Biochar-mediated remediation of nickel and copper improved nutrient availability and physiological performance of dill plants

Abstract

The presence of heavy metals, such as copper and nickel, in the rhizosphere reduces the physiological efficiency and growth of plants. This study evaluated the effects of plum tree biochar levels (0, 15, 30, and 45 g kg^-1 soil) with and without copper sulfate (200 mg kg^-1 soil), nickel sulfate (400 mg kg^-1 soil), and their combination on dill plants in a factorial experiment with a randomized complete block design in three replicates. The results indicated that the presence of copper and nickel in the soil had detrimental effects on the growth and physiological performance of dill. Specifically, copper stress alone reduced biomass by 31%, nickel stress by 27%, and their combined treatment by 37.7%. On the other hand, incorporating biochar into the soil decreased the uptake of heavy metals, oxidative stress, and the production of osmotic regulators in the plants, while enhanced nutrient uptake (N, K, Ca, Mg, Fe, and Zn), photosynthetic pigments, and plant biomass. Increasing biochar application rate in the soil did not have any additional beneficial effect on growth and physiological characteristics of plants. These results suggest that the low rate of biochar (15 g kg^-1) from agricultural wastes is an appropriate soil amendment to remediate copper and nickel pollutants in the rhizosphere to enhance nutrient availability and plant performance. Future research could focus on the long-term efficacy of biochar under diverse field conditions, soil types, and plant species to optimize sustainable agricultural practices.

Introduction

Plants are often sensitive to an abundance of certain heavy metals, because these elements have a significant impact on their physiological processes. These elements can exhibit high mobility and toxicity even at low concentrations^1,2. Heavy metals may disrupt photosynthesis, respiration, cell growth, nutrient and water absorption, as well as plant metabolism. Nickel (Ni) and copper (Cu) are essential micronutrients for plants, required only in small quantities³, with their excess being more detrimental than their deficiency⁴. The Ni has a role in various physiological and metabolic processes, including nitrogen metabolism, photosynthesis, and enzyme activation⁵. Low level of Ni reduced production of proteins, organic acids, and total nitrogen. This deficiency is associated with the accumulation of nitrate and specific amino acids in the plant⁶. The Cu is an essential component of proteins and enzymes that plays a key role in electron transfer and reduction reactions. It actively participates in various biological reactions as an enzyme cofactor and serves as an electron carrier in processes such as oxidative phosphorylation and photosynthesis⁷. However, an excessive amount of Cu can induce changes in plant cells, disrupting membrane permeability and enzyme activity associated with respiration and photosynthesis⁸.

Excessive Cu can have a negative impact on plant growth and development. It reduces the photosynthetic pigments, damages the photosynthetic apparatus, and alters the chloroplast structure. The Cu also alters the protein and lipid composition of the thylakoid membrane⁸. Additionally, an excess of both Ni and Cu affects the absorption and transfer of iron (Fe), zinc (Zn), and manganese (Mn). This interference may lead to deficiencies or imbalances in their absorption and disrupt water relations within the plant^9,10. The presence of both Ni and Cu in the soil may enhance their toxic effects on plants. In this context, the reactivity and behavior of Ni and Cu may be affected by their mutual interactions, complexation with other compounds, and the presence of additional substances in the soil system. Different plant species exhibit varying levels of tolerance to high concentrations of these metals. Moreover, soil pH, organic matter, and other properties can also influence a plant’s access to these metals^11,12. Soil pH specifically affects the solubility and mobility of Ni and Cu within the soil. Application of biochar may also alter the activity and behavior of Ni and Cu.

A cost-effective and eco-friendly method to minimize the harmful impact of heavy metal toxicity on plants is to add biochar to the rhizosphere¹³. This method can limit the entry of soil pollutants into plants¹⁴. Biochar is also beneficial as an additive in improving the physical and chemical properties of soil, reducing the mobility of heavy metals¹⁵, and limiting plant access to these metals. According to Farhangi-Abriz and Ghassemi-Golezani¹⁶, biochar is highly effective in releasing nutrients into the rhizosphere and facilitating better nutrient absorption by plants. Biochar, and especially chemically modified biochars, enhance cation exchange capacity that can facilitate nutrient uptake by plants. Various mechanisms, including ion exchange, and chemical interaction with functional groups on the surface of biochar, contribute to the adsorption of heavy metals¹⁷. Some studies have also reported potential negative effects of biochar on crop productivity and soil physicochemical properties¹⁸. The reason for the decline in crop productivity is linked to the existence of harmful substances and extremely volatile compounds in biochar, which can hinder plant growth and overall crop output¹⁹.

Dill (Anethum graveolensL.) is an aromatic and beneficial medicinal plant used as a medicine and vegetable. This plant belongs to the Umbelliferae family and contains numerous secondary metabolites with considerable medicinal value²⁰²¹,. One of the main reasons for selecting dill in this study is that soil pollutants, such as heavy metals, do not enter its essential oil, making it a safe and valuable product for commercial use even under polluted conditions²². However, these pollutants can interfere with nutrient uptake, affecting the growth and physiological performance of this plant. The objective of this research is to investigate the impacts of Cu and Ni pollutions on dill plants and to determine the ability of biochar to reduce the absorption of these metals by plants. Utilization of biochar is considered as an affordable and effective strategy for mitigating the negative impacts of heavy metals on plant growth. This study has the potential for practical, economical, and low-cost applications to help plant adaptation to the changing environment and pollution.

Materials and methods

Experimentation

This research was conducted in a greenhouse at the University of Tabriz in Iran. The experiment was arranged as a factorial based on a randomized complete block design with three replications. The first factor was heavy metals, including a non-polluted and polluted soil with copper sulfate (CuSO₄.5H₂O), nickel sulfate (NiSO₄.6H₂O), and a combination of both. The second factor involved varying levels of plum tree biochar, specifically 0, 15, 30, and 45 g/kg soil. The soil was loamy silt with a pH of 6.3, which was collected from the upper layers (0–30 cm) of the research farm of the University of Tabriz. The biochar was purchased from the Fifth Season Company in Shiraz. It was produced from the pyrolysis of pruned branches and leaves of plum trees at temperatures of 500 °C, with a heating rate of 5 °C per minute up to a final temperature, under oxygen-deficient conditions. Table 1 displays the properties of original soil and biochar. In this experiment, a total of 52 plastic pots were utilized. Of these, 48 pots were considered for sowing, while the remaining 4 pots with varying levels of biochar were left unsown to determine the control water status and the timing of irrigation. Each plastic pot (26 × 23 cm) was filled with approximately 5 kg of soil, which was then mixed with a specific amount of biochar based on the respective treatments. At least 30 seeds of Hungarian ecotype dill were sown at a depth of 5 mm in each pot. Then, 200 mg of nickel sulfate and 400 mg of copper sulfate per kilogram of soil were dissolved in the necessary amount of water to reach 100% field capacity, and then added to the pots. The water loss from the pots was compensated for by regularly weighing the unsown pots. After the seedlings were established, the plants were thinned, and only 10 plants were kept in each pot. Subsequently, 2 g per liter of NPK fertilizer was added to the pots. At the flowering stage (approximately 80 days after sowing), six plants were harvested from each pot to measure leaf pigments, phenols, proline, antioxidant enzymes, malondialdehyde (as an index of lipid peroxidation), hydrogen peroxide, and soluble sugars. At maturity (approximately 120 days after sowing), the remaining four plants were harvested to determine plant biomass, nutrient content, and Ni and Cu concentrations in shoots.

Table 1.

Some of physicochemical properties of the original experimental soil and biochar.

Soil		Biochar
pH	6.3	pH	8.2
CEC (cmol kg−1)	19.8	CEC (cmol kg−1)	20.6
Moisture (%)	< 1	Moisture (%)	< 1
N (%)	0.03	N (%)	0.79
P (mg kg−1)	32	P (%)	0.14
K (mg kg−1)	123	K (%)	0.17
Mg (mg kg−1)	28	Ca (%)	4.26
Ca (mg kg−1)	19.2	Mg (%)	5.77
Zn (mg kg−1)	32	C/N	10.20
Fe (mg kg−1)	4
Cu (mg kg−1)	11.6
Ni (mg kg−1)	22.4
EC (dSm−1)	2.5
OC (g kg−1)	12
Texture	Silty loam

EC: electrical conductivity, OC: organic carbon, CEC: cation exchange capacity.

Elements contents

The leaf samples were dried at laboratory temperature (20–25 °C) and powdered. The Kjeldahl method was applied to determine the nitrogen content²³. The levels of Ni, Cu, and other cations in the leaves were analyzed by digesting the samples with nitric acid (HNO₃). After drying the samples for ash at 550 °C for 7 h, the other metals (mg g⁻¹DW) were determined, using atomic absorption spectrophotometry²⁴.

Osmolytes

About 0.5 g of leaf tissue was powdered by liquid nitrogen, and subsequently mixed with 5 mL of 50 mM potassium phosphate buffer at a pH of 6.8. The resulting mixture was centrifuged at 12,000 rpm for 20 min at 4 °C. The supernatant obtained was used for measuring the protein content and antioxidant enzymes. The protein content in the leaf was determined using the Bradford method²⁵. A sub-sample of 200 μL from the extract was mixed with 300 μL of distilled water and 1500 μL of Bradford’s reagent. After 15 min of incubation in the dark, the absorption of the mixture was measured at 595 nm. The protein content was calculated as mg g⁻¹ FW.

The content of leaf proline was determined following the method described by Bates, Waldren, and Teare²⁶. A sample of fresh leaf tissue (500 mg) was powdered with liquid nitrogen and then extracted with 5 ml of 3% sulfosalicylic acid. The mixture was centrifuged at 2000 rpm for 5 min. Subsequently, 1 mL of the supernatant was poured into a test tube and mixed with 1 mL of distilled water, 1 mL of ninhydrin reagent, and 1 mL of glacial acetic acid. The mixture was heated in a water bath at 100 °C for an hour and then cooled to room temperature. Some amount of toluene (2 mL) was added to each sample. This resulted in the formation of two phases. The absorbance of the upper phase was recorded at 520 nm. The proline content in the samples was expressed as μg g⁻¹ FW.

The content of soluble sugars was determined using the phenol–sulfuric acid method²⁷. Fresh leaf tissue weighing 0.1 g was mixed with 5 ml of 70% ethanol and stored in darkness at a temperature of 4 °C in the refrigerator for one week. Then, 1 mL of the sample solution was taken and diluted with distilled water to a total volume of 2 mL. After adding 1 mL of 5% phenol and 5 mL of 60% sulfuric acid, the absorbance was measured at a wavelength of 485 nm. Finally, the content of soluble sugars in each sample was determined using the glucose standard curve, and the results were expressed in mg g⁻¹ FW.

Lipid peroxidation, H₂O₂ generation, and antioxidative activities

To assess the level of lipid peroxidation (Malondialdehyde content—MDA), 0.2 g of a leaf sample was homogenized in 2 ml of 0.1% trichloroacetic acid (TCA) using a mortar and pestle. The extract was then centrifuged at 13,000 rpm for 10 min. Then, 1 mL of 0.5% thiobarbituric acid (TBA) was added to 0.5 mL of the supernatant, and the resulting mixture was heated for 30 min at 95 °C. Afterward, it was centrifuged again at 10,000 rpm for 10 min. The supernatant liquid was collected, and the absorbance of the reaction mixture was measured at 532 nm and 600 nm²⁸. The difference between these absorbances was used to calculate the amount of MDA (nmol g⁻¹ of wet weight).

The content of hydrogen peroxide (H₂O₂) was measured using the Alexieva method²⁹, which involves the reaction of H₂O₂ with potassium iodide (KI). In this method, 0.5 g of fresh leaf tissue was homogenized in 0.1% trichloroacetic acid (TCA). The extract was centrifuged at 12,000 rpm for 15 min at 4 °C. Then, 0.5 mL of the supernatant was mixed with 0.5 mL of 100 mM potassium phosphate buffer (pH 7) and 2 mL of 1 M potassium iodide solution. After incubating the samples in darkness at room temperature for an hour, the absorbance was read at 390 nm. The H₂O₂ was quantified using a standard curve and expressed as µmol g⁻¹ FW.

Peroxidase (POX) activity was assessed by estimating its ability to increase the absorbance of guaiacol. The absorbance was read at 470 nm for 120 s, following the method described by Chance and Maehly³⁰. The enzyme activity was expressed as 1 mM of guaiacol oxidized per minute per mg of protein. Polyphenol oxidase (PPO) activity was measured by determining the rate of pyrogallol oxidation, following the method described by Raymond, Pakariyathan, and Azanza³¹. The absorbance was measured at 430 nm for two minutes.

Phenols

The leaf samples were crushed with 5 ml of 80% methanol in a mortar. After that, the mixture was spun in a centrifuge at 10,000 rpm at 4 °C for 5 min. The supernatant was collected to measure total phenol contents. Next, 2.8 mL of water, 2 ml of 2% sodium carbonate, and 100 μL of Folin-Ciocalteu reagent were mixed with 100 μL of the plant extract. After 30 min, the absorbance of the solutions was read at a wavelength of 720 nm³². The content of total phenol in the extract was reported as mg GAE g⁻¹ DW.

Leaf pigments

The levels of chlorophylls and carotenoids were measured according to Arnon³³and Maclachlan and Zalik³⁴, respectively. Each sample (approximately 0.5 g) was homogenized in 5 mL of 80% acetone and then centrifuged at 13,000 rpm for 20 min. The absorbance of the solution was measured at wavelengths of 645 nm and 663 nm (for chlorophylls a and b) and 470 nm (for carotenoids).

Plant biomass

The shoots of 4 remaining plants were cut and dried in the shade, and then the shoots were weighed, and the shoot mass per plant for each pot was calculated.

Statistical analysis

The data were analyzed using PROC GLM in SAS statistical software (version 9.4). The means were compared by Tukey’s HSD test at P ≤ 0.05. Figures and tables were prepared for the significant effects of individual factors or their interactions (ANOVA tables are presented in the supplementary file as appendices).

Results

Leaf Ni and Cu contents

The interaction between heavy metals and biochar treatments on Cu content of leaves was significant (Table S1). However, the Ni content of leaves was only affected by heavy metals and biochar treatments individually, with no significant interaction (Table S1). The addition of biochar to the soil reduced the Cu content in leaves by 22–24% in Cu treatment, and by 24–30% in combined treatment of Cu and Ni. However, none of the biochar application rates changed the Cu content of leaves in non- polluted and Ni polluted soil (Fig. 1). Soil contamination with Ni increased the endogenous content of this metal in plant leaves by about 110.4%. Conversely, biochar treatments resulted in a 31%—40% reduction in Ni content of plants (Table 2). All of the biochar rates had a similar effect on reducing Cu and Ni levels in leaf tissues.

Fig. 1.

The mean contents of copper in dill leaves affected by heavy metals × biochar interaction. Data represents the average ± standard error. Different letters between the treatments indicate significant difference at p ≤ 0.05.

Table 2.

The content of some elements (mg g⁻¹ DW) in dill leaves under heavy metals pollution and biochar treatments.

Treatments	Nickle	Nitrogen	Potassium	Calcium	Magnesium	Iron	Zinc
Heavy metals
Non-polluted	1.34 ± 0.11 c	31.98 ± 0.95 a	28.90 ± 0.63 a	11.83 ± 0.45 a	10.21 ± 0.37 a	2.62 ± 0.25 a	1.25 ± 0.11 a
Nickle	2.82 ± 0.17 a	24.77 ± 0.90 b	23.21 ± 0.55 bc	8.65 ± 0.42 b	8.49 ± 0.21 b	2.20 ± 0.29 ab	1.04 ± 0.07ab
Copper	1.78 ± 0.15 b	24.62 ± 0.72 b	22.42 ± 0.51 c	8.07 ± 0.50 b	7.82 ± 0.56 c	2.13 ± 0.26 b	0.96 ± 0.09 b
Nickle + Copper	2.75 ± 0.26 a	23.80 ± 0.66 b	24.27 ± 0.62 b	8.90 ± 0.46 b	8.75 ± 0.40 b	2.07 ± 0.25 b	0.99 ± 0.07 b
Biochar levels
Non-biochar	3.00 ± 0.29 a	24.23 ± 1.18 b	0.79 ± 0.90 b	7.21 ± 0.50 c	7.05 ± 0.14 b	1.02 ± 0.06 c	0.78 ± 0.06 b
15 g biochar kg−1 soil	2.07 ± 0.20 b	25.97 ± 1.33 ab	0.92 ± 1.03 a	9.99 ± 0.52 ab	8.89 ± 0.15 a	2.41 ± 0.13 b	1.02 ± 0.05 ab
30 g biochar kg−1 soil	1.81 ± 0.17 b	27.70 ± 1.19 a	0.94 ± 0.62 a	9.62 ± 0.52 b	9.97 ± 0.14 a	2.61 ± 0.22 ab	1.21 ± 0.11 a
45 g biochar kg−1 soil	1.80 ± 0.18 b	27.27 ± 1.20 a	0.95 ± 0.91 a	10.70 ± 0.47 a	9.35 ± 0.15 a	2.97 ± 0.16 a	1.22 ± 0.07 a

Data represents the average ± standard error.

Different letters between the treatments indicate significant difference at p ≤ 0.05.

Nutrients content in plant leaves

The impacts of heavy metals and biochar treatments on the levels of N, K, Ca, Mg, Fe, and Zn in plant leaves were significant (Table S1). However, the interaction between heavy metals and biochar treatments on these parameters was not significant (Table S1). Soil contamination with Cu and Ni reduced the nutrient content in plant leaves by up to 31%. In most cases, there was no significant difference in the nutrient content of leaves under various heavy metal treatments. However, the lowest level of potassium content in leaf tissues was observed when Cu was present in the soil (in the individual form), resulting in a 22.4% decrease compared to the non-polluted condition. The addition of biochar to the soil improved the N (7–14%), K (16–20%), Ca (33–48%), Mg (26–41%), Fe (136–191%), and Zn (30–56) contents in dill leaves compared to the non-biochar treatment (Table 2). These improvements were observed across all application rates compared to the non-biochar treatment. Almost all of the biochar application rates had a similar effect on increasing nutrient content of plant tissues. However, in some cases, e.g. Fe content, the addition of 45 g of biochar was the most effective treatment.

Osmolytes

The impact of heavy metals and biochar treatments on the protein content of leaves was not significant (Table S2). However, the levels of proline and soluble sugars were significantly affected by heavy metals and biochar treatments. The interaction of heavy metals and biochar treatments was only significant for proline content (Table S2). The levels of proline and soluble sugars significantly increased due to the addition of heavy metals to the soil, with an average increase of 189–220% for proline (Fig. 2) and 31–38% for soluble sugars (Table 3) compared to the non- polluted soil. All of the heavy metal treatments had a similar effect on the increase in proline and soluble sugars content in plant leaves. On the other hand, biochar treatments reduced the content of soluble sugars in plant leaves by up to 20%, compared to non-biochar plants. All of the biochar application rates had a similar effect on reducing the soluble sugar content in plant leaves. The reduction in proline content was only significant in response to biochar addition to the soil under combined form of heavy metals, where a decrease of up to 59.89% was observed.

Fig. 2.

The contents of proline (A) and malondialdehyde (B) in dill leaves affected by heavy metals × biochar interaction. Data represents the average ± standard error. Different letters between the treatments indicate significant difference at p ≤ 0.05.

Table 3.

The concentration of soluble proteins and carbohydrates (mg g⁻¹ FW) in dill leaves under heavy metals pollution and biochar treatments.

Treatments	Soluble proteins	Soluble carbohydrates
Heavy metals
Non-polluted	13.85 ± 0.05 a	38.39 ± 1.23 b
Nickle	13.57 ± 0.03 a	50.41 ± 1.96 a
Copper	13.90 ± 0.09 a	52.45 ± 2.68 a
Nickle + Copper	13.80 ± 0.05 a	53.27 ± 2.84 a
Biochar levels
Non-biochar	13.81 ± 0.06 a	55.11 ± 3.58 a
15 g biochar kg−1 soil	13.59 ± 0.06 a	49.03 ± 2.69 ab
30 g biochar kg−1 soil	13.83 ± 0.05 a	46.36 ± 2.02 b
45 g biochar kg−1 soil	13.66 ± 0.06 a	44.02 ± 1.85 b

Data represents the average ± standard error.

Different letters between the treatments indicate significant difference at p ≤ 0.05.

Lipid peroxidation and antioxidative activities

The MDA content in dill leaves was affected by the interactions of heavy metals and biochar treatments (Table S3), while the H₂O₂ content and PPO activity of leaves were only affected by the heavy metals and biochar treatments individually (Table S3). The activity of the POD enzyme was only affected by heavy metals, and the biochar treatments did not significantly alter this enzyme activity. Application of biochar treatments under non-polluted conditions did not change the MDA content of dill leaves. However, application of biochar in the presence of heavy metals in the soil led to a reduction in the MDA content of dill leaves by about 37.5% to 44.8% compared to the non-biochar treatment (Fig. 2). Soil contamination with heavy metals increased the generation of H₂O₂, while biochar treatments reduced the generation of H₂O₂ in plant leaves by about 25% to 30.8% (Table 4). There was no significant difference in the reduction of H₂O₂ in plant leaves among different levels of biochar application. The highest levels of PPO and POD activities were observed when a combination of the heavy metals was added to the soil (Table 4). The biochar treatments significantly reduced PPO activity in dill leaves by up to 22.4%, but had no effect on POD activity. All of the biochar rates had a similar effect on reducing PPO activity.

Table 4.

Changes in peroxidase and polyphenol oxidase activities (U g⁻¹ FW), H₂O₂ generation (µmol g⁻¹ FW) and phenolic compounds (mg gallic acid equivalent g⁻¹ FW) of dill leaves under heavy metals pollution and biochar treatments.

Treatments	Peroxidase	Polyphenol oxidase	H2O2	Phenols
Heavy metals
Non-polluted	6.69 ± 0.14 a	15.77 ± 0.61 c	0.16 ± 0.06 c	8.32 ± 0.79 a
Nickle	6.16 ± 0.12 b	28.91 ± 1.71 b	0.58 ± 0.05 a	10.06 ± 0.83 a
Copper	6.50 ± 0.16 ab	30.84 ± 2.06 ab	0.50 ± 0.04 ab	10.39 ± 0.71 a
Nickle + Copper	6.83 ± 0.14 a	32.93 ± 1.07 a	0.41 ± 0.04 b	9.05 ± 0.59 c
Biochar levels
Non-biochar	6.59 ± 0.17 a	31.86 ± 3.16 a	0.52 ± 0.83 a	11.49 ± 0.83 a
15 g biochar kg−1 soil	6.73 ± 0.13 a	26.87 ± 2.38 b	0.39 ± 0.59 b	8.55 ± 0.59 b
30 g biochar kg−1 soil	6.51 ± 0.17 a	25.00 ± 1.92 b	0.38 ± 0.61 b	8.67 ± 0.61b
45 g biochar kg−1 soil	6.35 ± 0.15 a	24.71 ± 1.65 b	0.36 ± 0.71 b	9.13 ± 0.71 ab

Data represents the average ± standard error.

Different letters between the treatments indicate significant difference at p ≤ 0.05.

Phenolic compounds

Phenolic compounds in dill leaves were significantly affected by biochar treatments, but not by heavy metals (Table S3). Application of biochar at all rates reduced the phenolic compounds in plant leaves (Table 4). However, the reduction by 45 g of biochar per kg of soil was not significant, compared to non-biochar treatment. In general, biochar treatments reduced the phenolic compounds in dill leaves by up to 25%. However, there was no significant difference between the biochar treatments in terms of reducing phenolic compounds in plant leaves.

Leaf pigments

Heavy metal and biochar treatments significantly affected the contents of chlorophylls a, b, total, and flavonoids in dill plants. Carotenoid content was only affected by heavy metal, and biochar treatments had no significant effect on photosynthetic pigments (Table S4). The effect of heavy metal in soil and biochar treatments was not significant on the anthocyanin content in dill plants. The interaction effects of heavy metal toxicities and biochar were not significant for photosynthetic pigments (Table S4). The Ni, Cu, and their combined forms reduced the contents of chlorophylls a and b, total chlorophylls, flavonoids, and carotenoids in dill leaves (Table 5). In most cases, the combined form of heavy metals caused the greatest reduction in photosynthetic pigments in plant leaves. For example, the combined form of heavy metals reduced the total content of chlorophylls in dill leaves by approximately 42%, compared to non-polluted plants. The reduction of chlorophyll a in response to the combined form of heavy metals was approximately 52%. Application of biochar improved the contents of chlorophylls and flavonoids, with no significant effect on anthocyanin and carotenoids. The different application rates of biochar did not show a significant difference in enhancing the contents of chlorophylls and flavonoids in dill leaves. Application of biochar resulted in about 20% increment in total chlorophyll content and a 25% increase in flavonoid content, compared to non-biochar treatment.

Table 5.

Leaf pigments (mg g⁻¹ FW) and biomass (g DW⁻¹ plant) of dill plants under heavy metals pollution and biochar treatments.

Treatments	Chlorophyll a	Chlorophyll b	Total chlorophyll	Flavonoids	Carotenoids	Anthocyanin	Biomass
Heavy metals
Non-polluted	0.85 ± 0.02 a	0.37 ± 0.01 b	1.23 ± 0.03 a	0.55 ± 0.02 a	0.62 ± 0.02 a	0.36 ± 0.010 a	0.77 ± 0.02 a
Nickle	0.57 ± 0.02 b	0.30 ± 0.01 a	0.87 ± 0.03 b	0.29 ± 0.01 b	0.33 ± 0.007 b	0.36 ± 0.007 a	0.56 ± 0.02 b
Copper	0.51 ± 0.02 b	0.30 ± 0.02 a	0.81 ± 0.02 b	0.30 ± 0.02 b	0.27 ± 0.008 c	0.36 ± 0.006 a	0.53 ± 0.03 bc
Nickle + Copper	0.40 ± 0.01 c	0.31 ± 0.01 a	0.71 ± 0.03 c	0.33 ± 0.02 b	0.34 ± 0.007 b	0.37 ± 0.006 a	0.48 ± 0.02 c
Biochar levels
Non-biochar	0.51 ± 0.06 b	0.28 ± 0.01 b	0.79 ± 0.07 a	0.31 ± 0.04 b	0.37 ± 0.14 a	0.36 ± 0.007 a	0.51 ± 0.03 b
15 g biochar kg−1 soil	0.59 ± 0.05 a	0.33 ± 0.01 a	0.92 ± 0.06 a	0.38 ± 0.04 ab	0.39 ± 0.15 a	0.37 ± 0.010 a	0.60 ± 0.03 a
30 g biochar kg−1 soil	0.61 ± 0.04 a	0.34 ± 0.02 a	0.94 ± 0.05 a	0.40 ± 0.03 a	0.39 ± 0.14 a	0.35 ± 0.006 a	0.62 ± 0.03 a
45 g biochar kg−1 soil	0.62 ± 0.05 a	0.33 ± 0.01 a	0.95 ± 0.05 b	0.39 ± 0.03 a	0.40 ± 0.15 a	0.35 ± 0.006 a	0.61 ± 0.04 a

Data represents the average ± standard error.

Different letters between the treatments indicate significant difference at p ≤ 0.05.

Plant biomass

The effects of heavy metal and biochar treatments were significant for plant biomass (Table S4). However, the interaction of heavy metal × biochar treatments was not significant for plant biomass. Dill biomass was reduced by Ni (by approximately 27%), Cu (by approximately 31%), and Ni + Cu (by approximately 37.7%) treatments. The lowest level of dill biomass was observed under the combined form of heavy metals, with no statistically significant effect of Cu treatment. Biochar addition to the soil at all application rates increased plant biomass. There were no significant statistical differences between the biochar treatments in terms of enhancing plant biomass. Application of biochar treatments resulted in an improvement of plant biomass by approximately 17–21% compared to plants grown without biochar treatments.

Discussion

The results revealed that the use of biochar in soils with elevated levels of heavy metals (Table 2 and Fig. 2) decreased the absorption of these pollutant by plants and enhanced the plants’physiological performance. The Ni competes with cations such as Ca, Mg, Fe, and Zn in absorption and translocation in plants due to its similar chemical properties. Due to this similarity, a high concentration of Ni in the soil, especially with low soil pH, can cause a significant increase in the soil’s Ni concentration. This increase in nickel concentration can lead to a deficiency of these nutrients in plants by inhibiting their absorption and movement^35,36. The reduction in the absorption of Ni and Cu elements by dill plants (Table 2 and Fig. 2) due to the application of biochar may be attributed to the decreased availability of these pollutants. Biochar is rich in functional groups that adsorb and immobilize heavy metals³⁷. Biochar is an organic material with a very large specific surface area that contains numerous active adsorption sites for the uptake of heavy metals³⁸, effectively immobilizing these elements when incorporated into the soil. These findings align with those of Ghassemi-Golezani, and Farhangi-Abriz³⁹ concerning the decrease in cadmium uptake by plant roots following biochar application.

The presence of heavy metals in the soil reduces the plant’s ability to absorb other nutrients (Table 2). Heavy metals can ultimately disrupt the absorption of nutrients in plants by altering the physiology and anatomy of the roots⁴⁰. In a recent study conducted by Ghassemi-Golezani, and Farhangi-Abriz³⁷, it was found that the presence of cadmium in the soil increases the production of lignin in the root, leading to a decrease in root activity and absorption of nutrients through the apoplastic pathway, ultimately reducing nutrient absorption by the plant. In addition, the presence of heavy metals in the root triggers the production of stress induced hormones such as jasmonic acid and abscisic acid, ultimately limiting root growth¹⁶. The inhibitory impact of Ni and Cu on the absorption of specific nutrients, such as Ni, K, Zn, Ca, Mg, and Fe, (Table 2) can be attributed to several factors. These factors include competition for transporters or common binding sites at the root surface⁴¹, inhibition of enzymes and proteins involved in the absorption, transport, and metabolism of nutrients^42,43, production of free radicals that can damage plant cells and tissues^42,43, and interference with metabolic processes⁴¹. On the other hand, biochar reduces the absorption of heavy elements by the roots, leading to an increase in growth hormones such as auxin in its tissues, ultimately improving root growth and nutrient absorption¹⁶. In addition to improving root properties, biochar plays a role in increasing the availability of nutrients in the soil through several mechanisms⁴⁴. Firstly, biochar can enhance the plant’s access to nutrients by increasing the nutrient content in the soil. Furthermore, biochar augments soil microbial activity and soil enzyme function, ultimately increasing the availability of nutrients for plants⁴⁵. Biochar also has positive effects on regulating soil pH and cation exchange capacity³⁹. In a research by Farhangi-Abriz and Ghassemi-Golezani¹³, it has been determined that the use of biochar increases the zeta potential of plant roots, thereby enhancing their absorption capacity. An increase in nutrient absorption resulting from the utilization of biochar has also been observed in other studies^46,47.

Osmotic stress is a common side effect of heavy metal exposure in plants, leading to the dehydration of plant cells and restricted water absorption. Biochar alleviates osmolytes production (Table 3 and Fig. 2A) in plants via reducing the absorption of heavy metals by plant roots. Additionally, biochar enhances water availability for plants, making it easier for plants to absorb water⁴⁸. Reducing the absorption of heavy metals and their accumulation in plant tissues ultimately improves the plant growth and reduces the production of osmotic regulators such as proline and soluble sugars, which are produced in response to stress in plant tissues. Proline, as an osmo-protectant, plays a role in maintaining the stability of proteins and membranes under environmental stress, such as drought⁴⁹. The accumulation of proline in dill under heavy metals appears to occur indirectly in response to nutritional imbalance, disruption in cell membrane function, and an increase in hydrogen peroxide levels. A decrease in the production of osmotic regulators has been reported as a result of using biochar in the presence of other heavy metals¹⁶. Application of biochar resulted in an increase in chlorophyll production in dill leaves. Both proline and chlorophyll are derived from the precursor glutamate⁵⁰, and an increase in the production of each one can decrease the other.

Oxidative stress is another harmful factor when heavy metals are present in the soil (Fig. 2B). Absorption of heavy metals in plants leads to widespread disruptions in the electron transfer system between photosystems, resulting in the formation of reactive oxygen species due to incomplete electron transfer. Plants enhance the activity of their antioxidant enzymes to cope with oxidative stress⁵¹. The occurrence of oxidative stress leads to the destruction of bioactive membranes, ultimately resulting in the degradation of cell organelles, such as chlorophyll, and a subsequent decrease in the amount of leaf chlorophyll⁵². The rise in phenols, soluble sugars, polyphenol oxidase, and peroxidase enzymes indicates the plant’s effective response to stress in order to alleviate the negative effects of heavy metals. These compounds play a role in defending against oxidative stress caused by heavy metals. According to the reports of some researchers⁵³, an excess Ni and Cu can lead to a significant decrease in chlorophyll and carotenoid content. Inhibition of crucial enzymes, such as 6-aminolevulinic acid dehydratase (ALA-dehydratase) and protochlorophyllide reductase which involved in chlorophyll biosynthesis, contributes to the reduction under heavy metal stress. By reducing the absorption and accumulation of heavy metals in plant leaves, biochar decreases the production of reactive oxygen species and the activity of antioxidant enzymes. In addition, incorporating biochar into the soil enhances the presence of nutrients including Mg, Fe, and N in plant tissues. These nutrients directly or indirectly contribute to the transfer of photosynthetic electrons, improving their transfer and reducing the production of active oxygen species³. Ultimately, this reduces oxidative stress and the activity of antioxidant enzymes.

The presence of heavy metals in rhizosphere causes osmotic and oxidative stresses in plants, that reduce plant growth (Table 5). On the other hand, the introduction of heavy metals into the soil leads to a substantial decrease in certain nutrients, such as N and K in plant tissues. This can potentially reduce plant growth and biomass production. The enhancement of plant growth resulting from the application of biochar in soil contaminated with heavy metals can be attributed to the decreased absorption of Ni and Cu by plants. This reduction ultimately alleviates oxidative and osmotic stresses in plants, leading to improved plant growth. Another reason for the growth of plants is the increased absorption of nutrients resulting from the utilization of biochar. The use of biochar in the presence of other environmental pollutants has also been found to enhance the growth of other plants^54,55.

Conclusions

The results of this research revealed the detrimental effects of excessive Cu and Ni content in plant tissues on the growth and physiological performance of dill plants. The presence of these heavy elements in the rhizosphere increased oxidative stress in plant tissues, ultimately reducing plant growth. Application of biochar at all rates not only reduced the absorption of heavy metals by the plants, but also decreased oxidative stress and the production of osmolytes in the plant tissues. On the other hand, biochar increased nutrient absorption by plants. All three levels of biochar application rates had nearly identical effects on enhancing plant growth and physiological efficiency. Utilization of biochar with a high rate did not cause further increase in plant growth, compared to a low application rate, indicating a preference for lower biochar application rates. Based on these findings, application of low rates of biochar from agricultural wastes could be suggested as an effective way to overcome heavy metals contaminations in soils for improving plant growth and productivity. Further studies may be needed for extensive use of biochar in modulation of heavy metals pollution in agricultural lands.

Author contributions

K.G.G. supervised and designed the experiment and carried out final editing, S.L. conducted the experiment, analyzed the data and wrote the initial manuscript, and S.F. helped data analysis and initial writing.

Data availability

The data is provided within the manuscript.

Declarartions

Competing interests

The authors declare that they have no competing interests.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-025-98646-0.