A reactive nitrogen budget of the Bohai Sea based on an isotope mass balance model

Shichao Tian, Birgit Gaye, Jianhui Tang, Yongming Luo 3, Wenguo Li, Niko Lahajnar, Kirstin Dähnke, Tina Sanders, Tianqi Xiong, Weidong Zhai, Kay-Christian Emeis 5 Institute for Geology, Universität Hamburg, Hamburg, 20146, Germany 5 Yantai Institute of Coastal Zone Research, Chinese Academy of Sciences, Yantai, China Institute of Soil Science, Chinese Academy of Siences, Nanjing, China Institute of Oceanography, Universität Hamburg, Hamburg, 20146, Germany Helmholtz-Zentrum Geesthacht (HZG), Institute for Coastal Research, Geesthacht, 21502, Germany Institute of Marine Science and Technology, Shandong University, Qingdao, China 10

7.4% to total nitrate. An unusually active interior nitrogen cycling contributes 59.1%-71.2% to total nitrate via nitrification.
Nitrogen is mainly trapped in the BHS and mainly removed by sedimentation (96.4%-96.9%) and only very little is exported to the YS (only 1.7%-2.0%). At present denitrification is only active in the sediments and removes 1.4%-1.7% of nitrate from the pool. A further eutrophication of the BHS could, however, induce water column hypoxia and denitrification as already observed -often seasonally off river mouths -in other marginal seas.

Introduction
Reactive or fixed nitrogen (Nr) is an essential nutrient of life on earth, but only few organisms can fix dinitrogen directly from the large atmospheric dinitrogen pool. Since the invention of the Haber-Bosch process the amount of fixed nitrogen (120 Tg N yr -1 ) has constantly grown and since 2010 exceeds the natural terrestrial sources of reactive N of 63Tg N yr -1 (Fowler et 35 al., 2013). Hotspots of agricultural N fertilizer application shifted from the US and western Europe in the 1960s to eastern Asia in the early 21 st century (Lu and Tian, 2017). In China, the Haber-Bosch process produces 37.1 Tg N yr -1 , which is almost 3 times of the biological N fixation of 12.0 Tg N yr -1 . An estimated 32.0 Tg N yr -1 are produced for fertilizer (Gu et al., 2015) and China accounted for 29% of the global ammonium production in 2018 (IFA, 2019). The leakage and volatilization of this man-made reactive nitrogen has strongly impacted limnic and marine ecosystems in China. Riverine reactive nitrogen 40 discharged to the ocean from China was estimated at 5.4 Tg N yr -1 (Gu et al., 2015). The total load of Chinese major estuaries to coastal seas was about 9% of the global river load for DIN and 1.5% of the global phosphate load, respectively (Smith et al., 2003;Liu et al., 2009).
The Bohai Sea (BHS) is a semi-enclosed basin with a surface area of 77×10 3 km 2 and an average depth of 18 m (Chen, 2009;Su, 2001) that is heavily impacted by human activities in one of the most densely populated terrestrial catchments of the 45 world. It exchanges salt water with the Yellow Sea (YS) through Bohai Strait and the Yellow River is a major source of freshwater to BHS (Chen, 2009). During the last fifty years, rising anthropogenic activity in the catchment induced severe environmental changes in the BHS, including increasing salinity, temperature, concentrations of dissolved inorganic nitrogen (DIN) and changes in stoichiometric nutrient ratios (Zhao et al., 2002;Zhang et al., 2004;Wang et al., 2019;Ning et al., 2010). DIN concentrations increased from 0.30μmol L -1 to 3.55μmol L -1 in the time from 1982-2009, while phosphate (from 0.76μmol 50 L -1 to 0.31μmol L -1 ) and silicate (26.6μmol L -1 to 6.60μmol L -1 ) concentrations significantly decreased, so that N/P increased dramatically (Zhang et al., 2004;Liu et al., 2011). Phytoplankton nutrient limitation in the BHS switched from nitrogen to phosphorus in the period of the 1980s to the 1990s and this limitation pattern persists until the present day (Xu et al., 2010;Liu et al., 2009;Wang et al., 2019).
The total annual water discharge of rivers into BHS is about 68.5×10 9 m 3 yr -1 , of which the YR accounts for more than 55 75% (Liu et al., 2011). Water exchange time of the YR estuary is only 0.1-0.2 days (Liu et al., 2009), which implies a fast transfer of nutrients into the open BHS and much of these are trapped in Laizhou Bay ( Fig. 1) (Zhang et al., 2004). The atmospheric deposition of nitrate (3.42×10 9 mol yr -1 ) in BHS was modelled to be less than riverine nitrate (7.25×10 9 mol yr -1 ), while more ammonium was supplied from atmospheric deposition (6.15×10 9 mol yr -1 ) than from riverine input (0.93×10 9 mol yr -1 ) in the 1990s (Zhang et al., 2004). BHS nitrate budgets reported during the last two decades were not completely 60 constrained, because crucial data, such as groundwater discharge or nitrification, were not available (Zhang et al., 2004;Liu et https://doi.org/10.5194/bg-2020-471 Preprint. Discussion started: 21 December 2020 c Author(s) 2020. CC BY 4.0 License. al., 2003;Liu et al., 2009;Liu et al., 2011). There are few published nutrient data from the BHS over the last decade, and the terms in the Nr budget of BHS concerning the quantities of Nr generated or eliminated by biogeochemical cycling within the basin have not been addressed.
The nitrogen budget and in particular the internal sources and sinks of nitrate can be constrained with a mass-based and dual stable-isotope approach based on  15 N and  18 O of nitrate. The combination of data permits tracking of nitrate and quantification of internal cycling of inorganic nitrogen (Sigman et al., 2005;Wankel et al., 2006;Sugimoto et al., 2009;DiFiore et al., 2006;Montoya et al., 2002;Emeis et al., 2010). Stable isotopes of reactive nitrogen have been used to explore nitrogen sources in the eastern Chinese seas (Umezawa et al., 2013;Wang et al., 2016;Liu et al., 2017;Wu et al., 2019;Liu et al., 2020) and the Changjiang Estuary (Yu et al., 2015;Wang et al., 2017;Yang et al., 2018;Chen et al., 2013).

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For this study we analyzed water, suspended matter and sediments in the Bohai Sea sampled during the spring and summer seasons for nutrient concentrations, carbon and nitrogen contents, dual isotopes ( 15 N and  18 O) of nitrate and  15 N of particulate nitrogen. Aim of the study is to characterize and quantify Nr sources and sinks, in particular those from internal cycling processes that have not been included in previous budgets (Zhang et al., 2004;Liu et al., 2003;Liu et al., 2009;Liu et al., 2011), to track the fate of Nr in the present Bohai Sea. The observation data presented here are the basis for a combined 75 mass and isotope balance model, results of which will be a basis for future studies on the rising impact of fast-growing megacities in the Bohai Sea catchment and their possible impact on the adjacent Yellow Sea.

Sample collection
Research cruises were carried out by R/V Dongfanghong 2 in spring and summer 2018 with 24 sampling sites in April 80 and 25 sites in August, respectively (Fig. 1). Water samples were taken from several depths by 12 L Niskin bottles attached to a CTD rosette (911plus, Seabird, USA). The water samples were filtered using nucleopore polycarbonate filters (0.4μm) with plastic Nalgene filtration units. The filtered water was collected in Falcon PE tubes (45mL), frozen immediately (-20℃) and kept frozen until analyses in the home laboratory in Germany. Between 1 to 8 L of water were filtered through pre-weighted GF/F filters (0.7μm, Φ=47mm, Sigma Aldrich) which had been pre-combusted at 450℃ for 4h. The filters were subsequently 85 dried on board under 45℃ for 24 hours. Surface sediments were taken with a box corer and surface samples were transferred into plastic bags with a metal spoon, frozen at -20℃ and were kept frozen until later analysis in the home lab.
Yellow River water samples were taken from the Kaiyuan floating bridge in Lijin, located 44 km upstream of the river mouth. Samples were taken in the middle of the river course with a plastic reversing water sampler at 1 meter under the surface.
The water samples were filtered immediately for nutrient analysis and collection of suspended particles, and subsequently were stored frozen until delivered to the home laboratory. Samples were taken monthly in May, July to November from Yellow River, and in November from Daliao River, Hai River, Luan River and Xiaoqing River (Fig. 1). Fig. 1 Sampling sites in Bohai Sea and rivers (Xiaoqing River not shown). Open red circles stand for sampling site in spring, open blue triangles stand for sampling sites in summer, names of the sites are marked nearby. Black arrows stand 95 for the most significant currents flows in and out of the Bohai Sea. Blue dashed lines strand for two main sections, black dashed line stand for the boundary of our study area.

Measurements of nutrients and nitrate isotopes
Nutrient concentrations were measured with an AutoAnalyzer 3 system (Seal Analytics) using standard colorimetric 100 methods (Grasshoff et al., 2009). The relative error of duplicate sample measurements was below 1.5% for NOx and phosphate concentrations, below 0.3% for ammonium. The detection limit was < 0.5 µmol kg −1 for NOx, > 0. injected into a suspension of the denitrifier Pseudomonas aureofaciens with injection volumes adjusted to yield 10 nmol N2O.

Measurements of suspended matters and sediments
The tared GF/F filters were weighed to calculate the amount of suspended particulate matter (SPM) per liter of water.
Total carbon and nitrogen concentrations in SPM and sediment samples were measured by a Euro EA 3000 (Euro Vector SPA) Elemental Analyzer, and SPM samples with high carbon and nitrogen contents and sediments were acidified to measure organic carbon content. The precision of total and organic carbon determination is 0.05%, that of nitrogen is 0.005%, and the standard 120 deviations are less than 0.08 for total and organic carbon and 0.02 for nitrogen. Nitrogen isotope ratios were determined with a FlashEA 1112 coupled to a MAT 252 (Thermo Fisher Scientific) isotope ratio mass spectrometer. The precision of nitrogen isotope analyses is better than 0.2‰, and the standard deviation less than 0.03.

Measurements of dissolved oxygen
The dissolved oxygen (DO) samples were collected, fixed, and titrated on board following the Winkler procedure at an 125 uncertainty level of <0.5%. A small quantity of NaN3 was added during subsample fixation to remove possible interferences from nitrite (Wong, 2012). The DO saturation (DO%) was calculated from field-measured DO concentration divided by the DO concentration at equilibrium with the atmosphere which was calculated from temperature, salinity and local air pressure, as per the Benson and Krause Jr (1984) equation.

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The regional three-dimensional hydrodynamic Hamburg Shelf Ocean Model, HAMSOM (Backhaus, 1985), was applied in the East China Seas (23°-45°N, 117°-131°E) to calculate the water and nutrient transport through the Bohai Strait for the year 2018. The spatial resolution of the model is 2' (approx. 3.7 km) with 20 layers in vertical direction, while the calculation time step is 3 minutes. The topography data (resolution of 2') were obtained from marine navigation charts. The meteorological forcing was derived from an hourly ERA5 dataset with a spatial resolution of 0.25° (CCCS, 2017). The open boundary SSH and the boundary T and S data and for the initial T and S fields were extracted from the daily Mercator-Ocean dataset (1/12

Hydrological properties
Averages of salinity and temperature in spring were 32.3±0.5 (n=72) and 4.7±0.8°C (n=72), respectively, and the water column was vertically mixed ( Fig. 2 and Fig. 3). The YR discharged relatively warm water and the lowest salinity was observed 150 in the southeast of YR estuary (site B68, T>6℃, S<31). Thus, the Yellow River Diluted Water (YRDW) is here defined as the water off the YR estuary with salinities lower than 31. In summer, averages of salinity and temperature were 31.6±0.8 (n=88) and 22.4±4.2°C (n=88), respectively, and the surface layer was stratified. The YRDW extended to an even larger area than in spring caused by high river discharge. The YRDW turned northeast towards LiaoDong Bay into the central BHS in the surface layer (T>27℃, S<31).

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The water column oxygen concentrations (see Supplement 1) in the study area in spring and summer were 10.27-11.47 mg L and 3.84-8.86 mg L -1 , respectively, and thus much higher than the threshold for water column denitrification (0.15 mg/L).
The detailed results of DO and other parameters are shown in Supplement 2.

Suspended particulate matter
In spring suspended particulate matter (SPM) concentrations were mostly vertically homogenous along both transects with

The discharge of the Yellow River
The water discharge of the year 2018 determined at the Lijin hydrography station was 333.8×10 9 m 3 which was by 14% higher than the multi-year average of 292.8×10 9 m 3 (1952-2015) (MWR, 2019). The monthly mean discharge was 27.80±20.21×10 9 m 3 month -1 (n=12), which was higher than the multi-year average value by 14%-51%, indicating that in YR basin 2018 was a flood year. The water discharge maximum was from July to October (Fig. 8). During May to November in

Nitrate exchange with the Yellow Sea
Based on current velocities and nutrient concentrations along the section crossing the BH Strait, the annual water and nutrient export from BHS to the Yellow Sea in the year 2018 was calculated to 1.26×10 -3 Sv (1 Sv=10 6 m 3 /s) and 0.9×10 9 mol 230 yr -1 , respectively. In this study, the exported nitrate are assumed with the average isotopes values of the BHS (δ 15 N=8.9‰ and δ 18 O=10.4‰).
Making use of the three-dimensional model (HAMSOM) results, it is also possible to determine a spatial distribution of the annual nutrient flux through the Bohai Strait section (Fig. 9). Positive values represent a nutrient flux out of the Bohai Sea, while negative ones indicate a flux into the Bohai Sea. The strongest nutrient export occurs at the southern part of the Bohai 235 Strait, while the major import takes place in the upper 15 m in the northern Bohai Strait (Fig. 9).

The hydrographic and nutrients characteristics in spring and summer
The sampling in early spring occurred during a season of low biological activity so that nutrients behaved almost conservatively. As is indicated by the negative correlations of

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N2 fixation is excluded as a significant input of Nr because of high N:P ratios.

The main sources and sinks of nitrate in the BHS
Most of the external and internal sources of nitrate to the BHS are characterized by distinct dual isotope values. These fingerprints combined with mass flux estimates are in the following used to constrain the mass and isotope budget of nitrate in the BHS. Specifically, the role of internal cycling processes can thus be quantified, which were lacking in previous budgets.

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In the following, each of the sources and sinks is described, along with isotope composition or isotope fractionation associated with cycling processes.

Riverine inputs
The main input from this source of BHS Nr is from the Yellow River and we calculated a nitrate input of YR of 7. based on water discharge and our nitrate measurements in YR, Hai River, Daliao River, Luan River and Xiaoqing River. Owing to a lack of data on nitrate concentrations of Shuangtaizi River, Daling River and Xiaoling River and considering that these 270 rivers drain basins adjacent to Daliao River, we assume that nitrate concentration of these rivers were same as those of the Daliao River. The total riverine input of nitrate summed up to 9.49×10 9 mol.
The mass-weighted average annual values for δ 15 N and δ 18 O of nitrate in these rivers were 10.0‰ and 1.3‰, respectively, taken here to represent the river nitrate isotopic composition discharged into BHS.

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The DIN supplied to BHS by submarine groundwater discharge (SGD) flux has been estimated to be 2-10 times the YR discharge (Luo and Jiao, 2016;Peterson et al., 2008;Wang et al., 2015). These fluxes of SGD are a mixture of submarine fresh groundwater discharge (SFGD) and recirculated saline groundwater discharge (RSGD) (Liu et al., 2011;Peterson et al., 2008), but only the freshwater component is relevant as a source for the budget.
The latest estimate of DIN flux for SFGD in Laizhou Bay amounts to 0.57-0.88 of YR input (Wang et al., 2015). We took 280 the average of the SFGD flux (Wang et al., 2015) and the average nitrate concentration of ground water around Laizhou Bay https://doi.org/10.5194/bg-2020-471 Preprint. Discussion started: 21 December 2020 c Author(s) 2020. CC BY 4.0 License.
(there is no data on nutrient concentrations of SFGD available so far) (Zhang et al., 2016) to calculate the SFGD inorganic nitrogen flux, which resulted in 3.62×10 9 mol year -1 . The ratio of NO3 -/( NO3 -+NH4 + ) is 0.75 in total SGD (Liu et al., 2011;Chen et al., 2007), so that the nitrate flux of SFGD is estimated at 2.73×10 9 mol. Due to lack of data on SFGD from the entire BHS, this number was used to represent the input from SFGD to the BHS, which may underestimate the factual value and is only approximately 10% of previous estimates of the input nitrate for SGD into BHS (Liu et al., 2011) due to the exclusion of RSGD.
Because of pollution and denitrification processes in soils, aquifers and groundwater Chen et al., 2007;Soares, 2000), the value of δ 15 N and δ 18 O of nitrate in SFGD is more enriched than those of river runoff and this is illustrated by the observed δ 15 N value of 20.2±9.0‰ (n=19) of on-land groundwater near the YR delta (Chen et al., 2007). As

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there are as yet no reported δ 15 N values of SFGD and RSGD inputs, we decided to take this value as the signal of nitrate δ 15 N imported by SFGD into the BHS. There are no data available for δ 18 O of nitrate of SFGD, and we will discuss possible constraints in the box model discussion (Sect. 4.3.2).

Atmospheric deposition
Combined atmospheric input by wet and dry deposition ranged from 3.14 ×10 9 mol yr -1 to 3.42±2.29 ×10 9 mol year -1 295 (Zhang et al., 2004;Liu et al., 2003) . We adopted the annual mass of NOx deposition for China of 6.2 Tg yr -1 (Zhao et al., 2017) and related this value to the area of the BHS, which results in an annual deposition of 3.6 ×10 9 mol yr -1 . Owing to a lack of directly measured data for atmospheric NOx the BHS, we adopt 3.42×10 9 mol yr -1 (Zhang et al., 2004) as the atmospheric nitrate flux.
The nitrate δ 18 O of PM2.5 in BHS ranged from 65.0‰ to 88.1‰ (seasonally) (Zong et al., 2017), the value in Beijing is 88.3±6.9‰ (Song et al., 2020)  The ammonium deposited from the atmosphere is assimilated by phytoplankton and is subsequently entrained into the N cycle via remineralization and nitrification or is nitrified directly in the water. Thus, the nitrified atmospheric ammonium is included here as a source bearing on  15 N of nitrate in the sea water. The ammonium deposition in BHS was 6.15×10 9 mol yr -1 , which is more than the nitrate deposition of 3.42×10 9 mol yr -1 (Zhang et al., 2004).

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The atmospheric ammonium has low δ 15 N values of -6.53 to -1.2‰ (Zhang et al., 2008;Chang et al., 2019). Given that the δ 15 N value of ammonium of the north China Plain is -1.2‰ (Zhang et al., 2007), and that there is no obvious accumulation of ammonium in the surface layer in the observations, we assume that this isotope value is identical to the δ 15 N value of nitrified atmospheric ammonium.

Nitrate diffusing from water to sediment
Benthic fluxes of nutrients have been investigated through incubation experiments and diffusion models (Zhang et al., 325 2004;Liu et al., 2011) and range from 63.3±296×10 6 mol/month nitrate diffusing from bottom water into the sediments (Liu et al., 2011) to sediment-water effluxes of 4.28×10 9 mol year -1 in the box model of Zhang et al. (2004). The difference of the estimates is probably due to different methods of calculation. For this study we adopted the annual flux from water to sediment as 0.76×10 9 mol year -1 (Liu et al., 2011). We assume that diffusion is not accompanied by isotope fractionation, so δ 15 N and δ 18 O of nitrate diffusing into the sediment was assumed to be the same as the nitrate pool in BHS (8.9‰ and 10.4‰, 330 respectively).

Ammonium diffusing from sediment to water
The processes of nitrogen cycling in sediments are complex and variable (Lehmann et al., 2004). The degradation of organic matter, nitrification and assimilation are acting under aerobic conditions, whereas denitrification, anammox and dissimilatory nitrate reduction to ammonium (DNRA) are observed under anaerobic conditions. When organic matter is 335 degraded in the surface sediments, part of the produced ammonium diffuses into the overlying bottom water and subsequently is nitrified to nitrite and nitrate under aerobic condition. For our purpose only the ammonium nitrified bears on the seawater nitrate pool. The mean δ 15 N value of sediment in BHS was 5.4‰ (n=20), and according to the fractionation factor during organic matter remineralization of 2‰ (Möbius, 2013) and subsequent nitrification (see above), the  15 N and δ 18 O of nitrate efflux from sediment are assumed to be 3.4‰ and 0.3‰, respectively. 340 https://doi.org/10.5194/bg-2020-471 Preprint. Discussion started: 21 December 2020 c Author(s) 2020. CC BY 4.0 License.

Sedimentation
The mass flux of Nr sedimentation is unknown. In terms of the effects of Nr sedimentation on nitrate dual isotopes, phytoplankton organisms that assimilate nitrate from the dissolved phase are the main source of sinking particles, so that the N and O will be removed from the nitrate pool following the assimilation fractionation factor. Sinking particles in the BHS have a δ 15 N of 5.2‰ (δ 15 Nsink), which integrates multiple processes such as photosynthesis of phytoplankton, heterotrophic synthesis of bacteria, and heterotrophic degradation (remineralization).
There is no observed data of δ 18 O of nitrate removed from the pool during assimilation (δ 18 Osink), but this value can be estimate by the assimilation fractionation factor ( 18 ε). The per mil fractionation factors  of N ( 15 ε) and O ( 18 ε) in nitrate during assimilation are generally assumed to be around 5‰, so that 15 ε: 18 ε=1:1. Here we adopt the average of 15 ε and 18 ε as 5‰ (Granger et al., 2010;DiFiore et al., 2009;Liu et al., 2017;Wu et al., 2019;Umezawa et al., 2013;Wang et al., 2016), so that the δ 18 O of nitrate removed from the pool during assimilation (δ 18 Osink) should be 5.0‰ according to the δ 18 O (10.0‰) of the dissolved nitrate pool.

The nitrate budget in the BHS
A box model of the nitrate budgets for the Bohai following the LOICZ approach (Zhang et al., 2004) balanced sources and sinks of nitrate in BHS and was updated by several other nitrate budgets for the BHS during last two decades (Zhang et 355 al., 2004;Liu et al., 2003;Liu et al., 2011;Liu et al., 2009). All were, in general, not completely constrained because of a lack of data on some important source or loss terms. We here associate the nitrate isotope compositions of pools, sources, and sinks of nitrogen with a box model of the BHS nitrate in order to improve the understanding of nitrate cycling in the BHS. Finally, based on the combined mass and isotope box model informed by new data on the isotopic composition of nitrate, surface sediment, and suspended particulate nitrogen in the water column discussed above, we propose an updated N-budget that is 360 internally consistent.

The nitrate budget based on mass fluxes and corresponding δ 15 N values
In our hypothesis, the sources of nitrate for BHS are river inputs, submarine fresh ground water input, atmospheric deposition, and remineralization. Most important sinks are net export to the YS, sediment denitrification and particulate matter sedimentation. Assuming the mass and N isotope of nitrate in the BHS are in steady state, the sources and sinks of nitrate 365 follow the Eq. (1) and Eq. (2): where the terms m with different subscripts refer to the corresponding nitrogen mass fluxes, refers to atmospherically 370 deposited nitrate,, refers to river nitrate, refers to nitrified ammonium deposited from the atmosphere, refers to nitrate in submarine fresh groundwater discharge, refers to nitrification in the water column. In terms of sinks, refers to the mass fluxes associated with net export of nitrate from BHS to the YS, refers to nitrate sedimenting from seawater as particulate N, and refers to denitrification in the sediment. The unit of the mass fluxes is 10 9 mol.
The "δ 15 N" refers to the δ 15 N value of the N mass flux which with the same subscripts. As mentioned previously, the mass

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indicating that the ammonium from PN mineralization is most likely completely converted to nitrate, so that there is no fractionation effect for this step. Thus, the δ 15 N value of newly nitrified nitrate from complete nitrification of ammonium generated by PN mineralization is 2.2‰. In the case of incomplete nitrification, especially under the thermocline in summer, the newly nitrified nitrate has a δ 15 N of 0.2‰, given a net fractionation factor of nitrification (ammonium to nitrate) of 2‰ (Sigman and Fripiat, 2019). In our model below, the assimilation of ammonium originating from SPM remineralization was 390 not included, as its proportion is unknown in the BHS. The simplified model may thus underestimate the input of 15 N-depleted nitrogen into the nitrate pool.
Ammonium diffusing out of the sediment will either be mixed into the euphotic layer and subsequently assimilated by the phytoplankton, or nitrified in the water column. The accumulating of ammonium beneath the thermocline was a significant process in summer, as shown by the high nitrite and ammonium concentrations beneath the thermocline. The ratio of these two 395 branching processes is not known in the BHS. If all of the ammonium from sediment is nitrified, the produced nitrate will have a δ 15 N of 3.4‰ (see above). If the ammonium is only partially nitrified (especially in summer beneath the thermocline), the produced nitrate will have a δ 15 N of 1.4‰ at a fractionation factor of nitrification of 2‰ (Sigman and Fripiat, 2019 As the only constraint, δ 18 OSFGD is expected to be higher than the value of nitrate imported from the rivers (δ 18 Or=1.3‰) due to the fractionation associated with denitrification in the anaerobic aquifers (see 4.2.2). The results can be summed up in three different cases: (1)When setting the value of δ 15 Nntr to 3.4‰, we obtain estimates for that range from 0.00 to 1.31×10 9 mol year -1 , for in the range of 32.57 to 38.69×10 9 mol year -1 and in the range of 47.87 to 52.68×10 9 mol year -1 . The corresponding values of δ 18 OSFGD range from 1.3‰ to 16.3‰, and the upper range of δ 18 OSFGD yields =0.0 due to the assumption that any mass flux must be equal or greater than 0.
(2) When we choose a δ 15 Nntr value of 3.2‰ and 3.0‰, respectively, to explore effects of the methodological error of δ 15 N 420 for our isotope method (0.2‰, see Sect. 2.2), again under the premise that the mass fluxes are positive numbers, results in , and estimates in a narrower range than when δ 15 Nntr is 3.4‰; these results are not shown.
Thus, reasonable solutions only are reached when δ 15 Nntr is between 3.4‰ and 3.0‰. The results of the budget are shown in Fig. 10 and Table 1, respectively.  The mass flux of nitrate ( ) originating from nitrification of atmospheric ammonium ranges from 0.00 to 3.18×10 9 mol year -1 and accounts for up to 51.7% of the total ammonium deposition (6.15×10 9 mol year -1 ; Zhang et al. (2004). This in turn implies that most of the atmospherically deposited ammonium is directly assimilated rather than nitrified to nitrate. This agrees with phytoplankton preference to assimilate ammonium rather than nitrate (Glibert et al., 2016). The bulk of internal sources 435 of nitrate originates from nitrification in the water column (from water column ammonification and ammonium diffusing from sediment). This single source ( ) accounts for 59.1%-71.2% of the total sources of nitrate in BHS and appears to be much more important than in other coastal environments. For example, between 15%-27% of productivity was supported by nitrified ammonium in the seawater in Monterey Bay (Wankel et al., 2007). Likewise, nitrification supplied 34% of the surface nitrate in the eastern Hainan Island, which like Monterey Bay is also an upwelling area (Chen et al., 2020). This indicates that nitrate 440 regeneration by nitrification may play a more important role in shallow and land-input dominated marginal seas than in upwelling dominated marine settings.

Assessment of model uncertainties
The

Biogeochemical implications of the box model
In other coastal eutrophic regions, such as the North Sea, a high δ 15 N of river nitrate is reflected in a halo of high δ 15 N in surface sediments in offshore areas . In the Bohai Sea, such an isotopic halo of river-borne eutrophication is not observed despite similar water exchange rates of 1-2 years Serna et al., 2010) and similarly isotopically 455 enriched river inputs. We speculate that the lack of a fingerprint of river nitrate in the d 15 N of sediments of BHS may be masked by active nitrification and atmospheric deposition that rapidly eradicate and homogenise spatial gradients.
Despite the uncertainties that are related to the box model approach, combining mass and isotope budgets of nitrate sources and sinks is clearly superior to solely nitrate mass balance considerations, especially when it comes to segregating the anthropogenic nitrate and the recycled nitrate inputs. It is of note that the Bohai Sea does not appear to pass on significant 460 https://doi.org/10.5194/bg-2020-471 Preprint. Discussion started: 21 December 2020 c Author(s) 2020. CC BY 4.0 License.
amounts of nitrate to the adjacent northern Yellow Sea, so that the effects of excessive loading of this shallow mixing zone between land and ocean with anthropogenic nitrogen are yet mitigated by internal cycling processes.

Conclusions
Rivers contributed 17.5%-20.6% and the combined terrestrial runoff (including submarine discharge of nitrate with fresh ground water) account for 22.6%-26.5% of the total Nr input to the BHS. Atmospheric input contributes 6.3%-7.4% of nitrate 465 to the BHS. Nitrification contributes 59.1%-71.2% of the total nitrate, indicating an unusually active interior nitrogen cycling of in the BHS. Nitrate was mainly trapped in the BHS and only very little was exported to the YS (only 1.7%-2.0%).
Furthermore, nitrate was rather assimilated than exported to the YS along the main transport pathway Lubei Coastal Current, effectively retaining Nr in BHS. Sedimentation trapped 96.4%-96.9% of nitrate inputs, whereas denitrification was only active in the sediments that removed 1.4%-1.7% of nitrate from the pool. Seasonal biogeochemical variations were observed in the 470 BHS in that dissolved inorganic nitrogen increased during summer under the thermocline, implying significant biological regeneration. If the interior cycling increases, for instance fueled by increased terrestrial and atmospheric Nr inputs, respiration coupled to organic matter and N recycling will increase and water-column hypoxia could consequently spread in the future and compromise ecosystems in the BHS. Whether this will invigorate water-column denitrification to balance the additional inputs is an open question, as is the capacity of BHS as a nitrate buffer between the growing source of Nr on land and the open 475 ocean.

Methods Appendix:
Nitrate was reduced to nitrite with a copperized cadmium column first. The nitrite ions reacted with sulfanilamide and N-1-naphthylethylendediamine (NEDD) to form red azo dye, and then measured at 520-560nm. Phosphate determination followed the method of Murphy and Riley (Murphy and Riley, 1962). Under acid conditions a phosphomolybdic complex was 480 formed of ortho-phosphate, antimony and molybdate ions (Wurl, 2009). Followed by the reduction of ascorbic acid, the blue colour complex was measured at 880 nm. The sample with ammonium is reacted with o-phthalaldehyde (OPA) at 75°C in the presence of borate buffer and sodium sulfite to form a fluorescent species proportional to the ammonia concentration. The fluorescence is measured at 460 nm following excitation at 370 nm (Kérouel and Aminot, 1997). Silicate is reacted with ammonium molybdate to silicomolybdate, and reduced in acidic solution to molybdenum blue by ascorbic acid (Grasshoff et 485 al., 2009).