w a t e r r e s e a r c h 4 7 ( 2 0 1 3 ) 2 6 6 1 e2 6 7 5

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The use of d15N and d18O tracers with an understanding of

groundwater flow dynamics for evaluating the origins and
attenuation mechanisms of nitrate pollution

Takahiro Hosono a,*, Takahiro Tokunaga b, Makoto Kagabu b, Haruhiko Nakata b,

Takanori Orishikida b, In-Tian Lin b, Jun Shimada b
a
Priority Organization for Innovation and Excellence, Kumamoto University, 2-39-1 Kurokami, Kumamoto 860-8555, Japan
b
Graduate School of Science and Technology, Kumamoto University, 2-39-1 Kurokami, Kumamoto 860-8555, Japan

article info abstract

Article history: During early 2000, a new analytical procedure for nitrate isotopic measurement, termed
Received 7 September 2012 the “denitrifier method”, was established. With the development of the nitrate isotope
Received in revised form tracer method, much research has been reported detailing sources of groundwater nitrate
4 February 2013 and denitrification mechanisms. However, a shortcoming of these tracer studies has been
Accepted 7 February 2013 indicated owing to some overlapping of isotope compositions among different source
Available online 19 February 2013 materials and denitrification trends. In order to reduce these uncertainties, we examined
nitrate isotope ratios within a frame of “regional groundwater flow dynamics” to eliminate
Keywords: unnecessary uncertainties in elucidating nitrate sources and behaviors. A total of 361
Groundwater samples were collected from the Kumamoto area: the circulated groundwater system with
Flow dynamics a scale of 103 km2 in southern Japan. Subsequently, the nitrate pollution was examined
Nitrate pollution within the above-mentioned framework. As a result, a reasonable identification of the
Stable isotope ratios sources and attenuation behaviors (both denitrification and dilution) of groundwater ni-
Denitrification trate pollution was obtained over the study area. This study demonstrates that the use of
nitrate isotope tracers efficiently improves with a comprehensive understanding of
groundwater flow dynamics. The approach emphasized in this study is important and
should be applicable in other areas.
ª 2013 Elsevier Ltd. All rights reserved.

1. Introduction Magaritz, 1986; Mariotti et al., 1988; Smith et al., 1991). By

Groundwater nitrate pollution is one of the most prevalent
and on-going global natural water environmental problems
(Rivett et al., 2008). The comprehension of nitrate pollution
sources and natural attenuation mechanisms is of prime
importance for better management of groundwater resources.
The stable isotope ratio of nitrogen in nitrate (d15 NNO3 ) has
been used since the 1970s as a powerful tracer revealing
concealed pollution sources and to understand the mecha-
nisms of denitrification (i.e. Kreitler, 1979; Kaplan and
the early 1990s, the combined use of d15 NNO3 with the stable
isotope of oxygen in nitrate ðd18 ONO3 Þ was proposed and
applied for groundwater study (for example Böttcher et al.,
1990; Wassenaar, 1995; Aravena and Robertson, 1998; Fukada
et al., 2003). In addition, the newly established analytical
procedure of nitrate isotopic measurement termed the
“denitrifier method” (Sigman et al., 2001; Casciotti et al., 2002)
enables the determination of dual d15 NNO3 and d18 ONO3 using a
smaller volume of water (<8 ml) with lower NO 3 concentra-
tion in quicker preparation time than previous methods. The

* Corresponding author. Tel./fax: þ81 96 342 3935.

E-mail address: hosono@kumamoto-u.ac.jp (T. Hosono).
0043-1354/$ e see front matter ª 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.watres.2013.02.020
2662 w a t e r r e s e a r c h 4 7 ( 2 0 1 3 ) 2 6 6 1 e2 6 7 5
nitrate isotope tracer method has become established as a
better tracing tool and has increasingly been applied in many
parts of the world (i.e. Umezawa et al., 2008; Otero et al., 2009;
Hosono et al., 2011a).
Within a nitrate isotope tracing study, the pollution sour-
ces can be estimated by isotopic comparison between the
water samples and possible source materials (Kendall, 1998).
The isotopic comparison can also be used as a denitrification
indicator by analyzing isotopic enrichment trends of water
samples (Kendall, 1998). However, source distinguishment
becomes difficult when different possible source materials,
such as manure and sewage, have a similar isotope signature
(Xue et al., 2009; Fenech et al., 2012; Minet et al., 2012). More-
over, if isotopic compositions overlap between the source
characteristics and denitrification trends, this distinction be-
comes difficult (Xue et al., 2009). These difficulties complicate
data interpretation. Therefore, it has recently been argued
that a new approach and concept are needed to reduce the
above mentioned shortcomings in nitrate isotope tracer
studies (Xue et al., 2009; Fenech et al., 2012; Minet et al., 2012).
In a circulated groundwater system, dissolved nitrate is
introduced to groundwater at the recharge zone and trans-
ported through the lateral flow zone to the discharge zone
within a frame of “groundwater flow dynamics” (Böhlke and
Denver, 1995; Böhlke et al., 2002; Hosono et al., 2011b).
Therefore, having a good understanding of the three-
dimensional groundwater flow pathway is very important
when tracing complicated nitrate sources and behaviors using
a nitrate isotope tracer. In addition, it is important to prepare
detailed land-use information for the target area, especially
for the assessment of potential impacts from manure and
sewage (Xue et al., 2009; Fenech et al., 2012; Minet et al., 2012).
The nitrate isotope tracer study method with a combined use
of the above mentioned control information appears to be an
efficient approach for reducing uncertainties in the past study.
However, emphasis has not been clearly placed on its
importance in previous articles.
Kumamoto City is located in the central part of the Kyushu
Islands in southern Japan. The city is known as the largest
groundwater utilization region in Japan where about one
million people in and around the city depend entirely on
groundwater for drinking purpose (Oshima, 2010; Shimada,
2012). However, the monitoring data for groundwater quality
show that nitrate concentrations have been increasing since
1970s (Tomiie et al., 2009, 2011). For example, NO 3 concen-
trations for some wells (Fig. 1a) increased by a factor of 2e7
during the last 40 years (Appendix 1). Although nitrate source
controls have been implemented since the 1990s, such as
control of fertilizer applications and the construction of bar-
riers around manure storage sites, groundwater NO 3 is still
increasing and becoming an increasingly serious environ-
mental issue (Kumamoto Prefecture and Kumamoto City,
2005; Kumamoto City Waterworks and Sewerage Bureau,
2008; Kumamoto City, 2009; Oshima, 2010). However, the
principal causes of the pollution and natural attenuation
mechanisms have not been fully understood.
This study presents a comprehensive amount of nitrate
isotope data (total of 361 samples) for groundwater (confined
and unconfined) and comparison water (spring and river
water) during different seasons, and possible source materials
(chemical fertilizers, manures, and sewage water) taken from
the Kumamoto area. These data were used within a tracer
study in the context of groundwater flow dynamics. We firstly
tried to understand the general view of the origins of ground-
water nitrate pollution, and then examined detailed attenua-
tion mechanisms along the flow paths by denitrification as
well as by mixing of the different source water. The results
from these investigations were used to emphasis the useful-
ness of the above approach for reducing uncertainties
encountered in previous studies, which is the prime objective
of this study. The approach could be applicable in future
studies for a better understanding of the sources and attenu-
ation mechanisms of groundwater nitrate pollution.
2. Outline of the research area
2.1. Topography and climate
The Kumamoto groundwater area is defined as an area
(945 km2) surrounded by the divide of the Shira River water-
shed to the north, the Midori River to the south, the Aso
Mountain (1592 m) to the east, and the Ariake Sea and Kinpo
Mountain (665 m) to the west (Fig. 1a). The altitude of this area
tends to decrease westward from land to ocean. Importantly,
volcano-pyroclastic and lava flow deposits derived at the time
of the Aso Caldera formation formed three major highlands
with altitudes around 100 m: the Kikuchi and Takayubaru
highlands extending westwards and the Ueki highlands to the
north of the Kumamoto area (Fig. 1a). In addition, volcanic
flow deposits of the Pre-Aso activity formed small hills in the
central area such as Tatsuta Mountain (Fig. 1a). Kumamoto
had an average annual temperature and precipitation of
16.9  C and 1986 mm, respectively during 1980e2010. The
rainy season occurs from JuneeJuly with around 40% of the
total annual precipitation occurring during this period.
2.2. Geology
The study area is composed of four major geological constit-
uents (Fig. 2): the Paleozoic basement of metamorphic and
sedimentary rocks; the Pre-Aso volcanic rocks of Tertiar-
yeQuaternary age; the Quaternary Aso volcanic rocks; and
overlaying alluvium deposits. Paleozoic basement rocks do
not outcrop at the surface within the study area. The Pre-Aso
volcanic rocks generally consist of lavas and tuff breccia (Ono
and Watanabe, 1985; Miyoshi et al., 2009), while the Aso vol-
canic rocks are composed of pyroclastic deposits and flow
lavas (Ono and Watanabe, 1985). The former rocks are present
at/around the Kinpo and Tatsuta Mountains (Fig. 1a), while
latter rocks are distributed around the eastern side of the
Kinpo Mountain and the western side of the Aso Caldera. Both
rock types have a wide chemical range between basalticer-
hyolitic compositions (Ono and Watanabe, 1985; Miyoshi
et al., 2009). The Aso volcanic rocks are subdivided into four
units according to eruption stages: Aso-1 (270 ka); Aso-2 (140
ka); Aso-3 (120 ka); and Aso-4 (89 ka) (Ono and Watanabe,
1985). Of these, the Aso-2 unit is unique in its composition
of andesitic lava with joints and porous structures. It is also
important to note that clay impermeable layers of lacustrine
 w a t e r r e s e a r c h 4 7 ( 2 0 1 3 ) 2 6 6 1 e2 6 7 5 2663

Fig. 1 e A map showing (a) topography and major groundwater flows and (b) land-use and sampling locations in the
Kumamoto area. Solid contour lines with numbers in Fig. 1a represent groundwater levels (m, above sea level) calculated
with a groundwater flow model using a surface and subsurface coupling flow simulator (GETFLOWS: Tosaka et al., 2000).
Details for groundwater flow model will be shown in separated paper.

sediments are widely distributed between the Aso-3 and Aso-
4 units (Fig. 2). The other clay layer of marine origin, the Ariake
clay layer, is distributed over the Kumamoto plain especially
within the coastal zone.
2.3. Hydrogeology
The highly permeable structure of Aso-4 pyroclastic deposits
at the Ueki, Kikuchi, and Takayubaru highlands result in these
areas becoming major groundwater recharge sources within
Kumamoto (Kumamoto Prefecture and Kumamoto City, 1995;
Taniguchi et al., 2003). From a hydrogeological point of view,
the Aso volcanic rocks and alluvial sedimentary deposits are
the major aquifers which overlie impermeable Paleozoic
basement and Pre-Aso volcanic rocks (Kumamoto Prefecture
and Kumamoto City, 1995) (Fig. 2). The aquifers are divided
into two main types: unconfined and confined aquifers (Fig. 2).
The former aquifer type is composed of unwelded Aso-4
2664 w a t e r r e s e a r c h 4 7 ( 2 0 1 3 ) 2 6 6 1 e2 6 7 5

Fig. 2 e Hydrogeological cross sections between AeA0 and BeB0 in Fig. 1. Arrow in the figures represents groundwater flow
direction at each grid obtained by the results of a groundwater flow model calculation using a surface and subsurface
coupling flow simulator (GETFLOWS: Tosaka et al., 2000). Size of arrow corresponds to relative velocity of groundwater flow.
Values for estimated enrichment factor ε are also shown in figures for each groundwater flow zone (see text for details).

pyroclastic and alluvial sedimentary deposits, which overlie
an aquiclude of impermeable lacustrine sediment layer. The
latter aquifer type consists of Aso-2 porous andesitic lava and
pyroclastic deposits of Aso-1, Aso-2, and Aso-3 units. As an
important consideration, the aquiclude is not continuously
distributed under the mid-stream area of the Shira River (Figs.
1a and 2). Due to the very high downward infiltration capacity
of this area (w500 mm d1), a large amount of water is
recharged from the river into the deep aquifer (Tanaka et al.,
2010) (Fig. 2). Groundwater is situated generally at w50 m
under the surface for unconfined and around 60e200 m for
confined aquifers. The groundwater levels for both aquifers
show 5e10 m seasonal change during a year. In general, the
high water season is from AugusteNovember and low water
season is from JanuaryeApril.
2.4. Groundwater flow and age
Groundwater flow systems are well understood within the
study area based on long-term water level monitoring and
groundwater modeling (Figs. 1a and 2). The most significant
flow system of confined groundwater is the flow AeA0 in Figs.
1a and 2. This main flow can be classified into 4 zones (Figs. 1a
and 2): A1- the flow from the recharge area of the Kikuchi
highland to the mid-stream area of the Shira River; A2- the
mixing zone of groundwater from zone A1, recharged water
from the Takayubaru highlands and the southwestern feet of
Aso Mountain, and the recharged water from the Shira River;
A3 e the southwestern lateral flow through the Aso-2 volcanic
lava aquifer toward the discharge area of the Ezu Lake with a
discharge rate of 1.5  108 m3 y1 (Shimada, 2012); and A4 e
the flow from the discharge area to the coastal stagnant area.
The flow BeB0 is the other important confined groundwater
flow system within the study area (Figs. 1a and 2), which can be
classified into 3 zones along the flow direction: B1 e southward
flow from the recharge area of the Ueki highlands to the point of
basement lifting; B2 e the flow across the basement uplifting
toward the entrance of plain area; and B3 e southwestward
flow to the coastal stagnant area. The number of the monitoring
wells for the unconfined groundwater is limited compared to
 w a t e r r e s e a r c h 4 7 ( 2 0 1 3 ) 2 6 6 1 e2 6 7 5
Fig. 3 e Comparison of NOL
3 concentrations among
groundwater, spring water, and river water samples from
the Kumamoto area.
that of confined groundwater. However, the general view of the
flow character is similar to each other, which is basically
controlled by regional geology and topography. According to
unpublished 3H, chlorofluorocarbon, SF6, and 85Kr age data
(Kagabu et al., personal communication), the groundwater ages
range from few years at the recharge zone to 25e30 years at the
discharge zone. The stagnant groundwater under the coastal
plain (zone A4 and B3) is thought to be old groundwater with an
age more than 50 years.
2.5. Land-use and groundwater use
Land-use in the study area is shown in Fig. 1b. The western
mountain foot of the Aso Mountain is widely covered by forest
and grassland, with anthropogenic loading being relatively
small in this area. Vegetable farming is the main land-use in
the Ueki, Kukuchi, and Takayubaru highlands, which has
been present since the early 1900s. In addition, livestock
farming has increased in this area since the 1970s. For
example, the numbers of livestock (cows and pigs) increased
by a factor of about 30 during the last 40 years (Tomiie et al.,
2011). On the contrary, paddy is distributed at lowland areas
along rivers and plain area (Fig. 1).
2665
The study area has population of about one million. People
in the area depend entirely on confined groundwater for do-
mestic water resources including drinking purpose (Oshima,
2010). A total of 8  107 m3 y1 of groundwater is pumped
from 58 pumping stations installed within the study area and
distributed to each house. The percentage of population with
access to municipal sewage treatment plant in the city is
about 86%. Treated water at several sewage treatment plants
is released directly to nearby rivers.
3. Sampling and methods
The sampling locations are shown in Fig. 1b. A total of 239
groundwater samples (83 for unconfined and 156 for confined
groundwater) were collected during November 2009eNovember
2011. To account for the high water season, groundwater sam-
ples were collected during November 2009, October 2010, and
OctobereNovember 2011, respectively. Representing the low
water season, samples were collected during JanuaryeMarch
2011 at the same wells as surveyed during the high water season
in OctobereNovember 2011. A total of 86 river and spring water
samples were also collected during April 2011 (low water sea-
son) and July 2011 (high water season), respectively. Further-
more, 16 sewage effluent samples for both pre-treated and
treated water from five representative sewage treatment plants,
ten chemically synthesized NPK fertilizer samples, and ten
manure samples were obtained for possible source materials.
Collected fertilizers are the most representative fertilizers
generally used over the study area for fruits, vegetables, and
rice. Manure samples were collected in the livestock farms at
Ueki, Kikuchi, and Takayubaru highlands.
Groundwater samples were collected using an internal or
portable pump (MP1, Grundfos). Prior to sampling of borehole
water, old water in the pipes (w5  103 l; the volume was calcu-
lated based on borehole depth and casing size) was pumped out
to collect fresh water as much as possible. Parameters such as
temperature, electrical conductivity (EC), pH, oxidation reduction
potential (ORP), and dissolved oxygen (DO) were continuously
monitored during pumping using field meters (HORIBA D-54) and
water samples were collected after these values became con-
stant. Collected water samples for analysis of nitrate concen-
trations and nitrate isotope ratios were filtered through 0.2 mm
cellulose-acetate filters before storing in 50 ml volume poly-
ethylene bottles. Filtered samples were kept immediately in ice
box after sampling, transported to laboratory within a day, and
stored in freezer with a constant controlled temperature of
15  C until just before analysis. Manures and chemical fertil-
izers were freeze dried and pulverized using an agate mortar.
NO3 concentrations were measured by ion chromatography
(Compact IC 761, Metrohm, Switzerland). The d15 NNO3 and
d18 ONO3 compositions were determined following the “denitrifier
method” of Sigman et al. (2001) and Casciotti et al. (2002). Briefly,
a sample containing NO 3 was converted to nitrous oxide (N2O)
by denitrifying bacteria that lack N2O-reductase activity. The
N2O was stripped from the sample vial using helium carrier gas,
purified using cryogenic trapping (Thermo Fisher Precon Sys-
tem), chromatographically separated (Thermo Fisher Gas
Bench), and analyzed using mass spectrometry (Thermo Fisher
Delta V Advantage). The ratios of 15N:14N and 18O:16O were
2666 w a t e r r e s e a r c h 4 7 ( 2 0 1 3 ) 2 6 6 1 e2 6 7 5

Fig. 4 e Seasonal comparison between the high and low water season for (a) NOL 15 18 15
3 , (b) d NNO3 , (c) d ONO3 , and (d) dual d NNO3
18
and d ONO3 compositions. Two solid lines in Fig. 4d represent isotopic fractionation trends during denitrification with
d15 NNO3 :d18 ONO3 of 1.3:1 and 2.1:1, respectively.

expressed relative to atmospheric nitrogen for nitrogen and
Vienna Standard Mean Ocean Water (V-SMOW) for oxygen.
Isotope values were calibrated using the United States Geological
Society (USGS) and International Atomic Energy Agency (IAEA)
internationally recognized nitrate standards: USGS-34, USGS-35,
and IAEA-N3. Based on replicate measurements of standards
and samples (n ¼ 288), the analytical precisions for d15 NNO3 and
d18 ONO3 were better than 0.2& and 0.3&, respectively. The
nitrogen isotope ratio for powdered samples was determined by
mass spectrometry interfaced with a CNS elemental analyzer
(Thermo Fisher Flash 2000). Analytical precision of the analysis
was better than 0.15&.
4. Results
4.1. Nitrate concentrations
The groundwater NO
3 concentrations ranged between 0 and
73 mg l1 and only 3 samples among the total analyzed
samples (n ¼ 239) exceeded the World Health Organization
limit of 50 mg l1. Therefore, current nitrate concentration
level was judged to be generally lower than environmental
criteria, although increasing trend of concentrations is a
threat to its drinking water supply in future for many bore-
hole sites (Tomiie et al., 2009, 2011) (Appendix 1). Similar
frequency patterns were observed for nitrate concentrations
between confined and unconfined groundwaters (Fig. 3). The
groundwater tended to be more NO 3 enriched than river
water but showed a similar concentration range to spring
water (Fig. 3). NO 3 concentrations basically were not
changed between water samples collected at high and low
water seasons (Fig. 4a). The highest NO 3 concentrations for
both confined and unconfined groundwater samples were
found in the recharge areas at the Ueki, Kikuchi, and
Takayubaru highlands where vegetable and livestock farms
are highly concentrated (Fig. 5a). It was also observed that
NO 3 concentrations clearly decreased along the ground-
water flow path from rechargeedischarge/stagnant zones
(Fig. 5a).
 w a t e r r e s e a r c h 4 7 ( 2 0 1 3 ) 2 6 6 1 e2 6 7 5 2667

4.2. Nitrate isotope ratios

The d15 NNO3 and d18 ONO3 of groundwater samples ranged be-
tween 6e46& and 3e48&, respectively (Fig. 6). An isotopic
compositional gap was not found between confined and un-
confined groundwaters (Fig. 6). Groundwater samples with
moderate to high NO 3 concentrations tended to have narrow
isotope compositions (d15 NNO3 ¼ 2e8& and d18 ONO3 ¼ 3e5&)
(Fig. 6), which are similar to the compositional ranges of
spring water samples. In contrast, the samples with the
lowest NO 3 concentrations tended to have higher isotopic
compositions (d15 NNO3 ¼ 8e46& and d18 ONO3 ¼ 5e48&), sug-
gesting the occurrence of denitrification. The spring and river
water d18 ONO3 compositions were similar to each other,
whereas d15 NNO3 compositions of river water were clearly
higher than those of spring water (Fig. 6).
Seasonal gaps of d15 NNO3 and d18 ONO3 were not observed for
all water samples, except groundwater samples showing
highly fractionated signatures (Fig. 4b and c). These ground-
water samples were unique in that the fractionation ratios of
d18 ONO3 to d15 NNO3 were highly variable (Fig. 4d). It has been
commonly thought that the fractionation ratio of
d15 NNO3 :d18 ONO3 during denitrification ranges within 1.3e2.1:1
(Aravena and Robertson, 1998; Mengis et al., 1999; Böttcher
et al., 1990; Fukada et al., 2003). However, some groundwater
samples collected in our study showed more variable ratios
than the reported range (Figs. 4 and 6). In fact, the cause of
variability of fractionation ratios is becoming recognized as a
matter of discussion (Kool et al., 2011), which mechanism has
not been solved yet. This will not be discussed in this paper;
however, results of this study suggest that d15 NNO3 and d18 ONO3
values of groundwater should be regarded as subject of vari-
ation during denitrification over different seasons. The
calculation of the average value is one of the approaches of
data treatment as shown in this study.
The nitrogen isotope ratios of the possible source materials
showed their own signatures with some compositional vari-
ations (Fig. 6). Remarkable isotopic compositional changes
within collected fertilizer samples were not found among
different production companies, type of farm products used
for, and sampling areas. Similarly, isotope compositions of
manure samples did not change depending on their sampling
areas. Moreover, no isotopic changing tendencies were
observed for sewage waters collected at different conditions.
It could be considered from these observations that nitrogen
isotope variation for each source material regards as isotopic
heterogeneity of material itself, but is not due to the factors of
space and probably of time.
The compositional range of d15N for chemical fertilizers
was similar to that of groundwater samples showing high
NO 1
3 concentrations (>30 mg l ), whereas sewage and
manure samples showed higher values (Fig. 6). The
groundwater samples tended to increase in d15 NNO3 (and
d18 ONO3 ) with decreasing of NO 3 and DO concentrations
along the flow direction from rechargeedischarge/stagnant
zones (Fig. 5). The pollutant input locations of manure with
high d15 NNO3 should be in/around the recharge highlands
area. The results indicated that increasing groundwater Fig. 5 e Distribution map for (a) NOL 15
3 , (b) d NNO3 , and (c) DO

d15 NNO3 in the plain and coastal areas is not due to the in the study area. Arrows indicate the major flow
contribution of manures. directions of the confined groundwater.
2668 w a t e r r e s e a r c h 4 7 ( 2 0 1 3 ) 2 6 6 1 e2 6 7 5
Fig. 6 e Nitrate isotope comparison for water samples with possible source references. Note that the data shown with an
asterisk are taken from Hirohata et al. (1999).
5. Discussion
5.1. Source of nitrate pollution in groundwater
It is clear from the observed nitrate distributions (Fig. 5a) that
groundwater nitrate for both confined and unconfined
groundwater with NO 3 concentrations >30 mg l
1
was
transported through the recharge area where vegetable and
livestock farms are widely distributed. This groundwater is
characterized by DO concentration values >6 mg l1 (Fig. 5c).
Under such conditions, it is commonly considered that deni-
trification would not preferentially occur (Spalding and Exner,
1993; Cey et al., 1999; McCallum et al., 2008). Importantly, such
high NO 1 15
3 groundwater (>30 mg l ) generally has d NNO3 and
18
d ONO3 compositions within 2e8& and -3e5&, respectively
with no significant isotopic fractionation between them
(Fig. 6). Therefore, it can be roughly estimated that the effects
of denitrification were not significant. From an isotopic com-
parison (Fig. 6), it is most likely that concentrated nitrate of
groundwater as well as those of spring water (where DO
concentration was in general >8 mg l1) was dominantly
originated from long-term application of chemical fertilizers
to the vegetable farms. However, with the fact that these high
nitrate water samples showed some d15 NNO3 variation (2e8&)
partial contributions of manures from livestock farms and
organic fertilizers cannot be neglected.
One confined groundwater sample at the recharge area
showed a high d15 NNO3 (14.9&) and NO 1
3 (31 mg l ) content
(Fig. 6). This isotope composition corresponds with that of
manure. However, from its elevated d18 ONO3 (8.7&) value and
depleted DO concentration value (1.3 mg l1), it can be sug-
gested that the sample properties were due to the denitrifi-
cation of the nitrate that originated from chemical fertilizers
under a sporadically anaerobic environment. The number of
houses is limited within the farming area. Even if some houses
are not connected to the sewer, domestic effluents generally
drain to nearby rivers through side ditches.
Leakage from sewage pipes is another possible trans-
portation source of domestic wastes in urbanized areas
(Lerner, 2002; Wakida and Lerner, 2005). In Kumamoto, the
downward flowing groundwater from the recharge area
generally did not display clear tendency of NO 3 concentration
increase with an increase of d15 NNO3 in urbanized areas (Fig. 5).
Therefore, it is likely that the contribution of sewage leaked
from sewage pipes is not the prime source of nitrate, although
 w a t e r r e s e a r c h 4 7 ( 2 0 1 3 ) 2 6 6 1 e2 6 7 5 2669

Fig. 7 e Changes of NOL 15 18 0 0

3 , DO, d NNO3 , and d ONO3 with groundwater flow (a) AeA and (b) BeB . Plots in Fig. 7 correspond to
groundwater samples collected at borehole sites within the solid lines in Fig. 1. The parallel axis indicates sampling points
in order of groundwater flow direction for AeA0 and BeB0 , respectively. The order was decided in Fig. 1 from the connecting
positions between the lines AeA0 (or BeB0 ) and the perpendicular line from each site. Compositional variation in each site
represents seasonal variation.

some contribution of it is suggested by the analysis of chem-
ical markers (artificial sweeteners such as acesulfame K)
(Nakata et al., personal communication), which is a more
sensible tracer of sewage source, and by the analysis with flow
systems (discussed later). Treated wastewater from sewage
treatment plants, which still show high NO 3 concentrations in
the order of few tens of mg l1, drain directly into nearby
rivers. As mentioned previously, river water (with DO con-
centrations >8 mg l1) showed higher d15 NNO3 compositions
than do spring water and groundwater with high NO 3 loads on the groundwater flow simulation model and by
concentrations > 30 mg l1 (Fig. 6). It is reasonable to conclude
that nitrate in river water was partly derived from wastewater
of sewage origin.
As a consequence, it can be concluded that long-term
application of chemical fertilizers is a prime cause of
groundwater nitrate pollution presently observed in Kuma-
moto. Minor contributions of manures from livestock farms
and organic fertilizers might also be suggested as secondary
important sources. It is however difficult to point out their
contribution rates for each sampling site due to the fact that
isotopic compositions of the source materials have their own
variations. Finer scale mixing signature of these sources
would be better expressed on a simulation model by putting
information of historical change of the land-use and nitrate
tuning between calculated results and observed concentra-
tion and isotopic data. We are preparing to show these results
in a separated paper. Our results taken in context with this
study imply that the effects of political action in the control-
ling of fertilizer applications enforced after the 1990s are not
2670 w a t e r r e s e a r c h 4 7 ( 2 0 1 3 ) 2 6 6 1 e2 6 7 5

Fig. 8 e Sample plots on the d15 NNO3 vs. NOL 3 diagram for groundwater with two different nitrate attenuation types: (a)
dilution and (b) denitrification, and (c) for the Midori River watershed named as zone C groundwater. Plots for dilution type
groundwater samples are also shown in Fig. 8b in a yellow color. (For interpretation of the references to colour in this figure
legend, the reader is referred to the web version of this article.)
 w a t e r r e s e a r c h 4 7 ( 2 0 1 3 ) 2 6 6 1 e2 6 7 5
yet evident. Groundwater nitrate monitoring along with
d15 NNO3 and d18 ONO3 analysis is important not only to check
pollution status but also to evaluate the effects of manure,
which might be the next additional concern as a pollution
source.
5.2. Two different attenuation mechanisms: dilution
and denitrification
The contrasting decline tendency in NO 3 concentration and
d15 NNO3 changes were observed along the flow direction be-
tween the two different groundwater flow systems: AeA0 and
BeB0 (Fig. 7). This implies that different attenuation processes
for nitrate pollution are occurring within each flow system.
This section describes the attenuation mechanisms for each
flow system in detail and compares their efficiencies in terms
of attenuation functions.
5.2.1. Groundwater flow AeA0
In zone A1, nitrate concentration found in confined ground-
water decreased along the flow direction (Fig. 7a). However,
the DO concentration values were still higher than 8 mg l1
and the d15 NNO3 change was very small (1e1.5&). Moreover,
isotopic fractionation was also unclear for d18 ONO3 (Fig. 7a). In
turn, it is well known that paddy irrigation water of the Shira
River water (NO 3 ¼ 5e6 mg l1, d15 NNO3 ¼ 7e10&,
1
DO ¼ 8e9 mg l ) recharges underneath groundwater at the
mid-stream area of the Shira River (Tanaka et al., 2010) (Fig. 2).
These observations suggest that the NO 3 concentration in
groundwater decreased along the flow direction due to
dispersion and/or dilution by irrigation water, but not by
denitrification. Water quality within the zone A2 largely var-
ied (Fig. 7a), mainly due to the zone A2 being a mixing zone
among several different types of water. However, the d15 NNO3
compositions were homogeneous (1.5e5.5&) with the range
almost equivalent to that of zone A1 (3.4e5.0&) (Fig. 7a).
Observed heterogeneity in NO 3 concentrations should not be
due to denitrification but to the mixing of different water of
varying NO 3 concentrations.
It is notable that NO 3 concentration in confined ground-
water gently decreased from 20 to 15 mg l1 with a small in-
crease of d15 NNO3 (w6&) through zone A3 flow (Fig. 7a). The DO
concentration values of groundwater averaged around
8 mg l1 throughout the zone, supporting the idea that deni-
trification is not a controlling factor in the decline of nitrate
concentrations. Nagai et al. (1983) revealed from water
chemistry data that confined groundwater in this zone is the
mixture of up-gradient groundwater and Shira River water
with a proportion of about 3e4:6e7. Considering our results in
the context of this fact, it can be concluded that the observed
decline in NO 15
3 concentrations and the increase in d NNO3 are
attributed to dilution by Shira River water of relatively lower
NO 15
3 concentration and higher d NNO3 value (average of
5.5 mg l1 and 7.9&, respectively; n ¼ 2), which is recharged at
the mid-stream area of this river. In Fig. 7a, water chemistry
and nitrate isotopic profiles from zone A1eA3 appear similar
between confined and unconfined groundwater, suggesting
that similar processes explained above were also taking place
for unconfined groundwater.
2671
Toward the end of zone A4, confined groundwater samples
displayed a decline of NO 3 concentration with a decreasing
of DO concentration (Fig. 7a). However, d15 NNO3 fractionation
was not observed and clear evidence of denitrification was
not confirmed. Therefore, this might be interpreted to be
owing to the weakening of pollution influence to the deep
part of stagnant groundwater. In contrast, unconfined
groundwater samples showed an increasing trend of d15 NNO3
with the decreasing of DO and NO 3 concentrations along
the flow direction (Fig. 7a). It was the only evidence of the
occurrence of denitrification within the groundwater flow
system AeA0 .
5.2.2. Groundwater flow BeB0
A clear decline of NO 1
3 concentrations (50e1 mg l ) with the
flow direction was confirmed for zone B1 confined ground-
water accompanied with a major decrease of DO concentra-
tion values (8e1 mg l1) and the increasing of d15 NNO3 values
(4e8&) (Fig. 7b). This indicates the progress of the denitrifi-
cation reaction (Knowles, 1982; Hiscock et al., 1991; Korom,
1992; Mariotti, 1986). At the entrance of zone 2B over the
basement uplifting point, the water NO 3 and DO concentra-
tions increased to 20 mg l1 and 7 mg l1, respectively, while
the d15 NNO3 decreased to 3& (Fig. 7b). It appears likely that
water with dissolved materials could be transported through
the thinning part of the aquifer and the discontinuity of the
aquiclude (Fig. 2). Subsequently, through the flow toward
beneath the Shira River, groundwater NO 3 declined again
(20e1.5 mg l1) with a very sudden depression of DO
(7e1 mg l1) and with a major increase of d15 NNO3 (up to 40& in
maximum) (Fig. 7b), showing clear evidence for the drastic
progress of the denitrification reaction. This zone is situated
under a topographical changing point where the terrestrial
hills meet with plain area covered by marine and flood sedi-
ments (Figs. 1a and 2). Considering that marine clays and flood
deposits are more enriched in organic materials than igneous
rocks and with its low DO feature, this area is regarded as a
denitrification hotspot similar to a riparian zone where sig-
nificant denitrification preferentially occurs (Böhlke and
Denver, 1995; Böhlke et al., 2002; Hoffmann et al., 2006). This
area is termed a “hills-plain border zone”.
After water passes this zone, the d15 NNO3 composition
returns again to low values between 0 and 10& at the entrance
of zone 3B (Fig. 7b). This indicates the scenario that the ma-
jority of nitrate from zone 1Be2B has been consumed by
denitrification and has not reached this point. The NO 3 con-
centration was low under the Shira River area (around
1 mg l1). Naturally occurring nitrate in the aquifer geology
might be the origin of this modestly concentrated nitrate. In
zone 3B, NO 3 concentrations in confined groundwater
decreased (w0 mg l1) with a significant increase of d15 NNO3 up
to 30& (Fig. 7b). Groundwater in this zone is in addition
characterized by low DO concentration values generally
<2 mg l1, suggesting the occurrence of denitrification. A
small influence of nitrate infiltration from the unsaturated
zone was confirmed in zone 3B; however, the general ten-
dency of changing patterns of NO 15
3 , DO, and d NNO3 for un-
confined groundwater was quite similar to that of confined
groundwater throughout the flow distance (Fig. 7b). Therefore,
similar processes should be affecting both groundwater types.
2672 w a t e r r e s e a r c h 4 7 ( 2 0 1 3 ) 2 6 6 1 e2 6 7 5
Fig. 9 e Estimation of the isotopic enrichment factor ε for
each zone.
5.2.3. Efficiencies of the two different attenuation functions
Fig. 8 compares the effects of the two different attenuation
types on groundwater nitrate pollution judged in the previ-
ous discussion: dilution (Fig. 8a) and denitrification (Fig. 8b).
Groundwater for both attenuation types showed highest
NO 1
3 concentrations around 50 mg l , which were largely
derived from chemical fertilizers. The NO 3 concentrations
decreased to 20e10 mg l1 for most of the samples attenu-
ated by the dilution with lower nitrate water (Fig. 8a). From
the plots distribution in Fig. 8a and previous discussion, it
could be suggested that the lower nitrate water is the
mixture of the least contaminated (or non-contaminated)
groundwaters and the recharged river waters moderately
contaminated by sewage origin nitrate. On the other hand,
NO 3 concentrations decreased to <1 mg l
1
by the action of
denitrification (Fig. 8b). This means that about 60e80% and
98% of the nitrate load was naturally purified by dilution and
denitrification, respectively. It is clear from this comparison
that denitrification is more efficient in terms of the nitrate
attenuation function for circulated groundwater systems.
Groundwater affected by dilution NO 3 attenuation is there-
fore, more vulnerable to the threat of additional pollution
such as in zone A3 (Figs. 1a and 8) especially at where
dilution water has already been contaminated in some
extent.
In a closed system, isotopic enrichment during denitri-
fication can be expressed by the following Rayleigh
equation:
d15 NNO3 ¼ d15 NNO3 i þ εlnðC=Ci Þ (1)
where ε is the isotopic enrichment factor (a parameter for
denitrification activity), d15 NNO3i and d15 NNO3 represent isotope
compositions of water at the initial time and sample time,
respectively, and Ci and C are the concentrations of nitrate in
water at the initial time and sample time, respectively.
Equation (1) can be used to estimate the enrichment factor ε by
plotting measured d15 NNO3 against ln(C). The ε value for
groundwater of each zone was estimated (Fig. 9), which was
judged as the denitrification type (Figs. 7 and 8b) under the
assumption that there is no additional nitrate input at the
intercept of the flow and dilution and dispersion rates were
constant through the flow. We used this value to compare the
relative efficiency of the denitrification activity at each flow
zone. The ε value for groundwater under the Midori River
watershed (shown as zone C) was also evaluated (Figs. 1 and
8c and 9). As a result, a relatively higher attenuation effect by
denitrification was found in the stagnant zone compared to
that in the recharge zone (Figs. 2 and 9). In addition, the
highest attenuation effect was confirmed in the hills-plain
border zone (Figs. 1 and 2; denitrification hotspot) even over
a short distance, except around the discharge zone. This zone
has the highest potential for natural attenuation of ground-
water nitrate pollution through circulated groundwater
systems.
6. Toward the better use of the d15N and d18O
tracer technique
Several approaches for tracing the source of groundwater ni-
trate pollution have recently been proposed, for instance, the
use of chemical markers (Fenech et al., 2012) and application
of other stable isotope tracers (Vengosh et al., 2002; Widory
et al., 2004; Xue et al., 2009; Hosono et al., 2010, 2011a) in
addition to d15 NNO3 and d18 ONO3 . These tracers are definitely
useful for achieving a comprehensive understanding of the
nitrate pollution mechanisms. However, in a strict sense these
tracers can be useful under the hypothesis that they can be
used as a proxy of nitrate. Therefore, seeking a more efficient
use of nitrate isotope tracers is still important as it is the only
direct fingerprint of tracing nitrate sources and recording the
occurrence of denitrification.
 w a t e r r e s e a r c h 4 7 ( 2 0 1 3 ) 2 6 6 1 e2 6 7 5
With the traditional way of utilizing d15N and d18O tracers
on d15 NNO3 vs. d18 ONO3 and NO 15
3 vs. d NNO3 diagrams (Fig. 6), it
was difficult to separate the two types of anthropogenic ni-
trate sources (chemical fertilizers and treated wastewater
from sewage treatment plants derived through recharging
river water) and also two types of attenuation processes
(dilution and denitrification) for Kumamoto groundwater,
since all samples are plotted within similar compositions
(Figs. 6 and 8). In contrast, the analysis taking into account
detailed groundwater flows enables us to separate the effects
of the mixing of different sources and denitrification (Fig. 8).
Our study clearly demonstrated that the nitrate isotopic
traceability significantly improves with the understanding of
groundwater flow dynamics.
7. Conclusions
Our study revealed that the application of chemical fertilizers
is a prime cause of groundwater nitrate pollution in Kuma-
moto. Nitrogen load from manure was not detected as being
the dominant source of pollution. However, this effect should
be checked during future monitoring programs, since the
number of livestock farms has increased in more recent years.
Polluted groundwater is attenuated along the flow distance
from recharge through lateral flow to the discharge (or stag-
nant) zone by two different processes: dilution and denitrifi-
cation. About 60e80% and 98% of nitrate of anthropogenic
origin were assumed to be purified by dilution and denitrifi-
cation, respectively. Denitrification was evaluated as the more
efficient attenuation function and the hills-plain border zone
has the highest attenuation potential over the area. In
contrast, the groundwater attenuated by dilution appears to
2673
be more vulnerable to additional pollution. Aspects regarding
to these natural attenuation potentialities should be infor-
mative for future groundwater resource exploration and
preservation. This study demonstrated that traceability of
nitrate isotope ratios can be improved by the combined use of
land-use and groundwater flow information. The proposed
approach should be applicable for future investigations in
other regions.
Acknowledgments
We thank Mr. Satoshi Sakaeda and Mr. Masaaki Hirohata of
the Kumamoto Prefectural Government for their help during
field surveys. We also thank Prof. Yasunori Kawagoshi of the
Department of Civil & Environmental Engineering, Kuma-
moto University, for a fruitful discussion. Groundwater flow
models used in this study were constructed under the
operation of the Geosphere Environmental Technology Cor-
poration, Tokyo, Japan. This study was funded by a CREST
Project (JST: Japan Science and Technology Agency) with
additional support from the Grant-in-Aid for Young Scien-
tists (A) (no. 24681007).
Appendix 1. Figure showing increaseL1
of nitrate
concentrations (vertical axis; mg l as NOL3 LN)
with time (horizontal axis; year) in confined
groundwater at selected borehole sites in
Kumamoto area (modified after Tomiie et al.,
2011).
2674 w a t e r r e s e a r c h 4 7 ( 2 0 1 3 ) 2 6 6 1 e2 6 7 5

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