Heavy metal tolerance and accumulation in the Brassica species (Brassica chinensis var. parachinensis and Brassica rapa L.): A pot experiment

Abstract

This study delves into the heavy metal tolerance and accumulation capabilities of Brassica chinensis var. parachinensis (B. chinensis) and Brassica rapa L. (B. rapa) in a pot experiment, specifically focusing on cadmium (Cd), chromium (Cr) and lead (Pb). Agricultural topsoils were spiked with varying concentrations of these heavy metals (0 mg/kg, 75 mg/kg, 150 mg/kg, 225 mg/kg and 300 mg/kg) for each element. The experiment involved cultivating 15 pots each of B. chinensis and B. rapa over 60 days. Results indicated that both Brassica species experienced delayed germination, with B. chinensis exhibiting a significant drop in germination percentage to 53 % at the highest concentration (300 mg/kg), while B. rapa showed a tendency for an increased germination percentage of up to 80 % at elevated metal concentrations; however, these differences were not statistically significant. Both B. chinensis and B. rapa demonstrated a stable decline in growth rate from 0.05 cm/day to 0.04 cm/day with increasing heavy metal concentrations, and the he reduction in relative growth rate was significant at the highest concentration compared to the control. The stress tolerance index revealed a significant decrease in plant heights for B. chinensis, in contrast to the stable performance of B. rapa, showcasing the tolerance of B. rapa to toxic conditions. Despite insignificant differences in fresh biomass due to metal treatments, B. chinensis consistently yielded higher biomass, yet it had a lower edible index due to its higher root biomass. Leaf areas increased significantly in both species at higher soil treatments, while root lengths remained unchanged, suggesting their resilience to elevated heavy metal concentrations. Analysis of plant tissues (leaves, stems and roots) using ICP-OES revealed that B. rapa accumulated the highest Cd concentration (864 mg/kg), whereas B. chinensis accumulated the highest Pb concentration (953 mg/kg) in root parts. Both species significantly accumulated Cr in roots, demonstrating a sequestration mechanism. These findings suggest that both species, particularly, B. rapa possess strong tolerance and accumulation capabilities for non-essential heavy metals, making them potential hyperaccumulators for green remediation techniques in toxic soil environments. Understanding the molecular mechanisms driving these responses and validating phytoremediation potential in real-world scenarios is essential for developing sustainable soil management practices.

1. Introduction

Heavy metals pose serious risks to plants and humans due to their high toxicity and persistence in natural ecosystems [[1], [2], [3]]. Their presence in plants can disrupt vital biochemical processes such as nutrient homeostasis, antioxidant and enzyme production, gas exchange components, and photosynthesis [4]. Various remediation techniques, including phytoremediation, chemical and physical methods, and microbial remediation, have been employed to mitigate heavy metal contamination in soil [5]. Phytoremediation, recognized as a cost-effective green technique, involves using plants to absorb and accumulate contaminants, such as heavy metals, through their root systems, effectively removing them from the environment [6,7]. Commonly employed phytoremediation procedures include phytodegradation, phytoextraction, phytostabilisation, phytovolatilisation, and rhizofiltration [8].

In phytoremediation, assessing different plant species' properties is crucial for understanding their tolerance and accumulation abilities against specific contaminants [9]. Phytotolerance analyses are essential to gauge metal tolerance levels, investigating the negative impacts of heavy metals on morphological, physiological, and biochemical activities in plants [10]. Monitoring stress tolerance during seed germination and seedling stages is particularly valuable, given the vulnerability of seeds to toxic environments compared to the vegetative stage [11,12]. Evaluating metal stress in seedlings provides insights into their growth performance under toxic conditions. Numerous studies have highlighted the effects of heavy metal-contaminated soils on germination, often resulting in reduced performance or seedling damage in various plant species [[13], [14], [15], [16]]. These effects can, in turn, impact physicochemical processes such as enzyme activities and photosynthesis [4,17].

Some recognized phytoaccumulators, like Ariplex halimus L. and Halimione portulacoides Aellen, are deemed suitable for accumulating cadmium (Cd), chromium (Cr), copper (Cu), mercury (Hg) and lead (Pb), contributing to phytostabilization in saline soils [[18], [19], [20], [21], [22]]. These hyperaccumulator plants release protons from their roots, increasing soil acidity and facilitating metal ion mobility, thereby enhancing metal uptake into roots and shoots [5]. Ideal hyperaccumulating plants exhibit traits such as high heavy metal concentrations, tolerance to heavy metals, adaptability to toxic environments, fast growth, high biomass, and harvestability [23]. Efficient metal translocation from roots to shoots is also crucial. Currently, only a few plant species, including those from Asteraceae, Brassicaceae, Caryophyllaceae, Cunoniaceae, Cyperaceae, Euphorbiaceae, Fabaceae, Flacourtiaceae, Lamiaceae, Poaceae, and Violaceae, meet these criteria [[24], [25], [26], [27]]. Given that different plant species accumulate various contaminants, careful plant selection for phytoextraction, a key phytoremediation method for heavy metals in soils, is essential [28]. While some studies have explored the use of woody plants like Populus tremula and Picea abies for soil decontamination, their limited extraction and accumulation of heavy metals pose disadvantages, restricting their use for phytoextraction [[29], [30], [31]].

Several non-woody plants, including Brassica species like Alyssum Bertolonii, Alyssum murale, Noccaeae caerulescens, Streptanthus polygaloides, Stanleya pinnata and Bornmuellera tymphaea, are known as metal accumulators [32]. The potential application of Brassica species for phytoextraction of heavy metals is mainly due to their underlying tolerance to heavy metals [33]. Brassica species are valued for their ability to tolerate heavy metals, particularly B. juncea, B. napus, B. oleracea, B. carinata and B. nigra, which have shown high tolerance to toxic metal concentrations in soils [34,35]. Studies have demonstrated the accumulation of metals like Cu, nickel (Ni), Pb, and zinc (Zn) in B. juncea compared to B. campestris, B. carinata, B. napus and B. nigra in a field or pot experiment, indicating its potential for phytoextraction [36]. B. juncea accumulated Cd, cobalt (Co), Ni, Pb and Zn in leaves, stems and roots, but only Cu and manganese (Mn), as well as Cr, in the stems and roots based on the metal transfer factor of more than 1 [37]. B. chinensis accumulated higher levels of Cd, Cr and Pb in all plant parts than B. rapa in a pot study using agricultural soils [38]. Further research is needed to explore metal uptake and tolerance in other Brassica species.

This study aims to determine the tolerance and accumulation rates of two Brassica species (Brassica chinensis var. parachinensis, B. chinensis hereafter, and Brassica rapa L., B. rapa hereafter) to Cd, Cr, and Pb, selected due to their prevalence in Brunei Darussalam's agricultural soils [38]. These species were chosen for their common presence in Brunei's farmland and their known high uptake capacities for these metals [38,39]. The study assesses the phytotolerance of B. chinensis and B. rapa to various concentrations of heavy metal contaminants, alongside evaluating their effects on growth performance. The specific objectives are as follows: (1) to monitor the growth of the two Brassica species from seed germination to mature stage at different concentrations of selected heavy metals contaminants (Cd, Cr and Pb), (2) to investigate the heavy metals tolerance levels of B. chinensis and B. rapa, and (3) to assess the phytoremediation potential of using B. chinensis and B. rapa to accumulate heavy metals in soils.

2. Materials and methods

2.1. Study area

The pot experiment with B. chinensis and B. rapa took place within a plant shade (6 m long x 4 m wide x 2 m high) which is located at the Faculty of Science, Universiti Brunei Darussalam, situated in Brunei Darussalam (4°58′31.1″ N, 114°53′48.6″ E) on the north-west side of the Borneo Island in Southeast Asia (Fig. 1). The plant shade was covered with a black shade netting (50 % light transmission) on the top and sides to shield the growing vegetables from direct sunlight, which could impede their early growth and survival. Additionally, a transparent plastic roof was used to cover the top of the plant shade, ensuring that the experiment remained unaffected by rainfall.

Fig. 1.

Location of plant shade at the Faculty of Science, Universiti Brunei Darussalam, Brunei Darussalam (4°58′31.1″ N, 114°53′48.6″ E). Source: https://www.google.com/maps; Accessed 12^th November 2023. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Brunei Darussalam experiences a humid equatorial climate. Throughout our study's growth phases from December 2021 to February 2022, the average monthly temperature ranged from 25.6 °C to 28.7 °C, with an average of 27.3 °C, while the average monthly rainfall varied from 2.1 mm to 31 mm, averaging at 8.7 mm (Brunei Darussalam Meteorological Department, unpublished data). Fig. 2 illustrates the average weekly temperature (°C) and rainfall (mm) over an 8-week period of this study.

Fig. 2.

Average weekly temperature (°C) and rainfall (mm) during the growth period of Brassica chinensis var. parachinensis and Brassica rapa L. from December 2021 to February 2022. Data was sourced from the Brunei Darussalam Meteorological Department (BDMD), Ministry of Transport and Infocommunication, Brunei Darussalam, located approximately 4 km from the study site.

2.2. Study species, sampling and sample pre-treatment

The chosen plant species, B. chinensis and B. rapa, are annual herbaceous plants which are known for their potential in soil heavy metal phytoremediation [40]. These Brassica species are versatile, with edible leaves and stems that can be consumed cooked or raw, and they are also utilized for seed oil production and as green manure [35,41]. Seeds of B. chinensis and B. rapa utilized in this study were sourced from local markets in Brunei Darussalam.

In July 2021, agricultural topsoil (120 kg) was collected from 10 random locations at the Bukit Agok agricultural farmland, located in the Brunei-Muara district of Brunei Darussalam (4°54′58.9″N, 114°47′44.4″E). These soil samples were carefully stored in labeled plastic basins and transported to the laboratory for pre-treatment and analysis. Before any treatment, the soil samples were air-dried and sieved through a 2 mm mesh sieve to remove stone pebbles and plant litter. Following drying, the soil samples (n = 10) were analyzed for physicochemical properties with triplicate reading. The soil analyses included texture classification [42], moisture content [43], pH [44], electrical conductivity [45], total organic carbon (OC) and organic matter (OM) content [46], total N, P, K, Ca, Mg and Na [47] and cation exchange capacity (CEC) [48]. The summarized data is presented in Table 1.

Table 1.

Physicochemical properties of agricultural soils at Bukit Agok, Brunei Darussalam, utilized in the pot experiments for assessing heavy metal tolerance in Brassica species. Values represent the mean with standard deviation (SD) for n = 10.

Soil parameters	Soil samples
Soil classification*	Nudinatric Solonetz
Textural class	Sandy loam
Sand (%) (0.05–2.00 mm)	52.4 (2.40)
Silt (%) (0.002–0.050 mm)	39.8 (2.40)
Clay (%) (<0.002 mm)	7.80 (0.60)
% moisture	15.3 (2.10)
pH	6.50 (0.02)
Electrical conductivity (dS/m)	0.16 (0.00)
Total organic C (%)	5.80 (1.10)
Organic matter (%)	9.90 (1.90)
Total N (mg/kg)	3195 (836)
Total P (mg/kg)	983 (63.1)
Ca (cmol/kg)	85.8 (27.4)
K (cmol/kg)	104 (45.4)
Mg (cmol/kg)	16.0 (3.20)
Na (cmol/kg)	136 (17.4)
CEC (cmol/kg)	342 (50.7)
Cd (mg/kg)	0.85 (0.77)
Cr (mg/kg)	1.64 (0.84)
Pb (mg/kg)	18.7 (1.62)

* Soil classification is based on the World Reference Base for Soil Resources [49].

In this study, a total of 30 polyethylene pots (22.5 cm diameter × 20.0 cm height) were arranged inside the plant shade. Following soil analysis, the soils were mixed and bulked. About 3 kg of air-dried soil was then distributed into each pot, spiked with uniform concentrations of Cd, Cr, and Pb at four levels: 75 mg/kg (T1), 150 mg/kg (T2), 225 mg/kg (T3), and 300 mg/kg (T4) of Cd (CdCl₂), Cr (CrCl₃.6H₂O), and Pb (Pb(CH₃COO)₂.3H₂O) as heavy metal pollutants, with triplicates for each treatment. The chosen concentration range is aligned with the maximum allowable limits set by the United States Environmental Protection Agency (USEPA) [50].

Soil spiking in each pot involved preparing individual concentration for each metal (Cd, Cr and Pb), followed by mixing to create the treatment concentrations. This was achieved by adding precise amounts of Cd, Cr and Pb salts dissolved in distilled water into each pot containing 3 kg of soil. Thorough mixing of the soils was conducted using clean plastic trowels, followed by a 2-week period for drying and stabilization at room temperature. This procedure was based on a modified protocol derived from Mkumbo [50] and Amin et al. [51]. Control pots (T0) were also set up with no addition of pollutants. The pot experiment involving varying concentrations of Cd, Cr, and Pb served to systematically explore the responses of B. chinensis and B. rapa to different levels of heavy metal contamination. This approach established a dose-response relationship, allowing researchers to identify threshold levels impacting germination, growth rates, and metal accumulation. By simulating realistic local soil conditions in Bukit Agok, Brunei Darussalam, the experiment enables the assessment of species-specific responses and evaluates the phytoremediation potential of these Brassica species. The controlled environment of the pot experiment enhances the reliability of observations, providing valuable insights into the adaptability and suitability of these plants for sustainable soil management practices.

The pots were arranged in a completely randomized design within the plant shade, resulting in 15 pots (3 pots x 5 treatments) for each species. Each pot was planted with a total of 20 seeds, yielding 60 seeds per treatment for each species. Germination progress for both the Brassica species was observed daily until day 10, with plants receiving irrigation of 100 mL distilled water three times weekly. After germination bioassay, seedlings were thinned to maintain three seedlings per pot, resulting in three B. chinensis or three B. rapa plants per pot. Daily weeding was performed to ensure the unhindered growth of the study species. Both B. chinensis and B. rapa typically germinated within 5 days and reached maturity in approximately 5 weeks (Fig. 3A0 – A4 and B0 – B4), following the BBCH scale which was used to assess the principal growth stages of the two species under various soil treatments, they can be described using numbers in ascending order [52]. In this experiment, no fertilizer was applied to ensure that the spiked soils remained the sole source of pollutants. After 60 days, both species were harvested in February 2022. Plants from each pot were bulked for chemical analysis in triplicates, resulting in three samples per treatment per species.

Fig. 3.

Phenological stages of (A): Brassica chinensis var. parachinensis at different heavy metal treatments at different days after planting (DAP) – A.0: Control or Treatment 0, A.1: Treatment 1, A.2: Treatment 2, A.3: Treatment 3 and A.4: Treatment 4, and (B): Brassica rapa L. at different treatments – B.0: Control, B.1: Treatment 1, B.2: Treatment 2, B.3: Treatment 3 and B.4: Treatment 4, according to the BBCH scale [52]: 12 (second true leaf unfolded), 13(third true leaf unfolded), 14 (fourth true leaf unfolded), 16 (sixth true leaf unfolded), 17 (seventh true leaf unfolded), 18 (eight true leaf unfolded), 19 (ninth true leaf unfolded), 33 (leaf rosette has reached 30 % of expected diameter), 35 (leaf rosette has reached 50 % of expected diameter), 41 (10 % of the leaf mass typical of the variety reached), 42 (20 % of the leaf mass typical of the variety reached), 43 (30 % of the leaf mass typical of the variety reached), 44 (40 % of the leaf mass typical of the variety reached, 45 (50 % of the leaf mass typical of the variety reached) and 47 (70 % of the leaf mass typical of the variety reached).

2.3. Data analysis

Seven plant growth measurements were conducted for each Brassica vegetable to assess their tolerance levels and ecotoxicological effects under varied soil treatments. These measurements included germination percentage, relative growth rate, stress tolerance index, fresh biomass, edible index, leaf area, and maximum root length.

2.3.1. Germination percentage

The germination percentage (G%) of B. chinensis and B. rapa was determined by calculating the ratio of germinated seeds to the total number of viable seeds sown in each metal-spiked treatment, following Eq. (1) [53], observed over a 10-day period:

$$Germinationpercentage\left(G\%\right)=\frac{\mathrm{G}}{{\mathrm{G}}_{\mathrm{o}}}x100\%$$ (Eq. 1)

where G and G₀ represent the number of germinated seeds in each treatment and the total number of seeds sowed in each treatment, respectively. In this study, 20 seeds were sown in each pot, resulting in a total of 300 viable seeds and 60 seeds per treatment for each species.

2.3.2. Relative growth rate of species

The relative growth rate (RGR) or efficiency index (EI) of both B. chinensis and B. rapa was assessed by measuring plant height under various metal-spiked soil treatments over a 60-day period, as per Eq. (2) [54]:

$$Relativegrowthrate\left(RGR\right)=\frac{\ln \left(\text{Final}\text{height}\left(\text{cm}\right)\right)-\ln \left(\text{Initial}\text{height}\left(\text{cm}\right)\right)}{\text{Day}\text{of}\text{final}\text{measurement}-\text{Day}\text{of}\text{initial}\text{measurement}}$$ (Eq. 2)

where Final height and Initial height are the heights of treated plant at the day of harvesting and the height of the treated plant during the initial measurement, respectively.

2.3.3. Stress tolerance index

The stress tolerance index (TI_ph) was evaluated for plant height by comparing the mean height of treated plants to that of control plants under various heavy metal treatments for both species. This comparison was conducted using Eq. (3) [55]:

$$Stresstoleranceindex\left(TIph\right)=\frac{\text{Height}\text{of}\text{treated}\text{plant}\left(\text{cm}\right)}{\text{Height}\text{of}\text{control}\text{plant}\left(\text{cm}\right)}x100\%$$ (Eq. 3)

2.3.4. Fresh biomass and edible index

Fresh biomass from each plant part (leaves, stems and roots) was determined from both the harvested species. Additionally, the edible index was calculated to assess the edibility of the plants grown in various metal-spiked soil treatments. This calculation involved measuring the total fresh biomass of the species and the fresh biomass of the edible plant parts (leaves and stems), following the method outlined by Mi et al. [56], as per Eq. (4):

$$Ediblerateofplant\left(ER\right)=\frac{{\text{FW}}_{\mathrm{e}}}{\text{FW}}$$ (Eq. 4)

where FW and FW_e are the total fresh biomass of plant species and the edible fresh biomass of plants, such as leaves and stems, respectively. The calculation is based on the percentage ratio of FW_e to FW.

2.3.5. Leaf area and root length

Leaf area (LA) and root length measurements were conducted to evaluate the potential impact of heavy metals on plant growth across various metal-spiked soil treatments. LA was determined by multiplying the maximum length (L) and width (W) of a leaf, as outlined in Eq. (5) [57]:

$$Leafarea\left(LA\right)=Maximumleaflength\left(L\right)\times Maximumleafwidth\left(W\right)$$ (Eq. 5)

The root length was estimated by measuring the maximum length of the primary or main root, from the base to the root tip using a standard ruler.

2.4. Analytical procedures

The extraction procedure used for analyzing heavy metals in B. chinensis and B. rapa samples was based on the USEPA method 3050B [58]. Each plant sample was divided into leaves, stems and roots and analyzed separately to investigate the accumulation of spiked heavy metals. Plant samples were oven-dried at 105 °C for 2 h to eliminate moisture [39]. Subsequently, the dried samples were homogenized and ground using a pestle and mortar before passing through a 2 mm sieve.

Oven-dried plant samples (1 g) were digested with 15 mL HNO₃ (Sigma Aldrich, ACS reagent, 65 %), 10 mL H₂O₂ (Sigma Aldrich, 30 %) and 10 mL HCl (Sigma Aldrich, ACS reagent, 35.4 %) for 2 h at 95 ± 5 °C. Following digestion, the samples were cooled down to ca. 24 °C and then diluted with distilled water. Subsequently, each sample was analyzed for Cd, Cr and Pb concentrations using an inductively coupled plasma-optical emission spectroscopy (ICP-OES Thermo Scientific iCAP 6000 Series, USA). Blank samples were prepared alongside each sample, and the instrument's accuracy was verified by analyzing the samples in triplicates. The operating conditions for ICP-OES used in this study are summarized as shown in Table 2.

Table 2.

Operating conditions, parameters, limit of detection (LOD) and limit of quantification (LOQ) of Inductively Coupled Plasma-Optical Emission Spectroscopy (ICP-OES) used for analyzing heavy metal concentrations in Brassica species samples collected from pot experiments.

ICP-OES parameter	Elements
	Cd (214.4)	Cr (283.5),	Pb (220.3)
LOD (ppb)	7.0	2.0	4.0
LOQ (ppb)	23.0	6.0	12.0
Output mode	Intensity
Plasma viewing mode	Axial
Spray chamber type	Glass cyclonic spray chamber
Instrument repeats	3
RF power	1150 W
Analysis pump rate	50 rpm
Auxiliary gas flow rate	0.5 L/min
Nebulizer gas flow rate	0.7 L/min
Flush pump rate	100 rpm
Pump stabilization time	15 s

2.5. Statistical analysis

Statistical analyses for all heavy metals and plant parameters in B. chinensis and B. rapa were performed using a one-way analysis of variance (ANOVA) and TukeyHSD tests at 5 % significance level using SPSS (IBM SPSS Statistics 22) [59]. Moreover, the heavy metal concentrations in both Brassica species were used to derive the principal component analysis (PCA) of metal elements across the three different plant parts using SPSS.

3. Results and discussion

3.1. Germination percentage

The study examined the germination percentage to gauge the viability and resilience of seeds in response to adverse environmental conditions induced by heavy metals. This bioassay aimed to offer insights into the early stages of growth and establishment of B. chinensis and B. rapa seeds under different metal-spiked soils (Fig. 4). In the control treatment, seeds of both species began to germinate around day 5 ± 1, while seeds in soils treated with metal exhibited a delay of at least 2 days (day 7 ± 1). This delay in germination time suggests the possibility of secondary induced dormancy in response to metal concentrations [60]. These current findings are consistent with those of Eze et al. [61], who similarly observed delayed germination in Cr-treated soils, with concentrations of up to 400 mg/kg compared to the control treatment, indicating that germination time increased with higher levels of metal contamination in soils.

Fig. 4.

Germination percentage (%) of Brassica chinensis var. parachinensis and Brassica rapa L. under different soil metal treatments: T0: control soil, T1: 75 mg/kg Cd, Cr and Pb, T2: 150 mg/kg Cd, Cr and Pb, T3: 225 mg/kg Cd, Cr and Pb, and T4: 300 mg/kg Cd, Cr and Pb. Different letters represent significantly different means for each species after analyzing using one-way ANOVA and TukeyHSD tests at 5 % significance level.

The germination percentages of B. chinensis and B. rapa were significantly affected by the heavy metal treatments (Fig. 4). The results indicated that B. chinensis maintained a consistent germination percentage ranging from 70 to 76 % across various treatments, except at 300 mg/kg (Treatment 4), where it significantly decreased to 53 %. Although the difference in germination percentage between the control and Treatment 4 was not significant, Treatment 4 exhibited a marked decrease compared to Treatment 2 (150 mg/kg Cd, Cr and Pb). Conversely, B. rapa displayed trend towards increased germination percentage, reaching levels up to 80 % at higher concentrations of Cd, Cr and Pb in the metal treatments, although these differences were not statistically significant.

The ANOVA results revealed contrasting germination percentages between the two Brassica species, highlighting their distinct sensitivity and tolerance to varying heavy metal concentrations in the soil [62]. This indicates that the germination of different plant seeds is influenced by different heavy metal concentrations [60]. The findings imply that elevated metal concentrations could impact the germination process of Brassica seeds. This study emphasizes the differential effects of heavy metal concentrations on the germination performance of Brassica seeds, consistent with findings from previous research [62].

3.2. Relative growth rate and stress tolerance index

The relative growth rate and stress tolerance index were assessed for both Brassica species to evaluate their growth performance under varying heavy metal treatments (Fig. 5). The relative growth rate offers a quantitative assessment of the species' overall growth performance under different soil treatments, reflecting how heavy metals influence their development from seedling to maturity. Similarly, the stress tolerance index offers valuable information regarding the species’ ability to withstand adverse conditions caused by heavy metal toxicity. A decline in the stress tolerance index indicates a reduction in plant height relative to control conditions, highlighting the detrimental effects of heavy metals on growth and development. While both relative growth rate and stress tolerance index of B. chinensis (Fig. 5A and B) were significantly impacted by metal treatments, only the relative growth rate of B. rapa showed a significant effect (Fig. 5A). In the various spiked treatments, the relative growth rate of both species exhibited a decline in growth performance from 0 mg/kg (control) to increasing metal concentrations up to 300 mg/kg of Cd, Cr and Pb in the soil. Both B. chinensis and B. rapa displayed a consistent decrease in growth rate from 0.05 cm/day to 0.04 cm/day with rising concentrations. However, the reduction in the relative growth rate for 300 mg/kg Cd, Cr and Pb (Treatment 4) was significantly different from the control treatment (T0) for both species.

Fig. 5.

(A) Relative growth rate (RGR) and (B) stress tolerance index (TI_ph) of Brassica chinensis var. parachinensis and Brassica rapa L. under different soil metal treatments: T0: control soil, T1: 75 mg/kg Cd, Cr and Pb, T2: 150 mg/kg Cd, Cr and Pb, T3: 225 mg/kg Cd, Cr and Pb, and T4: 300 mg/kg Cd, Cr and Pb. Different letters represent significantly different means for each species following statistical analysis using one-way ANOVA and TukeyHSD tests at 5 % significance level.

However, the stress tolerance index exhibited contrasting patterns between B. chinensis and B. rapa (Fig. 5B). B. chinensis displayed a steep decline in the stress tolerance index, plummerting from 100 % to 56.6 % in response to spiked treatments with 300 mg/kg metals, while the stress tolerance index for B. rapa remained relatively steady across various metal treatments. Only the stress tolerance index of B. chinensis under all metal treatments (T1 to T4) was significantly lower than T0 (control treatment), whereas this difference was not observed for B. rapa. This indicates that the relative growth rate of B. chinensis was significantly hindered by the presence of heavy metals, suggesting that metal stress was specific to this Brassica species, not B. rapa. B. rapa accumulated the highest concentrations of Cd and Pb, supporting the notion that it demonstrates high tolerance to elevated soil concentrations of Cd, Pb and Cr [38]. In B. chinensis, the transport of heavy metals from the roots to the shoots could disrupt cellular metabolism, contributing to the decline in growth rate and subsequent slowdown in growth performance [60]. Elevated concentrations of heavy metals in the soil have been shown to distress shoot development, affecting plant height, and potentially influencing root growth, thereby reducing nutrient and water uptake by the plants [63,64].

3.3. Fresh biomass and edible index

Fresh biomass of B. chinensis and B. rapa provided quantitative data on the total plant biomass, including the leaves, stems and roots (Fig. 6A and C), reflecting the overall growth and development of the species under different soil treatments. On the other hand, the edible index determined the proportion of edible biomass (leaves and stems) affected by varying levels of heavy metal contaminations (Fig. 6B and D). B. chinensis consistently exhibited higher total fresh biomass (18.2–22.6 g/plant) across all control and treated soils compared to B. rapa (15.2–18.6 g/plant). Furthermore, B. chinensis yielded greater root biomass (1.06–1.33 g/plant) across the various spiked treatments compared to B. rapa (0.38–0.58 g/plant), leading to a higher edible index of over 97.0 % in B. rapa compared to the edible index in B. chinensis (92.9–94.8 %). However, this study observed insignificant differences in the fresh biomass of leaf, stem and root parts, as well as edible indices of both species under various soil treatments. The observed decline in relative growth rate in both species (Fig. 5A) and stress tolerance index in B. chinensis did not correlate with a decrease in fresh biomass and edible index. It is possible that dry biomass could have been influenced by metal concentrations, as reported by studies by Amin et al. (2013). Unfortunately, this study did not evaluate dry biomass. Overall, these findings suggest that the two Brassica species, particularly B. rapa, might demonstrate tolerance to Cd, Cr and Pb heavy metals (Fig. 4, Fig. 5, Fig. 6), implying their potential ability to thrive in toxic soil environments [60].

Fig. 6.

Fresh biomass and edible indices of (A–B) B. chinensis var. parachinensis, respectively, and (C–D) B. rapa, L., respectively, under different spiked soil treatments: T0: control soil, T1: 75 mg/kg Cd, Cr and Pb, T2: 150 mg/kg Cd, Cr and Pb, T3: 225 mg/kg Cd, Cr and Pb, and T4: 300 mg/kg Cd, Cr and Pb. Different letters represent significantly different means for each species after analyzing using one-way ANOVA and TukeyHSD tests at 5 % significance level.

3.4. Leaf area and root length

The average leaf areas and root lengths of B. chinensis and B. rapa were measured to investigate the effects of heavy metals on the physiological growth of Brassica plants and the adaptation of species to grow effectively in contaminated environments (Fig. 7A and B). Leaf areas for B. chinensis and B. rapa showed an increase of 23.5 % and 11.8 %, respectively, in the highest soil treatment compared to the control soils, although these differences were not statistically significant (Fig. 7A). While there were no significant differences in leaf areas of B. chinensis, a trend toward larger sizes was observed with increasing soil-metal treatments, up to 300 mg/kg metals. Conversely, consistent leaf areas were observed for B. rapa across different soil treatments. No visible signs of toxicity were detected on the leaves of either species, suggesting potential tolerance or adaptive mechanisms to high metal concentrations, particularly in B. rapa [65].

Fig. 7.

(A) Leaf areas and (B) root lengths of Brassica chinensis var. parachinensis and Brassica rapa L. under different soil metal treatments: T0: control soil, T1: 75 mg/kg Cd, Cr and Pb, T2: 150 mg/kg Cd, Cr and Pb, T3: 225 mg/kg Cd, Cr and Pb, and T4: 300 mg/kg Cd, Cr and Pb. Different letters represent significantly different means for each species following analysis using one-way ANOVA and TukeyHSD tests at 5 % significance level.

The average maximum root lengths for B. chinensis and B. rapa across all soil treatments were 11.2 cm and 6.26 cm, respectively (Fig. 7B). However, neither species exhibited symptoms of metal contamination, and there were no significant differences in root lengths from the control soil (T0) to the highest soil treatment (T4). Additionally, there were no notable differences between root lengths for both species across different soil treatments, indicating their ability to grow and absorb nutrients normally under stress conditions. Despite a significant reduction in the relative growth rate for both species, this did not translate in significant differences in leaf areas and root lengths, mirroring the findings on fresh biomass and edible index. In theory, a decline in the relative growth rate should negatively impact fresh biomass, edible index, leaf area and root length [51]. Leaf area development and plant growth are typically influenced by the division and enlargement of plant cells, processes that could adversely affected by the presence of Cd and Pb [66]. However, the stimulatory effect of Cd in leaves could be attributed to the plants’ defense mechanism, where they synthesize stress proteins and secondary metabolites, alter antioxidant enzyme activity and reduce oxidative stress [[67], [68], [69]]. These results suggest that both B. chinensis (to a certain extent, given its low stress tolerance index) and B. rapa may exhibit tolerance to metal stress conditions, making them potential candidates for soil remediation techniques.

3.5. Heavy metals in plant species

Table 3 presents the concentrations of heavy metals in various plant parts (leaf, stem and root) of B. chinensis and B. rapa across different soil treatments (0 mg/kg – 300 mg/kg Cd, Cr and Pb). Heavy metal contents in different parts of Brassica plants were measured to understand how these plants absorb and accumulate toxins from the soil, assessing their ability to tolerate and potentially remediate contaminated environments. However, the duration of the pot experiment was only 60 days, which is relatively short to fully capture the long-term effects of heavy metal exposure on plant growth and development. Among the soil treatments (T1 – T4), B. rapa exhibited the highest accumulation of Cd, with 864 mg/kg Cd in root of T4, compared to B. chinensis, which accumulated 739 mg/kg Cd in root of T4. Notably, Cd tended to accumulate significantly in the roots of both species across all treatments, except in the control soils where no significant differences were observed. Previous studies have shown that Cd tends to distribute evenly across all plant parts in various species under natural conditions [38]. The sequestration of Cd in root vacuoles, particularly when exposed to high soil Cd levels, suggests the presence of Cd transporters as detoxification mechanisms in these Brassica species. These mechanisms facilitate the uptake and localization of Cd, minimizing its exposure in shoots [[70], [71], [72]]. Under toxic conditions, root vacuoles play a crucial role in storing ions and metabolites, contributing to the detoxification process and normal cell development [73]. These findings underscore the potential and tolerance of Brassica species, particularly B. rapa, to germinate and thrive in toxic environments.

Table 3.

Mean heavy metal concentrations (standard deviation, SD) in the different plant parts (leaf, stem and root) of Brassica chinensis var. parachinensis and Brassica rapa L. Different letters per heavy metal (row) represent significantly different means for the different parts (leaf, stem, and root) for each species following analysis using one-way ANOVA and TukeyHSD tests at 5 % significance level.

Heavy metals (mg/kg), dry weight	B. chinensis
	Cd			Cr			Pb
	Leaf	Stem	Root	Leaf	Stem	Root	Leaf	Stem	Root
Control (T0)	29.0b (19.7)	6.47a (1.92)	14.7ab (4.34)	17.3b (6.76)	9.95a (1.96)	38.5c (3.19)	72.2b (22.5)	44.2a (4.50)	169c (30.1)
Treatment 1 (T1)	47.5a (17.6)	31.5a (5.99)	139b (35.6)	21.1a (6.41)	16.3a (2.65)	84.6b (13.3)	123b (16.7)	61.7a (14.1)	314c (44.5)
Treatment 2 (T2)	65.0a (11.8)	52.4a (8.39)	343b (129)	19.1a (10.1)	24.5a (2.78)	164b (49.3)	202b (23.0)	86.2a (10.8)	542c (58.6)
Treatment 3 (T3)	92.3a (19.9)	69.1a (16.3)	456b (169)	22.1a (8.66)	33.6a (2.64)	130b (37.7)	279b (22.4)	116a (8.90)	620c (53.0)
Treatment 4 (T4)	106.5a (35.4)	78.7a (16.4)	739b (150)	39.9a (12.9)	44.5a (5.51)	161b (37.2)	406b (35.2)	152a (19.2)	953c (83.7)
B. rapa
Control (T0)	62.7a (43.1)	13.1a (6.65)	10.8a (10.4)	12.4a (2.89)	4.66a (2.11)	32.4b (12.2)	80.5b (25.1)	54.9ab (11.5)	23.6a (29.1)
Treatment 1 (T1)	86.5a (48.3)	47.4a (25.5)	115a (63.4)	13.2a (7.15)	19.1a (4.55)	72.0b (17.6)	105ab (21.2)	84.5a (10.3)	125b (35.0)
Treatment 2 (T2)	165a (54.8)	125a (11.9)	409b (70.5)	30.8a (12.4)	29.9a (2.08)	130b (34.1)	243b (84.9)	99.9a (6.09)	270b (71.2)
Treatment 3 (T3)	205a (47.7)	219a (61.1)	603b (67.6)	30.8a (10.5)	36.3a (6.40)	147b (37.6)	290b (32.5)	106a (8.50)	417c (48.9)
Treatment 4 (T4)	273a (75.3)	258a (39.2)	864b (383)	18.0a (9.42)	40.6b (2.50)	132c (23.1)	340b (19.5)	123a (9.52)	478c (42.2)

The patterns of Cr contents in both B. chinensis and B. rapa reflected the accumulations of Cd in different plant parts (leaf, stem and root) across the various soil metal treatments, with Cr significantly accumulating in the root part of both species. There were no significant differences observed between the leaf and stem parts in both the Brassica species, indicating minimal Cr uptake in the aerial parts compared to the roots. Both species accumulated similar amounts of Cr in different plant parts, with B. chinensis and B. rapa accumulating the highest amounts in the root parts at 161 mg/kg and 132 mg/kg, respectively. Cr, being a non-essential heavy metal, did not have any known biological function in plant physiology, leading to the absence of a specific uptake mechanism for Cr in plants [74,75]. The translocation and distribution of Cr are influenced by factors such as plant species, Cr concentration in the growth medium and its oxidation state and due to its low mobility in plant roots, the concentration of Cr is 100 times higher in roots than in aerial parts of plants [62,76,77]. In this study, Cr concentration was observed to be the highest in the roots, which could be attributed to the sequestration of Cr in the vacuoles of root cells as a protective mechanism [78], thereby naturally enhancing the tolerance of the two species to Cr toxicity [79].

Significant differences were observed in the various plant parts (leaf, stem and root) of B. chinensis and B. rapa (Table 3), with notable Pb accumulation detected in both the leaf and root parts of both species. This suggests that both species possess the capability to accumulate substantial amounts of Pb in their aerial parts. At spiked Pb concentrations of 300 mg/kg in soil (T4), both B. chinensis and B. rapa accumulated up to 406 mg/kg and 340 mg/kg Pb, respectively (Table 3), in the leaf part of the plants, indicating the high mobility of Pb in Brassica species. B. chinensis exhibited the highest Pb content (953 mg/kg Pb) in the root part of the plant compared to B. rapa. Some Brassica species may regulate the uptake of highly mobile Pb through intracellular detoxification involving sequestration and cell binding in plant cells to reduce the translocation of Pb to the aerial parts of the plants [62,[80], [81], [82]]. Although Pb tends to highly mobile at lower soil pH and is typically found in water-soluble complexes such as CdSO₄ compounds, CdCl⁺, CdHCO₃⁺ cations and Pb²⁺ [83,84], this study demonstrated that Pb could readily accumulate in the plant parts of Brassica. These findings suggest that both Brassica species have the potential to accumulate significant amounts of Cd, Cr and Pb and could be cultivated in contaminated soil environments containing up to 300 mg/kg of heavy metals. Future studies could also assess the potential impacts of heavy metal contamination on soil microbial communities, which could play a crucial role in nutrient cycling and plant health, to further understand the limitations of these species.

4. Conclusion

Previous studies have highlighted the phytoremediation potential of these species, but their specific responses to heavy metal treatments based on their morphological, physiological and biochemical traits have been relatively understudied. The findings indicate that B. rapa and, to some extent, B. chinensis possess the capacity to accumulate significant amounts of non-essential heavy metals (Cd, Cr and Pb) from soils, reflecting their tolerance to toxic soil conditions, which is crucial for soil remediation efforts. Germination percentages for both the Brassica species were consistent across various soil treatments, albeit with delays in more toxic environments. The relative growth rate notably decreased at higher soil treatments, highlighting the impact of heavy metals on Brassica growth rate, particularly evident at 300 mg/kg Cd, Cr and Pb concentrations. The stress tolerance index suggested a substantial decline in B. chinensis’ plant heights compared to controls, whereas B. rapa exhibited stability, suggesting its resilience to toxic conditions. Despite insignificant differences due to varied metal treatments, B. chinensis exhibited higher fresh biomass compared to B. rapa, with B. chinensis also displaying greater root biomass. Leaf areas increased with higher soil treatments for both species, while root lengths showed no significant differences, indicating their tolerance to high metal concentrations. B. rapa accumulated the highest amount of Cd (864 mg/kg Cd), while B. chinensis accumulated the most Pb (953 mg/kg Pb), primarily in the root parts. Both species similarly accumulated Cr in the roots. The hypothesis underlying this research posited that B. chinensis and B. rapa would exhibit varying degrees of tolerance and accumulation of Cd, Cr, and Pb in response to increasing concentrations of selected heavy metals in soil. Further investigations through long-term effects field studies can provide insights into the effects of heavy metal contamination on both soil and plant health in real-world agricultural settings. The study suggests that both Brassica species, particularly B. rapa, exhibit high tolerance to non-essential heavy metals and can thrive in toxic soil conditions, potentially serving as hyperaccumulators for green remediation techniques.

Data availability statement

Data will be made available on request.

CRediT authorship contribution statement

Adzrin Asikin Zunaidi: Writing – review & editing, Writing – original draft, Visualization, Validation, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization. Lee Hoon Lim: Writing – review & editing, Visualization, Supervision, Resources, Project administration, Funding acquisition, Data curation, Conceptualization. Faizah Metali: Writing – review & editing, Visualization, Supervision, Resources, Project administration, Funding acquisition, Data curation, Conceptualization.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.