Chemical soil characteristics
Chemical characteristics of the soils are shown in Table 1. Mine soils were developed on heterogeneous mixtures of host rocks, influenced by acid mine drainage and/or different waste materials, which consequently influenced the characteristics of the soils. Due to this heterogeneity of the materials, chemical characteristics of soils from mining areas presented, in general, a wide range of values.
The pH values of the soils from both mine areas are very acid-to-acid due to mine wastes from which they were developed. These pH values were significantly lower than those from Pomarão. Independently of the studied area, no significant differences were found among electrical conductivities as well as the concentrations of total N, organic C and extractable K. However, concentrations of extractable P in soils from São Domingos and Lousal mines were lower than in soils collected in Pomarão (Table 1).
The soils from São Domingos and Lousal mine had very high total concentrations of As (only São Domingos), Cu, Pb and Sb, which are in contrast with the total concentrations of the same elements in the soils collected in Pomarão. Besides, the highest total concentration of Zn was obtained in Lousal soils while the highest total concentrations of Mn were found in soils from Pomarão. No significant differences were observed between the concentrations of Cr, Ni and Cd in the different studied areas (Table 1).
According to different reference guidelines for metal(loid) levels in soils (CCME 2007; VROM 2009), the total concentrations of As, Sb, Cu, Cr, Pb and Sb in soils from both mine areas (Table 1) exceeded the intervention values and maximum permitted levels for the protection of ecosystems and human health as well as commercial and industrial land use. The total concentrations of metal(loid)s in the soils from Pomarão (reference area) did not exceed those levels, except for As and Cr (CCME 2007). Nonetheless, the concentrations of these elements are within the range of values for non-contaminated soils from region and developed on the same geological substratum (Abreu et al. 2008, 2012b; Santos et al. 2012; Tavares et al. 2008).
Although the total concentrations of the elements in the mine soils were higher, compared to those in Pomarão soils, the element concentrations in the available fraction of the soils were low (< 13.3% of the total concentrations) independently of the studied area. Moreover, no significant differences in the concentrations of As, Cd, Cr, Ni, Pb and Zn in the available fraction were obtained among the three studied areas, although some soils from São Domingos and Lousal can reach higher concentrations compared to Pomarão soils (Table 1). The concentrations of Cu and Sb in the available fractions of the mine soils were significantly higher than those in Pomarão soils. Besides, Mn concentrations in the available fraction of soils from Pomarão and Lousal were significantly higher than those in soils of São Domingos (Table 1).
The analysis of the PCA for soil characteristics (Fig. 1a) led to a reduction of the initial dimension of the dataset to two components, which explain 55.2% of the data variation (PC1 22.7%; and PC2 32.49% of the variance). The PC1 indicates that pH affects negatively the available contents of Cu, Sb and Pb in soils while available concentrations of Mn and Ni can be related to their total concentrations. Through PCA analysis, it was possible to obtain a clear separation of the studied areas. Thus, the soils from Pomarão, with high values of pH and extractable P contents as well as low concentrations of As, Cd and Ni in the total fraction and Cu, Cr, Ni, Pb and Sb in the available fraction, are differentiated from the mine soils, which have opposite characteristics. Within soil mines, Lousal soils are grouped especially by their high total concentrations of Ni and the concentrations of Cr and Ni in the available fraction, while São Domingos soils are distinguished mainly by their high total concentrations of As, Cd and Pb in the total fraction and the concentrations of As and Pb in the available fraction.
In general, concentrations of metal(loid)s in the total and available fractions as well as other chemical properties of the soils are in agreement with the range of values obtained in previous studies performed in the same areas (Abreu et al. 2008, 2012a, b; Alvarenga et al. 2012; Batista et al. 2017; Ferreira da Silva et al. 2005; Freitas et al. 2004; Pérez-López et al. 2014; Santos et al. 2012, 2014, 2016c).
Concentrations of metal(loid)s in plants
The concentrations of metal(loid)s in shoots and roots of C. monspeliensis are shown in Table 2. Independently of the area, the concentrations of the elements in roots and shoots were higher than the concentrations of the same elements in the available fraction of the soils (Table 1), except for Sb in roots. In general, the concentrations of metal(loid)s in shoots and roots in C. monspeliensis growing in both mines showed a great heterogeneity, as also observed for other Cistus species growing in mining areas from the IPB (e.g. Abreu et al. 2012a, b; Santos et al. 2012, 2014 and references therein).
Cistus monspeliensis from Pomarão showed the highest concentrations of Cr and Ni in roots and Ni, Cr and Mn in shoots. However, concentrations of As, Cu, and Sb in roots and As and Zn in shoots from São Domingos and Cd and Zn in shoots from Lousal were higher than those in Pomarão (reference area). Similar behaviour was observed in other species growing in contaminated and non-contaminated areas from the IPB, as Cistus ladanifer L. (As and Zn in shoots), Cistus salviifolius L. (e.g. As and Sb in shoots and roots) and Lavandula pedunculata (Mill.) Cav. (Abreu et al. 2012a; Santos et al. 2012, 2016c; Trigueros et al. 2012), as well as in Erica andevalensis (Cabezudo & J. Rivera) and Erica australis L. (Abreu et al. 2008; Pérez-López et al. 2014).
The PCA analysis (Fig. 1b) done to assess the possible relationship between the concentrations of metal(loid)s in the soil available fraction, and roots and shoots of C. monspeliensis can explain 52.9% of the data variation. The PC1, which explains 33.0% of the variance, shows that the concentrations of Pb and Sb in roots and shoots can be explained by the concentrations of the same elements in the available fraction of the soils. The same was obtained for Cd in PC2, which explains 19.9% of the variance. Also, PC2 shows a possible synergistic interaction Cd–Zn as reported by Kabata-Pendias (2011).
Intra- and inter-population differences were observed in the translocation behaviour (Table 3) of the elements in the plants. In general, plants from the three populations mainly translocated As, Cd, Cu, Mn, Ni, Sb and Zn from roots to shoots (Translocation coefficient > 1). This translocation behaviour differ to other species of the genus Cistus, such as C. populifolius, C. salviifolius and C. ladanifer, which mainly accumulated metal(loid)s in roots (Abreu et al. 2012a, b; Alvarenga et al. 2004; Santos et al. 2014). However, in general, the concentrations of the studied elements in C. monspeliensis shoots from the three populations were below the toxicity limit and/or within the range considered sufficient/normal for plants, except for As in plants from São Domingos, and Mn and Zn in plants from the three areas (Table 2) which present values considered as phytotoxic (Kabata-Pendias 2011). Despite these concentrations, no visual symptoms of toxicity were observed (data not shown). Moreover, an additional important aspect is that elemental concentrations in the shoots were below the toxicity limits for domestic animals (NRC 2005) and did not represent any environmental risk.
Otherwise, plants from Pomarão mainly stored Cr and Pb in roots (Translocation coefficient < 1). The storage in roots and/or decrease of the translocation of the potentially hazardous elements from roots to shoots can be considered a tolerance mechanism (Abreu et al. 2014; Hossain et al. 2012).
Taking into account the few published studies on the concentrations of potentially toxic elements in C. monspeliensis (Batista et al. 2017; De la Fuente et al. 2010; Freitas et al. 2004) (Table 2), C. monspeliensis shoots present a wide range of element concentrations. Nonetheless, most of the element concentrations obtained in the present study for C. monspeliensis are in the same range than for other species of the genus Cistus (e.g. As in C. salviifolius shoots from São Domingos, Cu in C. ladanifer roots from Lousal) growing in the same mine areas (Abreu et al. 2012a, b; Freitas et al. 2004; Santos et al. 2009, 2012, 2014).
Concerning the plant accumulation behaviour, evaluated by the soil–plant transfer coefficient (Table 3), plants from the three populations were Zn, Mn and Cd accumulators but not hyperaccumulators. For the other studied elements and independently of the population, the plants can be considered non-accumulators.
Concentration of pigments in leaves
Pigment concentrations in the leaves of C. monspeliensis are shown in Fig. 2a, b, c. In general, the excess of potentially hazardous elements in leaves can modify the concentration of pigments, which are usually associated to visual symptoms of plant disease and impaired photosynthetic activity (Kabata-Pendias 2011; Márquez-García and Córdoba 2009; Pang et al. 2003; Santos et al. 2016c; Tewari et al. 2008). However, independently of the population, no visual alteration in leaf colour was observed.
Although intra-population variation can be pointed out, no significant differences were obtained between the concentrations of chlorophylls (a, b and total), anthocyanins and carotenoids in the leaves from the three populations (Fig. 2a, b, c). Similar results were observed between contents of carotenoids in leaves of E. australis, C. ladanifer and L. pedunculata collected in different mining areas from IPB and in non-contaminated areas (Márquez-García and Córdoba 2009; Santos et al. 2013, 2016c).
A PCA was carried out to evaluate the possible influence of the contents of metal(loid)s on pigments in C. monspeliensis shoots (Fig. 1c), which was determined only for PC1 (43.99% of variance). The results showed that only Cd concentrations in shoots can affect negatively the concentrations of all studied pigments. Thus, the low contents of chlorophylls, anthocyanins and carotenoids in C. monspeliensis in the three studied areas might be attributed to the high level of solar radiation, air temperature and low humidity, stress factors associated to the Mediterranean conditions that occur in these areas (Correia 2002; Santos et al. 2013).
Concentration of H2O2
Hydrogen peroxide content in the shoots of C. monspeliensis is shown in Fig. 2d. Plants under normal physiological conditions produce significant amounts of H2O2 as a by-product of their metabolism and, under various stress factors, namely high concentrations of metal(loid)s, H2O2 levels tend to increase due to its speed of formation exceed the capacity for scavenging (Caverzan et al. 2012). On the other hand, plants can eliminate H2O2, through detoxification mechanisms, in order to limit the peroxidation reactions of the membrane lipids (Howlett and Avery 1997). The lowest levels of H2O2 in C. monspeliensis from mining areas, especially in some plants from São Domingos (Fig. 2d) can suggest the rapid elimination of this compound.
Comparing the studied populations, no significant differences were obtained due to the high variability of H2O2 concentrations in C. monspeliensis. Similar H2O2 concentrations were also reported in leaves of E. australis growing in mine wastes and uncontaminated soils from Spanish IPB (Márquez-García and Córdoba 2009). The PCA analysis indicates that this ecophysiological parameter is not explained by the concentrations of the studied metal(loid)s in the shoots.
Antioxidative enzymes and antioxidant molecules
Ascorbate and glutathione contents in the leaves of C. monspeliensis are shown in Fig. 3. No significant differences in the concentrations of ascorbate and glutathione were obtained among plants of the three studied populations. Similar concentrations of glutathione in leaves of P. lanceolata and C. arenosa from contaminated and non-contaminated areas were also reported by Nadgórska-Socha et al. (2013).
When assessing the levels of reduced and oxidised ascorbate (AsA and DAsA, respectively) in leaves of C. monspeliensis from the three studied areas (Fig. 3), the reduction state was high in all cases. Generally, the maintaining of a high percentage of AsA is essential for the proper scavenging of ROS in cells (Mittler 2002), so the results obtained for ascorbate are a good indication of the cell’s redox state. The percentages of AsA reduction in the three populations was in the same range varying between 73.1 and 97.1%. Nevertheless, the reduction state of glutathione (GSH) was generally low and the only parameter significantly lower in plants from Lousal (39.5–46.1%) and São Domingos (47.2–57.2%) than in plants collected in Pomarão (58.8–78.5%). These results can indicate that the plants from mines can be under oxidative stress that impaired the normal functioning of the reduction cycle of glutathione.
Activities of antioxidative enzymes in the leaves of C. monspeliensis are shown in Fig. 4. In general, under oxidative stress, plants can also stimulate the activity of antioxidative enzymes, which remove and neutralise ROS (Pang et al. 2003; Santos et al. 2009). However, no significant differences in the antioxidative enzyme activities were obtained among the studied populations. These results suggest that C. monspeliensis plants from the three studied areas are able to adapt their enzyme activities and concentrations of antioxidant molecules to the concentrations of metal(loid)s in their shoots, showing high tolerance to these elements. Therefore, the potential toxicity caused by toxic elements did not trigger the activities of antioxidative enzymes. Similar activities of some antioxidative enzymes were also observed in E. australis (e.g. CAT and APX), C. ladanifer (e.g. SOD), L. pedunculata (e.g. SOD) and P. lanceolata (e.g. SOD) and C. arenosa (e.g. SOD) growing in soils affected and not affected by multielemental contamination of the mining activity (Márquez-García and Córdoba 2009; Nadgórska-Socha et al. 2013; Santos et al. 2009, 2016c).