Nitrate stable isotopes: tools for determining nitrate...

Nitrate stable isotopes: tools for determiningnitrate sources among different land uses in theMississippi River Basin

Cecily C.Y. Chang, Carol Kendall, Steven R. Silva, William A. Battaglin,and Donald H. Campbell

Abstract: A study was conducted to determine whether NO3– stable isotopes (δ15N and δ18O), at natural abundance

levels, could discriminate among NO3– sources from sites with different land uses at the basin scale. Water samples

were collected from 24 sites in the Mississippi River Basin from five land-use categories: (1) large river basins(>34 590 km2) draining multiple land uses and smaller basins in which the predominant land use was (2) urban,(3) undeveloped, (4) crops, or (5) crops and livestock. Our data suggest that riverine nitrates from different land useshave overlapping but moderately distinct isotopic signatures. δ18O data were critical in showing abrupt changes in NO3

–

source with discharge. The isotopic values of large rivers resembled crop sites, sites with livestock tended to have δ15Nvalues characteristic of manure, and urban sites tended to have high δ18O values characteristic of atmospheric nitrate.

Résumé : Notre étude a cherché à déterminer si les isotopes stables de NO3– (δ15N et δ18O), aux niveaux naturels

d’abondance, permettent de distinguer les sources de NO3– provenant d’utilisations différentes des terres à l’échelle du

bassin hydrographique. Des échantillons d’eau ont été prélevés à 24 sites du bassin hydrographique du Mississipi danscinq catégories d’utilisation des terres: (1) de grands bassins hydrographiques de rivières (>34 590 km2) drainant desterres à utilisations multiples, ainsi que des bassins plus petits dans lesquels les terres étaient surtout (2) urbanisées,(3) non développées, (4) agricoles ou (5) agricoles et pâturables. Nos données laissent croire que les nitrates des riviè-res provenant de terres à usages différents possèdent des signatures modérément distinctes, bien qu’il y ait des chevau-chements. Les données de δ18O sont importantes pour indiquer les changements brusques de sources de NO3

– dans ledébit. Les valeurs isotopiques des grandes rivières ressemblent à celles des régions agricoles; les sites où il y a élevageont des valeurs de δ15N caractéristiques du fumier; les sites urbains ont de fortes valeurs de δ18O caractéristiques desnitrates atmosphériques.

[Traduit par la Rédaction] Chang et al. 1885

Introduction

The Mississippi Atchafalaya River Basin (MARB) is thethird largest river basin in the world, draining 41%(3 208 700 km2) of the conterminous United States. Nitro-gen (N) delivered from the MARB by the Mississippi Rivercontributes to the formation of a hypoxic zone in the Gulf ofMexico that can exceed 17 000 km2 (Rabalais et al. 1999).The Mississippi River delivers 1.6 million metric tons (t) ofnitrogen per year to the Gulf of Mexico, 61% as nitrate(Goolsby et al. 1999). Nitrate concentrations in the Missis-sippi River and the resulting N flux to the Gulf have tripledsince 1965 (Justic et al. 1995; Goolsby et al. 1999; Alexan-der et al. 2000). Between 1955 and 1996, the average flux ofnitrate increased by 19 000 t per year, with most of the in-

creases occurring between 1970 and 1983 (Goolsby et al.1999).

Nonpoint sources contribute ~90% of the nitrogen andphosphorus in the MARB. Fertilizer and mineralized-soil or-ganic N contribute ~50% of the total N flux to the Gulf ofMexico, with animal manure contributing ~15%. Though thecontribution of animal manure to total N flux has decreased,the dispersal pattern has changed from being highly dis-persed to being highly concentrated (Goolsby et al. 1999).Large portions of manure-derived nitrogen can be lost (20–70%) during storage, handling, and application (Goolsby etal. 1999).

Much (~35%) of the nitrogen carried by the MississippiRiver originates in the middle Mississippi Basin, which in-cludes Iowa, Illinois, and Indiana. This region composes

Can. J. Fish. Aquat. Sci. 59: 1874–1885 (2002) DOI: 10.1139/F02-153 © 2002 NRC Canada

1874

Received 9 November 2001. Accepted 29 October 2002. Published on the NRC Research Press Web site at http://cjfas.nrc.ca on10 January 2003.J16605

C.C.Y. Chang,1 C. Kendall, and S.R. Silva. U.S. Geological Survey, Water Resources Division, 345 Middlefield Road, MenloPark, CA 94025, U.S.A.W.A. Battaglin and D.H. Campbell. U.S. Geological Survey, Water Resources Division, Denver Federal Center, Denver, CO80225, U.S.A.

1Corresponding author (e-mail: [email protected]).

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only 9% of the total area but has the largest average total ni-trogen yields, ranging from 1000 to 3000 kg·km–2·year–1

(Goolsby et al. 1999). During flooding, the flux can doublebecause of the large reservoir of nitrogen stored in the soil.

The formation of the hypoxic zone in the Gulf of Mexicohas provided incentive for determining the fate of anthro-pogenic N at large spatial scales. At the basin scale, N fluxis strongly correlated to discharge and season. However, therelative contributions of fertilizer, sewage–livestock waste,and soil nitrate as functions of discharge, season, and nitrateconcentration are not well known.

Estimates of nitrate loading and whether nitrate concen-trations are responsive to changes in discharge can be ob-tained from concentration and discharge data; however, thesedo not provide information about sources. The δ15N of ni-trate can be used to distinguish between sources and evenbetter separation can be obtained if δ18O is used in conjunc-tion with δ15N (Fig. 1) (Kendall 1998). This is because theisotopic fingerprints of atmospheric, fertilizer, soil, and ma-nure-derived nitrate often have sufficiently distinct signa-tures to permit separation. Furthermore, analysis of δ18O canbe particularly useful in identifying denitrification, becausewhen it occurs, the remaining nitrate becomes heavier inboth δ15N and δ18O (2:1 for δ15N:δ18O ratio) (Amberger andSchmidt 1987; Böttcher et al. 1990).

Traditionally, isotope values are plotted against discharge,nitrate concentrations, and time to determine if there aresource-related changes associated with events (i.e., storms)and season, the latter of which may be related to anthro-pogenic activities such as fertilizer application and crop har-vest. If more than one isotope is used, then isotopes areplotted against each other (as shown in Fig. 1).

For example, both isotopes were used to show ammonia ni-trification (Aravena et al. 1993) and denitrification (Aravenaand Robertson 1998) in a septic plume. Nitrate sources andnitrification were identified in ground water adjacent topoultry farms and raspberry fields (Wassenaar 1995). Onslightly larger scales, seasonal and discharge-related changesin the contributions of microbial and atmospheric nitrate instreams and springs were observed in undisturbed water-sheds (Burns and Kendall 2002; Campbell et al. 2002;Sickman et al. 2003). On still larger scales, both isotopeswere used to compare watersheds draining forested, urban,and agricultural areas (Mayer et al. 2002).

The objective of this study was to determine whether theδ18O and δ15N of nitrate could discriminate between nitratesources from sites with different land uses. This is one of thefirst studies to apply these techniques at the basin scale. To-wards this goal, water samples were collected from 24 sitesin the Mississippi River Basin that represent five land-usecategories: (1) large river basins (>34 590 km2) draining mul-tiple land uses and smaller basins in which predominant landuse was either (2) urban, (3) undeveloped, (4) crops, or(5) crops and livestock (Fig. 2).

Materials and methods

Study sitesDischarge and other physical measurements were made in

1997 and 1998 from 24 sites located along streams andrivers in the MARB (Fig. 2). Most of the samples were col-

lected in 1998. Included were samples from the U.S. Geo-logical Survey (USGS) National Stream Quality AccountingNetwork (NASQAN) and National Water Quality Assess-ment (NAWQA) sites as well as from other USGS monitor-ing sites. For a complete description of the study plan, thereader is referred to Battaglin et al. (1997, 2001a).

The eight large river sites had mixed land uses (Fig. 3).Sixteen sites draining areas <2500 km2 were selected to rep-resent one of four land-use categories (Fig. 4a) (Battaglin etal. 2001a): land in row-crop agriculture, land with both live-stock and row-crop agriculture, urban land, and undevelopedland. Classification was made on the basis of dominant landcover (Fig. 4b) and manure production (Fig. 4c). Thoughland cover was more mixed and fragmented than these fourcategories, an “average” classification was necessary to pro-vide a sufficient number of samples in each category. For thepurposes of simplification, we grouped the 10 land coversinto four categories, e.g., rangeland, wetland, and fallowpasture are shown as undeveloped and row crop and live-stock are shown as agriculture in Fig. 4b. Sites where rowcrops were grown and where manure production was>3200 kg·km–2·year–1 were classified as livestock sites (Elk,Rattle, South Fork, and North Dry Creek) (Fig. 4a). Elk wasclassified as a livestock site because it had one of the highestmanure loads (4887 kg·km–2), despite that fact that the pre-dominant land cover was forested and undeveloped. Twosites were classified as being dominantly urban (ShingleCreek and Little Buck), two as dominantly undeveloped(Namekagon and Dismal River), and the remaining eight asdominantly row-crop sites (Fig. 4a).

Unfortunately, because of procedural problems, many ofthe 1997 samples were not processed for stable isotope anal-ysis. In 1998, four samples were collected at most sites. The

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Chang et al. 1875

Fig. 1. Isotopic signatures for different nitrate sources. FromKendall (1998).



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1876 Can. J. Fish. Aquat. Sci. Vol. 59, 2002

first set of samples was collected in spring, generally beforethe application of fertilizers to local fields. The second andthird sets of samples were collected in early summer after50% or more of the crops in the basin had been planted andduring significant runoff events (after rainfall), respectively.The fourth set of samples was collected in fall, generally af-ter crop harvest. Hence, the timing of sample collection var-ied across the study area as a function of planting season,climate, and weather.

Physical dataWhen appropriate, depth-integrated samples were collected

from three or more verticals per site using USGS protocols.Specific conductance, pH, discharge, and water temperaturewere measured at the time of sample collection. A completedescription of the sample collection methods, measurementsof physical properties, land-use data, and chemical analysis isgiven in Battaglin et al. (2001a).

Nutrients, major ions, and stable nitrate isotopesNitrate (actually nitrite and nitrate) was analyzed by auto-

mated colorimetric procedures with a N-detection limit of0.05 ppm. Nitrate stable isotope sample collection and analy-sis were done following the procedures of Chang et al. (1999)and Silva et al. (2000). Nitrogen isotope values (δ15N) are re-ported in per mil (‰) relative to atmospheric air defined as0‰. Oxygen isotope values (δ18O) are reported relative to theVienna standard mean ocean water (VSMOW), also definedas 0‰. The precision for analysis of laboratory standards(standard deviation) for δ15N of KNO3 is ±0.05‰ (n = 10)and for δ18O of AgNO3 is ±0.2‰ (n = 19). For simulatedfield samples (n = 12), the standard errors were ±0.07‰ forδ15N and ±0.36‰ for δ18O (Chang et al. 1999).

Eighteen pairs of concurrent replicated field samples (du-plicate and triplicate) were used to determine sampling andlaboratory precision for NO3

– concentration measurements,as well as for δ15N and δ18O analysis. The standard errors ofthe differences between the pairs of samples were ±0.11 ppmfor N-NO3

– concentration, ±0.22‰ for δ15N, and ±0.58‰ forδ18O. In this study, only samples with both δ15N and δ18O val-ues are presented. A comprehensive listing of all data can befound in Battaglin et al. (2001a).

Fig. 2. Site map showing the Mississippi River Basin, large river sites, and smaller basin sites. From Battaglin et al. (2001a).



Results

The range of δ18O values (+3.1 to +43.3‰) exceeded therange of δ15N values (–1.4 to +12.3‰) (Fig. 5). Several pat-terns emerged when data were coded by land use. Nitrateconcentrations of large river sites were low (<4 ppm), butnot as low as those at urban and undeveloped sites (<2 ppm)(Fig. 6). In contrast, nitrate concentrations at agriculturalsites ranged from near the detection limit (0.05 ppm) to18 ppm (Fig. 6). At sites where concentrations were always

<4 ppm, the range of δ15N and δ18O values were broad andcould not be predicted from nitrate concentration (Figs. 7and 8).

The isotopic values of sites with similar land use tended tocluster into overlapping but moderately distinct ranges (Figs. 5and 9). Excluding one high δ18O value (+43‰) from a cropsite, large river isotopic values (δ15N from +3 to +12‰; δ18Ofrom +3 to +24‰) resembled those of crop sites (δ15N from +3to +12‰; δ18O from +3 to +21‰) (Figs. 5 and 9). The isotopicvalues of agricultural sites were characteristic of mixtures offertilizer, soil, or manure-derived nitrate (Fig. 1). At livestocksites, the ranges of δ15N and δ18O values were similar (+5 to+13‰) (Fig. 5). A larger proportion (70%) of livestock siteshad nitrate concentrations <8 ppm compared with sites withonly crops (45%) and tended to have higher δ15N values for agiven nitrate concentration than “crop-only sites” (Fig. 7). Forexample, of the 10 agricultural samples with nitrate concentra-tions <2 ppm, four of the five highest δ15N values were fromElk, a livestock site. Of the 17 samples with nitrate concentra-tions ranging from 4 to 8 ppm, only three crop samples hadδ15N values equal to or higher than those from livestock sites.The nitrate concentrations of these samples were ~8 ppm: ontwo occasions at Little Cobb, δ15N = +12‰ (sampled in tripli-cate) and +9‰; Indian Creek, δ15N = +6.7‰ (sampled in dupli-cate). Higher δ15N values are characteristic of manure-derivednitrate, and these results suggest that at lower nitrate concentra-tions, the nitrate source at livestock sites is predominantly ma-nure, rather than fertilizer (Fig. 1). Finally, the range of δ15Nvalues for urban (–1.2 to +6‰) and undeveloped sites (–1.4 to+8.0‰) overlapped and was lower than for large river and ag-ricultural sites. The relatively low δ15N values of urban sitesand high range of δ18O values (+4 to +31‰) are characteristicof atmospheric nitrate (Fig. 1). Though synthetic nitrate fertil-izer could also produce relatively heavy δ18O (Amberger andSchmidt 1987), it is used much less commonly than ammo-nium fertilizers (Terry and Kirby 1999). These urban isotopicvalues are also inconsistent with nitrate produced from sewage.This is because nitrate δ18O should be in the range of –10 to+10‰ if two oxygens come from water (–25 to +4‰) and oneoxygen from the atmosphere (+23‰) (Amberger and Schimdt1987).

Large riversWhereas as NO3

– concentrations and discharge were higherin spring than at other times at Grand Chain and Thebes,there were no seasonal trends for δ15N or δ18O at any largeriver sites (data not shown). The range of isotopic values forlarge river sites was similar to those of crops sites (Figs. 5and 9) but showed no consistent correlation between NO3

–

concentrations and δ15N or δ18O values on a site-by-site ba-sis (Figs. 7 and 8). For example, although δ15N values ap-peared to increase as NO3

– concentrations increased at somesites (Yazoo, Grand Chain, Thebes, St. Francisville), each ofthem had one or two data points that did not follow this pat-tern. At other sites, δ15N values were relatively constant de-spite changes in NO3

– concentration (e.g., Hermann), or δ15Nvalues were chaotic (e.g., Clinton). Although the δ18O valuesof large river sites appear to decline as NO3

– concentrationsincreased (Fig. 8) and were better defined than any trend com-ing from a similar comparison between δ15N values and NO3

–

concentrations (Fig. 7), this correlation was weak (r2 = 0.34;

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Chang et al. 1877

Fig. 3. Large river (a) drainage area, (b) percent land cover, and(c) manure loads. Estimates of relative land cover and manureloads for Belle Chase (Belle) and Vicksburg (Vicks.) are notavailable. (b) Solid bars, agriculture; shaded bars, range; andopen bars, forest. Data are from Battaglin et al. (2001a). Num-bers above the drainage area bars indicate the actual drainagearea in km2. See Fig. 2 for full names.



regression analysis) and was even more poorly defined atindividual sites. The lack of correlation within a site demon-strates that apparent trends on lumped data sets can be mis-leading, and it is important to make comparisons on a site-by-site basis. Furthermore, we caution against averagingδ15N or δ18O values because the unique relation between thetwo isotopes used to identify the source of nitrate is lost.

Agricultural sitesTaken collectively, there was an inverse relation between

δ15N and NO3– concentrations for agricultural sites (crop and

crop and livestock sites) (r2 = 0.41) (Fig. 7). As NO3– con-

centration increased to 18 ppm, δ15N values declined from arange of +13.4 to +7.4‰ to a range of about ~+4 to +6‰. Astrong inverse correlation between δ15N and NO3

– concentra-tions at individual sites was apparent only at South Fork, alivestock site (r2 = 0.98), and Panther (r2 = 0.91). Wintersamples were collected at two sites, South Fork and LittleCobb (Fig. 10). At these sites, nitrate concentration and dis-charge were positively correlated, low in winter, increasing

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Fig. 4. Small basin (a) drainage area and land-use category, (b) relative land use, and (c) manure loads. Data are from Battaglin et al.(2001a). Numbers above the drainage area bars indicate the actual drainage area in km2. Though land cover was not always accuratelydescribed as being in one of the four categories (Fig. 4b), an “average” classification was necessary to provide a sufficient number ofsamples in each category. See Fig. 2 for full names.

Fig. 5. The distribution of δ18O or δ15N versus land use. Opensymbols, δ18O values; solid symbols, δ15N.



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Chang et al. 1879

at the end of March, and declining in August, though moreso at South Fork (n = 4; r2 = 0.81) than at Little Cobb (n =5; r2 = 0.65) (Fig. 7c). Only South Fork showed a seasonalpattern between nitrate concentration, discharge, δ15N, andδ18O. Discharge and NO3

– concentrations were low in winter(0.7 m3·s–1 and 4.3 ppm) (Fig. 10) and increased at the endof March (9.3 m3·s–1 and 12 ppm). In addition to the inversecorrelation of δ15N values and nitrate concentration at SouthFork, δ15N was also inversely correlated to discharge (r2 =0.87) and showed a seasonal trend, declining from +12.9‰in winter to +7.8‰ at the end of March (Fig. 10). Similarly,

δ18O values were inversely correlated with NO3– (r2 = 0.82)

and discharge (r2 = 0.60) at this site (Fig. 10). Otherwise,there was no consistent correlation between δ18O values andNO3

– concentration across all agricultural sites (r2 = 0.26)(Fig. 8). At some sites, δ18O values were relatively constantdespite large changes in nitrate concentration (Little Cobb,Walnut), whereas at other sites, δ18O was variable and nitrateconcentrations were not (Bogue Phalia, South Fabius, Rat-tle). There were also no seasonal correlations between δ18Oand discharge or time (Fig. 10).

The highly clustered isotopic values at Four Mile and

Fig. 6. Discharge versus nitrate concentrations (N-NO3–, ppm) for (a) all sites, (b) urban and undeveloped sites, (c) agriculture sites,

and (d) big river sites. In agriculture plots, solid diamonds, circles, squares, and triangles denote sites with both crops and livestock,and all other symbols denote sites with crops only. In urban and undeveloped plots, solid symbols denote undeveloped sites. See Fig. 2for full names.



Walnut (Fig. 6), small drainage basins (~52 km2) with simi-lar manure loads (Walnut ~2000 and Four Mile ~1400 kgN·km–2), suggest a consistent source of nitrate. The low val-ues (+3.0 to +5.5‰ for δ15N; +3.3 to +9.4‰ for δ18O) werecharacteristic of ammoniacal fertilizer and soil nitrate (Kendall1998). Sugar Creek, which drains a large basin (~243 km2),

had δ18O values in a similar range, though the δ15N values(+5 to +6.6‰) were slightly higher.

Urban and undeveloped sitesThere were no discernable correlations between stable iso-

topes and NO3– concentration, discharge, or time for urban

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Fig. 7. δ15N versus nitrate concentrations (N-NO3–, ppm) for (a) all sites, (b) urban and undeveloped sites, (c) agriculture sites, and

(d) big river sites. In agriculture plots, solid diamonds, circles, squares, and triangles denote sites with both crops and livestock, andall other symbols denote sites with crops only. In urban and undeveloped plots, solid symbols denote undeveloped sites. See Fig. 2 forfull names.



and undeveloped sites. This may be due to the small numberof samples collected for each site and the fact that NO3

–

concentrations were always low. Although the range of δ15Nvalues from undeveloped sites (–1.4 to +13.4‰) was similar

to that of urban sites, the range of δ18O values (+5.8 to+19.8‰) was lower. This suggests that atmospheric nitratewas relatively minor at undeveloped sites, perhaps becauseof a larger contribution of soil nitrate.

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Chang et al. 1881

Fig. 8. δ18O versus nitrate concentrations (N-NO3–, ppm) for (a) all sites, (b) urban and undeveloped sites, (c) crop sites, and (d) big river

sites. In agriculture plots, solid diamonds, circles, squares, and triangles denote sites with both crops and livestock, and all other symbolsdenote sites with crops only. In urban and undeveloped plots, solid symbols denote undeveloped sites. See Fig. 2 for full names.



© 2002 NRC Canada


An exception to the high δ18O values of urban sampleswas the low δ18O value (+3‰) for Little Buck in the spring(Fig. 6). This low value is more typical of soil than atmo-spheric NO3

– (Fig. 1), consistent with the observation thatthe sample, collected when there was a sixfold increase indischarge, was colored instead of clear. This suggests thatNO3

– was flushed out of the soil and that the water color

was derived from dissolved organic matter or fine soil parti-cles.

Discussion

Changes in nitrate sourceThe δ18O of nitrate was useful in revealing changes in ni-

Fig. 9. δ18O versus δ15N for (a) all sites, (b) urban and undeveloped sites, (c) agriculture sites, and (d) big river sites. In agricultureplots, solid diamonds, circles, squares, and triangles denote sites with both crops and livestock, and all other symbols denote sites withcrops only. In urban and undeveloped plots, solid symbols denote undeveloped sites.



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Chang et al. 1883

trate sources that accompanied high-discharge events. Anabrupt change in δ18O following a sudden storm occurred atBogue Phalia. This site had two of the highest δ18O valuesfor agricultural sites. Water does not easily penetrate theclay soil at Bogue Phalia and NO3

– concentrations werealways low (≤1 ppm). The δ18O values (+43, +21, and+8.8‰) were positively correlated with discharge (59, 6, and3.6 m3·s–1). The sample with the highest δ18O value was col-lected during a July thunderstorm, a common occurrence inthis area, when intense rains caused an order of magnitudeincrease in discharge. Thus, it is likely that the high δ18Ovalue of this sample and possibly the September sample(+21‰) reflect atmospherically derived nitrate. In contrast,values for the June sample (δ18O = +8.8‰; δ15N = +7.5‰)were more consistent with soil-derived nitrate.

Changes in nitrate composition associated with storm eventswere also reported by Ging et al. (1996), who investigatednitrate sources in two urban streams (each watershed was~34 km2). During low flow conditions, subsurface flowdominated and sewage was the predominant source of ni-

trate. In contrast, during storms, nitrate was from a mixtureof surface sources. Runoff from impermeable paved surfaceswas suggested as a significant mechanism for transportingnitrate into streams. Clearly, δ18O is a valuable tool for pro-viding information about nitrate sources and transformations.

The tendency of δ15N values to decline as nitrate concen-trations increased at agricultural sites suggests that manurewas the predominant source of nitrate at low concentrationsand soil-derived nitrate predominated at higher concentra-tions. This was best illustrated at South Fork. In winter, dis-charge and nitrate concentrations were low and δ15N valueswere high, and in spring, when discharge and nitrate concen-trations increased, δ15N values declined.

The lack of correlation between the isotopic values andnitrate concentration at large river sites can be explained bydilution and mixing of nitrate sources upriver. Because agri-cultural sites had the highest nitrate concentrations and maybe contributing significant amounts of nitrate to large rivers,it is not surprising that the isotopic values of large river sitesresembled those agricultural sites.

Fig. 10. Agriculture plots for (a) nitrate concentrations (N-NO3–, ppm) as a function of time, (b) δ15N as a function of time, (c) dis-

charge as a function of time, and (d) δ18O as a function of time. Solid symbols denote sites with both crops and livestock and opensymbols denote sites with crops only. No discharge measurements for Rattle in 1 August 1997 or Four Mile on 30 June 1997. Symbolsare enlarged for sites where trends occurred.



Seasonal trendsA hint of a seasonal trend was observed at agricultural sites

where discharge and nitrate concentrations were positivelycorrelated (lowest in fall and winter). At these sites, high win-ter δ15N values were characteristic of soil–manure nitrate orpossibly groundwater N that may have been denitrified. Asconcentrations and discharge increased in spring, δ15N val-ues became lower, which is consistent with a fertilizer–soilsource. The best example of this pattern was at South Fork,a livestock site. However, with a limited number of datapoints per site and the loss of the first year’s samples, furtherstatements about seasonal trends in this study are specula-tive. Although growing seasons vary with latitude and eleva-tion, the growing season was generalized to occur during thesummer months. Also, differences in timing of crop plantingand fertilizer application may mask trends.

Land useAlthough sites with different land uses had moderately

distinctive isotopic signatures, it was not possible to predicta particular isotopic value based on land use and nitrate con-centration alone. In our study, there was no correlation be-tween the percent of land used for agriculture and δ15N orδ18O values. This is probably because classifying land use aseither agriculture or nonagriculture is an oversimplificationof land use at the scale of this study. In fact, land use wasmore fragmented (e.g., urban, range, row crop, livestock,forest, fallow fields) and was changing at some sites. It isalso likely that the large scale of these drainage basins madeit more difficult to retain an agricultural fingerprint. In con-trast, high consumer δ15N values associated with domesticsewage and manure-fertilized areas (Cabana and Rasmussen1996; Hebert and Wassenaar 2001) and increases in δ15N ofnitrate with increasing percent of agricultural land (Harring-ton et al. 1998; Mayer et al. 2002 (r2 = 0.75)) have been re-ported by other investigators.

DenitrificationThough analysis of nitrate for δ18O and δ15N can be an im-

portant tool for discerning denitrification, it was not possibleto identify whether denitrification had occurred. This is be-cause (i) only by collecting Lagrangian samples can one de-termine if denitrification is taking place along a particularreach of a river, and (ii) a shift in δ15N and δ18O at a siteonly indicates that denitrification occurred somewhere in thebasin and not at that particular sampling site.

In a companion study, evaluation of a set of Lagrangiansamples from some of the same large river sites showed noevidence for denitrification in these rivers (Battaglin et al.2001b). This finding is consistent with the model of Alexan-der et al. (2000) and the 15N tracer studies of Peterson et al.(2001), which concluded that denitrification is more likely tooccur in small, shallow streams where there is more oppor-tunity for contact and exchange of stream water with thehyporheic zone. In effect, smaller streams and rivers are themajor sources of nitrogen in the MARB, and large rivers actas conduits for NO3

– export. Rapid uptake of inorganic ni-trogen and transformations occurred in small streams, whereretention and transformation of inorganic N was as much as50% of the nitrogen inputs (Peterson et al. 2001). As wasshown by Kellman and Marcel (1998), who used only δ15N

of nitrate, denitrification fingerprinting may be more effec-tive in small (5 km2), heavily fertilized watersheds.

Note that the lack of isotopic evidence for denitrificationdoes not mean that denitrification has not occurred. The 2:1denitrification slope can be obscured by low isotopic enrich-ment factors and continuous mixing of different nitrate sources(Mayer et al. 2002), if the denitrification rate is diffusion-limited, or by limiting nitrate concentrations. Finally, at theselarge scales, the isotopic signatures of denitrification are dilutedby the large reservoir of soil-derived nitrate.

In conclusion, data from this study suggest that at the ba-sin scale, δ15N and δ18O values of NO3

– can discern nitratesources and changes in composition. Riverine nitrate fromdifferent land uses had overlapping but moderately distinctsignatures. The inverse relation between nitrate concentra-tion and δ15N values at some agricultural sites suggests thatthe predominant source of nitrate is manure when dischargeand nitrate concentration are low (winter), and as dischargeand nitrate concentration increase, (spring–summer) soil-derived nitrate is introduced. Finally, shifts in δ18O related tohigh-discharge periods were useful for identifying changesin the dominant nitrate source.

Our results suggest that stable isotope analysis can addimportant information about nitrate beyond that available us-ing discharge and concentration data alone. Future work shouldincorporate multiple samples from a smaller number of sites,with similar climatic conditions, unless the objective is tocompare sources within different climatic zones. We alsorecommend that attention be paid to soil type (permeability),tile drains, and high and low flow events. Finally, it wouldbe of interest to determine the scale at which a site loses itscharacteristic land use related fingerprint and becomes an in-tegrator site.

Acknowledgments

We thank David Walters, Richard Bell, Jeff Martin, andRichard Coupe for their help in obtaining background data. Wealso thank Tom Jordan, Bill Showers, Robert Hesslein, and ananonymous referree for their reviews. We are also indebted toFred Murphy for his help in revising the manuscript.

References

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