Quantification was performed at 254 nm, while
plant tissue was watered daily so that field capac-
peak identities and purities were assessed by
ity was repeatedly exceeded. The leachate was
comparing sample and standard peak spectra and
collected and analyzed. The sum of all analytes
retention times. The detection limits for the ana-
from the leachates was compared to the total
lytes were approximately 0.1 mg/kg.
loading of explosives that was applied to the
Soil samples were taken by compositing the
plants.
contents of three 3/4-inch-diameter cores taken
For the contaminated soil experiments, kenaf
through the entire depth of soil in the pots. Root
seedlings were transplanted into pots with two
hairs were removed before analysis. Samples of
levels of explosives contamination and watered
plant material were taken by sacrificing individual
with tap water at intervals sufficient to maintain
plants, washing soil from the roots and cutting up
the soil at close to field capacity. One set of soil
the tissues with scissors, then air-drying. Samples
had TNT, RDX, and HMX at 1920, 6600, and 619
of soil weighing 2.00 g were added to 22-mL glass
mg/kg, respectively, and a second set had TNT,
vials; 10 mL of acetonitrile was added. Samples of
RDX, and HMX at 471, 1420, and 142 mg/kg,
tissue were of variable weights. The quantities of
respectively.
roots were small and most of each harvest could
Controls were kenaf plants grown in clean
sand and irrigated with tap water.
be extracted in one vial. As many stem and leaf
tissues as could be stuffed into a vial were extracted
with enough acetonitrile to cover them. After
Analytical methods
adding the acetonitrile, sample vials were sealed
Solvents used for extractions and analysis
with Teflon-lined caps and vortexed for one
were HPLC grade from Alltech (Deerfield, Illi-
minute. The vials were then placed in a cooled,
nois, USA). Acids, bases, and buffers were reagent
sonic bath and sonicated for 18 hours. Following
grade from Baker (Phillipsburg, New Jersey,
sonication, the extract was centrifuged and the
USA). Standards for HPLC analysis were made
supernatant decanted. A portion was mixed 1:1
from standard analytical reference material
with reagent-grade water containing 3.5 g/L of
(SARM) obtained from the U.S. Army Environ-
CaCl2, centrifuged again, and analyzed by HPLC.
mental Center (Aberdeen Proving Ground,
Maryland, USA). The diaminoNTs, azoxys, and
The acetonitrile extracts of stems and leaves were
nitroso-RDXs were supplied by Dr. Ronald
dark green. Most of the pigments were removed
by passing the extract through an Alumina-A solid-
Spanggord, SRI International (Menlo Park, Cali-
phase extraction cartridge prior to mixing with
fornia, USA). Solid-phase extraction cartridges
were Sep-PakRDX from Waters (Milford, Massa-
water.
chusetts, USA) and Alumina-A from Supelco
Residues from the acetonitrile extractions of
(Bellefonte, Pennsylvania, USA). Contaminated
soil and root tissues were repeatedly rinsed with
soils from several military sites were pooled to
acetonitrile/water mixtures, centrifuged, and
create a mixture that had sufficient TNT, RDX,
decanted until no more solvent-extractable anal-
and HMX for the experiments. A portion of this
ytes were detected. The exhaustively rinsed resi-
dues of soils and tissues were then air-dried in the
soil was extracted with acetone and was then
sample vials and 5 mL of 0.5 N NaOH added. The
diluted 1:1000 with reagent-grade water to create
samples were mixed for three minutes and soni-
the contaminated irrigation water.
cated overnight at 30C. Then, a 5-mL aliquot of
HPLC analysis was performed using a Waters
ice-cold, 50% H2SO4 was added, and the vials
system (717 autosampler, 616 pump, 600S con-
troller, 996 photodiode array detector, Millenium
were returned to the sonic bath for six hours.
workstation). A Phenomenex (Torrance, Califor-
After sonication, the contents of the vials were
nia, USA) Ultracarb 5 ODS(20) (4.6 mm 250 mm,
transferred to 125-mL flasks and neutralized to
5 m) reverse-phase column was used for the an-
pH 6.5 with approximately 75 mL of 1.0 M dibasic
alytical separations. The aqueous/ methanol
phosphate (pH = 8.6). The neutralized base/acid
(volume %/volume %) gradient elution time
digest was centrifuged and the supernatant
steps were as follows: start at 85/15, ramp to 65/
passed through a Sep-PakRDX solid-phase extrac-
35 at 8 minutes, ramp to 42/58 at 10 minutes and
tion cartridge, which retained the explosives and
hold for 13 minutes, ramp to 0/100 at 28 minutes
their transformation products. The cartridge was
and hold for 7 minutes, ramp down to 85/15 at 40
eluted with 5 mL of acetonitrile, which was then
minutes and hold for 10 minutes before the next
diluted 1:1 with reagent-grade water for HPLC
injection. The flow rate was 0.8 mL/minute.
analysis.
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