Analysis of soil-bound residues of C-13-labeled fungicide cyprodinil by NMR spectroscopy

C-13-NMR spectroscopy was applied to the evaluation of soil-bound residues of the fungicide cyprodinil (4-cyclopropyl-6-methyl-2-phenylaminopyrimidine). A mixture of the C-13- and C-14-labeled fungicide was used to obtain structural information as well as information on the quantitative distribution in the various fractions. Bound residues were accumulated by a 6-month incubation of the labeled compound with a clay loamy soil. Depending on the concentration of [C-13]cyprodinil (500, 250, 80, and 3 mg/kg), binding ranged from 18% to 54% of the initial radioactivity. After methanol extraction of soil (10 g dry weight)treated with 500 mg/kg (5.0 mg) of the fungicide, the amount of unextracted bound material was equivalent to 0.9 mg of C-13-labeled cyprodinil. Upon fractionation, 0.21 mg of the bound fungicide was found in the dialyzed humic acid, 0.13 mg in fulvic acid (after extraction with CH2Cl2), and 0.24 mg in humin. The methylene chloride extract from fulvic acid mainly contained unchanged cyprodinil (0.21 mg) that was apparently sequestered in soil by physical forces. The humic acid fraction was dissolved in a 1% solution of NaOD and examined by C-13-NMR. The NMR spectrum of the material from the control sample exhibited all the characteristic features of a typical humic acid. When the control humic acid was spiked with cyprodinil labeled uniformly with C-13 at the phenyl ring, four additional signals at 121.9, 124.4, 131.8, and 143.4 ppm could be distinguished in the NMR spectrum. However, when humic acid originated from the soil that was incubated with the phenyl-labeled fungicide, only two strong NMR signals, at 122.5 and 131.8 ppm, and two less significant signals around 142 and 162 ppm were observed. The difference in the signal pattern indicated cleavage of the cyprodinil molecule between the aromatic rings and independent binding of the phenyl and pyrimidyl moieties to humic acid.