Crab Shell Biochar and Compost Synergistically Mitigate Heavy Metal Toxicity in Soil–Plant System

Abstract

Addressing the threat of heavy metal contamination in agriculture, this study evaluated the efficacy of crab shell biochar (CB) and compost (CO) in immobilizing copper (Cu), zinc (Zn), and lead (Pb). The objective was to determine the impact of solitary and combined applications of CB and CO on soil physicochemical properties, nutrient availability, HMs bioavailability, subsequent growth, and oxidative stress responses in spinach plants. The experiment involved two soil types (clay loam and sandy clay loam) with differing initial properties, which were simultaneously spiked with 300 mg kg⁻¹ Cu, 500 mg kg⁻¹ Zn, and 400 mg kg⁻¹ Pb, aged for 30 days, and then treated with varying doses of CB and CO (e.g., 1% and 1.5% w/w). Key results demonstrated that the combined application of 1.5% CB + 1.5% CO was most effective, significantly (p < 0.05) increasing soil pH and reducing DTPA-extractable Cu (by 53–64%), Zn (42–50%), and Pb (57–59%) in both soil types. This treatment also led to a pronounced decrease in the bioaccumulation factor (BF) of HMs in spinach, coupled with improved plant growth parameters (height, fresh/dry weight, chlorophyll content) and reduced oxidative stress (as indicated by lower levels of MDA and antioxidant enzymes). We conclude that the synergistic interaction between CB and CO creates a multi-mechanistic immobilization system, offering a highly effective strategy for the remediation of heavy metal-contaminated soils and the safe cultivation of crops.

1. Introduction

Due to mining, smelting, and intensive fertilizer use, toxic elements like lead (Pb), copper (Cu), and zinc (Zn) persist in agricultural and industrial lands, disrupting soil function, lowering crop yields, and entering food chains through plant uptake, resulting in chronic diseases like cancer, neurological disorders, and renal dysfunction. Heavy metal (HMs)-contaminated soil poses a serious threat to human health and global ecosystems. The physicochemical and biological properties of soil are negatively impacted by HMs, which are persistent in the environment and can build up in soil and vegetables to toxic levels, resulting in adverse environmental issues [1]. Leafy vegetables are more likely to accumulate HMs than others; among them, spinach (Spinacia oleracea L.) accumulates HMs more readily. Approximately 24 million tons of spinach are produced annually worldwide [2]. Mohiuddin et al. (2022) reported that consuming spinach that contains high amounts of HMs has a direct impact on human health. Therefore, there are growing concerns regarding the presence of HMs in soil–plant systems and how they affect the quality of food [3]. HMs-polluted soils have been remedied using a variety of technologies, including physical treatments, electrokinetic techniques, and bio/phytoremediation [4]. Among these remediation techniques, the principle of in situ immobilization is a well-established strategy, with foundational research spanning several decades demonstrating its efficacy in reducing the bioavailability and phytoavailability of heavy metals in soil [5]. This long-standing approach is based on the use of amendments to stabilize metals, a concept that continues to be refined and optimized in contemporary research. According to Ghandali et al. (2024), applying stabilizing materials reduces heavy metals’ bioavailability and stops them from entering the food chain [6]. In recent decades, a number of immobilization/stabilization compounds, including lime, clay minerals, and biochar, have been developed and thoroughly investigated. Among these, organic amendments like biochar and compost can synergistically enhance soil–plant productivity. Biochar, a carbonized solid material produced through biomass pyrolysis under oxygen-limited conditions, exhibits physicochemical properties that depend on feedstock type and pyrolysis temperature [7]. It typically has a high pore volume, surface area, stable carbon, and aromatic compounds, all of which improve soil quality, lower metal pollution, and boost crop yields [8]. When applied to soil, biochar enhances soil’s ability to hold water, exchange cations (CEC), and absorb nutrients; it also improves soil physicochemical properties, microbial biomass, organic matter content, and pollutant adsorption [9]. According to Shyam et al. (2025), biochar treatment enhances soil quality, increases crop production, and promotes carbon sequestration [10]. Furthermore, biochar represents a sustainable waste management strategy by converting diverse waste biomass into a value-added product, providing global environmental advantages, as it can be produced from a variety of feedstock, including industrial waste [11], animal manures [12], sewage sludge [13], and agricultural wastes [14]. According to studies, the physical and chemical properties of biochar are influenced by the feedstock and pyrolysis process parameters, such as temperature and furnace residence time, which in turn affect the availability of soil nutrients and HMs remediation, resulting in effects on soil and plant growth [15]. Recent studies indicate that to maximize efficiency, biochar can be combined with other inorganic (such as metal hydroxides, natural minerals, and hydroxides) or organic (such as compost, garden soil, and microorganisms) materials [16]. Several studies have suggested that applying compost and biochar together increases the effectiveness of soil amendments in the remediation process [17]. Compost, a nutrient-rich organic amendment derived from decomposed organic matter, improves soil fertility and structure. Its application introduces organic substances that contribute to the immobilization of heavy metals [18]. In combination with biochar, compost provides essential nutrients that support plant growth in contaminated soils, while the biochar offers a porous structure and alkalinity for metal retention. This partnership creates a complementary system for enhanced soil remediation and plant development. Various studies have explained the effect of biochar on soil fertility and HMs immobilization. However, little is known about the residual effects of co-applying crab shell biochar (CB) and compost (CO) on the stabilization and uptake of HMs by plants in polluted sites. Therefore, in the current research, our hypothesis posits that the combined effect of CB and CO will effectively reduce Cu, Pb, and Zn uptake in spinach, as well as remediate multiple HMs-contaminated soils. In addition, the residual impact of these amendments on later crops may provide clearer information about the sustainable use of CB and CO to stabilize the long-term toxicity of HMs in polluted soil and their uptake by plants. The aims of our present study were as follows: (1) to investigate the residual impact of CB and CO on the stabilization of HMs in two different soil types; (2) to explore the impacts of both amendments on the physicochemical properties of the test soils; and (3) to investigate the residual impact of CB and CO on the growth and antioxidant response of spinach plants.

2. Materials and Methods

2.1. Soil Collection, Preparation, and Analysis

In this experiment, two contrasting soils, clay loam (Type 1, Hanjiang, Yangzhou, China) and sandy clay loam (Type 2, Liuhe, Nanjing, China), were collected from Jiangsu Province, China (avg. annual temp: 14–16 °C). Both soils were air-dried, crushed, and sieved (2 mm mesh). To obtain co-contaminated soils, the two soils were artificially contaminated with water-soluble salts of Zn, Cu, and Pb. Target concentrations were set at 500 mg kg⁻¹ Zn, 300 mg kg⁻¹ Cu, and 400 mg kg⁻¹ Pb, representing moderate-heavy pollution levels. Solutions of Zn (NO₃)₂ ·6H₂O, Cu(NO₃)₂ ·3H₂O, and Pb (NO₃)₂ were uniformly mixed into soils, adjusted to 70% water-holding capacity, and aged for 30 days (25 °C) with weekly mixing to ensure homogeneity. Post-aging, soils were air-dried and sieved through a 2 mm mesh. Standard protocols were employed to characterize their physicochemical properties. pH and electrical conductivity (EC) were determined in a 1:5 (soil/water) suspension [19]. Total carbon (TC) and total nitrogen (TN) were quantified using an elemental analyzer (Elementar vario EL cube). The total concentrations of lead (Pb), zinc (Zn), copper (Cu), calcium (Ca), magnesium (Mg), potassium (K), and phosphorus (P) in the soil samples were determined. This analysis was performed using a closed vessel microwave digestion system with a tri-acid mixture (typically HNO₃-HF-HClO₄) to ensure complete dissolution of the solid matrix. The elemental concentrations in the resulting digests were then quantified using Inductively Coupled Plasma Optical Emission Spectrometry [20]. The pre-amendment physicochemical properties of the soil are reported in Supplementary Table S1.

2.2. Compost Source and Characterization

Compost (abbreviated as CO) was sourced from a local nursery in Yangzhou. Before experimental use, the compost was air-dried and sieved through a 2 mm mesh. Its physicochemical properties were characterized using the same standard protocols as the soil, with pH and EC measured in a 1:5 (compost/water) suspension. TC, TN, and elemental concentrations (P, K, Ca, Mg) were determined as previously described. The key characteristics of the compost are summarized in Supplementary Table S2

2.3. Biochar Production and Characterization

Crab shells, sourced from Yangzhou market, were crushed, sun-dried, and pyrolyzed under N₂ at 550 °C to produce crab shell biochar (CB). For characterization, CB was sieved (2 mm). Morphology was analyzed by FESEM (SEM-EDS, Hitachi S–4800II, Tokyo, Japan), crystallinity by XRD (Bruker-AXS D8 Advance polycrystalline X- ray diffractometer, Billerica, MA, USA), and functional groups by FTIR (Fourier Transform IR, Bruker FTIR Spectrometer, FTIR Cary 610/670, Billerica, MA, USA). Total carbon (TC) and total nitrogen (TN) were quantified using an elemental analyzer (Elementar vario ELIII cube, Langenselbold, Germany). Metal concentrations (Ca, Mg, K, P, Cu, Zn, Pb) were determined via Inductively Coupled Plasma Optical Emission (ICP 720-ES, Varian, Palo Alto, CA, USA ) following tri-acid (HF-HNO₃-HClO₄) digestion [21]. pH and electrical conductivity (EC) were measured in a 1:10 (w/v) solid-to-water suspension after shaking [22]. The water-holding capacity of CB for soil was determined by adding 0.25 g of CB in soil to a 1:5 (soil/water) suspension, shaking at 180 rpm for 24 h, and then filtered using filter paper. The volume of the filtrate was used to calculate the water-holding capacity [23]. The physicochemical characteristics of CB are summarized in Supplementary Table S3.

2.4. Pot Experiment

A completely randomized pot experiment was conducted using spinach (Spinacia oleracea) as the test plant to evaluate the efficacy of amendments in heavy metal (HMs)-contaminated soil. Each pot (30 cm diameter, 22 cm height) contained either 2 kg of clay loam (Type 1) or sandy clay loam (Type 2) soil and amended with 0% (control), 1%, or 1.5% application rates of CB and CO, both individually and in combination. The processed soils and amendments were mixed properly. This experiment had a total of fourteen treatments (each with three replicates), including two controls (Supplementary Table S4). After a 2-day equilibration, seven seeds were sown per pot, and seedlings were thinned to three per pot after 12 days. Plants grew for 45 days under adequate water and sunlight. Post-harvest soil and plant samples were analyzed.

2.5. Agronomic Analysis

2.5.1. Parameter for Growth

After 45 days, several spinach plant growth metrics were examined for each treatment. A meter scale was used to measure the height of the plant. After the designated growth time, the plant’s fresh weight (W1) and dry weight (W2) were determined using a digital balance. W1 was measured immediately after harvest and W2 was measured after the entire plant was stored in an oven at 80 °C for one complete day [24].

2.5.2. Parameter for Chlorophyll Contents

Chlorophyll content in mature leaves was determined by extracting 0.5 g fresh tissue in 80% acetone overnight. After centrifugation, supernatant absorbance was measured at 663 nm and 645 nm. Chlorophyll a, b, and total concentrations were calculated through a spectrophotometer. The following formula was used to calculate chlorophyll a, b, and total chlorophyll content [25].

$$\mathrm{Chlorophyll} \mathrm{a}={\displaystyle \frac{[12.7\left(\mathrm{A}663\right)-2.69(\mathrm{A}645\left)\right]\times \mathrm{V}}{1000\times \mathrm{W}}}$$

$$\mathrm{Chlorophyll} \mathrm{b}={\displaystyle \frac{[22.9\left(\mathrm{A}645\right)-4.68(\mathrm{A}663\left)\right]\times \mathrm{V}}{1000\times \mathrm{W}}}$$

$$\mathrm{Total} \mathrm{chlorophyll}={\displaystyle \frac{[20.21 (\mathrm{A}645)+8.02 (\mathrm{A}663\left)\right]\times \mathrm{V}}{100\times \mathrm{W}}}$$

Here, V is the extract’s volume (mL), W is the weight of the leaves, and A-663 and A-645 are the absorbance of chlorophyll a and b, respectively.

2.5.3. Stress and Antioxidant Parameter Analysis

Malondialdehyde (MDA) content, was quantified using the TBA assay as defined by Senthilkumar et al. (2021). Fresh leaf samples (0.25 mg) were homogenized with 0.1% TBA, centrifuged (10,000 rpm), and the supernatants reacted with 20% TCA. After heating (95 °C, 30 min) and cooling, absorbance was measured at 532 nm (corrected at 600 nm), with MDA calculated using an extinction coefficient of 155 mM/cm. Antioxidant enzymes were extracted from leaves (0.5 g) in phosphate buffer (pH 7.8) containing PVP and EDTA, followed by centrifugation (10,000 g, 15 min, 4 °C) [26].

Superoxide dismutase (SOD): activity was determined by NBT reduction (Kumar and Tasleem, 2019) and by tracking inhibition of absorbance at 560 nm (units: μmol/min/mg protein) following the protocol of Kumar et al. [27].

Peroxidase (POD): activity was assessed using pyrogallol oxidation (Saxena and Shaiphali, 2023), expressed in μmol/min/mg protein [28].

2.5.4. Parameters for HMs Uptake

Following the addition of CB and CO, the total HMs absorption in spinach leaves was assessed through the acid extraction method, Plant samples were acid-digested in a 2:1 ratio of HNO₃ and HCLO₄. The Cu, Zn, and Pb content in solution were measured using an atomic absorption spectrometer (SP-IAA 4530, China) standardized with a series of standard solutions [29].

2.6. Post-Harvest Soil Analysis

The pH and electrical conductivity (EC) of post-harvest soil were measured in a 2:1 (water/soil) suspension. Total carbon (TC) and total nitrogen (TN) were quantified using an elemental analyzer. Available Ca, Mg, and K were extracted with 1 N ammonium acetate and measured by atomic absorption spectroscopy [29]. Available HMs were extracted using DTPA (pH 7.3) and quantified by AAS [30].

2.7. Bioaccumulation Factor

The bioaccumulation factor (BF) was computed to assess the effectiveness of each treatment in HMs accumulation.

BF = HM_plant/HM_soil

where HM_plant and HM_soil are heavy metal concentrations in the plant and soil, respectively.

2.8. Statistical Analysis

Using SPSS statistical 26.0 program (IBM Corp., Armonk, NY, USA), the effects of CB and CO on two different soil types and spinach plants were examined. Variance analysis was used to determine how the means of the samples differed. The means of three comparisons made in triplicate were compared using Duncan’s multiple range test. A 95% confidence interval was used to evaluate statistical significance, with a significance level of (p < 0.05). All of the soil–plant data were also subjected to Pearson correlation analysis. Origin Pro 2024 software was used to create a correlation map to standardize variables.

3. Result and Discussion

3.1. Biochar (CB) and Compost (CO) Key Characteristics

The CB was prepared from crab shells at 550 °C under anaerobic conditions, and its characteristics were examined using various parameters. Scanning electron microscopy (SEM) images (Figure 1a,b) revealed that CB produced at 550 °C exhibited a highly porous and irregular morphology characterized by an intricate network of interconnected macro- and mesopores. This complex structure arose from the combined effects of high-temperature pyrolysis: the carbonization of the chitinous organic matrix formed a rigid carbon skeleton with fine surface porosity, while the concurrent calcination of inherent calcium carbonate (CaCO₃) minerals generated gaseous CO₂, creating larger voids and channels. The resulting material demonstrated a well-developed pore architecture indicative of significant volatile matter release and mineral decomposition, yielding a carbon-mineral framework with high surface area and structural heterogeneity suitable for applications in adsorption, catalysis, or soil amendment [31].

The XRD spectra (Figure 2b) indicated that the analysis of CB synthesized at 550 °C confirmed the dominance of crystalline calcite (CaCO₃) with characteristic reflections at 29.4° and 39.4° 2θ, consistent with the natural mineral matrix of crustacean exoskeletons. Secondary phases include magnesite (MgCO₃), attributed to isomorphic substitution of Ca²⁺ by Mg²⁺, and trace quartz (SiO₂) from exogenous silicates. The sharp, high-intensity peaks denote extensive crystallinity induced by thermal decomposition of organic constituents [32], while the absence of chitin/protein reflections verifies complete carbonization at this pyrolysis temperature. FTIR analysis of CB pyrolyzed at 550 °C revealed a mineral-dominated spectrum (Figure 2b), with prominent carbonate vibrations at 1436, 872, and 714 cm⁻¹ attributed to residual CaCO₃ from the shell matrix. The peak at 1047 cm⁻¹ indicates C-O/P-O stretching, suggesting phosphates or residual carbohydrate derivatives [33], while the band at 3436 cm⁻¹ corresponds to trace O-H/N-H groups from adsorbed moisture or degraded chitin/proteins [34]. The minor peak at 2510 cm⁻¹ may arise from P-H or S-H functionalities, potentially linked to phospholipids or sulfur-containing biomolecules. These functional groups, particularly carbonates, phosphates, and hydroxyls, synergistically immobilize cationic and anionic heavy metals in contaminated soils through precipitation, ion exchange, and surface complexation mechanisms under alkaline conditions [35]. Table S3 (Supplementary file) shows the physicochemical characteristics of CB, the alkaline pH (10.6) of CB confirms its persistent carbonate mineral content, which synergizes with phosphate and hydroxyl groups to immobilize heavy metals in contaminated soils through precipitation and complexation. The compost (CO) used in the study was well-matured, a key characteristic evidenced by its optimal carbon-to-nitrogen (C/N) ratio (Supplementary Table S2). The CO exhibited a slightly acidic pH (5.7) and a low EC (0.6 dS m⁻¹). It was characterized by a high nutrient content, with total nitrogen (TN) at 2.5%, total potassium (K) at 11 g kg⁻¹, total phosphorus (P) at 3.2 g kg⁻¹, and substantial levels of calcium (Ca, 19.2 g kg⁻¹) and magnesium (Mg) at 5.3 g kg⁻¹. The high organic matter (OM) content of 34.5% underscores its significant potential to improve soil structure and contribute stable humic substances for metal complexation [18]. Critically, the compost showed no detectable (ND) levels of the target heavy metals (Cu, Zn, and Pb), confirming its purity and suitability for the remediation experiment. Water-holding or soil water-retention capacity refers to the soil’s maximal capability to hold or retain water. According to Table S1, the combined application of CB and CO (1.5% each) significantly increased the water-holding capacity, with a more pronounced effect observed in the sandy clay loam soil (Type 2) compared to the clay loam soil (Type 1). According to Ghorbani et al. (2023), this enhancement is attributed to the introduction of CB intra-porosity and the compost’s organic matter, which improved pore space distribution and water retention, particularly in the coarser-textured soil [36].

3.2. Effect of CB and CO on Development and Chlorophyll Contents of Plants

Spinach plant growth and chlorophyll contents changed with soil type and application of CB and CO. In comparison to the control, plants treated with CB and CO both separately and together, showed significant (p < 0.05) growth increases in both soil types (Figure 3a–c). The 1.5% CB + 1.5% CO treatment increased (47%) plant height in soil type 1 and (60%) in soil type 2, followed by 1% CB + 1% CO which increased (38%) in soil type 1 and (53%) in soil type 2. All remaining treatments showed a positive effect on plant length except for 1% CO alone, which had a non-significant effect in soil type 1 compared to the control (Figure 3a). In the case of plant fresh and dry weight, 1.5% CB + 1.5% CO significantly (p < 0.05) improved them by (37%), (43%) in soil type 1, and by (48%) and (50%) in soil type 2, respectively. This was followed by 1% CB + 1% CO which increased them by (33%), (39%) in soil type 1, and (39%) and (45%) in soil type 2. All other applications also increased fresh and dry weight in both soils, with a minimal effect observed for 1% CO in both soils compared to the control (Figure 3b,c). The enhanced plant growth observed in soils treated with CB and CO results from the combined benefits of these amendments. CB application improves soil health by increasing water-holding capacity and porosity, facilitating mineral transport, and contributing to greater plant height. Furthermore, CO provides essential nutrients vital for plant development [37]. Regarding fresh and dry weight, CB plays a significant role by enhancing soil ion availability, pH buffering, and nutrients and water retention, all contributing to improved plant biomass. Crucially, CB also immobilizes heavy metals (HMs), reducing their uptake into plant leaves and thereby further boosting spinach’s fresh and dry weight. CO complements this by improving soil nutrient status and directly supporting increased spinach growth, it specifically enhances plant fresh and dry weight by supplying readily available essential nutrients that fuel biomass production. Our findings are similar to those of Pandey et al. (2024), who reported that biochar in combination with compost improved spinach growth in Cd-contaminated soil [38]. The combined effect of CB and CO treatment was more pronounced in soil type 2 than in type 1, because the acidic condition may enhance the dissolution of beneficial minerals from CB, increasing nutrient availability and heavy metal immobilization efficiency [39]. To check the quality of the spinach, the chlorophyll content was measured. As seen in (Figure 3d–f), the chlorophyll content in spinach leaves increased after CB and CO applications. The combined application of CB and CO significantly increased chlorophyll a (Figure 3d), chlorophyll b (Figure 3e), and total chlorophyll (Figure 3f) compared to control. In soil type 1, chlorophyll a increased up to (52%) with 1.5% CB + 1.5% CO, and (61%) in soil type 2, respectively. A similar trend was observed for chlorophyll b and total chlorophyll, where the combined application significantly (p < 0.05) increased their content in spinach plants. The results showed that CB and CO synergistically increased spinach chlorophyll content in both soils by immobilizing toxins and enhancing nutrient availability. CB’s porous structure adsorbed heavy metals (e.g., Cu, Zn, Pb), reducing their uptake by plants and preventing disruption of chlorophyll synthesis and chloroplast damage. Critically, the magnesium-rich CB directly supplied substantial Mg²⁺, the central ion in chlorophyll molecules [40], while CO provided essential nitrogen (for chlorophyll’s structure) and improved soil health. Our finding are similar to those of Wang et al. (2022), who described that, biochar and mineral compost increase chlorophyll content in Brassica chinensis [41].

3.3. CB and CO Effect on Oxidant Activity and Antioxidant Enzymes of Spinach

The oxidant and antioxidant activity of spinach was considerably impacted by the addition of amendments. Figure 4a shows the oxidant activity (MDA); the use of CB and CO greatly reduced the MDA level in spinach leaves in contrast to the control (CK) treatment. The greatest decrease in MDA was found with 1.5% CB + 1.5% CO which was (50%, 60%) in both soils, followed by 1% CB + 1% CO (31%, 40%). All other applications of CB and CO also minimized the MDA level in spinach plant except for 1% CO, which showed a similar effect compared to control. Following CB and CO incorporation, there was a notable reduction in MDA contents, which may have resulted from lower heavy metal uptake, as evidenced by the decreased bioavailability of heavy metals. Similarly, Rehman et al. (2023) reported that MDA contents in kenaf plant were reduced in Pb- and Cd-polluted soil after being treated with biochar [42]. Furthermore, under metal stress, plants have their own oxidative defense systems to get rid of reactive oxygen species (ROS) and lessen the structural damage that ROS causes. Therefore, in response to ROS, plants activate several antioxidant enzymes (such as SOD and POD) and non-enzymatic antioxidants [43]. It has been observed that POD activity in several plants increased in HMs-contaminated soils. The present work indicated that the POD and SOD activity decreased significantly after CB and CO addition (Figure 4b,c). The POD activity in spinach plants was (37%, 30%) lower with 1.5% CB + 1.5% CO in both soils, which was followed by 1% CB + 1% CO (22%, 29%). A minimal effect was observed with 1% CO compared to the control (Figure 4b). Xu et al. (2019) discovered that after adding amendments, the pakchoi’s POD activity dropped, lessening the harmful impact of heavy metals on the plant’s growth [44]. Our current research showed that SOD activities also decreased with the individual and combined application of CB and CO, 1.5% CB + 1.5% CO decreased them by (34%, 36%), and 1% CB + 1% CO dropped them by (20%, 22%) in both soils. The 1% CO alone also declined SOD activities in both soils compared to the control (Figure 4c). In general, compared to control treatments, applying CB and CO to heavy metal-contaminated soils significantly lowers malondialdehyde (MDA), peroxidase (POD), and superoxide dismutase (SOD) levels in spinach. This occurs because the CB porous structure, alkaline properties (from CaCO₃), and chitin-derived functional groups (–NH₂, –OH) strongly adsorb cationic pollutants (e.g., Cu²⁺, Pb²⁺, Zn²⁺), reducing their bioavailability and uptake by plants. CO further immobilizes metals via humic acid chelation while supplying essential nutrients and organic compounds that support the plant’s antioxidant defense system. Together, they suppress reactive oxygen species (ROS) generation at the source by preventing metal uptake and diminish oxidative stress (reducing MDA) and the need for sustained high antioxidant enzyme activity (lowering POD/SOD).

3.4. Plant Heavy Metal (HMs) Content

Figure 5 shows Zn, Cu, and Pb content in the spinach plant, in comparison with control treatment (CK), all applications of CB and CO resulted in a notable decrease in the Cu concentration in spinach. The 1.5% CB + 1.5% CO significantly reduced Cu content by (71%, 64%) in both soils, followed by 1% CB + 1% CO (45%, 60%). The effect of 1% CO for Cu reduction was not significant in soil type 1 compared with the control (Figure 5a). Furthermore, all treatments of CB and CO reduced Zn accumulation in spinach plant, the 1.5% CB + 1.5% CO significantly impacted (p < 0.05) Zn content and reduced it by (63%, 65%) in both soils, followed by 1% CB + 1% CO with (54%, 58%). Individual applications of CB and CO also decreased Zn content, with 1% CO having a minimal effect compared with the control (Figure 5b). Similarly, in the case of Pb, all applications of CB and CO, both combined and individual, decreased Pb content in the spinach plant. The 1.5% CB + 1.5% CO treatment had the highest reduction (66%, 67%) in both soils, followed by 1% CB + 1% CO, which decreased Pb concentration equally by (60%) in both soils, the 1% CO in soil type 2 showed only a slight change in Pb content compared with the control (Figure 5c). Our current study shows that as the dosage of CB and CO increased, the HMs content in spinach was significantly reduced. Previous literature has revealed that higher biochar application rate reduces the contents of HMs in plants [45]. Skic et al. (2024) mentioned that biochar’s large surface area, porous structure, water-holding capacity, and variety of chemical groups aid in metal adsorption, which can lower their uptake in soil–plant systems [46]. Similarly, compost can potentially lower pollution and serve as an efficient organic amendment; their combined effect reduced HMs contents more efficiently than individual applications [16]. According to Sarraf et al. (2024), biochar lowers the metal concentration in the soil because of its alkaline nature, which changes the ions into a less mobile form and results in low metal concentrations in plant leaves [47]. In this study, CB and CO reduced HMs accumulation in spinach plant in both soils, but the effect was more significant in soil type 2. This may be due to an increase in soil pH, alteration in the physicochemical structure of soil, and reductions in the bioavailability of HMs with the addition of CB and CO.

3.5. CB and CO Impact on Post-Harvest Soil Properties

The physicochemical properties of heavy metal-contaminated soils were greatly improved by the application of CB and CO. The impact of these amendments on soil pH is shown in (Figure 6a). The 1.5% CB + 1.5% CO treatments considerably raised the pH of both soils. The greater absolute increase was observed in soil type 2 (from 5.0 to 6.4) compared to soil type 1 (from 6.5 to 7.5), which can be attributed to its initially higher acidity (Table S1), providing a greater buffering capacity for the alkaline CB to neutralize. Additionally, 1% CB + 1% CO and individual application of CB also increased the pH level in both soils. These findings indicate that CB was the primary driver for increasing soil pH. Prior to the amendment, the soils and CO had an acidic pH, however CB application led to a greater pH increase than CO alone. This is explained by Leng et al. (2021) who found that the high surface area and porosity of CB, improved the soil’s cation exchange capacity (CEC) and raised its pH [48]. According to Barrow et al. (2023), an alkaline pH of the soil aids in nutrient retention and encourages plant growth [49]. In contrast to its combined application with CB, the CO amendment alone proved ineffective in raising the soil’s pH. This might be explained by the lack of functional groups and carbonates, which are found in CB. The efficacy of CB is due to its inherent alkalinity and liming effect, as well as its high ash content, which is rich in carbonates and alkali metals that help raise the pH of contaminated soil [50]. A similar trend was observed for EC: the individual and combined applications of CB with CO increased the EC of both soils, while the individual application of CO did not have a significant effect compared to the control soils (Figure 6b). The 1.5% CB + 1.5% CO significantly (p < 0.05) increased the EC (53%, 58%) of both soils followed by 1% CB + 1% CO (46%) in soil type 1, while in soil type 2 the effect of 1.5% CB alone and 1% CB + 1% CO was similar (50%). The more pronounced relative increase in EC in soil type 2 can be attributed to its significantly lower initial background EC (0.4 dS m⁻¹) compared to soil type 1 (1.14 dS m⁻¹), making its soluble ion content more susceptible to change upon amendment addition. Study results revealed that the solitary application of CB increased EC because it is inherently rich in soluble salts, such as calcium carbonate, that readily release ions into the soil [50]. CO alone may not significantly increase EC if its nutrients are temporarily immobilized or bound within stable organic compounds. However, when combined, the alkaline nature of CB can alter the soil pH, which promotes the rapid chemical dissolution and release of the soluble salts contained within the CO. This reaction forces a quick flush of ions from both materials into the soil solution. The cumulative effect of this direct chemical release from both sources results in a measurable increase in EC that would not occur with either amendment applied alone.

The application of CB and CO also influenced the concentrations of available nutrients in contaminated soils (Figure 7a,b). Generally, the soil’s available nutrients increased as the CB and CO application rate increased; however, for the combined treatments of CB and CO, the soil’s available P and Ca contents decreased with an increased dose rate in soil type 2, while in soil type 1, both dosage rate had equal effects. This differential response in nutrient availability, particularly for P and Ca, is likely influenced by the distinct initial soil compositions; soil type 1 had higher native concentrations of P (0.61 g kg⁻¹) and Ca (13.2 g kg⁻¹) compared to soil type 2 (0.4 g kg⁻¹ P and 8.3 g kg⁻¹ Ca), which may have affected their buffering capacity and retention dynamics upon amendment. Additionally, the soil TC of combined CB and CO treatments was lower than in individual CO treatments, which may be attributed to enhanced nutrient uptake by spinach plants, which grew more vigorously in combined CB and CO treatments, resulting in lower residual soil nutrient contents in these treatments. Wang et al. (2022) reported that biochar and mineral compost addition can clearly alter the soil’s characteristics and the growth of Brassica chinensis [41]. In general, CB and CO have significant effects on the pH, available K, P, Ca, Mg, TN, and TC of the soil, which collectively impact the bioavailability of HMs and plant growth.

3.6. CB and CO Immobilized HMs (Cu, Zn, and Pb)

After harvesting the spinach plant, the post-harvested soil was examined to find the change in DTPA-extractable fractions of HMs. All applications of CB and CO immobilized the HMs in both soils. The combined application of 1.5% CB + 1.5% CO most significantly (p < 0.05) reduced Cu (53%, 64), Zn (42%, 50%), and Pb (57%, 59%) in both soils, followed by 1% CB + 1% CO which decreased Cu (40%, 50%), Zn (31%, 43%), and Pb (45%, 45%), respectively. All other applications of CB and CO also immobilized HMs in both soils except for 1% CO which had non-significant effect on Cu compared to the control in soil type 2 (Figure 8). When compared with the existing literature, the magnitude of immobilization for each metal underscores the effectiveness of our combined amendment. For instance, the 53–64% reduction in Cu observed here exceeds the 30–40% reduction commonly reported for biochar alone in acidic soils, likely due to the additional complexation with compost-derived organic matter [51]. Similarly, the >50% reduction for Pb aligns with the findings of Ahmed et al. (2023), who reported a 55% decrease in Pb bioavailability using biochar composite, attributed primarily to precipitation as Pb-phosphate and Pb-carbonate [52]. For Zn, the 42–50% reduction in our acidic soil type 2 is more pronounced than the <40% reduction observed by Ibrahim et al. (2022), in a neutral clay loam, highlighting the greater pH-correcting impact of CB in naturally acidic sandy soil [53]. The greater reduction in HMs observed in soil type 2 is attributed to its initially acidic pH and sandy texture, which naturally promoted greater metal solubility and mobility compared to soil type 1. Furthermore, the alkaline nature of the CB had a more pronounced pH-correcting effect in soil type 2, rapidly precipitating metals and enhancing adsorption. The observed immobilization of HMs in both soils can be explained by CB’s large surface area, alkalinity, and porosity, which can shift HMs from a highly exchangeable form to a less accessible state. Specifically, this shift involves multiple immobilization pathways, involving precipitation as low-solubility carbonates (e.g., PbCO₃ Zn₅ (CO₃)₂ (OH)₆) and phosphates, driven by the carbonates and phosphates released from the calcite-rich CB, surface complexation via coordination with oxygen-containing functional groups (-OH, -COO-) on both the CB- and CO-derived humic substances and ion exchange with cations such as Ca²⁺ and Mg²⁺ released from the CB matrix. These mechanisms collectively shift metals from the bioavailable, exchangeable fraction (measured by DTPA extraction) to more stable carbonate-bound, organic matter-bound, and residual fractions, thereby lowering their phytoavailability [54].

Beyond these individual pathways, the superior efficacy of the combined CB and CO treatment suggests direct synergistic interactions that enhance the proposed mechanisms. We speculate that the microbial community and organic acids (e.g., citric, oxalic) within the CO act as a “bio-activator,” accelerating the weathering of the crystalline calcite in the CB (identified via XRD in Figure 2b) This enhanced dissolution would speed up the release of Ca²⁺ and CO₃²⁻ ions, thereby amplifying the precipitation of HMs as low-solubility carbonates. Furthermore, the dissolved organic matter (DOM) from the CO may form an organo-mineral coating on the CB particles, as observed in other biochar–compost studies [55]. This coating likely functionalizes the CB’s porous surface (seen in SEM, Figure 1a,b) without blocking pores, introducing additional oxygen-containing functional groups for enhanced metal complexation and potentially modifying the sorption dynamics to create a superior composite sorbent. The efficacy of these synergistic mechanisms was contingent upon the use of a mature CO. The stable nature of the CO is indicated by its optimal C/N ratio (Table S2). This maturity ensured that its high organic matter content (~34.5%) contributed stable humic substances for effective metal complexation without the risks of phytotoxicity or nitrogen immobilization associated with immature composts [56]. According to several studies, biochar’s high pH, CEC, porous structure, and abundant functional groups enhance the immobilization of heavy metals [9]. According to Kamran et al. (2019), there is a clear inverse link between soil pH and bioavailable heavy metals [57]. This strong pH dependence, however, raises important considerations about long-term stability. While a significant decrease in soil pH could potentially remobilize a portion of the immobilized metals by dissolving carbonates, the synergistic CB + CO system offers enhanced resilience. Quan et al. (2020) reported that, the organo-mineral complexes formed with compost and the physical encapsulation within biochar’s porous matrix provide a more robust stabilization that is less susceptible to pH fluctuations compared to simple pH adjustment alone [58]. Therefore, this approach provides a reliable, long-term reduction in metal bioavailability, effectively removing them from biological cycles for extended periods. While the pH-dependent nature of carbonate precipitation poses a potential risk of metal remobilization under future soil acidification, the multi-mechanistic CB and CO system provides resilience. The alkaline nature of CB strongly buffers against pH drops, and the CO’s stable organic matter ensures metal complexation continues even under milder acidic conditions, thereby enhancing long-term stability beyond what a single amendment could achieve. The superior performance of the combined treatment in our experiment is in accordance with Bashir et al. (2021), who reported that applying compost and biochar together has a higher capacity in reducing soil metal pollution than applying them separately. According to the author, applying compost and biochar together at a rate of 3% w/w was successful in lowering soil Cr contamination [59].

The bioaccumulation factor (BF) values are given in (

Table 1. Bioaccumulation factor (BF) of two different soils (soil type 1 and type 2) under the influence of CB and CO.

Soils Type	Treatments	Bioaccumulation Factor (BF)
		Cu	Zn	Pb
Soil type 1	Control	0.32	0.55	0.075
	1% CB	0.27	0.44	0.072
	1.5% CB	0.25	0.40	0.070
	1% CO	0.32	0.53	0.073
	1.5% CO	0.32	0.47	0.072
	1% CB +1% CO	0.30	0.36	0.054
	1.5% CB +1.5% CO	0.29	0.34	0.052
Soil type 2	Control	0.38	0.46	0.061
	1% CB	0.35	0.36	0.059
	1.5% CB	0.33	0.36	0.056
	1% CO	0.38	0.44	0.061
	1.5% CO	0.36	0.40	0.060
	1% CB + 1% CO	0.30	0.34	0.052
	1.5% CB + 1.5% CO	0.21	0.32	0.048

Bioaccumulation factor: HMs-concentration in plant/HMs-concentration in soil.

). All BF values for HMs (Cu, Zn, Pb) were varied in both soils. BF values for HMs were reduced compared to control in both soils. The combined application of CB and CO reduced BF value more in soil type 2.

3.7. Analysis of Correlation for the Parameters Under Study

Pearson’s correlation analysis revealed a significant and noticeable impact of the applied treatments on all assessed attributes. Figure 9 displays the correlation matrix for the parameters under study. It was found that the post-harvest soil’s pH had a negative association with the concentration of HMs in both soil and spinach plant for the treatments of CB and CO. This may be explained by the characteristics of CB, which raised the pH of the soil and immobilized metals, which decreased their uptake. A strong positive correlation was observed between the DTPA-extractable HMs in the soil and their concentration in the plant, confirming the bioavailability of these metals. This indicates that the CB and CO amendments not only enhanced the soil and plant properties but also influenced the mobility of HMs. Furthermore, the DTPA-extractable HMs showed a negative correlation with soil physicochemical parameters (P, K, TN, Ca, Mg) and spinach growth characteristics (height, fresh/dry weight, chlorophyll content). Conversely, a positive correlation was found with plant stress and antioxidant parameters (MDA, SOD, POD), indicating that HMs stress activated the plant’s defense system. Lin et al. (2025) reported that, the use of biochar reduced cadmium stress in wheat plants and showed a significant relationship with MDA concentration and plant antioxidants [60]. The findings of Pearson’s correlation analysis were further supported by the PCA-biplot visualization. Each parameter appeared to be grouped in response to different treatment applications, as indicated by the PCA analysis (Supplementary Figure S1). Every treatment had a unique effect on the characteristics of the plants, either favorably or unfavorably. The PCA 1 and PCA 2 components contributed 80.4% and 9.2% of the variance in soil type 1, while 82.9% and 8.7% in soil type 2, respectively, accounting for 89.6% and 91.6% of the overall variance. Significant contributions to PCA1 were made by the concentrations of HMs in both soil and plant MDA, POD, and SOD, whereas other parameters were more oriented toward PCA 2. Both the PCA and correlation analyses confirm the results of our investigation. The research demonstrated a substantial relationship between HMs bioavailability and the plant defense system, leading to the conclusion that the combined application of CB and CO reduced oxidative stress and enhanced plant growth by immobilizing HMs. These findings are consistent with previous studies by Aqeel et al. (2021), who described that the concentration of HMs showed positive associations with MDA and other antioxidants but a significant negative association with yield and photosynthetic characteristics [61].

4. Conclusions

In conclusion, this study demonstrates that the combined application of CB and CO is a highly effective strategy for the remediation of heavy metal-contaminated soils and the mitigation of phytotoxicity in spinach. The synergistic effect of the amendments significantly enhanced soil health by increasing pH and water retention, which in turn dramatically reduced the bioavailability of Cu, Zn, and Pb (as evidenced by DTPA extraction) and their subsequent uptake by plants. This successful immobilization directly translated to improved plant physiological health, marked by increased biomass and chlorophyll content, alongside a significant reduction in oxidative stress, confirmed by lower malondialdehyde (MDA) levels and decreased activity of antioxidant enzymes (SOD, POD). The more pronounced results in the sandy clay loam soil (Type 2) are attributed to its inherently acidic pH and coarse texture, which initially favored higher metal solubility and bioavailability, thereby providing a greater potential for amendment-mediated immobilization. Therefore, the integration of CB and CO presents a sustainable and efficient approach to reclaiming contaminated agricultural land, ensuring both enhanced crop productivity and improved food safety.