Chlorination is the most popular method for disinfecting swimming pool water; however, although pathogens are being killed, many toxic compounds, called disinfection by-products (DBPs), are formed. Numerous epidemiological publications have associated the chlorination of pools with dysfunctions of the respiratory system and with some other diseases. However, the findings concerning these associations are not always consistent and have not been confirmed by toxicological studies. Therefore, the health effects from swimming in chlorinated pools and the corresponding stress reactions in organisms are unclear. In this study, we show that although the growth and behaviors of experimental rats were not affected, their health, training effects and metabolic profiles were significantly affected by a 12-week swimming training program in chlorinated water identical to that of public pools. Interestingly, the eyes and skin are the organs that are more directly affected than the lungs by the irritants in chlorinated water; instead of chlorination, training intensity, training frequency and choking on water may be the primary factors for lung damage induced by swimming. Among the five major organs (the heart, liver, spleen, lungs and kidneys), the liver is the most likely target of DBPs. Through metabolomics analysis, the corresponding metabolic stress pathways and a defensive system focusing on taurine were presented, based on which the corresponding countermeasures can be developed for swimming athletes and for others who spend a lot of time in chlorinated swimming pools.

Funding: This research was supported by the National Science Foundation of China (NSFC 21365013) and by the science project from the Jiangxi Provincial Education Department (GJJ 13240). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2015 Li et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Chlorination is the most popular method for disinfecting swimming pool water. However, although pathogens are being killed, many toxic compounds, called disinfection by-products (DBPs), are formed. Numerous publications have indicated that DBPs exposure may be related to several diseases [ 1 – 3 ], and Thomas Lachocki, the head of the National Swimming Pool Foundation of USA, has emphasized that the health benefits from swimming must be weighed against the risks of chemical exposure [ 4 ]. The epidemiological evidence for adverse health effects from swimming in chlorinated water primarily originate from studies concerning respiratory function and asthma, althoughVillanueva et al. reported a significant increased risk of bladder cancer for swimmers compared with nonswimmers [ 5 ]. The chlorination of pools has been associated with an increase in lung epithelium permeability [ 6 ], a risk of developing asthma [ 7 ], and with respiratory complaints [ 8 ]. Typically, trihalomethanes and trichloramines are blamed [ 4 ]. However, the findings regarding the association of chlorination with illness are not always consistent. Font-Ribera et al. reported that swimming did not increase the risk of asthma or allergic symptoms in British children [ 9 ] but was associated with slightly less respiratory tract symptoms [ 10 ], increased lung function and with a lower risk of asthma symptoms, particularly among children with preexisting respiratory conditions [ 9 ]. A meta-analysis performed by Goodman et al. demonstrated that the association between asthma and swimming could only be confirmed among competitive swimmers and could not be confirmed among non-competitive swimmers [ 11 ]. Extremely few toxicological studies have been performed in the area of swimming exposure and health thus far. Therefore, the health effects from swimming in chlorinated pools and the corresponding stress reactions occurring in our bodies are unclear. Generally, competitive swimmers are the most possible victims of DBPs exposure, because they have to do a lot of high intensive training in swimming pools for years. To reveal the health effects of DBPs exposure from swimming training, the experimental animals were trained in chlorinated water as competitive swimmers for twelve weeks in this study (according to the lifespan of the animals, twelve weeks for rats almost equals ten years for human being, which is a nessary period for an athlete to get a best performance). Their behaviors and appearances were observed during the training program, and then histopathological and metabolomic approaches were used to analyze the health effects and corresponding metabolic stress pathways.

Principal component analysis (PCA) was conducted on the urine metabolite data for pattern recognition using SIMCA-P+ software version 10.0 (Umetrics, Umea, Sweden). Before PCA, data were subjected to orthogonal signal correction (OSC) and unit variance scaling. Other statistical analyses were performed using IBM SPSS Statistics software version 20.0 (SPSS Inc., Chicago, IL, USA). The durations of the animals in swimming capacity test were expressed as the mean value ± standard deviation (SD) and compared using independent t-tests. The positive rates in histopathological analysis were evaluated by Chi-square (χ2) test. The significance level was set at 0.05.

All 1 H NMR Spectra were manually corrected for phase and baseline distortions and referenced to the TSP signal at 0 ppm using Top Spin software version 3.0 (Bruker Biospin, Germany). Integration was performed over a 10.00–0.02 ppm region, with a bucket width of 0.02 ppm. Regions corresponding to the spectrum signals of water and of urea (6.20–4.20 ppm) were excluded, and the integration of each region was normalized to the sum of the total spectrum to obtain the urine metabolite data ( S1 Table ).

Urine samples were thawed at room temperature, and then 400 μL urine was mixed with 200 μL phosphate buffer (pH 7.4,0.2 M NaH 2 PO 4 /Na 2 HPO 4 ), with 10% D 2 O as a field lock and with 0.05% sodium 3-trimethylsilyl-(2,2,3,3-2H4)-1-propionate (TSP) as a chemical shift reference. After centrifugation at 13000 g for 10 min, the supernatants were transferred into 5 mm NMR tubes and measured using a standard one dimensional 1 H pulse sequence with water suppression (Noesypresat) on a Bruker DRX400 spectrometer operating at 400.13 MHz 1 H resonance frequency and at 298 K.

The day after the swimming capacity test, animals were sacrificed with an overdose administration of pentobarbiturate (120 mg/kg). First, a gross anatomy dissection was performed on the animals, and tissues of their five major organs, i.e., heart, liver, spleen, lungs and kidneys, were collected and fixed with 4% formaldehydum polymerisatum. Twelve hours later, the fixed tissues were embedded in paraffin for sectioning, and then a regular histopathological analysis was performed using hematoxylin and eosin staining and optical microscopes.

The day before the swimming capacity test, 24-hour urine samples were collected with metabolic cages (NaN 3 preservation) and then centrifuged at 3000 r/min at 4°C for 10 min. Approximately 4 ml supernatant aliquots were transferred into 5 ml Eppendorf tubes and stored at −80°C for nuclear magnetic resonance (NMR) testing.

After the 12-week swimming training and one day of rest, a swimming capacity test was performed. The duration from entry into water until the exhaustion of each rat was recorded. The pool water used in the test was running water supplied by the municipal water company. To increase the intensity and to shorten the time, a 13.7 g screw nut was tied to the top end of the tail of each rat in the test. The exhaustion criterion was set such that rats remained submerged below the surface for ten seconds [ 13 ].

Two typical classes of chlorination DBPs, chloroform and chloramines, were measured at the 20th minute of every training session. Chloroform was measured using a gas chromatograph (SHIMADZU GC-2010) with a HP-5 chromatographic column (30 m×0.32 mm×0.25 μm) in accordance with the Chinese standard test methods for organic substances in drinking water (GB/T 5750.8–2006). Chloramines were measured using a colorimeter (HKM II, Guangdong Huankai Microbial Sci. & Tech. Co. Ltd.) based on the DPD method.

After acclimatization for one week, the 24 rats were randomly distributed into a control group (CG, n = 6) and an experimental group (EG, n = 18), and then a 12-week swimming training program was performed for both groups. Unfortunately, one of the rats accidentally drowned during swimming training; therefore, the final animal number of the EG was 17. The water for the EG was purified using a water purifier and then disinfected using calcium hypochlorite, similar to public swimming pools, whereas the water for the CG was only purified, not chlorinated. Free chlorine in the swimming water was monitored using the N, N-diethyl-p-phenylenediamine (DPD) method. The level of free chlorine in the water for the EG was adjusted to 1.4–1.6 mg/L before swimming training (the ideal level recommended by the World Health Organization for public pools [ 12 ]); no free chlorine was detected in the water for the CG. The training was performed once a day, five days a week. When training, a screw nut approximately 3% of their mean body weight was tied to the top end of the tail of each rat, and all rats were kept in the special pools with water of 60 cm depth (water temperature 25–30°C, pH 6.5–7.0) until fatigued (submerged below the surface for five seconds twice). The fatigued rats ceased training immediately, were removed from the water for a short break, showered with running water and then dried with hair dryers.

Animal welfare and experimental procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals (Ministry of Science and Technology of China, 2006) and were approved by the animal ethics committee of Jiangxi Normal University. Twenty-four Sprague-Dawley rats, which were three weeks old and weighed 207.1 ± 43.9 g, were commercially obtained from the Department of Laboratory Animal Science, Nanchang University, China. Throughout the study periods, all rats were housed in 590×380×200 mm plastic cages under the following conditions: 20–24°C room temperature, natural light, standard food and free water.

Results and Discussion

Concentrations of the typical chlorination DBPs Concentrations of the two typical classes of chlorination DBPs (chloroform and chloramines), which were measured at the 20th minute of the swimming training, were 0.7±0.05 μg/L and 1.05±0.12 mg/L in the water for the EG, and none of these chemicals were detected in the water for the CG. The 20th minute was approximately the middle point of the training session (the duration of each training session was approximately 40 minutes); thus, the concentrations of the DBPs at this point were selected to represent the exposure doses. The exposure doses of these two typical classes of DBPs for the EG rats were significantly higher than those for the CG rats, and this experiment was a typical chronic low-dose exposure experiment.

Behaviors and Appearances The final body weights of the rats measured before euthanasia were extremely close between the EG and the CG (344.34 ± 34.95 g vs. 337.07 ± 46.00 g, p>0.05). No significant behavior differences were observed between the two groups during the entire experimental period; however, some unusual appearance changes appeared in the EG rats. First, the skin around their eyes became increasingly red with the development of the experiment, and in the ending period of the experiment, bloodstains could be observed in the rims of most rats’ eyes. Second, from the third experimental week on, an increasing number of rats had bloodstains appearing at the tips of their noses; however, approximately two weeks later, this symptom gradually disappeared. Third, their fur became increasingly dry and lackluster, and significant signs of hair loss were observed during the last month. These results indicated that the fur, respiratory tracts and eyes of the EG rats were severely affected by chlorinated water, although their growth was essentially unaffected. According to our observations, the daily behaviors and sizes of the EG rats were normal, and their final body weights were even slightly heavier than those weights of the control group. Nevertheless, dried and lackluster fur, hair removal, bloody noses and eyes did occur in the EG rats and not to the CG rats. In fact, similar symptoms, red and swollen eyes, dried skin and nasal mucosal congestion, always appear after humans swim in a chlorinated pool; however, the long-term (12 weeks) and high-frequency (5 days a week) of the experimental swimming training caused even worse symptoms in these experimental rats. Additionally, an interesting phenomenon was observed by comparing the development of the bloody noses and bloody eyes. The bloody noses commonly appeared in the third and in the fourth week; however, approximately two weeks later, this symptom gradually disappeared. The significantly bloody eyes commonly appeared in the ending period of the experiment; however, this problem was becoming worse during the study, and no signs of improvement appeared. The bloody noses appeared first, suggesting that respiratory tracts may be more vulnerable to the irritants from the chlorinated water than eyes; the gradually disappearing symptom suggests that respiratory tracts may have some adaptability to chlorinated water possibly because of the protection from nasal mucous. In contrast, without the mucosal protection, the bloody eyes were becoming increasingly significant during the entire experiment, although this symptom appeared later than the bloody noses. Therefore, the eyes and skin may be the organs that must be the focus of concern regarding permanent damage induced by irritants from chlorinated water, rather than respiratory tracts, although respiratory symptoms were the most emphasized toxic risk of swimming exposure in recent decades.

Swimming capacity test The duration period from the entry into water until the rats reached exhaustion was significantly shorter for the EG rats compared with the CG rats (29.74±11.50 vs. 39.15±9.85 minutes, p<0.05), indicating that the training effects were significantly impaired by the chlorinated water.

Metabolism of chlorination DBPs Chlorine is a necessary element for our bodies, and nontoxic. HClO is the active ingredient of chlorination disinfectants, removing a variety of parasites, bacteria and viruses. However, although attacking microbes and viruses, HClO also reacts with many pool water substances and produces various DBPs. DBPs can be inhaled and ingested during swimming or absorbed dermally, and two classes of DBPs, chloroform and chloramines, have been the focus of most swimming pool studies thus far [26]. To better understand the corresponding metabolic stress pathways, the metabolism of these two classes of DBPs is illustrated in Fig. 5. PPT PowerPoint slide

PowerPoint slide PNG larger image

larger image TIFF original image Download: Fig 5. Metabolism of the representative chlorination disinfection by-products (DBPs) in swimming pools. (A) chloroform (CHCl 3 ), (B) Chloramines. CHCl 3 is primarily metabolized oxidatively to trichloromethanol and spontaneously decomposed to the electrophilic phosgene (COCl 2 ). COCl 2 is highly reactive, and binds covalently to cell components containing nucleophilic groups, and may be hydrolyzed by reacting with water, yielding CO 2 and HCl. Chloramines, including NH 2 Cl, NHCl 2 and NCl 3 , can change into one another easily while producing HClO and NH 3 . Overall, DBP-induced toxicity primarily originates from 4 reactive compounds, HClO, COCl 2 , HCl and NH 3 , which are marked with red in this figure. https://doi.org/10.1371/journal.pone.0119241.g005 Chloroform is primarily metabolized in the liver, which explains its hepatoxicity; however, chloroform metabolism also occurs in other tissues, such as the kidneys. Chloroform metabolism may occur via two pathways, oxidative and reductive, but primarily via the oxidative pathway (Fig. 5A), except under special conditions of high chloroform doses in preinduced animals [27]. Extensive rodent studies have demonstrated that chloroform may be metabolized oxidatively to trichloromethanol and spontaneously decomposed to the electrophilic phosgene (COCl 2 ) [28–29]. COCl 2 is highly reactive, and binds covalently to cell components containing nucleophilic groups, including proteins, reduced glutathione, and phospholipid polar heads [30–31], and may be hydrolyzed by reacting with water, yielding carbon dioxide and hydrochloric acid (HCl). Chloramines, including monochloramine (NH 2 Cl), dichloramine (NHCl 2 ) and trichloramine (NCl 3 ), are not persistent and can change into one another easily while producing HClO and NH 3 (Fig. 5B). Overall, DBP-induced toxicity primarily originates from 4 reactive compounds, HCLO, COCL 2 , HCl and NH 3 (Fig. 5). These compounds are all highly toxic to cells. These compounds can attack cells directly or indirectly by reacting with amino acids, destroying membranes, changing the construction and function of proteins and lipids, unbalancing the acid-base balance, blocking metabolism and by inducing respiratory burst.