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CHLORINE CONCENTRATION AND BACTERIAL
PRESENCE BETWEEN POOL CLEANINGS.
Samuel Hernandez and Robert McLaughlin. Department of Biological
Sciences, Saddleback College, Mission Viejo, CA, 92692.
Abstract
BackgroundIntroduction
Water is among one of the most valuable resources on earth. It is essential for life on
earth as we know it. For humans alone, it is required to sustain life, develop agriculture,
and even provide the popular recreational activity of swimming. The prevalent use and
dependence on water in society today also has the effect of increasing our risk of
exposure to waterborne illnesses. A report by Elizabeth Hilborn found that in the 20092010 year, 33 cases of water-associated outbreaks were reported to the CDC. These
outbreaks comprised more than 1000 cases of illness, almost 100 hospitalizations, and
even death (3). To reduce this the risk of water-associated outbreaks, regulations are in
place to ensure that water supplies are disinfected and are kept sanitary up until for use or
consumption; one such regulation is the chlorination of water for use in swimming pools.
Chlorine is currently the disinfectant of choice among recreational swimming pools.
A study by Rice, Clark, and Johnson in 1998 found that only a 1.1 mg/L of concentrated
chlorine, the average concentration in municipal water supplies, could deactivate E. coli
in a matter of minutes (4). Chlorine works by first forming hypochlorous acid (HOCl).
The hypochlorous acid then attacks lipids in the cell wall and enzymes inside the cell,
denaturing them and rendering them inoperable in the organism. According to the CDC,
current standards require chlorine concentrations to be between 1-3 mg/L and to have a
pH somewhere between 7 and 8 (5). Chlorine must also be regularly added into the
system, as organic contaminants or sunlight can extract free chlorine out of the system,
rendering it useless for disinfection.
Despite standardized regulations for recreational water disinfection, many public
waters are not maintained appropriately. According to a collection of studies reported by
the CDC in the Morbidity and Mortality Weekly Report in 2008, 15 state and local
agencies across the US inspected 120,020 swimming pools. Of those inspected, over 12
thousand had a violation due to inadequate disinfectant, and another 13,532 pools had to
be closed due to serious violations that threatened public health (1). A 2012 study in
Atlanta, Georgia of done on 161 swimming pool backwash filter samples found that 58%
had samples of nucleic acids belonging to E. coli, which strongly suggested fecal
contamination by swimmers (1).
Chlorine concentrations will be examined over time between one pool cleaning cycle,
and water samples will be collected over the period of time to test for the presence of E.
coli bacteria. Based on past research, it is predicted that as chlorine concentration in pool
water decreases, bacterial presence will increase.
Materials and Methods
The Solana community pool in Rancho Santa Margarita, California was chosen to be
the collection site of twelve water samples over a 24 hour period between pool cleanings
on November 24, 2013 and November 25, 2013. Sets of three 50 mL samples were
collected at four times a day at 9:00 AM, 3:00 PM, 9:00 PM, and on the next day at 7:00
AM, with each sample coming from a different site in the pool (North, South and East).
The samples were tested for chlorine concentration using SenSafe Chlorine Test Strips.
36 tubes of triple strength and 72 tubes of single strength Lactose broths were prepared in
and autoclaved to ensure sterility of equipment. Using aseptic technique, the following
volumes of sample were instilled into a lactose broth tube: 10 ml in each triple strength
tube, 1 ml in 36 single strength tubes, and 0.1 ml in the other 36 single strength tubes.
The lactose tubes were stored in an incubator for 4 days at 37oC. After the incubation
period days, the samples were removed from the incubator and examined for color
change from green to yellow, which would indicate a change in pH secondary to
fermentation, and gas production.
Results
Chlorine concentration was found to decrease over time (figure 1). Statistical analysis
by ANOVA found p to be less than 0.05, therefore showing statistical difference in the
results. The bonferroni correction found this significance to be when comparing between
samples 1 and samples 2 through 4, while no significant difference was found to exist
between samples 2, 3, and 4. Presumptive testing with the lactose broth solutions had one
sample belonging to group 4 turn yellow, suggesting pH change by fermentation. Gas
production however, was not observed in that or any other tube. The absence of
fermentation and gas production together strongly suggests that no E. coli was cultured in
the lactose broths, and the test was negative for E. coli.
Sample
collection
9:00 AM
Chlorine
concentration
(ppm)
1.40
Fermentation/Gas
Production
Fermentation/Gas
Production
Fermentation/Gas
Production
10 ml tubes
1.0 ml tubes
0.1 ml tubes
0
0
0
MPN
< 0.5
3:00 PM
0.73
0
0
0
< 0.5
9:00 PM
0.47
0
0
0
< 0.5
7:00 AM
0.43
0
0
0
< 0.5
1.80
Chlorine Concentration (mg/l)
1.60
1.40
1.20
1.00
Table
1. Chlorine concentrations demonstrating a decline in chlorine concentration
0.80
over time. No tubes demonstrated any form of fermentation and gas production
0.60
0.40
0.20
0.00
0
200
400
600
800
1000
Time (min)
1200
1400
1600
Figure 1. Decline in chlorine concentrations over time
Discussion
The initial drop in chlorine concentration was statistically significant, dropping below
the 1.0 mg/L line that is the current standard in pool disinfection by chlorination. Based
on prior research, this suggests it is possible that E. coli bacteria could colonize and grow
in the pool water. The presumptive test produced one test tube with a color change to
yellow due to pH change secondary to bacterial fermentation. The bacteria in this tube
did not produce any gas, making the presumptive test negative for E. coli. Based on the
results obtained, it does not support that as concentration decreases, bacterial presence
increases.
References Cited
1) CDC. Violations Identified from Routine Swimming Pool Inspections --- Selected
States and Counties, United States, 2008. Morbidity and Mortality Weekly
Report. May 21, 2010. 2013 Dec. 59(19);582-587.
http://www.cdc.gov/mmwr/preview/mmwrhtml/mm5919a2.htm
2) CDC. Microbes in Pool Filter Backwash as Evidence of the Need for Improved
Swimmer Hygiene — Metro-Atlanta, Georgia, 2012. Morbidity and Mortality
Weekly Report, May 17, 2013. 2013 Dec. 62(19);385-388.
http://www.cdc.gov/mmwr/preview/mmwrhtml/mm6219a3.htm?s_cid=mm6219a
3_w
3) Hilborn, Elizabeth D., D.V.M. 2013, Surveillance for Waterborne Disease
Outbreaks Associated with Drinking Water and Other Non-recreational Water United States, 2009-2010. Atlanta: U.S. Center for Disease Control, 2013.
4) Rice EW, Clark RM, Johnson CH. Chlorine Inactivation of Escherichia
coliO157:H7. Emerg Infect Dis [serial on the Internet]. 1999, Jun. 2013 Dec.
http://wwwnc.cdc.gov/eid/article/5/3/99-0322.htm
5) http://www.cdc.gov/safewater/publications_pages/chlorineresidual.pdf
Review Form
Department of Biological Sciences
Saddleback College, Mission Viejo, CA 92692
Author (s): Hernandez & McLaughlin
Title: CHLORINE CONCENTRATION AND BACTERIAL PRESENCE BETWEEN POOL
CLEANINGS
Summary
Summarize the paper succinctly and dispassionately. Do not criticize here, just show that you understood the
paper.
The authors investigated the change in concentration of chlorine and the presence of
coliform bacteria (E. coli) in Solana community pool. They hypothesized that based on
previous studies, as the chlorine concentration decreases over time, the conditions would
become suitable for bacterial growth, and therefore the growth of E. coli would increase.
The collected water samples were tested for chlorine concentration and also for the
presence of bacteria using the presumptive test. The results showed a significant decrease
in chlorine concentration but no gas or fermentation was observed, which lead to the
conclusion that there was no bacteria present even after the chlorine concentration
decreased.
General Comments
Generally explain the paper’s strengths and weaknesses and whether they are serious, or important
to our current state of knowledge.
Strengths:
The introduction was strong. By mentioning previous studies and the statistics revealing
the prevalence of water-borne disease, the authors successfully showed the urgency for
more studies on pools water. The methods and results sections were relatively strong. The
statistical analysis used was relevant and enough details were given in the methods section.
Improvement can be made by fixing the errors and adding the proper citations and figure
captions.
Weaknesses:
The discussion was the weakness of the paper. The results were not tied in to other studies
and data were just restated and not discussed. Some paragraphs and sentences need
revision as marked by the comments.
Technical Criticism
Review technical issues, organization and clarity. Provide a table of typographical errors,
grammatical errors, and minor textual problems. It's not the reviewer's job to copy Edit the paper,
mark the manuscript.
This paper was a final version
This paper was a rough draft
The paper was organized in the proper format. No major grammatical errors were present,
but the figure captions need revision. (l) that stands for liters needs to be changed to (L).
Sentences need to be reworded to improve clarity. Comments were made throughout the
text to help authors with revision.
Recommendation
 This paper should be published as is
 This paper should be published with revision
 This paper should not be published