The Netherlands:
The bbe FluoroProbe becomes the ‘Recommended’ Instrument as a Bathing Water
Early Warning System
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
Page 1
Tabel 3: bluegreen algae norms
cyanochlorophyll
µg/L
microcystins
µg/L
Cell density
cells/ml
biovolume
mm3/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**
Normen
*: 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
The idea: fluorescence of cyanobacteria can serve as an early warning system under worstcase 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
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Can Cell Counts do the Job?
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
59%
13%
36%
28%
standaard deviatie
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• 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.
A
450000
100000000
350000
celdichtheid [cellen/ml]
celdichtheid [cellen/ml]
400000
300000
250000
200000
150000
100000
10000000
1000000
100000
50000
0
10000
0
5
10
15
20
25
cyano-chlorofyl [µg/L]
The species varied in a series of tests
30
35
40
10
100
1000
10000
cyano-chlorofyl [µg/L]
The species remained the same in a series of tests (high values are
from cultures)
Page 5
• 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.
A
18.0
biovolume [mm3/L]
16.0
14.0
12.0
10.0
8.0
6.0
4.0
2.0
0.0
0
5
10
15
20
25
30
35
40
cyano-chlorofyl [µg/L]
Page 6
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.
Rijnland +Terranova
500
400
Chl_NEN
The bbe FluoroProbe Complies with the ISO/NEN
Standard
300
200
100
0
0
50
100
150
200
250
300
Chl_probe
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.
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Relationships between cyanobacteria and microcystin presence
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).
10000
microcystine [µg/L]
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.
A
1000
100
10
1
1
10
100
1000
10000
100000
cyano-chlorofyl [µg/L]
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).
B
microcystine [µg/L]
10000
1000
100
10
1
1
10
100
1000
10000
biovolume [mm3/L]
Page 8
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).
Tabel 4: blauwalgen normen volgens het NWO protocol 2010
Cyano
Cell
chlor
density
biovolume
Normen & acties
drijflaag
ocell
mm3/L
phyll
s/ml
µg/L
It is an option for blue-green algae risk analysis based on cell
density, biovolume or cyano-chlorophyll.
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
The risk is based on the values in the table.
Page 9
Page 11
parameter
unit
Drinking water
microcystin-LR µg.l-1
nodularin
µg.l-1
saxitoxin
µg.l-1
cyanobacteria
cells.ml-1
cyanobacteria
mm3.l-1
chlorophyll
µg.l-1
chlorophyll of µg.l-1
cyanobacteria
Recreational water
microcystin-LR µg.l-1
cyanobacteria
mm3.l-1
chlorophyll of
cyanobacteria
µg.l-1
WHO Canada
1
1.5
Australia
1.3
Australia short
exposure only
10
10
3
2000
20000
1
10
Ueno et al.
The
Netherlands
(1999)
1
0.01
0.33
2.3
20
6.6
(3.3)
46
(23)
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.
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Many Thanks for
your Attention
Page 13