IMarEST Compliance Testing Presentation. Evaluation of onboard methods
- 1. Compliance Testing METHODS FOR BALLAST WATER MONITORING Stephanie Lavelle MSc Marine Biology Chelsea Technologies Group
- 2. Overview • IMO Regulation D2 • Phytoplankton & Fluorescence Bulk Fluorescence Method Distribution Fluorescence Method (Portable) Staining Fluorescence Method • Zooplankton Detection • Bacterial Detection • Hollistic Approaches
- 3. 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).
- 4. 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 Regulations Onboard Cell Count Test Onboard Viability Test
- 5. Phytoplankton & Fluorescence It is assumed that when fluorescence is detected, the organism is photosynthetically active and therefore viable (Yentsch and Menzel, 1963; Maxwell, 2000).
- 6. 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
- 7. 1 mL sample Long pulse of light Photo Diode Fv/Fm Cells/mL Filtration Step Bulk Fluorescence Methods 1 minute
- 8. 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
- 9. 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
- 10. Distribution Fluorescence Method P P PS II C + + P StromaPhoton PS I Electrons donated from water molecules e Electron 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
- 11. 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.
- 12. Fluorescent Stain 100 mL stirred sample Photomultiplier Tube LED pulses Detection Point Stain Measurement Cells/mL Staining Fluorescence Methods 30 minutes
- 13. 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.
- 14. Bulk & Distribution Data CelldensitymL-1usingBulkFluorescence andDistributionFluorescenceMethods Microscope based estimate of cell density mL -1 Bulk Fluorescence Method Distribution Fluorescence 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)
- 15. 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)
- 16. 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.
- 17. 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.
- 18. 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.
- 19. 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.
Editor's Notes
- #3: This presentation discusses the difference in the onboard methods available for each type of organism within the D2, looking at the parameters they measure, their protocols and precision. The most predominant technologies to have emerged for onboard testing are fluorometry based instruments, due to their well understood and long standing methods. Such instruments have been the most widely applied to date due to their relatively straightforward use and rapid results.
- #4: To recap why the convention is so important; Rising trend in marine invasive sp. And it is estimated that every hour of every day 7,000 aliens are hitching a ride from ballast tanks to potentially conquer the world – this zooplankton ‘alien’ is what they based the alien in the movie on! 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). specific ballast discharge events have been held responsible for disasters such as outbreaks of deadly disease, complete collapse of fish stocks, mass blockages of internal waterways and eradication of species. These threats are made greater every day with the ever increasing number of journeys made by ships, so it is both economically and ecologically imperative that enforcement of the convention is widely understood, accepted and applied.
- #5: It seems to be completely accepted that the >50 µm size class represent zooplankton and the 10-50 µm represent phytoplankton, as this reflects any general aquatic ecosystem. Tools are available to measure number of cells and viability for all organisms, except zooplankton, where onboard viability testing remains the biggest challenge. All of the organisms outlined in the regulations can be tested for by count and/or culture methods in a laboratory, however, there are a very restricted number of trained specialists, such tests tend to be expensive, sample sizes are relatively small and organisms may be compromised between sampling and arrival at a test facility
- #6: Fluorescence can quite simply be described as the phenomena of a molecule absorbing light and re-emitting some of that light at a longer wavelength, known as the fluorescence emission, which is specific to different molecules (Lloyd, 1971). All photoautotrophic species, from trees to single-celled organisms, contain the pigment chlorophyll α to facilitate photosynthesis, which emits a natural red fluorescence which is intensified when excited with additional light (Maxwell, 2000). It is red because the plants reflect green and yellow light, giving them their natural colour, and absorb red and blue light for energy, the latter of which gets emitted as red fluorescence.
- #7: The most widely applied fluorometry method for indicative onboard testing have been bulk fluorescence methods. The basic principle for measuring bulk fluorescence is whereby the fluorescence is a sample before excitation is measured (Fo) and is a light source excites the sample and a photo diode sensor measures the overall maximum fluorescence intensity (Fm), which is typically directly proportional to the density of phytoplankton (Yentsch and Menzel, 1963). The difference in fluorescence values before and after excitation (Fo and Fm) is termed the variable fluorescence or Fv and is used to calculate the key parameter Fv/Fm.
- #8: To translate a fluorescent signal into a specific cell count of low cell densities has been a new objective for such implements, but It is important to align the fluorescence signature into a cell count so that it can be related to a D2 requirement. However, there are now a reasonably wide range of devices that have been developed to conduct onboard analyses, which are simplified in this diagram. Such procedures usually require sample preparation using filters to ensure just the 10-50 range is measured and then long pulses of light, often a laser, is used to excite the sample and a photo diode measures the general fluorescence signature. These tools tend to analyse the bulk fluorescence of around a 1 mL to produce an indicative cell count by analysing Fv/Fm.
- #9: This data shows how Fv varies between a cell close to the 10 micron boundary (D.salina) compared to a cell close to the 50 micron boundary – but the average Fv is about the same. Bulk fluorescence measurements have to assume a fixed level of Fv per cell, which may produce a cell count that can easily be erroneous by over two orders of magnitude, depending on the size of cells present. However, analysing this distribution of fluorescent signatures around the mean fluorescence, allows us to infer cell size.
- #10: The distribution method was developed to address challenges faced by using only a value for Fv/Fm in ballast water analysis (Silsbe et al., 2015). This method rapidly flashes 40 LED’s of complimentary wavelengths to chlorophyll pigment fluorescence, the emission of which is detected using a sensitive photomultiplier tube, acquiring and averaging 40 sequences at 40 Hz, to generate Fv values at 1 Hz (i.e. 1 data point a second averaged from 40 measurements), producing a graph similar to the previous slide. This allows for a lot of signal averaging every second, producing an excellent signal:noise ratio, and ultimately producing accurate measurements of Fv. It then analyses the Poisson distribution, which gets wider with increasing cell density, to infer cell size.
- #11: The quantum yield (ϕ) of photosynthesis (i.e. the minimum amount of photosynthesis) is defined as the ratio between oxygen released to photons absorbed in the process (Dubinsky & Berman, 1976), which occurs in PSII. Once PSII absorbs light and has accepted an electron, it is not able to accept another until it has passed the first onto a subsequent electron carrier. During this period, the reaction centre is said to be ‘closed’. If all reaction centres close it leads to an overall reduction in the efficiency of photochemistry and so to a corresponding increase in the yield of fluorescence. If quick flashes of light (around 400us) are used, we can measure each of these events without oversaturating the ETC. In contrast, bulk fluorescence methods use longer pulses of light (100-2000 ms), which causes multiple turnover events that do saturate the electron transport chain, resulting in a longer recovery time for cells which can lead to a 50% over-estimation in Fm (Kromkamp and Forster, 2003; Oxborough et al., 2012).
- #12: Better S:N ratio will also help cope with sediments that may be in the sample. This method can also be applied to flowing water due to the high sampling rate, which permits the option of an integrated continual monitoring system for ballast water discharge, allowing for a more representative result with no requirement to manually sample.
- #13: Fluorescein diacetate (FDA) and 5-Chloromethylfluorescein diacetate (CMFDA) are synthetic compounds that hydrolyse with active enzymes in cells, producing a green fluorescence emission when excited by a particular wavelength of light (Steinberg et al., 2011). It has been one of the most common laboratory procedures applied to ballast water testing, whereby viable cells absorb the stain and are counted. This protocol involves adding a staining reagent (FDA) to a 100 mL sample, which is stirred using a magnet to maintain a level of homogeneity. A photomultiplier tube is used to detect pulses of the fluorescence emission which, if the set threshold value is exceeded, is used to calculate a viable cell count. This test takes a little preparation time and a result can be produced in around 30 minutes. This test may also be applied to assess certain species of zooplankton, particularly soft bodied invertebrates which have been assessed in a laboratory using the staining method with a false positive error rate of 20%, however, hard bodied zooplankton have produced false positive error rates between 70-100% (Adams et al. 2014).
- #14: Challenges with this method occur as enzyme activity often continues in cells that are dead, as the metabolism can take a long time to shut down, which results in dead cells being mistaken for viable ones. Studies have found that using stains such as FDA and CMFDA may result in significant errors due to certain species lack of affinity with the stains. One recent study found that staining gave acceptable results for 8-10 out of 24 species tested (MacIntyre and Cullen, 2016) and some weakly staining species emitted less fluorescence intensity than dead cells. Other challenges include stain leaching from cells and the issue that microscopists are subjective. However, there is a portable staining method that addresses the latter problem at least. This test may also be applied to assess certain species of zooplankton, particularly soft bodied invertebrates which have been assessed in a laboratory using the staining method with a false positive error rate of 20%, however, hard bodied zooplankton have produced false positive error rates between 70-100% (Adams et al. 2014).
- #15: Comparison of bulk and distribution analyses with microscopy show are both accurate to a degree, but when analysing large and small cells – you can see on the graph how by analysing the distribution you can pull in all the data points to achieve a r2 correlation of nearly 9 with microscopy. The table shows specific number comparisons to illustrate how the average level of Fv might be very similar, but less than 10 big cells appear as more than 100 without analysing the distribution.
- #16: Here is some data that has been published to show that the portable staining method also has a very strong correlation (although very small sample size) with microscopy for both the 10-50 and >50 size classes – however it is important to remember only certain species in both classes stain.
- #17: Guidelines from IMO have been less clear about zooplankton.
- #18: However, there are tools in the healthcare industry that also determine the cell phase of the bacteria using spectrometry, which can sample up to 100 mL and reduce this timeframe to 0.5-2.0 hours, so there is the potential for this to be applied to ballast water analysis.
- #20: Over the past decade there has been a reasonable effort to test such methods, but due to a lack of BWTS in use it has been impossible to test any substantial amount of ballast water onboard. This is already starting to change since ratification, which has produced many more testing opportunities, but sharing data and more universal evaluations assessing the reliability of methods need to be encouraged. The increase in testing over the last 12 months alone, is showing strong evidence that onboard methods are highly likely to be assessed as more accurate than laboratory methods long term and that integrated monitoring systems may also be widely applied methods over the coming years.