Nitrogen stable isotopes of ammonium and nitrate in high mountain lakes of the Pyrenees

Nitrogen stable isotopes of ammonium and nitrate in high mountain lakes of the Pyrenees M. Bartrons, L. Camarero, and J. Catalan Limnology Unit (CSIC-UB), Centre for Advanced Studies of Blanes (CEAB-CSIC), Accés Cala St. Francesc, 14, Blanes, 17300, Spain Received: 20 November 2009 – Accepted: 25 November 2009 – Published: 11 December 2009 Correspondence to: M. Bartrons (mbartrons@ceab.csic.es) Published by Copernicus Publications on behalf of the European Geosciences Union.


Introduction
Stable isotopes of nitrogen are increasingly used to trace food web relationships and the flow of matter in aquatic ecosystems (Fry, 2006).Beyond discussions of the fractionation values at each trophic level, a key aspect that is still a source of major uncertainty is the understanding of the variability in primary producers (Vander Zanden and  prefer to use primary consumers as the trophic level for reference (Post, 2000).Much of the variability of the nitrogen stable isotope ratio in primary producers comes from the specific source of nitrogen used and the biogeochemical pathways behind it.Ammonium and nitrate are the predominant inorganic nitrogen forms in natural waters.They are the main nitrogen sources for algal and bacterial growth and, consequently, for the food web as a whole.In this study, we examine the patterns of variability of the nitrogen stable isotope ratio of dissolved ammonium (δ 15 N-NH + 4 ) and nitrate (δ 15 N-NO − 3 ) in high mountain lakes.
Atmospheric deposition is the dominant source of N compounds in most high mountain catchments (Wookey et al., 2009).Although nitrate and ammonium are deposited in similar proportions (Camarero and Catalan, 1996), nitrate is the major N form that enters lakes from catchments (Hood et al., 2003).Indeed, ammonium is either highly retained in the catchment, by biological assimilation and sorption in mineral soil horizons, or transformed into nitrate by bacterial nitrification (Campbell et al., 2000).Within the lake, ammonium is abundant in the sediment porewater, due to organic matter mineralization, and diffuses to overlying water.NH + 4 is also excreted by organisms living in the lake as a by-product of their metabolic activity.In general, NH + 4 is preferred over NO − 3 as the N source by primary producers, as it does not require internal reduction (Falkowski, 1983).However, it is advantageous to use NO  (Mariotti et al., 1981), denitrification (Choi et al., 2001), N uptake by osmotrophs (Hogberg, 1997), ammonia volatilization (Hogberg 1997), organic matter mineralization (Lehmann et al., 2002), atmospheric N 2 fixation (Shearer and Kohl, 1986), assimilation (Wada and Hattori, 1978;Doi et al., 2004) and diffusive processes between compartments (e.g., sediment-water (Owens, 1987)).
However, not all the potential processes are equally relevant in a specific ecosystem as sources of variability in nitrogen stable isotope ratios.On the one hand, some processes require particular environmental conditions, for instance, concerning oxygen or light conditions.On the other hand, fractionation generally occurs if there is a large pool of substrate and the amount actually used in the process is small compared to the pool size (Hoch et al., 1992;York et al., 2007).
In mountain lakes there are two main large pools of nitrogen that can supply the water column, namely, soil catchment and sediment pools.They have contrasting characteristics.Supply from the catchment is mostly in nitrate form (Campbell et al., 2002), which originates directly from deposition or from catchment nitrification of ammonium.
Talus landscapes show higher nitrification and mineralization rates (Campbell et al., 2000), and eventually higher nitrate loadings to the lakes, than flatter and more vegetated areas which retain more nitrogen from deposition.In contrast, sediments are rich in ammonium from recycled organic matter.Nitrogen stable isotope ratios in the water column (δ 15 N-NO − 3 and δ 15 N-NH + 4 ) largely depend on the relative importance of the contribution of these two large pools and the characteristics of their use and recycling within the water column.
High mountain lakes are essentially oligotrophic ecosystems.Variability in catchment characteristics mostly depend on the altitudinal position (Korner, 2007), which also has a large influence on lake thermal characteristics (Catalan et al., 2009b).Lake area or depth, which are highly correlated between them in these systems (Catalan et al., 2009b), provide an additional source of variability in lake dynamics.However, while lake changes in altitude are relatively gradual, depth mostly divides lakes into two types of contrasting characteristics (Catalan et al., 2009a): deep lakes (ca.>15 m depth) are characterised by a large volume hypolimnion with a zone where processes of mineralisation of organic matter permanently predominate over photosynthetic ones; whereas shallow lakes (ca.<15 m depth), show a relatively small hypolimnion, highly affected by metalimnetic and boundary layer transport, and mostly phototrophic because of the high transparency of the water in these lakes (Buchaca and Catalan, 2008).Therefore, to investigate the variability in δ + 4 concentrations and their nitrogen isotopic ratio in three compartments during stratification, namely, epilimnetic water (EW), deep chlorophyll maximum water (DCMW), which typically occurs in the hypolimnion in these lakes, and sediment porewater (SPW).In addition, we also measured the isotopic composition of both compounds in atmospheric deposition.The aims of our study were: 1. to assess how the two main phototrophic areas in these lakes (i.e., EW, DCMW) differentiate in δ 15 N-NO − 3 and δ 15 N-NH + 4 depending on the lake position in the altitudinal gradient, and lake size; and 2. to what extent in each case it may depend on catchment (mostly NO − 3 ) and sediment loadings (mostly NH + 4 ), and water column processes.

Study sites
The lakes are located in three catchments within an area of less than 15 km radius.All basins are on granodioritic bedrock of the Maladeta batholith in the Central Pyrenees (NE Spain) (Fig. 1).The eight lakes cover an altitudinal gradient of 1068 m (Table 1).
This gradient reflects a shift in land-cover type from bare rock and thin and poor soils in high altitude areas to extensive vegetation and well-developed soils at lower sites.The tree line is at ca. 2200 m a.s.l.Other general physical and chemical characteristics of the lakes are relatively similar (Table 1), with the exception of depth.The lakes are dimictic, covered by ice for 5 to 8 months a year, oligotrophic, and with low ionic content and acid-buffering capacity.After the snow and ice cover melting, deep water column mixes and ammonium and nitrate concentrations homogenise throughout the water column (Catalan, 1992).As soon as lake water reaches 4 • C, the water column stratifies and phytoplankton blooms.During summer stratification, the photic zone is deeper than the thermocline.Light penetration is high and the Secchi disk depth may reach up to 20 m in the deep lakes and usually reaches the bottom in shallow lakes.Deep chlorophyll maximum develops at about 1.5 times the Secchi disk depth or just a few centimetres above the bottom in shallow lakes (Catalan et al., 2002).We used dissolved reactive silica (DRSi) as a proxy for water column productivity during early summer, as it is a primary nutrient for diatoms, we assumed that the lower DRSi the higher the seasonally accumulated primary productivity (Catalan et al., 2002).

Sampling
Deposition sampling was carried out fortnightly from 1 June to 25 August 2006.Samples were collected at 2240 m a.s.l., which corresponds to the centre of the lake altitudinal distribution.Deposition at this point was considered representative, due to the previously observed lack of significant differences in the chemistry of bulk precipitation within the altitudinal range in this area (Camarero and Catalan, 1996).Water samples for δ 15 N-NO − 3 analysis were field-filtered (pre-ashed Whatman GF/F, 0.7 µm pore size) into clean polypropylene hermetic bottles and refrigerated (−20 • C) until analysis, following Spoelstra et al. (2004).With this method, Spoelstra et al. (2004) found no detectable nitrate production or assimilation in the samples during a two-week incubation period, and, that atmospheric nitrate isotopic ratios were preserved.
Lakes were surveyed during the first week of August 2004.Within each lake, EW, DCMW and SPW were sampled for NO − 3 , NH + 4 , δ 15 N-NO − 3 and δ 15 N-NH + 4 analyses.EW was sampled at 1 m depth, DCMW at 1.5 times the Secchi disk depth or, alternatively, from 1 m above the bottom when the Secchi disk was still visible at the lake bottom.These two water column samples were collected by means of a Ruttner bottle and treated in the same way as described for deposition samples.SPW was sampled using a gravity core.Immediately, the first five centimetres were extruded and stored in a polypropylene hermetic bag without air.This bag was transported cold to the lab where it was frozen.SPW was finally obtained after defrosting by high-pressure squeezing-filtration with Whatman GF/F 0.7 µm pore size.NO  1969).NO − 3 was determined using a Waters Quanta 4000 Capillary Electrophoresis system.pH was measured with an Orion Research model 720 A pHmeter, with a low ionic strength filling solution in the pH electrode (KCl1M).Sediment loss on ignition (LOI) was carried out at 550 • C, as an estimation of the organic content of the sediment samples (Heiri et al., 2001).Acid neutralising capacity (ANC) was determined by Gran titration.Conductivity was determined with an Instran-10 conductimeter.Dissolved inorganic carbon (DIC) was determined on a Shimadzu TOC-5000 analyzer by IR absorption.Total phosphorus was determined according to the Malachite green method, with previous acid persulphate digestion (Camarero, 1994).Dissolved reactive silica (DRSi) was determined with the blue silicon-molybdenum method (Grasshoff et al., 1983).
Stable isotopic ratios of NH + 4 and NO − 3 were determined by the alkaline headspace diffusion methods of Holmes et al. (1998) and Sigman et al. (1997), respectively.For the δ 15 N of NH + 4 , magnesium oxide (MgO) was added to increase the pH and to convert NH + 4 to NH 3 .A filter pack consisting of an acidified (KHSO 4 2.5 M) glass fibre filter sandwiched between two Teflon filters was placed in each sample bottle in the field.Back in the lab, the samples were incubated on a shaker table at 40 • C for 14 days, to promote diffusion of NH 3 towards the filter pack.To determine the δ 15 N of NO − 3 , samples were initially boiled, with MgO added, and vaporised up to 100 mL to concentrate and drive off NH + 4 as NH 3 .Thereafter, Devarda's alloy was added to samples, which were placed in an oven at 60 • C for 48 h to reduce NO against NIST Standard Reference Materials (IAEA-N1, IAEA-N2, IAEA-N3 and IAEA-CH7) (Gonfiantini, 1978).A sample's preliminary isotope ratio was measured relative to reference gases analysed with each sample.These preliminary values were finalised by adjusting the values for the entire batch based on the known values of the included laboratory standards.The precision of replicate analyses of standards was 0.2%.Corrections to isotope values were calculated according to Holmes et al. (1998) and Sigman et al. (1997).Standards were analysed concurrently, with every set of samples analysed for NO − 3 and NH + 4 δ 15 N.They were prepared by adding an ammonium stock solution of known isotopic composition to the same bottles to achieve an ammonium concentration similar to that expected.We used deviations between the known concentration and isotopic composition of the standards and the values obtained after the procedure to correct the sample results.δ 15 N-NO − 3 values were also corrected for any N added due to Devarda's alloy contamination (Devarda's blanks), as described in Sigman et al. (1997).Deviations due to the different bottle volumes used during the analysis were also corrected according to Holmes et al. (1998).

General patterns
There were very distinctive patterns in NO − 3 and NH + 4 concentrations and stable isotope ratios among the compartments studied (Fig. 2).The largest concentrations were found in deposition and porewater, but stable isotope ratios were contrasting between them.p-value<0.001and r 2 =0.56, p-value<0.05,respectively) but not with DCMW ratios (Fig. 3).The stable isotope ratios were not fully independent of the compound concentrations (Fig. 4).For NO − 3 , ratio and concentration were correlated for both EW and DCMW (r 2 =0.50, p-value<0.05and r 2 =0.75, p-value<0.05,respectively).In the case of NH + 4 only EW correlated (r 2 =0.68, p-value<0.05).

Conclusions
In high mountain lakes there are two large pools potentially supplying nitrogen to the water column: catchment soils through runoff, and sediments through diffusion and seasonal mixing.Catchment nitrogen loading is mostly in form of NO − 3 (Hood et al., 2003), whereas internal loading from sediments is mostly NH + 4 .Interestingly, these two main nitrogen sources show contrasting δ 15 N values and, as a consequence, their influence on the lake water column nitrogen pool can be traced.From our results, it is remarkable the scarce variability of NH + 4 sediment porewater among lakes, both in terms of concentration and δ 15 N, despite the large changes in catchment characteristics (i.e., altitudinal gradient) and lake size.The SPW constancy contrasts with the high variability in the water column values.This fact suggests a powerful buffering mechanism within the nitrogen cycling from the water column to sediments and back to water column.Preferential use of NH + 4 by primary producers could be a candidate mechanism; however, δ 15 N-NH + 4 variability in the water column is extremely large (Fig. 2).
Therefore, the SPW convergence among lakes has to occur during the process of organic matter mineralisation in the sediments and subsequent NH + 4 diffusion to the water column.Because diffusion is slow, mineralization builds up high NH + 4 concentrations in SPW, and thus fractionation during the diffusive process tends to be high.Lake primary productivity, oxygen conditions and bottom temperature in these lakes can provide N isotopic fractionation, e.g., nitrification

−
3 concentrations in SPW were very low.As a result, SPW NO − 3 isotopic composition could not be determined as it would have required about 4 L of porewater.according to Sol órzano ( packs from these analyses were analysed for the nitrogen isotopic ratio on a Europa Integra mass spectrometer (Sercon) at the University of California Davis Stable Isotope Facility.During analysis, samples were interspersed with several replicates of at least two different laboratory standards.These laboratory standards, which were selected to be compositionally similar to the samples being analysed, had been previously calibrated atmospheric deposition were 40±17 µmol L −1 and 47±29 µmol L −1 , respectively, differences between them being not significant (t=−0.6,df=10, p-value>0.5),which was also the case for δ 15 df=10, p-value>0.5).Therefore, a tentative weighted average value of −3.43±1.13‰was estimated for atmospheric δ 15 N in the area.SPW NH + 4 concentrations were remarkably high, 98±72 µmol L −1 , whereas SPW NO

Table 1 .
Physical and chemical characteristics of the lakes.