The objective of the work put forth by ER-2124 was to develop lines of evidence that environmental conditions at frontal boundaries enhanced 2,4,6, Trinitrotoluene (TNT) metabolism by natural microbial assemblages. This limited scope project primarily focused on demonstrating that frontal boundaries have enhanced rates of TNT mineralization, bacterial metabolism (i.e. heterotrophic production) and degradation of refractory organic matter (OM) (e.g. TNT, PAH, lignin). In addition, study of biogeochemical features of the interface that control these microbial rates and what cellular changes in the microbial assemblage occur in these environments (e.g. organotolerance) was initiated. If TNT metabolism rate can be related to heterotrophic production or OM transformation, then knowledge of general production and cycling rates may be applied to modeling TNT attenuation to predict the fate of TNT in coastal ecosystems. Three coastal water surveys and mixing experiments between water mass end members were performed to address this topic. By repeating these measurements and mixing experiments across other Department of Defense (DoD) relevant ecosystems, further identifying the specific areas and conditions associated with energetic degradation, researchers will increase the likelihood of public and regulatory acceptance of natural attenuation as a viable risk reduction alternative. Documenting and validating such natural pollution abatement mechanisms supports continued use of active DoD ranges.

Technical Approach

The goal of this limited scope project was to determine whether estuarine transition zones (e.g. fronts, convergences, salt wedges) are sites of enhanced 2,4,6,Trinitrotoluene (TNT) mineralization, bacterial metabolism and degradation of aromatic organic matter (e.g. petroleum hydrocarbons, lignin). Evidence was gathered to determine what biogeochemical features within these transition zones may contribute to such differences, and what cellular changes in the microbial assemblage may be indicative of these environments (e.g. organotolerance). Criteria for success of this investigation included measuring elevated rates of TNT mineralization and bacterial growth in mixing zones between water masses. These values will define the lability of TNT in natural ecosystems relative to other naturally-present forms of aromatic OM (e.g. lignin, humics, PAHs). Three coastal water surveys and mixing experiments between water mass end members were performed in 2011 to address these environmental topics: 1) Mississippi River/Gulf of Mexico (15-16 March; subtropical biome); 2) Charleston Harbor (25-26 June; temperate biome); and, 3) Kahana Bay (1 August; tropical biome).

Typically, water column (using CTD, Niskin bottles) and sediment samples (benthic grab, Wildco) were collected along salinity transects of these coastal systems with sampling concentrated across fronts between water masses and salt wedges.  Standard water quality measurements of dissolved oxygen (DO), salinity (practical salinity units, PSU), temperature (oC), pH were made with a hand-held YSI Multiprobeā„¢. 14C-TNT mineralization rates (TNT 14C-ring carbon conversion to 14CO2) were normalized to total heterotrophic carbon metabolism (3H-leucine incorporation) to account for differences in overall bacterial growth rate and transformation rates of other terrestrial aromatic organic matter (e.g. CDOM, lignin, naphthalene, phenanthrene).

Researchers expected that if there is no effect of water mass interface on TNT metabolism, then the relationship between salinity and TNT metabolism (i.e., mineralization) would be conservative.  That is, it would not be different from a linear mixing of end members with respect to salinity or DOC.  When there was metabolism different from that which was expected from conservative mixing, this was seen on a graph comparing these parameters with salinity or as a percentage mixture of two end members.  Those biogeochemical parameters that could be measured on similar spatial and temporal scales (e.g., DOC fluorescence, bacterial production, etc.) and that demonstrated a similar pattern of change with respect to salinity were considered candidates for further study, as they were putative factors that control TNT metabolism in nature.  Ultimately, the data was used to determine the likelihood of an energetic release impacting adjacent coastal waters and capacity of this ecosystem to attenuate these compounds.


Researchers found evidence that these water mass interfaces are sites of enhanced bacterial metabolism and TNT mineralization when examining salt wedges (Gulf of Mexico, Charleston Harbor), a frontal boundary (Charleston Harbor), and mixing experiments between freshwater and marine end members (Gulf of Mexico, Charleston Harbor, Kahana Bay). Two to 10-fold increase in bacterial growth rates across relatively short distances (meters) of a salt wedge (vertical fronts in Gulf of Mexico and Charleston Harbor) and a river convergence (horizontal front, Charleston Harbor) drove a large variation in degradation potential across stratified water masses. Likewise, TNT and phenanthrene mineralization rates were highest on both sides of the river convergence (Ashley and Cooper Rivers). Experimentally mixing freshwater and marine end members (Kahana Bay) resulted in higher rates of bacterial production (+62%), phenanthrene mineralization (+68%) and TNT mineralization (+33%) though the effect on production was time dependent (possibly a response to osmotic shock). These findings support the hypothesis that both overall heterotrophic metabolism and TNT mineralization are enhanced at frontal boundaries.

Measuring elevated TNT degradation rates amongst natural bacterial assemblages may reconcile the lack of detectable energetic concentrations in coastal sediment with their expected presence due to known long-term exposure to ordnance or surface runoff from a shore-side range. If TNT metabolism is related to overall bacterial growth on other aromatics, then a more extensive knowledge of environmental controls of natural aromatic biodegradation may be used to predict TNT fate in coastal ecosystems. The major outcome of this limited scope project was to demonstrate that energetics, such as TNT, are readily degraded by natural bacterial populations in estuarine and coastal environments that receive substantial terrestrial runoff rich in natural aromatic organic matter. By seasonally repeating these measurements and mixing experiments across other DoD-relevant sites and further identifying the specific areas associated with energetic degradation, researchers will increase the likelihood of public and regulatory acceptance of attenuation by natural bacterial assemblages as a viable risk reduction alternative.


Researchers found evidence that water mass interfaces are sites of enhanced bacterial metabolism and TNT mineralization in their examination. Mineralization of aromatic contaminants also appears closely related with natural aromatic OM indices. Moving forward with this proof of principle researchers could further examine the factors controlling TNT fate. In general, repeating these measurements seasonally and over different river flow regimes (at these sites and other Navy-relevant biomes) would make these important findings robust enough for publication in peer-reviewed scientific journals. Upon such validation, the applicability of such findings would be included to justify Monitored Natural Attenuation strategies or No Further Action decisions in cases where range emissions were exceeded by natural assemblage energetic metabolism. Understanding the relationships between biogeochemical parameters and TNT biodegradation amongst various ecosystem (biome) types may allow the determination of attenuation rates even at DoD sites with limited access to water and sediment samples due to the presence of Unexploded Ordnance (UXO).

Specific issues involving follow-on research the researchers would address include criteria for site selection, technical sampling issues involving the original hypothesis, and further developing findings from the mixing experiments. Ideally, the aggregate of study sites would be both DoD-relevant and comprise a large cross section of coastal environments and biome types. The collection of data could add to the body of knowledge of an ongoing or future site investigation, thereby reducing redundant chemical analyses and reference sampling cost to the DoD site manager. Seasonal sampling would address some of the natural variation in bacterial growth and mineralization rates in coastal ecosystems though this may be more important in temperate biomes than in tropical. Selecting a wide variety of ecosystems for study may allow the application of the findings to similar site where researchers have not yet collected data.

Technical sampling issues that would be addressed involve the spatial resolution of water column collection and determining the residence time of the mixed water mass. Relatively large heterogeneity (2-10 fold) was found with both bacterial production and TNT mineralization over small distances (m). Water collection bottles (20 L) on the shipboard Conductivity-Temperature-Depth Water Sampling Device (CTD) rosette were likely too large and vertically oriented to capture discreet water samples from the frontal interface (thus acting as mixing chambers themselves). A smaller, horizontal sampling bottle may be more useful in future water collection at both vertical and horizontal fronts.