Exploring the growth and biochemical response of canola varieties to cadmium and lead contamination

Abstract

Heavy metal (HM) contamination of agricultural soils by cadmium (Cd²⁺) and lead (Pb²⁺) pose a serious risk to the crop productivity and food security. However, a workable solution is required to reduce the impact of HM contamination in soils and improve productivity. Therefore, the current study aimed to investigate the tolerance of different canola varieties against the contamination of Cd²⁺ and Pb²⁺ under field conditions. The experimental site was previously contaminated with Cd²⁺ (6ppm) and Pb²⁺ (600ppm) where canola varieties were cultivated under the randomized complete bock design (RCBD) with three replicates of each treatment. The current study evaluated the growth, biomass accumulation, chlorophyll content and biochemical attributes of canola varieties. The results of this study revealed significant variability among varieties in response to the metal stress. Under Cd²⁺ contaminated conditions Oscar attained highest plant height (71.42 cm), root length (33.62 cm), shoot length (34.25 cm) however under Pb²⁺ contaminated soil Super Raya showed maximum plant height (39.43 cm), root length (13.45 cm) and shoot length (26.34). In contrast varieties like rainbow and Sandal Canola showed inhibited growth with a significant reduction in the root, shoot length and decline in the antioxidant enzyme activity. In addition, highest superoxide dismutase (SOD) 6.63 U/g Protein, catalase (CAT) 2.87 U/g Protein, peroxidase (POD) 43.26 U/g Protein, Malondialdehyde (MDA) 76.10 nmol /g FW, Ascorbate Peroxidase (APX) 74.20 U/g Protein, Ascorbic Acid (ASA) 70.52 µg/g FW and Glutathione (GSH) 73.71 µmol/g FW activity by Oscar under Cd²⁺ contaminated soil. In Pb²⁺ contaminated soil CONII represent its potential by showing highest SOD (5.78 U/g Protein), CAT (2.81 U/g Protein), POD (52.57 U/g Protein), MDA (72.77 nmol /g FW), APX (68.24 U/g Protein), ASA (74.49 µg/g FW) and GSH (72.76 µmol/g FW) activity. In conclusion, canola varieties exhibit differential tolerance to Cd²⁺ and Pb²⁺ contamination under field conditions, with certain varieties i.e., Oscar and Super Raya demonstrating superior growth and enhanced antioxidant defense mechanisms, making them suitable candidates for cultivation in heavy metal–contaminated soils.

Introduction

Cadmium (Cd²⁺) and Lead (Pb²⁺) are the major sources of heavy metal (HM) contamination in the soil and water [1]. The aforementioned contamination is an environmental issue that poses a health concern worldwide. These metals are non-essential plant elements that become toxic when their availability exceeds the permissible level [2]. The basic plant life-sustaining physiological processes, such as photosynthesis, nutrient uptake, and eventually growth, can be affected or even ceased altogether by the higher concentration of such metals [3]. The reduced and damaged physiological processes ultimately result in reduced yields, degrade the soil, and contaminate groundwater, which can cause serious health issues in humans. Along with other effects, the HM moves down the food chain, affecting consumers at all stages [4]. The contamination of HM, especially Cd²⁺ and Pb²⁺, in soil is due to a combination of anthropogenic and natural activities, such as improper industrial waste management, wastewater irrigation, and atmospheric deposition [5–7]. Awareness of HM contamination in agricultural soils has become more widespread in recent years. Many studies have reported elevated levels of Cd²⁺ and Pb²⁺ above safe limits set by the World Health Organization (WHO), and these activities are influenced by anthropogenic activities [8]. A recent study in Faisalabad reported the presence of nickel (Ni²⁺), Cd²⁺, Pb²⁺, and zinc (Zn²⁺) in agricultural soils, with concentrations of Pb ranging from 7.54 to 41.45 mg/kg [9] The toxic levels of HM in contaminated soils can be up taken by plants through the process of bioaccumulation and can be transported down the food chain and may enter into the human body through food consumption [10]. The World Health Organization has issued an advisory regarding the risks of HM contamination in the agricultural sector. It suggests thresholds of 85 mg/kg for Pb²⁺ and 1.30 mg/kg for Cd²⁺, indicating that levels above these limits would have negative effects on cereal, legumes, pulses, and field crops, as well as human health. WHO has suggested adopting sustainable agricultural practices to mitigate contamination of agricultural soils that has already occurred, while also ensuring food security [11]. The United States Environmental Protection Agency (US EPA) has also issued an advisory regarding elevated levels of Pb²⁺ and Cd²⁺. EPA suggested HM contamination caused by Pb²⁺ and Cd²⁺ has the potential to cause severe health effects, including neurotoxic effects, kidney damage, and even cancer [12]. The Toxic effects of Pb²⁺ and Cd²⁺ have been studied in detail in laboratories, but their effects in real field conditions still need proper evaluation [13, 14]. Therefore, the mitigation of HM contamination is the need of the time, which can be achieved through microbes, the use of some chemicals, adsorbents, and plant uptake. Among plants canola is very effective in uptake of HM from the soil which have been well documented in our previous research [15], while the performance of canola in field conditions is lacking where an integrated influence of soil physiochemical properties, climate, and all other factors provides a completely different scenario, which has not been thoroughly investigated. Moreover, field conditions involve additional complexities, and plant responses can differ significantly from those observed under controlled environments [16]. Canola (Brassica napus) has shown promising and valuable growth in a laboratory-controlled setting [15, 17], but its performance in field conditions in HM soil has yet to be evaluated. The study provides field-level screening of canola cultivars on a prolonged wastewater-contaminated soil, leading to the identification of cultivars with practical tolerance under real-world field conditions. The current research work aimed to screen out nine canola varieties at two different sites (a) naturally contaminated site by Cd²⁺ and Pb²⁺ with at least a 10-year history of untreated wastewater irrigation (b) uncontaminated site with similar physiochemical properties. The studied concentrations of both the HM were set based on the exposure level as reported by various studies to evaluate the response of canola at a specific contaminated site. The experiment was conducted in randomized complete block design (RCBD) with three replicates of each treatment, while the study focused on the assessment of morphological traits, physiological status of canola varieties and the biochemical stress response through the measurement of superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), ascorbate peroxidase (APX), malondialdehyde (MDA), ascorbic acid (ASA), and reduced glutathione (GSH) to compare the tolerance of canola varieties against Cd²⁺ and Pb²⁺ stress under field conditions. The results of this study provided an evidence base for the selection of canola varieties suitable to cultivate under Cd²⁺ and Pb²⁺ stress conditions.

Materials and methods

Description of study site and experimental setup

A naturally contaminated site was selected for field screening of Canola varieties to assess their uptake of HM (Cd²⁺ and Pb²⁺). At first, soil samples were collected from the selected site and brought to the laboratory for further processing. The soil samples were prepared using the AB-DTPA extraction procedure and analyzed using an atomic absorption spectrophotometer [18]. The selected site had a history of wastewater irrigation for 10 years, while the soil sample contained Cd²⁺ (6 mg/kg) and Pb²⁺ (600 mg/kg). Nine varieties of canola were selected for the experiment on Cd²⁺ and Pb²⁺ toxicity, which was previously screened. The selected varieties for Cd²⁺ included CONIII, Rainbow, Oscar, CONII, and Sandal Canola, while the varieties in the Pb²⁺ trial were CONII, Super Raya, Dunkeld, and Super Canola. The varieties were collected from the Oilseeds Research Institute and the Ayub Agricultural Research Institute in Faisalabad (31°24’15.0"N 73°03’04.5"E). In this study, the selected varieties were irrigated through the tube well. The treatments for Cd²⁺ included CONIII, Rainbow, Oscar, CONII, Sandal Canola, CONIII + 6 mg/kg Cd²⁺, Rainbow + 6 mg/kg Cd²⁺, Oscar + 6 mg/kg Cd²⁺, CONII + 6 mg/kg Cd²⁺ and Sandal Canola + 6 mg/kg Cd²⁺. In contrast, for Pb²⁺ the treatments were CONII, Super Raya, Dunkeld, Super Canola, CONII + 600 mg/kg Pb²⁺, Super Raya + 600 mg/kg Pb²⁺, Dunkeld + 600 mg/kg Pb²⁺ and Super Canola + 600 mg/kg Pb²⁺. In contrast, the control plots were selected separately, and Cd and Pb toxicity was not detected (ND), while the soil had the same texture and similar pH and ECe (Table 1). The experiment was conducted using a randomized complete block (RCBD) design with three biological or technical replicates per treatment.

Table 1.

Physicochemical properties of both the experimental sites (control vs. contaminated)

Parameter	Unit	Contaminated Site	Control Site
ECe	dSm− 1	2.87	2.53
pH	-	8.10	8.04
Organic matter	%	0.49	0.53
Texture	-	Clay	Clay
Saturation	%	36	38
Cd	mg/kg	6	*ND
Pb		600

*ND Not detected

Crop husbandry

The land preparation involved plowing, leveling, and application of organic matter content (OMC) with balanced nitrogen (N), phosphorus (P), and potassium (K) at the ratio of 35:35:25 kg/acre, as per the standard guidelines of the Government of Punjab Agriculture Department, Pakistan. It was cultivated during mid-October by using the mentioned varieties for each treatment. For irrigation, the treatment plan called for monitoring soil moisture to maintain it at 70% field capacity, using a moisture meter (YIERYI 4 in 1; Shenzhen, Guangdong Province, China). The moisture meter was calibrated to achieve an optimized moisture level within the Normal range of 65–70% of field capacity. In addition, irrigation was applied at the time of flowering and pod development. Weeds were controlled through manual weeding, while recommended pesticides were applied to control aphids. The crop was harvested when the pods turned yellow, which was around 65–70 days after sowing.

Data collection

The growth parameters like plant height (cm), root length (cm), shoot length (cm), root fresh weight (g), root dry weight (g), shoot fresh weight (g), shoot dry weight (g), leaves fresh weight (g) and leaves dry weight (g) were measured at the time of harvesting. Plant height and root and shoot lengths were measured with a measuring tape, and all samples were weighed on an analytical balance. In addition, the fresh leaves were preserved for analysis of antioxidant and chlorophyll content. At first, protein was determined in the enzyme extract as per the Bradford method using a BSA standard curve, and the enzymatic activities were expressed as U g^{− 1} protein. The biochemical analysis of leaf samples included measuring the activities of peroxidase (POD), catalase (CAT), and superoxide dismutase (SOD), and the concentration of malondialdehyde (MDA) using standard protocols. At first, POD was measured by incubating the sample for 5 min in a phosphate buffer (50 mM) at pH 7.0; afterward, sulfuric acid (H2SO4) was added, and the optical density (OD) was measured with a spectrophotometer at 470 nm [19]. The CAT activity was measured by taking the sample into a falcon tube and then 2.9 mL of phosphate buffer (50mM) at pH 7.0 was added to the solution along with 1.0 mL of H₂O₂ while the reduction in the concentration of H₂O₂ was measured by using a spectrophotometer at 240 nm [20]. In addition, the activity of SOD was also measured through the spectrophotometer, inhibiting the photochemical reaction between nitro blue tetrazolium (NBT) and superoxide radicals in a phosphate buffer mixture (50 mM) at pH 7.0, and monitoring absorbance at 560 nm [21]. The MDA content was measured by heating a mixture of 2.0 mL of TBA (0.67%) and 1 mL of acetic acid (20%) at 95–100 °C for 20 min, then determining the absorbance on a spectrophotometer at 532 nm [22]. Similarly, chlorophyll content was measured by cutting the leaves into small pieces and homogenizing them in a mortar and pestle with 80% acetone. The homogenized samples were further filtered through Whatman filter paper No. 42 to remove insoluble material and debris. Afterward, the filtrate was transferred into the test tube, followed by dilution with acetone to bring the volume to 5 mL. The absorbance of samples was measured on the spectrophotometer at two wavelengths (i) chlorophyll a = 663 nm and (ii) chlorophyll b = 645 nm, while the content was measured through Eqs. 1, 2, and 3.

1

2

3

Whereas W is the weight of the sample and V is the final volume of the sample after preparation.

The analysis of N, P, K and Na was performed after sample digestion with sulfuric acid and a digestion mixture (concentrated perchloric acid (HClO4) and concentrated nitric acid (HNO3) in a 2:1 ratio) for P, K, and Na [23]. After digestion, the samples of K and Na were measured on the flame photometer, while N samples were subjected to a distillation process, and P was measured on a spectrophotometer [24].

Statistical analysis

The collected data for the Cd²⁺ and Pb²⁺ trials were analyzed separately, as the field trials were conducted independently with different varieties. The effect of treatments was assessed through the two-way ANOVA and Fisher’s LSD test at a significant level of p ≤ 0.05 [25]. In addition, the visualization and graphical representation were conducted through OriginPro software [26].

Results

Influence of HM contamination on canola growth attributes

Plant height

Plant height showed significant variation among treatments in both field experiments. Under controlled conditions, Oscar variety outperformed Sandal Canola. Oscar variety showed the tallest plant of all other varieties, averaging 69.04 cm, followed by CONIII, which averaged 63.66 cm; Rainbow, 59.19 cm; CONII, 53.43 cm; and Sandal Canola, the smallest plant height of 48.94 cm. Under Cd (6 ppm) toxicity, Oscar produced the tallest plant and was the least performer. The tallest plant had an average height of 71.42 cm, followed by CONIII (58.34 cm), Rainbow (49.13 cm), and CONII (42.14 cm). Sandal Canola had the lowest plant height at 33.17 cm. This negative trend in plant height observed across all canola varieties strongly highlights the effect of Cd²⁺ contamination on canola height, while the degree of stress varied among species (Fig. 1a). The response to Pb²⁺ toxicity (600 ppm) in the field was significantly different among the treatments. Under the control conditions of this trial, Super Raya was the most tolerant variety, with the tallest plant height (average 39.43 cm) in both the control and contaminated field, compared to the lowest height. Furthermore, CONII was the second top performer, with a height of 34.98 cm, followed by Super Canola at 30.64 cm and Dunkled at 27.46 cm. The Super Raya managed to retain its superiority among other species having the tallest plants at an average of 39.39 cm even under Pb induced stress conditions (Fig. 1b).

Fig. 1.

Effect of Cd²⁺ (6 ppm) and Pb²⁺ (600 ppm) contamination on growth attributes of different canola varieties: (a) plant height under Cd²⁺ stress, (b) plant height under Pb²⁺ stress, (c) shoot length under Cd²⁺ stress, (d) shoot length under Pb²⁺ stress, (e) root length under Cd²⁺ stress, and (f) root length under Pb²⁺ stress. Different letters on column bars (means of 3 replicates ± SD) representing the significant changes at the p ≤ 0.05 compared by Tukey’s HSD test

Shoot length

Oscar variety outperformed and resulted in tallest shoot (34.25 cm) followed by CONIII (31.96 cm), Rainbow (30.97 cm), CONII (28.35 cm), and Sandal Canola reported the shortest shoots (26.84 cm). Under controlled conditions, Oscar variety outperformed Sandal Canola. Canola varieties showed a consistent increment trend under the Cd²⁺ contaminated soil (Fig. 1c). Contrastingly, field conditions under Pb²⁺ (600ppm) contamination represented that Pb²⁺ toxicity also effect the above ground plant portion, while the growth under the controlled conditions represented a significant variation among canola varieties. Super Raya had the longest shoots, with an average length of 26.34 cm, followed by CONII, with an average of 22.66 cm; however, Super Canola showed an average of 20.17 cm, and Dunkled showed an average of 17.82 cm. This variation can be traced to differences in inheritance among species. The shoot length increased in all tested canola varieties after exposure to Pb²⁺ toxicity under natural soil conditions. The least toxic effect of Pb²⁺ may help conclude that canola plants showed some degree of tolerance and specific adaptations to mitigate Pb2+-induced toxicity (Fig. 1d).

Root length

The normal growth plots under Cd2+ (6 ppm) contamination field conditions showed that Oscar had 57.8% higher results than Sandal Canola. Oscar produced the longest roots (34.79 cm) followed by CONIII (31.70 cm), Rainbow (28.22 cm), CONII (25.08 cm) and Sandal Canola (22.09 cm). However, exposure to Cd²⁺ resulted in reduction of root length among all the tested varieties. The Oscar variety still displayed the longest roots (33.62 cm) as compared to the least performing variety. Furthermore CONIII (27.91 cm), Rainbow (24.40 cm), CONII (15.62 cm), and Sandal Canola represented the shortest roots 12.60 cm (Fig. 1e). Pb²⁺ (600 ppm) contaminated soils revealed that, under controlled conditions, Super Raya had the longest root at 13.45 cm, followed by CONII at 12.58 cm, Super Canola at 11.18 cm, and Dunkled at 10.31 cm. The length of Super Raya was higher than that of Dunkled; however, the difference may be due to an inherited difference in growth patterns. Super Raya displayed a slight gain in root length with an average of 13.82 cm under Pb²⁺ stress, while CONII decreased root length to 11.77 cm, however Super Canola and Dunkled exhibited minor variations to 11.39 cm and 9.84 cm, respectively (Fig. 1f).

Shoot fresh weight

Shoot fresh weight (g) was found significant among varieties of canola in control and Cd²⁺ (6ppm) contaminated plots. The maximum shoot fresh weight was observed in Oscar (9.32 g), followed by CONIII (8.45 g), Rainbow (8.12 g), CONII (4.75 g), and Sandal Canola (4.45 g) in the control plots, with a difference of 109.8% between Oscar and Sandal Canola. While the Oscar (8.85 g) also outperformed under Cd²⁺ contaminated treatment followed by CONIII (7.85 g), Rainbow (5.27 g), CONII (4.32 g), and Sandal Canola with the lowest shoot dry weight of 2.87 g among Oscar and Sandal Canola (Fig. 2a). Contrastingly under controlled conditions of Pb²⁺ experiment Super Raya outperformed showing a highest shoot fresh weight (6.95 g) followed by CONII (6.21 g), Super Canola (4.32 g) and Dunkled (3.15 g) among Super Raya and Dunkled. These differences highlight inherent distinctions in the capacity of studied canola varieties to accumulate dry biomass under optimal growth conditions. Under Pb²⁺ (600ppm) contaminated soils all varieties decreased the dry weight as Super Raya exhibited higher fresh weight as compared to Dunkled, while having a maximum fresh weight of 6.25 g followed by CONII (6.00 g), Super Canola (4.32 g), and Dunkled (2.9 g) respectively (Fig. 2b).

Fig. 2.

Effect of Cd²⁺ (6 ppm) and Pb²⁺ (600 ppm) contamination on shoot biomass of different canola varieties: (a) shoot fresh weight under Cd²⁺ stress, (b) shoot fresh weight under Pb²⁺ stress, (c) shoot dry weight under Cd²⁺ stress, and (d) shoot dry weight under Pb²⁺ stress. Different letters on column bars (means of 3 replicates ± SD) representing the significant changes at the p ≤ 0.05 compared by Tukey’s HSD test

Shoot dry weight

The maximum shoot dry weight was observed in Oscar (4.69 g) followed by CONIII (4.50 g), Rainbow (4.13 g), CONII (3.86 g), and Sandal Canola (3.46 g) under the control plots. While the Oscar (3.63 g) also outperformed then the least performing variety. CONIII (3.31 g) was the second most promising canola followed by Rainbow (2.84 g), CONII (2.44 g), and Sandal Canola with the lowest shoot dry weight of 1.95 g (Fig. 2c). Similarly, under controlled conditions versus Pb²⁺ contamination field experiment, Super Raya performed better, showing the highest shoot dry weight (2.69 g), followed by CONII (2.19 g), Super Canola (1.45 g), and Dunkled (0.80 g). These differences highlight inherent distinctions in the capacity of studied canola varieties to accumulate dry biomass under optimal growth conditions. Upon exposure to Pb²⁺ (600 ppm) contaminated soils, all varieties decreased their dry weight, with Super Raya exhibiting the highest dry weight of 2.27 g, followed by CONII (1.14 g), Super Canola (0.94 g), and Dunkled (0.62 g) (Fig. 2d).

Root fresh weight

Under the control conditions, Oscar variety had the highest root fresh weight (2.89 g), followed by CONIII (2.75 g), Rainbow (2.58 g), CONII (2.39 g), and Sandal Canola (2.17 g). In contrast the root fresh weight of varieties under Cd²⁺ (6ppm) stress conditions decreased the root fresh weight while similar order as of non-contaminated soils were observed with Oscar having highest root fresh weight (1.90 g), followed by CONIII (1.82 g), Rainbow (1.72 g), CONII (1.50 g), and Sandal Canola with the lowest root fresh weight of 1.32 g, while showing a difference of 44.3% among Oscar and Sandal Canola (Fig. 3a). In the case of Pb²⁺, the Super Raya variety had the highest root fresh weight (2.83 g), followed by CONII (2.37 g), Super Canola (2.25 g), and Dunkled (1.89 g). These slight variations along the lineup of different canola varieties can be possible to inherit differences in growth patterns. Under the Pb²⁺ contaminated soil canola varieties showed a slight decrease in the root fresh weight. The Super Raya outperformed as compared to the Dunkled. It exhibited a marginal reduction in root fresh weight to 2.79 g, while CONII, Super Canola, and Dunkled showed comparable decreases to 2.38 g, 2.14 g and 1.80 g respectively (Fig. 3b).

Fig. 3.

Effect of Cd²⁺ (6 ppm) and Pb²⁺ (600 ppm) contamination on root biomass of different canola varieties: (a) root fresh weight under Cd²⁺ stress, (b) root fresh weight under Pb²⁺ stress, (c) root dry weight under Cd²⁺ stress, and (d) root dry weight under Pb²⁺ stress. Different letters on column bars (means of 3 replicates ± SD) representing the significant changes at the p ≤ 0.05 compared by Tukey’s HSD test

Root dry weight

Figure 3 represented the root dry weight (g) canola varieties which showed a similar order of variation for root dry weight under control conditions as Oscar had maximum root dry weight of (0.65 g) followed by CONIII (0.57 g), Rainbow (0.44 g), CONII (0.24 g), and Sandal Canola (0.16 g). While under Cd²⁺ (6 ppm) contaminated conditions, Oscar variety still outperformed (0.64 g), followed by CONIII (0.55 g), Rainbow (0.43 g), CONII (0.35 g), and Sandal Canola (0.25 g) among Oscar and Sandal Canola (Fig. 3c). In the case of Pb²⁺ (600 ppm), the varieties under control showed significant differences in root dry weight. Super Raya exhibited the highest root dry weight (0.63 g) followed by CONII (0.50 g), Dunkled (0.38 g) and Super Canola (0.20 g). When the underground dry biomass was monitored under Pb²⁺ (600 ppm) contamination, the variety Super Raya showed a lower weight (0.56 g), while CONII, Super Canola, and Dunkled displayed comparable reductions, with results of 0.37 g, 0.27 g, and 0.14 g, respectively (Fig. 3d).

Leaves fresh weight

Oscar again proved to have the highest leaves fresh weight, the CONIII (5.41 g), Rainbow (5.15 g), CONII (4.95 g), and Sandal Canola showed lowest leaves fresh weight (4.30 g). Similarly, varieties exposed to Cd²⁺ (6ppm) the leaves fresh weight had an overall reduction across all the varieties, while still the Oscar variety maintained the highest leaves fresh weight (5.84 g) followed by CONIII (4.76 g), Rainbow (3.79 g), CONII (3.43 g), and Sandal Canola (2.91 g) (Fig. 4a). Under normal field conditions, a distinct series of variation was observed, with Super Raya exhibiting the highest leaf fresh weight (2.89 g), followed by CONII (2.67 g), Super Canola (2.02 g), and Dunkled (1.42 g). Similarly, under Pb²⁺ (600 ppm), Super Raya maintained its top spot (2.53 g), while CONII, Super Canola, and Dunkled showed significant reductions, with leaf weights of 2.10 g, 1.30 g, and 1.04 g, respectively (Fig. 4b).

Fig. 4.

Effect of Cd²⁺ (6 ppm) and Pb²⁺ (600 ppm) contamination on leaf attributes of different canola varieties: (a) leaf fresh weight under Cd²⁺ stress, (b) leaf fresh weight under Pb²⁺ stress, (c) leaf dry weight under Cd²⁺ stress, (d) leaf dry weight under Pb²⁺ stress, (e) number of leaves under Cd²⁺ stress, and (f) number of leaves under Pb²⁺ stress. Different letters on column bars (means of 3 replicates ± SD) representing the significant changes at the p ≤ 0.05 compared by Tukey’s HSD test

Leaves dry weight

Oscar canola consistently performed with a maximum leaves dry weight (1.82 g) and similar to other parameters CONIII was remained after Oscar by resulting 1.63 g followed by Rainbow with a weight of 1.51 g which reduced in CONII having a weight of 1.27 g and Sandal Canola represented the lowest leaves dry weight of 1.11 g. Under Cd²⁺ contaminated sites overall decrease in leaves dry weight was observed while it resulted in a similar order as of any other parameter with Oscar variety still maintaining the highest leaves dry weight (1.81 g) followed by CONIII (1.41 g), Rainbow (1.26 g), CONII (1.00 g), and Sandal Canola (0.50 g) (Fig. 4c). In the Pb²⁺ the controlled conditions represented a better result as compared to contaminated sites. The control conditions showed variation in leaf dry weight across all varieties, with Super Raya (0.65 g) showing the highest value, followed by CONII (0.54 g), Super Canola (0.36 g), and Dunkled (0.27 g). The cause of these differences in leaves dry weight can be traced back to differences in inherited growth habits, when these test varieties were grown in Pb²⁺ stressed environment. Super Raya showed an increase in leaves dry weight (0.71 g), while CONII, Super Canola, and Dunkled displayed varying responses with leaves dry weights of 0.59 g, 0.43 g and 0.25 g (Fig. 4d).

Number of leaves

Number of leaves plays a crucial role in attaining a higher photosynthetic rate, which was appraised by chlorophyll Oscar variety outperforming (13.78), followed by CONIII (12.28), Rainbow (11.50), CONII (10.98), and Sandal Canola (10.10). Contrastingly, the contaminated plots showed that the average number of leaves decreased across all varieties, while a similar order was observed. Oscar resulted in highest number of leaves under Cd²⁺ contamination (11.77) followed by CONIII (10.61), Rainbow (9.51), CONII (7.18), and Sandal Canola (5.84) (Fig. 4e). The results of Pb²⁺ contamination trial also found significant as Super Raya exhibited the maximum number of leaves (5.09) followed by CONII (3.99), Super Canola (3.26) and Dunkled (2.66) under controlled conditions. The Pb²⁺ (600ppm) resulted in varying result as Super Raya showed an increase in the number of leaves (6.11), while CONII (5.04), Super Canola (4.29), and Dunkled (3.31) resulted in the different responses (Fig. 4f).

Biochemical analysis of canola

Influence of Cd²⁺ contamination under field conditions

The normal growth conditions (control) under Cd²⁺ contamination trial resulted that Oscar had maximum SOD activity (4.20 U/g protein), while CONIII found at the next position (4.08 U/g protein) followed by Rainbow (3.82 U/g protein), CONII (3.71 U/g protein), and Sandal Canola (3.53 U/g protein). The canola plants growing under Cd²⁺ (6ppm) soil resulted in higher SOD activity across all varieties and Oscar was the top performing variety maintaining the highest SOD (6.63 U/g protein). The highest POD activity (29.89 U/g protein) was resulted by Oscar followed by CONIII (25.24 U/g protein), Rainbow (22.52 U/g protein), CONII (19.77 U/g protein) and Sandal Canola with a least POD activity (17.03 U/g protein). In contrast the Cd²⁺ (6ppm) contaminated plots resulted that Oscar remaining at top spot and maintaining the highest POD activity of 43.26 U/g protein. The highest activity of CAT was also observed in Oscar (2.27 U/g protein) followed by CONIII (2.19 U/g), Rainbow (2.05 U/g protein), CONII (1.93 U/g protein), and Sandal Canola (1.70 U/g protein) under controlled conditions. However, Cd²⁺ (6ppm) resulted in that highest CAT activity was observed in Oscar (2.87 U/g protein). The MDA content under normal growth conditions were maximum (56.14 nmol/g) represented by Oscar followed by CONIII (47.69 nmol/g), Rainbow (38.74 nmol/g), CONII (31.54 nmol/g), and Sandal Canola with least MDA content (27.58 nmol/g). In contrast the Cd²⁺ contaminated plots showed had the maximum MDA content (76.10 nmol/g) under Oscar and the results followed a similar pattern of MDA. APX was maximum in Oscar under control (58.60 U/g protein) which followed a performing trend of CONIII (54.16 U/g), Rainbow (49.89 U/g), CONII (45.94 U/g), and Sandal Canola (42.67 U/g). Cd²⁺ exposure to canola varieties resulted Oscar had the highest activity (74.20 U/g), while Super raya came on the top of the charts with the highest APX activity (65.18 U/g Protein) followed by CONII (60.16 U/g Protein), Super Canola (54.87 U/g Protein) and Dunkled (39.21 U/g Protein) under controlled conditions. The ASA activity was also found significant among the varieties of control and Cd²⁺ contaminated soil. Oscar outperformed with the maximum ASA content (58.99 µg/g) followed by CONIII (50.57 µg/g), Rainbow (42.48 µg/g), CONII (31.13 µg/g) and Sandal Canola (25.12 µg/g) under the normal growth or controlled conditions of the trial. Contrastingly under exposure of Cd²⁺ Oscar maintained the maximum ASA content (70.52 µg/g). However, GSH under the controlled condition of also resulted in maximum GSH content (53.16 µmol/g) by Oscar followed by CONIII (46.55 µmol/g), Rainbow (41.73 µmol/g), CONII (38.63 µmol/g), and Sandal Canola (34.63 µmol/g). Contrastingly the varieties under Cd²⁺ exposure represented as Oscar still maintained the highest GSH content of 73.71 µmol/g (Table 2).

Table 2.

The effect of Cd²⁺ (6ppm) contamination on the biochemical attributes on canola varieties

Cadmium levels	Variety	SOD (U/g Protein)	POD (U/g Protein)	CAT (U/g Protein)	MDA (nmol /g FW)	APX (U/g Protein)	ASA (µg/g FW)	GSH (µmol/g FW)
0 mg/L	CONII	3.71 ± 0.02bc	19.77 ± 0.83a	1.93 ± 0.04 bcd	31.54 ± 1.98ab	45.94 ± 1.50ab	31.13 ± 2.76ab	38.63 ± 0.96 ab
	CONIII	4.08 ± 0.05 cd	25.24 ± 1.04 a	2.19 ± 0.02de	47.69 ± 2.21d	54.16 ± 1.84 cd	50.57 ± 3.80 cd	46.55 ± 1.17 cd
	Oscar	4.20 ± 0.07d	29.89 ± 2.84a	2.27 ± 0.03e	56.14 ± 2.92 e	58.60 ± 1.27d	58.99 ± 2.53d	53.16 ± 2.28 de
	Rainbow	3.82 ± 0.04bcd	22.52 ± 0.76a	2.05 ± 0.06cde	38.74 ± 3.65bc	49.89 ± 1.86bc	42.48 ± 2.99bc	41.73 ± 1.38abc
	Sandal Canola	3.53 ± 0.08b	17.03 ± 0.76a	1.70 ± 0.12b	27.58 ± 0.92a	42.67 ± 1.28a	25.12 ± 2.87a	34.63 ± 0.86 a
6 mg/L	CONII	3.67 ± 0.08b	29.70 ± 2.05 a	1.84 ± 0.15bc	44.07 ± 4.47 cd	55.04 ± 0.63 cd	45.44 ± 3.55c	43.59 ± 2.25 bc
	CONIII	5.38 ± 0.72e	38.84 ± 1.99 a	2.61 ± 0.05f	70.60 ± 2.19 f	66.97 ± 3.62e	60.08 ± 0.46de	66.21 ± 3.18f
	Oscar	6.63 ± 0.22f	43.26 ± 2.84a	2.87 ± 0.12f	76.10 ± 2.36f	74.20 ± 1.96f	70.52 ± 8.35e	73.71 ± 1.36 g
	Rainbow	3.83 ± 0.07bcd	34.25 ± 1.91a	2.17 ± 0.02de	62.32 ± 0.39e	58.76 ± 1.03d	52.97 ± 1.97 cd	55.09 ± 5.77 e
	Sandal Canola	3.19 ± 0.11a	26.89 ± 0.81a	1.30 ± 0.20a	38.13 ± 2.55bc	43.15 ± 1.66a	28.35 ± 4.74a	35.90 ± 0.27a
p value of main and interaction
Heavy metals (M)		< 0.01	< 0.01	< 0.01	< 0.01	< 0.01	< 0.01	< 0.01
Variety (V)		< 0.01	< 0.01	< 0.01	< 0.01	< 0.01	< 0.01	< 0.01
M × V		< 0.01	< 0.01	< 0.01	< 0.01	< 0.01	< 0.01	< 0.01

Values are the mean ± standard deviation (n = 3). Values with same letter (s) are statistically non-significant at p < 0.05. SOD: Superoxide dismutase, POD: Peroxidase, CAT: Catalase, MDA: Malondialdehyde, APX: Ascorbate Peroxidase, ASA: Ascorbic Acid, GSH: Glutathione

Influence of Pb²⁺ contamination under field conditions

The canola varieties were also tested under Pb²⁺ (600ppm) as its control resulted that Super Raya exhibited the highest SOD activity (5.68 U/g Protein) followed by CONII (4.84 U/g Protein), Super Canola (4.30 U/g Protein), and Dunkled (3.41 U/g Protein). Under contaminated plots this activity increased and Super Raya showed a slight increase in SOD (5.78 U/g Protein), while CONII exhibited an increase to 5.20 U/g Protein. In addition, Super Canola showed a decrease to 4.08 U/g Protein and Dunkled exhibited a similar SOD activity to the control conditions with 3.43 U/g Protein. Statistical analysis represent a non-significant variation among the SOD and varieties. Super Raya also resulted in the maximum POD activity (45.75 U/g Protein) followed by CONII (42.12 U/g protein), Super Canola (35.77 U/g protein) and Dunkled (24.39 U/g protein) under controlled conditions. Contrastingly a similar trend was observed under contaminated sites as Super Raya had maximum POD activity (52.57 U/g Protein), while CONII (45.15 U/g protein), Super Canola (40.08 U/g protein) and Dunkled (24.51 U/g protein) resulted in varying responses respectively. Super Raya also resulted in maximum CAT activity (2.81 U/g Protein) followed by CONII (2.24 U/g protein), Super Canola (1.99 U/g protein) and Dunkled (1.63 U/g protein) under non-contaminated plots. Additionally, Super Raya showed a slight decrease in CAT activity (2.56 U/g Protein) while CONII exhibited a similar decrease in activity (2.08 U/g protein). Super Canola decreased CAT (1.83 U/g Protein) and Dunkled slightly decreased of 1.59 U/g Protein, while significant variations were observed in MDA content of different canola varieties studied under Pb²⁺ exposed trial. Super Raya exhibited the highest MDA (60.33 nmol/g) followed by CONII (50.80 nmol/g), Super Canola (42.62 nmol/g) and Dunkled (35.24 nmol/g) under controlled conditions. While under Pb²⁺ (600ppm) contamination conditions Super Raya showed an increase in MDA content (72.77 nmol/g), while CONII (60.01 nmol/g), Super Canola (50.21 nmol/g), and Dunkled (38.69 nmol/g) respectively. Upon the exposure of canola to contaminated soils APX was maximum in Super Raya (68.24 U/g Protein), while CONII decreased to 57.81 U/g Protein. Super Canola decreased APX activity (48.16 U/g Protein) and Dunkled slightly increased to 41.88 U/g. Super Raya had the highest ASA content (62.25 µg/g), followed by CONII (55.98 µg/g), Super Canola (44.82 /g) and Dunkled (31.15 µg/g) under the normal growth conditions in the Pb²⁺ contaminated field trial. In addition, Super Raya also resulted in promising results and showed maximum ASA content (74.49 µg/g) followed by CONII (63.86 µg/g), Super Canola (56.55 µg/g), and Dunkled with a least ASA content (43.21 µg/g). While under control conditions of second trial Super Raya was the top performer with the GSH content (60.22 µmol/g) followed by CONII (52.06 µmol/g), Super Canola (48.89 µmol/g) and Dunkled (34.68 µmol/g). Upon exposure to Pb²⁺ contaminated conditions Super Raya showed increased GSH content (72.76 µmol/g) while Super Canola decreased to 43.90 µmol/g. Additionally Dunkled exhibited a slight increase to 38.13 µmol/g (Table 3).

Table 3.

The effect of Pd²⁺ (600ppm) contamination on the biochemical attributes on canola varieties

Cadmium levels	Variety	SOD (U/g Protein)	POD (U/g Protein)	CAT (U/g Protein)	MDA (nmol /g FW)	APX (U/g Protein)	ASA (µg/g FW)	GSH (µmol/g FW)
0 mg/L	CONII	5.68 ± 0.27d	45.75 ± 2.22 cd	2.81 ± 0.12d	60.33 ± 6.89 c	65.18 ± 0.4de	62.25 ± 1.69c	60.22 ± 6.77 cd
	Dunkled	4.30 ± 0.09abc	35.77 ± 2.59 b	1.99 ± 0.04ab	42.62 ± 1.09ab	54.87 ± 1.85bc	44.82 ± 5.88b	48.89 ± 3.00b
	Super Canola	3.41 ± 0.30a	24.39 ± 6.48 a	1.63 ± 0.32a	35.24 ± 5.16 a	39.21 ± 9.55a	31.15 ± 1.39 a	34.68 ± 0.85a
	Super Raya	4.84 ± 0.46bcd	42.12 ± 0.83bc	2.24 ± 0.14bc	50.80 ± 1.12bc	60.16 ± 2.39 cd	55.98 ± 3.69c	52.06 ± 0.82bc
600 mg/L	CONII	5.78 ± 0.19d	52.57 ± 2.78 d	2.56 ± 0.10 cd	72.77 ± 6.17d	68.24 ± 0.73e	74.49 ± 3.33d	72.76 ± 2.39e
	Dunkled	4.08 ± 0.52ab	40.08 ± 2.15bc	1.83 ± 0.11ab	50.21 ± 4.38 bc	48.16 ± 3.34ab	56.55 ± 2.24c	43.90 ± 1.35ab
	Super Canola	3.43 ± 0.25a	24.51 ± 3.11a	1.59 ± 0.11a	38.69 ± 3.06ab	41.88 ± 2.53a	43.21 ± 1.22b	38.13 ± 1.13a
	Super Raya	5.20 ± 0.3 cd	45.15 ± 1.43 cd	2.08 ± 0.11b	60.01 ± 3.06 c	57.81 ± 2.24 cd	63.86 ± 2.39c	63.66 ± 6.13de
p value of main and interaction
Heavy metals (M)		0.65	0.01	0.03	< 0.01	0.50	< 0.01	< 0.01
Variety (V)		< 0.01	< 0.01	< 0.01	< 0.01	< 0.01	< 0.01	< 0.01
M × V		0.01	0.01	0.01	0.01	0.01	0.01	< 0.01

Values are the mean ± standard deviation (n = 3). Values with same letter (s) are statistically non-significant at p < 0.05. SOD: Superoxide dismutase, POD: Peroxidase, CAT: Catalase, MDA: Malondialdehyde, APX: Ascorbate Peroxidase, ASA: Ascorbic Acid, GSH: Glutathione

Discussion

Cadmium (Cd²⁺) and Lead (Pb²⁺) are two of the major metals behind the HM contamination of agricultural soils. Cd²⁺ and Pb²⁺ contamination has emerged as a matter of great concern for ensuring food security [27]. The effects of Cd²⁺ and Pb²⁺ on the growth and development of canola remain poorly explored [28, 29]. The study aimed to reduce the gap by providing fact-based insights into how canola plants tolerate excessive concentrations of these toxic elements. The findings of current research represent the growth and development of canola affected by Cd²⁺ and Pb²⁺ toxicity, and also show that every canola variety has the ability to withstand certain levels of HM stress. The study revealed that two promising varieties, Oscar and Super Raya, showed relatively better morphological and physiological characteristics under stress, indicating their tolerance to Pb²⁺ and Cd²⁺. The aforementioned findingss are consistent with studies by Bilal et al. (2024), who observed that canola varieties can reduce the effects of metals and exhibit higher antioxidant activity, thereby performing better under metal stress. The study revealed that higher concentrations of Cd²⁺ and Pb²⁺ caused drastic changes, including reductions in root and shoot length and decreased biomass accumulation [30]. Similar results were reported by [31] who explained that Cd²⁺ interferes with nutrient uptake and effects cell division which ultimately result in decreased growth. The literature reports that Pb²⁺ is slightly less toxic which proved to have less severe effects on the plant growth as compared to Cd²⁺ [32]. The influence of genotypic variations among canola varieties serves a crucial role in the uptake of metal and reduces toxicity as Oscar was the most prominent variety, which interestingly showed a higher level of growth under the influence of increased concentrations of Cd²⁺ and Pb²⁺. The observed results could be attributed to the fact that some canola varieties have more effective mechanisms for metal sequestration or detoxification [33]. Similar results were noted by Zafar-ul-Hye et al. (2018), who reported that canola showed better growth because it improved the capability to retain HM in roots, thereby minimizing its translocation into the shoots. The increase in oxidative stress was responded to by canola varieties in the form of increased antioxidant enzyme activities. These findings suggest both have the most well-developed antioxidant defense mechanisms [34]. This aligns with the findings of those who documented the role of antioxidant enzymes in alleviating oxidative damage caused by HM. Both of the mentioned canola varieties seem to have a much more efficient ROS scavenging system. This greatly helps mitigate the toxic effects of Cd²⁺ and Pb²⁺. The varieties with lower ability to produce antioxidant enzymes, for example Rainbow and Sandal Canola, were more susceptible to ROS-related damage [35]. This metal-induced damage resulted in much lower biomass and chlorophyll content. These outcomes further validate the importance of antioxidants in mitigating metal toxicity, as demonstrated by Cuypers et al. (2010), who concluded that the antioxidant response is significant for plants exposed to metal stress. The impact of Cd²⁺ and Pb²⁺ on plant growth in the current investigation aligns with an expanding corpus of scholarly work. For instance [36] noted analogous declines in both growth and chlorophyll levels subjected to Cd²⁺ exposure. The majority of investigations regarding HM toxicity in canola have been carried out under regulated conditions, wherein parameters such as soil composition and climatic factors remain constant. This research carries a great significance in implications for agricultural practices as canola has the tendency to retain HM. The study aided in the identification of tolerant varieties of canola such as Oscar variety. Oscar Variety has proven to perform exceptionally well when compared to other tested varieties even under the stressed conditions [10]. The findings of this study underscore the need for further exploration of metal tolerance mechanisms in canola. The future research should be focused on working with canola on genetic level so specific varieties can be developed that can grow very effectively in metal affected soils this would uplift the sustainable agriculture and also improve the food security. The response observed in the current study may be attributed to the integrated detoxification and the defense mechanism under stress conditions. In our study the possible mechanism would be the neutralization of toxic metal through the chelation by the phytochelation and the related thiol containing compounds followed by the vacuolar sequestration which further limit their interaction with the metabolically active cellular compartments in the tissues. Moreover, the Phyto chelation mediated detoxification pathways supported by the glutathione metabolism that likely play a central role in the intracellular immobilization of metals. Similarly, metal stress also improve the ROS production which damages the limits and the membranes at a higher concentration which also function as signaling molecules that activate the antioxidant and defense pathways within the plant. The core differences among varieties might be associated with the variation in the studied metal, detoxification of ROS, efficiency of sequestration and stress adaptation capacity.

Heatmap analysis

Figure 5a, c left side panel displaying the variety-wise heatmap of Cd²⁺ and Pb²⁺ accumulation in canola varieties where higher score 0.5-1.0 indicates good-performing varieties, while right side panel (Fig. 5b, d) presenting the treatment wise heatmap comparing Cd²⁺ and Pb²⁺ level among control and treated (6ppm and 600ppm). The yellow shade represents higher accumulation of Cd²⁺ and Pb²⁺, while the blue shade indicates lower accumulation based on scores across treatments and the studied parameters (Fig. 5).

Fig. 5.

A variety wise heatmap of Cd²⁺ and Pb^2+, B Treatment wise heatmap of Cd²⁺ and Pb²⁺

based on Z-scores

Chord analysis

Chord diagram represents the connection among PH, RL, SL, RSW, RDW, SFW, SDW, LFW, LDW, No. of Leaves, MDA, SOD, CAT, APX, ASA, GSH and POD under Cd2+ and Pb2+ stress conditions. Each trait represents the color chord connecting it to the others; this shows the strength and direction of the association between the trait and the parameter. Similarly, the second chord diagram shows the influence of Pb2+ stress on the traits of canola varieties; moreover, the diagram's structure is similar, while the pattern of connections is different, which reflects that lead stress affected the physiological and biochemical traits of canola varieties (Fig. 6).

Fig. 6.

Chord analysis of Cd²⁺ and Pb²⁺ contamination on the traits of canola under field conditions

PCA analysis

PCA plot visualizes the variation in traits of canola under Cd²⁺ and Pb²⁺ stress across various varieties and treatments. The PCA plot in the top left of Fig. 7 shows the distribution of varieties under Cd²⁺ stress, with CONIII, Oscar, Rainbow, and Sandal Canola distinctly separated along PCA1 (70.7%) & PCA2 (24.3%). The traits represented by red arrow contributed towards the variation. Additionally, RFW, SFW, SDW, LFW, LDW, RL and PH found in quadrant 1 and showed the strong association with Oscar and CONIII. In contrast, other traits, such as RDW, SL, CAT, ASA, SOD, APX, GSH, MDA, and POD, showed strong association with similar cultivars in quadrant 2. The top right plot represented the PCA of Cd²⁺ stress vs. control conditions. The data points for each treatment distinctly separated which represented a clear distinction of control vs. contaminated fields. The PCA results showed that RDW, SDW, number of leaves, LFW, SFW, LDW, RL, and PH were strongly associated with the control plot in quadrant (1) However, RDW, SL, CAT, ASA, APX, POD, MDA, and GSH showed a strong association with the Cd²⁺ site in quadrant 2 (Fig. 7). PCA plot on bottom left of Fig. 7 shows the distribution of varieties under Pb²⁺ stress on varieties. CONIII, Super Raya, Super Canola and Dunkled distinctly separated along with the PCA1 (89.4%) & PCA2 (6.1%) as the as traits represented by red arrow contributed towards the variation. The SDW, LDW, RDW, CAT, RL, RFW and SFW found in quadrant 1 and showed the strong association with Super Raya and CONII. The biochemical trait SOD remained at the midline of the PCA, while other traits, such as plant height, shoot length, leaf dry weight, POD, GSH, MDA, ASA, and number of leaves, showed strong association with similar cultivars in quadrant (2) The bottom-right plot shows the PCA of Pb2 + stress vs. control conditions. The data points for each treatment distinctly separated which represented a clear distinction of control vs. contaminated fields. The PCA results showed that SDW, LFW, RDW, CAT, RL, APX, RFW, and SFW were strongly associated with the control plot in quadrant 1. However, PH, SL, LDW, POD, GSH, MDA, ASA, Number of leaves had the strong association with the Pb²⁺ site in the quadrant 2.

Fig. 7.

Principal component analysis (PCA) of Cd²⁺ and Pb²⁺ contamination on canola varieties under field conditions

Trait pair analysis

The traits pair plot display the relationship between physiological and biochemical traits of canola varieties under contaminated environment. The matrix represent the scatterplot comparing the pairs of traits. The diagonal histogram represents the distribution of each trail; however, the scatterplot off the diagonal shows the relationship between the pair of traits. The correlation coefficient is also displayed in each cell which reveal the strength of the relationships. The figure of Pb²⁺ revealed a positive correlation of PH & RL (0.94) which represent that taller plant tends to have long roots under contaminated sites. Similarly, shoot fresh weight, leaf fresh weight, root fresh weight, and leaf dry weight exhibited strong positive correlations (0.86–0.94), indicating a coordinated response in the accumulation of biomass across various parts of the canola plant. In addition, MDA correlated with SOD and POD, suggesting that higher oxidative stress led to improved antioxidant activity as the plant responded to Pb²⁺ contamination. Similarly, plant height and root length showed a strong positive correlation (r = 0.96) under Cd²⁺ exposure. However, root fresh weight, shoot fresh weight and leaf fresh weight represented a significant positive correlation (0.94–0.96) which revealed that aforementioned traits vary under contaminated environment. The correlation plot also revealed a moderate correlation among MDA, SOD, POD and CAD (0.57–0.79) which highlight the defensive response of canola to oxidative damage caused by the contamination of Cd²⁺ (Fig. 8).

Fig. 8.

Trait pair plot of Pb²⁺ and Cd²⁺ contamination on canola varieties under field conditions

Conclusion

It is concluded that Oscar and Super Raya were tolerant of Cd²⁺ and Pb²⁺ contamination under field conditions. Sandal Canola and Super Canola were found to be susceptible, while the remaining varieties were moderately resistant to Cd²⁺ and Pb²⁺ toxicity under field conditions. Growers facing the issue of Cd²⁺ and Pb²⁺ contamination are recommended to cultivate Oscar in Cd²⁺ and Super Raya in Pb²⁺ contaminated fields to achieve better growth and production. The validation trials are required to be conducted under field conditions to identify the top-performing and susceptible varieties against contamination by Cd²⁺ and Pb²⁺. Further investigations through field validation trials and genomic studies are warranted to reduce Cd²⁺ and Pb²⁺ toxicity under field conditions and ensure the best and most suitable utilization of land.

Author contributions

K.B.; N.N.E.; M.I.; contributed to the conceptualization and design of the study, as well as data collection, analysis, and interpretation. S.S.; T.A.; S.S.A; contributed to the statistical analysis; S.S.; M.D.A.; A.A.; M.A.; interpretation of the data. All authors have reviewed and approved the final version of the manuscript.

Funding

This work was supported by the Deanship of Scientific Research, Vice Presidency for Graduate Studies and Scientific Research, King Faisal University, Saudi Arabia (KFU261831). Princess Nourah bint Abdulrahman University Researchers Supporting Project number (PNURSP2026R355), Princess Nourah bint Abdulrahman University, Riyadh, Saudi Arabia.

Data availability

All data generated or analyzed during this study are included in this published article.

Declarations

Ethics approval and consent to participate

We all declare that manuscript reporting studies do not involve any human participants, human data, or human tissue. So, it is not applicable.

Consent for publication

Not Applicable.

Competing interests

The authors declare no competing interests.