Nadège Oustriere
email: oustriere.nadege@gmail.com

Lilian Marchand
email: marchand.lilian@gmail.com

Nathalie Lottier
email: nathalie.lottier@univ-orleans.fr

Mikael Motelica
email: mikael.motelica@univ-orleans.fr

Michel Mench
email: michel.mench@inra.fr

Ajit Sarmah

Long-term Cu stabilization and biomass yields of Giant reed and poplar after adding a biochar, alone or with iron grit, into a contaminated soil from a wood preservation site

Keywords: Arundo donax L, Populus nigra L, Soil pore water Copper contamination In situ stabilization Phytomanagement

Biochar with and without iron grit was added in a Cu-contaminated soil.

• Soil pore water: biochar with iron grit reduced its Cu 2 + and Cu contents in year 2 • Both amendments did not improve Giant reed and poplar growth.

• Shoot Cu concentration of poplar and

Giant reed reached common values. • Giant reed can increase the potential Cu leaching out of the root-zone.

Introduction

Soil Cu contamination at wood preservation sites frequently resulted from long-term use of Cu-based salts as wood preservatives and wood washings [START_REF] Bes | Remediation of copper-contaminated topsoils from a wood treatment facility using in situ stabilization[END_REF]Oustriere et al., 2016a). The functioning of ecosystems associated with such Cu-contaminated soils is generally affected, due to soil ecotoxicity and Cu dispersion through natural agencies [START_REF] Bakshi | Biochar amendment affects leaching potential of copper and nutrient release behavior in contaminated sandy soils[END_REF].

Out of gentle options for remediating Cu-contaminated soils, in situ stabilization is an alternative to physico-chemical methods. It aims at reducing the pollutant linkages by managing the contaminant pathways to protect the biological receptors [START_REF] Cundy | Developing effective decision support for the application of "gentle" remediation options: the GREENLAND project[END_REF]. It relies on incorporation of amendments into the soil to immobilize metal(loid)s in the stable solid phase by sorption, precipitation, complexation, ion exchange or redox process, thereby decreasing their mobility and bioavailability [START_REF] Kumpiene | Stabilization of As, Cr, Cu, Pb and Zn in soil using amendmentsa review[END_REF][START_REF] Bolan | Distribution and bioavailability of trace elements in livestock and poultry manure by-products[END_REF]. Amending soils with biochar can (1) increase the cation exchange capacity (CEC), pH, and retention of water and nutrients, (2) improve microbial soil habitats and (3) immobilize contaminants [START_REF] Zhang | Using biochar for remediation of soils contaminated with heavy metals and organic pollutants[END_REF]. Several studies have assessed the biochar effectiveness for in situ stabilization of Cucontaminated soils [START_REF] Beesley | The immobilisation and retention of soluble arsenic, cadmium and zinc by biochar[END_REF]Beesley et al., 2011). As suggested by [START_REF] Beesley | The immobilisation and retention of soluble arsenic, cadmium and zinc by biochar[END_REF], combining amendments with biochar may be more efficient than biochar alone for the remediation and revegetation of contaminated soils. Iron grit mainly consists of zerovalent Fe(0) and Mn and rapidly corrodes into the soil to form newly Fe/Mn oxi(hydro)xides. Such (hydro)xides can sorb metal(loid)s and reduce their (bio)availability [START_REF] Kumpiene | Assessment of aided phytostabilization of copper-contaminated soil by X-ray absorption spectroscopy and chemical extractions[END_REF][START_REF] Tiberg | Immobilization of Cu and as in two contaminated soils with zerovalent ironlongterm performance and mechanisms[END_REF]. In a previous pot experiment, a pine barkderived biochar (1% w/w) decreased Cu 2+ concentration in the soil pore water (SPW) of a contaminated soil (964 mg Cu kg -1 ) from a wood preservation site (Oustriere et al., 2016a). Adding this biochar with iron grit (1% w/w) decreased both total Cu and Cu 2+ concentrations in the SPW. However biochar alone and in combination did not promote the growth of dwarf bean (Phaseolus vulgaris L.). Moreover this study was limited to a 3-month reaction period followed by a 2week plant testing. Potential changes in the phytotoxicity of biocharamended soils over time must be considered. Long-term efficiency of soil amendments is pivotal to sustain metal(loid) immobilization in contaminated soils and to avoid their leaching out of the root zone.

Few long-term studies reported an increased immobilization of trace elements (TE) over time in biochar-amended soils (Cd, Pb: [START_REF] Bian | A three-year experiment confirms continuous immobilization of cadmium and lead in contaminated paddy field with biochar amendment[END_REF]Ni, Zn: Shen et al., 2016). Such long-term effects on TE immobilization however depend on biochar and soil types. After 3 years, hardwoodderived biochar gradually reduced the (CaCl 2 )-extractable Cd and Cu concentrations and uptake by rice, while these values did not change in the soil amended by corn-straw-derived biochar [START_REF] Li | The influence of biochar type on long-term stabilization for Cd and Cu in contaminated paddy soils[END_REF]. Eucalyptus wood-derived-biochar aged for 12 months into two (Cd/As)spiked soils increased Cd and As sorption in the Inceptisol whereas it decreased As sorption in the Oxisol [START_REF] Nagodavithane | Effect of ageing on surface charge characteristics and adsorption behaviour of cadmium and arsenate in two contrasting soils amended with biochar[END_REF]. The sorption capacity of aged biochar may depend on (1) metal(loid)s, (2) SPW pH, and (3) soil properties such as organic matter (OM) content and oxidation process through both abiotic and biotic mechanisms [START_REF] Nagodavithane | Effect of ageing on surface charge characteristics and adsorption behaviour of cadmium and arsenate in two contrasting soils amended with biochar[END_REF].

Among energy crops, poplar (Populus sp.,) and Giant reed (Arundo donax L.) used in short rotation coppices (SRC) have been tested for producing valuable feedstock for bioenergy productions on contaminated soils [START_REF] Lucas-Borja | Microbial activity in soil under fast-growing paulownia (Paulownia elongata × fortunei) plantations in Mediterranean areas[END_REF][START_REF] Evangelou | Assessment of suitability of tree species for the production of biomass on trace element contaminated soils[END_REF][START_REF] Kidd | Agronomic practices for improving gentle remediation of trace element-contaminated soils[END_REF][START_REF] Mola-Yudego | How much yield should we expect from fast-growing plantations for energy? Divergences between experiments and commercial willow plantations[END_REF][START_REF] Gonsalvesh | Valorisation of heavy metals enriched tobacco biomass through slow pyrolysis and steam activation[END_REF]. The lignocellulosic biomass of such plants can be either used to produce heat and electricity by direct combustion, transformed by pyrolysis and gasification into biofuels and biochars or used for producing derived bioproducts notably platform chemicals [START_REF] Bridgwater | Biomass for energy[END_REF][START_REF] Nsanganwimana | Arundo donax L., a candidate for phytomanaging water and soils contaminated by trace elements and producing plant-based feedstock, a review[END_REF]. Poplar is a fast growing tree, producing large yields, tolerant to high TE exposures, with low contaminant accumulation in the wood (Cd/ Zn are mainly accumulated in the leaves) and having high energy potential [START_REF] Calfapietra | Response and potential of agroforestry crops under global change[END_REF]. Arundo donax is a productive species, highly stress resilient (e.g. cold, drought, salinity, and extreme pH) [START_REF] Mantineo | Biomass yield and energy balance of three perennial crops for energy use in the semi-arid Mediterranean environment[END_REF], tolerant to high TE exposure [START_REF] Elhawat | Copper uptake efficiency and its distribution within bioenergy grass giant reed[END_REF], usable by many conversion chains, e.g. energy sector, litter, pulp and building materials [START_REF] Scordia | Bioconversion of giant reed (Arundo donax L.) hemicelluloses hydrolysate to ethanol by Scheffersomyces stipitis CBS6054[END_REF][START_REF] Nsanganwimana | Arundo donax L., a candidate for phytomanaging water and soils contaminated by trace elements and producing plant-based feedstock, a review[END_REF].

In situ stabilization combined with either Populus nigra L. or A. donax cultivation is a potential option to produce a valuable biomass while stabilizing Cu-contaminated soils at wood preservation sites. Therefore, we investigated the long-term aging effect of biochar, alone and combined with iron grit, in such Cu-contaminated soil cultivated with both plant species through (1) Cu and nutrient concentrations in the soil pore water, (2) biomass production of P. nigra and A. donax, and (3) shoot Cu concentrations and removals.

Material and methods

Soil, amendments and soil treatments

The wood preservation site (about 10 ha, Saint-Médard d'Eyrans, Gironde, SW France; 44°43.353′ N, 000°30.938′ W) has been used for over a century, with various Cu-based salts (mainly Cu-sulphate based on the duration and amount used) as wood preservatives [START_REF] Mench | Assessment of ecotoxicity of topsoils from a wood treatment site[END_REF]. Topsoil (Unt, 0-25 cm, Fluvisol -Eutric Gleysol, World Reference Base for soil resources) was collected (100 kg) at the P1-3 sub-site. This sandy soil (85.8% sand, 5.9% clay, and 8.3% silt; 1.3% OM; C/N 16), with a low CEC, is mainly contaminated by Cu (Table 1, Oustriere et al., 2016a), which largely exceeds its median and upper whisker background values in French sandy soils, but its total soil As and Cr are at background levels (Table 1, [START_REF] Baize | Guide des analyses en pédologie[END_REF][START_REF] Villanneau | Détection de valeurs anomaliques d'éléments traces métalliques dans les sols à l'aide du Réseau de Mesure de la Qualité des Sols[END_REF]. Soil was air-dried and homogenized after sieving through a 5 mm mesh.

Two treatments, a pine bark-derived biochar (B, pyrolysis of 420 °C for 180 s; not desalted; Florentaise, Saint-Mars-du-Désert, France) alone and combined with zerovalent iron grit (BZ), were trialed. Biochar was crushed, sieved at 2 mm and manually homogenized. Elemental composition, carbon (C) content and polycyclic aromatic hydrocarbon (PAH) concentrations were determined at the INRA Laboratoire d'Analyses des Sols (INRA LAS, Arras, France) with standard methods (Table 2). Zerovalent iron grit (Z, GH120, particle size b 0.1 mm) was obtained from Wheelabrator Allevard, France [START_REF] Bes | Remediation of copper-contaminated topsoils from a wood treatment facility using in situ stabilization[END_REF]. Amended soils were thoroughly homogenized in large plastic containers and individually prepared prior to use.

The pot experiment consisted in 3 soil treatments: (1) untreated Cucontaminated soil (Unt); (%, w/w) (2) Unt + 1% B (B), (3) Unt + 1% B + 1% Z (BZ). Each treatment was made in 10 replicates. Soils (3 kg) were potted (5 L, plastic pots) and placed in a greenhouse. Potted soils, with a bottom cup to avoid any leaching, were watered and weekly maintained to 70% of water holding capacity (WHC, 10% of air-dried

Plant testing

Stem cuttings of P. nigra and Giant reed (A. donax) (roughly 20 cm long) were collected in May 2013. Poplars were sampled on 4-year old trees growing at this wood preservation site. Giant reed plants were sampled from natural stand along a drainage ditch, San Remo, Italy, and cultivated since 2012 in a greenhouse. Stem cuttings were rooted in individual pots (9 * 8 * 9 cm 3 ) on perlite imbibed with a quarter-strength Hoagland nutrient solution (HNS, [START_REF] Marchand | Intra-specific variability versus constitutive-like tolerance to face Cu exposure in populations of six rooted macrophytes[END_REF] for one year in a greenhouse. For each treatment, one standardized plant of either P. nigra or A. donax was transplanted in potted soils (five replicates) and cultivated during 22 months from June 2014 to March 2016 in a greenhouse. The experiment consisted in 6 treatments:

(1) untreated Cu-contaminated soil planted with P. nigra (Pn) (2) B-amended soil planted with P. nigra (PnB) (3) BZ-amended soil planted with P. nigra (PnBZ) (4) untreated Cu-contaminated soil planted with A. donax (Ad) (5) B-amended soil planted with A. donax (AdB) (6) BZ-amended soil planted with A. donax (AdBZ).

Pots were arranged in a fully randomized block and maintained at 70% of WHC using deionized water without loss from drainage. Hoagland nutrient solution (250 mL) was applied each month in all pots to avoid nutrient deficiencies. Before the beginning of the 2015 growing season, in March, dry shoots of A. donax were harvested (Cut 1), 1 cm above the soil surface. In March 2016, the shoots and roots of P. nigra (Cut 1) and A. donax (Cut 2) were harvested. All harvested biomass were washed twice with deionized water, blotted with filter paper, placed in paper bags and oven dried at 60 °C to constant weight for 72 h and then weighed for determining the shoot and root DW yields.

Soil pore water and plant analysis

Dried shoots were ground (b 1.0 mm particle size, Retsch MM200) then weighed aliquots (0.5 g DW pot -1 ) were wet digested under microwaves (CEM Marsxpress 1200 W) with 5 mL supra-pure 14 M HNO 3 and 2 mL 30% (v/v) H 2 O 2 not stabilized by phosphates. Certified reference material (BIPEA maize V463) and blank reagents were included in all series. Mineral composition (Al, B, Ca, Cu, Fe, Mg, Mn, P, K, Na, and Zn) in digests was determined by ICP-MS (Thermo X series 200) at the INRA USRAVE laboratory, Villenave-d'Ornon, France. All elements were recovered (N95%) according to the standard values and standard deviation for replicates was b5%. Shoot Cu removal was calculated as: Cu (μg plant -1 ) = shoot DW yield (g plant -1 ) x shoot Cu concentration (μg g -1 DW).

23 months after the soil amendment, the soil pore water (SPW) was collected in all pots just before the harvest of P. nigra and A. donax (March 2016, three times 10 mL) using Rhizon MOM moister samplers (Eijkelkamp Agrisearch Equipment, The Netherlands) placed in January 2016 and samples kept at 4 °C prior to their analysis. The pH, electrical conductivity (EC), and Cu 2+ concentration in the SPW samples were determined using electrodes (Hanna instruments, pH 210, combined electrode Ag/AgCl-34, Tetracon 325 WTW, and Cupric ion electrode, Fischer Bioblock, USA), respectively. Aluminum, B, Ca, Cu, Fe, Mg, Mn, P, K, Na, and Zn were analyzed by ICP-OES (Varian Liberty 200). Dissolved organic carbon (DOC) in SPW samples was analyzed with a Shimadzu© TOC 5000A carbon analyzer. The measurement accuracy was checked by performing calibration with a standard reference solution of potassium hydrogen phthalate (KHP) at a concentration of 1000 mg C L -1 . Four DOC measures were performed for each SPW solution. The SPW composition was compared to previous values reported by Oustriere et al. (2016a) for the same unplanted soil treatments 3 months after soil amendment.

Statistical analysis

Influence of soil treatments on SPW parameters, shoot DW yields, shoot ionome and element removals were tested using one-way analysis of variance (ANOVAs). Shoot DW yields and shoot ionome of both plants were tested separately. Normality and homoscedasticity of residuals were met for all tests. When significant differences occurred between treatments, multiple comparisons of mean values were made using post-hoc Tukey HSD tests. Differences were considered statistically significant at p b 0.05. When element concentrations were below the detection limits in the Unt samples, influence of soil treatments were not statistically tested. All statistical analyses were performed using R software (version 3.0.3, Foundation for Statistical computing, Vienna, Austria).

Results

3.1. Soil pore water 3.1.1. pH, EC and nutrient concentrations

The Unt soil has a neutral SPW pH (Table 3). At month 3, biochar alone (B) and combined with iron grit (BZ) significantly increased the SPW pH (i.e. 7.3 ± 0.08 and 7.6 ± 0.05, respectively) and decreased the SPW EC (μS cm -1 ) albeit not significantly (i.e. 483 ± 51 and 477 ± 110 respectively). At month 22 with A. donax, the SPW pH and EC rose significantly in all soil treatments from 6.9 ± 0.1 (Unt) to 8.1 ± 0.2 (AdBZ) for the pH and from 1024 ± 117 (Unt) to 2037 ± 284 (AdBZ) for the EC. In the Pn and PnBZ treatments, the SPW pH did not differ from that of the Unt soil whereas it remained higher in the PnB soil (i.e. 7.5 ± 0.06). The SPW EC increased in the PnB soil (Month 22) as compared to the B soil (Month 3).

The SPW Ca concentration decreased in the B and BZ soils at month 3 and in all planted soils at month 22 as compared with the Unt soil. The SPW Fe and P concentrations remained below their detection limit, except for P in the B soil. At month 3, the SPW K concentration increased in the B and BZ soils but it decreased at month 22 for both plant species in all soils, and the SPW Mg concentration as well. The SPW Na concentration was significantly lower in the soils planted with A. donax as compared with the Unt soil.

DOM, Cu and Cu 2+

The DOM concentration in the SPW peaked in all soils cultivated with A. donax, being 3-5 fold higher than in the Unt soil (Fig. 1 A, B andC). In the Pn and PnB soils, the DOM increased significantly, i.e. (mg L -1 ) 24 ± 7 (Pn) and 23 ± 2 (PnB) relative to the Unt soil (13 ± 4). Total Cu concentration in the SPW (mg L -1 ) peaked in the Ad and AdB soils (i.e. 0.53 ± 0.18 and 0.68 ± 0.26, respectively) as compared to the Unt soil (i.e. 0.22 ± 0.05). Conversely, adding Z into the Unt soil significantly decreased the SPW total Cu concentration only in the AdBZ soil relative to Ad and AdB. All treatments, except the Pn soil, significantly decreased the SPW Cu 2+ concentration (mg L -1 ) as compared to the Unt soil (i.e. 14.9 ± 5.3).

Plants

Plant growth parameters

At month 22, the root and shoot DW yields of P. nigra and A. donax for the B-and BZ-amended soils did not differ from those for the Unt soil (Fig. 2 A andB). The root DW yields (mg DW plant -1 ) ranged from 17 ± 11 to 29 ± 9 for P. nigra and from 57 ± 12 to 84 ± 14 for A. donax whereas the shoot DW yields (mg DW plant -1 ) range from 19 ± 7 to 25 ± 11 for P. nigra and from 22 ± 4 to 26 ± 3 for A. donax. For A. donax, the shoot DW yield produced at month 10 (Cut 1) was lower than that harvested at month 22 (Cut 2).

Shoot ionome

Shoot Cu concentration of P. nigra was significantly lower for the PnBZ plants relative to the Pn plants (Table 4). For A. donax, the shoot Cu concentration was significantly higher for the AdB plants relative to the Ad plants. Shoot Cu removal slightly dropped for the PnBZ plants relative to the Pn plants and significantly increased for the AdB plants as compared to the Ad ones, mainly due to changes in shoot Cu concentration. Shoot Ca, Fe, K, Mg, Na, P, Zn and Mn concentrations of poplar and Giant reed were globally similar on the amended and untreated soils, except two significant cases: shoot Na concentration was lower for the PnBZ plants relative to the Pn ones, and shoot Mn concentration higher for the AdBZ plants as compared to Ad plants. For A. donax, shoot element concentrations for the Cut 1 were globally higher than those for the Cut 2. Only shoot Na and Mg concentrations were higher for the Ad and AdBZ plants of Cut 2 relative to plants harvested in Cut 1 (Supplemental material 1).

Discussion

Due to biogeochemical reactions between soil phases, amendments, and biological organisms, the behavior of chemical elements (e.g. Cu) in amended soils may change over time, which is referred as "aging" [START_REF] Kookana | The role of biochar in modifying the environmental fate, bioavailability, and efficacy of pesticides in soils: a review[END_REF]. Plant rhizodeposition and associated microorganisms may influence soil Cu speciation, especially when cultivated in a longtime basis [START_REF] Merino | Soil carbon controlled by plant, microorganism and mineralogy interactions[END_REF]. Here, we discuss the aging effect on the efficiency of biochar, alone and combined with iron grit, and how plants and amendments may influence Cu (im)mobilization and crop productivity for this Cu-contaminated soil from a wood preservation site.

Soil pore water

Composition at month 3

At short-term (3 months), the B amendment decreased Cu 2+ concentration while the BZ combination reduced both Cu 2+ and Cu concentrations in the soil pore water (Oustriere et al., 2016a). In the B soils, the decreased SPW Cu 2+ concentrations were associated with increased soil pH and SPW P and DOM concentrations and decreased SPW Ca concentrations. Based on X-ray absorption fine structure spectroscopy (XAFS), Cu 2+ sorption onto biochar is pH dependent [START_REF] Ippolito | Macroscopic and molecular investigations of copper sorption by a steam activated biochar[END_REF]. Free Cu 2+ may have (1) form inner sphere complexes with the active functional groups of biochar surface in line with SPW pH, (2) precipitate with phosphates and carbonates based on biochar composition (Table 2), and (3) form soluble Cu complexes with DOM increasing total Cu concentration in the SPW. Adding Z in combination with B increase soil pH. In the BZ treatment, free Cu 2+ ions may primarily be sorbed on Fe/Mn oxy-hydroxides, potentially by bidentate inner-sphere complexes with Fe (hydr)oxides (Oustriere et al., 2016a;[START_REF] Tiberg | Immobilization of Cu and as in two contaminated soils with zerovalent ironlongterm performance and mechanisms[END_REF].

Composition at month 22

Poplar and aging effect poorly affected pH, DOM and total Cu concentrations in the soil pore water (Table 3; Fig. 1), which agreed with previous studies: pH and total Cu in the SPW did not change over 36 months in a poly-contaminated soil at Aznalcázar, South West of Spain, cultivated with Populus alba L. [START_REF] Ciadamidaro | Growth of Populus alba and its influence on soil trace element availability[END_REF]; pH and total Cu in the SPW were similar after 5 years in the untreated and compost-amended field plots cultivated with Populus trichocarpa × deltoides cv. Beaupré at the P7 sub-site (soil contamination with Cu and PAHs) of this wood preservation site (Mench, unpublished data). Some studies on biochars supported the lack of aging effect on pH and DOM and total Cu concentrations in the soil pore water: in a PAHcontaminated soil, soil pH remained steady after artificial chemical, biological, and physical aging of corn stover residues-derived biochar [START_REF] Hale | Effects of chemical, biological, and physical aging as well as soil addition on the sorption of pyrene to activated carbon and biochar[END_REF]. The soil pH was kept between 7.9 and 8.1 over a 3year period in a (Ni/Zn)-contaminated soil, amended by broadleaf hardwood-derived biochar, Castleford, UK, [START_REF] Shen | Long-term impact of biochar on the immobilisation of nickel (II) and zinc (II) and the revegetation of a contaminated site[END_REF]. After 3 years, SOM and (CaCl 2 )-extractable Cu concentration were stable in a (Cu/Cd)-contaminated soil from a former Cu mine, amended by cornstraw-derived biochar [START_REF] Li | The influence of biochar type on long-term stabilization for Cd and Cu in contaminated paddy soils[END_REF]. At month 22, the SPW Cu 2+ concentration increased in the PnB and PnBZ soils as compared to the B and BZ ones (at month 3) (Fig. 2). This might be explained by: (1) less potential precipitation with phosphates in line with lower SPW P concentration in the Pn soil than in the B one, and (2) decrease of the sorption capacity of biochar over the time [START_REF] Martin | Marked changes in herbicide sorption-desorption upon ageing of biochars in soil[END_REF]. [START_REF] Rozada | Adsorption of heavy metals onto sewage sludge-derived materials[END_REF] suggested that Cu 2+ sorption on biochar surface was easily reversible. When biochar aged, the proportion of oxygen-containing acidic functional groups (e.g., COO -, COH and OH) increases on the biochar surface, while carboxyl groups slightly decrease [START_REF] Nguyen | Black carbon decomposition under varying water regimes[END_REF][START_REF] Guo | Mechanism of Cu(II) adsorption inhibition on biochar by ist aging process[END_REF], contributing to the negative charge of the biochar. Their association with cations (i.e. Ca, K and Mg), in line with the low SPW cation concentrations (Table 3), and strong affinity for water and DOM may decrease the sorption capacity of biochar for free Cu 2+ [START_REF] Li | The influence of biochar type on long-term stabilization for Cd and Cu in contaminated paddy soils[END_REF]. In addition, gradual coating and interactions with OM and inorganic phases [START_REF] Sorrenti | Biochar physico-chemical properties as affected by environmental exposure[END_REF] and microbial colonization may decrease the reactivity of the biochar surface. After 4 years, aging increased biochar skeletal density and reduced the water imbibition rate within fragments as a consequence of partial pore clogging [START_REF] Sorrenti | Biochar physico-chemical properties as affected by environmental exposure[END_REF]. The cation exchange capacity (CEC) and adsorption capacity of Cu(II) on the aged biochar were smaller than those of new biochar in a 300 day incubation time pot experiment [START_REF] Guo | Mechanism of Cu(II) adsorption inhibition on biochar by ist aging process[END_REF]. Here, aged biochar particles after cultivation could be further retrieved from the amended soils to assess their composition and reactivity and evidence the main mechanisms.

Enhanced SPW pH and DOM concentration in all amended soils planted with A. donax (Table 3; Fig. 1) agreed with an increase in soil pH (5-9%) after a 3-month cultivation of A. donax on red mudcontaminated soil [START_REF] Alshaal | Phytoremediation of bauxite-derived red mud by giant reed[END_REF]. Change in SPW pH may partly result from root activity, imbalance uptake of anions (e.g. NO 3 -) vs. cations increasing the rhizosphere pH in the basal root zone [START_REF] Hinsinger | Origins of root-mediated pH changes in the rhizosphere and their responses to environmental constraints: a review[END_REF][START_REF] Bravin | Rhizosphere alkalisationa major driver of copper bioavailability over a broad pH range in an acidic, coppercontaminated soil[END_REF][START_REF] Qasim | Rhizosphere effects of Populus euramericana Dorskamp on the mobility of Zn, Pb and Cd in contaminated technosols[END_REF]. Calcium uptake by plant, in line with low SPW Ca concentration (Table 3), may initiate a further cation desorption from the solid phase, promote H + sorption and increase soil pH. In addition, increased SPW pH may be related to the higher SPW DOM concentration (Fig. 1; [START_REF] Zhang | Changes in pH, dissolved organic matter and Cd species in the rhizosphere soils of Cd phytostabilizer Athyrium wardii (Hook.) Makino involved in Cd tolerance and accumulation[END_REF], which was previously reported in soils cultivated with A. donax [START_REF] Riffaldi | Comparison of soil organic-matter characteristics under the energy crop giant reed, cropping sequence and natural grass[END_REF]. DOM may originate from decayed plant litter, microbial and root depositions, and hydrolysis of insoluble SOM [START_REF] Haynes | Labile organic matter fractions as central components of the quality of agricultural soils: an overview[END_REF]. No leachate from plant litter was produced here as shoots were harvested, but A. donax formed a dense mat of roots and rhizomes in the soil (Fig. 2). Giant reed rhizodeposition can highly contribute to SOM available for microbial activity and the DOM pool [START_REF] Cattaneo | 13C abundance shows effective soil carbon sequestration in Miscanthus and giant reed compared to arable crops under Mediterranean climate[END_REF][START_REF] Nsanganwimana | Arundo donax L., a candidate for phytomanaging water and soils contaminated by trace elements and producing plant-based feedstock, a review[END_REF][START_REF] Monti | Sixteen-year biomass yield and soil carbon storage of giant reed (Arundo donax L.) grown under variable nitrogen fertilization rates[END_REF]. Enhanced microbial activity may stimulate both the degradation of SOM and production of microbial DOM [START_REF] Kiikkilä | Characterization of dissolved organic matter in decomposing Norway spruce and silver birch litter[END_REF][START_REF] Hagedorn | Tracking litter-derived dissolved organic matter along a soil chronosequence using 14C imaging: biodegradation, physico-chemical retention or preferential flow?[END_REF]. In A. donax cultivated soil, pH increased between 7.9 ± 0.2 (Ad) and 8.1 ± 0.2 (AdBZ) as compared to the Unt soil (i.e. 6.9 ± 0.1), which may promote DOM solubilization [START_REF] Fang | Comparative characterization of sewage sludge compost and soil: heavy metal leaching characteristics[END_REF]. At month 22, the SPW Cu concentration increased in the Ad and AdB soils as compared to the B and BZ ones (at month 3) (Fig. 2). Enhanced DOM concentration may form soluble complexes with Cu and increase its SPW concentration [START_REF] Beesley | Effects of biochar and greenwaste compost amendments on mobility, bioavailability and toxicity of inorganic and organic contaminants in a multi-element polluted soil[END_REF][START_REF] Karami | Efficiency of green waste compost and biochar soil amendments for reducing lead and copper mobility and uptake to ryegrass[END_REF]. Here, Giant reed-derived-DOM may act as competitive ligands preventing Cu retention on biochar surfaces [START_REF] Beesley | Assessing the influence of compost and biochar amendments on the mobility and toxicity of metals and arsenic in a naturally contaminated mine soil[END_REF]. In parallel, with aging, negative charges on biochar surface may form direct or indirect surface complexes with soil components. In addition, coating of DOM may gradually mask sorptive surfaces, thus limiting Cu sorption [START_REF] Pignatello | Effect of natural organic substances on the surface and adsorptive properties of environmental black carbon (char): attenuation of surface activity by humic and fulvic acids[END_REF].

In contrast, the SPW Cu 2+ and Cu concentrations in the AdBZ and PnBZ soils were lower than in the untreated and biochar-amendedsoils cultivated with A. donax and P. nigra, albeit not significantly for Cu 2+ as compared to AdB and PnB. The SPW Cu 2+ and Cu concentrations in the AdBZ and PnBZ soils at month 22 were as low as in the BZ soil at month 3 after soil amendment, albeit not significantly for Cu 2+ in the AdBZ soil. Cationic metal species such as Cu 2+ or DOM-Cu complex have high affinity for Fe oxides and can sorb on the newly formed Fe and Mn oxy-hydroxides after Z corrosion in the soil (Kumpiene et al., 2008; [START_REF] Komárek | Chemical stabilization of metals and arsenic in contaminated soils using oxides. A review[END_REF]. The sorption of Cu on Fe and Mn oxyhydroxides is pH-dependent with stronger sorption at high pH. The additional liming effect of biochar can favor the net negative surface charge of Fe and Mn oxy-hydroxides and enhance Cu sorption. [START_REF] Tiberg | Immobilization of Cu and as in two contaminated soils with zerovalent ironlongterm performance and mechanisms[END_REF] investigated Cu-contaminated soils collected in untreated and Z-treated plots at our sampling sub-site after 6 years: the bound Cu was primarily associated to SOM in the Unt soil whereas with Z addition and increased soil pH, Cu sorption shifted towards metal (hydr)oxides. Based on the Cu-ferrihydrite EXAFS spectrum, Cu was found to primarily bind as inner-sphere bidentate complexes with iron (hydr)oxide [START_REF] Tiberg | Immobilization of Cu and as in two contaminated soils with zerovalent ironlongterm performance and mechanisms[END_REF].

Plants

At month 3, this biochar alone and combined with iron grit did not promote the root and shoot yields of dwarf beans (Oustriere et al., 2016a). At month 22, this result was similar for P. nigra and A. donax (Fig. 2) and agreed with some studies: root and shoot biomass produced by Lolium multiflorum Lam. was unchanged in a (Cd, Zn, and Pb)-contaminated soil amended with 1% miscanthus-derived-biochar after 28 days and 56 days [START_REF] Houben | Mobility, bioavailability and pH-dependent leaching of cadmium, zinc and lead in a contaminated soil amended with biochar[END_REF]. After 2 years, grain yields and biomass of wheat were not affected by both rate 10 and 40 t ha -1 of wheat straw-derived biochar addition in a Cd contaminated paddy soil [START_REF] Cui | The reduction of wheat Cd uptake in contaminated soil via biochar amendment: a two-year field experiment[END_REF]. Germination of grass (mix of Festuca rubra L. and Lolium perenne L.) on site failed in a 3-year field experiment on a contaminated (Ni/Zn) soil amended with broad leaf hardwoodderived biochar [START_REF] Shen | Long-term impact of biochar on the immobilisation of nickel (II) and zinc (II) and the revegetation of a contaminated site[END_REF]. Here, despite HSN supply, Ca, Fe, K, Mg and P sub-deficiencies might be suggested as their concentrations in P. nigra and A. donax shoots were below or in the low ranges as compared to common values (Tremel-Schaub and Feix, 2005, Table 4). At month 22, Ca, Fe, K, Mg and P concentrations in the SPW were low as compared to common values in sandy soil (Table 3), likely due to (1) reaction with and amendment phases [START_REF] Cheng | Sorption properties for black carbon (wood char) after long term exposure in soils[END_REF], and (2) root uptake. Nutrient concentrations in A. donax shoot even decreased between the Cut 1 and the Cut 2 (Supplemental material 1). It may reflect a dilution effect as shoot DW yield increased. The shoot Cu concentrations of P. nigra and A. donax (mg kg -1 ) were similar across the treatments, ranging from 4.0 ± 0.5 (PnBZ) to 7.0 ± 2 (AdBZ) (Table 4), and in the range of common values (3-20 mg kg -1 , [START_REF] Tremel-Schaub | Contamination des Sols -Transferts des Sols vers les Plantes[END_REF]. Stem and foliar Cu concentrations (mg kg -1 ) reached 3.21 and 7.17 for A. donax on a poly-contaminated urban stream [START_REF] Bonanno | Heavy metal content in ash of energy crops growing in sewage-contaminated natural wetlands: potential applications in agriculture and forestry?[END_REF]. Shoot Cu concentration ranged between 7 and 17 mg kg -1 in P. alba grown for 3 years in a Cu-contaminated soil [START_REF] Ciadamidaro | Growth of Populus alba and its influence on soil trace element availability[END_REF]. Giant reed and poplars are Cu excluders accumulating Cu in their roots, which may explain their low shoot Cu concentration [START_REF] Kabata-Pendias | Trace Elements in Soils and Plants[END_REF][START_REF] Bonanno | Heavy metal content in ash of energy crops growing in sewage-contaminated natural wetlands: potential applications in agriculture and forestry?[END_REF][START_REF] Ciadamidaro | Growth of Populus alba and its influence on soil trace element availability[END_REF][START_REF] Elhawat | Phytoaccumulation potentials of two biotechnologically propagated ecotypes of Arundo donax in copper-contaminated synthetic wastewater[END_REF].

Practical implication

Effect of plant species: Giant reed was more efficient than P. nigra to decrease Cu 2+ exposure in its rhizosphere but increased total Cu concentration in the soil pore water, which may potentially enhance Cu leaching from the root zone (Fig. 1). After 2 years, P. nigra did not contribute to Cu immobilization (Fig. 1). Without soil amendment, both plant species were unable to stabilize Cu in excess.

Effect of soil amendment: combining iron grit with biochar promoted Cu stabilization but not the growth of A. donax and P. nigra. An additional fertilization may be required. Long-term field plots must investigate the sustainability of Cu stabilization, biomass production of non-food crops and the life cycling of such phytomanagement option.

Conclusion

After a 22-month reaction period, the SPW Cu 2+ concentration increased again in the B and BZ soils cultivated with P. nigra as compared to its value at month 3. Cultivation of A. donax incremented the DOM concentration in the soil pore water. Such high SPW DOM concentration in the Giant reed-planted soils, which may induce the formation of Cu-DOM complexes, matched with a decrease of Cu 2+ concentration but increased total Cu concentration in the soil pore water. Iron grit with biochar was more effective to stabilize soil Cu than biochar alone with both A. donax and P. nigra cultivation. Biochar alone and combined with iron grit did not promote the root and shoot yields of A. donax and P. nigra as compared to the untreated soil but the shoot Cu concentrations were in the common ranges for both plants.

Supplementary data to this article can be found online at http://dx. doi.org/10. 1016/j.scitotenv.2016.11.048.

Fig. 1 .

 1 Fig. 1. (A) DOM, (B) total Cu and (C) Cu 2+ concentrations in the soil pore water; Cu contaminated soil (Unt, white), amended with either biochar (B) or biochar plus iron grit (BZ) in the Cu contaminated soil cultivated with Populus nigra (Pn, grey) or Arundo donax (Ad, black), alone and amended with either biochar (PnB and AdB) or biochar plus iron grit (PnBZ and AdBZ). Mean values per treatment (n = 5, Unt: n = 6). Values with different letters differ significantly (one way ANOVA, p-value b0.05).

Fig. 2 .

 2 Fig. 2. (A) Root and (B) shoot DW yields of P. nigra (Pn, white, month 22) and A. donax (Ad, grey, and black) (mg DW plant -1 ) at month 22 in the Cu-contaminated soil (Unt) and in the soil amended with either biochar (PnB and AdB) or biochar plus iron grit (PnBZ and AdBZ). The grey bars correspond to the biomass month 10 (Cut 1). Mean values per treatment (n = 5). Values with different letters differ significantly (one way ANOVA, p-value b0.05).

Table 1

 1 Main physico-chemical soil parameters.

	Parameter	Mean ± SD	Background levels a
	pH	7 ± 0.09	6.6
	CEC	2.5 ± 0.2
	P 2 O 5 (g kg -1 )	0.03 ± 0.002
	Organic matter (g kg -1 )	13 ± 0.4
	Organic C (g kg -1 )	8 ± 0.3	14.5
	Total N (g kg -1 )	0.5 ± 0.01
	C/N	16 ± 0.2	10
	Texture (g kg -1 )
	Sand	858	≥650
	Silt	83	≤350
	Clay	59	≤180
	Total TE (mg kg -1 )
	Cr	21 ± 0.7	14-40
	Cu	964 ± 20	3.2-8.4
	Ni	5 ± 0.3	4.2-14.5
	Zn	37 ± 1.6	17-48
	As	7 ± 0.4	1-25 b
	a Frequent total concentrations in French sandy topsoils (Baize, 2000; Villanneau et al.,
	2008).

b Frequent total As concentrations for all French soil types

[START_REF] Bes | Remediation of copper-contaminated topsoils from a wood treatment facility using in situ stabilization[END_REF]

.

Table 2

 2 Composition of soil amendments. In bold, values exceeding the European Biochar Certificate V4.8 threshold values.

		B	Z	EBC a IBI b	French upper
						critical threshold
						values for organic
						amendments c
	pH	9.89		-	-
	CEC (cmol kg -1 )	1.23		-	-
	Major elements (% DW)
	H	0.8		-	-
	N	0.3		-	-
	S	0.03		-	-
	Cl	0.01		-	-
	C	90.3		N	Class 1: ≥
				50%	60%
					Class 2:
					[30% -60%]
					Class 3:
					[10% -30]
	Nutrients (g kg -1 )
	Ca	18.6		-	-
	K	13.8		-	-
	Mg	29.9		-	-
	Na	0.389		-	-
	P	0.105		-	-
	Elements (mg kg -1 )
	Al	1650 600	-	-
	As	0.461 70	-	12-100	18
	Cd	b0.5 0.03	1	1.4-39	3
	Cr	45.9	3500	80	64-1200	300
	Cu	230	1010	100 63-1500	120
	Mn	-	7710	-	-
	Fe	4470 973,000 -	-
	Hg	b0.1		1	1-17	2
	Ni	28.3	739	30	47-600	60
	Pb	2.61	20	120 70-500	180
	Zn	50.5	104	400 200-7000	600
	PAHs (mg kg -1 )
	Sum of 16 US EPA	163		4	6 -300
	PAHs d
	Fluoranthene	7.48		-	-	4
	Benzo(b)fluoranthene 0.149		-	-	2.5
	Benzo(a)pyrene	b0.2		-	-	1.5
	Naphtalene	101		-	-

a Following Switzerland's Chemical Risk Reduction Act (ChemRRV) on recycling fertilizers. b Range of Maximum Allowed Threshold values reflects different soil tolerance levels for these elements in compost, biosolids, or soils established by regulatory bodies in the US, Canada, EU and Australia (See Appendix 3 of the IBI Biochar Standards for further information). c French upper critical threshold values for organic amendments (NF U 44 051, Dec. 2010). d Corresponds to the PAH threshold values defined in the Swiss Chemical Risk Reduction Act (Chem RRV).

Table 3

 3 Comparison of physico-chemical parameters of soil pore waters, 3 and 22 months after soil amendment.

	Treatments			(μS cm -1 )	Nutrients (mg L -1 )
			pH	EC	Ca	Fe	K	Mg	Na	P
	SPW: Month 3
	Unt	(n = 6)	6.9 ± 0.1 f	1024 ± 117 cd 143 ± 39 a b0.02	14 ± 5 b	5 ± 2 a	12 ± 3 ab	b0.2
	B	(n = 3)	7.3 ± 0.08 def 483 ± 51 d	59 ± 5 b	b0.02	43 ± 11 a	3 ± 0.3 a	13 ± 2 a	0.3 ± 0.05
	BZ	(n = 3)	7.6 ± 0.05 bcd 477 ± 110 d	46 ± 20 b b0.02	53 ± 16 a	3 ± 1 ab	17 ± 6 a	b0.2
	SPW: Month 22
	Pn	(n = 5)	7.1 ± 0.1 ef	890 ± 171 cd	2.5 ± 0.5 c b0.02	4.1 ± 0.5 c	1.1 ± 0.2 c	9.7 ± 1.9 abc b0.2
	PnB	(n = 5)	7.5 ± 0.06 cde 1299 ± 118 bc 3.4 ± 0.3 c b0.02	6.3 ± 0.8 bc 1.4 ± 0.1 bc 12.9 ± 1.1 a	b0.2
	PnBZ	(n = 5)	7.2 ± 0.1 def	755 ± 231 cd	2.1 ± 0.6 c b0.02	4 ± 1.1 c	1.1 ± 0.2 c	5.7 ± 2.8 bcd b0.2
	Ad	(n = 5)	7.9 ± 0.2 abc	1737 ± 300 ab b0.5	b0.02	1 ± 0.3 d	0.8 ± 0.4 c	b 0.5	b0.2
	AdB	(n = 5)	8.0 ± 0.3 ab	1819 ± 529 ab 1.1 ± 0.5 c b0.02	1 ± 0.5 de	1.3 ± 0.2 bc 3.8 ± 3.9 cd b0.2
	AdBZ	(n = 5)	8.1 ± 0.2 a	2037 ± 284 a	0.8 ± 1.1 c b0.02	0.5 ± 0.3 e 1.1 ± 0.6 c	1 ± 1.5 d	b0.2
	Common values in sandy soils (Oustriere et al., 2016b) -	-	143 ± 66	0.05 ± 0.01 27 ± 44	21 ± 11	63 ± 12	5 ± 2

SPW: soil pore water; Mean value ± SD for each treatment. Values with different letters differ significantly (one way ANOVA, p-value b 0.05). Mean values followed by letters in bold are significantly different as compared to the Unt soil.

Table 4

 4 Shoot ionome and shoot Cu removal of P. nigra and A. donax at month 22 (n = 5).

										Shoot metal
	Treatment	Cu		Shoot nutrient concentrations (g kg -1 )			concentrations (mg kg -1 )
		Shoot Cu	Shoot Cu
		concentration	removal
		(mg kg -1 DW)	(µg plant -1 )	Ca	Fe	K	Mg	Na	P	Zn	Mn
	Pn	5.1± 0.7 a	2.6 ± 0.4 a	5.3 ± 1.1 a	0.019 ± 0.005 a 5.8 ± 0.3 a	0.87 ± 0.21 a 1.06 ± 0.28 a	1.24 ± 0.48 a	25 ± 6 a	8 ± 2 a
	PnB	4.3 ± 0.4 ab	2.2 ± 0.2 ab	5.1 ± 0.9 a	0.015 ± 0.002 a 6.6 ± 1.1 a	0.89 ± 0.12 a 0.68 ± 0.29 ab 1.2 ± 0.16 a	25 ± 8 a 10.4 ± 4 a
	PnBZ	4.0 ± 0.5 b	2.0 ± 0.2 b	4.8 ± 0.9 a	0.016 ± 0.003 a 6.5 ± 1 a	0.84 ± 0.12 a 0.47 ± 0.15 b	1.24 ± 0.19 a	32 ± 6 a	9.8 ± 2 a
	Ad	4.4 ± 0.7 b	2.2 ± 0.4 b	2.8 ± 1.6 a	0.015 ± 0.003 a 6.1 ± 1.2 a	1.49 ± 0.63 a 0.19 ± 0.05 a	0.16 ± 0.03 a	6 ± 4 a	13 ± 7b
	AdB	7.0 ± 2 a	3.5 ± 1.2 a	3.2 ± 0.8 a	0.016 ± 0.004 a 8.3 ± 1.4 a	1.68 ± 0.43 a 0.25 ± 0.1 a	0.25 ± 0.06 a	9 ± 5 a	14 ± 6 b
	AdBZ	5.2 ± 1 ab	2.6 ± 0.5 ab	3.2 ± 0.7 a	0.021 ± 0.007 a 8.7 ± 2.6 a	1.77 ± 0.28 a 0.2 ± 0.09 a	0.19 ± 0.03 a	7 ± 5 a	32 ± 2 a
	Common
	values *	3-20	-	1-50	0.02-0.3	20-50	1.5-3.5	-	1.6-6.0

Mean value ± SD for each treatment. Values with different letters differ significantly (one way ANOVA, p-value <0.05). Letters in bold indicated significant differences. *

(Tremel-Schaub and Feix,2005)

.

Acknowledgements

This work was supported by ADEME (French Agency for the Environment and Energy, PhD grant no 2013-ID5081 of N. Oustrière), the French National Research Agency (ANR CD2I, program PHYTOCHEM) and the ERA-Net FACCE SURPLUS (project INTENSE, no ANR-15-SUSF-0007-06; http://faccesurplus.org/research-projects/intense/). The UMR Biogeco is a member of the INRA Ecotoxicologist network, ECOTOX.


Thanh Thuy

Nguyen Tu

Céline Egasse

Christelle Anquetil

Florent Zanetti

Bernhard Zeller

Sylvain Huon

Sylvie Derenne

Thanh Thuy Nguyen Tu
email: thanh-thuy.nguyen_tu@upmc.fr

Céline Egasse b , Chr ist Elle Anquet

Flor Ent Zanet

Thuy Nguyen

Leaf lipid degradation in soils and surface sediments: A litterbag experiment

Keywords: diagenesis, biomar ker, lit t er bag, leaf lipids, soil, sur face sediment, GC-MS

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

1. I nt r oduct i on L ipid biomar ker s const it ut e efficient t ools for t r acing t he sources of organic mat t er (OM) in mar ine [START_REF] Fukuchi | Source of hydr ocar bons in mar ine sediment s in L üt zow-H olm bay, Ant arct ica[END_REF][START_REF] Aquilina | Biomar ker indicat or s for anaer obic oxidizer s of methane in br ackish -mar ine sediment s wit h diffusive methane fluxes[END_REF], fluvial [START_REF] Jaffé | The biogeochemist r y of t he r iver s of the Or inoco Basin[END_REF][START_REF] Scheffuß | Mult iple plant -wax compounds r ecor d different ial sour ces and ecosyst em st r uctur e I lar ge r iver cat chment s[END_REF] or lacust r ine envir onment s [START_REF] Rieley | The biogeochemist r y of Ellesmer e L ake U.K . -I : sour ce corr elat ion of leaf wax input s t o t he sediment ar y lipid recor d[END_REF][START_REF] Jacob | Cont r ast ed dist r ibut ions of t r it er pene der ivat ives in t he sediment s of L ake Caço reflect paleoenvir onment al changes dur ing t he last 20,000 yr s in NE Br azil[END_REF].

Quant ifying biomar ker var iat ion through sediment ar y sequences facilit at es r econst ruct ion of past envir onment al var iabilit y over var ious t imescales, fr om cent ur ies [START_REF] Farr Ingt On | The or ganic geochemist r y of Peru mar gin sur face sediment s: I I . Paleoenvironment al implicat ions of hydr ocar bon and alcohol pr ofiles[END_REF][START_REF] Sánchez-Palencia | L ipid biomar ker s in L ake Enol (Ast ur ias, Nor t her n Spain): Coupled natur al and human induced envir onment al hist or[END_REF] t o millions of year s [START_REF] Pear Son | Onocer ane and ot her tr it er penoids in L at e Cr et aceous sediment s fr om t he Upper Benue Trough, Niger ia: t ect onic and palaeoenvir onment al implicat ions[END_REF][START_REF] Tzor T Zaki | Biomar ker evidence for int er mit t ent phot ic zone euxinia in t he Apt ian-Albian or ganic sedimentar y r ecor d from t he I onian Zone (Epir us, Gr eece)[END_REF]. H owever , t he diagenet ic fat e of biomar ker s must be assessed befor e their dist r ibut ion and abundance can be used as quant it at ive pr oxies. Fr om their st r uct ur es, it would be ant icipat ed t hat differ ent biomarker s would exhibit var ying degr ees of suscept ibilit y t o degr adat ion, which would ult imat ely affect t heir pr eservat ion in sediment s (M eyer s and I shiwat ar i, 1993). Sever al st abilit y scales have been est ablished for lipids in sediment s; n-alkanes ar e gener ally considered as t he most st able component s, while funct ionalized compounds such as pigment s or n-alkanols as t he most r eadily degr aded [START_REF] Cr Anwell | Diagenesis of fr ee and bound lipids in t err est r ial detr it us deposit ed in a lacust r ine sediment[END_REF][START_REF] Colombo | L ipid biogeochemist r y in the L aur ent ian Though-I I . Changes in composit ion of fat ty acids, st er ols and aliphat ic hydr ocar bons dur ing ear ly diagenesis[END_REF]. St er ols and fat t y acids (FAs) exhibit int er mediat e st abilit y t hat var ies accor ding t o funct ionality and degr ee of unsat ur at ion [START_REF] Cr Anwell | Diagenesis of fr ee and bound lipids in t err est r ial detr it us deposit ed in a lacust r ine sediment[END_REF] M eyer s and I shiwar at i, 1993; [START_REF] Colombo | L ipid biogeochemist r y in the L aur ent ian Though-I I . Changes in composit ion of fat ty acids, st er ols and aliphat ic hydr ocar bons dur ing ear ly diagenesis[END_REF]. Environment al par amet er s in t he wat er column, at t he wat er -sediment int erface and wit hin t he sediment also influence lipid degr adat ion/pr eservat ion [START_REF]Or ganic geochemical pr oxies of paleoceanogr aphic, paleolimnologic, and paleoclimat ic processes[END_REF][START_REF] Ront Ani | Phyt ol degr adat ion pr oduct s as biogeochemical tr acer s in aquat ic envir onment s[END_REF].

Vascular plant s ar e major cont r ibut or s t o sediment ar y OM and plant biomar ker s oft en ser ve as proxies for cont inent al OM in mar ine sediment s [START_REF] Volkman | A r eview of st erol mar ker s for mar ine and t er r igenous organic mat t er[END_REF][START_REF] Oades | Compar at ive organic geochemist r y of soils and mar ine sediment s[END_REF]. The plant biomar ker signat ure in sediment s can be biased by differ ences in pr oduct ivit y among t axa, and biomar ker solubilit y or t r anspor t mode [START_REF] Diefendor F | A compar ison of t er penoid and leaf fossil veget at ion pr oxies in Paleocene and Eocene Bighorn Basin sediment s[END_REF]. Among polycyclic t er penoids, Gir i et al. (2015) showed t hat t hese pr e-sediment at ion pr ocesses t end t o favor dit er penoids at t he expense of t r it er penoids, leading t o an overest imat ion of conifer input vs. angiosper m input in paleoveget at ion r econst r uct ion.

Befor e deposit ion in mar ine sediment s, plant detr it us may remain for varying ext ent s of t ime in soil and/or in fr esh wat er. The cont r ast ing degr adat ion condit ions in these envir onment s may in t ur n affect t he plant biomarker signat ure. Sur face degr adat ion of a given plant on land was shown t o modify bulk leaf chemist r y t o a differ ent ext ent t han degr adat ion in bur ied condit ions [START_REF] Webst Er | Vascular plant br eakdown in fr eshwat er ecosyst ems[END_REF][START_REF] St Ear T | The chemical const r aint s upon leaf decay r at es: Taphonomic implicat ions among leaf species in Aust r alian t err est r ial and aquat ic environment s[END_REF]. Alt hough t he fat e of molecular biomar ker s was independent ly st udied in sever al envir onment s via var ious appr oaches (e.g. soil lit t er bags, soil/sediment pr ofiles/sect ions, labor at ory incubat ions; [START_REF] Cr Anwell | Diagenesis of fr ee and bound lipids in t err est r ial detr it us deposit ed in a lacust r ine sediment[END_REF]M eyer s and I shiwar at i, 1993;[START_REF] Rieley | L abor at or y st udies on sediment ary lipids pr eservat ion: differ ent pr eservat ion pot ent ial infer r ed for leaf and algal lipids[END_REF][START_REF] Zech | Effect of leaf lit t er degr adat ion and seasonalit y on D/H isot ope r at ios of nalkane biomar ker s[END_REF][START_REF] Wiesenberg | Use of molecular r at ios t o ident ify changes of M iscanthus  gigant eus (Greef et Deu.) soil dur ing a labor at ory exper iment[END_REF][START_REF] Wang | Chemical and car bon isot opic dynamics of gr ass or ganic mat t er dur ing lit t er decomposit ions: A lit t er bag exper iment[END_REF]), compar at ive st udies addr essing t he fat e of biomar ker s in diver se nat ur al envir onment s are r ar e. H owever, the plant biomar ker s r ecover ed fr om sediment ary OM may have undergone different pr e-bur ial hist or ies t hat may have influenced their r obust ness as a quant it at ive pr oxy.

As t he compar at ive fat e of plant lipids in differ ent envir onment s is not fully document ed, t he aim of t he pr esent st udy was t o compar e t he degr adat ion of lipids fr om a given plant species in two differ ing environment s: soil and t he wat er -sediment int er face.

The invest igat ion focused on leaf lipids, as epicut icular wax is the main sour ce of t he fat t y lipids commonly used as cont inent al biomarker s in mar ine sediment s. The st udy was based on a lit t er bag exper iment , in or der t o (i) allow the r ecovery of t he small leaf debr is gener ally obt ained aft er sever al months of degr adat ion and (ii) pr ecisely monit or t he ch anges undergone in t he leaves init ially int r oduced into t he bags, wit hout pot ent ial bias due t o cont aminat ion by ot her plant mat er ial. Eur opean beech (Fagus sylvatica L.) was chosen because it commonly occur s in t emper at e for est s. The lipid composit ion of t he leaves fr om two lit t er bag exper iment s conduct ed simult aneously in a soil and in a near by pond was invest igat ed t o t est whet her t hese differ ent envir onment s might lead t o differ ent biomar ker signatur es. The key quest ions wer e: Do differ ent lipid classes degr ade similar ly in t hese t wo environment s? I s the dist r ibut ion of fat ty li pids modified similar ly in the t wo envir onment s? 2. M at er i al and met hods 2.1. Exper imental site The exper iment was set up in the "Breuil -Chenue" exper iment al for est sit e, in Burgundy, Fr ance (47°18'10"N, 4°4'44"E). The for est is par t of the "Par c Nat ur el Régional du M orvan", a pr ot ect ed area wher e ur ban pollut ion is minimal. The exper iment al ar ea is on a shelf 650 m above sea level and exhibit s a slight nor t hwest facing slope. M ean annual t emper at ure and precipit at ion at the sit e ar e 9 °C and 1,280 mm, r espect ively. The bedrock is t he alkaline gr anit e of "L a Pierr e qui Vir e", cover ed by a t hin layer of eolian silt . The incubat ions t ook place in a 30 yr old Eur opean beech (Fagus sylvatica L ) st and plant ed aft er a clear -cut in 1976, and in a ca. 300 m 2 adjacent pond, r espect ively. The soil is classified as a dyst r ic cambisol (WRB, 2006) wit h moder -t ype humus. I t is acidic wit h pH 4-4.5 and has a poor ly sat ur at ed cat ion exchange complex. M or e infor mat ion on the sit e is available elsewher e [START_REF] Unier -L Amy | Effect of t r ee species subst it ut ion on organic mat t er biodegr adabilit y and miner al nutr ient availabilit y in t emper at e t opsoil[END_REF]Andr ianar isoa et al., 2010). The pond had a maximum dept h of 1.5 m. The main char act er ist ics wer e det er mined using a CTD (conduct ivit y -t emper at ur e -dept h) pr obe equipped wit h addit ional sensor s and ext ernal fluor omet er s (ASD sensor s). Dur ing t he cour se of th e st udy t he wat er column displayed near ly const ant t emper at ur e, electr ical conduct ivit y and pH with wat er dept h (Supplement ary Table A.1). The wat er had a slight ly acid pH (ca. 6.1), low dissolved ion load (low electr ical conduct ivit y, ca. 25.7 µS/cm), low t ur bidit y (FTU < 20), high CDOM cont ent (chr omolenic dissolved OM, > 200 µg/l) and sat ur at ed or close t o sat ur at ion dissolved O 2 concent r at ion (ca. 98%). Over all, t his is over all typical of sur face and subsur face wat er s of forest s soils on gr anit ic subst r at um (Beaucair e and M ichard, 1982).

Ther mal st r at ificat ion only occur s dur ing summer , when phot osynthet ic act ivit y is enhanced (high chlor ophyll -a concent r at ion and oversat ur at ion for dissolved O 2 ; Supplement ary Table A .1 and Fig. A.1).

L itter bag incubations

At t he end of Oct ober 2004, senescent leaves wer e collect ed fr om t he beech st and wher e the soil incubat i on t ook place. The soil lit t er bag exper iment has been descr ibed elsewher e [START_REF] Nguyen Tu | Ear ly degr adat ion of plant alkanes in soils: A lit t er bag exper iment using 13 C-labelled leaves[END_REF]. The leaves wer e collect ed fr om t he same height (ca.1.5 m) fr om t he br anches of sever al differ ent t r ees in or der t o pr ovide an aver aged composit ion and t o minimize bias linked t o int r apopulat ion var iabilit y. Aft er har vest ing, t he leaves wer e r insed wit h dist illed wat er t o remove ext r aneous par t icles and dr ied at 45 °C to const ant weight . Each bat ch was composed of 10-15 leaves and corr esponded t o ca. 1 g OM .

Bat ches wer e assembled using leaves fr om as many differ ent t r ees as possible t o provide equivalent bat ches.

L it t er bags (9.5  14.5 cm) wer e made of Al wir e; mesh size was 1.4  1.8 mm, i.e. sufficient t o ret ain small leaf lit t er debr is but sufficient ly large t o per mit fr ee ent ry of mesofauna (main funct ional group at t he sit e) and micr obial act ivit y (bact er ia and fungi).

Pond lit t er bags wer e ballast ed wit h glass st icks t o maint ain t hem at t he bot t om of the pond. Befor e being enclosed in lit t er bags, leaves were weighed and t hen r e-moist ened wit h dist illed wat er t o r ecr eat e nat ur al condit ions. The litt er bags wer e placed in t he field in December 2004. L it t er bags (in duplicat e) wer e then regular ly r et r ieved over 2.5 yr: aft er 5, 10,15,20,30,40,52,79 and 129 weeks for t he soil, and 9, 27, 66 and 100 weeks for t he pond. The soil lit t er bags cor r espond t o t he unlabeled leaves analyzed before for their specific alkane isot ope composit ion [START_REF] Nguyen Tu | Ear ly degr adat ion of plant alkanes in soils: A lit t er bag exper iment using 13 C-labelled leaves[END_REF].

L ipid extr action and fr actionation

The r ecover ed mat er ial was fir st r insed wit h dist illed wat er t o r emove obvious ext r aneous par t icles such as moss, r oot s or ar t hropods. L eaves wer e t hen dr ied at 45 °C t o const ant weight and weighed. Samples wer e st or ed dar k in Al foil at 5 °C unt il analysis. All t he leaves for each lit t er bag were combined and gr ound fine enough t o pass t hr ough a 500 m mesh. L eaf powder was ult r asonically ext r act ed for 20 min wit h CH 2 Cl 2 /M eOH (2/1; v/v).

The mixt ur e was cent r ifuged (10 min, 4,000 r pm) and t he lipids wer e r ecover ed in the supernat ant . The extr act ion procedure was r epeat ed 6 wit h t he cent r ifugat ion residue.

The combined ext r act s wer e concent r at ed via rot ar y evapor at ion and dr ied under N 2 . The ext r act was fr act ionat ed using column chr omat ogr aphy on alumina (Sigma-Aldr ich 507C, ca. 150 mesh) deact ivat ed t o Br ockmann gr ade I V by adding 0.1 wt % dist illed wat er . The non-polar fr act ion (1) was r ecover ed wit h hept ane. The slight ly polar fr act ion (2) was r ecover ed wit h t oluene. The medium polar it y fract ion (3) was r ecover ed wit h CH 2 Cl 2 and CH 2 Cl 2 /M eOH (9/1; v/v). Each fr act ion was concent r at ed via r ot ary evapor at ion and dissolved in ca. 3 ml hept ane, t oluene, and CH 2 Cl 2 , respect ively. The main component s of t he t hr ee fr act ions wer e quant ified using int er nal standar ds: t etr at r iacont ane for fr act ion 1 and t r icosan-1-ol for fr act ions 2 and 3.

Analysis

The hydr ocar bon fr act ion was analyzed as such, while t he ot her two fr act ions wer e conver t ed t o tr imet hylsilyl der ivat ives pr ior t o analysis. Aliquot s (30 µl) of the fr act ions wer e dr ied under N 2 and t hen silylat ed with 30 µl N,O-bis-(t r imet hylsilyl)tr ifluor oacet amide (BSTFA) for 1 h at 80 °C. Aft er cooling, t he aliquot was dr ied under a gent le st r eam of N 2 and dissolved in 30l of appr opr iat e solvent (t oluene for fr act ion 2 and CH 2 Cl 2 for fr act ion 3). Assignment and quant ificat ion of lipids wer e carr ied out using gas chr omat ogr aphy coupled-mass spect romet r y (GC-M S) and GC wit h flame ionizat ion det ect ion (FI D, 350 °C), r espect ively. Separ at ion was with an Agilent 6890N gas chr omat ogr aph equipped with a split /split less inject or (350 °C) and fit t ed wit h a fused silica column, coat ed wit h VF5-M S (50 m  0.32 mm i.d., 0.12 µm film thickness). The GC condit ions wer e: 80 °C t o 100 °C at 10 °C/min, and t hen t o 325 °C (held 30min) at 4 °C/min. He was the carr ier gas at a const ant 2 ml/min. For GC-MS an Agilent 5973N electr on impact mass spect r omet er (70eV, scan r ange m/z 35-800) was used. Component s wer e assigned accor ding t o libr ary and published mass spect r a. Quant ificat ion was achieved fr om the r esponse fact or in GC-FI D of the measur ed component s and compar ison of GC-FI D peak ar eas wit h t hose of t he int er nal st andar ds, int roduced to the fr act ions in known amount . Response fact or s wer e obt ained fr om calibr at ion curves of t he st andar d and t he component , or compound wit h similar st r uct ur e, polar it y and molecular weight , if no r eference compound was available. Coelut ion in GC-FI D occasionally pr event ed quant ificat ion of some lipid component s. Biomar ker cont ent was quant ified as µg/g dr y biomass and fur t her expressed in % r elat ive t o init ial cont ent in Fig. 1 The lipid fingerpr int of degr ading beech leaves r evealed no major qualit at ive changes dur ing 2 yr of diagenesis in t he soil or at t he wat er -sediment int er face. M ost of t he init ial component s were st ill pr esent at t he end of t he experiment and no degr adat ion product s wer e evidenced. Quant ificat ion of t he main component s of each lipid class for each degr adat ion st age showed that t he concent r at ion of all t he biomarker s st udied was (i) ver y var iable but (ii) subst ant ially r educed at t he end of the exper iment , alt hough t o var ying ext ent (Fig. 1).

Var iability in biomarker concentr ation

I n t his 2 yr exper iment, t he biomar ker cont ent of degr ading leaves exhibit ed subst ant ial var iabilit y among lit t er bags, even for a given t ime st ep wit hin a given envir onment al condit ion (Fig. 1). Ther efor e, we address t he pot ent ial or igin of such var iabilit y befor e discussing degr adat ion pat t erns. The differ ence in biomar ker cont ent bet ween r eplicat e lit t er bags maximized in soil at 70% and 100% of init ial leaf cont ent , for oleanolic acid and C 16 phyt yl est er , r espect ively. Ot her quant ified compounds (-Sit ost er ol, C 16 acid, C 28 alcohol and C 27 alkane) exhibit ed maximum differ ences between r eplicat e lit t er bags of around 40%. Such var iabilit y may r elat e t o (i) init ial var iabilit y of leaf lipid cont ent and/or t o (ii) var iabilit y in microenvironment al condit ions. The concent r at ion of leaf lipids is known t o vary subst ant ially between species, wit hin species, wit hin individual s and even wit hin a given leaf (H er bin [START_REF] Robins | Pat t erns of leaf variat ion and development in leaf wax alkanes[END_REF][START_REF] Gülz | Development al and seasonal var iat ions in t he epicut icular waves of Tilia tomentosa leaves[END_REF][START_REF] Gao | M ajor tr ends in leaf wax abundance, δ 2 H and δ 13 C values along leaf venat ion in five species of C 3 plant s: Physiological and geochemical implicat ions[END_REF]. I n agr eement wit h the lower var iabilit y of in vit ro exper iment s vs. field st udies, the var iabilit y in lipid decay is higher her e than r epor t ed for beech lipids in a labor at or y exper iment [START_REF] Rieley | L abor at or y st udies on sediment ary lipids pr eservat ion: differ ent pr eservat ion pot ent ial infer r ed for leaf and algal lipids[END_REF]. Soils, and especially for est soils, ar e r ecognized as highly het er ogeneous environment s (H edges and [START_REF] Oades | Compar at ive organic geochemist r y of soils and mar ine sediment s[END_REF][START_REF] Bélanger | Sampling for est soil . I n[END_REF][START_REF] L Ehmann | Spat ial complexity of soil or ganic mat t er for ms at nanomet re scales[END_REF]. A number of biogeochemical char act er ist ics of soils commonly vary wit hin a few decimet er s, so that differ ent decay pr ofiles bet ween lit t er bags separ at ed by ca. 20 cm is not unexpect ed (Wit kamp [START_REF] Olson | Energy st or age and t he balance of pr oduct s and decomposer s in ecological syst ems[END_REF][START_REF] Coe-K Lopat Ek | The effect s of lit t er qualit y and climat e on decomposit ion along an elevat ional gr adient[END_REF][START_REF] Suchewabor Ipont | Zonal var iat ion in leaflit t er decomposit ion in a secondar y mangr ove forest[END_REF]. Addit ionally, t he main dr iver s of lit t er decomposit ion in Br euil -Chenue exper iment al for est ar e sapr ophyt ic fungi , which are r epr esent ed mainly by thr ee species (M ycena inclinata, Mycena galopus and Phallus impudicus) t hat gener ally exhibit a het er ogeneous spat ial dist r ibut ion (Zeller et al., 2007). Decay condit ions within sediment s ar e believed t o exhibit smaller spat ial var iabilit y, alt hough leaf degr adat ion at the wat ersediment int er face under a subst ant ial wat er column has seldom been invest igat ed in t he field. Never theless, leaf decomposit ion in seawat er and st r eam wat er was shown t o be var iable over a few mont hs monit or ing and/or wit hin small ar eas [START_REF] Suber Kr Opp | Changes in t he chemical composit ion of leaves dur ing pr ocessing in a woodland st r eam[END_REF][START_REF] Rublee | Decomposit ion of t ur t legr ass (Thalassia testudinum K onig) in flowing sea-wat er t anks and lit t er bags: composit ional changes and compar ison with nat ur al par t iculat e mat t er[END_REF][START_REF] Webst Er | Vascular plant br eakdown in fr eshwat er ecosyst ems[END_REF][START_REF] Boult On | A r eview of met hodology used t o measur e leaf lit t er decomposit ion in lot ic envir onment s: Time t o t urn over an old leaf? M ar ine and Fr eshwat er Resear ch[END_REF][START_REF] Sant Os | Element al and st able isot ope composit ion of Zoster a noltii (H or neman) leaves dur ing t he ear ly phases of decay in a t emper at e mesot idal lagoon[END_REF][START_REF] Gonçalves | L eaf br eakdown in two tr opical st reams: Differences bet ween single and mixed species packs[END_REF]. H ydr ocar bon and FA cont ent of deciduous leaves degr ading in a blackwat er st r eam were shown t o vary subst ant ially over thr ee mont hs (M ills et al., 2001).

Occasional incr ease in biomar ker concentr ation

The lipids occasionally exhibit ed a higher cont ent in degr ading leaves t han in init ial senescent leaves. That was the case for 4 of t he 6 classes invest igat ed: -sit ost er ol r eached 112% of it s init ial cont ent in pond leaves aft er 27 weeks of degr adat ion, oleanolic acid had a cont ent of bet ween 114% and 141% for some lit t er bags in t he soil and in the pond bet ween 9 and 40 weeks of degr adat ion, 123% of t he init ial C 16 phytyl est er was det ect ed aft er 5 weeks in a soil lit t er bag, and the n-C 27 alkane reached 124% and 103% of it s init ial cont ent in pond lit t er bags aft er 27 and 103 weeks, r espect ively. L abor at or y incubat ion of beech leaves by an est uar ine microbial communit y [START_REF] Rieley | L abor at or y st udies on sediment ary lipids pr eservat ion: differ ent pr eservat ion pot ent ial infer r ed for leaf and algal lipids[END_REF] also showed an init ial st er ol incr ease t hat was at t r ibut ed t o t he release of fr ee compounds fr om a bound st at e. Gr ass degr adat ion in soil lit t er bags similar ly exhibit ed an occasional incr ease in n-alkane cont ent over 10 mont hs, r eaching up t o 230% of t he init ial cont ent [START_REF] Wang | Chemical and car bon isot opic dynamics of gr ass or ganic mat t er dur ing lit t er decomposit ions: A lit t er bag exper iment[END_REF]. I nit ial incr ease in alkane cont ent was also not ed in maple, ash and beech leaves dur ing a 2 year lit t er bag degr adat ion, and their deut er ium cont ent suggest ed a micr obial contr ibut ion [START_REF] Zech | Effect of leaf lit t er degr adat ion and seasonalit y on D/H isot ope r at ios of nalkane biomar ker s[END_REF]. I sot ope char act er izat ion ( 13 C) of t he alkanes from t he soil lit t er bags of t he pr esent st udy showed t hat micr obial alkanes also cont r ibut ed t o t he alkane pool in degr ading leaves [START_REF] Nguyen Tu | Ear ly degr adat ion of plant alkanes in soils: A lit t er bag exper iment using 13 C-labelled leaves[END_REF]. The occasional incr ease in some of t he biomarker s may thus r eflect eit her an ext ernal cont r ibut ion, especially for r at her ubiquit ous compounds such as fat t y lipids, and/or moiety release from bound st r uct ur es.

Biomarker degr adation pr ofiles

Degr adat ion at t he wat er -sediment int er face would be expect ed t o be less ext ensive t han in a soil. H owever, in t he present exper iment t he high var iabilit y in biomar ker concentr at ion pr ecludes such a conclusion (Fig. 1). I ndeed, this high var iabilit y pr event ed calculat ion of r epr esent at ive decay r at es for most biomar ker s. Consequent ly, no differ ence in degr adat ion r at es could be evidenced bet ween t he soil and t he pond lit t er bags. This lack of slower degr adat ion in t he pond than in the soil may also be par t ly due t o the oxic condit ions at t he bot t om of t he pond (Sect ion 2.1), which ar e known t o be mor e favor able for degr adat ion t han anoxic ones. The var iabilit y of t he pr esent r esult s calls for caut ion when compar ing decomposit ion pat t erns, but t he quant ified lipids seemed t o degr ade accor ding t o t hr ee main pr ofiles, at least dur ing t he fir st 6 months (Fig. 1).

-Phyt yl est er s were r apidly ext ensively degr aded, as shown by the phytyl hexadecanoat e cont ent , which dropped t o ≤ 10% aft er 15 weeks in t he soil, as well as in t he pond.

-Fat t y lipids (n-alkanes, n-acids and n-alcohols) under went exponent ial -like decr ease, r eaching ca. 7% of the init ial cont ent at t he end of the exper iment . M ass loss of bulk leaves is oft en appr oximat ed by an exponent ial decay model in soils and st r eams, as the r at e of mass loss is assumed t o be a const ant fr act ion of t he amount of mat er ial r emaining [START_REF] Olson | Energy st or age and t he balance of pr oduct s and decomposer s in ecological syst ems[END_REF][START_REF] Webst Er | Vascular plant br eakdown in fr eshwat er ecosyst ems[END_REF]. The pr esent r esult s may t hus suggest similar degr adat ion kinet ics for fat t y lipids.

-Polycyclic t r iter penoids exhibit ed a var iable degr adat ion pat t er n and r emained at  15% of t he init ial cont ent aft er 2 yr, as shown by t he -si t ost er ol and oleanolic acid profiles (Fig. 1). These r esult s mainly mat ch those repor t ed for a thr ee month labor at or y incubat ion of beech leaves, alt hough t he ment ioned labor at or y exper iment was mor e favor able for phytyl est er pr eservat ion and less for alkanes [START_REF] Rieley | L abor at or y st udies on sediment ary lipids pr eservat ion: differ ent pr eservat ion pot ent ial infer r ed for leaf and algal lipids[END_REF]. The pr esent r esult s also differ slight ly fr om t he st abilit y scales oft en deduced fr om sediment st udies since n-alkanes ar e gener ally consider ed as t he most st able lipid component s and funct ionalized polycyclic t er penoids as being moder at ely st able [START_REF] Cr Anwell | Diagenesis of fr ee and bound lipids in t err est r ial detr it us deposit ed in a lacust r ine sediment[END_REF][START_REF] Colombo | L ipid biogeochemist r y in the L aur ent ian Though-I I . Changes in composit ion of fat ty acids, st er ols and aliphat ic hydr ocar bons dur ing ear ly diagenesis[END_REF]. Our r esult s t hus emphasize t he complexit y and var iabilit y of l ipid degr adat ion in nat ur al envir onment s.

Distr ibution patter n of fatty lipids

Despit e t he subst ant ial degr adat ion of fat t y lipids, their main dist r ibut ion pat t ern at t he end of t he exper iment was similar t o the st ar t ing one ( [START_REF] Bol | Post -glacial var iat ions in dist r ibut ions, 13 C and 14 C cont ent s of aliphat ic hydr ocar bons and bulk or ganic mat t er in thr ee t ypes of Br it ish acid upland soils[END_REF][START_REF] Celer Ier | Dept h-r elat ed var iat ions in or ganic mat t er at t he molecular level in a loamy soil: r efer ence dat a for a longt er m exper iment devot ed t o t he car bon sequest r at ion r esearch field[END_REF][START_REF] Zech | Effect of leaf lit t er degr adat ion and seasonalit y on D/H isot ope r at ios of nalkane biomar ker s[END_REF][START_REF] Wiesenberg | Use of molecular r at ios t o ident ify changes of M iscanthus  gigant eus (Greef et Deu.) soil dur ing a labor at ory exper iment[END_REF], which wer e at t r ibut ed t o var ious causes, including cont r ibut ion from micr obial lipids, r oot lipids or else ant hr opogenic pollut ion. Never t heless, n-alkanes from sever al gr ass species in soil lit t er bags showed var iable degr adat ion pr ofiles but no change in ACL and CPI [START_REF] Wang | Chemical and car bon isot opic dynamics of gr ass or ganic mat t er dur ing lit t er decomposit ions: A lit t er bag exper iment[END_REF], similar ly t o the pr esent st udy. Nguyen Tu for t echnical assist ance. The wor k was funded by a gr ant fr om the Fond Nat ional pour la Science of Fr ance (ACI JC 10051 -not ificat ion 035214). Thanks ar e also due t o S. Feakins and an anonymous r efer ee, who provided const r uct ive r eviews.

Associate editor -K .G.J. N i er op

Fi gur e capt i ons

  for compar ison pur poses. 3. Resul t s and di scussi on 3.1. Biomarker behaviour The composit ion of lipids fr om senescent leaves of Fagus sylvatica has been descr ibed by Nguyen Tu et al. (2007). The lipids were dominat ed by st er ols, the most abundant being -sit ost er ol, and t o a lesser ext ent , by acyclic isopr enoids, including phyt adienes and phyt yl est er s (C 6 -C 20 , max. C 16 ). Oleanolic tr it er penoids, mainly oleanolic acid, and fat t y lipids wer e also abundant . The lat t er compr ise an homologous ser ies wit h even chain lengt h pr edominance: mainly n-alkan-1-ols (C 14 -C 30 , max. C 28 ), n-acids (C 8 -C 28 , max. C 16 , C 28 ) and, t o a lesser ext ent , n-aldehydes (C 20 -C 32 , max. C 28 ). Predominant ly odd number ed ser ies also occurr ed in subst ant ial amount s: mainly n-alkanes (C 12 -C 33 , max. C 27 ), and t o a lesser ext ent n-alkan-2-ols (C 23 -C 29 , max. C 27 ) and n-alkan-2-ones (C 25 -C 29 , max. C 27 ).

Fi g. 1 .

 1 Figure1

Table 1 ,

 1 Fig.2): they remained mar kedly dominat ed by a single homologue (n-C 28 for alcohols, n-C 16 for acids, and n-C 27 for alkanes), wit h a slight narr owing of t he dist r ibut ion r ange. Chain lengt h dist r ibut ion indexes r emained similar t o t he init ial values (Table1): t he aver age chain lengt h (ACL ) cent er ed ar ound the dominant homologue and the car bon pr efer ence index (CPI ) close t o zer o for acids and alcohols, and mar kedly >1 for alkanes. The only not iceable change in lipid ser ies dist r ibut ion concerns FAs, for which the long chain homologue (n-C 28 ) is r apidly degr aded (i.e. no longer det ect able aft er 5 weeks). H owever , such degr adat ion did not modify chain length dist r ibut ion indexes (Table1). On t he cont r ary, sever al st udies have r epor t ed ACL and CPI modificat ions t hr ough degr adat ion(Meyer s and I shiwar at i, 1993;

  Taken t ogether, t he r esult s suggest t hat, in spit e of degr adat ion and pot ent ial cont r ibut ion fr om ext ernal lipids, t he or iginal plant lipids can r emain the main sour ce of lipids in degr aded plant debr is and can t ypically r et ain their chain lengt h char act er ist ics. The result s t hus t end to validat e t he use of chain lengt h char act er ist ics of aliphat ic ser ies for source and pal eoenvir onment r econst ruct ion, at least on t he mid-t er m scale (i.e. a few year s). ant ial differ ence bet ween the soil and the pond could be det ect ed in biomarker degr adat ion pat t er ns dur ing t he 2 yr lit t er bag decay. All t he const it uent s init ially pr esent in the senescent leaves wer e degr aded, alt hough t o var iable ext ent. Phytyl est er s wer e the most ext ensi vely degr aded, while polycyclic t r it er penoids appear ed relat ively bet t er pr eser ved, wit h fat t y lipids exhibit ing int er mediat e degr adat ion pat t erns. Never t heless, t he main lipid const it uent s of init ial senescent leaves wer e st ill det ect ed aft er two 2 yr degr adat ion in bot h envir onment s. Dur ing t he exper iment , an occasional incr ease in t he abundance of sever al biomar ker s was not ed, implying moiet y r elease from bound st r uct ur es and/or an ext er nal cont r ibut ion. Alt hough fat t y lipids concentr at ions were markedly r educed, t he dist r ibution pat t erns wer e not subst ant ially affect ed, validat ing t heir use as source and envir onment al biomar ker s in ancient OM . The r esult s suggest that , for the mid-t er m, a plant biomar ker signat ure is not subst ant ially affect ed by differ ent ial degr adat ion in soil and at the wat er -sediment int er face under oxic condit ions, at least for a qualit at ive approach.

	4. Concl usi ons
	Compar at ive degr adat ion of leaf lipids in a soil and at t he wat er -sediment int er face
	of a well oxygenat ed pond r evealed that most of the biomar ker s exhibit ed highly var iable

cont ent , likely r eflect ing nat ur al microenvir onment al var iabilit y. Due t o t his var iabilit y and t he oxic condit ions in t he pond, no subst

Ack nowl edgement s

We ar e indebt ed t o J. Ranger for access t o the exper iment al sit e of Br euil-Chenue for est and D. Gelhaye, J.P. Calmet and P. Richar d for assist ance in t he field. Thanks ar e also due t o C. Fichot for access t o the pond. J. Br out in is acknowledged for encour agement and suppor t t o build the or ganic chemist r y labor at ory in his depar t ment . We ar e gr at eful t o B. David and S. L emet t r e for help in lipid pr epar at ion. We also t hank S. Peigné and S.

Table 1

 1 Distribution pattern of main fatty lipid series in F. sylvatica leaves.

		n-Alkanes		n-Acids		n-Alcohols
		C range (C max )	ACL a	CPI b	C range (C max C submax ) ACL a CPI b	C range (C max )	ACL a CPI b
	Initial leaves	C 12 -C 33 (C 27 )	26.4	9.9	C 9 -C 28 (C 16 , C 28 ) 16.1 0.0	C 14 -C 30 (C 28 )	25.3	0.0
	Pond	C 25 -C 29 (C 27 )	27.0 54.5	C 9 -C 24 (C 16 )	15.8 0.0	C 16 -C 30 (C 28 )	26.4	0.1
	Soil	C 12 -C 33 (C 27 )	26.9 14.7	C 9 -C 24 (C 16 )	16.6 0.1	C 16 -C 30 (C 28 )	26.2	0.0
	a n[C n ]) / [C n ]
	b [C 17+19+21+23+25+27+29+31+33 ]/[C 16+18+20+22+24+26+28+30+32 ]

Appendi x A. Suppl ement ar y dat a

Geochemical char act er ist ics of the wat er column ar e given: t he r ange of values measur ed in the wat er column (Table A.