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The freshwater green alga Scenedesmus opoliensis proves to be a suitable bioindicator …

Biology Articles » Hydrobiology » Stress-physiological reactions of the green alga Scenedesmus opoliensis to water pollution with herbicides » Materials and methods

Materials and methods
- Stress-physiological reactions of the green alga Scenedesmus opoliensis to water pollution with herbicides

Axenic monoalgal cultures of Scenedesmus opoliensis P. Richter, strain AICB 141, obtained from the culture collection of the Biological Research Institute in Cluj-Napoca [9, 22], were grown for 10 days in BBM nutrient media [1] supplemented, according to the different experimental variants, with 10 ìM diuron (DCMU), 10 ìM methylviologen (MV, Paraquat) or 10 ìM glufosinate (GF, Phosphinotricine). The herbicides were added to the sterile nutrient media from stock solutions, with filtration through a Millipore filter with pore diameter of 0.22 ìm in order to maintain the axenity of media. The control cultures were kept in the BBM medium without herbicide. All experimental setups had 5 repetitions. The initial pH of all the culture media was adjusted to 6.5 and the cell suspensions were illuminated continuously with fluorescent lamps at a photon flux density of 135 micromoles m-2s-1 on the surface of the cultures that were stirred continuosly (400 rpm) in an algal growth chamber, at 20 °C [10].

The dynamics of cell divisions was evaluated cytometrically with a light microscope. The initial cell density of all the cultures was set to 52x104 cells per milliliter. The dry algal biomass of the cultures was determined after 10 days of development of the algal populations, when the control cultures were at the end of exponential growth phase. Net photosynthetic oxygen production of the algal cultures was measured with an Oxy-Lab oxymeter at a constant light intensity of 110 ìM photons m-2 s-1 and 20 °C [1, 12].

For determination of lipid peroxidation after 10 days of exposure to herbicides, algal suspensions were centrifuged at 2500g for 10 minutes and pellets were weighed. To each pellet 0.1% (w/v) trichloroacetic acid (TCA) was added in 1:3 (g/ml) ratio. Algae were disrupted in a Constant Systems cell disrupter, than the extracts were centrifuged at 6300g for 10 minutes at 10 °C. 0.5% (w/v) 2-thiobarbituric acid (TBA) solution in 20% (w/v) TCA was added to the harvested supernatants in the ratio 1:4 (v/v), in 10 ml thermoresistant glass tubes. The extracts were heated for 25 minutes at 96 °C and, after lowering temperature on ice, they were centrifuged at 6300g for 10 minutes at 10 °C. Determination of thiobarbituric acid-reactive substances (TBARS), consisting mostly in malondialdehyde, was performed photometrically on the supernatants, at A532 – A600 nm, using at extinction coefficient of 159.2 mM-1 cm-1 [15].

Catalase activity was evaluated spectrophotometrically by determining the consumption of H2O2 associated with a change in absorbance at 240 nm. Algal cultures were centrifuged at 2500g for 10 minutes. The testing medium contained 750 ìl of sodium phosphate buffer (50 mM, pH 7.5), 100 ìl H2O2 (200 mM), and 150 ìl of algal extract with enzyme (5 ìg of protein) in a final volume of 1 ml. Proteins were extracted with 1.5 ml of 0.1 M sodium phosphate buffer (pH 7) and the extract was centrifuged at 2300g for 20 minutes at 5 °C [14]. Ascorbic acid content of the algal cells was determined titrimetrically. 25 ml algal suspension was centrifuged at 2500g for 10 minutes, the pellet was resuspended in 10 ml of 5% (v/v) metaphosphoric acid, filtered and completed with 20 ml of 5% metaphosphoric acid. This extract was titrated with 0.025% (w/v) 2,6-dichlorophenol indophenol until a persistent light pink color appeared. Ascorbic acid content was determined with the help of a standard curve obtained with titration of known concentrations of ascorbic acid [20].

Every measurement was repeated 5 times. The experimental data were evaluated statistically, the significance of the differences among the control and the treated cultures was tested using one-way ANOVA (after verification of variance homogeneity with the Levene test), followed by the multiple comparison Tukey test (P < 0.05) [30].

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