to be used.
Using lower capacity chlorine dioxide generation technology, in turn, would allow for greater numbers of
contractors that could employ CIO2 gas in the event of a wide area release of B. anthracis spores, in
which numerous structures would need to be decontaminated. For PAA fogging, the objective was to
determine the decontamination efficacy of a PAA fog (via a few screening tests, for proof of concept) for
the same mock duct system.
In addition to efficacy testing, other aspects relative to the HVAC system operation were examined,
including flow characterization tests (velocity measurements), which were conducted at several points
inside the unlined duct system, to determine if flow irregularities could affect decontamination efficacy.
Aeration time for the duct following fumigation was also assessed, because residual fumigant off-gassing
could affect efficacy. Lastly, visual qualitative effects of the decontaminants on the HVAC materials
(galvanized metal, fiberglass insulation) were observed and are noted in this report.
1.3 Experimental Approach
A closed loop duct was constructed and subjected to CIO2 decontamination under different operating
conditions. For the fogging tests with PAA, a modification was made to the supply duct so that the fogger
could be inserted. In both cases, 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 either the CIO2
decontamination technique or fogged with PAA.
Testing was conducted in test ductwork fabricated at EPA's Research Triangle Park facility. A test matrix
was developed at the start of the testing campaign, and this matrix was sequentially modified and built
upon as the results of completed tests were analyzed. In general, each test 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 B. 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 control coupon) at eight defined testing locations along the length of the ductwork.
These locations were chosen specifically to determine a) the potential effects of spatial 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 turns.
4. Application of a prescribed fumigation sequence. The CIO2gas was provided by a ClorDiSys Cloridox
-GMP Sterilization System. The target test condition (fumigant concentration, duct flow rate, and
exposure time) was set and controlled at the fan outlet of the ductwork. Relative humidity (RH) was
controlled at the inlet of the ductwork while temperature during testing was monitored but not
controlled. The CIO2 fumigant concentration was monitored continuously at three locations (inlet, mid-
duct, and at the end of the closed loop duct) to determine the concentration profile as a function of
distance from the injection point and the time in the duct. After the exposure time was reached, the
ductwork was aerated immediately until fumigant concentrations were low enough to allow safe
removal of the test coupons for analysis.
For the fogging tests, the hydrogen peroxide (H2O2) vapor concentration was monitored (as an
indicator for PAA, because PAA is produced in equilibrium with acetic acid and H2O2)6 continue
the same three locations (fan outlet, mid-duct, and at the fan inlet of the closed loop duct).
5. Transfer of test coupons, procedural blanks, and positive controls to the NHSRC Biocontaminant
Laboratory (Biolab) in sterile primary independent packaging within secondary containment
containing logical groups of samples for analysis. All samples were accompanied by a completed
chain of custody (COC) form.
6. Determination of surface decontamination efficacy (comparison of viable spore concentrations from
positive controls and test coupons).
In addition to the steps outlined above, all test activities were documented during the activity via
narratives in laboratory notebooks, real-time data acquisition, and the use of digital photography. The
documentation included, but was not limited to, any deviations from the test plans and physical impacts
on the materials.
-------
All tests were conducted in accordance with internal miscellaneous operating procedures (MOPs), to
ensure repeatability and adherence to the data quality validation criteria set for this project.
1.3.1 Definitions of Effectiveness
The sporicidal effectiveness (efficacy) of a decontamination technique is a measure of the ability of the
method to inactivate and/or remove the spores from a contaminated material surface (i.e., represented by
coupons in this study). Efficacy is evaluated by measuring the difference in the logarithm (Iog10) of the
measured colony forming units (CPU) before decontamination (determined from sampling the positive
control coupons) and after decontamination (determined from sampling the test coupons) for 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 sample location as defined in Equation 1-1.
(1-1)
I i AT AT
Jvc Jvx
where:
Surface decontamination effectiveness; the average log
reduction of spores on a specific sample location (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 CFUSk and the efficacy was reported as greater than or equal to the
value calculated by Eqn. 1-1. The choice of 0.5 CPU allowed differentiation between detect (1 CPU) and
non-detect, a vital distinction in a field event.
The standard deviation of the average log reduction (LR) of spores on a specific location (T\\) is
calculated by Eqn. 1-2:
Ns-l
(1-2)
-------
where:
_ Standard deviation of Y\\, the average log reduction of spores
Vi for a specific material location
„ _ The average log reduction of spores for a specific material
'' location (location designated by;)
The average of the log reduction from the surface of a test
•" k ~~
coupon (Eqn. 1-3)
Ns = Number of test coupons of a material surface type.
and
N,
where:
Represents the "mean of the logs" (geometric mean),
NC the average of the logarithm-transformed number of
Z^i\°g(CFUck) ^ viable spores (determined by CPU) recovered on the
\og(CFUc) = — control coupons (C = positive control coupons, Nc =
N
c 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.
(1-3)
In this report, decontamination efficacy is generally reported in terms of LR for a particular duct location.
We also occasionally report results by noting whether the average LR for a particular test is > 6.0, since a
decontaminant that achieves > 6 LR is considered effective7. Lastly, we also sometimes characterize
efficacy in terms of the number of sample locations in which no spores were detected, implying the
highest decontamination efficacy quantifiable and achievable.
-------
2 Materials and Methods
2.1 Facility Design
Testing was conducted in a test ductwork assembly that was fabricated at EPA's Research Triangle Park
facility. Figures 2-1 and 2-2 show a diagram of the test duct and a photograph of 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 building exhaust system). The design was chosen to maximize overall duct length, provide complex
flow regions including elbows, and fit inside the spray booth chamber. The test duct included both the
square 90° turns typical of many HVAC systems and radial turns included to reduce the total pressure
drop. A blower (Dayton Model 7C651, modified with a 1/4 horsepower (HP) inverter duty motor; Dayton
Electric Manufacturing, Miles, IL) provided recirculation of fumigant within the ductwork and a full dynamic
range of flow rates (with corresponding duct velocities typical of full-scale systems), when desirable. The
ductwork was made to be disassembled easily and to 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 duct 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)
-------
Figure 2-2. Photo of Actual Testing Facility
A modification was made to the duct system for the fogging tests (Figure 2-3) to allow for the fogger to be
positioned upright so that the fog could be directed initially upward through the riser duct. A T was
inserted at the elbow so that the fogger (Mini Dry Fog® System; Mar Cor Purification, Minneapolis, MN)
could be inserted into the duct. A damper was installed to section off the duetto prevent recirculation.
The fog was pulled through the ductwork and removed from the duct system via a line connected to the
air pump built into a STERIS VHP® generator (1000 ED, Steris Corporation, Mentor OH, USA). This
modified configuration allowed for the fogger to be positioned upright, but resulted in airflow in the
opposite direction than the air flow used in the CIO2 tests. (The air flow direction is arbitrary and change
in airflow direction was not expected to affect overall efficacy.)
-------
damper
closed
air supply
to fogger
VHP Return
for humidity and
chemical stripping
Figure 2-3. HVAC Duct Modification for Fogging Tests
-------
2.2 Decontamination Cycle
2.2.1 Chlorine Dioxide
Chlorine dioxide (CIO2)gas was generated using a Cloridox-GMP Sterilization System (ClorDiSys, Inc.,
Lebanon, NJ). The generator operates as a closed loop (Figure 2-4) providing an injection point and a
return sampling point. This closed loop construction enables the generator to monitor and control the
concentration of CIO2 in the duct testing facility. CIO2 was injected at the duct blower outlet, and the return
was located downstream but ahead of any duct fittings. In addition, two ClorDiSys Environmental
Monitoring Systems (EMS) CIO2 gas monitors were located at the duct mid- and endpoints. These gas
monitors were used for monitoring purposes only.
Air flow
EMS 2
photometer
(Location H)
EMS1
photometer
(Location D-E)
Cloridox-GMP
GMP photometer
(Location A)
Figure 2-4. Control Loop Schematic
The Cloridox-GMP was placed in the spray booth containing the duct system and was connected to a
control screen outside the booth. The booth exhaust system was then turned on as a safety measure.
• Pre-test Conditioning Phase: Utilizing a steam injection humidity bottle (Model HF-HBA, Fuel Cell
Technologies, Albuquerque, NM), the humidity was brought to the set point specified for the test.
The control sensor for humidity was located near the control for the CIO2 injection. Once the
humidity set point was reached and stablized, CIO2 injection was started.
Charge Phase: The GMP injected CIO2 until the target concentration was reached and stabilized.
-------
• Exposure Phase. The GMP injected sufficient CIO2 to maintain the target concentration.
2.2.2 Peracetic A cid Fog
Fogging with PAA was conducted as follows:
• The fogger was prepared with a specified initial amount of PAA solution and deionized (Dl) water.
(The capacity of the fogger reservoir is 500 ml.) The source of PAA was Minncare® Cold
Sterilant (Minntech Corp., Minneapolis, MN), which contains 4.5 % PAA and 22 % H2O2.
• The fogger was placed in the duct that was then sealed with a cap. Refer to Figure 2-3.
• An air supply was connected to the fogger.
• A line was connected at the end of the duetto a STERIS VHP® generator to facilitate airflow and
removal of decontaminant and humidity.
• A damper separated the fog injection point and the STERIS return line.
• During testing, the duct blower remained off.
• The sequence of operation was as follows:
1. The STERIS unit and the fogger were started and allowed to run until a maximum of 85 %
RH was reached on one of the sensors to keep the maximum RH in the duct between 75-85
%, in accordance with the fogger manufacturer's recommendation. The amount of water
added to the fogger reservoir will impact RH levels. Wood et al.6 provide further information
regarding operation of the fogger.
2. Once 85 % RH was reached, the STERIS unit (pump) and the fogger air supply were
stopped.
3. The system was allowed to dwell (i.e., pause in PAA fogging) for 15 minutes.
4. After the 15 minute dwell, the STERIS unit was restarted to reduce the humidity to 65 %, after
which time the fogger was also restarted.
5. Fogging continued until 85 % RH was reached again, at which time the STERIS unit and
fogger were both stopped and another dwell cycle began.
6. This sequence was repeated until the PAA/water solution was anticipated to be depleted in
the fogger. The fogger and STERIS unit were then turned off and the PAA was allowed to
dwell overnight.
10
-------
2.3 Coupon Preparation
2.3.1 Test Coupons
Test materials were 18 mm diameter coupons prepared from the same materials as the duct: galvanized
steel (18 gauge ,P/N 01170, Eastcoast Metal Distributors, Durham, NC) and liner (Knauf 1.5# 1"
fiberglass, Knauf Insulation, Shelbyville, IN). The liner coupon consisted of a 1 mm-thick slice of the liner
(including the inner 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, McMaster-Carr,
Atlanta, GA). The galvanized coupons were sterilized prior to use by steam autoclave. Liner coupons
were sterilized using ethylene oxide. All test procedures were performed in accordance with a pre-
approved Quality Assurance Project Plan (QAPP).8
A set of five coupons (four test coupons and one negative coupon) were collocated on a test coupon
holder (Figure 2-5) and inserted at each testing location in the duct immediately before the start of each
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 flush with the inner surface of the duct, thereby minimizing flow disruptions.
Magnet
Figure 2-5. Test Coupon 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
2-6. 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 spore inoculation levels were similar for
both positive controls and test coupons.
11
-------
Figure 2-6. Positive Control Coupon Holder
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 MDIs9 by the U.S. Army Edgewood Chemical Biological Center
(ECBC) according to a proprietary protocol.1011 Quality assurance (QA) documentation is provided by
ECBC with each batch of MDIs. Control checks for each MDI were included in the batches of coupons
contaminated with a single MDI.
2.3.4 Coupon Inoculation and Test Preparation
Coupons of the HVAC materials were inoculated (loaded) with spores of 8. subtilis using an MDI. The
inoculation procedure involved placing the coupon (18-mm diameter galvanized HVAC material with or
without duct liner attached) on a sterile stub (18-mm diameter SEM 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 was transferred to a new sterile stub, and the original inoculated stub was
discarded. This process was 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 (four total)
2. Inoculate the first four sets of four test coupons (16 total)
3. Inoculate the second set of four positive control coupons. Inoculate the second four sets of four test
coupons (16 total)
12
-------
4. Inoculate the last set of four positive control coupons (four total)
The MDIs are set to provide up to 200 discharges before the spore quantity per puff is expected to
diminish. The number of discharges per MDI was tracked so that use did not exceed this value.
Additionally, the weight of each MDI was determined after completion of the contamination of each
coupon. If an MDI weighed less than 10.5 g at the start of the contamination procedure, the MDI was
retired and a new MDI was used.
A log was maintained for each set of coupons that was dosed. 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 the
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 CIO2 fumigation or
fogging with PAA. 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 as fumigation concentration,
exposure time, and flow rate being necessitated as each test's results were reviewed and evaluated.
Table 2-1. Test Matrix
Test*
1
2
3
4
5
6
7
10
11
12
13
14
8
9
Fumigant
CIO2
CIO2
CIO2
CIO2
CIO2
CIO2
(Blank)
CIO2
CIO2
CIO2
CIO2
CIO2
(Blank)
CIO2
PAA
PAA
Concentration CIO2
(ppm) or PAA
quantity (mL)
200
200
3000
3000
200
0
3000
200
200
3000
0
3000
200
300
Exposure time (min)
240
480
180
360
600
600
360
240
480
180
480
180
Overnight dwell
Overnight dwell
Inverter
frequency
(blower speed
indicator)
15 Hz
15 Hz
15 Hz
15 Hz
15 Hz
15 Hz
15 Hz
15 Hz
15 Hz
15 Hz
15 Hz
15 Hz
OHz
OHz
RH
(%)
75
75
75
65
75
75
45
75
75
75
75
65
NA
NA
Lined
No
No
No
No
No
No
No
Yes
Yes
Yes
Yes
Yes
No
No
13
-------
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.
• 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 included fumigant concentration, velocity of air flow in duct, RH, and temperature.
Figure 2-7 shows all sampling and monitoring locations in the duct (letters indicate coupon locations). 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.
14
-------
A-H Sampling Locations
RH sensor —*
C1O2 injection
Figure 2-7. Duct Testing Facility (CIO2) with Sampling and Monitoring Locations Indicated by
Letters A-H
15
-------
Table 2-2. Frequency of Sampling Monitoring Events
Sample Type
Test coupon
Negative control
coupon
Positive control
coupon
Field blank coupons
Laboratory blank
coupons
Biolab material
blanks
CIC>2 monitors
CIC>2 wet chemistry
samples
hbCb monitors
Pressure of duct
RH/temperature
Sample
Number
4 per sampling
location, each
at a spatially
distinct height
within the duct
1 per sampling
location in duct
12 -a set of 4
inoculated at
the beginning,
middle, and
end of test
coupon
inoculations
3 coupons
which are co-
located with
control positive
coupons
3 sterile
coupons
3 per material
3 real-time
instruments
3 every hour
3 real-time
instruments
3
3
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
CIO2 fumigations
Once per port every
hour
Realtime during
PAA fogging
Logged every 10
seconds
Logged every 10
seconds
Sample Location
Shown in Figure 2-7 as
letters A-H
Shown in Figure 2-7 as
letters A-H. See also
Figure 2-5.
NA
Carried to test location
but not inserted into
duct or fumigated
NA
NA
Shown in Figure 2-4 at
three locations
Shown in Figure 2-7 at
three locations
At the inlet, mid-duct,
and outlet locations
(same as CIO2 sensors
shown in Figure 2-4)
Collocated with RH
sensors shown in
Figure 2-7 at 3
locations
Shown in Figure 2-7
(temperature measured
by RH sensor)
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
any degradation within the
duct
To verify proper operation
of CIO2 monitors
To determine exposure
experienced by the
coupons and to determine
any degradation within the
duct
For indication of airflow
To determine
environmental conditions
inside the duct
16
-------
Table 2-3. Critical and Non-Critical Measurements
Sample Type
Test coupon
Negative control coupon
Positive control coupon
Field blank coupons
Laboratory blank coupons
Biolab material blanks
CIC>2 monitors
H 262 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
CIC>2 concentration
hbCb component of PAA solution
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
NA
Temperature and RH of
duct
Pressure in duct, relative
to atmospheric pressure
NA
NA= Not applicable
2.5.1.2 CIO2 Concentration Measurement
Chlorine dioxide gas concentration within the duct system was monitored using three photometric
instruments (two ClorDiSys EMS instruments and a photometric instrument incorporated into the GMP
generator). The three photometric sensors were calibrated before each test using an optical reference
filter (Optek-Danulat, Inc., Germantown, Wl) at 7.04 mg/L.
To verify the EMS and GMP data, CIO2 levels in the duct were also measured each hour at each gas
sample location using a non-continuous gas sampling method. The gas is sampled through a series of
impingers containing a potassium iodide phosphate buffer (KIPB) solution. Gas samples were taken from
each of the three sampling ports every hour. Further details on this sampling and analytical method can
be found elsewhere
this "4500" method.
12
The CIO2 data shown in the results section of this report are based on the use of
2.5.1.3 Electrochemical Sensor for H2O2 Concentration Measurement
For the two tests using the PAA fog, H2O2 vapor concentration within the ductwork (in this case, the H2O2
is used as an indicator of the PAA because PAA is in solution in equilibrium with H2O2) was monitored
using an Analytical Technology Corp. (Collegeville, PA) electrochemical sensor (model B12-34-6-1000-1).
The sensors are factory-preset to measure from 0 to 1000 parts per million (ppm) H2O2 within an
17
-------
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 H2O2 solution.
2.5.1.4 Duct Flow Rate
Velocity measurement traverses were performed on the straight line duct in two locations using the
AIRDATA™ Multimeter ADM-860 electronic micromanometer from Shortridge Instruments, Inc.
(Scottsdale, AZ). This meter measures air duct velocities when used with a pitot tube and automatically
corrects for density variation due to local temperatures and barometric pressures.
At point A (in Figure 2.7), a sampling grid of 24 points was used: that is, 3 horizontal traverse lines at 6, 8,
and 10 inches from the bottom of duct (16 inches in the vertical direction) and 8 equally spaced sampling
points along the 3 horizontal lines. When sampling from point E to D (16 inches in the vertical direction), a
sampling grid of 48 points (3 vertical traverses, with 16 samples taken in each traverse) was used.
2.5.2 Microbiological A nalysis
The NHSRC Biolab located at the EPA facility in Research Triangle Park, NC analyzed samples either
qualitatively for spore presence (swab samples) or quantitatively for the number of viable spores per
coupon sample as CFU..
Details of the sampling 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, each18 mm test, procedural blank, and positive control coupon was
transferred aseptically into a clean 50 ml sterile vial. The sample vials were then transported to the
NHSRC Biolab, where 10 ml of sterile Phosphate Buffered Saline with Tween 20 (PBST) was aseptically
added. The sample vials were then sonicated for 10 minutes using an 8510 Branson Ultrasonic Cleaner
(Danbury, CT) at 44 kHz and 250 Watts. The sonication step was followed immediately with 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. A 0.1 ml aliquot of each dilution was inoculated onto trypic soy agar (TSA)
plates, spread with sterile beads and incubated at 35 ± 2 °C for 18-24 hours. CFU were counted
manually.
Any samples below countable criteria (30-300 CFU) on the primary dilution plates were subsequently filter
plated to reduce the detection limit. The filters were incubated at 35 ± 2 °C for 18-24 hours prior to
manual enumeration.
2.5.2.2 Swab Samples
For the first four tests, swab sampling was used for sterility checks of the ductwork prior to 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 2-7. Swabs were streaked onto TSA and incubated at 35 ± 2 °C for 18-24 hours prior to
qualitative growth analysis (presence / absence determination). After the fourth test, a negative (non-
inoculated) coupon was used at each sampling set location to address the potential issue of cross
contamination.
18
-------
2.5.2.3 Method Verification
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 Sample 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. 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.
2.6.2 Preventing Cross Contamination during Execution of the Decontamination
Process
The following management control was followed in an effort to minimize the potential for cross
contamination.
• For the first four tests, swab sampling was used for sterility checks of the ductwork. Thereafter,
negative control coupons were present for each test location. Growth on these coupons would
indicate contamination during fumigation or handling.
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.
• New sterile forceps were used for each sample.
• 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 control coupons.
19
-------
2.6.4 Preventing Cross Contamination during Analysis
General aseptic laboratory techniques were followed and are embedded in all procedures used by the
NHSRC Biolab to recover and plate samples. 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, 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 (taken after sterilization of the duct for the first four tests) were
placed in the sterile swab containers and then bagged in two individual sterile sampling bags as
secondary and tertiary containment.
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 NHSRC Biolab 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 Biolab also included, on each
plate, the date it was placed in the incubator.
20
-------
Table 2-4. Coupon Sample Coding
Coupon Identification: 65-TN-LC-RS
Category
65
TN
LC
Location Code
RS
Replicate Sample
Example
Code
65
1
B
BN
PA
PM
PZ
FB
LB
1
Work Assignment designation
Test Number (from Table 2-1 )
A through H as shown in Figure 2-7.
Negative at each location B
First set of positive controls (at beginning of puffing)
Second set of positive controls (at the middle of the
puffing)
Third set of positive controls(at end of puffing)
Field Blank
Laboratory blank
The replicate sample ID is dictated by the placement in
the holder or stage. The positive control RS is shown in
Figure 2-6, 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.
Biolab Plate Identification: 65-TN-LC-RS -R-D
65-TN-LC-RS
R
(Replicate)
D
(Dilution)
As above
R
1
A-C
Oto4, foMOEOto 10E4
2.6.8 Sample Preservation
Following transfer to the NHSRC Biolab, all samples were stored at 4 ± 2 °C until they were analyzed. All
samples were allowed to equilibrate at room temperature for one hour prior to analysis.
2.6.9 Sample Holding Times
After sample collection for a single test was complete, all biological samples were transported to the
NHSRC Biolab immediately, with appropriate COC form(s). Samples were stored no longer than five days
before the primary analysis. Typical hold time prior to analyses for most biological samples was < 2 days.
2.6.10 Sample Custody
Careful coordination with the NHSRC Biolab was required to achieve successful transfer of
uncompromised samples in a timely manner for analysis. Test schedules were confirmed with the Biolab
21
-------
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; or
• 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.
These protocols were required to be consistent with any protocols established by EPA.
In the transfer of custody, each custodian signed, recorded, and dated the transfer on the COC. Sample
transfer could be on a sample-by-sample basis or on a bulk basis. The following protocol was followed for
all samples as they were collected and prepared for distribution:
• A COC record accompanied the samples. When turning over possession of samples, the transferor
and recipient signed, dated, and noted the time on the record sheet. This record sheet allowed
transfer of custody of a group of samples from Highbay room H130-A to the NHSRC Biolab.
• If a sample custodian had not been assigned, the laboratory operator had the responsibility of
packaging the samples for transport. Samples were carefully packed and hand-carried between on-
site laboratories. The COC record showing the identity of the contents accompanied all packages.
2.6.11 Sample Archiving
All samples and diluted samples were archived for two weeks following completion of analysis. This time
allowed for review of the data to be performed to determine if any re-plating of selected samples was
required. Samples were archived by maintaining the primary extract at 4 ± 2 °C in a sealed 50 ml conical
tube.
22
-------
3 Results and Discussion
This section presents the results of decontamination of lined and unlined HVAC ductwork using either
CIO2 fumigation or fogging with PAA. Due to the boundary layer on duct walls created by airflow and the
turbulent nature of the airflow, decontamination of ductwork could be inherently difficult. The investigation
of the effectiveness of the CIO2 decontamination technique required some initial characterization of the
duct flow rate/flow pattern; 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 using CIO2 are
reported and discussed in Sections 3.2 and 3.3, respectively. The results of fogging with PAA are
reported in Section 3.5.
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 (FRNF50C1S-6U; Fuji Electric Co., Tokyo, Japan) was used to
control the blower speed to three different levels using three different frequencies (15 Hz, 30 Hz, and 60
Hz), resulting in calculated Reynolds (Re) numbers (a parameter used to characterize flow turbulence) for
the unlined duct all above 1 x105. These calculations showed that the overall bulk flow inside the duct was
highly turbulent at all tested flow rates.
3.1.1 Flow Velocity versus Blower Speed
The flow velocities inside the duct were characterized at the three different blower speeds (indicated by
the three variable frequency inverter settings) by performing pitot tube traverses on the straight line of the
duct.
The velocity profiles at Location A inside the duct are shown in Figure 3-1 for each blower speed
(indicated as inverter frequency) in the unlined duct. The results show that the air velocities vary linearly
with the speed of the blower. Note that the geometry of the duct did not provide a position with sufficient
length of straight flow, thus the standard US EPA Method 213 procedure for measuring flow could not be
followed. While this may have some impact on the accuracy of the velocity measurements, the general
trends of how velocity may vary by location or fan speed (the aim of these measurements) would not be
expected to be affected.
23
-------
1600
1400
1200
E 1000
0)
Q.
0)
0)
c
>.
800
o. 600
0)
400
200
15Hz(A),unlined
30Hz(A),unlined
60Hz(A),unlined
234567
sample location in inches from wall of duct
Figure 3-1. Post-Blower Velocity Traverses Inside Unlined Duct at Point A.
3.1.2 Flow Velocity Profile Near an Elbow
The flow pattern near a round elbow (traversing from Location E to Location D) was characterized at
three blower motor inverter frequency settings (15 Hz, 30 Hz, and 60 Hz),
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 was affected by the elbow upstream of Location E, with higher flow outside the
bend of the elbow (1 to 8 inches), decreasing on the inside of the bend (9 to 16 inches), causing flow
reversal and potential flow separation. While the total flux of fumigant across this plane of the duct is
proportional to the total flow rate of the system, the flux at any one point is unknown due to the flow
separation. The calculated bulk Re was greater than 4000, a benchmark for the transition from
intermediate 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.
24
-------
2500
2000
c 1500
-500
•15 Hz(D&E),unlined
•30 Hz(D&E),unlined
60 Hz(D&E),unlined
1 2 3 4 5 6 7 8 9 10 11 12 13 14
sample location in inches from wall of duct
Figure 3-2. Velocity Traverse Inside the Duct from Location E to D for Unlined Duct.
25
-------
3.2 CIO2 Fumigations - Unlined Duct Results
Table 3-1 shows the average CIO2 concentrations and RH during each unlined test.
Table 3-1. Average CIO2 Concentrations and RH during Unlined Duct Fumigation
Test
Target fumigation
concentration (ppm)
and RH (%)
Testl
(200)775 %
Test 2
(200)775 %
Test3
(3000)775 %
Test 4
(3000)765 %
Tests
(200)775 %
Test6
(0)75 %
Test?
(3000)745 %
CIO2 Concentration (ppm)
Location A
Location D-E
Location H
Average (±Standard Deviation)
215 (±4.5)
192 (±23)
2784 (±415)
3238 (±116)
179 (±14)
0
3347 (±61)
204 (± 6)
198 (±30)
2388 (±81 9)
31 39 (±87)
174 (±72)
0
31 65 (±22)
21 9 (±2)
110 (±43)
251 8 (±78)
3353 (±41)
186 (±77)
0
3454 (±27)
RH (%)
Location A
Location D-E
Location H
Average
74
75
75
65
74
75
45
76
74
73
65
75
75
47
76
75
75
66
80
76
48
Three sets of positive control coupons were inoculated alongside test coupon sets. These positive control
coupons were done at the beginning, middle, and the end of inoculations. The CPU recovered from these
sets of coupons are shown in Table 3-2.
Table 3-2. Positive Controls Inoculation Results for Unlined Fumigations (n = 4)
Testl
Test 2
Test3
Test 4
Tests
Test6
Test?
First Set
CFU/sample(± Standard
Deviation)
7.66x1 0s + 7.56x1 0s
1.15x107 + 6.35x106
2.68x107+ 1.81 x107
7.85 x 10s +1.83x1 0s
5.91 x 10s + 6.96 x 10s
1. 62 x107 + 8.43x1 0s
1. 02 x107 + 5.48x1 0s
Middle Set
CFU/sample
(± Standard Deviation)
8.65 x 105+ 5.76 x105
1.97x107+1.30x107
2.73 x107 + 2.21 x107
2.24 x 107 + 2.24 x107
1.13x107+7.71x106
1. 56 x107+ 6.32x1 0s
2.04 x107 + 6.86x1 0s
End Set
CFU/sample
(± Standard Deviation)
2.52x104+1.98x104
3.09 x 107 + 2.34 x107
6.00x1 0s +5.74x1 0s
5.92x1 0s + 2.21x1 0s
9.78x1 0s + 4.43x1 0s
2.25 x107+ 1.21 x107
1.44x107 + 3.12x106
26
-------
With the exception of Test 1, all of these values met the target dose QA requirements and allow for a six
LR. (Refer to Section 1.3.1 for further details regarding the relevance of a 6 LR.) The results suggest a
relatively steady inoculation level for all coupons in a test.
Table 3-3 summarizes the average CPU recovered from test coupons (four at each location) during
unlined duct tests. Many of the values are at or below the detection limit (indicated with a "<" symbol). The
detection limit varied due to the presence or absence of oxidation particles on the galvanized steel
coupon surfaces. Oxidation products (dark colored particles) were observed from the CIO2 fumigations,
but were especially noticeable at the 3,000 ppm tests. These particles made filter plate colony counting
difficult when relatively larger volumes (e.g., > 6 ml) of extraction solution were filter plated. Test 3 was
the only test with high concentration CIO2 for which large volume filter plates were performed. Filter plate
analysis was limited to 1 ml for subsequent tests when using a 3,000 ppm target level.
The dark colored particles were observed at the cut edge of the galvanized steel coupons. A white, flaky
substance was also observed on the surface of the coupons, presumably a reaction product between the
CIO2 gas and the galvanized surface, possibly zinc chloride.
Table 3-3. Average CPU Recovered from Test Coupons from Unlined Duct Sample Points (n = 4)
Test
Target
fumigation
concentration
andRH
ppmv
CIO2/RH
Testl
(200)775 %
Test 2
(200)775 %
Test3
(3000)775 %
Test 4
(3000)765 %
Tests
(200)775 %
Test?
(3000)745 %
Fumigation
Time
Minutes
240
480
180
360
600
360
Average CFU Recovered by Location
A
3 + 4.7
<1+0
<1+0
<5+0
<5+0
<5+0
B
1+0.3
<1+0
<1+0
<5+0
6+3
<5+0
C
1+0.4
5+10
2+2
<5+0
<5+0
<5+0
D
59+11
6
<1+0
1+0
<5+0
<5+0
<5+0
E
<1+0
<1+0
<1+0
<5+0
24+38
<5+0
F
<1+0
<1+0
<1+0
<5+0
<5±0
<5+0
G
<1+0
5+8
<1+0
<5+0
<5±0
<5+0
H
<1+0
5+10
<1+0
<5+0
<5±0
<5+0
Table 3-4 shows the log reduction as calculated by Equation 1. Note that LR values are a function of
positive control recovery and variations in detection limits.
27
-------
Table 3-4. Log Reduction during Testing of the Unlined Duct by Sample Location (n=4)
Test
Target fumigation
concentration in
ppm/RH
Testl
(200)775 %
Test 2
(200)775 %
Test3
(3000)775 %
Test 4
(3000)765 %
Tests
(200)775 %
Test?
(3000)745 %
Fumigation
Time
min
240
480
180
360
600
360
Locations
A
5.57+
0.63
>7.39
>7.35
>6.25
Ł6.12
>6.43
B
5.79+
0.17
>7.40
>7.35
>6.25
6.04+
0.15
>6.43
C
5.70+
0.20
7.02
+0.75
7.11 +
0.49
>6.25
Ł6.12
>6.43
D
5.14+
1.26
>7.40
>7.35
>6.25
>6.12
>6.43
E
>5.88
>7.40
>7.35
>6.25
5.82+
0.60
>6.43
F
>5.88
>7.39
>7.35
>6.25
>6.12
>6.43
G
>5.88
7.04+
0.71
>7.35
>6.25
>6.12
>6.43
H
>5.88
7.03+
0.76
>7.35
>6.25
>6.12
>6.43
Positive
Controls
Average
Log +SD
5.62 + 1.24
7.19 + 0.21
7.09 + 0.42
6.94 + 0.24
6.82 + 0.26
7.13±0.17
Note: Data in bright yellow cells and shown with ">" are based on detection limit values (no CPU detected) and had
SD values = 0.0.
Note from Table 3-3 that average spore levels greater than 10 were found on only two fumigation tests
(indicating less than ideal decontamination conditions), both at 200 ppmv CIO2 and 75 % RH. For most
test sites and fumigation conditions, CIO2 was an effective fumigant.
3.3 CIO2 Fumigations - Lined Duct Results
The HVAC duct internally lined with insulation presented a different fumigation scenario. Table 3-5 shows
the average CIO2 concentrations and RH during each lined duct test.
Table 3-5. Average CIO2 Concentrations and RH during Lined Fumigations
Test*
Target
fumigation
concentra-
tion (ppm)
and RH (%)
Test 10
(200)175 %
Test 11
(200)175 %
Test 12
(3000)/75 %
Test 14
(3000)/65 %
Fumigation Time
(min)
240
480
180
180
CIC<2 (ppm) by location (average ± SD)
A
206 (±27)
203 (±35)
1677 (±66)
2339 (±250)
D-E
21 5 (±29)
185 (±10)
1425 (±13)
1838 (±44)
H
21 7 (±23)
188 (±9)
1653 (±4)
2379 (±157)
Average RH (%)
A
62
73
75
65
D-E
67
72
80
69
H
67
75
76
67
28
-------
The fiberglass liner was expected to create a demand for the CIO2 gas, thus preventing the Cloridox-GMP
from achieving a target concentration of 3,000 ppm for Tests 12 and 14. Comparing Test 12 and Test 14,
lowering the target RH did enable the GMP to reach a higher concentration (due to less air being
introduced into the duct via the humidification system). No desorption of CIO2 was noted following
fumigation, in contrast to tests conducted with VHP®, in which significant desorption of VHP® occurred
when using lined duct3. The presence of fiberglass could require larger fumigant generators than required
for the duct alone. Lastly, no visual effect of the CIO2 fumigant on the insulation was observed.
Three sets of positive control coupons were inoculated alongside test coupon sets. These positive control
coupons were generated at the beginning, middle and the end of inoculations. The CPU recovered from
these sets of coupons are shown in Table 3-6.
Table 3-6. Positive Controls Inoculation Results for Lined Fumigations (n = 4)
Test 10
(200)/75 %
Test 11
(200)/75 %
Test 12
(3000)/75 %
Test 14
(3000)/65 %
First Set
CPU/sample
9.75 x 10s ±1.61 x106
9.54x106±5.75x106
1.44x107±1.96x106
1.44x107±1.59x106
Middle Set
CPU/sample
7.85x106±1.36x106
1.23x107±8.16x106
1.65x107±1.28x106
1.44 x107± 2.35 x 10s
End Set
CPU/sample
8.22 x 10s ±1.31 x106
8.31x106±2.38x106
2.36 x107± 7.02 x 10s
1.53x107±2.39x106
All of these values met the target dose QA requirements and allow for a potential 6 LR determination.
Table 3-7 shows the average CPU recovered from the test coupons in the lined duct tests.
29
-------
Table 3-7. Average CPU Recovered from Test Coupons from Lined Duct Sample Points (n = 4)
Test ID CIO2
(ppm)/RH (%)
Test 10
(200)175 %
Test 11
(200)/75 %
Test 12
(3000)175 %
Test 14
(3000)/65 %
Fumigation
Time
(minutes)
240
480
180
180
Location
A
29+28
<5
<5
<5
B
6+3
<5
<5
<5
C
10+7
29+20
<5
6+3
D
9+3
<5
19+28
<5
E
185+
324
230+
433
131 +
253
<5
F
9+3
<5
6+3
<5
G
*5.02x
104+
1.0x
105
6+3
<5
<5
H
13+9
<5
<5
6+3
Note: Data reported as "<" are based on detection limit values (no CPU detected) and had SD values = 0.0.
"Contamination or other inadvertent error is suspected for one of the coupons, since other coupons at this
location for Test 10 had CPU values ranging from 2-33.
Table 3-8. Log Reduction during Testing of the Lined Duct by Sample Location (n=4)
Test ID CIO2
(ppm)/RH
(%)
Test 10
(200)/75 %
Test 11
(200)/75 %
Test 12
(3000)/75 %
Test 14
(3000)/65 %
Fumigation
Time (min)
240
480
180
180
Location
A
5.64+
0.47
>6.26
>6.54
>6.46
B
6.15+
0.15
>6.26
>6.54
>6.46
C
6.00+
0.29
5.63+
0.45
>6.54
6.39+
0.15
D
6.00+
0.15
>6.26
6.28+
0.54
>6.46
E
5.43+
1.02
5.50+
1.06
6.04+
1.00
>6.46
F
6.00+
0.15
>6.26
6.54+
0.15
>6.46
G
4.10+
1.73
6.18+
0.15
>6.54
>6.46
H
5.93+
0.35
>6.26
>6.54
6.39+
0.15
Positive
controls
Average
Logs + SD
6.93 + 0.05
6.96 + 0.07
7.24 + 0.10
7.16 + 0.01
Note: Data in yellow cells are based on detection limit values (no CPU detected) and had SD values = 0.0.
With regard to comparing results for lined versus unlined duct results, there were three fumigation
conditions that were common to both the unlined and lined duct tests. In Tests 1 and 10, the fumigation
was conducted at 200 ppm, 75% RH, for 240 minutes. Tests 2 and 11 were both conducted at 200 ppm,
75% RH, but for 480 minutes. And Tests 3 and 12 were both conducted at 3000 ppm, 75% RH, for 180
minutes. However, comparing the efficacy results for the lined vs. unlined duct results is problematic, due
to other differences in test conditions. For example, the average spore loading for Test 1 was 5.62 log
CPU, while the spore loading for Test 10 was 6.93 log CPU. Further, even with comparable spore
loadings, the detection limit for the other unlined duct tests (Tests 2 and 3) was approximately a log lower
compared to the lined duct tests (Tests 11 and 12) . In any event, Tests 2 and 11 both resulted in having
30
-------
five sample locations with no spores detected. Tests 3 and 12 had six and five sampling locations with no
spores detected, respectively. In summary, although there are a limited number of tests to compare, and
there are some experimental differences between these tests, the tests indicate preliminarily similar or
comparable results for the two duct conditions.
3.4 Test Blank Runs - Lined and Unlined Duct
A test blank run for lined and unlined duct (no fumigant added) was added to the test matrix to evaluate
any non-fumigant related loss of CPU on the test coupons and demonstrate that the LR from fumigated
tests was indeed from the fumigation. The blank test was conducted and sampled the same way as the
other test runs. The data suggest that there is some small LR from simple operation of the duct or
manipulation of the sample holders (Table 3-9); however, the effect is very small.
Table 3-9 Log Reduction of the Blank Tests for Unlined and Lined Duct
Test ID
Unlined
(Test 6)
Lined
(Test 13)
Location
A
0.31
0.38
B
0.29
0.06
C
0.43
0.36
D
0.13
0.29
E
0.24
0.20
F
0.32
0.15
G
0.35
0.13
H
1.93
0.21
A statistical test was performed to compare the results for the blank test coupons to the results for the
positive controls. The p-value of the Student's t test was calculated and is shown in Table 3-10. The
results show that the CPU values for the blank test coupons are significantly less at the 95 % confidence
level than the positive controls.
Table 3-10. Results of t-Tests on Blank Tests
Test*
6
13
Average CPU from Test Coupons
8.71 x106
5.01 x106
SD
5.19x106
1.56x106
Average CPU from
Positive Controls
1.81 x107
8.43x1 0s
SD
8.98 x 10s
2.17x106
P Value
0.0042
0.0001
3.5 PAA fog
After completing the CIO2 testing on the unlined duct, two fogging tests were performed using PAA on the
unlined duct. These tests were conducted as "proof of concept", to determine if the technology
demonstrates any efficacy, and thus if further tests may be warranted. The ductwork was modified
(Figure 2-3) to allow for the insertion of the fogger.
31
-------
Table 3-11 presents some of the fogging operational parameters for the two tests. Different initial
amounts of PAA were added to the fogger for each test (200 ml for Test 8 and 300 ml for Test 9), and
then Dl water was added to fill the 500 ml reservoir to capacity. Based on previous experience with the
fogger, the 500 ml PAA/water solution was expected to be fogged in the amount of time the fogger was
operated, but this was not the case. For various unknown reasons (possibly due to inadequate air
pressure), only a portion of the fogging solution was fogged. The actual amount of PAA fogged was
calculated based on the volume of solution remaining. For Test 8, 158 ml of PAA was fogged, and for
Test 9, 66 ml PAA was fogged. After fogging, the system was allowed to dwell overnight. The following
morning, the duct was aerated.
Table 3-11. Conditions for PAA Screening Tests
Test#
8
9
Minncare
start
volume
(ml)
200
300
Dl
Water
volume
(ml)
300
200
Total
solution
volume
(ml)
500
500
Solution
volume
after
fogging
(ml)
104
390
Actual
Minncare
volume
fogged
(ml)
158
66
Fogger
operation time
(minutes)
51.5
75
System Dwell
(hours)
23
20
Figure 3-3 shows the H2O2 concentrations over time from the three H2O2 vapor sensors used during Test
9. The maximum H2O2 level of 197 ppm was reached at the sensor midway through the HVAC duct
circuit. The initial negative response of the sensors is not understood at this time, but may be an
interference response to the PAA. H2O2 vapor levels are not presented for Test 8 due to malfunction of
the data acquisition system.
32
-------
Q.
Q_
200-
175-
150-
125-
100-
75-
50-
25-
0-
-25-
-50
Is!
T—
CM
duct end point
duct mid point
duct start point
I
§
PI
d
Elapsed Time
Figure 3-3. H2O2 Levels During Fogging Test 9
Maximum RH levels for Tests 8 and 9 ranged from 81-90 % and from 74-86 %, respectively. The
average temperature during Test 8 was 17 °C, while for Test 9, the average temperature was 21 °C.
Table 3-12 shows the positive control data and average LR achieved for the two fogging tests. Although
only one sampling location per test showed complete kill (no spores detected), fogging of PAA provided a
high LR (over 6) for nearly all sample locations. This decontamination technology, thus, shows promise
for use in HVAC systems, although a more thorough investigation is warranted.
33
-------
Table 3-12. Average Log Reduction and Positive Control Levels for Testing of PAA on Unlined
Duct
Test#
8
9
A
7.16 +
0.46
7.18+
0.15
B
6.83+
1.09
6.85+
0.35
C
5.95+
0.26
5.11 +
1.45
D
>7.40
7.11 +
0.30
E
6.21 +
0.87
6.70+
1.13
F
6.84+
0.50
6.92+
0.54
G
6.49+
0.74
6.58+
0.87
H
6.57+1.48
>7.28
Average of
Positive
Controls
(Log value ±
SD)
7.20 + 0.15
7.07 + 0.12
Note: Data in yellow cells are based on detection limit values (no CPU detected) and had SD values = 0.0.
Lastly, we note that the impacts on coupons observed after CIO2 fumigation on the unlined duct
(corrosion seen on the edges of the coupons, as well as a white flaky material formed on the surface of
the coupons) were not observed after the two PAA fog tests.
34
-------
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)8
4.1 Sampling, Monitoring, and Analysis Equipment Calibration
There were standard operating procedures for the maintenance and calibration of all laboratory and
NHSRC Biolab equipment. All equipment was verified as being certified calibrated or having the
calibration validated by EPA RTP's on-site 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 National Institute of Standards and
Technology (NIST) Official U.S. time at
http://nist.time. aov/timezone.cai?Eastern/d/-5/iava
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
35
-------
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
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
inactivating spores inside an HVAC duct. This section discusses the 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)8 in place for this project was followed with deviations noted
as follows:
The original test matrix listed the airflows 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. Coupon holders and magnetic stubs were sterilized using
ethylene oxide rather than the autoclave to prevent the heat from the autoclave damaging the magnetic
material.
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 that
could cause cross contamination.
Supplies and consumables were acquired from reputable sources and were NIST-traceable when
available. Supplies and consumables were examined for evidence of tampering 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
personnel checked supplies and consumables prior to use to verify that they met specified task quality
objectives and did not exceed expiration dates.
36
-------
Quantitative standards do not exist for biological agents. Quantitative determinations of organisms in this
investigation did not involve the use of analytical measurement devices. Rather, CPU were enumerated
manually and recorded. Critical QC checks are shown in Table 4-3. The acceptance criteria were set at
the most stringent level that could be achieved routinely and are consistent with the data quality
objectives described in Section 4.4. Positive controls and procedural blanks were included along with the
test samples in the experiments so that well-controlled quantitative values were obtained. Background
checks were also included as part of the standard protocol. Replicate coupons were included for each set
of test conditions. Qualified, trained, and experienced personnel ensured data collection consistency.
When necessary, training sessions were conducted by knowledgeable parties, and in-house practice runs
were used to gain expertise and proficiency prior to initiating the research.
4.4 Acceptance Criteria for Critical Measurements
The Data Quality Objectives (DQOs) define the critical measurements (CMs) 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;
• RH measurements for fumigation conditions; and
• Exposure time.
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, then that sample is invalid. In contrast, for the real-time CIO2 measurements, some missing data
would not invalidate a test.
37
-------
Table 4-3. QA/QC Sample Acceptance Criteria
Sample Type
Negative control
coupons
Field blank coupons
Laboratory blank
coupons
Laboratory material
coupons
Blank ISA sterility
control
(plate incubated, but
not inoculated)
Positive control
coupons
4500-B wet chemistry
Fumigation extraction
blank samples
Post-test calibration of
RH sensors (Vaisala,
Helsinki, Finland)
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
Validate
concentration
Validated baseline
of extractive
techniques
Used to validate
sensor operation
Acceptance Criteria
No detectable spores
No detectable spores
No detectable spores
No detectable spores
No observed growth
following incubation.
1 x106CFU±0.5log
15 % of photometric
reading
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
on results with EPAWAM;
correct loading procedure
for next test and repeat
depending on decided
impact.
Repeat
Obtain new reagents
Reject results. Repeat test
as deemed appropriate.
Frequency
1 per sample
location
3 per test
3 per test
3 per material
per test
Each plate
1 2 per test
1 per location (3)
per hour
1 per test
1 per test
38
-------
Table 4-4. Critical Measurement Acceptance Criteria
Critical
Measurement
Plated volume
CFU/plate
CIO2 concentration
H2O2 concentration
Flow rate (velocity
pressure across
duct)
Fumigation Time
RH/temp of
fumigation
Measurement
device
Pipette
Hand counting
4500-B
ATI sensor
differential
pressure
transducer
Timer
Vaisala
HMD40Y
Accuracy
±2%
±10% (between 2
counters)
±1 5 % of photometric
value
±10% range
±1 %
±1 second
±5%
Precision
±1 %
±10%
±5%
±5%
±0.25 %
FS
± 1 second
±3%
Detection Limit
NA
1 CFU
10 ppm
1000 ppm
±0.5" WC
1 second
NA
Completeness
100%
100%
90%
90%
90%
100%
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 that demonstrate the ability of the NHSRC Biolab 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 Biolab;
• Laboratory material coupons: includes all materials, individually, used by the NHSRC Biolab in
sample analysis;
• Positive control coupons: coupons inoculated but not fumigated; and
• 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 CIO2 photometer calibrations were checked prior to each test and were within the factory
specifications during each fumigation.
39
-------
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.
40
-------
5 Summary and Recommendations
The primary objective of this investigation was to determine the efficacy of CIO2 fumigation and fogging
with PAA for inactivating bacterial spores (using 8. subtilis as a surrogate for 8. anthracis) inside a pilot-
scale HVAC system. The investigation focused on decontamination of either unlined galvanized metal
duct or galvanized metal lined internally with fiberglass duct insulation. Test samples were placed at
eight locations along the duct circuit to assess whether location within the system impacted results.
(Location within the duct system was generally not a factor in decontamination efficacy.) Tests were
conducted with varying operational parameters (e.g., contact time, decontaminant concentration, relative
humidity) to assess the effect of these parameters on decontamination efficacy.
Overall, CIO2 was found to be an effective decontaminant for use in both lined and unlined HVAC duct
systems for many of the test conditions tested. Although the overall decontamination efficacy was higher
at the higher concentration (3,000 ppm CIO2) compared to the lower concentration (200 ppm CIO2), as
expected, extending the contact time for the 200 ppm condition improved efficacy in most cases. For
example, average decontamination efficacy values of nearly 7.0 LR (when > 6.0 LR is considered
effective) were obtained for the 200 ppm CIO2 tests when contact times of eight hours or more were used.
Since some duct material degradation (evidenced by the presence of oxidation particles in coupon
extraction fluid) was observed when fumigating at 3,000 ppm CIO2, fumigating at lower concentrations but
longer contact times may be an effective decontamination approach and may mitigate potential impacts
on duct material.
Two tests were conducted to assess the feasibility and efficacy of fogging PAA in an HVAC (unlined)
system. Both of these screening tests resulted in average LR values greater 7.0 (LR > 6.0 considered
effective), although only a few of the sample locations were completely decontaminated. One benefit,
however, of using PAA fog was that no oxidation of duct materials was observed (an issue with high
levels of CIO2).
A modification was made to the HVAC duct system for the fogging tests to allow for the foggerto be
positioned upright so that the fog could initially be directed upward through the riser duct. This
modification to the HVAC duct system was required for the type of fogger we used in the study. Other
types of foggers may not require this type of modification or be required to be positioned in a certain
manner. Additional tests of PAA fog, including using different types of fogging devices, are recommended
to further assess operational or environmental factors that may enhance or diminish efficacy in HVAC
systems.
41
-------
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 U.S. Environmental Protection Agency. "Evaluation of H2O2 fumigation for HVAC decontamination.
Assessment and evaluation report." U.S. Environmental Protection Agency, National Homeland Security
Research Center, Research Triangle Park, NC. EPA/600/R-12/586. August 2012.
4 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.
5 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. 2009, 7(4): 405-412.
6 Wood, J.P., M.W. Calfee, M. Clayton, N. Griffin-Gatchalian, A. Touati, K. Egler. Evaluation of peracetic
acid fogging for the inactivation of Bacillus spores. Journal of Hazardous Materials. 2013, 250-251, 61-
67.
7 Determining the Efficacy of Liquids and Fumigants in Systematic Decontamination Studies for Bacillus
anthracis Using Multiple Test Methods. US EPA Report 600/R-10/088, December 2010.
8ARCADIS 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)
9 Lee, S.D., S.P. Ryan, E.G. Snyder. "Development of an aerosol surface inoculation method for Bacillus
spores," Applied and Environmental Microbiology. 2011, 77(5):1638-1645.
10Rastogi, 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," Applied and Environmental Microbiology.2009, 75(11):3688-3694.
11Rogers, J.V., C.L. Sabourin, Y.W. Choi, W.R. Richter, D.C. Rudnicki, K.B. Riggs, M.L. Taylor, J. Chang,
"Decontamination assessment of Bacillus anthracis, Bacillus subtilis, and Geobacillus stearothermophilus
spores on indoor surfaces using a H2O2 gas generator," Journal of Applied Microbiology. 2005, 99(4):
739- 748.
12Wood, J.P., S.P. Ryan, E.G. Snyder, S.D. Serre, A. Touati, M.J. Clayton. Adsorption of chlorine dioxide
gas on activated carbons. Journal Of the Air & Waste Management Association. 2010. 60(8): 898-906.
42
-------
13 US Environmental Protection Agency. Method 2 - Velocity - S-Type Pitot.
http://www.epa.gov/ttnemc01/methods/method2.html (accessed January 28, 2014).
43
-------
United States
Environmental Protection
Agency
PRESORTED STANDARD
POSTAGE & FEES PAID
EPA
PERMIT NO. G-35
Office of Research and Development (8101R)
Washington, DC 20460
Official Business
Penalty for Private Use
$300
------- |