Methodology and evaluation of a highly sensitive algae toxicity test based on multiwell chlorophyll fluorescence imaging

A new phytotoxicity bioassay based on chlorophyll fluorescence imaging of algae suspensions in multiwell plates is introduced. Phytotoxicity is quantified via inhibition of photosystem II quantum yield, Y(II), assessed with the saturation pulse method. The basics of this approach as well as the factors enhancing and limiting its performance are outlined. Compared to other established techniques the new system allows exceptionally rapid and accurate measurements of phytotoxicity using pulse-amplitude-modulation (PAM) fluorometry. While instrument related errors are negligibly small, optimal performance depends on appropriate choice of algae and illumination conditions. Illustrative examples for the response of Phaeodactylum tricornutum to diuron are presented. The standard deviation involved in the Y(II) determination of a single well amounts to the equivalent of 44 ng/L diuron. A decisive role is played by the light (measuring light, saturation pulses, actinic light) to which samples are exposed during the bioassay: (1) the inhibitor response is enhanced at high measuring light intensity. (2) Saturation pulses may be considered non-invasive only, if applied at low frequency and as long as physiologically healthy algae cultures are used. (3) Continuous actinic light may be problematic, as it induces complex physiological reactions that limit the performance of the approach; it is not required for assessment of diuron-type inhibitors at high measuring light intensity.