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Geosciences 2018, 8, 56 6 of 18 supply were characterised for Li content. Soil samples were thermally acid digested in aqua regia (HCl and HNO3) at a ratio of 1:1 for 2 h, filtered and then analysed for their Li content using FES. The x Li concentration measured in soil samples was 20.9 mg/kg SD = 8.2 mg/kg ranging from 9 to 34 mg/kg. Soil was dried and then sieved (4 mm) for pot experiments. Approximately 2 kg of soil was transferred to polyethylene pots (15 cm diameter and 15 cm depth). All plants in these trials were grown in a large greenhouse maintained at a relative humidity of 69.5% (day/night) and an air temperature of 26 ± 3 ◦C. Soils used in these experiments was artificially spiked with Li. Li as LiCl was made up in deionised water at each concentration of Li required (i.e., of 0, 20, 50, 100, 150, 200, 300, 400, 500 and 1000 mg/kg). Each plant species tested required 100 pots (i.e., 10 pots at each concentration). 10 pots required around 20 kg of soil. Groups of 20 kg of soil were saturated with a specific Li solution, mixed allowed to dry and then the process repeated until testing of the soil for Li concentration showed that the soil was ±5 mg/kg the desired concentration. These soils were then transferred to pots. All pots for each trial were treated this way. This method of ‘spiking’ the soil with Li was adapted from other works [81]. 2.4. Plant Trials Initial plant trials consisted of 100 plants of each of the 5 species (Brassica napus, Brassica oleracea var. capitata, Helianthus annuus, Solanum lycopersicum and Cardamine hirsuta) planted in Li amended soils. Plants were grown in soils amended with Li at levels of 20, 50, 100, 150, 200, 300, 400, 500 and 1000 mg/kg with 10 control plants grown in untreated soil. 10 plants were grown (one plant per pot) at each concentration, totalling 10 groups of 10 plants for each of the 5 species (N = 500 initial plants, N = 500 pots). Plants were grown for approximately 4 months to maturity and then harvested. The plants were watered on alternate days and fertilised once a week with nutrients in the form of Miracle grow® a commercially available water soluble all-purpose plant food. The study was conducted in a screened greenhouse at the Institute of Technology Carlow, Ireland. TomtechHC80 environmentally controlled research greenhouse facility, available from TOMTECH, Lincolnshire, England. Screens provided thermal protection at night and shading during the day. Plants were grown with a cycle of 16 h light and 8 h dark each day, with a constant light phase temperature of 26 ◦C (±3 ◦C) and dark phase temperature of 24 ◦C (±3 ◦C). The lighting system consisted of 18,400 watt lighting luminaries which supplemented natural daylight if insufficient. Light intensity threshold was set to 10 KL. Three of the original species progressed to chelator-induced accumulation trials Brassica napus, Brassica oleracea var. capitate (referred to herein after as Brassica oleracea) and Helianthus annuus. One hundred plants of each of the 3 species (N = 300) were planted in Li amended soil as before with 10 plants (one plant per pot) in each group. The plants were grown to maturity (circa, 4 months) then treated with EDTA in trial 1 and EDDS in trial 2 bidaily for 1 week prior to harvesting. In most induced metal accumulation experiments chelating agents are generally added to the soil at concentrations between 0.001 M and 0.02 M [35]. In these experiments EDTA and EDDS were added to the soil at a concentration of 0.05 M in aliquots of 100 mL during regular watering times [82]. The chelate concentration used was large to offset the co-complexation of other metals present in the soil with stronger chelating stability constants than Li. For example, the stability constant for Li-EDTA is 2.79 while the constant for Fe-EDTA is 25.1 and Ca-EDTA is 10.65, both of which typically have high concentrations in soil and compete for EDTA. At this high level of chelate addition the co-complexation of other more toxic elements from the soil used to the plants such as lead (Pb) is a real danger and was also considered as a contributing factor to plant mortality in this study. Another possible contributing factor to plant mortality included the toxic effect of salinity caused by the addition of large amounts of LiCl to the plants at high concentrations. The chosen chelate concentration also served to maintain the pH of the soil solution low enough for the Li to exist as ions.PDF Image | Induced Plant Accumulation of Lithium
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