Enhancing Wheat Productivity and Reducing Lead Uptake Through Biochar, Bentonite, and Rock Phosphate Integration

Abstract

Heavy metal (HMs) toxicity has severely impacted wheat production and is considered an emerging threat to human health due to bioaccumulation. The application of organic and inorganic amendments has proven effective in mitigating HM’s phytotoxicity by limiting their mobility in soil and plants. A pot experiment was conducted to evaluate the efficiency of biochar (BC), bentonite (BN), and rock phosphate (RP), both individually and in combination, in alleviating lead (Pb) toxicity and enhancing wheat growth, and physiological attributes. The present investigation revealed that BC, BN, RP, and their combined mineral biochar amendments (MBAs) at 1.5% level significantly enhanced wheat growth along with reducing DTPA-extractable Pb in soil by 30.0–49.8% and Pb uptake in roots by 15.7–37.5% and in shoots by 34.5–48.5%. Antioxidant enzymatic activities were improved, and stress indicators were reduced in roots and shoots of wheat under Pb stress, including hydrogen peroxide (H₂O₂) by 50.7 and 81.0%, malondialdehyde (MDA) levels by 16.0 and 74.9%, and proline content by 34.5 and 64.0%, respectively. The effectiveness of the treatments is described in descending order viz. MBA-1 > MBA-3 > MBA-2 > BC > RP > BN under Pb stress. In conclusion, the integration of biochar, bentonite, and rock phosphate is a promising strategy for sustainable and cleaner cereal crop production under heavy metal stress conditions.

1. Introduction

Heavy metal (HM) distribution and intensity in agricultural soils through geogenic and anthropogenic means have undermined sustainability and food safety in many parts of the world. Anthropogenic activities viz. mining, burning fossil fuels, chemical production, fertilizers and pesticides, and discharge of untreated industrial and domestic effluents have increased HMs and other toxic elements in the environment [1]. The toxic and persistent nature of a variety of HMs in agricultural production land have resulted in low soil fertility, deterioration of soil structure, and poor crop stand. The high level of HMs (e.g., Pb, Cd, As, Cr) in soil has disrupted plant physiological and biological functions and promoted the bioaccumulation of these metals in plant edible parts [2]. The uptake and bioaccumulation of these toxic metals by plants have implications for human health.

Lead (Pb) is a potent and persistent toxic element that enters the environment via anthropogenic activities, i.e., lead batteries, cable coatings, ceramic, fertilizer industries, paint, and refineries. Pb toxicity has decreased normal plant development, biomass production, and photosynthetic processes [3]. Elevated levels of Pb have been found to impair chlorophyll synthesis stopping photosynthesis, reducing respiration, inhibiting ATP synthase, and initiating oxidative stress, ultimately leading to plant death [4]. Its pollution also deteriorates soil fertility and chemical properties while posing significant challenges to the microbial community structure, which plays a crucial role in regulating essential nutrient cycling [5].

Wheat is an important staple food for many developing countries, but Pb toxicity has severely hindered its growth and causes a significant health risk due to high Pb contents in grain. Lead toxicity in cereal crops has been found to instigate reactive oxygen species (ROS), restricting attachment of functional groups and detachment of important nutrients from biomolecules [6]. The high levels of Pb in wheat crops increase H₂O₂ and MDA contents causing lipid peroxidation and inhibiting the initial growth stage of the crop [7]. Moreover, high Pb contents were observed in wheat’s upper parts, especially in grains, which compromised the quality and yield. Therefore, it is imperative to find a viable solution to restrict Pb phytotoxicity and its accumulation in cereal crops.

As per FAO/WHO permissible limits, the maximum Pb concentration in soil is 100 mg kg⁻¹ [8]. The upper limit in residential zones is 300 mg kg⁻¹ and for industrial areas up to 600 mg kg⁻¹. However, the toxic limit of Cd in wheat grains is set by FAO and WHO to 0.2 mg kg⁻¹ [9]. These higher soil (>600 mg kg⁻¹) concentrations of Pb in soils are responsible for several negative effects on crops, especially cereals, viz. reduction seed germination, root elongation, biomass development, nutrient uptake, chlorophyll synthesis, water status, photosynthesis, and protein synthesis ultimately led to yield reduction in crops. On the other hand, an increase in Pb-induced reactive oxygen species (ROS) production, lipid peroxidation, and enzyme inactivation led to cell death [10]. Rehman et al. [11] also found a significant decline in wheat yield (25–30%) in a pot experiment at 500 mg kg⁻¹ Pb concentrations. Moreover, our previous investigation, Noreen et al. [7], also suggested that the use of Fe modified biochar at 600 mg kg⁻¹ Pb significantly improved the root length (30%), shoot length (47%), superoxide dismutase (40%), peroxidase (20%), catalase (40%), and ascorbate peroxidase (13%) activities of wheat at 600 mg kg⁻¹ Pb concentration. On the other hand, it caused a reduction in malondialdehyde content (56%), Pb bioaccumulation (37%), soil to root translocation (9%), and root to shoot translocation (35%); that is why, in the present investigation, we used a higher level (700 mg kg⁻¹) to broaden the scope by integrating organic (biochar) and inorganic (bentonite and rock phosphate) amendments for bioremediation of Pb-contaminated soils. The application of organic and inorganic means to immobilize Pb in soil or reduce its toxicity to plants has been found to be a cost-effective and sustainable option. In this regard, biochar as an organic amendment has been widely used for its numerous benefits in metal-contaminated soils. The abundant functional groups, large surface area, and high nutrient ability owing to biochar have significantly immobilized Pb in soil [7,12]. The application of biochar to metal-contaminated soils has improved plant growth, photosynthesis, and antioxidant enzymatic activities to scavenge ROS and increase yield. The biochar improved soil physiochemical properties like aggregate stability, porosity, pH, CEC, and nutrient availability, which consecutively enhanced plant physiological and biochemical functions [13]. In addition to biochar, various inorganic minerals are increasingly being used to mitigate heavy metal contamination and promote crop growth. Bentonite was reported to increase wheat growth, stomatal conductance, and grain yield by improving soil fertility status [14]. Bentonite has proven highly effective in enhancing phytostabilization by reducing the translocation of heavy metals from lower to upper plant parts, improving soil fertility, and buffering soil pH in metal-contaminated soils [15].

Similarly, rock phosphate is another inorganic amendment being used to mitigate HM toxicity and enhance soil nutrient pollution. The combined application of rock phosphate with other additives was found to improve plant growth, yield, and soil functions [16]. The application of rock phosphate with Bacillus mycoides decreased HM uptake and promoted wheat growth, protein contents, and antioxidant enzymes in wheat crops [17]. Rock phosphate is a rich source of phosphorus, and the application of nano rock phosphate significantly enhances growth, grain yield, phosphorus nutrition, and soil enzyme activity [18].

The metals can be found in soil in different fractions like soluble, exchangeable, specifically adsorbed, complex with organic matter, and residual fraction responsible for its long-term persistence in soil [19]. The addition of organic amendments like biochar further enhances the organically complex fractions of metals by bonding it with functional groups attached on the surface and reduces the soluble, exchangeable, and residual fractions of Pb due to its slow degradation in soil, and reduces its uptake and thus protects plants against Pb toxicity under contaminated conditions [20]. The existing body of knowledge emphasizes the importance of biochar, bentonite, and rock phosphate as effective amendments for heavy metal (HM)-contaminated soils. However, limited research has explored the combined use of these materials to complement each other, enhance HM-removal efficiency, and promote plant growth in metal-contaminated soils. This study was, therefore, designed to evaluate the individual and combined effects of these amendments in Pb-contaminated soil. So, it can be hypothesized that the integration of biochar, bentonite, and rock phosphate may synergistically enhance wheat’s growth by not only improving nutrient availability, physiological attributes, and photosynthesis but also reducing lead (Pb) bioavailability in soil and uptake through synergistic immobilization of Pb in contaminated soils. The objectives of this study were (1) to check the wheat growth with single and combined addition of organic and inorganic amendments in Pb-polluted soil; (2) to evaluate the response of amendments on physiological and enzymatic activities of wheat in Pb-contaminated scenarios; and (3) to identify the best-suited amendment combination for Pb alleviation for promoting sustainable and cleaner wheat production.

2. Materials and Methods

2.1. Experimental Site and Material Collection

A pot experiment was conducted to check the effect of biochar and inorganic mineral amendments and their combination on wheat growth in Pb-contaminated soil at the wirehouse of the Department of Soil Science, The Islamia University of Bahawalpur, Pakistan (29.37° N and 71.77° E). The wirehouse protects pots against birds and rodents with natural weather conditions (no control of light, temperature, rain, and humidity). The soil samples were collected from the 0 to 30 cm layer with a shovel from the research area of the Department of Soil Science by removing the debris from the soil surface. The soil was homogenized by passing it through 2 mm mesh and stored in the dark. Later on, the soil was air-dried and sieved from 1 mm of mesh. The rice husk biochar (BC) pyrolysis at 550 °C for 3 h was purchased from a factory-scale reactor. The inorganic amendments like bentonite (BN) and rock phosphate (RP) were purchased from Sigma Aldrich company. The soil and biochar’s basic chemical and physical properties were measured by following the standard laboratory protocols as shown in

Table 1. Soil and biochar basic physical and chemical properties used in the study.

Parameters	Units	Soil	Biochar
Textural class	--	Silty Clay loam	--
Soil order	--	Aridisols	--
Sand	%	25	--
Silt	%	45	--
Clay	%	30	--
Bulk density	g cm−3	1.56	--
SAR	(mmol L−1)1/2	8.58	--
OM	%	0.75	--
pHs	--	8.45	8.32
ECe	dSm−1	3.87	0.
CEC	cmol c kg−1	5.2	--
Total Pb contents	(mg kg−1)	104	--
Available Cu	(mg kg−1)	8.52	3.55
Available Zn	(mg kg−1)	16.74	42.20
Available nitrogen	(mg kg−1)	0.98	0.75
Available phosphorus	(mg kg−1)	617.8	584.0
Available potassium	(mg kg−1)	86.94	0.015

.

2.2. Spiking of Lead and Application of Amendments

Based on our previous investigation with Pb concentration 600 mg kg⁻¹ (Noreen et al. [7]), the Pb concentration 700 mg kg⁻¹ was chosen as the test concentration under biochar application to broaden the scope of biochar as an amendment for bioremediation of soils with high Pb concentrations. The sieved (1mm mesh size) soil was spiked with lead nitrate (Pb(NO₃)₂). The soil was divided into small parts in plastic tubs and the required concentration of lead nitrate was added to achieve 700 mg kg⁻¹ level. The contaminated soils were then thoroughly mixed and stored in the dark for 1 month for aging by maintaining 30% water holding capacity (W.H.C). After 1 month, the contaminated soil was mixed with desired levels of BC (2%), BN (5%), RP (5%), and their consortia like MBA-1 (1.5%), MBA-2 (1.5%), and MBA-3 (1.5%), respectively. The detailed treatment plan and mixture levels of biochar and inorganic amendments are briefly described in

Table 2. Detailed layout of treatment levels of lead contamination (Pb) with and without addition of biochar (BC), bentonite (BN), rock phosphate (RP), MBA-1, MBA-2, and MBA-3 with different percentages to check the wheat (Triticum aestivum L.) growth in Pb-contaminated soil.

Treatments	Description	Levels of Pb	Amendment Levels
	-	(ppm)	(%)
CK	Control soil	0	0
Pb	Pb	700	0
BC	Pb + BC	700	2
BN	Pb + BN	700	5
RP	Pb + RP	700	5
MBA-1	Pb+ BC+ BN + RP	700	1.5
MBA-2	Pb+ BC+ BN + RP	700	1.5
MBA-3	Pb+ BC+ BN + RP	700	1.5
Biochar–Mineral Amendments	Contents		Mixing Ratio
MBA-1	Biochar + Bentonite + Rock phosphate		50% + 30% + 20%
MBA-2	Biochar +Bentonite + Rock phosphate		40% + 30% + 30%
MBA-3	Biochar + Bentonite + Rock phosphate		30% + 30% + 40%

. The prepared mixtures of soil and amendments were put into clay pots (18 inches in height and 9 inches in width), replicated 4 times, and shifted to the wire house. The pots were regularly irrigated through being distilled for 25 days before sowing at 40% W.H.C. The pots were arranged by completely randomized design (CRD) and were rotated every week to mitigate environmental effects.

2.3. Wheat Seedling Establishment and Growth Conditions

Wheat seeds were disinfected with 2.5% sodium hypochlorite solution and 10 seeds were sown in each moist clay pot with different treatments. After the 2-leaf stage, the healthy plants were selected, and remaining plants were uprooted and 5 plants per pot were maintained in the wirehouse. The recommended dose of N, P, and K (90, 60, and 60 kg ha⁻¹) fertilizers was applied in pots, and the growth of wheat plants was regularly checked by maintaining 40% W.H.C. As the climate of the study area is semi-arid, the temperature during the wheat season fluctuated between 23.2 to 34.4 °C from December 2023 to April 2024, respectively [21]. However, the relative humidity during the wheat season ranged between 74.5 and 46.2% [22].

2.4. Soil and Biochar Characterization

The soil and biochar’s basic chemical and physical properties were determined by standard methods of Zhang and Gong [23]. The soil particle size distribution and texture were measured by the hydrometer method [24]. Soil bulk density and organic matter content (oxidation with potassium dichromate) were determined by the core method and walked-black protocol [25,26]. The electrical conductivity (ECe) (in soil/water slurry at a ratio of 1:5 (w/w)) and pH (in water with the ratio of soil:water 1:2.5 (w/w)) of the soil were measured with an EC meter and pH meter (AB33 pH meter, OHAUS, Switzerland) [27]. ECe and pH in biochar samples were determined by EC and pH meter with biochar and water ratio of 1:2 following Rhoades’ [28] and Page et al.’s [29] methods, respectively. Nitrogen contents were calculated by the Kjeldahl method as described by Bremner and Mulvaney [30]. Soil and biochar available phosphorous contents were examined by adopting the method of Watanabe and Olsen [31] and analyzed by spectrophotometer (UV/VIS Cary 60, Model G6860A, Agilent Technologies, Mulgrave, Australia).

Soil and biochar potassium contents were measured via a flame photometer. The soil total Pb contents were measured by digesting 0.1 g soil samples by wet acid digestion (HNO₃, HF, and HCl) on a hot plate at 350 °C. The digested samples were filtered with Whatman no. 40 filter paper and shifted into a 50 mL glass flask with distilled water. The available Cu and Zn contents and DTPA extractable Pb in the soil and biochar samples were measured by atomic absorption spectrophotometer (Agilent Technologies, 200 series AA).

2.5. Plant Morphological Characteristics and Root Scanning

The wheat plants were harvested from clay pots after 2 months of growth. The plants’ shoots were cut 5 cm above the soil level, whereas plant roots were carefully isolated from the wet soil. The roots and shoots of harvested plants were washed with tap water and dried with paper towels. The plant shoot and root fresh weight was determined by using an electrical balance. The wheat plant leaf area was measured by using a leaf scanner (Win FOLIA Pro, STD 4800 Scanner, 2016, Regent Instruments, Québec City, QC, Canada). Root scanning parameters (total root volume, average root diameter, average root surface area, and number of root tips) involved using a root scanner (Win RHIZO Pro, STD4800, Scanner, Regent Instruments, Canada). The freshly harvested plant parts (root and shoot) were also stored at −80 °C to perform biochemical assays.

2.6. Measurement of Plant Physiological and Gas-Exchange Characteristics

Plant relative water contents (RWC) were analyzed with the Lazcano-Ferrat and Lovatt [32] method. Briefly, the fully expended youngest leaves of plants were cut at the base of the lamina and soaked in distilled water for 16–18 h at room temperature. The leaf turgid weight (TW) was determined and RWC was calculated with the following formula:

$$\mathrm{L}\mathrm{e}\mathrm{a}\mathrm{f} \mathrm{r}\mathrm{e}\mathrm{l}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{v}\mathrm{e} \mathrm{w}\mathrm{a}\mathrm{t}\mathrm{e}\mathrm{r} \mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{e}\mathrm{n}\mathrm{t}\mathrm{s} \left(\mathrm{\%}\right)=(\mathrm{F}\mathrm{W}-\mathrm{D}\mathrm{W})/(\mathrm{T}\mathrm{W}-\mathrm{D}\mathrm{W})\times 100$$

The plant membrane stability index (MSI) was calculated after crushing the shoot into small pieces, dipping them in distilled water, and heating them at 40 °C in a water bath, and electrical conductivity (EC) was measured (C1). The same plant material was immersed in boiling water and again EC was determined (C2) to calculate the membrane stability index (MSI) with following the formula:

$$\mathrm{M}\mathrm{S}\mathrm{I}=[1-(\mathrm{C}1/\mathrm{C}2\left)\right]\times 100$$

The chlorophyll and carotenoid contents were obtained by adopting Arnon’s [33] and Wellburn’s [34] methods. The crushed leaf samples (0.05 g) were extracted in 10 mL dimethyl sulphoxide (DMSO) and kept a 65 °C in an oven for 4 h. The plant extract absorbance was measured at 645 nm for chlorophyll a, 665 nm for chlorophyll b, and 470 nm for carotenoid concentration in a spectrophotometer ((UV/VIS Cary 60, Model G6860A, Agilent Technologies, Mulgrave, Australia)). The SPAD values were measured from the third upper leaf by SPAD meter (SPAD-502).

Wheat plant leaf gas-exchange attributes photosynthetic (Pn) and transpiration rate (Tr), stomatal conductance (Cn), and internal CO₂ concentration (Ci) were measured by using an infrared gas analyzer (Model IR-400, Yokogawa Electric Corporation, Tokyo, Japan). The youngest green leaf from each plant was placed in the leaf cuvette and the plant gas-exchange attributes were measured in the morning between 10:00 a.m. and 11:00 a.m. For the measurement of gas-exchange parameters, the relative humidity and photosynthetic photon flux density and CO₂ concentration in the chamber were maintained at 83%, 800 µmol mol⁻² s, and 600 µmol-mol, respectively, and at ambient air temperature (~25 °C).

2.7. Estimation of Oxidative Stress Indicator

The plant (root and shoot) malondialdehyde (MDA) contents were measured from the fresh root and shoot samples (0.5 gm) after grinding in 10 mL thiobarbituric acid (TBA) solution (0.25%) which was prepared in 10% trichloroacetic acid (TCA). The extracted material was heated at 95 °C for 30 min and then the reaction was stopped by ice cooling. The obtained material was centrifuged for 10 min at 10,000 rpm. The absorbance of the resultant supernatant was measured at 532 and 600 nm and subtracted for correction of non-specific turbidity and MDA contents were measured [35].

The plant proline contents were measured by slight modification in the protocol developed by Ye et al. [36]. Briefly, 0.5 g plant material (root and shoot) was mixed with 5 mL sulfosalicylic acid (3%) and 2 mL each of ninhydrin reagent and glacial acetic acid. The mixture was heated in the water bath for 10 min and cooled at room temperature. The cooled mixture was centrifuged for 10 min at 10,000 rpm, and the supernatant was collected. The readings for proline contents were taken at a 520 nm wavelength with a spectrophotometer, and proline concentration was calculated using known concentration proline standard curve. The hydrogen peroxide (H₂O₂) contents in the plant tissues (root and shoot) were measured by adopting the protocol of Velikova [37]. The 0.2 g plant tissues were dissolved in 1 mL of 0.1% TCA solution and centrifuged for 15 min at 8000 rpm to obtain the final supernatant. The aim with the reaction solution, composed of 10 mM K-P buffer solution (0.5 mL), 1 M potassium iodide (1 mL), and (0.5 mL) supernatant, was to obtain absorbance at 390 nm in a spectrophotometer to count the H₂O₂ contents in plant tissues.

2.8. Estimation of Antioxidant Enzymatic Activities

The root and shoot antioxidant enzymatic activities were measured to assess the role of amendments on wheat growth. The 0.5 g plant tissues were crushed with mortar and pestle and the extract was stored in ice-cold plaster. The extract was mixed with 2–3 mL of buffer solution (Na₂HPO₄·2H₂O (16.385 g) + NaH₂PO₄·2H₂O (0.663 g)) of pH 7.8 and distilled water to make 1000 mL volume. The samples were centrifuged at 8000–10,000 rpm for 20 min and supernatant was collected and stored in 5 mL centrifuge tubes. The plant superoxide dismutase (SOD) activity was measured by the method of Zhou et al. [38]. The 3 mL reaction mixture containing 50 mM potassium phosphate buffer (pH 7.8), 75 µM nitro-blue tetrazolium (NBT), 2.0 µM riboflavin, 13 mM methionine, and 0.1 mM EDTA was added to a 100 µL plant (root and shoot) extract and shaken to make the mixture homogenized. The SOD activity was then estimated at 560 nm in a spectrophotometer.

The peroxidase (POD) activity was measured by making a reaction solution consisting of 300 mM H₂O₂, 1.5% Guaiacol, and 50 mM phosphate (PBS). Now, 0.1 mL plant extract was mixed with 2.7 mL PBS + 0.1 mL Guaiacol + 0.1 mL H₂O₂ to measure POD activity. A blank sample consisting of the reaction solution was also run to obtain a zero reading. The solutions were gently shaken for 4–5 min and a reading was taken by using a spectrophotometer at 470 nm [39]. The catalase activity (CAT) was monitored by dissolving the 100 µL plant extract in 10 mM H₂O₂, 50 mM phosphate buffer solution (PBS), and 2 mM EDTA-Na₂. A blank sample was run by taking 2.8 mL of PBS + 0.1 mL of H₂O and 2 mM EDTA-Na₂ solution. The readings were observed at 240 nm by a spectrophotometer [40]. Similarly, the ascorbate peroxide (APX) activity was assayed by making the 3 mL reaction solution of 100 mM PBS (pH 7.0), 0.06 mM H₂O₂, 0.1 mM EDTA-Na2, 0.3 mM ascorbic acid (ASA), and 100 µL plant extract. The reading was obtained with a spectrophotometer at 290 nm after 30 s of H₂O₂ addition as described by Nakano and Asada [41].

2.9. Measurement of Plant Lead Contents

The plants were separated into root and shoot, washed with tap and distilled water, and dried with tissue papers. The plant material was soaked in 20 mM EDTA solution to remove adsorbed metals on the plant surfaces (root and shoot) and oven-dried at 60 °C. The plant (root and shoot) Pb concentration was measured by adopting the method of Wolf [42]. The 1.0 g well-dried and ground samples were digested on a hot plate at 350 °C with H₂O₂ and H₂SO₄ acids. The digested material was filtered with Whatman no. 40 filter paper and stored in 50 mL glass flasks. The Pb contents in the filter solution were measured by atomic absorption spectrophotometer (Agilent Technologies, 200 series AA). The plant biological concentration factor BCF was calculated by the equations devised by Chen et al. [43].

$$BCF={\displaystyle \frac{\mathrm{P}\mathrm{b} \mathrm{c}\mathrm{o}\mathrm{n}\mathrm{c}\mathrm{e}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n} \mathrm{i}\mathrm{n} \mathrm{r}\mathrm{o}\mathrm{o}\mathrm{t}}{\mathrm{P}\mathrm{b}\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{c}\mathrm{e}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n} \mathrm{i}\mathrm{n} \mathrm{S}\mathrm{o}\mathrm{i}\mathrm{l}}}$$

2.10. Statistical Analysis

The treatment means were calculated from the three replications for each treatment and evaluated for their significant differences by using a one-way analysis of variance (ANOVA) by adopting the LSD (least significant difference) test at p < 0.05 as per Steel et al. [44]. The standard error of means was computed using Microsoft Excel (Office 365). The data were analyzed by using a Windows-based SPSS-16.0 statistical package and graphical illustrations were performed on the Origin pro-2021 64-bit software. The cluster heat map and principal component analysis (PCA) were drawn through R-Studio Version 2024, 12.0+467.

3. Results

3.1. Growth, Biomass Production, and Leaf and Root Morphology of Wheat Plants

The wheat root and shoot fresh weight and leaf area under Pb toxicity and with the sole addition of organic and inorganic amendment and their combination are depicted in

Table 3. Effect of biochar (BC), bentonite (BN), rock phosphate (RP), and their mixtures (BMA-1, BMA-2 and BMA-3) on growth and roots parameters such as root and shoot fresh weights, leaf area, root surface area, root volume, root surface area, and number of root tips (NRT) of the wheat (Triticum aestivum L.) plants grown in Pb-contaminated soil.

Treatment	Root Fresh Weight (g)	Shoot Fresh Weight (g)	Leaf Area (cm2)	Root Surface Area (cm2)	Root Volume (cm3)	Number of Root Tips	Average Root Diameter (mm)
CK	3.92 ± 0.08 a	6.26 ± 0.09 a	80 ± 0.73 a	277 ± 3.94 a	7.33 ± 0.33 ab	2293 ± 22 a	2.94 ± 0.07 a
Pb	2.87 ± 0.09 e	3.02 ± 0.05 e	53 ± 0.74 e	157 ± 3.92 d	4.04 ± 0.20 e	1256 ± 38 e	1.17 ± 0.06 d
BC	3.28 ± 0.11 cd	5.60 ± 0.15 c	79 ± 0.45 ab	245 ± 7.69 b	6.66 ± 0.19 cd	1886 ± 34 c	2.67 ± 0.10 b
BN	3.10 ± 0.11 de	4.76 ± 0.07 d	75 ± 0.79 cd	224 ± 8.70 c	6.19 ± 0.13 d	1728 ± 28 d	2.40 ± 0.11 c
RP	3.11 ± 0.05 de	4.87 ± 0.08 d	73 ± 0.78 d	253 ± 5.01 b	6.92 ± 0.06 a–c	1949 ± 34 c	2.86 ± 0.11 ab
MBA-1	3.52 ± 0.07 b	6.18 ± 0.05 a	79 ± 1.20 ab	255 ± 3.71 b	7.45 ± 0.17 a	2047 ± 26 b	2.75 ± 0.07 ab
MBA-2	3.45 ± 0.06 bc	6.17 ± 0.03 a	78 ± 1.43 a–c	243 ± 6.88 b	6.88 ± 0.14 bc	1803 ± 35 d	2.32 ± 0.08 c
MBA-3	3.41 ± 0.08 bc	5.88 ± 0.04 b	77 ± 1.17 bc	253 ± 3.42 b	6.80 ± 0.15 bc	2062 ± 28 b	2.65 ± 0.09 b

Data are means ± standard deviation of 4 replications (n = 4). Treatments means sharing different letter(s) for each parameter are significantly different from each other according to least significance difference (LSD) test (p < 0.05).

. It was found that root and shoot fresh weight and leaf area (26.7%, 51.7%, and 33.7%, respectively) reduced under Pb toxicity while application of inorganic and organic amendment along with their combination significantly (p < 0.05) enhanced the wheat growth. The maximum results were observed for BC application compared to the sole application of BN and RP in Pb-contaminated soil. However, the results showed the highest increment in root and shoot fresh weight (18% and 51%) and leaf area (32%) with BMA-1 treatment in Pb-contaminated soil. Similarly, MBA-2 and MBA-3 applications also enhanced the shoot and root length (16.8% and 16%) and leaf (31.8% and 31%), respectively, compared to Pb-contaminated soil. Hence, from the data of Table 3, it can be deduced that combinations of organic and inorganic amendment to Pb-contaminated soil had a more pronounced effect on growth and leaf area than their sole application. Table 3 also presents the analyzed data of root morphology traits in Pb-contaminated soil with organic and inorganic amendments. It was observed that root parameters like root surface area, total volume, no. of root tips, and average diameter total significantly reduced (43.3%, 44.8%, 45.2%, and 60.3%) in Pb-contaminated soil. The application of MBA-1 (38.3%, 80%, 45.7%, and 32.6%), MBA-3 (38.7%, 40.6%, 40%, and 56%), MBA-2 (35.3%, 41.2%, 30.3%, and 46.7%), RP (29%, 41.6%, 35.1% and 59.2%), BC (35.3%, 39.3%, 33.4%, and 56.4%), and BN (29%, 34.7%, 27.3% and 51.4%) increased the root parameters, respectively, compared to Pb-contaminated soil. So, Table 3 shows that plant growth and root morphological parameters increased in the order MBA-1 > MBA-3 > MBA-2 > RP > BC > BN compared to Pb-contaminated soil, respectively.

3.2. Physiological and Gas-Exchange Activities of the Wheat Plant

Figure 1 represents the significant variation for leaf water relation and photosynthetic parameters under Pb-contaminated, organic, and inorganic amended soils. It was observed that leaf relative water contents significantly decreased in Pb-contaminated soil (49.8%) compared to control, while the application of organic and inorganic amended soils and their combination increased the relative water content in the order MBA-1 > MBA-3 > MBA-2 ˃ BC > RP ˃ BN (47.9%, 41.5%, 43.1%, 33.2%, 20.6%, and 29.5%, respectively). In Figure 1, it can also be observed that the membrane stability index significantly decreased in Pb-contaminated soil (47.52%) compared to control, while the application of organic and inorganic amended soils and their combination increased the relative water content in the order MBA-1 > MBA-3 > MBA-2 ˃ BC > BN ˃ RP (40.9%, 33.9%, 24.8%, 33.2%, 20.6%, and 29.5%, respectively) (Figure 1B). The results also revealed that the application of organic and inorganic amendments and their combination successfully mitigated the toxic effect of Pb by enhancing chlorophyll a and b contents of wheat plants grown in Pb-contaminated soil. The plant chlorophyll a and b contents significantly decreased to Pb exposure (54.15% and 81%) compared to control soil. MBA-1, MBA-3, MBA-2, BC, BN, and RP increased chlorophyll a (57.6%, 53.3%, 48.6%, 53.9%, 47.7%, 53.8 and 49.9%) and chlorophyll b (80.7%, 76.7%, 75.8%, 71.8%, 69.2%, and 53.3%) compared to Pb-contaminated soil (Figure 1C,D). The carotenoid contents of Pb-contaminated soil significantly decreased (73.68%) compared to control soil. However, applications of MBA-1, MBA-3, MBA-2, BC, BN, and RP significantly improved carotenoid content (67.8%, 55.7%, 59.2%, 51.9%, 50.1%, and 51.3%), respectively, compared to Pb-contaminated soil (Figure 1E). The leaf SPAD value increased to 40.3%, 36.8%, 32.8%, 33.9%, 25.8%, and 33.5% in MBA-1, MBA-3, MBA-2, BC, BN, and RP compared to Pb-contaminated soil (52.9%) (Figure 1F). Overall, combinations of organic and inorganic amendments (MBA-3, MBA-1, and MBA-2) had a more positive effect on changes in leaf–water relation and photosynthetic parameters of okra seedlings grown in Pb-contaminated soil. Wheat plant leaf gas-exchange attributes, i.e., photosynthetic rate (Pn), transpiration rate (Tr), stomatal conductance (Cs), and internal CO₂ concentration (Ci) were significantly altered by the imposition of lead stress and application of BC, BN, RP, MBA-1, MBA-2, and MBA-3 treatments improved these parameters (Figure 2). The maximum improvement was observed with MBA-2, MBA-1, and MBA-3 treatments where Pn (52.9%, 57.8%, and 46.7%), Tr (55.6%, 60.1%, and 47.8%), Cs (48.6%, 53.2%, and 37.6%), and Ci (59.1%, 63.9%, and 53.3%) significantly increased compared to Pb-contaminated soil. Thus, organic and inorganic combinations of BC, BN, and RP were prominent amendments to improve wheat gas parameters.

3.3. Oxidative Stress Indicators for the Wheat Plant

The data of oxidative damage in terms of H₂O₂, MDA, and proline contents to root and shoot of wheat seedlings grown in Pb-contaminated and organic and inorganic amended soil are presented in Figure 2A–C). The data represented that root and shoot H₂O₂ content (50.7%, and 81.0%), MDA contents (16.0% and 74.9%), and proline contents (34.5% and 64.0%) increased in Pb-contaminated soil compared to control, respectively. The results also revealed that application of BC (32.8%, 34%, and 40.7% and 31%, 24.3%, and 66.1%), BN (24.6%, 21.8%, and 44.1% and 22.4%, 19.3%, and 37.9%), RP (24.7%, 29.8%, and 31.5% and 27.6%, 23.7%, and 41.8%), OMA-1 (37.9%, 35.7%, and 48.3% and 38.7%, 30.8%, and 69.6%), MBA-2 (39.3%, 30.8%, and 39.5% and 37.2%, 26.7%, and 59.5%), and MBA-3 (31%, 21.4%, and 33.1% and 28.4%, 24%, and 55.6%), decreased the H₂O₂, MDA, and proline contents in wheat root and shoot, respectively. Thus, these findings revealed that organic and inorganic amendments showed a prominent effect in improving the oxidative machinery of wheat seedlings grown in Pb-contaminated soils.

3.4. Antioxidant Enzymatic Activities of Wheat Plant

It is well a well-proven fact that plants undergo certain changes to cope with oxidative injury posed by abiotic and biotic stress by modulating certain enzymatic activities. Figure 3, accurately truly representsed this fact by showing wheat seedling response to activate anti-oxidant defense enzymes such as CAT, POD, SOD, and APX in shoots and roots grown under in Pb-contamination conditions with organic &and inorganic amendments. The CAT, POD, SOD, and APX activities significantly reduced (46.7%, 35.7%, 27.3% and 40.8%) compared to control soil. However, organic and in-organic amended soil increased the root CAT, POD, SOD, and APX activities in order of in the order MBA-1 ˃ MBA-3 ˃ MBA-2 ˃ BC ˃ RP ˃ BN (44.4%, 39%, 30.6%, and 31%) ˃ (41.8%, 28.16%, 27.7%, and 19.7%) ˃ (40.2%, 32.7%, 25.4%, and 25.9%) ˃ (40.4%, 32.7%, 27.6%, and 27.5%) ˃ (35%, 27.8%, 22.7%, and 19.3%) ˃ (28.6%, 30.9%, 17.9%, and 14.4%), respectively, compared to Pb-contaminated soil. Similar results were also observed for shoot CAT activities (44.7%, 41.5%, 37.6%, 36.5%, 37.4%, and 38.7%) MBA-3, MBA-1, MBA-2, BC, RP, and BN amended soil. The POD activity was enhanced (65.2%, 60.9.%, 55.1%, 60.5%, 56%, and 56.3%) in MBA-1, MBA-3, MBA-2, BC, BN, and RP amended soil. The SOD and APX activities were improved (37.2%, 31.5%, 28.8%, 28.8%, 33.1%, 31.2%, and 26%) and (62.3%, 61.8%, 61.3%, 54.6%, 57.4% and 54%) in MBA-1, MBA-3, MBA-2, BC, BN, and RP amended soil compared to Pb-contaminated soil, respectively.

3.5. Lead Concentration in Root and Shoot in Wheat, and Soil DTPA Extractable Pb

The fate of Pb in the soil after the harvesting of seedlings under different amendments is depicted in Figure 4. It was found that availability of Pb significantly increased in Pb-contaminated soil, whereas organic and inorganic amendments reduced the Pb availability in soil. The amendments MBA-1, BMA-3, BMA-2, BC, RP, and BN decreased the DTPA extractable Pb by 45.1%, 34.9%, 33.8%, 49.8%, 33.8%, and 30%, respectively, compared to Pb-contaminated soil. The concentration of Pb in the root and shoot was recorded to be increased without the application of amendments. The results showed elevated levels of Pb in the root and shoot of wheat plants (77.9% and 80.9%) compared to control. The application of organic and inorganic amendments like MBA-1 (37.5% and 48.5%) MBA-3 (36.8% and 48.1%), MBA-2 (33.4% and 48.5%), BC (36.1% and 48.4%), RP (35.3% and 34.5%), and BN (15.7% and 41.1%) markedly decreases uptake of Pb by wheat in root and shoot, respectively (Figure 4B,C).

3.6. Principal Component Analysis and Pearson Correlation Analysis of Soil and Wheat Plant

A Principal Component Analysis (PCA) biplot demonstrated that applied amendments impacted growth, physiological and oxidative stress indicators, antioxidant enzyme activity, and Pb levels in soil, root, and shoot (Figure 5(left)). Dim₁ explained 77.9% of the variation, whereas Dim₂ explained 10.8%, accounting for 88.7%. Most treatments and variations in the parameters were positioned along Dim₁, with significant distributed variations. The patterns of the BC and CK treatments were comparable and clustered. The Pb treatment was fully isolated on Dim₁’s far-right side, differentiating it from the other treatments. Overlapping clusters, such as MBA-1 and MBA-2, indicated significant attribute interaction. MDAR, PbS, and DTPA extractable soil vectors were pointed in the Pb-treatment direction with greater arrow lengths, showing their major impact on this treatment. SOD, SCW, and LA, which were nearest to MBA clusters, were greatly enhanced under these treatments. The SPAD value and carotenoid concentrations were shorter vectors and contributed less to Dim₁ and Dim₂ variability. The lower-right quadrant RP treatment separation showed significant differentiation, whereas the BN treatment was moderately related to Dim₁. Key factor clustering in the middle region showed shared impacts across treatments, demonstrating the interconnection and impact of the observed attributes on the dataset.

The heatmap in Figure 5 represents the clustering for the impact of organic and inorganic amendments on different growth, physiological, biochemical, gas-exchange, ionic, and oxidative stress attributes of the plant under Pb stress on the vertical axis and the treatments (CK, Pb, BC, BN, RP, MBA-1, MBA-2, and MBA-3) on the horizontal axis. The treatments and parameters are clustered hierarchically to identify their differences and dependence on one another to represent the impact of different treatments on measured parameters. The heatmap includes the color code across the gradient as follows: red represents highly correlated, blue represents lower correlation, and white represents a basal level for all the measured parameters in plant samples. All the parameters in the cluster help to differentiate them according to their responses against different treatments, i.e., oxidative stress indicators (MDA, H₂O₂, proline) are grouped which represents a highly negative correlation with different growth, physiological, biochemical, and gas-exchange attributes. This is due to Pb stress and is related to oxidative injury. On the other hand, RWC, MSI, Chl a, Chl b, carotenoids, SOD, POD, CAT, APX, and leaf area are positively correlated with growth, physiological, and gas-exchange attributes.

4. Discussion

The results of the current study demonstrated the adverse effects of Pb contamination on wheat-growth parameters (Table 3). It was evident that Pb toxicity significantly impeded plant height, leaf area, and root morphology. Our results demonstrated the positive effect of organic and inorganic amendments on growth parameters like shoot and root lengths, leaf area, and root morphology in the order MBA-1 > MBA-3 > MBA-2 > BC > RP > BN compared to Pb-contaminated soil. Moreover, plant physiological and gas functions improved many folds with amendments compared to Pb-contaminated soil (Figure 2 and Figure 6). The toxic effect of HMs, especially Pb, significantly reduced the wheat yield and chlorophyll content while a 10% reduction was observed in gaseous exchange by Rehman et al. [45]. The combination of biochar, rock phosphate, and bentonite performed well compared to their sole application in alleviating Pb toxicity. The application of biochar showed a pronounced effect in improving plant growth in many studies. The application of biochar increased maize yield by enhancing plant height, biomass, soluble sugar, and protein along with SPAD values in a seven-year-old biochar amended trail [46]. Biochar plays a significant role in immobilizing Pb through adsorption on its surface due to the high surface area of adsorption, presence of functional groups; i.e., hydroxyl (-OH), carboxyl (-COOH), and phenolic (-OH) groups on biochar surfaces form strong complexes with Pb ions and being alkaline in nature biochar precipitates Pb by increasing soil pH and makes it unavailable for a plant’s uptake [47]. Moreover, hydroxyl (OH⁻¹) and silanol (SiOH) functional groups on bentonite form strong inner-sphere complexes with Pb²⁺; also, the entrapment of Pb²⁺ in the interlayers of montmorillonite clay reduces Pb mobility in soil [48].

The soil nutrient status, especially primary nutrients like N, P, and K, and water-holding capacity were increased in biochar-amended soil along with a reduction in soil pH and ultimately enhanced crop growth [49]. In another study, biochar promoted the root length by decreasing root senescence [50]. Similarly, biochar-positive effects were also found in metal-contaminated soils by improving soil nutrients, CEC, and water-holding capacity [51,52]. The plant root and shoot length dry weights and chlorophyll contents were significantly enhanced with biochar application in Cd- and Pb-contaminated soil compared to un-amended soil [53]. The addition of biochar under drought and Cd stress greatly improved photosynthetic pigments, transpiration rate, biomass, and yield of wheat crop [54], which was attributed to the abundance of various functional groups on the biocarbon fractions (biochar) effectively binding Pb on its surface. Its high pH reduces metal availability, while interactions with different elements promote metal precipitation. Additionally, incorporating biochar into metal-polluted soil enhances soil physicochemical properties, such as water-holding capacity and cation-exchange capacity (CEC), supports microbial communities, regulates soil pH, and releases essential nutrients in soil rather than Pb [55].

Inorganic clay minerals such as bentonite and rock phosphate were used to enhance plant growth by prompting soil chemical and physical properties. Bentonite amendment has been found to significantly improve soil water-holding capacity, biomass accumulation, photosynthesis, and SPAD values of plants grown due to improvement in soil aggregation, nutrient uptake and biodiversity [56]. The dual impact of organic matter and bentonite decreased Pb stress and enhanced plant growth. The rock phosphate, being a P source, was utilized to improve crop growth in metal-contaminated soils. Qu et al. [57] also advocated that phosphate rock and sepiolite significantly improved the soil P availability through better root proliferation and boosting the synthesis of photosynthetic pigments under Cd-contaminated soil. Recent studies also demonstrated the combined effect of biochar with clay minerals and found it to be the best strategy for crop production under metal-contaminated soils.

A significant effect on plant growth, physiology, and root morphology was found in BMA-1, BMA-3, and BMA-2 amendments, which clearly suggested that improvement in plant functions might be owing to the combined effect of biochar, bentonite, and rock phosphate. The results of our investigation are in line with the findings of Lahori et al. [58] who also demonstrated growth improvement and reduced oxidative damage in lentils by the integration of biochar and inorganic minerals (chitosan and bentonite) due to the presence of binding sites for metals on biochar and enhancement of the CEC of the soil by minerals to stabilize Ni in soil. Similarly, it was observed that a combination of Ca-bentonite (CB), tobacco biochar (TB), and zeolite (ZL) decreased the metal (Cu) uptake in the pak choi and maize plants compared to their solo applications; moreover, they also found maximum dry biomass, SPAD values, and chlorophyll contents by integration under Cu- and Pb-polluted soil [59]. Moreover, combinations of natural minerals (bentonite) with biochar were acknowledged to adsorb heavy metals and other pollutants from the metal-contaminated soil for cleaner production of agricultural products [60,61]. Bentonite along with compost application significantly (p < 0.05) increased the pak choi and Chinese cabbage biomass and reduced the DTPA-Cu, Cd, and Pb contents in soil due to their complexation with mineral fraction and functional groups on biochar [51,62]. Another study also described the co-application of biochar with bentonite to improve the growth of plants (physiological and biochemical parameters) in many folds in Pb- and Zn-contaminated soil [3]. The application of rock phosphate-loaded biochar increases the mustered growth and reduces the uptake of HMs from contaminated soils [63].

Figure 6 showed that Pb toxicity induced oxidative stress (H₂O₂, MDA, and proline) while organic and inorganic amendment along with their combination significantly decreased MDA, proline, and H₂O₂ levels by lowering the Cd-toxicity to crop, a clear indication of stress alleviation under integrated treatment [64]. Similarly, Figure 3 depicts the production of antioxidants like SOD, POD, APX, and CAT in roots and shoots. These enzymes were responsible for alleviating oxidative damage to plant membranes as SOD converted the superoxide radical into H₂O₂ and later H₂O₂ was split into H₂O and O₂ under the action of POD, APX, or CAT to safeguard lipid peroxidation and rupturing of the cell wall [65]. The organic amendment, especially biochar and its combination with inorganic amendment significantly enhanced the antioxidant activity in wheat plants. Other studies also supported the same fact under different abiotic stresses, especially in metal-contaminated soils owing to reduced metal bioavailability [51,66]. The positive effect of biochar on antioxidant capacity was linked with high biomass production, availability of soil nutrients, immobilization, and non-availability of heavy metals to plants [45]. Apart from biochar, inorganic amendments were also reported to enhance plant enzymatic activities by improving plant biomass and chlorophyll content, and enhancing micronutrient availability to plants through low uptake of heavy metals [67]. The oxalic acid-activated rock phosphate increased soil phosphate activity and restricted the Pb uptake in the root, ultimately reducing root and shoot Pb concentration in mung bean under Pb-contaminated soils [68]. The application of rock phosphate also activates the native P solubilizing bacteria in the soil and improves plant growth and photosynthesis, and reduces As availability and enhanced enzymatic activities (Catalase and SOD) in the S. lineare plant [69].

The results of this study exhibited that the application of amendments, especially their mixture, significantly decreased the Pb contents in plants and effectively immobilized Pb in soil by decreasing the available Pb concentration compared to Pb-contaminated soil (Figure 4). Similar results were found by other authors where biochar and inorganic amendments reduced the Pb phytotoxicity and decreased the soil-available Pb contents. Organic amendments like biochar and vermicompost reduce the bioavailable fractions of Pb and Cu and promote a metal-free production of food [70]. Similarly, hydroxyapatite, bentonite, and biochar significantly reduced the bioaccumulation of Pb in plants and DTPA-extracted Pb in soil by increasing the residual fraction [71]. Biochar and bentonite were found to increase SOC and pH, whereas TCLP-Cd and Pb and DTPA extractible Pb and Cd in vegetables were decreased with an order of hydroxyapatite > biochar > bentonite. This decrease in bioavailability, mobility, and bio-accessibility of HMs (Pb and Cd) was attributed to metal and phosphate co-precipitation, complexation, and surface adsorption [51,71]. Moreover, it was found that processes like sorption and precipitation might stabilize the Pb and Cu in soil and decrease their availability due to abundant functional groups, high total pore spaces, and surface area of biochar [72]. Apart from biochar, bentonite also improved wheat growth and decreased the Cd concentration in the shoot four times compared to contaminated control soil [73]. The combination of bentonite along with organic sources was an efficient technique to alleviate HM toxicity as bentonite with humic acid significantly improved the growth and decreased the Pb uptake in spinach [74]. Bentonite with different amendments minimized the bioaccumulation of HMs in Solanum melongena L. irrigated with tannery wastewater [75]. The rock phosphate on the other hand was also reported to improve crop growth by competing with Pb and other heavy metals for plant uptake and mitigating HM toxicity by converting the exchangeable fraction to a soil-stable fraction [76]. Moreover, a combination of bentonite and soluble phosphate to HM-contaminated soil was found to reduce the bioavailability of Pb [77]. The BC and RP combination significantly lowered the Cd and Pb toxicity by converting the exchangeable fraction to the residual fraction and promoted the immobilization of HMs in soil [78]. The results of our study elaborated that a mixture of biochar, bentonite, and rock phosphate significantly decreased soil and plant Pb levels in the order MBA-1 > MBA-3 > MBA-2 > BC > RP > BN. Thus, it would be suggested to use a combination of BC, BN, and RP (MBA-1) to regulate Pb phytotoxicity and bioaccumulation in wheat crops grown in Pb-contaminated soil.

5. Conclusions

The proposed study was devised to assess the Pb-contamination threshold on wheat growth, physiology, and biochemical assays under the sole and combined effects of biochar, bentonite, and rock phosphate. The elevated Pb level (700 mg kg⁻¹) significantly affected wheat growth and induced oxidative stress. The addition of amendments alleviated the Pb phytotoxicity by reducing the Pb uptake and its mobility in soil, in return improving plant growth, plant physiological, and biochemical attributes. The amendments showed significant results in the order MBA-1 > MBA-3 > MBA-2 > BC > RP > BN. Thus, it can be concluded that the integration of biochar, bentonite, and rock phosphate, especially MBA-1 (biochar 50%, bentonite 20%, and rock phosphate 20% used to attain 1.5%), should be employed for sustainable and cleaner production of cereals on metal-contaminated soils. This study also broadened our understanding of the potential of combining organic and inorganic amendments at appropriate rates to boost plant growth under high levels of metal contamination. However, the efficacy of MBA-1 should be evaluated through field trials across diverse agroecological zones, focusing on its molecular-level interactions in heavy metal co-contaminated soils to ensure environmental safety and assess potential implications. The study of Pb speciation in soil also needs to be explored. Similar treatment should also be explored for other heavy metals (Cd, Cr, As, etc.) to broaden the scope of technology.