The Sea:JOURNAL OF THE KOREAN SOCIETY OF OCEANOGRAPHY (한국해양학회지:바다)

The Korean Society of Oceanography (한국해양학회)
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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 (Ballast Water Center, Korea Institute of Ocean Science & Technology) ;
  • Shin, Kyoungsoon (Ballast Water Center, Korea Institute of Ocean Science & Technology) ;
  • Chung, Hansik (Ballast Water Center, Korea Institute of Ocean Science & Technology) ;
  • Choi, Seo-Yeol (Ballast Water Center, Korea Institute of Ocean Science & Technology) ;
  • Jang, Min-Chul (Ballast Water Center, Korea Institute of Ocean Science & Technology) ;
  • Lee, Woo-Jin (Ballast Water Center, Korea Institute of Ocean Science & Technology) ;
  • Choi, Keun-Hyung (Ballast Water Center, Korea Institute of Ocean Science & Technology)
  • 현봉길 (한국해양과학기술원 선박평형수센터) ;
  • 신경순 (한국해양과학기술원 선박평형수센터) ;
  • 정한식 (한국해양과학기술원 선박평형수센터) ;
  • 최서열 (한국해양과학기술원 선박평형수센터) ;
  • 장민철 (한국해양과학기술원 선박평형수센터) ;
  • 이우진 (한국해양과학기술원 선박평형수센터) ;
  • 최근형 (한국해양과학기술원 선박평형수센터)
  • Received : 2014.03.06
  • Accepted : 2014.09.03
  • Published : 2014.11.28
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Abstract

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.

선박평형수를 통한 외래종 확산을 방지하기 위해 국제해사기구(IMO)에서는 2004년 선박평형수관리협약을 채택하였다. 이 협약에 따르면 앞으로 대부분의 선박들은 선박평형수 처리시스템을 통해서 해양 생물을 사멸 또는 제거시킨 후 배출해야 하며, 이는 플랑크톤의 생사판별방법을 통하여 이루어진다. 본 연구에서는 국제적으로 선박평형수 처리후 생사판별법으로 널리 사용되고 있는 fluorescein diacetate assay (FDA) 염색방법의 제한성과 이의 대안으로 Neutral red (NR) 염색방법사용 가능성을 살펴보고자 하였다. FDA 염색법은 대부분의 플랑크톤 염색에 되어서 현재 가장 널리 사용되는 방법임에도 불구하고 본 연구에서는 Ditylum brightwellii 을 제외한 모든 식물플랑크톤 대해서 낮은 염색 효율(전체 평균 염색 효율 <50%)을 보였으며, 식물플랑크톤이 갖는 고유한 형광(적색)에 간섭을 많이 받는 것으로 나타났다. 또한 FDA는 형광파장에 노출된 후 빠르게 색이 바래지는 경향도 관찰되었다. 반면에 NR은 조사된 모든 동 식물플랑크톤 개체수에 대해서 90% 이상의 높은 염색 효율을 보였다. 두 염색법 모두 포르말린을 이용해서 사멸시킨 동 식물플랑크톤에 대해서는 염색이 되지 않았다. 본 연구결과를 통해서 NR 염색법을 이용한 동 식물플랑크톤 생사판별은 매우 효율적이라고 판단된다.

Keywords

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