Division 1.8.9 Incubation

Cap the test vessels with air-permeable stoppers. The vessels are shaken and placed in the culturing apparatus. During the test it is necessary to keep the algae in suspension and to facilitate transfer of CO₂. To this end, constant shaking or stirring should be used. The cultures should be maintained at a temperature in the range of 21 to 24 °C, controlled at ±2 °C. For species other than those listed in Appendix 1, e.g. tropical species, higher temperatures may be appropriate, providing that the validity criteria can be fulfilled. It is recommended to place the flasks randomly and to reposition them daily in the incubator.

The pH of the control medium should not increase by more than 1,5 units during the test. For metals and compounds that partly ionise at a pH around the test pH, it may be necessary to limit the pH drift to obtain reproducible and well defined results. A drift of < 0,5 pH units is technically feasible and can be achieved by ensuring an adequate CO₂ mass transfer rate from the surrounding air to the test solution, e.g. by increasing the shaking rate. Another possibility is to reduce the demand for CO₂ by reducing the initial biomass or the test duration.

The surface where the cultures are incubated should receive continuous, uniform fluorescent illumination e.g. of ‘cool-white’ or ‘daylight’ type. Strains of algae and cyanobacteria vary in their light requirements. The light intensity should be selected to suit the test organism used. For the recommended species of green algae, the light intensity at the level of the test solutions shall be selected from the range of 60-120 µE·m^–2·s^–1 when measured in the photosynthetically effective wavelength range of 400-700 nm using an appropriate receptor. Some species, in particular Anabaena flos-aquae, grow well at lower light intensities and may be damaged at high intensities. For such species an average light intensity in the range 40-60 µE·m^–2·s^–1 should be selected. (For light-measuring instruments calibrated in lux, an equivalent range of 4 440-8 880 lux for cool white light corresponds approximately to the recommended light intensity 60-120 µE·m^–2·s^–1). The light intensity shall not vary more than ±15 % from the average light intensity over the incubation area.