Marie-Paule Norini
email: mariepaule.norini@outlook.com

Hugues Thouin

Florie Miard

Fabienne Battaglia-Brunet

Pascale Gautret

Régis Guégan

Lydie Le Forestier

Domenico Morabito

Sylvain Bourgerie

Mikael Motelica-Heino

Mobility of Pb, Zn, Ba, As and Cd toward soil pore water and plants (willow and ryegrass) from a mine soil amended with biochar

Keywords:

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

Introduction

The exploitation of a large number of mining sites over hundreds of years has generated large amounts of wastes such as tailings resulting from crushing and ore concentration, and consisting of fine particles and/or fine sand or silt [START_REF] Li | A review of soil heavy metal pollution from mines in China: pollution and health risk assessment[END_REF]. Mine soils developing on these mine residues have particular physical and chemical properties that may lead to serious environmental problems due to the structure of the soils (e.g. diffusion of particles resulting from erosion) or to release of high metal(loid) concentrations [START_REF] Barrutia | Native plant communities in an abandoned Pb-Zn mining area of northern Spain: implications for phytoremediation and germplasm preservation[END_REF]. The main toxi-city problems of such contaminated soils are often related to metal(loid) s such as Cd, Pb, Hg, As, Cu, Zn, and Cr [START_REF] Ghosh | Wetland macrophytes as toxic metal accumulators[END_REF]. While the poisonous and/or carcinogenic effects of Pb, As and Cd no longer need to be proven, those related to Ba (different disorders and metabolic problems) remain to be defined [START_REF] Martin | Human health effects of heavy metals[END_REF][START_REF] Martin | Human health effects of heavy metals[END_REF][START_REF] Kravchenko | A review of the health impacts of barium from natural and anthropogenic exposure[END_REF]. Ba concentrations range from 13 to 2050 mg.kg -1 in the subsoil and from 30 to 1870 mg.kg -1 in topsoil phytostabilization) metal(loid)s, are interesting candidates for phytoremediation [START_REF] Sharma | Phytoremediation: role of terrestrial plants and aquatic macrophytes in the remediation of radionuclides and heavy metal con-taminated soil and water[END_REF][START_REF] Vamerali | Field crops for phytoremediation of metalcontaminated land. A review[END_REF]. Lolium perenne L. (perennial ryegrass) can accumulate and tolerate metal(loid)s without its growth being affected by high metal(loid) concentrations. Ryegrass is therefore a species commonly used for surface stabilization [START_REF] Arienzo | The potential of Lolium perenne for revegetation of contaminated soil from a metallurgical site[END_REF]. Salix viminalis L. (basket willow) has a different root system from that of ryegrass and therefore explores a different and deeper soil volume compared with the herbaceous plant. It is known that willow does not accumulate high concentrations of metal(loid)s [START_REF] Sytar | Plants used for biomonitoring and phytoremediation of trace elements in soil and water[END_REF], although some authors suggest an accumulating capability for Cd, Cr, Pb, and Hg [START_REF] Schmidt | Enhancing phytoextraction: the effect of chemical soil manipulation on mobility, plant accumulation, and leaching of heavy metals[END_REF] and a capability to survive on an As, Pb and Sb contaminated soil [START_REF] Bart | Phytostabilization of As, Sb and Pb by two willow species (S. viminalis and S. purpurea) on former mine technosols[END_REF]. Willow species are suitable candidates for remediation of metal con-taminated soils given their high resistance to many pollutants and their high biomass production and fast growth [START_REF] Greger | Use of willows in phytoextraction[END_REF].

Phytoremediation can be assisted and reinforced by adding organic or inorganic amendments to potentially enhance the organic content of degraded soils by increasing plant establishment and survival [START_REF] Fellet | Elements uptake by metal accumulator species grown on mine tailings amended with three types of biochar[END_REF] and also by reducing the mobility, transfer and bioaccu-mulation of metal(loid)s [START_REF] Ahmad | Biochar as a sorbent for contaminant management in soil and water: a review[END_REF]. BC appears to be one of the most promising amendments given its ability to improve both the physicochemical and biological properties of soils (Beesley et al., 2011). BCs are stable carbon-rich pyrolysis products from various organic matters (wood, manure, rice husk, etc.). With its singular porous structure and alkaline properties, BC helps to increase soil pH (neu-tralization of acidity) and redox potential (Eh) and also improves soil water retention capacity (reduction of leaching) [START_REF] Atkinson | Potential mechanisms for achieving agri-cultural benefits from biochar application to temperate soils: a review[END_REF] while enhancing cation exchange capacity (CEC) [START_REF] Cheng | Oxidation of black carbon by biotic and abiotic processes[END_REF]. Furthermore, BC can even reinforce soil enzymatic and microbial activities and diversity [START_REF] Ahmad | Biochar as a sorbent for contaminant management in soil and water: a review[END_REF][START_REF] Ahmad | Impact of soybean stover-and pine needle-derived biochars on Pb and as mobility, microbial community, and carbon stability in a contaminated agricultural soil[END_REF] thereby providing possible diverse niches with suitable conditions for microorganisms while improving plant growth [START_REF] Sohi | A review of biochar and its use and function in soil[END_REF][START_REF] Quilliam | Nutrient dynamics microbial growth and weed emergence in biochar amended soil are influenced by time since application and reapplication rate[END_REF]. With its large specific surface, BC may adsorb metal(loid)s through multiple interaction mechanisms such as electrostatic adsorption, ion exchange interactions or co-precipitation, and by the complexing function of oxygen functional groups and π electrons [START_REF] Sohi | A review of biochar and its use and function in soil[END_REF]Beesley et al., 2011;[START_REF] Ahmad | Biochar as a sorbent for contaminant management in soil and water: a review[END_REF]. More specifically, metal ca-tions may interact with active sites provided by BCs' functional groups (carboxylic, amine, hydroxyl, carbonyl, alcoholic and phenolic groups and some inorganic mineral species; [START_REF] Gul | Physico-chemical properties and microbial responses in biochar-amended soils: mechanisms and future directions[END_REF] leading to highly stable BC-metal cation complexes [START_REF] Baldock | Chemical composition and bioavailability of thermally altered Pinus resinosa (Red pine) wood[END_REF]. BC may also uptake metal cations by chemical precipitation with inorganic components [START_REF] Wang | Physicochemical and sorptive properties of biochars derived from woody and herbaceous biomass[END_REF]. Organic functional groups on BCs influence the pH increase in soil and can therefore improve the stability of metal cations. Moreover, negatively charged organic functional groups increase over time with their oxidation in the soil [START_REF] Cheng | Natural oxidation of black carbon in soils: changes in molecular form and surface charge along a climosequence[END_REF]. Several studies have evaluated the effect of BC amendments on assisted phytoremediation of contaminated mine soils [START_REF] Paz-Ferreiro | Use of phytoremediation and biochar to remediate heavy metal polluted soils: a review[END_REF] but few studies have aimed at determining the availability and behavior in mine soil pore water (SPW) and the absorption of metal (loid)s by plants [START_REF] Lebrun | Eco-restoration of a mine technosol according to biochar particle size and dose application: study of soil physico-chemical properties and phytost-abilization capacities of Salix viminalis[END_REF][START_REF] Bart | Phytostabilization of As, Sb and Pb by two willow species (S. viminalis and S. purpurea) on former mine technosols[END_REF][START_REF] Beesley | The immobilisation and retention of soluble arsenic, cadmium and zinc by biochar[END_REF].

The present study aims to determine the behavior of Pb, Zn, Ba, As and Cd in a contaminated mine soil amended with BC and planted with willow and ryegrass. We investigated the potential impacts of BC from hardwood feedstock (2% and 5% BC (w/w)) on: (1) physicochemical properties of SPW; (2) total dissolved concentrations of metal(loid)s in SPW; and (3) distribution of metal(loid)s in plant organs and their ca-pacities to promote plant growth in a mine soil. To our knowledge, there have been only a few reports on the behavior of Ba, and even fewer in the presence of BC. This metal has, accordingly, been the focus of special investigation.

Materials and methods

Study site and mine soil characterization

The study covered a mine soil formed by Ag-Pb ore mining. The mine soil was located in the mining district of Pontgibaud (Roure-Les-Rosiers, Massif Central region, France; 45°47′29″ N, 2°49′17″ E). The site is located at an average altitude of 750 m with annual rainfall of 579 mm and average temperature ranging from 1.3 °C (winter) to 16.6 °C (summer). Geologically, the site is located on metamorphic rocks of the migmatite type (metatexites) with nearby inclusions of basalts and amphibolites posterior to the emplacement of the meta-morphic soil [START_REF] Bouladon | Le faisceau filonien de Pontgibaud (Puy-de-Dôme)[END_REF].

The site has diversified mineralogy rich in secondary minerals (arsenopyrite, anglesite, barytine, pyrite, quartz, etc.). Quartz and/or barite gangues include sulphides. The site was formed through the deposition of laundry spills and slag from the ore processing and en-richment phases [START_REF] Dommanget | Ouvrages débouchant au jour et résidus miniers des concessions de Barbecot[END_REF]. Operation dates back to Gallo-Roman times (100-300 A.D.) and ceased in 1897, with a resumption of activities during the Second World War. Mining activity was most in-tense during the 19th century.

The samples were collected from a settling pond of the Ag-Pb ore mine. Sampling was systematic at a depth of 0-20 cm in the mine soil and was conducted using a 5 m mesh over an area of 300 m 2 . A total of 20 samples was collected and these were carefully mixed to form a single composite sample. Mining settling ponds were used mainly during the XIXth century to remove suspended solids from water pro-duced by a mining plant using physical treatment to concentrate metals. Due to the processed used at that time, the grain size of the tailings produced by the plant was rather coarse compared to more recent processes. Thus, in the pond the material deposited was manly sandy. After settling in the pond, the water was released in a nearby stream. The place from which the mine soil samples were taken for experiments was almost bare, with only very small patches of spontaneous vegeta-tion (trees and herbaceous) at the edge.

Mine soil and biochar

Composite soil samples were taken over a depth of 0-20 cm from the top layer of a mine soil from a settling pond and were air-dried, sieved through a 2-mm mesh, and stored at room temperature until analysis. The sandy mine soil had a pH of 4.7. The tailing material was dried at 40 °C, and crushed for sieving at 250 μm, according to standard NF ISO 11464. The tailing material (250 mg ± 0.5 mg) was then en-tirely solubilized with fluorhydric acid (5 mL) and perchloric acid (1.5 mL) by heating to 160 °C, and then taken up in hydrochloric acid (1 mL) according to NF X 31-147. The total concentrations of metal (loid)s in the mine soil measured by ICP/AES (detection limit (DL) in the mg.kg -1 range) were as follows: Pb = 12,519 mg.kg -1 , Zn = 143 mg.kg -1 , Ba = 1701 mg.kg -1 , As = 466 mg.kg -1 . Cd was below the DL of 2 mg.kg -1 . Analysis of major elements of the mine soil gave the following results: 2.54% K2O (ICP/AES), 0.01% MnO (ICP/ AES), 438 mg kg -1 P2O5 (ICP/AES), 1.12% SO4 (NF ISO 11048), and 84.8% SiO2, with CaO, Fe2O3, and MgO below DL (ICP/AES). The samples were extracted by a sequential extraction procedure for the fractionation of metal(loid)s [START_REF] Tessier | Sequential extraction procedure for the speciation of particulate trace metals[END_REF] after air drying and sieving through a 2-mm mesh. The water-soluble fraction and cation exchangeable fraction enabled determination of the phytoavailable part of the metal(loid)s, with the following results: 43.9 mg.kg -1 Pb, 7.5 mg.kg -1 Zn, 27.2 mg.kg -1 Ba, 0.2 mg.kg -1 Cd and 0.3 mg.kg -1 As. The cation exchange capacity (CEC), determined using the 0.05 N cobaltihexamine at soil pH method (standard NF ISO 31-130), was 4.56 cmol.kg -1 pH of the mine soil was 4.7 ± 0.01. Biochar (BC) from hardwood (source La Carbonerie, Crissey, France) was obtained after slow pyrolysis (500 °C) of platelets and chips of oak, beech and charm. It was ground (< 100 μm) for use as a soil amendment in pot experiments. Brunauer-Emmett-Teller (BET) surface area analysis, Barrett-Joyner-Halenda (BJH) pore size and volume and pH analysis were performed for this material. BC characterization showed a specific area of 242.4 m 2 .g -1 . Total pore volume was 0.17 cm 3 .g -1 with a mean pore diameter of 2.80 nm. The CEC de-termined using the 0.05 N cobaltihexamine at soil pH method (standard NF ISO 31-130) was 0.65 cmol.kg -1 pH of BC was 9.5 ± 0.01.

Measurements of total carbon, nitrogen and hydrogen were per-formed on the prepared mixtures of mine soil and BC prior to the pot experiment, using an elemental flash pyrolyser analyzer (Flash 2000; Thermo Fischer Scientific) on samples previously dried and crushed to obtain a fine powder < 70 μm. The total carbon concentrations were respectively 0.2%, 1.6%, and 4.7% for the mine soil without BC and for the mine soils with 2% and 5% BC. Total nitrogen concentrations of the mine soil were respectively 0.03% and 0.01% for the mine soil without BC and with 5% BC. No nitrogen was detected in the mine soil with 2% BC. Total hydrogen concentrations of the mine soil were 0.1% for the mine soil without BC and, respectively, 0.2%, and 0.2% for the mine soils with 2% and 5% BC. Elementary analysis of BC showed 81.2% carbon, 1.7% hydrogen, and 0.6% nitrogen.

Pot experiment

The experiment design is presented in Table 1. This was a pot experiment with full factorial design set up with 45 pots (3 amendments x 3 planting conditions x 5 replicates for each condition). Mine soil (equivalent to 400 g air-dried) was placed in 87 × 113 mm pots. BC was blended manually with mine soil at mass fractions (w/w) of 2% and 5% dry weight. Fifteen pots were planted with one non-rooted cutting (7.5 cm length x 1 cm diameter) of Salix viminalis L. (basket willow) (W) (source ONF, Guéméné-Penfao France) in mine soil without BC (5 pots), with 2% BC (5 pots) and with 5% BC (5 pots). Fifteen pots were sown with 30 seeds of Lolium perenne L. (perennial ryegrass) (R) (source Phytosem SAS, Gap, France) with the same soil treatments as for the willow. Fifteen pots were non-vegetated (NV), with the same treat-ments as for the willow and ryegrass.

Pots were kept for 5 weeks (ryegrass) or 7 weeks (willow) in a growth chamber with a 16 h day/8 h night photoperiod (20 ± 5 °C), 80% relative humidity and 800 μmol m -2 .s -1 of photosynthetically active radiation (PAR). Control pots were kept in the same conditions as the planted pots. Data from these controls correspond to analyses conducted at 5 and 7 weeks to match the duration of cultivation of the willow and of the ryegrass. The number of samples is not the same for each treatment because of mortality of certain plants, (see legend of Figures and Tables). The differences between the duration of cultivation of willows and of ryegrass are due to the fact that ryegrass showed signs of weakness. Pots with limited drainage were watered daily with a quantity of distilled water calculated to match 70% of the water holding capacity (WHC) previously measured for each blend of mine tailing-BC. The field capacity was measured using a pressure plate extractor to determine the water content of the soil considered to be ideal for plant Table 1 The experiment design is a pot experiment with full factorial design set up with 45 pots (3 amendments x 3 planting conditions x 5 replicates for each condition).The factors were (1) mine soil without biochar, (2) mine soil with 2% biochar, and (3) mine soil with 5% biochar. For each of the soil treatments: (a) 5 pots were non-vegetated, (b) 5 pots were planted with one non-rooted willow cutting, and (c) 5 pots were sown with ryegrass seeds. growth. The pF unit is derived from the soil moisture tension in cm H2O by taking the logarithm (pF = log (cm H2O)) [START_REF] Kirkham | Principles of Soil and Plant Water Retentions[END_REF]. Eva-luation of WHC allowed determination of the quantity of water to be provided (by weighing) for good plant growth in each of the conditions. A soil-moisture sampler (SMS) with a mean pore size of 0.15 μm (Rhizon, MOM model, Rhizosphere Research Products, Wageningen, The Netherlands) was placed at the center of each pot (at 45°) to collect the soil pore water (SPW) [START_REF] Cattani | Copper bioavailability in the rhizosphere of maize (Zea mays L.) grown in two Italian soils[END_REF]. The water yield with a pressure differential of 100 kPa was greater than 1 mL.min -1 [START_REF] Di Bonito | Overview of selected soil pore water extraction methods for the determination of potentially toxic elements in contaminated soils: operational and technical aspects[END_REF]. SPW (∼10 mL) was collected weekly, using syringes, two hours after watering. Given the SMS′ cut off threshold (0.15 μm), samples were not filtered. Each SPW sample was separated into several aliquots to allow a series of measurements and analyses. Measurements of pH, Eh, and electrical conductivity were performed directly on SPW samples, whereas dissolved organic carbon (DOC) and dissolved or-ganic nitrogen (DON) were analyzed the day of sampling after dilution of the aliquot. A portion of the aliquots was diluted and acidified with concentrated HNO3 to determine cation and metal(loid) concentrations, then stored (at 4 °C) until analysis.

2.4. Physicochemical analysis 2.4.1. Soil pore water analysis pH, Eh (platinum silver/silver chloride reference electrode) and electrical conductivity (EC) of SPW were measured at the initial and final steps with a Multi 197i portable multiparameter meter (WTW, Weilheim, Germany). DOC and DON concentrations were determined using a TOC 5050/SSM 5000-A elemental analyzer (Shimadzu, Kyoto, Japan) with C8H5KO4 (1000 mgCarbon.L -1 ) and KNO3 (100 mgNitrogen.L -1 ) used as standards. Ion chromatography (IC), using a 940 Professional IC Vario instrument (Metrohm, Switzerland) equipped with conductivity detectors, was used for major ion analyses. For anions (F -, Br -, Cl -, NO2 -, NO3 -, SO4 2-, and PO4 3-), a Metrosep A Supp 16 (150 mm × 4 mm) ionic resin column (Metrohm, Switzerland) and a Metrosep A Supp 4/5 guard column (Metrohm, Switzerland) were used, along with a self-regeneration suppressor (Model 833 suppressor unit, Metrohm, Switzerland). The eluent was Na2CO3 7.5 mM and NaOH 0.75 mM. For cations (Li + , Na + , NH4 + , K + , Ca 2+ , and Mg 2+ ), a Metrosep C6 (150 mm × 4 mm) separation column (Metrohm, Switzerland) was used with HNO3 1.7 mM and C7H5NO4 1.7 mM solutions as eluents. Some major ions were analyzed but were not detected in SPW (F -, Br -, NO2 -, PO4 3-, and Li + ).

Plant analysis

At harvest, the surviving plants were counted and each of the plant organs (leaves, stems, cuttings and roots) was isolated and washed for 30 min with 0.5 mM CaCl2 with slow agitation to remove adsorbed metal(loid)s [START_REF] Zacchini | Metal tolerance, accumulation and translocation in poplar and willow clones treated with cadmium in hydroponics[END_REF]; they were then rinsed thoroughly with doubly de-ionized water. CaCl2 was used to remove the adhering metal(loid)s from the cell wall and to avoid plasma membrane altera-tion. This desorption allows quantification of the intracellular fraction of metal(loid)s only, not of the metal(loid)s adsorbed on the plant surface. Plants organs were oven dried at 40 °C until constant weight.

Microwave-assisted digestion of plant organs was conducted with a pressurized closed-vessel microwave system (Multiwave 3000, Anton Paar GmbH, Germany). Around 200 mg ( ± 0.5 mg) of dry and pre-viously crushed sample were put into PFA-Teflon vessels with a mixture of 6 mL of 70% nitric acid and 3 mL of 37% hydrochloric acid. Samples were placed in the microwave and digested as follows: heating of samples over 15 min to a temperature of 180 °C; temperature of 180 °C maintained for 15 min; temperature allowed to drop over 15 min to reach 55 °C. Power was 850 W for all steps. The solution was then fil-tered at 0.45 μm (nitrocellulose filter, Sigma-Aldrich), transferred into a flask, and the volume adjusted to 50 mL with doubly de-ionized water. Blank controls using reagents (6 mL 70% nitric acid and 3 mL 37% hydrochloric acid) were treated in the same way.

Metal(loid) concentrations

Concentrations of metal(loid)s (Pb, Zn, Ba, As and Cd) in SPW and in plants were determined by inductively coupled plasma mass spec-trometry (ICP-MS; Finnigan Element XR, Thermo Electron, Germany) (DL in the μg.kg -1 range). The amounts of metal(loid)s found in plants were expressed either as a concentration (μg.g -1 ) in the dry material or as mineralomass (μg) (mineralomass = biomass of plant organs x [metal(loid)s]plant organs).

Metal(loid) transfer into plant organs

Three factors were determined to evaluate the capabilities of the metal(loid)s to transfer into the different plant organs. The transfer of metal(loid)s from the root to shoot parts (leaves or leaves and stems) was determined by the Translocation Factor (TF) (TF = [metal(loid) s]shoot/[ metal(loid)]root). The ability of the plant to accumulate metal (loid)s from mine soil in its tissues was determined by the Bioconcentration Factor (BCF) (BCF = = [metal(loid)]root/[ metal (loid)]SPW) and by the Biological Absorption (or Accumulation) Factor (BAF) (BAF = [metal(loid)]shoot/[ metal(loid)]SPW). The metal(loid) concentration in each plant part was obtained by calculating net bio-mass uptake.

Statistical analysis

All statistical tests were conducted in R 3.2.3 (R Development Core Team, 2014). After mortality of some plants, the corresponding samples were removed from the dataset, thereby reducing the number of re-plicates per treatment. The accuracy of the measurement of metal(loid) s, DOC and DON, assessed by the intra-species variance, was taken into account in the calculations of inter-species variance (total standard deviations). To analyze these data, inter-species averages were used. The other standard deviations were calculated directly from inter-spe-cies average. ANOVA and a post hoc test for homogeneity of variance were performed. Multiple comparisons were performed by the Dunnett test for comparing several treatments with a control with a 95% con-fidence level (mine soil without BC compared to mine soil with 2% and 5% BC). Multiple comparisons of means were performed by Tukey test for comparing treatments at levels *p < 0.05.

An unsupervised pattern recognition method by a hierarchical agglomerative cluster analysis (HCA) was performed on standardized data using the Ward method of linkage and using Euclidean distance be-tween samples as the similarity measurement. Correlations of metal (loid) concentration analyses were calculated using the non-parametric Spearman's correlation coefficients because most chemical parameters analyzed were not normally distributed and included outliers.

Results

Physicochemical parameters of soil pore water

Measurements of physicochemical parameters of soil pore water (SPW) (except dissolved organic carbon (DOC) and dissolved organic nitrogen (DON)) revealed changes in the composition of SPW due to biochar (BC) (Table 2). Overall, Eh dropped greatly while pH and electrical conductivity (EC) increased with the presence of BC. Con-centrations of major ionsespecially K + , Ca 2+ , Mg 2+ , Cl -, NO3 -, and SO4 2-were also influenced by BC amendment (mostly 5% BC) (SM 1). Some anions, such as NO2 -and PO4 3-, were not detected in SPW. The concentrations of major ions had decreased at the end of the ex-periment in both planted and non-planted pots, suggesting that this decrease is mainly attributable to time rather than to plant cultivation. For DOC, and especially for DON, variations among samples did not seem to be controlled by BC (Table 2). At the end of the experiment, pH and DOC values had fallen overall, whereas redox values had increased overall, except for R5. EC also decreased overall, except for NV5. At the final step there was almost no more DON. Closer examination of the Physicochemical properties of soil pore water (Redox potential (Eh), electrical conductivity (EC), pH, dissolved organic carbon (DOC), and dissolved organic nitrogen (DON)) at the beginning and at the end of experiment. I: average of initial values, NV: non-vegetated, W: willow, and R: ryegrass. 0, 2, and 5 for 0%, 2% and 5% biochar (BC). Data in parentheses indicate the standard error with n = 5 for NV0, NV2, NV5, R0 and R2; n = 4 for W2, W5, and R5; n = 3 for W0. ANOVA was followed by a post-hoc Dunnett test at level P < 0.05. Differences were evaluated comparing several treatments with a control (initial samples, I). Letters a, b, c refer to statistics on samples without BC, letters A, B, C to statistics on samples with 2% BC and letters α, β tor statistics on samples with 5% BC. For pairwise comparisons, analysis of variance was performed by a post-hoc Tukey test at different levels *P < 0.05 and n.s. for not significant statistics.

Eh

Electrical individual parameters showed that the duration of the experiment had more impact on the physicochemical parameters than the presence of plants (Table 2). For all treatments, physicochemical parameters were modified during the experiment. Redox potential increased between the beginning and the end of experiment for all treatments (by 57 mV for W2), except for NV0 and R0 which showed decreases (of 4 and 13 mV, respectively) and for R5, which remained constant. EC decreased during the experiment for all treatments (by 543.7 μS for NV2), with the exception of NV5 and W0 which showed increases (of 64.6 and 22.9 μS, respectively). pH values either remained constant compared to the average of initial values (for NV5 and R5) or decreased during experi-ment (drop of 0.4 for R2 and W5, and of 1 for W2). DOC decreased during the experiment whatever the treatment (respective losses of 3.2 mgC.L -1 and 5.6 mgC.L -1 for W5 and R2 compared to average of initial values). DON decreased for NV0 and W5 compared to initial values, with respective losses of 1.1 and 0.5 mgN.L -1 .

Metal(loid) concentrations in soil pore water

Total dissolved metal(loid) concentrations in SPW are given in Fig. 1. With the exception of Ba, concentrations of metal(loid)s de-creased with time. The presence of BC also induced a decrease in metal (loid) concentrations in SPW for Pb and Zn but had no significant effect on As and Cd. While [Pb]spw decreased during the experiment whatever the treatment, in the presence of plants significant differences were observed between the mine soils without BC and those with 2% BC. BC seemed to be a more influential parameter than plant presence (Fig. 1). Only the 2% BC and ryegrass combination showed a significant dif-ference, with higher [Pb]spw than the other treatments with 2% BC. It seemed that the behavior of [Zn]spw was similar to that of [Pb]spw. For the final samples, the addition of 5% BC tended to better reduce [Zn]spw whatever the treatment. As for Pb, BC did not have any influence on [Zn]spw in the presence of ryegrass. Multivariate statistical analysis of the sum of metal(loid)s accumulated by the plant (for each metal(loid) s), carried out using centered reduced principal component analysis Fig. 1. Total dissolved metal(loid) (Pb, Zn, Ba, As, and Cd) concentrations (μg.L -1 ) in the mine soil pore water at the beginning and at the end of experiment. BC: biochar, I: average of initial values, NV: non-vegetated, W: willow, and R: ryegrass. 0, 2, and 5 for 0%, 2% and 5% biochar. ANOVA was followed by a post-hoc Dunnett test at level P < 0.05 with n = 5 for NV0, NV2, NV5, R0 and R2; n = 4 for W2, W5, and R5; n = 3 for W0. Differences were evaluated comparing several treatments with a control (initial samples, I). Letters a, b, c refer to statistics on samples without BC, letters A, B, C to statistics on samples with 2% BC and letters α, β to statistics on samples with 5% BC. For pairwise comparisons, analysis of variance was performed by a post-hoc Tukey test at different levels *P < 0.05 and n.s. for not significant statistics. (PCA) (SM 2), showed close trends. The second principal component (F2) of PCA, which explained 29.1% of the total variability, did not separate the treatments, suggesting that [Pb]spw and [Zn]spw did not discriminate the samples. BC amendment seemed to significantly in-crease the [Ba]spw (Fig. 1). The presence of ryegrass also tended to increase [Ba]spw, especially for R2. PCA (SM 2) showed a similar trend. The first principal component (F1) of PCA explained 37.0% of the total variability, driven mainly by Ba concentration in SPW. Bi-dimensional plots which represent variable loadings and sample scores clearly se-parated the W0 samples from the R2 samples along the F1 axis (SM 2). No As and no Cd were detected in SPW at the end of experiment. Dif-ferences in initial metal(loid) concentrations were not due to BC amendment but to varying levels of contamination between samples.

The relationships between physicochemical parameters and metal(loid) concentration in soil pore water

The relations between all of the parameters measured in the SPW were shown by a heatmap built using Ward method of linkage and the Euclidean distance measurement (Fig. 2). This two-dimensional visua-lization of correlation was obtained from the 18 or 16 variables mea-sured, respectively, at the beginning and at the end of the experiment. The difference in the number of variables was due to the lack of de-tection of Cd and As in SPW at the end of the experiment. The heatmap yielded a combined dendrogram which clearly differentiates three groups of samples according to treatment (0% BC, 2% BC, 5% BC), at the beginning of the experiment (Fig. 2A) and at the end (Fig. 2B). The double hierarchical dendrogram reveals the correlation among treat-ments (column) and among chemical parameters (row). The column hierarchical dendrogram distinguishes treatments without BC, treatments with 2% BC and treatments with 5% BC, indicating that BC amendment was the most discriminating parameter acting on this group of variables. However, BC effect was not constant over time. At the beginning of the experiment, the treatments with 2% and 5% BC could be clearly distinguished from the treatment without BC, whereas at the end of the experiment, treatments with 5% BC exhibited a more marked difference (Fig. 2A). Over time, a 2% BC amendment no longer changed the composition of SPW. At the initial step, mine soil without BC was essentially characterized by high concentrations of metal(loid)s in SPW and by a low Eh. Variables influenced by the 5% BC amendment were a higher EC and ion concentrations (especially SO4 2-and Ca 2+ ) com-pared to mine soils without BC. Similarly, the dendrogram of variables indicated that at the beginning of the experiment Pb, Zn, and As con-centrations in SPW were related to Eh, DOC and Cl -, while Ba con-centration was linked to pH, EC and other major ions (Fig. 2A). Cor-relation matrices (Fig. 3) of aggregated data at initial step (Fig. 3A) showed that Pb and Zn concentrations in SPW were strongly positively correlated (0.83), and also negatively correlated with pH (-0.87 and -0.89 for Pb and Zn, respectively) and with EC (-0.77 and -0.71 for Pb and Zn, respectively). [Ba]SPW, [As]SPW, and

[Cd]SPW presented no correlations above 0.60. Correlation of aggregated data failed to high-light relations between the analyzed data. This showed that either the phenomena that govern the dynamics of these metal(loid)s were not highlighted by these correlations or that the aggregated data did not make it possible to highlight the correlations. It will be necessary to study correlations by treatment (SM 3). At the final step, samples amended with 5% of BC were ranked according to their pH and EC and also according to Ca 2+ , SO4 2-and Ba concentration (Fig. 2B). Thus, [Ba]SPW seemed to be linked to pH and conductivity whatever the time step considered, while Pb and Zn were linked to Eh at the beginning of the experiment and to Cl -at the end of the experiment. At the final step of the experiment, correlation of aggregated data (Fig. 3 B) showed a positive correlation between [Pb]SPW and [Zn]SPW (0.77). The con-centrations of these metal(loid)s were less related to the other para-meters than at the beginning of the experiment: correlation coefficient of [Pb]SPW and [Zn]SPW with pH (-65 and -0.50 for Pb and Zn, re-spectively) and EC (-0.41 and -0.37 for Pb and Zn, respectively). [Ba]SPW was correlated positively with pH (0.48 and 0.77 at the be-ginning and at the end of experiment, respectively) and correlated negatively with Eh (-0.57 and -0.78 at the beginning and at the end of experiment, respectively) (Fig. 3B). Regarding the study of correla-tions by treatment, the observation at the final step was different (SM 3). The positive correlation between [Pb]SPW and [Zn]SPW was sig-nificant only for certain treatments (1 for NV2, NV5 and W2, and 0.99 for R0).

Plant biomasses, metal(loid) concentrations in plant organs and mineralomasses

Willows and ryegrasses grown on mine soil without BC had no or Table 3 Survival rate and average biomasses dry weight (DW) (mg) W: willow, and R: ryegrass. 0, 2, and 5 for 0%, 2% and 5% biochar (BC). ANOVA was followed by a post-hoc Dunnett test at level P < 0.05. Differences were evaluated com-paring several treatments with a control (without BC namely W0 and R0) with n = 5 for NV0, NV2, NV5, R0 and R2; n = 4 for W2, W5, and R5; n = 3 for W0. Letters a, b, c refer to statistics on samples without BC, letters A, B, C to sta-tistics on samples with 2% BC and letters α, β to statistics on samples with 5% BC. For pairwise comparisons, analysis of variance was performed by a post-hoc Tukey test at different levels *P < 0.05; for not significant statistics, and n.d. not determined for unstated statistic (no data).

Survival rate

Biomasses poorly developed roots. For this reason, analyses were not conducted on the roots for mine soils without BC (Table 3, Table 4, Fig. 4, and Fig. 5).

Globally, the survival of the plants was improved by the presence of BC, especially for the 2% BC concentration (Table 3). Compared with the mine soil without BC, 2% and 5% BC favored higher plant biomasses, with significant differences depending on the species considered (Table 3). Thus, the global biomass of willow was, respectively, 14 times and 15 times higher for 2% and 5% BC; for ryegrass, overall biomass was, respectively, 2.4 times and 2.2 times higher for 2% and 5% BC. Growth difference was significant for all plant organs (Table 3). Metal(loid) concentrations in plant organs were determined at the end of the experiment. Concentrations are given in Fig. 4 and metal (loid) mineralomasses are presented in Fig. 5. The metal(loid) miner-alomass, which represents the total amount of metal(loid)s accumu-lated in the plants, can be used to evaluate metal(loid) removal effi-ciency by the plant organs on dry weight. For willow growing on mine soil with BC, Pb concentration in cuttings, stems and leaves, as well as in the roots, decreased significantly (P < 0.05) mainly for plants growing on mine soil with 5% BC. For ryegrass growing in the presence of BC, [Pb]plant also decreased, even if a significant decrease was ob-tained only for plants growing on mine soil with 2% BC, due to high heterogeneity of plants in samples without BC (Fig. 4B). However, Pb mineralomasses (Fig. 5) seemed to depend on plant organs, with more Pb in roots of willow or in leaves of ryegrass. A similar but less marked trend was observed for Zn. Plants growing in the presence of BC (re-gardless of its percentage), had less Zn in their tissues (Fig. 4). There was no difference in Zn concentrations in willow leaves for the different treatments (Fig. 4A). For ryegrass, only concentration of Zn in plants growing on mine soil amended with 2% BC showed a noticeable dif-ference (Fig. 4B). A similar finding was established for Zn mineralomass (Fig. 5). Ba appeared to behave differently. The plants growing in the mine soil with BC and in the mine soil without BC had similar Ba concentrations in their organs (Fig. 4) or similar Ba mineralomass (Fig. 5). The willows growing in mine soil with BC tended to have lower concentrations of As and less As mineralomass in their organs, except for leaves (Figs. 4 and5). For ryegrass growing on BC, a reduction of As in the leaves was noticeable although significant only for As mineralomass (Fig. 5A). When the willow grew on mine soils with BC, Cd concentration and mineralomass were increased whatever the quantity of BC (Figs. 4 and5).

Main contributing factors of F2 of the PCA (SM 2) were metal(loid)s in SPW (positively) and metal(loid)s accumulated in plants (nega-tively). This representation highlights opposing trends of concentra-tions of metal(loid)s in SPW and in plant organs, in particular for Pb and Zn (SM 2). This suggests that total metal(loid)s in plants depends on the level of concentration of metal(loid)s in SPW that the plant can absorb. These results were consistent with the influences of BC amendment on the amount of metal(loid)s in the SPW that can be up-taken by the plants.

Metal(loid) transfers into plant organs

The transfer of metal(loid)s was evaluated with the three factors: the translocation factor (TF), the bioconcentration factor (BCF), and the biological absorption (or accumulation) factor (BAF) (Table 4). These could not be determined in the absence of metal(loid)s in the SPW or when roots were nonexistent. The distribution of Pb in plant organs depended on the presence of amendments. For willow, Pb was mainly present in cuttings for plants growing on non-amended soil samples, whereas it was mostly present in the roots of plants growing on amended samples. Increasing BC content tended to reduce Pb con-centration in leaves of ryegrass growing on amended mine soils (Table 4). Regarding TF, none of the plants translocated Pb in aerial organs. The transport of Pb from SPW to aerial parts, expressed by the BAF, depended on the concentration of BC in the soil in which the Table 4 Metal(loid) transfer into plant organs (translocation factors (TF), bioaccumulation factor (BAF), and bioconcentration factor (BCF)). W: willow, and R: ryegrass. 0, 2, and 5 for 0%, 2% and 5% biochar (BC). ANOVA was followed by a post-hoc Dunnett test at level P < 0.05 with n = 5 for NV0, NV2, NV5, R0 and R2; n = 4 for W2, W5, and R5; n = 3 for W0. Differences were evaluated comparing several treatments with a control (without BC namely W0 and R0). Letters a, b, c refer to statistics on samples without BC, letters A, B, C to statistics on samples with 2% BC and letters α, β to statistics on samples with 5% BC. For pairwise comparisons, analysis of variance was performed by a post-hoc Tukey test at different levels *P < 0.05; n.s. for not significant statistics, and n.d. not determined for unstated statistics (no data). plants grew. Thus, BAF was higher for willows with 5% BC treatment (0.73) than for 2% BC (0.36). The level of transport of Pb from SPW to the root parts (expressed by BCF) was very high for all treatments planted with willow, whatever the BC concentration (15.73 and 28.52 respectively for plants growing in mine soils with 2% and 5% BC) (Table 4), whereas BCF was very low for ryegrass (0.01).

Pb

For Zn, only BAF and BCF calculated from [Zn]SPW had values greater than 1 (corresponding to W0 and R0, and R5 for BAFSPW and corresponding to R2 and R5 for BCFSPW) (Table 4). For Ba, the trans-location factors increased with the quantity of BC in which the plants grew. Ba translocation from the roots to the plants' aerial parts corre-lated positively with BC content. It was respectively 2 and 2.7 times higher for willow than for ryegrass in mine soils with 2% and 5% BC (Table 4). A high BAFSPW was obtained for plants growing in soil without BC (∼10.5 and ∼4 for willow and ryegrass, respectively). BCF calculated from the [Ba]SPW was also high for R2 and R5. For Cd, willow presented a TF > 1 (Table 4).

Discussion

Effect of biochar on physicochemical parameters of soil pore water

The application of biochar (BC) had a significant effect on the physicochemical parameters of soil pore water (SPW), especially at the beginning of the experiment. It has already been evidenced [START_REF] Ahmad | Biochar as a sorbent for contaminant management in soil and water: a review[END_REF] that BC can increase the values of pH, and electrical conductivity (EC) and Eh, and can provide several ions (NO3 -, SO4 2-, K + , Ca 2+ , Mg 2+ , Cl -) that can be quantified in SPW. The increase or decrease of dissolved organic carbon (DOC) depends on the BC used. Beesley et al. (2011) showed that BC contributes to increased soil or-ganic matter content, especially carbon and nitrogen, and also that it may react with metal(loid)s in soils more readily than native organic matter. However, our experiment did not show such an increase and the type of BC used in this experiment did not lead to increase in DOC (Table 2). This may be an obstacle for this experiment, but it has al-ready been demonstrated in other studies. As suggested by [START_REF] Ahmed | Insight into biochar properties and its cost analysis[END_REF], the carbon content of BC depends on the type of BC feedstock. According to [START_REF] Mukherjee | Organic carbon and nutrient release from a range of laboratory-produced biochars and biochar-soil mixtures[END_REF], BCs contain a range of nutrient forms with different release rates (depending on BC feedstock and on the conditions of production of the BC). The physicochemical changes linked to BC clearly distinguished the amended soils from the soil without BC at the beginning of the experiment. However, this effect decreased over time since, by the end of the ex-periment, only the mine soil with the highest BC content (5%) differed from the other two conditions (without and with 2% BC) (Figs. 2 and3).

Effect of biochar and plants on behavior of metal(loid)s

Effect of biochar and plants on availability of metal(loid)s

The study of SPW enables accounting for the concentration of watersoluble contaminants. This concentration corresponds in fact to the most labile fractions of metal(loid)s, which are the most bioavailable for living organisms but also the most ecotoxicologically hazardous [START_REF] Temminghoff | Effects of dis-solved organic matter on the mobility of copper in a contaminated sandy soil[END_REF]. Biogeochemical processes (e.g. precipita-tion, desorption, dissolution, adsorption, complexation, redox reac-tions) [START_REF] Adriano | Role of assisted natural remediation in environmental cleanup[END_REF] determine metal partitioning between the aqueous and solid phases and influence the availability and mobility of metal(loid)s in soil (Shahid et al., 2012a(Shahid et al., , 2012b)). As metal(loid)s such as Zn, Pb, and Cd are divalent cations, they could be adsorbed by the same sites and functional groups in soil and thus compete. The appli-cation of BC had variable effects depending on the metal(loid)s con-sidered. It is important to note that the application of BC used as an amendment in the experiment immediately reduced the availability of Pb and Zn in SPW. At the end of the experiment, the benefit of this amendment was less marked than at the beginning (Figs. 2 and3). This can be attributed to a decrease in sorption efficiency of BC. The Pb removal in aqueous solution by BC was diffusion-controlled (BC pore size dependent), starting with a rapid initial step followed by a slow-down as sorption approached equilibrium [START_REF] Mohan | Arsenic removal from water/wastewater using absorbants a critical review[END_REF][START_REF] Inyang | Removal of heavy metals from aqueous solution by biochars derived from anaerobically digested biomass[END_REF]. The availability of metals in the soil thereby de-creased with time [START_REF] Houben | Mobility, bioavailability and pH-dependent leaching of cadmium, zinc and lead in a contaminated soil amended with biochar[END_REF][START_REF] Burges | Impact of repeated single-metal and multimetal pollution events on soil quality[END_REF].

As plant uptake of metal(loid)s from soil is passive (mass flow of water into the roots) or active (transport across the plasma membrane of root epidermal cells), response to metal(loid)s depends on plant species [START_REF] Yoon | Accumulation of Pb, Cu, and Zn in native plants growing on a contaminated Florida site[END_REF]. L. perenne is a metal excluder, accumulation in the shoots being limited [START_REF] Nowack | Use of diffusive gradients in thin films (DGT) in undisturbed field soils[END_REF]. S. viminalis L. is able to survive in a soil contaminated with As, Pb and Sb [START_REF] Bart | Phytostabilization of As, Sb and Pb by two willow species (S. viminalis and S. purpurea) on former mine technosols[END_REF]. A difference in the growing times of the two species could be problematic when comparing the removal of metals by these plants. Metal(loid) concentrations in the plants expressed as mineralomasses put the metal(loid)s accumulated in the plants at the same level. However, the primary effect of BC application to mine soil was to greatly improve plant survival and yields (Table 3). BC input to the mine soil improved plant lifetimes and biomasses by releasing ions used by plants for their nutrition, as has already been shown in other work [START_REF] Fellet | Elements uptake by metal accumulator species grown on mine tailings amended with three types of biochar[END_REF]. This beneficial effect of BC is not always evidenced. Indeed, as suggested by Beesley et al. (2011), BC should be employed with a combination of composts, manures and other amendments be-cause some BC can retain nutrients useful for plants in degraded soils amended only with BCs. Without BC, neither ryegrass nor willow were able to develop a root system, as has already been observed for other mine soils [START_REF] Park | Role of organic amendments on enhanced bioremediation of heavy metal(loid) con-taminated soils[END_REF][START_REF] Lebrun | Eco-restoration of a mine technosol according to biochar particle size and dose application: study of soil physico-chemical properties and phytost-abilization capacities of Salix viminalis[END_REF].

Effect on Pb behavior

Pb was the major contaminant among the metal(loid)s present in our mine soil. When BC was provided the amount of Pb in SPW reduced drastically without any influence from plants (Fig. 1). This may indicate that the BC had the potential to immobilize Pb. The immobilization of Pb in soils is mostly due to the strong affinity of Pb for soil organic matter, where it is strongly sorbed (Kushwaha et al.2018). The increase of the mine soil pH related to addition of BC could be the main factor explaining Pb immobilization (Fig. 2). [Pb]spw and pH were correlated negatively at both the initial and final steps (Fig. 3). In addition, some authors have shown that BC application to water contributes to im-mobilization of metal(loid)s, and especially of Pb, through forces that are electrostatic (surface adsorption) and non-electrostatic (complexa-tion with functional groups) [START_REF] Jiang | Immobilization of Cu(II), Pb(II) and Cd(II) by the addition of rice straw derived biochar to a simulated polluted ultisol[END_REF][START_REF] Lu | Relative distribution of Pb2+ sorption mechanisms by sludge-derived biochar[END_REF]. These processes are strongly pH dependent. Indeed, as pH increases compe-tition between metal ions and protons decreases for binding sites on the BC or in the soil matrix, making adsorption more effective (Beesley et al., 2011). When BC is added to a soil (soil pH initially at about 4.7), it releases carbonates, phosphates and hydroxyl ions, promoting an overall liming effect [START_REF] Fellet | Elements uptake by metal accumulator species grown on mine tailings amended with three types of biochar[END_REF]. Metal(loid)s present in the soil compete for binding sites on BC. [START_REF] Namgay | Influence of biochar application to soil on the availability of As, Cd, Cu, Pb, and Zn to maize (Zea mays L.)[END_REF] have demon-strated that BC sorption strength decreases in the order Pb > Cu > Cd > Zn > As. Pb could have greater affinity for the carboxylic and phenolic functional groups situated on the surface of BCs [START_REF] Houben | Mobility, bioavailability and pH-dependent leaching of cadmium, zinc and lead in a contaminated soil amended with biochar[END_REF].

For willow and ryegrass growing on mine soil with 2% or 5% BC, Pb concentration in tissues was significantly lower than Pb concentration measured in plants growing in mine soil without BC. Ryegrass grown on mine soil with 2% BC influenced the behavior of Pb since only the 2% BC and ryegrass combination showed a significant difference with higher [Pb]spw than the other treatments. For both plants, even when growing on mine soil with BC, the majority of accumulated Pb was found in the roots (Figs. 4 and5). Translocation of metal(loid)s from root to shoot is effective when the translocation factor (TF) > 1 [START_REF] Baker | Terrestrials higher plants which hyper accumulate metallic elements. A review of their distribution, ecology and phytochemistry[END_REF]. Besides biomass production in the presence of metal (loid)s, metal(loid) bioconcentration efficiency is a parameter to eval-uate the feasibility of phytoremediation [START_REF] Mcgrath | Phytoextraction of metals and metalloids from contaminated soils[END_REF]. Ryegrass leaves also accumulated Pb. [START_REF] Cheraghi | Findings on the phytoextraction and phytostabilization of soils contaminated with heavy metals[END_REF] determined in their study that plants with a high biological absorption factor (BAF) (BAF > 1) are suitable for phytoextraction. Likewise, plants with a high bioconcentration factor (BCF) (BCF > 1) and low TF (TF < 1) have the potential for phytostabilisation. Even if the translocation factor was systematically below 1 for both plant species, this nonetheless leads to high phytoremediation efficiency parameters since BCFSPW is > 1 for willow in mine soil with 2% and 5% BC (Table 4). These results agree with those of [START_REF] Wahsha | Heavy metals accumulation in willows growing on Spolic technosols from the abandoned Imperina Valley mine in Italy[END_REF] who showed that willows have the ability to accumulate Pb in roots more than in the aerial parts. With a high bioconcentration factor and a low translocation factor (BCF > 1 and TF < 1) willow seem to have the potential for phytostabilization of PB in this mine soil and under these conditions.

Effect on Zn and Cd behavior

If the effect of BC was obvious in reducing Pb availability, the mechanisms linked to BC are less clear for other metal(loid)s present in the mine soil (Fig. 1). Although the Cd concentration was below the detection limit (< 2 ppm) in the mine soil, [Cd]SPW was detectable at the initial step (Fig. 1). These differences were mainly due to the de-tection limit of the analysis method used (see materials and methods part). The presence of Cd at the same time as Zn could be of some importance, as the presence of Zn in the mine soil studied may have affected the behavior of Cd. [START_REF] Kabata-Pendias | Trace Elements in Soils and Plants[END_REF] reported that Cd and Zn have similar ionic structures, electronegativities and chemical properties and Zn and Cd are water soluble, giving them high mobility in the soil. [START_REF] Beesley | The immobilisation and retention of soluble arsenic, cadmium and zinc by biochar[END_REF] have demonstrated the sorption of Zn onto BC particles, and that this retention was not im-mediately reversible.

Several authors have shown that Zn inhibits Cd uptake by plants. Zn competes with Cd because both metals are transported by a common carrier at the root plasma membrane [START_REF] Pandey | Phytoremediation of heavy metals from fly ash pond by Azolla caroliniana[END_REF]. Zn is more easily assimilated by plants than Cd, thus Cd phytoaccumulation decreases in the presence of Zn [START_REF] Hart | Zinc effects on cadmium accumulation and partitioning in near-isogenic lines of durum wheat that differ in grain cadmium concentration[END_REF]. Moreover, ryegrass induced a slight increase in Zn in SPW collected, in comparison with bared mine soil (Fig. 1). This may be due to the fact that the EC is higher in R samples than in NV samples (Table 2). [START_REF] Salimi | Influence of elec-trical conductivity on the phytoremediation of contaminated soils to Cd2+ and Zn2+[END_REF] highlighted that high EC levels of irrigation water increased Cd concentration and decreased Zn concentration in the shoots of Helianthus annuus. Plants growing in BC amended mine soil tended to have lower Zn uptake but, for Cd, plant concentration and mineralomass were increased for plants growing in BC amended mine soil. However, unlike Pb, Zn was easily transferred to willow shoots, as were Ba and Cd. Zn and Cd were transferred from roots to aerial parts for the willow but not for the ryegrass (Table 4). BAFSPW of Zn were > 1 for soil without BC but also with 5% BC, especially for ryegrass. This indicates a certain mobility of Zn but also an easy transfer into the aerial parts of the plants (Table 4). Most of the Zn was found in the leaves of the willows (Figs. 4 and5). Cd uptake depends on the exposure time and the species [START_REF] Grant | Cadmium accumulation in crops[END_REF]. Willows can tolerate and accumulate Cd in the root system: [START_REF] Landberg | Strategies of cadmium and zinc resistance in willow by regulation of net accumulation[END_REF] have demonstrated that S. viminalis accumulates Cd in leaves.

Effect on Ba behavior

Without BC, [Ba]SPW was relatively low and was not influenced by the presence of plants (Fig. 1). Even without plants, the mobility of Ba was less in mine soils without BC than in mine soils with BC, despite the more basic pH of SPW of the latter (Table 2). It would appear that in this study the BC contributed to Ba mobilization in SPW whereas pH of SPW increased (Table 2 and Fig. 1). The change in pH by adding BC was therefore not the factor that determined the mobility of Ba and its transfer to plants. These results agree with [START_REF] Mcbride | Concentrations of lead, cadmium, and barium in urban garden-grown vegetables: the impact of soil variables[END_REF], who reported no significant relationship between Ba in vegetables and soil pH. [START_REF] Lamb | Bioavailability of barium to plants and invertebrates in soils contaminated by barite[END_REF] and [START_REF] Myrvang | Geochemistry of barium in soils supplied with carbonatite rock powder and barium uptake to plants[END_REF] reported that the plant uptake of Ba was mainly controlled by the exchangeable Ba fraction in soil. [START_REF] Ball | WATEQ4F-user's Manual with Revised Thermodynamic Data Base and Test Cases for Calculating Speciation of Major, Trace and Redox Elements in Natural Waters[END_REF] noted that the concentration of free Ba in natural water is limited by equilibrium with barite (BaSO4) and witherite (BaCO3). For [START_REF] Suwa | Barium toxicity effects in soybean plants[END_REF], Ba solubility can be increased by the presence of other anions, except for SO4 2-. Thus, Ba solubilization was not controlled here by pH, but more probably by the ions released by BC (SM 1). This agrees with [START_REF] Bodek | Environmental Inorganic Chemistry: Properties, Processes and Estimation Methods[END_REF] who showed that the solubility of BaSO4 increases considerably in the pre-sence of chloride (Cl -) and other anions (NO3 -and CO3 2-). Ba may be mobile in soil, since it associates primarily with soil colloids by ion exchange [START_REF] Zhang | Adsorption of barium(II) on montmorillonite: an EXAFS study[END_REF]. [START_REF] Zhang | Adsorption of barium(II) on montmorillonite: an EXAFS study[END_REF] also showed that sorption of Ba 2+ on montmorillonite was not very sensitive to pH and that displacement of sorbed Ba increased with NaNO3 concentration. Here the study of correlations by treatment (SM 3) highlighted the in-fluence of major ions rather than that of pH.

Ryegrass growing on soil treated with 2% BC showed a significant difference in relation to the other treatments by an increase in the proportion of soluble Ba. Ba concentration in most plants ranged from 2 to 13 mg.kg -1 . [START_REF] Bowen | Strontium and barium in plants and soils[END_REF] have reported a range of 0.5-400 mg.kg -1 of Ba in different plant species growing in different soils. Ba concentrations of 200 mg.kg -1 have been found to be mod-erately toxic for plants, and an excess of 500 mg.kg -1 was considered to be toxic [START_REF] Pais | The Handbook of Trace Elements[END_REF]. Ba concentration or mineralomass for plants growing in BC amended mine soil was the same as for plants growing on mine soil without BC. Here the average concentrations (all organs combined) showed low Ba uptake by plant tissues (Fig. 4). Ba may accumulate in different parts of plants (IPCS, 1990), as shown here by translocation factors systematically above 1. Willows seemed to translocate more Ba than ryegrass (Table 4). Furthermore, even if [Ba]SPW was lower without BC (Fig. 1), BAFSPW was clearly above 1 (Table 4). Here, the willow tended to translocate Ba in its aerial parts while the ryegrass accumulated it in its roots (BCFSPW > 1 for the ryegrass only) (Table 4). Ba therefore showed the same plant-soil transfer mechanism as Ca [START_REF] Bowen | Strontium and barium in plants and soils[END_REF] and they may substitute for each other (Kabata-Pendias, 2011). Here, the cumulative effect of the Ba absorption with the other metal(loid)s may reinforce the harmful effect of the contamination in this mine soil and should be considered as a potential concern as it may impact Ba movement in the ecosystem [START_REF] Lamb | Bioavailability of barium to plants and invertebrates in soils contaminated by barite[END_REF].

Effect on As behavior

Arsenic can bind to anion exchange sites on soils (e.g. Fe, Al and Mn oxides and oxyhydroxides) [START_REF] Dzombak | Surface Complexation Modelling. Hydrous Ferric Oxide[END_REF][START_REF] Masscheleyn | Effect of redox potential and pH on arsenic speciation and solubility in a contaminated soil[END_REF][START_REF] Moreno-Jimenez | The fate of arsenic in the soil-plant system[END_REF]. Surprisingly, the addition of BC and an increase in the pH of the SPW did not increase the amount of As in SPW (Table 2 and Fig. 1). Nevertheless, high pH values can produce either a coprecipitation of As in the subsequently formed oxyhydroxides and sulfates [START_REF] García | Mobility of arsenic and heavy metals in a Sandy-loam textured and carbonated soil[END_REF], or a precipitate such as calcium arsenate in the presence of sulfates and carbonates. In our study, the [As]SPW was no longer detectable at the end of the experi-ment, whatever the presence of plants and amount of BC provided (Fig. 1). It can therefore be considered that neither the addition of BC nor the presence of plants seemed to be involved in this disappearance of As from the SPW, but that this may be due to physicochemical phenomena arising from the daily watering (probably leaching or im-mobilization by soil constituents). Several authors [START_REF] Fitz | Arsenic transformation in the soil-rhizosphere-plant system, fundamentals and potential application of phytoremediation[END_REF][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] have shown that competition may occur between DOC and As for soil retention sites. That work has linked the increase of DOC in SPW with the As concentration in solution (Table 2 and Fig. 1). Here, comparatively, DOC decreased during the experiment (Table 2), which may have reduced the competition between DOC and As for sorption sites. Finally, it can be assumed that the As present in the mine soil was not very mobile.

Concerning plant effects, BC addition generally significantly de-creased the mobility and availability of metal(loid)s in soil and reduced the phytotoxic concentrations of metal(loid)s [START_REF] Abdelhafez | Feasibility of biochar manufactured from organic wastes on the stabilization of heavy metals in a metal smelter contaminated soil[END_REF][START_REF] Lahori | Use of biochar as an amendment for re-mediation of heavy metal-contaminated soils: prospects and challenges[END_REF]. As concentration and mineralomass tended to be reduced for plants growing in BC amended soil. Even so, the effect of plant cultivation on mine soil with BC was not very clear regarding As concentration in plants and disagreement exists in the literature. [START_REF] Hartley | Arsenic mobility in brownfield soils amended with greenwaste compost or biochar and planted with miscanthus[END_REF] reported no increase of As concentrations and transfer in the plant, while a decrease of these parameters was observed elsewhere [START_REF] Namgay | Influence of biochar application to soil on the availability of As, Cd, Cu, Pb, and Zn to maize (Zea mays L.)[END_REF][START_REF] Beesley | Biochar addition to an arsenic contaminated soil increases arsenic concentrations in the pore water but reduces uptake to tomato plants (Solanum lycopersicum L.)[END_REF][START_REF] Lebrun | Eco-restoration of a mine technosol according to biochar particle size and dose application: study of soil physico-chemical properties and phytost-abilization capacities of Salix viminalis[END_REF]. If As was no longer detectable in SPW at the end of the experiment, it was absorbed by plants (Figs. 4 and 5). This suggests that the immobilization (or leaching) of As present in the water takes place gradually over time. Whereas BC had no visible effect on [As]SPW (Fig. 1), it induced a decrease in [As]plants, essentially in leaves (Figs. 4 and5). The reduction of As uptake by plants in the presence of BC may be due the sorption of As by BC [START_REF] Namgay | Influence of biochar application to soil on the availability of As, Cd, Cu, Pb, and Zn to maize (Zea mays L.)[END_REF]. Another hy-pothesis would be linked to pH: as BC induces an increase of soil pH, precipitation of iron oxides in the rhizosphere may be favored, and As could be retained in these iron sinks. The higher As concentrations in the roots in the presence of BC may support this hypothesis.

Conclusion

This work suggests that the addition of biochar (BC) to a con-taminated mine soil improved the nutrient status of this mine soil (especially 5% BC) and contributed to a better establishment of plants. It appears that BC enhances root development and promotes willow and ryegrass growth. The effects of BC on plant growth and on the behavior of metal(loid)s depend on the metal(loid)s considered. The effect of BC on Pb behavior is clearer than for the other metal(loid)s investigated. By increasing pH, BC reduced soluble concentrations of Pb and Zn but, surprisingly, it did not seem to have any influence on the behavior of As. The release of ions from the BC led to an increase in soluble concentrations of Ba. The beneficial effect of BC was evidenced for accumulation of metal(loid)s in plants, especially for As. The effect of plants on behavior of metal(loid)s was shown only for ryegrass grown on mine soil with 2% BC (and only for Ba and Pb). Zn, Ba and Cd were con-centrated in willow and ryegrass leaves of plants growing in mine soil with or without BC.

Future work should be focused on gaining a better understanding of metal(loid) phyotoavailability and of speciation of metal(loid)s in mine soil solid fractions (electron microscopy and X-photon spectroscopy and/or sequential extractions) to understand the complex mechanisms driving their sorption/desorption in the presence of BC. As the behavior of Ba in contaminated environments is not fully understood, it would be interesting to extend knowledge underlying the mechanisms involved in its availability and potential transfer into environmental compart-ments. The phytoremediation studied here showed that the contribu-tion of an amendment (BC) combined with plant cultures (willow, ryegrass) modified the behavior of certain metal(loid)s (Pb, Zn, and Ba) in soil pore water (SPW) while it only slightly influenced others (As, Cd). Special attention could be paid to understanding which ions and/ or what ion concentration makes Ba more mobile. Thus, in the assisted phytoremediation strategy using BC, precautions must be taken if soils have high concentrations of Ba. In that case, it is also important to know the plants' affinity for Ba.

Fig. 2 .

 2 Fig. 2. Heatmap analysis based on the correlation matrix of (A) 18 (initial step) or (B) 16 (final step) variables measured in the mine soil pore water at the beginning and at the end of the experiment. Heatmap analysis was obtained by hierarchical agglomerative clustering using Ward method of linkage and Euclidean distance as a measure of similarity. Warmer colors represent lower values and cooler colors represent higher values. The double hierarchical dendrogram reveals the correlation among treatments (column) and among chemical parameters (row). BC: biochar, I: average of initial values, NV: non-vegetated, W: willow, and R: ryegrass. 0, 2, and 5 for 0%, 2% and 5% biochar. Eh: Redox potential, EC: electrical conductivity, DOC: dissolved organic carbon, DON: dissolved organic nitrogen, and SPW: soil pore water. Heatmap analysis was obtained with n = 5 for NV0, NV2, NV5, R0 and R2; n = 4 for W2, W5, and R5; n = 3 for W0. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3 .

 3 Fig. 3. Matrices of Spearman's correlation coefficients for (A) 18 (initial step) or (B) 16 (final step) variables measured in the mine soil pore water at the beginning and at the end of the experiment. Warmer colors represent lower values and cooler colors represent higher values. Eh: Redox potential, EC: electrical conductivity, DOC: dissolved organic carbon, DON: dissolved organic nitrogen, and SPW: soil pore water. Correlation matrices were obtained with n = 5 for NV0, NV2, NV5, R0 and R2; n = 4 for W2, W5, and R5; n = 3 for W0. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 4 .

 4 Fig. 4. Total metal(loid) (Pb, Zn, Ba, As, and Cd) concentrations (μg.g -1 ) in plant organs for (A) willow and (B) ryegrass. W: willow, and R: ryegrass. 0, 2, and 5 for 0%, 2% and 5% biochar. ANOVA was followed by a post-hoc Tukey test at level P < 0.05 with n = 5 for NV0, NV2, NV5, R0 and R2; n = 4 for W2, W5, and R5; n = 3 for W0. For willow (A), letters a, b refer to statistics on roots, letters A, B to statistics on cutting, letters x, y to statistics on stem, and letters X, Y to statistics on leaves. For ryegrass (B), letters α, β refer to statistics on roots, and letters δ, ε to statistics on leaves.

Fig. 4 .

 4 Fig. 4. (continued)

Fig. 5 .

 5 Fig.5. Mineralomass (μg) of metal(loid)s (Pb, Zn, Ba, As, and Cd) from biomass for (A) willow and (B) ryegrass. W: willow, and R: ryegrass. 0, 2, and 5 for 0%, 2% and 5% biochar.ANOVA was followed by a post-hoc Tukey test at level P < 0.05 with n = 5 for NV0, NV2, NV5, R0 and R2; n = 4 for W2, W5, and R5; n = 3 for W0. For willow (A), letters a, b refer to statistics on roots, letters A, B to statistics on cutting, letters x, y to statistics on stem, and letters X, Y to statistics on leaves. For ryegrass (B), letters α, β refer to statistics on roots, and letters δ, ε to statistics on leaves.

Fig. 5 .

 5 Fig. 5. (continued)

Table 2

 2

Acknowledgements

The authors would like to acknowledge the financial support of Labex VOLTAIRE (ANR-10-LABX-100-01) and of the Centre-Val de Loire Region's PHYTOSELECT project (convention 2016-00108485). The authors gratefully acknowledge the financial support provided to the PIVOTS project by the Centre-Val de Loire Region: ARD 2020 program, CPER 2015-2020 and the European Union which invests in Centre-Val de Loire via the European Regional Development Fund. The authors wish to thank Louis De Lary de Latour (BRGM, DPSM) for in-formation on the site, Romain Nandillon (ISTO, IDEA) for character-ization data of the sampling site, Marielle Hatton (ISTO) for measure-ments of CHNS obtained with an elemental flash pyrolyser analyzer, Nathalie Lottier (ISTO) for measurements of DOC and DON obtained with a TOC 5050/SSM 5000-A elemental analyzer, and Louis De Lary de Latour (BRGM, DPSM) for information on the site. The authors are also grateful to JC Léger (La Carbonerie, Crissey, France) for providing biochars. The authors would like to acknowledge reviewers for their helpful comments and suggestions. A sincere thank you to Geoffrey Bird for diligent proofreading and language editing of this paper.

Appendix A. Supplementary data

Supplementary data to this article can be found online at https:// doi.org/10.1016/j.jenvman.2018.11.021.


Mathieu Ardyna
email: mathieu.ardyna@obs-vlfr.fr.

Hervé Claustre

Jean-Baptiste Sallée

Francesco D'ovidio

Bernard Gentili

Gert Van Dijken

Fabrizio D'ortenzio

Kevin Arrigo

Delineating environmental control of phytoplankton biomass and phenology in the Southern Ocean

Keywords: Phytoplankton ecology, Phytoplankton phenology, Biological pump, Climate Change, Southern Ocean, Remote sensing

Main point

Introduction

The Southern Ocean (SO) plays a key role in the global carbon cycle, absorbing 40% of the total oceanic inventory of anthropogenic CO 2 [START_REF] Khatiwala | Reconstruction of the history of anthropogenic CO2 concentrations in the ocean[END_REF]. However, current and predicted changes in ocean circulation and hydrology associated with climate variability might impact the efficiency of the SO carbon sink, although the extent of such changes and their specific impact on the biological pump are still vigorously debated [START_REF] Landschützer | The reinvigoration of the Southern Ocean carbon sink[END_REF][START_REF] Quéré | Impact of climate change and variability on the global oceanic sink of CO2[END_REF][START_REF] Munro | Recent evidence for a strengthening CO2 sink in the Southern Ocean from carbonate system measurements in the Drake Passage (2002-2015)[END_REF]. Such debate cannot advance without a better understanding of the factors that shape the complex biogeography of the SO, and what controls their time-scale of variability (i.e. daily, seasonal to decadal).

Delimiting marine bio-regions has proven highly valuable for disentangling multiple limiting growth factors affecting the efficiency of the biological pump [START_REF] Longhurst | Ecological Geography of the Sea (Second Edition)[END_REF] mediated by both PB and PH, and for ultimately underlining effects of climate change such as areal changes in trophic regime [START_REF] Polovina | Ocean's least productive waters are expanding[END_REF] or in fisheries stock and species [START_REF] Fossheim | Recent warming leads to a rapid borealization of fish communities in the Arctic[END_REF]. Changes in PB profoundly alter the efficiency of the biological pump, thereby modulating energy transfer to upper trophic levels and carbon export to deep water [START_REF] Legendre | Plankton and nutrient dynamics in marine waters[END_REF]. Changes in PH may also have important consequences for the marine food web, due to potential mismatch between primary and secondary producers and apex predators [START_REF] Ardyna | Recent Arctic Ocean sea-ice loss triggers novel fall phytoplankton blooms[END_REF][START_REF] Edwards | Impact of climate change on marine pelagic phenology and trophic mismatch[END_REF], and for the carbon cycle through export of unexploited carbon aggregates to the deep ocean [START_REF] Dall'olmo | Substantial energy input to the mesopelagic ecosystem from the seasonal mixed-layer pump[END_REF].

The efficiency of the SO's biological pump is strongly associated with phytoplankton biomass (PB) and its phenology (PH), which themselves are mediated by nutrient availability (e.g. mainly iron, and/or possibly nitrate and silicic acid), light, temperature and mortality factors [START_REF] Behrenfeld | Resurrecting the Ecological Underpinnings of Ocean Plankton Blooms[END_REF][START_REF] Boyd | Environmental factors controlling phytoplankton processes in the Southern Ocean[END_REF]. These factors are essentially controlled by vertical mixing, advection, seasonal fluctuations in solar irradiance, and the extent and typology of seaice cover. As a result, a variety of patterns of net primary production (NPP) are found in the SO [START_REF] Arrigo | Primary production in the Southern Ocean, 1997-2006[END_REF], with their own associated phytoplankton species succession, stock, and distribution [START_REF] Quéguiner | Iron fertilization and the structure of planktonic communities in high nutrient regions of the Southern Ocean[END_REF]. Delineating the interplay of environmental forcing on PB and PH remains challenging and requires novel approaches, which are available through satellite-derived observations. Using these integrative and multidisciplinary approaches (i.e. satellite-derived ocean color and altimetry, Argo floats, lagrangian modeling and biogeographic-derived analysis), we provide here a global understanding of the phytoplankton biomass and phenology in a potentially changing SO.

Material and methods

Satellite-derived, climatological and modeling products

Satellite-derived Level-3 data sets of chlorophyll-a concentration (chl a; mg m -3 ) and photosynthetically available radiation (PAR; E m -2 d -1 ) were obtained from the European Space Agency's GlobColour project (http://www.globcolour.info). Eight-day composite chl a concentrations using standard Case 1 water algorithms were used (i.e. OC4v5 for SeaWIFS, OC4Me for MERIS and OC3v5 for MODIS/VIIRS sensors; see O' Reilly et al. [2000] and [START_REF] Maritorena | Merged satellite ocean color data products using a bio-optical model: Characteristics, benefits and issues[END_REF] for details). The climatological annual net primary production (NPP) was derived from the adapted-SO NPP model of [START_REF] Arrigo | Primary production in the Southern Ocean, 1997-2006[END_REF]. A detailed description of the NPP model can be found in the supporting information [START_REF] Dobson | Bulk models of solar radiation at sea[END_REF][START_REF] Gregg | A simple spectral solar irradiance model for cloudless maritime atmospheres[END_REF][START_REF] Markus | Results from an ECMWF-SSM/I forced mixed layer model of the Southern Ocean[END_REF]. The climatologies of mixed layer depth and bathymetry were extracted respectively from [START_REF] Pellichero | The ocean mixed-layer under Southern Ocean sea-ice: seasonal cycle and forcing[END_REF] and the GEBCO, version 2014 (General Bathymetric Chart of the Oceans; http://www.gebco.net). The climatology of the length of the sea-ice cover was derived from the Special Sensor Microwave Imager (SSMI;1998[START_REF] Boyd | Environmental factors controlling phytoplankton processes in the Southern Ocean[END_REF]

Clustering K-means method

The bio-regions were defined here using a cluster K-means analysis (see the SI for more details), previously applied successfully in the Mediterranean Sea [START_REF] D'ortenzio | On the trophic regimes of the Mediterranean Sea: a satellite analysis[END_REF][START_REF] Mayot | Interannual variability of the Mediterranean trophic regimes from ocean color satellites[END_REF], in the North Atlantic [START_REF] Lacour | Phytoplankton biomass cycles in the North Atlantic subpolar gyre: a similar mechanism for two different blooms in the Labrador Sea[END_REF] and at the global scale [START_REF] D'ortenzio | Phenological changes of oceanic phytoplankton in the 1980s and 2000s as revealed by remotely sensed ocean-color observations[END_REF]. The analysis was performed on climatological and normalized annual chl a cycle, in order to statistically organize the GLOBcolour time series (1998)(1999)(2000)(2001)(2002)(2003)(2004)(2005)(2006)(2007)(2008)(2009)(2010)(2011)(2012)(2013)(2014) and to create clusters representing regions of similarity (i.e. annual chl a cycles). To maintain consistency in the seasonal data availability throughout the study area, the period study covers the period from September to March (as referred here to "annual"). This technique regroups pixels with seasonal cycles shapes -i.e. similar phenologies. A single characteristic seasonal cycle, that is statistically representative of the group as a whole, is then determined by calculating the center (average cycle) within each group or cluster. Each group representative of a characteristic seasonal cycle constitutes a phenological characteristic regime. A more detailed discussion of the cluster K-means analysis can be found in the supporting information [START_REF] Devred | Delineation of ecological provinces using ocean colour radiometry[END_REF][START_REF] Hartigan | Algorithm AS 136: A K-Means Clustering Algorithm[END_REF][START_REF] Ioccg | Partition of the Ocean into Ecological Provinces: Role of Ocean-Colour Radiometry[END_REF][START_REF] Lund | Revisiting Climate Region Definitions via Clustering[END_REF][START_REF] Milligan | An examination of procedures for determining the number of clusters in a data set[END_REF] A one-way analysis of variance by ranks (Kruskal-Wallis H test;[START_REF] Zar | Biostatistical Analysis[END_REF]) was performed to test whether eco-regions differed in their biological characteristics (i.e. annual mean chlorophyll a concentration, seasonality and timing of bloom). A significant result of the Kruskal-Wallis H test implies that at least one eco-region differs from all others.

Lagrangian modeling of horizontal iron fluxes

In order to estimate iron delivery due to horizontal stirring, we use an advection scheme based on altimetry, extending to the Southern Ocean a Lagrangian model used for predicting the development of the Kerguelen phytoplanktonic plume [START_REF] Sanial | Use of Ra isotopes to deduce rapid transfer of sediment-derived inputs off Kerguelen[END_REF]. The model has been extensively calibrated and validated in the Crozet and Kerguelen regions by integrating satellite data (altimetry and ocean color), lithogenic isotopes, iron measurements, and drifters [START_REF] Sanial | Use of Ra isotopes to deduce rapid transfer of sediment-derived inputs off Kerguelen[END_REF][START_REF] Sanial | Study of the phytoplankton plume dynamics off the Crozet Islands (Southern Ocean): A geochemical-physical coupled approach[END_REF][START_REF] Sanial | Use of Ra isotopes to deduce rapid transfer of sediment-derived inputs off Kerguelen[END_REF]. The main characteristics of the model were resumed here and we refer to d [START_REF] Ovidio | The biogeochemical structuring role of horizontal stirring: Lagrangian perspectives on iron delivery downstream of the Kerguelen Plateau[END_REF] for the full description of the model. The model seeds each open ocean location with a particle and finds the particle's most recent contact with a shallow bathymetry by a back-trajectory issued from altimetry data. It provides the time and the position at which the contact took place. An exponential scavenging relation (with a time constant based on in situ iron data from the KEOPS2 cruise) is used to estimate the decreasing of bio-available iron along the trajectory from the time of the contact with the potential iron source to the current position. Here, the model was applied to the entire Southern Ocean using a grid of 0.25°, a time window of 2004-2015 (one map every 4 days), potential sources of iron in the bathymetric band shallower than 500m, and a diffusion term of 40 m 2 s -1 .

Results and discussion

Based on satellite-derived observations, a large range of both annual mean chlorophyll a (chl a) concentration and net primary production (NPP) (>0.1 mg chl a m -3 /20 g C m -2 y -1 to more than 2 mg chl a m -3 /400 g C m -2 y -1 , respectively) were observed across the SO. It occurs at a variety of scales, including the latitudinal and the regional (i.e. Indian, Pacific and Atlantic; Fig. 1a) scales in the SO. The annual mean chl a concentration is associated with the amplitude of its seasonal cycle (Fig. 1a&c). In contrast, the timing of blooms (i.e. the maximum of the annual chlorophyll cycles, Fig. 1d) appears entirely decoupled from both large-scale patterns in annual chl a concentration and its seasonality (Fig. 1c), hence revealing a complex organization of phenological patterns of the chl a annual cycle.

Here, we disentangle the spatial complexity of chl a seasonal cycle using a K-mean clustering method [START_REF] D'ortenzio | Phenological changes of oceanic phytoplankton in the 1980s and 2000s as revealed by remotely sensed ocean-color observations[END_REF] with the goal of defining distinct bio-regions with similar large-scale patterns in PH (Fig. 2). In brief, this technique statistically gathers regions that exhibit similarly shaped seasonal chl a cycles (see the section 2.2 for more details). The seven distinct bio-regions so identified are distributed along a latitudinal gradient (Fig. 3a). At temperate latitudes, two bio-regions (i.e. 1 and 2) exhibit an early bloom in October. Moving south to 45-63°S, two other bio-regions (i.e. 4 and 5) display an annual chl a cycle with a delayed bloom in November/December. Finally, near the Antarctic continental shelf (< 63°S), the dominant bio-regions 6 & 7 were characterized by a late bloom in January/February. Interestingly, one bio-region (i.e. 3) differs drastically from the general phenological trend of a delayed bloom moving from subtropical to polar latitudes, having a late bloom at a moderate latitude (≈ 43°S, Fig. 2 &3d). Furthermore, the co-existence of bio-regions at a same latitude indicates variations in the seasonality of the annual chl a cycle (e.g., 1 & 2, 4 & 5 and 6 & 7) which are related to distinct levels of PB (Figs 3b-c).

From temperate to high latitudes, the latitudinal gradient in bloom timing clearly follows changes in light regime (expressed as the annual mean photosynthetically available radiation in the double boxplot; Fig. 4a). The unique bio-region (i.e., 3), characterized by a paradoxically late bloom with respect to the timing of light availability, is mainly located in the vicinity of the Antarctic Circumpolar Current. Interestingly, this region corresponds to the highest Southern Ocean wind stress and depth of the mixed layer (see SI, Fig. S1). Deep mixed-layers likely impose severe light limitation to phytoplankton growth in spring, hence delaying the bloom later in season. Close to the Antarctic shelf, as shown locally in the western Antarctic Peninsula [START_REF] Venables | Wintertime controls on summer stratification and productivity at the western Antarctic Peninsula[END_REF], the timing of the bloom (Fig. 2) was not related to winter sea-ice extent but more to the seasonal cycle of irradiance.

To address the potential causes of regional-scale variability in PB (Fig. 1a), we hypothesize that iron availability represents its first order driver [START_REF] Hutchins | Marine phytoplankton and the changing ocean iron cycle[END_REF]. Using integrative and multidisciplinary approaches, we disentangle the impact of four important iron supply mechanisms on PB. These surface-layer iron-sources are shallow plateaus (<500 m) which both (i) locally recharge the surface-layer in iron [START_REF] Arrigo | Environmental controls of marine productivity hot spots around Antarctica[END_REF][START_REF] Graham | Inferring source regions and supply mechanisms of iron in the Southern Ocean from satellite chlorophyll data[END_REF] and (ii) remotely recharge the surface layer by lateral advection of non-consumed iron [START_REF] Sanial | Use of Ra isotopes to deduce rapid transfer of sediment-derived inputs off Kerguelen[END_REF][START_REF] Graham | Inferring source regions and supply mechanisms of iron in the Southern Ocean from satellite chlorophyll data[END_REF]; (iii) sea-ice, which recharges the surface as ice melts [START_REF] Arrigo | Environmental controls of marine productivity hot spots around Antarctica[END_REF][START_REF] Lannuzel | Iron in sea ice: Review and new insights[END_REF]; and (iv) vertical mixing through deep convection, which can entrain iron from deep water [Tagliabue et al., 2014b]. Below, the potential impact of these four likely iron-sources on the intensity of PB is investigated. Note that the Aeolian iron deposition was not considered here as a major iron supply mechanism due to its intermittency and its unclear role in regulating PB [START_REF] Boyd | Mapping phytoplankton iron utilization: Insights into Southern Ocean supply mechanisms[END_REF][START_REF] Cassar | The Southern Ocean Biological Response to Aeolian Iron Deposition[END_REF][START_REF] Tagliabue | Evaluating the importance of atmospheric and sedimentary iron sources to Southern Ocean biogeochemistry[END_REF][START_REF] Tagliabue | The integral role of iron in ocean biogeochemistry[END_REF].

Clearly, the highest PB (> 1.2 mg m -3 ) are detected in shallow areas (< 500 m), on continental and island shelves where iron fluxes are expected to be significant [START_REF] Boyd | Mapping phytoplankton iron utilization: Insights into Southern Ocean supply mechanisms[END_REF]Tagliabue et al., 2014a]. Furthermore, we use a Lagrangian model based on altimetry in order to track which water parcels have been recently in contact with a shallow area -and when -to assess the probable locations of some of the iron plumes downstream of these iron sources and to estimate their loss of bio-available iron during advection by scavenging [START_REF] Sanial | Use of Ra isotopes to deduce rapid transfer of sediment-derived inputs off Kerguelen[END_REF].

We find a well-defined decline in PB (from 0.8 to 0.3 mg m -3 ) with distance and time from the most recent contact of the water parcel with a shallow area (Fig. 4b): the further away a waterparcel is from the initial shallow area iron source, or the longer after a water-parcel has been iron-enriched in shallow areas, the weaker is the PB signal. We note however that seamounts and submerged plateaus do not enhance local or downstream PB, as already observed north of Kerguelen plateau [START_REF] Graham | Inferring source regions and supply mechanisms of iron in the Southern Ocean from satellite chlorophyll data[END_REF] (Figs. 1a&4c), suggesting that not all shallow areas are active and/or bioavailable iron sources. Close to the Antarctic shelf and coastal polynyas, the potential role of the seasonal melting of sea-ice [START_REF] Arrigo | Environmental controls of marine productivity hot spots around Antarctica[END_REF][START_REF] Lannuzel | Iron in sea ice: Review and new insights[END_REF] as iron source is revealed with enhanced PB (≈0.4 mg m -3 ).

Moving away from potential iron delivery from shallow areas (either locally or downstream) and from sea-ice, we investigated the possible role of mixed-layer depth on regulating PB. If deepening of the mixed layer in winter can recharge the surface layer in nutrients (iron, but also nitrate and silicic acid in the temperate regimes) by deep convection, it can also limit PB by reducing light availability for phytoplankton growth during the growing season. Indeed, region of deep winter mixing in the Southern Ocean are mostly located directly north of the ACC, where strong winds and isopycnal tilts, are associated with an season-wide weak stratification, leading to deep winter mixed layer but also to relatively deep, compared to their surrounding, spring and summer mixed-layers.

Our findings clearly translate this complex interplay between availability of nutrient and light related to the winter mixed-layer depth. An increase in PB is observed with the deepening of the winter mixed-layer until a maximum winter depth of about 150 m. When the winter mixed layer becomes deeper, a decrease in PB is observed, likely resulting from a stronger light limitation of phytoplankton growth during the growth season (Sep-Mar; on average, winter mixed layer deeper than 150 m are associated to Sep-Mar mixed layer deeper than 110 m or so; Fig 4b). We thus depict here a tipping winter MLD at ~ 150m, which likely represents a shift between an iron-limited to a light-limited environment. Such threshold in the MLD is certainly dependent on the local structure of the iron profile [Tagliabue et al., 2014b]. Despite the HNLC (High Nutrient Low Chlorophyll) nature of the Southern Ocean, an intermediate mixing mode (i.e., with a winter MLD maximum from 120 to 200 meters) appears to enhance PB by supplying an optimal combination of both light and nutrient requirements for phytoplankton growth. This apparently modest effect of optimal mixing on PB may nevertheless have major implications due to the large areal extension of those zones relying on the vertical mixing to support phytoplankton iron requirements (Fig. 4c).

Deciphering the complexity of the SO's phytoplankton phenology and biomass is particularly critical to assess how climate variability and change might regionally impact the biological pump. Our results reveal two main scales of organization for both PB and PH, i.e. a large latitudinal and a regional scale. Alterations of stratification that are expected with global warming may modify light-mixing regimes, and thus potentially modulate the bloom timing.

However, since the phenology appears here to be strongly linked to the circumpolar seasonal cycle in irradiance, we suppose that only drastic changes in stratification could significantly alter bloom timing, and therefore the impact on the biological pump might be expected to be relatively minor.

Concerning the fate of phytoplankton biomass in the changing SO, no particular changes at local scale are expected in shallow bathymetry (i.e. where iron recharge from shallow plateaus (<500 m) are not prone to be affected). Potential changes also likely to remain minor in downstream transport from ACC, depending on whether the ACC has shifted locally in some regions over the past decades according to climate model [START_REF] Kim | On the Variability of Antarctic Circumpolar Current Fronts Inferred from 1992-2011 Altimetry[END_REF][START_REF] Sallée | Southern Ocean fronts and their variability to climate modes[END_REF] but not others [START_REF] Gille | Meridional displacement of the Antarctic Circumpolar Current[END_REF][START_REF] Shao | Properties of the Subantarctic Front and Polar Front from the skewness of sea level anomaly[END_REF]. Even if ACC changes remain unclear under next century climate change scenarios [START_REF] Meijers | Representation of the Antarctic Circumpolar Current in the CMIP5 climate models and future changes under warming scenarios[END_REF], past studies indicate that if there is a change, the expected position change should be small compared to the size of the bathymetry structure it interacts with to give rise to ocean surface iron plumes.

The main uncertainties regarding the fate of PB thus remains linked to the role of the mixed-layer depth and the sea-ice, which have a more direct response to climate variability and change. Climate modes have direct regional impact on both sea-ice and mixed-layer depth [START_REF] Sallée | Zonally asymmetric response of the Southern Ocean mixed-layer depth to the Southern Annular Mode[END_REF][START_REF] Simpkins | Seasonal Relationships between Large-Scale Climate Variability and Antarctic Sea Ice Concentration[END_REF]. In response to SAM and ENSO, sea-ice extends in some regions and is reduced in others, which, according to our results, would have regional impact on iron delivery and PB intensity. Similarly, SAM tends to deepen mixed-layer depth regionally but shallows it in other regions. Under next century climate change, the response is more regionally consistent, with an overall shallowing of the mixed-layer associated with increased stratification (freshwater input from glacier and sea-ice, and increased heat forcing from the atmosphere), and an overall reduction of sea-ice cover.

However, according to our findings, a shallowing (or deepening) of the mixed-layer is not expected to translate to the same PB response everywhere (see SI, Fig. S2). Indeed, the response would be state-dependent: a shallowing of a deep mixed-layer (typically deeper than 200 m) would result in an increase in PB, while a shallowing of an already relatively shallow mixedlayer (typically less than 120 m), would reduce PB. At the SO scale, a global shallowing of the mixed-layer depth appears to have negative consequences on PB (loss of ≈0.3 % m -1 ; see SI), mainly due to the areal increase of depleted-iron regions with less efficient vertical iron recharge.

Conversely, a positive response of PB (increase of ≈0.15 % m -1 ; see SI) would be observed with a global deepening of the mixed-layer until a critical threshold (+40 m compared to the actual mixed-layer depth climatology), from which severe light limitation damps phytoplankton growth and biomass. Given the more direct response of the MLD to climate variability and change in the SO, these results highlight the crucial role of the local vertical mixing and the associated subtle balance between light/nutrient availability on regulating PB in the majority of the HNLC areas of the SO. ), (c) the chlorophyll a seasonality (mg m -3 ), (d) the timing of the bloom (week) maximum based on the GLOBcolour time series (1998)(1999)(2000)(2001)(2002)(2003)(2004)(2005)(2006)(2007)(2008)(2009)(2010)(2011)(2012)(2013)(2014).

Figure captions

The climatological annual net primary production (b; mg C m -2 d -1 ) was derived from the adapted-SO NPP model of [START_REF] Arrigo | Primary production in the Southern Ocean, 1997-2006[END_REF]. Frontal positions calculated from MADT contours are shown for the STF (white), the SAF (red), the PF (yellow) and the SACCF (black).

Figure 1 :

 1 Figure 1: Phytoplankton biomass, net primary production and phenology in the Southern Ocean. Map showing the climatological (a) annual mean chlorophyll a concentration (mg m -3 ), (b) annual net primary production (g C m -2 d -1), (c) the chlorophyll a seasonality (mg m -3 ),(d)

Figure 2 :

 2 Figure 2: Biogeography of the Southern Ocean. Spatial distribution (a) of the 7 bio-regions (i.e. bio-regions 1 to 7) of the Southern Ocean obtained from the k-means analysis. The normalized annual chlorophyll cycles (continuous lines) of the centers of the clusters (b -h) obtained from the k-means analysis are presented, as well as their respective standard deviation.The absolute annual chlorophyll cycles (dashed lines) corresponding to the centers of the clusters (b -h) are also indicated.

Figure 3 :

 3 Figure 3: Latitudinal repartition of the bio-regions and their respective biological characteristics. Latitudinal proportion (a) of the different bio-regions. Box plots of the bioregions (1 to 7; x axis) against (b) annual mean chlorophyll a concentration (mg m -3 ), (c) the chlorophyll a seasonality (mg m -3 ), (d) the timing of the bloom maximum (month; y axis). The line in the middle of each box represents the region median. The top and bottom limits of each box are the 25th and 75th percentiles, respectively. The lines extending above and below each

Figure 4 :

 4 Figure 4: Environmental control of phytoplankton biomass and phenology in the Southern Ocean. Double boxplot (a) of the maximum bloom timing (weeks) versus the annual mean PAR (E m y -1 ) of the different bio-regions. The top/right and bottom/left limits of each box are the 25th and 75th percentiles, respectively. The numbers in each box are located at the median of both maximum bloom timing and annual mean PAR. Barplot (b) of the mean annual chlorophyll a concentration according (1) the shallow areas (< 500 m; red), (2) areas where iron delivery downstream take place (%; percent of iron remaining in a water parcel after scavenging in respect to its initial concentration acquired in shallow areas; red to blue), (3) areas characterized by a seasonal sea-ice cover (grey) and (4) areas where variations in the annual maximum MLD are analyzed (white). See the map (c) delineating the distinct areas listed just above.

  Figure 1.

Acknowledgments

The GLOBcolour data were made available by the European Space Agency's GlobColour project (http://www.globcolour.info). The altimetric data was provided by AVISO/CNES from their website http://www.aviso.altimetry.fr/fr/. We gratefully acknowledge the whole OMTAB team for constructive discussion on initial versions of the manuscript. We also thank Bernard Quéguiner and an anonymous reviewer for constructive comments on the manuscript. M.A. received a postdoctoral fellowship from the CNES (Centre National d'Études Spatiales) and the European Research Council (ERC) remOcean project (grant agreement 246777).