Heavy Metal Contamination in Soil, Water, and Biota: Sources, Impacts, Remediation Strategies, and the Emerging Role of Trifolium spp. with Valorized Eggshell and Bagasse Biosorbents

3.1. Lead (Pb)

Lead occurs mainly as Pb (II) compounds bound to airborne particulates, dissolved Pb²⁺ in aquatic systems, and strongly adsorbed mineral- or organic-bound forms in soils, where it exhibits high persistence and limited mobility (Angon et al., 2024; Kumar et al., 2020). Major anthropogenic sources include mining and smelting, legacy leaded gasoline, batteries, paints, contaminated fertilizers, sewage sludge, industrial effluents, and urban dust (Bouida et al., 2022; Gupta et al., 2024).

In soils, Pb contamination leads to long-term fertility degradation and suppression of microbial activity, while in plants it disrupts germination, photosynthesis, nutrient balance, and growth through oxidative stress. In humans and animals, Pb is a potent neurotoxin causing cognitive impairment, developmental disorders, renal dysfunction, and cardiovascular damage, with food-chain transfer via crops and forage representing a major health concern (Angon et al., 2024; Bouida et al., 2022).

3.2. Arsenic (As)

Arsenic is predominantly found as arsenite (As (III)) and arsenate (As(V)), with environmental speciation controlled by redox potential, pH, and microbial activity, strongly influencing its solubility, mobility, and toxicity in soils and water (He et al., 2025; Patel et al., 2023; Sinha et al., 2023). Sources include natural mineral weathering, mining and smelting, fossil fuel combustion, arsenical pesticides, wood preservatives, fertilizers, and industrial effluents, contributing to widespread groundwater and agricultural soil contamination.

Arsenic impairs plant growth by disrupting nucleic acid synthesis, enzyme activity, and antioxidant defense systems, leading to reduced biomass and yield. In humans, chronic exposure via contaminated water and food is associated with skin lesions, cancers, cardiovascular disease, and neurological disorders, establishing arsenic as a major global groundwater contaminant (He et al., 2025; Rajendran et al., 2024; Sinha et al., 2023).

3.3. Cadmium (Cd)

Cadmium primarily exists as Cd²⁺ in soil and water, forming soluble complexes with chlorides, sulfates, carbonates, and organic ligands, which enhance its mobility and bioavailability relative to less mobile metals such as lead (Angon et al., 2024; Haider et al., 2021). It is released through phosphate fertilizers, Ni-Cd batteries, PVC stabilizers, pigments, mining and smelting, coal combustion, wastewater discharge, and sludge application in agriculture (Genchi et al., 2020; Zulfiqar et al., 2022).

Cadmium readily accumulates in soils and is efficiently taken up by crops, posing threats to food safety and ecosystem integrity. In humans, Cd has no biological function and exhibits a prolonged biological half-life of 10 to 30 years, causing chronic kidney damage, bone demineralization, pulmonary toxicity, and increased cancer risk with long-term exposure (Genchi et al., 2020; Suhani et al., 2021; Xu et al., 2024).

3.4. Mercury (Hg)

Mercury occurs as elemental Hg⁰, inorganic Hg²⁺, and organic methylmercury (MeHg), with MeHg being the most toxic and bioaccumulative species, formed via microbial methylation in aquatic and anoxic environments (Beckers & Rinklebe, 2017; Gworek et al., 2020). Major sources include coal combustion, metal smelting, artisanal gold mining, chlor-alkali industries, waste incineration, industrial effluents, and mercury-containing products.

Mercury biomagnifies in aquatic and terrestrial food webs, causing phytotoxicity through reduced photosynthesis, enzyme inhibition, and oxidative damage in plants. In humans and animals, MeHg is a potent neurotoxin linked to cognitive impairment, developmental defects, and sensory dysfunction, while inorganic and vapor forms induce renal and neurological damage (Beckers & Rinklebe, 2017; Ray et al., 2025).

4.1 Conventional physicochemical approaches

Early heavy-metal remediation relied predominantly on physicochemical technologies such as chemical precipitation, coagulation-flocculation, membrane filtration, ion exchange, adsorption, soil washing, solidification/stabilization, and electrochemical treatments (Jadaa & Mohammed, 2023; Xu, Jin & Zeng, 2024; Zamora-Ledezma et al., 2021). These methods offer rapid and effective contaminant removal, particularly in water treatment systems and industrial effluent management.

Despite their effectiveness, long-term sustainability is constrained by high capital and operational costs, intensive energy consumption, extensive chemical usage, declining efficiency at low metal concentrations, secondary waste generation (e.g., metal-rich sludge), and disruption of soil structure during ex situ soil treatments. Critically, these approaches do not destroy heavy metals; they transfer contaminants into other matrices—sludge, concentrates, or spent sorbents—creating ongoing disposal and re-contamination risks (Zamora-Ledezma et al., 2021; Xu, Jin & Zeng, 2024).

4.2 Biological and nature-based remediation

In response to the environmental and economic constraints of conventional technologies, biological and nature-based remediation strategies have gained increasing attention. Phytoremediation techniques—including phytoextraction, phytostabilization, rhizofiltration, phytovolatilization, and phytofiltration—employ hyperaccumulator plants to uptake or immobilize metals in contaminated soils and waters (Nedjimi, 2021; Sharma et al., 2023; Yan et al., 2020). Microbial bioremediation involving plant growth-promoting rhizobacteria (PGPR) and mycorrhizal fungi further enhances metal bioavailability or immobilization while improving plant stress tolerance (Raklami et al., 2022; Karnwal et al., 2024).

Recent advances emphasize plant-microbe consortia and amendment-assisted phytoremediation, where biochar, compost, chelating agents, microbial inoculants, and plant root exudates improve metal uptake, stabilization, and detoxification while enhancing soil health (Tamma et al., 2025; Wan et al., 2024; Wei et al., 2025). Emerging biochemical strategies such as enzyme-induced carbonate precipitation (EICP) have demonstrated effective immobilization of Cd, Pb, and Zn through biomineralization into stable carbonate phases (Xu et al., 2025).

However, biological approaches are often limited by slow remediation rates, sensitivity to environmental conditions, root-zone constraints, and the requirement for safe management of metal-enriched biomass to prevent secondary contamination (Alengebawy et al., 2021; Srivastava et al., 2017).

4.3 Advanced and emerging technologies

To overcome the scalability limitations of both physicochemical and biological methods, advanced remediation strategies increasingly integrate engineered materials, nanotechnology, and biotechnology (Jadaa & Mohammed, 2023; Mitra et al., 2022; Xu, Jin & Zeng, 2024). Biochar-based remediation represents a major technological advancement, with engineered and functionalized biochars immobilizing heavy metals through adsorption, ion exchange, surface complexation, and mineral precipitation, while simultaneously improving soil fertility and microbial activity (Tamma et al., 2025; Wang et al., 2021; Wei et al., 2025).

Nanomaterial-assisted remediation—including magnetic nanoparticles, metal oxides, zeolites, polymers, chitosan, and metal-organic frameworks (MOFs)—offers high sorption capacity and selective metal removal, particularly in water treatment systems (Babu et al., 2025; Mohamed et al., 2025). Nevertheless, concerns remain regarding production cost, regeneration efficiency, long-term field stability, and potential nanotoxicity under large-scale deployment.

4.4 Modern green and hybrid remediation frameworks

Modern remediation increasingly favors integrated green hybrid systems that combine biological processes with advanced materials. In soil remediation, leading strategies include plant-microbe phytoremediation, biochar-assisted immobilization, iron-based stabilization, enzyme-mediated biomineralization, and nano-enabled sorbents deployed within in situ stabilization frameworks to minimize soil disturbance. For aquatic and water remediation, aquatic plant phytoremediation, biochar and natural zeolite sorbents, functionalized nanoadsorbents, and electrochemical or membrane-based systems have demonstrated high removal efficiency with reduced sludge production and chemical demand (Ali et al., 2020; Pang et al., 2023).

A consistent finding across all remediation categories is that heavy metals are rarely destroyed; they are transferred into secondary matrices—sludge, biomass, or immobilized soil fractions—that require secure long-term management. Consequently, the field is shifting toward integrated, low-impact, circular remediation models prioritizing in situ stabilization, plant-microbe synergies, engineered biochars, and resource-recovery concepts (Jadaa & Mohammed, 2023; Zamora-Ledezma et al., 2021).

5.1 Taxonomy, evolutionary diversity, and systematics

The genus Trifolium L. (clovers), belonging to the family Fabaceae, comprises approximately 250–260 herbaceous annual and perennial species distributed mainly across temperate regions of Europe, the Mediterranean, western Asia, North Africa, and the highlands of eastern Africa (Uslu & Babac, 2019; Ellison et al., 2006). Molecular phylogenetic analyses support the monophyly of Trifolium and indicate a probable Mediterranean origin during the Early Miocene, followed by dispersal to the Americas and sub-Saharan Africa. Classical taxonomic systems divided the genus into approximately eight sections based primarily on morphological characters; however, recent molecular systematics recognize two principal subgenera: subgenus Trifolium and subgenus Chronosemium (Ahmed et al., 2021).

Chromosomal evolution has played a significant role in diversification within the genus. Cytogenetic reconstructions suggest an ancestral basic chromosome number of 2n = 16, with repeated events of aneuploidy and polyploidy contributing to speciation and adaptive radiation (Ellison et al., 2006; Ahmed et al., 2021). Although more than 200 species have been described worldwide, only approximately 25 are of significant agricultural value; several, notably T. repens (white clover), T. pratense (red clover), and Trifolium alexandrinum (berseem clover), have achieved global prominence (McKenna et al., 2018).

5.2 White clover: agricultural importance and biological properties

T. repens L. stands out as one of the most economically and ecologically important species within the genus. Native to Europe, this perennial stoloniferous legume has attained a cosmopolitan distribution following agricultural introduction and naturalization across temperate regions worldwide (Carlsen & Fomsgaard, 2008). Its global success is attributed to grazing tolerance, vegetative propagation, environmental plasticity, and efficient symbiotic nitrogen fixation—commonly ranging from 100 to 300 kg N ha⁻¹ year⁻¹ under field conditions (Rodríguez-Navarro et al., 2021). In pasture-based livestock systems, white clover enhances forage quality, increases crude protein content, and reduces reliance on synthetic nitrogen fertilizers.

Several Trifolium species are also rich in proteins, flavonoids, isoflavones, saponins, and phenolic compounds, supporting their use in human nutrition, herbal medicine, and dietary supplements (Sabudak & Guler, 2009; Kołodziejczyk-Czepas, 2016). Trifolium pratense in particular is widely incorporated into nutraceutical formulations for managing menopausal symptoms and cardiovascular risk.

5.3. Phytoremediation capabilities

White clover has received substantial attention in phytoremediation research due to its documented heavy-metal uptake capacity, including cadmium, chromium, and lead, through rhizosphere-mediated mechanisms (Lin et al., 2021; Liu et al., 2021). It has also demonstrated effectiveness in the bioremediation of petroleum hydrocarbons and polycyclic aromatic hydrocarbons (PAHs) (Sawicka et al., 2023; Ahmad et al., 2025). Importantly, white clover stimulates soil enzyme activities including dehydrogenases, ureases, phosphatases, and catalases, while compatibility with plant growth-promoting endophytes such as Pseudomonas putida enhances performance under metal stress (Lin et al., 2021; Liu et al., 2021).

White clover exhibits a conservative accumulation profile with physiological regulation favouring phytostabilization, rather than hyperaccumulation. Its designation as a certified reference material (BCR-402) supports its widespread use as an analytical standard in biomonitoring studies (Ghiani et al., 2014). Among indigenous South African Trifolium species, comparative data indicate stronger accumulation tendencies: Trifolium burchellianum recorded elevated cadmium concentrations (0.5 mg kg⁻¹), while T. dubium accumulated high lead levels (up to 7.57 mg kg⁻¹), suggesting phytoextraction potential in regional remediation contexts (Gounden, 2017). T. africanum presents intermediate accumulation behaviour, and T. pratense exhibits a very conservative profile with arsenic, cadmium, and lead frequently below detection limits.

Collectively, white clover’s combination of regulated metal accumulation, strong rhizosphere stabilization, perennial stoloniferous growth, nitrogen fixation, and effectiveness against both inorganic and organic contaminants positions it as a versatile and low-risk species for integrated phytoremediation programmes. Nevertheless, indigenous South African Trifolium species remain substantially understudied at the rhizosphere level, representing a key knowledge gap for regional applications.

6.1 Rationale: the waste-to-resource paradigm

South Africa and other developing regions struggle with solid waste management, especially in rural and low-income communities where poor collection systems lead to illegal dumping and open burning. Turning organic waste into functional biosorbents supports both circular economy principles and waste reduction objectives. Chicken eggshells constitute approximately 11% of an egg’s weight and are composed predominantly of calcium carbonate (94–97%), with small amounts of magnesium carbonate, calcium phosphate, and organic material (Mignardi et al., 2020). Sugarcane bagasse, the fibrous residue after juice extraction, contains 32–45% cellulose, 20–32% hemicellulose, 17–32% lignin, and 1–9% ash, conferring a porous fibrous structure rich in hydroxyl groups suitable for adsorption and surface modification (Alokika and Singh, 2020).

6.2 Eggshell as a biosorbent: structure, mechanisms, and performance

Eggshell exhibits a complex hierarchical structure comprising cuticle, prismatic, palisade, and mammillary layers, with inner and outer membranes and a shell thickness of 280–400 μm featuring approximately 17,000 pores facilitating gas exchange (Mignardi et al., 2020). The predominant mineral phase is calcite (CaCO₃), providing reactive carbonate groups for metal binding. Functional groups, including hydroxyl (-OH), carboxyl (-COOH), and amino (-NH₂) groups, drive ion exchange, precipitation, and electrostatic attraction. While native shells may exhibit limited porosity, calcination, pyrolysis, or chemical modification substantially increases adsorption capacity ( Table 1).

Eggshell-mediated metal removal operates through multiple complementary mechanisms: (i) ion exchange, where Ca²⁺ from CaCO₃ exchanges with metal cations (Pb²⁺, Cd²⁺, Co²⁺) on surface sites; (ii) surface complexation through interactions with carboxyl and carbonate functional groups; and (iii) precipitation and co-precipitation, particularly in eggshell-derived hydroxyapatite systems where metal-phosphate minerals form (Mignardi et al., 2020; Harripersadth & Musonge, 2022) ( Table 1). The elevated pH induced by CaCO₃ dissolution promotes the formation of low-solubility metal hydroxides and carbonates ( Table 1).

6.3 Sugarcane bagasse: structure, mechanisms, and performance

Bagasse’s fibrous lignocellulosic structure forms a porous network that facilitates contaminant diffusion and entrapment. Abundant hydroxyl groups on polysaccharide chains serve as active sites for metal binding via surface complexation and physical adsorption. Chemical or thermal treatments expose more cellulose and hemicellulose while disrupting lignin resistance, increasing metal binding capacity. Bagasse-derived biochar demonstrates enhanced metal immobilization capacity, particularly when converted at 300–600 °C, developing a mesoporous structure (1.4–4.3 nm pore diameter) with surface areas of 180–198 m²/g (Jamilatun et al., 2022).

Comparative studies show that bagasse generally removes less Pb²⁺ than eggshell (31.45 vs. 277.8 mg/g), but performs equally well or better for Cd²⁺ (19.49 vs. 13.62 mg/g), highlighting the complementarity of combining both materials. Metal removal mechanisms on bagasse-based materials include complexation with oxygen-containing functional groups, π-electron interactions with aromatic structures, electrostatic attraction to charged surfaces, and mineral-associated precipitation.

6.4 Combined and co-pyrolyzed eggshell-bagasse systems

The combination of eggshell and bagasse exploits complementary adsorption mechanisms while valorizing two abundant waste streams. Eggshell provides CaCO₃-rich mineral phases that drive precipitation and ion exchange with strong affinity for Pb, Co, and As, while bagasse contributes lignocellulosic structures with hydroxyl and carboxyl groups particularly effective for Cd. Fixed-bed column studies demonstrated that all eggshell:bagasse mixing ratios (1:0 to 0:1) achieved 93–99% Pb²⁺ removal, with the 1:3 eggshell:bagasse mixture at 12 cm bed depth achieving 91% removal capacity at 28.27 mg Pb/g, while extending column service life through improved hydrodynamics (Harripersadth & Musonge, 2022).

True composite materials advance beyond physical mixtures: developed an eggshell-sugarcane bagasse composite that removed >90% of Cu²⁺ and Zn²⁺ from synthetic wastewater, with equilibrium data fitting the Freundlich isotherm and pseudo-second-order kinetics. Co-pyrolysis of eggshell and bagasse offers a route to engineered biosorbents with tunable properties; at 600 °C, the resulting biochar contains CaO distributed throughout an aromatic carbon matrix, yielding higher pH, greater surface area, and enhanced metal sorption through both carbon- and calcium-based mechanisms compared to physical mixtures alone (Jamilatun et al., 2022) ( Table 2).

6.5 Metal-specific removal efficacy and research gaps

Lead and cadmium removal by eggshell-bagasse systems represent the most extensively characterized application. Batch studies consistently demonstrate Pb removal capacities of 87–99.9% with maximum adsorption capacities up to 461 mg/g for eggshell-enhanced biochar composites. Cadmium removal efficiencies of 94–100% are achieved across various eggshell-based systems, with maximum capacities ranging from 125–265 mg/g.

Arsenic removal remains less thoroughly investigated. Calcined eggshells achieve maximum As(V) capacities of 91.05 mg/g, though phosphate ions compete strongly for binding sites. Fe₃O₄/bagasse-activated carbon magnetic composites exhibit As (III) capacity of 6.69 mg/g, outperforming plain bagasse but still modest compared to Pb/Cd systems. Mercury removal represents the most significant research gap; sparse data show low capacities for these specific materials, and methylmercury—the most toxic and bioaccumulative form—receives minimal attention. Future research should prioritize development of eggshell-bagasse carbons modified with iron or sulfur to improve mercury capture and stabilization.