1 Introduction

Heavy metal (HM) pollution has become a serious problem worldwide. The typical soil around nonferrous metal mining areas is liable to become seriously polluted by HMs emitted from the discharge of waste gas, water and residue during mining and smelting. According to a soil pollution survey by the Chinese government in 2014, in 16.1% of the sites nationwide, the soil was polluted, and HMs accounted for 82.8% of the pollution (MEP of China, 2014). HMs in soil can ultimately enter the human body through the food chain, causing high carcinogenic and noncarcinogenic risks to the public, especially to children and those living in severely contaminated regions (Li et al. 2014). Therefore, for the sake of food safety, human health, and sustainable development, it is necessary and urgent to remediate contaminated soils and control the ecological risk caused by HMs.

Compared with conventional physical and chemical remediation techniques, phytoremediation has been well recognized to decontaminate HMs in large areas due to advantages such as eco-friendliness, lower cost, in situ restoration of the biodiversity and soil functions (Ali et al. 2013; Gavrilescu 2022). Phytoremediation using fast-growing woody plants is one of the crucial measures to rapidly restore the vegetation of severely polluted soils and effectively inhibit the horizontal/vertical migration of soil HMs (Landberg and Greger 2022). Willow has been suggested as a superior phytoremediation candidate because of its fast growth, large biomass, developed root system and high tolerance and accumulation of multiple HMs (Wani et al. 2020; Unterbrunner et al. 2007).

Soil severely contaminated by HMs in mining area is often characterized by high levels of metals, abnormal pH, lack of nutrient elements, poor soil structure and other obstacles that seriously inhibit plant growth, resulting in a decreased plant survival rate, limited growth and weakened remediation efficiency (Tőzsér et al. 2018). For instance, El-Mahrouk et al. (2019) found that Salix mucronata might die when the concentrations of Cd, Cu and Pb in soil exceeded 80, 200, and 850 mg/kg, respectively, because its nutrient uptake was reduced and the antioxidant enzyme system was seriously damaged by oxidative stress under high HM contents. Moreover, due to the poor nutrient content of mining-contaminated soils, the growth rates of three Salix species (S. alba, S. viminalis and S. purpurea) on Pontgibaud technosol were 5.6, 4.4, and 4.3 times lower than those on garden soil, respectively (Lebrun et al. 2017). It is necessary to develop technology that can quickly reduce the toxicity of soil HMs, improve the overall physicochemical properties of soil, and promote the stable growth of plants and efficient recovery of vegetation in mine soil (Radziemska et al. 2019).

Biochar has been suggested as a promising soil amendment to decrease the mobility and bioavailability of HMs through HM precipitation, complex and ion exchange, and nutrient mineral supply and improve soil quality in mining areas (He et al. 2019; Chen et al. 2022). The stabilization of soil HMs by biochar is determined by feedstocks and their particle size. Compared with plant-derived biochar, bone biochar prepared from animal bones and other wastes is rich in carbon and oxygen-containing functional groups but also contains abundant hydroxyapatite (Ca10(PO4)6(OH)2; HAP) with a porous structure and nutrients such as calcium, phosphorus, and nitrogen (Vamvuka et al. 2018; Xiao et al. 2020). Bone biochar demonstrated a better passivation effect on HMs in soil and improved soil quality. Abdin et al. (2020) found that 1.5–3 wt% fishbone biochar greatly reduced the contents of Cd, Pb, Zn and Cu in water and soil environments.

Biochar with a smaller particle size exhibited a better stabilization effect on soil pore water and soil HM bioavailability than biochar with a larger particle size while increasing the amount of HM accumulation despite the insignificant effect on Salix growth. Lebrun et al. (2018a; 2018b) found that biochar with a smaller particle size was more effective in stabilizing Pb in the pore water than hardwood biochar with a larger particale size (< 0.1 mm, 0.2 ~ 0.4 mm, 0.5 ~ 1 mm and 1 ~ 2.5 mm) in Pb contaminated mining areas, while it had no effect on most of the plant growth parameters under addition 2% and 5% biochar. The accumulation of Pb by Salix viminalis was highest in the treatment of biochar with size of 1 ~ 2.5 mm, while Pb accumulation was significantly reduced in biochar treatment with size < 0.1 mm; Meanwhile, 3% bamboo biochar with different particle sizes (P1 < 0.149 mm, 0.149 mm < P2 < 0.25 mm, 0.25 mm < P3 < 0.5 mm) had no significant effect on bioavailable Cd in severely contaminated soil (~60 mg/kg), while the highest biomass, tissue Cd level and Cd amount in Salix psammophila were observed at treatment with biochar size of P2 (Li et al. 2021a). The addition of 1% to 5% bamboo biochar demonstrated little effect on plant growth, whereas rising the application mate to 7% significantly decreased biomass compared to the control (Li et al. 2021b). What’s more, the soil characteristics such as hetero aggregation and soil evaporation, which are dependent on the interaction of soil texture and biochar particle size, i.e., hetero aggregation, may occur between negatively charged nano biochar and the positive charged minerals in soil system due to the electrostatic interactions (Zhang et al. 2022). Wang et al. (2018) also found that larger biochar particles (0.25 ~ 2 mm) are better in reducing soil evaporation than smaller particles (< 0.25 mm). However, the effect of bone biochar and its size and application on Salix growth and HMs phytoextraction efficiency in clay soil heavily contaminated by HMs are not fully understood.

The present study explores the influence of bone biochar on Salix survival, growth and metal accumulation under different particle sizes and application rates in severely HM-contaminated clay soil. The objectives of the present study are (1) to explore the influence of bone biochar (BC) and ball-milled bone biochar (MBC) on HM bioavailability and soil physicochemical properties; (2) to investigate the effect of BC and MBC on the survival, growth, physiological response, and HM accumulation in Salix; and (3) to discuss the possible synergistic effect of BC/MBC and Salix on the remediation efficiency of heavily contaminated acid clay soil.

2 Materials and methods

2.1 Soil and biochar preparation

The clay soil (pH =5.6 ± 0.05) was collected from Fuyang District, Hangzhou, Zhejiang Province, which belonged to the typical clay soil in southern China with 37.3% silt, 44.2% clay, and 18.5% sand according to the USDA soil textural classes. The HMs, including Cd, Mn, Cu and Pb, were spiked in the soil and aged more than two years, with determined levels of the total contents: 1.65 ± 0.06 mg/kg, 1100.69 ± 32.20 mg/kg, 285.88 ± 8.48 mg/kg and 572.76 ± 16.52 mg/kg, respectively. The soil was heavily contaminated with Pb, Cu and Cd according to the soil environmental quality risk control standard for soil contamination of agricultural land (GB15618-2018) with the risk intervention value of Pb 500.0 mg/kg at 5.5 < pH ≤ 6.5 and the risk screening values Cu 50.0 mg/kg and Cd 0.3 mg/kg at 5.5 < pH ≤6. The natural air-dried soil was crushed and sieved through a 2-mm mesh screen to remove stones and other impurities.

The bone biochar (BC) and ball milled bone biochar (MBC) were prepared and described in detail in our previous work, which showed excellent adsorption capacities for HMs in solution (Xiao et al. 2020). Specially: (1) BC preparation: The cow bone meal was air-dried for 24 h at 80℃ and then pyrolyzed in a tube furnace with continuous N2 flow of 200 mL/min at 600℃ for 2 h. The obtained bone biochar was rinsed with DI water several times, dried at 80℃ for 12 h, and passed through a 200-mesh sieve; (2) MBC preparation: 3.30 g bone biochar, 330 g agate spheres and 60 g DI water were added to the agate jars, maintaining a mass ratio of agate spheres to bone biochar of 100:1. The ball milling machine was operated at a speed of 300 rpm for 12 h, and the direction of rotation was changed every 3 h. The MBC were collected after centrifugation for 5 min at 9000 rpm, dried at 80℃ for 12 h in the oven, ground and stored prior to use. The application rates of BC and MBC were 0 wt%, 0.5 wt%, 1.0 wt%, 2.0 wt% and 4.0 wt% (biochar to soil), which were labeled CK, B1, B2, B3, B4 and MB1, MB2, MB3, MB4, respectively (Gregory et al. 2014). Each treatment is consisted of three replicates.

The surface texture, morphology, elements-Mapping of BC and MBC were analyzed via Scanning electron microscopy-Energy dispersive spectroscopy (SEM-EDS, FEI Quanta 200FEG, Thermo scientific Quanta, USA). The BET specific area measurements were measured by a 3H-2000PM (Beishide Instrument Technology (Beijing) Co., Ltd.) at 77 K. The total surface area and average diameter and total pore volume of BC and MBC were calculated by multipoint Brunauer-Emmett-Teller (BET) method, T-Plot method and Barret-Joyner-Halenda (BJH) method, respectively.

2.2 Pot experiments

Salix jiangsuensis 172 (SJ-172) was selected as the test plant according to our previous work and displayed an outstanding capacity for HM accumulation (Cao et al. 2018). Plastic tubs (10 cm in diameter and 15 cm in height, each holding 1.10 kg soil) were used for planting. SJ-172 cuttings (1 year old, 10 cm in length and 0.5 cm in diameter) were planted and cultivated for 5 months (May to October 2019). One pot is set as a replicate, and three replicates were set for each treatment. The experiments were carried out in a greenhouse (temperature: 23–28 °C and mean relative humidity: 60–65%). The maximum water holding capacity of the tested soil was measured prior to experiment as described by Wilcox (1939). During the pot trial period, 200 mL tap water was used every 2 days to maintain the moisture of approximately 70% of the soil water holding capacity.

2.3 Physiological traits

The content of soluble protein in the leaves was determined by the Coomassie Brilliant Blue method (Xu et al. 2019). The concentrations of superoxide anion (O2∙−) in leaves were determined at 530 nm according to the modified protocol suggested by Ma et al. (2014). The malondialdehyde (MDA) content was analyzed at 450, 532 and 600 nm using a spectrophotometer (Lei et al. 2007). For antioxidant activity, crude enzymes were obtained from 1.0 g of leaves crushed with a 0.1 M Na2HPO4–KH2PO4 buffer solution at pH 7.0. The activities of the enzymatic antioxidants, including superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD), were determined according to Hashem (2014).

2.4 Plant harvest and analysis

The plants were harvested after recording the plant height and diameter. The leaves were sampled from three replicates for physiological testing. The plants were divided into stems, leaves, roots and cuttings and rinsed with deionized water, and the roots were washed with 0.2 mmol/L EDTA, oven-dried at 105 ℃ for 1.0 h and subsequently oven-dried at 70 ℃ to achieve a constant weight. The obtained samples were crushed and sieved (0.25-mm mesh) before chemical analysis. A portion of 0.10 g ground samples was digested with 6 mL HNO3 at 120 °C for 45 min and then 2.0 mL H2O2 for 30 min in a hot block system (ED36, Lab Tech, Germany). The concentrations of HMs (Cd, Mn, Cu and Pb) and mineral elements in solution were determined by induced coupled plasma–mass spectrometry (ICP‒MS; Agilent 7700x, USA).

2.5 Soil analysis

After plant harvest, the soils in each pot were collected, homogenized, air-dried, and ground for further chemical analysis. The soil organic carbon (SOC) content and pH were determined according to methods described by Lu (2000). Available K (AK) was extracted using an ammonium acetate solution (Lu 2000). The soil available N (AN) was extracted using a microdiffusion technique, and total N (TN) was measured according to the method suggested by Lu (2000). Soil available P (AP) was determined using a spectrophotometer after extraction with NaHCO3 solution (Lu 2000). The bioavailable HMs were extracted by 0.1 mol/L HCl, and the total HMs, total P (TP) and total K (TK) were determined by digesting the soils with HCl–HNO3 (mixture with 3:1 v/v ratio) using a hot block system (ED36, Lab Tech, Germany). The total and bioavailability concentrations of Cd, Mn, Cu, Pb, TP and TK were determined by ICP‒MS (Agilent 7700x, USA).

The modified European Community Bureau of Reference (BCR) continuous extraction method was applied to extract the chemical fraction of HMs (Rauret et al. 1999), namely, exchangeable, and weakly acid-soluble HMs (weak acid soluble state, F1), iron-manganese oxide-bound HMs (reducible state, F2), organic matter and sulfide-bound HMs (oxidizable state, F3) and HMs bound in the crystal lattice of soil minerals (residual state, F4).

2.6 Calculation and statistical analysis

The translocation factor (TF) was calculated to indicate the plant’s ability to translocate HMs from roots to aerial parts (Antoniadis et al. 2017):

$$TF = {C}_{aerial\;parts}/{C}_{roots}$$
(1)

where Caerial parts and Croots are the HM concentrations in the aboveground tissues (including leaves, stems and cuttings) and roots, respectively.

All data were statistically analyzed using Data Processing System version software (DPS13.01, Zhejiang University, Hangzhou, China) by two-way analysis of variance (ANOVA) followed by the least significant difference (LSD) test at a significance level of 0.05. The figures were plotted with Origin 2018 (OriginLab Corporation, Northampton, MA, USA).

3 Results

3.1 BCs characterizations, soil physicochemical properties and HM fractions

As depicted in the SEM patterns in Fig. 1 (a and b), the obvious change can be seen in the apparent morphology and particle size of the bone biochar before and after ball milling. The MBC showed small and homogeneous particles, while the BC demonstrated larger geometric irregularities. As shown in Fig. 1 (c and d), the specific surface area and average particle sizes of BC and MBC were 52.78 m2/g, 313.09 m2/g and 8.67, 6.46 nm, respectively, indicating that the wet ball-milling greatly improved more available active sites toward HMs (Xiao et al. 2020). The reduction of average particle sizes may attribute to the increase in the number of micro-pores of BC after ball milling. However, due to some mineral elements released into aqueous solution during the wet ball-milling, the concentration of mineral nutrients in BC and MBC such as K, Mg, and Na were decreased form 0.13, 0.18, 0.78 wt.% to 0.06, 0.07, 0.42 wt.%, respectively (Xiao et al. 2020). For the BC composition, EDX-Mapping and analysis showed that the bone biochar material contained main elements such as C, N, O, P and Ca (Fig. 1 e - l). In addition, the digital photographic images of the soil mixed with different ratios of BC and MBC showed in Fig. 1(m). The color of the soil became darken after MBC addition and became darker when rising the MBC dosage; unlike MBC, adding 0.5—4.0% BC displayed no significant change in soil color compared to the control.

Fig. 1
figure 1

the SEM images of BC (a) and MBC (a), (c) Low-temperature N2 adsorption-desorption isotherms of BC and MBCs; (e ~ k) Elemental mapping of the homogeneous dispersion of C, N, O, P and Ca elements in the as-prepared BC; (l) SEM-EDS spectra analysis of BC; (m) Digital photos of the mixture of BC, MBC and acidic HMs contaminated clay soil under different treatments. CK – 0%; B1 – 0.5% BC; B2 – 1.0% BC; B3 – 2.0% BC; B4 – 4.0% BC; MB1 – 0.5% MBC; MB2 – 1.0% MBC; MB3 – 2.0% MBC; MB4 – 4.0% MBC

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As shown in Table 1, BC and MBC significantly increased soil pH and showed a dose-dependent effect. Compared with the control (pH =5.93), the soil pH was significantly elevated to 6.86 and 6.63 at 4.0% BC and MBC (P < 0.05), which increased by 15.68% and 11.80%, respectively. Moreover, the SOC also gradually increased when the doses of BC and MBC increased, and the SOC increased by 299.63% and 479.78% at 4.0% BC and MBC, respectively, compared with the control.

Table 1 Effects of BC and MBC on physicochemical properties of soils
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Soil nutrients, including TN, TP, HN and AP, were also improved by BC and MBC application. The TN and TP were significantly increased by 42.35–340.0% and 31.76–382.35% and 153.17–856.14% and 246.78–1199.89%, respectively, for BC and MBC at application rates of 0.5–4.0% compared with the control (P < 0.05). However, the BC treatments did not change HN, while MBC at 2.0–4.0% significantly increased HN by 23.75–35.11% (P < 0.05). BC and MBC showed little effect on TK while significantly increased the AK contents by 18.42–196.62% and 3.38–61.47%, respectively (Table 1).

BC and MBC application enhanced HM immobilization by considerably decreasing the percentage of the F1 fraction while enhancing the percentage of the F3 and F4 fractions of Cd, Mn, Pb and Cu (Fig. 2). The stabilization effect of MBC on soil HMs was significantly better than those of BC. For example, the F1 fraction of Pb decreased from 9.32% (control group) to 3.77% and 0.59%, the F2 fraction of Pb decreased from 84.92% to 82.01% and 79.09%, and the F3 and F4 fractions of Pb increased from 3.76% to 5.45% and 7.46% and from 1.99% to 8.75% and 12.85% at 4.0% BC and MBC, respectively.

Fig. 2
figure 2

The HMs fraction in the soil: (a) Cd, (b) Mn, (c) Pb and (d) Cu. Different letters indicate significantly different values among the treatments and between the species (P < 0.05). CK – 0%; B1 – 0.5% BC; B2 – 1.0% BC; B3 – 2.0% BC; B4 – 4.0% BC; MB1 – 0.5% MBC; MB2 – 1.0% MBC; MB3 – 2.0% MBC; MB4 – 4.0% MBC

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The bioavailable content of HMs in acidic clay soil was considerably decreased by BC and MBC application (Fig. 3). Mn bioavailability in the acidic soil was dramatically decreased by BC and MBC compared with the control (P < 0.05). The bioavailable concentrations of Cd, Pb and Cu were reduced by 21.72.0% and 25.65%, 50.0% and 72.63%, and 25.81% and 53.87% at 4.0% BC and MBC compared with the control, respectively. MBC demonstrated better effects than BC in reducing the bioavailable concentrations of Cd, Pb and Cu in soil.

Fig. 3
figure 3

The effect of BC and MBC on the bioavailability of HMs: a Cd, b Mn, c Pb and d Cu. T, bone biochars dosage; S, bone biochar sizes; ns, not significant; *: p < 0.05; **: p < 0.01; ***: p < 0.001. Values present mean ± stand deviation (n=3). Different letters indicate significantly different values among the treatments and between the species (P < 0.05). CK – 0%; B1 – 0.5% BC; B2 – 1.0% BC; B3 – 2.0% BC; B4 – 4.0% BC; MB1 – 0.5% MBC; MB2 – 1.0% MBC; MB3 – 2.0% MBC; MB4 – 4.0% MBC

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3.2 Plant growth and biomass yield

BC and MBC had a large impact on the growth of SJ-172 in soil severely contaminated by HMs (Fig. 4). Interestingly, the cuttings of SJ-172 failed to survive in the pot trial, with a survival rate of 0% in the control and treatments with 0.5% BC and MBC (Fig. 4a), while the addition of BC and MBC at an application rate of 1.0 – 4.0% considerably increased the cutting survival rates to 100%. The plant growth parameters, including plant height and diameter, were improved by 1.0–4.0% BC and MBC (Fig. 4b and c). Compared with the 1.0% BC treatment, the plant height (60.59 cm) and stem diameter (2.17 mm) increased by 164.15% and 261.45%, respectively, in the 4.0% BC treatment (P < 0.05). In contrast, the plant height (52.48 cm) and stem diameter (1.63 mm) were significantly increased by adding 2.0% MBC (P < 0.05), while the 4.0% MBC treatment showed an insignificant effect compared to the 1.0% MBC treatment. BC (4.0%) was 16.15%, 31.76% and 24.88%, 19.81% superior to MBC (2.0%) and MBC (4.0%) in plant height and stem diameter of SJ-172, respectively.

Fig. 4
figure 4

The effect of BC and MBC on growth of SJ-172: a plant survival rate, b plant height, c stem diameter and the total dry weight (DW) of plant (d), the aboveground the plant (e) and the underground the plant (f). T, bone biochars dosage; S, bone biochar sizes; ns, not significant; *: p < 0.05; **: p < 0.01; ***: p < 0.001. Values present mean ± stand deviation (n=3). Different letters indicate significantly different values among the treatments and between the species (P < 0.05). CK – 0%; B1 – 0.5% BC; B2 – 1.0% BC; B3 – 2.0% BC; B4 – 4.0% BC; MB1 – 0.5% MBC; B2 – 1.0% MBC; B3 – 2.0% MBC; B4 – 4.0% MBC

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Applying 4.0% BC (6.34 g) significantly increased the total biomass by 223.24% compared to 1.0% BC (2.83 g) (P < 0.05), while 2.0% and 4.0% MBC (4.32 g and 4.31 g) significantly increased the total biomass by 89.47% (P < 0.05) (Fig. 4d). The total biomass of SJ-172 was 46.76% higher in the BC (4.0%) treatment than in the MBC (4.0%) treatment. The pattern of variation in root biomass and aboveground biomass was consistent with that of total biomass of SJ-172 in the treatments with 1.0–4.0% BC and MBC (Fig. 4d and e). Compared with MBC under the same dose, the root biomass of SJ-172 increased by 162.50%, 55.55% and 132.78% when BC was applied at 1.0%, 2.0% and 4.0%, respectively. In Fig. 4d, the biomass of the plant at 4.0% MBC showed no significant  changes compared with 2.0% MBC (P > 0.05), while the dry weight of the root of the plant at 4.0% MBC significantly decreased compared with 2.0% MBC (P < 0.05) (Fig. 4f). Thus, BC demonstrated a more promising effect than MBC on improving the soil quality of SJ-172 when growing in multimetal-polluted acid clay soil.

3.3 Physiological response of plants

The elevated doses (2%, 4%) of BC and MBC showed inhibitory effects on the production of soluble protein content in SJ-172 leaves compared with the 1% dose, and the inhibitory effect of MBC was more evident (Fig. 5a). High levels of HMs in soil induced the generation of O2–, the addition of 1.0–4.0% BC and MBC reduced the content of O2– in leaves (Fig. 5b), suggesting that BC and MBC can effectively alleviate the production of O2– in SJ-172. To scavenge the excess O2–, and the antioxidant system was triggered. The application of 1.0–4.0% BC and MBC first increased the SOD activity when it was below 2% and then decreased it at 4.0% (Fig. 5d), while it significantly improved the CAT and POD activities in SJ-172 (Fig. 5e and f) (P < 0.05). The addition of 1.0–4.0% BC and MBC had an inhibitory effect on the generation of MDA in SJ-172 leaves, and the effect became greater with increasing application rates, indicating that BC and MBC have a mitigating effect on the generation of MDA in SJ-172 (Fig. 5c). Moreover, applying MBC reduced the MDA content more effectively than applying BC at the same doses.

Fig. 5
figure 5

The effect of BC and MBC on the physiological characters of SJ-172 leaves: a soluble protein, b O2.-, c CAT, d SOD, e POD and f MDA. T, bone biochars dosage; S, bone biochar sizes; ns, not significant; *: p < 0.05; **: p < 0.01; ***: p < 0.001. Values present mean ± stand deviation (n=3). Different letters indicate significantly different values among the treatments and between the species (P < 0.05). CK – 0%; B1 – 0.5% BC; B2 – 1.0% BC; B3 – 2.0% BC; B4 – 4.0% BC; MB1 – 0.5% MBC; B2 – 1.0% MBC; B3 – 2.0% MBC; B4 – 4.0% MBC

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3.4 Mineral elements in plants

The contents of mineral elements in different organs of SJ-172 under BC and MBC treatments are listed in Table 2. The contents of Ca, P, Mg, K, S and Fe in all tissues in the MBC treatments were higher than those in the BC treatments under the same dose except for K and Fe in the leaves and roots. The leaf K content in the BC treatments was higher than that in the MBC treatments. MBC demonstrated a better promotion effect on P uptake in various tissues of SJ-172 than BC. The P content in each willow organ also increased with increasing application rates. The Mg content in all tissues in the MBC treatments was higher than that in the BC treatments.

Table 2 The effect of BC and MBC on the mineral nutrient elements in SJ-172
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3.5 Heavy metal accumulation and distribution in plants

The metal levels and distribution varied among element species and treatments (Table 3). The levels of Cd and Mn in leaves of SJ-172 were higher than those in roots, cuttings, and stems, while the levels of Cu and Pb in roots were much higher than those in leaves, cuttings, and stems. The aboveground Cd content was higher in the MBC treatments than in the BC treatments at the same application rates, while the aboveground Mn content in SJ-172 was higher in the BC treatments than in the MBC treatments at the same application rates. The Pb and Cu contents in the roots and cuttings of SJ-172 were significantly higher in the BC treatments than in the MBC treatments (P < 0.05). Conversely, the Cu contents in leaves and stems of SJ-172 in the MBC treatments were higher than those in the BC treatments at the same dose. Fig. 6 also suggests that Cd and Mn are more concentrated in the aboveground parts of plants, while Cu and Pb are more likely to accumulate in roots. BC amendment displayed better results than MBC amendment in promoting the total accumulation of Cd, Mn, Pb and Cu in SJ-172. The total accumulation of Cd, Pb, Mn and Cu in SJ-172 with 4.0% BC was significantly higher (115.23%, 161.82%, 285.23% and 219.29%, respectively) than that in SJ-172 with 4% MBC (P < 0.05).

Table 3 The effect of BC and MBC on the HMs concentration in various tissues of SJ-172
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Fig. 6
figure 6

The effect of BC and MBC on the accumulation of HMs of the SJ-172: a Cd, b Mn, c Pb and d Cu. T, bone biochars dosage; S, bone biochar sizes; ns, not significant; *: p < 0.05; **: p < 0.01; ***: p < 0.001. Values present mean ± stand deviation (n=3). Different letters indicate significantly different values among the treatments and between the species (P < 0.05). CK – 0%; B1 – 0.5% BC; B2 – 1.0% BC; B3 – 2.0% BC; B4 – 4.0% BC; MB1 – 0.5% MBC; B2 – 1.0% MBC; B3 – 2.0% MBC; B4 – 4.0% MBC

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BC and MBC application promoted the translocation of Cd from roots to shoots (Fig. 7). The TFs of Cd in the 4.0% BC and 4.0% MBC treatments were 1.75 times and 2.18 times those in the 1.0% BC and 1.0% MBC treatments, respectively. The maximum TF of Cd was observed at 2.0% MBC. In addition, the TFs of Mn, Pb and Cu were all reduced with increasing BC and MBC application rates.

Fig. 7
figure 7

The effect of BC and MBC on the TF of HMs in SJ-172: a Cd, b Mn, c Pb and d Cu. T, bone biochars dosage; S, bone biochar sizes; ns, not significant; *: p < 0.05; **: p < 0.01; ***: p < 0.001. Values present mean ± stand deviation (n=3). Different letters indicate significantly different values among the treatments and between the species (P < 0.05). CK – 0%; B1 – 0.5% BC; B2 – 1.0% BC; B3 – 2.0% BC; B4 – 4.0% BC; MB1 – 0.5% MBC; B2 – 1.0% MBC; B3 – 2.0% MBC; B4 – 4.0% MBC

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4 Discussion

4.1 Soil properties and HMs fractions

4.1.1 BCs increase soil pH, nutrient and SOM

As a unique carbon-based soil amendment, biochar derived from bone meal contains components such as HAP (Ca10(PO4)6(OH)2) and N-containing animal tissue protein. What’s more, BC and MBC amendments also have excellent pore structures and are rich in surface groups and mineral nutrient ions (P and N etc.), displaying a significant promoting effect on the basic physicochemical properties and the nutritional potential of soil (Xiao et al. 2020). The amelioration of mining soils contaminated by HMs was also observed by application of sheep bone biochar (Azeem et al. 2021). Compared with BC, MBC showed a better effect on increasing the AN, AP, TN and TP contents in the soil. This phenomenon may be attributed to the larger specific surface area and superior pore structure of MBC, which make it easier for nutrients to release into or transform in the soil. The difference in the concentration of TP in BC and MBC may be mainly caused by the digestion process of materials with different particle sizes, which agrees with the test results of TP content in HAP with different particle sizes (Cui et al. 2018). Conversely, compared with MBC, adding BC enhanced soil pH and the contents of TK and AK. The results might be related to several mineral nutrient elements (e.g., K, Mg, and Na) contained in MBC entering aqueous solution during the wet ball-milling process, which were then removed by centrifugation. Consequently, the decreased saline-alkali ions in MBC also reduced the ability for exchange with protons on the soil surface, weakening the effect on pH increase. Therefore, the results demonstrated that BC and MBC can both alleviate the basic soil chemical properties, especially for the improvement of N and P in the soil.

4.1.2 BCs enhance HMs stabilization

The stabilizing effect of biochar on HMs in soil is closely associated with the feedstock, surface properties and particle size of biochar. Plant-derived biochar mainly forms complexes with HMs through surface functional groups (e.g., hydroxyl groups, carboxyl groups, and ester groups), salt-based cation (e.g., Ca, Mg, K and Na ions) exchange, and physical fixation in biochar pores to reduce the bioavailability of HMs in soil (Lu et al. 2020; Palansooriya et al. 2022). In contrast, the BC containing the inorganic mineral component HAP or carbonate-bearing hydroxyapatite (CHAP) selected in the present study might not only immobilize soil HMs through the abovementioned pathways but also formed precipitation via PO43- and CO32- to enhance the stabilization of HMs in soil (Lei et al. 2020; Xiao et al. 2020). BC and MBC can transform the fractions of Cd, Pb, Cu and Mn in the soil from an unstable state to a stable state. The higher the doses of BC and MBC applied in the soil, the better the stabilization effects toward HMs, which was confirmed by the reduction in bioavailable concentrations of HMs in soil. The underlying mechanisms on HMs adsorbed by bone biochar are as follows: (1) presumably form precipitation with Cd and Pb by enriching PO43- and CO32-, other components (e.g., Ca2+ and Mg2+) in BC and MBC. Specifically, the high concentrations of total P and Ca in BC and MBC may facilitate the formation of associated phosphates/carbonates of Cd in soil, such as CdCO3 or Cd3(PO4)2 (Lei et al. 2020; Feng et al. 2021). Substantial HAP components may prompt Pb to form stable precipitates, i.e., pyromorphite (Pb5(PO4)3X; X = F−, Cl−, Br−, or OH−) and lead phosphate (Pb2P4O12), in the soil (Chen et al. 2006; Xue et al. 2020). (2) BC and MBC probably strengthen the stabilization and precipitation of HMs in the soil by elevating soil pH. Increasing soil pH can reduce the solubility of cationic HMs (Cd, Cu and Pb) (Chen et al. 2006; Chen et al.2019; Mei et al. 2022). The sharp decrease in the bioavailable content of Mn may be related to the increase in soil pH. (3) The O- and N-containing functional groups of BC and MBC possibly complex or exchange with HMs to reduce the mobility of soil HMs. Our previous study found that bone biochar derived from bone meal contains various groups, such as carboxyl, hydroxyl, ester, carbonyl, pyridyl, and pyrrole groups, which can interact well with HMs (Xiao et al. 2020). (4) The soil exchangeable cations in BC and MBC may exchange with HMs in soil. Pioneering studies demonstrated that HAP contains exchangeable Ca2+ and abundant mineral ions (i.e., Mg2+, K+, and Na+) in bone biochars (Srinivasan et al. 2006; Xiao et al. 2020). It is suggested that ion exchange with Ca2+ ions by Cu2+ was the main mechanism of Cu2+ adsorption by HAP (Corami et al. 2007; Wang et al. 2009). Lei et al. (2020) also found that ion exchange played a significant role in Cd immobilization in cattle-derived biochar (57.6–61.0%).

The MBC treatments resulted in a considerable reduction in the bioavailable contents of HMs in soil compared with the BC treatments at the same dose (Fig. 2). The results can be ascribed to ball-milling ground biochar into micro/nanoengineered powder, which increased the specific surface area and exposed more surface functional groups on MBC than on BC (Lyu et al. 2020; Luo et al. 2022). Thus, there are more available active sites on MBC, resulting in high efficiency in stabilizing HMs through complexation, ion exchange or precipitation. This result was confirmed by using tree biochar with different particle sizes (e.g., <3 mm, 3-6 mm and 6-9 mm) to stabilize soil Cd, Pb and Ni, which showed that biochar with a particle size < 3 mm was more favorable for reducing the bioavailability of HMs in the soil (Zeeshan et al. 2020). The application of empty fruit bunch biochar (< 50 μm) to Cd- and Pb-contaminated soil can immobilize HMs more efficiently than that of empty fruit bunch biochar (> 2 mm) (Fahmi et al. 2018).

4.2 Plant responses

4.2.1 Biochemical characteristics

Heavy metal stress can induce plants to produce superabundant free radicals, which leads to the disturbance of plant physiology and metabolism (Nagajyoti et al. 2010). Increased oxygen free radicals and membrane lipid peroxidation have been reported in different plants because of metal(loid) stress (Kumari et al. 2018; Cao et al. 2017). In our work, BC and MBC significantly alleviated the stress of soil HMs on plant leaves, such as O2.– and MDA, suggesting that BC and MBC played a great role in promoting SJ-172 adaptation to HM-polluted soils. Plants can adjust the activities of their antioxidant enzyme system (e.g., SOD, POD and CAT) to adapt to the impact of external adversity. Application of BC and MBC (1–2%) amendments in HM-polluted soil can improve the activities of SOD, POD and CAT in SJ-172, and the improvement effect is more evident when the addition dose increases. Ali et al. (2017) also reported that increasing biochar application rates favors a decline in the content of SOD and POD in Brassica leaves. The results suggested that BC and MBC changed the plant rhizosphere environment by reducing the bioavailability of HMs, reducing toxicity and alleviating stress. Moreover, the activity of SOD decreased at 4% BC and MBC, indicating that the rhizosphere of SJ-172 was better relieved, and the plant did not need to produce more SOD to decompose the abundant free radicals in SJ-172. Similarly, SOD levels declined in tomato leaves after increasing the biochar dose, which displayed the positive effect of a higher dose of biochar in reducing HM stress in smelter/mine-polluted soil (Li et al. 2015). It was observed that the MDA content of white willow seedlings was significantly reduced at 5.0% biochar application in multimetal-contaminated soil (Mokarram-Kashtiban et al. 2019). The maximum soluble protein contents were observed with the addition of 1.0% BC and MBC and then decreased with the increasing dose of amendments. This phenomenon might be attributed to the sufficient improvements in necessary mineral nutrients for plant protein synthesis, such as AP and HN at 1.0% BC and MBC. Thus, the enhancement of antioxidant defense systems in SJ-172 possibly originated from the following reasons: (1) BC and MBC can lower HM bioavailability in the plant rhizosphere, which consequently decreases HM toxicity and transfer to roots and lessens metal stress at the cellular level; and (2) BC and MBC can afford sufficient nutrients to support normal plant growth on HM-contaminated clay soils.

4.2.2 Mineral elements in Salix

Biochar can effectively increase the mineral nutrients in soil and improve the biomass production of plants. For instance, applying biochar derived from pelletized (4.0 mm) poultry manure and waste bamboo chips greatly increased the concentrations of P and K in plants (lettuce, maize, pak choi and Salix psammophila C.) (Gunes et al. 2015; Chen et al. 2020; Li et al. 2022). Compared to the control, bone char has been selected as a potential clean and renewable P fertilizer to enhance the supply of P in plants, such as lettuce, wheat, potato and pea (Pisum sativum) (Siebers et al. 2014; Mei et al. 2022). In present study, the same doses of BC and MBC had no significant effect on the contents of P, S and Fe in the shoots and leaves of SJ-172, which might be attributed to the sufficient P and relatively low Fe and S contents in BC and MBC. However, MBC can increase the contents of Ca and Mg in SJ-172 compared with BC, which may be attributed to MBC easily releasing Ca and Mg to soil which are then absorbed by SJ-172. In addition, the content of K in leaves was higher in the MBC treatments than in the BC treatments, indicating that the loss of K caused by wet ball-milled bone biochar affects the absorption of K by plants.

BC and MBC greatly improved the survival rate of SJ-172 in acidic clay soil contaminated by multiple metals. Comparing the two different sizes of bone biochar, the MBC is more beneficial in improving soil fertility (except for K+) and reducing bioavailable HMs than BC. Interestingly, under the same dose of BC and MBC, BC promoted the growth of SJ-172 better, such as in height, stem diameter and dry weight of biomass. Moreover, MBC had a serious impact on the belowground growth of SJ-172. The inhibition of Salix growth at 4% MBC may be caused at the early growth stage, which was also confirmed by Li et al. (2015) and Jia et al. (2019), indicating that excessive biochar can damage the membrane and DNA in roots or lead to root cell necrosis.

We concluded that the results might also be related to the micro/nano size of MBC and the typical clay properties of southern China, which might affect the physical nature of the system of soil and SJ-172 growth. Specifically, (1) the effect of BC on keeping the water holding capacity of mine clay soil may be stronger than that of MBC under the same conditions. Smaller MBC particles than soil particles may mechanically increase the compactness of fillers, enhancing the amount of soil water passing through in the soil and reducing the air pore space of the clay soil, resulting in resisting the plant root system to grow and decreasing the root respiration (Liu et al. 2016; Zhang et al. 2010; de Jesus Duarte et al. 2022). Moreover, for biochar particles with a size < 0.25 mm, increasing the biochar addition decreased the effective soil hydraulic conductivity and the transport of water to the surface of the soil in a very short duration process due to the creation of micropores (Wang et al. 2018); while smaller biochar particles would have a higher final cumulative evaporation than soil amended with the same amount of larger biochar particles for a higher initial water content and more efficient hydraulic conductivity in the later evaporation stage (Zhang et al. 2016; Abel et al. 2013). Similar findings were also reported for four sizes of biochar (< 0.1 mm, 0.2–0.4 mm, 0.5–1.0 mm and 2.5 mm) combined with Salix viminalis to remediate HM-polluted soil (Lebrun et al. 2018b). The results suggested that biochar can significantly improve the soil water-holding capacity except for biochar with a minimum size < 0.1 mm, and the optimal size of biochar to enhance plant growth was between 2.0% 0.2–0.4 mm. (2) Smaller biochar may promote water evaporation in clay soil (Wang et al. 2018; Kumar et al. 2020). Compared to BC, MBC had fine black particles (<1000 nm) after being fully mixed with red clay soil, and a darker soil enhanced the soil heat absorption capacity (Fig. 1m). Moreover, a larger dose of biochar accelerates the evaporation rate by forming connected water migration channels in clay soils (Zhang et al. 2020). (3) The hydrophobicity of MBC might decrease its ability to hold soil moisture, resulting in a decrease in available water for the plant to absorb and utilize (Lehmann et al. 2007). It is suggested that the available soil moisture decreased with increasing coal addition in clay soil, probably due to the hydrophobicity of the charcoal (Glaser et al. 2002). This result agrees with our findings that MBC can display good stability after 48 h when BC and MBC are dispersed in water, suggesting that MBC exposes more hydrophobic groups via ball-milling treatment (Xiao et al. 2020). (4) Compared with BC, adding 4% of N-doped biochar derived from the pyrrolic-N with high specific area, which may contain abundant free radicals in rhizosphere, results in root and shoot growth retardation and plasma membrane damage (Zhu et al. 2020; Zhang et al. 2022). The result is consistent with the observation of significant toxicity of persistent free radicals in biochars to crops (Liao et al. 2014). Therefore, the differences in soil rhizosphere caused by BC and MBC may alter the cycling and availability of soil nutrients, which in turn further influences plant growth.

4.3 Phytoremediation capacity

The phytoremediation potential for HMs is determined by both HM levels in plants and total plant biomass. BC demonstrated better performance for the accumulation of Cd, Mn, Pb and Cu in whole plants of SJ-172 than MBC at the same application rate (Fig. 5). In contrast, MBC was more beneficial than BC for enhancing the concentration of HMs in the aboveground parts of SJ-172. Thus, the large differences in the whole biomass of SJ-172 under the BC and MBC treatments may be caused by the particle size of the biochar and the corresponding clay soil texture at higher biochar application. In comparison to BC, applying MBC in combination with SJ-172 to remediate HM-contaminated soil may impact the basic physical structure of clay soils, such as, enhancing the evaporation of water, reducing the available water and impairing air circulation and root respiration in the clay soil. The above potential influencing factors may cause the abnormal growth of SJ-172 with MBC addition, significantly decrease biomass production and dramatically diminish the accumulation of HMs in comparison with BC. For instance, the dry weight of the root of the plant at 4.0% MBC significantly decreased compared with that at 2.0% MBC (P < 0.05) in this work (Fig. 4f). These findings are supported by previous works, i.e., Tryon (1948) found that adding biochar to sandy soils increased their effective moisture by 18%, while the effective moisture content decreased as the amount of biochar added increased in clay soils; both fresh and aged biochars decreased plant available water in the clay loam soil (Aller et al. 2017).

TF manifested that the capacity for contaminants in roots translocated HMs to vital aerial organs (Antoniadis et al. 2017). For Cd, BC and MBC enhanced the TF of Cd in SJ-172, while the TF significantly decreased for Mn, Pb and Cu with increasing doses of BC and MBC, implying that BC and MBC efficiently reduced metal translocation from the roots to shoots. The reasons for BC or MBC to reduce Cu-Pb translocation from roots to shoots in biochar-plant system can be ascribed to the followings: (1) Increasing the applied amount of biochar reduced Cu and Pb uptake. BC and MBC greatly reduced the contents of bioavailable metals in soil, and the reduction effect enhanced with biochar dosage. The results are consistent with Azeem et al. (2021); (2) The lower TF of Pb and Cu is ascribed to its accumulation in the cell membrane and vacuoles of the root, which is related to the stable attribute of Pb and Cu in the system of soil-plant (Cao et al. 2017; Castaldi et al. 2009). It’s believed that Pb as a toxic element can be recognized early by plant roots and immobilized in cell walls or vesicles to reduce its transfer to the aerial parts (Di Toppi et al. 1999). Under high copper levels in the soil, biochar reduced the translocation of Cu from root to shoot to alleviate the toxicity effect (Gonzaga et al. 2022).

It’s generally reported that biochar can reduce the bioavailable content of metals in soil and thus reduce the metal accumulation by plants especially for crops (Liu et al. 2020; 2021). The underlying mechanism of BC- and MBC-assisted phytoremediation for multi-HM-contaminated clay soil is concluded as follows (Fig. 8): (1) Based on the improvement of the soil environment such as reduced the bioavailable contents of HMs and the rich supply of nutrients (i.e., Ca, Mg, P, N, and K), BC and MBC can improve the survival rate of SJ-172 on acidic clay soil; (2) BC and MBC can improve the activity of plant antioxidant enzymes and the absorption of mineral nutrients. The previous studies also demonstrated that biochar can improve the efficiency of woody trees in remediating HMs-contaminated soil by enhancing soil enzyme activity and the activities of photosynthesis and transpiration (Li et al. 2021a; 2022). (3) Both BC and MBC can improve the growth SJ-172 and enhance its phytoremediation efficiency for heavily polluted soil. However, compared to BC, MBC with micro/nano size and higher hydrophobicity affecting these physical characteristics in the clay soil, which may enhance water evaporation, and cause a decrease in availability of air and water, has more toxicity of persistent free radicals, resulting in the decrease of the root respiration and abmormal growth and the decrease in SJ-172’s ability to accumulate HMs. Břendová et al. (2015) found that adding 5-15% coconut shell biochar to Cd-contaminated soil (43 mg/kg) in a mining area reduced 99% of Cd in soil leachate and promoted willow’s biomass and phytoremediation efficiency.

Fig. 8
figure 8

The possible mechanism of BC and MBC influenced on SJ-172 phytoremediation to HMs in severely contaminated clay soil

Full size image

4.4 Environmental impaction and economic analysis

The core of vegetation restoration is the improvement of rhizosphere environment for plant growth. Biochar prepared from waste biomass is cost effective and chemically stable, which has great potential for improving HM contaminated soil and vegetation restoration, while considering the long-term goals of environmental safety, efficient resource utilization and carbon neutrality. The Organization for Economic Cooperation and Development (OECD) reported that massive bone meal waste by the meat industry were produced because of a globally significant increase in meat consumption (OECD, 2018). Besides, the total meat production in 2022 reached 92.27 million tons in China, corresponding to about 9.22∼13.84 million tons of bone waste (National Bureau of Statistics of China, 2022; Grace 1983).

The bone biochar derived from bonemeal contained HAP and carbon component (~35%), which demonstrated great potential for the remediation of HM polluted soil and improvement of infertile soil (Xiao et al. 2020). Moreover, the stabilization of biomass carbon is the main contributor to carbon dioxide removal, biochar application can also deliver carbon dioxide removal by reducing the decomposition of SOC, lowering emission of nitrous oxide (N2O) from soil, and avoiding greenhouse gas emissions during the utilization of biomass residues such as animal carcasses and diseased livestock (Cowie et al. 2023; Joseph et al. 2021).

Economic cost is an important indicator of the competitiveness of a product in practical applications. The production cost of bone biochar is US $ 1200 ~ $1858.35 /ton (https://p4psearch.1688.com), which is slightly higher than that of biochars (US $ 670–1780/ton) while obviously lower than the commercial nano HAP (US $ 6040~69549/ton, https://p4psearch.1688.com) (Zhang et al. 2022; Arrenberg 2010). HAP has been shown to be a perfectly suitable and highly promising bio-inspired material for various environmental applications (Ibrahim et al. 2020). Thus, considering the stabilization effect of BCs on HMs and the enhancement of soil fertility which are far better than those of conventional biochar and the lower price, they are a promising soil amendament candidate in the near future for large-scale application in HM contaminated soil.

In present study, the reduction in particle size of bone biochar enhanced its stabilization performance against soil HMs, while caused a certain degree of limitation to normal plant growth in clay soil compared with BC. Hence, the environmental risk of micro-nano-sized biochar utilization also needs to be carefully evaluated, especially for different anionic forms of HMs and soil textures, etc. For instance, the nano-BC at a modest application rate of 10 mg‧L−1 is very toxic to Streptomyces, which can severely damage Streptomyces cells with only 68% survival rate (Liu et al. 2019). The smaller size of biochar can sorb HMs in contaminated soils and may act as a potential contaminant vector in terrestrial and aquatic ecosystems, and the evaluation of the stabilization of HMs in environmental aging and the longevity of soil microorganisms are also urgently needed (Zhang et al. 2022).

5 Conclusions

The application of BC and MBC had a significant effect on elevating soil pH, reducing the bioavailability of HMs by facilitating their transformation to stable forms, and ameliorating soil nutrients (e.g., Ca, P, N, and Mg). MBC was superior to BC in stabilizing soil HMs by exposing more active sites to fix HMs around plant roots. Both BC and MBC improved the survival rate, physiological indices and mineral element absorption of SJ-172 in acidic clay soil severely contaminated with HMs. BC facilitated the phytoremediation efficiency of SJ-172 in HM-contaminated acid soil more than MBC. This study provides a preliminary understanding of bone biochar with different sizes as an emerging eco-friendly amendment for improving phytoremediation capacity with fast-growing trees in multi-metal contaminated clay soils.