Analysis of Baccharis dracunculifolia and Baccharis trimera for Phytoremediation of Heavy Metals in Copper Mining Tailings Area in Southern Brazil

This study aimed to investigate the phytoremediation potential promoted by Baccharis dracunculifolia DC. and Baccharis trimera (Less.) DC. in copper mining tailing area, in the Southern part of Brazil. The plants were selected considering their spontaneous growth in tailing area. The phytoremediation indexes including translocation factor (TF), bioconcentration factor (BCF), metal extraction ratio (MER), and plant effective number (PEN) were assessed. Both species showed higher concentrations of heavy metals in the roots than to the shoots. B. trimera has potential for phytoextraction of Zn, Cd, Cr, and Pb and phytostabilization of Ba and Ni, whereas B. dracunculifolia demonstrated potential for phytoextraction of Pb and phytostabilization of Cu, Zn, and Ba. B. trimera showed higher potential in phytoremediation of the metals such as Cu > Zn > Cr > Ni and Cd than the B. dracunculifolia plants. A smaller number B. trimera plants was required to remove 1 g of Cu, Zn, Cr, Pb, Ni, and Cd than B. dracunculifolia plants, and implies that B. trimera is more efficient for decontamination of the metals. Both species showed potential for phytoremediation of metals in the mining tailing area under study.


Introduction
The wide range of mineral resources in Brazil is well known. Brazil has about 207 active mines, producing a wide variety of industrial minerals, including metals such as Al, Cu, Cr, Fe, Mg, Nb, Ni, Au, V, and Zn [1].
Mining is an essential economic activity for human societies. However, in the current years, the disruption of the dams of tailing of Brumadinho (2019) and Mariana (2015) brought a worldwide attention on Brazil due to the environmental contamination [2]. It was evident that the country has been facing problems regarding the proper control and management of its mining tailing areas.
Mine tailings are generated after the mineral processing, and consist of a mixture of rock (solid waste) with variable composition, depending on type of rock and mineral contents [3]. The mine tailings can impact environmental quality and contributes to high concentrations of heavy metals in soil, water, and other environments [4].
Due to the high content of toxic metals, tailings can contaminate the environment and must be chemically and physically stabilized. However, there is a wide range of techniques that can be used for remediation of these areas such as phytoremediation, which can stabilize both chemical and physical aspects [5].
Phytoremediation is a technique that uses plants for removing or immobilizing organic or inorganic contaminants [5]. They also have different mechanisms such as phytostabilization and phytoextraction, which the first is the uptake of contaminants from soil or water by the biomass of plants and the second is the translocation of the elements from the roots to the shoots [6]. Phytostabilization is using plants that bind toxic compounds in the roots or rhizosphere zone [7]. Both techniques are a way to investigate whether a species has potential for phytoremediation.
The Gazania rigens and Pelargonium hortorum plants grew spontaneously in a copper mining tailings area in Chile, and showed potential for phytoremediation of heavy metals [8]. The species B. trimera (Fig. 1a) and B. dracunculifolia (Fig. 1b) were found in some studies in copper and gold mining areas in the south of Brazil [9,10]. However, there is a lack of studies on the deployment of B. trimera and B. dracunculifolia for phytoremediation purposes. Belonging to the family Asteraceae, both species are rustic, perennial, and dioecious shrub, native to the southern and southeastern regions of Brazil [11,12].
B. dracunculifolia plays an important role in the colonization and regeneration of contaminated areas, and is considered in Brazil as secondary vegetation or involved in regeneration and advanced stages of regeneration that is resulting from natural processes of ecological succession [13]. A few studies indicated that both species have the ability to accumulate heavy metals in their biomass [9,10,14]. Thus, the aim of this study was to investigate phytoremediation potential and processes of phytoremediation in B. dracunculifolia and B. trimera in the copper mining tailings area in Southern Brazil.

Study Area
Camaquã Mines is the municipality of Caçapava do Sul in the state of Rio Grande do Sul located in the South of Brazil (Fig. 2). The region of Camaquã Mines is part of the Pampa Biome, characterized by vegetation of the wooded steppe type [15]. The climate is temperate and classified as humid mesotherm. The area is a part of the sub-basin of Arroyo João Dias [16]. The soils that comprise the Camaquã Mine region are classified as A-Chernozemic [17] and marked by what remains of the copper mining. The area is on the plane levels of mining tailings where the mined area has been filled.
The Camaquã Mines region contains one of the largest deposits of copper metals associated with Pb-Zn in the south of Brazil. The exploration of base metals (mainly copper) has occurred since the nineteenth century [18]. The region includes the tailings of Uruguay Mine (Cu) (closed in 1996), which is the specific area of this study.

Plant Samples and Elements Detection
Both species of B. trimera and B. dracunculifolia have grown spontaneously in the copper mining tailings of the Uruguay mine. B. trimera showed an average of the total dry biomass of 2.4 ± 0.7 g (roots), 4.8 ± 2.8 g (shoots), and an average height of plants of 42.0 ± 4.0 cm, while B. dracunculifolia showed an average height of 33.5 ± 12.0 cm and dry biomass of 0.9 ± 0.6 g (roots), 3.9 ± 2.2 g (shoots).
The plants (n = 12) were sampled in the copper mining tailings area in the summer season and the sampled material was packed, stored, and properly identified in paper bags and taken to the laboratory. The samples of plants were washed individually and divided into shoots and roots and fresh weight was determined. After, the biomass was dried in an oven at 70ºC for 48 h and ground to a homogeneous powder [19]. For the digestion process, concentrated nitric-perchloric acid (HNO 3 -HClO 4 ) was used at ratio of 3:1 (acid:plant powder) according to the methodology described by Tedesco et al. [20]. The determination of the elements was performed by ICP-OES (PerkinElmer®-version Optima™ 8300).

Chemical Analysis of Tailings
Sampling of the copper mining tailings was taken from a sampling composed of five replications at a depth of 0 to 20 cm, obtained mainly from the copper mining tailings of the Uruguay Mine (closed at 1998) in the region of Caçapava do Sul-RS, Brazil (Fig. 2).
The samples of waste were dried at room temperature, crushed, and sieved (3 mm) before analysis. The physico-chemical properties of the material were determined ( Table 1). The pH ratio in water is 1:1.5; (w/v) adapted from the methodology in Landon [21]; CEC: cation exchange capacity; P, Na, Cu, and Zn

Metal Accumulation
After determining the elements in the roots, shoots, and tailings, the phytoremediation indexes were carried out to evaluate the ability of plants to remediate the contaminated area.
Translocation factor (TF) and bioconcentration factor (BCF) are parameters to study the soil-to-plant transfer accumulation of heavy metals [28]. The parameters evaluate the phytoextraction potential of the plants.
Translocation factor value was represented according to the following Eq. 1. Translocation factor value greater than 1 indicates the translocation of the metal from root to aboveground part (tailings in this case) [29].
where [metal] shoots and [metal] roots are the metals concentrations in dry biomass (mg kg −1 ).
The bioconcentration factor (BCF) is used to calculate the distribution of heavy metals between sediment (tailings in this case) and the plant is defined by Eq. 2. According to Yoon et al. [6] and Kamari et al. [30], only plant species with both BCF and TF greater than 1 have the potential to be used for phytoextraction.
where [metal] roots is the metal concentration in the dry biomass (mg kg −1 ) and [metal] tailings is metal concentration in the tailings (mg kg −1 ).
The metal extraction ratio (MER) was also determined. This index expresses the capacity for accumulation of metal in the shoots of plants about that in the tailings [31] where [Cplant] is the concentration of the metal in the plant (mg kg −1 ); [Mplant] is the dry weight biomass; [Ctailings] is the concentration of the metal in the tailings (mg kg −1 ), and [zone Mrooted] is the tailings volume occupied by the plant (value adopted of 1 kg). To determine the number of plants required to extract 1.0 g of element or metal of interest, the PENs (plant effective number) were determined, which considers the biomass of the shoots of the plant and the PENt (plant effective number total) takes into the calculation the total biomass of the plant (shoots and roots) [32]. Both the PENs and PENt were determined considering the concentration of the target element present in the dry biomass of the plants as well as the dry weight biomass. The following equation was used: The phytoremediation potential (g ha −1 ) was estimated considering the total concentration of the target metal in the plant and the weight of biomass. For this, the dry biomass production of the plant was estimated considering studies by Santos et al. [33] for B. dracunculifolia (3.86514 t ha −1 ) and Amaral et al. [12] (10.3 t ha −1 ) for B. trimera, multiplied by the total concentration of each target metal in the plant (total mg kg −1 ).
The experimental design was completely randomized. The software Statistica® version 7.0 was used for the analysis of variance (ANOVA), and Tukey's test (p < 0.05) was carried out when ANOVA showed significant results.

Nutrients Uptake and Heavy Metal Content in Biomass
The behavior of both species of B. trimera and B. dracunculifolia into the concentration of macronutrients in the roots was similar. In B. trimera, the sequence of macronutrient concentrations was Ca > K > Mg > S > P, whereas in B. dracunculifolia, Ca > K > S > Mg > P sequence of macronutrient concentrations was observed. The sulfur concentration (1956.8 mg kg −1 ) was higher than the concentration of magnesium (1142.1 mg kg −1 ) in B. trimera (Fig. 3).
The concentrations of the elements in the shoots of B. trimera showed the following order, Fe > Na > Al > Mn > Cu > Zn > Ni > Co, whereas in the shoots of B. dracunculifolia, the order was Fe > Al > Na > Mn > Cu > Zn > Ni > Co (Fig. 4). Even with an inversion between the aluminum and sodium, the sequences between the species were similar in both species (Fig. 4). In the roots, these elements concentration followed the same order in both species B. trimera and B. dracunculifolia (Fe > Al > Na > Cu > Mn > Zn > Ni > Co).
The concentration of Cu in the shoots of B. dracunculifolia was almost half of the concentration showed by the B. trimera (Fig. 4). The same behavior was observed for the concentrations of Cu in the roots, being two orders of magnitude higher in B. trimera, when compared to B. dracunculifolia (Fig. 4).
The concentrations of Ni in the shoots were similar between the species (Fig. 4). The B. trimera showed higher concentrations of all elements than B. dracunculifolia for both shoots and roots, except for Zn and Ni (Fig. 4). Also, both species presented high concentrations of heavy metals in the roots than in the shoots, except for Cd and V (Fig. 5).

Phytoremediation Index
The translocation factor (TF) in B. dracunculifolia was higher than one (TF > 1) for Fe, Cd, Cr, Pb, and Zn. In B. trimera, a TF value higher than 1 was attained for only Pb   (Table 2). In B. dracunculifolia, the bioconcentration factors greater than one (BCF > 1) were detected for the metals Zn, Cd, Cr, Ni, Pb, and Ba; however, the B. trimera showed the BCF > 1 for Cu, Zn, Pb, and Ba (Table 3). Both species demonstrated TF > 1 for Pb and BCF > 1 for Zn, Pb, and Ba. The species B. dracunculifolia indicated BCF > 1 and TF > 1 for metals Zn, Cd, Cr, Pb, and BCF > 1 and TF < 1 only for Ba (Table 2). However, B. trimera showed BCF > 1 and TF > 1 only for Pb and BCF > 1 and TF < 1 for the metals Cu, Zn, and Ba (Table 3).
Both plants B. dracunculifolia and B. trimera exhibited high value of MER index for extraction of Zn and Ba metals (Tables 2 and 3). Although the MER index of Cr and Pb were not so high in both species, they were higher than one (MER > 1). Considering the copper element, only the B. trimera showed MER value higher than 1 ( Table 3).
The B. trimera species requires a smaller number of plants to remove 1 g of Cu, Zn, Cr, Pb, Ni, Cd, for both PENs and PENt compared to B. dracunculifolia ( Table 2).
Regarding the presented results, the B. trimera species showed a higher potential for phytoremediation of Cu > Zn > Cr > Ni > Cd than the B. dracunculifolia (Fig. 6). While B. trimera showed potential to remove 6039.60 g ha −1 of Cu, the B. dracunculifolia would remove 992.10 g ha −1 . Even though B. trimera displayed high potential for copper removal, the B. dracunculifolia also showed promising results for phytoremediation. B. trimera would remove more than two times as much Cd and Pb as B. dracunculifolia (Fig. 6).

Discussion
B. dracunculifolia and B. trimera plants grew very well in an area contaminated with copper mining tailings and displayed mechanisms of tolerance and resistance to heavy metals [9,10]. Species of the family Asteraceae are plants that are common in environments impacted with contaminants and undergoing regeneration. Melo-Júnior et al. [34] in a  survey in a degraded environment found more than 16 species of the Asteraceae family, among them, were B. dracunculifolia and B. crispa. Some species of the genus Baccharis are known as indicators of initial stages of environmental regeneration (B. trimera) and an indicator of primary vegetation of the middle and advanced stages of regeneration (B. dracunculifolia) according to the Brazilian legislation [13]. Depending on the ability of plants to adapt under adverse conditions, the species showed different phytoremediation mechanisms, capable of extracting or stabilizing metals, in the soil or in their system [35], as in the case of B. trimera and B. dracunculifolia, which demonstrated mechanisms of phytoextraction, phytoaccumulation, and phytostabilization of metals (Tables 2 and 3).
Both species seem to face difficulties in translocation of some metals to the shoots; this suggests resistance mechanisms, such as the exclusion of metals [36]. Thus, the transfer of the metal to the plant prevents its accumulation in the shoot's biomass as can be seen in Tables 2 and 3.
The concentration of metal accumulated in the shoots indicates that B. dracunculifolia is more efficient for translocation of Zn, Fe, Cd, Cr, and Pb than the B. trimera, which showed high efficiency only for Pb. Resistance mechanisms such absorption, permeability, and or active efflux of the metals by B. trimera may explain the low translocation of these metals [25].
A high concentration of metals in the roots may be due to a slow translocation considering the exposure to a high concentration of metals in copper mining waste [37]. Nevertheless, the B. dracunculifolia has potential for phytostabilization and phytoaccumulation for application in the recovery of areas contaminated with toxic metals [38,39].
When the metal is transferred from the soil to the plant's biomass, the plant uses mechanisms such as exudation, that send toxic compounds to isolate sites, such as apoplast, to avoid their entry into the symplast [40]. In this way, the plant restricts the absorption and translocation of the metals, demonstrating tolerance to the metal. Therefore, typical symptoms of intoxication were not shown in the B. dracunculifolia and B. trimera, since both species showed similar tolerance to high concentrations which were considered to be toxic for the metals Cu (20-100 mg kg −1 ), Cr (5-30 mg kg −1 ), and Ni (10-100 mg kg −1 ) [41].
Although Cu is essential for plants, passive absorption is likely to occur, especially in the toxic range of this metal in solutions. In the root tissue, Cu is almost entirely in complex forms [9]; however, it is more likely that the metal going into the root cells in dissociated forms [41].
B. dracunculifolia is a species that achieves a high survival in places with low nutrient concentration being indicated for environmental regeneration [13] because it presents a good adaptation in degraded areas [10]. This was shown in the study of Boechat et al. [9] in which B. trimera, in gold mining areas in Southern Brazil, obtained metal contents (Cu, Zn, Cd, Ni, Pb, Ba) higher than those found in this study for the same species (Figs. 3, 4, 5, and 6). Silva et al. [42] showed a low concentration of metals (Cd, Co, Cr, Cu, Pb, Zn) in the whole plant of the B. trimera. Although B. trimera presented some variations in terms of metal concentration in its biomass, it displayed TF higher than 1 for the metals such as Zn, Cd, Pb, and the BCF higher than 1 for Cu, Zn, Cd, and Ba [9]. This demonstrates its potential for phytoremediation.
On steel slag, A. thaliana had low biomass production and low BCF and TF, but MER was higher than 1% for Cd (MER = 7.7%), Cu (MER = 8.1%), and Pb (MER = 1.8%) [43]. The metal extraction rate is related to the plant biomass production; hence, more biomass promotes a higher metal extraction rate [36,42]. Thus, both B. trimera and B. dracunculifolia presented lower extraction rates for Cu, Pb, and Cd compared to A. thaliana, but presented very high rates for Zn and Ba.
A possibility that may also be related to the metal extraction rate of the plant is the soluble-exchangeable fraction of metals (Cu, Cd, Pb, Ni, Zn, Cr, and Ba) in the soil. It may be unavailable due to the presence of rhizobacteria resistant to heavy metals such as Kluyvera intermedia, Klebsiella oxytoca, and Citrobacter murliniae present in B. trimera [44]. Other factors such as age, species, pH (soil), influence of the sampling site, and the season of the year [41] can interfere with the metal accumulation in the plant. Seasonal variation can influence levels of metals in plants. In a study of a coal mine in the southern region of Brazil, an increase in the levels of metals in B. trimera during the winter was demonstrated [45].
Analyzing the phytoremediation factors in the B. trimera and B. dracunculifolia, it can be seen that B. trimera showed a potential for phytoextraction (TF > 1 and BCF > 1) for Zn, Cd, Cr, and Pb; and potential for phytostabilization (TF < 1 and BCF > 1) for Ba and Ni. On the other hand, the B. dracunculifolia showed potential for phytoextraction of Pb and phytostabilization of Cu, Zn, and Ba.
Some plants, although tolerant to high levels of metals in the soil (B. trimera and B. dracunculifolia), do not have a capacity to be hyper-accumulating as Verbascum thapsus L [46]. and C. tragacanthoides [47].
Even so, the potential of B. trimera and B. dracunculifolia for removal of heavy metals in toxic contaminated soils is higher than those found by Andreazza et al. [48] in their study of other species from the Asteraceae family. According to the authors, B. pilosa displayed Cu hyper-accumulation. In Cu contaminated soils, B. pilosa would need 83,001 shoots of plants for the copper removal (PENs in inceptisol) and 3,536 whole plants (PENt in mollisol).
The values of PENs for Cu were close to those estimated for B. dracunculifolia, but the PENs (Cu) and PENt (Cu) in B. trimera are much lower, meaning that a smaller number of plants are required for decontamination (Fig. 6).

Conclusions
The results showed that B. trimera demonstrated substantial potential for phytoextraction of Zn, Cd, Cr, and Pb, and phytostabilization of Ba and Ni; whereas B. dracunculifolia showed potential for phytoextraction of Pb and phytostabilization of Cu, Zn, and Ba in tailings area. Both species have a high ratio of Zn and Ba metals extraction.
For copper, only B. trimera demonstrated an MER index higher than 1, indicating a high potential to remove Cu. Besides, B. trimera requires a smaller number of plants to remove 1 g of Cu, Zn, Cr, Pb, Ni, and Cd, compared to B. dracunculifolia.
The values of PENs for copper are close to those estimated for B. dracunculifolia, but the PENs (Cu) and PENt (Cu) in the case of B. trimera are much lower, meaning that a smaller number of plants are required for decontamination. It is concluded that B. trimera showed a higher phytoremediation potential for Cu > Zn > Cr > Ni > Cd metals in relation to B. dracunculifolia, although both species have the potential to be applied in phytoremediation in tailings area because of their rapid and abundant growth. Nonetheless, further in situ experiments in other tailings areas are necessary to determine the feasibility of these plants for large-scale phytoremediation of tailing areas polluted with toxic metals.