Applied Geochemistry 23 (2008) 3597–3611

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Applied Geochemistry
journal homepage: www.elsevier.com/locate/apgeochem

Environmental isotopes (N, S, C, O, D) to determine natural

attenuation processes in nitrate contaminated waters:
Example of Osona (NE Spain)
Laura Vitòria a,*, Albert Soler a, Àngels Canals a, Neus Otero a,b
a
Grup de Mineralogia Aplicada i Medi Ambient, Departament de Cristal.lografia, Mineralogia i Dipòsits Minerals, Facultat de Geologia, Universitat de
Barcelona, Martí i Franquès, s/n, 08028, Barcelona, Spain
b
Departament de Geologia Ambiental, Institut de Ciències de la Terra ‘‘Jaume Almera”, CSIC C/Lluís Solé i Sabarís s/n, Barcelona 08028, Spain

a r t i c l e i n f o a b s t r a c t

Article history: Nitrate-contaminated groundwater from an aquifer in the Osona region (NE Spain) was
Received 25 May 2006 chemically and isotopically (d15 NNO3 ; d18 ONO3 ; d34 SSO4 ; d18 OSO4 , dD, d18 OH2 O and d13 CDIC ) char-
Accepted 4 July 2008 acterized. Diffuse- NO3 contamination reached values of 366 mg/L. Nearly 75% of the 37
Available online 30 August 2008
sampled sites had higher concentrations than the 50 mg/L in NO 3 limit for drinking water.

Editorial handling by R.S. Harmon

To identify the source of pollution d15 NNO3 and d18 ONO3 were used with results, for most
samples, in the range of pig manure NO 3 . Nitrification processes were evaluated by means
of the d18O of NO3 and water. Isotopic data suggested that natural attenuation of NO3 was


taking place. This process was confirmed using the d18 ONO3 coupled with the NO 3 =Cl ratio,
avoiding the influence of continuous NO 3 inputs. A further insight on denitrification pro-
cesses was obtained by analyzing the ions involved in denitrification reactions. Although
the role of organic matter oxidation could neither be confirmed nor discarded, this
approach showed a link between denitrification and pyrite oxidation. Therefore, in areas
with no adequate infrastructure (e.g. multipiezometers), such as the one studied, this
approach could be useful for implementing better water management.
Ó 2008 Elsevier Ltd. All rights reserved.

1. Introduction several aquifers have NO 3 concentrations up to 10 times

Groundwaters are one of the best water resources for
human consumption due to their normally high chemical
quality. However, there is increasing groundwater NO 3 region, and discriminate between the different sources of
pollution worldwide due to the intensive use of synthetic
and organic fertilizers, livestock piles, leaks from septic
tanks, and sewer systems in poor conditions. As for
Europe, in 1991, an EU Council Directive (91/676/EEC Offi-
cial Journal, 1991), known as the Nitrates Directive, was
formulated to protect waters against NO 3 contamination
in agricultural areas. Each State member must identify
its vulnerable zones – zones draining into water affected
by or vulnerable to N pollution – and establish a Codes
of Good Agricultural Practice. In Catalonia (NE Spain),
* Corresponding author. Fax: +34 93 402 13 40.
E-mail address: lvitoria@ub.edu (L. Vitòria).
higher than the 50 mg/L of NO3 threshold permitted for
human consumption (EC, 1998). The Catalan Water
Agency wants to characterise NO 3 contamination in the
pollution, in order to carry out the basic principle of the
European Union Treaty regarding communitarian policies
concerning environment – namely, that the ‘‘polluter
should pay” (EC, 1997).
Classical chemical water analyses only show the magni-
tude of NO3 contamination not the source of the pollution.
Therefore, in order to identify and/or quantify the origin of
this type of contamination new tools, such as stable iso-
topes, are needed. Over the last 30 a, different studies have
used the d15N and/or d18O of dissolved NO 3 in waters as
tracers of organic (e.g. animal and human wastes) and
inorganic (e.g. synthetic fertilizers) contamination with
relative success (Wassenaar, 1995; Kendall and McDonnell,

0883-2927/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.apgeochem.2008.07.018
3598 L. Vitòria et al. / Applied Geochemistry 23 (2008) 3597–3611
1998; Widory et al., 2004, among others). One of the con-
clusions that emerge from these studies is the need for a
clear chemical and isotopic characterization of the contam-
ination sources, as well as the identification of the fraction-
ation processes in order to interpret the isotopic data. For
example, denitrification takes place under specific phys-
ico-chemical conditions, where bacteria (Pseudomonas,
Bacillus, Thiobacillus, Propionbacterium and others) reduce
NO 15
3 to N2, increasing the isotopic composition (d N and
d18O) of the remaining NO 3 in waters (Böttcher et al.,
1990). This is considered a drawback in the application of
NO 3 isotopes as tracers of sources, nevertheless, it can be
applied to trace processes. Indeed, the isotopic composi-
tion of dissolved NO 3 is used to distinguish between
dilution and denitrification in areas where a decrease in
NO 3 concentration in groundwater samples is observed
(Grischek et al., 1998; Mengis et al., 1999; Cey et al,
1999). A further step in the investigation of denitrification
processes is to determine the factors controlling the reac-
tions. This has been done coupling chemical data with
the d15N and/or d18O of dissolved NO 3 and the isotopic
composition of the ions involved in denitrification reac-
tions such as d34S and d18O of dissolved SO2 4 , and/or
d13C, of dissolved inorganic C (Aravena and Robertson,
1998; Pauwels et al., 2000).
The aim of this study was to integrate chemical and sta-
ble isotope data of groundwater in the Osona region (NE
Spain) – one of the vulnerable zones – in order to evaluate
the state of the water resources and predict their evolution.
In the area, pig manure had been intensively used as or-
ganic fertilizer producing diffuse NO 3 contamination,
although synthetic fertilizers have also been occasionally
used. Therefore, the multi-isotopic approach (15N, 34S,
13
C, 18O, D) coupled with the chemical data, was applied
first to validate their use as tracers of NO 3 sources, and,
secondly as markers of N processes. In particular, the nitri-
fication process was evaluated using the d18 ONO3 value; the
amount of denitrification was estimated taking into con-
sideration both the chemical and the d15 NNO3 and d18 ONO3
data. Furthermore, the denitrification reactions were
determined to some extent applying a multi-isotopic ap-
proach (d34 SSO4 ; d18 OSO4 and d13 CDIC ). This knowledge is
important when designing water and agricultural control
and management plans.
2. Study area
The Osona region (NE Spain) is one of the areas classi-
fied as vulnerable to nitrate contamination from agricultural
sources by the European Union (EEC, 1991). The region is
situated to the North of Barcelona, at the East of the Ebro
basin, between the Montseny Mountain and the Pyrenees
range (Fig. 1). The Ter river is the major stream in the area,
with a mean discharge of 509 hm3/a at the gauging station
of Roda de Ter. The area has a sub-Mediterranean climate
with an annual mean temperature of 13 °C, precipitation
of around 530 mm/a, and potential evapotranspiration
greater than rainfall (740 mm/a). Therefore, Osona is a
dry area that mainly relies on ground water resources to
fulfil its agricultural and farm demand. Surface water from
the Ter River covers most of the urban demand. The region,
with its 1263.8 km2, has more than 60,000 sheep, 100,000
cows and around 1,000,000 pigs in more than 1000 pig
farms (IDESCAT, 1999), most of them concentrated in
small areas. This intensive activity produces great amounts
of organic animal wastes (more than 10,000 tones N/a),
mainly as pig manure. A small part (around 3% in 2001)
is processed in treatment plants and the rest is spread
out on the fields. As the application of pig manure is
banned near urban areas, due to the bad smells, synthetic
fertilizers are also used. The municipalities of the area
(Manlleu, Torelló and St. Hipòlit de Voltregà, with over
30,000 people) are connected to two sewage stations with
N elimination treatment designed to treat two and three
times more wastewater than they actually do. As a result,
the contribution of other N sources, such as domestic sew-
age, is negligible compared to agricultural sources. Resid-
ual waters are discharged into the Ter River that crosses
the area (Fig. 1).
From a geological perspective, this hydrogeological sys-
tem lies on Paleogene sedimentary materials overlaying
Hercynian crystalline (igneous and metamorphic) rocks.
The stratigraphic sequence primarily consists of carbonate
formations, with alternations of calcareous, marl and car-
bonate sandstone layers (Reguant, 1967). These up to
300 m-thick formations, show a quite uniform dip of about
7–10° W, and they only have an antiform structure at their
northern limit. It is also worth mentioning the presence of
framboidal pyrite in these formations (Urquiola, 1994).
Hydrogeologically, the system consists of a series of con-
fined aquifers located in the carbonate and carbonate-
sandstone layers, whose porosity is mainly related to the
fracture network. Marl strata act as confining layers, as ob-
served in boreholes and hydrogeological prospecting. Main
production wells for agriculture and farm demand usually
reach depths of more than 100 m, searching for the most
productive confined aquifers. Alluvial aquifers are scarce
and generally non-productive in the area, except those lo-
cated at the Ter river terraces.
3. Sampling and analysis
Thirty-seven groundwater samples (31 from private
parapet wells and 6 from natural springs) were collected
between 9 and 26 October 2001 (Fig. 1). Sampled ground-
waters were shallow, with piezometric depths from 0 to
45 m, and were collected with the pumps already installed
in wells or with portable pumps. In both cases, the sam-
pling was carried out after extracting the routine 3 well
volumes of water. The piezometric level, pH, conductivity
and water temperature were measured in situ. Samples
were kept at 4 °C for their subsequent chemical and isoto-
pic analyses. In the laboratory, pH and conductivity at
25 °C were measured again; NHþ 4 concentration was
analyzed by colorimetry (continuous flow analyzer, Tech-
nicon) and total organic C (TOC) by organic matter com-
bustion (TOC 500 SHIMADZU). Afterwards a split of the
sample was filtered through a 0.45 lm MilliporeÒ filter;
3 2
the anion (NO 
3 , NO2 , PO4 , SO4 and Cl) content was
measured by High Performance Liquid Chromatography
(WATERS 515 HPLC, with WESCAN and UV/VIS KONTROL
detectors); concentrations of Na, Ca, K, Mg, Sr, Fe, B, Zn
 L. Vitòria et al. / Applied Geochemistry 23 (2008) 3597–3611 3599

Fig. 1. Geological map of the study area, between the municipalities of Manlleu and Torelló. The October 2001 groundwater chemistry is represented with
Stiff diagrams.
3600 L. Vitòria et al. / Applied Geochemistry 23 (2008) 3597–3611

were measured by Inductively Coupled Plasma-Optical
Emission Spectrometry (ICP-OES, Thermo Jarell ASH 61-
E); Ni, Co, Mn, Ba, Zn, Cu, Cr, As, Se, Cd, V, Hg, Pb by Induc-
tively Coupled Plasma-Mass Spectrometry (ICP-MS, Perkin
Elmer Elan 6000); and alkalinity ðHCO 3 Þ by titration
(METROHM 702 SM Titrino). The quality of the chemical
analysis was checked by doing an ionic mass balance,
accepting an error lower than 5%.
For the d15 NNO3 and d18 ONO3 analysis, dissolved nitrates
were concentrated using anion-exchange columns Bio
RadÒ AG 1-X8(Cl) 100–200 mesh resin, after extracting
the sulphates and phosphates by precipitation with BaCl2
and filtration (Mayer et al., 2001). Afterwards dissolved ni-
trates were eluted with HCl and converted to AgNO3 by
adding silver oxide. The AgNO3 solution was then freeze-
dried to purify the AgNO3 for analysis (modified from Silva
et al., 2000). For the SO2 4 isotopic analysis (d34 SSO4 and
d18 OSO4 ), dissolved SO2
4 was precipitated as BaSO4 by add-
ing BaCl2  2H2O after acidifying the sample with HCl and
boiling it to prevent BaCO3 precipitation following stan-
dard methods (e.g. Dogramaci et al., 2001). Unfiltered
splits of samples were treated with NaOH–BaCl2 solution
to precipitate carbonates together with sulphates and
phosphates. Later, they were filtered at 3 lm and the
d13C of the total dissolved inorganic C, mainly HCO 3 , was
analyzed (Bishop, 1990).
Deuterium and O isotopes of water were analyzed in a
Finnigan Matt Delta S Isotope Ratio Mass Spectrometer
(IRMS) coupled to an automated line based on the equilibra-
tion between H-water and H2 gas with a Pt catalyst, and
between O-water and CO2 gas following standard methods
(Epstein and Mayeda, 1953). The d15 NNO3 ; d34 SSO4 and d13 CDIC
were analyzed in a Carlo Erba Elemental Analyzer (EA)
coupled in continuous flow to a Finnigan Delta C IRMS.
The d18 ONO3 and d18 OSO4 were analyzed in duplicate with a
ThermoQuest TC/EA (high temperature conversion-ele-
mental analyzer) unit coupled with a Finnigan Matt Delta
C IRMS using the method of Kornexl et al. (1999). Notation
is expressed in terms of d ‰ relative to the international
standards V-SMOW (Vienna Standard Mean Oceanic Water)
for dD and d18O; AIR (atmospheric N2) for d15N, V-CDT
(Vienna Canyon Diablo Troilite) for d34S; and V-PDB (Vienna
Peedee Belemnite) for d13C. Precision (1r) of the samples
calculated from international and internal standards sys-
tematically interspersed in the analytical batches was
±1.5‰, ±0.2‰, ±0.3‰, ±0.4‰, ±0.4‰, ±0.5‰ and ±0.2‰ for
dD, d18 OH2 O ; d15 NNO3 ; d18 ONO3 ; d34 SSO4 ; d18 OSO4 and d13 CDIC ,
respectively. A summary of international and internal
standards used for analysis and the assumed values is de-
tailed in Table 1. All chemical and isotopic analyses were
prepared in the Mineralogia Aplicada i Medi Ambient
Research Group laboratory and analyzed at the Scientific
Services of the University of Barcelona.
4. Results and discussion
4.1. Groundwater quality
Chemical results are presented in Table 2 (water
temperature, pH, conductivity and major elements) and
Table 3 (minor and trace elements). Water pH values were
all above 7.3, temperatures ranged from 14.3 to 18.8 °C and
conductivities varied from 801 for the most pristine to
2474 lS/cm for the most polluted in terms of NO 3.

Table 1
Standards used in the isotopic determinations

Ratio Scale Reference materials Matrix d (‰) Used for the determination of
2
H/1H VSMOW (Vienna Standard Mean Ocean Water) NEUa Water 71.3 dD–H2O
LAB 6a Water 48.0 dD–H2O
MARa Water 2.2 dD–H2O
13
C/12C VPDB (Vienna Peedee Belemnite) IAEA-CH6b Sucrose 10.43 ± 0.13 d13C–DIC
IAEA-CH7b Polyethylene 31.83 ± 0.11 d13C–DIC
USGS 24b C (Graphite) 15.99 ± 0.11 d13C–DIC
15
N/14N AIR-N2 (Atmospheric N2) IAEA-N1b (NH4)2SO4 +0.43 ± 0.07 d15N–NO3
IAEA-N2 (NH4)2SO4 +20.41 ± 0.12 d15N–NO3
IAEA-NO3b KNO3 +4.72 ± 0.13 d15N–NO3
USGS-25b (NH4)2SO4 30.25 ± 0.38 d15N–NO3
18
O/16O VSMOW (Vienna Standard Mean Ocean Water) NEUa Water 10.63 d18O–H2O
LAB 6ª Water 7.29 d18O–H2O
MARa Water +0.48 d18O–H2O
NBS127b BaSO4 +9.3 ± 0.4 d18O–NO3, d18O–SO4
YCEMa BaSO4 +17.6 ± 0.5 d18O–NO3, d18O–SO4
H2SO4a BaSO4 +12.9 ± 0.6 d18O–NO3, d18O–SO4
Camb 35-4c AgNO3 +0.5 d18O–NO3
Chilean KNO3c KNO3 +45.2 d18O–NO3
EGC-1c KNO3 +28.0 d18O–NO3
EIL61c KNO3 +11.0 d18O–NO3
EIL62c AgNO3 +20.5 d18O–NO
34
S/32S VCDT (Vienna Canyon Diablo Troilite) IAEA-S1b Ag2S 0.30 (Exact.) d34S–SO4
IAEA-S2b Ag2S +22.67 ± 0.15 d34S–SO4
IAEA-S3b Ag2S 32.55 ± 0.12 d34S–SO4
NBS127b BaSO4 +21.1 d34S–SO4
YCEMb BaSO4 +12.5 d34S–SO4
a
Internal standard (Serveis Cientificotecnics de la Universitat de Barcelona).
b
Internal Standard.
c
Environmental Isotopes Laboratory, Universitty of Waterloo, Canada.
Table 2
Water temperature, pH, conductivity, and major elements of sampled groundwater in Osona, October 2001

Sample Water Temp (°C) pH at 25 (°C) Cond. at 25 °C (lS/cm) NO

3 (mg/L) SO2
4 (mg/L) HCO3 (mg/L) Cl (mg/L) Na+ (mg/L) K+ (mg/L) Mg2+ (mg/L) Ca2+ (mg/L)
PO-001 16.3 7.9 1623 173.8 252.1 431.1 76.8 37.1 13.5 48.7 216.8
PO-004 17.0 7.8 1144 64.1 170.3 509.3 38.0 21.1 19.8 43.3 172.8
PO-012 17.3 7.7 1585 140.8 223.2 400.4 101.9 42.5 8.4 44.2 211.7
PO-020 15.1 7.5 1364 80.8 204.9 458.5 67.8 29.1 3.5 43.9 187.5
PO-036 16.1 7.5 1249 109.5 135.2 425.7 45.3 22.1 7.1 36.3 171.3
PO-037 18.5 7.5 1112 83.1 105.4 378.0 51.0 23.2 15.1 24.6 148.5
PO-043 16.7 7.6 675 15.6 101.6 265.9 3.0 4.3 2.7 18.8 97.4
PO-051 15.7 1.1 1098 58.7 162.5 387.1 24.7 16.8 3.9 37.7 138.9

L. Vitòria et al. / Applied Geochemistry 23 (2008) 3597–3611

PO-052 17.7 1.1 1011 30.5 90.6 465.1 21.9 13.1 1.8 24.8 148.5
PO-059 16.0 8.2 1514 77.3 239.3 471.3 74.9 53.0 7.0 50.3 163.8
PO-062 15.6 7.6 1265 96.8 140.0 445.2 52.7 18.4 4.1 38.0 169.1
PO-063 18.5 7.8 1486 107.3 214.0 491.7 57.9 26.6 10.3 52.7 185.0
PO-065 14.3 7.6 917 91.5 75.0 328.9 28.6 11.9 4.9 30.7 113.8
PO-070 16.9 7.3 1261 83.6 200.9 429.6 51.3 22.6 5.6 42.0 163.6
PO-084 16.8 7.6 1054 38.9 137.4 384.8 35.2 41.6 4.2 38.6 105.5
PO-086 17.9 7.5 1043 78.2 125.2 367.2 25.6 18.6 3.3 35.8 125.5
PO-100 16.7 7.5 2474 365.7 244.1 480.4 210.5 78.0 77.8 55.8 279.1
PO-101 18.8 7.8 989 68.7 156.0 253.0 47.3 27.4 3.6 17.3 136.5
PO-102 n.d. 7.8 791 18.8 78.7 281.0 38.9 23.5 6.0 20.4 90.5
PO-103 18.7 8.1 801 <0.2 152.5 373.6 17.2 44.4 2.8 35.5 92.6
PO-104 16.1 1.1 1155 55.5 194.0 418.1 25.7 23.9 5.8 39.5 144.4
PO-105 15.7 7.6 1405 95.2 234.0 435.0 49.5 66.2 5.3 50.0 141.8
PO-106 17.2 8.0 1097 12.5 140.0 409.7 40.9 174.9 4.8 16.3 31.0
PO-107 18.7 7.5 1055 52.2 149.3 388.3 27.9 23.1 6.0 35.3 140.0
PO-108 16.1 7.4 1365 176.9 146.0 372.1 70.1 29.0 10.4 34.1 184.6
PO-109 16.2 7.5 1225 75.4 197.6 391.6 36.0 31.2 3.4 42.7 144.9
PO-110 16.5 7.5 1463 127.8 188.2 457.1 68.1 37.3 6.6 43.5 178.0
PO-111 15.6 7.4 1157 105.1 95.0 439.4 37.6 20.5 3.5 24.0 174.2
PO-112 16.8 8.0 835 17.0 91.6 387.9 11.7 12.3 1.9 24.8 119.2
PO-113 16.9 1.1 879 46.5 103.7 343.9 19.2 12.6 1.8 24.6 123.8
PO-114 17.5 1A 1285 149.2 118.1 422.4 44.0 28.6 5.7 31.5 170.8
PO-115 15.6 7.3 1752 325.3 151.2 408.0 83.4 35.4 20.9 39.0 233.0
PO-116 16.3 7.4 976 28.9 154.6 392.1 11.3 8.8 4.1 31.5 140.2
PO-117 15.3 7.5 1400 72.8 252.7 396.3 53.1 46.9 7.3 41.1 171.0
PO-118 15.3 7.9 882 6.8 148.8 377.1 15.3 8.8 6.7 23.5 137.1
PO-119 17.8 1.1 1091 62.7 98.6 405.6 44.4 56.8 3.7 26.4 117.6
PO-120 17.6 7.9 1092 66.5 170.3 373.8 38.4 16.2 2.1 37.2 150.1
PO-121 18.6 7.8 1101 78.5 110.6 431.2 40.2 13.2 3.2 38.2 145.3
98/83/CEE treshold P6.5 and 69.5 <2500 at 20 °C 50 250 – 250 200 – – –

3601
3602 L. Vitòria et al. / Applied Geochemistry 23 (2008) 3597–3611

Table 3
Minor and trace elements of sampled groundwater in Osona (October 2001)

Site NHþ
4 (mg/L) NO
2 (mg/L) TOC (mg/L) Sr (mg/L) Ba (mg/L) Mn (mg/L) Cu (mg/L) Zn (mg/L) B (mg/L) Pb (mg/L)
PO-001 <0.1 <0.2 1.7 1.701 0.095 0.0004 0.006 0.031 <0.025 0.0002
PO-004 1.08 0.7 3.2 1.717 0.081 0.1812 0.004 0.008 <0.025 <0.0001
PO-012 <0.1 <0.2 1.3 1.652 0.094 0.0005 0.005 0.015 <0.025 <0.0001
PO-020 <0.1 <0.2 1.2 1.734 0.098 0.0289 0.004 0.007 <0.025 0.0002
PO-036 <0.1 <0.2 1.5 1.454 0.083 0.0003 0.002 0.002 <0.025 0.0005
PO-037 <0.1 <0.2 1.2 0.900 0.094 0.0002 0.005 0.008 <0.025 <0.0001
PO-043 <0.1 <0.1 5.3 0.839 0.026 0.0009 0.008 0.015 <0.025 0.0002
PO-051 <0.1 0.6 <1 2.007 0.102 0.0006 0.002 0.002 <0.025 0.0002
PO-052 <0.1 <0.1 1.2 0.751 0.053 0.0003 0.002 0.006 <0.025 0.0004
PO-059 <0.1 <0.2 1.2 3.060 0.080 0.0017 0.005 0.956 <0.025 <0.0001
PO-062 <0.1 <0.2 1.6 1.626 0.083 0.0007 0.002 0.009 <0.025 <0.0001
PO-063 <0.1 <0.2 1.0 2.192 0.071 0.0003 0.012 0.048 <0.025 0.0003
PO-065 <0.1 <0.1 <1 0.536 0.091 0.0002 <0.001 0.002 <0.025 <0.0001
PO-070 <0.1 <0.2 1.2 1.617 0.069 0.0003 0.004 0.016 <0.025 <0.0001
PO-084 <0.1 <0.2 2.4 4.770 0.034 0.0047 0.004 0.011 <0.025 0.0006
PO-086 <0.1 <0.1 3.2 3.728 0.226 0.0118 0.007 1.461 <0.025 0.0004
PO-100 <0.1 <0.3 4.1 1.480 0.127 0.0015 0.007 0.025 <0.025 <0.0002
PO-101 <0.1 0.4 1.6 0.826 0.072 0.0069 0.006 0.035 <0.025 0.0011
PO-102 <0.1 0.2 <1 0.817 0.041 0.0002 0.004 0.010 <0.025 0.0002
PO-103 <0.1 <0.2 <1 3.680 0.046 0.0087 0.002 0.005 <0.025 <0.0001
PO-104 0.14 0.3 1.6 5.037 0.042 0.0022 0.002 0.183 <0.025 <0.0001
PO-105 <0.1 <0.2 <1 12.065a 0.050 0.0067 0.002 0.018 <0.025 <0.0001
PO-106 <0.1 <0.2 <1 3.041 0.028 0.0014 0.002 0.041 0.061 <0.0001
PO-107 <0.1 <0.2 1.1 1.557 0.046 0.0019 0.009 0.030 <0.025 0.0005
PO-108 <0.1 <0.2 2.7 1.026 0.084 0.0013 0.003 0.008 <0.025 <0.0001
PO-109 <0.1 0.3 1.1 1.700 0.040 0.0006 0.002 0.005 0.062 <0.0001
PO-110 <0.1 <0.2 1.7 2.219 0.079 0.0025 0.004 0.009 0.052 <0.0001
PO-111 <0.1 <0.2 <1 0.654 0.078 0.0004 0.002 0.002 0.085 <0.0001
PO-112 <0.1 <0.1 <1 0.972 0.047 0.0007 0.002 0.447 <0.025 0.0004
PO-113 0.11 <0.1 1.5 0.822 0.051 0.0004 0.003 0.011 <0.025 0.0002
PO-114 0.21 0.9 4.3 1.419 0.068 0.0133 0.033 0.051 0.026 0.1525
PO-115 <0.1 0.2 12.6 1.174 0.142 0.0218 0.027 0.117 0.069 0.0017
PO-116 <0.1 <0.1 2.2 1.407 0.057 0.0011 0.005 0.013 <0.025 0.0001
PO-117 0.13 2.3 2.0 1.940 0.105 0.0014 0.004 0.007 0.079 0.0002
PO-118 <0.1 <0.1 5.5 0.654 0.038 0.0003 0.006 0.024 <0.025 0.0001
PO-119 <0.1 <0.2 1.5 1.387 0.056 0.0060 0.008 1.752 0.119 0.0023
PO-120 <0.1 <0.2 1.5 1.151 0.059 0.0001 0.003 0.008 <0.025 0.0001
PO-121 <0.1 <0.2 <1 0.875 0.053 0.0002 0.002 0.002 <0.025 0.0001
Analytical precision ±0.005 ±0.0001 ±0.0001 ±0.001 ±0.001 ±0.025 ±0.0001
98/83/CEE treshold 0.5 0.5 – – – 0.05 – – 1 0.01

PO4, V, Cr, Fe, Co, Ni, Cd, Hg, As and Se concentrations were under their detection limits (10, 0.004, 0.002, 0.005, 0.001, 0.01, 0.0002, 0.0004, 0.002 and 0.01,
respectively) except for Fe in sample PO-115 with 0.007 mg/L.
a
Analytical precision of ±0.025 mg/L.

Groundwaters in the area are mainly HCO3–Ca type, in
accordance with the lithology of the aquifer. Only 3 sam-
ples are HCO3–SO4–Ca, one HCO3–Cl–Ca and one HCO3–
Na water type (Fig. 1). Fig. 2a shows the piezometric
groundwater surface and Fig. 2b the NO 3 concentration
which varies from undetected up to 366 mg/L, with mean
values of 90 mg/L of NO3 . Around 75% of the studied sam-
ples had NO 3 concentration above the drinking water
threshold. Maximum NO3 contamination was concentrated
in small areas that could coincide with higher pig manure
application or, locally, with wells supplying shallower
groundwater, though no relationship was observed be-
tween well depth and NO 3 concentration. Ammonium con-
tent, detected in only 5 samples, was below the drinking
water threshold, except for one sample; NO 2 was unde-
tected in most samples, but exceeded the 0.5 mg/L thresh-
old in 4 samples. TOC ranged from undetected to 12.6 mg/
L, with a mean value of 1.9 mg/L. Nitrate showed a positive
tendency with Cl (Fig. 3a), Ca (Fig. 3b) and Ba – elements
found in high concentrations in both pig manure and syn-
thetic fertilizers (Vitòria et al., 2004a; Otero et al., 2005).
Sulphate content of waters ranged between 75 and
253 mg/L with a mean value of 157 mg/L, however, no
clear correlation was observed between SO2 4 concentra-
tions and other major or trace elements. No evaporite out-
crops exist in the studied area, and groundwater SO2 4 is
assumed to be related to oxidation of disseminated pyrite.
Potentially harmful elements (As, Cd, Co, Cr, Hg, Ni, Se and
V) were below the detection limit; only Cu, Zn, Mn and Pb
were detected. In a few samples Pb and Mn were found to
be slightly above the threshold given by Council Directive
98/83/EC (EC, 1998) (Table 3). Pig manure and synthetic
fertilizers had different trace element contents: higher
contents of Fe, Zn, Cu and Mn in manure, and higher con-
tents of As, Cd, Cr or U among others, in chemical fertilizers
(Vitòria et al., 2004a; Otero et al., 2005, and references
therein). Although the measured potentially harmful ele-
ment concentrations could suggest a link with pig manure,
trace element contents are not a conclusive tracer, as soil
processes and plant uptake were not evaluated. Conse-
 L. Vitòria et al. / Applied Geochemistry 23 (2008) 3597–3611 3603

Fig. 2. (a) Groundwater piezometric surface in meters above the sea level (masl) corresponding to October 2001. (b) Isoconcentrations of NO
3 in mg/L of the
same date.

a 225
b 300

200
250
175

150 200
r 2 = 0.70
Ca 2+ (mg/l)

r 2 = 0.71
Cl- (mg/l)

125
150
100

75 100

50
50
25

0 0
0 50 100 150 200 250 300 350 400 0 50 100 150 200 250 300 350 400
NO3- (mg/l) NO3- (mg/l)

Fig. 3. (a) NO  

3 versus Cl , (b) NO3 versus Ca.

quently, the water chemistry alone did not allow identifi-
cation of the source of N in these groundwaters.
Chemical data have also been used to outline processes,

e.g. NO
3 =Cl ratio is used to distinguish between dilution
and denitrification in groundwater samples (Altman and
Parizek, 1995). This approach could not be applied in the
studied area, where sampling relied on production wells,
and piezometers were not available. The lack of well cas-
ing, unnecessary from a construction point of view but
which brought about the simultaneous exploitation of
the different aquifer layers, made the interpretation of
chemical data difficult. Consequently, in this context stable
isotopes become an essential tool to determine the origin
and fate of N compounds.
4.2. Stable isotopes
The isotopic compositions of water (dD, d18 OH2 O ), dissol-
2
ved NO 15 18 34 18
3 (d NNO3 ; d ONO3 ), dissolved SO4 (d SSO4 ; d OSO4 )
and dissolved inorganic C (d13 CDIC ) are shown in Table 4.
Compositions of 2H and 18O fall on the local meteoric water
line LMWL (Fig. 4) calculated with data from the Global
Network of Isotopes in Precipitation (GNIP) from stations
0818001 and 0818002 located in Barcelona (IAEA/WMO,
2004). Groundwater dD and d18 OH2 O values in the studied
area corresponded to the actual meteoric waters, from lo-
cal recharge, and were not affected by appreciable evapo-
ration. Because the weighted local rain was sampled in
Barcelona, near the coast, it was shifted towards higher
isotopic compositions.
The d13 CDIC values ranged from 16.4‰ to 12.5‰ with
a mean value of 14.1‰ – values within the known range
of d13 CDIC for groundwater (16‰ to 11‰, Vogel and
Ehhalt, 1963). d34 SSO4 showed a wide range of values (from
20.2‰ to +5.8‰), with a mean value of 9.4‰, and
d18 OSO4 varied from 1.3‰ to +8.7 ‰, with a mean of
+3.3‰. The d34 SSO4 and d18 OSO4 confirmed an origin of
SO2
4 related to oxidation of disseminated pyrite. Most
3604 L. Vitòria et al. / Applied Geochemistry 23 (2008) 3597–3611

Table 4
Isotopic data of sampled groundwater in Osona, October 2001

Sample d2H % d18 OH2 O % d15 NNO3 % d18 ONO3 % d34 OSO4 % d18 SSO4 % d13 CHCO3 %
PO-001 41.2 6.7 12.8 7.0 0.4 4.0 14.7
PO-004 39.8 6.6 14.1 7.6 12.2 3.5 14.8
PO-012 40.2 6.7 16.5 7.1 3.2 3.3 13.2
PO-020 42.5 6.6 16.2 9.6 10.3 4.0 15.3
PO-036 41.1 6.6 13.0 6.9 9.5 1.5 15.1
PO-037 48.0 7.4 14.1 7.4 0.1 4.6 13.8
PO-043 37.5 6.0 2.2 7.3 13.8 1.1 13.7
PO-051 43.0 6.7 12.9 7.3 14.5 1.1 15.4
PO-052 42.6 6.7 12.0 7.7 14.4 2.0 15.4
PO-059 43.3 6.9 20.9 9.6 10.8 1.9 13.5
PO-062 42.0 6.5 12.8 7.7 9.4 1.9 14.4
PO-063 41.6 6.6 14.1 6.9 13.2 2.2 12.5
PO-065 41.6 6.6 7.2 4.7 0.8 3.0 12.5
PO-070 41.9 6.7 11.2 6.9 10.3 4.8 14.0
PO-084 42.5 6.7 16.6 7.8 16.2 4.2 13.1
PO-086 44.1 6.8 15.8 8.9 19.1 3.1 14.7
PO-100 43.3 6.7 13.8 4.7 5.8 6.2 13.1
PO-101 43.7 6.7 18.4 8.9 5.0 8.7 15.5
PO-102 47.4 7.7 10.5 5.6 2.7 7.5 13.3
PO-103 44.5 7.0 – – 17.9 3.9 11.6
PO-104 40.7 6.6 14.2 7.4 2.0 6.0 14.1
PO-105 40.4 6.6 16.3 8.4 16.8 3.1 13.1
PO-106 46.2 7.2 – – 12.1 5.0 11.0
PO-107 38.7 6.5 12.1 8.3 14.5 3.0 14.2
PO-108 39.0 6.5 12.8 6.5 5.4 -3.2 15.1
PO-109 41.4 6.8 13.8 7.2 11.3 2.9 14.5
PO-110 40.0 6.6 14.4 7.1 13.5 2.2 14.8
PO-111 41.8 6.8 10.4 4.6 4.1 4.2 13.4
PO-112 41.2 6.6 9.5 8.0 18.6 8.7 13.8
PO-113 44.9 7.0 7.6 5.4 13.0 2.4 14.0
PO-114 40.4 6.5 14.1 8.6 10.5 2.9 13.9
PO-115 36.6 5.9 15.0 8.0 3.3 3.6 16.4
PO-116 37.1 6.4 9.2 6.1 20.2 1.0 16.2
PO-117 41.6 6.7 10.3 9.7 14.9 0.7 14.4
PO-118 37.5 5.7 – – 14.8 3.7 13.7
PO-119 41.5 6.5 14.7 8.3 9.4 4.9 12.7
PO-120 43.7 6.8 12.1 7.9 10.3 3.8 13.4
PO-121 43.4 6.8 13.6 8.2 12.0 2.7 13.6
Analytical precision ±1.5 ±0.6 ±0.3 ±0.4 ±0.4 ±0.5 ±0.2

samples showed negative d34 SSO4 values, in accordance
with disseminated pyrite in the aquifer (d34 SFeS2 between
5‰ and 28‰; Viñals et al., 2002; Pierre et al. 1994),
coupled with low d18 OSO4 values. The positive d34 SSO4 sam-
ples could be related to anthropogenic SO2 4 sources, most
likely linked to agriculture: fertilisers and pig manure. Re-
sults are further discussed in Section 4.4.
Whereas the d15N of groundwater dissolved NO 3 ranged
between +2.2‰ and +20.9‰, with a mean value of +13‰,
d18 ONO3 showed less variability, ranging from +4.6‰ to
+9.7‰, with a mean value of +7.4‰. Results are plotted
in a d15N vs d18 ONO3 diagram (Fig. 5a), together with the
isotopic composition of NO 3 from different sources
(Mengis et al., 2001, and references therein). The main
sources of N in the area are linked to agriculture, and the
isotopic composition of locally used fertilisers and manure
were in agreement with literature values. Chemical fertilis-
ers commonly used in the area have d15 NNO3 between 8‰
and +7‰, with a median value of 0.0‰, and d18 ONO3 values
around +22‰ (Vitòria et al., 2004b). By law, in the studied
area pig manure can be stored in pits for no more than 4
months, after which it is used as an organic fertiliser. Dur-
ing the storage period, changes in both d15 NNH4 and ½NHþ 4 ,
caused by NH3 volatilisation result in d15 NNH4 from +8‰ to
+15‰ (Vitòria et al., 2004a), giving a narrower range of val-
ues than those found in the literature.
As for the NO 15
3 sources, most of the samples had d NNO3

values higher than +8‰, suggesting a NO3 origin linked to
pig manure (only 3samples had lower values, which could
be influenced by synthetic fertilizers, organic soil NO
3 , or a
mixture of the two sources). The d18 ONO3 values were high-
er than expected, and the samples showed a coupled in-
crease in d15 NNO3 and d18 ONO3 indicating the occurrence of
denitrification processes. However, to validate these
assumptions regarding NO 3 sources, and estimate the
occurrence of natural attenuation, the isotopic fraction-
ation affecting N compounds must be evaluated.
4.3. Evaluation of fractionation processes affecting N
compounds
When pig manure or ammoniacal synthetic fertilizers
were applied on the fields, hydrolysis of urea and NH3 vol-
atilization took place (Reaction (1)) increasing the isotopic
composition of the residual NHþ 4 (Letolle, 1980). Although
it is a known fact that the fractionation factor between
 L. Vitòria et al. / Applied Geochemistry 23 (2008) 3597–3611 3605

18
O (o/oo)
0
-14 -12 -10 -8 -6 -4 -2 0

Rains >20 mm
Rains <20 mm -20
Local Meteoric Water Line
Groundwaters Oct 01
Weighted Local Rain
-40

D ( o/oo)
46 -60
.32
+7
18 O -8 -7,5 -7 -6,5 -6 -5,5
·
8 97 -30
7.2 -80
D=
-35

-40 -100

-45

-120
-50

Fig. 4. Isotopic compositions (dD, d18O) of groundwater together with the Local Meteoric Water Line and the Weighted Local Rain, calculated from
precipitation values since 1985 at meteorological stations 0818001 and 0818002 of Barcelona, of the Global Network of Isotopes in Precipitation – GNIP –
Data Base (IAEA/WMO, 2004).

NH3 gas and NHþ 4 is 1.034 at 25 °C (Mariotti et al., 1981), to
try to give a fractionation rate for a study area of only
38 km2 is senseless. Volatilization fractionation will de-
pend on the d15 NNH4 applied, the air temperature, humid-
ity, wind speed, etc.
ð1Þ
As explained in the previous section, the expected d15 NNH4
values for pig manure, taking into account volatilization
processes during the storage period, ranged from +8‰ to
+15‰ (Vitòria et al., 2004a). Further volatilization could
occur once pig manure or fertilisers were applied on the
fields, resulting in higher d15N values. Except for 3 samples
(with d15N < 8‰), all the Osona samples showed d15N in
agreement with pig manure values, and a group of samples
showed even higher values (d15N > +15%). In order to
determine if the d15N increase is due to volatilization or
to denitrification processes, the behaviour of the d18 ONO3
values must be also verified.
In the unsaturated zone, under aerobic conditions, the
residual NH4+ was completely nitrified (Reaction (2)). For
this reason it was accepted that there was no difference be-
tween NH4–d15N and the newly formed NO 3 composition
(Heaton, 1986), which eventually becomes mixed with
the initial pool of NO 
3 , from other N inputs, such as NO3 -
fertilizers.
2NHþ4 þ 3O2 ! 2NO2 þ 2H2 O þ 4Hþ
ð2Þ
2NO2 þ O2 ! 2NO3
During Reaction (2), according to laboratory experiments
carried out by Anderson and Hooper (1983), Hollocher
(1984), among others, the d18 ONO3 followed Reaction (3),
where it is assumed that no fractionation occurred during
the incorporation of O into the new molecules of NO 3
(Mayer et al., 2001).
d18 OðNO3Þ ¼ 2=3d18 OðH2OÞ þ 1=3d18 OðO2Þ ð3Þ
Mayer et al. (2001) suggest that Reaction (3) takes place
during chimiolithoauthotrophic nitrification, not during
heterotrophic nitrification, when less than ? of the O came
from water. This can be observed in systems where the
NHþ 4 content is limited and when a low rate of nitrification
exists, but this was not the case in the study area, as pig
manure application on the fields provided an unlimited
amount of NHþ 4 . Therefore, Eq. (3) can be applied, and
NO 18
3 formed should have d ONO3 values between +2.7‰
and +4.0‰. Instead, all of the samples had higher isotopic
compositions (>+4.6‰) (Fig. 5a). This can only be explained
by: (a) a higher d18 ONO3 resulting from the nitrification
reaction, (b) a mixture with another source of NO 3 (e.g.
synthetic NO3-fertilisers, or atmospheric NO3), or (c) the
occurrence of denitrification processes.
Slight enrichments in d18 ONO3 could be explained taking
into account the nitrification reaction, that is, differences in
the d18O of the soil O2 and/or the d18O of soil water, or be-
cause during the nitrifying reaction water of a different ori-
gin and composition was involved. Bacterial respiration
could also enrich the soil O2 in 18O, with a fractionation
factor of 1.015 (Guy et al., 1993). This higher soil O2 com-
position could explain the higher d18 ONO3 in groundwater.
According to Gat (1996), Hsieh et al. (1998), among others,
water retained in soil as humidity is usually affected by
evaporation. Desert soils can show an increase in d18O in
soil water of up to 10‰ compared to the adjacent ground-
water; this difference can reach 8‰ in low rain areas and
3606 L. Vitòria et al. / Applied Geochemistry 23 (2008) 3597–3611

a 24
NO3- from
Springs
NO3- in Springs Low d18O
20 fertilisers
Wells
Wells Low d18O
16
δ O-NO3 ( ‰ )
Aluvial
N/ O= 2.1 (1)
12 N/ O= 1.3 (2)
18

4 NO3- from NO3- from

NH4+ in soil organic NO3- from manure or
fertilisers N sew age

0
-3 0 3 6 9 12 15 18 21 24
15
N-NO3 ( ‰ )

b 12
Springs
Springs Low d18O
10
Wells
Springs Low d18O
δ N-NO3 ( ‰ )

8
Aluvial

6
15

0
0 50 100 150 200 250 300 350 400
-
NO3 (mg/l)

Fig. 5. (a) d15N vs. d18 ONO3 diagram. Boxes represent the isotopic composition of NO3- from different sources (Mengis et al. 2001, and references therein),
lines represent the changes in the isotopic composition due to denitrification processes. The different slopes represent the extreme values from the
literature for eN/eO (1) Böttcher et al., 1995, (2) Fukada et al. (2003) (see explanation in the text). (b) Nitrate concentration vs d15N.

2–3‰ in rainy areas. Effectively, if for the study area an an-
nual enrichment of 2–3‰ in d18O-soil water is assumed,
the theoretical composition of NO 3 formed by nitrification
would range from +4‰ to +6‰ – closer to the lower
d18 ONO3 values found in the studied samples. Finally, the
presence of water of a different origin could come from
the pig manure itself, which is 70–90% water in volume.
Although the measured d18 OH2 O from an open-air manure
pit of the area was in the range of groundwater (5.0‰,
Vitòria et al., 2004a), the evaporation of pig manure stored
in pits under atmospheric conditions could increase its iso-
topic composition. A combination of these 3 causes could
explain why even the lowest d18 ONO3 values detected are
slightly above the calculated values using Eq. (3). Regard-
ing mixing or denitrification processes to explain the ob-
served d18O values (up to +9.7‰), a mixture between
manure and fertilisers or atmospheric N2 would produce
higher d18 ONO3 and lower d15 NNO3 . Available mean values
for these sources are close to 0‰ and 3‰, respectively,
for d15N, and around +20‰ and +45‰, respectively, for
d18 ONO3 (Kendall and McDonnell, 1998). Studied samples
show an opposite trend with a coupled increase in
d18 ONO3 and d15 NNO3 suggesting that denitrification reac-
tions could have occurred. Therefore, coupling d15N with
d18 ONO3 values, it became apparent that part of the variabil-
ity in the N isotopic values was due to volatilization, but
changes could also be caused by denitrification.
The coupled use of d15N and d18 ONO3 confirms a NO 3 ori-
gin linked to organic manure. Although most samples had
isotopic values in the range of pig manure NO 3 , one sample
had a clear signature of synthetic fertilisers and 4 showed
intermediate values, suggesting a mix between these two
sources. Furthermore, a significant number of samples
had high d18 ONO3 values coupled with high d15 NNO3 suggest-
ing that denitrification is taking place.
4.4. Evaluation of the natural attenuation: the denitrification
Several processes, such as sorption, dilution and/or
denitrification, can produce a natural attenuation of NO
3.
 L. Vitòria et al. / Applied Geochemistry 23 (2008) 3597–3611 3607


Whereas only denitrification causes a real attenuation, pared to Cl. Focusing on the NO 3 =Cl ratio instead of the
sorption and dilution produce a decrease of the concentra- NO 3 concentration dispenses with the effect of new NO3


tion but the total amount of contaminant remains in the
system. Denitrification requires anoxic conditions (Clark
and Fritz, 1997), where NO 3 is reduced to N2 or N2O by or-
ganic matter (Reaction (10)) or sulphide (Reaction (11))
oxidation. In both cases, the decrease in NO 3 concentration
is coupled with an increase in the d15N and d18O of residual
NO 3 with a eN/eO ratio that ranges from 1.3 (Fukada et al.,
2003) to 2.1 (Böttcher et al., 1990), (Fig. 5a). Intermediate
enrichment ratios were reported by Mengis et al. (1999),
Cey et al. (1999), DeVito et al. (2000). Sorption and dilution
processes produce no change in the d15N and d18O of NO 3,
or, if so, it is negligible (Clark and Fritz, 1997).
4NO3 þ 5C þ 2H2 O ! 2N2 þ 4HCO3 þ CO2
14NO3 þ 5FeS2 þ 4Hþ ! 7N2 þ 10SO2
4 þ 5Fe
2þ
þ 2H2 O
The Osona samples show a positive trend in a d15 NNO3 vs
d18 ONO3 diagram (Fig. 5a), in accordance with denitrifica-
tion, thus ruling out that 15N variations are only due to
NHþ 15
4 volatilization. A linear relationship between d NNO3
and d18 ONO3 as reported in previous studies was not ob-
served, neither was there a negative correlation between
d15 NNO3 and NO 3 concentration (Fig. 5b), because samples
were a mix from the distinct aquifer levels, and the initial
concentration and the d15N in the recharge area could dif-
fer depending on volatilization processes and rates of
application. In order to confirm the existence of this natu-
ral attenuation process, d18 ONO3 is used as a tracer of deni-
trification. Although a comparison between d18 ONO3 and
NO 3 concentrations could not be carried out due to the ini-
tial variability of the NO3 concentration and the different
application rates. A comparison between d18 ONO3 and the
NO

3 =Cl ratio shows a negative correlation for most sam-
ples (Fig. 6). Those with higher d18 ONO3 show a lower
 and, in an anaerobic media, by the nitrate reduction (Reac-
NO 
3 =Cl ratio pointing to a relative decrease in NO3 com-
Springs Springs Low d18O
12 Wells Wells Low d18O
Aluvial
input, hence, this correlation indicates that a denitrifica-
tion reaction is taking place, producing an increase in the
isotopic composition and a decrease in the concentration.
The reactions controlling the denitrification process Eq.
(4) and (5) can be revealed, to some extent, by studying the
isotopic composition of the solutes involved in the reac-
tions: (d15 NNO3 ; d18 ONO3 ; d13 CDIC ; d18 OH2 O ; d34 SSO4 and
d18 OSO4 ). Denitrification by oxidation of organic matter
(the d13C of one pig manure sample analyzed was
23.8‰, Vitòria, 2004, in agreement with values for d13C
organic matter – between 20‰ and 30‰ – reported
by Trumbore and Druffel, 1995) should result in a decrease
of NO 13
3 and d CDIC together with a DIC increase. However,
ð4Þ
in the present case, these relationships were not observed
(Fig. 7). Instead, the measured values were in the range of
ð5Þ d13 CDIC in groundwater – within Vogel’s range of 16‰ to
11‰ (Vogel and Ehhalt, 1963)—, possibly resulting from
equilibrium between soil CO2 and carbonate dissolution
(0‰; Urquiola, 1994). Because the d13C of the DIC in a
carbonated aquifer is buffered, no relationship was ob-
served. Although the organic matter could be playing a role
in the denitrification process, this can neither be confirmed
nor discounted with the available information. Detailed
sampling using multipiezometers, or temporal sampling,
should be applied to give further insight.
Although thermodynamically the reaction of denitrifi-
cation by organic matter oxidation is more favourable than
by pyrite oxidation, some authors (Pauwels et al., 2000;
Postma et al., 1991) have found that the kinetics of these
reactions facilitate the latter. In an aerobic media, S oxida-
tion can take place by the decrease of the dissolved O2 in
water
4FeS2 þ 14O2 þ 4H2 O ! 4Fe2þ þ 8SO2
4 þ 8H
þ
ð6Þ
tion (11). Because this last reaction consumes protons, it is
believed that it can not occur in carbonated aquifers,
where pH is higher than 7 (Moncaster et al., 2000). How-
ever, if the Fe2+ produced is oxidized, following Reaction

10 Springs Springs Low d18O

Wells Wells Low d18O
-10
Aluvial Low NO3
δ O-NO 3 ( ‰ )

8 -11

-12
δ C-DIC ( ‰ )

6 -13
18

-14
r 2 = 0.67
4 -15
13

-16
2
-17
0 1 2 3 4 5 6
- -
NO3 /Cl -18
200 250 300 35 400 450 500 550
 -
Fig. 6. NO
ratio vs. d18 ONO3 . A negative linear correlation is observed
3 =Cl
HCO3 (mg/l)
(outliers were not considered for the regression calculation), indicating
that the increase in d18 ONO3 is linked to denitrification (see explanation in Fig. 7. HCO 13 13
3 vs d CDIC . Samples are in the range of d CDIC in ground-
the text). water (16‰ to 11‰, Vogel and Ehhalt, 1963).
3608 L. Vitòria et al. / Applied Geochemistry 23 (2008) 3597–3611

(13), a global Reaction (14) can be written where protons
could locally overcome the ones consumed by carbonate
dissolution. If that is the case, denitrification by pyrite oxi-
dation could occur in a carbonate aquifer. Based on the
studies of Bottrell et al. (2000) regarding the reduction of
SO2
4 , and (Lawrence and Foster 1986) concerning the
reduction of NO 3 , the catalyst bacterial activity would take
place on the surface of carbonate fissures, as the pore
diameters of the carbonates are too small to allow the col-
onization of the matrix aquifer
10Fe2þ þ NO3 þ 14H2 O ! 10FeOOH þ N2 þ 18H2 O
6NO3 þ 2FeS2 þ 2H2 O ! 3N2 þ 2FeOOH þ
In the studied area, most samples had negative values of
d34 SSO4 (Table 3), matching the composition of pyrite dis-
seminated in the aquifer materials (d34 SFeS2 between 5‰
and 28‰; Viñals et al., 2002; Pierre et al. 1994). Further-
more, SO2 4 formed via sulphide oxidation must show a
relationship between d18 OSO4 and d18 OH2 O , as water is the
source of 50–100% of the SO2 4 oxygen. Van Stempvoort
and Krouse (1994) defined the area, in a d18 OSO4 vs.
d18 OH2 O diagram, where SO24 derived from sulphide oxida-
tion must fall. The lower limit of this area was d18 OSO4 ¼
d18 OH2 O , assuming no enrichment during the incorporation
of O from H2O (eH2O = 0); the upper limit, using experimen-
tal data of abiotic and biotic oxidation of sulphides, was de-
fined as d18 OSO4 ¼ 0:62  d18 OH2 O + 9. Fig. 8b shows the area
defined by Van Stempvoort and Krouse (1994) together
with the analyzed samples, showing that SO2 4 is derived
from sulphide oxidation in most samples.
Due to the absence of evaporite outcrops in the area,
positive S values could be explained by an anthropogenic
ð7Þ
input. In this case, the most likely source is related to
4SO2
4 þ 2H þ
agricultural activities: fertilisers or pig manure. In the
ð8Þ d34 SSO4 versus d18 OSO4 diagram (Fig. 8a), samples plot
over a mixing line between SO2 4 derived from pyrite oxi-
dation and pig manure SO2 4 . Local pig manure samples
had d34 SSO4 between 0% and +5% (Otero et al., 2007), in
the range of literature values (0.9‰ to 5.8‰, Cravotta,
1997). To the authors’ knowledge, no data reports in
the literature are available for d18O of pig manure dis-
solved SO2 4 , except for one, +5.6‰, offered by Otero
et al. (2007). The relationship between sulphide oxida-
tion and denitrification is evidenced in a d34 SSO4 vs.

a 10
Springs
Springs Low d18O
8 Wells
Wells Low d18O
Aluvial
6
δ O-SO4 ( ‰ )

Low NO3
Pig manure
Pig manure
4
18

0 Sulphates derived from

sulphide oxidation

-2
-22 -18 -14 -10 -6 -2 2 6 10
34
S - SO4(‰ )

16
b Springs
14 Springs Low d18O
Wells
12
Wells Low d18O
10 Aluvial
δ O-SO4 ( ‰ )

8 Low NO3

4
18

Experimental area of
2 sulphates derived from
sulphide oxidation
0

-2

-4
-12 -9 -6 -3 0
18
O - H2O (‰ )

Fig. 8. (a) d34 SSO4 vs. d18 OSO4 of dissolved SO4. The box represents the SO4 derived from sulphide oxidation, and the shadowed area the isotopic composition
of manure (Vitòria et al, 2004a; Cravotta, 1997, and references therein) (b) d18 OH2 O vs d18 OSO4 diagram. The experimental area of SO4 derived from sulphide
oxidation is defined by Van Stempvoort and Krouse (1994). See explanation in the text.
 L. Vitòria et al. / Applied Geochemistry 23 (2008) 3597–3611
Springs Springs Low d18O
Wells Wells Low d18O
8 Aluvial
denitrification
4 not linked to
pyrite oxidation
0 study area, the reactions controlling the denitrification
δ S-SO4 ( ‰ )
-4
-8
34
-12
-16
pyrite oxidation
not linked to
detnirification
-20
-24
2 4 6 8 10
18
O - NO3( ‰)
Fig. 9. d18 ONO3 vs. d34 SSO4 diagram. Samples with lower d18 ONO3 show
positive d34 SSO4 values, and samples with higher d18 ONO3 show a trend to
negative d34 SSO4 values, confirming the link between pyrite oxidation and
denitrification.
d18 ONO3 diagram (Fig. 9). Samples with higher d18 ONO3 ,
indicating a higher degree of denitrification, have lower
d34 SSO4 . It is worth noting that two samples have
d34 SSO4 below 12‰, and low d18 ONO3 indicating that,
for these samples, sulphide oxidation is not linked to
denitrification. Finally there is one sample showing high
d18 ONO3 , and positive d34S, this sample is from an alluvial
aquifer. In this case, the denitrification reaction would be
related to organic matter oxidation, though using DIC
and d13C this could not be confirmed nor discounted.
A quantification of the attenuation could be done by
taking into account that the denitrification reaction de-
scribes a Rayleigh distillation process (Reaction (9)) where
e is the isotopic enrichment factor that depends on the
aquifer materials and characteristics.
d15 Nresidual ¼ d15 Ninitial þ e lnð½NO3 residual =½NO3 initial Þ
Lehmann et al. (2003) presented a summary of the pub-
lished enrichment factors for both N and O in denitrifica-
tion processes in experiments with pure culture,
groundwater, and a marine environment. Values for the
eN ranged from 0‰ to 40‰, although in groundwater
studies the range is narrower, between 4.7‰ and
30‰. Only two values of eO are reported: 8‰ (Böttcher,
1990) and 18.3‰ (Mengis et al., 1999).
In the studied case, Eq. (9) cannot be applied, because
samples are a mix between different aquifer levels and a
flow path could not be determined. Therefore, only a rough
estimation of the degree of attenuation could be calcu-
lated. For example, taking an initial d15N composition be-
tween +8‰ and +15‰ for groundwater contaminated by
pig manure (Vitòria et al., 2004a), and using the extreme
enrichment factors from the literature, the natural attenu-
ation of contamination could be estimated varying be-
tween 20–80% for an e of 4.7‰ (Mariotti et al., 1988) or
between 5–25% for an e of 30‰ (Vogel et al., 1981). The
real enrichment factor for the Osona aquifer should be ob-
tained by studying the chemical and isotope data from a
3609
groundwater flow line, by means of a detailed temporal
study, or by using a comprehensive multipiezometer
network.
To predict the future evolution of contamination, it is
important to determine the enrichment factors in the
process, and the kinetics of these reactions. Moreover,
knowing the quantity of pyrite in the aquifer materials,
its availability to react through porosity, and its oxidizing
kinetics, should allow considering the possibility of in-
duced remediation techniques. Recent studies on chemi-
cal mediums in permeable reactive barriers for
groundwater nitrate remediation have shown that zero-
valent Fe can produce NHþ 4 ions under laboratory condi-
tions (Su and Puls, 2004). The present study showed the
12 potential use of pyrite for permeable reactive barriers, at
sites where the natural attenuation is limited. Future re-
search could focus on laboratory studies in order to
determine the kinetics of pyrite dissolution in the deni-
trification process.
5. Conclusions
In Osona, the use of pig manure as an organic fertiliser
has caused diffuse NO 3 pollution of groundwater with
NO 3 concentration up to 366 mg/L. Nearly 75% of the
sampled groundwater had NO 3 contents that exceed the
50 mg/L threshold for drinking water. The coupled use
of d15 NNO3 and d18 ONO3 confirmed the link between
groundwater NO 3 pollution and pig manure. While most
samples had isotopic values in the range of pig manure
NO 3 one sample had a clear signature of synthetic fertilis-
ers and 4 showed intermediate values, suggesting a mix
between these two sources. The study of d18O in nitrates,
groundwater and rain water has enabled evaluation of the
nitrification process and identification that there was a
pre-existing enrichment in 18O of the soil O2 and H2O in
the area, resulting from bacterial respiration and evapora-
ð9Þ
tion. Nitrate isotopic data suggested that denitrification
processes were taking place. Moreover, the occurrence
of this process was confirmed using d18 ONO3 coupled with

the NO 3 =Cl ratio, thus avoiding the influence of continu-

ous NO3 inputs. A further insight into denitrification pro-
cesses was obtained using a multi-isotopic approach,
analyzing the ions involved in denitrification reactions
(d15 NNO3 ; d18 ONO3 ; d34 SSO4 ; d18 OSO4 ; d18 OH2 O and d13 CDIC ). This
approach showed a link between denitrification and pyr-
ite oxidation; however, the role of organic matter oxida-
tion could not be confirmed nor discarded with the
available information. In order to quantify the natural
attenuation, to foresee the evolution of the contamina-
tion, and to implement induced remediation techniques,
it is necessary to determine the enrichment factor for
the aquifer and perform detailed studies on pyrite avail-
ability and its oxidation kinetics. To conclude, it should
be emphasized that in areas where aquifers are complex,
and with no good infrastructure (e.g. multi-piezometers)
to describe groundwater dynamics, isotopes provide
effective tools to trace sources of contamination and
study processes affecting N.
3610 L. Vitòria et al. / Applied Geochemistry 23 (2008) 3597–3611
Acknowledgements
This study was financed by the CICYT, project REN-2002-
04288 and CGL2005-08019-CO4-CO1/HID, granted by the
Spanish Government, and partially by the -SGR2005-0933
from the Catalan Government. We would like to thank the
Agencia Catalana de l’Aigua (Catalan Water Agency) for
their support (especially A. Ginebreda) and the Serveis
Cientificotècnics of the Barcelona University (Spain) for
their analytical support.
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