ORIGINAL ABSTRACT

1. Triple Resonance Isotope EDited nmr spectroscopy (TRIED) has been developed to detect and examine minute levels of glyphosate metabolites in microbial soil isolates. Using stable isotopic labelling (13C and 15N), TRIED allows the simultaneous detection of multiple metabolites in crude matrices at submicrogram levels. An improvement over earlier techniques where milligrams are needed, TRIED can detect 500 ng of triply labelled compound in a crude sample (1:14,000 mass ratio) in just hours. 2. TRIED is used here to compare the kinetics and metabolic pathways of glyphosate metabolism by two strains of Ochrobactrum anthropi, LBAA and S5. Both LBAA and S5 appear to metabolize glyphosate primarily via the aminomethylphosphonate (AMPA) pathway, since no detectable levels of glycine or sarcosine are observed in the media or lysates of either microbe. The formation of N-methylAMPA is common to the metabolism of both microorganisms, but N-acetylAMPA is observed only in LBAA. N-methylacetamide is detected predominantly in media and lysates of S5, although some evidence also points to the formation of this metabolite in LBAA. 3. Results are consistent with conventional radioactive tracer studies. TRIED nmr provides more specific structural information complementary to radiolabel methods. Both nmr and radioactivity studies show S5 glyphosate metabolism to be much slower than that of LBAA.