Absorption, Distribution, and Transformation of Radiolabeled Trinitrotoluene

Amy PalmerDr. A. Morrie CraigDepartment of Biomedical Sciences

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.

CH3

NO2O2N

NO2

Background

Background

The cost of decontamination as it stands now is $35 billion.

TNT toxicity has human health risks

Cataracts Hepatitis

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.

Strategy

Plants have been shown to break down TNTinto metabolites, but are only metabolizeddown to the monoamines are which are stilltoxic. Ruminal animals, such as sheep, areable to ingest the plants that take in the TNTand its metabolites and break down TNT intothe non-toxic metabolites with the help ofrumen microbes.

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.

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.

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

Methods

Determine the absorption, distribution, and transformation of [14C]-TNT from soil in three species of grasses

How: -- Verify uptake of [14C]-TNT through

autoradiography and HPLC

-- Quantify uptake and bioremediation of TNT by the grasses with HPLC

Methods

Soil preparation -- Each replicate had the same soil nutrients

that are measured carefully. -- The 14C- 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

ZnCu

TNT

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

Methods

Clippings

HPLCPlant

NutrientAnalysis

CombustionAnalysis

Each clipping was divided into five subgroups

Incubation in Rumen Fluid

Autoradiography

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

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.

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.

Acknowledgements

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