Nitrogen (N) and phosphorus (P) are key limiting factors determining autotroph growth in both terrestrial and aquatic ecosystems (Elser et al., 2007; Conley et al., 2009). The N:P ratio of reactive N and P anthropogenic inputs to the biosphere has dramatically increased during the past half century, inducing significant alterations in the organisms and ecosystems of Earth (Peñulas et al., 2012; Peñulas et al., 2013; Tong et al., 2020).

In shallow eutrophic lakes, in-lake TN:TP ratios are often low in summer due to the high denitrification and strong P release at high temperatures (Xu et al., 2010; Xu et al., 2021; Søndergaard et al., 2013; Søndergaard et al., 2017; Shatwell and Köhler, 2019). From a resource availability perspective, excess N loading could directly increase the summer phytoplankton biomass via alleviation of temporal N deficiency, and this has been confirmed by numerous bioassays and mesocosm experiments (Xu et al., 2010; He et al., 2015; Kolzau et al., 2018). Nitrogen loading may also influence the internal P loading via feedback mechanisms between phytoplankton blooms and sediment P release (Xie et al., 2003; Gao et al., 2014; Chen et al., 2018). That is, a higher phytoplankton biomass due to summer N enrichment augments the P release from P-rich sediments via various mechanisms including increasing diffusion, bacteria activity, pH, and likely also degradation of settled algae (Xie et al., 2003; Gao et al., 2014; Chen et al., 2018; Ma et al., 2018; Ma et al., 2021). This released P, in turn, hinders a change of the system towards P limitation at persistent external N loading and, therefore, it creates a positive feedback loop benefitting phytoplankton growth (Figure 1). Nonetheless, high doses of nitrate, may also prevent P release by improving oxidation of the sediment in shallow lakes (Andersen, 1982; Boström and Pettersson, 1982). For instance, a long-term investigation of 22 Danish shallow lakes revealed absence of P release if in-lake nitrate concentrations exceeded about 0.5 mg L⁻¹ (Andersen, 1982). However, this pattern was not supported in studies of four Danish shallow eutrophic lakes where summer nitrate additions increased the P release from the sediment (Jensen and Andersen, 1992). Thus, the dual effect of oxidized nitrogen on sediment P release makes prediction of the effects of summer N loading in shallow lakes uncertain.

Excess N loading may also favor phytoplankton growth via reducing the fitness and biomass of herbivorous zooplankton (Trommer et al., 2017; Lorenz et al., 2019). For instance, mesocosm experiments found that N addition caused stoichiometric shifts in the biomass of primary producers towards higher N:P and C:P ratios, which lowered cladoceran and total zooplankton biomasses but favored the dominance of rotifers (Lorenz et al., 2019). In shallow lakes, although P has traditionally been considered the primary nutrient causing harmful cyanobacterial blooms, recent findings from laboratory studies and lakes throughout the world demonstrate that N can be important in controlling the timing, density, and toxicity of some non-diazotrophic cyanobacterial blooms (reviewed by Gobler et al., 2016). Beaulieu et al. (2013) assessed cyanobacteria blooms in 1,147 lakes and reservoirs with differing trophic status across the U.S. and found that the best predictors of these events were TN and water temperature. Multi-year observations from western Lake Erie in United States demonstrate that microcystin concentrations peak in parallel with inorganic N concentrations and are significantly lower during years of reduced inorganic nitrogen loading and concentrations (Gobler et al., 2016). Thus, in cyanobacteria-dominated shallow lakes, it can be inferred that summer N loading might create high cyanobacterial biomass and cyanotoxin concentrations. As numerous studies have recorded that bloom-forming cyanobacteria generally are a poor food resource for both cladocerans and copepods due to their low nutritional value, e.g., insufficient amounts of essential lipids like PUFAs and sterols (Caramujo et al., 2008; Martin-Creuzburg et al., 2008), poor manageability (long filaments or aggregate colonies), and toxicity (DeMott and Moxter, 1991; Kirk and Gilbert, 1992), the feedback between high cyanobacteria blooms and low zooplankton grazing driven by summer N loadings may produce another “vicious cycle” that expectedly boosts eutrophication (Figure 1).

To obtain a better mechanistic understanding of the effects of summer N loading on the resource and grazing control of phytoplankton growth in shallow eutrophic lakes, we conducted a 70-days mesocosm experiment involving contrasting N loadings at the shore of Lake Taihu (China), one of China’s best studied lakes and known for its serious non-diazotrophic cyanobacterial (mainly Microcystis spp.) blooms (Guo et al., 2007; Qin et al., 2019). Previous bioassay experiments and field monitoring studies have revealed that the phytoplankton in Lake Taihu is generally N limited or N, P co-limited during summer and fall (Xu et al., 2010; Xu et al., 2021; Paerl et al., 2011). Thus, our hypothesis was that with increasing N loading in summer, the phytoplankton biomass would increase due to the alleviation of N deficiency and higher sediment P release. We also expected that N addition would increase the biomass and dominance of grazing-resistant cyanobacteria and thereby reduce zooplankton herbivory, which, in turn, would favor phytoplankton growth via release of grazing control.

Understanding the ecological mechanisms that underlie eutrophication in response to nutrient loading has long been an urgent objective in limnological research (Smith and Schindler, 2009). We found that summer N addition increased not only the N levels but also P availability for phytoplankton growth. We also found that N addition lowered the zooplankton-phytoplankton biomass ratio and promoted the phytoplankton yield, expressed as the chlorophyll a to TP and TN ratios, indicating that N addition favored phytoplankton growth through reduction of zooplankton grazing. Accordingly, our study suggests that in eutrophic shallow lakes, N loading may increase the phytoplankton biomass in summer via both resource and grazer effects. Our study thus showed that decreasing N loading may lead to reduced eutrophication in summer in shallow lakes, which is consistent with long-term field investigations conducted in temperate shallow waterbodies, e.g., Lake Müggelsee in Germany and shallow meres in England (Moss et al., 2005; Shatwell and Köhler, 2019).

As there was no external P loading in our study, the enhanced water TP concentrations induced by N loading can only be attributed to sediment P release, which was partially supported by the greater temporal reductions of sediment Total-P in the N addition treatments than in the control (Figure 3). Promotion by N addition of TP concentrations has been widely observed in eutrophic shallow waterbodies where sediments serve as an important P source (Jensen and Andersen, 1992; Xie et al., 2003; Sagrario et al., 2005; He et al., 2015; Ma et al., 2018). The mechanisms behind include increases in diffusion, bacteria activity, and pH and in the degradation of settled algae via the feedback between an N-induced high algal biomass and sediment P (Gao et al., 2014; Ma et al., 2018; 2021). Some of these mechanisms were also observed in our study. For instance, we found a clear decline in the contribution of TDP to TP after Day 42 and promotion of pH by N addition (Figure 2), indicating a high potential of diffusion (Xie et al., 2003; Chen et al., 2018; Ma et al., 2021) and desorption of PO₄ ^3- from suspended surface sediments (Koski-Vähälä and Hartikainen 2001; Søndergaard et al., 2003; Jin et al., 2006; Gao et al., 2014). This was also evidenced by the high reduction of NH₄Cl-P and NaOH-P caused by N addition in our study (Figure 2; Figure 3). Moreover, in our study, sediment P release may also have been enhanced by increased strength of sediment resuspension (Søndergaard et al., 2003; He et al., 2015) as the ISS concentrations in the N addition treatments were higher than in the control mesocosms (Figure 2). A possible explanation of the stronger resuspension by N loading, through speculative, is that fish had grown bigger in the N addition mesocosms due to the higher food production, as seen in the study by Wang et al. (2017). However, it is important to note that the reductions of Total-P in top 4 cm sediment were much larger than the increases of TP in the water. This means that only a small fraction of P in the sediment surface was released into the overlying water and most of the P was transported to the deeper sediment layers, likely due to the increased DO (Figure 2) and potential oxidized thickness by high NO₃ ⁻ addition (Jensen and Andersen, 1992). Overall, due to the simultaneous increase of water TP concentrations, persistent N addition did not significantly increase either the TN: TP or the PN: PP ratios, with phytoplankton growth likely being co-limited by N and P during the experimental period (Figure 2). The external N loading caused high internal P loading, thereby snowballing the eutrophication process via increased resources, as shown in Figure 1.

In our study, N addition caused a dominance replacement of diatoms by cyanobacteria (Figure 4D), as also seen in similar previous mesocosm experiments and lake investigations (Beaulieu et al., 2013; He et al., 2015; Gobler et al., 2016). The increased cyanobacteria dominance caused by N loading may be attributed to the increased competitive advantage by the shading-resistant cyanobacteria (e.g., Oscillatoria) in turbid and nutrient-enriched environments (Havens et al., 2003). Interestingly, N addition increased, not reduced, the dominance of Anabaena, an N₂-fixing taxon, in our study. This contrasted with the common view that declining N inputs would favor N₂-fixing cyanobacteria in lakes (Schindler et al., 2008), but concurred with observation from studies of a large number of Danish shallow lakes (Jensen et al., 1994; Jeppesen et al., 2005), suggesting that the mechanisms underlying the occurrence of N₂-fixing cyanobacteria in lakes are complex (Kolzau et al., 2018; Shatwell and Köhler, 2019). In our study, both TN: TP and PN: PP ratios were not obviously changed by N addition (Figure 2), which indicates that the growth of Anabaena in N addition mesocosms was unlikely caused by N deficiency.

Nutrient availability, however, was not the only reason why N addition increased the summer phytoplankton growth as it could not explain the observed elevation of the Chl-a: TP and Chl-a: TN ratios following N addition. The increased amount of chlorophyll a per unit of N and P suggests that factors other than resources affected the accumulation of phytoplankton biomass. Combined with the low total and herbivorous zooplankton to phytoplankton biomass ratios in the treatments with N addition, the results indicate that phytoplankton was also favored by the reduced herbivory. Similarly, Bergström et al. (2021) found much lower zooplankton to phytoplankton biomass ratios after whole-lake fertilization with N along a gradient of lake DOC concentrations. It could be argued that the increased Chl-a to TP and TN ratios by N addition were not due to changes in zooplankton herbivory, but instead were a result of changes of cellular pigment contents between phytoplankton groups (Nicholls and Dillon, 1978). However, this is not the case as we found a close relationship between phytoplankton biomass and Chl a concentration (Figure 4C).

In our study, the decline of zooplankton grazing by N addition could not be attributed to increased fish predation as both indicators, average zooplankton body mass and cladoceran to copepod biomass ratio, showed no obvious changes (Figure 6). Thus, changes in phytoplankton properties driven by N addition are the most likely explanation of the decreased zooplankton grazing. Supporting this view, an obvious N-driven substitution of diatoms by filamentous cyanobacteria was observed (Figure 4D). The latter are generally considered as low-quality food for both copepods and cladocerans (Caramujo et al., 2008; Martin-Creuzburg et al., 2008). For instance, laboratory feeding experiments revealed that when the freshwater harpacticoid copepod Attheyella trispinosa grew on cyanobacteria (Leptolyngbya foveolarum and Cylindrospermum stagnale), its survival was reduced, and the development from the first copepodite stage to adult lasted 26% longer than when they grew on diatoms (Achnanthes lanceolate and Nitzschia perminuta) (Caramujo et al., 2008). Moreover, cyanobacteria, e.g., Oscillatoria and Anabaena, may also resist zooplankton grazing because of poor manageability due to their long filaments and potential toxicity (DeMott and Moxter, 1991; Koski et al., 1999; Engström-Öst et al., 2015). Although feeding experiments have confirmed that Mesocyclops and other copepods may feed size-selectively on filaments and discriminate against toxic strains (DeMott and Moxter, 1991: Kumar and Ramakrishna Rao, 1999; Sarma et al., 2013), it is energetically costly and compensated for by a reduction in population size (DeMott and Moxter, 1991; Koski et al., 1999; Engström-öst et al., 2015).

In recent decades, increasing TN: TP ratios resulting from an imbalance between N and P supplies have been observed in many freshwater shallow lakes (Tong et al., 2020). However, the necessity of controlling N inputs remains a subject of debate (Schindler et al., 2008; Conley et al., 2009; Paerl et al., 2011; Shatwell and Köhler, 2019). Our results from a shallow eutrophic mesocosm study clearly showed that N inputs disproportionally increased the summer phytoplankton biomass (both Chl a and Chl a to TP or TN ratios) due to the combined effects of resource and grazing control. Our study therefore implies that N loading may not only physiologically support phytoplankton growth, it also modifies the abiotic and biotic environment and thereby creates conditions that are beneficial for phytoplankton growth. Thus, alleviation of eutrophication in shallow eutrophic lakes requires a balanced and strategic approach to appropriately control both nutrients (N and P).