Estuarine, Coastal and Shelf Science 86 (2010) 450–466

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Estuarine, Coastal and Shelf Science

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Modeling phytoplankton production in Ise Bay, Japan: Use of nitrogen isotopes

to identify dissolved inorganic nitrogen sources
Ryo Sugimoto a, b, *, Akihide Kasai a, Toshihiro Miyajima c, Kouichi Fujita d
a
Graduate School of Agriculture, Kyoto University, Oiwake, Kitashirakawa, Sakyo, Kyoto 606-8502, Japan
b
Field Science Education and Research Center, Kyoto University, Oiwake, Kitashirakawa, Sakyo, Kyoto 606-8502, Japan
c
Ocean Research Institute, University of Tokyo, Minimidai 1-15-1, Nakano, Tokyo 164-8639, Japan
d
Mie Prefecture Fisheries Research Institute, Shiroko 1-6277-4, Suzuka, Mie 510-0243, Japan

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

Article history: An important aspect of the nitrogen cycle in coastal environments concerns the source of the nitrogen
Received 29 January 2009 used in primary production. Phytoplankton production in Ise Bay, one of the most eutrophic embayments
Accepted 13 October 2009 in Japan, is supported by external nitrogen derived from rivers and the ocean, and regenerated nitrogen
Available online 30 October 2009
formed in hypoxic water within the bay. We evaluated the contribution of each source of dissolved
inorganic nitrogen (DIN) to phytoplankton production in Ise Bay. A unique three-dimensional ecosystem
Keywords:
model including nitrogen isotopes (d15N) was developed based on precise observations. Model results
ecosystem model
revealed that DIN (¼ammonium þ nitrate) consumption by phytoplankton exceeds the DIN supply from
estuarine circulation
Ise bay the rivers and ocean, indicating that a large amount of phytoplankton production in Ise Bay depends on
nitrogen isotopes regenerated DIN within the bay rather than on newly supplied DIN. However, the ratio of consumption to
phytoplankton production external supply differs seasonally. Distributions of simulated d15N clearly showed the source of nitrogen
seasonal variation incorporated by phytoplankton in each source. The intrusion depth of oceanic water changes from the
bottom to the middle layer in spring. Oceanic nitrate is transported into the euphotic layer by the middle
layer intrusion and stimulates phytoplankton production at the bay mouth. The subsurface chlorophyll
maximum layer then develops. In autumn, however, the intrusion depth of oceanic water changes from
the middle layer to the bottom layer. Regenerated NO 3 , which is accumulated in the hypoxic water mass,
is uplifted and continuously supplied to the euphotic layer. These results imply that phytoplankton
production in Ise Bay is mainly dominated by the internal cycle rather than the external supply.
Ó 2009 Elsevier Ltd. All rights reserved.

1. Introduction
Nitrogen is a key element in phytoplankton production and
subsequent ecosystem processes in the sea because it often limits
primary production. The form of nitrogen incorporated by phyto-
plankton determines the partitioning between new and regen-
erated production. Various nitrogen sources, including riverine,
oceanic, atmospheric, and regenerated nitrogen, maintain the
phytoplankton production in coastal ecosystems. Of these major
nitrogen sources, riverine input affected by anthropogenically
perturbed nitrogen flux from land areas to the coast has been
considered primarily responsible for eutrophication in coastal
zones (Vitousek et al., 1997). However, research has revealed that
a large amount of nitrogen, comparable to terrestrial input, is
* Corresponding author. Present address: Research Center for Marine Bioresources,
Fukui Prefectural University, 49-8-2 Katsumi, Obama-city, Fukui 917-0003, Japan
E-mail address: sugiryo@fpu.ac.jp (R. Sugimoto).
supplied from the adjacent marginal sea to the coastal region (e.g.,
Fujiwara et al., 1997c). Intrusion of nutrient-rich shelf water stim-
ulates phytoplankton production in coastal ecosystems (e.g.,
Sugimoto et al., 2009b). Moreover, seasonal variation in water
temperature affects the vertical structure of the water column. In
response to water-column stratification during the warm season,
oxygen concentrations in the lower water change seasonally, with
bottom waters often isolated and becoming hypoxic in summer
(e.g., Takahashi et al., 2000). As another source in a seasonal scale,
rich organic matter in the sediments and the water column in
eutrophic coastal ecosystems supply a large amount of regenerated
nitrogen, which accumulates in this oxygen-depleted water
mass. However, the quantitative contribution of each nitrogen
source to phytoplankton production in coastal environments is still
unclear.
In recent years, variations in the natural abundance of stable
isotopes of nitrogen (d15N) in marine ecosystems have attracted
considerable research attention. The ability to detect small differ-
ences in the 15N:14N ratio of various pools of nitrogen, combined

0272-7714/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.ecss.2009.10.011
 R. Sugimoto et al. / Estuarine, Coastal and Shelf Science 86 (2010) 450–466 451

with knowledge of kinetic isotope discrimination during chemical

and biological reactions, potentially provides novel ways to
monitor nitrogen fluxes in marine ecosystems on various temporal
and spatial scales. Utilization of d15N to study marine environments
requires biological and/or chemical knowledge of sources and sinks
and an understanding of isotope discrimination during sink and
source processes.
Knowledge of isotope discrimination would enable researchers
to use the 15N:14N signal of phytoplankton to estimate the extent
and/or source of nitrogen used by phytoplankton. The d15N value of
phytoplankton is principally determined by the substrate d15N and
isotope discrimination as demonstrated by several ocean studies
(e.g., Brandes et al., 1998; Sigman et al., 1999). However, the exact
relationship in coastal ecosystems is difficult to interpret because
the isotopic composition of the source nitrogen is variable and can
cause uncertainty when interpreting isotope discrimination,
especially if more than one form of nitrogen is present (e.g., NO 3
and NHþ 4 ), or if more than one source of nitrogen is available (e.g.,
riverine, oceanic, and regenerated nitrogen). Moreover, the
temporal and spatial variabilities of substrate nitrogen sources in
coastal environments are controlled by complex physical–biolog-
ical–chemical interaction processes associated with external
loading, advection/diffusion, and tidal mixing, as well as oceanic
water inputs.
Since these complicated processes are strongly non-linearly
coupled, studies on the relationships between nutrient behavior
and phytoplankton production have usually relied on physical-
ecosystem models that include inorganic and organic matter
transformation and utilization (e.g., Zheng et al., 2004; Kobayashi
and Fujiwara, 2008). However, ecosystem models have been criti-
cized in regard to reproducibility since they have a large number of
unknown parameters. Generally, these parameters are adjusted to Fig. 1. Study area. Circles indicate the sampling locations in the Kiso Rivers (Kiso,
reproduce observed data. Moreover, incubation experiments do not Nagara and Ibi Rivers), the central part of the bay, and the bay mouth (Irago Strait).
necessarily provide actual fluxes and rates. These experiments are
usually restricted to small spaces and short times, and conducting
nitrogen sources, the river input has been held primarily respon-
incubation experiments under actual conditions is difficult.
sible for the observed long-term eutrophication in Ise Bay. The
Phytoplankton production in Ise Bay is supported by nutrients
quantitative characterization of DIN sources to phytoplankton
derived from the river, ocean, and regeneration inside the bay. At
production is needed for the management of DIN input from the
the head of the bay, the surface chlorophyll maximum is observed
rivers.
near the river mouth. Phytoplankton blooms consume many
To address these issues, we developed a unique three-dimen-
terrestrial nutrients from rivers (Sugimoto et al., 2004). In contrast,
sional physical-ecosystem model coupled with d15N to evaluate the
the subsurface chlorophyll maximum (SCM) develops from the
contribution of the three major nitrogen sources to phytoplankton
center to the mouth of the bay (Fujiwara et al., 1997a; Kasai et al.,
production. In particular, we focused on reproducing accumulation
2007). The concentration of phytoplankton in the subsurface layer
of DIN in the lower layer and phytoplankton production in the
increases from spring to summer (Kasai et al., 2007). Fujiwara et al.
subsurface layer. Although regenerated DIN generally means NHþ 4
(1997a) suggested that the amount of phytoplankton in the SCM
rather than NO 3 in the ocean, here we refer the regenerated
layer may be larger than that in the river mouth. Possible NO 3
nitrogen to the recycled nitrogen within the bay, which includes
sources for the SCM are the nutrients supplied from the adjacent
NHþ 
4 and NO3 . In other words, the external DIN sources of river and
continental shelf (Kasai et al., 2007). Moreover, nutrients that
ocean are only the new nitrogen. The d15N values of major nitrogen
accumulate in the hypoxic water mass in the lower layer might be
sources (riverine, oceanic, and regenerated) in Ise Bay, Japan, were
another source for the SCM (e.g., Fujiwara et al., 1997a; Kakehi et al.,
identified by field observations. Such precise observations of d15N
2004). DIN to DIP ratio in the lower layer was high in winter (w20)
clarify the behavior of DIN and phytoplankton production. More-
and low in summer (w5), because of the nitrogen loss by denitri-
over, in our previous study, we reported on isotopic discrimination
fication and phosphorous addition by release from the sediments
for nitrification and denitrification in Ise Bay (Sugimoto et al.,
(Kakehi and Fujiwara, 2007). This indicates the nitrogen limits the
2008). Consequently, combining the ecosystem model with d15N
primary production in Ise Bay in summer seasons. The form of DIN
observations allows us to reveal the comprehensive flow of
also changes seasonally with the development of hypoxia. NHþ 4
nitrogen in the coastal ecosystem.
dominates in spring, and is transformed to NO 3 to summer (Sugi-
moto et al., 2008). In autumn, phytoplankton blooms in the surface
layer (e.g., Kakehi et al., 2005; Kasai et al., 2007). Water mixing 2. Study area
releases nutrients, which is accumulated in the lower layer in
summer, to the euphotic layer, and induces the autumn blooms of Ise Bay is one of the largest inner bays in Japan. It has a surface
phytoplankton. However, the quantitative contribution of different area of 1738 km2, volume of 33.9 km3, and mean depth of 19.5 m,
nutrient sources (river, ocean and regeneration) to primary with the deepest longitudinal depression extending over 35 m
production in Ise Bay is still unknown. Of these three major deep in the middle of the bay (Fig. 1). At its mouth, Ise Bay opens to
452 R. Sugimoto et al. / Estuarine, Coastal and Shelf Science 86 (2010) 450–466
the Pacific Ocean via the narrow Irago Strait (w10 km wide). Water
exchange between Ise Bay and the Pacific Ocean takes place
through Irago Strait. Three major rivers (the Kiso, Nagara, and Ibi
Rivers), known collectively as the Kiso Rivers, flow into the head of
the bay in the north and contribute w85% of the total freshwater
discharge into the bay. The discharge of the Kiso Rivers shows
seasonal variation, with large discharge in summer and small
discharge in winter, and an annual mean of w400 m3 s1. Semi-
diurnal constituents dominate the tide in Ise Bay. The typical
amplitude of tidal currents is 0.1 m s1 in the bay. At Irago Strait,
tidal flow converges, and its speed exceeds 0.8 m s1 during spring
tides (Fujiwara et al., 2002).
The estuarine gravitational circulation is often dominant in the
regions of freshwater influence; lighter water flows seaward in the
upper layer, while heavier water flows landward in the lower layer
(e.g., Fujiwara et al., 1997b; Kasai et al., 2000). However, recent
surveys in Ise Bay have shown that the longitudinal circulation does
not always follow the classical estuarine circulation pattern;
instead, the flow pattern has a bimodal character. The tidally mixed
strait water flows into the bay through the bottom layer in the
cooling season, while it flows through the middle layer in the
heating season (e.g., Takahashi et al., 2000). In winter, weak strat-
ification is maintained only by freshwater buoyancy near the bay
head. Sea surface cooling causes the freshwater to mix with the
deeper water, so that the water in the bay is lighter than that at the
strait. This density difference leads to bottom inflow in the cooling
season, following the conventional estuarine circulation pattern.
During summer, however, the effect of surface heating is restricted
to the upper layer by the strong thermocline and halocline in the
stratified area (inside the bay), while it extends to the bottom due
to the strong tidal currents in the well-mixed area around the bay
mouth (Irago Strait). The water temperature increases faster in the
strait than in the bottom water inside the bay, causing the bottom
bay water to become denser than the strait water. The well-mixed
strait water thus has a density equivalent to the middle layer inside
the bay and tends to flow through the middle layer when it intrudes
into the bay in the heating season (Takahashi et al., 2000). As
a consequence, relict water in the lower layer within the bay is
excluded from the exchange circulation and forms a cold water
mass (Takahashi et al., 2000; Fujiwara et al., 2002; Kasai et al.,
2002). The residence time of the isolated water mass was estimated
to be w50 days (Fujiwara et al., 2002). A pycnocline in the vertical
and strong fronts in the horizontal prevent oxygen supply from the
surrounding waters to the isolated water mass (Fujiwara et al.,
2002; Kasai et al., 2002). Consequently, the isolated water mass
becomes hypoxic with time as the oxygen consumption exceeds
the oxygen supply.
3. Field observations
3.1. Observations
Observations to obtain endmember values for each nitrogen
source and confirm the reproducibility of data were conducted
from May 2005 to July 2006. First, seasonal observations to identify
endmenber values of riverine nitrogen were conducted in the lower
parts of the Kiso, Nagara, and Ibi Rivers (Stn. R in Fig. 1). For
measurements of nutrient concentrations and nitrogen isotope
ratios of NHþ 
4 , NO3 , and particulate nitrogen (PN), surface water
samples were collected at the center part of the streams from
bridges using a clean plastic bucket. Water temperature and salinity
were measured using a temperature-salinity meter (Alec Elec-
tronics, ACT20-D). Second, the oceanic nitrogen endmembers were
determined by seasonal observations at the bay mouth (Stn. I). For
nutrient and isotopic analysis of mixed water at 30 m depth at Irago
Strait, samples were taken using 2.6-L Niskin bottles attached to
a rosette with a conductivity-temperature-depth (CTD) sensor (Sea
Bird, SBE-911 plus). Finally, water samples for reproducible data
and isotope discrimination were collected at 5-m intervals from the
surface to 1 m above the bottom (34 m) at the center of Ise Bay (Stn.
C). All water samples were filtered immediately through pre-
combusted (450  C, 4 h) glass fiber filters (Whatman GF/F). Filters
and filtrate samples were stored at 30  C until analyses.
3.2. Analyses
3
Nutrient concentrations (NHþ  
4 , NO2 , NO3 , and PO4 ) were
measured using an AutoAnalyzer (TRACSS 2000, Bran-Luebbe).
15
Measurement of d NNH4 was based on that of Holmes et al. (1998).
Filtrate samples were transferred to incubation bottles to which
NaCl, MgO, and NHþ þ
4 traps were added. The NH4 trap consisted of
an acidified 1-cm diameter GF/D filter sandwiched between two
Teflon membrane filters (2.5-cm diameter, 10-mm pore size). The
traps floated on the saline samples. Samples and references were
incubated for 10–14 days so that all NHþ 4 could diffuse out of the
solution and be trapped. During incubation, samples were shaken
gently and maintained at 40  C. After incubation, filter packages
were removed from the incubation bottles, dried, and stored in
a desiccator containing concentrated sulfuric acid for isotope
15
analysis. The procedure for d NNO3 followed the methods of Sig-
man et al. (1997). Filtrate samples were transferred to incubation
bottles with MgO and pre-incubated at 65  C for 5 days. After pre-
incubation, samples were boiled to concentrate NO 3 and to remove
NHþ 4 by volatilization. Devarda’s alloy was then added to the
samples and references to reduce NO þ
3 to NH4 . The subsequent
15
procedures were conducted in the same manner as for d NNH4 .
15
Values of d N were determined using an elemental analyzer con-
nected online to an isotope-ratio mass spectrometer (FLASH EA-
Conflo III-DELTAPLUS XP, Thermo-electron). Reproducibility was
better than 0.2&. These analyses were executed only for the
samples containing >1.0 mM of NHþ 
4 and NO3 .
The GF/F filters for elemental and isotopic analyses were treated
as follows. Each sample was dried and wrapped with a tin capsule.
PN concentration and its d15N were measured using a mass spec-
trometer (Delta S, Finnigan MAT) coupled with an elemental
analyzer (EA1108, Carlo Erba). The d15N values are expressed by the
usual d notation (&) relative to atmospheric N2. Reproducibility of
d15NPN was better than 0.3&.
3.3. Observed riverine and oceanic d15N
Fig. 2a shows the relationships between the d15N values in the Kiso
Rivers and the total river discharge of the Kiso Rivers. The riverine
d15N values are weighted average values of each nitrogen flux for the
15
three rivers. The d NNO3 of the Kiso Rivers decreases logarithmically
15
from 8 to 1& with the increase in river discharge (RD). Thus d NNO3
obeys a simple logarithmic decreasing relationship with the river
discharge (r2 ¼ 0.76, solid line in Fig. 2a) as follows:
d15 NNO3 ¼ 2:52  lnðRDÞ þ 18:88: (1)
15
Although d NPN rapidly decreases from 6 to 1& until
w400 m3 s1 of river discharge, d15NPN converges to w2& with
higher river discharge (>w400 m3 s1). An exponential equation of
the form y ¼ aebx þ c was fitted to the observed data, where y is the
d15NPN, x is the RD, a and b are constants, and c is an asymptotic
value of d15NPN. The best-fit curve (r2 ¼ 0.59, dashed line in Fig. 2a)
was calculated by the least-squares method as
d15 NPN ¼ 17:20e0:01RD þ 2:09: (2)
 R. Sugimoto et al. / Estuarine, Coastal and Shelf Science 86 (2010) 450–466 453

Fig. 2. (a) River discharge vs. d15N values of NO þ

3 (squares), NH4 (triangles), and particulate organic nitrogen (circles) at the Kiso Rivers (Stn. R). Solid and dashed lines are the best-
15
fitted curves for d NNO3 and d15NPN, respectively. (b) Concentrations vs. d15N values of NO 3 (squares) and particulate organic nitrogen (circles) at Irago Strait (Stn. I). The solid line is
15
the linear regression line for d NNO3 .

These relationships indicate that compositions of NO 3 and PN 4.2. Ecosystem model
are largely dependent on the discharge. However, no clear rela-
15
tionship was found between d NNH4 and the river discharge The ecosystem model has five compartments, the prognostic
(triangles in Fig. 2a), suggesting that characteristics of NHþ 4 are variables being concentrations of nitrogen (mmol L1). Fig. 4 illus-
more complex than those of NO 3 and PN. trates the nitrogen cycle with the compartments, namely NHþ 4,
Fig. 2b shows the relationship between concentrations and d15N NO3 , phytoplankton (PHY), zooplankton (ZOO) and detritus (DET).
values of NO 3 and PN of the mixing water at the strait from May Two other parameters, dissolved inorganic phosphorus (PO3 4 ) and
15
2005 to July 2006. The d NNO3 value at the strait would be influ- dissolved oxygen (DO), are also included. The Appendix describes
enced by the mixing between the two different sources at the shelf the formulation of PO34 and DO, following the method of Kakehi
15
region off Ise Bay; lower d NNO3 in the subsurface layer (w50 m) et al. (2005). The evolutions of these compartments are described
15
and higher d NNO3 in the lower layer (>100 m) (Sugimoto et al., with differential equations composed of biological source and sink
15
2009a,b). A strong correlation was thus found between d NNO3 and terms, external loading terms, diffusion terms, and advection
2
the NO 3 concentrations (r ¼ 0.71, solid line in Fig. 2b), given as terms. The mass conservation can be mathematically written as
h i
d15 NNO3 ¼ 1:19  NO
3  2:25; (3)      
vC vC vC vC v vC v vC v vC
þu þv þw ¼ Kh þ Kh þ Kz
where [NO 
3 ] is the concentration of NO3 . This relationship can be vt vx vy vz vx vx vy vy vz vz
adapted as the variable endmenber for the ecosystem model. There þ A þ B; ð4Þ
was no clear relationship between d15NPN and PN concentrations
(circles in Fig. 2b).
In our previous study (Sugimoto et al., 2008), we reported data
for isotope discrimination values by nitrification and denitrification
15
at the center of the bay (Stn. C). d NNO3 dramatically shifted from
8.5  2.0& in May to 8.4  0.7& in July, in response to the
development of a hypoxic water mass and oxidation from NHþ 4 to
NO 
3 . In particular, newly generated NO3 by nitrification involved
a large degree of isotope discrimination (¼24.5&), displaying
significantly 15N-depleted values during periods with an oxic water
column. Although prominent deficits of NO 3 in hypoxic bottom
waters due to denitrification were revealed in July, there was little
isotope discrimination of denitrification in the sediments (¼w1&).

4. Model description

4.1. Model domain

Fig. 3 presents an overall view of the computational grid used in

this study. The horizontal grid coordinates have 18 (x-axis)  21 (y-
axis) points, and the gird size is 20  20 (3052.16 m  3697.87 m).
Vertical layers are uniformly distributed every 4 m in depth with
a maximum 15 layers (60 m). The open boundary is set outside of
the bay mouth (bold line in Fig. 3), w70 km from the river mouth.
Freshwater from the Kiso Rivers empties into the surface box at the Fig. 3. Topography of the model basin. The bold line indicates the open boundary.
head of the bay (shaded area in Fig. 3). Fresh water flows into the shaded grid.
454 R. Sugimoto et al. / Estuarine, Coastal and Shelf Science 86 (2010) 450–466

Riverine
Loading

Grazing
Phytoplankton Zooplankton
Mortality

Assimilation

Excretion

Egestion
Mortality
Boundary
Flux

Assimilation
NO3- NH4+ Detritus
Nitrification Remineralization

Sedimentation
Denitrification

Release Sediment

Fig. 4. Schematic view of the ecosystem model. Boxes represent nitrogen-based standing stocks. Dashed and solid arrows indicate nitrogen flows with and without isotope
fractionation, respectively.

where C represents the specific compartments in the model, A is compartments, all processes described in Fig. 4 must be formulated.
the function that represents the internal source or sink, B is the The equations and parameters (Table 1) for the nitrogen cycle are
external loading, u and v are the horizontal velocity components in essentially based on the ecosystem model developed by Kawamiya
the x and y directions, respectively, and w indicates the vertical et al. (1995).
velocity components in the z direction. Kh and Kz are the horizontal Photosynthesis is determined by temperature (T,  C), [NHþ 4 ],
3
and vertical eddy diffusivity, respectively. [NO 2
3 ], [PO4 ], and light intensity (I, W m ), and is expressed as
The mathematical expressions for the internal sources or sinks
of each compartment are given as follows: ðPhotosynthesisÞ ¼ Vmax FT FN FI ½PHY; (10)
h i where Vmax is the maximum photosynthetic rate of phytoplankton,
v NO
3 and FN is the nutrient limitation factor that is assumed to follow the
¼ ðPhotosynthesisÞ  Fnew þ ðNitrificationÞ
vt Michaelis–Menten function, given as
 ðDenitrificationÞ; (5) ( h i
NO 3  h i
h i FN ¼ min h i exp  J NHþ 4

v NH4þ NO3 þ kNO3
¼ ðPhotosynthesisÞ  ð1  Fnew Þ þ ðExcretionÞ h i h i
vt )
NHþ 4 PO3
4
þ ðRemineralizationÞ  ðNitrificationÞ þ ðReleaseÞ; þh i ;h i ; (11)
3
(6) NHþ4 þ kNH4 PO4 þ kPO4

where kNO3 ; kNH4 and kPO4 represent half-saturation constants for

v½PHY NO þ 3 þ
¼ ðPhotosynthesisÞ  ðGrazingÞ  ðMortalityPHY Þ 3 , NH4 , and PO4 , respectively, and J is the NH4 inhibition
vt coefficient. To express the dependence on temperature (FT) and
þ ðSinkingPHY Þ; (7) light intensity (FI), the following formulae are used (Steel, 1962):
 
v½ZOO T T
¼ ðGrazingÞ  ðMortalityZOO Þ  ðEgestionÞ FT ¼ exp 1  ; (12)
vt Topt Topt
 ðExcretionÞ; (8)  
I I
FI ¼ exp 1  ; (13)
Iopt Iopt
v½DET
¼ ðMortalityPHY Þ þ ðMortalityZOO Þ þ ðEgestionÞ
vt
I ¼ I0 expðLzÞ; (14)
 ðRemineralizationÞ þ ðSinkingDET Þ
þ ðSedimentationÞ; (9) 1:45
L ¼ ; (15)
Square brackets indicate the concentration of each compart- Ds
ment, and Fnew (f-ratio) is the uptake ratio of NO
3 to total DIN by where Topt and Iopt are optimum temperature and light for phyto-
phytoplankton. To calculate the time evolution of the plankton growth, respectively, and I0 (W m2) is light intensity at
 R. Sugimoto et al. / Estuarine, Coastal and Shelf Science 86 (2010) 450–466 455

Table 1
Biological parameters.

Parameters Symbols Values Unit Remarks

Maximum photosynthetic rate Vmax 2.4 /day

Optimum temperature Topt 25 C
Optimum light intensity Iopt 104.7 W/m2
Half-saturation coefficient for NHþ4 kNH4 0.2 mmol/L
Half-saturation coefficient for NO3 kNO3 2.0 mmol/L
3
Half-saturation coefficient for PO4 kPO4 0.17 mmol/L
Ammonium inhibition coefficient j 1.5 L/mmol
Phytoplankton mortality rate at 0  C VMP 0.002 L/mg day
Temperature coefficient for phytoplankton mortality kMP 0.0693 / C
Zooplankton mortality rate at 0  C VMZ 0.0064 L/mg day
Temperature coefficient for zooplankton mortality kMZ 0.069 / C
Maximum grazing rate at 0  C Gmax 0.15 /day Tuning
Temperature coefficient for grazing kG 0.0693 / C
Ivlev constant l 0.1 L/mg day
Threshold value for grazing Chl* 0.602 mgN/L
Assimilation efficiency of zooplankton a 0.4
Growth efficiency of zooplankton b 0.3
Remineralization rate at 0  C VRem 0.03 /day
Temperature coefficient for remineralization kRem 0.0693 / C
Nitrification rate at 0  C VNit 0.0012 /day Tuning
Temperature coefficient for nitrification kNit 0.32 / C Tuning
Denitrification rate at 0  C VDenit 0.01 /day Tuning
Temperature coefficient for denitrification kDenit 0.14 / C Tuning
NHþ 
4 release rate at 0 C VRel 0.3 /day Tuning
Temperature coefficient for NHþ 4 release kRel 0.0693 / C Tuning
Sinking velocity of phytoplankton VSP 0.17 m/day
Sinking velocity of detritus VSD 1.73 m/day
Sedimentation rate of detritus VSED 0.3 /day Tuning

The above parameters were obtained from the studies of Kawamiya et al. (1995), Hayashi and Yanagi (2002), Yoshikawa et al. (2005), and Kakehi (2006).

n
the sea surface. The light dissipation coefficient (L) is calculated ðGrazingÞ ¼ Max 0; Gmax expðkG TÞ
using the transparency (Ds), given by Secchi disk measurements, n h  io o
following Walker (1980). Boundary conditions of I0 at the surface 1  exp l Chl*  Chl ½ZOO ; ð22Þ
water are described by
  ðExcretionÞ ¼ ða  bÞðGrazingÞ; (23)
p
I0 ¼ Imax ðidayÞ$cos $ðihour  12Þ ; (16)
day timeðidayÞ
ðEgestionÞ ¼ ð1  aÞðGrazingÞ; (24)
 
2p
Imax ðidayÞ ¼ 500 þ 300sin $ðiday  20Þ ; (17) ðRemineralizationÞ ¼ VRem expðkRem TÞ½DET; (25)
Ty

  where VMP and kMP are the phytoplankton mortality rate at 0  C and
2p
day timeðidayÞ ¼ 12 þ 1:5sin $ðiday  20Þ ; (18) temperature coefficient of phytoplankton mortality, respectively;
Ty
VMZ and kMZ are the zooplankton mortality rate at 0  C and
where Imax(iday), day_time(iday) and ihour are the maximum light temperature coefficient of zooplankton mortality, respectively;
intensity per iday, the insolation hour per day, and the calculation Gmax, kG, l, and Chl* are the maximum grazing rate of zooplankton,
hour in the day cycle, respectively. Ty and iday are the year cycle temperature coefficient of zooplankton grazing, Ivlev constant, and
(¼360 days) and the calculation day from the initial day threshold value for grazing, respectively; a and b are the assimi-
(1 March ¼ 0), respectively. lation efficiency and growth efficiency of zooplankton, respectively;
The f-ratio is calculated as and VRem and kRem are the remineralization rate of detritus to NHþ 4

h i at 0  C and temperature coefficient for detritus remineralization,

NO 3
 h i respectively.
h i exp  J NHþ 4 Nitrification may be influenced by many factors, including the

NO3 þ kNO3 substrate concentration, oxygen, light, suspended particulate
f  ratio ¼ h i h i :
matter, pH, and salinity (Herbert, 1999). However, Berounsky and
NO3  h i NHþ4
h i exp  J NHþ þh i Nixon (1990) reported that seasonal rates of nitrification are more
4
NO3 þ kNO3 NHþ 4 þ kNH4
strongly correlated with temperature than with other factors. They
found that the temperature coefficient of nitrification (kNit) in
(19) a water column was considerably higher than that in the sediments.
Mortality of phytoplankton and zooplankton, grazing, excretion, In Ise Bay, nitrification in the water column is more important
egestion, and remineralization are represented as: process in the nitrogen cycle, than that in the sediments (Sugimoto
et al., 2008). Nitrification in the water column would be governed
ðMortalityPHY Þ ¼ VMP expðkMP TÞ½PHY2 ; (20) by temperature and light intensity rather than DO concentration
(Sugimoto et al., 2009c). The nitrification rate in the aphotic water
column is thus assumed to be simply dependent on T and [NHþ 4 ] in
ðMortalityZOO Þ ¼ VMZ expðkMZ TÞ½ZOO2 ; (21) the aphotic water column.
456 R. Sugimoto et al. / Estuarine, Coastal and Shelf Science 86 (2010) 450–466

Tabel 2
ðNitrificationÞ ¼ VNit expðkNit TÞ½NH4 ; (26) Nitrogen isotope discrimination (3) in biogeochemical processes.

where VNit is the nitrification rate at 0  C. In photic layers, we Process Notation 3 (&) Remarks

negrected nitrification term. NO

3 assimilation 31 3 Field estimates
Denitrification in Ise Bay occurs in the sediments (Sugimoto et al., by phytoplankton 8–9 (Altabet et al., 1991)
4–6 (Sigman et al., 1999)
2008). The NO 3 in water overlying the sediments is used as the 5 (Altabet et al., 1999)
substrate for sedimentary denitrification (Herbert,1999). Kakehi et al. Culture estimates
(2005) suggested that denitrification occurs throughout the year. 1–23 (Wada and Hattori, 1978)
Potential of denitrification is high in summer, but in low in winter 0–15 (Montoya and McCarthy, 1995)
9 (Pennock et al., 1996)
(Kakehi and Fujiwara, 2007). The denitrification rate in the anoxic
4–5 (Waser et al., 1998a, 1998b)
sediment is thus assumed to be simply dependent on the tempera- 2–6 (Needoba et al., 2003)
ture and NO 3 concentration of the water overlying the sediments. NHþ
4 assimilation 32 8 Field estimates
by phytoplankton 9 (Cifuentes et al., 1989)
ðDenitrificationÞ ¼ VDenit expðkDenit TÞ½NO3 ; (27) 7–8 (Montoya et al., 1991)
Culture estimates
where VDenit and kDenit are the denitrification rate at 0 C
and 5–29 (Pennock et al., 1996)
temperature coefficient for the denitrification rate, respectively. 15–25(Waser et al., 1998a,b)
Excretion by 33 5 Field estimate
Release of NHþ4 from the organic-rich sediment into the over-
zooplankton 2–8 (Checkley and Miller, 1989)
lying water can be assumed to depend on the DO concentrations Egestion by 34 2 No data
and temperature in the overlying water. In general, NHþ 4 release is zooplankton
dominated by the strength of coupling process between the supply Nitrification 35 25 Field estimates (in waters)
(regeneration) and consumption (sedimentary nitrification) of NHþ 4
12–17 (Horrigan et al., 1990)
20 (Sebilo et al., 2006)
within the sediments. In Ise Bay, temperature and DO concentra- 25 (Sugimoto et al., 2008)
tions in bottom waters change seasonally. Seasonal changes of Field estimate (in sediments)
water temperature directly affect activities of heterotrophic 7 (Brandes and Devol, 1997)
bacteria, which decompose organic nitrogen to NHþ 4 , and nitrifying
Culture estimates
35 (Mariotti et al., 1981)
bacteria. Bacterial activity generally increases with increasing
14–38 (Casciotti et al., 2003)
temperature, while those in DO concentration affect directly Denitrification 36 1 Field estimates (in waters)
oxygen conditions in the sediments. This means that bottom 30–40 (Cline and Kaplan, 1975)
hypoxic waters in the stratified periods would inhibit sedimentary 22–30 (Brandes et al., 1998)
nitrification, because there is no oxygen supply from the water to 30–35 (Sutka et al., 2004)
Field Estimates (in sediments)
the sediment. In such a case, NHþ 4 is released from the sediment 0–3 (Brandes and Devol, 2002)
into the overlying water according to increasing water tempera- w5 (Sigman et al., 2003)
ture. Our model therefore assumes that NHþ 4 is released when DO w1 (Sugimoto et al., 2008)
concentrations decrease to below 6.5 mg L1 in the bottom waters: Culture Estimates
25–30 (Mariotti et al., 1981)
29 (Barford et al., 1999)
ðReleaseÞ ¼ VRel expðkRel TÞ  Sedfactor ; (28)
This table was complied largely from the report of Yoshikawa et al. (2005). 35 and 36
where VRel and kRel are the release rate at 0  C and temperature are values in Ise Bay observed by Sugimoto et al. (2008). 31, 32, 33, and 34 are the
coefficient for NHþ 4 release, respectively; and Sedfactor is the sedi- adjusted values. 3 values express substrate minus product.
ment factor (¼ 20), which is adjusted to reproduce the observed
[NHþ 
4 ] and [NO3 ] concentrations at the bottom water. the nitrogen concentration and the river discharge (Sugimoto,
Sinking and sedimentation processes are written as 3
unpublished data). Therefore, the [NO þ
3 ], [NH4 ], [PO4 ], and [DET]

v (zPN) of the Kiso Rivers are assumed to be constant values at 50 mM,

ðSinking PHY Þ ¼  VSP ½PHY; (29) 8 mM, 0.1 mM, and 5 mM, respectively.
vz

v 4.3. Computation of d15N

ðSinking DET Þ ¼  VSD ½DET; (30)
vz
The fractionation of nitrogen isotopes in the model occurs in the
ðSedimentationÞ ¼ VSED $½DET; (31) processes of assimilation (photosynthesis) of NO 3 and NH4 by
þ

phytoplankton, excretion and egestion by zooplankton, and nitrifi-

where VSP, VSD, and VSED are the sinking velocity of phytoplankton,
sinking velocity of detritus, and sedimentation rate, respectively.
Sinking is set to zero in the bottom layers, while sedimentation
occurs only in the bottom layers.
3
The external loading terms are as follows. [NO þ
3 ], [NH4 ], [PO4 ],
and Chl-a concentrations and the temperature and salinity observed
at Irago Strait are used as the open boundary conditions. The Chl-
a concentration is converted into [PHY] using the carbon to Chl-
a weight ratio (C/Chl-a ¼ 40) and Redfield mol ratio (C:N ¼ 106:16).
The concentrations of zooplankton are assumed to be 10% of [PHY].
[DET], assumed to be a constant (¼0.5 mM), was observed off Ise Bay
as the PN concentration (Sugimoto et al., 2009a). Although nitrogen
concentrations in the Kiso Rivers showed wide ranges when river
discharge was low, there were no clear relationships with between
cation and denitrification (dashed arrows in Fig. 4; their 3 values are
listed in Table 2). During photosynthesis, the lighter isotope (14N) is
more readily incorporated into the tissue of phytoplankton. Thus,
the d15N values of NO þ
3 and NH4 increase as phytoplankton take up
these nutrients (e.g., Waser et al.,1998a,b). The 3 value during uptake
of NO 3 by phytoplankton is estimated to range from 4 to 9& in the
field and from 0 to 23& in cultures. For NHþ 4 , the 3 value is estimated
to range from 7 to 9& in the field and from 5 to 29& in cultures. In
the laboratory, 3 values arising from nitrification are generally large
(14–38&), although 3 can vary substantially depending on the
specific nitrification enzymes involved (Mariotti et al., 1981; Cas-
ciotti et al., 2003). However, in the field, 3 values by nitrification
differ between in water and sediment. Apparent 3 values are esti-
mated to be 12–25& in water and 7& in the sediment.
 R. Sugimoto et al. / Estuarine, Coastal and Shelf Science 86 (2010) 450–466 457

Denitrification within the ocean water column can result in 3 values where RC is the ratio of 15N/14N for a specific compartment C. The
of 22–45&, which are similar to laboratory-based estimates of 25– d15N values for a specific compartment C are calculated by:
30&. However, denitrification in various sedimentary environments
!
has little effect on the net isotope enrichment (3 ¼ 0–5&). The NHþ 4 8 15
½ N 9
excreted by zooplankton is lighter (2–8&) in d15N than the bulk >
< 15 >
=
½N  ½ N
nitrogen of their bodies (Checkley and Miller, 1989). However, the d15 N ¼ ! C
1  1000; (38)
d15N of zooplankton fecal pellets is slightly heavier than that of the >
: 15
½ N
>
;
diet (Altabet and Small, 1990; Montoya et al., 1990). The net result of ½N  ½15 N
Atmospheric N2
these two processes is an increase in d15N of roughly 3& per trophic
level (Minagawa and Wada, 1984; Wu et al., 1997).
For the present model, 3 values of nitrification in the water where Atmospheric N2 is defined as the standard sample including
column and denitrification in the sediments were estimated by [15N] and equals 0.365%.
field observations (Sugimoto et al., 2008). Excretion and egestion by The d15N values of external loading nitrogen are as follows: d15N
zooplankton were cited from Yoshikawa et al. (2005). The other of NO3 and detritus (zPN) in the Kiso Rivers are described using
assimilation processes were adjusted to reproduce the observed Eqs. (1) and (2), depending on the river discharge. The average
d15N values based on the reference 3 values in Table 2. The model value (14&) is used as the d15N of NHþ 4 in the Kiso Rivers. At the
expresses each process from substrate to product, and thus the open boundary, d15N of NO 
3 is described by Eq. (3) with [NO3 ]. The
isotopic fractionation coefficient al (l ¼ 1, 2, ., 6) is assumed to be other d15N values are assumed to be 5&, which is the oceanic
  average (e.g., Altabet et al., 1999; Sigman et al., 1999).
3l
al ¼ exp ; (32)
1000 5. Model results
where 3l is the isotope discrimination listed in Table 2.
5.1. Reproducibility of seasonal variations of phytoplankton
All equations for the 15N cycle are based on the above equations
and DIN
for the nitrogen cycle. The prognostic variables for the 15N cycle are
calculated as a function of t using the following five governing
Initial temperature, salinity, and velocity fields were given as
equations:
those in March calculated by the physical model (detailed in the
Appendix). Initial concentrations and isotope ratios of all nitrogen
were set to zero. The equations were integrated for 48 months, and
v½15 NNO3 
¼ ðPhotosynthesisÞ  Fnew  RNO3  a1 the results in the last 12 months were used to analyze the prop-
vt
erties of seasonal variations.
þ ðNitrificationÞ  RNH4  a5  ðDenitrificationÞ Fig. 5 shows the seasonal variations in observed and calcu-
 RNO3  a6 ; lated concentrations of Chl-a, NHþ 
4 , and NO3 in the subsurface

(33) (10 m) and bottom (34 m) layer at the central part of Ise Bay
(Stn. C in Fig. 1). Calculated concentrations of each compartment
show seasonal patterns similar to observed pattern. The calcu-
v½15 NNH4  lated concentrations of Chl-a in the subsurface layer are high in
¼ ðPhotosynthesisÞ  ð1  Fnew Þ  RNH4  a2
vt spring (w4.5 mg L1) and autumn (w3.5 mg L1) and low in winter
þ ðExcretionÞ  RZOO  a3 þ ðDecompositionÞ (w1 mg L1). Chl-a maximum in spring well reproduces the SCM
 RDET  ðNitrificationÞ  RNH4  a5 þ ðReleaseÞ in Ise Bay. The concentrations of NHþ 
4 and NO3 and their
seasonal variation in the subsurface layer are smaller and lower
 Rsediment ;
than those in the bottom layer. The NO 3 concentration in the
(34) subsurface layer decreases to w1 mM during the spring bloom. In
the bottom layer, the NHþ 4 concentration peaks in spring
v½15 NPHY  (w7 mM), while NO 3 concentration peaks in summer (w12 mM).
¼ ðPhotosynthesisÞ  Fnew  RNO3  a1 This temporal shift from NHþ 
vt 4 to NO3 in the lower layer

þ ðPhotosynthesisÞ  ð1  Fnew Þ  RNH4  a2
 ðMortalityZOO Þ  RPHY  ðGrazingÞ  RPHY
þ SinkingPHY  RPHY ;
v½15 NZOO 
¼ ðGrazingÞ  RPHY  ðMortalityZOO Þ  RZOO
vt
ðEgestionÞ  RZOO  a4  ðExcretionÞ  RZOO  a3 ;
v½15 N DET 
¼ ðMortalityPHY Þ  RPHY þ ðMortalityZOO Þ  RZOO
vt
þ ðEgestionÞ  RZOO  a4  ðDecompositionÞ
 RDET þ SinkingDET  RDET þ Sedimentation
 RDET ;
clearly displays the effect of nitrification, indicating the accu-
mulation of regenerated NO 3 in the lower layer during the
stratified periods.
Fig. 6 shows the seasonal variations in observed and calculated
(35)
d15N values of PN, NHþ 
4 , and NO3 in the subsurface and bottom
layers at the central part of Ise Bay (Stn. C in Fig. 1). In this study,
d15N of phytoplankton was not observed in the field since it was
difficult to separate phytoplankton from organic detritus. Thus,
observed d15NPN was assumed to be the same as calculated d15NPN,
ð36Þ which is the weighted average value of phytoplankton,
zooplankton, and detritus. The calculated d15NPN value of PN in the
subsurface layer decreases from winter (w10&) to spring (w6&),
but increases from spring (w6&) to summer. This seasonal vari-
ation in calculated d15NPN is consistent with observations,
although the calculated d15NPN was slightly lower than the
observed d15NPN from summer to autumn. In the bottom layer,
seasonal variation in the calculated d15NPN successfully reproduces
that in the observed d15NPN throughout the year. In the bottom
ð37Þ layer, d15NPN shows a minimum in spring (w5&) and maximum in
458 R. Sugimoto et al. / Estuarine, Coastal and Shelf Science 86 (2010) 450–466

Fig. 5. Seasonal variation in observed and calculated concentrations of Chl-a, NHþ 

4 , and NO3 in the subsurface (10 m depth) and bottom waters (34 m depth) at the central part of Ise
Bay (Stn. C). Symbols and lines present observed and calculated values, respectively.

15
summer (w10&). Calculated d NNO3 in the bottom layer has d15 NNO3 in the bottom layer well reproduces the observed
15
a prominent minimum in spring (w6&) when the NHþ 4 seasonal variation. Calculated d NNH4 values in the bottom layer
concentration reaches its maximum (Fig. 5), indicating the isotope are low from winter to spring (<15&) and high from summer to
15
effect of nitrification in the water column. From spring to summer, autumn (>20&). Calculated and observed d NNH4 in spring also
d15 NNO3 increases to w8&. Seasonal variation in the calculated 15
have similar values. The developed d N coupled ecosystem model

Fig. 6. Seasonal variation in observed and calculated nitrogen isotope ratios (d15N) of particulate nitrogen (PN), NHþ 
4 , and NO3 in the subsurface (10 m depth) and bottom waters
(34 m depth) at the central part of Ise Bay (Stn. C). Symbols and lines are the observed and calculated values, respectively.
 R. Sugimoto et al. / Estuarine, Coastal and Shelf Science 86 (2010) 450–466 459

80 a 3.0 b

2.5

C o n s u m p t io n /S u p p ly
0

D IN f lu x ( tN /d a y )
2.0
-80
1.5

-160
1.0

-240 0.5
1 2 3 4 5 6 7 8 9 10 11 12 1 2 3 4 56 7 8 9 10 11 12
Month Month

Fig. 7. (a) Seasonal variation in the nitrogen budgets. Positive budgets indicate supply of DIN (¼NHþ 
4 þ NO3 ) from the rivers (thin dashed line) and ocean (solid bold line), while
negative budgets mean consumption of DIN by phytoplankton in Ise Bay (thin solid line). (b) Seasonal variation in the ratio of DIN consumption by phytoplankton to DIN supply
from the outer regions (rivers and ocean). A linear line of 1.0 indicates equilibrium between external DIN supply and internal DIN consumption.

represents the seasonal variation in the concentrations and d15N gradient associated with the f-ratio. On the other hand, a high f-
values of observed nitrogen. ratio (>0.7) indicates that phytoplankton take up NO 3 as the main
nitrogen source in the subsurface Chl-a maximum at the bay
5.2. Relationships between phytoplankton production and DIN mouth. However, riverine NO 3 has already been depleted (<1 mM)
sources in each season by surface production at the bay head. Under such conditions,
d15NPHY expresses d15 NNO3 values similar to the source NO 3 because
Fig. 7a shows the seasonal variation in DIN (¼NHþ 
4 þ NO3 ) fluxes there is little isotope effect. Possible NO 3 sources for the subsurface
of the new supply from the river and ocean, and the consumption Chl-a maximum must be regenerated NO 3 and/or oceanic NO3 . In

15
rates of DIN by phytoplankton within the bay. The riverine flux was the lower layer, NO 3 with a significantly low d N (<4&) accu-
calculated as a product of the DIN concentration and the river mulates, resulting from the isotope effect of nitrification. If phyto-
15
discharge. The flux of DIN from the outer ocean to the inner sea was plankton uses such negative d NNO3 as their main nitrogen source,
15
estimated through the transverse section at the open boundary the d15NPHY value should be negative. On the contrary, the d NNO3

(Fig. 3). The oceanic flux was computed by integration of northward (>2&) of oceanic NO3 at the bay mouth was clearly higher than
velocities multiplied by the concentration in each grid. Consump- that of regenerated NO 3 (<2&), and thus the most plausible source
tion rates rapidly increase from w70 to w200 tN d1 with the for the subsurface Chl-a maximum would be external nitrogen
phytoplankton bloom in spring (Fig. 5). In this period, the supply of from the outer ocean. The intrusion depth of oceanic water by
DIN from the shelf increases from w30 to w60 tN d1 because of estuarine circulation changes from bottom to middle in spring
15
the strengthened estuarine circulation. High assimilation continues (Takahashi et al., 2000). The d NNO3 distribution clearly displays
during summer (w200 tN d1), peaking in autumn (w220 tN d1). the middle layer intrusion of oceanic NO 3 . Thus, increased DIN flux
However, the DIN supply from the river and shelf shows decreasing from the outer ocean (Fig. 7a) and the shift of the intrusion depth
trends in autumn. In winter, consumption rates rapidly decreases to would induce the spring phytoplankton production at the bay head.
<80 tN d1. Fig. 7b presents the ratio of DIN consumption by However, the lower f-ratio (w0.5) in the upper layer also indicates
15
phytoplankton to the supply from the river and shelf (consump- that the regenerated NHþ 4 with lower d N by remineralization
tion/supply ratio). The ratio is higher than 1.0 throughout the year, within the upper layer is utilized and largely affects the primary
indicating that the external supply of DIN is insufficient and that production in the bay.
the phytoplankton production largely depends on the regenerated Water stratification is strengthened by surface heating and high
DIN within the bay. The consumption/supply ratio considerably freshwater discharge in summer (Fig. 8b). In the bottom layer, the
differs in spring and autumn. The lower ratio in spring (<1.5) cold water mass (<18  C) is isolated from the surrounding waters
suggests a larger contribution of the external DIN supply from the by the horizontal thermo and haloclines and the vertical bottom
river and ocean, while the higher ratio in autumn (w3) suggests the front. This cold water mass becomes hypoxic (DO < 3 mg L1). The
dominant contribution of regenerated DIN. In Ise Bay, physical Chl-a concentrations show two prominent maxima as in spring. As
15
properties change seasonally (e.g., Takahashi et al., 2000). This both NHþ 
4 and NO3 are depleted in the upper layer, the d NPHY of
seasonal difference in the relationships between phytoplankton the two Chl-a maxima expresses the d15N values of assimilated
and DIN sources is discussed later. nitrogen because there is little isotope fractionation. Near the river
In spring, seasonal thermo and haloclines largely divide the mouth, the slightly high d15NPHY (w7&) and low f-ratio (<0.6)
15
water properties into two layers: upper and lower layers (Fig. 8a). In indicate uptakes of riverine NHþ 4 , which has low d N values
þ
the upper layer, there are two prominent Chl-a maxima: a surface (<20&) compared to those of regenerated NH4 (>40&) in the
maximum at the bay head and a subsurface maximum at the bay lower layer. Moving from the bay head to the center of the bay,
mouth. The lower f-ratio at the bay head indicates that phyto- phytoplankton tends to use the NO þ
3 instead of NH4 . On the other
plankton mainly takes up NHþ 4 within 10 km from the river mouth. hand, at the bay mouth, the phytoplankton forming the subsurface
However, phytoplankton change their utilization rates of nitrogen Chl-a maximum takes up NO 3 as the major nitrogen source.
forms from NHþ 
4 to NO3 moving from the bay head to the bay However, the NO 3 source (oceanic and/or regenerated) cannot be
center, as evidenced by the f-ratio increase from 0.2 to 0.7. The determined because both types of NO 3 have similar isotope values
d15NPHY in the surface Chl-a maximum also shows the horizontal in summer. The consumption/supply ratio (w2, Fig. 7b) suggests
460 R. Sugimoto et al. / Estuarine, Coastal and Shelf Science 86 (2010) 450–466

15 15 15
Fig. 8. Vertical distributions of simulated temperature, salinity, f-ratio, [Chl], [NHþ 
4 ], [NO3 ], d NPN, d NNH4 , and d NNO3 along the longitudinal section in (a) spring (1 May),
(b) summer (1 August), (c) autumn (1 November), and (d) winter (1 February), respectively.

that the contribution of regenerated NO 3 is comparable to that of
external DIN supplied from the rivers and shelf. On the other hand,
the high (>0.6) f-ratio indicates the weak contribution of NHþ 4 to
15
phytoplankton production in the upper layer. The d NNH4 in the
upper layer is considerably lower than that in the lower layer. This
indicates that residual NHþ 4 is left by nitrification processes in the
lower layer, while the new NHþ 4 is produced by remineralization in
the upper layer. Mino et al. (2002) found that d15N values of sus-
pended matters decrease with the uptake of regenerated NHþ 4 regenerated NO
rather than the uptake of new NO 3 supplied from the deep layer in
the Atlantic Ocean. The relatively low d15NPHY values (<10&)
associated with lower f-ratios (<0.6) indicate that phytoplankton
do not take up the residual NHþ 4 by nitrification; rather, they take
up the newly generated NHþ 4 by remineralization in the upper layer.
Fluxes of newly regenerated NHþ 1
4 by remineralization (33.5 tN d )
and excretion (19.8 tN d1) in the upper layer (>12 m) was found to
be considerably larger than fluxes of NHþ 4 supplied from the lower
layer (<12 m) to the upper layer by advection (3.9 tN d1) and
diffusion (12.3 tN d1).
In autumn, thermal stratification is weakened, and saline water
(>33) intrudes into the bay through the bottom layer (Fig. 8c). In
contrast to spring, in autumn the intrusion depth of the oceanic
water changes from the middle to the bottom layer (Takahashi
15
et al., 2000). Thus, oceanic NO 3 with low d N (<5&) is supplied
into the lower layer by the bottom intrusion. Two maxima in Chl-
a concentration occur at the bay head and the bay mouth. The
higher f-ratio indicates that phytoplankton extensively take up NO 3 lower values (<0.3) at the bay head to higher values (>0.3) at the
as substrate nitrogen except for near the river mouth, where higher
d15NPHY (>7&) and lower f-ratio (<0.5) indicate the utilization of
riverine NHþ 
4 . Riverine NO3 is also consumed at the river mouth,

and thus NO3 is exhausted in the upper layer from the center to the
bay mouth. Moreover, the high consumption/supply ratio (w3,
Fig. 7b) means that the regenerated DIN is the dominant source for
phytoplankton production. The homogeneous vertical profile of the
d15 NNO3 in the center of Ise Bay clearly displays the supply of
3 into the euphotic layer by vertical mixing.
15
Although NO 3 is depleted (w1 mM) in the upper layer, d NNO3 is
comparable to that in the bottom layer. The finding of little vertical
15
difference of d NNO3 indicates that the regenerated NO 3 is
continuously supplied from the bottom layer into the euphotic
layer.
Water convection in winter creates vertically homogeneous
temperature and salinity (Fig. 8d). The consumption/supply ratio
(w1.5, Fig. 7b) suggests that two-thirds of phytoplankton produc-
tion depends on the external DIN supply rather than the regen-
erated DIN. Within the bay, Chl-a concentrations are considerably
low (<1 mg L1) because of low temperature and low DIN concen-
trations. d15NPHY at the bay head is higher than that at the bay
mouth. The concentrations and d15N values of NO-3 are homoge-
neous within the bay, indicating that the spatial difference of
d15NPHY is caused by the NHþ 4 assimilation by phytoplankton. The
low f-ratio also indicates that phytoplankton preferentially take up
NHþ 4 . However, the f-ratio shows slightly horizontal gradients from
 R. Sugimoto et al. / Estuarine, Coastal and Shelf Science 86 (2010) 450–466
15
bay mouth. In addition, d NNH4 at the bay mouth is higher than
that at the bay head. Therefore, d15NPHY at the head of the bay has
higher d15NPHY values.
5.3. Annual nitrogen budgets in Ise Bay
Fig. 9 shows the annual average nitrogen flux (tN d1) of each
process and of standing stocks (tN) in Ise Bay. The nitrogen supply
of 32 tN d1 from the river is composed of 24 tN d1 as NO
5 tN d1 of NHþ 4 , and 3 tN d
1
as detritus. The net nitrogen flux from
the ocean reaches 29 tN d1, which is comparable to the riverine
nitrogen flux. Dissolved forms, NO 1
3 (37 tN d ) and NH4
1
(11 tN d ), are supplied from the ocean to the bay, while particu-
late forms composed of phytoplankton (12 tN d1), zooplankton
(2 tN d1), and detritus (7 tN d1) flow out from the bay to the
ocean. A total of 77 tN d1 of DIN is supplied to the inner bay as new
nitrogen.
In contrast, the nitrogen flux for each process within the bay is
considerably larger than the new nitrogen flux from the outer
regions. Phytoplankton takes up 93 tN d1 of NO 3 and 67 tN d
1
þ 1
NH4 . The total flux of phytoplankton assimilation (160 tN d ) is
more than twice the total flux from the river and ocean. This result
means that more than half the amount of annual phytoplankton
production is supported by the regenerated DIN. However, high
consumption/supply ratios (>2.0) occur only in only periods from
September to November (Fig. 7b), suggesting that regenerated DIN,
which is accumulated in the hypoxic water mass during the strat-
ified season, contributes significantly to annual phytoplankton
461
Fig. 8. (continued).
production. Because a large amount of phytoplanktonic nitrogen is
transferred to detritus (94 tN d1) rather than zooplankton
(54 tN d1), phytoplankton mortality accounts for more than 70% of
the total detritus supply, which is one of the largest processes in Ise
Bay. Sedimentation of detritus is larger than the NHþ 4 release from
the sediments. Regenerated nitrogen from remineralization
(54 tN d1) and release from sediments (55 tN d1) also shows
large flux. These two fluxes account for w70% of the total NHþ 4
3, supply, suggesting that they are the most important sources for
phytoplankton production. Moreover, zooplankton excretion
accounts for 16% of the total NHþ 4 supply. A large supply of NO3 is

þ nitrification (80 tN d ), which accounts for w60% of the total NO
1
3
supply. The denitrification loss of 48 tN d1 is twice as large as the
NO 1
3 supply from the river (24 tN d ). The calculated denitrifica-
tion rate is comparable to that estimated from the Redfield stoi-
chiometry (42 tN d1; Kakehi et al., 2005) and larger than the
potential denitrification rate (19–28 tN d1; Sugawara, 2003).
These results indicate that phytoplankton production in Ise Bay is
mainly dominated by the internal cycle (DIN assimilation by
of phytoplankton, mortality of phytoplankton, remineralization of
detritus, release from the sediments, nitrification, and denitrifica-
tion) rather than the external supply (fluxes from rivers and the
ocean).
6. Summary and discussion
We developed an ecosystem model coupled with d15N to eval-
uate the seasonal variation in nitrogen dynamics, with particular
462 R. Sugimoto et al. / Estuarine, Coastal and Shelf Science 86 (2010) 450–466
Fig. 8. (continued).
focus on the relationship between phytoplankton production and
DIN behavior. DIN (¼NHþ 
4 þ NO3 ) consumption by phytoplankton
exceeds DIN supply from the river and ocean (Figs. 7 and 9), indi-
cating that phytoplankton production in Ise Bay largely depends on
regenerated DIN within the bay rather than on newly supplied DIN.
Similar results were reported from several other coastal environ-
ments. In Escambia Bay, Florida, the nutrient demand for phyto-
plankton production exceeds the terrestrial nutrient supply from
the rivers, implying that phytoplankton rely on recycled nutrients
to support growth, requiring that available nutrient pools be used
several times (Murrell et al., 2007). In Monterey Bay, California,
NO 3 regenerated by nitrification in the euphotic zone plays
a substantial role in gross productivity, ultimately supporting w30%
of the total NO 3 -based primary production (Wankel et al., 2007).
Modeling studies of eutrophic coastal environments in Japan have
also reported the significance of regenerated nitrogen (e.g., Hayashi
and Yanagi, 2002; Kittiwanich et al., 2006.
In Ise Bay, the formation of a cold water mass isolated from
surrounding waters during the stratified periods is key to the large
contributions of regenerated nitrogen to phytoplankton produc-
tion, because the cold water mass covers a large volume in the
lower layer (e.g., Fujiwara et al., 2002). The oxygen within the
isolated water mass is mainly consumed by remineralization of
organic matter in the waters and sediments, and thus a large
amount of DIN accumulates in the hypoxic water mass during the
stratified periods (Kakehi et al., 2005; Sugimoto et al., 2008).
Consequently, the standing stock of DIN in the lower layer is high
(w2500 tN) in summer, but low in winter (w1400 tN; Kakehi et al.,
2005). The consumption/supply ratio differs considerably by
season (Fig. 7b). The lower ratio in spring (<1.5) means that the
direct DIN supply from the rivers and ocean is more important than
that from regeneration, while the higher ratio in autumn (w3)
means that regenerated DIN is the major source for phytoplankton
production (Fig. 7b).
Although the riverine DIN stimulates phytoplankton production
at the bay head and forms a Chl-a maximum in the surface layer,
phytoplankton production at the bay mouth is largely controlled by
the estuarine circulation. In spring, the intrusion depth of oceanic
15
water changes from the bottom to the middle layer. d NNO3

distributions clearly show that oceanic NO3 is transported into the
euphotic layer by the middle intrusion and stimulates phyto-
plankton production at the bay mouth. Kasai et al. (2007) also
pointed out the importance of the middle intrusion of oceanic
water for the SCM layer. In autumn, the intrusion depth of oceanic
water changes from the middle layer to the bottom layer. Regen-
erated NO 3 , which is accumulated in the hypoxic water mass, is
vertically supplied into the euphotic layer. This vertical supply of
regenerated nitrogen induces the maximum DIN consumption rate
(w220 tN d1, Fig. 7a). These results show that seasonal shifts of the
intrusion depth of estuarine circulation induce spatial and
temporal differences in DIN behavior and subsequent phyto-
plankton production.
In recent years, ecosystem models incorporating d15N have
proven to be powerful tools for elucidating nitrogen dynamics in
both the past and modern ocean. Giraud et al. (2000, 2003)
developed a simple ecosystem model for simulation of d15N of
 R. Sugimoto et al. / Estuarine, Coastal and Shelf Science 86 (2010) 450–466
Fig. 8. (continued).
sediments in the Mauritanian upwelling region. Their model,
consisting of four compartments (phytoplankton, zooplankton,
detritus, and nutrient), was the first generalized ecosystem model
to include nitrogen isotopes. However, their model did not distin-
guish between NHþ 
4 and NO3 , although isotopic discrimination by
assimilation and nitrification of NHþ 4 is large (see Table 2). Yoshi-
kawa et al. (2005) developed a six-compartment (phytoplankton,
zooplankton, PON, DON, NHþ 
4 , and NO3 ) ecosystem model
including d15N and examined the seasonal variation in d15N of
sinking PN in the Sea of Okhotsk. From sensitivity analyses of
change in the isotope discrimination factor, they found that the
isotope effect of NO þ
3 and NH4 assimilation by phytoplankton and
nitrification is important for d15N variation in sinking PN. Yoshi-
kawa et al. (2006) adapted their model to the western and central
equatorial Pacific surface waters. However, the d15N-ecosystem
models developed by Giraud et al. (2000, 2003) and Yoshikawa
et al. (2005, 2006) were coupled with two dimensional physical
models (two and/or multi-layers models), because the physical
properties in their study regions are relatively simple. Moreover,
their studies focused on the modeling, and thus used relatively few
field-observed isotope data and 3 values.
On the other hand, the d15N coupling ecosystem model we
developed is fully three-dimensional and includes a multi-level
physical model, since the temporal and spatial distributions of
nutrients in Ise Bay are controlled by complex physical–biological–
chemical interaction processes associated with external loading,
advection/diffusion, oceanic water inputs, and sedimentary release.
Precise and seasonal observations of d15N of NO 3 , PN, and part of
463
NHþ 4 in the boundary conditions (river and ocean) and within the
bay and adoption of observed 3 values enhance the accuracy of the
ecosystem model. Therefore, our d15N coupling model provides
crucial information to distinguish DIN sources within the bay and to
evaluate the contribution of each source to primary production. In
15
particular, simulated d NNO3 distributions show the intrusion
pathway of oceanic NO 3 . These results strongly supported the
importance of oceanic NO 3 for phytoplankton production during
the stratified periods as reported by previous field studies (Kasai
et al., 2007; Sugimoto et al., 2009b). This work is the first attempt at
such an inclusive and integrative study is not only in coastal but
also in oceanic systems.
During the past three decades, researches have actively inves-
tigated the rate processes of biological nitrogen transformation in
the sea using the 15N tracer technique alone or in combination
with other chemical techniques. The information collected by
these investigations, together with data on nutrients, PON, DON,
and biomass, have improved our present understanding of
nitrogen dynamics in marine ecosystems. Many models have also
been presented to describe the kinetics of nitrogen trans-
formations and to identify factors that regulate the processes.
However, validation of transformation processes by only concen-
tration data is difficult. Furthermore, incorporation of rate data
estimated by incubation experiments does not necessarily provide
actual fluxes and concentrations. Since each approach has
temporal and/or spatial problems, combination studies provide
a more comprehensive understanding of nitrogen dynamics in
marine ecosystems. Our model successfully reproduced the
464 R. Sugimoto et al. / Estuarine, Coastal and Shelf Science 86 (2010) 450–466

Riverine
Loading

54
2
Phytoplankton=606 Zooplankton=159
94
12

Boundary
93 22 18 12 Flux
37

11
67
7
NO 3- =3313 NH 4+ =416 Detritus=452
24 80 54
5
3 48 55 66

Sediment

Fig. 9. Annual averages of nitrogen fluxes (tN d1) and standing stocks (tN) in Ise Bay. The magnitude of flux is visually shown by the thickness of lines. Each arrow corresponds to
those in Fig. 4.

observed concentrations and d15N values of NO þ

3 , NH4 , and PN help with observations, We also extend our thanks to the staff of
15
throughout the year. Because changes in each d N value occur the Center for Ecological Research, Kyoto University, and of the
during nitrogen transformation processes, validation of d15N Ocean Research Institute, University of Tokyo, where we conducted
values and adoption of observed 3 values allow us to estimate the isotope analysis.
more comprehensive reaction rates for each nitrogen trans-
formation process than those estimated by traditional ecosystem
models. For example, isotopic discrimination of nitrification in the Appendix
water column is the largest process in Ise Bay (Table 2). Our model
15
well reproduces the decrease of d NNO3 by the isotope effect of The physical model in this study was a fully three-dimensional
nitrification in spring. The temperature coefficient of nitrification (3-D) coastal ocean model. The objective of this model is to
adjusted in this model (¼0.32  C) is larger than that in Yoshikawa’s reproduce the seasonal variation in temperature, salinity, and
et al. (2005) model (¼0.0693  C), while similar to the observed velocities. The model is based on hydrostatic primitive equations.
value (¼0.29  C) at the Providence River estuary (Berounsky and Briefly, the equations for the 3D flow are as follows:
Nixon, 1990). However, further observation of d15N values of NHþ 4    
and sensitivity studies are needed to estimate more comprehen- vu vu vu vu 1 vP v vu v vu
þ u þ v þ w  fv ¼  þ Nh þ Nh
sive values. vt vx vy vz r0 vx vx vx vy vy
 
Our model shows that phytoplankton production in Ise Bay is v vu
þ Nz þ TSx ; ðA1Þ
mainly dominated by the internal cycle rather than by the external vz vz
supply. However, the river discharge of the Kiso Rivers can change
within short time periods, and floods frequently occur over daily    
vv vv vv vv 1 vP v vv v vv
time scales (e.g., Sugimoto et al., 2006). Therefore, our study might þ u þ v þ w þ fu ¼  þ Nh þ Nh
vt vx vy vz r0 vy vx vx vy vy
have underestimated the effect of riverine loadings. The abrupt  
increase in riverine nitrogen input may change nitrogen dynamics v vv
þ Nz þ TSy ;
in the bay. Unfortunately, the field observations made in this study vz vz
did not show the physical and biogeochemical characteristics of (A2)
high-discharge conditions. Further observations including flood
periods are needed to more precisely elucidate the effects of Z0
riverine loadings on the coastal environment. r0  r
P ¼ r0 g x þ r0 gdz; (A3)
r0
z

Acknowledgments
vu vv vw
þ þ ¼ 0; (A4)
vx vy vz
We thank Dr. T. Fujiwara of the Graduate School of Agriculture,
Kyoto University and Dr. S. Kakehi of the Tohoku National Fisheries
Zx ! Zx !
Research Institute, Fisheries Research Agency, for helpful discus- vx v v
sions and suggestions. We are grateful to the captain and crew of ¼  udz  v dz ; (A5)
vt vx vy
the R/V Asama of the Mie Prefecture Fisheries Research Institute for H H
 R. Sugimoto et al. / Estuarine, Coastal and Shelf Science 86 (2010) 450–466 465

     
vT vT vT vT v vT v vT v vT
þu þv þw ¼ K þ Kh þ Kz ; v½DO
vt vx vy vz vx h vx vy vy vz vz ¼ RFDO=Chl $ðPhotosynthesisÞ  DOdr ðDOdb Þ (A16)
vt
(A6)
where RFDO/PHY (¼0.139 mg mg1) is the ratio of oxygen and Chl-a of
Eq. (A1), and thus the first term on the right side indicates the
      oxygen supply by photosynthesis. The second and third terms give
vS vS vS vS v vS v vS v vS
þu þv þw ¼ Kh þ Kh þ Kz ; (A7) the oxygen consumption by remineralization in the water column
vt vx vy vz vx vx vy vy vz vz
and in the sediments. DOdr (g m3 d1) and DOdb (g m2 d1) are
where u and v are the horizontal velocity components in the x and y expressed as follows:
directions, respectively, and w is the vertical velocity component in  
2p
the z direction; t is time, P is pressure, f is the Coriolis parameter DOdr ¼ 0:062 þ 0:05sin $ðiday  30Þ ; (A17)
Ty
(¼0.8  104 s1), r0 is the mean density (¼1020 kg m3), r is
density, x is the height from the piezometric surface, g is the gravity
constant (¼9.8 m2 s1), H is the water depth, T is temperature, S is DOdb ¼ hDO $DOdr ; (A18)
salinity, Nh is horizontal eddy viscosity (¼2.5 m2 s1), and Kh is where hDO (¼7 m) is the factor for the unit changes, which is
horizontal eddy diffusivity (¼2.5 m2 s1). The vertical eddy adjusted to reproduce the observed DO concentrations (Kakehi,
viscosity (Nz) and vertical eddy diffusivity (Kz) are calculated as 2006).
pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi The mathematical expression for the internal sources or sinks of
Nz ¼ Kz ¼ 104 $ uT þ vT $H; (A8) PO3 1
4 concentrations (mmol L ) is given as follows (Kakehi, 2006):

where uT and vT are the tidal amplitude in x and y directions, h i

respectively, and Nz and Kz are 60  104 m2 s1 at the bay mouth v PO3
4 v½DO

and 2  104 m2 s1 at the bay center. Tidal stresses of the x and y ¼ RFPO4 =PHY $ þPO4 RF dev ; (A19)
vt vt
components are calculated using the tidal model (Kakehi, 2006):
where RFPO4 =PHY (¼ 0.225 mmol mg1) is the ratio of PO3
4 to Chl-a of
  Eq. (A1). PO4RF_dev (mM mo1) is the deviation from the Redfield
vu0 vu0 vu0
TSx ¼  u0 þ v0 þ w0 ; (A9) relationship such as by release and adsorption. PO4RF_dev is deter-
vx vy vz
mined by the temperature and DO concentration in the bottom
  waters and is expressed as
vv0 vv0 vv0
TSy ¼  u0 þ v0 þ w0 ; (A10) PO4RF ¼ hPO4 $ð  20:697 þ 0:0288$T  0:00321$DOÞ;
vx vy vz dev
(A20)
where overbars indicate the average per tidal cycle. u0 , v0, and w0 are
the velocity, calculated by the tidal model, in the x, y, and z direc- where hPO4 (¼10 m) is the factor for the unit changes, which is
tions, respectively. adjusted to reproduce the observed DO concentrations (Kakehi,
Boundary conditions are briefly determined as follows. Monthly 2006).
observed temperature and salinity at the open boundary at the
Irago Strait were obtained by from the Mie Prefecture Fisheries
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