EPA/600/C-10/010 | November 2010 | www.epa.gov/ord
United States
Environmental Protection
Agency
              Data Set:  Escherichia coli
              Persistence on Unlined Iron in
              Chlorinated and Chloraminated
              Drinking Water
Office of Research and Development
National Homeland Security Research Center

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                                on                  in
               U.S. ENVIRONMENTAL PROTECTION AGENCY
               OFFICE OF           AND DEVELOPMENT
               NATIONAL HOMELAND SECURITY
               CENTER
               CINCINNATI, OH

               CONTRACT NO. EP-C-04-034
Office of Research and Development
National Homeland Security Research Center

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                                                              Disclaimer
The U.S. Environmental Protection Agency through its Office of Research and Development funded
and collaborated in the research described herein under contract number EP-C-04-034 with Shaw
Environmental and Infrastructure, Inc. It has been subject to an administrative review but does not
necessarily reflect the views of the Agency. No official endorsement should be inferred. EPA does
not endorse the purchase or sale of any commercial products or services.
Questions concerning this document or its application should be addressed to:
Jeffrey Szabo
National Homeland Security Research Center (NG-16)
Office of Research and Development
United States Environmental Protection Agency
26 W. Martin Luther King Dr.
Cincinnati, OH 45268
(513)487-2823
szabo j eff@epa.gov
If you have difficulty accessing this PDF document, please contact Kathy Nickel
(Nickel.Kathy@epa. govl or Amelia McCall (McCall. Amelia@,epa.govl for assistance.

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Disclaimer	iii
List of Tables	vi
List of Acronyms	vii
Executive Summary	ix
1.0 Introduction 	I
2.0 Materials and Methods	3
    2.1 Experimental System	3
    2.2 Contamination	3
    2.3 E. coli Culturing and Analysis	4
    2.4 Comparison of the Colilert Assay with and without Corrosion Present	4
3.0 Results	5
    3.1 Chlorinated Reactor Results	5
    3.2 Chloraminated Reactor Results	5
    3.3 Colilert Comparison with and without Corrosion	5
4.0 References	9

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Table 1. Conditioning Period Water Quality	3
Table 2. E. coli Persistence Data (Chlorinated Water)	6
Table 3. E. coli Persistence Data (Chloraminatcd Water)	7
Table 4. Impact of Corrosion Material onE. coli Count with Colilert* Reagent	7

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                                               List
ATCC          American Type Culture Collection
BAR           Biofilm Annular Reactor
hr             Hour
MPN           Most Probable Number
NHSRC        National Homeland Security Research Center
rpm            Revolutions per minute
SD             Standard  deviation
%RSD          Relative Standard Deviation

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Data on Escherichia colt persistence are presented.  Unlined iron coupons were conditioned in chlorinated and
chloraminatcd water for two months in biofilm annular reactors (dynamic ring-shaped growth chambers for culturing
aggregates of microbes on solid surfaces) under controlled conditions. E. coli suspensions were pulse injected into
Hie reactors with the water dechlorinated, with the chlorine residual present, and with dechlorination during injection
but a disinfectant residual added thereafter. Colilerl® reagent and Quanti-Trays* were used to enumerate the numbers
of viable E. coli persisting on the coupons (excised surface samples) and in the bulk water phase. E. coli persistence
on the coupons was not observed for more than 3 days under the most favorable conditions  (no disinfectant residual).
When disinfectant residual was present, either after injection of E. coli or during the entire experiment, persistence of
less than one day was observed.

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                                                                                                 1.0
                                                                             Introduction
Concern over the risk for deliberate contamination of drinking water systems, and research aimed at mitigating this
risk, has intensified with the spate of global terrorist activity. The data in this report describes the persistence of
Escherichia coli on corroded iron surfaces in chlorinated and chloraminated drinking water. The data were generated
for a research project specifically aimed at developing a model for pathogen attachment and detachment in drinking
water distribution system infrastructure. The project was a collaborative effort between EPA/NHSRC and Pegasus
Technical Services (Contract EC-C-05-056).  Validation of attachment/detachment models requires experimental
data. Some of this data has been acquired from previous studies reported in the literature (Szabo et al. 2006; Szabo
et al, 2007).  However, it was found that the sampling intervals were too long to draw conclusions about the model's
representation of the data. Therefore, it was determined that controlled experiments would be performed under
conditions similar to Szabo et al. (2006, 2007), but with shorter sampling intervals.
The goal of this data report is two-fold: 1) summarize E. coli persistence data for the aforementioned project and 2)
provide the data and data collection methods to the broader research community. This document, containing both
the data collection methods and the data, is available online through the NHSRC Web site (www.epa.gov/nhsrcA.
Therefore, any publications generated from the aforementioned project can reference this data and the data is easily
accessed. In addition, the data provided in this open format is accessible to and available for use by other researchers
in the fields of microbiology, data modeling or water quality.

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                                                                                                2.0
                                                   Materials  and  Methods
2.1
Modified methods in Szabo et al. (2006, 2007) were utilized in this study. Drinking water distribution system
conditions were simulated using Biofilm Annular Reactors (BAR) (BioSurface Technologies Corporation, Bozernan,
Montana) (ring-shaped growth chambers for culturing aggregates of microbes on solid surfaces) under controlled
shear conditions. The BAR contains 20 flush-mounted rectangular polycarbonate slides attached to a rotating
polycarbonate cylinder, which is inside of a stationary glass outer cylinder. The  rotational speed of the inner drum
was set to 100 rpm for all experiments, which generates a shear on the inner drum equivalent 30.5 crn/s flow in a 10.2
cm diameter pipe.  However, this calculation is only valid for a smooth inner drum.  The biofilm and corrosion layers
protruded from the polycarbonate slide surface as they formed, thus the exact shear was not able to be determined at
the rough biofilm/corrosion surface. So, the value reported for shear is an estimate of the flow conditions.
Components of the B ARs were cleaned with soap and water and rinsed with tap  water before assembly. The
assembled reactors were filled with a 100 mg/L free chlorine solution, which was circulated by rotating the drum at
100 rpm for 2 hr.  The reactor was then drained and flushed with tap water.
A mixture of corrosion and biofilm was developed on iron coupons before contamination experiments. Coupons were
made of 99.5% pure iron, and each had a surface area of 1 cm2 and thickness of 0.05 cm. Coupons were attached to
the polycarbonate slides with acrylic cement (TAP Plastics, Oakland, California). The slides/coupons were installed
after reactor disinfection, and then the flow to the reactor commenced. Flow rate was maintained at 200-300 mL/min
during experimentation, so mean residence time in the reactor was approximately 3.3-5.0 min. This range keeps the
reactor well flushed, which minimizes the potential for re-adherence of spiked microorganisms after contamination.
Coupons were conditioned in this manner for two months.
Coupon conditioning and contamination experiments were conducted in both chlorinated and chloraminated water.
Chlorinated Cincinnati tap water was supplied from a tap in the laboratory. Chloraminated water was made by adding
ammonia to Cincinnati tap water in a C1,:N ratio of 4:1.  Water was fed to the reactors from a 15.2 cm diameter pipe
loop that was constantly supplied with chloraminated water. Key water quality parameters measured for the duration
of the study are summarized in Table 1 as the mean ± standard deviation. Free chlorine data was acquired from online
free chlorine sensors that recorded data continuously. Sensors were polled every two minutes, which resulted in
43,200 data points being used to calculate the mean and standard deviation over  the two month conditioning period.
Water quality data from the chloraminated pipe loop was acquired through daily  grab samples resulting in 60 data
points.
                               Table 1. Conditioning Period Water Quality
Parameter
FreeATotal Chlorine (mg/L)
pH
Temperature (T)
Free Chlorine
Mean ± SD
0.90±0.05
8.64±0.06
65.3±3.2
Chloramine
Mean ± SD
1 .95±0.36
8.89±0.31
72.4±2.0
2.2
Experiments were performed in BAR with chlorinated water and with chloraminated water. Reactor contamination
was performed three ways (Experiments 1, 2 and 3 described below) in the chlorinated reactors and two ways
(Experiments 2 and 3) in the chloraminated reactors.  In all contamination experiments. E. co/i was pulse injected at a
target initial density of 10" MPN/inL in the bulk water phase.  BAR rotational speed was 100 rpm.  Immediately before
E. coli pulse injection, flow to the reactor was stopped and it was ran in batch mode for 1 hr after injection (with the
inner drum rotating), after which flow was reintroduced.  This 1 hr period allowed the E. coli a chance to adhere to the
coupons in the presence of shear without being washed out of the reactor. Sampling intervals varied during the first
day after E. coli injection, but coupon samples were always removed 1 day after injection and further samples were
withdrawn at 1 day intervals, if necessary. Samples consisted of 10 mL of bulk phase water and one iron coupon.

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Experiment 1: Full Dechlorination
Tap water in the biofilm annular reactors was dechlorinated by co-injecting 1 mL of a 10% sodium thiosulfate solution
with E. coli before the 1 hr batch contact period. After flow was reestablished, a 10% sodium thiosulfate solution was
continuously injected with a syringe pump at 2 mL/hr for the rest of the experiment. Dechlorination occurred when
flow was present and during the 1 hr period of batch operation.  Coupons and bulk phase samples were extracted at 30
sec, 15 min, 30 min, 45 min, 1 hr, 2 hr, 4 hr,  8 hr and 24 hr after injection, and thereafter as necessary.
Experiment 2: Dechlorination During Injection
Biofilm annular reactors were dechlorinated or dechloraminated during injection by co-injecting 1 mL of a 10%
sodium thiosulfate solution with E. coli. Dechlorination or de chloramination only occurred during the 1 hr period of
batch operation. After the 1 hr batch operation, flow was re-established; disinfectant residual then returned within a
few minutes. Coupons and bulk phase samples were extracted at 30 sec, 1 hr, 2 hr, 4 hr and 8 hr after injection, and
thereafter as necessary.
Experiment 3: Full Chlorination/Chloramination
Tap water flowing through the biofilm annular reactors retained its chlorine residual for the entire experiment. This
includes the  1 hr batch operation period.  Coupons and bulk phase samples were extracted at 30 sec, 1  hr and 2 hr, and
thereafter as necessary.

2.3 E. coli Culturing and Analysis
E. coli  K-12 (ATCC 25204) was used in all experiments. E. coli was subcultured  in Terrific broth by inoculating the
broth and incubating at 37° C for 24 hr. E. coli was enumerated using Colilert® reagent for detection ofE. coli and
Quanti-Tray72000® (Idexx Corporation, Westbrook, Maine), which produces the data needed for the most probable
number (MPN) enumeration method Homogenized corrosion material suspended in 100 mL of sterile 0.05M KH2PO4
buffer (pH 7.2) and 10 mL of bulk water from the BAR were used for enumeration of the biofilm and bulk water
phases, respectively. Ten fold serial dilutions were made using sterile buffer. Positive tray wells were counted after
incubation. Positive wells for the Colilert reagent are defined by a change in color from clear (water white) to yellow.
The most probable number (MPN) was determined using the Quanti-Tray72000 MPN Table (http://www.idexx.com/
pubwebresources/pdf/en_us/water/qt97mpntable.pdf).
In all experiments, coupons and bulk samples were removed and assayed before injection to assure that no E. coli
were detected. Sterile buffer used for dilution was also assayed during each experiment to ensure that it was sterile.
All control samples had zero positive wells and are not included in the tables in the following section.

2.4 Comparison of the  Colilert Assay with and without Corrosion  Present
The question arose during the study whether the Colilert analysis was influenced by paniculate matter from the
coupon samples. Ten 100 mL bottles of sterile buffer were spiked with the E. coli to achieve a density of around 15
MPN/mL. Five of these bottles were analyzed after spiking using the Colilert method described in section 2.3.  The
other five bottles had 0.511-0.517 g of ground coupon corrosion material from a chlorinated BAR experiment added.
This corrosion material was not exposed to E. coli.  These samples were also enumerated using Colilert. Two negative
controls were performed.  One was the sterile buffer (no E. coli added) and the other was the buffer and corrosion
material (no E. coli added). Both showed no positive wells, which was the expected result.
The enumeration results of five bottles with and without corrosion were compared using a two tailed paired t-test
(a=0.05). The null hypothesis was that the mean values of the bottles with and without corrosion were equal.

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                                                                                                3.0
                                                                                      Results
3.1  Chlorinated
£ coli persistence data in the chlorinated BAR is presented in Table 2. E. coli was detected up to three days (72
hr) after injection on the coupons in Experiment 1 (full dechlorination). In Experiment 2 (dechlorinated during the
one hr batch operation after injection), most E. coli persisted for 8 hr and disappeared by one day after injection.
However, one positive well was detected on a coupon sampled at 24 and 48 hr after injection. In Experiment 3 (full
chlorination),  no £  coli was observed on the coupon after 1 hr past injection.
The discrepancy between the expected initial E. coli bulk phase density and actual measured density may be due to
three factors. First, the actual liquid volume of the BAR is difficult to estimate.  The water level in the BAR can vary
depending on the amount of flow, which changes the overall volume, and flow can vary over time as pressure in the
distribution system changes. The calculated initial density was based on a 1000 mL volume, but the actual volume
is likely between 950 to 1000 mL.  Second, E. coli adsorbed to the iron coupons in the reactor, but the amount of
adsorbed E. coli on the iron coupons does not account for all of the missing mass in solution. Adsorption also likely
occurred to other reactor surfaces (largely polycarbonate and glass) but the amount was not quantified. Finally, some
E. coli was inactivated by the disinfectant residual when it was present.

3.2  Chloraminated
E. coli persistence data in the chloraminated BAR is presented in Table 3. A fully dechlorinated experiment was not
performed since it was not expected that the results would differ from the  same experiment described in section 3.1.
The 1 hr dechlorination experiment shows a 3-log E.  coli reduction from the coupon surfaces 2 hr after injection. One
positive well was observed in one experiment at 24 hr. but no E. coli were detected at 48 hr. When chloramine was
present during the entire experiment, most E. coli was gone from the coupon surfaces by 2 hr, and none was detected
at 24 hr after injection.
The same analysis of bulk phase concentration discussed for the chlorinated reactors in section 3.1 is also true for the
chloraminated reactors.

3.3  Colilert                 with      without Corrosion
Results are presented in Table 4. The t-test results showed no difference between the means of the two data, sets
(/>=(). 91), so the null hypothesis was accepted.

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          Table 2. E. coll Persistence Data (Chlorinated Water)
Experiment 1-Full Dechlorination
Initial Bulk Phase (Calculated) = 7.4E+07 (MPN/mL)
Time
(h)
0.01
0.25
0.50
0.75
1.0
2.0
4.0
8.0
25.50
48.00
72.00
96.00
120.00
Coupon Density
(MPN/cm2)
Reactor 1 Reactor 2
1.14E+04 3.46E+04
4.35E+04 9.73E+04
3.10E+04 2.72E+05
3.10E+04 1.35E+05
3.66E+04 9.17E+04
1.60E+04 1.79E+04
1.57E+04 4.46E+04
1.48E+04 1.35E+05
1.09E+03 3.83E+02
5.75E+02 5.75E+02
O.OOE+00 2.00E+00
O.OOE+00 O.OOE+00
O.OOE+00 O.OOE+00
Bulk Phase Density
(MPN/mL)
Reactor 1 Reactor 2
1.95E+07 2.23E+07
1.59E+07 1.46E+07
1.26E+07 1.18E+07
1.59E+07 1.26E+07
1.03E+07 6.51E+06
1.20E+02 2.42E+02
7.94E+00 1.12E+02
4.79E+00 6.13E+01
2.79E+00 l.OOE-01
7.50E-01 O.OOE+00
O.OOE+00 O.OOE+00
O.OOE+00 O.OOE+00
O.OOE+00 O.OOE+00
Experiment 2-Partial Dechlorination
Initial Bulk Phase (Calculated) = 6.4E+07 (MPN/mL)
Time
(h)
0.01
0.50
1.00
2.00
4.00
8.00
24.00
48.00
72.00
Coupon Density
(MPN/cm2)
Reactor 1 Reactor 2
3.46E+04 8.65E+04
7.71E+04 6.15E+04
2.65E+04 1.95E+05
1.47E+02 6.07E+02
4.56E+01 3.50E+02
4.56E+01 1.22E+02
O.OOE+00 l.OOE+00
O.OOE+00 l.OOE+00
O.OOE+00 O.OOE+00
Bulk Phase Density
(MPN/mL)
Reactor 1 Reactor 2
1.74E+07 2.72E+07
2.25E+04 7.45E+05
2.80E+05 7.24E+05
O.OOE+00 O.OOE+00
O.OOE+00 O.OOE+00
O.OOE+00 O.OOE+00



Experiment 3-Full Chlorination
Initial Bulk Phase (Calculated) = 6.4E+07 (MPN/mL)
Time
(h)
0.01
1.00
2.00
24.00
48.00
Coupon Density
(MPN/cm2)
Reactor 1 Reactor 2
6.13E+02 3.45E+02
3.36E+01 4.79E+01
O.OOE+00 O.OOE+00
O.OOE+00 8.60E+00
O.OOE+00 O.OOE+00
Bulk Phase Density
(MPN/mL)
Reactor 1 Reactor 2
2.42E+02 2.42E+02
4.35E+01 3.87E+01
O.OOE+00 O.OOE+00
O.OOE+00 O.OOE+00
O.OOE+00 O.OOE+00
MPN, most probable number

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              Table 3. E. coll Persistence Data (Chloraminated Water)
Experiment 2-Partial Dechloramination
(During 1 hr injection period)
Initial Bulk Phase Density (Calculated) = 1. 35E+06 (MPN/mL)
Time (h)
0.01
1.00
2.00
4.00
6.00
25.99
48.00
Coupon Density
(MPN/cm2)
Reactor 1 Reactor 2
2.42E+05 2.42E+05
1.02E+06 2.61E+06
1.99E+03 9.70E+02
3.65E+02 3.65E+02
6.38E+01 2.61E+02
O.OOE+00 l.OOE+00
O.OOE+00 O.OOE+00
Bulk Phase Density
(MPN/mL)
Reactor 1 Reactor 2
2.42E+02 2.42E+02
2.42E+02 2.42E+02
4.10E-01 1.19E+01
2.00E-01 O.OOE+00
O.OOE+00 O.OOE+00
O.OOE+00 O.OOE+00
Experiment 3-Full Chloramination
Initial Bulk Phase Density (Calculated) = 1.35E+06 (MPN/mL)
Time (fr)
0.01
1.00
2.00
24.00
Coupon Density
(MPN/cm2)
Reactor 1 Reactor 2
6.87E+04 2.30E+04
3.10E+00 O.OOE+00
2.00E+00 4.10E+00
O.OOE+00 O.OOE+00
Bulk Phase Density
(MPN/mL)
Reactor 1 Reactor 2
2.42E+04 2.42E+04
O.OOE+00 O.OOE+00
6.30E-01 6.30E-01
O.OOE+00 O.OOE+00
Table 4. Impact of Corrosion Material on E. coll Count with Colilert® Reagent
Sample
Dilution Blank (Buffer)
Coupon Blank
(Buffer + coupon)
1 (Buffer + E. coli
1 (Buffer + E. coli)
3 (Buffer + E. coli)
4 (Buffer + E.coli)
5 (Buffer + E.coli)
6 (Buffer + E. coli + coupon)
7 (Buffer + E. coli + coupon)
8 (Buffer + E. coli + coupon)
9 (Buffer + E. coli + coupon)
10 (Buffer + E. coli + coupon)
Large
Wells
0
0
49
49
49
49
49
49
49
49
49
49
Small
Wells
0
0
43
42
42
42
45
40
42
43
45
44
Colilert
Count
(MPN/
100 mL)
0
0
1413.6
1299.7
1299.7
1299.7
1732.9
1119.9
1299.7
1413.6
1732.9
1553.1
liter (MPN)
(Sample Volume = 100 mL)
Sample Standard
Sample »» ™ . ,.
Mean Deviation
0
0
141360
129970
129970
129970
173290
111990
129970
141360
173290
155310


1.41E+05
1.42E+05


1.88E+04
2.35E+04
%RSD

13.3%
16.5%
t-test
P
(a=0.05)


0.91
Corrosion
Mass
Added(g)

0.5167

0.5112
0.5135
0.5116
0.5113
0.5131

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                                                                                      4.0
                                                                     References
Szabo. J.G.. Rice, E.W. and Bishop, P.L. (2006). Persistence of Klebsiella pneumoniae on simulated biofi 1m in a
    model drinking water system. Environmental Science and Technology, 40(16), 4996-5002.
Szabo. J.G.. Rice, E.W. and Bishop, P.L. (2007). Persistence and decontamination of Bacillus atrophaeus subsp.
    globigii spores on corroded iron in a model drinking water system. Applied and Environmental Microbiology,
    73(8), 2451-2457.

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