Isotope constraints on nitrogen dynamics in the upper water column of the South China Sea

The supply of nitrogen (N) from various external and internal sources into the euphotic zone, e.g., atmospheric N deposition (AND), upwelling, lateral intrusion, and remineralization, modulates the biogeochemical and climatic roles of oligotrophic oceans and complicates N dynamics in the upper water column (≤200 m). However, our ability to resolve the mechanisms controlling upper-ocean N cycling is limited by the lack of high-resolution vertical observations. Here, we analyzed concentrations and dual isotopes of nitrate (NO₃^–) in the upper 200 m of the oligotrophic South China Sea. By examining dual isotopic signatures of NO₃^– (δ¹⁵N_NO3 and δ¹⁸O_NO3) and multiple associated parameters vertically throughout the upper water column, we resolved the dominant N sources and processes, including AND/N₂-fixation, assimilative fractionation, and nitrification, and quantitatively evaluated their contributions in the vertical distribution of NO₃^–, which can be separated into the Δδ¹⁸O_NO3-positive (δ¹⁸O_NO3−obs−δ¹⁸O_NO3−200m>0) and Δδ¹⁸O_NO3-negative layers (δ¹⁸O_NO3−obs−δ¹⁸O_NO3−200m<0) according to the deviation in δ¹⁸O_NO3 at a given depth (δ¹⁸O_NO3-obs) from that at 200 m (δ¹⁸O_NO3-200m). In the Δδ¹⁸O_NO3-positive layer, the NO₃^– assimilated by phytoplankton was largely sourced from nitrification (39 ± 11%) and AND/N₂ fixation (17-28%), whereas these two processes accounted for 17 ± 10% and 7 ± 6% of the total NO₃^– pool in the Δδ¹⁸O_NO3-negative layer. Considering a substantial contribution of the regenerated (nitrification-sourced) NO₃^– to the total NO₃^– pool especially in the Δδ¹⁸O_NO3-positive layer, caution should be taken that the new production assessed by the rates of NO₃^– uptake may be significantly overestimated in the SCS. These findings not only highlight the importance of these biogeochemical processes to NO₃^– dynamics in the upper water column of marginal seas, but also with important implications for the estimation of biological carbon pump and/or the f-ratio.

1. Introduction

Nitrogen (N) supply limits productivity in the ocean (Moore et al., 2013), thereby connecting the N cycle with marine carbon sequestration (Falkowski, 1997; Buchanan et al., 2021). In oligotrophic regions, regenerated N is the main N source for phytoplankton growth in surface waters (<100 m), since inputs of subsurface (100-200 m) nitrate (NO₃^–) supply are generally limited by strong stratification (Yool et al., 2007; Van Oostende et al., 2017). However, in marginal seas, while diapycnal fluxes of N to the N-replete layer are sufficient to support the levels of export production, additional external sources of N from atmospheric N deposition/N₂ fixation (AND/N₂ fixation) can stimulate phytoplankton growth in the N-depleted layer (Kao et al., 2012; Du et al., 2017; Lu et al., 2019). In addition, lateral transport also influences N dynamics in the euphotic zone, as different water masses vary in their relative concentrations of N compounds and microbial communities that alter N dynamics physically and biologically (Du et al., 2013; Xu et al., 2018). Therefore, the co-influence of various external N sources and their differing magnitudes make N dynamics more complicated in the upper ocean of marginal seas (Kao et al., 2012; Liu et al., 2020).

The South China Sea (SCS) is one of the largest marginal seas in the world, with an area of 3.5×10⁶ km². It is a typical stratified and oligotrophic oceanic regime, showing extremely low N:P ratios (0.4-4.4) in surface waters (<60 m), and thus low levels of biomass primarily due to N limitation (Chen et al., 2004; Du et al., 2017). Although many studies have found various N sources involved in biological production within the upper ocean, such as diapycnal transport, lateral transport, AND/N₂ fixation (Kao et al., 2012; Du et al., 2013; Yang et al., 2014; Du et al., 2017; Yang et al., 2017; Lu et al., 2019), it remains unclear how these sources and subsequent processes imprint on the N cycle of the upper water column of the SCS. These processes contribute to the complexity of N dynamics in the region over temporal and spatial scales. Seasonally, reactive N deposition to the SCS varies from 48 ± 34 μmol N/m²/d in July to 99 ± 78 μmol N/m²/d in September (Yang et al., 2014). Regionally, the depth-integrated N₂ fixation rates vary from 50 ± 10 μmol N/m²/d in the basin to 463 ± 260 μmol N/m²/d in the Kuroshio-affected region (Lu et al., 2019). In oligotrophic water column, the vertical diapycnal N flux is three orders of magnitude larger in the nutrient-replete layer relative to the nutrient-depleted layer (Du et al., 2017). This study aims to decipher the N dynamics of multiple external sources and internal processes by using a NO₃^– dual isotopic approach (δ¹⁵N_NO3 and δ¹⁸O_NO3), which serves as an integral tracer of the N pool in the upper water column of the SCS. These results contribute to our understanding of N dynamics in marginal seas, and benefit the development and parameterization of N-driven physical-biogeochemical models.

The δ¹⁵N_NO3 and δ¹⁸O_NO3 provide useful constraints on its source (Sigman et al., 2005; Rafter and Sigman, 2016; Yang et al., 2022) and can be used to explore relevant N cycling processes (Emeis et al., 2010; Fawcett et al., 2015; Buchanan et al., 2021). The first study to utilize a dual isotopic approach (δ¹⁵N and δ¹³C) in the region focused on evaluating the role of N sources in zooplankton nutrition in the Vietnamese upwelling area (Loick et al., 2007). Additional work by Yang et al. (2017) examined N cycling using δ¹⁵N in NO₃^– and particulate phases in the northern SCS. However, these two isotope studies had difficulty assessing the influence of nitrification (a common process in the euphotic zone) due to the lack of δ¹⁸O_NO3 measurements. Recently, two studies have illustrated the spatial variations of NO₃^– sources and N cycling in the SCS using both δ¹⁵N_NO3 and δ¹⁸O_NO3 (Chen et al., 2019; Yang et al., 2022); however, they mainly focused on the whole water column, with only 2-4 measurable isotope samples in the top 200 m. Thus, it is difficult to make a comprehensive assessment of NO₃^– dynamics within the upper ocean. In this study, we performed high-resolution vertical observation of the concentrations and dual isotopic composition of NO₃^–, in order to decipher the critical elements influencing NO₃^– dynamics in the upper water column of the SCS including the relative importance of external N sources and internal processes to NO₃^– uptake and assimilative fractionation.

2. Materials and methods

2.1. Study area

The circulation patterns in the SCS vary seasonally as a function of the East Asia monsoon (Figure 1A). In winter, the northeast monsoon pushes the SCS Warm Current southwestward and drives a basin-wide cyclonic gyre (Hu et al., 2000; Liu et al., 2016). In summer, the SCS Warm Current shifts northeastward and the basin-wide cyclonic gyre shifts eastward under the influence of southwesterly monsoon (Liu et al., 2016). The surface water mass in the SCS changes based on the extent of intrusion of the Kuroshio Current through the Luzon Strait, which varies both throughout the year and over decadal time scales (Hu et al., 2000; Yuan et al., 2006; Nan et al., 2015). Previous studies showed lateral Kuroshio intrusion influences not only the heat and salt of the SCS, but also N and carbon biogeochemical cycles due to high dissolved organic carbon and low nutrient concentrations in the Kuroshio waters (Du et al., 2013; Nan et al., 2015; Wu et al., 2015).

FIGURE 1

Figure 1 (A) Study area and sampling stations in the SCS. Surface circulation patterns are shown with arrows synthesized from previous studies (Yuan et al., 2006; Nan et al., 2015; Liu et al., 2016): summertime surface currents (cyan), wintertime surface currents (white dashed), and the Kuroshio Current (black). The color bar indicates water depth. (B) Potential temperature-salinity diagram for all stations. The red and black triangles indicate typical SCS water and coastal water, respectively. The gray triangles represent the Kuroshio Current. (C–H) Depth and isopycnal profiles for all samples with NO₃^– concentrations ≥0.5 μmol/L: NO₃^– concentrations (C), δ¹⁵N_NO3 (D), δ¹⁸O_NO3 (E), N* (F), Δ(15-18) (G), and N/P ratio (H).

2.2. Field sample collections

Water samples were collected in the SCS onboard the R/V Dongfanghong II during March 2013, June 2014, May-June 2016, and onboard the R/V TAN KAH KEE during June 2017 (Figure 1A). Seawater was collected for analysis of the concentrations and isotopic composition of NO₃⁻ at 5-32 layers from the surface (5 m) to 200 m using 12 L Niskin bottles attached to a Seabird SBE-911 plus CTD-rosette sampling system. More details of the sampling layers/intervals in each station can be found in the Supplementary Table 1 and the datasets. Unfiltered seawater samples were collected in 125 mL acid-washed high-density polyethylene bottles that were rinsed thoroughly with in situ seawater prior to filling. Water samples were immediately frozen on board at –20 °C until analysis.

2.3. Concentration and isotopic analysis of nitrate

The concentrations of NO₃⁻ and nitrite (NO₂⁻) were measured using a Four-channel Continuous Flow Technicon AA3 Auto-Analyzer (Bran-Luebbe), with detection limits of 0.07 μmol/L for NO₃⁻ and 0.02 μmol/L for NO₂⁻ (Dai et al., 2008; Du et al., 2013).

The values of δ¹⁵N_NO3 and δ¹⁸O_NO3 (for samples with NO₃⁻ concentrations ≥ 0.5 μmol/L) were determined by the denitrifier method (Sigman et al., 2001; Casciott et al., 2002). Briefly, denitrifying bacteria lacking nitrous oxide (N₂O) reductase were used to quantitatively convert NO₃⁻ in samples to N₂O. Subsequently, the isotopic compositions of N₂O were measured via GasBench II-IRMS (Thermo Scientific DELTA V advantage) equipped with an on-line extraction and purification system. Four internationally recognized NO₃⁻ reference materials (International Atomic Energy Agency (IAEA)-NO₃⁻: δ¹⁵N = 4.7‰ and δ¹⁸O = 25.6‰, U.S. Geological Survey (USGS)-34: δ¹⁵N = –1.8‰ and δ¹⁸O = –27.9‰, USGS-35: δ¹⁸O = 57.5‰ and USGS-32: δ¹⁵N = 180‰) were used for δ¹⁵N and δ¹⁸O calibration (Böhlke et al., 2003). One of the NO₃⁻ reference materials (IAEA-NO₃⁻) was run in parallel to monitor bacterial conversion efficiency and mass spectrometer drift. In terms of sample replicates, the analytical precision of δ¹⁵N_NO3 and δ¹⁸O_NO3 measurements as better than ±0.2‰ and ±0.5‰, respectively (Yan et al., 2017; Yan et al., 2019). To avoid the influence of NO₂⁻ on δ¹⁵N_NO3 and δ¹⁸O_NO3 values, all samples were treated with sulfamic acid (Sigma, guaranteed reagent) to remove pre-existing NO₂⁻ following the procedure in Granger and Sigman (2009).

3. Results

3.1. Hydrological characteristics

From Figure 1B, we can see all potential temperature-salinity profiles merge at a salinity of 34.4 and temperature of 13 °C, suggesting the same subsurface water (~200 m) source among these stations. Generally, as the potential density anomaly (σ_ϴ=σ−1000, unit: kg/m³) decreased toward the surface (~5 m), the potential temperature rose while the salinity first increased and then decreased. However, distinct hydrological differences were found between offshore SCS water measured at the South-East Asian Time-series Study (SEATS) station, the Kuroshio Current and coastal water (station X5, σ_ϴ<25.5). At the same isopycnal surface, the Kuroshio Current had the highest potential temperature and salinity, followed by SEATS, and the coastal water. The hydrological characteristics at most stations were controlled by the isopycnal mixing between the Kuroshio Current and the coastal water. Thus, due to greater influence from Kuroshio intrusion near the Luzon Strait, datapoints were more scattered and skewed toward the typical Kuroshio characteristics in spring relative to those in summer.

3.2. Vertical distributions of nitrate and its isotopic composition

The vertical profiles showed NO₃^– concentrations increased with increasing water depth (Figure 1C), with values ranging from 5.2 to 21.9 μmol/L. The lowest values (~0.1 μmol/L) were found mostly at depths of <50 m, while the highest values appeared at 200 m. Thus, the vertical gradients of NO₃^– differed among stations. For example, at stations TS2, B5 and BC1 near the Luzon Strait, which were more influenced by Kuroshio intrusion, NO₃^– concentrations were lower and varied within a narrow range of 0.1-8.0 μmol/L. However, at the southernmost station C1 where upwelling appeared, the NO₃^– concentration showed a steep gradient, changing from 0.2 μmol/L at 50 m depth to a maximum of 21.9 μmol/L at 200 m. Clearly, the vertical distributions of NO₃^– concentrations exhibited distinct north-to-south differences. By defining the nitracline depth as that where NO₃^– concentrations reached 2.0 μmol/L (Wilson and Coles, 2005), we found it ranged between 24-154 m, with a mean value of 77 ± 30 m (n=22, Table 1). The nitracline depths at stations near the Luzon Strait were the deepest due to the influence of warm, nutrient-depleted waters from Kuroshio intrusion.

TABLE 1

Table 1 The isotope effect of NO₃^– assimilation estimated from the Rayleigh Model and the Open system Model at appropriate stations.

The δ¹⁵N_NO3 values showed strong gradients above the nitracline (<125 m), but weaker gradients below the nitracline (Figure 1D). They generally fell within a narrow range of 3.4-5.7‰ below the nitracline; however, they became enriched upward toward the surface (6.4-13.3‰, ~20 m), accompanied by lower NO₃^– concentrations. For example, at station A11, δ¹⁵N_NO3 reached a maximum of 13.3‰ at 70 m. However, the site with the shallowest nitracline (station D1) had a weaker δ¹⁵N_NO3 gradient varying between 4.9-7.0‰ (Table 1). In addition, the δ¹⁵N_NO3 values at several stations (A2, Q1, B5 and C1) showed minima (2.1‰, 3.5‰, 3.3‰, and 4.9‰, respectively) at 75-125 m depths.

The δ¹⁸O_NO3 values were relatively constant below the nitracline but increased sharply from depths of 100-125 m toward ~20 m (Figure 1E), and δ¹⁸O_NO3 variability (5.2-22.5‰) was larger than that of δ¹⁵N_NO3 above the nitracline. The maximum values of δ¹⁸O_NO3 above the nitracline differed among stations. At stations NS1 and C1, δ¹⁸O_NO3 reached maximum values of 19.0‰ and 22.6‰, respectively, around a depth of 75 m. However, δ¹⁸O_NO3 maxima were observed at shallower depths (24-55 m) at stations D1 and B5, reaching only 5.8‰ and 5.2‰, respectively. Spatially, δ¹⁸O_NO3 values in the southern stations (SS1, B1, B2, A2 and C1) were mostly higher than that at stations further north, especially at depths from 90-150 m. Notably, the vertical distribution patterns of δ¹⁸O_NO3 did not with synchronous change of δ¹⁵N_NO3. The δ¹⁸O_NO3 values had an overall range of 0.9-22.6‰, which was twice the range of δ¹⁵N_NO3 (2.1-13.3‰). Moreover, the minimum δ¹⁸O_NO3 values did not occur in the subsurface waters (75-125 m) at stations A2, Q1 and B5.

4. Discussion

4.1. Significance of external N inputs revealed by N* and Δ(15-18)

In the subsurface near the nitracline where regeneration occurs intensively, NO₃^– was depleted in δ¹⁵N, with lower δ¹⁵N_NO3 (2.1-4.9‰) found at stations 2014SEATS, SS1, B5, Q1, J1, and C1 relative to other stations (Figure 1D). Such a negative shift in δ¹⁵N_NO3 near the nitracline suggests an external input of isotopically light N likely derived from AND/N₂ fixation. Here, a quasi-conservative tracer N* (N*=[NO₃^–]–16×[PO₄^3–]) (Gruber and Sarmiento, 1997; Deutsch et al., 2001), is used as an indicator to reflect external N inputs (i.e. AND/N₂ fixation) since the non-Redfieldian addition of external N can increase seawater N* (Sigman et al., 2005; Yoshikawa et al., 2015). Although N* is negative throughout the upper 200 m water column (Figure 1F), an upward increase in N* is evident from 1000 m toward the surface (<100 m) (Supplementary Figure 1A), providing evidence of the effects of external N inputs supplying excess N in the subsurface waters between 100-200 m (Kim et al., 2014; Yang et al., 2022).

The cumulative signal of AND/N₂ fixation on the subsurface NO₃^– pool (100-200 m) can also be elucidated by using a complementary tracer, Δ(15–18) (=δ¹⁵N_NO3 – δ¹⁸O_NO3) (Rafter et al., 2013; Yoshikawa et al., 2018). Our results show that Δ(15–18) values vary widely between –11.6‰ and 3.5‰, with larger variations above the nitracline (–0.9 ± 3.3‰, n=41) than below the nitracline (1.1 ± 1.7‰, n=142) (Figure 1G). These Δ(15–18) values are also consistent with previous reports from the SCS (–0.5~4.0‰; Yang et al., 2022) and the western subtropical gyre of North Pacific (0.9 ± 1.3‰; Yoshikawa et al., 2018). Overall, Δ(15–18) values remain nearly constant below the nitracline but decreased upward to ~20 m (Figure 1G and Supplementary Figure 1B). The decreasing trend is well explained by the accumulation of AND/N₂ fixation (Tuerena et al., 2021; Yang et al., 2022) since both featured negative δ¹⁵N values mostly falling between –10‰ and 0‰ (Knapp et al., 2008; Yang et al., 2014; Shi et al., 2021). Isotopically light δ¹⁵N_NO3 relative to δ¹⁸O_NO3, which is influenced by δ¹⁸O in H₂O and O₂ via nitrification, may thus cause a negative shift in Δ(15–18). Several independent lines of evidences have been reported to support the significance of external N inputs around the study area, with total AND rates of 50-90 mmol N/m²/yr (Yang et al., 2014; Shi et al., 2021) and depth-integrated N₂ fixation rates of 18.2-169.0 mmol N/m²/yr in the SCS (Lu et al., 2019). Accordingly, both AND and N₂ fixation potentially contribute to the negative shift in Δ(15–18) with the same order of magnitude. Alternatively, the negative shift in Δ(15–18) can also result from internal processes, such as the coupling of incomplete NO₃^– assimilation and remineralization of newly fixed organic N and subsequent nitrification (Yoshikawa et al., 2018; Yang et al., 2022). Therefore, external N inputs from AND/N₂ fixation resulted in the upward increases in N* and decreases in Δ(15–18), confirming the significance of AND/N₂ fixation on modulating N dynamics in the upper water column of the SCS.

4.2. Nitrate isotopes reveal dominant N cycling processes

4.2.1. Nitrate assimilation and its isotope fractionation

NO₃^– assimilation is an important N cycling process in the euphotic zone, especially near the chlorophyll maximum (Rafter and Sigman, 2016; Wan et al., 2018; Tuerena et al., 2021). In our study area, NO₃^– uptake was evidenced by upward NO₃^– depletion and the synchronous elevation in δ¹⁵N_NO3 and δ¹⁸O_NO3 in the top 100 m (Figures 1C–1E) caused by the preferential uptake of ¹⁴N and ¹⁶O in the NO₃^– pool by phytoplankton (Granger et al., 2004; Sigman and Fripiat, 2018). This isotope shift was also supported by the highest NO₃^– uptake rates (56.8-132.7 nmol N/L/d) near nitracline at stations D1, 2014SEATS and NS1 (Wan et al., 2018, Tables 1, 2). The upward pattern of decreasing N/P ratios toward the surface (~20 m) further confirms NO₃^– assimilation by photosynthesis with removal of N and P at the Redfield ratio resulting in a decrease in the residual N/P ratios when N* was negative (Figures 1F, H, Supplementary Figure 1A) (Deutsch and Weber, 2012). Therefore, NO₃^– uptake was the dominant process modifying the vertical variations of δ¹⁵N_NO3 and δ¹⁸O_NO3 in the euphotic zone (Rafter and Sigman, 2016; Peng et al., 2018).

TABLE 2

Table 2 Summary of nitrification rates, NO₃^– uptake rates, the contributions of nitrification to NO₃^– uptake (F_nit/F_upt), and the contributions of external N inputs to NO₃^– uptake (F_atm-fix/F_upt) in the Δδ¹⁸O_NO3-positive layer.

To evaluate the fractionation factor of NO₃^– assimilation, we plotted δ¹⁵N_NO3 or δ¹⁸O_NO3 against the natural logarithm of NO₃^– concentrations (Figures 2A, B). The isotope effect of NO₃^– assimilation generally follows Eq. (1) according to the Rayleigh model (closed-system) and follows Eq. (2) using an open-system model (Umezawa et al., 2014; Sigman and Fripiat, 2018).

FIGURE 2

Figure 2 Values of δ¹⁵N_NO3 (A) and δ¹⁸O_NO3 (B) versus the natural logarithm of [NO₃^–] for all stations. These data overlay models of NO₃^– uptake based on the average NO₃^– concentration and isotope at a depth of 200 m (black square, [NO₃^–]=14.8 ± 4.0 μmol/L, δ¹⁵N_NO3 = 5.4 ± 0.9‰, δ¹⁸O_NO3 = 3.5 ± 1.4‰, n=23). The straight dashed lines represent the Rayleigh model while the concave-down lines represent the Open system model. The black arrows represent the dilution effect of Kuroshio intrusion with low concentrations of NO₃^–. The gray vertical lines denote the nitracline of 2.0 μmol/L. (C) The relationship between ¹⁸ϵ:¹⁵ϵ ratios and the nitracline depth. (D) Scatter plot of δ¹⁸O_NO3 versus δ¹⁵N_NO3 for all stations. The solid line is the best fitting curve for all datapoints. The gray dashed line represents a 1:1 NO₃^– isotope assimilation trend.

$\begin{array}{ll}{\text{δ}}^{15}{\text{N}}_{\text{NO}3}={\text{initialδ}}^{15}{\text{N}}_{\text{NO}3}-\text{ϵ}\times \text{ln}\left(f\right)& \text{, (1)}\end{array}$

$\begin{array}{ll}{\text{δ}}^{15}{\text{N}}_{\text{NO}3}={\text{initialδ}}^{15}{\text{N}}_{\text{NO}3}+\text{ϵ}\times \left(1-f\right)& \text{, (2)}\end{array}$

where f is the residual fraction of NO₃^– in the water column from the initial NO₃^– concentration, and ϵ is the isotope effect (in ‰ units) for NO₃^– assimilation.

Our results show that samples enriched in δ¹⁵N_NO3 and δ¹⁸O_NO3 can be better explained by the combination of the Rayleigh model and the Open system model than solely by the Rayleigh model or the Open system model (Figures 2A, B). An isotope effect of 1.0-6.0‰ produced by the Rayleigh model can explain the majority of the increase in δ¹⁵N_NO3 and δ¹⁸O_NO3. Yet, for those stations near the Luzon Strait, the Open system model better predicts isotope behavior. Although the above estimated ranges were close to the typical isotope fractionation induced by phytoplankton in culture (1.4-21.0‰) (Waser et al., 1998; Needoba et al., 2003; Granger et al., 2004) and field studies (4-11.9‰) (DiFiore et al., 2010; Rohde et al., 2015; Rafter and Sigman, 2016), they exhibit large variations that are associated with ambient environmental conditions (e.g., light intensity) and phytoplankton species composition (Needoba and Harrison, 2004; DiFiore et al., 2010; Rohde et al., 2015). Below we estimate the isotope effect specifically for each station since they may have experienced different hydrological conditions and/or different plankton community structures (Rafter & Sigman, 2016).

Here, the isotope effect was estimated for stations with obvious NO₃^– drawdown and sufficient measurements (≥3) to yield a fractionation trend. These calculations assumed that subsurface (~200 m) NO₃^– is the only NO₃^– source available for phytoplankton assimilation. The isotope effect yielded by the slope of the regression for the individual station is estimated (Table 1). Average isotope effects of 2.8 ± 1.2‰ (n=14) for ¹⁵ϵ and 5.0 ± 2.8‰ (n=15) for ¹⁸ϵ were obtained from 16 of the 23 stations using the Rayleigh model. The isotope effect was close to the mean isotope effect based on the Open system model (7 of the 23 stations), which yielded 3.2 ± 1.0‰ (n=4) for ¹⁵ϵ and 5.8 ± 2.7‰ (n=4) for ¹⁸ϵ. These values fall well within the overall isotope effect estimates of 1.0-6.0‰ in Figures 2A, B and 2.2-6.2‰ derived from culture studies (Needoba et al., 2003). Noteworthy is the N and O isotope effect at stations near the Luzon Strait that yielded lower values of 1.1-3.1‰ and 1.3-4.7‰ (Table 1), respectively. This may be a consequence of NO₃^–depleted water supplied to the surface (<100 m) from Kuroshio Current. When NO₃^–depleted Kuroshio Current waters mixes with NO₃^–replete SCS waters, it reduces NO₃^– concentration of the remaining N pool (Du et al., 2013) without changing its isotopic composition (Deutsch et al., 2004). Similarly, the relatively low isotopic values (δ¹⁵N_NO3<5.4‰, δ¹⁸O_NO3<3.5‰) that failed to yield a fractionation trend can also be explained by the dilution effect from the Kuroshio Current.

4.2.2. Nitrification

The ratio of O:N isotope effects (¹⁸ϵ:¹⁵ϵ) was 2.0 ± 0.6 (n=13) derived from the Rayleigh model and 2.5 ± 0.7 (n=3) from the Open system model (Table 1), showing disproportionate change in ¹⁵ϵ and ¹⁸ϵ. Such a high ¹⁸ϵ:¹⁵ϵ ratio differs from many field and culture studies that found a nearly equivalent isotope effect in N and O (Granger et al., 2008; Rohde et al., 2015; Rafter and Sigman, 2016). The input of newly fixed N from N₂ fixation can lower ¹⁵ϵ by introducing isotopically light N into the NO₃^– pool, which may partially explain the lower ¹⁵ϵ relative to ¹⁸ϵ. Nevertheless, similarly high ¹⁸ϵ:¹⁵ϵ ratios have been reported for marine diatom Thalassiosira weissflogii (¹⁸ϵ:¹⁵ϵ=1.4), and as high as 2.0 for cultured heterotrophic α-proteobacterial strains (Granger et al., 2010; Karsh et al., 2014). Additionally, our findings compare well with the slope of 1.8 ± 0.1 (R² = 0.72, p=0.01) derived from the scatter plot of δ¹⁸O_NO3 versus δ¹⁵N_NO3 (Figure 2C). Such a positive shift in δ¹⁸O_NO3 relative to δ¹⁵N_NO3 can result from nitrification in recycled NO₃^– (Wankel et al., 2009), is consistent with the reported nitrification rates in the study area (Wan et al., 2018; Xu et al., 2018). Moreover, such positive deviations are more evident at stations where the nitracline was deeper (Table 1), as indicated by the positive correlation between ¹⁸ϵ:¹⁵ϵ ratios and nitracline depth (Figure 2D). This supports the idea that nitrification contributed to the high ¹⁸ϵ:¹⁵ϵ ratios since the influence of nitrification increased with increasing water depth and the lessening of photoinhibition for nitrifiers (Wan et al., 2018). However, nitrification still cannot explain the majority of the highest δ¹⁵N_NO3 and δ¹⁸O_NO3 values measured (Figure 2C), revealing that NO₃^– assimilation was the main process regulating N cycling in the upper ocean, followed by nitrification. Therefore, the isotope effect in this study was a conservative estimate since the influence of nitrification and N₂ fixation cannot be separated from NO₃^– uptake.

4.3. Relative importance of nitrification and external N inputs

The above discussion implies the importance of external N inputs from AND/N₂ fixation on the N pool in the upper ocean. Meanwhile, the relative importance of external N inputs and internal processes, which could be imprinted on NO₃^– isotope values, may differ vertically throughout the water column. However, the relative importance of these inputs is difficult to assess accurately without high-resolution sampling. Below we separated the water column of each station (those with high-resolution vertical sampling during 2014 and 2017 cruises) into a δ¹⁸O_NO3-based two-layer structure, according to the deviation in δ¹⁸O_NO3 at a given depth (δ¹⁸O_NO3-obs) from that at 200 m (δ¹⁸O_NO3-200m). Samples with a negative deviation (δ¹⁸O_NO3-obs< δ¹⁸O_NO3-200m) were assigned to the Δδ¹⁸O_NO3-negative layer; otherwise, they were categorized as part of the Δδ¹⁸O_NO3-positive layer (δ¹⁸O_NO3-obs > δ¹⁸O_NO3-200m). Then the relative importance of various processes contributing to the isotope shifts in the Δδ¹⁸O_NO3-positive and Δδ¹⁸O_NO3-negative layers of those stations was assessed quantitatively.

4.3.1. The Δδ¹⁸O_NO3-positive layer

In the Δδ¹⁸O_NO3-positive layer where NO₃^– uptake dominated, the contribution of nitrification to NO₃^– uptake (F_nit/F_upt) was calculated directly from the reported nitrification to NO₃^– uptake rates at stations NS1, 2014SEATS and D1 (Wan et al., 2018). The results show that nitrification accounted for 2-98% of the NO₃^– uptake and its contribution increased with water depth in this layer (Table 2). To eliminate the influence of different sampling resolutions, F_nit/F_upt was first linearly interpolated at 1 m intervals, and then the mean depth-integrated value was calculated. Results show that the depth-integrated F_nit/F_upt was 34%, 53% and 32% for stations NS1, 2014SEATS and D1, respectively, with a mean of 39 ± 11% (n=3), confirming the importance of nitrification in supporting phytoplankton growth (Wan et al., 2018). Noteworthy, new production assessed by the NO₃^– uptake rates may be overestimated in this layer due to a substantial proportion of the NO₃^– assimilated by phytoplankton was produced from nitrification. The cumulative NO₃^– from nitrification should be considered as regenerated N rather new N (Dugdale and Goering, 1967). This finding has important implications for biological carbon pump and carbon cycling in the vast marginal seas.

To estimate the relative importance of AND/N₂ fixation to NO₃^– uptake (F_atm+fix/F_upt), a simplified one-dimensional model based on a N isotope mass balance (Bourbonnais et al., 2009) was applied (Supplementary Figure 2). Details of the calculations are provided in the Supplementary Text 1. Results show that F_atm+fix/F_upt was up to 100% at 79 m for station NS1 and at 44 m for station 2014SEATS (Table 2), suggesting the predominant role of external N inputs in supporting marine productivity at low-N depths. This is also consistent with the fact that extremely low diapycnal NO₃^– fluxes in the nutrient-depleted layer are observed in the SCS (Du et al., 2017). Combined with the very low F_nit/F_upt at these depths, our results also indicate that external N inputs from AND/N₂ fixation are rapidly consumed by phytoplankton without being remineralized. However, F_atm+fix/F_upt decreased rapidly with depth at stations 2014SEATS and D1, along with the increasing contribution of nitrification. The mean depth-integrated of F_atm+fix/F_upt was 28% and 17% at these two stations, respectively, which is lower than the proportion of nitrification (39 ± 11%). This suggests that nitrification and external N inputs make a significant contribution to NO₃^– uptake (~50%) in the upper water column (Bourbonnais et al., 2009; Yang et al., 2022).

4.3.2. The Δδ¹⁸O_NO3-negative layer

In the Δδ¹⁸O_NO3-negative layer, where NO₃^– uptake was limited by light and nitrification became more important, we estimated the proportion of regenerated NO₃^– (f_reg/tot) following the method mentioned by Granger et al. (2013) and Tuerena et al. (2021). Details of calculations are provided in the Supplementary Text 2. Results showed that f_reg/tot ranged from 0-86% in the Δδ¹⁸O_NO3-negative layer (Table 3), with large vertical and spatial variability. The highest depth-integrated mean proportion of 34% was observed at station C1 while the lowest fractions of 2-4% occurred at stations 2014SEATS and B1. Overall, the depth-integrated f_reg/tot was 17 ± 10% (n=10), revealing that on average ~17% of the NO₃^– pool was regenerated from nitrification in the Δδ¹⁸O_NO3-negative layer. This is consistent with many other field studies (15-27%, Wankel et al., 2007; Tuerena et al., 2021) and model results (~50%, Yool et al., 2007), indicating that nitrification plays a substantial role in the NO₃^– pool and its δ¹⁵N_NO3 and δ¹⁸O_NO3 signatures, as well as in oceanic productivity. Additionally, f_reg/tot in the Δδ¹⁸O_NO3-negative layer varies greatly vertically, generally increasing then decreasing with depth, and peaking at 123 ± 21 m (n=9) (Table 3). This is consistent with reported nitrification rates that peaked at around 50-100 m (Wan et al., 2018; Xu et al., 2018), further confirming the significance of nitrification in regulating the size of the NO₃^– pool and its dynamics in the Δδ¹⁸O_NO3-negative layer, where low light intensity and abundant NO₃^– enhanced the success of nitrifiers (Wan et al., 2018; Marconia et al., 2019).

TABLE 3

Table 3 Summary of the proportions of regenerated NO₃^– (f_reg/tot) and external N inputs from AND/N₂ fixation (f_atm-fix) in the Δδ¹⁸O_NO3-negative layer.

To assess the relative contributions of external N inputs to the NO₃^– pool (f_atm-fix/tot), a two-end-member mass and isotope balance was used following Yang et al. (2022). Details of the calculation are provided in the Supplementary Text 3. A vertically decreasing trend in f_atm-fix/tot was observed at most stations (Table 3), consistent with observations in the Atlantic Ocean (Knapp et al., 2008). f_atm-fix/tot also varied spatially, with higher proportions (8-33%) at southern stations (C1, A2) near the Nansha Island than northern stations (1-16%), implying a greater accumulation of external N in the south. The deeper nitracline in the southern SCS may hinder upwelling of subsurface (~200 m) NO₃^–, and thus favor the growth of diazotrophs, which would lead to higher f_atm-fix/tot. The depth-integrated mean value of f_atm-fix/tot was 7 ± 6% (1-22%, n=10) in the Δδ¹⁸O_NO3-negative layer, slightly lower than in the Δδ¹⁸O_NO3-positive layer (17-28%) but comparable to the previously reported proportion of 1-22% (Wong et al., 2007; Lu et al., 2019; Yang et al., 2022). Taken together, the above results imply the importance of AND/N₂ fixation to the total NO₃^– pool in the upper ocean, although their average contribution is lower than that supplied by nitrification (Knapp et al., 2008; Bourbonnais et al., 2009; Tang et al., 2019).

By deducting the total NO₃^– contribution of nitrification (17 ± 10%) and external N inputs (7 ± 6%), we can obtain the average contribution of upwelled NO₃^– to the total NO₃^– pool as 76 ± 12% in the Δδ¹⁸O_NO3-negative layer. Therefore, δ¹⁵N_NO3 and δ¹⁸O_NO3 signature in the Δδ¹⁸O_NO3-negative layer were similar to that at 200 m (Figures 1D, 1E), but also under the co-influence of nitrification and external N inputs. This finding suggests the complicated of N dynamics in the upper ocean of marginal seas with variable contributions from various N sources and processes.

5. Conclusions

All information collected regarding NO₃^– dynamics, external sources and processes in the upper water column of the SCS was assembled into a conceptual diagram (Figure 3). Vertically, NO₃^– concentrations increased with depth while its δ¹⁵N_NO3 and δ¹⁸O_NO3 covaried becoming higher towards the surface (<20 m) due to phytoplankton assimilation. The isotope effect during NO₃^– uptake was 2.8 ± 1.2‰ (n=14) for N and 5.0 ± 2.8‰ (n=15) for O, deduced from the Rayleigh model, with a ratio of 2.0 ± 0.6 (O/N, n=13). This high O/N ratio was attributable to nitrification and/or N₂ fixation. At the depth of ~100 m at some stations, a negative shift in δ¹⁵N_NO3 deviated significantly from the vertical pattern of δ¹⁸O_NO3, suggesting an addition of isotopically light N. The relative contributions of external N sources and internal processes was assessed by taking advantage of high-resolution observations, revealing clear vertical variations in their contributions in the δ¹⁸O_NO3-based two-layer structure. In the Δδ¹⁸O_NO3-positive layer, the NO₃^– assimilated by phytoplankton were largely sourced from nitrification (39 ± 11%) and AND/N₂ fixation (17-28%). In the Δδ¹⁸O_NO3-negative layer, the proportions of regenerated NO₃^– and external N inputs to the total NO₃^– pool were 17 ± 10% and 7 ± 6% (n=10), respectively. These findings suggests that in addition to upwelled NO₃^– from subsurface waters (~200 m), nitrification and external N sources (AND/N₂ fixation) also play an important role in modulating the NO₃^– pool, affecting its isotopic signatures and cycling dynamics in the upper water column of the SCS.

FIGURE 3

Figure 3 Conceptual diagram for NO₃^– dynamics in the upper water column of the SCS.

Data availability statement

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: the Zenodo deposit, https://www.zenodo.org/record/7219590 (Doi: 10.5281/zenodo.7219590).

Author contributions

S-JK and MD supervised and conceived this work. J-YY, MX, ET, ZZ and WZ contributed to the sample collection. XY conducted chemical and data analysis and wrote the manuscript. S-JK, XY and J-YY reviewed and edited the manuscript. All authors contributed to the article and approved the submitted version.

Funding

This research was supported by the National Natural Science Foundation of China (NSFC 42176046, 42106048, 41890802, 92058204, 41721005), the Basic and Applied Basic Research Foundation of Guangdong Province (2019A1515010611), the State Key Laboratory of Marine Resource Utilization in South China Sea (Hainan University; MRUKF2021017), and the Shantou University Scientific Research Foundation for Talents (NTF18013).

Acknowledgments

We are grateful to Lifang Wang, Tao Huang, Qiao Wu and Xianhui Wan for his assistance in sample collection and measurement during the cruise.

Conflict of interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Publisher’s note

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

Supplementary material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmars.2022.1104135/full#supplementary-material

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