Ecological potential of Epilobium dodonaei Vill. for restoration of metalliferous mine wastes

Ecological Engineering 95 (2016) 800–810

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Ecological potential of Epilobium dodonaei Vill. for restoration of metalliferous mine wastes – Dragana Randelovi c´ a,∗ , Gordana Gajic´ b , Jelena Mutic´ c , Pavle Pavlovic´ b , Nevena Mihailovic´ d , Slobodan Jovanovic´ e a University of Belgrade, Faculty of Mining and Geology, Department for Mineralogy, Crystallography, Petrology, and Geochemistry, Ðuˇsina 7, 11000 Belgrade, Serbia b University of Belgrade, Institute for Biological Research “Siniˇsa Stankovi´c”, Department of Ecology, Bulevar Despota Stefana 142, 11000 Belgrade, Serbia c University of Belgrade, Faculty of Chemistry, Department for Analytical Chemistry, Studentski Trg 12-16, 11000 Belgrade, Serbia d Institute for the Application of Nuclear Energy (INEP), Banatska 31b, 11080 Zemun, Serbia e University of Belgrade, Faculty of Biology, Institute of Botany and Botanical Garden Jevremovac, Takovska 43, 11000 Belgrade, Serbia

a r t i c l e

i n f o

Article history: Received 20 February 2016 Received in revised form 27 June 2016 Accepted 23 July 2016 Keywords: Mine waste Epilobium dodonaei Phytoremediation Metabolic response Ecological restoration Conservation biology

a b s t r a c t Metalliferous mine wastes represent one of the major sources of environmental contamination from mining activities. Bor region (Serbia) is one of the largest copper mine basins in Europe where long-term mining caused severe environmental deterioration and created one of the most degraded locations in Serbia and Europe. At the spontaneously colonized metalliferous mine wastes in Bor, plant species Epilobium dodonaei dominates in the mine slopes and mine waste surfaces. Epilobium dodonaei has the status of endangered and protected species in parts of European range (i. e. plant is included in the Red lists of the countries in the Carpathian mountains region), primarily due to losses of natural gravel habitats. The main focus of this research was physico-chemical characterization of mine waste, assessment of phytoremediation potential and plant metabolic stress response of Epilobium dodonaei at the hot spot metalliferous mine site in order to evaluate the possibility for application of endangered species in ecological restoration. The Bor mine wastes are characterized by coarse soil texture, various pH (4.58–8.30), and elevated concentrations of arsenic (44.5–271 mg kg−1 ) and copper (311–2820 mg kg−1 ) that exceed the Serbian limiting threshold and remediation values. Oxidation of metal-sulfide minerals on waste surface leads to increased acidity, followed by elevated metal mobility of the mine spoil solution. Content of arsenic, copper, lead and zinc in roots of E. dodonaei was correlated with pseudo-total and EDTAavailable concentrations in Bor mine spoils. Furthermore, the content of arsenic, copper, lead and zinc in roots (3.98 mg kg−1 , 140 mg kg−1 , 3.19 mg kg−1 , and 72.8 mg kg−1 , respectively) and shoots (4.69 mg kg−1 , 57.7 mg kg−1 , 1.17 mg kg−1 , and 59.3 mg kg−1 , respectively) of E. dodonaei reflected the multi-metal pollution at the investigated site. Epilobium dodonaei largely retains copper, lead and zinc in roots than in shoots and has the potential for phytoremediation of mine wastes. Epilobium dodonaei at Bor mine spoil had a high content of malondialdehyde in roots and leaves as well as reduced chlorophylls and carotenoids content in leaves, indicating great oxidative stress. However, elevated arsenic and copper content could promote biosynthesis of antioxidants in roots and leaves of E. dodonaei at mine spoil. Creation of an endangered species habitat on mine waste rocks of the Bor mining area and similar sites of Carpatho-Balkan metallogenic province could successfully contribute to the preservation of E. dodonaei. Development of practical procedures for the selection and application of endangered plant species in reclamation should create stronger link between ecological restoration and conservation biology. Finally, the application of endangered plant species should take a more prominent role in the restoration process and ecosystem design. © 2016 Elsevier B.V. All rights reserved.

1. Introduction ∗ Corresponding author. E-mail addresses: [email protected], – ´ [email protected] (D. Randelovi c). http://dx.doi.org/10.1016/j.ecoleng.2016.07.015 0925-8574/© 2016 Elsevier B.V. All rights reserved.

Restoration of ecosystems disturbed by human activities is an important field of ecological engineering that involves design, construction, and operation of new ecosystems (Mitsch and Jorgensen,

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1989; Kangas, 2005). Metalliferous mine wastes are unfavorable for plant development due to nutrient-poor conditions and elevated content of chemical elements. Environmental impacts may be significant, especially when sulfides or pyrite are present (Lottermoser, 2010). Incomplete technical reclamation of mine wastes often results in the construction of steep slopes that are devoid of vegetation and prone to erosion (Gentili et al., 2010). The amelioration of hostile environment is necessary for ecological restoration of mine waste site. Since plants pose capability to self-engineer or exert limited control over the rhizosphere and local biogeochemistry, selection and application of suitable plant species for the restoration of mining sites is one of the primary tools of ecological engineering design (McCutcheon and Schnoor, 2003; Simmons et al., 2007; Jørgensen, 2009). Use of plants to reduce the volume, mobility, or toxicity of contaminants in soil and water is operationalized through phytoremediation (USEPA, 2000). Phytoremediation is the cost-effective biotechnology for managing industrial wastes that represents an important part of the field of ecological engineering (McCutcheon and Schnoor, 2003). Phytostabilization is phytoremediation technique that stabilizes wastes and prevents exposure pathways via wind and water erosion, providing metal(loid) immobilization by adsorption or accumulation within the rhizosphere zone (Prasad and Freitas, 2003; Mendez and Maier, 2008). Successful establishment of the vegetation cover on mine spoil leads to pollutant control, slope stabilization, biodiversity, and aesthetic improvement (Wong, 2003). Generaly, many authors recommended conservation of rare or endemic metallophytes as a unique biological resource (Brooks et al., 1985; Dobson et al., 1997; Jordan et al., 1998; Young, 2000; Whiting et al. 2004; Boogert et al., 2006; Baker et al., 2010), but only few papers actually show that endangered or protected pioneering plants can be used to reclaim or stabilize mining and industrial sites (Brofas et al., 2007; Boisson et al., 2015). In this sense, Whiting et al. (2004) suggested systematic screening of plants on metalliferous sites (particularly those likely to be the focus of future mining) in order to identify priority candidates for conservation, implementing ecological restoration of mine sites, and the development of “green” technologies for removing metals from the soil. Plants that grow on mine wastes are exposed to toxicity of chemical elements, such as copper, lead, arsenic, and zinc (Conesa et al., 2006; Haque et al., 2008; Santos et al., 2009; NadgórskaSocha et al., 2013). Heavy metals and metalloids (pollutants) can cause oxidative stress generating reactive oxygen species which are known to damage membrane lipids, proteins, pigments, and nucleic acids, and disturb the basic physiological processes, such as photosynthesis, respiration, and mineralization (Mittler, 2002; Sharma and Dietz, 2009; Gajic´ et al., 2009; Hossain et al., 2012). Copper as an essential redox-active transition metal when present in excess can catalyze the production of hydroxyl radicals (OH• ) from superoxide (O2 •− ) and hydrogen peroxide (H2 O2 ) via the Haber-Wiess and Fenton reaction in plants (Halliwel, 2006). Arsenic leads to the generation of reactive oxygen species through the conversion of arsenate to arsenite in plants, induces lipid peroxidation, and enhances membrane permeability (Talukdar, 2013). Lead and zinc induce oxidative stress in plants causing lipid peroxidation of membranes (Pandey et al., 2009; Rossato et al., 2012). According to Weber et al. (2004) malondialdehyde is one of the final products of peroxidation of unsaturated fatty acids in membrane phospholipids, and is an indicator of the reactive oxygen species. Plants have different defense strategies to cope with toxicity of metals. Plant responses to pollutants that contribute to resistance and tolerance include exclusion (active process that prevents or reduces uptake of toxins), amelioration (active metabolic and detoxification processes that segregate or transform toxins to protect organs, tissues, and metabolic function), avoidance (restriction of the uptake of metals within root tissue by immobilizing

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metals through root exudates or binding metal in the cell wall), and evasion (removal of plant from the stressful environments) (McCutcheon and Mitsch, 1994; Medina et al., 2003). Plants have antioxidant mechanisms that control and scavenge the reactive oxygen species using non–enzymatic and enzymatic antioxidants (Smirnoff, 2005; Sharma et al., 2012; Hossain et al., 2012; Singh et al., 2016). In addition, phenolics are secondary metabolites which can directly scavenge reactive oxygen species, inhibit lipid peroxidation and chelate metal ions (Michalak, 2006; Sharma et al., 2012). Phenolics accumulation in plant leaves and roots occur under copper, lead, zinc, and arsenic stress (Gajic´ et al., 2009; Janas et al., 2010; Gajic´ et al., 2016). The alleviation of oxidative damage and increased resistance to metal stress is related to an effective antioxidant system, and plants with high antioxidant capacity show less sensitivity to metal toxicity (Hossain et al., 2012; Singh et al., 2016). According to Medina et al. (2003) understanding plant responses to pollution stress defines whether a plant has the potential to transform and tolerate a wide range of pollutants, which would enhance phytoremediation. Epilobium dodonaei Vill. [syn. Chamaenerion angustissimum (Weber) D. Sosn.; Ch. dodonaei (Vill.) Schur; Ch. palustre auct. mult., non (L.) Scop.; E. rosmarinifolium Haenke] is a herbaceous perrenial hemicryptophyte belonging to the Onagraceae family (Raven, 2001). The range of E. dodonaei is disjunct and confined to the Alpine areas and marginal of Europe, Asia Minor, and the Caucasus. Primarily, this species belongs to the vegetation class of mountain screes (Thlaspietea rotundifolii). In the central European temperate zone E. dodonaei often appears as a subthermophilous demontane species descending along the gravel alluvia of water courses, as well within plant communities developed in the warm and open habitats of gravel banks of the European (usually Alpine) streams (Valachoviˇc et al., 1997). Slavik (1986) notes the presence of this species in the both Czech Republic and Slovakia in river basins growing on diverse geological substrates: limestones, shales, calcareous sandstones and phyllite, diabase, and granite. Besides the primary occurrence of E. dodonaei in gravel bed habitats, apophytization has been taking place over the past hundred years in those industrial areas whose properties are closely related to the original habitats (Slavik, 1986; Smejkal, 1997). Thus, E. dodonaei has often been found on anthropogenic formations such as quarries, sandpits, gravel-pits, and mine waste dumps in various parts of ´ 2006; Brofas et al., 2007; HimmEurope (Koutecká and Koutecky, – c´ et al., 2014). The dispersal to suitable artificial ler, 2008; Randelovi habitats and extinction from the original habitats (due to natural loss or anthropogenic disturbance) changed the boundaries of species distribution. In some parts of Europe E. dodonaei has the status of endangered and protected species, primarily due to losses of natural habitats. This species is included in the Red List of the vascular plants of Luxembourg (Colling, 2005), Germany − Bavaria province (Scheuerer and Ahlmer, 2003); Poland (Mirek and PiekosMirkowa, 1992), Slovakia (Májeková et al., 2014), Czech Republic (Procházka, 2001), Romanian (Daraban, 2007), Hungary (Official Gazette, 128) and Red List of flora of the Serbian Republic within Bosnia and Herzegovina (Official Gazette, 124/12). Additionally, E. ´ 1973). dodonaei is very rare on natural habitats in Serbia (Diklic, Slavik (1986) recommends Epilobium dodonaei as an ideal model species for the study of the recent migration in time and space. Therefore, this species can be a good example for linking active conservation procedures with ecological restoration. The locations of E. dodonaei in areas with greater concentrations of metals have not been well mapped. The species has been investigated in the serpentine soils of Gjegjan in Albania, with a limited accumulation of nickel, zinc, cobalt and cromium in biomass (Shallari et al., 1998). Brofas et al. (2007) have been establish germination protocol for establishment of E. dodonaei in rehabilitation of calcareous mine spoils at bauxite mining region in Greece.

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We hypothesize that E. dodonaei has capability to grow at metalliferous mine wastes and it also has the potential for phytoremediation. The aims of this study were: 1) identification of the physico-chemical characteristics of mine waste near Bor; 2) determination of the phytoremediation potential of E. dodonaei growing at mine tailings; 3) assessment of the metal and metalloid induced oxidative stress and antioxidative response of E. dodonaei. This comprehensive study investigated metabolic stress response of pioneer plant species that colonizes mine wastes and needs to be conserved. This approach should create practical procedures for plant selection that link ecological restoration with conservation biology. 2. Materials and methods 2.1. Site description The study area is located in the Bor region (44◦ 04 25 North, 22◦ 05 26 East, East Serbia, South Eastern Europe). The area has a temperate continental climate and the surrounding landscape is covered by the Hungarian and Turkish oak forest communities (Quercetum frainetto-cerris Rud.). Bor is one of the largest active copper mine basins in Europe where century-long mining has caused severe degradation of environment, making the region one of the worst environmentaly degraded locations in Serbia and Europe (Dimitrijevic´ et al., 2009). The ore exploitation created large open pits, flotation tailings, and waste dumps. The contamination of air, soil, and water in the Bor area is mainly a consequence of copper mining, processing, and smelting that resulted in excessive polˇ lution with arsenic, copper, lead and zinc (Sajn et al., 2014). The copper in the soils of the Bor area greatly exceeds national limits and is the highest in Serbia (SEPA, 2007). The region of Bor is also one of the most arsenic polluted regions in Serbia and Europe ˇ ´ 2007; Serbula (Dangic´ and Dangic, et al., 2014), where the concentration of arsenic in the air exceeds the limits established by the European Union (EC, 2004) and Serbian legislation (Official Gazette, 19/06).

This study selected sampling sites on the Bor copper mine waste, labelled MWS (in Fig. 1). These sites are located within the industrial area of copper mining and smelting facilities, in the suburban area of the town Bor (Fig. 2A). Mine waste dumps are covering an area of approximately 150 ha and consist of volcanic rocks, where andesite and andesitic agglomerates and breccias prevail (Jankovic´ et al., 2002). The mine waste area was created by non-selective deposition of extracted material, which resulted in high heterogeneity of the surface rock material and, consequently, the creation of diverse gravel microsites (Fig. 2 B-D). Reclamation of this area resulted in the creation of steep slopes that were subjected to erosion and mostly unvegetated (Fig. 2B). The mine waste site was reclaimed ´ 1997). At in the 1980s with limited and variable success (Milijic, the spontaneously colonized mine waste sites, E. dodonaei dominates in mine slopes (Fig. 3D–F) and mine waste surfaces (Fig. 3G–I). – Randelovi c´ et al. (2014) showed that mine spoil texture was the governing edaphic factor for the development of diverse vegetation stages in reclaimed and non-reclaimed areas of the Bor mine wastes. The reference site (RS) with E. dodonaei was placed 15 km away from the mine waste site, in the vicinity of the limestone quarry pit (Figs. 1, 2E–G, 3A–C).

2.2. Soil and plant analyses Sampling was conducted at the Bor mine waste site and reference site in July 2014. The total of 23 composite soil and spoil samples were taken in the mine waste and reference soil from the upper 20 cm layer. Collected mine spoil (18 composites) and soil (5 composites) samples were packed into plastic bags and brought to the laboratory for the physic-chemical and elemental analysis. The corresponding number of composite plant samples was taken for the biochemical and elemental analysis. The samples for biochemical analysis were packed into plastic bags and put in refrigerated boxes and brought to the laboratory where were kept in a deep freezer (−80 ◦ C) for further analysis.

Fig. 1. Map of the investigated area showing the location of sampling sites (MWS – mine waste site, RS – reference site).

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Fig. 2. Copper mining and smelting facilities at the Bor mining area (A); Bor mine waste site, MWS (B–D); Reference site, RS (E–G).

The air-dried mine spoil and soil samples were sieved through a 2 mm plastic sieve. Soil texture classes (sand, silt, and clay) were examined using the international pipette-B method with sedimentation in water. The soil texture classifications are defined according to McDonald et al. (1998). Soil pH was measured in 1–2.5 ratio of soil to distilled water (pHH2O ) and 1 mol L−1 KCl solution (pHKCl ) using a pH meter (Iskra MA 5730) according to ISO (1994) 10390. Soil redox potential (Eh) was measured using the Iskra MA 5730 pH meter with a platinum electrode. The saturated calomel electrode (0.244 V at 25 ◦ C) was used as a reference electrode. Electrical conductivity (EC) was measured in 1–2.5 ratio of soil to distilled water solution using an electric conductometer CyberScan con 110. Total nitrogen content was determined by the Kjeldahl digestion (Benton and Jones, 2001), and available forms of phosphorus and potassium were analyzed using the standard AL-method (Egner et al., 1960). Organic carbon was measured by the method of Tjurin (1965). Sum of the base cations content was determined according to Kappen (1929). Exchangeable Ca and Mg were determined in 1 mol L−1 ammonium acetate extract (Thomas, 1982). The pseudo-total chemical element concentrations (maximum of potentially soluble or mobile content of metals, usually not bound in silicates, Rao et al., 2008) of arsenic, copper, lead, zinc, and sulfur in soils were determined by aqua regia digestion according to the USEPA method 3050 (Edgell, 1988). The available fractions of

elements were extracted in 0.05 mol L−1 EDTA solution according to McGrath (1996). This study divided collected composite plant samples into shoots and roots, then washed the samples with tap and distilled water, dried and digested with HNO3 and H2 O2 in a ratio of three to two. The measurements of elements in mine spoil, soils and plant material were determined using an inductively coupled atomic emission spectrometer, ICP-OES (Thermo Scientific, United Kingdom, model 6500 Duo). In order to check the accuracy and precision of instruments, the standard reference material ERM-CD281 (rye grass) JRC-IRMM from Belgium was tested. All measurements were done in triplicate.

2.3. Biochemical analyses Total chlorophyll and carotenoids were extracted with dimethyl sulfoxide (DMSO) according to Hiscox and Israelstam (1979). After centrifugation at 3000 x g for 10 min, Chl a, Chl b and carotenoids were determined by UV–vis spectrophotometry (Shimadzu UV160) at 663 nm, 645 nm, and 480 nm, respectively. Chlorophylls and carotenoids content was calculated using equations according to Arnon (1949) and Wellburn (1994), respectively. The amount of chlorophylls and carotenoids was expressed as mg g−1 of the dry weight. The anthocyanins in leaves were determined according to Creasy (1968) and Proctor (1974). The pieces of leaves were placed

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Fig. 3. Epilobium dodonaei at the reference site (A–C) and the Bor metalliferous mine site: mine waste slopes (D–F); mine waste surfaces (G–I).

in 1 ml DMSO and heated for 2 h at 65 ◦ C, then 0.5 ml of 2 mol L−1 HCl was added, and heated for 4 h at 65 ◦ C. The absorbance of anthocyanins was measured at 650 nm, 620 nm, and 520 nm spectrophotometrically. The amount of anthocyanins is expessed as mg g−1 of dry weight. The malondialdehyde in the leaves and roots was measured according to Heath and Packer (1968). Fresh leaf samples (0.5 g) were homogenized in 5 ml 80% ethanol, containing 0.05 ml of 2% butulated hydroxytoluene in order to prevent oxidation. The homogenate was centrifuged for 10 min at the speed of 3000 x g at 4 ◦ C. A solution of 1 ml of the supernatant, 0.5 ml of 0.65% thiobarbituric acid and 0.5 ml of 10% trichloacetic acid was heated for 15 min at 95 ◦ C, and then cooled on ice, and centrifuged for 10 min at 3000 x g. The absorbance of malondialdehyde was measured spectrophotometrically at 450 nm, 532 nm and 600 nm. The amount of malondialdehyde is expressed as nmol g−1 of fresh weight. Total phenolics (free and bound phenolics) were determined according to Djurdjevic´ et al. (2007). Highly soluble free phenolics were extracted from leaves and roots with 80% (v/v) boiling methanol solution (3 × 30 ml, 4 h) followed by ethyl acetate (3 × 30 ml, 4 h) in glass flasks of 100 ml volume at 90 ◦ C. The extraction was done in Soxlet apparatus under refluxing condition. After filtration, the pooled methanol and ethyl acetate extracts rotary were evaporated with an evaporator under N2 , the residue was dissolved in 10 ml of distilled water adjusted to pH 2.0 with 2 mol L−1 HCl and the phenolics were transfered to ethylacetate (3 × 30 ml). The ethylacetate phase was dehydrated with anhydrous Na2 SO4

and evaporated to dryness in a stream of nitrogen, and the residue was dissolved in 4 ml of 80% (v/v) methanol solution. Bound phenolics which are either ester, or bound to the polysaccharide matrices of the cell wall, or polymerized into lignin were prepared by boiling the insoluble residue that remained after first procedure, in 10 ml of 2 mol L−1 HCl for 60 min and transfer to ethylacetate (3 × 30 ml). An aliquot of the sample solution was taken and added 7 ml distilled water, 0.1 ml Folin-Ciocalteu reagent, and 0.2 ml of 20% Na2 CO3 . After boiling (at 90 ◦ C, 5 min) the samples were cooled at room temperature and diluted with distilled water to the volume of 10 ml. The absorbance of phenolics was measured spectrophotometrically at 660 nm according to Feldman and Hanks (1968). A standard curve was constructed with different concentrations of ferulic acid (99%, Serva, Heilderberg, Germany). The amount of phenolics is expressed as mg g−1 of dry weight of ferulic acid. The antioxidant capacity in plant leaves and roots was determined using the free radical (1,1-diphenyl-2-picrylhydrazyl, DPPH) according to Brand-Williams et al. (1995). Fresh samples of leaves and roots (0.5 g) were homogenized in 10 ml of 95% ethanol. Each sample was done in three concentrations (5, 25 and 50 ␮l) with the addition of 0.5 ml of DPPH. The solution was kept for 30 min in an oven at 27 ◦ C, and the absorbance was read at 517 nm spectrophotometrically. Low absorbance of the reaction solution indicates a high antioxidant activity, as can be seen from the color changes of the solution from purple to yellow. The total antioxidant capacity of each extract was calculated using the formula: TAC (%) = [(Ao −

– D. Randelovi´ c et al. / Ecological Engineering 95 (2016) 800–810 Table 1 Median and range values of physico-chemical mine spoil and soil parameters for the Bor mine waste site and the reference site. pHH2O – active acidity, pHKCl – potential acidity, H – hydrolitical acidity, Eh – oxido-reduction potential, EC – electrical conductivity, SB – sum of the base cations, C – content of organic carbon, K2 O – plantavailable potassium, N – nitrogen, P2 O5 –plant-available phosphorous. Statistically significant differences between sites are marked with *. Characteristic

Sand (%) Silt (%) Clay (%) pHH2O pHKCl H (cmol/kg) Eh (mV) EC (␮S) SB (meq/100 gr) C (%) Ca (mg/kg) K2 O (mg/100 g) Mg (mg/kg) Total N (%) P2 O5 (mg/100 g)

Mine Waste Site

Reference Site

Median Range

Median Range

97.8 0.96 1.27 6.85 6.00 1.33 266 135 46.8 1.55 4810 21.2 288 0.04 7.99

98.7 1.02 <0.01 7.78 6.82 0.35 201 66.3 98.4 1.97 20620 8.18 236 0.01 8.55

96.1–98.6 0.54–1.99 0.76–2.13 4.58–8.30 3.86–6.94 0.35–6.48 212–358 79.9–752 9.40–99.6 0.01–4.44 1900–27430 7.67–37 107–695 0.01–0.08 0.05–24

98.5–99.0 0.34–1.43 <0.01–0.03 7.58–8.00 6.80–6.87 0.35–0.35 191–213 66.0–68.6 98.3–98.6 1.22–2.11 20480–21220 5.80–8.67 223–248 0.01–0.02 7.99–9.30

Probability

0.009* 0.763 0.007* 0.044* 0.016* 0.016* 0.132 0.007* 0.016* 0.615 0.016* 0.021* 0.688 0.087 0.688

A1 )/Ao] x 100, where Ao is the apsorbance of the control and A1 is the absorbance of the antioxidant in the sample solution. 2.4. Data analyses This study conducted all data analyses using the Statistica StatSoft 8.0 software. Normality of data was tested using the Kolmogorov-Smirnov test. Group mean ranks were compared using the non-parametric Mann-Whitney U test with a significance level of p < 0.05. The Spearman correlation coefficient () was calculated. The bioconcentration factor (BCF) is calculated as a ratio of concentrations of elements in the root and in the soil, counted for both the pseudo-total content of elements in the soil, and the EDTAavailable content of elements in the soil (Yoon et al., 2006; Gilfedder and Lottermoser, 2008). The transfer factor (TF) is calculated as a ratio between concentrations of elements in the shoot versus in the root according to Yoon et al. (2006). Median element concentrations served to estimate these factors. Soil enrichment factors (Ef) were calculated as the ratio of median of element concentrations obtained in this study and median of element concentrations in European soils, as given by Salminen et al. (2005). 3. Results and discussion 3.1. Soil properties The physico-chemical parameters for the mine waste spoil and the reference site soil are presented in Table 1. Both the MWS and the RS are enriched with sand and limited with silt and clay, exhibiting a sand texture. The overburden pH at the MWS was significantly less than at the RS, varying from acid to alkaline as a consequence of the diverse mineralogical and geochemical characteristics of overburden rock. Electrical conductivity at the MWS was significantly less than at RS, indicating greater soluble salt concentrations. Oxide-reduction potential of mine spoils showed that oxidation prevails at the overburden surface. Sum of the base cations at the MWS was significantly less than at the RS, referring to generally less saturation with the base cations, possibly as a consequence of the leaching during the formation of acid mine drainage (Lottermoser, 2010). This acid mine drainage represents an important environmental issue associated with acid generation of copper mine waste rocks that contain sulfides upon exposure to air and water. The mine waste spoil and soil supporting E. dodonaei

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Table 2 Median and range of pseudo-total and EDTA-available element contents for the Bor mine waste site and the reference site. Statistically significant differences between sites are marked with *. Variable

Mine Waste Site

Reference Site

Median

Range

Median

Range

As (mg kg−1 ) Cu (mg kg−1 ) Pb (mg kg−1 ) S (mg kg−1 ) Zn (mg kg−1 )

101 856 107 2580 139

44.5–271 311–2820 62.9–201 1170–19560 76.7–278

4.21 47.0 0.22 136 30.9

3.01–7.11 40.3–55.9 0.17–0.82 85.0–136 25.6–33.7

0.007* 0.007* 0.007* 0.007* 0.007*

AsEDTA (mg kg−1 ) CuEDTA (mg kg−1 ) PbEDTA (mg kg−1 ) ZnEDTA (mg kg−1 )

8 452 23.8 34.5

0.10–47.8 121–1320 7.38–70 15.1–141

0.07 7.72 n.d. 0.50

0.06–0.07 5.41–9.12 n.d. 0.42–2.09

0.007* 0.007* 0.007* 0.007*

Probability

were deficient in nitrogen and organic carbon at the MWS and the RS, which resulted from limited naturally growing vegetation that reduced decomposition of litter. These mine spoils and soils were poor in plant-available phosphorous, while plant-available potassium was more available. Available phosphorous and potassium at the MWS and the RS originated from geological bedrock weathering and from the decomposition and mineralization of the present organic material. Table 2 presents the pseudo-total and EDTA-available fraction of arsenic, copper, lead, sulfur and zinc in mine spoils and soils at the MWS and the RS. Non-parametric Mann-Whitney U tests showed significant differences between the median of pseudo-total and EDTA-available content of all investigated elements between sites. The pseudo-total concentrations of arsenic, copper, lead, sulfur and zinc at the MWS were 24, 18, 486, 19, and 45 times greater, respectively, compared to the RS. In addition, the available concentrations of arsenic, copper and zinc at the MWS were 114, 58, and 69 times higher, respectively than at the RS. Enrichment factors calculated for the MWS spoils compared to the background for European soils were as follows: EfCu = 65.9, EfAs = 16.3, EfPb = 7.11, EfZn = 2.89. Pseudo-total arsenic and copper at the MWS were greater than the limiting threshold (As: 29 mg kg−1 ; Cu: 36 mg kg−1 ) and remediation values (As: 55 mg kg−1 ; Cu: 190 mg kg−1 ) proposed by Serbian national legislations (Official Gazette, 88/2010) and Kabata-Pendias (2011) (As: 4.7 mg kg−1 ; Cu: 14 mg kg−1 ), pointing out the strong metal pollution of the MWS. However, regulatory limits are based on pseudo-total metal content, while in reality only a certain fraction of these concentrations are readily mobile and available for uptake by plants and other biota and sorption to detritus. Therefore, an assessment of the available fractions is of the upmost importance for the estimation of phytoremediation potential and tolerance of the early colonizer plants. The relative availability and the mobility of the trace elements followed the order: Cu (52.8%) > Zn (24.9%) > Pb (22.3%) > As (7.9%) for the MWS and Cu (10.7%) > As (1.7%) > Zn (1.62%) > Pb (undetectable) for the RS. The greater degree of element mobility at the MWS was in accordance with findings which emphasize the increased mobility for the trace elements of anthropogenic origin to that of the geogenic origin (Chlopecka et al., 1996; Kabala and Singh, 2006; Kabata-Pendias, 2011). The significantly negative correlation between electrical conductivity and pH of mine spoil solutions ( = −0.57) pointed to the dissolution of sulfate salts that resulted from weathering of metal-sulfide minerals at the surface of the MWS (supported by a large correlation coefficient between the pseudo-total sulfur content and EC of  = 0.7), and was associate with an increase of soil acidity. A large correlation coefficient between available calcium and sum of the base cations ( = 0.88) suggests that calcium cations prevail within the sum of the base cations in the mine

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Table 3 Median and range of roots and shoots element contents for the Bor mine waste site and the reference site. Statistically significant differences between sites are marked with *. Variable

Mine Waste Site

Reference Site

Median Range

Median Range

Arsenicroot (mg kg−1 ) Copperroot (mg kg−1 ) Leadroot (mg kg−1 ) Sulfurroot (mg kg−1 ) Zincroot (mg kg−1 )

3.98 140 3.19 1688 72.2

0.10–6.99 76.4–333 0.30–19.9 1240–3570 26.9–124

0.03 6.40 4.01 872 35.11

0.02–0.05 5.37–8.66 0.85–4.55 543–971 24.0–38.9

0.007* 0.007* 0.615 0.007* 0.021*

Arsenicshoot (mg kg−1 ) Coppershoot (mg kg−1 ) Leadshoot (mg kg−1 ) Sulfurshoot (mg kg−1 ) Zincshoot (mg kg−1 )

4.69 57.7 1.17 3165 59.3

2.11–10.3 31.1–119 0.30–7.66 1450–6280 23.7–88.1

0.62 31.0 2.12 1929 33.7

0.34–0.7 21.9–34.6 1.68–2.12 1270–2200 32.1–37.6

0.007* 0.012* 0.421 0.016* 0.044*

Probability

spoil solution. The smaller pH of the mine spoil solution favors the precipitation of phosphorous complexes with aluminum and iron (Schlesinger, 1997; Whalen and Sampedro, 2010), decreasing phosphorous availability to plants, that is reflected through the correlation between available P2 O5 and pHH2O ( = 0.52) at the MWS. However, large correlations between soil available P2 O5 and available arsenic ( = 0.73) also occurred at the MWS. Soil oxidation favors arsenate speciation compared to arsenite (Masscheleyn et al., 1991). Given the similar speciation of arsenate and phosphate, the increased dissolved phosphate in the mine spoil and soil is associated with increased mobility and accessibility of arsenate (Signes-Pastor et al., 2007; Bolan et al., 2013). A well-known relation between reduced mobility of arsenic and soil acidity (Fritz and Wenzel, 2002) is reflected within the rizosphere of E. dodonaei trough a significant correlation of available arsenic contents with mine spoil pH ( = 0.7). Mine spoils exhibit significant correlations between pseudo-total content of arsenic and copper ( = 0.69), lead and copper ( = 0.76), and copper and zinc ( = 0.69). These elements are associated with main ore mineral paragenesis of the Bor copper mining area (Jankovic´ et al., 2002). The correlations between the pseudo-total and the EDTA-available concentrations of trace elements in mine spoils were significant in the case of copper ( = 0.84) and zinc ( = 0.85). 3.2. Concentrations of elements in plants Table 3 presents medians and ranges of element concentrations in plant roots and shoots collected from the mine waste and the reference site. Non-parametric Mann-Whitney U tests showed significant differences between the median of accumulated elements in E. dodonaei at the MWS and the RS, with the exception of lead in the roots and the shoots. The content of arsenic, copper, sulfur and zinc in roots of E. dodonaei at the MWS were 133, 22, 1.9, and 2 times greater, respectively, compared to the RS. In addition, the content of the same elements in shoots were 7.6, 1.9, 1.6, and 1.7 times greater, respectively at the MWS than at the RS. The magnitude elements analyzed in plant roots were copper > zinc > arsenic > lead, whereas the concentrations in shoots were zinc > copper > arsenic > lead. According to Kabata-Pendias (2011) the concentrations of arsenic (5–10 mg kg−1 ) and copper (20–100 mg kg−1 ) in plant shoots were toxic. The correlation coefficients between pseudo-total and EDTAavailable arsenic, copper, lead and zinc in mine spoils and the content of the same elements in E. dodonaei roots are:  = 0.49 and  = 0.52;  = 0.59 and  = 0.56;  = 0.55 and  = 0.58;  = 0.58 and  = 0.68, respectively. In this study, the contents of a single element in E. dodonaei roots and shoots at MWS also correlated positively: Cu ( = 0.60), Pb ( = 0.50) and Zn ( = 0.62). Additionally, copper and lead in roots of E. dodonaei at the MWS were significantly corre-

Fig. 4. Bioconcentration factors for pseudo-total (BCF) and EDTA-available fractions (BCFEDTA ) of elements at the mine waste site.

Fig. 5. Transfer factors (TF) of investigated elements at mine waste site.

lated ( = 0.72). Furthermore, arsenic and copper, arsenic and lead, copper and lead, and copper and zinc in shoots of E. dodonaei at the MWS were also highly correlated ( = 0.84,  = 0.94,  = 0.87,  = 0.60, respectively). Correlations observed in this study indicated that arsenic, copper, lead and zinc have similar origin and distribution within the MWS, which is primarily caused by anthropogenic ˇ et al., air deposition resulting from the ore smelting process (Sajn ˇ 2014; Serbula et al., 2014). 3.3. Bioconcentration factor and transfer factor The bioconcentration factor (BCF) for the pseudo-total and EDTA-available concentrations of elements at the MWS presents in Fig. 4. The estimated BCF for arsenic, copper, lead and zinc were less than 1, with the largest for zinc (0.51) and copper (0.15). Bioconcentration factors estimated for EDTA-available concentrations of elements (BCFEDTA ) were larger than 1 only in the case of zinc (2.1). Transfer factors (TF) for arsenic was 1.44, whereas for the other elements the TF was less than 1 (Fig. 5). The roots of E. dodonaei at MWS retained more copper, lead and zinc than the shoots, indicating the potential of this species in phytostabilization of mine wastes. 3.4. Biochemical plant response to stress Table 4 presents the chlorophylls, carotenoids and anthocyanins in the leaves of E. dodonaei at the MWS and the RS. The concentrations of Chl a, Chl a + b and total carotenoids were significantly lower in the leaves of E. dodonaei growing at the MWS than at the RS. There was no significant differences in Chl b and anthocyanins between sites. Positive correlations between Chl a, Chl a + b, and total carotenoids in the leaves of E. dodonaei and pHH2O at the MWS (␳ = 0.57, ␳ = 0.58, and ␳ = 0.53, respectively) are significant. However, the negative correlations between Chl a, Chl a + b and total carotenoids, and electrical conductivity were noted (␳ = −0.63, ␳ = −0.62, and ␳ = −0.57, respectively). The reduced Chl a, Chl a + b, and total carotenoids at the MWS in relation to the RS (52%, 47% and

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807

Table 4 Median and range of biochemical parameters for leaves and roots of E. dodonaei at the mine waste site and the reference site. Statistically significant differences between sites are marked with *. Parameters

Mine Waste Site

Reference Site

Probability

Median

Range

Median

Range

Leaves Chl a (mg g−1 ) Chl b (mg g−1 ) Chl a + b (mg g−1 ) Total Carotenods (mg g−1 ) Anthocyanins (mg g−1 ) Malondialdehyde (nmol g−1 ) Free phenolics (mg g−1 ) Bound phenolics (mg g−1 ) Total phenolics (mg g−1 ) Total antioxidant capacity (%)

1.03 0.75 1.77 0.53 0.69 1.75 91.8 67.7 161 93.4

0.77–2.31 0.47–1.66 1.25–3.97 0.18–1.00 0.52–1.65 1.19–2.51 63.5–144 49.5–109 134–225 86.2–94.8

2.14 1.19 3.33 0.94 0.79 0.78 67.6 39.9 110 51.3

1.57–2.32 0.78–1.50 2.35–3.82 0.70–1.08 0.68–1.01 0.67–0.87 62.0–78.6 39.4–48.0 102–127 47.2–66.3

0.016* 0.108 0.027* 0.021* 0.269 0.007* 0.027* 0.007* 0.007* 0.007*

Roots Malondialdehyde (nmol g−1 ) Free phenolics (mg g−1 ) Bound phenolics (mg g−1 ) Total phenolics (mg g−1 ) Total antioxidant capacity (%)

1.42 15.1 18.4 36.9 57.4

0.71–1.71 4.19–33.2 8.9–57 13.1–82.5 15.7–87.8

0.49 12.82 54.4 65.0 85.1

0.18–0.64 10.6–30.0 34.8–59.8 47.6–89.8 83.7–86.1

0.007* 1.000 0.021* 0.070* 0.035*

44%, respectively) could be the result of low pH and high electrical conductivity caused by the dissolution of sulfate and increased soil acidification, which consequently lead to higher mobility and uptake of toxic elements. Table 4 presents the malondialdehyde in the leaves and roots of E. dodonaei at the MWS and the RS. In this study, the greater content of malondialdehyde in the leaves and roots of E. dodonaei at the MWS compared to the RS (124%, 190%, respectively) indicated great oxidative stress. These could be a result of high electrical conductivity (␳ = 0.60), greater content of pseudo-total arsenic (␳ = 0.66), pseudo-total and available copper (␳ = 0.70, ␳ = 0.50, respectively) and zinc (␳ = 0.77, ␳ = 0.56, respectively) in mine spoils as well as elevated arsenic in shoots (␳ = 0.56) and roots (␳ = 0.57), and copper (␳ = 0.56) and zinc (␳ = 0.50) in roots. These results were in accordance with the results of other authors who also found increased malondialdehyde production with increased arsenic (Talukdar, 2013) and copper content (Singh et al., 2014) as well as high production of malondialdehyde in plants subjected to increased salinity (Fatma et al., 2014) and sulfur stress (Chandra and Pandey, 2014). However, the malondialdehyde in roots of E. dodonaei and electrical conductivity, and sulfur in mine spoils were significantly correlated (␳ = 0.58, ␳ = 0.51, respectively). Fatma et al. (2014) have shown that excess sulfur mediated alleviation of salt stress that is achieved through the synthesis of reduced sulfur compounds (glutathione, phytochelatine, and metalothionine) and increased activity of enzymatic and non-enzimatic antioxidants (Chandra and Pandey, 2014). Table 4 presents the phenolics content in the leaves and roots of E. dodonaei at the MWS and the RS. Significant correlations were found between free phenolics in leaves and arsenic (␳ = 0.48), and zinc (␳ = 0.54) in shoots, bound phenolics in leaves and arsenic in shoots (␳ = 0.52) and roots (␳ = 0.56), total phenolics in leaves and arsenic in shoots (␳ = 0.65) and roots (␳ = 0.51), and copper in shoots (␳ = 0.48). The greater content of free (35.9%), bound (69.7%) and total phenolics (48.4%) in the leaves of E. dodonaei at the MWS in comparison to the RS indicating that arsenic, copper and zinc in shoots can promote phenolics biosynthesis. The results from this study supported the results of other authors who found that excess arsenic and zinc induced large accumulation of flavonoids in plant leaves (Chandronitha et al., 2010; Santos et al., 2011). Relationship between free phenolics and sulfur in the shoots of E. dodonaei at the MWS (␳ = 0.46) could suggest that the large accumulation of sulfur

is potentially involved in the transport anthocyanins in vacuoles through glutathione S-transferase (Martinoia et al., 2007). Furthermore, increased content of soluble and cell wall-bound phenolics in the leaves of E. dodonaei at the MWS could be related to the large content of copper and induction of shikimate dehydrogenase biosynthesis which leads to the accumulation of flavonoids and lignin (Diaz et al., 2001). Significant correlation between malondialdehyde and bound phenolics was noted (␳ = 0.63). Mene-Saffrane et al. (2007) have demonstrated that trienolic fatty acid in leaves can act as the buffer of reactive oxygen species cellular pool and can be a signal for the gene expression of antioxidants during the oxidative stress (Weber et al., 2004). The results of this study indicated that bound phenolics in roots are reduced with decreased pH (␳ = 0.50) and increased electrical conductivity (␳ = −0.71) in mine spoil and greater sulfur in roots (␳ = −0.45). The levels of free phenolics, bound phenolics and total phenolics in roots were lower than in leaves (6.0, 3.7, and 4.4 times, respectively) indicated great root sensitivity to stress. Table 4 presents total antioxidant capacity in the leaves of E. dodonaei at the MWS and the RS. Correlations between the total antioxidant capacity in leaves and pseudo-total and available arsenic (␳ = 0.47 and ␳ = 0.50), copper (␳ = 0.49 and ␳ = 0.62), and zinc (␳ = 0.50 and ␳ = 0.57), and between the total antioxidant capacity and arsenic and copper in shoots (␳ = 0.45 and ␳ = 0.47) and roots (␳ = 0.54 and ␳ = 0.47), and zinc in roots (␳ = 0.51) were significant. Results show that total antioxidant activity in the leaves of E. dodonaei was significantly greater at the MWS than at the RS (82%) indicating increased biosynthesis and activity of the antioxidants as a response to metal and metalloid stress on the mine spoil. Increased antioxidant content (glutathione, proline, non-protein thiols, superoxide dismutase, peroxidase, and catalases) is a typical response to high levels of metals in plant species (NadgórskaSocha et al., 2013). Results of this study show a positive correlation between total antioxidant activity and total phenolics in leaves (␳ = 0.56) indicating that phenolics are important components of antioxidative system. However, Dutta and Maharia (2012) recorded low total antioxidant activity in the leaves of plants growing in copper mining areas. Significant correlations between total antioxidant capacity and pH were found (␳ = 0.74) indicating that low pH of the spoil of mine waste leads to a reduction in the total antioxidant activity. The lower total antioxidant activities in the roots of E. dodonaei at the MWS compared to the RS (1.5 times)

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can be attributed to the inhibition of the enzymes involved in antioxidant biosynthesis by toxic metal or the depletion of some antioxidants (glutathione) which are important in the biosynthesis of others (phytochelatines) (Madhava Rao and Sresty, 2000). The total antioxidant capacity in roots and sulfur in roots was negatively correlated (␳ = −0.45) suggesting that greater accumulation of sulfur can increased glutathione and other thiol-rich compounds which are crucial for the detoxification of arsenic, copper, lead and zinc (Shaw et al., 2006). Generally, species of genus Epilobium are characterized by large content of polyphenolics (Hevesi Tóth et al., 2008) and high total antioxidant activity (95.57%, leaves of E. ˇ angustifolium, Stajner et al., 2007), which is in accordance with our results. This indicates that E. dodonaei basically has high capacity to promote antioxidants biosynthesis as a response to metal and metalloid exposure.

3.5. Implications for ecological engineering Incorporation of the investigation results into reclamation methods could improve ecological engineering design, in order to achieve successful reclamation of diverse metalliferous mine wastes and similarly degraded sites. Reclamation based on the planting of pioneer plants stabilizes the slopes of excavated material, minimize erosion processes, and allows succession of more diverse plant and animal communities (Kuzovkina and Volk, 2009). Successful colonization of erosion prone steep slopes and growth in diverse rocky sites at the Bor mine area indicates that E. dodonaei is a suitable species for erosion control and reclamation of rocky industrial sites, as well as for the mine wastes. In the Bor mining area copper, arsenic, and coarse soil texture are the main factors affecting the vegetation development. Plant species appropriate for reclamation should be well adapted and ecologically resilient to these conditions. Epilobium dodonaei largely retain metals in roots than in leaves. The restricted metal accumulation in leaves means that detritus and dead decaying E. dodonaei represents a limited risk for poisoning herbivores and succeeding vegetation. Chemical analysis of mine spoil covered with E. dodonaei show the presence of organic material, nitrogen, phosphorous, and potassium as vital macronutrients important for biogeochemical cycling processes in a restored ecosystem. Metals stress response could be used to monitor success of the restoration project providing significant information about capability of plants to grow and survive at metalliferious sites (McCutcheon and Mitsch, 1994; Medina et al., 2003; Santos et al., 2009; Nadgórska-Socha et al., 2013). In addition, metabolic response of E. dodonaei to metal and metalloid stress can be used for selection plant species for ecorestoration of mine wastes. At mine wastes where restoration of the previous natural ecosystem is not always fully sustainable, restoration objectives can be achieved by artificially created analogues habitats of natural systems as an alternative for threatened plant species (Otte and Jacob, 2009; Lundholm and Richardson, 2010). Planting of E. dodonaei on gravel covered steep slopes should be applied over the broad geographical range of this pioneering plant to reclaim mining and other industrial sites. Use of E. dodonaei in reclamation should aid in conservation of this endangered species. The geographical range of E. dodonaei includes western parts of the Alpine-Himalayan orogenic belt associated with the Tethyan-Eurasian Metallogenic Belt and the provinces: Carpatho-Balkan, Serbo-Macedonian, Dinaric´ 1997). Special attention Hellenides and Greater Caucasus (Jankovic, should be given to the creation of artificial alternative habitats for E. dodonaei on metalliferous mine sites within Carpatho-Balkan metallogenic province, since the species is considered to be endangered and included in the Red lists of the countries in the Carpathian mountains region.

4. Conclusion Mine waste sites in Bor region (Serbia) covered by Epilobium dodonaei are characterized by coarse soil texture, greater range of pH values, smaller amounts of organic matter and macronutrients, and elevated concentrations of pseudo-total arsenic and copper. Oxidation and dissolution of metal-sulfide minerals on the surface of the Bor mine waste decrease pH of the spoils that is followed by the increase of metal mobility and electrical conductivity of the spoil solution. Content of elements in E. dodonaei roots correlates with pseudo-total and EDTA-available concentrations of elements in Bor mine spoils. Epilobium dodonaei growing at mine waste sites has higher arsenic and copper content in the shoots. However, this species largely retains copper, lead, and zinc in roots rather than in shoots and has potential for phytostabilization of metalliferous mine sites. Epilobium dodonaei at the mine waste sites has greater content of malondialdehyde in roots and leaves, as well as reduced chlorophyll and carotenoid content in leaves. Despite of great arsenic and copper content in roots and shoots, E. dodonaei has capability to increase biosynthesis of phenolics and antioxidants. Successful colonization of the steep slopes of the Bor mine wastes demonstrates that E. dodonaei can control erosion on gravelcovered industrial sites. Furthermore, the assessments of metabolic response to stress should be used in the selection of plant species for the reclamation designs. Epilobium dodonaei can be appropriate species for reclamation over a wider geographical range of different metallogenic provinces, especially within the Carpatho-Balkan metallogenic province where the species is considered endangered. Finally, when feasible, endangered plant species should be selected to preserve biodiversity.

Acknowledgement This study was supported by the Serbian Ministry of Education, Science, and Technological Development (project numbers 176016, 173030, 173018 and 172030). The suggestions and corrections by two anonymous reviewers were very helpful and are gratefully acknowledged.

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