重金属分析：
土壤总量（三酸消解高压罐法）
1. 植物总量（Digestion of the parts of the different plant species was developed in a graphite
heating block at high temperature (DP). 0.2 g of dry sample was weighed in a polypropylene tube and 5 mL of HNO3 and 2mL of H2O2 were added. The mixture was subjected to a temperature-time program using a temperature ramp to 90 °C which it maintained for 4 h. After cooling, digested samples were made up with ultrapure water to 25 mL and filtered through 0.45 μm filter before analysis.
Appropriate dilutions were made for the analysis. To ensure the quality of the results, the same procedure was applied to certified reference materials (CRM) of plants: (i) Virginia Tobacco Leaves (CRM-CTA-VTL-2), whose total As content is 949±72 μg·kg−1; (ii)Peach Leaves (CRM-GBW-08501), with a total As concentration of 340±60 μg·kg−1. The obtained results were 951±10 and 344± 21 μg·kg−1 respectively, in good agreement with the certified values. Analysis were carried out by ICP-MS. Regarding calibration, the slope obtained for normal calibration was compared with those of standard addition method. Results indicated no matrix effects for all samples, so external calibration was used throughout this study.）
2. 土壤形态分析：Available arsenic was considered as the sum of water-soluble and
phosphate-extractable fractions (Larios et al., 2010). 1）水溶态
Water-soluble extraction: 0.5 g of soil sample was weighed in a polypropylene tube, 30 mL of ultrapure water were added and vortexed. After shaking 24 h at 35 rpm in an end-over-end shaker samples were centrifuged at 5000 rpm for 15 min. Supernatants were taken with Pasteur pipette, filtered through 0.45 μm pore paper filters and made up to 50 mL. 2）有效态：Phosphate-soluble extraction: 40 mL of 0.5 M sodium phosphate at pH 8 were added to the solid residue obtained from the water-soluble extraction and shaking for 8 h. Again supernatants were taken with Pasteur pipette, filtered through 0.45 μm pore paper filters and made up to 50 mL. The extracts were stored at 4 °C until analysis by ICP-AES. 3. 3）结晶态、非结晶；4）连续提取
4. 植物形态分析：1）价态：According to Bohari et al. (2001) a 0.3 M orthophosphoric acid
solution was selected to extract arsenic species from plants without altering its speciation. Two extraction procedures were compared: microwave assisted extraction (MW) and extraction in a graphite digestor block at high temperature (DP). Extractions were conducted with the plant species Calluna Vulgaris leaves material with an arsenic concentration of 359±2 mg·kg-1. A mass of 0.2 g of dry sample was mixed with 25 mL of extracting agent in a polypropylene tube. After the extraction program, samples were allowed to cool to room temperature and centrifuged. After 20 min at 4000 rpm all the supernatants were diluted up to 50 mL. Following the recommendations given by Vergara-Gallardo et al. (2001) a tenfold secondary dilution was carried out to preserve the speciation. The samples were filtered through a 0.2 μm filter before chromatographic analysis. Digestion systems were programmed to heat the sample at a specific temperature and maintaining it for a specified time. Tested conditions for each procedure are included in Fig. 2. Although there is no reference material certified for arsenic speciation, the finally selected extraction method was also carried out with the CRM Virginia Tobacco Leaves CTA-VTL-2 to evaluate the analytical methodology. Total contents of the extracts were analyzed by means of ICP-MS.
The separation and determination of arsenic species in the orthophosphoric extracts was performed by HPLC-ICP-MS. The four more common arsenic species in plants (As (III), As (V), DMA and MMA) and the internal standard were completely separated in a Hamilton PRP-X100 anion-exchange column (250×4.1 mm, id 10 μm). The mobile phase and the elution program were selected and performed according to Milstein et al. (2002). Two TRIS acetate solutions, 30 and 100 mM, buffered at pH=7, were used as eluent and the gradient elution program: from 0 to 7 min, ramp from 30 mM to 100 mM; from 7 to 12 min, hold at 100 mM; at 12.5 min, step to 30 mM. Flow rate: 1.5 mL·min−1. Injected volume: 200 μL. All species were eluted in less than 13 min. Since the results achieved on speciation by
external calibration agree with those obtained by standard additions method, it was enough to use external calibration.
采样：
The most abundant plant species were collected from the studied mining sites, as well as their corresponding soils.
After collection, each plant sample was vigorously washed with tap water and rinsed with deionized water. They were divided in aboveground mass and roots; leaves and stalks were separated when it was possible (as was the case for Calluna vulgaris and Dryopteris affinis. In Calluna vulgaris woody and radicle roots were also sorted. Then, they were air-dried for 7 days to ensure constant weight and afterward ground using the stainless steel mill “IKA A 11 Basic” to pass a 0.5mm sieve.
Soils corresponding to each plant were collected at 25 cm deep when possible, and stored in polyethylene bags. Samples were disaggregated in the laboratory and the non-mineral material and rock fragments higher than 2 mm were discarded. Then, they were oven dried at a temperature below 40 °C to minimize loss of volatile elements (7–15 days), sieved and ground in an agate mortar to a size finer than 63 μm.
在矿区：筛选高积累和低积累植物及其微生物种群或丛植菌根
