2 Materials and Methods

Chemicals

TNT was obtained from Holston Army Ammunition Plant (AAP) in Kingsport, TN. 2,4,6-triaminotoluene trihydrochloride (TAT) was obtained from Chem Service, Inc., West Chester, PA. 2-amino-4,6-dinitrotoluene (2A46DNT), 4-amino-2,6-dinitrotoluene (4A26DNT), 2,4-diamino-6-nitrotoluene (24DA6NT), and 2,6-diamino-4-nitrotoluene (26DA4NT) were obtained from AccuStandard, Inc., New Haven, CT, and were 95 percent purity or greater.

Source of Inoculum for Biodegradation Studies

Biodegradation studies were carried out in serum bottles using sludge obtained from industrial and municipal wastewater treatment plants. The industrial wastewater treatment plant was located at the Holston AAP in Kingsport, TN. The treatment plant receives wastewater contaminated with RDX and HMX. Wastewater samples were collected from the beginning segment of the treatment plant (known as the anoxic filter) and were stored at 4°C until use. Anaerobic digester sludge from the municipal wastewater treatment plant for the city of Urbana, IL, was also used.

Serum Bottle Biodegradation Studies

Biodegradation of TNT was evaluated by comparing substrate disappearance in experimental bottles to that in sterile controls. TNT was added to serum bottles (160 ml total volume) containing 100 ml of a basal salts medium amended with 10 to 20 percent sludge (vol/vol). Sterile basal salts medium (steam sterilization, 121°C, 20 min) containing resazurin (0.0002 percent) was added to the serum bottles, followed by the addition of sludge. The basal salts medium consisted of the following per liter; NaCl, 0.8 g; NH₄Cl, 1.0 g; KCl, 0.1 g; MgSO₄ ^.·7H₂O, 0.02 g; KH₂PO₄, 1.35; K₂HPO₄, 1.75 g; NaHCO₃, 1.0 g; trace metal solution, 10 ml; vitamins, 10 ml. Trace metal and vitamin solutions were made as described by Tanner, McInerney, and Nagle (1989). The pH of the medium was adjusted to 7.2. The medium was prepared and dispensed using strict anoxic techniques as described by Shelton and Tiedje (1984). After addition of the sludge, the bottles were sealed with black butyl rubber stoppers and aluminum crimp seals. The headspace of the bottles was evacuated and replaced with a mixture of N₂:CO₂ (80:20) three times and then pressurized to 1.3 ATM. Sterile controls were prepared by adding HgCl₂ (5 mM final concentration) and autoclaving three times on three subsequent days (steam sterilization, 121 °C, 20 minutes). A stock solution of TNT (1,100 _M) was made in deionized water and added to the serum bottles to target concentrations ranging from 50 to 200 _M. The study was conducted in triplicate in the dark at room temperature.

Analytical Methods

TNT, 2A46DNT, 4A26DNT, 24DA6NT, and 26DA4NT were analyzed by reverse phase HPLC using a Waters LC Module 1 system or by capillary electro-phoresis as described by Chow et al. (1998).

For HPLC analysis, the following conditions were used initially for analyzing TNT, 2A46DNT, 4A26DNT, 24DA6NT, and 26DA4NT: mobile phase, 60:40 (acetonitrile:50 mM acetate buffer, pH 4.5); injection volume, 20 _l; flow rate, 1.1 ml/minute; wavelength, 254 nm; column, Lichrosphere C-18 reverse phase column (250 mm x 4.6 mm, 5 _m particles; Alltech Associates, Inc., Deerfield, IL). Because 2A46DNT and 4A26DNT co-eluted using this mobile phase, we were unable to quantitate these compounds.

Later analyses used a 50 percent methanol:50 percent acetate buffer (50 mM, pH 4.5) mobile phase with the other parameters identical to those listed above. This mobile phase resulted in the separation and quantitation of all TNT reduction products including 2A46DNT and 4A26DNT except triaminotoluene. Identification of unknown compounds was by comparison of their retention time with those of standards.

Triaminotoluene was analyzed using a Waters HPLC model 600E liquid chromatograph equipped with a 717 Plus autosampler and 991 photodiode array (PDA) detector. PDA data was collected from 200-310 nm, and extracted chromatograms were reproduced at 215 nm. HPLC analyses were performed with a Supelcosil LC-CN column (250 x 4.6 mm, 5 µm particles; Supelco, Bellefonte, PA) with a guard column of the same type. An HPLC gradient method retained triaminotoluene on the column in the beginning of the run, but eluted off the TNT reduction products toward the end of the run when acetonitrile made up a much higher portion of the mobile phase composition. The mobile phase consisted initially of 99 percent of 10 mM dibasic sodium phosphate buffer (pH 3.2, Aldrich Chemical Co.) and 1 percent of acetonitrile. The flow rate was 1.0 ml/min.

The following gradient mobile phase was used; the total elapsed time after each step gradient is given in parentheses. The initial conditions were 1 percent aceto-nitrile:99 percent phosphate buffer. After 5 minutes, acetonitrile was increased to 100 percent over a period of 1.5 minutes (6.5 min), maintained for 2 minutes (8.5 min), then decreased to 1 percent acetonitrile over 4.5 minutes (13 min) and maintained for 7 minutes (20 min). The total elapsed time between sample injections was 20 min.

The headspace of the serum bottles was monitored for the formation of CH₄ by gas chromatography. Gas samples were injected into a gas chromatograph (GC) equipped with a flame ionization detector and a Porapak-Q packed column (Alltech Associates, Inc., Deerfield, IL). The GC conditions were: helium flow rate, 30 ml/min; injector, oven, and detector temperatures, 75°C.

Hydrolysis Procedure

Transformation of TNT typically results in the formation of reduced TNT products, such as aminodinitrotoluenes and diaminonitrotoluenes. The reduction of the nitro group proceeds through the nitroso and hydroxylamino compounds (McCormick et al 1976). These intermediates are reactive and will bind to organic matter. The hydrolysis procedure, used for releasing TNT bound to organic matter, was carried out by a procedure developed by Thorne and Leggett (1997).

Sampling

Liquid samples were taken periodically from the serum bottles using a syringe and needle. Sample preparation for capillary electrophoresis consisted of centrifuging the samples at 12,000 x g for 4 minutes, followed by filtration (0.2 _m). Samples for HPLC analysis were mixed 1:1 with acetonitrile and stored at -20 °C until testing. After thawing, the samples were centrifuged (12,000 x g, 4 minutes). Due to the susceptibility of TAT to hydrolysis and abiotic degradation, samples were prepared as above, but run the same day they were taken.

Methane concentrations were determined by taking samples of the headspace gas (0.2 ml) using a 1-ml disposable syringe fitted with a 21-ga needle and injecting the sample directly into the GC as described above.