Amel Nouara

Christos Panagiotopoulos
email: christos.panagiotopoulos@mio.osupytheas.fr

Jérôme Balesdent

Kalliopi Violaki

Edouard Bard

Yoann Fagault

Daniel James Repeta

Richard Sempéré

Liquid chromatographic isolation of individual carbohydrates from environmental matrices for stable carbon analysis and radiocarbon dating

Keywords: Semi-preparative liquid chromatography, carbohydrates purification, carbohydrate-specific 13 C and 14 C analysis, EA-IRMS, EA-AixMICADAS

Carbohydrates are among the most abundant organic molecules in both aquatic and terrestrial ecosystems; however, very few studies have addressed their isotopic signature using compound-specific isotope analysis, which provides additional information on their origin (δ 13 C) and fate ( 14 C). In this study, semi-preparative liquid chromatography with refractive index detection (HPLC-RI) was employed to produce pure carbohydrate targets for subsequent offline δ 13 C and  14 C isotopic analysis. δ 13 C analysis was performed by elemental analyzer-isotope ratio mass spectrometer (EA-IRMS) whereas  14 C analysis was performed by an innovative measurement procedure based on the direct combustion of the isolated fractions using an elemental analyzer coupled to the gas source of a mini carbon dating system (AixMICADAS). In general, four successive purifications with Na + , Ca 2+ , Pb 2+ , and Ca 2+ cation-exchange columns were sufficient to produce pure carbohydrates. These carbohydrates were subsequently identified using mass spectrometry by comparing their mass spectra with those of authentic standards.

The applicability of the proposed method was tested on two different environmental samples comprising marine particulate organic matter (POM) and total suspended atmospheric particles (TSP). The obtained results revealed that for the marine POM sample, the δ 13 C values of the individual carbohydrates ranged from -18.5 to -16.8‰ except for levoglucosan and mannosan, which presented values of -27.2 a -26.2‰, respectively. For the TSP sample, the δ 13 C values a g f m -26.4 -25.0‰. The galactose and glucose  14 C values were 19 and 43‰ p c ly, for the POM sample.

On the other hand, the levoglucosan a ca b alu wa 33‰ f h SP ampl .

These results suggest that these carbohydrates exhibit a modern age in both of these samples. Radiocarbon HPLC collection window blanks, measured after the addition of phthalic acid ( 14 C free blank), ranged from -988 to -986‰ f h ab mentioned compounds, indicating a very small background isotopic influence from the whole purification procedure. Overall, the proposed method does not require derivatization steps, produces extremely low blanks, and may be applied to different types of environmental samples.

Introduction

Carbohydrates are among the most ubiquitous organic molecules and have been recorded in all geochemical systems, including terrestrial [1,[START_REF] Opsahl | Characterization of carbohydrates during early diagenesis of five vascular plant tissues[END_REF], marine [START_REF] Panagiotopoulos | Molecular level characterization of methyl sugars in marine high molecular weight dissolved organic matter[END_REF][START_REF] Repeta | Chemical characterization and cycling of dissolved organic matter[END_REF], and atmospheric organic matter [START_REF] Theodosi | Sugars in atmospheric aerosols over the Eastern Mediterranean[END_REF][START_REF] Fu | Seasonal variations of sugars in atmospheric particulate matter from Gosan, Jeju Island: Significant contributions of airborne pollen and Asian dust in spring[END_REF]. Although previous investigations have provided a wealth of information on their concentrations and distributions in all geochemical systems [START_REF] Oades | Soil organic matter and structural stability: mechanisms and implications for management[END_REF][START_REF] Panagiotopoulos | The composition and flux of particulate and dissolved carbohydrates from the Rhone River into the Mediterranean Sea[END_REF][START_REF] Pakulski | Abundance and distribution of carbohydrates in the ocean[END_REF][START_REF] Fu | Molecular characterization of marine organic aerosols collected during a round-the-world cruise[END_REF][START_REF] Sempéré | Distribution and bacterial availability of dissolved neutral sugars in the South East Pacific[END_REF], very less is known about their sources and fate, which have not been thoroughly studied using carbon isotopes. Such information, obtained from carbon isotope examination at the molecular level, may help trace the origin of the different components of organic matter and explain its reactivity during long-range transport [START_REF] Beaupré | The Carbon Isotopic Composition of Marine DOC[END_REF].

Bulk carbon isotope analysis generally reflects the average of the isotopic composition of the whole panel of organic molecules inside the sample [START_REF] Hobbie | Intramolecular, compound-specific, and bulk carbon isotope patterns in C 3 and C 4 plants: a review and synthesis[END_REF][START_REF] Hwang | Carbon isotope ratios of organic compound fractions in oceanic suspended particles[END_REF][START_REF] López-Veneroni | The stable carbon isotope composition of PM2.5 and PM10 in Mexico City Metropolitan Area air[END_REF][START_REF] Bataille | Influence of provenance and preservation on the carbon isotope variations of dispersed organic matter in ancient floodplain sediments[END_REF][START_REF] Zigah | An investigation of size-fractionated organic matter from Lake Superior and a tributary stream using radiocarbon, stable isotopes and NMR[END_REF][START_REF] Roland | Sources of molecularly uncharacterized organic carbon in sinking particles from three ocean basins: A c upl Δ14C a δ13C app ach Ma[END_REF]. Further extraction of the sample with acids or organic solvents produces "purified" fractions (e.g. sugar-or lipid-like fractions) and aids in the determination of the isotopic composition of the hydrophilic and hydrophobic components of the sample [START_REF] Roland | Sources of molecularly uncharacterized organic carbon in sinking particles from three ocean basins: A c upl Δ14C a δ13C app ach Ma[END_REF][START_REF] Wang | Radiocarbon studies of organic compounds classes in plankton and sediment of the northeastern Pacific Ocean[END_REF][START_REF] Hwang | Lipid-Like Material as the Source of the Uncharacterized Organic Carbon in the Ocean?[END_REF]. For example, previous studies on dissolved organic matter (DOM) have reported  13 C values for carbohydrate-like fractions in the ranges -29 to -25‰ in river estuaries [START_REF] Wang | Sources and transport of dissolved and particulate organic carbon in the Mississippi River estuary and adjacent coastal waters of the northern Gulf of Mexico[END_REF] and -21.5 to -20.3‰ h Atlantic and Pacific Oceans [START_REF] Loh | Variable ageing and storage of dissolved organic components in the open ocean[END_REF]. These  13 C values are typical for terrestrial and marine ecosystems, respectively. Moreover, the reported  14 C values for carbohydrate-like fractions in marine high molecular weight DOM showed a wide range of values spanning from 7 to -406‰, further implying that the age of carbohydrates spans from modern to very old (4180 yr BP) [START_REF] Loh | Variable ageing and storage of dissolved organic components in the open ocean[END_REF]. However, the bulk isotope analysis approach does not completely address the isotopic diversity of the individual molecules inside the sample.

In contrast with bulk isotope analysis, the compound-specific isotope analysis (CSIA) of the individual sugars offers valuable information on the origin ( 13 C) and age ( 14 C) of the single molecules [START_REF] Van Dongen | Carbon isotope variability in monosaccharides and lipids of aquatic algae and terrestrial plants[END_REF][START_REF] Glaser | Compound-specificstable-p (δ13C) a aly l c c[END_REF][START_REF] Sang | Stable carbon isotope ratio analysis of anhydrosugars in biomass burning aerosol particles from source samples[END_REF][START_REF] Zhu | Optimizing sample pretreatment for compound-specific stable carbon isotopic analysis of amino sugars in marine sediment[END_REF][START_REF] Repeta | Radiocarbon analysis of neutral sugars in highmolecular-weight dissolved organic carbon: Implications for organic carbon cycling[END_REF]. The CSIA technique is not a new approach; however, it requires high analytical skills for the purification and extraction of individual molecules from the sample. This step is crucial and might strongly affect the results. The two most commonly employed techniques for the stable carbon isotope analysis of carbohydrates are gas and liquid chromatography coupled with isotope ratio mass spectrometry (GC-IRMS and LC-IRMS, respectively). Since carbohydrates are not volatile, derivatization steps (silylation or alditol acetate derivatization) are required for GC-IRMS analysis. This further necessitates carbon corrections on the carbohydrate isotopic signatures [START_REF] Van Dongen | Carbon isotope variability in monosaccharides and lipids of aquatic algae and terrestrial plants[END_REF][START_REF] Derrien | Measurement of the 13C/12C ratio of soil-plant individual sugars by gas chromatography/combustion/isotoperatio mass spectrometry of silylated derivatives[END_REF][START_REF] Teece | Stable carbon isotope biogeochemistry of monosaccharides in aquatic organisms and terrestrial plants[END_REF][START_REF] Moerdijk-Poortvliet | Comparison of gas chromatography/isotope ratio mass spectrometry and liquid chromatography/isotope ratio mass spectrometry for carbon stable-isotope analysis of carbohydrates[END_REF]. Another disadvantage of the GC-IRMS technique is that two different monosaccharides (e.g. glucose and fructose) can produce the same alditol (e.g. glucitol) during the reduction step of the derivatization procedure, thereby causing a loss of compositional information [START_REF] Panagiotopoulos | Analytical methods for the determination of uga ma ampl : A h cal p p c and future directions[END_REF].

LC-IRMS is a good alternative to GC-IRMS because it does not require any derivatization steps [START_REF] Moerdijk-Poortvliet | Comparison of gas chromatography/isotope ratio mass spectrometry and liquid chromatography/isotope ratio mass spectrometry for carbon stable-isotope analysis of carbohydrates[END_REF][START_REF] Moerdijk-Poortvliet | LC/IRMS analysis: A powerful technique to trace carbon flow in microphytobenthic communities in intertidal sediments[END_REF][START_REF] Boschker | A versatile method for stable carbon isotope analysis of carbohydrates by high-performance liquid chromatography/isotope ratio mass spectrometry[END_REF][START_REF] Basler | Compound-p c f c δ13C a aly f m accha from soil extracts by high-performance liquid chromatography/isotope ratio mass spectrometry[END_REF]. However, this technique does not target all the sugar components of the sample; for example, neutral sugars, amino sugars, alditols, and anhydrosugars cannot be separated in a single run [START_REF] Rinne | Evaluation of a liquid chromatography method for compound-specific δ13C a aly f pla carbohydrates in alkaline media[END_REF][START_REF] Sevcik | Rapid analysis of carbohydrates in aqueous extracts and hydrolysates of biomass using a carbonatemodified anion-exchange column[END_REF]. Moreover, both the GC-IRMS and LC-IRMS techniques cannot be used for  14 C determination on single carbohydrates due to their low sensitivity toward the 14 C isotope. To date, very few radiocarbon data exist on single carbohydrates comprising neutral sugars, alditols, and anhydrosugars. Alditols and anhydrosugars are important tracers of terrestrial vegetation [START_REF] Bauer | Arabitol and mannitol as tracers for the quantification of airborne fungal spores[END_REF][START_REF] Medeiros | Sugars as source indicators of biogenic organic carbon in aerosols collected above the Howland Experimental Forest, Maine[END_REF] and burning biomass processes [START_REF] Simoneit | Biomass burninga review of organic tracers for smoke from incomplete combustion[END_REF], respectively. Thus, their isotopic study may help to evaluate their long-range transport from land to sea and more importantly, to assess their reactivity in long time scales (fate) in the marine environment. Although a significant amount of work was done on the compoundspecific radiocarbon analysis of individual lipids over the past 20 years [START_REF] Eglinton | Gas Chromatographic Isolation of Individual Compounds from Complex Matrices for Radiocarbon Dating[END_REF][START_REF] Eglinton | Composition, age, and provenance of organic matter in NW African dust over the Atlantic Ocean[END_REF][START_REF] Ingalls | Ten Years of Compound-Specific Radiocarbon Analysis[END_REF][START_REF] Makou | Plant Wax n-Alkane and n-Alkanoic Acid Signatures Overprinted by Microbial Contributions and Old Carbon in Meromictic Lake Sediments[END_REF] and more recently, on amino acids [START_REF] Bour | Radiocarbon Analysis of Individual Amino Acids: Carbon Blank Quantification for a Small-Sample High-Pressure Liquid Chromatography Purification Method[END_REF], a well-established technique for single carbohydrate purification from environmental samples for subsequent radiocarbon measurements has not been reported to date.

An interesting approach to produce pure carbohydrate targets for δ 13 C and  14 C analysis is the use of semi-preparative high-performance liquid chromatography with refractive index detection (HPLC-RI) on cation-exchange columns. Pure carbohydrates can be analyzed by an elemental analyzer-isotope ratio mass spectrometer (EA-IRMS) for δ 13 C and/or an accelerated mass spectrometer for  14 C analysis. HPLC-RI is a simple and well-established method for carbohydrate analysis; however, in environmental studies, it has been less explored [START_REF] Repeta | Radiocarbon analysis of neutral sugars in highmolecular-weight dissolved organic carbon: Implications for organic carbon cycling[END_REF][START_REF] Panagiotopoulos | Characterization of methyl sugars, 3-deoxysugars and methyl deoxysugars in marine high molecular weight dissolved organic matter[END_REF] than traditional GC-MS or high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) techniques [START_REF] Theodosi | Sugars in atmospheric aerosols over the Eastern Mediterranean[END_REF][START_REF] Panagiotopoulos | The composition and flux of particulate and dissolved carbohydrates from the Rhone River into the Mediterranean Sea[END_REF][START_REF] Fu | Molecular characterization of marine organic aerosols collected during a round-the-world cruise[END_REF][START_REF] Sempéré | Distribution and bacterial availability of dissolved neutral sugars in the South East Pacific[END_REF][START_REF] Panagiotopoulos | Analytical methods for the determination of uga ma ampl : A h cal p p c and future directions[END_REF][START_REF] Medeiros | Analysis of sugars in environmental samples by gas chromatography-mass spectrometry[END_REF][START_REF] Cheng | Simultaneous analyses of neutral carbohydrates and amino sugars in freshwaters with HPLC-PAD[END_REF].

This study employs HPLC-RI to demonstrate that four successive purifications on Na + , Ca 2+ , Pb 2+ , and Ca 2+ cation-exchange columns are sufficient to produce pure carbohydrates for compound-specific carbon isotope determination. In previous investigations, the isolated fractions were measured after combustion in evacuated quartz tubes and the produced CO 2 was purified on vacuum lines. This was followed by graphitization for solid measurements or transfer in glass ampoules for gas measurements. On the other hand, this study employs an alternative procedure [START_REF] Bard | AixMICADAS, the accelerator mass spectrometer dedicated to14C recently installed in Aix-en-Provence, France[END_REF] based on the direct combustion of isolated fractions using an elemental analyzer coupled to the gas source of a mini carbon dating system (AixMICADAS) for  14 C analysis. Compared to the time-consuming vacuum line method, this procedure is la ly fa a ca b a ap f ampl ma ≤10 g C with moderate precision.

The applicability of the method was tested on two different environmental samples, namely marine particulate organic matter (POM) and total suspended atmospheric particles (TSP).

Materials and Methods

Chemicals and reagents

All the carbohydrate standards used in this study were purchased from Sigma-Aldrich or Interchim at the purest available grade (>98% Three portions (17.34 cm 2 each) of the filter were extracted with 18 mL ultrapure water in an ultrasonic bath for 1 h and then filtered through a Pasteur pipette packed with quartz wool (both pre-combusted at 450 ˚C for 6 h) to remove any remaining particles [START_REF] Theodosi | Sugars in atmospheric aerosols over the Eastern Mediterranean[END_REF]. Finally, the sample was freeze-dried and stored in a freezer at -35 °C until chromatographic injection.

Chromatography

The carbohydrates were analyzed using an HPLC system (Thermo Scientific UltiMate 3000) equipped with a vacuum degasser and a 100-µL loop auto-injector. The carbohydrates were detected with a refractive index detector (Shodex RI-101) and eluted in isocratic mode with ultrapure water, which was previously degassed with high purity N 2 for 30 min.

Three cation-exchange analytical columns packed with a polymeric resin (sulfonated polystyrene-divinyl benzene) were used to purify the carbohydrates (Table S placed after the RI detector. The system was controlled via Chromeleon chromatography software (Thermofisher).

The detection limit of the HPLC-RI system was ~1 µM at a signal-to-noise ratio (S/N) of three for all the carbohydrates on the three tested columns; this was in agreement with previous results reported in the literature [START_REF] Cheng | On-line desalting and carbohydrate analysis for immobilized enzyme hydrolysis of waste cellulosic biomass by columnswitching high-performance liquid chromatography[END_REF]. The precision of the method was evaluated by calculating the relative standard deviation (RSD%) for six replicate HPLC-RI injections of the standard mixture of the 14 carbohydrates at the 5 µM level. The RSD was <10% for the peak area and <1% for the retention time for all the columns tested. Additional details on the system optimization are included in the supplementary information (section S1 and Fig. S1).

Isotopic measurements

EA-IRMS

Prior to EA-IRMS processing, the samples were acidified with HCl (final concentration, 0.01 M) to avoid any errors related to the isotopic signature of inorganic carbon mainly from atmospheric CO 2 absorbed in the sample [START_REF] Gordon | Sources and distribution of terrigenous organic matter delivered by the Atchafalaya River to sediments in the northern Gulf of Mexico[END_REF]. The samples were placed in a tin capsule (5 mm × 9 mm; light; Santis) and dried under a N 2 stream. The stable carbon isotope composition and the carbon content of the purified carbohydrates were measured using an elemental analyzer (Flash EA 1500; Thermo Finnigan, Germany) coupled with an isotope ratio mass spectrometer (IRMS Delta plus , Thermo Finnigan, Germany). Briefly, this technique measures the 13 C/ 12 C ratio of total carbon of the dried sample as follows: in a continuous helium flow, the sample is oxidized at 1000 °C in the presence of O 2 and catalysts; the resulting CO 2 is separated from the other combustion products and transferred by the helium flow to a gas source, magnetic sector, triple collector mass spectrometer. The latter determines the 13 C/ 12 C ratio of CO 2 -carbon. The stable carbon isotope composition is conventionally expressed as δ 13 C values according to the formula:

where VPDB is the Vienna Pee Dee Belemnite standard.

International Atomic Energy Agency (IAEA)-CH-7 polyethylene (δ 13 C = -32.2‰) and IAEA-CH-6 sucrose were used as the calibration standard and control, respectively [START_REF] Coplen | Af w ca a c a ch f h VPDB δ13C cal Rapid[END_REF]. The latter yielded a mean value of -10.5‰. The δ 13 C and carbon content of the samples were corrected for the contribution of carbon in the tin capsules. The δ 13 C standard deviations were determined from replicated measurements of the IAEA-CH-6 standard and were ± 0.2‰ ± 0.2‰ ± 0.4‰ and ± 0.7‰ f samples containing 20, 10, 5, and 2 µg C, respectively. The precision on the carbon content (precision on the absolute amount of carbon analyzed) was ± 0.1 µg C.

EA-AixMICADAS

Direct radiocarbon measurement of the CO 2 gas was carried out, after combustion, using an elemental analyzer (EA) coupled to the gas interface system (GIS) of the AixMICADAS system [START_REF] Bard | AixMICADAS, the accelerator mass spectrometer dedicated to14C recently installed in Aix-en-Provence, France[END_REF]. Briefly, the main characteristic of the EA is that it works with a combustion tube filled with tungsten oxide heated to 1050 °C to allow the introduction of silver boats containing the sample material. The purified extracts were recovered in ultrapure water without acidification, transferred into silver capsules (4 mm × 8 mm, Elemental Microanalysis Ltd), and dried at 80 °C on a hotplate under a N 2 stream. The silver capsules were baked at 800 °C for 3 h prior to use to eliminate any organic contamination [START_REF] Tuna | Development of small CO2 gas measurements with AixMICADAS[END_REF][START_REF] Fagault | Radiocarbon dating small carbonate samples with the gas ion source of AixMICADAS[END_REF]. The CO 2 produced by the EA is captured in the zeolite trap inside the GIS. The CO 2 is then released by heating the zeolite trap to 450 °C and is mixed inside the syringe with a helium flow in order to obtain 5% CO 2 in the gas mixture, which is sputtered into the ion source. The CO 2 is injected from the GIS into the ion source through a small fused silica capillary continuously fed by the syringe, which is driven by a stepping motor controlled by the GIS software. A carbon flow of 2.80 μg C min -1 keeps the total pressure constant inside the syringe (filled to 1300 mbar), allowing the ion source to produce stable currents. The tuning procedure and main operation parameters used in the gas configurations are described in the literature [START_REF] Bard | AixMICADAS, the accelerator mass spectrometer dedicated to14C recently installed in Aix-en-Provence, France[END_REF]. The measurements were normalized with the oxalic acid 2 standard (~100 g C;

OxA2 SRM 4990 C, National Institute of Standards and Technology) and corrected for blanks using phthalic anhydride acid (F 14 C = 0.0027; n = 3) prepared from the same protocol as that used for OxA2 (i.e. silver capsules measured by EA-GIS).

The samples were blank corrected with procedural blanks of the same size (section 3.3) and a conservative uncertainty of 30% of the blank value was propagated in the final error calculation. The samples were in the size range 50-150 g C, which translates to a precision of ~1% for a modern sample. However, samples down to 10 g C could be measured with less precision. The accuracy of 14 C measurements of small samples (< 100 µg C) with the gas ion source of AixMICADAS has been tested with numerous measurements on various standards (NIST 4990C, IAEA-C1, IAEA-C2, inhouse carbonate standards) [START_REF] Tuna | Development of small CO2 gas measurements with AixMICADAS[END_REF][START_REF] Fagault | Radiocarbon dating small carbonate samples with the gas ion source of AixMICADAS[END_REF]. For example, the analysis of oxalic acid NIST 4990C was replicated 132 times over 2.5 years, giving an average F 14 C of 1.3403 and a a a a (SD) f 0.0078 ( . . 6‰). h a hm c m a a a c a error (std error = 0.0007) are compatible with the weighted mean (1.3405) and the weighted error (0.0008), and closely agree with the NIST reference value of 1.3407 ± 0.0005.

The 14 C a aly w p a ∆ 14 C corrected for decay [START_REF] Stuiver | Reporting of C-14 data -Discussion[END_REF][START_REF] Reimer | Discussion: Reporting and calibration of post-bomb C-14 data[END_REF] according to the formula:

where A SN is the normalized sample activity, A ON is the normalized standard activity,

x is the year of formation or growth, and λ C = (1/8267) yr -1 .

Identification of purified monosaccharides

The identification of the isolated purified monosaccharides was checked with Liquid Chromatography coupled with Time-of-Flight Mass Spectrometry (LC-Q-TOF-MS)

Agilent 6500 system. The chromatographic separation was performed with a Luna HILIC c lum (100 mm × 2.00 mm I.D.

Results and discussion

Cation-exchange column selection and purification procedure

Cation-exchange chromatography allows the separation of poly-, oligo-, and monosaccharides based on their degree of polymerization [START_REF] Panagiotopoulos | Characterization of methyl sugars, 3-deoxysugars and methyl deoxysugars in marine high molecular weight dissolved organic matter[END_REF][START_REF] Angyal | The separation of sugars and of polyols on cation-exchange resins in the calcium form[END_REF]. The chromatographic resolution and selectivity can be modulated by changing the cation-exchange column.

The separation mechanism is based on the strength of the complex formed between the hydroxyl groups of the carbohydrate and the metal (e.g. K + , Na + , Pb 2+ , Ca 2+ , Cd 2+ , and Cu 2+ ).

In this study, Na + , Ca 2+ , and Pb 2+ columns were selected for optimum purification of the set of studied carbohydrates. The Na + column is ideal to separate polysaccharides from oligosaccharides and monosaccharides, whereas the Ca 2+ and Pb 2+ columns provide further and complementary separation among the different monosaccharides (e.g. neutral monosaccharides, alditols, anhydrosugars; Fig. 1 a-c). Compared to NH 2 columns (also employed for sugar preparative chromatography), Na + , Ca 2+ , and Pb 2+ columns have the advantage of running with ultrapure water as eluent instead of acetonitrile. This considerably reduces the organic carbon contamination delivered by the eluent, while ultrapure water is easily removed by evaporation with a minimum organic carbon background left behind.

Because carbohydrates in environmental samples can also be polymers (polysaccharides and oligosaccharides) and acid hydrolysis is not always 100% effective [START_REF] Panagiotopoulos | Spectrophotometric and chromatographic analysis of carbohydrates in marine samples[END_REF], an initial purification on a Na + column was necessary to separate the polymers from the monosaccharides [START_REF] Nobre | Comparison of adsorption equilibrium of fructose, glucose and sucrose on potassium gel-type and macroporous sodium ion-exchange resins[END_REF]. The next step was the sequential purification of the monosaccharides on the Ca 2+ , Pb 2+ and again, when necessary, on the Ca 2+ columns. This provided further and complementary separation among the different monosaccharides (e.g. neutral monosaccharides, alditols, anhydrosugars; Fig. 1 b-c) [START_REF] Vente | Comparison of sorption isotherms of mono-and disaccharides relevant to oligosaccharide separations for Na, K, and Ca loaded cation exchange resins[END_REF]. In summary the purification procedure flowchart proposed in this study is:

Na + → Ca 2+ → Pb 2+ → Ca 2+ .

Monosaccharide standards purification and recovery yields

The analytical procedure flowchart established in the previous section was tested on monosaccharide standards (neutral monosaccharides, alditols, and anhydrosugars; initial concentration of 50 µM) to evaluate the separation and peak isolation of the single monosaccharides. The monosaccharide standards were sequentially purified on Ca 2+ and Pb 2+ columns and if necessary, again on a Ca 2+ column. Note that for the purpose of this exercise the Na + column was not included because the polysaccharides and oligosaccharides were not considered in the standard (section 3.1).

The first separation was achieved on a Ca 2+ column (Fig. S2 It is worth noting that fractions in the time periods between the two adjacent peaks were also collected and their purity was checked after injection into the Pb 2+ column. The monosaccharide(s), if any, obtained in such a way was (were) further pooled with fraction(s) that contained the same monosaccharide to increase its recovery.

Following the second purification, 10 peaks were obtained corresponding to fractions F 1 -F 10 . The last purification was performed on a Ca 2+ column to ensure the collection of ultrapure monosaccharide targets (Fig. S2 c).

The obtained results revealed that the recovery yields after three successive purifications (Ca 2+ → Pb 2+ → Ca 2+ ) of the standard mixture ranged from 12.38 ± 0.01% to 36.32 ± 0.02% (n = 3), with the highest values observed for glucose, mannosan, levoglucosan, and mannitol and the lowest for arabinose (Table S2). Notably, for environmental sample analysis (section 3.5), the monosaccharides xylose, fucose, and rhamnose were also included in the standard (in total 14 monosaccharides) despite the fact that their recovery yield was not estimated via this exercise.

Chromatographic system blanks and background isotopic signature

The blanks were run on the HPLC-RI system with ultrapure water and the amount of carbon released from the chromatographic columns (column bleeding) and isotopic signature were evaluated (Table 1). Column blanks were recorded during the whole run for each column: 50 min for the Na + column, 30 min for the Ca 2+ column, and 45 min for the Pb 2+ column. Subsequently, the amount of carbon released from these three columns was measured via EA-IRMS. The obtained values were significantly close for the Ca 2+ and Pb 2+ columns (4.84 ± 0.25 and 2.80 ± 0.21 µg, respectively), while those of the Na + column were much higher (28.64 ± 1.57 µg). Despite these differences, all the blanks resulted in a similar δ 13 C signature ranging from -28.4 ± 0.4 to -27.8 ± 0.4‰

(Table 1).

The next step was to estimate the total carbon release after sequential purification on the Na + → Ca 2+ → Pb 2+ → Ca 2+ columns. The obtained results indicated that the purification procedure produced a blank that contained 19.76 ± 1.80 µg carbon (Table 1) with a δ 13 C value of -27.9 ± 1.1‰; these results were similar to those observed for each column. Blanks corresponding to the collection time window of the three monosaccharides (glucose, galactose, and levoglucosan) after Na + → Ca 2+ → Pb 2+ → Ca 2+ purification were also run. The respective carbon inputs and the δ 13 C signatures after Na + → Ca 2+ → Pb 2+ → Ca 2+ purification were 0.58 ± 0.004 µg a -24.4 ± 2.9 ‰ for glucose a 0.54 ± 0.15 µg a -26.7 ± 1.5‰ f l gluc a ( abl 1).

Because of the elevated cost of radiocarbon analysis, radiocarbon blanks were only run for the collection time trap of the three monosaccharides (glucose, galactose and levoglucosan) measured in this study. Moreover, as the amount of carbon delivered for the whole purification procedure (Na + → Ca 2+ → Pb 2+ → Ca 2+ columns) for each of the these monosaccharides was too small for a reliable  14 C measurement, phthalic acid ( 14 C free blank sample) was added to the final collected blank and the sample was processed as the monosaccharide sample (i.e. transferred into Ag capsules with ultrapure water and combusted by the EA coupled to the gas interface of AixMICADAS). The amount of phthalic acid was adjusted according to the size of the sample to correct for constant contamination offsets. The results revealed that the radiocarbon blanks exhibited values ranging from -988 to -986‰ ( abl 1).

These  14 C values are slightly higher than those observed for the phthalic acid samples ( -997‰) m a u c ly af a to the silver capsules (i.e. without any transfer from the collection tube to the silver cups). This suggests that little exogenous carbon was added to the sample from HPLC purification (column bleeding and organic residues), glassware contamination (collection vials, Pasteur pipets) and airborne particle deposition during sample collection and transfer. Moreover, the  14 C alu (-989.7 ± 3.5‰) and carbon amounts (0.52 ± 0.34 µg C) of the ultrapure water samples (10 mL corresponding to ~10 times the volume of the collection window of a pure monosaccharide) indicated that the addition of exogenous carbon from the eluent was also negligible. Ultrapure water (n = 3) 0.52 ± 0.34 -21.9 ± 5.9 -989.7 ± 3.5 ND: Not determined * Measured for the whole HPLC -RI run time for each column (Table S1) ** Measured for the whole purification procedure (Na + → Ca 2+ → Pb 2+ →Ca 2+ ) § n = 4 † Measured with EA-AixMICADAS ‡ n = 2 Despite the significantly low amount of contaminants (< 1% for both the glucose and levoglucosan samples from the estimated carbon content of the retention time window) and the low  14 C procedural blanks, the results presented in this study were blank corrected. Finally, it is worth noting that the direct combustion of the small silver cups via an elemental analyzer allows a low background (equivalent age of 46 000 yr BP) and is therefore a good alternative to the time-consuming conventional method based on purification with a vacuum line.

Overall, the above results clearly suggest that the proposed purification procedure does not induce any significant contamination in the samples or affect their isotopic signature.

Hydrolysis effects

The marine sample was submitted to hydrolysis (1 M HCl) prior to its chromatographic purification (Section 2) to release the monosaccharides from the biopolymer macrostructure. Moreover, additional experiments were performed on the standard mono-and polysaccharides to investigate whether the hydrolysis conditions affect the isotopic composition of the released monosaccharides. The three levoglucosan standard solutions (90 µg C each) presented similar δ 13 C levo values, namely -11.2 ± 0.2‰ (n = 3) and -11.2 ± 0.1‰ (n = 3) before and after hydrolysis, respectively. Similar results were obtained by Wang et al. [START_REF] Wang | Radiocarbon studies of organic compounds classes in plankton and sediment of the northeastern Pacific Ocean[END_REF] for the glucose standard (δ 13 C glc = -9.8‰ a δ 13 C glc = -9.9‰ before and after processing, respectively); however their study included additional purification steps comprising anion and cation-exchange resins.

In another set of experiments a polysaccharide standard (laminarin) containing glucose units was submitted to acid hydrolysis and the isotopic signature of the released glucose was compared to that of the original laminarin. The results indicated that glucose and laminarin exhibited similar isotopic signatures in terms of δ 13 C (δ 13 C glc = -10.4‰ a δ 13 C lam. = -13.0‰; n = 1) and  14 C ( 14 C glc = -85.4‰ a  14 C lam. = -84.90‰; n = 1) indicating few differences between the isotopic composition of the original polysaccharide and its monomeric constituent (Repeta; unpublished results).

Application to environmental samples

The purification procedure, including sample preparation, employed in this study is briefly summarized in Fig. 2. Two distinct environmental samples, namely, a marine POM and a TSP sample were considered. The choice of samples was made with respect to the opportunities and the logistics set to obtain them but more importantly of their high carbohydrate content.

Marine POM

The hydrolyzed marine sample was processed on a Na + column and the collected fractions (F 3 -F 6 ), corresponding to the monosaccharides, were further purified on Ca 2+ → Pb 2+ → Ca 2+ columns (Fig. 3). The results indicated that after four sequential purifications the major monosaccharides obtained were: galactose (368 µg), glucose (273 µg), mannose (65 µg), xylose (38 µg), and fucose/arabinose (16 µg). It is worth noting that levoglucosan (1.8 µg) and mannosan (1.5 µg) were also collected for the first time from marine POM; however, in very low amounts. The presence of the abovementioned anhydrosugars in the hydrolyzed marine sample was also confirmed by high-performance anion-exchange chromatography (Nouara et al., submitted). Finally, although rhamnose and galactosan were detected in the marine POM sample they yielded very small recovered amounts (< 0.5 µg), which were insufficient for isotopic measurement.

The results revealed that the marine POM sample exhibited a δ 13 C alu f -19.6 ± 0.6‰ which is typical of marine origin and consistent with the δ 13 C values reported for the surface coastal sinking POM (range: -23 to -20 ‰) [START_REF] Roland | Sources of molecularly uncharacterized organic carbon in sinking particles from three ocean basins: A c upl Δ14C a δ13C app ach Ma[END_REF][START_REF] Druffel | Cycling of dissolved and particulate organic matter in the open ocean[END_REF]. The δ 13 C signature of the POM hydrophilic extract (fraction comprising most of the hydrophilic components of the sample including monosaccharides) was slightly enriched (-17.2 ± 0.5‰) compared to that of the bulk POM and agreed well with the δ 13 C values recorded for glucose, galactose, mannose, xylose, a fuc / ham (-18.5 to -16.8‰; Table 2).

The δ 13 C signature recorded for these individual monosaccharides agrees well with the isotopic values measured for the individual monosaccharides in marine highmolecular-weight dissolved organic matter (HMWDOM) [START_REF] Repeta | Radiocarbon analysis of neutral sugars in highmolecular-weight dissolved organic carbon: Implications for organic carbon cycling[END_REF], and thereby pointing toward a marine origin. The slight depletion of the δ 13 C values of the bulk POM relative to its individual carbohydrate component may be due to the presence of other organic compounds (e.g. amino acids and lipids) in the sample, which may have a lighter stable carbon isotope signature than that observed for carbohydrates [START_REF] Hwang | Lipid-Like Material as the Source of the Uncharacterized Organic Carbon in the Ocean?[END_REF][START_REF] Loh | Variable ageing and storage of dissolved organic components in the open ocean[END_REF]. Regardless, further molecular level isotopic analysis on individual amino acids and/or lipids is warranted to test this hypothesis.

On the other hand, the levoglucosan (-27.2‰) a ma a (-26.2‰) isotopic signatures exhibited depleted δ 13 C values when compared to those of the other monosaccharides, indicating the different origin of these two monosaccharides (Table 2). Indeed, these sugars are well known tracers of terrestrial biomass burning processes [START_REF] Simoneit | Biomass burninga review of organic tracers for smoke from incomplete combustion[END_REF], and thus their presence in marine POM indicates an external terrestrial input probably via atmospheric deposition [START_REF] Theodosi | Sugars in atmospheric aerosols over the Eastern Mediterranean[END_REF][START_REF] Fu | Molecular characterization of marine organic aerosols collected during a round-the-world cruise[END_REF] from C3 land plant tissue (δ 13 C: -32 -20‰; mean: -27‰ [START_REF] O' | L a y Ca b I p Ph y h :F ac a ch ques may reveal new aspects of carbon dynamics in plants[END_REF]). The presence of levoglucosan and mannosan in the POM sample is not surprising since the sampling site was located a few kilometers offshore from the Lima area (section 2.2). The radiocarbon results of this study indicated that relatively to its hydrophilic fraction ( 14 C = 124 ± 5‰), the marine POM sample was depleted ( 14 C = 28 ± 8‰).

This agrees very well with previous investigations, which have reported similar results for a wide variety of environmental samples comprising sediments, sinking POM, planktons [START_REF] Wang | Radiocarbon studies of organic compounds classes in plankton and sediment of the northeastern Pacific Ocean[END_REF][START_REF] Wang | Radiocarbon and stable carbon isotope compositions of organic compound classes in sediments from the NE Pacific and Southern Oceans[END_REF] including riverine, and marine HMWDOM [START_REF] Loh | Variable ageing and storage of dissolved organic components in the open ocean[END_REF][START_REF] Wang | Variability in radiocarbon ages of biochemical compound classes of high molecular weight dissolved organic matter in estuaries[END_REF]. Intermediate radiocarbon values were recorded for glucose and galactose ( 14 C glc = 43‰ and  14 C gal = 19‰; Table 2) and are consistent with the radiocarbon monosaccharide signature reported for surface marine HMWDOM [START_REF] Repeta | Radiocarbon analysis of neutral sugars in highmolecular-weight dissolved organic carbon: Implications for organic carbon cycling[END_REF]. Overall, the above radiocarbon data indicate a modern age for the purified monosaccharides that further support the hypothesis that these monosaccharides are incorporated into a common family of polysaccharides (acylpolysaccharides) present in all terrestrial and aquatic ecosystems [START_REF] Aluwihare | A major biopolymeric component to dissolved organic carbon in surface sea water[END_REF][START_REF] Repeta | Chemical characterization of high molecular weight dissolved organic matter in fresh and marine waters[END_REF]. Nevertheless, more  14 C data on the individual monosaccharides including other environmental matrices (e.g. sediments, and riverine POM and DOM) are warranted before any generalizations can be made.

TSP

The purification of the atmospheric sample was performed, without prior processing, on the Na + column. This was due to the low complexity of the sample and to avoid further possible losses and contamination that may occur during additional manipulation. The purification was conducted in a Ca 2+ column, followed by a Pb 2+ column and again by a Ca 2+ column (Fig. 4). The obtained results indicated that after three purifications the major monosaccharides obtained were: levoglucosan (134 µg), fructose (40 µg), mannosan (38 µg), glucose (36 µg), and mannitol (4 µg). The abundance of these monosaccharides is in agreement with the results generally reported for PM 10 particles [START_REF] Theodosi | Sugars in atmospheric aerosols over the Eastern Mediterranean[END_REF][START_REF] Fu | Molecular characterization of marine organic aerosols collected during a round-the-world cruise[END_REF]. Similar to the abovementioned results galactose, arabinose and galactosan were detected in the TSP sample. However, these were present in very low amounts (< 0.5 µg) and no isotopic measurements were performed.

The δ 13 C value recorded for the TSP sample (-25.9 ± 0.0‰) was similar to that of its hydrophilic extract (-24.8 ± 0.1‰) and within the range of that of the isolated monosaccharides (-26.4 to -25.0‰; Table 2). These results reflect a dominant terrestrial origin from C3 vegetation and/or microorganisms, including fungal spores [START_REF] Fu | Seasonal variations of sugars in atmospheric particulate matter from Gosan, Jeju Island: Significant contributions of airborne pollen and Asian dust in spring[END_REF][START_REF] Bauer | Arabitol and mannitol as tracers for the quantification of airborne fungal spores[END_REF]. The δ 13 C values of levoglucosan and mannosan were in agreement with the values reported by Sang et al. [START_REF] Sang | Stable carbon isotope ratio analysis of anhydrosugars in biomass burning aerosol particles from source samples[END_REF] for hardwood and stalk plant combustion. The δ 13 C values reported in this study are also in good agreement with those measured for n- The final purified compounds (after Ca 2+ →Pb 2+ →Ca 2+ purification) are also indicated with arrows.

The radiocarbon signature of the TSP sample (Table 2) indicated that the bulk sample was highly depleted (-175 ± 5‰; 1545 yr) relative to its hydrophilic component (-64 ± 13‰; 530 yr). This is consistent with the characteristics already observed for the POM sample (section 3.5.1). The purified levoglucosan exhibited a modern radiocarbon age (33‰; Table 2), implying a very recent synthesis of this monosaccharide, which agrees with the radiocarbon signature of contemporary biosphere carbon ( 14 C = 0 to 200‰) and concurs with the radiocarbon monosaccharide data of the marine POM sample. Although this result is a single and unique up-to-date measure of the  14 C of levoglucosan, it can also explain, in part, the lack of stability of levoglucosan in the atmosphere over time since this compound has a modern age [START_REF] Fortenberry | Bulk and Molecular-Level Characterization of Laboratory-Aged Biomass Burning Organic Aerosol from Oak Leaf and Heartwood Fuels[END_REF][START_REF] Bertrand | Evolution of the chemical fingerprint of biomass burning organic aerosol during aging[END_REF]. The radiocarbon signature of the TSP sample (1545 yr) relative to those of the purified water extract (530 yr) and levoglucosan (modern), may be due to the contribution of aged organic material such as black carbon (>50,000 yr). This material is known to be ubiquitous in the atmosphere as a result of fossil fuel emissions. This observation is further supported by the location of the sampling site (Marseille, France), which is characterized by the high influence from anthropogenic emissions and is in agreement with previous radiocarbon data performed on fossil fuels, soot aerosols, and PM 2.5 particles in other areas [START_REF] Bonvalot | Estimating contributions from biomass burning and fossil fuel combustion by means of radiocarbon analysis of carbonaceous aerosols: application to the Valley of Chamonix[END_REF][START_REF] Niu | Source contributions to carbonaceous species in PM2,5 and their uncertainty analysis at typical urban, peri-urban and background sites in southeast China[END_REF][START_REF] Andersson | 14C-Based source assessment of soot aerosols in Stockholm and the Swedish EMEP-Aspvreten regional background site[END_REF].

However, more TSP radiocarbon data in conjunction with compound specific radiocarbon analyses (i.e. monosaccharides, lipids, polyaromatic hydrocarbons) are required to confirm this hypothesis.

Identification of purified monosaccharides: comparison with authentic standards

Isolated monosaccharides recovered after the whole extraction and purification procedure were compared with authentic standards to check their purity. Depending on the amount of carbon recovered and the number of subsequently performed replicate EA-IRMS analysis, only three monosaccharides were further explored: levoglucosan, glucose, and galactose. The results from this study indicated that the mass spectra of the isolated monosaccharides well matched those of authentic standards (Table S3), as revealed by liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (Q-TOF-LC/MS; Fig. S3). These results indicate that this proposed approach is specific, valid and reliable.

Conclusions and Outlook

The approach presented herein proved to be a powerful and robust tool for δ 13 C and  14 C determination of individual carbohydrates in the environmental matrices. Briefly, after four successive purifications on cation-exchange columns, the pure carbohydrates were produced and further assessed by EA-IRMS and/or EA-AixMICADAS. Particular attention was given to the procedural blanks, which were found to be extremely low during the whole cleanup procedure (0.5 μg C per carbohydrate collected).

The applicability of the proposed procedure was tested on two different environmental samples comprising marine POM and TSP and the results revealed that the isotopic compositions of the individual carbohydrates, in terms of δ 13 C, were in good agreement with the data reported in the literature. Unfortunately, the Δ 14 C values for individual carbohydrates are scarce in literature and to the best of our knowledge, only one study, on marine HMWDOM, has been reported to date. Therefore, we could not directly compare the obtained results to Δ 14 C values of similar samples.

Nevertheless, the obtained data suggested that the monosaccharides exhibit a modern age, further implying the recent synthesis of these compounds and their rapid cycling.

Applying this method to a wide variety of environmental samples comprising marine sediments, riverine organic matter, deep water HMWDOM, soils, and rain may substantially help to improve the understanding of the carbohydrate dynamics and organic matter cycling in all geochemical systems.

Finally, the overall approach highlights the high potential of preparative liquid chromatography for application to the purification of specific compounds after selection of the adequate column(s) for subsequent molecular-level isotopic measurements.

Moreover, future research employing two-dimensional cation-exchange chromatography may prove to be a very useful tool to speed up the whole purification procedure. The final purified compounds (after Ca 2+ →Pb 2+ →Ca 2+ purification) are also indicated with arrows. During the optimization step, a systematic large negative peak was always observed in the chromatograms at 18-20 min (Fig. S1). Previous studies indicated that this peak is probably due to the presence of dissolved oxygen in the sample and can mask the eluted compounds at this time window or cause an increase in the baseline noise [1]. The peak intensity somehow decreased after sample degassing (with pure He) but was still significant at low sample concentrations. We overcame this problem by installing a degassing device (RFIC eluent degasser, Thermo Fisher) before the chromatographic column. We found that this device increases neither the back-pressure of the system nor the retention time of the carbohydrates. To shorten the analysis time of the carbohydrates, no guard columns were used in this study. However, as a precaution, an online polyether ether ketone (PEEK) filter equipped with a stainless-steel frit (pore size 0.5 µm) was placed just before the online degasser. The filter frit was cleaned regularly by sonication in a methanol bath, followed by sonication in ultrapure water, once per month and between each sample purification.

Moreover, to maximize the collection efficiency of carbohydrates, the tubing between the RI detector and the fraction collector was minimized. The delay time between the detector and the fraction collector was calculated using 1 mM of vitamin B 12 without an analytical column. Further adjustments of the working flow rates (0.3 mL min -1 and 0.6 mL min -1 for Na + and Ca 2+ /Pb 2+ columns, respectively) were made with a glucose standard (50 µM). We found that the highest recovery (~80%) of the glucose was obtained with a delay time of 110 s for the Na + column, and 60 s for the Ca 2+ and Pb 2+ columns. These delay times were applied for all peak and fraction collections during the purification procedure either of standards or samples.

Fig. 1 .

 1 Fig. 1. Chromatogram of a standard monosaccharide mixture (1 mM each): (a) Na + column: F 1 (glucose + rhamnose + mannitol)/F 2 (xylitol + sorbitol + fructose + mannose + galactose + xylose)/F 3 (fucose + arabinose + galactosan)/F 4 (levoglucosan + mannosan); (b) Ca 2+ column: F 1 (glucose)/F 2 (xylose + galactose + mannose + rhamnose)/F 3 (fucose + fructose + arabinose)/F 4 (galactosan)/F 5 (mannitol)/F 6 (levoglucosan)/F 7 (sorbitol + xylitol)/F 8 (mannosan); and (c) Pb 2+ column: F 1 (glucose)/F 2 (xylose)/F 3 (galactose + rhamnose)/F 4 (galactosan)/F 5 (arabinose +

  a) and resulted in eight peaks corresponding to fractions F 1 -F 8 , which were collected into 6 mL pre-combusted (450 °C, 6 h) glass tubes. The obtained results indicated that at this stage of the purification process only the F 8 fraction (e.g. mannosan) was pure because it was eluted significantly after the other monosaccharides (Fig.S2 a). Therefore, this monosaccharide was not purified further. The collected fractions F 1 -F 7 were freeze dried, redissolved into 300 µL ultrapure water, and injected individually into the Pb 2+ column (Fig.S2 b).

Fig. 2 .

 2 Fig. 2. Procedural flowchart of this study with a simplified example of the purification

Fig. 3 .

 3 Fig. 3. Chromatogram of a marine particulate organic matter (POM) sample on a Na +

  alkanes (C 23 -C 33 : -26.7 to -28.5‰) n-alkanols (C 22 -C 32 : -23.9 to -30.4‰), and longchain n-alkanoic acids (C 22 -C 32 : -22.6 to -27.4‰) h h w Af ca u over the Atlantic Ocean [41].

Fig. 4 .

 4 Fig. 4. Chromatogram of the total suspended atmospheric particles (TSP) on a Ca 2+

Fig. 1 .

 1 Fig. 1. Chromatogram of a standard monosaccharide mixture (1 mM each): (a) Na +

Fig. 2 .

 2 Fig. 2. Procedural flowchart of this study with a simplified example of the purification

Fig. 3 .

 3 Fig. 3. Chromatogram of a marine particulate organic matter (POM) sample on a Na +

Fig. 4 .

 4 Fig. 4. Chromatogram of the total suspended atmospheric particles (TSP) on a Ca 2+

Fig. S1 .

 S1 Fig. S1. Comparison of chromatograms with and without online degasser 46

Fig. S1 .

 S1 Fig. S1. Comparison of chromatograms with and without online degasser

Fig. S2 :

 S2 Fig. S2: Example of a standard monosaccharide mixture (50 µM each) purification on (a) Ca 2+ column: F 1 (glucose)/F 2 (galactose + mannose)/F 3 (fructose + arabinose)/F 4

Fig. S3 .

 S3 Fig. S3. Extracted ion chromatograms corresponding to the analysis of underivatized

  ). The standard stock solutions of the individual (1 mM) and a mixture of 14 monosaccharides (glucose, galactose, mannose, fucose, rhamnose, arabinose, xylose, fructose, xylitol, sorbitol, mannitol, levoglucosan, mannosan, and galactosan, 1 mM and 50 µM each) were

	centrifugation (2000 rpm) was then transferred into a pre-combusted (450 °C, 6 h) glass
	vial and the acid was removed from the sample by three successive lyophilizations. The
	afforded dry powder was weighed (25.61 mg), redissolved in 1 mL ultrapure water and
	prepared by dilution with ultrapure water. The prepared solutions were stored in the filtered through a Pasteur pipette packed with quartz wool (both pre-combusted at 450
	a k a -15 °C until use. HCl (37%, Sigma-Aldrich), diluted with ultrapure water (final °C for 6 h) to remove any remaining particles prior to chromatographic injection.
	concentration of 1 M), was used for sample hydrolysis. The ultrapure water used in this 2.2.2. TSP
	work was produced by a Millipore Milli-Q system (Molsheim, France). The aerosol sample was collected on a pre-combusted (450 °C, 6 h) weighed
	2.2. Sampling and carbohydrate extraction Whatman quartz fiber filter (20.3 cm × 25.4 cm) using an automatic sampler (Tisch
	Environmental USA; flow rate 85 m 3 h -1 ). The sample was collected from the 10th to
		2.2.1. Marine POM
	17th March, 2016, from the rooftop of the Endoume marine station (Marseille; 43° 16'
	N -5° 21' E). After collection, the sample was dried for 24 h in a desiccator, weighed,
	and then stored in a freezer at -25 °C in pre-combusted aluminum foil (450 °C, 6 h).
	ga c ca b (OC) c	f h pa cl wa	h a g 20-29%. To obtain
	uff c	ma al f	p c a aly	f	p	(40-60 mg ach) f each of the
	five samples obtained from the respective collection chambers of the sediment trap were
	pooled together. This resulted in ~265 mg dry POM powder which was hydrolyzed with
	1 M HCl at 100 °C for 20 h [50]. The acid-soluble fraction recovered after

Sinking particles (marine POM) were collected over seven-day periods from January 6th to March 3th, 2013, in the upwelling system located offshore Lima (Peru) in the Pacific Ocean (12° 02' S -77° 40' W), using sediment traps (PPS3, Technicap) deployed in the oxycline/upper oxygen minimum zone (OMZ) layer at a depth of 34 m

[START_REF] Bretagnon | Modulation of the vertical particles transfer efficiency in the Oxygen Minimum Zone off Peru[END_REF]

. To avoid POM bio-degradation, a solution of seawater with 5% formaldehyde was added to the bottom of the collection chamber. After trap recovery, the living and dead swimmers were carefully removed so that only detrital particles remained in the sample. These detrital particles (marine POM) were stored in the dark at 4 °C in the initial chambers used in the PPS3. On land, the samples were filtered through 25 mm pre-combusted (450 °C, 6 h) Whatman GF/F filters (nominal retention size, 0.7 µm), freeze-dried, and subsequently stored in the dark at 4 °C until further analysis. The

Table 1 .

 1 Carbon content (µg), δ 13 C (‰) a  14 C(‰) alu f h p c u al bla k . Ph hal c acid was added to the collected time window of the three monosaccharide samples and the ultrapure water sample to make the measurement feasible. The mass of phthalic acid was adjusted according to the sample mass of the respective time window of the examined environmental samples.

	Blank	Carbon ± SD	δ 13 C ± SD	 14 C ± SD
	Na + column (n = 3) *	28.64 ± 1.57	-28.3 ± 0.8	ND
	Ca 2+ column (n = 3) *	4.84 ± 0.25	-28.4 ± 0.4	ND
	Pb 2+ column (n = 3) *	2.80 ± 0.21	-27.8 ± 0.4	ND
	Na + → Ca 2+ → Pb 2+ →Ca 2+ columns (n = 3)	19.76 ± 1.80	-27.9 ± 1.1	ND
	Retention time window of glucose (n = 3) **	0.58 ± 0.00	-24.4 ± 2.9	-986.2 ± 5.8 §
	Retention time window of galactose (n = 2) **	< 2 ± 2.0 †	ND	-988.2 ± 3.1
	Retention time window of levoglucosan (n = 3) **	0.54 ± 0.15	-26.7 ± 1.5	-988.2 ± 0.9 ‡

Table 2 .

 2 δ 13 C (‰) ± SD a ∆ 14 C (‰) ± SD values of the examined environmental samples. h ∆ 14 C values of galactose, glucose and levoglucosan are blank-corrected.

	Sample	Bulk OM §	Hydrophilic extract OM §	Pure monosaccharides
		δ 13 C / ∆ 14 C	δ 13 C/ ∆ 14 C		δ 13 C	∆ 14 C ± 1 
		(n = 3)	(n = 3)		(n = 3)
				Mannose	-17.6 ± 0.9
	POM	-19.6 ± 0.6 / 28 ± 8	-17.2 ± 0.5 / 124 ± 5	Xylose Fuc./Ara. § § Galactose Glucose	-16.8 ± 0.2 -18.5 ± 0.5 -16.9 ± 0.1 -17.6 ± 0.1
				Levoglucosan	-27.2 *
				Mannosan	-26.2 *

Table of Contents S 1. Optimization of the HPLC-RI system 44 S. 2. Supplementary figures and table legends 46

 of

Acknowledgments

This research was funded by the projects AIOLOS, TRACFIRE (Labex OT-Med; ANR-11-LABEX-0.061) and MANDARINE (grant No 2008-10372; Région Provence Alpes Côte d'Azur). The project leading to this publication has received funding from European FEDER Fund under project 1166-39417. The authors acknowledge the four anonymous reviewers for valuable comments and fruitful discussions. A. Nouara was supported by a Ph.D. grant from Aix-Marseille University.

Appendix A. Supplementary data

Supplementary data related to this article can be found at https://doi.org/10.1016/j.aca

Table and Figure captions

Table 1. Carbon content (µg), δ 13 C (‰) a  14 C(‰) values of the procedural blanks.

Phthalic acid was added to the collected time window of the three monosaccharide samples and the ultrapure water sample to make the measurement feasible. The mass of phthalic acid was adjusted according to the sample mass of the respective time window of the examined environmental samples. given in Table S3. [START_REF] Cheng | Simultaneous analyses of neutral carbohydrates and amino sugars in freshwaters with HPLC-PAD[END_REF] Table S1: Columns characteristics and analysis conditions used for the HPLC-RI system. 48

Table S2. Monosaccharide purification yields after three sequential purifications (Ca 2+ → Pb 2+ → Ca 2+ ) of a standard monosaccharide mixture (see text) at 50 µM (n = 3).

49

Table S3. Theoretical and measured masses (LC-Q-TOF-MS analysis) recorded as Na + and NH 4 + additives for levoglucosan, glucose and galactose. 49

References 50

S 1. Optimization of the HPLC-RI system

all spectra, the molecular ion was detected as a Na + and NH 4 + additive. More details are given in Table S3.


Sonia Loiacono

Grégorio Crini

Gilles Chanet

Marina Raschetti

Vincent Placet

Nadia Crini
email: nadia.crini@univ-fcomte.fr

Nadia Morin-Crini

Metals in aqueous solutions and real effluents: Biosorption behavior onto a hemp-based felt

Keywords: Hemp, Biosorption, Metals, Wastewater, Batch experiments

niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

The chemical contamination of water by a wide range of metals is a serious environmental problem involving potential human toxicity. Therefore, there is a need to develop technologies that can remove toxic substances including metals from wastewaters.

Biosorption using non-conventional natural materials has been proposed as an economical and effective method to adsorb metals present in aqueous solutions at trace levels over traditional treatment methods such as membrane filtration, evaporation, adsorption onto activated carbons, and ion-exchange or chelation using organic resins. [START_REF] Volesky | Biosorption of Metals[END_REF][START_REF] Volesky | Sorption and Biosorption[END_REF][START_REF] Wase | C Biosorbents for Metal Ions[END_REF][START_REF] Crini | Sorption Processes and Pollution[END_REF][START_REF] Dhankhar | Fungal biosorption -An alternative to meet the challenges of heavy metal pollution in aqueous solutions[END_REF][START_REF] Mudhoo | Removal of heavy metals by biosorption[END_REF][START_REF] Fomina | Biosorption: current perspectives on concept, definition and application[END_REF][START_REF] Gorska | An environmentally friendly method for removal and recovery of precious metals[END_REF][START_REF] Vandenbossche | Remediation of heavy metals by biomolecules: A review[END_REF] An important class of new materials is based on the use of hemp, an annual high yielding industrial crop grown for its fibers and seeds. Hemp is an interesting raw material because of its low-cost, readily availability, particular chemical composition of its fibres, mainly cellulose (over 75%), and for its versatility. [START_REF] Bouloc | Hemp: Industrial production and uses[END_REF] Applications are found in numerous sectors of industry, including pulp and paper, textiles, construction, automotive applications, composites (plastic, packaging), agrochemistry, food-chemistry, cosmetics and essential oils, pharmacy and medicine, adhesives and biotechnology. In recent years, hemp-based materials have been proposed for metal ion removal from aqueous synthetic solutions. Abundant data on this topic can be found in the literature. [START_REF] Tofan | Sorption studies of Ag(I), Cd(II) and Pb(II) ions on sulphydryl hemp fibers[END_REF][START_REF] Păduraru | Investigations on the possibility of natural hemp fibers use for Zn(II) removal from wastewaters[END_REF][START_REF] Tofan | Cobalt(II) removal from aqueous solutions by natural hemp fibers: batch and fixed-bed columns studies[END_REF][START_REF] Tofan | Fixed bed columns study on the removal of chromium (III) ions from aqueous solutions by hemp fibers with improved sorption performance[END_REF][START_REF] Tofan | Zinc remediation of aqueous solutions by natural hemp fibers: batch desorption/regeneration study[END_REF][START_REF] Pejić | Biosorption of heavy metal ions from aqueous solutions by short hemp fibers: Effect of chemical composition[END_REF][START_REF] Pejić | Mathematical modeling of heavy metal ions (Cd 2+ , Zn 2+ and Pb 2+ ) biosorption by chemically modified short hemp fibers[END_REF][START_REF] Rezić | Cellulosic fibers -Biosorptive materials and indicators of heavy metal pollution[END_REF][START_REF] Kyzas | Low-cost hemp biomaterials for nickel ions removal from aqueous solutions[END_REF] However, these published works focus on the removal of metals present in synthetic solutions using hemp in fibre form.

In this study, we propose the use of a hemp-based material in the form of felt to treat metals present in monometallic aqueous solutions or in polycontaminated effluents. The metal studied are: Al, Cd, Co, Cr, Cu, Fe, Mn, Ni, Pb and Zn. They are chosen for two reasons: firstly these metals (except Cd) are studied due to their common presence in discharge waters from the metal industry, and secondly, six metals (Cd, Cu, Cr, Ni, Pb and Zn) are included in the list of priority pollutants defined by the French Water Agency (French Circular, July 27, 2015). Here different controlling experimental conditions are investigated and discussed. The main parameters studied were initial metal concentration, effect of counter-ions (sulfate, nitrate and chloride metallic salts) biosorbent dosage, contact time and pH. We also report the performance of hemp-based felt to treat a mixture of metals present in industrial effluents.

This article is protected by copyright. All rights reserved.

Accepted Article

Material and methods

Material

The hemp-based material was provided by the French company Eurochanvre (Haute-Saône, France). The hemp felt was made of 100% fibre. Its characteristics are reported in Table 1. Before use, the material was extensively washed in reverse osmosis (RO) water (pH = 5.0 ± 0.1) and then dried at 60°C to constant weight. were dissolved in RO water to obtain a stock solution containing about 300 mg/L (except for Pb for which the concentration of the stock solution was 25 mg/L due to its poor solubility).

Solutions at lower concentrations (1, 10, 25, 50, 100, 150 and 200) were obtained by dilution of the stock solution. All the solutions were analyzed prior to each experiment.

Biosorption of metals by batch technique

Metal removal from solutions (spiked solutions or real effluents) by biosorption on hemp materials was studied by a batch method without changing the initial pH of the solution (pH = 5.0 ± 0.1). In each experiment, a fixed mass of felt was introduced into a given metal solution (100 mL) at a known concentration (for spiked solutions) in a tightly closed flask. The mixture was then mechanically stirred for a given time on a rotating shaker at constant speed (250 rpm) and at room temperature (21 ± 1°C). After treatment, the hemp felt was then easily removed and the concentration of the solution was determined by inductively coupled plasma atomic emission spectroscopy (ICP-AES, ThermoFisher, iCAP 6500, Courtaboeuf, France).

The biosorption performance was finally calculated and expressed as percentage abatement (%R). In controlled conditions, initial metal concentration (1, 10, 25, 50, 100, 150, 200 and 300 mg/L) in the solution, biosorbent dosage (0.25, 0.5, 1, 1.5 and 2 g) and pH solution (between 1 -6) were varied to investigate their effect on the biosorption capacity. Studies were also conducted for various time intervals (ranging from 1 -60 min) to determine when biosorption equilibrium was reached. All experiments (controlled conditions or real effluents)

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were run in triplicate and found reproducible (experimental error within 3%). Blanks were run without any material to determine the extent of metal removal by containers (no adsorption was observed). It was noted that, at the end of the biosorption process, a slight pH variation of between 0.5 and 0.8 units and between 0.6 and 0.7 units was observed for spiked solutions and real effluents in each experiment, respectively.

Analytical methods

Pollutants were assayed by a certified laboratory following a standard protocol and analyzed by ICP-AES [START_REF] Euvrard | Monitoring metal ions present in the effluent discharged from a surface treatment plant: Analytical results[END_REF] and results expressed in mg/L. When the concentrations were lower than the quantification limits (QL) or the detection limits (DL), the values of these limits were taken into account to calculate the percentage abatement. The pH was measured using a portable pH-meter (model 3110 WTW, Alès, France).

To evaluate changes in the surface morphology of the biosorbent, scanning electron microscopy images (ESEM FEI Quanta 450) with a tungsten filament voltage from 15 keV to 20 keV and low-vacuum conditions were performed. Elemental analysis of the fibre surfaces was also performed using a probe for energy-dispersive X-ray spectroscopy (EDS) and electron beam excitation (with a voltage from 15 keV to 20 keV).

Industrial effluents

Eight industrial effluents (abbreviated RE for real effluents) were collected for two sampling campaigns (four samples for each campaign taken on four consecutive days, representative samples of a daily activity) from the VMC Pêche metal-finishing factory located in Morvillars (Territoire de Belfort, France). This factory specializes in hook design and production and uses large volumes of water and chemical products, generating volumes of raw effluents about ∼40 m 3 per day, processed by a physicochemical treatment. This involves several successive steps (coagulation, precipitation, electrocoagulation, flocculation, settling and filtration). The characteristics of eight RE (collected after the first precipitation) are shown in Table 2. The values (mg/L) described in this table represent the minimal and maximal concentrations obtained for the 8 effluents studied. For this industry, discharge standards should be: Fe <5 mg/L; Al 5 mg/L; Zn 2 mg/L, Ni 1 mg/L; Cr 0.5 mg/L, and Cu 0.5 mg/L.

Table 2

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Ecotoxicological tests

To determine the toxicity of the effluents, a standardized bioassay based on the mobility of Daphnia magna was used. [START_REF] Druart | Chemical and ecotoxicological monitoring of discharge water from a metal-finishing factory[END_REF] The toxicity of five samples before and after treatment were studied. Their values were expressed by the EC50, effective concentration corresponding to 50% inhibition compared to controls (given in percentage of effluent).

Results and discussion

Effect of initial metal concentration

First, to study the effect of the initial metal concentration on biosorption performance, a series of freshly prepared metal ion solutions was used (solutions prepared with metal sulfate salts). Figure 1 shows the influence of initial metal concentration on the removal of 10 metals in mono-component system (concentrations of 1, 10, 25, 50, 100, 150, 200 and 300 mg/L for Al, Cd, Co, Cr, Cu, Fe, Mn, Ni and Zn, and only 1, 10 and 25 mg/L for Pb due to its poor solubility). The volume of solution, biosorbent dose and the contact time used in these experiments were 100 mL, 1 g and 60 min, respectively. The results described in Figure 1 show that, up to 25 mg/L, hemp exhibited interesting adsorption capacities (R > 60%) with the following affinity: Pb > Cd > Cu > Zn > Co ∼ Ni ∼ Fe ∼ Cr ∼ Al ∼ Mn. At 25 mg/L, the abatement values for Pb, Cd, Cu, Zn, Co, Ni, Fe, Cr, Al and Mn were 96, 88, 84.5, 77, 70, 68, 67, 67, 66 and 63, respectively. As expected, when the metal concentration was increased, biosorption efficiency decreased, due to the saturation of the biosorption sites on the material.

For example, for Cd and Cu, when the initial metal concentration was increased from 10 -300 mg/L, the removal decreased from 96 to 35% and 91 to 24%, respectively.

From Figure 1, at any concentration, the same order of abatement was obtained for all metals studied, so, the affinity was independent of metal concentration. For the concentration range studied (50 -300 mg/L), Cd showed the highest affinity (between 80% and 35%) whereas Mn demonstrated the lowest level of removal (between 48 and 13%). Cu also showed interesting affinities (between 78 and 24%). The difference in the degree of adsorption may be attributed to the physical and chemical characteristics of each metal such as ionic radius, electronegativity and molar mass (Table 3). The ionic radii, molar mass and Allred-Rochow electronegativities decrease in the order Pb > Cd > Mn > Zn ∼ Fe > Cu ∼ Co > Ni > Cr > Al; Pb > Cd > Zn > Cu > Ni ∼ Co > Fe > Mn > Cr > Al, and Cu ∼ Ni ∼ Co > Fe ∼ Mn ∼ Zn > Cr ∼ Pb > Cd ∼ Al, respectively. However, the two largest ions, lead and cadmium, for which This article is protected by copyright. All rights reserved.

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affinities were high, indicated that the size of ions, the molar mass and the electronegativity were not the only determining parameter in the adsorption process. Similar conclusions were reached by Pejić et al. [START_REF] Pejić | Biosorption of heavy metal ions from aqueous solutions by short hemp fibers: Effect of chemical composition[END_REF][START_REF] Pejić | Mathematical modeling of heavy metal ions (Cd 2+ , Zn 2+ and Pb 2+ ) biosorption by chemically modified short hemp fibers[END_REF] and Kyzas et al. [START_REF] Kyzas | Low-cost hemp biomaterials for nickel ions removal from aqueous solutions[END_REF] . Table 3 Figure 1.

Effect of counter-ions on metal biosorption

The effect of counter-ions in a biosorption system is an important issue as real effluents can contain anions other than sulfate, such as nitrate and chloride. The effect of counter-ions on the biosorption of Ni and Co by hemp is represented in Figure 2. For these two metals no significant differences in the abatement were observed for the three salts used (sulfate, chloride and nitrate). Similar results were obtained for other metals.

Effect of biosorbent dose

Metal biosorption on hemp material was studied by changing the amount of biosorbent (0.25, 0.5, 1, 1.5, and 2 g) added to sample at different initial metal concentrations (25, 50, 100, 150, 200 and 300 mg/L) while maintaining the volume (100 mL), the contact time (60 min) and the agitation speed (250 rpm) constant. The results (Figure 3) showed that metal removal increased with the mass of biosorbent reaching equilibrium between 1 g (for Cd, Cu and Pb) and 1.5 g (for Zn, Ni, Co, Mn, Fe, Cr, Al) of added material. As expected, this overall trend corresponds to the increase of the interactions between biosorbing material and metals, due to the higher biosorbent surface area, and thus the available active sites.

Effect of contact time

Biosorption of the metal ions on hemp was studied over a range of time (1-60 min) and three concentrations (5, 25 and 100 mg/L) whilst maintaining other parameters constant. The plots (Figure 4) representing the metal abatement can be split in three distinct regions: the first indicates instantaneous biosorption of the metal within 5 min of contact time suggesting rapid surface adsorption and external diffusion; the second shows a gradual equilibrium (between 5 -10 min) and the third (after 10 min) indicates the equilibrium state. The kinetics of biosorption were fast and independent of the metal concentration with equilibrium reached in This article is protected by copyright. All rights reserved.

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10 min for all metals. It is also interesting to note that the plots are single, smooth and continuous, leading to saturation, suggesting the possible monolayer coverage of metal on the surface of the biosorbent, and this could confirm the applicability of the Langmuir model. Indeed, interactions involved were analyzed through classical kinetic and equilibrium models used in the literature. The experimental data suit pseudo-second order kinetics with three biosorption stages (data not published). The Langmuir equilibrium model predicted the following maximum capacities (q max mg/g) of 27.47, 14.64, 10.59, 7.99, 7.85, 7.87, 6.53, 6.38 and 4.55 mg/g biosorbent for Cd, Cu, Zn, Co, Fe, Ni, Cr, Al and Mn, respectively. A direct comparison of literature data using different hemp materials is not possible since both biosorbents and experimental conditions are different. Nevertheless, it was observed that the q max values of the proposed biosorbent were comparable to those of the other available hempbased materials for metal removal (Table 4).

Effect of pH

The effect of solution pH on metal abatement was studied by varying the initial pH between 1 -6, keeping the other parameters constant. The pH behavior of all metals was similar (Figure 5). The percentage removal was strongly pH-dependent in the pH range 1-4 whereas the performances were independent between pH 4 and 6. At pH lower than 3, the abatements decreased drastically due to the competition of H + , except for Pb and Cu. A similar trend was reported for the adsorption of Ag, Cd, Co, and Zn by hemp fibres by Păduraru and Tofan [START_REF] Păduraru | Investigations on the possibility of natural hemp fibers use for Zn(II) removal from wastewaters[END_REF] , Tofan et al. [START_REF] Tofan | Cobalt(II) removal from aqueous solutions by natural hemp fibers: batch and fixed-bed columns studies[END_REF][START_REF] Tofan | Fixed bed columns study on the removal of chromium (III) ions from aqueous solutions by hemp fibers with improved sorption performance[END_REF] , Pejić et al. [START_REF] Pejić | Biosorption of heavy metal ions from aqueous solutions by short hemp fibers: Effect of chemical composition[END_REF][START_REF] Pejić | Mathematical modeling of heavy metal ions (Cd 2+ , Zn 2+ and Pb 2+ ) biosorption by chemically modified short hemp fibers[END_REF] , and Rezić. 18

Elimination and regeneration

Like all agricultural materials, after biosorption the felt as a low cost material can be incinerated to a much smaller volume of ash and/or recovery of biosorbed metal. [START_REF] Loiacono | Simultaneous removal of Cd, Co, Cu, Mn, Ni and Zn from synthetic solutions on a hempbased felt: Experimental design[END_REF] Regeneration is another solution: since the interactions between the metal and material were driven by weak interactions, acidic solutions can be used for the regeneration. Thus using a 0.1 mol/L HCl solution, the material was easily regenerated. After several adsorptionregeneration cycles (n = 5), the performance remained unchanged, showing the good reproducibility of the data.

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Test with real effluents

Table 3 reported the minimal and maximal values obtained during the two campaigns of sampling. For each campaign, four effluents, each representative of a whole day's activity of the plant, were sampled on 4 consecutive days. The pH values of the effluents were found to be relatively stable at around pH = 4.9 ± 0.1, which was the mean value for eight samples. Analytical monitoring of these effluents showed that 14 metals were found at least once: Al, Cd, Co, Cu, Cr, Fe, Li, Mn, Ni, Pb, Sn, Sr, Ti, and Zn. Of the 14 metals founds, 8 systematically occurred at detectable levels, i.e. Al, Co, Cu, Cr, Fe, Mn, Ni and Zn. Other metals such as Ag, As, Ba, Hg, Mo, Pd, Pt, Sb, Se, Tl, V, and W were below the QL. Eight other substances were identified and quantified for the two campaigns: Ca, K, Mg, Na, P, S, Si, and B. These results demonstrated that real effluents contained a high inorganic load. It is also important to note that the metal load was quantitatively variable (Table 3).

The concentrations of the substances obtained after treatment by biosorption are presented in Table 5. These results demonstrated that high levels of metal removal were attained with the use of hemp, allowing water quality criteria regarding industrial discharge waters to be achieved. This treatment led to the almost total elimination of metals such as Cd, Pb, Cu, and Ni. The treatment also lowered Fe, Zn, Co, Cr, Al, and Mn (campaign 1) by more than 85%, 84%, 69%, 68%, 43% and 26%, respectively. Certain substances such as Na, P, S and B were also eliminated by the process.

The performances of the based-hemp material toward pollutants present in real effluents can be explained by the presence of interactions between the metallic ions and the felt through physisorption (surface adsorption) and chemisorption (electrostatic interactions, precipitation) mechanisms. [START_REF] Păduraru | Investigations on the possibility of natural hemp fibers use for Zn(II) removal from wastewaters[END_REF][START_REF] Pejić | Biosorption of heavy metal ions from aqueous solutions by short hemp fibers: Effect of chemical composition[END_REF][START_REF] Kyzas | Low-cost hemp biomaterials for nickel ions removal from aqueous solutions[END_REF][START_REF] Loiacono | Simultaneous removal of Cd, Co, Cu, Mn, Ni and Zn from synthetic solutions on a hempbased felt: Experimental design[END_REF][START_REF] Loiacono | Simultaneous removal of Cd, Co, Cu, Mn, Ni and Zn from synthetic solutions on a hemp-based felt. 2: Chemical modification[END_REF][START_REF] Balintova | Sorption in acidic environment -Biosorbents in comparison with commercial adsorbents[END_REF][START_REF] Anirudhan | Removal of Cd(II) ions from aqueous solution using a cation exchanger derived from banana stem[END_REF][START_REF] O'connell | A modified cellulose adsorbent for the removal of nickel(II) from aqueous solutions[END_REF] It was noted that, at the end of the biosorption process, a slight pH variation between 0.6 and 0.7 units was observed in each experiment, indicating a chemisorption mechanism. To confirm this and to evaluate changes in the surface morphology of the biosorbent, SEM images and elemental analysis using EDS were performed. [START_REF] Loiacono | Simultaneous removal of Cd, Co, Cu, Mn, Ni and Zn from synthetic solutions on a hemp-based felt. 2: Chemical modification[END_REF] The SEM images indicated changes in the morphology of hemp after biosorption (Figure 6). SEM and EDS studies of raw and loaded biosorbent also confirmed the involvement of precipitation in the biosorption process. After biosorption, three of the ten metals (i.e. Al, Fe, and Zn) systematically encountered in the effluents and all the other substances except B (i.e. Ca, K, Mg, Na, P, S, Si) were detected using EDS at the surface of the fibres constituting the felt. In particular, it is interesting to note also the presence, in the EDS spectra (Figure 6c), of This article is protected by copyright. All rights reserved.

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agglomerate make up of S, a substance present in real effluents. Thus the felt was also useful to eliminate substances other than metals.

Table 5 Figure 6.

Ecotoxicological analysis of raw and treated effluents

Toxicity tests were carried out by applying a standard test to discharged industrial effluents and to the same discharged waters after biosorption treatment. The toxicity values of five effluents described in Table 6 showed that effluents were relatively toxic for Daphnia magna. EC50 values ranged from 5 for sample E2 to 44 for sample E3. According to a French guide on the use of bioassays for the quality assessment of environmental samples [START_REF] Crini | Sorption Processes and Pollution[END_REF][START_REF] Druart | Chemical and ecotoxicological monitoring of discharge water from a metal-finishing factory[END_REF] , the effluents were classified between toxic (EC50 < 50%) and very toxic (EC50 < 10%). After biosorption, the impact on D. magna was reduced indicated by higher EC values (Table 6). Thus removal of pollutants by the treatment obviously decreased the water toxicity. More experiments are in progress to confirm these chemical and biological results. Table 6.

Conclusion

In this study, the potential of a hemp-based felt for biosorption of metals from aqueous solutions and real effluents was explored in a batch system. In controlled conditions, the effect of process parameters such as biosorbent dose, metal concentration, contact time and pH was examined. The optimum values for biosorbent dosage, contact time and pH for metal biosorption were 1.5 g, 10 min and 5, respectively. High adsorption capacities were found using these conditions. The maximum adsorption capacities for Cd, Cu, Zn, Co, Fe, Ni, Cr, Al and Mn were 27.47, 14.64, 10.59, 7.99, 7.85, 7.87, 6.53, 6.38 and 4.55 mg/g of hemp, respectively. Good results were also obtained for real polymetallic effluents containing concentrations ranging from: Fe 6.9 to 24.5 mg/L; Ni 5.55 to 14.3 mg/L; Zn 3.3 to 11.6 mg/L; Al 1.1 to 9.6 mg/L; Cu 0.43 to 5.5 mg/L, and Cr 0.35 to 2.1 mg/L. The hemp was able to remove, in most cases 75-80% of metal load, allowing water quality criteria regarding industrial discharge waters containing these six metals to be achieved. Ecotoxicological tests also confirmed the efficiency of the biosorption to radically decrease the effluent toxicity. The study of the interactions involved in the biosorption process was studied by employing SEM This article is protected by copyright. All rights reserved.

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and EDS techniques which revealed that both surface interactions and precipitation participate in metal removal from aqueous solutions. The next step will consist of carrying out a semiindustrial scale test to investigate the feasibility of its implementation on an industrial scale.

Tables

Cd <QL-0.25 (0. This article is protected by copyright. All rights reserved. Adsorption capacities (q max in mg/g) for various metals using hemp. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

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Figure 2 .

 2 Figure 2.

Figure 3 .

 3 Figure 3.

Figure 4

 4 Figure 4. Table 4

Figure 5 .

 5 Figure 5.

Figure 1 .

 1 Figure 1. Effect of initial metal concentration on metal removal (expressed in % removal) onto hemp-based felt (solutions prepared with metal sulfate salts; conditions: biosorbent dose 1 g, volume of solution 100 mL, contact time 60 min, agitation speed 250 rpm, pH = 5, temperature 20°C, and n = 3).

Figure 2 .

 2 Figure 2. Effect of counter-ions on biosorption of Ni and Co ions (expressed in % removal) by hemp-based felt using different metal salts (conditions: biosorbent dose = 1 g, volume of solution = 100 mL, contact time = 60 min, agitation speed = 250 rpm, pH = 5, temperature = 20°C, and n = 3).

Figure 3 .

 3 Figure 3. Removal (%) vs. hemp dose at different initial metal concentrations (others conditions: volume of solution 100 mL, contact time 60 min, agitation speed 250 rpm, temperature 20°C, pH = 5, and n = 3).

Figure 4 .

 4 Figure 4. Kinetics of metal removal (%) by hemp at three different initial metal concentrations (others conditions: adsorbent dose 1 g, volume of solution 100 mL, agitation speed 250 rpm, temperature 20°C, pH = 5, and n = 3).

Figure 5 .

 5 Figure 5. Influence of pH on the abatement of ten metals (expressed in % removal) by hemp (conditions: adsorbent 1 g, volume of the solution 100 mL, initial metal concentration 25 mg/L).

Figure 6 .

 6 Figure 6. SEM images (left) and EDS (right) spectra of the felt before (a) and after (b,c) biosorption. The red arrow on the SEM image (c) shows agglomerates at the surface of the fibre.

Figure 1 .

 1 Figure 1. Effect of initial metal concentration on metal removal (expressed in % removal) onto hemp-based felt (solutions prepared with metal sulfate salts; conditions: biosorbent dose 1 g, volume of solution 100 mL, contact time 60 min, agitation speed 250 rpm, pH = 5, temperature 20°C, and n = 3).

Figure 2 .

 2 Figure 2. Effect of counter-ions on biosorption of Ni and Co ions (expressed in % removal) by hemp-based felt using different metal salts (conditions: biosorbent dose = 1 g, volume of solution = 100 mL, contact time = 60 min, agitation speed = 250 rpm, pH = 5, temperature = 20°C, and n = 3).

Figure 3 .

 3 Figure 3. Removal (%) vs. hemp dose at different initial metal concentrations (others conditions: volume of solution 100 mL, contact time 60 min, agitation speed 250 rpm, temperature 20°C, pH = 5, and n = 3).

Figure 4 .

 4 Figure 4. Kinetics of metal removal (%) by hemp at three different initial metal concentrations (others conditions: adsorbent dose 1 g, volume of solution 100 mL, agitation speed 250 rpm, temperature 20°C, pH = 5, and n = 3).

Figure 6 .

 6 Figure 6. SEM images (left) and EDS (right) spectra of the felt before (a) and after (b,c) biosorption. The red arrow on the SEM image (c) shows agglomerates at the surface of the fibre.

  [START_REF] Bugnet | Hemp decontamination of poly-metallic aqueous solutions[END_REF]

Table 1 2

 1 .2. Metals Al 2 (SO 4 ) 3 .16H 2 O, CdSO 4 .8H 2 O, CdCl 2 , CoSO 4 .7H 2 O, CoCl 2 .6H 2 O, Co(NO 3 ) 2 .6H 2 O, CrK(SO 4 ) 2 .12H 2 O, CuSO 4 .5H 2 O, CuCl 2 .2H 2 O, FeSO 4 , MnSO 4 .4H 2 O, MnCl 2 .2H 2 O, NiSO 4 .6H 2 O, NiCl 2 .6H 2 O, Ni(NO 3 ) 2 , PbSO 4 , and ZnSO 4 .7H 2 O were purchased from Sigma-Aldrich (France) and used without further purification. Appropriate weights of each metal

Table 1

 1 Characteristics of hemp-based material.

	Thick	about 5 mm
	Cellulose	75%
	Hemicellulose	15%
	Lignin	3%
	Pectins	5%
	%C	41.4
	%N	0.28
	%S	0.06

Table 2

 2 Minima and maxima (± standard deviation) for several pollutants over four real effluents (RE) during the two sampling campaigns (four samples taken on four consecutive days, representative samples of a daily activity; the results are expressed in concentration in mg/L; pH measured at 20°C).

	Parameter / Pollutant	Campaign 1	Campaign 2
	Samples	RE1, RE2 RE3, RE4	RE5, RE6 RE7, RE8

Table 3

 3 Metal ion characteristics.

		Pb(II)	Cd(II)	Cu(II)	Zn(II)	Ni(II)	Co(II)	Fe(II)	Cr(III)	Al(III)	Mn(II)
	Allred-Rochow EN 1	1.55	1.49	1.75	1.66	1.75	1.7	1.64	1.56	1.47	1.6
	MW 2	207.2	112.4	63.5	65.3	58.7	58.9	55.85	51.99	26.9	54.9
	Ionic radius 3	120	97	72	74	69	72	74	63	51	80
	1 Allred-Rochow electronegativity
	2 molar mass in g/mol
	3 in pm

Table 4

 4

Table 5

 5 Comparisons of the level of several pollutants present in the industrial effluents before (raw effluents) and after treatment (treated effluents) by biosorption on hemp (the results are expressed as a concentration range deviation minima and maxima (± standard deviation) and as a % mean abatement ± standard deviation for the 4 samples of the two campaigns; the concentrations are expressed in mg/L; pH measured at 20°C).

			Campaign 1			Campaign 2
	Samples	raw	treated		raw	treated
	Parameter / Pollutant	concentration	concentration	abatement	concentration	concentration	abatement
	pH	4.7-5.1 (0.2)	5.3-5.8 (0.2)		4.6-5.1 (0.2)	5.2-5.8 (0.3)
	Al	1.99-9.6 (3.5)	1.25-4.87 (1.7)	43±5	1.1-3.74 (1)	0.6-2.12 (1)	49±7
	Cd	<QL-0.25 (0.1)	<QL	100	<QL-0.16 (0.07)	<QL	100
	Co	3.36-7.55 (2.4)	0.8-3.36 (1.1)	69±6	7.3-13.34 (2.3)	2.17-5.01 (1.3)	64±5
	Cr	0.35-2.1 (0.8)	0.096-0.58 (0.3)	68±6	0.45-0.74 (0.1)	0.13-0.21 (0.1)	72±2
	Cu	0.97-5.5 (2.3)	0.035-0.51 (0.2)	92±3	0.43-1.61 (0.4)	0.02-0.24 (0.1)	92±4
	Fe	8.7-24.5 (7.6)	1.01-4.66 (71.8)	85±3	6.9-14.4 (3)	0.71-1.36 (0.3)	89±1
	Mn	4.8-22.3 (8.3)	3.14-17.9 (6.9)	26±7	3.99-10.1 (2.6)	0.31-0.52 (0.1)	23±10
	Ni	5.55-14.3 (4.1)	0.41-0.74 (0.1)	93±1	5.99-8.1 (0.8)	0.31-0.52 (0.1)	94±1
	Pb	<QL-0.11 (0.05) <QL-0.021 (0.01)	94±11	<QL-0.019 (0.02)	<QL	100
	Zn	5.9-11.6 (2.6)	0.78-1.81 (0.5)	86±1	3.3-9.1 (2.2)	0.57-1.97 (0.61)	84±5
	Li	<QL-0.52 (0.27) <QL-0.09 (0.01)	78±6	<QL-0.1 (0.04)	<QL	100
	Sn	<QL-1.95 (1.02) <QL-1.54 (0.8)	21±2	0.17-0.37 (0.08)	0.11-0.3 (0.08)	20±11
	Sr	<QL-0.91 (0.41) <QL-0.66 (0.2)	41±12	<QL-0.74 (0.27)	<QL-0.33 (0.09)	37±18
	Ti	0.44-3.25 (1.2)	0.21-1 (0.5)	61±13	<QL-0.47 (0.21)	<QL-0.2 (0.01)	41±15
	Ca	438-610 (89)	409-557 (79)	8±1	295-457 (57)	280-420 (57.3)	7±1
	K	22.7-44.6 (9.5)	22-43.9 (9.5)	2±1	19.7-34.3 (5.9)	18.9-33.7 (6.7)	4±1
	Mg	85-210 (64)	80-201 (61)	5±1	75-132 (22)	72-130 (25.9)	4±2
	Na	260-460 (104)	175-370 (102)	24±7	163-209 (16.7)	100-150 (22.5)	30±9
	P	2.98-7.91 (2.5)	1.35-4.11 (1.3)	47±10	1-1.97 (0.4)	0.22-0.77 (0.25)	65±9
	S	9.15-40.5 (12.9)	6.7-31.2 (10.2)	25±2	7.88-29.4 (7.9)	5.35-22.4 (7.3)	30±4
	Si	7.88-31.2 (10.2)	7.44-28.9 (9.4)	7±1	8.5-13.7 (1.9)	7.78-12.4 (1.9)	7±3
	B	21.2-105 (34.9)	24.9-90 (30.5)	18±4	21.2-40 (7.8)	11.8-31.6 (9.4)	33±11

Table 6 .

 6 Total of metals present in solution (in mg/L) before and after biosorption on hemp and toxicity values (expressed by EC50 in percentage of effluent) for Daphnia magna for five samples.

	Sample	Total of metals * present in solution in mg/L	EC50 in percentage of effluent
		raw effluent	treated effluent	raw effluent	treated effluent
	E1	44.51	13.69	31	70
	E2	98.93	35	5	44
	E3	37.7	9.74	44	>90
	E4	86.68	32.21	10	51
	E5	39.8	9.46	23	71

* Al+Cd+Co+Cr+Cu+Fe+Mn+Ni+Pb+Zn+Li+Sn+Sr+Ti

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Acknowledgments

Authors thank the Agence de l'Eau, Région Franche-Comté and FEDER for financial support (NIRHOFEX Program 2013-2017 -'Innovative materials for wastewater treatment').