the experiment. Individuals were exposed to the test concentrations
at 20 ± 2 C in a constant light dark photoperiod (16:8). After
96 h, the number of immobilized specimens (mortality) was determined
visually.
The toxicity tests with P. caudatum were carried out in a microplate
with wells containing 0.3 mL of the test dilution at 20 ± 2 C
and individuals were examined under a Laboval microscope (Carl
Zeiss, Jena). Ten individuals were exposed per well for 1 h, after
which the mortality was determined.
The germination experiments with R. sativus were carried out
on filter paper in Petri dishes. Five Petri dishes were used, each
of which was contained 5 seeds of radish. The test dilution
(5 mL) was placed into the dishes, and demineralized water was
used as a control. The dishes were placed in a germination chamber
22 ± 2 C at 6:18 dark-light regime. After 3 days, the root
lengths were measured.
For tests with P. caudatum, D. magna and R. sativus the percentage
inhibition (I, %) for each of the dilutions was determined by
comparing the number of immobilized or dead test organisms with
the number of organisms at the start. Subsequently, an inhibition
curve was fitted to calculate the lowest value of the dilution factor
of the water extract that exhibits 10% inhibition of a selected
biological response (LID10). For this, the inhibition values (I) for
each dilution were plotted against the corresponding dilution
factor. The desired values of EC10 were inferred from the intersection
of the straight lines with lines parallel to the abscissa at
ordinate values of 10%. Eluate was considered as not toxic if
LID10 was equal to 1.
In the test with oats (A. sativa) compost was added to the soil in
an amount of 10% by weight. The germination test was carried out
on gray forest soil (Haplic Greyzem). Each of 3 pots was sown with
10 oat seeds and after 10 days, the percentage germination and
root length were determined. The soil without added compost
was used as the control. The germination index was then calculated
according Zucconi (Zucconi et al., 1981).