EPA/600/R-17/211 August 2017
www.epa.gov/homeland-security-research
United States
Environmental Protection
Agency
Evaluation of Spray-Based, Low-Tech
Decontamination Methods under Operationally
Challenging Environments: Cold Temperatures
Office of Research and Development
National Homeland Security Research Center
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EPA/600/R-17/211
August 2017
EVALUATION REPORT
Evaluation of Spray-Based, Low-
Tech Decontamination Methods
under Operationally Challenging
Environments: Cold
Temperatures
U.S. Environmental Protection Agency
Research Triangle Park, NC 27711
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Disclaimer
The U.S. Environmental Protection Agency (EPA), through its Office of Research and Development's
National Homeland Security Research Center, funded and managed this investigation through Contract
No. EP-C-15-008 with Jacobs Technology, Inc. (Jacobs). This report has been peer- and administratively-
reviewed and has been approved for publication as an EPA document. This report does not necessarily
reflect the views of the EPA. No official endorsement should be inferred. This report includes photographs
of commercially available products. The photographs are included for the purpose of illustration only and
are not intended to imply that EPA approves of or endorses any products or manufacturers. EPA does not
endorse the purchase or sale of any commercial products or services.
Questions concerning this document or its application should be addressed to the following individual:
M. Worth Calfee, Ph.D.
Decontamination and Consequence Management Division
National Homeland Security Research Center
U.S. Environmental Protection Agency (MD-E343-06)
Office of Research and Development
109 T.W. Alexander Drive
Research Triangle Park, NC 27711
Telephone No.: (919) 541-7600
Fax No.: (919) 541-0496
E-mail Address: calfee.worth@epa.gov
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Acknowledgments
The principal investigator from the U.S. Environmental Protection Agency (EPA), through its Office of
Research and Development's National Homeland Security Research Center (NHSRC), directed this effort
with the support of a project team from across EPA and from Environment and Climate Change Canada
(ECCC). The contributions of the individuals listed below have been a valued asset throughout this effort.
Project Team
M. Worth Calfee, EPA, NHSRC/DCMD
Vladimir Blinov, ECCC
Leroy Mickelsen, EPA, OLEM/CMAD
Mike Nalipinski, EPA, OLEM/CMAD
Rich Rupert, EPA, Region 3
Shannon Serre, EPA, OLEM/CMAD
Konstantin Volchek, ECCC
EPA Quality Assurance
Eletha Brady-Roberts, NHSRC
Jacobs Technology. Inc.
Abderrahmane Touati
Madhura Karnik
Denise Aslett
Ahmed Abdel-Hady
Zora Drake-Richman
Peer Reviewers
Lori Miller, US Department of Agriculture
Joe Wood, EPA, NHSRC/DCMD
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Contents
Disclaimer i
Acknowledgments ii
Figures iv
Tables v
Acronyms and Abbreviations vii
Executive Summary 1
1 Introduction 1
1.1 Background 1
1.2 Project Objectives 1
2 Experimental Approach 3
3 EXPERIMENTAL MATERIALS AND METHODS 6
3.1 Coupon Preparation 6
3.1.1 Concrete Coupons 7
3.1.2 Glass Coupons 8
3.2 Sterilization of Materials 10
3.3 Test Organism and Coupon Inoculation 10
3.3.1 Bacillus atrophaeus var. globigii (Bg) 10
3.3.2 Coupon Inoculation 10
3.4 Concrete Material Evaluation 12
3.4.1 Effect of Concrete on pH of Decontamination Solutions 12
3.4.2 Concrete Liquid Retention Capacity 13
3.5 Decontamination Materials and Equipment 14
3.5.1 Environmental Test Chamber 15
3.5.2 NFB Decontamination Solutions 17
3.5.2.1 Initial Evaluation of pH, Redox Potential, and FAC (S1 through S8) 18
3.5.2.2 Short-Term Stability Testing of pH, Redox Potential, and FAC (S1 through S4) 19
3.5.2.3 Long-Term Stability Testing of pH, Redox Potential, and FAC (S1) 20
3.5.3 pAB Reference Solution 21
3.5.4 Application of Decontamination Solutions 21
3.5.4.1 Spray System Specifications 21
3.5.4.2 Spray System Operation 24
3.5.4.3 Spray System Functionality Test 25
3.5.4.4 Spray Pattern Test 26
3.6 Neutralizing Agents 26
3.6.1 Neutralization Agent Preparation 27
3.6.2 Neutralization Effectiveness Testing 27
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4 Sampling and Analytical Procedures 29
4.1 Microbiological Analysis 29
4.1.1 Sample Quantities 29
4.1.2 Sample Types 29
4.1.3 Sample Extraction and Analysis 30
4.2 Decontamination Solution Characterization 32
4.2.1 Free Available Chlorine (FAC) 32
4.2.2 pH 33
4.2.3 Redox Potential 33
5 Results and Discussion 34
5.1 Phase I Spray Optimization Tests 34
5.1.1 pAB Solution 34
5.1.2 pAB and S1 Solutions 35
5.2 Phase II Decontamination Efficacy Tests 36
5.2.1 Surface Decontamination Efficacy Results 36
5.2.1.1 Effect of Temperature and use of de-icing agents 37
5.2.1.2 Effect of NaCI as De-icing Agent 38
5.2.1.3 Effect of Increased Contact Time 39
5.2.2 Total Decontamination Efficacy Temperature Results 40
5.3 Results Summary 41
6 Quality Assurance and Quality Control 44
6.1 Criteria for Critical Measurements and Parameters 44
6.2 Data Quality Indicators 44
6.3 QA/QC Checks 45
6.3.1 Check of Integrity of Samples and Supplies 46
6.3.2 NHRSC Bio-laboratory Control Checks 46
6.3.3 QA Assessments and Corrective Actions 47
References 49
Figures
Figure ES-1. Surface Decontamination Efficacy for each NFB Formulation and pAB 2
Figure ES-2. Surface Decontamination Efficacy of NFB and pAB Solutions at Various
Temperatures 3
Figure ES-3. Total Decontamination Efficacy for NFB formulations and pAB 4
Figure 3-1. Coupon diagram of non-concrete and concrete and materials 6
Figure 3-2. Mold for Fabricating Concrete Coupons 7
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Figure 3-3. Cross Section of Final Concrete Coupon in Butter Board Mold 8
Figure 3-4. Concrete Coupon 8
Figure 3-5. Glass Coupon 9
Figure 3-6. Sterilization Envelope (A) and Bag (B) 10
Figure 3-7. MDI Canister (A) and Actuator (B) 11
Figure 3-8. Inoculation Process 11
Figure 3-9. Concrete Coupons After Double-Spray Tests 14
Figure 3-10. TestEquity ETC 16
Figure 3-11. Touchscreen Controller 16
Figure 3-12. Decontamination Solution Test Parameter Comparisons 18
Figure 3-13. Short-Term Stability Test Results (S1 through S4) 20
Figure 3-14. Long-Term Stability Test Results (for decontamination solution S1) 20
Figure 3-15. Spray System Schematic 21
Figure 3-16. Spray System Inside ETC 22
Figure 3-17. Decontamination Liquid Tank 22
Figure 3-18. Spray Nozzle Setup on CPVC Stand 23
Figure 3-19. Spray Nozzle Orientation during Phase I (A) and Phase II (B) Tests 23
Figure 3-20. Top View of Control Box 24
Figure 3-21. Kynar Tubing Distribution System 25
Figure 3-22. Spray Pattern Test Results 26
Figure 4-1. Bacterial Colonies on Spiral-Plated Agar Plate 30
Figure 4-2. Bg Colonies on Filter Plate 31
Figure 4-3. Bg Colonies on Spread Plate 31
Figure 5-1. Spray Optimization Test Results for pAB 35
Figure 5-2. Spray Optimization Test Results for pAB and S1 36
Figure 5-3. Surface Decontamination Efficacy of pAB and S5 through S7 at Various
Temperatures 38
Figure 5-4. Surface Decontamination Efficacy of S5 through S8 39
Figure 5-5. Surface Decontamination Efficacy vs. Contact Time 40
Figure 5-6. Total Decontamination Efficacy of pAB and S5 through S7 at Various Temperatures 41
Figure 5-7. Surface Decontamination Efficacy for pAB and S5 through S7 42
Figure 5-8. Total Decontamination Efficacy for S5 through S7 and pAB 42
Tables
Table 2-1. Test Matrix for Decontamination Solutions Evaluated for Efficacy 5
Table 3-1. Test Coupon Material Specifications 6
Table 3-2. Effect of Concrete Material on the pH of Decontamination Solution S1 13
Table 3-3. Decontamination Materials and Equipment 15
Table 3-4. Decontamination Solution Formulations 17
Table 3-5. Decontamination Solution Comparisons 18
Table 3-6. Short-Term Stability Test Results (S1 through S4) 19
Table 3-7. Long-Term Stability Test Results (S1) 20
Table 3-8. Volume Collected from Each Spray Nozzle in the Spray System 25
Table 3-9. DE Broth (± CaCh) Neutralization Effectiveness 28
Table 4-1. Sample Types and Numbers for Each Decontamination Solution 29
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Table 4.2. ORP (mV vs. Temperature) Orion Std. solution Estimated Readings 33
Table 5-1. Parameters for pAB Solution Spray Optimization Tests 34
Table 5-2. Parameters for pAB and S1 Efficacy Tests 35
Table 6-1. DQIs for Critical Measurements and Parameters 45
Table 6-2. Additional QC Checks for Biological Measurements 47
Table 6-3. QA/QC Assessment of Spore Recoveries (CFU) for Various Control Samples 48
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Acronyms and Abbreviations
|j|_ microliter(s)
jjm micrometer(s)
AC alternating current
ACS American Chemical Society
Avg average
BioLab NHSRC Research Triangle Park (RTP) Microbiology Laboratory
Bg Bacillus atrophaeus var. globigii
CaCb calcium chloride
CAS Chemical Abstract Services
CBRN Chemical, Biological, Radiological, and Nuclear
CFIA Canadian Food Inspection Agency
CFU colony-forming unit(s)
CH3COOH acetic acid
cm centimeter(s)
CMAD Consequence Management Advisory Division
CNC Computer numerical control
CPVC chlorinated polyvinyl chloride
DC direct current
DCMD Decontamination and Consequence Management Division
DE Dey Engley
Dl deionized
DQI data quality indicator
DQO data quality objective
DTRL Decontamination Technologies Research Laboratory
ECCC Environmental Climate Change Canada
Eh Target Redox Potential
EPA U.S. Environmental Protection Agency
ETC environmental test chamber
EtO ethylene oxide
FAC free available chlorine
FCC Food Chemicals Codex
ft3 Cubic feet
g/L gram(s) per liter
gpm gallon(s) per minute
HDPE high-density polyethylene
HSRP Homeland Security Research Program
ID identification
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in. Inch(es)
Jacobs Jacobs Technology, Inc.
LR log reduction
M Molar
MDI metered-dose inhaler
mg milligram(s)
mL milliliters)
mL/L milliliter(s) per liter
mm millimeter(s)
mV millivolt(s)
N Normal
NaCI sodium chloride
NaCIO sodium hypochlorite
NEM Nisshin Em Company, Tokyo, Japan
NFB non-freezing bleach
NHSRC National Homeland Security Research Center
NIST National Institute for Standards and Technology
NPT National Pipe Thread
OLEM Office of Land and Emergency Management
ORD Office of Research and Development
ORP Oxidation-reduction potential
pAB pH-adjusted bleach
PBST phosphate-buffered saline with 0.05% Tween® 20
PPE personal protective equipment
ppm part(s) per million
psi pound(s) per square inch
QA quality assurance
QC quality control
redox reduction-oxidation
RH relative humidity
RTP Research Triangle Park
SEM scanning electron microscope
SPDT Single-pole, double-throw
STD standard deviation
STS sodium thiosulfate
TSA tryptic soy agar
USP U.S. Pharmacopeia Convention
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Executive Summary
This project was conducted by the U.S. Environmental Protection Agency's (EPA) Office of Research and
Development's (ORD) National Homeland Security Research Center (NHSRC) and supports the mission
of the EPA's Homeland Security Research Program (HSRP) by providing relevant information pertinent to
the decontamination of contaminated areas resulting from a biological incident. The key objective of this
project is to estimate the potential reduction of viable bacterial spores (i.e., effectiveness) as a function of
the remediation activities applied under challenging situations and conditions in field operations. This
research effort specifically focused on the evaluation of bleach decontamination formulations, specifically
formulated to remain liquid at temperatures well below the freezing point for water. These formulations
could be beneficial when conducting remediation activities during cold weather conditions.
The project tests evaluated decontamination solution efficacy under challenging temperature conditions on
two common building material surfaces: concrete and glass. The test decontamination solutions were
applied with a low-tech spraying device. Low-tech applications were of particular interest for this project
because in the event of a widespread urban bioterrorist incident, use of relatively inexpensive off-the-shelf
materials could be critical for mobilizing an effective and swift remediation response.
The tests were conducted in an environmental test chamber (ETC) so that temperature conditions ranging
from -25 °C to 25 °C could be tightly controlled. An automated spray system that could fit inside the
environmental chamber and function under extreme environmental conditions was developed. The use of
this setup provides operationally relevant insights into the expected efficacy of the non-freezing bleach
solutions and further provides an opportunity to perform tests under accurate and repeatable conditions
(i.e. spray duration, spray pressure, volume of spray, relative temperature and humidity). The project results
provided insights into the expected efficacy of non-freezing bleach (NFB) solutions tested under various
environmental and operational conditions.
Eight non-freezing bleach-based formulations were prepared from recipes provided by the EPA-
Environment and Climate Change Canada (ECCC) working group. Each solution was evaluated for its
ability to inactivate Bacillus atrophaeus var. globigii (Bg) spores (a surrogate for Bacillus anthracis) on
building material surfaces. The solutions contained de-icing agents that depressed the freezing point of the
solutions below the target test temperatures so that the solutions could be spray-applied. In addition to the
NFB solutions, a pH-adjusted bleach (pAB) solution was included in the evaluations (when temperatures
permitted) as a reference decontamination agent.
Small coupons prepared from concrete and glass were inoculated with 1 x 107 Bg spores and then sprayed
for a prescribed exposure time with each bleach solution under various temperatures. Spray duration times,
spray pressures, number of reapplications, and solution contact times were varied among the tests. After
spraying, glass and concrete coupons were analyzed for potentially viable spores remaining on the coupon
surfaces. Overspray runoff (rinsate) samples were also analyzed for the presence of potentially viable
spores. In general, the decontamination efficacy for each solution was determined by comparing the mean
spore recovery results from positive control samples (unexposed to decontamination treatment) to the mean
spore recovery results from decontaminated coupon samples.
ES-1
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During laboratory evaluation, a decontaminant is considered effective when there is a log reduction (LR) >
6 for a 1 x 106 colony forming units (CFU) or greater challenge (USEPA. 2007). Laboratory test conditions
that achieve complete kill, i.e. no viable spores recovered following treatment, are considered highly
effective. Results for each NFB formulation were compared to a baseline pH-adjusted bleach (pAB)
solution, which is known to provide a LR > 6 on multiple material types (Calfee, et al. 2012). Results are
summarized in two ways: 1) surface decontamination efficacy and 2) overall spraying efficacy.
Surface decontamination efficacy indicates how effective each solution was at decontaminating the surface
of each material and was calculated as follows:
Mean (Log CFU positive control sample) - Mean (Log CFU test coupon sample)
Figure ES-1 summarizes the surface decontamination efficacy results for pAB and three of the most
efficacious NFB formulations. As the figure shows, pAB achieved a surface LR greater than 6 at
temperatures greater than 0 °C on both materials tested. None of the NFB formulations were as effective
as pAB.
Surface Decontamination Efficacy (0 °C - 25 °C)
i Glass Concrete
I
2.8
2.8
3.3
pAB
S5
S6
S7
Decontamination Formulation
Figure ES-1. Surface Decontamination Efficacy for each NFB Formulation and pAB
As the testing temperature was lowered, solution efficacy also tended to decrease. However, at no
temperature greater than 0 °C were the test solutions as effective as pAB, and none were observed to
provide a 6 LR, as shown in Figure ES-2. Decontamination efficacy data for the pAB solution were gathered
only to 0 °C because the freezing point of pAB was determined to be -8 °C.
ES-2
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pAB
S5
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3 4-fl ¦
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¦ Glass a Concrete
6.3
6.8
6 4
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c
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o
3 6.0
T3
m
Q£
5.0
g> 4.0
~ 3.0
^ 2-0
¦ Glass
Concrete
0DC 10°C 25°C
Temperature (°C)
-25 "c
25°C
Temperature (°CJ
S6
Concrete
10°C
25°C
Temperature (°C)
S7
u. 5 0
g> 4,0
— 3.0
>*
m Z0
TO
o -
c 1.0
- 0,
¦ Glass Concrete
r
2.4
I
1-2
jl 1
I"
¦25°C
•10°C 0°C 10°C
Temperature (°C)
il1!
5.0
25 C
^Denotes Full Surface Decontamination Based on Detection Limit
Figure ES-2. Surface Decontamination Efficacy of NFB and pAB Solutions at Various
Temperatures
The field applicability for a decontamination solution and its corresponding delivery technology depends on
factors that include the ultimate disposition (or fate) of the targeted spores. This information is required to
develop a comprehensive, site-specific remediation strategy. For example, if viable spores are washed off
materials, remediation field strategies may require not only consideration of contact times for primary
building materials but also soil collection and treatment. To assess the potential for viable spores to be
washed off the test surfaces, all liquids used in the decontamination test process were collected and
quantitatively analyzed. To provide a conservative estimate of spore fate and transport, rinsates were
neutralized immediately upon collection by pre-loading collection tubes with a neutralizing agent.
Consideration of both surface decontamination and viable spore relocation into the rinsate (recovery of
viable spores in rinsate) provided a measurement of the total decontamination efficacy, which was
calculated as follows:
Mean (Log CFU positive control sample) - Log (CFU test coupon sample + CFU liquid rinsate sample)
As observed with surface decontamination efficacy, the total efficacy for each tested NFB formulation was
less effective than pAB for temperatures greater than 0 °C. Results for the most effective solutions as
compared to pAB are summarized in Figure ES-3. As the figure shows, none of the NFB or pAB solutions
ES-3
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achieved a total decontamination efficacy > 2 LR. Generally, total decontamination efficacies were higher
on concrete coupons than on glass coupons, possibly because of the porosity of the concrete coupons,
allowing for greater adhesion of the Bg spores. The rinsate samples were immediately neutralized, which
explains the large differences between the surface and the total decontamination efficacies, and because
the rinsates were immediately neutralized, the viable spores captured therein provide an approximation of the
maximum amount of contamination that could be spread via runoff.
These results show that the spraying operation dislodged a large number of spores that were recovered
and quantified during analysis of the neutralized rinsate samples. Such action may be comparable to the
function of a neutralizing soil matrix in an actual field situation.
Total Decontamination Efficacy (0 °C - 25 °C)
— 2.5
c
o
o
2.0 ¦
3
T3
a)
0£
1.5 ¦
O)
o
—I
1.0 ¦
>
o
(0
o
0.5 ¦
£
HI
0.0 4—
i Glass Concrete
1.9
1.2
1.1
PAB S5 S6 S7
Decontamination Formulation
Figure ES-3. Total Decontamination Efficacy for NFB formulations and
pAB
Despite the NFB solutions demonstrating lower surface and total decontamination efficacies compared to
pAB, these solutions currently are the only NFB decontaminants evaluated against Bacillus spores. At
conditions below -8 °C, these solutions may be useful in reducing surface-bound spore concentrations
during remediation efforts. The testing results under this project provide an important baseline that further
work can build upon to develop and characterize new decontamination options under environmentally
challenging conditions such as freezing temperatures.
ES-4
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1 Introduction
This project was conducted by the U.S. Environmental Protection Agency (EPA) Office of Research and
Development's (ORD) National Homeland Security Research Center (NHSRC) and supports the mission
of the EPA's Homeland Security Research Program (HSRP) by providing relevant information pertinent to
the decontamination of contaminated areas resulting from a biological incident. The key objective of this
project is to estimate the potential reduction of viable bacterial spores (effectiveness) as a function of the
remediation activities applied under challenging situations and conditions in field operations. This research
effort focused on the evaluation of bleach decontamination solutions specifically formulated to remain liquid
at temperatures well below the freezing point for water. These solutions could be beneficial for remediation
activities conducted under cold-weather conditions.
This project evaluated the effectiveness of spray-based, "low-tech" decontamination methods for spores on
building materials (concrete and glass) applied under temperatures ranging from ambient indoor conditions
(approximately 25 °C) to cold-weather conditions (approximately -25 °C). The project addresses HSRP
strategic goals as described in detail in the Homeland Security Research Multi-Year Strategic Plan (USEPA
2012). Specifically, the project is relevant to Long-Term Goal 2, which states, "The Office of Land and
Emergency Management (OLEM) and other clients use homeland security research program products and
expertise to improve the capability to respond to terrorist attacks affecting buildings and the outdoor
environments." The following sections discuss the project background and objectives.
1.1 Background
The NHSRC 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. Further, NHSRC provides expertise and guidance on the selection and
implementation of decontamination methods that may ultimately provide the scientific basis for a significant
reduction in the time and cost of remediating contaminated sites.
This project addresses a direct need expressed by OLEM's Chemical, Biological, Radiological, and Nuclear
(CBRN) Consequence Management Advisory Division (CMAD). This need consists of identifying
"operationally challenging environments and situations" and evaluating the effectiveness of a set of
decontamination procedures, including spraying surfaces with sporicidal liquids under such conditions.
Decontamination at low temperature conditions is a critical technology gap identified by Environment and
Climate Change Canada (ECCC) and the Canadian Food Inspection Agency (CFIA). Recently, these
agencies began a review and analysis of relevant technical and scientific literature and confirm feasibility
of sub-zero temperature decontamination. The current study is a collaboration amongst the US EPA and
ECCC, to build upon ECCC, CFIA, and EPA's previous work, and address the gaps associated with
decontamination of surfaces under cold-weather conditions.
1.2 Project Objectives
The purpose of this project was to assess the effectiveness of a spray-based, "low-tech" decontamination
approach using liquid sporicides specially formulated to remain liquid at sub-freezing temperatures to
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reduce the levels of surface-bound bacterial spores under challenging environmental conditions. Low to
sub-freezing temperatures reflect one challenging environmental condition that could potentially occur
during such response activities. For the purpose of this effort, "low-tech" is defined as decontamination
approaches that do not require specialized materials or equipment (products used were available at a local
hardware store). Low-tech applications were of particular interest for this project because in the event of a
widespread urban bioterrorist incident, use of relatively inexpensive off-the-shelf materials could be critical
for mobilizing an effective and swift remediation response.
Bleach-based solutions prepared from recipes provided by the EPA-ECCC working group were evaluated
for their ability to inactivate Bacillus atrophaeus var. globigii (Bg) spores (a surrogate for Bacillus anthracis)
on building material surfaces. The solutions contained de-icing agents that depressed the freezing point of
the solutions below the target test temperatures so that the solutions could be spray-applied. In addition to
the non-freezing bleach (NFB) formulations, a pH-adjusted bleach (pAB) solution was included in the
evaluations (when temperatures permitted) as a reference decontamination agent.
Test coupons were prepared from typical urban building materials (glass and concrete), inoculated with the
target organism Bacillus atrophaeus var. globigii (Bg) using an aerosol deposition method, and sprayed
with test solutions under a range of test temperatures. Tests were conducted in an environmental test
chamber so that temperature conditions, ranging from -25 °C to 25 °C, could be tightly controlled. To
simulate large-scale outdoor operations, all test components (spray nozzles, coupons, runoff collection,
decontaminant reservoir, decontaminant supply tubing, etc.) were located within the chamber and were
acclimated to test temperature before testing began. The relative impacts of spray duration, spray
pressures, number of spray repeats, and solution contact times were evaluated. The surface
decontamination efficacy of each formulation was measured based on the reduction of viable agent
achieved on the surface of the test coupons. Relocation of viable spores from the contaminated coupon
surfaces into the overspray runoff during each decontamination event was also investigated.
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2 Experimental Approach
Testing was conducted at EPA's Research Triangle Park (RTP) facility in North Carolina. The general
experimental approach used to meet the project objectives is described below.
1. Preparation of representative coupons of test materials: Two common building materials,
concrete and glass, were tested. The test coupons were prepared from these materials as
described in Section 3.1.
2. Test material sterilization: The coupons and other test materials were sterilized as described
in Section 3 2.
3. Inoculation of test coupons with the target organism: The test coupons were inoculated using
an aerosol deposition method that delivered a known concentration of spores in a repeatable
fashion. Approximately 2 x 107 spores of Bg, a surrogate organism for Bacillus anthracis, were
deposited onto each coupon as discussed in Section 3 3.
4. Preparation of NFB decontamination solutions: Decontamination solutions were freshly
prepared on each test day. Eight bleach-based solutions, S1 through S8, were prepared from
recipes provided by the ECCC working group (Blinov. et al. 2015). De-icing agents were used to
depress the freezing point of the solutions to below the target test temperatures. In addition to the
NFB solutions, a pAB solution was also tested and used as a reference decontamination agent,
but de-icing agent was not added to the pAB. Section 3 5 2 discusses the preparation of the NFB
decontamination solutions, and Section 3.5.3 discusses the preparation of the pAB reference
solution.
5. Application of decontamination procedure to test materials: Test coupons (five coupons per
test material) were decontaminated using the test solutions in an Environmental Test Chamber
(ETC), described in Section 3.5.1. using an automated spraying system as discussed in Section
3 5 4.
6. Preparation of neutralizing agents: The neutralizing agents included sodium thiosulfate (STS)
and Dey Engley (DE) broth as discussed in Section 3.6. The neutralizing agents were applied to
stop the decontamination activity after a prescribed exposure time. After the prescribed exposure
times, coupons were collected and deposited into a tube containing the neutralizing agent. The
neutralizing agents were amended with a de-icing agent for low-temperature testing.
7. Collection of Runoff: Liquid runoff from each coupon was also collected through sterile funnels
into sample tubes that contained pre-determined volumes of neutralizer, and in cases of low
temperature testing, the tubes also contained a de-icing agent.
8. Sample extraction and analysis: Viable Bg spores were extracted from the test samples (coupon
and runoff), and aliquots were analyzed using an automated spiral plating system as discussed
in Sectio . Viable spore recovery was quantified in terms of colony forming units (CFU)
present in each sample.
9. Determination of decontamination efficacy: As discussed in Section 5.2, decontamination
efficacy was evaluated as a function of the: (1) decontamination procedure, (2) decontamination
solution, and (3) coupon material type. Decontamination efficacy was expressed as a log reduction
(LR) of the viable Bg spores recovered. Typically, for laboratory assessments of decontamination
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efficacy, a LR > 6 is considered effective, and when no viable spores are recovered after
decontamination treatment, the method is considered highly effective. Decontamination efficacy for
each coupon was determined by comparison to positive control sample results. The transfer of
viable organisms to post-decontamination liquid waste was evaluated through quantitative analysis
of decontamination procedure runoff
This project was conducted in two phases, as summarized below.
• Phase I Spray Optimization Tests: This phase was conducted to optimize and verify the
automated spray system inside the ETC. A series of method development tests were conducted to
identify test parameters (such as solution contact time, duration of spray, number of sprays, and
effective neutralizing agents) suitable for evaluating the decontamination solutions. During these
tests, the spray nozzles were arranged at a 30° angle to the test coupons. One NFB solution (S1)
was tested, and its effectiveness was assessed using Bg spores on concrete and glass. The
reference decontamination agent was pAB.
• Phase II Decontamination Efficacy Test: During this phase, the spray nozzles were situated
directly above the test coupons to maximize decontaminant delivery to coupon surfaces. Based on
test results from Phase I, two five-second sprays were used with a total contact time of 20 minutes.
To improve decontamination efficacy, new NFB solutions proposed by ECCC (S5 through S8) were
prepared by altering the S1 through S4 formulations. Decontamination solutions S5 through S8
were evaluated under the following temperature conditions: -25, -10, 0,10, and 25 °C. As in Phase
I, pAB was used as the reference decontamination agent down to 0 °C.
The pAB reference decontamination agent was selected based on published information indicating that
pAB can reduce bacterial spore populations (> 6 LR) under specific conditions related to concentration, pH,
contact time, and material (Calfee et al. 2012). EPA has issued crisis exemptions permitting the limited
sale, distribution, and use of EPA-registered bleach products against Bacillus anthracis spores at some
facilities and locations, including Capitol Hill, the U.S. Postal Service Processing and Distribution Centers
at Brentwood (Washington, DC) and Hamilton (Trenton, NJ), the Department of State, the General Services
Administration, and Broken Sound Boulevard (Boca Raton, FL) (FBI. 2001). Table 2-1 summarizes the test
matrix for pAB, and the three most efficacious NFB formulations (S5, S6, and S7).
4
-------
Table 2-1. Test Matrix for Decontamination Solutions Evaluated for Efficacy
Decontamination Solution
Temperature (°C)
Coupon Material
-25
-10
0
Glass
10
S5
25
-25
-10
0
Concrete
10
25
-25
-10
0
Glass
10
S6
25
-25
-10
0
Concrete
10
25
-25
-10
0
Glass
10
S7
25
-25
-10
0
Concrete
10
25
0
10
Glass
pAB
25
0
10
Concrete
25
5
-------
3 EXPERIMENTAL MATERIALS AND METHODS
This section describes the test materials, test facilities and equipment, general decontamination
approaches and test conditions, and the methods that were used to evaluate the data in accordance with
the project objectives. The following topics are discussed:
Coupon preparation
Sterilization of materials
Test organism and coupon inoculation
Concrete material evaluation
Decontamination materials and equipment
Neutralizing agents.
3.1 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. For this project,
representativeness was ensured by: (1) selecting test materials typically representative of building
materials, and (2) obtaining test materials from appropriate suppliers. Uniformity was ensured by preparing
test coupons using the same batch of material.
Coupons (18-mm diameter), shown in Figure 3-1, and summarized in Table 3-1, were prepared from two
target materials: concrete and glass. The glass coupon was made of an 18 mm glass disc which was
affixed to an aluminum stub using a carbon-based adhesive. The concrete coupon was fabricated from
Sakrete Top' N Bond Patcher, with a drywall nail in the center of the back for handling.
Glass disc
Carbon adhesive
Aluminum stub
¦
Concrete
cr
Non-concrete Material coupon
Drywall nail
Concrete material coupon
Figure 3-1. Coupon diagram of non-concrete and concrete and materials
Table 3-1. Test Coupon Material Specifications
Material
Concrete
Sakrete Top' N Bond Patcher 18-mm diameter, 6-mm height
Lowe's store,
Charlotte, NC
Glass
Glass discs, 18-mm diameter, 3-mm height
Prism Research
Glass, Raleigh, NC
Preparation of the concrete and glass coupons is discussed below.
6
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3.1.1 Concrete Coupons
Concrete coupons were fabricated from Sakrete Top' N Bond Patcher. Each coupon had a drywall nail in
the center of the back for handling. The following materials and equipment were used to prepare the
concrete coupons:
• Butter board measuring 6 by 12 by 2 inches (in.) (from McMaster Carr, Atlanta, GA, Catalog No.
86595K1)
• Computer numerical control (CNC) milling machine with 18-mm-diarneter mill cutter
• Drywall nails
• Sakrete Top' N Bond Patcher mix
• Suitable plastic container for mixing concrete
• Mixing stick
• Deionized (Dl) water
• Appropriate personal protective equipment (PPE), including gloves, safety glasses, and safety
footwear.
The procedure summarized below was used to prepare the concrete coupons.
1. Personnel preparing the coupons donned appropriate PPE.
Using the CNC milling machine, the butter board was drilled to produce the mold shown in
Figure 3-2.
Figure 3-2. Mold for Fabricating Concrete Coupons
2. In the plastic container, 1 pound of Sakrete Top' N Bond Patcher mix was mixed with 0.1 pint (50
milliliters [mL]) of clean water. The mixture was well-worked using a mixing stick.
3. Additional water was added as needed (not exceeding 0.6 mL or 0.13 pint total) to obtain a
workable, plastic-like consistency.
4. Insert the dry wall nail inside the concrete mixture
7
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5. The concrete fabrication mold (Figure 3-1) was filled with the concrete mix. The concrete at the
top of the mold was smoothed, using a mist of water during the drying process, to ensure a fiat
surface.
6. The concrete-filled mold was allowed to dry and cure indoors (at 70 °F or higher) for five days
before removal of the coupons from the mold.
Figure 3-3 shows a cross section of the final coupon mold, and Figure 3-4 shows the final concrete
coupon.
DRY WALL
NAIL
Figure 3-3. Cross Section of Final Concrete Coupon in Butter Board Mold
Figure 3-4. Concrete Coupon
3.1.2 Glass Coupons
The glass coupons were constructed from custom-made glass discs affixed to aluminum stubs using a
carbon-based adhesive. The following materials were used to prepare these coupons:
• Glass discs (18 mm in diameter, 3 mm thick) custom made by Prism Research Glass (Raleigh,
NC)
8
-------
• Scanning electron microscopy (SEM) stubs (18 mm in diameter, 8-mm pin length) from Ted Pella,
Inc. (Catalog No. 16119, Redding, CA)
• Double-sided adhesive carbon tape from Nisshin EM Co. Ltd. (NEM tape, Tokyo, Japan)
• Arch punch from C.S. Osborne & Co. (Catalog No. 01236, Harrison, NJ)
• Parafilm roll from Bemis Company, Inc. (Neenah, Wl)
• Tweezers
• Appropriate PRE, including gloves, safety glasses, and safety footwear.
The procedure summarized below was used to prepare the glass coupons.
1. Personnel preparing the coupons donned appropriate PPE.
2. A 10-in.-long strip of the NEM double-sided tape was cut and laid on a flat surface with the sticky
side up.
3. A 10-in.-long Parafilm strip was cut and placed on one sticky side of the NEM tape.
4. The arch punch was used to cut 18-mm-diameter stickers from the NEM and Parafilm strip.
5. The NEM tape cover from the underside of the NEM and Parafilm sticker was removed, and the
sticker was applied to a clean SEM stub.
6. Using a pair of tweezers, the Parafilm from the top of the NEM and Parafilm sticker was removed,
and a glass disc was attached to the stub.
7. The glass disc was mounted on the SEM stub.
Figure 3-5 shows the final glass coupon.
Figure 3-5. Glass Coupon
9
-------
3.2 Sterilization of Materials
The coupons, funnels, and stages for storing coupons and plastic spray bottles were sterilized using an
Andersen ethylene oxide (EtO) sterilizer system (EOGas®, Part No. 333, ANPRO, Haw River, NC), and a
sterilization kit (Kit #6, Part No. AN1006, ANPRO) that includes a cartridge, a humidichip®, a dosimeter,
and a bag. The sterilization procedure is summarized below.
1. All the items to be sterilized were packed in appropriate 12" x 15" EtO bags (Part No. AN2350)
(Figure 3-6A), and the bags were sealed properly.
2. Sealed EtO envelopes were placed in appropriate sterilization bags (Figure 3-6B) along with a
dosimeter, humidichip®, and EtO dispenser.
3. The sterilization bags were vacuum-sealed and loaded into the EtO sterilizer for an 18-hour
sterilization cycle.
Figure 3-6. Sterilization Envelope (A) and Bag (B)
3.3 Test Organism and Coupon Inoculation
The test organism and coupon inoculation are discussed below.
3.3.1 Bacillus atrophaeus var. globigii (Bg)
Bg, a surrogate for the spore-forming bacterial agent Bacillus anthracis, was used for this project. Like
Bacillus anthracis, Bg is a soil-dwelling, Gram-positive, aerobic microorganism, but unlike Bacillus
anthracis, Bg is non-pathogenic. Bg forms an orange pigment when grown on nutrient agar, a desirable
characteristic for detecting viable spores in complex environmental samples. Bg has a long history of use
in the biodefense community as a simulant for anthrax (Gibbons et al. 2011).
3.3.2 Coupon Inoculation
Each coupon was inoculated with approximately 2 x 107 spores of Bg (American Type Culture Collection
(ATCC) 9372, cultured, processed, and lyophilized at Dugway Proving Ground, Dugway, UT) using a
metered dose inhaler (MDI, canister Part No. BK0339783, Bespak, Hertfordshire, England). The MDI
10
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canister contained Bg spores suspended in ethanol solution and HFA-134A propellant (1,1,1,2-
tetrafluoroethane). The MDI canister was situated inside an actuator (Figure 3-7) so that each time the
actuator was depressed; a repeatable amount of spores was deposited on the coupon (Lee et al. 2011V
The MDI actuator is a small plastic tube into which the MDI canister is inserted.
Figure 3-7. MDI Canister (A) and Actuator (B)
Each MDI was charged with a volume of spore preparation plus propellant 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.
Each coupon was inoculated independently using the MDI canister and actuator. Figure 3-8 shows the
inoculation process.
Figure 3-8. Inoculation Process
The spores were allowed to settle on coupon surfaces for a minimum of 18 hours. During inoculation, each
MDI was weighed before and after inoculation to ensure proper discharge. For quality control of the MDIs,
an inoculation control coupon was included as the first, middle, and last coupon inoculated using a single
MDI in a single test.
11
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3.4 Concrete Material Evaluation
Concrete is a porous and highly alkaline material. This section discusses tests conducted to evaluate: (1)
the effect of concrete on the pH of decontamination solutions, and (2) the capacity of concrete to retain
liquid to determine the number and duration of sprays to be used for decontamination solution efficacy
testing.
3.4.1 Effect of Concrete on pH of Decontamination Solutions
Published studies have shown that the pH of an acidified bleach solution is an important factor affecting the
sporicidal activity of the solution (Wood et al. 20113). Decontamination efficacy is reduced as the pH
increases from neutral (pH 7). Therefore, this project included testing to determine if concrete, which has a
very alkaline pH, has any effect on the pH of the decontamination solutions. The test was performed as
summarized below.
1. The pH of each of five concrete coupons was measured using pH strips (Whatman pH indicator
paper, Catalog No. 2613991, GE, England) following ASTM International Method (ASTM F710-08.
2008). In accordance with this method, several drops of Dl water were placed on a clean concrete
surface to form a puddle approximately 1 in. (25 mm) in diameter. The puddle was allowed to set
for 60 ± 5 seconds, and then the pH paper was dipped into the Dl water. The paper was removed
immediately, and its color was compared to the pH chart to determine the pH reading.
2. Five specimen cups were filled with 25 mL of decontamination solution S1. The initial pH, reduction-
oxidation (redox) potential (in millivolts [mV]), and free available chlorine (FAC) of the solutions
were recorded.
3. One concrete coupon was transferred to each specimen cup, and the time was recorded.
4. Immediately after coupon transfer, each cup was tightly capped and gently shaken to allow
sufficient interaction between the decontamination solution and the concrete coupon surface.
5. After 10-minute, 30-minute, and 60-minute exposure times, the pH and redox potential (mV) of the
decontamination solution in each specimen cup were measured and recorded. FAC was measured
and recorded after 60 minutes only.
6. Additional decontamination solution (20.4 mL) was added to each sample after the 60-minute
exposure time in an attempt to reach the initial target pH value (±5%) of the prescribed solution.
7. The pH and redox potential were measured at 60-, 90-, and 120-minute time intervals after addition
of the additional decontamination solution. The FAC was measured at 60 and 120 minutes only.
Results for these tests are presented in Table 3-2. The concrete coupon had a time-dependent effect on
the pH of the solution. After 60 minutes of coupon exposure, the average pH increased from 4.73 to 6.02.
The addition of 20.4 mL of fresh decontamination solution, afterthe initial 60-minute exposure time, did little
to alter this effect, as the average pH was 5.85 after 180 minutes. The average FAC decreased slightly
after 60 minutes but showed an overall increase on the final reading. This is not the case for redox potential,
which remained relatively steady overtime and was unaffected by changes in the solution volume. These
results further suggest that the presence of the solution on the concrete coupon has the potential to move
the surface pH to a more neutral range and possibly adversely affect the efficacy of the solutions.
12
-------
Table 3-2. Effect of Concrete Material on the pH of Decontamination Solution S1
Parameter Variance
Parameters
Contact
Time (min)
0
0
10
30
60
120
(60 +60)
150
(90 + 60)
180
(120 + 60)
Redox (mV)
Average
NA
1320
1304
1286
1275
1290
1280
1273
Stdev
2.91
3.8
1.81
2.6
6.1
7.8
11.7
FAC (ppm)
Average
NA
2604
NA
NA
2404
2504
NA
3305
Stdev
419
419
354
274
PH
Average
10
4.73
5.5
5.64
6.02
5.67
5.78
5.85
Stdev
0
0.11
0.08
0.06
0.04
0.14
0.17
0.11
Temperature
(°C)
Average
NA
19
19
18
20
18
17
18
Stdev
0.1
0.18
0.24
0.2
0.3
0.2
0.2
Solution
volume (ml.)
Average
0
25
25
25
25
45.4
45.4
45.4
Stdev: Standard deviation; NA: Not available.
3.4.2 Concrete Liquid Retention Capacity
The porous nature of concrete has been shown to negatively impact the sporicidal efficacy of
decontamination solutions (Calfee, et al. 2011). Therefore, tests were conducted to determine the duration
and number of spray applications required to keep the concrete wet for at least 10 minutes. Five replicate
concrete coupons were used for each test. The liquid retention capacity test was conducted forthe following
spray parameters:
• 2-second spray at 30 pounds per square inch (psi) with single application
• 2.5-second spray at 30 psi with double application
• 5-second spray at 30 psi with single application
• 5-second spray at 30 psi with double application
Figure 3-9 shows the test results forthe 2.5-second double spray and the 5-second double spray after 10
minutes.
13
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Figure 3-9. Concrete Coupons After Double-Spray Tests
Visual observations of the coupons showed that the coupons were wet and retained the liquid over the
10-minute exposure test duration time.
3.5 Decontamination Materials and Equipment
Table 3-3 summarizes the decontamination materials and equipment used during testing.
14
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Table 3-3. Decontamination Materials and Equipment
Material or . ..
. Description
Equipment r
Environmental Test
Chamber
Model 1016H environmental test chamber, TestEquity (Moorpark, CA)
Automated sprayer
Assembled from the following parts:
• Nozzles: Full-cone spray nozzle, 303 stainless steel, 1/8 in. NPT connection, 0.5
gallons per minute (gpm) at 40 psi, McMaster-Carr No. 32885K52, Douglasville, GA
• Solenoid valves: Premium compact actuated on/off valve, 24-volt, 1/4 in. national pipe
thread (NPT) female connection, 150 maximum psi, 24-volt DC, McMaster-Carr No.
4639K783, Douglasville, GA
• Inlet for solenoid valves: Stainless steel Swagelok tube fitting, %-in. NPT male
connection tube outside diameter, Swagelok No. SS-400-1-4, Wake Forest, NC
• Pressure gauge and air regulator: Compact compressed air regulator, McMaster-Carr
No. 6763K81, Douglasville, GA
• Power supply: Alternating Current (AC) to DC, power supply, 24-volt, 5-ampere, 120
watts, Jameco Part No. 212266, Belmont, CA
• Timers: Off delay single time delay relay, 0.1 to 1.2 seconds, 1 to 12 seconds, SPDT,
24 to 48 volts AC/DC, Think Allied, Stock No. 70382111, Fort Worth, TX
Bleach
Clorox® Concentrated Germicidal Bleach, 8.3% NaCIO; <1% sodium hydroxide, Lowes, Cary, NC
Acetic acid
Acetic acid, glacial, grade Food Chemicals Codex (FCC)/US Pharmacopeia Convention (USP),
Chemical Abstract Service (CAS) No. 64-19-7, Pittsburgh, PA
Calcium chloride
Calcium chloride dehydrate (American Chemical Society [ACS] certified), FCC, CAS No. 10035-
04-8, Pittsburgh, PA
Sodium chloride
Sodium chloride (crystalline; ACS certified), FCC, CAS No. 7647-14-5, Pittsburgh, PA
Centrifuge tubes
The following different-capacity tubes were used:
• For coupon extraction: Falcon™ 50-mL conical centrifuge tubes, Catalog No. 14-959-
49A, Corning, NY
• For rinsate extraction: Corning™ 250-mL polypropylene sterile centrifuge tubes,
Corning, NY
The following sections discuss the ETC, NFB decontamination solutions, and application of
decontamination solutions.
3.5.1 Environmental Test Chamber
The ETC for this project was capable of achieving and maintaining the desired test temperatures ranging
from subfreezing to room temperature. The tests were conducted in a TestEquity (Moorpark, CA) Model
1016H ETC as shown in Figure 3-10. The working volume for this chamber is listed as 16 cubic feet (ft3)
(442 liters), and its internal dimensions are 30 in. (76 centimeters [cm]) high (h) by 30 in. (76 cm) wide (w)
by 30 in. (76 cm) deep(d). Chamber temperatures can be set as low as -73 °F (-58 °C) to as high as 175 °F
(79 °C), and the humidity can be programmed from 10% to 95% relative humidity (RH).
15
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Figure 3-10. TestEquity ETC
The chamber is equipped with a F4T touchscreen controller (Figure 3-11) that was used to regulate the
inner temperature of the chamber and achieve the desired test conditions.
1
A '•« «•«> I dl «
WOO RMtnri)
O^OOCO ^oSTaerature *u»o •
24.9 C
85.0 C
85.0 C
Humidity W» ;
82.5 %
85,0 % ^
85.0%
^^^¦1 *0%
PWftr „
PW«
¦ 0%
O »•-«' «ij© »-«• 0 ¦««*] u£l
A s
tD
High resolution touch screen display shows
profile running on a humidity chamber.
Figure 3-11. Touchscreen Controller
16
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3.5.2 NFB Decontamination Solutions
Eight decontamination solutions, S1 through S8, were freshly prepared on each test day. These bleach-
based solutions were prepared from recipes provided by the ECCC group Blinov et al. (2015'). De-icing
agents were used to depress the freezing point of the solutions to below the target test temperatures so
that the solutions did not freeze. In addition to the NFB solutions, a pAB solution was also tested to 0 °C
as a reference decontamination agent.
The decontamination solutions S1 through S8 were prepared in 200-mL volumes using the reagents
indicated in Table 3-4. Acetic acid (CH3COOH) concentrations were varied in each solution to provide a
desired redox potential. Calcium chloride (CaCh) was added to S1 through S7, and sodium chloride
(NaCI) was added to S8 to depress the freezing point to below the target test temperatures.
Table 3-4. Decontamination Solution Formulations
Decontamination Solution Composition
Formulation
ID
CaCI2-2H20
Clorox®
(NaOCI 8.3%)
Acetic Acid
(CH3COOH 99.9%)
Target Redox
Potential (Eh)
PH
g/L
mL/L
mL/L
mV
S1
409.3
60.27
4.01
1320
4.34
S2
409.3
60.27
3.15
1300
5.52
S3
409.3
60.27
0.23
1150
7.4
S4
409.3
60.27
0.02
1050
8.35
S5
409.3
120
6
1300
5.7
S6
409.3
180
6.5
1295
5.95
S7
409.3
240
7.5
1290
5.89
S8
780 g/L of NaCI
180
13.5
Not provided by ECCC
To prepare 100 mL of decontamination solution, the following approach was used:
1. Dissolve 30.9 g of dry CaCh {or 40.9 g of dihydrate salt (CaCl2*2H20)} in ~70 mL of water in
a 100-mL volumetric flask and agitate manually (until solution is warmed up).
2. Cool down to room temperature and add the required amount of acetic acid and agitate
manually.
3. Add the required volume of bleach (to get a concentration of 0.5 % NaOCI) in the volumetric
flask and agitate manually.
4. Fill with Dl-water to make the final volume 100 mL
5. Gently agitate by hand.
Each solution underwent initial evaluation for temporal stability of pH, redox potential, and FAC. The
following sections discuss this initial evaluation as well as short-term and long-term stability testing and the
pAB reference solution.
17
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3.5.2.1 Initial Evaluation of pH, Redox Potential, and FAC (S1 through S8)
Once the decontamination solutions were prepared, pH, redox potential, and FAC were measured and
recorded in triplicate. Results for pH and redox potential (mV) were compared to data provided by ECCC
to ensure that on-site solution preparations were consistent with the ECCC preparations. Table 3-5 and
Figure 3-12 summarize the data comparison results.
Table 3-5. Decontamination Solution Comparisons
Formulation
ID
PH
Redox Potential (mV)
FAC (ppm)
EPA
ECCC
EPA
ECCC
EPA
Avg
Stdev
Avg
Avg
Stdev
Avg
Avg
Stdev
S1
4.51
0.06
4.34
1329
4
1320
2945
122
S2
5.14
0.06
5.52
1308
3
1300
2941
245
S3
7.63
0.08
7.40
1119
7
1150
3450
87
S4
8.11
0.26
8.35
1074
14
1050
3510
71
Readings
S5
5.66
5.70
1290
1300
7952
S6*
5.71
5.95
1242
1295
10396
S7
6.01
5.89
1279
1290
13922
S8
4.24
ND
1308
Not Provided
by ECCC
3085
Decontamination Solutions
Figure 3-12. Decontamination Solution Test Parameter Comparisons
18
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3.5.2.2 Short-Term Stability Testing of pH, Redox Potential, and FAC (S1 through S4)
Short-term stability tests were performed, at ambient temperatures, on solutions S1 through S4 to
determine the stability of pH, redox potential and FAC over a three-hour period. The solutions were
prepared as specified in Table 3-4 Section 3.5.2. Table 3-6 and Figure 3-13 summarize the short-term
stability test results.
Table 3-6. Short-Term Stability Test Results (S1 through S4)
Solution ID
Sampling Time (hours)
0
1
2
3
0
1
2
3
0
1
2
3
PH
Redox Potential (mV)
FAC
ppm)
S1
4.47
4.48
4.48
4.57
1,333
1,335
1,327
1,325
2,824
3,185
3,085
2,925
S2
5.19
5.20
5.09
5.16
1,310
1,307
1,310
1,305
3,245
2,985
2,925
2,611
S3
7.52
7.59
7.56
7.70
1,113
1,113
1,120
1,128
3,539
3,432
3,432
3,338
S4
8.40
8.27
8.00
7.75
1,056
1,076
1,073
1,092
3,566
3,545
3,492
3,439
Notes:
ID = Identification
ppm = Parts per million
mV = millivolt
Redox Potential
I I FAC
_ pH-
—
<$*¦ <$*¦ <$*¦
S3 Solution
—
4*
S1 Solution
S4 Solution
19
-------
Figure 3-13. Short-Term Stability Test Results (S1 through S4)
3.5.2.3 Long-Term Stability Testing of pH, Redox Potential, and FAC (S1)
A long-term stability test was performed on solution S1 to determine the stability of pH, redox potential, and
FAC over a 24-hour period. Solution S1 was prepared as specified in Table 3-4 Section 3.5.2. Table 3-7
summarizes the long-term stability test results for each parameter, along with a percent variance calculation
performed as follows:
(Initial value - final value)/(initial value)
Table 3-7. Long-Term Stability Test Results (S1)
Parameter
Value
% Variance
Hold time (hours)
0
5.4
23.4
Not applicable
pH
4.4
-
4.6
5.4
Redox potential (mV)
1,323
-
1,281
-3.1
FAC (ppm)
2,904
1,923
2,063
-33.0
Note:
ppm = Parts per million
A 5.4% increase in pH was observed, while redox potential and FAC decreased 3.1 and 33.0%,
respectively. These results, illustrated in Figure 3-14, indicate that a key solution parameter, FAC, did not
remain steady over 24 hours, suggesting that the solutions should be used on the day of preparation to
avoid potential loss of sporicidal efficacy.
3000-
2500-
§ ^DO-
'S
CL
X
o
T3
0C.
1500-
T3
« 1000-
<:
500-
Redox Potential (mV)
FAC (ppm)
PH
I
Q.
5 10 15
Hold Time (Hours)
20
25
Figure 3-14. Long-Term Stability Test Results (for decontamination solution S1).
20
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3.5.3 pAB Reference Solution
The pAB reference solution contained the following:
• One-part bleach (with a 5.25 to 6% NaCIO concentration)
• One-part white vinegar
• Eight parts water.
Bleach and vinegar were not combined together directly. Water was first added to the bleach (two cups water
to one cup of bleach), then vinegar (one cup), and then the remaining water (six cups). The target FAC
concentration forthe pAB solution was between 5,000 to 6,000 ppm FAC, with a pH close to but not exceeding
7.
3.5.4 Application of Decontamination Solutions
A custom automated spraying system was designed and constructed for metered and precise applications
of the decontamination solutions onto the concrete and glass coupon test surfaces. The following sections
discuss the spray system specifications, operation, functionality test, and spray pattern test.
3.5.4.1 Spray System Specifications
The spray system was designed to fit inside the ETC, operate at low temperatures, and sustain repeated
exposure to bleach-based solutions. Figure 3-15 shows a schematic diagram of the spray system.
Figure 3-15. Spray System Schematic
21
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The automated spray system consisted of a pressurized decontamination liquid tank, a custom-made
spray nozzle stand, a coupon assembly stand, and connecting Kynar tubing (Part No. 5390K31,
McMaster Carr, Douglasville, GA). Figure 3-16 is a photograph of the spray system inside the ETC.
Kynar tubing
Coupon holding
funnel
Rinsate collection
vials
liquid tank
Coupon holding
stand
Figure 3-16. Spray System inside ETC
Major components and features of the spray system are detailed below.
Pressurized decontamination liquid tank: Figure 3-17 shows the decontamination liquid tank (Part No.
8624K58, McMaster Carr, Douglasville, GA), which was made of high-density polyethylene (HDPE) with a
stainless steel jacket. The tank lid included two tubes, an air inlet and a liquid outlet that connected the tank
to the rest of the system. A gasket between the tank and lid allowed an air-tight seal. A pressure regulator
was used to maintain the target pressure inside the tank and inside the liquid lines that transported the
solution to the sprayer apparatus
Stainless
steel
jacket with
gasket
HDPE liquid tank
Figure 3-17. Decontamination Liquid Tank
22
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Spray nozzles: The spray system had five stainless-steel, full-cone spray nozzles (McMaster-Carr, Catalog
No. 32885K52, Douglasviile, GA) rated at 0.5 gallons per minute (gpm) at 40 psi to achieve five replicate
applications at once. The spray nozzles were attached to normally closed stainless-steel solenoid valves
(McMaster-Carr, Catalog No. 4639K783, %-in. NPT female connection, 150 psi, 24-volt direct current [DC]).
Figure 3-18 shows the spray system assembly, which rested on a chlorinated polyvinyl chloride (CPVC)
stand that allowed adjustment of the height and angle of the spray nozzles as required.
Figure 3-18. Spray Nozzle Setup on CPVC Stand
Nozzle alignment: Two spray nozzle alignments were used during Phases I and II. During Phase I, the
spray optimization test, nozzles were placed at a 30° angle relative to the top of the coupons as shown in
Figure 3-19A. Solution S1 was evaluated using this design. During Phase II, the decontamination efficacy
test, the nozzles were aligned directly above the coupons so that the spray was delivered perpendicularly
(from above) to the coupon as shown in Figure 3-19B. This design was used to test solutions S5 through
S8.
Figure 3-19. Spray Nozzle Orientation during Phase I (A) and Phase II (B) Tests
23
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Spray system control box: The liquid spray system was equipped with a control box (Figure 3-20) that
regulated the pressure inside the decontamination liquid tank and the nozzle spray time. The unit was
equipped with a relief valve (Part No, 4639K783, McMaster Carr. Douglasville, GA), a pressure regulator
(part No. 6763K81, McMaster Carr, Douglasville, GA), a power supply (Part No. 212266, Jameco
Electronics, Belmont, CA), a push button timer controller (Part No. 26623, Jameco Electronics, Belmont,
CA, High-Polish 6" OD, 5.78" ID Sanitary Stainless Steel Tubing, Type 304L (Part No.4466K841,
McMaster Carr, Douglasville, GA), and toggle valves for the air lines (pressurization and relief).
Timer
Power supply
Pressure regulator
Figure 3-20. Top View of Control Box
3.5.4.2 Spray System Operation
The decontamination liquid tank was rinsed with Dl water and then filled with the target decontamination
solution. The spray system then was pressurized to the prescribed test condition. The pressure and spray
duration were adjusted using the pressure regulator and the timer, respectively. Activation of the time
controller switch allowed the test solution to flow from the decontamination liquid tank through the Kynar
tubing into the five solenoid valves connected to each of the five nozzles.
A thermocouple probe was used in the Kynar tubing liquid distribution system (Figure 3-21) to track the
temperature of the liquid reaching the nozzles. The spray action was performed only when the temperature
of the decontamination solution was within ± 0.5 °C of the target test temperature.
Time controller
button
24
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Thermocouple
probe
Figure 3-21. Kynar Tubing Distribution System
3.5.4.3 Spray System Functionality Test
The spray system functionality test was conducted to assess nozzle flow rate consistency and to estimate
the volume of liquid rinsate. A beaker was placed in front of each spray nozzle. The liquid tank was filled
with Dl water, and the spray system was primed with Dl water. The spraying action was performed at the
four combinations of time and pressure selected for sample testing as follows:
• One 2-second spray at 30 psi
• One 2-second spray at 60 psi
• One 5-second spray at 30 psi
• One 5-second spray at 60 psi
Table 3-8 summarizes the results.
Table 3-8. Volume Collected from Each Spray Nozzle in the Spray System
Nozzle ID
Volume Collected for Each Spray (mL)
2 seconds, 30 psi
2 seconds, 60 psi
5 seconds, 30 psi
5 seconds, 60 psi
1
21.0
36.0
36.3
53.0
2
21.5
36.0
36.8
52.5
3
21.5
37.0
38.0
54.5
4
23.5
38.0
40.0
57.0
5
23.0
37.5
39.8
56.0
Average volume collected
22.1
36.9
38.1
54.6
Standard Deviation
1,1
0.9
1.7
1.9
These results indicate that volumes collected from each of the five nozzles, for each spray condition, were
not significantly different. They also indicate a maximum precision of 5%, significantly less than the 10%
precision data quality indicator (DQI) for sprayer flow rate precision DQIs listed in Table 6-1. Also based
on these results, the minimum rinsate volume was estimated to be approximately 45 to 50 mL (total
including decontamination solution, neutralizer, and sterile Dl water used for rinsing the funnels) when the
25
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spray was administered as a single 2-second spray at 30 psi. The maximum rinsate volume would be
approximately 114 ml_ (total including decontamination solution, neutraiizer, and sterile Dl water) when the
spray was administered as a single 5-second spray at 60 psi.
The spray system functionality test was repeated to verify flow rates whenever any part of the system (such
as spray nozzles, solenoid valves, and Kynar tubing) was replaced. Also, a flow rate check of the spray
nozzles was conducted before each test using Dl water. Liquid was collected in five separate beakers after
five seconds of spraying at 30 psi and measured using a 100-mL graduated cylinder.
3.5.4.4 Spray Pattern Test
The goal of the spray pattern test was to qualitatively assess any differences in spray patterns of the five
spray nozzles in the spray system. Five pieces of construction paper were folded and attached to the
inner side of an acrylic orifice plate so that the paper replaced the coupons used during a normal spraying
operation. The spraying was conducted for five seconds at 30 psi. No significant differences in spray
pattern size or shape were noted across the five nozzles. Figure 3-22 shows the consistent spray pattern
for all five spray nozzles.
Figure 3-22. Spray Pattern Test Results
3.6 Neutralizing Agents
An adequate neutralizing agent is very important for properly assessing decontamination solution
efficacy (Calfee. et. al. 2011). If the neutraiizer does not fully quench the decontaminant, decontamination
effects may continue past the designated contact time and survivability assay results may overestimate test
solution effectiveness.
The neutralizing agents tested were STS and DE broth. The neutralizing agents were applied to stop the
decontamination activity after a prescribed exposure time. After the prescribed exposure times, coupons
were collected and deposited into a tube containing the neutralizing agent. The neutralizing agents were
26
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amended with a de-icing agent for low-temperature testing. Tests were conducted to ensure that an
effective neutralizing agent was used and to ensure that the addition of a de-icing agent did not affect
neutralizer performance. The following sections discuss neutralization agent preparation and effectiveness
testing.
3.6.1 Neutralization Agent Preparation
A 2 normal (N) solution of STS was prepared as described below.
1. STS pentahydrate (Na2S2C>3 5H2O, 496.4 grams) crystals were added to 1 liter of Dl water.
2. The solution was stirred until all the crystals dissolved completely.
3. The 2N STS solution was sterilized using a bottle-top filter (150-mL Corning Bottle Top Filter, 0.22-
micrometer [)jm] cellulose acetate, 33-mm neck, sterile, Catalog No. EK-680516, Corning, NY) and
a vacuum filtration system.
Each batch of STS was used within six months of preparation.
DE broth was prepared according to manufacturer instructions as summarized below.
1. Dehydrated DE broth media granules (39 grams) were added to 1,000 mL of Dl water in an
Erlenmeyer flask.
2. The flask was placed on a stir plate and gently heated to completely dissolve the granules.
3. The solution was autoclaved at 121 °C for 15 minutes.
Each batch of DE broth was stored below 8 °C, protected from direct light, and used within six months of
preparation.
3.6.2 Neutralization Effectiveness Testing
During Phase I spray optimization testing, STS was found to be ineffective in neutralizing the
decontamination reaction. Results for rinsate samples both with and without STS neutralizer were non-
detects, demonstrating that residual decontamination was occurring during sample storage.
A more effective neutralizing agent was investigated. Previous work with other Bacillus sp. showed that the
use of DE broth was an effective neutralizer for various antimicrobial agents (Dev and Enalev 1994). An
effectiveness test performed with solution S1 showed no residual decontamination effects when DE broth
was used as the neutralizing agent. Therefore, DE broth was used as the neutralizing agent throughout this
project.
During low-temperature testing, DE broth was observed to freeze prior to the collection of runoff. A test was
performed to evaluate neutralizing efficacy when CaCh was added to the 3.9% DE broth. For this
evaluation, solutions S5, S6, and S7 were prepared as detailed in Table 3-4 (Section 3.5.2). The neutralizer
effectiveness test was conducted as summarized below.
27
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1. 3.9% DE + 3.7 molar (M) CaCh: Five 50-mL conical tubes were prepared for each solution (S5,
S6, and S7), and 5 mL of 3.9% DE broth containing CaCh and 5 mL of decontamination solution
were added to each tube.
2. 3.9% DE: Five 50-mL conical tubes were prepared for each solution (S5, S6, and S7), and 5 mL of
3.9% DE broth plus 5 mL of decontamination solution were added to each tube.
3. Negative controls: DE broth (5 mL) was added to each of two conical tubes.
The coupon samples, except for the negatives, were inoculated with 6 * 106 CFU of Bg spores. After
inoculation, the samples were extracted and enumerated. Table 3-9 summarizes the results
Table 3-9. DE Broth (± CaCh) Neutralization Effectiveness
Solution ID
3.9% DE
+¦ CaCl2
3.9% DE Broth
Recovery (CFU)
Recovery(%)
Recovery (CFU)
Recovery (%)
Positive Control
6.0 x 10®
S5
5.80 x 10®
96.7
5.26 x 106
87.6
S6
5.79 x 10®
96.5
5.34 x 10®
89.0
S7
5.75 x 10®
95.8
5.23 x 10®
87.2
These results indicate that the addition of the CaCh to the DE broth (positive control for each solution) had
no impact on the neutralizing efficacy of the DE broth. The negative control results for the two solutions
were non-detects.
28
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4 Sampling and Analytical Procedures
The primary results from this study will be from the analysis of samples resulting in recovered CFUs per
sample expressed on a log-10 scale. Additional measurements prior to or during the decontamination
procedure application are also required in order to ensure quality control in the testing. These
measurements include quality control checks on the reagents and equipment being used in the
decontamination procedure.
A sampling data log sheet was maintained for each sampling event (or test) that included each sampling
event, the date, test name, sample IDs, and other test details such as test temperature, final rinsate volume,
and sample extraction time. The sample IDs were pre-printed on the sampling data log sheet before
sampling began. Digital photographs were taken to document activities throughout the test cycle.
4.1 Microbiological Analysis
This section discusses the project sampling and analytical procedures, including sample quantities, sample
types, and coupon sample extraction and analysis.
4.1.1 Sample Quantities
For each decontamination solution, there were five replicates of coupon samples, five liquid rinsate
samples, three positive control samples, one procedural blank and one negative control sample per material
and temperature. Table 4-1 lists the total numbers of samples of each type for each test.
Table 4-1. Sample Types and Numbers for Each Decontamination Solution
Sample Type
No. per Material and Temperature
Test coupon sample (decontaminated)
5
Liquid rinsate sample
5
Positive control sample
3
Procedural blank
1
Negative control sample
1
4.1.2 Sample Types
The three major sample types for this project are discussed below.
• Surface Test coupon samples: Each coupon sample was aseptically transferred, using sterile
forceps, from the stage in the ETC to a 50-mL conical tube containing 10 mL of phosphate buffered
saline with 0.05% Tween® 20 (PBST) and 1.5 mL of DE broth with or without CaCh, depending on
the temperature at which the test was conducted.
• Liquid rinsate samples: These samples were collected in 250-mL conical tubes to assess the
potential for viable microorganisms that were washed off the coupon surfaces. Samples were
collected from all liquid runoff during spray applications, and the collection funnels were
subsequently rinsed with sterile Dl water. Rinsate samples were collected in the same vials as
runoff and together constituted a single sample. Liquid runoff from each coupon also was collected
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through sterile funnels in sample tubes that contained pre-determined volumes of neutralizer. For
low-temperature testing, the tubes also contained a de-icing agent.
After collection, test coupon and liquid rinsate samples were sealed in secondary containment and
transported to the NHSRC Research Triangle Park (RTP) Microbiology Laboratory (BioLab) for quantitative
analysis.
4.1.3 Sample Extraction and Analysis
Both coupon (surface) and liquid rinsate samples were extracted by first sonicating each sample for 10
minutes. The coupons then were vortexed continuously for two minutes, and the liquid rinsate samples
were vigorously shaken for two minutes, each followed with an additional five minutes of sonication. After
this procedure, each sample was aliquoted and 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 iines
where the sample was deposited (Figure 4-1).
Figure 4-1. Bacterial Colonies on Spiral-Plated Agar Plate
The colonies on each plate were enumerated using a QCount® colony counter (Advanced Instruments Inc.,
Norwood, MA).
Positive control samples were diluted 100-fold (10 2) in PBST before spiral plating, and samples of unknown
concentration were plated undiluted and after a 100-fold dilution. Samples with known low concentrations
were plated undiluted. The QCount® colony counter automatically calculates the CFU/rnL 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 spreadsheet.
Only plates meeting the threshold of at least 30 CFU were used for spore recovery estimates. After
quantitation using the QCount® colony counter, glass coupon and rinsate samples with plate results below
the 30-CFU threshold were either spiral plated again with a more concentrated sample aliquot or filter-
30
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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. Bg Colonies on Filter Plate
Due to difficulties with filter plating the concrete coupon samples, concrete samples with plate results below
the 30-CFU threshold were either spiral plated again with a more concentrated sample aliquot or spread
plated in triplicate on TSA plates using 1-mL aliquots per plate to achieve a lower detection limit. The plates
were incubated at 35 ± 2 °C for 20 to 24 hours before manual enumeration. Figure 4-3 shows a spread
plate with colonies of Bg.
Figure 4-3. Bg Colonies on Spread Plate
31
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4.2 Decontamination Solution Characterization
Decontamination solution samples were collected from freshly prepared formulations before each test and
were evaluated for critical parameters (pH, redox potential, and FAC). This section discusses the
measurements of these critical parameters.
4.2.1 Free A vail able Chlorine (FA C)
The FAC was determined using an iodometric method for the determination of chlorine dioxide and
chlorite using a HACH® Hypochlorite Test Kit (Model CN-HRDT, Fisher Scientific, Waltham, MA). The kit
includes:
• HACH digital titrator
• Magnetic stir bar
• HACH starch indicator solution (cat. No. 349-32)
• Packet of potassium iodide (Kl) powder pillow
• Acid reagent packet
The FAC determination procedures consists of the following steps:
Sample Preparation
1. In a 250 mL beaker, add 5 mL of the sample and 150 ml of Dl water
2. Add the Kl pillow packet
3. Add 1 packet of acid reagent
4. Add a stir bar and place beaker on a stir plate
FAC Measurement
1. Reset the digital titrator counter to zero
2. Titrate with 2.26 N STS until the solution is pale yellow, then add 1 dropper of starch indicator
and continue titration until the solution becomes colorless. Record the number of digits required (B).
3. Calculate the volume of titrant delivered (VB): VB (mL) = B/ 800
FAC Calculation
In the following equations, 5 represents the sample size in mL, and 2.26 represents the normality of the
STS. The other constants are the equivalent weights (mg/eq) per electron, and Va and Vb are as defined
previously.
Bleach (ppm FAC) = VB * 2.26 *35453 / 5
32
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4.2.2 pH
The pH will be measured using an Oakton/Eutech PC 510 pH/conductivity meter (Vernon Hills, IL),
following the manufacturer's recommendations found
at http://www.4oakton.com/Manuals/pHORPIon/pH CON510mnlr3.pdf
4.2.3 Redox Potential
Steady oxidation-reduction (redox) values (mV) values and corresponding temperatures was recorded by
using Oxidizing Redox Potential (ORP) probe (Model No. 35805-13, Waltham, MA) along with the Oakton
pH 150 meter (Model No. 35614-30, Waltham, MA). Orion ORP standard solution (Cat. No. 13-641-210,
Waltham, MA) was used to calibrate the ORP probe and to recalculate the redox values of the
decontamination solution to hydrogen scale. The redox potential based on a hydrogen scale is calculated
as follows:
Redox value of the decontamination solution + Correction factor
The correction factors are calculated as follows:
Orion std. solution estimated reading (mV) for a given temperature* - Orion std. solution actual reading
(mV) for that same temperature)
The estimated Orion standard solution estimated readings were obtained from the following source:
http://fscimage.fishersci.com/cmsassets/downloads/segment/Scientific/pdf/WaterAnalysis/Log420RPStan
dardBenefits.pdf
Table 4.2. ORP (mV vs. Temperature) Orion Standard Solution Estimated Readings
Temp °C
mV
15
428
16
427.2
17
426.4
18
425.6
19
424.8
20
424
21
423.2
22
422.4
23
421.6
24
420.8
25
420
33
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5 Results and Discussion
This section summarizes the results for Phase I, the spray optimization tests, and Phase II, the
decontamination efficacy tests, for both the pAB and NFB solutions, followed by a results summary.
5.1 Phase I Spray Optimization Tests
The spray optimization tests were conducted using pAB and the S1 NFB formulation as discussed below.
5.1.1 pAB Solution
The pAB solution test was designed to optimize test parameters with regard to the number of sprays and
spray duration using pAB, a solution known to have high decontamination efficacy (Calfee. et al. 2012).
The objective of this test was to determine the number of sprays and the spray duration required to achieve
a 6 LR on both glass and concrete at 25 °C. These spray conditions then could be used for NFB formulations
to compare efficacies. The pAB solution was prepared as detailed in Section 3.5.3. Four spray schemes
were tested as shown in Table 5-1.
Table 5-1. Parameters for pAB Solution Spray Optimization Tests
Parameter
Test Details
Spray duration (seconds)
2
2.5
5
5
Number of sprays
1
2
1
2
Total contact time (min)
1C
I
Spray intensity (psi)
30
Test temperature (°C)
25
pH (acceptable range 6.5 to 7.0)
6.78
6.9
6.77
FAC (ppm)
6,030
6,370
6,049
The test was initiated by spraying concrete and glass procedural blank coupons, followed by the test
coupons (five replicates for each material). For coupons receiving two sprays, there was a five-minute
delay between the first and second sprays. After a 10-minute total contact time, coupons were immersed
in the neutralizing agent to quench the decontamination reaction. Viable spores were extracted from
coupon surfaces and enumerated.
Table A-1 and Table A-2 in Appendix A present the detailed test results. Figure 5-1 summarizes the test
results, which indicate that two sprays of five seconds each provided the highest overall surface
decontamination efficacy. Under these spray conditions, non-detects (> 6 LR) were observed for both test
materials.
34
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pAB Spray Optimization
Glass Concrete
1x2 1x5 2x2.5 2x5
Number of Sprays x Spray Duration (seconds)
*Denotes Full Surface Decontamination Based on Detection Limit
Figure 5-1. Spray Optimization Test Results for pAB
5.1.2 pAB and S1 Solutions
Based on results of the spray optimization test using pAB discussed above, a comparison test was
performed with pAB and NFB formulation S1 (prepared as detailed in Section 3.5.2). Table 5-2 lists the test
conditions.
Table 5-2. Parameters for pAB and S1 Efficacy Tests
Parameters Test Details
Solution
pAB
S1
pAB
S1
Spray duration (sec)
5
5
5
5
Number of sprays
1
1
2
2
Total contact time (min)
10
Spray intensity (psi)
30
Test temperature (°C)
25
The test was initiated by spraying concrete and glass procedural blanks, followed by the test coupons (five
replicates for each material). For coupons receiving two sprays, there was a five-minute delay between the
first and second sprays. After a 10-minute total exposure time, the coupons were immersed in the
neutralizing agent to stop the decontamination reaction. Potentially viable spores were extracted from the
coupon surfaces, plated on TSA, and enumerated. Table A-3 and Table A-4 in Appendix A present the
detailed test results. Figure 5-2 summarizes the test results.
35
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Application of Optimized Spray Conditions to pAB and S1
Glass
Concrete
pAB -1 x 5
pAB - 2 x 5
T
3.3
3.0
S1 - 2x5
Solution - Number of Sprays x Spray Duration (seconds)
*Denotes Full Surface Decontamination Based on Detection Limit
Figure 5-2. Spray Optimization Test Results for pAB and S1
These results indicate that two sprays of five seconds each again provided non-detect results (> 6 LR) for
both materials when pAB was used. Efficacies for S1 were significantly lower (LR = 3.3 and 3.0) than those
observed for pAB. But as observed with pAB, the spray scheme of two sprays at five seconds each resulted
in higher efficacies than one spray for both glass and concrete. Two sprays for five seconds each was
selected as the operational spray parameter for all remaining tests.
5.2 Phase II Decontamination Efficacy Tests
Because of the ineffectiveness of S1 compared to pAB, a decision was made to forego efficacy testing of
S2 through S4 and proceed to efficacy testing of S5 through S7. Decontamination efficacy was expressed
as a LR in viable Bg spores recovered. Typically, for laboratory assessments of decontamination efficacy,
a LR > 6 is considered effective, and when no viable spores are recovered after decontamination treatment,
the method is considered highly effective. Results for each NFB formulation also were compared to results
for the reference pAB solution, which is known to provide LR > 6 on multiple material types.
Decontamination efficacy results for this project included surface and total decontamination efficacy results
as discussed below.
5.2.1 Surface Decontamination Efficacy Results
Surface decontamination efficacy indicates how effective each solution was at decontaminating the surface
of each material and was calculated as follows:
Mean (Log CFU positive control sample) - Mean (Log CFU test coupon sample)
36
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Quantitative assessment of residual (background) contamination was performed by sampling procedural
blanks (non-inoculated coupons exposed to the same decontamination process as the test coupons). The
transfer of viable organisms to post-decontamination liquid waste also was evaluated through quantitative
analysis of decontamination procedure runoff samples.
The S5 through S7 NFB formulations were tested, and results were compared to pAB solution results. The
surface efficacy evaluations assessed the effect of temperature, de-icing agent, and contact time as
discussed below.
5.2.1.1 Effect of Temperature and use of de-icing agents
The effect of temperature on decontamination efficacy was tested in the ETC on the pAB solution at 0, 10,
and 25 °C and S5 through S7 solutions at -25, -10, 0, 10, and 25 °C. These tests included the use of a de-
icing agent as required to keep the tested solutions from freezing.
All NFB decontamination solutions were prepared as specified in Table 3-4 Section 3 5 2 and used on the
day of preparation. The solutions were checked to ensure that pH, redox potential, and FAC were within
the required ranges before use.
The tests were set up for a five-second spray duration, with one repeat application (two total applications).
The total solution contact time was increased to 20 minutes (10 minutes after the first spray and 10 minutes
after the second spray) for this set of tests, in order to improve upon (increase) the efficacies observed for
the tests with S1. After the spraying operation was complete, test coupons were immersed in a neutralizing
agent to quench the decontamination reaction. Potentially viable spores were extracted from the coupon
surfaces, plated on TSA, and enumerated using an automated counting system.
Figure 5-3 summarizes the surface decontamination efficacy results. Table A-5 through Table A-12 in
Appendix A provide the detailed test results. As the figure shows, pAB achieved a surface LR >6 regardless
of temperature (greater than 0 °C) and material used. S5 was the least efficacious of the three NFB
solutions, with an average surface LR of 3.2 and 2.3 for glass and concrete, respectively.
S6 was the most efficacious of the three solutions, with an average surface LR of 4.2 and 2.7 for glass and
concrete, respectively. S7 provided the highest surface LR on concrete, with a LR of 5.0 at 25 °C. However,
the effectiveness of this solution quickly diminished as the testing temperature was lowered. S6 showed
the highest surface LR on glass, with 5.9 at -10 °C. None of the other NFB solutions achieved surface LRs
greater than 5 on glass at any temperature. As the testing temperature was lowered, solution efficacy also
tended to decrease. Decontamination efficacy data for the pAB solution were gathered only for
temperatures greater than 0 °C because the freezing point of pAB was determined to be -8 °C.
37
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pAB
S5
¦ Glass Concrete
— 8.0
c
O 7 n
=> 6.0
"O
— 3.0
t 20
(J
it i.o
Hi
0.0
» Glass
Concrete
M
, i* B i"
o°c
10°C
Temperature (°c)
25°C
-25°C
-10°C
S6
¦ Glass Concrete
-25°C -10°C 0°C 10°C 25°C
Temperature (°C)
o°c
Temperature (°C)
S7
10'C
25°C
-25°C -1Q°C 0°C 10"C
Temperature (°C)
25°C
*Denotes Full Surface Decontamination Based on Detection Limit
Figure 5-3. Surface Decontamination Efficacy of pAB and S5 through S7 at Various Temperatures
5.2.1.2 Effect of NaCI as De-icing Agent
After testing of the S5 through S7 solutions, a new solution, S8, was developed and tested in an attempt to
increase decontamination efficacy. In S8, NaCI was substituted for CaCh as the de-icing agent. SS was
formulated as specified in Table 3-4 Section 3.5.2 Testing of S8 was performed at 25 °C only with a 20
minute total contact time, and the results were compared to the results for S5 through S7 at the same
temperature. Figure 5-4 summarizes the surface decontamination efficacy results for S5 through S8. Table
A-13 and Table A-14 in Appendix A provide the detailed test results. The results show that S8 performed
more consistently between the two materials compared to S5 through S7. However, the surface
decontamination efficacy of S8 was below a 5 LR for both material types.
38
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Surface Decontamination Efficacy
CaCI2 (S5, S6, S7) vs. NaCI (S8) at 25 °C
i Glass
5.0
Concrete
S6 S7
NFB Solution
4.3
S8
Figure 5-4. Surface Decontamination Efficacy of S5 through S8
5.2.1.3 Effect of Increased Contact Time
Contact time can significantly affect the efficacy of decontamination solutions. Therefore, the contact time
was increased from the previous test time of 20 minutes to 60 minutes. Testing was performed using pAB
and S7 at 0 °C. S7 was selected based on previous results showing that this solution had the highest
surface LR efficacy on concrete at 0 °C. Each coupon was sprayed twice, with spray durations of five
seconds each. The second spray occurred 30 minutes after the first spray. After 60 minutes from the initial
spray, the test coupons were immersed in a neutralizing solution. Spores were extracted and the viable
spores enumerated.
Figure 5-5 summarizes the surface decontamination efficacy results versus contact times of 20 and 60
minutes for S7 and pAB. Table A-5 and Table A-6 in Appendix A provide the detailed test results for pAB,
and Table A-11 and Table A-12 in Appendix A provide the detailed test results for S7. The results show
that although the efficacy of pAB did not change significantly with the longer contact time, S7 showed
increases of 1 LR and 0.6 LR for glass and concrete coupons respectively.
39
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o
+J
o
3
u
0)
o£
o
o
>«
o
9.0
8.0
7.0
6.0
5.0
4.0
3.0
Surface Decontamination Efficacy forS7 and pAB using
20 min or 60 min Contact Time at 0 °C
¦ Glass Concrete
s 2.0
UJ
1.0
0.0
S7 (20) S7 (60) PAB (20) PAB (60)
Solution and Contact Time (Minutes)
*Denotes Full Surface Decontamination Based on Detection Limit
Figure 5-5. Surface Decontamination Efficacy vs. Contact Time
5.2.2 Total Decontamination Efficacy Temperature Results
The field applicability for a decontamination solution and its corresponding delivery technology depends on
factors that include the ultimate disposition (or fate) of the targeted spores. This information is required to
develop a comprehensive, site-specific remediation strategy. For example, if viable spores are washed off
materials, remediation field strategies may require not only consideration of contact times for primary
building materials but also soil collection and treatment of liquid runoff. To assess the potential for viable
spores to be washed off the test surfaces, all liquids used in the decontamination test process were
collected and quantitatively analyzed. To provide a conservative estimate of spore fate and transport,
rinsates were neutralized immediately upon collection by pre-loading collection tubes with a neutralizing
agent. Consideration of both surface decontamination and viable spore relocation into the rinsate (recovery
of viable spores in rinsate) provided a measurement of the total decontamination efficacy, which was
calculated as follows:
Mean (Log CFU positive control sample) - Log (CFU test coupon sample + CFU liquid rinsate sample)
Figure 5-6 summarizes the total decontamination efficacy results. Table A-5 through Table A-12 in
Appendix A provide the detailed test results.
40
-------
¦a 2.5
9
5
d. t-S
y i.o
fa
E as
HI
ao
pAB
¦ Glass Conartt
T
1,*
1.7
»°C 19°C Z50e
Temperature (°C)
S6
¦ Glass.
CofioreM
MM
T
I
1,4
L
B
m
A
¦2S'C
-ItfX
rc
Temperature (°C)
10"C
2i*C
S5
S s. a
•o 2.5
9
i"
d. 1-5
>%
o 1-0
fit
0
E o.i
m
ao
¦ GIAS4 COllCf *Ml
J-
LI
1
T
!.•*
I
i I 1
(1—_
J4
14
-
1
OJ
ft*
J
M
-25*C
-1Q°C 0*C 1fl*C 25*C
Temperature (®G|
S7
3.5
3.0
a.a
2.0
1.5
1.0
0,5
0.0
• Glass C2 LR.
The pAB solution achieved >2 LR during only one test on concrete coupons at 10 °C. Generally, total
decontamination efficacies were higher on concrete coupons than on glass coupons, possibly because the
concrete coupons were not as smooth as the glass coupons, allowing for greater adhesion of the Bg spores.
Also, the rinsate samples were immediately neutralized, which explains the large differences between the
surface and the total decontamination efficacies, and. because the rinsates were immediately neutralized,
the viable spores captured therein provide an approximation of the maximum amount of contamination that
could be spread through runoff.
5.3 Results Summary
This project entailed evaluating the decontamination efficacy of NFB solutions developed by ECCC that
can function under extreme temperature conditions. The tests were conducted in an ETC so that
temperature conditions, ranging from 25 C to -25 °C, couid be tightly controlled. An automated spray
system was developed, which could fit inside the environmental chamber and function under extreme
environmental conditions. The use of this setup provides operationally-relevant insights into the expected
efficacy of the non-freezing bleach solutions. It further provides an opportunity to perform tests under
accurate and repeatable conditions (i.e., spray duration, spray pressure, volume of spray, relative
temperature and humidity). The project results provide insights into the expected efficacy of NFB solutions
tested under various environmental and operational conditions.
41
-------
Evaluation of the NFB solutions demonstrated lower surface and total decontamination efficacy compared
to pH amended Bleach (pAB) which was used as a benchmark solution due to published data which showed
it can reduce bacterial spore populations > 6 LR. Figure 5-7 summarizes the mean surface decontamination
efficacies for S5 through S7, and pAB for all temperatures 0 °C and above.
Surface Decontamination Efficacy (0 °C - 25 °C)
i Glass Concrete
I
2.8
I
2.8
3.3
pAB S5 S6
Decontamination Formulation
S7
Figure 5-7. Surface Decontamination Efficacy for pAB and S5 through S7
Figure 5-8 summarizes the mean total decontamination efficacy results for all temperatures 0 °C and above.
Total Decontamination Efficacy (0 °C - 25 °C)
— 2.5
c
o
"G 2.0
3
T3
a)
a: 1.5
O)
O
-J 1.0
>
o
S 0.5
£
HI
0.0 4—
Glass Concrete
1 1
PAB S5 S6
Decontamination Formulation
S7
Figure 5-8. Total Decontamination Efficacy for S5 through S7 and pAB
42
-------
It is important to reiterate that the rinsate samples were immediately neutralized, which explains the large
differences observed between the surface decontamination efficacies and the total decontamination
efficacies. Because the rinsates were immediately neutralized, the viable spores captured therein provide
an approximation of the maximum amount of contamination that could be spread via runoff.
Despite the NFB solutions demonstrating lower surface and total decontamination efficacies compared to
pAB, these solutions currently are the only NFB decontaminants evaluated against Bacillus spores. At
conditions below 0 °C, these solutions may be useful during remediation efforts. The testing results under
this project provide an important baseline that further work can build upon to develop and characterize new
decontamination options under environmentally challenging conditions such as freezing temperatures.
43
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6 Quality Assurance and Quality Control
All test activities were documented in laboratory notebooks and digital photographs. The documentation
included, but was not limited to, a record for each decontamination procedure, any deviations from the
quality assurance project plan, and physical impacts on materials. All tests were conducted in accordance
with established EPA Decontamination Technologies Research Laboratory (DTRL) and NHSRC RTP
Microbiology Laboratory (BioLab) procedures to ensure repeatability and adherence to the data quality
validation criteria set for this project.
The following sections discuss the criteria for the critical measurements and parameters, DQIs, and the
quality assurance (QA) and quality control (QC) checks for the project.
6.1 Criteria for Critical Measurements and Parameters
Data Quality Objectives (DQOs) are used to determine the critical measurements needed to address the
stated project objectives and specify tolerable levels of potential errors associated with simulating the
prescribed decontamination environments. The following measurements were deemed critical to
accomplish part or all of the project objectives:
• ETC temperature
• Flow rate of spray nozzles of the automated spray system
• Sample volume collected
• pH of the decontamination solutions
• Redox potential of the decontamination solutions
• NaCIO concentration (FAC) of the decontamination solutions
• Spray time
• Exposure time
• Temperature of the incubation chamber
• CFU counts
• Plated volume
• Neutralizer volume
• Pressure of the automated spray system
6.2 Data Quality Indicators
Table 6-1 lists the DQIs for the critical measurements and parameters. These DQIs were used to determine
if the collected data met the QA objectives. Volumes of components were measured as accurately as
possible using appropriate measuring equipment (such as volumetric flasks and graduated cylinders).
Commercial products such as Clorox® were used as a source of NaCIO. The concentration of each new
batch of Clorox® was evaluated. Glacial acetic acid (99%) was used for solution preparation.
44
-------
Table 6-1. DQIs for Critical Measurements and Parameters
Critical Measurement
Measurement Device
Accuracy or Precision
Target
Detection Limit
ETC temperature
Temperature control sensor
± 0.5 °C
-73 to +175 °C
Sprayer flow rate
Volume collected in
graduated cylinder per time
± 10%
1 mL per minute
Rinsate volume collected
Conical vial
12.5 mL
+ 0.1 mL
pH of decontamination solutions
National Institute for
Standards and Technology
(NIST)-traceable buffer
solutions
± 0.1 pH unit
0.1 pH unit
Redox potential of decontamination
solutions
ORP
ORP standard solution
(215 ± 0.2 mV)
NA
NaCIO concentration (FAC) of
decontamination solutions
HACH® Hypochlorite Test Kit
(volume measurement)
± 2%
+ 0.05 mL
Spray time
Timer
± 1 second
1 second
Exposure time
Timer
± 1 second
1 second
Temperature of incubation chamber
NIST-traceable thermometer
(daily)
O
o
C\J
+l
NA
CFU counts
QCount
Check of spiral plater
template within
1.82 x 104 to 2.30 x 104
1 CFU per plate
Plated volume
Spiral plater
NA
NA
Neutralizer volume
Serological pipette tips
0.1 mL
0.05 mL
Pressure of automated spray system
Compressed air regulator
± 1 psi
0 psi
6.3 QA/QC Checks
The following three parameters of the bleach solutions were measured and recorded before each use of
each decontamination solution:
• pH
• Redox potential (reduced to hydrogen scale)
• FAC (in ppm).
The critical measurements and parameters listed in Table 6-1 were measured before testing. If the
measurements obtained did not meet the DQI goals, the test was stopped. Tests proceeded only when the
DQI criteria were met.
Many QA/QC checks were used in this project to ensure that the data collected met all the critical
measurements listed in Table 6-1. The measurement and parameter criteria were set at the most stringent
levels routinely achievable. The acceptance criteria for the microbiological analysis also were set at the
most stringent levels routinely achievable, and decisions to accept or reject test results were based on
analytical judgment to assess the likely impact of the failed criterion on the conclusions drawn from the
data.
All the critical measurements and parameters met the DQI target acceptance criteria listed in Table 6-1.
Control samples and procedural blanks were included along with the test samples so that well-controlled
quantitative values were obtained. Background checks for the presence of bacterial spores were included
as part of the standard protocol. Replicate coupons of both materials were included for each set of test
conditions. Specific QC checks performed under this project included a check of the integrity of samples
45
-------
and supplies, NHRSC BioLab control checks and QA assessments and corrective actions are described
below.
6.3.1 Check of Integrity of Samples and Supplies
Samples were carefully maintained and preserved to ensure their integrity. Samples were stored away from
standards or other samples that could cross-contaminate them. In addition, project personnel carefully
checked supplies and consumables before use to verify that they met specified project quality objectives.
All pipettes were calibrated yearly by an outside contractor (Carter Calibrations, Wilmington, NC).
Incubation temperature was monitored using NIST-traceable thermometers, and the EPA Metrology
Laboratory calibrated the balances yearly.
6.3.2 NHRSC Bio-laboratory Control 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 CFU were considered
outside the quantitation range. If the CFU count did not fall within the acceptable quantitation range, the
sample was re-plated at a different volume or dilution 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 counted, a visual 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 by
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 routinely
achievable. 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:
• Positive control coupons: Coupons inoculated in tandem with the test coupons to demonstrate
the highest level of contamination recoverable from a particular inoculation event.
• Procedural blank coupons: Material coupons sampled in the same fashion as test coupons but
not inoculated with the surrogate organism before sampling.
• Blank TSA sterility controls: Plates incubated but not inoculated.
• Replicate plates of diluted microbiological samples: Replicate plates for each sample.
• Unexposed field blank: Material coupons sampled in the same fashion as test coupons but not
inoculated with the surrogate organism before sampling, or exposed to the decontamination
process.
46
-------
Table 6-2 lists the additional QC checks built into the NHRSC BioLab procedures designed to provide
assurances against cross-contamination and other biases in the microbiological samples.
Table 6-2. Additional QC Checks for Biological Measurements
Sample Type
Frequency
Acceptance Criterion
Information
Provided
Corrective Action
Positive control
coupons
Minimum of
three per test
1 x 107 for Bg,
50% relative standard
deviation (STD) 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
coupons
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
controls
Each plate
No observed growth after
incubation
Controls for sterility of
plates
All plates incubated before
use. Contaminated plates
discarded before use
Replicate plates of
diluted microbiological
samples
Each sample
Reportable CFU count of
triplicate plates within
100%; reportable CFU
counts between 30 and
300 CFU per plate
Used to determine
precision of replicate
plating
Re-plate sample.
Unexposed field blank
One per test
Non-detect
Level of contamination
present during
sampling
Clean up environment,
and sterilize sampling
materials before use.
6.3.3 QA Assessments and Corrective Actions
The QA assessments and corrective actions for this project were intended to provide rapid detection of
data quality problems. Mild contamination in QC procedural blank samples was observed after the
completion of testing. However, this contamination was very minimal and had little to no effect on the
project results. Project personnel were intimately involved with the data on a daily basis so that any data
quality issue became apparent soon after it occurred. Blank and negative samples in which spores were
present were at or near the detection limit.
Table 6-3 summarizes the QA/QC assessment of spore recoveries for the various control sample types.
47
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Table 6-3. QA/QC Assessment of Spore Recoveries (CFU) for Various Control Samples
Solution
Temperature (°C)
Procedural Blanks
Procedural Blank
Rinsates
Negative Controls
Concrete
Glass
Concrete
Glass
Concrete
Glass
25
ND
ND
7
8
ND
ND
10
ND
ND
4
2
ND
ND
S5
0
ND
ND
ND
ND
ND
7
-10
ND
ND
ND
ND
ND
ND
-25
ND
ND
1
ND
ND
2
25
ND
ND
1
ND
ND
ND
10
ND
ND
6
1
ND
62
S6
0
ND
ND
1
ND
ND
7
-10
ND
ND
ND
ND
ND
ND
-25
ND
ND
ND
ND
ND
ND
25
ND
ND
ND
ND
ND
ND
10
ND
ND
1
1ND
ND
ND
S7
0
ND
ND
ND
ND
ND
2
-10
ND
ND
ND
ND
ND
1
-25
ND
ND
ND
ND
1
ND
0 at 1 hour
1
ND
ND
ND
ND
ND
S8
25
ND
1
15
11
ND
ND
25
ND
ND
2
5
ND
ND
pAB
10
ND
ND
1
3
ND
62
0
ND
ND
4
3
ND
2
0 at 1 hour
ND
ND
ND
ND
ND
9
Note:
ND = Not detected
48
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References
ASTM F710-08 Standard Practice for Preparing Concrete Floors to Receive Resilient Flooring, ASTM
International, West Conshohocken, PA, 2008, https://doi.orq/10.1520/F0710-08
Blinov, V., K. Volchek, C.E. Brown, and E. Rohonczy. 2015. "Cold-temperature Decontamination
Formulations." Proceedings of the Thirty-Eighth Arctic and Marine Oil Spill Program (AMOP)
Technical Seminar. Environment Canada. Ottawa, ON. Pages 295 through 313.
Calfee, M.W., Y. Choi, J. Rogers, T. Kelly, Z. Willenberg, and K. Riggs. 2011. "Lab-Scale Assessment to
Support Remediation of Outdoor Surfaces Contaminated with Bacillus anthracis Spores." Journal
of Bioterrorism and Biodefense. 2(3). 2:110. Doi: 10.4172/2157-2526. 1000110.
Calfee, M.W., J. Wood, L. Mickelsen, C. Kempter, L. Miller, M. Colby, A. Touati, M. Clayton, N. Griffin-
Gatchalian, S. Payne, and R. Delafield. 2012. "Laboratory Evaluation of Large Scale
Decontamination Approaches." Journal of Applied Microbiology. 112(5). 874-882.
Dey, B.P., and F.B. Engley, Jr. 1994. "Neutralization of Antimicrobial Chemicals by Recovery Media."
Journal of Microbiological Methods. (19):1. 51-58.
Gibbons, H.S., S.M. Broomall, L.A. McNew, H. Daligault, C. Chapman, D. Bruce, M. Karavis, M. Krepps,
P.A. McGregor, C. Hong, K.H. Park, A. Akmal, A. Feldman, J.S. Lin, W.E. Chang, B.W. Higgs, P.
Demirev, J. Lindquist, A. Liem, E. Fochler, T.D. Read, R. Tapia, S. Johnson, K.A. Bishop-Lilly, C.
Detter, C. Han, S. Sozhamannan, and E.W. Skowronski. 2011. "Genomic Signatures of Strain
Selection and Enhancement in Bacillus atrophaeus var. globigii, a Historical Bio-warfare
Simulant." PLOS ONE. (6)3: e17836. doi: 10.1371/journal.pone.0017836.
Lee, S.D., S.P. Ryan, and E.G. Snyder. 2011. "Development of an Aerosol Surface Inoculation Method
for Bacillus Spores." Applied and Environmental Microbiology. 77(5). 1638-1645.
U.S. Environmental Protection Agency (EPA). 2007. "Guidance on Test Methods for Demonstrating the
Efficacy of Antimicrobial Products for Inactivating Bacillus anthracis Spores on Environmental
Surfaces." Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) Scientific Advisory Panel
(SAP) Meeting Minutes No. 2007-05. Arlington, VA.
U.S. Environmental Protection Agency (EPA), 2012. "Homeland Security, Strategic Research Action Plan
2012-2016." EPA 601/R-12/008. Office of Research and Development. Washington, DC.
U.S. Federal Bureau of Investigation (FBI) 2001. "Amerithrax or Anthrax Investigation."
https://en.wikipedia.org/wiki/2001 anthrax attacks#References. accessed June 6th, 2017.
Washington, DC.
Wood, J.P., M.W. Calfee, M. Clayton, N. Griffin-Gatchalian, and A. Touati. 2011a. "Optimizing Acidified
Bleach Solutions to Improve Sporicidal Efficacy on Building Materials." Letters in Applied
Microbiology. (53)6. 668-672.
49
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APPENDIX A: DATA REPORT
Table A-1. pAB Spray Optimization on Concrete 2
Table A-2. pAB Spray Optimization on Glass 3
Table A-3. pAB and S1 Surface Decontamination on Concrete 4
Table A-4. pAB and S1 Surface Decontamination on Glass 5
Table A-5. pAB Decontamination on Concrete by Temperature 6
Table A-6. pAB Decontamination on Glass by Temperature 7
Table A-7. S5 Decontamination on Concrete by Temperature 8
Table A-8. S5 Decontamination on Glass by Temperature 10
Table A-9. S6 Decontamination on Concrete by Temperature 12
Table A-10. S6 Decontamination on Glass by Temperature 14
Table A-11. S7 Decontamination on Concrete by Temperature 16
Table A-12. S7 Decontamination on Glass by Temperature 18
Table A-13. S8 Surface Decontamination on Concrete by Temperature 20
Table A-14. S8 Surface Decontamination on Glass by Temperature 20
Table Notes:
Avg = Average
CFU = Colony forming unit(s)
LR = Log reduction
pAB = pH-adjusted bleach
STD = Standard deviation
-------
Table A-1. pAB Spray Optimization on Concrete
No. of Sprays
and Duration
CFU
Log of CFU
Avg of Logs
Avg Surface LR
STD of LR
3.12E+04
4.5
2-second single
spray
4.73E+04
4.7
1.40E+05
5.1
4.56
2.7
0.86
1.42E+03
3.2
2.28E+05
5.4
5.50E+00
0.7
2.5-second
double spray
7.40E+03
3.9
5.50E+00
0.7
1.37
5.9
1.40
5.50E+00
0.7
5.50E+00
0.7
1.24E+03
3.1
5-second single
spray
3.08E+03
3.5
5.50E+00
0.7
1.76
5.5
1.40
5.50E+00
0.7
5.50E+00
0.7
5.50E+00
0.7
5-second double
spray
5.50E+00
0.7
5.50E+00
0.7
0.74
6.6
0.00
5.50E+00
0.7
5.50E+00
0.7
Positives during
1.37E+07
7.1
2-second single
1.31E+07
7.1
7.24
spray test
2.87E+07
7.5
Positives during
2.04E+07
7.3
2.5-second
1.74E+07
7.2
double spray
and 5- second
single and
double spray
2.37E+07
7.4
7.31
Note: 10-minute total contact time
A-2
-------
Table A-2. pAB Spray Optimization on Glass
No. of Sprays
and Duration
CFU
Log of CFU
Avg of Logs
Avg Surface LR
STD of LR
1.00E+00
0.0
2-second single
spray
1.00E+00
0.0
1.98E+03
3.3
1.65
5.9
2.33
8.76E+04
4.9
1.00E+00
0.0
1.00E+00
0.0
2.5-second
double spray
1.00E+00
0.0
1.00E+00
0.0
0.00
7.2
0.00
1.00E+00
0.0
1.00E+00
0.0
1.00E+00
0.0
5-second single
spray
1.00E+00
0.0
1.00E+00
0.0
0.00
7.2
0.00
1.00E+00
0.0
1.00E+00
0.0
1.00E+00
0.0
5-second double
spray
1.00E+00
0.0
1.00E+00
0.0
0.00
7.2
0.00
1.00E+00
0.0
1.00E+00
0.0
Positives during
3.97E+07
7.6
2-second single
3.25E+07
7.5
7.55
spray test
3.49E+07
7.5
Positives during
2.28E+07
7.4
2.5-second
1.29E+07
7.1
double spray
and 5- second
single and
double spray
1.40E+07
7.1
7.20
Note: 10-minute total contact time
A-3
-------
Table A-3. pAB and S1 Surface Decontamination on Concrete
No. of Sprays
and Duration
CFU
Log of CFU
Avg of Logs
Avg Surface
LR
STD of LR
1.24E+03
3.1
5-second pAB
single spray
3.08E+03
3.5
5.50E+00
0.7
1.76
5.5
1.40
5.50E+00
0.7
5.50E+00
0.7
5.50E+00
0.7
5-second pAB
double spray
5.50E+00
0.7
5.50E+00
0.7
0.74
6.6
0.00
5.50E+00
0.7
5.50E+00
0.7
2.01 E+05
5.3
5-second S1
5.91 E+04
4.8
4.85
2.0
0.31
single spray
4.24E+04
4.6
4.88E+04
4.7
1.90E+03
3.3
5-second S1
1.12E+04
4.0
3.92
3.0
0.49
double spray
7.57E+03
3.9
2.96E+04
4.5
Positives during
pAB testing
2.04E+07
7.3
1.74E+07
7.2
7.31
2.37E+07
7.4
Positives during
1.10E+07
7.0
6.87
S1 testing
4.99E+06
6.7
Note: 10-minute total contact time
A-4
-------
Table A-4. pAB and S1 Surface Decontamination on Glass
No. of Sprays
and Duration
CFU
Log of CFU
Avg of Logs
Avg Surface LR
STD of LR
1.00E+00
0.0
5-second pAB
single spray
1.00E+00
0.0
1.00E+00
0.0
0.00
7.2
0.00
1.00E+00
0.0
1.00E+00
0.0
1.00E+00
0.0
5-second pAB
double spray
1.00E+00
0.0
1.00E+00
0.0
0.00
7.2
0.00
1.00E+00
0.0
1.00E+00
0.0
1.54E+03
3.2
5-second S1
1.77E+04
4.2
3.42
3.6
0.62
single spray
5.98E+02
2.8
3.07E+03
3.5
4.39E+04
4.6
5-second S1
1.51E+04
4.2
3.67
3.3
1.23
double spray
7.34E+01
1.9
9.44E+03
4.0
Positives during
pAB testing
2.28E+07
7.4
1.29E+07
7.1
7.20
1.40E+07
7.1
Positives during
S1 testing
1.32E+07
7.1
9.25E+06
7.0
6.98
7.27E+06
6.9
Note: 10-minute total contact time
A-5
-------
Table A-5. pAB Decontamination on Concrete by Temperature
Temperature
CFU
Log of
CFU
Avg of
Logs
Avg
Surface
LR
STD of
Surface
LR
Avg Total
Decontamination
on LR
STD of Total
Decontamination
LR
5.75E+00
0.8
5.75E+00
0.8
25 °C
5.75E+00
0.8
0.76
6.8
0.00
1.8
0.12
5.75E+00
0.8
5.75E+00
0.8
5.75E+00
0.8
5.75E+00
0.8
10 °C
5.75E+00
0.8
1.19
6.4
0.96
2.2
1.09
5.75E+00
0.8
8.05E+02
2.9
5.75E+00
0.8
5.75E+00
0.8
0 °C
5.75E+00
0.8
1.10
6.3
0.77
1.6
0.15
5.75E+00
0.8
2.99E+02
2.5
5.75E+00
0.8
0 °C, 1-hour
contact time
5.75E+00
0.8
5.75E+00
0.8
0.76
6.6
0.00
1.5
0.06
5.75E+00
0.8
5.75E+00
0.8
Positives at
25 °C
2.88E+07
7.5
3.01 E+07
7.5
7.54
4.71 E+07
7.7
Positives at
10 °C
3.56E+07
7.6
3.48E+07
7.5
7.57
4.10E+07
7.6
Positives at
0 °C
2.67E+07
7.4
2.29E+07
7.4
7.39
2.45E+07
7.4
Positives at
2.20E+07
7.3
0 °C, 1-hour
3.05E+07
7.5
7.38
contact time
2.02E+07
7.3
5.92E+05
5.8
Rinsate
25 °C
4.56E+05
5.7
4.63E+05
5.7
5.7
8.93E+05
6.0
4.81 E+05
5.7
5.92E+05
5.8
Rinsate
10 °C
4.56E+05
5.7
4.63E+05
5.7
5.4
8.93E+05
6.0
4.81 E+05
5.7
8.06E+05
5.9
Rinsate
0 °C
7.79E+05
5.9
5.36E+05
5.7
5.8
6.21 E+05
5.8
3.44E+05
5.5
7.61 E+05
5.9
Rinsate at
7.41 E+05
5.9
0 °C, 1-hour
9.79E+05
6.0
5.9
contact time
8.29E+05
5.9
6.64E+05
5.8
Note: 20-minute total contact time, unless otherwise noted
A-6
-------
Table A-6. pAB Decontamination on Glass by Temperature
Temperature
CFU
Log of
CFU
Avg of
Logs
Avg
Surface
LR
STD of
Surface
LR
Avg Total
Decontamination
on LR
STD of Total
Decontamination
LR
7.18E+00
0.9
1.00E+00
0.0
25 °C
5.34E+00
0.7
0.32
7.1
0.44
1.7
0.26
1.00E+00
0.0
1.00E+00
0.0
1.00E+00
0.0
3.75E+00
0.6
10 °C
3.48E+00
0.5
0.53
6.9
0.35
1.2
0.06
9.39E+00
1.0
3.71 E+00
0.6
1.00E+00
0.0
1.47E+00
0.2
0 °C
1.00E+00
0.0
0.07
7.4
0.09
1.6
0.14
1.00E+00
0.0
1.47E+00
0.2
1.00E+00
0.0
0 °C, 1-hour
contact time
1.00E+00
0.0
7.57E+00
0.9
0.21
7.0
0.38
1.1
0.05
1.47E+00
0.2
1.00E+00
0.0
Positives at
25 °C
2.76E+07
7.4
2.36E+07
7.4
7.44
3.25E+07
7.5
Positives at
10 °C
3.25E+07
7.5
3.57E+07
7.6
7.47
2.20E+07
7.3
Positives at
0 °C
3.71 E+07
7.6
2.53E+07
7.4
7.48
2.94E+07
7.5
Positives at
1.25E+07
7.1
0 °C, 1-hour
1.29E+07
7.1
7.16
contact time
1.87E+07
7.3
1.64E+06
6.2
Rinsate
25 °C
5.11E+05
5.7
4.21 E+05
5.6
5.8
4.16E+05
5.6
4.16E+05
5.6
2.19E+06
6.3
Rinsate
10 °C
1.75E+06
6.2
1.60E+06
6.2
6.3
2.20E+06
6.3
1.78E+06
6.2
1.04E+06
6.0
Rinsate
0 °C
1.11E+06
6.0
6.37E+05
5.8
5.9
5.15E+05
5.7
9.04E+05
6.0
1.19E+06
6.1
Rinsate at
9.25E+05
6.0
0 °C, 1-hour
1.13E+06
6.1
6.0
contact time
1.07E+06
6.0
9.06E+05
6.0
Note: 20-minute total contact time, unless otherwise noted
A-7
-------
Table A-7. S5 Decontamination on Concrete by Temperature
Temperature
CFU
Log of
CFU
Avg of
Logs
Avg
Surface
LR
STD of
Surface
LR
Avg Total
Decontamination
on LR
STD of Total
Decontamination
LR
2.59E+04
4.4
1.67E+04
4.2
25 °C
8.81 E+03
3.9
4.3
3.2
0.23
1.1
0.17
2.86E+04
4.5
3.13E+04
4.5
3.70E+04
4.6
5.34E+04
4.7
10 °C
4.70E+04
4.7
4.8
2.8
0.21
1.3
0.08
5.12E+04
4.7
1.31E+05
5.1
1.85E+05
5.3
8.97E+04
5.0
0 °C
1.44E+05
5.2
5.2
2.3
0.13
1.3
0.11
1.98E+05
5.3
1.46E+05
5.2
2.29E+05
5.4
1.22E+06
6.1
-10 °C
1.44E+05
5.2
5.3
2.2
0.47
1.1
0.20
8.26E+04
4.9
1.00E+05
5.0
2.47E+06
6.4
2.13E+06
6.3
-25 °C
2.15E+06
6.3
6.4
1.0
0.08
0.9
0.07
3.25E+06
6.5
2.82E+06
6.4
5.75E+00
0.8
0 °C, 1-hour
contact time
5.75E+00
0.8
5.75E+00
0.8
0.76
6.6
0.00
1.5
0.06
5.75E+00
0.8
5.75E+00
0.8
Positives at
25 °C
2.88E+07
7.5
3.01 E+07
7.5
7.5
4.71 E+07
7.7
Positives at
10 °C
4.77E+07
7.7
2.85E+07
7.5
7.6
4.08E+07
7.6
Positives at
0 °C
2.58E+07
7.4
4.05E+07
7.6
7.5
2.28E+07
7.4
Positives at
-10 °C
2.98E+07
7.5
3.58E+07
7.6
7.5
3.14E+07
7.5
Positives at
-25 °C
1.95E+07
7.3
2.70E+07
7.4
7.4
2.71 E+07
7.4
Positives at
2.20E+07
7.3
0 °C, 1-hour
3.05E+07
7.5
7.38
contact time
2.02E+07
7.3
3.45E+06
6.5
Rinsate
25 °C
1.42E+06
6.2
3.41 E+06
6.5
6.4
3.50E+06
6.5
2.68E+06
6.4
Rinsate
1.86E+06
6.3
6.3
10 °C
1.85E+06
6.3
A-8
-------
Temperature
CFU
Log of
CFU
Avg of
Logs
2.33E+06
6.4
2.93E+06
6.5
1.96E+06
6.3
1.54E+06
6.2
Rinsate
0 °C
1.75E+06
6.2
1.36E+06
6.1
6.1
8.33E+05
5.9
1.69E+06
6.2
3.13E+06
6.5
Rinsate
-10 °C
2.16E+06
6.3
1.67E+06
6.2
6.3
2.46E+06
6.4
1.06E+06
6.0
3.58E+05
5.6
Rinsate
-25 °C
8.84E+05
5.9
5.19E+05
5.7
5.8
5.08E+05
5.7
8.72E+05
5.9
Avg
Surface
LR
STD of
Surface
LR
Avg Total
Decontamination
on LR
STD of Total
Decontamination
LR
Note: 20-minute total contact time, unless otherwise noted
A-9
-------
Table A-8. S5 Decontamination on Glass by Temperature
Temperature
CFU
Log of
CFU
Avg of
Logs
Avg
Surface
LR
STD of
Surface
LR
Avg Total
Decontamination
on LR
STD of Total
Decontamination
LR
1.73E+02
2.2
2.21 E+03
3.3
25 °C
1.01E+03
3.0
6.7
4.2
0.86
0.7
0.20
6.67E+02
2.8
3.58E+04
4.6
1.18E+04
4.1
9.09E+04
5.0
10 °C
9.04E+03
4.0
6.7
2.9
0.53
0.8
0.07
1.27E+05
5.1
7.16E+04
4.9
1.18E+04
4.1
9.56E+03
4.0
0 °C
1.41E+04
4.2
6.5
4.0
1.01
0.9
0.13
6.30E+01
1.8
1.04E+03
3.0
8.99E+04
5.0
1.77E+04
4.2
-10 °C
1.21E+04
4.1
6.3
2.9
0.63
0.8
0.12
3.09E+04
4.5
1.70E+03
3.2
2.90E+05
5.5
1.38E+05
5.1
-25 °C
5.30E+05
5.7
5.9
1.7
0.34
1.4
0.20
6.57E+05
5.8
1.08E+06
6.0
5.75E+00
0.8
0 °C, 1-hour
contact time
5.75E+00
0.8
5.75E+00
0.8
0.76
6.6
0.00
1.5
0.06
5.75E+00
0.8
5.75E+00
0.8
Positives at
25 °C
2.76E+07
7.4
2.36E+07
7.4
7.4
3.25E+07
7.5
Positives at
10 °C
2.91 E+07
7.5
4.54E+07
7.7
7.5
3.10E+07
7.5
Positives at
0 °C
2.10E+07
7.3
2.49E+07
7.4
7.4
3.06E+07
7.5
Positives at
-10 °C
1.03E+07
7.0
1.69E+07
7.2
7.1
1.41 E+07
7.1
Positives at
-25 °C
1.85E+07
7.3
2.27E+07
7.4
7.3
1.76E+07
7.2
4.77E+06
6.7
Rinsate
25 °C
2.57E+06
6.4
6.42E+06
6.8
6.7
8.39E+06
6.9
6.36E+06
6.8
7.39E+06
6.9
Rinsate
10 °C
5.02E+06
6.7
5.24E+06
6.7
6.7
4.95E+06
6.7
5.13E+06
6.7
A-10
-------
Temperature
CFU
Log of
CFU
Avg of
Logs
4.79E+06
6.7
Rinsate
0 °C
3.13E+06
6.5
2.41 E+06
6.4
6.5
2.27E+06
6.4
3.70E+06
6.6
2.02E+06
6.3
Rinsate
-10 °C
1.66E+06
6.2
1.21 E+06
6.1
6.3
1.75E+06
6.2
2.59E+06
6.4
2.93E+05
5.5
Rinsate
-25 °C
3.49E+05
5.5
1.56E+05
5.2
5.5
2.35E+05
5.4
4.96E+05
5.7
Avg
Surface
LR
STD of
Surface
LR
Avg Total
Decontamination
on LR
STD of Total
Decontamination
LR
Note: 20-minute total contact time
A-11
-------
Table A-9. S6 Decontamination on Concrete by Temperature
Temperature
CFU
Log of
CFU
Avg of
Logs
Avg
Surface
LR
STD of
Surface
LR
Avg Total
Decontamination
on LR
STD of Total
Decontamination
LR
3.85E+04
4.6
1.29E+04
4.1
25 °C
1.29E+04
4.1
4.3
3.3
0.20
1.1
0.08
1.91E+04
4.3
1.46E+04
4.2
3.99E+04
4.6
8.14E+04
4.9
10 °C
4.86E+04
4.7
4.9
2.7
0.25
1.0
0.13
1.47E+05
5.2
1.22E+05
5.1
1.92E+05
5.3
9.57E+04
5.0
0 °C
1.27E+05
5.1
5.1
2.4
0.13
1.4
0.26
9.11E+04
5.0
1.15E+05
5.1
1.68E+03
3.2
4.51 E+04
4.7
-10 °C
3.15E+03
3.5
4.1
3.4
0.73
1.6
0.20
3.82E+04
4.6
1.00E+05
5.0
1.11E+06
6.0
8.03E+05
5.9
-25 °C
4.67E+05
5.7
5.9
1.4
0.26
1.2
0.18
1.81E+06
6.3
4.46E+05
5.6
Positives at
25 °C
3.56E+07
7.6
4.77E+07
7.7
7.6
3.25E+07
7.5
Positives at
10 °C
3.56E+07
7.6
3.48E+07
7.5
7.6
4.10E+07
7.6
Positives at
0 °C
2.58E+07
7.4
4.05E+07
7.6
7.5
2.28E+07
7.4
Positives at
-10 °C
3.34E+07
7.5
4.48E+07
7.7
7.6
3.74E+07
7.6
Positives at
-25 °C
1.77E+07
7.2
2.59E+07
7.4
7.3
2.43E+07
7.4
2.33E+06
6.4
Rinsate
25 °C
3.03E+06
6.5
3.72E+06
6.6
6.5
3.26E+06
6.5
2.46E+06
6.4
4.48E+06
6.7
Rinsate
10 °C
2.64E+06
6.4
5.15E+06
6.7
6.5
3.50E+06
6.5
2.61 E+06
6.4
6.97E+05
5.8
Rinsate
0 °C
5.15E+05
5.7
2.69E+06
6.4
6.0
1.60E+06
6.2
1.06E+06
6.0
A-12
-------
Temperature
CFU
Log of
CFU
Avg of
Logs
Avg
Surface
LR
STD of
Surface
LR
Avg Total
Decontamination
on LR
STD of Total
Decontamination
LR
7.18E+05
5.9
Rinsate
-10 °C
8.68E+05
5.9
8.41 E+05
5.9
6.0
1.28E+06
6.1
2.26E+06
6.4
6.26E+05
5.8
Rinsate
-25 °C
3.06E+05
5.5
7.04E+05
5.8
5.7
7.56E+05
5.9
4.82E+05
5.7
Note: 20-minute total contact time
A-13
-------
Table A-10. S6 Decontamination on Glass by Temperature
Temperature
CFU
Log of
CFU
Avg of
Logs
Avg
Surface
LR
STD of
Surface
LR
Avg Total
Decontamination
on LR
STD of Total
Decontamination
LR
3.85E+04
4.6
1.29E+04
4.1
25 °C
1.29E+04
4.1
4.3
3.3
0.20
0.6
0.08
1.91E+04
4.3
1.46E+04
4.2
3.99E+04
4.6
8.14E+04
4.9
10 °C
4.86E+04
4.7
4.9
2.7
0.25
0.7
0.20
1.47E+05
5.2
1.22E+05
5.1
1.92E+05
5.3
9.57E+04
5.0
0 °C
1.27E+05
5.1
5.1
2.4
0.13
0.6
0.12
9.11E+04
5.0
1.15E+05
5.1
1.68E+03
3.2
4.51 E+04
4.7
-10 °C
3.15E+03
3.5
4.1
3.4
0.73
0.7
0.22
3.82E+04
4.6
5.95E+04
4.8
1.11E+06
6.0
8.03E+05
5.9
-25 °C
4.67E+05
5.7
5.9
1.4
0.26
1.5
0.22
1.81E+06
6.3
4.46E+05
5.6
Positives at
25 °C
3.56E+07
7.6
4.77E+07
7.7
7.6
3.25E+07
7.5
Positives at
10 °C
3.56E+07
7.6
3.48E+07
7.5
7.6
4.10E+07
7.6
Positives at
0 °C
2.58E+07
7.4
4.05E+07
7.6
7.5
2.28E+07
7.4
Positives at
-10 °C
3.34E+07
7.5
4.48E+07
7.7
7.6
3.74E+07
7.6
Positives at
-25 °C
1.77E+07
7.2
2.59E+07
7.4
7.3
2.43E+07
7.4
2.33E+06
6.4
Rinsate
25 °C
3.03E+06
6.5
3.72E+06
6.6
6.5
3.26E+06
6.5
2.46E+06
6.4
4.48E+06
6.7
Rinsate
10 °C
2.64E+06
6.4
5.15E+06
6.7
6.5
3.50E+06
6.5
2.61 E+06
6.4
6.97E+05
5.8
Rinsate
0 °C
5.15E+05
5.7
2.69E+06
6.4
6.0
1.60E+06
6.2
1.06E+06
6.0
A-14
-------
Temperature
CFU
Log of
CFU
Avg of
Logs
Avg
Surface
LR
STD of
Surface
LR
Avg Total
Decontamination
on LR
STD of Total
Decontamination
LR
7.18E+05
5.9
Rinsate
-10 °C
8.68E+05
5.9
8.41 E+05
5.9
6.0
1.28E+06
6.1
2.26E+06
6.4
6.26E+05
5.8
Rinsate
-25 °C
3.06E+05
5.5
7.04E+05
5.8
5.7
7.56E+05
5.9
4.82E+05
5.7
Note: 20-minute total contact time
A-15
-------
Table A-11. S7 Decontamination on Concrete by Temperature
Temperature
CFU
Log of
CFU
Avg of
Logs
Avg
Surface
LR
STD of
Surface
LR
Avg Total
Decontamination
on LR
STD of Total
Decontamination
LR
4.72E+02
2.7
2.30E+01
1.4
25 °C
1.64E+03
3.2
2.5
5.0
0.70
0.8
0.19
5.06E+02
2.7
5.87E+02
2.8
1.51E+04
4.2
1.07E+05
5.0
10 °C
5.04E+04
4.7
4.7
2.9
0.32
1.4
0.17
7.21 E+04
4.9
4.13E+04
4.6
4.97E+05
5.7
2.78E+05
5.4
0 °C
1.60E+05
5.2
5.3
2.0
0.24
1.2
0.16
1.21E+05
5.1
1.83E+05
5.3
1.13E+05
5.1
2.65E+05
5.4
-10 °C
1.83E+05
5.3
5.2
2.4
0.20
1.3
0.09
1.87E+05
5.3
7.98E+04
4.9
9.79E+05
6.0
1.08E+06
6.0
-25 °C
2.28E+06
6.4
6.1
1.2
0.15
1.1
0.12
1.23E+06
6.1
1.64E+06
6.2
5.95E+04
4.8
0 °C, 1-hour
contact time
4.08E+04
4.6
2.27E+05
5.4
4.9
2.6
0.28
1.5
0.18
1.01E+05
5.0
6.98E+04
4.8
Positives at
25 °C
2.31 E+07
7.4
4.82E+07
7.7
7.6
4.11 E+07
7.6
Positives at
10 °C
4.77E+07
7.7
2.85E+07
7.5
7.5
3.10E+07
7.5
Positives at
0 °C
2.66E+07
7.4
2.22E+07
7.3
7.4
2.29E+07
7.4
Positives at
-10 °C
3.67E+07
7.6
4.20E+07
7.6
7.6
3.86E+07
7.6
Positives at
-25 °C
2.76E+07
7.4
1.95E+07
7.3
7.4
2.37E+07
7.4
Positives at
3.18E+07
7.5
0 °C, 1-hour
3.37E+07
7.5
7.6
contact time
4.30E+07
7.6
4.30E+06
6.6
Rinsate
25 °C
4.12E+06
6.6
4.28E+06
6.6
6.8
9.38E+06
7.0
9.11E+06
7.0
A-16
-------
Temperature
CFU
Log of
CFU
Avg of
Logs
1.12E+06
6.0
Rinsate
10 °C
6.59E+05
5.8
1.63E+06
6.2
6.1
1.96E+06
6.3
1.59E+06
6.2
2.06E+06
6.3
Rinsate
0 °C
1.97E+06
6.3
1.01E+06
6.0
6.1
1.11E+06
6.0
1.19E+06
6.1
2.00E+06
6.3
Rinsate
-10 °C
2.16E+06
6.3
1.23E+06
6.1
6.2
1.69E+06
6.2
1.50E+06
6.2
1.74E+06
6.2
Rinsate
-25 °C
4.58E+05
5.7
8.08E+05
5.9
5.9
7.60E+05
5.9
5.12E+05
5.7
9.20E+05
6.0
Rinsate at
7.44E+05
5.9
0 °C, 1-hour
1.52E+06
6.2
6.1
contact time
2.14E+06
6.3
1.26E+06
6.1
Avg
Surface
LR
STD of
Surface
LR
Avg Total
Decontamination
on LR
STD of Total
Decontamination
LR
Note: 20-minute total contact time, unless otherwise noted
A-17
-------
Table A-12. S7 Decontamination on Glass by Temperature
Temperature
CFU
Log of
CFU
Avg of
Logs
Avg
Surface
LR
STD of
Surface
LR
Avg Total
Decontamination
on LR
STD of Total
Decontamination
LR
1.18E+05
5.1
4.99E+04
4.7
25 °C
1.44E+04
4.2
4.1
3.5
0.82
0.9
0.20
1.07E+03
3.0
4.05E+03
3.6
3.20E+03
3.5
1.36E+03
3.1
10 °C
8.66E+03
3.9
3.6
4.0
0.63
0.8
0.12
6.90E+02
2.8
2.55E+04
4.4
7.43E+05
5.9
1.53E+05
5.2
0 °C
5.38E+05
5.7
5.6
1.8
0.28
1.0
0.10
7.03E+05
5.8
3.80E+05
5.6
2.06E+03
3.3
3.48E+04
4.5
-10 °C
3.58E+03
3.6
4.2
3.2
0.69
0.4
0.07
7.48E+04
4.9
3.61 E+04
4.6
7.18E+04
4.9
8.63E+02
2.9
-25 °C
1.23E+06
6.1
4.9
2.5
1.21
1.4
0.22
3.80E+05
5.6
1.58E+05
5.2
3.99E+04
4.6
0 °C, 1-hour
contact time
2.84E+04
4.5
5.67E+03
3.8
4.6
2.8
0.58
0.8
0.16
1.78E+05
5.3
1.03E+05
5.0
Positives at
25 °C
5.01 E+07
7.7
4.08E+07
7.6
7.6
4.13E+07
7.6
Positives at
10 °C
2.91 E+07
7.5
4.54E+07
7.7
7.5
3.10E+07
7.5
Positives at
0 °C
3.74E+07
7.6
2.58E+07
7.4
7.5
2.80E+07
7.4
Positives at
-10 °C
2.37E+07
7.4
2.44E+07
7.4
7.4
3.02E+07
7.5
Positives at
-25 °C
2.83E+07
7.5
2.91 E+07
7.5
7.4
2.37E+07
7.4
Positives at
2.93E+07
7.5
0 °C, 1-hour
2.96E+07
7.5
7.4
contact time
2.34E+07
7.4
8.84E+06
6.9
Rinsate
25 °C
7.20E+06
6.9
5.36E+06
6.7
6.7
5.54E+06
6.7
2.69E+06
6.4
A-18
-------
Temperature
CFU
Log of
CFU
Avg of
Logs
5.62E+06
6.7
Rinsate
10 °C
3.69E+06
6.6
5.80E+06
6.8
6.7
5.96E+06
6.8
3.32E+06
6.5
2.24E+06
6.4
Rinsate
0 °C
3.42E+06
6.5
3.07E+06
6.5
6.4
1.59E+06
6.2
1.94E+06
6.3
1.09E+07
7.0
Rinsate
-10 °C
8.60E+06
6.9
1.23E+07
7.1
7.0
1.32E+07
7.1
1.08E+07
7.0
6.30E+05
5.8
Rinsate
-25 °C
5.58E+05
5.7
8.54E+05
5.9
5.8
6.78E+05
5.8
7.54E+05
5.9
5.17E+06
6.7
Rinsate at
4.63E+06
6.7
0 °C, 1-hour
4.49E+06
6.7
6.6
contact time
1.93E+06
6.3
4.06E+06
6.6
Avg
Surface
LR
STD of
Surface
LR
Avg Total
Decontamination
on LR
STD of Total
Decontamination
LR
Note: 20-minute total contact time, unless otherwise noted
A-19
-------
Table A-13. S8 Surface Decontamination on Concrete by Temperature
Temperature
CFU
Log of
CFU
Avg of
Logs
Avg
Surface
LR
STD of
LR
7.44E+02
2.9
9.20E+02
3.0
o
o
LO
CM
5.25E+02
2.7
2.9
4.3
0.65
1.34E+02
2.1
8.54E+03
3.9
Positives at
2.32E+07
7.4
1.26E+07
7.1
7.3
25 °C
2.01 E+07
7.3
1.36E+06
6.1
Rinsate at
25 °C
1.27E+06
6.1
1.10E+06
6.0
6.1
1.36E+06
6.1
1.13E+06
6.1
Note: 20-minute total contact time
Table A-14. S8 Surface Decontamination on Glass by Temperature
Temperature
CFU
Log of
CFU
Avg of
Logs
Avg
Surface
LR
STD of
LR
1.00E+03
3.0
3.75E+01
1.6
o
o
LO
CM
5.87E+02
2.8
2.5
4.9
0.54
3.45E+02
2.5
3.68E+02
2.6
Positives at
25 °C
1.61 E+07
7.2
2.67E+07
7.4
7.3
2.47E+07
7.4
1.67E+06
6.2
Rinsate at
25 °C
8.10E+05
5.9
1.60E+06
6.2
6.1
1.59E+06
6.2
8.55E+05
5.9
Note: 20-minute total contact time
A-20
-------
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United States
Environmental Protection
Agency
PRESORTED STANDARD
POSTAGE & FEES PAID
EPA
PERMIT NO. G-35
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