The MERE research group is part of a larger SDU network – BlueSDU – focused on strengthening and showcasing research broadly addressing water. The BlueSDU network has economists, biologists, lawyers, public health researchers, maritime archaeologists, biochemists, engineers, and others who are actively engaged in cross-disciplinary efforts concerning freshwater, marine and maritime issues.

A major concern as global shipping has increased over the past 2 centuries is that the ships have brought — and will continue to bring, both on their hulls and in their ballast water — new and potentially invasive species to economies and ecosystems that have suffered or will suffer significant damages from these introductions.

This week we will have a series of MERE and guest posts focused on the issue of ballast water regulation from a variety of disciplinary and interdisciplinary perspectives.

First, we hear from the biologists Henrik Holbech and Kim Lundgreen on this issue:

When and how can death be acknowledged?  The current state of verification challenges of organism viability in treated ballast water

Close up Razor clam
Close up Razor clam

Description of topic and related issues

The introduction of invasive species from ship’s ballast water discharge has for many years been a serious global matter1,2 – and it still is.3-5 Bioinvasion incidents such as from species pictured in this post have had large and detrimental consequences ecologically, socio-economically and in some places even caused near-extinction of endemic species.6,7 To prevent any further stress on fragile ecosystems and economics related to aquatic activities, high awareness and scientific focus of the issue is required. To protect aquatic ecosystem from invasive species, the United Nations IMO BW management convention (ratified by Denmark in 2012) sets up global regulations of ballast water (BW) discharge. All ships discharging BW must first apply a type approved BW management system (BWMS) to meet discharge standards related to number of viable organisms in defined size-classes. Similar rules became effective by 2012 in USA.

The recognized methods for determining viable cells are based on labor intensive direct microscopic counting of the number of live organisms. To facilitate counting, living cells are stained by fluorescent markers of esterase- or P450 activity and/or dead cells are stained with markers of plasma membrane damage.8-10 Ultraviolet (UV) radiation and electrolytic chlorination, frequently combined with filtration, represent the two prevailing treatment principles in type approved BWMS11. A special challenge for monitoring the effect of UV based technologies is that UV radiation causes damage to DNA in the cells12 which can either result in later death or in survival due to DNA repair. Current staining methods may therefore produce false positives because dead or dying organisms are recorded as viable13, 14. Effects on DNA cannot be measured by the methods currently prescribed8. The problem of identifying false positives and false negatives (recovering organisms) is currently solved by most probable number assays for measuring the algal re-growth in treated BW10, but these assays are time consuming and there is a need for a better characterization of the borderline cases. US Coast Guard have very recently rejected most probable number assays because the regulations specifically require ballast water treatment systems to be evaluated based on their ability to kill certain organisms. Further improvement of the current methods used for assessment of BWMS is therefore of high environmental as well as economic importance.

Due to this high level of import, Kim Lundgreen has just started a PhD to investigate solutions to the question of when you ‘know’ an organism is dead.

holbech ballast fish
invasiv art: sortmundet kutling (Neogobius melanostomus ) fanget i Bøgestrømmen


The PhD-project plans to test, develop and establish new standard methodologies and know-how for high quality assessments of planktonic organism viability in BWMS under different biotic and abiotic water conditions. The aim is that these analytical methods in the longer run can be applied and used as standard monitoring procedures for validation of implemented type approved BWMS. The applied methods and the science behind will support the development of cleaner and more efficient BWMS. They will help to ensure that treatment performance comply with discharge standards and the detection of insufficient BWMS and/or undetected BWMS failures.

The project will be divided into three sub-projects with main focus on sub-project (1):

  • Testing of novel staining methods: Flow cytometry for counting viable organisms works well for monocultures, but is limited when counting samples containing multiple species and colony forming/filamentous plankton.15 Comparative studies will be used to evaluate a number of novel markers and staining methods for practical viability assessment of planktonic organisms/cells from treated BWMS. Likewise to relate their resource requirements and quality. Results will be compared with existing methods. Besides high quality validation of the tested methods other characteristics such as cost, simplicity, resource and time demands will be considered. The project will investigate a number of live stains based on the presence of metabolic activity and also test efficiency of multi-labelling. Furthermore, the potential complementary use of advanced microscopy systems and image analysis techniques for a more automated and robust quantitative determination of live/dead organisms and species identification will be explored.
  • Staining challenges of phytoplankton: Algal cell structure challenges current staining methods as they are developed for cytotoxicity measurements in mammalian cells. Algal cells walls might prevent the entry of dyes into the cell or bind dyes non-specifically.15 The project will focus on if existing staining methods and flow cytometry analysis are also applicable on a variety of algal species.
  • Identification of robust key species: Some species tend to resist ballast water treatment.16-18 The sub-project will focus on the identification of robust key species and their attributes and on establishing dose-response relationships to different treatment processes with the aim to determine the best approach for their efficient removal from the discharged ballast water.
Shore Crab (Carcinus maenas)
Shore Crab (Carcinus maenas)


  • Ruiz GM, Carlton JT, Grosholz ED, Hines AH (1997). Global invasions of marine and estuarine habitats by non-indigenous species: mechanisms, extent and consequences. American Zoologist 37:621-32.
  • Molnar J, Gamboa RL, Revenga C, Spalding MD (2008). Assessing the global threat of invasive species to marine biodiversity. Frontiers in Ecology and Environment 6(9):485-492.
  • Thresher RE and Kuris AM (2004). Options for managing invasive marine species. Biological Invasions 6:295-300.
  • Al-Yamani FY, Skryabin V, Durvasula SRV (2015). Suspected ballast water introductions in the Arabian Gulf. Aquatic Ecosystem Health & Management, 18(3):282-289.
  • Available from Data of access 24/10/2015.
  • Hebert PDN, Muncaster BW, Mackie GL (1989). Ecological and genetic studies on Dreissena polymorpha (Pallas) – a new mollusk in the Great Lakes. Canadian Journal of Fisheries and Aquatic Sciences 46(9):1587–1591.
  • Berdnikov SV, Selyutin VV, Vasilchenko VV, Caddy JF (1999). Trophodynamic model of the Black and Azov Sea pelagic ecosystem: consequences of the comb jelly, Mnemiopsis leydei, invasion. Fisheries Research 42(3):261–289.
  • Agency, U. S. E. P. Generic protocol for the verification of ballast water treatment technology. (E.PA, 2010).
  • Guidelines for approval of ballast water management systems (G8). (2008).
  • Biological efficacy evaluation of ballast water management systems. (2013).
  • Technology, B. w. t., (Riviera Maritime Media Ltd., 2013).
  • Sinha RP & Hader DP (2002). UV-induced DNA damage and repair: a review. Photochemical & Photobiological Sciences 1:225-236.
  • Steinberg MK, Lemieux E, Drake LA (2011). Determining the viability of marine protists using a combination of vital, fluorescent stains. Biol. 158:1431-1437.
  • Zetsche EM & Meysman, FJR (2012). Dead or alive? Viability assessment of micro- and mesoplankton. Journal of Plankton Research 34:493-509.
  • Reavie ED, Cangelosi, AA, Allinger LE (2010). Assessing ballast water treatments: Evaluation of viability methods for ambient freshwater microplankton assemblages. Journal of Great Lakes Research 36(3):540-547.
  • Briski E, Linley RD, Adams JK, Bailey SA (2012). Evaluating efficacy of a ballast water filtration system for reducing spread of aquatic species in freshwater ecosystems. Management of Biological Invasions 5(3):245–253.
  • DHI (2012). Performance evaluation in landbased and ship-board test of the DESMI Ocean Guard ballast water management system DOG P40-300. DESMI Ocean Guard A/S Summary Report.
  • Gregg M, Rigby G, Hallegraeff GM (2009). Review of two decades of progress in the development of management options for reducing or eradicating phytoplankton, zooplankton and bacteria in ship’s ballast water. Aquatic Invasions 4(3):521-565.