Evaluating the response of freshwater organisms to vital staining

The unintentional introduction of nonindigenous species by ballast water discharge is one of the greatest threats to biodiversity in freshwater systems. Proposed international regulations for ballast water management will require enumeration of viable plankton in ballast water. In this study we analyze the efficacy of vital stains in determining viability of freshwater taxa. The efficacy of vital stains fluorescein diacetate (FDA) and FDA+5-chloromethylfluorescein diacetate (CMFDA) was evaluated with freshwater macroinvertebrates, zooplankton, and phytoplankton. Macroinvertebrates were cultured in laboratory, while plankton were collected from Hamilton Harbour and ballast tanks of commercial vessels. Organisms were subjected to various treatments (i.e., heat, NaClO, and NaOH) to establish efficacy of stains for viable and non-viable organisms. No significant difference in accuracy rate was found between stains, regardless of treatment, within groups of organisms, indicating that the addition of CMFDA is superfluous in the sample region studied. False positive errors, in which dead organisms fluoresced similarly to live organisms, occurred in most groups and were significantly different between test groups. False positive error rates were 2.3% for phytoplankton, 20% for ballast water zooplankton, 35% for Hamilton Harbour zooplankton and 47% for macroinvertebrates. Response to stains varied between taxonomic groups. Low (< 10%) false positive error rates were observed with phytoplankton, softbodied rotifers, oligochaetes, and Bosmina spp., while rates between 20% and 50% were observed for Daphnia spp., Hexagenia sp., and Chironomus riparius. False positive rates of copepods, Hyalella azteca, and Hemimysis anomala were between 70% and 100%. The FDA/FDA+CMFDA vital staining methods provide useful tools for viability analysis of freshwater phytoplankton, soft-bodied invertebrates and zooplankton, and may be used for viability analysis of the ≥ 10 μm to < 50 μm size fraction in compliance testing of ballast water. However, viability analysis of larger freshwater crustaceans with vital stains should be undertaken with caution.