Compliance TestingMETHODS FOR BALLAST WATER MONITORING

Stephanie Lavelle MSc Marine BiologyChelsea Technologies Group

Overview• IMO Regulation D2

• Phytoplankton & Fluorescence Bulk Fluorescence Method Distribution Fluorescence Method (Portable) Staining Fluorescence

Method

• Zooplankton Detection

• Bacterial Detection

• Hollistic Approaches

Aliens Attack!!• 10 billion tonnes of ballast water is transported around

the world every year (IMO, 1997).

• 20-30% of all introduced species worldwide cause a problem (Pimentel et al 2001).

• Invasive species have contributed to 40% of the animal extinctions that have occurred in the last 400 years (CBD, 2006).

• The total loss to the world economy as a result of invasive non-native species has been estimated at 5% of annual production (Pimentel et al, 2002).

Size Class D2 Standard

>50 μm <10 viable organisms per m3

10-50 μm <10 viable organisms per mL

Vibrio cholerae <1 colony forming unit (cfu) per 100 mL

Escherichia coli <250 cfu per 100 mL

Intestinal Enterococci <100 cfu per 100 mL

IMO D2 RegulationsOnboard Cell

Count TestOnboard

Viability Test

Phytoplankton & Fluorescence

It is assumed that when fluorescence is detected, the

organism is photosynthetically active and therefore viable (Yentsch and Menzel, 1963;

Maxwell, 2000).

Bulk Fluorescence Methods

• The overall fluorescence intensity is typically directly proportional to the density of phytoplankton.

• Fv/Fm is a measure of the maximum photosynthetic efficiency, but such values are traditionally translated into estimates of biomass, productivity and photosynthetic efficiency from relatively high cell densities.

Fv/Fm = maximum potential quantum efficiency of photochemistry

1 mL sample

Long pulse of light

Photo Diode Fv/Fm Cells/mLFiltration Step

Bulk Fluorescence Methods

1 minute

Bulk Fluorescence

• Individual cells fluoresce in proportion to their body size. For example, a 50 µm cell can easily emit 125 times more fluorescence than a 10 µm cell.

• Fluorescence can also be contributed from other sources, such as free chlorophyll, CDOM (dissolved coloured organic matter) or even dead cells.

Figure : Variable fluorescence signatures from T.punctigera and D.salina

20 mL stirred sample

Photomultiplier Tube

Specific array of LEDs rapidly flashing Detection Point

Distribution Analysis of Fv

Cells/mL

Distribution Fluorescence Method

1-8 minutes

Distribution Fluorescence Method

P

P

PS II

C

+ +

P

StromaPhoton

PS I

Electrons donated from water molecules

eElectron Transport Chain P

Oxygen molecules

Single Turnover Multiple Turnover

• Photosynthetic cycle of a cell (single turnover event) usually takes 400 µs

• ST captures each turnover event

• Longer pulses of light cause multiple turnover events

• MT can lead to a 50% over estimation of Fm

Distribution Fluorescence Methods

• The large amount of signal averaging provides for a much greater signal to noise ratio and consequently a more accurate value for Fv.

• This method analyses the Poisson distribution of the fluorescence signals around the mean value of Fv.

• Unique in that it considers cell size, as well as the specific fluorescence emission of chlorophyll.

• Accuracy decreases with higher cell densities and chain forming species also pose a challenge as they may be counted as 1 cell.

Fluorescent Stain

100 mL stirred sample

Photomultiplier Tube

LED pulsesDetection Point

Stain Measurement

Cells/mL

Staining Fluorescence Methods

30 minutes

Staining Fluorescence Methods

• Enzyme activity can continue in dead cells and therefore stain is still absorbed.

• Many species have a lack of affinity with stains. MacIntyre and Cullen (2016) found only out of 10/24 species they studied worked with staining.

• Portable method provides objective cell count.

• May also be applied to assess certain species of zooplankton.

Bulk & Distribution DataCe

ll de

nsity

mL

-1 u

sing

Bulk

Flu

ores

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d Di

strib

ution

Flu

ores

cenc

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Microscope based estimate of cell density mL -1

Bulk Fluorescence MethodDistributionFluorescence Method

Standard parameter Thalassiosira punnctigera

Dunaliella salina

Fv/Fm 0.111 0.940

Fv 0.254 0.263

Cells/mL

Bulk Fluorescence Method

111 (FAIL) 94 (FAIL)

Distribution Fluorescence Method

8.8 (PASS) 360 (FAIL)

Microscope Count 7.0 (PASS) 427 (FAIL)

Staining Fluorescence Data

Graphs: Correlation between (portable) Staining Method and microscope counting a) in the test using >50 µm and

b) 10-50 µm (Nakata et al., 2014)

Zooplankton Detection

• Zooplankton are the biggest challenge to measure onboard, as they do not fluoresce or stain as holistically as phytoplankton and they cannot be cultured as rapidly as bacteria.

• There are integrated monitoring methods that have been developed, which apply a combination of lasers and bulk fluorometry to assess both phytoplankton and zooplankton. However, viability of zooplankton is not an easy parameter to assess.

• Another method for indicating the presence of plankton onboard is flow cytometry, which uses imagery to count plankton cells that pass through its measurement cell to determine cells per volume concentration.

Bacteria Detection

• Culturing bacteria strains to grow to such a density that a difference in pressure due to respiration can be measured, is a common method applied in the healthcare industry.

• Portable systems have been developed that provide specific mediums to test for the strains outlined in the regulations, which apply an algorithm to translate the respiration measurement to a colony count.

• Such methods require some sample preparation and can currently take in excess of 24 hours to produce a result.

Holistic Approaches

Portable bulk ATP and DNA methods have been applied to ballast water monitoring to assess the cell densities of all

organisms in one test.

Assessing DNA produces a more qualitative presence/absence report and cannot determine viability.

ATP measurements produces a more quantitative result, however, it relies on a lot of assumptions of quantities of ATP

per cell, so can only provide an indicative result.

Conclusions

For compliance testing overall, there are a wide range of tools available that vary in the parameters they measure,

their precision and protocols.

MEPC has recommended that there be a two-three year trail period following entry into force with no penalties

to be applied to ships, in order for us to further test and understand which methods are robust and reliable in

practise.

Since ratification, BWTS are being turned on, so in depth testing is now starting to happen. Sharing of this data

and universal assessment protocols is imperative.

Questions?