United States

Environmental Protection

Agency

EPA/600/C-10/010 | November 2010 | www.epa.gov/ord

Data Set: Escherichia coli

Persistence on Unlined Iron in 

Chlorinated and Chloraminated

Drinking Water

Office of Research and Development

National Homeland Security Research Center

Data Set: Escherichia coli

Persistence on Unlined Iron in

Chlorinated and Chloraminated

Drinking Water

U.S. ENVIRONMENTAL PROTECTION AGENCY

OFFICE OF RESEARCH AND DEVELOPMENT

NATIONAL HOMELAND SECURITY RESEARCH 

CENTER

CINCINNATI, OH 45268

CONTRACT NO. EP-C-04-034

Office of Research and Development

National Homeland Security Research Center
ii
iii

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.jeff@epa.gov

If you have difficulty accessing this PDF document, please contact Kathy Nickel 

(Nickel.Kathy@epa.gov) or Amelia McCall (McCall.Amelia@epa.gov) for assistance. 
iv
v

Contents

Disclaimer...........................................................................................................................................iii

List of Tables.......................................................................................................................................vi

List of Acronyms...............................................................................................................................vii

Executive Summary...........................................................................................................................ix

1.0 Introduction .................................................................................................................................. 1

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
vi

List of Tables

Table 1. Conditioning Period Water Quality................................................................................................... 3

Table 2. E. coli Persistence Data (Chlorinated Water).................................................................................... 6

Table 3. E. coli Persistence Data (Chloraminated Water)............................................................................... 7

Table 4. Impact of Corrosion Material on E. coli Count with Colilert® Reagent............................................ 7
vii

List of Acronyms

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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ix

Executive Summary

Data on Escherichia coli persistence are presented. Unlined iron coupons were conditioned in chlorinated and 

chloraminated 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 

the reactors with the water dechlorinated, with the chlorine residual present, and with dechlorination during injection 

but a disinfectant residual added thereafter. Colilert® 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.
x
1

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/nhsrc/). 

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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3

2.0

Materials and Methods

2.1 Experimental System

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, Bozeman, 

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 cm/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 BARs 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 Cl2: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 Free Chlorine

Mean ± SD

Chloramine

Mean ± SD

Free/Total Chlorine (mg/L) 0.90±0.05 1.95±0.36

pH 8.64±0.06 8.89±0.31

Temperature (oF) 65.3±3.2 72.4±2.0

2.2 Contamination

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. coli was pulse injected at a 

target initial density of 107 MPN/mL 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 run 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.
4

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 37o C for 24 hr. E. coli was enumerated using Colilert® reagent for detection of E. coli and 

Quanti-Tray/2000® (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-Tray/2000 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 particulate 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 

(α=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 Reactor Results

E. 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 E. 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 Reactor Results

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 Comparison with and without Corrosion

Results are presented in Table 4. The t-test results showed no difference between the means of the two data sets 

(P=0.91), so the null hypothesis was accepted.
6

Table 2. E. coli Persistence Data (Chlorinated Water)

Experiment 1-Full Dechlorination

Initial Bulk Phase (Calculated) = 7.4E+07 (MPN/mL)

Time

(h)

Coupon Density

(MPN/cm2)

Bulk Phase Density

(MPN/mL)

Reactor 1 Reactor 2 Reactor 1 Reactor 2

0.01 1.14E+04 3.46E+04 1.95E+07 2.23E+07

0.25 4.35E+04 9.73E+04 1.59E+07 1.46E+07

0.50 3.10E+04 2.72E+05 1.26E+07 1.18E+07

0.75 3.10E+04 1.35E+05 1.59E+07 1.26E+07

1.0 3.66E+04 9.17E+04 1.03E+07 6.51E+06

2.0 1.60E+04 1.79E+04 1.20E+02 2.42E+02

4.0 1.57E+04 4.46E+04 7.94E+00 1.12E+02

8.0 1.48E+04 1.35E+05 4.79E+00 6.13E+01

25.50 1.09E+03 3.83E+02 2.79E+00 1.00E-01

48.00 5.75E+02 5.75E+02 7.50E-01 0.00E+00

72.00 0.00E+00 2.00E+00 0.00E+00 0.00E+00

96.00 0.00E+00 0.00E+00 0.00E+00 0.00E+00

120.00 0.00E+00 0.00E+00 0.00E+00 0.00E+00

Experiment 2-Partial Dechlorination 

Initial Bulk Phase (Calculated) = 6.4E+07 (MPN/mL)

Time

(h)

Coupon Density

(MPN/cm2)

Bulk Phase Density

(MPN/mL)

Reactor 1 Reactor 2 Reactor 1 Reactor 2

0.01 3.46E+04 8.65E+04 1.74E+07 2.72E+07

0.50 7.71E+04 6.15E+04 2.25E+04 7.45E+05

1.00 2.65E+04 1.95E+05 2.80E+05 7.24E+05

2.00 1.47E+02 6.07E+02 0.00E+00 0.00E+00

4.00 4.56E+01 3.50E+02 0.00E+00 0.00E+00

8.00 4.56E+01 1.22E+02 0.00E+00 0.00E+00

24.00 0.00E+00 1.00E+00

48.00 0.00E+00 1.00E+00

72.00 0.00E+00 0.00E+00

Experiment 3-Full Chlorination

Initial Bulk Phase (Calculated) = 6.4E+07 (MPN/mL)

Time 

(h)

Coupon Density

(MPN/cm2)

Bulk Phase Density

(MPN/mL)

Reactor 1 Reactor 2 Reactor 1 Reactor 2

0.01 6.13E+02 3.45E+02 2.42E+02 2.42E+02

1.00 3.36E+01 4.79E+01 4.35E+01 3.87E+01

2.00 0.00E+00 0.00E+00 0.00E+00 0.00E+00

24.00 0.00E+00 8.60E+00 0.00E+00 0.00E+00

48.00 0.00E+00 0.00E+00 0.00E+00 0.00E+00

MPN, most probable number
7

Table 3. E. coli 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)

Coupon Density

(MPN/cm2)

Bulk Phase Density

(MPN/mL)

Reactor 1 Reactor 2 Reactor 1 Reactor 2

0.01 2.42E+05 2.42E+05 2.42E+02 2.42E+02

1.00 1.02E+06 2.61E+06 2.42E+02 2.42E+02

2.00 1.99E+03 9.70E+02 4.10E-01 1.19E+01

4.00 3.65E+02 3.65E+02 2.00E-01 0.00E+00

6.00 6.38E+01 2.61E+02 0.00E+00 0.00E+00

25.99 0.00E+00 1.00E+00 0.00E+00 0.00E+00

48.00 0.00E+00 0.00E+00

Experiment 3-Full Chloramination

Initial Bulk Phase Density (Calculated) = 1.35E+06 (MPN/mL)

Time (h)

Coupon Density

(MPN/cm2)

Bulk Phase Density

(MPN/mL)

Reactor 1 Reactor 2 Reactor 1 Reactor 2

0.01 6.87E+04 2.30E+04 2.42E+04 2.42E+04

1.00 3.10E+00 0.00E+00 0.00E+00 0.00E+00

2.00 2.00E+00 4.10E+00 6.30E-01 6.30E-01

24.00 0.00E+00 0.00E+00 0.00E+00 0.00E+00

Table 4. Impact of Corrosion Material on E. coli Count with Colilert® Reagent

Sample Large 

Wells

Small 

Wells

Colilert 

Count 

(MPN/

100 mL)

Titer (MPN)

(Sample Volume = 100 mL) %RSD

t-test 

P

(α=0.05)

Corrosion 

Mass 

Added(g)

Sample Sample 

Mean

Standard 

Deviation

Dilution Blank (Buffer) 0 0 0 0

Coupon Blank 

(Buffer + coupon)

0 0 0 0 0.5167

1 (Buffer + E. coli 49 43 1413.6 141360

1.41E+05 1.88E+04 13.3%

0.91

2 (Buffer + E. coli ) 49 42 1299.7 129970

3 (Buffer + E. coli) 49 42 1299.7 129970

4 (Buffer + E. coli ) 49 42 1299.7 129970

5 (Buffer + E. coli ) 49 45 1732.9 173290

6 (Buffer + E. coli + coupon) 49 40 1119.9 111990

1.42E+05 2.35E+04 16.5%

0.5112

7 (Buffer + E. coli + coupon) 49 42 1299.7 129970 0.5135

8 (Buffer + E. coli + coupon) 49 43 1413.6 141360 0.5116

9 (Buffer + E. coli + coupon) 49 45 1732.9 173290 0.5113

10 (Buffer + E. coli + coupon) 49 44 1553.1 155310 0.5131
8
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4.0

References

Szabo, J.G., Rice, E.W. and Bishop, P.L. (2006). Persistence of Klebsiella pneumoniae on simulated biofilm 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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