The Biological Basis for Ballast Water Performance Standards: "Viable/Non-Viable" or "Live/Dead"?

The shipping industry is critical to international commerce; however, contemporary shipping practices involve uptake and discharge of ballast water, which introduces the potential for transfer of nonindigenous, invasive species among geographically distinct habitats. To counteract this hazard, regulations for ballast water management have been implemented by the International Maritime Organization (IMO) and by regulatory agencies such as the United States Coast Guard (USCG). IMO and USCG discharge standards are numerically identical, but involve different definitions of treatment end points, which are based on fundamentally different biological assays for quantification of ballast water treatment effectiveness. Available assays for quantification of the responses of organisms in the 10-50 μm size range include vital stains based on fluorescein diacetate (FDA), sometimes used in combination with 5-chloromethylfluorescein diacetate (CMFDA), observations of motility, and the most probable number dilution culture method (MPN). The mechanisms and implications of these assays are discussed relative to the Type Approval process, which quantitatively evaluates compliance with ballast water discharge standards (BWDSs) under controlled shipboard and land-based tests. For antimicrobial processes that accomplish treatment by preventing subsequent replication of the target species, the FDA/CMFDA and MPN methods can yield dramatically different results. An important example of a treatment process that is affected by the choice of assay is ultraviolet (UV) irradiation. Results of laboratory and field experiments have demonstrated UV-based technologies to be effective for accomplishing the objectives of ballast water treatment (inactivation of cellular reproduction), when the MPN assay is used as the basis for evaluation. The FDA, CMFDA, motility, and MPN methods are subject to well recognized sources of error; however, the MPN method is based on a response that is consistent with the objectives of ballast water management as well as the mechanism of action of UV-based inactivation. Complementary assays are available for use in compliance testing; however, the development of relevant indicative tests remains as a research priority. Historical lessons learned from applications of vital stains (and other indirect methods) for quantification of microbial responses to UV irradiation in other settings also support the use of assays that provide a direct measure of growth and reproduction, such as MPN. Collectively, these observations point to the use of MPN assays as the standard for type testing, especially when UV-based treatment is employed.