are formed in the atmosphere by photochemical processes Mu¨ller et al., 1996; Hashimoto et al.,
1998. It is readily absorbed by roots and leaves and primarily translocated via the transpiration
stream system. However small amounts are trans- ported via the symplast system Blanchard, 1954.
The initial step of plant injury caused by TCA is regarded as a modification of protein structure
and such changes could alter enzyme activity and membrane permeability Ashton and Crafts,
1981.
On the other hand, when plants are subjected to stress of many kinds physical, chemical, bio-
logical, bursts of active oxygen occur within min- utes after exposure Foyer et al., 1994. Although
the formation of toxic oxygen species is generally considered to be detrimental to cellular function,
these molecules are formed in normal cell metabolism and their production and destruction
is a regulated phenomenon Asada, 1993. An imbalance between oxidants and antioxidants in
favor of the oxidants, potentially leading to dam- age, is termed oxidative stress Sies, 1997. Up to
date, there is no report of active oxygen species associated with TCA toxicity.
Plants are well adapted for minimizing damage that could occur from toxic oxygen species. The
natural antioxidative defence system Elstner, 1982; Winston, 1990; Smirnoff, 1993; Sies, 1997
includes three general classes: a lipid soluble, membrane-associated antioxidants e.g. alpha-to-
copherol and beta-carotene; b water soluble reductants e.g. tripeptide glutathione and ascor-
bate; and c enzymatic antioxidants including superoxide dismutase, catalase, peroxidase and
the enzymes involved in the synthesis and regener- ation of the reduced forms of the antioxidants
e.g. enzymatic pool of glutathione. This system is present in both intra- and extra-cellular com-
partments. Since oxidative stress comprises a complex set of phenomena, it is highly unlikely
that a single response will provide a general marker for it, thus simultaneous increase in sev-
eral components of the antioxidative defence sys- tem would be necessary in order to obtain a
substantial increase in stress tolerance Foyer et al., 1994. Plant antioxidant enzyme activities
were considered as relevant endpoints in different stress situations, they have been investigated
mainly with air pollutants and metals Keller, 1974; Van Assche et al., 1988 and reviews were
published giving some theoretical rationale for using biomarkers Ernst and Peterson, 1994;
Vangronsveld et al., 1997.
In the present investigation, three terrestrial plants, oat A6ena sati6a, Chinese cabbage
Brassica campestris cv. chinensis and lettuce Lactuca sati6a, were exposed to different con-
centrations of herbicide TCA in a growth test according to guideline of Organization for Eco-
nomic Cooperation and Development 1984. The phytotoxic effects of soil contaminated with TCA
were measured by classical endpoints i.e. germi- nation rate and biomass and by biochemical
endpoints antioxidant enzyme activities. The purposes of this work were i to investigate more
sensitive endpoints for evaluating TCA contami- nated soils; ii to examine the ratios of sensitivity
between classical and biochemical endpoints car- ried out simultaneously according to the same
phytotoxicity protocol. As a matter of fact, the utilization of guidelines offers the advantage of
minimizing differences in the generation of results, and thus comparison of phytotoxicity results can
be more realistic and feasible.
It is proposed that changes in enzyme activities might be used as plant biomarkers in the evalua-
tion of the phytotoxicity of soils contaminated by the chlorinated aliphatic herbicides.
2. Materials and methods
2
.
1
. Chemical, plant material, soil, and growth conditions
The chemical used in the tests was TCA as NaTCA; Aloricu, lot c 19078-0. Plant species
were oat A6ena sati6a L., Chinese cabbage Brassica campestris L. cv. chinensis and lettuce
Lactuca sati6a L., and the seeds have been sup- plied by a private company Jardins, Nancy,
France. Assays were performed according to the guideline OECD c 208 OECD, 1984. A sum-
mary of the procedure is given below: the refer- ence control soil was a natural loamy soil clay
26, silt 52, and sand 22 and samples were taken from the 0 – 25 cm surface layer. Some
characteristics of the soil include: carbon 1.5, organic matter 3.0, pH = 6.6, and CN 8.2. The
soil was dried 110 – 130°C; 30 h before use. Test concentrations were drop-pippeted into the soil
surface at 66 of the maximum water-holding capacity. In this study, maximum water-holding
capacity was 37 ml water per 100 g soil; thus, water dropped to controls was 25 ml and the
different toxic test concentrations were dropped at the same volume 25 ml in corresponding pot
treatments.
Three tests were performed to each plant and for each test three pots disposable plastic, diame-
ter 7 cm, height 5 cm were used for each TCA treatment or controls. Sixteen seeds for oats and
40 seeds for Chinese cabbage and lettuce were sown in each pot containing 100 g of soil. Water
evaporation was determined by daily weighing of pots and compensated by addition of distilled
water. Plants were grown under controlled condi- tions at a temperature regimen of 25 9 2°C day
and of 17 9 2°C night, with irradiation flux of 72 mE m
− 2
s
− 1
from fluorescent tubes under a 168 lightdark photoperiod. Ten days after sow-
ing, the plants were cut at soil level and the wet weight of the plant material was immediately
determined using an analytical balance. For each pot, the total weight of the germinated plants
expressed in g wet weight was divided by the number of germinated plants plants that devel-
oped roots at least 20 mm long were considered germinated. The pH of the soil was measured at
the
beginning and
end of
each ten-day
experiment.
2
.
2
. Assay of enzyme acti6ities Fresh weight biomass oat = 0.125 g ml
− 1
buffer, Chinese cabbage = 0.137 g ml
− 1
buffer, lettuce = 0.100 g ml
− 1
buffer for each replicate was homogenized using a mortar and pestle under
ice-cold conditions in 4 ml of 50 mM potassium phosphate buffer pH = 7.6. The samples were
sonicated on ice three times for 20 s to avoid sample overheating, centrifuged 10 000 × g; 10
min; 4°C, and supernatants were stored at − 20°C until subsequent measurements were carried
out at 25°C using a Uvikon Spectrometer 930 Kontron Instruments.
Superoxide dismutase EC 1.15.1.1 activity was measured by an indirect method Paoletti and
Mocali, 1990 following the oxidation of reduced nicotinamide adenine dinucleotide NADH at
340 nm. Prior to the assay, each sample was poured into a Pharmacia column containing Sep-
hadex G-25 equilibrated with 100 mM tri- ethanolamine-diethanolamine TDB-HCl buffer
pH = 7.4. In a 2.13 ml reaction volume contain- ing 75 mM TDB, 0.28 mM NADH, 2.301.15 mM
ethylenediaminetetraacetic acid EDTAMnCl
2
and 0.1 ml sample, 0.93 mM of beta-mercap- toethanol was added to start the reaction. Super-
oxide dismutase SOD activity was expressed in units according to Asada method Asada et al.,
1974, where 1 unit was defined as the amount of enzyme required to inhibit the rate of NADH
oxidation of the control by 50. The results were reported as specific activities Asada Units.
Peroxidase EC 1.11.1.7 activity was measured using a slightly see below modified version of
published method Byl et al., 1994. Just prior to the assay, 0.7 ml of solution A 810 mg phenol
and 25 mg 4-aminoantipyrene in 50 ml distilled water and 0.2 ml of sample were added to a 3 ml
glass cuvette. A 0.75 ml aliquot of solution B 0.01 of hydrogen peroxide in 100 mM 4-2-hy-
droxyethyl-1-piperazineethane
sulfonic acid
HEPES buffer pH = 7.1 was mixed in order to start the reaction and the change in absorbance
was monitored at 510 nm. Catalase EC 1.11.1.6 activity was determined
by following the consumption of hydrogen perox- ide at 240 nm Beers and Sizer, 1951. This proce-
dure was slightly modified as follows: 1 ml of 0.15 buffered hydrogen peroxide in potassium
phosphate buffer, pH = 7.6 was added to 1 ml of 50 mM potassium phosphate buffer pH = 7.6
and 0.1 ml of diluted sample.
Glutathione reductase EC 1.6.4.2 activity was assayed following NADPH oxidation at 340 nm
Bergmeyer et al., 1983. The final reaction vol- ume
contained 66.6
mM hydroxymethyl
aminomethane TRIS buffer pH = 8.0, 0.2 mM Na
2
EDTA, 12 mM oxidized glutathione GSSG, 6 mM NADPH and 0.1 ml of sample.
Activities of all enzymes except SOD were expressed in enzyme units per mg of protein,
where 1 enzyme unit is defined as a change of 0.01 absorbance min
− 1
caused by the enzyme sample. Protein sample content was determined using
bovine serum albumin as a standard Bradford, 1976.
2
.
3
. Statistics Three tests were performed for each plant, and
data for enzyme activity, biomass and germina- tion rate were generated in triplicate for each
treatment or control, thus n = 9. No observed effect concentration NOEC and lowest observed
effect concentration LOEC values were deter- mined using Williams’ test P 5 0.05 Williams,
1971 after checking homogeneity of variances with the Hartley test. The software TOXSTAT
3.0 University of Wyoming, Laramie, WY, USA was used for these different calculations. The
coefficient of variation is representative of re- peatability triplicates and of reproducibility
tests realized at three different times.
3. Results
