Biochar-Assisted Phytostabilization for Potentially Toxic Element Immobilization

: In response to the growing threat to the quality of the soil environment, new technologies are being developed to protect and remediate contaminated sites. A new approach, namely, assisted phytostabilization, has been used in areas contaminated with high levels of potentially toxic elements (PTEs), using various soil additives. This paper determined the effectiveness of biochar-assisted phytostabilization using Dactylis glomerata L. of soil contaminated with high concentrations of the selected PTEs (in mg/kg soil): Cu (780 ± 144), Cd (25.9 ± 2.5), Pb (13,540 ± 669) and Zn (8433 ± 1376). The content of the selected PTEs in the roots and above-ground parts of the tested grass, and in the soil, was determined by atomic absorption spectrometry (AAS). The addition of biochar to the contaminated soil led to an increase in plant biomass and caused an increase in soil pH values. Concentrations of Cu, Cd, Pb and Zn were higher in the roots than in the above-ground parts of Dactylis glomerata L. The application of biochar signiﬁcantly reduced the total content of PTEs in the soil after ﬁnishing the phytostabilization experiment, as well as reducing the content of bioavailable forms extracted from the soil using CaCl 2 solution, which was clearly visible with respect to Cd and Pb. It is concluded that the use of biochar in supporting the processes of assisted phytostabilization of soils contaminated with PTEs is justiﬁed. the control soil. The biochar most signiﬁcantly reduced the total Cu (14%), Cd (31%), Pb (19%) and Zn (23%) contents in the soil, and the bioavailable forms of Cd and Pb extracted from the soil using CaCl 2 solution, as compared to the control series. Higher total contents of Cu, Cd, Pb and Zn were observed in the roots of Dactylis glomerata L. than in its above-ground parts. The biochar contributed to an increase in the total content of PTEs such as Cu, Pb and Zn in the roots. The results indicate that the application of biochar to soils contaminated with the trace elements Cu, Cd, Pb and Zn is justiﬁed and can be a beneﬁcial element of remediation due to its very good immobilization properties and positive effect on the development of Dactylis glomerata L.


Introduction
Increasing industrialization and traffic activities, the amount of deposited precipitation and the increasing use of chemicals have resulted in environmental pollution on a global scale [1,2]. One particular group of pollutants includes potentially toxic elements (PTEs), which may remain in the environment for many years, changing only the form in which they occur [3][4][5][6][7]. High PTE concentrations lead to a reduction in the number of microorganisms and in soil enzyme activity, and poor plant growth and yield, combined with impaired water and mineral management and photosynthetic and respiratory activity of plants [8,9]. PTEs can be released into the soil by the weathering of parent rocks, by precipitation of atmospheric dust and rain and by means of various anthropogenic sources [10]. In the case of natural sources, PTE concentrations in soils are generally not high, do not pose a threat and are considered natural and referred to as geochemical background [11]. However, relatively large areas are represented by soils with a higher content of PTEs in relation to the geochemical background, which may include steel disposal dumps [12]. These areas are characterized by a distinctive feature as remnants of industrial activity, and they attract attention mainly because of their characteristic buildings and visible degradation in the form of physical deformation of the earth's surface [13]. These sites are used as repositories for metal production waste, various types of metal products and demolished elements of building structures or wrecked vehicles and machines. Soil from these areas are characterized by the presence of significant PTE contamination.
In situ immobilization of heavy metals is a technology with the potential to reduce the remediation costs of degraded sites and the adverse environmental impact [14]. The combination of the application of plants and organic additives (called the "assisted phytostabilization technique"), such as biochar, makes it possible to restore plant cover and increase water retention, thus reducing metal leaching into the soil profile [15]. The role of plants used in phytostabilization is primarily to protect the surface of contaminated soil from water and wind erosion and to reduce the amount of leachate produced after rainfall [16]. This is only possible if the plants can develop a dense cover and a deep and highly branched root system [17]. They should also demonstrate considerable tolerance to PTEs, intensive transpiration and high accumulation of PTEs in roots accompanied by a low accumulation in above-ground parts [18]. Grass species including Dactylis glomerata L. tested by the authors are used in the phytostabilization technique owing to their perennial growth cycle, branched root system and ability to stabilize the soil structure [19]. Furthermore, the rapid greening effect of the ground cover achieved by grass cultivation prevents PTEs from penetrating deep into the soil profile.
Apart from the effective immobilization of PTEs, soil amendments should be characterized by ease of transport, safety during incorporation into the soil, lack of toxicity to plants and high availability. The application of amendments to soil using the assisted phytostabilization technique aims at transforming the ionic and easily soluble forms of PTEs into poorly or very poorly soluble forms, which leads to a reduced bioavailability of metals [20]. Therefore, it reduces the leaching of PTEs into the soil profile and the uptake of PTEs by plants. The use of biochar can be an effective alternative to chemical soil additives [21]. Biochar is characterized by its strong structure-forming effect towards a higher porosity and a large surface area, its sorptive properties towards organic and inorganic compounds, its carbon content in a stable form, its presence of some mineral components including N, P, Ca, Mg and K and its ability to retain water as well as a much slower decomposition rate than the original biomass (wood, corn husks or straw, poultry or animal manure, etc.) [22]. The type of starting biomass and the temperature at which the biochar products are made are the two most important production factors [23]. The application of biochar to the soil increases soil moisture and fertility, improves its structure and physico-chemical properties, increases the capacity of the sorption complex and provides favorable living conditions for soil microflora and fauna [24].
In view of the need to develop environmentally friendly methods for immobilization of PTEs, research was conducted to determine the potential of assisted phytostabilization of soils contaminated with selected trace elements, namely, Cu, Cd, Pb and Zn, using Dactylis glomerata L. as a test plant. The effectiveness of biochar-assisted phytostabilization was determined based on the yield and PTE content in the roots and above-ground parts of Dactylis glomerata L., and the physico-chemical composition of the soil.

Study Area, Soil Sampling and Sample Preparation
The PTE-contaminated soil, further referred to as the initial soil, was sampled from a site located in northeastern Poland where metal waste has been dumped directly into the ground since 1946 (Figure 1). The site is situated at an average altitude of 121 meters. For most of the year, the area receives polar maritime air from the temperate climate zone. Annual precipitation reaches 600 mm. The average temperature in July is about 18 • C, and in January, it is about −4 • C. The average annual temperature is about 7.6 • C. A prevalence of south-westerly and westerly winds is observed. Soil samples were collected with a stainless-steel shovel from the topsoil layer (0-25 cm) following a spot diagnosis of the state of the soil environment in the site under analysis. At each sampling site, a composite soil sample (approximately 50 kg) was obtained by thoroughly mixing four subsamples. All soil samples collected were carefully transferred to clean polyethylene bags before being transported to the laboratory. The collected soil samples were air dried at room temperature and were then sifted through a 2 mm sieve and stored in a 4 • C refrigerator for use in the phytostabilization experiments.

Biochar Characterization
The biochar used in the phytostabilization experiment was purchased from the Fluid S.A. company (Sędziszów, Poland) and was obtained through thermolysis (thermal treatment) of a biomass feedstock (willow chips) at 650 • C for 15 min, at a heating rate of about 3 • C/s, under no oxygen conditions [25]. Before the experiment, this additive was milled in a RETSCH SM-100 cutting mill to obtain a particle size less than or equal to 0.5 mm. Scanning electron microscopy with energy-dispersive X-ray spectroscopy (SEM-EDS) was performed on an EO Electron Microscopy Ltd. (model 1430 VP, England, with acceleration voltages of 5 kV and 28 kV) for examination of biochar morphology and EDS elemental microanalysis ( Figure 2). The biochar was characterized by an alkaline pH (10.4 ± 0.3), electrical conductivity of 2.9 ± 0.1 mS/cm, cation exchange capacity of 48.59 ± 2.3 cmol/kg, surface area of 313.73 m 2 /g and pore volume of 0.04 cm 3 /g. The content of individual PTEs amounted to (mg/kg biochar): Cd-0.2 ± 0.05; Cr-9.6 ± 0.7; Cu-3.9 ± 6.3; Ni-10.2 ± 0.1; Pb-1.1 ± 1.6; Zn-200.2 ± 10.7, determined by the atomic absorption spectrometry (AAS) method using a Varian AA28OFS (Mulgrave, Australia) spectrophotometer, performed in triplicates.

Biochar-Assisted Phytostabilization Experiment
In order to determine the effectiveness of the biochar-assisted phytostabilization experiment, 5.0 kg polyethylene pots were used in five replicates. Biochar was applied to the initial soil at a rate of 3.0% (w/w), which corresponds to a dose of 10 t ha −1 , in order to obtain biochar-amended soils, while soils without amendments (0.0%, w/w) were designated as the control series. The sampled, chemically degraded initial soil was characterized by a low moisture content (9.5%, measured gravimetrically), an alkaline pH (7.2 ± 0.10) and a relatively high cation exchange capacity of 57.5 ± 0.12 cmol/kg, and it contained large amounts of PTEs such as Cu, Cd, Pb and Zn Based on a particle size analysis, the initial soil was classified as loamy sand (86.4% sand, 11.4% silt and 2.2% clay). The soil with biochar was thoroughly mixed, and the control pots were placed in a dark room for over two weeks to equilibrate the soil mixture. In the pot experiment (control and biochar amended), Dactylis glomerata L. cv. Berta, in the amount of 5.0 g per pot, was cultivated under natural day/night conditions. During the day (14 h), the air temperature was 26 ± 3 • C, and it was~10 • lower (16 ± 2 • C) at night (10 h), with a relative humidity of 75 ± 5%. The plants were watered every second day with distilled water to 60% of the maximum water holding capacity of the soil. The soil moisture content for each pot was maintained at field capacity every three days. The plants were harvested after 52 days of planting. For the plant growth assays, no fertilizer was applied to avoid the interaction of these chemicals with the biochar.

Soil and Plant Analyses
After transport to the laboratory, the chemically degraded initial soil was air dried at room temperature for two weeks. Its pH value was then determined in distilled water extracts (1:2.5 w/v) using a pH meter (HI 221, NY, USA). The particle size of the soil was determined using a Mastersizer 2000 particle size analyzer. The cation exchange capacity of the initial soil and biochar was calculated as the sum of hydrolytic acidity (in 1M Ca(CH 3 COO) 2 ) and exchangeable bases (in 0.1 M HCl) (see Sections 2.2 and 2.3).
To determine the total content of PTEs in the initial, control and biochar-amended soils, the samples were mineralized in a mixture of concentrated HCl, HNO 3 and H 2 O 2 in a microwave oven (MARSXpress, CEM, Matthews, NC, USA). PTE contents (Cu, Cd, Pb and Zn) were measured by the AAS method. The exchangeable soil metal fractions were determined using 0.01M CaCl 2 (1:10 soil-extractant ratio) after agitation for 2 h at 20 • C. The extract was separated from the solid residue by centrifugation for 15 min. The quality of the analyses was assessed using a reference material (CRM 142 R), and the obtained recoveries ranged from 95% to 101%.
After the end of the experiment, the above-ground parts and roots of Dactylis glomerata L. were washed in tap water and then deionized water and air dried at room temperature. Before the analysis, the plants were powdered using an analytical mill (Retsch, ZM300, Hann, Germany). The roots and shoots were oven dried at 55 • C until they were completely dry, and the dry biomass was recorded. The plant material was mineralized in HNO 3 p.a. and 30% H 2 O 2 using a microwave oven (MARSXpress, CEM, Matthews, NC, USA). After filtration, the digestion samples were adjusted to a 100 mL volume with ultrapure water (Milli-Q System, Kenilworth, NJ, USA). Extracts were analyzed for PTE concentrations determined by the AAS method. All AAS analyses for the initial, control and biocharamended soils and plants were performed in triplicates.

Statistical Analysis
The statistical analysis of the obtained data was conducted using Statistica 13.3 software. The data were analyzed using one-way analysis of variance (ANOVA) or (when the ANOVA assumptions were not met) the Kruskal-Wallis test by ranks as the statistical method. For the data with significant differences identified between variables, further analyses were conducted following the application of Tukey's test (HSD).

Dactylis Glomerata L. Biomass after Biochar Application
PTE immobilization is one of the important elements determining the effectiveness of assisted phytostabilization, and, for this purpose, it is necessary to obtain a dense and compact vegetation cover on the contaminated land surface [26]. However, soils degraded by elevated levels of PTEs, including the selected trace elements Cu, Cd, Pb and Zn, are characterized by a low organic matter content and microbial abundance, as well as a generally low plant nutrient availability [27,28]. The consequence is a poorer vegetation cover in comparison to unpolluted soils [29]. Therefore, in this study, biochar was introduced into the soil to enhance plant cover development, which, as can be seen in Figure 3a, significantly (p < 0.05) differentiated the yield of Dactylis glomerata L. The results confirm the lower yield of the test plant in the control series (without biochar), which can be explained by the occurrence of increased contents of Cu, Cd, Pb and Zn in the soil. This relationship correlates with the results reported by Wyszkowski and Radziemska [30], and Shah and Daverey [31], who found that a lower level of biomass was observed at high PTE concentrations in the soil. It is worth emphasizing that the degree of tolerance of plants to excessive levels of PTEs in the soil depends primarily on the plant species, the soil pH value and its granulometric composition and the organic matter content [32,33]. The use of soil amendments such as biochar has a beneficial effect on the use of the ingredients contained in them, which can lead to an increase in plant biomass [34]. It is believed that the use of biochar leads to an increase in the content of many components (such as Corg., N, P, Ca, Mg and K), which significantly affects crop yields [35]. This was confirmed in research conducted in which the application of biochar resulted in a 28% increase in the yield of Dactylis glomerata L. as compared to the control series. Gonzaga al. [36] observed a significant increase in the yield of Brasica juncea L. following soil application of biochar from coconut husks and orange shells after the experiment. Radziemska et al. [12] also found that biochar from willow chips is an additive that is effective in increasing Lupinus luteus L. biomass.

Soil pH after Biochar Application
An important factor in the increase in PTE toxicity in soils is the pH [37]. An increase in the PTE share in the biogeochemical cycle is largely related to the acid pH of the soil [38]. Therefore, treatments to raise the pH value of the soil are a key element that affects PTE mobility and bioavailability. Biochar generally has a neutral or slightly alkaline pH, and its alkalinity increases with the pyrolysis temperature, typically carried out at 300 up to 600 • C, under little or no oxygen conditions [39]. The literature abounds with studies showing that biochar, as a soil additive, has the ability to raise the soil pH (that is, to neutralize its acidity) [40][41][42][43]. It has also been demonstrated that biochar contains many alkaline substances, including calcium carbonate in amounts ranging from 0.5% to 33%, and thus can increase the pH of soils [44]. The biochar used in the current experiment significantly increased the pH value (by 1.18 units) of the soil in comparison to the control soil in which this additive was not applied (Figure 3b). This tendency was previously confirmed in the research by Radziemska et al. [12], in which the addition of biochar to soil contaminated with PTEs also contributed to a significant increase in soil pH values. Yiang et al. [45] also observed that the addition of corn straw biochar to uncontaminated soil increased its pH.

The Total Content of Cu, Cd, Pb and Zn in Soil after Biochar-Assisted Phytostabilization
Excessive concentrations of PTEs are typical of soils from areas where various types of waste, including metal, are deposited. This phenomenon has been confirmed in the available literature [12,46,47] as well as in the results obtained during the current experiment. The PTE content in the soil before and after the establishment of the experiment is shown in Figure 4. The initial soil was characterized by higher PTE concentrations compared to the classification provided by the Regulation of the Polish Ministry of the Environment, Poland [48]. Compared with the ranges we obtained, it can be observed that the Cu content was exceeded by 24%, Cd by 41%, Pb by more than 20 times and Zn by more than 6.5 times. Among numerous technologies enabling the immobilization of PTEs in soils, phytostabilization is one of the most environmentally friendly. Soil amendments are used to enhance and strengthen the PTE immobilization effect in the soil. The presented results demonstrate that biochar application significantly affected the effectiveness of Cu, Cd, Pb and Zn immobilization in the soil. This may be due to the large specific surface area of these amendments and the occurrence of liming and adsorption effects. The latter phenomenon may include precipitation, co-precipitation, a reduction in metal species, complexation with functional groups, cation exchange and attractive electrostatic interactions [49]. As a result of the conducted experiment, significantly decreased total Cu (14%), Cd (31%), Pb (19%) and Zn (23%) concentrations in the soil were found after biochar application as compared to the control series. The effectiveness of PTE immobilization following the introduction of biochar into the soil was also confirmed by other studies. Jiang et al. [50] analyzed the effect of the introduction of rice straw biochar to soils contaminated with PTEs and proved that the Cu and Pb contents were 19% lower and 18% lower, respectively, than those in the control soils. Karim et al. [42] also proved that biochar significantly reduced Cu and Pb concentrations in their soils. In research conducted by Radziemska et al. [12], a significant reduction in the Cu, Ni, Cd, Pb, Zn and Cr content was observed in soil contaminated with high concentrations of these PTEs after the application of willow chip biochar. Figure 4. The total PTE content in the soil before beginning and after finishing the phytostabilization experiment. For a given PTE, different letters (a, b, c) indicate significant differences in the PTE content in the soil before the experiment (initial), and after (control and biochar-amended soils). ANOVA followed by Tukey's honest significant difference test, p < 0.05.

The CaCl 2 -Extractable Content of Cu, Cd, Pb and Zn in Soil after Biochar-Assisted Phytostabilization
The potential PTE availability to plants and mobility in the soil depend on their total concentration in the soil as well as their exchangeable bioavailable forms [51]. Substantially, the bioavailability of PTE exchangeable forms determines the toxicity of the soil and potential risk in entering the human food chain via groundwater. Biochar has good adsorptive removal efficiencies in single-metal systems but lower capacities in multiple-metal systems due to the competition between the PTE exchangeable forms present in the soil. Research found in the literature shows that competitive adsorption studies for biochar are necessary for an accurate estimation of adsorption mechanisms in natural environments [52][53][54]. Herein, experimentally determined concentrations of Cu, Cd, Pb and Zn as bioavailable forms in treatments using calcium chloride solution in control and biochar-amended soils were significantly lower than the determined PTE total contents. The CaCl 2 -extractable PTE concentrations in the soil, with and without biochar application, are presented in Figure 5. The application of biochar in the soil reduced the CaCl 2 -extractable contents of Cu, Cd, Pb and Zn by 18.2, 50, 37.5 and 14.3%, respectively, as compared to the control series. This result indicates that the forms of metals released during extraction of the CaCl 2 solution correspond to small changes in environmental conditions caused by biochar application to the soil; thus, the possibility of extraction using CaCl 2 solution allows for determining the current availability of metals, assuming that the time of 2 h was the optimal contact time to reach an extraction equilibrium in order to further consider the adsorption effect on the biochar. In adsorption studies, a likely explanation for the higher molar sorption capacity of metal ions into the solid surface (of various carbon-type materials) is the atomic radii of the adsorbed ions. Due to steric over-crowding, a larger ionic radius compared to a smaller radius is expected to induce a quick saturation of adsorption surface sites, resulting in lowering the molar sorption capacity, assuming the same ion concentration is present in the starting solution. Thus, the expected metal adsorption sequence is as follows: Zn (74 pm), Cu ((I) 77pm and (II) 73 pm), Cd (95 pm), Pb (119 pm) [55]. Moreover, in the case of the same valence, the metal adsorption sequence may follow the same order as the electronegativities, which are Pb (2.33), Cu (1.90), Cd (1.69) and Zn (1.65) [56]. Herein, two of the extracted PTE metals ions, that is, Cd and Pb, showed a much higher relative concentration decrease than the other two, Cu and Zn, in the CaCl 2 solution, probably resulting in a much greater adsorption ability of the biochar, which, conceptually, could be attributed to the presence of a specific active site on the surface area of the biochar used, which is retained by sorption of the selected metals [57][58][59]. This would explain the higher concentrations of the smaller ions Cu and Zn than the larger ions Pb and Cd in the extracted CaCl 2 solutions.

Content of Cu, Cd, Pb and Zn in Roots and Above-Ground Parts of Dactylis glomerata L.
Elevated levels of PTEs in vegetation growing in polluted areas may be detrimental due to the risk of PTEs by means of biomagnification. Assisted phytostabilization immobilizes potentially harmful PTEs in the plant root zone [60]. The results of the Cu, Cd, Pb and Zn contents in the roots and above-ground parts of Dactylis glomerata L. after the experiment with biochar-assisted phytostabilization are presented in Figure 6. Analysis of the literature indicates that PTEs taken up from the soil proportionally with an increasing soil content accumulate more in the roots than in the above-ground parts [9,61]. This relationship was also confirmed in the present study, in which the PTEs under analysis accumulated mainly in the roots of the test plant, especially when biochar was applied to the soil. After the biochar-assisted phytostabilization experiment, a higher concentration of PTEs in the roots of Dactylis glomerata L. as compared to the above-ground parts was most evident for Cu (more than 20-fold), Pb (35-fold) and Zn (43-fold). The studies available in the literature clearly show that the addition of biochar leads to the immobilization of PTEs in the soil, which, at the same time, leads to a decrease in the concentration of these elements in the above-ground parts of plants [62,63]. Radziemska et al. [12], comparing the Ni and Zn contents in the roots of Lupinus luteus L. after biochar application, found a significantly higher accumulation of these PTEs compared to the control. This can be explained by the fact that biochar can lead to immobilization of PTEs in soils while improving soil quality and can significantly reduce the uptake of PTEs by plants [64]. The results of the research by Zhang et al. [64] suggested that the accumulation of Zn, Cd and As in the roots of Festucaarun dinacea L. was significantly increased by adding 2% sawdust biochar. Karami et al. [60] observed that Cu amounts in the above-ground parts of Festuca rubra L. were significantly lower after biochar application. Figure 6. The PTEs in the above-ground parts and roots of Dactylis glomerata L. after finishing the phytostabilization experiment. For a given PTE, different letters (a, b, c) indicate significant differences in its accumulation in the above-ground parts or in the roots of Dactylis glomerata L. ANOVA followed by Tukey's honest significant difference test, p < 0.05.

Conclusions
Two of the key elements of biochar application as a soil amendment in assisted phytostabilization of PTE-contaminated soils are PTEs' immobilization, and enhancement of plant cover growth at degraded sites. As a result of the conducted research, it was confirmed that biochar is effective in developing Dactylis glomerata L. biomass since the yield of this plant was 28% higher after the assisted phytostabilization process. The application of biochar also contributed to a significant increase in the soil pH value, which, in this case, was 1.18 units higher for the biochar-amended soil in comparison to the control soil. The biochar most significantly reduced the total Cu (14%), Cd (31%), Pb (19%) and Zn (23%) contents in the soil, and the bioavailable forms of Cd and Pb extracted from the soil using CaCl 2 solution, as compared to the control series. Higher total contents of Cu, Cd, Pb and Zn were observed in the roots of Dactylis glomerata L. than in its above-ground parts. The biochar contributed to an increase in the total content of PTEs such as Cu, Pb and Zn in the roots. The results indicate that the application of biochar to soils contaminated with the trace elements Cu, Cd, Pb and Zn is justified and can be a beneficial element of remediation due to its very good immobilization properties and positive effect on the development of Dactylis glomerata L.