Manhattan Lebrun

Florie Miard

Nour Hattab-Hambli

Gabriella S Scippa

Sylvain Bourgerie

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

Effect of different tissue biochar amendments on As and Pb stabilization and phytoavailability in a contaminated mine technosol

Phytomanagement of metal(loid) contaminated soils is an important study of research nowadays.

However, such process often requires the application of amendments, i.e biochar, to improve soil condition and thus permit plant establishment and growth. However, biochar properties and effects on the soil and plants depend on several parameters, for example: feedstock type, particle size, pyrolysis conditions, and application rate. The aim of this study was to assess which tissue from the oak trunk (bark, sapwood, heartwood) was responsible for the positive effects observed in previous studies on biochars derived from wood. A mesocosm experiment was thus set up using a former mine soil, amended or not, using 2 % biochars produced from three oak tissues (bark, sapwood,

Introduction

Soil plays a crucial role in the environment. It supports many ecosystemic functions, such as providing a habitat for diverse microorganisms and supporting agriculture (Kabata-Pendias 2011).

However, those functions are threatened by soil contamination, which is one of the eight threats to soils, alongside erosion, organic matter decline, compaction, salinization, landslides, sealing and biodiversity decline [START_REF] Panagos | Contaminated sites in Europe: review of the current situation based on data collected through a European network[END_REF]. Soil contamination is mainly the result of anthropogenic activities, i.e mining, smelting, transport, fertilizer use [START_REF] Panagos | Contaminated sites in Europe: review of the current situation based on data collected through a European network[END_REF]. Worldwide, more than ten million sites are contaminated, of which more than 50% are by metal(loid)s [START_REF] Khalid | A comparison of technologies for remediation of heavy metal contaminated soils[END_REF]. uncontaminated site presented different properties. While [START_REF] Meng | Changes in heavy metal bioavailability and speciation from a Pb-Zn mining soil amended with biochars from co-pyrolysis of rice straw and swine manure[END_REF] produced several biochars by mixing rice straw and swine manure in different ratios, they found that biochar pH and electrical conductivity increased as the ratio of rice straw to swine manure increased. Similarly, [START_REF] Alburquerque | Effects of biochars produced from different feedstocks on soil properties and sunflower growth[END_REF] showed that the effect of biochar on soil variables depended on biochar type and application rate.

In previous studies, Lebrun et al. (2018 a, b) showed that biochars produced from hardwood biomass (oak, charm, beech) had the ability to improve the soil properties and immobilize Pb. However, due to the chemical composition and structure of the different tissues composing the biomass used, biochar properties can be fundamentally different. In addition, in order to reduce the price of biochar production, it would be better to use only a part of the tree trunk, and preferentially one that is not used for other materials or energy production. Tissue based biochars therefore need to be explored. Indeed, as shown by the sorption study of [START_REF] Mohan | Sorption of arsenic, cadmium, and lead by chars produced from fast pyrolysis of wood and bark during bio-oil production[END_REF], it can be hypothesized that biochars coming from tissues of different structure and composition will present different properties, and thus a different sorption capacity and contrasted effects on the soil and the plant. The objectives of the present study were to evaluate three biochar obtained from different oak trunk feedstock, bark, sapwood, heartwood, for their effects on soil properties, especially metal(loid) mobility and phytoavailability and determine if a specific tissue showed better results than the others. Specifically, the study aimed at assessing the effect of biochar produced from three different trunk parts, bark, sapwood and heartwood, on (i) the soil physico-chemical properties, (ii) plant growth and (iii) plant As and Pb accumulation. To the best of our knowledge, it is the first paper evaluating which biochars (prepared from different raw materials, i.e plant organs) are able to reduce As and Pb phytotoxicity in a mining soil context.

Material and Methods

Study site

A former silver-lead extraction mine site was studied. This site is located in Pontgibaud (Auvergne-Rhône-Alpes, France) and was very active during the nineteenth century. This high activity of silver and lead extraction led to huge amounts of contaminated wastes, mainly by Pb and As (Lebrun et al. 2018 a, b). A previous study measured pseudo-total concentrations of 1 g.kg -1 As and 23 g.kg -1 Pb at the same area, as well as an acidic pH (3.7) and a low organic matter content (2.60 %) [START_REF] Lebrun | Biochar effect associated with compost and iron to promote Pb and As soil stabilization and Salix viminalis L. growth[END_REF]).

Biochar preparation and characterization

Nine biochars were tested. They were provided by La Carbonerie (Crissey, France) and came from different tissues of oak trees: bark, sapwood and heartwood. The tissues were mechanically separated and subjected to the same pyrolysis program. Biomasses were first dried at 105 °C. The temperature was then increased from 105 °C to 150 °C over two hours. Finally, the biomass was pyrolysed at 500 °C (heating rate: 2.5 °C.min -1 ; residence time: 3 h). Following the pyrolysis, the material was sieved to obtain three particle sizes: between 0.2 and 0.4 mm, between 0.5 and 1 mm and between 1 and 2.5 mm. The nine biochars will be referred as Ba 0.2-0.4 (bark tissue, 0.2-0.4 mm particle size), Ba 0.5-1 (bark tissue, 0.5-1 mm particle size), Ba 1-2.5 (bark tissue, 1-2.5 mm particle size), Sa 0.2-0.4 (sapwood tissue, 0.2-0.4 mm particle size), Sa 0.5-1 (sapwood tissue, 0.5-1 mm particle size), Sa 1-2.5 (sapwood tissue, 1-2.5 mm particle size), He 0.2-0.4 (heartwood tissue, 0.2-0.4 mm particle size), He 0.5-1 (heartwood tissue, 0.5-1 mm particle size) and He 1-2.5 (heartwood tissue, 1-2.5 mm particle size).

The biochars were characterized for pH and electrical conductivity (EC) following the protocol described in Lebrun et al. (2018a). Five replicates were performed by biochar. In addition, specific surface area, total pore volume and mean pore diameter were determined by BET measurements (BelSorp Mini II, MicroTrac Bel) (LMI, Villeurbanne, France). Prior to analysis, the samples were degassed under vacuum at 150 °C for four hours. More exactly, the program of desorption was : (i) heating from ambient temperature to 100 °C at 3 °C.min -1 , (ii) 1 min isotherm at 100 °C, (iii) heating to 150 °C at 5 °C.min -1 , (iv) four hours isotherm at 150 °C. The specific surface area was determined using the Brunauer-Emmett Teller (BET) equation in the 0.05 ≤ P/P0 ≤ 0.35 interval of relative pressure and based on a value of 16.2 Å 2 for the cross sectional area of molecular N 2 .Finally, biochars were analyzed for carbon, hydrogen and nitrogen content, using a flash Thermo (2000).

Experimental set up

A mesocosm experiment was set up using Pontgibaud technosol (PG), either alone or amended with 2% (w/w) of each biochar, separately. In total, ten conditions were tested: (i) non amended Pontgibaud technosol (PG); (ii) PG amended with Ba 0.2-0.4 (PG + Ba 0.2-0.4); (iii) PG amended with Ba 0.5-1 (PG + Ba 0.5-1); (iv) PG amended with Ba 1-2.5 (PG + Ba 1-2.5); (v) PG amended with Sa 0.2-0.4 (PG + Sa 0.2-0.4); (vi) PG amended with Sa 0.5-1 (PG + Sa 0.5-1); (vii) PG amended with Sa 1-2.5 (PG + Sa 1-2.5); (viii) PG amended with He 0.2-0.4 (PG + He 0.2-0.4); (ix) PG amended with He 0.5-1 (PG + He 0.5-1) and (x) PG amended with He 1-2.5 (PG + He 1-2.5). Three pots (0.4 L) were prepared per treatment and sown with three pre-germinated seeds of Phaseolus vulgaris (Contender cultivar).

Phaseolus vulgaris was chosen because of its bio-indicator properties. Indeed, its growth is related to the soil toxicity, and its metal(loid) accumulation ability is linked to the phytoavailable metal(loid) concentrations in the soil [START_REF] Lebrun | Effect of Fe-functionalized biochar on toxicity of a technosol contaminated by Pb and As: sorption and phytotoxicity tests[END_REF][START_REF] Meers | Phytoavailability assessment of heavy metals in soils by single extractions and accumulation by Phaseolus vulgaris[END_REF], Kumpiene et al. 2014). Plants were grown for two weeks under the following conditions: 16h/8h photoperiod, 20 ± 2 °C temperature, 800 μmol m -2 s -1 light intensity and daily watering to field capacity using tap water.

Soil pore water (SPW) sampling and analysis

Soil pore waters were sampled in all pots (n=3) at the beginning (T0) and at the end (14 days) (TF) of the experiment, as described in [START_REF] Lebrun | Effect of biochar amendments on As and Pb mobility and phytoavailability in contaminated mine technosols phytoremediated by Salix[END_REF]. Soil pore waters were used directly to measure pH, EC and redox potential using a multimeter (Mettler-Toledo, Serveur excellence). Those parameters were measured due to their influence on metal(loid) behavior. Soil pore waters were then acidified (83.7 µL of concentrated HNO 3 in 5 mL of sample) before determining the major pollutant (As and Pb) concentrations by ICP-AES (Inductively Coupled Plasma-Atomic Emission Spectroscopy) (ULTIMA, HORIBA, Labcompare, San Francisco, USA).

Plant harvest and analysis

After 14 days of growth, Phaseolus vulgaris plants were harvested. Stem length and leaf width were measured; aerial and root parts were separated, dried (72 h at 60 °C) and weighted. Arsenic and Pb concentrations were then determined by ICP-AES after acid digestion as described in Bart et al. (2016).

Statistical analysis

Data was analyzed using R software version 3.1.2 (R Development Core Team, 2009), using the package PMCMR. Normality and homoscedasticity were assessed using Shapiro and Barlett tests, respectively; based on the normality or non-normality of the data, and means were compared using ANOVA or Kruskal tests, respectively followed by a post-hoc Tukey test. In addition, for the soil pore water data, the difference between T0 and TF was assessed using Student or Wilcox tests, based on normality and homoscedasticity test results. Differences were considered significant at p < 0.05.

Finally, global feedstock and particle size effects were evaluated using the first procedure.

Results

Biochar properties

The nine biochars used were analyzed for pH, EC, surface area, total pore volume and mean pore diameter (Table 1). The results showed that all biochars had an alkaline pH, ranging from 7.95 to 9.50 (Table 1). Out of the nine biochars, the three Sa biochars presented the highest biochar pH, with no difference between them, while the lowest pH was observed for He 1-2.5 biochar. When taken together, biochar particle size had no significant effect on pH whereas the feedstock effect was highly significant (p < 0.001). Indeed, sapwood biochars presented the highest pH values (pH 9.36 on average), followed by bark biochars (pH 8.84 on average) and heartwood biochars (pH 8.28 on average). Biochar EC ranged from 173 to 814 µS.cm -1 (Table 1) with the highest values found in the two fine Ba biochars, whereas the lowest EC was observed for the three He biochars. The particle size effect was globally non-significant whereas the feedstock effect was highly significant (p < 0.001).

However, contrary to pH, higher EC values were observed for bark biochars (725 µS.cm -1 on average), followed by sapwood biochars (617 µS.cm -1 ) and heartwood biochars (191 µS.cm -1 ).

BET measurements (specific surface area, total pore volume and mean pore diameter) were performed once on each biochar and showed high variation between feedstocks and particle sizes. Specific surface area was higher for the heartwood biochars (402.42 m².g -1 on average) compared to sapwood biochars (323.87 m².g -1 on average) and bark biochars (99.41 m².g -1 on average) (Table 1), and tended to decrease with increasing particle size. The same trend was observed for total pore volume, with the highest values observed for the heartwood biochars, followed by sapwood and bark biochars, while mean pore diameter was higher for bark biochars compared to sapwood and heartwood biochars. However, for these two parameters (total pore volume and mean pore diameter), particle size effect within each biochar feedstock was not as pronounced as for the other parameters.

Biochars elemental analysis showed that all biochars contained mainly carbon, more than 70 % (Table 1), followed by hydrogen and nitrogen. Moreover, when comparing biochars, carbon content did not show significant difference between biochar (Table 1). However, carbon content was affected by feedstock, with sapwood biochars having higher carbon content (85 % on average) than heartwood biochars (83 % on average) and bark biochars (69 % on average), and by particle size, with carbon content decreasing with the increase in particle size (Table 1). On the contrary, hydrogen content differed among biochars. In more detail, H content was the highest in Sa0.2-0.4 biochars and the lowest in the Ba biochars. In addition, H content was significantly affected by feedstock, with higher H content in He biochars (3 % on average), followed by Sa biochars (2.6 % on average) and Ba biochars (1.7 % on average), but not particle size. In the same way, N content was also the highest in Sa0.2-0.4 but the lowest values were found in the He biochars. Similarly to H content, N content was affected by feedstock, with Ba and Sa having higher H content (0.9 % on average) than He biochars (0.3 % on average) but not particle size (Table 1).

Soil pore water (SPW) physico-chemical properties

Soil pore water of the PG substrate was acidic at T0 (pH 3.7) (Table 2) and increased, between 0.2 and 2.9 unit, with all biochar applications (Table 2). However, feedstock showed a highly significant (p < 0.001) effect on SPW pH, while particle size effect was not significant. Indeed, pH rise was much higher when bark biochars were applied (2.6 pH units increase on average) compared to sapwood biochars (0.9 pH unit increase on average) and heartwood biochars (0.3 pH unit increase on average).

At the end of the experiment (TF), SPW of PG was still acidic (pH 4.4) (Table 2) but contrary to what was observed at T0, SPW pH was only higher in bark biochar (PG+Ba0.2-0.4, PG+Ba0.5-1 and PG+Ba1-2.5), and the two finest Sa biochars treatments (PG+Sa0.2-0.4 and PG+Sa0.5-1), compared to PG.

However, biochar made with the larger Sa particle size (PG+Sa1-2.5) and the three He biochars (PG+He0.2-0.4, PG+He0.5-1 and PG+He1-2.5) did not show a significant difference in terms of SPW pH compared to PG, whatever the particle size.

Regarding pH evolution with time, a pH rise between SPW pH at T0 and SPW pH at TF was observed in five conditions: PG+Ba1-2.5, PG+Sa0.5-1, PG+Sa1-2.5, PG+He0.5-1 and PG+He1-2.5. The pH increases ranged from 0.9 to 1.4 units (Table 2).

The substrate PG presented a low SPW EC at T0 and TF (306 and 757 µS.cm -1 , respectively) (Table 2), and only increased with Ba biochars (PG+Ba0.2-0.4, PG+Ba0.5-1 and PG+Ba1-2.5), at both times. At T0, the finest Ba biochar (PG+Ba0.2-0.4) led to a higher increase compared to the other two Ba biochars (PG+Ba0.5-1 and PG+Ba 1-2.5), while at TF, no difference was observed between the three Ba biochars. Globally, biochar feedstock effect was highly significant (p < 0.001) at both times.

When comparing SPW EC values at T0 and TF, higher values were observed at TF for PG, PG+Sa0.5-1, PG+Sa1-2.5 and for all He biochar treatments.

Redox potential of PG SPW was 449 mV at T0 (Table 2) and decreased with all biochar treatments, with a highly significant feedstock effect. Indeed, compared to PG, SPW redox potential was decreased by 27-30 %, 7-11 % and 5-7 % with bark, sapwood and heartwood biochars, respectively.

At TF, SPW redox potential was 347 mV on PG and it only decreased in treatments with bark biochars, between 29 % and 32 %, compared to PG.

Regarding time effect, a SPW redox potential decrease between T0 and TF was observed in all conditions.

Soil pore water As concentration of PG treatment at T0 was 0.06 mg.L -1 and decreased with all amendments except PG+Ba0.2-0.4 and PG+He1-2.5 (Table 2, Table S1). Biochar feedstock had a significant effect, with sapwood biochars (PG+Sa0.2-0.4, PG+Sa0.5-1 and PG+Sa1-2.5) inducing a larger decrease in SPW As concentrations compared to bark (PG+Ba0.2-0.4, PG+Ba0.5-1 and PG+Ba1-2.5) and heartwood biochars (PG+He0.2-0.4, PG+He0.5-1 and PG+He1-2.5). However, at TF, no difference was found between the different biochar-amended conditions and PG (Table 2).

Soil pore water PG presented a high Pb concentration at T0 (13.33 mg.L -1 ), which decreased when any biochars were added to PG (Table 2, Table S1). The amount by which it decreased was highly dependent on the biochar feedstock (p < 0.001). Indeed, bark biochars (PG+Ba0.2-0.4, PG+Ba0.5-1 and PG+Ba1-2.5) led to a higher decrease in SPW Pb concentrations (68-90 %) followed by sapwood biochars (PG+Sa0.2-0.4, PG+Sa0.5-1 and PG+Sa1-2.5) (41-51 %) and heartwood biochars (PG+He0.2-0.4, PG+He0.5-1 and PG+He1-2.5) (28-38 %) (Table S1). Concentration in Pb in the SPW of PG decreased at TF compared to T0, down to 7.43 mg.L -1 , but was lower in the bark and sapwood biochar amended treatments (PG+Ba0.2-0.4, PG+Ba0.5-1 and PG+Ba1-2.5, PG+Sa0.2-0.4, PG+Sa0.5-1 and PG+Sa1-2.5) (Table 2, Table S1). Heartwood biochars (PG+He0.2-0.4, PG+He0.5-1 and PG+He1-2.5) had no effect at TF. Similarly, at T0, Pb decrease was more important with the bark biochars (79-86 %) than with the sapwood biochars (13-25 %) (Table S1). Finally, SPW Pb concentrations on Baamended substrates showed no differences between T0 and TF, while they were lower at TF compared to T0 in the other conditions.

Plant growth parameters

Plant growth parameters, i.e stem length, leaf width and aerial and root dry weight, were determined on Phaseolus vulgaris plants after 14 days of growth on the different substrates. They all showed similar trends, with a highly significant feedstock effect and no particle size effect (Table 3 and Fig. 1). Indeed, only bark biochars (PG+Ba0.2-0.4, PG+Ba0.5-1 and PG+Ba1-2.5) improved the growth parameters, while the other two biochar feedstocks did not. In more detail, stem length was 7.7 cm on PG and increased by 1.6 times on average with Ba biochar application, while leaf width was 2.6 cm on PG and was doubled on average on Ba-amended substrates (Table 3, Table S1).

Phaseolus vulgaris plants produced a low biomass on PG: 0.10 g aerial biomass and 0.04 g root biomass (Fig. 1). When bark biochars were applied to PG, aerial dry weight was 3.1-4.2 fold higher while root dry weight was 2.3-3.8 fold higher, compared to PG (Table S1).

As and Pb concentrations in plants

Although Pb concentrations were much higher than As concentrations, they both behaved in the same way in response to biochar amendments. As for what was observed for the growth parameters, sapwood and heartwood biochars had no effect on As and Pb plant concentrations, whatever the organ and biochar particle size considered (Fig 2A). However, the application of Ba biochars (PG+Ba0.2-0.4, PG+Ba0.5-1 and PG+Ba1-2.5) reduced Pb concentrations in the roots of Phaseolus vulgaris plants, whereas aerial Pb and aerial and root As concentrations of bean plants were not affected. Lead root concentrations were decreased to similar levels (56 %) for the three Ba biochars (Fig. 2B, Table S1).

Discussion

Biochar properties

The nine biochars presented different values in terms of pH, EC, specific surface area, total pore volume and mean pore diameter. However, all parameter values, except for EC which was low, were in the range of what is usually observed [START_REF] Khan | The influence of various biochars on the bioaccessibility and bioaccumulation of PAHs and potentially toxic elements to turnips (Brassica rapa L.)[END_REF][START_REF] Mohan | Sorption of arsenic, cadmium, and lead by chars produced from fast pyrolysis of wood and bark during bio-oil production[END_REF][START_REF] Zheng | The influence of particle size and feedstock of biochar on the accumulation of Cd, Zn, Pb, and As by Brassica chinensis L[END_REF].

Indeed, biochar are usually characterized by an alkaline pH (7.5 to 11), a high electrical conductivity (3020 to 11 300 µS.cm -1 ) and a large specific surface area (2.04 to 25.4 m².g -1 ), with a total pore volume between 0.0033 and 1.06 cm 3 .g -1 and a pore diameter of several nanometers (3.82 to 10.31 nm) [START_REF] Khan | The influence of various biochars on the bioaccessibility and bioaccumulation of PAHs and potentially toxic elements to turnips (Brassica rapa L.)[END_REF][START_REF] Mohan | Sorption of arsenic, cadmium, and lead by chars produced from fast pyrolysis of wood and bark during bio-oil production[END_REF][START_REF] Zheng | The influence of particle size and feedstock of biochar on the accumulation of Cd, Zn, Pb, and As by Brassica chinensis L[END_REF]. Similarly, the contents of C, H and N observed in the study were in the range of what the previous studies observed: a high carbon content (20 to 83 %) and low contents in H (2 to 3 %) and N (0.3 to 2.8 %) [START_REF] Khan | The influence of various biochars on the bioaccessibility and bioaccumulation of PAHs and potentially toxic elements to turnips (Brassica rapa L.)[END_REF][START_REF] Mohan | Sorption of arsenic, cadmium, and lead by chars produced from fast pyrolysis of wood and bark during bio-oil production[END_REF][START_REF] Zheng | The influence of particle size and feedstock of biochar on the accumulation of Cd, Zn, Pb, and As by Brassica chinensis L[END_REF]). These parameters were mainly affected by feedstock. Such feedstock effect on biochar properties has already been observed in previous studies. Indeed, [START_REF] Dai | The potential feasibility for soil improvement, based on the properties of biochars pyrolyzed from different feedstocks[END_REF] showed that biochars produced from 20 different feedstocks presented large differences in terms of surface area and total pore volume, whereas [START_REF] Khan | The influence of various biochars on the bioaccessibility and bioaccumulation of PAHs and potentially toxic elements to turnips (Brassica rapa L.)[END_REF] characterized four biochars (sewage sludge, soybean straw, rice straw and peanut shell) and observed that the characteristics (pH, EC, specific surface area, pore volume and pore diameter) varied across the four feedstocks. In addition, Jindo et al. (2016) produced different biochars from two rice parts (straw and husk) at different temperatures. The biochars produced at 500 °C, which was the same pyrolysis temperature used for the biochar of the present study, had a pH of 8.99 and 9.82 and a specific surface area of 262 and 59.91 m².g -1 , for rice husk and rice straw biochars, respectively. Moreover, [START_REF] Mohan | Sorption of arsenic, cadmium, and lead by chars produced from fast pyrolysis of wood and bark during bio-oil production[END_REF] and [START_REF] Suliman | Influence of feedstock source and pyrolysis temperature on biochar bulk and surface properties[END_REF] studied biochars made from the bark and wood parts of trees (oak, pine Douglas fir), without distinguishing sapwood from heartwood. In each case, they found that the bark biochars presented higher values of pH, EC, specific surface area and total pore volume, except for the specific surface area of the pine biochars, which was higher in the wood biochar compared to the bark biochar. These differences can be directly related to the differing properties of the biomass feedstock used. Indeed, Dai et al. (2017) stated that biochar properties were mainly determined by the pyrolysis temperature and the feedstock used.

Soil pore water physico-chemical properties

Due to its high pH, which induces a liming effect, biochar is known to increase soil and SPW pH when applied to acidic soils [START_REF] Hmid | Olive mill waste biochar: a promising soil amendment for metal immobilization in contaminated soils[END_REF][START_REF] Egene | Impact of organic amendments (biochar, compost and peat) on Cd and Zn mobility and solubility in contaminated soil of the Campine region after three years[END_REF]. Also, biochar contains carbonates and oxides formed during pyrolysis, which can react with the H + ions and thus increase soil pH (Dai et al. 2017). Similarly, in the present study, the addition of alkaline biochars increased SPW pH. Moreover, the study showed a highly significant feedstock effect on SPW pH variations. This is consistent with previous studies [START_REF] Zheng | The influence of particle size and feedstock of biochar on the accumulation of Cd, Zn, Pb, and As by Brassica chinensis L[END_REF], Lebrun et al. 2018b). Moreover, at the end of the experiment (TF), there was no effect for coarse sapwood and heartwood biochar applications. Several studies showed that the effect of biochar depended on biochar and soil types. Indeed, [START_REF] Lima | Effect of biochar on physicochemical properties of a sandy soil and maize growth in a greenhouse experiment[END_REF] showed that coffee husk biochar application to a sandy soil increased soil pH, while the application of a coffee ground biochar did not. [START_REF] Sandhu | Analyzing the impacts of three types of biochar on soil carbon fractions and physiochemical properties in a corn-soybean rotation[END_REF] did not observe a biochar effect on soil pH after corn planting following the application of three different biochars (corn stover, pinewood and switchgrass). Finally, in this study, greater SPW pH increases were observed with the bark biochars compared to sapwood and heartwood biochars. However, the bark biochars did not have the highest pH values. Indeed, biochar pH decreased in the order Sa > Ba > He whereas the SPW pH increase induced by biochar application was highest with Ba, followed by Sa and He. Such findings contradict the study of [START_REF] Zong | Acidity, water retention, and mechanical physical quality of a strongly acidic Ultisol amended with biochars derived from different feedstocks[END_REF], which found that wood chip biochar was the best at improving soil pH due to its higher alkalinity. Therefore, the higher pH increases observed with Ba biochars could be related to other properties than just biochar alkalinity, such as buffering capacity, ash content, carbonate contents and porous structure [START_REF] Hmid | Olive mill waste biochar: a promising soil amendment for metal immobilization in contaminated soils[END_REF][START_REF] Nigussie | Effect of biochar application on soil properties and nutrient uptake of lettuces (Lactuca sativa) grown in chromium polluted soils[END_REF]). Finally, biochar particle size did not affect SPW pH, which could be related to the fact that biochar particle size did not affect biochar pH.

Biochar application to soil was shown to increase soil EC due to the elevated EC of biochar [START_REF] Hmid | Olive mill waste biochar: a promising soil amendment for metal immobilization in contaminated soils[END_REF], Lebrun et al. 2018a). However only bark biochars, which presented a high EC, increased SPW EC, while sapwood and heartwood biochars had no effect, probably due to their relatively low EC compared to bark biochar and other biochars in general [START_REF] Khan | The influence of various biochars on the bioaccessibility and bioaccumulation of PAHs and potentially toxic elements to turnips (Brassica rapa L.)[END_REF]. Biochars made from Heartwood even presented a lower EC value than the one measured in SPW of non-amended PG soil.

Moreover, biochar can also increase EC through the dissolution of salts and leaching of nutrients, which are favored by higher pH [START_REF] Lebrun | Biochar effect associated with compost and iron to promote Pb and As soil stabilization and Salix viminalis L. growth[END_REF]. Therefore, the increase in SPW EC only observed with Ba biochar could be related to the higher increase in SPW pH with Ba biochar in addition to its higher EC.

Biochar, when applied to soil, can decrease the redox potential of the soil by proton consumption [START_REF] Rinklebe | Amendment of biochar reduces the release of toxic elements under dynamic redox conditions in a contaminated floodplain soil[END_REF]. In addition, redox potential was shown to follow an opposite trend to that of the pH [START_REF] Husson | Redox potential (Eh) and pH as drivers of soil/plant/microorganism systems: a transdisciplinary overview pointing to integrative opportunities for agronomy[END_REF], which was the case in the present study: SPW redox potential decreased when SPW pH increased, and did not change when SPW pH was not modified. Indeed, SPW pH decreased in the order Ba > Sa > He, whereas SPW redox potential decreased in the order He > Sa > Ba at the beginning of the experiment, whereas at the end, redox potential only decreased with Ba biochar, similarly to SPW pH which increased mainly with Ba biochars. Moreover, plants can affect both pH and redox potential through root exudation [START_REF] Husson | Redox potential (Eh) and pH as drivers of soil/plant/microorganism systems: a transdisciplinary overview pointing to integrative opportunities for agronomy[END_REF]: indeed, root exudates can directly modify the pH and redox potential of the rhizosphere area, and can also stimulate specific microorganisms that affect pH and redox potential.

Biochars are not particularly known for sorbing As and reducing SPW As concentrations. However, [START_REF] Gregory | Biochar in co-contaminated soil manipulates arsenic solubility and microbiological community structure and promotes organochlorine degradation[END_REF] observed a decrease in water soluble As concentrations following willow biochar application while [START_REF] Mohan | Sorption of arsenic, cadmium, and lead by chars produced from fast pyrolysis of wood and bark during bio-oil production[END_REF] showed that oak bark char was able to sorb As(III).

Similarly, this study showed a decrease in SPW As concentration following biochar amendment at the beginning of the experiment. Such As immobilization could be attributed to a sorption of As on the biochar surface through ion exchange and surface complexation, as demonstrated in the studies of [START_REF] Niazi | Arsenic removal by Japanese oak wood biochar in aqueous solutions and well water: Investigating arsenic fate using integrated spectroscopic and microscopic techniques[END_REF] and [START_REF] Tabassum | Arsenic removal from aqueous solutions and groundwater using agricultural biowastes-derived biosorbents and biochar: a column-scale investigation[END_REF]. Moreover, in the present study, the decrease in SPW As concentration was only observed at T0, which could mean that As that may have sorbed onto the biochar surface was desorbed, due to biochar ageing and/or the effect of root exudates.

Several studies showed that biochar was able to immobilize Pb through two main mechanisms. First, biochar can sorb Pb on its surface [START_REF] Mohan | Sorption of arsenic, cadmium, and lead by chars produced from fast pyrolysis of wood and bark during bio-oil production[END_REF], through electrostatic interaction with the biochar functional groups [START_REF] Lebrun | Effect of Fe-functionalized biochar on toxicity of a technosol contaminated by Pb and As: sorption and phytotoxicity tests[END_REF][START_REF] Yu | Biochar amendment improves crop production in problem soils: A review[END_REF]) and calcite crystals, as well as surface complexation and precipitation [START_REF] Inyang | Removal of heavy metals from aqueous solution by biochars derived from anaerobically digested biomass[END_REF][START_REF] Ding | Pyrolytic temperatures impact lead sorption mechanisms by bagasse biochars[END_REF]. Second, biochar can induce an increase in soil pH (Forjan et al. 2016) which increases the surface charge in the Fe-, Al-and Mnoxides and subsequently increases metal chelation by organic matter or their precipitation as metal hydroxides, i. e Pb(OH) 3 [START_REF] Bolan | Remediation of heavy metal (loid) s contaminated soils-to mobilize or to immobilize?[END_REF]. Moreover, [START_REF] Husson | Redox potential (Eh) and pH as drivers of soil/plant/microorganism systems: a transdisciplinary overview pointing to integrative opportunities for agronomy[END_REF] showed that Pb concentrations increased with increasing redox potential. Therefore, the reduction of SPW redox potential induced by biochar application could have favored Pb immobilization. Bark, sapwood and heartwood biochars showed different abilities to reduce SPW Pb concentrations. Similarly, [START_REF] Mohan | Sorption of arsenic, cadmium, and lead by chars produced from fast pyrolysis of wood and bark during bio-oil production[END_REF][START_REF] Kabata-Pendias | Trace elements in soils and plants. 1st ed[END_REF] produced biochars from wood and bark tissues of different species (oak and pine). In these two studies, the authors observed that oak bark biochar presented a higher sorption capacity that the other biochars towards Pb(II), Cd(II) and As(III), and Cr, respectively. They attributed these results, at least partially, to the fact that this biochar had a higher surface area and pore volume, and a larger surface area, respectively, than the others. However, in the present study, the bark biochars showed the highest ability to decrease SPW Pb concentrations, while surprisingly they had the lowest surface area out of the three biochar feedstocks. Indeed, soil Pb immobilization capacity of biochar showed an opposite trend to their surface area. This indicates that the difference in effect observed between the biochar feedstocks was not only related to biochar surface area, as found in [START_REF] Abdelhadi | Production of biochar from olive mill solid waste for heavy metal removal[END_REF], but more possibly due to the higher pore volume of Ba biochars and the higher SPW pH rise induced, compared to the other two biochar feedstocks. Indeed, biochars with large surface areas and pore volume have a high capacity for metal(loid) sorption. Moreover, as shown by [START_REF] Rees | Mobilité des métaux dans les systèmes sol-plante-biochar[END_REF], metal(loid)s can precipitate with carbonates and calcite crystals, and biochars from the bark of trunks contain the highest quantities.

With time, SPW Pb concentrations decreased in Sa and He amended conditions while no change was observed with Ba biochars. Such results could be related to the fact that bark biochars had already reached their maximum sorption capacity, and also the SPW pH, which did not increase further in these conditions. In Sa and He amended conditions however, SPW pH increased with time, which could have increased Pb immobilization. Moreover, [START_REF] Houben | Mobility, bioavailability and pH-dependent leaching of cadmium, zinc and lead in a contaminated soil amended with biochar[END_REF] showed a slow secondary increase in metal retention with time, due to the diffusion, precipitation and/or adsorption of the metallic elements in the biochar micropores as biochar undergoes oxidation.

Plant growth parameters

Several previous studies showed the positive effect of biochar on plant growth parameters, such as Cassia alata height, root length and dry weight [START_REF] Huang | Shift of soil bacterial community and decrease of metals bioavailability after immobilization of a multi-metal contaminated acidic soil by inorganic-organic mixed amendments: A field study[END_REF], lettuce and cabbage stem length [START_REF] Carter | The Impact of Biochar Application on Soil Properties and Plant Growth of Pot Grown Lettuce (Lactuca sativa) and Cabbage (Brassica chinensis)[END_REF] or pak choi biomass [START_REF] Nie | Impact of sugarcane bagasse-derived biochar on heavy metal availability and microbial activity: A field study[END_REF]. Such plant growth improvements were attributed to the improvement in the soil conditions: reduction of metal(loid) stress, reduction of acidity and supply of nutrients [START_REF] Hmid | Olive mill waste biochar: a promising soil amendment for metal immobilization in contaminated soils[END_REF][START_REF] Huang | Shift of soil bacterial community and decrease of metals bioavailability after immobilization of a multi-metal contaminated acidic soil by inorganic-organic mixed amendments: A field study[END_REF]. However, other studies pointed out the non-effect of biochar on plant growth [START_REF] Cui | The reduction of wheat Cd uptake in contaminated soil via biochar amendment: a two-year field experiment[END_REF][START_REF] Jones | Biochar-mediated changes in soil quality and plant growth in a three-year field trial[END_REF], which shows that biochar's positive effect on plants depends on many parameters such as biochar type, soil types, plant species… Therefore, the non-effect of sapwood and heartwood biochars observed here could be related to their properties and the fact that their application to soil did not improve SPW physicochemical properties as much as bark biochars, and consequently probably not enough to sustain plant growth amelioration.

As and Pb concentrations in plants

Biochar is usually known to decrease metal(loid) concentrations in plants [START_REF] Zheng | The influence of particle size and feedstock of biochar on the accumulation of Cd, Zn, Pb, and As by Brassica chinensis L[END_REF][START_REF] Li | Biochars induced modification of dissolved organic matter (DOM) in soil and its impact on mobility and bioaccumulation of arsenic and cadmium[END_REF]) due to (i) a decrease in metal(loid) available concentrations and/or (ii) an increase in plant biomass production. Indeed, as shown in a previous section, biochar can reduce metal(loid) mobility through sorption on biochar or soil surface or precipitation at higher pH. Metal(loid)s are thus less available for plant uptake. However, [START_REF] Khan | The influence of various biochars on the bioaccessibility and bioaccumulation of PAHs and potentially toxic elements to turnips (Brassica rapa L.)[END_REF] stated that the level of reduction in metal(loid) concentrations was dependent on the biochar type. Indeed, they found that peanut shell biochar was the most effective in reducing metal(loid) concentrations together with the increase in soil pH. Moreover, [START_REF] Li | Biochars induced modification of dissolved organic matter (DOM) in soil and its impact on mobility and bioaccumulation of arsenic and cadmium[END_REF] observed that the decrease in toxic element accumulations in rice plants was linked to the lower pore volume, surface area and higher pore diameter of the biochar. All these arguments can explain why only the bark biochars reduced root Pb concentrations in plants.

Indeed, bark biochar treatments showed the highest pH increase and the three bark biochars were characterized by lower surface areas and total pore volumes, but higher mean pore diameters, compared to sapwood and heartwood biochars. Furthermore, the metal(loid) accumulation profiles can give an indication of the bioavailability of the metal(loid)s, as Phaseolus vulgaris is a bio-indicator plant [START_REF] Meers | Phytoavailability assessment of heavy metals in soils by single extractions and accumulation by Phaseolus vulgaris[END_REF]. The results showed that although bark biochars were very efficient in decreasing Pb and to a lesser extent As, neither metal(loid) concentration was significantly reduced in plants, except for root Pb concentrations, showing that bark biochars reduced As and Pb mobility but did not affect their plant availability.

Conclusion

A phytotoxicity test in mesocosm was set up as a preliminary experiment in order to evaluate which part of a wood trunk should be used for biochar production, based on the ability of the different biochars to decrease soil phytotoxicity, assessed through soil properties, bean growth and metal(loid) accumulation. The biochar feedstock and particle size effects were analyzed regarding their impact on soil physico-chemical properties and plant growth and metal(loid) accumulation capacity. The results showed that the nine biochars presented different properties, depending on the feedstock, while particle size had no effect in general. Such different properties induced diverse soil pore water and plant parameter modifications. The improvements in soil pore water physicochemical properties were higher with the bark biochars compared to sapwood and heartwood biochars, while plant parameters were only affected by the bark biochars. Therefore, bark, which isn't usually used in sawmills or other wood-based products, seems to be the best part of the trunk to use for producing a biochar that will allow soil physico-chemical improvements, Pb immobilization and better plant growth. It could therefore be used to produce a biochar for phytoremediation purposes. Indeed, its application to the soil could increase soil pH and immobilize Pb, improve plant growth, while not affecting plant metal(loid) accumulation capacity. Therefore, by using a plant with a high biomass production and a low metal(loid) translocation ability, the contaminated soil will present a plant cover which will prevent soil erosion while metal(loid) will be immobilized at the root zone. Finally, biochar particle size had no effects on soil parameters, plant growth and plant metal(loid) uptake. Therefore, for an application at big scale, a biochar of medium particle size, i.e 0.5 -1 mm could be produced, which will be easier to apply and less subjected to leaching and will not affect metal(loid) availability to plants. However, such preliminary results will need to be validated using higher plants and/or through a field study. In addition, mechanisms underlying the properties of biochar from bark will have to be investigated.

Fig 1 :

 1 Fig 1: Dry weight (g) of the aerial ( ) and root ( ) parts of beans determined at the end of the experiment on Pontgibaud technosol (PG) amended or not with biochar from three feedstocks (Ba = bark biochar; Sa = sapwood biochar; He = heartwood biochar) and harboring three particle sizes (0.2-0.4 mm, 0.5-1 mm, 1-2.5 mm). Letters indicate significant difference (p < 0.05) (n=9 ± SE). Feedstock and particle size effects: * p < 0.05, ** p < 0.01, *** p < 0.001, ns = non significant. PG PG + Ba PG + Sa PG + He 0.2-0.4 0.5-1 1-2.5 0.2-0.4 0.5-1 1-2.5 0.2-0.4 0.5-1 1-2.5

Table 1 :

 1 Biochar physico-chemical properties. EC = electrical conductivity (µS.cm -1 ); as,BET = specific surface area (m².g -1 ). Ba = bark biochar; Sa = sapwood biochar; He = heartwood biochar.

	Letters indicate

Table 2 :

 2 Soil pore water physico-chemical properties (pH, EC = electrical conductivity (µS.cm -1 ), [As] and [Pb] (mg.L -1 )) determined at the beginning (T0) and at the end of the experiment (TF) on Pontgibaud technosol (PG) amended or not with biochar from three feedstocks (Ba = bark biochar; Sa = sapwood biochar; He = heartwood biochar) and harboring three particle sizes (0.2-0.4 mm, 0.5-1 mm, 1-2.5 mm). For each biochar feedstock, letters indicate significant difference (p < 0.05) (n=3 ± SE). Feedstock, particle size and time effects: * p < 0.05, ** p < 0.01, *** p < 0.001, ns = non significant

			pH			EC (µS.cm -1 )		Redox potential (mV)		[As] (mg.L -1 )			[Pb] (mg.L -1 )
		T0	TF	Time effect	T0	TF	Time effect	T0	TF	Time effect	T0	TF	Time effect	T0	TF	Time effect
	PG	3.7 ± 0.0 e	4.4 ± 0.3 c	ns	306 ± 17 c	757 ± 29 b	***	449 ± 3 a	347 ± 11 a	**	0.06 ± 0.01 a	0.05 ± 0.01 a	ns	13.33 ± 0.15 a	7.43 ± 0.06 ab	***
	PG + Ba 0.2-0.4 6.5 ± 0.0 a	6.8 ± 0.1 a	ns	3971 ± 65 a	3508 ± 185 a	ns	303 ± 0 e	237 ± 2 b	***	0.04 ± 0.01 ac 0.05 ± 0.00 a	ns	1.47 ± 0.05 f	1.05 ± 0.10 d	ns
	PG + Ba 0.5-1 6.6 ± 0.1 a	6.9 ± 0.0 a	ns	2619 ± 249 b	2689 ± 436 a	ns	317 ± 1 de	247 ± 1 b	***	0.03 ± 0.00 cd 0.04 ± 0.01 a	ns	1.34 ± 0.65 f	1.64 ± 0.07 d	ns
	PG + Ba 1-2.5 5.7 ± 0.1 b	6.9 ± 0.0 a	**	1190 ± 160 b	2991 ± 355 a	ns	329 ± 8 d	244 ± 3 b	**	0.03 ± 0.01 cd 0.04 ± 0.00 a	ns	4.24 ± 0.87 e	1.58 ± 0.06 d	ns
	PG + Sa 0.2-0.4 4.6 ± 0.1 c	5.5 ± 0.4 b	ns	287 ± 3 c	596 ± 267 b	ns	400 ± 4 c	316 ± 15 a	*	0.01 ± 0.00 d	0.05 ± 0.01 a	ns	6.58 ± 0.10 d	5.58 ± 0.14 c	**
	PG + Sa 0.5-1 4.6 ± 0.2 c	5.6 ± 0.2 b	*	257 ± 11 c	655 ± 17 b	***	400 ± 8 c	315 ± 13 a	**	0.01 ± 0.00 c	0.03 ± 0.01 a	ns	7.47 ± 0.13 cd	5.90 ± 0.34 c	*
	PG + Sa 1-2.5 4.1 ± 0.1 cd 4.6 ± 0.1 bc	*	311 ± 24 c	701 ± 43 b	**	420 ± 4 bc	361 ± 4 a	***	0.02 ± 0.01 bc 0.03 ± 0.00 a	ns	7.89 ± 0.11 bcd 6.43 ± 0.12 bc	***
	PG + He 0.2-0.4 4.1 ± 0.0 d 4.8 ± 0.3 bc	ns	223 ± 3 c	581 ± 71 b	*	418 ± 3 bc	329 ± 13 a	*	0.02 ± 0.00 c	0.03 ± 0.00 a	*	8.31 ± 0.15 bcd 7.30 ± 0.15 ab	**
	PG + He 0.5-1 4.0 ± 0.0 d 4.9 ± 0.2 bc	*	246 ± 12 c	482 ± 38 b	*	423 ± 4 b	329 ± 14 a	*	0.03 ± 0.00 bc 0.03 ± 0.00 a	ns	8.93 ± 0.17 b	7.53 ± 0.29 a	*
	PG + He 1-2.5 3.9 ± 0.0 d 5.3 ± 0.2 bc	*	252 ± 5 c	495 ± 20 b	**	427 ± 1 b	322 ± 14 a	*	0.05 ± 0.01 ab 0.04 ± 0.01 a	ns	9.57 ± 0.11 bc	6.82 ± 0.36 ab	*
	Feedstock effect	***	***		***	***		***	***		*	ns		***	***
	Particle size
	effect	ns	ns		ns	ns		ns	ns		ns	ns		ns	ns

Table 3 :

 3 Plant stem length (cm) and leaf width (cm) determined at the end of the experiment on Pontgibaud technosol (PG) amended or not with biochar from three feedstocks (Ba = bark biochar; Sa = sapwood biochar; He = heartwood biochar) and harboring three particle sizes (0.2-0.4 mm, 0.5-1 mm, 1-2.5 mm). Letters indicate significant difference (p < 0.05) (n=9 ± SE). Feedstock and particle size effects: * p < 0.05, ** p < 0.01, *** p < 0.001, ns = non significant.

	Stem length (cm)	Leaf width (cm)
	PG

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 biochars. Finally we also thank Simon Chevolleau and Florent Beaumont for technical help.