Metabolism of the explosive hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) in a contaminated vadose zone

The aim of this study was to explore biodegradation potential of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) in a deep contaminated unsaturated zone over Israel's coastal aquifer. While anaerobic biodegradation potential was observed throughout the profile down to the water table at a depth of 45 m, aerobic biodegradation was limited to the surface of the unsaturated zone. Traces of nitroso-RDX intermediates were detected in the soil samples, indicating possible in situ activity. Polymerase chain reaction and denaturing gradient gel electrophoresis analysis revealed that the microbial population in the soil consisted of protobacteria, but no known RDX degraders were detected. However, a 16S rRNA gene sequence most similar to Sphingomonas sp. was detected at all depths. Biodegradation rates were faster in the surface (0 and 1m) versus deeper soil samples (22 and 45 m) and were not affected under anaerobic conditions by the presence of nitrate, indicating a concurrent reduction of both compounds. RDX half-life in the surface soil was mostly dependent on carbon content and to lesser extent on soil moisture. Biomineralization of RDX to CO(2) was confirmed by incubating surface soil with (14)C-labeled RDX. An aerobic RDX-degrading bacterium, identified as Gordonia sp., was isolated from the soil: it degraded RDX aerobically and produced 4-nitro-2,4-diazabutanal. This study, the first to explore RDX biodegradation in the deep vadoze zone, indicates biodegradation potential throughout the profile, which is likely to support natural attenuation.     

Quantifying RDX biodegradation in groundwater using δ15N isotope analysis

Isotope analysis was used to examine the extent of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) biodegradation in groundwater along a ca. 1.35-km contamination plume. Biodegradation was proposed as a natural attenuating remediation method for the contaminated aquifer. By isotope analysis of RDX, the extent of biodegradation was found to reach up to 99.5% of the initial mass at a distance of 1.15–1.35 km down gradient from the contamination sources. A range of first-order biodegradation rates was calculated based on the degradation extents, with average half-life values ranging between 4.4 and 12.8 years for RDX biodegradation in the upper 15 m of the aquifer, assuming purely aerobic biodegradation, and between 10.9 and 31.2 years, assuming purely anaerobic biodegradation. Based on the geochemical data, an aerobic biodegradation pathway was suggested as the dominant attenuation process at the site. The calculated biodegradation rate was correlated with depth, showing decreasing degradation rates in deeper groundwater layers. Exceptionally low first-order kinetic constants were found in a borehole penetrating the bottom of the aquifer, with half life ranging between 85.0 to 161.5 years, assuming purely aerobic biodegradation, and between 207.5 and 394.3 years, assuming purely anaerobic biodegradation.The study showed that stable isotope fractionation analysis is a suitable tool to detect biodegradation of RDX in the environment. Our findings clearly indicated that RDX is naturally biodegraded in the contaminated aquifer. To the best of our knowledge, this is the first reported use of RDX isotope analysis to quantify its biodegradation in contaminated aquifers.     

Sequential biodegradation of TNT,RDX and HMX in a mixture

We describe TNT's inhibition of RDX and HMX anaerobic degradation in contaminated soil containing indigenous microbial populations. Biodegradation of RDX or HMX alone was markedly faster than their degradation in a mixture with TNT, implying biodegradation inhibition by the latter. The delay caused by the presence of TNT continued even after its disappearance and was linked to the presence of its intermediate, tetranitroazoxytoluene. PCR–DGGE analysis of cultures derived from the soil indicated a clear reduction in microbial biomass and diversity with increasing TNT concentration. At high-TNT concentrations (30 and 90 mg/L), only a single band, related to Clostridium nitrophenolicum, was observed after 3 days of incubation. We propose that the mechanism of TNT inhibition involves a cytotoxic effect on the RDX- and HMX-degrading microbial population. TNT inhibition in the top active soil can therefore initiate rapid transport of RDX and HMX to the less active subsurface and groundwater.     

Impact of microbial activity on the hydraulic properties of fractured chalk

The impact of microbial activity on fractured chalk transmissivity was investigated on a laboratory scale. Long-term experiments were conducted on six fractured chalk cores (20 cm diameter, 23-44 cm long) containing a single natural fracture embedded in a porous matrix. Biodegradation experiments were conducted under various conditions, including several substrate and oxygen concentrations and flow rates. 2,4,6-Tribromophenol (TBP) was used as a model contaminant (substrate). TBP biodegradation efficiency depended mainly on the amount of oxygen. However, under constant oxygen concentration at the core inlet, elevating the flow rates increased the removal rate of TBP. Transmissivity reduction was clearly related to TBP removal rate, following an initial slow decline and a further sharp decrease with time. The fracture's transmissivity was reduced by as much as 97% relative to the initial value, with no leveling off of the clogging process. For the most extreme cases, reductions of 262 and 157 microm in the equivalent hydraulic apertures were recorded for fractures with initial apertures of 495 and 207 microm, respectively. The reductions in fracture transmissivity occurred primarily because of clogging by bacterial cells and extracellular polymeric substances (EPS) produced by the bacteria. Most of the biodegradation activity was concentrated near the fracture inlet, where the most suitable biodegradation conditions (nutrients and oxygen) prevailed, suggesting that the clogging had occurred in that vicinity. The clogging must have changed the structure of the fracture void, thereby reducing the active volume participating in flow and transport processes. This phenomenon caused accelerated transport of non-reactive tracers and doubled the fracture's dispersivity under constant flow rates.     

Isotopes as tracers in a contaminated fractured chalk aquitard

Clusters of industrial plants often generate contaminant plumes with several potential sources. Prevention of further pollution and designing suitable remedial measures require identification of the contributing source among all potential ones and the sorting of currently active sources from historical ones. In the study area, an industrial complex in the Negev desert, Israel, contaminants could not serve as indicators for the contamination sources because of their extensive spatial distribution across the site. However, stable isotopes of oxygen, hydrogen and sulfur, as well as tritium, proved to be efficient tools for this task. The isotopic characterization of the potential end members provided the criteria for constraining a contaminating source when several alternative sources appeared viable. The isotopic fractionation of oxygen and hydrogen isotopes could be tied to the various disposal phases of the industrial wastewater. The three case studies presented here confirm the important role of isotopes as tracers in contaminated sites.     

Particle transport in unsaturated fractured chalk under arid conditions

A series of field and laboratory experiments were conducted to study the mechanisms of particle detachment and transport from fractures in vadose chalk. Experiments of intermittent flow events along fracture surfaces were carried out in the laboratory. In the field, water was percolated from land surface via a discrete fracture into a compartmental sampler installed inside a horizontal corehole located I m below the surface. The mass, size distribution, and composition of the particles drained from the fracture voids were examined along with flow rates and salt dissolution. Two boreholes penetrating the underlying saturated zone were sampled and analyzed for colloidal concentration and composition. Most of the particle and solute release at the drained effluents occurred during the first several hours of flow, but erratic pulses of particles were still observed after long periods of time. Most of the detached particles had a mean diameter of >2 microm, while the mobile colloidal phase in the groundwater had a mean diameter of approximately 1 microm. Mineralogical composition of the groundwater colloids and the particles detached from the upper vadose fracture were similar. Laboratory observations demonstrated the importance of the existence of a coating layer, made of weathered particles and salts, on particle detachment. The results of this study suggest that: (1) particle detachment causes flow-rate variability in the unsaturated fracture; (2) the mechanisms of particle detachment and salt dissolution within the fracture are linked: and (3) although most of the detached particles are large and likely to accumulate inside fractures, some colloidal particles also eroded from the fracture void and are likely to be transported to the groundwater.     

Two-dimensional distribution of microbial activity and flow patterns within naturally fractured chalk

The two-dimensional distribution of flow patterns and their dynamic change due to microbial activity were investigated in naturally fractured chalk cores. Long-term biodegradation experiments were conducted in two cores ( approximately 20 cm diameter, 31 and 44 cm long), intersected by a natural fracture. 2,4,6-tribromophenol (TBP) was used as a model contaminant and as the sole carbon source for aerobic microbial activity. The transmissivity of the fractures was continuously reduced due to biomass accumulation in the fracture concurrent with TBP biodegradation. From multi-tracer experiments conducted prior to and following the microbial activity, it was found that biomass accumulation causes redistribution of the preferential flow channels. Zones of slow flow near the fracture inlet were clogged, thus further diverting the flow through zones of fast flow, which were also partially clogged. Quantitative evaluation of biodegradation and bacterial counts supported the results of the multi-tracer tests, indicating that most of the bacterial activity occurs close to the inlet. The changing flow patterns, which control the nutrient supply, resulted in variations in the concentrations of the chemical constituents (TBP, bromide and oxygen), used as indicators of biodegradation.     

Evaluation of soil flushing potential for clean-up of desert soil contaminated by industrial wastewater

The flushing potential of a desert loess soil contaminated by the flame retardant Tetrabromobisphenol A (TBBPA), chloride (Cl(-)) and bromide (Br(-)) was studied in undisturbed laboratory column experiments (20 cm diameter, 45 cm long) and a small field plot (2 x 2 m). While the soluble inorganic ions (Cl(-) and Br(-)) were efficiently flushed from the soil profile after less than three pore volumes (PV) of water, about 50% of the initial amount of TBBPA in the soil was also flushed, despite its hydrophobic nature. TBBPA leaching was made possible due to a significant increase in the pH of the soil solution from 7.5 to 9, which increased TBBPA aqueous solubility. The remaining TBBPA mass in the soil was not mobilized from its initial location in the topsoil due to the decrease in pH at this horizon. In situ soil flushing demonstrated that this method is a feasible treatment for reducing soil contamination at this site.