Viability assessed with the most probable number dilution culture method after chemical treatment of ballast water reveals the presence of false negatives from an approved vital stain method.

The present study compares the CMFDA/FDA + motility- and the Most Probable Number (MPN) Dilution Culture + Motility methods for testing the viability of ≥10-<50 µm organisms in chlorine treated ballast water. The results of both methods were within the regulatory compliance criterion <10 organisms/mL, but the MPN-method revealed that growth-outs did occur. While the CMFDA/FDA method showed <0.5 organisms/mL, the MPN-method gave approx. 6 organisms/mL. This demonstrated that false negatives, i.e. living but not stained organisms, may occur when using the CMFDA/FDA-method for compliance testing of chemical treated ballast water. Organisms surviving the treatment were primarily the dinoflagellate Scrippsiella sp. and various coccoid chlorophytes present in a brackish- and freshwater test, respectively. It is suggested that their resilience to the chemical treatment is the ability to transform into a temporary cyst (Scrippsiella sp.) or the presence of a chemical resistant cell wall (certain chlorophytes).

Inherent lack of CMFDA/FDA staining in certain algae and its implication for ballast water testing.

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.