The responses of periphyton to enrichment with nitrogen and phosphorus have been investigated in shallow lakes with low macrophyte biomass, but less is known about shallow lakes dominated by submerged plants. We examined the biomass and species composition of periphyton in 12 mesocosms dominated by Vallisneria natans (Lour.) Hara and subjected to different nitrogen and phosphorus enrichment regimes. Periphyton biomass measured as Chlorophyll a (Chl a) was higher in the nitrogen-enriched treatment relative to the controls. Chl a initially increased with phosphorus and nitrogen + phosphorus enrichment, but declined towards the end of the experiment. This delayed negative response of periphyton to nutrient enrichment appeared to be due to increased phytoplankton concentrations which inhibit light availability (nitrogen + phosphorus additions) and to increased growth of V. natans (phosphorus additions). Shifts in species composition of periphyton also occurred with different treatments. Our work demonstrated that periphyton are significantly affected by enrichment with nitrogen and phosphorus, and helped clarify the ecological role of these important primary producers.

Introduction

Eutrophication (nutrient enrichment) is one of the most common water-quality problems in freshwater systems worldwide (Schindler, 2006), with many lakes persisting in a eutrophic state for several decades. Studies of such systems often focus on relationships between aquatic primary production and the available nutrient pool. Periphyton (algae attached to aquatic plants) play a key role in nutrient cycling (McCormick et al., 1998) and are sensitive to changes in nutrient loading (Gaiser, 2009). Further influences of periphyton in shallow lakes include their interactions with other biota, including submerged plants (Phillips et al., 1978; Jones et al., 2002). Thus, changes in the biomass or taxonomic composition of periphyton affect many aspects of freshwater ecology (Dodds, 2003; Larned, 2010).

Periphyton biomass is positively correlated with nutrient concentrations. Enrichment with nitrogen (N) (Havens et al., 1999; Smith and Lee, 2006), and phosphorus (P) (Cattaneo, 1987; Vadeboncoeur and Lodge, 2000) is known to stimulate periphyton production. Excessive periphyton growth associated with elevated levels of both N and P has been also reported (Biggs, 2000; Dodds, 2003).

However, insignificant or negative responses of periphyton biomass to nutrient enrichments are also known. Some studies indicate that P enrichment may have a neutral or negative effect on periphyton biomass (Greenwood and Rosemond, 2005; Bowes et al., 2007), while work elsewhere suggests that periphyton do not respond to enrichment with N alone (Notestein et al., 2003; Scott et al., 2005). Combined enrichment with N and P can lead to a gradual loss of natural periphyton (McCormick et al., 2001). Thus, responses of periphyton to different nutrients appear to be variable.

Nutrient enrichment can lead to dramatic changes in the taxonomic composition of periphyton communities. In a study by Hawes and Smith (1993) periphyton in control groups and tanks enriched separately with either N or P were dominated by Microcoleus and Scytonema, whereas in an experimental group enriched with N + P together, Microcoleus, Nitzschia, and Cymbella were dominant. Nutrient enrichment can therefore be expected to alter the species composition of periphytic algal communities in shallow lakes, but the precise nature of these effects is variable.

Most of the research on periphyton responses to nutrient enrichment has focused on shallow lakes with little or no macrophyte biomass (Hansson, 1992; Havens et al., 1999; Biggs, 2000; Luttenton and Lowe, 2006). Interactions between nutrients, macrophytes and periphyton are complex (Phillips et al., 1978; Sand-Jensen and Borum, 1991), and relatively few studies have examined periphyton responses to N and P enrichment both individually and in combination in shallow lakes dominated by submerged plants (Liboriussen and Jeppesen, 2006; Smith and Lee, 2006). In this study, mesocosms dominated by Vallisneria natans (Lour.) Hara, a species frequently used in efforts to restore eutrophicated lakes (Wheeler and Center, 1996; Qiu et al., 2001; Xie et al., 2005), were used to evaluate the response of periphyton biomass and taxonomic composition to enrichment with N and P, both individually and in combination. The purpose of this study was to evaluate the periphyton response to N and P enrichments in shallow lakes dominated by submerged plants.

Materials and methods

Setup of a shallow aquatic ecosystem

The experiments were carried out in 12 bucket mesocosms, each with an upper diameter of 54 cm, a bottom diameter of 40 cm and a height of 60 cm. Sediment (total N, i.e. TN = 1.13 mg g−1; total P, i.e. TP = 0.56 mg g−1) was obtained from Ming Lake, a eutrophic shallow water body in Guangzhou City, air-dried and sieved (mesh size: 0.5 mm) to remove coarse debris. The sediment was homogenized and used to form a layer about 10 cm deep in each mesocosm. Mesocosms were then filled to the brim using lake water (TN: 2.15 mg l−1; TP: 0.06 mg l−1; soluble reactive P was very low) passed through a plankton net (mesh size: 0.064 mm) to remove debris and zooplankton. After the sediment and the lake water were added, the mesocosms were exposed to natural sunlight and allowed to equilibrate for two weeks.

Experiment design

After the equilibration period, nine rooted clonal individuals of V. natans (each of 18.2 ± 1.2 cm length) and two leaves of plastic artificial grass (Liboriussen and Jeppesen, 2006) of identical size (32.1 cm length × 1.5 cm width) were placed on the sediment surface of each mesocosm and replaced at 14-day intervals. Three treatment groups were established–N enrichment, P enrichment, and N + P enrichment–all in triplicate. An additional three replicate mesocosms received no nutrient additions and served as controls. The calculated doses of 1.5 mg N l−1 (as NaNO3), and 0.1 mg P l−1 (as NaH2PO4) were added weekly as solutions, prepared by dissolving nutrient compounds in distilled water. Nutrient solutions were stirred into the mesocosms to ensure complete mixing. Filtered lake water was added periodically to each mesocosm in order to maintain the water levels during the experiment. The experiment lasted from 21 August to 16 October 2010.

Sampling and analysis

Every two weeks, the artificial grasses were removed from the mesocosms and replaced with clean ones. One leaf from each tank was used to assess the biomass of accumulated periphyton (as Chl a normalized to m−2). The periphytic Chl a was measured spectrophotometrically after ethanol extraction at room temperature, as described by Jespersen and Christoffersen (1987). The second leaf was examined microscopically to identify the taxa present in the periphyton according to the methods of Hu and Wei (2006). Light intensity above the sediment surface was measured every two weeks between 0900 h and 1200 h using an underwater irradiance meter (ZDS-10W). Relative light intensity (RLI) was calculated using the formula RLI%  =  LI/li*100, where LI was light intensity in nutrient enrichment treatments and li was light intensity in the controls, accordingly.

Water samples for phytoplankton biomass (Chl a) analyses were collected every two weeks during the experiment before the addition of nutrients. Water samples (500 ml) were obtained from each mesocosm using a clean bottle to analyze phytoplankton Chl a biomass (Jespersen and Christoffersen, 1987), total nitrogen (TN) and total phosphorus (TP). TN and TP were analyzed after persulfate digestion (American Public Health Association [APHA], 1992). V. natans from each mesocosm was harvested at the end of the experiment (56 days). The plants were washed over a 1-mm-mesh sieve to remove attached sediment, debris and epiphytes; then oven-dried at 80°C until a constant weight was obtained. The dry mass of plant in each mesocosm was recorded.

Statistical analyses

Repeated measurement ANOVAs were used to test for significant differences in TN, TP, periphyton and phytoplankton biomass, and light intensity, under different enrichments, with time as the repeated factor. Where a significant difference was determined, an LSD test was used to detect treatments that differed. One-way ANOVA was performed to detect differences in the biomass between treatments on each date. If the difference was significant, an LSD test was used to detect which treatments differed. All results are presented as mean values ±SD.

Results

TN and TP concentrations

TN concentrations in the overlying water at the start of the experiment were 2.41 ± 0.42 mg l−1. TN was sharply elevated in the N-enriched treatment (repeated measurements ANOVAs, treatment effect; Figure 1) compared to the control group (p < 0.05) and the P or N + P enrichment groups, which did not differ significantly (p > 0.05).

TP concentrations in the overlying water during the initial stage were 0.14 ± 0.01 mg l−1. As the experiment progressed, dramatic differences in TP were observed between the enrichment treatments (repeated measurements ANOVAs, treatment effect; Figure 1). TP concentrations between the control group and the groups enriched with either N or P alone did not differ significantly (p > 0.05), but TP was higher (p < 0.05) in the N + P enrichment treatment than in the controls.

Periphyton biomass

Periphyton biomass varied with enrichment regime (repeated measurements ANOVAs, treatment effect; Figure 2) and over time (repeated measurements ANOVAs, time effect, p < 0.05). In the N-enriched group, Chl a concentrations were higher than in the controls, except at Day 14 (one-way ANOVA, treatment effect, p < 0.05; Figure 2), suggesting that N had stimulatory effect on periphyton biomass. P enrichment on the other hand, significantly increased Chl a concentrations during the early stages of the experiment, but levels declined at the end relative to the control group (One-way ANOVA, treatment effect, p < 0.05). In the N + P enriched treatment, the Chl a were elevated at Day 28 but declined at the end of the experiment compared to the controls (one-way ANOVA, treatment effect, p < 0.05; Figure 2).

Taxonomic composition of periphyton

Transitions in algal species composition (Table 1), including changes in dominant species of the periphyton community differed between treatments in the experiment. In the control and N-enriched groups, the periphyton was dominated by Closterium sp. Ulotrichales sp. and Synedra sp., whereas in the P–enriched group, periphyton was dominated by Ulotrichales sp., Closterium sp., Oscillatoria sp. and Synedra sp. In the N + P enrichment group, the dominant taxa were Ulotrichales sp., Scenedesmus sp., Cosmarium sp., Closterium sp. and Synedra sp.

Phytoplankton biomass

No significant differences were observed between enrichment treatments in terms of biomass of phytoplankton measured as Chl a (repeated measurements ANOVAs, treatment effect; p > 0.05). Phytoplankton Chl a varied significantly with time (repeated measurements ANOVAs, time effect, p < 0.05). Analyses of the effects of nutrient treatments on each date indicated that enrichment with N + P stimulated the growth of phytoplankton, with Chl a concentrations higher at the end of the experiment (Day 56) compared to the controls (one-way ANOVA, treatment effect, p < 0.05; Figure 3).

Light

No significant differences of light intensity at the sediment surface were observed between enrichment treatments (repeated measurements ANOVAs, treatment effect; p > 0.05). Analyses of the effects of nutrient treatments on each date indicated that light intensity at the end of the experiment (Day 56) was higher in the P enriched treatment and lower in the N + P enriched treatment than in the controls (one-way ANOVA, treatment effect, p < 0.05). Figure 5 showed the relative light intensity between enrichment treatments.

Discussion

This study suggests that N-enrichment has a significant stimulatory effect on periphyton growth in systems dominated by submerged plants. This is consistent with other studies showing that periphyton biomass often increases after N enrichment (Havens et al., 1999; Smith and Lee, 2006).

Previous studies have recorded both positive and negative responses of periphyton to P enrichment. A stimulatory effect of P on periphyton growth was recorded in the early stages of this experiment, perhaps because the nutrient conditions stimulated the increase in periphyton biomass (Phillips et al., 1978). However, there are several suggested mechanisms for periphyton losses associated with P enrichment (Sand-Jensen and Borum, 1991; Gaiser et al., 2006). Some researchers believe that such declines can be attributed to increased phytoplankton biomass leading to the inhibition of periphyton growth through shading (Sand-Jensen and Borum 1991); others implicate the death of submerged plants in reducing the area of substrate available for periphyton to attach (Steward and Ornes, 1975). P enrichment has also been shown to stimulate the growth of other algae and bacteria within periphyton capable of reducing mat growth through competition or other inhibitory interactions (e.g. allelopathy and lytic bacteria) (McCormick et al., 2001). Finally, some studies imply that P enrichment increases macrophyte growth, leading to a corresponding decline in periphyton biomass and productivity (Grimshaw et al., 1997). In the present experiment, concentrations of phytoplankton were not higher in the P-enriched mesocosms than in the unenriched control group (Figure 3). Similarly, low periphyton biomass in the P-enriched mesocosms could not be explained by competition for P because no significant difference (p > 0.05) in TP concentration (Figure 1) was observed compared with the unenriched controls. Since no plants died in this study, the most likely reason for the decline in the periphyton biomass at the end of the experiment is the increased growth of macrophytes (Figure 4).

There is strong evidence that N + P enrichment in shallow lakes often leads to excessive periphyton growth (Biggs, 2000; Dodds, 2003). In this study, periphyton biomass responded quickly to nutrient addition in the early stages of the experiment. However, significant increases in phytoplankton levels were recorded during the later part of the experiment (Figure 3) and shading caused by increased phytoplankton biomass may have contributed to the decline of periphyton observed towards the end of the experiment (Sand-Jensen and Borum, 1991).

We also found major changes in the composition of the algal periphyton community. Previous research has shown that algal species in periphyton differ in their nutrient requirements (Borchardt, 1996). Although we do not have information on the specific needs of particular taxa identified in this study, the observed shifts in species composition are evidence of variable periphyton responses to nutrient enrichments in macrophyte dominated shallow lakes (Dodds, 2003; Larned, 2010).

Periphyton plays a key role in many aquatic ecosystems (McCormick et al., 1998). Human activities currently exert powerful influences on global cycles of both N and P (Galloway et al., 1995; Vitousek et al., 1997), and excessive nutrients delivered to shallow lakes can both inhibit and enhance of the growth of periphyton and change their community composition. The implications for many aspects of freshwater ecology are likely to be profound (Zhang et al., 2013), and greater attention should be paid to the effects of both N and P on periphyton when assessing the effects of nutrient enrichment in lake ecosystems.

Conclusions

In conclusion, N has a stimulatory effect on periphyton biomass. P or N + P have a similar effect during the early stages, but inhibitory effect at the end of the experiment due to increased phytoplankton concentrations inhibiting light availability (N+P additions) and to increased growth of V. natans (P additions). Shifts in species composition of periphyton also occurred in different nutrient enrichments.

Funding

The study was sponsored by the National Natural Science Foundation of China (No. 31100339), the Special Program of China Postdoctoral Science Foundation (No. 2012T50494) and the State Key Development Program of Basic Research of China (No. 2008CB418104).

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