- Volume 19 Issue 4
DOI QR Code
Application of Neutral Red Staining Method to Distinguishing Live and Dead Marine Plankton for the Investigation of Efficacy of Ship's Ballast Water Treatment System
선박평형수 처리 시스템 효율 검증을 위한 해양 플랑크톤 생사판별시 Neutral red 염색법 적용 가능성 연구
- Hyun, Bonggil ;
- Shin, Kyoungsoon ;
- Chung, Hansik ;
- Choi, Seo-Yeol ;
- Jang, Min-Chul ;
- Lee, Woo-Jin ;
- Choi, Keun-Hyung
- Received : 2014.03.06
- Accepted : 2014.09.03
- Published : 2014.11.28
In order to prevent the spread of non-indigenous aquatic species through the ballast water in commercial ships, International Maritime Organization (IMO) adopted in 2004 the International Convention for Control and Management of Ship's Ballast Water and Sediments. The Convention mandates treatment of ballast water for most transoceanic voyages and its confirmation of treatment is made with plankton live/dead assay. Fluorescein diacetate assay (FDA), which produces bright green light for live phytoplankton, has been a de facto standard method to determine the survival of marine plankton, but its staining efficacy has been in dispute. In the present study, we examined the limitation of FDA, and compared its efficacy with Neutral red (NR) staining, another promising assay and widely used especially for zooplankton mortality. For all phytoplankton species studied in the present study, except Ditylum brightwellii, the staining efficiency was <50% with FDA. The green FDA fluorescence interfered with phytoplankton autofluorescence in most samples. In contrast, NR assay stained over 90% of both phytoplankton and zooplankton species tested in this study. FDA assay also showed that green FDA fluorescence rapidly faded when phytoplankton cells were exposed to microscope light. Both FDA and NR assay were negative on formalin-killed individuals of both phytoplankton and zooplankton species. Our results suggest that NR assay is more effective for determining the survival of marine plankton and can be applied to test the efficacy of ballast water treatment.
ballast water;International Maritime Organization (IMO);marine plankton;Neutral red (NR);fluorescein diacetate (FDA)
- Bentley-Mowat, J., 1982. Application of fluorescence microscopy to pollution studies on marine phytoplankton. Bot. Mar., 25: 203-204.
- Agusti, S., M.C. Sanchez, 2002. Cell viability in natural phytoplankton communities quantified by a membrane permeability probe. Limnol. Oceanogr., 47: 818-828. https://doi.org/10.4319/lo.2002.47.3.0818
- Baek, S.H. and K. Shin, 2009. Applicability of fluorescein Diacetate (FDA) and Calcein-AM to determine the viability of marine plankton. Ocean Polar Res., 31(4): 349-357. https://doi.org/10.4217/OPR.2009.31.4.349
- Bax. N., A. Williamson, M. Aguero, E. Gonzalez, W. Geeves, 2003. Marine invasive alien species: a threat to global biodiversity. Mar. Policy, 27(4): 313-323. https://doi.org/10.1016/S0308-597X(03)00041-1
- Bickel, S.L., K.W. Tang and H.-P. Grossart, 2009. Use of aniline blue to distinguish live and dead crustacean zooplankton composition in freshwaters. Freshwater Biol., 54: 971-981. https://doi.org/10.1111/j.1365-2427.2008.02141.x
- Crippen, R.W. and J.L. Perrier, 1974. The use of neutral red and Evans blue for live-dead determinations of marine plankton (with comments on the use of rotenone for inhibition of grazing). Biotech. Histochem., 49: 97-104. https://doi.org/10.3109/10520297409116949
- Chihara, M. and M. Murano, 1997. An illustrated guide to marine plankton in Japan, University of Tokai Press, Tokyo.
- Cupp, E.E., 1943. Marine plankton diatoms of the west coast of North America, University of California Press, Berkeley and Los Angeles.
- Dressel, D.M., D.R. Heinle and M.C. Grote, 1972. Vital Staining to Sort Dead and Live Copepods. Ches. sci., 13: 156-159. https://doi.org/10.2307/1351022
- Elliott, D.T. and K.W. Tang, 2009. Simple staining method for differentiating live and dead marine zooplankton in field samples. Limnol. Oceanogr. Methods, 7: 585-594. https://doi.org/10.4319/lom.2009.7.585
- Garvey, M., B. Moriceau and U. Passow, 2007. Applicability of the FDA assay to determine the viability of marine phytoplankton under different environmental conditions. Mar. Ecol. Prog. Ser., 352: 17-26. https://doi.org/10.3354/meps07134
- Endresen, O., H. Lee Behrens, S. Brynestad, A. Bjorn Andersen, R. Skjong, 2004. Challenges in global ballast water management. Mar. Pollut. Bull., 48: 615-623. https://doi.org/10.1016/j.marpolbul.2004.01.016
- Franklin, D., C. Cegres, O. Hoegh-Guldberg, 2006. Increased mortality and photoinhibition in the symbiotic dinoflagellates of the Indo-Pacific coral Stylophora pistillata (Esper) after summer bleaching. Mar. Biol., 149: 633-642. https://doi.org/10.1007/s00227-005-0230-z
- Fuhr, F., J. Finke, P.P. Stehouwer, S. Oosterhuis, M. Veldhuis, 2010. Factors influencing organism counts in ballast water samples and their implications. In Bellefontaine, N., Haag, F., Linden, O. et al. (eds), IMO-WMU Research and Development Forum. WMU Publications, Malmo, Sweden, pp. 253-259.
- Horobin, R.W., J.A. Kiernan, 2002. Conn's biological stains: A handbook of dyes, stains and fluorochromes for use in biology and medicine. (10th Ed) BIOS Scientific Publishers, Oxford.
- IMO, 2001. Report on the ballast water treatment standards workshop. In 1st International ballast water treatment standards workshop, IMO London, pp. 28-30 March 2001. http://globallast .Imo.org/workshopreport.htm.
- Onji, M., T. Sawabe, T. Ezura, 2000. An evaluation of viable staining dyes suitable for marine phytoplankton. Bull. Fac. Fish. Hokkaido Univ., 51(3): 153-157.
- Peperzak, L., C.P.D. Brussaard, 2010. Phytoplankton viability assay for oil compounds in water. In Timmis, K.N. (ed.) Handbook of hydrocarbon and lipid microbiology. Springer, Berlin Heidelberg, pp. 4499-4508.
- Pimented, D., R. Zuniga, D. Morrison, 2005. Update on the environmental and economic costs associated with alien-invasive species in the United States. Ecol. Econ., 52(3): 273-288. https://doi.org/10.1016/j.ecolecon.2004.10.002
- Tomas, C.R., 1997. Identifying marine phytoplankton, Academic Press, California.
- Reynolds, A., G. Mackiernan, S. Van Valkenburg, 1978. Vital and mortal staining of algae in the presence of chlorine-produced oxidants. Estuaries, 1(3): 192-196. https://doi.org/10.2307/1351463
- Steinberg, M., E. Lemieux, L. Drake, 2011. Determining the viability of marine protists using a combination of vital, fluorescent stains. Mar. Biol., 158: 1431-1437. https://doi.org/10.1007/s00227-011-1640-8
- Tang, Y.Z., F.C., Dobbs, 2007. Green autofluorescence in dinoflagellates, diatoms, and other microalgae and its implications for vital staining and morphological studies. Appl. Environ. Microbiol., 73: 2306-2313. https://doi.org/10.1128/AEM.01741-06
- USCG, 2010. Generic Protocol for the Verification of Ballast Water Treatment Technology. NSF International. 157pp.
- USEPA, 2013. Vessel general permit for discharges incidental to the normal operation of vessels. 193pp.
- Wait, T.D., J. Kazumi, P.V.Z. Lane, L.L. Farmer, S.G. Smith, S.L. Smith, G. Hitchcock, T.R. Capo, 2003. Removal of natural populations of marine plankton by a large-scale ballast water treatment system. Mar. Ecol. Prog. Ser., 258: 51-63. https://doi.org/10.3354/meps258051
- Zetsche, E.-M. and F.J.R. Meysman, 2012. Dead or alive? Viability assessment of micro- and mesoplankton. J. Plankton Res., 34: 493-509. https://doi.org/10.1093/plankt/fbs018
Supported by : 해양수산부