Nitrate sources and dynamics in the salinized rivers and estuaries – a δ 15 Nand δ 18 ONO − 3 isotope approach

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Introduction
Increasing population, extensive agricultural activities and rapid development of urbanization in coastal areas have dramatically increased N loading to rivers and coastal waters (Seitzinger and Kroeze, 1998;Jennerjahn et al., 2004;Umezawa et al., 2008).Estuaries play a prominent role for delivery of terrestrially derived N to coastal water through physical, chemical, and biological processes (Mulholland, 1992;Bernhardt et al., 2003;Sebilo et al., 2006;Hartzell and Jordan, 2012).
Many estuarine studies have focused on tracing N sources and assessing N dynamics in large estuarine systems, such as the Elbe Estuary (Dähnke et al., 2008) and the Atlantic coast (Middelburg and Herman, 2007) in Europe, the San Francisco Bay Estuary (Wankel et al., 2006), the Mississippi River Estuary (Rabalais et al.,1996), and the Mid-Atlantic coast (Dafner et al., 2007) in the United States, and the Yangtze River Estuary (Chai et al., 2009) and Pearl River Estuary (Dai et al., 2008) in China.Sel-Introduction

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Full 2 Material and method

Study area
The investigated three rivers are located in a coastal municipality, Tianjin, China (Fig. 1).The survey took place in three rivers along a salinity gradient, the Haihe River (HH River) on 7 November 2012, the Chaobaixin River (CB River) on 9 November 2012 and the Jiyun River (JY River) on 10 November 2012 (Fig. 1).Water samples were also taken along the estuary of the HH River (HH Estuary) and the mixing estuary of the CB River and the JY River (CJ Estuary) on 16 November 2012 to study reactive N transformation processes from the river to the estuary (Fig. 1).The river-path length from the upstream sampling location to the river mouth sampling location of the HH River was 72 km with a watershed area of 2066 km 2 (Liu et al., 2001).Since the separation by the floodgate, the upstream part of the HH River serves as a river-type reservoir for the purpose of supplying water to the residents living along the river bank.The other floodgate is located at the end of the HH River serves as flood discharging, tidal blocking and ship traffic.The CB River flows through the rural area with a total length of 81 km and the watershed area is about 1387 km 2 (Gburek et al., 1998).Animal manure could be a potential dominant NO − 3 source in the CB River as this watershed has important livestock breeding base for the municipality (Shao et al., 2010).The JY River flows through agricultural area and is considered as a significant water source for agricultural and domestic use.The total length of the Jiyun River is 144 km with a watershed area of about 2146 km 2 (Chen et al., 2000).

Sampling and analysis
Water samples were taken on transects spanning the freshwater portion of the rivers to coastal water of the Bohai Bay.Water samples were stored frozen in 1 L HDPE (High Density Polyethylene) bottles for determination of physico-chemical properties and δ 15 N-and δ 18 O-NO − Introduction

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Full 3 ), NO − 2 and NH + 4 concentrations were analyzed on a continuous flow analyzer (Auto Analyzer 3, Seal, Germany).

The δ
15 N-and δ 18 O-NO − 3 values were determined by the "Bacterial denitrification method" (Sigman et al., 2001;Casciotti et al., 2002;Xue et al., 2010) in the UC Davis Stable Isotope Facility of California University, which allows for the simultaneous determination of δ 15 N and δ 18 O of N 2 O produced from the conversion of NO − 3 by denitrifying bacteria, which naturally lack N 2 O -reductase activity.Isotope ratios of δ 15 N and δ 18 O are measured using a Thermo Finnigan GasBench + PreCon trace gas concentration system interfaced to a Thermo Scientific Delta V Plus isotope-ratio mass spectrometer (Bremen, Germany).The N 2 O sample is purged from vials through a double-needle sampler into a helium carrier stream (25 mL min −1 ) and CO 2 is removed using scrubber (Ascarite).By cryogenic trapping and focusing, the N 2 O is compressed onto an Agilent GS-Q capillary column (30 m×0.32 mm, 40 • C, 1.0 mL min −1 ) and subsequently analyzed by IRMS.Stable isotope data were expressed in delta (δ) units in per mil (‰) relative to the respective international standards: where R sample and R standard are the 15 N/ Full

Mixing model
Normally, a mixture (MIX) can be calculated via a basic mixing model (Liss, 1976): where C represents concentration, the subscripts R and M represent riverine and marine end-members, respectively; f represents the fraction of freshwater in each sample calculated from salinity (Dähnke et al., 2006): where MAX is taken as the maximum measured salinity of marine end-member for coastal water and MEA is taken as the measured salinity of the mixture.Isotopic values of mixed estuarine samples (δ MIX ) were calculated using concentration-weighted isotopic values for riverine and marine end-members, respectively (Fry, 2002;Dähnke, 2006): where C represents concentration, δ represents isotopic value, the subscripts R and M represent riverine and marine end-members, respectively; and f represents the fraction of freshwater in each sample.The salinity-based isotopic mixing does not follow linear conservative mixing but show curvilinear mixing that reflects concentration-based weighting of end-member isotopic contributions.Based on the mixing curves of DIN concentration against salinity, additional sources or cycling can be identified.When an enriched external source or biological transformation (e.g.mineralization, nitrification, etc.) contributes into the river, DIN distribution is expected to fall above the mixing line.In turn, when a depleted external source or the internal removal processes (e.g., denitrification, assimilation, etc.) appears in the river, DIN distribution is expected to fall below the mixing line (Wankel et al., 2006).Introduction

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Physicochemical properties
Table 1 summarizes the data of physicochemical properties collected in this study in the rivers and estuaries.Obviously, the salinities of the HH River (ranging from 0.7 to 4.9 with a mean value of 2.2) and its estuary (ranging from 18.6 to 24.1 with a mean value of 21.2) is higher than the rivers of CB (ranging from 0.5 to 0.6 with a mean value of 0.5) and JY (ranging from 0.6 to 0.8 with a mean value of 0.7) and the corresponding estuary (ranging from 2.0 to 20.0 with a mean value of 7.7), respectively.The municipality had been suffering multiple seawater intrusion and regression, which results in the salinization of the rivers and soil (Wang, 2004), while the greater salinization level of the HH River is also related to seawater intrusion over the floodgate until upstream of the HH River in a relatively long distance.The rivers and the estuaries showed similar pH values between 7.5 and 8.6.The temperature of the HH River varied around 12.3 • C slightly higher than the CB River (mean is 10.9 • C) and the JY River (11.5 • C).
The mean temperature of the HH Estuary (9.7) is also higher than that of the CJ Estuary (6.7).Dissolved oxygen (DO) concentrations were relatively enriched in this study (higher than 7.2 mg L −1 ), excluding the DO depleted area in the upstream of the HH River (lower than 5.0 mg L −1 ).

DIN species
Wide concentration variations were noticeable for DIN (NH + 4 , NO − 2 and NO − 3 ) species in To derive qualitative information on the predominant NO − 3 sources in the rivers and the corresponding estuaries, a classical dual isotope approach (δ 15 N-NO − 3 vs.δ 18 O-NO − 3 ) has been applied (Fig. 2).It is clear that the isotope signatures of all the sampling locations showed in a scattered distribution, indicating different NO − 3 source contamination in the rivers and the estuaries.Upstream of the HH River at a salinity of 1.0, a floodgate separates the river into two parts; and at the end of the river at the salinity of 4.9, the other floodgate controls the connection of the river to the HH Estuary.Hence, the δ 15 Nand δ 18 O-NO − 3 values of the HH River behaved quite differently, which moved from the overlapping area of the "NH + 4 fertilzier" and "soil N" source boxes for the majority of the upstream sampling locations, to the overlapping area of the "soil N" and "manure and sewage" source boxes at the end of the river.In this study, the majority of the sampling locations were potentially influenced by the source of "soil N" or "sewage" not the "mineral fertilizer", as the HH River flows through the municipality without agricultural activities.In addition, it can no be excluded the influence from salt water intrusion from the estuary, which showed similar isotopic values to that at the end of the HH River.The distribution of the HH Estuary does not show a landward trend due to the floodgate at the end of the HH River, but falls into the range of marine NO − 3 reported by Kendall et al. (2007).
Animal manure could be a potential dominant NO − 3 source in the CB River as this watershed plays the role of important livestock breeding base for the municipality (Shao et al., 2010).Furthermore, the δ 15 N-NO − 3 values were enriched and varied around 14 ‰, indicating anthropogenic NO − 3 derived from manure (Kendall et al., 2007;Xue et al., 2009).The isotope signatures of the JY River were mainly concentrated in the "soil N" source box.The δ A mixing line (HH1-E) was setup between the initial sampling location in the HH River and the last sampling location in the HH Estuary (Fig. 3).After the separation of the floodgate, the upstream of the HH River serves as a river-type reservoir.Thus, a new mixing line (HH2-E mixing line) was re-calculated between the sampling location after the floodgate and estuarine water (Fig. 3).The salinity gradient sampled in the HH River and its estuary showed a seaward decreasing trend in DIN (NH + 4 , NO − 2 and NO − 3 ) concentrations and an increasing trend in δ 15 N-NO − 3 and δ 18 O-NO − 3 values throughout the entire salinity gradient (Fig. 3).However, the DIN and isotopic trends did not behave conservatively, as most of the measured data deviated from the calculated mixing lines, especially from HH1-E between the riverine concentration and the coastal water.
It is clear that in the upstream part of the HH River before the floodgate 1, NO − 2 and NH + 4 were above (a source) while NO − 3 was below (a sink) the HH1-E mixing line.Hence, NO − 3 removal processes are expected to occur in the river.Denitrification has been considered as a dominant NO − 3 reduction pathway, which reduces NO − 3 to gaseous N compounds (NO, N 2 O and N 2 ) under anaerobic conditions.However, a large number of laboratory studies have reported that denitrification occurs under aerobic conditions in pure cultures of bacteria (Robertson and Kuenen, 1984;Ronner and Sorensson, 1985;Trevors and Starodub, 1987;Robertson et al., 1995).Various heterotrophic nitrifiers have been tested and found to be capable of simultaneous heterotrophic nitrification and aerobic denitrification (Castignetti and Hollocher, 1984; denitrification in nature, e.g. in Mississippi River Basin (Thomas et al., 2006) and in the central German Wadden Sea (Gao et al., 2010).Thus, the aerobic denitrification was expected under the relatively enriched DO (2.7-5.0 mg L −1 ) condition in this study.In addition, assimilation process can be ignored due to a non-rapid consumption of NH which is generally the preferred form of N (when NH + 4 was elevated compared to NO − 3 ) for phytoplankton uptake (Dugdale and MacIsaac, 1971;Dugdale and Hopkins, 1978;Dugdale et al., 2006).The NH + 4 species was accumulated as a source, potentially originating from organic matter decomposition not sewage discharge, as the δ For the HH2-E mixing line after the floodgate 1 (Fig. 3), salinity gradient sampled in the downstream of the HH River illustrated NO − 3 turned from a source (above the HH2-E mixing line) to a sink (below the HH2-E mixing line), while NO − 2 and NH + 4 turned from a sink (below the HH2-E mixing line) to a source (above the HH2-E mixing line) at the end of the river.Nitrate accumulation may be linked to an in-stream nitrification process, in which NO − 2 and NH + 4 were consumed to produce NO − 3 .In nitrification, the conversion of NH + 4 to NO − 2 and NO − 3 is accompanied by marked N isotope fractionation effects, resulting in 15 N depleted NO − 3 (Delwiche and Steyn, 1970;Mariotti et al., 1981;Macko and Ostrom, 1994).For δ 18 O-NO − 3 values, NO − 3 produced by nitrification in aquatic environments usually takes similar δ 18 O values to the ambient water (Casciotti et al., 2002;Sigman et al., 2005).There is evidence that O can exchange between H 2 O and intermediate compounds of nitrification (Andersson et al., 1982;DiSpirito and Hooper, 1986;Kool et al., 2007).Since the δ 18 O of estuarine water is expected to be higher than that of river water (Miyajima et al., 2009), δ 18 O-NO − 3 should increase along the salinity gradient when in situ nitrification is occurring.Thus, a decrease in δ 15 N-(4.6-3.9 ‰) and an increase in δ 18 O-NO − 3 (0.6-1.2 ‰) occurred downstream of

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Nitrate removal efficiency in the rivers and the estuaries
In this study, most of the measured data deviated from the calculated mixing lines, indicating rivers and estuaries becoming either a source or a sink.Thus, variation percentages of the measured data compared to the calculated mixing lines were computed to assess the NO − 3 removal efficiency for the rivers and estuaries (Fig. 5).Interestingly, in the upstream part of the HH River before the floodgate 1, NO − 3 was removed 30.9±22.1 % compared to the calculated mixing line.Denitrification could be the dominant NO − 3 removal process.This potentially results from the separation of the floodgate which limited water exchange with downstream water enriched in DO.Furthermore, the floodgate 1 might prolong water residence time in the upstream part to remove a significant part of riverine N loadings.The downstream part of the HH River between floodgate 1 and floodgate 2 showed an extremely weak NO Estuaries of rivers are considered as active sites of massive NO − 3 losses (Brion et al., 2004;Seitzinger et al., 2006), removing up to 50 % of NO − 3 (OsparCom, 2000).However, our data do not support this view as in the HH and the CJ estuaries.First, DO concentrations were higher than 10 mg L −1 not favorable for water column NO the estuaries by NO − 3 removing processes as reclamation projects for the regional and national economy leading to the hydrodynamics of circulation in Tianjin section disappearance (Qin et al., 2012).This phenomenon could reduce water residence time and force NO − 3 pollutants moving to the northern part of Bohai Bay, aggravating NO − 3 contamination.
Furthermore, this wintertime situation, with water temperature around 10 • C, ruled out most biological activity, and conservative mixing behavior in the HH River Estuary was not overly surprising.However, the CJ Estuary became a NO − 3 source, linking to sewage discharge of mooring ships.

Conclusions
The combined use of salinity, DIN concentrations and NO

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Full  Full Screen / Esc Printer-friendly Version Interactive Discussion Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Screen / Esc Printer-friendly Version Interactive Discussion Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper |were measured by a portable water quality probe (Thermo Orion, USA)samples were filtered through 0.45 µm membrane filters and stored at 4 • C until analysis.Nitrate (NO − Discussion Paper | Discussion Paper | Discussion Paper |

Table 1 .
In the HH River, the NH + 4 concentrations varied from 124.1 to 332.6 µmol L −1 , the NO − 3 concentrations varied from 62.5 to 219.0 µmol L −1 and the NO − 2 concentrations varied from 7.2 to 20.8 µmol L −1 .The DIN concentrations of the HH Estuary varied smoothly (5.6-6.7 µmol L −1 for NO − 2 , 7.1-25.7 µmol L −1 for NO − 3 , and 65.7-88.1 µmol L −1 for NH + 4 ) and were quite low compared to the HH River.Nitrate concentra-Discussion Paper | Discussion Paper | Discussion Paper | tions in the CB river were relatively elevated (120.0-171.5 µmol L −1 ) with a continuous accumulation along the entire salinity gradient, while NO − 2 concentrations decreased from 12.0 to 6.0 µmol L −1 .Ammonium concentrations in the CB River varied from 143.9 to 380.0 µmol L −1 .The JY River also showed NO − 3 accumulation (increased from 40.0 to 83.3 µmol L−1 ) along the entire salinity gradient, while a decreasing trend was ob-L −1 ) and NO − 3 (153.4-6.1 µmol L −1 ).Compared to the other river and estuaries, DIN results of this study are similar to that in the Pearl River Estuary(Dai et al.
Discussion Paper | Discussion Paper | Discussion Paper | peared in this estuary, due to sewage discharge of mooring ships in the vicinity of the sampling area.Thus, the influence of sewage and the CB River was considered as the dominant NO − 3 source.4.2 Nitrate dynamics in the salinized rivers and the corresponding estuaries 4.2.1 Nitrate dynamics in the HH River and its estuary Discussion Paper | Discussion Paper | Discussion Paper | ) were out of the sewage range.Aerobic denitrification could also be the potential process for NO − 2 accumulation in the upstream part of the HH River and the nitrification was potentially inhibited when NH + 4 concentrations were higher than 200 µmol L −1(Magalhães et al., 2005).
Discussion Paper | Discussion Paper | Discussion Paper | and the HH Estuary demonstrated a conservative behavior with respect to NO − 3 .In contrast, a significant source of NO − 3 is present in the CB (36.6 ± 25.2 %) and JY (34.6 ± 35.1 %) rivers compared to the calculated mixing line, explained by external N source addition and in-stream nitrification, respectively.Moreover, the CJ Estuary demonstrated higher NO − 3 accumulation efficiency (82.1 ± 78.8 %) as a result of an external N source input.Great variation percentages were observed between the sampling points from the same river or estuary, possibly resulting from different N dynamics and/or external source input.
, dredging and diking work to deepen the ship channel decreased the sediment area (where denitrification mainly occurred) that is in contact with the overlying water column in the rivers (Dähnke et al., 2008), thus the NO − 3 removal ability was reduced.Third, water residence time is not long enough to remove N loadings in Discussion Paper | Discussion Paper | Discussion Paper | dynamics in the salinized rivers of HH, CB and JY and elucidated mixing patterns of NO − 3 in the corresponding estuarine system.The HH River demonstrated a significant NO − 3 sink appeared in the upstream part of the HH River by aerobic denitrification process.This potentially results from the separation of the floodgate 1 which limited water exchange with downstream water enriched in DO and prolong water residence time in the upstream to remove a significant part of riverine N loadings.The downstream of the HH River showed an extremely weak NO − 3 removal tendency from active NO − 3 turnover processes.In contrast, a significant source of NO − 3 is present in the rivers of CB and JY, linking to external N source addition and in-stream nitrification, respectively.We found that the estuarine mixing behavior is mostly conservative excluding the point source input appearing in the CJ Estuary.Data indicate that the rivers and their corresponding estuaries have lost their natural capacity of NO Discussion Paper | Discussion Paper | Discussion Paper | Dafner, E. V., Mallin, M. A., Souza, J. J., Wells, H. A., and Parsons, D. C.: Nitrogen and phosphorus species in the coastal and shelf waters of Southeastern North Carolina, Mid-Atlantic US coast, Mar.Chem., 103, 289-303, 2007.Dähnke K., Bahlman, E., and Emeis, K.: A nitrate sink in estuaries?An assessment by means of stable nitrate isotopes in the Elbe estuary, Limnol.Oceanogr., 53, 1504-1511, 2008.
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Table 1 .
Physicochemical properties and isotopic composition of NO − 3 for the three investigated rivers and the corresponding estuaries.
a represents river; b represents estuary.Introduction