268 (NIP-1115-998200) Aquatic toxicity of ordnance compounds: an overview.
Start time: 8:00 AM
Nipper, M.¹, Carr, R.², ¹ Texas A&M University-Corpus Christi, Corpus Christi, TX, USA² USGS, CERC, Marine Ecotoxicology Research Station, Corpus Christi, TX, USA
The environmental implications of contamination with explosives became the subject of intensified research with the decommissioning of military installations in the USA and overseas in the 1990s and potential use of such grounds for non-military activities. Explosives have been detected in surface water of numerous military installations, including bays and estuaries in addition to freshwater bodies, sometimes at exceedingly elevated concentrations. The fate and biological effects of energetic chemicals to marine and freshwater organisms has been reviewed and will be discussed. A relatively broad variety of aquatic toxicity studies exists for nitro-substituted phenol, toluene and benzene explosives and related compounds, but very little toxicological information is available for tetryl, cyclic nitramines such as RDX and HMX, and other energetic compounds such as nitrocellulose, nitroguanidine, nitroglycerin, pentaerythritol tetranitrate (PETN), and dethyleneglycol dinitrate (DEGDN). Sublethal assessments with different categories of explosives suggest their usefulness for the interpretation of their potential long-term environmental effects. Concentrations of a variety of explosives and related compounds associated with reproductive effects were substantially lower than those promoting mortality of the cladocerans Daphnia magna and Ceriodaphnia dubia, and the marine polychaete, Dinophilus gyrociliatus. The toxicity of nitroaromatic isomers, e.g., para, ortho and meta-dinitrotoluene, is dependent on the position of the nitro groups. Unicellular organisms such as freshwater microalgae and zoospores of the marine macroalga Ulva fasciata were not only more sensitive than invertebrates and fish to a variety of ordnance compounds, but their toxicological response was influenced differently by the position of the nitro group in nitroaromatic compounds. Very little information is available on the toxic mode of action of energetic materials, and research on this subject should be encouraged.

269 (LOT-1117-839335) Toxicokinetics, biotransformation, dietary uptake and critical body residues of explosives in aquatic animals.
Start time: 8:20 AM
Lotufo, G¹, Lydy, M², ¹ U.S. Army Engineer Research and Development Center, Vicksburg, MS, USA² Department of Zoology, Southern Illinois University, Carbondale, IL, USA
The biological effects of explosives and related compounds in aqueous exposures have been extensively investigated. However, despite the high environmental relevance of those compounds, reports on their bioaccumulation potential in relevant aquatic animals are scarce. Explosive compounds are weakly hydrophobic and therefore their predicted bioaccumulative potential is very low, as confirmed empirically. The bioconcentration factors (BCFs) for TNT and aminodinitrotoluenes ranged from 0.8 to 14.8 ml/mg for a variety of invertebrates and fish species. Similar BCFs (2.1-13.1 ml/mg) were derived for aminodinitrotoluenes, dinitrotoluenes and dinitrobenzenes but lower BCFs (0.5 to 2.1 ml/mg) were determined for RDX, HMX and 2,4-diaminonitrotoluene. The elimination rate constants determined for TNT, aminodinitrotoluenes, diaminonitrotoluenes, RDX and HMX in aquatic invertebrates and fish were high (), and led to steady-state bioaccumulation within hours. The dietary uptake of the explosives TNT and RDX has been examined using two species of fish and different routes of contaminant delivery. Steady-state bioaccumulation factors (concentration in fish relative to concentration in the diet) and all its breakdown products, determined using radioactivity, were low for TNT (up to 0.02 g/g) and RDX (up to 0.01 g/g). Nitroaromatic explosives and related compounds undergo fast transformation in soils and sediments. Efficient biotransformation of TNT has been reported for a variety of aquatic invertebrates and fish. Exposure to TNT resulted in the formation of aminodinitrotoluenes as well as non-identified solvent-extractable and non-extractable transformation products in invertebrate and fish species. The ability of aquatic animals to biotransformation other explosives and related compounds is currently unknown. Lethal body residues in fish and aquatic invertebrates were 4 to 20 nmol/g for TNT (sum concentration of TNT), 268 for 2-aminodinitrotoluene, and 41 nmol/g for RDX exposures. Because of the fast elimination rate and exceedingly low bioaccumulative potential, exposure to explosives at contaminated sites are unlikely to pose unacceptable risks to aquatic animals.

270 (CON-1117-762533) Fate and Toxicity of Explosives in Sediments.
Start time: 8:40 AM
Conder, J¹, Lotufo, G², ¹ ENVIRON International Corporation, Irvine, CA, USA² U.S. Army Corps of Engineers, Vicksburg, MS, USA
Contamination of sediment and surface water with explosives (e.g., TNT, TNB, RDX, HMX, picric acid, and tetryl) is associated with military activities at ammunition production sites and military training facilities. We present an overview of the fate and toxicity of sediment-associated explosives and related compounds, including a review of the adverse biological effects of explosive compounds and their transformation products to benthic invertebrates exposed to spiked sediments and corresponding pore waters. The assessment of the fate and effects of explosives that undergo rapid transformation when added to sediment in laboratory toxicity experiments has been challenging, complicating development of accurate reference and screening values for use in ecological risk assessment. Extrapolating ecological risks at contaminated sites from laboratory-based toxicity benchmark values to field situations is burdened with high uncertainty due to the unknown fate and toxicity of aged explosives in field sediments. Although there are reports of high levels of sediment contamination by explosives, lack of information on spatial distribution, bioavailability, and toxicity of sediment-associated explosives in historically-contaminated military and active training sites precludes an accurate evaluation of the local and global environmental significance of the presence of explosives in aquatic systems. Future studies should be designed to characterize the temporal nature of the dose-response relationship in spike-sediment studies with explosives, establish experimental data that enables comparisons of the bioaccumulation and toxicity of explosive compounds to benthic invertebrates belonging to different functional groups, and provide data that allows comparisons of the toxicity of explosives in spiked and field-collected sediments. The use of biomimetic devices (e.g., solid phase microextraction fibers) as tools in meeting many of these challenges will be discussed.

271 (ROB-1117-656835) Bioaccumulation and transformation of RDX in the earthworm Eisenia andrei.
Start time: 9:00 AM
Robidoux, PY¹, Sarrazin, M¹, Hawari, J¹, Sunahara, GI¹, ¹ National Research Council Canada, Montreal, Quebec, Canada
Energetic compounds can be released into the environment at munitions production and processing facilities, military firing ranges as well as through field use and disposal practices. Based on laboratory studies, 1,3,5-trinitro-1,3,5-triazacyclohexane (RDX) has sublethal effects on earthworms at environmentally relevant concentrations in soil. Little information is available describing the bioccumulation and transformation of RDX in soil organisms. In preliminary experiments, the potential bioaccumulation of RDX was assessed using acetonitrile extraction (USEPA Method 8330A) of earthworms exposed to RDX in different substrates. After 72 h exposure on RDX on filter paper, maximum uptake of RDX in earthworms was 506 g/g dry tissue. Exposure to RDX-amended artificial soil for 28 d gave a similar maximum uptake (538 g RDX/g dry tissue). Time-course studies using two different RDX amended natural soils (Sassafras sandy loam, or a Canadian sandy soil) showed that maximum uptake can be attained after 2 d exposure. In addition, earthworm exposure to RDX amended soil led to a time-dependent increase in levels of the RDX metabolite hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine (MNX), reaching up to 13.7 g/g dry tissue. Acid combustion of [¹⁴C]-RDX exposed earthworms gave similar tissue RDX uptake levels as those using acetonitrile-extracted worms exposed to non-radiolabelled RDX. This information can be used to develop bioaccumulation models that relate soil exposure concentrations to potential bioaccumulation of RDX in the earthworm.

(58185) Break.
Start time: 9:20 AM

272 (ROB-1117-659142) Can bioavailability of metals and EM in contaminated soils be appraised by chemical bioaccessibility ?
Start time: 10:00 AM
Berthelot, Y^{1, 2}, Auroy, A^{1, 2}, Savard, K^{1, 2}, Robidoux, PY^{1, 2}, ¹ Biotechnology Research Institute, National Research Council Canada, Montreal, Quebec, Canada² Research Centre for Environmental Toxicology (TOXEN), University of Quebec at Montreal, Montreal, Quebec, Canada
Bioavailability is now recognized as a main ecotoxicological concern for all media. The challenge is however that it constitutes a multifaceted parameter, which can be tackled with several approaches. In military Energetic Compounds (EC)-contaminated soils, exposure of organisms such as earthworms to various metals and EC either single or in combination may result in deleterious impacts. Those toxic effects are expected to be modulated by toxicant bioaccessibility and bioavailability. This paper reports on the suitability of chemical measures of metals and EM bioaccessibility for the scope of bioavailability assessment. In this study, the earthworm Eisenia andrei was exposed experimentally for 28 days to sublethal concentrations of Pb, HMX and mixtures of Pb + HMX spiked in a forest soil with high organic matter content. Standard endpoints (survival, growth and reproduction parameters) were determined. A suite of different biomarkers has also been used. Bioaccessibilities of Pb and HMX were measured using KNO₃ aqueous extracts at the beginning and the end of the exposure, both in presence and absence of worms. Data showed that HMX and mixtures of Pb+HMX decreased fertility and destabilized lysosomal membranes of earthworm coelomocytes. Because of the high organic carbon content in soil, bioaccessibility of Pb was low when compared to HMX. The regression analysis revealed a significant negative relationship between initial bioaccessible HMX concentrations and either number of juveniles (R²=-0.81, p≤0.05) or total number of cocoons (R²=-0.87, p≤0.05) or number of hatched cocoons (R²=-0.83, p≤0.05). Also, a significant incidence of earthworm activity on bioaccessible Pb concentrations was evidenced but not for HMX. This paper highlights the relevance of jointly using chemical and biological tools for a better bioavailability and toxicity assessment.

273 (SAM-1117-816958) Development of soil-to-plant tissue bioaccumulation models for energetic compounds and metabolites.
Start time: 10:20 AM
Sample, B.E.¹, Tsao, C.L.¹, Johnson, M.S.², ¹ CH2M HILL, Sacramento, CA, USA² US Army Center for Health Promotion and Preventive Medicine, Aberdeen Proving Ground, MD, USA
Concentrations of contaminants in tissue are needed to estimate exposure of birds and mammals. In the absence of measured data, models are used. Empirical models based on measured relationships are better than models based on chemical characteristics (i.e., Kow). We developed soil-to-plant tissue bioaccumulation models for 10 energetic compounds [HMX, perchlorate, RDX, tetryl, TNB, TNT, and its metabolites (2-ADNT, 4-ADNT, 2,4-DNT, and 2,6-DNT] representing 4 plant tissue types (leaf, root, fruit, and seed). These models were based on data from journal papers and the gray literature. The studies were pooled by chemical and tissues to determine whether linear relationships between chemical concentrations in soil and in plant tissues existed. Six chemicals (2-ADNT, 2,6-DNT, HMX, RDX, and TNT) had significant log-linear relationships between soil to at least one plant tissue compartment; inversely proportional relationship were found for TNT and 4-ADNT in soil to plant root. For the remaining chemical-to-tissue combinations, summary statistics of BAFs (ratio of soil to tissue) were generated.. Only 2,4-DNT and 4-ADNT had median soil-to-plant foliage BAFs of less than one; the remaining chemicals (2,6-DNT, 2-ADNT, HMX, perchlorate, RDX, tetryl, TNB, TNT and its metabolites) all had median foliage uptake values greater than one. Three chemicals had soil-to fruit uptake values (RDX, tetryl, and TNT). RDX is the only chemical that had a median uptake value of soil-to-fruit greater than one. For soil-to-root uptake values, RDX, tetryl, and TNT had median uptake values greater than one. Among the four chemical uptake data from soil-to-seed (perchlorate, RDX, tetryl, and TNT), only RDX had median uptake value greater than one.

274 (GON-1117-839355) Neurotoxicity of CL-20 in earthworm Eisenia fetida.
Start time: 10:40 AM
Gong, Ping¹, Inouye, L², Perkins, E², ¹ Analytical Services, Inc, Vicksburg, MS, USA² US Army ERDC, Vicksburg, MS, USA
Recent studies suggest that CL-20, a new energetic compound in contention for replacing current explosives, or its transformation products, have unforeseen negative impacts on terrestrial and aquatic ecosystems. CL-20 has been found highly toxic to soil invertebrates with EC20 values as low as 0.02 mg/kg soil. However, little is known about its toxicological mechanism. Based on previous observations of neurotoxicological symptoms, we hypothesized that CL-20 or its transformation products acted as neurotoxins to earthworm central nervous system. A noninvasive electrophysiological technique was employed to (1) assess the impacts of acute sublethal exposures on impulse conduction in the worms medial (MGF) and lateral (LGF) giant nerve fiber pathways, (2) to detect whether such effects were generalized or localized within various body regions, and (3) to evaluate the reversibility of neurotoxic effects. Earthworms (E. fetida) were exposed to 0.2 to 4 microgram/cm2 of CL-20 for one day on moistened filter paper. Conduction velocities of MGF and LGF were recorded on a digital oscilloscope before and after exposure. Results showed that even at the lowest exposure level worms exhibited physiological impacts such as retardation, stiffness and body shrink. There was no significant differentiation between different body regions, suggesting that neurotoxic impacts of CL-20 were generalized. Both MGF and LGF conduction velocities were negatively correlated with increasing doses of CL-20 (do you have a value for this correlation?). However, such neurotoxic effects were alleviated or even eliminated in 1-3 days after exposed worms were transferred to uncontaminated environment, depending on exposure doses. This indicates that the neurotoxicity is reversible after short-term exposure. However, the reversibility of chronic exposure needs to be further investigated.

275 (BAR-1117-658447) Purification of Japanese quail and rabbit cytosolic glutathione S-transferases capable of biotransforming CL-20.
Start time: 11:00 AM
Bardai, G¹, Hawari, J¹, Spear, P², Grosse, S¹, Hoang, J¹, Sunahara, G¹, ¹ Biotechnology Research Institute, National Research Council of Canada, Montreal, Quebec, Canada² Research Center for Environmental Toxicology (TOXEN), University of Quebec at Montreal, Montreal, Quebec, Canada
Hexanitrohexaazaisowurtzitane (CL-20), a highly energetic polycylic nitramine is an emerging environmental contaminant. Our earlier studies showed that adult quail exposed to CL-20 led to increased liver weight and liver aspartate aminotransferase activities, and yet CL-20 was not detectable in quail brain, heart, spleen, or liver. The possibility that adult quail liver may biotransform CL-20 was considered. Liver homogenates were prepared from adult female Japanese quail (Coturnix coturnix japonica) prior to the CL-20 biotransformation assays and the glutathione S-transferase (GST) purification procedures. Data indicates that the disappearance of CL-20 using whole cytosol was inhibited in vitro by either ethacrynic acid or the glutathione (GSH) analogue, s-octylglutathione, and suggested the involvment of GST. Using size exclusion, and affinity chromatography, we purified and characterized a cytosolic GST from quail liver capable of biotransforming CL-20. The molecular masses of the purified proteins were about 28 and 27 kDA. Partial N-terminal sequence analysis showed both alpha and mu classes of GST, having 100% homology to chicken and quail GST described in the literature. For comparative purposes, we also purified a rabbit GST enzyme. With the requirement of GSH, purified enzyme preparations from quail and rabbit livers biotransformed CL-20 at the rates (SD) of 0.27 (0.05) and 0.13 (0.02) nmol/min/mg protein, respectively. Data suggest that the purified protein can biotransform CL-20, as evidenced by the concomitant formation of nitrite (NO₂^-) with CL-20 removal. These data suggest that GST purified from female quail liver is capable of biotransforming CL-20 in-vitro, and may explain the absence of CL-20 in liver in-vivo.

276 (PAL-1117-689975) Examination of the potential effects of Instantaneous pressure changes on larval rainbow trout (Onchorhynchus mykiss).
Start time: 10:20 AM
Palace, V¹, Cott, P², Godard, D¹, Wautier, K¹, CLayton, D³, Wong, A⁴, Monita, D⁵, Hannah, B⁶, Morris, W⁷, Wright, D¹, ¹ Department of Fisheries and Oceans, Winnipeg, Manitoba, Canada² Department of Fisheries and Oceans, Yellowknife, NWT, Canada³ ConocoPhillips, Calgary, Alberta, Canada⁴ Encana Corporation, Calgary, Alberta, Canada⁵ Aquatics Environmental Services, Calgary, Alberta, Canada⁶ Alaska Department of Natural Resources, Fairbanks, Alaska, USA⁷ Fisheries Joint Management Committee, Inuvik, NWT, Canada
Industry and regulatory agencies in Canada acknowledge that the use of explosives under ice in fish bearing waters is potentially harmful to fish. The Department of Fisheries and Oceans, Western Arctic Area (DFO), has been closely monitoring explosives use and have been refining conditions and regulations surrounding their use. However, major gaps in our understanding of the effects of seismic activities using explosives on fish remain. For example, proven mitigation to minimize the pressure from shallow buried charges and methodologies that allow for more predictable pressures from set charges are absent. In addition, identifying the pressures that cause harm to northern over-wintering fish has become an issue of contention. Instantaneous pressure changes (IPC) from explosive charge detonation can have harmful effects on fish including damage to soft tissues from expansion beyond their elastic capacities. DFO has developed the guidelines for the use of explosives in waters such that maximum peak pressure not exceed 100kPa. This value was derived from the literature as an LD50 value for fish species in general. By virtue of being an LD50 derived value, it is not precautionary. In order to more accurately delineate IPC from explosive detonations levels that cause negative effects to fish early life stages of rainbow trout (Onchorhynchus mykiss) were subjected to a range of IPCs from explosive detonation (7 to 280kPa). Fish were held in cages suspended below ice and IPCs were monitored using seismographs and hydrophones capable of measuring a minimum of 16,000s-1 per channel. Exposed rainbow trout larvae and eggs were assessed for evidence of effects to craniofacial features, spinal curvatures, as well as soft tissues (ie. swimbladder) using microscopic and histological methods.