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

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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)

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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)

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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)

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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

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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

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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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