Nitrate Regeneration and Loss in the Central Yellow Sea Bottom Water Revealed by Nitrogen Isotopes

The Yellow Sea (YS) is an epicontinental sea framed by the densely populated mainland of China and the Korean peninsula. Human activities over the last decades resulted in heavily increasing discharge of reactive nitrogen into the YS, which created numerous ecological problems. To elucidate the role of central YS in the cycling of reactive nitrogen, specifically the Yellow Sea Cold Water Mass (YSCWM), we determined nutrient concentrations, dual stable isotopes of nitrate (δ15N-NO3- and δ18O-NO3-), and stable isotopes of particulate and sedimentary nitrogen in spring and summer, i.e., in biologically inactive and active periods. The nitrate concentration in spring was higher than that in summer in the northern part of the YSCWM, Nitrate increased in the southern part accompanied by a decrease in δ15N-NO3- and δ18O-NO3-, which are indicative of nitrification that was a significant source of recycled nitrate in the south part of YSCWM. To quantify this regenerated nitrate, we use a mixing model with end members of preformed nitrate in spring and regenerated nitrate in summer, both with their distinct dual isotope values. The results suggest that only 35% nitrate was a residual of nitrate preformed in spring and 65% in summer in the southern branch of YSCWM was regenerated. The northern part of YSCWM has low concentrations of dissolved inorganic nitrogen, mainly because of denitrification in sediments. In contrast, the southern pool of YSCWM is a growing reservoir of regenerated terrestrial reactive nitrogen, the addition of which compensates the removal by co-occurring sediment denitrification. In consequence, the southern branch of YSCWM is facing a higher ecological risk than the northern branch, when excess reactive nitrogen discharge from Changjiang River continues at present levels or even increases.


INTRODUCTION
The small area of the coastal seas compared to the vast open ocean hosts around 20% of all marine primary production (Jahnke, 2010) and plays a key role in processing nutrients and contaminants that enter the ocean via continental runoff and rivers (Levin et al., 2015). River input and atmospheric deposition are the largest contributors and supply more than half of the reactive nitrogen (N r ) to the coastal sea areas (Voss et al., 2013). Anthropogenic activities have doubled or tripled N r supply from natural processes at a global scale (Galloway et al., 2013), so that human activities on land are more and more impacting ecosystems in marginal seas (Levin et al., 2015). An example are the seas bordering mainland China. The contribution of Chinese major estuaries to global coastal seas has been estimated at 5.4 Tg N yr −1 (Gu et al., 2015), which is about 10% of the global river input for dissolved inorganic nitrogen (DIN) (Smith et al., 2003;Liu S. et al., 2009). The increased input of anthropogenic N r in the last decade resulted in a shift from N-deficiency to P-deficiency in the coastal area of Yellow Sea (YS) and East China Sea (ECS) Moon et al., 2021).
The YS with an area of about 3.8 × 10 5 km 2 and an average depth of 44 m is a semi-closed marginal sea surrounded by mainland China and the Korean peninsula. It connects to the Bohai Sea in the northwest by the narrow Bohai Strait and with the ECS in the south. Changjiang River (Yangtze River), the largest river in China, discharges on average 9.25 × 10 11 m 3 of water per year into the ECS, of which about 20% is advected into the YS (Fan and Song, 2014;Liu et al., 2020). Changjiang Diluted Water (CDW) is a main nutrient source of YS in summertime, while Yellow Sea Warm Current water (YSWC), the sub-branch of the Kuroshio Current, in wintertime is another major source .
The concentrations of nitrate and DIN in the south YS have been continuously increasing from the 1980s Wei et al., 2015;Wang et al., 2020b). The most rapid increase of NO 3 − and DIN occurred after the mid-1990s  and the level of the annual NO 3 − concentration in 2012 was over 5 times that of 1984 . A large upward spike in the N/P ratio was apparent in 2008, and the dissolved oxygen (DO) concentration decreased sharply in the same year . The increase of N input has been held responsible for increasing incidences of harmful algae blooms and macroalgal blooms in recent years (Li et al., 2017b;Wang et al., 2018;Xiao et al., 2019).
The Yellow Sea Cold Water Mass (YSCWM) fills the central YS trough and forms due to the unique basin topography and the impact of both the seasonal evolution of the thermocline and the circulation system in the YS (Zhang S. et al., 2008). YSCWM forms by subsidence of winter cold water (Lee and Beardsley, 1999;Zhang S. et al., 2008), and water of YSCWM could not be distinguished from other ambient waters based on the oxygen isotope of seawater (Kang et al., 1994). It is isolated from the direct influence of major river inputs so that the internal remineralized nutrients would play important roles instead (Chen et al., 2012;Sun et al., 2013;Li et al., 2017a). A strong linkage of hypoxia and acidification with organic matter mineralization and stratification was observed in the YSCWM . This water mass is a significant pool of nutrients, supplying them to the south YS by vertical diffusion/upwelling processes (Su et al., 2013;Wei et al., 2016;Wang et al., 2020c). Loss of nitrate was reported in the YSCWM (Liu S. et al., 2003;Duan et al., 2016) which was due to benthic processes rather than to water column denitrification (Zhang et al., 2018).
However, the portions of regenerated nitrate in the YSCWM have not been quantified, and the dynamics of internal nitrogen cycling are not clear. The internal cycling generally includes the assimilation and remineralization (including ammonification and nitrification) of N r (Sigman and Boyle, 2000;Brandes and Devol, 2002). In the euphotic layer, DIN is assimilated by phytoplankton, and the organic detritus is decomposed below the euphotic layer. During decomposition, ammonium is first released and subsequently transformed to nitrite and nitrate via nitrification. These biogeochemical processes can be traced by stable isotopes due to their unique fractionation effect (Altabet, 2006;Casciotti, 2016;Liu S. M. et al., 2017;Wang et al., 2017;Sigman and Fripiat, 2019;Wu et al., 2019). In our study, we measured the nutrients, dual stable isotopes of nitrate, and stable N-isotope composition of particulate and sedimentary nitrogen in two seasons in the YS. Results of the isotopic investigation quantify the shares of preformed and newly regenerated nitrate in the YSCWM. Seasonal comparison of DIN quantity and isotopic character in the northern and southern YSCWM furthermore give insights into how the regional and global nutrient sources affect the YSCWM in the central YS trough.

Sample Collection
Research cruises were carried out by R/V Dongfanghong 2 in spring and summer 2018 with 42 sampling sites in April and 45 sites in August, respectively (Figure 1). Water samples were taken from several depths by 12 L Niskin bottles attached to a CTD rosette (911plus, Seabird, United States). The water samples for nutrient and isotope analysis were filtered using nucleopore polycarbonate filters (0.4 µm) with plastic Nalgene filtration units. The filtered water was collected in Falcon PE tubes (45 mL), frozen immediately (−20 • C) and kept frozen until analyses in the home laboratory. For suspended particulate matter (SPM) and organic particle analysis, between 0.5 and 8 L of water were filtered through preweighed GF/F filters (0.7 µm, = 47 mm, Sigma-Aldrich) which had been pre-combusted at 450 • C for 4 h. The filters were subsequently dried on board at 45 • C for 24 h. Surface sediments were taken with a box corer and surface samples FIGURE 1 | Sampling stations of spring and summer cruises are marked by red circles and black triangles, respectively. Note that most of the stations were sampled on both expeditions. The southern and northeastern boundary of the Yellow Sea is marked by the black dashed line. Yellow Sea Warm Current (YSWC) and Changjiang Diluted Water (CDW) are important external waters in spring and summer, respectively. These currents are drawn according to their general positions and our data of 2018. Sampling stations clustering in the north Cold Water mass (nCW) and south Cold Water (sCW) stations are marked by blue dashed lines, nCW and sCW are water masses defined in this study, they both are part of the Yellow Sea Cold Water Mass (YSCWM). (base map sourced by Ocean Data View, Schlitzer, Reiner, Ocean Data View, https://odv.awi. de, 2021).
were frozen at −20 • C and were kept frozen until analysis in the home lab.

Measurements of Nutrients and Nitrate Isotopes
Nutrient concentrations were measured with an AutoAnalyzer 3 system (Seal Analytics) using standard colorimetric methods (Grasshoff et al., 2009). The relative error of duplicate sample measurements was below 1.5% for NO x and phosphate concentrations, below 0.3% for ammonium. The detection limit was <0.05 µmol L −1 for NO x , >0.01 µmol L −1 for PO 3− 4 , and >0.013 µmol L −1 for ammonium. The pooled standard deviation is 0.004 µmol L −1 for NO x , 0.006 µmol L −1 for PO 3− 4 , and 0.01 µmol L −1 for ammonium.  (Sigman et al., 2001;Casciotti et al., 2002). Only the samples with nitrate concentrations >1.7 µmol L −1 were analyzed and δ 15 N and δ 18 O were analyzed in one sample run. Water samples were injected into a suspension of the denitrifier Pseudomonas aureofaciens with injection volumes adjusted to yield 10 nmol N 2 O. The N 2 O gas was purged by helium into a GasBench 2 (Thermo Finnigan) for purification. Afterward the N 2 O gas was analyzed by a Delta V Advantage and a Delta V Plus mass spectrometer. Samples were measured in duplicate and the two international standards IAEA-N3 (δ 15 N-NO 3 − = +4.7 , δ 18 O-NO 3 − = +25.6 ) and USGS-34 (δ 15 N-NO 3 − = −1.8 , δ 18 O-NO 3 − = −27.9 ) and an internal potassium nitrate standard were measured in each batch. The data were corrected by applying a bracketing correction  and the standard deviations of the international and in-house standards was found to be ≤0.2 for δ 15 N and ≤0.5 for δ 18 O. The standard deviations of duplicate samples were in the same range. Nitrite affects the results and was removed following the protocol of Granger and Sigman (2009)

Measurements of Suspended Matters and Sediments
Loaded filters were weighed to calculate the amount of 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. Organic carbon was measured after acidifying samples for three times. The precision of total and organic carbon determination is 0.05%, that of nitrogen is 0.005%, and the standard 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 uncertainty level of <0.5% (Xiong et al., 2020). A small quantity of NaN 3 was added during subsample fixation to remove possible interferences from nitrite (Wong, 2012). The DO saturation 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 (1984) (Anderson, 1995) is used to estimate the regenerated nutrients (Rafter et al., 2013;Wu et al., 2019). The regenerated nitrate ([NO − 3 ] r ) can be calculated following Eq. 1 and Eq. 2, Apparent Oxygen Utilization (AOU) and  in these equations with subscript sp and sm stand for data of spring and summer, respectively. The seasonal variation of nitrate ( NO − 3 ) is calculated by Eq. 3.

Hydrological Conditions
In spring the water column in the YS was vertically well mixed with salinity and temperature both decreasing northwards. The Yellow Sea Warm Current (YSWM) entered from the outer shelf northwestward into the central YS trough, transporting relatively warm and saline water (T > 7.0 • C, S > 32.5) and its northernmost tip reached 37 • N (station B08) (Figures 1, 2A-D). The coastal water in the south YS (sYS) was characterized by relatively high temperatures and low salinities (T > 8.0 • C, S < 32.5). In the north YS (nYS), temperatures decreased to around 4.0 to 6.0 • C and salinities were in the range of 32.2 to 32.5. Sea water in the YS can thus be roughly considered as a mixture of nutrient depleted nYS water with nutrient abundant YSWC and sYS coastal water (Figure 3). In summer, the water column was stratified. The Yellow Sea Cold Water Mass (YSCWM; T < 10.0 • C) was found beneath the thermocline in the deep trough of the central YS ( Figure 4B). The YSCWM can be divided into the northern Cold Water (nCW, 32.0 < S < 32.5) and southern Cold Water (sCW, 32.5 < S < 33.0) (Figure 3). While the nCW is mostly derived from the spring nYS water, the sCW inherited the features of the YSWC water (Supplementary Figure 1). The upper layer of the YSCWM had relatively low salinities (31.0 < S < 32.0) and high temperatures (23.0 • C < T < 30.0 • C). Changjiang Diluted Water (CDW) in the southwest YS is characterized by low salinities and high temperatures (S < 30.0, T > 25.0) and appeared in the north and northeast of the Changjiang estuary.

Nitrate and Phosphate Concentrations
The maxima of nitrate and phosphate in bottom waters in spring match the tongue of YSWC (Figures 2D,F,H). Nitrate and phosphate were enriched ([NO − 3 ] > 6 µmol L −1 , [PO 3 − 4 ] > 0.4 µmol L −1 ) along the south most transect at 32 • N. sYS coastal water found further north than 34 • N near Jiangsu coast was relatively nutrient depleted ([NO − 3 ] < 4 µmol L −1 , [PO 3 − 4 ] < 0.1 µmol L −1 ), probably due to a lack of external replenishment of nutrients in spring. In nYS, nitrate and phosphate concentrations were high in the central area (stations B14 and B23) whereas they were depleted in the northern part (stations B16, B18, and B19) (Figures 2E-H).
In summer, nutrients were depleted of the upper layer of the YS, except for the CDW and in the coastal areas of Jiangsu province. CDW had nitrate concentrations > 14 µmol L −1 at phosphate concentrations of only around 0.2 µmol L −1 in the surface (stations H34 and H35), but nitrate decreased to 12 µmol L −1 and phosphate increased to 0.4 µmol L −1 in the bottom water. Nitrate and phosphate were enriched in the sCW ([NO 3

Seasonal Variation of Dissolved Inorganic Nitrogen in the Central Yellow Sea Bottom Waters
We divide the YSCWM into nCW and sCW as described in Section "Hydrological Conditions" for a seasonal comparison of nutrient dynamics. Although YSCWM was not formed in spring, temperature and salinity was almost constant in the central YS basin during these two seasons (Supplementary Figure 1). This is in line with the origin of YSCWM that forms by subsidence of winter cold water (Lee and Beardsley, 1999;Zhang S. et al., 2008), and makes the following seasonal comparison feasible. The spring samples were grouped in the same way as summer samples, although the YSCWM had not formed yet in spring. Nitrate concentration of sCW increased from spring to summer, whereas nitrate of nCW decreased ( Figure 5). Considering that the hydrological properties of nCW and sCW are quite similar, these differing seasonal alterations of nitrate (or DIN) concentrations are remarkable and suggest different modes of operation of these two basins with respect to reactive nitrogen recharge. Ammonium concentration of sCW were relatively low in both seasons ( Figure 5) and accounted for 6 and 4% of DIN in spring and summer, respectively. In the nCW, on the other hand, ammonium accounted for 15-57% of DIN in spring and summer, respectively. Nitrite concentrations were low with average concentrations <0.30 µmol L −1 accounting for less than 6% of DIN ( Figure 5 and Table 1) in both seasons. Phosphate average concentrations were almost uniform in the bottom waters in both seasons except slight increases in the sCW ( Table 1). The phosphate concentration of sCW was significantly higher than that of the nCW (p < 0.05, ANOVA). The average values of DIN/P ratio were relatively high in the sCW ( Table 1). In the nCW, our results are lower than the historical data in the two seasons but are in accord with the decreasing trend of DIN and nitrate from spring to summer. Likewise, in the sCW, our results are lower than the previous results, but consistent with the increasing tendency from spring to autumn ( Table 2). The relatively low concentrations in the year of 2018 were probably affected by the annual nutrient fluctuation Yang et al., 2018;Wang et al., 2020b). The variable and water-mass specific patterns of N/P suggest different mechanisms of N-cycling for the two branches of the deep water mass. N * is a tracer that reflects the combined effects of denitrification and nitrogen fixation, atmospheric deposition and river inflow on the ratio of N to P (Gruber and Sarmiento, 1997 , the most commonly used stoichiometric ratio of these two essential nutrients for phytoplankton assimilation. This tracer was first used in studies of the open ocean where nitrate is predominant, whereas nitrite and ammonium have to be accounted for as well in our study area (Zheng and Zhai, 2021) . N * decreased from spring to summer in the nCW and sCW from −1.4 ± 0.3 to −2.5 ± 1.1 and from −1.6 ± 0.5 to −2.7 ± 0.7, respectively. The decrease in N * suggests that DIN is lost in both nCW and sCW between the spring and summer sampling campaigns by mechanisms different from phytoplankton assimilation. Nutrient distribution was affected by vertical mixing in spring and by stratification of the water column in summer. Water column integration for nutrients can help us to compare the seasonal variations of inventories by eliminating the effect of stratification. In our study, nCW and sCW areas are 7.9 × 10 3 km 2 and 37.5 × 10 3 km 2 , respectively, in the proportions of 2 and 10% of the whole YS. Water column integration for total DIN, nitrate, ammonium and phosphate were carried out for every individual station, following interpolation by a gridding method (by ArcGIS). The amount of DIN, nitrate, ammonium and phosphate are shown in Table 1. DIN, nitrate and phosphate decreased by 52, 69, and 82%, respectively, in summer in the nCW and ammonium increased by 69%, reflecting loss of DIN and phosphate to biomass and sediment from spring to summer, and their partial recycling. In the sCW, DIN, nitrate and phosphate in summer decreased by 5, 11, and 18%, respectively, whereas ammonium increased by 137%. These estimates indicate that although DIN is lost in the whole water column, this loss was compensated by production of DIN in the sCW. Less relative loss of nitrate in sCW than in the nCW points to nutrient replenishment from external sources (see Section "Sources of Excess Nitrogen in the Southern Part of Central Yellow Sea Bottom Water").

Isotopic Composition of Nitrate and Particulate Nitrogen
The δ 15 N of nitrate (δ 15 N-NO − 3 ) in spring was on average 8.3 ± 2.2 and ranged from 3.6 to 14.2 , and the corresponding δ   (Table 1).
N % of SPM in spring was on average 1.15% ± 1.03% and YSWC surface water and nYS surface water had the highest N % (N % > 2.0%), whereas suspended particulate matter in southern coastal water had the lowest N-concentrations (a) Only one valid data point.
(N % < 0.5%). N % of SPM in summer was on average 0.40% ± 0.21% with N % in surface layer higher than those in the bottom layer. In summer the CDW area had highest values (N % > 0.8%). The N % of bottom waters in summer were lower than in spring, indicating stronger decomposition of organic particles, resuspension is less likely the major reason because the weaker water mixing in summer. Average N % of sCW (N % = 0.16% ± 0.03%) was significantly lower than nCW (p < 0.05) ( Table 1), probably indicating strong remineralization of particles in the sCW. δ 15 N of particulate nitrogen (δ 15 N-PN) in spring was on average 4.3 ± 1.8 . YSWC water had the lowest values (δ 15 N-PN < 3.0 ) whereas nYS had the highest values (δ 15 N-PN > 5.0 ). In the sea surface, incomplete phytoplankton assimilation results in increased δ 15 N in the residual nitrate pool, and produces organic particles with relatively low δ 15 N (Sigman and Boyle, 2000;DiFiore et al., 2009). Studies in the YS and ECS proposed an isotope fractionation factor of approximately 5 during phytoplankton assimilation (Umezawa et al., 2013;Liu S. M. et al., 2017;Wu et al., 2019;Liu et al., 2020). The corresponding high N %, high values of nitrate dual isotopes and low δ 15 N-PN suggest phytoplankton assimilation of nitrate and its conversion to biomass in the surface of YSWC. In summer, δ 15 N-PN was on average 4.7 ± 1.5 , but the range of values of δ 15 N-PN in the surface was quite wide due to co-occurrence of diverse processes, such as phytoplankton assimilation and nitrification. Assimilation and nitrification were both revealed by nitrate isotopes in this area (Liu S. M. et al., 2017;Wu et al., 2019), which is the likely reason for the wide range of δ 15 N. The values converged to between 4 and 6 with increasing water depth and at >40 m water depth approached the δ 15 N of nitrate and sediment ( Table 1).

Characteristics of Sediments
The sediment under different water masses are characterized by similar water content, nitrogen content (N %), organic carbon content (C org %) and δ 15 N (δ 15 N-sed) of sediment under nCW and sCW. There are no statistically significant differences between other groups of sediments due to low sample numbers, but the distribution (Supplementary Figure 2) and average values suggest that the sediment under coastal water masses had relative low water content, N %, C org %, and δ 15 N-sed, whereas they had relatively high C/N ratio.

DISCUSSION
The inter-seasonal variations of nitrate and ammonium are different for the two branches of deep water in the Yellow Sea (nCW and sCW), although DIN and nitrate loss occurred in both water masses. Nitrate concentration decreased in the nCW in summer, whereas it increased in the sCW. In contrast, ammonium concentrations increased in the nCW, but decreased in the sCW. In the following, we aim to explain and quantify DIN and nitrate loss in the central YS bottom water and shed light on different seasonal dynamics of nitrate and ammonium cycling in nCW and sCW. The key processes, namely denitrification and nitrification, are discussed and the proportion of regenerated nitrate in summer is quantified by an isotope mass balance approach in the sCW. The excess nitrate in sCW compared to the nCW is analyzed as well to elucidate its sources.

Nitrate Loss in Central Yellow Sea Bottom Water
The decrease of DIN in the water columns of both nCW and sCW areas from spring to summer (Table 1) requires an N loss process such as denitrification or sedimentation of organic matter and associated reactive N.
The YSCWM with its two offshore water masses considered here (nCW and sCW) is isolated from any euphotic assimilation by summer stratification. As these cold water masses are located below the thermocline there is no air-sea exchange of oxygen, and DO is continuously consumed once the thermocline is formed. Thus, Apparent Oxygen Utilization (AOU) represents the net change due to respiration (Xiong et al., 2020). In the YS, approximately 90% of the organic carbon derived from primary productivity is decomposed in the water column Zheng and Zhai, 2021) and the positive AOU values in summer indeed suggest that respiration is the dominant process. The [NO − 3 ] r expected in nCW and sCW from AOU is higher than the actually measured NO − 3 between summer and spring sampling ( Table 3), implying that regenerated nitrate was removed from the water masses.
A likely reason for nitrate loss in both the nCW and sCW is denitrification. Water column denitrification can be ruled out, because the lowest value of DO in nCW and sCW was 218.3 µmol L −1 (station HS1, at 73 m) in summer, which is much too high to allow denitrification in the water column that can only proceed when DO is below 5 µmol L −1 (Altabet, 2006). In addition, the dual isotope values of residual nitrate in that case must show the fractionation effect that enriches 15 N and 18 O during water column denitrification (Wankel et al., 2006), which is not observed in our data. Instead, the dual nitrate isotope ratios were lower in summer than in spring ( Table 1).
Sedimentary denitrification is a strong candidate for the observed nitrate loss in the nCW and sCW. Continental shelf sediment are reported to account for >50% of global nitrate and DIN loss (Bohlen et al., 2012), and sediment denitrification normally is associated with little or no isotope fractionation due to the limitation of nitrate supply to the reactive sediment zones (Brandes and Devol, 1997;Lehmann et al., 2004;Devol, 2015). In the sediments of YS, nitrate is transported from the sediments to the overlying water at most sampling stations, whereas transport into the sediments was observed in the YSCWM area (Liu S. M. et al., 2017;Wu et al., 2019).
In the sediment of the YS, denitrification is a dominant process compared with anammox as evident by abundance of corresponding genes (Gao et al., 2017;Zhang et al., 2018;Cai et al., 2019). For instance, denitrification removes 54-98% N in the BHS and nYS (Zhang et al., 2018). In the surface sediment of the YS, richness and diversity of denitrifiers in mud deposition zones of YS (i.e., in the area of sCW in our study) were lower than the ones in the Yangtze Estuary (Zheng et al., 2015), and higher than the ones in the surface sediment of the South China Sea (Katsuyama et al., 2008;Gao et al., 2017). High concentration of organic matter facilitates the enrichment of denitrifiers (Hill and Cardaci, 2004) and other heterotrophic bacteria (Ren et al., 2020). In the YS, the mud sedimentation area (under nCW and sCW) has relatively high organic carbon content (C org %). Previous studies revealed that C org % is positively correlated with the abundance of the functional genes of denitrification (Gao et al., 2017;Zhang et al., 2018), implying that N loss in nCW and sCW is prominently driven by microorganisms.
In the nYS, the abundance of denitrifiers was higher in summer than in spring and autumn, and denitrification flux in summer was about 2 times higher than those in spring and autumn . All this is in accord with significant N loss to denitrification during summer in the nCW. In the sYS, the bacterial production in summer was on average 3 times that of spring, and the YSCW area had the highest bacterial production (Zhao et al., 2010), probably associated with the N loss in CWs in summer.
The assimilation of nitrate by benthic foraminifera and subsequent intercellular denitrification has been proposed as one reason for N loss in the sCW in summer (Xu et al., 2017;Wu et al., 2019). The distribution of benthic foraminifera was correlated to the median grain size of the sediment and it was abundant in the mud area in the central YS trough (Sun et al., 2009). Sediment denitrification is thus the most plausible reason for the observed nitrate loss in the sCW. The conversion of ammonia and nitrite to N 2 by the anammox process cannot be excluded based on our data in hand, but denitrification likely is predominant (Zhang et al., 2018).
Another possible reason of nitrate loss is nitrate vertical diffusion. Upwelling at the boundaries of the YSCWM (Wei et al., 2016) and vertical diffusion can both supply nutrients from deep water to the euphotic layer (Su et al., 2013). Vertical diffusion from the near bottom water in summer has been estimated to supply 4,945 µmol m −2 d −1 of DIN and 236 µmol m −2 d −1 of P to the thermocline in the sCW (Su et al., 2013). Although the upwelling is generally not considered as a cause for changes in the stoichiometry of nutrients, the N:P ratio of the diffusion flux of 21 implies that DIN is consumed more than P.
In summary, the nutrients were regenerated from sinking organic matter or sediments in summer in both the nCW and sCW. However, nitrate concentrations in summer were much lower than the AOU-based estimate of regenerated [NO − 3 ] r in both nCW and sCW. Denitrification is the main factor resulting in nitrate loss in the nCW and sCW, and stronger N depletion in the nCW compared to the sCW was observed.

Quantification of Regenerated Nitrate in sCW
The regenerated nitrate is an important source of YSCWM in summer, the proportion can be estimate by its distinctive δ 18 O value. δ 15 N of newly nitrified nitrate bears the signal of the particulate nitrogen (PN) source and the fractionation effect nitrate cycling to PN and back to nitrate. Differing from this, 3 is a good tracer of nitrification because δ 18 O of newly nitrified nitrate bears the signal of ambient water and dissolved oxygen without contribution of preformed nitrate, regardless of its origin from particulate or dissolved organic nitrogen. For quantification of the portions of preformed and regenerated nitrate in summer, a simple mixing model can be set up between newly nitrified and preformed nitrate with distinctive δ 18 O values of each source. The portion of preformed nitrate in spring and newly nitrified nitrate in summer can be calculated following Eq. (4) and Eq. (5). The calculations below are only based on the sCW due to the fact that nitrate dual isotopes in the nCW were near the detection limit.  (Schmidt et al., 1999), this must be the lower limit of δ 18 O-H 2 O for newly nitrified nitrate.
(2) Assuming that 5/6 of oxygen atoms are from water (Mayer et al., 2001) and the remaining 1/6 has a δ 18 O-O 2 of 23.5 (Kroopnick and Craig, 1972) the δ 18 O ntr can be expected to be 3.3 ± 0.2 . (3) In the open ocean, δ 18 O ntr is around 1.1 higher than in the ambient water . Assuming the same systematic difference would result in δ 18 O ntr of 0.4 in the YS. In summary, the δ 18 O ntr is theoretically in the range of −0.7 to 3.3 in the YS.
To further constrain the value of δ 18 O ntr , an iterative mixing simulation is employed based on our observed data. This mixing model can be simplified by several assumptions: (1) Reaction rates of nitrification and denitrification in sCW are stable during the study period. (2) Nitrate concentration in sCW is the function of time (in days). As our spring and summer cruises were in April and August, the whole simulation period is about 120 days. (3) Nitrification, mixing and removal of nitrate proceed simultaneously. Because decomposition of organic particulate nitrogen in the water column accounted for around 90% of total nitrogen loss, the predominant nitrate flux should be directed from water to the sediment so that water column nitrification probably supplies nitrate for sediment denitrification. Ammonium assimilation in the sCW is not implemented in the simulation due to the fact that most of it occurs in the euphotic layer. Thus, the mixing model can be simplified, as nitrate is a mixture of preformed and newly nitrified nitrate, which afterward is removed by denitrification or diffusion (Eq. 6 and Eq. 7). δ 18 O loss in Eq. 7 can be calculated by Eq. 8 to Eq. 10.
where n denotes the number of steps (=days) of the simulation (in d, 1 ≤ n ≤ 120).  Figure 6. 18 O ntr is 0.9 and in good agreement with our initial assumption. Combining this value with Eq. 4 and Eq. 5 suggests that nitrate in summer in the sCW consists of 35% preformed and 65% regenerated nitrate. Furthermore, the δ 15 N of regenerated nitrate ( 15 N r ) is 3.3 that was calculated following Eq. 11. Different from the result of 18 O ntr , the value of 3.3 for 15 N r reflects not only the δ 15 N of regenerated nitrate, but also the potential fractionation effects on N cycling.
Overall, regenerated nitrate had a δ 15 N r of 3.3 and an δ 18 O ntr of 0.9 and contributed 65% to the nitrate pool of sCW. Because nitrate is depleted in the upper layer in summer, 10.3 × 10 6 mol nitrate of the sCW water column (Table 1) can be considered as the inventory of the sCW. This means that 3.6 × 10 6 mol nitrate were performed in spring and 6.7 × 10 6 mol nitrate was regenerated.
In the sCW, lower N % and higher AOU than those of nCW suggest that the decomposition of particulate nitrogen was stronger. The low levels of ammonium and accumulation of nitrate imply that nitrification converting ammonium to nitrate is a major process in the sCW. To our knowledge, there has not been research on nitrification in the YS water column. On the other hand the distribution of two ammonia-oxidizing microorganisms was positively correlated with δ 15 N-sed in sediment of the southern YS , which is consistent with the relatively high δ 15 N-sed of the nCW and sCW. We interpret this as evidence for a fundamental role of microbiological nitrification in the nCW and sCW.
We introduce the molar ratio of nitrate/DIN ([NO − 3 ]/[DIN]) for simply representing the degree of nitrification. As nitrification proceeds, ammonium is converted to nitrate, resulting in increased [NO  Buchwald et al., 2012;Casciotti, 2016). The same is evident in the values of δ 15 N-NO − 3 in the sYs of 5.0 in autumn before winter mixing . Besides, sediment nitrogen isotope under the sCW was also close to isotope values of particles and nitrate (δ 15 N-sed = 5.2 ), these similar delta-values imply that the fractionation of δ 15 N of PN, nitrate and sediment nitrogen was almost in equilibrium. Further data like phytoplankton abundance, Chl a and dissolved organic nitrogen will be helpful for constructing the internal nitrogen cycling in the area.

Influences of Atmospheric Deposition
Atmospheric deposition is probably responsible for much of the increase of nitrate concentrations in the YS and in the northwest Pacific Ocean in recent decades (Kim et al., 2011;Moon et al., 2021). This input contributed more than 2 times the nitrate flux of rivers to the YS (Liu S. M. et al., 2017). Sporadic events like dust storms accompanied by precipitation have been observed to stimulate phytoplankton blooms in the YS . It has been estimated that atmospheric nitrogen approximately supported 17-37% of the annual new production in the YS (Chung et al., 1998). The flux of atmospheric deposition of nitrate was around 23.4 × 10 9 mol year −1 which contributed 47% of external sources (atmospheric depositions and riverine influx) (Han et al., 2013). Other estimates quantify the share of nitrate atmospheric deposition among external inputs (atmospheric deposition, river input, and water exchange) to 71% (appr. 25.4 × 10 9 mol year −1 ) (Zhang et al., 2007;Liu S. M. et al., 2017), which was more than 2 times the nitrate flux of rivers to the YS (Liu S. M. et al., 2017).
The ratios of N:P of atmospheric deposition in the YS was 119 to 832 (Zhang et al., 2007), which is much higher than that in the sea water. Besides, during the last two decades, atmospheric deposition accounted for 71% of the increase of N excess in the YS (Zheng and Zhai, 2021). However, the atmospheric deposition would not reach the CWs before they are assimilated in the euphotic layer.
There are no directly measured data of nitrate isotopes in atmospheric deposition in the YS. δ 15 N-NO − 3 of precipitation in northern China and the Japan Sea ranged from −2.5 to +0.9 Chang et al., 2019;Kim et al., 2019;Li et al., 2019), whereas δ 15 N-NO − 3 values of PM2.5 (fine particulate matter suspended in the air) ranged from 3.5 to 17.8 in northern China (Zong et al., 2017;Fan et al., 2019;Zhang et al., 2019;Song et al., 2020). The ratio of wet to dry nitrogen deposition is close to 1:1 in China on land  and 1.9:1 in the eastern YS (Kim et al., 2010). Taking the δ 15 N value of 8.2 reported from in Bohai Strait for dry deposition (Zong et al., 2017), and the δ 15 N value of 2.35 for precipitation  results in an estimate for the δ 15 N-NO − 3 of nitrate deposited from the atmosphere of 1.3-2.9 . These values are slightly lower but similar to the 3.3 of δ 15 N r , implying that atmospheric deposition is an important external nitrate source to the sCW from the perspective of nitrate isotopes. Likewise, if we take the δ 18 O-NO − 3 of 65.0 ± 5.8 for dry deposition (Zong et al., 2017) and 57.80 ± 4.23 for precipitation  then the δ 18 O-NO − 3 for total deposition is 60.3-61.4 . δ 18 O of newly nitrified nitrate, however, only bears the signal of ambient water FIGURE 8 | Different nitrate budgets in the nCW and sCW. In both water masses, nitrate consists of the preformed nitrate and regenerated nitrate. The sCW is impacted by the inflow of Yellow Sea Warm Current (YSWC) in spring, the input from atmospheric deposition and inflow of the Changjiang River in summer, while nitrate is lost mainly to benthic denitrification. The nCW is impacted by the input of nitrate by atmospheric deposition only and nitrate loss mainly to benthic denitrification, the prevalence of which makes the area filled by nCW a net sink for nitrate in summer. Proportions for regenerated and preformed nitrate are estimated by the isotopic mixing only for the sCW because of a lack of isotope values in the nCW. , δ 15 N and δ 18 O are labeled by different subscript, "sp," "sm" denotes the corresponding average values observed in spring and summer, respectively, "r/ntr" and "loss" denotes regeneration and loss, respectively, "ntr_res" and "sp_res" denotes the residual portion of the preformed nitrate in spring and of the residual portion of regenerated nitrate in summer, respectively. and dissolved oxygen, so that the high δ 18 O is not suitable for tracing the atmospheric deposition to δ 18 O-NO − 3 in the bottom water. Nevertheless, the high δ 18 O of atmospheric deposition may enhance the δ 18 O in the upper layer and may subsequently change the δ 18 O in the whole water column if vertical mixing is more intense, like in spring.
Most of the studies considered the difference of the atmospheric deposition between the nYS and sYS as not significant (Zhang et al., 2007(Zhang et al., , 2010Shi et al., 2012;Han et al., 2013;Qi et al., 2013). We surmise that atmospheric deposition is an important external nitrate source to the YS, but less likely to be the reason for the higher nitrate concentration in the sCW.

Sources of Excess Nitrogen in the Southern Part of Central Yellow Sea Bottom Water
In spring, nitrate concentrations in the sCW were higher than those in the nCW. The reason is the strong impact of the YSWC, the sub-branch of Kuroshio Current, on the sCW area, reflected in higher concentrations in the south and low nutrient concentrations near 36 • N before reaching the nYS in spring (Figure 2). The δ 15 N-NO − 3 in the water column correspondingly increased northward due to the fractionation effect during nitrate assimilation when YSWC flows northward (Umezawa et al., 2013;Liu S. M. et al., 2017). Specifically, δ 15 N-NO − 3 of Kuroshio Current to the east of Taiwan, the upstream of the Kuroshio Current, was in the range of 4.9-6.7 , while δ 15 N-NO − 3 and δ 18 O-NO − 3 of the Kuroshio Subsurface water downstream (200 m < depth < 300 m) was 5.5-6.0 and 3.5-4.0 , respectively (Umezawa et al., 2013). Before reaching sYS, the Kuroshio invasion water (200 m < depth < 400 m) has δ 15 N-NO − 3 and δ 18 O-NO − 3 of 5.5 ± 2.0 and 3.4 ± 0.5 , respectively . These data indicate that the δ 15 N-NO − 3 of the Kuroshio Current before mixing with the continental water mass in the YS is almost stable in the range from 5 to 6 . δ 15 N-NO − 3 of sCW and nCW had higher values of 8.6 ± 2.2 and 9.6 ± 1.5 , respectively.
In summer 2018, Changjiang River discharged 8.0 × 10 11 m 3 of fresh water into the YS and ECS (MWR, 2019) and approximately 14-28% of the waters from Changjiang enter the southern YS during summer by the northward transported CDW which dominated surface circulation (Liu S. et al., 2003;Fan and Song, 2014). Historical nitrate concentration in the Changjiang Estuary was 20.5 µmol L −1 in the 1960s (Zhou et al., 2008), but reached 66.1-84.1 µmol L −1 in recent decades Li et al., 2010;Yu et al., 2015;Wang et al., 2016;Zhong et al., 2020). During the longer period of 1900-2010, the Changjiang River N input to the YS and ECS increased from 337 to 5,896 Gg N year −1 (i.e., 0.3 and 5.9 Tg N year −1 ) according to a recent model simulation . The area of highest nutrient concentrations emerged in the south or southwestern part of the southern YS during the 1980s-2012 (Li et al., 2017b). The supply of CDW is suggested to lead to major oxygen consumption and even hypoxia, inducing up to 70% of the total DO consumption of the Changjiang River estuary . Besides, it also partially accounts for the magnitude of macroalgal blooms in the southern YS in recent years (Li et al., 2017b). DIN/P ratios of CDW in the range of 48.3-106.4 (in the salinity range from 24.8 to 28.4) during our survey were much higher than those of sCW. Formation of the thermocline prevents CDW from being a direct nutrient source to the bottom water. In the northeastward CDW frontal area, nitrate concentration increased with water depth, whereas nitrate isotope values and N % of particles decreased. This indicated a CDW-sourced nitrate supply to the sCW by the production and subsequent decomposition of sinking particles and nitrate regeneration.
δ 15 N-NO − 3 in the Changjiang and its estuary have δ 15 N values in range of 2.0-6.6 Chen et al., 2013;Yu et al., 2015;Wang et al., 2016;Liu et al., 2020;Zhong et al., 2020). Taking 5.8 ± 2.0 as the end member of the Changjiang River ) the calculated value above from the mixing model of regenerated nitrate of δ 15 N r 3.3 is lower. Two possible reasons should account for this difference: (1) during decomposition of PN CDW there is a fractionation effect, and (2) there are other external sources with lower δ 15 N-NO − 3 values. In the nCW the river influence on the N-pool was low. Yalu River has an annual average water discharge of 3.20 × 10 8 m 3 (Li et al., 2017a) which is about 89% of river input from the Liaodong Peninsula (Chen et al., 2012). It was restricted to the coastal area northwest of the nYS (Duan et al., 2016;Yang et al., 2018). Although 70-80% of this input occurs during the flood season (July and August) (Li et al., 2017a), resulting in a negative correlation between DIN and salinity in coastal area of nYS (Duan et al., 2016), there was no low salinity water during our survey, so that the influence of rivers to central nYS must have been quite limited. The difference of river impact on nYS and sYS in summer is the reason for higher nitrate concentration in sCW.
In addition, the organic carbon content, nitrogen content and δ 15 N of sediments in central areas of both the nYS and sYS are very similar (Table 1), sediments in both nYS and sYS are mostly clayey silt . This implies that reflux from sediment probably did not account for the different nitrate concentration in nCW and sCW, specifically as 90% of organic particles are decomposed in the water column.
Overall, the higher nitrate concentration in the sCW compared to the nCW is due to the stronger inflow of nitrate via YSWC to the sYS in spring, and more river input via the Changjiang River in summer (Figure 8). Atmospheric deposition is an important source of the YS, but it is not the reason for higher nitrate concentrations in the sCW. The δ 15 N value of newly regenerated nitrate to the sCW of 3.3 combines the isotope fingerprints of nitrate from Changjiang River and atmospheric deposition.
The ecosystem of the YS has responded to this excess nitrogen in the past decades. Red tides have been observed 36 times covering areas of up to 1,460 km 2 in 2018 (MEE, 2019). A positive relationship was reported between the mean HAB frequency and DIN concentration in the coastal areas of China . The most eutrophic seawaters are found in the large estuaries, including the Changjiang River , and correspondingly, the HABs occurred most frequently in coastal water (Fu et al., 2012), especially in the Zhejiang and Guangdong coastal area where Changjiang River and Pearl River discharge into the sea Xiao et al., 2019). Because the imbalance of nutrients induced by excess N, HAB occurrence in the coastal area of China may increase in the future . On the other side, reduced ammonium loads from rivers led to a decrease of HAB frequency on a decadal scale in the Laizhou Bay, Bohai Sea (Jiang et al., 2018).
Green tides in the YS have been reported since 2007 (Xia et al., 2009;Liu et al., 2013), covering areas of 18,237 km 2 in the YS in 2018 (MEE, 2019). These green tides generally appeared in the Jiangsu coast and drift northwards . Because the occurrence of green tides has a positive correlation with DIN concentration Li et al., 2017b), the central YS is less affected than the coastal area so far. Although the impact of human perturbations on the YS was mainly concentrated in the coastal areas in the sYS, the nYS and the central part of sYS will face a higher risk of eutrophication if nitrogen input continuously increases.

CONCLUSION
Based on analyses of nutrient concentrations and nitrate isotope composition in water, particulate matter and sediments in spring and summer, we distinguish differences in nitrogen cycling in two prominent sub-thermocline water masses in the Yellow Sea. Although the hydrographic conditions were quite similar in the northern part (nCW) and southern part (sCW) of the YSCWM, nitrate concentrations of nCW were much lower than those in the sCW in both spring and summer. In spring, nitrate concentrations of nCW were lower than in sCW due to weakening northward transport of YSWC that is expressed as a nutrient gradient between the nYS and the sYS. In summer, the influx of nitrate with Changjiang Diluted Water (CDW), its assimilation into biomass and subsequent nitrification enhanced the nitrate in sCW, but the central area of nYS was hardly impacted by terrestrial influx. DIN pool decreased by 52 and 5% in nCW and sCW, respectively, sediment denitrification and vertical diffusion accounted for DIN loss in the central YS bottom water. However, substantial nitrification in sCW was revealed by dual nitrate isotopes, resulted in DIN in sCW increased in summer opposite to the nCW, A simple mixing model based on δ 18 O-NO − 3 suggested that nitrate of sCW in summer is the mixture of nitrate preformed in spring and regenerated nitrate in summer, co-occurring with DIN loss. Preformed and regenerated nitrate was estimated to amount to 35 and 65% of the total nitrate in the sCW, respectively. Sedimentary denitrification proceeded beneath both water masses gradually reduced the pool of DIN in nCW, whereas higher rates of nitrate regeneration than of denitrification increased the sub-thermocline nitrate pool in sCW. These diverging trends suggest that the sYS is more sensitive to deoxygenation than the nYS. Although the bottom water is still oxic in the south-central YS, the ecological risk of continued and rising excess nitrogen from Changjiang River should not be ignored.

DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

AUTHOR CONTRIBUTIONS
BG and K-CE designed the study. ST collected the samples on board. JT and YL supported the sampling on cruises and field trips. ST, NL, and TS analyzed samples. TX and WZ supplied the dissolved oxygen data. ST, BG, K-CE, and KD interpreted the data. ST prepared the manuscript with input from all coauthors. All authors contributed to the article and approved the submitted version.

FUNDING
The study was part of the project FINGBOYEL (03F07686B) carried out within the MEGAPOL consortium and financed by the German Federal Ministry of Education and Research (BMBF).

ACKNOWLEDGMENTS
We thank the crew of the Chinese research vessel Dongfanghong 2 for their support of our work on board. We thank Markus Ankele and Frauke Langenberg for their support of the nutrient and stable isotope analyses. We also thank Chuancheng Fu from Yantai Institute of Coastal Zone Research for help on the using of ArcGIS.