Outline. Sampling purpose D-2 standard Ballast water biology Indicative vs detailed analysis Organisms detection methods Viability Minimum dimension

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2 Outline Sampling purpose D-2 standard Ballast water biology Indicative vs detailed analysis Organisms detection methods Viability Minimum dimension

3 Sampling Purpose Compliance with D-2 (Ballast Water Performance Standard) <10 viable organisms/m³ >50 µm <10 viable organisms/ml <50 & > 10µm Indicator microbes Escherichia coli <250 cfu in 100 ml Enterococci <100 cfu in 100 ml Vibrio cholerae <1 cfu in 100 ml or in 1 gr ww zooplankton

4 Ballast Water Biology Almost all types of organisms Dominant are crustaceans, molluscs, worms and phytoplankton 15 cm long fishes found in tanks Harmful algae

5 Ballast Water Biology

6 Two Different Approaches Indicative analysis A quick and dirty check for gross exceedence; e.g. 100s orgs = non-compliance Detailed analysis A detailed analysis; e.g. 10 orgs = non-compliance

7 Summaries of Organism Detection Methods EMSA Study 2010 Interreg IV B Ballast Water Opportunity 2012 BALMAS 2014

8 Possible analysis methods An evaluation of the following was done: Accuracy Reliability Time to a result Expertise Portability Costs Use onboard or in lab

9 Indicative Analysis Methods D-2 organism groups Viable organisms less than 50 and greater than or equal to 10 micrometres in minimum dimension 12 methods considered Viable organisms greater than or equal to 50 micrometres in minimum dimension 10 methods considered Methods for bacteria analysis 13 methods considered

10 Indicative Analysis Methods Organisms <50 and 10 µm in minimum dimension Presence/absence (no viability, no counts) e.g. DNA, ATP, Chl a methods deliver results in less than 60 minutes Viability (and estimated counts) Pulse-amplitude modulated (PAM) fluorometry Flow cameras or flow cytometry (< 60 minutes, not portable, viability stain needed)

11 Indicative Analysis Methods Organisms <50 and 10 µm in minimum dimension Best compromise: Pulse-amplitude modulated (PAM) fluorometry portable, easy to use, low expertise needed viability in less than 10 minutes onboard (by PSC) no real counts, but biomass and Chl a indication

12 Number of smaller organisms / ml Pulse-Amplitude Modulated Fluorometry (PAM) PAM measures phytoplankton biomass and viability No direct counts Clear relation of biomass and viability measurements with organism numbers Suitable tool to show that D-2 was not met Detection 100 limit is 50 1 org / ml 0 (calculated) Total number of smaller organisms 0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 Organism viability

13 New PAM Phytoplankton Tools Welschmeyer Turner Hach bbe This is now considered a commercially interesting market Validation experiments ongoing

14 Indicative Analysis Methods Organisms 50 µm in minimum dimension Presence/absence methods (no viability, no counts) e.g. DNA, ATP methods deliver results in less than 60 minutes Counts (no viabilty) Hand-held flow cameras (less than 30 minutes) Best compromise: Stereomicroscope (counts & viability) in laboratory results in less than 20 minutes portable, easy to use, but high expertise needed

15 Indicative Analysis Methods D-2 Bacteria Presence/absence methods (no cfu and/or counts) e.g. DNA, ATP methods deliver results in less than 60 minutes All methods to determine cfu require incubation time of >8 hours

16 Indicative Analysis Methods New Developments New indicative ATP method is available since June 2014, validation ongoing ATP signal calculated into organism counts For all D-2 organisms (filtration) Laboratory analysis New ballast water test kit (2015) Includes mini-incubator and microscope For all D-2 organisms (filtration) Uses stains Validation status unclear

17 Indicative Analysis Methods Suggestions Start with one method to evaluate one organism group in D-2 Should this show presence or high numbers, take result as indication of a failure to meet D-2 Should this show absence or low numbers, continue with second (and third) D-2 organism group to confirm results The easiest to start with may be the analysis for phytoplankton (Pulse-Amplitude Modulated fluorometry) on board and if needed followed by zooplankton (stereomicroscope) in laboratory

18 Indicative Analysis Approach Organisms <50-10 µm (PAM) on board D-2 met Organisms 50 µm (stereomicroscope) lab D-2 not met D-2 not met D-2 non-compliance action D-2 met D-2 compliance action

19 Detailed Analysis Work according to good laboratory practice standards Most accurate analysis technologies should be used At best consider living samples Keep time between sample taking and sample analysis as short as possible Unlikely to have one detailed sample processing standard world-wide

20 Viability G8: Viable Organisms are organisms and any life stages thereof that are living Phytoplankton viability stain Fluorescein DiAcetate (FDA) Zooplankton initiate movement with light under stereomicroscope gentle poking with a needle Bacteria colony forming means viable

21 Viability stain Fluorescein DiAcetate (FDA) Stains living phytoplankton Cell-permeant esterase substrate documents enzymatic activity (needed to activate its fluorescence) and cell-membrane integrity (needed for retention of fluorescent product in cell) Problem with UV treated water

22 Minimum dimension Look at the organisms from all axes Select the smallest axes Measure the smallest dimension Not always possible! Use smallest visible axes

23 Colony vs. single organism Several algae form colonies or chains What if a colony or chain is above 50 µm in minimum dimension and the single individual below 50 µm? Measure and count the single individual Because D-2 refers to viable organisms and it is the individual being viable and not the colony 200 µm 40 µm

24 Detailed Analysis Suggested methods Organisms <50 and 10 µm Phytoplankton - Epifluorescence microscope Zooplankton Microscope Organisms 50 µm Phytoplankton - Microscope Zooplankton Stereomicroscope Bacteria Selective media Consider routine bacteria analysis methods (drinking / bathing water quality standards)

25 Detailed Analysis Methods D-2 organism <50 and 10 µm Epifluorescence microscopy Fluorescein diacetate (FDA) 1 ml Sedgewick-Rafter chamber with grid of 1000 squares Grid lines = µm Count vertical transects Sedgwick-Rafter chamber

26 Detailed Analysis Methods D-2 organisms 50 µm Microscopic analysis Stains don t work 100% Living dead judgement by a scientist 50 µm beads or mesh for size Stephan counting chamber

27 Detailed Analysis Methods D-2 methods for bacteria colony forming units to identify not all bacteria form colonies incubation needed selective media

28 Analysis Methods Suggestions Consider to equip a van with organism detection technology Drive from vessel to vessel in a port Send sampling team onboard and deliver the samples as soon as possible to van for analysis In this scenario the organism detection tools would not need to be carried onboard Sampling team only to board the vessel, no need to bring organism detection team onboard as well

29 Analysis Methods, Summary Organism 50 µm Indicative Stereomicroscope (in laboratory) < 50 and 10 µm PAM (on board method) Bacteria Developing Organism 50 µm Detailed Stereomicroscope (needle for poking) < 50 and 10 µm Epifluorescence microscope with FDA stain Bacteria Selective media

30 Conclusions Methods exist to proof D-2 compliance Harmonized approach needed, not that one vessel is compliant in one port, but not in another because of different sample processing methods used An indicative sample analysis may be followed by a detailed analysis of the same sample

31 Contacts ><(((( > GoConsult Stephan Gollasch GoConsult, Hamburg, Germany Matej David Dr. Matej David Consult d.o.o. Korte, Slovenia

32 Thank you very much for your attention