Human activities have significantly increased soil contamination on a global scale, notably through processes such as mining, industrialization and urban expansion. These activities promote the accumulation of persistent heavy metal (HM) in the environment, including Pb, Cd, Cu and Zn (Zhao et al., 2023). This waste typically has low fertility, low organic matter content and high concentrations of toxic metals conditions that limit vegetation establishment and pose long-term ecological and health risks to ecosystems (Hernández-Acosta  et al., 2025).
       
Given this scenario, phytoremediation emerges as a sustainable strategy for the remediation of contaminated soils. This approach relies on the use of plants capable of tolerating, immobilizing, or extracting HM, thereby reducing their mobility and bioavailability in the environment (Putra et al., 2022). Among the species used, grasses are particularly well-suited due to their rapid growth, fibrous root systems and high biomass production. In this same vein, efficiency can also be increased through the application of arbuscular mycorrhizal fungi (AMF). Given that these organisms have been shown to play an important role in nutrient uptake, increased tolerance to abiotic stress and the modification of heavy metal bioavailability in soil (Maity et al., 2025).
       
Despite advances in knowledge regarding the role of AMF in phytoremediation processes, there is still limited information about their influence on the simultaneous phytoextraction of Pb, Cd, Cu and Zn in MT through their combination with Lolium perenne L. Therefore, this study aimed to evaluate the effect of inoculating L. perenne seeds with AMF on the phytoextraction of these metals, as well as its impact on accumulation and translocation patterns within the plant. It was hypothesized that AMF inoculation significantly influences the accumulation and translocation of Pb, Cd, Cu and Zn in L. perenne grown in MT and that these responses vary depending on the inoculation rate and the metal evaluated.

Collection of contaminated soil samples
 
Samples of contaminated waste were collected at mine tailings (MT), located in the municipality of Hidalgo del Parral, Chihuahua, Mexico (26°55'N, 105°40'W), during the first week of October 2025. Sampling was conducted in accordance with the guidelines of standard NMX-AA-132-SCFI-2006 (DOF, 2006). Subsequently, the subsamples were homogenized and placed in pre-labeled polyethylene bags, then transported to the laboratory for analysis.
 
Research conduct
 
The experimental activities related to the preparation and production of plant material were carried out in the greenhouse area of the Antonio Narro Autonomous Agrarian University, Laguna Campus, located in Torreón, Coahuila, Mexico (25°33'N, 103°22'W). The experiment was carried out in a non-automated greenhouse without environmental control systems. For this reason, temperature and relative humidity were not systematically monitored or recorded during the experimental period. The remaining experimental procedures were carried out in the soil laboratory of the same institution.
 
Determination of heavy metals in mine tailings
 
The samples were allowed to dry at room temperature for one week, then sieved through a 2-mm mesh for physical and chemical analysis. To determine metal concentrations, a subsample was finely ground using a high-speed pulverizer (model XZ-6B, China) to obtain a homogeneous material prior to the digestion process, as well as to determine the concentration of total organic matter present in the tailings (Table 1).


 
Preparation of the growing medium
 
During the second week of October, the substrate was prepared by homogeneously mixing MT and perlite in a 50:50 (v/v) ratio. This combination was used to improve the physical properties of the medium, particularly aeration, structure and drainage (Oğuztürk  et al., 2025).
 
Selection of plant material
 
Commercial seeds of L. perenne, rye grass linn variety (El Trébol Semillas, Mexico), with 90% germination and certified grade, were used. 0.6 g of seed per pot was used, keeping this amount constant in all experimental units. The amount of seed was established considering the commercial sowing recommendation and the germination percentage declared by the supplier.

Treatments, irrigation and harvest
 
Inoculation was performed once during sowing by directly applying a commercial biofertilizer (RAIDEN®, Colima, Mexico) to the seed. In this regard, four application rates were used: 0, 10, 20 and 30 g of biofertilizer per kilogram of substrate, identified as T0, T10, T20 and T30, respectively. The experimental units were maintained under a controlled irrigation regimen in a greenhouse, with 250 mL of water applied every three days over an eight-week period (Ramirez-Gottfried  et al., 2019).
       
At the end of the eight-week growth period, the plant material was harvested. Each plant was then manually divided into two parts: the aerial fraction, consisting of leaves and stems and the root system. Both fractions were placed in clean trays and left to dry at room temperature for 5 days. Once dry, the samples were stored in pre labeled brown paper bags for later analysis.
 
Preparation of digests
 
For the extraction of metals present in the biomass, the dry digestion technique was used, following the methodology described by Ramirez-Gottfried  et al. (2019).
 
Determination of heavy metals in plant material
 
The determination of HM concentration was performed by atomic absorption spectrophotometry using a Perkin-Elmer Model 2380 instrument (Norwalk, USA). For Cu and Zn, a Perkin-Elmer Lumina™ multielement lamp was used, while individual lamps were used for Pb and Cd. As part of the analytical quality control, the readings of the samples were performed in triplicate. Analytical accuracy was evaluated by the coefficient of variation. The concentrations of the metals in the plant material were expressed in mg kg^-1 based on the dry weight of the biomass analyzed.
 
Bioaccumulation factor and biological translocation factor
 
The bioaccumulation factor (BAF) was calculated as:
 

 

 
Where,
Cplant= The HM concentration in plant tissue.
Csubstrate= The HM concentration of MT in the substrate.
       
BAF values > 1 indicate accumulation capacity. Biological translocation factor (BTF) was calculated as:
  

 

Where,
CPA= The HM concentration in the aboveground parts.
CR= The HM concentration in the root.
       
BTF values > 1 indicate efficient translocation to the aboveground parts (Elik and Gül, 2025).
 
Statistical analysis
 
The experiment was conducted using a completely randomized design considering AMF inoculation rate as the single experimental factor, with four levels (0, 10, 20 and 30 g kg^-1 of substrate; T0, T10, T20 and T30, respectively), with four replicates per treatment, resulting in a total of 16 experimental units. The data obtained were analyzed using analysis of variance. This analysis allowed us to determine the effect of the treatments on the evaluated variables. When differences were evident (P≤0.05), a comparison of means was performed using Tukey’s test.

Plant material
 
The concentration of HM in the aboveground parts of L. perenne varied depending on the AMF treatments. Differences (P≤0.05) were observed in the accumulation of Pb, Cd, Cu and Zn among the evaluated treatments.
       
Table 2 shows the average of heavy metal concentrations (ppm) in the aboveground biomass of L. perenne under different AMF treatments in mining tailings substrate.


       
Based on the results obtained, the hypothesis proposed in this study is not rejected, because inoculation with AMF influenced the uptake and distribution of HM in the plant, depending on the applied dose and the HM evaluated. This was achieved by quantifying HMs through biomass analysis and the calculation of bioaccumulation and translocation factors, a methodology widely used to study the symbiosis of AMF in contaminated environments (Boorboori and Zhang, 2022). In this regard, inoculation with AMF modified the above-ground accumulation of metals in L. perenne, although the response was dependent on the HM and the applied dose (P≤0.05).
       
In addition, the results indicated that lead concentrations were highest in the T30, suggesting that, under MT conditions, a high dose of AMF may promote its mobilization into harvestable tissues. This behavior is consistent with what has been reported for L. perenne, where Pb accumulation in aboveground biomass depended on substrate conditions and interactions with edaphic factors (Wang et al., 2024). In the case of Cd, the differences between treatments were numerical, as there were no statistically significant differences between treatments (P>0.05), suggesting stricter control of its transport to the aerial parts. This result is consistent with studies indicating that L. perenne tends to regulate Cd absorption and distribution through detoxification and changes in gene expression under metal stress conditions (Bai et al., 2024).
       
Regarding Cu, similar to Cd, no significant differences were observed, although numerically the highest concentrations were recorded in T20. Similar results have been reported in this species, where Cu accumulation remains stable and depends more on soil conditions than on external treatments. For Zn, the highest concentrations were calculated in T20, while in T30 the Zn concentration decreased, indicating that intermediate doses of AMF favored metal uptake and mobilization, but more intense colonization may have limited their internal transport. These results are similar to those described by Sarathchandra (2024), who observed that the above-ground content of Cu and Zn in L. perenne varied with substrate characteristics. This pattern also agrees with other phytoremediation studies using L. perenne, where Zn accumulation depended on the balance between metal availability and biological activity in the rhizosphere (Saldarriaga et al., 2023).
       
Table 3 shows the concentration of heavy metals in the roots of L. perenne, revealing differences among the AMF treatments (P≤0.05). In general, greater root accumulation was observed compared to the aboveground parts for some metals, suggesting a retention effect in the root system associated with mycorrhizal inoculation.


       
The results obtained show that inoculation with AMF significantly influences the accumulation of HM in the roots of L. perenne, which is consistent with studies highlighting the role of AMF as key modulators in the dynamics of metal absorption and distribution in plants grown in contaminated soils (Zhao et al., 2024). In the case of Pb, the higher concentration at T0 and its decrease with AMF suggest an immobilization effect, associated with retention in fungal structures and the formation of stable compounds in the soil (Cáceres-Mago et al.,  2025). For Cd accumulation in roots, a dose dependent response was observed, indicating that the plant AMF interaction is not linear and depends on system factors; furthermore, AMF can reduce its translocation to the aboveground parts by retaining it in the root (Zhao et al., 2024). Regarding Cu and Zn, the decrease in their concentration with higher doses of AMF suggests a root retention mechanism, characteristic of phytostabilization processes (Putra et al., 2024).
 
Bioaccumulation factor and biological translocation factor
 
The BAF results show that L. perenne exhibits a high capacity for Pb accumulation (BAF>1 in all treatments) (Table 4). This indicates efficient uptake from the substrate into plant tissues.  Specifically, BAF values for Pb ranged from 2.18 to 4.76. Compared to the non-inoculated control (T0), the BAF of Pb decreased 10.9% at T10, 54.2% at T20 and 3.2% at T30. Although T20 presented the greatest reduction from the control, its BAF remained above 1, indicating that the accumulation of Pb from the substrate was maintained despite the decrease associated with this dose of AMF (Table 4). This behavior is consistent with studies indicating that AMF can increase or modulate metal accumulation depending on the plant species and soil conditions, favoring both phytoextraction and phytostabilization processes (Şahin and İnci, 2026).


       
In the case of Cd, BAF values < 1 in all treatments indicate a limited capacity for accumulation, suggesting that inoculation with AMF promotes exclusion or immobilization mechanisms. Compared to T0, the BAF of Cd decreased by 51.1% at T10, 40.4% at T20 and 34.0% at T30, with the greatest reduction being observed with the dose of 10 g kg^-1 of AMF. This behavior has been reported in studies where AMF reduce Cd bioaccumulation through changes in the metal’s availability and its retention in the rhizosphere. Likewise, it has been demonstrated that Cd bioaccumulation in plants depends heavily on soil conditions and the plant microorganism system (Vallejos-Torres et al., 2023). For Cu, BAF values ranged from 0.63 to 0.81 remaining below 1 in all treatments. Compared to T0, Cu BAF increased 28.6% at T10, 14.3% at T20 and 12.7% at T30, with the highest value being observed with the 10 g kg^-1 dose of AMF (0.81). Despite these increases, BAF remained below 1, indicating a moderate accumulation of Cu. This pattern can be attributed to the ability of AMF to modify metal availability through interactions in the rhizosphere, such as the adsorption of metal ions and the activation of microbial processes that affect their mobility (Hu et al., 2024).
       
Regarding Zn, BAF values < 1 in all treatments indicate low accumulation efficiency, suggesting that this metal is preferentially retained or immobilized in the soil root system. This behavior is consistent with studies reporting that AMF act as a biological barrier that limits the transfer of metals to plant tissues, reducing their bioaccumulation and toxicity (Dhalaria et al., 2020). Taken together, the results show that the L. perenne AMF association allows for differential modulation of BAF depending on the metal, favoring accumulation in the case of Pb and limiting it for Cd, Cu and Zn. This behavior has been documented in MT systems, where AMF can reduce the BAF of certain metals while improving plant tolerance and system stability (Putra et al., 2022; Banerjee et al., 2025).
       
Table 5 shows the average BTF values in L. perenne, revealing variations among treatments with AMF (P≤0.05). The BTF values obtained indicate that L. perenne ability to translocate metals from the root to the aboveground parts depends on the type of metal and the level of AMF inoculation, reflecting differential regulation of internal transport. This behavior has been associated with the ability of mycorrhizal fungi to modify both transport gradients and the intracellular distribution of metals in the host plant (Zhao et al., 2024).


       
In the case of Pb, BTF values < 1 in all treatments indicate limited translocation to the aboveground parts, even with mycorrhizal inoculation. Compared to T0, the BTF of Pb decreased 6.0% in T10, but increased 22.0% and 28.0% in T20 and T30, respectively. This suggests that Pb is retained in the roots, which is consistent with studies reporting that AMF favor the immobilization of this metal through its accumulation in extraradical hyphae, reducing its mobility within the plant. This behavior is characteristic of phytostabilization mechanisms rather than phytoextraction. For Cd, a notable increase in BTF was observed in the T10 (BTF>1), indicating greater efficiency in translocation to the aboveground parts under inoculation conditions. For Cd, BTF increased from 0.55 at T0 to 1.34 at T10, which corresponds to an increase of 143.6% compared to the control without inoculation. At T20, BTF decreased to 0.64, while T30 reached 1.01. Therefore, the greatest increase in Cd translocation occurred with the 10 g kg^-1 dose of AMF, where the concentration of Cd in the aerial part was approximately 1.34 times that recorded in the roots. This result suggests that, at intermediate doses, AMF may facilitate Cd transport, possibly by activating specific transporters or modifying the ionic balance in the root. However, at higher doses, this capacity tends to decrease, which has been reported as a regulatory effect where AMF limit the mobility of highly toxic metals to protect photosynthetic tissues (Putra et al., 2022).
               
In the case of Cu, BTF values remained below 1 in all treatments. Compared to T0, BTF decreased 43.0% at T10, increased 5.1% at T20 and decreased 12.7% at T30. These values indicate a limited translocation of Cu to the air tissues in all treatments. This pattern suggests that Cu, being an essential micronutrient, exhibits finer control over its transport within the plant, where AMF can both facilitate and restrict its mobility depending on physiological needs. Studies have shown that AMF actively participate in the regulation of Cu through chelation and vesicular storage processes, preventing its toxicity in aerial tissues (Agouni et al., 2025). For Zn, BTF showed a progressive dose-dependent increase. These values represent increases of 78.9, 263.2 and 342.1% compared to the control without inoculation, for T10, T20 and T30, respectively. Therefore, the higher dose of AMF increased the Zn translocation factor approximately 4.4 times compared to T0. This behavior suggests a potential for phytoextraction of this metal under high inoculation conditions. This phenomenon has been documented, where AMF increases the mobility of essential metals such as Zn by improving root absorption and their subsequent transport to aerial organs (Boorboori and Zhang, 2022).

Inoculation with AMF significantly influenced the uptake, accumulation and distribution of HM in L. perenne, demonstrating a response dependent on the metal and the applied dose. The results showed that Pb exhibited high accumulation (BAF>1) but limited translocation (BTF<1), indicating a predominant phytostabilization behavior. In contrast, Cd exhibited a dose-dependent response, with greater translocation in intermediate treatments, while Cu maintained stable dynamics associated with its role as a micronutrient. Zn showed greater mobility at high AMF doses, suggesting potential for phytoextraction under specific conditions. Overall, the L. perenne AMF association allows for the modulation of metal fate within the plant, directing processes toward phytoextraction or phytostabilization depending on the metal and the applied dose, positioning this species as a viable alternative for the remediation of contaminated sites.

The authors thank the UAAAN staff for the support provided and for facilitating access to the laboratory facilities necessary for the development of this study.
 
Disclaimers
 
The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.
 
Informed consent
 
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The authors declare that there are no conflicts of interest regarding the publication of this article. No funding or sponsorship influenced the design of the study, data collection, analysis, decision to publish, or preparation of the manuscript.