Development of a novel automated analytical method for viability assessment of phytoplankton used for validation of ballast water treatment systems
To limit the spreading of aquatic invasive species, regulations require ships’ ballast water to be treated before discharge. To validate ballast water treatment system (BWTS) performance, treated water is analyzed for living organisms in different size classes. Quantitative assessment of the size class 10–50 μm (mainly phytoplankton) is carried out using the vital stain method, which requires labor-intensive manual microscope counts of fluorescent (i.e., living) cells. The method is slow, demands specialized personnel, and is challenged by subjectivity and mobile organisms. Using a high-content screening platform (HCS-Platform) and image analysis, we developed an automated, objective and faster quantification method. The automated method neutralized subjectivity by using fixed cell recognition parameters for image analysis. The implementation of membrane filters gently manipulated the organisms into a 2D plane that reduced mobility. Quantifications were performed at different concentrations using monocultures of slow-moving Rhodomonas salina, highly mobile Tetraselmis suecica and natural algae. Results were compared to the standard manual counting procedure. Automated counts of monocultures were comparable to manual counts at low and medium concentration levels. Manual counts of T. suecica at high concentration levels were significantly lower compared to automated counts stressing the challenge to count mobile cells in 3D. Natural algal counts were similar for both counting approaches, but accuracy was challenged by colony forming species and high number of algal species ~ 10 μm. Automated counts were significantly faster than manual counts. In conclusion, the HCS-Platform showed promising results as an alternative quantitative phytoplankton assessment method for BWTS validation.
KeywordsAdvanced microscopy Algae Invasive species, ballast water Image analysis Monitoring Ballast water treatment systems
The work was supported by the Danish Maritime Fund (Project 2016-046) and University of Southern Denmark. We would like to thank Annette Duus and the staff at DHI Ballast Water Center, Denmark for technical support.
- Baek SH, Shin K (2015) A staining method to determine marine microplanktonic organism viability and investigate the efficacy of a ship's ballast water treatment system. J Korea Acad Indust Coop Soc 16:4328–4334Google Scholar
- Berek M (1927) Grundlagen der Tiefenwahrnehmung in Mikroskop. Sitzungsber Gesellsch Beförd gesamten Naturwissensch Marburg 62:189–223Google Scholar
- Bradie J, Broeg K, Gianoli C, He J, Heitmüller S, Lo Curto A, Nakata A, Role M, Schillak L, Stehouwer P, Vanden Byllaardt J, Veldhuis M, Welschmeyer N, Younnan L, Zaaje A, Bailey S (2018) A shipboard comparison of analytic methods for ballast water compliance monitoring. J Sea Res 133:11–19CrossRefGoogle Scholar
- Gollasch S, Cangelosi A, Peperzak L (2012) Testing of ballast water treatment systems performance regarding organisms below 10 micron in minimum dimension. Final report Prepared for Interreg IVB North Sea Ballast Water Opportunity project 17:1–25Google Scholar
- IMO (2004) Convention BWM/CONF/36 international convention for the control and management of ship’s ballast water and sediments. International Maritime Organization (IMO): [S.l.]. pp 1-36Google Scholar
- IMO (2016a) International Maritime Organization Marine Environment Protection Committee, Resolution MEPC.279(70). 2016 Guidelines for approval of ballast water management systems (G8). International Maritime Organization (IMO). London, United Kingdom. pp 1–42Google Scholar
- IMO (2016b) Review of the guidelines for approval of ballast water management systems (G8): analysis methods for determining the viability of organisms in the 10 to 50 μm size class. Submitted by Denmark and Norway. IMO PPR 4/7. International Maritime Organization (IMO), London, United Kingdom. pp 1-3Google Scholar
- Liebich V (2013) Invasive plankton, implications of and for ballast water management. PhD dissertation. University of HamburgGoogle Scholar
- Outinen O, Lehtiniemi M (2017) Literature review for the indicative ballast water analysis methods. Trafi Research Reports. Finnish Transport Safety Agency (Trafi), Finnish Environment Institute SYKE, Helsinki. Pp 1–55Google Scholar
- Peperzak L, Zetsche E-M, Gollasch S, Artigas LF, Bonato S, Creach V, de Vré P, Dubelaar GBJ, Henneghien J, Hess-Erga O-K, Langelaar R, Larsen A, Maurer BN, Mosselaar A, Reavie ED, Rijkeboer M, Tobiesen A (2018) Comparing flow cytometry and microscopy in the quantification of vital aquatic organisms in ballast water. J Mar Eng Technol:1–10. https://doi.org/10.1080/20464177.2018.1525806
- Schulze K, Tillich UM, Dandekar T, Frohme M (2013) PlanktoVision - an automated analysis system for the identification of phytoplankton. BMC Bioinformatics 14. doi:Artn11510.1186/1471-2105-14-115
- van der Star I, Liebich V, Stehouwer PP (2011) The forgotten fraction: the importance of organisms smaller than 10 μm when evaluating ballast water treatment systems. In: Ballast Water Management Systems: Proceedings of the Global R&D Forum on Compliance Monitoring and Enforcement – The Next R&D Challenge and Opportunity. Istanbul, Turkey 2011Google Scholar