Effects of disinfection by-products in swimming pool environments on the immunological mechanisms of respiratory diseases

Swimming in pools is a popular recreational activity enjoyed by millions of people worldwide, and it offers several health benefits (Zwiener et al. 2007). However, there has been growing concern regarding the potential adverse health effects associated with exposure to disinfection byproducts (DBPs) in chemically treated pool water, primarily due to the increased indoor exposure time to commonly used disinfectants and DBPs (Zheng et al. 2020; Lou et al. 2021; Zheng et al. 2021). This has resulted in frequent cases of disinfectant toxicity (Chang et al. 2020). Moreover, the diverse nature and potential interactions between chemical substances upon exposure can cause significant public health issues (Dominici et al. 2010; Carlin et al. 2013; Rider et al. 2013). Currently, the most common method for pool water disinfection, which is similar to that of drinking water treatment, is ‘chlorine disinfection’ (Richardson et al. 2007). Chlorine disinfection is indispensable for effective bacteria control, ease of operation, and cost-effectiveness, making it the preferred oxidant and disinfectant in swimming pools (Park et al. 2010; Li et al. 2013; Kim et al. 2017; Sun et al. 2019). Disinfection is crucial for preventing infectious diseases; however, it leads to the formation of DBPs, which are regulated in drinking water worldwide because of their adverse health effects (Fantuzzi et al. 2001; Chu & Nieuwenhuijsen 2002; Glauner et al. 2005; Sciera et al. 2008; Kanan & Karanfil 2011; Fischer et al. 2012). Pool operators face the challenge of maintaining precise chlorine levels to ensure microbial safety and minimize DBP formation by adjusting the chlorine levels thrice a day (Yang et al. 2016). Despite maintaining a free available chlorine (FAC) concentration between 1 and 5 mg/L, which is generally used to control pathogenic organisms in swimming pools, an increase in DBPs can result in a higher chlorine demand.

Compared to drinking water, which is consumed by many individuals, the risk of DBP exposure through inhalation and dermal routes is higher in swimming pools (Villanueva et al. 2004; Caro & Gallego 2007). This exposure is associated with respiratory symptoms such as allergies, asthma, and increased inflammation (Martin et al. 2003; Kaydos-Daniels et al. 2008; Florentin et al. 2011; Kim et al. 2014; Del Giacco et al. 2015). A hypothesis linking respiratory diseases to the chemical and biological agents in indoor swimming pools has been proposed since 1953; it is supported by subsequent studies (Bernard et al. 2007; Del Giacco et al. 2015; Couto et al. 2021). However, the challenges in designing research to distinguish between the effects of chemical and biological agents make it difficult to determine the role of indoor swimming in the occurrence of respiratory diseases (Bowen et al. 2007). Some studies have reported an association between increased asthma and exposure to chlorinated irritants in swimming pools (Varraso et al. 2002; Kohlhammer et al. 2006); however, these associations were based on a limited number of cases. Conflicting results from various studies make it challenging to confirm the hypothesis that asthma occurs solely because of DBP exposure, considering that asthma is a complex respiratory disease influenced by multiple environmental factors (Del Giacco et al. 2015). Furthermore, most studies have not thoroughly investigated the potential range of DBP concentrations in indoor swimming pool environments or statistically separated the effects of swimming activities. Therefore, independent evidence supporting immune responses associated with DBPs is lacking (Bowen et al. 2007), and these results require careful interpretation (Gleeson et al. 2011; Vlaanderen et al. 2017). It is important to consider that other chemical and biological agents present in the indoor swimming pool environment could affect the respiratory system and contribute to adverse health outcomes, along with the exposure duration (Westerlund et al. 2015). Concerns regarding asthma and swimming without any association can hinder the disinfection of indoor pool water. Addressing this issue requires close collaboration and research among experts from various fields (Westerlund et al. 2015).

This study is the first investigation to provide independent evidence of the inflammatory response related to DBPs, while specifically excluding swimming activities in an indoor swimming pool setting.

Six-week-old male C57BL/6 mice (Dongnam Institute of Radiological and Medical Sciences Animal Inc., Busan, Korea) were used, following a 1-week quarantine and acclimatization period. The mice were housed in a room maintained at 23 ± 2 °C, with a relative humidity of 50 ± 5%. The lighting schedule consisted of artificial light from 08:00 to 20:00; the air within the room underwent 13 − 18 air changes per hour. The mice were provided a standard laboratory diet and had unlimited access to water (12 mice/each group). All experimental procedures adhered to the National Institute of Health Guidelines for the Care and Use of Laboratory Animals and were performed according to a protocol approved by the Institutional Animal Care and Use Committee of the Dongnam Institute of Radiological and Medical Sciences (Permit Number: DI-2022-021). Animal welfare was ensured in compliance with the regulations stipulated by the National Animal Welfare Law of Korea.

To induce the generation of DBPs, a constant-temperature stirrer (IKA C-MAG HS7) and a reaction tank measuring 30 × 30 × 40 cm (width × length × height) were prepared. The residual chlorine concentration following chlorination was measured in the swimming pool using a residual chlorine analyzer (HANNA HI97771C); an initial residual chlorine concentration of 5 mg/ml or higher was ensured. After the reaction, the final residual chlorine concentration was maintained at 1 mg/ml or less. Sodium hypochlorite (MAGIC-POOL, 7510490409, Korea) was employed as the chlorine-based bleach for generating DBPs. An ammonium chloride solution (12125-02-9, Korea) mimicking bodily fluids was synthesized with appropriate ratios of elements, such as creatinine, histidine, hippuric acid, uric acid, citric acid, L-arginine, and glycine (Neslihan et al. 2019). The synthesized solution was loaded into a syringe (NE-1000 Programmable Single Syringe Pump) for usage.

An in vivo model was established to investigate the effects of DBP exposure from swimming pools on respiratory diseases.

The mice were euthanized 48 h after the final challenge through an intraperitoneal injection of Alfaxan (0.5 mg/kg; Australia), and a tracheostomy was performed. To obtain the bronchoalveolar lavage fluid (BALF), ice-cold PBS (0.5 ml) was infused into the lungs thrice and withdrawn each time through tracheal cannulation, resulting in a total volume of 1.5 ml. The total number of inflammatory cells was determined by counting the cells in at least five squares of a hemocytometer after excluding dead cells using trypan blue staining. Differential cell counts in the BALF were determined using Systemex ADVIA 2120 (Siemens Healthcare Diagnostic Inc., IL, USA) following the manufacturer's instructions. The numbers of macrophages, neutrophils, and lymphocytes were calculated by multiplying the percentages obtained from the total yield. The slides were imaged using a digital camera attached to a microscope (Nikon Eclipse 80i; Nikon Corporation, Tokyo, Japan).

The amounts of IL-2, -4, -6, and -10 and interferon (IFN)-γ secreted in the serum of the controlled mice were quantitatively measured using a commercially available enzyme-linked immunosorbent assay (ELISA) kit (BD OptEIA^™ Mouse IL-2 (#555148), 4 (#555232), 6 (#555240), 10 (#555252), and IFN-γ (#555138) ELISA Kit II) according to the manufacturer's instructions (BD Biosciences, San Diego, CA, USA).

The isolated cells were resuspended in 100 μl of 1% FBS solution in PBS and incubated with anti-CD3 (PE-Cy7-conjugated, BD Ms T Lym Subset Ab Cktl, #558391), anti-CD4 (PE-conjugated, BD Ms T Lym Subset Ab Cktl, #558391), anti-CD8 (FITC-conjugated, BD Ms T Lym Subset Ab Cktl, #558391), and anti-CD25 (APC-conjugated, BD MS CD25 APC, #558643) antibodies. The cell pellets were resuspended in 400 μl of 1% FBS solution in PBS and analyzed using flow cytometry (FACSAriaⅡ cell sorter, BD Biosciences, USA).

Statistical differences between groups were analyzed using Student's two-tailed t-test for comparisons between two groups and one-way ANOVA for comparisons between more than two groups. All calculations were performed using GraphPad Prism software (5.0). Significance was set at p < 0.05.

The use of disinfectants in swimming pools significantly impacts public health worldwide (Dehghani et al. 2018). The management of widespread toxicity from DBPs in swimming pool environments is becoming increasingly important, along with the increasing number of people engaging in swimming activities. A review presented various components of DBPs and showed their risks (Shakhawat et al. 2014). Among them, it is known that trihalomethanes (THMs), haloacetic acids (HAAs), and aldehydes are mainly present. The review suggested risks in swimming pool environments due to the complex actions of DBPs. The removal and regulation of DBPs remain challenging because of the multifactorial nature of swimming pools (Peng et al. 2023). In recent years, studies on the association between DBP exposure from swimming pools and asthma resulted in conflicting results without conclusive evidence (Weisel et al. 2009; Font-Ribera et al. 2014; Voisin et al. 2014). Individuals in occupations related to swimming pools, such as athletes, workers, and cleaners, who are frequently exposed to chlorinated environments, are concerned about the development of asthma and respiratory allergies (Medina-Ramón et al. 2005; Jacobs et al. 2007). The connection between these occupational groups and respiratory symptoms is established (Lévesque et al. 2006; Goodman & Hays 2008). Therefore, considering these factors, it is important to assess the relationship between exposure to swimming pool environments and the occurrence of asthma (Lévesque et al. 2006; Goodman & Hays 2008).

Cytokines, signaling molecules secreted by immune cells, play a crucial role in maintaining homeostasis between cell-mediated and humoral immune responses as well as in regulating the inflammatory process (Couto et al. 2021). Therefore, to understand the changes in the immune response, we analyzed the levels of cytokines in the blood. No statistically significant differences in IL-2, IL-4, IL-10, and IFN-γ levels were observed. However, the level of IL-6a was marginally decreased in the EG; IL-6a promotes inflammatory responses. IL-4 is an immunosuppressive cytokine with a role in antitumor responses; in addition, it significantly enhances antiviral control by promoting eomesodermin (Eomes) expression in CD8+ T cells and inducing IFN-γ production (Lee et al. 2013; Park et al. 2016; Rolot et al. 2018). Cytokines such as IL-2, IL-4, and IFN-γ, which are beneficial for protective immune responses in disease improvement, are greatly influenced by physical activities such as swimming (Lee et al. 2019a). IL-6, an indicator of the inflammatory response, is affected by exercise (Lee et al. 2019b).

The possible mechanisms through which exercise exerts its anti-inflammatory effects include the release of IL-6 into the bloodstream from contracting muscle fibers, leading to subsequent increases in the circulating levels of IL-10 and IL-1 receptor antagonists. Exercise also promotes an increase in the number of circulating IL-10-secreting regulatory T cells. In addition, it downregulates Toll-like receptor expression in monocytes and inhibits downstream responses such as pro-inflammatory cytokine production, antigen presentation, and co-stimulatory molecule expression. Exercise reduces the number of circulating pro-inflammatory monocytes and inhibits the infiltration of monocytes and/or macrophages into adipose tissue. Regular moderate exercise is associated with a lower incidence of infection compared to that with a completely sedentary lifestyle; however, elite athletes who undergo long hours of intense training are more susceptible to infections (Gleeson et al. 2011).

Exercise protocols, timing, and duration are significant factors influencing the immune response (Cai et al. 2007). However, interpreting the effectiveness based solely on a minor decrease in the inflammatory cytokine IL-6 without a significant increase in IFN-γ, which inhibits IL-6, would not be accurate. IL-6, which promotes eosinophil infiltration, is strongly associated with various immune disorders, inflammatory diseases, and lymphatic tumors. Therefore, any significant increase or decrease observed in the T cells in the BALF, lymph nodes, spleen, and thoracic glands through FACS analysis should be considered at minimal expression levels to ascertain the presence of an inflammatory response (asthma or allergy). A study evaluating the frequency of exposure to DBPs resulting from regular attendance at swimming pools in 5,738 individuals showed no significant association with bronchial hyperresponsiveness and a lower risk for increased pulmonary function and asthma symptoms (Font-Ribera et al. 2011). This finding aligns with another meta-analysis that reported an unclear association between indoor swimming pool DBP exposure and asthma diagnosis (Goodman & Hays 2008; Valeriani et al. 2017). A study conducted on 3,223 participants to assess the relationship between respiratory symptoms and the frequency of exposure to swimming pool environments found no significant associations (Font-Ribera et al. 2009). However, a recent study confirmed a high prevalence of exercise-induced bronchospasm (EIB), a respiratory symptom, among athletes, especially swimmers (Boulet et al. 2017). This phenomenon is commonly observed in sports but is particularly pronounced in swimmers. The underlying mechanism involves increased airway resistance during exercise, leading to the release of inflammatory mediators, such as histamine, prostaglandins, and leukotrienes, from the immune cells. Increased airway ventilation enhances exposure to environmental stressors, such as air pollutants, allergens, and other stimuli, resulting in functional and structural changes in the respiratory epithelium and subsequent epithelial damage and remodeling, leading to chronic airway inflammation (Kanikowska et al. 2018). These changes promote the penetration of harmful substances, including allergens and viruses, creating a local microenvironment that perpetuates Th2-mediated inflammation and contributes to respiratory epithelial damage, remodeling, and induction of chronic bronchial inflammation (Holgate 2011; Kanikowska et al. 2018). In contrast, a higher prevalence of specific respiratory symptoms was observed in individuals occupationally exposed to swimming pool environments (excluding professional swimmers); however, chronic effects have not been reported and the causality owing to DBP exposure remains uncertain (Goodman & Hays 2008; Villanueva & Font-Ribera 2012). Considering that swimming is a recommended sport for individuals with respiratory conditions such as asthma (Uyan et al. 2009), it is important to elucidate the association between DBPs and respiratory diseases.

Based on the results and comprehensive analysis of previous studies, it is evident that the health benefits of swimming far outweigh the potential respiratory health risks associated with chemical contamination such as DBPs. When swimming activities are performed according to the individual's fitness level, considering the type of swimming, intensity, and duration, the risk of immune-related respiratory reactions from DBP inhalation is low. However, long-term exposure to DBPs, as highlighted in several previous studies, may increase concerns about their potential adverse health effects. Therefore, limiting exposure by maintaining proper ventilation to dilute the DBPs generated from chlorine-containing disinfectants and fostering an environment considering hygiene is essential for reaping the health benefits.

The swimming pool environment is reported to potentially impact respiratory health due to continuous chlorine disinfection and the presence of high levels of DBPs in both water and air, resulting from organic materials. However, the hypothesis that respiratory diseases are caused by exposure to DBPs has been a subject of diverse opinions in various studies to date. Furthermore, it has been challenging to statistically isolate the exposure effects related to physical activities such as swimming. Therefore, this study is the first to construct a simulation model of an indoor swimming pool to analyze independent evidence of the association between DBPs in swimming pool environments and immune-inflammatory responses related to respiratory diseases, excluding from physical activities such as swimming. The impact of DBPs in swimming pool environments on the immunological mechanisms of respiratory diseases was examined, excluding physical activities such as swimming. Exposure to DBP in swimming pool environments, excluding the effects of swimming activities, did not induce respiratory diseases. The changes observed in this study were minimal when compared to those observed with exposure to chlorinated irritants in swimming pools in previous studies. Asthma, one of the most common respiratory diseases, occurs because of the complex interplay of various environmental factors and cannot be solely attributed to DBP exposure. However, considering the excessive respiratory symptoms observed in previous studies among swimmers and other individuals attending swimming pools, it is highly unlikely that these observations are coincidental or biased. Therefore, future research should investigate the specific types, intensity, and duration of exercise that may have a significant impact on immune-inflammatory responses in the presence of DBP exposure.

We thank Yonghae Son and Koanhoi Kim (Pusan National University) for help with editing this manuscript.

B.-A.L. did all the work in this study. The author has read and agreed to the published version of the manuscript.

This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2021R1I1A1A01059491).

All relevant data are included in the paper or its Supplementary Information.

The authors declare there is no conflict.