Effect of Storage on the IsotopicComposition of Nitrate in BulkPrecipitationJ O H N S P O E L S T R A , * , † S H E R R Y L . S C H I F F , †

D E A N S . J E F F R I E S , ‡ A N DR A Y G . S E M K I N ‡

Department of Earth Sciences, University of Waterloo,Waterloo, Ontario, Canada N2L 3G1 and EnvironmentCanada, National Water Research Institute, Burlington,Ontario, Canada L7R 4A6

Stable isotopic analysis of atmospheric nitrate is increasinglyemployed to study nitrate sources and transformationsin forested catchments. Large volumes have typically beenrequired for δ18O and δ15N analysis of nitrate in precipitationdue to relatively low nitrate concentrations. Havingbulk collectors accumulate precipitation over an extendedtime period allows for collection of the required volumeas well as reducing the total number of analyses neededto determine the isotopic composition of mean annualnitrate deposition. However, unfiltered precipitation left incollectors might be subject to microbial reactions thatcan alter the isotopic signature of nitrate in the sample.Precipitation obtained from the Turkey Lakes Watershedwas incubated under conditions designed to mimic unfilteredstorage in bulk precipitation collectors and monitored forchanges in nitrate concentration, δ15N, and δ18O. Results ofthis experiment indicated that no detectable nitrateproduction or assimilation occurred in the samples duringa two-week incubation period and that atmosphericnitrate isotopic ratios were preserved. The ability to collectunfiltered precipitation samples for an extended durationwithout alteration of nitrate isotope ratios is particularly usefulat remote study sites where daily retrieval of samplesmay not be feasible.

IntroductionHuman activities such as the burning of fossil fuels and theexcessive use of inorganic nitrogen (N) fertilizers by theagriculture sector have been implicated in recent increasesin atmospheric nitrogen deposition across the globe (e.g.,Galloway et al. (1)). While pristine areas experience nitrogendeposition levels of less than 5 kg N‚ha-1‚yr-1, the easternUnited States receives about 28 kg N‚ha-1‚yr-1 (2). Heavilypolluted areas in Europe have nitrogen deposition in excessof 75 kg N‚ha-1‚yr-1 (3). One of the consequences of theseelevated deposition levels is that forested ecosystems thathave historically been thought of as nitrogen-limited arebecoming nitrogen-saturated (4, 5). Nitrogen saturation candecrease forest health through increased water stress, reducedfrost tolerance, soil acidification, nutrient leaching, anddecreased fine root biomass (5).

Concerns over the long-term effects of elevated nitrogendeposition have led to an increase in the number of studiesusing stable isotope analysis to trace the fate of atmosphericnitrate deposition in forested ecosystems. The two sourcesof nitrate in most forested watersheds are (1) nitrate fromatmospheric deposition and (2) nitrate produced by nitri-fication in soils (microbial nitrate). Atmospheric and mi-crobial nitrate are isotopically distinct and therefore isotopicratios, particularly 18O/16O, can be used to study nitratesources and cycling in forested catchments (6-12).

Even at sites receiving elevated nitrate deposition, severalliters of water are often required for isotopic analysis ofatmospheric nitrate using the methods of Chang et al. (13)and Silva et al. (14). In pristine areas, 10 L or more ofprecipitation may be needed to determine both δ15N andδ18O values of nitrate. Typical bulk precipitation collectorsdesigned to sample water for chemical analyses might notaccumulate sufficient water from individual rain events todetermine nitrate isotope ratios; therefore, multiple collectorsare often required. However, recently developed nitrateisotope methods, which use denitrifying bacteria to convertnitrate to nitrous oxide (N2O) for determination of δ15N andδ18O values, reduce the required sample size by two ordersof magnitude (15, 16). Therefore, these new techniques willsignificantly decrease the collection period needed to ac-cumulate sufficient precipitation.

For many long-term catchment studies, it would beimpractical to analyze nitrate isotope ratios for each pre-cipitation event. A significantly less expensive and less labor-intensive method of determining the isotopic signature ofannual nitrate deposition is to combine individual precipita-tion samples acquired over an extended time interval. Thus,a mass-weighted mean δ15N and δ18O value is determinedfor the period of collection.

At remote sites, or where resources are limiting, bulkcollectors may accumulate precipitation for several days orweeks before filtering and preservation occurs. With thisapproach, microbial reactions that consume or producenitrate might alter the concentration and isotopic signatureof nitrate in unfiltered samples. Maximum alteration of nitratewould be expected during summer months when elevatedtemperatures promote greater microbial activity. Microbialalteration would lead to the determination of an erroneousisotopic composition for atmospheric nitrate, thus affectingsubsequent source-contribution calculations.

Several studies have investigated the temporal stabilityof ion concentrations in unfiltered bulk precipitation, withvarying conclusions with respect to nitrate. Galloway andLikens (17, 18) found no change in the ionic composition ofprecipitation samples stored at 25 °C for seven months andattributed the results to the preservation effect of low pH(<4.5). Karlsson et al. (19) did not see a change in nitrateconcentration over a seven-week period for samples kept at4 °C. In contrast, other studies have shown significant changesin nitrate levels after much less time for precipitation storedunder a similar range of conditions (20-22).

To date, the isotopic stability of nitrate in precipitationsamples has not been assessed. The goal of this paper wasto determine if a two-week storage period at ambienttemperatures alters the nitrate isotope composition ofunfiltered bulk precipitation samples.

Experimental SectionBulk precipitation samples were collected at the Turkey LakesWatershed (TLW), located about 50 km north of Sault Ste.Marie, Ontario, Canada. The TLW is located in the Great

* Corresponding author phone: (519)888-4567 ext. 7277; fax:(519)746-7484;e-mail: jspoelst@uwaterloo.ca.

† University of Waterloo.‡ National Water Research Institute.

Environ. Sci. Technol. 2004, 38, 4723-4727

10.1021/es030584f CCC: $27.50 2004 American Chemical Society VOL. 38, NO. 18, 2004 / ENVIRONMENTAL SCIENCE & TECHNOLOGY 9 4723Published on Web 08/14/2004

LakessSt. Lawrence forest region (23) and has been intenselymonitored since 1980 to investigate the biogeochemicalconsequences of human disturbance to a Canadian Shieldecosystem. The TLW is jointly operated by EnvironmentCanada, the Canadian Forest Service, and the Departmentof Fisheries and Oceans, in cooperation with the OntarioMinistry of Natural Resources.

The TLW climate is influenced by its leeward locationwith respect to Lake Superior. The TLW receives ap-proximately 1200-1300 mm of precipitation annually (1981-1997 mean) (24), about 35% as snow (25). Bulk atmosphericdeposition of nitrate to the basin averaged 5.0 kg N‚ha-1‚yr-1

from 1982 to 1996 with a mean nitrate concentration of 0.38mg N/L (26). A comprehensive physical, chemical, andbiological description of the TLW is presented by Jeffries etal. (27).

For the longer-term study of nitrate stable isotopes ratiosat the TLW (8), precipitation has been collected using aTeflon-coated, stainless steel funnel (2500 cm2) that drainsinto a 20-L bottle kept in a dark chamber below the collector.In this study, a custom-built bulk collector was used to acquirelarge volume precipitation samples from discrete rain eventsbetween May 2000 and June 2002. The collector consistedof a 110 × 90 cm plastic sheet that was suspended 70 cmabove the ground using a frame constructed of 1/2" PVCpipe. A 3-cm-diameter drain in the center of the plastic sheetdirected intercepted rain into a 20-L jug. The rain collectorwas erected in an open area prior to forecasted rain events.

Immediately following sample collection, a subsamplewas filtered to 0.45 µm and retained for chemical analysis.The remaining water was divided into three aliquots,providing a control sample and two samples for incubationexperiments. Control samples were immediately filtered to0.45 µm to remove particulate matter and bacteria and thenfrozen until processing to determine unaltered atmosphericnitrate isotope ratios. Filtered (0.45 µm) and unfilteredaliquots were incubated to evaluate the effect of microbialreactions on nitrate concentration and isotopic composition.Incubated samples were kept at 25-28 °C for two weeks tomimic storage conditions in bulk collectors during thesummer. The bottles were covered in a manner that preventedcontamination by dust, while allowing gaseous exchange withthe atmosphere. Following incubation, samples were filteredto 0.45 µm, subsampled for chemical analysis, and frozenuntil processing to determine nitrate isotope ratios.

The methods used to isolate nitrate and subsequentlyconvert it to silver nitrate were adapted from Chang et al.(13) and Silva et al. (14). Once thawed, sample volumes werereduced to less than 500 mL by evaporating at 90 °C so theycould be processed through the anion resin within oneworking day. Nitrate was isolated by dripping samplesthrough columns containing 2 mL of Bio-Rad, AG 1-X8, 100-200 mesh, anion-exchange resin in the chloride form at arate of approximately 2 mL/min. Dissolved organic matter(DOM) contains both oxygen and nitrogen and can thereforeinterfere with the isotopic analysis of nitrate. Chang et al.(13) recommended passing samples through a cation-exchange resin to protonate the DOM, thereby reducing itsaffinity for the anion resin. For TLW samples, we found thatmore DOM was removed without using the initial cationresin because the DOM was strongly retained by the anionresin, even during subsequent elution of the nitrate with 3M HCl. The solution containing the eluted nitrate wasneutralized with silver oxide. Following filtration to removethe resulting silver chloride precipitate, an excess of bariumchloride was added to precipitate oxygen-bearing anions suchas sulfate and phosphate. The elutant was refrigeratedovernight to allow the precipitate to develop, then filteredto 0.45 µm the following day. The filtrate was passed througha column containing 2 mL of Bio-Rad, AG 50W-X8, strong

cation-exchange resin (H+ form) at a rate of approximately2 mL/min to remove excess barium ions. The resultingsolution was re-neutralized with about 1 g of silver oxide andfiltered to 0.2 µm to remove the silver chloride precipitateand excess silver oxide. The final solution of silver nitratewas freezedried to yield a silver nitrate salt that was storedin amber vials to prevent photodegradation.

Nitrogen isotope ratios were determined using a slightlymodified version of the elemental analysis-isotope ratio massspectrometry (EA-IRMS) method described by Silva et al.(14). To determine 15N/14N ratios, 1 mg of silver nitrate samplewas combined with 2 mg of sucrose in tin capsules and loadedinto the autosampler of a Carlo Erba elemental analyzercoupled to a Micromass Isochrom mass spectrometer.Isotope analyses were performed at the EnvironmentalIsotope Lab (EIL) at the University of Waterloo and resultsreported in delta notation in units of per mil (‰) relative toatmospheric N2. Within each run, samples were bracketedby sets of three internal silver nitrate standards (δ15N ) +1.0,+13.8, and +18.4‰) that were previously calibrated againstinternational δ15N standards. Repeat analysis of standardsand selected samples yielded a precision of (0.3‰.

Oxygen isotope ratios were determined using an elementalanalysis-pyrolysis method modified from Mengis et al. (28).A Eurovector EA coupled to a Micromass Isoprime massspectrometer at the EIL was used to analyze 0.2 mg ofdesiccated silver nitrate loaded into tin capsules. The sampleswere pyrolyzed at 1290 °C in a ceramic pyrolysis tubecontaining glassy carbon and the resulting CO was analyzedfor 18O/16O isotope ratios. Repeat analysis of standards andsamples gave a precision of (0.8‰ or better. Multiple setsof three internal silver nitrate standards (δ18O ) +11.0, +28.0,and +45.2‰), which had been previously calibrated to IAEA-N3 using a value of +23.0‰), were run with each batch ofsamples and the results reported relative to Vienna StandardMean Ocean Water (VSMOW).

Results and DiscussionFollowing the two-week incubation, all treatments, filteredor unfiltered, showed no significant difference in nitrateconcentrations compared to initial values for each sample(Table 1). However, the isotopic composition of samples couldhave shifted without a change in nitrate concentration ifnitrate production and consumption occurred at the samerate.

Bacteria could produce nitrate in unfiltered precipitationsamples by nitrification of atmospheric ammonium (NH4

+)

TABLE 1. Nitrate Concentrations and Nitrate Isotope Ratiosfor Control and Incubated Bulk Precipitation Samples.Analytical Precisions for Nitrate Concentration, δ15N, andδ18O-nitrate Are (0.02 mg N/L, (0.3‰, and (0.8‰,Respectively

nitrate (mg N/L) isotope ratios

sample treatment initial final δ15N (‰) δ18O (‰)

BP-1-C filtered, frozen 0.80 -4.6 60.7BP-1-F filtered, incubated 0.80 0.76 -4.9 60.4BP-1-U unfiltered, incubated 0.80 0.80 -4.8 61.6BP-2-C filtered, frozen 0.27 -5.7 52.0BP-2-F filtered, incubated 0.27 0.28 -5.5 56.5BP-2-U unfiltered, incubated 0.27 0.27 -5.4 55.8BP-3-C filtered, frozen 0.23 -7.3 51.1BP-3-F filtered, incubated 0.23 0.22 -7.3 49.0BP-3-U unfiltered, incubated 0.23 0.24 -7.4 52.6BP-4-C filtered, frozen 0.34 -7.6 51.1BP-4-F filtered, incubated 0.34 0.35 -7.8 54.8BP-4-U unfiltered, incubated 0.34 0.33 -7.4 54.8BP-5-C filtered, frozen 0.26 -5.8 53.9BP-5-F filtered, incubated 0.26 0.26 -5.9 55.4BP-5-U unfiltered, incubated 0.26 0.26 -5.7 53.9

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present in the sample or ammonium produced from dissolvedor particulate organic matter (DOM or POM) decomposedduring the incubation period. Nitrifying bacteria stronglyfractionate against 15NH4

+ (ε ) -12 to -29‰) (29); therefore,the initial nitrate produced is markedly depleted in 15Ncompared to the ammonium source. However, the overallδ15N of nitrate produced by nitrification depends on severalfactors, including the δ15N of the ammonium source and thefraction of ammonium nitrified. As a larger proportion isnitrified, the δ15N of that nitrate approaches the δ15N of theoriginal ammonium pool. Atmospheric ammonium is gen-erally depleted in 15N compared to coexisting nitrate inprecipitation (30, 31). However, without nitrogen isotope datafor ammonium or dissolved organic nitrogen (DON) in TLWprecipitation, we cannot be sure that nitrification nitrate willbe significantly depleted compared to the original atmo-spheric nitrate.

Interpretation of δ18O values, in conjunction with δ15N,more reliably assesses the effects of nitrification. Nitrateproduced by nitrification is significantly depleted in 18Orelative to atmospheric nitrate. During the nitrification ofammonium, oxygen is added to the nitrogen molecule fromO2 and from water (32-34). Atmospheric O2 has a δ18O valueof +23.5‰ (35) or +24.2‰ if dissolved in water (36). Theδ18O of the water contribution largely depends on the δ18O-H2O of local precipitation. Given these controls, nitrateproduced by the nitrification of ammonium is expected tohave δ18O values ranging from about -5 to +15‰ (37).Therefore, the overall result of gross nitrification occurringin unfiltered precipitation samples would be (1) an increasein nitrate concentration and (2) a shift to lower δ18O (andlikely δ15N) values. Since nitrate concentrations did notchange and none of the treatments had 18O-depleted nitrateisotope ratios compared to the controls (Figure 1), weconclude that nitrate production by nitrification was neg-ligible during the incubation.

Loss of nitrate could also result in isotopic fractionation.Microbes that use nitrate as a nitrogen source must firstreduce it to ammonium. Assimilative nitrate reductionrequires energy, and thus ammonium is generally preferredby microorganisms as an initial nitrogen source (38). Am-monium and nitrate concentrations in the precipitationsamples were similar (Table 2). Therefore microbial am-monium assimilation could occur in samples; however, thisprocess would not affect nitrate concentrations or isotoperatios. Compared to nitrate, the isotopic composition ofatmospheric ammonium is probably more susceptible tomicrobial alteration during sample storage.

Several types of microorganisms have been shown tofractionate against 15N to varying degrees during nitrateassimilation (e.g., Hubner (39)). Although little informationexists on the associated isotopic discrimination for δ18O-NO3

- during nitrate assimilation, biochemical reactionsgenerally discriminate against heavy isotopes (37). Therefore,nitrate assimilation by microbes is expected to preferentiallyutilize isotopically light nitrate, progressively enriching theresidual nitrate in 15N and 18O. The overall effect of this typeof microbial activity in bulk precipitation samples would bea (1) decrease in nitrate concentration over time and a (2)likely concomitant increase in the δ15N and δ18O values ofthe residual nitrate.

Denitrification, a dissimilatory nitrate reduction pathway,converts nitrate to nitrous oxide and dinitrogen gas underanaerobic conditions. This microbial reaction causes adecrease in nitrate concentration while increasing δ15N andδ18O values of the remaining nitrate in a characteristic ratioof approximately 2:1 (40-45). However, denitrification wasnot expected to be a significant process affecting samples inbulk precipitation collectors because development of anaer-

obic conditions was limited by low DOC and POC concen-trations and the fact that oxygen could freely diffuse into thecollection vessels.

Isotopic analysis did not reveal a concomitant increaseof δ15N and δ18O values for atmospheric nitrate following thetwo-week incubation (Figure 1). In fact, none of the incubatedsamples had δ15N values that differed from the controls, whichwere filtered and frozen immediately after collection. Theabsence of a reduction in nitrate concentration, in conjunc-tion with no increase in δ15N values, strongly indicates thatneither assimilative nitrate reduction nor denitrificationoccurred in any of the incubated samples. We suggest thatthe approximately 4‰ difference between the δ18O valuesof the control and the treatments for BP-2 and BP-4 is theresult of slight DOM contamination of the silver nitrateproduced from the controls of both these samples.

Natural DOM consists of approximately 40% oxygen byweight (46) and is isotopically depleted in 18O (δ18O ) +8.2to +25.3‰) (47) relative to atmospheric nitrate. DOM alsocontains nitrogen, but at only about 1 wt % (46). Small

FIGURE 1. Nitrogen and oxygen isotope ratios of control (4), filteredand incubated (b), and unfiltered and incubated (9) bulk precipita-tion samples for five separate rain events. Horizontal and verticalbars indicate the precision of δ15N ((0.3‰) and δ18O-nitrate((0.8‰) analyses.

TABLE 2. Selected Chemical Parameters for Bulk PrecipitationSamples at the Time of Collection

sample NH4+ (mg N/ L) NO3

- (mg N/L) SO42- (mg/ L) DOC (mg C/ L)

BP-1 0.74 0.80 4.12 1.40BP-2 0.31 0.27 1.51 1.52BP-3 0.27 0.23 1.76 0.60BP-4 0.40 0.34 1.68 3.27BP-5 0.29 0.26 1.51 2.24

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amounts of DOM contamination that interfere with δ18O-nitrate analysis would not necessarily contain enoughnitrogen to shift δ15N-nitrate values. Other common oxygen-bearing ions such as sulfate, phosphate, and bicarbonate,which could cause a shift in the measured δ18O withoutaffecting δ15N values, are quantitatively removed duringnitrate collection by anion exchange and its subsequentconversion to silver nitrate (14).

The effectiveness of DOM removal from water samplesprior to nitrate isotope analysis is dependent on the physicaland chemical properties of the DOM and therefore can varywith sample type and research site (13). Approximately 85%of the DOM in samples collected from the TLW is removedby the combined effects of permanently binding to the anion-exchange resin and flocculation during conversion of aqueousnitrate to a silver nitrate salt (Spoelstra, unpublished data).The incubation period might have altered the chemical andphysical characteristics of the DOM in the treatment samples,resulting in a higher proportion of the DOM being removedand less or no contamination of the resulting silver nitratefor incubated samples compared to the controls. BP-4 andBP-2 had the highest and third-highest initial DOC concen-trations of the five precipitation samples, 3.3 and 1.5 mgC/L, respectively (Table 2). The difference between δ18Ovalues of treatment and control samples for BP-2 and BP-4,without a change in nitrate concentrations or δ15N values,is not consistent with a microbial effect. As a result, we suspectthat the controls have been shifted slightly to lower δ18Ovalues relative to incubated samples. Our results furtherunderscore the need for DOM removal prior to δ18O analysisof atmospheric nitrate, even at relatively low DOC concen-trations.

Since 1995, bulk precipitation samples have been collectedand processed for nitrate isotope ratios as part of a studyusing stable isotope techniques to investigate nitrate sourcesand cycling at the TLW (8). After a two-week collection period,samples are retrieved, filtered, and frozen until furtherprocessing at the University of Waterloo to determine δ15Nand δ18O values of nitrate. Although precipitation is in thedark while in the collector, the sample remains unfiltered atdaytime temperatures that often exceed 25 °C during thesummer. Results of the incubation experiment presented inthis paper confirm that the isotope signature of atmosphericnitrate is not compromised by current precipitation collectionmethods employed at the TLW.

The length of time that unfiltered precipitation can remainin collectors without alteration of nitrate isotope ratios islikely a function of temperature and initial sample chemistry.Precipitation chemistry can be influenced by local pollutionsources and land use. Winds at the TLW are typically fromthe west to southwest, coming across Lake Superior, andland dominated by forest cover. Therefore, low nutrient levelsin TLW precipitation could be one reason nitrate concentra-tion and isotopic composition did not change during thetwo-week incubation. Bacterial growth in TLW bulk pre-cipitation might also be limited by elements such asphosphorus and labile organic carbon and by the low pH(mean of 4.3) (48). Sites that are downwind of agriculturalareas with disturbed soils likely receive higher concentrationsof nutrients, especially in particulate form, and thereforecould be more susceptible to microbial alteration (e.g., Pedenand Skowron (20)). Windblown soil particles are expected tohave high bacteria counts that could inoculate bulk pre-cipitation samples and expedite nitrogen transformations.Precipitation collected as throughfall is enriched in certainnutrients and labile organic carbon because trees, especiallyconifers, are efficient collectors of dry deposition (49).Michalzik et al. (22) found that nitrate and ammoniumconcentrations in bulk precipitation were more stable thanthose of throughfall samples.

The effects of storage on the isotopic composition of otherions in bulk precipitation remains to be investigated. Isotopicdata for nitrate presented in this paper, in conjunction withprevious studies showing little or no change in sulfateconcentrations (17-21, 50), suggest that the isotopic ratiosof less biologically active ions such as sulfate also remainunmodified after extended storage times. Conversely, theresults of previous concentration-based studies (19, 22, 50,51) indicate that nutrient compounds such as ammoniumlikely require prompt preservation to avoid isotopic alterationby microorganisms.

The ability to accumulate precipitation over an extendedtime period without the alteration of nitrate δ15N or δ18Ovalues reduces the total number of samples to be processedand is especially relevant for precipitation collection at remotesites where immediate sample retrieval is not always possible.We caution that nitrate isotope ratios may be more suscep-tible to alteration where higher nutrient concentrations inprecipitation are less limiting to bacterial growth or wheretemperatures exceed those used in this study.

AcknowledgmentsWe thank R. Elgood, Environmental Geochemistry Lab (EGL),University of Waterloo, and G. LaHaie and R. Neureuther,Environment Canada, for logistical support. We also ac-knowledge the assistance of K. Sentance, J. Smith, and P.Bryk, EGL, during sample collection and preparation. Fundingfor this research was provided by Environment Canada -National Water Research Institute (NWRI) and through aNatural Sciences and Engineering Research Council ofCanada (NSERC) Discovery grant to Dr. S. L. Schiff.

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Received for review August 7, 2003. Accepted July 1, 2004.

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