Screening of native plant species for phytoremediation potential in Pb-Zn mines in Iran

Industrial mining activities are one of the significant sources of contamination and their deleterious effects impact soils, water bodies, wildlife habitats, and quality of life far from their location. Contaminated soil threatens the human health as heavy metals can be transferred into the food chain, especially when these sites are converted to agricultural land or pastureland (Dickinson, 2017). Therefore, it is necessary to reclaim the contaminated sites and reduce the risk of transfer of contaminants along the food chain.

Phytoremediation is a technique wherein metal tolerant plants are used to decontaminate the environment. Some plants, called hyper-accumulators, can accumulate significant quantities of heavy metals in their tissues, and are suitable for phytoremediation. The metal accumulation ability of hyper-accumulator plants is approximately 100–1000-fold higher than that of normal plants growing in soil with normal concentrations of metals, and 10–100-fold higher than that of plants growing in metal-contaminated soils (McGrath, Zhao, & Lombi, 2002). Hyper-accumulators are described as plants can accumulate more than 10000 mg kg^-1 of Zn or Mn, or more than 1000 mg kg^-1 of Co, Cu, Cr, Pb, or Ni, or more than 100 mg kg^-1 of Cd or other rare metals, measured in dry weight (Baker and Brooks, 1989; cited in Koleli et al., 2015). Moreover, hyper-accumulators are characterized by a shoot-to-root metal concentration quotient, i.e., translocation factor (TF) of more than one (Baker, 1981), and a shoot-to-soil metal concentration quotient, i.e., bio-concentration factor (BF), of more than one (Brooks, 1998).

In a study on the Angouran Pb-Zn mine in Iran, Chehregani, Noori, & Yazdi (2009) reported Noea mucronata as a Pb hyper-accumulator; after two years of phytoremediation using plant colonies of N. mucronata, the concentrations of Pb in the experimental pots decreased considerably. Boojar and Tavakkoli (2011) investigated two dominant plant species, Zygophyllum fabago and Peganom harmala, that naturally colonized the region around the Angouran Pb-Zn mine in Iran. They measured the accumulation of Pb, Zn, and Cu, and the chlorophyll and biomass contents of the plant tissues in metal-contaminated and non-contaminated sites. As markers of oxidative stress, the malondialdehyde and dityrosine contents of the plant tissues were measured. However, none of the plants absorbed significant amounts of the elements in the abovementioned sites, and the contents of chlorophyll, biomass, malondialdehyde, and dityrosine in the two plant species did not vary markedly between the two zones studied. In another study carried out by Mahdavian, Ghaderian, and Torkzadeh-Mahani (2015), in a Pb-Zn mine in Yazd, a city in the center of Iran with a semi-arid climate, 40 plant species and the associated soil samples were collected and analyzed for Pb, Zn, and Ag; TF and BF values also were calculated; they tentatively used 1 mg kg^-1 in plant dry maters as a threshold for Ag hyper-accumulator; some of the plant species including Colchicum schimperi, Londesia eriantha, Lallemantia royleana, Bromus tectorum, Hordeum glaucum, and Thuspeinantha persica reached this threshold, with Ag concentrations in shoots ranged between 0.5-3.8 mg kg^-1, TF values above one (1.3.-4.2), and BF values almost above one (0.7-3.1). Hesami, Salimi, and Ghaderian (2018) also researched a Pb-Zn mine in Isfahan-Iran to find locally adapted species for phytoremediation purposes, 69 vascular plant species and associated soils were collected; concentrations of the soluble, exchangeable and total amount of Pb, Zn, Cd, Ca, and Mg in the soil samples were measured; for plants concentration of Pb, Zn and Cd in shoots and roots were measured; TF and BF factors were calculated; they also measured Ca/Mg ratio as a factor that helps plants to uptake less Pb and Zn and reduces toxicity in plants. In a survey conducted on cultivated rice (Oryza sativa), Kim, Yang, & Lee (2002), showed a high concentration of Ca blocked Pb uptake into roots and high concentration of both Ca and Mg block Cd uptake in roots which reduce toxicity caused by high Pb and Cd in plants.

Still, the search for hyper-accumulator species compatible with the surrounding environment is necessary. The selection of true hyper-accumulators is essential for the remediation of contaminated soils. It is desirable that hyper-accumulator plants be identified and examined in their natural habitats (van der Ent, Baker, Reeves, Pollard, & Schat, 2013). The use of native plants for phytoremediation reduces the risk of invasive species that could threaten indigenous plant diversity (Mohanty, Dhal, Patra, Das, & Reddy, 2010). In the present work, the aim was to determine native plant species that can tolerate high concentrations of heavy metals in their above-ground tissues.

Materials and methods

Site description and collection of samples

The study was carried out in a former Pb-Zn mine that had been mined from 1923 until 1971. The mine has nine holes at different elevations, all of which had been abandoned for over 40 years; mining activity at the site was resumed in 2017. The Marjanabad Pb-Zn mine is located at a high elevation in the Manjil region in the Guilan Province of Iran (Fig. 1). The geographic coordinates of the sampling site are as follows: longitude, 49° 28ʹ 18ʺ; latitude, 36° 46ʹ 57ʺ; and altitude, 1700 m above sea level. The region generally has a dry climate with average annual precipitation of about 165 mm. The average annual temperature is about 17.5°C. The warmest month is August with a maximum temperature of about 38.5°C. By contrast, January is usually the coldest month with a minimum temperature of about -5°C.

Figure 1. Location of survey area in Guilan province

The mine is placed in hill forest and dominated by trees and shrubs, such as olive, cypress, beech, oak, hornbeam, checker tree, juniper, ilex, and hawthorn, that have grown owing to the flow of groundwater. There is a village downstream with a population of around 2600 people. The main job of villagers is agriculture and animal husbandary, and the chief agricultural product of the region is olive.

In the course of field survey, an open area was found at the top of the mountain that had undergone significant changes. The soil there had been extensively excavated, the trees had been cut, and consequently, herbs were growing well in the region. Therefore, the study was conducted there and one sample site was chosen. In order to have a general vision of physiochemical characteristics of soil, four soil samples were randomly collected and placed in plastic bags and sent to the laboratory for further analysis. Specimens of plants, shrubs and trees that were more common were randomly collected. Sampling was carried out in August 2017. Healthy, mature plants of the same physiological age and appearance were collected. Plants were avoided that were affected by diseases, insect damage, and physical or chemical injury. A total of twelve plant samples which were the most common species in the area were collected, including five herbaceous plant species belonging to five different families, and three species of shrubs, and four species of trees. For trees and shrubs only leaves were taken with a clean scissor, and for herbaceous species whole plants were excavated and stored in paper bags. Three replicates of each plant were collected, two of which were employed for the purpose of identification and one for the measurement of heavy metal contents. Soil samples were collected around the roots of the plants gathered. Some plants were growing in the same place, so ten soil samples were taken. The plants were divided into roots and shoots. The samples were carefully washed with tap water, rinsed with distilled water, then washed with 0.1 mol L^-1 HCl to ensure there were no soil particles adhering to the plants, and rinsed with distilled water several times. The samples were dried at 70°C for three days, ground to pass a 2 mm mesh sieve, placed in labeled bags, and transported to the environmental laboratory for further analysis. The soil samples were dried in air, stored in plastic bags, and transported to the environmental laboratory for further analysis.

Soil and plant analyses

In the laboratory, the physical and chemical properties of soil were determined via several methods. The soil acidity (pH) and electrical conductivity (EC) values were measured via the saturated extraction method as described by Warncke (1986), and read using a pH meter and EC meter, respectively, while the soil texture was analyzed via the hydrometer method as described by Gee and Bauder (1986). The cation-exchange capacity (CEC) was determined using the Bower method (Bower, Reitemeier & Fireman, 1952). The organic carbon content was determined using the Walkley and Black procedure (Nelson & Sommers, 1982).

For the analysis of heavy metals in soil, the soil samples were pulverized using a low-Chrome stainless steel mortar and pestle and passed through a 0.075 mm mesh plastic sieve. The total metal contents of the soil samples were determined via multi-acid digestion of 250 mg of soil using HF, HCl, HClO_4, and HNO₃. The resultant solutions were analyzed via inductively coupled plasma mass spectrometry (ICP-MS) with detection sensitivities of 0.1 mg kg^-1 for As, Cd and Mo, and 1 mg kg^-1 for Co, Cr, Cu, Ni, Pb and Zn, and 0.5 mg kg^-1 for Sb.

Extractable metals were measured by adding 50 ml DTPA (diethylene triaminepenta acetic acid) to 25 g of the pulverized soil and shaking for 2-hr, samples were filtered through Whatman filter paper, and the resulting solution was analyzed by using inductively coupled plasma optical emission spectrometry (ICP-OES). The detection limit was 0.05 mg kg^-1 for all heavy metals.

For the analysis of heavy metals in the plants, a mixture of HNO₃/HClO₄ was added to 250 mg of dry plant tissues. The cell containing the samples and reagents was placed in a microwave, after the plant samples were diluted with, and dissolved in, distilled water. The resultant solutions were analyzed via ICP-MS. The detection limits were 0.01 mg kg^-1 for As, Cd, Co, Cr, Mo, Ni, Sb and Pb, and 1 mg kg^-1 for Cu and Zn. Then, the BF and TF values were determined. The BF value was expressed as the quotient of metal concentration in the shoot to that in the soil, and the TF value was expressed as the quotient of metal concentration in the plant shoot to that in the root (Deng, Ye, and Wong, 2004).

Results

Soil properties

The physiochemical characteristics of the soil samples are presented in Table 1. All the soil samples were alkaline, and the pH values were approximately 8. The pH value is one of the most important factors controlling the bioavailability of metals in soil. The soil samples were classified based on their EC values as follows: less than 2, non-saline; 2–8, moderately saline; 8–16, very saline; and exceeding 16, extremely saline (Boulding, 1994). The soil samples had low EC values (< 2 dS m^-1) and were non-saline. The CEC value shows the ability of the soil to retain exchangeable cations, and is an important factor in sites polluted by heavy metals. In general, soils with high CEC can absorb significantly more quantities of heavy metals than those absorbed by soils with low CEC. Almost all the soil samples in the present study had high CEC values (20–33 me.100^-1). The CEC of soil is primarily driven by its organic matter content. The organic carbon contents of the soil samples were moderate (2–7%), and the texture was mostly clay loam.

Table 1. Physiochemical characteristics of soil samples

OC: Organic carbon

EC: Electrical conductivity

CEC: Cation-exchange capacity

Heavy metals in soils

As seen in Table 2, the concentrations of As, Cd, Pb, Zn and Sb in the soils were mostly outside the ranges of maximum allowable concentration (MAC) and trigger action values (TAVs) of trace metals in agricultural soils, as established by Kabata-Pendias and Mukherjee (2007). The average As concentrations in the soils were relatively high and in the range 26–760 mg kg^-1. The Cd concentrations in the soils varied from a minimum of 0.3 mg kg^-1 to a maximum of almost 48 mg kg^-1. The soils were extremely contaminated with Pb and the Pb concentrations were in the range 450–24226 mg kg^-1. Similarly, the average Zn concentrations were high and varied in the range 138–37100 mg kg^-1. The concentration of Sb varied from 6 to 153.

Table 2. Total metal concentration of soil samples

<DL: Below detection limit; MAC, ranges of Maximum Allowable Concentration and TAV, Trigger Action Value for trace metals in agricultural soils (mg/kg)  (A. Kabata-Pendias & Mukherjee, 2007).

DTPA extraction of heavy metal was used to find out the bioavailability of heavy metals for plants uptake. The concentrations of DTPA-extractable for As, Co, Cr, Cu, Mo, Sb, and Ni were considerably low or undetectable (Table 3).

The available Cd concentrations were less than 10 mg kg^-1, the available Pb concentration ranged from 120 to 1552 mg kg^-1, and the available Zn concentration varied between 2.5 to 573 mg kg^-1.

Table 3. Available metal concentrations in soil samples

<DL: Below detection limit

Heavy metals in plants

The plant species collected were identified based on the references provided by Flora of Iran (Ghahreman, 1978-2003), and the trees and shrubs were identified based on Trees and Shrubs of Iran (Mozaffarian, 2004). The names of the plant species and their families as well as the heavy metal concentrations in different plant parts are summarized in Table 4. The calculated BF and TF values are presented in Table 5.

As seen in Table 4, the concentrations of As, Pb, and Zn in Stachys byzantina, Thlaspi hastulatum, and Urtica dioica were in the ranges deemed ‘excessive’ or at ‘toxic levels’ for plants based on the classification established by Kabata-Pendias (2001).

In S. byzantina, the average concentrations of Pb in the shoot and root were quite high, with the former exceeding 1000 mg kg^-1 in dry weight. The average concentrations of As in the shoot and root were significant although not high enough for the species to qualify as an As hyper-accumulator. The TF values of the plant species were more than one except Cu, which indicated good ability to translocate most elements studied from the root to shoot, although its BF values for all the elements investigated were less than one. In the case of T. hastulatum, the average concentrations in the root of the almost all
elements studied were higher than those in the shoot, except Mo which is slightly higher in the shoot. None of the elements were present in adequately high levels for the plant to qualify as a hyper-accumulator. Its TF and BF values were less than one for almost all elements. T. hastulatum appears to be an excluder since the heavy metal concentrations in its roots are higher than those in its shoots. In the case of U. dioica, As, Pb and Zn were in excessive concentration levels, but none of these values were high enough for the species to qualify as a hyper-accumulator. Its TF values were more than one for all the elements studied, except Cr while the BF values were less than one for all elements studied.

The concentrations of all studied elements in Colchicum speciosum and Carduus accanthoides were not significant and were lower than the toxic levels established by Kabata-Pendias (2001). None of the trees and shrubs accumulate significant amount of heavy metals. Almost all the heavy metals in trees and shrubs including Crataegus microphylla, Quercus castaneifolia, Sorbus torminalis, Junperus communis, Carpinus betulus, Fagus orientalis, Ilex spinigera were generally below the toxic levels in plants established by Kabata-Pendias (A. e. Kabata-Pendias, 2001).

Table 5. Bioaccumulation Factor (BF¹) and Translocation Factor (TF²) for plants samples

¹ BF, bioaccumulation factor, (ratio of metal concentration in shoot to that in soil)

² TF, translocation factor, (ratio of metal concentration in shoot to that in root)

Discussion

The goal of this survey was a primary assessment of local plant species useful for phytoremediation. Phytoremediation technique allows the soil to be decontaminated by successive cropping and harvesting. Metal enriched plants can be disposed of as hazardous material or, if economically feasible, can be recycled.

The only plant that exhibited the characteristics of hyper-accumulators was S. byzantina, that accumulated a significant amount of Pb in its above-ground tissues (1797 mg kg^-1), which met the criteria of Pb concentration in dry shoot matter exceeding 1000 mg kg^-1. Pb is the most common soil contaminant. Hyper-accumulators of Pb are rare mainly owing to the low solubility of Pb for plant uptake from soil (McGrath et al., 2002). Even in contaminated sites, the uptake of Pb by plants are low and mostly less than 50 mg kg^-1 (Cunningham et al., 1995 cited in McGrath et al., 2002).

4. byzantine had the TF value exceeding one (4.84), however, the BF value was less than one (0.12), which could be due to the high level of Pb in the soil (15472 mg kg⁻¹). Besides, the soil was alkaline, which rendered significant quantities of elements in the soil unavailable for plant uptake. Metals are more soluble in acidic conditions than in alkaline conditions (Wang, Angle, Chaney, Delorme, & Reeves, 2006; Zeng et al., 2011).

Given the high concentrations of heavy metals in the soil, the BF value is not significant and does not represent the significant ability of the plant to accumulate elements. In agreement with our research, Bech et al. (2012) reported Senecio sp. as a Pb hyper - accumulator with Pb concentration in its aerial part exceeding 4000 mg kg^-1, TF value exceeding one, and BF value of less than one; the average Pb concentration in the soil was more than 13000 mg kg^-1. Similar observations have been reported from other mining areas. In a study by Rotkittikhun et al. (2006) on an extremely contaminated Pb mining site in Thailand (Pb concentration in the range 6420–142400 mg kg^-1), 26 of the 48 plant species investigated accumulated more than 1000 mg kg^–1 of Pb in their shoots, 11 species had more than 1000 mg kg^-1 of Pb accumulated in their shoots as well as TF values of more than one, while none had a BF value exceeding one. Similar results were obtained by Bech, Duran, Roca, Poma, Sánchez, Barceló, et al. (2012) who did not regard the BF value as a reliable index in the case of soils with extremely high levels of metals; in their study, Plantago orbignyana accumulated a significant amount of Zn (8290–11560 mg kg^-1), with TF value of more than one, although the BF value was less than one mainly owing to the high Zn concentration in the soil (30565 mg kg^-1).

Additionally, according to Reeves (2006), plants that accumulate remarkable quantities of metals and whose BF values are less than one are interesting candidates for phytoremediation; by contrast, plants that accumulate insignificant quantities of metals but whose BF values are more than one owing to the low concentrations of metals in soil are not useful for phytoremediation. In our study, the only plant that has the BF value more than one is Carduus accanthoides. Although it has the BF value more than one (1.01) for Cd, the concentration of Cd in its shoot is so trivial (0.3 mg kg^-1), and the reason it had the BF value more than one is that the amount of Cd in the soil is negligible (0.3 mg kg^-1) too. Therefore, the BF value is not a good representative of the extraordinary accumulation of heavy metals in such plants.

Conclusion

The S. byzantina plant species has the potential to function as a Pb hyper-accumulator owing to its significant capacity for the uptake and transfer of Pb to aerial parts as well as its ability to grow in the presence of toxic metals. One of the main limitations of our work was lack of replicate samples, thus further study is necessary to thoroughly assess the entire potential of S. byzantina. Our future work will entail additional investigation into this plant in controlled environments too to ensure that it exhibits healthy growth and to determine any symptoms of phytotoxicity owing to the high quantities of heavy metals.