Quantitative and enantioselective analyses of non-extractable residues of the fungicide metalaxyl in soil

The remobilization of non-extractable residues (NER) is a potential source for enhanced ecotoxicity and bioavailability in aquatic and terrestrial systems. The objective of this study is to investigate the influence of microbial and extracellular enzyme-assisted transformation of the fungicide metalaxyl and the formation of its non-extractable residues with special interest on the enantioselectivity of these processes. Soil sterilization using mercuric chloride treatment and chloroform fumigation allowed us to inhibit microbial activity without substantial alteration of the extracellular enzyme activity. Under sterilized and non-sterilized conditions, incubation experiments with rac-metalaxyl were carried out for a short-term (10 days) and a long-term (92 days) period. Particle size fractionation delivered sand, organo-silt, and organo-clay fractions of the agricultural soil. Extractable residues (water and solvent soluble) were separated from insoluble proportion of the organo-mineral components (organo-clay, organo-silt). The latter were subdivided into humic subfractions (fulvic acids, humic acids, and humin) and non-humic substances. All these subfractions were subjected to alkaline hydrolysis treatment delivering hydrolyzable proportion of NER of metalaxyl. All fractions were analyzed quantitatively by gas chromatography-mass spectrometry (GC/MS), and the enantioselectivity was determined by means of chiral-GC/MS. In all experimental set-ups, most of the metalaxyl residues were found in the water-soluble fraction. The amount of total extractable metalaxyl residue decreased in the time period of 10-92 days from approximately 95 to 75 % under sterilized and non-sterilized conditions. Chiral analyses showed the dissipation of R-metalaxyl (enantiomer fraction (EF) > 0.5) after 92 days even in sterilized samples, indicating a direct influence of not only microbial but also extracellular enzymatic activity in the enantioselective fungicide degradation processes. The metabolite metalaxyl acid was predominantly found in the water-soluble fraction (30 %) and in hydrolyzable NER fraction (2 %) under non-sterilized condition. Chiral-GC/MS showed an enrichment of the previously dissipated R-metalaxyl in these fractions (EF < 0.5). There is evidence of microbial as well as extracellular enzyme-assisted formation of non-extractable metalaxyl residues. Higher availability of the transformation product metalaxyl acid allowed also higher amounts of hydrolyzable NER in the non-sterilized samples. Hereby, a strong incorporation of the acid metabolite was evident. Chiral analyses provide insights into the stereoselectivity not only of the metabolic transformation processes but also of the formation of NER of the fungicide metalaxyl.