Determining the viability of marine protists using a combination of vital, fluorescent stains
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Determining the viability of protists and small microzooplankton has long been a focus of studies in marine biology and ecology. It is especially relevant in the issue of shipborne invasive species, and impending international guidelines and various national regulations on the allowable concentrations of organisms in discharged ballast water have spurred the growth of an industry that develops and manufactures ballast water management systems. The success of management systems and ability of ships to meet ballast water discharge standards is determined by the number of viable organisms in treated water. Here, we propose combining two vital, fluorescent stains (fluorescein diacetate [FDA] and 5-chloromethylfluorescein diacetate [CMFDA]) with direct microscopic observation to enumerate viable organisms ≥10 and <50 μm in minimum dimension (nominally protists). This approach was validated in four locations in the United States to determine the efficacy of the stains. Although the accuracy of the stains varied by geographic location and the taxonomic composition of the planktonic assemblage, combining fluorescent stains is a robust, powerful tool that can be optimized for the species present at each location. While this method was developed for analyzing viable organisms in treated ballast water, it may also be used or adapted for any field of research that examines a broad taxonomic range of autotrophic and heterotrophic plankton.
KeywordsDinoflagellate Ballast Water International Maritime Organization Viable Organism Plankton Assemblage
This research was supported by the United States Coast Guard (contract #HSCG23-09-X-MMS028) and does not represent official USCG policy. Many thanks to Mr. Scott Riley, Ms. Stephanie Robbins-Wamsley, and Dr. Matthew First at the Naval Research Laboratory in Key West, Florida and to Dr. Richard Everett for providing feedback that greatly improved this manuscript. Additional thanks to Dr. Mario Tamburri, Mr. Timothy Mullady, Mr. George Smith, and Ms. Janet Barnes from the Maritime Environmental Resource Center; Dr. Andrea Copping, Dr. Dana Woodruff, and Mr. William Pratt from the Pacific Northwest National Laboratory; and Dr. Michael Sieracki and Dr. Nicole Poulton from Bigelow Laboratory for Ocean Sciences for their generous assistance during this project.
- Carlton JT (1985) Transoceanic and interoceanic dispersal of coastal marine organisms: the biology of ballast water. Oceanogr Mar Biol Ann Rev 23:313–371Google Scholar
- International Maritime Organization (2004) Convention BWM/CONF/36 Adopted 13 February 2004; “International Convention for the Control and Management of Ships’ Ballast Water and Sediments”Google Scholar
- International Maritime Organization (2005) G8 Resolution MEPC 125(53) Adopted 22 July 2005; “Guidelines for approval of Ballast water management systems (G8)”Google Scholar
- Onji M, Sawabe T, Ezura Y (2000) An evaluation of viable staining dyes suitable for marine phytoplankton. Bull Fac Fish Hokkaido Univ 51:153–157Google Scholar
- Register Federal (2009) Standards for living organisms in ships’ Ballast water discharged in US waters; draft programmatic environmental impact statement, proposed rule and notice, 74 FR 44631–44672 (28 August 2009). National Archives and Records Administration, Washington, DCGoogle Scholar
- Throndsen J (1978) The dilution culture method. In: Sournia A (ed) Phytoplankton manual. Unesco, Paris, pp 218–224Google Scholar
- Tjallingii F (2001) Global market analysis released. Ballast Water News 6:6–8Google Scholar
- Wright D, Dawson R, Orano-Dawson C (2007) Shipboard trials of menadione as a ballast water treatment. Mar Tech 44:68–76Google Scholar
- Yentsch C, Menzel D (1963) A method for the determination of phytoplankton chlorophyll and phaeophytin by fluorescence. Deep Sea Res 10:221–231Google Scholar