Plant Tissue Extraction Method for Complexed and Free Cyanide
A method for free cyanide and strongly-complexed cyanide measurement within plant tissue was developed to study uptake and movement of cyanide species separately from cyanide metabolism and metabolite movement by a willow plant (Salix eriocephala var. Michaux). Spike recoveries from solutions with and without plant tissue, using various solvent combinations, and background control tissue contributions were investigated to obtain an accurate and precise extraction method for measurement of complexed and free cyanide concentrations within plant tissue. The optimum extraction technique involved the freezing of plant tissue with liquid nitrogen to facilitate homogenization prior to extraction. Homogenized willow tissue samples, 1 to 1.5 g-fresh weight, were ground a second time under liquid nitrogen followed by grinding in slurry with 2.5 M NaOH. The slurry was brought to 100 mL volume, sonicated for 5 min, extracted in the dark for 16 h, and analyzed without filtration for total and free cyanide by acid distillation and microdiffusion respectively. Sample tissue extraction controls found recoveries of 89% and 100% for 100 μg L−1 CNT as KCN and K4Fe(CN)6 spiked in willow tissue slurries. Methanol, hexane, and 2-octanol inclusion in the solvent matrix with 2.5 M NaOH interfered with the cyanide analytical technique while chloroform reacted with NaOH and free cyanide in solution. Filtration was not included due to increased cyanide loss, and analysis of control tissue showed minimal release of cyanide or interference of plant tissue with the cyanide analytical method. Tissue cyanide concentrations from hydroponically-exposed tissue using the optimal extraction method agreed with tissue cyanide stable isotope (15N) results.
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