Waqas A Cheema

Tarek Manasfi

Kamilla M S Kaarsholm

Henrik R Andersen

Jean- Luc Boudenne
email: jean-luc.boudenne@univ-amu.fr

Effect of medium-pressure UV-lamp treatment on disinfection byproducts in chlorinated seawater swimming pool waters

Keywords: trihalomethane, haloacetonitrile, haloacetic acid, post-UV chlorination, chlorine reactivity

Several brominated disinfection by-products (DBPs) are formed in chlorinated seawater pools, due to the high concentration of bromide in seawater. UV irradiation is increasingly employed in freshwater pools, because UV treatment photodegrades harmful chloramines. However, in freshwater pools it has been reported that post-UV chlorination promotes the formation of other DBPs. To date, UV-based processes have not been investigated for DBPs in seawater pools. In this study, the effects of UV, followed by chlorination, on the concentration of three groups of DBPs were investigated in laboratory batch experiments using a medium-pressure UV lamp. Chlorine consumption increased following post-UV chlorination, most likely because UV irradiation degraded organic matter in the pool samples to more chlorine-reactive organic matter. Haloacetic acid (HAA) concentrations decreased significantly, due to photo-degradation, but the concentrations of trihalomethanes (THMs) and haloacetonitriles (HANs) increased with post-UV chlorination. Bromine incorporation in HAAs was significantly higher in the control samples chlorinated without UV irradiation but decreased significantly with UV treatment. Bromine incorporation was promoted in THM and HAN after UV and chlorine treatment. Overall, the accumulated bromine incorporation level in DBPs remained essentially unchanged in comparison with the control samples. Toxicity estimates increased with single-dose UV and chlorination, mainly due to increased HAN concentrations. However, brominated HANs are known in the literature to degrade following further UV treatment.

Introduction

The use of seawater in swimming pools was first developed for therapeutic purposes to treat sore throats, digestive problems, rheumatic arthritis, musculoskeletal injury, development disorders in children, respiratory problems (asthma and bronchitis), endocrine imbalances and skin diseases (e.g. psoriasis) [START_REF] Charlier | The Healing Sea: A Sustainable Coastal Ocean Resource: Thalassotherapy[END_REF][START_REF] Shani | Penetration of selected Dead Sea minerals through a healthy rabbit skin, from a sustained-release transparent varnish, as a prospective treatment for psoriasis[END_REF]. This practice, called 'thalassotherapy', was defined by the French Medical Academy in 1960 as being the use of seawater, seaweed, sea mud or other sea resources and/or the marine climate for the purpose of medical treatment with a medicinal effect and is today largely present in Western (Great Britain, France, Germany, Spain) and Eastern Europe and along the Atlantic Ocean, Channel, Mediterranean Sea, Baltic Sea and Black Sea coasts [START_REF] Crecente | A multicriteria approach to support the location of thalassotherapy (seawater therapy) resorts: Application to Galicia region, NW Spain[END_REF]. Nowadays, seawater swimming pools are found not only in thalassotherapy centres but also in various (health) tourism and leisure sites worldwide (hotels, campsites, aquatic centres) located near to the coast [START_REF] Lee | Opportunities and Issues in the Health Tourism Industry: Deep Sea Water Development in Taiwan[END_REF][START_REF] Wyer | Relationships between human adenoviruses and faecal indicator organisms in European recreational waters[END_REF] or in pools and hot tubs on ships (WHO, 2006). Among the natural constituents of seawater, high levels of bromide (Br -) may be problematic, mainly when chlorination is applied to disinfect such waters. The average level of bromide in the sea is around 67 mg/L [START_REF] Summerhayes | Oceanography : an illustrated guide[END_REF], with levels varying from 15 mg/L in the Baltic Sea [START_REF] Feistel | Density and Absolute Salinity of the Baltic Sea 2006-2009[END_REF] to 85 mg/L in the Mediterranean Sea and up to 5.2 g/L in the exceptional case of the Dead Sea [START_REF] Salameh | Origin of high bromide concentration in the water sources in Jordan and in the Dead Sea water[END_REF] Currently, chlorine is used as a disinfectant in swimming pools. However, chlorine reacts with organic and inorganic matter present in pool water and forms disinfection by-products 1 (DBPs), which results in negative health effects such as irritations (eye, skin, nose and throat) [START_REF] Guglielmina | Prevalence of ocular, respiratory and cutaneous symptoms in indoor swimming pool workers and exposure to disinfection by-products (DBPs)[END_REF], bladder cancer [START_REF] Villanueva | Bladder cancer and exposure to water disinfection by-products through ingestion, bathing, showering, and swimming in pools[END_REF] and reproductive anomalies [START_REF] Hinckley | Late pregnancy exposures to disinfection by-products and growth-related birth outcomes[END_REF]. Furthermore, some DBPs are reported to have genotoxic and carcinogenic effects [START_REF] Richardson | Occurrence, genotoxicity, and carcinogenicity of regulated and emerging disinfection by-products in drinking water: A review and roadmap for research[END_REF]. The most commonly known DBPs reported in swimming pool water are haloamines, trihalomethanes (THMs), haloacetic acids (HAAs) and haloacetonitriles (HANs) [START_REF] Chowdhury | Disinfection byproducts in swimming pool: Occurrences, implications and future needs[END_REF]Manasfi et al., 2017a). However, less frequently reported and unregulated groups are halodiacids, haloaldehydes, haloketones, haloamides, halophenols, halobenzoquinones and N-nitrosamines [START_REF] Richardson | What's in the pool? A comprehensive identification of disinfection by-products and assessment of mutagenicity of chlorinated and brominated swimming pool water[END_REF][START_REF] Teo | Chemical contaminants in swimming pools: Occurrence, implications and control[END_REF][START_REF] Wang | Halobenzoquinones in swimming pool waters and their formation from personal care products[END_REF]. Abbreviations. DBP, disinfection by-products. HAA, haloacetic acids. HAN, haloacetonitriles. THM, trihalomethanes. TOC, total organic carbon.

In chlorinated seawater pools, bromine is the most prevalent disinfectant and oxidant, as bromide ions react with chlorine to form hypobromous acid [START_REF] Bougeard | Comparison of the disinfection byproduct formation potential of treated waters exposed to chlorine and monochloramine[END_REF][START_REF] Ged | Effect of seawater intrusion on formation of bromine-containing trihalomethanes and haloacetic acids during chlorination[END_REF][START_REF] Wang | Decomposition of two haloacetic acids in water using UV radiation, ozone and advanced oxidation processes[END_REF]. These pools are expected to have a high content of bromide and are likely to generate more brominated DBPs [START_REF] Manasfi | Identification of disinfection by-products in freshwater and seawater swimming pools and evaluation of genotoxicity[END_REF][START_REF] Parinet | Exposure levels to brominated compounds in seawater swimming pools treated with chlorine[END_REF]. Furthermore, it is also known that brominated DBPs are more toxic than the chlorinated DBP (Manasfi et al., 2017b;[START_REF] Muellner | Haloacetonitriles vs. regulated haloacetic acids: Are nitrogen-containing DBFs more toxic?[END_REF][START_REF] Plewa | Comparative mammalian cell toxicity of N-DBPs and C-DBPs[END_REF].

An established method employed to reduce combined chlorine in swimming pool water is direct photolysis by UV (Hansen et al., 2013a). The combined application of UV and chlorination provides opportunities for the formation of hydroxyl radicals as reactive intermediates [START_REF] Feng | Photolysis of aqueous free chlorine species (HOCl and OCl-) with 254 nm ultraviolet light[END_REF]. However, reaction pathways promote the formation of some DBPs with UV, followed by chlorination. [START_REF] Spiliotopoulou | Secondary formation of disinfection by-products by UV treatment of swimming pool water[END_REF] described that DBPs are only formed in secondary reactions after chlorine addition during the post-UV chlorination treatment of freshwater pools. [START_REF] Cimetiere | Effects of UV-dechloramination of swimming pool water on the formation of disinfection by-products: A lab-scale study[END_REF]) also reported increased the formation of some DBPs with UV treatment followed by chlorination in freshwater pools during the post-chlorination step. However, no data have been reported for reaction pathways leading to an increase in the formation of THMs and some other DBPs in seawater. [START_REF] Liviac | Genotoxicity of water concentrates from recreational pools after various disinfection methods[END_REF] and [START_REF] Plewa | Differential toxicity of drinking water disinfected with combinations of ultraviolet radiation and chlorine[END_REF] reported UV treatment for the reduction of genotoxicity and cytotoxicity in chlorinated swimming pool water and drinking water treatment systems. Furthermore, several studies have reported that mediumpressure UV lamps are more efficient than low pressure UV lamps for the photo-degradation of THMs [START_REF] Kristensen | Testing of different treatment technologies in swimming pools[END_REF][START_REF] Zare Afifi | Effects of UV-based treatment on volatile disinfection byproducts in a chlorinated, indoor swimming pool[END_REF].

Water characteristics in seawater pools can influence DBP formation and result in the formation of different DBPs than those found in freshwater pools. Furthermore, the higher pH of seawater pools than freshwater pools will also have an effect on DBP formation (Hansen et al., 2012b).

Therefore, the effect of UV treatment followed by chlorination on DBP formation was studied using laboratory experiments. Water from three different indoor chlorinated seawater pools was exposed to light from a standard medium-pressure UV lamp, followed by chlorination. Three groups of DBPs were investigated, namely THMs, HANs and HAAs. The DBP level measured after post-UV chlorination was compared to a dark control sample which was not subjected to UV exposure. Bromine substitution was investigated to analyse its effects on the formation of DBPs. Finally, overall cytotoxicity and genotoxicity were estimated for the toxic potency of compounds, before and after treatment.

2 Material and method 2.1 Pool water

Sample collection

This study included samples from three indoor seawater swimming pools in two thalassotherapy establishments located in the Cote d´Azur region in south-eastern France. The first establishment had an indoor pool (pool-1) filled with seawater, while the second establishment had two indoor pools (pool-2, pool-3) filled with seawater. These seawater pools were filled with water originating from the Mediterranean Sea.

The disinfection treatment involved the automatically controlled addition of sodium hypochlorite to maintain a constant level of free residual chlorine equivalents. These pools were equipped with sand filters to remove particulars and pollutants. Sampling was undertaken during March 2016. Physicochemical parameters were measured on-site while the samples were collected in 2-litre amber glass bottles and kept at 4°C until treated the same day.

Water quality parameters

Parameters including pH, conductivity and temperature were determined on-site, using a multi-electrode multimeter (WTW Multiline P4 multimeter, Weilheim, Germany). Residual chlorine and total chlorine were measured on-site via the colorimetric DPD method, using a spectrophotometer (AQUALYTIC-AL 800, Germany), and total organic carbon (TOC) was measured by catalytic oxidation at a high temperature (Multi N/C 2100, Analytic Jena, Germany).

Disinfection by-product analysis

Samples were extracted by liquid-liquid extraction (LLE) with methyl tert-butyl ether (MTBE) of Chromosolv, HPLC grade (Sigma-Aldrich, Germany) and analysed by gas chromatography coupled to an electron capture detector (GC-ECD) (Perkin Elmer Clarus 580 system, Norwalk, CT, USA). The analysis of THMs and HANs was conducted according to U.S.EPA 551.1 [START_REF] Munch | Determination of Chlorination Disinfection Byproducts, Chlorinated Solvents, and Halogenated Pesticides/Herbicides in Drinking Water by Liquid-Liquid Extraction and Gas Chromatography With Electron-Capture Detection-Method 551[END_REF] along with a number of modifications as described previously by [START_REF] Manasfi | Identification of disinfection by-products in freshwater and seawater swimming pools and evaluation of genotoxicity[END_REF] and detailed description can be found in supporting information (Section 3.1, SI). To measure HAAs, samples were treated and analysed based on U.S.EPA 552.3 [START_REF] Domino | Method 552.3 Determination of haloacetic acids and dalapon in drinking water by liquid-liquid microextraction, derivatization, and gas chromatography with electron capture detection[END_REF], again along with slight modifications as described previously by [START_REF] Manasfi | Identification of disinfection by-products in freshwater and seawater swimming pools and evaluation of genotoxicity[END_REF] and detailed description can be found in supporting information (Section 3.2, SI). Sample aliquots (50 mL) were adjusted to pH 0.5 or less, by adding sulphuric acid (analytical grade reagent, Fisher Scientific, UK), and then extracted with MTBE (5 mL). Next, the organic phase containing the HAAs was treated with acidified methanol at 50°C for 2 h to form the methyl ester derivatives of HAAs.

The derivatised HAAs were analysed with GC-ECD. The limits of quantification (LOQ) were 0.2 µg/L for dibromochloromethane, bromoform, dichloroacetonitrile and dibromoacetonitrile; 0.3 µg/L for bromodichloromethane, trichloroacetonitrile and bromochloroacetonitrile; 0.5 µg/L for trichloroacetic acid, bromochloroacetic acid, dibromoacetic acid, dibromochloroacetic acid and dichloroacetic acid; 0.6 µg/L for bromodichloroacetic acid; 0.7 µg/L for dichloroacetic acid; 0.8 µg/L for chloroform and 1.0 µg/L for tribromoacetic acid, monochloroacetic acid and monobromoacetic acid.

UV treatment

Batch experiments were conducted by using a thermostatically controlled cylindrical reactor with a standard medium-pressure UV lamp (P=700 W, Peschl Ultraviolet) further details about reactor layout and the lamps spectral emission can be found in supporting information. In this work, the UV dose was determined according to the method described by Hansen et al. (2013a). A UV exposure cylindrical reactor setup was correlated to the real flow-through system on the pool by using combined chlorine as an actinometer. A UV system requires 1.0 kWh/m 3 to remove 90% of combined chlorine and 0.61 kWh/m 3 to remove monochloramine (Hansen et al., 2013a). The required radiation time for the cylindrical reactor setup to remove 90% of the monochloramine from the pool sample was 4.2 min (2.1 J/cm 2 ). In order to compare the experimental UV dose to a realistic treatment level, the UV system in the Gladsaxe swimming pool's hot water basin was used. This system consists of four UV lamps using a total of 2800W and operating 24 hours per day on a total pool volume of 50 m 3 [START_REF] Kristensen | Full scale test of UV-based water treatment technologies at Gladsaxe Sportcentre -with and without advanced oxidation mechanisms[END_REF]. Therefore, the applied electrical energy dose from UV was 1.34 kWh/m 3 per day, and so it can be calculated that the dose equivalent to one day of treatment is achieved after 9.5 min radiation (4.7 J/cm 2 ). Furthermore, using monochloramine as a second actinometer [START_REF] Cheema | Combined UV treatment and ozonation for the removal of by-product precursors in swimming pool water[END_REF], the UV dose was recalculated for our system to correspond to a 247 mJ/cm 2 UV dose delivered per minute from a low pressure UV lamp, based on the data published in [START_REF] Li | UV photodegradation of inorganic chloramines[END_REF].

Chlorination

The formation of DBPs as a result of chlorination was investigated using a standardised DBP formation assay. Similar tests have been used in other studies investigating the potential for the formation of NCl 3 [START_REF] Schmalz | Trichloramine in swimming pools -Formation and mass transfer[END_REF], THM and HAA in swimming pool water [START_REF] Kanan | Occurrence and formation of disinfection by-products in indoor swimming pools water[END_REF], THM, HAN and HAA from synthetic body fluid (Hansen et al., 2012b) and particles from pools (Hansen et al., 2012a). The effect of chlorine concentration in the assay was also recently investigated by Hansen et al. (2013b). The concentration of free chlorine was adjusted to 3.0 ± 0.05 mg/L by adding a sodium hypochlorite solution, and then the sample reacted for 24 h at 25°C. Chlorination was performed in quadruplicate, with three samples used for DBP analysis and one for the determination of residual chlorine. Samples for DBP analysis were dosed with an ammonium chloride solution (50 mg/L), to quench free chlorine, which neither affects the already formed DBP [START_REF] Kristiana | To add or not to add: The use of quenching agents for the analysis of disinfection by-products in water samples[END_REF] nor increases N-DBP formation [START_REF] Hua | Disinfection byproduct formation from lignin precursors[END_REF]. The samples were extracted and analysed for DBPs the same day.

Experiments

In this study, batch experiments were performed in the laboratory to ensure controlled experimental conditions. The dark control samples were directly chlorinated for DBP analysis, to investigate the formation potential of pool water without UV treatment. These samples were passed through the UV reactor without UV light to ensure the same experimental conditions (retention time, stirring). All samples from three different pools were collected on the same day and used for experiments immediately upon arrival in the laboratory (within three hours of collection). Collected samples were divided into four equal subsamples from which the first subsample was used for initial analysis, the second for dark control, the third for treatment with a low UV dose and the fourth for a high UV dose. Samples were transferred into glass vials for analysis, without headspace.

Calculation of parameters 2.6.1 Bromine incorporation

Bromine incorporation was calculated as a percentage of the incorporated halogen, comprising bromine for each group of DBPs, by adding molar concentrations of each group species multiplied by the associated number of incorporated bromines divided by the total amount of incorporated halides. For example, the incorporation for THM was estimated by summing up threefold the molar concentration of bromoform, twofold the molar concentration of dibromochloromethane and the molar concentration of bromodichloromethane, which was then divided by three times the total THM.

Toxicity

Toxicity was estimated as reported by Hansen et al. (2012b). Based on the measured concentration of the different DBPs, cytotoxicity and genotoxicity were estimated as the sum of the concentration of each compound divided by its EC 50 (Equation ( 1)):

Toxicity = ∑ C EC , (1)
All EC 50 values were used as reported in the literature [START_REF] Muellner | Haloacetonitriles vs. regulated haloacetic acids: Are nitrogen-containing DBFs more toxic?[END_REF][START_REF] Plewa | Comparative mammalian cell toxicity of N-DBPs and C-DBPs[END_REF].

Results and discussion

To evaluate the effect of post-UV chlorination, samples were included in the investigation from three indoor chlorinated seawater pools. As batch experiments were conducted in a laboratory, the DBP formation may vary from continuous UV treatment carried out over a longer time scale. Furthermore, pool water does not receive doses equivalent to several days of treatment at once in a real treatment system, and thus there might be some reactions between chlorine and photolysis by-products for the first UV dose until water enters the UV reactor a second time. [START_REF] Cheema | Combined UV treatment and ozonation for the removal of by-product precursors in swimming pool water[END_REF][START_REF] Spiliotopoulou | Secondary formation of disinfection by-products by UV treatment of swimming pool water[END_REF], and so no further investigations were included in this study. Bromide content varied between 75 and 88.5 mg/L in the three pools, representing a higher bromide content than found in the raw seawater used to fill these pools (72 mg/L). This discrepancy may be explained by the low frequency of water renewal, thereby leading to the upconcentration of bromide through water evaporation.

Chlorine consumption

All treatments were analysed for residual chlorine after chlorination for 24 h (Fig 1). Low chlorine consumption was observed in dark control samples; however, it increased following UV treatment in all pools. Furthermore, the increase in chlorine consumption was proportional to consumption in the dark control samples for each pool, e.g. pool-1, high consumption in dark control and high increase in consumption by treatment; pool-2, lowest consumption in dark control and lowest increase in consumption by treatment and pool-3, highest consumption in dark control and highest increase in consumption after UV treatment. Moreover, high UV exposure resulted in high consumption in all pools, while chlorine consumption was higher in pool-3 compared to the other pools, most likely due to the higher TOC in this pool.

A likely explanation for the increase in chlorine consumption is that UV irradiation degraded organic matter in the pool samples to species more reactive to chlorine. A similar trend has been reported in freshwater pool studies [START_REF] Cheema | Combined UV treatment and ozonation for the removal of by-product precursors in swimming pool water[END_REF][START_REF] Cimetiere | Effects of UV-dechloramination of swimming pool water on the formation of disinfection by-products: A lab-scale study[END_REF][START_REF] Spiliotopoulou | Secondary formation of disinfection by-products by UV treatment of swimming pool water[END_REF] and also in drinking water, where it has been observed that UV treatment increases the fraction of low molecular organic matter, which in turn increases chlorine consumption [START_REF] Choi | The effects of UV disinfection on drinking water quality in distribution systems[END_REF]. Furthermore, hypobromous acid (HBrO) and hypobromite (BrO -) in seawater might be photolysed by UV radiation into hydroxyl (HO • ) and bromine radicals (Br • ) [START_REF] Feng | Photolysis of aqueous free chlorine species (HOCl and OCl-) with 254 nm ultraviolet light[END_REF][START_REF] Watts | Chlorine photolysis and subsequent OH radical production during UV treatment of chlorinated water[END_REF], which then might react with organic matter present in the pool water. Thereby, non-chlorine reactive molecules may become highly reactive after UV treatment, thus leading to an observed increase in chlorine consumption. Nonetheless, in a recent study, increased chlorine reactivity caused by radical oxidation was found to be insignificant compared with the increase resulting from UV radiation [START_REF] Spiliotopoulou | Secondary formation of disinfection by-products by UV treatment of swimming pool water[END_REF].

I n i t i a l 2 D C , C l 2 , C l 2 / C l 1 / 2 d U V 2 , C l 2 / C l 1 d U V I n i t i a l 2 D C , C l 2 , C l 2 / C l 1 / 2 d U V 2 , C l 2 / C l 1 d U V I n i t i a l 2 D C , C l 2 , C l 2 / C l 1 / 2 d U V 2 , C l 2 / C l 1 d U V 0 1 2 3 4 5 POOL -1 POOL -2 PO OL -3 NA NA NA
Chlorine consumption (mg/L) Figure 1 Chlorine consumption after 24 h at 25°C. Three pool samples were investigated as pool-1, pool-2 and pool-3. 'DC', 'UV 1/2 ' and 'UV 1d ' represents dark control, half-day UV-dose and one-day UV dose, respectively. 'UV/Cl 2, Cl 2 ' represents a pre-chlorine-adjusted pool water sample treated with UV followed by chlorination. 'NA' is not applicable and indicates no incubation with chlorine, i.e. no consumption.

3.2 Formation of DBPs in post-UV chlorination

Haloacetic acids (HAAs)

Initial concentrations of HAA were higher in pool-2 compared with the other two pools (Figs. 2f,2g,2h,2i).

All pool water samples had formation potential for HAA, as increased concentrations were observed after chlorination for 24 h. The highest formation potential (dark control) of HAA was observed in pool-3, which had the lowest initial HAA concentration, whereas the lowest formation potential (dark control) was noticed in the pool water (pool-2) with the highest initial HAA concentrations. The high increase in the formation potential (dark control) of pool-3 is likely due to the high TOC in this water. Furthermore, dibromoacetic acid and tribromoacetic acid were observed as predominant haloacetic acids, while bromochloroacetic acid and dibromochloroacetic acid were the sparsest of the detected compounds.

The formation of total HAA (THAA) decreased in all pool water samples with post-UV chlorination (Fig 2).

In fact, in pools 1 and 2, concentrations after UV treatment and chlorination were lower than the initial concentrations. The highest decrease (almost 60%) was observed in pool-2, while the lowest decrease (20%)

was found in pool-1. Additionally, concentrations decreased further as a result of high UV exposure.

However, concentration levels in pool-3 remained higher after treatment in relation to the initial concentration level.

Bromochloroacetic acid concentration remained unaffected by post-UV chlorination, while dibromoacetic acid and dibromochloroacetic acid concentrations decreased significantly. Dibromoacetic acid concentration decreased to a level of 10 µg/L and dibromochloroacetic acid to 1 µg/L in all pools. Furthermore, the highest decrease in concentration was observed in tribromoacetic acid by post-UV chlorination, where the concentration level decreased to the quantification limit.

Overall, concentrations of HAA decreased by post-UV chlorination, which is likely due to the direct photolysis of the HAA, as all HAA found in the pool water contained bromine, and bromine containing DBP in swimming pool water is generally easily photolysed by UV (Hansen et al., 2013a). Studies with other water types have also described that brominated HAA is photolysed by UV treatment [START_REF] Jo | Simultaneous degradation of disinfection byproducts and earthy-musty odorants by the UV/H2O2 advanced oxidation process[END_REF].

Trihalomethanes (THMs)

Initial concentrations for THMs were higher in pool-2 compared with pool 1 and pool-3 (Figs. 2a,2b,2c).

However, low formation potential (dark control) was observed in pool-1 and pool-3, while no formation potential (dark control) was noted in pool-2. Furthermore, bromoform was the most predominant THM followed by dibromochloromethane, whereas chloroform was the least prevalent in all pools.

Concentration increased for total THM (TTHM) with UV, followed by chlorination (Fig 2j). Furthermore, a low increase in concentration was observed in pool-2, while a high increase occurred in pool-1 and pool-3.

Additionally, high UV exposure increased TTHM concentration further in all pools.

Bromoform and dibromochloromethane concentration increased following UV and chlorination treatment and increased further with high UV exposure. However, as chloroform concentrations were close to or under the quantification limit, no definite trend was observed for chloroform. [START_REF] Spiliotopoulou | Secondary formation of disinfection by-products by UV treatment of swimming pool water[END_REF] reported that UV irradiation does not create chloroform in freshwater swimming pool waters, with or without free chlorine present, while [START_REF] Cassan | Effects of medium-pressure UV lamps radiation on water quality in a chlorinated indoor swimming pool[END_REF], [START_REF] Cimetiere | Effects of UV-dechloramination of swimming pool water on the formation of disinfection by-products: A lab-scale study[END_REF] and [START_REF] Spiliotopoulou | Secondary formation of disinfection by-products by UV treatment of swimming pool water[END_REF] all reported chloroform formation as a result of chlorination following UV irradiation in freshwater pools.

An increase in bromoform and in dibromochloromethane is quite surprising; indeed, previous studies have shown that both low and medium-pressure UV lamps are efficient to remove brominated THMs [START_REF] Cimetiere | Effects of UV-dechloramination of swimming pool water on the formation of disinfection by-products: A lab-scale study[END_REF][START_REF] Nicole | Etude de la degradation des trihalomethanes en milieu aqueux dilue par irradiation UV -determination du rendement quantique de photolyse a 253,7 nm[END_REF]. In the present case, the observed increase in the concentrations of these two THMs in post-chlorinated water suggests that the amount formed during post-chlorination was higher than the amount photo-decomposed in the UV reactor.

Overall, concentration increased for THMs with UV followed by chlorination. A possible explanation is that the breaking of large organic molecules, which increased the chlorine reactivity of dissolved organic matter, also promoted precursors in the pool water for THM formation. Previous studies on freshwater pools have also reported similar trends regarding an increase in THM concentration through UV irradiation [START_REF] Cimetiere | Effects of UV-dechloramination of swimming pool water on the formation of disinfection by-products: A lab-scale study[END_REF]. However, [START_REF] Spiliotopoulou | Secondary formation of disinfection by-products by UV treatment of swimming pool water[END_REF] reported that UV treatment accelerates THM formation instead of increasing it, since pool water without UV treatment had the same increase in THM formation with a high chlorine level. The study also reported that THM concentration decreased with high UV dose treatment followed by chlorination which was likely due to photodegradation of THM and their precursors at high UV dose. Moreover, Cheema et al. ( 2017) also reported a decrease in THM concentration with high UV dose treatment followed by chlorination which was likely due to decrease in TOC level by oxidation at high UV dose and hence less amount of precursors were available for DBP formation.

Therefore, it is likely that repeating UV treatment followed by chlorination in cycles with even lower doses might decrease the formation of THM and their precursors in seawater pools.

Haloacetonitriles (HANs)

Initial concentrations were similar for HANs in all pools (Figs. 2d,2e), and no formation potential (dark control) for HANs was observed in the pool water. Furthermore, dibromoacetonitrile was the main HAN, followed by bromochloroacetonitrile.

Concentrations increased for total HANs with UV, followed by chlorination (Fig 2k). Furthermore, a high increase was observed in pool-3, while lower increases were observed in pool-1 and pool-2. However, high UV exposure had no significant effect on concentrations. Bromochloroacetonitrile concentration increased to a similar level in all pools with UV, followed by chlorination, whereas a high increase in dibromoacetonitrile concentration was observed in pool-3 and lower increases in pool-1 and pool-2.

HANs are the most toxic group investigated in this study [START_REF] Plewa | Comparative mammalian cell toxicity of N-DBPs and C-DBPs[END_REF] and have also been reported as contributing most in terms of toxicity to chlorinated pool water [START_REF] Kramer | Haloacetonitriles -another importent group of disinfection byproducts in swimming pool water[END_REF]. [START_REF] Cimetiere | Effects of UV-dechloramination of swimming pool water on the formation of disinfection by-products: A lab-scale study[END_REF] reported an increase in the concentration of dichloroacetonitrile and bromochloroacetonitrile via the post-UV chlorination of freshwater pools, which is similar to the findings in this study in relation to seawater pools. However, a recent study has reported HANs degradation in freshwater pools with UV treatment by repeating experiments in cycles [START_REF] Cheema | Combined UV treatment and ozonation for the removal of by-product precursors in swimming pool water[END_REF]. et al. (2015) reported bromine substitution shifted from the larger brominated molecules to smaller volatile compounds via UV treatment, followed by the chlorination of freshwater pool waters, which resulted in increased bromine substitution in THM. However, bromine incorporation was expected to be unaffected by UV treatment followed by the chlorination of water from seawater pools, due to the presence of high bromide levels of seawater in this study. Furthermore, previous studies with drinking water have shown increased DBP levels in the presence of bromide, as well as the formation of brominated DBPs [START_REF] Chang | Effects of bromide on the formation of THMs and HAAs[END_REF][START_REF] Hua | Effect of Bromide and Iodide Ions on the Formation and Speciation of Disinfection Byproducts during Chlorination[END_REF], which is likely due to the oxidation of bromide by hypochlorous acid to form hypobromous acid [START_REF] White | Guidelines for safe recreational water environments. Volume 2, Swimming pools and similar environments[END_REF], the latter of which reacts with the dissolved organic matter and forms brominated DBPs. A higher level of brominated DBPs has been reported by several studies for pools filled with seawater [START_REF] Chowdhury | Disinfection byproducts in swimming pool: Occurrences, implications and future needs[END_REF][START_REF] Manasfi | Identification of disinfection by-products in freshwater and seawater swimming pools and evaluation of genotoxicity[END_REF]. Therefore, increased bromide levels in seawater pool can increase concentrations of brominated DBPs, which in turn can increase health risks to swimmers. Bromine incorporation was estimated before and after treatment to evaluate the effect of UV treatment on DBP formation in the seawater pool.

Bromine incorporation

Spiliotopoulou

THM concentration increased, as bromine incorporation increased by post-UV chlorination. Furthermore, HAN concentration increased, while bromine substitution levels decreased with UV followed by chlorination. However, HAA concentration significantly decreased, as bromine incorporation decreased with UV treatment followed by chlorination. Overall, the bromine incorporation level goes up and down in a single treatment but is expected to decrease after multiple or continuous UV treatments.

Predicted toxicity

Seawater contains high concentrations of bromide which resulted in a high fraction of brominated DBPs that are generally more toxic than their chlorinated analogs [START_REF] Muellner | Haloacetonitriles vs. regulated haloacetic acids: Are nitrogen-containing DBFs more toxic?[END_REF][START_REF] Plewa | Comparative mammalian cell toxicity of N-DBPs and C-DBPs[END_REF].

Cytotoxicity and genotoxicity were estimated to evaluate the effect of DBPs formation on bather's health (Fig 3a,3b). Although brominated DBPs were detected for all the investigated groups (Fig S3, SI), the HANs had the highest contribution to the overall toxicity (Table S2, SI).

Potential health effects of swimming pool water have been reported by previous studies of freshwater pools. [START_REF] Villanueva | Bladder cancer and exposure to water disinfection by-products through ingestion, bathing, showering, and swimming in pools[END_REF] reported increased bladder cancer risk after swimming in freshwater pools, whilst [START_REF] Liviac | Genotoxicity of water concentrates from recreational pools after various disinfection methods[END_REF] reported the higher genotoxicity of freshwater pools in comparison to supply water, due to DBP formation in the pool water. Furthermore, DBP regulations in swimming pool water have mostly focused only on THMs and chloramines around the world. However, previous studies have shown that other DBPs, such as HANs, HAAs and cyanogen halides, are generally more toxic than regulated DBPs [START_REF] Glauner | Elimination of swimming pool water disinfection by-products with advanced oxidation processes (AOPs)[END_REF][START_REF] Zwiener | Drowning in disinfection byproducts? Assessing swimming pool water[END_REF]. Therefore, in order to evaluate the effect of DBP formation on swimmers' health, the cytotoxicity and genotoxicity of DBPs were estimated as described in the materials and method section, using Equation 1.

The calculated initial cytotoxicity was lower in pool-3 than in pool-1 and pool-2 (Fig. 3a). In general, the cytotoxicity increased in the chlorinated samples (dark control) expect for pool-2 where it was unchanged.

The cytotoxicity was further increased in the samples which were UV treated and chlorinated and the highest calculated cytotoxicity was found for pool-3. Furthermore, HANs contributed most to toxicity, followed by

HAAs and with THMs contributing the least. Calculated genotoxicity (Fig. 3b) followed similar trends as cytotoxicity where increased genotoxicity was found in UV treated and chlorinated samples compared with the initial samples. The HANs had the highest contribution to the calculated genotoxicity and HAAs only had a minor contribution. The THMs did not contribute to calculated genotoxicity as they are not genotoxic [START_REF] Muellner | Haloacetonitriles vs. regulated haloacetic acids: Are nitrogen-containing DBFs more toxic?[END_REF][START_REF] Plewa | Comparative mammalian cell toxicity of N-DBPs and C-DBPs[END_REF].

Overall, HANs were the main toxicity contributor and most potent DBP group investigated in this study. A previous study on freshwater pools also reported HANs' higher potency of cytotoxicity and genotoxicity than HAAs and THMs [START_REF] Plewa | Comparative mammalian cell toxicity of N-DBPs and C-DBPs[END_REF]. Furthermore, [START_REF] Kramer | Haloacetonitriles -another importent group of disinfection byproducts in swimming pool water[END_REF] reported that two HANs, dibromoacetonitrile and bromochloroacetonitrile, were the main contributors to the overall toxicity of pool water in freshwater pools. However, a recent study reported HANs degradation in freshwater pools following repeated UV treatment [START_REF] Cheema | Combined UV treatment and ozonation for the removal of by-product precursors in swimming pool water[END_REF]. As this study was conducted in a single time batch experiment, it is likely that the toxicity would indeed decrease in seawater pools following repeated or continuous UV treatment.

1

 1

Figure 2 .

 2 Figure 2. Concentration of investigated DBPs in different experimental conditions for samples from three different pools, where a), b) and c) represent THMs; d) and e) represent HANs; f), g), e) and h) represent HAAs and j), k) and l) represent total THM, total HAN and total HAA, respectively. All treatments are followed by post-chlorination except for the initial analysis. The error bars represent the standard deviation of three replicates. Percentage (%) indicates the amount of bromine incorporation. The dotted line indicates the limit of quantification (LOQ), and error bars are standard deviation among replicates.

  acid, tribromoacetic acid). However, some of the DBPs were not detected during the analysis, for example bromodichloromethane, dichloroacetonitrile, trichloroacetonitrile, monochloroacetic acid, monobromoacetic acid, dichloroacetic acid, trichloroacetic acid and bromodichloroacetic acid.Water quality parameters from the three different pools sampled in this study are listed in Table1. These three pools are among pools studied in previous papers :[START_REF] Manasfi | Identification of disinfection by-products in freshwater and seawater swimming pools and evaluation of genotoxicity[END_REF] found a TOC value of 11 to 12 mg/L (due to heavy use and high frequentation in summer) and[START_REF] Parinet | Exposure levels to brominated compounds in seawater swimming pools treated with chlorine[END_REF] reported a TOC value of 2.0 to 3.6 mg/L (due to lower frequentation in winter), these latter being similar to ours (sampling in March).Furthermore, similar TOCs were reported by several studies on UV in freshwater pools, for example[START_REF] Cheema | Combined UV treatment and ozonation for the removal of by-product precursors in swimming pool water[END_REF], 1.8 mg/L; Cimetiere and De Laat. (2014), 2.75 mg/L and Spiliotopoulou et al. (2015), 2.1 mg/L. Moreover, TOC concentration was not significantly affected by post-UV chlorination, as reported in previous studies of freshwater pools

	Three classes of DBPs were investigated, including THMs (chloroform, bromodichloromethane,
	dibromochloromethane,	and	bromoform),	HANs	(dichloroacetonitrile,	trichloroacetonitrile,

bromochloroacetonitrile, dibromoacetonitrile) and HAAs (monochloroacetic acid, monobromoacetic acid, dichloroacetic acid, trichloroacetic acid, bromochloroacetic acid, dibromoacetic acid, bromodichloroacetic acid, dibromochloroacetic

Table 1 .

 1 Water quality parameters

	Parameters	Pool-1	Pool-2	Pool-3
	Temperature ( º C)	32.0	30.2	33.3
	pH	8.13	8.07	8.22
	Conductivity (mS/cm)	61.3	62.0	70.8
	Br -(mg/L)	88.5	75.0	78.3
	O 2 (%)	104	100	104
	TOC (mg/L)	2.72	3.14	3.92
	Free chlorine (mg/L)	0.57	0.52	0.91
	Total chlorine (mg/L)	0.72	0.76	1.11

Conclusions

The present study is the first to investigate the fate of brominated DBPs, submitted to a medium-pressure UV lamp followed by post-chlorination, in real seawater swimming pool samples. The results show that UV followed by chlorination does not lead to real abatement in most of the DBPs. Concentration of HAA i.e.

dibromoacetic acid and dibromochloroacetic acid significantly decreased while concentration of THM i.e.

bromoform and dibromochloromethane and concentration of HAN i.e. bromochloacetonitrile and dibromoacetonitrile increased with UV followed by chlorination. Moreover, as HAN were the main contributor to the estimated toxicity thus the toxicity increased with UV treatment followed by chlorination.

Therefore, this study suggests that UV treatment used for dechloramination could result in increased formation of some brominated DBPs in seawater swimming pools. Further studies would be required to interpret these findings including the influence of water composition, UV dose rate and chlorine dose on the kinetics of brominated DBP formation. Another key factor to investigate in follow up experiments is the effect of repeated treatments which could reduce TOC level and most importantly, the concentration of DBPs in seawater pools.

Acknowledgements

The authors would like to thank the public pools' staff for their cooperation. Waqas A. Cheema is thankful to National University of Sciences & Technology (NUST) and Technical University of Denmark (DTU) for their cooperation and support of this work.


J Brisswalter
email: jeanick.brisswalter@wanadoo.fr

; C Hausswirth

; F Vercruyssen

; M Collardeau

; J M Vallier

; R Lepers

; C Goubault

Carbohydrate ingestion does not influence the change in energy cost during a 2-h run in well-trained triathletes

Keywords: running, Fatigue, Carbohydrate feeding, Triathlon

The aim of this study was to examine whether the increase in the energy cost of running (C r ), previously reported to occur at the end of a prolonged run, could be influenced by the ingestion of either an artificially sweetened placebo (Pl) or a 5.5% carbohydrate (CHO) solution. Ten well-trained triathletes completed three testing sessions within a 3-week period. The aim of the first session was to determine maximal oxygen uptake (V 0 2max) and the velocity associated with ventilatory threshold (v VT ). The second and the third sessions were composed of two submaximal treadmill runs (20 min long, 0% grade, performed at v VT ), before and after an 80-min overground run, also conducted at v VT . During these submaximal tests, the subjects ingested (in a random order) either a Pl or CHO solution prior to the first submaximal run and every 20 min after that. During the first session, Ventilatory threshold (VT) occurred at [mean (SD)] 81.2 (2.5)% V0 2max and 16.5 (0.6) km • h -1 . A significant effect of exercise duration was found on C r (∆Cr) at the end of the run, whatever the solution ingested (∆C r = 5.7% and 7.01% for CHO and Pl, respectively). A reduction in the respiratory ex-change ratio (from 0.98 to 0.90) was observed only at the end of the Pl trial. In this study, C r seems to be affected only to a minor extent by substrate turnover. Moreover, the increase in the demand for oxygen, estimated from the increase in ventilation, accounted for only a minor proportion of the increase in C r (11% and 17% for CHO and Pl, respectively). No correlation was found between the changes in C r and the changes in the other physiological parameters recorded. These results suggest, indirectly, that C r increases during a 2-h run at 80% V0 2max in well-trained subjects can be explained mainly by alterations in neuromuscular performance, which lead to a decrease in muscle efficiency.

Introduction

Triathlon is an endurance activity in which athletes compete sequentially in swimming, cycling and running. In addition, the main constraint placed upon competitors during a triathlon race is the exercise duration, which ranges from 1 h to 10 h (Sprint to Ironman, respectively). Numerous studies have tried to identify those physiological Variables that are the most likely to result in successful triathlon performance. For exercise lasting more than 2 h, successful performances are dependent upon various physiological, biomechanical and psychological factors. Variables such as maximum oxygen uptake (V02 max), the maximal fraction of VO2 max that can be sustained and the energy cost of locomotion are often cited (e.g., [START_REF] O'toole | Applied physiology of triathlon[END_REF]. At the end of a triathlon, recent studies have observed a significant increase in energy cost that is related to a variation in some physiological or biomechanical parameters [START_REF] Guezennec | The energy cost of running increases at the end of a triathlon[END_REF][START_REF] Hausswirth | Relationships between running mechanics and running efficiency at the end of a triathlon and a marathon[END_REF].

The energy cost of running (C r ), defined as the amount of energy spent per unit distance (di Prampero 1986), depends upon both physiological and biomechanical factors, and several non-exclusive mechanisms have been proposed to explain the increase in C r (e.g., [START_REF] Martin | Biomechanical considerations for economical walking and running[END_REF]. Therefore, the issue of the effect of exercise duration on the increase in energy cost remains to be established in this area of research, which is essentially interdisciplinary. On the one hand, factors such as changes in running pattern and neuromuscular performance have been suggested to influence C r through an alteration in running mechanics [START_REF] Brisswalter | Variability in energy cost and walking gait during a racewalking in competitive race walkers[END_REF][START_REF] Hausswirth | Relationships between running mechanics and running efficiency at the end of a triathlon and a marathon[END_REF][START_REF] Nicol | Fatigue effects of marathon running on neuromuscular performance: changes in force, integrated electromyographic activity and endurance capacity[END_REF]. On the other hand, previous studies on long-distance events have implicated the role of physiological factors such as thermoregulation and substrate turnover in this increase in C r [START_REF] Bosch | Physiological differences between black and white runners during a treadmill marathon[END_REF][START_REF] Brisswalter | Variability in energy cost and walking gait during a racewalking in competitive race walkers[END_REF][START_REF] Bruckner | The energy cost of running increases with distance covered[END_REF][START_REF] Guezennec | The energy cost of running increases at the end of a triathlon[END_REF]. It has been suggested that muscle glycogen depletion, which occurs in active muscle fibers during prolonged exercise, affects C r [START_REF] Amstrong | Muscle fiber activity as a function of speed and gait[END_REF][START_REF] Guezennec | The energy cost of running increases at the end of a triathlon[END_REF]. Earlier studies have clearly shown that the significant alteration in C r observed after a race longer than 15 km is concomitant with a significant decrease in the respiratory exchange ratio (R) to values close to 0.85. [START_REF] Bosch | Physiological differences between black and white runners during a treadmill marathon[END_REF][START_REF] Brisswalter | Variability in energy cost and walking gait during a racewalking in competitive race walkers[END_REF][START_REF] Bruckner | The energy cost of running increases with distance covered[END_REF]. This decrease indicates that a long exercise duration leads to glycogen depletion and an increase in free fatty acid oxidation (e.g., [START_REF] Ahlborg | Substrate turnover during prolonged exercise in man. Splanchnic and leg metabolism of glucose, free fatty acids and amino acids[END_REF]. Thus, about half of the demand for energy is accounted for by the oxidative breakdown of lipid substrates, and the low efficiency of lipid oxidation can partly explain the increase in C r (e.g., [START_REF] Sahlin | Metabolic changes limiting muscle performance[END_REF]).

Classically, it has been reported that glycogen sparing during prolonged exercise is linked with the metabolism of the exogenous supply of carbohydrate (CHO: e.g., [START_REF] Tsintzas | Carbohydrate ingestion and single muscle fiber glycogen metabolism during prolonged running in men[END_REF]. Ingesting a CHO solution during prolonged submaximal exercise delays the onset of fatigue and improves endurance performance, especially in exercise lasting 2 h or more (e.g., [START_REF] Coyle | Timing and method of increased carbohydrate intake to cope with heavy training, competition and recovery[END_REF]. In addition, in some studies it has been observed that running performance is higher at the end of a 30-km run when subjects ingested a CHO-electrolyte solution than when they drank water [START_REF] Tsintzas | Influence of carbohydrate-electrolyte drinks on marathon running performance[END_REF][START_REF] Williams | Influence of carbohydrate fluid intake on endurance running performance[END_REF]). To the best of our knowledge, the effect of CHO ingestion during exercise lasting longer than 1 h has been studied mostly for exercise of moderate intensity (<75% VO2max). Nevertheless, endurance triathletes have been reported to be able to run at high submaximal intensities VO2max) corresponding to their ventilatory threshold (VT), at the end of a triathlon [START_REF] Sleivert | Physical and physiological factors associated with success in the triathlon[END_REF]. The aim of the current investigation was therefore to compare the change in C r (∆C r ) of well-trained triathletes during a 2-h run completed at VT, when ingesting a CHO solution or a sweetened placebo solution (Pl).

Methods

Subjects

The subjects were ten well-trained triathletes who were competing regularly at inter-regional or national level. All subjects were experienced treadmill runners and were injury free. The physical characteristics of the subjects are given in Table 1. Before participating in this study, the subjects were fully informed about the protocol, and informed consent was obtained prior to all testing, according to local ethical committee guidelines.

Protocol

Each subject completed three testing sessions in a 3-week period. Subjects were asked to stop their training program 24 h before each testing session. The first session was used to determine VO 2max and oxygen uptake (VO2) at the VT. The second and the third sessions were composed of two submaximal treadmill runs, which were performed before and after an 80-min overground run. All of these submaximal tests were conducted at the velocity that elicited VT (v VT ). According to previous studies [START_REF] Coyle | Timing and method of increased carbohydrate intake to cope with heavy training, competition and recovery[END_REF][START_REF] Gisolfi | Fluid balance for optimal performance[END_REF][START_REF] Tsintzas | Influence of carbohydrate-electrolyte drinks on marathon running performance[END_REF][START_REF] Tsintzas | Carbohydrate ingestion and single muscle fiber glycogen metabolism during prolonged running in men[END_REF], during these two sessions subjects had to ingest (in a random order), either an artificially sweetened PI drink, or a 5.5% CHO solution (glucose, fructose, malto-dextrins). The P1 and CHO solutions used contained the same concentration of electrolytes (sodium: 20 mEq, potassium: 5 mEq). Furthermore, subjects were required to record their normal diet and to consume exactly the same diet for 2 days before each of the submaximal sessions.

VO2max Session 1: evaluation of VO2max

VO2max was determined in a first session during an incremental protocol on a treadmill, according to the criteria described by [START_REF] Howley | Criteria for maximal oxygen uptake: review and commentary[END_REF]. After familiarization on the treadmill at 10 km • 11 -1 and 3% grade, the speed of walking was increased by 1 km • h -1 every 1 min until the subject reached volitional exhaustion. V0 2 max was deemed to have been reached when the following criteria had been achieved: a plateau in VO 2 , an R value of 1.15 or greater, and a post-exercise blood lactate level above 8 mmol • l -1 . The VT was estimated from the non-linearity of a plot of carbon dioxide output against VO 2 [START_REF] Sherill | Using smoothing splines for detecting ventilatory thresholds[END_REF]).

Sessions 2 and 3: overground run and submaximal tests

During the second and the third sessions, an overground run was undertaken on a 400-m track very close to the laboratory. The running speed of each subject was checked and regulated every 400 m, so that it was kept constant over the 80-min period. Before and after this overground test, each subject completed a submaximal test. Before the first submaximal test (Test 1), the subjects performed a 10min treadmill familiarization run at 10 km • h -1 .

After a 4-min rest period, the first 20-min, 0% grade submaximal run was carried out. The same test was performed immediately after the overground run (Test 2; Fig. 1). Throughout the test, the treadmill speed was controlled continuously by a photoelectric cell.

According to the study of [START_REF] Tsintzas | Carbohydrate ingestion and single muscle fiber glycogen metabolism during prolonged running in men[END_REF], after the accommodation run, 8 ml • kg -1 body weight of fluid had been ingested, and thereafter 2 ml • kg -1 body weight was ingested every 20 min.

Experimental measurements and calculations

At rest and during the treadmill exercise, respiratory gases were collected using a breath-by-breath system (CPX Medical Graphics). The interval between measurement of respiratory parameters was set at 10 s. Five periods were analyzed: the 3rd (T 3 ), the 10th (T10), and the 20th (T 20 ) min of Test 1, and at the beginning (T 100 ) and the end (T 120 ) of Test 2. The analyzers were calibrated before and after each subject's session by using gases of known concentration. Heart rate (HR) was recorded continuously (Baumann 5000). Fingertip samples of arterialized capillary blood were obtained before exercise and immediately at the end of each submaximal test (T 20 and T 120 ). The samples were analyzed for blood glucose concentration with an automated glucose oxidase method, and for lactate concentration (YSI, Yellow Spring Instruments). At the end of each submaximal treadmill test, the subjects were asked to report their rating of perceived exertion (RPE) using the Borg 15-grade scale [START_REF] Noble | The Borg scale: development, Administration and experimental use[END_REF]. Body mass (BM) was measured before and immediately after the submaximal run in order to observe a possible mass loss at the end of the prolonged run.

For each period, the C r was calculated from the VO 2 values recorded, according to the equation (di Prampero 1986): C r [(VO2 -V 02rest )v -1 ) • 60], where C r is expressed in ml • k-g -1 km 1, VO 2 in ml • kg -1 • min -1 , and v in km • h -1 . VO 2rest is resting VO 2 and was recorded during the 4 min preceding T1; the last 2-min values were used.

Statistical analysis

All data are expressed as the mean (SD). Two-way analyses of variance (CHO vs Pl) with repeated measures were performed using C r , minute ventilation (V E ), frequency of breathing (f b ), R (five periods), lactate concentration, plasma glucose concentration (three periods), RPE and BM (two periods) as dependant variables. Newman-Keuls post hoc tests were used to compare specific means.

Relationships between variables were analyzed using Spearmanrank correlation coefficients. For all of the statistical analyses, the level of significance was set at P < 0.05.

Results

The mean data for the maximal parameters recorded during the first session are presented in Table 1. During this test, VT occurred at 81.2 (2.5)% V02max and 16.5 (0.6) km • h -1 . Table 2 shows the mean Values of C r V E , f b , and R measured at T3 and T120 for both conditions (Pl vs CHO). In our study, the first period (T 3 ) was used to control intra-individual variability (i.e., coefficient of variation, cv) in physiological parameters among sessions. The results show no significant variation in C r or the other variables between sessions [for C r : cv = 2.5 (1.4)%, P > 0.05].

A significant effect of period was found for Cr, whereas no significant interaction effect of CHO or Pl ingestion was observed. Whatever the condition, we have found a slight but significant increase in C r , where ∆C r , = [(T 120 -T 3 )/T 3 ] • 100, at the end of the run (∆C r , = 5.7% and 7.01% for CHO and Pl, respectively). The mean Values of V0 2 represented 81.4% and 86.8% V02max •f or T3 and T 120, respectively < 0.05). Conversely, a significant interaction effect was found for R Values. At the end of the run, R decreased significantly only during the Pl trial (0.98 and 0.90, for T 3 and T 120 , respectively), whereas a non-significant trend was observed during the CHO trial (T 3 vs T 120 : 0.97 vs 0.95, respectively, P > 0.05).

During the first submaximal test, no significant difference was found between the C r values calculated at T 3 , T 10 and T 20 , and a significant increase in plasma glucose concentration was observed during the CHO trial at T 20 .

Significant variations in C r and R were observed at the beginning of the second test [∆C r (T 3 vs T 100 ) = 4.6% vs 4.7%, respectively, and ∆R (T 3 vs T 100 ) = -6.8% Vs -1.05%, respectively: Fig. 2]. Furthermore, a significant increase in V E , HR, and f b , were found with exercise duration, without any interaction effect of the solution ingested.

No effect of CHO ingestion was observed for RPE. Between the two submaximal tests, the RPE increased significantly from "very light" [9.7 (0.8) Vs 10.1 (0.9) for the Pl and CHO conditions, respectively] to "somewhat hard" [13.3 (1.2) vs 12.6 (0.8) for Pl and CHO trials, respectively]. Moreover, a significant increase in lactate concentration was observed between T 20 and T 120 (Table 3). Finally, for both conditions no significant changes in BM or plasma glucose concentration were found after the 2-h run.

The mean significant Variations expressed as a percentage of Test 1 [∆ = RT 120 -T 3 )/T 3 ] • 100} observed between the two tests were: ∆VE= 15.2 (12)%, ∆f b = 10.3 (9.8)%; ∆HR = 6.2 (4.6)%; ∆RPE = 30.6 (8.4)%; and ∆ lactate concentration = 18 (5)%. No significant correlations were found between these variations, and a wide inter-individual variability in these changes was observed for C r (range: -2% to 12%), V E (range: -8% to 38%), and RPE (range: 18% to 44%).

Discussion

The main finding of this study was that the significant increase in C r observed at the end of a 2-h run in well-trained triathletes seemed to occur independently of whether the CHO or Pl solution was ingested during exercise. This result can be compared with those of previous studies indicating similar results for exercise of a shorter duration (i.e., <90 min; [START_REF] Below | Fluid and carbohydrate ingestion independently improve performance during 1 hour of intense exercise[END_REF][START_REF] Sproule | The influence of either no fluid or carbohydrateelectrolyte fluid ingestion and the environment (thermoneutral vs hot and humid) on running economy after prolonged high intensity exercise[END_REF] or lower intensities (i.e., <75% VO 2max; [START_REF] Blomstrand | Effect of branched-chain amino acid and carbohydrate supplementation on the exercise-induced change in plasma and muscle concentration of amino acids in human subjects[END_REF][START_REF] Mitchell | Influence of carbohydrate dosage on exercise performance and glycogen metabolism[END_REF][START_REF] Tsintzas | Carbohydrate ingestion and single muscle fiber glycogen metabolism during prolonged running in men[END_REF].

Like previous studies conducted with triathletes [START_REF] Guezennec | The energy cost of running increases at the end of a triathlon[END_REF][START_REF] Hausswirth | Relationships between running mechanics and running efficiency at the end of a triathlon and a marathon[END_REF], the present study examined the effect of a 2-h run on the Variability in C r . While for a shorter exercise duration an increase in C r is not always reported to occur (e.g., [START_REF] Bruckner | The energy cost of running increases with distance covered[END_REF][START_REF] Dressendorfer | Acute reduction in maximal oxygen uptake after long-distance running[END_REF][START_REF] Sproule | The influence of either no fluid or carbohydrateelectrolyte fluid ingestion and the environment (thermoneutral vs hot and humid) on running economy after prolonged high intensity exercise[END_REF], this increase seems to occur systematically when the exercise duration is longer than 90 min (e.g., [START_REF] Bruckner | The energy cost of running increases with distance covered[END_REF][START_REF] Guezennec | The energy cost of running increases at the end of a triathlon[END_REF][START_REF] Hausswirth | Relationships between running mechanics and running efficiency at the end of a triathlon and a marathon[END_REF]Tsintzas et al. 1996 Xu and[START_REF] Xu | Effect of prolonged exercise at 65 and 80% of V02max on running economy[END_REF]. In our study, the mean increase in C r increase ranged from 5.7% to 7.01%. These Values are in agreement with those reported previously. [START_REF] Bruckner | The energy cost of running increases with distance covered[END_REF] observed a 5% increase in C r at the end of a marathon, and [START_REF] Guezennec | The energy cost of running increases at the end of a triathlon[END_REF] and [START_REF] Hausswirth | Relationships between running mechanics and running efficiency at the end of a triathlon and a marathon[END_REF] noted a 7% increase in C r in triathletes after a 135-min run.

Most of these studies have associated the increase in C r observed at the end of a long-duration running exercise with glycogen depletion and the progressive use of the fatty acids to replace CHO as an energy source. Classically, it has been suggested that glycogen depletion in working muscle is related to fatigue in athletes during exercise ranging in duration and intensity from 1 to 3 h and 65% to 85% V 0 2 max respectively (e.g., [START_REF] Coyle | Timing and method of increased carbohydrate intake to cope with heavy training, competition and recovery[END_REF]. Within this framework, CHO feedings during exercise improved endurance performance and delay, but did not prevent fatigue (e.g., [START_REF] Coyle | Muscle glycogen utilization during prolonged strenuous exercise when fed carbohydrate[END_REF]). It has been suggested that during prolonged cycling exercise, CHO ingestion maintains plasma glucose concentrations late in exercise. Subjects would therefore be able to maintain a higher exercise intensity at the end of exercise [START_REF] Coggan | Carbohydrate ingestion during prolonged exercise: effects on metabolism and performance[END_REF]. In the present study, no hypoglycemic state was observed at the end of exercise during any of the treatments, and no effect of treatment was observed on plasma glucose concentration after 2 h. This is in agreement with the work of [START_REF] Tsintzas | Carbohydrate ingestion and single muscle fiber glycogen metabolism during prolonged running in men[END_REF], who found no significant difference between a P1 and a CHO trial from 40 min into a 2-h run [132 (12.3) min]. For running events, the effect of CHO ingestion on endurance response and/or glycogen sparing seems less clear, and is different than for cycling (e.g., [START_REF] Derman | Fuel kinetics during intense running and cycling when fed carbohydrate[END_REF]. For example, an improvement in endurance running performance is not systematically observed with CHO ingestion [START_REF] Noakes | Carbohydrate ingestion and muscle glycogen depletion during marathon running and ultramarathon racing[END_REF]). Furthermore, it has been suggested that glycogen depletion, and thus the importance of CHO availability, is less significant if the duration of the run is not longer than 2 h (e.g., [START_REF] Coggan | Carbohydrate ingestion during prolonged exercise: effects on metabolism and performance[END_REF]. It is therefore possible that in well-trained athletes, a 2-h run is not long enough to observe, on the one hand, a significant decrease in whole-muscle glycogen levels and, on the other hand, a significant effect of CHO ingestion on glycogen sparing. This assumption may be illustrated by the higher, but not significant, variation in C r observed during the P1 trial when compared to the CHO trial between T 100 and T 120 [∆C r , (T 120 -T 100 ) = 2.5% and 1.2% for the Pl and CHO trials, respectively]. Furthermore, we observed a significant decrease in R from T 100 to T 120 during the P1 trial only. Classically, a decrease in R is reported to occur during prolonged exercise, leading to a lower energy equivalent of V0 2 . This decrease is often used to explain increases in C r that occur as a result of the effect of substrate turnover. When this effect is slight, earlier results have indicated that the variability in C r could be independent of the reduction in R. For example, it has been reported that running economy could remain constant for 1 day after a 30-min run at 85% V02max despite a lower R [START_REF] Morgan | Effects of a prolonged maximal run on running economy and running mechanics[END_REF]). In the study presented here, we could make the hypothesis that in well-trained athletes, during a 2-h run the decrease in R was not sufficient to be associated with a significant rise in C r . These results seem, therefore, to minimize the role of metabolic changes in the increase in C r that occurs during high-intensity submaximal runs that range in duration from 1 to 2 h (e.g., [START_REF] Sproule | The influence of either no fluid or carbohydrateelectrolyte fluid ingestion and the environment (thermoneutral vs hot and humid) on running economy after prolonged high intensity exercise[END_REF]).

In the present study, several other factors could be responsible for the increase in C r . For one, we observed a systematic increase in the relative intensity of exercise with exercise duration, with a variation in the mean values of VO 2 that represented from 81.4% to 86.8% VO 2 max • Furthermore, we have found a significant increase in lactate concentration and in RPE between the submaximal tests. It is well known that when exercise is associated with a concomitant lactic acid accumulation (heavy intensity), a slow rise in VO 2 , known as the VO 2 slow component, occurs during constant-intensity prolonged exercise (e.g., [START_REF] Barstow | Characterization of VO 2 kinetics during heavy exercise[END_REF][START_REF] Poole | Determinants of oxygen uptake: implications for testing[END_REF]. While no significant effect of exercise duration on C r was found between the 3rd and the 20th min of exercise, the continuous additional rise in C r observed from the 100th min could be related to the mechanism underlying the VO 2 slow component. Several factors, such as the work of breathing, thermal stress, or the role of the exercising muscle, could contribute to this phenomenon [START_REF] Casabury | Influence of work rate on ventilatory and gas exchange kinetics[END_REF][START_REF] Poole | Role of exercising muscle in slow component of V0 2[END_REF]). On the one hand, thermal stress could be involved in the increase in V E observed at the end of the 2-h run [START_REF] Hanson | Determinants and consequences of ventilatory responses to competitive endurance running[END_REF]. Therefore, an increase in the work of breathing could be responsible for one part of the VO 2 slow component. For mean group values, our results indicate a significant increase of V E at the end of the test. Based on the equation proposed by [START_REF] Coast | Ventilatory work and oxygen consumption during exercise and hyperventilation[END_REF], the VO 2 demand estimated from the increase in the work of breathing represents, in our study, 39.5 (0.5) m10 2 • min -1 and, therefore, could only explain 11% and 17% of the increase in C r observed in the Pl and CHO trials, respectively. When individual adaptations are considered, no significant correlations were found between increases in V E and increases in C r (Fig. 3).

On the other hand, biomechanical factors are often attributed to the VO 2 slow component. [START_REF] Poole | Role of exercising muscle in slow component of V0 2[END_REF] suggested the important link between changes in the pattern of activity of the working muscle and the additional increase in V 0 2. Prolonged exercise is commonly associated with changes in electromyographic activity and/or changes in fiber recruitment. For instance, progressive increases in exercise intensity toward "heavy intensity" exercise would lead to a larger recruitment of type II fibers. Therefore, the resulting decrease in thermodynamic muscle efficiency (e.g., Woledge 1998) could explain, at least in part, the increase in the energy cost of locomotion. Furthermore, recent studies have observed that the reduction in neuromuscular performance previously reported to occur during prolonged cycling exercise (e.g. [START_REF] Capelli | Effects of prolonged cycle ergometry on maximal muscle power and oxygen uptake in humans[END_REF]) is more relevant after a marathon than after a triathlon [START_REF] Hausswirth | Relationships between running mechanics and running efficiency at the end of a triathlon and a marathon[END_REF][START_REF] Lepers | Reduction of neuromuscular performances after prolonged exercise: comparison of marathon and triathlon[END_REF]. While the relationship between increases in C r and mechanical changes with fatigue remains unclear, a major portion of the alteration in C r could be attributed to the increase in mechanical constraints (i.e., eccentric muscle work) that exists during prolonged running at high, but submaximal intensity.

In conclusion, the results of this study confirm that C r increases during a 2-h run. The increase in C r observed here was not influenced by CHO ingestion during exercise or by a reduction in R. These results appear to minimize the role of metabolic changes or increases in the work of breathing during prolonged exercise at 80% V0 2 max suggesting (indirectly) that the increase in C r observed in our welltrained subjects can be explained mainly by a decrease in muscle efficiency.

Figure 1 Figure 2 Figure 3

 123 Figure 1 Experimental protocol. (v VT -Ventilatory threshold)

Table 1

 1 Physical characteristics and maximal physiological data of the subjects (n = 10). (V02ma x Maximum oxygen uptake, H Rmax maximum heart rate, v max maximum velocity) V max represents the speed that elicits VO 2max .

	Parameter	Mean (SD)	Minimum	Maximum
	Age (years)	24.5 (3.2)	21	31
	Body mass (kg)	70.2 (7.9)	61	86
	Height (cm) VO2max (m1-kg -1 • min -1 )	179.7 (7.8) 66.8 (3.9)	173 59.2	195 73.4
	HR max (beats • min -1 ) v max (km • h -1 ) a	183 (9.7) 20.1 (1.45)	177 18	201 22

a

Table 2

 2 Mean group values of the energy cost of running (Cr), minute ventilation (VE), respiratory frequency (f b ) and respiratory exchange ratio (R), recorded at the 3rd min of Test 1 (T 3 ) and at the end of Test 2 (T 120 ), while ingesting either a placebo (Pl) or a carbohydrate (CHO) solution

	Variable	Solution	T 3		T 120
			Mean	SD	Mean	SD
	Cr (ml • kg -1 • km -1 )	Pl	185.9	8.7	200.6*	9.2
		CHO	183.5	9.7	195.1*	9.1
	V E , (1 • min -1 )	P1	92.1	15.6	112.1*	17.7
		CHO	92.2	13.8	107.8*	14.8
	f b (cycles • min -1 )	P1	42.4	3.5	46.9*	4.1
		CHO	42.1	3.7	46.1*	4.2
	R	Pl	0.98	0.02	0.90*	0.04
		CHO	0.97	0.03	0.95**	0.03

* Significant difference between T 3 and T 120 ; ** significant difference between trials (Pl vs CHO)

Table 3

 3 Parameters recorded at rest and at the end of each submaximal test exercise while ingesting either a Pl or a CHO solution

	Parameters	Solution	Rest		T 20		T 120
				Mean	SD	Mean	SD	Mean	SD
	Body	mass	Pl	70.2	5.9			69.5	5.4
	(kg)		CHO	70.2	5.9			69.7	6.2
	Lactate		P1	1.1	0.1	1.8	0.2	4.2*	0.5
	concentration (mmol • 1 -1 )	CHO	0.9	0.1	1.6	0.2	3.9*	0.4
	Plasma		Pl	4.7	0.2	5.1	0.2	4.9	0.4
	glucose (mmol • 1 -1 )	CHO	4.9	0.3	5.8* , **	0.3	5.1*	0.5

* Significant difference between periods; ** significant difference between trials (Pl vs CHO)