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PRIMARY RESEARCH PAPER
Effect of chlorophyll sampling design on water qualityassessment in thermally stratified lakes
Peeter Noges • Sandra Poikane • Toomas Koiv •
Tiina Noges
Received: 27 December 2009 / Revised: 5 March 2010 / Accepted: 15 March 2010 / Published online: 9 May 2010
� Springer Science+Business Media B.V. 2010
Abstract In order to adequately assess the ecolog-
ical status of thermally stratified lakes based on
chlorophyll, the sampling must cover all productive
layers of the water column. Missing the deep chloro-
phyll maxima (DCM) that often occur in the meta- or
hypolimnion of transparent lakes supported by suffi-
cient illumination and good nutrient availability may
cause serious underestimation of the productivity and
lead to misclassification of the lake ecological status.
There is no commonly accepted sampling design for
stratified lakes, and various monitoring guides suggest
controversial designs. Our aim was to find some robust
criteria to assess the probability of occurrence of a
DCM and estimate the differences in measured mean
chlorophyll concentrations caused by various sam-
pling designs. Our theoretical model showed that the
probability of occurrence of a DCM increases with
increasing water transparency and decreasing lake
size. Empirical data from Italian and Estonian strat-
ified lakes confirmed the results. Testing of different
sampling designs on lakes with full measured chloro-
phyll profiles available showed that taking only
surface layer samples will lead with a high probability
to an underestimation of the chlorophyll concentration
in the trophogenic layer. In order not to miss the Chl
peak in stratified lakes, in most cases it would be more
precautious not to limit the sampling to the well-mixed
epilimnion but to extend it to the whole euphotic layer.
Sampling the epilimnion instead of the euphotic zone
could cause up to a 70% underestimation of the
chlorophyll concentration, an error that would cause a
misclassification of the lake by one or even two status
classes in a 5-class assessment system. In most cases,
the 2.5 * Secchi depths proved a suitable criterion of
the sampling depth and only in the case of surface
scums, would sampling of a 3 * Secchi depth layer be
recommended in order not to miss the deep chloro-
phyll maximum.
Keywords Ecological status �Deep chlorophyll maximum � Euphotic depth �Metalimnion � Thermocline
Introduction
Measuring primary production could give a good
estimate to the trophic and ecological status of lakes.
In practice, however, primary production is not a
Handling editor: Luigi Naselli-Flores
P. Noges (&) � T. Koiv � T. Noges
Centre for Limnology, Institute of Agricultural and
Environmental Sciences, Estonian University of Life
Sciences, 61120 Rannu, Tartumaa, Estonia
e-mail: peeter.noges@emu.ee
S. Poikane � T. Noges
Rural, Water and Ecosystem Resources Unit, TP-483,
European Commission, Joint Research Centre, Institute
for Environment and Sustainability, Via E. Fermi 2749,
21027 Ispra, VA, Italy
123
Hydrobiologia (2010) 649:157–170
DOI 10.1007/s10750-010-0237-4
convenient indicator for ecological status mostly
because of its strong dependence on weather condi-
tions, diurnal and seasonal variations, and a number
of methodological difficulties (Schindler, 1972; Lik-
ens, 1975; Schroder, 1994). Moreover, at the same
productivity level, the visible external features such
as water transparency or bloom occurrence may vary
strongly depending on species and efficiency of the
food chain. Obviously due to above-mentioned
reasons, phytoplankton abundance (and not primary
production), composition, and bloom frequency were
selected as obligatory phytoplankton characteristics
to be used in ecological status assessment according
to Water Framework Directive (WFD; Directive,
2000). In order to guarantee a Europe-wide compa-
rability of chlorophyll results, a standardization
of sampling protocols is needed to minimize the
methodological bias.
In moderately deep and deep lakes, a seasonal
thermal stratification divides the water column into
epi-, meta-, and hypolimnion, often with contrasting
values of water quality parameters. For such lakes, a
well-planned sampling design is required as moni-
toring results strongly depend on the selection of
sampling depths. Although it is generally agreed to
take integrated samples over the depth range of
interest to avoid under- or over-representation of
vertical distribution peaks of the variables, the depth
ranges themselves are often vaguely defined.
In its simplest form as described in many hand-
books (e.g., Bass et al., 2001), thermal stratification
of lakes creates a layer of warm, relatively light
surface water called the epilimnion and a cold, denser
layer called the hypolimnion at the bottom. These two
layers are separated by a transition layer (metalim-
nion or thermocline) with a strong temperature
gradient that prevents the epilimnion from mixing
any deeper, thus isolating the hypolimnion waters
from the water body’s surface.
The middle or transitional zone between the well-
mixed epilimnion and the stagnant hypolimnion
layers is loosely defined depending on the shape of
the temperature profile. The word ‘‘thermocline’’ was
proposed by Birge (1897) and has been defined as the
region in which the temperature gradient is equal to
or greater than 1�C m-1 depth. Depending on the
lake type and climatic region, however, the criteria
could be different. Therefore, for instance, in the deep
tropical Lake Malawi, a temperature gradient of only
0.01�C m-1 over 25 m formed an effective barrier to
mixing and thus the layer constituted the metalimnion
(Eccles, 1974). Wetzel (2001) proposed a graphical
definition of the mixing depth by the intersection of
two lines fitted to the thermal profile: one through the
upper part of the epilimnion and the other through the
metalimnion. In general, shallow metalimnia charac-
terize the lakes of higher latitudes, whereas in warm
lakes of lower latitudes deeper metalimnia were
recorded (Tafas et al., 1997).
There is still some confusion with the terms of
metalimnion and thermocline. In some sources (e.g.,
U.S. EPA, 2006), they are considered as synonyms.
In most cases, however, metalimnion is understood as
the whole transitional zone, which may extend
vertically to several or even several tens of meters
in deep lakes and which contains the planar thermo-
cline—an infinitely thin layer of the maximum
temperature gradient (Hutchinson, 1957).
The depth of the metalimnion as well as the
steepness of the temperature gradient depends on lake
morphometry, water clarity, and the climatic region.
Maximum effective length (MEL), maximum depth,
and chlorophyll a contribute significantly to the
prediction power of regression models, but are of
secondary importance in the presence of humic
substances (Perez-Fuentetaja et al., 1999). One of
the most important factors affecting the mixing depth
is the wind fetch, i.e., the maximum length (ML) of
open water over which the wind can blow. The larger
the lake, the longer the wind fetch. The amount of
energy transferred from the wind to the water
increases together with the increasing timespan and
the speed of the blowing wind. Hanna (1990), who
compared 17 empirical models that related mixing
depth to lake morphometric variables, found the MEL
of a lake to be the best single predictor of thermocline
depth (Zthc). For regularly shaped lakes, MEL
corresponds to ML, whereas for irregularly shaped
lakes ML may be curvilinear, and thus greater than
MEL.
In highly transparent lakes and reservoirs, light
may penetrate into the meta- and hypolimnion, and
support photosynthesis there. Due to ample nutrient
availability in these layers, deep chlorophyll maxima
(DCM) may develop in such lakes. It is utmost
important that the sampling would reach the DCM if
such exists in a lake. In order not to miss the DCM
layer, it is recommended that the vertical sample
158 Hydrobiologia (2010) 649:157–170
123
would cover not only the well-mixed epilimnion, but
the whole euphotic zone. According to the most
common definition, the photic or euphotic zone (Zeu)
is limited by the depth where photosynthetically
active radiation (PAR) drops to 1% of its surface
value. Thus, Zeu is a measure of water clarity or
transparency similar to Secchi depth (Zs). Several
attempts have been made to relate these two param-
eters to assess the thickness of the euphotic layer by
the simple Secchi measurement. The main conclusion
from these studies (e.g., Grobbelaar & Stegmann,
1976; Tilzer, 1988) is that the relationship between
Zeu and Zs is nonlinear, and consequently, euphotic
depth cannot be correctly calculated from Secchi
depth by multiplying it by a certain coefficient, (e.g.,
2.5).
Based on studies in Lake Constance, Tilzer (1988)
showed that the ratio of Zeu/Zs varied from 1.5 to[5
and increased with water turbidity. The coefficient
2.5 often recommended (Poikane, 2009) would fit if
Zs is around 4 m but would under- or overestimate
Zeu at lower or higher transparencies.
A number of guidelines exist both on national and
international levels on how to set up monitoring
programmes (Table 1). Although some protocols are
based on surface samples (U.S. EPA, 2007), most of
the guidelines for sampling phytoplankton and asso-
ciated chemical parameters recommend depth inte-
grated samples over the epilimnion (Chapman, 1996)
or euphotic zone defined as 2–2.5 Secchi depths
(OECD, 1982). It is generally acknowledged that
sampling depth should not be constant from lake to
lake and not even from visit to visit, but should be
decided on every occasion according to changing
mixing depth or Secchi depth.
In Europe in course of the implementation of
Water Framework Directive (WFD; Directive, 2000)
a large variety of national assessment methods needs
to be harmonized in a special Intercalibration Exer-
cise (EC, 2005). Although an agreed standard exists
for the determination of the chlorophyll a concentra-
tion (ISO, 1992) that is applicable across Europe,
paradoxically, no standard sampling design for lake
phytoplankton and chlorophyll has been established.
The WFD Monitoring guidance (EC, 2003) recom-
mends integrated or discrete samples from the water
column (i.e., epilimnion, euphotic depth, metalim-
nion). As a consequence, countries have developed
(or are developing) different monitoring methods
ranging from sampling from the surface (UK,
Finland) to taking integrated euphotic zone or
epilimnetic samples (Table 2). In some cases, there
are marked differences even within one country. Still
this situation is in line with the spirit of the WFD,
which accepts different monitoring systems as far as
they can be successfully intercalibrated.
Woods (1986) and Hanna & Peters (1991), who
earlier have analysed the methodological bias in
chlorophyll results caused by different sampling
designs, concluded a general robustness of this
Table 1 Overview of sampling guidances
Reference to guidance document Recommended sampling design
EC (2003) Integrated or discrete samples in the water column (i.e., epilimnion,
euphotic depth, metalimnion)
EC (2009) Not specified
ISO (1987) Depends on the information required and local circumstances
OECD (1982) Euphotic zone (surface waters)
Chapman (1996) Depth integrated samples over epilimnion
Bartram & Ballance (1996) Integrated sample for the productive water layer (euphotic zone)
EEA (2009) Depth average (epilimnion or euphotic zone)
US EPA (2007) Upper 2 m of the water column; If euphotic zone \2 m, only euphotic zone
Elias et al. (2008) Integrated sample 0–2 m
MDEQ (2001) Integrated over euphotic zone (2 * Secchi depths)
MPCA (2004) Integrated sample 0–2 m
Carlson & Simpson (1996) Surface sample, integrated sample (epilimnion, photic zone, the
whole column, fixed depth)
Hydrobiologia (2010) 649:157–170 159
123
indicator regarding the protocol but acknowledged
the effect of the DCMs on the results. Their data,
however, covered oligotrophic to moderately eutro-
phic lakes within a geographically limited range.
In our study, we analyzed (1) how the occurrence
of a DCM could be predicted based on lake size and
Secchi depth and (2) how different sampling designs
would affect the estimates of chlorophyll concentra-
tions in different types of stratified lakes including
hypertrophic ones. First, we constructed a theoretical
model to assess the effect of lake size on Zeu/Zthc
ratio and to estimate how important it is to consider
the nonlinearity of the relationship between the
Secchi depth and the euphotic depth. As a second
step, based on chlorophyll profiles from a number of
stratified lakes of different size and trophic state, we
compared the mean chlorophyll concentrations
obtained if the vertical sampling was limited to:
1. the upper first meter (just a surface sample),
2. the upper boundary of the metalimnion (the layer
where the vertical temperature gradient reaches
1�C m-1),
3. the thermocline depth (layer of the maximum
vertical temperature gradient),
4. euphotic depth where PAR drops to 1% of its
surface value,
5. euphotic depth estimated as 2.5 * Zs.
On the basis of this comparison, one can judge upon
the comparability of chlorophyll data collected by
different sampling strategies and avoid common
pitfalls in sampling design of thermally stratified lakes.
Table 2 Overview of sampling methods in the WFD phytoplankton assessment systems
Country Sampling depth Reference
Austria Euphotic depth or alternatively epilimnion (if
epilimnion [ euphotic depth)
Wolfram & Dokulil (2009)
Carinthia Integrated sample from the upper 6-m layer Wolfram, pers.com. (2009)
Salzburg Integrated sample from the upper 12-m layer for most lakes
throughout the year, from the upper 6 meters for some small
lakes
Wolfram, pers.com. (2009)
Upper Austria Euphotic depth (2.5 * Secchi). Maximum sampling depth 20 m
independently of Secchi depth (Zs)
Wolfram, pers.com. (2009)
Belgium Integrated samples from epilimnion and metalimnion Poikane (2009)
Estonia Depending on stratification, 2–3 samples from distinct depths
(0.5 m, thermocline or sometimes oxycline, hypolimnion) from
the deepest point.
Poikane (2009)
Finland Integrated surface sample (0–2 m) Lepisto et al. (2004);
Lepisto et al. (2006)
France (lakes) Integrated whole water column sample or integrated surface
sample (1–2 m depth)
Poikane (2009)
France (reservoirs) Euphotic zone (2.5 * Zs) Poikane (2009)
Germany Epilimnion or euphotic zone (2.5 * Zs in clearwater lakes) Mischke & Nixdorf (2008)
Hungary Integrated sample from euphotic zone, the whole water column in
shallow lakes (\3.5 m)
Padisak et al. (2006)
Italy Layer between 0 and 20 m (euphotic depth) Salmaso et al. (2006)
Italy Euphotic zone (2.5 * Zs) Marchetto et al. (2009)
Netherlands Epilimnion Poikane (2009)
Norway Integrated surface sample (0–6 m) Ptacnik et al. (2009)
Poland Epilimnion Pasztaleniec & Poniewozik (2009)
Portugal Euphotic zone (2.5 * Zs) Poikane (2009)
Spain Euphotic zone (2.5 * Zs) Poikane (2009)
Sweden Epilimnion or surface sample (0.5 m) SEPA (2007)
UK Surface sample 0.3 m UKTAG (2008)
160 Hydrobiologia (2010) 649:157–170
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Materials and methods
Definitions
In this article, we used consistently the same
definitions for the layers both for the modeling
approach and for empirical data.
1. The boundary between epi- and metalimnion
(Zepi) was defined as layer where the tempera-
ture gradient reaches 1�C m-1.
2. The thermocline depth (Zthc) is the depth of the
layer with maximum vertical temperature gradi-
ent. If the gradient remained\1�C m-1, the lake
was considered non-stratified. In some cases
when during hot days a temporary secondary
thermocline was formed, the main thermocline
was decided on the basis of its strength and
seasonal development of the profiles.
3. Euphotic depth defined as 1% of the PAR level
just under the surface layer (Zeu1%).
4. Euphotic depth estimated as 2.5 * Zs (Zeu2.5S).
Theoretical model
Using Secchi depth (Zs) to derive a proxy for
euphotic depth (Zeu), and MEL for that of the
thermocline depth (Zthc), we constructed a nomo-
gram (Fig. 1) to see when the Zeu/Zthc ratio exceeds
the value of one, i.e., when there will be a risk of
underestimation of the lake’s trophic state by sam-
pling only the water layer above thermocline. We
used two ways to derive the Zeu from Zs: the rough
linear approximation used, e.g., by Chorus & Bartram
(2003)
Zeu2:5S ¼ 2:5 �Zs ð1Þ
and the step relationship between 1% PAR depth and
Secchi depth described by Tilzer (1988) in Lake
Constance
Zeu1% ¼ 4:7064 �Zs0:5626: ð2Þ
We applied the relationship by Hanna (1990) to
calculate the thermocline depth:
Log Zthc ¼ 0:336 Log MEL � 0:245: ð3Þ
Empirical data
We used two data sets: one measured in seven small
stratified lakes of Estonia in summer and the second
collected seasonally from lakes Varese and Monate in
Lombardy, Northern Italy. The main physical charac-
teristics of the lakes are given in Table 3. The Estonian
lakes were visited on two occasions in July 1998 and
July 1999. The data consisted of Secchi depths,
temperature profiles, and chlorophyll concentrations
measured at eight depths (two in epilimnion, four in
metalimnion, and two in hypolimnion). Chlorophyll
concentrations were measured spectrophotometrically
in acetone extract and calculated according to Jeffrey
& Humphrey (1975). In Italian lakes, temperature and
chlorophyll fluorescence profiles were measured over
2 years (2008 and 2009) with a 1-m depth resolution
weekly during the stratified period (April–October)
and fortnightly during the rest of the time using the
Hydrolab DS5X probe.
The fluorescence readings were regularly
calibrated against spectrophotometrically measured
chlorophyll and expressed in concentration units
(Chl, lg l-1). For further analysis, the integrated
Fig. 1 Euphotic to mixing
depth ratio (Zeu/Zthc) in
different size classes of
lakes (by maximum
effective length, MEL) at
different Secchi depths.
Euphotic depth calculated
from Secchi depth
according to power function
(A; Tilzer, 1988) and
constant coefficient
of 2.5 (B)
Hydrobiologia (2010) 649:157–170 161
123
mean values of Chl were calculated over the depth
ranges of interest.
Secchi depth was measured on each sampling
occasion and light profiles were registered using a
spherical Li-Cor dual-PAR sensor with a surface light
reference. We used the surface reference light intensity
only for correcting the light profiles for cloud
variability and calculated the 1% value from light
intensities just under the water surface. As the
uppermost light measurement was often noisy because
of waves, we recalculated the surface value from 1-m
depth reading by applying the average vertical light
attenuation coefficient calculated for the upper two
meters.
Results
Model results
According to our model, in very large lakes
(MEL * 100 km), the euphotic depth remained
always smaller than the mixing depth, independent
of the Secchi depth and the way the euphotic depth was
calculated (power function by Tilzer (1988) or a
constant of 2.5). In medium size lakes (MEL *10 km), if the Secchi depth is\5 m, the euphotic depth
should mostly remain above the thermocline, but in
transparent lakes its depth exceeds the mixed layer.
Among small lakes (MEL * 1 km), only very turbid
ones are mixed deeper than the euphotic depth and in
very small lakes (MEL * 0.1 km) sufficient light
levels for photosynthesis almost always reach deeper
than the mixing depth. According to the size and
transparency ranges, the model showed that in Lake
Monate the euphotic zone is expected to extend always
below the thermocline while in Lake Varese it is the
case only in periods when the Secchi depth is 5 m or
more. Most of the Estonian lakes were expected to
develop a DCM because of the small size and thus a
small mixing depth of the lakes if the light penetration
is not strongly limited by low transparency of about
1.5 m or less.
Empirical data analysis
Despite considerable size differences between the
lakes, the measured Secchi depths plotted against the
euphotic to thermocline depth ratio ranged within a
narrow band (Fig. 2). In all the cases where light
penetrated below the thermocline (Zeu/Zthc [ 1), we
found a DCM (filled circles) manifested by higher
chlorophyll concentrations in the metalimnion com-
pared to the epilimnion. In two cases, we found a
metalimnetic chlorophyll peak even in very turbid
lakes.
In Italian lakes, the thermocline was first formed
close to the surface in February but showed small
temperature gradients not reaching 1�C m-1(Fig. 3).
Stabile thermal stratification lasted from April to
October during which the thermocline lowered with
the metalimnion layer becoming narrower. In accor-
dance to the modeling results, in the more transparent
Lake Monate, the euphotic depth reached almost
constantly deeper than the thermocline. In Lake
Varese, this phenomenon occurred only in spring
when the water was still transparent and the thermo-
cline located closer to the surface. With the deepening
Table 3 Main characteristics of the lakes studied
Lake Area (ha) Maximum
depth (m)
Maximum effective
length (m)
Trophic type Secchi depth
in July (m)
Varese 1,480 26 7,880 Eutrophic 0.5–2.3
Monate 251 36 2,850 Oligotrophic 6.5–9.5
Verevi 13 11 723 Hypertrophic 0.7–2.7
Tsolgo Mustjarv 9 30 380 Dyseutrophic (mixotrophic)
softwater
0.7–2.8
Pindi Karnjarv 8 26 494 Dyseutrophic hardwater 1.6–2.3
Nohipalu Valgjarv 6 12 394 Oligotrophic 4.0–4.5
Vellavere Kulajarv 5 25 261 Hypertrophic 1.1–1.7
Peta 4 25 422 Eutrophic hardwater 1.7–1.9
Kaussjarv 2 22 197 Eutrophic hardwater 1.5–1.8
162 Hydrobiologia (2010) 649:157–170
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of the thermocline and decreasing transparency in
summer, the euphotic zone was limited above the
thermocline. The 2.5 * Zs criterion for calculating the
euphotic depth estimated in most cases slightly deeper
euphotic zone compared to the 1% surface PAR
criterion and the difference increased with increasing
water transparency. At low water transparencies,
especially if caused by a surface scum of cyanobac-
teria, the 1% PAR penetrated deeper than 2.5 * Secchi
depth. A step function describing the relationship
between the euphotic depth and the Secchi depth in
two lakes together (Eq. 4) was rather similar to Eq. 2
found by Tilzer (1988):
Zeu1% ¼ 4:300 �Zs0:600; R2 ¼ 0:65;n ¼ 122:
ð4Þ
The chlorophyll maximum had three typical positions
relative to temperature and light gradients (Fig. 4). In
several small lakes, the DCM developed at the lower
boundary of the euphotic layer (2.5 * Zs) at different
distances from the thermocline. In some lakes with
high water transparency such as Lake Monate
(Fig. 4A) or the Estonian Lake Nohipalu Valgjarv,
the DCM was temporarily pushed down even to the
hypolimnion. In Lake Varese, the chlorophyll peak
was located mostly above the thermocline (Fig. 4B,
C), but the 2.5 * Secchi depth did not always capture it
(Fig. 4C).
These differences in chlorophyll distribution had
clear implications on the mean integral Chl values
(Fig. 5). In all the lakes, Chl values measured in the
upper 1 m were the smallest and differed significantly
(P \ 0.05) from all other approaches. Due to the
existence of the DCM in Lake Monate and in
Estonian lakes, the mean Chl values increased with
increasing the sampling depth. In these lakes, the Chl
values of euphotic layer exceeded all other results. In
the Italian lakes where two euphotic depth criteria
were used, there was no significant difference in
mean Chl concentrations for the layers defined as
Zeu2.5S and Zeu1%. Also the two estimates for the
mixed layer Chl (until Zthc and Zepi) were rather
similar in all lakes. In Lake Varese, the different
sampling approaches (except the surface sampling)
yielded comparable results.
The difference between the epilimnetic and
euphotic layer mean chlorophyll decreased during
the vegetation period in Lake Monate (Fig. 6). The
underestimation of the mean chlorophyll concentra-
tion if sampling was limited to epilimnion instead of
the euphotic depth was around 70% in April and
dropped to zero in autumn due to the seasonal
deepening of the thermocline. In Lake Varese, the
difference between epilimnetic and euphotic layer
Fig. 2 Measured Secchi depth (Zs) plotted against the
euphotic to thermocline depth ratio (Zeu/Zthc) in July. The
two Italian lakes are indicated by name, the rest are Estonian
small lakes. Filled circles the occurrence of deep chlorophyll
maximum (DCM), open circles no DCM. The line indicates the
boundary above which DCM can be expected
Fig. 3 Seasonal
development of thermal
stratification and changes in
the euphotic layer thickness
in lakes Varese and Monate
over the years 2008–2009
Hydrobiologia (2010) 649:157–170 163
123
mean chlorophyll fluctuated around zero without
showing any particular pattern. In some cases, during
periods of deeper mixing, e.g., in October, the mean
chlorophyll concentration in the euphotic layer could
be smaller than that in the mixed layer (Fig. 4C),
resulting in up to 50% underestimation of Chl by the
2.5 * Zs criterion for sampling depth.
Discussion
Depending on the balance of light and nutrient
limitation, various vertical patterns of algal distribu-
tion could be found in poorly mixed aquatic ecosys-
tems (Klausmeier & Litchman, 2001). At low nutrient
levels, algae might form a benthic layer; at interme-
diate nutrient levels, a deep chlorophyll maximum
(DCM) might occur; and at high nutrient levels, a
surface scum might form. DCM is quite common in
stratified oligotrophic and mesotrophic lakes where
epilimnetic nutrient depletion allows enough light to
penetrate to the metalimnion and upper hypolimnion
(Reynolds, 1992). Certain adaptive features such as
diel vertical migration, accessory pigments, and
tolerance to hydrogen sulfide allow several phyto-
plankton species to inhabit meta- and hypolimnion
taking advantage of higher nutrient availability and
lower grazing pressure. DCMs have been reported
Fig. 4 Three typical chlorophyll profiles in relation with the
temperature and light observed in the studied Italian lakes.
A Deep chlorophyll maximum located at the lower boundary of
the euphotic zone (2.5 * Secchi) far below the thermocline;
B chlorophyll maximum above the thermocline at the lower
boundary of the euphotic zone; C chlorophyll maximum above
the thermocline but outside the euphotic zone
Fig. 5 Mean chlorophyll concentration within differently
defined layers for sampling. To fit to the scale, chlorophyll
values from Estonian lakes were divided by 10. Zeu1%—
integrated samples from the euphotic layer defined by light
attenuation, Zeu2.5S—same defined by Secchi depth, Zthc—
integrated samples from the layer above the planar thermo-
cline, Zepi—integrated samples from the mixed layer above
the temperature gradient value of 1�C m-1, Z1m—surface
samples from the upper 1-m layer
164 Hydrobiologia (2010) 649:157–170
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mostly for temperate lakes of North America (Fahn-
enstiel & Glime, 1983; Jackson et al., 1990; Knapp
et al., 2003) and Europe (Kasprzak et al., 2000;
Gervais et al., 2003; Grigorszky et al., 2003; Camacho,
2006), as well as Argentina (Perez et al., 2002). In most
cases, the phytoplankton and microbial communities
forming the DCM are taxonomically distinguishable
from those in the epilimnion. DCMs are built up by
autotrophic organisms such as photosynthetic sulfur
bacteria (Chapin et al., 2004; Noges & Solovjova,
2005), cryptophytes (Gervais, 1998), dinophytes (Gri-
gorszky et al., 2003), chlorophytes (Reichwaldt &
Abrusan, 2007), cyanobacteria (Padisak et al., 2003) or
a combination of those. Chlorophyll concentration in
the DCM layer may exceed that of the epilimnion by
one or even two orders of magnitudes.
In order to cope with each particular vertical
distribution pattern, a proper phytoplankton/chloro-
phyll sampling strategy should be designed for
evaluating the concentration in lakes. None of the
techniques in use (Table 4) that range from single
surface sample to integrated samples (epilimnion,
euphotic zone, or fixed depth) as well as volume-
weighted composite samples has achieved general
acceptance among limnologists because each of them
has advantages and disadvantages, either because of
limnological, logistical, or statistical considerations.
Sampling at water surface is the easiest and less
expensive and requires the least equipment. However,
surface samples might overestimate the concentra-
tions due to surface scums formed by blue-green
algae (Soranno, 1997) or underestimate them due to
subsurface chlorophyll maxima which are common in
many lakes (Pick et al., 1984) and can account for an
important part of the net primary production and
biomass of planktonic algae (Moll & Stoermer, 1982;
Gasol et al., 1992; Camacho et al., 2001). Our present
study demonstrated convincingly that in stratified
lakes Chl values measured in the upper 1-m water
layer underestimate significantly mean Chl concen-
trations. Therefore, sampling only surface water layer
should be avoided.
Epilimnetic sampling integrates plankton from a
thicker water layer and reduces the bias caused by
vertical heterogeneity. However, the logistical diffi-
culty of epilimnion sampling is related to the need to
measure the detailed temperature profile and estimate
the stratification structure before the sampling. In
certain cases when there is a double thermocline or a
continuous vertical gradient in temperature, it is not
Fig. 6 Percent underestimation of the mean chlorophyll
concentration if sampling was limited to epilimnion (Zepi)
instead of euphotic depth (Zeu2.5S). Calculated as (Chleu2.5S -
Chlepi) * 100/Chleu2.5S
Table 4 Overview of advantages and drawbacks of different sampling methods
The sampling method Advantages Disadvantages
Surface samples Simple, inexpensive, requires little
training and no special sampling
equipment
High variability. May cause overestimation due to algal
scums or underestimation due to deep Chl maxima
Epilimnion sampling Reduces variability caused by vertical
heterogeneity
Ignores meta- and hypolimnetic chlorophyll maxima.
Requires a detailed temperature profile
Euphotic sampling Reduces variability caused by vertical
heterogeneity, covers the zone of
photosynthesis
Estimation based on Secchi depth is a potential source of
error. If extending into an anaerobic hypolimnion the
measurement of chlorophyll a, nutrients and cell counts
can be disturbed
Vertically integrated
volume-weighted
composite samples
The best for whole-lake concentrations if
combined with morphometric data
Complicated and labor-intensive sampling, requires detailed
knowledge of lake morphometry
Hydrobiologia (2010) 649:157–170 165
123
easy to decide upon the right sampling depth in field
conditions, and that can be an additional source of
errors. Moreover, epilimnetic sampling is not suffi-
cient for deep and transparent water bodies as Lake
Monate in our study, which exhibit maximum
chlorophyll levels in the meta- or hypolimnion. Our
study showed that in such lakes epilimnetic sampling
could cause up to 70% underestimation of Chl
concentration. Considering the ecological status class
boundaries by Chl set for this lake type (L-AL3;
‘‘high’’/‘‘good’’ 2.2–2.7 lg l-1, ‘‘good’’/‘‘moderate’’
3.8–4.7 lg l-1; EC, 2008), an error of such size
would cause a misclassification of the lake by one or
even two status classes.
Several manuals recommend sampling of the
euphotic zone, which is usually estimated as a
multiple of the Secchi depth. Although conceptually
reasonable, this adds a potential error due to the fact
that the euphotic zone is not necessarily 2- or 2.5-fold
Secchi depth, although this is a useful rule of thumb.
For example, Davies-Colley & Vant (1988) reported
coefficients ranging from 1.16 to 2.3, Preisendorfer
(1986) from 1.8 to 2, Lind (1979) stated a value of 2.7
while Tilzer (1988) showed that the relationship
between Secchi depth and euphotic depth is nonlin-
ear. As this coefficient varies as a function of a
number of uncontrollable environmental variables,
the euphotic depth assessed by Secchi depth is a very
rough estimate. Despite these uncertainties, our study
showed 2.5 * Zs criterion for calculating the euphotic
depth could be applied for monitoring purposes. This
criterion estimated in most cases a slightly deeper
euphotic zone compared with the 1% surface PAR
criterion but this did not cause significant differences
in the estimate of the mean Chl estimate.
The possible occurrence of autotrophic sulfur
bacteria containing bacteriochlorophyll below the
thermocline in lakes with anaerobic hypolimnia may
create problems for euphotic sampling causing over-
estimation of chlorophyll a with spectrophotometric
methods (Noges & Solovjova, 2005). High perfor-
mance liquid chromatography (HPLC) can be used to
distinguish between these two types of chlorophyll if
microscopic analysis reveals the occurrence auto-
trophic bacteria. No example of such lake type was
included in this article.
A disadvantage of both the integrated epilimnion
and euphotic zone sampling is that they do not
provide an adequate measure of the whole lake
concentration because the volume differences of lake
layers are not considered. Large metalimnetic chlo-
rophyll peaks would have an exaggerated impact on
the estimate of the average concentration. Collection
of vertically integrated volume-weighted composite
samples (Blomquist, 2001) could be the best method,
but involves too much labor and requires a proper
knowledge of lake morphometry (Carlson & Simp-
son, 1996; Blomquist, 2001).
Our study demonstrated that in order not to miss the
Chl peak in stratified lakes, it would be more precautious
to sample euphotic layer. The MEL of the lake and the
Secchi depth give a useful indication of the probability of
DCM occurrence and for the need to implement the
measures to secure its capture by sampling.
However, on a few occasions in Lake Varese, the
largest among the studied lakes, the Chl peak
developed above the thermocline but below the
2.5 * Zs depth and therefore could not be captured
by euphotic layer sampling. There could be different
reasons for the formation of this intermediate peak. A
simple explanation could be that 2.5 * Zs underesti-
mated the real euphotic depth. In fact, this was the
obvious reason in several cases in summer when a
surface scum in Lake Varese considerably reduced
the Secchi reading but the underlying water layers
were less turbid resulting in the 1% surface irradiance
to penetrate deeper than 2.5 * Zs. Sampling in these
cases a 3 * Zs layer could help to overcome the bias
in the euphotic depth estimate caused by the surface
scum. Not always, however, a surface scum could
be found when the 2.5 * Zs did not reach the
chlorophyll peak in Lake Varese. In the case shown
in Fig. 4C, for example, the 1% surface PAR reached
exactly the 2.5 * Zs. As it was close to the end of the
vegetation period, we suppose that in this case the
Chl peak was formed by settling algae, whose flux
was slowed down by the steep density gradient at the
thermocline, a phenomenon described, for example,
by MacIntyre et al. (1995) and Haberyan & Porter
(2003). In stratified oligo- to mesotrophic lakes and
reservoirs, a shade tolerant cyanobacterium Plankto-
thrix rubescens can be the main primary producer and
dominant taxon causing DCMs below the 2.5 * Sec-
chi depth (Dokulil & Teubner, 2000; Ernst et al.,
2009). Due to potential toxicity, the occurrence of
P. rubescens in freshwater resources requires special
attention when designing site inspection and sam-
pling procedures for the correct risk assessment and
166 Hydrobiologia (2010) 649:157–170
123
management of cyanobacterial blooms in the field
(Paulino et al., 2009). In such cases, the euphotic
depth should be changed to 3 * Secchi depth,
Several studies have evaluated the effect of
vertical sampling strategies on estimates of lake
concentrations. Woods (1986) used a large data base
of chlorophyll a measurements from Big Lake, in
south-eastern Alaska, to illustrate the effect of three
different sampling designs—euphotic zone, epilim-
nion or surface sampling—on designation of the lake
to a trophic state category. Applying the OECD
(1982) trophic state classification system, the epilim-
nion and surface chlorophyll values indicated an
oligotrophic state, whereas results from the euphotic
zone indicated a mesotrophic state. The larger
chlorophyll a concentrations obtained from the
euphotic zone were attributable to a DCM located
within the upper metalimnion.
Hanna & Peters (1991) assessed six different
methods of sampling lake chlorophyll a concentrations
by comparing Chl values for 16 lakes in southern
Quebec. Like us they found smallest Chl in subsurface
samples, intermediate in integrated epilimnetic sam-
ples, and the highest in the integrated samples of the
euphotic zone (estimated as 2 * Zs). Still the inter-lake
differences remained greater than those caused by
different sampling techniques, meaning that any of
the techniques could distinguish between lakes. The
authors concluded that subsurface samples having the
lowest values and the greatest daily variation should be
avoided in inter-lake surveys. They recommended
integrated sampling over euphotic zone (2 * Zs)
because of the simplicity of its identification and good
representativeness of lake productivity.
Conceptually, it is not always clear how the
occurrence of non-epilimnetic chlorophyll maxima
should be taken into account in the overall ecological
status assessment of lakes. Although accounting for a
part of the primary productivity of lakes, DCMs
appear to be publicly less perceptible as deterioration
compared to increased epilimnetic chlorophyll con-
centrations (Ryding & Rast, 1989). Assessment of the
ecological status of lakes, however, is not aiming to
reach a full compliance with public perception
(which could be an argument, for instance, for
bathing waters). In deep stratified lakes, the epilim-
nion may be rapidly depleted of nutrients already
during the spring bloom after which the effect of the
production pulse can be traced only in deeper layers.
In this way, the eutrophication of these lakes may be
detected earlier in the deep layers where the signal is
more persistent while the surface layers may still
maintain an unimpacted look during most time of the
vegetation period.
Conclusions
In order to guarantee a better comparability of the
ecological status of lakes across Europe and to assess
the effects of management efforts, a greater stan-
dardization of chlorophyll a sampling protocol for
stratified lakes is needed. As a contribution to this
scope, the following practical conclusions can be
drawn based on our study:
1. Taking only surface layer samples will lead with
a high probability to an underestimation of the
chlorophyll concentration in the trophogenic
layer of the lake and, hence, to an underestima-
tion of the lake primary productivity.
2. In order not to miss the Chl peak in stratified lakes,
in most cases it would be more precautious to
extend sampling to the euphotic layer. Only in
cases when the mixing depth exceeds the euphotic
depth, could a mixed layer sample be considered
more representative of lake productivity.
3. Using the MEL of thermally stratified lakes and
the Secchi depth gives an easy tool to assess the
probability of occurrence of a DCM.
4. In most cases, 2.5 * Secchi depths proved a
suitable criterion for defining sampling depth. Its
simplicity compared to determining the thermo-
cline depth is a big advantage in field conditions.
5. When surface scums are present that disturb
more the Secchi reading than affects the light
attenuation, the 2.5 * Secchi depth may under-
estimate the real euphotic depth. In order to
overcome this bias, sampling 3 * Secchi depth
layer would be recommended in cases of surface
scums. The same can be recommended for lakes
where a DCM can be expectedly caused by the
dim light specialist, Planktothrix rubescens,
which extends the euphotic layer beyond the
1% surface irradiance limit.
Acknowledgements The study was supported by the EU FP7
grant 226273 (WISER), by JRC institutional exploratory
project of the Action EEWAI, and by SF 0170011508 from
Hydrobiologia (2010) 649:157–170 167
123
Estonian Ministry of Education. Special thanks go to Michela
Ghiani, Bruno Paracchini, Joaquin Pinto Grande and Veljo
Kisand for field measurements on Italian lakes, and to Fabrizio
Sena for chlorophyll analysis. The study of Estonian lakes was
supported by the core grant 0370208s98 of the Estonian
Ministry of Education and by grant 3579 of the Estonian
Science Foundation.
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