Algal toxicity tests with volatile and hazardous compounds in air-tight test flasks with CO2 enriched headspace

The application of an air-tight test flasks with a CO₂ enriched head space has been found useful for toxicity testing of volatile and hazardous chemicals with algae. The procedure has been demonstrated to perform well with the green algae Selenastrum capricornutum in tests of one or two days duration for a pH range of 7.0 - 8.5. The procedure makes it possible to maintain a constant pH as well as a constant and well defined concentration of rest compound during the entire test. To ensure a sufficient carbon supply for exponential growth of the algae through the whole test period and to avoid pH increase due to bicarbonate utilization, the headspace of the test flask is enriched with 1% or 2% CO₂ depending on the test duration, the algal growth rate and the inoculated algal density. The test medium is buffered with HCO₃- which is added to a (precalculated) concentration in equilibrium with the partial pressure of CO₂ in the head space at the end of the test, at the desired test pH. With 1% CO₂, approximately 12.5 mM of [HCO₃-] (which is high) must be added to obtain the pH of 8.1 prescribed in the ISO 8692 standard procedure. Therefore it is suggested to perform the closed test at pH of 7. Results with the method are shown for phenanthrene, a moderate volatile PAH compound (H = 3.6.10^-5 atm.m3/mole). The obtained EC10 and EC50 values were 10 times lower than those obtained with open test systems.