Flow cytometry as a diagnostic tool for the effects of polyphenolic allelochemicals on phytoplankton

Abstract

We investigated the impact of the polyphenol tannic acid (TA) on the green algae Desmodesmus armatus and Scenedesmus vacuolatus and the diatom Stephanodiscus minutulus in order to find new diagnostic tools for allelopathic effects on phytoplankton. Esterase activity, membrane integrity and production of reactive oxygen species (ROS) were tested using flow cytometry with specific fluorescent markers. For comparison, growth rate and photosynthesis, two variables known to be affected by TA, were evaluated. Algae were exposed to TA concentrations between 0.6 and 30μmolL−1 for 3, 14 and 24h. A significant inhibition of esterase activity was detected at every time point in all three tested algal species at 30μmolL−1 TA and in most other treatments when TA concentrations exceeded 3μmolL−1. A significant production of ROS could also be detected in all three algal species, but only after a longer exposure period. Changes in membrane rigidity revealed no consistent patterns of enhancement or inhibition when tested with different TA concentrations, algal species and exposure time. Growth rates of all algae were significantly inhibited after 24h, whereas D. armatus was the only species for which the photosynthetic yield did not decline.The effects on esterase activity and ROS production indicate a general influence of polyphenolic allelochemicals on phytoplankton, but also reveal patterns which vary between species, concentrations and exposure times. Changes in esterase activity were the most sensitive variable, and could be detected after short exposure periods and at naturally occurring concentrations. Thus, esterase activity may be a suitable variable for future investigations into the allelopathic effects of submerged macrophytes on phytoplankton.