To prepare the biochar, Wedelia trilobata was collected from roadsides near Hainan University, Haikou, Hainan, China. The collected material was manually cleaned to remove visible dust and washed thoroughly with ultrapure water to eliminate residual soil particles. The cleaned plants were then dried in an oven at 60°C for 24 hours until completely dehydrated. Once dried, the plants were cooled to room temperature and finely ground using a grinder to obtain a homogeneous powder, which was stored in clean Ziplock bags for later use. A portion of the powder was wrapped in tinfoil and placed in a crucible, which was then heated in a muffle furnace at 250°C for 2 hours (Yang et al., 2021). One of the methods for preparing biochar torrefaction is value-added at lower temperatures (200-300°C) and the temperature range belongs to the category of low-temperature waste heat (<300°C) (Lin et al., 2023). Besides, low-temperature waste heat recovery could be a potential energy source, and its development belongs to a relatively energy-saving technology (Chen et al., 2022; Lin et al., 2023). After heating, the crucible was left to cool down gradually to room temperature over 24 hours. Once cooled, the charred material was removed, reground to ensure uniformity, and stored in clean, sealed bags for further analysis.

The surface morphology of the prepared Wedelia trilobata biochar was examined using scanning electron microscopy (SEM, Verios G4 UC, Thermo Fisher (FEI), Czech Republic). Physical phase analysis and crystallinity determination were carried out using X-ray diffraction (XRD, Smart Lab, Rigaku, Japan). The chemical structure and functional groups were analyzed through Fourier transform infrared spectroscopy (FTIR, Thermo Nicolet iS50, Thermo Scientific, USA). Qualitative and quantitative elemental analyses, including elemental distribution and species content (with permissible relative error), were performed using energy-dispersive spectroscopy (EDS) integrated with SEM (Verios G4 UC, Thermo Fisher (FEI), Czech Republic). The specific surface area and porosity were analyzed using nitrogen adsorption-desorption isotherms at 77 K on a BET analyzer.

Cabbage seeds (Beijing No. 3, Brassica rapa L. ssp. pekinensis) were sourced from the Hebei Qingxian Vegetable Seed Breeding Base (http://www.xingyunzhongye.com). Prior to planting, the seeds were washed with DI water and subjected to a screening process to remove floating or damaged seeds. The selected seeds were surface sterilized by soaking in a 30% hydrogen peroxide solution for 15 minutes, followed by thorough rinsing three times with ultrapure DI water to ensure sterility (Yin et al., 2024). After rinsing, the seeds were placed on clean cheesecloth in plastic trays to maintain adequate humidity for germination. The trays were then incubated in a climate-controlled growth chamber set at 25/20°C (day/night temperature), with a relative humidity of 70% and a 14/10-hour light/dark cycle (Yin et al., 2024). Germination proceeded for 10 days, during that time the seeds were monitored to ensure proper growth conditions. Healthy and uniform seedlings were selected and transplanted into six-hole hydroponic boxes with a hole diameter of 20 mm and a total volume of 1 L, providing an optimal setup for cabbage cultivation.

The cabbage seedlings were carefully placed into the designated holes in the hydroponic boxes, ensuring that the roots were fully immersed in the Hoagland’s nutrient solution. To stabilize the plants and prevent floating or tilting, planting cotton was used to secure the seedlings. The hydroponic boxes with the plants were then placed in a climate-controlled incubator. The nutrient solution levels were monitored regularly to ensure that the roots remained submerged. Hoagland’s nutrient solution was replaced every 3-4 days to meet the growth requirements of the plants. Poorly growing plants were removed promptly to maintain uniformity, and plant health was monitored throughout the experiment. After ten days, cabbage seedlings with consistent growth were selected for further experimentation.

The experiment was designed with one control group (CK) and five treatment groups (T1, T2, T3, T4, and T5), each replicated three times. The experimental setup was as follows: Control group (CK): Hoagland’s nutrient solution (prepared by adding Hoagland’s reagent to ultrapure water according to standard guidelines). Pre-screened cabbage seedlings were transplanted into the solution, and the containers were labelled as (CK (Cr = 0 mg/L)) to indicate no chromium or biochar was added. T1: Hoagland’s nutrient solution was mixed with a Cr solution to achieve a final Cr concentration of 50 mg/L. The solution was stirred thoroughly, and pre-screened cabbage seedlings were transplanted. Containers were labelled as [T1 (Cr = 50 mg/L, BC = 0 g/L)]. T2: A mixture of chromium solution and Hoagland’s nutrient solution was prepared as in T1. Wedelia trilobata biochar was added to achieve a final concentration of 0.1 g/L. The solution was mixed well, and pre-screened cabbage seedlings were transplanted. Containers were labelled as [T2 (Cr = 50 mg/L, BC = 0.1 g/L)]. T3, T4, and T5: Following the same procedure as T2, Wedelia trilobata biochar concentrations were adjusted to 0.5 g/L, 1 g/L, and 3 g/L, respectively, with containers labelled as [T3 (Cr = 50 mg/L, BC = 0.5 g/L); T4 (Cr = 50 mg/L, BC = 1 g/L); and T5 (Cr = 50 mg/L, BC = 3 g/L)].

Biochar has demonstrated potential to enhance plant growth and nutrient uptake in hydroponic systems when its distribution is properly managed (Awad et al., 2017). One challenge is sedimentation, which can lead to non-uniform exposure of roots to biochar’s beneficial properties. To mitigate this, we adopted manual stirring as a practical strategy: each container was gently stirred twice daily for approximately 10 minutes, ensuring a uniform suspension and minimizing particle settling throughout the experimental period. This approach is consistent with prior studies suggesting manual agitation as an effective technique for maintaining homogeneous particle distribution in liquid systems (Meng et al., 2021). The experiment was conducted under controlled conditions in a climatic incubator, matching the environmental parameters used during cabbage cultivation: 25/20°C (day/night), 70% relative humidity, and a 14/10-hour light/dark cycle (Yin et al., 2024). This setup ensured consistent environmental conditions across all groups for reliable comparisons.

At the conclusion of the experiment, plants were thoroughly rinsed with ultrapure deionized (DI) water to remove any residual particulate matter. The plant surfaces were then blotted dry. Key growth parameters, including total plant biomass, root and shoot lengths, and root and shoot biomass, were measured and recorded. In addition, the pH and electrical conductivity (EC) of the nutrient solution in each hydroponic container were assessed.

To quantify chromium (Cr) concentrations in the plant roots and leaves, the procedure outlined in the GBT5009.123-2014 National Standard for Food Safety: Determination of Chromium in Foodstuffs by Atomic Absorption Graphite Furnace Method was followed. Total nitrogen content in the plant tissues was determined using an automatic nitrogen determination instrument. Total phosphorus and potassium concentrations were quantified using the methodology described by Bao Shidan in the third edition of Soil Agrochemical Analysis (China Agriculture Press). Plant samples underwent acid digestion with a mixture of strong acids, followed by the determination of calcium and magnesium content via atomic absorption spectroscopy.

Biochemical parameters were extensively analyzed in both shoot and root tissues, including malondialdehyde (MDA), hydrogen peroxide (H₂O₂), superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), proline, soluble proteins, and soluble sugars. Photosynthetic pigments, such as chlorophyll a, chlorophyll b, total chlorophyll, and carotenoids, were also quantified. The following assay kits were employed for these determinations: SOD activity was measured using the Superoxide Dismutase-WST-8 Method Activity Assay Kit (G0101W); CAT activity was assessed using the Catalase Activity Kit (G0105W); POD activity was measured with the Catalase Kit (G0107W); MDA content was quantified using the Malondialdehyde Content Kit (G0109W); soluble sugar content was determined with the Soluble Sugar Content Kit (G0501W); proline content was quantified using the Proline Content Assay Kit (G0111W); soluble protein content was analyzed with the Protein Content Assay Kit by Coomassie Brilliant Blue Method (G0417W); chlorophyll content was measured using the Chlorophyll Content Assay Kit (G0601W); and H₂O₂ content was quantified with the Hydrogen Peroxide Content Assay Kit (G0168W). These comprehensive analyses were performed to gain a detailed understanding of the effects of varying concentrations of Wedelia trilobata biochar on the growth and biochemical responses of cabbage plants exposed to chromium-contaminated nutrient solutions. This thorough evaluation aims to elucidate the potential of biochar to mitigate chromium-induced stress in hydroponic cultivation systems.

In this study, robust statistical methodologies were implemented to ensure the precision and reproducibility of the findings. Analysis of variance (ANOVA) was performed using IBM SPSS Statistics 23 (NY, USA) to compute the mean and standard deviation (± SD) of the dataset. To assess the influence of biochar application, the least significant difference (LSD) test was utilized, with a significance threshold set at p ≤ 0.05. Furthermore, multiple comparisons across treatment groups were conducted using Tukey’s Honestly Significant Difference (HSD) test, establishing a significance level of p < 0.05. Data visualization and graphical representation were executed using Origin 2019b (9.65) (Origin Lab Corporation, Northampton, USA). These analytical techniques facilitated a comprehensive evaluation of treatment differences, offering a scientifically rigorous foundation for assessing the effects of varying biochar concentrations on cabbage growth under chromium (Cr) stress.

Figure 1 presents scanning electron microscope (SEM) images of Wedelia trilobata biochar, highlighting its microstructure and surface morphology. After the pyrolysis process at 250 °C, it can be seen that the fiber structure of the plant powder has undergone obvious changes, resulting in obvious fragmentation, including cracks, collapses, surface roughness, and the formation of a clear pore structure ( Figure 1 ). Slow pyrolysis results in the release of volatile organic compounds, hemicellulose, and lignin, as well as shrinkage, melting, and cracking, thereby increasing the porosity of the material (Batista et al., 2018). At a scale of 40 μm ( Figure 1a ), the biochar’s outer contour exhibits a clear, typical tubular pore structure with rougher pores, and the cavity structure of the plant conduits is prominently visible, reflecting its porous characteristics. Upon magnification to 30 μm ( Figure 1b ), 10 μm ( Figure 1c ), and 5 μm ( Figure 1d ), the pores progressively become more regular and denser. The walls of the tubular structures thin out, and regularly arranged small pores appear on the inner surfaces of the tubes. Additionally, multi-diameter pores emerge as the pyrolysis progresses. These observations suggest that the microstructure of Wedelia trilobata biochar evolved during the pyrolysis process, resulting in the formation of a distinctive porous structure feature. Due to the presence of a highly porous structure, Wedelia trilobata biochar may exhibit a greater affinity for heavy metals (Li et al., 2017).

Scanning Electron Microscopy combined with Energy Dispersive X-ray spectroscopy (SEM-EDX) is a powerful technique for investigating both the morphological and chemical characteristics of materials (Allegretta et al., 2022). Supplementary Figure S1 illustrates the results of SEM-EDX analysis for Wedelia trilobata biochar. ( Supplementary Figure S1a ) displays the overall elemental profile spectra corresponding to the localized area in ( Supplementary Figure S1b ), which provides a detailed view of the biochar sample’s surface. ( Supplementary Figures S1c, d ) depict the spatial distribution of carbon (C) in yellow and oxygen (O) in green within the analytical scanning range. The total elemental distribution map at a scale of 100 μm reveals the presence of carbon (C), oxygen (O), and aluminum (Al) in Wedelia trilobata biochar. Quantitatively, the weight percentages of C and O are 67.0% and 32.4%, respectively, at a dissociation cross-section (σ) of 1.0, while the weight percentage of Al is measured at 0.5% with a dissociation cross-section (σ) of 0.2. In general, the C content of biochar is inversely proportional to the biochar yield, that is, the higher the C content, the lower the biochar yield (Mahimairaja and Shenbagavalli, 2012). At the same time, the production of biochar by pyrolysis is a carbonization process in which the carbon content increases with temperature while the oxygen and hydrogen content decreases (Angin et al., 2013). Carbon is the primary element in condensed aromatic structures, which dominate the organic phase of biochar; while O is the key element in many polar organic functional groups on biochar surfaces, which influence biochar reactivity (Bakshi et al., 2020).

Fourier Transform Infrared Spectroscopy (FTIR) is an effective technique for analyzing functional groups in biochar, with spectra recorded in the range of 500-4000 cm^-1. As shown in Figure 2 , the broad absorption peak at 3337.211 cm^-1 corresponds to O-H stretching vibrations, indicating the presence of hydroxyl groups derived from glucose molecules in cellulose and hemicellulose, as well as phenolic functional groups in lignin (Chen et al., 2024; Pasumarthi et al., 2024). Peaks at 2924.520 cm^-1 and 2855.095 cm^-1 are associated with symmetric C-H stretching vibrations, revealing the presence of saturated hydrocarbon chains and aliphatic hydrocarbons, respectively (Gu et al., 2021; Pasumarthi et al., 2024). The peak at 1591.949 cm^-1 reflects the stretching vibrations of C=C double bonds, indicating the presence of aromatic structures. As shown in Figure 2 , the peak at 1416.459 cm^-1 is linked to C=N stretching vibrations, which may correspond to primary amides, while the peak at 1052.943 cm^-1 is associated with C=O stretching vibrations, typically found in alcohols, ethers, or esters (Wang et al., 2021). In addition, the peak at 781.029 cm^-1 indicates the presence of aromatic compounds (Dalavi and Patil, 2016). These diverse functional groups, including hydroxyl, aliphatic, aromatic, and oxygen-containing groups, provide a strong basis for the adsorption properties of Wedelia trilobata biochar.

X-ray Diffraction (XRD) was employed to identify the crystalline phases present in the Wedelia trilobata-derived biochar sample, as shown in Figure 3 . The XRD pattern exhibits several sharp and well-defined peaks, indicating a high degree of crystallinity in the material (Nanda et al., 2013) (Pasumarthi et al., 2024). Prominent diffraction peaks were observed at 2θ values of approximately 24.482°, 28.345°, 40.507°, 50.169°, 58.640°, and 66.381°, which correspond to the (111), (200), (220), (222), (400), and (420) crystal planes, respectively. These reflections closely match the standard pattern of potassium chloride (KCl), as referenced by the Powder Diffraction File PDF#41-1476. The presence of KCl may be attributed to the retention of native plant mineral components during pyrolysis, potentially enhancing the ionic exchange capacity and nutrient availability of the biochar when applied in hydroponic or soil systems.

The nitrogen adsorption-desorption isotherm and Barrett-Joyner-Halenda (BJH) pore size distribution curve of Wedelia trilobata-derived biochar (WBC) are presented in ( Figures 4a, b ), respectively. The isotherm exhibits a typical Type IV hysteresis loop, which is characteristic of mesoporous materials according to the IUPAC classification. This loop confirms the presence of mesopores and capillary condensation phenomena, indicating a porous structure well-suited for adsorption applications. The gradual increase in adsorption quantity with relative pressure (P/P_o) and the sharp rise near P/P_o ≈ 1.0 suggest the presence of a combination of micro- and mesopores. The pore size distribution inset ( Figure 4b ) reveals that the majority of the pores fall below 10 nm, further supporting the mesoporous nature of WBC. The narrow distribution centered around ~3-5 nm indicates uniform pore development, which enhances surface accessibility for metal ions like Cr(VI). These findings are consistent with earlier studies highlighting the significance of surface area and mesoporosity in determining the sorption efficiency of biochar materials (Razali and Kamarulzaman, 2020, 2022; Wening and Herdyastuti, 2021). Thus, WBC’s structure-function relationship underscores its potential as a promising, sustainable amendment for heavy metal remediation and agricultural applications.

Potassium is one of the important nutrients for plant growth and metabolism (Kumar et al., 2020). Potassium is present in biochar in various forms, with water-soluble and non-water-soluble potassium being more common. Related studies have pointed out that preparation conditions such as pyrolysis temperature have a significant effect on the morphology and solubility of potassium present in biochar (Xiu et al., 2023). Biochar prepared at lower temperatures increases the proportion of water-soluble and exchangeable potassium (Bilias et al., 2023). It is widely believed that biochar contains large amounts of water-soluble potassium (Hossain et al., 2020). Water-soluble potassium dissolves rapidly in soil solution, providing a short-term source of easily absorbed potassium to meet the rapid needs of plants at specific growth stages. However, it is prone to migration in the soil solution and water loss to deeper layers of the soil or to the surrounding environment, resulting in reduced utilization (Cheng et al., 2023). In the study by (Kong et al., 2014) the water-soluble potassium content in biochar reached at least 47% of the total potassium content. The presence of insoluble potassium in biochar increases its slow-release capacity and reduces the leaching of potassium (Piash et al., 2021).

Insoluble potassium gradually releases potassium ions for plant uptake through slow dissolution or reaction with soil substances in the soil, so that the biochar can provide potassium to plants stably for a long time and reduce the problem of frequent fertilization due to rapid loss of potassium. From XRD analysis, the crystal structure of potassium-related minerals (e.g., KCl) in biochar is closely related to the solubility and release characteristics of potassium. The crystal structure integrity, the nature of crystal faces and the crystal grain size all affect the dissolution and release of potassium in biochar. When the crystal structure of potassium minerals in biochar is stable, potassium dissolution needs to overcome the higher energy barrier, showing a lower solubility and a slow-release rate.

The correlation matrix analysis highlighted strong interrelationships among physiological, biochemical, and growth traits in Brassica rapa under chromium (Cr) stress and biochar treatments, revealing that shoot weight (WeightS) was positively correlated with shoot length, macronutrient levels (N, P, K), and photosynthetic pigments (TChl, Chla, Chlb) ( Figure 9 ), suggesting that improved nutrient availability and photosynthetic efficiency contributed significantly to biomass accumulation consistent with previous findings on biochar-enhanced nutrient uptake under heavy metal stress (Verlinden et al., 2018; Chtouki et al., 2022). Antioxidant enzymes (SODR, CATR, PODR) were positively intercorrelated, indicating coordinated oxidative defense, though their weak or negative correlation with shoot biomass suggests they act as stress indicators rather than growth promoters, while oxidative markers like malondialdehyde (MDAS) and hydrogen peroxide (H₂O₂) were negatively associated with growth traits, confirming their role in stress-related damage. Additionally, osmolytes such as proline and soluble sugars showed moderate positive correlations with antioxidants and pigments, hinting at their dual role in ROS detoxification and photoprotection under Cr stress. Secondary nutrients like calcium and magnesium also correlated positively with shoot biomass and chlorophyll content, reinforcing their contribution to structural and metabolic resilience (Jin et al., 2023), while phosphorus emerged as a key driver of growth recovery under Cr exposure. Collectively, these correlations support the conclusion that Wedelia trilobata-derived biochar enhances nutrient assimilation and modulates stress-responsive metabolic pathways, reducing oxidative damage and promoting growth, thereby positioning it as an effective and sustainable amendment for Cr-contaminated hydroponic systems.

Principal component analysis (PCA) was conducted to evaluate the multivariate variation among treatments, with PC1 and PC2 accounting for 43.07% and 28.67% of the total variance, respectively (cumulative 71.74%). The PCA biplot revealed clear separation between treatments, with distinct clustering patterns and labeled centroids indicating that higher WBC concentrations (T4 and T5) were associated with more favorable physiological and biochemical traits compared to the chromium-only treatment (T1). The Cr-only treatment (T2) clustered far from the control (T1) ( Supplementary Figure S7 ), reflecting the detrimental effects of Cr toxicity on plant health, such as reduced biomass, impaired nutrient uptake, and elevated oxidative stress. In contrast, treatments with Wedelia trilobata-derived biochar (T3-T5) progressively shifted toward the control cluster, suggesting a dose-dependent alleviation of Cr-induced stress through improvements in antioxidant enzyme activity, photosynthetic pigment content, and nutrient assimilation. These results support the role of biochar in mitigating abiotic stress by modulating plant metabolic pathways and improving soil or hydroponic nutrient dynamics, consistent with previous findings (Barzana et al., 2021). The PCA thus confirms biochar’s potential as a sustainable amendment to restore plant functionality under heavy metal stress.