CHLORINE CONCENTRATION AND BACTERIAL PRESENCE BETWEEN POOL CLEANINGS. Samuel Hernandez and Robert McLaughlin. Department of Biological Sciences, Saddleback College, Mission Viejo, CA, 92692. Abstract Background 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 study 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 risk, regulations are in place to ensure water supplies are disinfected and kept sanitary up until use or consumption. One such regulation is the chlorination of water for use in swimming pools. Chlorine is (the most common) 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 13 mg/l and 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(facilities) 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 161 swimming pool backwash filter samples found that 58% had samples of nucleic acids belonging to E. coli, which strongly suggests 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 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 4 times of day: 9:00 AM, 3:00 PM, 9:00 PM, and 7:00 AM the next day, 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. Lactose broths were prepared in triple strength (36 tubes) and single strength (72 tubes) 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 37 oC. After 4 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. Result Chlorine concentration was found to decrease over time (figure 1). Statistical analysis by ANOVA found statistical significance. The bonferroni correction found this significance to be when comparing between sample 1 to samples 2 through 4, while no significant difference was found to exist between samples 2(two, not 2… for the rest as well), 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 Chlorine concentration (ppm) Fermentation/Gas Production Fermentation/Gas Production Fermentation/Gas Production 10 ml tubes 1.0 ml tubes 0.1 ml tubes MPN 9:00 AM 1.40 0 0 0 < 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 Table 1. Chlorine concentrations demonstrating a decline in chlorine concentration over time. No tubes demonstrated any form of fermentation and gas production 1.80 Chlorine Concentration (mg/l) 1.60 1.40 1.20 1.00 0.80 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 ( I just italicized it )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 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 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=mm6219a3_w 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. 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 http://www.cdc.gov/safewater/publications_pages/chlorineresidual.pdf Delete Replace Move The paper was well written, there are very few correction I found, the only thing is if you are citing the source at the end of the sentence, it is overkill to also cite it at the beginning. You should be able to just start a sentence, the reader will know its from that article. Also, there is different spacing in your paper, its on 1.0 and 1.15, I’m changing it to 1.0 all around. Furthermore, the Methods, results, into… should be in the center of the page according to Teh. Lastly, you don’t need to number your sources. Just indent the ones after the first line. Is the last one officially cited? Review Form Department of Biological Sciences Saddleback College, Mission Viejo, CA 92692 Author (s): Samuel Hernandez and Robert 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. This paper was about chlorine concentrations in pools and how effective cleaning is. Many pools are not cleaned properly, so they collected data at a pool to see how much chlorine was found in the pool between cleaning it, and they found E. coli in the pool, and there was an insufficient amount of chlorine to clean the pool after the first sample of data. General Comments Generally explain the paper’s strengths and weaknesses and whether they are serious, or important to our current state of knowledge. There were no grammatical errors that I caught, the paper was well written. The methods were clear to me, and I could follow everything you said. Weaknesses found were minor with formatting. The references were not done correctly, and the into, methods, results and discussion needed to be centered on the page. 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. X This paper was a final version This paper was a rough draft The paper was well written, there are very few correction I found, the only thing is if you are citing the source at the end of the sentence, it is overkill to also cite it at the beginning. You should be able to just start a sentence, the reader will know its from that article. Also, there is different spacing in your paper, its on 1.0 and 1.15, I’m changing it to 1.0 all around. Furthermore, the Methods, results, into… should be in the center of the page according to Teh. Lastly, you don’t need to number your sources. Just indent the ones after the first line. Is the last one officially cited? Recommendation This paper should be published as is This paper should be published with revision This paper should not be published