Richard Sempere
email: richard.sempere@mio.osupytheas.fr

Bruno Charrìère

Javier Castro-Jiménez

Kimitaka Kawamura

Christos Panagiotopoulos

Richard Sempéré

Bruno Charrière

Occurrence of α,ω-dicarboxylic acids and ω-oxoacids in surface waters of the Rhone River and fluxes into the Mediterranean Sea

Keywords:

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Introduction

Low-molecular-weight (LMW: C 2 -C 9 ) a, x-dicarboxylic acids (diacids) and related polar compounds such as x -oxoacids (or ketoacids) hereafter called BCAs (bi-functional carboxylic acids = diacids + ketoacids) are ubiquitous water-soluble organic compounds. These chemicals have been detected in a variety of environmental compartments and regions, including atmospheric aerosols and precipitation [START_REF] Sempéré | Low molecular weight dicarboxylic acids and related polar compounds in the remote marine rain samples collected from Western Pacific[END_REF][START_REF] Kawamura | A review of dicarboxylic acids and related compounds in atmospheric aerosols: molecular distributions, sources and transformation[END_REF], snowpacks and ice cores [START_REF] Narukawa | Dicarboxylic acids in the Arctic aerosols and snowpacks collected during ALERT 2000[END_REF][START_REF] Kawamura | A Greenland ice core record of low molecular weight dicarboxylic acids, ketocarboxylic acids, and adicarbonyls: a trend from Little Ice Age to the present (1540 to 1989 A.D.)[END_REF], marine sediments [START_REF] Gogou | Marine organic geochemistry of the Eastern Mediterranean: 1. Aliphatic and polyaromatic hydrocarbons in Cretan Sea surficial sediments[END_REF], lakes [START_REF] Bertilsson | Photochemical transformation of dissolved organic matter in lakes[END_REF][START_REF] Brinkmann | Photoformation of lowmolecular-weight organic acids from brown water dissolved organic matter[END_REF] and seawaters [START_REF] Goldstone | Reactions of hydroxyl radical with humic substances: bleaching, mineralization, and production of bioavailable carbon substrates[END_REF][START_REF] Tedetti | Determination of low molecular weight dicarboxylic and ketocarboxylic acids in seawater samples[END_REF]. BCAs can originate in the atmosphere from several natural processes, such as direct biogenic emission [START_REF] Kawamura | Mid-chain ketocarboxylic acids in the remote marine atmosphere: Distribution patterns and possible formation mechanisms[END_REF], ozonolysis, as well as photooxidation of precursor organic compounds [START_REF] Sempéré | Trans-hemispheric contribution of C 2 -C 10 a, xdicarboxylic acids, and related polar compounds to water-soluble organic carbon in the western Pacific aerosols in relation to photochemical oxidation reactions[END_REF][START_REF] Kawamura | Photochemical production and loss of organic acids in high Arctic aerosols during long-range transport and polar sunrise ozone depletion events[END_REF][START_REF] Rinaldi | Evidence of a natural marine source of oxalic acid and a possible link to glyoxal[END_REF]. These chemicals can also be released to the atmospheric compartment at important levels by several anthropogenic sources, such as incomplete combustion of fossil fuels and biomass burning [START_REF] Kawamura | High abundances of water-soluble dicarboxylic acids, ketocarboxylic acids and a-dicarbonyls in the mountaintop aerosols over the North China Plain during wheat burning season[END_REF]. In streams and seawater, BCAs may be produced by phytoplankton photorespiration [START_REF] Steinberg | Oxalic, glyoxalic and pyruvic acids in eastern Pacific Ocean waters[END_REF] photochemical degradation of dissolved organic matter (DOM) [START_REF] Kirk | Optics of UV-B radiation in natural water[END_REF][START_REF] Pullin | Effects of sunlight and hydroxyl radical on dissolved organic matter: Bacterial growth efficiency and production of carboxylic acids and other substrates[END_REF] and microbial degradation of long-chain lipids [START_REF] Kester | Diterminal oxidation of long-chain alkanes by bacteria[END_REF]. BCAs can also reach to seawater by diffusion from surface sediments (especially for oxalic acid) [START_REF] Peltzer | Low molecular weight a-hydroxy carboxylic and dicarboxylic acids in reducing marine sediments[END_REF], inputs of terrigenous material from rivers [START_REF] Pullin | Effects of sunlight and hydroxyl radical on dissolved organic matter: Bacterial growth efficiency and production of carboxylic acids and other substrates[END_REF] and wet and dry depositions processes [START_REF] Sempéré | Trans-hemispheric contribution of C 2 -C 10 a, xdicarboxylic acids, and related polar compounds to water-soluble organic carbon in the western Pacific aerosols in relation to photochemical oxidation reactions[END_REF].

Aerosol particles enriched with BCAs can play important roles in several biogeochemical and physical processes, contributing to the radiative forcing at the Earth's surface. The BCA hygroscopic properties make these chemicals capable of acting as cloud condensation nuclei [START_REF] Kerminen | Low-molecular-weight dicarboxylic acids in an urban and rural atmosphere[END_REF]. BCAs also participate in many biological processes in aquatic systems, such as by serving as important intermediates in the tricarboxylic acid and glyoxylate cycles as well as in the catabolism and anabolism of amino acids [START_REF] Steinberg | The determination of a-keto acids and oxalic acid in sea-water by reversed phase liquid chromatographic separation of fluorescent quinoxilinol derivatives[END_REF]. In addition, BCAs could represent a non-negligible fraction of LMW DOM and have a potential role in the organic carbon cycle in the natural environment [START_REF] Tedetti | Determination of low molecular weight dicarboxylic and ketocarboxylic acids in seawater samples[END_REF]. However, because of analytical difficulties, studies on the BCA occurrence in seawaters are limited to a few measurements performed in coastal waters from the Pacific Ocean where one dicarboxylic acid, i.e., oxalic acid, and two ketocarboxylic acids (glyoxylic and pyruvic) were detected (Steinberg andBada, 1982, 1984) and the Sargasso Sea and coastal waters off Florida, where ketocarboxylic acids (glyoxylic and pyruvic) and dicarbonyls (methylglyoxal and glyoxal) were measured [START_REF] Mopper | Sources and sinks of low molecular weight organic carbonyl compounds in seawater[END_REF][START_REF] Kieber | Photochemical formation of glyoxylic and pyruvic acids in seawater[END_REF][START_REF] Zhou | Photochemical production of low-molecular-weight carbonyl compounds in seawater and surface microlayer and their air-sea exchange[END_REF]. Even fewer data exist for semi-enclosed marine environments, such as the Mediterranean Sea. Only one study reported the surface water concentrations of a homologous series of BCA from samples collected in 2005 at levels of approximately 50 mg l À1 in the northwestern (NW) Mediterranean Sea [START_REF] Tedetti | Determination of low molecular weight dicarboxylic and ketocarboxylic acids in seawater samples[END_REF].

The Mediterranean Sea is under strong anthropogenic pressure (e.g. industrial, urban, agriculture, and intense maritime traffic (The MERMEX group, 2011 and references therein, UNEP/MAP, 2012) and it has been reported to be largely impacted by persistent organic pollutants (POPs) and related anthropogenic contaminants, some of which have similar atmospheric sources as BCA, such as polycyclic aromatic hydrocarbons (PAHs) and polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs) (The MERMEX group, 2011;[START_REF] Berrojalbiz | Biogeochemical and physical controls on concentrations of polycyclic aromatic hydrocarbons in water and plankton of the Mediterranean and Black Seas[END_REF][START_REF] Guigue | Occurrence and distribution of hydrocarbons in the surface microlayer and subsurface water from the urban coastal marine area off Marseilles, Northwestern Mediterranean Sea[END_REF][START_REF] Guigue | Spatial and seasonal variabilities of dissolved hydrocarbons in surface waters from the Northwestern Mediterranean Sea: results from one year intensive sampling[END_REF][START_REF] Castro-Jiménez | PCDD/F and PCB multi-media ambient concentrations, congener patterns and occurrence in a Mediterranean coastal lagoon (Etang de Thau, France)[END_REF], 2010, 2012). For example, common anthropogenic sources of BCA and PAHs to the atmosphere are direct combustion processes such as fossil fuels and biomass burning [START_REF] Kawamura | High abundances of water-soluble dicarboxylic acids, ketocarboxylic acids and a-dicarbonyls in the mountaintop aerosols over the North China Plain during wheat burning season[END_REF][START_REF] Castro-Jiménez | Polycyclic aromatic hydrocarbons (PAHs) in the Mediterranean Sea: Atmospheric occurrence, deposition and decoupling with settling fluxes in the water column[END_REF], as well as photochemical oxidation of precursor aromatic hydrocarbons during long range transport in the atmosphere (Kawamura and Bikkina, 2016 and references therein). In addition to atmospheric deposition processes, riverine inputs are major entry pathways for organic species to the Mediterranean coastal areas, especially the Gulf of Lions (the NW Mediterranean Sea), which is under the influence of the Rhone River [START_REF] Sempéré | Carbon inputs of the Rhone River to the Mediterranean Sea: biogeochemical implications[END_REF][START_REF] Panagiotopoulos | The composition and flux of particulate and dissolved carbohydrates from the Rhone River into the Mediterranean Sea[END_REF]. Although little is known about its chemical composition, riverine DOM influences the bacterioplankton community structure and function [START_REF] Crump | Circumpolar synchrony in big river bacterioplankton[END_REF]. At a global scale, previous studies showed that riverine DOC, that contains both degraded remains of aged and recent vascular plant materials [START_REF] Hedges | What happens to terrestrial organic matter in the ocean?[END_REF], is modern and younger than particular organic carbon (POC) [START_REF] Raymond | Flux and age of dissolved organic carbon exported to the Arctic Ocean: a carbon isotopic study of the five largest arctic rivers[END_REF][START_REF] Spencer | Dissolved organic carbon and chromophoric dissolved organic matter properties of rivers in the USA[END_REF]. Such apparent degraded biochemical signature of riverine DOM, along with modern radiocarbon ages of the DOC, has been reported in systems from the Arctic to the Equator [START_REF] Raymond | Flux and age of dissolved organic carbon exported to the Arctic Ocean: a carbon isotopic study of the five largest arctic rivers[END_REF][START_REF] Spencer | Dissolved organic carbon and chromophoric dissolved organic matter properties of rivers in the USA[END_REF]. Compared to the rest of global ocean, the flux of riverine DOM to the Mediterranean Sea consti-tutes a major source of reduced carbon to the Mediterranean Sea [START_REF] Sempéré | Carbon inputs of the Rhone River to the Mediterranean Sea: biogeochemical implications[END_REF]. The occurrence of BCA in riverine surface waters and relative inputs of BCA to Mediterranean waters are poorly characterized, and to best of our knowledge no data exist for the Rhone River which is the main supplier of organic carbon to the whole Mediterranean Sea.

The objectives of this study are: (1) to provide the first assessment on the concentrations of a, x-dicarboxylic acids and xoxoacids in surface waters from the Rhone River as well as their relative abundance within the dissolved organic carbon (DOC) pool, (2) to estimate the fluxes of a, x-dicarboxylic acids and xoxoacids from the Rhone River into the Mediterranean Sea and

(3) to investigate the possible organic matter and BCA sources in relation to the degradation mechanisms occurring in the area.

Experimental

Sampling area description

The Rhone River is 816 km long with a drainage basin area of 100 Â 103 km 2 and average annual discharge of 53 Â 10 9 m 3 of water and 2-10 Mt of sediments [START_REF] Iban ˇez | Characterization of the Ebre and Rhone estuaries: A basis for defining and classifying salt-wedge estuaries[END_REF][START_REF] Sempéré | Carbon inputs of the Rhone River to the Mediterranean Sea: biogeochemical implications[END_REF][START_REF] Pont | Medium-term changes in suspended sediment delivery to the ocean: consequences of catchment heterogeneity and river management (Rhone River, France)[END_REF][START_REF] De Madron | Nutrients and carbon budgets for the Gulf of Lion during the Moogli cruises[END_REF][START_REF] Rabouille | Comparison of hypoxia among four river-dominated ocean margins: the Changjiang (Yangtze), Mississippi, Pearl, and Rhone rivers[END_REF][START_REF] Eyrolle | Consequences of hydrological events on the delivery of suspended sediment and associated radionuclides from the Rh??ne River to the Mediterranean Sea[END_REF][START_REF] Sadaoui | Controls, budgets and variability of riverine sediment fluxes to the Gulf of Lions (NW Mediterranean Sea)[END_REF]. The river stage fluctuates by 2-6 m annually, and it has a peak discharge in early spring and late autumn and a minimum in the summer [START_REF] Pont | Medium-term changes in suspended sediment delivery to the ocean: consequences of catchment heterogeneity and river management (Rhone River, France)[END_REF]. The Rhone Delta is divided into two outlets, the Petit Rhone and the Grand Rhone, corresponding to 20% and 80% of the water flow, respectively [START_REF] Iban ˇez | Characterization of the Ebre and Rhone estuaries: A basis for defining and classifying salt-wedge estuaries[END_REF]. The water samples analyzed in this study were collected between June 2006 and June 2009 at the town of Arles (Fig. 1), which is located on the Grand Rhone (47.5 km upstream of the Mediterranean Sea). The collection site was chosen from routinely studied sites over the last two decades by French scientific community [START_REF] Sempéré | Carbon inputs of the Rhone River to the Mediterranean Sea: biogeochemical implications[END_REF][START_REF] Pont | Medium-term changes in suspended sediment delivery to the ocean: consequences of catchment heterogeneity and river management (Rhone River, France)[END_REF][START_REF] Sicre | Poly-aromatic hydrocarbon (PAH) inputs from the Rhone River to the Mediterranean Sea in relation with the hydrological cycle: Impact of floods[END_REF][START_REF] Ollivier | Seasonal variations of physical and chemical erosion: a three-year survey of the Rhone River (France)[END_REF][START_REF] Panagiotopoulos | The composition and flux of particulate and dissolved carbohydrates from the Rhone River into the Mediterranean Sea[END_REF]. Water samples were collected at the SORA observatory station near the Compagnie Nationale du Rhone (CNR; http://www.cnr.tm.fr/fr/) gauging station in Arles at 7 m from the right bank and 0.5 m under the surface. During the sampling period, the Rhone water discharge varied from 450 to 3821 m 3 s À1 (mean = 1787 ± 835 m 3 s À1 ).

Sample collection

Rhone River water (1-2 L) samples were collected between February 2006 and June 2009 (Table 1) using pre-cleaned glass bottles (rinsed with a 2% HCl solution and baked for 6 h at 450 °C). The bottles were rinsed three times with the sample, closed with Teflon-lined screw caps, and kept in the dark at 4 °C until filtration. The samples were brought to the laboratory on dry ice (< 24 h) and filtered through 0.7-lm filters (GF/F-47 mm precombusted for 6 h at 500 °C; P < 50 mm Hg) to remove the water particulate phase. Prior to filtration, the 0.7-lm GF/F filters were flushed with (100 ml) Milli-Q water and a small volume of the sample to minimize contamination [START_REF] Yoro | Dissolved organic carbon contamination induced by filters and storage bottles[END_REF]. Triplicate sub-samples were collected for DOC analyses and transferred into 10-ml glass ampoules (Wheaton Ò ), which were previously precombusted for 6 h at 450 °C and rinsed 3 times with sample. Then, 20 lL of H 3 PO 4 acid was added as a preservative (final pH $ 2), and the ampoules were flame-sealed. The ampoules were stored in darkness at 4 °C until further analyses. The remaining sample was used for dissolved BCA analyses and stored in precombusted (6 h, 450 °C) Pyrex bottles with a bactericide HgCl 2 (10 mg l À1 ), in dark at À4 °C.

Rhone River plume

Gulf of Lions

Table 1

The Rhone River water discharge (Q); dissolved organic carbon (DOC); bi-functional carboxylic acids, including the sum of the dicarboxylic acid and ketoacid (BCA) concentrations and fluxes; the fraction of carbon in BCAs (BCA-C) concentrations and the BCA-C/DOC ratios measured in the Rhone River during the period from February 2006 to June 2009.

Date

Q (m 3 s À1 ) DOC (mgl À1 ) BCAs (mgl À1 ) BCA's Fluxes (t d À1 ) BCA-C (mgl

Diacids and ketoacids determination

a, x -dicarboxylic acids (or diacids) and x-oxoacids (or ketoacids) (Fig. 2) were determined by the method of [START_REF] Tedetti | Determination of low molecular weight dicarboxylic and ketocarboxylic acids in seawater samples[END_REF]. Briefly, 100 ml of the sample was first concentrated to 3-4 ml using a rotary evaporator under vacuum. The sample was then transferred into a 25-ml pear-shaped flask, further concentrated by the rotary evaporator, and finally concentrated to almost dryness under a nitrogen stream. A total of 0.25 ml of 14% BF 3 /1butanol was immediately added to the sample, and the flask was sealed with a glass stopper, Teflon tape, and clamp. The organic acids and reagent were mixed under ultrasonication for 1 min and then heated at 100 °C for 30 min to derive dibutyl esters for the carboxyl group and dibutoxy acetal for the aldehyde group.

The derivatives were extracted with 5 mL of n-hexane after adding 3 ml of Milli-Q water and 0.2 ml of acetonitrile. The hexane layer was further washed with Milli-Q water (3 Â 3 ml). The derivatives were dried using a rotary evaporator, and a nitrogen stream, and were finally dissolved in 100 ml of n-hexane.

A volume of 1 ml of the n-hexane solution containing the derivatives was injected onto an Agilent 6850 gas chromatograph equipped with a 7683B split/splitless auto-injector that was used in the pulsed splitless mode. The GC was interfaced with an Agilent 5975 C mass spectrometer that was configured in the electron impact (EI) mode. Chromatographic separation of dibutyl esters was accomplished with a fused-silica capillary Agilent J&W GC HP-5MS column (Agilent Technologies, 30 m, phase: (5%-phenylmethylpolysiloxane). Compounds were identified by comparison of the retention times and mass spectra with those of standards and quantified using a spike experiment of diacids in a natural Rhone sample by GC-MS selected ion monitoring mode (SIM) [START_REF] Tedetti | Determination of low molecular weight dicarboxylic and ketocarboxylic acids in seawater samples[END_REF]. Samples were analyzed in duplicate, and all identified compounds showed good reproducibility, with relative standard deviation (RSD) less than 10%. Blanks had Milli-Q water as the sample; the blanks for oxalic acid were <5% of real samples. The blank values were subtracted for all samples. Concentrations of BCAs in water samples were determined using spike experiments.

For each spike experiment, a standard solution containing a known amount of the targeted compounds was added to one Rhône sample. From these five experiments we established a linear regression between spikes concentration of BCAs and the measured peak areas. The method responds linearly to the increasing concentrations of BCAs in Rhône water for all the compounds. The slopes of these linear regressions, that are significantly different from zero (r2 = 0.95 and their slopes and y-intercepts are significantly (p < 0.01) different from zero (t-test), were used to determine the concentrations of the targeted compounds in unknown water samples. Different ranges of spiked concentrations were used depending on the initial concentration of the compound in seawater.

DOC determination

The DOC was measured using a Shimadzu (Model TOC-V total) carbon analyzer with a quartz combustion column filled with 1.2% Pt on silica pillows [START_REF] Sempéré | Distribution and bacterial availability of dissolved neutral sugars in the South East Pacific[END_REF]. Prior to analysis, samples were sparged for 10 min by CO 2 -free pure air to remove inorganic carbon. A four-point calibration curve was conducted daily using standards prepared by diluting a stock solution of potassium hydrogen phthalate in Milli-Q water. Then, 100 ml of sample was injected 3-4 times for each sample and standard; the analytical precision of the procedure was an average of 2%. The precision of the TOC analysis was determined by the standard deviation of triple or quadruple analysis of the same sample.

C C C C C C C C C C C 2 ) 5 C ( d i c a c i r a t u l G ) 4 C ( d i c a c i n i c c u S ) 2 C ( d i c a c i l a x O
Malonic acid (C3) The average DOC concentrations in the DAW and LCW reference standards were 45 ± 2 mMC, n = 24 and 1 ± 0.3 mM C, n = 24, respectively. The nominal analytical precision of the procedure was within 1%.

Results and discussion

Water discharge and DOC concentrations in the Rhone River

General information regarding the Rhone River annual discharge as well as the bulk parameter features (water discharge, total suspended matter, organic carbon content of particles, and POM concentrations) during the sampling period (Feb. 2006-June 2009) are described in detail in [START_REF] Panagiotopoulos | The composition and flux of particulate and dissolved carbohydrates from the Rhone River into the Mediterranean Sea[END_REF]. Briefly, during the sampling period, water discharges ranged from 450 to 3820 m 3 s À1 , with peak values occurring during the ''flood" events of April 2008 (3244 m 3 s À1 ), May 2008 (3821 m 3 s À1 ), September 2008 (2983 m 3 s À1 ), and October 2008 (3817 m 3 s À1 ) (Table 1). It is worth noting that the events that occurred in May and September 2008 were not related to physical phenomena resulting in floods (i.e., artificial water release from dam); therefore, they were not considered in the annual fluxes estimates [START_REF] Panagiotopoulos | The composition and flux of particulate and dissolved carbohydrates from the Rhone River into the Mediterranean Sea[END_REF]. The DOC concentrations were generally >1000 mg l À1 , averaging 1640 ± 372 mg l À1 ; however, their maximum values did not always coincide with the high water discharge values, as one would expect (Table 1). Such DOC values are slightly lower, but in good agreement with previous studies carried out in the Rhone River [START_REF] Sempéré | Carbon inputs of the Rhone River to the Mediterranean Sea: biogeochemical implications[END_REF].

BCA concentrations, BCA/DOC ratios, and fluxes

The BCA concentrations (as sum of compounds) exhibited a wide range of values from 13.2 mg l À1 (March 2008) to 71.2 mg l À1 (December 2007) (Fig. 3). The arithmetic mean concentration is 32.4 mg l À1 (sum of concentrations Ci/n = 23) while the weighted mean concentration ( P Qi Ci/ P Qi) would be 35.5 mg l À1 . The contribution of carbon from BCAs to the DOC pool (BCA-C) accounted for 0.28-1.42% of DOC (Table 1, Fig. 4). Individual BCA concentrations are presented in Table 2. The a, x -dicarboxylic acids (C 2 -C 9 ), and two x-oxocarboxylic (C 2 and C 4 ) acids concentrations averaged (20.7 ± 10.6 mg l À1 ) and (11.7 ± 6.2 mg l À1 ), respectively and they were detected for the first time in the Rhone River (Table 2). Although no seasonal trend was evident during the study period, the results showed, as for the DOC, that the highest BCA concentrations did not always follow high water discharges (flood events). The highest values were found in winter 2007 and, to a lower extent by the end of winter 2008 (Fig. 3). This is the first study to report a complete series of homologous dicarboxylic and ketoacids in River waters. The Rhone River BCA levels are close to those reported for Mediterranean coastal seawaters (15.9 to 58.3 mg l À1 ; 1-2.5% DOC; [START_REF] Tedetti | Determination of low molecular weight dicarboxylic and ketocarboxylic acids in seawater samples[END_REF] and wet deposition (22.6-1040 mg l À1 ; 1-5% of DOC; [START_REF] Sempére | Comparative distributions of dicarboxylic acids and related polar compounds in snow, rain and aerosols from urban atmosphere[END_REF][START_REF] Sempéré | Low molecular weight dicarboxylic acids and related polar compounds in the remote marine rain samples collected from Western Pacific[END_REF]. It is worth noting that our BCA-C/DOC values are comparable to those recorded for the water-soluble fraction of marine aerosol particles (2-15% WSOC; [START_REF] Sempéré | Trans-hemispheric contribution of C 2 -C 10 a, xdicarboxylic acids, and related polar compounds to water-soluble organic carbon in the western Pacific aerosols in relation to photochemical oxidation reactions[END_REF]. A comparison with other identified organic compounds within the DOC pool for the Rhone River indicated that our BCA concentrations are two to three orders of magnitude higher than the reported values of dissolved water phase PAHs (0.003-0.12 mg l À1 ), one of the most abundant classes of organic contaminant in the marine environment [START_REF] Bouloubassi | Dissolved, particulate and sedimentary naturally derived polycyclic aromatic hydrocarbons in a coastal environment: geochemical significance[END_REF][START_REF] Sicre | Poly-aromatic hydrocarbon (PAH) inputs from the Rhone River to the Mediterranean Sea in relation with the hydrological cycle: Impact of floods[END_REF], and/or the sum of dissolved water phase phthalates (0.61 mg l À1 ; [START_REF] Paluselli | Occurrence of phthalate acid esters (PAEs) in the northwestern Mediterranean Sea and the Rhone River[END_REF]. Our BCA values are on the same order of magnitude as natural compounds, such as polysaccharides (180-558 mg l À1 ; [START_REF] Panagiotopoulos | The composition and flux of particulate and dissolved carbohydrates from the Rhone River into the Mediterranean Sea[END_REF] measured in the Rhone River or glycolic acid in the western Mediterranean Sea (24-89 mg l À1 ; [START_REF] Leboulanger | Diel variability of glycolate in the eastern tropical Atlantic Ocean[END_REF]. These BCA levels are close to the concentration of the sum of some mono-(acetic, formic) and dicarboxylic/ketoacids acids (pyruvic, oxalic, malonic and succinic acids: equal to 34 mg l À1 ) recorded in Bog Lake drain in Germany [START_REF] Brinkmann | Photoformation of lowmolecular-weight organic acids from brown water dissolved organic matter[END_REF], and they fall within the lower range of values reported for pyruvic (75-6680 mg l À1 ) and oxalic (50-1510 mg l À1 ) acids in sediment pore waters [START_REF] Xiao | Characteristics and distribution of low molecular weight organic acids in the sediment porewaters in Bosten Lake[END_REF]. These compounds accounted for 6.4% and 0.7-18% of the DOC, respectively in Bog Lake drain, Germany [START_REF] Xiao | Characteristics and distribution of low molecular weight organic acids in the sediment porewaters in Bosten Lake[END_REF], and in sediment pore waters in Bosten Lac, China [START_REF] Xiao | Characteristics and distribution of low molecular weight organic acids in the sediment porewaters in Bosten Lake[END_REF], and they agree with the present study.

During the sampling period (Feb. 2006-June 2009), the Rhone River discharge varied from 450 to 3821 m 3 s À1 , which corresponds to daily flux levels of the DOC and BCA on the order of 62.1-668 and 1.37-16.36 t d À1 , respectively (Table 1). Assuming a mean water discharge during our study of 1787 m 3 s À1 , a broad estimate suggests that Rhone River provides 750-4000 t yr À1 BCAs to the Mediterranean Sea [load = BCA concentrations (min or max in mg. l À1 ) ⁄ mean flow rate (Q in m 3 yr À1 )]. The estimates of BCA concentration ranges are based on 23 daily samples, representative of the Rhone River flow. These fluxes estimates mainly depends of the annual river flow which, on a 10 years study [START_REF] Sempéré | Carbon inputs of the Rhone River to the Mediterranean Sea: biogeochemical implications[END_REF], vary from 1300 to 2000 m 3 s À1 (our calculation is based on a mean river flow of 1787 m 3 s À1 during our study period).

The mean annual flux, calculated as the BCAs weighted mean concentration (35.5 mg l À1 ) multiplied by the mean flow rate (1787 m 3 s À1 ) over the studied period is estimated to 2003 t yr À1 . Compared to other identified compounds within the Rhone in the same period of sampling, the above BCAs fluxes are approximately one order of magnitude lower than those of dissolved carbohydrates (10-31 kt yr À1 ) [START_REF] Panagiotopoulos | The composition and flux of particulate and dissolved carbohydrates from the Rhone River into the Mediterranean Sea[END_REF].

Molecular composition and relative abundance of BCAs

Among diacids, short-chain diacids were found to be the most abundant compound class in the Rhone River. Oxalic acid (C 2 ) was most abundant (27.1 ± 7.0%) diacid, followed by fumaric (unsaturated C 4 ), malonic (C 3 ) and succinic (C 4 ) acids. On the other hand, two oxoacids glyoxylic acid (xC 2 ) and 4-oxobutanoic acid (xC 4 ), were detected, and xC 2 was always predominant (Table 2, Fig. 5 a,b; Fig. 6). Other oxoacids (xC 3 -xC 9 ) were not detected or found at trace levels, and they were not quantified or presented in this study. Short-chain diacids (C 2 -C 4 ) are more abundant than longer-chain diacids, which is a common trend with aerosol and rainwater molecular distributions [START_REF] Kawamura | A review of dicarboxylic acids and related compounds in atmospheric aerosols: molecular distributions, sources and transformation[END_REF] and references therein). Surprisingly, xC 2 is usually the most abundant BCA (18 out of 23 followed by oxalic and fumaric acids) (Table 2; Fig. 5 a, b, and6). Interestingly, such a molecular distribution differs from that usually reported in the atmosphere (Fig. 7) for which a predominance of oxalic acid is usually more abundant than xC 2 which is followed either by malonic (C 3 ) or succinic (C 4 ) acid according to aging of the aerosols [START_REF] Sempéré | Trans-hemispheric contribution of C 2 -C 10 a, xdicarboxylic acids, and related polar compounds to water-soluble organic carbon in the western Pacific aerosols in relation to photochemical oxidation reactions[END_REF][START_REF] Fu | Dicarboxylic acids, ketocarboxylic acids and glyoxal in the marine aerosols collected during a round-the-world cruise[END_REF][START_REF] Kawamura | A review of dicarboxylic acids and related compounds in atmospheric aerosols: molecular distributions, sources and transformation[END_REF]. Long-chain diacids are generally less abundant, while there are similar proportions of glutaric (C 5 ), adipic (C 6 ) and azelaic (C 9 ) acids. It is of interest that such a predominance of glyoxylic acid over oxalic acid was also reported for a seawater study based on a limited number of coastal seawater samples [START_REF] Tedetti | Determination of low molecular weight dicarboxylic and ketocarboxylic acids in seawater samples[END_REF].

This lower relative abundance of oxalic acid might also be due to a lower efficiency of photochemical oxidation reactions producing oxalic acid in surface waters than in the aqueous phase of the atmosphere. Indeed hydroxyl radical (ÅOH) a strong oxidant in natural waters, involved in oxalic acid formation, react preferentially with scavengers such as bromide in sea water (i.e. 93% of ÅOH react with Br-in sea water [START_REF] Mopper | Hydroxyl radical photoproduction in the sea and its potential impact on marine processes[END_REF])) while ÅOH scavenger in atmospheric waters are dissolved organic compounds, which are poorly characterized [START_REF] Arakaki | A general scavenging rate constant for reaction of hydroxyl radical with organic carbon in atmospheric waters[END_REF]. Moreover [START_REF] Warneck | In-cloud chemistry opens pathway to the formation of oxalic acid in the marine atmosphere[END_REF] and [START_REF] Ervens | CAPRAM 2.4 (MODAC mechanism): an extended and condensed tropospheric aqueous phase mechanism and its application[END_REF] and [START_REF] Ervens | A modeling of study of aqueous production of dicarboxylic acids: 1. Chemical pathways and speciated organic mass production[END_REF] evidenced the photochemical production of oxalic acid in marine atmosphere from acetylene, ethylene and aromatic compounds with glyoxal as an intermediate compound of oxidation reactions. Oxalic acid is the main dicarboxylic acid since it represents the end product of the oxidation of several organic chemical compounds [START_REF] Ervens | A modeling of study of aqueous production of dicarboxylic acids: 1. Chemical pathways and speciated organic mass production[END_REF]. Two aliphatic unsaturated diacids were also detected in Rhone River samples, maleic (cis configuration and) and fumaric acid (trans configuration). The cis/trans ratios are, for most samples, lower than 1 (0.16 ± 0.2). The highest values were found in December 2006 (0.6) and December 2007 (1.1), indicating a large predominance of fumaric over maleic acid. Notably, the trans configuration is usually more abundant than the cis configuration in the marine atmosphere, whereas the opposite trend was reported for continental samples [START_REF] Kawamura | Seasonal changes in the distribution of dicarboxylic acids in the urban atmosphere[END_REF][START_REF] Sempéré | Trans-hemispheric contribution of C 2 -C 10 a, xdicarboxylic acids, and related polar compounds to water-soluble organic carbon in the western Pacific aerosols in relation to photochemical oxidation reactions[END_REF]. In the atmosphere, the predominance of the precursor cis configuration has been explained by the photochemical oxidation of benzene or toluene, whose cis configuration is preserved in the structure of the oxidation product. The maleic acid may further be photochemically isomerized to the trans configuration (fumaric acid) in the atmosphere under solar radiation [START_REF] Kawamura | Seasonal changes in the distribution of dicarboxylic acids in the urban atmosphere[END_REF]. These results sug- gest that the predominance of fumaric acid over maleic acid cannot only be explained by photochemical isomerization of maleic acid. Because the highest relative abundance of fumaric acid was found in December, photochemical oxidation reactions alone cannot explain the variations in the M/F ratios. Fumaric acid is also observed in fungi and lichen, and it can also carried from the land through the Rhone River runoff. Moreover, as an intermediate in the citric acid cycle that is used by prokaryotes and eukaryotes, fumaric acid is formed by the oxidation of succinate with succinate dehydrogenase [START_REF] Krebs | Krebs' citric acid cycle: half a century and still turning[END_REF]. Although a strict origin of fumarate is difficult to determine, the biological origin of fumarate cannot be precluded. These data suggest that biological processes largely contribute to the molecular distribution of BCAs in aquatic systems.

Relative abundance (%)

Origin of BCAs in the Rhone River

To the best of our knowledge these are the first data on the baseline concentrations and distributions of BCAs in surface river water, yet little is known about the origin and degradation of such compounds in freshwater ecosystems. The present data set does not allow for a comprehensive study of the origin of BCA in the study area, but some hypothesis about their potential sources can be drawn as follows:

(1) Atmospheric emissions, formation and deposition. The incomplete combustion of fossil fuels [START_REF] Kawamura | Dicarboxylic acids generated by thermal alteration of kerogen and humic acids[END_REF] and biomass burning [START_REF] Andreae | Influence of plumes from biomass burning on atmospheric chemistry over the equatorial and tropical South Atlantic during CITE 3[END_REF][START_REF] Stephanou | Oxocarboxylic and.alpha.,omega.-dicarboxylic acids: photooxidation products of biogenic unsaturated fatty acids present in urban aerosols[END_REF][START_REF] Novakov | Cloud condensation nucleus activity of the organic component of biomass smoke particles[END_REF] could represent important emissions of BCAs to the atmosphere in an area that is largely urbanized and under a strong anthropogenic pressure such as the Gulf of Lions. This airborne release of BCAs, together with their direct formation (as water-soluble fraction of the aerosols), may generate BCA atmospheric stock subject to dry and wet deposition processes which likely contributed to the BCA stock of riverine DOM [START_REF] Sempéré | Trans-hemispheric contribution of C 2 -C 10 a, xdicarboxylic acids, and related polar compounds to water-soluble organic carbon in the western Pacific aerosols in relation to photochemical oxidation reactions[END_REF]. This is consistent with riverine 14 C-DOC studies demonstrating that some part of riverine DOM may be delivered from anthropogenic aerosols in European rivers [START_REF] Stubbins | Anthropogenic aerosols as a source of ancient dissolved organic matter in glaciers[END_REF]. In addition, the high levels of hydrocarbons present in the air-water interphase in the study area [START_REF] Berrojalbiz | Biogeochemical and physical controls on concentrations of polycyclic aromatic hydrocarbons in water and plankton of the Mediterranean and Black Seas[END_REF][START_REF] Guigue | Occurrence and distribution of hydrocarbons in the surface microlayer and subsurface water from the urban coastal marine area off Marseilles, Northwestern Mediterranean Sea[END_REF][START_REF] Guigue | Spatial and seasonal variabilities of dissolved hydrocarbons in surface waters from the Northwestern Mediterranean Sea: results from one year intensive sampling[END_REF][START_REF] Castro-Jiménez | Polycyclic aromatic hydrocarbons (PAHs) in the Mediterranean Sea: Atmospheric occurrence, deposition and decoupling with settling fluxes in the water column[END_REF] could support the indirect in situ formation of BCAs and enhance their atmospheric deposition. The oxidation of precursor aromatic hydrocarbons and/or unsaturated molecules (e.g., long chain unsaturated fatty acids) has been reported as a potential source of BCAs in the atmosphere [START_REF] Kawamura | A review of dicarboxylic acids and related compounds in atmospheric aerosols: molecular distributions, sources and transformation[END_REF]. Available estimates show that approximately 15 tons of PAHs (dissolved + particle water phase) can be delivered annually by the Rhone River to the NW Mediterranean Sea [START_REF] Sicre | Poly-aromatic hydrocarbon (PAH) inputs from the Rhone River to the Mediterranean Sea in relation with the hydrological cycle: Impact of floods[END_REF]. This PAH riverine input is small when compared with our estimates of BCA fluxes from the Rhone (750-4000 t y À1 ), suggesting that the indirect BCA formation from PAHs offers a minor contribution to the amounts measured in surface waters. However, we compare fluxes that were estimated with different methodologies and based on measurements performed in different years, resulting in large variability. In addition, there is no information on the potential formation of BCA from PAHs on the photic zone, where photodegradation of potential precursors of BCAs, such as PAHs or aliphatic hydrocarbons, could also occurs. (2) Riverine DOM from terrestrial origin. The Rhone River, like other rivers, carries the DOM of autochthonous origin, as well as the DOM of terrestrial origin, including the degrading remains of vascular plant materials in aged-soils [START_REF] Hedges | What happens to terrestrial organic matter in the ocean?[END_REF][START_REF] Raymond | Event controlled DOC export from forested watersheds[END_REF] and the DOC derived from recent vascular plant production [START_REF] Spencer | Dissolved organic carbon and chromophoric dissolved organic matter properties of rivers in the USA[END_REF]. Such a material can undergo a variety of biogeochemical processes during transport to a coastal area and exhibits an acidic/aldehyde ratio that increases with the diagenetic state [START_REF] Opsahl | Photochemical reactivity of dissolved lignin in river and ocean waters[END_REF] although this trend could also be due to abiotic processes, such as adsorption and dissolution of DOM [START_REF] Hernes | Fractionation of lignin during leaching and sorption and implications for organic matter ''freshness[END_REF]. Soil organic matter includes a variety of exudates released from macro-and microorganisms such as simple monocarboxylic and oxalic acids [START_REF] Schnitzer | Chapter 1 Humic Substances: Chemistry and Reactions[END_REF] and other organic acids, which are commonly present at a relatively high concentration around plant roots [START_REF] Drever | Role of soil organic acids in mineral weathering processes[END_REF]. These substances can easily be leached by runoff and then transported towards river water. Other organic compounds can be decomposed by prokaryotes during river transport producing LMW organic acids, including BCAs. Note that these molecules are known to be readily metabolized by bacteria and their residence time in the soil is likely to be short (a matter of days of less). The concentrations of the molecules are maintained by continuous production from the biota. (3) Autochthonous production. Although no report has evaluated the local biological production in freshwaters, the autochthonous origin of BCAs in the Rhone cannot be precluded. When considering marine organism metabolism linked to BCA production, it is also well known that tri-and dicarboxylic acids are involved in the citric acid cycle during carbohydrate degradation. Then, prokaryotic and eukaryotic activities are probably one of the sources of BCAs. Microbial degradation of long chain lipids [START_REF] Kester | Diterminal oxidation of long-chain alkanes by bacteria[END_REF] that have been reported for marine waters, is also probably a source of BCAs in freshwaters. These observations strongly support an autochthonous production of BCAs in the Rhone River samples. (4) Photochemical degradation processes of riverine DOM. DOM degradation processes could play an important role in the BCA formation in the water column and may support the production of BCAs in surface waters from the Rhone River.

Previous studies indicated that photochemical oxidation of marine DOM by solar irradiation may provide LMW compounds, such as formaldehyde, pyruvate, oxalate and other LMW organic acids [START_REF] Kirk | Optics of UV-B radiation in natural water[END_REF][START_REF] Kieber | Photooxidation of triglycerides and fatty acids in seawater: implication toward the formation of marine humic substances[END_REF][START_REF] Bertilsson | Photochemically produced carboxylic acids as substrates for freshwater bacterioplankton[END_REF]Bertilsson and Tranvick, 2000;[START_REF] Pullin | Effects of sunlight and hydroxyl radical on dissolved organic matter: Bacterial growth efficiency and production of carboxylic acids and other substrates[END_REF], and these processes are probably the source of BCAs in freshwater. Similarly, [START_REF] Tedetti | Hydroxyl radical-induced photochemical formation of dicarboxylic acids from unsaturated fatty acid (oleic acid) in aqueous solution[END_REF] reported high production of C 2 -C 9 diacids (particularly C 9 di) and C 2 -C 9 oxooacids from ( . OH)-induced photochemical oxidation of oleic acid (cis-9-octadecenoic) diluted in Milli-Q water in laboratory-controlled conditions. Photochemical oxidative reactions may also occur on terrestrial DOM, which is rich in carboxyl-rich aliphatic molecules [START_REF] Hertkorn | Characterization of a major refractory component of marine dissolved organic matter[END_REF]. For instance lignin is susceptible to produce BCAs, as previously shown in the atmosphere [START_REF] Kawamura | A review of dicarboxylic acids and related compounds in atmospheric aerosols: molecular distributions, sources and transformation[END_REF]. Direct photochemical reactions can be enhanced through sensitizer production of hydroxyl ( . OH) radicals, which are the most reactive oxidants in aquatic systems (E = 1.83 V) [START_REF] Zafiriou | Photochemistry of natural waters[END_REF] and are essentially produced by nitrate and nitrite photolysis with initiation by UV-B and UV-A (315-400 nm) [START_REF] Vaughan | Photochemical formation of hydroxyl radical by constituents of natural waters[END_REF][START_REF] Mack | Photochemistry of nitrite and nitrate in aqueous solution: a review[END_REF][START_REF] Zhou | Determination of photochemically produced hydroxyl radicals in seawater and freshwater[END_REF]. Both the high solar radiation encountered in this area [START_REF] Sempéré | Variability of solar radiation and CDOM in surface coastal waters of the Northwestern Mediterranean Sea[END_REF] and the high level of nitrates/nitrites carried by the Rhone River [START_REF] Moutin | The input of nutrients by the Rhone River into the Mediterranean Sea: recent observations and comparison with earlier data[END_REF] suggest that photochemical oxidation reactions of DOM may induce BCA production in the Rhone River. It is important to note that in situ phytoplanktonic activity and photo-oxidation of biogenic emissions from the ocean producing azelaic acid (C 9 di) has already been suggested [START_REF] Kawamura | Mid-chain ketocarboxylic acids in the remote marine atmosphere: Distribution patterns and possible formation mechanisms[END_REF].

The occurrence of BCAs in the surface waters of the Rhone River is likely driven by a combination of the afore-mentioned sources, and there is a predominant role of DOM in their formation. Further research is needed to ascertain the major BCA sources in the area.

Summary and conclusion

Most diacids and two oxoacids were detected for the first time in riverine surface waters. The concentrations of BCAs are lower than those carbohydrates, but they are two to three orders of magnitude higher than those of representative waterborne anthropogenic compounds such as PAHs or phthalates. The relative abundance of BCAs in surface waters were found to be different from previous results reported for atmospheric samples, and glyoxylic acid was more abundant than oxalic acid in most samples. Different processes might generate these organic acids. The results indicated that the Rhone River delivers significant levels of diacids and oxoacids to the northwestern Mediterranean Sea. A similar situation could be expected for other rivers that drain the Mediterranean Sea, but experimental confirmation is needed. The presented findings contribute to a better understanding of the sources and production/degradation mechanisms of these molecules and more generally, of organic matter in aquatic systems, although predominance of glyoxylic acid over oxalic acid merits further investigations. The environmental implications and potential impacts of this stock of BCA in Mediterranean waters remain unknown. In addition, the links between BCA and other important anthropogenic compounds (e.g., PAHs) present in the DOC pool in marine and freshwater ecosystems as well as possible transfer of BCAs from surface waters toward lower atmosphere should be further investigated.

Fig. 1 .

 1 Fig. 1. Rhone River mouth area and sampling location (Arles). Gd Rhone and Pt Rhone indicate ''Grand Rhone" and ''Petit Rhone", respectively, which are the two functional arms of the river.

Fig. 2 .

 2 Fig. 2. Chemical structure of dicarboxylic acids and ketoacids detected in the Rhone River water. Cn means dicarboxylic acid with n carbons, and M and F are abbreviations for the unsaturated diacids maleic and fumaric acids, respectively. Cn x indicates x -oxocarboxylic acid with n carbons and Gly means glyoxylic acid.

Fig. 4 .

 4 Fig. 4. Contribution of the BCA-C (BCA-C are the contribution of carbon from BCAs) to the DOC content (%) in the Rhone River during the period from February 2006 to June 2009.

Fig. 3 .

 3 Fig. 3. The total BCA concentrations (mg l À1 ) and water discharge (m 3 s À1 ) in the Rhone River during the period from February 2006 to June 2009.

Fig. 5 .

 5 Fig. 5. The Mean BCA's concentration (mgl À1 ) distributions (a) and Oxalic (C 2 ) and Glyoxylic acids (xC 2 ) concentrations (mg l À1 ) (b) in the Rhone River during the period from February 2006 to June 2009.

Fig. 6 .

 6 Fig. 6. The Relative BCA distribution (%) in the Rhone River during the period from February 2006 to June 2009.

Fig. 7 .

 7 Fig.7. Mean relative distribution of BCA's in three contrasted environments: Mediterranean Sea waters[START_REF] Tedetti | Determination of low molecular weight dicarboxylic and ketocarboxylic acids in seawater samples[END_REF], Rhone River waters (this study) and Marine aerosols (data from[START_REF] Fu | Dicarboxylic acids, ketocarboxylic acids and glyoxal in the marine aerosols collected during a round-the-world cruise[END_REF] that are representative of the global Ocean marine aerosols including Mediterranean.

Table 2

 2 Concentration ranges and average concentrations of individual dicarboxylic acids and ketoacids collected in the Rhone River (n = 23) during the period from February 2006 to June 2009.

		Concentrations (mgl À1 )		Relative abundance (%)
		Range	Average	Range	Average
	Oxalic acid (C 2 )	3.35-23.01	9.08 ± 5.54	15.9-42.6	27.1 ± 7.0
	Malonic (C 3 )	1.05-5.98	2.56 ± 1.50	4.0-16.2	8.0-3.2
	Maleic acid (C 3 )	0.16-8.04	0.86 ± 1.70	0.82-11.29	2.12 ± 2.65
	Succinic Acid (C 4 )	0.88-4.18	2.00 ± 0.88	4.3-11.9	6.4 ± 1.6
	Fumaric acid (C 4 )	1.41-9.68	4.73 ± 2.16	10.3-21.4	14.7 ± 2.7
	Glyoxylic acid (xC 2 )	3.77-26.43	11.37-6.20	14.1-50.7	35.8 ± 10.3
	Glutaric acid (C 5 )	0.11-0.70	0.35 ± 0.15	0.65-2.11	1.13 ± 0.38
	Adipic acid (C 6 )	0.15-3.00	0.62 ± 0.57	0.89-7.33	1.98 ± 1.44
	4-oxobutanoic acid (xC 4 )	0.10-2.98	0.31 ± 0.58	0.29-7.16	0.95 ± 1.37
	Pimelic acid (C 7 )	0.00-0.23	0.07 ± 0.08	0-0.81	0.19 ± 0.25
	Suberic acid (C 8 )	0.05-0.44	0.16 ± 0.10	0.22-1.08	0.49 ± 0.24
	Azealic acid (C 9 )	0.08-0.79	0.32 ± 0.18	0.46-3.77	1.06 ± 0.71

* Flood event.

Acknowledgments

This study was conducted as part ofthe WP3/WP4 MERMEX/ MISTRALS project and is a contribution to the internationalSOLAS, IMBER and LOICZ program and supported by Pole Mer Méditerranée. We acknowledge the financial support from Hokkaido University, Sapporo Japan for R. Sempéré. The authors thank to Michel Fornier from SAM-M I O technical platform for sampling and Natascha Schmidt for drawing Fig. 1. This research also contributes to the Labex OT-Med (no. ANR-11-LABX-0061) funded by the French Government ''Investissements d'Avenir" (ANR) through the A⁄MIDEX project (no ANR-11-IDEX-0001-02). The authors are grateful to two anonymous referees for their useful and constructive comments during the review process.