Effects of phosphorus and nitrogen enrichment on the phytoplankton in a tropical reser- voir (Lobo Reservoir, Brazil)*

R. Henry’, J. G. Tundisi2 & P. R. Curi ‘Department of Zoology and 3Biostatistics, Instituto Bhico de Biologia Mkdica e Agricola, Universidade Estadual Paulista, Campus de Botucatu, SP, Brazil 2Laboratory of Limnology, Department of Biological Sciences, Universidade Federal de &To Carlos, Sa”o Carlos, SP, Brazil

Keywords: enrichment, phosphorus, nitrogen, phytoplankton, tropical reservoir

Abstract

The effects of enrichment with phosphate (O-500 pg. 1-t) and forms of nitrogen (nitrate, nitrite, ammonia and urea) (o-3500 pg. 1-t) on the phytoplankton growth of Lobo Reservoir (Brazil) were studied in July, 1979. Suspended matter, chlorophyll a, cell concentrations and the carotenoid:chlorophyll ratio were estimated following 14 days of ‘in situ’ incubation. Phosphate alone caused no significant effects, but enrichment with nitrogen caused a substantial increase on the growth of phytoplankton. Comparison between the different forms of nitrogen showed insignificant effects after their additions with 350 pg. 1-t and in combination with phosphate. However, when nitrogen was added in large quantities (3 500 pg.lm*), significant differences between the nitrogeneous forms were found, with urea causing the strongest effect. In July, nitrogen is the main limiting nutrient to phytoplankton growth of Lobo Reservoir.

Introduction

Some studies (Golterman, 1975; Clasen & Bern- hardt, 1974; Halman & Elgavish, 1975; Jordan & Bender, 1973; Schindler, 1977; Schindler et al., 1978; Welch et al., 1978) have shown a limiting effect of phosphorus for phytoplankton growth in temperate lakes and reservoirs, but in the tropics nitrogen also seems to be a critical nutrient. This was demonstrated for Lake Victoria (Africa) by Talling (1966) from a seasonal study of phytoplank- ton composition and its interaction with physical and chemical environmental factors. However, Evans (196 1) showed in the same lake major growth for the cultures of diatom Melosira sp. when phos- phate was added. In water bodies of Malawi, Moss (1969) observed that nitrate and the interaction nitrate-phosphate were of major importance to algal growth. In a study in Rhodesia, Robarts &

*Supported by CNPq and FAPESP.

Hydrobiologia 118, 177-185 (1984). 0 Dr W. Junk Publishers. Dordrecht. Printed in the Netherlands

Southall (1977) recorded that phosphorus should be considered as primary and iron and nitrogen as secondary nutrients to algal growth. In more eutro- phic environments, light penetration also played an important role. Toerien & Steyn (1975) observed that in two impoundments (Haarbeespoort and Rietvlei Dam) nitrogen was the primary limiting nutrient to growth of Selenastrum capricornutum (used as test organism). In another reservoir (Vaal Dam), nitrogen and phosphorus, dependent on the season, were important to algal growth. In Roode- plaat Dam, phosphorus was the primary nutrient during the greater part of the year.

Some studies on phytoplankton, primary pro- duction and limnology in Lobo Reservoir (Sgo Paulo, Brazil) were started in 1971. In their first report of the physical and chemical factors of the water at Lobo Reservoir, Tundisi et al. (1972a, b) concluded that the environment can be divided in two compartments: one more eutrophic in the upper reservoir and the other more oligotrophic in

178

the lower reservoir. In the eutrophic region, dia- toms (for instance, Anomoensis serians, Cymbella presila, C. amphicephala, Frustulia romboides, Melosira italica, Navicula pupula, N. grimmei, N. cryptocephala, Eunotia sp, Nitzchia sp, Pinnularia sp, Surirella sp) are more frequent (Tundisi & Hino, 1981). In the oligotrophic region, the most common genera are Melosira sp, Ankistrodesmus sp, Botry- coccus sp, Cosmarium sp, Straurastrum sp, Oocys- tis sp, Pediastrum sp, Arthrodesmus sp, Dictyos- phaerium sp, Tetraedron sp and Scenedesmus sp. The seasonal cycle of phytoplankton composition is controlled by two climatological factors: rainfall and wind action. High rainfall in the summer cause an input of nutrients and suspended matter in the reservoir (Moraes, 1978). In August-September, the wind action is very important, because produce an introduction of filaments of Melosira italica and nutrients from the bottom to the water column (Lima et al., 1979; Marins, 1981). Tundisi et al. (1979) reported that the primary production of phy- toplankton is probably limited by inorganic nu- trients, particularly nitrogen. The seasonal cycle of nanno and microphytoplankton primary produc- tion was also studied (Tundisi et al., 1978). The monthly average values showed that the nannophy- toplankton contributes to more than90% of photo- synthetic activity. The greater values of primary production were recorded from September to No- vember (about 25 to 40 mgC. m-3 *d-t). A slight increase in the microphytoplankton primary pro- duction occurs during the spring (September to November). Considering that the nutrients are probably the main limiting factors on phytoplank- ton growth and primary production, a research schedule was started in Lobo Reservoir to study the effects of enrichment by nutrients on phytoplank- ton (Henry, 1981).

Evaluation of nutrients limiting phytoplankton growth and primary productivity has usually been done using enrichment experiments of water sam- ples. This can be done by single btoassays (Welch et al., 1978; McDiffett, 1980; and others) or multiple addition bioassays (Smayda, 1971; Robarts & Sou- thall, 1977; Maslin & Boles, 1978). In Lobo Reser- voir, single addition experiments were chosen be- cause the use of multiple addition bioassays does not show any possible synergistic effects (de Haan et al., 1982). In January 1979, synergistic effects due to the nitrate and phosphate additions to water

samples were observed on the chlorophyll a and phytoplankton cell numbers (Henry & Tundisi, 1982a). In other experiments (Henry & Tundisi, 1982b), the results showed that molybdenum and nitrogen enrichment caused a synergistic effect on the chlorophyll a and cell number. The enrichment with phosphate alone did not stimulate the phyto- plankton growth. In February 1980, the addition of nitrate and phosphate to water samples produced significant effects on the chlorophyll a and cell counts, while EDTA enrichment acted only on the cell production (Henry & Tundisi, 1983). The aim of the present work was to enrich the phyto- plankton with various forms of nitrogen (nitrate, nitrite, ammonia and urea) and with phosphorus, and to compare the effects on the suspended matter, chlorophylla, carotenoid:chlorophyll ratio and cell concentration.

Material and methods

Surface water samples containing natural phyto- plankton, were collected in 20 1 buckets on 13/07/ 1979 in the central region of the Lobo Re- servoir (Station 1, Fig. I). The samples were dis- tributed between 40 Erlenmeyers flasks (2 1 capaci- ty) to which had been added inorganic nutrients and 0.5 gel-* Tris Buffer (Vieira & Tundisi, 1979). The reagents used were potassium di-acid phos- phate, potassium nitrate, sodium nitrite, ammonia sulphate and urea. Two phosphorus concentrations were studied: Ps unenriched; P, 500 c(g.l-t. Three concentration levels were tested for all nitrogen froms (N, unenriched; Nt 350 Fg. 1-t and N2 3 500 pg. 1-t). These concentrations are 10 and 100 times higher than the natural nitrate and phosphate contents in the Lobo Reservoir (Moraes, 1978). Ten treatments with four replicates (Table 1) were studied and the flasks were incubated at surface water for 14 days. The following phytoplankton responses to enrichment were then measured: sus- pended matter (Teixeira & Kutner, 1962), chloro- phyll a (Golterman & Clymo, 1969) (not corrected for phaeophytin), the carotenoid: chlorophyll ratio (Margalef, 1964) and cell concentration determined with a Sedgwick-Rafter cell. The same variables and some selected nutrients were also measured in the surface water on 13/07/ 1979 at the start of the experiment.

179

Table I. Reagents, concentrations and experimental design of enrichment carried out in July, 1979 at the Lobo Reservoir.

Symbol Concentration (/Jg.l ‘)

Reagents Treatments (T)

Number Addition with Number of replicates

PO PI

No NI N2

NI

N2

Nl

N2

NI

N2

0 500

KH2P04

0 350 KN%

3500

350 3500

NaN02

350 NH&O4

3500

350 NH2CONH2

3500

TI PO No T2 PI No

T3 T4

T5

T6

T7 PI NI Ammonia 4 TU PI N2 4

T9 PI NI

J-IO PI N2

PI NI

PI N2

PI N2

PI N2

Nitrate

Nitrite

Urea 4 4

MORPHOMETRIC DATA

MaxImum length (km) 7 5

Maxlmum breadth (km) 2 2 Maxlmum depth (m ) 12 0 Mean depth (ml 30 Volume (m’) 22x106 Surface area (km’1 6.6

\

Cdrrego do Gerald0

Co’rrego do Ltmoelro

ltaquerl

I km

\ Rbeiroo do Lobo

Fig. I. Lobo Reservoir (SZo Paulo, Brazil): stations at which water samples were collected (1) and incubated (II).

180

These responses to enrichment were submitted to statistical analysis. Means, standard deviations and coefficients of variation of the four replicates were calculated for each of the ten treatments. For cell counts, the square root transformation was used to adjust the results to normality (Cochran & Cox, 1957). Possible differences between the means were determined through multivariate tests according to Morrison (1967). This showed the differences be- tween the treatments either considered as a whole (multivariate values of F) or isolated (univariate values of F). Possible interactions between the phy- toplankton responses to enrichment were shown through calculation of the correlation between the pairs of variables.

Eleven statistical hypotheses were made to study: 1) the effects of enrichment with phosphate (PI); 2) the effects of enrichment with phosphate (PI) and nitrate (N,) in relation to the control treatment (P,N,); 3) the effects of enrichment with increasing nitrate concentrations (N,, N1 and N2) and with phosphate (P,) in all the treatments; 4) the effects of enrichment with phosphate (PI) and nitrite (N,) in relation to control (P,N& 5) the effects of enrich- ment with increasing nitrite concentrations (N,, N, and N2) and with phosphate (PI) in all treatments; 6) the effects of enrichment with phosphate (PI) and ammonia (N,) in relation to control (N,P,,); 7) the effects of enrichment with increasing ammonia concentrations (No, N, and N2) and with phosphate (P,) in all treatments; 8) the effects of enrichment with phosphate (P,) and urea (N,) in relation to control (P,N,); 9) the effects enrichment with in- creasing urea concentrations (N,, NI and N2) and with phosphate (P,) in all treatment; 10) the com- parative effects of enrichment between the treat- ments with different forms of nitrogen (nitrate, ni- trite, ammonia and urea), all in the concentration (N,) and with phosphate (PI) and 11) the compara- tive effects enrichment between the treatments with different forms of nitrogen (nitrate, nitrite, ammo- nia and urea), all in the concentration (N2) and with phosphate (P,).

Results

Table 2 and Fig. 2 present the amount of sus- pended matter, chlorophyll a concentration, the carotenoid: chlorophyll ratio and some dissolved

inorganic nutrients concentrations of the water sample at the beginning of the experiment (13/07/1979), Station I. The means (four repli- cates), standard deviations and coefficients of vari- ation for each of the responses in the ten treatments are also indicated.

All the mean values for suspended matter in the treatment were higher than the values obtained at the beginning of the experiment including the con- trol treatment (T,). The coefficient of variation was low, not exceeding 20%.

Chlorophyll increased in eight of the ten treat- ments where nitrogen had been added. Pigment decreases, compared to the value in the surface water at the beginning of the experiment (5.39 pg. Chl.a.l-I), were observed only in the control treatment (T,) and in the phosphate enriched treatment (T2). The coefficients of variation of chlorophyll were higher than those obtained for suspended matter. The highest coefficients were obtained in T, and T3 treatments.

Enrichment caused an increase in the cell number when phosphate alone was added to the cultures. However, phosphorus addition with nitrogen, for all forms and concentrations, caused a higher cell production. This increase was up to four times the control value.

The carotenoid:chlorophyll ratio showed a de- crease of its mean values from the starting value. A different response was observed in two treatments (T, and T2) where higher mean values of the pig- ment ratio were recorded compared to before incu- bation. The coefficients of variation of this re- sponse were the lowest of the four variables studied.

The multivariate (the four variables together) and univariate values of F (for each of the variables separately) are shown in Table 3. With the excep- tion of hypothesis 10, all the multivariate values of

Table 2. Chemical conditions of the surface water in the Lobo Reservoir. Station I, 13/07/ 1979.

Variables Concentration (mg. 1-I)

Nitrate <O.lOO Nitrite < 0.050 Ammonia <O.lOO Phosphate <O.lOO Magnesium 0.210 Silicate 3.080

181

6 2401 12

: 200

? 160 z 120

5 9

5 0.01

!llluk

28

24

20

16

12

8

4

0

IO.

9. 4 4

8. 5

7. IO +

6. -Jr

5.

4,

3.

2.

I,

6 II 13 6

F + 18

a T5’qN, b;FiN* T,‘qN, Te’lNe :

‘E NITRITE AMMONIA

8

1 ‘9: - UREA

Fig. 2. Means, standard deviations (I) and coefficients of variation (numbers above the bars) of suspended matter, chlorophyll a, carotenoid:chIorophyll ratio and cell counts obtained in treatments (T, see Table I for individual discrimination) after 14 days incubation in the Lobo Reservoir.

182

7hbk 3. Multivariate and univariate values of F for each response of the phytoplankton to enrichment.

Hypothesis F F Univariate Multivariate

Suspended matter

Chlorophyll Carotenoid: Cells 0 chlorophyll number/ ml

ratio

8 9

10 II

4.77s 1.12 25.79: 9.65* 27.93* 5.13* 19.39* 5.87* 12.25; 2.44 17.95* 7.62* 16.688 4.68* 45.92* 18.20* 48.22* 12.09*

2.29 0.69 25.48* 7.22*

0.22 0.48 1.51 12.58: 7.92* 12.37* 10.04* 3.30: 9.04* 13.40* 9.87* 18.31* 1 I .02* 6.77* 12.12* 11.01* 10.39s 8.88* 8.74* 7.14* 4.32*

20.138 15.90* 11.57* 17.01* I I .74* 6.01* 0.98 1.30 0.69 3.12* I .28 4.62*

*p < 0.05.

F were statistically significant, that is, differences existed between the treatments tested in each hy- pothesis for the four responses together. A discre- pancy between the multivariate value of F and re- spective univariate values for hypothesis 1 (exa- mining the effects of enrichment with phosphate) was observed. When analysed separately, the addi- tion of phosphate alone did not have any significant effects in each of the responses. Complete coheren- cy was obtained in the multi- and univariate values of F in all other hypotheses (except hypothesis 5 for suspended matter and 11 for the carotenoid:chlo- rophyll ratio). There were no significant differences in the responses to enrichment when comparing the different forms of nitrogen (in the concentration N,) with phosphate in all treatments (hypothesis 10).

Table 4 shows the correlation between the re-

Table 4. Matrix of correlations among the four responses of phytoplankton to enrichment.

Variables Suspended Chlorophyll Carotenoid: matter (I chlorophyll

ratio

Chlorophyll 0 0.18 Carotenoid: chlorophyll ratio 0.04 -0.81* Cells number/ ml 0.32* 0.92* -0.73*

*p < 0.05.

sponses taken two by two. A negative correlation was seen between both chlorophyll and cell concen- tration and the carotenoid:chlorophyll ratio. Posi- tive correlations were observed between suspended matter and cell concentration and between chloro- phyll and cell concentration.

Discussion

The existence of interrelations between the re- sponses to enrichment was clearly shown and justi- fied the use of an analysis taking into account this dependency structure. Negative correlation between chlorophyll and carotenoid:chlorophyll ratio was shown by Hallegraeff (1976) and Vince & Valiela (1973). A significant positive correlation between chlorophyll and total particle volume was shown by Hallegraeff (1977), who also verified the existence of a correlation between chlorophyll and seston material. De Haan et al. (1982) observed in enrich- ment experiments that changes in dry weight of seston were weakly correlated with changes in tur- bidity or chlorophyll a. These data are confirmed by our results (Table 4). Consequently, suspended matter can not be considered appropriate to illus- trate enrichment effects.

All algae, with few exceptions, can assimilate ammonia or nitrate if added in adequate concentra- tion (Syrett, 1962; Morris, 1974). Nitrogen can also be used in the form of nitrite but its toxicity in high

183

concentrations makes it less suitable than ammonia or urea.

Algae preferentially use ammonia, i.e., when ammonia and nitrate are added to the algae, nitrate is used only after total consumption of ammonia (Syrett, 1962). However, there are exceptions. Le Cohu & Guene (1976) showed that nitrate was the best form of nitrogen for the growth of desmid Staurastrum polymorphum. They also observed that ammonia chloride and ammonia nitrate (in increased concentrations) had limiting effects.

In addition to the preferential use of ammonia, nitrate and sometimes nitrite, organic compounds may be substituted for inorganic nitrogen. Among them, urea can be used by a wide range of algae (Syrett, 1962). Carpenter et al. (1972), studying the growth of seven marine algae, observed that six were able to grow on urea, but McCarthy (1972), verified that more than 50% of his species did not grow in cultures containing this form of nitrogen.

In the Lobo Reservoir, the phytoplankton popu- lations responded positively to the additions of whichever form of nitrogen by increasing the chlo- rophyll a, cell concentration and suspended matter and decreasing the carotenoid:chlorophyll ratio. The addition of phosphorus alone had no signifi- cant effect on phytoplankton in July. The compara- tive study of the enrichment effects between the treatments with different forms of nitrogen(nitrate, nitrite, ammonia and urea), all added in the concen- tration N, and in the presence of phosphate showed no significant differences in the four responses con- sidered as a whole (multivariate values of F) or as individual level (univariate values of F, hypothesis 10, Table 3). However, when the same comparison was made between the treatments with different forms of nitrogen added in concentration Nz, three of the four responses to enrichment (suspended matter, chlorophyll a and cells concentration) pres- ented significant differences, which vary according to the form of nitrogen added. Generally, the addi- tion of urea at the concentration N, caused higher means of the responses to enrichment (see Fig. 2).

The Lobo Reservoir is situated in a bassin with typical savanna (‘cerrado’) soils. These soils are characterized by a high nutritive defficiency, main- ly phosphate, nitrogen and potassium (Goodland & Ferri, 1979). This shortage of nutrients is due to the great acidity of the soils (below pH 5.0). Lobo Reservoir has a water column well mixed by wind

(Tundisi, in press). Therefore, the vertical distribu- tion of dissolved oxygen is dependent on this mix- ing process. Generally, the water column presents level of dissolved oxygen of the order of 80%. Fu- kuhara et al. (unpublished) demonstrated that re- lease of phosphate from the bottom sediments was very low in the natural conditions of the reservoir. On the other hand, in anoxic conditions (experi- mental conditions) high levels of phosphate were found in the water column. Since Lobo Reservoir is well mixed during the whole year, the contribution of phosphorus from the bottom sediments is neglig- ible. This probably explains the limitation of phos- phorus during most the year.

Inputs of nitrogen and phosphorus occur mainly during the summer, with a runnoff by rainfall. Oth- er input of nitrogen and phosphorus occurs during other periods of the year, mainly throughout the two main rivers (Lobo and Itaqueri). These inputs are absorved by macrophytes in the upper reservoir (Tundisi et al., 1977). This can to explain the low nutrient concentrations in the lower reservoir dur- ing most of the year and as a result a stimulation of phytoplankton growth was observed after the en- richment.

A seasonal pattern of the phosphorus limitation for the phytoplankton growth occurs at Lobo Re- servoir. The results (Henry & Tundisi, 1982a, b; Henry & Tundisi, 1983; and this experiment) showed that phosphorus was essential for increas- ing the chlorophyll a and the cell concentration in some months (January 1979 and February 1980) but not in others (July 1979 and January 1980). These different responses depend on the nutritional quality of enriched water, on the physiological con- ditions of the phytoplankton and on the storing capacity of phosphorus by algae. The limiting ac- tion of nitrogen was well demonstrated in all the experiments and particularly in July. So, nitrogen seems to be the primary limiting factor and phos- phorus the secondary for the phytoplankton growth in Lobo Reservoir.

Summary

The importance of four different forms of nitro- gen and of phosphorus to phytoplankton growth was studied in the Lobo Reservoir (Brazil). Surface water samples collected in July 1979 (Station I, Fig.

184

1) were placed in 2 1 Erlenmeyers to which phos- phorus (in two concentrations) and nitrogen (in three concentrations, for each of the forms: nitrate, nitrite, ammonia and urea) had been added. Ten treatments with four replicates each, were exam- ined (Table 1). After 14 days in situ incubation (Station II, Fig. l), the following variables were measured: suspended matter (by gravimetry), chlo- rophyll a (spectrophotometry), cell counts (in Sedg- wick - Rafter cell) and the carotenoid:chlorophyll ratio. Enrichment effects were tested through mul- tivariate analysis of eleven hypotheses. The addi- tion with phosphate alone had no significant effect on the phytoplankton growth. The addition of phosphorus and nitrogen (for all its forms and con- centrations) caused significant effects on the phy- toplankton growth when compared with the con- trol treatment (Fig. 2). An significant increase of suspended matter, Chlorophyll a and cell number, in all treatments (except Tz), was observed. Com- pared with control, the addition of these nutrients caused a significant decrease in the carotenoid: chlorophyll ratio. Comparative study of enrichment effects with different forms of nitrogen, added in low concentrations and in the presence of phos- phate, did not show significant differences. When a increasing addition of nitrogen was added, three of the four responses (suspended matter, chlorophyll and cell number) showed significant differences, which varied dependent on the form of nitrogen added. Generally, the addition of urea (in high con- centration) caused stronger effects. Statistically significant positive between chlorophyll and phy- toplankton cell number and negative correlations between the carotenoid:chlorophyll ratio and both chlorophyll and cell number, were obtained. The data showed that nitrogen had a positive effect on phytoplankton growth. This did not occur when phosphorus was added alone. In July, nitrogen clearly was the primary limiting factor to algae growth in Lobo Reservoir.

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Received I April 1983; in revised form 20 October 1983; accept- ed 18 January 1984.