A simple in vitro fluorescence method for biomass measurements in algal growth inhibition tests

Abstract

The estimation of biomass concentrations in algal growth inhibition tests from measurements of pigment fluorescence in extracts of 20% sample (final v/v) prepared by direct addition to dimethylsulfoxide/acetone solvent offers several advantages compared to currently used direct or indirect methods. The extraction stops the electron transfer and other processes which interact with chlorophyll fluorescence when measured in vivo. As a result the response is stabilized and the sensitivity improved. The injection method is very fast, has a high potential for automation, allows storage of samples and is suitable for small sample volumes (e.g., 0.2 ml). The typical initial cell density in standard toxicity tests of 10 4 cells ml −1 of Selenastrum capricornutum was measured precisely with a standard fluorimeter set-up, and 10 3 cells ml −1 of S. capricornutum was measured reliably with a sensitive fluorimeter. At low levels of toxicity by the model test compound potassium dichromate, the proposed fluorescence method resulted in very similar inhibition figures as obtained with electronic particle counting. At high levels of toxicity, on the other hand, biomass determinations from pigment fluorescence readings were markedly affected by toxicant-induced changes of the algal physiology. The low effect part of a dose response curve is normally that one of major interest, and biomass estimation errors associated with fluorescence measurements on extracts are thus considered acceptable in most situations. When the entire dataset was applied for endpoint estimation by the Weibull model, EC-1 estimates were markedly affected by the curve fitting to data in the high inhibition range, while EC-10 and EC-20 were less and EC-50 almost unaffected. The method is expected to be less suitable for toxicity testing of herbicides specifically inhibiting photosynthesis.