Hexahydro-1,3,5-trinitro-1,3,5-triazine is a widely used military explosive that is more commonly known as Royal Demolition Explosive (RDX). Because of concerns with the potential toxicity of RDX, increasingly stringent regulations are anticipated for wastewater discharges from munitions manufacturing facilities. At the Holston Army Ammunition Plant (HAAP), where RDX is currently produced in the U.S., the treatment sequence includes an anoxic filter prior to aerobic filters and activated sludge reactors. The intent of the anoxic filter is to remove the high levels of nitrate that are often present in munitions wastewater, as well as RDX and other nitrated energetic compounds. However, RDX removal across the filter is typically not adequate to meet the anticipated regulatory target, and RDX tends to persist under aerobic conditions. The objective of this study was to evaluate the reason for poor RDX removal in the anoxic filter. Microcosms were set-up with HAAP wastewater and biofilm scraped from the anoxic filter. Nitrate-reducing conditions were readily established, with organics present in the wastewater as well as acetate serving as electron donors. However, as long as nitrate was present, no decrease in RDX (15 μM) occurred. As soon as nitrate was depleted and some primary substrate was still available, RDX was rapidly biotransformed to metabolites that included mononitroso, dinitroso, and trinitroso derivatives. The disappearance of nitrate, followed by biotransformation of RDX, coincided with a decrease in redox potential to below −200 mV. These results were confirmed with an ethanol-grown nitrate-reducing enrichment culture that was inoculated with biofilm from the HAAP anoxic filter. The presence of RDX had no apparent effect on nitrate reduction, whereas RDX biotransformation was completely inhibited until all of the nitrate was consumed. Nitrate removal was achieved by repeated additions of ethanol. Thus, in order to ensure efficient biotransformation of RDX, nitrate removal must be complete, which requires an electron donor supply that exceeds the acceptor capacity of the nitrate.

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