Zebrafish embryo toxicity of anaerobic biotransformation products from the insensitive munitions compound 2,4‐dinitroanisole

Abstract

2,4-dinitroanisole (DNAN) is an emerging insensitive munitions compound that readily undergoes anaerobic nitro-group reduction to 2-methoxy-5-nitroaniline (MENA) and 2,4-diaminoanisole (DAAN), followed by subsequent formation of unique azo-dimers. Currently there is scarce knowledge on the ecotoxicity of DNAN (bio)transformation products. In this work, mortality, development, and behavioral effects of DNAN (bio)transformation products were assessed using zebrafish (Danio rerio) embryos. We tested individual products, MENA and DAAN, as well as dimer and trimer surrogates. As pure compounds, 3-nitro-4-methoxyaniline and 2,2’-dimethoxy-4,4’-azodianiline caused significant effects with lowest observable effect levels (LOEL) at 6.4 µM on one or two developmental endpoints, respectively. The latter had six-additional significant developmental endpoints with LOELs of 64 µM. Based on light/dark swimming behavioral tests, DAAN (640 µM) caused reduction in swimming, suggestive of neurotoxicity. No significant mortality occurred (≤ 64 µM) for any of the individual compounds. However, metabolite mixtures formed during different stages of MENA (bio)transformation in soil were characterized using high-resolution mass spectrometry in parallel with zebrafish embryo toxicity assays, which demonstrated significant mortality during the onset of azo-dimer formation. Overall the results indicate that several DNAN (bio)transformation products cause different types of toxicity to zebrafish embryos.

Aquatic toxicity of photo‐degraded insensitive munition 101 (IMX‐101) constituents

“…Alternatively, the toxicity of the DNAN transformation product dinitrophenol is 2-to 3-fold greater than that of DNAN to C. dubia [15] and thus could be a contributor to the photo-induced toxicity. The DNAN biotransformation products 2,2 0 -dimethoxy-4,4 0azodianiline (an intermediate), 3-nitro-4-methoxyaniline, and 2-4 diaminoanisole caused behavioral and developmental effects in Danio embryos [22]. However, these compounds were not formed through photodegradation, and the photoinduced toxicity of DNAN is relatively minor relative to that of NQ.…”

“…It is important to understand the potential for UV-induced phototoxicity and generation of degradation compounds for both the individual constituents and the mixture. Ecotoxicological information was only recently published on DNAN biotransformation products to zebrafish [22], but that study did not investigate photo-induced toxicity. Whereas some toxicity reference values are available on the photo-induced toxicity of NQ [13], photo-induced hazard information is limited for NTO, DNAN, and IMX-101.…”

“…Previous conclusions were that any level of discharge of photo-degraded NQ may be hazardous [18], but the impact of initial concentration or actual environmental exposure potential was not considered. Ecotoxicological information was only recently published on DNAN biotransformation products to zebrafish [22], but that study did not investigate photo-induced toxicity. We are not aware of studies investigating the relative photo-induced ecotoxicity of all 3 IMX-101 constituents and the IMX-101 mixture itself to any single National Pollutant Discharge Elimination System permit-relevant species; this is needed for direct conclusions comparing the relative photoinduced toxicity of insensitive munition constituents and their contribution to the IMX-101 mixture itself.…”

Ecotoxicity of the insensitive munitions compound 3-nitro-1,2,4-triazol-5-one (NTO) and its reduced metabolite 3-amino-1,2,4-triazol-5-one (ATO)

“…The lethal concentration for 50% of the organisms (LC 50 ) was estimated at 0.10 mM, and the concentration causing vertebral column deformities (EC 50 ) was estimated at 0.09 mM. The same protocol described in this work was used to evaluate the toxicity of DNAN transformation products [40]. The aromatic amines MENA and DAAN did not cause mortality or developmental toxicity to the test-organisms at concentrations as high as 640 and 64 µM, respectively.…”

3D Visualization of Developmental Toxicity of 2,4,6-Trinitrotoluene in Zebrafish Embryogenesis Using Light-Sheet Microscopy

“…Zebrafish ( Danio rerio ) embryos are ideal model vertebrates for assessing the acute toxicity of environmental samples and newly developing compounds [15,16]. Their development has been extensively characterized and embryos remain transparent until late pharyngula stages, allowing unobstructed imaging of key morphological events.…”