Algal Bioassay
Selenastrum capricornutum , (a coccoid unicellular green algae) was cultivated in a nutrient stock solution containing macronutrients: NaNO₃ 25.5 mg L-1, MgCl2.6H2O 12.2 mg L-1, CaCl2.2H2O 4.41 mg L-1, MgSO₄.7H2O 14.7 mg L-1, K2HPO₄ 1.04 mg L-1, and micronutrients: H₃BO₃ 185 µg L-1, MnCl2.4H2O 416 µg L-1, ZnCl2 3.27 µg L-1, CoCl2.6H2O 1.43 µg L-1, CuCl2.2H2O 0.012 µg L-1, Na2MoO₄.2H2O 7.26 µg L-1, FeCl3.6H2O 160 µg L-1, Na2SeO₄ 2.39 µg L-1, at 25±1º C under continuous (250 µE m-2 s-1) ”Cool-White” fluorescent light and 3 cm3/s aeration flow rate, and shake 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. Culture medium was buffered with NaHCO₃ 15 mg L-1. The culture medium containing microalgae at a stationary phase was centrifuged at 3000g, supernatant was discarded and pellets introduced to P-free nutrient growth medium Severe phosphorus deficiency was estimated to occur when cells started to turn yellow (about 7 d)., allowing cells to grow for an additional 3 d to ensure that the culture was starved (Figure. 2).
The test begins when the algae are added to the test flasks. Mix the inoculum well, and add 1 mL to the test solution in each flask. Flask positions in the incubator was randomly rotated each day to minimize possible spatial differences in illumination. Three replicates of each sediment were prepared by adding 100 mg of homogeneous sediment to 50 mL of P-free nutrient growth medium in separate 250-mL Erlenmeyer flasks, autoclaved at 121ºC and 15 psi to avoid microbial contaminations. Flasks were inoculated with P-starved cell to give an initial flasks concentration 3×10⁴ cell mL-1, incubated at a constant temperature of 23±1º C, light intensity of 110 µE m-2s-1 and stirred manually twice a day. Cell counting was done using a hemocytometer counting chamber for 14 d.