A Comparison Between Freshwater and Seawater ...

A Comparison Between Freshwater and Seawater Swimming Pools: From Disinfection By-products Profile to Genotoxicity Tarek Manasfia, Michel De Méob, Bruno Coulomba, Carole Di Giorgiob, J.-L. Boudennea a

Aix Marseille Université, CNRS, LCE FRE 3416, Laboratoire Développements Métrologiques et Chimie des milieux, 13331 Marseille, France (E-mail: [email protected]; [email protected]; [email protected]) b Aix Marseille Université, CNRS, IRD, Avignon Université, IMBE UMR 7263, Laboratoire de Mutagénèse Environnementale, 13385, Marseille, France (E-mail: [email protected]; [email protected])

Abstract Despite the growth in the sector of thalassotherapy where pools are filled with seawater and disinfectants are added, no data about the genotoxicity of water in these pools have been reported. In the present study, a comparison was established between seawater swimming pools of two thalassotherapy centers and a freshwater swimming pool. This comparison aimed at the determination of disinfection by-products (DBPs) qualitatively and quantitatively as well as the evaluation of genotoxic effects induced by water samples using Ames test. Generally, the predominant DBPs found in freshwater were chlorinated while those of seawater pools were brominated. The freshwater pool was found to possess the highest content in DBPs due to high frequentation rate of this pool in contrast to the seawater pools. The analysis of the content of the pools in DBPs allowed understanding the results of the mutagenesis assay. This assay conducted on Salmonella typhimurium strain TA100 showed that the samples of freshwater pool were more mutagenic than those of the tested seawater pool despite the existing evidence concerning brominated DBPs known to be more genotoxic than chlorinated ones. These findings highlight the importance of limiting the introduction of organic matter to pools by swimmers, especially in pools with high levels of frequentation.

Keywords Thalassotherapy; swimming pools; seawater; freshwater; chlorination; disinfection by-products (DBPs); mutagenesis; genotoxicity

INTRODUCTION As medical and wellness tourisms are growing and gaining increasing attention in countries around the world, fundamental questions arise about how to ameliorate the conditions in these establishments to ensure the wellbeing of curists and wellness seekers. Among the rapidly growing sectors of medical and spa tourism is thalassotherapy (Crecente J. et al., 2012). This sector dating back to the late 1800s, involves the medical use of seawater and the marine environment as a source of therapy. Modern thalassotherapy centers welcome diverse classes of attendees encompassing curists as well as those seeking wellness merely (Schwartz, 2005). In these centers, seawater swimming pools should be treated with disinfectants similarly to freshwater swimming pools in order to eliminate biological hazards and prevent outbreaks of diseases caused

by infectious agents (WHO, 2006). In France, regulations impose a residual free chlorine level between 0.4 and 1.4 mg.L-1 in swimming pools disinfected by bleach or chlorine gas irrespective the type of water alimenting the pool (ANSES, 2010). These treatments give rise to the formation of unintended chemicals known as disinfection by-products (DBPs). DBPs mainly derive as a result of reactions taking place in the pool between disinfectants and organic matter originally present in the water or introduced by pool users themselves such as sweat, urine, soap residues, and personal-care products (Chowdhury, 2014). Several studies have suggested evidence of the formation of mainly brominated DBPs upon the addition of chlorine to bromide-rich water (Bougeard et al., 2010; Wang et al., 2010). Chlorinating seawater that has a relatively high content of bromide (around 65 mg.L-1) leads to the appearance of predominantly brominated DBPs. However, only few studies have investigated these DBPs and assessed their toxicity. Brominated DBPs were shown to display more important toxicities in comparison to chlorinated DBPs (Hsu et al., 2001; Richardson et al., 2007; Fabricino and Korshin, 2009 and Lee et al., 2009). Thus taking into consideration the public and occupational health concerns, it is important to analyze qualitatively and quantitatively DBPs in seawater pools of thalassotherapy centers as well as to assess the mutagenicity of this water in comparison to pools alimented with freshwater. In the present study, we conducted an investigation on four pools located in two spas in Southeast France during summer period. This investigation involved the identification of DBPs qualitatively and quantitatively in the four pools as well as the assessment of the genotoxic effect of water in two swimming pools one alimented with seawater (thalassotherapy pool) and the other with freshwater.

MATERIALS AND METHODS Sampling Four swimming pools in two thalassotherapy centers designated E1 and E2 located in Southeast France were studied. In these centers, water was filtrated before being alimenting the pools and chlorine (sodium hypochlorite, NaOCl) was added to the pools in an automated manner to conserve an approximately constant residual free chlorine level. One of the centers (E1) contained an outdoor pool alimented with freshwater and an indoor thalassotherapy pool alimented with seawater while the other center (E2) included two indoor pools alimented with seawater. Samples for DBPs’ analysis were collected from the four pools in 65-mL glass bottles filled without headspace and tightly sealed with a PTFE-lined screw cap. Prior to sampling, ascorbic acid was added in the glass bottles to quench residual chlorine present in samples to avoid the alteration of the concentrations of DBPs due to continuous reactions (Kristiana et al., 2014). Samples were also collected in 1-L glass bottles for the analysis of total organic carbon (TOC) and total nitrogen (TN). Samples of 30 L were collected from the two pools (seawater and freshwater) of E1 for mutagenesis testing. All collected samples were stored at 4°C away from light until extraction. Physicochemical and DPBs Analysis Physicochemical parameters including pH, conductivity and salinity, redox potential and dissolved oxygen were determined on-site at the time of sampling using specific

electrodes from WTW (SenTix 41-3 pH electrode, WTW Pt 4805/S7 electrode, TetraCon 325 redox electrode, and CellOx 325 electrode, respectively). Residual chlorine was measured on-site also by the DPD colorimetric method using Merck Spectroquant NOVA 60 (Darmstadt, Germany). Total organic carbon (TOC) and total nitrogen (TN) were measured by catalytic oxidation at high temperature (Multi N/C 2100, Analytic Jena, Germany). To determine DPBs including trihalomethanes (THM), haloacetonitriles (HAN), haloketones, haloacetyaldehydes and halophenols, sample aliquots (50 mL) were extracted by liquid-liquid extraction (LLE) with 5 ml of methy tert-butyl ether (MTBE) and analyzed by gas chromatography with an electron capture detector (GC-ECD) using conditions derived from U.S.EPA 551.1 (1995). To analyze haloacetic acids (HAA), 50mL sample aliquots were adjusted to a pH of 0.5 or less and extracted with MTBE (5 mL). The organic phase containing the haloacetic acids was treated with acidified methanol to convert HAA to their methyl esters. Extracts containing the esters were then neutralized by adding a saturated sodium bicarbonate solution submitted and then submitted to GC-ECD analysis (U.S.EPA 552.3, 2003). Procedural standard calibration was used to quantify target molecules. 2,3-dibromopropionic acid was used as a surrogate and 1,2,3-chloropropane was used as an internal standard. Mutagenesis Assay The second part of this study consisted of the evaluation of mutagenesis induced by the most chemically charged seawater swimming pool and the freshwater swimming pool by Ames test (Salomnella assay). Sample aliquots of 30 L from each of the two tested pools were concentrated on a column containing two overlapping layers of cleaned Amberlite XAD-8 over XAD-2 resins respectively. The column was eluted with 350 mL ethyl acetate. Eluents were then treated with sodium sulfate (Na 2SO4) to remove water incorporated in the organic phase and then concentrated to 1.5 mL. A 0.5 mL aliquot of the resultant concentrate underwent solvent exchange to 0.5 mL dimethylsulfoxide (DMSO). The 20,000x DMSO concentrates (0.5 mL of DMSO corresponding to the equivalent of 10 L of the swimming pool water) were tested in triplicate using a micromethod of Ames test in Salmonella typhimurium strain TA100 (HisG46 base uvrB rfa pKM101) in the presence and absence of rat liver enzymes-containing metabolic fraction S9 mix. Four doses of each extract were tested. Sodium azide and benzo(a)pyrene were used as positive controls in experiments without and with S9 mix respectively. After 48 h incubation, revertants were counted on each plate with a colony counter equipped with a bacterial enumeration program.

RESULTS AND DISCUSSION Physicochemical parameters and DBPs Table 1 lists the results of physicochemical analyses and the levels of DBPs found in the four studied pools. All the pools involved in this study had approximately similar levels in free chlorine with a mean value of 1.28 mg.L-1. TOC values were generally of the same order in the four swimming pools. The profile of DBPs, whether chlorinated or brominated, varied according the type of water alimenting the swimming pool. Although in all swimming pools chlorine was used as a disinfectant, brominated DBPs were

predominant in seawater pools while chlorinated DBPs were predominant in freshwater pool. Such findings are compatible with previous studies concerning the formation of bromine and therefore, brominated DBPs, after the addition of chlorine to bromide-rich water containing organic matter (Wang et al., 2010). The most abundant DBPs were mainly among HAAs, THMs and HANs in seawater and freshwater pools. In the latter, haloketones were also found as major DBPs with 1,1,1-trichloropropanone reaching 71.88 µg/L. Chloroform was the most abundant THM in freshwater pools versus bromoform in seawater pools. THM levels in the pools were generally close one to another with the exception of seawater pool (2) of E2 that showed relatively lower levels. This might be due to the lower rate of frequentation in this pool. However, freshwater pool contained significantly higher levels of HANs (total HAN 87.37 µg/L) and HAAs (total HAA 501.18 µg/L) in comparison to the three seawater pools with average levels of 19.91 µg/L and 117.14 µg/L for total HAN and total HAA respectively. The higher levels of DBPs found in the freshwater swimming pool can be attributed to the elevated rate of frequentation to this outdoors pool. That means more organic matter is introduced to the pool by the swimmers to react with the available chlorine present in the pool. This characteristic can also explain the discrepancies in the abundance of different categories of DBPs and particularly between THM and HAA. The higher values of HAA in contrast to THM can be explained in part by their physicochemical properties since THMs are volatile compounds and are thus mainly found in air (Richardson et al., 2010) while HAAs are highly soluble in water and resist degradation in the presence of high chlorine residual (Kanan and Karanfil, 2011). Moreover, the nature of organic matter reacting with chlorine at the origin of forming DBPs plays a role in the dominance of one category of DBPs on the other. Organic matter brought by bathers is mainly constituted of nitrogen-containing compounds (such as ammonia, urea, amino acids) (De Laat et al., 2011) that react with chlorine to form more HAAs than THMs, as measured in numerous studies carried out in real swimming pools (Kanan and Karanfil, 2011; Lakind et al., 2010; Parinet et al., 2012).

Table 1: Results of physicochemical and DBPs analyses in the four pools. Establishment Code

E1 Freshwater Seawater Pool Pool

E2 Seawater Seawater Pool (1) Pool (2)

Physicochemical Parameter T ( °C) pH Salinity (PSU) TOC (mg/L) Free/Residual Chlorine mg/L

29.40 7.00 1.15 11.52

33.20 8.54 51.87 11.88

30.90 8.46 44.55 10.88

33.40 8.32 44.32 11.82

1.55

1.39

1.16

1.05

Chloroform Bromodichloromethane Dibromochloromethane Bromoform Total THM

69.82 7.86 1.93 0.62 80.23

3.84 62.45 66.29

5.17 86.55 91.72

1.56 48.83 50.38

Dichloroacetonitrile Trichloroacetonitrile Bromochloroacetonitrile Dibromoacetonitrile Total HAN

74.59 1.13 9.16 2.49 87.37

0.90 16.22 17.12

0.99 27.59 28.58

0.91 13.13 14.04

Haloketone

1,1-Dichloropropanone 1,1,1-Trichloropropanone

20.48 71.88

Halophenol

2-Bromo-4-chlorophenol 2,4,6-tribromophenol

3.78

4.32 0.24

1.30 0.24

4.21 0.17

HAA

Monochloroacetic acid Monobromoacetic acid Dichloroacetic acid Trichloroacetic acid Bromochloroacetic acid Dibromoacetic acid Bromodichloroacetic acid Dibromochloroacetic acid Tribromoacetic acid Total HAA

1.72 1.28

0.99

0.93

0.35 3.53 72.04 0.40 3.50 53.19 134.29

0.45 3.09 62.75 0.15 2.60 38.97 109.00

0.37 4.36 63.50 0.35 3.00 35.62 108.13

2.22

1.01

0.35

DBPs (µg/L) THM

HAN

Other DBPs

Chloropicrine Bromal hydrate

23.02 461.12 2.44 1.07 7.31 2.74 1.76 501.18 4.45

Mutagenicity Figure 1 shows that the sample from the freshwater swimming pool were significantly more mutagenic (3.7 rev/mL-eq) than the sample from seawater swimming pool chosen to be tested (E2 Pool 1) which was weakly mutagenic (0.4 rev/mL) in Ames test. Adding the metabolic fraction S9 mix decreased mutagenicity of the extracts (1.8 and 0.3 rev/mL for the freshwater and seawater swimming pools respectively). For the first instance these findings might seem surprising especially that brominated DBPs are known to be mutagenic and genotoxic (DeMarini et al., 1997; Kogevinas et al., 2010) and there is evidence suggesting that they are more genotoxic than the chlorinated ones (Richardson et al., 2007; Fabricino and Korshin, 2009; Lee et al., 2009). However, the chemical analyses performed in this study have shown that the examined freshwater pool had a more important content in DBPs than the other swimming pools with many of the occurring DBPs known to be mutagenic in Salmonella (Richardson et al., 2007). That means the more pronounced genotoxic effect exerted by freshwater can be linked to the richer content in DBPs despite the potential presence of certain more genotoxic DBPs in seawater. The higher content of DBPs in freshwater swimming pool can be explained by the higher frequentation rate responsible for the more important load of organic matter being introduced to the pool in comparison to seawater swimming pools which were characterized by lower frequentation. These findings raise concerns over the importance of reducing organic matter brought by swimmers in order to diminish or eliminate chemical risks.

Figure 1: Mutagenicity in Salmonella TA100 of freshwater and seawater pools samples.

CONCLUSION This study, to our knowledge, is the first to assess the genotoxicity of water in thallasotherapy pools (alimented with seawater) and compare it with that of freshwater pools. DBPs of fours swimming pools in two spas located in Southeast France were analyzed qualitatively and quantitatively with the levels of bromal hydrate being reported for the first time in modern thalassotherapy establishments. This study demonstrated the mutagenicity of swimming pool water having an important content of DBPs as a result of high frequentation rates and permanent addition of disinfectant. Brominated DBPs were dominant in seawater swimming pools in contrast to freshwater swimming pools where chlorinated compounds were dominant. Presently, many of the identified compounds are not regulated by authorities. These findings stress the need for broader regulations concerning swimming pools. Also, they highlight the importance of educating swimmers about their partial responsibility in reducing chemical risks by following hygienic instructions (such as a shower before swimming) and behaving accordingly.

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