Characterization of the bound residues of the fungicide cyprodinil formed in plant cell suspension cultures of wheat

The non-extractable residues of the fungicide cyprodinil formed in heterotrophic cell suspension cultures of wheat were studied by application of [2-pyrimidyl-C-14] or [2-pyrimidyl-C-13]cyprodinil. The main objective was to examine whether solid-state and liquid C-13 NMR spectroscopy can be used to examine plant bound residues of pesticides. For 14C experiments, wheat suspensions grown on glucose as carbon source were treated with 10 mg litre(-1) of C-14-cyprodinil. After incubation for 12 days, 20% of applied C-14 was detected as non-extractable residues. The cell debris were treated with 0.1 M HCl (reflux), 1.0M HCl (reflux), buffer, or 2 M NaOH (50degreesC; Bjorkman lignin and acidolysis lignin fractions were also prepared from the debris. Radioactivity liberated and solubilized by these procedures was examined by thin-layer chromatography and high-performance liquid chromatography. The results showed that cyprodinil and primary metabolites contributed to the fungicide's bound residues. Most of the residues (12% of applied C-14) remained associated with polar or polymeric/oligomeric endogenous cell materials in a stable manner. For the study with C-13-cyprodinil, wheat suspensions were cultivated on C-13-depleted glucose for four growth cycles, resulting in maximum C-13 depletion of the natural cell components to about 0.10%. During the fourth cycle, C-13-labelled cyprodinil was applied, and cells were incubated (12 days). Cell debris was prepared and examined by solid-state C-13 NMR spectroscopy. Debris was then treated as described above in the C-14 experiment. Solubilized fractions were analyzed by liquid C-13 NMR spectroscopy. However, none of the C-13 NMR spectra recorded gave utilizable or unambiguous results, and all exhibited large inconsistencies, especially concerning the data from the conventional C-14 experiment. (C) 2003 Society of Chemical Industry