Freshximler Biology (1993) 30, 369-376

A seasonal study of phosphorus deficiency in a eutrophicreservoir

i NEDOMA,* PETRA PORCALOVA, JAROSLAVA KOMARKOVA AND V O J T E C HVYHNALEKHydwbiological Institute, Academy of Sciences of Czech Republic, Na sddkdch 7, CS-37005 Ceskd Bud^jovicc, Czech

Republic

*Autbor to whom correspondence should be addressed

SUMMARY

1. Seston elemental composition (C, N, P) and the orthophosphate uptake rate constant,k{upi), were measured in the epilimnion of the eutrophic fomov Reservoir in the periodMarch-August 1989.2. Seasonal series of log C/P, log N/P and log [/:(upt)] values were highly correlated (r' inthe range 0.84—0.93), but, based on literature-derived criteria, fc(upt) values indicatedstronger P defidency than did the C/P values.3. The results showed moderate to strong phosphorus deficiency in summer, moderatedefidency at the end of the spring peak of phytoplankton, and no defidency early in thespring or during the spring clear-water period.

Introduction

Nutrient defidency is an important factor infiu-endng seasonal development of phytoplanktonin aquatic ecosystems. Its relative importance, com-pared with other factors such as grazing pressure,light intensity or water column stability, differsamong lakes and changes considerably during theannual cycle (Reynolds, 1984; Sommer et al., 1986;Vanni & Temte, 1990). The most frequently defidentnutrient in temperate freshwater lake ecosystems isphosphorus (Dillon & Rigler, 1974).

In the Rimov Reservoir, a eutrophic water bodywith a high load of nutrients from its main tributary(the Mal§e river —about 5gm"^year~' of total phos-phorus and about 200gm"^year~^ of total nitrogen),the seasonal development of plankton generallyfollows the pattern described for other eutrophicwater bodies: a spring phytoplankton maximum, aclear-water period and a second peak of phytoplank-ton in summer (Sommer et al., 1986). Some evidenceindicates, however, that mechanisms controlling thisdevelopment in the fomov Reservoir could differfrom those generally proposed for eutrophic systems(the PEG model; Sommer et al., 1986): despite the

high nutrient input, a possible role for phosphoruslimitation in spring phytoplankton developmenthas been suggested (Komarkova, 1989; Vyhnaiek,1989; Brandl et ah, 1989a,b; Vyhnaiek ct al., 1991).In summer, P deficiency in the fomov Reservoirmay be occurring, but little direct evidence has beenavailable (Brandl ct al., 1989b).

The actual degree of P defidency at a given localitycan be estimated, among other methods, by measuringthe molar ratio of organic carbon to total phosphorusin seston (Healey & Hendzel, 1980; Gage & Gorham,1985), the turnover time of dissolved orthophosphate(Rigler, 1964; Lean et al, 1983) or the rate constant oforthophosphate uptake (Currie, 1990), which is aparameter closely related to the turnover time.

In this study, two independent methods to assessphosphorus status (seston C/P, N/P ratios; ortho-phosphate uptake rate constant) were used andcompared to identify periods of phosphorus defidencyin the eutrophic fomov Reservoir.

Material and Methods

The fomov Reservoir (South Bohemia, Czech Repub-lic) is a eutrophic, dimictic water body, positively

370 /. Nedoma et al.

stratified from March to October. It has a maximumdepth of 43 m (mean 16 m), maximum volume of34 X 10*'m^ and a theoretical retention time of 92 days.For more detailed characterization of the reservoirsee Hejzlar & Straskraba (1989). Integrated samples(0-2 m) were obtained with a tube sampler at sevensites across the reservoir near the dam. The sampleswere filtered immediately through a 100 im sieveand transported to the laboratory for further analyses.

Soluble reactive phosphorus (SRP) was measuredin Whatman GF/C filtered water samples according toStephens (1963). Total phosphorus (TP) was analysedas SRP after HCIO4 digestion (Popovsky, 1970).Samples for total dissolved phosphorus (TDP) wereWhatman GF/C filtered and preserved by addition ofH2SO4 (0.1% final concentration) before analysis. Toestimate hot water extractable phosphorus (HWEP)in seston, duplicate 250-1000 ml subsamples werefiltered through Whatman GF/C filters. Filters werestored at -20°C before analyses. HWHP was esti-mated as SRP released during 1-hour boiling of filtersplaced in 50 ml of double-distilled water (Fitzgerald& Nelson, 1966).

For analyses of elemental composition of seston,seston from 10—221 of water sample was concentratedusing a modified Foerst centrifuge, and freeze-dried. Organic carbon, nitrogen and hydrogen weremeasured with a CHN-CORDER NT-3 analyser(Yanaco). Total phosphorus was determined as above.

To determine the rate constant of orthophosphateuptake /f(upt), duplicate 100 ml aliquots of watersample were supplemented with 0.1 MBq of carrier-free [ 2p].HPO3 (ROTOP, Dresden, Germany), andincubated at tbe in situ temperature for 2-8b. Atappropriate time intervals, 2 ml subsamples werefiltered through Whatman GF/C filters. Radioactivityretained on the filters was measured with a BeckmanLS 7000 spectrometer as Cherenkov scintillation. Totalactivity was measured in unfiltered 2 m] subsamplesand did not change markedly (more than 10%) in thecourse of experiments. The 'c(upt) was determinedfrom the time course of ^ P incorporation into theparticles retained on the filters as the initial slope ofincorporation, using the method of Currie & Kalff(1984). The second degree polynomial equation:

P(%) = Po -h 100 k{upt)t + at^

where P(%) is filter-retained •'"P activity at time texpressed as per cent total • P activity, and P(, isfilter-retained •' P activity at zero time, was fitted to

the data using non-linear regression (Statgraphics).Data points where incorporation was higher than70% of the apparent equilibrium (plateau) level wereexcluded from the analysis (Currie, 1990). The poly-nomial model was chosen in preference to the twocompartment exchange (exponential) model (Currie &Kalff, 1984), because it gave better estimation of k{upi)in situations of slow PO4 uptake (^(upt) < 0.05h~^)when only a few per cent of total activity was incor-porated in the course of the experiment. When theuptake was rapid (/:(upt) >0.1 h^'), however, thetime courses of incorporation fitted the exponentialmodel extremely well.

Concentration of chlorophyll a (chl.a) was de-termined by the in vivo fiuorescence technique(Strickland & Parsons, 1968) during the spring whencyanobacteria were not present, or by the spectro-photometric method (Lorenzen, 1967) during thesummer when c}'anobacteria formed an importantpart of the phytoplankton biomass.

Samples for analyses of spedes composition ofphytoplankton were preserved with Lugol iodinesolution and cells were counted and measured usingKolkwitz's chambers and an inverted microscope(Olympus IMT 2) (Lund, 1951).

Zooplankton was sampled using an Apstein sam-pler, with 200 |im netting, by vertical hauls throughthe whole depth of the water column (Brandl et at.,1989a). Cladocera were separated by the flotationmethod according to StraSkraba (1964), and theirprotein biomass was determined by the biuret methodaccording to Blazka (1966).

Results

The development of chl.o concentration and phyto-plankton composition in the fti'mov Reservoir duringthe study period is shown in Fig. la and Table 1. Earlyin the spring and during the spring phytoplanktonmaximum (in April, maximum chl.a concentration =23 [xg 1 ~ ), phytoplankton was composed of flagellates(Chlorophyceae, Cryptophyceae and Chrysophyceae)and diatoms. During the subsequent clear-waterperiod (in May, chl.fl concentration = 1.4—4.5 ngl~'),diatoms and flagellates (Cryptophyceae) prevailed.Cyanobacteria occurred at the end of the clear-waterperiod, however no colonies were present and allchl.fl passed through the 1(X) fim mesh. Later in thesummer some phytoplankton particles greater than100 ^m in size appeared (usually less than 20% of total

March April Moy June

1989

July August

Fig. 1 Epilimnetic seasonal patterns of selected parametersin the ftimov Reservoir in 1989. (a) Chlorophyll n concentration(chin; squares) and dadoceran biomass (asterisks), (b) Totaldissolved phosphorus (TDP; triangles), soluble reactivephosphorus (SRP; filled squares) and hot water extractablephosphorus (HWEP; empty squares) concentrations.

Phosphorus deficiency in a eutrophic reservoir 371

chl.fl; Table 1); these were not included in analyses.The summer phytoplankton peak was formed byc)'anobacteria, diatoms and coccoid green algae; themaximum total chl.o concentration reached 45^gr^(maximum chl.fl< 100(im concentration was 37M.gr').

A broad spring peak in cladoceran biomass oc-curred in the second half of April and in May, whilein June the biomass decreased and remained ratherstable for the rest of the sample season (Fig. la).Daphnia galenta was the dominant cladoceran.

The concentration of SRP was relatively high(15-20 fig r ) in the spring, with a temporary decreaseduring the phytoplankton peak (Fig. lb). In summerit decreased to l-5[Lgr\ The TDP concentraHonchanged roughly in parallel with SRP concentration,being higher by about 5 fig 1"' in spring and by about10(igr' in summer (Fig. lb). Seston HWEP concen-tration was 5-6 ugl"' early in the spring; during thephytoplankton peak it decreased and remained withone exception (20 July) at a level of 1 -2 .g 1"' for therest of the sampling season (Fig. lb).

Examples of time courses of tracer '' P-PO4 incor-poration into particles are given in Fig. 2. Initialincorporation rate changed dramatically throughoutthe season. The orthophosphate uptake rate constant.

Table 1 Seasonal variations in concentration of chlorophyll a in particles <100|im (jigl '; per cent of total chl.n), in sestonelemental composition (per cent of seston dry weight), and in phytopiankton composition (per cent of total algal biovolumerepresented by genera forming more than 10% of total biovolume), in I imov Reservoir in 1989. n.a. = not available

Dominant phytoplankton

Chlamydomonas (20%), Rhodomonas (18%), Crj'ptomonas (10%)Cryptomonas (40%), Chlamydomonas (11%)Synedra (53%), Chiamydomonas (11%)Chlamydomonas, Pascherina (36%), Chrj'sochromulina (10%)Chrysochromulina (22%), Stephamxliscus (18%)Pianktospbaeria (17%), Rhodomonas (13%), Stephanodiscus (10%)Chlamydomonas (26%), Cryptomonas (19%)Stephanodiscus (707,.), Rhodomonas (14%)Synedra (26%), Stephanodiscus (24%), Rbodomonas (13%)Ankyra (39%), Synedra (14%), Cryptomonas (11%)Cryptomonas (38%), Anabaena (36%)Asterionella (78%)Asterionella (52%), Pediastrum (14%)Aulacoseira (44%), Willea (19%)Cryptomonas (27%), Staurastrum (21%)Staurastrum (31%), Volvox (27%)Anabaena (29%), Staurastrum (15%), Microcystis (IT/o)Microcystis (36%), Pianktospbaeria (14%) 'Aphanizomenon (41%), Microcj'stis (16%)Aphanizomenon (54%), Microc\'stis (247o)

Date

21 Marcb28 March4 April

13 April20 April26 April4 May

11 May16 May25 May

I June8 June

15 June22 June29 June6 July

13 July20 July11 August31 August

Chl.fl <

l^gl"'

13.58.32.0

23.315.416.45.62.61.44.52.6

10.17.39.14.82.55.9

11.837.117.6

100 nm

% total

10010010010010010010010010010010074

10054963992868293

Seston

%C

24.924.918.930.526.226.420.014.8n.a.n.a.n.a.18.822.922.332.9n.a.34.934.037.824.4

DW composition

%N

4.514.303.145.544.794.474.463.11n.a.n.a.n.a.3.413.733.244.68n.a.4.784.926.114.37

%P

0.800.720.530.670.720.460.740.41n.a.n.a.n.a.0.400.520.290.46n.a.0.340.520.40n.a.

%H

3.903.943.234.654.214.223.182.55n.a.n.a.n.a.3.223.723.665.00n.a.5.224.965.613.79

372 /. Nedoma et al.

50-

•D 40-«

k "S 30-a.

° 20-

300

120 i80

Time (min)

240 500

Fig. 2 Time courses of the Incorporation of carrier-free•''-P-PO4 into the particles retained on Whatman GF/C filters insamples collected from tbe epilimnion of the ftimov Reservoirin 1989. Sampling dates and the values of orthophospbateuptake rate constants, calculated as tbe initial slope of the datausing tbe second order )x>lynomial, are given for eachexperiment.

/c(upt), varied from 0.0016 h ^ during the culminationof the spring clear-water period to 10.6 h^ in sum-mer, tending to increase with decreasing SRP andwith increasing chl.fl concentrations (Fig. 3a, Table 2).When achieved (in summer experiments, whenk{upi) > 0.5 h"'), the apparent equilibrium level(plateau) of ^ P-PO4 incorporation occurred whenonly 35-65% of total added ^ P-PO4 was incorporated(Fig. 2-data on 29 July).

Seston elemental composition is shown in Table 1.Organic carbon content varied between 14.8% and38.8% of seston dry weight (DW), increasing withchl.fl concentration. In spring the sestonic molar C/Pratio varied in the range 70-121, with a temporary

March April May June July August

1989

Fig. 3 Epilimnetic seasonal patterns of nutrient statusestimators in the ftimov Reservoir in 1989. (a) Orthophosphateuptake rate constant (t(upt); triangles), seston C/P molarratio (squares); dashed line indicates the Redfield ratio level,C/P = 106:1. (b) Seston N/P (triangles) and N/C (squares)molar ratios; dashed line indicates tbe Redfield ratio level,common for N/P scale (16:1) and N/C scale (16:106 = 0.151).

increase to 148 at the end of the spring phytoplanktonbloom. In summer the ratio increased to valuesbetween 169 and 265 (Fig. 3a). The sestonic molarN/P raho changed in parallel with the C/P ratio inthe range 12.4-33.7, whereas the N/C ratio variedless, in the range 0.117-0.191 (Fig. 3b).

As examplified in Fig. 4 for k{upi) and C/P, relation-ships among the seasonal series of /r(upt), C/P, N/P

log |lc(upt)]

Temperature

log chl.fl

log SRP

log N/P

log C/P

+0.916*"(14)+0.850***(14)+0.294 NS(15)-0.869"*(15)+0.966***(15)

log N/P

+0.922*"(14)+0.847***(14)+0.353 NS(15)-0.829*"

(15)

log SRP

-0.875***(19)-0.657"(19)-0.482*(20)

log cht.ii

+0.536*(19)+0.059 NS(19)

Temperature

+0.702***(19)

Table 2 A summary of correlationsamong tbe seasonal series oforthophosphate uptake rate constants[Jf(upt)], seston C/P and N/P molarratios, soluble reactive pbospborusconcentrations (SRP), chlorophyll aconcentrations (cbl.fl) and temperatures,measured in the epilimnion of theftimov reservoir in 1989. Number ofobservations in parentheses. Statisticalsignificance: NS = not significant,*P < 0.05, " P < 0.01, ***P < 0.001

Fig. 4 Relationship between epilimnetic values of seston C/Pmolar ratio and ortbophosphate uptake rate constant, it(upt),simultaneously determined in tbe ftimov Reservoir in 1989.Vertical lines (at the levels of 130 and 260) represent respectiveboundaries between no, moderate and extreme P deficiency asindicated by the C/P ratio. Horizontal lines (at the levels of0.1 h"' and of 1 h"') represent respective boundaries betweenno, moderate and extreme P deficiency as indicated by the/c(upt) method.

and SRP were roughly linear on a log-log scale.Correlation coefficients are given in Table 2.

Discussion

Method reliability

For estimation of the orthophosphate uptake rateconstant, fc(upt), we used Whatman GF/C filters (ofeffective pore size about 0.8 nm) to separate free andparticle-incorporated ^"P-PO4. Judging from thesummer experiments, in which uptake was rapidenough to reach a plateau level (Fig. 2), a high pro-portion of the total added -'-P was incorporated intosmall cells, presumably mostly bacteria, passingthrough the filters. Consequently, our /r(upt) valuescannot be related to phosphorus uptake by all theseston, but rather by algae plus some bacteria retainedon the GF/C filters. Consistent with size fractionationof ^ P-PO4 uptake observed in eutrophic or mesotro-phic lakes (Rai&Jacobsen, 1990; Taylor & Lean, 1991),this accounts for 35-65% of the seston phosphorusuptake capacity. The resulting underestimation ofthe k{upt), however, is not large when compared

Phosphorus deficiency in a eutrophic reservoir 373

with the four-order-of-magnitude changes in Jt(upt)occurring throughout the sampling season.

For seston stoichiometry determinations we usedthe continuous-flow centrifugation technique. Incomparison to the collecting of seston on filters, thecentrifugation method is substantially less effectivein collecting cyanobacteria due to their buoyancy.In our study, this could have significance for theperiod of cyanobacterial dominance (from the secondhalf of July).

Both methods used in this study for the estimationof seston P status are thus subject to various sourcesof potential error. However, measurements of •* P-PO4uptake kinetics and of seston elemental compositionare completely independent of each other, each beinginfluenced by different kinds of error. The reliabilityof both methods is supported by virtually parallelseasonal courses of log fc(upt) values, molar N/P, andC/P ratio values (Fig. 3), which are also bome out bytight correlations of their seasonal series (Table 2,Fig. 4).

Indication of phosphorus deficiency

According to Lean, Abbott & Pick (1987), values ofk{npt) in excess of 0.1 h"^ and 1 h"' indicate moderateand extreme phosphorus deficiency, respectively.Application of these criteria to our data indicates nophosphorus deficiency early in spring or during theclear-water period, moderate phosphorus deficiencyduring the spring phytoplankton maximum, andmoderate to extreme P deficiency in summer in theepilimnion of the Ri'mov Reservoir in 1989 (Fig. 3).Applying the C/P ratio values of 130 and 260, sug-gested as respective boundaries for moderate andsevere P deficiency (Healey & Hendzel, 1980; Gage& Gorham, 1985), to our data indicated the samepattern; no deficiency in early spring or during theclear-water period, moderate P deficiency during thespring phytoplankton maximum, and moderate tosevere P deficiency in summer (Fig. 3).

Thus, on the basis of criteria derived from theliterature, our data on ' P uptake kinetics and onseston stoichiometry can be interpreted in termsof phosphorus deficiency in a generally consistentmanner. In summer, however, the P uptake kineticsindicated higher P deficiency than did the C/P ratio(Fig. 4). The reason for such a discrepancy is notclear, but it is worth noting, because in many studies

374 /. Nedoma et aJ.

conclusions about the degree of phosphorus deficiencyare based on one of these methods only. Simultaneousseasonal measurements of "P uptake rate and sestonstoichiometry were reported by Lean et al, (1983)from Lake Erie, but the temporal trends of the twoparameters were not consistent in that case. Moredata from different t\'pes of lakes seem to be necessaryto intercalibrate the two methods.

Another parameter reported to be of potential useas an indicator of P status, hot water extractablephosphorus (HWEP) (Fitzgerald & Nelson, 1966;Petterson, 1985), appeared to be of only limited usein this study, due to its modest temporal dynamics(Fig. 1). No significant correlation of HWEP with anyother P limitation estimator was found.

Phosphorus status and phytoplankton development

The overall pattern of seasonal development of phyto-plankton biomass and spedes composifion in theRimov Reservoir in 1989 (Table 1) was in accordancewith generalized patterns of the seasonality of phyto-plankton in temperate eutrophic freshwater lakesas described, for example, by Reynolds (1984) andSommere/ al. (1986).

In the early-spring period preceding the springphytoplankton bloom (up to the middle of April1989), the fomov Reservoir phytoplankton wasphosphorus saturated, judging from C/P ratio valuesin the range 80-92, the high concentration of HWEP,and the low values of the orthophosphate uptake rateconstant, A"(upt). Most probably, phytoplankton wascontrolled at this time by physical factors such aslight availability (deep mixing at a time when positivestratification was not yet stabilized), low temperature,or sedimentation.

During the spring peak of chl.fl concentration(in the second half of April), stable stratificationdeveloped, leading to the rapid depletion of SRPin the epilimnion. At the end of the spring phyto-plankton peak (26 April), moderate phosphorusdeficiency was detected (C/P = 147; fc(upt) nearly lh" 'indicating rapid orthophosphate turnover). Thereis controversy about the occurrence of P limitationduring the spring chl.fl peak and about the role that itplays in the triggering of the subsequent clear-waterperiod. While in some lakes the role of zooplanktongrazing has been well documented (Lampert ct al.,1986; Lampert, 1988), in others the spring phyto-

plankton depression seems to be primarily causedby P limitation (Van Donk, Mur & Ringelberg, 1989;Weise ct al., 1990). In the ftimov Reservoir, inductionof clear-water period by P deficiency was probablein 1986 (Brandl et al., 1989b) and in 1990 (Vyhnalek,Sed'a & Nedoma, 1993).

During the clear-water period, no P deficiency wasobserved. Phytoplankton was controlled by otherfactors, presumably by zooplankton (Lampert et al.,1986).

In summer, moderate to strong P deficiency wasdetected, which was, however, not stable throughoutthe whole period: several fluctuations of epilimnionP status were observed, reflected by parallel changesof all measured P deficiency estimators.

Summer P limitation, common in eutrophic lakes,is in many lakes gradually replaced by nitrogenlimitation during late summer (Sommer et al., 1986),but this does not seem to be the case of the fomovReservoir. Although the N/C molar ratio in summerdeCTeased to slightly below 0.125 (the border fornitrogen limitation suggested by Healey & Hendzel,1980), the high N/P ratios measured concurrently(21-34) indicate that phosphorus was much moredeficient in seston than nitrogen, and that limitationby nitrogen is not probable (Fig. 3). A certain decreaseof N content in P-Umited and strongly P-deficientcultures of the alga Sclenastrum minutum has alsobeen reported by Elrifi & Turpin (1985), who haveeven proposed that a certain decrease in the cellquota of the non-limiting nutrient in parallel to thedecrease in the cell quota of the limiting nutrientcould be a common occurrence.

It has been suggested that nitrogen deficiencyfavours development of blooms of N2-fixing cyano-bacteria (Smith, 1983). In August 1989, however, aheavy bloom of Aphanizornefwn fios-aquae appearedin the Rimov Reservoir under conditions of per-sistent high nitrogen availability and of increasingphosphorus deficiency. As pointed out by Hrbac ek(1964) and Komarkova (1983), resistance to zoo-plankton grazing and the ability to regulate buoyancyprobably give sufficient competitive advantage toAphanizomenon flos-aquae colonies to outgrow edibleand/or non-migrating species. Such a view seems tobe supported by the fact that Aphanizomenon wasaccompanied by another colonial but non-N2-fixingcyanobacterium, Microcystis aeruginosa, in the late-summer ftimov Reservoir phytoplankton.

Acknowledgments

We thank our colleagues L. Prochazkova, J. Kopa^ekand J. Hejzlar for providing the data about nutrientloading in the ftfmov Reservoir, and Z. Prachaf forexcellent technical assistance during zooplanktonsampling and protein detenriination. We are indebtedto Professor C. R. Townsend (University of Otago)and to an anonymous reviewer for helpful commentsand suggestions on an earlier version of this paper.

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{Manuscript accepted 4 June 1993)