In vitro anaerobic trinitrotoluene (TNT) degradation with rumen fluid ...

Service. Two diaminomononitrotoluene congeners (24DA6NT and 26DA4NT) were
obtained from International Chem. Two monoaminodinitrotoluene congeners
(2A46DNT and 4A26DNT) and TAT were purchased from Aldrich Chemical Co. All
chemicals for experimentation were HPLC (high performance liquid chromatography)
grade.
2.3.2 Medium
The rumen contents for the study of in vitro TNT biotransformation were col-
lected from healthy rumen-cannulated ewes at the Animal Isolation Lab, Oregon State
University, Corvallis, OR. Approximately 40 grams of combined ruminal solids and
liquor were removed and transported to the laboratory. The ruminal contents were
anaerobically blended with a 90% buffer solution for three minutes in a mechanical
blender. The blended ruminal contents were transferred into 150 ml serum bottles
under an argon gas flow, and 10 mg of TNT in ethanol was added to each serum
bottle containing 100 ml of growth medium and stoppered with a rubber stopper. The
in vitro assay buffer contained 4.0 grams of Na2CO3, 0.57 grams of KC1, 9.3 grams of
Na2HPO4, 0.47 grams of NaCl, 0.1 grams of MgSO4 7H20, 0.04 grams of CaC12
2H20, 2.64 grams of (NH4)2SO4, and 0.002 grams of resazurin placed in a one-liter
container of deoxygenazed water. Bottles containing a 10 % rumen fluid mixture and
100 ppm of TNT were incubated at 38°C and sampled at time intervals. Aliquots of a
sample were indexed and frozen at -4°C until analysis by HPLC or other methods.
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