&EPA
United States
Environmental Protection
Agency
EPA/600/R-15/146 I April 2016
www.epa.gov/homeland-security-research
Water Security Test Bed Experiments
at the Idaho National Laboratory
Office of Research and Development
Homeland Security Research Program
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April 2016
EP A/600/R-15/146
WATER SECURITY TEST BED EXPERIMENTS AT THE
IDAHO NATIONAL LABORATORY
EPA Contract No. EP-C-14-012
Work Assignment No. 0-08
CB&I DN: 500204-QA-RP-000103
Prepared for:
U.S. Environmental Protection Agency
National Homeland Security Research Center
26 West Martin Luther King Drive
Cincinnati, Ohio 45268
Prepared by:
CB&I Federal Services LLC
5050 Section Avenue
Cincinnati, Ohio 45212
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Disclaimer
The U.S. Environmental Protection Agency through its Office of Research and Development
funded the research described here under Interagency Agreement (IA) DW-89-92381801 with
the Department of Energy and under contract EP-C-14-012 with CB&I Federal Services LLC. It
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, products, or services does not convey official EPA approval, endorsement, or
recommendation.
Questions concerning this document or its application should be addressed to:
Jeff Szabo, Ph.D., P.E.
National Homeland Security Research Center
Office of Research and Development
U.S. Environmental Protection Agency
26 W. Martin Luther King Drive
Cincinnati, OH 45268
szabo.ieff@epa.gov
John Hall
National Homeland Security Research Center
Office of Research and Development
U.S. Environmental Protection Agency
26 W. Martin Luther King Drive
Cincinnati, OH 45268
hall. i ohn@epa. gov
li
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Acknowledgements
Contributions of the following organizations to the development of this document
acknowledged:
CB&I
Idaho National Laboratory
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Table of Contents
Disclaimer ii
Acknowledgements iii
Abbreviations vi
Executive Summary vii
1.0 Introduction 1
1.1 WSTB Description 1
2.0 Description of Experiments 8
2.1 Dye Test 8
2.2 Dechlorination and Triggered Flushing 8
2.3 Contamination/Decontamination Tests 13
2.3.1 Injection of Contaminant (addition of B. globigii spores to WSTB) 16
2.3.2 Decontamination (addition of chlorine dioxide for a specified contact time) .17
2.3.3 Post-decontamination flushing and monitoring 18
2.4 The Waterstep Mobile Water System 19
2.4.1 WaterStep M-100 Water Treatment Experiments 19
3.0 Analysis of Decontamination and Water Treatment Results 22
3.1 Contamination and Decontamination Experiment 22
3.2 Water Treatment Experiment 28
4.0 Conclusions and Future Work 30
5.0 References 32
Appendix A - Detailed Experimental Protocols and Quality Assurance Criteria 33
Appendix B: T&E SOP 304, Heterotrophic Plate Count (HPC) Analysis Using IDEXX
SimPlate® Method 46
Appendix C: T&E SOP 309, Preparation and Enumeration of Bacillus globigii Endospores 47
Appendix D: Operation of the Water Sample Concentrator 48
List of Figures
Figure 1. Schematic overview of the water security test bed 2
Figure 2. Aerial View of the water security test bed (WSTB) 2
Figure 3. Injection setup for water security test bed 3
Figure 5. Pipe coupon 4
Figure 6. Water security test bed as-built (view from upstream-end) 5
Figure 7. Flushing hydrant (view from downstream-end) 5
iv
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Figure 8. Instrument panel water quality analyzers (inside view) 6
Figure 9. Water security test bed system flow regulator 6
Figure 10. Water security test bed discharge lagoon 7
Figure 11. Injected dye mix (left) and diluted dye exiting the water security test bed system flow
regulator (right) 8
Figure 12. Online monitoring of free chlorine (denoted F-Ch in mg/L) during testing 9
Figure 13. Flushing hydrant in operation 10
Figure 14. Downstream free chlorine free chlorine (denoted F-Ch in mg/L) response and
flushing hydrant operation 11
Figure 15. Free chlorine (denoted F-Ch in mg/L) response and UV-determined total organic
carbon (TOC) to change in hydraulic condition (downstream) 12
Figure 16. Free chlorine (denoted F-Ch in mg/L) response to change in hydraulic condition
(upstream) 13
Figure 17. WaterStep M-100 chlorinator 20
Figure 18. WaterStep setup at the water security test bed lagoon 20
Figure 19. Bulk water 5. globigii (BG) and chlorine dioxide results over time after spore
injection (hr) 23
Figure 20. Coupon B. globigii (CFU/in2) and bulk water chlorine dioxide (mg/L) results over
time after spore injection (hr). Note: to determine the microbial density on the coupons in
CFU/cm2, divide by 6.45 25
Figure 21. Extended timeline bulk water B. globigii (BG) and chlorine dioxide (mg/L) results
over time after spore injection (hr) 26
List of Tables
Table 1. Background Sampling Activity 14
Table 2. Background Heterotrophic Plate Count (HPC) Data 14
Table 3. Contamination Sampling Activity 16
Table 4. Decontamination Sampling Activity 17
Table 5. Flushing Sampling Activity 18
Table 6. Lagoon Sampling Activity 21
Table 7. Bulk Water and Water Sample Concentrator Sampling Results 22
Table 8. Coupon Sampling Results 24
Table 9. Lagoon Sampling Results 28
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Abbreviations
AC Alternating Current
AWBERC Andrew W. Breidenbach Environmental Research Center
BTEX benzene, toluene, ethylbenzene and xylene
BWS Bulk Water Sample
CB&I CB&I Federal Services LLC
CFU/in2 Colony Forming Units per square inch
CFU/mL Colony Forming Units per milliliter
CP Coupon
DC Direct Current
EPA U.S. Environmental Protection Agency
gpm gallons per minute
HPC Heterotrophic Plate Count
INL Idaho National Laboratory
IP Instrument Panel
MPN/mL Most Probable Number per milliliter
MWS Mobile Water System
NHSRC National Homeland Security Research Center
PVC Polyvinyl Chloride
T&E Test and Evaluation
TOC Total Organic Carbon
UV Ultraviolet
WSC Water Sample Concentrator
WSTB Water Security Test Bed
VI
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Executive Summary
The EPA's National Homeland Security Research Center partnered with the Idaho National
Laboratory (INL) to build the water security test bed (WSTB) at the INL facility in Idaho Falls,
Idaho. The WSTB was built using an 8 inch (20 cm) diameter cement-lined drinking water pipe
that had been previously taken out of service. The pipe was exhumed from the INL grounds and
oriented in the shape of a 448 ft (137 m) long drinking water system (see Section 1.1 for a detailed
description). Effluent from the pipe is captured in a lagoon. The WSTB can support drinking water
distribution system research related to drinking water treatment including biofilms, water quality,
sensors and homeland security related contaminants. Because the WSTB is constructed of real
drinking water distribution system pipes, research can be conducted under conditions similar to
those in a real drinking water system.
After the construction of the WSTB was completed in late September 2014, the following
experiments were performed using the WSTB:
1. A dye test (tracer) to evaluate travel times and system flows
2. Dechlorination of the water in the WSTB and triggering of an automated hydrant-based
flushing device
3. A contamination (using Bacillus globigii) and decontamination (using chlorine dioxide)
test
4. Evaluation of the WaterStep mobile water system (MWS) for its ability to disinfect spores
in the lagoon water that was flushed from the WSTB pipe
The following is a summary of conclusions based on the testing performed at the INL WSTB:
• The dye-testing confirmed the theoretical flow velocity and travel time of water flowing
down the pipe. There was some visible mixing and dispersion/diffusion of the dye slug
observed during testing.
• Sodium thiosulfate (a surrogate contaminate) removed free chlorine from the water and
successfully triggered the hydrant-based flushing device. Some of the dispersed sodium
thiosulfate was lodged in dead end pipes and was released with subsequent hydraulic
changes.
• The test results indicated a 5 to 8 logio reduction of the contaminant (B. globigii) in bulk
water upon decontamination with an initial chlorine dioxide at 110 mg/L for 24 hours (the
chlorine dioxide decayed to 35 mg/1 over 7 hours). The testing results also indicate a less
effective removal (~2 log reduction based on highest observed B. globigii density before
decontamination) of the contaminant from the pipe surface. These data are different than
decontamination results generated in a pilot-scale pipe system developed by EPA, where
chlorine dioxide was much more effective.
• The WaterStep MWS was ineffective at treating water in the lagoon contaminated with B.
globigii spores. However, the treatment unit was operated in manner different than its
original design, which may have affected its performance.
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1.0 Introduction
The EPA's National Homeland Security Research Center (NHSRC) partnered with the Idaho
National Laboratory (INL) to build the water security test bed (WSTB) at the INL facility 50 miles
west of Idaho Falls, Idaho. The WSTB was built using an 8 inch (20 cm) diameter cement-lined
drinking water pipe that was previously taken out of service. The pipe was exhumed from the INL
grounds and oriented in the shape of a small drinking water distribution system (see Section 1.1
for a detailed description). Effluent from the pipe is captured in a lagoon. The WSTB can support
drinking water distribution system research on a variety of topics including biofilms, water quality,
sensors and homeland security related contaminants. Because the WSTB is made of previously
used drinking water distribution system pipes, research can be conducted under conditions that
simulate those in a real drinking water system. This is important to note since the
contamination/decontamination experiments described in this report had been previously
conducted on the bench and pilot scale, and were being conducted again to verify their results in a
real-world setting.
EPA/NHSRC led the experiments described in this study with technical support from CB&I
Federal Services LLC (CB&I) under contract. Testing and analyses described in this report were
conducted by CB&I. Under direction from EPA, CB&I personnel conducted a dechlorination-
triggered flushing test, a contamination/decontamination experiment and assessed the
effectiveness of a mobile water treatment device using the WSTB at INL in October 2014. Bacillus
globigii spores, which are a surrogate for pathogenic B. anthracis, were used as the contaminant
in the decontamination and treatment experiments. This report summarizes the data and results
obtained from this testing.
1.1 WS TB Description
The WSTB consists primarily of an 8 inch (20 cm) diameter drinking water pipe oriented in the
shape of a small drinking water distribution system. The WSTB contains ports for simulating
water demands from service connections and a 15 ft (5 m) removable coupon (extracted samples)
section designed to sample the pipe interior. Figure 1 schematically depicts the main features of
the WSTB.
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Legend
FM Flow Meter
PG Pressure Gauge
IP1 Instrument Panel 1 (Upstream, Cellular)
IP2 Instrument Panel 2 (Downstream, Radio)
[X] Valve, Open
M Valve, Closed
O* Valve, Partly Open
£ Fire Hydrant
| Flushing Hydrant
—3 Blind Flange
Pressure Reducing Valve
f\J Check Valve/Backflow Preventer
—»- Service Connector (Closed)
Existing Fire Hydrant
Fire Hose
Start of WSTB |-
Injection Port -CXI—
Not to Scale
0-exf-
15-tt Coupon Section
—X—j-—X—j-—X-
Bulk Water Sample Tap
-X—
Drinking Water
from
INL Pumphouse
Parking
Area
Drainage
Ditch
Figure L Schematic overview of the water security test bed.
Figure 2 shows the aerial view of the WSTB. The lower right corner shows the upstream and
system inlet; the upper left corner shows the lagoon.
Lagoon
WSTB End
WSTB Start
Figure 2. Aerial View of the water security test bed (WSTB).
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As depicted in Figure 1, drinking water was supplied to the WSTB through an existing fire hydrant.
Drinking water was chlorinated ground water that also supplied the surrounding INL facilities.
The WSTB incorporates approximately 448 ft (137 m) of 8 inch (20 cm) diameter cement-lined
pipe. The 8 inch (20 cm) pipe system is constructed directly over the lined drainage ditch for spill/
leak containment. The total volume of the WSTB is estimated to be -1,150 gallons (4,353 L). A
positive displacement pump was used to inject the target contaminant at the beginning (-10 ft (3
m) from the pipe start) of the 448 ft (137 m) WSTB system. Figure 3 shows the injection setup.
Figure 3. Injection setup for water security test bed.
A 15 ft (5 m) polyvinyl chloride (PVC) pipe-segment unique to this pipe system was designed and
fabricated to contain 10 sets of duplicate removable coupons (totaling 20 coupons) made from the
cement-lined pipe used to construct the rest of the WSTB. The coupons allow measurement of
biofilm growth, contaminant persistence on pipe material, and the effectiveness of
decontamination. Placement of the coupons on the top section of the pipe allowed for coupon
removal without entirely draining the pipe. Figure 4 shows a portion of the coupon section.
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Coupons
Figure 4. Removable PVC coupon section.
The pipe material for the 20 small coupons (22/32 of an inch (1.8 cm) in diameter and 0.371 square
inches (2.4 square centimeters) in area) were cut from the cement mortar-lined iron pipe obtained
from INL and set into threaded plugs that were inserted into the PVC-coupon section of the pipe.
Figure 5 shows a picture of the threaded coupon that was inserted into the pipe main. The twenty
coupons were individually numbered CP-0/CP-0D through CP-9/CP-9D in duplicate.
Figure 5. Pipe coupon.
Figure 6 shows a picture of the WSTB as-built (view from the upstream-end of the WSTB), with
the upstream red fire hydrant and the instrument panel (IP 1) in the view.
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Instrument Panel 1 (Upstream)
Upstream Hydrant
Flow
Direction
Figure 6. Water security test bed as-built (view from upstream-end).
The second yellow fire hydrant (downstream location) was installed and paired with the Hydro-
Guard®1 HG-6 hydrant-based automatic flushing system (the red box). The downstream hydrant
setup and the associated instrument panel (IP2) are shown in Figure 7.
Instrument Panel 2 (Downstream)
Hydro-Guard
Flushing Unit
Downstream
Hydrant
Figure 7. Flushing hydrant (view from downstream-end).
The WSTB instrumentation panels (IP1 and IP2) were equipped with sensors that continuously
measure two basic water quality parameters: free chlorine and total organic carbon (TOC). One
Hach® CL-17 chlorine analyzer (Hach Co., Loveland, CO) and one RealTech M4000 TOC
analyzer (Real Tech Inc., Whitby, ON, Canada) were included in each of the instrumentation
panels. The Hach CL-17 chlorine analyzer uses colorimetric DPD chemistry to monitor water
continuously for free chlorine. The RealTech M4000 uses the ultraviolet (UV) 254 nanometer
1 Mueller Co., 633 Chestnut Street, Suite 1200, Chattanooga, TN, USA
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wavelength (i.e., UV254) for determining the TOC content. UV254 instruments are often used as
an inexpensive indicator of TOC in water. UV254 measurements are known to have some bias
towards aromatic organics; however, they are relatively inexpensive to maintain and to operate
when compared to the traditional UV-persulfate based TOC analyzers. Figure 8 depicts the inside
of one of the instrument panels.
Figure 8. Instrument panel water quality analyzers (inside view).
When active experimentation was not taking place, the WSTB system was operated at a low flow
rate of around 2.5 gallons per minute (gpm) (10 L/min), resulting in a total of 25,200 gallons
(95,382 L) discharged per week into the lagoon. The valve near the end of WSTB along with the
flow meter (shown in Figure 9) was used to regulate and maintain flow.
Figure 9. Water security test bed system flow regulator.
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During experiments, the system was operated at a higher flow rate (-15 gpm or 57 L/min)) to
reduce travel time and manage sampling activities. The flow had to be regulated because of the
limited discharge capacity of the lagoon. The lagoon (Figure 10) has a water storage capacity of
28,000 gallons (105,980 L). The water and any contaminant or decontaminating agent used during
experimentation were conveyed via the drainage ditch and discharged to the lagoon. The
discharged water was trucked out for disposal on an as-needed basis.
Figure 10. Water security test bed discharge lagoon.
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2.0 Description of Experiments
2.1 Dye Test
Prior to contamination/decontamination tests, a simple dye tracer study (using non-toxic
biodegradable dye from Bright Dyes - www.brightdves.com) was performed to visually confirm
the theoretical calculations of tracer travel times and system flows. This dye testing was performed
on October 1, 2014 at 9:00 AM. A 5-minute injection of red dye was performed. The dye mixture
was prepared by dissolving 3 tablets of the red-colored Bright Dye in 2 gallons (8 L) of water using
the injection setup (previously shown in Figure 3). The dye mixture injection rate was 1,500
mL/min. At 10:12 AM, the dye was visibly noticed at the outlet flow control rotameter. The
theoretical calculated travel time (based on plug flow travel, total pipe length of 429 ft (131m)
from the injection port to end, and a flow rate of 15 gpm (57 L/min)) is 1 hour and 15 minutes.
The exit flow rotameter is located slightly upstream of the end-of-pipe and the actual travel time
was 1 hour and 12 minutes. The testing confirmed the theoretical flow velocity and travel time
calculations. As expected, there was some visible mixing, dispersion and diffusion of the dye slug
observed during testing because the dye color was visible for a period of 10 minutes in the flow
meter, whereas the injection only lasted 5 minutes. Figure 11 shows the injected dye mix (left) and
diluted dye exiting the WSTB system flow regulator (right).
Figure 11. Injected dye mix (left) and diluted dye exiting the water security test bed system
flow regulator (right).
2.2 Dechlorination and Triggered Flushing
The purpose of the dechlorination and flushing experiments was to demonstrate the feasibility of
using online water quality sensors in concert with an automated flushing hydrant to intelligently
divert and remove contaminants from water distribution systems. Sodium thiosulfate was used to
dechlorinate the drinking water in the WSTB pipe, and the resulting loss of chlorine level was used
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to trip the automated flushing device. Ten grams of thiosulfate was mixed with 2 gallons (8 L) of
water for a 5-minute injection (similar to the dye tracer study). The mixture was injected into the
WSTB on October 1, 2014, at 12:48 PM for 5 minutes. Figure 12 shows the online chlorine data
(mg/L) over time from the upstream and downstream instrument locations.
INL Sodium Thiosulfate Injection October 1, 2014
1.2
1
0.8
0.4
0.2
0
fNl
T—I
fNl
rsi
^ Time
m
ro
ro
H
Upstream F-CI2 mg/L Downstream F-CI2 mg/L ^—Thiosulfate Injection Start Thiosulfate Injection Stop
Figure 12. Online monitoring of free chlorine (denoted F-Ck in mg/L) during testing.
Based on the data, the slug appears to reach the downstream instrument panel in 1 hour and 2
minutes. The shape of the response on the downstream chlorine monitor shows that the 5-minute
injected slug dispersed to a 12-minute slug by the time it reached the downstream location. From
the chlorine level signals, it is clear that they can be used to trigger a flushing device.
Figure 13 shows the flushing hydrant in operation during the testing. The signal from the
downstream Hach CL-17 chlorine monitor was used to trigger the flushing hydrant to open and
flush. When the chlorine level measured by the Cl-17 dropped below 0.05 mg/L, the flushing
hydrant valve triggered open (Figure 14). After the chlorine value recovers (i.e., > 0.05 mg/L), the
flushing valve was set to automatically trigger to a closed position.
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Figure 13. Flushing hydrant in operation.
The flushing hydrant tripped open at 13:50 and stayed open till 14:05. The grab sample collected
at 13:50 from the downstream system flow regulator confirmed that the chlorine level had dropped
below the trigger point of 0.05 mg/L. Figure 14 shows the downstream chlorine levels in
conjunction with the automated flushing operation. The 15-minute flushing hydrant operation
period corresponds to the 12-minute spread of the slug (based on the dye-tracer study) and a 2.5
to 3.5 minute delay between sensor measurement and the triggered response. The data plot
indicates that the automated flushing hydrant operated as designed.
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1.2
1
0.8
3 0.6
0.4
0.2
0
m
*—I
m
m
I
Time
^—Downstream F-CI2 mg/L ¦^—Flusher Opens Flusher Closes
Figure 14. Downstream free chlorine free chlorine (denoted F-Ck in mg/L) response and
flushing hydrant operation.
Figure 15 shows the real time instrumentation from both TOC (as measured by UV absorbance)
and chlorine instruments at the downstream location. The data show the first dip at 13:50 that
caused the flusher to open and a second dip in chlorine level at 14:42. The chlorine level dips are
also matched by a peak in RealTech UV TOC response at the downstream location. The peak in
TOC is likely due to absorbance exhibited by thiosulfate in the UV 254 wavelength. At 14:40, the
system flow was stopped (near the downstream location) for several minutes to collect a coupon
sample. This change in hydraulic condition appears to have caused a release of some residual
thiosulfate that remained in the system (possibly at pipe dead ends) and was not flushed out during
the automated flushing operations.
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^—Downstream F-CI2 mg/L Flow Change (15 to 2.5 gpm) ^—Flusher Opens ^—Flusher Closes uvTOC
Figure 15. Free chlorine (denoted F-Ch in mg/L) response and UV-determined total
organic carbon (TOC) to change in hydraulic condition (downstream).
A dip in chlorine similar to the downstream sensor was noted in the upstream location (Figure 16)
when the flusher was opened and closed. The change in hydraulic condition appears to have caused
a release of some residual thiosulfate that remained in the system (possibly at dead ends).
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CM O 00 rs VD
rH O M O M
m T. uS uS
tH *h *h lime *H «—I *H
Upstream F-CI2 mg/L ^^—Flusher Opens ^^—Flusher Closes
Figure 16. Free chlorine (denoted F-Ck in mg/L) response to change in hydraulic condition
(upstream).
These data appear to indicate that while automated flushing operations remove the bulk of the
injected test contaminants, some of the dispersed contaminants lodged in the dead-ends are
released later on when the system hydraulic conditions change. Therefore, any automated flushing
operations should take into account any dead-end zones trapping the dispersed/diffused
contaminants that may be re-suspended due to hydraulic changes in the system. Also a stop and
restart protocol during the flushing operation warrants further consideration and testing.
2.3 Contamination/Decontamination Tests
These experiments involved contamination of the WSTB using B. globigii spores and the
subsequent decontamination using chlorine dioxide as the decontaminating agent. The
contamination/decontamination experiment consisted of the following main steps:
1. Injection of contaminant (addition of B. globigii spores to WSTB)
2. Decontamination (addition of chlorine dioxide for a specified contact time)
3. Post-decontamination flushing and monitoring
On October 6, 2014, at 8:15 AM prior to contamination/decontamination testing, the online
instrumentation data was reviewed for the presence of stable free chlorine concentration recorded
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as 0.69 mg/L at the upstream instrument panel location and as 0.5 mg/L at the downstream
instrument panel location of the WSTB. The stable value served as an indication of stabilization
of pipe wall chlorine demand. Biofilm formation was confirmed by collecting baseline bulk water
sample (BWS-0) and duplicate coupon (CP-0/CP-0D) samples. The water and coupon samples
were analyzed in the laboratory for the presence of heterotrophic plate count (HPC). In addition,
BWS-B was collected during the sampling event as a quality assurance check. The BWS-B sample
consisted of a sterile buffer bottle that was opened and placed in the vicinity of the coupon and
bulk water sampling area. Any contamination that was in the air or aerosolized by bulk water or
coupon sampling would be collected by BWS-B.
The coupon sampling event was performed by isolating the coupon section of the main pipe by
closing the upstream flanking gate valve (previously shown in Figure 1), removing the duplicate
coupon plugs (CP-0 and CP-OD) and replacing the coupon plugs with blank plugs. The detailed
sampling procedures, sample containers, sample preservation, sample labeling, sample shipping
and analytical methods are described in Appendix A. The specific sampling activities are described
in Table 1.
Table 1. Background Sampling Activity
Sample ID
Sample Description
Date/Time and System
Flow
BWS-0
(Control)
Bulk water sample collected from the pipe
prior to injection of B. globigii
October 6, 2014-8:50 AM,
Flow at 2.5 gpm (10 L/min)
BWS-B
(Background)
Background control collected at the same
time as BWS-0
October 6, 2014-8:50 AM,
Flow at 2.5 gpm (10 L/min)
CP-0 and
CP-OD
Coupon sample collected at the same time
as BWS-0
October 6, 2014-8:50 AM,
Flow at 2.5 gpm (10 L/min)
CP, coupon; BWS, bulk water sample; gpm, gallons per minute; L/min, liters per minute; 0, sample taken before
injection; B, background sample (an open bottle of sterile water sitting beside the pipe to detect any aerosolized BG);
D, duplicate
The resulting HPC values from the background and baseline samples collected are reported as
most probable number per milliliter (MPN/mL) in Table 2. The same background samples were
also analyzed for the absence of B. globigii spores which are reported separately along with other
experimental data. The CP-0 and CP-OD values indicate that a biofilm was established on the pipe
section prior to the contaminant injection.
Table 2. Background Heterotrophic Plate Count (HPC) Data
Sample ID
HPC Counts
BWS-0
299 MPN/mL
BWS-B
0 MPN/mL
CP-0
6.87E+03 MPN/in2
CP-OD
5.12E+03 MPN/in2
BWS, bulk water sample; CP, coupon; MPN, most probable number; 0, sample taken before injection; B, background
sample (an open bottle of sterile water sitting beside the pipe to detect any aerosolized BG); D, duplicate; Note: to
determine the microbial density on the coupons in MPN/cm2, divide by 6.45.
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2.3.1 Injection of Contaminant (addition ofB. giobigii spores to WSTB)
Offsite Preparation of the Contaminant Stock - The B. giobigii spores for this study were obtained
from NHSRC's laboratory at the Andrew W. Breidenbach Environmental Research Center
(AWBERC) in Cincinnati, Ohio. A culture of B. giobigii vegetative cells were mixed with generic
sporulation media and incubated by gentle shaking at 35C for 7 days at the EPA Test and
Evaluation (T&E) Facility in Cincinnati, OH. The concentration of B. giobigii stock was
determined following the method described by Rice et al. (1994). The B. giobigii stock was heat-
shocked to remove any remaining vegetative cells and analyzed using the spread plate method and
membrane filtration as described in Appendix A. Thirty-eight liters of the spore stock was prepared
over several weeks and shipped in individual one-liter containers (preserved at 4±2°C) to the site.
Onsite Preparation and Injection of the Contaminant Stock - On October 6, 2014, the above
referenced B. giobigii stock suspensions were mixed in three five gallon (19 L) buckets and
prepared for injection. At 10:00 AM, the mix was introduced into the WSTB using a positive
displacement pump (injection setup shown previously in Figure 3) to achieve a target bulk water
concentration between 105 and 106 Colony Forming Units/milliliter (CFU/mL) in the pipe. During
the injection period, the WSTB was operated at 15 gpm (57 L/min) to allow for a minimum contact
time of approximately 1 hour (and to accommodate for travel time through the system). The
injection was stopped at 11:01 AM so that there was at least 1 hour of contact time and to ensure
the bolus of B. giobigii suspension had reached past the coupon section of the pipe.
Sample Collection - Bulk Water Samples (BWS) and coupon (CP) samples were collected at
11:10 AM, which allowed for the initial pass-through of the contaminant through the coupon
section. Coupon samples (CP-1, CP-ID, CP-2 and CP-2D) were extracted (scraped) immediately
after removal as described in Appendix A (Specific Sampling Procedures). Another BWS/CP
sampling event was performed at 11:30 AM. During this sampling period, one sample was
collected and analyzed onsite to confirm that chlorine dioxide was not detectable in the
background. Chlorine dioxide was measured onsite using the Hach DR/890 pocket colorimeter as
described in Appendix A (Specific Sampling Procedures). The CIO2-O sample was measured as 0
mg/L which confirmed the absence of chlorine dioxide in the WSTB prior to the introduction of
chlorine dioxide in the decontamination step. The contamination step-related sampling activities
are summarized in Table 3.
Table 3. Contamination Sampling Act
tivity
Sample IDs
Sample Description
Date/Time and System
Flow
BWS-1, CP-1
and CP-ID
Collected after the injection of B. giobigii
reaches the coupon section and prior to
the introduction of chlorine dioxide
October 6, 2014, 11:10 AM
Flow at 15 gpm (57 L/min)
BWS-2, BWS-
B2, CP-2, CP-
2D, and CIO2-O
Collected after the injection of B. giobigii
and prior to the introduction of chlorine
dioxide
October 6, 2014, 11:30 AM
Flow at 15 gpm (57 L/min)
BWS, bulk water sample; CP, coupon; gpm, gallons per minute; L/min, liters per minute; B, background sample (an
open bottle of sterile water sitting beside the pipe to detect any aerosolized BG); D, duplicate; C102, chlorine dioxide
sample; 1, 2, etc., sequential sample number
-------
2.3.2 Decontamination (addition of chlorine dioxide for a specified contact time)
Preparation of Decontaminant Agent Stock - Prior to injection, chlorine dioxide decontaminant
stock solution was prepared onsite at INL using the G02™ kit. Components A and B from the
G02™ (G02 International, 6700 Caballero Blvd., Buena Park, CA 90620, USA) kit were mixed
in 25 L of tap water (-0.5 mg/L free chlorine) in a covered carboy in an outside ventilated area.
The stock concentration yield was expected to be around 4,000 mg/L and the targeted in-pipe
concentration of chlorine dioxide was a minimum of 25 mg/L (based on previous pilot-scale testing
performed at the EPA Test & Evaluation Facility in Cincinnati, Ohio). The stock concentration
can be variable depending upon the water temperature and reaction time of the two kit components.
Decontamination Test Protocol - Prior to injection of the decontaminant, the flow to instrument
panels was cut-off to protect the instruments from the potentially detrimental effect of chlorine
dioxide. Subsequently, on October 6, 2014 at 12:00 PM, the chlorine dioxide stock solution (as
prepared above) was injected into the WSTB to achieve a minimum target in-pipe bulk water
chlorine dioxide concentration of 25 mg/L. At 12:45 PM, a chlorine dioxide sample was collected
from the pipe (first air release valve near inlet) and reported a value of 150 mg/L; another sample
collected at 12:57 PM at a downstream location reported a value of 105 mg/L. At 12:57 PM, the
chlorine dioxide injection was completed. All of the injectable prepared stock was used, except for
a small amount in the bottom of the 25-L carboy. At 1:12 PM, the flow through the WSTB was
stopped to hold the decontaminant in the pipe for the next 24 hours. The sampling activities for
the decontamination step are summarized in Table 4.
Table 4. Decontamination Sampling Activity
Sample ID
Sample Description
Date/Time and System
Flow
BWS-3, CP-3,
CP-3D, and
C102-l
• Collected after 80 minutes of injection/~20
minute contact time.
October 6, 2014, 1:20 PM
Flow at 0 gpm (0 L/min)
BWS-4 and
CIO2-2
• Collected after 140 minutes of the
introduction of chlorine dioxide and 80
minute contact time.
October 6, 2014, 2:20 PM
Flow at 0 gpm (0 L/min)
BWS-5 and
C102-3
• Collected after 200 minutes of the
introduction of chlorine dioxide and 140
minute contact time.
October 6, 2014, 3:20 PM
Flow at 0 gpm (0 L/min)
CP-4 and CP-
4D
C102-4
• Collected after 260 minutes of the
introduction of chlorine dioxide and 200
minute contact time.
October 6, 2014, 4:20 PM
Flow at 0 gpm (0 L/min)
BWS-6 and
C102-5
• Collected after 320 minutes of the
introduction of chlorine dioxide and 260
minute contact time.
October 6, 2014, 5:20 PM
Flow at 0 gpm (0 L/min)
BWS-7, CP-5,
CP-5D, and
CIO2-6
• Collected after 1400 minutes of the
introduction of chlorine dioxide and 1340
minute (-22 hours) contact time.
October 7, 2014, 11:20 AM
Flow at 0 gpm (0 L/min)
B WS, bulk water sample; CP, coupon; gpm, gallons per minute; L/min, liters per minute; D, duplicate; C102, chlorine
dioxide sample; 1, 2, 3, etc., sequential sample number
17
-------
2.3.3 Post-decontamination flushing and monitoring
At 12:00 PM on October 7, 2014, following collection of the last sample shown in Table 4, the
flow through the WSTB was resumed at 15 gpm (57 L/min) for the purposes of flushing the
decontaminant from the WSTB. The system was flushed with fresh water at 15 gpm (57 L/min)
for 2 hours to clear the chlorine dioxide. The flow was then reduced to 5 gpm (19 L/min) at 2:00
PM. Bulk water samples, chlorine dioxide, and coupon samples were collected following the
procedures described in Appendix A (Specific Sampling Procedures).
In addition to these samples, large volume samples (20 L Cubitainers® [Hedwin Division of Zacros
America, Inc., Baltimore, MD]) of water were also collected for analysis using the water sample
concentrator (WSC). Briefly, the WSC is a device that uses ultrafiltration membranes to
concentrate low numbers of biological agents from large volumes of water into a smaller volume
of water. This increases the chance that a biological agent can be detected in a large volume water
sample and lowers the detection limit of the analytical method used for enumeration (spread
plating). A description of the WSC, how it is operated and enumeration methods are included in
Appendix A. Additional large WSC-related samples were taken at 7 and 14 days after the initial
flushing by INL personnel. The post flushing sampling activities are summarized in Table 5.
Table 5. Flushing Sampling Activil
ty
Sample ID
Sample Description
Date/Time and System
Flow
BWS-8, CP-6,
CP-6D and
C102-7
• Collected 180 minutes after the start of
flushing
October 7, 2014, 3:00 PM
Flow at 5 gpm (19 L/min)
BWS-9, CP-7,
CP-7D and
CIO2-8
• Collected 1,440 minutes after the start of
flushing
October 8, 2014, 12:00 PM
Flow at 5 gpm (19 L/min)
BWS-10, CP-
8, CP-8D and
WSC-1
• Collected 1,440 minutes after the start of
reconditioning
• Collected WSC sample of 20 Liters 1440
minutes after the start of reconditioning
October 9, 2014, 12:00 PM
Flow at 2.5 gpm (10 L/min)
BWS-11, CP-
9, CP-9D and
WSC-2
• Collected 1,440 minutes after the start of
reconditioning
• Collected 20 Liters 1,440 minutes after the
start of reconditioning
October 10, 2014, 12:00 PM
Flow at 2.5 gpm (10 L/min)
WSC-3
• INL collected 20 Liters 7 days after the
start of reconditioning
October 15, 2014, 12:00 PM
Flow at 2.5 gpm (10 L/min)
WSC-4
• INL collected 20 Liters 14 days after the
start of reconditioning
October 22, 2014, 12:00 PM
Flow at 2.5 gpm (10 L/min)
BWS, bulk water sample; CP, coupon; D, duplicate; C102, chlorine dioxide sample; 1, 2, 3, etc., sequential sample
number; gpm, gallons per minute; L/min, liters per minute; INL, Idaho National Laboratory; WSC, water sample
concentrator
The results from the samples collected are presented in Section 3.0.
18
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2.4 The Waterstep Mobile Water System
The WaterStep ((WaterStep, Louisville, KY)) emergency management mobile water system
(MWS) was used to assess disinfection capability of a mobile treatment unit. The self-contained
WaterStep MWS ships in a pallet/skid for easy deployment. The WaterStep MWS is contained in
one locking, rolling storage cart with the following components:
1. The WaterStep M-100 chlorinator (an onsite chlorine generator)
2. Pumps: Circulating pump (12V DC), distribution pump (120V AC) and a hand pump.
3. Electrical Components: connectors and cords for equipment needing a power supply
including: ground fault interrupter, one 12V DC, deep cycle battery, storage case, a solar
panel, one 10/2/50 ampere automatic battery charger.
4. Plumbing Components: tubing and quick-connect cam-lock fittings for all water
connections.
5. Filter: Disc Filter (100 and a 25 micron - not used).
6. Storage: Collapsible potable water bladders with protective ground cover (not used)
2.4.1 WaterStep M-100 Water Treatment Experiments
The WaterStep M-100 chlorinator uses salt (sodium chloride) and the process of electrolysis using
direct current from a 12 volt (car) battery to produce chlorine gas and sodium hydroxide
(WaterStep, 2013). Table salt purchased from a grocery store was used in this experiment. The
system runs an electrical current between the two electrodes, separated by a membrane, in a
solution of sodium chloride. Electrolysis breaks up the salt molecules and frees chlorine gas from
the brine. The chlorine gas is used as the disinfectant. Only a small amount sodium hydroxide is
generated which needs to be disposed separately.
The chlorine gas is introduced into the water stream using a venturi connected to the M-100. A
pressure pump (a shallow well pump with bladder tank and a pressure switch) is used to draw
water from the source and circulate it through the venturi using a garden hose. As the water passes
through the venturi, it creates a vacuum which draws the chlorine gas out of the M-100. As the
water is mixed with the chlorine gas, it flows through and returns to the source or bladder tank for
storage and disinfection contact time. This process is typically continued until the free chlorine
concentration in the water reaches the desired level.
The WaterStep MWS has the capability to pump water into 10,000 gallon (37,850 L) portable
bladders, where the contaminated water is temporarily stored to provide contact time for
disinfection and then treated water is disposed. These bladders were not used during tests at INL.
Instead, the WaterStep MWS was set-up to pump contaminated water directly from the lagoon
through the chlorinator and then recirculated back into the lagoon for storage/disinfection. During
planning of the water treatment experiments, it was felt that pumping water from the lagoon
directly into the WaterStep unit (and bypassing the bladders) would be a more accurate
representation of how the unit would be deployed during an emergency water treatment scenario.
The enclosed lagoon was expected to provide the necessary contact time and storage. Figure 17
shows the WaterStep M-100 chlorinator in operation at WSTB.
-------
onw"
Figure 17. WaterStep M-lOO chlorinator.
Operationally, water was drawn from near the lagoon inlet (the presumed point of highest
contamination in the lagoon) into the WaterStep MWS. The chlorinated effluent from the
WaterStep was pumped back into the far end of the lagoon, away from the inlet near the WSTB
piping. It was hoped that this configuration would increase or promote mixing within the lagoon
which is not mechanically mixed. Figure 18 shows the operational setup of the WaterStep system.
Chlorinated Water
Outlet
Inlet to WaterStep
Recirculation Water
Outlet
Figure 18. WaterStep setup at the water security test bed lagoon.
20 | P a g e
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On October 6, 2014, at 12:50 PM, the WaterStep M100 unit was put in operation to disinfect the
water present in the lagoon. At 4:00 PM, additional salt (a "handful" or approximately 50 to 100
grams) was added to replenish the chlorinator. Subsequently, at 5:30 PM, the chlorinator was shut
off (-280 minutes of operation). The unit operated for 4 hours and 40 minutes. Throughout this
period, samples from the chlorinated water outlet were collected and analyzed for free chlorine
using a pool-kit. The numbers reported were consistently above 5 ppm (the kit can only report
values up to 5 ppm). Field dilution was not performed because this was simply a check to determine
if chlorine was being generated by the system. Grab samples were collected from the lagoon to
evaluate the chlorine levels and submitted offsite for analysis of B. globigii to determine if
disinfection was being accomplished. The lagoon sampling activities are summarized in Table 6.
Table 6. Lagoon Sampling Activity
Sample ID
Sample Description
Date/Time and System
Flow
LG-1, LG-1D
• Lagoon samples for Bacillus globigii
• Onsite sample for free chlorine, pH paper,
and temperature
10/6/2014
2:20 PM
Flow at 0 gpm (0 L/min)
LG-2, LG-2D
• Sample collected 150 minutes after the
WaterStep was turned on.
• Lagoon samples for B. globigii
• Onsite sample for free chlorine, pH paper,
and temperature
10/6/2014
3:20 PM
Flow at 0 gpm (0 L/min)
LG-3, LG-3D
• Sample collected 245 minutes after the
WaterStep was turned on. The WaterStep
unit was turned off at 5:30 PM (-35
minutes after this sample was collected).
• Lagoon samples for B. globigii
• Onsite sample for free chlorine, pH paper,
and temperature
10/6/2014
4:55 PM
Flow at 0 gpm (0 L/min)
LG-4, LG-4D
• Lagoon samples for B. globigii
• Onsite sample for free chlorine, pH paper,
and temperature
• Sample collected 1,230 minutes after the
WaterStep was turned on and 950 minutes
after the unit was turned off.
10/7/2014 (Tuesday)
9:20 AM
Flow at 0 gpm (0 L/min)
LG-5, LG-5D
• Sample collected 1,350 minutes after the
WaterStep was turned on and 1070 minutes
after the unit was turned off.
• Lagoon samples for B. globigii
• Onsite sample for free chlorien, pH paper,
and temperature
10/7/2014 (Tuesday)
11:20 AM
Flow at 0 gpm (0 L/min)
gpm, gallons per minute; L/min, liters per minute; LG, water sample from the lagoon; D, duplicate; 1, 2, 3, etc.,
sequential sample number
21
-------
3.0 Analysis of Decontamination and Water Treatment Results
3.1 Contamination and Decontamination Experiment
The field samples were packaged, shipped and analyzed as described in Appendix A. The
experimental phase-specific bulk water and water sample concentrator sampling results for B.
globigii are summarized in Table 7 and color categorized according to the experimental phase. In
Table 7, log reduction of spore in the water is calculated as LR = -Log(N/No) where LR = log
reduction, N = number of surviving spores at a particular time point, No = initial number of
spores. The initial number of spores in the water was the sample taken at 11:30 am as this was
the highest number observed.
Table 7. Bulk Water and Water Sample Concentrator Sampling Results
Sample Time
Elapsed
Elapsed
Coupon
Sample
B.
Log
Experiment
Time
Time
contact
ID
globigii
Reduct
Phase
After
After
time
spore
-ion
Spore
CI02
with
density
in
Injection
Injection
CI02
in water
spore
Start
Start
(min)
(CFU/100
density
(min)
(min)
mL)
10/6/14 8:50 AM
0
-
-
BWS 0
2.7E+00
-
Pre-injection
Baseline
10/6/14 11:10 AM
70
-
-
BWS 1
7.0E+07
-
B. globigii
10/6/14 11:30 AM
90
-
-
BWS 2
1.0E+08
-
injection
10/6/14 1:20 PM
200
80
20
BWS 3
2.0E+03
4.70
Decontaminat
10/6/14 2:20 PM
260
140
80
BWS 4
3.3E+01
6.48
ion with
10/6/14 3:20 PM
320
200
140
BWS 5
1.0E+02
6.00
chlorine
dioxide
10/6/14 4:25 PM
385
265
205
BWS5A
1.4E+00
7.85
10/6/14 5:20 PM
440
320
260
BWS 6
1.7E+00
7.78
10/7/14 11:20 AM
1,520
1,400
1340
BWS 7
5.0E+00
7.30
10/7/14 3:00 PM
1,740
1,620
N/A
BWS 8
5.0E+00
7.30
Flush WSTB
10/8/14 11:50 AM
2,990
2,870
N/A
BWS 9
5.0E+00
7.30
10/9/14 12:00 PM
4,440
4,320
N/A
BWS 10
ND
>8
Return to
10/10/14 12:00 PM
5,880
5,760
N/A
BWS 11
5.0E+02
5.30
Baseline
10/9/14 12:00 PM
4,440
4,320
N/A
WSC-1
2.1E+00
7.68
10/10/14 12:00 PM
5,880
5,760
N/A
WSC-2
7.9E-01
8.10
10/15/14 12:00 PM
13,080
12,960
N/A
WSC-3
3.2E-01
8.50
10/22/14 10:00 PM
23,760
23,640
N/A
WSC-4
1.7E+00
7.76
BWS, bulk water sample; cfu, colony forming units; WSC, water sample concentrator; WSTB, water security test
bed; ND, none detected
The experimental phase-specific average bulk water B. globigii values shown in Table 7 along
with the associated chlorine dioxide concentrations in their color category are plotted in Figure 19.
22 | Page
-------
Please note that the pre-injection baseline phase is a single point and does not appear color-coded
in Figure 19.
1.0E+08
1.0E+07
§ l.Ub+Ob
1.0E+05
l.(Jfc+04
0
03 1.0E+03
D£
LU
1.0t+02
l.Ut+Ul
Shading:
Orange = B. globigii injection phase
Green = Decontamination (Flow Stopped)
phase
Blue = Flushing phase
Grey = Return to Baseline
120
80
40
20
30 40 50 60 70
TIME AFTER SPORE INJECTION (HR)
80
90
100
• BG spores in water (cfu/100 ml)
-Chlorine Dioxide Concentration (mg/L)
Figure 19. Bulk water B. globigii (BG) and chlorine dioxide results over time after spore
injection (hr).
The trends in Figure 19 indicate that the chlorine dioxide level drops within minutes;
correspondingly, the B. globigii values also drop in the bulk water phase. A minimum of 5-log
reduction was observed throughout the decontamination phase. However, some B. globigii appears
during the flushing phase and the return to baseline phase of the experiment. The results also
indicate that the WSTB flushing phase is successful in removing chlorine dioxide from the system .
The corresponding experimental phase-specific coupon sampling results for B. globigii are
summarized in Table 8 and color categorized according to the experimental phase.
23 | P a g e
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Table 8. Coupon Sampling Results
Sample Day/Time
Sample
ID
Average B.
globigii (CFU/in2)
attached to
Coupon Surface
Range
between
coupons
(CFU/in2)
Log
Reduction
Experiment Phase
10/6/14 8:50 AM
CP0
6.7E+00
1.3E+01
NA
Pre injection
Baseline
CP 0D
10/6/14 11:10 AM
CP 1
9.5E+03
1.5E+04
NA
B. globigii injection
CP ID
10/6/14 11:30 AM
CP 2
1.3E+06
1.7E+06
0
CP 2D
10/6/14 1:20 PM
CP 3
1.3E+04
2.7E+03
2.0
Decontamination
with chlorine
dioxide
CP 3D
10/6/14 4:30 PM
CP 4
1.8E+04
1.5E+04
1.8
CP 4D
10/7/14 11:20 AM
CP 5
3.4E+04
1.6E+04
1.6
CP 5D
10/7/14 3:00 PM
CP 6
9.4E+03
1.1E+04
2.1
Flush WSTB
CP 6D
10/8/14 12:00 PM
CP 7
1.6E+04
2.8E+04
1.9
CP 7D
10/9/14 12:00 PM
CP 8
7.7E+03
1.0E+04
2.2
Return to Baseline
water quality
CP 8D
10/10/14 12:00 PM
CP 9
2.6E+03
2.9E+03
2.7
CP 9D
CFU, colony forming units; CP, coupon; WSTB, water security test bed; NA, not applicable (pre-injection); Note: to
determine the microbial density on the coupons in CFU/cm2, divide by 6.45
The experimental phase-specific average coupon B. globigii values shown in Table 8 along with
the associated chlorine dioxide concentrations in their color category are plotted in Figure 20.
Please note that the pre-injection baseline phase is a single point and does not appear color-coded
in Figure 20. The range between the duplicate coupons samples that make up the average number
is also displayed. In most cases, there is variability between the duplicate samples, which indicates
that spore adhesion was spatially heterogeneous.
24 | Page
-------
1.0E+07
1.0E+06
i
5 1.0E+05
Li.
o
>-
(/>
jjj 1.0E+04
O
HI
O
Q_
^ 1.0E+03
CQ
0
LU
1
o
< 1.0E+02
Shading:
Orange = B. globigii injection phase
Green = Decontamination (Flow Stopped)
phase
Blue = Flushing phase
Grey = Return to Baseline
1.0E+01
1.0E+00
•
30 40 50 60 70
TIME AFTER SPORE INJECTION (HR)
¦ Attached BG Spore Density (cfu/in2) • Chlorine Dioxide Concentration (mg/L)
Figure 20. Coupon B. globigii (CFlJ/in2) and bulk water chlorine dioxide (mg/L) results
over time after spore injection (hr). Note: to determine the microbial density on the
coupons in CFU/cm2, divide by 6.45
The trends in Figure 20 indicate that the chlorine dioxide level drops within minutes; however, the
coupon B. globigii values stabilize (~ order of magnitude 104 CFU/in2) after an initial ~ 2 log
reduction indicating that the chlorine dioxide decontamination procedure was not as effective in
removing B. globigii from the pipe wall. The limited effectiveness of the chlorine dioxide may be
due to demand from the pipe walls or surface adhesion and layering effects. Although the chlorine
dioxide concentration was high in the bulk phase, demand from the pipe wall may have limited
the penetration of the chlorine dioxide into the cement-mortar matrix and thus contact with the
attached spores. B. globigii from the pipe wall or those trapped in dead end sections of pipe appear
to release into the bulk water during the flushing phase and return to baseline phase of the
experiment as shown previously in Figure 19 and the extended timeline plot in Figure 21.
25 | P a g e
-------
1.0E+08
g 1.0E+06
T-
D
Li.
o
>- 1.0E+05
K
CO
z
LU
Q
W 1.0E+04
O
Q_
to
o
03 1.0E+03
OZ
LU
s*; 1.0E+02
_l
D
CO
1.0E+01
1.0E+00
i
~
Shading:
Orange = B. globigii injection phase
Green = Decontamination (Flow Stopped)
phase
Blue = Flushing phase
Grey = Return to Baseline
,
,
\
\
y
h
120
100
80
60 2
40
cm
0
—I
1
o
TIME AFTER SPORE INJECTION (HR)
»BG spores in water (cfu/100 ml)
-Chlorine Dioxide Concentration (mg/L)
Figure 21. Extended timeline bulk water B. globigii (BG) and chlorine dioxide (mg/L)
results over time after spore injection (hr).
One of the key benefits of using the WSTB to conduct research is the ability to generate data on a
realistic field scale. This data can then be compared to other data generated using smaller bench
or pilot scale systems. Comparing bench and/or pilot scale data with data obtained from the WSTB
will allow EPA to understand whether using a bench or pilot scale system for research yields results
similar to those obtained from a realistic field scenario. Alternatively, if data generated from the
WSTB is different from data obtained from a smaller research scale, it is likely due to the fact that
the WSTB simulates real world drinking water system attributes that cannot be simulated on a
smaller scale.
The data show in Figures 22 and 23 compare data on contamination and decontamination of
drinking water infrastructure obtained from a pilot-scale research system at EPA's T&E facility
(Figure 22) and the WSTB (Figure 23). Data in both figures comes from experiments where B.
globigii spores were injected into an experimental pipe system and allowed to come into contact
with cement-mortar drinking water infrastructure surfaces. The surfaces with adhered spores were
then decontaminated with chlorine dioxide. The pilot scale system at the T&E facility system used
a six inch (15 cm) diameter pipe loop with cement-mortar coupons installed in the same manner
as the WSTB and Cincinnati tap water flowing at a velocity of 1 ft/sec (0.3 m/sec). A detailed
description of the pilot scale system at the T&E facility can be found in Szabo et al., 2012.
26 | Page
-------
1.0E+07
1.0E+06
=>
LI—
(J
>-
1.0E+05
^ 1.0E+04
LU
DC
° 1.0E+03
i/)
(J
CO
9, 1.0E+02
u
<
1.0E+01
1.0E+00
»BG spores attached to
coupons (cfu/in2)
¦ Chlorine Dioxide
Concentration (mg/L)
Shading:
Orange = B. globigii injection phase
Green = Decontamination (Flow Stopped) phase
Blue = Flushing phase
Grey = Return to Baseline
120
100
80
-------
There are two key differences in the decontamination data obtained from the field scale WSTB
and the pilot scale pipe system at the T&E facility. First, in the pilot scale system, chlorine dioxide
at 25 mg/L achieved a greater than 4 logio reduction of the adhered spores within 2 hours of contact
time. The number of spore dropped below the detection limit of the analytical method. In the
WSTB, 110 mg/L of chlorine dioxide was initially achieved, and this concentration remained
above 50 mg/L for the first six hours of decontamination. However, only 2 logio reduction was
achieved in the WSTB, and the spores were still easily detectable on the coupons.
The second key difference between the experiments is the chlorine dioxide concentration. In the
pilot-scale test at the T&E facility, a chlorine dioxide concentration of 25 to 30 mg/L was easily
achieved over 24 hours. In the WSTB experiment and initial chlorine dioxide concentration of
110 mg/L dropped to 70 mg/L at 4 hours after decontamination began, 35 mg/L at 8 hours and 12
mg/L after 24 hours of contact time. This suggests that the pipe material exerted a significant
disinfectant demand. If the pipe exerted significant demand, is possible that the chlorine dioxide
was not effectively penetrating into the cement-mortar matrix where the spores were adhered. The
presence of spores remaining after the chlorine dioxide decontamination phase suggests that pipe
demand was a factor in their persistence.
The key message from the decontamination experiment in the WSTB is that drinking water
infrastructure decontamination is more challenging in a real world field environment. Future
decontamination research efforts will require a higher chlorine dioxide concentration or a different
decontamination approach altogether (for example, physical removal through pigging). However,
these results underscore the importance of conducting research at a real world experimental facility
like the WSTB, which can yield results more relevant to the real world than a bench or pilot scale
system.
3.2 Water Treatment Experiment
The results from the treatment of B. globigii spore contaminated water flushed into the lagoon
using the WaterStep treatment unit are tabulated in Table 9.
Table 9. Lagoon Sampling Results
Date/Time
Sample ID
B. globigii (CFU/mL)
Average B. globigii (CFU/mL)
10/6/14 1:40 PM
LG-0
2.2E+05
2.2E+05
10/6/14 1:40 PM
LG-0D
2.1E+05
10/6/14 2:20 PM
LG-1
3.0E+05
2.8E+05
10/6/14 2:20 PM
LG-1D
2.7E+05
10/6/14 3:30 PM
LG-2
2.5E+05
2.5E+05
10/6/14 3:30 PM
LG-2D
2.6E+05
10/6/14 4:30 PM
LG-2A
1.9E+05
1.5E+05
10/6/14 4:30 PM
LG-2AD
1.2E+05
10/6/14 5:20 PM
LG-3
8.9E+04
1.0E+05
10/6/14 5:20 PM
LG-3D
1.2E+05
10/7/14 9:20 AM
LG-4
1.1E+05
1.1E+05
10/7/14 9:20 AM
LG-4D
9.9E+04
CFU, colony forming units; LG, water sample from the lagoon; D, duplicate; 1, 2, 3, etc., sequential sample number
28 1
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The results in Table 9 indicate that each of the average B. globigii values reported from the lagoon
samples were greater than 105 CFU/mL. The field data (previously reported in Section 3.1,
reporting chlorine values consistently above 5 ppm) proved that the WaterStep unit was successful
in producing chlorine as designed. However, field methodology for delivering the chlorine
disinfectant to the lagoon without the bladders was ineffective. The highest free chlorine residual
detected in the lagoon was 0.03 mg/L, but the highest total chlorine residual detected was 1.71
mg/L. This indicated that the free chlorine being generated by the WaterStep unit was being
transformed into total (or combined) chlorine once it entered the lagoon. The large exposed surface
area of the lagoon, in combination with shallow depth, and intense sunlight, may all have
contributed to the rapid degradation of the chlorine delivered to the lagoon. Other confounding
factors include: high organic load from the dusty lined lagoon and the presence of growth media
carried over from flushing.
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4.0 Conclusions and Future Work
The following is a summary of conclusions based on the testing performed at the INL WSTB:
• The dye-testing confirmed the theoretical flow velocity and travel time calculations (~ 1
hour travel time). As expected, there was some visible mixing and dispersion/diffusion of
the dye slug observed during testing.
• Sodium thiosulfate (a surrogate contaminant) removed free chlorine from the water and
successfully triggered the hydrant-based flushing device. Some of the dispersed sodium
thiosulfate was lodged in dead end pipes and was released with subsequent hydraulic
changes. The experiment showed that changes in water quality resulting from
contamination can trigger a flushing hydrant and remove contaminated water from a
distribution pipe.
• The decontaminant (chlorine dioxide) targeted in-pipe concentration of >25 mg/L was also
achieved. The highest observed chlorine dioxide concentration was 110 mg/L, but the
chlorine dioxide decayed to 35 mg/1 over the next 7 hours.
• The contaminant (B. globigii) targeted in-pipe bulk water concentration (107 and 108
CFU/100 mL) was achieved. The water sampling results indicated a 5 to 8 log reduction
of the contaminant in bulk water over the course of 24 hours (chlorine dioxide ranging
from 110 mg/L down to 18 mg/L. The sampling results also indicate a less effective
removal (~2 log reduction based on highest observed B. globigii density before
decontamination) of the contaminant from the coupon surface over the same 24 hour
period.
• Comparison of the decontamination results from the WSTB and those from pilot scale
decontamination research studies performed in EPA facilities suggests that
decontamination of biological agents in a real world field setting is more challenging than
the data from the pilot scale studies had indicated. Certain aspects of a real water
distribution system that could influence the effectiveness of a decontamination method,
such as pipe wall disinfectant demand and dead end spaces, are difficult to simulate on the
pilot scale. Therefore, future decontamination research should ideally be performed at the
field scale instead of the bench or pilot scale, because a realistic setting will provide a truer
picture of decontamination effectiveness.
• The lagoon/WaterStep decontamination procedure was ineffective as performed. In the
future, the temporary storage bladders will need to be used to provide sufficient contact
time, reduce surface area, remove the adverse effects of sunlight on the disinfection
process, and reduce the impact of the organic load from the lagoon. Operationally, the unit
could not run much over 3 hours without replenishing the sodium chloride solution in the
generator. Because the membrane will burn out if the salt solution gets too low, this system
is not suitable for an unmanned operation.
Overall, the WSTB was operated without issues and enabled EPA NHSRC to perform the study.
Specifically, the following operational observations were made during the performance of the EPA
study:
• The actual pressure drop matched the theoretical pressure gradient drop of ~3 psi across
the system.
• The WSTB pipe system (linear pipe length of 448 ft (137 m)) operated leak-free during the
testing.
30 1
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• The WSTB maintained pressure throughout the testing, including the flushing event where
large amounts of water were withdrawn from the system.
• A stable free chlorine residual was maintained through the length of the pipe when active
testing was not occurring at a baseline flow of 2.5 gpm (10 L/min).
• The flow monitoring and flow control device (i.e., rotameter) operated successfully.
• The lagoon volume was sufficient to perform a complete test.
• The instrument panels were installed and continuous monitoring data was successfully
telemetered via cellular/radio back to the EPA Test and Evaluation Facility in Cincinnati,
Ohio.
• The injection system successfully delivered a controlled volume of tracer/contaminant/
decontaminant.
• The automated flushing hydrant functioned as expected.
• The PVC pipe coupon section functioned as expected. The isolation valves allowed for the
removal and replacement of coupons during the experiment without draining the WSTB.
• The lined trench under the pipe directed the flow to the lagoon as intended.
Future research using the WSTB will focus on answering some of the remaining questions and
filling data gaps in this report, as well as addressing other outstanding EPA National Homeland
Security Research Center needs.
• Decontamination with chlorine dioxide will be reattempted in the spring of 2015. The
results from experiments described in this report indicate that the WSTB was not
thoroughly decontaminated, and Bacillus spores remain attached to the pipe. The WSTB
will be decontaminated again using chlorine dioxide. An increased contact time with the
pipe wall will be implemented and the dead end portion of the WSTB will be thoroughly
flushed by adding flow ports to the dead-end portions of the pipe.
• Treatment of water in the lagoon contaminated with Bacillus spores will be attempted with
additional commercially available water treatment units. The goal will be gathering data
on the field performance of commercially available water treatment units.
• Crude oil will be injected into the WSTB and the persistence of constituents in the crude
oil such as benzene, toluene, ethylbenzene and xylene (BTEX) will be assessed.
Decontamination approaches such as flushing or adding surfactants will be studied. This
study is warranted because crude oil spilled into a water body that feeds a drinking water
treatment plant could make it through the treatment works and into the distribution system.
Should this happen, first responders will need to know if the BTEX constituents persist on
the distribution system infrastructure and the effectiveness of decontamination methods.
31
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5.0 References
Rice, E. W., Fox, K. R., Miltner, R. J., Lytle, D. R. and Johnson, C. H. (1994). A microbiological
surrogate for evaluating treatment efficiency. In Proc. of AWWA/Water Quality Technology
Conference, San Francisco, October, 1994, p. 2035
Szabo, J. G., Muhammad, N., Heckman, L., Rice, E. W. and Hall, J. S. (2012). Germinant-
enhanced decontamination of Bacillus spores adhered to iron and cement-mortar drinking water
infrastructure. Applied and Environment Microbiology, 78(7): 2449-2451.
WaterStep (2013). Instruction Manual for the M-100 Chlorine Generator. WaterStep, 625 Myrtle
St., Louisville, KY 40208 (www.waterstep.org)
32
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Appendix A - Detailed Experimental Protocols and Quality Assurance Criteria
33
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Contamination/Decontamination Tests
These experiments involve contamination of the water security test bed (WSTB) using Bacillus
globigii spores and the subsequent decontamination of WSTB using chlorine dioxide as the
decontaminant. Each contamination/decontamination experiment consists of the following main
steps:
Step 1 - Pipe Conditioning (cultivation of biofilm)
Step 2 - Instrumentation Panel/Injection Setup and Dye Tracer Study
Step 3 - Injection of Contaminant (addition of B. globigii spores to WSTB)
Step 4 - Decontamination (chlorine dioxide/flushing)
Step 5 - Post-Decontamination Flushing, Reconditioning and Monitoring
Step 1 - Pipe Conditioning (Cultivation of Biofilm)
To effectively study the adsorption of contaminants on pipe walls, it is essential to ensure that
there is a viable biofilm. The biofilm could influence adsorption of the contaminant on the pipe
wall in addition to metabolism, biodegradation, or detoxification of the contaminant.
Previously under EPA Contract EP-C-09-041, CB&I Federal Services LLC (CB&I) performed a
literature review of biofilm cultivation and identified four primary techniques that could
potentially be used for cultivating biofilm within the WSTB:
1. Sequential batch fermentation and introduction into the WSTB
2. Using the WSTB as a reactor by passing water with low concentrations of carbon,
nitrogen, and salts
3. Use of an external annular reactor
4. Natural biofilm cultivation by passing water through the WSTB
The fourth option, natural cultivation of biofilm, has been chosen as the cultivation procedure for
testing of the WSTB. This will be accomplished by passing Idaho National Laboratory (INL) tap
water through the WSTB continuously over a period of time (estimated to be 4 weeks - starting
early to mid September 2014). After initial flushing to remove any debris, the flow rate will be
set at 2.5 gallons per minute (gpm) (10 L/min) with a total discharge of 25,200 gpm (95,382 L)
to the lagoon which allows for weekly trucking and disposal of the accumulated discharge.
Step 2 - Instrumentation Panel/Injection Setup and Dye Tracer Study
Late September 2014, the CB&I team will arrive at INL to install the instrument panel and
injection pump. A simple dye tracer study (using non-toxic biodegradable dye such as Bright
Dyes®, Kingscote Chemicals, Miamisburg, OH; www.brightdves.com) will be performed to
visually confirm the theoretical calculations of travel times and system flows.
The presence of stable free chlorine concentration and temperature at the downstream instrument
panel location of the WSTB will indicate stabilization of pipe wall chlorine demand and biofilm
formation. If the measured free chlorine levels are stable, a coupon in duplicate and a bulk water
sample (BWS) will be collected. The heterotrophic plate count (HPC) concentration on the
coupon surface will be measured to determine the presence of viable biofilm in the WSTB. The
heterotrophic colony count (HPC) coupon and BWS sample can be collected anytime between
one day and 1 week before injection of contaminant (Step 3).
34
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This HPC sampling will be performed by isolating the coupon section of the main pipe by
closing the upstream flanking gate valve, removing the duplicate coupon plugs (CP-0 and CP-
OD) and replacing the coupon plugs with blank plugs. The biofilm sample will be collected from
the duplicate coupon plugs as described in Sampling Procedures section (later in this Appendix)
to determine the formation of biofilm on the coupons and measure HPC concentration. At the
same time, a BWS background sample as described in the Sampling Procedures section (later in
this Appendix) will be collected to serve as the background control. The sampling activities are
described in Table Al.
Table Al. Bac
iground Sampling Activity
Sample ID
Sample Description
Estimated Timeline &
System Flow
BWS-0
(Control)
Collect at ~1 week prior to injection of B.
globigii
Late September 2014
Flow at 2.5 gpm (10 L/min)
BWS-B
(Background)
Background control collected at the same time
as BWS-0
Late September 2014
Flow at 2.5 gpm (10 L/min)
CP-0 and
CP-OD
Collect at the same time as BWS-0
Late September 2014
Flow at 2.5 gpm (10 L/min)
BWS, bulk water sample; CP, coupon; gpm, gallons per minute; L/min, liters per minute
Step 3 Injection of Contaminant (Addition of B. globigii Spores to WSTB)
Preparation of Spores (Contaminant Stock) - The B. globigii spores for this study were
originally obtained from EPA NHSRC at the Andrew W. Breidenbach Environmental Research
Center (AWBERC) in Cincinnati, Ohio. A culture of B. globigii vegetative cells will be mixed
with generic sporulation media and incubated by gentle shaking at 35°C for 7 days at the EPA
T&E Facility. The concentration of B. globigii stock will be determined following the method
described by Rice et al. (1994). The B. globigii spores will be heat-shocked and analyzed using
the spread plate method and membrane filtration. After the stock is ready, 40 liters of prepared
stock will be shipped in separate 1 liter containers (preserved at 4±2°C) to the site.
Injection of Contaminant Test Protocol - In early October, the B. globigii suspension will be
introduced into the WSTB using a positive displacement pump to achieve a target bulk water
concentration between 105 and 106 CFU (Colony Forming Units)/mL in the pipe. The WSTB will
be operated at 15 gpm (57 L/min) under this condition with a minimum contact time of
approximately 1 hour (to accommodate for travel time). Injection duration is also estimated to
be 1 hour so that there is a contact of 1-hour after the bolus of B. globigii suspension reaches the
coupon section of the pipe.
BWS and coupon samples will be collected at 5 and 60 minutes after initial pass-through of the
contaminant at the coupon section. A sample for chlorine dioxide will also be collected at 60
minutes after initial pass-through of the contaminant at the coupon section. Coupon samples (CP-
1, CP-ID, CP-2 and CP-2D) will be extracted (scraped) immediately after removal as described
in the Sampling Procedures section (later in this Appendix). The CIO2-O sample serves as
background for chlorine dioxide (prior to the introduction of chlorine dioxide in the
decontamination step). The WSTB operations will be continued at the same rate until the
35
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contaminant bolus has passed through the system (estimated to be ~ 1 hour and 5 minutes based
on theoretical calculations and the dye tracer travel time confirmation). This is necessary to
remove the contaminant from the bulk phase of water. A BWS will be collected after the bolus is
estimated to have passed through the system. The sampling activities are described in Table A2.
Table A2. Contamination Sampling Activity
Sample IDs
Sample Description
Estimated Timeline &
System Flow
BWS-1, CP-1
and CP-ID
Collect after 5 minutes of the injection of B.
globigii reaches the coupon section and prior
to the introduction of chlorine dioxide
Early October 2014
Flow at 15 gpm (57 L/min)
BWS-2, CP-2,
CP-2D and
CIO2-O
Collect after 60 minutes of the injection of B.
globigii and prior to the introduction of
chlorine dioxide
Early October 2014
Flow at 15 gpm (57 L/min)
BWS, bulk water sample; CP, coupon; gpm, gallons per minute; L/min, liters per minute
Step 4 - Decontamination (Chlorine dioxide/flushing)
Preparation of Decontaminant Agent Stock - Chlorine dioxide decontaminant stock solution
will be prepared in advance onsite at INL using the G02™ kit (G02 International, Buena Park,
CA). Components A and B from the G02™ will be mixed in 25 L of deionized water using two
buckets, covered, and kept static for 3 hours in an outside ventilated area. Chlorine dioxide stock
concentration will be measured using the Hach® DR/890 pocket colorimeter (Hach Inc.,
Loveland, CO). The expected stock concentration yield is about 4,000 mg/L.
Decontamination Test Protocol - Approximately 5 minutes following the 60 minute sample
collection (shown in Table A2), the prepared chlorine dioxide stock solution will be injected into
the WSTB to achieve a target bulk concentration of approximately 25 mg/L. Concentration of
the chlorine dioxide stock will be verified before injection. Injection of chlorine dioxide will
continue until the chlorine dioxide has reached the end of the pipe (estimated to be
approximately 1 hour and 5 minutes based on theoretical calculations and the dye tracer travel
time confirmation). Injection will be stopped, online instrumentation will be stopped, and water
flow out of the WSTB will be stopped for 18-24 hours so that the water containing disinfectant
will be stagnant in the pipe to perform disinfection. Duplicate coupon samples will be collected
at 0 min, 180 min and 1,200-1,440 minutes (20-24 hours) after the decontaminant injection is
shut down and disinfection is occurring. Each coupon will be extracted (scraped) immediately
after removal (see Sampling Procedures section later in this Appendix) and the resulting
suspension stored in a cooler at 4°C. BWS and CIO2 samples will be collected at the same time
coupons are removed, and analyzed for B. globigii and chlorine dioxide, respectively.
Additional chlorine dioxide samples will be collected at 60 min, 120 min and 240 min after
decontamination injection is shut down. The sampling activities are described in Table A3.
Table A3. Decontamination Sampling Activity
Sample ID
Sample Description
Estimated Timeline &
System Flow
BWS-3, CP-3,
CP-3D, and
Collect after 0 minutes of the introduction of
chlorine dioxide.
Early October 2014
Flow at 15 gpm (57 L/min)
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C102-1
Allow chlorine dioxide to reach the end of the
pipe - estimate 65 minutes. Stop flow.
Early October 2014
Flow at 15 gpm (57 L/min)
BWS-4 and
CIO2-2
Collect after 60 minutes of the introduction of
chlorine dioxide
Early October 2014
Flow at 0 gpm (0 L/min)
BWS-5 and
C102-3
Collect after 120 minutes of the introduction of
chlorine dioxide
Early October 2014
Flow at 0 gpm (0 L/min)
CP-4 and CP-
4D
Collect after 180 minutes of the introduction of
chlorine dioxide
Early October 2014
Flow at 0 gpm (0 L/min)
BWS-6 and
C102-4
Collect after 240 minutes of the introduction of
chlorine dioxide
Early October 2014
Flow at 0 gpm (0 L/min)
BWS-7, CP-5,
CP-5D, and
C102-5
Collect after 1,200 - 1,440 minutes (20 - 24
hrs.) of the introduction of chlorine dioxide
Early October 2014
Flow at 0 gpm (0 L/min)
BWS, bulk water sample; CP, coupon; gpm, gallons per minute; L/min, liters per minute
Step 5 - Post-Decontamination Flushing, Reconditioning, and Monitoring
Following collection of the 1,440 minutes samples (shown in Table A3), the WSTB will be
flushed with fresh water for approximately 1 hour at 15 gpm (57 L/min) to clear the chlorine
dioxide. The flow will then be reduced to 5 gpm (19 L/min). BWS, chlorine dioxide and coupon
samples will be collected following the procedures described in the Sampling Procedures section
(later in this Appendix) at 180 min and 1,200-1,440 min (20-24 hours) after the start of flushing.
The sampling activities are described in Table A4.
Table A4. Flushing Sampling Activity
Sample ID
Sample Description
Estimated Timeline &
System Flow
BWS-8, CP-6,
CP-6D and
CIO2-6
Collect after at 180 minutes from the start of
flushing
Early to mid October 2014
Flow at 5 gpm (19 L/min)
BWS-9, CP-7,
CP-7D and
C102-7
Collect after at 1,200 - 1,440 minutes from the
start of flushing
Early to mid October 2014
Flow at reset at 2.5 gpm (10
L/min)
BWS, bulk water sample; CP, coupon; gpm, gallons per minute; L/min, liters per minute
Large volume samples of 20 L for water sample concentrator (WSC), BWS, and coupons will be
removed at 1 and 2 days after flushing is initiated. Additional large volume 20 L WSC samples
will be taken at 7 and 14 days after flushing stops by INL personnel. The sampling activities are
described in Table A5.
Table A5. Return to Baseline Sampling Activity
Sample ID
Sample Description
Estimated Timeline &
System Flow
BWS-10,
CP-8, CP-8D
and WSC-1
Collect after 1,440 minutes of the start of
reconditioning
Collect WSC sample 20 Liters after 1440
Mid October 2014
Flow at 2.5 gpm (10 L/min)
37
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minutes of the start of reconditioning
BWS-11,
CP-9, CP-9D
and WSC-2
Collect after 1,440 minutes of the start of
reconditioning
Collect 20 Liters after 1,440 minutes of the start
of reconditioning
Mid October 2014
Flow at 2.5 gpm (10 L/min)
WSC-3
INL will collect 20 Liters after 7 days after the
start of reconditioning
Mid October 2014
Flow at 2.5 gpm (10 L/min)
WSC-4
INL will collect 20 Liters after 14 days after the
start of reconditioning
Late October 2014
Flow at 2.5 gpm (10 L/min)
BWS, bulk water sample; CP, coupon; gpm, gallons per minute; L/min, liters per minute; WSC, water sample
concentrator
New coupons will be inserted into the WSTB and flow will resume through the WSTB at 2.5
gpm (10 L/min) to restart the biofilm cultivation process (Step 1) for the next test. If an
experiment is the last test before the WSTB is shut down for the winter, blank coupons will be
inserted and the WSTB will be drained. Given the project schedule, it is anticipated that only
one test condition will be completed using the cement mortar-lined iron pipe from INL (test
material), B. globigii (contaminant), and chlorine dioxide (decontaminant) in 2014.
Dechlorination/Flushing Experiments
The purpose of these experiments is to demonstrate the feasibility of using online water sensors
in concert with flushing hydrants to intelligently divert and remove contaminants from water
distribution systems. For these experiments, the Hach CL-17 and the Real Tech instruments
(Real Tech Inc., Whitby, ON, Canada) will be used to signal a flushing hydrant to open and flush
the injected contaminant from the WSTB. For this purpose, a test will be performed using
sodium thiosulfate as the "injected contaminant" to de-chlorinate the system for approximately
30 minutes. A set point or trigger value of measured chlorine level (e.g., 0.05 mg/L or lower)
using the Hach CL-17 at the upstream location will be used for opening the flushing hydrant
valve. After the chlorine value at the upstream recovers (e.g., >0.5 mg/L) to the background
value, the valve will be automatically triggered to close. Grab samples will be collected and
analyzed for free chlorine residuals at a downstream location from the flushing hydrant. The
purpose of this grab sample will be to determine if dechlorinated water is able to "jump" across
the "tee" to the flushing hydrant and proceed downstream. Free chlorine levels for these grab
samples will be measured using the Hach DR/890 pocket colorimeter (same instrument used to
measure chlorine dioxide). The triggered flushing experiment will be independent of the
contamination/ decontamination experiment and will occur after the
contamination/decontamination experiment if time and weather permits.
For the second objective, the Hach CL-17 will be used to trigger a flushing event based on
chlorine concentrations, as described above. The RealTech M4000 TOC instrument's ability to
trigger events based on organic concentrations will be evaluated at a later date.
Disinfection of Large Water Volumes
This experiment will assess the ability of a portable disinfection unit to disinfect a large volume
of water containing Bacillus spores. During the contamination and decontamination experiment
described above, there will be an 18-24 hour time period where water is not flowing into the
lagoon (see steps 3 and 4). After contamination, the contaminant bolus will be allowed to flow
38
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out of the WSTB and into the lagoon. Chlorine dioxide will then be added to the WSTB. Once
the WSTB is full of chlorine dioxide, flow will be stopped for 18-24 hours to allow disinfection
to occur. This 18-24 period of no flow will be ideal for testing a field portable disinfection
device since the contaminant bolus will be contained in the lagoon, but flow from the WSTB will
not be diluting it.
The WaterStep mobile water system (MWS) will be used to assess disinfection capability of a
mobile treatment unit. The MSW has the capability to pump water into 10,000 gallon (37,850 L)
bladder where it is disinfected and then dispensed once it has been disinfected. These bladders
will not be used during tests at INL. Instead, water will be pumped from the lagoon into the
disinfection unit and then dispensed back into the lagoon after disinfection. This process will
occur during the 18-24 hour period of no flow from the WSTB. Samples will be removed
directly from the lagoon when water from the WSTB is shut off (time zero). Once the
disinfection unit starts working, samples will be removed from the lagoon at 1, 2, 4, 18, 20 and
24 hours (if applicable). Samples will be removed in duplicate from the lagoon at each sampling
time.
SAMPLING PROCEDURES
Site-Specific Factors
Contamination/decontamination and flushing experiments will be conducted at INL. Samples
will be shipped to the EPA T&E Facility for analysis. A summary of the experimental sampling
strategy (including the number of samples) is presented in Table A6.
Table A6. Summary of Experimental Sampling Strategy
Sample/
Sampling Location
Measurement
Total No.
Matrix
Measurement
Location
Sampling Frequency
of
Samples
Contamination -
Biofilm
HPC
T&E Facility
1 sample in duplicate
2
Decontamination Tests/
Biofilm
B. globigii
T&E Facility
9 sample in duplicate
18
WSTB
Water
B. globigii
T&E Facility
18 100 mL samples in duplicate
Four 20 L samples in duplicate
36 (100 ml)
4 (20L)
Water
Chlorine
Dioxide
Field Site
7 samples
7
Water
Free Chlorine
Field Site
~2 samples
2
HPC, heterotrophic plate count; WSTB, water security test bed
Specific Sampling Procedures
Extraction of Biofilm and Spores from Coupon Surface for HPC and B. alobiaii analyses
The coupons will be collected from the WSTB carefully without touching the surface that was
exposed to WSTB water. The biofilm and spores will be scraped from the surface using a
disposable sterile surgical scalpel. The extracted material will be collected in a sterile sample
bottle with a sodium thiosulfate tablet and 100 mL of pre-filled carbon-filtered water. The
extracted sample will be transferred to a cooler at 4±2°C. The samples will be shipped overnight
to the EPA T&E Facility and analyzed upon receipt.
39
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Samples for HPC Concentration Measurement
The BWS for HPC concentrations (BWS-0) will be collected using the grab sampling technique
in 100-mL sterile sample bottles with a sodium thiosulfate tablet. The BWS sampling port will
be opened and the water will be drained for 15 seconds prior to collection of 100 ml of water
from the WSTB. The extraction of biofilm from the coupon surface (CP-0/CP-0D) will be
conducted as described in the previous paragraph. The samples will be transferred to a cooler at
4±2°C. The samples will be shipped overnight to the EPA T&E Facility and analyzed upon
receipt.
BWS for B. globigii spores
The BWS for B. globigii concentrations (BWS-1 through BWS-9) will be collected using the
grab sampling technique in 100 mL sterile sample bottles with a sodium thiosulfate tablet. The
BWS sampling port will be opened and the water will be drained for 15 seconds prior to
collection of 100 ml of water from the WSTB. For larger samples, 20 L will be collected
flexible plastic bladders (Cubitainers®, Hedwin Division of Zacros America, Inc., Baltimore,
MD) with sodium thiosulfate tablets (0.01% w/v). A sample of water will be removed from the
Cubitainers to ensure that no free chlorine residual is present. The samples will be transferred to
a cooler at 4±2°C. The samples will be shipped overnight to the EPA T&E Facility and analyzed
upon receipt.
To estimate the BWS background (BWS-B), a sample bottle containing sterile buffer solution
will be exposed to background air while the actual BWS is being collected to serve as the
background control.
Samples for Chlorine Dioxide - Field Measurement
During the decontamination step, with every bulk water sample collected using the grab
sampling technique and a laboratory beaker (CIO2-O through C102-7) will be used to draw a
sample for chlorine dioxide measurement. The sample will be immediately processed for
measurement using the Hach Method 10126 (pocket Colorimeter) in the field.
Samples for Free Chlorine - Field Measurement
During the dechlorination/flushing experiments, grab samples will be collected from a
downstream location of the flushing hydrant using the grab sampling technique and a laboratory
beaker and analyzed for free chlorine. The sample will be immediately processed for
measurement using the Hach Method 10102 (pocket colorimeter) in the field.
Water Sample Concentrator
Once received at the EPA T&E Facility, the 20 L water samples (labeled WSC) will be subjected
to concentration using the water sample concentrator. The water sample concentrator will be
operated according to EPA NHSRC's Water Sample Concentrator Standard Operating Procedure
(SOP) 030 (Automated Concentrator Ultrafiltration Protocol), which is in Appendix D of this
report. The resulting concentrated sample will be placed into sterile 100 mL sample bottles and
analyzed in the same manner as all other B. globigii BWS.
WaterStep Mobile Water System
B. globigii water samples will be removed from the lagoon to assess the disinfection capability of
40
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the MSW. A sampling position in the lagoon will be chosen so that is away from the MSW
effluent entering the lagoon. This will allow for some mixing in the lagoon to occur. Samples
will be collected using the grab sampling technique in 100 mL sterile sample bottles with a
sodium thiosulfate tablet. Samples will be removed from the lagoon with a sterile pipette, and
the contents of the pipette dispensed into the bottle. The pipette tip will be lowered 4 to 6 inches
(10 to 15 cm) below the water surface when collecting a sample. The samples will be transferred
to a cooler at 4±2°C. The samples will be shipped overnight to the EPA T&E Facility and
analyzed upon receipt.
Sampling Containers and Quantities
Sample containers and quantities are shown in Table A7.
Sample Preservation and Holding Times
Sample preservation and holding times are shown in Table A7.
Sample Labeling
Sample identification is summarized below.
Samples collected for analysis will be identified by type (BWS, CP, or Grab), collection interval
(-0, -1,-2, etc.), analysis (B. globigii, HPC, chlorine dioxide (CIO2), free chlorine), and date
collected. Duplicate coupons will be identified using a "D" after the collection interval.
Table A7. Grab Sampling and Analytical Procedures
Measurement
Sampling Method
Analysis Method
Sample Container/
Quantity of
Sample
Preservation/
storage
Holding
times
Chlorine Dioxide
See Specific
Sampling Procedures
Hach Method 10126
Glass beaker (~50
mL)
None
Immediate
Free Chlorine
See Specific
Sampling Procedures
Hach Method 10102
Glass beaker (~50
mL)
None
Immediate
B. globigii spore
See Specific
Sampling Procedures
CB&I T&E SOP
309 (Appendix C)
100 mL sterile
sample bottles
20 L Cubitainers1
The bottles
contains sodium
thiosulfate tablet.
Cool 4 ± 2°C
Analyze
upon receipt
at the EPA
T&E
Facility.
HPC
See Specific
Sampling Procedures
CB&I T&E SOP
304 (Appendix B)
100 mL sterile
sample bottles
The bottles
contain sodium
thiosulfate
tablets. Cool 4 ±
2°C
48 hours
HPC, heterotrophic plate count; SOP, standard operating procedure; T&E, Test and Evaluation
'The 20 L Cubitainer samples will be concentrated via the water sample concentrator and placed into the 100 mL
sterile sample bottles for analysis.
Sample Packaging and Shipping
The biofilm samples, BWS, and WSC samples will be preserved in coolers with ice and shipped
to the EPA T&E Facility overnight. Chain of custody forms will be completed and shipped with
the samples.
41
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MEASUREMENT PROCEDURES
ANALYTICAL METHODS
The analysis methods are shown in Table A7. The microbiological methods are further
discussed below.
HPC determinations will follow T&E SOP 304, Heterotrophic Plate Count (HPC) Analysis
UsingIDEXXSimPlate Method. This method is based on multiple enzyme technology which
detects viable bacteria in water by testing for the presence of key enzymes known to be present
in these organisms. It uses multiple enzyme substrates that produce a blue fluorescence when
metabolized by bacteria. The sample and media are added to a SimPlate® plate (Idexx
Laboratories, Inc., Westbrook, ME), incubated, and then examined for fluorescent wells. The
number of fluorescing wells corresponds to a most probable number (MPN) of total bacteria in
the original sample. This method is included as Appendix B in this document.
Preparation and analysis of B. globigii will follow T&E SOP 309, Preparation and Enumeration
ofB. globigii Endospores. B. globigii is an aerobic spore-forming bacteria used as a surrogate
for evaluating the performance of water treatment systems for removal of bacterial endospores.
In analyzing spores, the indigenous vegetative cells are inactivated by heat treatment. The
surviving bacterial spores in the sample are analyzed by culturing that permits the spores to
germinate and produce bacterial cells. Tryptic soy agar will be used for culturing B. globigii.
This method is included as Appendix C in this document.
The samples are diluted, as necessary, depending on the expected concentration of cells/spores in
the sample. For example, the expected initial concentration of spores in this study is 106
spores/mL. The initial samples will be diluted up to 105 fold. Duplicate plates using 0.1 mL of
the 104 and 105 fold diluted samples will be analyzed using the spread plate method. If the
number of colonies is too many to count in more than one plate, the sample will be diluted and
re-analyzed. If the number of colonies is too many to count for one measurement, the remaining
plates will be considered for enumeration of spore concentration for the sample.
CALIBRATION PROCEDURES
The calibration procedures, linearity checks, and continuing calibration checks are included in
the T&E SOPs or the instrument manuals for the analysis methods referenced in Table A7.
QUALITY METRICS (QA/QC CHECKS)
QC Checks
Instruments/equipment will be maintained in accordance with the SOPs and analysis methods
listed in Table A7, and for field instruments, in accordance with the manufacturer's instructions.
Table A8 presents the QA/QC checks to be implemented for the measurement of the specific
parameters.
Table A8 lists the QA/QC checks that will be used to verify the validity of the analyses
conducted on grab samples conducted during this study. Table A9 summarizes the QA/QC
42
-------
requirements for the optical devices used in this study.
The RPD is calculated for duplicate analyses based on the following:
(C1-C2)
^=05(C. + C,)X'00%
where:
RPD = Relative Percent Difference
CI = Larger of two values
C2 = Smaller of two values
If calculated from three or more replicates, the relative standard deviation (RSD) will be used
according to the following equation:
RSD = 100% ^
3^
where:
RSD = relative standard deviation (%)
5 = standard deviation
yaVe = mean of the replicate analyses
Standard deviation is defined as follows:
where:
V (y> ~ yave)2
^ n-1
5 = standard deviation
yt = measured value of the ith replicate
yaVe = mean of the replicate measurements
n = number of replicates
Table A8. QA/Q
>C Checks for Grab Samples
Measurement
QA/QC Check
Frequency
Acceptance
Criteria
Corrective Action
B. globigii
Positive control
using stock
Once per
experiment
±10 fold of the
spiking suspension
Investigate laboratory
technique. Change
stock organisms and
use new set of media
plates. Re-analyze the
spiking suspension and
change it if necessary.
B. globigii
Negative Control
using sterile buffer
Once per
experiment
0 CFU/plate
Investigate laboratory
technique. Use a new
lot. Re-analyze.
43
-------
B. globigii
Negative control
for heat shock
Once per
experiment
0 CFU of
vegetative
cell/plate
Investigate the hot
water bath. Heat
samples for longer
period.
B. globigii
Duplicate
Once per
experiment
<20% variation
Consider other
dilutions. Reanalyze.
B. globigii
Field blank (an
open bottle of
sterile water in the
vicinity of the
BWS location)
Every 5 BWS
0 CFU/plate
Determine if
background values
impact results.
HPC
Negative Control
Before every set
of measurements
No fluorescent
wells
Re-analyze sterile
buffer and change it if
necessary.
HPC
Positive Control
Once per
experiment
Fluorescent wells
Investigate laboratory
technique. Re-analyze.
HPC
Duplicate
Once per
experiment
Duplicate plates
much agree within
5%
Investigate laboratory
technique. Re-analyze.
Chlorine Dioxide/
Free Chlorine
Manufacturer DPD
color standards kit
Once per
experiment
As specified by the
color standards kit
Clean the colorimeter
measuring cell. Clean
the DPD standards vials
and recheck.
BWS, bulk water sample; CFU, Colony Forming Unit; HPC, heterotrophic plate count
Table A9. Quality Assurance/Quality Control (QC) Checks for Online Ec
Instrument/
Measurement
Calibration/QC
Alternative
Frequency
Acceptance
Criteria
Corrective Action
RealTech UV254/
TOC
Custom Zero using
deionized water
One time per
quarter according to
instrument O/M
manual
N/A
Clean the quartz
windows using 5%
bleach solution.
HachCL-17/
Free Chlorine
Factory calibration
- do not change.
Perform a one-point
check against a
DPD colorimetric
method calibration
based on DPD
method
Quarterly
±10%
Clean colorimeter
and check the
instrument flow.
uipment
TOC, total organic carbon; UV, ultraviolet; N/A, not applicable
DA TA ANAL YS/S, INTERPRET A TION, AND MANAGEMENT
Data Reporting Requirements
All data generated during the study will be presented in tabular/spreadsheet format. Table A10
identifies the reporting units for the various parameters.
Table A10. Reporting Units for Measurements
Measurement
Units
44
-------
Bacillus globigii
CFU/ mL
HPC (heterotrophic plate count)
MPN/ mL
Chlorine Dioxide
mg/L
Free Chlorine
mg/L
TOC (total organic carbon)
mg/L
CFU, colony forming unit; MPN, most probable number
Data Validation Procedures
Calculations will be carried out on a computer and will be checked initially by the analyst for
gross error and miscalculation. The calculations and data entered into computer spreadsheets
will be checked by a peer reviewer for accuracy, and checking the calculation by hand or
checking entries of data from the original. Detected errors will be corrected and other data in the
same set investigated before it is released to the EPA work assignment contract officer's
representative (WACOR).
Data Summary
All sample data will be presented by CB&I in tabular/spreadsheet format and submitted to the
EPA WACOR for evaluation. Tabular data summaries will be included in the main discussion of
the reports and raw data will be included as appendices.
Data Storage
Laboratory records will be maintained in accordance with Section 13.2, Paper Laboratory
Records, of the Office of Research and Development (ORD) Policies and Procedures Manual.
Controlled access facilities that provide a suitable environment to minimize deterioration,
tampering, damage, and loss will be used for the storage of records. Whenever possible,
electronic records will be maintained on a secure network server that is backed up on a routine
basis. Electronic records that are not maintained on a secure network server will be periodically
backed up to a secure second source storage media, transferred to an archive media (e.g.,
compact discs, optical discs, magnetic tape, or equivalent), or printed. Electronic records that are
to be transferred for retention will be transferred to an archive media or printed, as directed by
EPA.
45
-------
Appendix B: T&E SOP 304, Heterotrophic Plate Count (HPC) Analysis Using
IDEXX SimPlate® Method.
46
-------
,
SKawr
Shaw Environmental & Infrastructure, Inc.
EPA T&E Contract Technical
Standard Operating Procedure
Heterotrophic Plate Count (HPC) Analysis Using IDEXX SimPlate Method
T&E SOP 304
Revision Number: 1
Revision Date: 02/08/2012
-------
SOP 304, Heterotrophic Plate Count Analysts
Revision Number: 1
Date: 02/08/2012
Page 2 of 10
SOP Approval
E. Raciha Krishnan, P.E,
Program Manager
l •• y'A-'-s ] v ^ ^ fr.v h'\' } ^!J
Signature ~ " Date
Steven Jones, ASQ CQA/CQE
Quality Assurance Manager
Signature
Date
-------
SOP 304, Heterotrophic Plate Count Analysis
Revision Number: 1
Date: 02/08/2012
Page 3 of 10
Revision Summary
Revision
Name
Date
Description of Change
0
Nur Muhammad
01/31/2006
Developed SOP.
1
Nancy Shaw/
Steven Jones
01/25/2012
Revised Sections 1,2,4, 6, 7, 8,
9.2, 10 and 12. Added
Attachments A and B.
-------
SOP 304, Heterotrophic Plate Count Analysis
Revision Number: 1
Date: 02/08/2012
Page 4 of 10
TABLE OF CONTENTS
SECTION
SECTION TITLE
PAGE
NUMBER
NUMBER
1.0
Scope and Applicability
5
2.0
Summary of Method
5
3.0
Definitions
5
4.0
Health and Safety Warnings
5
5.0
Cautions
5
6.0
Interferences
5
7.0
Personnel Qualifications
5
8.0
Equipment and Supplies
6
9.0
Procedures
6
9.1 Sample Collection, Handling, and Analysis
9.2 Media Preparation and Sample Analysis
10.0 Data and Records Management
11.0 Quality Control and Quality Assurance
12.0 References
Attachment A MPN Tables
Attachment B Datasheet for Heterotrophic Plate Count Analysis
6
6
7
7
7
8
10
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SOP 304, Heterotrophic Plate Count Analysis
Revision Number: 1
Date: 02/08/2012
Page 5 of 10
1.0 Scope and Applicability
The method described in this standard operating procedure (SOP) is applicable to the enumeration
of heterotrophic bacteria, generally known as heterotrophic plate counts (HPC), in water and
wastewater samples.
2.0 Summary of Method
IDEXX SimPlate method for quantification of HPC is based on multiple enzyme technology which
detects viable bacteria in water by testing for the presence of key enzymes known to be present in
these organisms. It uses multiple enzyme substrates that produce a blue fluorescence when
metabolized by bacteria. The sample and media are added to a SimPlate plate, incubated and then
examined for fluorescing wells. The number of fluorescing wells corresponds to a Most Probable
Number (MPN) of total bacteria in the original sample.
3.0 Definitions
3.1 HPC - Heterotrophic Plate Count
3.2 IDEXX - Biological system and reagent developing company.
3.3 SimPlate - Registered trademark of BioControl Systems Inc., and is used by IDEXX under
license from BioControl System Inc.
4.0 Health and Safety Warnings
4.1 Standard laboratory personal protective equipment (i.e., laboratory coat, gloves, and safety
glasses) is required. In addition, any chemical-specific or project-specific protective gear
required will be described in the project-specific Health and Safety Plan (HASP).
4.2 If using an ultraviolet (UV) light system without a viewing chamber, wear UV protective safety
glasses and direct light away from eyes.
4.3 Special precautions, such as wearing heat-resistant gloves, are required for autoclaving.
5.0 Cautions
Samples collected for analysis in accordance with this Standard Operating Procedure (SOP) shall
be preserved at 4±2 °C after collection and processed preferably within 48 hours after sample
collection.
6.0 Interferences
6.1 Contamination during analysis affects the results. Aseptic technique should be followed
during analysis.
6.2 Chlorinated samples should be treated with sodium thiosulfate prior to testing.
7.0 Personnel Qualifications
The techniques of a first time analyst shall be reviewed by an experienced analyst prior to initiating this
SOP alone. During this review, the new analysts will be expected to demonstrate their capability to
perform this analysis.
-------
SOP 304, Heterotrophic Plate Count Analysis
Revision Number: 1
Date: 02/08/2012
Page 6 of 10
8.0 Equipment and Supplies
8.1 IDEXX multi dose sterile media
8.2 IDEXX sterile SimPlate plates with lids
8.3 10 ml sterile disposable pipettes
8.4 Sterile dilution buffer (90 ml vials) from Hardy Diagnostics (www.hardvdiaqnostics.com: Cat #
D690)
8.5 UV light set (6 watt, 365 nm) with viewing chamber
8.6 Incubator capable of maintaining a temperature of 35±0.5 °C
8.7 SimPlate® For HPC Most Probable Number (MPN) Table (supplied with the IDEXX media
and plates)
8.8 100 ml sampling bottles with sodium thiosulfate (0.01% w/v) (Fisher Scientific, Cat..No. 09
730 91)
8.9 Autoclave capable of sterilizing with fast, liquid, and dry cycles
9.0 Procedure
9.1 Sample Collection, Handling, and Analysis
9.1.1 Use 100 ml sampling bottles containing sodium thiosulfate for sample collection.
9.1.2 Samples should be transported to the laboratory immediately and stored at 4±2 °C
until processed.
9.1.3 Samples should be processed within 48 hours of sample collection.
9.2 Media Preparation and Sample Analysis
9.2.1 Open the IDEXX multi dose media vessel and add 100 ml sterile dilution buffer. Re-
cap the vessel and shake to dissolve the media properly.
9.2.2 Prepare serial dilutions of the sample if necessary.
9.2.3 Pipette 1 ml sample and then 9 ml of the re-hydrated IDEXX multi dose media onto
the center of an IDEXX SimPlate plate base.
9.2.4 Cover the SimPlate plate with lid and gently swirl to distribute the sample into all the
wells.
9.2.5 Tip the plate 90 - 120° to drain excess sample into the absorbent pad.
9.2.6 Invert the plate, and incubate for 45 - 72 hours at 35±0.5 °C.
9.2.7 Remove cover and put the plate in the UV system viewing chamber. Turn the UV
light (Section 8.5) on 5 inches above the plate, and count the number of fluorescent
wells.
-------
SOP 304, Heterotrophic Plate Count Analysis
Revision Number: 1
Date: 02/08/2012
Page 7 of 10
9.2.8 Refer to the SimPlate® For HPC Most Probable Number (MPN) Table (see
Attachment A) to determine the MPN of heterotrophic plate count bacteria in the
original sample. Report the MPN to reflect the dilution used. For example, if 1 mL of
a 1:10 dilution of the original sample was tested, then the reported MPN is the table
number multiplied by 10 and the result is reported as MPN per 10 mL.
9.2.9 Record the analysis date, dilutions, number of fluorescence wells and heterotrophic
bacterial counts on Attachment B, Datasheet for Heterotrophic Plate Count Analysis.
9.2.10 Autoclave the plates to sterilize, and dispose of the plates.
9.2.11 Refrigerate any unused rehydrated media and discard after 5 days if not used.
9.2.12 Store dehydrated media in the dark at room temperature.
10.0 Data and Records Management
10.1 All original analysis documentation generated and prepared for the U.S. Environmental
Protection Agency (EPA) shall be controlled in accordance with Shaw T&E SOP 101, Central
Files.
10.2 All data packages shall be assembled and reviewed per Shaw T&E SOP 102, Data Review
and Verification.
11.0 Quality Control and Quality Assurance
11.1 Negative Control - test a negative control following the test procedure using 10 ml re-
hydrated media before every set of measurements. No wells should fluorescence after
incubation. In case of failure, use a new media vessel and dilution buffer.
11.2 Positive Control - test a positive control following the test procedure using 10 mL
dechlorinated tap water to rehydrate the media. An acceptable positive control should yield
10-30 fluorescent wells (21 - 74 MPN) or more. To dechlorinate, add tap water to 100 ml
sampling bottle containing sodium thiosulfate (Section 8.8).
11.3 Duplicate - for verification purposes, perform tests in duplicate per sample dilution and for
each positive control. Counts from duplicate plates must agree within 5%.
12.0 References
12.1 IDEXX. Instructional Manual for SimPlate for HPC Multi Dose, Maine, USA.
12.2 Shaw Environmental & Infrastructure, Inc., 2011. EPA T&E Contract Administrative SOP
101, Central Files.
12.3 Shaw Environmental & Infrastructure, Inc., 2011. EPA T&E Contract Administrative SOP
102, Data Review and Verification.
12.4
Standard Methods for the Examination of Water and Wastewater, 20th edition, 1998. Method
9215 A, Heterotrophic Plate Count. American Public Health Association.
-------
SOP 304, Heterotrophic Plate Count Analysis
Revision Number 1
Date: 02/08/2012
Page 8 of 10
Attachment A - MPN Tables (Page 1 of 2)
Unit-Dose
SimPlate® For HPC
Most Probable Number (MPN) Table
# Positive
MPN
95% confidence limits
Wells
lower
upper
0
<0.2
<0.03
<1.4
1
0.2
0.03
1.4
2
0.4
0.1
1.6
3
0.6
0.2
1.9
4
0.8
0.3
2.2
5
1.0
0.4
2.5
6
1.2
0.6
2.7
7
1.5
0.7
3
8
1.7
0.8
3.3
9
1.9
1
3.6
10
2.1
1.1
3.9
11
2.3
1.3
4.2
12
2.6
1.5
4.5
13
2.8
1.6
4.8
14
3.0
1.8
5.1
15
3.3
2.0
5.4
16
3.5
2.2
5.8
17
3.8
2.3
6.1
18
4.0
2.5
6.4
19
4.3
2.7
6.7
20
4.5
2.9
7
21
4.8
3.1
7.4
22
5.1
3.3
7.7
23
5.3
3.5
8.0
24
5.6
3.8
8.4
25
5.9
4
8.7
26
6.2
4.2
9.1
27
6.5
4.4
9.4
28
6.8
4.7
9.8
29
7.1
4.9
10.2
30
7.4
5.1
10.6
31
7.7
5.4
10.9
32
8.0
5.6
11.3
33
8.3
5.9
11.7
34
8.6
6.2
12.1
35
9.0
6.4
12.6
36
9.3
6.7
13.0
37
9.7
7
13.4
38
10.0
7.3
13.9
39
10.4
7.6
14.3
40
10.8
7.9
14.8
41
11.2
8.2
15.2
42
11.6
8.5
15.7
# Positive
MPN
95% confidence limits
Wells
lower
upper
43
12.0
8.8
16.2
44
12.4
9.1
16.7
45
12.8
9.5
17.3
46
13.2
9.8
17.8
47
13.7
10.2
18.3
48
14.1
10.6
18.9
49
14.6
10.9
19.5
50
15.1
11.3
20.1
51
15.6
11.7
20.7
52
16.1
12.1
21.3
53
16.6
12.5
22.0
54
17.1
13.0
22.7
55
17.7
13.4
23.4
56
18.3
13.9
24.1
57
18.9
14.4
24.9
58
19.5
14.9
25.7
59
20.2
15.4
26.5
60
20.9
15.9
27.3
61
21.6
16.5
28.2
62
22.3
17.1
29.2
63
23.1
17.7
30.2
64
23.9
18.3
31.2
65
24.8
19.0
32.3
66
25.7
19.7
33.5
67
26.6
20.4
34.7
68
27.6
21.2
36.1
69
28.7
22.0
37.5
70
29.9
22.9
39.0
71
31.1
23.8
40.7
72
32.4
24.8
42.5
73
33.9
25.8
44.4
74
35.5
27.0
46.6
75
37.2
28.2
49.1
76
39.2
29.6
51.9
77
41.4
31.1
55.1
78
44.0
32.8
58.9
79
47.0
34.8
63.6
80
50.7
37.1
69.5
81
55.5
39.8
77.5
82
62.3
43.2
89.9
83
73.8
47.6
114.6
84
>73.8
>47.6
>114.6
MPN is per ml of sample (pour-off is accounted for).
-------
SOP 304, Heterotrophic Plate Count Analysis
Revision Number 1
Date: 02/08/2012
Page 9 of 10
Attachment A — MPN Tables (Page 2 of 2)
Multi-Dose
SimPlate® For HPC
Most Probable Number (MPN) Table
# Positive
MPN
95% confidence limits
Wells
lower
upper
0
<2
<0.3
<14
1
2
0.3
14
2
4
1
16
3
6
2
19
4
8
3
22
5
10
4
25
6
12
6
27
7
15
7
30
8
17
8
33
9
19
10
36
10
21
11
39
11
23
13
42
12
26
15
45
13
28
16
48
14
30
18
51
15
33
20
54
16
35
22
58
17
38
23
61
18
40
25
64
19
43
27
67
20
45
29
70
21
48
31
74
22
51
33
77
23
53
35
80
24
56
38
84
25
59
40
87
26
62
42
91
27
65
44
94
28
68
47
98
29
71
49
102
30
74
51
106
31
77
54
109
32
80
56
113
33
83
59
117
34
86
62
121
35
90
64
126
36
93
67
130
37
97
70
134
38
100
73
139
39
104
76
143
40
108
79
148
41
112
82
152
42
116
85
157
# Positive
AWN
95% confidence limits
Wells
lower
upper
43
120
88
162
44
124
91
167
45
128
95
173
46
132
98
178
47
137
102
183
48
141
106
189
49
146
109
195
50
151
113
201
51
156
117
207
52
161
121
213
53
166
125
220
54
171
130
227
55
177
134
234
56
183
139
241
57
189
144
249
58
195
149
257
59
202
154
265
60
209
159
273
61
216
165
282
62
223
171
292
63
231
177
302
64
239
183
312
65
248
190
323
66
257
197
335
67
266
204
347
68
276
212
361
69
287
220
375
70
299
229
390
71
311
238
407
72
324
248
425
73
339
258
444
74
355
270
466
75
372
282
491
76
392
296
519
77
414
311
551
78
440
328
589
79
470
348
636
80
507
371
695
81
555
398
775
82
623
432
899
83
738
476
1146
84
>738
>476
>1146
MPN is per ml of sample (pour-off is accounted for).
-------
SOP 304, Heterotrophic Plate Count Analysis
Revision Number: 1
Date: 02/08/2012
Page 10 of 10
Attachment B - Datasheet for Heterotrophic Plate Count Analysis
Analysis Date: Work Assignment:
Sterile Dilution Buffer (for negative control) Lot #: Exp. Date:
Sodium Thiosulfate Bottle (for positive control) Lot #: Exp. Date:
Sample ID
Dilution
Factor
# of
Fluorescent
Wells
Heterotrophic
Bacteria
(MPN/mL)
Heterotrophic
Bacteria x
dilution factor
(MPN/mL)
Quality Control
Negative control buffer analyzed? Q Yes Q No
Negative control results acceptable (no yellow or fluorescent wells)? Q Yes [Q No
Positive control results acceptable (5 - 30 fluorescent wells)? Q Yes Q No
Comments:
Analyst: Date:
Reviewed by:
Date:
-------
Appendix C: T&E SOP 309, Preparation and Enumeration of Bacillus globigii
Endospores.
47
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Shaw Environmental & Infrastructure, Inc.
EPA T&E Contract Technical
Standard Operating Procedure
Preparation and Enumeration of B. globigii Endospores
T&E SOP 309
Revision Number: 2
Revision Date: 11/12/2012
-------
SOP 309, B. globigii Endospores
Revision Number: 2
Date: 11/12/2012
Page 2 of 11
SOP Approval
K. Radha Krishuan, P.1C.
Program Manager
\teib, k
Si<>na(uiv
'I 2 /2-& I 2
Date
Steven Jones, ASQ CQA/CQE
Quality Assurance Manager
Signature Date
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SOP 309, B. globigii Endospores
Revision Number: 2
Date: 11/12/2012
Page 3 of 11
Revision Summary
Revision
Name
Date
Description of Change
0
Nur Muhammad
03/24/2010
Developed SOP.
1
Nancy Shaw /
Steven Jones
07/27/2012
Revised Sections 4, 5, 6, 8, 9, 12 and
Attachment A to update SOP.
2
Lee Heckman/
Gune Silva
11/12/2012
Revised Attachment A datasheet to
incorporate columns for volume plated,
average plate count, and reported results to
provide additional information for peer review
of the data.
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SOP 309, B. globigii Endospores
Revision Number: 2
Date: 11/12/2012
Page 4 of 11
TABLE OF CONTENTS
SECTION
NUMBER
SECTION TITLE
PAGE
NUMBER
1.0
Scope and Applicability
5
2.0
Summary of Method
5
3.0
Definitions
5
4.0
Health and Safety Warnings
5
5.0
Cautions
5
6.0
Interferences
6
7.0
Personnel Qualifications
6
8.0
Equipment and Supplies
6
9.0
Procedure
7
9.1
Preparation of Generic Sporulation Media
7
9.2
Preparation of Nutrient Agar
7
9.3
Controls
8
9.4
Preparation of Endospores
8
9.5
Purification of Endospores
8
9.6
Enumeration of Stock/Sample Concentration
9
10.0
Data and Records Management
10
11.0
Quality Control and Quality Assurance
10
12.0
References
10
Attachment A Datasheet for Bacillus globigii Endospores 11
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SOP 309, B. globigii Endospores
Revision Number: 2
Date: 11/12/2012
Page 5 of 11
1.0 Scope and Applicability
The method described in this standard operating procedure is applicable to the preparation and
purification of Bacillus globigii (B. globigii) endospores for use in research studies. This method is
also applicable for the enumeration of stock and sample concentrations of B. globigii.
2.0 Summary of Method
B. globigii is an aerobic spore-forming bacteria used as a surrogate for B. anthracis. For the
preparation of B. globigii stock, a culture of vegetative cells (stock) is mixed with generic sporulation
media and incubated by gentle shaking (~150 rpm) at 35 C for five days. The presence of spores is
confirmed using phase-contrast microscopy (<0.1% vegetative cells). The spores are purified using
gradient separation (Section 9.5) and preserved in 40% ethanol in a refrigerator at 4 ± 2 C until use.
The B. globigii stock is heat-shocked and analyzed using standard membrane filtration for
determining the stock concentration.
3.0 Definitions
3.1 Biosafety Levels - The degree of containment (or the combinations of standard and special
practices, safety equipment, and facility design criteria) appropriate for the operations
performed and the biohazardous agents used within the laboratory.
3.2 CFU - Colony Forming Units
3.3 Negative Control - A sample that does not contain the desired analyte. It ensures that a test,
its components, or the environment do not cause undesired effects, or produce incorrect test
results.
3.4 Positive Control - A sample that contains a known concentration of the desired analyte. It
ensures that a test and/or its components are working properly and producing expected
results.
4.0 Health and Safety Warnings
4.1 Standard laboratory personal protective equipment (i.e., laboratory coat, gloves, safety
glasses) is required. In addition, any project-specific protective gear is required as described
in the project-specific Health and Safety Plan.
4.2 B. globigii are saprophytic organisms, whose principal habitat is soil. They do not pose a
public health risk. Standard Biosafety Level 1 laboratory precautions apply for its
microbiological analysis.
4.3 Special precautions, e.g., wearing heat-resistant gloves, are required for autoclaving the
accessories.
5.0 Cautions
Spores prepared in accordance with this SOP require preservation in a refrigerator at 4±2°C
immediately after preparation.
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SOP 309, B. globigii Endospores
Revision Number: 2
Date: 11/12/2012
Page 6 of 11
6.0 Interferences
Contamination interferes with accurate and precise enumeration and measurement. Preventive
measures guarding against contamination include maintaining sterility throughout the processing
procedure by using aseptic techniques and sterile containers when processing samples.
In addition, the stock concentration may decline by germination if kept at room temperature.
Declining concentration is prevented by transferring the stock to a refrigerator at 4±2o0C
immediately after use.
7.0 Personnel Qualifications
7.1 The techniques of a first-time scientist/ technician shall be reviewed by a senior scientist/
technician prior to initiating this SOP alone.
8.0 Equipment and Supplies
8.1 Non-consumable Equipment
8.1.1 Sterile sampling bottles (100 mL) with Sodium Thiosulfate (Corning Inc.)
8.1.2 Sterile Erlenmeyer flasks (250 mL) with screw-caps
8.1.3 Thermostatically controlled water bath with a 250 mL pilot flask and thermometer
capable of registering temperature with a range of 37 to 90°C
8.1.4 Incubator (35 - 37°C)
8.1.5 Autoclave
8.1.6 UV Sterilizer
8.1.7 Ice bath
8.1.8 Hotplate
8.1.9 Colony counter
8.1.10 Tweezers
8.1.11 1-L glass bottles/flask
8.1.12 Fixed-angle rotor centrifuge capable of operating at 5,860 rcf
8.1.13 Swinging-bucket rotor centrifuge capable of operating at 5,860 rcf
8.1.14 50 mL Centrifuge tube with conical bottom
8.1.15 Phase Contrast Microscope
8.2 Consumable Equipment
8.2.1 Individually packed sterile plastic pipettes
8.2.2 60 x 15 mm Petri dishes with loose lids
8.2.3 47 mm, 0.45 jjm porosity sterile membranes
8.2.4 Membrane filtration apparatus
8.2.5 Membrane filters with 47 mm diameter and a 0.45 micron pore size
8.3 Consumable Reagents
8.3.1 Sterile dilution phosphate buffer (90 mL bottles)
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SOP 309, B. globigii Endospores
Revision Number: 2
Date: 11/12/2012
Page 7 of 11
8.3.2
8.3.3
8.3.4
8.3.5
8.3.6
8.3.7
8.3.8
8.3.9
8.3.10
8.3.11
8.3.12
8.3.13
Procedure
9.1 Preparation of Generic Sporulation Media
9.1.1 Measure 8 g nutrient broth, 40 mg manganese sulfate, and 100 mg calcium chloride
into a 1 L glass bottle or flask, and add 1 L distilled water. Stir the contents until
dissolved.
9.1.2 Aliquot 100 mL into ten 250 mL flasks.
9.1.3 Sterilize in the autoclave for 15 minutes at 15 psi and 121°C.
9.1.4 Store the media at room temperature.
9.2 Preparation of Nutrient Agar
9.2.1 Measure and pour 5 grams (g) peptone, 3 g beef extract, 15 g agar and 0.015 g
Trypan blue dye in a 1-liter (L) glass bottle and add 1-L distilled water. Heat to
boiling using a hot plate and a stir bar for complete mixing.
9.2.2 Sterilize in the autoclave for 15 minutes at 15 psi and 121°C.
9.2.3 After cooling to 25°C, pour 5 to 6 mL of agar media in each Petri dish. Leave the
plates covered at room temperature until solidified. Store the plates in a refrigerator
at 4±2 °C.
9.2.4 Bring the required number of plates out of the refrigerator and warm them to room
temperature for one hour before use.
9.2.5 If any type of growth other than the desired culture is observed, the plates are
considered to be contaminated. If contamination is found, discard the plates and
prepare new ones.
Peptone
Beef extract
Trypan blue dye
Agar
Nutrient broth
Manganese sulfate
Calcium chloride
Deionized water
B. globigii vegetative cells/stock
Either Enterococcus faecium (ATCC 35667), Escherichia Coii (ATCC13706) or
Pseudomonas aeroginosa (ATCC27853)
Ethanol (95%)
Hypaque solution
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SOP 309, B. globigii Endospores
Revision Number: 2
Date: 11/12/2012
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9.3 Controls
9.3.1 Positive control is prepared by spiking a known concentration of B. globigii into sterile
water followed by membrane filtration and incubation at 35 - 37 C for 24 hours.
Formation of colonies at the appropriate concentration is the acceptance criteria for a
B. globigii positive control. If the acceptance criteria are not obtained, the result of
the test is used as the true starting count of the stock and future positive controls are
spiked with known concentrations based on this starting count. If the positive control
is negative or contaminated with vegetative cells, the entire test (controls plus
unknown samples) are repeated with new plates and controls.
9.3.2 Negative control for heat-shock is obtained by spiking one of the following into sterile
water: Enterococcus faecium (ATCC 35667), Escherichia Coli (ATCC13706) or
Pseudomonas aeroginosa (ATCC27853). The spiked sample is then heat-shocked
and subjected to membrane filtration and incubation at 35 to 37 C for 24 hours.
Formation of colonies indicates inadequate heat shock for killing the vegetative cells.
The temperature of the heat shock water bath and the sterility capabilities of the
autoclave will be monitored and the entire test (controls plus unknowns) will be
repeated if the negative control produced growth (is positive).
9.3.3 Negative control for buffer is obtained by filtering 100 mL of sterile buffer and
incubating the membrane at 37 C for 24 hours followed by membrane filtration and
incubation at 35 to 37 C for 24 hours. Formation of colonies indicates contaminated
dilution buffer and the entire test (controls plus unknowns) will be repeated using
fresh buffer of a different lot.
9.4 Preparation of Endospores
9.4.1 Inoculate a flask of generic sporulation media (100 mL) with 1 mL of B. globigii
vegetative cells/stock.
9.4.2 Incubate with continuous gentle shaking (~ 150 rpm) at 35 C for at least five days
(120 hours).
9.4.3 Check solution for the presence of spores with a wet mount slide preparation using
phase contrast microscopy.
9.4.4 When the slide preparation reveals an adequate spore suspension (approximately 1
x 109 cfu/mL), proceed with the purification.
9.5 Purification of Endospores
9.5.1 Aseptically transfer the contents of each flask into sterile 35 mL centrifuge tubes.
Balance the tubes and centrifuge at approximately 5,860 relative centrifugal force
(rcf) for 20 minutes, using a fixed-angle rotor.
9.5.2 Pour off the supernatant into a discard beaker. Add 30 mL of cold, sterile deionized
water to each tube. Vortex each tube until the spores are completely resuspended in
the water. Centrifuge again at approximately 5,860 rcf for 10 minutes. Discard the
supernatant and resuspend in 30 mL of cold, sterile deionized water per tube.
Centrifuge for another 10 minutes as before and discard the supernatant. Autoclave
the contents of the discarded supernatant and discard appropriately.
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SOP 309, B. globigii Endospores
Revision Number: 2
Date: 11/12/2012
Page 9 of 11
9.5.3 Combine the contents of the tubes into one tube or into multiples that will allow ease
of centrifugation. Aseptically add 30mL of cold, sterile deionized water to each tube
and resuspend the spores.
9.5.4 Combine 58 mL of Hypaque solution with 42 mL of sterile, deionized water. Mix well.
Add 12 mL of the Hypaque solution to clean, sterile 35 mL centrifuge tubes. Pipette
the spore suspension, carefully layering it on top of the Hypaque solution. Centrifuge
at approximately 5,860 rcf for 30 minutes using a swinging bucket rotor.
9.5.5 Pour off and discard the supernatant. Add 30 mL of cold, sterile deionized water to
the pellet in each tube and resuspend the spores. Centrifuge at 5,860 rcf for 15
minutes using a fixed-angle rotor.
9.5.6 Discard the supernatant and resuspend the spores in 30 mL of cold, sterile deionized
water. Wash the spores by centrifuging at 5,860 rcf and resuspending twice more.
Centrifuge again, discard the supernatant, and resuspend the spores in a 40% (v/v)
ethanol solution. Store in a refrigerator at 4±2 C.
Enumeration of Stock/Sample Concentration
9.6.1 Prepare a 90 C water bath using a 50:50 mixture of deionized water and tap water.
Add enough water to the bath so that when the samples are immersed, the water
level meets the sample volume level.
9.6.2 Prepare serial dilutions of the stock/samples using sterile buffer and select at least
two dilutions for analysis. Shake the diluted stocks/samples vigorously 25 times and
transfer to appropriately labeled 250 mL Erlenmeyer flasks.
9.6.3 Place the samples and the pilot flask (containing distilled water) into the 90 C shaker
water bath. Shake the samples at 60 to 80 rpm. Monitor the temperature in the pilot
flask until it is 80 C.
9.6.4 Set a timer and incubate the samples and pilot flask for 10 minutes after the water
reaches 80 C.
9.6.5 Quickly remove the samples and pilot flask from the water bath and place them into a
slurry of ice and water to reduce temperature to approximately room temperature as
soon as possible.
9.6.6 Use the Membrane Filtration apparatus and filter the samples according to the
following procedure:
9.6.6.1 Turn on the Millipore vacuum system.
9.6.6.2 Wet a membrane support if necessary with 5 mL of sterile phosphate buffer.
9.6.6.3 Dip a pair of metal tweezers into methanol and flame them until the
methanol is consumed. Using the sterilized tweezers, lay a membrane on
to the support, avoiding any contamination by refraining from touching the
membrane to any other surface.
9.6.6.4
Rinse the funnel with 10 mL sterile buffer and drain it.
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SOP 309, B. globigii Endospores
Revision Number: 2
Date: 11/12/2012
Page 10 of 11
9.6.6.5 Add 10 mL of sterile buffer to the funnel. Dispense 10 mL of sample with a
sterile disposable pipette and filter.
9.6.6.6 Rinse and drain the funnel twice using 10 mL aliquots of sterile phosphate
buffer.
9.6.6.7 Unscrew the funnel and place it in the UV sterilizer.
9.6.6.8 Carefully remove the membrane from the support, avoiding contamination,
and place it on a labeled nutrient agar Petri plate.
9.6.6.9 Place the membrane supports and tweezers in the UV sterilizer.
9.6.6.10 Sterilize the whole assembly for at least five minutes.
9.6.6.11 Conduct duplicate analysis for each dilution of the stock/samples.
9.6.7 Incubate plates at 35 to 37 C for 24 hours.
9.6.8 The aim is to have 10 to 80 colonies per plate; adjust the dilution of the samples if
necessary.
9.6.9 Count every colony as a spore forming organism using a colony counter, and report
results of the analysis on Attachment A - Datasheet for Bacillus globigii Endospores.
10.0 Data and Records Management
All original laboratory data records shall be maintained in accordance with T&E SOP 101, Central
Files.
11.0 Quality Control and Quality Assurance
11.1 It is necessary to carry out the positive and negative control tests prior to every set of
analysis. If the positive control fails, change the stock organisms and use a new set of media
plates. If the negative control fails, re-run the test, heating the sample for a longer period. In
the case of negative control for buffer failure, use a new lot.
11.2 Duplicate analysis is required for each sample. A 20% variation is acceptable for duplicate
samples. However, in case of failure of a duplicate sample, consider the other dilutions for
enumeration of bacteria. If duplicates for all dilutions fail, discard the sample and re-run the
experiment.
11.3 If the number of colonies counted is less than 30, filter a greater volume of sample; if the
number of colonies count is greater than 80, further dilute the sample.
12.0 References
12.1 Shaw Environmental & Infrastructure, Inc. (2011). EPA Test and Evaluation Contract
Administrative SOP 101: Central Files.
12.2 Shaw Environmental & Infrastructure, Inc. (2012). EPA Test and Evaluation Contract
Technical SOP 301: Enumeration of Bacillus Subtilis in Water Samples.
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SOP 309, B. globigii Endospores
Revision Number: 2
Date: 11/12/2012
Page 11 of 11
ATTACHMENT A - DATASHEET FOR BACILLUS GLOBIGII ENDOSPORES
Analysis Date: Work Assignment:
Stock Concentration: Stock ID:
Dilution Buffer Lot No.: Expiration Date:
Sample ID
Volume
Plated
Colony Counts
Average Plate
Count
Dilution
Factor
B. globigii
(CFU / mL)
Plate
#1
Plate
#2
Control Samples:
Positive control acceptable (colonies observed)?
~
Yes
~
No
Negative control for heat shock acceptable (no growth observed)?
~
Yes
~
No
Negative control buffer acceptable (no growth observed)?
~
Yes
~
No
All duplicate results within 20% RPD?
~
Yes
~
No
Comments:
Analyst:
Date:
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Appendix D: Operation of the Water Sample Concentrator
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SOP No:
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NHSRC 030
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Jan 21, 2009
Introduction:
There is a need for detection of biological contaminants that could be used as weapons of
terrorism against the nation's water supplies. Ultrafiltration is a method of concentrating a large
volume of water to detect microorganisms. This procedure outlines the basic Ultrafiltration
process. Changes have been made and are to be made to this process as research has been
progressing.
Materials:
Appropriate PPE
1. Disposable lab gown
2. N95 Particulate respirator
3. Disposable nitrile gloves
4. Safety glasses
Reagents
1. 2.5 % Bovine Serum Albumin - SOP NHSRC 001
2. Diluent - SOP NHSRC 002
3. Backwash - SOP NHSRC 003
4. Filter Block - SOP NHSRC 004
5. 10 % Thiosulfate - SOP NHSRC 005
6. Eluting Solution (0.001 % Tween 80) - SOP NHSRC 006
7. 10 % Bleach - SOP NHSRC 007
8. 70% Ethanol - SOP NHSRC 028
1. Tube cutter (Cole Parmer catalog number EW-0638-10 orequivalent)
2. Sample carboy [For example, a 50-liter autoclavable polypropylene (pp) carboy with handles,
with pp leak proof screw cap, without tubulation, Fisher Scientific catalog number 02-960
20B, Nalgene Nunc International No. 2250-0130; however, depending on the specific logistics
or application, other carboys may be more appropriate.]
3. Filter, Rexeed 25S [Rexbrane Membrane High-Flux 2.5ml/2; manufactured by Asahi Kasei
Medical America Inc. Henry Schein Item #6292966]
4. Tubing (all tubing is listed with US Plastics 2009 productnumber):
a. Masterflex Tygon (R-3603 formulation) tubing, inside diameter outside diameter
V2\ USP# 57111
b. Masterflex Tygon (R-3603 formulation) tubing, inside diameter 3/8", outside diameter
5/8"; USP# 57117
c. Tygon Sanitary Silicone (3350 formulation) tubing, inside diameter outside
diameter 7/16"; USP# 57297
d. Tygon Sanitary Silicone (3350 formulation) tubing, inside diameter Vi",outside
Equipment
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diameter 3/8"; USP# 57296
e. Tygon Sanitary Silicone (3350 formulation) tubing, inside diameter 3/8", outside
diameter 5/8"; USP# 57302
5. Retentate vessel: Heavy-duty bottles, pp, 1-liter capacity, with pp leak proof screw caps (Cole
Parmer catalog number EW-06257-10 or equivalent)
6. Vented cap for retentate bottle: Filling/Venting cap, pp, size 53B (Cole Parmer catalog
number EW-06258-10 or equivalent).
7. PendoTECH PressureMAT Single-Use Sensor, Luer Fitting, Polycarbonate [used as
disposable water pressure transducer in the automator] Cole-Parmer 19406-32.
8. Reusable tubing connectors, hose clamps, and filtered vent; disposable cableties.
NOTE Alternative fitting combinations are possible. A functionalfitting combination is given
below. The fitting combination above represents the currently used fitting combination and seems
to function better than the one that follows.
a. High density polyethylene Tee fitting with barbed ends; 3/8" x 3/8" x 3/8". USP#
62065
b. Natural polypropylene reduction coupler 3/8"x Vi". USP# 64374
c. Hose clamps; SNP-10 and SNP-3
d. Check Valve with Vi" x 5/16" barb connectors by Smart Products (Part# 304305PS
0050S000-1402).
e. DIN Adapter 1/4" barb and luer [connects tubing to filter],
f. Polypropylene extra-flow coupling quick disconnects (inserts & bodies); USP# 60661
elbow insert with Vi" barb (3), 60653 body with %" barb (3), 60654 body with 3/8"
barb (1), 60657 strait insert with Vi" barb (1).
g. Whatman HEPA-Vent Filter; Fisher Scientific# 09-744-79
h. Polypropylene Tee connector with Vi" barbs and luer lock for pressure transducer.
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SOP No: MI SRC 030
Revision: 1
Date: Jan 21, 2009
Figure A: An aerial few of the tubing set up inside the automated concentrator.
Key to Figure A:
1. Silicone tubing with ID V* \ OD 7/16". Length varies according to sampling location.
2. Silicone tubing with ID Vi", OD 7/16". Section length: 8.5"
3. TygonR-3603 tubing with ID 3/8", OD 5/8". Section length: 17.25"
4. TygonR-3603 tubing with ID Va \ OD Iff'. Section length: 25" (wrapped into coil with
4.8-4.9" inner diameter.
5. TygonR-3603 tubing with ID W>: OD Vz\ Section length: 3.5"
6. Silicone tubing with ID OD 3/8". Section length 10.125"
7. Silicone tubing with ID Vi", OD 3/8". Section length9.25"
8. Silicone tubing with ID Vi\ OD 7/16". Section length 10.5"
9. Silicone tubing with ID 3/8", OD 5/8". Section length3.25"
10. Silicone tubing with ID W, OD 3/8". Section length 14"
11. Silicone tubing with ID 3/8", OD 5/8". Section length6.75"
12. Silicone tubing with ID Va \ OD 7/16". 3X Section lengths 1.5" each
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Jan 21, 2009
Protocol:
A. Set up of tubing andfilter apparatus within the automated concentrator; use Figure A photo as a
reference when setting up:
1. Attach tube 2 to check valve (at 5/16" barb) and to the Tee connector with 3/8" barbs.
2. Attach tube 3 to the opposite barb of the Tee connector with 3/8" barbs, as well as the reducer,
at the 3/8" barb. At the reducer, attach an SNP-10 hose clamp where the tubing connects just
past the barb.
3. Attach tube 8 to the final barb of the tee connector with 3/8" barbs; (setup thus far is in a
horizontal plane).
4. Wrap tube 4 into a loop (secure with cable ties), such that the inner diameter of the loop is
between 4.8"- 4.9", leaving one open end slightly longer than the other (see Figure A).
5. Attach the loop in a vertical/perpendicular plane with the rest of the previously attached
tubing, and attach to the Vi" barb of the reducer; secure tubing connection with a cable tie at
the barb.
6. Attach the polypropylene tee connector with the luer lock and Vi" barbs to the free end of the
loop, such that when the apparatus is in the concentrator, the luer is facing upward. Attach the
pressure transducer to the luer lock, and attach the transducer adapter to the control panel
connection cable. Secure the tubing connection with the barb using a cable tie.
7. On the filter, unscrew the small white cap attached to the red cap (bottom); attach DIN adapter
by screwing in the luer. Observe the Figure A for the orientation of the filter in the
concentrator, and attach tube 5 to the barb of the DIN adaptor such that the free end of the tube
will be able to connect to the remaining barb from step six. Attach cable ties to secure the
tubing at both connectors.
8. After making certain all solenoid valves are in the "open" position, place currently assembled
apparatus into the automated concentrator as shown in Figure A, and clamp the pump head
down on tube 3. Be sure tube 3 is correctly secured in the pump head.
9. Remove the small white cap on attached to the blue cap of the filter (top), and attach DIN
adapter.
10. Roll one end of tube six back over itself, and attach this end to the DIN adapter; use a SNP-3
hose clamp to secure the tubing connection to the barb. Attach a quick disconnect body
(female connection) with a Vi" barb to remaining end of the tube 6, and gently push tubing into
its solenoid valve. Check that the tubing is not pinched on either side of the solenoid, such
that water flow will not be obstructed.
11. Ensure that the vented 1L polypropylene bottle (retentate bottle) has a short length of silicone
tubing with a small tee connector attached to the inside right barb, and a piece of Teflon tubing
extending to nearly the bottom of the bottle on the inside left barb, with the inside of the down
pointing vent having nothing attached to it.
12. Gently place the retentate bottle on the load cell, and attach tube 12 (three separate pieces) to
each barb, and connect an quick disconnect elbow insert (male connector) on each tube.
13. Attach the quick disconnect body on tube 6 to the insert attached to the lower right barb (make
sure there is a "click" when they connect).
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14. Attach tube seven to another quick disconnect body with a 'A" barb, then attach a Whatman
HEPA Vent filter to the opposite end, and gently push to the tube into the corresponding
solenoid. Connect the quick disconnect body to the insert on the barb pointing towards the
15. Attach another quick disconnect body with a 'A" barb to tube 8, and attach to the remaining
quick disconnect insert on the top of thebottle.
16. On the filter, remove the large white cap on the upper side of the filter, and attach tube 9, with
a quick disconnect body with a 3/8" barb on the opposite end.
17. Role back the end of tube 10 on itself (as was done with tube 6), attach to a quick disconnect
strait insert with a Vi" barb, and then attach insert to the body on tube 9. Gently push tube 10
into the corresponding solenoid, and attach to the free end to the bottom of the flow meter.
18. Attach tube 11 to the top of the flow meter and to the quick disconnect leading to the effluent.
19. Push the end of tube 1 (influent line) into the automated concentrator and connect to the check
valve.
20. Double check all tubing connections.
* Additional set up associated with the influent will vary depending on method and purpose of
sampling. The length of the effluent line will also vary depending on sampling location.
B. Influent Options
Influent Option 1: Sampling from the distribution system or Seeding with BSL-1 organisms
For either sampling method, attach adisposable lOmL serological pipette (with cotton
removed, and fine point tip broken off), to the influent line. During the concentrator run, this line will
go directly into the sample reservoir.
Influent Option 2: Seeding a water sample with BSL-2 organisms using a syringe injection port from
within a biological safety cabinet.
For seeding water with a BSL-2 organism that may require a biological safety cabinet for
manipulation, run the influent line from the automated concentrator outside the BSC to a tee
connector with a luer lock (the same size and type used for the pressure transducer) that is secured
with a ring stand in the cabinet. Attach an additional length of the same tubing type used for the
influent line to the other end of the tee connector, and run the back out of the biological safety cabinet,
attach a 10 mL pipette (as described above), and use as needed.
Influent Option 3: Seeding a water sample with BSL-2 organisms a disposable cubitainer as an air gap
from within a biological safety cabinet.
For seeding water with a BSL-2 organism that may require a biological safety cabinet for
manipulation, attach a 10 mL pipette (as described above) to the influent line and run the influent line
from the automated concentrator outside the BSC to a cubitainer inside the BSC. Take an additional
length of tubing (length varies; use the same size used a tube 9 in the above setup), and place one end
filter.
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into the cubitainer. Place the tubing into an additional pump head, and attach a 25mL serological
pipette in the same fashion as the lOmL pipette described above. Place this new influent line into the
water reservoir.
C. Running the Automated Concentrator, using Influent Option 3:
AUTOMATED ULTRAFILTRATION CONCENTRATOR PROGRAM
Manual Pump Speed
SYSTEM STATUS
PR0CI55IN6 WATER
PAUSE
, : 7.25
Flapped Time (mill)
— I
Slop Volume (I)
0.900 -
0.800
II./INI
Manual
Stop
Reteritate
Level (L)
0.600 7
ij : uu
0.742
UCver2.0-082608
ABORT
0,300-
0.20U-
'I I I'M
FORWARD
Waste
Fk? wrote
[ml/min
Total System
Throughput (1}
0.924
Figure B: View of the Automated Concentrator monitoring
screen as seen during an ultrafiltration run.
1. Once the influent and effluent lines are attached and set up, remove the influent line with
the 10 mL pipette from the cubitainer and place it in 1L of prepared filter block solution.
2. Using the pump attached to the large influent line, pump water into the cubitainer tothe
desired level to create a reservoir and air gap, and stop thepump.
3. Turn on the automated concentrator using the switch on the above right of pump head. It
is imperative that this is done before opening the automated concentrator program.
4. On the computer, open the concentrator program.
5. When the program is pulled up, a prompt will ask if the retentate bottle is in place and
empty inside the concentrator. If it is, click yes to proceed; if it is not, click no to abort
the program and secure the bottle or empty it.
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SOP No:
Revision:
Date:
NHSRC 030
1
Jan 21, 2009
6. Click "Concentrate Water" to begin the ultrafiltration process. After this, the program
can be stopped completely at any point throughout the run by clicking the "Abort" button
on the monitoring screen.
7. Another series of prompts will ask if the influent line has been placed in 1L of filter block
solution, and to click yes to start concentration.
8. The automated concentrator will then draw up an appropriate about of block, andgo
through a three minute re-circulation of the block solution through the UF system.
9. After the re-circulation, the concentrator will draw down the level of water in the
retentate bottle and stop when it reaches its programmed level. At this time, remove the
lOmL pipette influent from the bottle of filter block solution and place it in thecubitainer.
A prompt from the program will tell the analyst to do this, and to click "OK" after it has
been done.
10. Once the "OK" button has been clicked, the concentrator will fill the retentate bottle to a
programmed level, and then begin the ultrafiltration of water. At this point, the program
can also be paused by clicking the "Pause" button at any point if need be. The program
monitoring screen will look like Figure B shown above.
11. At this point, monitor the cubitainer to keep a level of water as constant as possible by
increasing or decreasing the speed of the pump delivering water to the container. It is
imperative to constantly monitor the screen and the cubitainer because the speed and flow
rate of water in the automated concentrator can vary, depending on the water conditions.
Do not allow the cubitainer to over flow or run dry.
12. After allowing about 10L to pass through the system (the flow meter reading on the
concentrator screen will provide the analyst with this information), seed the organisms
being analyzed into the cubitainer using a sterile pipette.
13. Once the cubitainer has been seeded, the analyst should observe the screen throughout the
run, monitoring the water level in the retentate bottle and the pressure readings. If there is
a sudden large drop in pressure and water level in the retentate bottle, it could indicate a
leak, at which point the program should be aborted.
14. Once the volume of water desired has been processed through the system, the analyst will
click the "Manual Stop" button. At this point, the concentrator will stop drawing
influent, and allow the water level in the retentate bottle to draw down to a programmed
level.
15. A prompt will tell the analyst to place the influent line in 1L of elution solution. To do
this, place a bottle of elution solution inside the BSC, and place the influent line with the
lOmL pipette into the bottle. Click "OK" to start the elution procedure.
16. During the elution procedure, the concentrator will carry out four forward wash and draw
down steps, then carry out a 3 minute recirculation. After the recirculation, the
concentrator will fill the retentate bottle to programmed volume.
17. A final prompt will appear on the screen to allow the analyst to quit the program;
however that analyst should first record the volume of retentate in the bottle before
exiting the program.
18. At this point the program will close completely, and it will be safe to open the
concentrator and remove the retentate bottle by disconnecting the quick disconnects. The
analyst should wrap a 10% bleach wipe around the quick disconnects as they are being
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SOP No:
Revision:
Date:
NHSRC 030
1
Jan 21, 2009
disconnected to catch any contaminated water that may leak out. Place the retentate
bottle in a BSC.
19. Remove the quick disconnects from the retentate bottle along with the short lengths of
tubing (tube 12), and connect them to spare empty retentate bottle.
20. Place the spare bottle back in the concentrator, and reconnect the quick disconnects, and
disconnect the effluent quick disconnect.
D. Disinfection of the Automated Concentrator
1. After the spare bottle has been connected to the concentrator system, open the
concentrator program again. The same first prompt will come up; click yes.
2. Instead of entering the concentration mode, click "TestMode."
3. The analyst will few a similar screen to that seen during the concentration procedure,
however this screen will give the user freedom to manipulate the solenoid valves (green
dot signifies "open", red dot signifies "closed"), pump on/off, and pump speed.
4. Open all solenoid valves; place the influent pipette in a 1L bottle of 10% bleach
solution (made according to NHSRC SOP 007), and slowly manipulate the pump to
draw up bleach into the system and fill the retentate bottle to 700mL. Quickly close the
influent solenoid valve, and allow the bleach to re-circulate for 10 minutes. After the
recirculation, stop the pump, and exit the Test Mode to allow the bleach to hold in the
system for an additional 50 minutes.
5. Once the disinfection holding time is complete, reattach the effluent quick disconnect,
but remove it from the current effluent line and connect it to a new effluent line that
passes directly into a sink or drain. This step must be done so that bleach solution does
not pass through the flow meter gauge.
6. Reopen the Test Mode, open all solenoid valves, but close the influent solenoid. Turn
on the pump to remove the bleach from the system. When the tubing appears foamy or
mostly full of air, turn of the pump, exit the Test Mode, and close theprogram.
7. Switch off the automated concentrator. Disassembly of the tubing may now begin.
8. Dispose of the tubing and the UF filter. Disinfect all connectors in 10% bleach for an
additional half hour, then rinse with water, and allow to dry.
Refer to separate SOPs for sample processing.
10
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SOP No: NHSRC030
Revision: 1
Date: Jan 21, 2009
References:
U.S. Environmental Protection Agency April 13, 2006. Quality Assurance Project Plan for
Development of Sampling and Analytical Procedures for Detection of Targeted Biological Threat
Agents in Tap Water Rev 1.2.a
11
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vvEPA
United States
Environmental Protection
Agency
PRESORTED STANDARD
POSTAGE & FEES PAID
EPA
PERMIT NO. G-35
Office of Research and Development (8101R)
Washington, DC 20460
Official Business
Penalty for Private Use
$300
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