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1. Aquat. Sci. (2010) 72:277–281 DOI 10.1007/s00027-010-0132-0 Aquatic Sciences RESEARCH ARTICLE The use of TN:TP and DIN:TP ratios as indicators for phytoplankton nutrient limitation in oligotrophic lakes affected by N deposition Ann-Kristin Bergstro ¨m Received: 26 June 2009/Accepted: 4 February 2010/Published online: 3 March 2010 ? Springer Basel AG 2010 Introduction Abstract chemistry affects nutrient limitation among phytoplankton. I show how TN:TP and DIN:TP ratios vary in oligotrophic lakes of Europe and the USA affected by different amounts of N deposition, and evaluate whether the DIN:TP ratio is a better indicator than the TN:TP ratio for discriminating between N and P limitation of phytoplankton. Data were compiled from boreal and low to high alpine lakes, and comprise epilimnetic lake water chemistry data (106 lakes) and results from short-term nutrient bioassay experiments (28 lakes). A large share (54%) of the oligotrophic lakes in the study had low TN:TP mass ratios (\25). DIN:TP ratios showed higher variability than TN:TP ratios. Variability in DIN:TP ratios was related to N deposition, but also to catchment characteristics. Data from short-term bioassay experiments with separate addition of N and P showed that the DIN:TP ratio was a better indicator than the TN:TP ratio for N and P limitation of phytoplankton. Phyto- plankton shift from N to P limitation when DIN:TP mass ratios increase from 1.5 to 3.4. High DIN:TP ratios, indi- cating P limitation of phytoplankton, were generally found in alpine lakes with low to moderate N deposition and in boreal lakes with high to very high amounts of N deposition. The stoichiometric composition of lake water In lakes, nitrogen (N) or phosphorus (P) often limits phy- toplankton growth. The absolute concentrations as well as the balance between fractions of N and P affect phyto- plankton growth rate and abundance (Guildford and Hecky 2000; Wetzel 2001) as well as community composition (Smith 1982; Hecky and Kilham 1988). Whereas the tro- phic status of lakes generally increases with increases in their total N (TN) and total P (TP) concentrations (Wetzel 2001), the balance between TN and TP (the TN:TP ratio) vary along the trophic gradient. For example, in a global analysis (data from 221 lakes in 14 countries) of the rela- tionship between the TN:TP ratio in the epilimnion and lake trophic status, Downing and McCauley (1992) found that the TN:TP mass ratio was as high as 240 in oligo- trophic lakes and as low as 0.5 in eutrophic lakes because of different TN:TP supply ratios. Oligotrophic lakes may have either high or low TN:TP ratios (Bergstro ¨m et al. 2005). Differences in ratios among lakes can be a reflection of high atmospheric N deposition, which raises TN:TP ratios without any significant influence on TP concentration (Kopa ´c ˇek et al. 1995; Bergstro ¨m et al. 2005; Elser et al. 2009a). Catchment characteristics also are critical determinants of N:P ratios in lakes. The extent of the vegetative cover, soil depth, hydrology, and climate affect nutrient uptake in terrestrial biomass and catchment retention of dissolved inorganic N (DIN), and thus deter- mines DIN transport to lakes (Kopa ´c ˇek et al. 2000; Hood et al. 2003; Burns 2004). These variables also determine fluxes of leached terrestrial dissolved organic carbon and N (DON), and associated inorganic P (Kopa ´c ˇek et al. 2000; Hood et al. 2003; Karlsson et al. 2005). The TN:TP ratio has often been used in determining whether phytoplankton growth in aquatic systems is mainly Keywords Boreal ? Alpine/high mountain ? Phytoplankton Nutrient limitation ? DIN:TP ? TN:TP ? A.-K. Bergstro ¨m (&) Department of Ecology and Environmental Science, Umea ˚ University, 901 87 Umea ˚, Sweden e-mail: ann-kristin.bergstrom@emg.umu.se

2. A.-K. Bergstro ¨m 278 were very similar across studies. Epilimnetic water was passed through a net to remove macrozooplankton. Bio- assay experiments were then conducted on the same day as the water was collected. The experimental design incor- porated four treatments (each with three replicates; i.e. control, N only, P only, and N ? P). Transparent con- tainers (1L, Vrede and Tranvik 2006); (10L, Morris and Lewis 1988) (20L, Bergstro ¨m et al. 2008) were incubated either in an indoor incubator (Vrede and Tranvik 2006) or in situ (Morris and Lewis 1988; Bergstro ¨m et al. 2008) under light saturated conditions. The duration of the incubations ranged from 4 to 7 days. The response of the phytoplankton was determined by analysis of the chloro- phyll a concentrations of each treatment at the end of the incubation period. Enrichment experiments may be interpreted either from category of response (i.e. yes or no for growth response in a given treatment), or may be based on magnitude of response (i.e. amount of chlorophyll produced relative to controls) (Elser et al. 2007, 2009a). The categorical approach is used here (Morris and Lewis 1988). Binary logistic regression analysis was used to estimate the probability of N and P limitation along a gradient with increasing TN:TP or DIN:TP mass ratios in the lakes. Thus, only incubations with responses to either N or P additions alone relative to the controls in the bioassays, and where lake concentrations of DIN, TN, and TP were available, were included. Based on these selection criteria, the dataset then comprised 44 incubations from 28 lakes (Morris and Lewis 1988; Vrede and Tranvik 2006; Bergstro ¨m et al. 2008, cf. Table 1). In total, 57 incubations were conducted in these lakes and 78% of the experimental results were consequently used in the analyses. In the 13 excluded incubations, there were no data on lake water chemistry, or there were no response to nutrient additions or only a response to N and P in combination. The TN:TP and DIN:TP ratios were used as covariates, and N or P limitation was the dependent variable, where N limita- tion = 0 and P limitation = 1. The threshold value for the analyses was set to 0.5 (i.e. 50% probability for N or P limitation). The analyses were further performed with the assumption that lake concentrations (i.e. DIN, TN, and TP) were proportional to supply rates of limiting resources. The regression analyses were performed with log-transformed data using SPSS Statistics 17.0. N or P limited (Downing and McCauley 1992; Guildford and Hecky 2000; Elser et al. 2009a). Because phyto- plankton can use not only dissolved inorganic P, but also P derived from enzymatic hydrolysis of dissolved organic P and stored P (Wetzel 2001), bioavailable pools of P have been assumed to correspond to TP (Lewis and Wurtsbaugh 2008). For N, highly available forms are ammonium and nitrate, which comprise the bulk of DIN (Wetzel 2001). Even in oligotrophic lakes, significant amounts of TN often consist of refractory DON compounds (Wetzel 2001; Lewis and Wurtsbaugh 2008). Therefore, variation in the ratio of DIN to DON may cause TN to be a poor indicator of bioavailable N. Thus, use of TN:TP ratios as indicators of N and P limitation for phytoplankton can be questioned (Lewis and Wurtsbaugh 2008). In this study, concentrations and mass ratios of TP, TN, and DIN were evaluated in oligotrophic lakes affected by varying degrees of atmospheric N deposition. Short-term bioassay experiments had been performed in some of the lakes and were used to evaluate whether TN:TP or DIN:TP ratios can be used as indicators for discriminating between N and P limitation of phytoplankton. The following hypotheses were tested: (1) oligotrophic lakes can have low TN:TP ratios, (2) DIN:TP ratios show higher variability than TN:TP ratios, and (3) the DIN:TP ratio is a better indicator than the TN:TP ratio for discriminating between N and P limitation of phytoplankton. Materials and methods Lake and stream water chemistry Lake water chemistry data were compiled for 106 lakes from oligotrophic boreal, sub-alpine lakes (Vrede and Tranvik 2006; Bergstro ¨m et al. 2008), low to high alpine Swedish lakes (Karlsson et al. 2001; Vrede and Tranvik 2006), and high mountain lakes in the Tatra Mountains, Slovakia and Poland (Kopa ´c ˇek et al. 2000) and in the Rocky Mountains, Colorado, USA (Morris and Lewis 1988) (Table 1). Nutrient concentrations are reported for the epilimnion as summer means or for a single month. Data on annual wet DIN deposition for each lake are from Bergstro ¨m and Jansson (2006) and Bergstro ¨m et al. (2008). TN:TP and DIN:TP ratios and phytoplankton nutrient limitations Results In the short-term bioassay experiments, nutrients were added to water from unproductive boreal to sub-alpine Swedish lakes (Vrede and Tranvik 2006; Bergstro ¨m et al. 2008) and high mountain lakes in the Rocky Mountains, Colorado, USA (Morris and Lewis 1988). The experiments The lakes ranged between 46–676 lg L-1TN and 1–20 lg L-1TP (Table 1) and TN was positively related to TP (r2= 0.26, F = 38, P\0.05, n = 133). The lakes in the Tatra Mountains, with highest N deposition, had much

3. TN:TP and DIN:TP ratios and nutrient limitation 279 Vrede and Tranvik (2006) higher TN concentrations than the other lakes. In Swedish boreal lakes (Dalsland, Bergslagen, and Va ¨sterbotten), TN:TP mass ratios declined with declining N deposition toward the north (from 30 to 16). The Swedish sub- to high alpine lakes in Norrbotten, with very low N deposition, had TN:TP mass ratios ranging between 21 and 24, whereas those located in the southern Swedish mountain area in Ja ¨mtland (boreal to sub-alpine lakes), with higher N deposition, had higher TN:TP ratios (36). The TN:TP ratios of the high Rocky Mountains lakes (24) were similar to those of the Norrbotten lakes, although they had somewhat higher N deposition. In the Tatra Mountains, high alpine rocky lakes had considerably higher TN:TP ratios (240) than forested lakes at lower elevation (25). In 54% of the lakes, the TN:TP ratios were less than 25. The DIN concentrations ranged from 3 to 609 lg L-1. There were generally higher DIN:TN ratios in alpine/high mountain lakes (0.14–0.90) in comparison to boreal/for- ested lakes (0.05–0.11). Among boreal/forested lakes, DIN concentrations were more than three times higher in the Tatra forested region (41 lg L-1) than in forested lakes of Sweden (Dalsland, Bergslagen, and Va ¨sterbot- ten). The alpine and high mountain lakes (Tatra, Rocky, Ja ¨mtland and Norrbotten), had higher DIN concentrations than many of the boreal lakes, although N deposition in these areas was lower, with the exception of the Tatra region. As a result, the DIN:TP ratios of alpine/high mountain lakes were higher than in many of the boreal/ forested lakes. The DIN and the DIN:TP ratios showed a higher variability than the TN and the TN:TP ratios (Table 1; Fig. 1). The binary logistic regression analyses indicated that the TN:TP mass ratio corresponding to a threshold of 50% probability for N or P limitation was 28, but the predict- ability of the regression model was poor (Nagelkerke r2= 0.20). The 25 and 75% probabilities for P limitation (or 75 and 25% probabilities for N limitation) corre- sponded to estimated TN:TP mass ratios of 19 and 41 (Fig. 1a). When using the DIN:TP ratios, the predictability of the model increased r2= 0.72). The DIN:TP mass ratio corresponding to an estimated 50% probability for P limitation (or N limitation) was 2.2, and the ratios corresponding to 25 and 75% probabilities for P limitation (or 75 and 25% probabilities for N limitation) were 1.5 and 3.4, respectively (Fig. 1b). Morris and Lewis (1988) Bergstro ¨m et al., (2008) Bergstro ¨m et al. (2008) Bergstro ¨m et al. (2008) Bergstro ¨m et al. (2008) Kopa ´c ˇek et al. (2000) Kopa ´c ˇek et al. (2000) Karlsson et al. (2001) Reference (kg N ha-1 deposition year-1) \1–1.5 \1–1.5 \2–2.5 13–15 13–15 DIN 2–3 2–3 8–9 4–7 Table 1 Chemical characteristics of the oligotrophic lakes and wet inorganic nitrogen (DIN) deposition of the lake regions (means with ± SD in parentheses) May, June, and Aug samples May, June, and Aug samples Jun and Aug samples Jun and Aug samples Month and samples Summer means Jun–Oct means Sept means Sept means Aug 3.8 (±3.9) 4.9 (±6.6) 2.3 (±2.1) 1.4 (±1.2) 1 (±0.5) 4 (±3.8) 1.6 (±1) DIN:TP 219 2.1 21 (±10) 24 (±10) 36 (±21) 30 (±11) 16 (± 4) 20 (±5) 29 (±7) TN:TP 240 25 609 (±110) 19 (±21) 41 (±20) 49 (±52) 22 (±16) 29 (±22) 14 (±11) 12 (±10) 15 (±9) (lg L-1) DIN 447 (±172) 173 (±120) 674 (±91) 260 (±61) 153 (±55) 120 (±40) 208 (±62) 217 (±61) 240 (±63) (lg L-1) TN 20 (±15) 3 (±2) 8 (±4) 14 (±4) 7 (±3) 4 (±2) 8 (±5) 9 (±2) 14 (±6) (lg L-1) TP points Data 18 1 24 23 20 1 8 9 9 considerably (Nagelkerke No. of lakes 21 12 12 12 11 8 9 12 9 Low to High Alpine Boreal to Sub-alpine TP total phosphorus, TN total nitrogen High Mountain Alpine rocks Lake type Subalpine Boreal Boreal Boreal Forest Discussion Rocky mountains Tatra mountains Tatra mountains A majority of the lakes had lower TN:TP ratios than the oligotrophic lakes reported in the global analysis by Down- ingandMcCauley(1992).Asinotherstudies(Kopa ´c ˇeketal. 1995; Bergstro ¨m et al. 2005; Elser et al. 2009a), some of the Va ¨sterbotten Bergslagen Norrbotten Norrbotten Ja ¨mtland Dalsland Region

4. A.-K. Bergstro ¨m 280 (A) relatively high N deposition, were fairly low, implying effective DIN retention in forested catchments. Thus, in addition to N deposition, catchment variables influenced DIN concentrations in the lakes. The DIN:TP ratios of the alpine/high mountain lakes were in many cases also higher than in boreal/forested lakes. Other studies have shown that TP concentrations in oligotrophic lakes generally increase with temperature, weathering rate, and terrestrial export associated with lea- ched organic C and N (Kopa ´c ˇek et al. 2000; Karlsson et al. 2005). In high elevation catchments, lake DIN concentra- tions and DIN:TP ratios decline with declining elevation as DIN becomes increasingly bound by terrestrial and aquatic biota. At the same time, organic N and TP release to water increase with enhanced leaching from more vegetated terrestrial surroundings (Kopa ´c ˇek et al. 2000; Hood et al. 2003). This explains why lakes in this study, which are from various elevations and climate regions with different N deposition, have similar TN:TP ratios but show a broad gradient in DIN:TP ratios (Table 1; Fig. 1). Because the TN:TP mass ratios explained less than 30% of the variation in bioassay growth response, it was a poor predictor for discriminating between N and P limitation of phytoplankton. Downing and McCauley (1992) reported that phytoplankton in lakes are significantly more fre- quently N than P limited when the TN:TP mass ratio is below 14. However, many of the lakes they considered had TP concentrations exceeding 30 lg L-1, which is above the lakes assessed in this study. Using the threshold TN:TP mass ratios for high probability of N limitation estimated by Guildford and Hecky (2000: 9); Downing and McCau- ley (1992: 14) and this study (19) as proxies for identifying N-limited conditions for phytoplankton, only 0, 16, and 30%, respectively, of the lakes examined here would be considered to be N limited (Fig. 1a), which is a consider- able underestimate (Fig. 1, phytoplankton in 66% of the lakes were N limited). In contrast to the TN:TP mass ratios, the DIN:TP mass ratios (Fig. 1b) explained a large part ([70%) of the var- iation in bioassay response. The results indicated that phytoplankton moved from clear N to clear P limitation with an increase in DIN:TP mass ratios from 1.5 to 3.4, in approximate accordance with observations in oligotrophic lakes in the USA (Morris and Lewis 1988; Axler et al. 1993). The DIN:TP mass ratio therefore appears to be a better predictor than the TN:TP mass ratio for discrimi- nating between N and P limited conditions, probably because it more closely reflects the actual amounts, and the supply rates, of bioavailable pools of N and P in the lakes (Lewis and Wurtsbaugh 2008). These threshold values should, however, be tested more by, for example, using response sizes as response variable (cf. Elser et al. 2007, 2009a), and preferentially by using 2 Predicted P limitation 1.5 Log TN:TP Predicted N limitation 1 P limited N limited 0.5 0.0 0.5 1.0 Log TP (µg L-1) 1.5 2.0 2.5 3.0 (B) 2 1.5 Log DIN:TP 1 Predicted P limitation 0.5 0 Predicted N limitation -0.5 P limited N limited -1 0.0 0.5 1.0 Log TP (µg L-1) 1.5 2.0 2.5 3.0 Fig. 1 Relationship between a TN:TP and, b DIN:TP and TP concentrations (lg L-1) in epilimnetic waters of the oligotrophic lakes for which bioassay data are available. In a dotted, solid, and dashed lines show TN:TP ratios corresponding to 25, 50, and 75% probabilities for P limitation from the binary regression analysis (TN:TP mass ratios of 19, 28, and 41, respectively). In b dotted, solid, and dashed lines show DIN:TP ratios corresponding to 25, 50, and 75% probabilities for P limitation, from the binary regression analysis (DIN:TP mass ratios of 1.5, 2.2, and 3.4, respectively). Note different scales for y-axis a and b variationinTN:TPratiosbetweenlakescouldberelatedtoN deposition (Table 1). Inthealpine/highmountainlakes,theDINcontributionto TNwasgenerallyhigherthaninboreal/forestedlakes.These findings reflect the poor retention of DIN in high-elevation catchments, where soils are generally poorly developed and vegetation cover is sparse (Williams et al. 1996; Kopa ´c ˇek et al. 2000; Hood et al. 2003). DIN concentrations were highest in the Tatra region with very high N deposition, whereas the DIN concentrations in the Swedish boreal lakes (Dalsland, Bergslagen, and Va ¨sterbotten), also with

5. TN:TP and DIN:TP ratios and nutrient limitation 281 Bergstro ¨m A-K, Jonsson A, Jansson M (2008) Phytoplankton response to nitrogen and phosphorus enrichment in unproductive Swedish lakes along a gradient of atmospheric nitrogen depo- sition. Aquat Biol 4:55–64 Burns DA (2004) The effects of atmospheric nitrogen deposition in the rocky mountains of Colorado and southern Wyoming, USA––a critical review. Environ Pollut 127:257–269 Downing JA, McCauley E (1992) The nitrogen:phosphorus relation- ship in lakes. Limnol Oceanogr 37:936–945 Elser JJ, Bracken MES, Cleland EE et al (2007) Global analysis of nitrogen and phosphorus limitation of primary producers in freshwater, marine and terrestrial ecosystems. Ecol Lett 10:1135–1142 Elser JJ, Andersen T, Baron JS et al (2009a) Shifts in lake N:P stoichiometry and nutrient limitation driven by atmospheric nitrogen deposition. 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Freshw Biol 20:315–327 Smith VH (1982) The nitrogen and phosphorus dependence of algal biomass in lakes: an empirical and theoretical analysis. Limnol Oceanogr 27:1101–1112 Vrede T, Tranvik LJ (2006) Iron constraints on planktonic primary production in oligotrophic lakes. Ecosystems 9:1094–1105 Wetzel R.G. (2001) Limnology: lake and river ecosystem. Academic Press, San Diego, pp 1006 Williams MW, Baron JS, Caine N et al (1996) Nitrogen saturation in the Rocky Mountains. Environ Sci Technol 30:640–646 data on lake water chemistry and results from bioassay experiments from a larger number of lakes. Based on the selection criteria used here, ca. 80% of the amount of available data was used in creating the binary regression models. Since analyses like these should involve testing of the model by applying it to data not used in creating the model, it remains to be evaluated how applicable these thresholds values are on a larger scale to lakes. When applied on the whole lake dataset in this study (Table 1), lakes with high probability of P limitation of phytoplankton (DIN:TP ratios [3.4, [75% probability) were mainly alpine and high mountain lakes with generally low to moderate enhanced N deposition, and boreal lakes with high to very high N deposition. Because the natural nutrient concentrations of oligo- trophic lakes are low, these lakes are very sensitive to small changes in supply of either N or P (Downing and McCauley 1992). The naturally low N:P ratios of oligo- trophic lakes can rise sharply if they receive high amounts of N deposition, and phytoplankton in lakes may conse- quently switch from being mainly N to mainly P limited. Evidence of such switches has been documented for lakes in the Colorado Front Range, USA (Williams et al. 1996; Baron et al. 2000; Elser et al. 2009b), the Sierra Nevada, USA (Fenn et al. 2003), the Tatra Mountains (Kopa ´c ˇek et al. 1995, 2000), Sweden (Bergstro ¨m et al. 2005, 2008) and at larger scales, many oligotrophic lakes in the north- ern hemisphere (Bergstro ¨m and Jansson 2006; Elser et al. 2009a). Acknowledgments ary regression analyses and other statistics, and Anders Jonsson and Mats Jansson for valuable comments on the manuscript. This study was supported by the Faculty of Science and Technology, Umea ˚ University. I thank Martin Berggren for help with the bin- References Axler RP, Rose C, Tikkanen CA (1993) Phytoplankton nutrient deficiency as related to atmospheric nitrogen deposition in northern Minnesota acid-sensitive lakes. 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