Popular wood and sugarcane bagasse biochars reduced uptake of chromium and lead by lettuce from mine-contaminated soil

Abstract

As a result of metal mining activities in Pakistan, toxic heavy metals (HMs) such as chromium (Cr) and lead (Pb) often enter the soil ecosystem, accumulate in food crops and cause serious human health and environmental issues. Therefore, this study examined the efficacy of biochar for contaminated soil remediation. Poplar wood biochar (PWB) and sugarcane bagasse biochar (SCBB) were amended to mine-contaminated agricultural soil at 3% and 7% (wt/wt) application rates. Lactuca sativa (Lettuce) was cultivated in these soils in a greenhouse, and uptake of HMs (Cr and Pb) as well as biomass produced were measured. Subsequently, health risks were estimated from uptake data. When amended at 7%, both biochars significantly (P<0.01) reduced plant uptake of Cr and Pb in amended soil with significant (P<0.01) increase in biomass of lettuce as compared to the control. Risk assessment results showed that both biochars decreased the daily intake of metals (DIM) and associated health risk due to consumption of lettuce as compared to the control. The Pb human health risk index (HRI) for adults and children significantly (P<0.01) decreased with sugarcane bagasse biochar applied at 7% rate relative to other treatments (including the control). Relative to controls, the SCBB and PWB reduced Cr and Pb uptake in lettuce by 69%, 73.7%, respectively, and Pb by 57% and 47.4%, respectively. For both amendments, HRI values for Cr were within safe limits for adults and children. HRI values for Pb were not within safe limits except for the sugarcane bagasse biochar applied at 7%. Results of the study indicated that application of SCBB at 7% rate to mine impacted agricultural soil effectively increased plant biomass and reduced bioaccumulation, DIM and associated HRI of Cr and Pb as compared to other treatments and the control.

1. Introduction

Contamination of soil ecosystems with heavy metals (HMs) is one of the major global environmental issues that affect human health and agricultural productivity (Neilson and Rajakaruna, 2015; Mishra et al., 2019). HMs such as chromium (Cr) and lead (Pb) enter the environment through natural processes like volcanic eruption and weathering of rocks while some of the anthropogenic sources include mining, land application of biosolids, wastewater irrigation, fuel production, use of pesticides, fertilizers, smelters and power transmissions (Rehman et al., 2017; Nawab et al., 2018). HMs are persistent in nature, remain in soil ecosystems for a long time period and bioaccumulate in crops, affecting their growth, productivity and quality with implication for food security and human health (Chen et al., 2018; Afanasyeva et al., 2019). The health of children is particularly of concern due to the associated risks of Cr and Pb (Pan et al., 2016; Cao et al., 2016: Francová et al., 2020). Pb and Cr are ranked 2^nd and 17^th in the list of top 20 hazardous contaminants respectively (Wilbur et al., 2000; ATSDR, 2007).

Cr is easily detectable in the earth’s crust and is an important micronutrient for biological life. However, it is toxic, carcinogenic, mutagenic and teratogenic above critical levels (Kamran et al., 2017; Ali et al., 2019). Vegetation grown in Cr contaminated soils have the potential for uptake of Cr and accumulation in their tissues (Nagajyoti et al., 2010; Khan et al., 2020). Cr reduces plant’s production and causes toxicity in nutritional contents (Sharma et al., 2020). Similarly, physical impacts of Pb are severe such as gastro-intestinal distress, neurological, oncogenic effects, and act as a neurotoxin that affects every organ in human body, damages kidneys and reproductive systems, reduces cognitive development in children, causes headache and irritability, lung and stomach cancer and abdominal pain (ATSDR, 2007; Li et al., 2014; Fang et al., 2014). Increased Pb exposure causes severe impacts on children’s health (Cherfi et al., 2014). Low level, long term exposures to HMs through contaminated food consumption has detrimental impacts on human health that appears after several years of exposure (Bortey-Sam et al., 2015). The exposure can occur through multiple routes, such as inhalation of soil particles and ingestion of food contaminated with Pb. Food intake is the most critical pathway because crops and vegetables are important sources of human nutrition (Chen et al., 2016; Cao et al., 2016; Mehri, 2020). Therefore, crops and vegetables cultivated in agricultural field close to mining activities might be a matter of concern due to metals accumulation in their tissues (Neilson and Rajakaruna, 2015). Hence, immobilization of HMs in agricultural soil and restoration of such sites by using economically feasible technologies is necessary (Sarwar et al., 2017).

Several conventional methods are in use for HM-contaminated soil remediation such as solidification, soil replacement, washing, and electro kinetic extraction (Khalid et al., 2017). Immobilization of metals through the addition of highly sorptive materials is a recent economically viable remediation technique. Organic amendments such as biochar can be prepared from different feedstocks, like wood chips, manure, rice husks, and municipal solid waste which can be used for remediation of HM contaminated soil (Ahmad et al., 2017; Wei et al., 2019). Biochars are carbon rich materials, with some unique properties that help to reduce HM bioavailability, such as high pH, strong sorption capacity, large surface area and high porosity (Hunter et al., 2017; Yu et al., 2019). Biochar application can increase soil fertility, soil water retention capacity, plant growth and yield, retain essential nutrients, improve soil physical and biological characteristics, immobilize bioavailable HMs in soil and reduce plant HMs uptake (Qi et al., 2017; Khan et al., 2018; Yu et al., 2019).

Most mining sites in Pakistan are situated in hilly rural areas. People in these areas often live in extreme poverty with no awareness of the harmful effects of mining and disposal of mining waste, and do not have the option to grow food elsewhere. In these situations, remediating the soil is imperative to reducing food contamination and protecting human health. Arid environments present unique growing conditions and soil properties such as high temperatures, high irrigation rates, and elevated pH and/or electrical conductivity (EC). These properties likely influence the remediation efficacy of biochar, but research in this area is limited. In this study, biochars produced from two feedstocks were applied to mine-impacted agricultural soil and it is hypothesized that biochars may improve these soils, increase crop yield, and reduce crop uptake of HMs. Thus, biochar is predicted to contribute to agriculture sector sustainability. The present study investigates the influence of biochar on mine-contaminated agricultural soil, lettuce biomass, bioaccumulation of Cr and Pb in lettuce, daily intake of metal (DIM) and human health risk index (HRI) related to consumption of lettuce.

2. Materials and methods

2.1. Collection, preparation and extraction of soil

Surface soil samples (50 Kg in total) from 0–20 cm depth were collected using an auger from different agricultural fields adjacent to multiple mine-impacted sites (chromite and manganese) of a selected area and brought to the laboratory for further processing. Soil was homogenized by thoroughly mixing to prepare a composite sample of contaminated soil (CS). All pebbles, stones, organic debris and other undesired particles were removed by hand. Then, the soil was air dried at room temperature and passed through a 2 mm sieve.

A sub-sample was taken from the composite CS for the determination of physicochemical properties. Soil characteristics were determined sing standard procedures adopted by Waqas et al. (2014). Hydrogen ion concentration was measured using 105 Ion analyzer pH meter in a soil: water ratio of 1:2.5. EC was determined through Electrochemical Analyzer (EC meter 4510, Jenway, UK) in 1:5 (w/v) soil water suspension. Fritsch Analysette 3 RPO sieve machine was used to perform particle size distribution as procedure adopted by Khan et al. (2010). Textural class was identified through the USDA textural triangle after determining the percentage of clay, silt and sand using Fritsch Analysette 3 PRO sieve machine. Cation exchange capacity (CEC) was determined by the ammonium acetate (NH₄OAc) method (Chapman, 1965). Total concentration of Cr and Pb were analyzed using standard procedure adopted by Khan et al. (2008). In brief, 0.5 g soil samples were digested with 15 ml of aqua regia (HNO₃, H₂SO₄, and HClO_4; ratio of 5:1:1) at 160°C in a digestion chamber. Upon completion of the digestion process, samples were cooled down at room temperature, filtered and raised their volume up to 50 ml in corning tubes using double deionized water. HM concentrations in samples were quantified using Atomic Absorption Spectrophotometry (AAS) (Perkin Elmer Model 700, USA).

2.2. Extraction of soil samples for bioavailable Cr and Pb

Bioavailable Pb and Cr in soil were determined by the standard procedure developed by Soltanpour and Schwab (1977). Soil samples of 10 g were placed in 125 ml Erlenmeyer flasks with 20 ml extracting solution AB-DTPA. The flasks were then shaken for 15 min at 180 rpm on a reciprocal shaker. Concentrated HNO₃ (100 µl) was added to the extract and analyzed for bioavailable HMs using AAS.

2.3. Biochar preparation

Biochars were prepared from poplar wood (PWB) and sugar cane bagasse (SCBB) feedstocks, respectively at 550°C. These feedstocks were chosen based on their availability in Pakistan. Previously, Puga et al. (2015) and Melo et al. (2013) used sugar cane feedstock for the preparation of biochar to immobilize HMs in contaminated soil. Poplar wood was dried in the open sun and chopped into small pieces of 2.5 cm. Biochar was prepared from the poplar wood pieces in a stainless-steel tube furnace by a slow pyrolysis process at 550°C under very low O₂ conditions and residence times ~8 h as described by the Kloss et al. (2012). For SCBB production the feed stock was milled and placed in sealed reactor to inhibit O₂, then was heated to 550°C. This temperature was maintained for ~1 h, then the reactor was slowly cooled to room temperature. This temperature was chosen based on prior research showing that biochars at higher temperatures (>400°C) are more effective at removing HMs compared with those produced at lower temperatures, due to increased surface area and adsorption capacity (Ding et al., 2014; Lu et al., 2014; Xie et al., 2015; Inyang et al., 2016; Patra et al., 2017). Both biochars were ground and passed through a 2 mm sieve before applying to the soil.

Basic properties of the soil, PWB and SCBB are shown in Table 1.

Table 1.

Physicochemical properties of mine impacted soil and biochars (PWB and SCBB)

Parameters	CS (dry weight basis)	PWB 550°C	SCBB 550°C	SEPA limits (1995)
pH	7.2	7.33	7.76
EC (µS/cm)	328	340	395
SOM (%)	2.8
CEC (cmol/kg)	6.02
Texture	Sandy Loam
Clay (%)	22
Silt (%)	30.7
Sand (%)	47.3
N (%)	0.42±0.1	1.08 ± 2	2.2±0.4
P (mg/kg)	125±10	2450±30	130.6±12.4
Cr total (mg/kg)	545±2	0.77±0.01	0.72±0.01	250
Pb total (mg/kg)	44±0.8	0.14±0.01	0.18±0.1	350
Cr bioavailable (mg/kg)	25±05	0.03 ±0.01	0.02 ±0.01
Pb bioavailable (mg/kg)	7±1.5	0.02 ±0.01	0.03±0.02

CS: contaminated soil; PWB: poplar wood biochar; SCBB: Sugar cane bagasse biochar.

2.4. Experimental Design

A greenhouse experiment was conducted to study the effect of PWB and SCBB at 3% and 7% application rates on Cr and Pb and its uptake by lettuce grown in mine impacted soil. Soil for the greenhouse experiment was collected from Qala area of Mohmand District, Khyber Pakhtunkhwa, Pakistan. Agricultural soil of the selected area is highly contaminated due to mining practices and atmospheric deposition (Nawab et al., 2016).

For the greenhouse experiment, round pots of 20 cm height and 15 cm diameter were filled with 2.5 kg mine impacted soil. PWB and SCBB were ground into a fine powder and mixed thoroughly with contaminated soil (CS) with 3% and 7% (wt/wt) application rate. Pots having no biochar served as controls. Doses of NPK were applied to ensure maximum nutrient supply (NH₄NO₃ 120 mg N kg⁻¹ soil, K₂HPO₄ 30 mg P kg⁻¹ soil and 75.7 mg K kg⁻¹ soil) following the procedure adopted by Khan et al. (2013). Pots for all treatments were prepared in triplicates, irrigated with deionized water to keep the moisture content at 70% and then kept in complete randomized order for 15 days (d) at room temperature (25±3 °C) to reach equilibrium. Lettuce was cultivated in a full factorial design in triplicate (1 crop x 1 soil x 5 treatments) x 3 replicates = 15 pots in total, while control, PWB3, PWB7, SCBB3, and SCBB7 represent soil amended respectively with: no biochar, 3% PWB, 7% PWB, 3% SCBB and 7% SCBB. The high rate of 7% biochar was used to ensure detection of the biochars’ effects at the laboratory scale.

Lettuce seeds were brought from Tarnab Agriculture Farm, Peshawar, Pakistan, thoroughly washed with H₂O₂ and then with deionized water and grown in seedling trays with potting soil (no biochar present). Seedlings were kept in controlled environment i-e photoperiod (dark/light) of 12/12 h with 25 ± 3°C temperature and 70% relative humidity. After 20 d of germination, healthy seedlings were transplanted to each pot (potting soil removed from roots). Plants were grown for 60 d to ensure the maximum uptake of selected HMs.

2.5. Plant analysis

2.5.1. Plant biomass assessment and sample preparation

After 60 d of the experiment, lettuce plants were harvested, brought to the laboratory, rinsed with double deionized water and weighed for the determination of biomass (fresh weight without roots). The lettuce was then dried in an oven for ~72 h at 70°C and weighed for dry weight. Samples were ground into powder and stored in labelled polyethene bags for further chemical process.

2.5.2. Wet digestion

For wet digestion, the procedure adopted by Khan et al. (2008) was used. Briefly, 0.5 g of powdered sample was digested with 10 ml concentrated HNO₃ at 80°C. The temperature was then raised to 130°C for ~20 h. Then, 5 ml of HNO₃ was added and heated until the solution became transparent. The volume of the filtered solution was raised to 50 ml with deionized water for further analysis. Cr and Pb were quantified through AAS.

2.6. Analytical procedures

All chemicals used for sample preparation and analysis were of analytical grade having high purity of 99.9% Merck Darmstade, (Germany). Digested samples were analyzed in triplicates under standard condition, within confidence level of 95%, for data quality assurance.

2.7. Quality control

The reagents blanks and standard reference materials for plant (GBW07603-GSV-2) and soil (GBW07406-GSS-6) were used to determine the accuracy and precision of the extraction and subsequent processes. The recovery rates of selected HMs were good, 94 ± 4.3% and 92.3 ± 2.6% for plants and soil, respectively.

2.8. Concentration Index (CI)

Concentration index (CI) was calculated for lettuce with the equation No 1, as given below:

$$CI=\frac{\text{concentration}\hspace{0.17em}\text{of}\hspace{0.17em}\text{metals}\hspace{0.17em}\text{in}\hspace{0.17em}\text{treated}\hspace{0.17em}\text{plant}}{\text{concentration}\hspace{0.17em}\text{of}\hspace{0.17em}\text{metals}\hspace{0.17em}\text{in}\hspace{0.17em}\text{control}\hspace{0.17em}\text{plant}}$$ (1)

2.9. Health risks

Health risk assessment was a significant parameter for the analysis of this study through various computer-based software and formulae. Exposure through oral pathways were mainly considered in the study. Daily intake of metals (DIM) and HRI were measured for adults and children in lettuce grown in CS and amended soils to quantify the human health risks present in the agricultural soil of the study area with following formulae.

2.9.1. Daily intake of metal (DIM)

Daily intake of metal (DIM) was calculated using formula (Equation No. 2) as adopted by Khan et al. (2008) and Jan et al. (2010).

$$DIM=\frac{\text{Cm}\times \text{Cf}\times \text{IRveg}}{\text{Bw}}$$ (2)

Cm represents concentration of metals in vegetables (mg/kg), Cf is a conversion factor (0.085), IR is ingestion rate for adults and children as 0.345 and 0.232 kg/person/day, respectively (Khan et al., 2008), Bw is body weight for adult and children as 73 kg and 32.7 kg, respectively (Khan et al., 2013).

2.9.2. Health risk index (HRI)

To investigate the human health risk index (HRI) of any toxic pollutant, it is necessary to estimate the exposure level by assessing the possible pathways of exposure of a contaminant to the target organism. HRI was determined for Cr and Pb using the formula adopted by Khan et al. (2008) and Jan et al. (2010).

$$HRI=\frac{\text{DIM}}{\text{RfD}}$$ (3)

HRI represents human health risk index, DIM is the daily intake of metal, and R_fD is the reference dose of the metal. Oral toxic reference dose (R_fD) values for Cr and Pb are 1.5E-0 and 3.5E-03 mg/kg/day, respectively mentioned by US-EPA (2005). If HRI is less than 1, the exposed population is safe from the harmful effects of element (Muhammad et al., 2011).

2.10. Data analysis

The statistical software package SPSS (version 21) was used for interpretation of data. Sigma plot 10.0 was used for plotting graphs and ANOVA was used for significant difference.

3. Results and discussion

3.1. Physicochemical properties of soil and organic amendments

The contaminated soil was a sandy loam with pH 7.20, EC 328 µS/cm, SOM 2.80%, CEC 6.02 cmol/kg, bulk density 1.53 g cm⁻³ and field capacity 0.178 cm³ water per cm³ soil. Total Cr concentration in the soil was very high (545 mg/kg) and above the permissible limit (250 mg/kg) of the State Environmental Protection Administration of China (SEPA, 1995). Pb concentration (44 mg/kg) was within the SEPA permissible limit (350 mg/kg). Bioavailable concentrations of Cr and Pb in soil were 25 and 7 mg/kg, respectively (Table 1).

An increase in the pH of biochar amended soils were observed. pH increased from 7.2 to 7.3, 7.2 to 7.4, 7.2 to 7.5, 7.2 to 7.5, and 7.2 to 7.8 for control, PWB3, PWB7, SCBB3 and SCBB7, respectively (Table 2). The highest increase in pH was observed for SCBB7 as compared to control. Lu et al. (2017) reported increases in soil pH (0.24 unit) with 5% bamboo biochar application. Increase in the soil pH generally decreases the bioavailability of the HMs present in the contaminated soils (Liu et al., 2016). EC values for PWB and SCBB were also increased as compared to control. Biochar contains alkalis and salts which can raise soil pH and EC respectively (Al Wabel et al. 2015; Fidel et al. 2017).

Table 2.

Characteristics of mine impacted soil after amendment with biochar. Mean concentration on dry weight basis (n=3)

Parameters	Control soil	PWB3 Soil	PWB7 Soil	SCBB3 soil	SCBB7 soil
pH	7.3±0.01	7.40±0.11	7.48±0.06	7.51±0.02	7.85±0.03
EC (µS/cm)	318±2.06	395±3.55	415±4.20	435±3.56	470±4.09
CEC (cmol/kg)	5.03	6.44	6.92	6.68	7.97
Cr(bioavailable) (mg/kg)	23±0.21	22.6±0.32	19.2±1.00	17.3±0.64	12.4±0.92
Pb(bioavailable) (mg/kg)	6.07±0.1	5.65±0.08	4.95±0.03	3.72±0.04	2.84±0.02

Control soil: soil without amendment; PWB soil: Poplar wood biochar treated soil; SCBB soil: Sugarcane bagasse biochar treated soil; 3 and 7: % application rates

Both amendments (PWB and SCBB) with different rates decreased the available HMs concentrations as compared to the control soil (Table 2). A decrease in bioavailable concentrations of Cr (9.6, 23.2%) and Pb (19.28, 29.3%) were observed after harvesting in PWB3 and PWB7 amended soil, respectively. Similarly, the decrease due to SCBB amendment (3 and 7%) addition were also found for Cr (30.8, 50.4%) and Pb (46.9, 59.4%), respectively. These reductions can be attributed to the biochars’ higher adsorption characteristic as reported by Jindo et al. (2014). Ahmad et al. (2012) reported that, Pb was immobilized in contaminated soil with the application of oak wood biochar due to rise in pH and surface adsorption. Exchange sites are present on the surface of the biochar which influence the elements bioavailability and their retention capacity (Fellet et al., 2014).

Based on the above results, it can be concluded that SCBB (3% and 7%) reduced HMs concentrations in mine-impacted soil more than PWB. One reason might be the higher pH value and increased CEC of SCBB biochar as compared to PWB. Previous studies showed that the increase in pH decrease the HMs mobility in soil (Khan et al. 2008; Shen et al. 2016).

3.2. Biochar application effects on yield

Aboveground fresh and dry weight of lettuce significantly (P < 0.01) increased at all biochar application rates: PWB3, PWB7, SCBB3 and SCBB7 as compared to the control (Fig. 1). Further detailed data of fresh weight and dry weight of lettuce are given in Supporting Information (SI) (Table S1). The most pronounced effect was observed for the SCBB7 that significantly (P < 0.01) increased (152.6%) lettuce aboveground mass as compared to the control. No significant difference was observed in fresh weight of lettuce among the PWB3 and SCBB3. Similarly, greater yield results with biochar application were also observed in greenhouse studies conducted by Jones et al. (2016) and Singh et al. (2018). The increase in the above ground biomass of lettuce with the application of biochar was also reported by Carter et al. (2013). Uzoma et al. (2011) reported 98–150% increase in the yield of maize with manure biochar. Similarly, a study conducted on poplar wood biochar observed 111% increase in lettuce biomass (Viger et al., 2015). Puga et al. (2015) also reported increased in biomass with the application of higher ratio of sugar cane derived biochar. However, the findings of this study are not in agreement with Alburquerque et al. (2013) who observed slight or no response to crop yield with the application of biochar as compared to the control.

Fig. 1.

Fresh weight and Dry weight of lettuce grown in control soil and biochar (PWB3, PWB7, SCBB3 and SCBB7) amended soils. Control: Soil without biochar, PWB3 and PWB7 represent poplar wood biochar applied at 3% and 7%, repectively, while SCBB3 and SCBB7 represent sugarcane bagasse biochar applied at 3% and 7%, respectively. The different letters represent significant differences and similar letters represent no significant differences between the treatments and control.

Both amendments increased the lettuce growth and height (Fig. 2). In the 1^st week, there was no significant difference in heights between all treatments and control. But at the 4^th week amended soil lettuce heights were significantly greater than the control. After the 8^th week the lettuce height was observed as 59.9% greater for SCBB7 relative to the control. The height of lettuce grown in amended soil at 7% application rate were significantly (P < 0.01) different from control at the time of harvesting.

Fig. 2.

Height of lettuce grown in control soil and biochar (PWB3, PWB7, SCBB3 and SCBB7) amended soils. Control: Soil without Biochar, PWB3 and PWB7 represent poplar wood biochar applied at 3 and 7%, while SCBB3 and SCBB7 represent sugarcane bagasse biochar applied at 3 and 7%.

Various mechanisms can be responsible for the increase in plant aboveground biomass that can alter essential nutrients (N, P, K) availability and change the basic properties of the soil (Khan et al., 2014). Biochar increases some fundamental properties such as pH, cation exchange capacity (CEC), and water content, that could improve the availability of nutrients to plants, subsequently resulting in an increase of biomass (Sohi et al., 2010; Scotti et al., 2015; Puga et al. 2015). Biochar increased the pH of soil, which could also stimulate the growth of lettuce (Clough et al., 2010). Beeslay et al. (2011) observed that alkalization of soil with biochar positively affects the dissolved organic carbon (DOC) content and can increase plant biomass.

Higher CEC and decreased bioavailability of HMs helped plants to grow and survive in contaminated soils, as mine soils are commonly found with limited nutrient availability (Fellet et al., 2014). Biochar addition increased CEC, soil organic matter, and pH buffering capacity, resulting in reduced activity of hazardous metals in soil (Ahmad et al., 2010). Limiting contaminants’ activity in soil decreases the phytotoxicity which leads to significant increase in yield (Tlustos et al., 2006).

3.3. Effects of biochar on Cr and Pb bioaccumulation

Effects of biochars (PWB and SCBB) application at 3 and 7% rate on Cr and Pb uptake in lettuce are presented in Fig. 3. Results indicated that both biochars reduced Cr and Pb concentrations in amended soil and lettuce. Concentrations of Cr and Pb in lettuce above ground mass were reduced significantly (P < 0.01) with PWB7 and SCBB7 amendments as compared to control. PWB as compared to control at both application rates (3% and 7%) decreased the accumulation of Cr and Pb (44.4% and 57.1%), (26.3% and 47.4%) respectively in lettuce. PWB significantly (P < 0.05) reduced the Cr, Pb uptake in lettuce. SCBB significantly (P < 0.01) reduced Cr and Pb uptake in lettuce with both application rates. For SCBB (3% and 7%) application to contaminated soil reduced Cr, Pb uptake in lettuce (49.2% and 69.8%) and (42.1% and 73.7%) respectively. Results have clearly demonstrated that the amendments (PWB3, PWB7, SCBB3 and SCBB7) reduced Cr and Pb bioaccumulation in lettuce. Among the biochars, SCBB reduced concentrations of selected metals the most relative to the control. At the 3% application rate, SCBB increased immobilization of Cr and Pb in soil, reduced their uptake by 8.8% and 21.4%, respectively in lettuce as compared to the PWB3. Similarly, reduction in bioaccumulation of Cr and Pb was higher (29.7% and 50.0%, respectively) in SCBB at 7% application rate as compared to PWB. Detailed data is provided in SI (Table S3).

Fig. 3.

Bioaccumulation of Cr and Pb in lettuce grown in control soil and biochar (PWB3, PWB7, SCBB3 and SCBB7) amended soils. Control: Soil without biochar, PWB3 and PWB7 represent poplar wood biochar applied at 3% and 7%, repectively, while SCBB3 and SCBB7 represent sugarcane bagasse biochar applied at 3% and 7%, respectively. The different letters represent significant differences and similar letters represent no significant differences between the treatments and control.

The results indicate that SCBB at both application rates has more efficiently suppressed the Cr and Pb uptake in lettuce. Puga et al. (2015) also observed immobilization of HMs with the application of sugar cane biochar. Kim et al. (2015) reported a 60% decrease of Pb uptake relative to control in lettuce with the application of 10% biochar. Zheng et al. (2012) also reported 72% decrease in Pb accumulation in rice with the application of rice derived biochar. Miscanthus biochar at 5 and 10% showed decreased available concentration of Pb in contaminated soil (Houben et al., 2013). Jiang et al. (2019) reported that biochar effectively reduced HMs from contaminated soil. Khan et al. (2020) also reported 75% and 50% reduction in Cr and Pb uptake in spinach by using 3% hardwood biochar. The above results are also in agreement with the previous studies conducted by Skjemstad et al. (2002) and Cheng et al. (2003), reporting that Bamboo biochar efficiently adsorbs Cr from soil.

Ahmad et al. (2012) reported a 92.5% decrease in Pb bioavailability by increasing the soil pH applying biochar to military shooting range contaminated soil. Increase in soil pH and adsorption capacity were the mechanism for reduction of Pb. High pH (8.08) of chicken manure biochar has efficiently reduced bioavailable Pb in naturally contaminated soil by immobilizing Pb up to 100 to 94.6% while low pH biochar was not effective for the immobilization of Pb (Park et al., 2013). Thus, SCBB used in this study has high pH (7.76) as a result, its immobilization of Pb efficiency was more than PWB.

HMs are more bioavailable in acidic soil condition (Soltan et al., 2019). By increasing pH, HMs bioavailability is reduced as Karami et al. (2011) reported 1 unit increase in soil pH decreased bioavailability of HMs. Wnetrzak et al. (2014) and Qian et al. (2016) observed higher Cr sorption capacity by biochar having high pH. Cr sorption is dependent on pH, and pH is directly proportional to sorption (Pan et al., 2016). Reduced Cr concentration in lettuce grown in amended soil is due to the higher Cr adsorption capacity of biochar (Nigussie et al., 2012). According to Park et al. (2013) Pb might be removed not only by sorption but by precipitation too. Biochar’s strong adsorption affinity for various ionic solutes was also reported by Radovic et al. (2001). Minimum bioaccumulation of Cr and Pb from biochar-amended soil could also be attributed to an increase in soil CEC due to the additions of biochar (Table 2). HMs adsorption to soil increases with the increase in pH and CEC of soils (Choppala et al. 2010). Jefferson et al. (2010) also reported increase sorption capacity of walnut shell biochar for HMs especially Pb. Other possible reason for the HMs suppression by SCBB might be its larger surface area. Reduced availability of Cr and Pb can be because of exchange sites presence on the biochar surfaces, that are responsible for elements retention resulting in low bioavailability (Fellet et al., 2014). Biochars effects on the HM bioavailability to plants was previously also reported by Khan et al. (2018).

3.4. Concentration index

Cr and Pb concentration index (CI) in lettuce under different organic amendments with different treatments are shown in Table 3. Both amendments significantly affected the CI of Cr and Pb. SCBB decreased the CI more prominently as compare to PWB. SCBB application significantly (P < 0.01) decreased the CI of selected metals as compared to control. SCBB7 decreased Cr by 69.8% and Pb by 73.7% in lettuce.

Table 3.

Concentration Index (CI) of Cr and Pb in lettuce under SCBB and PWB treatments

Treatments	Cr	Pb
PWB3	0.55	0.73
PWB7	0.42	0.52
SCBB3	0.5	0.57
SCBB7	0.3*	0.26*

^*Significantly different at P<0.01 from the control, PWB: poplar wood biochar; SCBB: sugarcane bagasse biochar; 3 and 7% application rates

3.5. Daily intake of metals (DIM) and human health risk index (HRI)

The DIM and HRI values for Cr and Pb oral consumption via lettuce, grown in CS and amended soils are given in Table 4. Both amendments significantly (P < 0.01) decreased the DIM for metals in lettuce at 7% application rate as compared to the control. Decrease in DIM_adl (adults) of Cr for SCBB7 and PWB7 amendments were 69.8% and 57.1%, respectively. Among the amendments the SCBB7 showed 29.7% more immobilization of Cr as compared to PWB7. The DIM for Pb was observed significantly (P < 0.01) reduced for both amendments at 7% application rate as compared to control. At 7% application SCBB reduction of DIM_adl for Pb was 73.7% and PWB was 47.4%. SCBB7 immobilization of Pb was observed to be 50.0% higher than PWB7. DIM values of Cr and Pb in lettuce were observed less than 1 in all treated soils. Results reveal that DIM of both metals was efficiently decreased by SCBB as compared to PWB and control. The findings of this study agree with Khan et al. (2014), who reported 66.0% reduction in DIM in rice grown in mining impacted agricultural soil due to biochar addition. Khan et al. (2020), also observed reduction (Cr 86% and Pb 98%) in daily intake of metals in rice grown on Mn-Cr mine contaminated soil with the application of 3% HWB.

Table 4.

Daily dietary intake (mg kg⁻¹ day⁻¹), Health risk index associated with selected metals in lettuce grown in amended soil as compared to control.

HMs	DIM and HRI	Control	PWB		SCBB
			PWB3	PWB7	SCBB3	SCBB7
Cr	Cr (Conc)	63	35	27	32	19
	DIM (Adl)	2.53E-02	1.41E-02	1.08E-02	1.29E-02	7.63E-03
	DIM (Chl)	3.80E-02	2.11E-02	1.63.E-02	1.93E-02	1.15E-02
	HRI (Adl)	1.69E-02	9.37E-03	7.23E-03	8.57E-03	5.09E-03
	HRI (Chl)	2.53E-02	1.41E-02	1.09E-02	1.29E-02	7.64E-03
Pb	Pb (Conc)	19	14	10	11	5
	DIM (Adl)	7.63E-03	5.62E-03	4.02E-03	4.42E-03	2.01E-03
	DIM (Chl)	1.15E-02	8.44E-03	6.03E-03	6.63E-03	3.02E-03
	HRI (Adl)	2.18E+00	1.61E+00	1.15E+00	1.26E+00	5.74E-01
	HRI (Chl)	3.27E+00	2.41E+00	1.72E+00	1.90E+00	8.62E-01

HMs: heavy metals; DIM: daily intake of metals; HRI: human health risk index; PWB: poplar wood biochar; SCBB: sugar cane bagasse biochar; Adl: adults; Chl: children

HRI values for Cr and Pb through consumption of lettuce was calculated in both amended and control soil, and efficiency of amendments were investigated. HRI values for Cr obtained for children and adults in control and both amendments are within the safe limits (< 1). The lowest value of HRI was obtained for SCBB7 and highest was obtained for Control. HRI values for Pb were not within safe levels (˃ 1) both for adults and children in control and amended soil, except SCBB7. High values of HRI indicate higher concentration of Pb in soil which might be due to the mining activities and open dumping of mining wastes. HRI values were observed in the safe limit (< 1) with the application of SCBB7 representing no health risks for children and adults. Both the amendments efficiently reduced the HRI for both metals as compared to control. Results of HRI of this study are consistent with Singh et al. (2010). Hamid et al. (2016) also reported lower HRI for Cr in vegetables. Nawab et al. (2018) also reported HRI<1 for vegetables in mine impacted agricultural soil. From CS the Pb enter the food chain (Wang et al., 2011) and its continuous exposure effected several organs, such as kidneys, liver, lungs and spleen.

SCBB has a high pH and high adsorption capacity for Pb (Qian et al., 2016), possibly explaining its efficiency for reducing HRI in adults and children. However, the HRI method considers only exposure to HMs through consumptions of vegetables, and does not address other pathways like inhalation, dermal contact, and factors like presence of herbicides and agrochemicals.

It can be concluded from the results that the use of PWB and SCBB amendments for agricultural soils located near mining sites reduced the bioaccumulation and associated risk of toxic HMs and can increase food quality and health safety. To monitor long term immobilization and stability of HMs in mine impacted soil, their bioaccumulation in vegetables should be subjected to further research work with aims to reduce human health risks especially in developing countries’ agricultural systems. Metals dietary intake through food results in long-term low-level accumulation of HMs in the human body and its harmful impacts may only become apparent after several years of exposure.

4. Conclusion

This study concluded that application of biochar in mine-impacted soils resulted in increased plant biomass and decreased bioaccumulation of selected HMs in lettuce resulting in a projected reduction in related health risks. Plant biomass was increased under treatments as compared to control. SCBB7 showed promising results, by increasing 7% more fresh biomass than PWB7. The concentrations of HMs (Pb, Cr) in soil and their bioaccumulation in lettuce, grown on organic amendments (PWB and SCBB), decreased as compared to control. The application of SCBB7 significantly (P < 0.01) decreased the CI of Cr and Pb in lettuce. The DIM and HRI via lettuce consumptions in adults and children were decreased by the PWB and SCBB efficiently at both ratios (3%, 7%) as compared to control. SCBB7 increased the plant biomass, decreased the values of CI, DIM and HRI for selected HMs more efficiently than PWB7. Hence, it can be concluded that the amendments of SCBB and PWB have significant impacts on mine-contaminated agricultural soils, could enhance biomass of vegetables and nutrient availability, and can play a vital role in sustainable farming and reducing human health risk by minimizing HM bioaccumulation in vegetables. Monitoring for HMs on a regular basis in the vegetables and other food crops, especially those cultivated in agricultural soils near mining areas, are essential to avoid their excessive accumulation in the food chain.