Phytoremediation and Compost-Assisted Phytoremediation of a Heavy-Metal-Contaminated Soil: A Sustainable Approach Using Waste-Derived Amendments

Abstract

Soils contaminated with heavy metals including cadmium, lead, zinc, copper, and chromium continue to represent a significant environmental issue, particularly in areas affected by industrial activities. In this context, the present study aimed to assess the feasibility and efficiency of an integrated bioremediation technique that combines, in a synergistic approach, phytoremediation with the use of natural amendments in order to reduce soil pollution with heavy metals. In addition, the potential for heavy metal recovery was investigated. The experiments were conducted under field conditions in the vicinity of the CET II Holboca power plant, using two plant species, Trifolium repens and Brassica napus, as bioaccumulators, while biochar was applied as a natural amendment. The analyses focused on metal concentrations, translocation factors, the degree of heavy metal recovery, and morpho-structural characteristics. The results indicated a high accumulation of metals in plant roots, particularly in soils treated with higher doses of biochar (4905.93 mg/kg iron for B. napus), and a significant growth stimulation (root elongation increases of up to 78% in T. repens and up to 29% in B. napus). B. napus exhibited greater translocation of metals to the aerial parts (with values up to 0.83 for zinc), whereas T. repens predominantly retained metals within the root system. The highest recovery efficiency values were observed in the case of lead, reaching 224.7% in T. repens and 86.7% in B. napus in soil amended with increased amounts of biochar. Overall, biochar application stimulated plant growth and enhanced metal uptake efficiency, suggesting a viable and practically applicable method for the ecological reconversion of contaminated land.

1. Introduction

The expansion of industrial activities, mining, and improper waste management has led to the widespread accumulation of heavy metals in soils. These persistent contaminants increase soil toxicity, disrupt ecosystem functions, and can enter food chains, posing significant threats to biodiversity and human health [1]. As the rate of pollutant release increasingly exceeds the natural capacity of ecosystems to recover, sustainable and nature-based remediation strategies are urgently needed to restore soil health, protect biodiversity, and ensure long-term environmental resilience [2]. In this context, phytoremediation is increasingly recognized as a sustainable solution that combines natural plant processes with innovative technologies to reduce soil contaminants, limit their mobility, and promote ecosystem recovery [3]. Phytoremediation involves several distinct strategies, such as phytoextraction and phytostabilization, and its success depends on plant species, pollutant type, and soil conditions [4,5]. Among these, Trifolium repens has demonstrated the ability to tolerate and accumulate metals such as cadmium, lead, copper, iron, and zinc, supporting both soil remediation and the conservation of plant species diversity in impacted ecosystems [6,7].

Plant species belonging to the families Brassicaceae and Fabaceae have been widely recognized for their tolerance to contaminated soils and their capacity to accumulate or stabilize heavy metals, making them suitable candidates for phytoremediation and post-remediation biodiversity recovery [8]. Consequently, the use of such plant species contributes not only to pollution control but also to habitat restoration and the long-term conservation of plant species diversity in impacted ecosystems [9].

Conventional ex situ bioremediation involves excavating soil for treatment in controlled systems and is often used at heavily polluted sites for rapid contaminant removal [10,11]. However, these methods are costly, disrupt soil, and may cause secondary pollution, threatening ecosystem function and biodiversity [12]. In contrast, phytoremediation relies on higher plants to extract, accumulate, transform, or stabilize pollutants directly in situ. This approach represents a sustainable and environmentally friendly remediation strategy, as it minimizes soil disturbance and preserves its physical structure [13]. At the same time, phytoremediation contributes to ecological restoration by improving soil quality, supporting the recovery of microbial communities, and enhancing overall biodiversity within the affected ecosystems [14].

Members of these families include both hyperaccumulator species and high-biomass plants suitable for remediation approaches that support soil recovery and later recolonization by native flora. Phytoremediation performance depends on plant physiological and structural traits, soil characteristics, and amendments that influence metal availability and uptake [15,16]. Heavy metals such as Cd, Pb, Cu, Fe, and Zn are persistent and toxic pollutants that contaminate soils and waters through industry, agriculture, and poor waste management [17,18].

Biochar is a carbon-rich, stable material produced through pyrolysis of biomass under limited oxygen conditions, used as a soil amendment to enhance carbon sequestration, improve soil properties, and immobilize contaminants. It is widely regarded as a durable soil amendment that supports long-term carbon storage and helps mitigate climate change by lowering CO₂ emissions [19]. Its stability in soil allows it to function as a persistent organic carbon pool, slowing decomposition and enhancing soil carbon accumulation [20]. Modified forms can also protect organic matter from microbial breakdown and reduce carbon losses through erosion and runoff [21]. Biochar’s carbon-rich composition and reactive surface groups enable it to adsorb heavy metals like lead, supporting phytoremediation by reducing pollutant bioavailability and improving soil contaminant removal [18].

In this context, the present study aims to evaluate the phytoremediation potential of Brassica napus L. (Brassicaceae) and Trifolium repens L. (Fabaceae) through a comprehensive assessment of their physiological and anatomical responses under different soil amendment treatments. By examining metal accumulation patterns and plant tolerance mechanisms, this research seeks to determine the suitability of these species for the remediation of contaminated soils and their role in habitat restoration and the conservation of plant species diversity in impacted ecosystems. These insights are of particular relevance for ecosystem restoration initiatives, where the combined use of tolerant plant species and natural amendments like biochar may enhance soil recovery, promote biodiversity, and support long-term ecological resilience.

2. Materials and Methods

The experiments were designed to identify the morphological, physiological, and histo-anatomical effects induced by the presence of heavy metal concentrations in soil. The evaluated parameters included a range of analyses and determinations aimed at highlighting the relevance of the selected plant species and providing insights into their potential application in an in situ bioremediation program.

2.1. Study Area

This study was conducted in the proximity of a large industrial Holboca (Iasi, Romania, 47°09′01.2″ N 27°43′06.4″ E) during a season of vegetation (May–June) (Figure 1). The annual mean temperature is from 9.7 to 9.8 °C, and the annual precipitation ranged from 570 to 572 mm [22]. The elevation ranged from 35 to 38 m a.s.l.

2.2. Soil Analysis

This study focused on sampling points in the study area, with samples collected at a distance of 80 m from each other [23]. The positioning of the sampling points on the map was achieved using GPS coordinates recorded in the field, using the AndroidTS GPS (Version 1.48) test application. The recorded coordinates were then exported in KML format, and the resulting data were transposed onto the map. The graphs were created in Microsoft Excel 2021, and the map was created in QGIS version 3.34.

2.3. Materials

Spruce bark (Picea abies L., H. Karst.), a by-product of the wood-processing industry, was collected from a sawmill located in Vatra Dornei. After air-drying at room temperature under normal ventilation conditions, the bark was ground to a particle size of 1–1.5 mm and stored in a desiccator until further analysis.

Soil amendment consisted of a biochar produced in the laboratory through the pyrolytic conversion of Picea abies bark. The spruce bark was subjected to a slow pyrolysis process at 550 °C [18,24].

Both the raw spruce bark and the biochar obtained through thermochemical conversion were physically, chemically, morphologically, and structurally characterized within the research group. These materials exhibited multiple functional groups, including hydroxyl (–OH) and carboxyl (–COOH) groups (biochar), as well as aromatic methoxyl (C–H) and carbonyl (C=O) groups (spruce bark).

For the biochar produced by slow pyrolysis, analyses revealed a specific surface area of 62.3 m² g⁻¹, an adsorption volume of 0.09 cm³ g⁻¹, and a micropore volume of 0.008 cm³ g⁻¹. Porosity and specific surface area are key properties of adsorbent materials and, together with surface functional groups, contribute significantly to pollutant retention capacity [24].

The seeds of Trifolium repens and Brassica napus used in the experiments were purchased from the company Jelitto Staudenzamen GmbH (Schwarmstedt, Germany) (https://www.jelitto.com).

2.4. Experimental Methodology

Soil collected from the investigated site was prepared for the control treatment by sieving through a 2 mm mesh and weighing 150 g per vegetation pot. Distilled water was added to reach 50% soil moisture. The soil was then incubated at 25 ± 1 °C and 65 ± 5% relative humidity in the dark for 10 days.

After the 10-day pre-treatment period, the soil was amended. The experimental treatments were coded as follows: S (control soil), LB (soil amended with low biochar content—3 g/150 g soil), HB (soil amended with high biochar content—7.5 g/150 g soil), and GSB (soil amended with ground spruce bark—3.5 g/150 g soil), corresponding to the treatments applied in the vegetation pots. Soil moisture was maintained at 50%, and the amended soils were incubated for an additional 30 days.

After the 30-day soil incubation period, seeds of the two selected hyperaccumulator plant species—B. napus a cultivated crop plant, and T. repens, a species naturally occurring in this study area’s vegetation—were sown. Each treatment was performed in triplicate, with three seeds manually sown in each vegetation pot. The cultivated soils were irrigated daily with 15 mL of distilled water.

After 40 days of growth, plants were harvested and separated into roots, petioles, and leaves, which were subsequently subjected to characterization analyses to assess the effects of cultivation conditions (the plant species and soil amendments applied) on plant growth and development, as well as on the retention of heavy metals from contaminated soil.

2.4.1. Gravimetric and Biometric Assessment

To determine growth differences in terms of elongation of vegetative organs (root, stem, and leaves), the biometric method was used. This method was applied 10 days after sowing, and measurements were performed using a digital caliper. The vegetative organs studied were the root, stem, and leaves.

The determination of fresh plant biomass accumulation was carried out using the gravimetric method with an analytical balance. Inhibitory effects were evaluated as percentages compared to the control variant.

2.4.2. Determination of Metal Ion Concentration

The analysis of the concentration of heavy metal ions was carried out according to the SR ISO 11047:1999 standard [25]. The soil samples, dried and finely ground, were subjected to a mineralization process by disaggregation with a mixture of nitric acid (65%), hydrochloric acid (35%) and deionized water, in a ratio of 4:12:5 (v/v/v), heated at 150 °C, for 7 h. After cooling, the samples were filtered with cellulose acetate membranes with a porosity of 0.45 µm and then diluted to the desired volume. The determinations were carried out in triplicate, and the concentrations of heavy metals were evaluated using the GBS Avanta atomic absorption spectrometer.

The analysis was carried out using a 99.95% pure air/acetylene flame, with triplicate readings with background correction. Calibration curves with a minimum allowable determination coefficient of R² > 0.995 were accepted prior to each analysis. The materials were passed through 0.45 µm porosity cellulose acetate membrane filters before being subjected to AAS-flame analysis.

Plants were harvested 40 days after cultivation and separated into roots, stems, and leaves. The plant material was dried at 28 °C for one week, weighed, and then calcined at 550 °C for 2 h. Each vegetative organ was transferred into 250 mL reaction vessels, where 0.5 mL bidistilled water, 10.5 mL of 12 mol/L HCl, and 3.5 mL of 15.8 mol/L HNO₃ were added dropwise under constant agitation to minimize foaming.

The resulting mixture was left for 2 h at room temperature to allow for slow oxidation of organic matter, then heated on an electric hot plate under reflux conditions for 2 h, ensuring that the condensation zone did not exceed one-third of the condenser height. After cooling, 10 mL of 0.5 mol/L HNO₃ was added to rinse the condenser, followed by filtration and dilution of the filtrate [26].

Metal ion concentrations in different plant parts were used to calculate the translocation factor and recovery rate, defined as follows:

• Translocation factor (TF) = metal ion concentration in aerial parts/metal ion concentration in roots.
• Recovery rate (%) = metal concentration in roots, stems, and leaves/metal ion concentration in the growth medium (soil) [27].

All determinations were performed in triplicate, and heavy metal concentrations were measured using a GBS Avanta atomic absorption spectrometer (GBC Scientific Equipment Pty Ltd., Braeside, Victoria, Australia).

2.4.3. SEM Analysis of Vegetative Organ Structure

Plant material (leaves, petioles, and roots) was fixed and stored in 70% ethanol. Selected vegetative organs were sectioned under a stereomicroscope using a razor blade. Following dehydration through a graded ethanol series (80%, 90%, and 100%) and acetone, the samples were dried using CO₂ critical point drying (K850 Critical Point Dryer, EMS, Hatfield, PA, USA). The dried material was sputter-coated with a thin gold layer (30 nm) using an EMS 550X coating system and subsequently examined by scanning electron microscopy (SEM) (Tescan Vega II SBH, TESCAN, Brno, Czech Republic) at an accelerating voltage of 30 kV [28].

2.4.4. Statistical Analysis

Differences among soil treatments (S, GSB, LB, and HB) were evaluated using one-way analysis of variance (ANOVA). When the ANOVA indicated significant treatment effects, pairwise comparisons of the means were performed using Tukey’s Honestly Significant Difference (HSD) test. Statistical significance was established at p < 0.05.

All analyses were carried out in the R Statistical Software (version 4.1.4; R Core Team, 2024) [29].

3. Results

3.1. Biometric Determinations

In B. napus, one-way ANOVA indicated that soil type significantly affected root, stem, and leaf elongation (p < 0.001). Tukey’s HSD tests showed that plants grown in GSB soil had consistently lower growth compared with those in LB and HB soils. No significant differences in root length were observed among S, LB, and HB treatments, whereas stems and leaves of B. napus were significantly longer in HB than in S. B LB and HB soils stimulated vegetative growth in B. napus (up to 29% for roots and 20% for leaves), while GSB limited plant development (Figure 2A).

In T. repens, soil type significantly influenced the elongation of all analyzed organs. Root growth in T. repens was significantly greater in LB and HB soils than in S, with root elongation increasing up to 78%, while GSB caused notable reductions, especially in leaves (−38%). Stem elongation was lowest in GSB, while HB promoted greater growth than S. Leaf elongation in T. repens differed significantly among most soil treatments, except between LB and HB, and overall the species showed the best growth performance in LB and HB soils (Figure 2B).

Regarding the number of leaves (Figure 3), an increase was observed in both B. napus and T. repens in the LB and HB treatments.

3.2. Gravimetric Determinations

In both B. napus and T. repens, an increase in fresh biomass was observed across all experimental variants. In all experiments, relatively consistent stimulation values of fresh biomass accumulation were recorded for each vegetative organ. The results indicate a pronounced stimulation of fresh biomass growth in both B. napus and T. repens, particularly in the LB and HB treatments (Figure 4).

3.3. Variation of Heavy Metal Ion Concentrations in Vegetative Organs

The analysis of cadmium, copper, iron, lead, and zinc concentrations in roots, stems, and leaves showed that amending the growth media with 7.5 g of biochar (HB) significantly enhanced the bioaccumulation of heavy metals. The addition of 3 g (LB) and 7.5 g (HB) of biochar to polluted soils stimulated heavy metal accumulation in both B. napus and T. repens in the LB and HB treatments.

In B. napus, cadmium accumulation occurred predominantly in the roots across the four soil types tested (S, GSB, LB, and HB) (Figure 5). The highest root concentration was observed in HB soil (0.71 mg/kg), followed by LB (0.50 mg/kg), while leaves contained no detectable cadmium. Stems showed only minimal accumulation, with 0.23 mg/kg in LB and 0.26 mg/kg in HB (Figure 5).

Copper primarily accumulated in the roots of both species, with higher concentrations in biochar-amended soils. In B. napus, the maximum values in HB soil were 15.24 mg/kg (root), 4.99 mg/kg (stem), and 1.70 mg/kg (leaf) (Figure 6). T. repens exhibited even higher concentrations, reaching 21.69 mg/kg in HB-treated roots (Figure 6).

Iron accumulation was substantial in the roots of both species, peaking in HB soil at 4905.93 mg/kg for B. napus and 4355.81 mg/kg for T. repens (Figure 7). Stems and leaves also showed elevated levels, particularly in LB and HB soils, with B. napus presenting significantly higher leaf concentrations (205.36 mg/kg) compared to T. repens (87.03 mg/kg) (Figure 7).

Lead was mainly accumulated in the roots, with maximum values in HB soil: 41.55 mg/kg in B. napus and 44.78 mg/kg in T. repens. Translocation to stems and leaves was more efficient in LB and HB treatments. In leaves, T. repens showed slightly higher lead concentrations than B. napus (3.67 vs. 3.20 mg/kg in HB), indicating slightly greater mobility of lead in this species (Figure 8).

Zinc also accumulated predominantly in roots, reaching 17.60 mg/kg in B. napus and 18.33 mg/kg in T. repens in HB soil. Translocation to stems and leaves was more pronounced in LB and HB treatments. B. napus displayed higher leaf concentrations (3.88 mg/kg in HB) compared to T. repens (2.22 mg/kg) (Figure 9).

3.4. Translocation Factor of Various Heavy Metals in Vegetative Organs of Brassica napus and Trifolium repens

Translocation factors for B. napus indicated an increased mobility of zinc (Zn), with values up to 0.83 on HB soil, followed by copper (Cu), which reached 0.43. Cadmium (Cd) showed a generally low translocation, with a peak on LB soil (0.45). Iron (Fe) and lead (Pb) had the lowest translocation factors, remaining below 0.2–0.36. Biochar soils (LB and HB) clearly favoured the transfer of metals to the aerial parts of the plant, especially in the case of Zn and Cu.

In T. repens, the translocation factors indicated a relatively high mobility of zinc (Zn) within the plant, with values ranging between 0.25 and 0.62. Copper (Cu) also showed noticeable mobility, reaching a value of 0.33 on LB soil. Cadmium (Cd) presented a moderate level of translocation, with a maximum value of 0.53, while lead (Pb) and iron (Fe) displayed lower translocation factors, remaining below 0.32. The highest values for these elements were generally recorded on LB and HB soils, suggesting that the presence of biochar in the substrate may enhance the movement of certain metals within the plant. In particular, this effect appeared more evident for Zn and Cu, which were more efficiently translocated to the aerial organs of the plant under these soil conditions (Figure 10).

3.5. The Recovery Degree of Different Heavy Metals in the Vegetative Organs of Brassica napus L. and Trifolium repens L.

Following the calculation of the recovery degree (%), obtained by relating the concentration of heavy metals (Zn, Cu, Cd, Pb, and Fe) in the roots, stems, and leaves to the concentration of these metals in the growth medium [27], it was observed that the addition of organic amendments, namely spruce bark and biochar, contributed to an improvement in the recovery of heavy metals from the growth medium. This trend was evident in the HB experiment and was recorded for both plant species studied, B. napus and T. repens. The presence of these organic materials in the substrate appears to influence the interaction between the plants and the growth medium, facilitating a more effective uptake and recovery of heavy metals by the plant tissues. As a result, the amended growth medium supported a more pronounced recovery response compared with the unamended conditions, highlighting the positive role of spruce bark and biochar in enhancing the recovery of heavy metals in the HB experimental setup for both species.

The degree of cadmium recovery was higher in T. repens, especially in the HB soil, where values reached 21.6% in roots and 8.2% in stems. In B. napus, the maximum recovery was 14.2% in roots and 5.2% in stems, also on HB soil. In both species, cadmium was not translocated to the leaves (Figure 11).

The degree of copper recovery was higher in T. repens, particularly in plants grown on HB soil, where the highest values were observed in the roots and stems. In this case, recovery reached 8.03% in roots and 2.24% in stems, indicating a pronounced accumulation of copper in the below-ground plant organs and a moderate transfer to the stems. In B. napus, the maximum recovery values were lower, reaching 2.45% in roots and 0.80% in stems. Unlike T. repens, B. napus also showed a moderate transfer of copper to the leaves, with a value of 0.27% recorded on HB soil. These results suggest differences between the two species in the distribution and translocation of copper within plant tissues (Figure 12).

The degree of iron recovery was higher in B. napus, particularly in the case of plants grown on HB soil, where the highest values were recorded in the roots, followed by the stems and leaves. Specifically, the recovery reached 0.54% in roots, 0.08% in stems, and 0.022% in leaves. In T. repens, the maximum recovery was observed mainly in the roots, reaching 0.46%, while the stems showed a value of 0.073%, and the leaves presented lower recovery values compared with the other plant organs. These results indicate that, for both species, iron accumulation occurred predominantly in the root system, with progressively smaller amounts translocated to the aerial parts of the plants (Figure 13).

The degree of lead recovery was significantly higher in T. repens, with a maximum of 224.7% in roots on HB soil, followed by 53.3% in stems and 18.4% in leaves. In B. napus, the values were lower, with a peak of 86.7% in roots, 24.3% in stems, and 6.6% in leaves (Figure 14).

The Pb recovery rate exceeded 100% in some treatments (particularly in T. repens roots under the influence of HB) because the indicator used is a concentration ratio (organ concentration/soil concentration), not a mass balance recovery. In contaminated soils, Pb distribution can be heterogeneous at small spatial scales, meaning that the measured soil concentration may underestimate the actual concentration encountered by the roots during growth [30]. In addition, biochar amendment can modify soil conditions (e.g., pH, CEC—Cation Exchange Capacity) and rhizosphere processes, influencing the fraction of Pb available to plants over the course of the experiment, which may increase the calculated ratio [31].

The degree of zinc recovery was slightly higher in T. repens, particularly in the roots and stems of plants grown on HB soil, where values of 3.01% and 1.51%, respectively, were recorded. In contrast, B. napus showed higher recovery values in the leaves, reaching 0.52%. Across all soil types analyzed, both plant species exhibited a consistent increase in zinc recovery from S to HB. Moreover, comparable values were generally observed in the roots and stems for both species, indicating a similar pattern of zinc uptake and distribution within these plant organs (Figure 15).

3.6. Histo-Anatomical and Micro-Morphological Aspects of the Analyzed Species Under the Action of Natural Amendments

Histo-anatomical features of the vegetative organs of the seedlings of the two species were analyzed on transverse sections taken through the leaf blade (made at its midpoint), the petiole (sectioned in the median region), and the root (at 1 cm from the tip). The sections were analyzed using a scanning electron microscope, as their fragile structure did not allow for processing by classical techniques commonly used in plant histology.

3.6.1. Histo-Anatomical Aspects of Trifolium repens Seedlings

In cross-sections, the leaf was amphistomatic and bifacial (Figure 16A), with a unilayered palisade parenchyma beneath the upper epidermis, a multilayered spongy tissue with large intercellular spaces, and simple vascular bundles lacking mechanical elements (Figure 16B).

The upper epidermis consisted of large cells elongated tangentially, with thin cell walls, while the lower epidermis had a similar structure. Numerous stomata were present in both epidermises; the stomatal apparatus was anomocytic, the guard cells being surrounded by ordinary epidermal cells without specialized subsidiary cells (Figure 16C). The external walls of the upper epidermal cells were covered by a very thin cuticle bearing epicuticular wax formed by fine anastomosed microtubules, and in surface view the epidermal cells appeared polygonal and nearly isodiametric.

The palisade tissue was formed of a single layer of cells 2–3 times longer than wide, arranged perpendicular to the epidermis, while the spongy tissue consisted of 4–6 layers of smaller rounded or slightly elongated cells with large intercellular air spaces (Figure 16B). The petiole showed a cordiform transverse section with a single-layered epidermis, homogeneous parenchymatous ground tissue, and three open collateral vascular bundles arranged at the corners of the section. The petiole had a central aeriferous cavity (Figure 16D).

The root transverse section was circular and showed a developed secondary structure, with a central vascular system consisting of a thick phloem ring surrounding a central xylem body, and the formation of a thin periderm.

In the seedlings grown on GSB soil, the general structure of the vegetative organs was similar to that described in the previous variant. The palisade tissue was well developed, with elongated cells oriented perpendicular to the epidermis (Figure 16E). The petiole ground parenchyma was compact, lacked in air spaces, and contained three vascular bundles, with a sclerenchyma sheath visible at the phloem pole.

In the seedlings grown on LB soil, the leaf blade was thinner, with the reduction in thickness resulting from a mesophyll composed of fewer cell layers; the palisade tissue was reduced, and its cells became nearly isodiametric, resembling those of the spongy tissue.

Both epidermises retained thin cell walls. The cuticle on the upper epidermis was partially eroded in the central region of the cells, while the lower epidermis showed a smooth cuticle with areas where epicuticular wax was partially detached (Figure 16F); occasionally multicellular glandular hairs oriented parallel to the epidermal surface could be observed.

In the petiole, four vascular bundles were observed—three large and one smaller, additional to those in the control—and the ground parenchyma persisted in the central region, with no aeriferous space as seen in the control.

In the plants grown on HB soil, the petiole had a compact structure with four vascular bundles and a homogeneous, parenchymatous cortical parenchyma lacking aeriferous spaces (Figure 16G). The leaf blade showed a structure similar to that of the control, but with a better-developed midrib and vascular bundles containing a higher number of xylem and phloem elements (Figure 16H). The upper epidermis was composed of slightly papillose cells, conical in cross-section; for this reason, the stomatal cells were located below the level of the epidermal cells, resulting in a shallow suprastomatic chamber (Figure 16I). The root also exhibited secondary structure, with a more consolidated periderm leading to exfoliation of the rhizodermis and outer cortex, and an increased number of xylem vessels compared to the control.

3.6.2. Histo-Anatomical Aspects of Brassica napus Seedlings

In control, the leaf blade was bifacial with a palisade layer under the upper epidermis with a prominent midrib and rare unicellular tector hairs on both epidermises (Figure 17A,B). The petiole had a V-shaped structure with a flattened adaxial surface, poorly developed ground parenchyma, and 7–8 vascular bundles of varying sizes (Figure 17A,B).

In the HB soil variant, the palisade tissue was formed of a single layer of relatively short cells, and the spongy tissue consisted of 4–5 layers of rounded cells, giving the mesophyll a normal dorsiventral organization (Figure 17D,E). The epidermises observed in cross-section consisted of larger, tangentially elongated cells in the upper epidermis and smaller, nearly isodiametric cells in the lower epidermis. In surface view, both epidermises showed isodiametric cells among which numerous stomata were present; the stomatal apparatus was anisocytic, typical for the family Brassicaceae, consisting of two larger subsidiary cells and one smaller cell.

The petiole was V-shaped in the cross-section, with a compact parenchyma, with five vascular bundles (three large centrally and two smaller in the arms) (Figure 17F). The epidermis was composed of small, isodiametric cells with slightly thickened outer walls, with stomata uniformly distributed and located at the same level as the surrounding epidermal cells; rare long unicellular tector hairs with pointed tips were also present. The ground parenchyma was homogeneous and compact, consisting of rounded cells with small intercellular air spaces.

The leaf blade was amphistomatic. The cuticle covering both epidermises was finely reticulated (Figure 17G).

The midrib was prominently visible on the abaxial (lower) side in both soil variants, while in the LB soil variant, it was strongly raised also on the adaxial (upper) side. In the LB variant, a collenchymatous mechanical tissue began to form beneath the upper epidermis along the midrib. The palisade tissue in the LB variant was better developed, consisting of more elongated cells with smaller intercellular air spaces, and the vascular bundles in the leaf blade were also more developed than in the HB variant, containing more xylem vessels and a few protective mechanical elements at the phloem pole (Figure 17H).

As in the leaf blade, the vascular bundles of the petiole were better developed in the LB variant (Figure 17I).

At the analyzed stage, the root tissues were predominantly secondary in origin, produced by cambial activity, while the phellogen had not yet differentiated. The vascular tissues consisted of a thin ring of secondary phloem at the exterior and a massive central secondary xylem body in which isolated vessels and numerous libriform elements with thickened and lignified walls could be observed. The cambium was active and consisted of 2–3 layers of flattened cells that divided intensively and produced mainly secondary xylem elements.

The LB soil variant (amended with 3 g biochar) favored vegetative development of the seedlings, the resulting individuals being more vigorous and showing better developed assimilatory and conducting tissues.

4. Discussion

The results obtained highlight significant differences between the two analysed species, Brassica napus and Trifolium repens, depending on the type of metal, the plant organ analysed, and the applied treatment. In the case of cadmium, predominant accumulation in the roots and its absence in the leaves confirm its low mobility in plants, as well as its high toxic potential for aerial organs. Its retention within the root system is considered beneficial, as it reduces the risk of translocation to the edible parts of the plant. Recent studies have emphasized the essential role of roots in Cd immobilization (through chelation and vacuolar sequestration mechanisms), thereby preventing its systemic effects on aerial organs [32,33].

Regarding copper, an essential micronutrient involved in enzymatic processes but toxic in elevated concentrations, T. repens demonstrated a more efficient mobilization, with higher recovery rates. This behavior is supported by recent findings highlighting the ability of legumes to tolerate metal stress through intracellular chelation mechanisms and the activation of antioxidant systems [34]. In contrast, B. napus exhibited a more balanced translocation of Cu to the leaves, suggesting an efficient systemic redistribution. Bortoloti and collaborators [32] reported that species of the Brassicaceae family possess a remarkable adaptive capacity for metal management through the regulation of membrane transporters and cellular detoxification mechanisms.

The iron concentrations were noticeably higher in B. napus, particularly in the aerial organs, indicating an enhanced capacity for active transport through xylem vessels. This phenomenon was promoted by the application of biochar, which, according to Chen and collaborators [35], can positively influence iron bioavailability by modifying soil pH and redox potential as well as stimulating rhizosphere microbial activity.

In the case of lead, both species accumulated high concentrations at the root level. However, T. repens exhibited more pronounced translocation to stems and leaves, especially in soils amended with HB biochar. This capacity was associated with a root recovery index exceeding 200%, confirming the species’ potential for the remediation of Pb-contaminated soils. Rajendran and collaborators [36] and Yan and collaborators [5] demonstrated that organic amendments can increase lead solubility and enhance its uptake by altering root exudation patterns and associated microbial communities.

Regarding zinc, B. napus recorded the highest concentrations in leaves, indicating superior efficiency in Zn translocation to aerial biomass. This characteristic makes the species well-suited for phytoremediation via phytoextraction, particularly in moderately contaminated areas. The valorization of biomass obtained from such crops through phytomining or energy conversion, thereby adding economic value to the remediation process, is supported by previous studies [4,37]. A central aspect of this study is the role of biochar as an organic amendment. In all cases, soils treated with higher biochar concentrations resulted in significant increases in plant biomass and metal accumulation. These findings are consistent with the results of Chen and collaborators [35], who reported that biochar not only reduces metal toxicity through immobilization and adsorption processes, but also improves nutrient availability and soil biological activity.

Although some quantitative and local anatomical differences were recorded among the soil variants, these changes did not translate into major structural modifications capable of limiting organ functionality in either species. The overall preservation of leaf mesophyll differentiation, together with the adequate development of vascular tissues, indicates that photosynthetic efficiency and assimilates and water transport was not adversely affected. Moreover, the maintenance of a stable histo-anatomical organization can be interpreted as part of the adaptive capacity of plants to tolerate heavy metal presence, allowing for resistance to metal-induced stress without structural impairment, as reported for other species exposed to contaminated soils [33,38].

A comparative evaluation of T. repens and B. napus highlights some differences in the practical applicability of the results obtained in laboratory studies, especially from the perspective of the cost-effectiveness of phytoremediation at field level. B. napus is a species with a rapid and high biomass production, which gives it increased possibilities of extracting heavy metals per unit area. Its rapid growth and well-developed root system make it suitable for short-term phytoremediation strategies, when rapid removal of large quantities of pollutants from the environment is necessary [39]. However, it should also be taken into account that this species has high nutritional requirements and special agricultural work, which can lead to increased costs when cultivated on a large scale for phytoremediation.

In contrast, T. repens has a slower growth rate but is more adaptable to different soil types, including compacted or nutrient-poor soils. In our study, these were solonthes—on recent fluvial and fluvial-lake deposits. The species ability to fix atmospheric nitrogen at the root nodule level leads to lower fertilizer requirements and lower agricultural costs, making it more desirable, from a cost perspective, for long-term phytoremediation activities [40]. Also, its low soil quality requirements make it useful in the decontamination of degraded soils.

When considering cost effectiveness, B. napus may involve higher upfront cultivation and management expenses, yet it can achieve faster contaminant reduction where intensive short-term remediation is required. T. repens, while less aggressive in terms of metal uptake on a per cycle basis, offers a more sustainable and lower input option for prolonged remediation projects or for integration into multifunctional land use systems. Overall, the choice between the two species should be guided by site specific objectives, available resources, and the desired balance between remediation speed and long-term economic sustainability.

The complementary properties of the two analyzed species, one characterized by high efficiency in belowground metal accumulation (T. repens), and the other by superior aerial translocation capacity (B. napus) provides a strong basis for the development of integrated phytoremediation strategies.

One limitation of the present study is the absence of long-term field monitoring data, as the experiment was conducted over a relatively short evaluation period. Consequently, the long-term stability of metal uptake, plant persistence, and the sustained effects of biochar amendments under natural environmental conditions could not be fully evaluated. Future research should therefore include extended field trials and multi-season monitoring to assess the durability of phytoremediation performance and the long-term interactions between plants, soil and metal mobility.

5. Conclusions

This study showed that Trifolium repens has an increased capacity to accumulate cadmium and lead in its roots, with increased recovery values especially in soil treated with high amounts of biochar. The species also demonstrated efficient mobilization of metals and their translocation to aerial organs, indicating good tolerance and adaptability to contaminated substrates. Brassica napus displayed a superior capacity for translocation of iron and zinc to aerial organs, recording the highest concentrations in leaves and stems, especially in soils amended with high doses of biochar (HB). This pattern suggests an efficient internal transport of metals toward harvestable organs, highlighting the species’ suitability for phytoextraction approaches where removal of contaminated biomass is targeted. For short-term phytoremediation of highly contaminated soils, B. napus species in combination with biochar (in different concentrations) is recommended due to its rapid accumulation of biomass which helps to remove heavy metals from the soil. On the other hand, for situations where the phytoremediation process is of increased duration, the use of T. repens species is recommended, which performs better in compact and nutrient-poor soils, even in the presence of lower amounts of additives. In conclusion, B. napus is preferred in situations requiring rapid decontamination, while T. repens can be used for long-term soil stabilization.