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.