&EPA
United States
Environmental Protection
Agency
I: PA/600/R 16/i 62
www.epa.gov/homeland-security-research
Effectiveness of Spray-Based
Decontamination Methods for Spores
and Viruses on Heavily Soiled Surfaces
Office of Research and Development
Homeland Security Research Program
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EPA 600/R-16/162
Effectiveness of Spray-Based
Decontamination Methods for Spores
and Viruses on Heavily Soiled Surfaces
Assessment and Evaluation Report
M.Worth Calfee, Ph.D.
National Homeland Security Research Center
Office of Research and Development
U.S. Environmental Protection Agency
Research Triangle Park, NC 27711
Abderrahmane Touati, Ph.D., Snigdha Gayatri Pongur, Timothy McArthur,
and Barbara Wyrzykowska-Ceradini, Ph.D.
Jacobs Technology Inc.
Research Triangle Park, NC 27709
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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-09-027, Work Assignment (4-6)-52 with ARCADIS U.S., Inc. (ARCADIS), and 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 Environmental Protection Agency document. It
does not necessarily reflect the views of the Environmental Protection Agency. No official endorsement
should be inferred. This report includes photographs of commercially available products. The
photographs are included for purposes of illustration only and are not intended to imply that EPA
approves or endorses the product or its manufacturer. 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
This effort was initiated following the identification of knowledge gaps by US Department of Homeland
Security's Agricultural Defense Branch-led subcommittee on Foreign Animal Disease Threats (FADTs),
Decon, Disposal, and Depopulation (3D) Working Group, which is co-membered by the US EPA and the
US Department of Agriculture (USDA). Emergency response and remediation following a foreign animal
disease (FAD) incident will involve numerous federal agencies (particularly those listed), as well as state,
local, and private entities. This project addresses closing gaps in our ability to decontaminate and
remediate facilities following an agro-terrorism incident. Funding support by the US Department of
Homeland Security (DHS) to complete this effort is greatly appreciated.
This effort was directed by the principal investigator from EPA's Office of Research and Development's
(ORD's) National Homeland Research Center (NHSRC), utilizing the support of a project team consisting
of staff from across the US EPA, DHS, and USDA. The contributions of the following individuals have
been a valued asset throughout this effort:
• R. Leroy Mickelsen (US EPA/OLEM/CMAD)
• Joseph P. Wood (US EPA/ORD/NHSRC)
• Michelle Colby (DHS)
• Lori Miller (USDA)
Additionally, the authors would like to thank the following peer reviewers for their significant contributions:
• Nathan Birnbaum (USDA/APHIS)
• Elise Jakabhazy (US EPA/OLEM CMAD)
• Lukas Oudejans (US EPA/ORD/NHSRC)
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Contents
Disclaimer i
Acknowledgments ii
Figures vii
Tables ix
Acronyms and Abbreviations xi
Executive Summary xiv
Introduction 1
1.1 Background 1
1.2 Project Description and Objectives 1
Experimental Approach 2
Experimental Methods and Materials 5
3.1 Preparation of Coupons 5
3.1.1 Large Coupon Preparation 6
3.1.1.1 Plywood Large Coupons 6
3.1.1.2 Concrete Large Coupons 6
3.1.1.3 Stainless Steel Large Coupons 7
3.1.2 Small Coupon Preparation 8
3.1.2.1 Plywood Small Coupons 8
3.1.2.2 Concrete Small Coupons 9
3.1.3 Sterilization of Coupons 12
3.1.3.1 Large Coupon Sterilization 12
3.1.3.2 Small Coupon Sterilization 13
3.2 Grime Formulation, Preparation, and Application 13
3.2.1 Agricultural Grime Formulation 14
3.2.1.1 Particulate Soil 15
3.2.1.2 Animal Sebum 17
3.2.1.3 Animal Impurities 19
3.2.2 Grime Preparation 23
3.2.2.1 Raw Materials 23
3.2.2.2 Preparation of Individual Components 23
3.2.2.3 Mixing of Grime 25
3.2.3 Grime Application 26
3.2.3.1 Application of Grime on Large Coupons 26
3.2.3.2 Application of Grime on Small Coupons 28
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3.3 Test Organisms 29
3.3.1 B. atrophaeus Surrogate for B. anthracis 29
3.3.1.1 B. atrophaeus Spore Preparation 30
3.3.1.2 B. atrophaeus Spore Inoculations 30
3.3.2 Bacteriophage MS2 - Surrogate for Viral Agents 30
3.3.2.1 MS2 Preparation 31
3.3.2.2 MS2 Inoculations 32
Decontamination Approach 34
4.1 Decontamination Materials and Equipment 34
4.2 Decontamination Agents 34
4.2.1 pAB Solution 35
4.2.2 Spor-Klenz® RTU Solution 37
4.2.3 2% Citric Acid Solution 37
4.3 Decontamination Testing Approach 38
4.4 Large Coupon Decontamination Testing 39
4.4.1 Test Chamber for Large Coupons 39
4.4.2 Application of Decontaminants Using Sprayers for Large Coupons 40
4.4.2.1 Backpack Sprayer 41
4.4.2.2 Chemical Sprayer 43
4.4.3 Post-Decontamination Rinse for Large Coupons 43
4.4.4 Decontamination Chronology for Decontamination Testing (Large Coupons) 44
4.5 Small Coupon Decontamination Testing 46
4.5.1 Spray Apparatus for Small Coupons 46
4.5.2 Decontamination Procedure for MS2 Testing (Small Coupons) 48
Neutralizing Agents for Extracted Samples 50
5.1 Neutralization Agent Preparation 50
5.2 Neutralization for Large Coupons 51
5.2.1 Surface Neutralization Tests 51
5.2.2 Neutralization Tests for Liquid Effluents 52
5.3 Neutralization for Small Coupons 53
5.3.1 DE Broth Neutralizer Effectiveness, Test 1 53
5.3.2 Neutralizer Buffer Effectiveness, Test II 55
5.3.3 Suitable Extraction Buffer and Inoculation Hold Time, Test III 55
5.3.4 Method Development Test for Neutralizer Volume Determination 57
Sampling Approach 59
6.1 Sampling Strategy for Large Coupons 60
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6.1.1 Sample Types 60
6.1.2 Sample Quantities 60
6.1.3 Sampling and Monitoring Points 61
6.1.4 Frequency of Sampling and Monitoring Events 61
6.2 Sampling Methods 61
6.2.1 Wipe Sampling 61
6.2.1.1 Wipe Sampling Preparation 61
6.2.1.2 Wipe Sampling Procedure 63
6.2.2 Runoff and Rinsate Sampling 63
6.2.3 Aerosol Sampling 64
6.2.4 QA/Q C Sampling 65
6.3 Sampling Strategy for Small Coupons 66
6.3.1 Sample Types 66
6.3.2 Sample Quantities 66
6.3.3 Sampling and Monitoring Points 66
6.3.4 Frequency of Sampling and Monitoring Events 67
6.4 Sample Handling 67
6.4.1 Sample Containers for Large Coupons 67
6.4.2 Sample Containers for Small Coupons 67
6.4.3 Sample Preservation for Large Coupons 68
6.4.4 Sample Preservation for Small Coupons 68
Analytical Procedures 69
7.1 Analytical Procedures for Microbiological Analyses 69
7.2 Filtration and Plating of Bacteria from Liquid Extracts 69
7.3 Plating of MS2 from Liquid Extracts 70
7.4 Data Reduction 71
Results and Discussion 72
8.1 B. atrophaeus Decontamination Testing 72
8.1.1 Extraction Efficacy from Neat and Heavily Grimed Surfaces (14-in x 14-in
Coupons) 72
8.1.2 Neutralizing Agent Testing for Extracted Samples 73
8.1.3 B. atrophaeus Decontamination Testing Using pAB and Spor-Klenz® RTU 74
8.2 MS2 Decontamination Testing 77
8.2.1 DE Broth Neutralizer Effectiveness Test I Results 77
8.2.2 DE Broth Neutralizer Effectiveness Test II Results 79
8.2.3 Suitable Extraction Buffer and Inoculation Hold Time Test III Results 80
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8.2.4 MS2 Decontamination Testing on Small Coupons Using pAB and 2% Citric
Acid Formulation 82
8.2.5 MS2 Decontamination Testing Using pAB and 2% Citric Acid Formulation on
Large Coupons 84
Quality Assurance and Quality Control 88
9.1 Criteria for Critical Measurements/Parameters 88
9.2 Data Quality Indicators 89
Summary 92
References 94
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Figures
Figure 3.1-1. Curing of Large Concrete Coupons 7
Figure 3.1-2. Small (18-mm diameter) Plywood Coupon 9
Figure 3.1-3. Mold for Fabricating Small Concrete Coupons 10
Figure 3.1-4. Cross Section of Final Small Concrete Coupon in Butterboard Mold 11
Figure 3.1-5. Concrete Small Coupon 11
Figure 3.1-6. Stainless Steel Stage 13
Figure 3.2-1. Raw Dried Manure (a), and Homogenized Sample of Manure (b) 24
Figure 3.2-2. Final Grime Solution Sterilization (a), and Final Batch of Solidified Grime (b) 25
Figure 3.2-3. Grime Aliquot Preparation 26
Figure 3.2-4. Coupons Readied for Grime Application 27
Figure 3.2-5. Grime Application Procedure (First Application) 27
Figure 3.2-6. Heavily Grimed Large Coupon Surface after Second Grime Application 28
Figure 3.2-7. Heavily Grimed Small Coupon Surface after Second Grime Application 29
Figure 3.3-1. Droplet Pattern Used forMS2 Inoculations on Large Coupons 33
Figure 4.4-1. Decontamination Test Chamber for Large Coupons 40
Figure 4.4-2. Spraying Through Center-Aligned Port in the Test Chamber Door 41
Figure 4.4-3. Electric Backpack Sprayer 41
Figure 4.4-4. Spray Pattern for Backpack Sprayer 42
Figure 4.5-1. Front View of Spray Apparatus with Orifice Plate 47
Figure 4.5-2. Side View of Spray Apparatus with Orifice Plate 47
Figure 4.5-3. Front View of Spray Apparatus without Orifice Plate 48
Figure 6.2-1. Sampling Template Centered on Heavily Grimed Large Plywood Coupon 62
Figure 6.2-2. Test Chamber Exhaust Duct (white arrow shows sampling point location) 65
Figure 7.2-1. Dilution Plate (Left) and Filter Plate (Right) Showing Colonies ofB. atrophaeus 70
Figure 7.2-2. Dilution Plate Showing PFU of MS2 70
Figure 8.1-1. Bacillus atrophaeus Recoveries on Pre-Decontaminated Inoculated Surfaces 74
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Figure 8.1-1. Bacillus atrophaeus Decontamination Efficacy 76
Figure 8.2-1. MS2 Recoveries Using DE Broth as an Extraction Buffer 78
Figure 8.2-2. MS2 Extraction Efficacy with and without DE Broth, after 0 and 4 Days 80
Figure 8.2-3. MS2 Recoveries from Positive Control, Procedural Control, and Decontaminated
Test Coupons 83
Figure 8.2-4. MS2 Recoveries from Large Coupon Tests 86
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Tables
Table 3.1-1. Test Coupon Materials Specifications 5
Table 3.2-1. Synthetic Agricultural Grime Composition 14
Table 3.2-2. Particulate Soil Composition 15
Table 3.2-3. Relative Composition of Non-Polar Lipids on the Skin Surfaces of Various
Species 18
Table 3.2-4. Manure Components and Their Composition 19
Table 3.2-5. Swine Fresh Manure Characteristics 20
Table 3.2-6. Cattle Fresh Manure Characteristics 22
Table 3.2-7. Raw Materials for Grime Preparation 23
Table 4.1-1. Decontamination Materials and Equipment 34
Table 4.4-1. B. atrophaeus and MS2 Decontamination Test Matrix for Large Coupons 39
Table 4.5-1. MS2 Decontamination Test Matrix for Small Coupons 46
Table 5.2-1. Neutralization Tests for Extractive Samples 51
Table 5.3-1. Test I Matrix 53
Table 5.3-2. Test II Matrix 55
Table 5.3-3. Test III Matrix 56
Table 6-1. Sampling Materials and Equipment 59
Table 6.1-1. Sample Types and Numbers for Each Decontamination Test 60
Table 6.1-2. Sampling Frequencies 61
Table 6.1-3. Sample Types and Numbers for Each Decontamination Test 66
Table 6.1-4. Sampling Frequencies 67
Table 7.1-1. Analytical Procedures 69
Table 8.1-1. B. atrophaeus Recovery from Grimed and Neat Surfaces 72
Table 8.1-2. Neutralization Test Results for B. atrophaeus Extracted Samples 73
Table 8.1-3. B. atrophaeus Decontamination Results 75
Table 8.1-4. Fate of B. atrophaeus Spores during Decontamination Procedures 76
Table 8.2-1. MS2 - DE Neutralizer Broth Effectiveness Test I Results 78
Table 8.2-2. MS2 - DE Broth Neutralizer Effectiveness Test II 79
Table 8.2-3. Suitable Extraction Buffer and Inoculation Hold Time Test III Results for MS2 81
Table 8.2-4. MS2 Recoveries from Positive Control, Procedural Control, and
Decontaminated Test Coupons 84
Table 8.2-5. MS2 Recoveries from Runoff Samples from Small Coupons 84
Table 8.2-6. MS2 Recoveries during Large Coupon Testing 85
Table 8.2-7. Fate of MS2 during Decontamination Procedures 87
Table 9.2-1. DQIs for Critical Measurements 89
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X
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Acronyms and Abbreviations
% percent
2 M 2 molar
2 N 2 normal
|j|_ microliter(s)
jjm micrometer(s)
ACS American Chemical Society
ADA aerosol deposition apparatus
APWMC Animal and Poultry Waste Management Center
ARCADIS ARC AD IS U.S., Inc.
ASTM American Society for Testing and Materials, now ASTM International
ATCC American Type Culture Collection
B. Bacillus
BOD biochemical oxygen demand
BSC biological safety cabinet
CAS Chemical Abstract Services
CBRN Chemical, Biological, Radiological, and Nuclear
CFU colony-forming unit(s)
cm centimeter(s)
cm2 square centimeter(s)
CMAD Consequence Management Advisory Division
COD chemical oxygen demand
db dry basis (% of dry matter)
DE Dey Engley
DHS Department of Homeland Security
Dl deionized
DQI data quality indicator
DQO data quality objective
DTRL Decontamination Technologies Research Laboratory
EPA U.S. Environmental Protection Agency
EtO ethylene oxide
FAC free available chlorine
FAD Foreign Animal Disease
FADT Foreign Animal Disease Threat
FIFRA Federal Insecticide, Fungicide, and Rodenticide Act
FMDV foot and mouth disease virus
g gram(s)
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g/L
gram(s) per liter
Gpm
gallon(s) per minute
GRAS
generally recognized as safe
in
inch(es)
in2
square inch(es)
IUPAC
International Union of Pure and Applied Chemistry
H2O2
hydrogen peroxide
HSRP
Homeland Security Research Program
Jacobs
Jacobs Technology, Inc.
kcal/g
kilocalorie(s) per gram
LB
Luria-Bertani
lb/day
pound(s) per day
Ib/day/head
pound(s) per day per head
lb/ft3
pound(s) per cubic foot
lb/ton
pound(s) per ton
Lowe's
Lowe's Home Improvement
Lpm
liter(s) per minute
LR
log reduction
MDI
metered-dose inhaler
mg
milligram(s)
mg/100 cm2
milligram(s) per 100 square centimeters
mg/kg
milligram(s) per kilogram
mg/L
milligram per liter
mg/mL
milligram(s) per milliliter
mL
milliliter(s)
mm
millimeters)
NCSU
North Carolina State University
NHSRC
National Homeland Security Research Center
NIST
National Institute of Standards and Technology
NPT
National Pipe Taper
NSF
National Sanitation Foundation
obs
observed
OCSPP
Office of Chemical Safety and Pollution Prevention
OLEM
Office of Land and Emergency Management
ORD
Office of Research and Development
PAA
peracetic acid
pAB
pH-adjusted bleach
PAH
polycyclic aromatic hydrocarbon
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PARTNER
Program to Align Research and Technology with the Needs of Environmental
Response
PBS
phosphate-buffered solution
PBST
phosphate-buffered saline with 0.05% Tween® 20
PFU
plaque-forming unit
PPE
personal protective equipment
psi
pound(s) per square inch
PPm
part(s) per million
ppmv
part(s) per million by volume
PVC
polyvinyl chloride
QA
quality assurance
QAPP
Quality Assurance Project Plan
QC
quality control
RNA
ribonucleic acid
rpm
revolution(s) per minute
RSD
relative standard deviation
RTU
Ready-to-Use
SEM
scanning electron microscopy
SM
magnesium salt
SSL
sebum saturation level
STD
standard deviation
STS
sodium thiosulfate
TKN
total Kjeldahl nitrogen
TSB
tryptic soy broth
USDA
US Department of Agriculture
USP
U.S. Pharmacopeia
VHP
vaporized hydrogen peroxide
wb
wet basis (as is)
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Executive Summary
The objective of this project was to assess the effectiveness of spray-based common decontamination
methods for inactivating Bacillus (B.) atrophaeus (surrogate for B. anthracis) spores and bacteriophage
MS2 (surrogate for foot and mouth disease virus [FMDV]) on selected neat or heavily soiled (i.e., with a
model agricultural grime loaded on the surface) test surfaces (concrete and treated wood). Relocation of
viable viruses or spores from the contaminated coupon surfaces into aerosol or liquid fractions during the
decontamination methods was investigated. This project was conducted to support jointly held missions
of the U.S. Department of Homeland Security (DHS) and the U.S. Environmental Protection Agency
(EPA). Within the EPA, the project supports the mission of EPA's Homeland Security Research Program
(HSRP) by providing relevant information pertinent to the decontamination of contaminated areas
resulting from a biological incident.
The effectiveness of removing target microorganisms from the surfaces of the coupons provided critical
information regarding the effectiveness of each decontamination procedure. However, field applicability
depends on several other factors, including the ultimate disposition (or fate) of the spores or viruses. This
information is required to develop a comprehensive, site-specific remediation strategy. For example, if
viable spores or viruses are washed off materials, remediation field strategies may require rinsate
collection and treatment. If spores or viruses are detected in air samples, spread of contamination or
recontamination of previously decontaminated surfaces must be considered in determining the overall
remediation approach. To obtain critical information on the fate of the spores or viruses, several additional
samples were collected. To assess the potential for viable spores or viruses to be washed off the
surfaces, all liquids used in the decontamination process were collected and quantitatively analyzed
(runoff and rinsate samples). To assess the potential for aerosolization of spores or viruses from coupon
surfaces during spraying, aerosol samples were collected from the decontamination chamber during
spraying activities.
The effectiveness of removing/inactivating two target microorganisms was assessed for three different
decontamination solutions. pH-Amended Bleach (pAB) and Spor-Klenz® Ready-to Use (RTU) were
evaluated against B. atrophaeus spores, and 2 percent (%) weight/volume (w/v) citric acid in sterilized
deionized (Dl) water and pAB were evaluated against MS2. Three application methods (handheld
sprayer, backpack sprayer, and a chemical sprayer) were utilized throughout the testing to deliver
decontaminants to the test surfaces. The evaluation was conducted on two test material surfaces
(concrete and plywood), with and without agricultural grime. The handheld application method was
conducted using a bench-scale test spray apparatus to evaluate the pAB and citric acid spray-based
decontamination methods for 18-millimeter (mm) coupons (both grimed and neat) contaminated with
MS2. The backpack and the chemical sprayer application methods were conducted to simulate field
operations. For all the tests, a wetted surface contact time of 30 minutes was used, followed by a surface
rinse with water. The fate of the microorganisms in the runoffs generated during the decontamination
procedure and in the subsequent rinse step, as well as their potential re-aerosolization in the air, were
also investigated.
Decontamination tests with B. atrophaeus spores indicated that higher efficacies were achieved on neat
materials than on grimed materials, independent of the type of material or application method. pAB was
found more effective than Spor-Klenz® RTU for decontaminating neat concrete materials, while the latter
decontaminant was more efficient with neat plywood materials independent of application method
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(backpack sprayer versus chemical sprayer). Viable spore levels found in rinsate samples were higher for
the backpack sprayer tests than for the chemical sprayer tests, potentially because the chemical sprayer
was more effective at physically removing spores before the rinse step. Relatively high aerosolization
(greater than 1 * 103 colony forming units [CFU] per test) was observed during some tests with both the
backpack and chemical sprayers.
Decontamination tests with MS2 indicated that 2% citric acid was not effective on concrete and plywood.
However, pAB was found to be efficacious against MS2, with full decontamination on neat or grimed
concrete and limited efficacy for neat or grimed plywood. Further, few viable viruses were detected in the
runoff from pAB tests, unlike for the 2% citric acid formulation, which had almost complete wash-off of the
viruses from the all coupon types. Finally, no viable MS2 aerosol formation/emission was observed in any
of the conducted tests, independent of the type of decontamination solution used. However, it should be
noted that the Via-Cell® bio-aerosol cassette sampling method, used in this study, was not validated for
MS2 sampling or recovery.
Effectiveness was measured by determining the log reduction (LR) in viable spores or viruses. In this
report, data are frequently presented as the average log reduction (LR) for a particular test. In laboratory
tests, if a particular set of decontamination conditions achieves > 6 LR (against a 6-7 log challenge), the
decontamination is generally considered "effective." This benchmark is consistent with sporicidal efficacy
tests used to register sporicides under the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA).
Achieving complete kill (no viable agent recovered following the decontamination treatment) is considered
"highly effective."
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Introduction
This report discusses a project that evaluated the effectiveness of spray-based decontamination methods
for spores and viruses on heavily soiled surfaces. The project was conducted to support jointly held
missions of the U.S. Department of Homeland Security (DHS) and the U.S. Environmental Protection
Agency (EPA). Within the EPA, the project supports the mission of EPA's Homeland Security Research
Program (HSRP) by providing relevant information pertinent to the decontamination of contaminated
areas resulting from a biological incident. The project addresses HSRP strategic goals as described in
detail in the Homeland Security Research Multi-year Strategic Plan1. 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." This project addresses a
direct need expressed by OLEM's Chemical, Biological, Radiological, and Nuclear (CBRN) Consequence
Management Advisory Division (CMAD). In addition the project is relevant to EPA's Office of Chemical
Safety and Pollution Prevention (OCSPP) crisis exemption process and the OCSPP's regulatory function
under the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA). The following sections discuss
the project background and the project description and objectives.
1.1 Background
Contamination incidents may result from intentional or accidental releases of biological materials or
human or animal disease outbreaks. All scenarios pose significant challenges with regard to determining
the extent of contamination, containing the contaminant spread, and remediating the event so that re-
occupancy or reuse can occur. The project that is the subject of this report supports multiagency
objectives of better understanding and preparing for the remediation of heavily soiled surfaces after a
biological contamination incident.
1.2 Project Description and Objectives
The purpose of this project was to evaluate common decontamination methods for inactivating Bacillus
(B.) atrophaeus (surrogate for B. anthracis) spores and bacteriophage MS2 (surrogate for foot and mouth
disease virus [FMDV]) on selected test surfaces (with or without a model agricultural grime). Coupons
loaded with the model agricultural grime reflect challenging environments expected during agricultural
facility decontamination events. The coupons were then loaded with the target organisms (B. atrophaeus
and MS2) using an aerosol deposition or liquid inoculation method. The coupons were then treated using
the selected decontamination methods, and the effectiveness of each method was measured based on
the reduction of viable agent (spores or viruses) achieved. Relocation of viable viruses or spores from the
contaminated coupon surfaces into aerosol or liquid fractions during the decontamination methods also
was assessed.
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Experimental Approach
The general experimental approach used to meet the project objectives is described below.
1. Preparation of representative coupons of test materials: Coupons were prepared using two
porous materials common to agricultural facilities: treated plywood and unpainted (smooth finish)
concrete.
2. Agricultural grime formulation and optimization of its manufacturing and application
methods: A model agricultural grime formulation and its application method were developed.
The grime was applied to two types of common agricultural facility materials (concrete and
treated plywood). The grime was added to the test materials to simulate the challenging
environments expected during decontamination efforts in agricultural settings and to assess the
impact of surface-associated grime on decontamination efficacy. In addition, the compatibility of
the grime was tested for the two target organisms: B. atrophaeus (surrogate for B. anthracis) and
bacteriophage MS2 (surrogate for FMDV), prior to testing.
3. Contamination of coupons using standardized inocula of target organisms: Coupons were
contaminated using an aerosol deposition (Bacillus spores) or liquid inoculation (MS2) methods.
A known quantity of the surrogate organism (1 * 107 B. atrophaeus CFU (colony-forming units) or
1 x io9 PFU (plaque-forming units) bacteriophage MS2) was deposited onto the coupons,
followed by quantitative assessment of pre-decontamination loading by sampling positive control
(non-decontaminated) coupons (n = 3 per test).
4. Decontamination of test coupons: Test coupons (n = 5 coupons per each decontamination
procedure tested) were decontaminated using the following decontamination agents: pH-adjusted
bleach (pAB), Spor-Klenz® Ready-To-Use (RTU), and 2 percent (%) citric acid solution. Each
decontamination agent was applied using either a backpack, chemical, or handheld sprayer,
followed by quantitative determination of viable B. atrophaeus spores or MS2 particles remaining
on the coupons. Recoveries from test coupons subjected to the decontamination treatment were
compared to positive control coupons. In addition, quantitative assessment of residual
(background) contamination was performed by sampling negative controls (non-inoculated
coupons, not subjected to the decontamination process) and procedural blanks (non-inoculated
coupons that went through the same decontamination process as the test coupons). The transfer
of viable organisms to post-decontamination liquid waste and air was evaluated through
quantitative analysis of decontamination procedure residues (such as decontamination solution
runoff and rinse water waste) and analysis of air samples collected during the decontamination
process. An understanding of the transfer of viable organisms to post-decontamination liquid
waste and air is important for determining fugitive emissions, latent infection and health risks, and
overall decontamination effectiveness.
5. Decontamination effectiveness: Decontamination effectiveness, as a function of the
procedure/decontaminant and material type was measured as log reduction (LR) in viable
spores/plaques. Typically, for laboratory assessments of sporicidal efficacy a LR > 6 (>
99.9999% reduction), when a titer of 1 x 107 challenge organism is used, is considered effective.
For virucidal efficacy assessments, a LR > 3 is considered effective against a 1 x 104 challenge.
In the current study however, since both spore and virus challenge titers were > 7 log; a 6 LR was
2
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considered "effective" against either organism. Complete kill (no viable agent recovered following
the decontamination treatment) was considered "highly effective."
Post-decontamination results and the physical impact of decontamination on the test materials were
assessed through visual inspection and documented in laboratory notebooks and by digital photographs.
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This project was conducted in three phases, as summarized below.
1. Phase I: The effectiveness and operational parameters for decontamination of B. atrophaeus on
grimed and neat concrete and treated plywood using pAB or Spor-Klenz® RTU deployed using a
backpack or chemical sprayer was determined. For these tests, a wetted surface contact time of
30 minutes was used, followed by a surface rinse with water. The rinse step was used to simulate
field operations in which rinsing may be used to minimize collateral damage to facilities resulting
from extended contact with harsh decontamination chemicals.
2. Phase II: The effectiveness and operational parameters for decontamination of MS2 on grimed
and neat concrete and treated plywood using pAB or 2% citric acid deployed using a backpack or
chemical sprayer was determined. For these tests, a contact time of 30 minutes for a wetted
surface was used, followed by a surface rinse with water.
3. Phase III: Due to inconsistencies encountered during the extraction process for MS2 using
phosphate-buffered saline with 0.05% Tween®20 (PBST) in Phase II, an extensive series of
method development tests was conducted using smaller coupons (18 millimeters [mm], 0.07 inch
[in] diameter) to determine the best buffer solution to maximize recoveries. The buffer solutions
investigated were Dey Engley (DE) neutralizing broth, deionized (Dl) water, PBST, phosphate-
buffered saline (PBS), and tryptic soy broth (TSB). Further, a series of tests was conducted as
control testing to evaluate the performance of the pAB and 2% citric acid solutions for
decontaminating MS2 on 18-mm round coupons using a handheld sprayer.
4
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Experimental Methods and Materials
This section describes the experimental testing and materials, including the preparation of coupons;
grime formulation, preparation, and application; and the test organisms.
3.1 Preparation of Coupons
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 typical of those found in agricultural
animal husbandry and farming facilities that meet industry standards and specifications, and (2) obtaining
these materials from appropriate suppliers. Material uniformity means that all test materials are
equivalent. Uniformity was maintained by obtaining and preparing a quantity of material sufficient to allow
the preparation of multiple test samples with presumably uniform characteristics (that is, test coupons
were cut from the interior rather than the edge of a large piece of material).
Coupons of two building materials, concrete and treated plywood, were prepared onsite for
decontamination testing. Control coupons of stainless steel were also prepared for use as inoculation
controls. Table 3.1-1 lists the test coupon materials, suppliers or manufacturers, and preparation
methods.
Table 3.1-1. Test Coupon Materials Specifications
Material
Plywood
ACQ-D pressure-treated plywood
% in thick measuring 4 by 8 feet
(Catalog No. CCX34T25C)
Lowe's Home
Improvement
(Lowe's) store
1. Remove wood particles using soft-bristle brush.
2. Sterilize using vaporized hydrogen peroxide
(VHP).
Concrete
QUIKRETE® sand/topping mix
QUIKRETE®
Companies and
Lowe's store
1. Remove particles by power washing.
2. After power washing, allow to air dry in climate-
controlled environment for at least five days.
3. Sterilize in an autoclave.
Stainless
Steel
Multipurpose stainless steel 0.036
in thick measuring 48 by 48 in,
type 304, #2B mill (unpolished)
McMaster-Carr
1. Remove lubricant and grease using acetone,
and wipe dry.
2. Remove particles and dust by wiping clean with
water and wipe dry.
3. Sterilize in an autoclave.
The coupons were made in two sizes: (1) large coupons measuring 35.6 centimeters (cm) x 35.6 cm (14
in x 14 in) for bench-scale decontamination testing during Phases I and II, and (2) smaller round coupons
with a diameter of 18 mm (0.07 in; surface area 1.58 square inches [in2]) for method development and
Phase III testing. The preparation of the large and small coupons is discussed below.
5
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3.1.1 Large Coupon Preparation
This section discusses the preparation methods for the plywood and concrete coupons for
decontamination testing and the stainless-steel control coupons.
3.1.1.1 Plywood Large Coupons
The following materials and equipment were used to prepare the large plywood coupons:
• ACQ-D pressure-treated plywood % in thick measuring 4 by 8 feet (ft)
• Table saw
• Appropriate personal protective equipment (PPE, including gloves, safety glasses, hearing
protection, and safety footwear and dust masks if needed)
The procedure summarized below was used to prepare the large plywood coupons.
1. Personnel preparing the coupons donned appropriate PPE and put up necessary warning signs
around the work area.
2. A table saw was used to cut each 14 by 14-in plywood coupon.
3.1.1.2 Concrete Large Coupons
The following materials and equipment were used to prepare the large concrete coupons:
• QUIKRETE® sand/topping mix
• Water source
• Mixing trough
• Trowel
• Leveling board
• Plastic covering for curing process
• Appropriate PPE (including gloves, safety glasses, and safety footwear)
The procedure summarized below was used to prepare the large concrete coupons.
1. Personnel preparing the coupons donned appropriate PPE and put up necessary warning signs
around the work area.
2. Custom 14 by 14-in forms were manufactured for these coupons.
3. The concrete mix was prepared according to instructions on the package using a trough and garden
hose for the water supply.
4. The concrete mix was poured into the custom forms.
6
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5. A trowel was used to smooth the coupon surface, and each coupon was allow to dry in the form
overnight.
6. After drying, plastic was laid over the coupons, and the coupons were allowed to cure for at least
five days (see Figure 3.1-1).
Figure 3.1-1. Curing of Large Concrete Coupons
3.1.1.3 Stainless Steel Large Coupons
The following materials and equipment were used to prepare the large stainless steel coupons:
• Multipurpose stainless steel 0.036 in thick measuring 48 by 48 in, type 304, #2B mill (unpolished)
• Heavy-duty hydraulic shears
• Appropriate PRE (including gloves, safety glasses, and safety footwear)
The procedure summarized below was used to prepare the large coupons.
1. Personnel preparing the coupons donned appropriate PPE and put up necessary warning signs
around the work area.
2. Heavy-duty power hydraulic shears were used to cut metal into 14 by 14-in coupons.
7
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3.1.2 Small Coupon Preparation
This section discusses the preparation methods for the plywood and concrete coupons for method
development and Phase III decontamination testing.
3.1.2.1 Plywood Small Coupons
The following materials and equipment were used to prepare the small plywood coupons:
• ACQ-D pressure-treated plywood % in thick measuring 4 by 8 ft
• Table saw
• Drill press
• 22-mm hole saw without pilot bit
• Scanning electron microscope (SEM) aluminum stubs with 18-mm diameter and 8-mm pin length
(Ted Pella, Inc., Redding, CA, Catalog No. 16119)
• Double-sided adhesive carbon tape (NEM tape, Nisshin Em. Co., Ltd., Tokyo, Japan)
• Parafilm roll (Bemis Company, Inc., Neenah, Wl)
• Tweezers
• Arch punch (C.S Osborne & Co., Harrison, NJ, Catalog No. 01236)
• Appropriate PPE (including gloves, safety glasses, and safety footwear)
The procedure summarized below was used to prepare the small plywood coupons.
1. Personnel preparing the coupons donned appropriate PPE and put up necessary warning signs
around the work area.
2. Strips of plywood measuring 1.0-in2 were cut using a table saw.
3. Using a hole saw in the drill press, rounds were drilled to a depth of approximately 0.7 in each.
The rounds were not drilled all the way through, so each strip was still in one piece.
4. The table saw guide was set to 1.0 cm.
5. The plywood strip from Step 3 was turned on its edge and cut to make plywood cylinders each
measuring 18 mm in diameter, with a height of 1 cm.
6. A 10-in-long strip of NEM tape was cut and laid on a flat surface with the sticky side up. A
parafilm strip of the same size as the NEM tape was cut and placed on the NEM strip.
7. Stickers measuring 18 mm in diameter were punched out from the NEM and parafilm strip using
the arch punch.
8. The film from the underside of each NEM and parafilm sticker was removed and stuck onto an
SEM stub.
8
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9. Using a pair of tweezers, the parafilm was carefully removed from the top of the sticker, and the
plywood cylinder was attached to the SEM stub. The plywood cylinder mounted on the SEM stub
constituted the small plywood coupon (see Figure 3.1-2).
Figure 3.1-2. Small (18-mm diameter) Plywood Coupon
3.1.2.2 Concrete Small Coupons
The following materials and equipment were used to prepare the small concrete coupons:
• Butterboard measuring 6 by 12 by 2 in. (from McMaster Carr, Atlanta, GA, Catalog No. 86595K1)
• CNC milling machine with 18-mm mill cutter
• SEM stubs with 18-mm diameter and 8-rmm pin length (from Ted Pella, inc., in Redding, CA,
Catalog No. 16119)
• QUIKRETE® sand/topping mix
• Suitable plastic container for mixing concrete
• Mixing stick
• Dl water
• Appropriate PRE (including gloves, safety glasses, and safety footwear)
9
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The procedure summarized below was used to prepare the small concrete coupons.
1. Personnel preparing the coupons donned appropriate PPE and put up necessary warning signs
around the work area.
2. Using the CNC milling machine, the butterboard was drilled to produce a moid as shown in
Figure 3.1-3.
Figure 3.1-3. Mold for Fabricating Small Concrete Coupons
3. A clean SEM stub was placed in each mold hole so that the pin of the SEM stub fit through the
smaller hole in the mold.
4. In the plastic container, 1 pound (lb) of QUIKRETE® sand/topping was mixed with 0.1 pint (50
milliliters [ml.]) of clean water. The mixture was well-worked using a mixing stick.
5. Additional water was added (not exceeding 0.6 mL or 0.13 pint in total) to obtain a workable,
plastic-like consistency.
6. The concrete fabrication mold (Figure 3.1-3) was filled with the concrete mix. The top of the mold
was smoothed to ensure a flat surface.
7. The concrete-filled mold was allowed to dry and cure indoors (70 °F or higher) for five days
before removal of the coupons from the mold.
10
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Figure 3.1-4 shows a cross section of the final coupon mold, and Figure 3.1-5 shows the final small
concrete coupon.
SEM STUB
PIN
Figure 3.1-4. Cross Section of Final Small Concrete Coupon in Butterboard Mold
Figure 3.1-5. Concrete Small Coupon
11
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3.1.3 Sterilization of Coupons
This section discusses the sterilization of the large and small coupons.
3.1.3.1 Large Coupon Sterilization
The large coupons were individually enclosed in VHP-permeable sterilization bags (General Econopak,
Inc., Steam Component Autoclave Bag, white, 20 by 20 in, Item No. 62020TW) before sterilization. The
stainless steel coupons were wrapped in aluminum foil, before being placed in the VHP-permeable
sterilization bags.
Plywood Coupons-These coupons were sterilized using 250 parts per million (ppm) hydrogen peroxide
(H2O2) vapor, also referred to as VHP, for four hours using a STERIS VHP ED1000 generator (STERIS
Corporation, Mentor, OH). Biological indicators designed for H2O2 were included in each fumigation to
identify systematic problems. Sterility was evaluated by swab sampling one coupon from each sterilization
batch. Prior to use, the coupons treated with VHP were incubated at 30 to 35 °C for two days or at room
temperature for 14 days to force off-gassing of H2O2 from the coupons, as suggested by Calfee et al.2 to
prevent biocidal activity.
Bagging of the plywood coupons and VHP sterilization was performed after the coupons were deemed
sufficiently dry (that is, constant mass was observed for three sample coupons for a period of 48 hours as
determined gravimetrically every 12 to 18 hours). The coupons were sterilized in batches. The number of
coupons per batch was limited so that all coupons in the chamber were exposed to the VHP without
shielding (no physical overlap of coupons) and so that appropriate mixing of the H2O2 occurred in the
chamber.
After the VHP cycle, plywood coupons were stored in a vertical position using racks or other types of
spacers to prevent the formation of mold after sterilization. The coupons were then placed into a sterile
container for storage prior to transport to the testing location. The container was marked with the
contents, including the sterilization date. The sterility of the coupons was verified through the analysis of
laboratory blank control samples.
Aerosol deposition apparatus (ADA) pyramids also were sterilized with 250 parts per million by volume
(ppmv) VHP for four hours using a STERIS VHP ED1000 generator.
Concrete and Stainless Steel Coupons- these coupons were sterilized using a large STERIS Amsco
Century SV 120 Scientific Pre-vacuum Sterilizer using a one-hour 121°C gravity cycle.
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3.1.3.2 Small Coupon Sterilization
The small coupons were sterilized using an Andersen ethylene oxide (EtO) sterilizer system (PN: 333
EOGas®, Haw River, NC, USA). The sterilization procedure is summarized below.
1. The coupons were loaded into stainless-steel stages (see Figure 3.1.6).
Figure 3.1-6. Stainless Steel Stage
2. The stage loaded with the coupons was placed in a glass Petri dish and loosely covered with a
crystallization dish (see Figure 3.1-6).
3. Each Petri dish was placed into an appropriate sterilization bag.
4. The sterilization bags were loaded into a cabinet for sterilization using EtO.
5. The sterilization bags were removed from the EtO cabinet with the crystallization dishes still
covering the Petri dishes to maintain coupon sterility.
3.2 Grime Formulation, Preparation, and Application
No universal grime substrate is representative of the many types of grime present at various animai
production and farming facilities. For this project, the composition of grime substrate was based on
scientific literature review and its applicability for use in evaluating the performance of decontamination
methods. The grime formulation was intended to challenge decontamination methods for heavily soiled
surfaces. Other formulations of agricultural grime may yield different results. This section discusses grime
formulation, preparation, and application.
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3.2.1 Agricultural Grime Formulation
The agricultural grime surrogate was designed using the following criteria:
• Constituent representativeness criteria:
¦ Must include the general grime component (also known as "particulate soil"), a mixture of
general outdoor dusts, soils, oils, soot, etc.;
¦ Must include a surrogate of agricultural grime-specific components such as animal sebum or
animal fat;
¦ Must include a surrogate of agricultural grime-specific impurities that are potentially
chemically and biologically active (such as nutrient-rich manure)
Note: Impurities that are not chemically or biologically active (such as animal hair) were not considered
essential for grime composition.
• Functional and operational applicability criteria:
¦ All components must be easily homogenized;
¦ All components must be suitable for sterilization, either by steam autoclave, heating or
boiling, irradiation, or application of EtO; and
¦ At least one component must be a carrier of the other constituents. For example, if the carrier
is liquefied, constituents must be mixed into the liquid carrier, and then the complete grime
formulation is spread onto coupon surfaces
Table 3.2-1 shows the composition of the agricultural grime.
Table 3.2-1. Synthetic Agricultural Grime Composition
Component
Particulate
soil
Natural humus
Paraffin oil
Used crankcase motor oil
Portland cement
Iron oxide
Silica
Kaolin clay
Carbon black
Stearic acid
Oleic acid
Synthetic particulate surrogate soil for testing cleaning
performance of products intended for use on resilient
flooring and washable walls
3
Animal
sebum
Lanolin
Wool grease secreted by sheep sebaceous glands;
surrogate for animal sebum
4
Animal
impurities
Standardized manure
Dry, homogenized cow manure; surrogate for animal
impurities
5
The following sections discuss each component, its significance and function, and information on sources
of standardized individual constituents.
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3.2.1.1 Particulate Soil
The particulate soil grime component was prepared using a modified recipe for particulate soil preparation
adapted from the American Society for Testing and Materials (ASTM) International Method D4488-
95(2001)e1, "Standard Guide for Testing Cleaning Performance of Products Intended for Use on Resilient
Flooring and Washable Walls."3 This ASTM International method provides techniques for soiling,
cleaning, and evaluating performance of detergent systems under controlled but practical hard-surface
cleaning conditions, where soil is defined as foreign matter on a hard surface, and the soiled surface
being cleaned is defined as a substrate.
The types of soils in this method were used for evaluating the cleaning performance of solutions of
soluble powdered detergent, dilutions of concentrated liquid detergent, or products intended for full-
strength use (such as foams, sprays, liquids, or pastes for cleaning hard surfaces). The method
emphasizes that the soils recommended for evaluating general cleaning performance are not a universal
soil/substrate combination representative of the many soil removal tasks required for a given type of
cleaner under actual use conditions. Choice of soil/substrate and cleaning conditions should be by
agreement between the testing laboratory and those using the data to evaluate cleaning performance
relative to user experience3. The particulate soil recipe adopted for use in this project from ASTM Method
D4488-95(2001)e1 is summarized in Table 3.2-2.
Table 3.2-2. Particulate Soil Composition
Constituent
% Weight
Natural humus
38.0
Paraffin oil
1.0
Used crankcase motor oil
1.5
Portland cement
17.7
Silica
18.0
Carbon black
1.5
Iron oxide
0.3
Kaolin clay*
18.0
Stearic acid
2
Oleic acid
2
Kaolin clay replaces bandy black clay from the ASTM International Method D4488-95(2001)
particulate soil recipe. Mineral composition is similar for both clays, but an intense
pigmentation typical for bandy black clay is not necessary for biological contamination testing.
This soil formula has numerous components typical of natural soils (top organic layer of soil, numerous
common earth minerals, carbon black, oils, fats, etc.). These components are likely to be found in soils
typical of agricultural farming and animal facilities. The organic top layer of soil (natural humus) and the
soil mineral components (silica, iron oxide, and kaolin clay) can easily be tracked by humans or cloven-
hoofed animals, and Portland cement can be expected in dust from concrete flooring. Impurities from
agricultural equipment (used crankcase motor oil and carbon black), mineral oil (paraffin oil), and fatty
acids (stearic acid and oleic acid) common in animal and plant fats also are expected to be present in
agricultural grime. More information on each soil constituent is given below.
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• Natural humus - Humus is an organic layer of soil formed during the decomposition of plant
litter. Humus has a characteristic black or dark-brown color, is organic due to an
accumulation of organic carbon, and may act as a carbon source for microorganisms that
subsequently produce acids and contribute to weathering. Soil nutrients (nitrogen,
phosphorus, potassium, calcium, magnesium, manganese, iron, zinc, cadmium, and copper)
are also present in humus at levels from micrograms (jjg) to milligrams (mg) per gram (g)
soil.6
• Paraffin oil (mineral oil) - Paraffin oil, often referred to as mineral oil, is a mixture of liquid
hydrocarbons from petroleum. It does not have an exact chemical composition but is a
mixture of alkanes with the general formula CxH2x+2, with the value of "x" typically between 10
and 18. Mineral oils are used to produce animal feeds. Premixing micronutrients with mineral
oil, a suitable carrier, is common to ensure the proper distribution of nutrients in the final feed.
The carrier's purpose is to physically accommodate finely powdered micro-ingredients and
provide uniform distribution in the process. Mineral oils are chemically and biologically stable
and do not support bacterial growth.7
• Used crankcase motor oil - Used mineral-based crankcase motor oil is another name for
used motor oil or used engine oil. It is similar to unused oil except that it contains additional
chemicals produced or that build up in the oil when it is used as an engine lubricant. Used
mineral-based crankcase motor oil has many of the characteristics of unused oil. It smells
similar to unused oil and contains the chemicals found in unused oil, including straight-chain
(aliphatic) hydrocarbons, aromatic or polycyclic aromatic hydrocarbons (PAH) distilled from
crude oil, and various additives that improve the performance of the oil in the engine.
In addition to the chemicals found in unused oil, used mineral-based crankcase motor oil also
contains chemicals formed when the oil is exposed to the high temperatures and pressures
inside an engine. It also contains metals such as aluminum, chromium, copper, iron, lead,
manganese, nickel, silicon, and tin from engine parts as they wear down. In addition, used
mineral-based crankcase motor oil contains small amounts of water, gasoline, antifreeze, and
chemicals from gasoline when it burns inside the engine. These stay in the environment for a
long time, and can build up in plants, animals, soil, sediments, and non-flowing surface
water.8
• Portland cement (often referred to as "OPC," from "ordinary Portland cement") - OPC is the
most common type of cement in general use around the world.9. It is a basic mixture of
ingredients of concrete, mortar, stucco, and most non-specialty grout. It usually originates
from limestone. It is a fine powder produced by grinding. ASTM C150 defines Portland
cement as "hydraulic cement (cement that not only hardens by reacting with water, but also
forms a water-resistant product) produced by pulverizing clinkers consisting essentially of
hydraulic calcium silicates, usually containing one or more of the forms of calcium sulfate as
an inter-ground addition".
• Silica (silicon oxide) - Silicon and oxygen are the earth's two most abundant elements, and
together, they make silica, one of the earth's three most common rock-forming minerals.
Silica occurs in three main crystalline forms. The principal occurrence is as the mineral
quartz, but silica also occurs in other rarer mineral forms known as tridymite and cristobalite.
16
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It is a very durable mineral resistant to heat and chemical attack, and these properties have
made it industrially interesting to man.10
• Carbon black - Carbon black is a material produced by the incomplete combustion of heavy
petroleum products. It is a fine black powder consisting of nearly pure elemental carbon.
Carbon black is a form of Para-crystalline carbon that has a high surface area-to-volume
ratio, although lower than that of activated carbon. Unlike soot, carbon black has a much
higher surface area-to-volume ratio and significantly lower (negligible and non-bioavailable)
PAH content. Still, it is widely used as a model compound fordiesel soot fordiesel oxidation
experiments. Carbon black is used mainly as a reinforcing agent in vehicle tires and rubber
automotive products. Other common everyday products also often contain carbon black,
including inks, paints, plastics, and coatings.11
• Iron oxide - Iron oxides and oxide-hydroxides are widespread in nature, play an important
role in many geological and biological processes, and are widely used by humans (for
example, as iron ores, pigments, and catalysts). Common rust is a form of iron (III) oxide. Iron
oxides are widely used as inexpensive, durable pigments in paints, coatings, and colored
concretes.12
• Kaolin clay (aluminum silicate hydroxide, bolus, and hydrated aluminum silicate) - Kaolin is
a type of rock rich in kaolinite, a common layered silicate clay mineral, part of the group of
industrial minerals with the chemical composition Al2Si20s(0H)4. Kaolin clay occurs in
abundance in soils formed from the chemical weathering of rocks in hot, moist climates.
Kaolin is the most common mineral in clays. Kaolin is important in the production of ceramics
and porcelain. It also is used as a filler for paint, rubber, and plastics because it is relatively
inert and long lasting. But the greatest demand for kaolin is in the paper industry for
producing glossy papers such as those used in most magazines.
• Stearic acid (octadecanoic acid) - Stearic acid is a saturated fatty acid with an 18-carbon
chain. The International Union of Pure and Applied Chemistry (IUPAC) gives stearic acid the
name "octadecanoic acid." Stearic acid is a waxy solid occurring in many animal and
vegetable fats and oils, but it is more abundant in animal fat (up to 30%) than vegetable fat
(typically <5%).
• Oleic acid - Oleic acid is a monounsaturated omega-9 fatty acid abbreviated with a lipid
number of 18:1 cis-9. Oleic acid occurs naturally in various animal and vegetable fats and
oils. It is an odorless, colorless oil, although commercial samples may be yellowish. The term
"oleic" means related to or derived from oil or olive oil, an oil predominantly composed of oleic
acid.
3.2.1.2 Animal Sebum
Animal sebum was the major component (95% by weight in the final product) of the synthetic animal
grime used in this project. It also served as a liquid carrier for grime applications onto coupon surfaces.
In most animals, main wax production is associated with the sebaceous glands of the skin. Sebaceous
glands usually are associated with hair follicles, but there are also related structures on the eyelids called
"Meibomian glands." Sebaceous glands secrete mainly non-polar lipids in the form of sebum onto the skin
17
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surface. The rate of sebum excretion varies between species and often is measured using the sebum
saturation level (SSL), which represents the maximum amount of lipid that can accumulate on the skin
surface. Sebum production may also be affected by metabolism, environmental factors, and gender. For
example, in one study, sebum saturation levels for two breeds of cattle were 16.4 and 13.5 mg per 100
square centimeters (mg/100 cm2) at a thermo-neutral temperature (24 °C) and 31.2 and 67.2 mg/cm2 at a
constant high environmental temperature (32 °C).13 An animal with a skin surface area of 1 to 4 square
meters (m2) and a sebum excretion rate of approximately 20 mg/100 cm2 per day would excrete 200 mg
to 8 g of sebum a day).13
The composition of animal sebum varies between species. Although relatively few species have been
studied in detail, it is apparent that a wide range of lipid classes are present in the sebum of different
animal species. There also may be variation with age. The composition of human sebum differs
appreciably from that of other species, especially in the high content of triacylglycerols and in fatty acid
composition. Human sebum is unique in containing cis-6-hexadecenoic acid (6-16:1 or"sapienic" acid),
accompanied by an elongation and desaturation product, 5,8-octadecadienoic acid ("sebaleic" acid).
Sapienic acid is formed in the sebaceous glands by a distinctive A6 desaturase and has powerful
antibacterial properties. The skin of mammals also contains a wide range of more polar lipids based on
the ceramide backbone.14
Table 3.2-3 lists the relative composition (as a percentage of weight) of the non-polar lipids on the skin
surfaces of various species.
Table 3.2-3. Relative Composition of Non-Polar Lipids on the Skin Surfaces of Various Species14
Total % Weight
Species
Squalene
Sterols
Sterol
Esters
Wax
Esters
Diesters
Glyceryl
Ethers
T riacyl-
glycerols
Free
Acids
Free
Alcohols
Human
12
1
3
25
41
16
Sheep
12
46
10
21
11
Rat
1
6
27
17
21
8
1
Mouse
13
10
5
65
6
For this project, lanolin was selected as an animal sebum surrogate. Lanolin is the purified secretory
product of the sheep sebaceous gland. The raw material is referred to as "Adeps lanae," "wool wax,"
"wool fat," or "wool grease." Raw lanolin comprises 10 to 25% of the weight of sheared wool.14
Lanolin is a complex mix of fatty acids and alcohols, sterols (including cholesterol and lanosterol),
hydroxy acids, diols, and aliphatic and steryl esters.4 Because lanolin predominantly is composed of high-
molecular-weight esters, it is classified chemically as a wax, not as a fat.
Pure anhydrous lanolin is a semi-solid, clear to very slightly hazy, waxy substance. According to the U.S.
Pharmacopeia, lanolin is insoluble in water but mixes without separation with approximately twice its
weight of water.
For this project, pure pharmaceutical-grade lanolin was purchased from Sigma-Aldrich USA (see Section
3.2.2.1 for details). In a series of preliminary tests, the lanolin was confirmed to be free of the surrogate
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test organisms (B. atrophaeus and MS2) chosen for this study. In a series of additional solution-based
tests, pure lanolin was also shown to be mildly bacteriostatic and to affect the growth of MS2 negatively.
However, its overall compatibility with the surrogate test organisms did not hinder the pursuit of further
testing with a lanolin-based grime.
3.2.1.3 Animal Impurities
Manure is organic matter used as fertilizer in agriculture. There are two classes of manures in soil
management: green manures and animal manures. Green manures are used for crops grown for the
express purpose of plowing them under to increase soil fertility through the nutrients and organic matter
returned to the soil. Animal manure is the animal excreta (feces or excrement) of plant-eating mammals
(herbivores) and plant material (often straw) that has been used as bedding for animals and thus is
heavily contaminated with feces and urine (see Table 3.2-4).
Livestock manure has a variable composition, with solid and liquid portions as well as organic and
inorganic components. The composition of animal manure varies with livestock type, age, size, nutrition,
housing, and bedding as well as the nature and amount of materials (such as bedding and wastewater)
added to it.15 Table 3.2-4 lists manure components and their possible composition.
Table 3.2-4. Manure Components and Their Composition
Manure Component
Possible Composition
Feces
Undigested feed
Other bodily wastes
Pathogens
Pharmaceuticals
Organic forms of nutrients and organic acids
Inorganic forms of nutrients and salts
Urine
Water
Acids and salts
Nutrients (such as nitrates)
Bedding
Straw and wood fiber
Wasted solid feed
Water
Drinking water
Leaking or spilled water
Water from eaves, troughs, precipitation, and snow melt
Wash water and runoff
Facility wash water
Milking parlor wash water
Runoff from yards, stored feed, and manure
Animal manures are rich in nutrients and macro-elements (such as phosphorus, potassium, calcium,
magnesium, sodium) and contain some trace elements (such as iron, cobalt, selenium, manganese,
aluminum, arsenic, zinc, copper, chromium, and cadmium). Fresh manure is also a habitat for bacteria,
fungi, protozoa, nematodes, earthworms, insects (such as springtails and dung beetles), and other
arthropods (such as centipedes, millipedes, and pill bugs). Cow manure is rich in humus, the bulky and
fibrous material from undigested plant matter.
19
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For this project, the animal manure used was 50/50% dried homogenized swine/cattle manure prepared
by The Animal and Poultry Waste Management Center (APWMC) at North Carolina State University
(NCSU) in Raleigh, NC, USA.
Tables 3.2-5 and 3.2-6 summarize the comprehensive physical, biological, and chemical characteristics
of typical raw swine and cattle manure, respectively. The analytical data are from the Biological &
Agricultural Engineering Department of NCSU's Agronomic Division, North Carolina Department of
Agriculture & Consumer Services.5
Table 3.2-5. Swine Fresh Manure Characteristics
Parameter Concentration
Parameter Mass
Parameter
Unit
Obs.
Min
Max
Median
Mean
STD
lb/day/
head
STD
Manure (total feces and
urine)
lb/135
lb/day
81
4.0
23
11
11
3.3
11
82
Urine (total urine expressed
as part of manure)
%
manure
10
37
68
52
50
8.5
5.6
42
Density
lb/ft3
23
59
65
62
62
1.4
-
-
Total solids (dry matter)
% weight
78
2.9
28
10
10
4.7
1.1
8.5
Total suspended solids
%db
6
57
80
76
72
8.8
0.83
6.1
Volatile solids
%db
53
53
93
82
80
7.1
0.92
6.8
Volatile suspended solids
%db
2
66
68
67
67
0.76
0.77
5.7
Total alkalinity
mg/kg
1
250
250
250
250
-
0.0028
0.021
BOD
mg/kg
34
23480
48736
32868
37134
6020
0.41
3.1
COD
mg/kg
50
45500
243011
90528
102710
39307
1.1
8.4
Inorganic carbon
mg/kg
2
4970
7120
6045
6045
1075
0.067
0.50
Total organic carbon
mg/kg
7
9460
120866
24800
38699
35144
0.43
3.2
Volatile acids
mg/kg
4
1870
4270
4205
3638
1022
0.040
0.30
pH
2
7.0
8.1
7.5
7.5
0.57
-
-
TKN (as N)
lb/ton
61
3.2
27
12
12
4.6
0.068
0.50
NH3N (ammoniacal nitrogen
as N)
%TKN
15
35
93
58
62
19
0.042
0.31
N03N (nitrate nitrogen as N)
lb/ton
1
0.057
0.057
0.057
0.057
-
0.00032
0.0024
P205 (total phosphate)
lb/ton
56
2.4
24
8.0
9.3
4.4
0.052
0.38
P04 (orthophosphate)
%P205
1
69
69
69
69
-
0.036
0.27
K20 (potash)
lb/ton
55
2.4
19
8.9
8.8
4.1
0.049
0.36
Aluminum
lb/ton
3
0.074
0.097
0.097
0.089
0.011
0.00050
0.0037
Arsenic
lb/ton
1
0.017
0.017
0.017
0.017
-
0.000093
0.00069
Boron
lb/ton
6
0.060
0.096
0.084
0.082
0.011
0.00046
0.0034
Calcium
lb/ton
25
4.1
18
5.7
8.1
4.3
0.045
0.33
Cadmium
lb/ton
3
0.00018
0.0016
0.00018
0.00065
0.00067
0.0000036
0.000027
Chloride
lb/ton
2
5.0
7.5
6.2
6.2
1.2
0.035
0.26
20
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Parameter Concentration
Parameter Mass
Parameter
Unit
Obs.
Min
Max
Median
Mean
STD
lb/day/
head
STD
Cobalt
lb/ton
4
0.00040
0.0011
0.0011
0.00090
0.00029
0.0000050
0.000037
Copper
lb/ton
23
0.010
0.089
0.023
0.029
0.020
0.00016
0.0012
Iron
lb/ton
16
0.17
1.0
0.46
0.44
0.21
0.0024
0.018
Magnesium
lb/ton
25
1.0
4.4
1.6
1.8
0.77
0.010
0.074
Manganese
lb/ton
12
0.018
0.079
0.038
0.043
0.018
0.00024
0.0018
Molybdenum
lb/ton
5
0.000053
0.0020
0.00040
0.00066
0.00072
0.0000037
0.000027
Sodium
lb/ton
11
0.32
3.9
1.1
1.6
1.3
0.0089
0.066
Nickel
lb/ton
Estimated
-
-
-
0.0019
-
0.000011
0.000079
Lead
lb/ton
3
0.0016
0.0022
0.0022
0.0020
0.00028
0.000011
0.000082
Sulfur
lb/ton
14
0.24
2.9
1.7
1.8
0.96
0.010057
0.074
Zinc
lb/ton
24
0.074
0.31
0.11
0.12
0.056
0.00069
0.0051
Acid detergent fiber
%db
1
18
18
19
19
-
0.21
1.6
Crude fiber
%db
8
15
24
15
18
3.8
0.20
1.5
Crude protein
%db
9
20
35
24
25
3.9
0.28
2.1
Crude fat (ether extract)
%db
9
6.6
11
8.0
8.3
1.5
0.095
0.71
Nitrogen-free extract
%db
7
38
48
38
40
3.5
0.46
3.4
Total digestible nutrients
%db
2
48
68
58
58
10
0.67
4.9
Total protein
%db
2
16
16
16
16
0.18
0.18
1.3
Gross energy
kcal/g db
5
2.9
4.6
4.3
4.2
0.64
2158
15988
Enterococcus bacteria
col/100 g
2
5.50E+08
8.40E+08
6.95E+08
6.95E+08
1.45E+08
3.50E+10
2.59E+11
Escherichia coliform bacteria
col/100 g
1
1.00E+07
1.00E+07
1.00E+07
1.00E+07
-
5.03E+08
3.73E+09
Fecal coliform bacteria
col/100 g
6
6.50E+07
3.40E+08
3.30E+08
2.46E+08
1.23E+08
1.24E+10
9.17E+10
Fecal streptococcus bacteria
col/100 g
4
3.40E+08
8.40E+09
8.40E+09
6.39E+09
1.49E+09
3.21 E+11
2.38E+12
Streptococcus bacteria
col/100 g
2
3.00E+06
8.50E+07
4.40E+07
4.40E+07
4.10E+07
2.22E+09
1.64E+10
Total coliform bacteria
col/100 g
3
2.00E+08
1.10E+09
3.30E+08
5.43E+08
1.97E+08
2.74E+10
2.03E+11
Source:5
% - Percentage
BOD - Biochemical oxygen demand
COD - Chemical oxygen demand
col/100 g - Colonies per 100 grams
db - Dry basis (% of dry matter)
kcal/g - Kilocalorie per gram
lb/day - Pounds
per day
Ib/day/head - Pound per day per head
lb/ft3 - Pound per cubic foot
lb/ton - Pound per ton
mg/kg - Milligrams per kilogram
Obs - Observations
STD - Standard deviation
TKN - Total Kjeldahl nitrogen
21
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Table 3.2-6. Cattle Fresh Manure Characteristics
Parameter
Total No.
Obs
Parameter Concentration
Parameter Mass
STD Ib/da^hea gjp
d
Total solids (dry matter)
%wb
57
8.2
24
15
15
3.9
7.1
8.9
Total suspended solids
%db
3
73
83
73
76
4.9
5.5
6.8
Volatile solids
%db
36
53
99
85
82
10
5.8
7.3
Volatile suspended solids
%db
1
58
58
58
58
-
4.1
5.2
BOD
mg/kg
21
12750
49085
25004
28082
10180
1.4
1.7
COD
mg/kg
42
72917
239000
127095
130232
38382
6.3
7.9
Total organic carbon
mg/kg
3
40000
81496
58200
59899
16983
2.9
3.6
PH
4
6.5
7.3
7.0
7.0
0.34
-
-
Total Kjeldahl nitrogen (as N)
lb/ton
50
8.2
19
11
12
2.4
0.29
0.36
NH3N (ammoniacal nitrogen
as N)
%TKN
3
22
42
33
33
8.3
0.094
0.12
N03N (nitrate nitrogen as N)
lb/ton
2
1.3
1.6
1.4
1.4
0.13
0.035
0.043
P205 (total phosphate)
lb/ton
53
2.7
12
7.1
7.3
2.1
0.18
0.22
P04 (orthophosphate)
%P205
1
32
32
32
32
-
0.057
0.072
K20 (potash)
lb/ton
51
3.8
17
8.8
8.9
2.3
0.22
0.27
Boron
lb/ton
2
0.029
0.033
0.031
0.031
0.0022
0.00076
0.00095
Calcium
lb/ton
14
1.7
12
3.3
4.4
2.7
0.11
0.13
Cadmium
lb/ton
Estimated
-
-
-
0.00050
-
0.000012
0.000015
Chloride
lb/ton
Estimated
-
-
-
3.9
-
0.094
0.12
Copper
lb/ton
7
0.0045
0.018
0.0097
0.011
0.0044
0.00027
0.00033
Iron
lb/ton
9
0.077
0.67
0.24
0.29
0.21
0.0070
0.0088
Magnesium
lb/ton
16
0.85
3.0
1.7
1.7
0.53
0.041
0.051
Manganese
lb/ton
7
0.015
0.070
0.032
0.040
0.018
0.00097
0.0012
Molybdenum
lb/ton
1
0.0015
0.0015
0.0015
0.0015
-
0.000035
0.000044
Sodium
lb/ton
6
0.26
2.6
0.86
1.1
0.81
0.025
0.032
Nickel
lb/ton
Estimated
-
-
-
0.0069
-
0.00017
0.00021
Lead
lb/ton
Estimated
-
-
-
0.00056
-
0.000014
0.000017
Selenium
lb/ton
5
1.3
1.7
1.6
1.5
0.18
0.037
0.046
Zinc
lb/ton
8
0.027
0.060
0.030
0.034
0.010
0.00083
0.0010
Specific conductance
umhos/cm
1
3067
3067
3067
3067
-
-
-
Acid detergent fiber;
%db
6
31
47
42
41
5.2
2.9
3.7
Crude fiber
%db
7
17
38
31
27
8.0
1.9
2.4
Crude protein
%db
10
12
20
15
16
3.0
1.1
1.4
Crude fat (ether extract)
%db
6
2.3
6.5
2.8
3.4
1.4
0.24
0.31
Nitrogen-free extract
%db
4
33
53
49
46
17
3.3
4.1
Total digestible nutrients
%db
4
42
48
47
46
2.5
3.3
4.1
Gross energy
kcal/g db
3
4.1
4.8
4.7
4.5
0.30
14676
18345
Total anaerobic bacteria
col/100 g
1
2.40E+10
2.40E+10
2.40E+10
2.40E+10
-
5.28E+12
6.60E+12
Escherichia coliform bacteria
col/100 g
1
2.95E+11
2.95E+11
2.95E+11
2.95E+11
-
6.49E+13
8.11E+13
Enterococcus bacteria
col/100 g
4
1.10E+08
1.00E+09
4.23E+08
4.89E+08
3.70E+08
1.08E+11
1.34E+11
Fecal coliform bacteria
col/100 g
5
2.30E+07
1.10E+09
2.70E+08
4.83E+08
4.73E+08
1.06E+11
1.33E+11
Fecal streptococcus bacteria
col/100 g
4
1.00E+07
1.90E+09
1.30E+08
5.43E+08
7.85E+08
1.19E+11
1.49E+11
Total bacteria
col/100 g
3
1.50E+09
6.54E+12
1.00E+11
2.22E+12
3.06E+12
4.87E+14
6.09E+14
Total coliform bacteria
col/100 g
6
2.89E+07
2.50E+09
7.90E+08
1.09E+09
1.01E+09
2.41 E+11
3.01 E+11
Source:5
% - Percentage
|jmhos/cm - Micromhos per centimeter
BOD - Biochemical oxygen demand
COD - Chemical oxygen demand
col/100 g - Colonies per 100 grams
db - Dry basis (% of dry matter)
kcal/g - Kilocalorie per gram
Ib/day/head - Pound per day per head
lb/ton - Pound per ton
mg/kg - Milligram per kilogram
Obs - Observations
STD - Standard deviation
TKN - Total Kieldahl nitrogen
umhos/cm - Micromhos per centimeter
wb - wet basis (as is)
22
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3.2.2 Grime Preparation
This section discusses the raw materials of the grime, preparation of individual components of the grime,
and grime mixing.
3.2.2.1 Raw Materials
Raw materials for grime preparation either were obtained in their standardized or pure form from national
suppliers or prepared by trained personnel using standardized methods. Table 3.2-7 lists the raw
materials for grime preparation, trade names, manufacturers, and other information.
Table 3.2-7. Raw Materials for Grime Preparation
Raw Material
Trade Name or Composition
Manufacturer
CAS No.
Product No.
Natural humus
Ancient Forest 0.5 CF Humus
Soil Amendment
General Organics, USA
Not available
GH3200
Paraffin oil
Paraffin oil; puris
Sigma-Aldrich USA
8012-95-1
18512-1L
Used crankcase
motor oil
Not applicable
Local automobile service station
Not available
Not available
Portland cement
QUIKRETE® Portland Cement
QUIKRETE®, USA
Not available
1124
Silica
About 99% silicon dioxide, 0.5
to 10 micrometers (|jm)
Sigma-Aldrich USA
14808-60-7
S5631-1 KG
Carbon black
Raven 401
Powder Technology Inc., PTI, USA
Not available
Not available
Oleic acid
Oleic acid
technical grade, 90%
Sigma-Aldrich USA
112-80-1
364525-1L
Kaolin clay
Kaolin
Sigma-Aldrich USA
1332-58-7
18672-2.5KG
Iron oxide
Ferric oxide
Sigma-Aldrich USA
1309-37-1
310050-500G
Stearic acid
Stearic acid >95%
Sigma-Aldrich USA
57-11-4
W303518-1KG-K
Lanolin
Lanolin
Sigma-Aldrich USA
8006-54-0
L7387-1KG
Manure
Dried homogenized 50/50%
swine/cattle manure
APWMC at NCSU
Not available
Not available
3.2.2.2 Preparation of Individual Components
As summarized in Table 3.2-1, the grime was composed of particulate soil, lanolin, and manure. This
section discusses the preparation of each component.
Particulate Soil Preparation
Natural humus was dried and homogenized before use as a particulate soil component. The humus was
placed in a shallow tray and dried at 40 °C until a constant mass was achieved. After drying, the material
was sieved through a 3/8-in (9.5-mm) screen to extract large pieces (such as wood sticks and stones).
Then, the material was mixed and placed in a 150-mL container with three plastic balls and mixed in a
ball mill (SPEX SamplePrep dual mixer/mill, Metuchen, NJ, USA) for five to six minutes. The final product
was sieved through a 35-mesh (0.5-mm) screen. All other components were used as purchased.
In a plastic 250-mL Nalgene bottle, particulate soil was prepared by adding the ingredients listed in
Table 3.2-2 in the following order:
23
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• Natural humus (after sieving through 35-mesh screen): 38.0% by weight
• Paraffin oil: 1.0% by weight
• Used crankcase motor oil: 1.5% by weight
• Portland cement: 17.7% by weight
• Silica: 18.0% by weight
• Carbon black: 1.5% by weight
• Iron oxide: 0.3% by weight
• Kaolin clay: 18.0% by weight
• Stearic acid: 2.0% by weight
• Oleic acid: 2.0% by weight
After weighing, one part water of the mix volume was added, with a final volume of slurry not exceeding
approximately 60% of the container volume. Five plastic balls were added to the 250-mL container. The
slurry was then mixed in a ball mill (SPEX SamplePrep) for 30 minutes. After mixing, the slurry was
transferred to a shallow tray and dried overnight at 40 °C until a constant mass was achieved. The
material was turned over occasionally. After drying, the material was pulverized using a mortar and pestle
and then milled to pass a 35-mesh (0.5-mm) screen.
Lanolin Preparation
Immediately before preparation of the grime, neat lanolin was placed on a laboratory hot plate and
liquefied at 50 °C in its original amber glass container.
Manure Preparation
Standardized manure was prepared by the APWMC at NCSU from a 50/50% mix of fresh swine/cattle
manure. A representative sample of fresh manure was dried at 40 °C to constant mass and milled to pass
a 0.5-mm sieve. Figure 3.2-1 a shows a sample of the raw dried manure mix, and Figure 3.2-1 b shows the
final sample of dried homogenized manure.
Figure 3.2-1. Raw Dried Manure (a), arid Homogenized Sample of Manure (b)
24
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3.2.2.3 Mixing of Grime
Grime was prepared in a commercial paint shaker (Red Devil 5400, Red Devil Equipment, Plymouth, MN,
USA) using a two-step process: (1) preparation of concentrated grime, and then (2) preparation of final
grime. The two-step process simplified the process by reducing the amount of time required for working
with the liquefied lanolin.
First, the required amount of liquefied lanolin (50% by weight), particulate soil component (40% by
weight), and standardized manure (10% by weight) was added to a one-pint, paint-shaker-compatible
can. The can was capped loosely and placed in a 50 °C water bath for one hour. After one hour, the can
was closed tightly and mixed using the paint shaker for one hour. To keep the lanolin warm (in liquid
form), the primary container was placed in another bigger container filled with heated sand. After mixing
the concentrated grime solution, the cans were transferred to a chemical hood. The final grime was
prepared in a one-gallon can by mixing one part of the liquid concentrated grime (10% by weight) with
nine parts of liquid lanolin carrier (90% by weight) in the paint shaker for 30 minutes. The final grime
solution then was sterilized on a hot plate by gentle boiling at 106 ±,2 °C for 30 minutes (Figure 3.2-2a).
Then, the final grime solution was allowed to cool until solid (Figure 3.2-2b). The can then was tightly
closed and refrigerated.
Figure 3.2-2. Final Grime Solution Sterilization (a), and Final Batch of Solidified Grime (b)
2014/03/23
25
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3.2.3 Grime Application
This section discusses grime application on the large and small coupons.
3.2.3.1 Application of Grime on Large Coupons
Grime was applied onto building materials coupons using the liquefaction-solidification procedure
described below.
First, a large batch of sterile grime was liquefied at 80 to 100 °C. The grime was allowed to cool to
approximately 50 to 60 °C, and then individual 50-mL aliquots of liquid grime were aseptically transferred
to 50-mL, pre-weighed sterile conical tubes (Figure 3.2-3).
Figure 3.2-3. Grime Aliquot Preparation
Each conical tube was allowed to cool, and its weight was recorded to establish the amount of grime (in
grams) in each tube. After weighing, the grime aliquots were refrigerated until used in grime application
onto the coupons. Immediately before grime application, each batch of grime was subjected to a
10-minute-long heat shock in a hot water bath at 100 °C to ensure sterility.
Grime was applied onto the coupons in a pre-cleaned, Type II biological safety cabinet (BSC). Sterile
coupons of building materials were aseptically assembled in the BSC (Figure 3.2-4).
26
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Figure 3.2-4. Coupons Readied for Grime Application
Grime was applied to each coupon using a sterile paint roller. Rollers were labeled, pre-weighed, and
sterilized with EtO before use.
Prior to application, a batch of grime aliquots (in 50-mL conical tubes) was again liquefied and then kept
in a warm (50 to 60 °C) water bath to prevent the grime from solidifying. This temperature achieved the
optimal grime viscosity to allow even spreading but prevent runoff from the coupon edges.
Each coupon received the contents of two 50-mL conical tubes of grime. First, the entire content of one
conical tube was gently poured in the central part of each coupon and immediately spread using a paint
roller until the entire surface of the coupon was covered with grime (Figure 3.2-5). This step was
performed quickly to prevent premature solidification of the grime.
Figure 3.2-5. Grime Application Procedure (First Application)
27
-------
After application of the first tube of grime, the roller was placed in an empty, sterile specimen cup next to
the grimed coupon. The weight of the empty conical tube was recorded on a Grime Application Tracker
Form. This procedure was repeated for each coupon. The second aliquot (50 ml_) of grime was applied
30 minutes after the first application onto each coupon using the coupon-specific roller used for the first
application on that coupon. The weight of the second empty conical tube also was recorded. In addition,
the final weight of the roller after the second application was also recorded. Figure 3.2-6 shows a heavily
grimed concrete coupon after the second application of grime.
Figure 3.2-6. Heavily Grimed Large Coupon Surface after Second Grime Application
The average amount of grime applied onto concrete and plywood coupons using the procedure discussed
above was 54.98 g per 14- by 14-in coupon. The application method had high repeatability of grime
delivery for both materials (relative standard deviation [RSD] 8 and 13% for concrete and plywood
coupons, respectively).
3.2.3.2 Application of Grime on Small Coupons
Grime was applied on small coupons using the same liquefaction-solidification procedure described in
Section 3.2.3.1. Because of the small area of the coupon, 0.4 mL of the grime was poured into the central
area of the coupon using a sterile, 1-mL Finntip™ Flex Filter Pipette Tip (or equivalent). The grime was
allowed to spread over the entire surface of the coupon as shown in Figure 3,2-7.
28
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1
Figure 3.2-7. Heavily Grimed Small Coupon Surface after Second Grime Application
3.3 Test Organisms
Two types of test microorganisms were used for this project.
• B. atrophaeus, a surrogate for spore-forming bacterial agent B. anthracis
• Bacteriophage MS2, a surrogate for small, non-enveloped viral agents such as FMDV.
3.3.1 B. atrophaeus Surrogate for B. anthracis
B. atrophaeus is a soil-dwelling, non-pathogenic, aerobic, gram-positive spore-forming Bacillus species
related to B. subtilis. This bacterial species was formerly known as B. subtilis var. niger and subsequently
B. globigii. For more than six decades, this organism has played an integral role in the biodefense
community as a simulant for biological warfare and bioterrorism events. B. atrophaeus is commonly
referred to by its military two-letter designation "Bg." The taxonomic placement of B. atrophaeus has
changed dramatically over the years. Originally isolated as B. globigii in 1900 by Migula as a variant of
B, subtilis, it was at first distinguished from B. subtilis by the formation of a black-tinted pigment on
nutrient agar and by low rates of heterologous gene transfer from B. subtilis. Other than the formation of
the dark pigment, it is virtually indistinguishable from B. subtilis by conventional phenotypic analysis, and
the lack of distinguishing metabolic or phenotypic features has contributed to the confusion in the
taxonomy of this organism. Low interspecies DNA transfer frequencies suggest substantial divergence.
Based on analysis of comparative DNA hybridization, phenotypic tests, and biochemical tests, Gibbons et
al.16 advocate that pigment-producing 6. subtilis-\\ke isolates should be classified as a distinct species
termed B. atrophaeus. Recently, more sensitive typing methods such as amplified fragment length
polymorphism analysis show that B. atrophaeus strains could be classified into two major biovars: var.
globigii encompassing the classical commonly used Bg isolates and var. atrophaeus encompassing other
closely related yet genetically distinct strains.16
29
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The B. atrophaeus used for this project was a powdered spore preparation of B. atrophaeus 9372
(American Type Culture Collection [ATCC], product ATCC® 9372) and silicon dioxide particles purchased
from the U.S. Army's Dugway Proving Ground Life Sciences Division in Dugway, UT, USA. B. atrophaeus
spore preparation and inoculations are discussed below.
3.3.1.1 B. atrophaeus Spore Preparation
After 80 to 90% sporulation, the B. atrophaeus suspension was centrifuged to generate a preparation of
approximately 20% solids. A preparation resulting in a powdered matrix containing approximately 1 * 1011
viable spores per gram was prepared by dry blending and jet milling the dried spores with fumed silica
particles (Degussa, Frankfurt am Main, Germany). The powdered spore preparation was loaded into
metered-dose inhalers (MDIs) in accordance with an EPA proprietary protocol. The initial weight of each
MDI was verified using an Ohaus Adventurer Pro balance ARC120 (Ohaus Corporation, Parsippany, NJ,
USA). Ongoing control checks for each MDI were included in the batches of coupons contaminated using
a single MDI. The ongoing checks during use were performed using a Mettler-Toledo PL303 balance
(Mettler-Toledo, Inc., Columbus, OH, USA).
3.3.1.2 B. atrophaeus Spore Inoculations
Coupons were inoculated (loaded) with spores of B. atrophaeus using MDIs. Each coupon was
contaminated independently by placing it into a separate dosing chamber (ADA)17 designed to fit one
1.17- by 1.17-foot coupon of any thickness. The MDI was discharged once into the dosing chamber. The
spores were allowed to settle onto the coupon surfaces for a minimum of 18 hours.
The MDIs are claimed to provide 200 discharges per MDI. The number of discharges per MDI was
tracked so that use did not exceed this value. Additionally, each MDI was weighed after completion of the
contamination of each coupon. If an MDI weighed less than 10.5 g at the start of the contamination
procedure, it was retired and a new MDI was used. For quality control (QC) 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. The contamination control coupon was a stainless steel coupon measuring 1.17 by 1.17 feet and
inoculated, sampled, and analyzed.
3.3.2 Bacteriophage MS2 - Surrogate for Viral Agents
MS2 is an icosahedral ribonucleic acid (RNA) bacteriophage with triangulation number of T = 3 whose
capsid is formed by 180 copies of the coat protein, folded as seven antiparallel p-strands and two
helices18. Each face of the icosahedron is formed by trimers of coat protein. The virus shell has one copy
of an additional protein, A, associated with it. Similar to FMDV, MS2 is a small (25 to 30 nanometers),
non-enveloped, single-strand RNA virus.
MS2 was purchased from ATCC (product ATCC® 15597-B1 ™ in ATCC® Medium 271: Escherichia
medium). ATCC® 15597-B1 ™ uses ATCC® 15597™ Escherichia coii strain C3000 as the host. When
grown in presence of the E. coii, MS2 forms very hazy plaques with large halos in the Luria-Bertani (LB)
agar. Plaques vary in size. The phage was inoculated on LB agar before the E. coii and LB agar overlay.
30
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The host range of this phage has not been tested. It has been reported that the titer of the MS2 rapidly
drops if the MS2 is kept at refrigerator temperatures. The MS2 stock was stored in a refrigerator at 2 to 8
°C for short-term storage and -20 °C for long-term storage. The titer was tested before each use.
MS2 preparation and inoculations are discussed below.
3.3.2.1 MS2 Preparation
E.coli and MS2 preparation and plating are discussed below.
Preparation ofE. coli (ATCC 15597) Cell Stock
In a BSC, a vial of E. coli was aseptically removed from its packaging. A micropipette was used to
aseptically add 0.4 mL of LB broth to the vial containing the freeze-dried pellet. The pellet was suspended
by swirling or flicking the vial. Once mixed, the entire volume of the mixture was transferred to a sterile
tube containing 5 to 6 mL of LB broth. The tube containing the LB broth and E. coli mixture was incubated
at 35 ± 2 °C for 24 hours.
After incubation, two to three sterile tubes containing 10 mL of LB broth were inoculated with
100 microliters (|jL) of the E. coli overnight culture. These inoculated tubes were then placed in an
incubator overnight at 35 ± 2 °C. Once the incubation period was complete, a serological pipette was
used to aseptically combine the contents of the 10-mL tubes into a sterile 50-mL centrifuge tube.
The 50-mL centrifuge tube was placed in the centrifuge at 2,500 revolutions per minute (rpm) for
15 minutes. (The rpm speed and centrifugation time depend on the centrifuge and size of centrifuge tube
used to prepare the stock.). The supernatant from the centrifuge was decanted and then discarded. The
remaining cells in the tube were resuspended with 15 mL 10-millimolar magnesium sulfate and stored at
2 to 8 °C.
Preparation ofMS2 (ATCC 15597-B1) Stock
An active growing broth culture of E. coli (ATCC 15597-B1) was prepared in a BSC by inoculating a
sterile tube containing 5 mL of LB broth with 100 |jL of the E. coli bacterial cell stock prepared as
discussed in the paragraph above. The active growing broth culture of E. coli was incubated for 4 to
6 hours at 35 ± 2 °C.
After incubation, 100 |jL of the active growing broth culture of E. coli was inoculated into a tube containing
5 mL of LB top agar. The LB top agar and inoculum were quickly mixed, and care was taken not to
introduce air and create bubbles in the agar. After mixing, the contents of the tube were immediately
poured onto an LB agar plate. The LB top agar was spread evenly by gently swirling the plate. The vial
containing the freeze-dried pellet of MS2 was carefully and aseptically opened, and 0.5 mL of LB broth
was aseptically added to the freeze-dried pellet. The LB broth then was mixed with the pellet by carefully
swirling or flicking the vial until pellet was suspended. Then the surface of the LB agar was covered with
0.5 mL of the phage suspension. The plate with the phage suspension was incubated at 35 ± 2 °C for 24
hours.
31
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After incubation, the soft agar layer was scraped into a 50-mL centrifuge tube, and 15 mL of SM buffer
was added to the centrifuge tube. The tube containing the soft agar and magnesium salt (SM) buffer was
centrifuged at 7,000 rpm for 15 minutes. (The rpm speed and centrifugation time depend on the
centrifuge and size of centrifuge tube used to prepare the stock.) After centrifugation, the supernatant
was removed using a micropipette. The supernatant then was filtered through a 0.2-jjm filter.
Plating of Samples Containing MS2 (ATCC 15597-B1)
All sample dilutions were prepared in the same manner used for the B. atrophaeus samples. Three LB
agar plates were labeled for each dilution (plated in triplicate) and labeled using the same sample
identification number. Before the plating of the dilutions, the dilution tubes were vortex-mixed for
10 seconds, and then a portion of the dilution (100 |jL to 500 |jl_, depending on the target dilution) was
immediately inoculated onto the surface of an LB agar plate. A new pipette tip was used for each set of
replicate dilutions.
The sample inoculated on the LB agar was spread over the surface of the LB agar plate using a cell
spreader and a circular motion starting from the center of the plate and working outwards to the edge of
the plate. A tube containing 5 mL of LB top agar and 100 |jL of active growing E. coli solution was
removed from the water bath, swirled gently to homogenize it, and immediately poured onto the LB agar
plate. The top agar was evenly distributed by gently swirling the plate. The plates were allowed to sit
undisturbed on a level surface for a few minutes until the top agar layer solidified. All samples were
incubated at 35 ± 2 °C for 18-24 hours.
After incubation, all PFU were enumerated manually.
3.3.2.2 MS2 Inoculations
The MS2 was propagated as described in Section 3.3.2.1. Before use for experimental testing purposes,
MS2 concentrations were confirmed through plating.
After confirmation of the MS2 concentration, the MS2 stock was used to inoculate the coupons. The
sterile coupons, either in a grimed or neat state, were carefully and aseptically placed in a BSC. The MS2
inoculum was homogenized using a vortex mixer immediately prior to inoculation and again for every 10-
second time lapse that occurred during coupon inoculation (i.e., after each row of droplets was
dispensed). Using a standard 20- to 250-jjL positive-displacement micropipette and starting at the top
right of the coupon, twenty x 100 |jL droplets were applied onto each large coupon surface in accordance
with the pattern shown in Figure 3.3-1. For the small coupons, one 100 |jL droplet was applied at the
center of the surface of the using a positive displacement pipette (100-|jL droplet). The target surface
concentration for MS2 experiments was 1 * 108 PFU.
32
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4"
o o o o o
3"
~o o o o o
3"
o o o o o
o o o o o
< ~
2"
4"
Figure 3.3-1. Droplet Pattern Used for MS2 Inoculations on Large Coupons
For QC purposes, one stainless steel control coupon was inoculated in addition to test material coupons.
The time of inoculation was recorded. Sampling of the stainless-steel inoculation control was performed
within 10 minutes after inoculation (± 2 minutes).
33
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Decontamination Approach
This section discusses the decontamination materials and equipment, decontamination agents,
decontamination testing approach, large coupon decontamination testing, and small coupon
decontamination testing.
4.1 Decontamination Materials and Equipment
Changes in technique during the project could introduce variability and bias, thereby leading to erroneous
conclusions. Therefore, the decontamination materials and equipment summarized in Table 4.1-1 were
used in an attempt to provide as much standardization as possible.
Table 4.1-1. Decontamination Materials and Equipment
Material or Equipment
Description
Backpack sprayer
SHURflo ProPack™ SR600 Rechargeable Electric Backpack Sprayer, Cypress, CA
Chemical sprayer
Model UAG-1003HU, Pro-Chlorine Gas Powered Chemical Sprayer, Ultimate Washer Inc.,
Jupiter, FL
Handheld sprayer
RL FloMaster Model No. 56HD, Lowell, Ml
Bleach
Ultra Clorox® Concentrated Germicidal Bleach (EPA Reg. No. 67619-8); 8.3% sodium
hypochlorite; <1% sodium hydroxide
Vinegar
5% v/v technical grade acetic acid
Citric acid
Sigma-Aldrich USA No. 251275, American Chemical Society (ACS) grade, >99.5% pure,
CAS No. 77-92-9
Spor-Klenz®
STERIS Spor-Klenz® RTU liquid decontaminant (EPA Reg. No. 1043-119); 1% H2O2,
0.08% peracetic acid (PAA), <10% acetic acid
Nozzle
Standard brass, adjustable-flow garden hose nozzle 4 in long
Garden hose
75 feet long, 5/8-in diameter heavy duty rubber hose
Pressure regulator
Bronze pressure regulator, plumbing code-rated, standard, %-in National Pipe Taper (NPT)
female, 25 to 75pounds per square inch (psi)
Bucket of Dl water
3 gallons in a 5-gallon plastic bucket
Carboy container
5.25-Gallon (20-L) heavy-duty Nalgene plastic polypropylene carboy container,
autoclavable, leak proof, for full vacuum applications up to eight hours, U.S. Pharmacopeia
Convention (USP) class VI, vacuum-rated for intermittent vacuum use only, 83B closure
size
Pump
National Sanitation Foundation (NSF)-certified rotary vane pump forwaterwith motor,
brass, maximum capacity 4.3 gallons per minute (gpm), 3/4 horsepower
4.2 Decontamination Agents
This project focused on providing operationally feasible decontamination methods for the cleaning of
farming and animal facilities after contamination with a bacterial or viral agent. The study concentrated on
the following two classes of commonly used disinfectants:
• Oxidizers including pAB and Spor-Klenz® RTU; and
• Acids including 2% citric acid.
The following sections discuss each solution of decontamination agent used in this project.
34
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4.2.1 pAB Solution
Sodium hypochlorite (bleach) is a registered antimicrobial pesticide under the authority of FIFRA for use
as a sanitizer or disinfectant to kill bacteria, fungi, and viruses in households, food-processing plants,
agricultural settings, animal facilities, hospitals, and human drinking water supplies. However, bleach is
not a registered sterilant under FIFRA and does not have a registration claim to inactivate bacterial
spores (including B. anthracis). Published scientific data demonstrate that pAB reduced bacterial spore
populations under specific conditions related to concentration, pH, and contact time. Therefore, EPA has
issued crisis exemptions permitting the limited sale, distribution, and use of EPA-registered bleach
products against B. anthracis spores at a number of facilities and locations, including Capitol Hill, the U.S.
Postal Service Processing and Distribution Centers at Brentwood (Washington, DC, USA) and Hamilton
(Trenton, NJ, USA), the Department of State, the General Services Administration, and Broken Sound
Boulevard, Boca Raton, FL, USA. The application of bleach under crisis exemptions was limited to
specific buildings or treatment sites, and the specific conditions summarized below applied.
• Only hard, nonporous surfaces could be treated.
• A bleach solution close to but not above pH 7 (neutral), verified using a paper test strip at a
concentration of 5,000 to 6,000 ppm was prepared by mixing the following:
¦ One part bleach (with a 5.25 to 6% sodium hypochlorite 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).
• Treated surfaces had to remain in contact with the bleach solution for 30 minutes.
• Repeated applications were necessary to keep the surfaces wet.
• Treated PPE and containers removed from a treatment area required only 10 minutes contact
time with the bleach solution.
Although the chlorine content of the solution affects the time required for inactivation or overall
effectiveness, the pH of the solution has a much greater impact. Therefore, the comparative effectiveness
of alternative formulations (such as Clorox® Outdoor bleach having a higher sodium hypochlorite
concentration) is not easy to predict. The bleach formulation used in this study is the one that EPA used
previously under the crisis exemptions.
The concentration of household bleach and the strength of white vinegar can vary by batch and storage
time. Therefore, the formulation listed above can vary in pH and chlorine concentration depending on the
starting reagents. This source of variation can complicate a laboratory study such as this project by
skewing data, potentially leading to erroneous conclusions. To reduce the impact of "natural" variations in
the bleach solution for this project, the pH and chlorine content were measured at the start and monitored
throughout each test. The frequency of pH measurement was at a minimum at the start of testing of each
35
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coupon set. Data quality indicators (DQI) for the bleach solution are discussed in Section 9.2. The
solution had a mean pH close to but not above neutral (>6.5 and <7.0) and a mean total chlorine content
of 6,000 to 6,700 ppm. The temperature of the solution was 18 to 24 °C (64 to 75 °F). Any solution having
a pH, chlorine content, or temperature falling outside these ranges at anytime was discarded and a fresh
pAB solution was prepared.
The chlorine content was measured by titrating 5 mL of solution with a Hach high-range bleach test kit
(Method 10100). The pH and temperature were measured using an Oakton pH probe (OKPH502; pH5).
Dl water was used as the base for all solutions.
The pAB solution was prepared just before the initiation of testing on each day and was used within
three hours from the time of preparation. After three hours, the bleach solution was discarded and a fresh
pAB solution was prepared. However, a single preparation was used within a single coupon set. The pAB
solution was prepared as summarized below.
1. The concentration of the concentrated germicidal bleach tr 8.3% sodium hypochlorite) was
measured using a Hach test kit titration approach. If the calculated value was below 7.0%, the
feedstock was discarded and replaced with new bleach.
2. The pH-adjusted bleach consisted of 80% Dl water, 10% germicidal bleach (prepared in Step 1),
and 10% acetic acid. For example, 10 L of solution consisted of 1 L of prepared regular
germicidal bleach, 2 L of Dl water, 1 L of acetic acid, and 6 L of Dl water (in the order listed). The
solution was prepared in a container that accommodated the total volume of solution using a
funnel if necessary. The total volume was recorded as "Vstart" in the laboratory notebook.
3. The mixing container was sealed and gently agitated for mixing. The pH probe was placed into
the solution, and the pH was measured (target pH = 6.8). If the pH was above 7.0, small
increments of acetic acid were added. If the pH was below 6.5, germicidal bleach was added. The
Quality Assurance Project Plan (QAPP) discusses permitted adjustments. The volume required
for adjustment was recorded as "Vadd," and "Vtotai" was calculated as Vstart + Vadd in the laboratory
notebook.
4. The free available chlorine (FAC) was measured. The target FAC was 6,350 milligrams per liter
(mg/L). The acceptable range was 6,000 to 6,700 mg/L.
a. If the FAC exceeded the acceptable range, the total volume was diluted with Dl water by
the percent difference between the target FAC and the actual FAC.
Dilution volume = [(actual - target) target] x (Vtotai)
b. If the FAC was less than the acceptable range, bleach was added according to the
following equations:
Additional volume of bleach = (target - actual)/ target x Vtotai
Vtotai was recalculated for all the additions, and Steps 3 and 4 were repeated until both
parameters were met.
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4.2.2 Spor-Klenz® RTU Solution
Spor-Klenz® RTU is a broad-spectrum liquid disinfectant and sporicide registered with the EPA under
FIFRA (Registration No. 1043-119). Spor-Klenz® is a mixture of 1.0% H2O2, 0.08% PAA, and 98.92%
inert proprietary ingredients. The RTU variety of Spor-Klenz® was used for this study instead of the
concentrate (Registration No. 1043-120) to reduce the variation between experiments. Preparation of
diluted Spor-Klenz® from the concentrate for each day of testing would introduce unwanted variation.
Spor-Klenz® RTU requires no dilution before use. A new 3.2 Liter bottle of Spor-Klenz® RTU was used for
each day of testing. Unused Spor-Klenz® RTU was neutralized with STS, and discarded through the EPA
chemical services. Because Spor-Klenz® RTU is produced under manufacturer quality assurance (QA)
criteria, only temperature was a critical measurement for this liquid. The concentrations of H2O2 and PAA
in Spor-Klenz® RTU were verified during preliminary testing to help determine the amount of neutralizer
needed for quenching decontamination in liquid effluents from decontamination (see Section 5.2.2).
Spor-Klenz® is a sterilant and sporicide for nonporous surfaces when a 5.5-hour contact time (at 20 °C) is
used. This contact time far exceeds the planned contact times for this project because the study was
conducted to evaluate technologies under conditions realistic for their use in homeland security-related
remediation events. Maintaining a 5.5-hour contact time in a farming or animal facility would likely be
unrealistic for the amount of surface area requiring decontamination. Consistent with a previous study of
Spor-Klenz® RTU, a contact time of 30 minutes was therefore used. Spor-Klenz® RTU was applied using
a backpack sprayer and a chemical sprayer.
4.2.3 2% Citric Acid Solution
Citric acid is not sporicidal and therefore was used only for decontamination tests with MS2. Citric acid
occurs naturally in plants and in animal tissues and fluids and can be extracted from citrus fruit and
pineapple waste. Citric acid contains three carboxylic acid functional groups and has a molecular formula
of H3C6H5O7. Citric acid is an active ingredient in pesticide products registered for residential and
commercial use as disinfectants, sanitizers, and fungicides. These products, which contain citric acid in
combination with other active ingredients, are used to kill odor-causing bacteria, mildew, pathogenic
fungi, certain bacteria, and some viruses, and to remove dirt, soap scum, rust, slime, and calcium
deposits. Citric acid products are used in bathrooms and on dairy and food processing equipment. It can
be produced on an industrial scale by mold-based fermentation of carbohydrates such as molasses.
The first pesticide products containing citric acid as an active ingredient were registered in the early
1970s. Currently, three products containing citric acid and other active ingredients are registered for use
as fungicides and bactericides as described above. Citric acid is "generally recognized as safe," or GRAS
(see Section 21 of the Code of Federal Regulations, 182.1033). Acidic disinfectants function by
destroying the bonds of nucleic acids and by precipitating proteins.
Acids also change the pH of the environment, making it detrimental to many microorganisms. Numerous
studies have demonstrated the antimicrobial efficacy of acetic acid, citric acid, and sodium bicarbonate
using suspensions of bacteria and recovery from treated hard surfaces, and in meat-rinsing and produce-
washing operations. In studies, the efficacy of citric acid as demonstrated in suspensions (in the absence
37
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or presence of organic matter) was drastically different from its efficacy demonstrated on produce20. Study
results suggest that vinegar (acetic acid) exhibits the most antimicrobial efficacy, followed by lemon juice
(citric acid) and baking soda (sodium bicarbonate)21. Typically, gram-negative bacteria such as Shigella
sonnei, Salmonella spp., E. coli, Pseudomonas aeruginosa, and Yersinia enterocolitia are more
susceptible to organic acids (such as acetic acid and citric acid) than gram-positive bacteria such as
Staphylococcus aureus and Listeria monocytogenes. The highly cross-linked cell walls of gram-positive
bacteria are believed to impair the diffusion of the organic acids into the cell, preventing antimicrobial
action.
Acids are generally highly virucidal. With the correct choice of acid or acid mixture, they can be used
under a wide variety of conditions, including residential cleanup. Citric acid is a milder acid available in
solid form that is active against acid-sensitive viruses and can be used safely for personnel and clothing
decontamination. It is particularly useful when added to detergents for the inactivation of the FMDV.
Solutions of 0.2% citric acid at 30 minutes contact time are suggested as a safe decontamination option
for clothes and the body, especially for FMDV decontamination. Previous studies on birch wood carriers
have shown that 2% citric acid is an effective disinfectant for FMDV.22
For this project, citric acid was purchased from Sigma-Aldrich USA and its 2% (weight per volume)
solution was prepared using sterilized Dl water. The concentration of citric acid was confirmed before
each experiment through titration with sodium hydroxide. A total contact time (visibly wetted surface) of
30 minutes was used for citric acid.
4.3 Decontamination Testing Approach
Key operating parameters of the decontamination procedure included the following:
• Type of decontaminant
• Presence of grime or organic matter
• Application mode of decontaminant diluted pAB solution using a pressurized (chemical or
backpack)sprayer
• Contact time of 30 minutes, followed by a rinse step.
Each test included five test coupons for each material type, three positive control coupons, one
procedural blank coupon, and one negative control coupon. Therefore, a total of 10 coupons were
required for each material type per test. The additional stainless steel coupons were used during the
inoculation phase as inoculation controls. Procedural blanks (coupons of each material not contaminated
with the test organisms) were run first, followed by the test coupons. The test chamber was cleaned both
before and after the procedural blank test.
After the decontamination steps, the coupon surfaces were sampled to determine the efficacy of the
combination of operational parameters and decontamination approaches. Moistened sterile non-cotton
wetted wipes were used to conduct surface sampling of the coupons, as described in Section 6.2.1.
Liquid effluent (runoff) samples, rinse water samples, and air samples collected during the
decontamination process also were analyzed to determine the fate of the test organisms (See Section
6.2.2).
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4.4 Large Coupon Decontamination Testing
Table 4.4-1 below shows the test matrix for B. atrophaeus and MS2 testing of the large coupons.
Table 4.4-1. B. atrophaeus and MS2 Decontamination Test Matrix for Large Coupons
Test
Decontamination
Decontamination
Micro-
Material
Coupon
Total No. of
Application Method
Liquid
organism
Type
Condition
Coupons*
1
Concrete
Neat
10
2
pAB
Grimed
10
3
T reated
Neat
10
4
Backpack Sprayer
Plywood
Grimed
10
5
Concrete
Neat
10
6
Spor-Klenz® RTU
Grimed
10
7
CO
^3
CD
T reated
Neat
10
8
CO
•C
Plywood
Grimed
10
9
J2
0Q
Concrete
Neat
10
10
pAB
Grimed
10
11
T reated
Neat
10
12
Chemical Sprayer
Plywood
Grimed
10
13
Concrete
Neat
10
14
Spor-Klenz® RTU
Grimed
10
15
T reated
Neat
10
16
Plywood
Grimed
10
17
Concrete
Neat
10
18
pAB
Grimed
10
19
T reated
Neat
10
20
Backpack Sprayer
CM
CO
Plywood
Grimed
10
21
2
Concrete
Neat
10
22
2% (v/v) citric acid
Grimed
10
23
T reated
Neat
10
24
Plywood
Grimed
10
I *Five test coupons: three positive control coupons, one procedural blank coupon, and one negative control I
| coupon.
I
This section discusses the test chamber, application of decontaminants using sprayers, post-
decontamination rinse, and decontamination chronology for the large coupon tests.
4.4.1 Test Chamber for Large Coupons
Decontamination testing for large coupons was conducted in a spray chamber located at EPA's Research
Triangle Park facility in North Carolina. Briefly, sets of the building material coupons were inserted in a
vertical position in the test coupon holders of the spray test chamber as shown in Figure 4.4-1.
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Figure 4,4-1. Decontamination Test Chamber for Large Coupons
The test chamber measured 4 feet high by 4 feet wide by 4 feet deep and was designed to accommodate
three large coupons at a time in a horizontal or vertical position. For this project, only the vertical
assembly was used. The chamber was constructed of solid stainless steel except for the front face and
top, which were constructed of clear acrylic plastic.
The reverse-pyramid design of the chamber bottom allowed the collection of runoff from the coupons
during the decontamination procedure through a central drain 3 in in diameter. The bottom of the
chamber had a collection capacity of 189 L (50 gallons).
The chamber was fitted with connections allowing air to exit through a readily accessible connection to
the facility's air handling system. Aerosol samples were collected from the chamber exhaust duct using
Via-Cell® bioaerosol cassettes (Zefon International; Ocala, FL). The sampling points were eight diameters
downstream and two diameters upstream of any flow disruptions.
4.4.2 Application of Decontaminants Using Sprayers for Large Coupons
Decontaminant was applied using a backpack sprayer (SHURflo ProPack™ SR600 rechargeable electric
backpack sprayer, SHURflo Inc., Cypress, CA, USA) or chemical sprayer (Model UAG-1003HU, Pro-
Chlorine gas powered chemical sprayer, Ultimate Washer, Inc., Jupiter, FL, USA). The acrylic door of the
test chamber was fitted with three ports, one per coupon, allowing insertion of the sprayer wand into the
central area in front of each vertical coupon, as shown in Figure 4.4-2. Each sprayer type and its use for
applying decontaminants is discussed below.
40
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Figure 4.4-2. Spraying Through Center-Aligned Port in the Test Chamber Door
4.4.2.1 Backpack Sprayer
The decontamination solution was prepared inside a four-gallon electric backpack sprayer (see Figure 4.4-
3).
Figure 4.4-3. Electric Backpack Sprayer
The sprayer was rinsed with Dl water and filled with the decontamination solution. The flow rate of each
sprayer was verified before each test using a 500-mL graduated cylinder and a stopwatch (liquid collected
and volume recorded from 10 seconds of spraying time). The spray pattern was tested by spraying at the
appropriate distance of 1 foot onto a piece of 14- by 14-in blue construction paper mounted in the test
41
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chamber in the vertical orientation corresponding to a test coupon. The spray was discharged onto the
center of the paper, and the pattern was visually assessed for consistency.
To apply the decontamination solution to the coupons, a set of three replicate coupons was installed
vertically in the test chamber. After confirmation that all QC requirements were within specifications, the
coupons were sprayed through the center port of the chamber door. The sprayer wand was inserted
through the port, keeping the doors closed to minimize the exposure of the worker to toxic fumes from the
decontamination solution during application. The spray nozzle was kept a distance of approximately 1
foot (± 2 in) from the coupon surface for the backpack sprayer tests. A spray pressure of 35psi was
maintained by the backpack sprayer. At this constant pressure, the target flow rate of the
decontamination solution was set to 1.2 liters per minute (Lpm), with a cone spray pattern having a 14-in
diameter at 1 foot from the surface of the coupon.
The flow and spray pattern were checked at the start and end of each set of spray applications. Figure
4.4-4 shows the spray pattern and pass order. Each pass was set to five seconds, for a total spray time of
15 seconds per application. The spray pattern shown in Figure 4.4-4 was performed in one continuous
application, starting in the top left corner and ending in the bottom right corner. The passes are shown
separately in Figure 4.4-4 only for illustration purposes and to show total coverage.
Pass 1 —Top left corner to top right corner
"
Pass 2 - Middle right side to middle left side
¦4
Pass 3 - Bottom left corner to bottom right corner
~
I II I
Figure 4.4-4. Spray Pattern for Backpack Sprayer
42
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The spray wand was moved back and forth as evenly as possible to cover the surface of all three
coupons completely. This step was repeated as often as necessary to satisfy the required spray duration
time of 15 seconds per application. After a 15-minute exposure time, the decontaminant was re-sprayed
using the method described above (15-second spray time). After the second application of
decontamination solution, another 15-minute exposure time was provided, for a total exposure time of 30
minutes.
4.4.2.2 Chemical Sprayer
The chemical sprayer uses a low-pressure diaphragm pump for spraying corrosive solutions such as
those containing sodium hypochlorite. The gasoline-operated pump requires an external source of the
chemical to be sprayed. The decontamination solution was prepared inside a source container. During
operation, chemical not being sprayed was returned to the source container.
The source container was rinsed with Dl water and filled with the decontamination solution. The flow rate
of each sprayer was verified before each test using a 500-mL graduated cylinder and a stopwatch (to
verify 10 seconds of spraying time). The spray pattern was tested by spraying at the appropriate distance
of three feet onto a piece of 14- by 14-in blue construction paper mounted in the test chamber in the
vertical orientation corresponding to a test coupon. The spray was discharged onto the center of the
paper, and the pattern was visually assessed for consistency.
To apply the decontamination solution to the coupons, a set of three replicate coupons was installed
vertically in the test chamber. After confirmation that all QC requirements were within specifications, the
coupons were sprayed through the center port of the chamber door. The sprayer wand was inserted
through the port, keeping the doors closed to minimize the exposure of the worker to toxic fumes from the
decontamination solution during application. The spray nozzle was kept a distance of approximately three
feet (± two in) from the coupon surface for the chemical sprayer tests. The chemical sprayer is reported
by its manufacturer to achieve a pressure of 200 psi. At this constant pressure, the flow rate was
maintained at 11 L/min (2.9 gal/min), with a cone spray pattern having a 14-in-diameter at three feet from
the surface of the coupon. The flow and spray pattern were checked at the start and end of each set of
spray applications.
A set of three replicate coupons was installed vertically in the test chamber, and the coupons were
sprayed using the same procedure, pattern, and exposure time discussed in Section 4.4.2.1 for the
backpack sprayer.
4.4.3 Post-Decontamination Rinse for Large Coupons
After the 30-minute exposure time, the large coupons were rinsed with Dl water using the standard
garden hose nozzle listed in Table 4.4-1. The rinse step was used to simulate field operations in which
rinsing may be used to minimize collateral damage to facilities resulting from extended contact with harsh
decontamination chemicals. The water was supplied to the nozzle through the garden hose listed in Table
4.4-1 from a 60-gallon tank reservoir by the pump listed in Table 4.4-1 to provide a pressurized stream.
The head pressure was constantly maintained at approximately 60 psi using the pressure regulator listed
in Table 4.4-1. An Oakton pH probe was maintained in the Dl water reservoir to monitor the pH and
temperature continually.
43
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The three coupons were rinsed in the same manner as the decontamination solution was applied, with
side-to-side strokes using the pattern shown in Figure 4.4-4 from the central port of the test chamber. The
spray was controlled using the nozzle to have a one-foot diameter measured at threex feet from the
nozzle. Each rinse was conducted for 10 seconds. The start time and duration of rinsing were recorded
and performed as consistently as possible across all coupons.
After rinsing, the coupon positions were recorded on a Coupon Tracker form. Then each coupon was
removed from its position in the test chamber and placed on a drying pan. Each drying pan containing a
coupon then was moved to a designated storage cabinet and allowed to dry overnight. Negative control
coupons were transferred to the blank coupon cabinet, and decontaminated coupons were transferred to
the decontaminated coupon cabinet.
The volume, FAC, pH, and temperature of the rinsate were recorded, and rinsate samples were collected
for microbiological analyses as discussed in Section 6.4.2.
4.4.4 Decontamination Chronology for Decontamination Testing (Large
Coupons)
The backpack and chemical sprayers were used to apply the pAB, Spor-Klenz® RTU, and 2% (v/v) citric
acid solutions to the large coupons. The decontamination chronology for each microorganism testing for
the large coupons is summarized below.
Day 0
Coupon Inoculation
• Five test and three positive control coupons were inoculated with spores of B. atrophaeus
through aerosol deposition using an MDI or with MS2 through liquid inoculation.
• Coupons were then moved to coupon storage cabinets. Non-contaminated control coupons
were transferred to a blank coupon cabinet, and contaminated coupons were transferred to a
positive coupon cabinet. Positive control coupons and material sterility blank coupons were not
used for decontamination testing and remained in their respective cabinets until sampling.
Day 1
Test Chamber Preparation and Air Sampling Cassette Assembly
• The test chamber was sterilized using pAB solution prepared as a 1:10 dilution of bleach in Dl
water, pH-adjusted to ~6.8 using glacial acetic acid.
• The gaskets used during the inoculation procedure were cleaned via fumigation with a STERIS
VHP sterilization cycle, which maintained a constant H2O2 concentration of 250 parts per million
by volume (ppmv) in a decontamination chamber for four hours.
• Via-Cell® bioaerosol cassettes for air sampling were assembled prior to testing.
Coupon Assembly in the Test Chamber
44
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Three coupons at a time were inserted into the test chamber. All coupons were tested in the
vertical orientation.
Coupons were inserted into the test chamber using sterile gloves, taking care not to touch the
coupon surface.
Liquid Decontaminant Solution Preparation
The procedure for preparing pAB solution is discussed in Section 4.2.1. Critical operational
parameters consisted of FAC, pH, and temperature measurements.
As discussed in Section 4.2.2, the Spor-Klenz® RTU formulation was used as is without
dilution. The temperature and pH of the Spor-Klenz® RTU solution were recorded, but only
temperature was considered a critical measurement.
Runoff and Rinse Water Collection
A clean, sterile carboy (Carboy No. 1) loaded with neutralizer was placed under the drain of the
test chamber to collect liquid effluent type 1 (runoff of liquid decontaminant) during the
decontamination testing. A second clean sterile carboy (Carboy No. 2) loaded with neutralizer
was placed under the drain of the test chamber to collect liquid effluent type 2 (water from
rinsing of the liquid decontaminant). Approximate volumes of material-specific runoff and
rinsate were assessed during liquid effluent characterization tests (Section 5.2.2). The amount
of neutralizer needed for each material type was determined in preliminary testing (see Section
5.2. for details).
Decontaminant Solution Application
Application of liquid decontaminant was performed using either a backpack or chemical sprayer
as discussed in Section 4.4.2.
Post-Decontamination Coupon Rinse
The post-decontamination rinse of the large coupons was conducted as discussed in
Section 4.4.3.
Assessment of Surface Damage on Coupons
The surfaces of the procedural blank and positive control coupons were visually inspected side-
by-side before and after decontamination of the procedural blanks. Differences in color,
reflectivity, and roughness were qualitatively assessed, and observations were documented in
the laboratory notebook. Time- and date-stamped digital photographs were taken to document
any observed surface change.
45
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Day 2
Sampling of Test Coupons
• After a minimum of 18 hours and when all coupons surfaces were visibly dry, the coupons were
sampled using wipe sampling as discussed in Section 6.4.1.
Transfer of Samples for Microbiological Analysis
• Samples were transferred in sterile, primary, independent packaging within sterile, secondary
containment in logical groups for analysis. All samples were accompanied by a completed
chain of custody form.
4.5 Small Coupon Decontamination Testing
Table 4.5-1, below, shows the test matrix for MS2 testing of the small coupons.
Table 4.5-1. MS2 Decontamination Test Matrix for Small Coupons
Decontamination
Application
Method
Decontamination
Liquid
Exposure
Time
Material
Type
Coupon
Condition
Total No. of
Coupons*
Total No. of
Effluents**
Handheld sprayer
2% citric acid
30 minutes
Concrete
Neat
13
8
Grimed
13
8
pAB
30 minutes
T reated
Plywood
Neat
13
8
Grimed
13
8
*Five test coupons: three procedural positive controls, one procedural blank, and one negative control.
**Eight effluents: Five test rinsate samples and three procedural positive rinsate samples.
This section discusses the spray apparatus, decontamination procedure, and control testing for MS2
testing for the small coupons.
4.5.1 Spray Apparatus for Small Coupons
A bench-scale test spray apparatus was used to evaluate the pAB and citric acid spray-based
decontamination methods for the small coupons (both grimed and neat) contaminated with MS2. Figures
4.5-1 through 4.5-3 show the apparatus used.
46
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^ wi i li^
Figure 4.5-1. Front View of Spray Apparatus with Orifice Plate
Figure 4.5-2. Side View of Spray Apparatus with Orifice Plate
47
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Figure 4.5-3. Front View of Spray Apparatus without Orifice Plate
In this apparatus, each test coupon was attached to a specially designed funnel connected to a conical
50-rnL collection vial (Fisher Scientifics, BD Falcon, Catalog No. 352098) to retain runoff generated
during spraying with the target decontaminant fluid. The coupon was attached horizontally though a
custom-made connector.
The decontamination solution was applied using a 1,75-L chemical- and break-resistant, adjustable,
commercial handheld sprayer (RL FloMaster Model No. 56HD) made of high-density polyethylene with a
Viton seal. The handheld sprayer includes a pump trigger that can provide controlled delivery and was
adjusted to deliver a fine mist to minimize runoff and dripping of the decontaminant. The sprayer also was
fitted with a pressure gauge (<30 psi). The entire spray bottle was disinfected by rinsing and purging with
decontaminant solution three times before the bottle was filled with the final decontaminant solution of the
target formulation.
The spray pattern was controlled by placing an orifice between the spray bottle and the sample to confine
the spray as tightly as possible to the coupon. The spray nozzle was maintained at a distance of six in
from the surface of the coupon. Pre-test method development trials were conducted to determine the
spray duration required to fully wet the coupon surface yet minimize decontaminant solution runoff and
overspray. These spray conditions and resulting decontaminant volumes were then used to determine the
volumes of neutralizer required for pre-loading in the collection vials to fully quench decontaminant
activity and achieve a precise contact time.
4.5.2 Decontamination Procedure for MS2 Testing (Small Coupons)
The small coupon efficacy testing for MS2 included 2% citric acid as well as pAB. Each coupon was
inoculated with 1 x 108 PFU of MS2. Test samples were tested in quintuplicate, with exposure contact
time of 30 minutes. Four material types (grimed concrete, grimed plywood, neat concrete, and neat
plywood) were tested during this phase. The decontamination procedure for MS2 on small coupons is
summarized below.
48
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1. The commercial handheld sprayer was rinsed with decontamination solution three times to
sterilize and disinfect the sprayer.
2. After triple-rinsing of the sprayer, the decontamination solution was discarded, and then the
sprayer was filled with at least 500 mL of decontamination solution (the minimum volume
required in the sprayer to ensure consistent spraying).
3. The sprayer was pressurized by pumping to 20 psi.
4. The conical tube was pre-loaded with the required amount of DE broth and PBST neutralizer
solution.
5. The runoff collection vials were preloaded with the required amount of neutralizer solution.
6. The inoculated coupons were aseptically installed in the decontamination spray apparatus with
50-mL conical tube runoff collection vials.
7. The sprayer was held six in from the spray apparatus orifice plate, and each coupon was
sprayed for 10 seconds. The five test coupons and one procedural control coupon were sprayed.
The resulting volume of decontaminant used on each coupon was recorded.
8. The coupons were allowed to sit for the required 15-minute initial exposure time, and were then
sprayed again for five seconds. The coupons were then allowed to sit for an additional 15-minute
exposure time.
9. Each decontaminated coupon was aseptically extracted into its respective coupon collection vial
after a total exposure time of 30 minutes.
10. The funnels were rinsed with sterile Dl water, and this rinsate was collected in the rinsate
collection vials. The total volume in each runoff collection did not exceed 35 mL.
11. Samples were transferred into sterile, primary, independent packaging within sterile, secondary
containment in logical groups for analysis. All samples were accompanied by a completed chain
of custody form.
49
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Neutralizing Agents for Extracted Samples
A series of tests was conducted to identify the optimal neutralizing agent, if any, for each decontaminant
and to determine its effectiveness in neutralizing (quenching decontaminant activity) and maintaining the
integrity of the samples potentially containing viable B. atrophaeus spores or MS2. For B. atrophaeus,
sodium thiosulfate (STS) was used as the neutralizing agent for pAB and potassium carbonate was used
as the neutralizing agent for Spor-Klenz® RTU. For MS2, DE broth was used as the neutralizing agent for
2% citric acid and pAB.
This section discusses neutralization agent preparation, neutralization for large coupons, and
neutralization for small coupons.
5.1 Neutralization Agent Preparation
STS was prepared in 1 L batches, in a 1-L volumetric flask. The solution was prepared at a 2 Normal (2
N) strength as described below.
1. STS pentahydrate (Na2S2C>3 5H2O, 496.4 g) crystals were added to 1 L of Dl water in an
Erlenmeyer flask.
2. The solution was stirred until all the crystals dissolved completely.
3. Once the crystals dissolved completely in water, the solution was transferred to a 1-L volumetric
flask. Additional Dl water was added if needed until the lower meniscus of the solution aligned
with the gradation line of the volumetric flask.
4. The 2 N STS solution was then sterilized using a bottle-top filter (150-mL Corning Bottle Top
Filter, 0.22-jjm cellulose acetate (CA), 33-mm neck, sterile, Catalog No. EK-680516, Corning,
NY, USA). The liquid was poured 150 mL at a time into the top part of the filtration unit, which
was connected to a sterile bottle to collect the filtrate. Vacuum was used to pull the liquid through
the filter.
5. Each batch of STS solution prepared in-house was used within six months of preparation.
Potassium carbonate was prepared in 1 L batches, in a 1-L volumetric flask. The solution was prepared at
a 2 Molar (2 M) strength as described below.
1. Potassium carbonate (K2CO3, 276.41 g) crystals were added to 1 L of Dl water in an Erlenmeyer
flask.
2. The solution was stirred until all the crystals dissolved completely.
3. Once the crystals dissolved completely in water, the solution was transferred to a 1-L volumetric
flask. Additional Dl water was added if needed until the lower meniscus of the solution aligned
with the gradation line of the volumetric flask.
4. The 2 M K2CO3 solution then was sterilized using a bottle-top filter (150-mL Corning Bottle Top
Filter, 0.22-jjm CA, 33-mm neck, Sterile, Catalog No. EK-680516, Corning, NY, USA). The liquid
was poured 150 mL at a time into the top part of the filtration unit, which was connected to a
sterile bottle to collect the filtrate. Vacuum was used to pull the liquid through the filter.
50
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5. Each batch of potassium carbonate solution prepared in-house was used within six months after
preparation.
DE broth was prepared in 1 L batches, in a 1-L volumetric flask. The solution was prepared at a 1X
(100%) strength as described below.
1. Dehydrated DE broth media granules (1,000 g) were added to 1,000 mL of Dl water in an
Erlenmeyer flask.
2. The flask was placed on a heated stir plate and heated gently to dissolve the broth granules
completely.
3. The solution was carefully transferred to an autoclave-safe glass bottle and autoclaved at 121 °C
for 15 minutes.
4. Each batch of DE broth prepared in-house was stored below 8 °C, protected from direct light.
5.2 Neutralization for Large Coupons
Neutralization of the sample extraction buffer for the large coupons required surface neutralization tests
and neutralization tests for liquid effluents as discussed below.
5.2.1 Surface Neutralization Tests
Table 5.2-1 summarizes the tests conducted on large coupons necessary to investigate the need for
neutralizer within sample extraction buffers after post-decontamination sampling during pAB orSpor-
Klenz® RTU testing with B. atrophaeus.
Table 5.2-1. Neutralization Tests for Extractive Samples
Test
Decontaminant*
Material Type
Total No. of Coupons
A1
pAB
Concrete
3
A2
Spor-Klenz® RTU
Concrete
3
A3
None (water only)
Concrete
3
A4
pAB
Plywood
3
A5
Spor-Klenz® RTU
Plywood
3
A6
None (water only)
Plywood
3
| *Decontaminant applied using sprayer |
These tests were performed before the testing of each decontamination solution using representative sets
of concrete and plywood coupons. Completion of the test matrix was expected to provide information on
whether or not neutralization of samples after collection was necessary.
Representative non-inoculated coupons were subjected to decontamination procedures and allowed to
dry for approximately 18 hours before undergoing surface sampling. After sampling, the sample extracts
were spiked with 1 * 107 CFU of B. atrophaeus spores. Recoveries for samples collected from
decontaminated coupons were compared with recoveries for samples collected from extracted blank (not
decontaminated) coupons (Tests A3 and A6). If a statistically significant difference existed for each
coupon type between the two populations (CFU for decontaminated coupons vs. CFU from blanks
51
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sprayed with water only), then it was determined that the samples required neutralization after collection.
If needed, the various neutralizing solutions discussed in Section 5.2.1 were tested to confirm that the
presence of the neutralizer did not bias recovery negatively. The spore inocula were enumerated in
triplicate at the beginning, middle, and end of spiking.
5.2.2 Neutralization Tests for Liquid Effluents
The continued action of the decontamination agents (pAB, Spor-Klenz® RTU [H2O2 and PAA], and citric
acid) in liquid effluents collected during the decontamination process (in runoff and rinsate water) may
bias recovery. Therefore, as discussed in Section 4.4.4, carboys used to collect liquid effluents were pre-
loaded with liquid neutralizer solution, to prevent loss of viable spores/plaques after the prescribed
contact time.
Neutralization tests were performed before decontamination testing using concentrations of active
ingredients determined in runoff and rinsate water during characterization tests of process-specific liquid
effluents as described below.
Liquid Effluent Type 1
1. Blank coupons were assembled in the test chamber.
2. Three coupons of concrete or plywood material sprayed using a backpack or chemical sprayer
were evaluated to account for inter-material differences of the decontamination procedure
(material demand for sporicide and amount of runoff for each material, depending on porosity).
3. Clean Carboy No. 1 triple-rinsed with Dl water was placed under the drain of the test chamber to
collect liquid effluent type 1 (runoff of liquid decontaminant).
4. Liquid decontaminant was applied using a sprayer as described in Section 4.4.2.
5. A clean, sterile carboy (Carboy No. 1) loaded with neutralizer was placed under the drain of the
test chamber to collect liquid effluent type 1 (runoff of liquid decontaminant) during the
decontamination testing. A second clean sterile (Carboy No. 2) loaded with neutralizer was
placed under the drain of the test chamber to collect liquid effluent type 2 (water from rinsing of
the liquid decontaminant). Approximate volumes of material-specific runoff and rinsate were
assessed during liquid effluent characterization tests (Section 5.2.2). The amount of neutralizer
needed for each material type was determined in preliminary testing (see Section 4.9.2. for
details).
Liquid Effluent Type 1
1. To characterize liquid effluent type 2 (water from rinsing of the liquid decontaminant), Steps 1
through 4 above were repeated using a clean Carboy No. 2 triple-rinsed with Dl water placed
under the drain of the chamber to collect liquid effluent type 2 (rinsate of liquid sporicide).
2. The volumes of the runoff or the rinsate were collected as composite samples from the spraying
of the three coupons and determined using a graduated bucket of appropriate size.
52
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3. The concentration of the decontamination agent (pAB, Spor-Klenz® RTU [H2O2 and PAA], and
citric acid) was determined through titration using the appropriate methods. The pH and
temperature measurements of runoff solution were recorded.
5.3 Neutralization for Small Coupons
The following series of tests was conducted on the small (18-mm -diameter) coupons:
• Test I to determine the DE broth neutralizer effectiveness to quench decontamination activity of
the 2% (v/v) citric acid solution in Dl water against MS2 inoculated on the small coupons with
and without grime on their surfaces.
• Test II to determine the neutralizer buffer effectiveness of DE broth for samples that do not
receive decontamination solution.
• Test III to determine a suitable extraction buffer for the samples and determine the maximum
post-inoculation hold time for the coupons.
Each test is discussed below, followed by a discussion of method development test to determine the
neutralizer volume.
5.3.1 DE Broth Neutralizer Effectiveness, Test I
Test I was designed to determine the neutralizer effectiveness in quenching the activity of the
decontamination solution at a desired contact time on a coupon. Table 5.3-1 summarizes the test matrix.
Table 5.3-1. Test I Matrix
Material
Concrete
Test coupon
Yes
Yes
Yes
5
(grimed)
Positive control coupon
No
Yes
No
3
Procedural blank coupon
Yes
No
Yes
1
Negative control coupon
No
No
No
1
Concrete and
plywood
Runoff liquid test coupon
tube
Yes
Yes
Yes
Five each for
concrete and
plywood
Runoff liquid positive
control coupon tube
Yes
Yes
No
Three each for
concrete and
plywood
Runoff liquid negative
control coupon tube
Yes
No
No
One each for
concrete and
plywood
Test runoff collection
tube
Yes
No
No
Five total
I Plywood (grimed)
Test coupon
Yes
Yes
Yes
5
Positive control coupon
No
Yes
No
3
Procedural blank coupon
Yes
No
Yes
1
Negative control coupon
No
No
No
1
53
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A known quantity of surrogate organism (MS2) was deposited on small coupons of grimed materials:
unpainted (smooth finish) concrete and pressure-treated plywood. For each decontamination test
sequence set, five test and three positive control coupons were inoculated with MS2 inoculum using a
positive displacement pipette (100-|jL droplets). The target surface concentration for these experiments
was 1 x 108 PFU.
The test coupons and 1 mL of 2% citric acid solution (the decontamination agent) were added
simultaneously to 50-mL conical tubes, each containing 1 mL of the sterile DE broth and 10 mL of sterile
PBST. The same operation was performed with positive control coupons that were transferred to 50-mL
conical tubes containing only 10 mL of PBST. Procedural blank coupons underwent the same process as
the test coupons.
Runoff liquids were also tested with and without neutralizerto determine their effectiveness. For these
tests, five mL of citric acid decontamination solution (corresponding to the runoff from the spray-down
decontamination method) was added to test runoff collection vials (with DE broth) inoculated with 1 * 108
PFU of MS2. The samples were extracted for two hours, and 18 hours, after inoculation, and then plated
the same day for Day 0 testing, and re-plated 18 hours later for Day 1 testing. Table 4.5-1 summarizes
the matrix for the runoff liquid tests. The procedure summarized below was used for this test.
1. Ten 50-mL conical tubes were prepared, each containing one mL of sterile DE broth and 10 mL
of PBST. These tubes were the test coupon collection vials: five for the concrete test coupons
and five for the plywood test coupons.
2. Six 50-mL conical tubes were prepared, each containing one mL of the sterile DE broth and 10
mL of sterile PBST. These tubes were the positive control coupon collection vials (that is, they
were inoculated with MS2): three for the concrete positive control coupons and three for the
plywood positive control coupons.
3. Two more 50-mL conical tubes were prepared, each containing one mL of the sterile DE broth
and 10 mL of sterile PBST. These tubes were not inoculated with MS2 and served as negative
control coupon tubes (one each for concrete and plywood).
4. Five 50-mL conical tubes were prepared, each containing five mL of the sterile DE broth and five
mL of sterile Dl water. These tubes were the test runoff collection vials.
5. To each test coupon collection tube, one mL of the 2% citric acid solution was added to a test
coupon collection tube. Simultaneously, a grimed concrete coupon was aseptically transferred to
a test coupon collection tube. This step was conducted on all the concrete and plywood test
coupon tubes. The neutralizerto decontamination solution ratio was 1:1.23
6. A sterile concrete coupon was aseptically transferred to a positive control coupon tube. This step
was conducted on all concrete and plywood positive control and negative control coupon tubes.
7. To each of the test runoff collection vials, five mL of citric acid solution was added (corresponding
to the runoff from the spray-down decontamination method).
8. The sample tubes were transported to the analytical laboratory with appropriate chain of custody
for inoculation and microbial analysis.
54
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9. Each tube was inoculated with MS2 inoculum using a positive displacement pipette (100-|jL
droplets). The target surface concentration for these experiments was 1 * 108 PFU.
5.3.2 Neutralizer Buffer Effectiveness, Test II
The Test II evaluations were conducted to determine the effectiveness of DE broth as a neutralizing
buffer for samples that did not receive decontamination treatment. This test was designed to determine
how the reaction between traditionally used PBST buffer and the DE broth (without the presence of citric
acid) affects recoveries of MS2. Table 5.3-2 summarizes the test matrix.
Table 5.3-2. Test II Matrix
Material
Concrete (Grimed)
Test coupon
Yes
Yes
Yes
5
Positive control coupon
Yes
Yes
No
5
Negative control coupon
No
No
No
1
Plywood (Grimed)
Test coupon
Yes
Yes
Yes
5
Positive control coupon
Yes
Yes
No
5
Negative control coupon
No
No
No
1
The procedure summarized below was used for Test II.
1. Ten 50-mL conical tubes were prepared, each containing one mL of the sterile DE broth and 10
mL of sterile PBST. These tubes were the test coupon collection vials.
2. Twelve 50-mL conical tubes were prepared, each containing 10 mL of sterile PBST. Ten of these
tubes were the positive control coupon collection vials, and two were the negative control coupon
collection vials.
3. A coupon was aseptically transferred to each test and each positive control coupon collection
tube. This step was conducted for all concrete and plywood test coupons.
4. The sample tubes were transported to the analytical laboratory with appropriate chain of custody
for inoculation and microbial analysis.
5. Each tube was inoculated with MS2 using a positive displacement pipette (100-|jL droplets). The
target surface concentration for these experiments was 1 * 108 PFU
5.3.3 Suitable Extraction Buffer and Inoculation Hold Time, Test III
The Test III evaluations were conducted to determine the optimal extraction buffer for the samples and
determine the post-inoculation hold time for the coupons. The following four extraction buffers were tested
and compared:
• Dl water
55
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• PBST
• PBS
• TSB.
The following materials were tested:
• Concrete (grimed and neat)
• Pressure-treated plywood (grimed and neat)
The samples were analyzed at two different time points:
• Two hours after inoculation (Day 0)
• One day after inoculation (Day 1)
Table 5.3-3 summarizes the test matrix.
Table 5.3-3. Test III Matrix
Material
Type
Buffer
No. of Day 0
No. of Day 1
Replicates
Replicates
Concrete
Grimed
Dl water
2
2
PBST
2
2
PBS
2
2
TSB
2
2
Neat
Dl water
2
2
PBST
2
2
PBS
2
2
TSB
2
2
Plywood
Grimed
Dl water
2
2
PBST
2
2
PBS
2
2
TSB
2
2
Neat
Dl water
2
2
PBST
2
2
PBS
2
2
TSB
2
2
The procedure summarized below was used for Test III.
Day 0
1. The grimed and neat concrete 18-mm coupons were inoculated with 0.1 mL of the MS2
suspension and allowed to dry for two hours.
56
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2. Eight 50-mL conical tubes were prepared, each containing one mL of the sterile DE broth and
nine mL of sterile Dl water. These tubes were the Dl water test coupon collection vials.
3. Eight 50-mL conical tubes were prepared, each containing one mL of the sterile DE broth and
nine mL of sterile PBST. These tubes were the PBST test coupon collection vials.
4. Eight 50-mL conical tubes were prepared, each containing one mL of the sterile DE broth and
nine mL of sterile PBS. These tubes were the PBS test coupon collection vials.
5. Eight 50-mL conical tubes were prepared, each containing one mL of the sterile DE broth and
nine mL of sterile TSB. These tubes were the TSB test coupon collection vials.
6. One coupon was aseptically transferred to each of the test collection vials previously prepared.
7. The sample tubes were transported to the analytical laboratory with appropriate chain of custody
for inoculation and microbial analysis.
Day 1
1. The grimed and neat concrete coupons were inoculated with 0.1 mL of the MS2 suspension and
allowed to dry overnight (18 hours).
2. After the drying period, eight 50-mL conical tubes were prepared, each containing one mL of the
sterile DE broth and nine mL of sterile Dl water. These tubes were the Dl water test coupon
collection vials.
3. Eight 50-mL conical tubes were prepared, each containing one mL of the sterile DE broth and
nine mL of sterile PBST. These tubes were the PBST test coupon collection vials.
4. Eight 50-mL conical tubes were prepared, each containing one mL of the sterile DE broth and
nine mL of sterile PBS. These tubes were the PBS test coupon collection vials.
5. Eight 50-mL conical tubes were prepared, each containing one mL of the sterile DE broth and
nine mL of sterile TSB. These tubes were the TSB test coupon collection vials.
6. One coupon was aseptically transferred to each of the test collection vials previously prepared.
7. The sample tubes were transported to the analytical laboratory with appropriate chain of custody
for inoculation and microbial analysis
5.3.4 Method Development Test for Neutralizer Volume Determination
A series of tests was performed to determine the required volume of the neutralizer broth and PBST
needed to be preloaded into the 50-mL conical coupon collection vials (BD Falcon, Catalog No. 352098)
(described in Section 4.3) to achieve complete quenching of the decontaminant. This testing was
conducted as summarized below.
1. The spray apparatus was aseptically set up with coupons and rinsate collection vials. The runoff
collection vials, coupon collection vials, and coupons were weighed before installation. Three
replicates per coupon material were used.
2. Each coupon was sprayed with the decontamination solution and allowed to sit for an initial
exposure time of 15 minutes.
57
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3. After the initial 15-minute exposure time, the coupon was again sprayed with the
decontamination solution and allowed to sit for an additional 15 minutes.
4. After a total of 30 minutes of exposure time, the coupon was aseptically placed into a coupon
collection vial for extraction.
5. The rinsate and coupon collection vials were weighed, and the difference in weight was
recorded. The recorded weights were used to calculate the volume of decontamination solution
as collected on the coupon and as rinsate.
6. An equivalent portion of neutralization solution (1:1 neutralizer-to-citric-acid volume ratio) was
determined for the highest sprayed volume of rinsate collected. For example, if three mL of runoff
was collected, three mL of DE neutralizing broth was determined as the volume of the
neutralization solution.
7. Similarly, depending on the amount of decontamination solution collected on the coupon, the
corresponding neutralizer volume was determined using a 1:1 ratio.
58
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Sampling Approach
A sampling event log sheet was maintained for each sampling event (or test) that included each sampling
team member's name, the date, run number, and all sample codes with corresponding coupon codes.
The coupon codes were pre-printed on the sampling event log sheet before sampling began. Digital
photographs of selected coupons with a noticeable change due to the decontamination procedure were
taken after the completion of sampling for all coupons during an event or test. Pre- and post-
decontamination photographs were taken for three representative coupons of each material type.
Table 6-1 lists the materials and equipment used for sampling.
Table 6-1. Sampling Materials and Equipment
Material or Equipment
Description
Non-powdered, sterile
surgical gloves
KIMTECH PURE* G3 Sterile Nitrile Gloves, Kimberly-Clark (VWR P/N HC61110 for extra-
large, VWR P/N HC61190 for large, and VWR P/N HC61180 for medium)
Non-powdered, non-
sterile surgical gloves
Examination gloves (Fisherbrand Powder-Free Nitrile Exam Gloves, Fisher P/N 19-130-
1597D for large and 19-130-1597C for medium)
Dust masks
3M Particulate Respirator 8271, P95
Disposable laboratory
coats
Kimberly-Clark Kleenguard A10 Light Duty Apparel, P/N 40105
PBS
PBS with PBST (Sigma Aldrich USA, P/N: P3563-10PAK)
50-mL conical tubes
BD Falcon® BlueMax Graduated Tubes, 15-mL (Fisher Scientific P/N 14-959-70C)
Sterile sampling bags
Fisherbrand Sterile Sampling Bags (TWIRL'EM) Overpack Size 10- by 14-in
Inner bag size: 5.5- by 9-in (wipe)
Sample bag size: 5.5- by 9-in
Bleach wipes
Dispatch® Bleach Wipes (Chlorox Co., Oakland, CA)
Wipes
Kendall Curity Versalon absorbent gauze sponge, 2- by 2-in, sterile packed
(ravon/polvester blend) (httD://www.mfasco.com/. last accessed June 14. 2016)
Swabs
Bacti Swab® (httD://www.remelinc.com/lndustrial/CollectionTransDort/BactiSwab.asDX. last
accessed June 14. 2016)
Carboys (2)
Nalgene autoclavable carboys with tabulation
(20 L) (Fisher Catalog No. 02-690-23)
Analytical filter units
150-mL Nalgene analytical filter units (0.2-|jm cellulose acetate) (Fisher Catalog No. 130-
4020)
Vacuum pump
Gast oil-free vacuum pump with adjustable suction (Fisher Cat# 01-092-25)
Tubing
Fisher polyvinyl chloride (PVC) clear tubing (1/2-in-inside diameter, 1/16-in thick) (Fisher
Catalog No. Cat# 14-169-7J)
Fisher PVC cleartubing (3/8-in-inside diameter, 1/16-in thick) (Fisher Catalog No. 14-169-
7G)
Fisher PVC cleartubing (vacuum tubing), (3/8-in-inside diameter, 1/8-in thick) (Fisher
Catalog No. 14-169-7H)
Filter cassettes
Via-Cell® Bioaerosol Samplinq Cassette (http://www.zefon.com/store/via-cell-bioaerosol-
samDlina-cassette.html. last accessed June 14. 2016 )
Sampling pump
Isokinetic Method 5 Source SamDlina Console (httD://www.aDexinst.com/Droduct/xc-50-
method-5-source-samplinq-console)
The following sections discuss the sampling strategy, sampling methods, and sample handling.
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6.1 Sampling Strategy for Large Coupons
The following sections discuss the sample types, sample quantities, sampling and monitoring points, and
frequency of sampling and monitoring events.
6.1.1 Sample Types
The three major sample types are discussed below.
• Surface samples include surface wipe samples from each material collected in sets of five,
positive control surface samples collected in sets of three, and procedural blank surface samples.
• Liquid runoff and rinsate samples were collected to assess the potential for viable
microorganisms to be washed off the surfaces. Samples were collected from all liquid runoff and
rinsates from the decontamination process and from post-decontamination chamber cleaning.
These samples were analyzed quantitatively for each test. In addition, liquid decontamination
solution was sampled and verified before each decontamination procedure began.
• Aerosol samples were collected using Via-Cell® bioaerosol cassettes during each
decontamination and procedural blank test. Results for these samples were used to estimate the
occurrence and magnitude of fugitive emissions of viable B. atrophaeus or MS2 during the
decontamination process.
6.1.2 Sample Quantities
For each coupon, only one wipe sample was taken. The liquid waste samples were composite samples
collected from a set of five (5) test coupons (liquid runoff and rinsate samples were collected separately).
One aerosol sample per material was collected for the entire test decontamination procedure. Table 6.1-1
lists the total numbers of samples of each type for each test.
Table 6.1-1. Sample Types and Numbers for Each Decontamination Test
Sample Type
Positive control
3
Inoculation control
3
Test sample (decontaminated)
5
Procedural blank
1
Laboratory blank
1
Wipe field blank
1
Liquid samples
3
Aerosol
1
Swab
2 per item
60
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6.1.3 Sampling and Monitoring Points
The front face of each coupon was the only surface sampled in this study. All coupons were sampled
using wipes. The liquid runoff from a coupon set was collected during the application of the
decontamination solution. Each runoff sample was from a combination of all five coupons. In addition to
runoff samples, an aerosol sample was collected during the active spraying phase for each coupon set.
Aerosol samples were collected from the bulk volume of the chamber containing the coupons for each
decontamination method.
6.1.4 Frequency of Sampling and Monitoring Events
Table 6.1-2 summarizes the frequency of sampling and monitoring events for each decontamination
testing sequence as well as the measurement method or equipment and the sampling ranges.
Table 6.1-2. Sampling Frequencies
Testing
Sequence
Measurement Method or Equipment
Range
Frequency
Decontamination Formulation
2% citric acid
solution
Titration
2%
Once before and after testing
Oakton pH probe
1.98 to 2.02 pH unit
| Decontamination Testing |
For each
decontamination
test
Decontamination solution negative
controls (PFU, CFU)
0 to 200 PFU/CFU
per sample per filter
One sample per material type
Laboratory blank solution (PFU, CFU)
One sample per neutralization
test
Positive controls (PFU, CFU)
0 to 1 x 108 PFU or
CFU per coupon
material, 20 to 200
per plate
Three samples per material
type
Test samples (PFU, CFU)
Five samples per material type
6.2 Sampling Methods
The following sections discuss the methods used for wipe, runoff and rinsate, aerosol, and QA/QC
sampling.
6.2.1 Wipe Sampling
6.2.1.1 Wipe Sampling Preparation
For B. atrophaeus testing, the large coupons were allowed to dry for 18 hours before sampling. For MS2
testing, the coupons were sampled 15 minutes after decontamination (15 minutes after completion of the
water rinse). All coupons were placed horizontally for sampling regardless of their orientation during the
decontamination process. Sample volumes, time of day, and observations were recorded in laboratory
notebooks.
61
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The general approach for wipe sampling was to use a moistened, sterile, non-cotton gauze pad to wipe
the area to recover bacteria or viruses. A three-person team was used, employing an aseptic technique
throughout. The team consisted of a sampler, coupon handler, and support person.
The surface area sampled was one square foot. Wipe samples were collected within an area measuring
12- by 12-in using a sampling template centered on the coupon as shown in Figure 6.2-1. The outer 1 .Cl-
in around each coupon was not sampled to avoid unrepresentative edge effects.
Figure 6.2-1. Sampling Template Centered on Heavily Grimed Large Plywood Coupon
Before the sampling event, all materials needed for sampling were prepared using aseptic techniques.
Table 6-1 lists all the materials used for sampling. Non-powdered surgical gloves were used during
sampling. Individually wrapped pre-moistened bleach wipes (Hype-Wipe - current technologies,
Indianapolis, Indiana), used for sample bag decontamination, were placed in sterile sampling bags.
Alternatively, Dispatch® bleach wipes were used. A sampling material bin was stocked for each sampling
event based on the sample quantity. The bin contained enough wipe sampling kits to accommodate all
required samples for the specific test. Five additional kits also were on hand for backup. Enough prepared
packages of gloves and bleach wipes were included in the bin as well as extra gloves and wipes.
Paper sampling templates each measuring 1.17- by 1.17-feet with an interior opening of 12- by 12-in
were prepared, sterilized, and packaged in sterile bags (10 templates per bag). These bags of templates
were included in the sampling kits. A sample collection bin was used to transport samples to the NHSRC
RTP Microbiology Laboratory. The exterior of the transport container was decontaminated by wiping all
surfaces with bleach wipes ortowelettes moistened with a solution of pAB before transport to the NHSRC
RTP Microbiology Laboratory.
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6.2.1.2 Wipe Sampling Procedure
A three-person team was used, employing aseptic technique throughout. The team consisted of a
sampler, sample handler, and support person who followed a strict sampling protocol to avoid any
potential cross-contamination among coupons, or among samples. Throughout the procedure, the
support person logged anything deemed to be significant into the laboratory notebook and handled the
sampling kits (pre-moistened all-purpose sponge, conical tube, sampling bags, etc). The sample handler
handled the sample coupon and placed it on the sampling area, being careful to handle the coupon only
around the edges. The sampler conducted the sampling as follows:
• Wipe the surface of the sample horizontally using S-strokes to cover the entire sample area of the
coupon using a consistent amount of pressure.
• Fold the all-purpose sponge concealing the exposed side and then wipe the same surface
vertically using the same technique.
• Fold the all-purpose sponge over again and roll up the folded sponge to fit into the conical tube.
• Carefully place the all-purpose sponge into the 50 mL conical tube that the support person is
holding, being careful not to touch the surface of the 50 mL conical tube or plastic sterile sampling
bag.
For each single test, surface sampling of the materials was completed for all procedural blank coupons
before sampling of any test material. Positive controls were sampled.
6.2.2 Runoff and Rinsate Sampling
Rinsate samples were collected during the decontamination procedure. Liquid effluents from the
decontamination process were collected into sterilized carboys. The two types of liquid effluent samples
summarized below were collected.
• Runoff is defined as excess liquid decontaminant applied to the coupon surface that flowed from
the surface. Runoff samples were collected as one composite sample for each decontamination
test (composite of five test coupons subjected to the decontamination procedure).
• Rinsate is defined as water used to remove residual decontaminant from the coupons after active
decontamination (post-decontamination rinsate).
Note: Neutralizer was prepared in the collection vessel before collection of liquid samples so that the
active (sporicidal) ingredient was neutralized as the sample was being collected, not after collection.
Before decontamination, Carboy No. 1 (used for runoff collection, sterile and pre-weighed) was charged
with enough neutralization solution to neutralize all residual decontamination solution collected. After the
active decontamination concluded, Carboy No. 2 (pre-weighed carboy) was charged with enough
neutralization solution to neutralize all decontamination solution collected from the water rinsing step.
Therefore, any residual decontaminant was neutralized upon collection.
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The runoff and rinsate from the coupons was collected throughout the entire decontamination procedure
for a given coupon set (material type or all blanks).
After collection, 1,000-mL aliquots were collected using the aseptic technique summarized below.
• For each carboy, the total mass of liquid collected was recorded to compare the final versus the
initial weight.
• The contents of the carboy were agitated to homogenize.
• The carboy cap was removed.
• Using a new 100-mL sterile serological pipette, 10 x 100 mL (1 L total) aliquots of sample were
aseptically withdrawn into a sterile 1-L container.
• Sterile bags were used as secondary and tertiary containment during sample storage and
transport to the NHSRC RTP Microbiology Laboratory for analysis at the conclusion of the entire
test. Samples were processed immediately. If, due to the test schedule, liquid samples must be
stored, they were refrigerated at 4 °C ± 2 °C until processed (within 24 hours).
6.2.3 Aerosol Sampling
The use of high-pressure sprayers was expected to generate aerosols that could contain viable spores or
viruses removed from the coupon surfaces. Therefore, aerosol samples were collected during the entire
decontamination process. A single composite aerosol sample was collected for all five coupons. Aerosol
samples were collected from the chamber exhaust duct using Via-Cell® bio-aerosol cassettes.
A 4-in diameter, 44-foot long, flexible galvanized duct was attached to the test chamber to allow precise
flow measurements and sampling. The duct was attached to the chamber using a coupling and a 90-
degree elbow. The sampling port was located 32 in (eight diameters) downstream from the 90-degree
elbow connected to the chamber and 12 in (three diameters) from the bend in the duct connected to the
main exhaust. Figure 6.2-2 shows the sampling point location with a white arrow symbol.
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Figure 6.2-2. Test Chamber Exhaust Duct (white arrow shows sampling point location)
The flow through the Via-Cell® bioaerosol cassettes was isolated as much as possible to minimize
potential contamination from the laboratory environment.
6.2.4 QA/QC Sampling
The additional QA/QC samples summarized below were collected.
• Swab samples for were used for sterility checks on coupons and equipment before use in the
testing. A single, pre-moistened swab sample was collected from each item and coupon.
• Material samples and field samples for biological DQIs were collected. Results from these
samples provided information on the level of contamination possibly present during sampling due
to contaminated materials. These samples were referred to as unexposed field blank samples.
Blank plating of microbiological supplies was conducted to provide controls for testing the sterility
of supplies used in dilution plating.
• Grime samples were collected and analyzed for each batch of grimed coupons as a sterility
check.
• Decontamination solution samples for chemical DQIs were evaluated before each
decontamination event.
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6.3 Sampling Strategy for Small Coupons
The following sections discuss the sample types, sample quantities, sampling and monitoring points, and
frequency of sampling and monitoring events.
6.3.1 Sample Types
The two major sample types are discussed below.
• Surface samples included coupon samples from each material collected in sets of five for
surface extraction samples (as discussed in Sections 4.4 and 4.5), positive control surface
samples collected in sets of three, and procedural blank surface samples.
• Liquid rinsate samples were collected to assess the potential for viable microorganisms to be
washed off the surfaces. Samples were collected from all liquid runoff and the funnels were
rinsed with sterile deionized water where rinsate was collected in the same vials as runoff
collected as a single composite sample. These samples were analyzed quantitatively for each
test. In addition, liquid decontamination solution was sampled and verified before each
decontamination procedure began.
6.3.2 Sample Quantities
For each coupon, only one wipe sample was taken. The liquid waste samples were composite samples
collected from a set of five (5) test coupons (liquid runoff and rinsate samples were collected together as
a composite sample). Table 6.1-3 lists the total numbers of samples of each type for each test.
Table 6.1-3. Sample Types and Numbers for Each Decontamination Test
Sample Type
Positive control
3
Test sample (decontaminated)
5
Procedural blank
1
Laboratory blank
1
Liquid runoff/rinsate
5
Decontamination Solution
1
6.3.3 Sampling and Monitoring Points
All coupons were extracted in a vial containing PBST and DE Broth. The liquid runoff/rinsate from a
coupon set was collected during the application of the decontamination solution. The run/rinsate vials
were pre-loaded with DE Broth.
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6.3.4 Frequency of Sampling and Monitoring Events
Table 6.1-4 summarizes the frequency of sampling and monitoring events for each decontamination
testing sequence as well as the measurement method or equipment and the sampling ranges.
Table 6.1-4. Sampling Frequencies
Testing
Sequence
Measurement Method or Equipment
Range
Frequency
| Decontamination Formulation |
2% citric acid
solution
Titration
2%
Once before and after testing
Oakton pH probe
1.98 to 2.02
| Decontamination Testing |
For each
decontamination
test
Decontamination solution negative
controls (PFU, CFU)
0 to 200 PFU/CFU
per sample per filter
One sample per material type
Laboratory blank solution (PFU, CFU)
One sample per neutralization
test
Positive controls (PFU, CFU)
Oto 1 x 108
PFU/CFU per
coupon material, 20
to 200 per plate
Three samples per material
type
Test samples (PFU, CFU)
Five samples per material type
6.4 Sample Handling
This section discusses the sample containers and sample preservation.
6.4.1 Sample Containers for Large Coupons
For each wipe sample and grime sample, the primary containment container was an individual sterile 50-
mL conical tube. Secondary and tertiary containment consisted of sterile sampling bags.
For aerosol sampling, each Via-Cell® bioaerosol cassette was placed into a sterile foil bag and zipped
closed. The red safety seal label was applied over the top of the foil bag opening to ensure sample
integrity until analysis. The foil bag containing the cassette was then placed inside a pre-labeled 5.5- by
15-in sterile bag for tertiary containment.
Liquid effluent samples were collected in individual sterile specimen cups or Nalgene bottles placed
inside pre-labeled sterile bags for secondary containment.
A large plastic container was used for storage of sampling kits in the decontamination laboratory during
testing and for transport of kits post-collection to the NHSRC RTP Microbiology Laboratory.
6.4.2 Sample Containers for Small Coupons
For each coupon, a runoff/rinsate sample was extracted in an individual sterile 50-mL conical tube.
A large plastic container was used for storage of sampling kits in the decontamination laboratory during
testing and for transport of kits post-collection to the NHSRC RTP Microbiology Laboratory.
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6.4.3 Sample Preservation for Large Coupons
After sample collection for large coupons, sample integrity was maintained by storing the samples in four
containers (one sample collection container, one sterile inner bag, one sterile outer bag with the exterior
sterilized during the sample packaging process, and one sterile container holding all samples from a test).
All individual sample containers remained sealed while in the decontamination laboratory and during
transport.
6.4.4 Sample Preservation for Small Coupons
After sample collection for small coupons, the individual sample vials remained sealed while in the
decontamination laboratory and during transport to the NHSRC RTP Microbiology Laboratory.
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Analytical Procedures
This section discusses analytical procedures for microbiological analyses, filtration and plating of bacteria
from liquid extracts, and plating of MS2 from liquid extracts.
7.1 Analytical Procedures for Microbiological Analyses
Table 7.1-1 lists the analytical procedures used for this project.
Table 7.1-1. Analytical Procedures
Matrix
B. atrophaeus
inoculated
surfaces
CFU/area
Wipe sampling
Filtration and
plating of 6.
atrophaeus from
liquid extracts
Triple-
bagged
Refrigeration
72 hours
MS2 inoculated
surfaces
PFU/area
Wipe sampling
Filtration and
plating of MS2
from liquid
extracts
Triple-
bagged
Refrigeration
1 hour
Surface sterility
checks
Growth or no
growth
Swab
sampling
Filtration and
plating of 6.
atrophaeus and
MS2 from liquid
extracts
Swab
container
Refrigeration
1 to 72
hours
Grime
CFU/volume or
PFU/volume
Aseptic bulk
collection
Filtration and
plating of 6.
atrophaeus and
MS2 from liquid
extracts
Sterile
specimen
cup and
double-
bagged
Refrigeration
1 to 24
hours
Solid sample
roller
CFU/sample or
PFU/volume
Aseptic bulk
collection
Filtration and
plating of 6.
atrophaeus and
MS2 from liquid
extracts
Double-
bagged
Refrigeration
1 to 24
hours
7.2 Filtration and Plating of Bacteria from Liquid Extracts
The NHSRC RTP Microbiology Laboratory analyzed all samples for presence (sterility check samples)
and to quantify the CFU per sample. For all sample types, PBST was used as the extraction buffer. After
the extraction procedure, the buffer was subjected to a five-stage serial dilution (10-1 to 10-5). The
resulting samples were plated in triplicate and incubated overnight (minimum of 18 hours) at 35 °C ± 2 °C.
After incubation, CFU were enumerated manually. If the number of CFU on all three plates did not fall
between 30 and 300 CFU and/or was less than 30 CFU, filter plating or re-plating procedures were
conducted in an attempt to quantify recoveries at the lowest level possible. Figure 7.2-1 shows a dilution
and a filter plate with colonies of B. atrophaeus.
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Figure 7.2-1. Dilution Plate (Left) and Filter Plate (Right) Showing Colonies of B. atrophaeus
7.3 Plating of MS2 from Liquid Extracts
The NHSRC RTF Microbiology Laboratory analyzed all samples for presence (sterility check samples)
and to quantify the PFU per sample. For all sample types, PBST was used as the extraction buffer. After
the extraction procedure, the buffer was subjected to a five-stage serial dilution (10 ' to 10 5). The
resulting samples were plated in triplicate and incubated overnight (minimum of 18 hours) at 35 °C ± 2 °C.
After incubation, PFU were enumerated manually. Due to the limited stability of MS2 in sample storage,
additional dilutions of samples were plated during initial plating. Figure 7.2-2 shows a dilution plate
showing plaques of MS2.
Figure 7.2-2. Dilution Plate Showing PFU of MS2
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7.4 Data Reduction
Data reduction was performed on measurements of the total spores (CFU) or virus (PFU) recovered from
each replicate coupon; average recovered CFU or PFU and standard deviation (STD) for each group of
coupons. The groups of coupons included the following for each combination of material type and
extracted sample type:
• Positive control areas (replicates, average, STD)
• Test areas (replicates, average, STD)
• Procedural blank coupons.
Efficacy is defined as the extent (by LR) to which the agent recovered from the surface of the coupons
after the decontamination procedure has been reduced from the positive control areas (not exposed to
the decontamination procedure). Efficacy was calculated using Equation 7-1, below, for each material
within each combination of decontamination procedure (i) and test material (j).
LR„ = S '08(XFUJ' N„c "I log(XFU,J > Ntk i
C-1 k=\
where:
the average log reduction of spores on a specific material
surface
the average of the logarithm of the number of viable spores
(determined by CFU) or viral particles (PFU) recovered on the
control coupons [C = control, j = coupon number, and Nc = the
number of coupons (1,7)]
the average of the logarithm of the number of viable spores
(determined by CFU or PFU) remaining on the surface of a
decontaminated coupon [S = decontaminated coupon, k =
coupon number, and Nt = the number of coupons tested (1, k)]
X = C for colony, and X=P for plaque
When no viable surrogates were detected, the detection limit of the sample was used, and the efficacy
was reported as greater than or equal to the value calculated using Equation 9-1.
The cumulative standard deviation for the LR is calculated as follows: Let SUnand STr denote the standard
deviations of the log reduction values for the untreated carriers (positive controls) and the treated carriers
(post-decontamination samples), respectively. Then, the cumulative standard deviation is calculated as
follows:
SlR = [(S2Un / nun) + (S2TR / riTr)]1/2 7-2
where nun and Prr designate, respectively, the number of control and post-decontamination samples.
LR,
Z^XFU^'Nc
Y}°n(XFU si' N,
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Results and Discussion
This section discusses the results of the tests evaluating common decontamination methods for
inactivation of B. atrophaeus and MS2 on two test material surfaces (concrete and plywood), with and
without agricultural grime. The test materials were loaded with an agricultural grime surrogate that reflects
challenging environments expected during agricultural facility decontamination. The decontamination
solutions investigated were pAB and Spor-Klenz® RTU for B. atrophaeus-contaminated large coupons
and a solution of 2% (v/v) citric acid in Dl water and pAB against MS2 inoculated on small and large
coupons. The following sections discuss the results for B. atrophaeus and MS2 testing.
8.1 B. atrophaeus Decontamination Testing
This section discusses the testing results for extraction efficacy from neat and heavily grimed surfaces for
the large coupons, neutralizing agent testing for extracted samples, and B. atrophaeus decontamination
testing using Spor-Klenz® RTU and pAB.
8.1.1 Extraction Efficacy from Neat and Heavily Grimed Surfaces (14-in x 14-in
Coupons)
During scoping tests, grimed and neat test coupons were inoculated with 1 * 107 spores of B. atrophaeus
by aerosol deposition. After the settling period, each coupon was wipe-sampled. The recoveries from the
grimed and neat test coupons were compared to recoveries from the neat stainless steel inoculation
controls. Table 8.1-1 summarizes the results. The results suggest that the addition of the grime affected
the recovery of the surrogate spores by almost one order of magnitude. The presence of grime
prevented the target recovery of 6 logs from being achieved on wood and concrete.
Table 8.1-1. B. atrophaeus Recovery from Grimed and Neat Surfaces
Test Results
Log CFU
CFU
Average
STD
Recovery1 (%)
Neat Stainless Steel Inoculation Control (
inoculated @ E07)
Control coupon 1
7.4
2.28E+07
2.59E+07
2.95E+06
100
Control coupon 2
7.5
2.87E+07
Control coupon 3
7.4
2.63E+07
Neat Concrete Control Coupons
Test coupon 1
6.1
1.29E+06
3.41 E+06
1.85E+06
13.2
Test coupon 2
6.7
4.66E+06
Test coupon 3
6.6
4.29E+06
Grimed Concrete Coupons
Test coupon 1
5.2
1.54E+05
2.02E+05
6.76E+04
0.78
Test coupon 2
5.2
1.73E+05
Test coupon 3
5.4
2.79E+05
Concrete Procedural Blanks (not inoculated)
Procedural blank 1
Non-detects
72
-------
Test Results
Log CFU
CFU
Average
STD
Recovery1 (%)
Neat Wood Control Coupons
Test coupon 1
6.4
2.77E+06
3.45E+06
1.11E+06
13.3
Test coupon 2
6.7
4.74E+06
Test coupon 3
6.5
2.85E+06
Grimed Wood Coupons
Test coupon 1
5.5
3.30E+05
3.99E+05
6.07E+04
1.54
Test coupon 2
5.6
4.21 E+05
Test coupon 3
5.6
4.45E+05
Wood Procedural Blanks (not inoculated)
Procedural blank 1
Non-detects
1 Material Recovery was calculated as percent of neat Stainless Steel Recovery (Control Coupons)
8.1.2 Neutralizing Agent Testing for Extracted Samples
A series of tests was conducted to identify if a neutralizing agent is warranted to neutralize residual
decontaminant on the surfaces of the coupons after a decontamination event. The coupons were allowed
to dry overnight before sampling using wipes. The results from these decontaminated coupons were
compared with results for spiked samples from extracted blank (not decontaminated) coupons (Tests A3
and A6).
The results presented in Table 8.1-2, and illustrated in Figure 8.1.1 show no reduced recoveries resulting
from residual decontaminant, for both decontaminants (pAB and Spor-Klenz®RTU), suggesting there is
no need to neutralize the coupon surfaces after decontamination. However, STS (at 2 N [normal]) and
potassium carbonate (at 2 M [molar]) were used as neutralizing agents for pAB and Spor-Klenz® RTU in
runoff and rinsate samples.
Table 8.1-2. Neutralization Test Results for B. atrophaeus Extracted Samples
Test
Decontaminant*
Material Type
B. atrophaeus Recovery
(CFU)
Average
STD
A1
pAB
Concrete
1.69E+07
2.14E+06
A2
Spor-Klenz® RTU
Concrete
2.12E+07
4.74E+06
A3
None (water only)
Concrete
1.65E+07
1.96E+06
A4
pAB
Plywood
1.66E+07
3.27E+06
A5
Spor-Klenz® RTU
Plywood
2.15E+07
1.69E+06
A6
None (water only)
Plywood
2.07E+07
3.69E+06
* Decontaminant applied using sprayer
73
-------
10 -=
o
^ 106
0
0
a:
« 105
0
CO
_c
CL
2
CO 1Q4
CCi
10J
I Concrete
] PlywDod
pAB Spor-Klenz® RTU
Decontaminant Type
None (water only)
Figure 8.1-1. Bacillus atrophaeus Recoveries on Pre-Decontaminated Inoculated Surfaces
8.1.3 B. atrophaeus Decontamination Testing Using pAB and Spor-Klenz® RTU
Testing was conducted to evaluate the decontamination efficacy of pAB and Spor-Klenz® RTU against B.
atrophaeus spores on selected grimed and neat surfaces (concrete and plywood). Table 8.1-3
summarizes the results for the surface decontamination results, and Figure 8.1-1 illustrates the results.
Table 8.1-4 summarizes the results for liquid effluents (runoff and water rinsates) and air samples
collected during the decontamination process.
The decontamination efficacies encompassed a wide range of LR values from roughly 2.0 to 7.4 (Table
8.1-3). pAB was found to be more effective than Spor-Klenz® RTU for decontaminating concrete, while
the latter decontaminant was more effective on neat plywood, independent of application method
(backpack sprayer versus chemical sprayer). Both decontaminants were less effective on grimed
materials, compared to neat, with LR values on grimed surfaces ranging from 2.1 to 4.6, independent of
the material/application method.
A greater number of viable spores were found in rinsate samples during tests conducted with the
backpack sprayer than with the chemical sprayer, potentially because the chemical sprayer was more
effective at physically removing spores before the rinse step (during the decontamination step). Also, for
the backpack sprayer, rinsates from grimed coupons had higher viable spore levels than neat coupons.
Relatively high aerosolization (over 1 * 103 per sample) was observed during some tests with both the
backpack and chemical sprayers.
74
-------
Table 8.1-3. B. atrophaeus Decontamination Results
Decontamination
Application
Method
Material
Decontamination
Coupon
Positive Controls
(CFU)
Test Coupons
LR
(CFU)
Type
Liquid
Condition
Average
STD
Average
STD
Average
STD
1
Backpack sprayer
Concrete
pAB
Neat
1.63E+07
1.67E+06
ND
-
7.3
0.02
2
Grimed
1.02E+06
1.77E+05
1.24E+03
8.78E+02
3.0
0.36
3
Backpack sprayer
Treated
pAB
Neat
2.92E+06
1.08E+06
1.99E+02
3.65E+01
6.6
0.90
4
plywood
Grimed
6.46E+051
3.01 E+05
6.36E+02
5.99E+02
3.3
0.64
5
Backpack sprayer
Concrete
Spor-Klenz0 RTU
Neat
7.21 E+06
3.72E+06
2.67E+02
2.03E+02
4.6
0.62
6
Grimed
1.24E+04
1.51E+03
1.01 E+02
9.22E+01
2.4
0.66
7
Backpack sprayer
T reated
plywood
Spor-Klenz0 RTU
Neat
1.59E+07
7.09E+06
ND
-
7.4
0.01
8
Grimed
1.27E+06
5.26E+05
1.88E+03
2.20E+03
3.1
0.53
9
Chemical sprayer
Concrete
pAB
Neat
2.01 E+06
1.46E+06
ND
ND
6.4
0.01
10
Grimed
1.66E+051'2
1.44E+05
4.65E+02
4.03E+02
3.5
0.52
11
Chemical sprayer
Treated
pAB
Neat
6.73E+06
2.72E+06
1.27E+00
9.33E-01
6.8
0.27
12
plywood
Grimed
4.29E+051
2.05E+05
1.96E+02
3.40E+02
3.9
0.79
13
Chemical sprayer
Concrete
Spor-Klenz0 RTU
Neat
4.94E+041
2.39E+04
5.10E+02
3.33E+02
2.5
1.31
14
Grimed
1.51 E+06
2.80E+05
3.60E+01
3.78E+01
4.8
0.43
15
Treated
Neat
9.58E+06
3.09E+05
ND
_
7.1
0.14
Chemical sprayer
Spor-Klenz0 RTU
plywood
16
Grimed
| Samples were exposed to exccess heat during heat shock process |
1 Positive control recoveries below 6 logs, prevent achievement of 6 LR
2Some replicates were too contaminated to enumerate.
75
-------
c
o
'ts
~u
-------
* Spor-klenz effluents could not be neutralized with STS alone, therefore rinsate and runoff data were not of sufficient
quality to report. Method development was later performed with potassium carbonate to neutralize SK
8.2 MS2 Decontamination Testing
This section discusses the testing results for extraction efficacy from neat and heavily grimed surfaces for
the small and large coupons, neutralizing agent testing for extracted samples, and MS2 decontamination
testing using citric acid solution and pAB.
Preliminary scoping experiments performed on large coupons showed inconsistencies during the
extraction process for MS2 using PBST. As a result, the following tests were conducted as discussed in
Section 5.3:
• Test I to determine the neutralizer effectiveness of DE broth for the 2% (v/v) citric acid solution in
Dl water and MS2 inoculated on small coupons with and without grimed surfaces
• Test II to determine the neutralizer buffer effectiveness of DE broth for samples that do not
receive decontamination treatment
• Test III to determine a suitable extraction buffer for the samples and determine the inoculation
hold time for the coupons
The results of these tests are discussed in sections 8.2.1 through 8.2.3
Decontamination testing was conducted on small and large coupons (see Sections 4.4 and 4.5) to
evaluate the decontamination efficacy of pAB and 2% (w/v) citric acid against MS2 on selected grimed
and neat surfaces (concrete and plywood). The results of these tests are discussed in section 8.2.4 and
8.2.5 for small control coupons and large coupons, respectively.
8.2.1 DE Broth Neutralizer Effectiveness Test I Results
Table 8.2-1 summarizes the Test I results, and Figure 8.2-1 illustrates the results for these tests. The
results demonstrate that the DE broth acts not only as a stabilizer but also as a buffer that increases the
MS2 extraction efficiency (test coupons with DE broth demonstrated greater recoveries than positive
controls with no DE broth) but also maintains the integrity of the sample test coupons (coupon Day 4
versus Day 0), which is not the case for the positive coupons that received PBST alone.
77
-------
Table 8.2-1. MS2 - DE Neutralizer Broth Effectiveness Test I Results
Material
Sample Type
(Number of
Coupons)
Received DE
Broth
Inoculated
Day Plated
Recovery
(Log PFU)
STD
(Log PFU)
Concrete
(grimed)
Test coupon (5)
Yes
Yes
Day 0
8.1
0.02
Day 4
7.5
0.12
Positive control
coupon (3)
No
Yes
Day 0
3.5
5.4
Day 4
ND
-
Negative control
coupon (1)
No
No
Day 0
ND
-
Day 4
ND
-
Concrete
and
plywood
Runoff liquid (5)
Yes
Yes
Day 0
7.7
0.04
Day 4
0.3
0
Plywood
(grimed)
Test coupon (5)
Yes
Yes
Day 0
7.9
0.18
Day 4
7.6
0.16
Positive control
coupon (3)
No
Yes
Day 0
7.2
1.0
Day 4
1.6
3.4
Negative control
coupon (1)
No
No
Day 0
ND
-
Day 4
ND
-
=>
Ll_
CL
O
O)
i>
8
Q?
9-
Concrete Day 0 plating
Concrete Day 4 Plating
Plywood Day 0 Plating
Plywood Day 4 Plating
Run Off Day 0 Plating
Run Off Day 4 Plating
Test Samples Positive Control Negative Control
Types of Samples
r
RunOff
Figure 8.2-1. MS2 Recoveries Using DE Broth as an Extraction Buffer
78
-------
8.2.2 DE Broth Neutralizer Effectiveness Test 11 Results
Table 8.2-2 summarizes the Test II results, and Figure 8.2-2 illustrates the results for these tests. The
results suggest that the addition of DE broth to the grimed positive controls (concrete and plywood)
increased the extraction efficiency of MS2 compared to the positive controls without DE broth. Further,
the results suggest that delaying the plating process negatively affected recoveries for samples plated
using PBST alone and, to a lesser extent plating process negatively affected recoveries for samples
containing PBST alone, and to a lesser extent samples in PBST/DE broth solutions.
Table 8.2-2. MS2 - DE Broth Neutralizer Effectiveness Test II
Concrete test coupon
1 mL of sterile DE broth and 10
mL of sterile PBST
Yes
Day 0
4.47E+07
7.90E+06
Day 4
1.07E+07
3.16E+06
Concrete positive
control coupon
10 mL of sterile PBST
Yes
Day 0
1.7E+04
4.5E+03
Day 4
8.8E+02
2.27E+03
Concrete negative
control coupon
None
No
Day 0
ND
-
Day 4
ND
-
Plywood test coupon
1 mL of sterile DE broth and 10
mL of sterile PBST
Yes
Day 0
3.0E+07
1.1E+07
Day 4
4.39E+06
1.36E+06
Plywood positive
control coupon
10 mL of sterile PBST
Yes
Day 0
2.4E+04
3.1E+04
Day 4
2.84E+03
6.21 E+04
Plywood negative
control coupon
None
No
Day 0
Not detected
Not detected
Day 4
Not detected
Not detected
*Four out of five test results were non-detects
79
-------
Concrete Day 0 plating
Concrete Day 4 Plating
Plywood Day 0 Plating
Plywood Day 4 Plating
PBST & D/E Broth PBST
Extraction Buffer Solution
Figure 8.2-2. MS2 Extraction Efficacy with and without DE Broth, after 0 and 4 Days
8.2.3 Suitable Extraction Buffer and Inoculation Hold Time Test III Results
Table 8.2-3 summarizes the Test III results, using DE broth as a neutralizer with different extraction
buffers. The results suggest the following:
• DE broth increased recovery of MS2,
• The PBST/DE broth neutralization/extraction buffer achieved the highest recoveries, although
Dl water/DE broth was comparable as an extraction combination,
• Grimed samples provided better recovery compared to neat samples,
• Same-day inoculation (10-minute hold time for neat samples and 2-hour hold time for grimed
samples) resulted in higher MS2 recoveries compared to overnight inoculation (18- to 24-hour
hold time), and
• PBST with DE broth was the most effective neutralizer/extraction buffer (except for
grimed concrete samples where Dl water was more effective).
Each sample was plated on the day of extraction (Day 0) and one day after extraction (Day 1). Although
samples from Day 1 had lower recoveries than samples from Day 0, the average log difference between
Day 0 and Day 1 recoveries for PBST/DE broth buffer was approximately 0.5 log.
80
-------
The overall results suggest that neat inoculated coupons should be tested within minutes after inoculation
and that grimed coupons should be tested within one day after inoculation. Neutralized test samples and
positive samples should be extracted in a DE broth-PBST neutralizer/buffer solution to achieve sufficient
recoveries and maintain the viability of MS2. The sample extracts can be then plated within four days
without any substantial reduction in recovery
Table 8.2-3. Suitable Extraction Buffer and Inoculation Hold Time Test III Results for MS2
Test Type
Coupon Material
Buffer Used
Day Plated
Recovery
(Log PFU)
Dl Water
Day 0
6.9
Day 1
7.1
PBST
Day 0
6.9
Concrete
Day 1
7.3
PBS
Day 0
3.6
Day 1
0.7
TSB
Day 0
6.9
Neat - 2-hour
Day 1
6.7
inoculation hold
Dl Water
Day 0
5
Day 1
ND
PBST
Day 0
5.9
Plywood
Day 1
5.9
PBS
Day 0
0.7
Day 1
ND
TSB
Day 0
5.6
Day 1
5.3
Dl Water
Day 0
0.7
Day 1
ND
PBST
Day 0
0.7
Concrete
Day 1
ND
PBS
Day 0
0.7
Day 1
ND
TSB
Day 0
0.7
Neat -1 -day
Day 1
ND
inoculation hold
Dl Water
Day 0
0.7
Day 1
ND
PBST
Day 0
0.7
Plywood
Day 1
ND
PBS
Day 0
0.7
Day 1
ND
TSB
Day 0
0.7
Day 1
ND
Dl Water
Day 0
7.7
Day 1
7.6
PBST
Day 0
7.9
Concrete
Day 1
6.3
Grimed - 2-hour
PBS
Day 0
7.6
inoculation hold
Day 1
0.7
TSB
Day 0
7.2
Day 1
7.2
Plywood
Dl Water
Day 0
7
Day 1
6.8
81
-------
Test Type
Coupon Material
Buffer Used
Day Plated
Recovery
(Log PFU)
PBST
Day 0
8.2
Day 1
8.1
PBS
Day 0
8
Day 1
0.7
TSB
Day 0
7.5
Day 1
6.8
Dl Water
Day 0
7.8
Day 1
7.4
PBST
Day 0
6.6
Concrete
Day 1
6.2
PBS
Day 0
4.2
Day 1
0.7
TSB
Day 0
5.8
Grimed - 1-day
Day 1
3.5
inoculation hold
Dl Water
Day 0
7.7
Day 1
7.3
PBST
Day 0
7.3
Plywood
Day 1
6.7
PBS
Day 0
0.7
Day 1
ND
TSB
Day 0
6.6
Day 1
6.3
8.2.4 MS2 Decontamination Testing on Small Coupons Using pAB and 2% Citric
Acid Formulation
The decontamination solutions were tested for effectiveness in quintuplicate for an exposure time of
30 minutes for all material types using 18-mm coupons. Control testing was conducted to determine the
physical removal of MS2 due to the liquid spraying. To do this, a separate set of coupons were sprayed
with Dl water using the same methods used with pAB and citric acid. These test coupons were
designated as procedural positive control coupons. For each test, there was one negative test control
(non-inoculated coupon) that underwent the same approach as the test coupons and three positive test
controls. All the coupons were extracted in DE broth/PBST solutions.
Coupons of four material types (grimed concrete, grimed plywood, neat concrete, and neat plywood) were
tested, each inoculated with 1 * 108 PFU MS2.
The results for MS2 recovery (PFU) on the surfaces of all materials tested are illustrated in Figure 8.2-3
and summarized in Table 8.2-4. The results show that the liquid spraying with water had low efficacy
(determined by comparing results for the procedural positive controls to the non-sprayed positive
controls), suggesting that physical removal during spraying is minimal.
Table 8.2-4 shows the decontamination efficacy (positive controls PFU compared to log PFU remaining
after decontamination) for all the materials. The results of these tests suggest that the 2% (v/v) citric acid
formulation is not effective against MS2 on the concrete and plywood test materials. However, pAB was
found to be efficacious against MS2 for concrete, with full decontamination on neat concrete material and
82
-------
near full decontamination on grimed concrete material (four out of five samples with non-detects).
However, limited efficacy was observed for neat or grimed plywood materials.
to
z>
8
0
OH
CM
CO
q8J 2% at
°7"i b
°'-i
05-j
°1
°3"i
°1
014J—B,J—
1
n
0-, —
04
0-1 _
°6"i M
1
°3-i
°21 Hnd
n1 —
B
r
rh
Neat Concrete Grimed Concrete Neat Plywood Grimed Plywood
Positive Control
Procedural Positive Control
Decontaminated Material
Material Type and Condition
Figure 8.2-3. MS2 Recoveries from Positive Control, Procedural Control, and Decontaminated Test
Coupons
83
-------
Table 8.2-4. MS2 Recoveries from Positive Control, Procedural Control, and Decontaminated Test
Coupons
Decon
Agent
Material
Positive Coupon
PFU
Procedural Positive
Control Coupon
Test Coupon
PFU
Surface Log Reduction
(LR)
Average
STD
Average
STD
Average
STD
Average
Cumulative STD
Neat
concrete
6.77E+06
2.68E+06
8.84E+06
8.54E+06
ND
-
7.1
0.12
pAB
Grimed
concrete
2.99E+07
2.59E+07
1.53E+07
1.75E+06
2.83E+05
6.34E+05
6.4
1.3
Neat
plywood
1.37E+08
7.97E+07
7.01 E+05
8.05E+05
4.54E+05
1.46E+05
2.4
0.19
Grimed
plywood
4.91 E+07
7.36E+07
1.10E+08
5.22E+07
8.57E+05
9.86E+05
3.7
1.7
Neat
concrete
3.68E+07
1.24E+07
5.22E+07
6.00E+06
1.39E+07
7.93E+06
0.46
0.15
2%
Citric
acid
Grimed
concrete
6.17E+07
1.03E+08
7.96E+07
3.95E+07
4.99E+06
4.21 E+06
1.1
1.1
Neat
plywood
6.21 E+07
1.12E+07
3.10E+05
3.93E+05
3.52E+04
3.83E+04
3.5
0.25
Grimed
plywood
6.35E+07
8.05E+07
1.37E+08
9.44E+07
7.88E+07
6.96E+07
0.08
0.56
The fate of MS2 was assessed by collecting and analyzing runoff samples to address the potential
physical removal of the virus from the surface of the coupons during the decontamination procedure.
These results are shown in Table 8.2-5 and demonstrate that most of the runoff from pAB effluent had no
detectable MS2, which was not the case for the 2% citric acid formulation, which showed almost complete
wash off of viable viruses from the coupons independent of type of materials exposed was observed. This
result confirms that the citric acid formulation does not have the desired biocidal effect on the MS2.
Table 8.2-5. MS2 Recoveries from Runoff Samples from Small Coupons
Positive Coupon
Runoff
Decon Agent
Material
(PFU)
(PFU)
Average
STD
Average
STD
Neat concrete
6.77E+06
2.68E+06
ND
-
pAB
Grimed concrete
2.99E+07
2.59E+07
ND
-
Neat plywood
1.37E+08
7.97E+07
6.05E+01
8.17E+01
Grimed plywood
4.91 E+07
7.36E+07
ND
-
Neat concrete
3.68E+07
1.24E+07
4.82E+03
2.87E+03
2% Citric acid
Grimed concrete
6.17E+07
1.03E+08
1.44E+07
1.50E+07
Neat plywood
6.21 E+07
1.12E+07
1.32E+04
1.62E+04
Grimed plywood
6.35E+07
8.05E+07
5.89E+04
9.72E+04
8.2.5 MS2 Decontamination Testing Using pAB and 2% Citric Acid Formulation
on Large Coupons
Testing was conducted to evaluate the decontamination efficacy of pAB and citric acid against MS2 on
selected grimed and neat surfaces (concrete and plywood). A backpack sprayer was used to spray the
84
-------
decontamination solution on large 14-in x 14-in coupon materials. Table 8.2-6 summarizes the results for
the surface decontamination results, and Figure 8.2-4 illustrates these results.
During these large coupon tests, pAB was found to be effective at inactivation of MS2 on grimed
materials (complete kill, and > 6 LR for grimed concrete and grimed plywood, respectively). The lower
decontamination efficacies on grimed surfaces observed during tests with B. atrophaeus were not
observed during the tests with MS2. Complete kill of MS2 was also achieved with pAB on neat concrete.
Viable MS2 was recovered following pAB treatment from neat wood. A greater occurrence of non-detects
(complete kill) following decontamination during large coupon tests as compared to small coupon tests
could be explained by the lower efficiency of wipe sampling (large coupons) as compared to extraction-
based sampling (small coupons). This disparity is evident in the higher recoveries from positive control
coupons during small coupon testing. Consistent with the small coupon tests, the 2% citric acid
formulation was found to be ineffective for neat concrete materials (LR ~ 0.2), and more effective with the
grimed concrete material (LR ~ 4.3). However, complete kill was not achieved for either neat or grimed
concrete, when 2% citric acid was the decontaminant. Testing of 2% citric acid against MS2 on neat and
grimed plywood have not been completed at the time of report preparation.
Table 8.2-6. MS2 Recoveries during Large Coupon Testing
Decon
Positive Coupon
(PFU)
Test Coupon
(PFU)
Surface Log Reduction
(LR)
Agent
Material
Average
STD
Average
STD
Average
Cumulativ
e STD
Neat Concrete
2.46E+04
6.61 E+03
ND
-
4.7
0.06
pAB
Grimed Concrete
1.54E+06
2.65E+05
ND
-
6.2
0.04
Neat Plywood
3.64E+06
-
9.78E+01
4.44E+01
4.8
0.33
Grimed Plywood
4.70E+06
4.71 E+04
ND
-
7.0
0.00
2%
Citric
Acid
Neat Concrete
6.20E+03
6.74E+03
2.89E+03
1.98E+03
0.20
0.36
Grimed Concrete
8.36E+05
3.26E+05
1.15E+02
1.06E+02
4.3
0.35
85
-------
I
a:
eg
CO
10'
10s
105
104
103
102
101
107
10s
105
104
103
102
101
Neat Concrete
Grimed Concrete
ND
ND
pAB
2% Citric Acid
Neat Concrete Grimed Concrete Neat Plywood Grimed Plywood
I Positive Controls
I Decontaminated Material
Material Type and Condition
Figure 8.2-4. MS2 Recoveries from Large Coupon Tests
Liquid effluent (runoff) samples, rinse water samples, and air samples collected during the
decontamination process also were analyzed to determine the fate of the test organisms. The results of
these tests are shown in Table 8.2-7. The effluents from the pAB experiments show no-detects of MS2
independent of material type or condition (neat or grimed), while runoffs from the 2% citric acid
formulation tests resulted in substantive amounts of the MS2. These results confirm the ineffectiveness of
the 2% citric acid formulation, observed during the small coupon testing. No viable MS2 was observed in
any of the aerosol samples (Table 8.2-7). No impact of grime on MS2 recoveries was observed in runoff
or rinsate samples.
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Table 8.2-7. Fate of MS2 during Decontamination Procedures
Test
Material
Decontamination
Coupon
Runoff
CFU /
Sample
Rinsate
Aerosol
Type
Liquid
Condition
CFU/ Sample
CFU/Sample
1
Concrete
Neat
ND
ND
ND
2
pAB
Grimed
ND
ND
ND
3
T reated
Neat
ND
ND
ND
4
plywood
Grimed
ND
ND
ND
5
2% citric acid
Neat
2.20E+03
2.42E+03
ND
6
Concrete
formulation
Grimed
ND
4.50E+02
ND
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Quality Assurance and Quality Control
All test activities were documented via narratives in laboratory notebooks and the use of digital
photography. The documentation included, but was not limited to, a record for each decontamination
procedure, any deviations from the QAPP, and physical impacts on materials. All tests were conducted in
accordance with established EPA Decontamination Technologies Research Laboratory (DTRL) and
NHSRC RTP Microbiology Laboratory procedures to ensure repeatability and adherence to the data
quality validation criteria set for this project
9.1 Criteria for Critical Measurements/Parameters
The data quality objectives (DQOs) are used to determine the critical measurements needed to address
the stated objectives and specify tolerable levels of potential errors associated with simulating the
prescribed decontamination environments. The following measurements were deemed critical to
accomplish part or all of the project objectives:
• pH and temperature of the pAB solution
• Sodium hypochlorite concentration (FAC) of the pAB decontamination solution
• Citric acid concentration of the 2% citric acid decontamination solution
• Temperature of incubation
• CFU or PFU abundance per plate
• Neutralizer volume
• Mass of grime applied onto test coupons
• Backpack sprayer spray diameter at one foot
• Chemical sprayer spray diameter at three feet
• Flow rate of backpack sprayer, chemical sprayer, and water hose
• Pressure of backpack sprayer and garden hose.
The following measurements were non-critical, but were monitored and recorded throughout the entire
testing schedule:
• Temperature and pH of the Spor-Klenz® RTU and citric acid liquid sporicide solutions and of
the rinse water
• Head pressure for the rinse water.
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9.2 Data Quality Indicators
The data quality indicators (DQIs) for the critical measurements listed in Table 9.2-1 were used to
determine if the collected data met the quality assurance objectives. If a measurement method or device
resulted in data that did not meet these goals, the data derived from the critical measurement were
rejected. Decisions to accept or reject test results were based on engineering judgment used to assess
the likely impact of the failed criterion on the conclusions drawn from the data. The acceptance criteria
were set at the most stringent levels that can routinely be achieved. All the DQIs were within the target
acceptance criteria set for this project as shown in Table - 9.2-1.
Table 9.2-1. DQIs for Critical Measurements
Measurement Parameter
Analysis Method
Accuracy
Acceptance Criteria
Mean Value / Pass or Fail
Test
Mass of grime
Gravimetric
0.1 g
± 10% of target value
30% RSD between test set
56.5 g
(Pass)
FAC and pH in pAB solution
Na2S203/KI titration pH meter/NIST-
traceable buffer solutions
±0.06 g/L
±0.01 pH units
6,000 to 6,700 mg/mL
6.5
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9.3 Quality Control Checks
Many QA/QC checks were used in this project to ensure that the data collected met all the critical
measurements listed in Table 9.2-1. The measurement/parameter criteria were set at the most stringent
level that can routinely be achieved. The integrity of the sample during collection and analysis was
evaluated. 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 were included for each set of test
conditions. Specific quality control checks that were performed in this project are described in the
following sections.
9.3.1 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 possibly cross-contaminate them.
Project personnel carefully checked supplies and consumables prior to use to verify that they met
specified project quality objectives. All pipettes were calibrated yearly by an outside contractor (Calibrate,
Inc.), incubation temperature was monitored using NIST-traceable thermometers, and balances were
calibrated yearly by the EPA Metrology Laboratory.
9.3.2 NHRSC Biolab Control Checks
Quantitative standards do not exist for biological agents. Quantitative determinations of organisms in this
investigation did not involve the use of analytical measurement devices. Rather, the CFU were
enumerated manually and recorded. If the CFU count for bacterial growth did not fall within the target
range, the sample was either filtered or re-plated. For each set of results (per test), a second count was
performed on 25 percent of the plates within the quantification range (plates with 30 - 300 CFU). All
second counts were found to be within 10 percent of the original count.
9.4 QA/QC Sample Acceptance Criteria
The acceptance criteria for the critical CFU measurements were set at the most stringent level that could
be achieved routinely. Positive controls and procedural blanks were included along with the test samples
in the experiments so that well-controlled quantitative values were obtained. Background checks were
also included as part of the standard protocol. Replicate coupons were included for each set of test
conditions. 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 materials used in this effort did not themselves
contain spores. The checks included the following:
• Negative control coupons: sterile coupons that underwent the same sampling process without
spore deposition.
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• Field blank coupons: sterile coupons carried to the decontamination location but not
decontaminated.
• Laboratory blank coupons: sterile coupons not removed from NHSRC Biolab.
• Laboratory material coupons: includes all materials, individually, used by the NHSRC Biolab in
sample analysis.
• Stainless steel positive control coupons: coupons inoculated but not decontaminated.
QA/QC acceptance criteria are shown in Table 9.4-1. These criteria provide assurances against cross-
contamination and other biases of microbiological samples.
Table 9.4-1. Additional DQIs Specific to Microbiological Data
Coupon or
Sample Type
Positive control
coupons
sample from
material coupon
contaminated with
biological agent and
sampled using the
wipe method
1 x 107forS.
atrophaeus
1 x108for MS2
30% RSD between
coupons in each test
set
Shows viability of wipe sampling
technique and plate's ability to
support growth of B. atrophaeus
and MS2
Identify and remove source
of variability if possible
Pass
Procedural blank
coupon without
biological agent that
underwent the
sampling procedure
Non-detect
Controls for sterility of materials and
methods used in the procedure
Analyze data with
procedural blank results as
test minimum; identify and
remove source of
contamination if possible
Pass
Material blank
grime, roller, and
sterilized coupon of
each material
Non-detect
Controls for sterility of materials and
methods used in the procedure
Analyze data with
procedural blank results as
test minimum; identify and
remove source of
contamination if possible
Pass
Blank plating of
microbiological
supplies
No observed growth
after incubation
Controls for sterility of supplies used
in dilution plating
Sterilize or dispose of
source of contamination;
replate samples.
Pass
Blank tryptic soy
agar sterility control
Plate incubated but
not inoculated
No observed growth
after incubation
Controls for sterility of plates
All plates incubated before
use, so contaminated plates
discarded before use
Pass
Exposed field blank
samples; a wipe kit
will be handled
Non-detect
Level of contamination present
during sampling
Clean up environment;
sterilize sampling materials
before use
Pass
Unexposed field
blank samples; a
wipe kit will be
transferred without
handling
Non-detect
Level of contamination present
during sampling
Clean up environment;
sterilize sampling materials
before use
Pass
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Summary
The objective of this study was to assess the effectiveness of spray-based common decontamination
methods for inactivating Bacillus (B.) atrophaeus (surrogate for B. anthracis) spores and bacteriophage
MS2 (surrogate for foot and mouth disease virus [FMDV]) on selected test surfaces (with or without a
model agricultural grime). Relocation of viable viruses or spores from the contaminated coupon surfaces
into aerosol or liquid fractions during the decontamination methods was also investigated.
The effectiveness of removing/inactivating two target microorganisms was assessed for three different
decontamination solutions. pH-Amended Bleach (pAB) and Spor-Klenz® Ready-to Use [RTU] were
evaluated for their effectiveness against B. atrophaeus spores, and 2% (w/v) citric acid in sterilized Dl
water and pAB were evaluated against the bacteriophage MS2. Three application methods (handheld
sprayer, backpack sprayer, and a chemical sprayer) were utilized throughout the testing to deliver
decontaminants to the test surfaces. The evaluation was conducted on two test material surfaces
(concrete and plywood), with and without a model agricultural grime on the surface. The handheld
application method was conducted using a bench-scale test spray apparatus to evaluate the pAB and
citric acid spray-based decontamination methods for 18-mm coupons (both grimed and neat)
contaminated with MS2. The backpack and the chemical sprayer application methods were performed to
simulate field operations. For all the tests, a wetted surface contact time of 30 minutes was used, followed
by a surface rinse with water. Method developments were conducted to determine the most effective
extraction buffer, if any, for each decontaminant and to determine its effectiveness in neutralizing
(quenching decontaminant activity) and maintaining the integrity of the samples potentially containing
viable B. atrophaeus spores or MS2
Testing conducted to evaluate the decontamination efficacy against B. atrophaeus spores on selected
grimed and neat surfaces (concrete and plywood) using pAB and Spor-Klenz® RTU indicated that higher
efficacies were achieved on neat materials than on grimed materials, independent of material type
(concrete or wood) or decontaminant application method (backpack sprayer versus chemical sprayer for
large coupons and handheld sprayer for small coupons). pAB was found to be more effective than Spor-
Klenz® RTU for decontaminating neat concrete materials, while the latter decontaminant was more
efficient at decontamination of neat plywood materials, independent of application method. Viable spore
levels found in rinsate samples were higher for the backpack sprayer tests than the chemical sprayer
tests, potentially because the chemical sprayer was more effective at physically removing spores before
the rinse step (during the decontaminant application step). Relatively high aerosolization (greater than 1 *
103 CFU per test) was observed during some tests with both the backpack and chemical sprayers.
Tests conducted to evaluate decontamination efficacy against MS2 on selected grimed and neat surfaces
(concrete and plywood) using pAB and 2% (v/v) citric acid formulation indicated that 2% citric acid was
not effective against MS2 on these test materials. Conversely, pAB was found to be efficacious against
MS2, with full decontamination (complete kill) on neat or grimed concrete and limited efficacy for neat or
grimed plywood. No apparent effects of grime on decontamination efficacy were observed during MS2
tests. Further, it was demonstrated that few viable viruses were detected in the runoff from pAB tests,
unlike for the 2% citric acid tests, which had almost complete wash off of viable viruses from all coupon
types. Finally, no viable MS2 aerosol formation/emission was observed in any of the conducted tests,
independent of the type of decontamination solution used. However, the Via-Cell® bio-aerosol cassette
92
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sampling method used in this study was not validated for MS2 collection and subsequent analytical
methods. Lack of recovery could be indicative of low or no viral aerosol formation during tests, poor
collection efficiency of the method, or loss of viability of viral particles due to desiccation following
collection but prior to analysis.
From the neutralizer optimization tests, sodium thiosulfate (STS) at 2 N (normal) and potassium
carbonate at 2 M (molar) were found to be suitable neutralizing agents for pAB and Spor-Klenz® RTU.
PBST was also found to be a very effective extraction buffer for B. atrophaeus spores,
but not for MS2. For testing involving MS2, a phosphate-buffered saline with 0.05% Tween® 20
(PBST)/Dey Engley (DE) broth combination was found to act not only as a stabilizer but also as a
neutralizer/extraction buffer that increased the efficiency of MS2 recovery.
The results of this study may help emergency responders select decontamination chemicals and
application methods that are effective yet feasible.
93
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vvEPA
United States
Environmental Protection
Agency
PRESORTED STANDARD
POSTAGE & FEES PAID
EPA
PERMIT NO. G-35
Office of Research and Development (8101R)
Washington, DC 20460
Official Business
Penalty for Private Use
$300
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