*3.2. Toxicity Assay*

Acute toxicity was determined according to the Spirotox assay procedure [1]. Briefly, the assay was performed in 24-well polystyrene microplates. Five 2-fold dilutions were prepared directly in the multi-well plate. Each well contained 1 mL of the test solution and 10 protozoan cells. The microplates were incubated at 25 ◦C in darkness. Toxic e ffects (lethality, sublethal responses such as shortening, bending of the cell, immobilization) were noted after 1, 2, and 7 days of incubation. LC50, EC50 and EC20 values were calculated on the basis of lethal response (L) and all toxic e ffects (lethal and sublethal) (E), respectively. The toxicity values were expressed in mg/<sup>L</sup> on the basis of the initial concentrations of the tested compounds. The LC50, EC50 and EC20 values were determined by graphical interpolation of test response versus toxicant concentration (log scale) [3]. As the diluent and control, Tyrod solution [1] bu ffered with NaH2PO4 and Na2HPO4 (50 mM) was used. The toxicity of each compound was tested at pH 6.0; 6.5, and 7.4. All tests were performed in quadruplicate.
