The approved method for testing the efficacy of ballast water management systems with respect to killing 10–50 μm organisms uses movements of the organisms or the vital stains CMFDA/FDA. The present study demonstrates that certain freshwater coccoid chlorophytes, known or suspected to contain a highly resistant cell wall component (algaenan), stain poorly with CMFDA/FDA, resulting in false negatives. The staining rates for the most dominant species were determined and were approx. 3–70 %. The use of Crystal Violet as an indicator for the presence of algaenan gave inconclusive results. The number of the 10–50 μm organisms in a small pond was found to be 10,183 organisms/mL (Lugol's fixed sample) vs. 2335 organisms/mL (CMFDA/FDA-stained sample). Using the staining rates obtained, it was estimated that the number of false negatives could make 40–50 %. The implications for biological performance evaluation of ballast water management systems are discussed.