Impact of biochar and root-induced changes on metal dynamics in the rhizosphere of Agrostis capillaris and Lupinus albus

Chemosphere xxx (2014) xxx–xxx

Contents lists available at ScienceDirect

Chemosphere journal homepage: www.elsevier.com/locate/chemosphere

Impact of biochar and root-induced changes on metal dynamics in the rhizosphere of Agrostis capillaris and Lupinus albus David Houben a,b,⇑, Philippe Sonnet a a b

Earth and Life Institute, Université catholique de Louvain, Croix du Sud 2/L7.05.10, 1348 Louvain-la-Neuve, Belgium HydrISE, Institut Polytechnique LaSalle Beauvais, rue Pierre Waguet 19, 60026 Beauvais Cedex, France

h i g h l i g h t s  Biochar application has a liming effect in bulk soil.  Adding biochar shift metals from exchangeable pool to carbonate-bound pool in bulk soil.  Root-induced acidification counteracts the liming effect of biochar in rhizosphere.  Metals shifted to carbonate-bound pool were re-mobilized in rhizosphere.  Limiting rhizosphere acidification is suggested to optimize the effects of biochar.

a r t i c l e

i n f o

Article history: Received 22 July 2014 Received in revised form 17 November 2014 Accepted 12 December 2014 Available online xxxx Handling Editor: Chang-Ping Yu Keywords: Biochar Rhizosphere Heavy metal Phytoremediation Sequential extractions Immobilization

a b s t r a c t Rhizosphere interactions are deemed to play a key role in the success of phytoremediation technologies. Here, the effects of biochar and root-induced changes in the rhizosphere of Agrostis capillaris L. and Lupinus albus L. on metal (Cd, Pb and Zn) dynamics were investigated using a biotest on a 2 mm soil layer and a sequential extraction procedure (Tessier’s scheme). In the bulk soil, the application of 5% biochar significantly reduced the exchangeable pool of metals primarily due to a liming effect which subsequently promoted the metal shift into the carbonate-bound pool. However, metals were re-mobilized in the rhizosphere of both A. capillaris and L. albus due to root-induced acidification which counteracted the liming effect of biochar. As a result, the concentrations of metals in roots and shoots of both plants were not significantly reduced by the application of biochar. Although the study should be considered a worst-case scenario because experimental conditions induced the intensification of rhizosphere processes, the results highlight that changes in rhizosphere pH can impact the effectiveness of biochar to immobilize metals in soil. Biochar has thus a potential as amendment for reducing metal uptake by plants, provided the acidification of the rhizosphere is minimized. Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction Anthropogenic activities have left a toxic footprint originating from mineral exploration and mining to ore beneficiation and metal refining. Because there is no natural attenuation of heavy metals in the environment, there is a statutory requirement to remediate polluted sites in order to prevent exposure to humans and ecosystems. Increasingly, phytostabilization is suggested as an in situ cost-effective and environmentally friendly approach ⇑ Corresponding author at: HydrISE, Institut Polytechnique LaSalle Beauvais, rue Pierre Waguet 19, 60026 Beauvais Cedex, France. Tel.: +33 (0)3 44 06 93 45; fax: +33 (0)3 44 06 25 26. E-mail addresses: [email protected], [email protected] (D. Houben).

for the remediation of heavy metal-contaminated soils (Bolan et al., 2011; Lambrechts et al., 2011b). This technique consists in installing a vegetation cover on contaminated areas for containing the contaminant within the Vadoze zone. The plant canopy limits aeolian dispersion while the roots prevent water erosion and leaching of metals (Bolan et al., 2011). Phytostabilization is usually aided by the in situ application of soil amendments that are effective both in the immobilization of metals and the supply of material conditions that promote plant growth and stimulate ecological restoration (Bolan et al., 2011; Houben et al., 2012). Classical amendments include liming material, phosphate minerals, inorganic clay minerals, Fe-, Mn-, Albased materials and organic matter. These additives potentially reduce exposure by one or more of the following processes: sorption, redox reaction, precipitation, co-precipitation, ion exchange,

http://dx.doi.org/10.1016/j.chemosphere.2014.12.036 0045-6535/Ó 2014 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Houben, D., Sonnet, P. Impact of biochar and root-induced changes on metal dynamics in the rhizosphere of Agrostis capillaris and Lupinus albus. Chemosphere (2014), http://dx.doi.org/10.1016/j.chemosphere.2014.12.036

2

D. Houben, P. Sonnet / Chemosphere xxx (2014) xxx–xxx

complexation and excess of competing elements (Mench et al., 2006). More recently, among the numerous amendments proposed for phytostabilization, biochar has gained a growing attention (Beesley et al., 2011; Houben et al., 2013b; Paz-Ferreiro et al., 2014). Besides its attractive interest for replenishing C stocks and improving C sequestration on the long term in soils (Woolf et al., 2010), biochar can act as a soil conditioner because its application may rapidly increase the soil fertility and plant growth by supplying and retaining nutrients while improving soil physical and biological properties (Sohi et al., 2010). Moreover, recent studies have shown that biochar application to contaminated soils may not only improve soil biochemical properties (Lu et al., 2015) but also immobilize heavy metals and reduce their bioavailability (Park et al., 2011a; Houben et al., 2013c; Fellet et al., 2014; Lu et al., 2014). Although research on the effects of amendments on metal fate has so far mostly focused on changes caused to the bulk soil, it is increasingly recognized that, in order to improve the phytostabilization processes, a solid understanding of the complex interactions in the rhizosphere is required (Wenzel, 2009; Hashimoto et al., 2011). Indeed, the rhizosphere is a microenvironment where physico-chemical conditions may drastically differ in many respects from those in the bulk soil (Marschner and Römheld, 1996), thereby having a considerable effect on the solid speciation and, ultimately, the bioavailability of heavy metals (Hinsinger, 2001; Bravin et al., 2012; Houben and Sonnet, 2012). In particular, root-induced changes in pH are recognized as a critical mechanism influencing metal solubility in the rhizosphere (Loosemore et al., 2004; Houben et al., 2014b). Moreover, the application of soil amendments for phytostabilization purposes may also modify the rhizosphere response. As reviewed by Park et al. (2011b), this may in turn impact the transformation, mobility and bioavailability of metals notably due to change in soil pH, rhizodeposition and microbial community. Since biochar incorporation into contaminated soils is irreversible, it is thus essential to elucidate its effects on the rhizosphere response prior to implement it at field scale for phytostabilization purposes. Therefore, the objective of this study was to gain a better insight about how the biochar application to contaminated soil affects the rhizospheric response and, in turn, the speciation and the bioavailability of heavy metals (Cd, Pb and Zn). Agrostis capillaris L. (=A. tenuis Sibth.) and Lupinus albus L. were selected as the model plants because they are commonly used in phytostabilization schemes. A. capillaris is a metal-tolerant plant that can, in a temperate climate, successfully cover any Pb-/Zn-contaminated soil and metallurgical or mining waste disposal site, irrespective of whether it is calcareous or acidic (Tordoff et al., 2000; Houben et al., 2013a). L. albus is considered an excellent candidate for phytostabilization because it has several characteristics that allow it to thrive in poor and contaminated soils, including its nitrogen-fixation capacity, adaptability to poor acidic soils, tolerance to lime excess as well as to high salinity, elevated metal content in soils and several other biotic and abiotic stresses (Vazquez et al., 2006; Martínez-Alcalá et al., 2010; Houben et al., 2012). 2. Materials and methods 2.1. Soil and biochar The study soil was collected at Sclaigneaux (50°300 0300 N, 5°020 5600 E; Namur province, Belgium). Although this 55 ha site is now a natural reserve accessible to the public, from the 1850s to the 1970s, it was intensively subjected to Cd-, Zn-, and Pb-bearing atmospheric fallouts originating from an adjacent Zn and Pb smelting plant (De Nul, 2010). The site is located in the alluvial plain of the Meuse River at an altitude of about 170 m. The climate is

temperate oceanic and the soil is a sandy loam Luvisol. A total mass of 250 kg of surface soil (0–14 cm) was obtained by composite sampling of a 20  20 m2 area that was colonized by metal-tolerant plant species (Rumex acetosa L., Festica nigrescens Lam., A. capillaris L.). After drying at ambient temperature and sieving (2 mm), the soil was amended with biochar on a 5% w/w basis (thereafter called ‘‘biochar-5%’’). Biochar derived from miscanthus (Miscanthus  giganteus) straw and was obtained from Pyreg GmbH (Dörth, Germany) using an industrial pyrolysis reactor (30 m of residence time and 600 °C of end temperature of pyrolysis). Some properties of this biochar can be found in a previous study (Houben et al., 2014a). Untreated (thereafter called ‘‘control’’) soil was also part of the experimental design. Four 200-g pots were prepared per each treatment and were incubated at field capacity in darkness over a period of 56 d (for details, see Houben et al., 2013c). The soils were then removed from the pots, dried at ambient temperature and stored in plastic bottles prior to be characterized and used for the biotest experiment. The pH (pH-CaCl2) of the incubated soils was potentiometrically measured in a 1:5 (w/v) suspension of 0.01 M CaCl2. The cation exchange capacity (CEC) and the concentration of exchangeable cations were assessed by leaching the soil with 1 M ammonium acetate at pH 7 (for Ca2+, Mg2+, K+ and Na+) and then 1 M KCl (for CEC) (Chapman, 1965). Available P content was determined according to the Mehlich 3 procedure (Mehlich, 1984). Organic C content was measured following the Walkley and Black procedure (Page et al., 1982). Ammonium-(NH4–N) and nitrate–nitrogen (NO3–N) contents in the soils were determined following Bremner and Keeney (1965). Metal (Cd, Pb and Zn) concentration in the soils was determined by inductively coupled plasma-atomic emission spectroscopy (ICP-AES; Jarrell Ash) after calcination at 450 °C followed by acid digestion (HNO3, HClO4 and HF), as described in Lambrechts et al. (2011a). The main physical and chemical characteristics of the soils are presented in Table 1.

2.2. Biotest experimental device The design of the biotest used in this study was based on that of Lambrechts et al. (2011a) and is illustrated in Fig. 1. The principle of this device is similar to that used by other authors (Chaignon and Hinsinger, 2003; Loosemore et al., 2004; Bravin et al., 2010,2012) and consists in separating plant roots from soil with a 20-lm polyamide mesh to facilitate the collection of roots and rhizosphere. Plant and soil compartments have an external diameter of 80 and 90 mm and a height of 15 and 30 mm, respectively. Briefly, 6 or 0.08 g of sterilized seeds (10 min in 2 M H2O2) of L. albus or A. capillaris respectively per pot were sown in the plant compartment and germinated in demineralized water for three

Table 1 Characteristics and total metal content of the soil (control) and the soil amended with 5% biochar (biochar-5%).

pH-CaCl2 CEC Exchangeable-Ca Exchangeable-K Exchangeable-Mg Exchangeable-Na Mehlich 3-P Organic C NO3–N NH4–N Total Cd Total Zn Total Pb

cmolc kg1 cmolc kg1 cmolc kg1 cmolc kg1 cmolc kg1 mg kg1 g kg1 mg kg1 mg kg1 mg kg1 mg kg1 mg kg1

Control

Biochar-5%

5.66 7.38 3.65 0.12 0.80 0.01 20.8 190 40.6 3.5 24 3080 2690

6.24 7.92 3.99 1.06 0.94 0.07 27.5 207 10.5 10.5 25 2940 2590

Please cite this article in press as: Houben, D., Sonnet, P. Impact of biochar and root-induced changes on metal dynamics in the rhizosphere of Agrostis capillaris and Lupinus albus. Chemosphere (2014), http://dx.doi.org/10.1016/j.chemosphere.2014.12.036

D. Houben, P. Sonnet / Chemosphere xxx (2014) xxx–xxx

3

Fig. 1. (a) Biotest experiment design (see Lambrechts et al., 2011a, for more details), plant compartment at the completion of the pre-growth stage with (b) Agrostis capillaris and (c) Lupinus albus, and (d) whole experimental setup.

days in darkness. Then, the demineralized water was replaced with nutrient solution and the seedlings were allowed to grow for 7 d under a photoperiod of 16 h (photon flux density of 120– 180 lmol m2 s1), constant temperature (20 °C) and relative humidity (95%). The chemical composition of the nutrient solution expressed in mM was: 1 Ca(NO3)2.4H2O, 0.5 KCl, 0.25 K2SO4, 0.05 MgCl2, 0.05 MgSO4, 0.05 NaH2PO4, and 0.08 H3BO3. This pregrowth period allowed the formation of a dense root mat at the surface of the 20 lm-polyamide mesh. At the completion of this pre-growth stage, plant compartments were transferred onto a thin layer of pre-incubated soil mixture (about 2-mm thick, 7.5 g dry soil). The soil was not treated with any chemical fertilizers. The whole system was placed on a polystyrene plate floating on a volume of 1-dm3 water. A polyamide cloth below the soil layer dipped into the demineralized water reservoir in order to maintain constant soil moisture by capillarity. After 21 d of contact, the soil layer under plants was considered as rhizosphere. The contact duration was adapted from other studies (Chaignon and Hinsinger, 2003; Lambrechts et al., 2011a; Bravin et al., 2012) that found it suitable to account for rhizosphere processes when assessing metal availability. Similar to Bravin et al. (2012), unplanted control treatments, in which the soil had been incubated in similar devices without plants (thereafter called bulk soil), were carried out. In total, 24 such devices were implemented: 2 treatments (control or biochar-5%)  3 crop conditions (A. capillaris, L. albus, and uncropped/bulk soil)  4 replicates.

2.4. Soil analyses At the completion of the biotest experiment, cropped soils (rhizosphere) and uncropped soils (bulk soil) were collected and dried at ambient temperature. Soil pH was measured in H2O (soil to water ratio of 1:5) after shaking for 1 h. The fractionation of Cd, Zn and Pb in soils was determined according to the sequential extraction scheme proposed by Tessier et al. (1979) which consists of five steps to extract heavy metals bound to the following operationally defined fractions: exchangeable (EXCH), bound to carbonates (CARB), bound to Fe and Mn oxides (OXI), bound to organic matter (ORG) and residual (RES). All metal concentrations are expressed on a dry matter basis (i.e., dried at 105 °C). 2.5. Statistical analyses Statistical analyses to compare the average results of sequential extractions and pH were performed using a one-way analysis of variance (ANOVA) followed by Fisher’s test (p < 0.05). Paired comparisons between the control and the biochar-5% treatment were carried out using Student’s t-test. Prior to statistical analyses, homogeneity of variances was tested using Levene’s test. Pearson’s correlation coefficients (r) were calculated to determine the relationships between the heavy metal concentrations in sequential extraction fractions and the soil pH. Three levels of significance were considered: p < 0.05, p < 0.01 and p < 0.001. All statistical analyses were carried out using XLSTAT (Addinsoft, ver. 2010.5.08).

2.3. Plant analyses 3. Results and discussion At harvest, shoots and roots were separated and roots were gently rinsed with deionized water. Shoots and roots were then dried at 60 °C, weighed and crushed. The metal (Cd, Zn and Pb) concentration in shoots and roots was determined by ICP-AES after mineralization by HNO3 and aqua regia digestion.

3.1. Bulk soil and rhizosphere pH In the bulk soil, the application of biochar showed a significant liming effect as the pH raised from 5.58 to 6.70 (Fig. 2), probably

Please cite this article in press as: Houben, D., Sonnet, P. Impact of biochar and root-induced changes on metal dynamics in the rhizosphere of Agrostis capillaris and Lupinus albus. Chemosphere (2014), http://dx.doi.org/10.1016/j.chemosphere.2014.12.036

4

D. Houben, P. Sonnet / Chemosphere xxx (2014) xxx–xxx

due to the dissolution of metal oxides, hydroxides and carbonates (e.g. CaO, CaCO3) admixed with biochar as well as the presence of – COO and –O groups at the surface of biochar which can contribute to alkalinity through association with H+ (Yuan et al., 2011; Hass et al., 2012). However, such a liming effect was not observed in the rhizosphere of both plants as their rhizopheric pH in the presence and in the absence of biochar were similar and significantly lower than the bulk soil pH (Fig. 2). Using titration curves previously measured for the bulk soils (Houben et al., 2013c), we calculated that the quantity of H+ necessary to decrease bulk soil pH to those measured in the rhizosphere was 0.026 and 0.017 mmol g1soil for A. capillaris and L. albus respectively in control and 0.052 and 0.038 mmol g1soil for A. capillaris and L. albus respectively in biochar-5% treatment. This indicates that the net H+ production in the rhizosphere increased twofold in the presence of biochar, both for the first and for the second plant. The main process explaining the difference in pH between the rhizosphere and the bulk soil is the release of H+ or OH by roots to compensate charge imbalance due to unequal uptakes of cations and anions (Nye, 1981; Hinsinger et al., 2003). Thus, a possible explanation is that the high supply of exchangeable cations (Table 1) by the biochar promoted their absorption by plants and, in turn, increased the rhizosphere acidification. For instance, Loss et al. (1993) showed that in K-enriched media, such as biochar-5% (Table 1), the K uptake by Lupinus angustifolius L. was exacerbated thereby leading to the secretion of large amounts of H+ to compensate the charge imbalance in solution. It is also well-known that the rhizosphere pH is greatly influenced by the N uptake by roots and that a higher uptake of NH4–N relative to NO3–N increases the release of H+ from roots (Hinsinger et al., 2003). Consequently, the higher NH4–N content while lower NO3–N content in biochar-5% (Table 1) contributed likely to stimulate rhizosphere acidification. Taghizadeh-Toosi et al. (2011) proposed that one possible mechanism for the reduced NO3–N content is the stronger adsorption of NH+4 by the biochar which reduced the soil NH4–N pool available to nitrifiers. In addition, enhanced release of H+ under Fe deficiency has been described as a strategy for Fe uptake by the plant and has been found in all dicotyledonous species (Marschner, 1995). For L. albus, the liming effect consecutive to the biochar application may have decreased the availability of Fe, and thus enhanced the H+ efflux by the roots. N2-fixation by L. albus could also contribute to rhizosphere acidification (Sas et al., 2002). However, no nodules could be visually detected on L. albus roots, neither in the absence nor in the presence of biochar, suggesting that this process did not contribute to increase the H+

a

7 6.5

Control Biochar-5%

bc

6

pH

b cd

5.5 d

d

5 4.5 4

Bulk Bulk soil soil

A.capillaris A. capillaris L. L. albus albus

Bulk Bulksoil soil A.capillaris A. capillaris L.L.albus albus

Fig. 2. pH in the bulk soil and in the rhizosphere of Agrostis capillaris and Lupinus albus in the control and biochar-5% treatment. Values are average (n = 4) ± standard deviations. Columns with the same letter do not differ significantly at the 5% level according to the Fisher’s multiple comparison test.

production observed in the L. albus rhizosphere. Finally, proteoid roots of L. albus can also acidify the rhizosphere by releasing citrate, malate and protons, especially under P deficiency (Sas et al., 2001). However, it is unlikely that this process occurred in the rhizosphere of the biochar-amended soil because the application of biochar substantially increased the available P (Mehlich 3-P) content (Table 1). 3.2. Effects of biochar on heavy metal fractionation in bulk soil The fractionation of metals in soils was assessed using sequential extraction procedure according to the Tessier’s scheme (Tessier et al., 1979). Although sequential extractions provide results on fractions which are operationally-defined and suffer from several drawbacks such as lack of selectivity and element redistribution during extraction, they have been used extensively by researchers to elucidate the fractionation of heavy metal in soils, including the rhizosphere (Bacon and Davidson, 2008). According to Hammer and Keller (2002), their use is particularly justified when the aim is to compare differences generated in the same soil by different treatments, such as the effect of amendment or plant growth on metal distribution. Results of chemical fractionation of metals in the bulk soil show that the biochar application depressed the amount of Cd and Zn in the EXCH pool while it increased that in the CARB pool (Fig. 3). The other Cd and Zn fractions were not significantly affected. The shift of Cd and Zn from the EXCH to CARB fraction induced by biochar application was most likely the result of the increase in soil pH which promoted the precipitation of metals (Lindsay, 1979). Our results are consistent with those of Xian and In Shokohifard (1989) which showed that when the soil pH was changed, Cd and Zn were chiefly shifted between exchangeable and carbonate forms, while the other fractions remained almost unchanged. Similarly, several studies (Jiang et al., 2012; Rees et al., 2014) have indicated that metal precipitation as a result of the pH increase brought about by biochar application was one of the mechanisms involved in the metal immobilization. As for Cd and Zn, the biochar application decreased significantly the EXCH pool of Pb in the bulk soil (Fig. 3). However, it was not possible to determine which pool received the Pb from the EXCH pool. Even though the CARB and RES pools were slightly increased, the differences were not statistically significant (p > 0.05) compared to the control (Fig. 3). 3.3. Effects of root-induced changes on heavy metal fractionation In both the control and the biochar-5% soils, the growth of both A. capillaris and L. albus decreased the Cd amount in the CARB fraction (Fig. 3). This decrease can be attributed to the rhizosphereinduced acidification (Fig. 2) which is well-known to favor the release of metals associated with carbonates (Marschner and Römheld, 1996). The strong positive correlation between pH and Cd concentration in the CARB pool (Fig. 4) confirms the predominant role that the pH played in the control of the carbonate-bound Cd solubility. A similar trend was noticed for Zn, the CARB pool in the rhizosphere of both control and biochar-5% soils being significantly lower than that in the bulk soil (Fig. 3), likely as a result of the lower pH (Fig. 2). Irrespective of the treatments, the pool of Zn bound to Fe- and Mn-oxides (OXI) was also significantly depleted in the presence of plants. Since the metals associated with oxides can be released under acidic conditions (Xian and In Shokohifard, 1989), this Zn depletion can also be explained by the root-induced acidification (Marschner and Römheld, 1996), as mirrored by the strong positive correlation found between pH and Zn bound to Fe- and Mn-oxides pool (Fig. 4). Unlike Cd, the EXCH pool of Zn in the presence of biochar was significantly higher in the rhizosphere of both A. capillaris and L. albus than in the bulk soil

Please cite this article in press as: Houben, D., Sonnet, P. Impact of biochar and root-induced changes on metal dynamics in the rhizosphere of Agrostis capillaris and Lupinus albus. Chemosphere (2014), http://dx.doi.org/10.1016/j.chemosphere.2014.12.036

5

Cd (mg kg-1)

D. Houben, P. Sonnet / Chemosphere xxx (2014) xxx–xxx

18 16 14 12 10 8 6 4 2 0

a bc

b d cd

cd

ab b b a b

EXCH

d

cd

a

ab ab

a a a a

a a

bc b

a a a a a a

CARB

OXI

ORG

RES

1600

Zn (mg kg-1)

1400

ab

1200 1000

a

800

c

bc

a

bc

bc

a

a a a

b c

600

d

d

e

400

b

a a

a c c

c

bc

200

ab b ab ab a ab

0

EXCH

CARB

OXI

1600

a a a a

1400

Pb (mg kg-1)

ORG

RES

ab b

1200 1000 800 600 400 200

a bc

a bc

ab

a a

a

a a a

a

c

d a a

0

EXCH

a

a a

CARB

OXI

a

a

a aa

ORG

RES

Control

Biochar-5%

Control ++A.A.tenuis capillaris

Biochar-5% ++A. A.tenuis capillaris

Control ++L.L.albus albus

Biochar-5% ++L.L.albus albus

Fig. 3. Fractionation of Cd, Zn and Pb in the bulk soil and in the rhizosphere of Agrostis capillaris and Lupinus albus in the control and biochar-5% treatments (EXCH: exchangeable; CARB: bound to carbonate; OXI: bound to Fe and Mn oxides; ORG: bound to organic matter; RES: residual). Values are average (n = 4) ± standard deviations. For each fraction, columns with the same letter do not differ significantly at the 5% level according to the Fisher’s multiple comparison test.

(Fig. 3). This suggests that, contrarily to Cd, a fraction of Zn solubilized from the CARB pool due to root-induced acidification was not taken up by plants but redistributed onto the exchange sites of soil and biochar particles. In agreement with Loosemore et al. (2004), the concentration of Cd and Zn in the EXCH pool is thus the final result from the concurrent dynamics of pH change and metal uptake induced by roots. In both treatments, the rhizosphere activity of A. capillaris and L. albus increased the Pb amount in the EXCH pool compared to the bulk soil (Fig. 3). The reason of this Pb enrichment in the EXCH pool was probably also related to the root-induced acidification as suggested by the strong negative correlation between the amount of Pb in this pool and the soil pH (Fig. 4). The strong positive correlation between the amount of Pb in the CARB pool and the soil pH (Fig. 4) also suggests that root-induced acidification promoted the dissolution of carbonate-bound Pb pool and this may have contributed to the increase of Pb in EXCH pool. However, this interpretation should be regarded with caution as the Pb amounts in the CARB pool were not statistically significantly lower in the rhizosphere (Fig. 3). Our results are in line with those Hashimoto et al. (2011) which demonstrated that Pb immobilization in a hydroxyapatite-amended was negatively affected by rhizosphere processes because of the root-induced acidification which increased the solubility of Pb.

3.4. Effects of biochar and root-induced changes on metal uptake by plants Except for Cd concentration in A. capillaris shoots and L. albus roots, the application of biochar did not significantly reduce the concentration of heavy metals in both roots and shoots of plants (Fig. 5). The inefficacy of biochar application to reduce metal uptake by plants was probably related to the effects of root-induced changes on metal distribution in the rhizosphere. Results of sequential extractions revealed that the main mechanism of Cd, Zn and to a lesser extent Pb immobilization after introducing biochar was metal precipitation due to the pH increase. However, subsequent rhizosphere activity suppressed the liming effect of biochar and lowered the rhizosphere pH down to a level which was similar to that measured in the rhizosphere of plants grown in the absence of biochar. This root-induced acidification dissolved or hindered the formation of metal precipitates in the rhizosphere and thus counteracted the short-term biochar-induced immobilization effect observed in the bulk soil. As a result, metal bioavailability at the soil-root interface was most likely similar regardless of the presence or the absence of biochar. These results confirm the predominant role of pH in the control of metal bioavailability in biochar amended soils and coincide with our previous findings (Houben et al., 2013c) which showed that the release of Cd, Zn and Pb under

Please cite this article in press as: Houben, D., Sonnet, P. Impact of biochar and root-induced changes on metal dynamics in the rhizosphere of Agrostis capillaris and Lupinus albus. Chemosphere (2014), http://dx.doi.org/10.1016/j.chemosphere.2014.12.036

16 12 8 r = 0.46 5.8

6.3

6.8

200 0

r = -0.70** 4.8 5.3 5.8 6.3 6.8

4 3 2 1

r = 0.97**

0 4.8

5.3

5.8

6.3

6.8

600 400 200 r = 0.98***

0

4.8 5.3 5.8 6.3 6.8

8 6 4 r = 0.95**

0 4.8

5.3

5.8

6.3

1600 1200 800 400 0

6.8

r = 0.94**

ORG-Zn (mg kg-1)

ORG-Cd (mg kg-1)

1 0.8 0.6 0.4 r = -0.48

0 4.8

5.3

5.8

6.3

6.8

RES-Zn (mg kg-1)

RES-Cd (mg kg-1)

6 4 2 r = -0.43 4.8

5.3

5.8

200

r = -0.93**

0

4.8 5.3 5.8 6.3 6.8

pH 800 600 400 200

r = 0.98***

0

4.8 5.3 5.8 6.3 6.8

6.3

6.8

1200 800 400 0

r = 0.40

pH

80 60 40 r = -0.32

0

1600

pH 100

20

pH

4.8 5.3 5.8 6.3 6.8

150 120 90 60 30 0

r = -0.54

4.8 5.3 5.8 6.3 6.8

4.8 5.3 5.8 6.3 6.8

pH

pH

pH

0

400

4.8 5.3 5.8 6.3 6.8

pH

0.2

600

pH OXI-Zn (mg kg-1)

OXI-Cd (mg kg-1)

pH

2

800

pH CARB-Zn (mg kg-1)

CARB-Cd (mg kg-1)

pH

CARB-Pb (mg kg-1)

5.3

400

OXI-Pb (mg kg-1)

4.8

600

ORG-Pb (mg kg-1)

0

800

1200 800 400 0

r = -0.83*

RES-Pb (mg kg-1)

4

1000

EXCH-Pb (mg kg-1)

20

EXCH-Zn (mg kg-1)

D. Houben, P. Sonnet / Chemosphere xxx (2014) xxx–xxx

EXCH-Cd (mg kg-1)

6

800 600 400 200 0

r = 0.87*

4.8 5.3 5.8 6.3 6.8

4.8 5.3 5.8 6.3 6.8

pH

pH

pH

Control

Control+ Control +A. A.tenuis capillaris

Control Control++L.L.albus albus

Biochar-5%

Biochar-5%+A. Biochar-5% + A.tenuis capillaris

Biochar-5% + L. albus Biochar-5%+L. albus

Fig. 4. Relationship between soil pH and metal fractions in soil (EXCH: exchangeable; CARB: bound to carbonate; OXI: bound to Fe and Mn oxides; ORG: bound to organic matter; RES: residual). Values are average (n = 4) ± standard deviations. Marked r correlation coefficient is the Pearson’s correlation coefficient. ⁄p < 0.05; ⁄⁄p < 0.01; ⁄⁄⁄ p < 0.001.

identical acidic conditions was similar regardless of the presence or the absence of biochar into the soil. However, our results contrast with the results from Lu et al. (2014) who found different levels of Cd bioavailability in soils amended with biochar or with lime. The lack of significant reduction of metal concentration in plant parts after biochar application appears to disagree with results obtained by other studies (Park et al., 2011a; Houben et al., 2013b,c; Lu et al., 2014). However, by restricting the roots in small

volume of soil, the biotest used in this study exaggerated somewhat the effects of the roots on the soil. High root density intensified the effects of root-induced mobilization mechanisms, leading probably to an overestimation of rhizosphere effects. Moreover, since the thin layer of soil was the sole source of nutrients for plants, this restricted reservoir most likely induced the intensification of rhizosphere processes. This is consistent with Chaignon and Hinsinger (2003) who, using similar biotest devices,

Please cite this article in press as: Houben, D., Sonnet, P. Impact of biochar and root-induced changes on metal dynamics in the rhizosphere of Agrostis capillaris and Lupinus albus. Chemosphere (2014), http://dx.doi.org/10.1016/j.chemosphere.2014.12.036

7

D. Houben, P. Sonnet / Chemosphere xxx (2014) xxx–xxx

Agrostis capillaris

Lupinus albus Control Biochar-5%

*

Cd (mg kg-1)

80 60

*

0.2 0.1

40

0

20

Shoots

0

4000

4000

3000

3000

Zn (mg kg-1)

Zn (mg kg-1)

Roots

2000 1000

2000 1000 0

0

Roots

Roots

Shoots

400

400

300

300

Pb (mg kg-1)

Pb (mg kg-1)

Shoots

200 100

Shoots 3 2 1 0

200

Shoots

100 0

0

Roots

Roots

Shoots

Shoots

Fig. 5. Concentration of Cd, Zn and Pb in roots and shoots of Agrostis capillaris (left) and Lupinus albus (right) in the control and biochar-5% treatments. Values are average (n = 4) ± standard deviations. ⁄Significant at the 0.05 probability level.

showed that Cu bioavailability was systematically higher when the plants grew in the presence of water instead of nutrient solution. Results of the present study should thus be regarded as the results of the worst-case scenario. They allow defining the limitations for the use of biochar for phytostabilization strategies. In studies conducted using larger soil volume (e.g. pot or field experiment), we might expect a slower root-induced metal mobilization due to stronger acid buffering capacity and larger nutrient supply. 4. Conclusion Biochar application to the bulk soil significantly reduced the exchangeable pool of Cd, Zn and Pb due to liming effect which subsequently promoted the metal shift into the carbonate-bound pool. However, concentrations of metals in roots and shoots of both A. capillaris and L. albus were generally not reduced in the presence of biochar. This was due to the root-induced acidification of the rhizosphere which counteracted the liming effect of biochar and, in turn, suppressed short-term metal immobilization. Although the study should be considered a worst-case scenario because experimental conditions induced the intensification of rhizosphere processes, the results highlight that potential changes in rhizosphere pH need to be accounted prior to implement phytostabilization with biochar (or other amendments) in the field. Preventing soil acidification in the rhizosphere to avoid metal re-mobilization could also be achieved, notably by increasing the acid buffering capacity (e.g. by liming), supplying N as nitrate instead of ammonia and selecting less acidifying plants. Additional results of this study also suggest that, among the two tested plants, preference should be given to L. albus over A. capillaris as the first species displayed lower root-induced acidification.

Acknowledgements D. Houben was supported by the ‘‘Fonds pour la formation à la Recherche dans l’Industrie et dans l’Agriculture’’ (FRIA) of Belgium. We thank P. Populaire for his technical assistance and C. Givron and A. Iserentant for their analytical assistance. References Bacon, J.R., Davidson, C.M., 2008. Is there a future for sequential chemical extraction? Analyst 133, 25–46. Beesley, L., Moreno-Jiménez, E., Gomez-Eyles, J.L., Harris, E., Robinson, B., Sizmur, T., 2011. A review of biochars’ potential role in the remediation, revegetation and restoration of contaminated soils. Environ. Pollut. 159, 3269–3282. Bolan, N.S., Park, J.H., Robinson, B., Naidu, R., Huh, K.Y., 2011. Phytostabilization: a green approach to contaminant containment. In: Donald, L.S. (Ed.), Adv. Agron. Academic Press, pp. 145–204. Bravin, M.N., Garnier, C., Lenoble, V., Gérard, F., Dudal, Y., Hinsinger, P., 2012. Rootinduced changes in pH and dissolved organic matter binding capacity affect copper dynamic speciation in the rhizosphere. Geochim. Cosmochim. Acta 84, 256–268. Bravin, M.N., Michaud, A.M., Larabi, B., Hinsinger, P., 2010. RHIZOtest: a plant-based biotest to account for rhizosphere processes when assessing copper bioavailability. Environ. Pollut. 158, 3330–3337. Bremner, J.M., Keeney, D.R., 1965. Steam distillation methods for determination of ammonium, nitrate and nitrite. Anal. Chim. Acta 32, 485–495. Chaignon, V., Hinsinger, P., 2003. A biotest for evaluating copper bioavailability to plants in a contaminated soil. J. Environ. Qual. 32, 824–833. Chapman, H.D., 1965. Cation exchange capacity. In: Black, C.A. (Ed.), Methods of soil analysis: Part 1, physical and mineralogical methods. American Society of Agronomy and Soil Science Society of America, Madison, pp. 891–901. De Nul, R., 2010. Les minéraux des scories métallurgiques de Sclaigneaux (Andenne). In: Goemaere, E. (Ed.), Terres, pierres et feu en vallée mosane. Service géologique de Belgique and Institut royal des Sciences naturelles de Belgique, Bruxelles (Belgium), pp. 349–372. Fellet, G., Marmiroli, M., Marchiol, L., 2014. Elements uptake by metal accumulator species grown on mine tailings amended with three types of biochar. Sci. Total Environ. 468–469, 598–608.

Please cite this article in press as: Houben, D., Sonnet, P. Impact of biochar and root-induced changes on metal dynamics in the rhizosphere of Agrostis capillaris and Lupinus albus. Chemosphere (2014), http://dx.doi.org/10.1016/j.chemosphere.2014.12.036

8

D. Houben, P. Sonnet / Chemosphere xxx (2014) xxx–xxx

Hammer, D., Keller, C., 2002. Changes in the rhizosphere of metal-accumulating plants evidenced by chemical extractants. J. Environ. Qual. 31, 1561–1569. Hashimoto, Y., Takaoka, M., Shiota, K., 2011. Enhanced transformation of lead speciation in rhizosphere soils using phosphorus amendments and phytostabilization: an X-ray absorption fine structure spectroscopy investigation. J. Environ. Qual. 40, 696–703. Hass, A., Gonzalez, J.M., Lima, I.M., Godwin, H.W., Halvorson, J.J., Boyer, D.G., 2012. Chicken manure biochar as liming and nutrient source for acid Appalachian soil. J. Environ. Qual. 41, 1096–1106. Hinsinger, P., 2001. Bioavailability of trace elements as related to root-induced chemical changes in the rhizosphere. In: Gobran, G.R., Wenzel, W.W., Lombi, E. (Eds.), Trace Elements in the Rhizosphere. CRC Press, Boca Raton, Fl, pp. 25–41. Hinsinger, P., Plassard, C., Tang, C.X., Jaillard, B., 2003. Origins of root-mediated pH changes in the rhizosphere and their responses to environmental constraints: a review. Plant Soil 248, 43–59. Houben, D., Couder, E., Sonnet, P., 2013a. Leachability of cadmium, lead, and zinc in a long-term spontaneously revegetated slag heap: implications for phytostabilization. J. Soils Sediments 13, 543–554. Houben, D., Evrard, L., Sonnet, P., 2013b. Beneficial effects of biochar application to contaminated soils on the bioavailability of Cd, Pb and Zn and the biomass production of rapeseed (Brassica napus L.). Biomass Bioenerg. 57, 196–204. Houben, D., Evrard, L., Sonnet, P., 2013c. Mobility, bioavailability and pH-dependent leaching of cadmium, zinc and lead in a contaminated soil amended with biochar. Chemosphere 92, 1450–1457. Houben, D., Pircar, J., Sonnet, P., 2012. Heavy metal immobilization by cost-effective amendments in a contaminated soil: effects on metal leaching and phytoavailability. J. Geochem. Explor. 123, 87–94. Houben, D., Sonnet, P., 2012. Zinc mineral weathering as affected by plant roots. Appl. Geochem. 27, 1587–1592. Houben, D., Sonnet, P., Cornelis, J.-T., 2014a. Biochar from Miscanthus: a potential silicon fertilizer. Plant Soil 374, 871–882. Houben, D., Sonnet, P., Tricot, G., Mattielli, N., Couder, E., Opfergelt, S., 2014b. Impact of root-induced mobilization of zinc on stable Zn isotope variation in the soil-plant system. Environ. Sci. Technol. Jiang, J., Xu, R.-K., Jiang, T.-Y., Li, Z., 2012. Immobilization of Cu(II), Pb(II) and Cd(II) by the addition of rice straw derived biochar to a simulated polluted Ultisol. J. Hazard. Mater. 229–230, 145–150. Lambrechts, T., Couder, E., Bernal, M.P., Faz, A., Iserentant, A., Lutts, S., 2011a. Assessment of heavy metal bioavailability in contaminated soils from a former mining area (La Union, Spain) using a rhizospheric test. Water Air Soil Pollut. 217, 333–346. Lambrechts, T., Gustot, Q., Couder, E., Houben, D., Iserentant, A., Lutts, S., 2011b. Comparison of EDTA-enhanced phytoextraction and phytostabilisation strategies with Lolium perenne on a heavy metal contaminated soil. Chemosphere 85, 1290–1298. Lindsay, W.L., 1979. Chemical Equilibria in Soils. John Wiley and Sons, New York. Loosemore, N., Straczek, A., Hinsinger, P., Jaillard, B., 2004. Zinc mobilisation from a contaminated soil by three genotypes of tobacco as affected by soil and rhizosphere pH. Plant Soil 260, 19–32. Loss, S.P., Robson, A.D., Ritchie, G.G.P., 1993. H+/OH- excretion and nutrient uptake in upper and lower parts of lupin (Lupinus angustifolius L.) root systems. Ann. Bot. 72, 315–320. Lu, H., Li, Z., Fu, S., Méndez, A., Gascó, G., Paz-Ferreiro, J., 2014. Can biochar and phytoextractors be jointly used for cadmium remediation? PLoS ONE 9, e95218. Lu, H., Li, Z., Fu, S., Méndez, A., Gascó, G., Paz-Ferreiro, J., 2015. Combining phytoextraction and biochar addition improves soil biochemical properties in a soil contaminated with Cd. Chemosphere 119, 209–216.

Marschner, H., Römheld, V., 1996. Root-induced changes in the availability of micronutrients in the rhizosphere. In: Waisel, Y., Eshel, A., Kafkafi, U. (Eds.), Plant Roots The Hidden Half. Marcel Dekker Inc, New York, USA, pp. 557–580. Martínez-Alcalá, I., Walker, D.J., Bernal, M.P., 2010. Chemical and biological properties in the rhizosphere of Lupinus albus alter soil heavy metal fractionation. Ecotoxicol. Environ. Saf. 73, 595–602. Mehlich, A., 1984. Mehlich 3 soil test extractant: a modification of Mehlich 2 extractant. Commun. Soil Sci. Plant Anal. 15, 1409–1416. Mench, M., Vangronsveld, J., Bleeker, P., Ruttens, A., Geebelen, W., Lepp, N., 2006. Phytostabilisation of metal-contaminated sites. In: Morel, J.-L., Echevarria, G., Goncharova, N. (Eds.), Phytoremediation of Metal-Contaminated Soils. Springer, Dordrecht, The Netherlands, pp. 109–190. Nye, P.H., 1981. Changes of pH across the rhizosphere induced by roots. Plant Soil 61, 7–26. Page, A.L., Miller, R.H., Keeney, D.R., 1982. Methods of Soil Analysis. Part 2— Chemical and Microbiological Properties, second ed. America Society of Agronomy, Madison. Park, J., Choppala, G., Bolan, N., Chung, J., Chuasavathi, T., 2011a. Biochar reduces the bioavailability and phytotoxicity of heavy metals. Plant Soil 348, 439–451. Park, J.H., Lamb, D., Paneerselvam, P., Choppala, G., Bolan, N., Chung, J.-W., 2011b. Role of organic amendments on enhanced bioremediation of heavy metal(loid) contaminated soils. J. Hazard. Mater. 185, 549–574. Paz-Ferreiro, J., Lu, H., Fu, S., Méndez, A., Gascó, G., 2014. Use of phytoremediation and biochar to remediate heavy metal polluted soils: a review. Solid Earth 5, 65–75. Rees, F., Simonnot, M.O., Morel, J.L., 2014. Short-term effects of biochar on soil heavy metal mobility are controlled by intra-particle diffusion and soil pH increase. Eur. J. Soil Sci. 65, 149–161. Sas, L., Rengel, Z., Tang, C., 2001. Excess cation uptake, and extrusion of protons and organic acid anions by Lupinus albus under phosphorus deficiency. Plant Sci. 160, 1191–1198. Sas, L., Rengel, Z.E.D., Tang, C., 2002. The effect of nitrogen nutrition on cluster root formation and proton extrusion by Lupinus albus. Ann. Bot. 89, 435–442. Sohi, S.P., Krull, E., Lopez-Capel, E., Bol, R., 2010. Chapter 2 – A Review of Biochar and Its Use and Function in Soil. In: Donald, L.S. (Ed.), Adv. Agron. Academic Press, pp. 47–82. Taghizadeh-Toosi, A., Clough, T.J., Condron, L.M., Sherlock, R.R., Anderson, C.R., Craigie, R.A., 2011. Biochar incorporation into pasture soil suppresses in situ nitrous oxide emissions from ruminant urine patches. J. Environ. Qual. 40, 468– 476. Tessier, A., Campbell, P.G.C., Bisson, M., 1979. Sequential extraction procedure for the speciation of particulate trace metals. Anal. Chem. 51, 844–851. Tordoff, G.M., Baker, A.J.M., Willis, A.J., 2000. Current approaches to the revegetation and reclamation of metalliferous mine wastes. Chemosphere 41, 219–228. Vazquez, S., Agha, R., Granado, A., Sarro, M.J., Esteban, E., Penalosa, J.M., Carpena, R.O., 2006. Use of white lupin plant for phytostabilization of Cd and As polluted acid soil. Water Air Soil Pollut. 177, 349–365. Wenzel, W., 2009. Rhizosphere processes and management in plant-assisted bioremediation (phytoremediation) of soils. Plant Soil 321, 385–408. Woolf, D., Amonette, J.E., Street-Perrott, F.A., Lehmann, J., Joseph, S., 2010. Sustainable biochar to mitigate global climate change. Nat. Commun. 1, 56. Xian, X., In Shokohifard, G., 1989. Effect of pH on chemical forms and plant availability of cadmium, zinc, and lead in polluted soils. Water Air Soil Pollut. 45, 265–273. Yuan, J.-H., Xu, R.-K., Zhang, H., 2011. The forms of alkalis in the biochar produced from crop residues at different temperatures. Bioresour. Technol. 102, 3488– 3497.

Please cite this article in press as: Houben, D., Sonnet, P. Impact of biochar and root-induced changes on metal dynamics in the rhizosphere of Agrostis capillaris and Lupinus albus. Chemosphere (2014), http://dx.doi.org/10.1016/j.chemosphere.2014.12.036