Covalent binding of reduced metabolites of [N-15(3)]TNT to soil organic matter during a bioremediation process analyzed by N-15 NMR spectroscopy

Evidence is presented for the covalent binding of biologically reduced meta bolites of 2,4,6-N-15(3)-trinitrotoluene (TNT) to different soil fractions (humic acids, fulvic acids, and humin) using liquid N-15 NMR spectroscopy. A silylation procedure was used to release soil organic matter from humin a nd whole soil for spectroscopic measurements. TNT-contaminated soil was spi ked with 2,4,6-N-15(3)-trinitrotoluene and C-14-ring labeled TNT, before tr eatment in a soil slurry reactor. During the anaerobic/aerobic incubation t he amount of radioactivity detected in the fulvic and humic acid fractions did not change significantly(11-16%), whereas the radioactivity bound to hu min increased to 71%. The N-15 NMR spectra of the fulvic acid samples were dominated by a large peak that corresponded to aliphatic amines or ammonia. In the early stages of incubation, N-15 NMR analysis of the humic acids in dicated bound azoxy compounds. The signals arising from nitro and azoxy gro ups disappeared with further anaerobic treatment. At the end of incubation, the NMR shifts showed that nitrogen was covalently bound to humic acid as substituted amines and amides. The NMR spectra of the silylated humin sugge st formation of azoxy compounds and imine linkages. Bound metabolites posse ssing nitro groups were also detected. Primary amines formed during the ana erobic incubation disappeared during the aerobic treatment. Simultaneously, the amount of amides and tertiary amines increased. Nitro and azoxy groups of bound molecules were still present in humin at the end of the incubatio n period. Formation of azoxy compounds from partially reduced TNT followed by binding and further reduction appears to be an important mechanism for t he immobilization of metabolites of TNT to soil.