Current World Environment

Introduction

Increasing anthropogenic environmental heavy metals (HMs) pollution is a serious global concern due to its adverse environmental effects.While HMs are natural components of soil, human activities have increased their concentration beyond permissible limits.¹Waste from tanneries, mining, agricultural and metal industries, waste water irrigation, sewage sludge, and waste dumping contaminate both soil and groundwater. HMcontamination frequently includes Arsenic, Cadmium, Chromium, Copper, Lead, Nickel, etc. The use of untreated sewage and wastewater leads to significantHM deposition in agricultural land;these metals are subsequently absorbed by crops and consumed by humans and other animals.²Long-term exposure of HMs isdeleterious to biological systems- including plants, animals and humans-when concentrations exceed specific threshold levels.³ Reducing HMs bioavailabilityis essential not only to mitigate health risks but also to facilitate the total removal of toxic metals from contaminated soil. Various soil remediation techniques, such as phytoextraction, chemical stabilization, and soil washing, have been extensively employed by researchers.⁴ The integration of various natural and artificial amendments, including phosphate fertilisers, clay minerals, etc., enhances phytostabilization by promoting the root-mediated sequestration and the absorption of HMs.⁵

In phytotransformation or phytovolatilization-based remediation, plants uptake pollutants and utilize specialized enzymes or metabolic processes to transform the contaminants into volatile derivatives,which are eventually discharged into the atmosphere.⁶In phytofiltration, plants are initially acclimatedto contaminated water prior to field transfer for site-scale remediation,once the roots reach pollutant saturation, the plants are harvested.⁷In addition to other remediation techniques,phytoextraction is used to clean up the HMs from contaminated soil,where metals are absorbed by roots and translocated to the aerial parts of the plant.⁸ These plants are subsequently harvested and safely disposed. Certain plant groups can containor reduce HM pollution through phytoremediation,offeringanenvironmentally sustainable and eco-friendly long-term solution for soil reclamation.²Chemical immobilisation involves the application of soil amendments to contaminated soil; that reacts with HMs to form less toxic, insoluble complexes, thereby reducing their bioavailability and effectively hindering their absorption by the plants. Among the amendments that adsorb HMs and decrease their bioavailability and uptake, biochar has proved to be cost-effective⁹due to its unique physicochemical properties.^10,11Biochar is a stable, carbon-densematerial derived from the pyrolysis of agricultural wastes, making it a sustainable and environmental friendly material.¹² Biochar's porous structure, containing over 60% carbon, makes it a good adsorbent that improves soil structure andquality.¹³ Additionally, biochar has been found to enhance nutrient retention, water holding capacity, and microbial activity in the soil, creating a favourable environment for plant growth and remediation processes.^14,15

Nickel is recognised as one of the 23 heavy metal pollutants posing a significant threat to both the environment and human health;^16-18however, its stress effect on plants is not yet fully understood.The concentration of Ni in soil varies significantly, ranging from 0.2 to 450 mg kg^-1,depending primarily on the underlying soil formation processes.^19-21 Nickel has long been known as an important plant micronutrient.^22,23While small concentrations of Ni are essential for normal plant growth, elevated concentrationscause deleterious effects on plant development and trigger Ni toxicity symptoms,such as inhibition of germination, reduced root and shoot growth, chlorosis, foliar necrosis, leaf spotting, etc.^24-26

Ni reaches the soil from various sources,^27-30Ni contamination in soil results in widespread exposure across plants and is consumers, affecting various stages of life cycles.According to the World Health Organization (WHO),the target value of Ni in soil is 35 mg kg^-1, while the permissible value in plants or crops is 10 mg kg^-11.Addressing this issue requires innovative and sustainable solutions to reduce the toxic effects of Ni, especially Ni (II),among the four oxides ofdivalent Nickel.^31-33

Experimental results demonstrated that biochar-amended soil achieves substantial removal efficiencies for Ni.³⁴ The addition of biochar to contaminated soil facilitates the sequestration of HMs, effectively reducing their bioavailability and limiting plant uptake. This process is governed by several complex factors, including specific metal type, soil physical and chemical properties, amount of biochar used, pyrolysis temperature, porosity, and surface area, etc.^9,10Therefore,the remediation of HMs from polluted soil is a challenging process.The selection of appropriate HM-tolerant plant species is the most important criterion for successful phytoextraction.³⁵ Native plants are generally selected as a medium for phytoextraction because these plants are evolutionary products of their specific environment. One species used for this purpose is the economically important food crop maize (Zea mays L.). Assisted phytoextraction has become an increasingly prevalent technique for the management of contaminated sites. Rice husk biochar pyrolyzed at 400 ^oC may enhance the plant’s growth, development and phytoextracting ability of maize.³⁶Rice husk biocharis particularly effective due toits high silica content.The optimum pyrolysis temperature creates negatively charged binding sites for Ni (II) via cation exchange capacity (CEC),providingadditional sites for metal binding.^37,38Therefore, in our study,we selected biochar derived from pyrolysis of rice huskat 400^oC under oxygen-limited conditions as a soil amendment to improve phytoextraction by maize.Converting rice husk, an agricultural waste, into biochar is a multi-win strategy to preserve soil carbon, improve soil fertility, and immobilize organic and HMs pollutants.³⁹

To evaluate the efficacy of biochar in the phytoremediation of Ni, a pot experiment was conducted using maize grown in soil contaminated with 100 mg kg^-1 and 300 mg kg^-1 of Ni, with a control set maintained for comparative analysis. The effect of biochar on Ni sequestration in various maize plant parts was also assessed. We expect biochar to increase the germination percentage and growth of maize under high Ni-stress conditions while simultaneouslyenhancing its bioaccumulating capacity.

Materials and Methods

Preparation of soil sample

Garden soil samples were collected, air-dried at room temperature,and passed through a 2mm sieve to remove debris.⁴⁰Sandy clay loam soil, free from exogenous metal contamination, was used for the experiment. The soil used for treatment had a pH range between 7.0 and 8.0, and awater-holding capacity of80% (Table 1).

Preparation of Contaminated Soil and Pot Experiment

Nickel Chloride Hexahydrate (NiCl₂.6H₂O) solutions were prepared by dissolving salts equivalent to 100 mg and 300 mg of Ni in 1litre of distilled water, respectively. Prior to sowing,the Ni solution was added tothe soil. To ensure uniformity, the air-dried soil was thoroughly mixed with the metal salts through repeated agitation before being distributed into three replicate pots per treatment.Pots were arranged in a completely randomized design, and metal concentration analyses were blinded to treatment identity.After 10 days of incubation, maize seedlings were raised in a 2-inch layer of soil contaminated with Ni(100 mg and 300 mg kg^-1).^41-43Experimental controlswere established using three pots of uncontaminated soil. In addition, specific soils were amended with 1% (w/w) biochar.⁴⁴ Each concentration set and biochar treatment was executed in three replicates.

Maize seeds (VNR 4226) were purchased from the Bidhan Chandra Krishi Viswavidyalaya (BCKV), West Bengal, India. Thisvariety ispartially Ni-tolerant and commonly cultivated by local farmers. The seeds were surface sterilised for 2 minutes in ethanol, followed by 10 minutes insodium hypochlorite solution (0.75% of Cl)⁴⁵ and subsequently washed six times withdistilled water.⁴⁶The plants were grown under natural light at ambient temperature andharvested 10 days aftersowing. The number of germinated seeds was recorded daily to determine growth parameters,including the germination percentage. Seeds were considered germinated upon the visible protrusion of a 2mm plumule.⁴⁷The equation for determining germination percentage is as follows (1)⁴⁸:

Preparation of Biochar

1kg of rice husk was collected from rice fieldspost-harvest. After being cut and washed, the husk was air-dried under direct sunlight for 48 hours. The samples were then ground and sieved to obtain a particle size of 0.25 mm. The ground rice husk biochar was prepared by slow pyrolysis in a high-temperature programmable Muffle Furnace (Labocon, LMF-1200). Biochar was produced by heating the biomass at a rate of 10^oC/min to a peak temperature of 400^oC for 2 hours under oxygen-limited conditions.^49,38

Characterization of Biochar

Different characterization techniques were employed to analyse the surface morphology and modification feasibility of the rice husk biochar. To ensure the presence of appropriate functional groups required for HM sequestration, Fourier transform infrared spectroscopy (FTIR) analysis was conducted using a Shimadzu FTIR 8400 instrument, over a spectral range of 4000 to 400cm^-1(Fig. S1). Additionally, the surface morphological structure of biochar was characterised using a Carl Zeiss SIGMA 300 Scanning Electron Microscope (SEM).Biochar pH, CEC, and surface area were not measured, thereby limiting comparison with other studies.

Table 1: Physio-chemical properties of soilfrom different heavy metal concentrations.

Impact of biochar amendment on the Bioavailability and tissue specific accumulation of heavy metals in maize

The study assessed the distribution of HMs by analysing their accumulation levels within the soil and plant parts. The concentration of Nickel in the digested soil, rootsand shootsextractswas determined by the Dimethylglyoxime (DMG) test.^50,51The absorbance ofresulting mixture was measuredat 540nm usinga JASCO V-630 UV-Vis spectrophotometer. Standard curve was preparedusing NiCl₂ solutionsranging from 0.1 to 10 mgL^-1 (R² = 0.9758).

Metal uptake in plant

To evaluate the phytoextraction potential of maize, the bioconcentration factor (BCF) and translocation factor (TF) were determined. The value of BCF of hyperaccumulator plants is greater than 1, indicating that the plant is a potential accumulator for the HMs.⁵² A translocation factor (TF) greater than 1signifies the plant’s potential as a hyperaccumulator and metal transport to the shoots; conversely, TF<1 indicates a strategy of phytostabilization, where HMs are effectively sequestered within the root system.⁵³

Bothbioconcentration Factor and translocation factor were calculated as by the equation (2) and (3), respectively.⁵⁴

Where C_plant – metal concentrations in the tissues of plant parts, i.e. shoot and root, C_soil – initial concentration of metal in the soil. In the case of TF, C_shootis the heavy metal content in the aerial part and C_root is the total heavy metal content in the underground.

Statistics

All tests were performed in triplicate.Significant differences in plant germination and accumulation parameters with reference to the application of biochar in Ni-contaminated soil were evaluated using Student’s t-test. Results were summarised by the mean and standard deviation.

Results

Characterization of Biochar

The detailed surface morphology of the rice husk biochar has been demonstrated through  SEM images, suggesting a high potential for HM adsorption. At a magnification of×1080, the biochar exhibitsseveral irregular voids and a diverse composition, a well-developed porous and rough texture in the first image (Fig. 1A), resulting from the pyrolysis of lignocellulose precursor. These characteristics point to a large surface area, which is advantageous for adsorption and facilitates physical sequestration of Ni.⁵⁵ Sharp, ridge-like structures that seem to protrude from the surface of the biochar are highlighted in the second image (Fig. 1 B), which has a greater magnification of ×1860. These sharp edges probably improve the biochar's reactive surface characteristics and mechanical stability, and act as a secondary sink for heavy metals, where ions are immobilised to mitigate phytotoxicity.

Figure 1: Scanning electron microscope (SEM) images of 0.25 mm sieved biochar particles. SEM images from the same sample were taken (A) at ×1080 magnification and(B) at×1860 magnification. Click here to view Figure

Effect on germination percentage

Germination percentage was higher in the control setup, whichis 73% as compared to soil contaminated with Ni (33%) at varying concentrations (p < 0.005). Although germination percentage increased to about 48±7.70 percent (Fig. 2) when contaminated soil was amended with biochar. A significant effect of biochar was observed on the germination of maize growing at 300 mg kg^-1 concentration of Ni (p =0.03).

Figure 2: Germination of potted maize seeds across different Ni concentrations in soil treatments 100 to 300 mg kg-1 with and without biochar. The error bar represents standard deviation. Click here to view Figure

Nickel accumulation in soil and plant parts

Various concentrations of Ni were used in a pot experiment, treated with and without biochar. Bioavailable Ni was significantly reduced in the soil treated with biochar (p<0.005) than without the application of biochar (Fig. 3). At 100 mgkg^-1biochar, the accumulation of Ni in roots was significantly reduced (p < 0.01), but had no significant effect on Ni deposition in roots at higher concentrations(p=0.6). It has no significant effect on Ni accumulation in the shoot across all treatments (Fig. 4).

Figure 3: Bioavailable concentrations of Nickel (mg kg-1) across different Ni treatments in soil 100 and 300 mg kg-1 with and without biochar. The error bar represents the standard deviation. Click here to view Figure

Figure 4: Accumulated Ni concentration in maize plant parts, (A) root and (B) shoot across different Ni soil treatments 100 to 300 mg kg-1 with and without biochar. The error bar represents standard deviation. Click here to view Figure

BCF of the maize plant growing in control soil was 0.8. In comparison, at a concentration of 100 mgkg^-1, BCF was 0.6 without biochar, which was enhancedto1.2 with the application of biochar. At a higher concentration of Ni at 300 mg kg^-1, BCF doubled to 2.4from 0.9 by the addition of biochar.

BCF significantly increased with application of biochar at both 100 mg kg^-1 (Ni 95% CI [0.5315, 0.8560]; Ni+Biochar95% CI [0.8275, 1.6292],Cohen's d = 4.3431, p = 0.02) and at 300 mg kg^-1(Ni 95% CI [0.8834, 1.0610]; Ni+Biochar95% CI [1.2977, 3.5631],Cohen's d = 4.5088, p=0.03)(Fig. 5). In all cases TF remains <1 (Fig. 6); biochar has no significant effect on TF.

Figure 5: Bioconcentration of Ni in maize plant across different soiltreatments from100 to 300 mg kg-1 with and without biochar. The error bar represents standard deviation. Click here to view Figure

Figure 6: Translocation of Ni in maize plant across different soil treatments 100 to 300 mg kg-1with and without biochar. The error bar represents standard deviation. Click here to view Figure

Discussion

Ni is an essential metal needed for thegrowth of plants,but become highly toxic when plants are grown in soil with excessive Ni concentrations.²⁰One of the signs of Ni toxicity is the inhibition of germination and a delay in germination time.²⁵ Some authors suggest that elevated Ni concentration in the growth medium impairs the germination of rice seeds by triggering Ni stress;thisrestricts the hydrolysis process and delays themobilisation of reserve protein within the endosperm.²⁰The phytotoxic impact of excessive Ni can significantly reduce the germination percentage.⁵⁶In our study, treatment with Ni at 100 mg kg^-1 and 300 mg kg ^-1 decreased thegermination percentage compared to the control set. At optimum concentrations, Ni is known to improve seed germination in many species.^57,58Therefore,a high percentage of maize seeds germinated in the control set. According to the WHO,the regulatory limit of Ni in soil is 35 mg kg^-1 and the permissible value in crops is 10 mgkg^-11. In this study, bioavailable Ni in soil did not exceed the WHO permissible limit in all treatment cases;however, at the higher concentration, i.e. 300mg kg^-1,the Ni concentration in the roots exceeded the permissible limit.Seminal work on Ni uptake kinetics shows that the bioavailability of Ni in soil and its uptake by plants is influenced by several factors,including the soil Ni concentration,⁵⁶soil or soil solution acidity,⁵⁹ and plant species,²⁰ etc.Nearly half of the accumulated Ni is retained within the roots,⁵⁶which is attributed to its sequestration at the cation exchange sites of the vessel walls and xylem parenchyma;this process effectively immobilises the metal⁵⁶.The soil used in our experiment was slightly alkaline (pH: 7.6), which may result in immobilisation of Ni, thereby reducing its bioavailability to the plant.²⁰

The addition of biochar further increases soil alkalinity (pH: 8.0).This reduces metal solubility by promoting precipitation into hydroxides or carbonates and enhancing binding withsoil particles.⁶⁰This mechanism represents an indirect metal immobilisation by biochar amendments, which reduces the bioavailability of Ni and renders the soil less toxic to plants.This findingis supported by several studies.^61-63

Although maize is often cited as a potential hyperaccumulator,^64,65the BCF of maize in all our experimental cases remained<1,withoutbiochar application. Biochar amendments improved plant growth and enhanced the phytoextractionof Ni;consequently, the BCF of the biochar-treated maize plants at both concentrations increased to>1. However, no significant differences in shoot Ni concentrations were observed between the plants treated with and without biochar. Furthermore, the translocation factor was also lower than 1in all cases of treatment except forthe control set.Studies describe phytostabilization as stabilization and accumulation of HM by the root but outside in the soil and rhizosphere,⁶⁶ whereas phytoextraction refers to the accumulation of HMs inside root tissue.⁶⁷ Since most of the Ni is accumulated in the roots, here maize is referred to as a phytoextracting plant.

A similar result hasbeen reported in studies with the hyperaccumulatorAlyssum murale growing at Ni rich environment.Here, shoot Ni concentration was also not affected by thebiochar amendments, despite the enhancementofgermination and growth.^68,9,69,70

The ultrastructural image of biochar prepared by the pyrolysis of rice husk at 400^oC exhibits a highly heterogeneous surface characterised by micro and mesopores, with a rugged and fractured appearance. This structure issuitable for providinga high surface area for Ni binding sites and helps in mitigating Ni toxicity by CEC.^37,38A study with cottonseed hull biochar obtained within a temperature range of 200-800^o C demonstrated that the soil matrix controls metal sequestration (Nickel, Copper, Lead and Cadmium) through the surface functional groups of the biochar.⁷¹Thus, the prevalence of oxygen-containing functional groups in low- temperature biochar makes it a superior adsorbent for inorganic contaminants, facilitating sequestration through electrostatic attraction and precipitation.³⁹

As expected, the addition of biochar improved phytoextraction of Ni in contaminated soil by maize (Zea mays L.). Asan amendment,it improves the plant germination and survivability byrestricting shoot Ni uptake and sequesteringthe majority of Ni within the maize roots. However, the effectiveness of biochar as a phytoremediating agent of heavy metals is influenced by several factors³⁹and further studies are required to evaluate its long-term efficacy.

Conclusion

Although maize is considered a hyperaccumulator plant,the BCF of Ni in maize for both treatments (100 and 300 mg kg^-1) without biochar application was less than1,with metals primarily sequestered in the roots of maize. Therefore, the present study indicates that maize (Zea mays. L) does not efficiently extract Ni from highly contaminated soil independently.Our findingssuggest that assisted phytoremediation using biochar is a feasible approach for Ni-contaminated soil. Although heavy metal in maize roots decreased, biochar application resulted in an increase in the bioconcentrationfactor (BCF >1), especially at higher concentrations, but with limited translocation (TF < 1).The positive effect on germination percentage following biochar application indicates that usingZea mays L.in conjunction with biochar could be a suitable strategy for reclaimingsoil contaminated with heavy metals like Ni.

Thermochemical conversionof rice husk into biochar offers astrategic approach for the productive repurposing of agricultural wastes, mitigating the disposal challenges.The application of cost-effective biochar for the remediation of HM-contaminated soil offers a dual environmental advantage by simultaneously immobilising contaminants and facilitating carbonsequestration.

Care should be taken when generalizing these findings to field application or to biochar derived from different feedstocks and production methods. A comprehensive evaluation of the long-term sustainability of these assisted phytoremediation techniques is essential for their transition from laboratory to field-scale application. Supplementing contaminated sites with biochar or chemical fertilizers can drive the continuous biomass production essential for the effective and large-scale extraction of toxic Ni from the environment.

Acknowledgement

The authors are thankful to Guru Nanak Institute of Pharmaceutical Science and Technology, West Bengal, for providing the necessary instruments and equipment forthe experiment.

Funding Sources

This author(s) received no financial support for the research, authorship, and/or publication of this article.

Conflict of Interest

The authorsdo not have any conflict of interest.

Data Availability Statement

The authors will provide the primary data if and whenever required.

Ethical Approval

This research did not involve human participants, animal subjects, or any material that requires ethical approval.

Informed Consent Statement

This study did not involve human participants, and therefore, informed consent was not required.

Permission to reproduce material from other sources

Not Applicable

Author Contributions

MunmunKundu:  Conceptualization, Methodology, Data collection, Analysis, Visualization, Writing- Original draft

SouparnaChakrabarty: Analysis, Visualization, Writing- Review and Editing

Swati Chakraborty: Conceptualization, Supervision

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