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Dutch Investigations into the bbe FluoroProbe, Chlorophyll-a, Biovolume, Cell Numbers, Chlorophyll Determination by ISO methods and Content of Mycrocystins . Reported by Ron van der Oost, Waternet, Amsterdam.

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  1. Dutch Investigations into the bbe FluoroProbe, Chlorophyll-a, Biovolume, Cell Numbers, Chlorophyll Determination by ISO methods and Content of Mycrocystins Reported by Ron van der Oost, Waternet, Amsterdam The Netherlands: The bbe FluoroProbe becomes the ‘Recommended’ Instrument as a Bathing Water Early Warning System Page 1

  2. Tabel 3: bluegreen algae norms Normen microcystins µg/L Cell density cells/ml biovolume mm3/L cyano-chlorophyll µg/L Norm 1: Increased Awareness - 20.000 1 5 Norm 2: Watch out 10* 100.000** 5 25 Norm 3: Negative swimming advice 20* 200.000*** 10 50** *: CIW norm **: WHO norm for high risk ***: WHO norm cell density high risk, adjusted by the work group ‘cyanobacteria’ The WHO determines a limit for the amount of cyanobactaria chlorophyll but there must be no other algae classes present to a higher degree. The interesting thing: only the FluoroProbe can tell you which part of the total chlorophyll really belongs to the group of cyanobacteria !! This means the WHO rule can be applied all the time Page 2

  3. The idea: fluorescence of cyanobacteria can serve as an early warning system under worst-case scenarios • every cyanobacteria cell contains toxins • the toxin concentration is related to cyanobacteria chlorophyll (WHO directives) • the probe can identify the cyanobacteria chlorophyll in a mixture •  the bbe FluoroProbe is the choice application Page 3

  4. Tabel 1: blauwalgen celtelling (cellen/ml) door 3 verschillende laboratoria Difficult cell counts: 3 different laboratories were unable to determine cell numbers of cyanobacteria samples code MYC UvA PLA UvA ANA UvA APH UvA Lab 1 614000 481000 788000 188000 Lab 2 1325000 565000 674000 303000 Lab 3 2267000 438000 1293000 193000 Gemiddelde 1402000 494000 918000 228000 standaard deviatie 59% 13% 36% 28% Can Cell Counts do the Job? Page 4

  5. • There exists a weak significant relationship between chlorophyll fluorescence and microscopically determined blue algae cell density. The relationship between these parameters is insufficient for reliable prediction of the cyanobacteria cell density using cyano-chlorophyll. The species remained the same in a series of tests (high values are from cultures) The species varied in a series of tests Page 5

  6. • There is a highly significant linear correlation between the fluorescence of some cyano-chlorophyll and microscopic cyanobacteria biovolume. This provides a good basis for a reliable prediction of the cyanobacteria biovolume to be carried out under cyano-chlorophyll. Page 6

  7. 5.1 Conclusions • Based on tests using the linearity and reproducibility, it can be concluded that the reliability of fluorescence analysis with the bbe Moldaenke FluoroProbe is good within the range of 0 to 400 micrograms chlorophyll per litre. There is a highly significant linear relationship with chlorophyll fluorescence and some total chlorophyll spectrophotometrically according to NEN 6520 (~ISO 15839). The relationship between these two chlorophyll levels is 1.6.Calibration with NEN chlorophyll seems a good basis for results from different laboratories with different equipment to compare fluorescence. The bbe FluoroProbe Complies with the ISO/NEN Standard Page 7

  8. Relationships between cyanobacteria and microcystin presence The biggest problem is that these organisms can produce cyanobacteria toxins (cyanotoxins). A bloom can lead to dangerously high toxin levels that are a risk for recreational water during swimming in blue algae. Often the symptoms of the cyanotoxins are difficult to distinguish from other causes, but mild symptoms such as skin irritation, diarrhea and nausea seem to exist for quite general swimmers after contact with cyanobacteria. An epidemiologic study of 852 recreationals showed a higher number of complaints about diarrhea, vomiting, fever and irritated eyes, ears and skin seen within one week after exposure to cyanobacteria. The health problems are related to exposure duration and density of cyanobacteria (Pilotto et al, 1997). The datasets for the present study were analyzed, there was no correlation between the levels of microcystin (the most measured cyanotoxin) and with parameters obtained using fluorescence or microscopy (cyano-chlorophyll, cyanobacteria cell density and biovolume). Page 8

  9. The National Water Authorities (NWO) produced in March 2010 a new blue-green algae protocol setting out a number of recommendations (from the draft version of) this report (already included here). It is an option for blue-green algae risk analysis based on cell density, biovolume or cyano-chlorophyll. The risk is based on the values in the table. Tabel 4: blauwalgen normen volgens het NWO protocol 2010 Normen & acties drijflaag Cell density cells/ml biovolume mm3/L Cyanochloro-phyll µg/L 1. Increased alertness (frequent monitoring, microscopic investigation) categorie 1* - - - 2: Warnings categorie 2 50.000 2.5 12.5 3: Negative swimming advice issued categorie 3 300.000 15 75 Page 9

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  11. Looking at drinking water, it becomes even more important that you know about the contribution of the cyanobacteria chlorophyll in the water. This can easily be done using the bbe FluoroProbe, the bbe AlgaeTorch or a bbe online instrument. Page 12

  12. Many Thanks foryour Attention Page 13

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