Influence of different nitrogen to silica ratios and artificial mixing on the structure of a summer phytoplankton community from the Swedish west coast (gullmar fjord)

159

Journal of Sea Research 35 (1-3): 159-167 (1996)

INFLUENCE OF DIFFERENT NITROGEN TO SILICA RATIOS AND ARTIFICIAL MIXING ON THE STRUCTURE OF A SUMMER PHYTOPLANKTON COMMUNITY FROM THE SWEDISH WEST COAST (GULLMAR FJORD) EVA SCHOLLHORN and EDNA GRANt~LI Department of Marine Ecology, University of Lund, S-22362 Lund, Sweden

ABSTRACT A natural summer phytoplankton community from the Gullmar Fjord (Skagerrak, Swedish west coast) was diluted 10 times with filtered seawater in twelve 300-dm 3 polyethylene cylinders. Nutrients were added to produce two different molar ratios of nitrogen to silicon (N/Si 3.2/3.2 I~M=I and 12.8/3.2 l~M=4). Three cylinders of each nutrient treatment were carefully mixed while the other three of each nutrient treatment remained unmixed. Diatoms (Nitzschia and Chaetoceros species) were favoured by low dissolved N/Si molar ratios (N/Si 1) and by mixing. Diatoms increased as a percentage of total phytoplankton biomass from 11% initially to about 32% in the unmixed and to 46% in the mixed cylinders. At high N/Si ratios (N/Si 4) small flagellates (1-6 pm) became dominant and diatoms never constituted more than 20% of total phytoplankton biomass.

Key words: phytoplankton, eutrophication, nitrogen, silicon, nutrient ratios

1. INTRODUCTION Increasing nutrient inputs to coastal marine environments have been reported in most parts of the world. Eutrophication has therefore been the focus of many studies. In Sweden nitrogen and phosphorus loads have also increased sharply during recent decades due to anthropogenic activities (Larsson et al., 1985; Rosenberg et al., 1990). The resulting increase in primary production is, however, not always passed on to higher trophic levels, and thus fish production is not necessarily increased by eutrophication. Instead the increased primary production often produces a large phytoplankton biomass, which, when decomposed in deeper water layers or at the sediment surface, causes oxygen depletion. Low oxygen concentrations cause all the widely reported negative effects of 'eutrophication' on benthos and fish communities (Baden et aL, 1990; Hansson & Rudstam, 1990). It has also been discussed whether eutrophication could be the cause of the increase in toxic phytoplankton blooms which have been observed in marine coastal waters during recent decades (Smayda, 1990). Ryther & Officer (1981) divided phytoplankton into species which are good or bad from a human point of view. The 'good' type of algae grows

fast, and is rapidly decomposed. It is also a good food organism in terms of digestibility and nutritional value. The 'bad' type has all the opposite characteristics and may even be toxic to other organisms. It remains to be determined which of all the factors influencing phytoplankton succession is/are changed by eutrophication and can cause a shift from a community dominated by 'good' species to a community dominated by 'bad' algae. Diatoms, especially centric diatoms, are generally regarded as beneficial while dinoflagellates and bluegreen algae are often toxic or in some other way (size, taste etc.) inedible for zooplankton (Gran61i et aL, 1993). Flagellates (except dinoflagellates) are for practical reasons often placed together in one group, although they are taxonomically quite heterogeneous. The 'flagellate' group might thus contain 'good' species as well as 'bad' species such as Chrysochromulina polylepis. A diatom-dominated phytoplankton community would therefore be desirable. In contrast to other algal groups, diatoms (and silicoflagellates) need significant amounts of silicon. The silica requirements of single diatom species are quite variable (Paasche, 1973; Olsen & Paasche, 1986; Sommer, 1986, 1988a and b, 1991; Nelson & Treguer, 1992). On average, however, a diatom community will need N and Si in

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The objective of our study was to test how different nitrogen to silica ratios (N/Si) influence the community structure of a natural summer phytoplankton popula• tion from the Swedish west coast. In particular we wanted to determine whether high N/Si ratios can cause a succession towards a flagellate and/or dinoI jan-92 flagellate dominated community. Many dinoflagellates and other flagellates are known to be sensitive to turbulence (White, 1976; Pollingher & Zemel, 1981) and the development of dinoflagellate blooms is often connected with periods of calm weather. In most experi1993 ments the phytoplankton is mixed to keep algal cells evenly distributed. We therefore also tested how mixing influences the development of the phytoplankton community under different nutrient regimes. 1991

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Fig. 1. Molar ratios of inorganic nitrogen and inorganic silica in the Gullmar Fjord 1991 and 1993. Ratios were calculated from mean nutrient concentrations from 1 to 10 m depth (unpublished data from Lindahl and colleagues). equal amounts (Redfield et al., 1963; Brzezinski, 1985). Silicon influx to coastal environments has not changed in the same way as nitrogen and phosphorus influx during recent decades. Thus anthropogenic nutrient enrichment may enhance diatom growth to the extent that dissolved silica becomes seasonally exhausted. This may cause a succession from diatoms with high Si demands to those with lower demands and finally a shift to a plankton community dominated by less beneficial algal groups, as has been shown for freshwater communities (Kilham, 1971; Tilman et al., 1986; Kilham, 1986). Schelske & Stoermer (1971) and Schelske (1988) have shown that eutrophication of a lake system does not only influence the Si budget seasonally. A considerable amount of Si is annually buried in the sediment, because of the incomplete dissolution of diatom frustules at the sediment surface. As the Si inputs to the lake cannot compensate for these losses, yearly average Si concentrations are decreasing. Similar patterns of decreasing Si concentrations and changes in species composition have been shown for marine environments (Conley & Malone, 1992; Van Bennekom et al., 1975), although most marine diatoms have lower demands for Si than freshwater species (Paasche, 1980; Sommer, 1988b; Conley et aL, 1989). The delay of Si remineralization in eutrophicated marine coastal areas caused by increasing aluminium fluxes may also contribute to decreasing Si availability in these areas (Van Beusekom & Weber, 1992). Long-term decreases in silica concentrations have been observed in the Baltic (Sanden et al., 1991; Wulff & Rahm, 1988) and other areas (see review by Smayda, 1990).

2. MATERIALS AND METHODS In August 1991 surface water from the Gullmar Fjord (Swedish Skagerrak coast) was pumped into twelve polyethylene cylinders (diameter 70 cm, height 103 cm), each with a capacity of 300 dm 3. The natural phytoplankton community was diluted 10 times by filtering 270 dm 3 of the 300 dm 3 through a system of filter cartridges with a pore size of about 1 #m. From the remaining 30 dm ° larger grazers were removed, by filtering through a 160 #m mesh size plankton net. This was done in order to decrease the side effect that grazing might have on a natural phytoplankton assemblage. To keep temperature close to in situ levels (18-18.5 °C), the cylinders were placed in a swimming pool into which seawater was pumped continuously. The lids of the cylinders reduced the natural light intensity to about 80-90% of the ambient level. Initially, the seawater in the cylinders contained 0.16 #M phosphate and 1.54 p.M nitrogen (ammonia and nitrate/nitrite). The silica concentration was below the detection limit of the standard method used for nutrient analyses (i.e. <0.03 #M). The experiments were run as batch cultures and nutrients were added once at the beginning of the experiment to increase the concentration of phosphorus, nitrogen and silicon to the following final concentrations: Nutrient (#M): Treatment 1 Treatment 2

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The additions were made to produce two different N:Si ratios 1:1 and 4:1 (later referred to as 'N/Si 1' and 'N/Si 4'). The nitrogen to phosphorus ratio was kept at 16 ('Redfield ratio'). Phosphate was added as K2HPO4, nitrogen as NaNO 3, and silicon was added as Na203Si * 5H20. This single, relatively low nutrient addition was chosen to keep experimental conditions as close as possible to natural conditions. As shown in Fig. 1, N/Si ratios between 1 and 4 are not uncom-

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mon in the Gullmar Fjord. The dilution of the natural phytoplankton community was made in an attempt to keep nutrient ratios constant at least during the first few days of the experiment. The experiment lasted for 10 days and samples were taken every day. Before sampling, three cylinders of each nutrient treatment were carefully mixed several minutes with a Secchi-disk, while the other three remained undisturbed (later called 'mixed' and 'unmixed', respectively). Water was sucked out from all layers in the cylinders by slowly lowering a tube connected to a vacuum pump, into the cylinders. This procedure was repeated several times until about 2 dm 3 were collected from each cylinder. Since water was taken from all layers, the resulting samples contained plankton from the whole water column including settled cells on the bottom of the cylinders. Subsamples were taken from these samples for nutrient analyses, in vivo fluorescence measurements and cell counts. In vivo fluorescence was measured daily with a Turner Fluorometer (Model 112). In vivo fluorescence was then transformed to chlorophyll a after analysis of extracted chlorophyll using the method of Jeffrey & Humphrey (1975). The calibration was made at the beginning and at the end of the experiment; since the measurements did not differ significantly a mean value was used for the transformation. Phytoplankton cell counts were made using an inverted microscope (Nikon) according to the method of Uterm6hl (1958). About 400 individuals of the dominant species were counted. Biomass was calculated from cell counts and measurements of linear dimensions as by Edter (1979). Nutrient analyses (phosphate, nitrate/nitrite, ammonium, and silicon) were performed on seven occasions during the experiment using standard methods (UNESCO, 1983) modified for use with an autoanalyser (TRAACS). 3. RESULTS

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Fig. 3. Development of chlorophyll a concentrations in the four treatments (in #g.dm-3; error bars =_+ standard deviation). 3.2. CHLOROPHYLL In the 'N/Si 1' cylinders chlorophyll a concentrations reached a maximum of about 4 #g-dm "3 on the third day of the experiment and subsequently decreased slowly. The 'N/Si 4' cylinders reached a maximum chlorophyll a concentration of about 20 #g.dm -3 on the fifth day (Fig. 3). In the 'N/Si 4' treatments chlorophyll a concentrations differed significantly (Mann-Whitney U-test, p<0.05) between the 'mixed' and 'unmixed' cylinders from day 4 to 7 with higher concentrations in the unmixed cylinders. In the 'N/Si 1' treatment the difference between the 'mixed' and 'unmixed' cylinders was significant only on the last day. 3.3. PHYTOPLANKTON SPECIES COMPOSITION The changes in cell densities of the most abundant species of each algal group in the different treatments are shown in Fig. 4. Chaetoceros sp. and Leptocylindrus minimus reached maximum cell densities in the 'N/Si 4' treatments on day 4 of the experiment. This was probably because the silica concentration in the 'N/Si 4' treatment only slowly dropped to growth-limiting levels, from above 2 pM on day 3, to 1 #M on day 5, and to below detection limit on day 7, while nitrogen was probably already limiting in the 'N/Si 1' treatments from the second or third day of the experiment. At the end of the experiment, however, Chaetoceros sp. numbers were significantly higher in the 'N/Si 1' cylinders. For Leptocylindrus minimus and Nitzschia c.f. Iongissima the differences between the two nutrient treatments at the end of the experiment were not so clear (see Fig. 4). With the exception of Nitzschia in the 'N/Si 4' treatment, diatoms were positively influenced by 'mixing'. Cell densities of the small flagellates (<3 pm and 3-6 pm) were highest in the 'N/Si 4' treatments and

were negatively affected by 'mixing'. In the 'N/Si 1' treatment there was no clear difference between 'mixed' and 'unmixed'. Other flagellates such as Pyramimonas sp. and Chrysochromulina sp. also showed a similar response to the treatments, but as their cell densities were quite low they are not shown here. The dinoflagellates Prorocentrum micans and Gyrodinium sp. (heterotrophic) reached higher cell densities in the 'N/Si 4' treatments, but seemed to be unaffected by the mixing process. In the undiluted seawater at the beginning of the experiment diatoms contributed about 11% of the total phytoplankton biomass. The dominant diatom species were Leptocylindrus minimus and Nitzschia c.f. Iongissima. Dinoflagellates contributed about 37%, of which a Gymnodinium species predominated, but Prorocentrum micans was also common. The 'flagellate' group constituted about 52% of total phytoplankton biomass. In this group small, unidentified species (<6 #m) dominated. In the cylinders receiving nitrogen and silicon at a ratio of 1 ('N/Si 1') the percentage of the total phytoplankton biomass constituted by diatoms increased during the experiment to about 46% in the 'mixed' cylinders and to 32% in the 'unmixed' cylinders (Fig. 5). The proportion of flagellates increased in all treatments during the initial days of the experiment. This might have been a result of the filtration process, which removed only about 90% of the small flagellates (<6 #m). They were thus less diluted and might have had an initial population advantage compared with the other algae. From the third day of the experiment the absolute cell numbers of flagellates in the 'N/Si 1' treatment were quite constant, while as a proportion of the total phytoplankton biomass, they decreased slightly. Except for some Gyrodinium species that grew well during the first few days, all other dinoflagellates decreased in the 'N/Si 1' cylinders. In the 'N/Si 4' cylinders flagellate contribution to total phytoplankton biomass increased to about 80% in the 'mixed' cylinders and to about 90% in the 'unmixed' cylinders (Fig. 5). Diatom numbers increased rapidly at the beginning of the experiment, but diatoms never exceeded 20% of the total phytoplankton biomass and decreased during the experiment to about 10% in the 'mixed' cylinders and to about 5% in the 'unmixed'. A two-way factorial analysis of variance (ANOVA) of the data showed that both flagellate and dinoflagellate biomass significantly increased in the 'N/Si 4' treatment (p<0.001). A significant negative effect of 'mixing' was shown for flagellate biomass on the last day of the experiment (p<0.001). Although diatom biomass was initially positively influenced by the higher nitrogen addition in the 'N/Si 4' treatment, there was no significant difference between the two nutrient treatments at the end of the experiment. In the 'mixed' cylinders diatoms reached a significantly higher biomass (p<0.05) than in the 'unmixed' cylin-

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biomass increased, while flagellate biomass decreased in the mixed cylinders relative to the unmixed cylinders. This might have been caused by direct physical damage of fragile flagellates through the 'mixing' process, but it is more likely that 'mixing' enhanced the efficiency of nutrient uptake of the non-mobile diatoms by removing the nutrient-depleted microzones around the cells, as it has been suggested by Munk & Riley (1952) for sinking diatom cells. This might have increased the nutrient availability for resuspended diatoms, not only during the short 'mixing' procedure, but also while the cells were slowly sinking towards the bottom again. Thus 'mixing' might have increased the competitive abilities of diatoms. Dinoflagellates seemed, as did flagellates, to be negatively affected by the 'mixing' process, but the differences between the two treatments were not significant. This contradicts the findings of White (1976) and Pollingher & Zemel (1981) about dinoflagellate sensitivity to turbulence. Estrada et al. (1987), working with mesocosm experiments and different degrees of turbulence, found that dinoflagellates, as a group, do not always respond in the same way to increasing turbulence. Thus dinofiagellate sensitivity to turbulence might be a species-specific feature and not general for all species.

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NITROGEN TO SILICA RATIOS AND PHYTOPLANKTON

The 'mixing' method used in the experiment differs in several aspects from turbulence or mixing events in natural environments. First, turbulence in the sea occurs on a longer time scale and is not restricted to single events of a few minutes as in our experiments. Thus the positive influence on diatoms and the negative influence on flagellates might be more pronounced in natural environments than in our experiment. Second, in natural environments mixing events are responsible for the distribution of phytoplankton cells in the water column. Diatoms might sink out of the euphotic layer during periods of calm weather and low water turbulence. It has also been shown that diatom sinking rates can increase drastically during times of nutrient limitation (Smayda, 1970; Bienfang et al., 1982; Smetacek, 1985). As sinking rates are species-specific they might also be important for phytoplankton succession (Brzezinski & Nelson, 1988). In the 'unmixed' cylinders, samples were integrated from the surface to the bottom covering the whole water column. Thus the diatoms were not lost from the system and might therefore have had an advantage over natural systems. Thirdly, in natural environments nutrient availability is often increased by turbulence as nutrient-rich water from deeper layers is mixed with surface water. In our experiment, however, nutrient concentrations were not changed by the 'mixing' process. In summary, turbulence in natural environments might have an even stronger influence on species composition than shown in this experiment. 4.2. NITROGEN TO SILICA RATIO All phytoplankton groups responded positively to the high nitrogen addition in the 'N/Si 4' treatments. Diatoms also initially grew best in the 'N/Si 4' treatments (see Fig. 4 and Fig. 6). In the 'N/Si 1' treatment all added nitrogen was taken up by the phytoplankton during the first two days of the experiment. In spite of this, diatoms continued to grow, even during the second period of the experiment, when net growth rates of all phytoplankton groups in the 'N/Si 4' treatments were close to zero or negative (Fig. 6). This indicates that the diatoms present were better competitors for low nitrogen concentrations, as long as silicon was not limiting. According to Tilman's competition theory (Tilman, 1982), each species, due to its different requirements for nitrogen and silica, has an optimum N/Si ratio for growth. Thus species can be ranked along a resource ratio gradient. When environmental conditions are stable long enough, there will be a succession towards the dominance of the species with an optimum ratio close to the prevailing nutrient ratio, provided that nutrient concentrations are above certain thresholds. This has been shown for diatom distribution along a N/Si ratio gradient in the Antarctic (Sommer & Stabel, 1986; Sommer, 1988a) and for Lake Michigan phytoplankton along a gradient of

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phosphorus to silica ratios (Tilman, 1981; Kilham, 1986). In our experiment, however, the same diatom species were more or less abundant in both nutrient treatments. The ten days our experiment lasted may have been too short to allow a succession within the group of diatoms to take place, especially as many diatom species might already have been very rare before the dilution process, due to low silica concentrations in the Gullmar Fjord several weeks before our experiment. One also has to keep in mind that the ratios we are referring to reflect the nutrient ratios at the beginning of the experiment, and that they were not constant during the experiment. Theoretically the best way to study the response of phytoplankton species to different N/Si ratios would have been to use chemostat systems. However, many flagellates and dinoflagellates will not grow very well in chemostat cultures and it might be difficult to draw any conclusions about their competitive abilities from such experiments. As a proportion of total phytoplankton biomass diatoms showed a clear response to the nutrient treatments. In the 'N/Si 1' cylinders the proportion of diatoms increased from 11% to about 32% in the unmixed and to 46% in the mixed cylinders, while the proportion in the 'N/Si 4' treatment never exceeded 20% and decreased in the second period of the experiment (Fig. 5). Apart from diatoms, only some Gymnodiniales showed a positive net growth rate in the 'N/Si 1' treatment (see Fig. 6), but only during the first period of the experiment. The Gymnodiniales were probably heterotrophic and grazing on small flagellates. Other dinofiagellates (such as Prorocentrum, Gyrodinium, Ceratium, and Heterocapsa) only grew in the 'N/Si 4' treatment, but they hardly reached their initial cell densities before dilution (Fig. 4). This contrasts with the general view that dinoflagellates predominate phytoplankton communities in stratified and nutrient-poor waters (Margalef, 1978). Perhaps characteristics other than low nutrient requirements allow dinoflagellates to build up high cell densities in nutrient-poor waters. One possible reason is that dinoflagellates have lower loss rates. Many dinoflagellates are, because of their size, taste or toxicity, hardly grazed by zooplankton and their motility prevents them from sinking out of the euphotic zone (Huntley et aL, 1987; Gran~li et al., 1993). Thus high dinoflagellate numbers might be more the result of accumulation over time than of high growth rates. Another possibility is that dinoflagellates might use their motility to exploit nutrient sources that are unavailable to other algal species, such as deeper, nutrient-rich water layers (Olsson, t990) or micropatches caused by zooplankton excretion (Lehman & Scavia, 1982). In the experimental cylinders, where larger grazers were removed, water depth was too low for any algae to sink out of the euphotic layer, and without nutrient-rich deeper water layers, dinoflagellates lost all

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their possible advantages for survival in nutrient-poor environments. This might be the reason for the poor performance of dinoflagellates in the experiment. In the 'N/Si 4' treatment flagellates dominated the phytoplankton community constituting 80-90% of total phytoplankton biomass. Similar results were obtained in an earlier experiment with phytoplankton from the same area (Sch611horn & Graneli, 1993). In the 'N/Si 1' treatment the flagellate group showed no net growth but still constituted 45-60% of phytoplankton biomass (Fig. 5). This was probably due to their predominance in seawater at the beginning of the experiment and the fact that they were less diluted than the other algae because they were not completely removed by the filtration. Our experiment shows that diatoms may be an important part of the phytoplankton even during summer. In natural environments diatoms may experience high loss rates during summer, due to grazing and sinking. But as long as they are not strongly nutrient-limited they may be able to balance these high loss rates by a high growth potential (Furnas, 1990). Egge & Aksnes (1992) found that diatoms dominated the phytoplankton community in large, mixed outdoor mesocosm experiments, independent of season, as long as silica concentrations were kept over 2 p.M. Diatom growth can influence the nutrient regime of other algal groups in two ways. Firstly by direct competition for limiting nutrients and secondly by exporting considerable amounts of the available nutrient pool either by sinking out from the euphotic zone or by transfer of nutrients to higher trophic levels through grazing. In the Gullmar Fjord in 1991 and 1993 silica concentrations in the upper 10 m of the water column during summer were often under the limit of 2 #M, which was postulated by Egge & Aksnes (1992) for diatom dominance. N/Si ratios also often increased above the optimal ratio of 1 for diatom growth (see Fig. 1). The increase of nitrogen concentration relative to silicate concentration might cause a successional change during summer in favour of flagellates/dinoflagellates which can use the surplus of nitrogen left by silica limited diatoms. Eutrophication and the resulting changes in nutrient ratios might therefore be one important factor behind the increase of flagellate/dinoflagellate blooms observed in the Gullmar Fjord during the last 15 years.

Acknowledgements.--We wish to thank Bodil Andersson for helping us with the nutrient analyses, Odd Lindahl and his colleagues for the nutrient data from the Gullmar Fjord, and Christer Nylander for statistical assistance. Per Carlsson and Wilhelm Graneli and three referees made valuable comments on the manuscript. Roger Finlay kindly corrected the English. Financial support was given by the Swedish Environmental Protection Agency (SNV).

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