Journal of Plankton Research V0LI8 no.9 pp.1557-1566, 1996
Biomass-pigment relationships in potamoplankton
Jean-Pierre Descy and Arnaud M6tens
Unit of Freshwater Ecology, Department of Biology, FUNDP, rue de Bruxelles
61, B-5000 Namur, Belgium
Abstract During most of the growing season of 1994, pigment content, as determined by HPLC analysis of algal sample extracts, was followed in the River Meuse (Belgium) potamoplankton. The concentration of some algal pigments (chlorophylls a and b, fucoxanthin, lutein, echinenone and alloxanthin)
was related to biomass estimates of total phytoplankton and of major taxonomic components
(diatoms, green algae, cyanobacteria and cryptomonads). Highly significant linear regressions were
obtained for chlorophyll o-total biomass, fucoxanthin-diatoms, lutein-green algae, chlorophyll
fr-green algae. However, no relationship was found for cyanobacteria or cryptomonads and their
specific pigments, which may be attributed to poor accuracy of biomass estimates for these nondominant algae. In conclusion, the good relationship found for dominant algae and their specific pigments confirms the value of pigments as quantitative markers of phytoplankton, as detected in other
marine and freshwater environments.
Introduction
Since the introduction of HPLC for analysis of natural phytoplankton extracts
(Gieskes and Kraay, 1983; Mantoura and Llewellyn, 1983), concentrations of
specific algal pigments (chlorophylls and xanthophylls) and pigment profiles have
been used for studies of phytoplankton composition, production, sedimentation,
and predation by herbivorous zooplankton (reviewed by Millie et al., 1993a).Technical improvements and automation of analysis render these methods well suited
for large-scale surveys in marine and continental waters (Millie etai, 1993a,b). On
a smaller scale, detailed analysis of algal pigments as biomarkers in the investigation of trophic relationships yields rich information on the effect of zooplankton predation on algal communities (Buma et al, 1992; Gieskes et al, 1988;
Johnson et al, 1992; Klein and Riaux Gobin, 1991; Wright and Jeffrey, 1987), as
well as on the contribution of zooplankton categories to total grazing rates
(Bautista and Harris, 1992; Perressinetto, 1992; Wong et al., 1992; Barlow et al.,
1993a; Quiblier et al., 1994; Quiblier-Lloberas, 1994).
However, the use of pigments as organic markers or algal biomass indicators
needs validation, which was not carried out in all studies. It is well known that the
carbon to chlorophyll a (C.CMa) ratio may vary over a rather wide range according to light, nutrients (especially nitrogen concentration) and the physiological
state of phytoplankton. Critics of the pigments approach often point out that
phptoacclimation subsequent to variation of the light climate at the seasonal scale,
or at even smaller time scales, should change the ratio Crpigments, which casts
some doubt on the conclusions of the pigment-based studies. This problem has
been circumvented by some authors, who used the ratio markenChla as an estimate of the relative contribution of the corresponding algal group to total algal
biomass (Wilhelm et al., 1991; Barlow et al., 1993b; Soma et al., 1993; Letelier et al.,
1993). The obvious underlying rationale is that if photoacdimation affects the
cell's pigment content, all photosynthetic pigments are affected in the same way,
O Oxford University Press
1557
J.-P.Descy and AMitens
so that the pigment ratio remains unchanged or at least rather stable (Brunet et
al., 1992). On the other hand, it seems that cell concentrations of various
xanthophylls may respond less to changes of light climate than do chlorophylls,
which makes these pigments more reliable estimators of algal biomass (Ridout
and Morris, 1985; Foy, 1987; Strom and Welschmeyer, 1991; Millie et al., 1993a).
However, it must be recognized that the pigment-biomass relationship is not
always well established in numerous studies, probably because classic determination of phytoplankton biomass is cumbersome and may yield inaccurate data.
Still, some calibration remains a necessary step in a pigment-based study.
In the present work, we aimed to verify the validity of specific pigments as estimators of the biomass of algal groups in a large river environment (River Meuse,
Belgium), by parallel studies of phytoplankton composition carried out by the classic
counting method under the microscope and the HPLC determination of specific
xanthophyll concentrations, throughout the growth period of the potamoplankton.
Method
Study site
The River Meuse rises in the east of France and flows through Belgium and The
Netherlands, where it meets the lower Rhine, forming the Dutch Delta, which
opens into the North Sea. The total length of the river is 885 km and its catchment
area is -36 000 km2,40% of it being in the Belgian territory. In all of its Belgian
course, the River Meuse has been regulated for navigation, with weirs and locks
distributed along its length.
The site studied (La Plante) is situated 537 km from the source. At this site, the
mean depth is 3.95 m and the mean width is 100 m.The River Meuse has alkaline
nutrient-rich waters. Some variations in the nutrient content occur over an annual
cycle, due to inputs from the drainage area (N, Si), from sewage (mostly P) and to
uptake by primary producers. However, nutrients are not depleted to levels where
they may be limiting for phytoplankton growth. More detail can be found in Descy
(1987).
Sampling and data acquisition
Whenever possible, sampling was carried out on a weekly basis during the period
of potamoplankton development in the river in 1994, i.e. from the end of March
to the end of September. Various environmental parameters were measured
throughout the study: temperature, discharge, surface irradiance, vertical light
attenuation in the water column, dissolved nutrients (N, P, Si), particulate carbon
and nitrogen, chlorophyll a [by the standard spectrophotometric technique developed by P6char (1987)] and other general measurements. The methods have been
described elsewhere (fee, for example, Descy and Gosselain, 1994).
Phytoplankton were collected from the surface with a 3 1 Van Dorn bottle and
carried back to the laboratory within 1 h. Three 250 ml aliquots were filtered on
Whatman GF/C filters and deep frozen for subsequent extraction and HPLC
analysis, while 11 aliquots were preserved with Lugol's solution.
Examination under the microscope was carried out on subsamples obtained by
1558
Potamophuikton biomass-pigmetit relationships
sedimentation of the Lugol-preserved samples in cyUnders of decreasing volumes,
allowing 24 h settling in each cylinder. The subsamples were mounted in a Btirker
cell and examined with a Leitz Laborlux D standard microscope equipped with a
graduated eyepiece, under a 10 X 40 optical combination most of thetime.As accurate biomass estimates were needed, it was necessary to measure the size of phytoplankton units in every sample. For this purpose, the data acquisition was by means
of a computer running Hamilton's (1990) program for the enumeration of plankton
samples, which enables the recording and processing of the number and dimensions
of phytoplankton units. The measurement precision was 0.5 um and the unit biovolume was calculated by reference to the closest simple geometrical shape. The
level of taxonomic ascription varied from the species to the genus—according to
practical considerations—except for unicellular centric diatoms which were counted
as one unit The latter category encompasses several taxa (species and varieties of
Stephanodiscus, Cyclotella, Cyclostephanos and Thalassiosira), likely to be similar to
those reported by Gosselain et al. (1994) for the sameriver.The number of specimens counted varied between 200 and -500, depending on algal concentration and
diversity. For each sample, final calculations of biomass per unit used unit abundance
(numberml"1) X mean unit biovolume(jun3), followed by conversion to carbon (jtg
C I"1) by means of the Eppley equations (in Sournia, 1978), with the appropriate
factors for sample volume conversions. The results were then expressed by phylogenetic groups. Occasional replicates yielded very similar results (not shown).
HPLC analysis
Extraction of the deep-frozen samples was achieved following the protocol of
Wright et al. (1991), modified in order to optimize carotenoid extraction (Webb et
al., 1992; Downes et al., 1993). The filters were ground for 90 s with a Teflon pestle
in 5 or 2.5 ml 100% acetone, sonicated for 30 s, then left in the solvent for 4 h
before being filtered on Gelman Acrodisc filters prior to injection. All manipulations were carried out at 4°C under dim light. Before injection, the extracts were
diluted to obtain a solution of 90:10 acetone extract:2% ammonium acetate. The
standard HPLC system comprised a Waters pump 600 equipped with a manual
injector U6K, a Nova-Pak C18 column (4.6 X 250 mm, particle size 4 um, porosity
60 A) and two detectors: an absorbance detector (detection wavelength 436 nm)
and a fluorescence detector (excitation wavelength 420 nm; detection wavelength
650 nm). The data were stored and processed on a PC compatible computer
running the Millennium software.
The solvent gradient followed Wright et al. (1991), without modification.
However, a residual absorbance of ethyl acetate by the detector did not allow
correct integration of chlorophyUides, including chlorophylls c, and peridinin.The
other pigments were resolved and their identification was verified on another
system equipped with PDA detection and comparison with reference pigment profiles from pure algal cultures: Dictyosphaerium ehrenbergianum N2g. for chlorophytes, Planktothrix agardhii Gom. for cyanobacteria, Cyclotella meneghiniana
Klltz. for diatoms and Cryptomonas ovata var. palustris Ehr. for cryptophytes.
Quantitation of pigment concentration was based on chlorophyll a standards
1559
X-P.Descy and AMUeas
prepared from pure chlorophyll a from Sigma, using the ratio of the specific
absorption coefficient of a given pigment to that of chlorophyll a at 440 nm [values
given in Mantoura and Llewellyn (1983)]. The calculation of the quantity of
pigment p (<2p) was:
where a is the slope of the chlorophyll a calibration curve, . E ^ is the specific
absorption coefficient of chlorophyll a at 440 nm, Ep is the specific absorption
coefficient of pigment p at 440 nm and Ap is the area of pigment p peak.
This calibration was verified for chlorophyll b with solutions of pure pigment
from Sigma. Extracts of pure cultures and chlorophyll a standards were analysed
before every analytical series to verify calibration and retention times.
The following pigments were used as phylogenetic markers: lutein and chlorophyll b for chlorophytes, echinenone for cyanobacteria, alloxanthin for cryptomonads and fucoxanthin for diatoms (chrysophytes scarcely reached significant
biomass in the River Meuse phytoplankton). Epimers and allomers of chlorophylls were grouped with the typical pigment form at the quantitation step; similarly cisltrans isomers of xanthophylls were grouped with the typical xanthophyll.
Results
Variations in the environmental conditions are presented in Figure 1. In 1994, discharge and temperature patterns were quite typical for the River Meuse: they
should have allowed phytoplankton development from spring to autumn, without
significant interruption. Owing to water turbidity, the daily average of light energy
in the water column varied within the rather narrow range of 100-300 uE nr 2 s"1.
All nutrients (results not shown) were always at saturating levels for algal growth.
The phytoplankton composition in the studied section of the River Meuse is
characterized by overall dominance of diatoms. In 1994 (Figure 2), these algae
appeared early in the growing season: at that time they were mostly represented
by unicellular small centrics. Chlorophytes are the second group by importance,
but they achieved a significant biomass only during summer. Both types of algae
declined to low levels in the middle of the summer, then recovered toward the end
of the summer, and declined again at the end of the growing season. The community of the second part of the summer changed greatly, exhibiting larger phytoplankton units (large unicells, colonies, filaments). For instance, the late summer
diatom assemblage was dominated by Aulacoseira species, contrasting with the
'small centrics' assemblage of the spring.
Chlorophyll a and the characteristic pigments of the two main algal groups
followed a seasonal trend very similar to that of algal biomass (Figure 3), showing
good correspondence between, respectively, chlorophyll a-total phytoplankton
biomass, fucoxanthin-diatom biomass and lutein-green algae biomass. For the
other algal groups and their specific markers, the data were too scattered to derive
any significant relationship from our results.
Linear regression equations were calculated for the pigments-biomass couples
cited above (Figure 4): in the three cases, the coefficient of determination is highly
1560
PotamoplanktoD bfomass-ptgrnent relationships
600
enn
400
30
.
-
Fig. L (A) Variations in daily discharge and temperature in the River Meuse at La Plante (Namur) in
1994. (B) Variations in the daily average of light energy in the River Meuse at La Plante (Namor) in
1994, calculated from the daily mean surface irradiance, divided by the depth of the water column and
by the vertical extinction coefficient, measured once per week and extrapolated for the following days.
significant. The concentration of chlorophyll b also followed green algal biomass
(not shown), but less closely than did lutein.
Discussion and conclusions
The rise and fall of potamoplankton in a given river section, and its composition,
are usually determined by the discharge pattern, light, temperature and meteoro^
logical events. In fact, these factors control algal growth in the upstream sections,
1561
J.-P.Descy and AJVtttens
2000
o
Oi 1500
a Cyanobactena
B Cryptomonads
m Dinophyceae
D Chlorophytes
a Diatoms
Fig. 2. Variations in the biomass (jig C t~') of the main taxonomk groups of algae in the potamoplankton of the River Meuse at La Plantc (Namur) in 1994.
whose morphology and hydrodynamic behaviour is of great importance [see, for
example, for the River Meuse, Descy (1987) and Descy and Gosselain (1994); for
more general considerations, see Reynolds and Descy (in press)]. However, it is
clear from the data presented above that the midsummer decline of the potamoplankton cannot be fully explained by variations of these physical factors. In fact,
other processes are obviously involved, in which zooplankton grazing may play a
major role (Gosselain et al., 1994; see also Garnier et al., 1995). The extent of
grazing control and its influence on the biomass and composition of the Meuse
phytoplankton in 1994 will be considered in another paper. The scope of this paper
is the use of pigments as biomass markers, i.e. as substances which may be utilized
to trace algal biomass and its fate in a variety of aquatic environments. Our data
confirm the value of xanthophyll concentrations as estimators of the absolute
biomass of the two major algal groups in large rivers. For the two remaining
markers, alloxanthin and echinenone, we attribute a failure to relate them to
cryptomonad and cyanobacterial biomass, respectively, to the low contribution of
these two algal groups to the River Meuse potamoplankton. The low numbers of
those organisms in the samples made it difficult to establish reliable biomass estimates by the counting procedure, suitable for the dominant taxonomic groups.
Clearly, the degree of reliability of fucoxanthin and lutein for assessing the
biomass of diatoms and greens is at least equivalent to that of chlorophyll a for
the total algal biomass, in the environmental conditions encountered in the River
Meuse and probably in other large eutrophic rivers. The reason why the
biomass:pigment ratio in planktonic riverine algae is rather stable over the
growing season may be that the light climate is relatively constant in those turbid
environments. Turbulent mixing in a turbid water column considerably reduces
1562
PotamopUnkton blorjuu-pigment relationships
Algal biomass- chlorophyll a
Diatom biomass - fucoxatrthln
2000
Orwis biomass - lutaln
600
4 T
Fig. 3. Variations in the biomass (jig C h 1 ) of the main taxonomic groups of algae in the potamoplankton of the River Meuse at La Plante (Namur) in 1994, overlaid to variations of the specific
pigment of the group.
1563
J.-PJ>escy and AM&eta
Relation betw—n c/i/a and biomass (from counts)
Woman =18.9*ehl»-115.9 (R*=0.82)
2500
a
~- 2000
o
«
u
1500
^b a
I 1000
KQ
I
ci
a
500
£1
V
0
20
40
60
80
100
120
Relation between fucoxmnthln mnd dfetoai btomass
bkrniBM » 28.3TUccx-4.78 (R^.74)
2000
1600
D
120
°
800
1
a
^
a
a
400
0
0
10
20
30
40
50
f u c o x a n t h l n Qtg I 1 )
60
Rotation bttw—n lutetn and grams biomass
btooiOM • 232.2*!utein-63.7 (1^=0.87)
600
0
Csoo
§
400
300
!
•
D
200
1 100
Fig. 4. Linear regressions of the specific pigment content (p.g l~') versus the biomass (|xg C r~') of the
corresponding algal group; data from analysis of the polamoplankton of the River Meuse at La Plante
(Namur) in 1994.
1564
Potamoplankton biomao-pigment relationships
the light available to the algal cells, whose growth is consequently extremely light
dependent. As pointed out by various authors (e.g. Reynolds, 1994), algae in a
large river or in a mixed shallow lake experience exposure to a steep light gradient (i.e. often from effective darkness to subsurface sunlight and back to darkness)
over short time scales (minutes), but are not subject to large variations over the
greater time scales which typically induce a chromatic adaptation (Falkowski,
1984; Falkowski and Laroche, 1991). Consequently, algal cells in those environments need a permanently high concentration of photosynthetic pigment per cell
to absorb photons during their short periodic exposure to light. For instance, the
C:Chlfl ratios are typically low (30-45 is a frequently reported range), rather constant seasonally, but tend to decrease as the depth of the mixed layer or turbidity
increases (see, for example, Descy and Gosselain, 1994).
As a conclusion, our results validate the use of fucoxanthin and lutein as quantitative markers of diatoms and green algae in regulated large rivers similar to the
River Meuse. This validation is a first step toward the utilization of xanthophylls
in studies of grazing by zooplankton in those freshwater environments.
Acknowledgements
We are thankful to M.Dieu, Department of Biology, FUNDP, who provided technical help for HPLC-PDA, and to the two referees for significant improvements
to the final manuscript.
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Received on December 23,1995; accepted on April 1,1996
1566