Abstract
Commercial development of the fungicide prochloraz for the control of pre- and post-harvest diseases in fruit crops has been restricted by the occasional occurrence of 'musty' taints. The primary aims of this research were to identify the cause of taint and and its means of formation. Sensitive and selective residue methodology based upon determination by gas chromatography with mass selective detection was devised, validated and used to establish 2,4,6-trichloroanisole (2,4,6-TCA) as the direct cause of taint by analysis of field-treated samples and studies involving 14C-radiolabelled prochloraz. Evaluation of analytical results in conjunction with corresponding sensory data led to proposals of flavour threshold levels ranging between 0.04 and 1.0mug/kg for 2,4,6-TCA in different fruit types. Formation of the anisole in pre-harvest treatment situations was shown to proceed via the minor plant metabolite 2,4,6-trichlorophenol (2,4,6-TCP) as the direct precursor; under post-harvest conditions, however, metabolism of prochloraz was minimal, and 2,4,6-TCA taints arose primarily from 2,4,6-TCP as an impurity (measured at 0.9% w/w) in the commercial emulsifiable concentrate formulation. Detailed investigation of the relationship between 2,4,6-TCP and 2,4,6-TCA under controlled conditions demonstrated that certain fruit types possess a capacity for direct O-methylation of the phenol to the anisole, whereas others do not. These findings were generally consistent with occurrences of taints associated with prochloraz. Kinetic studies of the active O-methyltransferase system in apple homogenates indicated the same Vmax value for 2-chlorophenol, 2,4-dichlorophenol, 2,6-dichlorophenol and 2,4,6-TCP. Reactivity in this series was found to be closely related to the ability of the molecules to to form effective nucleophiles for reaction with s-adenosylmethionine as the assumed methyl donor, and a linear relationship was established between chlorophenol pKa and log(apparent Km). The O-methylation of 2,4,6-TCP, whilst inhibited competitively by other o-chlorophenolic substrates, remained unaffected by the addition of representative plant o-diphenolics previously reported as substrates for O-methylation in plants.