Exploring the Phytoremediation Capability of Athyrium filix-femina , Ludwigia peruviana and Sphagneticola trilobata for Heavy Metal Contamination

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). Heavy metals are currently considered to be major pollutants because of their persistence, toxicity, and inability to be degraded by biological processes; furthermore, the accumulation of heavy metals in soil presents a risk to agricultural production on a worldwide scale (Long et  Phytoremediation is thought to have various advantages over other approaches for lowering the concentration of contaminants in soil. Some of these advantages include a low cost, in-situ remediation, respect for the environment, and improvement of the landscape ( The distribution of heavy metals in the shoots and roots of herbaceous species such as A. filix-femina, L. peruviana, and S. trilobata has not been entirely clarified; therefore, it is unclear which of these species or tissue organs is a bioaccumulator, phytoextractor, or phytostabilizer. Hence, the main contribution of this research is centered on the assessment of the concentration of Al, Ag, Cd, Cr, Ga, and Sr concentration in shoots and roots of three herbaceous plant species. According to the translocation and bioconcentration factors of the examined heavy metals, the evaluation of phytoextraction and bioaccumulation capabilities was presented further.

Sampling
The sampling site of the three native dominant plant species A. filix-femina, L. peruviana, and S. trilobata was located on the banks of the Carrizal River (Bolívar-Ecuador) ( Figure 1). According to the bioclimatic map of Ecuador, this area has characteristics of tropical climate

Pot experiment
In a plastic flowerpot, the A. filix-femina, L. peruviana, and S. trilobata species (Fig. 2) were propagated (diameter: 30 cm; height: 40 cm). The pots were filled with 2 kg of loamy soil (pH 7.6). A mix of a heavy metal solution (Merck, USA) was added, containing: 12 mg/ kg Ag, 15000 mg/kg Al, 1 mg/kg Cd, 30 mg/kg Cr, 8 mg/kg Ga, and 90 mg/kg Sr. The content of heavy metals was measured thirty days after plant species were sown. Each measurement was replicated three times.

Elements analysis
The plants were harvested carefully for element analysis after one month of planting in the pots. The harvested plants were separated gently into roots and shoots. These parts were treated by high-temperature desiccation below 105 °C for 30 minutes and dried in an oven at 65 °C to obtain a constant weight; the dry tissues were ground to a fine powder and placed in polyethylene bags for further analysis (Alves et al., 2022). Soil samples were air-dried, ground to a particle size of less than 0.147 mm and stored in polyethylene bags until analysis. The content of heavy metals, micro-and macronutrients was determined by inductively coupled plasma mass spectrometry (ICP-MS/MS ion trap, Perkin Elmer, USA).
Analysis of variance (ANOVA) and Tukey range tests (p<0.05) were conducted using Rproject and R-studio with ggplot2 package (R Core Team 2022; Wickham 2016).

Bioconcentration and translocation factors
The translocation factor (TF) establishes the relationship of the concentrations of elements in the shoot and root parts of the plant; whereas, in bioconcentration factor (BCF) it is the proportion of concentrations of elements in plant tissues and soil, which represents the ability of plants to accumulate soil elements (Ding et (1) where: C p -concentration of the element in the plant; C s -concentration of the element in the soil.
where: C za -concentration of the element in the shoot zone of the plant; C r -concentration of the metal en the root of the plant.

Effect on micro-and macronutrients
The concentration of micro-and macronutrients varied depending on the plant species. According to Figure 3a, in the root system, A. filix-femina accumulated K as the highest concentration (6656.30 mg/kg). In shoots, for A. filixfemina, the lowest concentration was for Co (0.00 mg/kg) and Ni (0.02 mg/Kg), while the highest concentration was for Va (923.50 mg/kg) and Ca (4041.62 mg/kg). In general, greater accumulation of Co and Ni occurred in rhizomes than in fronds, suggesting limited mobility and translocation of these minerals once absorbed by ferns (Samecka-Cymerman et al. 2011; Drăghiceanu et al. 2019). The mostly transported minerals from rhizomes to fronds were Ca, Fe, Mg, P, Va, Si, Mn, B and Cu (Fig. 3a).
L. peruviana accumulated high concentrations of Fe (571.13 mg/kg), Ca (868.82 mg/kg), and K (5345.87 mg/kg) in the root system. In the shoots, L. peruviana accumulated the lowest concentration of Co (0.00 mg/kg), Ni (0.01 mg/Kg), Va (0.10 mg/Kg) and Cu (1.31 mg/Kg); while the highest concentration was for K (8259.96 mg/kg), Ca (4581.15 mg/kg), P (949.41 mg/kg) and Mg (823.67 mg/kg). Greater accumulation of V and Co occurred in the root system than shoots. The mostly transported minerals from roots to shoots were K, Ca, Mg, Fe, P, S, Na, Si, Mn, B, and Cu (Fig. 3b).
Mg is an important macronutrient for plants because it is part of the chlorophyll structure and protects the molecular structure of ribosomes (Farhangi-Abriz and Ghassemi-Golezani 2021). A sufficient level of Mg is vital for plant salt tolerance. K, being a primary macronutrient, is the most abundant inorganic cation and is important to ensure optimal plant growth; it is an activator of enzymes for protein synthesis, sugar transport, N and C metabolism as well as photosynthesis (Xu et al., 2020). In the complex interactions that occur in soil, Na replaces K which can lead to nutrient deficiencies (Artiola et al. 2019).
Plants have developed highly specific and highly efficient mechanisms for obtaining essential micronutrients from the environment with the help of the root´s chelating agents whereas the pH changes induced by redox reactions, can solubilize and absorb micronutrients of very low levels in the soil, even from almost insoluble precipitates (Usman et al. 2020). Plants have also developed highly specific mechanisms for moving and storing micronutrients, which are involved in the uptake, translocation and storage of toxic elements, the chemical properties of which simulate those of essential elements (Steliga and Kluk 2021). In this regard, phytoremediation is rather interested in micronutrient absorption pathways. Figure 4 shows the overall count concentration of different elements concentrations (mg/ kg) as stack bars of L. peruviana, A. filix-femina and S. trilobata on soil, shoot and root system. Bioavailability of micronutrients, macronutrients, and heavy metals varies amongst plant systems. According to the findings, the rate of absorption and bioaccumulation is higher in roots compared to shoots. The three plant species accumulate Ba, Cd, Cr, Sr, Al, Li, Ag, Ga, In, Te, As, and Sr in shoot and root systems. Elements like Cr are heavy metals that are considered micronutrients but become toxic when consumed in large amounts (Bhat et al. 2022). On the other hand, Cd, Hg, Al, Pb, Sr, Te are non-essential heavy meals that are lethal to living organisms (Ali and Khan 2019). Heavy metals inhibit physiological processes, such as respiration, photosynthesis, cell elongation, the plant-to-water relationship, and metabolism. It has been reported that soil infertility, associated with its acidification, is mainly caused by aluminum, an element capable  A silver initial concentration of 12 mg/kg was taken up by A. filix-femina shoots in 52% and roots in 40%. Ag accumulation was up to 1.3 times higher in shoots than in roots in the or- Cadmium accumulated to a greater degree in the shoots (14 and 19%) of A. filix-femina and L. peruviana, respectively, compared to their roots (8% and 12%). The opposite was observed for S. trilobata. Its roots took up in the most Cd, with 27%, but its shoots only accumulated 14%. It is considered that L. peruviana is a good accumulator of heavy metals such as Cu, Zn, Pb, and Cd, and it is one of the most important genera of wetland plants (Chowdhury et al. 2013; Anyinkeng et al. 2020). Furthermore, S. trilobata accumulates more than 100 mg/kg of Cd and has the tolerance mechanisms that make it a viable candidate for phytoremediation of this element (Pernía et al. 2019).
Chromium had an initial concentration in soil of 30 mg/kg, although the permissible limits for Cr in soil is 3.8 mg/kg (Vodyanitskii 2016). In all plant species, it was found that the root system accumulated the highest concentrations of Cr in the following order S. trilobata > A. filix-femina > L. peruviana. According to results, A. filix-femina and S. trilobata uptaked up to 19% of Cr in their roots, and up to 2% in their shoots. The toxic effects of Cr on plants result in Gallium is an emerging contaminant that is used in advanced industries and is considered as toxic to humans and produces an inhibition effect on crops (Chen et al. 2022;Syu et al. 2021). Shoots of S. trilobata accumulated up to 24%, whereas roots accumulated up to 18%. From an initial Ga concentration of 8 mg/kg, S. trilobata was shown to accumulate the most Ga of any plant species in this investigation.
Strontium was accumulated by A. filix-femina in a concentration of 15% in shoots and 31% in roots. The L. peruviana and S. trilobata shoots took up 26% of Sr, whereas the roots 20%. Strontium limits plant calcium uptake and is extremely toxic, producing metabolic imbalances in the tissues (Mikhailovskaya and Pozolotina 2020).
According to Table 1, there were statistical differences in the heavy metal accumulation in the shoots and roots (Factor A). However, the accumulation response to heavy metals did not depend on the three plant species (Factor B).
Tukey´s test (Table 2) shows that a significant difference exists among soil and plant organs in the accumulation of all tested heavy metals. In this case, the heavy metal content in roots was much higher than shoots for all plant species. Heavy metals are considered to be transferred from roots to shoots via root pressure and transpiration (Wu et al. 2021). Membrane transporters, which are proteins enclosed in the membrane phospholipid bilayer, enable higher plants to efficiently absorb accessible metal ions from the soil (DalCorso et al. 2013).   Bioconcentration factor (BCF) and translocation factor (TF) According to Table 3, L. peruviana shows a potential as phytoextractor of Sr, Ag and Ga. The process of phytoextraction is distinguished by the rapid production of a significant amount of biomass in a short amount of time, as well as a high rate of water intake and transpiration, and a robust root system. Accordingly, A. filix-femina shows a potential as phytoextractor of Ag, while S. trilobata shows a potential as phytoextractor of Ag and Ga. Several variables, including soil characteristics, pH, agroclimatic conditions, cultivation techniques, and soil microbial populations, and the total concentration of metals in the soil, affect the process of heavy metal transport to plant tissues (

CONCLUSIONS
The key findings of the study showed that L. peruviana, A. filix-femina, and S. trilobata have the ability to transport Cd, as well as strong phytoextraction and bioaccumulation potentials for this element. Additionally, the plant species have the potential for Sr, Ag, and Ga phytoextraction. There was a clear difference between the heavy metal concentration of roots and shoots across all plant species. The plants that accumulated Cd showed no signs of diminished growth or yield. The rhizosphere of these plants is capable of extracting and accumulating heavy metals. Furthermore, these species are common perennial herbs that may establish, grow, and reproduce rapidly, resulting in a significant biomass yield. The contribution of this work was to use phytoextraction to safely remove metals from shallow contaminated soil and translocate them into plant tissues as soluble compounds. The roots and fast growth rates of the herbaceous plants tested here indicated a potential for survival in the soils contaminated with heavy metals.