Interactive Suitability of Rice Stubble Biochar and Arbuscular Mycorrhizal Fungi for Improving Wastewater-Polluted Soil Health and Reducing Heavy Metals in Peas

Abstract

Arable soils irrigated with wastewater (SIWs) cause ecological and human health issues due to the presence of heavy metals (HMs). Burning rice stubble (RS) poses severe environmental and human health hazards. Converting RS into rice stubble compost (RSC) and rice stubble biochar (RSB) can overcome these issues. Here, we considered the role of RS, RSC, and RSB as individual soil amendments and combined each of them with arbuscular mycorrhiza fungi (AMF) to observe their effectiveness for HM immobilization in SIW, their uptake in pea plants, and improvements in the physicochemical properties of soil. The results revealed that adding RSB and AMF reduced the bioavailable concentrations of Pb, Cd, Ni, Cu, Co, and Zn in SIW by 35%, 50%, 43%, 43%, 52%, and 22%, respectively. Moreover, RSB+AMF treatment also reduced Pb, Cd, Ni, Cu, Co, and Zn concentrations in grain by 93%, 76%, 83%, 72%, 71%, and 57%, respectively, compared to the control. Improvements in shoot dry weight (DW) (66%), root DW (48%), and grain yield (56%) per pot were also the highest with RSB+AMF. RSB+AMF treatment enhanced soil health and other soil attributes by improving the activity of urease, catalase, peroxidase, phosphatase, β-glucosidase, and fluorescein diacetate by 78%, 156%, 62%, 123%, 235%, and 96%, respectively. Interestingly, RSB+AMF also led to the strongest AMF–plant symbiosis, as assessed by improved AMF root colonization (162%), mycorrhizal intensity (100%), mycorrhizal frequency (104%), and arbuscular abundance (143%). To conclude, converting RS into RSB can control air pollution caused by RS burning. Moreover, adding RSB with AMF to SIW can reduce HM uptake in plants, improve soil health, and thus minimize ecological and human health issues.

1. Introduction

Freshwater accounts for 3% of the total available water on Earth, but less than 1% of this fraction is available for utilization [1]. However, climate change, intense population growth, several anthropogenic activities, and socio-economic problems have resulted in a scarcity of freshwater [2]. In developing countries, farmers have no choice except to irrigate their fields with wastewater (WW) [3]. In Pakistan, about 30% of the total WW generated irrigates approximately 32,500 h of agricultural land [4,5]. Unfortunately, WW contains toxic heavy metals (HMs), such as Pb, Cd, Zn, Ni, Hg, and As. Soil irrigated with WW (SIW) results in HM accumulation in the food chain, causing several human health issues [6,7]. Irrigating soil with WW negatively influences its structure, water-holding capacity (WHC), infiltration rate, and hydraulic conductivity [8]. Moreover, alterations in pH values, microbial activities, cation exchange capacity (CEC), and OM concentrations of soil have also been reported, all of which negatively influence soil productivity [1].

After the harvesting of rice crops, a massive quantity of rice straw and stubble is produced annually in the world (up to 800−1000 million tons) and in Asia (600−800 million tons) [9]. Unfortunately, rice stubble (RS) remains intact in the fields [10,11] and is incinerated in situ, releasing PM_2.5, PM₁₀, volatile organic compounds (VOCs), CO₂, CH_4, and NO_x, thereby causing air pollution. As a result, several health issues, such as asthma, lung capacity loss, chronic obstructive pulmonary disease (COPD), bronchitis, emphysema, and even cancer, have been reported in humans [12].

Due to several limitations, it is practically impossible to remediate SIW and restore its properties using harsh remediation strategies [13]. Contrarily, an in situ immobilization approach involving the use of various cheap soil amendments can remediate SIWs and improve their properties [13,14]. For instance, the use of compost (COM) obtained from composting residues of different plants and horse manure has proven successful in remediating HM-polluted soils [7,15]. Moreover, improvements in the physicochemical properties of soil and plant growth in SIW with COM application have also been reported [7]. Interestingly, biochar (BR), a carbon-based material, can have agronomic benefits (soil fertility and plant growth) and remediate HM-polluted soil [16,17]. The production method and mineral composition of feedstock define the features of BR. The slow pyrolysis of feedstock produces a BR with an acidic pH (due to various acidic functional groups, phenolic compounds, and organic acid formation), high CEC value, and lower surface area [18,19]. Hence, using such BR could enhance the bioavailability of HMs in soils and their accumulation in plants [20]. In contrast, the BR produced at high temperatures has a greater specific surface area due to the cleavage of cellulose and lignin substances in feedstock. Moreover, it has high pH values because of the detachment/disappearance of alkali salts (Ca, Na, K, and Mg) from cellulose and lignin structures. Such a type of BR also has a high ash content because it has higher inorganic minerals. Enhanced bioavailability of HMs in soil and their uptake in plants have been reported after incorporating such a type of BR in soil [19,20]. Moreover, supplementing soil with nutrients such as boron (B) and rhizospheric microorganisms that it lacks is a promising method that helps crops resist HM stress and improves plant growth [21,22,23]. Arbuscular mycorrhizal fungi (AMF) exist in symbiotic relationships with several plant species and have the remarkable ability to decontaminate HM-polluted soils and improve several soil functions [22,24]. Moreover, enhancements in plant growth and plant tolerance against HM stress are also linked with AMF, highlighting their significance for ecological restoration and food security [25,26].

Unfortunately, the area of SIW and the practice of RS burning are increasing in underdeveloped countries. We hypothesize that RS can be converted into rice stubble compost (RSC) and rice stubble biochar (RSB). Furthermore, these RS products (RSC and RSB), in combination with AMF, can remediate SIW, improve its properties, and enhance plant growth. Previous research exhibits a gap in reporting the significance of RS, its products (RSC and RSB), and AMF for the remediation of HM-polluted SIW and food safety. Thus, we performed a pot experiment to observe the effects of RS, RSC, RSB, and AMF as individual soil additives and combinations of RS, RSC, and RSB with AMF on (1) HM immobilization in SIW and HM uptake in pea plants, (2) various physicochemical properties of SIW, (3) growth and yield of pea plants, and (4) activities of soil enzymes.

2. Materials and Methods

2.1. Materials for Soil Incorporation

The RS was brought from an agriculture field in Faisalabad (31°23′45.0528″ N, 72°55′42.6504″ E) after the rice crop harvest and carefully flushed with sterile water. Following open-air drying, these RS samples were initially chopped into small pieces (~3 cm) and later crushed to 2 mm size. Rice stubble compost was prepared by adopting the protocol of Kausar et al. [27]. Briefly, the RS was sprinkled with water to obtain 60% moisture content. Later, the RS was placed in a plastic container (length = 44 cm, width = 32 cm, and height = 21 cm). The container was covered with a black polyethylene sheet and placed in an aerated place. At the start, the content of the container was mixed every alternate day (for three weeks) for its proper homogenization and aeration, and later, the content of the container was mixed weekly. Warm air convection was maintained through perforated PVC pipes. The moisture content of the composting material was maintained by sprinkling water when needed. The temperature of the content was regularly monitored with a thermometer. The process of preparing RSC was terminated when the temperature of the content was cooled to ambient temperature. For the preparation of RSB, the RS was pyrolyzed in the tube furnace (Kejia S50IC, Zhengzhou, China) at 500 °C with a heating rate of 10 °C min^–1, under a nitrogen gas (N₂) flow of 10 mL min^–1 for 2 h, and pressure = 0.1 MPa. The tube furnace was cooled to room temperature under N₂-flow conditions to prevent oxidation before RSB samples were collected. Later, RSB was cooled at room temperature. The ash content of RSB was 37.6%, as determined by weight loss of 1 g of RSB after it was heated at 750 °C for 6 h [28]. RS, RSB, and RSC were ground to 2 mm mesh size before their utilization in the soil experiment. The physicochemical properties of RS, RSB, and RSC are given in

Table 1. Physicochemical characteristics of rice stubble, rice stubble compost, and rice stubble biochar.

Property	Unit	Rice Stubble	Rice Stubble Compost	Rice Stubble Biochar
pH	−	7.60	7.91	9.11
EC 1	dS m−1	0.31	1.15	2.67
CEC 2	cmolc kg−1	11.3	38.4	68.4
Surface area	m2 g−1	1.34	17.4	234.1
C	%	37.5	34.5	45.1
H	%	5.38	4.10	4.43
O	%	35.6	19.2	22.4
N	g kg−1	25.5	32.4	18.7
P	g kg−1	2.27	2.91	2.52
K	g kg−1	23.0	31.0	29.2
Ca	g kg−1	12.6	14.8	13.4
Mg	g kg−1	5.90	7.32	6.90
S	g kg−1	1.33	1.62	1.01
Fe	mg kg−1	187.1	263.8	241.2
Zn	mg kg−1	67.7	80.6	72.8
Mn	mg kg−1	113.4	139.5	125.2
Cu	mg kg−1	49.5	63.4	54.9
B	mg kg−1	23.9	27.1	30.6

¹ EC = electrical conductivity, ² CEC = cation exchange capacity.

.

An AMF inoculum, having nine AMF species, named “BioOrganics™ Micronized Endomycorrhizal Inoculant” was procured from BioOrganics, 2799 Creamery Road, New Hope, PN, USA (www.bio-organics.com, accessed on 11 September 2022). This consortium contains Glomus aggregatum, Glomus etunicatum, Glomus clarum, Glomus deserticola, Glomus intraradices, Glomus monosporus, Glomus mosseae, Gigaspora margarita, and Paraglomus brasilianum.

2.2. Gathering of SIW and Analysis

The SIW was obtained from an agricultural farm irrigated with untreated WW for several years in Faisalabad, Pakistan. The soil samples from the top layer (0−20 cm) were randomly gathered from different locations within the same site. Then, the composite sample was prepared, shifted to the laboratory, air-dried, and screened (2 mm). A sub-sample of soil was analyzed to determine its various physicochemical characteristics with methodologies elaborated in our publication [29]. These physicochemical characteristics of SIW are given in

Table 2. Physicochemical characteristics of SIW.

Characteristic	Unit	Value
Texture	−	Clay loam
Sand	%	39.5
Silt	%	31.0
Clay	%	29.5
pH	−	6.71
EC	dS m−1	1.46
CEC	cmolc kg−1	5.86
OM 1	%	0.61
Total C 2	%	0.637
CaCO3	%	2.67
Total N	mg kg−1	0.71
NH4−N 3	mg kg−1	8.38
NO3−N 4	mg kg−1	51.5
Bioavailable Ni	mg kg−1	2.57
Bioavailable Cd	mg kg−1	1.64
Bioavailable Pb	mg kg−1	3.31
Bioavailable Cu	mg kg−1	1.92
Bioavailable Co	mg kg−1	1.15
Bioavailable Zn	mg kg−1	1.24
Total Ni	mg kg−1	81.7
Total Cd	mg kg−1	13.1
Total Pb	mg kg−1	518.7
Total Cu	mg kg−1	209.4
Total Co	mg kg−1	20.1
Total Zn	mg kg−1	130.2

¹ OM = organic matter; ² Total C = total carbon; ³ NH₄−N = ammonium nitrogen; ⁴ NO₃−N = nitrate nitrogen.

.

2.3. Plant Experiment

The SIW was mixed with the AMF, RS, RSC, and RSB to formulate eight soil treatments. For this aim, the calculated amounts of AMF, RS, RSC, and RSB were blended into a small quantity of soil in a plastic container using a plastic scooper. Further, these blends were mixed into the soil left behind to make the following experimental treatments: control, RS, RSC, RSB, AMF, RS+AMF, RSC+AMF, and RSB+AMF. The treatment where no additive was mixed in the soil was referred to as control (

Table 3. Treatments for the pot study.

Soil Treatment	Rice Stubble and Its Products	Inoculum of AMF
	(% w/w of Soil)	(g pot−1)
No soil additive	−	−
Rice stubble	5	−
Rice stubble compost	5	−
Rice stubble biochar	5	−
Arbuscular mycorrhizal fungi	−	1.70
Rice stubble+arbuscular mycorrhizal fungi	5	1.70
Rice stubble compost+arbuscular mycorrhizal fungi	5	1.70
Rice stubble biochar+arbuscular mycorrhizal fungi	5	1.70

No soil additive = control; rice stubble = RS; rice stubble compost = RSC; rice stubble biochar = RSB; arbuscular mycorrhizal fungi = AMF; rice stubble+arbuscular mycorrhizal fungi = RS + AMF; rice stubble compost+arbuscular mycorrhizal fungi = RSC + AMF; rice stubble biochar + arbuscular mycorrhizal fungi = RSB + AMF.

). The formulated treatments were moistened (WHC = 65%) and secured in a dark area (25 °C, 45 days). Succeeding, plastic nursery pots (24 cm in height and 18 cm in diameter) containing drainage holes at the bottom were filled with 3 kg of soil. There were three repeated pots kept for every treatment. These pots were conveyed to a botanical site in Government College University Faisalabad, Pakistan, and randomly settled. Seeds of the pea variety “Hilltop” were bought from an online store named Plants Universe, Pakistan (www.plantsuniverse.com, accessed on 8 September, 2022), and germinated in perlite. Three healthy seedlings (seven days old) were planted in each pot. The soil was irrigated using sterile water during the experiment. A recommended dose of a balanced fertilizer, “MILAGRO”, having NPK at a 20-20-20 ratio and micro-nutrients (B = 0.001%, Mn = 0.002%, Cu = 0.001%, Fe = 0.003%, and Zn = 0.001%), bought from Starlet International, Lahore, Pakistan (www.starlet-international.com, accessed on 26 September, 2022), was applied to the plants. On the 70th day, the pods from every pot were collected, and the number of pods plant⁻¹ was counted. These pods were de-hulled, and the grain yield and number of seeds plant⁻¹ were calculated. After the segregation of plant biomass into shoots and roots, it was washed with distilled water, and the lengths of shoots and roots were scaled using a folding meter stick. Then, this fresh biomass was oven-dried (70 °C, Memmert laboratory oven, Schwabach, Germany) to determine the plant dry weight (DW). Meanwhile, substantial quantities of the harvested soil from every pot were packed in zipper polythene bags and kept for analysis.

2.4. Soil Analysis

2.4.1. Properties of SIW and Bioavailable HMs in It

To estimate plant-available metals, a 10 g mass of the soil was extracted with 5 mM diethylenetriaminepentaacetic acid (DTPA) extracting solution (1:2 w/v ratio) [30]. After the extract was filtered with Whatman No. 42 filter paper, the HMs in the extract were measured using ICP−MS. Various soil physicochemical attributes were measured by using standard techniques. Soil pH, CEC, soil aggregate stability, particle density, bulk density, available water content, pore size distribution (wide, medium, and fine pores), and the contents of soil OM and organic C were measured according to the methods explained in detail in our publication [29].

2.4.2. Arbuscular Mycorrhizal Fungi and Soil Enzyme Responses

An extraction method presented by Voroney et al. [31] was adopted to quantify microbial biomass carbon (MBC) in SIW. To assess total glomalin-related soil protein (T−GRSP), 1 g of the soil was extracted using 8 mL of citrate buffer (50 mM) and autoclaved (60 min), and pH of the sample was maintained to 8 [32]. Following centrifugation, the supernatant was removed, and a second extraction was performed. A Bradford protein assay was used to estimate the concentration of T−GRSP within the extracts. Similarly, mycorrhizal intensity (MI) and mycorrhizal frequency (MF) were calculated using the following Formulas (1) and (2) proposed by Derkowska et al. [33] and Giovannetti and Mosse [34], respectively:

MI (%) = (95n5 + 70n4 + 30n3 + 5n2 + n1)/N

(1)

where n = the number of root fragments allocated with the number 0 (no), 1 (trace), 2 (less than 10%), 3 (11 to 50%), 4 (51 to 90%), or 5 (more than 91%) of colonization.

MF (%) = 100 × (N − NO)/N

(2)

where N = the number of observed root fragments and NO = the number of non-mycorrhizal root fragments.

For the assessment of arbuscules, vesicles, and AMF root colonization, 1 g of fine roots was collected from each plant. Later, these roots were washed (with water), immersed (at 80 °C, 60 min) in 10% potassium hydroxide (KOH), acidified (5%, HCl), and stained with 0.05% trypan blue [35]. The slide intersection method (at 200× magnification) was used for assessing the vesicles, arbuscules, and AMF colonization while examining 100 intersections per sample [36].

For estimating viable spores and the number of spores in soil, a wet sieving and sucrose density gradient centrifugation procedure was used to extract spores from the pot culture [37]. Counting of spores was carried out at 40 × magnification with stereo microscope. Spore viability was assessed after staining them with MTT stain (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) [38]. The spores with red or purple color were viable, while those with a blue, black, or colorless appearance were nonviable. The determination of color and counting of spores were carried out using a microscope (40× magnification). The results were recorded as the percentage of viable spores after all spores were checked. In the post-harvest soil, urease, catalase, peroxidase, phosphatase, and β-glucosidase activities were measured using different standard protocols precisely explained in our previous publication [39]. Fluorescein diacetate was measured using the method of Adam and Duncan [40].

2.5. Plant Analysis for HM Estimation in Shoots, Roots, and Grain

Ground biomasses of roots, shoots, and grain (each weighing 0.5 g) were digested in 5 mL of a nitric–perchloric acid blend (HNO₃ + HClO₄), v/v = 2/1 [41]. Then, the total concentrations of Ni, Cd, Cr, Pb, Cu, and Co in the digested solutions were estimated using ICP−MS. The bio-concentration factor (BCF) and translocation factor (TF) for HMs in grain were calculated according to Wang et al. [42].

2.6. Statistical Analysis

This experiment was performed in a completely randomized design (CRD) with three replicates for each treatment. Computations of means and standard errors (SEs) values were done in Microsoft Office Excel 2019^®. The responses of plant and soil parameters were analyzed using one-way analysis of variance (ANOVA) at p < 0.05) in Statistix version 8.1.1 (Analytical Software, Tallahassee, FL, USA) [43]. Error bars indicate the SEs computed from the means (n = 3), while different alphabetic letters written with the values (in the case of tables) or on the top of bars (in the case of figures) represent significant (p < 0.05) differences.

3. Results

3.1. Arbuscular Mycorrhizal Fungi Responses

Data related to AMF root colonization, MBC, T−GRSP content, mycorrhizal intensity, mycorrhizal frequency, arbuscules, vesicles, spore number, and viable spores were in the ranges of 21.5 to 56.5% of root colonized, 115.6−230.1 mg kg⁻¹ soil, 1.17−2.26 µg g⁻¹ soil, 12.3−24.6% of the root system, 27.7−56.6% of the root system, 5.91−14.3% of the root system, 2.10−4.62% of the root system, 99.8−336.2 spores 100 g⁻¹ soil, and 10.6−40.6% of total spores, respectively, in all treatments (Figure 1). RS did not significantly improve AMF parameters, except MBC content and arbuscules in the root system, compared to the control. Other treatments had significant impacts on improving all other AMF parameters compared to the unamended control. Interestingly, RSB+AMF treatment improved AMF root colonization, MBC, T−GRSP content, mycorrhizal intensity, mycorrhizal frequency, arbuscules, vesicles, spore number, and viable spores, which were promoted by 162%, 99%, 93%, 100%, 104%, 143%, 120%, 237%, and 282%, respectively, compared to unamended pots.

3.2. Soil Physicochemical Attributes

Data of CEC, pH, bulk density, particle density, available water content, organic C, soil OM, soil aggregate stability, wide pores, medium pores, and fine pores in the soil were within the ranges of 5.88−8.51 cmol_c kg⁻¹, 6.73−7.74 units, 1.28−1.69 g cm⁻³, 1.38−1.80 g cm⁻³, 16.0−21.1%, 0.35−0.49%, 0.61−0.85%, 7.12−12.8%, 16.9−28.0%, 9.94−16.2%, and 8.25−15.1%, respectively (Figure 2). In contrast to the control, significant influences on bulk density, particle density, pH, CEC, soil aggregate stability, organic C, wide pores, medium pores, and fine pores were observed upon the application of soil treatments, excluding AMF. Likewise, except for RS and AMF treatments, the remaining treatments noticeably promoted the soil OM and the available water content in the soil, compared to unamended pots. Specifically, the RSB+AMF treatment led to the highest increments for CEC, soil aggregate stability, soil OM, organic C, medium pores, and fine pores, by 45%, 80%, 40%, 41%, 63%, and 83%, over the control treatment. Interestingly, compared to the control, the available water content in RSC+AMF and RSB+AMF treatments showed the biggest improvements, by 25% and 32%, respectively. Moreover, the lowest bulk density, particle density, and wide pores were found in the soil of RSC+AMF and RSB+AMF treatments and were reduced by up to 19% and 24%, 19% and 23%, and 35% and 40%, correspondingly over control. In addition, RSB and RSB+AMF resulted in the highest increase in pH of 0.58 and 0.99 units, respectively, relative to the control.

3.3. HMs in SIW and Their Plant Uptake

Pb, Cd, Ni, Cu, Co, and Zn availability in post-harvest soil were in the ranges from 2.13 to 3.30, 0.82−1.64, 1.46−2.55, 1.09−1.91, 0.55−1.15, and 0.97−1.24 mg kg⁻¹ soil, respectively (Appendix A,

Table A1. Effects of arbuscular mycorrhizal fungi (AMF) inoculum, rice stubble (RS), rice stubble compost (RSC), and rice stubble biochar (RSB) on the concentrations of Pb, Cd, Ni, Cu, Co, and Zn in soil−DTPA extracts. Values of each column are averages computed from three replicates of each treatment (mean ± standard error (SE)). In each column, different lowercase letters indicate significant differences among treatments analyzed by LSD test (at p < 0.05) based on one-way ANOVA.

Treatment	HMs in Soil–DTPA Extract
	Pb	Cd	Ni	Cu	Co	Zn
	Concentrations (mg kg−1 Soil)
Control	3.30 ± 0.08 a	1.64 ± 0.04 a	2.55 ± 0.07 a	1.91 ± 0.05 a	1.15 ± 0.03 a	1.24 ± 0.03 a
RS	3.07 ± 0.08 bc	1.50 ± 0.04 b	2.35 ± 0.06 bc	1.78 ± 0.05 b	1.05 ± 0.03 b	1.22 ± 0.03 a
RSC	2.85 ± 0.07 d	1.30 ± 0.03 c	2.16 ± 0.06 d	1.57 ± 0.04 c	0.84 ± 0.02 d	1.17 ± 0.03 ab
RSB	2.30 ± 0.06 e	1.04 ± 0.03 d	1.77 ± 0.05 e	1.28 ± 0.03 d	0.71 ± 0.02 e	1.08 ± 0.03 bc
AMF	3.21 ± 0.08 ab	1.59 ± 0.04 ab	2.43 ± 0.06 ab	1.86 ± 0.05 ab	1.10 ± 0.03 ab	1.23 ± 0.03 a
RS+AMF	2.95 ± 0.08 cd	1.38 ± 0.04 c	2.24 ± 0.06 cd	1.64 ± 0.04 c	0.96 ± 0.02 c	1.18 ± 0.03 a
RSC+AMF	2.76 ± 0.07 d	0.92 ± 0.02 e	1.62 ± 0.04 ef	1.19 ± 0.03 de	0.64 ± 0.02 f	1.03 ± 0.03 cd
RSB+AMF	2.13 ± 0.05 e	0.82 ± 0.02 f	1.46 ± 0.04 f	1.09 ± 0.03 e	0.55 ± 0.01 g	0.97 ± 0.02 d
LSD0.05	0.22	0.10	0.16	0.12	0.07	0.09

). Except for AMF, all treatments significantly lowered Pb, Cd, Ni, Cu, and Co in DTPA solution relative to the control. Moreover, significant reductions in extractable Zn were only noted in the RSB, RSC+AMF, and RSB+AMF treatments. Compared to the control, the RSC+AMF and RSB+AMF treatments resulted in the highest decrease in concentrations of Ni, by 37% and 43%; Zn, by 17% and 22%; and Cu, by 38% and 43%, respectively. The topmost reductions in Pb concentration, by 30% and 35%, were noted in RSB and RSB+AMF treatments compared to the control. In contrast, the lowest Cd and Co concentrations of 50% and 52% were noted with the RSB+AMF treatment compared to the control.

The Pb, Cd, Ni, Cu, Co, and Zn varied in the ranges of 59.9 to 190.2, 1.68−4.73, 8.61−30.6, 32.2−86.1, 3.39−8.30, and 57.7−97.5 mg kg⁻¹, respectively, in shoots. Furthermore, in roots, Pb ranged from 169.1 to 403.4, Cd from 4.62 to 11.9, Ni from 26.4 to 72.7, Cu from 96.7 to 190.2, Co from 10.6 to 18.8, and Zn from 78.8 to 128.2 mg kg⁻¹. The Pb, Cd, Ni, Cu, Co, and Zn concentrations varied in the ranges of 4.31 to 64.1, 0.47−2.02, 1.68−9.98, 13.1−46.9, 0.84−2.94, and 21.5−50.1 mg kg⁻¹, respectively, in grain (Figure 3). Each metal concentration in roots and the Cd, Ni, and Zn concentrations in shoots and grain were not significantly reduced with AMF, than control. Moreover, compared to the control, RS treatments did not reduce Pb concentration in shoots. In the remaining cases, the concentration of each metal in every plant part was remarkably decreased when compared with unamended pots. The greatest decreases in the concentrations of Pb, Cd, Ni, and Co in roots (58%, 61%, 63%, and 43%), shoots (68%, 64%, 71%, and 59%), and grain (93%, 76%, 83%, and 71%) were recorded with RSB+AMF treatment, compared to the control. Zn concentrations were maximally reduced with RSB+AMF and RSC+AMF treatments in shoots (41% and 35%) and grain (50% and 58%), respectively. Cu concentration, compared to unamended pots, was maximally reduced by 49% and 62% in roots and shoots, respectively, with BSB+AMF treatment. However, the greatest decreases in grain Cu concentration, by 64% and 72%, were recorded with RSC+AMF and RSB+AMF treatments, compared to the control.

The values of TF for Pb, Cd, Ni, Cu, Co, and Zn in the grain were from 0.07 to 0.34, 0.28−0.43, 0.20−0.33, 0.41−0.54, 0.25−0.35, and 0.37−0.51, respectively. Grain BCF values for Pb, Cd, Ni, Cu, Co, and Zn were in the ranges of 0.008 to 0.12, 0.04−0.16, 0.02−0.12, 0.06−0.22, 0.04−0.15, and 0.17−0.39, respectively (Appendix A,

Table A2. Effects of arbuscular mycorrhizal fungi (AMF) inoculum, rice stubble (RS), rice stubble compost (RSC), and rice stubble biochar (RSB) on the BCF values for Pb, Cd, Ni, Cu, Co, and Zn in grain and their TF values. Values of each column are averages computed from three replicates of each treatment (mean ± standard error (SE)). In each column, different lowercase letters indicate significant differences among treatments analyzed by LSD test (at p < 0.05) based on one-way ANOVA.

Treatment	BCF						TF
	Pb	Cd	Ni	Cu	Co	Zn	Pb	Cd	Ni	Cu	Co	Zn
Control	0.12 ± 0.002 a	0.16 ± 0.008 a	0.12 ± 0.006 a	0.22 ± 0.01 a	0.15 ± 0.004 a	0.39 ± 0.01 a	0.34 ± 0.01 a	0.43 ± 0.005 a	0.33 ± 0.004 a	0.54 ± 0.01 a	0.35 ± 0.004 a	0.51 ± 0.01 a
RS	0.10 ± 0.002 c	0.13 ± 0.004 b	0.10 ± 0.003 b	0.17 ± 0.005 c	0.12 ± 0.003 c	0.34 ± 0.01 b	0.27 ± 0.004 c	0.40 ± 0.005 b	0.32 ± 0.004 a	0.51 ± 0.01 cd	0.33 ± 0.004 b	0.48 ± 0.01 b
RSC	0.05 ± 0.001 e	0.10 ± 0.003 c	0.08 ± 0.002 d	0.12 ± 0.003 e	0.08 ± 0.002 d	0.27 ± 0.01 d	0.19 ± 0.002 e	0.39 ± 0.004 c	0.30 ± 0.003 b	0.47 ± 0.01 e	0.28 ± 0.003 d	0.44 ± 0.005 c
RSB	0.03 ± 0.001 f	0.07 ± 0.002 d	0.05 ± 0.001 e	0.10 ± 0.003 f	0.07 ± 0.002 e	0.22 ± 0.01 e	0.16 ± 0.002 f	0.36 ± 0.004 d	0.28 ± 0.003 c	0.52 ± 0.01 bc	0.31 ± 0.004 c	0.41 ± 0.005 d
AMF	0.11 ± 0.002 b	0.14 ± 0.007 a	0.11 ± 0.007 a	0.20 ± 0.01 b	0.13 ± 0.004 b	0.36 ± 0.01 a	0.32 ± 0.01 b	0.42 ± 0.005 a	0.32 ± 0.004 a	0.53 ± 0.01 ab	0.34 ± 0.004 b	0.50 ± 0.006 a
RS+AMF	0.06 ± 0.001 d	0.11 ± 0.003 b	0.09 ± 0.002 c	0.15 ± 0.004 d	0.11 ± 0.003 c	0.29 ± 0.01 c	0.23 ± 0.003 d	0.39 ± 0.004 bc	0.31 ± 0.003 b	0.49 ± 0.006 d	0.33 ± 0.004 b	0.45 ± 0.005 c
RSC+AMF	0.02 ± 0.0003 g	0.05 ± 0.002 e	0.04 ± 0.001 f	0.08 ± 0.002 g	0.06 ± 0.002 f	0.19 ± 0.01 f	0.10 ± 0.001 g	0.32 ± 0.004 e	024 ± 0.003 d	0.44 ± 0.005 f	0.26 ± 0.003 e	0.40 ± 0.005 d
RSB+AMF	0.008 ± 0.0001 g	0.04 ± 0.001 f	0.02 ± 0.001 g	0.06 ± 0.002 g	0.04 ± 0.001 g	0.17 ± 0.005 g	0.07 ± 0.001 h	0.28 ± 0.003 f	0.20 ± 0.002 e	0.41 ± 0.005 g	0.25 ± 0.003 f	0.37 ± 0.004 e

). With very few exceptions, the BCF and TF values of each HM were significantly reduced with applied treatments than the control. Notably, compared to control, the highest depreciations in TF values of Pb, Cd, Ni, Cu, Co, and Zn, by 79%, 35%, 41%, 24%, 29%, and 27%, were noted in the RSB+AMF treatment. Moreover, this treatment also resulted in the highest decline in the BCF values of Cd, Ni, Co, and Zn, by 77%, 83%, 72%, and 57%, compared to the control. The RSB+AMF and RSC+AMF treatments exhibited the highest reduction in the BCF values of Pb, by 93% and 86%, and Cu, by 71% and 64%, respectively, than control.

3.4. Plant Growth Performance

Measured data of shoot length, root length, shoot DW, root DW, No. of pods, grain yield, and No. of seeds varied in the ranges of 53.8 to 74.7 cm, 17.0−24.8 cm, 3.98−6.62 g plant⁻¹, 1.15−1.70 g plant⁻¹, 6.00−15.3 plant⁻¹, 2.75−4.30 g plant⁻¹, and 13.7−26.7 plant⁻¹, respectively (

Table 4. Responses of arbuscular mycorrhizal fungi (AMF) inoculum, rice stubble (RS), rice stubble compost (RSC), and rice stubble biochar (RSB) on growth performance of peas. Values of each column are averages computed from three replicates of each treatment (mean ± standard error (SE)). In each column, different lowercase letters indicate significant differences among treatments analyzed by LSD test (at p < 0.05) according to one-way ANOVA.

Treatment	Shoot Length	Root Length	Shoot DW	Root DW	No. of Pods	No. of Seeds	Grain Yield
	cm		g Plant−1		No. Plant−1		g Plant−1
Control	53.8 ± 1.38 f	17.0 ± 0.44 f	3.98 ± 0.10 f	1.15 ± 0.03 e	6.00 ± 0.58 f	13.7 ± 0.33 e	2.75 ± 0.07 f
RS	57.4 ± 1.47 ef	18.0 ± 0.46 ef	4.41 ± 0.11 e	1.25 ± 0.03 de	7.67 ± 0.33 de	17.3 ± 0.33 d	3.03 ± 0.08 e
RSC	59.4 ± 1.52 de	18.7 ± 0.48 de	5.04 ± 0.13 d	1.32 ± 0.03 d	9.00 ± 0.58 d	18.3 ± 0.33 d	3.35 ± 0.09 d
RSB	65.2 ± 1.67 bc	20.8 ± 0.53 bc	5.78 ± 0.15 bc	1.49 ± 0.04 bc	13.0 ± 0.58 bc	21.3 ± 0.67 c	3.76 ± 0.10 bc
AMF	56.3 ± 1.44 ef	17.8 ± 0.45 ef	4.31 ± 0.11 ef	1.22 ± 0.03 de	7.33 ± 0.33 ef	17.0 ± 0.58 d	2.92 ± 0.07 ef
RS+AMF	62.7 ± 1.61 cd	20.2 ± 0.52 cd	5.46 ± 0.14 c	1.44 ± 0.04 c	12.0 ± 0.58 c	21.0 ± 0.58 c	3.53 ± 0.09 cd
RSC+AMF	69.1 ± 1.77 b	22.0 ± 0.56 b	6.09 ± 0.16 b	1.57 ± 0.04 b	14.0 ± 0.58 ab	24.0 ± 0.58 b	3.93 ± 0.10 b
RSB+AMF	74.7 ± 1.91 a	24.8 ± 0.64 a	6.62 ± 0.17 a	1.70 ± 0.04 a	15.3 ± 0.67 a	26.7 ± 0.67 a	4.30 ± 0.11 a
LSD0.05	4.81	1.54	0.41	0.11	1.62	1.58	0.27

). Solo RS and AMF treatments had no significant effects on shoot length, root length, and root DW, while shoot DW, No. of pods, and grain yield were not affected by AMF, compared to the control. The highest responses of shoot length, root length, shoot DW, root DW, grain yield, and No. of seeds plant⁻¹ were achieved in the RSB+AMF treatment, in which they were augmented by up to 39%, 46%, 66%, 48%, 56% and 95%, respectively, compared to control pots. Likewise, relative to unamended pots, the RSC+AMF and RSB+AMF treatments resulted in the highest increments, by 133% and 156%, in the No. of pods plant⁻¹, respectively.

3.5. Soil Enzyme Activities

Urease, catalase, peroxidase, phosphatase, β-glucosidase, and fluorescein diacetate activities in the soil were in the ranges of 1.59 to 2.83 µg N−N (H₄⁺ kg⁻¹ h⁻¹), 0.23−0.59 Vol of 0.1 M KMnO₄ g⁻¹ of soil, 2.85−4.61 mol g⁻¹ h⁻¹, 29.1−65.0 μg PNP g⁻¹ soil h⁻¹, 25.5−85.5 μg PNP g⁻¹ soil h⁻¹, and 32.0−62.9 μg fluorescein g⁻¹ dry soil h⁻¹, respectively (

Table 5. Effects of arbuscular mycorrhizal fungi (AMF) inoculum, rice stubble (RS), rice stubble compost (RSC), and rice stubble biochar (RSB) on the enzymatic activities in the soil. Values of each column are averages computed from three replicates of each treatment (mean ± standard error (SE)). Lowercase letters in every column show significant differences among treatments analyzed by LSD test (at p < 0.05) according to one-way ANOVA.

Treatment	Urease	Catalase	Peroxidase	Phosphatase	β-Glucosidase	Fluorescein Diacetate
	µg N−N(H4+ kg−1 h−1)	Vol. of 0.1 M KMnO4 g−1 of Soil	mol g−1 h−1	µg PNP g−1 Soil h−1	µg PNP g−1 Soil h−1	µg Fluorescein g−1 Dry Soil h−1
Control	1.59 ± 0.04 f	0.23 ± 0.01 g	2.85 ± 0.07 f	29.1 ± 0.75 f	25.5 ± 0.65 g	32.0 ± 0.82 f
RS	1.82 ± 0.05 e	0.30 ± 0.01 f	3.13 ± 0.08 e	34.8 ± 0.89 e	39.0 ± 1.00 f	36.5 ± 0.93 e
RSC	2.01 ± 0.05 d	0.36 ± 0.01 e	3.48 ± 0.09 d	41.8 ± 1.07 d	49.0 ± 1.25 e	41.6 ± 1.07 d
RSB	2.41 ± 0.06 b	0.44 ± 0.01 c	3.95 ± 0.10 bc	52.7 ± 1.35 c	66.3 ± 1.70 c	48.7 ± 1.25 c
AMF	1.78 ± 0.05 e	0.28 ± 0.01 f	3.04 ± 0.08 ef	32.5 ± 0.83 ef	35.4 ± 0.91 f	35.6 ± 0.91 e
RS+AMF	2.22 ± 0.06 c	0.40 ± 0.01 d	3.77 ± 0.10 c	45.3 ± 1.16 d	54.1 ± 1.39 d	43.8 ± 1.12 d
RSC+AMF	2.56 ± 0.07 b	0.50 ± 0.01 b	4.21 ± 0.11 b	58.5 ± 1.50 b	76.5 ± 1.96 b	56.9 ± 1.46 b
RSB+AMF	2.83 ± 0.07 a	0.59 ± 0.02 a	4.61 ± 0.12 a	65.0 ± 1.67 a	85.5 ± 2.19 a	62.9 ± 1.61 a
LSD0.05	0.17	0.03	0.28	3.58	4.40	3.53

). Except for AMF treatment in the case of peroxidase and phosphatase, significant enhancements in the activities of all enzymes were noted with each treatment, than control. The most remarkable enhancements in urease, catalase, peroxidase, phosphatase, β-glucosidase, and fluorescein diacetate activities, by 78%, 156%, 62%, 123%, 235%, and 96%, respectively, were found in RSB+AMF treatment, compared with control pots.

4. Discussion

4.1. Arbuscular Mycorrhizal Fungi Responses

In soil, the presence of HMs reduced spore density, root colonization, and proliferation of AMF [22,44]. The most prominent responses of AMF-related parameters were found in the RSB+AMF treatment (Figure 1). Recently, BR has been reported to enhance AMF root colonization, MBC, and secretion of glomalin in HM-polluted soil [25]. Improved mycorrhizal frequency and mycorrhizal intensity in Zea mays were also seen with BR addition in soil [45]. Moreover, BR application in soil enhanced the number of AMF spores in soil (5.34%), mycelium (11.79%), vesicles (41.80%), and arbuscules in the root system (29.72%) [46]. AMF inoculation in the soil increased glomalin secretion, mycorrhizal colonization, arbuscules, intra- and extra-radical hyphae, spores, and vesicles [47]. The improvements in AMF biomass and its symbiosis with pea roots are because of RSB addition, as BR (1) improves soil health via enhancing soil moisture retention, nutrition, and C content and (2) provides a favorable niche for AMF in its porous structure [26,48]. Moreover, BR improves AMF activities by (1) altering AMF and bacterial interactions, (2) improving plant–AMF signals, (3) providing shelter to AMF against AMF grazers [26], and (4) reducing the HM toxicity to the AMF via immobilization [25]. Interestingly, AMF upregulate the diversity of soil microbes (bacteria and fungi) by providing them C and nutrients in the form of glomalin [44]. The hyphae of AMF drive P from beyond the reach of roots to the host plant, thus, in return, receiving C from the host plant for efficient hyphal growth [49]. Moreover, root exudates, the initial plant signal sensed by AMF, positively strengthen the symbiotic relationship of AMF through enhanced spore germination [50].

4.2. Soil Physicochemical Attributes

Irrigation of soil with WW negatively influences soil physicochemical properties [1,2]. Contrarily, BR addition increased micropores and reduced macropores in soil [51]. Adding BR in HM-polluted soil increased soil pH, organic C, OM, soil aggregate stability, available water content, CEC, and medium and fine pores while reducing the wide pores, bulk density, and particle density [29]. Interestingly, AMF improved OM content and increased soil pH while reducing the bulk density of soil [24]. The improvements in the soil properties could be related to the various features of BR as (1) the porous structure of BR enhances the porosity, WHC, and aggregation while reducing the bulk density of soil, and (2) the high surface area of BR also tends to improve the WHC and aggregation of soil [52]. The presence of minerals and functional groups in BR is reported to (1) increase the soil pH and CEC, and (2) the high C content of BR enhances the soil organic C content [52,53]. Additionally, a positive shift in soil pore size distribution with BR addition could be due to (1) the attraction of labial OM onto BR surfaces, providing a substrate for microbiota that support the formation of soil aggregates via mucilage [54] and (2) the re-arrangement of fine sand particles because of surface oxidation at the interface of BH–soil particles [55]. Moreover, AMF improve soil physicochemical properties through various mechanisms such as (1) high AMF root colonization with a vast hyphal network grips soil particles via physical entanglement [22] and (2) releasing glomalin, which holds soil particles firmly; improves soil structure; decreases bulk density; and increases organic C, available water content, and porosity [44,56]. Moreover, AMF promote the abundance and activities of soil microbes in the mycorrhizosphere. These microbes produce OM, which behaves like a cementing agent, improving soil aggregate stability and organic C [44,56,57]. In soil, AMF proliferation occurs in the pores and on particle surfaces, affecting pore size and altering the physicochemical characteristics by releasing biochemical compounds promoting particle re-distribution [44,58].

4.3. Bioavailability of HMs in Soil and Their Plant Uptake

Irrigation of soil with WW significantly increased HM concentrations in Raphanus sativus L. [6] and Lycopersicon esculentum [59]. Furthermore, Mawof et al. [7] observed that adding BR in SIW reduced the uptake of various HMs in potatoes. Inoculation with AMF significantly reduced the concentrations of Cd in Trifolium repens and soil Cd bioavailability [48]. The lowest concentrations of HMs in roots, shoots, and grain (Figure 3); lowest concentrations of HMs in DTPA extract (Appendix A, Table A1); and lowest grain BCF and TF values (Appendix A, Table A2) were noted with the RSB+AMF treatment. RSB was alkaline (pH = 9.1) and increased the soil pH (Figure 2C), which reduced the bioavailability of HMs through (1) enhancing net negative charges in soil and (2) precipitating HMs as hydroxyl bond species (HMOH⁺) [26]. Moreover, enormous negative sites, a high surface area, and multiple functional groups also facilitated HM immobilization with BR through adsorption, surface interaction, precipitation, and complexation [25,53] and thus reduced their uptake by pea plants. AMF also contributed to reducing soil HMs’ bioavailability and their uptake in pea plants by (1) retaining HM ions in their spores, arbuscules, hyphae, vesicles, and mycelia; (2) releasing EPSs (extracellular polymeric substances) having carboxyl, amine, phosphoric, and hydroxyl functional groups, which effectively adsorb HMs; and (3) creating a protective barrier around the plant roots against HM uptake [22] The fungal cell wall contains enormous functional groups (amino, imidazole carboxyl, and free hydroxyl groups), which adsorb HMs [60]. AMF produce not only organic acids (i.e., malic, citric, and oxalic acids) but also other compounds like glomalin, metallothioneins, and phytochelatins, which efficiently bind HM ions, leading to their reduced soil bioavailability and plant uptake [61,62]. In the soil–plant system, the uptake of HMs and the accumulation of HMs in grain are assessed using the BCF and TF values of grain (Appendix A, Table A2). A value > 1 indicates the potential of a plant for HM phytoextraction, while a value < 1 refers to excluder plants for HMs [63]. Grain TF refers to the ratio of HM concentration in grain and HM concentration in shoots, while grain BCF is the ratio of HM concentration in grain and HM concentration in soil [42]. The lowest values of BCF and TF for each HM were found with the RSB+AMF treatment, representing the effectiveness of the combined application of RSB and AMF for reducing HMs’ bioavailability in soil and their accumulation in edible parts of peas.

4.4. Plant Growth Performance

Previously, higher concentrations of Ni, Cu, Cr, Co, Cd, and Zn in SIW adversely affected the biomass and yield of Pisum sativum L. [64]. Contrarily, BR improved the growth traits of Trifolium repens [48] and Zea mays [26] in HM-polluted soil. Moreover, AMF addition in HM-polluted soil also promoted the biomass of white clover [65]. The possible mechanisms of RSB for enhancing the growth and yield of peas are as follows: (1) BR improves porosity, bulk density, nutrient retention, and water status of soil [7,65]; (2) BR reduces HMs’ toxicity to the plant through immobilizing them; and (3) being rich in nutrients, BR acts like a slow-release plant fertilizer [39]. Reduced plant oxidative stress with BR also contributed to improving plant growth and yield [25]. Interestingly, AMF improved the growth and yield of peas by boosting plant mineral uptake through (1) their better acquisition via the vast AMF hyphal network [25], (2) enhancing nutrient bioavailability [49,66], and (3) improving soil aggregate stability, which minimizes nutrient leaching [21]. Moreover, glomalin released by AMF supports soil microbes in secreting growth hormones that improve plant growth [44,66]. Reducing HM toxicity and disease attack and improving plant water relations are other contributing factors that improve plant growth and yield with AMF [21,22].

4.5. Soil Enzyme Activities

Nutrient cycling in the soil is facilitated by soil enzymes. Therefore, they play important functions in soil fertility and availability of nutrients to plants. However, variations in soil quality, especially the presence of HMs, negatively affect microbial activities and enzymes secreted by microbes [16]. Formerly, the application of BR in HM-polluted soils was reported to increase the activities of soil enzymes [29]. Moreover, AMF enhance the dehydrogenase and alkaline phosphatase activities in soil [47]. Enhanced soil enzymatic activities in RSB+AMF-treated soil are due to several mechanisms associated with RSB and AMF. Biochar improves the secretion of soil enzymes by the microbes by (1) supporting the settlement, growth, and abundance of soil microbes (including AMF) by providing them with a niche in its porous structure and (2) acting as a source of essential nutrients for them [25,39]. Apart from other soil microbes, AMF also secrete urease, catalase, phosphatase, and β-glucosidase in soil [25]. Interestingly, glomalin released by AMF also accelerates the catabolic functions of soil microbes, improving their secretion of catalase, peroxidase, phosphatase, and fluorescence diacetate [57]. Furthermore, the alleviation of HM toxicity to soil microbes due to HM immobilization by BR and AMF also improved the microbes’ capacities to secrete more enzymes [26,67].

5. Conclusions

Soils irrigated with wastewater (SIWs) have poor health and high concentrations of heavy metals (HMs). Moreover, burning rice stubble (RS) causes air pollution, thus affecting human health in many countries. Converting RS into biochar (RSB) is a practical way to use this type of waste to remediate SIW. In this study, we tested the efficacy of RS, rice stubble compost (RSC), RSB, and arbuscular mycorrhiza fungi (AMF) as solo soil treatments and the combinations of RS, RSB, and RSC with AMF in reducing HMs’ bioavailability in SIW, their uptake in pea plants, and their effect on soil health. The RSB+AMF treatment was most effective in immobilizing Pb, Cd, Ni, Cu, Co, and Zn in SIW and resulted in reducing their concentrations by 93%, 76%, 83%, 72%, 71%, and 57%, respectively, in pea grain compared to the control. Interestingly, this treatment also improved soil health by enhancing the activities of soil enzymes, i.e., urease (78%), catalase (156%), peroxidase (62%), phosphatase (123%), β-glucosidase (235%), and fluorescein diacetate (96%) activities, and other physicochemical properties. The incorporation of RSB+AMF supported plant–AMF symbiosis and enhanced shoot dry weight (DW), root DW, and grain yield by 66%, 48%, and 56%, respectively, compared to the control. Due to the massive availability of RS after the rice harvest in many countries, instead of burning this valuable feedstock, it can be converted into RSB and coupled with AMF. Adding RSB+AMF to SIW can successfully (i) minimize HM uptake in food crops, (ii) improve plant growth and yield, and (iii) augment soil health.