Comparison of heavy-metal bioaccumulation properties in Pinus sp . and Quercus sp . in selected European Cu deposits

Heavy-metal contamination of Pinus pinaster, P . sylvestris, Quercus robur, and Q. rotundifolium was studied in four abandoned historic Cu deposits from Italy (Libiola, Caporciano), Portugal (São Domingos), and Slovakia (L’ubietová). The highest Cu and Mn contents in anthropogenic soil were described in Libiola and Caporciano whereas the highest Pb, Zn, As, and Sb contents in São Domingos. The anthropogenic soil in L’ubietová shows the highest Co contents. The area of São Domingos is the most acidified. There are important differences between the bioaccumulation of plants from individual deposits. Bioavailability of the heavy metals is generally independent of the pH values. The high Ca and Mg contents in soil are able to block the transport of heavy metals to the plant tissues. The bioconcentration factor values of all plant taxa, in all deposits, indicate a predominant strategy of excluders. Only Ag shows excellent bioconcentration ability. In L’ubietová, Pinus sylvestris has a strategy as an accumulator of Pb (2.43) and Zn (2.49); Pinus pinaster of Mn (4.97), Cd (1.85), and Co (5.62) and Quercus rotundifolium of Mn (3.54) in São Domingos. The predominantly low translocation factor values indicate that in most cases the heavy metals are accumulated in roots; only in a few rare cases do they migrate to shoots (e.g. Zn in Pinus sp. from all localities, Co in P . pinaster in São Domingos).


Introduction
Mine dump fields are the specific habitats, because they have specific ecological conditions, which limit plant growth.Mainly, it is deficiency of soil substrate, nutrients, and water.The soils are rocky, strongly skeletal with deficiency or absence of humus layer, and have increased content of heavy metals in soils naturally rich in their content.The long-term influence of the stress factors caused, in the case of some plants, physiological and genetical tolerance necessary for their survival (Viehweger, 2014).It is a global evolution strategy (Herrera-Estrella et al., 1999;Ramírez-Rodríguez et al., 2007).This adaptation from metallotolerant ecotypes to genotypes is well known from heavy-metal tolerance evolution (MacNair and Baker, 1994), and their direct utilization is applied in phytoremediation.Many authors study the ac-cumulation of metals in plants as the results of this study have practical importance for bioindication, biomonitoring, and phytoremediation (e.g.Remon et al., 2013;Kleckerová and Dočekalová, 2014;Paz-Ferreiro et al., 2014;Stefanowicz et al., 2015).
The transfer and accumulation of the heavy metals into the plant organs is usually substantially influenced by various factors: primarily by heavy-metal content in soil and by soil characteristics (e.g. the mould and clay minerals content), by soil reaction, by cation exchange capacity, or by Ca / Mg content ratio (Kavamura and Esposito, 2010;Fijalkowski et al., 2012;Čurlík et al., 2015).The effect of pH on the metal mobility is a variable but very important factor (Alkorta et al., 2004;Vamerali et al., 2010).Mobility of metals in soils with low pH decreases in the order Cd > Ni > Zn > Mn > Cu > Pb (Vamerali et al., 2010).Highly contaminated sites are usu- ally poor in organic matter content.Organic matter can work as a factor which causes heavy-metal release but also can immobilize heavy metals.This is the reason for the use of recycled organics in agriculture, forestry, and mine rehabilitation (Kelly, 2008).
The potential of oxidation reduction of soil significantly determines participation in the form of a mobile element, which can enter the biological cycle, in relation to the total element content.Lack of oxygen in the soil causes start-up and increases the mobility of the large part of heavy metals (Kavamura and Esposito, 2010).
Also forms of heavy-metal occurrence in soil significantly affect their mobility.The most mobile elements are Cd, Zn, and Mo, while the least mobile are Cr, Ni, and Pb (Prasad and Freitas, 2003).Our study also pointed out the Mn high mobility.
Uptake and accumulation of selected metals is primarily dependent on the plant species, its inherent controls, and the soil quality (Chunilall et al., 2005).
Four abandoned Cu deposits having historical importance in Europe were compared (Fig. 1).First data about the heavymetal contamination of plants in L'ubietová are discussed by Andráš et al. (2007).Information about the heavy-metal contamination of some plants in Libiola and Caporciano is presented by Buccheri et al. (2014) and Dadová et al. (2015), and in area of São Domingos mining district was studied by numerous authors (Freitas et al., 2004;Abreu and Magalhães, 2009;Abreu et al., 2012).
Among the plants identified in the study areas we focused on some autochthon-related species and simultaneously dominant pioneer trees with relatively high coverage that can be compared to each other with respect to their bioconcentration properties.These are the same strategies regarding the colonization of mining heap, with the same role in the process of spontaneous succession.They belong to two genera -Pinus and Quercus.Each is represented by two species: Pinus sylvestris L. and Quercus robur L. in Slovakia, Pinus pinaster Aiton and Quercus rotundifolia Lam. in Italy and Portugal.This paper addresses the following questions: i.Is there a negative correlation between soil acidity and bioavailability of heavy metals in the investigated species?
ii.Is the heavy-metal input to plants influenced by Ca and Mg content or by Ca / Mg ratio in sorption complex?
iii.Are the bioaccumulation characteristics of these dominant pioneer trees similar and usable in remediation?
2 Material and methods

Subjects of scientific studies
L'ubietová (Slovakia) was one of the most important mining areas in central Europe during the Middle Ages.The Cu content in the ore ranged from 4 to 10 %, and the Ag content was about 70 g t −1 (Andráš et al., 2012;Kharbish et al., 2014).In 500 years about 25 000 t of copper was produced.The mining activities were finished at the end of the 19th century (Ilavský et al., 1994).
The stratiform Libiola deposit is situated at the western border of northern Apennines mountains in Gromolo stream valley near Sestri Levante (Liguria, Italy).The Caporciano near Montecatini Val di Cecina (Tuscany, Italy) was the most important European Cu deposit during the 19th century.The exploitation finished in 1907, and the mine was closed (Orlandi, 2006).During the last mining activity 30 000 t of copper was exploited.Both Libiola and Caporciano had been exploited since Etruscan times.
São Domingos (Baixo Alentejo province, Portugal) was exploited even in pre-Roman times.The deposit belongs to the most important massive Cu mineralization of massive pyrite-chalcopyrite metallogenic province in the Iberian Pyrite Belt, where the resources of the ore are estimated at about 1700 Mt (Sáez et al., 1999).The mining ended by 1966 (Matos et al., 2010).

Data sampling and analysis
During the growing season in 2012 (in Slovakia), 2013 (in Portugal), and 2014 (in Italy) 15 plant samples were taken (separately roots, branches, leaves/needles, bore out of the tree trunk at a height of 1 m respectively just below the treetops) and 15 soil samples from their root ball from a depth up to 10 cm from every species and every locality.The soil and plant samples were dried at laboratory temperature.The individual parts of the plants were analysed separately.The average value was calculated for all species at each of the four localities.The values of heavy-metal content in shoots were calculated as the average of values of single above-ground plant parts.Both the soil and plant samples were analysed by ICP-MS in ACME Laboratories in Vancouver, Canada.The rinse and paste pH of soil was measured according to the methodology by VanReeuwijk (1995) and calculated for standard hydrogen electrode.Rinse and paste pH was in soil samples measured according to Sobek et al. (1978).
As two important parameters for assessing input and accumulation of heavy metal by plants are bioconcentration factor (BCF) and translocation factor (TF). BCFs were calculated following Eq.( 1) according to Mehes-Smith et al. ( 2013) and TF according to Singh et al. (2010) following Eq.( 2): BCF = content of heavy metal in shoots / content of heavy metal in corresponding soil (mg kg −1 ), (1) TF = content of heavy metal in shoots / content of heavy metal in corresponding roots (mg kg −1 ). (2) If the BCF < 1, the plant is an excluder; if BCF = 1, the plant is an indicator.If the BCF > 1, the plant is an accumulator to hyperaccumulator (Baker, 1981).
There are important differences between the bioaccumulation of plants from individual deposits.The BCF values of all plant taxa, from the studied deposits, indicate a strategy of excluders, especially in the case of oaks.This means there are no suitable species for phytoextraction.They could be used only in the process of phytostabilization, since heavy metals accumulate preferentially in their roots.A similar effect was confirmed also in Picea abies (L.) H. Karst.and Populus tremula L., which were exposed long-term to metalcontaminated soils (Brunner et al., 2008).Only Ag shows excellent bioconcentration ability (Table 1), especially in Pinus pinaster (BCF = 57.51-73.75)and Quercus rotundifolium (BCF = 43.79;66.59).In L'ubietová, Pinus sylvestris has a strategy of an accumulator of Pb and Zn.While the zinc is translocated into leaves (TF = 2.95), Pb is fixed in roots (TF = 0.18).The translocation of Zn is often restricted due to the ability of this element to create zinc-phytochelatin  The predominantly low TF values indicate also that in most cases the heavy metals are accumulated in the roots, which is known in many other scientific studies (Tamás and Kovács, 2005;Tomaškin et al., 2013;Parzych, 2016).Only in a few rare cases do they migrate to shoots (Andráš et al., 2013), e.g.Mn and Zn in all species (accepted Quercus robur), and Co in Pinus pinaster (TF = 16.38) in São Domingos.

Conclusions
Obtained results show various pictures that are dependent on the object and the type of rock formations.Different plant species and even the same plant species at different localities show very variable results of bioavailability for individual metals.Bioavailability of the heavy metals is at the studied localities generally independent of the pH values.The studied areas are poor in organic matter content.The high Ca and Mg contents in soil are able to block the transport of heavy metals to the plant tissues.Most of the plants are excluders (BCF < 1) for individual elements.Only Ag shows excellent bioconcentration ability.Also BCF data for Pinus sylvestris from L'ubietová reflect moderate bioavailability of Pb and Zn and migration of Zn predominantly to the needles (at all localities).In the rest of the plants, the metals are accumulated in roots, with few exceptions.None of the studied plant species are suitable for phytoextraction.As the localities are strongly contaminated by heavy metals and their area has great extent, the only possible phytoremediation method is phytostabilization.Finding the suitable plant for such a solution will be the aim of the next study.

Figure 1 .
Figure 1.Localization of the studied deposits.

Figure 5 .
Figure 5. Correlation between pH and BCF of Mn, Zn, Cd, and Cu (in mg kg −1 ) in Pinus pinaster from São Domingos.

Table 1 .
Bioconcentration factor and translocation factor calculated for plants from studied localities.