R Nandillon Abc

O Lahwegue

F Miard

M Lebrun

M Gaillard

S Sabatier

F Battaglia-Brunet

D Morabito

S Bourgerie

Potential use of biochar, compost and iron grit associated with Trifolium repens to stabilize Pb and As on a multi-contaminated technosol

Keywords: Phytomanagement, Biochar, Compost, Iron grit, metal(loid)s, Trifolium

Vegetation cover can be used in the phytomanagement of polluted areas by adding value to abandoned sites and reducing the dispersion of pollutants by erosion. Appropriate amendments, that allow both efficient plant growth and the immobilization of contaminants in the soil must be chosen in order to optimize the efficiency of this process. We used a mining technosol mainly contaminated by arsenic (1068 mg.kg -1 ) and lead (23387 mg.kg -1 ) to study the effect of three amendments (biochar, compost and iron grit) on (i) physico-chemical properties of the soil and soil pore water, (ii) metal(loid) mobility, bioavailability and bioaccessibility (CaCl2 and Simple Bioaccessibility Extraction Test (SBET)), and (iii) the capability of Trifolium repens to germinate and grow. All the amendments used increased the pH and electrical conductivity of the SPW, resulting in a 90% decrease in the concentration of lead in the soil pore water (SPW). We also demonstrated a decrease in Pb phytoavailability.

The amendments allowed the establishment of a plant cover, although the addition of iron grit alone did not allow any clover germination. For the Pontgibaud technosol, the combination of the three amendments resulted in a significant decrease in As and Pb concentrations in clover tissues, mainly in the aerial organs. The amendments also made it possible for some of them to halve the phytoavailable fraction of arsenic. However, for compost, both the As concentrations in the SPW, and the bioavailable fraction of As increased. All the amendments used had contrasting effects on the bioaccessible fractions of metal(loid)s. The most efficient amendment combination was the addition of 5 % biochar and 5 % compost.

Introduction

After more than 200 years of industrialization, the European Union now faces a major problem with metal and organic contaminated soils (EEA, 2007). It was estimated that approximately 342,000 sites are currently contaminated, and that industrial activities, including mining, are among the main sources, representing 33% of the contamination [START_REF] Panagos | Contaminated sites in Europe: review of the current situation based on data collected through a european network[END_REF].

To the extent that this industrial waste contain organic and inorganic pollutants, they participate as source of pollution for soil, water and air. Thus, these wastes can have negative health consequences through direct ingestion or inhalation [START_REF] Uzu | Characterization of lead recycling facility emissions at various workplaces: major insights for sanitary risks assessment[END_REF] or by entering the food chain [START_REF] Schreck | DECA: a new model for assessing the foliar uptake of atmospheric lead by vegetation, using Lactuca sativa as an example[END_REF].

To reduce these risks, remediation methods such as controlled backfilling, soil fixation, or leaching can be very useful. However, these methods are generally expensive, and some may even have negative effects on the biodiversity, biological activities, soil structure, or fertility [START_REF] Ali | Phytoremediation of heavy metalsdconcepts and applications[END_REF]. In contrast, environment-friendly remediation options, such as "aided phytostabilisation", could improve soil functionality and allow a vegetation cover to be established, at the same time as reducing the mobility and availability of pollutants [START_REF] Cundy | Developing principles of sustainability and stakeholder engagement for "gentle" remediation approaches: the European context[END_REF][START_REF] Cundy | Brownfields to green fields: realising wider benefits from practical contaminant phytomanagement strategies[END_REF]. Plant cover may also reduce contaminant leaching [START_REF] Houben | Heavy metal immobilization by cost-effective amendments in a contaminated soil: effects on metal leaching and phytoavailability[END_REF], as well as stabilizing soils, and it controls water and wind erosion [START_REF] Reubens | The role of fine and coarse roots in shallow slope stability and soil erosion control with a focus on root system architecture: a review[END_REF]). The implementation of plants will stabilize contaminants by adsorption or accumulation in roots [START_REF] Vangronsveld | Phytoremediation of contaminated soils and groundwater: lessons from the field[END_REF]. However, the translocation to the shoot system should remain limited to avoid the transfer of contaminants into the food chain through grazing [START_REF] Henry | Phytotechnologies: preventing exposures, improving public health[END_REF][START_REF] Pérez-De-Mora | Phytostabilization of semiarid soils residually contaminated with trace elements using by-products: sustainability and risks[END_REF][START_REF] Kidd | Trace element behaviour at the root-soil interface: implications in phytoremediation[END_REF].

The selection of an appropriate metal tolerant vegetation cover is critical, as it affects the efficiency of the phytoremediation process.

Trifolium repens has characteristics which have led to it being selected for restoring plant cover. Clover mainly stores metals and metal(loid)s in its roots [START_REF] Bidar | Seasonal and annual variations of metal uptake, bioaccumulation, and toxicity in Trifolium repens and Lolium perenne growing in a heavy metal-contaminated field[END_REF], and it enriches poor soils by fixing atmospheric nitrogen, which allows the development of other plant species. In addition, it has a high germination rate and good resistance to environmental stress conditions. It appears that its stoloniferous growth makes Trifolium repens able to colonize bare spaces in lawns and it can therefore be used in various revegetation strategies [START_REF] Bidar | Behavior of Trifolium repens and Lolium perenne growing in a heavy metal contaminated field: plant metal concentration and phytotoxicity[END_REF]. Previous studies using Trifolium repens in contaminated zones have focused on metal uptake and remediation [START_REF] Bidar | Behavior of Trifolium repens and Lolium perenne growing in a heavy metal contaminated field: plant metal concentration and phytotoxicity[END_REF][START_REF] Bidar | Seasonal and annual variations of metal uptake, bioaccumulation, and toxicity in Trifolium repens and Lolium perenne growing in a heavy metal-contaminated field[END_REF][START_REF] Lopareva-Pohu | Influence of fly ash aided phytostabilisation of Pb, Cd and Zn highly contaminated soils on Lolium perenne and Trifolium repens metal transfer and physiological stress[END_REF], and have shown that Trifolium repens could therefore play a positive role in the metal phytoremediation strategy.

However, the extreme conditions encountered on polluted sites, i.e the high concentrations of toxic elements, the low pH, and the low organic matter content, do not usually allow plant growth. It is therefore essential to add mineral and organic amendments that could reduce the mobility of soil pollutants, and therefore their (phyto)availability [START_REF] Houben | Heavy metal immobilization by cost-effective amendments in a contaminated soil: effects on metal leaching and phytoavailability[END_REF][START_REF] Kumpiene | Stabilization of As, Cr, Cu, Pb and Zn in soil using amendments a review[END_REF][START_REF] Lambrechts | Comparison of EDTA-enhanced phytoextraction and phytostabilisation strategies with Lolium perenne on a heavy metal contaminated soil[END_REF][START_REF] Mench | Progress in remediation and revegetation of the barren Jales gold mine spoil after in situ treatments[END_REF], at the same time as increasing soil fertility in order to improve plant development [START_REF] Lopareva-Pohu | Influence of fly ash aided phytostabilisation of Pb, Cd and Zn highly contaminated soils on Lolium perenne and Trifolium repens metal transfer and physiological stress[END_REF].

Biochar, used as an organic amendment, has received increasing attention in recent years. It is a calcined carbon material produced by heating biomass in a closed system with a limited oxygen supply, using thermochemical technologies such as pyrolysis, gasification, or hydrothermal technologies [START_REF] Xu | Removal of Cu, Zn, and Cd from aqueous solutions by the dairy manure-derived biochar[END_REF]. In degraded soils it is used for its potentially beneficial effects on the environment. This includes increased soil fertility through improvement of the soil microbial activity, and reduction of pollutant mobility. It can also help reduce the greenhouse effect by long-term sequestration of carbon in the soil [START_REF] Inyang | A review of biochar as a low-cost adsorbent for aqueous heavy metal removal[END_REF]. Biochar can adsorb pollutants from the soil due to its microporous structure, high pH, cation exchange capacity, and its surface 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] Ding | Multiple Characterization for Mechanistic Insights of Pb(II) Sorption onto Biochars Derived from Herbaceous Plant, Biosolid, and Livestock Waste[END_REF]. However, biochar does not generally contain enough nutrients to allow plant growth on polluted soils, which are often characterized by low nutrient content [START_REF] Fischer | Synergisms between Compost and Biochar for Sustainable Soil Amelioration[END_REF]. Extra nutrients, such as those found in compost must therefore be added alongside the biochar. The addition of compost will allow plant growth as it contains N, P, K, Ca, Mg, and S, as well as humus and different kinds of microorganisms [START_REF] Fischer | Synergisms between Compost and Biochar for Sustainable Soil Amelioration[END_REF][START_REF] Gil-Loaiza | Phytostabilization of mine tailings using compost-assisted direct planting: Translating greenhouse results to the field[END_REF]. Moreover, it can reduce the phytoavailability of metal(loid)s due to their association with organic matter (OM), carbonates, or metal oxides [START_REF] Mench | Capacity of soil amendments in lowering the phytoavailability of sludge-borne zinc[END_REF], resulting in a reduction in the plant uptake of metals. But the effect of compost on metal bioavailability depends on the nature of the organic matter, the mineral species content, the potential degradability by microbiota, the pH and redox potential of the soil, and the soil type and metal(loid)s concerned [START_REF] Walker | The effects of soil amendments on heavy metal bioavailability in two contaminated Mediterranean soils[END_REF].

Although the application of biochar and compost has been shown to improve soil agronomic characteristics, as well as its ability to sorb and reduce metal cation availability such as Pb in soil, neither amendment is effective in decreasing the mobility of metal anions, such as arsenic. They even tend to increase anion soil mobility due to the rise in soil pH [START_REF] Beesley | A review of biochars' potential role in the remediation, revegetation and restoration of contaminated soils[END_REF]. This can cause problems in soils polluted with both cationic and anionic metal(loid)s. In such polluted sites, one possible strategy would be to combine compost and biochar with iron-rich amendments, or Mn oxides for metal(loid) immobilization [START_REF] Ruttens | Phytostabilization of a metal contaminated sandy soil. II: Influence of compost and/or inorganic metal immobilizing soil amendments on metal leaching[END_REF]. Among these amendments, zero-valent iron, in the form of iron grit, is the most relevant type of iron [START_REF] Kumpiene | Assessment of zerovalent iron for stabilization of chromium, copper, and arsenic in soil[END_REF], because its high adsorption potential has proved valuable in immobilizing inorganic contaminants [START_REF] Lefèvre | Reducing leachability and bioaccessibility of lead in soils using a new class of stabilized iron phosphate nanoparticles[END_REF][START_REF] Satapanajaru | Remediation of Atrazine-contaminated Soil and Water by Nano Zero valent Iron[END_REF][START_REF] O'day | Mineral-Based Amendments for Remediation[END_REF]. In soil, iron grit is known to be transformed to reactive iron (hydr)oxydes (e.g. ferrihydrite) [START_REF] Komárek | Chemical stabilization of metals and arsenic in contaminated soils using oxidesea review[END_REF].

Moreover, iron (hydr)oxydes capacity to sorb ions is modulated according to soil pH. A strong sorption of anions as arsenic is implemented at low pH due to a higher net positive surface charge on iron, whereas at high pH a stronger sorption of cations will be possible. In addition to sorption reactions, co-precipitation of metals and metalloids with iron oxides is another important mechanism for stabilizing metal and metalloid elements in amended soils [START_REF] Komárek | Chemical stabilization of metals and arsenic in contaminated soils using oxidesea review[END_REF]. Thus, it has been shown that the formation of relatively insoluble mineral precipitates Fe(II/III)-As (e.g. FeAsO4 -H2O, FeAsO4 -2H2O and Fe3(AsO4)2) influences the immobilization of arsenic and its bioavailability in soils [START_REF] Kumpiene | Stabilization of As, Cr, Cu, Pb and Zn in soil using amendments a review[END_REF][START_REF] Drahota | Secondary arsenic minerals in the environment: a review[END_REF].

Thus, to demonstrate the effectiveness of amendments and revegetation, it is essential to study the metal(loid) concentrations in the soil, soil pore water and plants continuum.

However, as Lebrun et al. (2018a) observed, the metal(loid) concentration in soil pore water did not always reflect the amount available for plants. It is therefore interesting to measure this phytoavailable quantity by simple extraction tests. These plant availability tests can be performed using extractant salts to determine the more or less mobile fraction of metal(loid)s, to understand their availability for plants [START_REF] Qasim | Potentially toxic element phytoavailability assessment in Technosols from former smelting and mining areas[END_REF][START_REF] Black | Evaluation of soil metal bioavailability estimates using two plant species (L. perenne and T.aestivum) grown in arrange of agricultural soils treated with biosolids and metal salts[END_REF].

However, the metal(loid) phytoavailability is not the only parameter to be measured, as human exposure to pollutants must also be taken into account. This exposure can include inhalation, oral ingestion or dermal absorption. One of the most common exposure pathways is the accidental ingestion of polluted soils [START_REF] Luo | Incorporating bioaccessibility into human health risk assessments of heavy metals in urban park soils[END_REF]. Such risk of metal(loid) exposure is often evaluated by a conservative approach in which the total metal(loid) content of the soil is measured. However, it is often pointed out that the risk for the exposed individual is only related to a small fraction of the total metal(loid)s, which is referred to as the bioaccessible fraction. To measure the bioaccessible fraction, various in vitro methods have been developed. They are usually simple and quick to manage, relatively inexpensive and do not raise ethical concerns [START_REF] Deshommes | Experimental determination of the oral bioavailability and bioaccessibility of lead particles[END_REF]. Some of these methods evaluate the fraction of ingested metal(loid)s that are soluble in the gastrointestinal tract [START_REF] Mendoza | Evaluation of the bioaccessible gastric and intestinal fractions of heavy metals in contaminated soils by means of a simple bioaccessibility extraction test[END_REF][START_REF] Wijayawardena | Evaluation of relative bioaccessibility leaching procedure for an assessment of lead bioavailability in mixed metal contaminated soils[END_REF] and therefore available in the blood flow. The Simple Bioaccessibility Extraction Test (SBET) procedure consists of an acid extraction that imitates the gastric compartment where the bioaccessibility is the strongest [START_REF] Ruby | Estimation of lead and arsenic bioavailability using a physiologically based extraction test[END_REF][START_REF] Casteel | Bioavailability of lead to juvenile swine dosed with soil from the Smuggler Mountain NPL site of Aspen, Colorado[END_REF].

The objectives of this study were to evaluate: (1) the capacity of biochar, compost and iron grit to reduce As and Pb phytoavailability when added to a multi-contaminated technosol by measuring their concentrations in soil pore water and by using a CaCl2 neutral salt extractant; (2) whether clover can be used to set up a vegetative cover and (3) the bioaccessibility of Pb and As in soil and in the aerial part of clover plants grown on amended soil, using the SBET approach.

Materials and methods

Origin of contaminated soil (Pontgibaud site)

The studied site in Roure-les-Rosiers (France) is a former silver-lead extraction mine, located in the Massif Central near Clermont-Ferrand (Saint-Pierre-le-Chastel (63)), in the Pontgibaud mining district (Ps), Lambert II coordinates are: X: 638147.54 Y: 2087962.79.

The mine has been in operation since Gallo-Roman times, and was industrially exploited only during the 19th century and ceased in 1897, with a resumption of activities during the Second World War. Mining activities stopped definitively in 1947.

The old mining site is subject to a semi-continental climate, with snowy winters, and hot summers, which can be marked by severe and localized thunderstorms. On average, the Roure-les-Rosiers sector receives about 770 mm of rainfall per year, and is located at around 700 meters above sea level.

Preparation of amended soils -Seed sowing and plant biomass

The soil chosen for this study came from the slag heaps of the former Pontgibaud mine site. Six different soil samples were tested: polluted soil (Ps), (Ps) amended with 5 % (w/w) of hardwood-derived biochar (PsB), (Ps) amended with 5 % (w/w) of compost (PsC), (Ps) amended with 1.5 % (w/w) of iron grit (PsI); (Ps) amended with 5 % (w/w) of hardwoodderived biochar and 5 % (w/w) of compost (PsBC) and (Ps) amended with 5 % (w/w) of hardwood-derived biochar and 1.5 % (w/w) of iron grit (PsBI). Biochar, was provided by La Carbonerie (Crissey, France). It was obtained by the slow pyrolysis of hardwood biomass (Quercus sp., Carpinus sp. and Fagus sp.) at 500 °C (residence time: 3 h, heating rate: 2.5 °C.min -1 ), followed by a sieving to obtain a particle size between 0.2 and 0.4 mm. Physico-chemical properties of amendments including biochar is provided in table 1 SM.

Three replicates of the different mixtures were placed in rectangular pots with a capacity of 5 liters, and a surface area of 360cm 2 (24cm x 15cm). The bases of the pots were covered with clay beads to keep the soil mixture in the pots when watering. Each pot was divided into 2 compartments: one non-vegetated (NV), and the other vegetated (V), with 500 clover seeds, which had a measured germination rate of 91%. The experiment was carried out in a controlled growth chamber under the following conditions: day/night temperatures (25°C/16°C), 16 hours of light/8 hours of darkness and with a light intensity of 800 μmol.m - 2 .s -1 .

The experiment lasted 30 days. The plants were then separated from the soil. The roots were washed thoroughly with tap water and rinsed with distilled water to remove soil particles attached to the roots. Next, the plants were divided into two: the root system and the aerial parts (shoots and leaves). Finally, the plant organs were dried in an oven at 55 °C (until they were a constant weight), and the dry weight of the organs (DW) was determined.

Soil pore water (SPW) sampling and analysis

SPW was collected using Rhizon® (Rhizosphere Research Product, Wageningen, The Netherlands), from each pot. 20mL of SPW was sampled to determine the pH (pH meter, FE20/EL20, Mettler-Toledo AG 2007), electrical conductivity (EC) (conductimeter, SOLEA, Tacussel électronique, Lyon, France), dissolved organic carbon (DOC) concentration (Pastel UV spectrophotometer, SECOMAM, Ales, France) and metal(loid) concentrations (ICP-AES, ULTIMA 2, HORIBA, Labcompare, San Francisco, USA), according to [START_REF] Motelica-Heino | Phytostabilization of As, Sb and Pb by two willow species (S.viminalis and S. purpurea) on former mine technosols[END_REF].

The sampling was done before sowing the clover seeds (T0) and at the end of the greenhouse experiment (TF) (30 days).

Metal(loid) quantification in tested soils and in plant organs

Metal(loid) contents (As, Fe and Pb) of soil samples and plant organs were determined by aqua regia digestion treatment (HNO3 (65%) : HCl (37%), 1:3 v/v) [START_REF] Zhang | Reaction and transport of arsenic in soils: equilibrium and kinectic modeling[END_REF], with a solid/liquid ratio of 1/45. The digestion program consisted of a gradual increase in temperature to 180°C in 15 minutes, a 15 minutes digestion step at 180°C followed by a 15 minutes cooling step. The mineralized samples were then adjusted to a volume of 50 mL with ultrapure water (18 MΩ.cm -1 ) and filtered through a nitrocellulose membrane at 0.45 μm.

Arsenic, iron and lead concentration were measured by Inductively Coupled Plasma Atomic Emission Spectroscopy (ICP-AES, Ultima 2, HORIBA, Labcompare, San-Francisco, USA).

The accuracy of the assay technique was verified by enriching the samples analyzed with a solution whose concentration of the element to be assayed was known.

Soil metal(loid) simple extraction procedures (CaCl2 treatment)

The phytoavailable fractions of As and Pb were obtained as described by [START_REF] Qasim | Potentially toxic element phytoavailability assessment in Technosols from former smelting and mining areas[END_REF], using CaCl2 extractant at 0.01M with a solid: liquid ratio 1: 10. Metal(loid) concentrations were determined by ICP-AES (ULTIMA 2, HORIBA, Labcompare, San Francisco, USA).

Metal(loid) bioaccessibility test for soil samples (SBET) and plant organs

According to the method developed by [START_REF] Mendoza | Evaluation of the bioaccessible gastric and intestinal fractions of heavy metals in contaminated soils by means of a simple bioaccessibility extraction test[END_REF], crushed dry material from tested soils or plant upper parts were put in a glycine solution 0.4 mol.L -1 adjusted to pH 1.5 at a concentration of 1%. The suspension was shaken for 1 h at 37 ± 1 °C, then filtered through a 0.45 μm pore size membrane with syringe filter and stored at 4 °C until analysis.

Metal(loid) concentrations were determined by ICP-AES (ULTIMA 2, HORIBA, Labcompare, San Francisco, USA).

Results and discussions

Effect of amendments on soil characteristics

All tested amendments apart from iron induced an increase in WHC, averaging 10% for compost, and 30% for biochar when added alone at 5% (w/w) to Pontgibaud soil. Biochar showed real efficiency in retaining water due to its porous structure, its hydrophobicity and high surface area [START_REF] Pietikäinen | Charcoal as a habitat for microbes and its effect on the microbial community of the underlying humus[END_REF][START_REF] Warnock | Mycorrhizal responses to biochar in soil-concepts and mechanisms[END_REF]Molnar et al., 2016).

However, these results are inconsistent with those obtained when measuring water holding capacity (WHC) on isolated compost and biochar, which were 299% and 183% respectively (Table 1SM). This difference is certainly related to the fact that the compost is compressed when it is added to Pontgibaud soil, meaning it is no longer able to expand in order to retain such a large quantity of water. Biochar is less subject to compressive stress when added to the Pontgibaud soil due to its porous and rigid structure, and therefore maintains its water retention characteristics.

None of the treatments affected the As pseudo-total concentrations compared to Ps. The iron concentration in Pontgibaud soil was 6325 mg kg -1 , and the addition of amendments, apart the iron grit, did not induce a significant change in iron soil concentration. The iron grit addition in PsI and PsBI caused the pseudo-total Fe soil concentration to increase by approximately 3 times. Lastly, the pseudo-total Pb soil concentrations in the different tested soils were the same for Ps, PsI, PsC, PsB and PsBI, whereas for PsBC, Pb concentration decreased by 30% compared to Ps due to a dilution effect produced by the addition of compost and biochar (Table 1).

Effect of amendments and clover plants on SPW characteristics

At the beginning of the experiment, neither time nor the presence of clover significantly modified the SPW pH of Pontgibaud soil (Ps) which was approximately 4.08 (Table 2).

Iron grit application to Ps allowed a 0.5 unit increase in SPW pH, whereas time and clover growth induced a significant decrease in SPW pH (0.5 units). Compost (C), biochar (B), biochar + iron grit in combination (BI) and biochar + compost in combination (BC) increased Ps SPW pH by 2.3 units, 2.7 units, 2.6 units and 3.3 units, respectively. It is noted that clover growth impacted SPW pH for PsB and PsBC by 0.45 units, whereas for these treatments on non-vegetated pots, time did not allow any significant pH modification. The alkalinization of SPW due to the addition of biochar has already been demonstrated by Lebrun et al. (2018a) when 5% pinewood biochar was added to Pontgibaud soil, inducing a 2.7 pH unit increase. Moreover, [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] showed that the SPW pH of a contaminated soil increased by more than three units when compost combined with biochar was added. The improvement in pH following the application of biochar or compost can be attributed to: (i) the alkalinity of the biochar (Table 1SM) which induced a liming effect [START_REF] Lebrun | Effect of biochar amendments on as and Pb mobility and phytoavailability in contaminated mine technosols phytoremediated by Salix[END_REF]; (ii) the dissolution or addition of alkali metals, and (iii) the presence of functional groups on the surface of the biochar, such as carbonyls, phenols, carboxyls and pyrones [START_REF] Marks | Unintended effects of biochars on shortterm plant growth in a calcareous soil[END_REF]Lebrun et al., 2018a).

Finally, for PsBI the pH decreased by two units over the time period of the treatment. This can be explained by the fact that the iron was oxidized during the experiment thus inducing the SPW pH decrease.

At T0, the SPW electrical conductivity (EC) for Ps and PsI was 0.220 mS.cm -1 and 0.315 mS.cm -1 respectively. For the other treatments, at T0, EC value was approximately 1.1 mS.cm -1 . At the end of the experiment, an increase in EC was observed compared to T0 on all tested non-vegetated soils, and corresponded to 4.2 fold, 2.5 fold, 2 fold, 2.4 fold, 3 fold and 2.1 fold for Ps, PsI, PsC, PsB, PsBI, PsBC, respectively. The presence of clover had no effect on Ps, PsI and PsBC, while for PsC, PsB and PsBI it caused a 1.3 fold EC decrease when compared to the corresponding non-vegetated pots. Overall, biochar and compost significantly increased SPW EC (p <0.05), which is consistent with previous studies [START_REF] Lebrun | Effect of biochar amendments on as and Pb mobility and phytoavailability in contaminated mine technosols phytoremediated by Salix[END_REF](Lebrun et al., , 2018a)). Indeed, organic matter and mineral compounds such as Ca 2+ , Mg 2+ , K + or inorganic carbonates present on biochar and compost can be released into the soil and thus increase SPW electrical conductivity [START_REF] Chintala | Effect of biochar on chemical properties of acidic soil[END_REF]. [START_REF] Kloss | Trace element concentrations in leachates and mustard plant tissue (Sinapis alba L.) after biochar application to temperate soils[END_REF] demonstrated an increase in EC from 0.04 dS.cm -1 to 0.19 dS.cm -1 when 3% straw biochar was applied to a planosol. Finally, iron grit must be solubilized and mostly precipitated in the form of iron oxide on soil particulates, which is why it did not induce a large increase in the specific EC value.

The DOC content of SPW at T0 (Table 2) for Ps was 5.7 mg.L -1 , which is identical to that of PsBI. Moreover, the DOCs for PsB and PsBI were 2.8 fold and 1.2 fold smaller than Ps. On the contrary, DOC for PsC and PsBC were 3.7 and 1.9 fold higher than for Ps. It should be noted that neither the time nor the presence of clover modified the DOC.

A decrease in DOC was observed in previous studies when biochar was added to various soils [START_REF] Lu | Effect of bamboo and rice straw biochars on the bioavailability of Cd, Cu, Pb and Zn to Sedum plumbizincicola[END_REF][START_REF] Kloss | Trace element concentrations in leachates and mustard plant tissue (Sinapis alba L.) after biochar application to temperate soils[END_REF][START_REF] Jain | Kinetic leaching of high sulphur mine rejects amended with biochar: buffering implication[END_REF]. This has been explained by the structure of the biochar, which contains sorption sites able to bind soluble organic compounds [START_REF] Hass | Chicken manure biochar as liming and nutrient source for acid appalachian soil[END_REF]. Treatments containing compost (i.e PsC and PsBC) showed higher levels of DOC, which could be explained by the fact that even though the compost is composed of refractory humified material, labile fresh organic matter is also present (Lefevre, 2015).

SPW metal(loid) concentrations and phytoavailable metal(loid)s

At T0 (Table 2), no As content was detected for Ps, PsI, PsB and PsBI SPW whereas for PsC and PsBC, As concentrations were 0.09 mg.L -1 and 0.06 mg.L -1 , respectively. Both the passage of time, and the presence of clover plants increased As SPW for these last two treatments, by 2 fold and 4 fold respectively. For Ps, at TF, As SPW concentration could be quantified, and corresponded to 0.03 mg.L -1 and 0.02 mg.L -1 , for TF-NV and TF-V, respectively. The As soil phytoavailable fractions (CaCl2-extractable fraction) for Ps and PsC at T0 were not significantly different, and corresponded on average to 0.11 % of the total soil As concentration (Table 1). Whereas for the other treatments PsI, PsB, PsBI and PsBC, As phytoavailability was 1.9 times smaller than Ps. Arsenic is usually found as an oxyanion in solutions, and presents specific challenges to sanitation, because its mobility increases when pH increases [START_REF] Beesley | A review of biochars' potential role in the remediation, revegetation and restoration of contaminated soils[END_REF][START_REF] Nandillon | Effect of biochar associated with various amendments (compost, garden soil and slag) on the phytotoxicity and phytoavailability of Pb and As in a mining technosol CLEAN-Soil[END_REF]. It was shown that the addition of compost may increase the mobility and leacheability of metal(loid)s, particularly arsenic [START_REF] Mench | Progress in remediation and revegetation of the barren Jales gold mine spoil after in situ treatments[END_REF]. In the present study, a significant increase in arsenic SPW concentration was observed when compost was added to soils, probably because DOC competed with arsenic for sorption sites, such as those present in iron oxides, resulting in its increased mobility [START_REF] Bolan | Remediation of heavy metal(loid)s contaminated soils or to immobilize?[END_REF]. Compost with a high degradable organic matter content, associated to a neutral pH, present an active microbial activity which uses the organic matter as energy source [START_REF] Balasoiu | Partitioning and speciation of chromium, copper, and arsenic in CCA-contaminated soils: influence of soil composition[END_REF]. This means that compost and its microbiota could reduce the soil redox potential, and consequently support the transformation of As(V) to As(III), which is more mobile [START_REF] Kim | Stabilization of available arsenic in highly contaminated mine tailings using iron[END_REF]. This high mobility could also be explained by the presence of phosphorus. Indeed, [START_REF] Fresno | Aided phytostabilisation of As-and Cu-contaminated soils using white lupin and combined iron and organic amendments[END_REF] showed that phosphorus-Olsen had a significant effect on mobilizable As, as phosphate and arsenate have similar chemical properties, and they compete for organic and inorganic binding sites [START_REF] Bolan | Remediation of heavy metal(loid)s contaminated soils or to immobilize?[END_REF][START_REF] Moreno-Jiménez | Arsenic and selenium mobilisation from organic matter treated mine spoil with and without inorganic fertilisation[END_REF][START_REF] Adriano | Trace Elements in Terrestrial Environments[END_REF]. Other authors [START_REF] Clemente | The use of a halophytic plant species and organic amendments for the remediation of a trace elements-contaminated soil under semi-arid conditions[END_REF][START_REF] Moreno-Jiménez | Arsenic and selenium mobilisation from organic matter treated mine spoil with and without inorganic fertilisation[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 already reported that competition for mineral sorption sites between these anions can be expected, which will lead to arsenic mobilization from less labile soil fractions and increase its availability. It is important to note that the phytoavailable fraction of arsenic was identical for the Ps and PsC treatments, suggesting that compost influenced the easily mobilizable fraction found in pore water, but not the CaCl2 extractable fraction. Moreover, the decrease in As concentration observed between PsB and PsBC both at T0 and at TF (Table 2) could be explained by: (i) the mobile part of arsenic potentially being trapped in the biochar structure; (ii) DOC decrease after biochar addition resulting in less competition between dissolved organic matter and arsenate; and (iii) the calcite present in the biochar could form stable spherical compounds with arsenate anions on the biochar's surface [START_REF] Alexandratos | Arsenate uptake by calcite: macroscopic and spectroscopic characterization of adsorption and incorporation mechanisms[END_REF][START_REF] Yin | Varying effect of biochar on Cd, Pb and As mobility in a multi-metal contaminated paddy soil[END_REF].

At T0, the SPW iron concentration (Table 2) for Ps was 0.05 mg.L -1 . No soluble Fe was quantified in SPW for PsC, PsC and PsBC. As expected, Fe concentration in PsI and PsBI treatments were 450 times and 50 times higher than Ps, respectively. Moreover, for PsI and PsBI, no significant difference in SPW Fe concentration was observed at TF between nonvegetated and vegetated pots. Although at TF, SPW Fe concentration increased by 2.4 fold for PsI and 38 fold for PsBI. The values for the Fe-CaCl2-extractable fraction (Table 1) for Ps, PsI and PsBI, were not significantly different, and were all between 0.06 % and 0.08 %. Finally, compost or biochar added to Ps soil, alone or in combination (PsB, PsC and PsBC), led to a significant decrease in the Fe-CaCl2-extractable fraction (3.5 fold).

As expected, lead was the most prevalent ion in Pontgibaud SPW (11.2 mg.L -1 ), at T0, and on top of that, no significant difference was measured between T0 and TF for Ps, or any of the other studied treatments. The SPW Pb concentrations for PsI, PsC, PsB, PsBI and PsBC were 4.8 mg.L -1 , 0.3 mg.L -1 , 0.6 mg.L -1 , 1.4 mg.L -1 and 0.12 mg.L -1 , respectively. For the Pb-CaCl2-extractable fraction (Table 1) four different significant groups were identified PsI 2.75 %; Ps 2.15 %; PsB and PsBI 1.6 %; and PsC and PsBC 0.15 %.

Compost and biochar + compost appeared to be the treatments that decreased the phytoavailable Pb fraction the most significantly, suggesting that the high pH and organic content of these amendments reduce the lead mobility in the soil. As is well known, SPW metal mobility is strongly influenced by organic carbon, which is brought to the soil by the biochar and compost [START_REF] Bolan | Remediation of heavy metal(loid)s contaminated soils or to immobilize?[END_REF]. Furthermore, higher pH values are known to increase the surface charges of soil particles including amendments, which induces the retention of metal (Perez-Esteban et al., 2014;[START_REF] Boisson | Evaluation of hydroxyapatite as a metal immobilizing soil additive for the remediation of polluted soils. Part 1. Influence of hydroxyapatite on metal exchangeability in soil, plant growth and plant metal accumulation[END_REF][START_REF] Mench | Physico-chemical aspects and efficiency of trace element immobilization by soil amendments[END_REF][START_REF] Mench | Capacity of soil amendments in lowering the phytoavailability of sludge-borne zinc[END_REF].

These amendments can immobilize metal(loid)s via three mechanisms [START_REF] Mench | Capacity of soil amendments in lowering the phytoavailability of sludge-borne zinc[END_REF]:

(i) adsorption of metals to highly accessible sites on the surface of modified aluminosilicates, soil components and of biochar or compost through interactions with oxygenated functional groups (Lebrun et al. 2018b); (ii) precipitation with Al, Fe and Mn oxides [START_REF] Oustrière | Woodderivedbiochar combined with compost or iron grit for in situ stabilization of Cd, Pb, and Zn in a contaminated soil[END_REF]; (iii) formation of minerals (such as metal silicates) and diffusion through mineral surfaces [START_REF] Lee | Comparison of biochar properties from biomass residues produced by slow pyrolysis at 500°C[END_REF] and (iv) precipitation with phosphates and carbonates contained by the biochar [START_REF] Lomaglio | Effect of biochar amendments on the mobility and (bio)availability of As, Sb and Pb in a contaminated mine technosol[END_REF].

Metal(loid) bioaccessibility in soil

The SBET was used to assess the oral bioaccessibility of As, Fe and Pb in soil samples [START_REF] Mendoza | Evaluation of the bioaccessible gastric and intestinal fractions of heavy metals in contaminated soils by means of a simple bioaccessibility extraction test[END_REF]. This method simulates the human gastric phase, and was validated by the EPA (USEPA, 2012). The results for the present study are presented in Table 3.

The bioaccessible arsenic concentration for Ps was 3.2% and decreased for PsI and PsBI treatments by 2.1 and 1.8 times respectively, compared to Ps. This is because the iron oxide formed strongly adsorbed As. Many studies [START_REF] Martin | In situ remediation of arsenic in contaminated soils[END_REF][START_REF] Subacz | Decreasing arsenic bioaccessibility bioavailability in soils with iron amendments[END_REF] have shown that use of Fe-rich amendments (FeCl3, FeCl2.4H2O, Ferrihydrite, Fe 0 and FeBr3) could be an effective strategy for reducing bioavailability and bioaccessibility, and for remediating soils contaminated with As. [START_REF] Cui | Assessment of In Situ Immobilization of Lead (Pb) and Arsenic (As) in Contaminated Soils with Phosphate and Iron: Solubility and Bioaccessibility[END_REF] demonstrated that the addition of FeSO4 to a mining technosol decreased gastric and intestinal bioaccessibility of arsenic in all treatments. Finally, the bioaccessible arsenic fraction for PsC, PsB and PsBC treatments was almost 1.4 times higher than for Ps. This could be explained by the fact that anions such as bicarbonate, phosphate, silicate, and organic acids present in the soil solution due to the addition of compost and biochar to Ps could compete with As for chemisorption sites, and thus mobilize As to the aqueous phase. The links between biochar and arsenic, and between compost and arsenic are weak and therefore easily broken by the glycine present in SBET solution. This would explain the greater As accessibility for PsC, PsB and PsBC.

The iron bioaccessible fraction for Ps was 1.3 % , which demonstrates its high stability in soil. When compost and biochar were added to Ps (PsC and PsB), Fe accessibility was unchanged. As expected, the addition of iron to Ps or PsB led to an increase in accessible-Fe, by 1.9 and 3.1 fold, respectively. Interestingly, for PsBC, the iron bioaccessible fraction was almost twice the value measured for Ps. This was probably due to the destabilization of iron oxides by the increase in organic ligands following the addition of biochar and compost.

Finally, the bioaccessible Pb proportion was high regardless of the treatment. For the Ps treatment, just over half of the total lead (51 %) was bioaccessible, which shows its low stability in soil. This could be explained by the low pH (pH = 1.5) and the presence of organic ligands in the gastric solution which may have favored the dissolution and desorption of the minerals containing lead [START_REF] Cui | Assessment of In Situ Immobilization of Lead (Pb) and Arsenic (As) in Contaminated Soils with Phosphate and Iron: Solubility and Bioaccessibility[END_REF]. Iron did not affect the Pb accessible fraction when added to Ps or PsB. Similarly, a recent study showed that a highly adsorbent Mn oxide added in large quantities (10% by weight) to a Pb polluted soil produced no change to the Pb bioaccessible fraction [START_REF] Mccann | Remediation of a historically Pb contaminated soil using a model natural Mn oxide waste[END_REF]. When compost or biochar were added to Ps, whether alone or in combination, the Pb accessible fraction increased between 1.2 and 1.4 times . This result was probably due to the establishment of even weaker links between the lead and compost and lead and biochar than those that may exist between lead and soil particles in the technosol Ps.

Plant growth and metal(loid) uptake

After 30 days of growth, the clover dry weight (DW) and the metal(loid) concentrations in the roots and aerial parts were measured for the different treatments. Firstly, it was noted that when iron was added alone to Ps the seeds could not germinate. This was probably due to the high iron concentration in the soil pore water at T0. Indeed, when just iron grit was added to Ps the concentration of Fe in the SPW was 22.5 mg L -1 , whereas when iron grit was added to Ps at the same time as biochar (PsBI), the Fe SPW concentration was 2.5 mg L -1 due to the biochar's physico-chemical characteristics. Iron alone seemed to have an acidifying effect, certainly due to the oxidation of Fe(II) followed by the precipitation of Fe(OH)3. Thus, the combination of high Fe supply and acidity induced a high solubility and Fe availability. In the presence of biochar, the pH effect was attenuated. However, it was noted that the smallest increase in the biomass of aerial parts was observed for PsBI treatment (0.623 g). For the Ps treatment, the dry weights of the roots and aerial parts were smaller, and corresponded to 0.124 g and 0.170 g, respectively. For the roots, PsC, PsB and PsBC DW were not significantly different and corresponded to four times the value for Ps.

For PsC, PsB and PsBC, the average DW of the aerial parts was 1.24 g, i.e. 7 times the value measured in Ps pots. The same beneficial effect for the DW of Salix viminalis and Populus euramericana cuttings was obtained by Lebrun et al. (2018a) when adding biochar produced from different feedstocks (hardwood, lightwood and pinewood, at 2% and 5% w/w) to Pontgibaud technosol. [START_REF] Fresno | Aided phytostabilisation of As-and Cu-contaminated soils using white lupin and combined iron and organic amendments[END_REF] showed that iron combined with bio-char on an As and Cu contaminated technosol slightly increased lupine shoot biomass. In our case, even though biochar allowed a better biomass production of clover aerial parts, the compost addition to biochar improved DW production by 30%. Indeed, organic matter, provided by the compost improved the N, P, K nutrient cycle [START_REF] Marques | Application of manure and compost to contaminated soils and its effect on zinc accumulation by Solanum nigrum inoculated with arbuscular mycorrhizal fungi[END_REF][START_REF] Abbas | Biochar application increased the growth and yield and reduced cadmium in drought stressed wheat grown in an aged contaminated soil[END_REF], allowing better plant growth, as observed by [START_REF] Marques | Application of manure and compost to contaminated soils and its effect on zinc accumulation by Solanum nigrum inoculated with arbuscular mycorrhizal fungi[END_REF] 1.8 times for arsenic and between 1.8 and 3.8 times for lead, whereas As and Pb concentrations in Ps plants were 396 mg kg -1 and 4577 mg kg -1 , respectively. In roots, As concentrations were only decreased by PsB and PsBC treatments, and corresponded to 500 mg kg -1 , whereas for Ps, PsC and PsBI, As concentrations were approximately 812 mg kg -1 . For Pb concentrations in roots, Ps and PsBI demonstrated the same accumulation i.e 13300 mg kg -1 , whereas PsC, PsB and PsBC decreased root Pb concentration by 2.4 fold. As expected, iron concentration was highest in clover plants grown on the PsBI soil, with concentrations of 5000 mg kg -1 and 17000 mg kg -1 in aerial parts and roots, respectively. Whereas for Ps, PsC, PsB and PsBC treatments Fe concentrations in aerial parts were 2500, 1200, 1300 and 800 mg kg -1 , respectively. Finally, Fe concentrations in roots for Ps, PsC, PsB and PsBC were not significantly different, and corresponded to 3100 mg kg -1 , which was 5.5 times lower than the Fe concentration measured in roots of plants grown on the PsBI soil. In the present study, for all treatments, As, Fe and Pb concentrations were higher in the roots than in the aerial parts of clover. This is consistent with the results of [START_REF] Bidar | Behavior of Trifolium repens and Lolium perenne growing in a heavy metal contaminated field: plant metal concentration and phytotoxicity[END_REF][START_REF] Bidar | Seasonal and annual variations of metal uptake, bioaccumulation, and toxicity in Trifolium repens and Lolium perenne growing in a heavy metal-contaminated field[END_REF] and [START_REF] Lopareva-Pohu | Influence of fly ash aided phytostabilisation of Pb, Cd and Zn highly contaminated soils on Lolium perenne and Trifolium repens metal transfer and physiological stress[END_REF] for Trifolium repens growing on metal(loid) contaminated soil. However, it is difficult to distinguish between the elements fixed to the root surface and those that are absorbed. We observed, a significant As, Fe and Pb concentration decrease in the aerial parts for all treatments, apart from for plant Fe accumulation levels on the PsBI treatment. For Pb and Fe, this decrease could be associated to a significant diminution of the two ions in the soil pore water, correlated to the amendments. Interestingly we demonstrated that compost being added to PsC and PsBC soils was associated with an increase in As concentrations in the soil pore water, whereas As concentrations in the aerial parts decreased compared to Ps. Such results could be associated to a probable change in the chemical form of As, which is certainly less assimilable for clover plants. In fact, the plant metal(loid) absorption depends on different parameters, relative both to the plant (such as root exudations), and to the soil characteristics (such as oxidation-reduction processes in the soil, pH, cation exchange capacity, dissolved oxygen, and temperature), which modify the metal(loid) speciation [START_REF] Cheng | Heavy metals in plants and phytoremediation[END_REF].

The high concentration of As in the roots for the PsBI treatment compared to the PsB and PsBC treatments, which have similar biomasses and phytoavailable arsenic, could be due the mobilization of As by root exudates in soils treated with iron [START_REF] Fresno | Effect of Lupinus albus L. root activities on as and Cu mobility after addition of iron-based soil amendments[END_REF]). Another explanation could be that the precipitation of iron oxides on the roots causes an indirect increase in arsenic on the roots. The SBET bioaccessible fraction (Table 4) was only measured for the aerial parts of clover plants grown on amended soil. We were not able to do these measurements for aerial parts of clover grown on Pontgibaud soil (Ps) due to poor plant biomass.

The bioaccessible As fraction was significantly larger for clover plants grown on soils containing compost (Table 4). Thus, the SBET bioaccessible As fraction for clover plants collected on PsC and PsBC soils was 8.2 % and 9.5 %, respectively. These values were twice as high as those calculated for clover aerial parts collected on PsB and PsBI soils. The PsB and PsBI soils were the treatments with the lowest As SBET bioaccessible fraction. Finally, we demonstrated that the PsBI soil allowed the highest Fe and Pb bioaccessible fractions for clover aerial organs. On average this increase was 6.6 and 1.7 times the average value measured for the other amendments tested, respectively. It was also noted that lead was the most accessible element measured in the clover aerial organs. Between 34 % and 64% of all lead was part of the bioaccessible fraction and could therefore potentially be released into the gastric tract. In addition, our results for lead were significantly higher (2 to 6 times higher), than those obtained in the gastric phase for edible parts of spinach, lettuce, radish and carrot plant grown on a poly-metallic polluted soil [START_REF] Intawongse | Use of the physiologically-based extraction test to assess the oral bioaccessibility of metals in vegetable plants grown in contaminated soil[END_REF].

It has been explained that the SBET bioaccessible fraction of ions in plant organs is correlated to the chemical form, location and concentration of the ion, as well as the compounds to which it is potentially associated within the plant tissues, such as fibers, polyphenols and phytates [START_REF] Labronici-Bertin | Mineral composition and bioaccessibility in Sarcocornia ambigua using ICP-MS[END_REF][START_REF] Machado | In vitro bioaccessibility study of As, Cd, Cu, Fe, Ni, Pb and Zn from raw edible artichoke heads (Cynara cardunculus L. subsp. Cardunculus)[END_REF]. These specific tissue locations for As and Pb are linked to plant metal(loid) tolerance. Indeed, [START_REF] Chou | Recent progress in protein subcellular location prediction[END_REF] and [START_REF] Fu | In vitro digestion/Caco-2 cell model to estimate cadmium and lead bioaccessibility/bioavailability in two vegetables: The influence of cooking and additives[END_REF] proposed that metal(loid)s are mainly found in the vacuole and cell wall, which contains polysaccharides and proteins which are composed of many oxhydryl, carboxyl, aldehyde and phosphate groups, which show affinity with metal(loid)s. Therefore, these groups could form bonds to reduce metal(loid) activity, which could modify their bioaccessible fraction.

Conclusion

In the present study, we demonstrated that the addition of amendments (compost, biochar or iron grit, alone or combined) to an As and Pb polluted soil could be efficient in reducing As and Pb soil mobility and availability. In addition, the amendments allowed the establishment of a clover cover on a soil that had previously been resistant to any plant development. The most appropriate treatment for the studied technosol was 5 % biochar combined with 5 % compost. It resulted in a decrease in As and Pb concentrations in both clover roots and aerial parts. However, while the Pb concentration in SPW decreased significantly, the As concentration increased, although it did remained within environmentally acceptable values. It should also be noted that the bioaccessible As and Pb soil fractions increased significantly following the addition of amendments. In our case, the use of iron grit did not give the expected positive results.

Finally, field tests using the selected combination of amendments are currently being undertaken to demonstrate the role of clover leaf cover in stabilizing soil particles, and preventing the dispersion of arsenic and lead into the environment by wind or surface runoff.

  on Solanum nigrum and Gregory et al. (2014) on Lolium perenne grown on polluted soils Concerning the concentrations of ions in clover organs (Fig 2), we noticed that the amendments systematically decreased the As and Pb concentrations in aerial parts by about

Figure 1 :Figure 2 :

 12 Figure 1: Trifolium repens dry weight (DW) (g) of organs collected at the end of the experiment (30 days); white columns: aerial parts, black columns: roots. Results are obtained from 500 seeds sown on tested soils. Ps: polluted soil, PsI: Ps + iron grit (I) 1.5%, PsC: Ps + compost (C) 5%, PsB: Ps + biochar (B) 5%, PsBI: Ps + B 5% + I 1.5%, PsBC: Ps + B 5% + C 5%. Results are expressed as the mean value and bars indicate standard error (n=5); letters indicate significant difference (p<0.05). NG: no germination.

Table 1 :

 1 Physico-chemical characteristics of tested soils at the beginning of the experiment (T0). WHC: water holding capacity (% mass), TOC: total organic carbon (% mass) and soil As, Fe and Pb concentrations (mg.kg -1 ) and CaCl2-extractable fraction (%). Ps: polluted soil, PsI: Ps + iron grit (I) 1.5%, PsC: Ps + compost (C) 5%, PsB: Ps + biochar (B) 5%, PsBI: Ps + B 5% + I 1.5%, PsBC: Ps + B 5% + C 5%. Results are expressed as the mean value ± standard error (n = 5). Letters indicate treatment significant difference (p < 0.05).

		WHC (%)	TOC (%)	Pseudo-total concentrations (mg.kg -1 )	CaCl2-extractable fraction (%)
	treatments			[As]	[Fe]	[Pb]	[As]	[Fe]	[Pb]
	Ps	29.80±0.47 e	0.04	1068±20 a	6325±114 b	23387±1020 a	0.11±0.01 a	0.06±0.02 a	2.15±0.19 b
	PsI	28.11±0.38 f	0.04	1007±52 a	23248±4341 a	18420±1422 ab	0.07±0.01 b	0.08±0.01 a	2.75±0.06 a
	PsC	32.75±0.49 d	0.87	906±44 a	5407±205 b	18882±670 ab	0.13±0.01 a	0.02±0.01 b	0.12±0.03 d
	PsB	40.11±0.48 b	3.28	1003±18 a	5960±103 b	18927±555 ab	0.05±0.01 b	0.01±0.00 b	1.59±0.06 c
	PsBI	36.83±0.26 c	4.03	990±17 a	20090±2410 a	18903±435 ab	0.05±0.01 b	0.06±0.01 a	1.60±0.09 c
	PsBC	44.55±2.06 a	3.89	842±41 a	4973±296 b	16419±295 b	0.06±0.01 b	0.02±0.01 b	0.17±0.04 d

Table 2 :

 2 Physico-chemical characteristics of the soil pore water of tested soils at the beginning of the experiment (T0) and after 30 days (TF). Non-vegetated soils corresponded to TF-NV and vegetated soils corresponded to TF-V. EC: Electrical Conductivity (mS.cm -1 ), DOC: dissolved organic carbon (mg.L -1 ) and As, Fe and Pb concentrations (mg.L -1 ). Ps: polluted soil, PsI: Ps + iron grit (I) 1.5%, PsC: Ps + compost (C) 5%, PsB: Ps + biochar (B) 5%, PsBI: Ps + B 5% + I 1.5%, PsBC: Ps + B 5% + C 5%. Results are expressed as the mean value ± standard error (n = 5). For each treatment, letters indicate time and vegetation effect (p < 0.05). Amendments effect is represented by asterisk, **: p < 0.01, ***: p < 0.001.

	treatments		pH	EC (mS.cm -1 ) DOC (mg.L -1 ) [As] (mg.L -1 )	[Fe] (mg.L -1 )	[Pb] (mg.L -1 )
		T0	3.98±0.20 a 0.220±0.09 a	5.7±0.3 a	0.00±0.00 a	0.05±0.02 a	11.25±0.41 a
	Ps	TF-NV	4.02±0.16 a 0.916±0.063 b	5.9±0.6 a	0.03±0.00 b	0.09±0.02 a	8.08±1.60 a
		TF-V	4.24±0.18 a 0.761±0.067 b	5.4±0.1 a	0.02±0.00 b	0.02±0.01 a	5.46±1.52a
		T0	4.51±0.03 a 0.315±0.025 a	4.8±0.2 a	0.00±0.00 a	22.47±0.45 a	5.92±0.25 a
	PsI	TF-NV	4.02±0.03 b 0.797±0.044 b	4.9±0.4 a	0.00±0.00 a	59.03±6.04 b	5.06±0.51 a
		TF-V	4.09±0.06 b 0.822±0.039 b	4.8±0.5 a	0.00±0.00 a	50.16±14.61 b	3.46±0.52 a
		T0	6.32±0.15 a 0.926±0.095 a	23.4±0.2 a	0.09±0.01 a	0.01±0.01 a	0.61±0.12 a
	PsC	TF-NV	6.97±0.19 a 1.824±0.085 b	21.9±6.8 a	0.43±0.09 b	0.00±0.00 a	0.18±0.06 a
		TF-V	6.95±0.11 a 1.295±0.134 a	24.9±6.7 a	0.34±0.11 b	0.01±0.01 a	0.15±0.07 a
		T0	6.70±0.04 a	1.176±0.079 a	2.0±0.2 a	0.00±0.00 a	0.00±0.00 a	0.58±0.04 a
	PsB	TF-NV	7.04±0.13 ab 2.785±0.042 b	2.1±0.5 a	0.00±0.00 a	0.00±0.00 a	0.54±0.01 a
		TF-V	7.16±0.13 b 2.433±0.082 c	1.9±0.1 a	0.00±0.00 a	0.00±0.00 a	0.61±0.07 a
		T0	6.60±0.10 a 1.088±0.035 a	5.7±0.5 a	0.00±0.00 a	2.51±0.33 a	0.88±0.21 a
	PsBI	TF-NV	4.53±0.09 b 3.196±0.047 b	5.8±0.4 a	0.00±0.00 a	107.64±16.24 b	1.78±0.18 a
		TF-V	4.47±0.04 b 2.634±0.064 c	5.6±0.4 a	0.00±0.00 a	83.09±23.34 b	1.44±0.13 a
		T0	7.31±0.09 a 1.232±0.094 a	10.7±0.2 a	0.06±0.01 a	0.00±0.00 a	0.15±0.02 a
	PsBC	TF-NV	7.64±0.15 ab 2.573±0.019 b	10.6±0.9 a	0.15±0.06 b	0.00±0.00 a	0.06±0.01 a
		TF-V	7.75±0.13 b 2.255±0.199 b	10.8±0.8 a	0.12±0.05 b	0.00±0.00 a	0.15±0.04 a
	amendments effect T0	***	***	***	***	***	***
	amendments effect NV	***	***	***	***	***	***
	amendments effect V	***	***	**	***	***	***

Table 3 :

 3 As, Fe and Pb bioaccessible fractions (%) in tested soils using SBET (Simplified Bioaccessibility Extraction Test). Ps: polluted soil, PsI: Ps + iron grit (I) 1.5%, PsC: Ps + compost (C) 5%, PsB: Ps + biochar (B) 5%, PsBI: Ps + B 5% + I 1.5%, PsBC: Ps + B 5% + C 5%. Results are expressed as the mean value ± standard error (n = 5). Letters indicate treatment significant difference (p < 0.05).

		SBET bioaccessible fractions (%)
	treatments	[As]	[Fe]	[Pb]
	Ps	3.24±0.07 c	1.32±0.21 b	51.12±0.61 d
	PsI	1.58±0.09 d	2.43±0.24 c	53.00±2.36 cd
	PsC	4.38±0.20 b 2.12±0.14 bc	60.54±1.04 b
	PsB	4.12±0.15 b	1.68±0.07 b	59.52±2.04 bc
	PsBI	1.97±0.12 d	4.14±0.32 a	54.08±2.79 cd
	PsBC	5.11±0.14 a	2.46±0.10 c	69.49±1.18 a

Table 4 :

 4 As, Fe and Pb bioaccessible fractions (%) in Trifolium repens aerial parts collected at the end of the experiment (30 days) on amended soils. Results are obtained from 500 seeds sown on tested soils. PsC: Ps + compost (C) 5%, PsB: Ps + biochar (B) 5%, PsBI: Ps + B 5% + I 1.5%, PsBC: Ps + B 5% + C 5%. Results are expressed as the mean value ± standard error (n=5); letters indicate significant difference (p<0.05).

		SBET bioaccessible fractions (%)
	treatments	As	Fe	Pb
	PsC	8.2±0.5 a	2.3±0.2 b	34.6±2.2 b
	PsB	5.5±0.7 b	2.3±0.4 b	34.7±1.8 b
	PsBI	3.6±0.2 b	16.4±3.0 a	64.2±3.7 a
	PsBC	9.5±0.6 a	2.9±0.2 b	42.5±2.6 b

Acknowledgments:

We would like to thank Ingrid Girardeau and Louis De Lary De Latour (DPSM / BRGM) for giving us the authorization to study the former Pontgibaud mine and Mr. JC. Léger (La Carbonerie) for the supply of biochar. We also wanted to thank the National Association of Research and Technology (ANRT) for funding R. Nandillon's thesis.

Data processing and statistical analysis

Data was analyzed using R statistical software Version 3.1.2 (R Development Core Team, 2009). The normality and homogeneity were tested using Shapiro and Bartlett tests, and the means were compared using a parametric Anova test for normal data and the nonparametric Kruskal-Wallis test for non-normal data. Differences were considered significant when p<0.05.


Romain Nandillon

Manhattan Lebrun

Florie Miard

Gaillard Marie

Stéphane Sabatier

Marc Villar

Sylvain Bourgerie

Domenico Morabito
email: domenico.morabito@univ-orleans.fr

M Gaillard

Capability of amendments (biochar, compost and garden soil) added to a mining technosol contaminated by Pb and As to allow poplar

Keywords: Phytomanagement, Amendments, Biochar, Metal(loid)s, Seeds, Populus nigra, As, Pb

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

Introduction

Out of all the inorganic soil pollutants, metal(loid)s are the main cause of soil contamination on a global level. In the European Union, metal(loid) contamination accounts for more than 37% of the polluted areas, followed by mineral oils (33.7%) and polycyclic aromatic hydrocarbons (PAHs, 13.3%) (EEA 2007). Localised contaminated soils, known as brownfields (French et al. 2006), are frequently associated to abandoned industrial areas or to urban soils (EEA-UNEP 2000). Mining activities are the main sources of environmental contamination as they release large amounts of metallic and metalloid elements through erosion caused by runoff or wind [START_REF] Razo | Arsenic and heavy metal pollution of soil, water and sediments in a semi-arid climate mining area in Mexico[END_REF].

Many industrialised countries are now focusing on using regulations to reduce the impact of pollution. For example, the European Pollutant Release and Transfer Register (Regulation 166 EC 2006) lists potentially hazardous activities and pollutants and establishes the thresholds for releases to air, water and soil for all major contaminants [START_REF] Vamerali | Phytoremediation trials on metaland arsenic-contaminated pyrite wastes (Torviscosa, Italy)[END_REF]. Most of the remediation methods developed for metallised soils are physical or chemical approaches, such as soil washing, excavation and rewetting [START_REF] Wang | A review on In situ phytoremediation of mine tailings[END_REF]. Phytoremediation uses plants to absorb or to stabilise metals in the roots and in the associated rhizosphere and is a low-cost technique carried out directly on the contaminated spot [START_REF] Salt | An annual review of plant physiology and plant molecular biology[END_REF][START_REF] Vamerali | Phytoremediation trials on metaland arsenic-contaminated pyrite wastes (Torviscosa, Italy)[END_REF]. Phytoremediation could help maintain or even improve soil structure [START_REF] Mleczek | Biomass productivity and phytoremediation potential of Salix alba and Salix viminalis[END_REF]. [START_REF] Vamerali | Phytoremediation trials on metaland arsenic-contaminated pyrite wastes (Torviscosa, Italy)[END_REF] and [START_REF] Ali | Phytoremediation of heavy metals-concepts and applications[END_REF] defined this strategy as an alternative method which is positively perceived by the population due to its significantly reducing metal(loid) availability without using chemical processes. It also creates a favourable environment around the root system by providing organic and mineral nutrients, resulting in a decrease in the amount of pollutants released into the environment through erosion. Phytoremediation is a general term comprising different techniques, with the two major methods being phytoextraction and phytostabilisation.

Phytostabilisation is based on root characteristics such as biomass, exchange surface, exudates production and the associated microorganism activities. Root metal(loid) accumulation is also indirectly linked to plant transpiration, which could allow greater ion absorption. Finally, when fixed in the root and the rhizosphere, metal(loid) leaching is then controlled [START_REF] Sarwar | Phytoremediation strategies for soils contaminated with heavy metals: modifications and future perspectives[END_REF]. Many abandoned or post-operative mining dams are sterile or have minimal natural vegetative colonisation due to limited soil physicochemical characteristics such as acidic or alkaline pH, high ion content, lack of required nutrients, metal toxicity, high bulk density, lack of structure, low water retention capacity and, sometimes, low air permeability [START_REF] Lebrun | Biochar effect associated with compost and iron to promote Pb and As soil stabilization and Salix viminalis L. growth[END_REF]. To overcome these limitations, amendments can be added, which can be considered as an aided phytostabilisation (Alvarenga et al. 2009a, b;[START_REF] Galende | Field assessment of the effectiveness of organic amendments for aided phytostabilization of a Pb-Zn contaminated mine soil[END_REF]. An e ff i c i e n t w a y o f e s t a b l i s h i n g a n e ff i c i e n t phytostabilisation may therefore involve the addition of organic matter to soil, which will improve its structure and fertility, as well as its ability to retain water or create niches favourable to the development of microorganisms [START_REF] Pérez-Esteban | Phytostabilization of metals in mine soils using Brassica juncea in combination with organic amendments[END_REF][START_REF] Cha | Production and utilization of biochar: a review[END_REF][START_REF] Lebrun | Biochar effect associated with compost and iron to promote Pb and As soil stabilization and Salix viminalis L. growth[END_REF].

The selection of suitable plant species is crucial for a successful phytostabilisation. Such plants must be able to develop both an extensive root system and a large amount of biomass in the presence of metal(loid)s while mainly compartmentalising metal(loid)s in the root zone and keeping their translocation from the roots to the shoots as low as possible. This will limit their spread to the food chain or their return to the ground when the leaves fall [START_REF] Henry | Phytotechnologies: preventing exposures, improving public health[END_REF].

Several hundred different plant species could be used in a phytoremediation process [START_REF] Moosavi | Phytoremediation: a review[END_REF]. But, many of these plants are small (small leaf area and small root system) and would not be efficient, due to their evaporating limited amounts of water, and their roots being unable to explore a large volume of soil. To overcome these disadvantages, woody species with rapid growth, high biomass production and high metal(loid) storage capacity within a deep root system are attractive options. Among the woody species, willow and poplar have already been proposed as phytoremediating plants for metal-contaminated soils [START_REF] Marmiroli | Growth, physiological and molecular traits in Salicaceae trees investigated for phytoremediation of heavy metals and organics[END_REF]. Poplar, in particular, has several interesting features rendering it a good c an d i d at e f o r p hy t o r e m e d i a t i o n ( R ad o jč i ć Redovniković et al. 2017). For instance, Populus nigra, along with a few other poplar species, are recognised for both their root water purification abilities, which improve soil water quality. They are known, in particular, for their highly developed root system, which acts as a natural filter by trapping organic and inorganic pollutants [START_REF] Chamaillard | Efficience d'utilisation de l'eau chez le peuplier noir (Populus nigra L.): variabilité et plasticité en réponse aux variations de l'environnement. Biologie végétale[END_REF][START_REF] Houda | Comparative study of plant growth of two poplar tree species irrigated with treated wastewater, with particular reference to accumulation of heavy metals (Cd, Pb, As, and Ni)[END_REF][START_REF] Radojčić Redovniković | Poplar response to cadmium and lead soil contamination[END_REF]) and allows a good cohesion and fixation of soil materials [START_REF] Foussadier | Les systèmes racinaires des arbres de la ripisylve: effets des contraintes physiques et exemples[END_REF][START_REF] Rodrigues | In-channel woody vegetation controls on sedimentary processes and the sedimentary record within alluvial environments: a modern example of an anabranch of the River Loire, France[END_REF]. Poplar have a large geographical distribution zone from Western Europe to the western tip of China through a narrow North African fringe [START_REF] Euforgen | Distribution map of black poplar (Populus nigra)[END_REF], demonstrating a large adaptability to different soils and climates. In addition, its capacity to be used in short rotation coppices allows its use in biomass production. [START_REF] Baldantoni | Different behaviours in phytoremediation capacity of two heavy metal tolerant poplar clones in relation to iron and other trace elements[END_REF] showed that two poplar clones (Populus alba AL22 and P. nigra N12) cultivated on a soil collected from an urban and industrial area have a remarkable ability to absorb and accumulate Cd and Zn and to phytostabilise Cu, Fe and Pb.

Among the available amendments used in phytomanagement, biochar obtained mainly by carbon-rich biomass pyrolysis has attracted attention in recent years due to its beneficial effects on soils by increasing electrical conductivity, soil pore water (SPW) pH, microorganism activities [START_REF] Beesley | A review of biochars potential role in the remediation, revegetation and restoration of contaminated soils[END_REF]) and heavy metal sorption [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][START_REF] Lebrun | Effect of biochar amendments on as and Pb mobility and phytoavailability in contaminated mine technosols phytoremediated by Salix[END_REF]. However, the integration of biochar into the soil may have some disadvantages such as (i) SPW carbon release, which could increase arsenic mobility, and (ii) cationic nutrient immobilisation. When these two phenomena occur, they interact negatively with plant growth, especially if soil nutrient capacities are already low. It therefore seems necessary to combine biochar with other amendments such as compost, allowing the addition of micronutrients [START_REF] Beesley | A review of biochars potential role in the remediation, revegetation and restoration of contaminated soils[END_REF]. Various studies discuss using combinations of amendments [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] Lebrun | Effect of biochar amendments on as and Pb mobility and phytoavailability in contaminated mine technosols phytoremediated by Salix[END_REF], in which various types of organic amendments (garden soil and biochar) have been tested alone or associated in order to ameliorate plant growth and decrease SPW Pb concentration. Many other articles also reported the use of several types of organic amendments, such as compost and/or biochar [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][START_REF] Pérez-Esteban | Phytostabilization of metals in mine soils using Brassica juncea in combination with organic amendments[END_REF][START_REF] Yin | Varying effect of biochar on Cd, Pb and As mobility in a multi-metal contaminated paddy soil[END_REF][START_REF] Pardo | Phytostabilisation of severely contaminated mine tailings using halophytes and field addition of organic and inorganic amendments[END_REF].

Lastly, all previous experiments using poplar as phytoremediator plants have been undertaken using cuttings, which is an efficient way of producing monoclonal short rotation coppices [START_REF] Ruttens | Short rotation coppice culture of willow and poplar as energy crops on metal contaminated agricultural soils[END_REF][START_REF] Van Slycken | Field evaluation of willow under short rotation coppice for phytomanagement of metalpolluted agricultural soils[END_REF][START_REF] Janssen | Phytoremediation of metal contaminated soil using willow: exploiting plant-associated bacteria to improve biomass production and metal uptake[END_REF]. To our knowledge, no experiments have been conducted on poplar trees originating from seed germination with the aim of screening potential efficient phytoremediator genotypes and vegetating and remediating soils contaminated by metal(loid)s. We hypothesise that by planting poplar seeds instead of cuttings in an open field, we will (1) observe spontaneous poplar recolonisation on polluted sites by adding amendments when necessary and (2) perpetuate spontaneous plant propagation by improving the agronomic capacity of technosols while maintaining the biochar's ability to immobilise metal(loid)s.

The aims of our study were (i) to evaluate, in mesocosm, the germination capacity of black poplar seeds (Populus nigra L.) on a mining technosol, with and without the presence of amendments (biochar, compost or garden soil), and (ii) to determine the best combination of amendments (garden soil, compost and biochar). For this purpose, six different conditions were studied: mining technosol (P), mining technosol and hardwood-derived biochar (P + B), mining technosol and garden soil (P + G), mining technosol and compost (P + C) and combinations of different amendments: (P + G + B) and (P + C + B). The soil pore water physicochemical properties (pH, electrical conductivity (EC), As and Pb) were measured in tested soils. Germination rate, plant growth and metal(loid)s accumulated into plant organs were determined.

Materials and methods

Soil sampling

The studied site in Roure-les-Rosiers corresponds to a former silver-lead extraction mine and is located in the Massif Central near Clermont-Ferrand (Saint-Pierre-le-Chastel (63, France)), in the Pontgibaud mining district (P). Two streams cross the site: the Veyssière, which constitutes the southern edge of the deposits, and the Faye, which borders the northwest of the deposits. The location of the study area in Lambert II coordinates is as follows: X = 638,147.54 and Y = 2,087,962.79. The old mining site has a semi-continental climate, with hot summers, which can be marked by severe and localised thunderstorms and snowy winters. On average, the Roure-les-Rosiers sector receives about 770 mm of rainfall per year and is located at around 700 m above sea level. The area consists mainly of deposits of silverbearing lead ore that still contain toxic elements (mainly lead and arsenic). Surface soils (0-20 cm) were sampled with a stainless steel shovel. The soil samples collected were dried in open air, screened using a 2-mm mesh sieve to remove crude plant material (Qasim and Motelica-Heino 2014) and homogenised manually. One fraction was used to determine the physicochemical soil properties, another one was used for the metal(loid) analysis and the third fraction was used to conduct germination tests.

Soil physicochemical analysis

The As and Pb concentrations were determined using Xray fluorescence (XRF) (DELTA Portable XRF Analyzer Model; Olympus, Hannover, Germany). The signal provided by the pXRF was previously calibrated using a curve obtained after total dissolution in fluorhydric acid and inductively coupled plasma (ICP)-MS quantification of the original contaminated soil. The pH and EC were determined according to AFNOR NF T01-013, using a combined pH EC meter (ProfiLine 1970i; WTW, Germany), calibrated using pH 4.0 and pH 7.0 standards and a KCl standard solution with an electrical conductivity of 1430 μS cm -1 . Water holding capacity (WHC) was determined according to [START_REF] Lebrun | Effect of biochar amendments on as and Pb mobility and phytoavailability in contaminated mine technosols phytoremediated by Salix[END_REF].

Soil preparation

Three different organic amendments were added to Pontgibaud technosol, either individually or in combination: (i) a garden soil collected from the University of Orléans, Parc de la Source, France; (ii) a compost, which was a commercial product mix of potting soil and horse manure; and (iii) a biochar, made from tree wafer and chip feedstock (oak, beech and hornbeam) pyrolysed at 500 °C (La Carbonerie, Crissey, France). The main physicochemical properties of the various amendments are presented in Table 1. All amendments as well as Pontgibaud contaminated soil were homogenised and sieved to 2 mm. The mixtures tested corresponded to Pontgibaud contaminated soil (P), P amended with 10% garden soil (PG), P amended with 10% compost (PC), P amended with 2% hardwood-derived biochar (PB) and combinations of different amendments: PB + 10% garden soil (PBG) and PB + 10% compost (PBC). For each tested treatment, 300 g of soil were distributed individually in 400-mL pots, and 6 pots were prepared per treatment. The pots were then placed outside for 30 days for soil equilibration, at which point the poplar seeds were sown (T0) on the different soils tested. The growth of the young seedlings was measured after 28 days.

Soil pore water metal(loid) concentrations

To evaluate the effects of amendments on soil As and Pb leachability, soil pore waters were collected at day 0 and at day 28, using Rhizon samplers (Rhizosphere Research Product, Wageningen, The Netherlands). The Rhizon samplers were inserted transversely over the height of the pots. Samples were collected in plastic tubes, after conductivity and pH measurements according to [START_REF] Bart | Phytostabilization of As, Sb and Pb by two willow species (S. viminalis and S. purpurea) on former mine technosols[END_REF]. The samples were then acidified with 1.6% (v/v) of 65% HNO 3 and stored at 4 °C. Total dissolved metal(loid) concentrations (As, Pb) were determined by ICP atomic emission spectroscopy (AES) (ULTIMA 2, HORIBA; Labcompare, San Francisco, USA).

Seed germination

At day 0, twelve Populus nigra seeds were sown per pot and allowed to germinate and grow for 28 days. Pots were placed in a controlled chamber under a 16-h light regime at 23 °C ± 2 °C. The soil WHC was kept between 55% and 65% using deionised water. The germination rate was determined by the number of seedlings that had developed leaf and root systems at the end of the experiment (day 28).

Plant analysis, dry weight and metal(loid) concentrations

The seedlings were harvested 28 days after sowing. They were rinsed thoroughly with double deionised water, then divided into roots, stem and leaves before being dried at 40 °C, for 72 h, at which point their dry weight was measured. Three replicates per treatment and per organ were digested in a Regal water solution using a pressurised vacuum microwave system (Multiwave 3000; Anton Paar GmbH, Germany). The digestion program consisted of a gradual increase of temperature for 15 min up to 180 °C, followed by a digestion step of 15 min at 180 °C, then a cooling step to 55 °C over 15 min. Once cooled to room temperature, the samples were diluted in 30 mL of ultra-pure water (18 MΩ cm) and then filtered on a 0.45-μm filter nitrocellulose. Samples were measured three times using ICP-AES from plant digestions to determine the concentrations of As and Pb in different organs of Populus nigra.

Statistical analysis

As described by [START_REF] Lebrun | Effect of biochar amendments on as and Pb mobility and phytoavailability in contaminated mine technosols phytoremediated by Salix[END_REF], R statistical software (version 3.1.2) was used to analyse data. Parametric data was analysed using ANOVA and Tukey HSD tests, and non-parametric data were compared with the Kruskal-Wallis test per pair.

Results

Effects of amendments on seed germination and soil and SPW properties

The germination rate on the control medium was obtained by sowing the seeds on a substrate composed of only sand. The germination rate for this case was 92%. We showed that Pontgibaud contaminated soil (P) was unsuitable for spontaneous seed germination (Fig. 1). All the amendments allowed the germination of black poplar seeds without any real difference between treatments. Germination rates were between 43 and 69%. The pH of the P soil used in this study was 4.55 ± 0.06, its electrical conductivity was low (54 μS cm -1 ± 2 μS cm -1 ) and metal(loid) concentrations were 728 mg kg -1 ± 26 mg kg -1 for As and 9944 mg kg -1 ± 227 mg kg -1 for Pb. For the WHC (Table 2), the Pontgibaud soil plus garden soil (PG) treatment showed no improvement in WHC, whereas the other treatments improved soil water retention by 5%, 6%, 8% and 10% for PBG, PB, PC and PBC, respectively.

SPW pH and EC increased significantly (p < 0.05) after the addition of amendments (Table 2), but no significant differences were observed between T0 and T28. The pH of Pontgibaud SPW was 4.8, whereas for the amended soils, the pH was between 7.24 (PC) and 7.84 (PBC). EC was also positively affected by the addition of amendments; its value was multiplied by at least 2.3-fold for PBC, whereas when biochar was added alone, the EC value was three times higher than that in P (~600 μS cm -1 ).

The various amendments induced a rise in SPW As concentrations for all cases (Table 2). The PC (4.161 ± 0.556 at T0) treatment showed the largest increase, with a value 400 times higher than that in P (0.010 ± 0.007 at T0). For all the other treatments, the increase was smaller but significant (p < 0.05), with values ranging between 1.8 and 50 times for PG and PBC, respectively. In contrast, SPW Pb concentration was decreased by adding amendments. In non-amended soil (P), SPW Pb concentration was 12.3 mg L -1 , whereas for all amended treatments, SPW Pb concentration was at least by 93% lower. No significant differences were found between T0 and T28 (p < 0.05).

Seedlings' dry weight and metal(loid) uptake

Measurements for the dry biomass of the different seedling organs (Fig. 2) showed that the highest biomass overall was found for the PC treatment. The highest root biomass was observed on PG and PC modalities, with 1.31 mg and 1.26 mg, respectively. Root biomasses for PB, PBG and PBC treatments were measured with values of 0.63 mg, 0.58 mg and 0.66 mg, respectively. Stem biomass was measured for all modalities; PB and PBG treatments showed values of 1.76 mg and 1.89 mg, respectively, while PG and PBC treatments showed intermediate values of 2.8 mg; the highest value was for the PC treatment with 3.6 mg. The highest foliar biomass was also obtained for plants grown on PC with 14.88 mg, with the lowest on PB with 6.46 mg. The remaining values for foliar biomass corresponded to 10.46 mg, 7.04 mg and 7.83 mg for PG, PGB and PBC, respectively.

For the five treatments allowing germination, As (Fig. 3) and Pb (Fig. 4) concentrations were higher in the root parts than in the aerial parts (leaves and stems) of the poplar, except for the Pb concentration in the stem of the PBC treatment, which was twice that of the roots. Moreover, in terms of aerial organs, the stem had higher As and Pb concentrations than leaves. Arsenic concentrations in the leaves were not significantly different between PG, PC, PB and PBC modalities, with values of 0.035 mg g -1 , 0.026 mg g -1 , 0.043 mg g -1 and 0.020 mg g -1 , respectively. The lowest value was obtained when P soil was amended with biochar and garden soil (PBG) and corresponded to 0.002 mg g -1 . In the stems, arsenic concentrations for PG, PC and PB treatments were not significantly different, and their values were 0.12 mg g -1 , 0.06 mg g -1 and 0.08 mg g -1 , respectively. The lowest value was 0.01 mg g -1 and was observed for the PBG treatment, whereas the PBC treatment had the highest As concentration value (1.33 mg g -1 ). For the root parts, the highest As concentration was calculated for the PC treatment with a value of 4.10 mg g -1 , and the other treatments had an arsenic concentration of 2.60 mg g -1 , 2.47 mg g -1 , 1.80 mg g -1 and 0.99 mg g -1 for PG, PBC, PB and PBG, respectively (Fig. 3). Pb concentration in PG leaves was 0.55 mg g -1 whereas for PBC, PB, PBG and PC, the concentrations were 0.39 mg g -1 , 0.22 mg g -1 , 0.22 mg g -1 and 0.11 mg g -1 , respectively. The PBC treatment demonstrated the highest Pb concentration in the stems and corresponded to 6.50 mg g -1 , whereas for PB, PBG, PG and PC, lead concentrations were 1.09 mg g -1 , 0.92 mg g -1 , 0.60 mg g -1 and 0.41 mg g -1 , respectively. For the root parts, the highest concentrations of Pb were obtained for PG and PB treatments and corresponded to 9.11 mg g -1 and 8.46 mg g -1 , respectively, while for PBG, PC and PBC, lead concentrations were 5.98 mg g -1 , 4.11 mg g -1 and 3.55 mg g -1 , respectively (Fig. 4). The six different treatments were as follows: polluted soil (P), P amended with 10% garden soil (PG), P amended with 10% compost (PC), P amended with 2% hardwood-derived biochar (PB) and a combination of different amendments: PB + 10% garden soil (PBG) and PB + 10% compost (PBC) (mean n = 5; ±SE). Letters indicate a significant difference (p < 0.05)

Discussion

Physicochemical characteristics of soils and soil pore waters

In this study, all tested amendments increased SPW pH and EC values. Amendments reduced the mobility of Pb but increased arsenic mobility for the same tested modalities. After the addition of amendments (biochar, compost or garden soil), and in particular for biochar, pH increase can be explained by two mechanisms: (i) the alkaline pH of biochar induced a liming effect [START_REF] Bian | A three-year experiment confirms continuous immobilization of cadmium and lead in contaminated paddy field with biochar amendment[END_REF][START_REF] Lebrun | Effect of biochar amendments on as and Pb mobility and phytoavailability in contaminated mine technosols phytoremediated by Salix[END_REF]) and (ii) the presence of functional groups such as COO -and O -on the amendments was able to bind protons [START_REF] Houben | Impact of biochar and rootinduced changes on metal dynamics in the rhizosphere of Agrostis capillaris and Lupinus albus[END_REF]. The SPW electrical conductivity of Pontgibaud technosol (P) was very low and significantly increased by biochar addition. Similar results were also observed by [START_REF] Lomaglio | Effect of biochar amendments on the mobility and (bio)availability of As, Sb and Pb in a contaminated mine technosol[END_REF] who described an increase in EC when 2% or 5% of hardwood-based biochar was added to a mining technosol contaminated mainly by arsenic.

SPW Pb concentration in amended soils was always lower than that in P, suggesting that the high pH of amendments reduced soil Pb mobility, as shown by the negative correlation (-0.97) (Table 3) between the SPW Pb concentration and the pH values at T28. Indeed, a high pH value is known to increase the negative surface charges of soil particles and thus the retention of metals in soil [START_REF] Pérez-Esteban | Phytostabilization of metals in mine soils using Brassica juncea in combination with organic amendments[END_REF]. Moreover, as already described, metals such as Cd, Pb and Cu could be immobilised not only by the organic C content of biochar but also by humic acids contained in compost [START_REF] Bolan | Remediation of heavy metal(loid)s contaminated soils or to immobilize?[END_REF]. The mechanisms by which the amendments immobilise metal(loid)s are based on different properties, via different mechanisms [START_REF] Boisson | Immobilization of trace metals and arsenic by different soil additives: evaluation by means of chemical extractions[END_REF][START_REF] Mench | In-situ inactivation and phytorestoration of metal contaminated soils Physicochemical aspects and efficiency of trace element immobilization by soil amendments[END_REF][START_REF] Mench | Capacity of soil amendments in lowering the phytoavailability of sludge-borne zinc[END_REF]: (i) adsorption of metals to highly accessible sites located on the surface of biochar through interactions with oxygenated functional groups [START_REF] Lebrun | Effect of Fefunctionalized biochar on toxicity of a technosol contaminated by Pb and As: sorption and phytotoxicity tests[END_REF], as well as on the humic substances contained in the compost [START_REF] Lebrun | Biochar effect associated with compost and iron to promote Pb and As soil stabilization and Salix viminalis L. growth[END_REF], and (ii) precipitation with phosphates and carbonates contained by the biochar [START_REF] Lomaglio | Effect of biochar amendments on the mobility and (bio)availability of As, Sb and Pb in a contaminated mine technosol[END_REF].

Arsenic, unlike cationic metals (Cu, Zn, Pb, Cd), is known to be mainly present in the form of an oxyanion. Moreover, increasing soil pH allows an increase in As release from the solid phase to the soil pore water [START_REF] Beesley | A review of biochars potential role in the remediation, revegetation and restoration of contaminated soils[END_REF]. The effect of organic residues on the mobility and bioavailability of metal(loid)s in the soil depends on the type of soil and the characteristics of the amendment (EC, pH and degree of humification). This means that conditions such as a pH increase, induced by the addition of biochar to soils, could increase As mobility and therefore outweigh the capacity of biochar to sorb metal(loid)s. Finally, the organic and mineral residues brought about by the amendments are known to improve the agronomic potential of the new formed technosol, which should allow for better plant growth [START_REF] Abbas | Biochar application increased the growth and yield and reduced cadmium in drought stressed wheat grown in an aged contaminated soil[END_REF].

As observed by [START_REF] Mench | Progress in remediation and revegetation of the barren Jales gold mine spoil after in situ treatments[END_REF] at the Jales gold mine after adding municipal compost in combination with other amendments, the addition of compost to P increased the mobility and leaching of metal(loid)s, particularly arsenic. In the present study, the large increase in SPW arsenic concentration for the PC treatment was probably due to the fact that compost is able to discharge high quantities of dissolved organic carbon into the soil solution, which is known to compete with arsenic for sorption sites such as iron oxide, resulting in an increase in arsenic mobility [START_REF] Bolan | Remediation of heavy metal(loid)s contaminated soils or to immobilize?[END_REF][START_REF] Moreno-Jiménez | Arsenic and selenium mobilization from organic matter treated mine spoil with and without inorganic fertilization[END_REF]. Compost could also contain a significant amount of soluble phosphate which may have displaced arsenic from organic and inorganic binding sites [START_REF] Bolan | Remediation of heavy metal(loid)s contaminated soils or to immobilize?[END_REF]). However, the lower As concentrations observed in PBC SPW compared to PC suggest a possible and nonnegligible As entrapment by biochar.

Plant growth and metal(loid) tolerance

The unamended Pontgibaud soil is unsuitable for poplar seed germination. This certainly explains why the plot selected to sample the soil used for t h i s s t u d y i s c u r r e n t l y u n v e g e t a t e d . T h e improvement to Pontgibaud soil quality through the addition of amendments clearly allows better seed germination and the production of young seedlings. This can be explained (i) by the improvement in soil physicochemical properties (WHC, pH, EC) as well as the supply of nutrient matter [START_REF] Abbas | Biochar application increased the growth and yield and reduced cadmium in drought stressed wheat grown in an aged contaminated soil[END_REF] and by (ii) the decrease in SPW lead concentration. Interestingly, we observed concomitantly that although the highest dry weight production was when compost was added to P, the SPW As concentration for this treatment was higher than for the other amendments tested. This probably means that, even though As was found in high concentrations in the SPW in such conditions, the As was probably less phytoavailable. However, this increase in As in soil pore water could cause environmental problems for other organisms; therefore, this increase should be avoided or at least controlled.

It should be noted that the three lowest plant organ's dry weights were measured for PB, PBG and PBC treatments. This could be attributed to the presence of biochar, which probably partially adsorbed the nutrients present in soil pore water to its surface. The highest concentrations of Pb and As were found in poplar seedling root tissues for all amendments added to the Pontgibaud technosol. This was also observed by [START_REF] Vamerali | Phytoremediation trials on metaland arsenic-contaminated pyrite wastes (Torviscosa, Italy)[END_REF] who tested several species of Populus and Salix in metal-and arsenic-contaminated pyrite wastes (containing Co, Cu, Pb and Zn) and found that metal(loid) concentrations were higher in roots than in the upper organs. As demonstrated by [START_REF] Gupta | Lead tolerance in plants: strategies for remediation[END_REF], this root containment could prevent metal toxicity. Indeed, [START_REF] Borišev | Phytoextraction of Cd, Ni, and Pb using four willow clones (Salix spp.)[END_REF] proposed that willow metal(loid) exclusion mechanisms from the upper parts may protect the photosynthetic pathway, for example through complexation and sequestration of these metals in root cell vacuoles [START_REF] Lasat | Molecular physiology of zinc transport in the Zn hyperaccumulator Thlaspi caerulescens[END_REF]. Moreover, it was also proposed from a cellular point of view that mechanisms governing metal(loid) tolerance in plant cells correspond to (i) cell wall binding, (ii) active transport of ions into the vacuoles and (iii) chelation through the induction of metal-binding peptides and the formation of metal complexes (Hazrat et al. 2013).

Conclusion

The present work showed that the addition of compost, garden soil and biochar as soil amendments allows poplar seed germination and seedling development in a soil which was previously unfavourable to any vegetation. Such an approach could be proposed to remediate a soil, allowing it to be recolonised by the seeds present in the surrounding natural environment. Moreover, we demonstrated that such amendments could stabilise Pb in the soil effectively, allowing a decrease in concentrations found in the SPW, which, in fine, will protect the groundwater table. It is, however, important to precise that in the case of As, an increase in its mobility was observed for all amendments used and, in particular, when compost was added to the technosol. Further study into the behaviour of arsenic will be required, in particular concerning the choices for new amendments which will be efficient in stabilising it in the soil.

  Fig.1Germination (%) of Populus nigra seeds at 28 days under 7 different treatments: control, polluted soil (P), P amended with 10% garden soil (PG), P amended with 10% compost (PC), P amended with 2% hardwood-derived biochar (PB) and a combination of different amendments: PB + 10% garden soil (PBG) and PB + 10% compost (PBC) (mean n = 6; ±SE). NG non-germinated. Letters on bar graphs indicate a significant difference (p < 0.05)

  ± 0.03 d 7.87 ± 0.03 d 1396 ± 80 c 1367 ± 32 c 0.562 ± 0.133 f 0.591 ± 0.110 f 0.105 ± 0.013 f 0.092 ± 0.012 f

Fig. 2 Fig. 3

 23 Fig.2Dry weight (mg) of the different Populus nigra organs (leaves (white), stem (grey), root (black)) at 28 days under 5 different treatments: polluted soil (P), P amended with 10% garden soil (PG), P amended with 10% compost (PC), P amended with 2% hardwood-derived biochar (PB) and a combination of different amendments: PB + 10% garden soil (PBG) and PB + 10% compost (PBC) (mean n = 3; ±SE). Letters on bar graphs indicate a significant difference (p < 0.05)

Fig. 4

 4 Fig.4Lead concentration (mg g -1 ) in the different Populus nigra organs (leaves (white), stem (grey), root (black)) at 28 days under 5 different treatments: polluted soil (P), P amended with 10% garden soil (PG), P amended with 10% compost (PC), P amended

Table 1

 1 Amendments of physicochemical properties

	Granulometry (mm)	Compost (ND)	Biochar (0.2-0.4)	Garden soil (< 2)
	pH	7.4 ± 0.02	8.98 ± 0.02	6.5 ± 0.01
	EC (μS cm -1 )	801 ± 24	432 ± 2	253 ± 2
	WHC (%)	51 ± 1	183 ± 3	31 ± 2
	Specific area (m 2 g -1 )	ND	43.91	ND
	Total pore volume (cm 3 g -1 )	ND	0.0364	ND
	Mean pore diameter (nm)	ND	3.32	ND
	EC electrical conductivity (μS cm -1 ), WHC water holding capacity (% mass), ND no data

Table 2

 2 Technosol water holding capacity (WHC) and SPW physicochemical characteristics (pH, electrical conductivity (EC) and As and

	Pb concentration) at T0 (date of sowing poplar seeds) and at T28 (28 days) under 6 different treatments
		Soil WHC	SPW
		(%)
			pH		EC (μS cm -1 )	[As] (mg L -1 )		[Pb] (mg L -1 )
			T0	T28	T0	T28	T0	T28	T0	T28
	P	34 ± 0.3 a	4.80 ± 0.18 a 4.87 ± 0.11 a 599 ± 11 a 607 ± 2 a	0.01 ± 0.007 a 0.01 ± 0.003 a 12.307 ± 1.156 a 12.404 ± 1.003 a
	PG 33 ± 1.

Table 3

 3 Correlations between pH, electrical conductivity (EC), organs' dry weight (DW) and metal(loid)s in soil pore water (SPW) and in

	Populus nigra organs
		pH EC	SPW		DW			[As]			[Pb]
				[As]	[Pb]	Leaves	Stem	Root	Leaves	Stems	Roots	Leaves	Stems	Roots
	pH	1	0.87 0.12 -0.97 0.63	0.71	0.6	0.45	0.38	0.56	0.57	0.46	0.67
	EC		1	0.16 -0.93 0.69	0.66	0.74	0.69	-0.04 0.59	0.52	0.04	0.91
	SPW
	[As]			1	-0.27	0.74	0.65	0.53	0.18	-0.09 0.76	-0.27	-0.13 -0.16
	[Pb]				1	-0.78	-0.82 -0.76 -0.58	-0.28 -0.71 -0.58	-0.34 -0.73
	Weight
	Leaves					1	0.96	0.94	0.52	0.05	0.95	0.37	0.03	0.44
	Stems						1	0.91	0.5	0.31	0.96	0.52	0.29	0.4
	Roots							1	0.64	0	0.89	0.6	-0.04 0.62
	[As]
	Leaves								1	0.04	0.62	0.48	0.04	0.74
	Stems									1	0.23	0.44	0.99	0.17
	Roots										1	0.39	0.19	0.35
	[Pb]
	Leaves											1	0 . 4 2	0.61
	Stems											1	-0.11
	Roots											1
	Significant correlations are represented in italics

Acknowledgements

The authors wish to thank the Bureau de Recherche Géologique et Minière (BRGM) for giving access to the technosol and La Carbonerie (Léger J. C.) for providing the biochar.