1874

Nitrate stable isotopes: tools for determining

nitrate sources among different land uses in the
Mississippi 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 uses
have 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 δ15N
values 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 dans
cinq catégories d’utilisation des terres: (1) de grands bassins hydrographiques de rivières (>34 590 km2) drainant des
terres 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 le
débit. Les valeurs isotopiques des grandes rivières ressemblent à celles des régions agricoles; les sites où il y a élevage
ont des valeurs de δ15N caractéristiques du fumier; les sites urbains ont de fortes valeurs de δ18O caractéristiques des
nitrates atmosphériques.

[Traduit par la Rédaction] Chang et al. 1885

Introduction creases occurring between 1970 and 1983 (Goolsby et al.

The Mississippi Atchafalaya River Basin (MARB) is the
third 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 River
contributes to the formation of a hypoxic zone in the Gulf of
Mexico that can exceed 17 000 km2 (Rabalais et al. 1999).
The Mississippi River delivers 1.6 million metric tons (t) of
nitrogen 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 tripled
since 1965 (Justic et al. 1995; Goolsby et al. 1999; Alexan-
der et al. 2000). Between 1955 and 1996, the average flux of
nitrate increased by 19 000 t per year, with most of the in-
1999).
Nonpoint sources contribute ~90% of the nitrogen and
phosphorus in the MARB. Fertilizer and mineralized-soil or-
ganic N contribute ~50% of the total N flux to the Gulf of
Mexico, with animal manure contributing ~15%. Though the
contribution 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 et
al. 1999).
Much (~35%) of the nitrogen carried by the Mississippi
River originates in the middle Mississippi Basin, which in-
cludes Iowa, Illinois, and Indiana. This region composes

Received 9 November 2001. Accepted 29 October 2002. Published on the NRC Research Press Web site at http://cjfas.nrc.ca on
10 January 2003.
J16605
C.C.Y. Chang,1 C. Kendall, and S.R. Silva. U.S. Geological Survey, Water Resources Division, 345 Middlefield Road, Menlo
Park, CA 94025, U.S.A.
W.A. Battaglin and D.H. Campbell. U.S. Geological Survey, Water Resources Division, Denver Federal Center, Denver, CO
80225, U.S.A.
1
Corresponding author (e-mail: ccchang@usgs.gov).

Can. J. Fish. Aquat. Sci. 59: 1874–1885 (2002) DOI: 10.1139/F02-153 © 2002 NRC Canada
Chang et al. 1875

only 9% of the total area but has the largest average total ni- Fig. 1. Isotopic signatures for different nitrate sources. From
trogen yields, ranging from 1000 to 3000 kg·km–2·year–1 Kendall (1998).
(Goolsby et al. 1999). During flooding, the flux can double
because of the large reservoir of nitrogen stored in the soil.
The formation of the hypoxic zone in the Gulf of Mexico
has provided incentive for determining the fate of anthro-
pogenic N at large spatial scales. At the basin scale, N flux
is strongly correlated to discharge and season. However, the
relative contributions of fertilizer, sewage–livestock waste,
and soil nitrate as functions of discharge, season, and nitrate
concentration 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, these
do not provide information about sources. The δ15N of ni-
trate can be used to distinguish between sources and even
better separation can be obtained if δ18O is used in conjunc-
tion with δ15N (Fig. 1) (Kendall 1998). This is because the
isotopic fingerprints of atmospheric, fertilizer, soil, and ma-
nure-derived nitrate often have sufficiently distinct signa-
tures to permit separation. Furthermore, analysis of δ18O can
be particularly useful in identifying denitrification, because
when it occurs, the remaining nitrate becomes heavier in
both δ15N and δ18O (2:1 for δ15N:δ18O ratio) (Amberger and
Schmidt 1987; Böttcher et al. 1990).
Traditionally, isotope values are plotted against discharge,
nitrate concentrations, and time to determine if there are
source-related changes associated with events (i.e., storms)
and season, the latter of which may be related to anthro- lected in 1998. Included were samples from the U.S. Geo-
pogenic activities such as fertilizer application and crop har- logical Survey (USGS) National Stream Quality Accounting
vest. If more than one isotope is used, then isotopes are Network (NASQAN) and National Water Quality Assess-
plotted against each other (as shown in Fig. 1). ment (NAWQA) sites as well as from other USGS monitor-
For example, both isotopes were used to show ammonia ni- ing sites. For a complete description of the study plan, the
trification (Aravena et al. 1993) and denitrification (Aravena reader is referred to Battaglin et al. (1997, 2001a).
and Robertson 1998) in a septic plume. Nitrate sources and The eight large river sites had mixed land uses (Fig. 3).
nitrification were identified in ground water adjacent to Sixteen sites draining areas <2500 km2 were selected to rep-
poultry farms and raspberry fields (Wassenaar 1995). On resent one of four land-use categories (Fig. 4a) (Battaglin et
slightly larger scales, seasonal and discharge-related changes al. 2001a): land in row-crop agriculture, land with both live-
in the contributions of microbial and atmospheric nitrate in stock and row-crop agriculture, urban land, and undeveloped
streams and springs were observed in undisturbed water- land. Classification was made on the basis of dominant land
sheds (Burns and Kendall 2002; Campbell et al. 2002; cover (Fig. 4b) and manure production (Fig. 4c). Though
Sickman et al. 2003). On still larger scales, both isotopes land cover was more mixed and fragmented than these four
were used to compare watersheds draining forested, urban, categories, an “average” classification was necessary to pro-
and agricultural areas (Mayer et al. 2002). vide a sufficient number of samples in each category. For the
The objective of this study was to determine whether the purposes of simplification, we grouped the 10 land covers
δ18O and δ15N of nitrate could discriminate between nitrate into four categories, e.g., rangeland, wetland, and fallow
sources from sites with different land uses. This is one of the pasture are shown as undeveloped and row crop and live-
first studies to apply these techniques at the basin scale. To- stock are shown as agriculture in Fig. 4b. Sites where row
wards this goal, water samples were collected from 24 sites crops were grown and where manure production was
in the Mississippi River Basin that represent five land-use >3200 kg·km–2·year–1 were classified as livestock sites (Elk,
categories: (1) large river basins (>34 590 km2) draining mul- Rattle, South Fork, and North Dry Creek) (Fig. 4a). Elk was
tiple land uses and smaller basins in which predominant land classified as a livestock site because it had one of the highest
use was either (2) urban, (3) undeveloped, (4) crops, or manure loads (4887 kg·km–2), despite that fact that the pre-
(5) crops and livestock (Fig. 2). dominant land cover was forested and undeveloped. Two
sites were classified as being dominantly urban (Shingle
Materials and methods Creek and Little Buck), two as dominantly undeveloped
(Namekagon and Dismal River), and the remaining eight as
Study sites dominantly row-crop sites (Fig. 4a).
Discharge and other physical measurements were made in Unfortunately, because of procedural problems, many of
1997 and 1998 from 24 sites located along streams and the 1997 samples were not processed for stable isotope anal-
rivers in the MARB (Fig. 2). Most of the samples were col- ysis. In 1998, four samples were collected at most sites. The

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1876
Fig. 2. Site map showing the Mississippi River Basin, large river sites, and smaller basin sites. From Battaglin et al. (2001a).
first set of samples was collected in spring, generally before
the application of fertilizers to local fields. The second and
third sets of samples were collected in early summer after
50% or more of the crops in the basin had been planted and
during 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 data
When appropriate, depth-integrated samples were collected
from three or more verticals per site using USGS protocols.
Specific conductance, pH, discharge, and water temperature
were measured at the time of sample collection. A complete
description of the sample collection methods, measurements
of physical properties, land-use data, and chemical analysis is
given in Battaglin et al. (2001a).
Nutrients, major ions, and stable nitrate isotopes
Nitrate (actually nitrite and nitrate) was analyzed by auto-
Can. J. Fish. Aquat. Sci. Vol. 59, 2002
mated colorimetric procedures with a N-detection limit of
0.05 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 as
0‰. Oxygen isotope values (δ18O) are reported relative to the
Vienna standard mean ocean water (VSMOW), also defined
as 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 simulated
field samples (n = 12), the standard errors were ±0.07‰ for
δ15N 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 and
laboratory precision for NO3– concentration measurements,
as well as for δ15N and δ18O analysis. The standard errors of
the differences between the pairs of samples were ±0.11 ppm
for N-NO3– concentration, ±0.22‰ for δ15N, and ±0.58‰ for
δ18O. In this study, only samples with both δ15N and δ18O val-
ues are presented. A comprehensive listing of all data can be
found in Battaglin et al. (2001a).
© 2002 NRC Canada
Chang et al.
Fig. 3. Large river (a) drainage area, (b) percent land cover, and
(c) manure loads. Estimates of relative land cover and manure
loads for Belle Chase (Belle) and Vicksburg (Vicks.) are not
available. (b) Solid bars, agriculture; shaded bars, range; and
open bars, forest. Data are from Battaglin et al. (2001a). Num-
bers above the drainage area bars indicate the actual drainage
area in km2. See Fig. 2 for full names.
Results
The range of δ18O values (+3.1 to +43.3‰) exceeded the
range of δ15N values (–1.4 to +12.3‰) (Fig. 5). Several pat-
terns emerged when data were coded by land use. Nitrate
concentrations of large river sites were low (<4 ppm), but
not as low as those at urban and undeveloped sites (<2 ppm)
(Fig. 6). In contrast, nitrate concentrations at agricultural
sites ranged from near the detection limit (0.05 ppm) to
18 ppm (Fig. 6). At sites where concentrations were always
1877
<4 ppm, the range of δ15N and δ18O values were broad and
could not be predicted from nitrate concentration (Figs. 7
and 8).
The isotopic values of sites with similar land use tended to
cluster into overlapping but moderately distinct ranges (Figs. 5
and 9). Excluding one high δ18O value (+43‰) from a crop
site, large river isotopic values (δ15N from +3 to +12‰; δ18O
from +3 to +24‰) resembled those of crop sites (δ15N from +3
to +12‰; δ18O from +3 to +21‰) (Figs. 5 and 9). The isotopic
values of agricultural sites were characteristic of mixtures of
fertilizer, soil, or manure-derived nitrate (Fig. 1). At livestock
sites, the ranges of δ15N and δ18O values were similar (+5 to
+13‰) (Fig. 5). A larger proportion (70%) of livestock sites
had nitrate concentrations <8 ppm compared with sites with
only crops (45%) and tended to have higher δ15N values for a
given nitrate concentration than “crop-only sites” (Fig. 7). For
example, of the 10 agricultural samples with nitrate concentra-
tions <2 ppm, four of the five highest δ15N values were from
Elk, a livestock site. Of the 17 samples with nitrate concentra-
tions ranging from 4 to 8 ppm, only three crop samples had
δ15N values equal to or higher than those from livestock sites.
The nitrate concentrations of these samples were ~8 ppm: on
two 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-derived
nitrate, 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 δ15N
values 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 sites
and high range of δ18O values (+4 to +31‰) are characteristic
of atmospheric nitrate (Fig. 1). Though synthetic nitrate fertil-
izer could also produce relatively heavy δ18O (Amberger and
Schmidt 1987), it is used much less commonly than ammo-
nium fertilizers (Terry and Kirby 1999). These urban isotopic
values 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 one
oxygen from the atmosphere (+23‰) (Amberger and Schimdt
1987).
Large rivers
Whereas as NO–3 concentrations and discharge were higher
in spring than at other times at Grand Chain and Thebes,
there were no seasonal trends for δ15N or δ18O at any large
river sites (data not shown). The range of isotopic values for
large river sites was similar to those of crops sites (Figs. 5
and 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 some
sites (Yazoo, Grand Chain, Thebes, St. Francisville), each of
them 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 δ15N
values were chaotic (e.g., Clinton). Although the δ18O values
of large river sites appear to decline as NO3– concentrations
increased (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;
© 2002 NRC Canada
1878 Can. J. Fish. Aquat. Sci. Vol. 59, 2002

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 accurately
described as being in one of the four categories (Fig. 4b), an “average” classification was necessary to provide a sufficient number of
samples in each category. See Fig. 2 for full names.

Fig. 5. The distribution of δ18O or δ15N versus land use. Open
symbols, δ18O values; solid symbols, δ15N.
regression analysis) and was even more poorly defined at
individual 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 averaging
δ15N or δ18O values because the unique relation between the
two isotopes used to identify the source of nitrate is lost.

Agricultural sites
Taken 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 a
range of +13.4 to +7.4‰ to a range of about ~+4 to +6‰. A
strong inverse correlation between δ15N and NO3– concentra-
tions at individual sites was apparent only at South Fork, a
livestock site (r2 = 0.98), and Panther (r2 = 0.91). Winter
samples were collected at two sites, South Fork and Little
Cobb (Fig. 10). At these sites, nitrate concentration and dis-
charge were positively correlated, low in winter, increasing

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Chang et al. 1879

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. 2
for full names.

at the end of March, and declining in August, though more
so at South Fork (n = 4; r2 = 0.81) than at Little Cobb (n =
5; r2 = 0.65) (Fig. 7c). Only South Fork showed a seasonal
pattern between nitrate concentration, discharge, δ15N, and
δ18O. Discharge and NO3– concentrations were low in winter
(0.7 m3·s–1 and 4.3 ppm) (Fig. 10) and increased at the end
of March (9.3 m3·s–1 and 12 ppm). In addition to the inverse
correlation of δ15N values and nitrate concentration at South
Fork, 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 and
NO3– concentration across all agricultural sites (r2 = 0.26)
(Fig. 8). At some sites, δ18O values were relatively constant
despite large changes in nitrate concentration (Little Cobb,
Walnut), whereas at other sites, δ18O was variable and nitrate
concentrations were not (Bogue Phalia, South Fabius, Rat-
tle). There were also no seasonal correlations between δ18O
and discharge or time (Fig. 10).
The highly clustered isotopic values at Four Mile and

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1880 Can. J. Fish. Aquat. Sci. Vol. 59, 2002

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, and
all other symbols denote sites with crops only. In urban and undeveloped plots, solid symbols denote undeveloped sites. See Fig. 2 for
full names.

Walnut (Fig. 6), small drainage basins (~52 km2) with simi-
lar manure loads (Walnut ~2000 and Four Mile ~1400 kg
N·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) were
characteristic of ammoniacal fertilizer and soil nitrate (Kendall
1998). 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 sites
There were no discernable correlations between stable iso-
topes and NO3– concentration, discharge, or time for urban

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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 symbols
denote sites with crops only. In urban and undeveloped plots, solid symbols denote undeveloped sites. See Fig. 2 for full names.

and undeveloped sites. This may be due to the small number to that of urban sites, the range of δ18O values (+5.8 to
of samples collected for each site and the fact that NO3– +19.8‰) was lower. This suggests that atmospheric nitrate
concentrations were always low. Although the range of δ15N was relatively minor at undeveloped sites, perhaps because
values from undeveloped sites (–1.4 to +13.4‰) was similar of a larger contribution of soil nitrate.

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1882 Can. J. Fish. Aquat. Sci. Vol. 59, 2002

Fig. 9. δ18O versus δ15N 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.

An exception to the high δ18O values of urban samples was derived from dissolved organic matter or fine soil parti-
was the low δ18O value (+3‰) for Little Buck in the spring cles.
(Fig. 6). This low value is more typical of soil than atmo-
spheric NO3– (Fig. 1), consistent with the observation that Discussion
the sample, collected when there was a sixfold increase in
discharge, was colored instead of clear. This suggests that Changes in nitrate source
NO3– was flushed out of the soil and that the water color The δ18O of nitrate was useful in revealing changes in ni-
© 2002 NRC Canada
Chang et al. 1883

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 open
symbols denote sites with crops only. No discharge measurements for Rattle in 1 August 1997 or Four Mile on 30 June 1997. Symbols
are enlarged for sites where trends occurred.

trate sources that accompanied high-discharge events. An
abrupt change in δ18O following a sudden storm occurred at
Bogue Phalia. This site had two of the highest δ18O values
for agricultural sites. Water does not easily penetrate the
clay soil at Bogue Phalia and NO3– concentrations were
always low (≤1 ppm). The δ18O values (+43, +21, and
+8.8‰) were positively correlated with discharge (59, 6, and
3.6 m3·s–1). The sample with the highest δ18O value was col-
lected during a July thunderstorm, a common occurrence in
this area, when intense rains caused an order of magnitude
increase in discharge. Thus, it is likely that the high δ18O
value 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 events
were also reported by Ging et al. (1996), who investigated
nitrate sources in two urban streams (each watershed was
~34 km2). During low flow conditions, subsurface flow
dominated and sewage was the predominant source of ni-
trate. In contrast, during storms, nitrate was from a mixture
of surface sources. Runoff from impermeable paved surfaces
was suggested as a significant mechanism for transporting
nitrate 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 manure
was the predominant source of nitrate at low concentrations
and 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 values
were high, and in spring, when discharge and nitrate concen-
trations increased, δ15N values declined.
The lack of correlation between the isotopic values and
nitrate concentration at large river sites can be explained by
dilution and mixing of nitrate sources upriver. Because agri-
cultural sites had the highest nitrate concentrations and may
be contributing significant amounts of nitrate to large rivers,
it is not surprising that the isotopic values of large river sites
resembled those agricultural sites.
© 2002 NRC Canada
1884
Seasonal trends
A hint of a seasonal trend was observed at agricultural sites
where discharge and nitrate concentrations were positively
correlated (lowest in fall and winter). At these sites, high win-
ter δ15N values were characteristic of soil–manure nitrate or
possibly groundwater N that may have been denitrified. As
concentrations and discharge increased in spring, δ15N val-
ues became lower, which is consistent with a fertilizer–soil
source. The best example of this pattern was at South Fork,
a livestock site. However, with a limited number of data
points per site and the loss of the first year’s samples, further
statements 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 the
summer months. Also, differences in timing of crop planting
and fertilizer application may mask trends.
Land use
Although sites with different land uses had moderately
distinctive isotopic signatures, it was not possible to predict
a 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 or
δ18O values. This is probably because classifying land use as
either agriculture or nonagriculture is an oversimplification
of land use at the scale of this study. In fact, land use was
more fragmented (e.g., urban, range, row crop, livestock,
forest, fallow fields) and was changing at some sites. It is
also likely that the large scale of these drainage basins made
it more difficult to retain an agricultural fingerprint. In con-
trast, high consumer δ15N values associated with domestic
sewage and manure-fertilized areas (Cabana and Rasmussen
1996; Hebert and Wassenaar 2001) and increases in δ15N of
nitrate 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.
Denitrification
Though analysis of nitrate for δ18O and δ15N can be an im-
portant tool for discerning denitrification, it was not possible
to 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 particular
reach of a river, and (ii) a shift in δ15N and δ18O at a site
only indicates that denitrification occurred somewhere in the
basin and not at that particular sampling site.
In a companion study, evaluation of a set of Lagrangian
samples from some of the same large river sites showed no
evidence 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 to
occur in small, shallow streams where there is more oppor-
tunity for contact and exchange of stream water with the
hyporheic zone. In effect, smaller streams and rivers are the
major sources of nitrogen in the MARB, and large rivers act
as conduits for NO3– export. Rapid uptake of inorganic ni-
trogen and transformations occurred in small streams, where
retention and transformation of inorganic N was as much as
50% of the nitrogen inputs (Peterson et al. 2001). As was
shown by Kellman and Marcel (1998), who used only δ15N
Can. J. Fish. Aquat. Sci. Vol. 59, 2002
of nitrate, denitrification fingerprinting may be more effec-
tive in small (5 km2), heavily fertilized watersheds.
Note that the lack of isotopic evidence for denitrification
does not mean that denitrification has not occurred. The 2:1
denitrification 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 these
large scales, the isotopic signatures of denitrification are diluted
by 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 nitrate
sources and changes in composition. Riverine nitrate from
different land uses had overlapping but moderately distinct
signatures. The inverse relation between nitrate concentra-
tion and δ15N values at some agricultural sites suggests that
the predominant source of nitrate is manure when discharge
and nitrate concentration are low (winter), and as discharge
and nitrate concentration increase, (spring–summer) soil-
derived nitrate is introduced. Finally, shifts in δ18O related to
high-discharge periods were useful for identifying changes
in the dominant nitrate source.
Our results suggest that stable isotope analysis can add
important information about nitrate beyond that available us-
ing discharge and concentration data alone. Future work should
incorporate multiple samples from a smaller number of sites,
with similar climatic conditions, unless the objective is to
compare sources within different climatic zones. We also
recommend that attention be paid to soil type (permeability),
tile drains, and high and low flow events. Finally, it would
be of interest to determine the scale at which a site loses its
characteristic land use related fingerprint and becomes an in-
tegrator site.
Acknowledgments
We thank David Walters, Richard Bell, Jeff Martin, and
Richard Coupe for their help in obtaining background data. We
also thank Tom Jordan, Bill Showers, Robert Hesslein, and an
anonymous referree for their reviews. We are also indebted to
Fred Murphy for his help in revising the manuscript.
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