The main message of this study is that the edibility of R. alpinus can be questionable, considering the accumulation and distribution of risk elements in different organs of this species. The intensity of elements accumulation by R. alpinus is considerably site-specific. The release of the trace and risk elements relates to the lithogenic and anthropogenic (e.g., metallurgy) sources resulting in the subsequent accumulation in different departments of R. alpinus. Moreover, the accumulation of risk elements such as As, Cr, Ni, Pb, and Cd was affected by season. In addition to acidic soil, the dissolution by precipitation H2O during the autumn contributed to the release of elements (Truog 1947), reflected in higher concentrations in the organs during autumn compared to summer in localities of the Krkonoše Mountains.
4.1. Chemical characterization of soil
The reduced soil acidity in all the localities was due to the high concentrations of Ca and Mg resulting predominantly from the geological substrates. The acidity of the soils can contribute to the release of elements for plant uptake.The concentrations of total Zn and Cu were below the permissible limits (220 for Zn and 70 mg kg-1 for Cu) for most agricultural soils (Kabata-Pendias and Pendias 2001). Meanwhile, the average value of Zn in agricultural soils in the Czech Republic ranges from 105 - 120 mg kg-1 (Poláková et al. 2016). Thus, the limit exceeded all the localities of the Krkonoše Mountains. The high total concentration of Zn in the soils from the Krkonoše Mountains relates to the historic mining and smelting of Zn (Kafka 2003). The average concentration of Cu for agricultural soils ranges from 1 - 50 mg kg-1 (Adriano 2001) and corresponds with the investigated localities. Additionally, a higher total concentration of Mn was recorded in the localities of the Krkonoše Mountains and DCH. According to Kabata-Pendias and Pendias (2001), the average total Mn concentration in podzols is 270 mg kg-1 and this exceeded more than twice. The high Mn is due to the acidic conditions and redox potential of the soils. Limited uptake of Mn by plants can occur after organic fertilization, where Mn is oxidized in the soil, thereby reducing its solubility and availability to plants. Moreover, the reduction in the solubility of Mn and its availability can result from a lack of H2O precipitation (Vaněk et al. 2012, Fig. 7d).The total concentrations of Al, Cr, Ni, Pb, and Cd were within the reported range in most agricultural soils (Kabat-Pendias and Pendias 2001). In the localities of the Krkonoše Mountains, the concentrations of these elements were higher compared to the Alps, which relate to mining influence from the past (Lokvenc 2003, 2007, Tásler 2012). Cr concentration in soils is directly related to parent rocks (geogenic elements), with an average value of 50 mg kg-1 (Adriano 2001). The average total concentration in the Czech Republic is approximately 41 mg kg1 (Tásler 2012), which indicates that the limit was exceeded in VT, PC, and HM, resulting from the anthropogenic activities. The average Ni concentration in the soil is generally considered to be about 20 mg.kg-1 (Adriano 2001), the results show that it was again exceeded in the Krkonoše localities. Notably, contaminated agriculture areas in the Czech Republic have values of Ni > 200 mg kg-1 (Poláková et al. 2016). The permissible limit of Pb in agricultural soils is 100 mg kg-1: thus, there was no exceedance of Pb in the examined localities (Kabata-Pendias and Pendias 2001). According to Pugh et al. (2002), Cd concentration in uncontaminated soils ranges from 0.05 – 2 mg.kg-1, thus, in VT and LB, Cd exceeded this range in rhizome during autumn. The concentrations of As, was above Czech legislative limits (20 mg kg-1) at LB, VT, HM, and MD, indicating a risk to the safety of food or feed, direct danger to human or animal health in contact with soil, and a negative impact on the production function of agricultural land (Kabata-Pendias and Pendias 2001). In the Krkonoše localities, the soils remain contaminated by As probably due to arsenopyrite mining in the past (Tásler 2012).
4.2 Distribution of Fe, Zn, Cu, Mn, Al, As, Cr, Ni, Pb, and Cd in the organs
The selection of adequate plant species with accumulative characteristics is crucial for successful phytoextraction processes. Hyperaccumulating plants frequently reach low biomass, pest management, or harvesting practices as main drawbacks for their utilization for metal phytoextraction (Wenzel et al. 1999). The amount of risk elements extracted depends on the concentrations in the harvestable parts of plants and other plant biomass. Mean concentrations of Fe, Zn, Cu, and Mn in all organs of R. alpinus were found to be relatively low and within ranges of geochemical background values given by Kabata-Pendias and Pendias (2001). The variability in the concentrations of microelements in the organs of the R. alpinus relates to the compartmentalization and translocation in the vascular system (Hänsch and Mendel 2009, Vondráčková et al. 2014). Zinc and Fe ions enter the plant xylem through the symplastic pathway (Lu et al., 2009, She et al. 2018). The concentration of Zn was highest in the rhizome, consistent with the finding of Bohner (2005). Moreover, the concentration of Fe was lowest in the stem, which is consistent with the studies by Gaweda (2009) and Vondráčková et al. (2014). The highest concentration of Fe was in senescent leaves, and this finding corresponds with the study by Bohner (2005). The same pattern as Fe exhibited in the case of Mn compared to Bohner (2005), who reported elevated concentration of Mn in mature leaves of R. alpinus. We recorded the lowest concentration of Mn in mature leaves and highest in senescent leaves. Gaweda (2009) reported the highest concentration of Fe and Mn in Rumex acetosa in the below-ground biomass. Nonetheless, Fe and Mn concentrations in the senescent leaves were higher than the other organs, which suggests that the plant is probably supersaturated (Baker 1981) by these elements due to the high level in the soil. However, the Fe and Mn concentrations in the leaves (emerging and mature leaves) are within the normal recorded range according to Levy et al. (1999) and Mahler (2004) (see Table S1). A different distribution of Cu was in emerging and mature leaves, which is not consistent with the distribution of Cu in below-ground organs of Rumex acetosa (Gaweda 2009). The high concentration of Cu in the emerging and mature leaves explains why the higher part of Cu localize in the growing section of the tissues and chloroplasts, respectively (Vaněk et al. 2012).
Several studies have investigated plants with the mechanisms to tolerate high concentration of Al (Tolrà et al. 2004, Arunakumara et al. 2013, Vondráčková et al. 2015). In this study, the mean total concentration of Al was lowest in emerging leave and highest in the petiole, followed by the rhizome, due to low transport from below-ground organs to leaf: a defensive mechanism against high concentration of Al in plants (Poschenrieder et al. 2008). In comparison with the experiment by Vondráčková et al. (2015), concerning R. obtusifolius, the recorded values in the organs of this study were low, which indicates that R. alpinus prevents the intake of Al. The mean total concentration of As in individual organs ranged from 1-1.8 mg kg-1, which slightly exceeds the limit according to Kabata-Pendias and Pendias (2001). Arsenic concentration in the leaves was lower and higher in petiole and senescent leaves, which indicates that the plant tried to get rid of it. The mean total concentration of Cr in R. alpinus organs of this study was lower compared to the results of Rumex obtusifolius, according to Vondráčková et al. (2014). While their results showed the highest Cr concentration in the leaves, we obtained the lowest in emerging, mature leaves and the highest in stem and rhizome. However, these results are comparable to Gaweda (2009), who studied Rumex acetosa. Notably, the different results in varying degrees relate to the species, anthropogenic activities, and the soil chemical properties.
Meanwhile, Pb usually is taken up by plants from air pollution because of anthropogenic activities (Kabata-Pendias and Pendias 2001). The mean total concentration of Pb and Ni in all the organs was approximately the same and in the normal range (Allen 1989, Pugh et al. 2002). Compared to Rumex acetosa, Rumex crispus, and Rumex K-1 (Zhuang et al. 2007, Gaweda 2009), where the highest concentration of Pb was in below-ground organs, we recorded Pb higher in above-ground organs. According to Pawlak et al. (2007), Pb accumulates mainly in roots with an affinity to galacturonic acid. The concentration of Cd was lower in above-ground organs and highest in the below-ground organ. Zn and Fe may have an antagonistic effect on the absorption of Cd in plants, leading to the inhibition of Cd absorption (Li et al. 2004). Zinc and Fe compete with Cd for plant surface absorption sites, thereby affecting the absorption of Cd in the plant tissue. Fei et al. (2018) reported that the young leaf protection mechanism with Cd preferentially distributed to the senescent leaves to avoid Cd toxic effect in the emerging at mature leaves (She et al. 2018, Wang et al. 2019). However, the highest concentration was in the rhizomes, which indicates that R. alpinus save the above-ground organs against Cd (Zhuang et al. 2007, Gaweda 2009).
4.3 Transport and accumulation of elements by R. aphinus
From the results of this study, R. alpinus has a strategy to exclude Fe, Mn, and Pb, although the concentration of some of the elements was high (e.g., Fe) in the soil. R. alpinus accumulated Mn only in MD and HM, even with the lowest plant-available concentration in the soil, perhaps because the plants used this element as essential. The situation was similar for Zn. In this case, the studied species exclude Zn in localities with high availability in the soil (e.g., in PC) and vice versa in localities with the lowest available Zn, rending them accumulators (Zhao et al. 2003). In localities with a low concentration of Zn in the soil, Zn levels in the plant organs were lower than localities with higher concentration: a similar result reported by Barrutia et al. (2009). R. alpinus seemingly exhibits accumulating strategy for Cu, but the trend was not the same in all localities. Notwithstanding, in LB and PC R. alpinus are accumulators only in the leaves during summer. However, in the remaining localities, accumulation occurred almost in all the organs, except for petioles in VT and MD. Precisely, BF of Cd in the emerging leaves have excluding strategy supporting accumulating strategy in the rhizome. Hence, this is a strategy to protect the most metabolic organ before toxicity, which can reduce biomass production, cause inhibition of cell elongation and division (Anton and Mathe-Gaspar 2005, Chen and Wong 2006, Barrutia et al. 2009, Vondráčková et al. 2014). In HM and VT, R. alpinus accumulated As in the organs in both terms, although BF was not significantly affected by the soil. A similar trend occurred in DCH. Meanwhile, at LB with the highest soil As concentration, accumulation occurred only during summer in the petiole. R. alpinus has tolerance for a wide range of soil chemical properties with generally a high As availability (Lorestani et al. 2011). According to the As concentration in individual organs, As moved the most in the petiole and senescent leaves. Hence, the plant can absorb and transport metals and store them in their senescent (above-ground biomass), the declining part (Baker 1981, Lorestani et al. 2011). In connection with the results of Vondráčková et al. (2015), where R. obtusifolius is a hyperaccumulator of Al, we expected a similar strategy with R. alpinus. However, the results showed that R. alpinus is an excluder of Al and can exudate chelating ligands, form a pH barrier at the rhizosphere, cell wall immobilization, and selective permeability of the plasma membrane.
The TF represents a phytoextraction parameter used to evaluate the capacity of each accession to translocate metals from rhizome to leaves. In our study, the mean TF found from all localities was higher than 1 for Zn, Cu, Mn, As, Ni and Pb. The BF results suggest an accumulation strategy for Cr, which accumulated in both terms at all localities except the petiole and rhizome at MD. However, TF for Cr was below 1, following the high concentration of these risk elements in the rhizome. Probably, high tolerance by R. alpinus to Cr has not confirmed accumulation strategy in all localities except MD.