Identifying Toxic Biotransformation Products of the Insensitive Munitions Compound, 2,4-Dinitroanisole (DNAN), Using Liquid Chromatography Coupled to Quadrupole Time-of-Flight Mass Spectrometry (LC-QToF-MS)

As insensitive munitions (IMs) replace conventional explosives, releases of 2,4-dinitronanisole (DNAN) -an IM compound- are expected to increase in the environment. DNAN is readily biotransformed in soils, and while toxicity studies have evaluated DNAN, little attention has been given to its biotransformation products. In this work, we elucidated and semiquantitated product mixtures formed during anaerobic biotransformation of DNAN using high-resolution mass spectrometry techniques. DNAN underwent nitroreduction, and at later bioconversion stages, formed azo-dimers, accounting for the majority of end-products from DNAN anaerobic biotransformation. The chemical analyses were complemented by microbial (methanogenesis and Aliivibrio fischeri bioluminescence) and zebrafish embryo toxicity assays on mixtures of products formed, as well as individual compounds (biotransformation products and model azo-oligomers). Methanogens were severely inhibited during nitroreduction stages, but recovered at longer incubation times when azo-dimers were predominant. On the other hand, A. fischeri bioluminescence decreased at later biotransformation stages. When tested individually, the most toxic products to the microbial targets were the azo-oligomer models, while the least toxic species were 2,4-diaminoanisole and its N-acetylated analog. Zebrafish embryos showed few active developmental endpoints for the individual compounds tested, but 3-nitro-4-methoxyaniline and the model azo-dimer 2,2 Œ-dimethoxy-4,4 Œ-azodianiline had a lowest observable adverse effect level (LOAEL) of 6.4 ÊM. Overall, the interdisciplinary experimental design allowed to identify key transformation processes and products that alter toxicity beyond the parent compound.