Copper phytoremediation potential of wild plant species growing in the mine polluted areas of Armenia

Environmental Pollution 249 (2019) 491e501

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Copper phytoremediation potential of wild plant species growing in the mine polluted areas of Armenia* Karen Ghazaryan a, *, Hasmik Movsesyan a, Naira Ghazaryan a, Beatriz Amanda Watts b a b

Chair of Ecology and Nature Protection, Faculty of Biology, Yerevan State University, Alex Manoogian St. 1, 0025, Yerevan, Armenia €tsallee 1, D-21335, Lüneburg, Germany Leuphana University, Universita

a r t i c l e i n f o

a b s t r a c t

Article history: Received 29 June 2018 Received in revised form 11 March 2019 Accepted 17 March 2019 Available online 21 March 2019

Nowadays the pollution of soil by trace metals from the mining industry is one of the biggest threats to ecosystems and human health. In this study, sixteen native wild plant species growing in Cu contaminated soils of mining region in Armenia were investigated to reveal their phytoremediation potential for restoration of soils in this area. During the investigation soil main characteristics affecting the Cu accumulation capability of plants were also determined. In roots (dry weight) of dominant plant species growing in Cu contaminated areas the content of copper varied between 55 mg/kg (Hypericum perforatum) and 775 mg/kg (Thymus kotschyanus), and in shoots of plants - in the range from 33 mg/kg (Teucrium orientale) to 243 mg/kg (Phleum pratense). Since the Cu accumulation capability of plants depends both on physiological peculiarities of plants and on the content of Cubioavailable in the soil, the studies were carried out in this direction and it was found that the high contents of organic matter and clay in the soil facilitated the decrease of the ratio Cubioavailable/Cutotal and as a result - the decrease of Cu accumulation capability of plants. Thymus kotschyanus, Phleum pratense, and Achillea millefolium had the highest phytostabilization potential from all studied plant species due to high bioconcentration factor of root (BCFroot) and low translocation factor (TF) values registered in these plants, and further field and laboratory experiments are planned to confirm this useful ability. The detection of phytoremediation potential of wild plant species growing in areas polluted by trace metals will enable us to use ecofriendly and cost-effective remediation methods, utterly required to clean up the soils in the mining regions of Armenia. © 2019 Elsevier Ltd. All rights reserved.

Keywords: Mine pollution Copper accumulation Native wild plants Phytoremediation Armenia

1. Introduction In these times mining activities are known to have considerable adverse environmental effects (Hudson-Edwards et al., 1995; Li et al., 2007; Romero et al., 2007; Samarghandi et al., 2007). They generate many waste rocks, ore dust, and tailings, which often contain various trace metals in concentrations reaching harmful levels (Callender, 2003; Li, 2005). Considering the pace of economic development, particularly of the mining industry, toxic metals contamination had gradually become a significant problem not only worldwide (Chopin and Alloway, 2007; Farmaki and Thomaidis, 2008; Moreno et al.,

* This paper has been recommended for acceptance by Natalia Rodríguez Eugenio. * Corresponding author. E-mail address: [email protected] (K. Ghazaryan).

https://doi.org/10.1016/j.envpol.2019.03.070 0269-7491/© 2019 Elsevier Ltd. All rights reserved.

2007; Peng et al., 2009) but also in Armenia. Areas near mining activities can become polluted by the transfer of trace metals, that have strong toxic impacts both on ecosystems (AleksanderKwaterczak and Helios-Rybicka, 2009; Bech et al., 2012; Chen et al., 2005; Zabowski et al., 2001) and on human health directly (through contaminated soil, air and water) and indirectly (through food by food chains) (Guidotti, 2005; Kamnev and van der Lelie, 2000; Sing and Sing, 2010; Vaxevanidou et al., 2008). With swift mining industry development without enough attention paid to ecological problems, the soils of risky regions in developing countries such as Armenia were polluted with trace metals including copper and posed different ecological problems in last years (Ghazaryan et al., 2016). Now in Armenia, the soils polluted by trace metals make up to 50000 ha, and 20000 ha from these soils have a medium and substantial degree of pollution. Consequently, limiting the risks of mining regions affected by trace metal pollution has become an urgent ecological problem. In view of previously mentioned the remediation of soils

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contaminated by trace metals is an actual environmental and social problem in Armenia. Traditional remediation methods (physical, chemical and so forth), such as soil excavation, burial, replacement, dilution, organic fertilizing, use of absorbents (activated carbon, zeolite), washing with chelators and strong acids (Baylock and ndez-Caliani and BarbaHuang, 1999; Cheng et al., 2015; Ferna Brioso, 2010; Garcia-Sanchez et al., 2002; Theodoratos et al., 2000), cannot remediate trace metals fully from the soil, moreover in certain cases they can be environmentally destructive and degrade the biodiversity, structure, and fertility of soil (Baran cíkov a et al., 2004; Chen et al., 2000; Mench et al., 1994; Muddarisna et al., 2013). Therefore, the traditional methods of soil remediation must be replaced by alternative eco-friendly (green) and sustainable methods such as phytoremediation (Abreu et al., 2008; Ghosh and Singh, 2005; Hursthouse, 2001; Lasat, 2002; Mulligan et al., 2001; Scullion, 2006). Green methods are acceptable by the society, do not require heavy expenses and at the same time can have a positive impact on the stability of ecosystem, preventing soil erosion, improving the soil structure and biodiversity. Phytoremediation uses the ability of plants to concentrate and metabolize different trace metals from the soil in their tissues; it appears to be very useful for the remediation of contaminated soils (Alkorta and Garbisu, 2001; Zhi-xin et al., 2007). There are five main phytoremediation processes for remediation of soils polluted by different pollutants: phytoextraction, phytodegradation, phytovolatilization, rhizofiltration and phytostabilization (Adriano et al., 2004; Kucharski et al., 2005). For remediation of soils polluted by trace metals phytostabilization and phytoextraction are mainly used (Garbisu and Alkorta, 2003; Pinto et al., 2016). It is a fact that some plants such as Indian mustard (Brassica juncea), common sunflower (Helianthus annuus), smilograss (Piptatherum  and miliaceum), shortpod mustard (Hirschfeldia incana) (Barcelo Poschenrieder, 2003; García et al., 2004; Gisbert et al., 2006; Pichtel and Salt, 1998), cuprophyte (Haumaniastrum katangense, Lamiaceae) (Brooks, 1998), Asiatic dayflower (Commelina communis) and Shiny Elsholtzia (Elsholtzia splendens) (Wang et al., 2005; Wang et al., 2004) can absorb and accumulate trace metals in their tissues. For this purpose, hyperaccumulator plants are generally used. Hyperaccumulator species have been identified based on accumulation ability of different trace metals in their tissues, specifically of copper (up to 1000 mg/kg in dry matter), as well as on bioconcentration factor index (BCF), and shoot/root ratio (S/R) (in case of phytoextraction, in particular), when these indices are greater than 1.0 (Baker, 1981; Brooks et al., 1998; Rotkittikhun et al., 2006). Also, for high effectiveness fast-growing and high biomass developing plants are required. For the phytostabilization purpose, the plants generating strong rootage are needed with shoot/root ratio lower as much as possible (Alvarenga et al., 2008). Although hyperaccumulating plants can accumulate trace lez and metals, they are not suitable for each mining region (Gonza vez, 2006). High polluted areas may support the Gonz alez-Cha growth of specific species of metallophyte plants, which can potentially be used for phytoremediation purposes (Robinson et al., 2009). This capability is mainly developed in local plants because these species are frequently good adapted to local climatic conditions and various properties of soil (Golia et al., 2008). The absorption and allocation of trace metals in different tissues of the plant are significant aspects of estimating their remediation ability of polluted soils. Taking into account the fact that mining activities have been carried out for about 70 years in the studied area and that this territory is significantly polluted by trace metals, in particular by copper, it was obvious, that wild plants already adapted to pollution

and at the same time having a phytoremediation potential could grow in this area. Therefore, the objectives of the present study were as follows: (1) to identify the Cu accumulation capability of native wild plant species grown in contaminated soils of mining region, (2) to determine the characteristics of soil appreciably affecting Cu accumulation capability of plants, (3) to ascertain the plant species that are more acceptable for phytostabilization and that are more suitable for phytoextraction purposes depending on the kind of tissue where they accumulate Cu. 2. Materials and methods 2.1. Site description The study area is situated in the south part of Armenia, near Kajaran town in the surroundings of Zangezur Copper and Molybdenum Combine. First geological investigations in Kajaran were implemented in 1880th and continued up to 1931, when the implementation of wide-ranging drilling programs started in the region. The combine delivered its first production in 1951. It is operated by the open-cut method. Currently, approximately 22 million tons of ore is being processed annually. The combine extracts and processes the copper and molybdenum ore, further processing of which yields copper and molybdenum separate concentrates. The soils of the study region belong to mountain cambisol. This soil type in Armenia is distributed on 500e1700 m above seа level and оn southern dry slopes it extends up to a height of 2400 m (Edilyan, 1990). The relief in the area of distribution of mountain cambisol is multifarious and is characterized by many heights (mountain ranges and water-divider mountain peaks) as well as trenches which descend to canyons and floodplains. The degraded structures of porphyrites, dolomites, limestone, conglomerates, sand, granodiorites are the main soil-producing rocks of mountain brown forest soils (Edilyan et al., 1976). They are mainly represented by weathered carbonic and strong basic sand clay, rarely by clay, the layer of which can reach 1.5e2 m. In this region, the mean annual air temperaturе is 8e12
C, in August it can increase up to 37
C and in January decrease to 23
C. Annual mean precipitation ranges to 450e560 mm (Baghdasaryan, 1971). Warm, mild and variable damp climate, long duration of the soil formation active period, the availability of supportable internal drainage system and seasonal changes of interflow directions contribute to deep and intensive weathering of primary minerals and formation of secondary mineral substances, as well as of rather strong clayey soils. 2.2. Soil and plant sampling and analysis Based on our previous studies the high-risk three sites (Q-F-01 with coordinates N 39
090 36,900 and E 46
080 4300 ; Q-OM-03 with coordinates N 39
090 13,500 and E 46
070 87,900 ; Q-F-11 with coordinates N 39
090 24,100 and E 46
080 52,000 ) and one control (unpolluted) site (Q-CONT with coordinates N 39
130 01,800 and E 46
130 96,000 ) were chosen for investigation. Sampling strategy engaged the collection of soils and plants. Soil sampling was performed from the topsoil (0e25 cm depth) directly nearby the plants. All soil samples were air-dried at room temperature (20e22
C) for two weeks, then were ground and passed through 0.15 mm mesh. For determination of copper total content the soil samples were digested with mixture HNO3 þ HClO4 þ HF (5:1:1, v:v:v) (Baker and Amacher, 1982). Cu content was measured by Atomic Absorption Spectrometer PG990 (PG Instruments LTD). Exchangeable, water, and acid soluble (e.g., carbonates) trace

K. Ghazaryan et al. / Environmental Pollution 249 (2019) 491e501

metals are generally called “bioavailable”. For determination of bioavailable copper in the soil the acetic acid was used, that is, 1 g of ground soil sample (prepared as was mentioned) were placed in 50 ml tubes, mixed in a stepwise fashion with 40 ml of 0.11M CH3COOH, and the suspensions were equilibrated for 16 h at room temperature (He et al., 2013). Then the mixtures were filtered, and the content of copper was measured in filtrates by Atomic Absorption Spectrometer. Plant sampling was done in vigorous growth stage (in June). Three replicates were done for each plant species within the sampling area. Ten individual plants were collected for each one and combined to give a composite whole plant sample. Whole plant samples were thereafter throughly washed with flowing tap water, then twice with deionized water for deletion of any particles from the plants. The plants were separated into root and shoot parts and then weighed. Hereupon the underground and aboveground tissues of plants were dried at 70
C to reach the constant weight. The dried roots and shoots were weighed, after which all samples were grounded into powder and sieved through 0.15 mm nylon meshes. Then the samples were digested by mixture of HNO3 and HClO4 (4:1, v:v) in closed vessels at 150
C for 200 min (0.1 g roots or shoots of plants by 10 ml of acid mixture) (Qu et al., 2008;  et al., 2006). In the resulting solution, the content of Zemberyova total copper was determined by atomic absorption spectrometric method. Chlorophyll content index (CCl) in plants was determined in a field through the instrumentality of CCM-200 plus Chlorophyll Content Meter immediately during the plant sampling. The CCI was measured in median and apical leaves of ten individual plants of each plant species set aside for the determination of copper content. For each species thirty measurements were done. The measurement average was immediately calculated by Chlorophyll Content Meter, following which the mean value of CCI of ten plants was counted. 2.3. Phytoremediation potential of plants Two indices, the bioconcentration factor of the root (BCFroot) and translocation factor (TF), were determined to assess phytoremediation potential of plants (Mertens et al., 2005; Wang et al., 2007). BCFroot may be used to assess the copper accumulation capacity of the root. BCF of root was calculated by the following equation (Marchiol et al., 2013): BCFroot ¼ Curoot/Cusoil (1) where Curoot is the copper concentration in harvested plant roots and Cusoil is the bioavailable copper concentration in soils. The TF was calculated using the following equation: TF ¼ Cushoot/Curoot (2) where Cushoot is the copper concentration in harvested plant shoots and Curoot is the copper concentration in harvested plant roots. If values of BCFroot are above 1.0, it indicates that the plant species has copper accumulation potential and can be used for phytoremediation purposes. If values of BCFroot are below or equal to 1.0, the plant species are not applicable for phytoremediation. In case of TF, the plants with TF values higher than 1.0 can be used for phytoextraction purpose, but they are not suitable for phytostabilisation. And vice versa, if TF value of the plant is less than 1.0, it means that the plant can be used for phytostabilisation and that it is

493

not practical for phytoextraction purpose. 2.4. Statistics Copper concentrations in roots and shoots are represented for three separate replicates. Further evaluation was done via Duncan's multiple range tests. The statistical analysis was performed using SPSS software, version 15. 3. Results 3.1. Soil characteristics of study areas The description of each soil section and their general properties clarified during the field studies are presented in Table 1. The data of field studies indicated that decalcified subtypes of mountain cambisol were the main soils in the studied area. The data of Table 1 show that comparatively good vegetation cover was observed at the Q-OM-03 (95%) site of soil sampling. Almost in all sites, the main vegetation was presented by herbage. The most stoniness was observed at the site Q-F-11. The Q-OM-03 (95%) site of soil sampling was weakly eroded while at the sites Q-F-01 and Q-F-11 the soil was heavily eroded that could be explained by the joint impact of several factors, namely disposition of slopes, steepness of slopes, sparse vegetation and so on. From all soil samples studied only the sample taken at the site Q-OM-03 had a good texture. The texture of the rest of the soil samples was moderately bad. Findings of investigation of soil samples from four study areas reveаled that pH of soils fluctuated in the range 7.6e8.2 that was sufficient for healthy growth and development of vegetation (Table 1). The highest value of pH was observed in soil sample of area Q-F01 where the minimal content of organic matter (OM) was registered (1.8%). OM content in studied territories was fluctuating in the range 1.8e6.48%. The maximum value (6.48%) was observed in a soil sample from the control area, where the mining industry did not have any negative impact both on soil and plants growing in this territory. The lowest content of sand and the greatest content of clay were observed in a soil sample from area Q-OM-03 which could be explained by the northern location of sampling site slope and slight development of erosion processes conditioned by such location. As far as the deposite in the study area is a copper-molybdenic mine and copper is the primary pollutant of soils in this territory, the contents of total and bioavailable copper in soil samples were also determined. In the studied three risky areas, the highest contents of the total as well as bioavailable copper were registered in the site Q-F-11 that is situated at a distance of approximately 300 m from ore mills of processing plant (the ore dust spread to surrounding territories) and is subjected to adverse influence of mining industry the most. The next also high extent of pollution was observed in the area Q-F-01 that is located at a distance of around 600 m from ore mills of the processing plant. In this site due to the relatively large distance from pollution source the content of total copper in the soil decreased by approximately 44% and the content of bioavailable copper e by 70% as compared with their contents in the soil of the area Q-F-11. The lowest level of pollution but still exceeding the total content of copper of the control site approximately 12 times and the content of bioavailable copper 25 times was observed in soils at a distance of 300 m from open mine (Q-OM-03).

7.9 ± 0.3

3.61 ± 0.40

40 ± 4

39 ± 3

21 ± 1

3480.3 ± 209.8 494.4 ± 24.1

3.2. Copper accumulation by dominant plants in three high-risk and one control sites in surroundings of Zangezur Copper and Molybdenum Combine

Decalcified mountain cambisol

Decalcified mountain cambisol

Decalcified mountain cambisol

Q-F-01

Q-OM03

Q-F-11

* 0 - uneroded, 1- weakly eroded, 2 e moderately eroded, 3 e heavily eroded, 4 e very heavily eroded.

moderately bad herbage - 50%, naked 3 soil - 20%, stones - 30% 30


sandy loam

82.4 ± 8.9 888.8 ± 71.4 39 ± 4 45 ± 5 16 ± 2 4.34 ± 0.34 7.6 ± 0.2 good clay loam herbage - 95%, naked soil - 5% 10


1

1951.3 ± 184.7 148.4 ± 10.8 20 ± 2 35 ± 3 45 ± 4 1.80 ± 0.22 8.2 ± 0.2 moderately bad sandy loam herbage - 70%, naked soil - 25%, stones - 5% 22


3

71.6 ± 11.0 30 ± 3 39 ± 4 31 ± 4 6.48 ± 0.38 7.8 ± 0.1 moderately bad sandy clay loam 2 herbage - 80%, naked soil - 20%

Voghji N 39
130 river 01,800 E 46
130 96,000 Voghji N 39
090 river 36,900 E 46
080 4300 Voghji N 39
090 river 13,500 E 46
070 87,900 Voghji N 39
090 river 24,100 E 46
080 52,000 Q-CONT Decalcified mountain cambisol

35


Cutotal Clay Silt pH Humus content Sand Texture Texture classification (mean ± SE) (mean ± SE), % (mean ± SE), (mean ± SE), (mean ± SE), (mean ± SE), % % % mg/kg Erosion degree (0 e4) * Sampling Surface Soil surface cover coordinates gradient Basin Sample Soil type and number subtype

Table 1 General characteristic of sampling area and of plant root zone soils in study sites.

3.2 ± 0.5

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Cubioavailable (mean ± SE), mg/kg

494

Copper concentrations in dominant plant shoots and roots collected from three high-risk and one control sites in surroundings of Zangezur Copper and Molybdenum Combine are shown in Fig. 1. The common eight species of plants per area were gathered in three high-risk study areas: golden locoweed (Astragalus aureus), lacustrine locoweed (Astragalus uraniolimneus), oriental germander (Teucrium orientale), common yarrow (Achillea millefolium), mugwort (Artemisia vulgaris), timothy-grass (Phleum pratense), thyme (Thymus kotschyanus) and small tumbleweed mustard (Sisymbrium loeselii) in area Q-F-01, Artemisia vulgaris, red clover (Trifolium pratense), Achillea millefolium, yellow sweet clover (Melilotus officinalis), dog rose (Rosa canina), coltsfoot (Tussilago farfara), Sisymbrium loeselii and St John's wort (Hypericum perforatum) in area QOM-03, Phleum pratense, Thymus kotschyanus, Astragalus aureus, Astragalus uraniolimneus, feverfew (Tanacetum parthenium), Achillea millefolium, Caucasian stonecrop (Sedum caucasicum) and oriental chervil (Astrodaucus orientalis) in area Q-F-11. Areas Q-F-01 and Q-F-11 resemble by vegetation that is conditioned by similar soil characteristics as well as by their natural-climatic peculiarities. Only one common plant species (Achillea millefolium) was found in all three risky areas. Among the plant species sampled in the area Q-F-01 the highest concentration of copper in root was found in Thymus kotschyanus (718 mg/kg), while the highest concentration of copper in the shoot was found in Phleum pratense (243 mg/kg). In the area Q-OM-03 the highest concentration of copper in roots of plants was revealed in Achillea millefolium (325 mg/kg) and in shoots e in Trifolium pratense (100 mg/kg). In the area Q-F-11 the highest concentrations of copper were found in roots of Thymus kotschyanus (775 mg/kg) and shoots of Tanacetum parthenium (127 mg/kg), respectively. Ten plant species (in total) found at least in one from high-risk studied areas were sampled in the area Q-CONT for the comparison of this unpolluted territory with three high-risk areas. Only Achillea millefolium was found in all studied areas (Fig. 2). In roots and shoots of all plants sampled in the unpolluted area, the content of copper was lower than in the same plant species growing in any of risky areas. This difference is conditioned not only by high contents of copper in the soils of risky areas but also by other properties of soil as well as by physiological peculiarities of plants indeed. The appreciable exceeding of copper content in shoots as compared with control was registered in Tanacetum parthenium (3 times, area Q-F-11), in Achillea millefolium (2.85 times, area Q-F-11) and in Sisymbrium loeselii (2.53 times, area Q-F01). This difference became more evident in roots of plants, particularly in Thymus kotschyanus (18.55 times in area Q-F-01 and 20.03 times in area Q-F-11). 3.3. Relationship between plants and soil Sixteen plant species were sampled from the three high-risk and one control areas. The abundance of diversity of these species in four studied areas, where significant differences in copper contents were also registered, is presented in Fig. 3. According to their tolerance to copper content in the soil the sixteen plant species were clustered in three different categories: moderately Cu tolerant (up to 1000 mg/kg of Cu in the soil), tolerant (up to 3000 mg/kg of Cu in the soil) and highly tolerant (more than 3000 mg/kg of Cu in the soil). According to such division, the eight plants sampled in area Q-F-11 are considered highly tolerant because they can stand such high contents of copper. In the next part of our studies the correlation analysis between

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Fig. 1. Copper concentrations in shoots and roots (dry matter) of dominant plants collected from three high-risk sites of Zangezur Copper and Molybdenum Combine surroundings (a - Q-F-01; b - Q-OM-03; c - Q-F-11).

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K. Ghazaryan et al. / Environmental Pollution 249 (2019) 491e501

Fig. 2. Copper concentrations in shoots and roots of some plant species growing in the three high-risk and one control sites.

Fig. 3. Allocation of 16 species of plants according to concentration of total copper in the soil Accordance between numbers on X axis of the diagram and plant species names is as follows: 1 - Artemisia vulgaris, 2 - Rosa canina, 3 - Tanacetum parthenium, 4 - Achillea millefolium, 5 - Melilotus officinalis, 6 - Sisymbrium loeselii, 7 - Thymus kotschyanus, 8 - Trifolium pratense, 9 - Hypericum perforatum, 10 - Tussilago farfara, 11 - Astragalus aureus, 12 - Astragalus uraniolimneus, 13 - Teucrium orientale, 14 - Phleum pratense, 15 - Sedum caucasicum, 16 Astrodaucus orientalis.

the soil copper total and bioavailable concentrations and copper concentrations in shoots and roots was performed (Fig. 4). A positive correlation was found in all four cases. Such dependence was more pronounced in case of Curoot - Cusoil-total, where a moderate correlation was observed (r ¼ 0.54; P < 0.05), followed by cases Curoot - Cusoil-bioavailable (r ¼ 0.48; P < 0.05), Cushoot - Cusoil-total (r ¼ 0.44; P < 0.05) and Cushoot - Cusoil-bioavailable (r ¼ 0.37; P < 0.05) with weak correlation.

3.4. Chlorophyll content index (CCI) variation in dominant plant species in three high-risk and one control sites In dominant plant species of all studied territories (three highrisk and one control) the CCI values were determined because the chlorophyll concentration indicated the plant growth and tolerance to metal stress in the environments. CCI value is an indicator that is typical for particular plant species, and it can be changed along

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497

in them, has decreased. And, for example, the reduction of CCI values in Rosa canina (1.11 times), Achillea millefolium (1.29 times maximum) and Thymus kotschyanus (1.19 times maximum) was relatively small that indicated the high adaptation of these plant species to soil pollution by copper. 4. Discussion 4.1. Dependence of copper bioavailability on some soil characteristics

Fig. 4. Root and shoot copper concentrations in plant species as a function of total and bioavailable soil copper concentrations (a - Curoot - Cusoil-total, b - Curoot - Cusoilbioavailable, c - Cushoot - Cusoil-total, d - Cushoot - Cusoil-bioavailable).

with the fluctuations of environmental conditions (it may increase in favorable conditions, and it may decrease in unfavorable conditions). Almost in all plants growing in three polluted sites, the study revealed a decrease in CCI values as compared with plants from the control site (Table 2). Drastic decrease of CCI values was observed in some plant species, in particular in Tanacetum parthenium (3.19 times), Melilotus officinalis (2.25 times) and Trifolium pretense (2.14 times). This fact indicated that the potential growth of mentioned plants in copper-polluted soils, specifically capacity to absorb light

The strong toxic impact of trace metals, particularly of copper, on ecosystems and human health is caused primarily by the content of their bioavailable forms and not by their total contents. Such impact is conditioned by the fact that plants can absorb only the bioavailable forms of trace metals passed consequently to human organisms through food chains (Ghazaryan et al., 2017). To identify factors affecting the alteration in the content of bioavailable copper the correlation analysis was performed. Results of the analysis are shown in Fig. 5. It was revealed that the content of bioavailable copper had a strong positive correlation (r ¼ 0.983; P < 0.05) with the copper total content including both bioavailable copper (exchangeable, water and acid soluble) and the forms bounded to iron and manganese oxides, organic matter and soil matrix (residual fraction). It is logical because the bioavailable form of copper is a constituent of total copper. The lowest ratio was registered in area Q-CONT and made up to 4.5% of the total copper content, while the highest ratio was observed in area Q-F-11 constituting 14.2% of the total content. But then the bioavailable copper generally is formed from the other components of total copper since they can turn into bioavailable form due to various biological and chemical processes in the soil (Rieuwerts et al., 1998; Tak a c et al., 2009). The increase in the portion of bioavailable copper on the total content of copper in the soil or the transition of the biologically not accessible form of copper to its bioavailable form depends on the variety of soil characteristics (Table 3). From the correlation analysis using soil variables, it was found out that the change of the portion of bioavailable copper content on the total content of copper (Cubioavailable/Cutotal) was in moderate negative correlation (r ¼ 0.564; P < 0.05) with OM content in the soil. This negative correlation can be explained by the fact that the high content of OM leads to the binding of trace metals (Young, 2013), in this case to the formation of the compounds of copper with organic matter and hereupon to decrease of its biological availability. The high content of OM also promotes the development of soil biota which in turn absorbing the bioavailable copper causes a decrease of its content in the soil (Rieuwerts et al., 1998). The ratio Cubioavailable/Cutotal has also a weak negative correlation (r ¼ - 0.390; P < 0.05) with the content of clay in the soil. The negative correlation is conditioned by the ability of copper to be incorporated into the crystal lattice of clay and become biologically not available (Young, 2013). A very weak positive correlation was found with pH that could be dependent of the chemical speciation of the copper in the soil and interactions of copper with other ions in the soil-water interface (Oorts, 2013). 4.2. The identification of phytoremediation potential of wild plant species Phytoremediation is considered a harmless approach for removal of pollutants, especially trace metals, from the environment or for their conversion into forms that are unavailable for other organisms (Cunningham and Berti, 1993). It should be observed that the low cost of implementing activities, and the fact

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K. Ghazaryan et al. / Environmental Pollution 249 (2019) 491e501

Table 2 Chlorophyll Content Index (CCI), bioconcentration factor of root (BCFroot) and translocation factor (TF) of 16 plant species growing in study sites. Plant species

Artemisia vulgaris Rosa canina Tanacetum parthenium Achillea millefolium Melilotus officinalis Sisymbrium loeselii Thymus kotschyanus Trifolium pratense Hypericum perforatum Tussilago farfara Astragalus aureus Astragalus uraniolimneus Teucrium orientale Phleum pratense Sedum caucasicum Astrodaucus orientalis

Q-CONT

Q-F-01

CCI

CCI

BCFroot

TF

Q-OM-03 CCI

BCFroot

TF

Q-F-11 CCI

BCFroot

TF

5.8 5.2 5.1 1.8 43.2 24.3 1.9 41.5 e e e e e e e e

6.7 e e 1.4 e 28.5 1.8 e e e 1.5 1.5 1.4 1.5 e e

1.45 e e 0.86 e 1.44 4.84 e e e 1.28 1.60 0.77 4.64 e e

0.39 e e 0.61 e 0.47 0.08 e e e 0.42 0.84 0.29 0.35 e e

3.3 4.7 e 1.9 19.2 7.8 e 19.4 1.4 46.5 e e e e e e

0.87 0.73 e 3.94 0.79 1.21 e 1.87 0.67 0.75 e e e e e e

0.70 0.71 e 0.16 0.77 0.60 e 0.65 1.27 0.84 e e e e e e

e e 1.6 1.4 e e 1.6 e e e 1.7 1.2 e 1.9 7.8 1.7

e e 0.51 0.37 e e 1.57 e e e 0.63 0.36 e 1.11 0.75 0.12

e e 0.51 0.59 e e 0.08 e e e 0.34 0.42 e 0.15 0.31 1.19

Fig. 5. Correlation between concentrations of bioavailable and total soil Cu.

Table 3 Correlation analysis of some characteristics of plant root zone soils in three high-risk and one control sites. Cubioavailable/Cutotal

Cubioavailable/Cutotal

pH

OM, %

Sand, %

Silt, %

Clay, %

1 0,692 0932 0,954 0,893

1 0,539 0448 0565

1 0,953 0,990

1 0900

1

1 pH OM, % Sand, % Silt, % Clay, %

0,123 0,564 0,262 0,031 0,390

that this ecofriendly method has social acceptance and quite effectively prevents the subsequent pollution of air and soil are the main reasons for using of plants for remediation concerns (Wong, 2003). Moreover, depending on the ability of the plant to accumulate trace metals in its below ground or aerial parts the phytoextraction and phytostabilization capacity of a given plant could be revealed. BCFroot and TF are the important indices for the revelation of phytoremediation potential. A high bioconcentration factor of the root is a significant characteristic of plant species for phytostabilization suitability. When BCFroot value is < 1 it means that defence mechanisms involved in metal immobilization are not

enough developed in plant, but when BCFroot > 1 the absorption of bioavailable copper occurs actively due to immobilization processes and such plant species have the potential to be used for phytostabilization purposes. The immobilization processes include the complexation of metal ions in the rhizosphere, the sequestration in several subcellular compartments (cell wall, cytosol, vacuole), immobilization with chelating agents (e.g., organic acids and €mer and aminoacids), peptides or proteins, export of metal etc. (Kra phane and Lebrun, 2006; Peer et al., 2006; Clemens, 2006; Ste Andresen et al., 2018). BCFroot values for 16 dominant plant species from three high-risk areas are shown in Table 2. The highest BCFroot

K. Ghazaryan et al. / Environmental Pollution 249 (2019) 491e501

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Fig. 6. Thymus kotschyanus root copper concentrations as a function of bioavailable copper concentration in soils.

values were registered in Thymus kotschyanus (4.84) and Phleum pratense (4.64) growing in area Q-F-01 as well as in Achillea millefolium (3.94) growing in area Q-OM-03. These plant species are quite developed in mentioned areas and can be used for phytostabilisation purposes. TF low values are also observed in them. This fact indicates that only small amounts of copper transfer to aboveground parts of these plants and that the probability to be included into food chains is low. The use of these plants will lead to the decrease of the content of bioavailable copper in the soil which in turn will reduce the pollution of ground and surface waters as well as will limit the transfer of copper to human in toxic quantities. It should be noted that the BCFroot indices for the same plant species had various values in different areas. For example, in Achillea millefolium in area Q-OM-03 a high value of BCFroot was registered while in area Q-F-11 it was low. This fact can be explained by significant differences in soil characteristics of these two areas that had a direct impact on plant growth and development while BCFroot different values in Thymus kotschyanus growing in areas Q-F-01 and Q-F-11 with similar characteristics of soil can be explained by physiological peculiarities of the particular plant species. In particular, the copper contents in the given plant species in both Q-F-01 and Q-F-11 areas were 718 mg/kg and 775 mg/kg, respectively, but in the area Q-CONT, where the content of bioavailable copper in soil was low the copper content in the plant amounted barely to 38.7 mg/kg (Fig. 6). Based on data obtained from these three stations the conclusion was drawn that in case of relatively low concentrations of bioavailable copper in the soil rapid growth in copper accumulation by roots is observed simultaneously with the increase of copper content. During the further increase of bioavailable copper content the accumulation of copper becomes slower due to changes in physiological processes in plant roots. In other words, root specific defensive mechanisms are operated so that the copper has no toxic effects on the plant. Specifically, reduction of copper uptake is implemented possibly by extracellular exudates or mycorrhiza action (Cuypers et al., 2013). Nevertheless, for testing of this hypothesis kinetic studies under controlled laboratory conditions are needed. High translocation factor is a significant characteristic of plant species for phytoextraction suitability, but for phytostabilization suitability а low translocation factor is required. The highest TF values (>1) were observed in Hypericum perforatum (1.27) and Astrodaucus orientalis (1.19). This fact indicates that mentioned plants by active mechanisms (mediated by ATPases, also called

‘CPx-ATPases’ because of a characteristic conserved cysteineeproline motif in their sequence or ATP-binding cassette transporters (ABC transporters) that in contrast to the CPx-ATPases, do not transport the metal (loid) ion in a free state, but bound to a ligand such as glutathione or phytochelatin (Andresen et al., 2018)) transfer the copper to their aboveground parts and it can be removed from the soil through harvesting. However, in Astrodaucus orientalis very low value of BCFroot (0.12) was observed which limits the use of this plant species for phytoextraction purposes. But in Hypericum perforatum BCFroot is 0.65 and for the identification of potential for its use in phytoextraction purposes further laboratory studies are required to reveal the factors that may contribute to the increasing of BCFroot and TF values. In this way, it is possible to remove bioavailable copper from the soil and prevent its transfer to the food chain and further toxic effects on human health and other living organisms. In Thymus kotschyanus and Phleum pratense growing in area QF01 and Achillea millefolium from area Q-OM-03, which had high values of BCFroot, low TF values were observed (0.08, 0.35 and 0.16, respectively) which is a sufficient condition for application of these plants in phytostabilization purposes. It should be noted that in plant species Thymus kotschyanus and Achillea millefolium growing in polluted sites a slight decrease of CCI values was registered, that pointed to the ability of mentioned plants to grow in copper polluted soils. Tussilago farfara from studied plant species had not very high values of BCFroot and TF. At the same time, this plant grew very good and spread quickly in low nutrient substrata which would be a significant advantage in the revegetation of waste rock piles and open mine areas not in use. Such ability of this plant species also would reduce the costs of this process, because it can grow well without fertilizer. 5. Conclusion Studies have shown that the content of bioavailable copper in the soil primarily depends on copper total soil content and the dependence of the ratio Cubioavailable/Cutotal on different characteristics of soil made the following descending series: OM ˃ clay ˃ sand ˃ pH ˃ silt. In some cases this dependence was negative (OM, clay), and in other cases - positive (sand, pH, silt). Studies of copper content in below ground and aboveground tissues of 16 dominant plant species from three high-risk areas

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revealed that investigated plant species very differed in their capability to accumulate copper. Thymus kotschyanus, Phleum pratense and Achillea millefolium demonstrated higher copper accumulation in their root tissues (depending on soil characteristics this feature was displayed in Achillea millefolium in different ways) than the other 13 plant species, all of which well grew in copper contaminated areas. Phytostabilization potential in Thymus kotschyanus and Phleum pratense is greater as far as they are perennial plants and develop a strong root system. A slight decline of CCI values observed in Thymus kotschyanus and Achillea millefolium at the copper polluted sites as compared with the control site is the indication of high adaptation of these plant species to soil pollution by this metal. Study results have led us to the conclusion that Thymus kotschyanus, Phleum pratense and Achillea millefolium are suitable for use in remediation of copper contaminated soils due to their good phytostabilization potential. Based on our findings, it is planned to increase subsequently the phytoremediation potential of studied plants by the application of non-toxic and environmentally harmless chelating agents and to use these plants for soil restoration in the mining regions of Armenia. Acknowledgments This work was supported by the State Committee of Science MES RA, in the frames of research project SCS No 18RF-077 and by a research grant from the Ministry of Education and Science of the Republic of Armenia, Armenian National Science and Education Fund based in New York, USA (research project N
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