Potential of organic and inorganic amendments for stabilizing nickel in acidic soil, and improving the nutritional quality of spinach

Contamination of soils by nickel (Ni) has become a serious environmental problem throughout the world, and this substance wields dangerous effects on the ecosystem and food chain. A pot experiment was conducted to examine the effect of rice straw (RS), rice straw biochar (BI), and calcite (CC) at 1% and 2% application rates in a Ni-contaminated soil. The objective was to potentially stabilize Ni and reduce its bioavailability to spinach (Spinacia Oleracea L.). Spinach plants were grown in a Ni-contaminated Ultisol (commonly known as a red clay soil). Plant growth parameter results indicated that a BI 2% application rate significantly increased the root and shoots dry biomass increased by 1.7- and 6.3-fold, respectively, while essential nutrients were enhanced in the spinach plant compared to those in the untreated soil (CK). Moreover, adding amendments significantly decreased CaCl2 extractable Ni by 62.5% 94.1%, and 87.2%, while the toxicity characteristics leaching procedure (TCLP) fell by 26.7%, 47.8%, and 41.7% when using RS, BI, and CC, respectively, at 2% compared to CK. The Ni concentrations in the spinach roots declined by 51.6%, 73.3%, and 68.9%, and in the shoots reduced by 54.1%, 76.7%, and 70.8% for RS, BI, and CC, at a 2% application rate, respectively. Bio-concentration factor (BCF) and translocation factor (TF) dropped significantly by as much as 72.7% and 20%, respectively, for BI 2% application rate. Results of the present study clearly indicated that biochar potential soil amendments for Ni stabilization, thereby reducing its bioavailability in the Ni-contaminated soil. This process enhanced the safety of food to be consumed and mitigated security risks.


Introduction
Potential toxic elements (PTEs) that contaminate soil constitute one of the world's major problems regarding food security and human health (Xu et al. 2015;Shaheen et al. 2019). Contamination of soils by nickel (Ni) has become a serious environmental concern/threat throughout the world, as it reduces plant growth and poses detrimental effects on plant and animal's health when it enters in the food chain. It is released into the environment due to increased globalization of economic systems, modern industrialization, household use, municipal effluents, and too much application of pesticides and fertilizers (Ramzani et al. 2016;Shahzad et al. 2018).
In south and central China, Ni has existed as a key pollutant in agriculture for some decades (MEEPRC 2018;Ali et al. 2020), where the mean concentration of Ni is 226 mg kg −1 and maximum 1000 mg kg −1 in the arable lands of Jinchang City. This municipality is also famously known as "Nickel City" in Gansu Province, which has the highest Ni concentration than permissible value, that is > 40 mg kg −1 (MEEPRC 2018).
Consequently, at low levels, Ni plays a significant role in seedling growth and development (Rizwan et al. 2018;Shahzad et al. 2018). However, elevated Ni concentrations in soil constitute a major challenge because Ni typically has high availability, mobility, and toxicity (Shahbaz et al. 2018b;Shaheen et al. 2019), and is easily accumulated by leafy vegetables including spinach and cabbage (Salam et al. 2019;Bashir et al. 2020). Elevated Ni can in turn cause physiological disorders in plants and curtail crop growth (Mosa et al. 2016;Sehrish et al. 2019).
Spinach (Spinacia Oleracea L.) is one of the most popular leafy vegetables and it is grown throughout the world. In 2014, China became the world's largest spinach producer and in fact produced 22.1 million tonnes, representing 85% of the global supply (Pariona Ameber 2017; Boostani et al. 2019). Compared to cereal crops, a leafy vegetable such as spinach exhibits a great potential to accumulate Ni from the soil, resulting in compromised growth, smaller yield, and poorer quality (Younis et al. 2015;Nawab et al. 2019). Khan et al. (2017) noted that larger amounts of essential nutrients and physiological characteristics of plant leaves are necessary to achieve proper spinach growth under conditions of Ni stress, during which amendments may have to be added. Therefore, minimizing or decreasing Ni toxicity from the soil while at the same time ensuring reduced Ni uptake by spinach are important processes for retaining a healthy environment and healthy people. Ensuring the nutritional quality of spinach requires the incorporation of various amendments in soil, and this can be achieved by minimizing Ni uptake in spinach and immobilizing/stabilizing Ni in contaminated soil.
Use of traditional techniques such as soil excavation, land filling, and soil washing for soil remediation have been developed but they are time-consuming, expensive, and disturb the environment (Naeem et al. 2017). For these reasons, it is important to develop techniques that can immobilize/stabilize Ni in situ and the phytoremediation remediation technique are effective, cost-effective, and eco-friendly (Azhar et al. 2019). Alternatively, using organic amendments could be considered for the immobilization of Ni soils by curtailing Ni availability in polluted soils (Naeem et al. 2017;El-Naggar et al. 2018).
In China, the amount of annual residues of rice straw (RS) is estimated to be approximately 3.80 billion tonnes Ali et al. 2020). One important strategy is to apply RS into soil to increase soil organic matter, nutrient contents, and improving crop yields and also decrease the availability of PTEs including Ni in contaminated soils (Feng et al. 2014). Meanwhile, the RS incorporation into soils may maintain a few months due to rapid organic matter decomposition by soil microorganisms Gao et al. 2020) The pyrolyzed form (i.e., biochar) of RS application to soils has emerged as being better than RS (Naeem et al. 2017;Khan et al. 2019). Biochar (BI) can serve as an effective organic amendment that possesses the ability to adsorb PTEs in soil by electrostatic interaction, complexation, ion exchange, precipitation, and chemo-sorption Ali et al. 2019). Different biochars have been widely incorporated in contaminated soils and they can effectively decrease the Ni availability and especially Ni uptake by spinach (Younis et al. 2015;Khan et al. 2019), significantly reduced root and shoot accumulation. Similarly, maize (Shahbaz et al. 2018b), wheat and Chinese cabbage (Rehman et al. 2017;Salam et al. 2019) significantly decreased the toxic metals uptake, enhanced growth, and physiological parameters. More recently, Shaheen et al. (2019) reported that incorporation of BI in contaminated soils with PTEs: firstly, prominently enhances plants' tolerance of PTEs including Ni stress; and secondly, improves the soil's physicochemical properties.
As well as this, incorporation of inorganic amendments, i.e., calcite (CC) into contaminated soils can transform its mobile form to the most stable (residual) portion through a variety of processes such as adsorption, complexation, and precipitation of PTEs in soil (Li et al. 2014;Mahar et al. 2017). According to Hamid et al. (2019), the application of CC as an inorganic amendment in polluted soil may have proved to be effective for PTEs mobility and bioavailability. According to above discussions, it is evident that most studies have focused on cereals crops; there have been limited information is available regarding the application and effects of RS, BI, and CC on leafy vegetable growth especially in acidic soil. Therefore, the aim of the current pot experiment was to assess the effectiveness of amendments on (1) mitigation of Ni availability in Ultisol soil; (2) reducing Ni accumulation by Chinese spinach; and (3) enhancing the growth and nutritional quality of spinach.

Soil and amendments collection, preparation and characteristics
The soil used in this experiment is a brown-red type (Ultisol). The soil sampling site is located in Jiangxia district, Wuhan City, Hubei Province, China (30°17.804′ N, 114°19.246). The region's climate is humid subtropical monsoon with an average temperature of 15.8-17.5°C and 1269 mm annual rainfall, of which > 60% occurs from June to August ). The soil sample was transferred to the laboratory, dried at room temperature and ground to pass through a 2 mm mesh sieve. Its physicochemical properties were analyzed ( Table 1). The soil's pH and electrical conductivity (EC) were determined in 1:2.5 and 1:5 soil: water (m/V) ratio, respectively , while the wet oxidation method served for analyzing the soil organic matter (Lu 1999). Particle size distribution was determined by the pipette method (Gee et al. 1986), whereas soil cation exchange capacity (CEC) was determined by the NH 4 OAC (pH 7.0) method.
Three amendments-RS, BI, and CC-were employed in the present study's experiment. Rice straw was collected from an agriculture field at Huazhong Agriculture University, Wuhan, China. All the samples were air-dried, and then the air-dried RS was chopped into pieces smaller than 1.5 mm. Pyrolysis of collected RS was done in a high temperature furnace with a temperature control program. To ensure the pyrolysis worked successfully, the crucible filled with a precursor was sealed with tin foil beforehand. Following this, the pyrolysis was done by raising the temperature to 500°C at a rate of 20°C min −1 and maintained for 2 h (Gao et al. 2020). Meanwhile, CC was purchased from Sinopharm Chemical Reagent Co. Ltd, Shanghai, China. Total concentration of Ni in soil and amendments were determined after digesting the samples with aqua regia (HCl-HNO 3 -HClO 4 ). This was done by adopting the open Pyrex flask digestion procedure as recommended by Lu (1999) and further measured on flame atomic absorption spectrophotometry (FAAS) (Varian AA240FS). Table 2 summarizes the physicochemical characteristics of the amendments. The amendments pH and EC were determined in 1:10 and 1:5 solid:water (m/V) ratio using a Mettler-Toledo FE20 pH meter and EC meter, respectively (Gao et al. 2020).

Pot experiment
A pot experiment was carried out in the warehouse of Huazhong Agricultural University, employing a completely randomized design (CRD) with replications done in triplicate and seven treatments as follows: (1) untreated soil (CK), (2) RS 1%, (3) RS 2%, (4) BI 1%, (5) BI 2%, (6) CC 1%, and (7) CC 2%. In total, 2 kg air-dried soil sieved (2 mm) was put into plastic pots (14 cm height × 18 cm diameter). Soil was spiked with a nickel sulfate (NiSO 4 .6H 2 O) salt to obtain 100 mg Ni kg −1 concentration in soil. Soil samples were incubated at 25 ± 1°C and constant humidity (70% of water holding capacity) for 60 days. Each pot was lined with a plastic bag in order to prevent metals and nutrients leaching. The Ni-contaminated soil was thoroughly mixed with RS, BI, and CC at the application rate of 1% and 2% (w/w) based on previous studies (Houben et al. 2013;Ali et al. 2019) and no amendment in the controlled soil and again incubated in a dark room at 25°C for more than 60 days. Spinach seeds were disinfected by immersing them in H 2 O 2 (2%, V/V) for a period of time, and then rinsed with distilled water (DI) 3-4 times. After seed drying, eight seeds were cultivated in 21 pots. All pots were kept in a warehouse with a transparent roof and nylon net walls. The warehouse's average temperature was retained at a maximum 15°C and minimum 2°C, while humidity ranged between 76 and 78% throughout the experiment. After 14 days of seed germination, only four similar plants were maintained in each pot. During the experiment, pots were regularly irrigated with deionized water three times a week to avoid drought stress to plants and regularly changing the pots' position until harvesting. Moreover, N:P:K fertilizer at the rate of 0.19:0.11:0.13 g kg −1 was applied in urea, superphosphate, and potassium sulfate form, respectively.

Physiological and growth parameters of spinach
Prior to harvesting (60 days), the physiological parameters such as photosynthetic rate (A) and transpiration rate (E) were observed in the youngest, fully grown two healthy leaves using a Portable Infra-Red Gas Analyzer (IRGA) (Li-6400 Li-COR, Lincoln, NE, USA). These parameters were measured according to Hafeez et al. (2019). Briefly, gas exchange measurements were taken on a clear sunny day between 10:00 and 11:00 h a.m. with the following conditions: photosynthetic photon flux density of 1500 μmol m −2 s -1 , 380 μmol mol −1 CO 2 concentration; 1.2-1.5 kPa vapor pressure gradient Silt (g kg −1 ) 5 0 7 . 2 Clay (g kg −1 ) 3 6 8 . 2 Organic matter (g kg −1 ) 1 4 . 7 9 CEC (cmolc kg −1 ) 1 2 . 7 5 Total Ni (mg kg -1 ) 3 5 Available phosphorus (mg kg −1 ) 8 . 7 4 Available potassium (mg kg −1 ) 1 5 0 (VPD); relative humidity 50-65%; and leaf temperature 26-32°C. Fully matured spinach was harvested and divided into roots and shoots. The fresh biomass of each treatment was recorded with a digital weighing balance. Roots and shoots were washed carefully with tap and distilled water, after which they were oven-dried to at 70°C to achieve a consistent weight. Next, the oven-dried root and shoot samples were ground separately using a stainless-steel grinder for further analysis.

Assessment of Ni and nutrients concentration in plant tissues and post-harvest soil analysis
The ground plant samples (root and shoot) were digested with a mixture of HNO 3 and HClO 4 at a ratio of (3:1V/V) (Houben et al. 2013). Briefly, shoot and root samples were separately taken in a 100 mL conical flask and 10 mL of di-acid mixture was added and kept overnight. Next day, the digestion was done using a hot plate at 150-180°C until 2-3 mL suspension remained, then suspension was diluted to 25 mL with DI. Then, diluted supernatants were filtrated and stored at 4°C and their concentrations of Ni were determined using FAAS. The Ni concentration of the spinach roots and shoots were used to calculate several factors as described by Bashir et al. (2018b) The total concentration of phosphorus (P) and potassium (K) in plant and soil digest samples were measured on inductively coupled plasma mass spectrometer (ICP-MS-7890A), while the total nitrogen (N) concentrations were determined using a flow injection system (Auto-analyzer, Seal, Germany) (Lu 1999). After plants were harvested, the leachable Ni was assessed in the rhizosphere soil that existed in each treatment through the toxicity characteristic leaching procedure (TCLP). This has been recently described in the paper by Bashir et al. (2020). Briefly, 1 g of soil was weighed into 50 mL centrifuges tube, and 20 mL of glacial acetic acid solution (pH 2.88) was added. The mixtures were put into a shaker for 18 h. Similarly, bioavailable Ni was examined by extracting the mixture with 0.01M CaCl 2 (2:20 m/V) suspensions as recommended by Houben et al. (2013). The mixtures were then shaken in a thermostatic reciprocating shaker for 2 h. Once the mixtures were equilibrated, liquids were separated by centrifuge at 3900 rpm for 20 min and then each solution was filtered and determined for Ni concentration (mg kg −1 ) using FAAS.

Statistical analysis
The one-way analysis of variance (ANOVA) of three replicates was employed on data obtained from the experiment employing Statistic 8.1 (Analytical Software, USA). Tukey's honestly significant difference (HSD) test was used to highlight significant differences between the mean of treatment at P < 0.05.

Physicochemical properties of soil and amendments
The basic physicochemical properties of soil, organic and inorganic amendments are summarized in Tables 1 and 2. Soil presented a silty clay loam texture, acidic nature (pH 5.3), and a small amount of organic matter (14.79 g kg −1 ). Moreover, all the amendments were alkaline in nature. Biochar had the highest C content 470.7 (g kg −1 ) followed by RS 364.0 (g kg −1 ) and CC 121.2 (g kg −1 ) (Tables 1 and 2).

Effect of amendments on soil's post-harvest chemical properties
The current pot study confirmed that the incorporation of RS, BI, and CC significantly (P < 0.05) improved the chemical properties of post-harvest contaminated soil. Changes in postharvest, i.e., soil pH, total N, P, and K contents significantly (P < 0.05) differed among used amendment types and their application rates ( Fig. 1 and Table 3). The highest soil pH value Fig. 1 Effect of amendments applied to the Ni contaminated soil on soil pH after spinach was grown. Treatments: untreated soil (CK), rice straw (RS), biochar (BI), and calcite (CC). Error bars depict standard deviations (SDs) of the means (n = 3). Different letters indicate significant differences among treatments at P < 0.05 was observed in CC at 2%. The pH of the original soil was 5.8 and it rose to 6.04, 6.49, and 6.94 with 1% RS, BI, and CC addition, respectively. However, the soil pH increased to 6.47, 6.52, and 7.1 with 2% RS, BI and CC addition, respectively.
Similarly, the concentrations of total N, P, and K were in the following respective ranges: 0.22 to 0.44, 0.36 to 0.46, and 8.016 to 9.02 g kg −1 , respectively (Table 3). Interestingly, the concentrations of total N, P, and K in post-harvest were significantly (P < 0.05) increased by adding RS and BIc. Soil N rose by 68% and 100% when RS and BI were applied at the 2% application rate, respectively. Similarly, P rose by 5.55%, 27.77%, and 16.66%, and K by 7.36%, 12.60%, and 9.23% with the application of RS, BI, and CC at 2%, respectively, relative to CK.

Effect of amendments on Ni bioavailability
After harvesting spinach plants, the bioavailable (CaCl 2 extractable) Ni concentration significantly (P < 0.05) decreased in the presence of amendments (Fig. 2a). The lowest soil Ni concentration was noted with BI 2% (1.05 mg kg −1 ) relative to control (18 mg kg −1 ). In the presence of RS, BI, and CC at an application rate of 2%, the reductions reached 62.5%, 94.1%, and 87.2%, respectively, compared to CK. Overall, the maximum reduction in CaCl 2 extractable Ni concentration was noted in 2% BI application.
Likewise, TCLP extractable Ni concentration trend was similar to CaCl 2 extractable Ni. The TCLP Ni concentration in spinach in post-harvested soil varied from 28 to 14.6 mg kg −1 soil, respectively (Fig. 2b). The TCLP extractable Ni also significantly decreased after the incorporation of RS, BI, and CC. In the presence of RS, BI, and CC 2%, the reductions reached 26.7%, 47.8%, and 41.7%, respectively, compared to the CK. Overall, the maximum reduction of TCLP extractable Ni concentration was observed in 2% BI application.

Effect of amendments on spinach leaves' physiological and growth parameters
Effects of soil amendments on the physiological and growth parameters of spinach are illustrated in Figs. 3 and 4. All the amendments significantly (P < 0.05) enhanced physiological parameters, i.e., photosynthetic rate and transpiration rate in spinach leaves when compared with (CK) (Fig. 3A and B). The recorded ranges of photosynthetic rate and transpiration rate were 4-18.6 μmol m 2 s −1 and 1.7-8.9 mmol m 2 s −1 , respectively. The BI 2% application to soil caused the highest increase (4.6-fold) in photosynthetic rate of spinach followed by CC 2% (3.24-fold) and RS 2% (2.21-fold), respectively (Fig. 3A).
Similarly, the maximum transpiration rate was recorded with BI application, while the minimum was documented for CK. The BI 2% followed by CC 2% and RS 2% enhanced transpiration rate of spinach relative to CK, i.e., 2-, 5-, and 3.2fold, respectively (Fig. 3B). Generally, in all amendments, the values of photosynthetic and transpiration rate improved in the following order: BI>CC>RS>CK.
Plant growth parameters, i.e., biomass (shoot and root fresh and dry weight) and total yield of spinach were increased significantly (P < 0.05) through the application of organic and inorganic amendments relative to the control plants ( Fig.  4 and Table 4). The fresh biomass of shoots and roots ranged from 9.3-31.19, and 1.87-3.05 g pot −1 , while the dry biomass of shoot and roots ranged from 2.4-4.2 and 0.32-1.90 g pot −1 , respectively, in the current experiment (Fig. 4). These parameters were observed to reach their maximum in biochar-treated soil. Relative to CK, fresh shoot biomass was enhanced by 2.4-fold, 3.4-fold, and 2.5-fold, and fresh root biomass by 1.4fold, 1.6-fold, and 1.4-fold with RS, BI, and CC at 2% application rate over the CK, respectively. Conversely, dry shoot biomass of spinach increased by 1.4-fold, 1.8-fold, 1.5-fold, and dry root increased by 2.2-fold, 5.9-fold, and 2.8-fold with RS, BI, and CC at 2% application rate over the CK, Table 3 Effect of amendments applied to the Ni contaminated soil on nutrients in the soil and plant after spinach was grown. Treatments: untreated soil (CK), rice straw (RS), biochar (BI), and calcite (CC). Error bars depict standard deviations (SDs) of the means (n = 3)

Treatment
Soil Plant N g kg −1 P g kg −1 K g kg −1 N g kg −1 P g kg −1 K g kg −1 Different letters indicate significant differences among treatments at P < 0.05 respectively (Fig. 4). As well, total dry biomass (TDM) of spinach enhanced by 43.9%, 136.3%, and 66.05% in the presence of RS 2%, BI 2%, and CC 2% application rate, respectively, when compared to CK (Table 4). In all the amendments, the values of growth parameters were enhanced in the following order: BI>CC>RS>CK.

Effect of amendments on essential nutrient accumulation by plants
Incorporation of amendments in Ni acidic contaminated soil significantly (P < 0.05) increased N, P, and K concentrations in spinach in comparison to CK (Table 3). The highest N, P, and K concentrations were observed in BI 2% application rate. Spinach N content was increased from 4.1 to 11.9, 10.1, and 11.3 g kg −1 DW, respectively, when BI, RS, and CC were applied at the 2% application rate. Contrasted to this, spinach P concentrations were enhanced from 1.38 to 3.42, 1.94, and 2.86 g kg −1 DW, and K were enhanced by 25.4 to 56.05, Fig. 4 Effect of amendments applied to the Ni contaminated soil on shoot fresh weight (A), root fresh weight (B), shoot dry weight (C), and root dry weight (D) after spinach was grown. Treatments: untreated soil (CK), rice straw (RS), biochar (BI), and calcite (CC). Error bars depict standard deviations (SDs) of the means (n = 3). Different letters indicate significant differences among treatments at P < 0.05

Effects of amendments on Ni concentrations in roots and shoots, bioconcentration factor, and translocation factor
Nickel concentrations in the roots and shoots of spinach had the lowest values in amended soil when compared to CK ( Fig.  5A and B). The maximum Ni concentration in spinach was found in plants growing in untreated soil. However, the highest Ni reduction in roots (73.3%) and shoots (76.7%) were noted in BI 2% application rate compared to other treatments and CK. Incorporation of the amendments in the contaminated soil decreased the Ni concentration in roots by 51.6%, 73.3%, and 68.9% with RS, BI, and CC at 2%, respectively, over the untreated soil. Overall, spinach roots' Ni content decreased in the following order: BI 2% > CC 2% > BI 1% > RS 2% > CC 1% > RS 1% > CK. Similarly, the maximum decreases in shoot Ni concentration of spinach were recorded with BI 2% application rate, and the minimum spinach Ni content was measured in the untreated soil (Fig. 5B). Application of RS, BI, and CC at 2% decreased spinach shoots' Ni content by 54.1%, 76.7%, and 70.8%, respectively, over the untreated soil. The potential efficiency of incorporated amendments to reduce spinach shoots' Ni concentration was recorded in the following order: BI 2% > CC 2%, BI 1%, RS 2%, CC 1%, RS 1% > CK.
The bioconcentration factor (BCF) and translocation factor (TF) represents the contents of Ni in spinach root and shoot to that applied in the contaminated soil. The BI 2% application to soil caused the highest reduction (72.7%) in BCF-shoot of spinach followed by CC 2% (63.6%) and RS 2% (45.4%), respectively, relative to CK (Table 4). Compared to CK, BCF-root was reduced by 44.8%, 65.5%, and 62% in RS 2%, BI 2%, and CC 2% treated spinach, respectively, whereas the maximum TF value fell (20%) in BI 2% application rate when compared to CK (Table 4).

Discussion
The incorporation of organic and inorganic as soil amendments are promising solution for PTEs including Ni immobilization/stabilization within soils (Shahbaz et al. 2019;Shaheen et al. 2019). The main purpose of the current study was to assess the efficiency of RS, its derived BI and CC, in order to quantify their effectiveness in stabilizing Ni in soil, reducing Ni concentration by spinach, and improving its nutritional quality and yields. Adjusting the pH to counteract soil acidity is essential for sustainable agriculture (Shaaban et al. 2018). Soil pH management can markedly alter soil properties, such as availability of nutrients and decreased bioavailability of toxic metals in acidic soil (Bashir et al. 2018a). In the present study, RS, BI, and CC application significantly Fig. 5 Effect of amendments applied to the Ni contaminated soil on Ni concentration in roots and shoots after spinach was grown. Treatments: untreated soil (CK), rice straw (RS), biochar (BI), and calcite (CC). Error bars depict standard deviations (SDs) of the means (n = 3). Different letters indicate significant differences among treatments at P < 0.05 (P < 0.05) increased soil pH and reduced Ni bioavailability from the acidic soil due to their alkaline nature ( Table 2). The dissolution of these alkaline amendments into oxides, hydroxides, and excessive calcium ions may increase soil pH. The present study's findings are consistent with what other analyses documented, for example Shahbaz et al. (2018a) and Salam et al. (2019). They revealed that high mineral ash content, and alkaline substances, like hydroxides and carbonates of amendments especially biochar, might increase soil pH. Similarly, CC has a low surface area but high pH and sufficient amounts of calcium which could increase soil pH and promote metal carbonate through precipitation . Ali et al. (2019) and Shaheen et al. (2019) concluded that adding different amendments in Ni-contaminated soil could enhance soil pH compared with untreated soil. Several studies have discovered that BI is a competent amendment for increasing soil pH and inducing a liming effect on contaminated acidic soil Bashir et al. 2020). Similarly, Houben et al. (2013) tested CC 5% addition in contaminated soil by growing rapeseed in post-harvested soil and the pH levels increased remarkably compared to biochar (10% application rate) and untreated soil.
According to the current study findings, amendments specially the addition of biochar to polluted soil could enhance soil and plant nutrients, which might be due to the stability of BI in soil and the resistant to further degradation ). Meanwhile, RS may have a relatively high decomposition rate due to its lower lignin, cellulose, and hemi-cellulose contents as compared to biochar (Naeem et al. 2017). Khan et al. (2019) used wheat straw and its derived biochar and they revealed that as compared to conversion of straw into biochar seems to be an efficient solution that could justify both the solutions of agriculture residual disposal and valuable nutrients enhancement at the same time. Ding et al. (2016) revealed that BI has potential nutrients source and can release considerable amounts of N, P, and K. Therefore, our results concluded that BI can potentially increase total N, P, and K content in soil and plant when compared to other amendments (Table 3). Similar results were observed in the work by Bashir et al. (2018a), who indicated that incorporation of rice straw biochar in contaminated soil significantly increased the total amounts of N, P, and K by 75.7%, 79.5%, and 40.4%, respectively, relative to the CK. Among all amendments, BI application increased total essential nutrients stability due to COOH groups under short-term experiments and organic carbon fixed on BI surface (Feng et al. 2014) Our results concur with the findings reported by Khan et al. (2017) and Khan et al. (2019), where spinach and tomato growth and nutrient contents rose in the presence of straw and biochar application. Overall, the application of BI to Ni acidic contaminated soil has great potential for improving soil fertility and promoting spinach growth in soils with high concentrations of Ni. The present study used biochar properties that are almost similar to those employed by Kamran et al. (2020) and Gao et al. (2020).
The remarkable changes in post-harvest soil Ni in CaCl 2 and TCLP extract following the amendments' incorporation may also influence Ni immobilization/stabilization. We detected a significant decrease in CaCl 2 and TCLP-extractable Ni concentration when RS, BI, and CC were applied at the 1% and 2% application rates ( Fig. 2A and B). The significant increase in Ni stabilization with the significant increase in soil pH. Salam et al. (2019) examined that high pH and mineral ash contents of applied amendments could increase immobilization through adsorption and precipitation. Specifically, BI at 2% significantly curtailed the extractability of Ni by discouraging Ni metal toxicity via immobilization in the soil. Similar studies have documented that PTEs stabilization increased with different rates of BI and CC through different mechanism such as adsorption and precipitation due to its porous structure, high surface area (Table 2), and plenty of functional groups on BI surface play a vital role in decreasing the available PTEs by adsorbing the metals on its surface Hamid et al. 2019;Ali et al. 2019). Bashir et al. (2018b) noted a 73% and 38% reduction in CaCl 2 and TCLP extractable soil concentration, respectively, when adding rice straw biochar in contaminated soil and comparing this to the untreated soil. The amount of Ni adsorption on BI, CC, and RS was increased with increasing the solution pH, because at low pH, PTEs including Ni can compete with hydrogen ions for sorption sites (Ramzani et al. 2016;Mahar et al. 2017;Rehman et al. 2017). Conversely, our findings concur with those of Houben et al. (2013) who in their rapeseed study suggested that incorporating BI and CC at various rates in contaminated soil: firstly, significantly decreased Cd, Pb, and Zn availability; and secondly, improved plant growth and biomass. Recently, Salam et al. (2019) found that the incorporation of organic amendments, such as rice straw biochar in naturally acidic contaminated soil improved leafy vegetable growth as well as significantly decreased TCLP and CaCl 2 extractable Pb and Cu.
Physiological and growth parameters are considered to be key indicators that can help evaluate the damage caused by Ni toxicity to plants (Xu et al. 2015;Shahzad et al. 2018;Azhar et al. 2019). We discovered a remarkable increment physiological and growth parameter in amended pot. Likewise, the most significant results were noted in BI 2% treated plants (Figs. 4 and 5 and Table 4). This increase in above parameters can be attributed to Ni stabilization and enhanced nutritional quality after added RS, BI, and CC. This finding agrees with previous analyses where Ni stress caused a decline in physiological parameters in red clover plant (Shahbaz et al. 2018a). Similar results regarding Ni-induced reductions in physiological and growth parameters in different plants have been reported by Rehman et al. (2016) and Sehrish et al. (2019). The decline in biomass and physiological parameters might be due to Ni-induced reduction in nutrient uptake and ultra-structural changes in plants species (Rizwan et al. 2018;Shahzad et al. 2018). These results agreed with Boostani et al. (2019), who reported the adding BI at 2.5% increased the shoots' dry weight of spinach by about 46%, compared to the respective Ni-contaminated soil. Increase in spinach biomass was a key factor when judging the effects of Ni on plant growth. In our findings, we observed that all factors play vital role in enhancing plant dry biomass, which was consistent with Younis et al. (2015), who investigated that incorporation of cotton sticks biochar could increase biomass of Spinacia oleracea fresh and dry biomass shoot and root by 78% and 58%, respectively, compared to the control. The incorporation of RS and CC elevated soil pH, and reduction in Ni solubility in soil solution which could increase plant growth and soil health and our finding was in accordance with that documented by Houben et al. (2013), who suggested that CC amendments have sufficient amounts of calcium ions, which offered more benefits by, firstly, increasing soil pH and better rapeseed growth and, secondly, decreasing toxic metal mobility. The possible mechanism for the improvement in above parameters of spinach as a leafy vegetable, through the application of organic and inorganic amendments, could be due to physiological parameters being enhanced via chlorophyll content, facilitation of electron transport and better water mobility in spinach leaves via the xylem process (Xu et al. 2015;Rizwan et al. 2018).
The content of Ni in spinach shoots and roots was significantly (P < 0.05) different among all amended pots. Applying amendments reduced Ni content by spinach, which must indicate the poorer bioavailability of Ni in contaminated soil with increasing rates of amendments and especially BI rates (Shen et al. 2016;Shaheen et al. 2019). Rice straw biochar may have the highest absorption capacity because it is alkaline in nature and has a larger surface area as compared to CC and RS in Table 2. These characteristics suggest that application of BI could enhance Ni absorption on BI-amended soil, which might be one of the mechanism/reasons for minimizing Ni availability to spinach relative to CK. The minimum absorption of Ni by spinach tissues is due to the lowest concentration values of Ni mobility and leachability in soil, which was directly related to the increment of soil pH in biochar amended soil. Our findings agree with the work conducted by Rehman et al. (2016), who stated that Ni decreased significantly when the rate of adding biochar increased from 1 to 2%. Therefore, the incorporation of BI significantly reduced the Ni absorbed by plant tissues, and this proved to be consistent with Khan et al. (2017), who observed that application of miscanthusderived biochar significantly reduced Ni content in spinach by up to 58%, relative to CK. Similar findings were observed by Salam et al. (2019), who concluded that the application of biochar remarkably reduced toxic metal content by up to as much as 37.81% when employing Brassica chinensis.
Similarly, the addition of CC decreased Ni content in spinach tissue. Inorganic amendment especially CC is reported to raise soil pH in acidic soil (Hamid et al. 2019). This elevated soil pH with CC liming amendment may be attributed to reduced bioavailability and increased metal immobilization in postharvest contaminated soil (Houben et al. 2013). The possible mechanisms for this resultant concentration of Ni in spinach root and shoot are the formation of stable BI complexes with Ni either by surface sorption onto its different functional groups. This could be due to the exchange of Ni with various cations associated with BI and CC (Shen et al. 2016;Lahori et al. 2017).
The BCF refers to the ratio of metal in a plant compared to its content in the soil, whereas TF is another important factor that can assess the ability of spinach plants to transfer Ni from roots to shoots (Mosa et al. 2016;Boostani et al. 2019). In the current experiment, the BCF and TF values were noted to be highest in roots compared to shoots (Table 4). Meanwhile, the extent of BCF in shoots and roots were recorded in CK. The uptake of Ni in shoots is possible due to there being more Ni in the roots. Nawab et al. (2019) and Salam et al. (2019) investigated the distribution ratio of metals' concentrations in the total amount of metals in leafy vegetable (spinach) and Chinese cabbage in root. They noted that this may play an important role in the Ni concentration in plant shoots. The BCF and TF values calculated in the present study were lower than the unity in all amended soil samples. The reduced BCF and TF values which were less than unity in spinach under amended soil conditions, confirming that BI and CC could reduce Ni uptake by spinach. Our findings are virtually the same as those of other studies by Herath et al. (2015) and Bashir et al. (2018a), who noted the BCF and TF values after CC and BI application may have resulted from their great ability to minimize plants' uptake of metal substances. In addition, the BCF and TF factors were the chief components that made it possible to assess the ability of plants to survive in contaminated soils.

Conclusion
Our findings demonstrated that incorporation of rice straw, biochar, and calcite significantly affected the spinach physiological and growth parameters, Ni contents, and soil postharvest properties. Soil and plant total essential nutrients were highest in BI followed by RS. The Ni concentration in shoot and root were minimum in BI 2% compared to all other amendment and unamended soil. Meanwhile, the lowest BCF and TF values for Ni in spinach were observed in BI 2%, compared to untreated soil, respectively. The amendments increased the spinach yields and reduced the CaCl 2 and TCLP extractable Ni. These findings suggest that the application of biochar at 2% would be a potential for reducing Ni uptake, and can reduce toxicity to plants, and reduce bioavailability and leachability in the acidic soil. It is further suggested that combined effect of BI, RS, and CC is needed to elucidate the Ni immobilization under field conditions.