Herbicides applied in agriculture or weed control are regularly found in samples of surface waters. Bioassays with algae can be used to assess the effects of these herbicides to non-target aquatic organisms. For routine monitoring, such bioassays should be able to screen large amounts of low volume samples and give reproducible and accurate results within a short time period.
For this purpose Escher & Rutishauser (2007) developed a 24h-biotest using 96-well-microplates for simultaneously assessing the photosynthesis inhibition and effects on growth in green algae measured as optical density (OD). For herbicides inhibiting the cell division of algae, such as chloroacetanilide herbicides, this test might have a blind spot since OD detects effects on biomass but is less sensitive for effects on cell division. To analyze the quantitative importance of this blind spot the above mentioned biotest was expanded to the additional endpoint inhibition of reproduction (measured by means of cell number). Two substances were selected as model compounds for this study: the phenylurea herbicide diuron, which is a known photosynthesis inhibitor and is already used as a model substance in the 24h biotest, and the chloroacetanilide herbicide metazachlor.
Comparing the EC₅₀ values of the different endpoints photosynthesis inhibition (by means of quantum yield measurements), growth inhibition (by means of biomass measurements) and inhibition of reproduction identifies inhibition of reproduction to be the most sensitive endpoint for metazachlor, followed by growth and photosynthesis. The EC₅₀ determination for photosynthesis and growth inhibition was constrained by non-sigmoidal concentration-response relationships. For diuron, the endpoint photosynthesis inhibition is most sensitive, followed by inhibition of reproduction. Cell division inhibitors like metazachlor are not the only herbicides occurring in environmental samples, thus the effect of metazachlor has been assessed in mixtures with diuron. The effect concentrations of diuron/metazachlor mixtures showed good agreement with predictions based on the independent action concept for the endpoint inhibition of reproduction. For the photosynthesis and growth inhibition endpoints, the influence of the non-sigmoidal concentrationresponse relationship of metazachlor led to deviations in EC₅₀ values between experimental data and predictions. By predicting the EC₅₀ value of mixtures with various diuron:metazachlor ratios it could be shown that for ratios of 1:1.66 or higher, the combined algae test obtains sensitive results by measuring photosynthesis inhibition. When the metazachlor fraction is higher, measuring inhibition of reproduction obtained a more sensitive EC₅₀ value. The application of these findings on measured environmental concentrations in samples from two different running waters in the canton of Zurich suggests that in 96 % and 80 % of all samples sensitive EC50 values would be obtained by measuring photosynthesis inhibition. Thus, the combined algae test seems to be a suitable choice to assess the toxicity of environmental samples in the majority of cases. The general inclusion of the cell number measurement in order to calculate reproduction inhibition is not necessary since this endpoint is usually less sensitive than effects on the photosystem II. Such, an additional workload of at least 1.5 h per triplicate can be avoided. For the toxicity assessment of single cell division inhibitors or mixtures containing high fractions of such herbicides, it is advisable to extent the combined algae test to the endpoint inhibition of reproduction.