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Environmental
Monitoring
Cite this: J. Environ. Monit., 2011, 13, 110
www.rsc.org/jem PAPER
Performance evaluation of phycocyanin probes for the monitoring of
cyanobacteria
 ıse Veilleux,a Christian Deblois,a Annabelle Warrenb
Christian Bastien,*a Richard Cardin,a Elo€
b
and Isabelle Laurion
Received 20th July 2010, Accepted 20th September 2010
DOI: 10.1039/c0em00366b
Published on 26 October 2010. Downloaded on 30/10/2014 12:48:38.

The performance of two field probes (YSI 6600 and TriOS), used for the measurement of in vivo
phycocyanin fluorescence, was compared and validated in the laboratory in 2008 and 2009 with
cultures of Microcystis aeruginosa and field samples. The background noise of the two probes was low
and the detection limits were estimated at 1500 cells mL1 for the YSI and 0.69 mg PC L1 for the TriOS.
The linearity and repeatability of both probes have been excellent. Strong relationships were observed
between the in vivo fluorescence and the total cyanobacterial biovolume (R2 ¼ 0.82 YSI; 0.83 TriOS) or
the abundance (R2 ¼ 0.71 YSI; 0.75 TriOS) of cyanobacteria. However, the difference between cell
densities determined by microscopy and measured by the YSI can be very large and has been associated
to the variability of cell volume among cyanobacteria. This last observation makes the YSI a qualitative
tool if a post-calibration is not done. The analysis of filtrated samples showed that dissolved
phycocyanin (extracellular) may represent a significant fluorescence signal. No relationship could be
established between the abundance, the total cyanobacterial biovolume or the in vivo fluorescence of
phycocyanin and the concentrations of cyanotoxins (R2 # 0.22).

Introduction support this management plan, identification and enumeration

The proliferation of cyanobacteria in lakes of Quebec (Canada)
is of growing concern given the large number of blooms and
degradation of many lakes reported in recent years, and the
ability of cyanobacteria to produce toxins potentially at risk for
human health.1 In order to protect public health, to improve
knowledge related to cyanobacteria issues and to improve the
water quality, the Government of Quebec has established the
Management Plan 2007–2017.2 From 2007 to 2009, an average of
148 lakes have been affected by blooms in Quebec.3 In order to
a
Centre d’expertise en analyse environnementale, MDDEP, 2700 rue
Einstein, Qu ebec (Qc), G1P 3W8, Canada. E-mail: christian.bastien@
mddep.gouv.qc.ca; Fax: +1 418 643-9023; Tel: +1 418 643-8225 ext. 227
b
Institut National de la Recherche Scientifique—Eau Terre Environnement,
490 de la Couronne, Qu ebec (Qc), G1K 9A9, Canada
of cyanobacteria are performed and analysed for cyanotoxins on
a regular basis. These tests are essential to provide reliable data
for assessing the state of lakes and take decisions on the basis of
management thresholds expressed as cyanotoxin concentrations
and abundance of cyanobacteria. However, these relatively time-
consuming methods performed on grab samples do not allow
monitoring of cyanobacteria with sufficient spatio-temporal
resolution. In this context, field probes measuring the in vivo
fluorescence of phycocyanin may represent an interesting
approach for monitoring cyanobacteria in bringing a rapid tool
to locate and follow the bloom forming cyanobacteria and for
the selection of samples to be analyzed in the laboratory.
Phycocyanin (PC) is a blue pigment which belongs to a group
of light harvesting proteins named phycobiliproteins (PBPs). The
major biological role of PBPs is photosynthetic light harvesting.

Environmental impact
Field probes for the measurement of in vivo fluorescence of phycocyanin have been proposed as a complementary tool for moni-
toring cyanobacteria with the more classical laboratory analysis. Our paper discusses in detail the reliability of those probes and the
limit to the interpretation of results. The impact of cell volume on results expressed in cells mL1 is especially considered. In our
knowledge it is the first report that discriminates the contribution of dissolved (extracellular) and intracellular phycocyanin to the
fluorescence signal. We have also established a relationship between potentially harmful cyanobacteria and actual detection of
cyanotoxins. We think that the paper brings original and useful information for those, like public organizations, who are questioning
the practical application of these probes.

110 | J. Environ. Monit., 2011, 13, 110–118 This journal is ª The Royal Society of Chemistry 2011

PBPs are water-soluble and strongly fluorescent.4 PC is found as
a major pigment (>10% of total pigments) in cyanophyceae and
cryptophyceae, and as a trace pigment (<1% of total pigments) in
rhodophyceae.5,6 It absorbs red and orange light at wavelengths
between 610 and 630 nm (absorption peak at 620 nm) and emits
fluorescence in the band of wavelengths from 600 to 700 nm
(emission peak at 647 nm). In freshwaters, the PC is mainly
associated with cyanobacteria and can be used as an indicator of
their biomass.7 Previous research has demonstrated a very good
relationship between the PC measured with TriOS probe and the
cell density determined by microscopy on nearly 800 samples
(R2 ¼ 0.73).8 Field probes measuring PC fluorescence can then
potentially be used to perform spatio-temporal monitoring of
cyanobacteria blooms and to optimize sampling for laboratory
analyses. Vertical profiles of PC could also be done to evaluate
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the proximity of cyanobacteria to drinking water intakes. In this
situation, the use of such probes could provide early warning
signals.9–12
The objectives of this study are to evaluate the reliability
(background noise, detection limit, quantification limit, linearity,
repeatability and accuracy) of two field probes, the YSI 6600 and
the TriOS microFlu-blue, in controlled conditions in the labo-
ratory, and to determine their potential for a use in the context of
monitoring blooms of cyanobacteria.
Material and methods
Measurement of the in vivo phycocyanin fluorescence
The YSI 6600 probe, equipped with the optical sensor
BGA-PC 6131, uses an orange light emitting diode for the
excitation (peak at 610 nm) and a photodiode of high sensi-
tivity for the detection of fluorescence emitted between
600 and 700 nm. The probe is designed to transform the
fluorescence of the PC into an equivalent cell density of
Microcystis aeruginosa. If the probe is used with the manu-
facturer’s default calibration, its putative measuring range is
between 0 and 280 000 cells mL1. Cyanobacteria density here
expresses the concentration of cells of a known cell volume,
equivalent to dimensions of the species used for calibration
(for M. aeruginosa: cell diameter z 3.5 mm and cell volume z
38 mm3). Three types of calibration can be used with that
probe: the manufacturer’s default calibration (which is done
with a culture of M. aeruginosa), a calibration with rhodamine
to estimate the temporal drift in the calibration and a two-
point home calibration performed with a culture of
cyanobacteria (e.g., 0 and 5000 cells mL1). The two-point
calibration with cyanobacteria is considered the optimal way
to calibrate the probe.13 In 2008 and 2009, tests were made
with manufacturer’s calibration and two-point calibration with
a culture of M. aeruginosa for the determination of back-
ground noise and linearity (see section below). For all others
measurements, the YSI probe had a two-point calibration with
a culture of M. aeruginosa at a cell density of
20 000 cells mL1.
The TriOS microFlu-blue probe has an excitation peak at
620 nm and reads the fluorescence emitted between 650 and
660 nm. This probe provides a result in terms of concentration of
PC and its putative measuring range is between 0 and 200 mg L1.
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The probe allows measurements to be made in two ranges of
sensitivity, from 0 to 20 mg L1 and from 0 to 200 mg L1.14 The
manufacturer’s calibration was used for all tests and was vali-
dated with a standard solution of PC (see section below). The
TriOS probe was tested only in 2009. Values of cyanobacteria
biomass (mg PC L1) measured with the TriOS must be converted
to density equivalent (cells of 38 mm3 mL1) or biomass
(mg chl-a L1) to evaluate risk associated to cyanobacteria on the
basis of established guidelines.1,15
Culture of M. aeruginosa
A culture of M. aeruginosa (UTCC 299) was maintained in
a BG-11 medium16 with continuous agitation under controlled
light conditions (20 mmol m2 s1), temperature (26  C  1) and
photoperiod (16 h light and 8 h dark). Under these conditions,
the culture remained unicellular (without colony formation).
The cell density and average cell volume of the culture were
determined using a particle counter Coulter Multisizer 3. The
results of counts were regularly validated using an inverted
microscope (Nikon Eclipse TE 2000S) with the Uterm€ ohl’s
method.17
Standard solution of phycocyanin
A standard solution of PC was made with the C-phycocyanin
extracted from Spirulina (P2172 Sigma) in phosphate buffer at
pH 7.6. The PC concentration was determined by spectropho-
tometry using the following equation:18
 
  A615  ð0:474  A652 Þ
PC mg mL1 ¼
5:34
where A is the absorbance at 615 or 652 nm.
Background noise
The average background noise of probes was determined with
phosphate buffer at pH 7.4 and with raw water from St Charles
River (near Quebec City) filtered through a nitrocellulose
0.22 mm filter (Millipore GSWP). The values of background
noise were subsequently subtracted from all measures that
followed.
Method detection limit and method quantification limit
The method detection limit (MDL) and the method quantifica-
tion limit (MQL) were considered, respectively, as the concen-
tration equivalent to 3 times and 10 times the standard deviation
of 10 measures done on 10 replicates of a sample at low density.19
For the YSI probe, the MDL and MQL were determined with
the culture of M. aeruginosa maintained in the laboratory at
concentrations of 1500 and 4000 cells mL1. The MDL and MQL
of the TriOS probe were determined using a standard solution of
PC at a concentration of 3.1 mg L1 and also with M. aeruginosa
at 4000 cells mL1.
Linearity
The relationship between the in vivo fluorescence of PC measured
with the YSI probe and the cell density of the culture of
M. aeruginosa was determined with calibrations done with two
J. Environ. Monit., 2011, 13, 110–118 | 111

cyanobacteria cell densities (5000 and 25 000 cells mL1) and for
the manufacturer’s calibration. A culture of M. aeruginosa at
high cell density (1  106 cells mL1, 15%) was quantified with
a particle counter and validated by microscopy. The microscopy
measured values were used as reference points and subsequently,
a dilution series was performed to obtain aliquots between 0 and
100 000 cells mL1 (15%). Some of the dilutions were then
validated by microscopy.
With the TriOS probe, the relationship was established
between the in vivo fluorescence and a standard solution of PC
(from 0 to 150 mg L1). The relationship was also determined
with a culture of M. aeruginosa (from 0 to 100 000 cells mL1,
15%).
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Repeatability and accuracy
The repeatability and the accuracy of the YSI probe were
determined by taking fluorescence measures of 10 replicates of
a culture of M. aeruginosa at cell densities of 30 000 and
65 000 cells mL1. The repeatability of the TriOS probe was
determined by measuring 10 replicates of a culture of M. aeru-
ginosa at densities of 30 000 and 75 000 cells mL1 and its
accuracy was determined by compiling measures of 10 replicates
of two concentrations (20 and 75 mg L1) of the PC standard
solution.
The repeatability was calculated as follows:19
tð0:975; n1Þ  SD
pffiffiffi
n
where t is the Student’s t with a probability of 0.975 and SD is the
standard deviation.
The accuracy was calculated as the difference between the
‘‘true’’ value and the measured value. In the case of the YSI, the
‘‘true’’ value is the cell density determined by microscopy, while
for the TriOS probe, it is the PC concentration determined by
spectrophotometry. The accuracy was calculated as follows:
ðVm  Vt Þ
Relative error ð%Þ ¼  100
Vt
accuracy (%) ¼ 100  relative error (%)
where Vm is the average of 10 measures and Vt is the ‘‘true’’ value.
Table 1 Background noise of the YSI and TriOS probes. N ¼ number of replicates; SD ¼ standard deviation; Min ¼ minimum value measured; and
max ¼ maximum value measured
Mean
YSI probe N Cells mL1
Manufacturer’s calibration 10 307
Calibration with 10 219
5000 cells mL1
of M. aeruginosa
Mean
TriOS probe N mg PC L1
Manufacturer’s calibration 9 0.56
112 | J. Environ. Monit., 2011, 13, 110–118
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Validation of probes with field samples
The 91 samples used for this part of the study were taken from
20 lakes at the surface (portion 0 to 1 metre depth) or in surface
scum between May and November in 2008 and 2009. Samples
were preserved on ice during transport and at 4  1  C in the
laboratory for a maximum storage time of 5 days before fluo-
rometric measurement. A parallel experiment, conducted to
ensure that the storage time of samples was appropriate, indi-
cated no significant loss of PC (a < 0.05) (data not shown). All
in vivo fluorescence measurements were performed in the labo-
ratory. It should also be noted that the samples analyzed in this
study were obtained from several lakes visited upon alerts given
by citizens and were not following a time sequence monitoring.
In 2008, only the fluorescence of total PC (intra and extra-
cellular) was measured with the YSI probe. In 2009, the in vivo
fluorescence was measured with both probes before and after the
filtration of samples through a 0.22 mm filter (Millipore GSWP).
This was done in order to discriminate the signal contribution by
the intracellular PC and the dissolved or extracellular PC as well
as the effect of other possible sources of interference. The iden-
tifications, enumerations and estimations of total cyanobacterial
biovolume were made from sub-samples preserved with 1%
Lugol solution and analyzed using inverted microscopy. All
identifications were done on the basis of reference books.20–23 The
cyanotoxins (microcystin LR, RR, YR, LA, LY, LW, LF and
anatoxin-a) were analyzed using liquid chromatography coupled
to mass spectrometers in tandem (HPLC-MS/MS).
Statistical analysis
All results were transformed into logarithms to meet the
requirements of normality and homoscedasticity for the statis-
tical treatment. The statistical analysis was performed using the
software Sigma Plot 2001 (version 7101) and coefficients of
determination (R2) are presented.
Results and discussion
Background noise
The background noise of both probes was low. The average
background noise of the YSI probe was less than 310 cells mL1,
no matter the type of calibration used, and it was estimated at
SD Min Max
58 213 387
350 1034 364
SD Min Max
0.32 0.24 1.0
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Table 2 Method detection limit (MDL) and the method quantification limit (MQL) of the YSI and TriOS probes. N ¼ number of replicates and SD ¼
standard deviation

Mean SD MDL (3  SD) MQL (10  SD)

YSI probe N Cells mL1

1500 cells mL1 of M. aeruginosa 10 1688 407 1221 4070

4000 cells mL1 of M. aeruginosa 10 4374 594 1782 5940

Mean SD MDL (3  SD) MQL (10  SD)

TriOS probe N mg PC L1

Standard solution of PC at 3.1 mg L1 10 3.2 0.31 0.93 3.1

4000 cells mL1 of M. aeruginosa 10 3.8 0.23 0.69 2.3
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0.56 mg PC L1 for the TriOS probe (Table 1). Negative numbers
were only recorded when the YSI probe was calibrated with
M. aeruginosa.
Method detection limit and method quantification limit
The method detection limit (MDL) and the method quantifica-
tion limit (MQL) of the YSI probe were determined at two cell
densities (Table 2). The average MDL obtained with these two
tests was 1500 cells mL1, which is higher than the 220 cells mL1
reported by the manufacturer.13 The MDL of the TriOS probe
was estimated at 0.93 mg PC L1 with the standard solution of PC
and at 0.69 mg PC L1 with a suspension of M. aeruginosa. These
MDL expressed in PC concentrations represent respectively
around 1170 and 870 cells mL1 of M. aeruginosa. The variability
in the response of the TriOS probe was lower in the culture of
cyanobacteria which has contributed to a lower MDL. The YSI
showed an average MQL of 5000 cells mL1 for the two dilutions
of M. aeruginosa tested and the TriOS showed a MQL of 2.3 mg
PC L1 (or 2900 cells mL1) with the suspension of
M. aeruginosa.
A MQL between 3000 and 6000 cells mL1 is acceptable as it
allows quantitative detection of cyanobacteria with a pure
culture of M. aeruginosa. This limit is also low enough for an
application in the context of monitoring raw and treated water
for human consumption. However, the MDL and MQL may be
higher where the colonial or filamentous forms of cyanobacteria
are dominating, as in the natural environment.24

Fig. 1 Relationships between cell density of M. aeruginosa measured by microscopy and the YSI probe calibrated (a) by the manufacturer or (b) with
cyanobacteria suspensions (filled circle with 5000 cells mL1 and empty circles with 25 000 cells mL1), and relationships between (c) a standard PC
solution and the TriOS probe or (d) relationship between cell density measured by microscopy and the TriOS probe (the dotted lines indicate 1 : 1
relationships).

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 View Article Online

MDL of 102 and 103 cells mL1 is reported in the literature for
M. aeruginosa measured in the laboratory using a spectropho-
tometer.25,26 MDL ranging from 103 to 107 cells mL1 for other
species of cyanobacteria was also reported.25 An MDL of 102
cells mL1 was found in a study with the fluoroprobe designed by
BBE Moldaenke.7 In another study done on a reservoir affected
mainly by M. aeruginosa, a MDL of 1113 cells mL1 was
obtained using the fluorometer Turner Designs 10-AU-005.10
In order to release the PC, the use of a fluorometer coupled to
an ultrasonic device to break the colonies and filaments and even
cause cell lysis has been proposed in which a sonication of
20 minutes would increase by ten the fluorescence response at low
cell density.25 The sonication of samples prior to the reading of
fluorescence could thus substantially decrease the MDL. Such an
approach has the potential to increase sensitivity and reduce
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cyanobacteria significantly improved the accuracy beyond
5000 cells mL1 by comparison to the manufacturer’s calibration.
The stability of the YSI calibration was followed during the
summer 2009 by repeated measurements using a solution of 100
mg L1 of rhodamine. This monitoring has shown an excellent
stability of the calibration over the entire period of the evaluation
(N ¼ 40, mean ¼ 289 000 cells mL1, and SD ¼ 10 789 cells
mL1).
The linearity of the TriOS probe determined with a standard
solution of PC was highly significant (R2 ¼ 0.998, P < 0.0001;
Fig. 1c). Moreover, the results are near the nominal concentra-
tions expected. The differences between the probe and the 1 : 1
relationship were of 30% or less in the concentration range 5 to
150 mg L1. When the exercise was done with a culture of M.
aeruginosa, the linearity was also highly significant

variability related to the morphological diversity of cyanobac- (R2 ¼ 0.999, P < 0.0001) for intracellular PC concentrations of
teria. However, sonication is not easily applicable in the field. about 1 ng per cell (Fig. 1d).

Repeatability and accuracy

Linearity
Overall, the linearity of both probes was excellent with
M. aeruginosa cultures in controlled conditions in the laboratory.
With the manufacturer’s calibration, the linear regression
between cell density measured by microscopy in the range 0 to
100 000 cells mL1 and the density determined with the YSI
probe was highly significant (R2 ¼ 0.996, P < 0.0001; Fig. 1a).
The linearity was also very good even if only the range
0 to 10 000 cells mL1 was considered (R2 ¼ 0.951, P ¼ 0.0001;
data not shown). However, this calibration of the YSI probe
leads to results that are largely underestimated (by 70 to 93%) in
comparison to values determined by microscopy.
When the YSI probe was calibrated with cyanobacteria at
densities of 5000 and 25 000 cells mL1, the linear regressions
were again highly significant in the range 0 to 100 000 cells mL1
(R2 > 0.993, P < 0.0001; Fig. 1b). When considering the rela-
tionship in the range 0 to 10 000 cells mL1 only, R2 was similar
as for the entire range for the calibration done at 5000 cells mL1.
However, it was slightly lower for the calibration done at
25 000 cells mL1 (R2 ¼ 0.815, P ¼ 0.0009; data not shown).
According to our results, the probe seems more efficient when it
is calibrated at 5000 cells mL1 in comparison with a calibration
at 25 000 cells mL1. Moreover, the calibration with
The repeatability of measurements was excellent for both probes
(Table 3). The coefficients of variation (CV) of the YSI probe
calibrated by the manufacturer are of 2.0 and 1.1% depending on
the cell density measured. However, the results show relatively
large differences between the probe and the nominal values
determined by microscopy (the probe overestimated cell density
by 38 and 59%, respectively at 30 000 and 60 000 cell mL1). The
CV of the TriOS probe varied between 1.2 and 4.8% depending
on the type of sample used. Although the TriOS probe response
was more variable than that of the YSI probe, it proved to be
more accurate (with differences with ‘‘true’’ values lower or equal
to 15%) in comparison to the YSI probe even if this last one was
calibrated with M. aeruginosa. Considering this aspect, the TriOS
probe appears more efficient than the YSI probe, although it is
also possible that the use of pure PC extracts artificially
improved its accuracy.
Validation of probes with field samples
In 2008 and 2009, the in vivo fluorescence of PC measured by
both probes was strongly related to the cell density and to
the total cyanobacterial biovolume measured by microscopy
(R2 > 0.705, P < 0.0001; Fig. 2). However, the differences

Table 3 Repeatability and accuracy of the YSI and TriOS probes. N ¼ number of replicates; SD ¼ standard deviation; and CV ¼ coefficient of
variation

Mean SD CV Repeatability Accuracy

YSI probe N Cells mL1 % Cells mL1 %

30 000 cells mL1 of M. aeruginosa 10 41 401 832 2.0 44 401  540 +38
65 000 cells mL1 of M. aeruginosa 10 103 451 122 1.1 103 451  802 +59

Mean SD CV Repeatability Accuracy

TriOS probe N mg L1 of PC % mg L1 of PC %

30 000 cells mL1 of M. aeruginosa 10 30 0.49 1.6 30  0.32 na

75 000 cells mL1 of M. aeruginosa 10 59 0.71 1.2 59  0.51 na
Standard solution of PC at 20 mg L1 10 23 1.1 4.8 23  0.71 +15
Standard solution of PC at 75 mg L1 10 69 2.4 3.5 69  1.6 8.4

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Fig. 2 Relationships between the YSI probe in 2008–2009 for field samples and (a) the cell density measured by microscopy; (b) the biovolume
measured by microscopy; and relationships between the TriOS probe in 2009 and (c) the cell density measured by microscopy; and (d) the biovolume
measured by microscopy (the dotted line indicates 1 : 1 relationship).
between YSI probe and microscopy results varied from 97 to
1239% in 2008 and from 93 to 890% in 2009. For the TriOS
probe, the relationships had slightly higher R2 by comparison to
the YSI probe. TriOS accuracy with field samples cannot be
assessed because the PC concentrations were not determined by
a reference method (e.g., with pigment extraction and spectros-
copy).
The samples for which density was underestimated by the YSI
probe contained in most cases less than 30 000 cells mL1 while
the overestimated densities were observed in samples having
more than 30 000 cells mL1 (Fig. 2a). The underestimated
samples generally exhibited diverse communities of cyanobac-
teria, including picocyanobacteria that are often dominant in
terms of abundance. Inversely, the overestimated samples often
contained bloom-forming species dominated by one or two
genera and where picocyanobacteria were rarely identified. This
dominance of picocyanobacteria in samples of low density is also
reflected in the relationship between the in vivo fluorescence
measured with the YSI probe and the total cyanobacterial bio-
volume in 2008–2009 (Fig. 2b) where nearly 15% of samples
containing a total cyanobacterial biovolume that was very small.
It is possible that picocyanobacteria have been underestimated in
samples with the presence of bloom-forming species. Moreover,
no false-positives of PC have been observed in this study but
some authors have reported false-positives in the presence of
high turbidity.27
In order to better understand the effect of picocyanobacteria on
the relationships observed, linear regressions have also been
applied after the picocyanobacteria genera quantified by
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microscopic analysis (Aphanothece, Aphanocapsa, Cyanodictyon,
Merismopedia, Planktolyngbya, Limnothrix, Phormidium and
Synechococcus) have been removed from the data series. It was
found that the withdrawal of these genera did not affect R2 values
between the total cyanobacterial biovolume and both probe
estimations, suggesting their small contribution to the total cya-
nobacterial biovolume. However, for the relationships with cell
density, the withdrawal of the picocyanobacteria genera dimin-
ished the R2 values for both probes (R2 ¼ 0.655 YSI, 0.610 TriOS)
(data not shown). This diminution is clearly due to samples
dominated by picocyanobacteria. In this regard, it may be wise to
use the YSI raw data (in relative fluorescence units) instead of the
result transformed in cells per mL since the raw data are directly
related to the quantity of PC and then to the total cyanobacterial
biovolume, the same way as for the TriOS probe. In addition to
the variability in cell volume of different genera of cyanobacteria
and the possible effect of turbidity, there are several other factors
that may influence the in vivo fluorescence of PC. They comprise
the growth stage of cyanobacteria,28 the level of light saturation,29
the aggregation of cells into colonies more or less voluminous,25,30
and the historical conditions of light and nutrients.31,32
Because in vivo PC fluorescence measurements are qualitative
in the absence of data correction following post-calibration, it
can be difficult to evaluate whether or not a decisional threshold
is reached. However, some authors suggested management
thresholds based on PC levels for the determination of the
appropriate sampling frequency.8 Some also suggested using
direct PC thresholds (measured in the laboratory with a spec-
trophotometer) but they warn of the need to give special
J. Environ. Monit., 2011, 13, 110–118 | 115

attention to samples having an in vivo fluorescence near the
management threshold given the variability between probes in
terms of reliability and calibration.33 In the context of optimi-
zation of field sampling, the PC in vivo fluorescence could be used
as criteria to decide whether or not a sample should be taken for
identification and enumeration of cyanobacteria by microscopic
analysis in the laboratory. Given the qualitative aspect of the
measures of PC, a threshold corresponding to a density of 15 000
cells mL1 of cyanobacteria or about 15 mg PC L1 seems
appropriate as decision criteria, as one of the management
thresholds is 20 000 cells mL1. However, a more precise vali-
dation is needed near the threshold value of 20 000 cells mL1,
especially for the YSI probe. Also, the combined use of PC in vivo
fluorescence and cyanotoxins screening tests (microcystins strip
test34) seems optimal by avoiding false negative (i.e., detectable
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presence of cyanotoxins with low density of cyanobacteria or
conversely high density of cyanobacteria and no cyanotoxins
detection). The latter immunochromatographic strip test is being
already tested in the Government of Quebec Management Plan.
The simultaneous variations of both probes with the cell
densities and total cyanobacterial biovolume measured by
microscopy are illustrated in Fig. 3. The curves show a close
agreement between probe estimations and the total cyano-
bacterial biovolume. However, differences sometimes exceeding
an order of magnitude are seen between cell densities estimated
with the YSI probe and those measured by microscopy. The
samples for which the YSI probe presented a good accuracy were
Fig. 3 Comparison between the cell density of field samples and the biovolume measured by microscopy: (a) the YSI probe for 2008–2009 and (b) the
TriOS probe for 2009.
116 | J. Environ. Monit., 2011, 13, 110–118
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dominated by Microcystis or a mixture of Anabaena and Apha-
nothece. The latter potentially results in an average cell volume
similar to the one of M. aeruginosa used for the calibration of the
instrument in the laboratory.
In 2009, measurements obtained on filtrated samples indicated
the presence of extracellular PC in many samples. There are
significant relationships between the fluorescence signal in the
filtrate measured by the YSI probe and the cell density
(R2 ¼ 0.600, P < 0.0001; data not shown) or total cyanobacterial
biovolume (R2 ¼ 0.733, P < 0.0001; Fig. 4a), as well as for the
TriOS probe signal with cell density (R2 ¼ 0.570, P < 0.0001; data
not shown) or total cyanobacterial biovolume (R2 ¼ 0.725,
P < 0.0001; Fig. 4b). On average, 21 to 25% of the fluorescence
signal was related to the filtrate, and therefore to extracellular
PC. The variations of the filtrate fluorescence signal were
important between samples and the ratio of extracellular PC to
intracellular PC varied from 1 to 100%. In some cases, the whole
PC signal was therefore extracellular. Interferences caused by
varying concentrations of chromophoric dissolved organic
matter may have contributed to the fluorescence of filtrates.
However, the relationship between the filtrate PC signal and total
cyanobacterial biovolume suggests that the fluorescence was
indeed related to the extracellular PC released by lysed cells of
cyanobacteria. This observation raises questions on the expres-
sion of the YSI results in terms of cells mL1 because the extra-
cellular PC is included in the computed abundance.
Measurement of the in vivo fluorescence must be interpreted in
This journal is ª The Royal Society of Chemistry 2011

Fig. 4 Relationships between the fluorescence of the filtrate of field samples and the biovolume measured by microscopy for (a) the YSI probe in 2008–
2009 and (b) the TriOS probe in 2009.
Published on 26 October 2010. Downloaded on 30/10/2014 12:48:38.
terms of total PC and the subtraction of a field blank obtained
from filtered samples will allow an estimate of intracellular PC.
The presence of extracellular PC has already been raised as
a hypothesis to explain some data showing no apparent rela-
tionship with cell density.8 No relationship could be established
between the presence of extracellular PC and the cyanobacteria
genera observed or the time of the year. However, in some cases
where the extracellular PC was elevated, high densities of the
genus Anabaena were observed.
During the two years of the study, cyanotoxins have been
identified in 36 of the 91 samples. When all samples were
considered, no relationship was established between the
concentration of cyanotoxins and the cell density (R2 ¼ 0.235) or
the total cyanobacterial biovolume (R2 ¼ 0.198) or with the
probe estimations (R2 ¼ 0.172 and 0.161, respectively for the YSI
and the TriOS probes). This result confirms that there is no direct
link between the presence of cyanobacteria and the cyanotoxin
concentrations measured. It is known that the gene expression
for microcystin or other toxin production may vary from one
strain to another.35 The activation of these genes is also strongly
influenced by environmental conditions, such as the concentra-
tions of nitrogen and phosphorus.36–38 Moreover, there is a wide
range of cyanotoxin types39 and many are not currently quanti-
fied. This could also contribute to the weakness of the relation-
ship between in vivo PC fluorescence and the concentrations of
cyanotoxins. A genomic approach has been proposed to distin-
guish toxic and non-toxic genotypes of Microcystis.40
It is important to note that the 36 samples with detectable
concentrations of cyanotoxins contained in most cases high
densities of cyanobacteria. The presence of cyanobacteria
remains therefore an indicator of risk even if a quantitative
relationship cannot be established.
Conclusion
In vivo PC fluorescence measurements using field probes present
an interest for spatio-temporal monitoring of algal blooms in
natural environments, for the optimization of sampling strategies
and for the early detection of cyanobacteria in drinking water
intakes. The relationships observed with the total cyanobacterial
biovolume and cell density are significant and the quantification
limits of fluorescence probes are low enough for the purposes of
such monitoring. However, the results for the YSI probe are
This journal is ª The Royal Society of Chemistry 2011
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qualitative if no correction is applied following a post-calibra-
tion, as suggested by the manufacturer.
The TriOS probe appears interesting for monitoring cyano-
bacterial blooms because of its ease of use, its reliability and its
expression of the results in terms of PC concentration which is
not biased by the variability of cell volume. However, thresholds
generally used in management plans are in cells per mL which
disadvantages the TriOS probe as this provides results in terms of
concentration of PC. The advantage of using a multi-probe
system such as proposed by YSI, where for example data on
water temperature, dissolved oxygen, total chlorophyll-a and
PC are collected simultaneously, is certainly considerable and
should also be taken into account in monitoring studies.41
Improvement of converting equations provided by manufac-
turers is needed. The lack of quantitative relationship between
cyanobacteria and cyanotoxin concentrations shows that these
two parameters must be used in a complementary way to manage
risk appropriately.
Acknowledgements
The authors would like to thank Ga€elle Triffault-Bouchet for her
support to the revision of the document. They also want to thank
all the people from the different regional divisions of the ministry
for their contribution to the field sampling.
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