®EPA
EPA 542-R19-001
May 2017
Low-Concentration Hydrogen Peroxide
(LCHP) Vapor for Bioremediation
Assessment and Evaluation Report
Contract EP-C-15-008
Office of Land and Emergency Management (OLEM)
Consequence Management Advisory Division (CMAD)

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Low-Concentration Hydrogen Peroxide
(LCHP) Vapor for Bioremediation
Assessment and Evaluation Report
EPA/OLEM/CMAD Technical Lead: R. Leroy Mickelsen
Prepared by:
Abderrahmane Touati, Ph.D., Francis Delafield, Denise Aslett, Ph.D., and
Ahmed Abdel-Hady
Jacobs Technology, Inc.
Research Triangle Park, NC 27709
JACOBS
Contract EP-C-15-008, WA 1-150
Office of Land and Emergency Management (OLEM)
Consequence Management Advisory Division (CMAD)

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Disclaimer
The U.S. Environmental Protection Agency (EPA), through its Office of Land and Emergency
Management/ Consequence Management Advisory Division (CMAD) funded and managed this
investigation through an EPA's Research Laboratory Support Contract No. EP-C-15-008 (Work
Assignment (WA) 1-150) with Jacobs Technology, Inc. (Jacobs). This report will undergo peer and
administrative reviews and will be approved for publication as an EPA document. This report does not
necessarily reflect the views of the EPA. No official endorsement should be inferred.
Questions concerning this document or its application should be addressed to the following individual:
R. Leroy Mickelsen
Consequence Management Advisory Division (CMAD)
Office of Land and Emergency Management
U.S. Environmental Protection Agency (MD-E343-06)
109 T.W. Alexander Drive
Research Triangle Park, NC 27711
Telephone No.: (919) 541-1356
Fax No.: (919) 541-0496
E-mail Address: mickelsen.lerov@epa.gov

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Acknowledgments
This research effort is part of EPA's Chemical, Biological, Radiological, and Nuclear (CBRN)
Consequence Management Advisory Division (CMAD) and National Homeland Security Research Center
(NHSRC) to develop "low-tech" techniques for Bacillus anthracis remediation operations. Additional focus
has been placed on correlating the effectiveness of decontamination efficiency with Bacillus anthracis
surrogates and technology operating parameters for the most promising technologies through systematic
decontamination studies.
The goal of this research effort is to demonstrate the efficacy of the fumigant distribution method in a full-
scale structure and the variables that affect the fumigant distribution effort. This research effort evaluates
the sporicidal efficacy of low-concentration hydrogen peroxide (LCHP) vapor in a typical residential home.
The LCHP vapor was generated from a 3 to 4% hydrogen peroxide (HP) aqueous solution in water
placed in commercial off-the-shelf (COTS) humidifiers.
R. Leroy Mickelsen from EPA's CBRN CMAD, the principal investigator, directed this research effort with
support from a project team consisting of staff from across the EPA. In addition, the individuals listed
below contributed to this research effort.
EPA
Shawn P. Ryan, Ph.D., Division Director
Decontamination and Consequence Management Division
National Homeland Security Research Center
Telephone No.: (919) 541-0699
E-mail Address: rvan.shawn@epa.gov
William Nichols, EPA Quality Assurance (QA) Representative
Office of Emergency Management
Regulation and Policy Development Division
Washington, DC
Telephone No.: (202) 564-2625
E-mail Address: nichols.nick@epa.gov
Shannon Serre, Ph.D.
Consequence Management Advisory Division (CMAD)
Office of Land and Emergency Management
Telephone No.: (919) 541-3817
E-mail Address: serre.shannon@epa.oov
Joseph Wood
Decontamination and Consequence Management Division
National Homeland Security Research Center
Telephone No.: (919) 541-5029
E-mail Address: wood.ioe@epa.qov
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M. Worth Calfee, Ph.D.
Decontamination and Consequence Management Division
National Homeland Security Research Center
Telephone No.: (919) 541-7600
E-mail Address: calfee.worth@epa.gov
Timothy Boe
Decontamination and Consequence Management Division
National Homeland Security Research Center
Telephone No.: (919) 541-2617
E-mail Address: boe.timothv@epa.gov
John Archer, MS, CIH, Research Industrial Hygienist
Decontamination and Consequence Management Division
National Homeland Security Research Center
Telephone No.: (919) 541-1151
E-mail Address: archer.iohn@epa.gov
Jayson Griffin
Consequence Management Advisory Division (CMAD)
Office of Land and Emergency Management
Telephone No.: (919) 541-2110
E-mail Address: griffin.iavson@Epa.gov
Doug Hamilton
ORISE Research Participant
Decontamination and Consequence Management Division
National Homeland Security Research Center
Telephone No.: (919) 541-0496
E-mail Address: hamilton.douglas@epa.gov
Kenneth Rhame
On-Scene Coordinator
EPA Region 4
Telephone No.: (919) 475-7397
E-mail Address: rtiame.kenneth@epa.gov
Benjamin Franco
On-Scene Coordinator
EPA Region 4
Telephone No.: (404) 562-8578
E-mail Address: franco.beniamin@epa.gov
Natalie Koch
Consequence Management Advisory Division (CMAD)
Office of Land and Emergency Management
Telephone No.: (859) 594-6528
E-mail Address: koch.natalie@Epa.gov
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Elise Jakabhazy
Consequence Management Advisory Division (CMAD)
Office of Land and Emergency Management
Telephone No.: (617) 918-1228
E-mail Address: Jakabhazv.elise@Epa.gov
Jacobs Technology. Inc.
Abderrahmane Touati, Ph.D.
Department Manager/Project Manager
Jacobs Technology, Inc.
Telephone No.: 919-541-3662
E-mail Address:touati.dahman@epa.gov
Rob Delafield
Work Assignment Leader
Jacobs Technology, Inc.
Telephone No.: (919) 541-1066
E-mail Address: francis.delafield@Jacobs.com
Denise Aslett, Ph.D.
Senior Microbiologist
Jacobs Technology, Inc.
Telephone No.: (919) 541-3059
E-mail Address: aslett.denise@epa.oov
Lee Brush
Technical Support
Jacobs Technology, Inc.
Telephone No.: (919) 541-4683
E-mail Address: lee.brush@iacobs.com
Ahmed Abdel-Hady
Scientist II
Jacobs Technology, Inc.
Telephone No.: (919) 541-2423
E-mail Address: abdel-hadv.ahmed@epa.qov
Brian Sechrest
Technical Support
CSS Dynamac
Telephone No.: (919) 541-2352
E-mail Address:bsechrest@css-dvnamac.com
Additionally, the authors would like to thank Dan Freeland and the peer reviewers for their significant
contributions.
Dan Freeland
Raleigh Marriott
4500 Marriott Drive Valley
Raleigh, NC 27612
Phone: 919-781-7000
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Contents
Disclaimer	iii
Acknowledgments	iv
Figures	viii
Tables	x
Acronyms and Abbreviations	xi
Executive Summary	xiii
1	Introduction	1
1.1	Project Background	1
1.2	Project Description and Objectives	2
2	Experimental Approach	3
3	Experimental Methods and Materials	4
3.1	Cary Test House	4
3.2	Experiment Set-up	5
3.3	Air Infiltration Estimates	6
3.4	Coupon Preparation	7
3.4.1	Coupon Sterilization	7
3.4.2	Coupon Inoculation	8
3.5	Biological Indicator discs	9
3.6	Decontamination Approach	9
3.7	Test Matrix	10
4	Testing and Measurements	13
4.1	Hydrogen Peroxide Sensors and Monitoring Points	13
4.2	Analytical Procedures	13
4.2.1	Coupon Analysis	13
4.2.2	Biological Indicator Disc Analysis	15
4.2.3	Data Reduction	15
5	Results and Discussion	17
5.1	Test 0 (Humidity Adjustment Only)	17
5.1.1	Living Area Fumigation Conditions	17
5.1.2	Temporal Environmental Conditions in Crawl Space and Attic	20
5.1.3	Temporal Survivability of Spores	22
5.2	Test 1	23
5.2.1	Living Area Fumigation Conditions	24
5.2.2	Crawl Space and Attic Fumigation Conditions	27
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5.2.3 Decontamination Efficacy	29
5.3	Test 2	32
5.3.1	Living Area Fumigation Conditions	34
5.3.2	Crawl Space and Attic Fumigation Conditions	36
5.3.3	Decontamination Efficacy	39
5.4	Test 3	42
5.4.1	Living Area Fumigation Conditions	45
5.4.2	Decontamination Efficacy	46
5.5	Test 4	50
5.5.1	Living Area Fumigation Conditions	52
5.5.2	Decontamination Efficacy	54
5.6	Test 5	57
5.6.1	Living Area Fumigation Conditions	59
5.6.2	Decontamination Efficacy	61
6 Quality Assurance and Quality Control	63
6.1	Project Documentation	63
6.2	Integrity of Samples and Supplies	64
6.3	Instrument Calibration	64
6.4	Critical Measurements	64
6.5	QC and NHRSC BioLab Checks	65
6.6	QA Assessments and Response Actions	67
References	67
Figures
Figure 3-1. CTH Floor Plan	4
Figure 3-2. Humidifier Placement in Kitchen	5
Figure 3-3. Typical Sampling Set with Coupons in Plastic Holders, Temperature and RH Monitor,
Three Bl discs in Tyvek Envelopes, HP Test Strip, and DragerTube	6
Figure 3-4. Stainless-Steel Stage for Coupon Sterilization	8
Figure 3-5. MDI and MDI Actuator	9
Figure 4-1. Bacterial Colonies on Spiral-plated Agar Plate	14
Figure 4-2. Bacterial Colonies on Filter Plate	14
Figure 4-3. Bl Turbidity Analysis: Left - TSB Turbid (Growth); Right - TSB Clear (No Growth)	15
Figure 5-1. CTH Test Set-up	18
Figure 5-2. Test 1 Temporal Air Exchange Rates in Living Areas	19
Figure 5-3. Baseline Test Temporal RH in Living Areas	20
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Figure 5-4. Baseline Test Temporal Environmental Conditions in Crawl Space	21
Figure 5-5. Baseline Test Temporal Environmental Conditions in Attic	22
Figure 5-6. Test 1 Temporal HPV Concentration in Living Areas	25
Figure 5-7. Test 1 Temporal RH in Living Areas vs. Outdoors	26
Figure 5-8. Test 1 Temporal Temperature in Living Areas vs. Outdoors	26
Figure 5-9. Test 1 Temporal HPV Concentration in Crawl Space and Attic	27
Figure 5-10. Test 1 Temporal RH in Crawl Space vs. Outdoors	28
Figure 5-11. Test 1 Temporal RH in Attic vs. Outdoors	29
Figure 5-12. Bl Disc Package Placement at Periphery of Crawl Space	31
Figure 5-13. Post-Decontamination DragerTube in Living Area	32
Figure 5-14. CTH Equipment and Sampling Locations for Test 2	33
Figure 5-15. Test 2 Temporal HPV Concentration in Living Areas	34
Figure 5-16. Test 2 Temporal RH in Living Areas	35
Figure 5-17. Test 2 Temporal Temperature in Living Areas	36
Figure 5-18. Test 2 Temporal HPV Concentration in Crawl Space and Attic	37
Figure 5-19. Test 2 Temporal RH in Crawl Space	38
Figure 5-20. Test 2 Temporal RH in Attic	39
Figure 5-21. Furniture Added to CTH for Test 3	42
Figure 5-22. Test 3 Placement of Bl discs Between Couch Cushions	43
Figure 5-23. Test 3 Placement of Bl discs Under Carpet	43
Figure 5-24. Test 3 Placement of Bl discs in Bathroom Drawer (closed during testing)	43
Figure 5-25. Test 3 Placement of Bl discs Under One and Five Pieces of Paper	44
Figure 5-26. Test 3 Temporal HPV Concentration in Living Area	45
Figure 5-27. Test 3 Temporal RH in Living Areas	46
Figure 5-29. CTH Equipment and Sampling Locations for Test 4	50
Figure 5-30. Test 4 Bl discs Inside Book (closed during the test)	51
Figure 5-31. Test 4 Bl disc Inside Coat Pocket in Entry Closet	51
Figure 5-32. Test 4 Bl discs Behind Light Switch Plate	51
Figure 5-33. Test 4 Temporal HPV Concentration in Living Areas	53
Figure 5-34. Test 4 Temporal RH in Living Areas	54
Figure 5-35. Test 4 LR Values in Spore Counts for Coupons in Living Areas	56
Figure 5-36. CTH Equipment and Sampling Locations for Test 5	58
Figure 5-37. Test 5 Temporal HPV Concentration in Living Areas	60
Figure 5-38. Test 5 Temporal RH in Living Areas	61
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Tables
Table 3-2. Test Matrix (Humidifiers operations and locations)	11
Table 5-1. CTH Air Exchange Rates	19
Table 5-2. Test 0 (Humidity Adjustment Only) Recoveries on Test Coupons and Bl discs	22
Table 5-3. Test 1 Fumigation Conditions	24
Table 5-4. Test 1: Post-Decontamination Recoveries on Test Coupons and Bl discs	30
Table 5-5. Test 2 Fumigation Conditions	33
Table 5-6. Test 2: Post-Decontamination Recoveries on Test Coupons and Bl discs	40
Table 5-7. Test 3 Fumigation Conditions	44
Table 5-8. Test 3: Post-Decontamination Recoveries on Test Coupons and Bl discs	47
Table 5-9. Test 3 Post-Decontamination Recoveries on Hard-to-Reach Bl discs	49
Table 5-10. Test 4 Fumigation Conditions	52
Table 5-11. Test 4 Post-Decontamination Recoveries on Test Coupons and Bl discs	55
Table 5-12. Test 4 Post-Decontamination Recoveries on Hard-to-Reach Bl discs	57
Table 5-13. Test 5 Fumigation Conditions	59
Table 5-14. Test 5: Post-Decontamination Recoveries on Test Coupons and Bl discs	61
Table 5-15. Test 5 Post-Decontamination Results for Hard-to-Reach Bl discs	63
Table 6-1. Instrument Calibration Frequencies and Expected Tolerances	64
Table 6-2. DQIs and Acceptance Criteria for Critical Measurements	65
Table 6-3. Additional Quality Checks for Biological Measurements	66
Table 6-4. QA/QC Assessment of Spore Recoveries for Various Sample Types (CFUs/Sample)	67
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Acronyms and Abbreviations
Ba	Bacillus anthracis
Bg	Bacillus atrophaeus var. globigii
Bl	biological indicator
BioLab	NHSRC Research Triangle Park (RTP) Microbiology Laboratory
CBRN	Chemical, Biological, Radiological, and Nuclear
CFU	colony-forming unit
CMAD	Consequence Management Advisory Division
COTS	commercial off-the-shelf
CT	concentration-time
CTH	Cary Test House
DQI	data quality indicator
DTRL	Decontamination Technologies Research Laboratory
EPA	U.S. Environmental Protection Agency
EtO	ethylene oxide
FIFRA	Federal Insecticide, Fungicide, and Rodenticide Act
ft2	square foot
GM	galvanized metal
G/NG	growth or no growth
Gs	Geobacillus stearothermophilus
HP	hydrogen peroxide
HPV	hydrogen peroxide vapor
HVAC	heating, ventilation, and air conditioning
IDLH	immediately dangerous to life and health
LCHP	low-concentration hydrogen peroxide
LR	log reduction
MDI	metered dose inhaler
mL	milliliter
mm	millimeter
ND	Non detect
NHSRC	National Homeland Security Research Center
NIST	National Institute of Standards and Technology
OEM	Office of Emergency Management
OLEM	Office of Land and Emergency Management
PARTNER	Program to Align Research and Technology with the Needs of Environmental
Response
PBST	phosphate-buffered saline with 0.05% Tween® 20
ppmv	part per million by volume
QA	quality assurance
QAPP	Quality Assurance Project Plan
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QC
quality control
RH
relative humidity
SD
standard deviation
TSA
tryptic soy agar
TSB
tryptic soy broth
WA
work assignment

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Executive Summary
The U.S. Environmental Protection Agency's (EPA) Chemical, Biological, Radiological, and Nuclear
(CBRN) Consequence Management Advisory Division (CMAD) of the Office of Emergency Management
(OEM) and the National Homeland Security Research Center (NHSRC) are providing information to the
response community on decontamination technologies for restoring sites contaminated with biological,
chemical, or radiological agents. For biological remediation technologies, the EPA has focused on
evaluating "low-tech" solutions for decontaminating building materials contaminated with Bacillus
anthracis Ames strain (Ba: anthrax) spores. Additional focus has been placed on correlating the
effectiveness of decontamination efficiency with Bacillus anthracis (Ba) surrogates and technology
operating parameters for the most promising technologies through systematic decontamination studies.
The goal of this research effort was to demonstrate the efficacy of the fumigant distribution method in a
full-scale structure and identify the variables that affect the fumigant distribution. This research effort
evaluated the sporicidal efficacy of low-concentration hydrogen peroxide (LCHP) vapor in a typical
residential home. The LCHP vapor was generated from a 3 to 4% hydrogen peroxide (HP) aqueous
solution in water placed in commercial off-the-shelf (COTS) humidifiers. The potential application of this
method would be for homes and small businesses near the Exclusion Zone but not in it. These structures
may be contaminated with low amounts of Ba spores. Decontamination resources will be occupied inside
the EZ leaving homes and small businesses near the contaminated area without access to typical
remediation options.
The technical approach for this study involved systematic field research on the efficacy of
decontaminating building materials inoculated with Bacillus spores as a function of operating conditions,
materials present, and fumigation method. Tests were conducted using spores of Bacillus atrophaeus var.
globigii (Bg) Gibbons, et.al. (2011) and Geobacillus stearothermophilus (Gs), both non-pathogenic
surrogates for Bacillus anthracis. Bg and Gs, like Bacillus anthracis, are gram-positive, spore-forming
bacteria. Coupons and biological indicator (Bl) discs containing Bg and Gs spores, respectively, were
placed in a typical single-family home (the Cary Test House [CTH]), which was subsequently fumigated.
The laboratory technique developed for systematic evaluation of the efficacy of biological warfare agents
was adapted forthis effort. The technique consisted of (1) inoculating coupons of typical home materials
(carpet and galvanized metal [GM]), (2) exposing the coupons (inoculated with Bg) and Bl discs (pre-
inoculated with Gs) to a range of decontamination conditions, and (3) evaluating the viable spores
recovered from the coupons and Bl discs after decontamination and comparing the results to positive
controls (inoculated but non-exposed coupons and Bl discs).
The results of this study show that using LCHP vapor generated from HP aqueous solution in water
disseminated by COTS humidifiers can be an effective sporicidal surface decontamination technique to
help reduce indoor Bacillus anthracis contamination. In some configurations, test coupons exhibited a log
reduction (LR) of 6 in recovered spores.
The results of this study are summarized below.
• LCHP vapor was efficacious under certain specific operating conditions for full
decontamination (complete spore kill) of indoor living areas.
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•	LCHP vapor was efficacious (6 LR) for carpet and GM coupons inoculated with Bg and Bl
discs inoculated with Gs (Bg and Gs are surrogates for Bacillus anthracis).
•	The efficacious dose was 12 gallons of 3 to 4% HP for the inside of a 1,200-square-foot (ft2),
9,600 cubic feet home (1 gallon per 800 ft3).
•	There were several ways to distribute the humidifiers to obtain efficacious results. Placement
of the humidifiers near the heating, ventilation, and air conditioning (HVAC) air return ducts
and setting the HVAC fan to operate continuously was an effective LCHP vapor distribution
method.
•	LCHP vapor penetrated through sheets, thin clothing, a bedding comforter, five sheets of
paper, and some closed drawers, resulting in full decontamination of Gs spores on Bl discs.
•	LCHP vapor did not penetrate into light switch boxes, thick clothing, books, rugs, furniture
cushions, 10 sheets of paper, and some closed drawers, resulting in less efficacious
decontamination of Gs spores on Bl discs.
•	The attic and to a lesser extent, the crawl space, were the most challenging spaces for the
decontamination method because vents in these locations allowed outside air exchange.
•	The results of this study may not be directly applicable to any given site, and spore
inactivation results on the tested Bl discs and coupons may not apply to other site-specific
materials and conditions.
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1 Introduction
The U.S. Environmental Protection Agency's (EPA) Chemical, Biological, Radiological, and Nuclear
(CBRN) Consequence Management Advisory Division (CMAD) of the Office of Emergency Management
(OEM) and the National Homeland Security Research Center (NHSRC) are providing information to the
response community on decontamination technologies for restoring sites contaminated with biological,
chemical, or radiological agents. For biological remediation technologies, the EPA has focused on
evaluating "low-tech" solutions for decontaminating building materials contaminated with B. anthracis
Ames strain spores. Additional focus has been placed on correlating the effectiveness of decontamination
efficiency with Ba surrogates and technology operating parameters for the most promising technologies
through systematic decontamination studies.
Specifically, the Office of Land and Emergency Management (OLEM) CBRN CMAD scaled up the Office
of Research and Development's NHSRC laboratory research findings to a full-scale venue that may be
directly applicable in a real-life emergency situation. Results and documents generated from this study
may be directly applicable for real-world recovery operations.
This report discusses the efficacy of the fumigant distribution method in a full-scale structure and the
variables that affect fumigant distribution. This research effort evaluated the sporicidal efficacy of low-
concentration hydrogen peroxide (LCHP) vapor in a typical residential home. The LCHP vapor was
generated from a 3 to 4% hydrogen peroxide (HP) aqueous solution in water placed in commercial off-
the-shelf (COTS) humidifiers.
The technical approach for this study involved systematic field research on the efficacy of
decontaminating building materials inoculated with Bacillus spores as a function of operating conditions,
materials present, and fumigation method. Tests were conducted using spores of Bacillus atrophaeus var.
globigii (Bg) and Geobacillus stearothermophilus (Gs), both surrogates for Ba. Bg and Gs, are gram-
positive, spore-forming bacteria. Coupons and biological indicator (Bl) discs inoculated with Bg and Gs
spores respectively were placed in a typical single-family home (the Cary test house [CTH]), which was
subsequently fumigated.
The following sections discuss the project background and the project description and objectives.
1.1 Project Background
The EPA's Homeland Security Research Program strives to provide expertise and products that can be
widely used to prevent, prepare for, and recover from public health and environmental emergencies
arising from terrorist threats and other contamination incidents. OLEM, through its Special Teams, which
includes the CBRN CMAD, supports the emergency response functions carried out by EPA Regional
Offices. The Office of Chemical Safety and Pollution Prevention supports the decontamination effort by
providing expertise on biological agent inactivation and ensuring that the use of pesticides and other
inactivation agents in such efforts is conducted in accordance with the Federal Insecticide, Fungicide, and
Rodenticide Act (FIFRA). Close collaboration between the different program offices having homeland
security responsibilities is required to increase EPA's capabilities in helping the United States recover
from a terrorist event involving the intentional release of CBRN materials. Such collaboration is fostered
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through efforts such as the Program to Align Research and Technology with the Needs of Environmental
Response (PARTNER)1 (EPA 2014).
Contamination incidents may result from intentional or accidental releases of biological materials or
human or animal disease outbreaks. All scenarios pose significant challenges with regard to determining
the extent of contamination, containing the contaminant spread, and remediating the event so that re-
occupancy or reuse can occur. The project that is the subject of this report supports multiagency
objectives of better understanding and preparing for the remediation of Bacillus anthracis in homes after a
biological contamination incident.
Resources for responding to a large Bacillus anthracis (anthrax) release are limited. After a large Bacillus
anthracis release, there will be a high demand for response and recovery and few resources to meet that
demand. Limited resources result in longer recovery times and higher negative impacts. Many efficacious
remediation products produce highly toxic environments and require very specialized equipment and
expertise. For example, chlorine dioxide, an unstable chemical, must be produced on location. In addition,
chlorine dioxide, methyl bromide, and typically HP are used at concentrations 20 to 100 times higher than
the concentration that is immediately dangerous to life and health (IDLH) (TLVS and BEIS. 2017),
requiring specialized equipment and expertise for safe handling.
HP typically is used at a concentration of 400 parts per million by volume (ppmv) for 4 hours
(concentration * time = 1,600 ppm-hours). Wood, et al. (2016) conducted a laboratory experiment using
test coupons of small sections of common materials such as carpet, concrete, ceiling tiles, metal, and
drywall. In this experiment, a lower concentration of HP vapor (HPV) (5 parts per million [ppm] over 4 to 7
days = 480 to 840 ppm-hour) resulted in a six-log reduction of spores on all materials tested with the
exception of concrete. Wood, et al. (2016) also demonstrated that the LCHP vapor could be disseminated
using COTS humidifiers.
Based on these laboratory findings, this project was developed to evaluate the efficacy of LCHP vapor to
clean indoor areas of residential homes contaminated with surrogates of Bacillus anthracis (the causative
agent of anthrax) spores. Low HPV concentrations (less than 25 ppm) and easy-to-deploy techniques
were targeted for this effort. The higher the fumigant concentration deployed, the more expertise required
for deployment. Conversely, the lower the fumigant concentration, the safer the technique is to deploy.
For HP the IDLH is 75 ppm, and the threshold limit value and the permissible exposure limits are both 1
ppm (CDC. NIOSH. OSHA). HP levels used in this study were all below the IDLH and are less hazardous
than typical HP fumigations (400 ppm). Even at these low HP concentrations, HP exposure should be
avoided. The project that is the subject of this report supports multi-agency objectives of better
understanding and preparing for the remediation of a typical residential structure using LCHP vapor.
1.2 Project Description and Objectives
The purpose of this project was to evaluate the efficacy of the LCHP vapor fogging approach in a full-
scale structure. Specifically, this project evaluated the sporicidal efficacy of LCHP vapor in a typical
residential home represented by an EPA test house, the CTH. The LCHP vapor was generated from 3 to
4% liquid HP placed in COTS humidifiers.
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Tests were conducted using spores of Bg and Gs, both surrogates for Ba. The laboratory technique
developed by Wood, et al. (2016) for systematic evaluation of the efficacy of remediation of biological
warfare agents was adapted for this effort. This technique consisted of (1) inoculating coupons
constructed of typical household materials (carpet and GM), (2) exposing the coupons and Bl discs pre-
inoculated with Bg and Gs, respectively, to a range of decontamination conditions, and (3) evaluating the
viable spores recovered from the coupons and Bl discs before and after decontamination. The coupons
and Bl discs containing the test spores were placed in a typical single-family home (the CTH). Carpet and
GM were selected for this study because these materials were the most difficult (excluding concrete) to
decontaminate in the laboratory using LCHP vapor, based on findings reported by Wood, et al. (2016).
2 Experimental Approach
The overall experimental approach consisted of preparing coupons of carpet and galvanized metal (GM)
and inoculating each coupon with Bg spores. In addition, Bl steel discs pre-inoculated with Gs spores
were used. The coupons and Bl discs were placed throughout a 1,200-ft2, three-bedroom residential
home. The home was decontaminated using LCHP vapor generated by seven COTS humidifiers charged
with a 3 to 4% HP aqueous solution. The humidifiers were set to run continuously until all liquids were
dispensed.
HP vapor sensors were placed in various locations throughout the CTH in living areas, the attic, and the
crawl space. The CTH's heating, ventilation, and air conditioning (HVAC) system was used during testing
(thermostat placed on "heat" setting) to maintain the household temperature at 70 °F, and the HVAC
circulation fan operated continuously. Oscillating fans also were used to aid in air mixing. A real-time
relative humidity (RH) and temperature meter was set up to monitor the CTH throughout each test. A
meteorology station was set up outside the CTH to monitor wind velocity, temperature, RH, and rainfall so
that these data combined with the data inside the house could be used to estimate air exchange rates.
After fumigation, the test coupons and Bl discs were collected and analyzed for spore survival using a
plating and colony-counting methodology. Inoculated but unexposed carpet and GM coupons and Bl
discs were used as positive controls.
The general experimental approach used to meet project objectives is described below.
•	Preparation of representative coupons of test materials: Coupons of carpet and GM with a
diameter of 18 mm each were prepared as discussed in Section 3.4.
•	Sterilization of the coupon materials: Prior to use, the GM and carpet coupons were sterilized
for 18 hours using ethylene oxide (EtO) as discussed in Section 3.4.1.
•	Inoculation of coupons: Positive control and test coupons were inoculated using the aerosol
deposition method described in Section 3.4.2. Briefly, a known quantity of the surrogate organism
(2 x 1 o7 colony-forming units [CFU] of Bg spores) was deposited onto the center of each coupon
using a metered-dose inhaler (MDI). Inoculation was conducted for both types of coupons on the
same day, and then the coupons were placed into the CTH for fumigation within 48 hours after
inoculation.
•	Decontamination: The decontamination approach consisted of fumigating the CTH indoor area
using 3 to 4% HP liquid fumigant placed in COTS humidifiers as discussed in Section 3.6. LCHP
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fumigation typically lasted 24 to 48 hours, with subsequent off-gassing and aeration continuing for
the remainder of the week.
• Decontamination Evaluation: As discussed in Section 4.2. after fumigation, coupon samples
were analyzed for viable spores (CFUs), and Bl discs pre-inoculated with >1 x 106 Gs spores
were cultured and analyzed for growth or no growth (G/NG). The fumigation atmosphere for HPV
concentration, RH, and temperature were tabulated and summarized as discussed in Section 5.
Decontamination effectiveness was measured as an LR value for viable spores on the inoculated
coupons exposed to fumigation compared to inoculated but un-fumigated control coupons.
Decontamination effectiveness for the Bl discs was evaluated based on G/NG results, with NG
results indicating effectiveness.
This section describes the experimental testing methods and materials, including the CTH, experiment
set-up, determination of baseline conditions, coupon preparation, Bl discs, decontamination, and the test
matrix.
3.1 Gary Test House
The CTH is a three-bedroom, ranch-style house with a crawl space, a central forced-air heating system
that uses natural gas, and an electric air-conditioning system. The total floor area is 1,200 ft2, and the
house has a total volume of approximately 9,600 cubic feet. Figure 3-1 shows the CTH floor plan.
3 Experimental Methods and Materials
Figure 3-1. CTH Floor Plan
4

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3.2 Experiment Set-up
Fifteen sampling locations were selected to place test coupons to evaluate the efficacy of the
decontamination process over the entire CTH, Variables included locations of humidifiers, amount of HP
solution used to charge the humidifiers, and whether or not spaces were sealed (such as the crawl space
and attic). Initial conditions called for placing one humidifier in each of the following areas: kitchen, den,
and the three bedrooms; two humidifiers in the living room; and three humidifiers each in both the attic
and crawl space, for a total of 13 humidifiers. Humidifiers, as shown in Figure 3-2, generally were placed
in or near the center of the room. Each humidifier was charged with 2 gallons of 3 or 4% liquid HP
solution. All windows and exterior doors were closed, and the attic and crawl space were loosely sealed.
The door for each room was left open during the decontamination event. An oscillating fan (model 2521,
Lasko Products, Inc., West Chester, PA) was placed in each room that did not have a ceiling fan to aid in
mixing.
Figure 3-2. Humidifier Placement in Kitchen
The garage was used for personnel and instrument staging and was not fumigated. An HPV sensor
capable of detecting 0 to 10 ppm HPV was placed in the garage and set to alarm at 0.5 ppm in case of
accidental release of fumigant to this area.
The typical sampling set (as shown in Figure 3-3) for each location consisted of the following items:
(1)	Three carpet and three GM inoculated coupons.
(2)	One carpet and one GM un-inoculated (blank) coupon - designated locations only
(3)	One Bl disc was co-located with each coupon set. Two additional Bl discs were located nearby
in the same room in hard-to-reach places.
(4)	a HOBO® RH/T Onset Data Logger (Model U12, Onset Computers, Bourne, MA) to monitor
temperature and RH.
5

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(5)	an SPS Medical GPS-250R VH202 Chemical indicator Strip (SPS Medical Supply Corp., Rush,
NY) to monitor exposure to HP.
(6)	a Drager HP tube (Drager™ Short-Term Detector Tubes - Hydrogen Peroxide, Cat. No
8101041, Drager, Inc., Houston, TX) with one end open to monitor (similar to chemical indicator
test strips) HPV diffusion into the tube.
Figure 3-3. Typical Sampling Set with Coupons in Plastic Holders, Temperature and RH Monitor,
Three Bl discs in Tyvek Envelopes, HP Test Strip, and Drager Tube
Drager tubes are designed to estimate the HP concentration in an atmosphere based on pulling a known
volume of atmosphere through the tube. For this study, the Drager tubes were re-purposed by opening
only one end of the tube and allowing the fumigant to diffuse into the tube, with the objective of
developing a correlation between the Drager tube cumulative diffusion reading and fumigant efficacy. A
total of 16 Drager tubes were used for each test run, one at each coupon location.
3.3 Air Infiltration Estimates
A meteorology station (Vantage Pro2, Product # 6152, Davis Instruments, Hayward, CA) and a data
acquisition system (WeatherLink®, Windows, USB, Product#651OUSB. Davis Instruments, Hayward,
CA) were placed outside the CTH to monitor wind velocity, temperature, RH, and rainfall. The weather
data outside and temperature inside the CTH were used to estimate the air infiltration rate (N) using
Equation 3-1 below. Equation 3-1 and constants A, B, and C were developed in a previous CTH study
conducted in 1995 by Guo. Sparks, and Bero. The CTH configuration may differ slightly from the test
house used in the previous study, so the calculated N values are gross estimations.
N = A + BAT + C V	(3-1)
6

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Where
N = Air infiltration rate (hour1)
AT = Air temperature difference (°C)
V = Outdoor wind speed (meter per second)
A = 0.184 ±0.05
B = 0.0129 ±0.0004
C = 0.0882 ± 0.003
3.4 Coupon Preparation
The representativeness and uniformity of test materials are essential in achieving defensible evaluation
results. Materials are considered representative if they are typical of materials currently used in facilities
and buildings in terms of quality, surface characteristics, and structural integrity. Material uniformity
means that all test materials are equivalent. Uniformity was maintained by obtaining and preparing a
quantity of material sufficient to allow the preparation of multiple test coupons with presumably uniform
characteristic. Test coupons were cut from the interior rather than the edge of a large piece of material.
These material coupons were randomly selected for use as test, blank, and positive control coupons.
For this study, 18-mm-diameter coupons made of carpet and GM were used. GM was punched from 18-
gauge galvanized steel (P/N 01170, Eastcoast Metal Distributors, Durham, NC). Discs with an 18-mm-
diameter were cut from carpet (Multiplicity 54594, Shaw Industries Group, Dalton, GA). The coupons then
were mounted to 18-mm aluminum stubs (P/N 16119, Ted Pella, Inc., Redding, CA) using double-sided
adhesive tape (P/N 16073-2, Ted Pella, Inc., Redding, CA). The coupons were sterilized and inoculated
as summarized below.
3.4.1 Coupon Sterilization
The coupons were sterilized using an Andersen EtO sterilizer system (PN:333 EOGas®, Haw River, NC).
The sterilization procedure is summarized below.
1.	The coupons were randomly selected and loaded into stainless-steel stages (see Figure 3-4).
2.	The stage loaded with the coupons was placed in a glass petri dish and loosely covered with a
crystallization petri-dish (see Figure 3-4).
3.	Each petri dish was placed into an appropriate sterilization bag (PN:333 EOGas®, Haw River, NC).
4.	The sterilization bags were loaded into a cabinet for sterilization using EtO.
5.	The sterilization bags were removed from the EtO cabinet with the crystallization dishes covering
the petri dishes to maintain coupon sterility.
7

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Figure 3-4. Stainless-Steel Stage for Coupon Sterilization
3.4.2 Coupon Inoculation
The test organism for coupon inoculation was a powdered spore preparation of Bacillus atrophaeus var.
globigii, mixed with silicon dioxide particles obtained from the U.S. Army Dugway Proving Ground Life
Sciences Division. The preparation procedure is described in Brown, et al. (2007). After 80 to 90%
sporulation, the suspension was centrifuged to generate a preparation of approximately 20% solids. A
preparation resulting in a powdered matrix containing approximately 1 * 1011 viable spores per gram was
prepared by dry blending and jet milling the dried spores with fumed silica particles (Degussa, Frankfurt
am Main, Germany).
Positive control and test coupons were inoculated on the same day with approximately 2 x 107 aerosolized
spores using an aerosol deposition method. Briefly, each coupon was placed inside a chamber and
positioned in front of an MDI canister containing Bg spores suspended in ethanol solution and HFA-134A
propeiiant gas. The MDI is situated inside an actuator such that each time the actuator is depressed a
repeatable amount of spores is deposited on the coupon. (Lee, Ryan, and Snyder 2011). The MDI actuator
is a small plastic tube in which the MDI is inserted (see Figure 3-5). Each MDI was charged with a volume
of spore preparation plus propeiiant sufficient to deliver 200 discharges of 50 microliters (pl_) per discharge.
MDI use was tracked so that the number of discharges did not exceed 200. Additionally, MDIs selected for
testing were required to weigh more than 10.5 grams. MDIs weighing less than 10.5 grams were retired
and no longer used. Test and positive control coupons were inoculated a maximum of 48 hours before
decontamination.
8

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j \
\ 1
MDI actuator
Figure 3-5. MDI and MDI Actuator
3.5	Biological Indicator discs
The commercial Bl discs used for this study were purchased from Mesa Laboratories Inc. (P/N HMV-091,
Lakev/ood, CO). Each 0.35-inch diameter x 0.008-inch-thick stainless steel (Grade 304) disc was
inoculated with a minimum of 1 x 106 Gs spores and came from the manufacturer enclosed in a Tyvek
pouch.
3.6	Decontamination Approach
Whole-house COTS humidifiers (Model HCN-6009, Honeywell, Morristown, NJ) were charged with 3 to
4% HP solution instead of water. This type of humidifier uses a small fan to push air through a sponge
that wicks up the reservoir liquid. Each humidifier holds 3.4 gallons. For this study, 13 humidifiers were
deployed throughout the CTH living areas, crawl space, and attic. The HVAC system was set to hold the
CTH indoor temperature at 70 °F, and the fan was set to run continuously. A real-time RH and
temperature meter (Model HMD53, Vaisala, Helsinki, Finland) was set up to monitor the CTH inside RH
and temperature.
Seven days after beginning the decontamination process, a sampling team verified that the HPV
concentration was below 1 ppm using a Drager HP tube (Drager™ Short-Term Detector Tubes -
Hydrogen Peroxide, Cat. No 8101041, Drager, Inc., Houston, TX). The sampling team then entered the
CTH to collect samples. Positive control and blank coupons that remained outside of the CTH were
collected at the same time.
9

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3.7 Test Matrix
•	Test 0 (Humidity Adjustment Only)
As part of a scoping test, the humidity profile for the CTH was determined during baseline testing (Test 0,
Humidity Adjustment only). The temporal effect of high humidity (no HPV presence) on spore survivability
also was assessed.
To evaluate the efficacy of the LCHP vapor decontamination technique, five tests were run underthis
project. For Test 1, test conditions were scaled up from efficacious laboratory results for Wood, et al.
(2016) and then adjusted for subsequent tests based on whether or not a 6 LR was achieved for all
materials at all locations. Based on test results, site-specific changes were made during Test 2 by
changing the placement of fans, the number or placement of humidifiers, or the amount of liquid HP
solution deployed. When a 6 LR was achieved, then new and more challenging test conditions, such as
adding furniture or reducing the amount of liquid HP deployed, were utilized during Tests 3 through 5.
Unless otherwise noted, the test procedure was as summarized below.
1.	Start the HPV monitoring equipment.
2.	Ensure the HVAC system controls were set properly.
3.	Place the coupons, temperature and RH monitor, Bl discs, HP test strip, and Dragertube at each
sampling location throughout the CTH.
4.	Fill the humidifiers, measure the volume of filled solution, and place the humidifiers.
5.	Start the fans.
6.	Turn on the humidifiers.
7.	Secure the CTH until fumigation is completed (after 3 to 7 days).
The conditions for each of the five tests are described below. Table 3-2 summarizes the operating
conditions of the overall test matrix.
•	Test 1
o One humidifier in the kitchen, one in the den, two humidifiers in the living room, one
humidifier in each of the three bedrooms, and three humidifiers each in both the crawl space
and attic.
o Each humidifier charged with 2 gallons of 3% liquid HP solution,
o HVAC fan on continuously to circulate air.
o HVAC system on to maintain the household temperature at 70 °F.
o Humidifiers, fans, test coupons, and Bl discs located throughout the CTH as shown in Figure
5-1 in Section 5.
•	Test 2
o Same conditions as Test 1 with the following modifications:
¦	Humidifiers in crawl space loaded with 3.4 gallons of 3.8% HP aqueous solution; all other
humidifiers charged with 2 gallons of 3.8% HP aqueous solution.
¦	One living room humidifier moved to hallway under the air return; other humidifier
remained in living room.
10

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¦	One fan added to master bedroom, and one fan removed from den.
¦	Additional test coupons added to both den and living room.
o Humidifiers, fans, test coupons, and Bl discs located throughout the CTH as shown in Figure
5-14 in Section 5.
•	Test 3
o Same conditions as Test 2 with the following modifications:
¦	Humidifiers in crawl space loaded with 3.4 gallons of 3% HP aqueous solution; all other
humidifiers charged with 2 gallons of 3% HP aqueous solution.
¦	Furniture and clothing added as shown in Figure 5-21.
¦	Additional Bl discs placed in furniture and hard-to-reach places as shown in Figure 5-22
through Figure 5-25.
¦	Added extra test coupons to den and living room to be collected after 3 days.
•	Test 4
o For this test, several adjustments made to fan placement; in the corner and middle bedrooms,
fans moved closer to closets, and fan added near closet in living room (see Figure 5-29).
o Number of humidifiers in living areas reduced from seven to two and placed in hallway under
air return; both humidifiers filled to maximum capacity of 3.4 gallons, and run sequentially 24
hours apart; all humidifiers (including ones in crawl space and attic) filled to maximum
capacity of 3.4 gallons with 3.0% HP aqueous solution,
o Additional Bl discs placed in furniture and hard-to-reach places as discussed in Section 5.5
and shown in Figure 5-30 through Figure 5-32.
•	Test 5
o Same conditions as Test 4 except the two humidifiers were refilled after 72-hours of
operation: one with 3.2 gallons and the other with 2 gallons of 3% HP aqueous solution,
o Additional Bl discs placed in furniture and hard-to-reach places as discussed in Section 5.6.
o Humidifiers, fans, test coupons, and Bl discs located throughout the CTH as shown in Figure
5-36 in Section 5.
Table 3-2. Test Matrix (Humidifiers operations and locations)
Test 0 (Humidity Adjustment Only)
Location
Di-Water

Number of Humidifiers Volume Spent (gallons)
Concentration (%)
Master Bedroom
1
2.0
No HP
Den
1
2.0
Corner Bedroom
1
2.0
Middle Bedroom
1
2.0
Kitchen
1
2.0
Living Room
1
3.8
Crawl Space Periphery
3
1.6
Crawl Space Central Location
Attic Periphery
3
6.0
Attic Central Location
11

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Test 1
Location

HP Aqueous Solution

Number of Humidifiers
Volume Spent (gallons)
Concentration (%)
Master Bedroom
1
2.0
3.0
Den
1
1.9

Corner Bedroom
1
2.0

Middle Bedroom
1
2.0
3.0
Kitchen
1
1.9

Living Room
2
3.8

Crawl Space Periphery
3
5.4

Crawl Space Central Location
3.0
Attic Periphery
3
5.5
Attic Central Location

Test 2
Location
Number of Humidifiers
Volume Spent (gallons)
Concentration (%)
Master Bedroom
1
2.0
3.8
Den
1
1.9

Corner Bedroom
1
1.9

Middle Bedroom
1
1.9

Kitchen
1
1.9

Living Room
1
1.8
3.8
Hallway Air Return
1
2.0
Crawl Space Periphery
3


Crawl Space Central Location


Attic Periphery
3
5.5

Attic Central Location

Test 3
Location
Number of Humidifiers
Volume Spent (gallons)
Concentration (%)
Master Bedroom
1
2.0
3.0
Den
1
1.9

Corner Bedroom
1
1.9

Middle Bedroom
1
1.9

Kitchen
1
1.9

Living Room
1
2.0
3.0
Hallway Air Return
1
2.0
Crawl Space Periphery
3
8.3

Crawl Space Central Location

Attic Periphery
3
9.6

Attic Central Location

Test 4
Location
Number of Humidifiers
Volume Spent (gallons)
Concentration (%)
Hallway Air return
2
6.6

Crawl Space Periphery
3
8.2

Crawl Space Central Location
3.0
Attic Periphery
3
9.8

Attic Central Location

Test 5
Location
Number of Humidifiers
Volume Spent (gallons)
Concentration (%)
Hallway Air Return
2
11.6

Crawl space Periphery
3
9.8

Crawl Space Central Location
3.0
Attic Periphery
3
9.9

Attic central Location

12

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4 Testing and Measurements
The fumigated areas of the CTH were monitored continuously for HP concentration, RH, and temperature
using the HP sensors and monitoring points discussed in Section 4.1. Coupon samples were analyzed for
the number of CFUs, and Bl discs were analyzed for G/NG as discussed in Section 4.2.
4.1	Hydrogen Peroxide Sensors and Monitoring Points
Three types of electrochemical HP sensors were used during fumigation: one HP sensor capable of
detecting 0 to 10 ppm HP (Analytical Technology Inc. (ATI) Model B12-34-1 -0010-1, Collegeville, PA) for
low concentrations and safety monitoring, three HP sensors capable of detecting 0 to 25 ppm HP (ATI,
Model B12-34-5-0025-1), and seven HP sensors capable of detecting 0 to 100 ppm HP (ATI, Model
B120100-1). Each sensor was wired into the data acquisition system and had a variable output monitored
in real time. The three HP sensors capable of detecting 0 to 25 ppm HP were placed in the center of the
CTH, in the crawl space, and in the attic during fumigation, along with one real-time RH and temperature
probe (Vaisala, Model HMD53, Vaisala, Helsinki, Finland) in the center of the house. The seven 0- to
100-ppm sensors were distributed throughout the CTH. In addition, as discussed in Section 3.2, RH and
temperature sensors were placed at each coupon location.
4.2	Analytical Procedures
The following sections discuss coupon analysis, Bl disc analysis, and data reduction.
4.2.1 Coupon Analysis
The sampling team collected coupons from each sampling location in the CTH using gloves and handling
only the outside of the coupon holder. Sterile forceps were used to aseptically transfer individual coupons
to sterile 50-milliliter (mL) conical tubes. Positive control coupons were transferred last to prevent cross
contamination. All sample tubes were transported to the NHSRC RTP Microbiology Lab (BioLab) for
quantitative analysis of the number of viable spores recovered per sample (CFUs). Test coupons (positive
controls), Bl discs, and field blanks (negative control coupons) were deployed and collected after
fumigation in separate containers to reduce cross contamination among the different sample types. The
extraction and analysis of the coupon samples are discussed below.
To each 50-mL conical tube containing a coupon, 20 mL sterile phosphate-buffered saline with 0.05%
Tween® 20 (PBST) was added. The tubes then were sonicated for 10 minutes and vortexed continuously
for 2 minutes. Aliquots were plated in triplicate using a spiral plater (Autoplate 5000, Advanced
Instruments Inc., Norwood, MA), which deposits the sample in exponentially decreasing amounts across
a rotating agar plate in concentric lines to achieve three 10-fold serial dilutions on each plate. Plates were
incubated at 35 ± 2 °C for 16 to 19 hours. During incubation, the colonies develop along the lines where
the sample was deposited (see Figure 4-1). The colonies on each plate were enumerated using a
QCount® colony counter (Advanced Instruments Inc., Norwood, MA).
13

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Figure 4-1. Bacterial Colonies on Spiral-plated Agar Plate
Positive control samples were diluted 100-fold (102) in PBST before spiral plating, while samples of
unknown concentration were plated with no dilution and with a 100-fold dilution. Samples with known low
concentrations were plated with no dilution. The QCount® colony counter automatically calculates the
CFU/mL in a sample based on the dilution plated and the number of colonies that develop on the plate.
The QCount® records the data in an MS Excel (2007, Version 6.1.7600) spreadsheet.
Only plates meeting the threshold of at least 30 CFUs were used for spore recovery estimates. After
quantitation with the QCount® colony counter, samples with plate results below the 30-CFU threshold
were either re-spiral plated with a more concentrated sample aliquot or filter-plated to achieve a lower
detection limit. The filter plate volume was based on the CFU data from the QCount® result. The filters
were placed onto tryptic soy agar (TSA) plates and incubated at 35 ± 2 °C for 20 to 24 hours before
manual enumeration. Figure 4-2 shows a filter plate with colonies of Bg.
Figure 4-2. Bacterial Colonies on Filter Plate
14

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4.2.2 Biological Indicator Disc Analysis
Bl discs containing Gs were aseptically transferred to 15-mL polypropylene culture tubes (USA Scientific
Inc., P/N 169897, Ocala, FL) containing up to 10 mL of tryptic soy broth (TSB). The tubes were statically
incubated at 55 ± 2 °C. After 7 days, the medium in each tube was visually inspected for turbidity (Figure
4-3). To confirm the qualitative culture tube results and to verify that the turbidity was caused by the target
organism, a 0.1-mL aliquot was plated to confirm that the growth was from the target organism (based on
colony morphology). All sample tubes with no turbidity also were plated (0.1 mL) to confirm that there was
no target organism growth.
Figure 4-3. Bl Turbidity Analysis: Left - TSB Turbid (Growth); Right - TSB Clear (No Growth)
4.2.3 Data Reduction
For the coupon sample results, data reduction was performed using measurements of the total viable
spores (CFUs) recovered from each replicate coupon to calculate average recovered CFUs and standard
deviation (SD) for each group of coupons. The groups of coupons and Bl discs included the following for
each combination of material type and extracted sample type at each sampling location:
•	Positive control coupons (replicates, average, SD)
•	Test coupons and Bl discs (replicates, average, SD)
•	Procedural blank coupons
Efficacy is defined as the extent (based on LR) to which the agent recovered from the surfaces of the
coupons after decontamination has been reduced compared to the agent recovered from the positive
control coupons (not exposed to the decontamination procedure). Efficacy was calculated using Equation
4-1 below for each material within each combination of decontamination procedure (/) and test material (J).
15

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S(lo8io Cjc)
c=1
log.o^j
{ \
(4-1)
Where
Cjc = Number of viable organisms recovered from C positive control coupons foryth test material
Njc = Number of positive control coupons for the yth test material
Njk = Number of viable organisms recovered on the Wh replicate coupon for the yth test material
If no viable spores were detected, then the detection limit of the sample was used and the efficacy
reported as greater than or equal to the value calculated using Equation 4-2 below. The detection limit of
a sample depends on the analysis method and so may vary. The detection limit of a plate was assigned a
value of 0.5 CFU, but the fraction of the sample plated varies. For example, the detection limit of a 0.1-mL
plating of a 20-mL sample suspension would be100 CFUs, but if all 20 mL of the sample is filter plated,
the detection limit would be 0.5 CFU.
The standard deviation (SD) of LR is calculated by Equation 4-2:
jk
SD
(4-2)
Where:
LR .
J is the average LR of spores on a specific material surface, and
is the average of the LR of a decontaminated coupon calculated using Equation 4-3.
X IS i°s(CFU,)' Nc - '°s(CFU,»
(4-3)
k C= 1
NJk
16

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5 Results and Discussion
This section discusses the results for the baseline conditions and Tests 1 through 5. For each test, the
following operational parameters are discussed: living area fumigation conditions (including temporal HPV
concentration, temporal RH, and temporal temperature), crawl space and attic fumigation conditions
(including temporal HPV concentration, temporal RH, and temporal temperature), and decontamination
efficacy.
5.1 Test 0 (Humidity Adjustment Only)
As part of a scoping test, the humidity profile for the CTH was determined during baseline testing. The
temporal effect of high humidity (no HPV presence) on spore survivability also was assessed. Seven
humidifiers were used in the living area: one in the kitchen, one in the den, two in the living room, and one
in each of the three bedrooms. In addition, the crawl space and attic contained three humidifiers each.
Each humidifier was charged with 2 gallons of de-ionized water.
5.1.1 Living Area Fumigation Conditions
This section discusses the environmental conditions in the living areas for the initial CTH set-up shown in
Figure 5-1, including air infiltration estimates, and temporal RH in each section of the living areas.
Further, the CTH environmental conditions were evaluated to determine if high RH affects spore viability
on the coupons. The humidifiers operated continuously until the fumigant was depleted. After a 1-week
baseline testing period, the coupons were collected and analyzed. During the baseline testing, the HVAC
fan continuously circulated air throughout the testing sequence, which lasted 7 days, and the temperature
in the living areas was constantly maintained at 70° F.
17

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mum
v iV 2Tj>i
1	X
Master
Bedroom
1HHIH
%1
Clos Clos^ Return
Clos aI Air
ClosA
A
a*a
Corner
Bedroom
&
11111111
Clos
Utility
Middle
ABedroom
&

iiiiim
Den
^ nrrrm\
fTTTTm
Kitchen
¦
Living
Room
ir
i
j
CIOST
iimiii
Instruments
Garage
^ Fan Humidifier • Test Coupon ^ BI | HP Sensor amm = Registers
Figure 5-1. CTH Test Set-up
5.1.1.1 Gary test House Air Infiltration Rates
A cyclical pattern was observed for the CTH air infiltration rates throughout the testing sequence. The air
exchange was highest during the cooler nights, and fell off during the days, as shown in Figure 5-2 for
Test 1. These fluctuations were controlled solely by the outside temperature, since the living areas were
temperature controlled. Table 5-1 summarizes the exchange rates over the five 7-days testing events.
The average air exchange for this house was found to be 0.32 hr1, with maximum air exchanges less
than 0.55 hr1, which shows that this home is relatively airtight.
18

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0.45
0.40-
0.35-
S 0.30-
00
£=
O
"-I—»
CT3
0.25-
£=
0.20-
<
0.15-
0.10
0
20
40
60
80
100 120 140 160 180
Time (hours)
Figure 5-2. Test 1 Temporal Air Exchange Rates in Living Areas
Table 5-1. CTH Air Exchange Rates
Air Infiltration Rate (hr1)
Test Number
Min
Mean
Max
Test 1
0.14
0.29
0.42
Test 2
0.17
0.32
0.47
Test 3
0.19
0.35
0.52
Test 4
0.19
0.36
0.55
Test 5
0.16
0.30
0.43
5.1.1.2 Temporal RH in Living Areas
Figure 5-3 shows the effect of the humidifiers on RH throughout the CTH living areas for eight locations
(master bedroom, master bathroom, corner bedroom, middle bedroom, hallway bathroom, kitchen, den,
and living room).
19

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100
to
CO
I
O)
c
5
0
_£Z
_c
'g
'E
X
5
90-
80-
70-
60-
50-
40-
30-
20-
0
a:
TK 10-
Master Bedroom
Master Bathroom
Corner Bedroom
Middle Bedroom
Hallway Bathroom
Kitchen
Den
Living Room
-r
20
40
60
80
100 120 140 160 180
Fumigation Time (Hours)
Figure 5-3. Baseline Test Temporal RH in Living Areas
The results show that the RH increased rapidly from about 54% to greater than 75% in all living areas,
even in the master bathroom, where no humidifiers were placed. The humidification process resulted in
greater than 75% RH within 2 hours and maintained an elevated RH level (especially for 48 hours when
the humidifiers were still generating vapor) for the 7 days of the baseline testing period. The HVAC fan
continuously circulated air, transporting humidity from locations with humidifiers to other locations with no
humidifiers such as the bathrooms. The observed oscillations observed for the RH inside the living areas
may be due to diurnal cycling of the outdoor temperature/RH.
5.1.2 Temporal Environmental Conditions in Crawl Space and Attic
5.1.2.1 Crawl Space
The effect of the three humidifiers on the RH and temperature inside the crawl space was monitored for
more than 7 days at three locations (east side, central location, and west side). Figure 5-4 shows the RH
results for the crawl space. These results indicate that the RH increases rapidly from about 50 to 55% to
greater than 80% uniformly throughout the crawl space in less than 1.5 hours and that this RH level was
maintained for more than 1 week. The temperature is relatively stable through the testing sequence.
20

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65-
CD
< 60
100
90-
=5 70-
E
x 60-
(D
I 50-
Crawl Space East Locations
Crawl Space Central Locations
Crawl Space West Locations

1
20
T
40
T
60
r
80
T
100
120
140
160
Fumigation Time (Hours)
Figure 5-4. Baseline Test Temporal Environmental Conditions in Crawl Space
5.1.2.2 Attic
The effect of the three humidifiers on the RH/temperature in the attic was monitored for more than 7
days at three locations (east side, central location, and west side). Figure 5-5 shows the RH results for
the attic. These RH results show a cyclical process between days and nights, regardless of sampling
location. The oscillations observed for the temperature/RH inside the attic may be due to the cycling of
the outdoor temperature/RH between day and night. The cyclical process includes RH ranges well below
50% that could affect decontamination efficacy and fumigation effectiveness for HP.
21

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120
LL 110
o
a> 100
1 90
a>
E 80

-------
Test Coupon Recovery (CFUs) and Bl disc Recovery (G/NG)
Location Sample Type Rep 1	Rep 2	Rep 3
Corner Bedroom
Floor
Carpet coupon
2.46 x107
3.88 x107
1.91 x 107
GM coupon
5.88x10®
3.92x10®
3.03x10®
Bl disc
G
G
G
Middle Bedroom
Floor
Carpet coupon
2.52 x107
2.99 x107
2.64 x107
GM coupon
6.59x10®
4.80x10®
1.16 x 107
Bl disc
G
G
G
Kitchen Floor
Carpet coupon
2.50 x107
2.13 x 107
2.78 x107
GM coupon
3.92x10®
2.43x10®
4.70x10®
Bl disc
G
G
G
Living Room
Floor
Carpet coupon
2.30 x107
2.47 x107
2.04 x107
GM coupon
4.03x10®
3.41 x 10®
2.42x10®
Bl disc
G
G
G
Crawl Space
Under Corner
Bedroom
Carpet coupon
2.07 x107
2.49 x107
2.72 x107
GM coupon
1.59x10®
7.40 x105
1.04 x 10®
Bl disc
G
G
G
Crawl Space
Under Kitchen
Carpet coupon
2.66 x107
2.88 x107
2.08 x107
GM coupon
8.46 x105
6.07x105
5.53 x10s
Bl disc
G
G
G
Crawl Space
Under Den
Carpet coupon
3.06 x107
2.57 x107
2.56 x107
GM coupon
1.05x10®
5.20 x105
8.47 x105
Bl disc
G
G
G
Attic Over Master
Bath
Carpet coupon
2.60 x107
1.79 x107
2.28 x107
GM coupon
7.05x10®
4.72x10®
9.06x10®
Bl disc
G
G
G
Center of Attic
Carpet coupon
2.42 x107
2.18 x 107
2.70 x107
GM coupon
8.00x10®
3.91 x 10®
5.02x10®
Bl disc
G
G
G
Attic over Den
Carpet coupon
2.72 x107
2.66 x107
2.88 x107
GM coupon
8.44x10®
4.19x10®
5.67x10®
Bl disc
G
G
G
5.2 Test 1
Test 1 was the reference test for this project. The test layout was similar Test 0 (Humidity Adjustment
only). Each humidifier was charged with 2 gallons of 3% off-the-shelf HP aqueous solution. The HVAC
fan continuously circulated air throughout the testing sequence, which lasted 7 days, and the temperature
in the living areas was constantly maintained at 70 °F.
The average results for the living areas, attic, and crawl space fumigation conditions: HPV concentration,
(ppm), overall calculated HPV exposure (concentration-time [CT] in ppm-hours), and RH (%) are
tabulated in Table 5-3. The temporal HPV concentration, RH, and temperature are discussed in Sections
23

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5.2.1.1 through 5.2.1.3 for living areas, and in Sections 5.2.2.1 through 5.2.2.3 for the crawl space and
attic. Finally, the decontamination efficacy of the test coupons inoculated with Bg spores, and growth/no
growth assessments of Bl discs co-located with the coupons are discussed in Section 5.2.3.
	Table 5-3. Test 1 Fumigation Conditions	
Test 1 - Fumigation and Relative Humidity Conditions by Location
Location
HPV Max
(ppm)
CT
(ppm-hour)
RH
Average
(%)
RH Max
(%)
Master Bedroom
43
1310
72
86
Master Bathroom
Not Measured
74
85
Hallway Bathroom
7.9
270
72
86
Center of Den
38
1260
72
86
Corner Bedroom
Not Measured
69
85
Middle Bedroom
Not Measured
72
88
Kitchen
Not Measured
69
86
Living Room Floor
73
2030
71
88
Air return
32
1060
65
82
Crawl Space Center
18
787
82
92
Crawl Space North East
0.7
18
83
94
Crawl Space South West
81
92
Center of Attic
37
1077
78
90
Attic Over Master Bath
5.5

77
90
Attic over Den
177
78
90
5.2.1 Living Area Fumigation Conditions
5.2.1.1 Temporal HPV Concentration
The real-time HPV concentration was monitored at five locations using HP sensors at the locations shown
in Figure 5-1: master bedroom, bathroom, hallway air return, living room, and den (see Figure 5-6). The
highest HPV concentration was found in the living room (73 ppm), which contained two humidifiers, and
the lowest HPV concentration was in the hallway bathroom, with a maximum of 7.9 ppm, which contained
no humidifiers. The hallway air return location showed a comparable HPV concentration to the bedrooms,
living room, and den, which had one humidifier each.
24

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— ¦ — Master Bedroom
—• — Bathroom
—Hallway Return Air
—Living Room
— Den
70
CL
CL
60
50
40
CL
30
20
10
0
20
40
60
80
100 120 140 160 180
Fumigation time (Hours)
Figure 5-6. Test 1 Temporal HPV Concentration in Living Areas
5.2.1.2	Temporal RH
The effect of the humidifiers on the RH in the CTH living areas was monitored during the fumigation
sequence at eight locations (master bedroom, master bathroom, corner bedroom, middle bedroom,
hallway bathroom, kitchen, den, and living room), as shown in Figure 5-7. The outside RH measurements
were monitored via a weatherstation and are illustrated in Figure 5-7. RH increased rapidly to values
greater than 70% in all living areas, even in the hallway bathroom, where no humidifiers were placed. The
HVAC fan continuously circulated air, transporting humidity from the locations with humidifiers to other
locations with no humidifiers. Furthermore, the RH was maintained above 60%, even when the HPV
concentration decreased to less than 10 ppm. The high swings in outdoor RH seem to have little or no
effect on the indoor living areas.
5.2.1.3	Temporal Temperature
The temperature inside the living areas was monitored during the fumigation sequence at eight locations
(master bedroom, master bathroom, corner bedroom, middle bedroom, hallway bathroom, kitchen, den,
and living room), as shown in Figure 5-8. The outside temperature measurements were monitored via a
weather station, and illustrated also in Figure 5-8. The central AC unit was set to "heat" with a setpoint of
70 °F, and the fan was set to run continuously. As expected, the temperatures in the living areas were
uniform and were affected neither by the humidification process, nor by fluctuations in the outdoor
temperature.
25

-------
30-
20-
> 10-
-r~
20
-r-
40
-r~
60
-r~
80
Master Bedroom
Master Bathroom
Corner Bedroom
Middle Bedroom
Hallway Bathroom
Kitchen
Den
Living Room
¦ Outdoor
100
—i—
120
—I—
140
160
Fumigation Time (Hours)
Figure 5-7. Test 1 Temporal RH in Living Areas vs. Outdoors
ro 50

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5.2.2 Crawl Space and Attic Fumigation Conditions
5.2.2.1 Temporal HPV Concentration
The temporal HPV concentrations of the crawl space and attic area were monitored at their central and
periphery locations (Figure 5-9). The highest HPV concentrations were detected in the central locations of
the crawl space and attic, and these concentrations were comparable to those observed in indoor rooms.
The peripheral locations had much lower HPV concentrations which may have been caused by leaks of
fresh air through the vents.
— ¦ — Crawl Space Periphery
—• — Crawl Space Central Location
—•'—Attic Central Location
—t—Attic Periphery
cl 35-
*¦—r
100 120
180
Fumigation time (Hours)
Figure 5-9. Test 1 Temporal HPV Concentration in Crawl Space and Attic
5.2.2.2 Temporal RH and Temperature
Crawl Space
The effect of the humidifiers on the RH and temperature was monitored during the fumigation sequence
at three locations in the crawl space: east, central, and west locations (see Figure 5-10). Despite large
fluctuations in the outdoor conditions, between days and nights, the temperatures in the crawl space,
were unaffected and remained constant throughout the testing sequence. The outside RH appeared to
have little to no impact on the crawl space RH, as it stayed relatively high (above 70%) even after the
HPV concentration decreased to less than 10 ppm.
27

-------

Vi
Crawl Space East Locations
Crawl Space Central Locations
Crawl Space V\fest Locations
— Outdoor
20 40 60 80 100 120 140 160
Fumigation Time (Hours)
Figure 5-10. Test 1 Temporal RH in Crawl Space vs. Outdoors
Attic
The effect of the humidifiers on the RH was monitored during the fumigation sequence at two locations in
the attic: east and west locations (see Figure 5-11V The temperature and RH inside the attic mimic the
cyclical pattern of the outdoor environmental conditions. This would suggest that the driving forces for the
attic conditions are the outdoor environmental conditions.
28

-------
80-
ll 70- rr
o
/ ;ui
f: W
60-
2
d)
Cl
E
50-
100
90-
80-
70-
60-
Attic East Locations
Attic West Locations
Outdoor
50-
20
40
60
80 100 120 140 160
Fumigation Time (Hours)
Figure 5-11. Test 1 Temporal RH in Attic vs. Outdoors
5.2.3 Decontamination Efficacy
Test coupons inoculated with Bg spores exhibited at least a 6 LR in viable spore count, despite growth of
spores for some of the test coupons placed in living areas, crawl spaces, and the attic. These results
were mostly independent of test coupon material type, location, or decontamination conditions (HPV
concentration, RH, and temperature). Most of the detected results were observed for the GM coupons (8
detections out of 45), with co-located carpet coupons experiencing almost full decontamination (44 non-
detects out of 45). Table 5-4 summarizes spore recovery results for the test coupons (three replicates
each) and G/NG results for the Bl discs (three replicates each).
29

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Table 5-4. Test 1: Post-Decontamination Recoveries on Test Coupons and Bl discs
Test Coupon Recovery (CFUs) and Bl disc Recovery (G/NG)	^
Location	Sample Type Rep 1 Rep 2	Rep 3
Master Bedroom
Floor
Carpet coupon
ND
ND
ND
7.45
GM coupon
35
ND
53
7.04
Bl disc
NG
NG
NG
Not Applicable
Master Bathroom
Floor
Carpet coupon
ND
ND
ND
7.45
GM coupon
1
ND
6
7.04
Bl disc
G
G
NG
Not Applicable
Bathroom Sink
Carpet coupon
ND
ND
ND
7.45
GM coupon
ND
ND
ND
7.04
Bl disc
NG
G
NG
Not Applicable
Center of Den
Carpet coupon
ND
ND
ND
7.45
GM coupon
ND
ND
ND
7.04
Bl disc
NG
NG
NG
Not Applicable
Corner Bedroom
Floor
Carpet coupon
ND
ND
1
7.45
GM coupon
ND
ND
54
7.04
Bl disc
NG
NG
NG
Not Applicable
Middle Bedroom
Floor
Carpet coupon
ND
ND
ND
7.45
GM coupon
ND
ND
ND
7.04
Bl disc
NG
NG
NG
Not Applicable
Kitchen Floor
Carpet coupon
ND
ND
ND
7.45
GM coupon
ND
6
ND
7.04
Bl disc
NG
NG
G
Not Applicable
Living Room Floor
Carpet coupon
ND
ND
ND
7.45
GM coupon
ND
ND
ND
7.04
Bl disc coupon
NG
NG
NG
Not Applicable
Crawl Space
Under Corner
Bedroom
Carpet coupon
ND
ND
ND
7.45
GM coupon
ND
ND
ND
7.04
Bl disc
G
G
G
Not Applicable
Crawl Space
Under Kitchen
Carpet coupon
ND
ND
ND
7.45
GM coupon
ND
ND
ND
7.04
Bl disc
G
G
G
Not Applicable
Crawl Space
Under Den
Carpet coupon
ND
ND
ND
7.45
GM coupon
ND
ND
ND
7.04
Bl disc
G
G
G
Not Applicable
Attic Over Master
Bathroom
Carpet coupon
ND
ND
ND
7.45
GM coupon
ND
ND
ND
7.04
Bl disc
NG
NG
NG
Not Applicable
Center of Attic
Carpet coupon
ND
ND
ND
7.45
GM coupon
ND
ND
ND
7.04
Bl disc
NG
NG
NG
Not Applicable
Attic Over Den
Carpet coupon
ND
ND
ND
7.45
GM coupon
16
ND
ND
7.04
Bl disc
NG
NG
NG
Not Applicable
A/C Duct
Carpet coupon
ND
ND
ND
7.45
GM coupon
ND
9
ND
7.04
Bl disc
NG
NG
NG
Not Applicable
30

-------
Test Coupon Recovery (CFUs) and Bl disc Recovery (G/NG)
Location
Sample Type
Rep 1
Rep 2
Rep 3
Positive Controls
Carpet coupon
3 x 107
3 x 107
3 x 107
GM coupon
8x10s
8 x10s
3 x 107
Bl disc
G
G
G
LR (CFUs)
ND = Non-detect
NG = No growth (Bl)
G = Growth (Bl)
Most of the Gs Bl discs recovered after the fumigation events showed no growth in the living areas except
in locations such as the master bathroom (two out of three Bl discs showed growth). HP concentration
was not monitored in the master bathroom. Also, one out of three Bl discs placed in the hall bathroom
sink showed growth, and this one positive may be due to the low HPV concentration (maximum
concentration of 8 ppm) in the hall bathroom. No humidifiers were used in or near this location. One Bl
disc out of three in the kitchen also showed growth.
All the Gs Bl discs in the crawl space showed growth despite the observed full decontamination of the co-
located test coupons. Three of those Bl discs were co-located with the coupons, while other Bl discs were
placed in the periphery of the crawl space as shown in Figure 5-12. The periphery of the crawl space had
very low HPV concentrations (less than 1 ppm), and therefore less HPV exposure.
Figure 5-12. Bl Disc Package Placement at Periphery of Crawl Space
31

-------
The Test 1 results demonstrated significant differences in decontamination efficacy between Bg
inoculated on the test coupons and purchased Gs Bl discs. These results are consistent with the findings
of previous studies (Klapes and Veslev 1990; Kokubo, Inoue, and Akers 1998), where Gs spores were
found to be more resistant to HP fumigation than Bg spores. The Gs Bl discs were not enumerated and
"growth" can be observed even when a disc's residual spore quantity is very low.
This low concentration HPV decontamination approach can be considered an effective sporicidal surface
decontamination treatment because L.R values exceeded 6 in the entire CTH, regardless of location and
material type. For example, even though the HPV concentration was less than 8 ppm in the hallway
bathroom, where no humidifier was deployed, two out of three Bl discs showed no growth.
A Drager HP tube with one end open was used to determine that HPV diffused into the tube in the living
areas. Figure 5-13 shows a typical Drager tube. Drager tube results generally were less than 3 ppm,
regardless of location. Since the Drager tubes were used as passive samplers, and no flow was pulled
through them, per manufacturer's instructions, these results should be considered qualitative at best.
However, the Drager tube results can be used as indicators of HPV presence in various test areas.
Figure 5-13. Post-Decontamination Drager Tube in Living Area
5.3 Test 2
For Test 2, the HP concentration in the aqueous solution was increased from 3 to 3.8%, but all other
fumigation conditions remained the same as Test 1 The humidifiers in the crawl spaces were loaded with
3.4 gallons of HP aqueous solution, and all other humidifiers were charged with 2 gallons. One humidifier
from the living room was moved to the hallway under the air return vent. Further, one fan was added to
the master bedroom, and one fan was removed from the den. Additional test coupons were added to both
the den and the living room shown in light green in Figure 5-14.
32

-------
Master
E^r corn
*
"V Master
y.>' Bath^A
imiiTi
Clos I Clp3
CI03A
Bath
I a
_J\U?
Clos
mA
Corner
Bedroom
-yjjjps |U'fity
Middle
Bedroom
4b
A
•A
Den
GiifV*
Living
Room
i
A
~
Clos

4
¦»
Instruments
m>ents
Garage
¦ Humidifier (COTS)	| HP Sensor	
-------
5.3.1 Living Area Fumigation Conditions
5.3.1.1 Temporal HPV Concentration
The HPV concentration was monitored using HP sensors at the five following locations: master bedroom,
bathroom, hallway air return, living room, and den (see Figure 5-15). The increase in concentration from 3
to 3.8% of the HP aqueous solution resulted in an overall increase in HPV concentration throughout the
CTH. The results indicate that moving one humidifier to the hallway resulted in an overall increase in HPV
concentration and CT ppm-hours in the hallway, bathroom, and air return areas.
180-
E
Q.
Q.
160-
° 140-
120-
c
s
c
(3 100
0
Q.

-------
5.3.1.3 Temporal Temperature
The temperature inside the living areas was monitored throughout the fumigation sequence at eight
locations as shown in Figure 5-17. As observed during Test 1, the temperature inside the living areas was
uniform and was affected neither by the humidification process nor by the outside environmental
conditions.
100-
o
o
0
T3
C
CD
(/)
CD
1
O)
g
'>
Lj
CD
r 30-
20-
c.
>?
'E
X
.i
to,
a:
90-
80-
70-
60-;
50-
40-
10-
o-
rv\
—i—1—r~
20 40
-r~
60
-r~
80
Master Bedroom
Master Bathroom
Corner Bedroom
Middle Bedroom
Hallway Bathroom
Kitchen
Den
Living Room
— Outdoor
100
—I—
120
—I—
140
160
Fumigation Time (Hours)
Figure 5-16. Test 2 Temporal RH in Living Areas
35

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<1)
£
"e
£2
0
&

-------
40
— ¦ — Crawl Space Periphery
—• — Crawl Space Central Location
—¦'—Attic Central Location
—t—Attic Periphery	
& 35
30
25
20
CL
15
10
0
20
40
60
80
100 120 140 160 180
Fumigation time (Hours)
Figure 5-18. Test 2 Temporal HPV Concentration in Crawl Space and Attic
5.3.2.2 Temporal RH
Crawl Space
The effect of the humidifiers on the RH and temperature was monitored during the fumigation sequence
at three locations in the crawl space: east, central, and west locations (see Figure 5-19). Despite large
fluctuations in the outdoor conditions, between days and nights, the temperatures in the crawl space,
were mostly unaffected and remained relatively constant throughout the testing sequence. Likewise, the
wide fluctuations observed for the outdoor conditions appear to have little effect on the crawl space RH.
After the humidifiers were depleted offumigant, an overall decrease in RH was observed but the RH
stayed high (near 70%) even when HPV concentration levels decreased to single-digit ppm levels. These
results are similar to the Test 1 results.
37

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a. 40
30-
20-
	Crawl Space East Location
—	¦ — Crawl Space Central Locations
—	¦ — Crawl Space West Locations
—	— Outdoor Locations
—¦—i—1—i—1—i—1—i—1—r
CO „

-------

80

70


LL
o
60


(D

3
50
2


-------
Table 5-6. Test 2: Post-Decontamination Recoveries on Test Coupons and Bl discs
Test Coupon Recovery (CFUs) and Bl disc Recovery (G/NG)
Location	Sample	Rep 1	Rep 2	Rep 3
LR (CFUs)
Master Bedroom
Floor
Carpet coupon
ND
ND
ND
7.62
GM coupon
ND
ND
ND
7.08
Bl disc
NG
NG
NG
Not Applicable
Master Bathroom
Floor
Carpet coupon
ND
ND
ND
7.58
GM coupon
ND
ND
ND
7.08
Bl disc
NG
NG
NG
Not Applicable
Bathroom Sink
Carpet coupon
ND
ND
ND
7.59
GM coupon
ND
ND
ND
7.10
Bl disc
NG
NG
NG
Not Applicable
Center of Den
Carpet coupon
ND
ND
ND
7.61
GM coupon
ND
ND
ND
7.09
Bl disc
NG
NG
NG
Not Applicable
Corner Bedroom
Floor
Carpet coupon
ND
ND
ND
7.61
GM coupon
ND
ND
ND
7.10
Bl disc
NG
NG
NG
Not Applicable
Middle Bedroom
Floor
Carpet coupon
ND
ND
ND
7.61
GM coupon
ND
ND
ND
7.10
Bl disc
NG
NG
NG
Not Applicable
Kitchen Floor
Carpet coupon
ND
ND
ND
7.61
GM coupon
ND
ND
ND
7.11
Bl disc
NG
NG
NG
Not Applicable
Living Room
Floor
Carpet coupon
ND
ND
ND
7.61
GM coupon
ND
ND
ND
7.10
Bl disc
NG
NG
G
Not Applicable
Living Room
Floor (a)
Carpet coupon
ND
ND
ND
7.62
GM coupon
ND
ND
ND
7.10
Living Room
Floor (b)
Carpet coupon
ND
ND
ND
7.60
GM coupon
ND
ND
ND
7.08
Crawl Space
Under Corner
Bedroom
Carpet coupon
ND
ND
ND
7.59
GM coupon
ND
ND
ND
7.10
Bl disc
G
G
G
Not Applicable
Crawl Space
Under Kitchen
Carpet coupon
ND
ND
ND
7.61
GM coupon
ND
ND
ND
7.11
Bl disc
NG
NG
NG
Not Applicable
Crawl Space
Under Den
Carpet coupon
ND
ND
ND
7.53
GM coupon
ND
ND
ND
7.11
Bl disc
NG
G
G
Not Applicable

Carpet coupon
ND
ND
ND
7.60
40

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Test Coupon Recovery (CFUs) and Bl disc Recovery (G/NG)
LR (CFUs)
Location
Sample
Rep 1
Rep 2
Rep 3

Attic Over Master
GM coupon
9.80 x 103
8.86 x 104
3.73 x103
2.79
Bathroom
Bl disc
NG
NG
G
Not Applicable

Carpet coupon
ND
ND
ND
7.62
Center of Attic
GM coupon
ND
ND
ND
6.27

Bl disc
NG
NG
NG
Not Applicable

Carpet coupon
6.70 x 105
2.36 x 105
1.18 x 104
2.39
Attic over Den
GM coupon
1.07 x 107
3.83 x 10®
5.48 x 10®
0.14

Bl disc
G
G
NG
Not Applicable
A/C Duct
Carpet coupon
ND
ND
ND
7.60
GM coupon
ND
ND
ND
7.11
Center of Den (a)
Carpet coupon
ND
ND
ND
7.62
GM coupon
ND
ND
ND
7.00
Center of Den (b)
Carpet coupon
ND
ND
ND
7.59
GM coupon
ND
ND
ND
7.07

Carpet coupon
7.50 x 107
1.14 x 107
3.32 x 107

Positive 1
GM coupon
6.38 x 10®
6.81 x 10®
1.13 x 107


Bl disc 1,2,3
G
G
G

Positive 2
Carpet coupon
1.64 x 107
1.97 x 107
2.64 x 107

GM coupon
4.74 x 10®
1.11 x 107
9.99 x 10®

ND = Non-detect NG = No growth (Bl) G = Growth (Bl)
The GM test coupons placed in the attic spaces over the den and over the master bathroom showed LR
values of less than 2.8 to almost no decontamination, despite the increase in HPV concentration. Three
out of nine Bl discs in the attic and five out of nine Bl discs placed in the crawl space exhibited growth
after the decontamination process.
In summary, the Test 2 results confirm the Test 1 results, suggesting that this low concentration HPV
decontamination approach may be considered an effective sporicidal surface decontamination treatment
because LR values exceeded 6 in the living areas of the CTH, regardless of location and material type.
LR values exceeded 6 even when the exposure time for Test 2 was 3 days in the living areas.
In the crawl space, LR values exceeded 6 when there was a 7-day exposure time. Despite some growth
observed for Bl discs in the crawl space, full decontamination was observed on the co-located coupons
suggesting that a LR of 6 to 7 can be achieved in much of the crawl space. However, for Bl discs
(providing non-quantitative assessments) placed near ventilation inlets, some spores survived the HPV
treatment. Gs spores have been shown to be more hardy than Bg spores during HPV exposures (Klapes
and Veslev 1990; Kokubo, Inoue, and Akers 1998),
41

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For the attic, very limited decontamination occurred over? days. The attic is not well sealed (the ridge
vent remained open during fumigation) and the roof is not insulated. The RH data showed a cyclical
fluctuation between days and nights.
5.4 Test 3
For Test 3, the humidifiers, fan, test coupons, and Bl disc placements were kept the same as Test 2. All
humidifiers were loaded with 3.0% HP aqueous solution. The humidifiers in the crawl space and attic
were each loaded with 3.4 gallons of HP aqueous solution, and the humidifiers in the iiving areas were
charged with only 2 gallons of this solution . Further, for this test, furniture was added to the CTH,
including three beds with bedding and pillows, clothing, five rugs, two couches, two chairs, and three
boxes of paper and books. Figure 5-21 shows the placement of some of these items in the CTH.
Master	Den	Living
Figure 5-21. Furniture Added to CTH for Test 3
Furthermore, Bl discs were placed in the following hard-to-reach areas to test the efficacy of the
fumigation process:
•	Between couch cushions (Figure 5-22)
•	Under carpet (jute-backed) (Figure 5-23)
•	Inside a closed bathroom drawer (Figure 5-24)
•	Under one and also under five pieces of paper (Figure 5-25)
42

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w
Figure 5-22. Test 3 Placement of Bi discs Between Couch Cushions
Figure 5-23. Test 3 Placement of BI discs Under Carpet
Figure 5-24. Test 3 Placement of BI discs in Bathroom Drawer (closed during testing)
43

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Figure 5-25. Test 3 Placement of Bl discs Under One and Five Pieces of Paper
The average results for the living areas, attic, and crawl space fumigation conditions which include HPV
concentration (ppm), overall calculated HPV exposure (concentration-time [CT] in ppm-hours), and
average RH (%) are tabulated in Table 5-7. The temporal HPV concentration, RH, and temperature are
discussed in Sections 5.4.1.1 through 5.4.1.2 for living areas, and the decontamination efficacy of the test
coupons inoculated with Bg spores, and growth/no growth assessments of Bl discs co-located with the
coupons are discussed in Section 5.4.2.
Table 5-7. Test 3 Fumigation Conditions
Test 3 - Fumigation and Relative Humidity Conditions by Location
Location
HPV Max
(ppm)
CT
(ppm-hour)
RH Average
(%)
RH Max
(%)
Master Bedroom
46
1480
60
86
Master Bathroom
Not Measured
61
83
Hallway Bathroom
17
655
55
81
Center of Den
30
1090
62
85
Corner Bedroom
Not Measured
59
85
Middle Bedroom
Not Measured
59
83
Kitchen
Not Measured
61
89
Living Room
116
2780
58
84
Air return
35
1210
50
78
Crawl Space Center
27
2130
73
92
Crawl Space North East
9.9
503
75
95
Crawl Space South West

78
97
Center of Attic
18
1620
82
94
Attic North East
11
643
83
92
Attic South West
82
93
44

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5.4.1 Living Area Fumigation Conditions
5.4.1.1 Temporal HPV Concentration
The HPV concentration was monitored using HP sensors at the same five locations used during Test 2:
master bedroom, bathroom, hallway air return, living room, and den (see Figure 5-26). Adding furniture
did not significantly increase the demand for HP aqueous solution or change the CT (ppm-hour) results,
and the maximum HPV concentrations in the living areas were not much different than the Test 1 results.
There was better distribution of HP to the hall bathroom.
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— Den
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0 20 40 60 80 100 120 140 160 180
Fumigation time (Hours)
Figure 5-26. Test 3 Temporal HPV Concentration in Living Area
5.4.1.2 Temporal RH and Temperature
The effect of the humidifiers on the RH in the CTH living areas was monitored during the fumigation
sequence at eight locations (master bedroom, master bathroom, corner bedroom, middle bedroom,
hallway bathroom, kitchen, den, and living room), as shown in Figure 5-27. The outside RH
measurements was monitored via a weatherstation, and illustrated also in Figure 5-27. The results show
that the RH was maintained above 70% for the first 3 days of the fumigation event, and slowly decreased
after the humidifiers were depleted of fumigant. Temperature (data not shown), as expected, was steadily
maintained near 70 °F by the CTH HVAC system.
The large fluctuations in the outdoor conditions seem to have a negligible effect on the indoor conditions.
These results suggest that the driving force for the high RH in the living areas is the continuous
45

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fumigation in the living areas. As shown in Figure 5-27, the RH dropped below 70% after about 70 hours,
when the fumigant was depleted below 10 ppm in the living areas.
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Master Bedroom
Master Bathroom
Comer Bedroom
Middle Bedroom
Hallway Bathroom
Kitchen
Den
Living Room
- Outdoor
-r~
20
-r~
40
-r~
60
-r~
80
100
120
—I—
140
160
Fumigation Time (Hours)
Figure 5-27. Test 3 Temporal RH in Living Areas
5.4.2 Decontamination Efficacy
The addition of furniture to the CTH had little to no effect on the decontamination efficacy in the CTH
living areas. Full decontamination with a LR value of at least 6 was observed for all the test coupons in
the living areas, except for the coupons placed in the living room hallway and on the master bathroom
floor (where no humidifier was located), but even these samples resulted in low CFU counts after
fumigation. During Test 3, three extra sets of coupons (sample set "b") were deployed in the den,
bathroom sink, and living room along with the primary set of coupons. On the third day of the 7-day test
period, the "b" coupons were recovered and extracted. The "b" sample set results were similar to results
for the primary samples that underwent the full 7-day decontamination period. These samples also
exhibited full decontamination, indicating that 3 days of decontamination is sufficient, for those two
locations, to achieve full decontamination in the living areas of the CTH. Table 5-8 summarizes spore
recovery results for the test coupons and G/NG results for the Bl discs.
46

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Table 5-8. Test 3: Post-Decontamination Recoveries on Test Coupons and Bl discs
Test Coupon Recovery (CFUs) and Bl disc Recovery (G/NG)
Location	Sample Type Rep 1	Rep 2	Rep 3
Master Bedroom
Floor
Carpet coupon
ND
ND
ND
7.56
GM coupon
ND
ND
ND
7.13
Bl disc
NG
NG
NG
Not Applicable
Master Bedroom
Floor (b)
Carpet
ND
ND
ND
7.60
GM
ND
ND
ND
7.15
Bl disc
NG
Not measured
Not Applicable
Master Bathroom
Floor
Carpet coupon
ND
ND
ND
7.56
GM coupon
3
220
ND
6.10
Bl disc
NG
NG
NG
Not Applicable
Bathroom Sink
Carpet coupon
ND
ND
1
7.47
GM coupon
ND
ND
ND
7.14
Bl disc
NG
NG
NG
Not Applicable
Bathroom Sink
(b)
Carpet coupon
ND
ND
1
7.48
GM coupon
ND
ND
ND
7.15
Bl disc
NG
Not measured
Not Applicable
Center of Den
Carpet coupon
2
ND
ND
7.46
GM coupon
ND
ND
ND
7.13
Bl disc
NG
NG
NG
Not Applicable
Center of Den (b)
Carpet coupon
ND
ND
ND
7.48
GM coupon
ND
ND
ND
7.15
Bl disc
NG
Not measured
Not Applicable
Corner Bedroom
Floor
Carpet coupon
ND
ND
ND
7.56
GM coupon
ND
ND
ND
7.12
Bl disc
NG
G
NG
Not Applicable
Middle Bedroom
Floor
Carpet coupon
ND
ND
ND
7.56
GM coupon
ND
ND
ND
7.13
Bl disc
NG
NG
NG
Not Applicable
Kitchen Floor
Carpet coupon
ND
ND
ND
7.57
GM coupon
ND
ND
ND
7.12
Bl disc
NG
NG
NG
Not Applicable
Living Room /
Hallway
Carpet coupon
ND
2
1
7.37
GM coupon
ND
ND
ND
7.13
Bl disc
NG
NG
G
Not Applicable
Living Room
Floor (b)/
Dinning Area
Carpet coupon
ND
ND
ND
7.58
GM coupon
ND
ND
ND
7.04
Bl disc
NG
NA
NA
Not Applicable
Crawl Space
Under Corner
Bedroom
Carpet coupon
ND
ND
ND
7.56
GM coupon
1
8.80 x102
1.56 x 103
4.98
Bl disc
G
G
G
Not Applicable
47

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Test Coupon Recovery (CFUs) and Bl disc Recovery (G/NG)
Location
Sample Type
Rep 1
Rep 2
Rep 3
l_r\
Crawl Space
Under Kitchen
Carpet coupon
ND
ND
ND
7.59
GM coupon
6.30 x102
ND
ND
6.15
Bl disc
NG
G
G
Not Applicable
Crawl Space
Under Den
Carpet coupon
ND
ND
ND
7.56
GM coupon
ND
ND
ND
7.14
Bl disc
NG
G
G
Not Applicable
Attic Over
Master Bathroom
Carpet coupon
ND
3
7.82 x 105
5.36
GM coupon
1.97 x105
1.78 x 105
1.07 x 105
1.77
Bl disc
NG
NG
NG
Not Applicable

Carpet coupon
ND
ND
ND
7.56
Center of attic
GM coupon
1.29x103
1.79 x 104
8.60 x 103
3.20

Bl disc
NG
NG
NG
Not Applicable

Carpet coupon
6
1.36 x 102
1.38 x 101
6.09
Attic over Den
GM coupon
3.40 x105
5.21 x 105
3.88 x 105
1.35

Bl disc
G
NG
G
Not Applicable

Carpet coupon
ND
ND
ND
7.55
A/C Duct
GM coupon
ND
ND
ND
7.13

Bl disc
NG
NG
NG
Not Applicable

Carpet coupon
2.94 x107
2.79 x 107
3.10 x 107

Positive 1
GM coupon
6.19x10s
1.20 x 107
1.07 x 107


Bl disc 1,2,3
G
G
G

Positive 2
Carpet coupon
1.94 x107
2.75 x 107
3.15 x 107

GM coupon
1.11 x 107
6.53 x 10®
8.73 x 10®

ND = Non-detects
NG = No growth (Bl)
G = Growth (Bl)
NA: Bl Not deployed in replicates
Consistent with the results for Tests 2 and 3, the GM coupons placed in the attic showed LR values of
less than 3.2 to almost no decontamination. On the other hand, the carpet coupons showed more than a
5 LR to full decontamination.
For the crawl space, LR values of 6 to full decontamination were observed for the carpet coupons, but not
for the GM coupons. Additionally, seven out of the nine Bl discs in the crawl space showed growth,
indicating that full decontamination was not achieved, particularly for locations near vents in the crawl
space. For the coupons in the crawl spaces, the GM coupons were less prone to LCHP vapor
decontamination than carpet coupons, as shown in Figure 5-28. This finding highlights potential
differences between real world conditions and those obtained when Rogers, et. al (2005') treated seven
different surface materials for 20 minutes with > 1,000 ppm HPV in a closed chamber and observed better
inactivation of Bacillus spores on non-porous surfaces than on porous surfaces. See also Ryan, et al.
(2008).
48

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91
8-
Carpet Coupons
Galvanized Steel Coupons
Sampling Locations
Figure 5-28. Test 3 Decontamination Efficacy on Carpet and GM Coupons
For the hard-to-reach places, the decontamination efficacy depended on the placement of the Bl discs
and the type of materials shielding them. The Bl discs placed between the couch cushions and under the
rug showed little, if any, decontamination, while the Bl discs placed under one and five sheets of paper
and inside the closed bathroom drawer showed full decontamination. The jute-backed carpet and couch
cushions evidently prevented the HPV from reaching and affecting the spores on the Bl discs. Table 5-9
summarizes the spore count and G/NG results for the Bl discs in hard-to-reach places.
Table 5-9. Test 3 Post-Decontamination Recoveries on Hard-to-Reach Bl discs
Location
Rep 1
Quantitative Analysis of Bl discs (CFUs)
Rep 2 I Rep 3 I Rep 4
Rep 5
Between couch cushions
2.0 x 10®
2.1 x 10®
2.1 x 10®
2.2 x10s
1.9x10s
Under rug
1.3 x 10s
2.7 x 105
1.3 x 105
2.1 x 105
8.3 x105
Positives
2.1 x 10®
2.2 x 10®
2.3 x 10®
NA
NA
Location
Qualitative Analysis of Bl discs
Between couch cushions
NG
G
G
NG
NG
Under rug
G
G
G
G
G
Under one piece of paper
NG
NG
NG
NA
NA
Under five pieces of paper
NG
NG
NG
NA
NA
In closed bathroom drawer
NG
NG
NG
NG
NG
NA: Bl Not deployed in replicates
NG= No growth (Bl) G = Growth (Bl)
49

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5.5 Test 4
For Test 4, several adjustments were made to the placement of fans. In the corner and middle bedrooms,
the fans were moved closer to the closets, and a fan was added near the closet in the living room. The
number of humidifiers in the living area was reduced from seven to two. The two humidifiers were placed
in the hallway under the air return vent. The first humidifier was started while the second was on a delay
timer set to turn on 24 hours after the start of first one. This approach allowed time for the first humidifier
to fully dispense its contents and shut off. All the humidifiers, including the ones in the crawl space and
the attic, were filled to maximum capacity with 3.4 gallons of 3.0% HP aqueous solution. Figure 5-29
shows the test layout.
Master

Master
Bedroom
Bath
11 HI! 11
Bath
I A
Clos f Clos
Return
Clos
Corner
Bedroom
Mi^e
A Bedroom
Den
{]

-------
Figure 5-30. Test 4 B! discs Inside Book (closed during the test)

Figure 5-32. Test 4 Bl discs Behind Light Switch Plate
51
Figure 5-31. Test 4 Bl disc Inside Coat Pocket in Entry Closet

-------
The average results for the living areas, attic, and crawl space fumigation conditions including: HPV
concentration (ppm), overall calculated HPV exposure (concentration-time [CT] in ppm-hours), and RH
(%) are tabulated in Table 5-10. The temporal HPV concentration, RH, and temperature are discussed in
Sections 5.5.1.1 through 5.5.1.2 for the living areas. The decontamination efficacy of the test coupons
inoculated with Bg spores, and growth/no growth assessments of Bl discs co-located with the coupons
are discussed in Section 5.5.2.
	Table 5-10. Test 4 Fumigation Conditions	
Test 4 - Fumigation and Relative Humidity Conditions by Location
Location
HPV Max
(ppm)
CT
(ppm-hour)
RH Average
(%)
RH Max
(%)
Master Bedroom
20
788
41
58
Master Bathroom
Not Measured
40
55
Hallway Bathroom
13
525
31
46
Center of Den
13
432
47
55
Corner Bedroom
Not Measured
38
50
Middle Bedroom
Not Measured
47
61
Kitchen
Not Measured
43
56
Living Room
18
873
45
55
Air return
36
1320
44
55
Crawl Space Center
51
2465
62
81
Crawl Space North East
2.9
113
73
90
Crawl Space South West
68
86
Center of Attic
134
6130
63
81
Attic North East
17
800
63
82
Attic South West
66
83
5.5.1 Living Area Fumigation Conditions
5.5.1.1 Temporal HPV Concentration
The HPV concentration was monitored using HP sensors at the same five locations used during Test 2:
master bedroom, bathroom, hallway air return, living room, and den (see Figure 5-33). Reducing the
number of humidifiers in the living area from seven to two and placing them in the hallway under the air
return vent decreased the maximum concentration of HPV in the living room by more than 84% when
compared to Test 3, but decreased the HPV concentration in the other rooms much less. The HPV
concentration in the whole house decreased rapidly to about 5 ppm after 24 hours of fumigation before
increasing again with the start of the second humidifier.
52

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Master Bedroom
Bathroom
Hallway Return Air
Living Room
Den
CL
CL
CL
'¦dl
O)
0
20
40
60
80
100 120 140 160 180
Fumigation time (Hours)
Figure 5-33. Test 4 Temporal HPV Concentration in Living Areas
5.5.1.2 Temporal RH and Temperature
The results shown in Figure 5-34 illustrate fumigation RH conditions, using only two humidifiers run
sequentially 24 hours apart, throughout the decontamination testing. The RH for most of the living areas,
except for the den, remained below 50% for most of the fumigation period. The temperature, as expected,
remained steady near 70 F as maintained by the CTH HVAC system (data not shown). As observed for
the prior tests, outdoor conditions appear to have little bearing on the RH conditions observed for the
living areas inside the CTH. As shown in Figure 5-34, outdoor RH widely fluctuated, but the indoor RH
had a more even trend and remained mostly under 50% throughout the test period.
53

-------
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Middle Bedroom ~ Hallway Bathroom < Kitchen
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20
-r~
40
-r~
60
-r~
80
—I—
100
120
—I—
140
Fumigation Time (Hours)
160
Figure 5-34. Test 4 Temporal RH in Living Areas
5.5.2 Decontamination Efficacy
Reducing the number of humidifiers in the living areas from seven to two greatly affected decontamination
efficacy (see Table 5-11 and Figure 5-35V The humidifiers were placed in the hallway under the air
return vent and run sequentially 24 hours apart. Most of the test coupons in the living areas did not
achieve LR values of 6. The HPV concentrations were lower than the HPV concentrations observed in the
prior three tests.
During Test 4, three extra sets of coupons (sample set "b") were deployed in the master bedroom closet,
the outside wall of the den, and the middle bedroom closet along with the primary set of coupons. On the
third day of the 7-day test period, the "b" coupons were recovered and extracted. The "b" sample set
results were similar to results for the primary samples that underwent the full 7-day decontamination
period, demonstrating that extending the exposure time beyond 3 days did not improve the
decontamination efficacy. Table 5-11 summarizes spore recovery results for the test coupons and G/NG
results for the Bl discs. Interestingly, there were many NG for Bis with Gs spores which are reported to be
more difficult to inactivate than Bg, but a different conclusion is indicated here.
54

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Table 5-11. Test 4 Post-Decontamination Recoveries on Test Coupons and Bl discs
Location
Sample Type
Test Coupon Recovery (CFUs) and
Bl disc Recovery (G/NG)
LR (CFUs)


Rep 1
Rep 2
Rep 3
Average
SD
Master Bedroom
Closet
Carpet coupon
2.20 x 105
6.11 x 105
6.09 x 105
1.9
0.3
GM coupon
1.71 x 103
4.30 x 103
5.06 X 103
3.7
0.3
Bl disc
NG
NG
NG
Not Applicable
Master Bedroom
Carpet coupon
3.29 x 10®
4.36 x 105
7.66 x 104
1.9
0.8
Closet (b)
GM coupon
1.14 x 104
4.35 x 103
5.00 x 104
3.0
0.5
Master Bathroom
Floor
Carpet coupon
2.27 x 105
1.37 x 105
2.39 x 105
2.3
0.1
GM coupon
1.81 x 104
1.78 x 103
9.79 x 103
3.3
0.5
Bl disc
NG
NG
NG
Not Applicable

Carpet coupon
4.35 x 105
3.95 x 104
1.25 x 104
2.8
0.8
Bathroom Sink
GM coupon
3.57 x 103
ND
1
6.0
2.1

Bl disc
G
NG
NG
Not Applicable

Carpet coupon
4.88 x 105
2.09 x 104
6.99 x 104
2.6
0.7
Center of Den
GM coupon
1.39 x 103
2.67 x 102
3
5.2
1.4

Bl disc
NG
NG
NG
Not Applicable
Outside Wall of
Carpet coupon
2.08 x 105
8.64 x 104
1.59 x 104
2.7
0.6
Den (b)
GM coupon
4.44 x 103
3.81 x 103
1.92 x 103
3.7
0.2
Corner Bedroom
Floor
Carpet coupon
2.74 x 105
1.24 x 105
1.39 x 105
2.3
0.2
GM coupon
9.07 x 101
1.77 x 103
4.20 x 102
4.6
0.6
Bl disc
NG
NG
NG
Not Applicable
Middle Bedroom
Closet
Carpet coupon
2.51 x 105
2.73 x 105
5.44 x 104
2.4
0.4
GM coupon
3.18 x 104
1.55 x 104
1.87 x 104
2.9
0.2
Bl disc
G
NG
NG
Not Applicable
Middle Bedroom
Carpet coupon
4.91 x 105
3.31 x 10®
2.45 x 10®
1.4
0.4
Closet (b)
GM coupon
3.35 x 104
4.24 x 103
4.08 x 103
3.3
0.5

Carpet coupon
1.09 x 10®
1.11 x 10®
4.73 x 105
1.6
0.2
Kitchen Floor
GM coupon
6.14 x 104
3.35 x 105
2.39 x 103
2.6
1.1

Bl disc
G
NG
NG
Not Applicable

Carpet coupon
4.45 x 105
6.99 x 104
1.12 x 105
2.4
0.4
Living Room Floor
GM coupon
9.93 x 104
3.40 x 102
ND
4.7
2.6

Bl disc
NG
NG
G
Not Applicable

Carpet coupon
3
ND
ND
2.0
0.5
Entry Closet
GM coupon
7.00 x 101
ND
2.84 x 104
3.3
0.4

Bl disc
NG
NG
NG
Not Applicable
Duct
Carpet coupon
3.58 x 105
1.08 x 105
5.13 x 104
2.5
0.4
GM coupon
ND
2.50 x 101
ND
6.8
0.9
Crawl Space
Under Corner
Carpet coupon
3
ND
ND
7.5
0.3
GM coupon
7.00 x 101
ND
2.84 x 104
5.1
2.3
Bedroom
Bl disc
NG
G
NG
Not Applicable
Crawl Space
Under Kitchen
Carpet coupon
ND
ND
1.11 x 104
6.3
2.4
GM coupon
ND
ND
ND
7.3
0.0
Bl disc
NG
G
G
Not Applicable
55

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Location
Sample Type
Test Coupon Recovery (CFUs) and
Bl disc Recovery (G/NG)
LR (CFUs)


Rep 1
Rep 2
Rep 3
Average
SD
Crawl Space
Under Den
Carpet coupon
2
5.56 x 105
8.50 x 102
4.6
2.8
GM coupon
ND
ND
ND
7.3
0.0
Bl disc
G
G
G
Not Applicable
Attic Over Master
Bathroom
Carpet coupon
8.30 x 102
ND
ND
6.7
1.8
GM coupon
1
ND
ND
7.2
0.2
Bl disc
NG
NG
NG
Not Applicable

Carpet coupon
ND
4.18 x 104
9.26 x 104
4.4
2.8
Center of Attic
GM coupon
ND
ND
ND
7.3
0.0

Bl disc
NG
NG
NG
Not Applicable

Carpet coupon
1.29 x 107
1.42 x 104
1.28 x 107
1.4
1.7
Attic Over Den
GM coupon
4.83 x 104
4.21 x 105
1.18 x 105
2.1
0.5

Bl disc
NG
G
NG
Not Applicable

Carpet coupon
4.81 x 107
3.72 x 107
4.25 x 107


Positive 1
GM coupon
1.63 x 107
1.46 x 107
1.18 x 107



Bl disc
G
G
G


Positive 2
Carpet coupon
2.43 x 107
Not
available
3.55 x 107



GM coupon
1.55 x 107
1.62 x 107
3.35 x 107


ND = Non-detect
NG = No growth (Bl)
G = Growth (Bl)
Carpet Coupons
Galvanized Steel Coupons



Living Areas Locations
Figure 5-35. Test 4 LR Values in Spore Counts for Coupons in Living Areas
56

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In the living areas there were higher LR values for the GM coupons than the co-located carpet coupons.
For the Bl discs, 23 out of the 27 samples showed no growth. One possible suggestion for this result may
be that the reduction of RH did not hinder the inactivation of spores on GM but did for spores on carpet.
In the crawl space, where the fumigation conditions were maintained the same as during the previous
tests, 11 of 18 coupons exhibited full decontamination. In the attic, 8 of 18 coupons exhibited full
decontamination (greater than 6 LR). For the hard-to-reach places, 21 of 24 Bl discs exhibited growth,
demonstrating that Test 4 was not very efficacious. Table 5-12 summarizes G/NG results for the Bl discs
in hard-to-reach places.
Table 5-12. Test 4 Post-Decontamination Recoveries on Hard-to-Reach Bl discs
Location
Qualitative Analysis of Bl disc (CFUs)
Rep 1
Rep 2
Rep 3
Inside Closed Book
G
G
G
Master Bedroom Pants Pocket
NG
G
G
Inside Coat Pocket in Entry Closet
G
G
G
Dining Room Behind Light Switch Plate
G
G
G
Master Bedroom Window East Wall
G
NG
NG
Front Door Jamb
G
G
G
Den Deck Door Jam
G
G
G
Den Behind Switch Plate on outside wall
G
G
G
NG = No growth (Bl)
G = Growth (Bl)
5.6 Test 5
Figure 5-36 shows the layout for Test 5 with the equipment placement the same as for Test 4. Test 5
used two humidifiers, each filled and operated in a staggered sequence as follows:
•	Day 0:
>	Two humidifiers (Humidifiers 1 and 2) both filled with 3.4 gallons of 3.0% HP aqueous
solution placed in hallway under the air return
>	Humidifier 1 started, and Humidifier 2 set to start 24 hours later
•	After Day 1:
>	Humidifier 2 automatically started after 1 -day delay
•	After Day 3:
>	Humidifier 1 refilled with 3.2 gallons of 3.0% HP aqueous solution and started
>	Humidifier 2 refilled with 2 gallons of 3.0% HP aqueous solution and set to start 24 hours later
Collection of "b" sample sets
57

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Furthermore, Bl discs were placed in the following hard-to-reach areas to test the efficacy of the
fumigation process:
Between couch cushions
Under one piece of carpet (jute-backed)
Under one piece of paper
Under five pieces of paper
Under 10 pieces of paper
Under door mat, entry way
Inside a pants pocket, Master bedroom closet
Inside a coat pocket, entry closet
inside a book
Behind light switch plate, dining room
Behind light switch plate, exterior porch
Inside a pillowcase, middle bedroom
Between bed sheets, middle bedroom
Under comforter, middle bedroom
Inside light fixture, middle bedroom
Hall linen closet with closet door closed
Closed drawer, kitchen
Open drawer, kitchen
Window, master bedroom
Door jambs (front and back)
®j V Master
..•* Bath^A
A B
EFT
Master
Bedroom
A
Bath
I a
. J •*
CI03 I'cios r Return I lim
-3STT 1w

Oos
Corner
Bedroom

Middle
* Bedroom


G*
Oen
D


Kitchen
Living
Room
'dos

1

Instruments
• A
Garage
HP Sensor
Humidifier (COTS)
Test coupon	A 61
Figure 5-36. CTH Equipment and Sampling Locations for Test 5
asm Register
Fan
58

-------
The average results for the living areas, attic, and crawl space fumigation conditions including HPV
concentration (ppm), overall calculated HPV exposure (concentration-time [CT] in ppm-hours), and RH
(%) are tabulated in Table 5-13. The temporal HPV concentration, RH, and temperature are discussed in
Sections 5.6.1.1 through 5.6.1.2 for living areas, and the decontamination efficacy of the test coupons
inoculated with Bg spores, and growth/no growth assessments of Bl discs co-located with the coupons
are discussed in Section 5.6.2.
Table 5-13. Test 5 Fumigation Conditions
Test 5 - F
"umigation and Relative Humidity Conditions by Location

Location
HPV Max (ppm)
CT (ppm-hour)
RH Average (%)
RH Max (%)
Master Bedroom
16
1217
49
57
Master Bathroom
Not Measured
51
60
Hallway Bathroom
9
767
52
61
Center of Den
29
1239
52
63
Corner Bedroom
Not Measured
49
57
Middle Bedroom
Not Measured
45
60
Kitchen
Not Measured
52
63
Living Room
36
2274
50
61
Crawl Space Center
44
3304
70
83
Crawl Space North East
4
219
76
89
Crawl Space South West
74
86
Center of Attic
167
7537
47
68
Attic North East
29
881
48
71
Attic South West
51
77
5.6.1 Living Area Fumigation Conditions
5.6.1.1 Temporal HPV Concentration
The HPV concentration was monitored using HP sensors at the same five locations used during Test 4:
master bedroom, bathroom, hallway air return, living room, and den (see Figure 5-37). The results show
that staggering the operation of the humidifiers over 4 days increased the exposure time while reducing
the maximum HP concentration reached during testing as compared to Tests 1 and 3.
59

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¦ Master Bedroom
•	Bathroom
*	Hallway Return Air
» Living Room
Den
30
10
CO
0
20 40 60
80 100 120 140 160 180
Fumigation time (Hours)
Figure 5-37. Test 5 Temporal HPV Concentration in Living Areas
5.6.1.2 Temporal RH and Temperature
The results shown in Figure 5-38 illustrate fumigation RH conditions throughout the decontamination
testing, using the staggered start and refilling humidifier operation, at eight locations: master bedroom,
master bathroom, corner bedroom, middle bedroom, hallway bathroom, kitchen, den, and living room.
The staggered start and refilling humidifier operation was able to maintain indoor RH above 50%
throughout the entire fumigation period, exceeding the Test 4 overall humidification process. The large
swings in the outdoor environmental conditions, as for all other tests, had minimal effects on the indoor
environmental conditions. Temperature, as expected, remained steady near 70 F as maintained by the
CTH HVAC system (data not shown).
60

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100-
o
o
0
T3
C
CD
w
CD
1
O)
c
5
CD
90-
80-
70-
60-
50-
~ 30-|
'E
X
%
20-
10-
ro
<*>,
a:
^ o-
J\
1 i
/" j I i j y
T**- 7 !*•»»»	~
; * 1 !
* I \ f w
*4 ~
"i
¦J
ii j
k i
1 ?
kl
U
Master Bedroom • Master Bathroom •» Comer Bedroom
Middle Bedroom ~ Hallway Bathroom < Kitchen
Den • Living Room —¦—Outdoor
-r~
20
-r~
40
-r~
60
-r~
80
—I—
100
120
—I—
140
160
Fumigation Time (Hours)
Figure 5-38. Test 5 Temporal RH in Living Areas
5.6.2 Decontamination Efficacy
The increase in exposure time resulted in LR values greater than 6 for 46 of 54 test coupons placed in the
living areas. Results for carpet coupons in the master bedroom closet did not show full decontamination
for either sample set "b" recovered and extracted after the third day of the test or for two of the three
samples recovered and extracted on the seventh day of the sampling event. For 26 of 27 Bl discs placed
in the living areas, no growth was observed. LR values greater than 6 were observed for test coupons
located in the crawl space, but six of nine Bl discs exhibited growth. In the attic, only 11 of 18 coupons
exhibited greater than a 6 LR. Table 5-14 summarizes spore recovery results for the test coupons and
G/NG results for the Bl discs.
Table 5-14. Test 5: Post-Decontamination Recoveries on Test Coupons and Bl discs


Test Coupon Recovery (CFUs)
LR (CFUs)
Location
Sample Type
and Bl disc Recovery (G/NG)


Rep 1
Rep 2
Rep 3
Average
SD
Master Bedroom
Closet
Carpet coupon
1.06 x 103
ND
2.58 x103
5.42
1.96
GM coupon
ND
ND
ND
7.25
0.00
Bl disc
NG
NG
NG
Not Applicable
Master Bedroom
Carpet coupon
3.79 x 104
1.01 x 104
1.18 x 104
3.30
0.31
Closet (b)
GM coupon
9.80 x 102
ND
ND
6.16
1.80
Master Bathroom
Floor
Carpet coupon
5.20 x 103
ND
ND
6.35
2.21
GM coupon
ND
ND
ND
7.23
0.04
Bl disc
NG
NG
NG
Not Applicable
61

-------
Location
Test Coupon Recovery (CFUs)
Sample Type	and Bl disc Recovery (G/NG)
Rep 1	Rep 2 Rep 3
LR (CFUs)
Average SC
Bathroom Sink
Carpet coupon
ND
4.60 X102
ND
6.70
1.60
GM coupon
ND
ND
ND
7.22
0.06
Bl disc
NG
NG
NG
Not Applicable |
Center of Den
Carpet coupon
6
ND
ND
7.34
0.49
GM coupon
ND
ND
ND
7.23
0.02
Bl disc
NG
NG
NG
Not Applicable |
Outside Wall of
Den (b)
Carpet coupon
ND
2
ND
7.54
0.23
GM coupon
ND
ND
ND
7.26
0.03
Corner Bedroom
Floor
Carpet coupon
ND
ND
6.50 x 102
6.66
1.70
GM coupon
ND
ND
ND
7.24
0.04
Bl disc
NG
NG
NG
Not Applicable |
Middle Bedroom
Closet
Carpet coupon
ND
1
1.12 x 103
6.48
1.75
GM coupon
2.00 x 101
ND
ND
6.75
0.85
Bl disc
NG
NG
NG
Not Applicable |
Middle Bedroom
Closet (b)
Carpet coupon
1.16 x 104
4.31 x 104
ND
4.64
2.58
GM coupon
ND
ND
ND
7.25
0.03
Kitchen Floor
Carpet coupon
ND
7.13 x 103
ND
6.30
2.29
GM coupon
ND
ND
ND
7.23
0.03
Bl disc
NG
NG
NG
Not Applicable |
Living Room
Floor
Carpet coupon
ND
ND
6
7.34
0.51
GM coupon
ND
ND
ND
7.27
0.02
Bl disc
G
NG
NG
Not Applicable |
Entry Closet
Carpet coupon
9.30 x 102
ND
ND
5.96
1.56
GM coupon
ND
ND
ND
7.26
0.02
Bl disc
NG
NG
NG
Not Applicable |
Entry Closet (b)
Carpet coupon
1.29 X 102
1
9.34 x 103
5.43
1.90
GM coupon
ND
6.90 x 102
ND
6.23
1.73
Crawl Space
Under Corner
Bedroom
Carpet coupon
ND
ND
ND
7.65
0.01
GM coupon
2.00 x 101
ND
ND
6.75
0.85
Bl disc
NG
G
G
Not Applicable |
Crawl Space
Under Kitchen
Carpet coupon
ND
ND
ND
7.67
0.01
GM coupon
ND
ND
ND
7.24
0.03
Bl disc
NG
G
G
Not Applicable |
Crawl Space
Under Den
Carpet coupon
3.33 x 101
ND
ND
7.10
0.96
GM coupon
ND
2.00 x 101
ND
6.76
0.86
Bl disc
NG
G
G
Not Applicable |
Attic Over Master
Bathroom
Carpet coupon
1.35 x 103
ND
ND
6.55
1.88
GM coupon
ND
ND
ND
7.24
0.04
Bl disc
G
NG
NG
Not Applicable |
Center of Attic
Carpet coupon
ND
ND
3
7.45
0.34
GM coupon
1
ND
1
7.06
0.17
Bl disc
NG
NG
NG
Not Applicable |
Attic Over Den
Carpet coupon
8.51 x 105
4.82 x 105
7.98 x104
2.01
0.54
GM coupon
4.46 x 104
1.81 x 104
5.11 x 104
2.53
0.24
Bl disc
G
NG
NG
Not Applicable |
Positive 1
Carpet coupon
3.33 x107
3.83 X107
3.97 x 107

GM coupon
6.95 x10s
8.58X10®
1.44 x 107
Bl disc
G
G
G
Positive 2
Carpet coupon
3.17 x 107
2.68 x 107
2.59 x 107
GM coupon
1.68 x107
1.45 x 107
9.58 x 10®
ND = Non-detects
NG = No growth (Bl) G = Growth (Bl)
62

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For the hard-to-reach places, once again efficacy was reduced when Bl discs were placed under
cushions, under carpet, inside clothing, or behind walls. Table 5-15 summarizes qualitative results for the
Bl discs in hard-to-reach places.
Table 5-15. Test 5 Post-Decontamination Results for Hard-to-Reach Bl discs
Test Location
Bl disc Recovery (G/NG)
Rep 1
Rep 2
Rep 3
Between couch cushions
G
G
G
Under carpet (jute-backed)
G
G
G
Under one piece of paper
NG
NG
NG
Under five pieces of paper
NG
NG
NG
Under 10 pieces of paper
G
G
G
Under entry door mat
G
G
G
Master bedroom pants pocket
NG
NG
NG
Inside coat pocket in entry closet
G
G
G
Inside book
G
G
G
Switch plates (interior)
G
G
G
Switch plates (exterior)
G
G
G
Inside pillowcase in middle bedroom
NG
NG
NG
Between sheets in middle bedroom
NG
NG
NG
Under comforter in middle bedroom
NG
NG
NG
Inside light fixture in middle bedroom
NG
NG
NG
Closed hall linen closet
NG
NG
NG
Closed drawer in kitchen
G
G
G
Open drawer in kitchen
NG
NG
NG
Window in master bedroom
NG
NG
NG
Door jambs (front)
G
G
G
Door jambs (back)
G
G
G
6 Quality Assurance and Quality Control
This section discusses the quality assurance (QA) and quality control (QC) procedures for this study,
including project documentation, integrity of samples and supplies, instrument calibration, critical
measurements, QC and NHSRC BioLab checks, and QA assessments and response actions.
6.1 Project Documentation
This project was performed under the approved Category III Quality Assurance Project Plan (QAPP)
entitled, "Cary Test House Low-Concentration Hydrogen Peroxide Vapor Decontamination Tests,"
prepared by Jacobs and approved by EPA on September 23, 2015. All test activities are documented in
laboratory notebooks, digital video footage, and digital photographs. The documentation includes a record
for each sampling procedure and any deviations from the QAPP. All tests were conducted in-house in
accordance with developed Decontamination Technologies Research Laboratory (DTRL) and NHSRC
BioLab procedures to ensure repeatability and adherence to the data quality validation criteria for this
project.
63

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6.2	Integrity of Samples and Supplies
Samples were carefully maintained and preserved to ensure their integrity. Samples were stored away
from standards and other samples to prevent cross contamination. Supplies and consumables were
acquired from reputable sources and were National Institute of Standards and Technology (NIST)-
traceable whenever possible. Supplies and consumables were examined for evidence of tampering or
damage upon receipt and before use as appropriate. Supplies and consumables showing evidence of
tampering or damage were discarded. All examinations were documented, and supplies were
appropriately labeled.
6.3	Instrument Calibration
For this project, established and approved operating procedures were used to maintain and calibrate all
laboratory equipment. All laboratory measuring devices were certified as having been recently calibrated
or were calibrated by the on-site EPA Metrology Laboratory at the time of use. Table 6-1 summarizes the
instrument calibration frequency. Any deficiencies were noted and the instrument replaced or repaired as
needed to meet calibration tolerances.
Table 6-1. Instrument Calibration Frequencies and Expected Tolerances
Equipment
Calibration/Certification
Expected Tolerance
Thermometer
Compare to independent NIST thermometer (a thermometer
recertified annually by either NIST or an International
Organization for Standardization [ISO]-17025 facility) value
once per quarter
± 1 °F
RH meter
Three-point calibration using NIST-traceable salt cells
performed before and after each test
± 5%
HP sensor
Annual calibration provided by the manufacturer
± 1% full scale
Clock
Compare to office U.S. time at time.aov everv 30 davs
± 1 minute / 30 days
Pipettes and
Burets
Check calibration gravimetrically once a quarter
± 3%
6.4 Critical Measurements
The following measurements were deemed critical in achieving the project objectives:
•	Volume or mass of HP
•	Concentration of liquid HP decontaminant
•	Runtime
•	HPV concentration
•	RH
•	Temperature, including incubation temperature
•	Plated volume
•	CFU counts
Table 6-2 lists the data quality indicators (DQI) for the critical measurements. These DQIs were used to
determine if the collected data met the project QA objectives.
64

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Table 6-2. DQIs and Acceptance Criteria for Critical Measurements
Measurement Parameter
Analysis Method
Acceptance
Criteria
Actual
Pass or Fail Test
Mass of decontaminant (HP)
Scale
Accuracy: 0.1 g
0.05 g
Pass
Volume
Serological pipette tips
0.1 mL
± 10% of target value
Pass
Concentration of liquid HP
decontaminant
Preparation of 3% HP
solution in batches
using titration
(Envirotech 2013)
Precision: ± 10%
of target value
Test 1: 3%;
Test 2: 3.8 + 0.13%;
Test 3: 3 + 0.2%;
Test 4: 3 + 0.13%;
Test 5: 3 + 0.12%;
Pass
Run time
NIST-calibrated
stopwatch
± 1 minute per
hour
± 2 minutes (2*±1
min)
Pass
HPV Concentration
Pre- and post-
calibration by
manufacturer
Bias: ± 10%
Bias: ± 7.7%
Pass
RH
Vaisala
Bias: ± 5%

Pass
Temperature
NIST-traceable
thermometer (daily)
+ 2°C
Not applicable
Pass
Plated volume (liquid)
Pipette
2%
± 1%
Pass
CFU counts per plate
QCount® colony
counter
1.82 x 104 < QC
plate < 2.3 x 104
Within range of QC
plate, Part #510014,
Spiral Biotech, Inc.
Pass
Decisions to accept or reject test results were based on engineering judgment used to assess the likely
impact of the failed criterion on the conclusions drawn from the data. The acceptance criteria were set at
the most stringent levels that can routinely be achieved. All the DQIs were within the target acceptance
criteria set for this project as shown in Table 6-2.
Several QC checks were used for the measurement instruments to ensure that the data collected met the
criteria listed in Table 6-2. Sample integrity was evaluated during collection and analysis. Qualified,
trained, and experienced personnel utilized validated operating procedures to ensure data collection
consistency. When necessary, knowledgeable parties conducted training, and in-house practice runs
were used to gain expertise and proficiency before research began. The QC checks performed for this
project are detailed in Section 6.5.
In addition to the measurement instrument checks, positive control samples and procedural blanks were
included along with the test samples, so that optimal spore recovery and unintentional contamination of
test coupons could be assessed. Replicate coupons were included for each set of test conditions to
assess the variability of each test procedure.
6.5 QC and NHRSC BioLab Checks
Quantitative standards do not exist for biological agents. Viable spores were counted using an Advanced
Instruments QCount® colony counter. Counts greater than 300 or less than 30 CFUs were considered
outside of the targeted range. If the CFU count for bacterial growth did not fall within the target range, the
sample was re-plated., and then re-counted.
Before each batch of plates was enumerated, a QC plate was analyzed, and the result was verified to be
within the range indicated on the back of the QC plate. As the plates were being counted, a visual
65

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inspection of colony counts made by the QCount® colony counter was performed. Obvious count errors
made by the software were corrected by adjusting the settings (such as colony size, light, and field of
view) and recounting using an edit feature of the QCount® software that allows manual removal of
erroneously identified spots or shadows on the plate or by adding colonies that the QCount® software
may have missed.
The acceptance criteria for the critical CFU counts were set at the most stringent level that could routinely
be achieved. Positive controls were included along with the test samples so that spore recovery from the
different surface types could be assessed. Background checks also were included as part of the standard
protocol to check for unanticipated contamination. Replicate coupons were included for each set of test
conditions to characterize the variability of the test procedures.
Further QC samples were collected and analyzed to check the ability of the NHSRC BioLab to culture the
test organism as well as to demonstrate that the test materials used did not contain pre-existing spores.
The checks included the following:
•	Procedural blank coupons: Material coupons sampled in the same fashion as test coupons but
not contaminated with the surrogate organism before sampling
•	GM and carpet positive control coupons: coupons inoculated in tandem with the test coupons to
demonstrate the highest level of contamination recoverable from a particular inoculation event
Table 6-3 lists the additional QC checks built into the BioLab procedures designed to provide assurances
against cross-contamination and other biases in the microbiological samples.
Table 6-3. Additional Quality Checks for Biological Measurements
Sample Type
Frequency
Acceptance Criteria
Information
Provided
Corrective Action
Positive control coupon:
material coupon sample
contaminated with biological
agent and sampled using
extraction method
Minimum
of three
per test
1 x 107 for Bg,
50% relative standard
deviation between
coupons in each test set
Used to determine
extent of recovery
of inoculum on
target coupon type
If outside range, discuss in
the results section of this
report
Procedural blank
coupon (without biological
agent) that underwent
sampling procedure
One per
test
Non-detect
Controls for sterility
of materials and
methods used in
the procedure
Analyze extracts from
procedural blank without
dilution; identify and
remove source of
contamination if possible
Blank TSA sterility control
(plate incubated but not
inoculated)
Each plate
No observed growth after
incubation
Controls for sterility
of plates
All plates incubated before
use; contaminated plates
discarded before use
Replicate plating of diluted
microbiological samples
Each
sample
Reportable CFU count of
triplicate plates within
100%; reportable CFU
counts between 30 and
300 CFUs per plate
Used to determine
precision of
replicate plating
Re-plate sample
Unexposed field blank sample
One per
test
Non-detect
Level of
contamination
present during
sampling
Clean up environment;
sterilize sampling
materials before use
66

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6.6 QA Assessments and Response Actions
The QA assessment and corrective action procedures of this project were intended to provide rapid
detection of data quality problems. No contamination in QC procedural blank samples was observed after
the completion of testing. Table 6-4 summarizes the QA/QC assessment of spore recoveries for the
various sample types. Project personnel were intimately involved with the data on a daily basis so that
any data quality issue became apparent soon after it occurred.
Table 6-4. QA/QC Assessment of Spore Recoveries for Various Sample Types (CFUs/Sample)
Test No.
Sample
Positive Controls
Negative Controls
Material
Average
SD
Average
1
Carpet
2.82 x 107
3.96 x10s
ND
GM
1.09 x 107
4.62 x10s
ND
2
Carpet
3.04 x107
2.32 x107
ND
GM
8.39 x 10®
2.76x10®
ND
3
Carpet
2.78 x 107
4.41 x 10®
ND
GM
9.21 x 10®
2.45x10®
ND
4
Carpet
3.69 x 107
8.11 x 10®
ND
GM
1.52 x 107
1.87x10®
ND
5
Carpet
3.26 x 107
5.70x10®
ND
GM
1.18 x 107
3.95x10®
ND
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