and of 5 µ 0 in the particle size range 20-75y 0. Data pro- cessing was done on a PDP-8 computer taking into ac- count corrections for a blank and coincidence .
Hydrobiologia vol . 55,
2,
pag. 145-t65, 1977
A COMPARISON OF DIFFERENT METHODS USED FOR THE QUANTITATIVE EVALUATION OF BIOMASS OF FRESHWATER PHYTOPLANKTON Gustaaf M . HALLEGRAEFF Limnological Laboratory, University of Amsterdam, Kruislaan
320,
Amsterdam, The Netherlands .
Received January 15, 1977 Keywords : Biomass of freshwater phytoplankton . Comparison of methods . Chlorophyll-a concentration, seston dry weight, total particle concentration .
Abstract Although in a strict sense the term 'phytoplankton biomass' only refers to living algal material, in aquatic ecology the term has been associated with a variety of biological and biochemical procedures used to quantify the particulate matter suspended in natural waters . Relative merits of different `biomass' characteristics have been studied in three Dutch freshwater lakes with great differences in absolute biomass . Parallel determinations have been made of seston dry weight and supplementary elementary and caloric analyses of seston, of chlorophyll-a concentration and supplementary paper chromatographic analyses of pigment extracts, of particle concentration and particle size distribution as studied with an electronic particle counter, and of phytoplankton cell volume as calculated from the results of microscopic enumeration and sizing of algae . In this way an attempt was made to create a detailed picture of the nature of the seston of the three freshwater lakes . Different analytical techniques give strikingly different information, the accuracy of any method is largely dependent on the circumstances present, and different biomass characteristics therefore are only of value in limited spheres . It is suggested to distinguish between total seston characteristics (e .g. seston dry weight, particulate organic carbon, total particle volume) and strictly algological biomass characteristics (e .g. chlorophyll-a concentration, phytoplankton cell volume). The pattern of growth of phytoplankton populations shown by e .g . chlorophylla concentration may differ markedly from that indicated by e .g. total particle volume or seston dry weight . Also, to more or less extent the wax and wane of phytoplankton populations may go undetected among the total seston . Apparently, there is no one method of estimating biomass and no conversion factor that may serve for general purposes . In general, unambiguous information on the nature of the seston of natural waters may only be obtained by estimating total seston characteristics and algological biomass characteristics simultaneously . Depending on the
Dr. W. Junk b. v . Publishers - The Hague, The Netherlands
objective of the investigation supplementary component analyses should be carried out to guarantee the correct interpretation of the results .
Introduction The quantitative assessment of phytoplankton populations is a fundamental requirement in the study of almost all aspects of aquatic ecosystems . Phytoplankton 'biomass' (i .e . the total weight of all living phytoplankton organisms present in a unit area at a given time) has widely been used as a base of reference in primary productivity studies, as a measure of food available for zooplankton, and as an index of lake fertility in general . In addition to living organisms (phytoplankton, zooplankton, bacteria) the particulate matter suspended in natural waters (seston) also includes non-living organic detritus and particulate inorganic material, however . In order to obtain qualitative and quantitative knowledge of the various components of the seston, the aquatic ecologist will find a great variety of biological and biochemical procedures at his disposal . Unfortunately the different analytical techniques not necessarily produce comparable results . The simplest measure of biomass to obtain is wet weight, but since water content of algae is variable it has generally been replaced by dry weight . In samples which are contaminated with inorganic particles or which contain heavily silicified diatoms, ash-free dry weight (organic weight, `loss on ignition') is more appropriate (Nalewajko, t966 ; Hallegraeff, in press) . Determinations of 1 45
dry weight are of particular interest in studies requiring an estimate of biomass which may be interpreted in terms of energy . In this context carbon content has been demonstrated to be closely related to energy content of the dry weight biomass (Platt & Irwin, 1973 ; Hallegraeff, in press) . Carbon content has also been favoured as an expression of phytoplankton biomass in studies on primary productivity measured by the C 14 -technique . In current aquatic ecology there is a need for rapid routine biomass determinations, and as such there has been some interest in electronic particle counters (Sheldon & Parsons, 1967a ; Mulligan & Kingsbury, 1968 ; Evans & McGill, 1970) . It is obvious that all such methods of estimating phytoplankton biomass may be seriously impaired by the presence of non-algal suspended matter . On the other hand, it should be borne in mind that all this material is potentially available as food for zooplankton (Melchiorri-Santolini & Hopton, 1973 ; Poulet, 1976) . Because chlorophyll-a is a unique component of plant matter and essential for photosynthesis, the determination of chlorophyll-a concentration generally has been recognized as one of the most manageable means of estimating the biomass of the algal fraction of the seston (Harvey, 1934; Krey, 1958) . Unfortunately, it is hard to distinguish between chlorophyll-a in living algae and in detritus (Hallegraeff, 1976a ; 1977) and chlorophyll content per unit organic matter may vary widely according to species composition, cell age, nutrient availability, light intensity and temperature (see Strickland, 196o ; Steele & Baird, 1961, 1962, 1965) . Apparently with all analytical techniques mentioned, supplementary microscopic examination of the samples will be needed to guarantee the correct interpretation of the results . Also, information on species composition of the phytoplankton biomass may only be obtained by microscopic enumeration and sizing of algae . This technique is tedious and time-consuming, however, and because only a limited number of samples can be treated microscopic counts may be encumbered with large statistical errors (Kutkuhn, 1958 ; Lund et al., 1958) . The objective of the present investigation has been to judge relative merits of different analytical techniques used for the quantitative evaluation of biomass of natural populations of freshwater phytoplankton . Particular emphasis has been placed on testing the comparability of different biological and biochemical procedures . A comparison has been-made between estimates of seston dry weight and supplementary elementary and caloric analyses of seston, determinations of chlorophyll-a concentra1 46
tion and supplementary paper chromatographic analyses of pigment extracts, determinations of particle concentration and particle size distribution made with an electronic particle counter, and results obtained by microscopic enumeration and sizing of algae . By performing all these analyses on portions of the same water samples an attempt was made to create a detailed picture of the nature of the seston of three Dutch freshwater lakes . The utility of conversion factors between different biomass parameters was tested and seasonal as well as lake-to-lake variations have been analyzed . The data have been collected in conjunction with investigations on primary productivity and zooplankton grazing which have been studied from early spring until the end of summer in the three Dutch freshwater lakes Barlosche Kolk, Pool 't Hammetje and Lake Maarsseveen .
Description of the lakes The three lakes selected to study annual phytoplankton succession are all enclosed water-bodies with negligible inlet and outlet . Some physical, chemical and biological characteristics of the lakes have been summarized in Table 1 . If the three lakes are ranked on the basis of decreasing lake fertility, the sequence is : Pool 't Hammetje, Barlosche Kolk, Lake Maarsseveen (see also Hallegraeff, 1976b) . The three lakes have been- sampled from early spring until the end of summer, at 5-weekly intervals in the Barlosche Kolk, 1973, at 4-weekly intervals in Pool 't Hammetje, 1974, and at 2-3-weekly intervals in Lake Maarsseveen, 1975 . In the Barlosche Kolk and Pool 't Hammetje samples have been collected at sunrise, midday, and sunset throughout one or two 24 hr periods, in Lake Maarsseveen samples have been collected only at midday . Samples from seven different depths were obtained from a single station in the deepest portion of the lakes, by means of a modified Friedinger-sampler (2 liter capacity) in the Barlosche Kolk and Pool 't Hammetje, and by pumping water into plastic jars (30 liter capacity) in Lake Maarsseveen . If necessary a preliminary filtration of the water samples was made through a 150 µ-gauze to remove the larger zooplankton . Quantitative determinations of seston dry weight, chlorophyll-a concentration and total particle volume have been performed for almost all samples . Concurrent supplementary elementary analyses of the seston, paper chromatographic pigment analyses and microscopic phytoplankton analyses have been carried out for the midday samples only . Results of
Table 1 . Some physical, chemical, and biological characteristics of the three lakes . The range has been indicated of values encountered throughout the year . Values in brackets refer to extreme conditions . BARLOSCHE KOLK max . depth, m surface area, ha . 1% light depth, m .
6 3 .5 (1 .90)4 .00 - 4 .95+
POOL 'T HAMMETJE 8 3 .5 1 .90 - 3 .65++
LAKE MAARSSEVEEN 30 70 (6 .00)7 .50 - 13 .00++
nutrients in surface waters PO ; , mg/l NO3 - , mg/l
0 .03 - 0 .28 0 - 1 .19
0 .04 - 0 .40++ + 0 .18 - 7 .00+++
0 .01 - 0 .16+++ 0 .80 - 2 .00+++
chlorophyll-a, µg/l total particle volume, mm' /1 seston dry weight, mg/1
2 .0 3 .1 1 .2
6 .1 - 159 .9 4 .1 - 49 .4 3 .3 - 18 .8
0 .4 1 .0 0 .4
- 95 .5 - 25 .5 - 17 .0
- 8 .3 - 7 .6 - 1 .4
+ data kindly supplied by Mr . Robert Lingeman ;++ data kindly supplied by Mr . Dick de Zwart ;+++data supplied (in part) by the Research Institute for Nature Management (RIN)
analyses on samples from seven different depths have been integrated over the water column under i m` water surface . In the Barlosche Kolk, Pool 't Hammetje and Lake Maarsseveen this corresponded to a water column of 5m, 8m and 23m depth, respectively .
Methods Analysis of seston dry weight
In the Barlosche Kolk and Pool 't Hammetje sufficient quantities of particulate matter for the estimate of seston dry weight (mg/1) could be obtained by filtering 500 mlduplicate samples over predried and preweighed 0 .45 µmembrane filters (Sartorius, 0 47 mm) . In the less productive Lake Maarsseveen it was necessary to collect particulate matter from at least 25-50 liters of water by continuously centrifuging at 7000 g (M SE high speed 18 centrifuge ; continuous action rotor; flow rate i liter/minute) . Subsequently, the concentrates so obtained were collected on filters as indicated above . After 24-48 hrs drying in an oven at 6o' C and subsequent cooling in a desiccator the filters were weighed on a Sartorius analytical balance . Samples from the hypolimnion, which on the basis of visual inspection were suspected to be contaminated with lake sediment, have been omitted for analysis . Methods and results of concurrent supplementary caloric analyses and elementary analyses for %ash, %C, %H, %N and %P (on the basis of dry weight) have been described in detail elsewhere (Hallegraeff, in press) .
Analysis ofphytoplankton pigments
In the Barlosche Kolk and Pool 't Hammetje algae from 250 ml-duplicate samples were concentrated under slight suction on 0 .45 .r Gelman-filters (AN-45o, 0 47 mm) . These acrylic acropor filters have proved to be particularly suitable for the quantitative collection of algae from small quantities of water taken from productive freshwater lakes . In the less productive Lake Maarsseveen 3 liter-duplicate samples were filtered through Whatman GF/C-filters . These glass-fiber filters have a much higher filter capacity and they were precovered with a small amount of MgCO 3 to act as a filter aid . The filters were dissolved in 2 ml loo% acetone (Uvasol) and ground after 12-24 hrs in a darkened refrigerator . The acetoneextract was homogenized and after the addition of 4 ml acetone centrifuged . The absorption spectrum (330-780 nm) of the clear supernatant was recorded with an Aminco Chance Recording Spectrophotometer (splitbeammode) in a 1 cm-cell . Readings from the recorder chart were made relative to the absorbance at 750 run, a wavelength at which no absorption by pigments is known to occur . From readings of the absorption peak at about 665 nm, chlorophyll-a concentration (µg/1) has been computed according to the simplified formula of Tailing & Driver (1963) . Methods and results of supplementary paper chromatographic analyses of pigment extracts have been described in detail elsewhere (Hallegraeff, 1976a,1977) . Electronic analysis ofparticles
Particle concentration and particle size distribution have been measured with a Coulter counter model ZB, which 1 47
in a later stage of the investigations was used in combination with a Coulter-Channelyzer (model C-looo) . A detailed description of the Coulter counter has been given by Sheldon & Parsons (i 967a) . After calibration with Lycopodium spores and preliminary experiments with paper mullberry spores, puff ball spores and unialgal cultures, the electronic particle counter was applied to natural samples as follows . In order to avoid blocking of the aperture by larger particles, immediately after collection 20o ml-samples were poured through a 150 µ nylon screen and subsequently the samples were fixed with io ml particle-free neutralized 37% formalin (cf . Kubitschek, 1969 ; Evans & McGill, 1970) . To create a proper electrolyte medium, directly before counting 9.4 ml particle-free 20% NaCl-solution was added to give a final concentration of o.9% in the counting solution (cf. ElSayed & Lee, 1963) . To obtain a count within the optimum range of the instrument the samples from Pool 't Hammetje have been diluted with prefiltered destilled water, whereas for the Barlosche Kolk and Lake Maarsseveen nondiluted samples have been counted . In between counts the particles were kept in suspension by means of an electrically driven stirrer . A 200U aperture tube was chosen to cover a particle size range of 4 up to 75 µ diameter (of spheres of an equivalent cell volume), which size range included most of the phytoplankton . Manometer volume control was set at 2 ml, and the samples were counted at 25 threshold settings corresponding with size intervals of i µ 0 in the particle size range 4-20 µ 0, and of 5 µ 0 in the particle size range 20-75y 0. Data processing was done on a PDP-8 computer taking into account corrections for a blank and coincidence . Microscopic analysis of the phytoplankton
To facilitate identification of the algae, portions of the samples have been microscopically examined in fresh condition, either directly or after being concentrated by centrifugation . For counting purposes the samples were fixed with neutralized formalin up to a final concentration of 2% (cf . Unesco, 1974) and subsequently the samples were concentrated by centrifugation . The effluent of the centrifuge was frequently checked for algae . Depending on the density of the algae subsamples of 2,5, or 1o ml were sedimented in counting chambers (after Utermohl, 1958) and were counted with an inverted ReichertMeF2 microscope at magnification 6.3 x 100 or if necessary at higher magnification 1o x 10o with a Wild M-4o inverted microscope supplied with phase contrast objectives . A portion of the bottom area of the counting cham148
ber was counted for common forms and the whole bottom area of the counting chamber was enumerated for rare and large species . If algal cells were suspected to be dead, as witnessed by the absence of pigmentation, they were omitted from counting . Cell counts of different phytoplankton species have been transformed to estimates of population volume by determining average cell dimensions for each species on projections of the image of the inverted microscope on a ground glass screen, and by approximating cell shape to the closest geometric solid configuration (sphere, cylinder, rectangle, cone, ellipsoid) . Principal interest was directed towards the species composition of the phytoplankton biomass in the entire water column, and therefore counting has been focused to vertical sample series rather than intensely treating the individual samples . Estimates of cell volume of the entire phytoplankton community were obtained by adding the concentrations of the different phytoplankton species present in each sample and integrating the values for samples from seven different depths over the water column under i m 2 water surface .
Results and discussion Precision of quantitative biomass estimates
The reliability of a quantitative estimate of the phytoplankton biomass present in a unit area at a given time is governed by errors arising from the analytical procedure employed as well as by errors arising from the process of lake sampling . Plankton populations may exhibit marked spatial and temporal variations and as a result in plankton studies sampling errors may comprise the largest component of error (Verduin, 1951 ; Lund et al., 1958 ; Cassie, 1962; Platt et al., 1970) . In the study of the three Dutch freshwater lakes it has tentatively been assumed that measurements taken at a single station were adequate to describe major seasonal trends (cf . Platt et al., 1970) . If at a given station the phytoplankton populations are homogeneously distributed with depth and in time (e .g . fig. ic, Pool 't Hammetje : February, May 1974) it is obvious that a reliable estimate of phytoplankton biomass may be obtained from a limited number of samples only. On the other hand, an extensive sample program will be needed to characterize (1) vigorously growing algal populations (e .g . fig . 1c, Pool 't Hammetje : March), (2) algal populations which are able to perform active vertical migrations (e .g. Ceratium hirundinella populations, Barlosche Kolk : summer months), and (3) stratified phytoplankton communities (e .g . Lake Maars-
seveen : summer months ; compare fig . 4) . It is beyond the scope of this study to judge problems posed by variations in the diurnal, vertical or horizontal distribution of the biomass parameter to be estimated . The following discussion will be restricted mainly towafds the relative merits of the different analytical techniques . Errors arising from the analytical procedure employed include (i) errors arising from the instrumental technique, (2) errors arising from sample treatment, and (3) errors arising from the natural variability of the plankton material itself. The first type of error can be estimated only from a careful review of the technique and by comparing the results obtained with those furnished by comparable techniques . The second type of error is amenable to statistical treatment and can be estimated by performing replicate analyses on identical homogeneous samples . In this way for several series of ten replicate analyses on seston samples of widely different nature, standard errors of the mean were obtained of 1 .5-2 .8% for chlorophyll-a determinations, of 8-11% for determinations of seston dry weight, of o .6-3 .7% (up to 6% upon sample storage) for Coulter countings, and of 3-12% for microscopic estimates of total algal volume, respectively . The high level of error associated with determinations of dry weight probably is the result of variations by the drying procedure and the difficulty of collecting sufficient seston dry weight to be accurately weighed relatively to the rather high background of filter dry weight . The precision of the microscope method is limited because the technique is subject to much larger subsampling errors than the other analytical techniques which deal with larger quantities of algae . On the other hand, the microscope method may be the best method if phytoplankton densities are too low to allow for an accurate determination of e .g . chlorophyll-a concentration or dry weight biomass . In general, from a viewpoint of rapidity and low analytical error, among the biomass characteristics studied chlorophyll-a concentration and total particle volume estimated by the Coulter counter would be most suitable to be applied in routine biomass determinations . The implications of the variability of the seston material itself will be discussed in the subsequent sections . A comparison of quantitative biomass determinations by chlorophyll-a concentration, total particle volume and seston dry weight.
Analyses on samples collected from seven different depths and throughout the year indicated chlorophyll-a concentrations in the range 2 .0-95 .5 jug/ 1 in the Barlosche
Kolk, 6 .1 - 159 .9 µg/l in Pool 't Hammetje, and 0 .4-8 .3 yg/l in Lake Maarsseveen, respectively . Values for total particle volume ranged from 3 .1-25 .5 mm 3 /1 in the Barlosche Kolk, 4 .1-49 .4 mm3 /1 in Pool 't Hammetje, and 1 .0-7 .6 mm 3 /l in Lake Maarsseveen, respectively . Determinations of seston dry weight gave values in the range 1 .2-17 .0 mg/l in the Barlosche Kolk, 3 .3-18 .8 mg/I in Pool 't Hammetje, and 0 .4-1 .4 mg/l in Lake Maarsseveen, respectively (Table i) . The variability of conversion factors between chlorophyll-a concentration, total particle volume and seston dry weight may be attributed to a large extent to (i) variation in physiological condition of the algal populations, (2) variation in the taxonomic composition of the phytoplankton community, and (3) changes in the proportion of living algae and detritus in the seston . To demonstrate the impact of variations in physiological condition of algal populations, fig . 1 presents results of a laboratory study on a batch culture of the green alga Scenedesmus dimorphus as well as of measurements made during a natural diatom bloom in Pool 't Hammetje, where in spring 74-94% of the algal biomass was made up by Cyclotella comta (compare Table 4) . At the start of the exponential growth phase both algal populations demonstrated a prompt increase in chlorophyll-a concentration (figs . ia, b) . With the natural diatom population during this phase marked day-to-day increases in chlorophyll-a concentration were found (fig . ic, March) . Diel changes in chlorophyll usually did not correspond with changes in total particle volume (fig . 1 b) . Unlike the studies of Yentsch & Scagel (1958) and Ryther et al . (1958), for the majority of the sample series analyzed no consistent diel pattern of variation of chlorophyll content per unit particle volume was found . In both algal populations depicted in figs ia, b, after the exponential phase of growth when evidence was found that nutrients became exhausted (unpublished results), the chlorophyll-a content of the cells rapidly decreased . Particularly with the natural diatom bloom at this time changes in the physiological state of the algal population were prominently reflected in a loss of buoyancy of the algal cells (fig . 1c, April) . It is a well-documented phenomenon that in algal cultures of limited volume storage products accumulate at the onset of the declining phase of growth (Fogg, 1965) and eventually this also may result in an enlargement of algal cells (Prakash et al., 1973) . Most likely this would explain the maximum values for total particle volume and of dry weight biomass during the declining phase of growth of the Scenedesmus culture (cf. Steele & 1 49
0
NATURAL
8
W 6
a
2 1- 4
_ 2
00
DIATOM 10
20
chlorophyll -a
6/5
50
4/2
C
9/5
7/2
- 7/3
1974
1/4 - 4/4
4/3
100
POOL 'T HAMMETJE
3 0)J9/1
BLOOM
Fig. I . Quantitative changes of different biomass characteristics throughout the typical time-course pattern of growth of two planktonic algae . A . Changes in Chlorophyll-a, total particle volume (Coulter counter) and algal dry weight shown by a batch culture of Scenedesmus dimorphus; grown at 15°C and 6000 Lux (14 h L : 1o h D) in a medium after Rodhe (1948) ; mass culture in a transparent perspex tube (145 cm high, 2o cm 0) in which circulation was provided by stirring and aeration . B . Chlorophyll-a, total particle volume (Coulter counter) and seston dry weight in the water column of Pool 't Hammetje, 1974, throughout the diatom spring bloom (almost exclusively Cyclotella comta) . Analyses on samples collected at sunset, sunrise and midday during two consecutive 24 hr periods . The numbering indicates the time sequence of sampling . C . The vertical distribution of chlorophyll-a (uncorrected for breakdown products) during the diatom spring bloom in Pool't Hammetje, 1974. Sample sequence as in fig . iB .
SCENEDESMUS- CULTURE
150
Baird, 1962) . With the natural diatom bloom, however, the seston dry weight remained high also after chlorophyll and total particle volume rapidly dropped . Most likely this is the result of the accumulation of non-living materials which were too small or too big to be detected in Coulter counter analysis (compare Table 2) . With both the Scenedesmus-culture and the Cyclotella comtabloom the time-course pattern of growth caused an about 6-fold variation in the chlorophyll-a content per unit of particle volume . It is obvious from fig . 1 that the pattern of growth shown by chlorophyll-a concentration may differ markedly from that indicated by total particle volume or seston dry weight . In order to examine the effect of taxonomic composition of the phytoplankton on variations in conversion factors between chlorophyll-a concentration, total particle volume, and seston dry weight, in Table 2 the results of analyses carried out on portions of the same water samples collected from early spring until the end of summer from the Barlosche Kolk, Pool 't Hammetje, and Lake Maarsseveen have been grouped according to species composition and the presence of extraneous matter in the seston . The data have been grouped on the basis of the results of supplementary paper chromatographic pigment analysis, microscopic phytoplankton analysis, and elementary analysis of seston (figs . 3, 6) . In Table 2 correlation coefficients and linear regression statistics
together with their probabilities according to a t-test are presented for paired components of the seston of the Barlosche Kolk, Pool 't Hammetje and Lake Maarsseveen . In general, the scatter around the regression lines is very considerable . More or less significant positive correlations have usually been obtained between chlorophyll-a concentration and total particle volume . To some extent this may be due to the low analytical error involved in such biomass determinations . No correlation was found for the samples collected from Pool 't Hammetje in September 1974, when due to circulation of water masses large amounts of flocculent detritus had been resuspended into the water column . The slope of the regression equations between chlorophyll-a concentration and total particle volume would indicate chlorophyll-a content of the particles, whereas positive Y-intercepts would indicate the presence of chlorophyll-a in particles that somehow were missed in Coulter counter analysis (< 4µ, > 75 a 0) . The highest chlorophyll-a content was recorded for the diatom plankton of Pool 't Hammetje (3 .4 µg/mm') . In Lake Maarsseveen negative Y-intercepts would be indicative of the presence of particles without chlorophyll, which occurred particularly in summer months (positive X-intercept : 0 .96 mm 3 /1) . Seston dry weight usually did not correlate with total particle volume . Rather poor correlations have been obtained only for the dinoflagellate plankton at the end of
Table 2 . Statistics for grouped data of paired components of the seston of the Barlosche Kolk, 1973, Pool't Hammetje, 1974, and Lake Maarsseveen, 1975 .
lake
month
Barlosche Kolk Barlosche Kolk Pool 't Hammetje Pool 't Hammetje Pool't Hammetje Lake Maarsseveen Lake Maarsseveen
Feb, Apr, May Jul, Aug, Sep Feb-May ; Oct Jun, Jul, Aug Sep Jan-Jun ; Nov Jul-Oct
Barlosche Kolk Barlosche Kolk Pool 't Hammetje Pool 't Hammejte Lake Maarsseveen
Feb, Apr, May Jul, Aug, Sep Feb-Aug Sep, Oct Feb-Oct
Barlosche Kolk Barlosche Kolk Pool 't Hammetje Pool 't Hammetje Pool t Hammetje Lake Maarsseveen
Feb, Apr, May Jul, Aug, Sep Feb-May ; Oct Jul, Jun, Aug Sep Feb-Oct
range Y
range X
N
Pr
regression analysis slope Y-intercept
Ph
predominant seston component
50 .s . At the smaller size range throughout the year the water column integrated particle size spectra were characterized by prominent maxima at 4-10 µ 0 . Lake
Table 5 . List of cell volumes and equivalent spherical diameters calculated for the most abundant phytoplankton species of the Barlosche Kolk (B), Pool 't Hammetje (H), and Lake Maarsseveen (M) . In the case of considerable intraspecific size variation the range of values has been indicated .
volume (µ 3 ) CHLOROPHYTA Closterium acutum Closterium aciculare Coelastrum microporum . . . coenobium Phacotus lenticularis Sphaerocystis schroeteri . . . colony Tetraedron minimum
equivalent diameter (µ)
lake
570 3500 - 7500
10 19 - 24
B M
1500 -7500 450-- 1100
14 - 24 9 .5 - 13
H B, H, M
14000 - 27000 200-320
30 -37 7-8 .5
M H
EUGLENOI'HYTA Trachelomonas sp .
1400 - 2200
14 - 16
H
DIATOMEAE A sterionella formosa . . . colony Cyclotella comta Fragilaria crotonensis . . . colony/I cm Melosira italica . . . colony/I cm Nitzschia sp . Stephanodiscus astraea
200 - 45 0 2000 - 7000 750 - 5000 600- 1800 . . .10 5 150-500 . . .10' 220 500--10000
7 -9 .5 16 - 24 11 -21 10 .5 - 15 . . .125 7-10 . . .27 7 10 - 27
CHRYSOPHYCLAF. Dinobryon divergens Chrysococcus sp .
500-750 50
10-11 4 .5
B,M H
CRYPTOPHYCEAE: Cryptornonas erosa Cryptornonas ovata
400-500 2200 - 4600
9-10 16 -21
B H, M
27 - 66 14 .5 - 17 42-51
B, M M B,M
PYRRO13HYTA Ceratium hirundinella Gymnodiniurn sp . Peridinium cinctum CYANOPHYTA Aphanizomenon flos-aquae . . . filament/250 p . . . trichome aggregation Aphanocapsa delicatissima . . . colony Aphanothece clathrata . . . colony Chrooccocus turgidus . . . colony Microcystis aeruginosa . . . colony Oscillatoria tenuis . . . filament/250 .s
10' - 1 .5 x 10 5 1600- 2500 5x104 -7x10'
B, M B, H, M B, M M B B, M
17 . . .58
B
5x104 -2x10 5
45-73
M
10'-2x10'
27-73
M
2500 . .10 5
15000 5 x 10 4 - 10 6 2500
M
31 46 - 125 17
B
Maarsseveen is a rather large exposed lake which is sub-
vertical differentiation, in Lake Maarsseveen in summer
ject to considerable wave action . In spring these particle
a pronounced thermocline developed (fig . 4c) . At that
maxima therefore most likely refer to the presence of
time the hypolimnion samples of this lake markedly
abundant small resuspended sediment particles . In con-
differed from epilimnion samples by the dominance of
0; fig.
trast to the shallow Barlosche Kolk and Pool 't Hamme-
small-size particles (4-10A
tje where the particle size spectra usually showed little
copic examination of hypolimnion samples revealed
4b) . Concurrent micros-
159
abundant minute non-algal particles, which also would explain the low chlorophyll-a content per unit particle volume and the relative importance of phaeopigments in the hypolimnion (fig . 4a) . Quantitatively non-algal particulate matter was less abundant in Lake Maarsseveen than in the two other lakes (compare Table 2) . However, compared to the low phytoplankton densities, the non-algal suspended particulate matter made up a major part of the total seston in this lake . As a result, in the seston size distribution of this lake, the algae were masked by the high background of non-algal particles to a large extent (fig . 5) . The seasonal variation of total particle volume of the seston in the water column of the Barlosche Kolk, 1973, Pool 't Hammetje, 1974, and Lake Maarsseveen, 1975, has been summarized in fig . 6 . Total particle volume has arbitrarily been grouped in size classes of 4-10 µ, 10-20 µ
BARLOSCHE KOLK
and > 20 µ diameter . In the Barlosche Kolk the development of the dinoflagellate summer bloom is conspicuous owing to the predominance of the > 20µ-fraction . In Pool 't Hammetje the importance of Cyclotella coma is reflected in the predominance of the to-2o IA-fraction in spring and autumn, whereas throughout summer months the development of small Chlorococcales species together with the accumulation of small non-algal particles, gave rise to a large 4-1o /A-fraction. In Lake Maarsseveen the 4-10 IA-fraction made up an important part of the total seston, in spring when the lake was subject to considerable wave action and throughout summer months when hypolimnion particles made a major contribution . In this lake the co-occurrence of large diatom colonies in spring and of voluminous blue-green algae colonies in summer is reflected in the co-dominance of the > 20 µ-fraction .
POOL 'T HAMMETJE
LAKE MAARSSEVEEN
28-2-1973
V111\
9-4
14-5
2-7 C 0 3 2
u
E 1013 13-8
7
u
u
1012
u24-9
5 10 15 20
30 40 50 8011 equivalent spherical diameter
1013
10
10
12
11
Fig. 5 . Size frequency distributions of the total suspended particulate matter on different dates in the Barlosche Kolk, 1973, Pool 't Hammetje, 1974, and Lake Maarsseveen, 1975 . Water column integrated particle size spectra have been drawn with the logarithm of particle concentration (by volume) along the ordinate and equivalent spherical diameter on a linear scale on the abscissa . Compare with Tables 4 and 5 . 16o
Species composition of the phytoplankton biomass
In fig . 6 a comparison is made between the seasonal variation of total particle volume and that of calculated algal volume in the water column of the Barlosche Kolk, Pool 't Hammetje and Lake Maarsseveen . The contribution of the most abundant algal groups Diatomeae, Pyrrophyta, Chlorophyta, and Cyanophyta has been indicated . The percentile contribution of the predominant algal species to total phytoplankton volume has been summarized in Table 4. In general, there was considerable seasonal and lake-to-lake variation in the cell volumes of the different species (Table 5) and therefore for the major species cell dimensions have been determined on each occasion . In all three lakes in spring the phytoplankton standing crop was almost exclusively composed of diatoms (629o% in the Barlosche Kolk, 79-97% in Pool 't Hammetje, 79 - 95% in Lake Maarsseveen) . Dinoflagellates were the major component of the summer phytoplankton of the Barlosche Kolk (74-85%), and to less extent they also contributed to the summer phytoplankton crop in Lake Maarsseveen (6-45%) . Dinoflagellates were practically absent in Pool 't Hammetje . In this lake Chlorophyta made up the major part of the summer phytoplankton volume (31-63%) . Chlorophyta only reached 13% of the summer phytoplankton crop of the Barlosche Kolk, and 22% in Lake Maarsseveen . Cyanophyta developed up to 21% at the end of summer in the Barlosche Kolk, only up to 6% in Pool 't Hammetje, and up to 88% of total phytoplankton volume at the end of summer in Lake Maarsseveen . In the Barlosche Kolk the seasonal variation of total algal cell volume more or less followed that of total particle volume (fig . 6), except for somewhat unexpected higher microscope values at the diatom peak (April) and at the dinoflagellate peak (August) . Similarly, in Pool 't Hammetje throughout the diatom spring bloom algal volume and total particle volume closely agreed, except again at the diatom peak (February) when the microscope method indicated unexpected higher values . In this lake throughout summer months total particle volume steadily increased in contrast to total algal volume which remained at a rather low level . The discrepancy between total particle volume and algal cell volume became most prominent after September when due to circulation of water masses, important amounts of organic detritus were resuspended into the water column . As a result in Pool 't Hammetje in September algal volume accounted only for 30% of total particle volume . In Lake Maarsseveen total particle volume and algal volume showed a
more or less comparable pattern of seasonal variation except for some dissimilarities at the end of summer . In this lake the high background level of non-algal particles markedly smoothed the pattern of wax and wane of the phytoplankton populations . The extremely low phytoplankton densities in this lake only contributed 5-32% to total particle volume in the entire water column . Such low participation of phytoplankton in total seston is evident also from seston size distribution (fig . 5) . Similar extremely low participation of algae in total suspended particulate matter has been reported, for instance, by Riley et al. (1964) for tropical and subtropical oceanic surface waters, where estimates of organic aggregates greatly exceeded that of the living phytoplankton . Conclusions on the nature of the seston of the three lakes
In fig . 3 a comparison is made between the seasonal variation of seston dry weight and of phytoplankton chloroplast pigments in the water column of the Barlosche Kolk, Pool 't Hammetje and Lake Maarsseveen . The results of qualitative analyses of seston dry weight and of phytoplankton chloroplast pigments have been summarized . In fig. 6 a comparison is made between the seasonal variation of total particle volume and of calculated algal volume. Information on particle size distribution and on species composition of the phytoplankton biomass has been included as well . In this way, by simultaneously observing the suspended particulate matter through four completely different `windows', the following picture has emerged with regard to the nature of the seston of the three lakes . In the highly eutrophicated Pool 't Hammetje in spring a unispecific mass diatom bloom gave rise to the highest chlorophyll-a concentrations encountered throughout the present investigation . At that time important amounts of fucoxanthin have been detected by paper chromatography, and diatom cells could easily be recognized in total seston size distribution . In Pool 't Hammetje the wane of the diatom bloom resulted in the accumulation of important amounts of non-living particulate matter, thus causing a marked discrepancy between seston dry weight and algal biomass . In this lake important amounts of Mg-containing chlorophyll-a derivatives have been detected throughout summer months, when lutein and chlorophyll-b were indicative of the incidence of important amounts of green algae . The abundance of small Chlorococcales species was reflected in the dominance of small-sized particles in total seston size distribution . In Pool 't Hammetje large amounts of 161
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Fig. 6 . Seasonal variation of total particle volume and of calculated algal volume in the water column of the Barlosche Kolk, 1973, Pool 't Hammetje, 1974, and Lake Maarsseveen, 1975 . Above : total seston particle volume and the contribution of the size fractions 4-1o µ 0, 1o-2o µ 0, and > zo µ 0. Below : calculated algal volume and the contribution of the most abundant algal groups Diatomeae, Pyrrophyta, Chlorophyta, and Cyanophyta .
flocculent organic sediment were resuspended into the water column due to circulation of water masses in September, thus causing a marked discrepancy between algal biomass and total particle volume . At that time phytoplankton algae only made up 30% of total particle volume and spectrophotometrically estimated chlorophylla included only 32% intact chlorophyll-a . In the moderately eutrophic Barlosche Kolk a small multispecific diatom spring bloom was reflected by the importance of fucoxanthin, whereas at the end of summer the mass development of the dinoflagellate Ceratium hirundinella gave rise to important amounts of peridinin . The dinoflagellate could easily be recognized in seston size distribution, which then predominantly was made up of large-size particles . In this lake in summer the seston dry weight included relatively low amounts of ash (23% of dry weight) . In the Barlosche Kolk green algal pigments have been detected only in minute amounts in early summer . The more or less oligotrophic Lake Maarsseveen is a rather exposed lake in which a distinct thermocline developed at the end of summer . The seston dry weight included relatively important amounts of ash (up to 57% in spring, 48-53% during summer in hypolimnion samples) . Throughout the year seston size distribution indicated abundant small-size particles . In contrast to the two other lakes, where the occurrence of `phaeopigments' was correlated with the development of the zooplankton, in this lake Mg-voided chlorophyll-a breakdown-products regularly were encountered throughout the year . The extremely low algal concentrations in this lake only contributed 5-32% to total particle volume, and in the seston size distribution the algal fraction was masked to a large extent by the relatively high background level of non-algal particles . In spring the successive development of several diatom species was reflected in the importance of fucoxanthin . At the end of summer the detection of peridinin indicated the development of dinoflagellates, and the development of poorly pigmented large colonies of blue-greens, which made up as much as 88% of total algal volume, gave rise to minute amounts of myxoxanthophyll . Small amounts of green algal pigments have been encountered in Lake Maarsseveen throughout the year .
Summary In a strict sense, the term `phytoplankton biomass' would refer only to the living algal material present in a unit area and at a given time (cf . Westlake, 1965 ; Vollenweider et al., 1974) . In aquatic ecology, however, the term commonly has been associated with a great variety of biological and biochemical procedures which are used to quantify the particulate matter suspended in natural waters . Consequently, the term 'biomass' has been referred to living material as well as non-living material (cf. Margalef, 1968 ; Odum, 1969) . In this context, the need was felt to study relative merits of different biomass characteristics . In three Dutch freshwater lakes with great differences in absolute biomass, parallel determinations have been made of seston dry weight and supplementary elementary and caloric analyses of seston, of chlorophylla concentration and supplementary paper chromatographic analyses of pigment extracts, of particle concentration and particle size distribution as studied with an electronic particle counter, and of microscopic enumeration and sizing of algae . Different analytical techniques give strikingly different information : carbon content would indicate energy content of the 'biomass', chlorophyll-a would reflect photosynthetic potential, particle size distribution would characterize the availability of particles as a food-resource for zooplankton etc . The accuracy of any method is largely dependent on the circumstances present, and different biomass characteristics are only of value in limited spheres . It is suggested to distinguish between total seston characteristics (e .g . seston dry weight, particulate organic carbon, total particle volume) and strictly algological biomass characteristics (e .g . chlorophyll-a concentration, algal cell volume) . The pattern of growth of phytoplankton populations shown by e .g . chlorophyll-a concentration may differ markedly from that indicated by e.g. total particle volume or seston dry weight . After a phytoplankton outburst when chlorophyll-a may rapidly decrease, total seston biomass may remain at a high level for longer or shorter times, due to the accumulation of non-living material . To a certain extent the wax and wane of algal populations may go undetected among the total seston . Regression statistics between quantitative data on chlorophyll-a concentration, total particle volume, and seston dry weight showed considerable scatter . There was no uniform effect of taxonomic composition of the samples on conversion factors between different biomass characteristics . Apparently, there is no one method of 1 63
estimating biomass and no conversion factor that may serve for general purposes . Calorimetric and elementary analyses of seston dry weight have indicated the presence of important amounts of ash and of low caloric organic detritus in the seston . Paper chromatographic pigment analyses have indicated the imprecise nature of spectrophotometric determinations of chlorophyll-a concentration . Coulter counter analyses did allow only for very crude information on particle size distribution, and in the size frequency distributions the individual species frequently overlapped in size or were masked by non-algal particles . In general, unambiguous information on the nature of the seston of natural waters may only be obtained by estimating total seston characteristics and algological biomass characteristics simultaneously . Preferably, depending on the objective of the investigation, supplementary elementary, chromatographic or microscopic component analyses should be carried out to guarantee the correct interpretation of the results .
Acknowledgements I wish to express my sincere gratitude to Dr . J . Ringelberg for his interest and encouragement throughout this study . It is a pleasure to thank Mr . B . J . G . Flik, Mr . R. Lingeman, Mr . F. Verkley, Miss C. Winkelman, Mr . F . Icke and Miss M . H . M . Van der Riet, who kindly assisted in the field work under all climatological conditions . Mr . Th. G . N . Dresscher generously checked species identifications, Mr . H . Pieters (Laboratory of Organic Chemistry, University of Amsterdam) performed the many organic micro-analyses, and Dr . A . Jensen (University of Trondheim, Norway) gave advice on chromatographic pigment analysis. The manuscript has greatly benefited from criticism and comments by Dr . J . Ringelberg, Prof. Dr. C . v . d . Hoek, Mr . F. Colijn, Dr . W . W . C. Gieskes and Mr . R . Lingeman. The investigations were supported by research-grant 14-85-002 from the Foundation for Fundamental Biological Research (BION), which is subsidized by the Netherlands Organization for the Advancement of Pure Research (ZWO) .
1 64
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