Evaluación fisiológica y bioquímica de Lippia alba quimiotipo carvona en ambientes contaminados con Cu(II) y Zn(II): un enfoque hacia la remediación ambiental

Resumen

Los metales pesados son contaminantes inorgánicos caracterizados por su gran persistencia en el medio ambiente y sus efectos tóxicos, incluso a concentraciones traza. Estos contaminantes son especialmente relevantes en zonas agrícolas periurbanas, donde diversas actividades domésticas, industriales y agrícolas actúan como fuentes de contaminación, lo que puede comprometer la seguridad alimentaria. La fitorremediación es un enfoque sostenible y respetuoso con el medio ambiente que utiliza plantas nativas para extraer metales pesados o mitigar su toxicidad en entornos contaminados. Además, ofrece la posibilidad de obtener fitoproductos de valor, facilitando así la restauración y transformación de estos sitios en paisajes productivos. Este estudio evalúa la respuesta de Lippia alba (Mill.) quimiotipo carvona, una planta aromática nativa, ante altas concentraciones de Zn(II) y Cu(II), analizando su crecimiento, bioacumulación y sus respuestas fisiológicas y bioquímicas. Ambos metales afectaron negativamente el crecimiento de la planta, provocando un aumento en la fuga de electrolitos y una reducción en el contenido de clorofila, carotenoides y proteínas. A pesar de estos efectos, L. alba mostró una tolerancia significativa sin registrarse mortalidad, respaldada por mecanismos de defensa antioxidante como la acumulación de prolina y compuestos fenólicos. La bioacumulación de ambos metales superó los umbrales de fitotoxicidad, alcanzando niveles entre 1,8 y 5,6 veces superiores para el Zn(II) y entre 10,1 y 15,2 veces superiores para el Cu(II). La planta acumuló los metales principalmente en las raíces, con una translocación mínima hacia la parte aérea. Estos resultados sugieren que L. alba podría utilizarse eficazmente en la revegetación y fitorremediación de suelos moderadamente contaminados con Zn(II) y Cu(II), actuando como agente fitoestabilizador.

INTRODUCTION

According to United Nations (UN) projections, by 2050, two-thirds of the population in developing countries are expected to live in urban areas. This demographic shift suggests that cities will need to ensure not only an adequate food supply but also a healthy environment for their inhabitants. However, the rapid pace of urbanization has established cities as significant contributors to climate change and global pollution (Cittadino et al., 2020). In response to these challenges, peri-urban agriculture has been promoted as a multifunctional strategy that simultaneously addresses food security, employment and environmental sustainability. It also preserves essential ecosystem services, thereby enhancing the resilience of urban systems. Despite its numerous benefits, this activity faces risks associated with environmental contamination, which could compromise food safety, particularly due to exposure to pollutants whose significance is often underestimated (Buscaroli et al., 2021).

Peri-urban areas are characterized by the coexistence of intensive agricultural activities, diverse industrial developments and continuous urban expansion, making them potential hotspots for contamination. Among emerging pollutants, heavy metals (HM) have gained prominence due to their high environmental persistence and high toxicity even at trace concentrations. These inorganic pollutants cannot be degraded through physical, chemical or biological processes, facilitating their absorption, accumulation and biomagnification within food chains (Briffa et al., 2020). This process can result in toxic concentrations that negatively affect both ecosystems and human health. Despite their negative connotation, certain HMs like Zn and Cu are essential nutrients, but they can become toxic at elevated concentrations(Hu et al., 2023).

The primary sources of HM pollution include industrial and domestic effluents from tanneries, metallurgical and petrochemical industries, open dumpsites, vehicular emissions and energy production. Also, intensive agricultural activities, such as the excessive use of pesticides and synthetic fertilizers, the application of untreated manures and the improper disposal of agricultural plastics substantially contribute to the accumulation of HM in soils (Mehmood et al., 2019).

HM-polluted soils remediation presents significant technical and economic challenges. Traditional physicochemical methods, while effective for the rapid extraction of contaminants, often result in irreversible alterations to soil properties. Such changes severely limit the suitability of treated soils for subsequent use (Rajendran et al., 2022).

In response to these limitations, bioremediation techniques have gained prominence as sustainable approaches to environmental decontamination. Among these, phytoremediation (utilizing plants and their associated microorganisms to immobilize, extract or detoxify contaminants) stands out for its numerous advantages. This approach is particularly cost-effective, with expenses up to 80% lower compared to physicochemical methods. Furthermore, it minimizes disruption to soil properties, facilitating habitat restoration and the preservation of biodiversity (Ansari et al., 2020).

Phytoremediation not only helps reduce environmental contamination but also offers additional benefits, such as creating green spaces, enhancing landscapes, ecological services and producing commercially valuable phytoproducts. Consequently, dual-purpose plants (those capable of remediating soils while yielding safe, marketable phytoproducts) have become increasingly sought after. Among these phytoproducts are biofuels, biosurfactants, bioplastics, biosynthetic nanomaterials, pharmaceutical compounds and essential oils (Muthusaravanan et al., s.f.).

Particularly, aromatic plants have been proposed for the recovery of sites contaminated with HMs. Furthermore, the use of native species is encouraged, as it minimizes ecological risks associated with introducing exotic species, which could disrupt local ecological dynamics (Mishra y Chandra, 2022). Native plants are often well-adapted to the local climate, soil conditions and stress factors, such as endemic pests. Additionally, employing native species facilitates habitat restoration after remediation (Futughe et al., 2020).

Lippia alba (Mill.) N. E. Brown, an aromatic native plant of the Verbenaceae family, is widely cultivated in Brazil, Argentina, Colombia, Uruguay, Paraguay and Mexico for its medicinal properties and essential oils. To date, no studies have assessed the potential of L. alba as a phytoremediation species. Therefore, this study aimed to evaluate the physiological and biochemical responses of L. alba chemotype carvone under elevated concentrations of Zn(II) and Cu(II), along with its capability for bioaccumulating these elements. Furthermore, phytoremediation strategies were analyzed to determine its suitability for remediating environments contaminated with HMs in the CHP.

MATERIALS AND METHODS

EXPERIMENTAL PROCEDURE

This experiment was conducted between April and December in a greenhouse located in La Plata, Buenos Aires, Argentina (34° 54' 45.57" S, 57° 55' 51.38" W). L. alba plants were obtained through vegetative propagation using stem cuttings from mother plants identified and located at the Experimental Station Ing. Agr. Julio Hirschhörn, which belongs to the Facultad de Ciencias Agrarias y Forestales (FCAyF) of the Universidad Nacional de La Plata (UNLP), La Plata, Buenos Aires, Argentina (34° 59' 7.85" S, 57° 59' 48.66" W).

Stem cuttings, with an average length of approximately 20 cm, a diameter of 15 ± 2 mm, and an initial fresh biomass of 1243 ± 9 mg, were treated with 50 ppm naphthalene acetic acid (NAA) solution for 24 hours. Subsequently, they were placed in trays filled with a soil-sand substrate (1:1 ratio) that had been thoroughly irrigated prior to planting. The cuttings were allowed to root for approximately two months. All successfully rooted cuttings were individually transplanted into 3 L black plastic pots filled with a soil-sand substrate (2:1). The plants were irrigated weekly with tap water (pH 6.7 ± 0.2).

After five months of growth (a period established to allow the plants to reach sufficient biomass for the assays), ten homogeneous plants per treatment, selected based on their height (1 m ± 10 cm), were chosen for metal exposure. Zn(II) and Cu(II) were applied as aqueous solutions of ZnSO₄·7H₂O and CuSO₄·5H₂O, respectively, to obtain the following treatments:

• Control: no Zn(II) or Cu(II) addition.
• Zn(II) treatments: 1000 ppm, 2000 ppm and 3000 ppm Zn(II).
• Cu(II) treatments: 500 ppm, 1000 ppm and 1500 ppm Cu(II).

The metals were applied by immersing the pots in their respective solutions, ensuring constant agitation to prevent precipitation. After 24 hours, the pots were removed from the solutions and allowed to percolate. 21 days post-application, the plants were harvested for further analysis. This period was established as sufficient to allow the manifestation of physiological and biochemical responses to metal exposure, as well as detectable differences in metal accumulation within plant tissues.

PHYSIOLOGICAL AND BIOCHEMICAL PARAMETERS MEASURED

At harvest, the dry weight of roots and shoots was determined by oven-drying the plant material at 80°C until a constant weight was achieved. Total chlorophyll and carotenoids contents were quantified from 5 mm diameter leaf disks following the method of (Wellburn, 1994), with absorbance measured at λ=647, 664, and 480 nm. Electrolyte leakage was evaluated as a measure of membrane stability, based on the method described by (Lutts, 1996), using 200 mg of fresh leaf and root tissues immersed in distilled water, with electrical conductivity (dS m^-1) measured via a Jenco 3173 conductivity meter. Lipid peroxidation was assessed by determining malondialdehyde (MDA) content, following the thiobarbituric acid reaction method of (Heath y Packer, 1968); for this, 200 mg of fresh leaf and root tissues were processed, and absorbance was measured at λ=532 nm, with corrections for nonspecific turbidity at λ=600 nm. Soluble protein content was quantified using the (Bradford, 1976) method, with 100 mg of fresh leaf and root tissues and absorbance measured at λ=595 nm to calculate protein concentrations based on a standard curve prepared with bovine serum albumin (BSA) (SiFMa Chemical Co.). Proline content was determined using the (Bates et al., 1973) method, with 100 mg of fresh leaf and root tissues processed and absorbance measured at λ=520. Total phenolic compounds content was quantified using 500 mg of fresh leaf and root tissues, following the Folin-Ciocalteu reagent method described by (Singleton y Rossi, 1965). Absorbance measurements for the afore mentioned parameters were determined using a Shimadzu UV-160 spectrophotometer (Kyoto, Japan).

METALS TOLERANCE ASSESSMENT AND BIOCONCENTRATION ANALYSIS

Dried root, shoot and substrate samples (500 mg each) were ground into a fine powder. Digestion of the plant samples was performed using a 3:1 mixture of HNO₃ and HClO₄ on a hot plate set to 120°C for 4 hours, while the substrate material was digested with 20 mL of 1N HCl on a shaker at 30°C for 24 hours, following the method described by (Boudet et al., 2011). Zn and Cu concentrations in substrate and biomass samples were measured using a Shimadzu AA6650F Atomic Absorption Spectrophotometer (Japan). Standard stock solutions for HMs were certified reference materials from J.T. Baker^® and Chem-Lab^®. Reagent blanks were prepared and analyzed alongside the samples under identical conditions. The limits of detection (LOD) and quantification (LOQ) for the spectrophotometer were consistent across substrate and biomass samples. The LODs were <0.005 mg kg^-1 for Zn and <0.02 mg kg^-1 for Cu; and the LOQs were <0.02 mg kg^-1 for Zn, <0.06 mg kg^-1 for Cu.

The tolerance index (TI), bioavailability factor (BAF), accumulation factor (AF) and translocation factor (TF) for plants were calculated using the equations outlined by (Gonzalez et al., 2024).

• TI = [DW_roots of control treatment] / [DW_roots of HM treatment]
• BAF = [HM_roots] / [HM_substrate]
• AF = [HM_shoots] / [HM_substrate]
• TF = [HM_shoots] / [HM_roots]

Where [HM_shoots], [HM_roots]and [HM_substrate] represent the HMs concentrations in shoots, roots and substrate, respectively, while [DW_roots] represent the dry weight of roots.

EXPERIMENTAL DESIGN AND DATA ANALYSIS

The experimental design was fully randomized, consisting of 7 treatments with 10 replicates per treatment (n=10). The data were analyzed using InfoStat (2019) software. The Shapiro–Wilk test was applied to assess normality. For data meeting the assumptions of normality, analysis of variance (ANOVA) was performed (for dry weight, pigment content, phenolic compound content, malondialdehyde content, tolerance index and heavy metal concentrations). For non-normal data, the Kruskal–Wallis test was applied (electrolyte leakage, soluble protein content, and proline content). In both cases, comparisons were made at a significance level of 5% (p < 0.05). Pearson correlations were determined using R software version 4.3.1. All results were expressed as mean values with corresponding standard deviations.

RESULTS

PHYSIOLOGICAL AND BIOCHEMICAL PARAMETERS

Figure 1 shows that the dry weight of shoots (p<0.0001 and p<0.0001 for Zn(II) and Cu(II) respectively) and roots (p<0.0001 and p<0.0001 for Zn(II) and Cu(II) respectively) decreased significantly with increasing concentrations of Zn(II) and Cu(II). At the highest treatment levels, dry weight reductions of 49% and 58% were observed in shoots, and 31% and 45% in roots, for Zn(II) and Cu(II), respectively.

Figure 1

Effect of Zn(II) and Cu(II) concentrations on the dry weight of L. alba.

Figure 2 shows that this decline could be associated with the reduction observed in total chlorophyll content (p<0.0001 and p<0.0001 for Zn(II) and Cu(II) respectively), which decreased by 24% and 29% at the highest concentrations of Zn(II) and Cu(II), respectively. For carotenoids content, no significant changes were observed under Cu(II) treatments (p=0.5943). In Zn(II) treatments, carotenoids levels began to decline (p=0.0114;) at 2000 ppm, ultimately reaching values 17% lower than the control. Despite these negative effects, no plant mortality was observed in any treatment.

Figure 2

Effect of Zn(II) and Cu(II) concentrations on chlorophylls and carotenoids contents of L. alba.

In Figure 3, root electrolyte leakage increased significantly even at the lowest concentrations of both HMs, showing values approximately 4 times those of the control. Root electrolyte leakage reached values 4.5 and 6.1 times that of the control for 3000 ppm Zn(II) and 1500 ppm Cu(II), respectively (p<0.0001 and p<0.0001 for Zn(II) and Cu(II) respectively). Conversely, leaf electrolyte leakage decreased at the lowest concentrations of both HMs (p=0.0461 and p=0.0019 for Zn(II) and Cu(II) respectively). At higher concentrations, leaf electrolyte leakage values were comparable to those of the control, with no statistically significant differences detected.

Effect of Zn(II) and Cu(II) concentrations on the electrolyte leakage of L. alba.

Figure 3

Effect of Zn(II) and Cu(II) concentrations on the electrolyte leakage of L. alba.

Figure 4 shows that MDA levels in leaves significantly increased under high Zn(II) stress, peaking at 2000 ppm Zn(II) with values 38% higher than the control. However, at 3000 ppm Zn(II), MDA levels decreased to control levels, a trend similarly observed for Cu(II) treatments (p<0.0001 and p<0.0001 for Zn(II) and Cu(II) respectively). In roots, MDA levels under Zn(II) treatments showed a significant decline as concentrations increased, reaching values 70% lower than the control at 3000 ppm Zn(II) (p<0.0001). For Cu(II) treatments, root MDA levels did not follow a linear trend with increasing concentrations. Values differed significantly from the control at 500 and 1000 ppm (p<0.0001), with a decrease of approximately 34% at 1000 ppm Cu(II), whereas at the highest concentration root MDA levels were not statistically different from the control.

Figure 4

Effect of Zn(II) and Cu(II) concentrations on the malondialdehyde (MDA) content of L. alba.

Figure 5 shows that soluble proteins content in leaves decreased with increasing Zn(II) concentrations, showing a pronounced reduction at the highest concentration, where levels were 63% lower (p<0.0001) than the control. For Cu(II) treatments, a similar trend was observed, with the minimum value recorded at 1000 ppm Cu(II) (p<0.0001). In roots, increasing concentrations of both HMs caused a significant decline in soluble protein content (p<0.0001 for both Zn(II) and Cu(II)). Minimum values were approximately 44% and 71% lower than the control for Zn(II) and Cu(II), respectively, and soluble protein levels remained significantly lower than those of the control at the highest concentrations.

Figure 5

Effect of Zn(II) and Cu(II) concentrations on the soluble proteins content of L. alba.

In Figure 6, proline content exhibited distinct patterns under Zn(II) and Cu(II) treatments. In leaves, concentrations nearly doubled (p<0.0001) at the highest Zn(II) level compared to the control. In roots, levels increased consistently across all Zn(II) treatments (p<0.0001), peaking at 2000 ppm with values 2.4 times that of the control. At the maximum concentration (3000 ppm), values were significantly lower than those at 2000 ppm but remained significantly higher than the control. Under Cu(II) exposure, a significant increase was observed in leaves at the highest concentration (p<0.0001), with values reaching approximately seven times those of the control. In roots, the content showed a gradual rise (p<0.0001) with increasing Cu(II) concentrations, reaching a maximum at 1500 ppm, approximately 3 times that of the control.

Figure 6

Effect of Zn(II) and Cu(II) concentrations on the proline content of L. alba.

Figure 7

Effect of Zn(II) and Cu(II) concentrations on the phenolic compounds content of L. alba.

ZN(II) AND CU(II) BIOACUMULATION AND TOLERANCE ASSESSMENT

Figure 8 shows that L. alba plants accumulated both HMs predominantly in the roots, as indicated by translocation factor values consistently lower than 1 across all tested concentrations of Zn(II) and Cu(II) (p<0.0001 and p<0.0001 for Zn(II) and Cu(II) respectively), as detailed in Table 1. Maximum bioaccumulation values in roots were 1333 mg kg-1 for Zn(II) and 1007 mg kg-1 for Cu(II), which were 38 and 28 times greater than those of the control, respectively. This trend is further supported by the bioavailability factor values, which were higher than the accumulation factor values, indicating that the plant is more efficient at extracting metals from the soil and accumulating them in its root biomass.

Table 1 also shows that tolerance assessment, based on the tolerance index values, revealed a significant reduction in L. alba tolerance to both metals at higher concentrations (p<0.0001 and p<0.0001 for Zn(II) and Cu(II) respectively). The lowest tolerance values were observed at the highest concentrations, with values being lower for Cu(II) than for Zn(II), reaching a 60% of tolerance for both metals.

Figure 8

Zn(II) and Cu(II) concentrations in shoots, roots and substrate of L. alba.

Table 1

Zn(II) Treatment	Factor
		TI	BAF	AF	TF
Zn(II) control	-	4.19±0.36 c	13.94±1.64 c	3.32±0.14 c
1000 ppm Zn(II)	95%±10% b	1.03±0.04 b	0.53±0.02 b	0.51±0.01 b
2000 ppm Zn(II)	66%±8% a	0.90±0.04 a	0.49±0.02 b	0.54±0.01 b
3000 ppm Zn(II)	69%±6% a	0.97±0.02 ab	0.41±0.02 a	0.42±0.03 a
Cu(II) Treatment	TI	BAF	AF	TF
Cu(II) control	-	6.93±0.21 c	5.97±0.49 c	0.86±0.09 c
500 ppm Cu(II)	65%±5% ab	1.99±0.37 a	0.58±0.08 a	0.29±0.02 b
1000 ppm Cu(II)	73%±12% b	6.68±0.54 c	0.92±0.06 b	0.14±0.01 a
1500 ppm Cu(II)	55%±11% a	2.12±0.14 b	0.65±0.05 a	0.31±0.01 b
TI: Tolerance index, BAF: Bioavailability factor, AF: Accumulation factor, TF: Translocation factor

Phytoremediation factors L. alba for each Zn(II) and Cu(II) treatment.

CORRELATION ANALYSIS

Figure 9 presents the complete correlation analysis between metal bioaccumulation and the evaluated physiological and biochemical parameters in L. alba. Zn(II) content in the biomass exhibited significant negative correlations (p<0.0001) with both shoots and roots dry weight, with a stronger correlation observed with shoots dry weight. Additionally, Zn(II) bioaccumulation was negatively correlated (p<0.0001) with total chlorophyll, carotenoids (p<0.0001) and roots phenolic compounds contents (p<0.0001). However, it showed positive correlations with proline (p<0.0001) and phenolic compounds levels in leaves (p<0.0001), suggesting the potential activation of stress response mechanisms.

In contrast, Cu(II) content in the biomass exhibited much stronger negative correlations with shoots dry weight (p<0.0001) and total chlorophyll content (p<0.0001), but no significant (p=0.563) correlation with carotenoids content. It also showed pronounced negative correlations with leaf (p<0.0001) and root (p<0.0001) soluble proteins content, as well as with root phenolic compounds content (p=0,0008 and p<0.0001 for shoots and roots respectively). Interestingly, Cu(II) bioaccumulation exhibited a strong positive correlation with proline levels in both leaves (p<0.0001) and roots (p<0.0001), but, unlike with Zn(II), it showed negative correlations with leaf phenolic compounds content (p<0.0001), suggesting a distinct and more pronounced stress response to Cu(II) compared to Zn(II).

Figure 10

Pearson correlation matrix of all physiological and biochemical parameters measured in L. alba under Zn(II) and Cu(II) stress. Negative correlations are represented by varying shades of red, while positive correlations are depicted in varying shades of blue. Crossed correlations ("X") denote non-significant correlations (p>0.05).

DISCUSION

L. alba bioaccumulated high concentrations of both metals. Typically, plants require Zn(II) concentrations in shoots of approximately 15-30 mg kg^-1 and Cu(II) concentrations within a range of 5-20 mg kg^-1. However, concentrations exceeding 100-300 mg kg^-1 for Zn(II) and 20-30 mg kg^-1 for Cu(II) are considered phytotoxic (Fischer y Fischer-García, 2023). In L. alba, shoots HMs concentrations ranged from 307 to 559 mg kg^-1 for Zn(II) and 53 to 303 mg kg^-1 for Cu(II), exceeding the phytotoxic thresholds even at the lowest tested concentrations. These bioaccumulation levels were 1.8 to 5.6 times higher for Zn(II) and 10.1 to 15.2 times higher for Cu(II) than the established phytotoxic limits.

(Navarrete Gutiérrez et al., 2018) reported that L. alba growing in serpentine soils with 38 to 166 mg kg^-1 of Zn(II), bioaccumulated 14.2 mg kg^-1 of Zn(II) in its shoots. However, no other studies to date have examined the accumulation capacity nor the physiological and biochemical responses of this species to such stress. Additionally, only a limited number of investigations have focused on other Lippia species. For instance, (Artwell et al., 2017) reported 15.32 mg kg^-1 of Cu(II) in shoots of L. javanica (Burm.f.) Spreng., although the metal concentration in the growing medium was not specified. The bioaccumulation values in our study are significantly higher due to the elevated metal concentrations applied.

Our findings suggest that L. alba exhibits traits of a phytostabilizer. This is evidenced by translocation factor values consistently below 1 across all tested concentrations (Gonzalez et al., 2021). Notably, the species exhibited a greater phytostabilization capability for Cu(II) than for Zn(II), as evidenced by lower translocation factor values and higher bioavailability factors for Cu(II).

From an applied perspective, the preferential retention of both metals in the root system, together with the limited translocation to aerial tissues, reinforces the potential relevance of L. alba for phytostabilization-based approaches. Such strategies aim to reduce metal mobility and bioavailability in contaminated soils rather than promoting metal extraction. In this context, the differential responses observed between Zn(II) and Cu(II) further highlight the importance of considering metal-specific behavior when evaluating plant performance under multi-metal stress scenarios.

A reduction in growth was observed, with more pronounced effects in shoots compared to roots. (Bibbiani et al., 2018) similarly reported growth reduction in Tetradenia riparia (Hochst.) Codd exposed to high Zn(II) concentrations, showing a comparable trend of more significant growth inhibition in shoots than in roots. They also observed that metal sensitivity in plant organs did not always directly correspond to the amount of metal accumulated, as more Zn(II) was sequestered in roots than in shoots, a pattern consistent with our findings. In contrast, (Goswami y Das, 2016) observed a uniform reduction in dry weight across shoots and roots in Calendula officinalis L. exposed to high Cu(II) concentrations, despite higher Cu(II) accumulation in shoots compared to roots, which differs from the findings in our study. However, correlation analysis in our study revealed a significant negative correlation between the bioaccumulation of both HMs and the dry weight of shoots and roots, suggesting that higher metal concentrations in one organ not only impact that organ but also affect others.

Decreased dry weight is one of the most reported symptoms of HMs stress. Roots, being the first organ in direct contact with HMs, are typically the initial site of toxic effects, which impair water and nutrient uptake (Ghori et al., 2019). Excess Zn(II) and Cu(II) have been shown to induce cytotoxicity and genotoxicity by interfering with cell division processes and causing chromosomal aberrations, negatively impacting plant cell development and growth. Specifically in roots, these metals can also cause damage such as cuticle disruption, reduced root hair proliferation and, in severe cases, significant deformation of root structure (Kaur y Garg, 2021; Mir et al., 2021). These effects ultimately disrupt the overall metabolism of the plant, further exacerbating the reduction in dry weight.

Once metals are translocated to the shoots, they can directly affect the photosynthetic apparatus. (Li et al., 2023) reported a decrease in total chlorophyll content in Triticum aestivum L. plants exposed to high concentrations of Cu(II), while carotenoids content remained unaffected, similarly to our findings. Carotenoids are essential photosynthetic pigments with various functions in plants, such as protection against photooxidation and defense against oxidative stress. This type of stress is caused by reactive oxygen species (ROS), which, at high concentrations, trigger a cascade of reactions that lead to the degradation of lipids and proteins, causing extensive damage to DNA and cellular membranes. This suggests that carotenoids could have a protective role against oxidative stress induced by Cu(II) excess in L. alba. In contrast, it has been reported that HMs can replace the central Mg in chlorophyll molecules, thereby completely inhibiting its function, inhibit enzymes involved in chlorophyll biosynthesis and increase chlorophyllase activity, ultimately leading to reduced chlorophyll levels (Rai et al., 2016).

Oxidative stress-induced membrane damage is associated with elevated electrolyte leakage and MDA levels, a byproduct of lipid peroxidation of cellular membranes. (Dobrikova et al., 2021) reported an increase in MDA concentration of Salvia sclarea L. plants exposed to high concentrations of Zn(II), while (Aqeel et al., 2023) observed increased electrolyte leakage in Mentha arvensis L. exposed to high Cu(II) concentrations. These authors emphasize that cell membranes are the first structures to be affected by HMs, with damage induced by factors such as protein oxidation and alterations in the composition and fluidity of plasma membrane lipids, driven by lipid peroxidation. However, in our study, we did not find significant positive correlations between these parameters. For both metals, electrolyte leakage increased predominantly in roots, while leaf values remained largely unchanged. In contrast, MDA levels decreased in roots. In leaves, MDA levels varied with concentration, showing higher values at lower exposures and returning to control levels at higher concentrations. This suggests that while the permeability of root cell membranes may be compromised, lipid peroxidation could not be its primary cause in L. alba. (Yuan et al., 2024) suggested that HM ions can interact with S and N groups in membrane proteins, leading to alterations in membrane ionic channels, including disruptions to H⁺-ATPase pumps and protein degradation. These alterations could lead to an increase in electrolyte leakage without inducing lipid peroxidation and, consequently, without an associated rise in MDA levels.

This could be linked to the decrease in soluble proteins levels observed in L. alba under exposure to both metals, as soluble proteins content showed a strong negative correlation with electrolyte leakage. Similarly, (Sidhu et al., 2020) reported a reduction in soluble proteins content in Coronopus didymus (L.) Sm. under Zn(II) stress, attributing it to decreased nitrate reduction caused by Zn(II)-induced inhibition of nitrate reductase activity. Additionally, HM ions can directly denature proteins by binding to their structure, thereby altering their function or synthesis. Indirectly, they can induce ROS production, which promotes proteolysis and disrupts metabolic pathways critical for protein synthesis (Georgiadou et al., 2018).

Despite these negative effects, some defense mechanisms were observed in L. alba, such as the increase in proline concentration, particularly in leaves under Cu(II) stress, and the increase in phenolic compounds, especially in leaves treated with Zn(II). Similarly, (Nazir et al., 2019) reported an increase in proline content in Solanum lycopersicum L. exposed to Cu(II) stress, while (Marichali et al., 2016) observed an increase in both proline and phenolic compounds levels in Nigella sativa L. under Zn(II) stress. Both compounds are part of the non-enzymatic antioxidant defense mechanism, acting as ROS scavengers. Additionally, proline plays a critical role in osmoregulation and osmoprotection (Reddy et al., 2024). These findings suggest that L. alba may have stronger or more efficient non-enzymatic antioxidant defense mechanisms in leaves compared to roots, or that the activation of these mechanisms results in more effective protection in leaves than in roots.

These mechanisms have likely contributed to the ability of L. alba to tolerate high concentrations of HMs, as reflected in its tolerance indexes, which exceeded 60% at all levels except at the highest concentration of Cu(II). (Goswami y Das, 2016) observed tolerance indexes exceeding 60% in C. officinalis exposed to Cu(II) stress, identifying this threshold as the acceptable limit for growth reduction to classify a species as tolerant. Notably, L. alba exhibited a tolerance index of 95% at 1000 ppm Zn(II), suggesting that this species demonstrates greater tolerance to high Zn(II) concentrations compared to Cu(II).

Although the present study focused on non-enzymatic antioxidant responses, the observed accumulation of proline and phenolic compounds suggests that additional defense pathways may be involved in the tolerance of L. alba to metal stress. Further research addressing enzymatic antioxidant systems would be necessary to obtain a more comprehensive understanding of the mechanisms underlying stress mitigation in this species. Moreover, evaluating how metal exposure affects the yield and chemical composition of L. alba essential oils would be particularly relevant, as these phytoproducts could add economic value and improve the feasibility of phytoremediation or revegetation programs.

CONCLUSIONS

High concentrations of Zn(II) and Cu(II) negatively affected the growth of L. alba, as evidenced by reductions in biomass, soluble protein content, and total chlorophyll levels. Despite these effects, the species demonstrated a high capacity to bioaccumulate both metals, reaching concentrations above phytotoxic thresholds without exhibiting plant mortality. Metal accumulation occurred predominantly in roots, with limited translocation to aerial tissues, indicating a phytostabilization-oriented response.

Overall, L. alba exhibited greater tolerance to Zn(II) than to Cu(II) and activated non-enzymatic antioxidant responses, including proline and phenolic compound accumulation, particularly in leaves. These findings highlight the potential of L. alba for use in phytostabilization and revegetation strategies in soils moderately contaminated with Zn and Cu.

It should be acknowledged that the experimental design was based on artificially contaminated substrates, in which metals were supplied in highly soluble forms. While this approach does not fully reflect the complexity of field-contaminated soils (where metals undergo stabilization, adsorption, and redistribution among soil fractions) it allows for a controlled assessment of tolerance thresholds and physiological responses under conditions of high metal availability. Therefore, the results obtained here should be interpreted within this experimental framework and considered as a first step toward understanding metal stress responses that warrant validation under more realistic soil conditions.

ACKNOWLEDGMENTS

The authors would like to thank Cecilia Bernardelli (CINDEFI-CONICET) for technical assistance. Financial support for this study was provided by the Proyecto de Incentivos a la Investigación (A376) of the Facultad de Ciencias Agrarias y Forestales (UNLP), Argentina.

Authors´ contribution

Colaboracíon académica
Author´s name	1	2	3	4	5	6	7	8	9	10	11	12	13	14
Matias Alberto Gonzalez			X	X	X	X	X	X	X	X	X		X	X
Valeria Bernardo			X					X					X	X
Sebastián Garita			X					X					X	X
Laura Wahnan			X					X					X	X
Josefina Plaza Cazón			X					X					X	X
Cecilia Arango			X					X					X	X
Marcela Ruscitti	X	X	X	X	X	X	X	X	X	X		X	X	X

Referenc es . 1-administra tion of the project , 2-adquisi tion of funding 3-a formal, analsys 4-conceptuali zation , 5- data curation, 6-writing, review and editing, 7-investigation, 8-methodology, 9-resources, 10-drafting, original draft, 11-software, 12-supervision, 13-validation, 14-visualization.

Referencias

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Gonzalez, M. A., Bernardo, V., Garita, S., Cazón, J. P., Arango, C., Hernández, M. P. y Ruscitti, M. (2024). Morphophysiological and biochemical responses of Schedonorus arundinaceus to Zinc (II) excess: insights from biomarkers and elemental accumulation. Revista de la Facultad de Ciencias Agrarias UNCuyo, 56(2), 34-47. https://doi.org/10.48162/rev.39.135 ↑

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Mehmood, K., Ahmad, H. R., Abbas, R., Saifullah y Murtaza, G. (2019). Heavy metals in urban and peri-urban soils of a heavily-populated and industrialized city: Assessment of ecological risks and human health repercussions. Human and Ecological Risk Assessment: An International Journal, 26(6), 1705-1722. https://doi.org/10.1080/10807039.2019.1601004 ↑

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