Chitosan with Bentonite and Biochar in Ni-Affected Soil Reduces Grain Ni Concentrations, Improves Soil Enzymes and Grain Quality in Lentil

: Ecological and human health risks associated with Ni-affected soils are one of the major attention seeking issues nowadays. The current investigation is based on the usage of biochar (BR), chitosan (CN), bentonite (BE), and their mixture to immobilize Ni in a Ni-polluted soil and accordingly contracted Ni distribution in lentil plant parts, improved grain nutritional quality, antioxidant defense system, and soil enzymatic activities. The soil was initially amended with CN, BE, and BR and later lentil was grown in this soil in pots. Results depicted the highest signiﬁcance of BE+CN treatment in terms of reducing the Ni distribution in the roots, shoots, grain, and DTPA-extractable fractions, relative to control treatment. Contrarily, the BR+CN treatment displayed the minimum oxidative stress and the utmost plant growth, chlorophyll contents in the leaves, relative water content (RWC), micronutrient concentrations, and grain biochemistry. The BR+CN indicated the highest activities of soil enzymes. Based on the results, we recommend BE+CN treatment to reduce the Ni distribution in the lentil plant. Although, improvement in plant growth, grain quality, soil enzymes, and a signiﬁcant reduction in plant oxidative stress can only be gained with BR+CN.


Introduction
Nickel is a pernicious heavy metal that originates from numerous anthropogenic and geogenic inputs [1][2][3]. Unregulated release of effluents from industries such as electroplating [4], batteries, Ni mining, and smelter [5] is a leading anthropogenic route of Ni pollution in the soils of Pakistan [4,5]. The permissible limit of Ni concentration in the soil is 50 mg kg −1 soil [6][7][8]. Recent investigations have reported that the soil Ni concentrations in the different cities of Pakistan were as follows: Kasur 46.4 mg kg −1 soil [9], on biomass, biochemical attributes, oxidative stress and antioxidant enzymes in lentil and enzymatic activities in soil and (3) to identify the most efficient combination of these amendments in terms of curtailing Ni toxicity to lentil plants grown under Ni stress.

Collection and Analysis of Ni-Affected Soil
The topsoil portion (0-15 cm) of Ni-affected soil was collected nearby an industrial zone receiving untreated effluents discharged from a Ni electroplating industry in the outskirt of Lahore, Punjab, Pakistan. The plastic soil sampling bags were filled with Ni-affected soil and transported to the laboratory. Before going to use the soil in this experiment, standard procedures were adopted to estimate the physicochemical characteristics of this Ni-affected soil. To this end, the soil was dried in shade by spreading it on a polyethylene sheet and sieved using a 2 mm sieve to remove stones and other impurities. The hydrometer method [40] was used to determine soil texture. To achieve it, 40 g of the soil was thoroughly mixed with 60 mL of sodium hexametaphosphate (NaPO 3 ) 6 in the beaker and placed overnight. The next day, the de-ionized water was added into the formulated amalgamate and shaken for 8 h. Later, this blend was carefully transferred into a 1 L graduated cylinder, filled to the standard mark, and a hydrometer was used to determine proportions of sand, silt, and clay. Likewise, electrical conductivity (EC) and soil pH were determined using calibrated EC meter (Thermo Orion 4 Star (Thermo Scientific, Waltham, MA, USA) and standardized pH meter (Thermo Orion 4 Star pH ISE Benchtop Meter, Cole-Parmer Co., Vernon Hills, IL, USA) respectively, at 25°C in a saturated soil paste that was prepared by agitating soil-water mixture (1:2.5, soil: deionized water) for approximately 1 h. The CEC of Ni-affected soil was assessed by adopting the procedure endorsed by Rhoades [41]. To achieve this, the cation exchange sites of the soil were saturated with Na through equilibrating a soil sample by a formulated amalgamate of 0.4 M sodium acetate (NaOAc) and 0.1 M sodium chloride (NaCl) in 60% ethanol. Next, this Na saturated soil was extracted by 0.5 M magnesium nitrate [Mg(NO 3 ) 2 ] to quantify the total fraction of exchangeable Na. For determining the CEC of the soil, the quantity of total Na in the extract was estimated on ICP−MS (PerkinElmer's NexION ® 2000, PerkinElmer, Inc., Waltham, MA, USA). In a similar context, the organic matter was determined employing the Walkley-Black procedure as suggested by Jackson [42]. For this purpose, 1 g of air-dried soil was blended with 10 mL of 1 N potassium dichromate (K 2 Cr 2 O 7 ) and 20 mL of sulphuric acid (H 2 SO 4 ). Next, this formulated concoction was amalgamated with 10 mL of concentrated orthophosphoric acid (H 3 PO 4 ) and 200 mL of de-ionized water and further titrated with 0.5 M ferrous ammonium sulphate (FeH 8 N 2 O 8 S 2 ). Similarly, the content of plant-available P was acquired from the soil by adopting a standard method proposed by Olsen [43]. To this end, 2.5 g of soil was amalgamated with 0.5 M sodium bicarbonate (NaHCO 3 ) (adjusted pH = 8.5) solution and shaken for 30 min. After filtration of this amalgamate, the orthophosphate (PO 4 3− ) in the filtrate was estimated on a spectrophotometer (Analytik Jena SPECORD 200 PLUS, Analytik Jena AG, Jena, Germany) by reacting it with ammonium molybdate [(NH 4 ) 6 Mo 7 O 24 ]. The amount of exchangeable K was valued by following the method put forwarded by Richards [44]. For this, a soil sample (5 g) was mixed with 33 mL of 1 N ammonium acetate (NH 4 OAc). This amalgamate was centrifuged to achieve a transparent supernatant. After filtration, the NH 4 OAc extract was diluted with de-ionized water and the concentration of K was measured at 767 nm on a flame photometer (BWB Model BWB-XP, 5 Channel). Likewise, the total N in the soil was determined by a standard protocol suggested by Bremner and Mulvaney [45]. The content of CaCO 3 was also determined in Ni-affected soil via dissolution of carbonate using 0.5 M (hydrochloric acid) HCl and titration of extra acid by 0.2 M sodium hydroxide (NaOH) [46]. For the estimation of plant available Ni in the soil, it was extracted with 0.005 M diethylenetriaminepentaacetic acid (DTPA) solution. To prepare this solution, 1.97 g of DTPA and 1.1 g of calcium chloride (CaCl 2 ) were mixed within a beaker and dissolved with de-ionized water up to 1 L volume. Separately, 14.92 g triethanolamine (TEA) was dissolved with de-ionized water and made a volume of 900 mL within a 1-L flask. Then, 6 N HCl was used to adjust the pH (at 7.3) of this solution. After taking the extract, the concentration of Ni was measured on ICP−MS [47]. A subsequent quantity of soil (5 g) was deployed for its digestion in aqua regia mixture [HCl: nitric acid (HNO 3 ), 3:1 v/v) to determine the amount of total Ni in the soil [48] and later examined on ICP−MS.

Pot Trial
Prior to using the soil to conduct this pot trial, the soil was mixed with BR, CN, and BE at various proportions. These immobilizing amendments (as an individual dose at 2% and their combination at 1% each) were homogenized with extreme care in the Ni-affected soil as per the experimental plan (Table 1). Afterward, the plastic pots (25.4 cm width, 33.0 cm height) were filled with 5 kg un-amended soil as control and modified soil with immobilizing amendments having three replicates. Later, the position of filled pots was switched to a greenhouse depicting exemplary environmental conditions and kept in the randomized design. Further, lentil seeds (15 seeds pot −1 ) were sown in each pot. After one week of seed sprouting in the pots, two plants per pot were maintained. The pots were irrigated with deionized water two times per week to maintain optimum moisture levels (65% WHC). After the 100 days of plant growth, the lentil pods became mature which was the sign that the plants were ready to harvest.

Reaping of Lentil Plants and Analysis
Right afore plant gleaning, the plant height was estimated with the help of a measuring tape. After 100 days, lentil plants were carefully clipped near the soil surface with the help of a sharp cutter. After the harvest of aerial biomass, the soil from the individual pot was profited with ultimate care to eradicate roots. Thereafter, the plant shoots were comprehensively rinsed with deionized water to abolish aerially fallen dust flecks. Whereas, the roots were initially washed with deionized water to remove the soil adhered to them and later in 0.05 M CaCl 2 solution in an ultrasonic bath for the removal of metal ions from the apparent free space of root tissues, correspondingly.
The leaf fresh weight (FW), turgid weight (TW), and dry weight (DW) of the plants were employed to calculate relative water content [RWC (%)] with the help of an Equation (1). To this end, the fresh leaves of lentil plants were weighed to estimate the FW. To determine the TW, these fresh leaves were put in test tubes, filled with distilled water, kept in dark at room temperature (25 • C) for 24 h, and later weighed. Coming after, the fresh leaves were oven-dried (60 • C, 48 h) till the perpetual DW was achieved. Afterward, the RWC was estimated [49]. (1)

Determination of Ni in Soil and Plant Parts and Micro-Nutrients in Grain
For the estimation of plant-available Ni, the soil was extracted with 0.005 M DTPA solution [DTPA + TEA + CaCl 2 , pH = 7.3] and later the concentration of Ni in the extract was analyzed on ICP−MS. Since the soil used in this experiment was calcareous, the extractability of Ni is best determined with DTPA extractant [50]. Previously, it has been well reported that the fractions of Ni extracted with DTPA extractant from the calcareous soils were well correlated with the Ni concentrations in the plants [5,51,52].
To achieve a constant DW of plant biomass, the harvested plant material was initially air-dried and later oven-dried in an oven (Memmert, Beschickung-loading, model 100-800, Schwabach, Germany) for 72 h at 70 • C. The concentrations of micro-nutrients in grain and Ni concentrations in the shoots, and roots and grain were assessed in digested plant parts. To this end, 1 g of plant DW (roots, shoots, and grain) was taken, pulverized in a mill, and passed from 2 mm mesh. Subsequently, these ground samples of plant parts were digested by way of a di-acid concoction [HNO 3 : perchloric acid (HClO 4 ), 2:1, v/v) for the evaluation of Ni in the roots, shoots, and grain, as well as essential micro-nutrients in grain [53]. Later, the concentrations of these cations were measured on ICP−MS.

Biochemical Spectrum of Grain
Numerous biochemical compounds were also estimated in the grain to gain an insight view of grain nutritional quality as affected by different amendments. The contents of fiber and fat were determined by following the Association of Official Analytical Chemists (AOAC) protocol [54]. Similarly, the content of total soluble protein was estimated by following the protein-dye binding procedure after the addition of the subsequent amount of buffer into the protein reagent. Later, bovine serum albumin as the stock was employed to record the interference of components present in the sample buffer with Bradford assay [55]. The Folin Ciocalteu method was used to analyze polyphenols in grain by calculating an absorbance coefficient curve of gallic acid at 760 nm on a spectrophotometer.

Activities of Antioxidants and Oxidative Injury in the Leaves
For the estimation of the antioxidants activities and ROS contents in lentil leaves, 0.5 g weight of fresh leaf was taken, comprehensively mixed in 4 mL potassium phosphate (K-P) buffer (50 mM, pH = 7) possessing 5 mM β-mercaptoethanol, 1 mM ascorbic acid (AsA) prepared from 10% w/v and 100 mM potassium chloride (KCl). Afterward, the concoction was centrifuged at 11,500× g to acquire liquid. Later, the response of antioxidants was evaluated in the liquid. The SOD activity was assessed by taking a particular amount (1 mL) of reaction mixture consisting of 10 mM pyrogallol, 100 mM ethylene diamine tetraacetic acid (EDTA), 50 mM sodium phosphate (Na-P) buffer with pH 7.8, and 50 mL enzyme extract. The spectrophotometer was employed to recognize the absorbance in the reaction concoction at 420 nm [56]. In a similar context, Cakmak and Marschner [57] protocol was deployed for observing the CAT response in leaves. To prepare an exclusive reaction mixture, H 2 O 2 (10 mM, 1 mL) was comprehensively assorted with enzyme extract [2 mL diluted with 50 mM buffer (pH = 7)] and a spectrophotometer was employed to note the absorbance in the reaction concoction at 420 nm. A reaction concoction (0.5 mM AsA, 0.5 enzyme extract, 0.25 mL EDTA, 0.1 mM H 2 O 2 , and Na-P buffer) was prepared to assess APX activity in lentil leaves. The alteration in absorbance was recorded at 290 nm. The particular APX activity was calculated in a reaction concoction with the help of an extraction factor at 40 mM −1 cm −1 [58].
The ROS such as the contents of H 2 O 2 and MDA and O 2 − generation rate were also examined in leaves by performing recommended methods. For the estimation of H 2 O 2 content, 0.5 g leaf FW was taken and mixed in 5 mL K-P buffer. Afterward, to obtain a supernatant, the enzyme mixture was centrifuged at 10,000× g for approximately 15 min. Afterward, 5 mL of 0.1% w/v trichloroacetic acid (TCA) mixture was taken, homogenized in 10 mM K-P buffer (pH at 7) containing 1 mL of 1 M potassium iodide (KI). Later, the absorbance value of H 2 O 2 in the reaction solution was noted at 390 nm by using a spectrophotometer [59]. The MDA was estimated in leaves by taking the extraction of fresh leaf tissue (0.5 g) dissolved in 5 mL of TCA (0.1%). For obtaining a 2.5 mL supernatant, the mixture was well homogenized and later centrifuged at 10,000× g for a time interval of 15 min. Afterward, a particular amount (1 mL) of TCA (20%) and thiobarbituric acid (TBA) (0.5%-w/v) were blended, heated at 95 • C, and allowed to cool for 30 min to acquire the reaction mixture. Thereafter, an absorbance at 532 and 600 nm was taken with the help of a spectrophotometer. Finally, the MDA contents in the reaction solution were recorded by observing the differences in absorbance at 532 and 600 nm ensuing Beer and Lambert's expression [60]. Likewise, the reduction rate of nitroblue tetrazolium (NBT) was calculated to estimate the generation rate of O 2 − in leaves at 25 • C (pH = 7) in a 5 mL supernatant mixture obtained from 0.5 g leaf tissue [61]. For each assay, the reaction assay was comprised of 50-200 µg protein, 50 mM nicotinamide adenine dinucleotide (NADH), 100 µM NBT, 100 mM KCl, 100 µM EDTA and 50 mM phosphate. The generation of O 2 − radicals in the reaction assay was recorded by calculating a reduction rate of NBT after 500-1000 units of SOD addition. Afterward, the pure mixture of xanthine oxidase was further added for calibrating the feedback of NBT to the synthesis of O 2 − within the observed variations arising betwixt the reaction of NBT and O 2 − [61].

Soil Enzymatic Activities
A handsome amount of soil from each experimental pot was taken to estimate the activities of soil enzymes. For this purpose, the soil was passed through a mesh (2 mm) to remove impurities. The response of β-glucosidase in post-harvested soil was also estimated [62]. Synthetic substrate (ρ-nitrophenyl-β-D-glucopyranoside) was added in a 1 g soil sample and incubated (37 • C) for about 1 h. Later, to terminate the reaction of β-glucosidase, further addition of Tris (pH = 12, 0.02 mol L −1 ) was made. A spectrophotometer was used at 464 nm to record the reduction rate of the ρ-nitrophenyl substrate. Similarly, catalase activity was assessed based on the content of H 2 O 2 consumed by the soil. A soil sample (5 g) was taken, homogenized with 25 mL H 2 O 2 (3%) ensuing incubation at 4 • C for approximately 30 min. Thereafter, the reaction mixture was filtered and 25 mL of 1 M H 2 SO 4 was added to the filtrate. Afterward, 5 mM potassium permanganate (KMnO 4 ) was taken and the reaction mixture was titrated [63]. Similarly, the response of phosphomonoesterase was also assessed in post-harvested soil [64]. Phosphomonoesterase activity was computed by plotting a standard calibration curve and it was noticed that reaction was substrate-dependent. Similarly, for the estimation of acid phosphatase activity, soil (1 g) was taken, incubated at 37°C for 1 h, and homogenized in 0.1 M of acetate buffer (pH-5.4). Later, ρ-nitrophenyl phosphate was adjusted [65].

Statistical Analysis of Experimental Data
The current pot study was executed in a CRD (completely randomized design) and the treatments were performed in three replicates. Later, the data were interpreted using Statistix 8.1 ® (Analytical Software, Tallahassee, FL, USA) through a one-way analysis of variance (ANOVA). Similarly, the least significant difference (LSD) test [66] was employed to calculate the significant difference (P < 0.05) between treatments.
Nickel concentrations were computed from 34.8 to 72.9, 13.7−43.2, and 2.4−19.8 mg kg −1 DW in roots, shoots, and grain, correspondingly, for lentil while the plant available Ni was estimated from 1.05 to 3.06 mg kg −1 soil ( Figure 1). Nickel concentrations in roots, shoots, and grain, as well as plant-available Ni in the soil, were significantly lessened by the mixing of BR, CN, and BE (as an individual dose at 2% and consolidation at 1% each) in a Ni-affected soil, comparative to untreated soil. The treatment BE+CN resulted in the least Ni concentrations to significant extents in grain, shoots, roots, and plant available Ni in the soil that was statistically lower up to 87%, 68%, 52%, and 66%, respectively, over control treatment.

Biomass, Growth, Chlorophyll Contents, and RWC of Lentil
The response of plant height, shoot, and root dry biomass was in extent from 45.4 to 76.4 cm, 3.1−5.7, and 1.2−1.9 g pot −1 , respectively, in whole treatments ( Table 2). Application of BR, CN, and BE in a Ni-affected soil significantly improved biomass and growth of lentil. Interestingly, in contrast to plants in control, the maximal data of biomass and growth was acknowledged in the plants grown in the soil tainted with BR+CN that was statistically augmented up to 68%, 86%, and 58%, respectively. The outcome of Chl-a, Chl-b in leaves of lentil, and RWC was recorded from 0.65 to 0.86, 0.53−0.83 mg g −1 FW, and 73.8−91.7%, respectively (Table 2). Every single treatment represented a significant rise in the contents of Chl-a (except BE treatment) and Chl-b (except both BE and CN treatments) in leaves over control. However, the highest Chl-a (in BR+CN treatment) and Chl-b (in BR+CN treatment followed by BR+BE) were recorded in leaves of lentil which were significantly higher for Chl-a till 51% and Chl-b till 56% and 46%, respectively. The relative water content of lentil plants in all treatments was calculated from 73.8 to 85.4% ( Table 2). Incorporation of BR, CN and BE into the soil, excluding BE treatment, significantly increase RWC of lentil, compared to control. However, statistically, the greatest RWC values of lentil plants were calculated in BR+CN treatment followed by BR+BE and BE+CN which were increased by 24%, 20%, and 15%, respectively while comparing these findings with control.

Biomass, Growth, Chlorophyll Contents, and RWC of Lentil
The response of plant height, shoot, and root dry biomass was in extent from 45.4 to 76.4 cm, 3.1−5.7, and 1.2−1.9 g pot −1 , respectively, in whole treatments ( Table 2). Application of BR, CN, and BE in a Ni-affected soil significantly improved biomass and growth of lentil. Interestingly, in contrast to plants in control, the maximal data of biomass and growth was acknowledged in the plants grown in the soil tainted with BR+CN that was statistically augmented up to 68%, 86%, and 58%, respectively.

Biochemical Compounds and Micronutrients in the Grain and Soil pH
Data regarding biochemical attributes (protein, fiber, fat, total sugar, and polyphenols) in lentil grain were measured in the ranges from 17.6 to 24.9, 3.9−7.0, 0.9−1.5, 1.9−3.1, and 3.3−5.9 mg g −1 FW, correspondingly in whole treatments (Table 3). Adding BR, CN, and BE into a Ni-affected soil statistically improved protein (excluding BE and CN treatments), fiber, fat, and total sugar, and while diminished polyphenols in grain over plants in unamended soil. The BR+CN treatment demonstrated the greatest fiber and total sugar in the grain which was significantly improved up to 78% and 61%, respectively. Statistically, the highest fat content in the grain was found in BR+CN treatment followed by BR+BE and BE+CN up to 58%, 50%, and 45%, respectively, compared to plants of control. Similarly, the BR+CN and BR+BE treatments demonstrated the maximal protein (improved by 42% and 30%, respectively) while minimal polyphenols (diminished by 45% and 38%, respectively) in the grain in contrasting with plants of the untreated control.  Table 3). Application of BR, CN, and BE increased the concentrations of Zn, Mg, and Ca in the grain over control. Interestingly, significant concentrations at their peaks for Zn (in BR+CN treatment by 63%), Mg (in BR+CN, BR+BE and BR treatments by 32%, 27% and 23%, respectively) and Ca (in BR+CN and BR treatments by 38% and 32%, respectively) in grain, relative to plants in control, were reported. Likewise, all treatments exhibited significant upgradation in concentrations of Mn and Na (with exception of BE treatment), and Fe (with exception of BE and CN treatments) in the grain over plants in control. Surprisingly, the BR+CN treatment demonstrated the highest advancement in concentrations of Mn, Na, and Fe in grain up to 71%, 32%, and 42%, respectively, compared to control.
Likewise, The values of pH were ranged from 7.03 to 7.78 in the post-harvest soil of entire treatments (Table 3). Except for CN and BR+CN, the further treatments were able to expressively improve the pH values of post-harvest soil, relative to the control. Significantly, the highest pH values were found in the soils treated with BE, BR+BE, and BE+CN that were 0.75, 0.61, and 0.51 units greater than the control treatment. − by 45% and 40%, respectively with respect to control. In the instance of MDA, the highest reduction in leaves was resulted due to the BR+CN treatment which was statistically 56% lower, compared to control. Similarly, SOD and APX in leaves, in contrast with control, could be noticed in BR+CN, BR+BE, and BE+CN treatments which presented as significantly topmost bolstered up SOD by 36%, 29%, and 25%, respectively, and APX by 41%, 35%, and 32%, respectively. For CAT, the maximum activity was found in leaves of plants grown in BR+CN treatment that was significantly upgraded by 75%, relative to un-amended control.

Enzymatic Activities in Soil
Data of enzymatic activities such as β-glucosidase, phosphomonoesterase, catalase, and acid phosphatase in soil were calculated in the ranges from 1.  (Figure 3). All treatments significantly enhanced phosphomonoesterase and catalase in the soil over control treatment. However, the maximum activities of both phosphomonoesterase and catalase were estimated in the soil of BR+CN treatment that was conspicuously incremented up to 200% and 162%, correspondingly, over control. Application of BR, CN, and BE in the soil, except BE treatment, significantly raised β-glucosidase and acid phosphatase in a Ni-affected soil compared to untreated soil. Interestingly, the BR+CN treatment showed statistically the highest improvement in β-glucosidase by 0.72% and acid phosphatase by 65% in the soil, compared to control. Application of BR, CN, and BE in the soil affected by Ni significantly controlled the generation of O2 − , and the contents of H2O2 and MDA, whereas, upgraded APX, CAT, and SOD (except BE treatment) activities in leaves over control. However, non-significantly the highest decline of H2O2 and O2 − in leaves of lentil grown in BR+CN and BR+BE treatments was seen. These BR+CN treatment reflected maximal curtailment in H2O2 and O2 − by 45% and 40%, respectively with respect to control. In the instance of MDA, the Minerals 2021, 11, x. https://doi.org/10.3390/xxxxx www.mdpi.com/journal/minerals the soil over control treatment. However, the maximum activities of both phosphomonoesterase and catalase were estimated in the soil of BR+CN treatment that was conspicuously incremented up to 200% and 162%, correspondingly, over control. Application of BR, CN, and BE in the soil, except BE treatment, significantly raised β-glucosidase and acid phosphatase in a Ni-affected soil compared to untreated soil. Interestingly, the BR+CN treatment showed statistically the highest improvement in β-glucosidase by 0.72% and acid phosphatase by 65% in the soil, compared to control.

Nickel Distribution in Lentil Plant, Bioavailable Ni in Soil, and Soil pH
We found that application of BR, CN, and BE in the Ni-affected soil considerably diminished Ni distribution in lentil (roots, shoots, and grain) and bioaccessible Ni in the soil after plant reap. These data are more noticeable in lentil plants grown in the soil of BE+CN treatment (Figure 1). This significant scaling of Ni concentrations in the plant portions, as well as Ni bioavailable fraction in the soil, has already been reported by several studies after the application of BN and CN in Ni-affected soils [28,29,38]. Conceivable reasoning behind this significant drop in Ni concentrations in shoots, roots, and grain as well as plant available Ni in the soil is due to the adsorption of Ni onto the large surface area of BE [36,38] and CN [12,28,29] through ion exchange, chemisorption, and complexation in the soil, and therefore, restricted Ni uptake by the plants [28,29,32,38]. Chitosan exhibits numerous hydroxyl and amino groups that support Ni chelation through inter or intramolecular binding [12,28,29]. Additionally, the presence of alumina, silica, and oxides (i.e., Al2O3, MnO, Fe2O3, CaO, and MgO, etc.) in BN also supports Ni immobilization via cation exchange and complexation mechanisms [67]. All these mechanisms supported Ni immobility in the soil and its reduced uptake and distribution in lentil plant parts.
The bioavailability of Ni in the soil is directly controlled by the soil pH [67,68]. According to our data, significantly the highest pH values of the post-harvested soil were found in BE, BR+CH, and BE+CH treatments, compared to control (Table 3). Previously, the significant rise in the pH values of post-harvest soils by 1.5 units [69], 0.84 unit [70], and 1.38 units [71] with the application of BR derived from woodchip, lignin, and rice straw feedstocks, respectively, were reported. Furthermore, the rise in pH values of different metal-contaminated soils after the addition of BE has been well documented [72][73][74]. During the charring process of feedstock to manufacture BR, basic cations (Mg, K, Na, and Ca) existing in the feedstock are transformed into corresponding carbonates, oxides, and hydroxides [75,76] which escalate soil pH after their dissolution upon BR addition in the soil [77]. Furthermore, the rise in soil pH after the addition of BE is associated with its alkaline nature [67,73]. A rise in soil pH ropes the development of insoluble oxides, hydroxides, carbonates, and phosphate of Ni and therefore, triggers Ni bioavailability in the soil [67,68,78].

Biomass, Growth, Chlorophyll Contents, and RWC of Lentil
We commenced that growth (plant height, shoot and root dry biomass), Chl-a, Chl-b, and RWC parameters (with few exceptions) were significantly enhanced by the incorporation of BR, CN, and BE in a Ni-affected soil, relative to control. Surprisingly, data associated with these parameters were found the most significant with BR+CN treatment ( Table 2). Retardation in the growth and biomass of lentil [26], sunflower [28], red clover [4], and maize [5] grown in soil contaminated with Ni has been previously addressed by several authors. Similarly, Ni-affected soils significantly curtailed the Chl contents in the leaves of lentil [25] and RWC in green buttonwood [79]. The improvement in the growth, biomass, and chlorophyll contents of lentil after the application of BR and CN in Ni-affected soil is in conformity with the outcomes of preceding studies [4,5,28,29]. The possible mechanism behind this upgradation of growth and biomass in lentil might be because of the occurrence of BR in the soil which raised soil pH (Table 3) through the existence of CHNO bearing functional groups, carbonates, and −OH groups as well as the release of basic cations like Mg, Ca, Si, etc. [76]. The rise in soil pH is known to improve plant growth by relieving Ni stress to them through transforming the bioavailable Ni fractions into its nonsoluble compounds i.e., oxides, hydroxides, carbonates, and phosphate [67,68]. Another justification could be that the mixing of BR and CN in Ni polluted soil promoted overall soil health via improving porous structure, moisture retention, plant-water relationship, and CEC [11,28,29] and thereby, upgraded plant growth. Furthermore, improvement in biomass, photosynthesis, and RWC of lentil could be concomitant with improved plant health via a sharp decline in bioavailable Ni after its adsorption onto carboxyl, hydroxyl, phenolic, and aromatic stretches of BR plus free amino and hydroxyl groups CN [12,28,29]. Remarkably, BR acts as a slow-release fertilizer and provides imperative nutrients to the plants following its addition to the soil [67,80]. Interestingly, the adsorption of Ni onto BR surfaces releases an equal quantity of cations (Ca 2+ , Na + , Mg 2+, and K + ) as a mechanism of cation exchange. These released cations are uptaken by the plants which improve their growth and biomass [67,80].

Biochemical Compounds and Micronutrients in the Grain
With few exceptions, the biochemical compounds (protein, fiber, fat and total sugar) and micronutrients (Fe, Zn, Mn, Mg, Ca, and Na) concentrations in grain were significantly boosted by incorporating BR, CN, and BE into a Ni-affected soil over control (Table 3). However, the most significant data related to these parameters were found in the plants of BR+CN treatment. The toxicity of Ni to the plants has been reported to reduce the biochemical compounds in red clover [4] and brinjal [29] as well as micro-nutrients in sunflower [28] and brinjal [29]. Surprisingly, after BR and CH application in Ni-affected soil, enhancement in the biochemical compounds in maize, sunflower, spinach, brinjal, and wheat has been previously reported [4,5,11,24,28,29]. Likewise, an increment in the status of micronutrients in lentil grain is in alignment with the findings of recent studies where advancement in micro-nutrients in maize [31] and quinoa [30] grown in BR amended soil as well as in cereal in CN amended soil [81], was reported. The enhancement in the contents of biochemical compounds as well as micronutrients in grains might be because of the augmentation in the soil WHC, porosity, health, and enzymatic activities after amending with BR [11] and CN [34,67] which ultimately enhanced nutrient bioavailability and transferability in plants [82] as well as a higher generation of protein, carotenoids, sugar, starch, and amino acids through improvement in plant metabolism via efficient water mobility through the xylem to the leaves [83]. Moreover, the presence of BR in the soil supports the plant nutrition and quality via (i) the slow release of nutrients from mineralization of BR as well as adsorbed nutrients from its surfaces [84,85] and (ii) set free mineral nutrients from BR surfaces during the adsorption of heavy metals through cation exchange [67,85].

Oxidative Injury and the Status of Antioxidant Defense Machinery in Lentil
To overcome Ni stress, the plants generate antioxidant enzymes (such as CAT, SOD, APX, etc.) which help plants to cope with oxidative stress like the excessive generation of ROS [15,86] via inhibition of oxidizing chain reaction [87]. Resultantly, this oxidative pressure destroys primary organelles such as lipid, protein, and DNA through the inactivation of enzymes which as a result, causes the death of cells [24,28,29]. In our research trial, we found that the incorporation of BR, BE, and CN into Ni-stressed soil significantly (including some exceptions) lessens the production of ROS and boosted up antioxidants enzymes in leaves. Interestingly, these results were more conspicuous in BR+CN treatment ( Figure 2). The data of our study is coherent with conclusions of preceding research where enhancement of antioxidant enzymes whereas lowering oxidative pressure in sunflower, maize, and brinjal grown in a Ni stressed soil with the incorporation of BR and CN, were reported [24,28,29]. Application of BR and CN in Ni stressed soils intensify the generation of antioxidants and reduced ROS in the plants due to mitigation of Ni stress to them via reduced Ni bioavailability in the soil after its immobilization on the colossal surface area of BR and CN as well as on their active functional groups [4,5,28,29,88,89]. Chitosan displays the capability to provoke vital nutrients via altering the osmotic pressure in the cell as well as declining the load of noxious free radicals [90], which leads to augment antioxidant enzymes [91]. Moreover, CN has a large surface area encompassing reactive functional groups which equivalently implant antioxidants on their skeleton [91,92]. Additionally, the presence of CN in the soil is known to shrink ROS generation and boost the immune response in the plants by gene expression of CAT and SOD [67].

Enzymatic Activities in the Soil
The enzymatic activities in the soil are considered as a biological indicator that demonstrates the whole quality of the soil specifically for assessing the influence of Ni contamination in soil because the elevated concentration of Ni in the soil inhibits soil enzymatic activities [11,93]. In our investigation, soil enzymatic activities (β-glucosidase, phosphomonoesterase, catalase, and acid phosphatase) were significantly affected in a Ni-affected soil amended with BR, BE, and CN, compared to the untreated soil ( Figure 3). Remarkably, these findings were more pronounced in the soil of BR+CN treatment. Similar data regarding enzymatic activities in the soil was previously demonstrated by researchers who have amended soil using BR [11,94] and CN [35]. In our experiment, the rise in the activities of soil enzymes is owing to the alleviation of Ni stress to the soil microbes after Ni was immobilized on BR and CN surfaces [67]. Furthermore, BR and CN inclusion in the soil leads to better soil conditions which are favorable for boosting the microbial biomass and their activities [67,68,70]. It has been well documented that BR, as a slow-release fertilizer, provides essential nutritional elements to the soil microbes which increase their abundance and diversity leading to the secretion of higher concentrations of soil enzymes by them [70].

Conclusions
The current investigation depicts the exclusive role of BR, CN, and BE to render the bioavailability of Ni in a Ni-affected soil and resultantly lessen uptake by lentil. Each treatment significantly abridged the Ni uptake in the roots, shoots, and grain, relative to control. However, the most significant outcomes were found in BE+CN in terms of Ni concentrations in these plant parts. Whereas, BR+CN treatment indicated the minimum oxidative stress and the utmost plant growth, Chl contents, RWC, grain micronutrients, and biochemical compounds and soil enzymes. We suggest using BE+CN treatment in Ni-affected soil to reduce Ni concentrations in plant parts whereas, BR+CN treatment can improve plant growth, grain nutrition and biochemistry, soil enzymes, and oxidative stress to plant.