1. Absorption, Distribution, and Transformation of Radiolabeled Trinitrotoluene Amy Palmer Dr. A. Morrie Craig Department of Biomedical Sciences

2. Relevance  Nitroaromatic compounds such as TNT contaminate soil and groundwater across the US.   -- 700,000 cubic yards of  soil  -- 10 BILLION gallons of  groundwater  Most of the contamination occurs on more than 16,000 Department of Defense facilities. CH 3 NO 2 O2NO2N

3. Background

4.  The cost of decontamination as it stands now is $35 billion.  TNT toxicity has human health risks Cataracts Hepatitis

5. Past Work  Plants alone: There have been many experiments on different types of plants, from the lower forms to higher plants, each showing that plants are able to handle only certain levels of TNT.  Microbes: There have been different types of microbe studies from bioslurries to rumen fluid microbes which have been performed by Dr. Craig previously.

6. Strategy Plants have been shown to break down TNT into metabolites, but are only metabolized down to the monoamines are which are still toxic. Ruminal animals, such as sheep, are able to ingest the plants that take in the TNT and its metabolites and break down TNT into the non-toxic metabolites with the help of rumen microbes.

7. Purpose  Determine if cool season grasses take in TNT from the contaminated soil and which of the three grasses does it the best  Determine if the plant had broken down the TNT to other metabolites and what those metabolites are.

8. Hypothesis  Cool season grasses such as tall fescue, perennial ryegrass, and orchard grass, will take in TNT contaminated soil and break it down into other metabolites.

9. Three Candidates Tall Fescue www.nps.gov/plants/alien/fact/loar1.htm Perennial Ryegrass www.allergyclinic.co.nz/guides/26.html Orchard Grass www.kiowacd.org/.../color pic orchardgrass.htm

10. Methods  Determine the absorption, distribution, and transformation of [ 14 C]-TNT from soil in three species of grasses  How:  -- Verify uptake of [ 14 C]-TNT through autoradiography and HPLC  -- Quantify uptake and bioremediation of TNT by the grasses with HPLC

11. Methods  Soil preparation  -- Each replicate had the same soil nutrients that are measured carefully.  -- The 14 C- TNT mixture with cold TNT that is dissolved in acetone was then S added.  Planting of the three types of seed (Tall Fescue, Perennial Ryegrass, Orchardgrass)  -- 24 pots of soil  -- Each pot yields between 20 to 50 individual seedlings K P N B Mg Zn Cu TNT

12. Methods  Harvesting of plant materials -- collect clippings every 60 days or when the seedlings are 6 inches tall 6 in. Cut at 2-3 in. above soil

13. Methods Clippings HPLC Plant Nutrient Analysis Combustion Analysis Each clipping was divided into five subgroups Incubation in Rumen Fluid Autoradiography

14. Methods  At each harvest and at the beginning of the experiment a plug of soil had been taken from two pots of each species designated for destructive soil sampling. The extraction had followed a modified EPA method 8330.   -- Concentrations of TNT were monitored  -- TNT had been determined by the radioactivity of the carbon

15. Results to date  During the method development for soil extraction, TNT was extractable in minute amounts.  These amounts of TNT show an accurate measurement of ppb that would be found in most contaminated areas.  The plants have now been planted and are starting to sprout seedlings.

16. Future Work  The plant experiment will continue through 4-5 harvests and then the results will be compiled.  The next step is to combine the plant portion of the experiment with the animal portion on actual sites.

17. Acknowledgements  Howard Hughes Medical Institute  Dr. A. Morrie Craig  Dr. Jennifer Duringer  USDA  Dr. Craig’s Lab  Dr. Kevin Ahern