Microsoft Word - Duresova et al 2015_NBeC_FINAL.doc 176 Nova Biotechnologica et Chimica 14-2 (2015) DOI 10.1515/nbec-2015-0025 © University of SS. Cyril and Methodius in Trnava COMPARISON OF Cd AND Zn ACCUMULATION IN TISSUES OF DIFFERENT VASCULAR PLANTS: A RADIOMETRIC STUDY ZUZANA DÜREŠOVÁ1, ANNA ŠUŇOVSKÁ1, MIROSLAV HORNÍK1, MARTIN PIPÍŠKA1, MARCELA GUBIŠOVÁ2, JOZEF GUBIŠ2, JURAJ LESNÝ1 1Department of Ecochemistry and Radioecology, Faculty of Natural Sciences, University of SS. Cyril and Methodius in Trnava, Nám. J. Herdu 2, Trnava, SK-917 01, Slovak Republic (hornik@ucm.sk) 2Plant Production Research Institute, National Agricultural and Food Centre, Bratislavská cesta 122, Piešťany, SK-921 68, Slovak Republic (gubisova@vurv.sk) Abstract: The aim of the present work was to compare the accumulation and translocation of Cd and Zn in plants of tobacco (Nicotiana tabacum L.), celery (Apium graveolens L.), maize (Zea mays L.), giant reed (Arundo donax L.), and alpine pennycress (Noccaea caerulescens L.) under conditions of short-term hydroponic experiments using nutrient solutions spiked with radionuclides 109Cd or 65Zn, and direct gamma- spectrometry. It was found that the time-course of metals accumulation in studied plants was not different in terms of target metal, but it was significantly different on the level of plant species. The highest values of Cd accumulation showed plants of giant reed, whereby the accumulation decreased in the order: giant reed > tobacco > alpine pennycress >> maize and celery. On the basis of concentration ratios (CR) [Me]shoot / [Me]root calculation for both metals, it was found that Cd and Zn were in prevailing part accumulated in the root tissues and only partially accumulated in the shoots, where the amount of accumulated Cd and Zn increased from the oldest developed leaves to the youngest developed leaves. The CR values corresponding to these facts were calculated in the range 0.06 – 0.27 for Cd and for Zn 0.06 – 0.48.In terms of plant species, the CR values obtained for Cd decreased in the order: maize > celery > tobacco and giant reed > alpine pennycress. The similarity between studied objects – individual plant species on the basis of the obtained variables defining Cd or Zn accumulation at different conditions of the experiments as well as the relationships between obtained variables and conditions of the experiments were subjected to multivariate analysis method – cluster analysis (CA). According to the findings and this analysis, it can be expected that plants of tobacco and giant reed will dispose with similar characteristics as plants of alpine pennycress, which are classified as Zn/Cd hyperaccumulators, in terms of Cd or Zn accumulation and other positive parameters for their utilization in phytoremediation processes and techniques. Key words: cadmium, zinc, plants, accumulation, translocation, speciation modelling, cluster analysis 1. Introduction Heavy metals are well-known risk elements for the environment as non-degradable and persistent contaminants, which can be harmful for humans and the environment. The pollution of the environment with heavy metals predominantly comes from anthropogenic sources. Despite the fact that Hg, Pb, and Cd emissions from the non- ferrous metal industry decreased in Europe over the last 50 years, the increasing industrial activities in other continents (Asia, Africa) could significantly affect the global emissions of metals (ETTLER, 2015). The biota may require some of these Bereitgestellt von Slovenská poľnohospodárska knižnica | Heruntergeladen 17.01.20 10:05 UTC Nova Biotechnologica et Chimica 14-2 (2015) 177 elements considered to be essential (e.g. iron, zinc, copper or molybdenum) in trace quantities, but at higher concentrations they may become toxic. However, metals like cadmium, lead or aluminium are non-essential and toxic even at very low levels (IANNONE et al., 2015). Cadmium (Cd) represents one of the most dangerous of the trace elements not only because of its significant occurrence in the environment together with zinc (Zn) as its chemical analogue, but also because of its negative accumulation, impact on living organisms, and being the fourth most toxic element to vascular plants as well. According to the Agency for Toxic Substances and Disease Registry Cd is actually ranked at the 7th place within the priority list of hazardous substances based on their frequency of occurrence in the environment, toxicity and adverse potential to affect human health (ATSDR, 2013). First, second, and third places in the mentioned priority list belong to arsenic (As), lead (Pb), and mercury (Hg), respectively. Cadmium is also considered as a priority pollutant by the European Community, within the Water Framework Directive (EC, 2001) and is released into the environment mainly by power stations, heating systems, metal working industries, waste incinerators, urban traffic, cement factories, and as a by-product of phosphate fertilisers (HAWRYLAK-NOWAK et al., 2014). The free ionic form Cd2+ is the most bioavailable and consequently more toxic for organisms and plants, but it can complex with oxides and organic compounds, and it is not soluble above pH 7.5. The content of Cd in crop plants varies in a wide range from 5 up to 400 μg/kg of dried weight (KABATA-PENDIAS and MUKHERJEE, 2007). Even at low concentrations, Cd is toxic for most plants, while at concentrations greater than 5 – 10 mg/kg of leaf dry weight it leads to plant death (WHITE and BROWN, 2010). Cd is easily taken up by plant roots and transported to aerial parts, thus entering into the food chain causing health problems in animals and humans (BERNARD, 2008). Cd-induced toxicity in plants can be described by: growth reduction, chlorosis and necrosis of leaves, wilting, red- brown colouration of leaf margins or veins (GILL et al., 2013). Moreover, Cd can disturb the water balance, mineral nutrition, photosynthesis, respiration as well as general plant development (WÓJCIK and TUKIENDORF, 2005). However, the biochemical and physiological basis of Cd phytotoxicity is not always clear (HAWRYLAK-NOWAK et al., 2015). Cadmium, like other heavy metals, can accept a pair of electrons from a coordinate covalent bond, react with –S- groups, –OH- groups, amino groups, and carboxylic acid termini, and thus affect sulfhydryl groups and N atoms in proteins causing their inactivation (LAMBERS et al., 2008). Soil-to-plant transfer of Cd is the major exposure pathway for humans with regards to soil contamination (LIANG et al., 2013). Food chain contamination is the main source of Cd entry to human, especially non-smoking general population, and is the main constraint for food safety and agricultural land quality (ATAFAR et al., 2010). Excessive dietary intake of Cd is accumulated in the body throughout life, and can lead to kidney malfunction and other diseases (MCLAUGHLIN et al., 1999). Besides the ability to take up essential elements, plants are able to absorb and accumulate other metals, even those of a toxic character or with unidentified metabolic functions (HAWRYLAK-NOWAK et al., 2014). Toxic trace heavy metals, Bereitgestellt von Slovenská poľnohospodárska knižnica | Heruntergeladen 17.01.20 10:05 UTC 178 Dürešová, Z. et al. such as Cd, have chemical properties similar to essential micronutrients, such as Zn, and are generally transported in plants by the same transporters as those essential micronutrients (TAVAREZ et al., 2015). These mechanisms responsible for toxic metals uptake, translocation and accumulation in plant tissues represent serious risks related to their entering into the food chain. On the other hand, these mechanisms can be utilized for removal of heavy metals and radionuclides from contaminated environment, thus for remediation of contaminated environment by plants. Phytoremediation, the use of plant systems to remove the contaminants from the soils and water has recently attracted a great deal of attention as an alternative means of soil decontamination, since it is a cost-effective, environment-friendly approach, applicable to large areas (BAUDDH et al., 2015). The capability of the plants to uptake contaminants, survive in contaminated soil and the low bioavailability of the contaminants in the soil are some of the limiting factors that influence phytoremediation efficiency (CHIRAKKARA and REDDY, 2015). The plenty of plant species (including agricultural crops, grasses, shrubs, water plants, alpine plants a.o.) were subjected to characterization their potential for phytoremediation techniques. Some of them showed an ability to tolerate and accumulate high concentrations of heavy metals and therefore were defined as hyperaccumulators. In order to fully utilize the contaminated sites and to overcome the disadvantage of phytoremediation, a new strategy of combining phytoremediation with energy crops cultivation, with a view to achieving low cost decontamination of soil through the production of biofuels (BAUDDH et al., 2015; PANDEY et al., 2016). A further current area of interest, at least for those metal ions essential for the human diet, also lies in the prospect of transferring the trait to crop plants as a way of combatting dietary microelements deficiencies (VISIOLI and MARMIROLI, 2013). Our previous works studied the accumulation of Zn, Co, Cd or Cs from nutrient solutions by roots of hydroponically cultivated plants of sunflower (Helianthus annuus L.), tobacco (Nicotiana tabacum L.), celery (Apium graveolens L.), lettuce (Lactuca sativa L.), and giant reed (Arundo donax L.) plants (HORNÍK et al., 2005; HORNÍK et al., 2007; HORNÍK et al., 2008; HORNÍK et al., 2009; GULDANOVÁ et al., 2010; ŠUŇOVSKÁ et al., 2012; DÜREŠOVÁ et al., 2014). The aim of this work was to compare the uptake, translocation, and accumulation of heavy metals Cd and Zn in various plants including tobacco (N. tabacum L.), celery (A. graveolens L.), maize (Zea mays L.), giant reed (Arundo donax L.), and alpine pennycress (Noccaea caerulescens L.) under conditions of short-term hydroponic experiments. A quantitative description of Cd and Zn accumulation was carried out using nutrient solutions spiked with radionuclides 109Cd or 65Zn, and direct gamma-spectrometry. The selection of the chosen plants included a typical model plant (tobacco), root crop (celery), agricultural crop (maize), potential energy crop (giant reed), and alpine plant (alpine pennycress) classified as a hyperaccumulator of heavy metals. In addition, the similarity between individual plant species on the basis of obtained variables defining the studied metals accumulation in plants at different conditions of the experiments, and the relationships between obtained variables and conditions of the experiments, were evaluated applying multivariate analysis method – cluster analysis (CA). Bereitgestellt von Slovenská poľnohospodárska knižnica | Heruntergeladen 17.01.20 10:05 UTC Nova Biotechnologica et Chimica 14-2 (2015) 179 2. Materials and methods 2.1 Plant material Seeds of tobacco (Nicotiana tabacum L.) were obtained from Plant Production Research Institute in Piešťany (Slovak Republic). Seeds of celery (Apium graveolens var. rapaceum Mill.) and maize (Zea mays convar. saccharata Koern.) were used as commercially available seeds from the company SEMO, Inc. (Czech Republic). Seeds of alpine pennycress (Noccaea caerulescens L.) were purchased from B&T World Seeds (France). All mentioned seeds were germinated and grown in pots filled with granulated perlite as an inert carrier and watered with 25 % or 50 % strength nutrient medium according to HOAGLAND (1920). The composition of the full strength Hoagland medium (100 % HM) was (in mg/dm3) MgSO4.7H2O – 370; KNO3 – 404; CaCl2 – 444; NaH2PO4.2H2O – 292; Na2HPO4.12H2O – 46.5; FeSO4.7H2O – 17.9; NaNO3 – 340; NH4Cl – 214; NH4NO3 – 160; H3BO3 – 8.5; Na2MoO4.2H2O – 0.06; MnSO4.5H2O – 5.0; ZnSO4.7H2O – 0.66; CuSO4.5H2O – 0.8. Table 1. Conditions of the experiments evaluating the accumulation of Cd and Zn in plants of tobacco (N. tabacum L.), celery (A. graveolens L.), maize (Z. mays L.), giant reed (A. donax L.), and alpine pennycress (N. caerulescens L.). Plant Metal Conditions of the experiments Cd 25 % HM; pH 6.0; 10 μmol/dm 3 CdCl2; 40.5 kBq/dm 3 109CdCl2; 12h/12h day/night (2 000 lx); 60 % relative humidity; 22±2 °C Tobacco Zn 25 % HM; pH 6.0; 10 μmol/dm 3 ZnCl2; 36.4 kBq/dm 3 65ZnCl2; 12h/12h day/night (2 000 lx); 60 % relative humidity; 22±2 °C Cd 50 % HM; pH 5.5; 10 μmol/dm 3 CdCl2; 45.0 kBq/dm 3 109CdCl2; 12h/12h day/night (2 000 lx); 60 % relative humidity; 22±2 °C Celery Zn 25 % HM; pH 5.5; 10 μmol/dm 3 ZnCl2; 50.0 kBq/dm 3 65ZnCl2; 12h/12h day/night (2 000 lx); 60 % relative humidity; 22±2 °C Cd 50 % HM; pH 5.5; 10 μmol/dm 3 CdCl2; 45.0 kBq/dm 3 109CdCl2; 12h/12h day/night (2 000 lx); 60 % relative humidity; 22±2 °C Maize Zn 50 % HM; pH 5.5; 5.9 μmol/dm 3 ZnCl2; 49.0 kBq/dm 3 65ZnCl2; 12h/12h day/night (2 000 lx); 60 % relative humidity; 22±2 °C Cd 25 % HM; pH 6.0; 10 μmol/dm 3 CdCl2; 95.5 kBq/dm 3 109CdCl2; 12h/12h day/night (4 000 lx); 60 % relative humidity; 25/22 °C Giant reed Zn 25 % HM; pH 6.0; 1 μmol/dm 3 ZnCl2; 74.4 kBq/dm 3 65ZnCl2; 12h/12h day/night (4 000 lx); 60 % relative humidity; 25/22 °C Cd 25 % HM; pH 6.0; 10 μmol/dm 3 CdCl2; 168 kBq/dm 3 109CdCl2; 16h/8h day/night (11 450 lx); 60 – 80 % relative humidity; 28/15 °C * Alpine pennycress Zn 25 % HM; pH 6.0; 10 μmol/dm 3 ZnCl2; 164 kBq/dm 3 65ZnCl2; 16h/8h day/night (11 450 lx); 60 – 80 % relative humidity; 28/15 °C * * Conditions provided by a plant growth chamber (KBWF 720, Binder, Germany). Plants of giant reed (Arundo donax L. var. versicolor) were obtained by vegetative propagation under in vitro conditions. Shoots development from lateral buds was induced in an MS medium (MURASHIGE and SKOOG, 1962) with an addition of 2– Bereitgestellt von Slovenská poľnohospodárska knižnica | Heruntergeladen 17.01.20 10:05 UTC 180 Dürešová, Z. et al. 4 mg/dm3 of BAP (6-benzylaminopurine) as a cytokinin-type growth regulator. For rooting of regenerated shoots and in vitro storage of giant reed plants, MS medium without an addition of growth regulators was used. Subsequently, giant reed plants were transferred and pre-cultivated in a 25 % HM. After 4 – 8 weeks of plant pre-cultivation, seedlings were gently removed from perlite and roots were washed free of any adhering perlite granules by deionized water and used in the accumulation experiments. 2.2 Accumulation experiments and conditions of the experiments Plants from the pre-cultivation phase were transferred into Erlenmeyer flasks (100 – 250 cm3) with a cover to protect plant roots against lights and cultivated for 8 days in a 50 – 150 cm3 (according to plant) Hoagland medium (HM) containing Cd (in the form of CdCl2) or Zn (in the form of ZnCl2 or ZnSO4) and spiked with 109CdCl2 or 65ZnCl2. The conditions of the individual experiments are detailed in the Table 1. In time intervals, samples of nutrient solution were taken and 109Cd or 65Zn radioactivities were measured by gamma-spectrometry. At the end of the experiments, plants were harvested, roots were carefully rinsed in deionized water and incorporated radioactivity in roots, stems or leaves was measured. Individual parts of plant were then oven dried (at 60 °C for 24 hours) and dry weights were determined. 2.3 Radiometric analysis All nutrient solutions used in the experiments were spiked with 109CdCl2 and 65ZnCl2 radioactive solutions obtained from the Czech Metrological Institute (Czech Republic). The radiometric analysis in terms of volume or specific radioactivity of 109Cd and 65Zn in the samples of nutrient solutions or individual parts of plant was realized using a well type NaI(Tl) scintillation gamma-spectrometer 54BP54/2-X or 76BP76/3 (Scionix, The Netherlands or Envinet, Czech Republic) and the data processing software ScintiVision-32 (Ortec, USA). For energy and efficiency calibration, a library of radionuclides was built by selecting characteristic γ-ray peaks for 109Cd (Eγ = 88.04 keV), 137Cs (Eγ = 661.66 keV) or 65Zn (Eγ = 1115.52 keV), and for the calibration, standard solutions of 109CdCl2, 137CsCl or 65ZnCl2 with known radioactivity including the half-life of radionuclides ( 109Cd: T1/2 = 462.6 d; 137Cs: T1/2 = 11 019 d, and 65Zn: T1/2 = 243.9 d) were used. 2.4 Speciation modelling The qualitative and quantitative proportion of the ion forms of Cd and Zn in nutrient solutions were calculated by the software MINEQL+ ver. 4.62 (Environmental Research Software, USA). This software employs chemical equilibrium models to calculate metals speciation and solubility equilibria in the laboratory or natural aqueous systems under a variety of conditions including Bereitgestellt von Slovenská poľnohospodárska knižnica | Heruntergeladen 17.01.20 10:05 UTC Nova Biotechnologica et Chimica 14-2 (2015) 181 a gas phase with constant partial pressure, pH, temperature, ionic strength, and concentrations of cations and anions. 2.5 Statistical analysis All analytical determinations were performed in triplicate. The statistical significance of differences in calculated values of Cd or Zn accumulation in plant tissues were evaluated by a multiple range test to ascertain differences between individual groups. The level of significance p was 0.05 in all cases. To evaluate the similarity degree between the objects – individual plant species on the basis of obtained variables and conditions of the experiments as well as to evaluate the relationships between the obtained variables and conditions of the experiments, the cluster analysis (CA) was applied. Parameters describing the obtained variables, such as Cd or Zn accumulation in plants (in μmol/g; fresh wt.) or concentration ratio (CR) defined as the ratio of the concentration of Cd or Zn accumulated in shoots to concentration of Cd or Zn accumulated in roots ([Me]shoot / [Me]root), and the conditions of the experiments, such as the initial concentrations of Cd2+ or Zn2+ ions (in μmol/dm3) predicted by the speciation modelling, the pH value of the nutrient solutions, the strength of Hoagland medium (25 % or 50 % HM) or conditions of the plant cultivation (temperature, relative humidity, day/night photoperiod or intensity of illumination) were utilized in this analysis. Statistical analysis, evaluation, and graphical interpretation of the obtained data were carried out using programs OriginPro ver. 8.5 (OriginLab Corp., USA), STATGRAPHICS Centurion ver. 15 (StatPoint Technologies Inc., USA), SYSTAT ver. 13 (Systat Software Inc., USA), and SigmaPlot ver. 12 (Systat Software Inc., USA). 3. Results and discussion Fig. 1 depicts the kinetics of Cd and Zn accumulation in plants of tobacco (N. tabacum L.), celery (A. graveolens L.), maize (Z. mays L.), giant reed (A. donax L.), and alpine pennycress (N. caerulescens L.) during 8 days of plant cultivation under hydroponic conditions. The conditions of the experiments were partly different (see Table 1) in terms of the initial concentrations of Cd (in the form of CdCl2) and Zn (in the form of ZnCl2 or ZnSO4), the pH of nutrient solutions and the strength of Hoagland medium (HM) as well as from the point of view of conditions of plant cultivation (temperature, relative humidity, day/night photoperiod or intensity of illumination). To carry out a calculation of free Cd2+ and Zn2+ ionic forms proportion and concentration in nutrient solutions as the most bioavailable metal forms for plants, the program MINEQL+ was used. However, this prediction has to be considered with caution, because the modelling program does not include the participation of the plant. Therefore, it can be used for the evaluation of initial conditions of the experiments (without plants). According to in silico speciation analysis by mentioned equilibrium modelling software, it was found that Cd present in a 25 % or 50 % HM representing the model Bereitgestellt von Slovenská poľnohospodárska knižnica | Heruntergeladen 17.01.20 10:05 UTC 182 Dürešová, Z. et al. of soil solution can occur in 15 different inorganic chemical forms within the range of pH 3 – 12. In the case of Zn, 12 different inorganic chemical forms were calculated under the same conditions. At the pH 5.5 or 6.0, 25 % or 50 % HM, and initial concentration of CdCl2 10 μmol/dm 3, which were included in the experiments, the proportion of Cd2+ form from the total amount of Cd was in the range 71.7 % to 77.1 %. In the case of Zn and the given initial conditions, the proportion of Zn2+ form represented 92.2 % – 94.8 %. 0 1 2 3 4 5 6 7 8 0,00 0,05 0,10 0,15 0,20 0,25 Time [day] C d ac cu m ul at io n [μ m ol /g ]; fr es h w t. Tobacco Celery Maize Giant reed Alpine pennycress 0 1 2 3 4 5 6 7 8 0,00 0,05 0,10 0,15 0,20 0,25 Time [day] Z n ac cu m ul at io n [μ m ol /g ]; fr es h w t. Tobacco Celery Maize Giant reed Alpine pennycress Fig. 1. Kinetics of Cd (A) and Zn (B) accumulation in plants of tobacco (N. tabacum L.), celery (A. graveolens L.), maize (Z. mays L.), giant reed (A. donax L.), and alpine pennycress (N. caerulescens L.) during 8 days of plant cultivation under hydroponic conditions. Conditions are detailed in Table 1. Data are presented as arithmetic means of triplicate experiment. From the Fig. 1, it is evident that the curves describing the Cd accumulation (Fig. 1A) in tobacco, celery, maize, giant reed, and alpine pennycress plants show a relatively similar trend with the curves obtained for Zn (Fig. 1B). This result corresponds with the fact that Cd and Zn are mutual physico-chemical analogues as well as that Cd is transported within the plants via the Zn pathways, but the mechanisms responsible for cellular Zn tolerance cannot detoxify Cd effectively (ZHAO et al., 2006). However, there were significant differences between plant species in time-course of Cd and Zn accumulation and in the obtained values of accumulation (μmol/g; fresh wt.). The most different trend of the curve describing the Cd and Zn accumulation showed plants of giant reed, when the two-phase process given by a slow Cd or Zn accumulation within the first 3 days of plant cultivation followed by exponential increase of accumulation within the next 5 days was observed. At the other studied plant species, the typical curves with gradual increasing the Cd or Zn accumulation in plants were found. The phytotoxic symptoms at used Cd concentrations were not observed in either case. The Fig. 2 compares the differences in the obtained values of Cd and Zn accumulation in individual plant species on the basis of multiple range test at the level of p < 0.05. It was found that the highest value of Cd accumulation showed plants of giant reed and the accumulation decreased in the order: giant reed > tobacco > alpine pennycress >> maize and celery. QUEZADA- HINOJOSA et al. (2015) also found the variability in Cd contents among the six different perennial plant species growing in a wooded pasture of the Swiss Jura Mountains, where the soils are geogenically enriched in Cd. In the case of Zn, when A B Bereitgestellt von Slovenská poľnohospodárska knižnica | Heruntergeladen 17.01.20 10:05 UTC Nova Biotechnologica et Chimica 14-2 (2015) 183 the different initial concentrations of Zn (see Table 1) were used, the order of plants for Zn accumulation was: tobacco and alpine pennycress > celery > maize >> giant reed. It should be mentioned that the nutrient solution for giant reed contained a 10-fold lower concentration of Zn in comparison with plants of tobacco and alpine pennycress. From these results, it can be concluded that tobacco and giant reed showed a similar Cd accumulation efficiency as plants of alpine pennycress, which are classified as Zn/Cd hyperaccumulators. In the case of celery and maize, as typical agricultural crops, the Cd and Zn accumulation reached significantly lower levels. 0,00 0,05 0,10 0,15 0,20 0,25 0,30 0,35 12 1 c b ab M et al a cc um ul at io n [μ m ol /g ]; fr es h w t. Cd Zn Alpine pennycress Giant reedMaizeCeleryTobacco a c 1 2 3 Fig. 2. Comparison of Cd (A) and Zn (B) accumulation in plants of tobacco (N. tabacum L.), celery (A. graveolens L.), maize (Z. mays L.), giant reed (A. donax L.), and alpine pennycress (N. caerulescens L.) after 8 days of plant cultivation under hydroponic conditions. Conditions are detailed in Table 1. Mean values from triplicate experiments with the same letter (for Cd) or number (for Zn) above columns are not significantly different at the p < 0.05 level based on multiple range test. In terms of phytoremediation technologies, it is important to mention that toxic metals or radionuclides have to be accumulated mainly in above-ground parts of plants. In general, significant differences in distribution of Cd and Zn within shoots and roots of studied plant species and higher percentage distribution of Cd into the shoots from the total accumulated amount of Cd in comparison with the data obtained for Zn were found as well (Fig. 3). The highest proportion of translocated Cd into shoots was observed in plants of tobacco, celery and alpine pennycress, whereby the distribution of Cd for account of shoots decreased in the order: tobacco, celery and alpine pennycress > maize > giant reed. In the case of Zn, when the different initial concentrations of Zn (see Table 1) were used, the percentage distribution into shoots decreased in the order: giant reed >> alpine pennycress > celery > tobacco > maize. Within the shoots, the direct gammaspectrometric determinations revealed that the amount of accumulated Cd and Zn (in specific radioactivities Bq/g of dry wt. or Bq/cm2) increased from the oldest developed leaves to the youngest developed leaves (data not shown). This result was observed in all studied plant species. ZHANG Bereitgestellt von Slovenská poľnohospodárska knižnica | Heruntergeladen 17.01.20 10:05 UTC 184 Dürešová, Z. et al. et al. (2015) reported that Cd accumulation within the plants of two castor cultivars (Ricinus communis L.) decreased in the order: root > stem > young leaf > old leaf. 0 20 40 60 80 100 Zn A lp in e pe nn yc re ss G ia nt r ee d M ai ze C el er y 323 23 21 3 233 2 c abb a ab c b aa D is tr ib ut io n of M e [% ] Root Shoot a 1 T ob ac co A lp in e pe nn yc re ss G ia nt r ee d M ai ze C el er y T ob ac co Cd Fig. 3. Distribution of Cd and Zn in roots and shoots of tobacco (N. tabacum L.), celery (A. graveolens L.), maize (Z. mays L.), giant reed (A. donax L.), and alpine pennycress (N. caerulescens L.) after 8 days of plant cultivation under hydroponic conditions. Conditions are detailed in Table 1. Mean values from triplicate experiments with the same letter (for Cd) or number (for Zn) above columns are not significantly different at the p < 0.05 level based on multiple range test. Also, for the evaluation of Cd and Zn mobility in conductive tissues of studied plants in the term of their translocation efficiency it was established a non-dimensional concentration ratio (CR) which represents the ratio of metal concentration in the above-ground part of plants [Me]shoot (µmol/g; dry wt.) to metal concentration in roots [Me]root (µmol/g; dry wt.). From the data mentioned in Table 2, it is evident that on the basis of these concentration ratios [Me]shoot / [Me]root Cd and Zn were in prevailing part accumulated in the root tissues and only partially accumulated in shoots. In general, the obtained values of CR were very similar for both target metals Cd and Zn. This result is in agreement with the above mentioned fact that Cd and Zn are mutual physico-chemical analogues and that Cd is transported via the Zn pathway as well. Some present differences in the obtained CR values between Cd and Zn were probably caused by lower concentrations of Zn in nutrient solutions. The CR values were calculated in the range 0.06 – 0.27 for Cd and for Zn 0.06 – 0.48. Similar concentration ratios found POŁEĆ-PAWLAK et al. (2005) for plants of Arabidopsis thaliana cultivated under hydroponic conditions during 14 days in HM containing 25 or 50 μmol/dm3 of Cd2+. From the point of view of plant species, the CR values obtained for Cd decreased in the order: maize > celery > tobacco and giant reed Bereitgestellt von Slovenská poľnohospodárska knižnica | Heruntergeladen 17.01.20 10:05 UTC Nova Biotechnologica et Chimica 14-2 (2015) 185 > alpine pennycress. These results suggest that plants of celery and maize with a lower ability to accumulate Cd and Zn from nutrient solutions showed higher values of CR. On the other side, plants, such as tobacco, giant reed and alpine pennycress, with a higher ability to accumulate Cd and Zn from nutrient solutions showed lower values of CR. This fact can be related to ability of these plants to bind and detoxicate of metals, such as Cd and Zn, in root cells and cellular compartments (e.g. vacuoles). Thus, in this case the root system acts as a protective barrier to further metals movement into the above-ground parts of plants. This character results in a high tolerance to metals and metals accumulation in plants or in given plant tissues. SALT et al. (1999) reported that Zn was mostly complexed to histidine in roots, transported as Zn2+ in the xylem sap, and complexed to organic acids in leaves. It can be expected that mentioned processes are also involved in Cd accumulation. Some works reported that the application of chelating agents, such as ethylenediaminetetraacetate (EDTA) or citric acid (CA), can increase not only the concentration of metals in soil solutions, but also the translocation of metals within the plants (HORNÍK et al. 2008; HORNÍK et al. 2009; GULDANOVÁ et al., 2012; EHSAN et al., 2014). Table 2. Comparison of the values of non-dimensional concentration ratio (CR) calculated for Cd and Zn after 8 days cultivation of plants of tobacco (N. tabacum L.), celery (A. graveolens L.), maize (Z. mays L.), giant reed (A. donax L.), and alpine pennycress (N. caerulescens L.) under hydroponic conditions. Conditions are detailed in Table 1. Mean values from triplicate experiments with the same letter (for Cd) or number (for Zn) above columns are not significantly different at the p < 0.05 level based on multiple range test. CR ([Me]shoot / [Me]root) ± S.D. Plant Cd Zn Tobacco 0.12±0.02c 0.06±0.014 Celery 0.16±0.02b 0.14±0.013 Maize 0.27±0.03a 0.18±0.022; * Giant reed 0.11±0.01c 0.48±0.201; ** Alpine pennycress 0.06±0.01d 0.06±0.014 * Initial concentration of Zn 5.9 μmol/dm3 in a nutrient solution; ** Initial concentration of Zn 1.0 μmol/dm3 in a nutrient solution. The realized experiments were deliberately designed for different initial conditions of the experiments and conditions of the plant cultivation (see Table 1). This fact allows to evaluate the relationships between variables obtained at the end of experiments and these conditions as well as to find the similarity between objects – individual plant species. For these purposes, the method of multivariate analysis – cluster analysis (CA) was applied and parameters describing the obtained variables, such as Cd or Zn accumulation in plants (in μmol/g; fresh wt.) and concentration ratio (CR) defined as the ratio of the concentration of Cd or Zn accumulated in shoots to concentration of Cd or Zn accumulated in roots ([Me]shoot / [Me]root) and the conditions of the experiments, such as the initial concentrations of Cd2+ or Zn2+ ions (C0 Cd 2+ or C0 Zn 2+; in μmol/dm3) predicted by the speciation modelling, the pH value of nutrient solutions, the strength of Hoagland medium (25 % or 50 % HM) or conditions of the plant cultivation (temperature, relative humidity, day/night photoperiod or intensity of illumination) were utilized in this analysis. Bereitgestellt von Slovenská poľnohospodárska knižnica | Heruntergeladen 17.01.20 10:05 UTC 186 Dürešová, Z. et al. 0 4 8 12 16 20 24 D is ta nc e T ob ac co C el er y M ai ze G ia nt r ee d ne p en ny cr es s 0 4 8 12 16 20 24 D is ta nc e C 0 C d fr ee ac cu m ul at io n C d H M C d T R C on di tio nspH 0 4 8 12 16 20 24 D is ta nc e T ob ac co C el er y M ai ze G ia nt r ee d pe nn yc re ss 0 4 8 12 16 20 24 D is ta nc e C 0 Z n fr ee C on di tio ns pH cc um ul at io n Z n H M Z n T R Fig. 4. Dendrogram of cluster analysis including squared Euclidean distance and Ward’s method as the aggregation criterion for the evaluation of the similarity degree between objects – individual plant species on basis of obtained variables and conditions of the experiments (A, C) as well as the relationships between obtained variables and conditions of the experiments (B, D) for Cd (A, B) and Zn (C, D). Conditions of the experiments are detailed in Table 1. From CA including squared Euclidean distance and Ward’s method as the aggregation criterion for the evaluation of the similarity degree between objects represented by studied plant species, it was found that in the case of Cd plants of tobacco, giant reed, and alpine pennycress, these formed separate cluster (Fig. 4A, No. 1). It means that these plants showed similar characteristics from the point of view of Cd accumulation and relationships between conditions of the experiments and obtained data describing Cd accumulation. Thus, it can be expected that plants T ob ac co G ia nt re ed A lp in e p en ny cr es s C el er y M ai ze D is ta nc e D is ta nc e D is ta nc e D is ta nc e T ob ac co G ia nt re ed A lp in e pe nn yc re ss C el er y M ai ze C 0 C d2 + pH C d ac cu m ul at io n C d C R C 0 Z n2 + pH Z n ac cu m ul at io n C on di tio ns of c ul tiv at io n % H M Z n C R A B C D % H M C on di tio ns of c ul tiv at io n 1 2 1 2 1 2 1 2 Bereitgestellt von Slovenská poľnohospodárska knižnica | Heruntergeladen 17.01.20 10:05 UTC Nova Biotechnologica et Chimica 14-2 (2015) 187 of tobacco and giant reed will dispose with similar characteristics as plants of alpine pennycress, which are defined as Zn/Cd hyperaccumulators, in terms of Cd accumulation and other positive parameters for their utilization in phytoremediation processes and techniques. LIU et al. (2016) also classified the plants of tobacco (N. tabacum L.) as hyperaccumulators of Cd on the basis of realized experiments under hydroponic conditions and characterized the effect of conditions of the experiments (chemical inhibitors, temperature or illumination) on Cd accumulation. On the other side, plants of celery and maize as agricultural crops are significantly different from this group, because they formed a second separate cluster (Fig. 4A, No. 2). Within the evaluation of relationships between the conditions of the experiments and obtained values for Cd accumulation (Cd accumulation and CR for Cd), the correlations between C0 Cd 2+, pH and Cd accumulation (Fig. 4B, No. 1) as well as between the strength of HM (representing the different concentrations of macro- and microelements and ionic strength) and CR for Cd (Fig. 4B, No. 2) were found. From these results, it can be concluded that Cd accumulation through the root system of studied plants is strongly dependent on initial concentration of free Cd2+ ions and the value of pH. On the other hand, the concentration ratio CR describing the Cd translocation from roots-to-shoots is primarily affected by the strength of HM corresponding with concentrations of macro- and microelements in nutrient (soil) solutions. In the case of Zn, on the basis of the similarity degree, two separate clusters were also formed. The first cluster was represented by plants of tobacco and alpine pennycress (Fig. 4C, No. 1) and the second cluster formed plants of celery, maize, and giant reed (Fig. 4C, No. 2). Similarly, as in the case of Cd, the two logical correlations between C0 Zn 2+ and Zn accumulation (Fig. 4D, No. 1) and between the strength of HM and CR for Zn (Fig. 4D, No. 2) were found. 4. Conclusions The obtained results showed that the time-course of Cd accumulation in plants of tobacco, celery, maize, giant reed, and alpine pennycress cultivated under conditions of short-term hydroponic experiments had a relatively similar trend with curves obtained for Zn. This result supports the fact that Cd and Zn are mutual physico-chemical analogues as well as that Cd is transported within the plants via the Zn pathways. On the other side, a significant difference in time-course of Cd and Zn accumulation and in the obtained values of accumulation (μmol/g; fresh wt.) between plant species existed as well. The differences in the obtained values of Cd and Zn accumulation in individual plant species were evaluated by multiple range test at the level of p < 0.05. The highest values of Cd accumulation showed plants of giant reed and the accumulation decreased in the order: giant reed > tobacco > alpine pennycress >> maize and celery. From the point of view of Cd and Zn distribution within the plants, the highest proportion of translocated Cd into shoots was observed in plants of tobacco, celery, and alpine pennycress. A similar result was also found in the case of Zn. Within the shoots, the amount of accumulated Cd and Zn increased from the oldest developed leaves to the youngest developed leaves. On the basis of concentration Bereitgestellt von Slovenská poľnohospodárska knižnica | Heruntergeladen 17.01.20 10:05 UTC 188 Dürešová, Z. et al. ratios (CR) [Me]shoot / [Me]root calculation for both metals, it was found that Cd and Zn were in prevailing part accumulated in the root tissues and only partially accumulated in shoots. The CR values corresponding to these facts were calculated in the range 0.06 – 0.27 for Cd and for Zn 0.06 – 0.48. From the point of view of plant species, the CR values obtained for Cd decreased in the order: maize > celery > tobacco and giant reed > alpine pennycress. This result suggests that plants of tobacco, giant reed, and alpine pennycress showed the ability to bind and detoxicate of metals, such as Cd and Zn, in root cells and cellular compartments in larger extent than studied agricultural crops (maize and celery). The similarity between studied objects – individual plant species on the basis of obtained variables and conditions of the experiments as well as the relationships between obtained variables and conditions of the experiments were subjected to multivariate analysis method – cluster analysis (CA). According to this analysis, it can be expected that plants of tobacco and giant reed will dispose with similar characteristics as plants of alpine pennycress, which are defined as Zn/Cd hyperaccumulators, in terms of Cd accumulation and other positive parameters for their utilization in phytoremediation processes and techniques. Also, it was found that Cd and Zn accumulation through the root system of studied plants is strongly dependent on initial concentration of free Me2+ ions and the value of pH. On the other hand, the concentration ratios CR describing the Cd or Zn translocation from roots-to- shoots are primarily affected by concentrations of macro- and microelements in the nutrient (soil) solutions. Acknowledgement: This work was supported by the Slovak Research and Development Agency under the contract No. APVV-0380-12. References ATAFAR, Z., MESDAGHINIA, A.R., NOURI, J., HOMAEE, M., YUNESIAN, M., AHMADIMOGHADDAM, M., MAHVI, A.H.: Effect of fertilizer application on soil heavy metal concentration. Environ. Monit. Assess., 160, 2010, 83-89. ATSDR (Agency for Toxic Substances and Disease Registry): Priority list of hazardous substances, U.S. Department of Health and Human Services, Atlanta, GA, USA, 2013. Available from: . BAUDDH, K., SINGH, K., SINGH, B., SINGH, R.P.: Ricinus communis: A robust plant for bio-energy and phytoremediation of toxic metals from contaminated soil. Ecol. Eng., 84, 2015, 640-652. BERNARD, A.: Cadmium & its adverse effects on human health. Indian J. Med. Res., 128, 2008, 557-564. CHIRAKKARA, R.A., REDDY, K.R.: Biomass and chemical amendments for enhanced phytoremediation of mixed contaminated soils. Ecol. Eng., 85, 2015, 265-274. CLEMENS, S., AARTS, M.G.M., THOMINE, S., VERBRUGGEN, N.: Plant science: the key to preventing slow cadmium poisoning. Trends Plant Sci., 18, 2013, 92-99. Bereitgestellt von Slovenská poľnohospodárska knižnica | Heruntergeladen 17.01.20 10:05 UTC Nova Biotechnologica et Chimica 14-2 (2015) 189 DÜREŠOVÁ, Z., ŠUŇOVSKÁ, A., HORNÍK, M., PIPÍŠKA, M., GUBIŠOVÁ, M., GUBIŠ, J., HOSTIN, S.: Rhizofiltration potential of Arundo donax in Cd and Zn removing from contaminated wastewaters. Chem. Pap., 68, 2014, 1452-1462. EC (European Commission): Decision No. 2455/2001/EC of the European Parliament and of the Council of 20 November 2001 establishing the list of priority substances in the field ofwater policy and amending Directive 2000/60/EC. Off. J. Eur. Commun., L 331, 2001, 1-5. EHSAN, S., ALI, S., NOUREEN, S., MAHMOOD, K., FARID, M., ISHAQUE, W., SHAKOOR, M.B., RIZWAN, M.: Citric acid assisted phytoremediation of cadmium by Brassica napus L. Ecotoxicol. Environ. Saf., 106, 2014, 164–172. ETTLER, V.: Soil contamination near non-ferrous metal smelters: A review. Appl. Geochem., 2015 (in Press). GILL, S.S., HASANUZZAMAN, M., NAHAR, K., MACOVEI, A., TUTEJA, N.: Importance of nitric oxide in cadmium stress tolerance in crop plants. Plant Physiol. Biochem., 63, 2013, 254-261. GULDANOVÁ, J., HORNÍK, M., MAREŠOVÁ, J., PIPÍŠKA, M., AUGUSTÍN, J.: Bioaccumulation and distribution of 137Cs in tobacco cultivated under hydroponic conditions. Nova Biotechnol., 10-2, 2010, 95-106. GULDANOVÁ, J., HORNÍK, M., MAREŠOVÁ, J., PIPÍŠKA, M., AUGUSTÍN, J., LESNÝ, J.: Uptake of zinc organic complexes by roots of vascular plants. In: BEHL, R.K., BONA, L., PAUK, J., MERBACH, W., VEHA, A. (Eds.), Crop science and technology for food security, bioenergy and sustainability, Agrobios, Jodhpur, India, 2012, 301–308. HAWRYLAK-NOWAK, B., DRESLER, S., WÓJCIK, M.: Selenium affects physiological parameters and phytochelatins accumulation in cucumber (Cucumis sativus L.) plants grown under cadmium exposure. Sci. Hortic., 172, 2014, 10-18. HAWRYLAK-NOWAK, B., DRESLER, S., MATRASZEK, R.: Exogenous malic and acetic acids reduce cadmium phytotoxicity and enhance cadmium accu- mulation in roots of sunflower plants. Plant Physiol. Biochem., 94, 2015, 225-234. HOAGLAND, D.R.: Optimum nutrient solution for plants. Science, 52, 1920, 562- 564. HORNÍK, M., GULDANOVÁ, J., PIPÍŠKA, M., MAREŠOVÁ, J., AUGUSTÍN, J.: Effect of chelating agents on phytotoxicity and bioaccumulation of heavy metals in vascular plants. Nova Biotechnol., 9-3, 2009, 271-278. HORNÍK, M., PIPÍŠKA, M., VRTOCH, Ľ., AUGUSTÍN, J., LESNÝ, J.: Bioaccumulation of 137Cs and 60Co by Helianthus annuus. Nukleonika, 50, 2005, S49-S52. HORNÍK, M., PIPÍŠKA, M., SEKÁČOVÁ, J., AUGUSTÍN, J.: Determination of long distance transport of Cs+, Co2+ and Zn2+ ions in vascular plants by autoradiography and gamma-spectrometry. Nova Biotechnol., 7-1, 2007, 33-40. HORNÍK, M., PIPÍŠKA, M., VRTOCH, Ľ., SEKÁČOVÁ, J., AUGUSTÍN, J., LESNÝ, J.: Influence of complexing ligands on Zn uptake and translocation in tobacco and celery plants. Acta Agron. Ovar., 50, 2008, 65-71. IANNONE, M.F., GROPPA, M.D., BENAVIDES, M.P.: Cadmium induces different biochemical responses in wild type and catalase-deficient tobacco plants. Environ. Exp. Bot., 109, 2015, 201-211. Bereitgestellt von Slovenská poľnohospodárska knižnica | Heruntergeladen 17.01.20 10:05 UTC 190 Dürešová, Z. et al. KABATA-PENDIAS, A., MUKHERJEE, A.B.: Trace elements from soil to human, First Edition, Springer-Verlag, Berlin Heidelberg, Germany, 2007, 550 p. LAMBERS, H., CHAPIN III, F.S., PONS, T.L.: Plant physiological ecology, Second Edition, Springer-Verlag, New York, USA, 2008, 605 p. LIANG, Z., DING, Q., WEI, D., LI, J., CHEN, S., MA, Y.: Major controlling factors and predictions for cadmium transfer from the soil into spinach plants. Ecotoxicol. Environ. Saf., 93, 2013, 180-185. LIU, H., WANG, H., MA, Y., WANG, H., SHI, Y.: Role of transpiration and metabolism in translocation and accumulation of cadmium in tobacco plants (Nicotiana tabacum L.). Chemosphere, 144, 2016, 1960-1965. MCLAUGHLIN, M.J., PARKER, D.R., CLARKE, J.M.: Metals and micronutrients – food safety issues. Field Crops Res., 60, 1999, 143-163. MURASHIGE, T., SKOOG, F.: A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol. Plant. 15, 1962, 473-497. PANDEY, V.C., BAJPAI, O., SINGH, N.: Energy crops in sustainable phytoremediation. Renew. Sust. Energ. Rev., 54, 2016, 58-73. POŁEĆ-PAWLAK, K., RUZIK, R., ABRAMSKI, K., CIURZYŃSKA, M., GAWROŃSKA, H.: Cadmium speciation in Arabidopsis thaliana as a strategy to study metal accumulation system in plants. Anal. Chim. Acta, 540, 2005, 61-70. QUEZADA-HINOJOSA, R., FÖLLMI, K.B., GILLET, F., MATERA, V.: Cadmium accumulation in six common plant species associated with soils containing high geogenic cadmium concentrations at Le Gurnigel, Swiss Jura Mountains. Catena, 124, 2015, 85-96. SALT, D.E., PRINCE, R.C., BAKER, A.J.M., RASKIN, I., PICKERING, I.J.: Zinc ligands in the metal hyperaccumulator Thlaspi caerulescens as determined using X-ray absorption spectroscopy. Environ. Sci. Technol., 33, 1999, 713-717. ŠUŇOVSKÁ, A., HORNÍK, M., MAREŠOVÁ, J., PIPÍŠKA, M., AUGUSTÍN, J.: 137Cs uptake and translocation in leafy vegetable: A study with Lactuca sativa L. grown under hydroponic conditions. Nova Biotechnol. Chim., 11, 2012, 153-166. TAVAREZ, M., MACRI, A., SANKARAN, R.P.: Cadmium and zinc partitioning and accumulation during grain filling in two near isogenic lines of durum wheat. Plant Physiol. Biochem., 97, 2015, 461-469. VISIOLI, G., MARMIROLI, N.: The proteomics of heavy metal hyperaccumulation by plants. J. Proteomics, 79, 2013, 133-145. WHITE, P.J., BROWN, P.H.: Plant nutrition for sustainable development and global health. Ann. Bot., 105, 2010, 1073-1080. WÓJCIK, M., TUKIENDORF, A.: Cadmium uptake, localization and detoxificationin Zea mays. Biol. Plant., 49, 2005, 237-245. ZHAO, F.J., JIANG, R.F., DUNHAM, S.J., MCGRATH, S.P.: Cadmium uptake, translocation and tolerance in the hyperaccumulator Arabidopsis halleri. New Phytol., 172, 2006, 646-654. ZHANG, H., GUO, Q., YANG, J., SHEN, J., CHEN, T., ZHU, G., CHEN, H., SHAO, C.: Subcellular cadmium distribution and antioxidant enzymatic activities in the leaves of two castor (Ricinus communis L.) cultivars exhibit differences in Cd accumulation. Ecotoxicol. Environ. Saf., 120, 2015, 184-192. Bereitgestellt von Slovenská poľnohospodárska knižnica | Heruntergeladen 17.01.20 10:05 UTC