Algal Bioassay
Selenastrum capricornutum , (a coccoid unicellular green algae)
was cultivated in a nutrient stock solution containing macronutrients
NaNO₃, MgCl2.6H2O,
CaCl2.2H2O, MgSO₄.7H2O,
and K2HPO₄ (25.5, 12.2, 4.41, 14.7 and 1.04 mg
L-1, respectively), and micronutrients: H₃BO₃,
MnCl2.4H2O, ZnCl2,
CoCl2.6H2O,
CuCl2.2H2O,
Na2MoO₄.2H2O,
FeCl3.6H2O, and Na2SeO₄
(185 416, 3.27, 1.43, 0.012, 7.26, 160 and 2.39 µg
L-1, respectively). Light source intensity was 250 µM
m-2 s-1 of photosynthetically active
radiation (PAR). Temperature was kept at 21°C during the day. The
culture medium was aerated and disturbed twice daily by hand until cells
reached a stationary growth phase (10 d). To improve test performance
Na2EDTA.2H2O 300 µg
L-1 was added. The medium was buffered with NaHCO₃ 15
mg L-1. At the stationary phase, the medium was
centrifuged at 3000g, supernatant was discarded and pellets introduced
to P-free nutrient growth medium. When the algae cells started to turn
yellow (about 7 days), additional three days were allowed for ensuring
that the medium was suffered from starvation (Figure. 2).
There were three replicates for each sediment suspension by adding 100
mg of sediment to 50 mL of P-free nutrient culture medium. The
suspension was autoclaved at 121ºC and 15 psi to avoid microbial
contaminations. The algal bioassay commenced when the P-starved algae
was added to the test flask (250 mL erlnmeyer). The suspension of
sediment and P-free medium was inoculated by Selenastrum
capricornutum faced with P starvation to bring an initial flasks
concentration to 3×10⁴ cell mL-1. The the
cotton-stoppered flasks were incubated at a constant temperature and
light intensity (see above) and stirred manually twice a day to minimize
possible spatial variability in light radiation. Cell counting was
carried out using a hemocytometer counting chamber at the end of
experiment.