Bioaccumulation potential of selected plant species of the family Brassicaceae from serpentine habitats in Serbia

Ultramafics ('ultrabasic' or 'serpentine') represent magmatic or metamorphic rocks which are characterized by high concentrations of Mg, Fe, Ni, Cr and Co and low concentrations of Ca, P, and K and contain less than 45% silica (SiO2). Serpentine substrates commonly support vegetation and flora that is sparser and more stunted than that of other nearby soils. Many plant species are incapable of growing on serpentine substrates because they are extremely poor in essential nutrients and have a low Ca/Mg ratio. On the basis of heavy metal uptake, plants which grow on ultramafic soils can be divided into ‘normal’ and metal hyperaccumulators. While most serpentine plants are able to grow on these soils without excessive uptake of elements, hyperaccumulators take up more than 1000 mg kg-1 Ni and accumulate it into their leaf dry matter. To date, about 360 hyperaccumulators of Ni from ultramafic soils have been identified. They are mostly found in Brassicaceae family in temperate areas (especially Mediterranean and Turkey). By far the greatest number of nickelaccumulating species within any genus is found in Alyssum L. Besides the Alyssum species, nickel hyperaccumulation was also discovered in species that belong to other genera of the Brassicaceae family of Europe and Turkey. So far only the data about Ni accumulation in A. murale from the central Serbia was published, but there has been no information about the hyperaccumulative potential of the species from the ultramafic areas of Serbia that belong to some other genera (Isatis, Cardamine, Aethionema, Rorippa, Erysimum) of the Brassicaceae family. Hence, it is necessary to obtain more information about metal accumulation capacities of the plants from the Brassicaceae family growing on serpentine soils to evaluate their potential for phytoremediation including metal extraction (phytoextraction). Therefore, the aims of this thesis were to: 1) determine the basic habitat characteristics of the selected taxa from the Brassicaceae family, including information on the type of vegetation and/or community, altitude, slope, geological and pedological substrate and climate; 2) identify the basic physical (texture) and chemical (pH in H2O, pH in 1N KCl, percent of organic matter, contents of P2O5 and K2O) features of serpentine soil of the study sites; 3) determine the concentrations of the macro (Ca, Mg, Fe) as well as trace elements (Ni, Mn, Zn, Cu, Cr, Pb, Co, Cd) in soils that are developed over serpentinite substrates at several locations in the northwestern, western, southwestern and central Serbia; 4) examine the concentrations of the macro and trace elements in underground and aboveground parts (stems and/or leaves) of plants of the family Brassicaceae that inhabit serpentinite habitats in Serbia; 5) based on the obtained results, the assessment of the capacity of some plant species of the family Brassicaceae for tolerance and/or bioaccumulation of certain trace elements (with special attention to nickel and zinc); 6) identificationn and discovery of new potential hyperaccumulator species in the family Brassicaceae from Serbia. Soil and Brassicaceae plant samples were collected in 2010 and 2011 from 30 sampling points within 9 wider areas of the NW, W, SW and C Serbia. The following 11 plants were analyzed: Erysimum linariifolium Tausch, Erysimum carniolicum Dolliner, Thlaspi kovatsii Heuffel, Thlaspi praecox Wulfen, Alyssum murale Waldst. & Kit., Alyssum montanum L., Alyssum repens Baumg., Cardamine plumieri Vill., Rorippa lippizensis (Wulfen) Reichenb., Aethionema saxatile (L.) R. Br., Isatis tinctoria L. Soil analysis – particle size distribution was determined by sieving and sedimentation after samples were soaked in the dispersant sodium hexametaphosphate in 1:2 soil–water suspensions. The sand is fractionated by dry sieving. The pipette method was used for the determination of clay and silt fractions. Soil actual and exchangeable pH was determined in distilled water and in 1 M KCl solution respectively, in a solid-liquid (S/L) ratio of 1:2,5 ml g-1. Available P and K were measured in AL solution extract (S/L 1:20). Phosphate concentration was determined by molybdenum blue method and potassium concentration was determined using flame emission spectrophotometry atomic absorption spectrophotometer. Exchangeable Ca and Mg were determined in 1 M ammonium acetate extract by AAS. Organic matter concentration was determined by dichromate digestion based on the FAO procedure. Extraction of available metals in soil was performed by 0.1 M HCl (S/L 1:10). Total metal extraction was done by HCl and HNO3 digestion. Metal concentrations in both extracts were determined using atomic absorption spectrophotometry. Plant analysis – dried and ground plant material was digested by slightly modified wet procedure described by ISO 6636/2, using a boiling mixture of nitric and sulphuric acids. Phosphorus concentration was determined by modified the molybdenum blue method. Potassium concentration was determined using flame emission spectrophotometry. Metal concentrations were determined by using atomic absorption spectrophotometry. On the basis of the analyses of physical soil characteristics that were done on 30 different ultramafic sites (within nine locations) in NW, W, SW and C Serbia it can be concluded that textural classes of the studied samples range from silty loam to sandy loam. Silt and clay contents in serpentine soils are generally minimal but in our study almost one third of the soil samples were of silty loam classes. The pH in H2O of the serpentine soil samples varied from moderately acidic to moderately alkaline, while percent of organic matter in soils was low to moderate which indicate a relatively good humidization. Soils collected at Serbian sampling sites contained low level of P2O5, K2O and Ca and high Fe and Mg contents which is also general characteristics of ultramafic soils. The serpentine soils of 30 Serbian sites were characterized by generally elevated total concentrations of trace elements such as Ni, Mn, Co and Cr, while most of the total Cu, Zn, Cd and Pb concentrations in analyzed serpentine soil samples fall within the ranges for normal soils. Concerning the available concentration of major elements, the soils were more or less of typical ultramafic composition, with the moderate to high concentration of available Mg and low to high available Ca content. The Ca/Mg quotients for the available fraction in 23 samples were relatively low; the content of the available Fe was high for the loamy and alkaline soils. Available concentrations of Ni, Zn and Mn were high in soil samples. Available Cr contents were low which confirms low mobility of Cr in surface soil layer and its limited availability to plants. Generally speaking, the content of Ca was higher than Mg content in all plant tissues and the highest Ca/Mg ratio was noticed in A. murale leaves. In the roots and shoots of A. montanum, A. repens, E. linariifolium and only in shoots of R. lippizensis and T. praecox the Ca/Mg ratios > 1 were noticed. In the plant tissues of the rest of investigated Brassicaceae species, the contents of Ca were lower than Mg. Contents of Fe in the most of Brassicaceae plant tissues were elevated in relation to the natural Fe content of fodder plants, and it has been concluded that high Fe content of plants is may probably be applicable to all the serpentine flora of the Balkans. The total quantity of Ni in tissues of all nine A. murale samples studied was high to extremely high and in some cases, the ratio between concentration of Ni in leaves and in the corresponding soil was 67:1. The Ni concentrations in the shoots of Thlaspi species were high to extremely high, while in A. montanum and A. repens Ni contents were high and very unusual for the species that belong to section Alyssum. Actually, this is the first time that such high Ni content was measured within any species that belongs to this section. All the rest of Brassicaceae species studied here had Ni concentrations in their roots and shoots lower than 100 mg kg−1, confirming that no nickel hyperaccumulating but tolerant species were recorded among these plants. Comparing to low Zn content in soil samples, in the roots of all A. murale samples Zn concentrations were the highest. Also, certain Thlaspi species studied here was noteworthy for containing Zn at concentrations 1000 mg kg−1 . Although Mn content in soil samples was relatively high, its concentration in plant tissues was noticeably lower; it is assumed that species growing in serpentine soils generally act as excluders of Mn and can restrict transport of this element to the shoots. In plant tissues of almost all the Brassicaceae plants Cu, Cr and Co were found only in traces. Correlation of Ni accumulating capacity with soil properties lead to a conclusion that weakly acidic soils, poor in P and K, with low Ca/Mg ratio and with low contents of Mn, Cr and Co, are most favourable for the development of A. murale specimens with high hyperaccumulating capacity. If hyperaccumulators are defined as those plants which contain in their tissue more than 1000 mg kg−1 dry weight of Ni or more than 10000 mg kg−1 dry weight of Zn or Mn, only A. murale, T. kovatsii and T. praecox belong to the group of Ni hyperaccumulators. Strong accumulators can accumulate 100–999 mg kg−1 Ni in leaves and based on their accumulation properties, some populations of A. montanum, A. repens and I. tinctoria can be classified as Ni strong accumulators. Accumulators show a tendency or ability to translocate and accumulate high metal levels in above-ground plant parts from both low and high soil metal concentrations without toxicity symptoms; their shoot/root metal concentration quotient is >1.0. If we apply this criterion for the selection of accumulator Brassicaceae species from Serbian serpentine sites, some populations of A. montanum, E. linariifolium, T. kovatsii and T. praecox can be