BIOACCUMULATION KINETICS OF THE CONVENTIONAL ENERGETICS TNT AND RDX RELATIVE TO INSENSITIVE MUNITIONS CONSTITUENTS DNAN AND NTO IN RANA PIPIENS TADPOLES GUILHERME R. LOTUFO,*y JAMES M. BIEDENBACH,y JERRE G. SIMS,y PORNSAWAN CHAPPELL,z JACOB K. STANLEY,y and KURT A. GUSTy yUS Army Engineer Research and Development Center, Vicksburg, Mississippi, USA zBadger Technical Services, San Antonio, Texas, USA (Submitted 19 September 2014; Returned for Revision 10 December 2014; Accepted 15 December 2014) Abstract: The manufacturing of explosives and their loading, assembling, and packing into munitions for use in testing on training sites or battlefields has resulted in contamination of terrestrial and aquatic sites that may pose risk to populations of sensitive species. The bioaccumulative potential of the conventional explosives 2,4,6-trinitrotoluene (TNT) and hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) and of the insensitive munitions (i.e., less shock sensitive) compound 2,4-dinitroanisole (DNAN) were assessed using the Northern leopard frog, Rana pipiens. Trinitrotoluene entering the organism was readily biotransformed to aminodinitrotoluenes, whereas no transformation products were measured for RDX or DNAN. Uptake clearance rates were relatively slow and similar among compounds (1.32–2.19 L kg À1 h À1 ). Upon transfer to uncontaminated water, elimination rate was very fast, resulting in the prediction of fast time to approach steady state (5 h or less) and short elimination half-lives (1.2 h or less). A preliminary bioconcentration factor of 0.25 L kg À1 was determined for the insensitive munitions compound 3-nitro-1,2,4-trizole-5-one (NTO) indicating negligible bioaccumulative potential. Because of the rapid elimination rate for explosives, tadpoles inhabiting contaminated areas are expected to experience harmful effects only if under constant exposure conditions given that body burdens can rapidly depurate preventing tissue concentrations from persisting at levels that may cause detrimental biological effects. Environ Toxicol Chem 2015;34:880–886. # 2014 SETAC Keywords: Amphibians; Bioconcentration; Energetics; Toxicokinetics INTRODUCTION Manufacturing of explosives and their loading, assembling, and packing into munitions for use in testing on training sites and use in the battlefield has resulted in contamination of terrestrial and aquatic sites [1,2]. Some sites have been reported to contain explosives and associated compounds in soil, sediment, groundwater or surface water at concentrations that span several orders of magnitude [3,4]. Explosives compounds released from discarded shells as well as fragments of explosives formulations remaining following incomplete detonations may be present in surface soils and in aquatic habitats. Underwater unexploded and discarded munitions may also contribute to contamination of the water column from the slow release of explosives from corroded and breached shells [5–7]. Research on the ecotoxicity of explosive compounds has focused on munitions compounds, which have been heavily used by the military for many decades and continue to be used around the world [8–10]. Several insensitive munitions compounds that are chemically stable enough to withstand mechanical shocks without unintentional detonation have been developed and are being evaluated for future weapon systems to replace more sensitive explosives. Among these are 2,4- dinitroanisole (DNAN) as a substitute of 2,4,6-trinitrotoluene (TNT) and 3-nitro-1,2,4-trizole-5-one (NTO) as a replacement for hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), a polynitr- amine compound [11]. Little is known about the aquatic toxicity and bioaccumulation potential associated with the presence of DNAN and NTO in the environment. A primary objective of the present research effort was to comparatively assess the bioaccumulation potential of the insensitive munitions compounds (DNAN and NTO) relative to the conventional munitions compounds (TNT and RDX) to determine potential differences in bioconcentration using a novel amphibian test species. Bioconcentration is the process by which a chemical is absorbed by an organism from the water through its respiratory and dermal surfaces. It is the net result of competing rates of uptake and elimination, including biotrans- formation [12]. In addition, the octanol–water partition coefficient (K OW ) is widely used as an indicator of hydropho- bicity and thus the partitioning of a chemical from water into lipids and other organic phases [13]. Relatively low log K OW values have been reported for TNT (1.86 [14]), RDX (0.90 [15]), and DNAN (1.61 [16]). According to predictive models (e.g., Arnot and Goas [17] and Meylan et al. [18]), those explosives are weakly hydrophobic and therefore have low potential to bioconcentrate. Reports on the bioconcentration potential of explosives in fish and aquatic invertebrates, which investigated only a relatively small number of species, confirmed the expected low potential to bioconcentrate [8,9]. Moreover, studies examining uptake from water, as well as elimination kinetics and biotransformation potential, were conducted for only a few compounds and even fewer aquatic species [8,9]. Report of studies investigating the bioaccumu- lation, bioconcentration, or toxicokinetics of explosives in larval amphibians were not found in the available literature. Aquatic toxicology studies using larval amphibians are limited to evaluations of the lethal effects of TNT to Xenopus laevis [19] and to Rana catesbeiana [20]. Given the broad public * Address correspondence to guilherme.lotufo@usace.army.mil. Published online 18 December 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/etc.2863 Environmental Toxicology and Chemistry, Vol. 34, No. 4, pp. 880–886, 2015 # 2014 SETAC Printed in the USA 880