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Geosciences 2018, 8, 56 7 of 18 2.5. Sample Preparation and Analysis Plants were harvested and washed with deionised water. The leaves from each plant were separated from the rest of the plant, and then dried in a forced air oven (WiseVen® Am Bildacker 16, 97877 Wertheim, Germany) at 60 ± 1 ◦C to a constant weight overnight. One g of leaf material from each plant was grinded and then acid digested. A reliable, conventional digestion method was used to digest all plant leaf samples. The method involved the pre-digestion of samples (overnight) in a mixture of HNO3 and H2O2 at a ratio of 4:1. Further digestion took place in an open vessel, heated on a hot plate (WiseStir® Am Bildacker 16, 97877 Wertheim, Germany) in the same mix at the same ratio for approximately 30 to 40 min until the plant material was brought into solution. This method cannot be said to constitute a complete digestion of materials but was internally consistent being replicated with all samples in the study. The digest was then filtered first using Whatman® (800 Centennial Avenue, Building 1, Piscataway, NJ 08854-3911, USA) grade 1 filter paper and then using a 0.45 μm syringe filter. The filtrate was brought to a constant volume of 100 mL in a volumetric flask with deionised water i.e., giving a dilution factor of 1/100 when analysed. The FES (Flame Emission Spectroscopy) instrument used was a Sherwood 410 Flame Photometer using a mixture of natural gas and air, operating at a temperature of 1700 to 1800 ◦C, suitable for Li, Na and K analysis. Li is routinely analysed by FES. Limit of Detection (LOD) for FES analysis was 0.13 mg/L while limit of quantification (LOQ) was 0.44 mg/L. After every 20 samples, the instrument was recalibrated using blank samples and working standards. Typical readings obtained from blank samples were 0.0001 to 0.0003 mg/L of Li. Li in plants and animals interacts with K and Na [69]. In this study we determined the K and Na content in all plants both as internal consistency standards and to investigate their intrinsic concentration. The accuracy of Li determinations using FES (670.8 nm) are not affected by the presence of K and Na which have intense spectrum lines at 589 and 767 nm respectively [83]. 2.6. Data Analysis Statistical analysis was carried out using SPSS® (Statistical Package for the Social Sciences, version 23.0, IBM Corp, Armonk, NY, USA) and Microsoft Excel® (2016 MSO 16.0.8625.2121). Where significant differences were found, a post-hoc t-test was used to identify significant differences between sample means. A p-value of 0.05 was considered significant. Bonferroni corrections were used as appropriate. 3. Discussion and Results 3.1. Germination Trials The results from the general germination trials were used as a means of selecting suitable plant species for the plant trials. In all cases, as the concentration of Li increased the germination rate decreased. Of the original 34 species, nine had total germination rates above 90%, germinating in deionised water along with increasing Li concentrations. These nine species were selected to continue to further germination trials, Brassica napus, Brassica oleracea var. capitate, Helianthus annuus, Solanum lycopersicum, Brassica hirta, Brassica rapa subsp. chinensis, Raphanus raphanistrum, Lepidium sativum and Cardamine hirsuta. These germination trials were carried out as before but included the addition of EDTA and EDDS along with Li. In the presence of EDTA germination was poor with only three species showing germination rates above 10%. In the presence of EDDS four species had germination rates above 90% and the other five above 55%. Based on the results of these trials, five species were selected to continue to plant trials, Brassica napus, Brassica oleracea var. capitate, Helianthus annuus, Solanum lycopersicum, and Cardamine hirsuta. 3.2. Plant Pot Trials Three main pot trials were carried out using the selected plant species. In each trial the soil of each group was amended with increasing concentrations of Li, to identify any potential natural LiPDF Image | Induced Plant Accumulation of Lithium
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