Microcosm experiments on the effect of nutrient enrichment and a soil layer on the development of freshwater plankton

Limnologica 30 (2000) 9-19 http://www.urbanfischer.de/j ournals/limno

LIMNOLOGICA © by Urban & Fischer Verlag

Department of Zoology, University of Delhi, Delhi, India

Microcosm Experiments on the Effect of Nutrient Enrichment and a Soil Layer on the Development of Freshwater Plankton S. NANDINI& T. RAMAKRISHNARAO With 5 Figures Key words: Microcosms, phosphorus, nitrogen, phytoplankton, zooplankton

Abstract We investigated the response of phytoplankton and zooplankton to experimental alteration of nitrate and phosphate levels in outdoor enclosures. Experiments were conducted in summer and winter and in the absence and presence of a layer of soil. The tubs (12 in all) except the two plain water controls were manured initially with a mixture of fresh cowdung (50g I 1), mustard oil cake (25 g 1 i) and poultry wastes (25 g 11; mostly excreta), prior to enrichment. Water samples were collected from the experimental tubs twice a week to measure selected physico-chemical and biological variables. Water temperature in the summer experiments ranged from 20-30 °C and during the winter experiments from 11-15 °C. The pH values ranged from 8.0 to 9.5 and the dissolved oxygen levels from 8.2-10.0 mg 1-1. The levels of soluble reactive phosphorus and nitrate nitrogen ranged from undetectable levels to 1800 pg 11 and 6000 lag 1-1,respectively. The increase in chlorophyll-a following enrichment was rapid (3-7 days) during summer, but slower in winter (7-14 days). The predominant phytoplankton species observed in the tubs belong to the genera Sphaerocystis, Chlorella, Scenedesmus, Cosmarium, Ulothrix, Zygnema, Gonium and Pandorina. The rotifer species observed were Brachionus calyciflorus, Rotaria neptunia, Lecane bulla, L. hma, L. unguitata, Euchlanis dilatata, Asplanchna intermedia, Pseudoharringia spp., Eosphora spp., Lepadella ovalis, Epiphanes brachionus, Hexarthra mira and Cephalodella gibba. The cladocerans observed were Macrothrix spp. and Alona spp.

Introduction On an evolutionary time scale, all inland water bodies experience the natural process of eutrophication primarily due to an increase in the input of nutrients particularly phosphorus and nitrogen. Studies conducted on temperate lakes show that phosphorus most often limits algal growth (ELSER et al. 1990); in fact, the trophic status of many aquatic ecosystems is often assessed in terms of phosphorus levels alone.

Although in many temperate lakes, primary productivity is limited more by phosphorus than nitrogen, this need not always be so (WHITE et al. 1982). With high levels of phosphates, nitrates could become limiting. In tropical and subtropical freshwaters and marine ecosystems, nitrogen could play an important role as a limiting nutrient (PRISCU & PRISCU 1984). Nitrogen is available to autotrophs as ammonium ions, nitrites and nitrates. Nitrate levels above 300 lag 1-1 indicate eutrophic conditions and under heavy anthropogenic influence, the levels could exceed 1000 ~tg 1-1.When point source organic pollution (sewage) is diverted, nitrate levels in the water are known to decline faster than phosphate levels, resulting in nitrate limitation (EDMONDSON 1970). Much less is however known about nitrogen limitation and recycling in freshwaters as compared to phosphorus (PRISCU & PRISCU 1984; LEAN & KNOWLES 1987). Since autotrophs differ widely in their requirements of nitrogen and phosphorus and in their efficiency of utilisation of these two nutrients, it is expected that the overall response of an autotroph community in a waterbody to nutrients is better understood by considering the relative proportions of nitrogen and phosphorus than their absolute concentrations. N:P ratios have therefore become important in predicting the consequences of eutrophication and nutrient limitation (WELCH et al. 1975). Changing the N:P ratio could influence the outcome of competition among algal species and thereby the diversity of zooplankton (KmHAM & KILHAM 1984). The general consensus appears to be that an N:P ratio of less than 10 will result in nitrogen limitation and values greater than 17 in phosphorus limitation. N:P values in the range of 10-17 could lead either to phosphorus or nitrogen limitation, depending on other factors (FoRSBERG et al. 1978). More recent studies (LATHORP 1988; CANFIELDet al. 1989) however 0075-9511/00/30/01-009 $ 12.00/0

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suggest that complete reliance on N:P ratios to explain the relative abundance of different autotroph groups may not be justified. Sediments bind nearly half the phosphorus load, their retention capacity being stronger in oligotrophic systems (SCHINDLER 1977; STEVENS & NI~LSON 1987). This sedimentary phosphorus is released back, slowly but continuously into the water column above, contributing to the 'internal' phosphorus load. Being more readily available and maximally utilisable (~80%), this internal phosphorus load could play a more important role than the external phosphorus load in governing the productivity of aquatic ecosystems, particularly oligotrophic waters (NORNBERG & PETERS 1984). The rate at which bound phosphorus in the sediments is released into the water above is influenced by abiotic and biotic factors. The abiotic factors include: i) oxygen concentrations anoxic conditions facilitate maximal release (RILEY & PREPAS 1984); ii) pH value (HOLDREN & ARMSTRONG 1980); iii) temperature (BOSTROM & PETTERSSON 1982); iv) turbulence (Tw~NCH & PETERS 1984); V) nitrate levels in the water (NORNBERO et al. 1986) and quality of the sediments (KLoTZ 1991). Biotic factors influencing the rate of phosphorus release from the sediments include: i) bacterial densities in the anoxic sediments (BOSTROM et al. 1988); ii) bioturbation caused by benthivorous fish (NAKASHIMA & LEGGET 1980; JANA et al. 1992); iii) presence of macrophytes (REDDY et al. 1988), and iv) the size structure of the zooplankton community (GOLDMAN 1986; ELSER et al. 1988). In light of the above information we hypothesise that temperature and the presence of a sediment layer to bind nutrients and facilitate their slow and steady release into the water column would have a considerable impact on plankton. Therefore, in this study, we have investigated the response of phytoplankton and zooplankton to experimental alteration of nitrate and phosphate levels in outdoor enclosures. Experiments were conducted in summer and winter. The effect of a soil layer on the process was examined by conducting experiments during both seasons in the absence and presence of a layer of soil.

Materials and Methods Twelve rectangular cement tubs, each with a capacity of 225 1 and a depth of 45 cm, were arranged in three rows, each of 4 tubs, in an unshaded open space within the premises of the Department of Zoology, University of Delhi, India. The tubs were thoroughly scrubbed clean at the beginning of each experiment, filled with tap water (2001) which was left for a day or two for dechlorination. All the tubs except the two plain water controls were manured initially. Prior to the experimental alteration of the N:P ratio, traditional manure as used to increase productivity of fish ponds in various parts of India was introduced into the tubs. This manure ensured certain minimal levels of the essential nitrogen and phosphorus into the tubs and was also conducive to the growth of algae and zooplankton without inoculation. The manure used was a mixture of 10

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fresh cowdung (50 g kz), mustard oil cake (25 g 1-~) and poultry wastes (25 g kl; mostly excreta). The mixture was first added to 161 of water in a bucket and stirred daily for a week to allow for complete mineralisation by microbial action. After discarding any solids present, one litre of the concentrate was added to each of the test tubs. A sample of this concentrate was set aside for determining the nitrate nitrogen and soluble reactive phosphorus concentrations. For enrichment trials, four different N:P ratios, namely 1:5, 1:25, 5:1 and 25:1, based on the molecular weight, were chosen. The desired ratios were achieved by adding to the tubs requisite amounts of laboratory grade KNO3 or KH2PO4. In addition to the replicated treatments, two tubs with plain water and two enriched with only manure (but not KNOB or KH2POD served as controls. Nutrients were added only once at the begining of each experiment. The treatments as well as controls were completely randomised among the 12 tubs using a random number table. All the tubs were covered with galvanised iron screens (mesh size: 2.5 ram) to exclude external inputs of organic matter (such as falling leaves). A slight reduction in light intensity caused by the presence of these screens was considered negligible as far as photosynthesis was concerned. In summer months periodic replenishment of water was necessary to make up the loss by evaporation. Water from every tub was sampled twice a week to measure temperature, pH, dissolved oxygen concentration, nitrates, phosphates (soluble reactive phosphorus and total phosphorus) and chlorophyll-a. pH value was measured with a pH meter. Dissolved oxygen (DO) concentrations and temperature were recorded using a DO meter (Yellow Springs Instruments, Model 54 ARC). On a few occasions when the instrument failed, water samples were fixed in BOD bottles and the dissolved oxygen determined by the modified Winkler's method (APHA 1989). Nitrate levels were estimated using the phenol disulphonic acid method (TAYLOR1958). The soluble reactive phosphorus [referred to as filtrable reactive phosphorus (APHA 1989)] as well as the total phosphorus levels were measured using the ammonium molybdatestannous chloride method (APHA 1989). In order to estimate the chlorophyll-a content of the phytoplankton in the samples, 50-100 ml of unfiltered water was filtered through a GFC filter. The filter paper was then placed in a centrifuge tube with 10 mI of a mixture of chloroform and methanol (2:1 v/v). Analysis was done following the method of WOOD (1985). Zooplankton samples were collected twice a week by filtering 51 of water from each tub, through a nytex sieve (53 gm), returning the filtered water to the tubs. The samples, preserved immediately with buffered formalin (8%), were later analysed in the laboratory after appropriate dilution. A 100 ml sample of water from each tub was collected for qualitative analysis of phytoplankton which was done after the plankton settled for two weeks on the addition of Lugol's solution. The dominant phytoplankton (to the genus level wherever possible) and zooplankton (mostly rotifers, cladocerans and copepods, to the species level wherever possible) was identified. Phytoplankton taxa were not individually quantified since it was considered that the chlorophyll-a values provide a usable measure of the abundance of phytoplankton. The zooplankton taxa were quantified separately by examining under microscope, 2-4 replicate samples on a Sedgewick-Rafter Cell. Each experiment was terminated after four weeks. In order to understand the role of sediments in maintaining the nutrient levels in the water which in turn affects phytoplankton and therefore zooplankton, experiments were done both in the absence

as well as in the presence of a 10 cm thick loam soil layer, free from pebbles and stones. To understand the possible seasonal variations in the response of plankton to nutrient enrichment, the experiments were conducted during the warm months of March-May and during the cold months of November-January. Correlation coefficients were calculated between the various parameters measured, with emphasis on nutrient levels, concentration of chlorophyll-a, and the density of rotifers and cladocerans.

Results Physico-chemical variables During the summer (March-May) experiments, the water temperature in the experimental tubs remained in the range of 20-30 °C and during the winter experiments in the range of 11-15 °C. Starting at about 8, the pH reached -9.5 during the course of the experiment, more rapidly in summer than in winter. The dissolved oxygen levels, initially at 8.2-10.0 mg 1-1, dropped to -3.5 mg 1-1 soon after manuring, but returned gradually to high levels, occasionally exceeding 11 mg 1-1. Nutrients: Changes in the levels of soluble reactive phosphorus (SRP) and nitrate nitrogen (NO3-N) in the control and enriched tubs are shown in Figs. 1 and 2 respectively. The levels of soluble reactive phosphorus and nitrate nitrogen ranged from undetectable levels to 1800 pg 1-1 and 6000 btg 1-1 respectively.The plain water controls (PW) had very low, almost negligible levels of SRP and NO3-N, during both summer and winter experiments. In the manured controls (C) and enriched tubs the SRP levels increased soon after treatment, followed by a gradual decrease to near plain water levels over a period of 3-28 days depending on the temperature and levels of phosphorus enrichment. The trends, but not the levels were comparable in both the summer and winter experiments. The levels of NO3-N were low in the plain water as well as the manured controls particularly during the summer months. Following nitrogen enrichment however, the initial NO3-N values increased and then decreased gradually. The return of NO3-N levels in the water to near control levels with any treatment was slower in winter than in summer (Fig. 2). Soil layer: The presence of a soil layer at the bottom of the tubs had a significant effect on the nutrient levels particularly after enrichment, pointing to the importance of benthic substratum on the nutrient dynamics and cycling in natural, relatively shallow water bodies. In phosphorus enriched tubs, the SRP levels in the water were lower in the presence of soil than in its absence in the summer experiments; in the winter experiments however, the soil layer helped to keep the SRP concentrations in the water higher, particularly in the tubs treated with a N:P of 1:25 (Fig. 1). In nitrogen enriched tubs also, in both summer and winter, the NO3-N levels following the initial rise, declined at a slower rate. NO3-N remained for a longer period of time in the water column in the tubs with soil layers than in those without (Fig. 2).

Chlorophyll-a The response of phytoplankton to enrichment is reflected quantitatively in the chlorophyll-a levels in the treated tubs (Fig. 3). Although the controls (PW and C) also supported some phytoplankton, the positive effects of enrichment on chlorophyll-a were obvious and significant. The increase in chlorophyll-a following enrichment was very rapid (3-7 days) during summer, but much slower in winter (7-14 days). The peak levels during summer experiments occurred within 3-10 days but much later (> 20 days) in winter experiments. Nitrogen enrichment (both 5:1 and 25:1 ratios) contributed to significantly higher chlorophyll-a levels than phosphorus enrichment did; this was true in both winter and summer experiments. The levels of chlorophyll-a were not only higher, but they were also sustained for a longer duration than with phosphorus enrichment. In tubs enriched with phosphorus at 1:5 and 1:25, the chlorophyll-a levels dropped drastically after 7 days, whereas in the nitrogen enriched tubs, the levels started declining only after 14 days. A minor peak of chlorophyll-a was observed around the 24th day in nearly all the tubs, but in the summer experiments only.

Phytoplankton and zooplankton The tubs in all these experiments were neither seeded nor inoculated with any planktonic species. The species of phytoplankton or zooplankton that developed, did so naturally from spores or resting eggs introduced from the manure, soil or air. The predominant phytoplankton species observed in the tubs during the summer experiments belonged to the genera

Sphaerocystis, Chlorella, Scenedesmus, Cosmarium, Ulothrix and Zygnema and during the winter experiments to Cosmarium, Gonium and Pandorina. Among these the dominant species in summer were Chlorella and Scenedesmus, and during winter Pandorina. There was no significant difference in the species diversity due to enrichment. The trends with chlorophyll-a in terms of the soil layer were similar to those with the nutrients, nitrogen and phosphorus (Fig. 3). Among zooplankton, rotifers and cladocerans were nearly the exclusive taxa that developed in the tubs except for the occurrence of unidentified copepod nauplii during one of the summer experiments. Rotifers: The diversity as well as the density of the dominant species in the tubs was significantly higher in the summer than in the winter (Fig. 4). Both, in summer and winter, the earliest species to come up following manuring and enrichment were Brachionus calyciflorus and Rotaria neptunia. Other species recorded in summer experiments were

Lecane bulla, L. luna, L. unguitata, Euchlanis dilatata, Asplanchna intermedia, Pseudoharringia spp., Eosphora spp., Lepadella ovalis, Epiphanes brachionus, Hexarthra mira and Cephalodella gibba. In winter the species observed were Brachionus calyciflorus, Rotaria neptunia, Lecane luna, L Limnologica 30 (2000) 1

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L i m n o l o g i c a 30 (2000) 1

unguitata, Lepadella ovalis, Epiphanes brachionus, Hexarthra mira and Cephalodella gibba. Although the diversity of benthic species was higher, it were normally high densities of planktonic rotifers that led to blooms. Enrichment effects on rotifer densities, achieved or sustained, were significant only in the summer experiments. The extremely low (< 50 1-1) rotifer densities observed in winter experiments might have been due to the very low levels of chlorophyll-a during the first two weeks in all the tubs (Fig. 3). In the summer experiments, enrichment with an N:P of 1:5 resulted in rotifer densities which were much higher (2000-6000 1-1) in tubs with the soil layer than in those without soil (500-2500 l-t). In the former, a rotifer bloom with a density of more than 38000 1-~ was recorded. High densities were also sustained for a longer period with a soil layer present. The higher level of phosphorus enrichment (1:25) did not lead to a proportionate increase in rotifer densities. Despite low phosphorus enrichment, high densities of rotifers (> 300 1-1), were sustained longer in the presence of soil. Enrichment with nitrogen also resulted in a dramatic increase in rotifer densities, with trends nearly similar to those found in phosphorus enriched tubs, except that in the former the average densities sustained were much higher. Again, as in the phosphorus enriched tubs, the soil layer had a significant role in maintaining generally a higher level of rotifer densities. Cladocerans: The only cladocerans which developed naturally in the tubs and that also only during the summer, were Macrothrix spp. and Aloha spp. In none of the tubs in the winter experiments cladocerans developed during the four-week test period (Fig. 5). The general trends in the summer experiments were similar to those observed for rotifers in that: i) compared to controls, enrichment resulted in higher densities; ii) the highest densities were achieved in tubs enriched with phosphorus at 1:25 and with nitrogen at 5:1; iii) nitrogen enrichment stimulated a higher level of cladoceran densities than phosphorus enrichment did, and iv) in controls as well as enriched tubs, presence of a soil layer at the bottom had a profound effect on the sustenance of cladoceran populations during the test period.

Statistical Correlations Correlation coefficients were calculated and the levels of significance checked to examine the strength of correlations among various parameters measured in the tub experiments - particularly the correlation of nutrient levels to chlorophyll-a and of the latter with rotifers and cladocerans. Only the following correlations were statistically significant: • in summer experiments: NO3-N and chlorophyll-a in phosphorus enriched (1:5) tubs (p < 0.05); chlorophyll-a and rotifers in the controls PW (p < 0.01) and C (p < 0.05); rotifers and chlorophyll-a in phosphorus enriched (1:25) tubs with soil layer at the bottom (p < 0.05), rotifers and NO3-N in nitrogen enriched (5:1) tubs with soil (p < 0.01);

in winter experiments between chlorophyll-a and rotifers in PW (p < 0.01), and in nitrogen enriched (25:1) tubs without soil (p < 0.05); between rotifers and chlorophyll-a in phosphorus enriched (1:5) tubs with soil (p < 0.01) and in nitrogen enriched tubs with soil (p < 0.01). Correlations among all the other pairs of parameters were extremely weak and statistically insignificant.

Discussion Judging by the criteria normally used in limnological studies (chlorophyll-a, levels of nitrogen and phosphorus), the trophic status of many freshwater bodies in India is eutrophic to hypertrophic (DAKSHINI & GUPTA 1984; ZAFAR 1986; JANA & KUNDU 1993). Seasonal fluctuations notwithstanding, the chlorophyll-a and nutrient levels particularly SRP remain high, making the assigned trophic status a perennial one. The relation between nutrient levels and phytoplankton biomass (most often in terms of chlorophyll-a) has been investigated in many studies, mostly in oligotrophic and mesotrophic temperate waters. These show a significant and positive correlation between chlorophyU-a and nutrient levels, especially of phosphorus (OSTROFSKY& RIGLER 1987; TRIMBEE& PREPAS 1987; CANFIELDet al. 1989; PRAIRIEet al. 1989). In this study however, we did not find significant correlations between chlorophyll-a and phosphorus and nitrogen in all the treatments. This result is not totally unexpected. The relation between phosphate levels and chlorophyll-a is direct and proportional only upto a limit (100-125 pg 1 ~P) beyond which the response is asymptotic (STRASKRABA 1980; CANFIELD 1983; PRAIRIEet al. 1989). In phosphorus rich waters, phosphorus concentration is a poor predictor of chlorophyll-a levels (MCCAULEYet. al. 1989). Nitrogen limitation further weakens the correlation between phosphorus and chlorophyll-a (E~SER et. al. 1990). Nitrogen limitation is common in shallow sub-tropical and tropical ponds and lakes. This is because: i) the inputs of phosphorus are substantially more than those of nitrogen; ii) because of high temperatures (> 25 °C for nearly 8 months of a year) and shallowness (depth: 1.5-2.5 m), both phosphorus regeneration and recycling are significantly faster, and iii) loss of dissolved inorganic nitrates is greater due to the higher rates of denitrification and ammonia volatilisation (WELCH 1992; MEYBECKet. al. 1989). Our study clearly indicates that nitrogen enrichment results in higher and sustained levels of chlorophyll-a which confirms the possibility of nitrogen limitation. Zooplankton species do not respond directly to ambient nutrient levels but to changes caused by them in terms of the composition and abundance of phytoplankton. Of the nine significant correlations observed in this study, eight were between rotifer densities and chlorophyll-a at various treatments. This indicates that enrichment results in increased Limnologica 30 (2000) 1

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concentrations of phytoplankton which in turn, being utilised by the rotifers, led to a concomitant increase in their biomass (NELSON & EDMONDSON 1955). The tub experiments clearly show that the fate and consequences of nutrient alterations are not identical in winter and summer. Following enrichment by N or P, the return to pretreatment levels was significantly faster in summer than in winter. The response of chlorophyll-a to enrichment was higher and reached faster in summer than in winter, and in turn, the response of rotifers to phytoplankton was similar. The underlying mechanisms are the effect of temperature on nutrient uptake by phytoplankton, nutrient regeneration rates and on the metabolic and reproductive rates of zooplankton (RHEE & GOTHAM 1981; MAY 1983). Nitrogen depletion rates were faster in summer and in the absence of a soil layer. Besides being taken up rapidly by the nitrogen limited autotrophs, nitrate is also depleted faster in shallow eutrophic waters because of the increased rates of denitrification at the oxygen depleted sediment water interface (LEAN & KNOWLES 1987). This is the main reason why eutrophic systems cannot store nitrogen long enough to carry over to the next growing season (BARICA 1990). It has been shown that selective nitrogen enrichment with increasing N:P ratios can lead to replacement of cyanobacteria by the more edible green algae (ScHINDLER 1974; BARICA et al. 1980). In several water bodies in India, perennial inedible cyanobacterial blooms are found (ZAFAR 1986). Nitrogen enrichment at frequent intervals (since nitrogen depletion is faster due to high temperatures, shallow water depth and other factors) may be a solution to the problem and may result in higher secondary productivity. The use of mesocosms and microcosms allows ideas generated in laboratory studies to be effectively tested in the field. They also allow optimal utilisation of resources and the advantage of replicability (CARPENTER 1996; DRAKE et al. 1996). The major disdavantage in using micro- or mesocosms is the high degree of variability inherent in natural systems which may mask the results despite their presence (DRAKE et. al. 1996). Nonetheless, the use of microcosms and mesocosms is a valuable tool in aqatic ecology and can be effectively used to answer questions pertaining to plankton dynamics.

Acknowledgements: One of us (S.N.) thanks the Council of Scientific and Indutrial Research (CSIR, New Delhi, India) for a Senior Research Fellowship. We thank the reviewer and Dr. S.S.S. SARMA for making critical comments on the manuscript.

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