EPA/600/R-18/157 | July 2018
www.epa.gov/homeland-security-research
United States
Environmental Protection
Agency
Evaluation of Diperadipic Acid as a
Surface Decontaminantfor Spore-
Forming Biological Agents
Office of Research and Development
Homeland Security Research Program

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Evaluation of Diperadipic Acid as a Surface
Decontaminant for Spore-Forming Biological
Agents
Evaluation Report
Authors:
M. Worth Calfee, Ph.D.
US Environmental Protection Agency
Office of Research and Development
National Homeland Security Research Center
Decontamination and Consequence Management Division
Abderrahmane Touati, Ph.D., and Madhura Karnik
Jacobs Technology, Inc.
Research Triangle Park, NC 27709

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Notice/Disclaimer Statement
The U.S. Environmental Protection Agency (EPA), through its Office of Research and
Development's (ORD's) National Homeland Security Research Center (NHSRC), directed and managed
this work in partnership with the U.S. Department of Homeland Security, Science and Technology
Directorate. This report has been peer and administratively reviewed and has been approved for
publication as an EPA document. The views expressed in this report are those of the authors and do not
necessarily reflect the views or policies of the Agency. Mention of trade names or commercial products
does not constitute endorsement or recommendation for use of a specific product.
Questions concerning this document or its application should be addressed to:
M. Worth Calfee, Ph.D.
National Homeland Security Research Center
Office of Research and Development
U.S. Environmental Protection Agency
Mail Code E343-06
Research Triangle Park, NC 27711
919-541-7600
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Table of Contents
Notice/Disclaimer Statement	ii
Figures	iv
Tables	iv
Acronyms and Abbreviations	v
Executive Summary	1
1	Introduction	1
1.1	Background	1
1.2	Proj ect Obj ectives	1
2	Experimental Approach	3
2.1	Overall Experimental Approach	3
2.2	Test Matrix	4
3	Sampling and Analytical Procedures	5
3.1	Microbiological Analysis	5
3.1.1	Sample Quantities	5
3.1.2	Sample Types	5
3.1.3	Sample Extraction and Analysis	6
3.2	Data Reduction	6
4	Results and Discussion	8
4.1	Neutralization Results	8
4.2	Decontamination Results	9
5	Quality Assurance and Quality Control	11
5.1	Criteria for Critical Measurements and Parameters	11
5.2	Data Quality Indicators	11
CFU, colony forming unit; ETC, environmental test chamber; NIST, National
Institute of Standards and Technology; NA, Not Applicable	12
5.3	Quality Assurance/Quality Control Checks	12
5.3.1	Check of Integrity of Samples and Supplies	13
5.3.2	Microbiology Laboratory Control Checks	13
5.3.3	QA Assessments and Corrective Actions	14
References	16
iii

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Figures
Figure 2-1. Coupon diagram of non-concrete and concrete materials	3
Figure 4-1. Neutralizer effectiveness	9
Figure 4-2. Test sample recoveries for concrete and glass following a DPAA
decontamination event	10
Tables
Table 2-1. Test Matrix	4
Table 3-1. Sample Types and Numbers for Each Decontamination Solution	5
Table 4-1. Results of Neutralizer Effectiveness Tests	8
Table 4-2. Sample Types and Numbers for Each Decontamination Solution	10
Table 5-1. Data Quality Indicators for Critical Measurements and Parameters	11
Table 5-2. Additional Quality Control Checks for Biological Measurements	14
Table 5-3. Quality Assurance/Quality Control Assessment of Spore Recoveries (CFU) for
Various Control Samples	15
iv

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Acronyms and Abbreviations
BioLab
NHSRC Research Triangle Park (RTP) Microbiology Laboratory
Bg
Bacillus atrophaeus var. globigii
CFU
colony forming unit(s)
DE
Dey Engley
DHS
U.S. Department of Homeland Security
DI
deionized
DPAA
diperadipic acid
DQI
data quality indicator
DQO
data quality objective
EPA
U.S. Environmental Protection Agency
ETC
environmental test chamber
EtO
ethylene oxide
HSRP
Homeland Security Research Program
ID
identification
LR
log reduction
NHSRC
National Homeland Security Research Center
NIST
National Institute for Standards and Technology
ORD
Office of Research and Development
pAB
pH-adjusted bleach
PBST
phosphate-buffered saline with 0.05% Tween® 20
QA
quality assurance
QC
quality control
RH
relative humidity
RSD
relative standard deviation
RTP
Research Triangle Park
STS
sodium thiosulfate
TSA
tryptic soy agar
V

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Executive Summary
An environmental contamination incident involving an infectious or contagious
biological agent may pose significant risks to human health. Currently, there are a
limited number of sporicidal chemicals that are approved for use against Bacillus
anthracis, the causative agent of anthrax. Characterization of potential decontamination
options, ahead of a contamination incident, are important to ensure response and
remediation operations initiate promptly and are effective. The current laboratory-scale
study was undertaken to evaluate the decontamination efficacy of diperadipic acid, under
application conditions that were found to be effective for pH-adjusted bleach (a common
sporicidal liquid). The results showed that diperadipic acid demonstrated >6 Log
Reduction on glass surfaces, but <2 Log Reduction on concrete. Like studies with pH-
adjusted bleach, viable spores were found in runoff samples, indicating that relocation of
contaminants from surfaces may be possible during decontamination.
ES-1

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1 Introduction
This project was conducted by the U.S. Environmental Protection Agency (EPA) Office of
Research and Development's (ORD's) National Homeland Security Research Center (NHSRC)
and supports the mission of the EPA's Homeland Security Research Program (HSRP) by
providing relevant information pertinent to the decontamination of contaminated areas resulting
from a biological incident. The key objective of this project is to estimate the potential reduction
of viable bacterial spores (effectiveness) as a function of the remediation activities applied under
challenging situations and under conditions in field operations.
This study was conducted to evaluate the decontamination efficacy of a diperadipic acid
formulation (Lvnntech. 2.017). when spray-applied to relevant building materials (concrete and
glass) contaminated with Bacillus anthracis surrogate spores. (Bacillus anthracis is the causative
agent for anthrax.) Test conditions (spray duration, spray flow rate, spray distance, contact time,
temperature, materials, test chamber, etc.) previously shown to be effective when pH-adjusted
bleach was evaluated were selected for the current evaluation of diperadipic acid (DPAA).
1.1	Background
The EPA HSRP provides expertise and products that can be widely used to prevent, prepare for,
and recover from public health and environmental emergencies arising from terrorist threats and
other contamination incidents. To carry out the HSRP mission, the NHSRC conducts research to
provide expertise and guidance on the selection and implementation of decontamination methods
that could provide the scientific basis for a significant reduction in the time and cost of
remediating contaminated sites.
This project addresses a direct need expressed by the EPA's Office of Land and Emergency
Management's Chemical, Biological, Radiological, and Nuclear Consequence Management
Advisory Division (CMAD). This need consists of evaluating the effectiveness of innovative
decontamination formulations and procedures, including spraying surfaces with sporicidal
liquids under such conditions.
1.2	Project Objectives
The present effort, described in this data report, evaluated the effectiveness of a spray-based
"low-tech" decontamination method to inactivate/remove spores from building materials
(concrete and glass) using a DPAA formulation developed by Lynntech, Inc. The DPAA
biological agent decontamination formulation is a stabilized powder form, claimed to be
sporicidal and effective against a wide range of microorganisms including aerobic and anaerobic
bacteria and algae. For this effort, "low-tech" is defined as a decontamination approach that can
use off-the-shelf material or equipment, readily available at a local hardware store.
Test coupons were prepared from typical urban building materials (glass and concrete),
inoculated with the target organism Bacillus atrophaeus var. globigii (Bg) using an aerosol
deposition method (U.S. EPA. 2.017). and sprayed with the test solution at 25 °C. Tests were
conducted in an environmental test chamber (ETC) so that the environmental conditions
(temperature and relative humidity [RH]) could be tightly controlled. To simulate large-scale
ES-1

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outdoor operations, all test components (spray nozzles, coupons, runoff collection,
decontaminant reservoir, decontaminant supply tubing, etc.) were located within the chamber
and were acclimated to test conditions prior to testing. The surface decontamination efficacy of
each formulation was measured based on the reduction of viable spores on the surface of the test
coupons. Relocation of viable spores from the contaminated coupon surfaces into the overspray
runoff during each decontamination event was also investigated. Spray pressure, volumes,
duration, and angle of application were selected based upon conditions previously shown
effective (complete kill) when using pH-adjusted bleach (U.S. EPA. 2017).
2

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2 Experimental Approach
2.1 Overall Experimental Approach
Testing was conducted at EPA's Research Triangle Park (RTP) facility in North Carolina. The
general experimental approach used to meet the project objectives is described in the previously
published report entitled "Evaluation of Spray-Based, Low-Tech Decontamination Methods
under Operationally Challenging Environments: Cold Temperatures," (Lee et. al.. 2017) and is
summarized below.
1. Test Coupon Preparation: The 18-mm diameter coupons shown in Figure 2-1 were
prepared from two target materials: concrete and glass. The glass coupon was made of an
18-mm glass disc that was affixed to an aluminum stub using a carbon-based adhesive.
The concrete coupon was fabricated from Sakrete® Top' N Bond Patcher (Sakrete,
Cincinnati, OH), with a drywall nail in the center of the back for handling.
Glass
Carbon adhesive
Aluminum stub
Concrete-
Non-concrete
Dry
wall
Concrete
Material
Figure 2-1. Coupon diagram of non-concrete and concrete materials.
4.
Test Material Sterilization: The coupons, funnels, and stages for storing coupons and
plastic spray bottles were sterilized using an Andersen ethylene oxide (EtO) sterilizer
system (EOGas®, Part No. 333, ANPRO, Haw River, NC), and a sterilization kit (Kit #6,
Part No. AN1006, ANPRO) that includes a cartridge, a humidichip®, a dosimeter, and a
bag.
Test Material Inoculation: The test coupons were inoculated using an aerosol
deposition method (U.S. EPA 2017) that delivered a known concentration of spores in a
repeatable fashion. Approximately 1 x 107 spores of Bg, a surrogate organism for
Bacillus cmthracis, were deposited onto each coupon.
Decontamination Solution Preparation: A powdered concentrate of DPAA,
manufactured by Lynntech, Inc. (College Station, TX), and provided by the Department
of Homeland Security (DHS), was dissolved in deionized (DI) water (130 grams of
DPAA in 1 liter of DI water), and stored at 4 °C before use.
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5.	Decontamination Procedure: Test coupons (five coupons per test material) were
decontaminated using the ETC, equipped with an automated spraying system.
6.	Sample neutralization: Dey Engley (DE) broth (Dev and Englev. 1994) was selected as
the neutralizing agent, as discussed previously (U.S. EPA. 2017. Section 3.6). The
neutralizing agent was applied to stop the decontamination activity after a prescribed
exposure time. After the prescribed exposure times, coupons were collected and
deposited into a tube containing the neutralizing agent. Sodium thiosulfate (STS) was
also evaluated as a neutralizer.
7.	Runoff Collection: Liquid runoff from each coupon was also collected through sterile
funnels into sample tubes that contained pre-determined volumes of neutralizer.
8.	Sample extraction and analysis: Viable Bg spores were extracted from the test samples
(coupon and runoff), and aliquots were analyzed using an automated spiral plating system
(Autoplate 5000, Advanced Instruments Inc., Norwood, MA). Viable spore recovery was
quantified in terms of colony forming units (CFU) present in each sample.
9.	Determination of decontamination efficacy: Decontamination efficacy was expressed
as log reduction (LR) of viable spores recovered. Decontamination efficacy for each
coupon was determined by comparison to positive control sample results. The transfer of
viable organisms to post-decontamination liquid waste was evaluated through
quantitative analysis of decontamination procedure residues (such as decontamination
solution runoff samples).
2.2 Test Matrix
The test matrix for this effort is detailed in Table 2-1. The test conditions denote the temperature
and humidity conditions surrounding the coupons at the time of the spray test. The test chamber
temperature was set at 25 °C, while the RH inside the chamber was recorded at the set
temperature with no corrections. The decontaminant application conditions were set to a
duration, flow rate, and reapplication frequency that achieved >6 LR when pH-adjusted bleach
(pAB) was used as the decontaminant in the same test chamber under the same environmental
conditions (U.S. EPA. 2017). This test design allows results from tests with DPAA and other
decontaminants, to be compared with the baseline performance of a well-characterized
decontaminant (pAB). Test coupons were sprayed with a 5-second spray at time zero, followed
by another 5-second spray at the 10-minute mark. Two 10-minute contact times, one after each
spray, were rendered. The total exposure time (wetted contact time) was 20 minutes.
Table 2-1. Test Matrix
Test
Decontaminant
/Formulation*
Spray
Number/Duration
Contact
time
(Minutes)
Temperature
(°C)
Humidity
(%RH)
Material
Type
1
Diperadipic Acid
5-second spray,
one at 0 minutes,
one at 10 minutes
20
25
Ambient
Concrete
2
Glass
RH, relative humidity
4

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3 Sampling and Analytical Procedures
The primary results from this study will be from the analysis of samples resulting in recovered
viable spores (measured as CFU) per sample expressed on a log-10 scale. Additional
measurements prior to or during the decontamination procedure application are also required to
ensure quality control in the testing. These measurements include quality control checks on the
reagents and equipment being used in the decontamination procedure.
A sampling data log sheet was maintained for each sampling event (or test) that included each
sampling event, the date, test name, sample IDs (identifications), and other test details such as
test temperature, final rinsate volume, and sample extraction time. The sample IDs were pre-
printed on the sampling data log sheet before sampling began. Digital photographs were taken to
document activities throughout the test cycle.
3.1 Microbiological Analysis
This section discusses the project sampling and analytical procedures, including sample
quantities, sample types, and coupon sample extraction and analysis.
3.1.1 Sample Quantities
For each decontamination solution, there were five replicates of coupon samples, five liquid
rinsate samples, three positive control samples, one procedural blank and one negative control
sample per material. Table 3-1 lists the total numbers of samples of each type for each test.
Table 3-1. Sample Types and Numbers for Each Decontamination Solution
Sample Type
Number of
Samples
Test coupon sample
(decontaminated)
5
Liquid rinsate sample
5
Positive control sample
3
Procedural blank
1
Negative control sample
1
3.1.2 Sample Types
The three major sample types for this project are discussed below.
• Surface test coupon samples: Each coupon sample was aseptically transferred,
using sterile forceps, from the stage in the ETC to a 50-mL conical tube containing
10 mL of phosphate buffered saline with 0.05% Tween® 20 (PBST) (TWEEN®,
Croda International PLC, Snaith, UK) and 1.5 mL of DE broth.
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• Liquid rinsate samples: These samples were collected in 250 mL conical tubes,
which were pre-loaded with a set amount of neutralize^ to assess the potential for
viable microorganisms that were washed off the coupon surfaces. Samples were
collected from all liquid runoff during spray applications, and the collection funnels
were subsequently rinsed with sterile DI water. Rinsate samples were collected in
the same vials as runoff and together constituted a single sample. After collection,
test coupon and liquid rinsate samples were sealed in secondary containment and
transported to the NHSRC Research Triangle Park (RTP) Microbiology Laboratory
(BioLab) for quantitative analysis.
3.1.3 Sample Extraction and Analysis
The EPA RTP BioLab analyzed all samples for the presence of spores (sterility check samples)
and quantified the number of viable spores per sample (test coupon and liquid runoff samples).
For all sample types, PBST was used as the extraction buffer. After the extraction procedure,
each sample was aliquoted and plated in triplicate using a spiral plater (Autoplate 5000,
Advanced Instruments, Inc., Norwood, MA), which deposits the sample in exponentially
decreasing amounts across a rotating agar plate in concentric lines to achieve three 10-fold serial
dilutions on each plate. Plates were incubated at 35 ± 2 °C for 16 to 19 hours. The colonies on
each plate were enumerated using a QCount® colony counter (Advanced Instruments, Inc.,
Norwood, MA).
Positive control samples were diluted 100-fold (10"2) in PBST before spiral plating, and samples
of unknown concentration were plated undiluted and after a 100-fold dilution. Samples with
known low concentrations were plated undiluted. The QCount® colony counter automatically
calculates the CFU/mL in a sample based on the dilution plated and the number of colonies that
develop on the plate. The QCount® records the data in an Microsoft® Excel® spreadsheet.
Only plates meeting the threshold of at least 30 CFU were used for spore recovery estimates.
Samples below the 30-CFU threshold were either spiral-plated again with a more concentrated
sample aliquot, filter-plated, or spread-plated in triplicate on tryptic soy agar (TSA) plates using 1-
mL aliquots per plate to achieve a lower detection limit. The plates were incubated at 35 ± 2 °C for
20 to 24 hours before manual enumeration.
3.2 Data Reduction
The overall effectiveness of a decontamination technique is a measure of the ability of the
method to inactivate and/or remove the spores from material surfaces while considering viable
spores that might be relocated from the test surface. Such fugitive biological emissions could
result in secondary contamination that would necessitate additional remediation strategies.
Data reduction was performed on measurements of the total viable spores (measured as CFU)
recovered from each replicate coupon. The average recovered viable spores and standard
deviation for each group of coupons was determined. The groups of coupons included the
following for each combination of material type and extracted sample type:
• Positive control areas (replicates, average, standard deviation)
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•	Test areas (replicates, average, standard deviation)
•	Procedural blank coupons
7

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4
Results and Discussion
This section summarizes the test results for the decontamination efficacy (surface and total) of
the DPAA decontaminant. Relocation of viable spores from the contaminated coupon surfaces
into the overspray runoff during each decontamination event is also reported.
4.1 Neutralization Results
The presence of the decontamination solution on the sample surface or in the liquid effluent
following a decontamination event could negatively bias recovery and efficacy results. Based on
previous studies, sodium thiosulfate (STS) (Calfee et al.. 2011). and DE broth were shown to be
effective neutralizers for various antimicrobial agents. Both STS and DE broth were evaluated
for their respective effectiveness in neutralizing the DPAA solution using glass and concrete
building materials.
The volume of DPAA solution used for the test coupons in this test was derived from the results
obtained for previously performed volume determination test. Table 4-1. indicates the volumes
of DPAA solution and neutralizers used in this test.
The results of the neutralization test series, shown in Table 4-1, and illustrated in Figure 4-1 for
the liquid effluent, confirm the effectiveness of both neutralizers and the residual
decontamination of the DPAA formulation, when not neutralized. To remain consistent with
concurrent studies undertaken by EPA to evaluate low tech/low cost decontamination
formulation effectiveness to inactivate spores, DE broth was used as a neutralizer for the
decontamination testing.
Table 4-1. Results of Neutralizer Effectiveness Tests
Description
Neutralizer
Material
Average
DPAA
Volume
(mL)
Average
Neutralizer
Volume (mL)
Spore Recovery
Avg.
CFU
RSD (%)
Test
coupon
DE Broth
Concrete
0.6
1.5
3.48E+06
18.79
STS
0.6
0.4
3.90E+06
8.23
DE Broth
Glass
0.2
1.5
8.16E+05
8.60
STS
0.2
0.2
1.14E+06
5.44
Rinsate
DE Broth
-
5.0
5.0
2.76E+06
15.54
STS
5.0
3.7
1.58E+07
18.86
DE Broth
5.0
10.0
1.13E+07
56.11
STS
5.0
7.3
1.43E+07
18.60
No
neutralizer
5.0
0.0
8.66E-01
15.24
Inoculum
Control
1.18E+07
CFU, colony forming units; DE, Dey Engley; DPAA, diperadipic acid; RSD, relative standard deviation; STS,
sodium thiosulfate
8

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107
^ 10
=>
ll
O 105
CD
O
=L 104
> 103
o
0
a: 102
o
CL
E 101
CC
CO
10°
10"1
DE Broth
STS
No Neutralizer
Inoculum Control	Test Samples
Test Samples
Figure 4-1. Neutralizer effectiveness.
4.2 Decontamination Results
Decontamination efficacy (Figure 4-2 and Table 4-2) was expressed as a log reduction (LR) of
the viable Bg spores (CFU) recovered. Typically, for laboratory assessments of decontamination
efficacy, for a 1 x 106 CFU) or greater, the LR > 6 is considered effective (U.S. EPA. 2007). and
when no viable spores are recovered (complete kill) after decontamination treatment, the method
is considered highly effective. Decontamination efficacy for each test material was determined
by comparison to positive control sample results, and calculated as follows:
Decontamination efficacy = Mean (Log CFU positive control sample) - Mean
(Log CFU Post Decontamination test coupon sample)
Quantitative assessment of residual (background) contamination was performed by sampling
procedural blanks (non-inoculated coupons exposed to the same decontamination process as the
test coupons). The transfer of viable organisms to post-decontamination liquid waste also was
evaluated through quantitative analysis of decontamination solution runoff samples.
The tests were set up for a five-second spray duration, with one repeat application (two total
applications). The total solution contact time was 20 minutes (10 minutes after the first spray and
10 minutes after the second spray). After the spraying operation was complete, test coupons were
immersed in a neutralizing agent to quench the decontamination reaction. Samples were then
sent to the BioLab for analysis. The results show that the formulation is very effective for
9

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nonporous glass material but much less effective with the more alkaline, porous concrete
material.
To assess the potential fate of the spores, immediately after a decontamination event, all liquids
used in the decontamination test process were collected and quantitatively analyzed. To provide
a conservative estimate of spore fate and transport, rinsates were neutralized immediately upon
collection by pre-loading collection tubes with a neutralizing agent. As expected, the post-
decontamination spore recoveries in the rinsates were on the same order of magnitude for both
the concrete and glass and are results of a physical removal of the spores from the materials.
10°
10' -i
S io6-i
Ll_
o
g>105-
5 104H
>
o
u 10H
Q- 9
E 10H
cc
CO
101 -I
Positive Controls
Test Coupons
Rinsates
T
Concrete
Glass
Material Type
Figure 4-2. Test sample recoveries for concrete and glass following a DPAA
decontamination event.
Table 4-2. Sample Types and Numbers for Each Decontamination Solution
Material
Type
Positive Control
Recovery (CFU)
Test Coupon Recovery
(CFU)
Rinsate Recovery
(CFUs)
Surface Decon
Efficacy(LR)
Average
Standard
Deviation
Average
Standard
Deviation
Average
Standard
Deviation
Average
Standard
Deviation
Concrete
1.07E+07
3.29E+06
2.18E+05
1.83E+05
1.20E+06
4.80E+05
1.83
0.42
Glass
6.76E+06
5.67E+06
1.10E+00
9.17E-01
1.80E+06
7.02E+05
6.78
0.27
CFU, colony forming units; LR, log reduction
10

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5 Quality Assurance and Quality Control
All test activities were documented in laboratory notebooks and digital photographs. The
documentation included, but was not limited to, a record for each decontamination procedure,
any deviations from the quality assurance project plan, and physical impacts on materials. All
tests were conducted in accordance with established EPA Decontamination Technologies
Research Laboratory and BioLab procedures to ensure repeatability and adherence to the data
quality validation criteria set for this project. These procedures are maintained with the facility
manual.
The following sections discuss the criteria for the critical measurements and parameters, data
quality indictors (DQIs), and the quality assurance (QA) and quality control (QC) checks for the
project.
5.1	Criteria for Critical Measurements and Parameters
Data Quality Objectives (DQOs) are used to determine the critical measurements needed to
address the stated project objectives and specify tolerable levels of potential errors associated
with simulating the prescribed decontamination environments. The following measurements
were deemed critical to accomplish part or all of the project objectives:
•	ETC temperature
•	Flow rate of spray nozzles of the automated spray system
•	Sample volume collected
•	Exposure time
•	Temperature of the incubation chamber
•	CFU counts
•	Plated volume
•	Neutralizer volume
5.2	Data Quality Indicators
Table 5-1 lists the DQIs for the critical measurements and parameters. These DQIs were
used to determine if the collected data met the QA objectives. Volumes of components
were measured as accurately as possible using appropriate measuring equipment (such as
volumetric flasks and graduated cylinders).
Table 5-1. Data Quality Indicators for Critical Measurements and
Parameters
Critical Measurement
Measurement Device
Accuracy or
Precision Target
Detection
Limit
ETC temperature
Temperature control
sensor
± 0.5 °C
-73 to+175 °C
Sprayer flow rate
Volume collected in
graduated cylinder per
time
± 10%
1 ml_ per
minute
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Critical Measurement
Measurement Device
Accuracy or
Precision Target
Detection
Limit
Rinsate volume collected
Conical vial
12.5 mL
+ 0.1 mL
Spray time
Timer
± 1 second
1 second
Exposure time
Timer
± 1 second
1 second
Temperature of incubation
chamber
NIST-traceable
thermometer
±2 °C
NA
CFU counts
QCount
Calibration of spiral
plater with
instrument standard
2.0 x 104 must yield
QCount output of
1.82 x 104 to 2.30 x
104
20 CFU/plate
Plated volume
Spiral plater
NA
NA
Neutralizer volume
Serological pipette tips
0.1 mL
0.05 mL
Pressure of automated spray
system
Compressed air
regulator
± 1 psi
0 psi
CFU, colony forming unit; ETC, environmental test chamber; NIST, National Institute of Standards and
Technology; NA, Not Applicable
5.3 Quality Assurance/Quality Control Checks
The critical measurements and parameters listed in Table 5-1 were measured before testing. If
the measurements obtained did not meet the DQI goals, the test was stopped. Tests proceeded
only when the DQI criteria were met.
Many QA/QC checks were used in this project to ensure that the data collected met all the
critical measurements listed in Table 5-1. The measurement and parameter criteria were set at the
most stringent levels routinely achievable. The acceptance criteria for the microbiological
analysis also were set at the most stringent levels routinely achievable, and decisions to accept or
reject test results were based on analytical judgment to assess the likely impact of the failed
criterion on the conclusions drawn from the data.
All the critical measurements and parameters met the DQI target acceptance criteria listed in
Table 5-1. Control samples and procedural blanks were included along with the test samples so
that well-controlled quantitative values were obtained. Background checks for the presence of
bacterial spores were included as part of the standard protocol. Replicate coupons of both
materials were included for each set of test conditions. Specific QC checks performed under this
project included a check of the integrity of samples and supplies, BioLab control checks and QA
assessments and corrective actions are described below.
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5.3.1	Check of Integrity of Samples and Supplies
Samples were carefully maintained and preserved to ensure their integrity. Samples were stored
away from standards or other samples that could cross-contaminate them. In addition, project
personnel carefully checked supplies and consumables before use to verify that they met
specified project quality objectives. All pipettes were calibrated yearly by an outside contractor
(Calibrate, Inc., Carrboro, NC). Incubation temperature was monitored using National Institute
of Standards and Technology (NIST)-traceable thermometers, and the EPA Metrology
Laboratory calibrated the balances yearly.
5.3.2	Microbiology Laboratory Control Checks
Quantitative standards do not exist for biological agents. Viable spores were counted using an
Advanced Instruments QCount® colony counter. Counts greater than 300 or less than 30 CFU
were considered outside the quantitation range. If the CFU count did not fall within the
acceptable quantitation range, the sample was re-plated at a different volume or dilution and then
re-counted.
Before each batch of plates was enumerated, a QC plate was analyzed, and the result was
verified to be within the range indicated on the back of the QC plate. As the plates were counted,
a visual inspection of colony counts made by the QCount® colony counter was performed.
Obvious count errors made by the software were corrected by adjusting the settings (such as
colony size, light, and field of view) and by recounting using an edit feature of the QCount®
software that allows manual removal of erroneously identified spots or shadows on the plate or
by adding colonies that the QCount® software may have missed.
The acceptance criteria for the critical CFU counts were set at the most stringent level routinely
achievable. Positive controls were included along with the test samples so that spore recovery
from the different surface types could be assessed. Background checks also were included as part
of the standard protocol to check for unanticipated contamination. Replicate coupons were
included for each set of test conditions to characterize the variability of the test procedures.
Further QC samples were collected and analyzed to check the ability of the BioLab to culture the
test organism as well as to demonstrate that the test materials used did not contain pre-existing
spores. The checks included the following:
•	Positive control coupons: Coupons inoculated in tandem with the test coupons to
demonstrate the highest level of contamination recoverable from a specific
inoculation event.
•	Procedural blank coupons: Material coupons sampled in the same fashion as
test coupons but not inoculated with the surrogate organism before sampling.
•	Blank TSA sterility controls: Plates incubated but not inoculated.
•	Replicate plates of diluted microbiological samples: Replicate plates for each
sample.
•	Unexposed field blank: Material coupons sampled in the same fashion as test
coupons but not inoculated with the surrogate organism before sampling, or
exposed to the decontamination process.
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Table 5-2 lists the additional QC checks built into the BioLab procedures designed to provide
assurances against cross-contamination and other biases in the microbiological samples.
Table 5-2. Additional Quality Control Checks for Biological Measurements
Sample Type
Frequency
Acceptance
Criterion
Information
Provided
Corrective Action
Positive control
coupons
Minimum of
three per
test
1 x 107for6g,
50% relative standard
deviation (RSD)
between coupons in
each test set
Used to determine
extent of recovery
of inoculum on
target coupon type
If outside range,
discuss in the results
section of this report.
Procedural blank
coupons
One per test
Non-detect
Controls for
sterility of
materials and
methods used in
the procedure
Analyze extracts from
procedural blank
without dilution.
Identify and remove
source of
contamination, if
possible.
Blank TSA sterility
controls
Each plate
No observed growth
after incubation
Controls for
sterility of plates
All plates incubated
before use.
Contaminated plates
discarded before use
Replicate plates of
diluted
microbiological
samples
Each
sample
Reportable CFU
count of triplicate
plates 100% RSD;
reportable CFU
counts between 30
and 300 CFU per
plate
Used to determine
precision of
replicate plating
Re-plate sample.
Unexposed field
blank
One per test
Non-detect
Level of
contamination
present during
sampling
Clean up
environment, and
sterilize sampling
materials before use.
Bg, Bacillus atrophaeus var. globigii; CFU, colony forming units; RSD, relative standard deviation; TSA,
tryptic soy agar
5.3.3 QA Assessments and Corrective Actions
The QA assessments and corrective actions for this project were intended to provide rapid
detection of data quality problems. Mild contamination in QC procedural blank samples was
observed after the completion of testing. However, this contamination was very minimal and had
little to no effect on the project results. Project personnel were intimately involved with the data
on a daily basis so that any data quality issue became apparent soon after it occurred. Blank and
negative samples in which spores were present were at or near the detection limit. Table 5-3
summarizes the QA/QC assessment of spore recoveries for the various control sample types.
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Table 5-3. Quality Assurance/Quality Control Assessment of Spore
Recoveries (CFU) for Various Control Samples
Procedural
Blanks
Procedural Blank
Rinsates
Negative
Controls
Concrete
Glass
Concrete
Glass
Concrete
Glass
6
ND
6
6
ND
5
CFU, colony forming units; ND, non-detect
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References
Calfee MW, Choi Y, Rogers J, Kelly T, Willenberg Z and Riggs K. 2011. Lab-Scale
Assessment to Support Remediation of Outdoor Surfaces Contaminated with
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Dey BP and Engley FB Jr. 1994. Neutralization of Antimicrobial Chemicals by Recovery
Media. Journal of Microbiological Methods 19(1):51-58.
Lee SD, Ryan SP, Snyder EG. 2011. Development of an Aerosol Surface Inoculation
Method for Bacillus Spores. Applied Environmental Microbiology 77(5): 1638-
1645.
U.S. Environmental Protection Agency (U.S. EPA). 2007. Guidance on Test Methods for
Demonstrating the Efficacy of Antimicrobial Products for Inactivating Bacillus
anthracis Spores on Environmental Surfaces. Federal Insecticide, Fungicide, and
Rodenticide Act (FIFRA) Scientific Advisory Panel (SAP) Meeting Minutes No.
2007-05. July 17-19, 2007. Arlington, VA.
U.S. EPA. 2017. Evaluation of Spray-Based, Low-Tech Decontamination Methods Under
Operationally Challenging Environments: Cold Temperatures. EPA 600/R-
17/211. Washington, DC: U.S. Environmental Protection Agency, 2017.
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