O
2
N
CH
3
NO
2

NO
2
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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.

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


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.

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.


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.


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.

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




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

TNT
P
N






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
Mg
Zn
Cu
K
B


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

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


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




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.


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.

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