United States
Environmental Protection
Agency
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EPA/600/R-10/026 April 2010 www.epa.gov/ord
INVESTIGATION REPORT
Persistence Testing of
Brucella suis on Outdoor Materials
UNITED STATES ENVIRONMENTAL PROTECTION AGENCY
RESEARCH TRIANGLE PARK, NORTH CAROLINA 27711
Office of Research and Development
National Homeland Security Research Center, Decontamination and Consequence Management Division
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Disclaimer
The U.S. Environmental Protection Agency (EPA), through its Office of Research
and Development, National Homeland Security Research Center, funded and directed
this investigation through a Blanket Purchase Agreement under General Services
Administration contract number GS23F0011L-3 with Battelle. This document has been
subjected to the Agency's review and has been approved for publication. Note that approval
does not signify that the contents necessarily reflect the views of the Agency.
Mention of trade names or commercial products in this document or in the methods
referenced in this document does not constitute endorsement or recommendation for use.
Questions concerning this document or its application should be addressed to:
Joseph P. Wood
U.S. Environmental Protection Agency
Decontamination and Consequence Management Division
Mail Code E343-06
Research Triangle Park, NC 27711
(919) 541-5029
wood.ioseph(@,epa.gov
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Acknowledgments
Contributions of the following individuals and organizations to the development of this document are
acknowledged.
United States Environmental Protection Agency (EPA)
Office of Research and Development, National Homeland Security Research Center
Alan Lindquist
Sanjiv Shah
Sarah Taft
Deborah McKean
Frank Schaefer
Office of Solid Waste and Emergency Response
Curtis Snook (Office of Emergency Management, National Decontamination Team)
David Wright (Office of Superfund Remediation and Technology Innovation, Environmental
Response Team)
EPA Regions
Jon Rauscher (Region 6)
Richard Rupert (Region 3)
Randy Schademann (Region 7)
United States Department of Energy
Paula Krauter (Sandia National Laboratories)
Battelle
Karen Riggs
James Rogers
Harry Stone
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Contents
Disclaimer iv
Acknowledgments v
Abbreviations/Acronyms viii
Executive Summary ix
1.0 Introduction 1
2.0 Investigation Approach 3
2.1 Biological Agent 3
2.2 lest Materials 3
2.3 Spiking Coupons 4
2.4 Environmental Conditions 4
2.5 lest Matrix 6
2.6 B. suis Recovery 7
3.0 Quality Assurance/Quality Control 9
3.1 Instrument/Equipment Testing, Inspection, and Maintenance 9
3.2 Inspection/Acceptance of Supplies and Consumables 9
3.3 Data Management 9
3.4 Assessment and Response Actions 9
3.4.1 Technical Systems Audit 9
3.4.2 Performance Evaluation Audit 9
3.5 Data Quality Audit 9
3.6 Reports to Management 9
3.7 Data Review 10
3.8 Performance Criteria 10
3.9 Deviation 10
4.0 Test Results 13
4.1 Method Demonstration - B. suis Recovery 13
4.2 Persistence Results 13
4.2.1 Aluminum 13
4.2.2 Concrete 15
4.2.3 Glass 15
4.2.4 Soil 18
4.2.5 Wood 20
5.0 Summary 21
6.0 References 23
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Figures
Figure 2-1. Spiking coupon using a multichannel pipette 4
Figure 2-2. Refrigerator configured with ultraviolet-A/B lamps 5
Figure 2-3. Schematic (top view) of ultraviolet radiation sampling locations 6
Figure 4-1. B. suis persistence on aluminum 15
Figure 4-2. B. suis persistence on glass 18
Figure 4-3. B. suis persistence on soil 20
Tables
Table ES-l. Summary of persistence test conditions andB. suis recoveries x
Table 2-1. Test materials 3
Table 2-2. Ultraviolet radiation levels 6
Table 2-3. B. suis persistence test matrix 7
Table 3-1. Performance evaluation audits 10
Table 3-2. Spike and positive control recovery data from persistence testing 11
Table 4-1. Recovery data from method demonstration 13
Table 4-2. B. suis persistence on aluminum 14
Table 4-3. B. suis persistence on concrete 16
Table 4-4. B. suis persistence on glass 17
Table 4-5. B. suis persistence on soil 19
Table 4-6. B. suis persistence on wood 20
Table 5-1. Summary of B. suis persistence 22
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Abbreviations/Acronyms
BHI brain heart infusion
°C degrees Celsius
CPU colony-forming unit(s)
cm centimeter
EPA U.S. Environmental Protection Agency
g gram
mL milliliter
NHSRC National Homeland Security Research Center
nm nanometer
OD optical density
PBS phosphate-buffered saline
QA quality assurance
RH relative humidity
TSA technical systems audit
uL microliter
UV ultraviolet
UV-A ultraviolet radiation within the wavelength range of 320 to 400 nanometers
UV-A/B ultraviolet radiation within the wavelength range of 290 to 400 nanometers (i.e., UV-A and UV-B)
UV-B ultraviolet radiation within the wavelength range of 290 to 320 nanometers
UV-C ultraviolet radiation within the wavelength range of 180 to 290 nanometers
uW microwatt
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Executive Summary
The persistence of biological agents is influenced by
environmental conditions and the materials with which
these biological agents are in contact. The generation of
scientifically defensible persistence data is useful for the
proper planning of decontamination efficacy tests and helps
formulate response or remediation plans in preparation
for possible natural occurrences or intentional releases of
biological agents. This report presents the results of an
investigation to evaluate Brucella suis persistence on five
materials (typically found in the outdoor environment) under
various environmental conditions and exposure durations.
Persistence (recovery of viable organisms) was assessed for
B. suis spiked onto five materials (aluminum, concrete, glass,
soil, and wood). The spiked materials were then exposed
to controlled environmental conditions. The environmental
conditions comprised moderate temperature (about 22 °C) or
low temperature (about 5 °C), and the absence or presence
of ultraviolet (UV) radiation within the wavelength range
of 290 to 400 nm (UV-A/B, which was intended to simulate
natural sunlight). Relative humidity (RH) was ambient and
not controlled. The persistence investigation lasted up to
28 days. Persistence was determined by the recovery of
B. suis as colony-forming units (CPU) from the materials at
the completion of the exposure duration.
The persistence investigation results are summarized in
Table ES-1. In the absence of UV-A/B, B. suis persisted on
aluminum, glass, and soil for at least 28 days (the longest
duration tested), at both low and moderate temperatures. On
concrete, B. suis was only recovered from the seven day test
at the "low temperature, no UV" environmental condition.
Because B. suis showed low persistence on concrete, the
concrete coupons were replaced with wood coupons for the
longer persistence tests. Persistence testing with B. suis on
wood resulted in recoveries after 21 and 28 days of exposures
to the "low temperature, no UV environmental condition,
but B. suis was not recovered from wood after 21 and 28
days of exposures to the "moderate temperature, no UV
environmental condition.
In general B. suis had higher CPU recoveries (persisted
longer) at low temperature than at moderate temperature.
In the presence of UV-A/B, the length of time that B. suis
persisted on aluminum and glass was reduced, so that
the longest exposure durations from which B. suis were
recovered ranged from 2 to 7 days. Such reductions in the
duration of B. suis persistence were not as pronounced when
B. suis was spiked onto soil and exposed to UV-A/B. In the
presence of UV-A/B, B. suis was recovered from soil at the
longest exposure durations tested (for at least 14 days at
"moderate temperature, UV-A/B" and for at least 14 days
at "low temperature, UV-A/B"). When exposed to UV-A/B,
B. suis was not recovered from concrete. Testing was not
conducted with B. suis on wood in the presence of UV-A/B.
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Table ES-1. Summary of persistence test conditions and B, suis recoveries
Duration (Days)
7
14
21
28
Temperature (°C)t
23
23
23
23
BiffE
39
29
39
39
'MM ^Ei^^n^^^^l
Moderate Temperature
7.71 x 104
2.29 x 104
1.38x 104
5.19x 102
Moderate Temperature,
1
2
4
7
10
14
22
23
22
22
22
23
50
50
50
50
50
48
NT
NT
l.SOx 102
2.27 x 102
ND
ND
, NoUV
0
0
NT
NT
UV-A/B§
ND
ND
ND
ND
NT
NT
• rr*i-i it
. suis (LrU/cc
Glass
3.32 x 104
7.36 x 103
6.26x 102
7.87x 102
2.53x 102
ND
ND
ND
NT
NT
>upon) by Material*
Soil
2.66x 104
9.29x 103
6.88x 102
1.13x 102
NT
NT
5.03x 104
3.49 x 103
1.67x 103
4.33 x 102
Wood
NT
NT
ND
ND
NT
NT
NT
NT
NT
NT
Low Temperature, No UV
7
14
21
28
5.3
5.8
4.1
4.4
52
50
11
12
8.75x 106
1.81 x 106
9.82 x 105
3.48 x 105
5.32 x 101
ND
NT
NT
2.23x 106
2.07x 105
7.75x 105
3.05x 105
9.67x 104
5.41 x 104
9.35 x 104
4.14x 104
NT
NT
1.73x 105
1.57x 105
Low Temperature, UV-A/B5
1
2
2#
5
5#
14
6.5
7.6
4.9
4.9
4.5
4.2
53
46
58
59
58
57
4.10 x 103
2.20 x 102
8.68 x 101
ND
1.32 x 101
NT
ND
ND
ND
ND
NT
NT
4.23 x 103
4.21 x 102
ND
ND
NT
NT
4.24 x 106
4.05 x 106
4.57 x 106
1.01 x 106
7.69x 105
7.11 x 103
NT
NT
NT
NT
NT
NT
#
Spike controls ranged from 5.67 x 107 to 1.22 x 108 CPU/coupon.
Mean temperature and RH values based on continuous monitoring at 1 to 2 minute intervals.
"ND" indicates that no viable organisms were recovered from any of the replicate coupons. The detection limit for a given coupon with triplicate plating is
approximately 33 CPU/coupon (see Section 2.6).
UV-A/B exposures were cyclical (12 hours on, 12 hours off) to simulate diurnal conditions.
Tests were repeated to improve the quantification of the UV measurements.
"NT" is a condition/material that was not tested.
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1.0
Introduction
The U.S. Environmental Protection Agency (EPA) is
investigating the persistence of biological and chemical
agents in the absence of decontamination. For biological
agents, persistence reflects the extent to which viability
is retained over a defined period of time. Some biological
agents are unstable and lose viability within minutes of
their release, thereby diminishing the risk to human health
and the environment; other agents can remain viable for
weeks, months, or years. The persistence of biological
agents is influenced by environmental conditions and the
materials with which they are in contact. The generation of
scientifically defensible persistence data is useful for the
proper planning of decontamination efficacy tests and helps
formulate response plans in preparation for possible natural
occurrences or intentional releases of biological agents.
This investigation focused on the persistence ofBmcella
suis, a bacterium that can cause a debilitating influenza-like
illness in humans. The intent was to determine the length of
time that B. suis remained viable on various materials found
in the outdoors (aluminum, concrete, glass, soil, and wood)
under various environmental conditions, primarily while
exposed to moderate or low temperatures and in the presence
or absence of ultraviolet (UV) radiation levels typical of
those associated with sunlight.
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2.0
Investigation Approach
This report describes the investigation of the persistence of
B. suis on various materials exposed to various temperature
and UV radiation conditions. Briefly, B. suis was spiked
onto materials (aluminum, concrete, glass, soil, and wood)
that could become contaminated in the outdoor environment
and then be exposed to environmental conditions for
controlled exposure durations. Persistence was then assessed
by quantifying the recovery of B. suis as colony-forming
units (CPU) from each tested combination of material,
environmental condition, and exposure duration. All testing
was performed in accordance with the peer-reviewed and
EPA-approved Quality Assurance/Test Plan for Persistence
Testing ofBrucella suis on Outdoor Materials.^
2.1 Biological Agent
The biological agent B. suis is a gram-negative, aerobic,
non-spore-forming coccobacillus.(2) For this investigation,
B. suis biotype I (Battelle BRU163) was used. The B. suis
biotype I was originally obtained from American Type
Culture Collection (Manassas, VA) and maintained in pure
culture by Battelle. Fresh B. suis culture was prepared in
advance of each day that coupons were spiked by transferring
colonies from a streak plate (freshly growing or stored less
than two weeks at 2 °C to 8 °C) into 10 mL of brain heart
infusion (BHI) broth (BD Diagnostic Systems, Sparks,
MD) and incubated overnight at 37 °C ± 2 °C on an orbital
shaker set to 200 revolutions per minute, until an increase in
turbidity was observed. The bacterial culture (late log phase
of growth) was diluted with BHI broth to an optical density
(OD) reading at 600 nm (OD600 nm) of approximately 0.1
to 0.2 OD units. The 0.1 to 0.2 OD600 nm has previously
Table 2-1. Test materials
been shown to yield average CFU/mL that would meet
the stock suspension spore concentration requirements for
this investigation (1 x 107 - 1 x 109 CFU/mL). The viable
B. suis bacteria in the stock suspension were enumerated
to determine CFU/mL ("spike control") by analyzing serial
dilutions (serial 1:10 dilutions) of the stock suspension
prepared using phosphate-buffered saline (PBS) and plated
onto BHI agar for CPU determination.
2.2 Test Materials
Materials typically found in the outdoors that were used
for B. suis persistence testing are described in Table 2-1.
Test coupons of the outdoor materials were generally cut
to the sizes indicated in Table 2-1 from larger pieces of
stock material. Concrete coupons were poured into molds
rather than being cut to size. Soil "coupons" consisted of
3.5 cm diameter Petri dishes with a height of 1 cm lined
with Parafilm® and filled with equal masses (7 g ± 1 g) of
uncompacted soil. Coupons were sterilized by autoclaving or
gamma irradiation (no pre-cleaning of the coupons occurred
prior to sterilization). The selected approach, as shown in
Table 2-1, was based on cost-effectiveness and minimization
of physical alterations of the material. Autoclaving followed
Battelle's standard operating procedure.(3) Gamma-irradiation
at 40 kilogray was conducted by STERIS Isomedix Services
(Libertyville, IL). Prior to gamma irradiation, coupons were
sealed in 6 mL Urine® poly tubing (Urine, Chicago, IL) to
preserve sterility until the coupons were ready for use. Test
coupons were each visually inspected prior to being used
in any experiment or test. Coupons with anomalies on the
application surface were discarded and not used.
Material
Aluminum
(finished)
Concrete
(unpainted)
Glass
Soil (topsoil)
Wood
(untreated pine)
Lot, Batch,
or Observation
Aluminum alloy 2024
5 parts sand:
2 parts cement
American Society for
Testing and Materials
International C1036
Batch No. PY1A0597
Generic molding
Soil "coupons" consisted of a 3.5 cm diameter Petri
• Manufacturer or
Supplier Name
Adept Products, Inc.,
West Jefferson, OH
Wysong Concrete,
Fairfield, OH
Brooks Brothers Glass and
Mirror Service,
Columbus, OH
GardenScape, Inc.,
Eau Claire, PA
West Jefferson Hardware,
West Jefferson, OH
dish with a height of 1 cm (or equivalent)
Coupon Size,
Width x Length
1.9 cm x 7.5 cm
1.9 cm x 7.6 cm
1.9 cm x 7.5 cm
*
1.9 cm x 7.5 cm
lined with Parafilm8 and
Coupon
Thickness
0.2 cm
1.3cm
0.3 cm
*
1 cm
filled with 7 g ± 1 g c
Material
Preparation
Autoclave
Autoclave
Autoclave
Gamma
irradiation
Gamma
irradiation
if uncompacted soil.
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2.3 Spiking Coupons
The stock suspension (approximately 1 x 108 CFU/mL,
prepared as described in Section 2.1) was used to spike the
coupons. Test and positive control coupons were placed
lying flat in the cabinet and spiked with a 100 uL aliquot
of stock suspension of approximately 1 x 108 CFU/mL of
B. suis generally using a multichannel micropipette as two
rows of five droplets (10 uL per droplet) across the surface
of the coupons (Figure 2-1); soil coupons were spiked using
a single channel pipette to apply ten 10 uL droplets across
the material surface. The 100 uL aliquot of stock suspension
yielded approximately 1 x 107 CPU/coupon. Spiked coupons
were immediately exposed to the test conditions without a
drying period.
Spiking of test coupons with B. suis was performed in a
Class III biological safety cabinet. To ensure further
cleanliness and prevent contamination of test surfaces, sterile
techniques following Battelle policies and guidelines(4"6)were
exercised during all phases of handling the test coupons.
Prior to spiking, each coupon was assigned a unique identifier
code by the test personnel for traceability. The identifier code
was placed on the coupons and test tubes in indelible ink.
2.4 Environmental Conditions
The four environmental conditions tested were based on
temperature (moderate and low) and UV radiation (absent or
present). UV radiation, within the wavelength range of 290
nm to 400 nm (UV-A/B) was intended to simulate natural
sunlight. Relative humidity (RH) was ambient; efforts
were not taken to achieve an especially low, high, or stable
RH level during any of the tests. The four environmental
conditions included:
• Moderate temperature, ambient (about 22 °C), no UV
• Moderate temperature, ambient (about 22 °C), UV-A/B
(12-hour on/off cycle)
• Low temperature, about 4 °C, no UV
• Low temperature, about 7 °C, UV-A/B (12-hour on/off
cycle).
The target temperature for the "low temperature, UV-A/B"
environmental condition was 7 °C, rather than 4 °C which
was the target temperature for the "low temperature, no UV
environmental condition. The 7 °C target temperature was
selected to avoid potential freezing/thawing cycles that might
have been generated with the use of heat-producing lamps
inside a refrigerator.
Persistence testing was conducted inside a compact glove
box (Plas Labs, Inc., Lansing, MI) for moderate temperature
tests and inside a refrigerator modified with glove ports for
the low temperature tests. In the low temperature testing,
temperature and RH were monitored (every one to two
minutes) using a Yokogawa DX2010 data logger (Yokogawa
Electric Corporation, Tokyo) connected to an Omega
HX93 AC temperature/RH probe (Omega Engineering Inc.,
Stamford, CT); for moderate-temperature testing a HOBO
U10 data logger (Onset Computer Corporation, Bourne, MA)
was used to measure temperature and RH. Although RH
was monitored, efforts to manipulate the RH level were not
undertaken. The actual temperatures and RH levels observed
during testing are documented in the test matrix (Table 2-3)
presented in Section 2.5.
Fluorescent UV-A/B lamps inside the glove box or
refrigerator were used to generate the UV-A/B (see Figure
2-2 for a photograph of the refrigerator configured with the
UV-A/B lamps). UV radiation conditions were designed
to approximate the intensity and wavelengths of UV
radiation (especially UV-B, with UV radiation within the
wavelength range of 290 to 320 nm) encountered in natural
sunlight at the earth's surface. UV-B is reported to be the
primary component of sunlight which inhibits biological
activities.(7) The spectrum and intensity of terrestrial UV
radiation is variable and is affected by time of day, day of
year, geographical location, atmospheric pollution, and
clouds. Naturally- occurring UV-B levels, observed around
noon, range from 19.5 to 150 uW/cm2.(8-n) The target UV-B
level of 70 uW/cm2 was generated using ReptiSunTM 10.0
Linear Fluorescent UV-B lamps (Zoo Med Laboratories, Inc.,
San Luis Obispo, CA). The amount of UV-A (UV radiation
within the wavelength range of 320 to 400 nm) generated
Figure 2-1. Spiking coupon using a multichannel pipette
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during testing was approximately 110 uW/cm2, which was
within the range of UV-A observed in natural sunlight (0 to
4,500 uW/cm2).(12) The level of UV-C (UV radiation within
the wavelength range of 180 to 290 nm) generated during
testing was 0 uW/cm2; solar UV-C does not generally reach
the earth's surface/12'13) UV radiation was conducted for
12 hours (UV-A/B lamps on), and then the UV-A/B lamps
were turned off for 12 hours to simulate diurnal conditions.
UV radiation was measured with Solarmeter® Model 5.7
for total UV radiation, Model 6.2 for UV-B, and Model 8.0
for UV-C, from Solartech, Inc. (Harrison Township, MI).
UV radiation measurements were made from five positions
beneath the UV-A/B lamps (Figure 2-3). UV radiation
measurements were taken beneath the UV-A/B lamps at the
same distance the test coupons were from the UV-A/B lamps.
UV-B, UV-C, and total UV radiation were generally
monitored twice a day (at least four hours apart) during
persistence testing when the UV-A/B lamps were operating
(UV-A was calculated as total UV radiation minus the
UV-B and UV-C levels). UV radiation measurements were
generally monitored twice daily (at least four hours apart)
during the persistence testing when the UV-A/B lamps were
operating. UV radiation was not measured on Saturdays,
Sundays, or at night. In only one instance, testing was
initiated in the afternoon and therefore only one UV radiation
measurement event occurred on the first test day.
During the low temperature tests a downward drift in the
UV-B measurements was discovered. This drift was evident
as a decreasing trend in measured UV-B levels during the
five-day test at low temperature. After the test, once the UV
radiation meters were allowed to warm to room temperature,
the measured irradiation inside the refrigerator showed
typical UV-B levels (approximately 70 uW/cm2). Therefore,
the downward trend in measurements was attributed to the
prolonged exposure of the UV radiation meters to the low
temperature conditions. Subsequent low temperature tests
were conducted with UV radiation measurements taken only
pre- and post-test, which enabled the UV radiation meters
to be stored at room temperature when not in use. Although
all of the UV radiation levels measured during each test
are provided in Table 2-2, as footnoted, some of the low
temperature measurements are believed to be inaccurate.
The mean and range calculated included all irradiation
measurements taken during a given persistence test. For
example, the seven-day test shown in Table 2-2 resulted in
10 measurement events with five individual measurements
taken at each event; the mean and range in Table 2-2 were
for all fifty measurements (10 measurement events x five
individual measurements per event = 50 measurements).
Figure 2-2. Refrigerator configured with ultraviolet-A/B lamps
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Work surface
(top view)
Five points measured for UV-B, UV-C and total
UV radiation during persistence testing
All coupons located within dashed line (irradiated area)
Figure 2-3. Schematic (top view) of ultraviolet radiation sampling locations (not to scale)
Table 2-2. Ultraviolet radiation levels*
Duration (Days)
UV Radiation Measurement Events
UV-A (uW/cm2)'
Moderate Temperature, UV-A/B*
1
2
4
7
10
14
1
2
2
5
5
14
2 (twice daily)
4 (twice daily)
4 (twice daily)
10 (twice daily)
16 (twice daily)
20 (twice daily)
Low Temperature,
2 (pre- and post-testing)
4 (twice daily)
2 (pre- and post-testing)
9 (twice daily)
2 (pre- and post-testing)
2 (pre- and post-testing)
94 ±7.4
94 ±5. 8
96 ±5. 5
97 ±5. 7
96 ± 5.3
96 ± 5.3
UV-A/B*
110±6.0
113 ±5.6
109 ±6.9
101 ± 12
111 ±7.6
110±6.9
UV-B (uW/cm2)* 1
68 ± 4.2
68 ± 4.4
68 ± 4.0
69 ±4.1
68 ±4.1
69 ± 4.0
67 ±5.9
63 ± 8.3§
67 ±7.1
60 ± 8.8§
67 ±5.0
66 ±5.6
* The target UV-C level was 0 pW/cm2 and all measurements were 0 pW/cm2.
' Data are expressed as mean ± standard deviation.
* The target UV-A level was 110 pW/cm2 and target UV-B level was 70 pW/cm2; UV-A/B exposures were cyclical (12 hours on, 12 hours off) to simulate diurnal
conditions.
8 These measurements are believed to be inaccurate, due to the effect of low temperature on the meter. The actual UV-B levels are believed to be in the range of the
tests in which pre- and post-testing measurements were conducted.
2.5 Test Matrix
The test matrix for persistence testing with B. suis is provided
in Table 2-3; the order of the testing was not always done in
the sequence shown in the table. After spiking the coupons,
at least four non-zero exposure durations were monitored
for each environmental condition. The range of exposure
durations was determined in consultation with the EPA Task
Order Project Officer and was based on the outcome of initial
persistence test results. Positive controls (coupons spiked
with B. suis and extracted at time-zero, i.e., immediately
after spiking) and blanks (coupons not spiked with B. suis)
were also run for each material, environmental condition,
and exposure duration combination. A brief method
demonstration ensured that the methods previously used(14)
were adequate to obtain sufficient recovery of B. suis from
the materials, with the exception of aluminum for which
adequate recoveries were already established. Testing with
wood was only occasionally conducted (i.e., in some cases
B. suis would not persist on concrete, so persistence was
tested on wood as an alternative material).
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2.6 B. suis Recovery
For sample extraction, test coupons, positive controls, and
blanks were transferred aseptically to sterile individual 50
mL conical vials containing 10 mL of sterile extraction buffer
(i.e., PBS). For the soil "coupon", soil and the Parafilm
liner (which prevented soil from adhering to the Petri dish)
were removed from the Petri dish and placed into the vial
containing the extraction buffer. The vials were agitated
on an orbital shaker for 15 minutes at approximately 200
revolutions per minute at room temperature. Following
extraction from the coupons, the extracts were removed and a
series of dilutions (serial 1:10 dilutions) were prepared using
PBS. During this investigation, B. suis was not recovered
from any of the associated blanks.
An aliquot (0.1 mL) of the selected dilutions and, when
necessary, the undiluted extract were plated onto BHI
agar (BD Diagnostic Systems, Sparks, MD) in triplicate.
The cultures were incubated for up to 72 hours at 37 °C ±
2 °C. The colonies were counted manually and the CPU/
mL determined. Traditionally plates having colony counts
between 25 and 250 are typically used for calculating the
CFU/mL. However, under certain circumstances (i.e.,
poor recovery, reduced persistence over time, efficient
decontamination, etc.) there were less than 25 colonies per
plate from the undiluted extract. In these cases the number
of colonies was counted and recorded even if there were 25
colonies or less per plate. The CPU/coupon were calculated
Table 2-3. B, suis persistence test matrix
by multiplying the CFU/mL by the volume of the extraction
buffer used for each coupon (10 mL per coupon). The total
CPU extracted from a coupon was calculated as:
Total CPU/coupon = [(mean CPUplate count x I/dilution
factor)'/plated volume] x (extraction buffer volume) (1)
Where:
Mean CPU plate count
Plated volume
Dilution factor
Extraction buffer volume
= average number of colonies
counted in three replicate
plates
= volume that was applied
to each plate, in this case
0.1 mL
= portion of the total extraction
buffer that was used to
prepare the dilutions
= volume of extraction buffer
used to extract coupon, in
this case 10 mL.
A single viable bacterium present in a plated aliquot of
sample would be expected to be observed as a CPU.
Considering one CPU observed on one of the three plates
of undiluted extract, the individual coupon detection limit is
approximately 33 CPU/coupon based on Equation 1. Since
only a portion (i.e., 0.1 mL aliquot per plate) of undiluted
extract is cultured, viable bacteria could be present in the
Target Environmental Condition
Moderate temperature (22 °C),
ambient RH, no UV
Moderate temperature (22 °C),
ambient RH, UV-A/B*
Low temperature (4 °C), ambient
RH, noUV
Low temperature (7 °C), ambient
RH, UV-A/B*
Material
Aluminum, concrete*,
glass, soil, and wood§
Aluminum*, concrete11,
glass11, and soil*
Aluminum, concrete*,
glass, soil, and wood§
Aluminum, concrete,
glass, and soil1
Duration (Days)
7
14
21
28
1
2
4
7
10
14
7
14
21
28
1
2
2
5
5
14
Temperature (°O*
22.8 ±0.35
22.8 ±0.33
23.3 ± 0.83
23.3 ± 0.83
22.1 ±0.92
22.6 ± 1.25
22.3 ±0.95
22.3 ±0.96
22.4± 1.15
22. 7 ± 1.21
5.29 ±0.95
5.79± 1.00
4.08 ±3. 97
4.43 ±3. 25
6.47 ±2. 22
7. 56 ±4.30
4.87 ±2.38
4.92 ± 2.47
4.48 ±2. 61
4.22 ± 2.94
RH (%)*
38. 6 ±22. 5
29. 2 ±21.3
38. 8 ±2.88
38. 8 ±2.83
49. 9 ±2.48
50.4 ±4.82
50.1 ±4.36
50. 2 ±3.94
49. 6 ±3.66
48.4 ±4.15
52. 0± 15.0
50. 0± 14.0
10. 7 ±5.87
11.5±5.59
53.0 ±24.0
45. 5 ±23.0
58.4 ±25. 7
58. 7 ±20.9
58. 2 ±25. 7
56. 6 ±25.9
* Data are presented as mean ± standard deviation.
' The target UV-A level was 110 pW/cm2 and target UV-B level was 70 pW/cm2; UV-A/B exposures were cyclical (12 hours on, 12 hours off) to simulate diurnal
conditions.
* Concrete was tested only on days 7 and 14.
8 Wood was tested only on days 21 and 28.
* Aluminum and soil were tested on days 4, 7, 10, and 14.
1 Concrete and glass were tested on days 1, 2, 4, and 7.
Only soil was tested on day 14.
-------
extract that were not collected for plating. However, given combination by dividing the total number of viable organisms
the number of replicate coupons (five) and replicate plates extracted from all five test coupons by the number of
(three) per undiluted coupon extract it is unlikely that the replicate coupons (i.e., five). Persistence (recovery) curves,
presence of viable bacteria would go undetected. out to 28 days, were also developed for B. suis for several
The recovery of B. suis bacteria (quantified as mean CPU/ material wd environmental condition combinations, by
coupon ± standard deviation) was calculated for each ^P1^ B' suis recovered (quantified as CPU/coupon)
material/environmental condition/exposure duration agams ime.
-------
3.0
Quality Assurance/Quality Control
Quality assurance (QA)/quality control procedures
were performed in accordance with the program quality
management plan(15) and the test/QA plan(1) for this
investigation. QA/quality control procedures are summarized
below.
3.1 Instrument/Equipment Testing, Inspection, and
Maintenance
All equipment (e.g., pipettes, incubators, biological safety
cabinets) used was verified as being certified, calibrated, or
validated at the time of the investigation and was maintained
and operated according to the quality requirements of the
Battelle Biomedical Research Center.
3.2 Inspection/Acceptance of Supplies and
Consumables
Supplies and consumables were acquired from reputable
sources and were National Institute of Standards and
Technology-traceable when possible. The source and purity
were documented in the investigation records. Supplies and
consumables were examined for evidence of tampering or
damage; coupons were examined for anomalies on the test
surface. Any suspect material was not used. In addition,
expiration dates were noted and recorded. Solutions were
prepared following Battelle Biomedical Research Center
protocols and documented in reagent preparation forms.
3.3 Data Management
Data acquisition during the investigation included proper
recording of the procedures used in the testing to assure
consistency in the investigation and adherence to the test/QA
plan(1); documentation of sampling/testing conditions; and
recording of analytical results and investigation conditions.
Data acquisition was carried out electronically by the
data logger (e.g., temperature, RH, and time) or manually
by Battelle test personnel. Manually-acquired data were
recorded immediately in a consistent format throughout
the investigation. All written records were in ink and any
corrections to recorded data were made with a single line
through the original entry and the correction entered,
initialed, and dated by the person making the correction.
Non-obvious corrections included a reason for the correction.
Whether collected manually or electronically, relevant data
were entered into an electronic spreadsheet set up to organize
the data in a clear and consistent manner. The accuracy of
entering manually recorded data into the spreadsheets was
checked at the time the data were entered, and a portion of
the data was checked by the Battelle QA Manager as part of
the data quality audit.
3.4 Assessment and Response Actions
3.4.1 Technical Systems Audit
Battelle QA staff conducted a technical systems audit (TSA)
during April, May, and August 2009 to ensure that the
investigation was being conducted in accordance with the
test/QAplan(1) and associated amendments and the quality
management plan.(15) As part of the TSA, test procedures
were compared to those specified in the test/QA plan and
data acquisition and handling procedures were reviewed.
Observations and findings from the TSA were documented
and submitted to the Battelle Task Order Leader for response.
None of the findings of the TSA required corrective action.
TSA records were permanently stored with the QA Manager.
3.4.2 Performance Evaluation Audit
Performance evaluation audits were performed for those
measurements that factored into the data used in quantitative
analysis. Table 3-1 summarizes the performance evaluation
audits that were performed. The spectrophotometric
absorbance was audited using a SpectraTest™ Absorbance
Validation Package (MDS, Inc., Toronto, Canada) which
provides a National Institute of Standards and Technology-
traceable solution for validating optical performance.
The test measurements and calculations are performed by
SoftMax® Pro software (MDS, Inc., Toronto, Canada). No
performance evaluation audit was performed for B. suis
because quantitative standards for this biological material do
not exist; however, the associated spike and positive controls
and blanks form the basis of support for conclusions drawn
from the investigation.
3.5 Data Quality Audit
At least 10% of the data acquired during the investigation
were audited. A Battelle QA auditor traced the data from the
initial acquisition, through reduction and statistical analysis,
to final reporting to ensure the integrity of the reported
results. All calculations performed on the data undergoing the
audit were checked.
3.6 Reports to Management
Each audit was documented in accordance with the quality
management plan.(15) The results of the TSA and data quality
audit were submitted to EPA.
-------
Table 3-1. Performance evaluation audits
Volume
Spectrophotometric
absorbance
Temperature
RH
Total UV radiation
UV-B
UV-C
Time
f^n iV^Q
Micropipettes checked by gravimetric evaluation
SpectraTest™ Absorbance Validation Package
with calculations by SoftMax® Pro software
Compared to independent calibrated
thermometer value
Compared to independent calibrated
hygrometer value
Compared to independent calibrated total UV
radiation value
Compared to independent calibrated UV-B value
Compared to independent calibrated UV-C value
Compared to independent clock or watch value
RIIF ^Erat
± 10%
± 10% OD
±2°C
± 10% (full scale)
± 10%
± 10%
± 10%
2 seconds/hour
Actual Tolerance
Done 3 times, all <0.3%
Done 7 times,
all <10% OD
Done 4 times, all <0.4 °C
Done 4 times,
all <1. 7% (full scale)
Done 4 times, all <0.6%
Done 4 times, all <1.4%
Done 4 times, all = 0.0%
Done 4 times,
all = 0 seconds/hr
3.7 Data Review
Records generated in the investigation received a quality
control/technical review and a QA review before they were
used to calculate, evaluate, or report investigation results. All
data were recorded by Battelle staff. The person performing
the review was involved in the experiments and added his/
her initials and the date to a hard copy of the record being
reviewed. This hard copy was returned to the Battelle staff
member who stored the record.
3.8 Performance Criteria
As shown in Table 3-2, except for one stock suspension
(shown in bold), all spiked amounts were within the target
range of 1 x 107 - 1 x 109 CFTJ/mL (1 x 106 - 1 x 108 CPU/
coupon). One stock suspension slightly exceeded the target at
1.22 x 109 CFU/mL. The concentrations of stock suspensions
ranged from 4.90 x 107 - 1.22 x 109 CFU/mL.
As shown in Table 3-2 for the persistence tests, percent
recoveries of viable and culturable bacteria from positive
controls were within the target range for all materials except
for some concrete samples (shown in bold). Although
the recovery of B. suis from concrete during the method
demonstration tests was adequate (see Table 4-1), the
recovery from concrete positive controls was less than 5% for
most of the subsequent persistence tests; the reason for this
difference is not known. Nevertheless an appreciable amount
of B. suis was recovered (generally >1 x 105 CPU/coupon)
from concrete to assess persistence over time.
No CPU were recovered from any blank coupons. No results
were excluded as outliers.
3.9 Deviation
The test/Q A plan specified that the RH was to be ambient
conditions, and monitored, but not controlled. However,
the data quality objectives specified in the test/QA plan
set an allowable test measurement tolerance for RH in
the chamber of ±20% (full scale). Because the RH range
exceeded this specification, the deviation is noted. The
level of RH may impact persistence, but RH was not a
controlled or independent variable for this investigation.
Although the RH range was higher than anticipated, mean
RH levels were generally similar and ranged from 38.6% to
58.7% for all tests except three: the 14-day test at "moderate
temperature, no UV" (29.2% RH) and the 21- and 28-day
tests at "low temperature, no UV" (10.7% RH and 11.5%
RH, respectively).
-------
Table 3-2. Spike and positive control recovery data from persistence testing*
Material Spike Control (CPU/Coupon)
Aluminum 6.33 x 107
1.22 x 108
6.27 x 107
8.30 x 107
5.73x 107
l.OOx 108
7.07 x 107
4.90 x 107
7.60 x 107
8.07 x 107
5.67 x 107
Concrete 6.33 x 107
8.30 x 107
6.27 x 107
5.73x 107
7.07 x 107
4.90 x 107
7.60 x 107
8.07 x 107
Glass 6.33 x 107
1.22 x 108
8.30 x 107
6.27 x 107
5.73x 107
l.OOx 108
7.07 x 107
4.90 x 107
7.60 x 107
8.07 x 107
Soil 6.33 xlO7
1.22 x 108
6.27 x 107
8.30 x 107
5.73x 107
l.OOx 108
7.07 x 107
7.60 x 107
8.07 x 107
5.67 x 107
6.13 x 107
Wood 1.22 x 108
l.OOx 108
BIRliIiHSHlHHIliHWHHnBM
from Positive Controls
7.32 ±0. 67 x 107
9.99 ±0. 78 x 107
5. 76 ±0.41 x 107
9.07 ± 1.69x 107
3.53± 1.79x 107
9.42 ± 1.99x 107
5.74+ i.44x 107
4.78 ±0. 53 x 107
5.71 ±0.64x 107
7.27 ± 1.22x 107
5.33 ± 1.75x 107
9.27 ±20. 5 x 106
3.32 ±7. 04 x 106
1.91 ±2.64x 105
3. 54 ±7. 64 x 106
1.81 ±3.49x 103
2.07 ±2. 94 x 105
1.64 ±2.31 x 105
2. 73 ±6. 08 x 106
6.49 ±0. 98 x 107
8.65 ± 0.49 x 107
1.03 ±0. 20 x 108
6.30 ± 0.49 x 107
5.18± 1.60x 107
9.86 ± l.SOx 107
5.57± l.OOx 107
6.13±0.79x 107
6.37 ±0. 70 x 107
8.33 ±0. 75 x 107
6.09 ±0. 65 x 107
7.74±0.53x 107
5.52±0.99x 107
8.24 ±0. 80 x 107
3.02 ±2. 03 x 107
6.93 ± 1.63x 107
4.44 ± 1.53x 107
5.98 ±0. 59 x 107
7.67 ±0. 69 x 107
4.01 ±0.48x 107
4.31 ±0.42x 107
3.60 ±2. 70 x 107
3.34 ± 2.47 x 107
Recovery as Percentage of
116%
82%
92%
109%
62%
94%
81%
98%
75%
90%
94%
15%
4.0%
0.3%
6.2%
0.003%
0.4%
0.2%
3.4%
103%
71%
124%
100%
90%
99%
79%
125%
84%
103%
96%
63%
88%
99%
53%
69%
63%
79%
95%
71%
70%
30%
33%
*Spike and positive control recovery data associated with the method demonstration are presented in Table 4-1.
Bolded values are those outside of the performance criteria ranges of 1 x 106 - 1 x 108 CPU/coupon based on spike control values or mean CPU >5% of spike levels
recovered from the positive control coupons.
-------
-------
4.0
Test Results
For this investigation, persistence data were generated for
B. suis in contact with up to five different materials exposed
to four environmental conditions for controlled exposure
durations. Persistence curves were also generated, where
applicable, by graphing the B. suis CFU/mL derived from
bacteria recovered from each material against time for each
set of environmental conditions. The following sections
summarize the results of the method demonstration (tests
conducted initially to ensure sufficient B. suis recovery
from each spiked material) and the persistence testing
investigation.
4.1 Method Demonstration - B. suis Recovery
As noted in Section 2.5, a brief method demonstration
was performed to ensure that previously used methods for
extracting biological agents from materials were applicable
for the combinations of B. suis and material. Results of the
method demonstration are presented in Table 4-1. The B. suis
recoveries, enumerated as CPU/coupon, attained the recovery
performance criterion (mean CPU/coupon >5% of the spiked
level) specified in the test/QAplan.(1) Recoveries of >5% of
the bacteria spiked onto the coupon (i.e., >5.0 x 104 CPU)
allow for a sufficient amount of initial bacteria to assess
persistence.
4.2 Persistence Results
Persistence results for each material/environmental condition
combination are summarized in Tables 4-2 through 4-6
and Figures 4-1 through 4-3. Where spike controls are
identical, the coupons were spiked with the same stock
suspension on the same day. An "ND" for mean recovered
CPU/coupon in the result tables indicates that no CPU were
detected in the undiluted extract sample plated for any of
five replicate coupons. A "0" was used in the calculations
of mean recovered CPU/coupon for any replicate coupon
having no CPU detected. A single viable bacterium present
in the plated extract would be expected to be observed as a
CPU. Also note, testing at the "low temperature, UV-A/B"
environmental condition was repeated for the durations
of 2 and 5 days because the low temperatures appeared to
adversely affect the performance of the UV meters.
4.2.1 Aluminum
The results obtained for B. suis persistence on aluminum are
summarized in Table 4-2 and Figure 4-1. In the absence of
UV-A/B, B. suis persisted for at least 28 days (the longest
duration tested) at the "moderate temperature, no UV" and
"low temperature, no UV environmental conditions; the
associated bacteria recoveries were higher at low temperature
(e.g., 3.48 x 105 CPU/coupon at 28 days) than at moderate
temperature (e.g., 5.19 x 102 CPU/coupon at 28 days).
The duration of B. suis persistence on aluminum decreased
in the presence of UV-A/B. At the "moderate temperature,
UV-A/B" environmental condition B. suis persisted less than
10 days, and B. suis did not persist or persisted at a relatively
low level, i.e., 1.32 x 101 CPU/coupon, following the five-
day duration at the "low temperature, UV-A/B" condition.
Table 4-1. Recovery data from method demonstration
Material
Concrete
Glass
Soil
Wood
B. suis Spike Control (CPU/coupon)
9.33 x 107
(all spiked the same day with the same stock suspension)
(CPU/coupon)*
7.98 ± 10.4 x 107
4.50 ± 0.55 x 107
4.29 ± 2.48 x 107
2.29 ± 1.77 x 107
Recovered B. suis
85.6 ± 112
48.2 ±5.87
46.0 ±26.6
24.5 ± 18.9
' Data are expressed as mean ± standard deviation of five replicate coupons.
-------
Table 4-2. B, suis persistence on aluminum
Duration (Days)*
Spike Contr
(CFU/coupo
1 IVIcdM rxcCOVci 6Q D, SUt
n) Positive Control*
fsiuru/couponr
Test Coupon§
Moderate Temperature, No UV
7
14
21
28
6.33 x 107
6.33 x 107
1.22 x 108
1.22 x 108
7.32 ±0. 67 x 107
7.32 ±0. 67 x 107
9.99 ±0. 78 x 107
9.99 ±0. 78 x 107
7.71 ±3.03x 104
2.29± 1.15x 104
1.38± 1.95x 104
5. 19 ± 4.09 x 102
Moderate Temperature, UV-A/B*
4
7
10
14
7
14
21
28
1
2
2
5
5
6.27 x 107
6.27 x 107
8.30 x 107
8.30 x 107
5.73x 107
5.73x 107
l.OOx 108
l.OOx 108
7.07 x 107
4.90 x 107
7.60 x 107
8.07 x 107
5.67 x 107
5. 76 ±0.41 x 107
5. 76 ±0.41 x 107
9.07 ± 1.69x 107
9.07 ± 1.69x 107
Low Temperature, No UV
3.53± 1.79x 107
3.53± 1.79x 107
9.42 ± 1.99x 107
9.42 ± 1.99x 107
Low Temperature, UV-A/B*
5.74+ i.44x 107
4.78 ±0. 53 x 107
5.71 ±0.64x 107
7.27 ± 1.22x 107
5.33 ± 1.75x 107
1.80 ± 2.06 x 102
2.27 ± 4.88 x 102
ND
ND
8. 75 ± 2.89 x 106
1.81 ±0.49x 106
9.82 ± 1.18x 106
3.48 ±0. 57 x 105
4.10 ± 5.60 x 103
2.20 ±3.31 x 102
8.68 ± 12.4 x 101
ND
1.32 ± 1.81 x 101
* Durations were determined in consultation with the EPA Task Order Project Officer and were based on the outcome of initial persistence test results; the order of
testing was not always conducted sequentially.
' Data are expressed as mean ± standard deviation of five replicates.
* Positive control coupons are spiked and extracted at time zero (i.e., immediately after spiking); one set of positive controls was used for test durations initiated at the
same time.
8 Test coupons are spiked and exposed to the environmental condition for the exposure duration.
* UV-A/B exposures were cyclical (12 hours on, 12 hours off) to simulate diurnal conditions.
"ND" indicates that no viable organisms were recovered from any of the replicate coupons. The detection limit for a given coupon with triplicate plating is
approximately 33 CPU/coupon (see Section 2.6).
-------
0 2 4 6 S 10 i: 14 16 IS 20 22 24 26 2S 30
Duration (Days)
^^—Moderate Temperature.Xo UV ^^^Moderate Temperature. UY-A B
A Lew Temperature. No UV ') ( Lcnv Temperature. UY-A B
Notes:
Only the highest persistence results are graphed for the replicate testing on days two and five for "Low Temperature, UV-A/B"
* No CPU were detected on days 10 and 14 for "Moderate Temperature, UV-A/B".
Figure 4-1. B. suis persistence on aluminum
4.2.2 Concrete
The results obtained for persistence of B. suis on concrete
are summarized in Table 4-3. On concrete, B. suis persisted
only at the "low temperature, no UV" environmental
condition, and, at that condition, B. suis was recovered
(mean of 5.32 x 101 CPU/coupon) only from the seven-day
exposure duration.
4.2.3 Glass
The results obtained for persistence of B. suis on glass are
summarized in Table 4-4 and Figure 4-2. In the absence of
UV-A/B, B. suis persisted for at least 28 days (the longest
duration tested) at the "moderate temperature, no UV" and
"low temperature, no UV" environmental conditions; the
associated bacteria recoveries were higher at low temperature
(e.g., 3.05 x 105 CPU/coupon at 28 days) than at moderate
temperature (e.g., 7.87 x 102 CPU/coupon at 28 days).
The duration of B. suis persistence on glass decreased in the
presence of UV-A/B. At the "moderate temperature, UV-
A/B" environmental condition B. suis persisted on glass less
than 2 days, and B. suis persisted less than 5 days at the "low
temperature, UV-A/B" environmental condition.
-------
Table 4-3. B, suis persistence on concrete
.
Duration (Days)
7
14
1
2
4
7
7
14
1
2
2
5
Spike Control mean Kecoverea °-
(CPU/coupon) Positive Control
Moderate Temperature, No UV
6.33 xlO7 9. 27 ±20. 5 x 106
6.33 xlO7 9. 27 ±20. 5 x 106
Moderate Temperature, UV-A/B*
8.30 xlO7 3.32 ± 7.04 x 106
8.30 xlO7 3.32 ± 7.04 x 106
6. 27 xlO7 1.91 ±2.64 x 105
6. 27 xlO7 1.91 ±2.64 x 105
Low Temperature, No UV
5. 73 xlO7 3. 54 ± 7.64 x 106
5. 73 xlO7 3. 54 ± 7.64 x 106
Low Temperature, UV-A/B*
7. 07 xlO7 1.81 ±3.49 x 103
4.90 xlO7 2. 07 ± 2.94 x 105
7. 60 xlO7 1.64 ±2.31 x 105
8. 07 xlO7 2. 73 ± 6.08 x 106
suis luru/coupon;1
Test Coupon5
ND
ND
ND
ND
ND
ND
5.32 ± 10. 2 x 101
ND
ND
ND
ND
ND
Durations were determined in consultation with the EPA Task Order Project Officer and were based on the outcome of initial persistence test results; the
order of testing was not always conducted sequentially.
Data are expressed as mean ± standard deviation of five replicates.
Positive control coupons were spiked and extracted at time zero (i.e., immediately after spiking); one set of positive controls was used for test durations
#
initiated at the same time.
Test coupons were spiked and exposed to the environmental condition for the exposure duration.
UV-A/B exposures were cyclical (12 hours on, 12 hours off) to simulate diurnal conditions.
"ND" indicates that no viable organisms were recovered from any of the replicate coupons. The detection limit for a given coupon with triplicate plating is
approximately 33 CPU/coupon (see Section 2.6).
-------
Table 4-4. B, suis persistence on glass
Duration (Days)*
Kffil ffffla
'coupon) Positive Control"'"
suis (CPU/coupon)1
Moderate Temperature, No UV
7
14
21
28
1
2
4
7
6.33 x 107
6.33 x 107
1.22x 108
1.22x 108
6.49 ± 0.98 x 107
6.49 ± 0.98 x 107
8.65 ± 0.49 x 107
8.65 ± 0.49 x 107
3.32 ± 2.37 x 104
7.36 ± 14.0 x 103
6.26 ± 2.99 x 102
7.87± 11.2 x 102
Moderate Temperature, UV-A/B*
8.30 x 107
8.30 x 107
6.27 x 107
6.27 x 107
1.03±0.20x 108
1.03±0.20x 108
6.30 ± 0.49 x 107
6.30 ± 0.49 x 107
2. 53 ± 3.04 x 102
ND
ND
ND
Low Temperature, No UV
7
14
21
28
5.73x 107
5.73x 107
l.OOx 108
l.OOx 108
5.18± 1.60x 107
5.18± 1.60x 107
9.86 ± 1.80 x 107
9.86 ± 1.80 x 107
2.23± 1.22 x 106
2.07±0.59x 105
7. 75 ±3. 57 x 105
3.05± 1.27 x 105
Low Temperature, UV-A/B*
1
2
2
5
7.07 x 107
4.90 x 107
7.60 x 107
8.07 x 107
5.57± l.OOx 107
6.13±0.79x 107
6.37 ±0. 70 x 107
8.33 ±0. 75 x 107
4.23 ±9. 16 x 103
4.21 ±9.22x 102
ND
ND
* Durations were determined in consultation with the EPA Task Order Project Officer and were based on the outcome of initial persistence test results; the
order of testing was not always conducted sequentially.
' Data are expressed as mean ± standard deviation of five replicates.
* Positive control coupons were spiked and extracted at time-zero (i.e., immediately after spiking); one set of positive controls was used for test durations
initiated at the same time.
8 Test coupons were spiked and exposed to the environmental condition for the exposure duration.
* UV-A/B exposures were cyclical (12 hours on, 12 hours off) to simulate diurnal conditions.
"ND" indicates that no viable organisms were recovered from any of the replicate coupons. The detection limit for a given coupon with triplicate plating is
approximately 33 CPU/coupon (see Section 2.6).
-------
10 12 14 16 IS 20 22 24 26 IS 30
Duration (Days)
•Moderate Temperature. Xo UV
v Temperature.Xo UV
HModerate Temperature. UV-A B
^Low Temperature. UV-A B
Notes: Only the highest persistence results are graphed for the replicate testing on day two for "Low Temperature, UV-A/B".
* No CPU were detected on days two, four, and seven for "Moderate Temperature, UV-A/B" or on day five for "Low Temperature,
UV-A/B".
Figure 4-2. B. suis persistence on glass
4.2.4 Soil
The results obtained for persistence of B. suis on soil are
summarized in Table 4-5 and Figure 4-3. On soil, B. suis
persisted under all environmental conditions for the longest
durations tested: 28 days at the "moderate temperature,
no UV" and "low temperature, no UV" environmental
conditions, 7 days at the "moderate temperature, UV-
A/B" environmental condition, and 14 days at the "low
temperature, UV-A/B" environmental condition.
-------
Table 4-5. B, suis persistence on soil
Duration (Days)*
Spike Control (CPU
s luru/cuupun;
icai Coupon
Moderate Temperature, No UV
7
14
21
28
6.33 x 107
6.33 x 107
1.22 x 108
1.22 x 108
6.09 ± 0.65 x 107
6.09 ± 0.65 x 107
7.74±0.53x 107
7.74±0.53x 107
2.66± 1.46x 104
9.29 ± 14.2 x 103
6.88 ±3. 78 x 102
1.13±0.90x 102
Moderate Temperature, UV-A/B*
4
7
10
14
6.27x 107
6.27x 107
8.30 x 107
8.30 x 107
5. 52 ± 0.99 x 107
5. 52 ± 0.99 x 107
8.24 ± 0.80 x 107
8.24 ± 0.80 x 107
5.03 ± 4.36 x 104
3.49 ± 1.14x 103
1.67 ± 1.08 x 103
4.33 ± 1.05x 102
Low Temperature, No UV
7
14
21
28
5.73x 107
5.73x 107
l.OOx 108
l.OOx 108
3.02 ± 2.03 x 107
3.02 ± 2.03 x 107
6.93 ± 1.63x 107
6.93 ± 1.63x 107
9.67 ± 1.28 x 104
5.41 ± 1.72 x 104
9.35 ± 2.09 x 104
4.14 ± 1.29 x 104
Low Temperature, UV-A/B*
1
2
2
5
5
14
7.07x 107
4.90 x 107
7.60x 107
8.07x 107
5.67x 107
6.13 x 107
4.44 ± 1.53x 107
4.57 ± 0.35 x 107
5.98 ±0. 59 x 107
7.67 ± 0.69 x 107
4.01 ±0.48x 107
4.31 ±0.42x 107
4.24 ± 1.28 x 106
4.05 ±2. 12 x 106
4.57 ± 1.28 x 106
1.01 ±0.46x 106
7.69± 1.01 x 105
7.11 ± 1.05x 103
* Durations were determined in consultation with the EPA Task Order Project Officer and were based on the outcome of initial persistence test results; the
order of testing was not always conducted sequentially.
' Data are expressed as mean ± standard deviation of five replicates.
* Positive control coupons were spiked and extracted at time-zero (i.e., immediately after spiking); one set of positive controls was used for test durations
initiated at the same time.
8 Test coupons were spiked and exposed to the environmental condition for the exposure duration.
* UV-A/B exposures were cyclical (12 hours on, 12 hours off) to simulate diurnal conditions.
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0 2 4 6 S 10 i: 14 16 IS 20 22 24 26 2S 30
Duration (Days}
^^—Moderate Temperature.Xo UV ^^^Moderate Temperature. UV-A B
A Lo^v Temperature. No UV ) ( Low Temperature. UV-A B
Note:
Only the highest persistence results are graphed for the replicate testing on days two and five for "Low Temperature, UV-A/B"
Figure 4-3. B, suis persistence on soil
4.2.5 Wood
The results obtained for persistence of B. suis on wood are
summarized in Table 4-6. Persistence testing of B. suis on
wood was conducted only occasionally. Wood coupons
were used in place of the concrete coupons in the longer
persistence tests because of the lack of persistence of B. suis
on concrete at the shorter time points. At the "moderate
temperature, no UV" environmental condition, B. suis was
not recovered from wood after a 21-day exposure duration
(the shortest duration tested), but B. suis was recovered
after 28 days of exposure to the "low temperature, no UV"
environmental condition.
Table 4-6. B. suis persistence on wood
Duration (Days)'
21
28
Spike Control (CPU/coupon)
Moderate Temperature,
1.22 x 108
1.22 x 108
Mean Recovered B.
NoUV
3.60 ± 2.70 x 107
3.60 ±2. 70 x 107
suis (CPU/coupon^
ND
ND
Low Temperature, No UV
21
28
l.OOx 108
l.OOx 108
3.34 ± 2.47 x 107
3.34 ± 2.47 x 107
1.73± 1.43 x 105
1.57 ± 1.22 x 105
* Durations were determined in consultation with the EPA Task Order Project Officer and were based on the outcome of initial persistence test results; the
order of testing was not always conducted sequentially.
' Data are expressed as mean ± standard deviation of five replicates.
* Positive control coupons were spiked and extracted at time-zero (i.e., immediately after spiking); one set of positive controls was used for test durations
initiated at the same time.
8 Test coupons were spiked and exposed to the environmental condition for the exposure duration.
"ND" indicates that no viable organisms were recovered from any of the replicate coupons. The detection limit for a given coupon with triplicate plating is
approximately 33 CPU/coupon (see Section 2.6).
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5.0
Summary
The B. suis persistence results are summarized in Table 5-1.
These data denote the longest exposure duration (days) that
B. suis was recovered (persisted) and the shortest exposure
duration that B. suis was not recovered to bracket the length
of time that B. suis remained viable for each material and
environmental condition. The shortest duration without
B. suis recovery provides, where possible, an upper bound
on the persistence of B. suis per materials and environmental
conditions tested. In general, greater B. suis persistence
occurred at lower temperatures (especially without exposure
to UV), and B. suis persisted longer without exposure to
simulated sunlight (although UV had less of an effect on the
persistence of B. suis on soil).
On aluminum, glass, and soil, B. suis persisted for at least
28 days (the longest duration tested) under the "moderate
temperature, no UV" and the "low temperature, no UV
environmental conditions. On concrete, B. suis did not persist
following a 7-day exposure duration (the shortest duration
tested) at the "moderate temperature, no UV environmental
condition, but B. suis on concrete did persist 7 days when
exposed to the "low temperature, no U V environmental
condition. On wood, B. suis did not persist following a 21-
day exposure duration (the shortest duration tested) at the
"moderate temperature, no UV environmental condition, but
B. suis did persist on wood for 28 days when exposed to the
"low temperature, no UV environmental condition.
The incorporation of UV-A/B into the environmental
conditions shortened the duration that B. suis persisted on
aluminum and glass, but had less of an effect on soil. At the
"moderate temperature, UV-A/B" environmental condition,
the longest exposure durations associated with recovered
B. suis were 7 days for aluminum and one day for glass. At
the "low temperature, UV-A/B" environmental condition, the
longest durations from which B. suis was recovered were 2
days on glass and 5 days on aluminum.
When exposed to UV-A/B, B. suis persisted on soil for
14 days (the longest duration tested) at the "moderate
temperature, UV-A/B" environmental condition, and
B. suis persisted on soil for 14 days (the longest duration
tested) at the "low temperature, UV-A/B" environmental
condition. On concrete, B. suis was not recovered at any of
the environmental conditions that incorporated UV-A/B.
Persistence testing with B. suis on wood was not conducted
in the presence of UV-A/B.
-------
Table 5-1. Summary of B, suis persistence
iviaienai ana Lnvironmeniai
Condition
Aluminum
Moderate temperature, No UV
Moderate temperature, UV-A/B
Low temperature, No UV
Low temperature, UV-A/B
Concrete
Moderate temperature, No UV
Moderate temperature, UV-A/B
Low temperature, No UV
Low temperature, UV-A/B
Glass
Moderate temperature, No UV
Moderate temperature, UV-A/B
Low temperature, No UV
Low temperature, UV-A/B
Soil
Moderate temperature, No UV
Moderate temperature, UV-A/B
Low temperature, No UV
Low temperature, UV-A/B
Wood
Moderate temperature, No UV
Moderate temperature, UV-A/B
Low temperature, No UV
Low temperature, UV-A/B
Longesi uurauon luays;
with B. suis Recovery
28
7
28
5*
ND
ND
7
ND
28
1
28
2t
28
14
28
14
ND
Not tested
28
Not tested
5SBB
without B. suis Recovery
AD
10
AD
5*
7
1
14
1
AD
2
AD
2t
AD
AD
AD
AD
21
Not tested
AD
Not tested
AD = B. su/swas detected at all durations tested.
ND = B. su/swas not detected at the shortest duration tested.
* B. su/swas recovered during the 5-day test conducted 9/24/09 - 9/29/09 but not during the 5-day test conducted 8/23/09 - 8/28/09.
• B. su/swas recovered during the 2-day test conducted 9/2/09 - 9/4/09 but not during the 2-day test conducted 9/18/09 - 9/20/09.
-------
6.0
References
1. Technology Testing and Evaluation Program Quality Assurance/Test Plan for Persistence Testing of
Brucella suis on Outdoor Materials, Version 1, Battelle, Columbus, Ohio, October 2008.
2. Sarinas, P.S.A. and R.K. Chitkara, Brucellosis. Semin. Respir. Infect, 2003(18): p. 168-182.
3. Battelle, MREF Standard Operating Procedure for the Operation and Maintenance of Primus
General Purpose Steam Sterilizer Model: PSS5-A-MSSD, 2006.
4. Battelle, MREF Facility Safety Plan Annex 12 to Appendix B, Guidelines for the Use of Class II and
Class III Biological Safety Cabinets in the MREF Biofacility, July 2006.
5. Battelle, FSP Annex 5 to Appendix B, Guidelines for Safe Handling and Storage of Etiologic Agents
at the MREF, July 2006.
6. Battelle, FSP Annex 7 to Appendix B, Guidelines for Disinfection/Decontamination of Etiological
Agents at the MREF Biofacilities, July 2006.
7. Weinbauer, M.G., et al., Photoreactivation Compensates for UV Damage and Restores Infectivity to
Natural Marine Virus Communities. Appl. Environ. Microbiol., 1997(63): p. 2200-2205.
8. Balasaraswathy, P., et al., UVA and UVB in Sunlight, Optimal Utilization of UV Rays in Sunlight for
Phototherapy. Indian J. Dermatol. Venereol. Leprol., 2002(68): p. 198-201.
9. Jeanmougin, M. and J. Civatte, Dosimetry of Solar Ultraviolet Radiation. Daily and Monthly
Changes in Paris. [Article inFrench] Ann. Dermatol. Venereol., 1987(114): p. 671-676.
10. Kolari, P.J., et al., Midsummer Solar UV-Radiation in Finland Compared with the UV-Radiation from
Phototherapeutic Devices Measured by Different Technique. Photodermatol., 1986(3): p. 340-345.
11. McNamara, A.E. and W.R. Hill, UV-B Irradiance Gradient Affects Photosynthesis and Pigments but
Not Food Quality of Periphyton. Freshwater Biology, 2000(43): p. 649-662.
12. Diffey, B.L., Sources and Measurement of Ultraviolet Radiation. Methods, 2002(28): p. 4-13
13. Qui, X., et al., Survival ofShewanella oneidensis MR-1 after UV Radiation Exposure. Appl. Environ.
Microbiol., 2004(70): p. 6435-6443.
14. Technology Testing and Evaluation Program Test/QA Plan for Systematic Investigation ofFumigant
Technologies for Decontamination of Biological Agents from Contaminated Building Materials,
Version 1, Battelle, Columbus, Ohio, May 2007.
15. Quality Management Plan (QMP) for the Technology Testing and Evaluation Program (TTEP),
Version 3, Battelle, Columbus, Ohio, January 2008.
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