RP186 (LIU-1117-729748) Extraction and Analysis of HMX in Eggs by Liquid Chromatography-Mass Spectrometry.
Start time: 8:00 AM
Liu, J¹, Severt, S¹, Pan, X¹, Smith, P¹, Cobb, G¹, ¹ The Institute of Environmental and Human Health, Texas Tech University, Lubbock, TX, USA
An efficient sample extraction and cleanup method was developed for determination of trace amounts of Cyclotetramethylenetetranitramine (HMX) in eggs. The method included solvent extraction of HMX from eggs followed by cleanup using Florisil and Styrene-Divinyl Benzene (SDB) Solid Phase Extraction (SPE) cartridges. Egg aliquots were weighted in centrifuge tubes and 10 mL acetonitrile was added. After homogenization, the samples were sonicated for 1 hour. Each sample was centrifuged and all liquid was collected for cleanup to remove endogenous interfering compounds. After filtering, the extract was analysed by liquid chromatography coupled with a mass spectrometry (LC-MS) using a RP C-18 column, with a mobile phase containing 60% methanol and 40% water + 1 mM acetic acid. Studies were performed using eggs containing both fortified and incurred HMX at levels of 0.05-500 ng/mL. The limit of quantification is 0.1 ng/mL. The average recoveries of HMX from 1 g of eggs containing 10, 50, 500, 1000, 2000 ng/mL were 82-112%.

RP187 (SMI-1117-827929) Reproductive Effects of the Anaerobic RDX Metabolite TNX in Deer Mice.
Start time: 8:00 AM
Smith, J¹, Pan, X¹, Stormberg, A², Smith, E¹, Gentles, A¹, Cox, S¹, Cobb, G¹, ¹ The Institute of Environmental and Human Health, Texas Tech Univeristy² Idaho National Laboratory
Royal Demolition Explosive (RDX) contamination has been identified at areas of explosive manufacturing, processing, storage, and usage; and thus, potential exists for exposure to N-nitroso compounds (MNX (hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine, DNX (hexahydro-1,3-dinitroso-5-nitro-1,3,5-triazine), and TNX (hexahydro-1,3,5-trinitroso-1,3,5-triazine)) formed via anaerobic transformation of RDX. Following exposure to TNX, reproductive toxicity was evaluated in three litters of deer mice (Peromyscus maniculatus) offspring. TNX was administered ad libitum via drinking water at four doses-100 g/L, 10 g/L, 1 g/L, and control (0 g/L). Endpoints investigated include: reproductive success, offspring survival, offspring weight gain, offspring organ weights, liver TNX residue levels, and DNA microsatellite analysis. Data from this study indicate that TNX bioaccumulates in the liver and is associated with dose-dependent postpartum mortality, decrease in body weight from birth to weaning, and decrease in kidney weight in deer mice offspring. Currently, a follow up study is underway, and additional data may become available.

RP188 (CHE-1117-747525) Analysis of perchlorate in urine, milk, and plasma: comparison of LC-ESI-MS and IC-SCD.
Start time: 8:00 AM
Cheng, Qiuqiong¹, Cobb, George¹, Anderson, Todd¹, ¹ The Institute of Environmental and Human Health, Texas Tech University, Lubbock, TX, USA
Perchlorate has been shown to disrupt normal thyroid function. Perchlorate contamination in the environment has been widely reported in the U.S. Due to possible hazard that animals may experience with perchlorate exposure, perchlorate contamination monitoring has become important. Perchlorate residue in biological matrices such as urine, milk, and plasma can serve as non-lethal biomarkers for environmental perchlorate monitoring. However, there are usually interfering components in these samples, resulting high background conductivity and possible co-elution. Thus, the sensitivity and precision in perchlorate analysis by traditional ion chromatography with suppressed conductivity detection (IC-SCD) may not be satisfactory even with efficient cleanup methods for these matrices. Application of mass spectrometry to these analyses can solve this problem. Here we present a method developed for perchlorate determination in these complicated biological matrices using liquid chromatography electrospray ionization mass spectrometry (LC-ESI-MS), and a comparison of IC-SCD and LC-ESI-MS in analysis of perchlorate in these three biological fluids. Perchlorate in these matrices responded similarly for LC-ESI-MS. The instrument detection limit for the matrices under study was below 3 ng/mL (S/N=3). Good linearity (r² > 0.990) was obtained in the range of 2-200 ng/mL in all perchlorate-spiked matrices using the MS method. Unlike IC-SCD, no interference or high background problems were encountered with the MS method. The LC-ESI-MS method described here offers a significant improvement over IC-SCD with better precision (especially at low concentrations), perchlorate confirmation, and lower detection limits for perchlorate analysis. perchlorate, mass spectrometry, ion chromatography, urine, milk, plasma

RP189 (TRA-1117-652008) Formation and fate of the products formed from remediation of explosives using zero-valent iron.
Start time: 8:00 AM
Tratnyek, Paul¹, Johnson, Richard¹, Bandstra, Joel^{1, 2}, Bae, Bumhan^{1, 3}, ¹ Oregon Health & Science University, Portland, OR² Pennsylvania State University, University Park, PA³ Kyungwon University, Korea
In the presence of zero-valent iron (nano-sized, conventional reagent grade, and construction grade), explosives such as TNT and RDX are rapidly reduced. Thus, the kinetics of removal of the parent compounds is unlikely to be the limiting factor in design of treatment for these explosives using permeable reactive barriers. Even small quantities of nano-sized iron may be adequate to degrade explosives, and this could be deployed by injection into (deep) wells. With respect to products formed from these treatments, however, the situation is more complex because the products are hard to identify and quantify, dependent on conditions in complex ways, and potentially hazardous. By iterating between batch, column, field, and modeling approaches to studying the formation and fate of these products, we have gradually converged on a comprehensive understanding of the most important processes operating in this system. Along the way, we have had to reconcile our understanding with a number of at first surprising experimental observations. One of these surprises was that the products observed under batch conditions are quite different from those obtained under column of field conditions because of the effect that iron concentration has on branching among product formation pathways. Another is that the major product of TNT reduction, triaminotoluene, is quite stable under relevant conditions, even thought it is characteristically unstable under most laboratory and environmental conditions. Finally, we have found that the rate and products of RDX degradation by nano-sized iron is strongly affected by the presence of putative electron shuttles like fulvic acid, even though only very modest shuttle effects have been observed with other contaminants.

RP190 (AAA-1117-842826) Reproductive effects TNX (hexahydro-1,3,5-trinitroso-1,3,5-triazine) in deer mice (Peromyscus maniculatus): Testicular histopathology and hormonal effects.
Start time: 8:00 AM
Viņas, R¹, Gentles, A¹, Smith, J¹, Pan, X¹, Cobb, G¹, Kendall, R¹, Smith, E¹, ¹ The Institute of Environmental and Human Health, Texas Tech University, Lubbock, Texas, USA.
Hexahydro-1,3,5-trinitroso-1,3,5-triazine (TNX), a metabolite of royal demolition explosive (RDX, hexahydro-1,3,5-trinitro-1,3,5-triazine) has been used as an explosive for both military purposes and civilian applications worldwide. Data on the toxicologic effects of TNX in wildlife are currently limited. Thus, this study investigated the toxic effects of TNX on the reproductive system of developing male deer mice. Breeding pairs of laboratory deer mice were exposed to TNX during gestation, lactation, and directly to the pups for 24 days, during the post-lactation period via drinking water. The exposure concentrations were 100 g/L, 10 g/L, 1 g/L, and control (0 g/L). The animals were euthanized on postnatal day 45. Body weight and testicular weights were recorded. Plasma total testosterone concentration was determined by RIA and testicular morphology was evaluated using histological techniques. There were no significant effects on body weight or testicular weight. Hexahydro-1,3,5-trinitroso-1,3,5-triazine did not significantly alter total testosterone concentration. At this point the implication for long-term chronic exposure to TNX, following gestational and lactational exposure beyond PND 45 is not clear and needs to be evaluated. The impact of Hexahydro-1,3,5-trinitroso-1,3,5-triazine on testicular morphology following gestational, lactational, and post-lactational exposure will be presented.

RP191 (FRE-1117-834670) Effects of RDX and TNT metabolites on soil invertebrates.
Start time: 8:00 AM
Freitag, Christina¹, Anderson, Todd¹, ¹ Texas Tech University, Lubbock, TX, USA
A variety of energetic materials are produced and used by the DoD. These explosives can contaminate the environment at munitions testing grounds and at production facilities across the United States. First and second generation explosives and their metabolites may affect the environment in a variety of ways. While there are many research reports describing the effects of the parent compounds, few studies have been concerned with the metabolites of the parent materials. In the environment, RDX can be degraded to metabolites such as TNX and MNX. Trinitrotoluene (TNT) breaks down aerobically into a variety of metabolites including 2 amino-DNT, 4 amino-DNT, and 2,4-DNT. The house cricket (Acheta domestica) was used to initially identify whether these metabolites had an adverse effect egg development. TNX caused a significant decrease in cricket egg hatching success in sand at concentrations above 1 ug/g and completely inhibited hatching at 100 ug/g. Cricket egg hatching success in sand was not affected by MNX until concentrations reached 100 ug/g. The effects of the metabolites on pill bugs (Armadillidium vulgare) was also investigated in order to determine the bioavailability of the TNT metabolites in soil. The ability to characterize invertebrate exposure to these metabolites is important in beginning to assess the potential biological effects of explosive metabolites in soil.

RP192 (WIN-1117-833178) The development of regression formulas for explosive bioaccumulation and the utility of short-term screening bioassays.
Start time: 8:00 AM
Winfield, L¹, Kiker, G¹, Farrar, D¹, Clarke, J¹, Ragan, D², Graham, T², ¹ US Army Engineer Research & Development Center, Vicksburg, MS, USA² Alcorn State University, Lorman, MS, USA
Inadequate data on the impact of explosives to terrestrial plants contribute to the high level of uncertainty and resultant costs associated with risk assessments and related activities. The development of regression formulas for RDX and HMX bioaccumulation by terrestrial plants and rapid screening bioassays to assess for their bioavailability or toxicity would provide valuable tools to address this issue. Studies were conducted to assess the impacts of short-term (<12 days) and long-term (7-13 weeks) RDX and HMX exposures. Two explosive sensitive plants, sunflower and sanfroin, were germinated and grown in soil amended with graded concentrations of RDX or HMX. Indicators of phytotoxicity were developed during all experiments. Several exposure durations were used during the long term experiments to determine the optimal exposure needed to develop regression formulas for RDX and HMX bioaccumulation and to evaluate plant uptake of both explosives. HMX was taken up and translocated to plant leaves at slower rates by both plants. However, greater numbers of adverse developmental effects were observed at lower HMX (5 mg g ¹ concentrations when compared with RDX (50 mg g ¹). RDX bioaccumulation regression formulas from these studies have been included in a Questions and Decisions (QnD) model developed to facilitate linkages between risk characterization information, remediation/management options, and stake-holder values. HMX bioaccumulation regression formulas are scheduled for future incorporation. The short-term bioassays are cost-effective screening tools that are potentially valuable for risk assessments, site characterizations, and monitoring activities.

RP193 (KOL-1117-808946) Reproducibility of Standard Curves for Analytical Determination of Explosives by HPLC.
Start time: 8:00 AM
Kolakowski, J¹, Checkai, R¹, Simini, M¹, Kuperman, R¹, Phillips, C¹, Kurnas, C¹, ¹ US Army Edgewood Chemical Biological Center, Aberdeen Proving Ground, Maryland, USA
A modified USEPA Method 8330 was used to analyze environmental media in order to analytically determine the concentrations of six military explosives by high performance liquid chromatography (HPLC) in support of various soil toxicity tests. More than 4,500 individual samples were analyzed over a 4-year period. The explosives for which we regularly prepared standard curves included the cyclic nitramine energetic materials (EMs) hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), and octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX), as well as the nitroaromatic EMs 2,4,6-trinitrotoluene (TNT), 2,4-dinitrotoluene (2,4-DNT), 2,6-dinitrotoluene (2,6-DNT), and 1,3,5-trinitrobenzene (TNB). Concentrations of explosives in samples were calculated using linear regression analyses of the peak areas produced by 7 and 8 point calibration curves, obtained by analyzing standard solutions ranging in concentration between 0 and 30 parts per million (ppm; mg/L). The overall mean r-squared value for the standard curves of each of the EM analytes analytically determined was r2 >0.99. Standard curve slopes were 2.998 (11.5 %RSD) for RDX (666 standards analyzed), r2 =0.9990 +/- 0.0039; 2.940 (21.0 %RSD) for HMX (303 standards analyzed), r2 =0.9989 +/- 0.0014; 6.359 (5.6 %RSD) for TNT (830 standards analyzed), r2 =0.9995 +/- 0.0007; 5.839 (27.5 %RSD) for 2,4-DNT (183 standards analyzed), r2 =0.9987 +/- 0.0022; 2.860 (7.6 %RSD) for 2,6-DNT (99 standards analyzed), r2 =0.9997 +/- 0.0007; and 5.205 (17.8 %RSD) for TNB (143 standards analyzed), r2 =0.9968 +/- 0.0046.

RP194 (SIM-1117-649634) Toxicity of RDX to the earthworm Eisenia fetida in soils with contrasting characteristics.
Start time: 8:00 AM
Simini, M¹, Checkai, R¹, Kuperman, R¹, Phillips, C¹, Kolakowski, J¹, ¹ U.S. Army Edgewood Chemical Biological Center, Aberdeen Proving Ground, Maryland, USA
We investigated the toxicity of the cyclonitramine, hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) to the earthworm Eisenia fetida in five natural soils that vary in organic matter, clay content, and pH: Sassafras sandy loam (SSL), Teller sandy loam (TSL), Richfield clay loam (RCL), Kirkland clay loam (KCL), and Webster clay loam (WCL). According to Ecological Soil Screening Level (Eco-SSL) criteria, relative bioavailability scores for organic chemicals were considered high for SSL and TSL, medium for RCL and KCL, and low for WCL soil. We adapted standardized toxicity tests using soils amended with RDX, and then subjected to moist/dry cycles (10-14 weeks) to simulate field conditions. We analyzed earthworm reproduction data using nonlinear regression models to determine RDX concentrations causing a 20% (EC₂₀) or 50% (EC₅₀) reduction in measurement endpoints. Preliminary results showed that RDX toxicity to earthworm production of both cocoons and juveniles was, from greatest to least: RCL > TSL > SSL > KCL > WCL. Results of regression analyses will be presented. Toxicity of RDX to earthworm reproduction in TSL, SSL, KCL, and WCL soils appeared to follow relative bioavailability scoring criteria. Contaminated WCL, with low relative bioavailability, was least toxic of the five soils tested. RDX toxicity to earthworm reproduction was relatively high in the high relative bioavailability soils that have lowest organic matter, clay content, and pH (SSL and TSL). However, RDX toxicity to earthworm reproduction was much greater than expected in RCL based on the relatively high organic matter and clay content of this soil. Characteristics unique to RCL soil may have been responsible for increased toxicity. Results of these studies, including the toxicity benchmarks derived, will be submitted to the Eco-SSL Task Group to undergo quality assurance before inclusion in the Eco-SSL database, and subsequent use in developing an Eco-SSL for RDX and soil invertebrates.

RP195 (ROS-1117-819479) Toxicity and bioaccumulation of explosive compounds in the marine mussel, Mytilus galloprovincialis.
Start time: 8:00 AM
Rosen, Gunther¹, Lotufo, Guilherme², Wild, William¹, ¹ Space and Naval Warfare Systems Center San Diego, San Diego, CA, USA² U.S. Army Engineer Research and Development Center, Vicksburg, MS, USA
The toxic and bioaccumulative potential of the explosive compounds, 2,4,6-trinitrotoluene (TNT), hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), and octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) were assessed in separate water only exposures with the Mediterranean mussel (Mytilus galloprovincialis). Two toxicity endpoints were assessed, including normal development of larvae and adult survival in 48 and 96-h exposures, respectively. Larvae were an order of magnitude more sensitive to TNT compared to adults, with EC50 values of 0.92 and 11.51 mg/L, respectively. Neither life stage exhibited a response to RDX or HMX at the highest concentrations tested (30 and 2 mg/L, respectively). Kinetically derived bioconcentration factors (BCFs) in adults were 1.61, 0.87, and 0.44, for TNT, RDX, and HMX, respectively, confirming the expected low bioaccumulative potential of these weakly hydrophobic compounds. Uptake and elimination rates for each compound will be presented. Based on these data, it appears that exposure conditions likely associated with the presence of explosives in the marine environment are unlikely to pose unacceptable risks to mussels, and suggests that potential for trophic transfer is quite low.

RP196 (PAN-1117-665562) Analysis of Octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) by Liquid Chromatography / Electron Spray Ionization / Mass Spectrometry (LC-ESI-MS).
Start time: 8:00 AM
Pan, XP¹, Tian, K¹, Cobb, G¹, ¹ Texas Tech University, Lubbock, TX, USA
We have developed a simple, sensitive LC-ESI-MS method to analyze octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) in environmental samples. HMX per se is difficult to ionize using an electrospray ionization (ESI) source. We investigated the HMX adduct formation efficiencies of several compounds (formic acid, acetic acid, propionic acid, dichloropropionic acid, ammonium nitrate, ammonium chloride, ammonium acetate, sodium nitrate, sodium nitrite). Acetic acid gave the most intensive and stable adduct ions, and thus was used as an additive for HMX analysis. Optimized conditions were as follows: separation was performed with a reverse phase (RP) C18 column (4.6 * 250 mm, 0.005 mm packing); the mobile phase, containing 0.4 mM acetic acid in the mixture of methanol, isopropanol, and water (50:10:40, v/v/v), was delivered at flow rate of 0.5 ml/min; the MS was operated in negative ion mode. The method detection limit was 0.78 pg for HMX in standard solutions using selected ion monitoring (SIM). Linearity (R2>0.990) was obtained at low concentrations (0.1-100 ng/ml). The repeatability and accuracy of the method were acceptable over a wide concentration range. The system optimization included HPLC and MS operation conditions along with selection of various eluent additives. This method performed well in analysis of aqueous waste samples from ongoing animal dosing studies.

RP197 (BEN-1117-828374) Development and Characterization of a Prototype Explosives-Contaminated Soil Reference Material to Support Environmental Measurements.
Start time: 8:00 AM
Benner, Jr., B.¹, MacCrehan, W.¹, ¹ NIST, Gaithersburg, Maryland, USA
The demilitarization of proving ground sites will certainly continue into the future and associated with this activity will be efforts to remediate sites contaminated with explosives. Therefore, an explosives contaminated soil reference material characterized for energetics and degradation products is needed to validate the analytical measurements that verify the effectiveness of remediation of previously contaminated sites. Toward that goal, a portion of a bulk explosives-contaminated soil was dried, sieved, homogenized and irradiated with a ⁶⁰Co source to minimize microbial degradation of the inherent explosives (particularly 2,4,6-trinitrotoluene). This material was then extracted by pressurized fluid extraction using acetone at 100 °C and 2000 psi and characterized for energetics and degradation products by gas chromatography/ negative ion chemical ionization mass spectrometry (GC/NICI-MS) and liquid chromatography/ mass spectrometry (LC/MS) using isotopically-labeled energetic standards. The energetics measured included 2,4,6-trinitrotoluene (TNT), 1,3,5-trinitro-1,3,5-triazacyclohexane (RDX), and 1,3,5,7-tetranitro-1,3,5,7-tetrazacyclooctane (HMX), and the TNT degradation products included dinitrotoluene and microbially-produced amino-dinitrotoluene isomers. This presentation will discuss challenges encountered during the preparation and chemical analyses of this prototype material, including the assessment of the stability of some of the analytes one year after ⁶⁰Co irradiation.

RP198 (ZHA-1117-140268) Acute Toxicity and Bioavailability of Hexahydro-1,3,5-trinitro-1,3,5-triazacyclohexane (RDX) Metabolites to the Earthworm, Eisenia fetida.
Start time: 8:00 AM
Zhang, B¹, Anderson, T¹, ¹ Texas Tech University, Lubbock, TX, USA
MNX and TNX are two important metabolites of the common explosive RDX. Although several studies have demonstrated that RDX is toxic to a number of organisms, including humans, the toxicity of MNX and TNX is not known. In this study, acute toxicity and bioavailability of MNX and TNX to Eisenia fetida was evaluated. Results indicate that both MNX and TNX had lethal and sublethal effects on earthworms. Exposure to MNX- or TNX-contaminated soil caused a significant concentration-dependent decrease in survival and growth of earthworms. Earthworms were more sensitive to TNX than to MNX. MNX and TNX were more toxic in sandy loam soil than in silt loam soil. The Lowest Observed Lethal Concentration (LOLC) for both MNX and TNX was 100 mg/kg in the sandy loam soil, and 200 mg/kg in the silt loam soil. At 7 days of exposure, the Lowest Observed Effect Concentration (LOEC) for earthworm growth was 50 mg/kg for TNX and 100 mg/kg for MNX in both soil types. The kinetics of MNX and TNX uptake into passive sampling devices (PSDs), and the ability of PSDs to serve as surrogates for evaluating bioavailability of RDX metabolites were also investigated. MNX and TNX absorption into PSDs was best fitted with a polynomial curve model: y = ax2 + bx + c (y: amount of MNX or TNX absorbed into PSD; x: incubation time of PSDs in soil), with an excellent correlation coefficient (r2 > 0.95). A linear relationship between PSD uptake and earthworm uptake was observed. The correlation coefficients were > 0.90 in almost all test soils spiked with MNX or TNX. These data indicate that C18 PSDs may be used as a surrogate for soil organisms such as earthworms and provide a simple chemical test for assessing the bioavailability of contaminants in soils.

RP199 (RIC-1117-835444) Human exposure to perchlorate in milk depends on its concentrations in dairy feed.
Start time: 8:00 AM
Rice, C.¹, Abbott, L.², Hapeman, C.¹, McCarty, G.¹, Baldwin, R.³, Capuco, A.³, Hare, W.⁴, Paape, M.³, McConnell, L.¹, Van Tassell, C.³, ¹ U.S. Department of Agriculture, Environmental Quality Lab, Beltsville, MD, USA² USDA, Office of Risk Assessment and Cost Benefit Analysis, Washington D.C., D.C., USA³ USDA, Bovine Functional Genomics Laboratory, Beltsville, MD, USA⁴ USDA, Veterinary Service, Beltsville, MD, USA
The concern for human exposures to perchlorate has been a subject of intense debate for several years. Adding to this debate are recent discoveries of low levels of perchlorate in milk. Determining the extent to which perchlorate in feed is transferred to milk will aid in managing human exposure. We conducted an experiment where 16 lactating dairy cows were dosed with ruminally-infused perchlorate. From these data, a dose-response relationship was established. The concentrations in the milk was directly related to the levels in the feed; however, especially important was the discovery that the efficiency of transfer to the milk diminished in a regular and predictable pattern as the concentration in the food increased. Using USDA Food and Nutrition Survey data and USDA Nutritional Information data, potential perchlorate exposures from milk were determined. The findings emphasize the need to better understand how perchlorate cycles through the environment and ends up in dairy feed.