Christopher I. Olivares

Pathways of reductive 2,4‐dinitroanisole (DNAN) biotransformation in sludge

As the use of the insensitive munition compound 2,4‐dinitroanisole (DNAN) increases, releases to the environment may pose a threat to local ecosystems. Little is known about the environmental fate of DNAN and the conversions caused by microbial activity. We studied DNAN biotransformation rates in sludge under aerobic, microaerophilic, and anaerobic conditions, detected biotransformation products, and elucidated their chemical structures. The biotransformation of DNAN was most rapid under anaerobic conditions with H2 as a cosubstrate. The results showed that the ortho nitro group in DNAN is regioselectively reduced to yield 2‐methoxy‐5‐nitroaniline (MENA), and then the para nitro group is reduced to give 2,4‐diaminoanisole (DAAN). Both MENA and DAAN were identified as important metabolites in all redox conditions. Azo and hydrazine dimer derivatives formed from the coupling of DNAN reduction products in anaerobic conditions. Secondary pathways included acetylation and methylation of amine moieties, as well as the stepwise O‐demethylation and dehydroxylation of methoxy groups. Seven unique metabolites were identified which enabled elucidation of biotransformation pathways. The results taken as a whole suggest that reductive biotransformation is an important fate of DNAN leading to the formation of aromatic amines as well as azo and hydrazine dimeric metabolites. Biotechnol. Bioeng. 2013; 110: 1595–1604.

Microbial toxicity of the insensitive munitions compound, 2,4-dinitroanisole (DNAN), and its aromatic amine metabolites

2,4-Dinitroanisole (DNAN) is an insensitive munitions compound considered to replace conventional explosives such as 2,4,6-trinitrotoluene (TNT). DNAN undergoes facile microbial reduction to 2-methoxy-5-nitroaniline (MENA) and 2,4-diaminoanisole (DAAN). This study investigated the inhibitory effect of DNAN, MENA, and DAAN toward various microbial targets in anaerobic (acetoclastic methanogens) and aerobic (heterotrophs and nitrifiers) sludge, and the bioluminescent bacterium, Aliivibrio fischeri, used in the Microtox assay. Aerobic heterotrophic and nitrifying batch experiments with DAAN could not be performed because the compound underwent extensive autooxidation in these assays. DNAN severely inhibited methanogens, nitrifying bacteria, and A. fischeri (50% inhibitory concentrations (IC50) ranging 41-57μM), but was notably less inhibitory to aerobic heterotrophs (IC50>390 μM). Reduction of DNAN to MENA and DAAN lead to a marked decrease in methanogenic inhibition (i.e., DNAN>MENA≈DAAN). Reduction of all nitro groups in DNAN also resulted in partial detoxification in assays with A. fischeri. In contrast, reduction of a single nitro group did not alter the inhibitory impact of DNAN toward A. fischeri and nitrifying bacteria given the similar IC50 values determined for MENA and DNAN in these assays. These results indicate that reductive biotransformation could reduce the inhibitory potential of DNAN.

(Bio)transformation of 2,4-dinitroanisole (DNAN) in soils

Recent studies have begun to assess the environmental fate and toxicity of 2,4-dinitroanisole (DNAN), an insensitive munition compound of interest to defense agencies. Aerobic and anaerobic DNAN biotransformation in soils was evaluated in this study. Under aerobic conditions, there was little evidence of transformation; most observed removal was attributed to adsorption and subsequent slow chemical reactions. Under anaerobic conditions, DNAN was reductively (bio)transformed and the rate of the transformation was positively correlated with soil organic carbon (OC) up to a threshold of 2.07% OC. H2 addition enhanced the nitroreduction rate compared to endogenous treatments lacking H2. Heat-killed treatments provided rates similar to the endogenous treatment, suggesting that abiotic factors play a role in DNAN reduction. Ten (bio)transformation products were detected by high-resolution mass spectrometry. The proposed transformation pathway involves reduction of DNAN to aromatic amines, with putative reactive nitroso-intermediates coupling with the amines to form azo dimers. Secondary reactions include N-alkyl substitution, O-demethylation (sometimes followed by dehydroxylation), and removal of an N-containing group. Globally, our results suggest that the main reaction DNAN undergoes in anaerobic soils is nitroreduction to 2-methoxy-5-nitroaniline (MENA) and 2,4-diaminoanisole (DAAN), followed by anaerobic coupling reactions yielding azo-dimers. The dimers were subsequently subject to further (bio)transformations.

Arsenic (III, V), indium (III), and gallium (III) toxicity to zebrafish embryos using a high-throughput multi-endpoint in vivo developmental and behavioral assay

Gallium arsenide (GaAs), indium gallium arsenide (InGaAs) and other III/V materials are finding increasing application in microelectronic components. The rising demand for III/V-based products is leading to increasing generation of effluents containing ionic species of gallium, indium, and arsenic. The ecotoxicological hazard potential of these streams is unknown. While the toxicology of arsenic is comprehensive, much less is known about the effects of In(III) and Ga(III). The embryonic zebrafish was evaluated for mortality, developmental abnormalities, and photomotor response (PMR) behavior changes associated with exposure to As(III), As(V), Ga(III), and In(III). The As(III) lowest observable effect level (LOEL) for mortality was 500 μM at 24 and 120 h post fertilization (hpf). As(V) exposure was associated with significant mortality at 63 μM. The Ga(III)-citrate LOEL was 113 μM at 24 and 120 hpf. There was no association of significant mortality over the tested range of In(III)-citrate (56-900 μM) or sodium citrate (213-3400 μM) exposures. Only As(V) resulted in significant developmental abnormalities with LOEL of 500 μM. Removal of the chorion prior to As(III) and As(V) exposure was associated with increased incidence of mortality and developmental abnormality suggesting that the chorion may normally attenuate mass uptake of these metals by the embryo. Finally, As(III), As(V), and In(III) caused PMR hypoactivity (49-69% of control PMR) at 900-1000 μM. Overall, our results represent the first characterization of multidimensional toxicity effects of III/V ions in zebrafish embryos helping to fill a significant knowledge gap, particularly in Ga(III) and In(III) toxicology.

Oxidation of reduced daughter products from 2,4-dinitroanisole (DNAN) by Mn(IV) and Fe(III) oxides

Abiotic transformation of anthropogenic compounds by redox-active metal oxides affects contaminant fate in soil. The capacity of birnessite and ferrihydrite to oxidize the insensitive munitions compound, 2,4-dinitroanisol (DNAN), and its amine-containing daughter products, 2-methoxy-5-nitro aniline (MENA) and 2,4-diaminoanisole (DAAN), was studied in stirred reactors at controlled pH (7.0). Aqueous suspensions were reacted at metal oxide solid to solution mass ratios (SSR) of 0.15, 1.5 and 15 g kg-1 and solutions were analyzed after 0-3 h by high performance liquid chromatography coupled with photodiode array or mass spectrometry detection. Results indicate that DNAN was resistant to oxidation by birnessite and ferrihydrite. Ferrihydrite did not oxidize MENA, but MENA was susceptible to rapid oxidation by birnessite, with nitrogen largely mineralized to nitrite. This is the first report on mineralization of nonphenolic aromatics and the release of mineralized N from aromatic amines following reaction with birnessite. DAAN was oxidized by both solids, but ca. ten times higher rate was observed with birnessite as compared to ferrihydrite at an SSR of 1.5 g kg-1. At 15 g kg-1 SSR, DAAN was removed from solution within 5 min of reaction with birnessite. CO2(g) evolution experiments indicate mineralization of 15 and 12% of the carbon associated with MENA and DAAN, respectively, under oxic conditions with birnessite at SSR of 15 g kg-1. The results taken as a whole indicate that initial reductive (bio)transformation products of DNAN are readily oxidized by birnessite. The oxidizability of the reduced DNAN products was increased with progressive (bio)reduction as reflected by impacts on the oxidation rate.

Environmental Fate of 14C Radiolabeled 2,4-Dinitroanisole in Soil Microcosms

2,4-Dinitrosanisole (DNAN) is an insensitive munitions component replacing conventional explosives. While DNAN is known to biotransform in soils to aromatic amines and azo-dimers, it is seldom mineralized by indigenous soil bacteria. Incorporation of DNAN biotransformation products into soil as humus-bound material could serve as a plausible remediation strategy. The present work studied biotransformation of DNAN in soil and sludge microcosms supplemented with uniformly ring-labeled 14C-DNAN to quantify the distribution of label in soil, aqueous, and gaseous phases. Electron donor amendments, different redox conditions (anaerobic, aerobic, sequential anaerobic-aerobic), and the extracellular oxidoreductase enzyme horseradish peroxidase (HRP) were evaluated to maximize incorporation of DNAN biotransformation products into the nonextractable soil humus fraction, humin. Irreversible humin incorporation of 14C-DNAN occurred at higher rates in anaerobic conditions, with a moderate increase when pyruvate was added. Additionally, a single dose of HRP resulted in an instantaneous increased incorporation of 14C-DNAN into the humin fraction. 14C-DNAN incorporation to the humin fraction was strongly correlated (R2 = 0.93) by the soil organic carbon (OC) amount present (either intrinsic or amended). Globally, our results suggest that DNAN biotransformation products can be irreversibly bound to humin in soils as a remediation strategy, which can be enhanced by adding soil OC.

Continuous treatment of the insensitive munitions compound N-methyl-p-nitro aniline (MNA) in an upflow anaerobic sludge blanket (UASB) bioreactor

N-methyl-p-nitroaniline (MNA) is an ingredient of insensitive munitions (IM) compounds that serves as a plasticizer and helps reduce unwanted detonations. As its use becomes widespread, MNA waste streams will be generated, necessitating viable treatment options. We studied MNA biodegradation and its inhibition potential to a representative anaerobic microbial population in wastewater treatment, methanogens. Anaerobic biodegradation and toxicity assays were performed and an up-flow anaerobic sludge blanket reactor (UASB) was operated to test continuous degradation of MNA. MNA was transformed almost stoichiometrically to N-methyl-p-phenylenediamine (MPD). MPD was not mineralized; however, it was readily autoxidized and polymerized extensively upon aeration at pH = 9. In the UASB reactor, MNA was fully degraded up to a loading rate of 297.5 μM MNA d(-1). Regarding toxicity, MNA was very inhibitory to acetoclastic methanogens (IC50 = 103 μM) whereas MPD was much less toxic, causing only 13.9% inhibition at the highest concentration tested (1025 μM). The results taken as a whole indicate that anaerobic sludge can transform MNA to MPD continuously, and that the transformation decreases the cytotoxicity of the parent pollutant. MPD can be removed through extensive polymerization. These insights could help define efficient treatment options for waste streams polluted with MNA.