Algal growth inhibition test: does shading of coloured substances really matter?

Coloured substances are often assumed to behave differently in the algal growth inhibition test due to shading of the algae. We have investigated EU-notifications of new substances and found no significant differences in algal test results between colours and non-colours. A modified method to differentiate between shading and toxicity is not satisfactory, because the method used to determine the shading effect is incorrect. Due to the shaking of the test flasks in the algal test intermittent light conditions are achieved which lead to a higher growth rate than expected from proposed tests with constant reduced light conditions. This will result in overestimation of the shading effect and thus to underestimation of chemical toxicity. Furthermore, we showed in experiments with the reference substance potassium dichromate that reduced light can reduce the measurable toxicity of chemicals. Comparing our results with approaches proposed in the literature, a simple approach to reduce effects of undesired shading when testing coloured substances is to achieve light saturation for the algae by increasing illumination at least to the upper limit given in test guidelines (120 microEs(-1)m(-2)) and, optionally, to reduce the light path by choosing smaller test volumes.