Biochar as a Stimulator of Zea mays Growth and Enzyme Activity in Soil Contaminated with Zinc, Copper, and Nickel

Abstract

Biochar continues to attract growing interest as a promising soil amendment, particularly in areas contaminated with heavy metals. The present experiment was conducted on soil contaminated with zinc (Zn), copper (Cu), and nickel (Ni) in the following treatments: contamination with a single heavy metal (Zn, Cu, or Ni) and with a combination of heavy metals (ZnCu, ZnNi, CuNi, and ZnCuNi). The analysis was performed in soil samples with and without biochar addition. The biochar dose was 15 g kg⁻¹ soil. The biochar was produced from sunflower husks, with the following composition: ash—7.49%; organic carbon (C_org)—83.92%; total nitrogen (N_total)—0.91%; hydrogen—2.56%; sulfur—0.02%; oxygen—3.30%; and pH—9.79. Nickel, followed by Cu, induced the greatest decrease in Zea mays yields, whereas the smallest decline in yields was observed in response to Zn contamination. The combined application of the tested heavy metals had more damaging effects, in particular by decreasing maize yields. The values of the heavy metal impact index (IF_Hm) confirmed that heavy metals exerted a negative impact on the biochemical activity of soil. Copper applied alone and in combination with other heavy metals had the most inhibitory effect on soil enzyme activity. The toxicity of the analyzed heavy metals for plants and soil enzymes was reduced by biochar. This is confirmed by the tolerance index (TI) values for copper and nickel in Zea mays. The TI values for copper increased from 0.318 in soil without biochar to 0.405 in soil with biochar. For nickel, the TI values increased from 0.015 to 0.133. The values of the biochar impact index (IF_CB) also suggest that biochar stimulated enzyme activity in all treatments. Biochar also improved the chemical and physicochemical properties of soil, including the content of C_org and N_total and soil pH.

1. Introduction

Biochar is produced by the thermochemical conversion of biomass into a stable form of carbon, and it can considerably influence soil properties and plant growth and health [1,2,3,4]. One of the key characteristics of biochar is its unique porous structure. As a result, biochar can significantly increase the water-holding capacity of soil, which is a particularly important consideration in areas affected by water deficits [1,5]. Biochar can also absorb and gradually release nutrients, which promotes nutrient use efficiency by plants. Stable nutrient supply contributes to uniform plant development under more sustainable conditions, and thus biochar’s positive role is increasingly recognized in agriculture and environmental protection [6,7,8]. Due to its unique physicochemical properties, biochar is not only an effective tool for carbon sequestration, but it is also a promising amendment for improving soil quality [9,10,11]. Biochar can play a crucial role in soil remediation, particularly in areas contaminated with heavy metals, and it can promote the growth of many crops, including maize [12,13]. Maize is one of the major and most economically important cereal crops in the world. Due to growing levels of environmental pollution, the search for effective methods supporting maize growth is not only a necessity but also a considerable challenge for researchers and agricultural producers [14,15]. Maize is sensitive to heavy metals, and its growth and ability to absorb essential nutrients can be compromised in contaminated areas [16,17].

Biochar can considerably enhance plant growth by stimulating the activity of soil enzymes [18,19]. Enzymes such as dehydrogenases, phosphatases, and urase play an essential role in the biogeochemical cycle of nutrients, and they are sensitive markers of soil fertility [20,21]. Biochar stimulates the activity of soil enzymes by improving physicochemical conditions and minimizing oxidative stress in soil-dwelling microorganisms exposed to heavy metals [22]. In consequence, higher soil enzyme activity increases the ability of plants to absorb nutrients, which is the key to their health and productivity [23].

Heavy metal contamination poses a serious environmental problem. Heavy metals can be introduced to soil from various sources, including industrial facilities, transport, fertilizers, and plant protection products [24,25,26]. Heavy metals such as zinc (Zn), copper (Cu), and nickel (Ni) can affect the physical and chemical properties of soil as well as plant health [27,28]. Zinc and Cu are classified as essential plant minerals [29]. Zinc plays a crucial role in numerous enzymes and proteins and affects the synthesis and metabolism of nucleic acids as well as the production of plant hormones such as auxins. Optimal Zn levels in the soil promote photosynthesis and the growth and development of plants [30,31,32]. In turn, Cu plays a role in photosynthesis, cellular respiration, and antioxidant systems. Copper is a component of many enzymes and proteins that participate in various metabolic processes, including lignin synthesis, which promotes the structural integrity of plant cells [33,34]. In comparison with Zn and Cu, the role of Ni has been less extensively researched; however, this heavy metal is an essential component of urease, an enzyme that catalyzes the hydrolysis of urea to ammonia and carbon dioxide, which is important for the nitrogen cycle in plants [35,36]. According to the available studies [28,37,38], an optimal zinc content in soil is considered to be between 5 and 50 mg kg⁻¹, while values above 100 mg kg⁻¹ are considered harmful to the environment. The optimum range for copper is 5–30 mg kg⁻¹, while levels above 40 mg kg⁻¹ often lead to detrimental changes in plants and soils. The nickel content in soil is considered acceptable when it is between 5 and 30 mg kg⁻¹, but it becomes dangerous when it exceeds 100 mg kg⁻¹, leading to plant toxicity and the disruption of the uptake of other nutrients, as well as having negative effects on soil biology [39]. Despite the above, excess heavy metal concentrations in the soil may have negative consequences. Above all, excessive Zn supply can inhibit plant growth and lead to leaf chlorosis or even necrosis. Excess Zn also disrupts the assimilation of other micronutrients, including iron and Cu, which further compromises plant health. In turn, excess Cu can lead to metabolic disorders; it can inhibit root and shoot growth and decrease iron and molybdenum absorption in plants. Due to its toxic effects, Cu can disrupt photosynthesis and cellular respiration, which can contribute to a significant, long-term decrease in crop yields. Excessive Ni supply is less frequently noted, but it also poses a threat to plants. High Ni concentrations can inhibit plant growth, contribute to leaf and root deformities, and reduce photosynthetic efficiency [40,41]. Therefore, effective solutions are needed to minimize the negative impact of heavy metals. Biochar is a promising soil amendment that can stimulate plant growth and increase soil fertility, thus addressing contemporary challenges in environmental protection and sustainable agriculture [40,41,42]. In addition, biochar supports plants in their adaptation to changing environmental conditions. Biochar can also promote the adaptation of maize plants to water deficits and extreme weather events [43]. Effective adaptation can increase plant tolerance to abiotic stresses, such as droughts and high temperatures, and biotic stresses such as pests and pathogens. This contributes to increased biomass production, photosynthetic efficiency, crop yield, and food security [44,45,46].

Previous research [47,48,49] has focused mainly on conventional materials for biochar production such as wood or waste biomass. In contrast the present study analyzes innovative resources, such as waste products from the agri-food sector, e.g., sunflower seed hulls. This approach supports the more sustainable and environmentally friendly use of biochar in agricultural practice. This study contributes to existing research by determining the impact of biochar on the toxicity of heavy metals, as well as on the physicochemical parameters and biochemical properties of the soil, as well as plant growth and yields. In contrast, previous studies were often limited to a specific set of parameters, without examining the broader context.

The following research hypotheses were formulated: (1) The use of biochar stimulates the growth and development of Zea mays. (2) Biochar increases soil activity. (3) Biochar improves the physicochemical properties of the soil. Therefore, the aim of this study was to (1) determine the responses of Zea mays plants to Zn, Cu, Ni, and biochar and (2) determine the effect of Zn, Cu, Ni, and biochar on the physicochemical properties of the soil and soil enzyme activity. The results of this study can contribute to the development of new, innovative solutions in agricultural biotechnology and environmental protection technology.

2. Materials and Methods

2.1. Soil and Biochar Characteristics

Soil samples for this study were collected at a depth of 0–20 cm from arable land in the Agricultural Experiment Station in Tomaszkowo in north-eastern Poland. The experimental soil was classified as Eutric Cambisol developed from sandy loam [50]. Its granulometric composition was as follows: sand—69.41%; silt—27.71%; and clay—2.88%. This soil had the following characteristics: C_org content—7.80 g kg⁻¹; N_Total—1.20 g kg⁻¹; pH_KCl—5.80; hydrolytic acidity (HAC)—13.50 mmol(+) kg⁻¹; exchangeable base cations (EBCs)—31.00 mM(+) kg⁻¹; total cation exchange capacity (CEC)—44.50 mM (+) kg⁻¹; and base cation saturation ratio in soil (BCSR)—69.65%.

Biochar, supplied by NTP Sp. z o.o. (Poland), was produced from sunflower seed husks processed by pyrolysis at a temperature of 450 °C for 2 h in an anaerobic environment. The biochar had the following composition: ash—7.49%, C_org—83.92%, N_total—0.91%, hydrogen—2.56%, sulfur—0.02%, oxygen—3.30%, and pH—9.79.

2.2. Pot Experiment

A pot experiment was conducted in the greenhouse of the University of Warmia and Mazury in Olsztyn, Poland. The research was carried out between 1 June and 21 July 2022. The experiment lasted for 50 days, and the experimental conditions were monitored regularly. The mean air temperature was 17.5 °C; the humidity was 76.8%, and the daylight duration ranged from 15 h 5 min to 17 h 30 min. The effects of two experimental factors (heavy metal interactions and biochar addition) were investigated in 16 treatments with four replicates each (

Table 1. Experimental design.

Number	Treatment	Biochar Dose, g kg−1 Soil	Heavy Metal Dose, mg kg−1 d.m.
1	C	0	0
2	Zn		420
3	Cu		420
4	Ni		420
5	ZnCu		210 (Zn) + 210 (Cu)
6	ZnNi		210 (Zn) + 210 (Ni)
7	CuNi		210 (Cu) + 210 (Ni)
8	ZnCuNi		140 (Zn) + 140 (Cu) + 140 (Ni)
9	C	15	0
10	Zn		420
11	Cu		420
12	Ni		420
13	ZnCu		210 (Zn) + 210 (Cu)
14	ZnNi		210 (Zn) + 210 (Ni)
15	CuNi		210 (Cu) + 210 (Ni)
16	ZnCuNi		140 (Zn) + 140 (Cu) + 140 (Ni)

).

The analyzed macronutrients were added to soil in doses corresponding to the nutrient requirements of maize (in mg kg⁻¹ soil): N—150, P—70, K—120, and Mg—15. In the following step, biochar and heavy metal chlorides (ZnCl_2, CuCl₂, and NiCl₂·7H₂O) were applied according to the experimental design presented in Table 1. This study used heavy metals in the form of chlorides. Aqueous solutions containing ZnCl₂ at a concentrations of 61.282 g dm⁻³, CuCl₂ at a concentrations of 62.204 g dm⁻³, and NiCl₂·7H₂O at a concentrations of 119.060 g dm⁻³ were applied to provide Zn, Cu, or Ni doses per kg of soil, according to the test scheme presented in Table 1. After adding the solutions, the soil was thoroughly mixed and packed into pots. The heavy metal concentrations were selected on the basis of an analysis of previous studies in which various levels of exposure to heavy metals were tested [29,51,52,53]. This information formed the basis for our decision to set a critical threshold of 420 mg kg⁻¹. All ingredients were thoroughly combined with the soil and placed in plastic pots (diameter—20 cm and depth—25 cm). Maize cultivar LG 32.58 was sown in pots. After emergence, seedlings were thinned, and four seedlings were left in each pot. The soil moisture content was maintained at 50% maximum water-holding capacity throughout the experiment. Maize biomass was harvested in stage 39 of the BBCH scale (Biologische Bundesanstalt, Bundessortenamt und Chemische Industrie). At the same time, soil samples were collected for laboratory analyses. The leaf greenness index (SPAD) of maize plants was measured in BBCH stage 33. The measurements were conducted in five replicates with the use of a Spectrum Technologies chlorophyll meter (Konica Minolta, Inc., Chiyoda, Japan).

2.3. Chemical, Physicochemical, and Biochemical Analyses

2.3.1. Soil

Soil enzyme activity was determined in soil samples collected after maize harvest. Soil properties were tested on samples that had been sieved through a 2 mm diameter sieve prior to the experiment. Physicochemical analyses included determining the following metrics: pH_KCl [54], hydrolytic acidity (HAC) [55], exchangeable base cations (EBCs) [55], the total cation exchange capacity of the soil (CEC) [56], the base cation saturation ratio in the soil (BSR) [56], and the chemical content for C_org [57] and N_Total [57], as well as Cu, Zn, and Ni. This was achieved by mineralizing the soil using a microwave mineralizer (UltraWAVE, Milestone, Sorisole, Italy). An inductively coupled plasma emission spectrometer (iCAP 7000 Series ICP-OES, Thermo Scientific, Newington, CT, USA) was used to determine the heavy metal content. Biochemical analyses included determining the activity of dehydrogenases (Deh) [58], catalase (Cat) [59], urease (Ure) [60], acid phosphatase (Pac) [60], alkaline phosphatase (Pal) [60], β-glucosidase (Glu) [60], and arylsulfatase (Aryl) [60]. Detailed procedures for determining the activity of soil enzymes were described in the authors’ previous studies [61,62].

2.3.2. Plant

The Cu, Zn, and Ni contents were determined in both the roots and the above-ground parts. A total of 0.5 g of the plant material was weighed and mineralized in 5 cm³ of 65% HNO₃ using a microwave mineralizer (UltraWAVE, Milestone, Sorisole, Italy). The mineralized samples were then diluted with demineralized water to a final volume of 100 cm³. Elemental concentrations were measured using an inductively coupled plasma emission spectrometer (iCAP 7000 Series ICP-OES, Thermo Scientific, Newington, CT, USA).

2.4. Calculations and Statistical Analysis

Based on maize yield, the tolerance index (TI) of maize plants was calculated using the formula below:

$$\mathrm{T}\mathrm{I}={\displaystyle \frac{{\mathrm{Y}}_{\mathrm{H}\mathrm{m}}}{{\mathrm{Y}}_{\mathrm{C}}}}$$

(1)

where

Y_Hm—the yield of above-ground parts/roots in soil contaminated with heavy metals (g kg⁻¹);

Y_C—the yield of above-ground parts/roots in uncontaminated soil (g kg⁻¹).

The tolerance index (TI) assesses the ability to tolerate stressful conditions, including elevated concentrations of heavy metals in the environment. TI values greater than 1 suggest tolerance to pollution, while values less than 1 indicate sensitivity and the potential for negative health effects in plants.

To determine the effect of heavy metals and biochar on soil biochemical activity, the heavy metal impact index (IF_Hm) and the biochar impact index (IF_CB) were calculated based on the activity of dehydrogenases, catalase, urease, acid phosphatase, alkaline phosphatase, β-glucosidase, and arylsulfatase using the following formulas:

$${\mathrm{IF}}_{\mathrm{Hm}}={\displaystyle \frac{{\mathrm{A}}_{\mathrm{Hm}}}{{\mathrm{A}}_0}}-1$$

(2)

where

IF_Hm—the heavy metal impact index;

A_Hm—the enzyme activity in soil contaminated with heavy metals;

A₀—the enzyme activity in uncontaminated soil.

$${\mathrm{IF}}_{\mathrm{CB}}={\displaystyle \frac{{\mathrm{A}}_{\mathrm{CB}}}{\mathrm{A}}}-1$$

(3)

where

IF_CB—the biochar impact index;

A_CB—the enzyme activity in soil with biochar addition;

A—the enzyme activity in soil without the addition of biochar.

An impact factor (IF) above 1 indicates a positive effect of the tested variables (e.g., heavy metals or biochar) on soil enzyme activity, while an IF below 1 indicates a negative effect. The results were processed by two-way analysis of variance (ANOVA). Homogeneous groups were identified by Tukey’s HSD test at a significance level of p ≤ 0.05. Statistical analyses were conducted in the Statistica program [63]. The greenness index (SPAD) of maize leaves and the indices of the impact of heavy metals (IF_Hm) and biochar (IF_CB) on the biochemical activity of the soil were visualized using the TBtools-II version 2.202 software [64].

3. Results

3.1. The Effect of Heavy Metals and Biochar on the Chemical and Physicochemical Properties of Soil

The first thing that stands out is the reduction in the heavy metal content in uncontaminated soil after the addition of biochar (CB), compared to soil without biochar addition (CBw) (

Table 2. The heavy metal content of the soil mg kg⁻¹.

Treatment	Heavy Metal Tested	CBw	CB
C	Zn	22.762 i ± 1.020	11.579 j ± 1.011
	Cu	3.974 k ± 0.080	2.472 k ± 0.052
	Ni	6.022 jk ± 0.093	2.806 k ± 0.067
Zn	Zn	375.520 b ± 4.070	385.261 b ± 2.228
Cu	Cu	406.119 a ± 5.120	395.320 b ± 2.478
Ni	Ni	415.202 a ± 4.152	308.419 c ± 3.121
ZnCu	Zn	161.615 e ± 2.223	191.695 de ± 1.975
	Cu	201.646 d ± 2.026	211.529 d ± 1.999
ZnNi	Zn	194.430 de ± 1.268	188.732 de ± 1.853
	Ni	195.699 de ± 1.732	194.927 cd ± 1.278
CuNi	Cu	204.011 d ± 2.098	182.323 de ± 1.645
	Ni	186.535 de ± 1.365	167.257 e ± 1.874
ZnCuNi	Zn	78.252 h ± 1.786	108.232 g ± 1.765
	Cu	88.951 h ± 1.259	157.465 ef ± 1.899
	Ni	81.529 h ± 1.223	146.107 f ± 1.367

CBw—without biochar; CB—with biochar; C—uncontaminated soil; Zn, Cu, and Ni at a dose of 420 mg kg⁻¹ d.m. soil; ZnNi and CuNi at a dose of 210 mg kg⁻¹ d.m. soil; ZnCuNi at a dose of 140 mg kg⁻¹ d.m. soil. The same letters (a–k) refer to homogeneous groups.

). In particular, a significant reduction in Zn content was observed at Zn-contaminated sites, decreasing from 22.762 mg kg⁻¹ without biochar to 11.579 mg kg⁻¹ with biochar application. In the case of soil contaminated with heavy metals, whether they were used individually or in combination, the effect of biochar was less evident. At sites where heavy metals were present individually, applying the fertilizer increased the Zn content of the soil while decreasing the Cu and Ni contents. Applying the fertilizer to soil contaminated with a combination of three metals (Zn, Cu, and Ni) resulted in a significant increase in the content of the tested metals.

In uncontaminated soil, biochar addition significantly increased the content of organic carbon in soil (

Table 3. Physicochemical properties of soil contaminated with heavy metals and amended or not amended with biochar.

Treatment	Corg		Ntotal		pHKCl		HAC		EBC		CEC		BCSR
	CBw	CB	CBw	CB	CBw	CB	CBw	CB	CBw	CB	CBw	CB	CBw	CB
C	7.605 i ± 0.011	11.795 f ± 0.062	1.230 c ± 0.011	1.385 b ± 0.013	5.73 e ± 0.05	5.98 e ± 0.05	15.56 a ± 0.19	13.31 b ± 0.19	31.00 g ± 0.26	35.00 f ± 0.34	46.56 f ± 0.20	48.31 e ± 0.27	66.49 i ± 0.37	72.38 f ± 0.41
Zn	7.965 gh ± 0.010	13.085 c ± 0.054	1.325 c ± 0.010	1.350 bc ± 0.011	5.76 e ± 0.04	6.08 d ± 0.05	15.45 a ± 0.08	12.75 c ± 0.28	29.50 gh ± 0.23	27.00 h ± 0.31	44.95 g ± 0.25	39.75 k ± 0.31	65.63 i ± 0.29	67.79 h ± 0.32
Cu	7.345 k ± 0.013	12.220 d ± 0.047	1.305 d ± 0.013	1.420 a ± 0.012	5.97 de ± 0.05	6.16 c ± 0.02	9.19 i ± 0.19	12.75 c ± 0.26	65.00 a ± 0.50	28.00 h ± 0.28	74.19 a ± 0.39	40.75 j ± 0.33	87.62 a ± 0.39	68.22 h ± 0.29
Ni	7.115 l ± 0.023	13.785 ab ± 0.042	1.340 c ± 0.012	1.385 b ± 0.012	6.35 b ± 0.03	6.55 a ± 0.01	9.75 g ± 0.04	9.38 h ± 0.28	30.50 g ± 0.30	46.00 b ± 0.38	40.25 jk ± 0.35	55.38 c ± 0.41	75.75 e ± 0.36	82.99 b ± 0.38
ZnCu	7.765 h ± 0.033	13.845 a ± 0.045	1.195 f ± 0.012	1.275 e ± 0.011	5.95 de ± 0.02	6.10 cd ± 0.02	13.69 b ± 0.18	11.81 de ± 0.16	31.00 g ± 0.33	31.00 g ± 0.32	44.69 g ± 0.33	42.81 h ± 0.39	69.36 g ± 0.28	72.40 f ± 0.42
ZnNi	7.485 j ± 0.027	12.035 e ± 0.036	1.275 e ± 0.011	1.345 b ± 0.013	6.13 c ± 0.04	6.30 b ± 0.03	11.44 e ± 0.17	10.31 f ± 0.15	36.00 f ± 0.35	45.00 bc ± 0.29	47.44 f ± 0.39	55.31 c ± 0.42	75.86 e ± 0.43	81.31 b ± 0.44
CuNi	8.165 g ± 0.030	13.645 b ± 0.027	1.385 b ± 0.010	1.385 b ± 0.012	6.10 cd ± 0.03	6.30 b ± 0.01	12.19 d ± 0.18	11.81 ef ± 0.18	48.00 b ± 0.32	41.00 e ± 0.35	60.19 b ± 0.40	52.81 d ± 0.37	79.73 c ± 0.46	77.63 d ± 0.47
ZnCuNi	7.295 j ± 0.029	13.825 a ± 0.029	1.235 f ± 0.011	1.435 a ± 0.013	6.03 d ± 0.02	6.30 b ± 0.03	12.38 cd ± 0.28	10.31 f ± 0.09	44.00 c ± 0.25	33.00 fg ± 0.36	56.38 c ± 0.42	43.31 h ± 0.35	78.02 c ± 0.48	76.18 e ± 0.37

C_org—total organic carbon; N_total—total nitrogen; HAC—hydrolytic acidity; EBC—total exchangeable base cations; CEC—total cation exchange capacity; BCSR—base cation saturation ratio; CBw—without biochar; CB—with biochar; C—uncontaminated soil; Zn, Cu and Ni applied at a dose of 420 mg kg⁻¹ d.m. soil; ZnNi and CuNi applied at a dose of 210 mg kg⁻¹ d.m. soil; ZnCuNi applied at a dose of 140 mg kg⁻¹ d.m. soil. The letters a–l denote homogeneous groups, separately for each parameter.

). Biochar increased the C_org content from 7.605 mg kg⁻¹ to 11.795 mg kg⁻¹ in uncontaminated treatments. In the Zn, Cu, Ni, ZnCu, ZnNi, CuNi, and ZnCuNi treatments, the C_org content was also higher in biochar-amended soil than in soil without biochar addition. Biochar had a similar effect on the N_total content. In biochar-amended soil, N_total increased from 1.230 mg kg⁻¹ to 1.385 mg kg⁻¹. Moreover, in treatments contaminated with one or more heavy metals, biochar had a positive influence on N_total, but the observed increase was less spectacular than in the case of C_org.

Biochar also modified the physicochemical properties of the soil (Table 3). In uncontaminated treatments, biochar decreased hydrolytic acidity but increased soil pH, total exchangeable base cations (EBCs), soil sorption capacity, and the total base cation saturation ratio (BCSR). Biochar exerted similar effects on soil pH and hydrolytic acidity in soil contaminated with heavy metals and in the control soil. In turn, biochar’s impact on EBC and soil sorption capacity varied across treatments. However, biochar did not improve soil sorption capacity in contaminated treatments.

3.2. The Effect of Heavy Metals and Biochar on Maize

Soil contamination with individual heavy metals (Zn, Cu, and Ni) and combinations of two (ZnCu, ZnNi, CuNi) or three heavy metals (ZnCuNi) decreased the yield of above-ground parts (APs) and roots (Rs) in maize plants grown in treatments with and without biochar addition (Figure 1a). In treatments contaminated with heavy metals, the growth of maize plants was accelerated in biochar-amended soil relative to soil without biochar addition. In uncontaminated soil, maize biomass growth reached 30.88 g d.m. pot⁻¹ in the absence of biochar and 36.03 g d.m. pot⁻¹ after biochar addition. Significant differences in maize yields were observed between treatments contaminated with the tested heavy metals. In treatments contaminated with Zn, the maize yield reached 34.15 g in the absence of biochar and 36.10 g after biochar addition. In treatments contaminated with Cu, the maize yield was significantly lower at 9.8 g and 14.60 g, respectively. The yield of above-ground plant parts was lowest in treatments contaminated with Ni, and it was determined at 0.450 g in the absence of biochar and 4.78 g after biochar addition. These results indicate that biochar can help alleviate the negative impact of heavy metals and promotes plant adaptation to unfavorable conditions. The yield of above-ground plant parts also decreased in response to contamination with two heavy metals. In ZnCu treatments, maize yields increased from 6.53 g (without biochar) to 20.16 g after biochar application. In ZnNi and CuNi treatments, the yields of above-ground plant parts were lower, but biochar significantly alleviated the negative impact of heavy metals. Biochar also induced a five-fold increase in biomass yield in treatments contaminated with all three heavy metals.

Similar trends were observed in maize root yields (Figure 1b). In uncontaminated treatments, root yields ranged from 12.10 g in soil without biochar to 18.21 g in soil with biochar addition. In treatments not amended with biochar, the addition of Zn, Cu, and Ni decreased root yields by 10.59%, 68.24%, and 98.55%, respectively. In treatments contaminated with Zn and Ni, biochar increased root yields by 57% and 88.89%, respectively, relative to the soil without biochar addition. Biochar induced the smallest increase in root yield (2.93%) in soil subjected to Cu pressure. Biochar’s positive effects were even more pronounced in treatments contaminated with combinations of heavy metals. The root yield increased from 4.98 g to 7.55 g in ZnCu treatments, from 0.45 g to 3.77 g in ZnNi treatments, and from 0.39 g to 2.16 g in CuNi treatments. Biochar exerted a particularly positive impact in ZnCuNi treatments, where root yield increased more than ten-fold. These observations indicate that Zea mays yields were most severely compromised by Ni, followed by Cu, and were least affected by Zn.

The tolerance index (TI) provides important information about biochar’s role in promoting plant adaptation to Cu, Zn, and Ni stress (Figure 2). The TI is a key parameter for assessing plant development in soil contaminated with heavy metals. In Zea mays, the TI of above-ground plant parts was higher in biochar-amended soil than in soil without biochar. Maize grown in Zn treatments was the only exception, where the TI decreased from 1.106 g (without biochar) to 1.002 g (with biochar). In contrast, the TI increased from 0.318 (without biochar) to 0.405 (with biochar) in Cu treatments and from 0.015 (without biochar) to 0.133 (with biochar) in Ni treatments. The increase in TI values after biochar addition suggests that this soil amendment enhanced maize growth and development. Biochar also exerted a positive impact on the TI of above-ground plant parts in treatments contaminated with more than one heavy metal. In ZnCu treatments, the TI increased more than two-fold, from 0.211 to 0.560. In ZnNi and CuNi treatments, the TI increased from 0.028 to 0.114 and from 0.033 to 0.100, respectively. The analyzed parameter increased from 0.048 (without biochar) to 0.216 (with biochar) in treatments contaminated with all three heavy metals (ZnCuNi).

The TI of Zea mays roots responded differently to heavy metal contamination and biochar application. In Zn treatments, the TI increased from 0.807 (without biochar) to 0.841 (with biochar), but it decreased from 0.395 to 0.255 in response to Cu contamination. In turn, no significant differences in TI values were observed between Ni treatments with and without biochar addition. In ZnCu, ZnNi, and CuNi treatments, biochar application led to an increase in TI values from 0.412 to 0.415, from 0.037 to 0.207, and from 0.032 to 0.119, respectively. Similar observations were made in ZnCuNi treatments, where the TI increased from 0.046 to 0.326 after biochar application. These results suggest that biochar reduces the harmful effects of heavy metal contamination.

The leaf greenness index (SPAD) of maize in the control treatment without biochar application was 31.10 and increased to 33.820 after biochar addition (Figure 3). Biochar application decreased SPAD values from 34.11 to 31.77 in soil contaminated with Zn and from 35.33 to 33.68 in soil contaminated with Cu. The SPAD values for the objects containing Ni were not analyzed, as Ni inhibited maize growth almost completely. The yield in the plots without biochar was 0.450 g d.m. pot⁻¹, while in the plots with biochar, it was 4.780 g d.m. pot⁻¹. In ZnCu treatments, SPAD was 33.18 in the absence of biochar and 31.94 after biochar addition. In contrast, in ZnNi and CuNi treatments, SPAD values were higher in soil amended with biochar than in soil without biochar addition. In treatments contaminated with all three heavy metals (ZnCuNi), SPAD values also increased from 22.46 (without biochar) to 25.34 (with biochar).

The lowest heavy metal content was found in the above-ground parts and roots of corn grown in uncontaminated soil (

Table 4. The heavy metal content in the above-ground parts (APs) and roots (Rs) of maize, mg kg⁻¹.

Treatment	Heavy Metal Tested	AP		R
		CBw	CB	CBw	CB
C	Zn	7.106 h ± 0.071	6.433 h ± 0.048	6.418 g ± 1.020	6.831 g ± 0.078
	Cu	2.507 h ± 0.023	2.231 h ± 0.031	2.745 g ± 1.020	6.790 g ± 0.055
	Ni	3.292 h ± 0.035	1.512 h ± 0.016	2.117 g ± 1.020	2.043 g ± 0.025
Zn	Zn	338.325 c ± 4.571	314.242 c ± 1.386	419.970 b ± 3.769	510.949 a ± 4.039
Cu	Cu	313.369 cd ± 3.524	331.435 c ± 2.125	354.623 c ± 2.534	372.265 bc ± 1.904
Ni	Ni	408.228 b ± 2.989	440.135 a ± 3.187	465.322 ab ± 2.776	492.658 a ± 2.597
ZnCu	Zn	304.755 c ± 2.256	255.199 d ± 1.870	343.656 c ± 3.026	376.423 bc ± 2.034
	Cu	111.981 ef ± 1.897	116.288 ef ± 1.098	326.657 c ± 2.732	303.389 cd ± 2.026
ZnNi	Zn	252.661 d ± 1.798	191.788 de ± 1.788	309.218 cd ± 2.098	317.824 c ± 2.123
	Ni	341.060 c ± 2.265	176.302 de ± 1.487	378.479 bc ± 2.111	332.689 c ± 2.059
CuNi	Cu	146.696 e ± 1.567	112.115 ef ± 1.275	171.383 e ± 1.137	167.872 e ± 1.754
	Ni	189.981 de ± 1.246	123.429 e ± 1.524	234.164 d ± 1.273	269.995 d ± 1.673
ZnCuNi	Zn	111.603 ef ± 1.452	76.548 f ± 1.071	116.676 f ± 1.153	175.928 e ± 1.561
	Cu	97.064 f ± 1.015	99.665 f ± 1.039	106.885 f ± 1.211	106.378 f ± 1.275
	Ni	136.206 e ± 1.125	45.348 g ± 1.011	194.887 e ± 1.327	177.301 e ± 1.325

CBw—without biochar; CB—with biochar; C—uncontaminated soil; Zn, Cu, and Ni at a dose of 420 mg kg⁻¹ d.m. soil; ZnNi and CuNi at a dose of 210 mg kg⁻¹ d.m. soil; ZnCuNi at a dose of 140 mg kg⁻¹ d.m. soil. Identical letters (a–h) indicate statistically homogeneous groups, distinguished separately for the above-ground parts and the roots.

). Biochar had a positive effect on reducing heavy metal concentrations in the ZnCu, ZnNi, and CuNi samples in the above-ground parts but only had a similar effect on the Ni content in the ZnCuNi sample. Biochar did not significantly modify the heavy metal content in the above-ground parts of Zea mays in plots where Zn, Cu, and Ni were applied individually. In most cases, regardless of the type of object, the content of the tested metals was higher in the roots than in the above-ground parts.

3.3. The Effect of Heavy Metals and Biochar on Soil Enzyme Activity

The biochemical activity of the soil was significantly modified in response to contamination with heavy metals, applied either alone (Zn, Cu, and Ni) or in combination (ZnCu, ZnNi, CuNi, and ZnCuNi), as well as biochar addition (

Table 5. Enzyme activity in soil contaminated with heavy metals.

Treatment	Dehydrogenases μmol TFF kg−1 d.m. h−1		Catalase mol O2 kg−1 d.m. h−1		Urease mmol N-NH4 kg−1 d.m. h−1		Acid Phosphatase		Alkaline Phosphatase		β-Glucosidase		Arylsulfatase
							mmol PNP kg−1 d.m. h−1
	CBw	CB	CBw	CB	CBw	CB	CBw	CB	CBw	CB	CBw	CB	CBw	CB
C	3.040 b ± 0.027	3.684 a ± 0.007	0.378 e ± 0.007	0.407 bc ± 0.004	0.364 h ± 0.028	1.015 b ± 0.030	1.491 c ± 0.014	1.865 a ± 0.014	0.868 f ± 0.027	1.075 d ± 0.015	0.418 e ± 0.003	0.699 a ± 0.007	0.667 h ± 0.013	0.913 g ± 0.042
Zn	1.655 d ± 0.040	2.059 c ± 0.014	0.360 f ± 0.004	0.400 b ± 0.006	0.252 i ± 0.027	0.733 f ± 0.025	1.274 e ± 0.009	1.550 b ± 0.014	0.957 e ± 0.027	1.153 c ± 0.014	0.425 e ± 0.005	0.429 e ± 0.003	1.174 e ± 0.087	1.934 a ± 0.055
Cu	0.156 n ± 0.007	0.378 k ± 0.014	0.364 f ± 0.008	0.387 de ± 0.007	0.255 i ± 0.029	0.172 j ± 0.027	0.952 i ± 0.002	1.436 d ± 0.100	0.759 h ± 0.027	0.759 h ± 0.003	0.426 e ± 0.003	0.474 d ± 0.004	1.159 e ± 0.007	0.901 g ± 0.027
Ni	0.892 h ± 0.007	1.138 g ± 0.004	0.398 d ± 0.004	0.435 a ± 0.003	1.049 a ± 0.028	0.885 de ± 0.032	1.219 g ± 0.012	1.492 c ± 0.038	0.710 i ± 0.027	0.823 f ± 0.005	0.376 f ± 0.003	0.420 e ± 0.002	1.485 c ± 0.027	1.713 b ± 0.018
ZnCu	0.315 l ± 0.014	0.312 l ± 0.074	0.382 e ± 0.004	0.392 c ± 0.004	0.342 h ± 0.028	0.226 i ± 0.024	1.098 h ± 0.083	1.274 e ± 0.064	0.948 e ± 0.027	1.090 d ± 0.034	0.520 c ± 0.006	0.628 b ± 0.005	0.869 g ± 0.019	1.075 f ± 0.058
ZnNi	1.222 f ± 0.014	1.388 e ± 0.027	0.398 d ± 0.003	0.401 c ± 0.004	1.000 b ± 0.027	0.878 e ± 0.036	1.343 d ± 0.021	1.433 d ± 0.056	1.295 a ± 0.027	1.217 b ± 0.041	0.378 f ± 0.004	0.456 d ± 0.003	0.463 j ± 0.055	1.322 d ± 0.018
CuNi	0.260 m ± 0.054	0.679 i ± 0.043	0.393 d ± 0.005	0.416 b ± 0.006	0.713 f ± 0.029	0.490 g ± 0.040	1.241 f ± 0.060	1.345 d ± 0.025	0.711 i ± 0.027	0.807 fg ± 0.050	0.690 a ± 0.006	0.546 c ± 0.003	1.114 f ± 0.072	0.577 i ± 0.045
ZnCuNi	0.261 m ± 0.081	0.410 j ± 0.051	0.386 de ± 0.004	0.411 b ± 0.005	0.944 c ± 0.029	0.911 d ± 0.051	1.239 f ± 0.015	1.417 cd ± 0.024	0.989 d ± 0.027	1.038 d ± 0.023	0.464 d ± 0.004	0.685 ab ± 0.006	1.553 c ± 0.088	1.356 d ± 0.081

CBw—without biochar; CB—with biochar; C—uncontaminated soil; Zn, Cu, and Ni applied at a dose of 420 mg kg⁻¹ d.m. soil; ZnNi and CuNi applied at a dose of 210 mg kg⁻¹ d.m. soil; ZnCuNi applied at a dose of 140 mg kg⁻¹ d.m. soil. The letters a–n denote homogeneous groups, separately for each enzyme.

). Dehydrogenase activity was highest in treatments contaminated with Zn and lowest in treatments contaminated with Cu. In soil contaminated with more than one heavy metal, dehydrogenase activity was highest in ZnNi treatments. In all treatments, the analyzed parameter was higher in soil amended with biochar (CB) than in soil without biochar addition (CBw). Heavy metals, applied either alone or in combination, did not induce an equally severe decrease in catalase activity. Catalase activity was highest in Zn and Ni treatments and in soil amended with biochar. Urease activity also increased in response to biochar, but considerable differences in this parameter were observed between treatments contaminated with heavy metals. Urease activity was lowest in Cu treatments, both with and without biochar addition. In turn, urease activity was higher in Ni, ZnNi, CuNi, and ZnCuNi treatments than in the control treatment without biochar. The activity of acid phosphatase and alkaline phosphatase also increased in response to biochar. In the group of the tested heavy metals, Cu exerted the most inhibitory effect on these enzymes. However, alkaline phosphatase activity was higher in Zn, ZnCu, ZnNi, and ZnCuNi treatments than in uncontaminated treatments, both with and without biochar. Biochar also had a positive effect on β-glucosidase activity. The negative impact of heavy metals was most evident in Ni and ZnNi treatments. The greatest differences were observed in arylsulfatase activity, depending on the applied heavy metals, their combinations, and biochar addition. Treatments contaminated with Zn were characterized by the highest arylsulfatase activity, which reached 1.934 mmol PNP kg⁻¹ d.m. h⁻¹ in the presence of biochar and 1.174 mmol PNP kg⁻¹ d.m. h⁻¹ in the absence of biochar.

Dehydrogenases play an important role in soil redox reactions, and they are sensitive indicators of the viability of soil microorganisms (Figure 4). The obtained results indicate that heavy metals exert a negative effect on dehydrogenase activity. In treatments contaminated with Zn, the IF_Hm was determined at −0.456 (CBw) and −0.441 (CB), which suggests that this parameter was not significantly affected by biochar addition. In turn, the IF_Hm reached −0.949 (CBw) and −0.897 (CB) in treatments contaminated with Cu, which shows that this heavy metal strongly inhibits soil enzyme activity regardless of biochar addition. The values of IF_Hm in CuNi and ZnCu treatments indicate that these heavy metals suppress dehydrogenase activity both in the presence and absence of biochar. Similar observations were made in ZnCuNi treatments, where IF_Hm values confirmed the inhibitory effect of heavy metals on dehydrogenase activity, particularly in soil not amended with biochar. Catalase plays a key role in the decomposition of hydrogen peroxide, and it is an indicator of oxidative stress in soil. In contrast to dehydrogenase, catalase activity is not significantly inhibited by all heavy metals. Urease, which catalyzes the reaction of urea with water to produce ammonia and carbon dioxide, responds differently to heavy metals. Both Zn and Cu decreased urease activity, particularly in the presence of CB, whereas Ni in combination with other heavy metals, such as ZnNi (1.747 CBw), can even enhance urease activity.

Acid phosphatase responded negatively to Zn, as demonstrated by IF_Hm values which reached −0.146 for CBw and −0.169 for CB (Figure 4). Copper exerted a more inhibitory effect on the activity of this enzyme, and IF_Hm values were −0.362 for CBw and −0.230 for CB. Alkaline phosphatase responded in a more ambiguous manner to the same factors. Copper clearly decreased the activity of this enzyme, and IF_Hm values reached −0.126 for CBw and −0.294 for CB. In treatments contaminated with Ni, IF_Hm values of −0.182 for CBw and −0.234 for CB indicate that this metal has a negative impact on alkaline phosphatase activity. An analysis of IF_Hm values revealed that the examined metals had a minor influence on β-glucosidase. The activity of this enzyme increased in ZnCu treatments (IF_Hm = 0.244 for CBw) but decreased under exposure to Zn (IF_Hm = −0.102 for CB). Arylsulfatase activity increased considerably in response to ZnCuNi and biochar (IF_Hm = 1.328 for CBw) but decreased after the application of Cu (IF_Hm = −0.322 for CB). Complex interactions were observed in treatments exposed to all three heavy metals (ZnCuNi) and biochar. In ZnCuNi treatments, enzyme activity was higher in soil without biochar (IF_Hm = 1.328 for CBw) than in soil with biochar addition (IF_Hm = 0.485 for CB).

The data in Figure 5 clearly indicate that biochar enhanced dehydrogenase activity. In treatments contaminated with Zn, Cu, and Ni, IF_CB values reached 0.244, 1.423, and 0.276, respectively, and were higher than in the control treatment (0.212). The values of IF_CB were also higher in CuNi and ZnCuNi treatments than in uncontaminated soil. In turn, the IF_CB decreased in ZnCu treatments. Catalase also responded positively to biochar, but its IF_CB values were lower in comparison with dehydrogenases. In contrast to dehydrogenases and catalase, biochar did not alleviate the negative effects of heavy metals on urease activity. The only exception was the Zn treatment, where the IF_CB increased relative to the control treatment. Biochar had a positive influence on acid phosphatase activity, particularly in the Cu treatment, where IF_CB reached 0.508 and was two times higher than in the control treatment. Alkaline phosphatase activity was not significantly modified by biochar, and IF_CB values were lower in contaminated than uncontaminated treatments. An analysis of β-glucosidase activity produced similar results, and the IF_CB was lower in contaminated treatments than in the control treatment. Arylsulfatase activity increased considerably in response to Zn and ZnNi, as shown by high values of IF_CB. In turn, negative IF_CB values in Cu, CuNi, and ZnCuNi treatments suggest that biochar addition did not mitigate the adverse effects of these heavy metals on arylsulfatase activity.

4. Discussion

This study demonstrated that the development of maize plants is influenced by the presence of heavy metals in soil. Maize plants grown in soil contaminated with a single metal were characterized by lower yields of above-ground parts and roots. Copper and Ni exerted more negative effects than Zn, which could be due to their higher toxicity for biological processes in plants [40,41]. High soil contamination with Zn and Cu can affect plants not only directly but also indirectly through imbalances in the uptake of other necessary nutrients for proper plant functioning [65,66]. The growth of maize plants was most strongly suppressed under the combined influence of all three heavy metals (ZnCuNi). Biochar promoted plant growth in contaminated soils. A greater increase in biomass yield was observed in soil that was amended with biochar than in non-amended soil, which suggests that biochar effectively alleviated heavy metal stress [67,68,69]. Biochar was particularly effective in treatments contaminated with more than one heavy metal, which could be attributed to biochar’s ability to immobilize heavy metals and decrease their bioavailability to plants [70,71]. This reduced the toxic stress exerted by heavy metals on the root system and promoted nutrient absorption and biomass growth [16,17]. From a practical point of view, biochar applied in maize cultivation can increase yields and improve plant health. A stronger root system also promotes more effective utilization of water and soil nutrients, which additionally enhances plant development [6,72,73]. The results of the study indicate various interactions between heavy metals and biochar, as well as the accumulation of these metals in different parts of Zea mays. The low heavy metal content observed in the above-ground parts and roots of maize grown in uncontaminated soil emphasizes the importance of a healthy soil environment in reducing the bioaccumulation of metals. This suggests that environmental factors, such as soil quality, are crucial for the health and survival of plants in the presence of contaminants [41]. The observation that heavy metal concentrations were higher in the maize roots than in the above-ground parts is also consistent with the results of numerous studies documenting the tendency of plants to accumulate metals in their root systems. Plants often bind metals in their roots as a defense mechanism to limit their transfer to the above-ground parts, thus protecting photosynthesis and overall growth [74,75,76].

The analysis of soil enzyme activity also produced interesting results. In our studies, enzymes activity tended to decrease under the influence of the heavy metals used. However, some exceptions were observed: urease activity increased in the presence of nickel and decreased in the presence of copper. Nickel acts as a cofactor in the urease produced by microorganisms. It plays a key role in urease activity and forming a coordination bond with the enzyme’s functional groups [77]. Conversely, copper plays a different role in urease: if it is present in excess in the environment, it can impair substrate binding [78]. Biochar stimulates soil enzymes, thus enhancing soil microbial activity and soil fertility [79]. Biochar influences soil enzyme activity by interacting with soil microbiota. This soil amendment may create a favorable environment for microbial growth, increase microbial biodiversity, and stimulate the growth of microbial populations that produce enzymes. Therefore, biochar can support soil detoxification by enzymes [80,81,82]. Enzymes such as dehydrogenases, urease, acid phosphatase, and alkaline phosphatase play a key role in biogeochemical processes by decomposing organic matter and promoting the release of nutrients that are essential for plant growth [21,23]. When these processes are intensified, soil quality improves and the growth and development of plants is accelerated [43,46]. Biochar significantly contributes to soil fertility [83], in particular by exerting a positive influence on soil pH, EBC, and the total cation exchange capacity (CEC) of the soil [83,84]. The results of this study corroborate these findings. An increase in the pH of soil acidified by heavy metals creates a more supportive environment for plant development by increasing the availability of numerous nutrients [76]. Total EBC responded differently to biochar addition, but its value increased in most cases, which may suggest that biochar increases the retention of calcium and magnesium ions, thus improving the structural properties of soil. In turn, an increase in CEC promotes nutrient retention in soil and significantly contributes to the stability of crop yields [85]. The present study also demonstrated that biochar significantly increased the content of organic carbon in soil. Due to its structural stability, biochar is resistant to microbial degradation, and carbon is retained in soil for a long period of time [84]. In turn, higher C_org content improves soil structure, water-holding capacity, and the biological properties of soil [79].

The mechanisms for mitigating the negative effects of heavy metals in soil are based on a number of physicochemical properties of biochar that are crucial to its effectiveness. The first mechanism of biochar’s action is its ability to absorb heavy metals. Biochar is characterized by its large specific surface area and a porous structure, which increases its ability to bind heavy metals. The physical and chemical sorption by biochar reduces the bioavailability of heavy metals for plants and soil organisms, thus minimizing their toxicity. The efficiency of this process depends on the pH value and composition of the biochar as well as its structure and surface charge [86,87,88,89]. Another important property of biochar is its effect on soil pH. As most biochar is alkaline, it can raise the pH of acidic soils. A higher pH promotes the precipitation of heavy metals in the form of compounds that are less soluble in water and therefore less bioavailable. This limits their mobility and uptake by plants [71]. In addition, biochar stimulates the development of soil microorganisms, which in turn contribute to soil detoxification and the immobilization of heavy metals [18,19]. Our own research has shown that the addition of biochar to soil can reduce the mobility of zinc, copper, and nickel. Biochar can also influence the formation of metal complexes with organic substances, thereby further reducing the toxic effect of heavy metals [41,84]. The effect of biochar on the mobility of heavy metals is related to the physicochemical properties of the soil, and depending on their levels, the impact of biochar may vary across different soil types [41,90]. In general, biochar adsorbs heavy metals and contributes to their accumulation on its surface, thereby reducing their availability to plants [91]. On the other hand, depending on the chemical and physicochemical properties of the soil, biochar may also activate forms of heavy metals that were previously unavailable to plants, making them more susceptible to leaching and uptake. This process can disrupt the balance between water-soluble and insoluble forms of heavy metals. The former are bioavailable to plants, whereas the latter are not. For these reasons, the influence of biochar on the total content of heavy metals may vary between different soils [92].

The results of the current study suggest that biochar can effectively alleviate the negative impact of heavy metal contamination on maize yields. Biochar not only promotes the growth of plants exposed to environmental stress but also improves soil quality by inducing positive, long-term changes in the structure and biological activity of the soil. Despite these promising results, further research is needed to validate biochar’s properties under field conditions and assess the potential profits resulting from large-scale application of biochar in agricultural practice.

5. Conclusions

Soil contamination with Zn, Cu, and Ni as well as combinations of two (ZnCu, ZnNi, and CuNi) and three heavy metals (ZnCuNi) decreased the yields of above-ground parts and roots in Zea mays plants and induced negative changes in the biochemical, chemical, and physicochemical properties of the soil. This study revealed that soil ecology is affected not only by the type and concentration of a given heavy metal but also by total contamination levels. Soil contamination with all three heavy metals applied at 140 mg kg⁻¹ soil each exerted more toxic effects on Zea mays than contamination with Zn alone or Cu alone at a dose of 420 mg kg⁻¹ soil. Nickel exerted the most inhibitory effects on the growth and development of maize plants, whereas Cu induced the most undesirable changes in the biochemical properties of the soil. These results emphasize the complexity of the interactions between heavy metals and their subsequent impact on both plant health and soil quality. Biochar application created a more favorable environment for plant growth in soils contaminated with heavy metals by decreasing the acidity, increasing the content of organic carbon and total nitrogen, and stimulating soil enzyme activity. These changes suggest that the negative impact of Zn, Cu, and Ni is being offset, which highlights the effectiveness of biochar in supporting the revitalization of soils contaminated with heavy metals. These findings indicate that biochar is an effective amendment that can help restore the fertility of soils contaminated with heavy metals, thus facilitating their management.