Elsevier

Journal of Hazardous Materials

Volume 262, 15 November 2013, Pages 281-287
Journal of Hazardous Materials

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

https://doi.org/10.1016/j.jhazmat.2013.08.046Get rights and content

Highlights

  • The microbial toxicity of DNAN and its metabolites was assayed.

  • DNAN caused severe inhibition of methanogens, nitrifiers, and Aliivibrio fischeri.

  • The aromatic amine metabolites were generally less toxic than DNAN.

Abstract

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 41–57 μM), 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.

Introduction

The defense industry is currently developing insensitive munitions (IM). Utilization of these energetic chemicals is expected to reduce accidental explosions since IMs have a high detonation temperature and enhanced resistance to shocks [1]. 2,4-Dinitroanisole (DNAN) is an IM compound that is being considered as a replacement for the widely used nitroaromatic, 2,4,6-trinitrotoluene (TNT) [2]. While DNAN is less sensitive, other technical properties of this material compare favorably with those of TNT and make it advantageous for the manufacturing explosives formulations [2].

An assessment of the environmental fate and toxicity potential of DNAN is required since DNAN production and usage is expected to increase. Many nitroaromatic compounds are toxic and mutagenic to different types of organisms, including bacteria, algae, plants, invertebrates, and mammals [3], [4]. DAAN has also been shown to be mutagenic and a potential carcinogen in various assays [5], [6], [7], [8]. However, data on the inhibitory potential of DNAN toward microorganisms are very scarce. Microbial toxicity could impact biological treatment of effluents containing DNAN and impair the function of natural microbial populations in contaminated soil, which in turn could compromise the effectiveness of soil bioremediation efforts.

DNAN has been reported to undergo microbial transformation to 2-methoxy-5-nitroaniline (MENA) in aerobic conditions [9], and to 2,4-diaminoanisole (DAAN) in anaerobic conditions [10]. Recently we have also reported that microorganisms present in conventional wastewater treatment systems can reduce the nitro group in DNAN under aerobic, microaerophilic and anaerobic conditions [11]. Both MENA and DAAN were identified as important microbial metabolites in all redox conditions. Initially, the ortho nitro group in DNAN is regioselectively reduced to yield MENA. Subsequently, the para nitro group in MENA is reduced to form the diamino compound, DAAN. Microbial transformation of DNAN could alter the potential toxic impact of this aromatic compound. Unfortunately data on the microbial toxicity of the reduced metabolites of DNAN are largely unavailable.

The objective of this study was to evaluate the inhibitory effect of DNAN and its reduced intermediates MENA and DAAN to microorganisms commonly found in the environment under different redox conditions, namely anaerobic methanogens, aerobic heterotrophs and nitrifying bacteria. The inhibitory impact of these compounds was also evaluated using the Microtox assay, a method that relies on bioluminescence measurements in cultures of the bacterium Aliivibrio fischeri. The results obtained will contribute to a better understanding of the environmental impact of DNAN and will facilitate the development and optimization of efficient bioremediation technologies for the removal of this nitroaromatic compound.

Section snippets

Microbial inocula

Methanogenic sludge, aerobic return activated sludge (RAS), and nitrifying sludge were used as inoculum. The methanogenic sludge was obtained from a full-scale anaerobic bioreactor treating brewery wastewater (Mahou, Guadalajara, Spain). RAS and the nitrifying inoculum were collected from local municipal wastewater treatment plants; Ina Road and Randolph Park Wastewater Reclamation Facilities (Tucson, AZ, USA), respectively. All sludge samples were stored at 4 °C. The volatile suspended solid

Methanogenic inhibition

Fig. 1A illustrates the time course of methane production in methanogenic activity assays amended with DNAN. The specific methanogenic activities determined in these assays were normalized based on the activity of the DNAN-free control. In each case, the activity was determined during the time period when the control displayed maximum methane production rate (i.e., time 0–31 h). The normalized methanogenic activity as a function of the initial DNAN concentration is shown in Fig. 2A. The same

Discussion

As previously observed for TNT [15], [16], significant environmental emissions of DNAN could occur during compound manufacture and use in testing and training grounds. Environmental emissions of DNAN are of concern because many nitroaromatic compounds exhibit high toxicity to microorganisms [17], [18], [19], [20], and to higher aquatic and terrestrial organisms, including mammals [3], [19], [21], [22], [23], [24]. TNT and related nitroaromatic compounds have also been found to be mutagenic and

Conclusions

Taken as a whole these results indicate that DNAN causes strong acute cytotoxicity in methanogenic and nitrification microbial populations. Preliminary results suggest that microbial reductive transformation may reduce the inhibitory impact of DNAN. Additional toxicity studies with secondary dimeric metabolites of DNAN and their soil conjugates are needed to better characterize the hazard associated with this emerging energetic compound. In addition to cytotoxicity, there is a need to

Acknowledgements

This study was supported by the Strategic Environmental Research and Development Program (SERDP project ER-2221). JL was supported by the China Scholarship Council and CO by the Mexican National Council for Science & Technology (CONACyT). We thank Dr. Wenjie Sun for advice on bioassay methodologies.

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