Environmental Engineering Reference
In-Depth Information
water content of the plants was determined on the basis of fresh and dry mass by
using Drazic and Mihailovic's equation (Drazic and Mihailovic
2005
).
WC
=
(
FM DM DM
−
) /
(4.1)
(WC—water content; FM—fresh mass, DM—dry mass; in g/g DM).
Simultaneous phytotoxicity and mutagenicity assay was carried out on plant spe-
cies
Vicia sativa
L. var. Klára according to Miadoková et al. (
2001
,
2005
). After
24 h of soaking at 25 °C in distilled water or solution with metal concentration
equal to IC
50
value, the seeds of
V. sativa
were allowed to germinate in Petri dishes
(diameter = 18.5 cm) with filter paper soaked with the same concentration of tested
metal as that used for soaking. Phytotoxicity was assayed after 72 h of the dark cul-
tivation in the thermostat at 25 °C by the same way as for
S. alba
. The seedling roots
and shoots of
V. sativa
were measured and percent growth inhibition was assessed.
The seedling roots used for chromosome and genome mutability evaluation were
fixed and permanent slides were prepared by the Feulgen method. Chromosome
aberrations were determined at least in 500-anatelophases. For statistic analysis the
Student's t-test was used.
The procedures for maintaining the
Tradescantia
plants and for analyzing mi-
cronuclei frequency in the tetrads have been described by Mišík et al. (
2006
,
2007
).
Tradescantia paludosa
clone 03 was cultivated at the Department of Botany, Fac-
ulty of Natural Sciences, Comenius University in Bratislava, Slovakia. Inflores-
cences were harvested at the 8-10-bud stage and immersed into 500 mL of tested
metal solutions (100 mg/L CrO
3
and NiCl
2,
1,000 mg/L Cr(NO
3
)
3
) for 12 h. As
control tap-water was used. The 24 h reconvalescence, during which inflorescence
peduncles were dipped in 500 mL of tap-water, succeeded to 12 h exposure. Then
the buds were fixed for 24 h in ethanol: acetic acid (3:1). The fixed material was
stored in 70 % ethanol. Slides were prepared from the fixed material using the ace-
to-carmine squash technique. Micronuclei were scored in the early tetrad stages of
pollen mother cells. In the present study, 15-20 inflorescences were in a sample.
Three hundred tetrads were scored from each of five slides prepared from a treat-
ment sample for a total of 1,500 tetrads per plot. Data were recorded as the number
of micronuclei (MCN) per 100 tetrads. A change of frequency of MCN/100 tetrads
was considered statistically significant (at
P
< 0.05) if the difference between the
mean of the control population and the mean of the treated population was at least
twice as large as the standard error of the difference between the two means (Mišík
et al.
2007
; Ma et al.
1994
).
The salts of tested metals, NiCl
2
.6H
2
O, Cr(NO
3
)
3
.9H
2
O and CrO
3
of analytical
grade p.a., were obtained from Lachema, Brno, Czech Republic.
All experiments were set up in a completely randomized design with three rep-
licates. Chronic toxicity was assessed as inhibition of root and shoot growth and
the results were evaluated by the Gryck-Haustein method and IC
25
, IC
50
and IC
75
concentrations were determined. The results were statistically evaluated by using
the Toxicity program. For statistical evaluation of biomass production, statistical
program STATISTICA 8.1 was used.
