The Potential of Cistus salviifolius L. to Phytostabilize Gossan Mine Wastes Amended with Ash and Organic Residues.

The São Domingos mine is within the Iberian Pyrite Belt, a mining district with large concentrations of polymetallic massive sulfide deposits. Mine waste heaps are considered extreme environments, since they contain high total concentrations of potentially hazardous elements (PHE), which contribute to inhibiting the development of most plants. Autochthonous plant species, such as Cistus salviifolius L., are able to grow naturally in this degraded environment, and may contribute to minimizing the negative chemical impacts and improving the landscape quality. However, the environmental rehabilitation processes associated with the development of these plants (phytostabilization) are very slow, so the use of materials/wastes to improve some physicochemical properties of the matrix is necessary in order to speed up the process. This work studied the effectiveness of the phytostabilization with C. salviifolius of gossan mine wastes from the mine of São Domingos amended with organic and inorganic wastes in order to construct Technosols. The mine wastes have an acid pH (≈3.5), high total concentrations of PHE and low concentrations of organic C and available nutrients. The best vegetative development occurred without visible signs of toxicity in the Technosols containing a mixture of agriculture residues. These treatments allowed the improvement of the soil-plant system providing a better plant cover and improved several chemical properties of mine wastes, helping to speed up the environmental rehabilitation.

Use of combined tools for effectiveness evaluation of tailings rehabilitated with designed Technosol.

Soil and water characteristics and biogeochemical processes can be improved by the application of an integrated technology based on circular economy: designed Technosol. The evaluation of the effectiveness of the superficial application of a designed Technosol, with andic and eutrophic properties, on the rehabilitation of sulfide tailings of a uranium mine (Fé mining area, Spain) was the aim of this study. After 20 months of the Technosol application, the tailing rehabilitation status (Rehabilitated tailing) was compared to a non-rehabilitated tailing (Tailing). To assess the rehabilitation of these systems, several properties were analyzed: chemical characteristics of the materials and their leachates, soil enzymatic activities (dehydrogenase, ß-glucosidase, acid phosphatase and urease), basal respiration and several plant endpoints from direct and indirect bioassays and pot experiment using Lolium perennse L. and Trifolium pratense L.. Potentially toxic concentrations of Co, Mn and Ni were identified in both available fraction and leachates, pointing out the serious environmental risk posed by the tailing. The improvement of overall physicochemical properties in the rehabilitated tailing materials (e.g., decrease of the hazardous element concentrations in leachates and available fraction, and improvement of the fertility and structure) allowed a quick plant cover with pasture species and provided a suitable habitat for active microbial community (evaluated by increasing dehydrogenase activity and basal respiration). This improvement in the rehabilitated tailing contributed to a significant decrease in the ecotoxicological risk and the spread of hazardous elements. The field application of this specific Technosol was a promising and lasting solution for rehabilitation of this type of tailings.

Influence of Seed Source and Soil Contamination on Ecophysiological Responses of Lavandula pedunculata in Rehabilitation of Mining Areas.

Mining activities have turned many areas of the Iberian Pyrite Belt (IPB) into extreme environments with high concentrations of metal(loid)s. These harsh conditions can inhibit or reduce the colonization and/or development of most vegetation. However, some species or populations have developed ecophysiological responses to tolerate stress factors and contaminated soils. The main objectives of this study are: (i) to assess the differences in germination, growth, development and physiological behaviour against oxidative stress caused by metal(loid)s in Lavandula pedunculata (Mill.) Cav. from two different origins (a contaminated area in São Domingos mine, SE of Portugal and an uncontaminated area from Serra do Caldeirão, S of Portugal) under controlled conditions; and (ii) to assess whether it is possible to use this species for the rehabilitation of mine areas of the IPB. After germination, seedlings from São Domingos (LC) and Caldeirão (L) were planted in pots with a contaminated soil developed on gossan (CS) and in pots with an uncontaminated soil (US) under controlled conditions. Multielemental concentrations were determined in soils (total and available fractions) and plants (shoots and roots). Germination rate, shoot height, dry biomass and leaf area were determined, and pigments, glutathione, ascorbate and H2O2 contents were measured in plant shoots. Total concentrations of As, Cr, Cu, Pb and Sb in CS, and As in US exceed the intervention and maximum limits for ecosystem protection and human health. The main results showed that L. pedunculata, regardless of the seed origin, activated defence mechanisms against oxidative stress caused by high concentrations of metal(loid)s. Plants grown from seeds of both origins increased the production of AsA to preserve its reduction levels and kept the contents of GSH stable to maintain the cell's redox state. Plants grown from seeds collected in non-contaminated areas showed a high capacity for adaptation to extreme conditions. This species showed a greater growth capacity when seeds from a contaminated area were sown in uncontaminated soils. Thus, L. pedunculata, mainly grown from seeds from contaminated areas, may be used in phytostabilization programmes in areas with soils with high contents of metal(loid)s.

Improvement of chemical quality of percolated leachates by in situ application of aqueous organic wastes on sulfide mine tailings.

One of the major environmental concerns in the mining industry is the generation of acid leachates from tailings deposits, which are highly concentrated in potentially hazardous elements. The continuous processing of these leachates in treatment plant is unsustainable, so the in situ chemical improvement of the mine wastes and their leachates, mainly with another waste produced in the mining area, can reduce treatment and operational costs. This study aimed to evaluate the effect of two types of domestic wastewaters (DWW) on the improvement of the chemical characteristics of the leachates generated from mine wastes containing sulfides. A mesocosm assay was performed under greenhouse and controlled conditions with mine wastes collected in La Zanja mining area (Peru). Three irrigation treatments were tested: untreated DWW, treated DWW and water as control. Percolated leachates of each treatment were collected once per week, for a period of 10 weeks. Electrical conductivity, pH and multi-elemental concentration were analysed. During the assay, the mine wastes generated acid leachates (≈4) with significant concentrations of elements (mg/L; Al: 1.4-30.0; Cd: 0.05-0.19; Cu: 5.7-22.1; Fe: 1.6-19.4; Mn: 2.6-26.0; Zn: 1.2-9.2) and sulfates (204.3-997.8 mg/L), which exceed the thresholds established by Peruvian legislations. After DWW application, pH in the leachates increased to ≈7 and concentrations of several studied elements (e.g. Al, As, Fe, Cu, Zn, Cd, Pb, Ni, Mn) and sulfates decreased (>70% depending on DWW type, element and sampling) compared to the control. This fact allowed that the Environmental Quality standards from Peru (except for Cu and Mn) were reached. However, an enrichment of Na and K was obtained at the same leachates. At short term, the DWW application (especially untreated) on the mine wastes containing sulfides was effective in the improvement of the general chemical quality of their leachates. Moreover, the combined management of these two studied wastes (domestic wastewater and mine wastes) represents a promising cost-effective strategy during mining operation.

Rehabilitation of mining areas through integrated biotechnological approach: Technosols derived from organic/inorganic wastes and autochthonous plant development.

In order to restore the plant cover, improve ecosystem services and decrease the environmental risk of two mine wastes (gossan and sulfide-rich wastes), an integrated biotechnology was tested at long-term and greenhouse conditions. This integrated biotechnology involves the natural isolation of sulfide-rich wastes through an alkaline barrier, covered by designed Technosols (both of them derived from mining and agro-industrial wastes) and a plant cover with Lavandula pedunculata and Cistus ladanifer. Technosols allowed significant germination (L. pendunculata: 16-18%; C. ladanifer: 5-11%) and biomass production of both species (g FW/pot; Roots: 16.3-30.9, Shoots: 41.2-76.4 depending on species and Technosol). In the control was reached the lowest germination (<3%) and seedlings died after 40 days, so the improvement of the chemical characteristics of the surface layer, i.e. the implementation of the designed Technosols, is essential to ensure good vegetative development. No visual symptoms of nutritional deficiency and phytotoxicity neither element concentrations above hazardous levels for domestic animals intake were observed in those plants species. The alkaline barrier's components stabilise the sulfide-rich wastes by decreasing the oxidation and capillary rise of acid solutions that are rich in metals/metalloids. The limestone gravel showed, in some places, a thin layer of salts from alunite-jarosite group and metal-oxyhydroxides. As an outcome, the biotechnology was efficient and sustainable allowing the combined rehabilitation of both mine wastes at long-term.

Unraveling the crucial role of the ascorbate-glutathione cycle in the resilience of Cistus monspeliensis L. to withstand high As concentrations.

Cistus monspeliensis L. is a species that grows spontaneously in contaminated mining areas of the Iberian Pyrite Belt. This species can accumulate high concentrations of As in the shoots without visible signs of phytotoxicity. In order to understand the physiological mechanisms underlying this tolerance, C. monspeliensis was grown in an Arenosol irrigated with aqueous nutrient solutions containing increasing concentrations of As (0, 1500, 5000, 10000, 15000 µM) and the effects of this metalloid on plant development and on the defence mechanisms against oxidative stress were monitored. Independently of the treatment, As was mainly retained in the roots. The plants with the highest concentrations of As in the shoots (> 5000 µM) showed toxicity symptoms such as chlorosis, low leaf size and decrease in biomass production and also nutritional deficiencies. Most of the studied physiological parameters (pigments, glutathione, ascorbate and antioxidative enzymes) showed significant correlation with As concentration in roots and shoots. Pigments, especially anthocyanins, were negatively affected even in the treatments with the lowest As concentrations. Glutathione increased significantly in roots at low As levels while in shoots this increase occurred in all As treatments. Ascorbate decreased in both tissues with As addition. The highest concentrations of As in shoots of C. monspeliensis triggered defence mechanisms against oxidative stress, namely by inducing the expression of genes coding antioxidative enzymes.

Cistus monspeliensis L. as a potential species for rehabilitation of soils with multielemental contamination under Mediterranean conditions.

The Iberian Pyrite Belt (IPB; SW of the Iberian Peninsula) is one of the most important volcanogenic massive sulphide ore deposits in the world. Cistus monspeliensis L. is a native woody shrub that grows spontaneously in non-contaminated soils as well as in soils with multielemental contamination from the IPB. In this study, different ecophysiological parameters of C. monspeliensis growing in soils with different levels of metal(loid)s were evaluated to assess the potential of this species for revegetation of degraded areas. Composite samples of plants and rhizosphere soils were sampled in São Domingos and Lousal mines and in a reference area without soil contamination (Pomarão, Portugal) (Portuguese sector of IPB). Classical characterisation of the soils and quantification of their total and available metal(loid) concentrations were done. Multielemental concentration was determined in plants (shoots and roots). Ecophysiological parameters were also determined in shoots: concentrations of pigments (chlorophylls, anthocyanins and carotenoids), antioxidants (glutathione and ascorbate) and hydrogen peroxide as well as activities of several antioxidative enzymes. Although mining soils present high total concentrations of potentially hazardous elements, their available fractions were low and similar among studied areas. Soil pH as well as concentrations of extractable P, total concentrations of As, Cd and Ni and concentrations of Cu, Cr, Ni, Pb and Sb in the soil available fraction differentiate the studied areas. Only concentrations of Cd, Pb and Sb in roots and shoots were explained by the concentrations of the same elements in the soil available fraction. Although the majority of elements were translocated from roots to shoots, the shoots concentrations were below the toxic values for domestic animals and only As, Mn and Zn reached phytotoxic concentrations. Ecophysiological parameters were similar independently of the studied area. Due to its adaptability, tolerance and standard plant features, C. monspeliensis is a good choice for rehabilitation of soils with multielemental contamination under similar climatic characteristics.

Mycorrhizal Inoculation Differentially Affects Grapevine's Performance in Copper Contaminated and Non-contaminated Soils.

Plant inoculation with arbuscular mycorrhizal fungi (AMF) is increasingly employed to enhance productivity and sustainability in agricultural ecosystems. In the present study, the potential benefits of AMF inoculation on young grapevines replanted in pots containing vineyard soil with high Cu concentration were evaluated. For this purpose, one-year-old cv. Touriga Nacional grapevines grafted onto 1103P rootstocks were further inoculated with Rhizoglomus irregulare or Funneliformis mosseae, or left non-inoculated, and maintained in a sterilized substrate under greenhouse conditions for three months. After this time, half of the plants were transplanted to containers filled with an Arenosol from a vineyard which had been artificially contaminated or not with 300 mg kg-1 of Cu. At the end of the growing season, soil nutrient concentration, soil dehydrogenase activity and mycorrhizal colonization rate were analyzed. Grapevine performance was assessed by measuring several vegetative growth and physiological parameters as well as nutrient concentrations in leaves and roots. In the non-contaminated soil, R. irregulare- and F. mosseae-inoculated plants had significantly greater root biomass than the non-inoculated ones. However, the opposite effect was observed in the Cu-contaminated soil, where non-inoculated plants performed better regarding shoot and root development. Concerning nutrient levels, an increase in Cu, Mg and Mn concentrations was observed in the roots of plants growing in the contaminated soil, although only Mn was translocated to leaves. This led to a large increase in leaf Mn concentrations, which was significantly higher in non-inoculated and F. mosseae- inoculated plants than in the R. irregulare- inoculated ones. Copper contamination induced a general decrease in leaf N, P and Fe concentrations as well as chlorosis symptoms. The largest decrease in N and P was observed in F. mosseae- inoculated plants, with 73 and 31.2%, respectively. However, these plants were the ones with the least decrease in Fe concentration (10% vs. almost 30% in the other two inoculation treatments). In conclusion, this study indicates that soil Cu levels can modify the outcome of AMF inoculations in young grapevines, disclosing new AMF-plant associations potentially relevant in vineyards with a tradition of Cu-based fungicide application.

The physiological mechanisms underlying the ability of Cistus monspeliensis L. from São Domingos mine to withstand high Zn concentrations in soils.

Cistus monspeliensis L. is a species that grows spontaneously in contaminated mining areas from the Iberian Pyrite Belt. This species can have high concentrations of Zn in the shoots without visible signs of phytotoxicity. In order to understand the physiological mechanisms underlying this tolerance, C. monspeliensis was grown at several concentrations of Zn(2+) (0, 500, 1000, 1500, 2000µM) and the effects of this metal on plant development and on the defence mechanisms against oxidative stress were evaluated. Independently of the treatment, Zn was mainly retained in the roots. The plants with the highest concentrations of Zn showed toxicity symptoms such as chlorosis, low leaf size and decrease in biomass production. At 2000µM of Zn, the dry biomass of the shoots decreased significantly. High concentrations of Zn in shoots did not induce deficiencies of other nutrients, except Cu. Plants with high concentrations of Zn had low amounts of chlorophyll, anthocyanins and glutathione and high contents of H2O2. The highest concentrations of Zn in shoots of C. monspeliensis triggered defence mechanisms against oxidative stress, namely by triggering antioxidative enzyme activity and by direct reactive oxygen species (ROS) scavenging through carotenoids, that are unaffected by stress due to stabilisation by ascorbic acid.

Mutielemental concentration and physiological responses of Lavandula pedunculata growing in soils developed on different mine wastes.

This study aimed to: i) evaluate the accumulation and translocation patterns of potentially hazardous elements into the Lavandula pedunculata and their influence in the concentrations of nutrients; and ii) compare some physiological responses associated with oxidative stress (concentration of chlorophylls (Chla, Chlb and total), carotenoids, and total protein) and several components involved in tolerance mechanisms (concentrations of proline and acid-soluble thiols and total/specific activity of catalase (CAT) and superoxide dismutase (SOD)), in plants growing in soils with a multielemental contamination and non-contaminated. Composite samples of soils, developed on mine wastes and/or host rocks, and L. pedunculata (roots and shoots) were collected in São Domingos mine (SE of Portugal) and in a reference area with non-contaminated soils, Corte do Pinto, with the same climatic conditions. São Domingos soils had high total concentrations of several hazardous elements (e.g. As and Pb) but their available fractions were small (mainly <5.8 % of the total). Translocation behaviour of elements was not clear according to the physiological importance of the elements. In general, plant shoots from São Domingos had the highest elements concentrations, but only As, Mn and Zn reached phytotoxic concentrations. Concentration of Chlb in shoots from São Domingos was higher than those from Corte do Pinto. No significant differences were obtained between concentrations of Chla, total protein, proline and acid-soluble thiols in shoots collected in both areas, as well as SOD activity (total and specific) and specific CAT activity. Total CAT activity varied with population being lower in the shoots of the plants from São Domingos, but no correlation was obtained between this enzymatic activity and the concentrations of the studied elements in shoots. Lavandula pedunculata plants are able to survive in soils developed on different mine wastes with multielemental contamination and low fertility showing no symptoms (visible and physiological) of phytotoxicity or deficiency.

Evaluation of chemical parameters and ecotoxicity of a soil developed on gossan following application of polyacrylates and growth of Spergularia purpurea.

The aim of this study was to evaluate the chemical characteristics and ecotoxicity of a mine soil developed on gossan materials and amended with hydrophilic polyacrylate polymers after a growth cycle of Spergularia purpurea. Different acute bioassays (Daphnia magna immobilization; microalgae growth inhibition; germination and growth of lettuce and oat) were carried out with simulated leachates, pore water and soil samples. The germination and growth of native shrubs (Cistus ladanifer and Lavandula sampaioana) were also evaluated in the lysimeters where S. purpurea had grown. The soil had high total concentrations (g/kg) of Al (3.50-8.60), As (2.55-2.73), Cu (0.13-0.91) and Pb (4.48-6.16). However, the percentages of elements in aqueous extracts (simulating leachates, pore water, and the conditions of the rhizosphere soil) were small when compared to their total soil concentrations (less than 9% except for Na in leachates). Growth of S. purpurea and other natural colonization of plant species (Poaceae, Fabaceae and Asteraceae families) improved chemical characteristics but the application of the polyacrylate polymers contributed to a further improvement of soil quality. However, this was not sufficient to ensure the growth of a large number of shrubs despite a great germination rate. Among the several species used on the ecotoxicological assessment, the D. magna test was the only bioassay that showed a clear toxicity of soil leachates, suggesting the importance of using several ecotoxicological tests to assess the environmental risk of soil contamination and its rehabilitation. Although the studied soil can be considered contaminated taking into account the total soil concentrations of Al, As, Cu and Pb, the low concentrations of the same chemical elements in extractable solutions, that simulated the fractions really available for organisms, did not demonstrate a substantial toxic effects in the organisms and, consequently, negative impact on the environment.

Trace elements and activity of antioxidative enzymes in Cistus ladanifer L. growing on an abandoned mine area.

The Mediterranean shrub Cistus ladanifer grows naturally in São Domingos (Portugal), an abandoned copper mine. High levels of trace elements in plants can generate oxidative stress increasing the activity of antioxidant enzymes. The aim of this work was to evaluate and compare As, Cu, Pb and Zn concentrations and the activity of the soluble and cell wall ionically bounded forms of the enzymes catalase, peroxidase and superoxide dismutase in leaves of C. ladanifer, collected in spring and summer, growing on São Domingos mine and on a non-contaminated area (Pomarão). São Domingos soils showed high total concentrations of As (2.6 g kg(-1)) and Pb (7.3 g kg(-1)) however the available fraction represented less than 1.5% of the total. C. ladanifer population from mine showed tolerance to Pb and Zn, which attain in leaves concentrations considered toxic for plants. The enzymatic activity of catalase, peroxidise and superoxide dismutase varied with plant populations and seasons, although with no particular trend, being specific to each trace element and enzyme cell localization. Catalase activity was evenly distributed between the soluble and ionically bounded forms, whereas the ionically bounded form of peroxidase predominated relatively to total activity, and the opposite was observed for superoxide dismutase. Spring and summer leaves from the two areas presented enzymatic activities in both fractions except to peroxidase soluble activities in leaves collected in summer. C. ladanifer enzymatic activity seems to be related with the co-existence of different stress factors (trace elements concentration, temperature, UV radiation and drought). The survival and growth of this species on contaminated mining soils is due to the presence of effective antioxidant enzyme-based defence systems.