EPA/600/R-16/148 | September 2016
www.epa.gov/homeland-security-research
United States
Environmental Protection
Agency
oEPA
Pilot-Scale Decontamination of
Surrogate Radionuclides in a
Pilot-Scale Drinking Water
Distribution System
Office of Research and Development
Homeland Security Research Program

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Pilot-Scale Decontamination of Surrogate Radionuclides in a Pilot-Scale
Drinking Water Distribution System
U.S. Environmental Protection Agency
Cincinnati, Ohio 45268
September 7, 2016

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Disclaimer
The U.S. Environmental Protection Agency (EPA), through its Office of Research and
Development's National Homeland Security Research Center (NHSRC), funded and managed
this project under contract EP-C-14-012 with CB&I Federal Services LLC. This report has been
peer and administratively reviewed and has been approved for publication as an EPA document.
It does not necessarily reflect the views of the EPA. Mention of trade names or commercial
products does not constitute endorsement or recommendation for use of a specific product.
Questions concerning this document or its application should be addressed to:
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. i eff@epa. gov
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Acknowledgements
Contributions of the following organizations to the development of this document are
acknowledged:
CB&I Federal Services LLC

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Table of Contents
Disclaimer	2
Acknowledgements	3
List of Figures	5
List of Tables	5
List of Acronyms	5
Executive Summary	6
1.0 Introduction	7
2.0 Pilot Scale System and Methods	7
2.1	Testing Overview	7
2.2	Experimental Apparatus	8
2.3	Experimental Protocol	9
2.4	Sampling Protocols	12
2.4.1	Bulk Water Sample Collection	12
2.4.2	Coupon Sample Collection	12
2.4.3	Analytical Methods	13
2.5	Quality Assurance/Quality Control	13
3.0 Results	15
3.1	Heterotrophic Plate Count	15
3.2	Cesium Persistence and Decontamination	16
3.3	Cobalt Persistence and Decontamination	19
3.4	Strontium Persistence and Decontamination	21
4.0 Conclusions	24
5.0 References	25
Appendix A: T&E SOP 210: Biofilm Sample Collection from Coupons in the Distribution System Simulator
(DSS) Pipe-Loop System	27
Appendix B: T&E SOP 304: Heterotrophic Plate Count (HPC) Analysis Using IDEXX SimPlate® Method.... 35
Appendix C: Safety Data Sheet for EDTA	45
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List of Figures
Figure 1: Drinking water distribution system simulator at the EPA T&E facility	9
List of Tables
Table 1: Pilot Scale Radiological Surrogate Test Protocol	11
Table 2: Bulk Water Sample Storage and Preservation Procedures	12
Table 3: Sample Preparation and Analytical Method Summary	13
Table 4: QA/QC criteria and controls for HPC, cesium, cobalt and strontium analyses	14
Table 5: Cesium Contamination and Decontamination Duplicate Results	17
Table 6: Cobalt Contamination and Decontamination Duplicate Results	19
Table 7: Strontium Contamination and Decontamination Duplicate Results	23
List of Acronyms
BWS
bulk water sample
DI
deionized
DSS
distribution system simulator
EDTA
ethylenediaminetetraacetic acid
EPA
U.S. Environmental Protection Agency
HPC
heterotrophic plate count
MPN
most probable number
NHSRC
National Homeland Security Research Center
ORP
oxidation-reduction potential
PVC
polyvinyl chloride
QA
quality assurance
QC
quality control
SOP
Standard Operating Procedure
T&E
Test and Evaluation
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Executive Summary
The U.S. Environmental Protection Agency's (EPA's) Homeland Security Research Program
conducts research to provide the nation's drinking water utilities with tools that would help them
recover from contamination of distribution system infrastructure. Accidental or intentional
contamination of water systems could result in contaminated infrastructure that would need
remediation before the system was returned to service. This study examined decontamination of
non-radioactive cesium, strontium and cobalt in a pilot scale model of a drinking water
distribution system. The system was outfitted with coupons (excised pipe wall samples) of
corroded iron and cement-mortar, which are common distribution system pipe materials. Results
from this study were compared with similar decontamination data generated in smaller bench
scale experimental systems. The primary findings are as follows:
•	Cesium was not persistent on corroded iron in the pilot scale system, similar to the bench scale
studies. These results are consistent with the literature that shows that cesium reversibly
adsorbs to iron oxides. The bench scale results suggest that clean water flushing alone would
remove cesium from corroded iron. Clean water flushing may be effective at removing adhered
cesium from iron, but addition of potassium chloride could enhance the decontamination.
Cesium was more persistent on cement-mortar relative to iron, but potassium chloride was an
effective decontaminant. Past bench scale data and some of the pilot-scale coupons showed
that clean water flushing removed cesium from cement-mortar coupons. Pipe surface
characteristics variability can influence contaminant adherence and decontamination
effectiveness. If decontamination of cesium from water infrastructure in the field is necessary,
the data suggests that flushing and application of potassium chloride are effective
decontamination methods. Potassium chloride ions can compete with the cesium on the pipe
material surface.
•	Cobalt adhered to corroded iron and cement-mortar water infrastructure, and EDTA was an
effective decontaminant for both surfaces, a finding which was consistent with past bench scale
decontamination data. On the pilot scale, complete dissolution of EDTA in the bulk water
phase was a challenge, which resulted in inconsistent levels of decontamination on the
coupons. The lack of full EDTA dissolution was attributed to a drop in pH, which affected
EDTA solubility. When implementing EDTA decontamination in a real distribution system,
addition of a basic compound (i.e., sodium hydroxide) may be necessary for complete
dissolution.
• Strontium adhered to both corroded iron and cement-mortar, but the amount of initial
persistence on cement-mortar was 20 to 25 time higher than on iron. Ammonium acetate
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(0.01 M) did remove the adhered strontium, but not to the extent observed in bench scale
studies using 0.2 M. Due to the large amount of ammonium acetate needed to achieve 0.2 M
in a real water distribution system, adequate decontamination of adhered strontium may
require a physical removal operation (e.g., pigging) in addition to flushing and ammonium
acetate injection.
1.0 Introduction
The U.S. Environmental Protection Agency's (EPA's) Homeland Security Research Program
conducts research to provide the nation's drinking water utilities with tools that would help them
recover from contamination of distribution system infrastructure. Accidental or intentional
contamination of water systems could result in contaminated infrastructure that would need
remediation before the system was returned to service. Development of strategies to manage
contaminated infrastructure requires an understanding of the persistence of radionuclides on
drinking water infrastructure and the removal, or decontamination, of the adsorbed radionuclides.
Currently, this is a topic with little published data. The purpose of this study is to examine the
persistence of surrogate radionuclides on drinking water infrastructure and the decontamination
in a pilot scale model of a water distribution system.
A pilot-scale drinking water distribution pipe system was constructed with removable coupons
(excised pipe wall samples) of unlined, corroded iron and cement-mortar lined iron, which are
common distribution system materials. Contamination and decontamination experiments were
conducted by injecting solutions of non-radioactive cesium, cobalt or strontium salts. These salts
acted as chemical surrogates for radioactive cesium-137, cobalt-60 and strontium-90.
Decontamination was conducted by adding chemical decontaminants followed by flushing with
water. The results presented in this report provide data on the effectiveness of decontamination
agents and flushing on a pilot-scale. Data from this report are also compared to contamination
and decontamination data from smaller bench scale studies. This comparison provides insight
into whether data from simpler bench scale studies is scalable to larger, yet more realistic, pilot
scale studies.
2.0	Pilot Scale System and Methods
2.1	Testing Overview
During the tests, cesium chloride (99.99% pure, Acros Organics, Thermo Fisher Scientific,
Waltham, MA), cobalt chloride (97% pure, anhydrous, Acros Organics), or strontium chloride
(99.99% pure, anhydrous, Acros Organics) solutions were introduced into the distribution system
simulator (DSS) (described in section 2.2) through the recirculation tank to achieve target "in-
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pipe" concentrations between 4 mg/L and 10 mg/L. Subsequent to the completion of the
contamination protocol (described later under the "Experimental Protocol" section of the report),
decontamination strategies (e.g., flushing, chemical agents) were used to remove the radiological
contamination from the bulk liquid phase as well as the DSS pipe walls. The decontamination
agents used included: potassium chloride (99% pure, ACS certified, Thermo Fisher Scientific,
Waltham, MA), ethylene-diamine-tetraacetic acid (EDTA) (99.5% pure, Thermo Fisher
Scientific), and ammonium acetate 98% pure, HPLC grade, Thermo Fisher Scientific). The
specific concentration of each contaminant and decontaminant are presented in Tables 4, 5 and 6
in Section 3 (Results).
2.2 Experimental Apparatus
Experiments were conducted at the EPA's Test and Evaluation (T&E) Facility in Cincinnati, Ohio
using a DSS pipe loop. Figure 1 depicts a schematic 3-D overview of the DSS. The arrows depict
flow during normal operation. The main components of the DSS are a large reservoir to supply
water, approximately 75 ft (23 M) of 6 in (15.2 cm) diameter polyvinyl chloride (PVC)
interconnected main pipe, a 100 gallon (378.5 L) re-circulation tank (in-line with the main pipe),
water pumps, associated valves/fittings and small-diameter interconnecting pipes, and electronic
control devices necessary to operate the system. The total volume of the DSS (including the 85
gallons [322 L] in the recirculation tank) is approximately 240 gallons (908 L). The interior surface
area of the loop, including the recirculation tank (available for adsorption), is approximately
25,000 in2 (161,250 cm2). Operation of the DSS system with the in-line recirculation tank causes
an injected contaminant to homogeneously mix within a few minutes in the main pipe. The DSS
is also equipped with sensors that continuously measure the basic water quality parameters such
as pH, conductivity, temperature, free chlorine and oxidation-reduction potential (ORP).
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Coupons
Conductivity/Resistivity Sensor
pH Sensor
ORP Sensor
Figure 1: Drinking water distribution system simulator at the EPA T&E facility.
Globe Valve
Recirculation Tank
(open to atmosphere)
Drain
Overflow
2.3 Experimental Protocol
The test protocol consists of the following steps:
1)	Cultivation of Biofilm - Establish background conditions and biofilm
2)	Contamination Phase - Contaminant introduction
3)	Decontamination Phase - Decontaminant introduction
4)	Flushing - Flushing with clean tap water
Online water quality instruments are used to monitor pH, ORP, conductivity, temperature, flow,
and pressure during each step. Grab samples for free and total chlorine are collected along with
bulk water samples (BWS) and coupon samples as described in the individual step descriptions.
Step 1 - Cultivation of Biofilm:
Cultivation of biofilm in the DSS was accomplished by passing Cincinnati tap water continuously
through the DSS and measuring the heterotrophic plate count (HPC) concentration of both the bulk
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water and inside pipe surface. The water in the loop was circulated using a centrifugal pump
(operating at 88 gallons per minute (gpm) [333 L/min] achieving 1 ft/sec [0.3 m/sec] velocity) to
facilitate biofilm formation over a 4-week period. Fresh tap water was added at the rate of 0.8 gpm
(6.2 L/min) during the cultivation period to maintain a residual free chlorine concentration in the
loop.
A series of 30 coupons (1 in2 [6.5 cm2]) with specific test materials (15 ductile iron and 15 cement)
set in threaded plugs were inserted into the removable section of the pipe loop (previously shown
in Figure 1). The coupons were removed at specific times during the test (i.e., examination of
biofilm prior to the test for background, contamination phase, decontamination phase and flushing
phase).
Prior to each test, a set of coupons were collected and analyzed for HPC and the selected chemical
contaminant. The coupons were collected by shutting off the pump and segregating the removable
section of PVC-pipe by closing the flanking butterfly valves, removing the coupons and replacing
them with a blank plug. The background HPC/biofilm sample was collected by scraping the
coupon surface using a sterilized surgical scalpel. The scraped material was suspended in sterile
buffer, homogenized and analyzed for HPC to determine the formation of biofilm on the coupons.
Step 2 - Contamination Phase:
During this step, the radiological surrogate was introduced into the DSS through the recirculation
tank to achieve a target concentration between 4 mg/L and 10 mg/L in the loop. A bulk water
sample (BWS) was collected after 5 minutes of mixing at 88 gpm (333 L/min). Then, the DSS
operating flow rate was reduced and the contaminant was recirculated for 2 hours at 10 gpm (37.9
L/min). After this 2-hour period, the contaminated bulk water was sampled again and a coupon
sample set was collected to evaluate the adsorption of chemical onto the pipe surface.
At the end of this step, the DSS was drained and filled with tap water twice to remove as much of
the remaining contaminant from the bulk phase as possible.
Step 3 - Decontamination Phase:
The selected chemical decontaminant was introduced into the DSS and re-circulated continuously
at 88 gpm (333 L/min) for 20 minutes. After this time, the recirculation pump was turned off, the
back-pressure valve was opened, and loop contents were kept static during the rest of the
decontamination phase (22 hours). One set of coupons was removed from the DSS at 30, 60, 90,
120, 180, 240, 360, 1,200 and 1,320 minutes. A BWS was collected at each of these coupon
sampling times with an additional BWS collected at 15 minutes after the introduction of
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decontaminant. A duplicate BWS was collected at 60 minutes. The BWSs were preserved in
Nalgene™ (Thermo Fisher Scientific), with 2 drops of concentrated nitric acid.
The coupon removal process was similar to what was described in Step 1. One notable exception
is that the scraped material was suspended in deionized (DI) water (instead of sterile buffer),
homogenized and analyzed for metals. At the end of this step, the DSS was drained and filled with
tap water twice to remove the decontaminant present in the bulk phase.
Step 4 - Flushing:
The tap water-filled loop was recirculated at approximately 88 gpm (333 L/min) (~1 ft/sec [-0.30
m/sec]) with a fresh water influx at a 10 gpm (37.9 L/min) rate to provide the flushing action.
Coupon samples were removed at 120, 240 and 1,320 minutes after the 10 gpm (37.9 L/min)
flushing action is initiated. A duplicate coupon sample was collected at 120 minutes. The coupon
removal process was similar to what was described in Step 3. The scraped material was suspended
in DI water, homogenized and analyzed for metals.
BWSs were collected for metals analyses at each of the aforementioned coupon sampling times,
with an additional BWS sample collected at 15 minutes after the initiation of the flushing action.
Similar to Step 3, the BWSs were preserved in Nalgene™ bottles with 2 drops of concentrated
nitric acid.
After the completion of the flushing protocol, the DSS was drained and filled with tap water and
fitted with new coupon materials. This initiates the cultivation of biofilm (Step 1) for the next
study.
A summary of the 4-stage protocol is presented in Table 1:
Table 1: Pilot Scale Radiological Surrogate Test Protocol
Experimental
Phase
PVC Loop
Condition
Duration
Loop
Flow
(gpm)
Fresh
Water
(gpm)
Sample
No.
Sampl
e
Type*
Cultivation of
Biofilm
Tap water-
biofilm growth
7-28 days
(minimum)
88
0.8
(addition)
1
DI
1
CL
Contamination
Phase
Radiological
surrogate addition
to tap water and
mix
2 hours
10
0

BWS
1
DI
1
CL
Decontamination
Phase
Drain and fill
twice.
Decontaminant
addition to tap
water and mix
20 min
10
0
1
BWS
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Experimental
Phase
PVC Loop
Condition
Duration
Loop
Flow
(gpm)
Fresh
Water
(gpm)
Sample
No.
Sampl
e
Type*

Static
22 hours
0
0
10
BWS
9
DI
9
CL
Flushing
Drain and fill
twice. Begin
flushing/recirculat
ion with a portion
of the water to
discharge
22 hours
88
10
(discharge)
4
BWS
3+1
duplicate
DI
3+1
duplicate
CL
DI = Ductile iron, CL = Cement lined, BWS = Bulk Water Sample
2.4 Sampling Protocols
2.4.1 Bulk Water Sample Collection
The BWSs were collected using a grab sampling technique into 125 ml Nalgene™ sample bottles
that contain two drops of nitric acid to preserve the sample. The sampling port was opened and
flushed prior to collection of the samples. Table 2 summarizes the BWS storage and preservation
procedures.
Table 2: Bulk Water Sample Storage and Preservation Procedures
Measurement
Sample Container/
Quantity of Sample
Preservation/Storage
Holding
Times
Cesium
Nalgene plastic or
glass/ 125mL
pH <2 with HNO3.
Refrigerate between 4 ± 2 °C
6 months
Cobalt
Nalgene plastic or
glass/ 125mL
pH <2 with HNO3.
Refrigerate between 4 ± 2 °C
6 months
Strontium
Nalgene plastic or
glass/ 125mL
pH <2 with HNO3.
Refrigerate between 4 ± 2 °C
6 months
2.4.2 Coupon Sample Collection
The individual coupon sample collection process was described in the experimental protocol
section of this report. Once collected, the coupon surface was scraped using a scalpel. The debris
scraped from the surface was put directly into a sample bottle containing DI water. Scraping of
the iron coupons resulted in all corrosion material being removed, so that only bare, un-oxidized
iron was present. Cement mortar coupons were scraped until the substratum was visible. The
coupon surface and the scalpel were then rinsed using the least amount of DI water possible. The
rinsate was also put directly into the sample bottle. Sample bottles are labeled and refrigerated.
The collection of biofilm for HPC and metals analysis from the coupons is further described in
CB&I T&E SOP [Standard Operating Procedure] 210: Biofilm Sample Collection from Coupons
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in the Distribution System Simulator (DSS) Pipe-Loop System (Appendices A and B,
respectively).
2.4.3 Analytical Methods
After the completion of the specific experiment, the samples were prepared and analyzed in
accordance to the methods identified in Table 3.
Table 3: Sample Preparation and Analytica
Method Summary
Measurement
Sample Preparation Method
Analysis Method
HPC
CB&I T&E SOP 210 (Appendix A)
CB&I T&E SOP 304 (Appendix
B)
Cesium
BWS Samples: USEPA Method 3015A;
Coupon Samples: USEPA Method 3051A
USEPA Method 200.9 (GFAA)
Cobalt
BWS Samples: USEPA Method 3015A;
Coupon Samples: USEPA Method 3051A
USEPA Method 6010C/
USEPA Method 200.7 (ICP
OES)
Strontium
BWS Samples: USEPA Method 3015A;
Coupon Samples: USEPA Method 3051A
USEPA Method 6010C/
USEPA Method 200.7 (ICP
OES)
HPC, heterotrophic plate count; ICP, inductively coupled plasma; OES, optical emission spectra; GFAA, graphite furnace atomic
adsorption
Sources: USEPA Method 200.7, Revision 5 (2001): Determination of Metals and Trace Elements in Water and Wastes by
Inductively Coupled Plasma-Atomic Emission Spectrometry.
USEPA Method 200.9, Revision 2.2 (1994): Determination of Trace Elements by Stabilized Temperature Graphic Furnace Atomic
Absorption.
USEPA Method 3015A (SW846), Revision 1 (2007): Microwave Assisted Acid Digestion of Aqueous Samples and Extracts.
USEPA Method 3051A (SW846), Revision 1 (2007): Microwave Assisted Acid Digestion of Sediments, Sludges, Soils, and Oils.
USEPA Method 6010C (SW846), Revision 3 (2000): Inductively Coupled Plasma - Atomic Emission Spectrometry.
2.5 Quality A ssurance/Quality Control
All methods and protocol described throughout Section 2.0 were performed in accordance with
the EPA Quality Assurance (QA) program. The methods used to analyze HPC, cesium, strontium
and cobalt are referenced in Table 3. The controls, QA/Quality Control (QC) criteria, frequency
of the control and acceptance criteria are listed in Table 4. No significant deviations from the
QAPP were encountered, although two observations should be noted. First in the Results section
(Section 3), it is noted in Tables 5 and 6 that some samples were lost during processing, and no
data are reported for those samples. Second, it should also be noted that in experiments with cobalt
and strontium, concentrations less than the target concentration were detected in the bulk phase
immediately after contaminant injection. This was attributed to sorption of the contaminants to
the DSS pipe wall soon after injection, which is supported by the data in Tables 6 and 7. However,
due to the adsorption, the initial target concentrations could not be achieved.
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Table 4: QA/QC criteria and controls for HPC, cesium, cobalt and strontium analyses
Measurement
QA/QC Check
Frequency
Acceptance
Criteria
Corrective Action
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.
Cesium
Calibration
Daily
R2> 0.998
Investigate issues.
Prepare new standards if
necessary and recalibrate
Cesium
Quality Control
Sample (QCS)
(Second Source,
external to the
laboratory)
After
Calibration
±10%
Reanalyze. If fails:
Prepare again and reanalyze.
If fails:
Prepare new calibration
standards and recalibrate
Cesium
Instrument
Performance
Check (IPC)
Calibration
Verification
After
Calibration,
every 10
samples and at
the end of the
analysis batch
±10%
Reanalyze. If fails:
Prepare again and reanalyze.
If fails:
Reanalyze samples not
bracketed by appropriate
oc
Cesium
Laboratory
Reagent Blank
(LRB)
One per batch
Analyte
Concentration
must be less than
10% of the lowest
Calibration
Standard
Investigate source of
contamination and reanalyze
Cesium
Laboratory
Fortified Blank
(LFB)
One per batch
±15% Spiked
Recovery
Investigate source of
problem (interference) and
reanalyze
Cesium
Laboratory
Fortified Matrix
(LFM)
One per 10
samples
±30% Spiked
Recovery
Investigate source of
problem (interference) and
reanalyze
Metals (Cobalt and
Strontium)
Calibration
Daily
R2> 0.998
Investigate issues.
Prepare new standards if
necessary and recalibrate
Metals (Cobalt and
Strontium)
Initial Calibration
Verification
(ICV) (This is a
mid-range 2nd
source standard)
After
Calibration
±10%
Reanalyze. If fails:
Prepare again and reanalyze.
If fails:
Prepare new standards and
recalibrate
Metals (Cobalt and
Strontium)
Calibration Blank
(CB)
After
Calibration,
every 10
samples and end
of analysis
batch
Analyte
Concentration
must be less than
10% of the lowest
Calibration
Standard
Reanalyze. If fails:
Prepare again and reanalyze.
If fails:
Reanalyze samples not
bracketed by appropriate
oc
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Measurement
QA/QC Check
Frequency
Acceptance
Criteria
Corrective Action
Metals (Cobalt and
Strontium)
Low Level
Calibration
Verification
(LLCV)
After
Calibration and
at the end of the
analysis batch
±30%
Reanalyze. If fails:
Prepare again and reanalyze.
If fails:
Reanalyze samples not
bracketed by appropriate
QC
Metals (Cobalt and
Strontium)
Continuing
Calibration
Verification
(CCV)
After every 10
samples and end
of analysis
batch
±10%
Reanalyze. If fails:
Prepare again and reanalyze.
If fails:
Reanalyze samples not
bracketed by appropriate
oc
Metals (Cobalt and
Strontium)
Method Blank
One per sample
batch
Analyte
Concentration
must be less than
10% of the lowest
Calibration
Standard or lowest
Sample
Concentration
Reanalyze. If fails:
Prepare again and reanalyze.
If fails:
Prepare entire sample batch
again and reanalyze.
Metals (Cobalt and
Strontium)
Laboratory
Control Sample
(LCS)
One per sample
batch
±20% Spiked
value
Reanalyze. If fails:
Prepare again and reanalyze.
If fails:
Prepare entire sample batch
again and reanalyze
Metals (Cobalt and
Strontium)
Matrix spike,
unspiked
duplicate and/or
matrix spike
duplicate
(MS/Dup or
MS/MSD)
One per sample
batch
20% RSD for
duplicates
±25% Spike value
recovery
Investigate for interferences
or error.
Reanalyze.
3.0	Results
3.1	Heterotrophic Plate Count
HPC values on the cement-mortar coupons ranged from 200 to 1350 most probable number
(MPN)/in2 (31 to 209 MPN/cm2), with an average of 650 MPN/in2 (101 MPN/cm2). HPC values
on the ductile iron coupons ranged from 800 to 41,000 MPN/in2 (124 to 6357 MPN/cm2), with an
average of 11,600 MPN/in2 (1799 MPN/cm2). The primary reason for measuring HPC on the
coupons was to establish that microorganisms had colonized the coupons after one month of
conditioning. HPC was generally higher on the ductile iron coupons, which was not surprising
since the surface is visibly rougher than the cement-mortar surface. More roughness increases the
15

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surface area with deep recesses that experience less shear from the flow, and decreased or no
disinfectant residual due to the iron surface consuming it.
3.2 Cesium Persistence and Decontamination
Table 5 shows the results from duplicate contamination/decontamination experiments with cesium
in the DSS. Cesium concentration in the bulk water phase and the amount adhered to corroded
iron and cement-mortar infrastructure coupons are displayed. Cesium adhered to corroded iron,
but to a lesser degree than cement-mortar. This is consistent with previously reported results
showing that cesium reversibly adsorbs to iron oxides, but the adsorption is weak and cesium will
desorb in presence of clean water (Ebner et al., 1994; Todorovic et al., 2001). In both experiments,
cesium was undetectable on the coupons after application of potassium chloride and flushing. The
duplicate experiment had an initial cesium concentration of 21.2 mg/L compared to 13.4 mg/L in
the first experiment. However, this difference in initial concentration did not appear to influence
the persistence of cesium on either coupon type.
Potassium chloride is a competing ion that will replace cesium adhered to the coupon surface.
Since potassium chloride was applied soon after contamination, it is difficult to determine if the
cesium removal is due to fresh water being pumped into the DSS pipe between the contamination
and decontamination phase, or due to the presence of potassium chloride. However, in both
experiments, cesium was detected on the iron coupons after decontamination began, but before
becoming undetectable, which suggests that the potassium chloride played a role. In this
experiment, it was difficult to separate the effect of flushing alone from that with the potassium
chloride.
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Table 5: Cesium Contamination and Decontamination Duplicate Results

12.7 mg/L Cesium chloride (10.0 mg/L
Cesium) decontaminated with 0.001 M
KC1 (74.6 mg/L)
12.7 mg/L Cesium Chloride (10.0 mg/L
Cesium) decontaminated with 0.001 M
KC1 (74.6 mg/L)
Experimental
Activity
Elapsed Time
(hrs)
Bulk water Cs
concentration
(mg/L)
Ductile iron
coupon Cs
concentration
(mg/kg)
Cement lined
coupon Cs
concentration
(mg/kg)
Bulk water Cs
concentration
(mg/L)
Ductile iron
coupon Cs
concentration
(mg/kg)
Cement lined
coupon Cs
concentration
(mg/kg)
Control
():()()
ND
—
—
\l>

—
Contaminant
Injection
00:00







0:05
13.4
—
—
21.2
—
—

2:00
12.9
3.16
26.7
::.r'
2.38
75.9
1 X'contaminant
Phase
0:00







0:15
0.574
—
—
0.612
—
—

0:30
0.434
ND
152.5
0.549
1.44
158.4

1:00
0.54
1.58
137.1
0.503
1.22
119.8

1:30
0.125
ND
80.6
0.763
ND
174.2

2:00
0.102
ND
ND
0.581
ND
90.5

3:00
0.154
ND
ND
0.579
NAa
210.5

4:00
0.0600
ND
ND
0.637
ND
ND

6:00
0.228
ND
ND
0.648
ND
72.4

20:00
0.227
ND
38.3
0.412
ND
ND

22:00
0.247
ND
ND
0.311
ND
ND
Flushing Phase
0:00







0:15
ND
—
—
ND
—
ND
ND

2:00
ND
ND
31.6
ND
ND

2:00 (dup)
—
ND
9.7
—
ND

4:00
ND
ND
34.9
ND
ND
39.6

22:00
ND
ND
21.5
ND
ND
ND
NAa:Sample lost during processing; ND: Not Detected; —: sample not taken
Cesium adherence to cement-mortar was higher than on iron oxides, which is consistent with the
literature showing that cesium does adsorb to cement-mortar matrices (Apak et al., 1996). In both
experiments with cement mortar coupons, a trend is present where the amount of cesium present
on the coupon was higher early in the decontamination phase compared to the end of the
contamination phase. This suggests that adherence of the cesium to the cement mortar continued
after the last contaminant injection sample was removed (elapsed time of 2 hours). This
17

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phenomenon is also supported by studies showing that cesium adsorption to clays is slow and not
instantaneous (Atkinson and Nickerson, 1988; Chorover et al., 2003). Application of potassium
chloride did result in removal of cesium, but cesium removal was not consistent over time. Cesium
decreased to undetectable levels on some coupons after decontamination started, but was still
detected on other coupons. Cesium was also detectable on coupons after flushing began. Some
of this may be due to cesium trapped in dead end spaces being remobilized during flushing and
subsequently re-adhering to the coupon surfaces.
Two previous studies directly focused on cesium adherence to drinking water infrastructure (Szabo
et al., 2009; USEPA, 2014). In Szabo et al., 2009, cesium was not detected on corroded iron
coupons in bench scale biofilm anular reactors, after spiking and subsequent flushing with clean
water. These results are consistent with the results in this study in the sense that strong cesium
adsorption to corroded iron (containing iron oxides) was not observed. The bench scale results
suggest that flushing alone would remove cesium from iron coupons, while pilot scale results from
this study suggest that addition of potassium chloride enhanced decontamination. In practice,
clean water flushing may be effective at removing adhered cesium, but addition of potassium
chloride could enhance the decontaminating effect of clean water. It should also be noted that
decontamination with 0.001 M KC1 required that 0.15 lb (67 g) be dissolved in the pilot scale pipe
loop. If this volume were extrapolated to a 400 ft (122 m), 6 inch (15.2 cm) water main between
two fire hydrants, 0.4 lb (165.8 g) would be necessary. This mass is easy to handle, and KC1 is
highly soluble, so this decontamination technique should be implementable in the field.
In USEPA, 2014, cesium initially adhered to cement-mortar coupons when spiked into biofilm
annular reactors, but persistence was not observed. Furthermore, simulated flushing in the annual
reactors at 1.6 to 2.5 ft/sec (0.50 to 0.75 m/sec) was effective at removing adhered cesium to
undetectable levels. Application of potassium chloride and then flushing was effective for some
coupons in the pilot scale system, but not others. This result may reveal one of the challenges
associated with decontamination on a large scale. The coupons in biofilm annular reactors are
consistent in their content and surface smoothness. The coupons in the pilot scale DSS are also
consistent in their sand and cement content, but their surface roughness and shape can vary as they
are handmade. Cement mortar coatings on pipes in distribution systems should be consistent in
their sand and cement content, but different pipes can have varying surface characteristics due to
their age or the way they were manufactured. Varying surface characteristics (including biofilm
presence) can influence how a contaminant adheres or how a decontaminant interacts with the
surface. The inconsistent results observed in the pilot scale DSS may be reflective of the
challenges associated with decontaminating real world drinking water infrastructure surfaces.
18

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3.3 Cobalt Persistence and Decontamination
Table 6 shows the results from duplicate contamination/decontamination experiments with cobalt
in the DSS.
	Table 6: Cobalt Contamination and Decontamination Duplicate Results


10 mg/L Cobalt (11) chloride (4.48
mg/L Cobalt) decontaminated with
0.01 M EDTA (2,922 mg/L)
10 mg/L Cobalt (11) chloride (4.48
mg/L Cobalt) decontaminated with
0.01 M EDTA (2,922 mg/L)
Experimental
Activity
Elapsed Time
(hrs)
Bulk water Co
concentration
(mg/L)
Ductile iron
coupon Co
concentration
(mg/kg)
Cement lined
coupon Co
concentration
(mg/kg)
Bulk water Co
concentration
(mg/L)
Ductile iron
coupon Co
concentration
(mg/kg)
Cement lined
coupon Co
concentration
(mg/kg)
Control
()():()()
ND

—
ND

—
Contaminant
Injection
()():()()







0:05
3.553
—
—
3.944
—
—

2:00
3.057
80.17
52.74
3.618
69.59
131.09
Dcconlaminanl
Phase
0:00






0:15
0.753
—
—
0.298
—
—

0:30
0.756
5.18
10.3
0.291
ND
16.48

1:00
0.751
12.40
7.91
0.280
ND
NAa

1:30
0.751
7.73
7.19
0.284
ND
ND

2:00
0.747
15.37
8.08
0.276
ND
ND

3:00
0.747
13.27
7.24
0.273
ND
ND

4:00
0.746
13.38
6.58
0.282
ND
ND

6:00
0.750
12.23
5.84
0.280
ND
ND

20:00
0.731
14.34
3.94
0.282
ND
ND

22:00
0.736
14.86
ND
0.282
ND
ND
Mushing Phase
0:00







0:15
ND
—
—
ND
—
—

2:00
ND
22.48
ND
ND
ND
ND

2:00 (dup)
—
7.28
4.14
—
ND
ND

4:00
ND
6.65
ND
ND
ND
ND

22:00
ND
25.82
3.76
ND
ND
ND
NAa:Sample lost during processing; ND: Not Detected; —: sample not taken
Cobalt adhered to both corroded iron and cement-mortar drinking water infrastructure coupons.
In past studies, it has been observed that when soluble cobalt chloride is introduced into chlorinated
water, the soluble cobalt(II) oxidizes to insoluble cobalt(III), and this insoluble material
precipitates on and sticks to drinking water infrastructure surfaces (Szabo et al., 2009; USEPA,
19

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2014). Precipitation and adherence of the cobalt was also observed in the pilot scale study. For
both corroded iron and cement mortar, EDTA was an effective decontaminating agent. In one of
the experiments, cobalt was reduced to non-detectable levels (>99.99% removal), while in the
other 80% to 93% cobalt decontamination was observed. In the experiments where EDTA did not
reduce cobalt to undetectable levels, flushing after application of EDTA did not result further
removal of cobalt.
The discrepancy between the two replicate experiments can be explained by the inability to
completely dissolve EDTA in solution. EDTA can be dissolved up to 0.26 M (Appendix C), and
was introduced into the DSS at 0.01 M. However, EDTA dissolves slowly, and it can be influenced
by pH. EDTA is more soluble at pH of 4 to 6 or above. The pH of the Cincinnati tap water used
in the DSS ranged from 8.0 to 8.5, and it was expected that it would have enough buffering capacity
to keep the pH in the 4.0 to 6.0 range after introduction of EDTA. However, after introduction of
EDTA, pH dropped to between 3.0 and 3.5 and remained in that range for the duration of the
decontamination experiment. As a result, precipitated EDTA was observed sitting at the bottom
of the DSS pipe. Some EDTA was dissolved, but the dissolved amount in contact with the coupons
was likely different in each experiment.
In Szabo et al., 2009, precipitated cobalt was extracted from iron coupons in bench scale biofilm
annular reactors with 0.36 M sulfuric acid, which removed over 90% of the adhered cobalt. In the
pilot scale studies, 0.01 M EDTA resulted in 80 to 93% cobalt removal in one experiment and
>99.99% (non-detectable level of cobalt) in a second identical study. EDTA at 0.01 M compares
favorably with acid treatment. Although pH dropped to a range between 3.0 and 3.5 during EDTA
treatment, this pH level is preferable than the intensely corrosive conditions resulting from sulfuric
acid treatment, which removed cobalt by dissolving the iron surface. However, achieving 0.01 M
EDTA in the pipe loop required 5.8 lbs (2,655 g) of EDTA. If this mass were extended to a 400
ft (122 m), 6 inch (15.2 cm) water distribution pipe between two fire hydrants, 14 lbs of EDTA
would be required.
The results in USEPA, 2014 showed that cobalt was persistent on cement-mortar coupons in bench
scale biofilm annular reactors. Flushing at 1.6 to 2.5 ft/sec (0.50 to 0.75 m/sec) had no effect on
the adhered cobalt. Treatment with 0.1 M EDTA removed 95% of the cobalt adhered to cement
mortar. There was no mention of whether EDTA precipitation was observed in USEPA, 2014.
The results of the pilot scale study reported here compare favorably to those of USEPA, 2014.
This is especially noteworthy given that a lower concentration of EDTA was used in the pilot
studies. Together, the bench and pilot scale results indicate that EDTA is a good decontamination
agent for cobalt adhered to cement mortar. However, when implementing EDTA decontamination
in a real distribution system, care will have to be taken to ensure that full dissolution of the EDTA
20

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occurs. Co-injection of a basic compound like sodium hydroxide could help keep the pH in the
4.0 to 6.0 range (or higher), which could facilitate EDTA dissolution.
3.4 Strontium Persistence and Decontamination
Table 7 shows the results from duplicate contamination/decontamination experiments with
strontium in the DSS. Like cesium, strontium adhered more to cement-mortar than corroded
iron. Strontium was detected on both materials after decontamination with 0.01 M ammonium
acetate and subsequent flushing. For ductile iron, decontamination effectiveness in the two
experiments was 23% to 31% in the 30 minutes after ammonium acetate was applied, and up to
50% to 87%) by the end of the treatment phase (22 hrs). For cement-mortar, decontamination
ranged from 13% to 28% in the first experiment and 11% to 94% in the second experiment. Like
in the cesium experiments, the variability of strontium concentration on the coupons during
decontamination is attributed to variability in the surface characteristics such as roughness and
the presence of biofilm. It is presumed that in reality, the same type of pipe material can vary in
surface roughness and composition of cement/sand or impurities in iron due to different pipe
ages and manufacturers.
Strontium adsorption to iron oxides, which make up most of the iron corrosion matrix on water
pipe interiors, has been studied in small, bench scale experiments. In general, these bench scale
studies have found that strontium adsorption is transient and reversible, particularly if iron oxides
are exposed to clean water after application of strontium (Axe et al., 1998; Gerke et al., 2014;
Small et al., 1999). However, strontium is far more persistent on calcium carbonate (calcite),
which can deposit on water infrastructure (Carroll et al., 2008). Strontium and calcium are
neighbors in the alkaline earth metals column of the periodic table and share many similar
properties. Strontium exchange with calcium carbonate compounds may also promote strontium
adhesion on goethite and other iron oxides. These phenomena may explain the persistence of
strontium on the corroded iron coupon and the inability of ammonium acetate to completely
remove it.
USEPA, 2014 contains persistence and decontamination results for strontium on cement-mortar
water infrastructure. Like the results of this pilot scale study, the results in USEPA, 2014 in ARs
show that strontium readily adheres to cement mortar infrastructure. The results also show that
strontium is persistent on cement-mortar, but flushing at 1.6 to 2.5 ft/sec (0.50 to 0.75 m/sec)
removes approximately 40% of the adhered strontium. Application of ammonium acetate at 0.2
M removed 90% of the adhered strontium. The results of this study support those generated
from bench scale biofilm annular reactor studies. More strontium was removed from cement-
mortar in the bench scale study, but 20 times more ammonium acetate was applied on the bench
scale compared to the pilot scale experiments.
21

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The difference in results between the bench scale and pilot scale studies highlight one of the
challenges of translating bench scale data to a pilot scale setup or real distribution system.
Ammonium acetate was an effective decontamination agent when applied at 0.2 M in bench
scale experiments, but less so when applied at 0.01 M (20-fold less) in the pilot scale
experiments. Achieving 0.01 M ammonium acetate in the pilot scale pipe loop required
dissolving 1.5 lbs (681 g) of the chemical in the entire volume. A concentration of 0.2 M would
require 30 lbs (13,620 g), which was deemed too much for this experiment. For comparison,
consider a 400 ft (122 m), 6 inch (15.2 cm) diameter pipe between two fire hydrants.
Decontaminating this volume with ammonium acetate at 0.2 M would require 75 lb (34,050 g),
while 0.01 M would require 3.7 lb (1,680 g). Therefore, ammonium acetate may be a good
decontamination method for strontium adhered to cement-mortar, but the amount that would
need to be added to a real distribution system would be large enough that its application may be
prohibitive over a large area. Adequate decontamination of adhered strontium may require a
physical removal operation (e.g., pigging) in addition to flushing and ammonium acetate
injection.
22

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Table 7: Strontium Contamination and Decontamination Duplicate Results

10 mg/L Strontium chloride (5.46 mg/L
Strontium) decontaminated with 0.01
M Ammonium acetate (770.8 mg/L)
10 mg/L Strontium chloride (5.46 mg/L
Strontium) decontaminated with 0.01
M Ammonium acetate (770.8 mg/L)
Experimental
Activity
Elapsed Time
(hrs)
Bulk water Sr
concentration
(mg/L)
Ductile iron
coupon Sr
concentration
(mg/kg)
Cement lined
coupon Sr
concentration
(mg/kg)
Bulk water Sr
concentration
(mg/L)
Ductile iron
coupon Sr
concentration
(mg/kg)
Cement lined
coupon Sr
concentration
(mg/kg)
Control
()():()()
0.172

—
0.318

—
Contaminant
Injection
00:00







0:05
3.900
—
—
5.924
—
—

2:00
3.732
69.99
1,459.0
5.381
96.80
2,433.4
Deeontaminanl
Phase
0:00







0:15
0.330
—
—
0.317
—
—

0:30
0.342
48.23
1,077.5
0.274
56.57
1,470.7

1:00
0.332
46.41
1,107.3
0.315
74.77
262.7

1:30
0.328
43.49
1,045.0
0.322
51.87
2,152.6

2:00
0.326
41.10
1,103.7
0.333
46.29
239.7

3:00
0.334
40.65
1,274.4
0.315
12.59
1,386.7

4:00
0.344
43.94
1,318.2
0.387
66.59
195.8

6:00
0.334
41.81
1,276.3
0.274
45.64
1,264.6

20:00
0.342
38.00
1,269.6
0.258
24.01
862.9

22:00
0.336
34.68
1.553.2
0.267
28.15
125.1
Mushing Phase
0:00







0:15
0.170
—
—
0.167
—
—

2:00
0.166
36.73
1,340.4
0.168
32.14
119.7

2:00 (dup)
—
41.16
1,008.1
—
33.94
116.6

4:00
0.166
37.89
1,202.5
0.157
9.81
443.2

22:00
0.162
44.62
996.6
0.172
34.52
2,046.2
23

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4.0 Conclusions
This study produced pilot-scale decontamination data for non-radioactive cesium, cobalt and
strontium adhered to corroded iron and cement-mortar drinking water infrastructure. This pilot-
scale data was also compared to decontamination data generated on the bench scale under similar
conditions. The key findings are as follows:
•	Cesium was not persistent on corroded iron in the presence of 0.001 M potassium chloride on
the pilot scale. The bench scale results suggest that flushing alone would remove cesium from
corroded iron. In practice, clean water flushing may be effective at removing adhered cesium
from iron, but addition of potassium chloride could enhance the decontamination effect of
clean water. Cesium was more persistent on cement-mortar relative to iron, but 0.001 M
potassium chloride was an effective decontaminant. Bench scale data showed that flushing
with clean water removed cesium from cement-mortar coupons. If decontamination of cesium
from water infrastructure in the field is necessary, the data suggests that flushing and
application of potassium chloride are effective decontamination methods. It should be noted
that the pilot scale results showed that not all coupons were decontaminated equally, which
may also hold could true in a real water distribution system with pipe materials of different
ages and from different manufacturers.
•	Cobalt adhered to corroded iron and cement-mortar water infrastructure, and 0.01 M EDTA
was an effective decontaminant for both surfaces. These results are consistent with
decontamination data generated on the bench scale. However, in the pilot scale experiments,
it was noticed that EDTA did not fully dissolve in the pipe loop. This may have affected the
concentration of EDTA in the pipe, which resulted in inconsistent level of decontamination
between experiments. The lack of full EDTA dissolution was attributed to a drop in pH, which
negatively affected EDTA solubility. When implementing EDTA decontamination in a real
distribution system, care will have to be taken to ensure that full dissolution of the EDTA
occurs. Co-injection of a basic compound like sodium hydroxide could help keep the pH in
the 4.0 to 6.0 range (or higher), which could facilitate EDTA dissolution and make it more
potent.
•	Strontium adhered to both corroded iron and cement-mortar, but the amount of initial
persistence on cement-mortar was 20 to 25 time higher than iron. Ammonium acetate (0.01
M) did remove the adhered strontium, but not to the extent observed in bench scale studies
using 0.2 M. Decontaminating the pilot scale pipe loop required dissolving 1.5 lbs (681 g) of
the chemical in the entire volume. A concentration of 0.2 M would have require 30 lbs
(13,620 g). Extrapolated to a 400 ft (122 m), 6 inch (15.2 cm) diameter water pipe, 0.2 M
would require 75 lb (34,050 g), while 0.01 M would require 3.7 lb (1,680 g) of ammonium
acetate. Due to the large amount of ammonium acetate needed to achieve 0.2 M in a real
water distribution system, adequate decontamination of adhered strontium may require a
24

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physical removal operation (e.g., pigging) in addition to flushing and ammonium acetate
injection.
5.0 References
Apak R, Atun G, GU9IU K, and Tutem E. (1996). Sorptive removal of cesium-137 and strontium -
90 from water by unconventional sorbents II: Usage of coal fly ash. JNucl Sci Technol.
33(5), 396^102.
Atkinson A and Nickerson AK. (1988). Diffusion and sorption of cesium, strontium, and iodine
in water-saturated cement. Nucl Technol. 81(1), 100-113.
Axe L, Bunker GB, Anderson PR and Tyson TA. (1994). An XAFS analysis of strontium at the
hydrousferric oxide surface. J Colloid Interface Sci. 199 (1), 44-52.
Carroll SA, Roberts SK, Criscenti LJ and O'Day PA. (2008). Surface complexation model for
strontium sorption to amorphous silica and goethite. Geochem Trans. 9(2), 1-26.
Chorover J, Choi S, Amistadi MK, Karthikeyan KG, Crosson G and Mueller KT. (2003).
Linking cesium and strontium uptake to kaolinite weathering in simulated tank waste
leachate. Environ Sci Technol. 37(10), 2200-2208.
Ebner AD, Ritter JA and Navratil JD. (2001). Adsorption of cesium, strontium, and cobalt ions
on magnetite and a magnetite-silica composite. IndEng Chem Res. 40(7), 1615-1623.
Gerke TL, Little BJ, Luxton TP, Scheckel KG, Maynard BJ and Szabo JG. (2014). Strontium
adsorption and persistence with iron corrosion products from a model and actual drinking
water distribution system. J Water Supply Res Technol-Aqua. 63(6), 449-460.
Small TD, Warren LA, Roden EE and Ferris FG. (1999). Sorption of strontium by bacteria,
Fe(III) oxide, and bacteria-Fe(III) oxide composites. Environ Sci Technol. 33(24), 4465-
4470.
Szabo JG, Impellitteri CA, Govindaswamy S and Hall JS. (2009). Persistence and
decontamination of surrogate radioisotopes in a model drinking water distribution
system. Water Research. 43(20), 5005-5014.
Todorovica M, Milonjica SK, Comor JJ and Gal IJ. (1992). Adsorption of radioactive ions 137Cs,
85Sr2+, and 60Co2+ on natural magnetite and hematite. Sep Sci Technol. 27(5), 671-679.
USEPA (2014). Radiological Contaminant Persistence and Decontamination in Drinking Water
Pipes. Washington, DC: U.S. Environmental Protection Agency. EPA/600/R-14/234
25

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26

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Appendix A: T&E SOP 210: Biofilm Sample Collection from Coupons in the
Distribution System Simulator (DSS) Pipe-Loop System
EPA T&E Contract Technical
Standard Operating Procedure
BIOFILM SAMPLE COLLECTION FROM COUPONS IN THE
DISTRIBUTION SYSTEM SIMULATOR (DSS) PIPE-LOOP SYSTEM
T&E SOP 210
Revision Number: 0
Date: 11/24/2014

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SOP 210, Coupon Biofilm Sample Collection
Revision Number: 0
Date 11/24/2014
Page 2 of 8
SOP Approval
1. E. Radha Krishnan, P.E.
Program Manager
11/24/2014
Date
2. Steven Jones, ASQ CQA/CQE
Quality Assurance Manager
Signature
		11/24/2014
ignature	Date

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SOP 210, Coupon Biofilm Sample Collection
Revision Number: 0
Date 11/24/2014
Page 3 of 8
Revision Summary
Revision
Name
Date
Description of Change
0
Nicole Sojda/
Lee Heckman
11/24/2006
Developed SOP.

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SOP 210, Coupon Biofilm Sample Collection
Revision Number: 0
Date 11/24/2014
Page 4 of 8
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
6
7.0

Personnel Qualifications
6
8.0

Equipment and Supplies
6
9.0

Media and Reagents
6
10.0

Procedure
6

10.1
Sample Collection, Handling, and Preservation
6

10.2
Sample Storage
7

10.3
Analysis
7
11.0

Data and Records Management
7
12.0

Quality Control and Quality Assurance
7
13.0

References
8

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SOP 210, Coupon Biofilm Sample Collection
Revision Number: 0
Date 11/24/2014
Page 5 of 8
1.0 SCOPE AND APPLICABILITY
1.1	The method described in this standard operating procedure (SOP) is applicable to the
collection of biofilm samples from coupons in the Distribution System Simulator (DSS) pipe-
loop system for purposes of determining the viability of biofilm growth in the DSS and
determining the effectiveness of various decontamination methods on different interior pipe
surfaces.
1.2	Coupon samples are fabricated from different pipe materials, such as ductile iron, concrete,
and PVC. Coupon fabrication and installation in the DSS are discussed in T&E SOP 208,
Operation of the Distribution System Simulator (DSS) Pipe-loop System.
2.0 SUMMARY OF METHOD
Coupons of different pipe materials are inserted into the DSS, and naturally occurring biofilm is
allowed to accumulate for approximately thirty (30) days. Water containing contaminants of interest
are introduced into the DSS and recirculated, allowing them an opportunity to adhere to the
coupons. The DSS is then decontaminated. Coupons of the different pipe materials are removed
from the DSS at various times to evaluate the effectiveness of the decontamination process. The
surface of each coupon is scraped using a sterilized surgical scalpel to collect biofilm sample for
microbiological analysis. Extracted materials are collected in 100mL coliform sample vials with a
sodium thiosulfate tablet, and 90mL of sterile phosphate buffer. Large pieces of coupon material
are ground using a sterile metal rod. Coupon samples can be pasteurized immediately, or held at
4°C for later pasteurization.
3.0 DEFINITIONS
3.1	Biofilm - An aggregation of microorganisms that forms a thin coating on a substrate.
3.2	Coupon - A replaceable plug that is used as the substrate upon which biofilm is grown.
4.0 HEALTH AND SAFETY WARNINGS
4.1	Standard laboratory personal protective equipment (PPE) (i.e., lab coat, gloves, safety
glasses, and steel toed boots) is required.
4.2	The biohazards and the risk of infection by pathogens associated with this method are
minimal.
4.3	Exposure to ultra-violet (UV) radiation is minimal. An interlock device in the Millipore UV
sterilizer switches off the UV lamps when the lid is opened.
5.0 CAUTIONS
5.1	Avoid touching coupon surfaces prior to scraping to prevent biofilm loss.
5.2	Ensure scalpels and metal rods are exposed to UV light at least two minutes before use.
This will prevent cross-contamination from prior uses.
5.3	Do not use the scalpel/metal rod pair designated for ductile iron coupons on cement-lined
coupons, and vice versa. There are dedicated scalpels and metal rods for each coupon type.
For example, scalpels marked "I" for ductile iron should only be used for ductile iron coupons,
and scalpels marked "C" should only be used for concrete coupons.
5.4	To prevent spilling sample, gently grind large pieces of coupon material with the appropriate
metal rod.

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SOP 210, Coupon Biofilm Sample Collection
Revision Number: 0
Date 11/24/2014
Page 6 of 8
6.0 INTERFERENCES
None anticipated.
7.0 PERSONNEL QUALIFICATIONS
The techniques of a first-time analyst must 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 procedure.
8.0 EQUIPMENT AND SUPPLIES
8.1	100mL coliform sample bottles with sodium thiosulfate tablet.
8.2	Scalpels.
8.3	Metal rods.
8.4	Millipore UV sterilizer.
8.5	Petri plates.
9.0 MEDIA AND REAGENTS
9.1	Dilution blanks containing 90 mL phosphate buffer with magnesium chloride.
9.2	Deionized water in squeeze bottle.
10.0 PROCEDURE
10.1 Sample Collection, Handling, and Preservation
10.1.1	After removing coupons from DSS, place each in one half of a petri plate for transport
to the BSL-2 Laboratory.
10.1.2	Use one 100 mL coliform sample bottle per sample. Pour approximately half of a 90
mL dilution blank into the coliform sample bottle. The remaining portion of the
dilution blank may be used for a second sample.
10.1.3	Remove the scalpel from the UV sterilizer designated for the coupon type to be
scraped. Close the UV sterilizer.
10.1.4	If the surface of the coupon appears dry, wet the surface with deionized water from
the squeeze bottle while holding coupon above the open coliform sample bottle to
collect any contact water runoff.
10.1.5	Holding the coupon over the open pre-labeled coliform sample bottle, gently scrape
the coupon surface with a scalpel into the 100 mL coliform sample bottle.
•	Ductile iron coupons should have a coating of iron oxide. This may fall off in
pieces, or in a single piece when scraped.
> Rinse the iron coupon surface with deionized water, and scrape the exposed
iron. Rinse with deionized water.
•	Cement-lined coupons may have cement on the periphery of the coupon. This is
to be collected by scraping it into the buffer.

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SOP 210, Coupon Biofilm Sample Collection
Revision Number: 0
Date 11/24/2014
Page 7 of 8
> Scrape the surface of the coupon's cement plug. A slight change in color
may be observed. Rinse with deionized water.
• Rinse the scalpel with deionized water into the 100 mL coliform sample bottle.
10.1.6	Place the scalpel back in the UV sterilizer. Remove the metal rod designated for
grinding extracted coupon material of the coupon type. The large diameter rod is to
be used for ductile iron. The small diameter rod is to be used for cement-lined
coupons.
10.1.7	Close the UV sterilizer. Grind the extracted coupon material so that no splashing
occurs.
10.1.8	Rinse the metal grinding rod with deionized water into the 100 mL coliform sample
bottle.
10.1.9	Place the metal grinding rod into the UV sterilizer and close the lid.
10.1.10	Fill the coliform sample bottle with deionized water to the 100 mL fill line.
10.2	Sample Storage
10.2.1	Extracted coupon samples can to be stored at 4°C up to 24 hours. They must then
be pasteurized/heat shocked.
10.2.2	Pasteurized/heat shocked samples may be stored at 4°C up to 48 hours before
analysis using the spread plate method. Pasteurization/heat shocking is detailed in
Section 9.6 of T&E SOP 309, Preparation and Enumeration ofB. globigii
Endospores.
10.3	Analysis
10.3.1 Conduct microbiological sample analysis in accordance with the analysis-specific
SOP or method.
11.0 DATA AND RECORDS MANAGEMENT
11.1	All original analytical documentation generated and prepared
in accordance with T&E SOP 101, Central Files.
11.2	All data packages shall be assembled and reviewed per T&E
Verification.
12.0 QUALITY CONTROL AND QUALITY ASSURANCE
12.1 One field duplicate coupon is collected from the DSS at a frequency of one per experiment.
Acceptance criteria for the field duplicate is <20% variation or as specified in the project-
specific Quality Assurance Project Plan (QAPP).
for the EPA shall be controlled
SOP 102, Data Review and
12.2 Analysis-specific QA/QC requirements are specified in the analysis-specific SOP or method.

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SOP 210, Coupon Biofilm Sample Collection
Revision Number: 0
Date 11/24/2014
Page 8 of 8
13.0 REFERENCES
13.1	EPA, March 2001. Guidance for Preparing Standard Operating Procedures (EPA QA/G-6),
EPA/240/B-01/004, Office of Environmental Information.
13.2	CB&I Federal Services LLC, 2011. EPA T&E Contract Administrative SOP 101, Central
Files.
13.3	CB&I Federal Services LLC, 2011. EPA T&E Contract Administrative SOP 102, Data Review
and Verification.
13.4	CB&I Federal Services LLC, 2010. EPA T&E Contract Technical SOP 208, Operation of the
Distribution System Simulator (DSS) Pipe-loop System.
13.5	CB&I Federal Services LLC, 2012. EPA T&E Contract Technical SOP 309, Preparation and
Enumeration of B. globigii Endospores.

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Appendix B: T&E SOP 304: Heterotrophic Plate Count (HPC) Analysis
Using IDEXX SimPlate® Method
A
SKavr
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

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SOP 304, Heterotrophic Plate Count Analysis
Revision Number: 1
Date; 02/08/2012
Page 2 of 10
SOP Approval
E. Raciha Krishnan, P.E,
Program Manager
| ' -Y,.	Vs- t, t ¦ ^	t\J ! i'} tx/ J
Signature ~~	Date
Steven Jones, ASQ CQA/CQE
Quality Assurance Manager
Signature
Date

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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.

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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.

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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.

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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.

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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
3i5
7.7
23
5.3
35
8.0
24
5.6
3a
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).

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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 UPC
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
MPN
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).

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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:

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Appendix C: Safety Data Sheet for EDTA
3050 Spruce Street
Saint Louis, Missouri 63103 USA
Telephone 800-325-5832 • (314) 771-5765
Fax (314) 286-7828
email: techserv@sial.com
sigma-aldrich.com
SIGMA
Ethylenediaminetetraacetic acid disodium salt dihydrate
Product Number E5134
Store at Room Temperature
Product Description
Molecular Formula: CioHi4N2Na208 • 2H20
Molecular Weight: 372.2
CAS Number: 6381-92-6
Melting Point: 248 °C
pKa: 2.0, 2.7, 6.2, 10.31
Synonyms: EDTA, (Ethylenedinitrilo)tetraacetic acid
This product is designated as Molecular Biology grade
and is suitable for molecular biology applications. It
has been analyzed for the presence of nucleases and
proteases.
EDTA is an inhibitor of metalloproteases, at effective
concentrations of 1-10 |iM. EDTA acts as a chelator
of the zinc ion in the active site of metalloproteases,
and can also inhibit other metal ion-dependent
proteases such as calcium-dependent cysteine
proteases. EDTA may interfere with biological
processes which are metal-dependent.2
For use as an anticoagulant, disodium or tripotassium
salts of EDTA are most commonly used. The optimal
concentration is 1.5 mg per ml of blood. EDTA
prevents platelet aggregation and is, therefore, the
preferred anticoagulant for platelet counts.3 Using a
2% EDTA solution, 1-2 drops per ml of whole blood
can be used as an anticoagulant.
A procedure for a chromogenic assay of EDTA has
been published.4
Precautions and Disclaimer
For Laboratory Use Only. Not for drug, household or
other uses.
Productlnformation
Preparation Instructions
This product is slowly soluble in water at room
temperature up to 0.26 M, which is approximately
96 mg in a final volume of 1 ml. The pH of this
solution will be in the range of 4 to 6. EDTA salts are
more soluble in water as the pH increases: the more
EDTA there is in the salt form, the higher the pH of a
water solution, and therefore, the higher the room
temperature solubility. This can be achieved by a
gradual addition of concentrated sodium hydroxide
solution to the EDTA solution.
Storage/Stability
A stock solution of 0.5 M at pH 8.5 is stable for months
at 4 °C.2
Solutions of EDTA may be autoclaved.
References
1.	Data for Biochemical Research, 3rd ed., Dawson,
R. M. C., et al., Oxford University Press (New
York, NY: 1986), p. 404.
2.	Proteolytic Enzymes: A Practical Approach, 2nd
ed., Beynon, R. and Bond, J. S., eds., Oxford
University Press (Oxford, UK: 2001), p. 322.
3.	Clinical Hematology: Principles, Procedures,
Correlations, ed. Lotspeich-Steininger, C. A., et
al., Lippincott (Philadelphia, PA: 1992), p. 18.
4.	Sorensen, K., An Easy Microtiter Plate-based
Chromogenic Assay for
Ethylenediaminetetraacetic Acid and Similar
Chelating Agents in Biochemical Samples. Anal.
Biochem., 206(1), 210-211 (1992).
GCY/RXR 11/02
Sigma brand products are sold through Sigma-Aldrich, Inc.
Sigma-Aldrich, Inc. warrants that its products conform to the information contained in this and other Sigma-Aldrich publications. Purchaser
must determine the suitability of the product(s) for their particular use. Additional terms and conditions may apply. Please see reverse side of
the invoice or packing slip.

-------
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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