Dual isotopic evidence for nitrate sources and active biological transformation in the Northern South China Sea in summer

Fajin Chen; Xin Zhou; Qibin Lao; Shuangling Wang; Guangzhe Jin; Chunqing Chen; Qingmei Zhu

doi:10.1371/journal.pone.0209287

Abstract

Nitrate (NO₃^-) concentrations and their dual isotopic compositions (δ¹⁵N-NO₃^- and δ¹⁸O-NO₃^-) were measured to constrain N sources and their cyclic processes in summer using samples from the water column of the northern South China Sea (NSCS). Our data revealed that higher NO₃^- concentrations and δ¹⁵N-NO₃^- values were observed in the upper waters of the coastal areas near the Pearl River Estuary (PRE). The Bayesian stable isotope mixing model was used to calculated the proportion of nitrate sources, the results indicated that the nitrate in the upper waters of the coastal areas near PRE were mainly influenced by manure and sewage (63%), atmospheric deposition (19%), soil organic nitrogen (12%) and reduced N fertilizer (6%). For the upper waters of the outer areas, low NO₃^- concentrations and δ¹⁵N-NO₃^- values, but high δ¹⁸O-NO₃^- values, reflected that NO₃^- was mainly influenced by Kuroshio water intrusion (60%), atmospheric deposition (32%) and nitrogen fixation/nitrification (8%). Complex processes were found in bottom waters. Nitrification and phytoplankton assimilation may be responsible for the higher nitrate concentrations and δ¹⁵N-NO₃^- values. Our study, therefore, utilizes the nitrate dual isotope to help illustrate the spatial variations in nitrate sources and complex nitrogen cycles in the NSCS.

Citation: Chen F, Zhou X, Lao Q, Wang S, Jin G, Chen C, et al. (2019) Dual isotopic evidence for nitrate sources and active biological transformation in the Northern South China Sea in summer. PLoS ONE 14(1): e0209287. https://doi.org/10.1371/journal.pone.0209287

Editor: Jianlin Shen, Chinese Academy of Sciences, CHINA

Received: August 22, 2018; Accepted: December 3, 2018; Published: January 2, 2019

Copyright: © 2019 Chen et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the manuscript.

Funding: This work was supported by the National Natural Science Foundation of China (41476066, 41466010, 41676008), Guangdong Natural Science Foundation of China (2016A030312004), International Science and Technology Cooperation Project (GASI-IPOVAI-04), National Key Research and Development Plan (2016YFC1401403), the Project of Enhancing School with Innovation of Guangdong Ocean University (GDOU2014050201, GDOU2013050215, GDOU2013050315, GDOU2013050201), and the Fund of Key Laboratory of Global Change and Marine-Atmospheric Chemistry, SOA, (GCMAC1503) to FC. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Nitrogen (N) is a pivotal element in regulating marine primary productivity and plays a prominent role in marine biogeochemistry [1]. Among the various forms of N, nitrate (NO₃^-) is the most important form of bioavailable N in the ocean. Recently, anthropogenic activities have greatly increased the input of N to estuaries and adjacent coastal areas and have led to a host of environmental problems, such as eutrophication, hypoxia and harmful algal blooms [2–5]. Therefore, improving our ability to trace N sources, its turnover processes, and dispersal behaviours in estuaries and coastal oceans offers broad benefits.

The South China Sea (SCS) is an oligotrophic marginal sea in the Pacific, and the Pearl River Estuary (PRE) is an interface connecting the Northern SCS (NSCS) and the mainland of South China. Dissolved inorganic nitrogen (DIN) input into the river from the mainland of south China would be finally discharged into the PRE and adjacent coastal areas. On the other hand, the SCS is also frequently influenced by Kuroshio intrusion through the Luzon Strait [6–9]. The Kuroshio Current is characterized by warm and saline surface waters and carries the most oligotrophic water in the world’s oceans into the NSCS [7, 10, 11]. By encountering the warm, saline and oligotrophic from the Kuroshio Current and experiencing the influence of the Pearl River diluted water input, the NSCS becomes a highly complicated and dynamic system. Under such complex hydrodynamic conditions, complex N cycles and sources are present; the spatial NO₃^- distribution controlled by the dynamics of the biogeochemistry is still unclear across the NSCS.

The abundance of ¹⁵N and ¹⁸O in nitrate (δ¹⁵N-NO₃^- and δ¹⁸O-NO₃^-) have proven to be useful in identifying N sources in marine ecosystems [2, 12–16]. For example, NO₃^- originating from inorganic fertilizers is characterized by modest δ¹⁵N-NO₃^- values (-4 ~ +4‰) [16]. However, NO₃^- originating from sewage and manure is usually enriched in δ¹⁵N-NO₃^- values (+7 ~ +25‰) due to ammonia volatilization, which may cause enriched δ¹⁵N in residual nitrate [17, 18]. During the N cyclic processes, depleted δ¹⁵N-NO₃^- can result from the preferential biological uptake of light N isotopes (¹⁴N) during nitrification or minimal isotope fractionation during N₂ fixation [19]. Using the difference in isotopes in nitrate between various sources, Ye et al. [2] suggested that municipal sewage and remineralized soil organic N were the major sources of NO₃^- in the PRE. However, N loss processes, such as assimilation and denitrification, can confound the distinction of NO₃^- sources. The preferential uptake of the lighter isotope in NO₃^- during these processes can lead to ¹⁵N enriched in the remaining NO₃^- pool [13, 20]. In addition, the proportion of ¹⁸O in nitrate (δ¹⁸O-NO₃^-) is a powerful tool to distinguish the NO₃^- sourced from atmospheric deposition, which has higher values (>50‰) [21, 22] compared to NO₃^- generated biologically in water and soil (0.8 ~ 5.8‰) [23, 24]. The dual isotope approach can therefore serve as an important tool in many investigations [2, 16, 19, 25] and help deconvolute multiple nitrate sources and biological processes in coastal areas.

In this study, δ¹⁵N-NO₃^-, δ¹⁸O-NO₃^-, NO₃^- and NO₂^- concentrations and other physiochemical parameters were measured in the NSCS during the summer (June 2017). The dual isotope approach enables us to qualitatively characterize the predominant sources of nitrate in coastal areas and understand the controlling factors influencing nitrate distributions.

Materials and methods

Field sampling

A sampling cruise was carried out in the summer (June) of 2017. Twenty-seven sampling stations were visited in five representative transects (A, B, C, D, and E) to investigate the spatial distribution of NO₃^- and its dual isotopic composition within the NSCS; the sampling sites are presented in Fig 1. No specific permissions were required for the sampling area, because the sampling area did not belong to the nature reserve and not involve endangered or protected species.

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Fig 1. Sampling sites in the NSCS during the summer cruise.

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At each site, water samples were collected from various depths (2m, 25m, 50m, 75m, 100m, including surface, middle and bottom water) in the water column in 12 L Niskin bottles with a conductivity-temperature-depth (CTD) metre (SBE911, Seabird). Samples for chlorophyll a (Chl-a) were filtered using glass-fibre filters (Whatman, 0.7 μm, GF/F) and stored at -20 °C before analysis. For nutrients and the dual isotopic NO₃^- analysis, water samples of 500 ml were filtered through pre-combustion (450 °C, 4 h) GF/F membranes (47 mm diameter, Whatman), and the filtrate was placed into an acid-washed polyethylene bottle and stored at -20 °C for home lab analysis. This study did not make any damage to the endangered or protected species. This study was carried out without any ethics problem.

Chemical analyses

The concentration of DO was measured on-site using Winkler titration [26], and the precision was 0.07 mg L^-1. Chl-a was extracted using 90% acetone and analysed spectrophotometrically [27]. For the nutrients, NO₃^- and NO₂^- were determined using a San++ continuous flow analyser (Skalar, Netherlands). The data quality was monitored by intercalibrations, and the detection limits for NO₂^- and NO₃^- were 0.1 μmol L^-1. For the dual isotopes of nitrate, nitrite was removed by sulfamic acid, and then isotopic analyses of δ¹⁵N-NO₃^- and δ¹⁸O-NO₃^- were conducted following a chemical conversion method, which was modified by Mcllvin and Altabet [28]. According to this method, NO₃^- was reduced to NO₂^- with spongy cadmium and further reduced to N₂O with sodium azide in an acetic acid buffer. Finally, N₂O was separated, purified and analysed for N and O isotopes with a GasBench II-MAT 253. δ¹⁵N and δ¹⁸O were calibrated by the international standard IAEA-N3. The analysis deviation for the standard was < 0.2‰ for δ¹⁵N and < 0.5‰ for δ¹⁸O. The reproducibility of the duplicate sample analyses was < 0.3‰ for δ¹⁵N (average±0.1‰) and < 0.6‰ for δ¹⁸O (average±0.3‰).

Data analysis

Nitrate from the physical mixing (N_mix) of marine and Pearl River water end-members was calculated for the coastal areas near the PRE using the conservative mixing model of Liss [19], as fllowing: (1) (2) (3) (4) where q₁ and q₂ represent the fractional contribution of riverine and marine end-members, respectively. The terms S₁ and S₂, N₁ and N₂, δ₁ and δ₂ represent the parameters of salinity, nitrate and the nitrate dual isotope (δ¹⁵N-NO₃^- and δ¹⁸O-NO₃^-) of riverine and marine end-members. The terms S_mix, N_mix and δ_mix represent the salinity, nitrate and isotopes of a sample in a mixture in the two end-members, respectively. According to the above formula: (5) (6)

Under steady-state conditions, nitrate concentration varies linearly along the mixing gradient, whereas the salinity-based isotopes show curvilinear behaviors, which reflect the concentration-weighted volumes of the two end-members. However, deviations of measured nitrate concentrations or δ¹⁵N-NO₃^- and δ¹⁸O-NO₃^- values from the conservative curves may indicate addition of nitrogen from other sources or/and occurrence of nitrogen processing [2]. In this study, the riverine end-member in the upper estuary and the subsurface water from the NSCS were used as the riverine and marine end-member (Table 1), respectively, which were also used by previous studies [2, 19]. Because the subsurface water of the NSCS is less effected than other waters by the addition of variable new nitrogen sources, e.g. nitrogen fixation, phytoplankton assimilation and atmospheric deposition. The common phenomena in the NSCS that wind-induced upper water mixing in winter and off-shore upwelling in summer can provide the subsurface nitrate as the ultimate marine end-member for the coastal surface water [2]. The data of the two end-members (Table 1) was reported in the study of Pearl River Estuary by Ye et al. [2].

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Table 1. Definitions of marine and riverine end-members.

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SIAR mixing model

SIAR (stable isotope analysis in R) is a software package to conduct the Bayesian stable isotope mixing model, which is used to calculate the relative proportion of several nitrate sources. In the mixing model, the Bayesian framework is utilized to calculate the probability distribution proportion of nitrate sources. The model framework as following: (7) (8) (9) (10) where X_ij is the isotope values (j = 2, δ¹⁵N-NO₃^- and δ¹⁸O-NO₃) of the sample i (i = 1, 2, 3, …, N); S_jk is the isotope value j of the source k (k = 1, 2, 3, …, K) and normally distributed with average μ_jk, and standard deviation ω_jk; P_k is the proportion of source k, which is calculated by the SIAR model; c_jk is the fractionation factor for j on source k, and c_jk is normally distributed with average λ_jk and standard deviation τ_jk; ε_jk is the residual error of the additional unquantified variation between individual samples, and normally distributed with average 0 and standard deviation σ_j. A more detailed description of the model can be found in Moore and Semmens [28], Zhang et al. [29] and Xue et al. [30].

Results

Physiochemical parameters

The hydrographic characteristics, including the temperature and salinity, exhibited stratified distributions in the NSCS (Fig 2). The temperature and salinity ranged from 19.28 °C to 30.74°C (average of 25.15 °C) and 29.52 to 34.68 (average of 33.46), respectively. Strong thermal stratification was observed in the sampling areas, with a higher temperature in the upper waters and lower temperature in the bottom waters. In contrast, a higher salinity was observed in the bottom waters, whereas a lower salinity was observed in the upper waters of the coastal areas. High temperature and low salinity were observed in the upper waters near the coastal areas of the PRE (Transect A, B and C). However, high temperature (>28.0 °C) and high salinity (33.6) water-column conditions were observed in the outer areas of Transect B, C, D and E. Concentrations of Chl-a ranged from 0.06 to 3.71 μg L^-1, and higher concentrations were observed in the coastal areas in Transect A and B and the outer areas of Transect C and D.

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Fig 2. Spatial distributions of temperature, salinity and Chl-a in the NSCS.

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Nutrient concentrations

The concentrations of NO₃^- and NO₂^- ranged from 0.06 to 7.36 μmol L^-1 (average of 1.50 μmol L^-1) and ND (no detection) to 0.88 μmol L^-1 (average of 0.13 μmol L^-1), respectively. The distribution patterns of NO₂^- were similar to those of NO₃^-. As shown in Fig 3, they all exhibited higher levels in the upper water of the nearshore areas in Transect A, B and C. Combined with the distributions of salinity and temperature, these abundant nutrients are probably influenced by riverine input. However, it might not be transported towards the east since low NO₂^- and NO₃^-concentrations were observed in Transect D and E. In addition, low concentrations of NO₂^- and NO₃^- were observed in the outer areas and mid-water column. Combined with high temperature and high salinity in the outer areas, this may indicate the influence of the Kuroshio intrusion.

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Fig 3. Spatial distributions of NO3- and NO2- in the NSCS.

This is the Fig 3 legend.

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Isotopic compositions of nitrate

The isotopic compositions of nitrate, namely δ¹⁵N-NO₃^- and δ¹⁸O-NO₃^-, ranged in value from 1.49‰ to 11.11‰ and 9.96‰ to 27.86‰, respectively. Similar to the distributions of nutrients, the higher δ¹⁵N-NO₃^- values were observed in the upper waters near the coastal areas of Transect A, B and C (Fig 4). However, lower values of δ¹⁵N-NO₃^- were found in the water column of the outer areas in Transect C, D and E. Extremely high values of δ¹⁸O-NO₃^- were observed in the upper and mid-water column, particularly in the outer areas. The observed high δ¹⁸O-NO₃^- values (ranged from 9.96‰ to 27.86‰, average of 20.27) in our study areas are at the high end of the range of the δ¹⁸O-NO₃^- values to be reported in marine environments (-5.0 ~ 33.9‰) [16, 17, 19, 31, 32, 33, 34], and the results are similar to those reported in the lower estuary of the Pearl River (0.7~25.6‰) [2].

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Fig 4. Spatial distributions of δ¹⁵N-NO3- and δ¹⁸O-NO3- values in the NSCS.

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Discussion

Sources of nitrate in the upper waters of the coastal areas near the PRE

As shown in Fig 3, a higher temperature and lower salinity in upper waters near coastal areas in Transect A, B and C (stations S46, S48, S56, S57, S59, S61, S63, S77, S75, S73, and S71) were observed and accompanied by higher NO₃^- concentrations compared to those at other stations. This suggests that the coastal areas near the PRE might be influenced by the Pearl River diluted water and/or the diluted water from the cities adjacent to our study area. This would also be supported by the linear correlation between NO₃^- concentration and salinity (Fig 5). However, the contribution of the Pearl River diluted water can be ruled out, since the Pearl River diluted water near the mouth flows along the west shore due to the Coriolis effect [19]. Because the low temperature and high salinity in the water column were observed in the middle and bottom waters, respectively, the strong thermocline and halocline layer prevented the vertical mixing of water. Thus, the diluted water from the cities adjacent to our study area only influenced the upper waters near the coastal areas in Transect A, B and C.

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Fig 5. Linear relationship between NO3- concentration, salinity, δ¹⁵N-NO3- and the natural logarithm of NO3- concentrations in the upper waters near the coastal areas in Transect A, B and C.

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As shown in Figs 3 and 4, the upper waters of the coastal areas near the PRE are characterized by heavier δ¹⁵N-NO₃^- and δ¹⁸O-NO₃^- values (δ¹⁵N-NO₃^- of 6.78–11.11‰ and δ¹⁸O-NO₃^- of 14.03–19.42‰) and higher NO₃^- concentrations, which may be associated with biogeochemical processes (such as assimilation and denitrification) and mixing from the upper estuary. However, denitrification are not thought to be responsible for the heavier dual isotopes of nitrate values because high DO level (ranged from 5.01 to 7.07 mg L^-1, average of 6.09 mg L^-1) was found. If assimilation was the dominant factor for the heavier δ¹⁵N-NO₃^- and δ¹⁸O-NO₃^-, NO₃^- would be consumed during the process, leading to low NO₃^- concentrations. Our data are not consistent with this expected pattern. Furthermore, phytoplankton assimilation would be constrained by high turbidity near the coastal areas; a low Chl-a level (0.10–0.34 μg L^-1) was observed in the coastal areas near the PRE (Fig 2). Thus, higher NO₃^- concentrations and δ¹⁵N-NO₃^- values would be associated with the diluted water from the cities adjacent to our study area.

From the calculated results of the mixing model, the diluted water from the cities adjacent to our study area in the coastal areas near the PRE comprised 12% of the total, and the subsurface water of the NSCS was 88%. In the diluted water from the cities adjacent to our study area, NO₃^- originated from synthetic NO₃^- fertilizers and atmospheric deposition, and the nitrification of NH₄⁺ from manure and sewage, reduced N fertilizer, soil organic nitrogen [14, 35]. However, the contribution of synthetic NO₃^- fertilizers can be ruled out, since it accounts for less than 2% of the nitrogen fertilizer applied in China [14]. The slightly higher δ¹⁸O-NO₃^- values may be influenced by atmospheric deposition and synthetic NO₃^- fertilizers, which have heavier isotopic values (more than 50‰ for atmospheric deposition and +17–+25‰ for synthetic fertilizer) [36, 37]. Since synthetic NO₃^- fertilizers have been ruled out above, atmospheric deposition may be responsible for the slightly higher δ¹⁸O-NO₃^- values. Previous studies also suggested its importance in providing nitrate for new production in the PRE and the SCS and the higher contribution that was found in the outer-most station [2, 38]. To quantify the contribution of the sources to nitrate in the coastal areas, the Bayesian isotopic mixing model was used in this study. Our results suggest that the fraction of atmospheric deposition (AD) in the coastal areas near the PRE ranged from 16% to 23% (average of 19%), sources from manure and sewage (M&S) ranged from 50% to 76% (average of 63%), soil organic nitrogen (SON) from 0% to 25% (average of 12%) and reduced N fertilizer (RNF) from 0% to 15% (average of 6%) (Fig 6). This result was different from the Pearl River diluted water that the nitrification of reduced N fertilizer was a significant nitrate source, due to most of the land in the Pearl River Basin being used for agriculture [2, 14]. In our study, the cities adjacent to our study area include Hongkong, Shenzhen, Huizhou, which are the most rapid developing cities in China. This would lead that the predominant NO₃^- source in the diluted water from the adjacent cities was manure and sewage. We realize that the uncertainty is large and hard to constrain with the present data. However, such an estimate can at least provide an insight of nitrate sources in the diluted water from the adjacent cities during wet seasons.

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Fig 6. Values of δ¹⁵N-NO3- and δ¹⁸O-NO3- in various nitrate sources reservoirs (boxes) [39] and in upper water near the PRE (black dots); the results of the proportion of potential nitrate sources (atmospheric deposition (AD), manure and sewage (M&S), soil organic nitrogen (SON) and reduced N fertilizer (RNF) calculated by Bayesian isotopic mixing model.

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Impact of the Kuroshio intrusion on nitrate in the upper and mid-waters in the outer areas

Except for Transect A, observations from the vertical distributions of the hydrographic characteristics (Fig 2) indicate a high temperature and high salinity in the upper and mid-waters of the outer areas, and the high-salinity water extended towards to the bottom water in the coastal areas, where the salinities were higher than 33.8, even near the coastal areas. These water column conditions are believed to originate from the Kuroshio Current intruding through the Luzon Strait. Similar intrusion patterns have been reported previously [6, 7, 9]. Thus, this phenomenon may introduce low-nutrient waters into the NSCS, during the later intrusion. Our results show significantly low concentrations of NO₃^- and NO₂^- in the water column of the outer areas. Those low-nutrient waters extended to the mid-waters of the coastal areas (Fig 3), which was consistent with the high-salinity distribution patterns. Previous studies also suggested that the Kuroshio intrusion significantly influenced the nutrient distribution in the NSCS and that the nutrient inventory was overall negatively correlated with the fraction of Kuroshio water [7]. However, the distribution patterns of NO₃^- concentration in Transect E were different from those in other transects, which exhibited a higher NO₃^- level along the transects. This nutrient increase may have originated from the Taiwan Strait, which might be less influenced by the Kuroshio Current and exhibit higher NO₃^- concentrations [40].

Interestingly, the higher Chl-a level and lower δ¹⁵N-NO₃^- values were found in the water column of outer areas in Transect C, D and E (Figs 2 and 3). As mentioned above, oligotrophic water from the Kuroshio intrusion may dilute the nitrate inventory in the water column of the SCS, potentially reducing new production and promoting nitrogen recycling [7, 9]. Nitrification and nitrogen fixation may be responsible for the lower δ¹⁵N-NO₃^- values in the water column of outer areas. Although the Kuroshio water was characterized by low inorganic nutrients, the dissolved organic nitrogen (DON), dissolved organic carbon (DOC) and total organic carbon (TOC) inventories in the upper 200 m were significantly higher than those in the NSCS [7, 9]. Thus, the high dissolved organic matter (DOM) brought by Kuroshio waters may be bioavailable for microbes, which may activate remineralization and ammonification of DOM [9]. Xu et al., [9] used the ¹⁵N-NH₄⁺ to investigated ammonia oxidation (AO) in the NSCS, also indicating that the Kuroshio intrusion enhances NH₄⁺ regeneration and subsequent oxidation, to complicate conventional new production. Therefore, nitrification would be an important biogeochemical process in outer areas. In addition to nitrification, nitrogen fixation may be another key process in outer areas, as a high abundance of Trichodesmium and high N₂ fixation rates were observed in the path of the Kuroshio Current [41–43]. The high abundance of Trichodesmium would stimulate the N fixation in the water column of NSCS, which would increase phytoplankton productivity. The enhancement of nitrogen fixation in the Kuroshio intrusion route would also result in the increase of organic matter mineralization and be further nitrified for the formation of nitrate. In addition, the isotopic feature of δ¹⁵N-NO₃^- in the Kuroshio Current is approximately 5.6‰ [44, 45], and the value is consistent with the average of the oceanic water over the global ocean (5.0‰) [46]. However, our results from these outer stations (average of 4.9‰) were significantly lower than those of the Kuroshio Current and the average of oceanic water. This further demonstrated that nitrogen fixation and nitrification occurred in the water column of outer areas, which would lead to a lighter isotopic value of nitrate.

However, the extremely high δ¹⁸O-NO₃^- values were observed in the water column of outer areas, and the middle waters extended into the coastal areas, indicating that it may also be influenced by atmospheric deposition because both nitrification and nitrogen fixation cannot cause the increase in δ¹⁸O-NO₃^- values. In addition, the mixing with other water masses from the Kuroshio Current and NSCS is not significant enough to lead to δ¹⁸O-NO₃^- values greater than 10‰ [2, 19]. Since the sampling period was during the rainy season in the NSCS, this would bring high δ¹⁸O-NO₃^- via atmospheric deposition into the oceanic water. To quantify the contribution of atmospheric nitrate deposition to the water column of the outer NSCS, a simple isotope mass balance based on δ¹⁸O-NO₃^- was utilized. According to the discussion above, the nitrate sources in the outer NSCS were mainly from nitrogen fixation, nitrification (including the remineralization of organic matter formed by nitrogen fixation), Kuroshio intrusion and atmospheric deposition. The δ¹⁸O-NO₃^- values of nitrogen fixation, which were further mineralized and nitrified, were calculated to be approximately 7.8‰ [14], and the δ¹⁸O-NO₃^- values of the Kuroshio Current water were close to the average oceanic water (7.8‰) [41]. Thus, the isotopic feature of nitrogen fixation and nitrification are consistent with the Kuroshio waters, and the input of atmospheric deposition changes the values. To calculate the contribution of atmospheric deposition, we use the following formula: (11) where C represents the δ¹⁸O-NO₃^- measured value, A represents the δ¹⁸O-NO₃^- value for wet deposition (58.8‰)[22], B represents the δ¹⁸O-NO₃^- value (including nitrification, nitrogen fixation and the Kuroshio Current (7.8‰)), and X represents the contribution rate of wet deposition. Our estimates suggest that the fraction of atmospherically derived nitrate in the outer NSCS ranged from 28%–39% (average of 32%). The nitrate source from atmospheric deposition in the outer areas was higher than that the coastal areas near the PRE (19%) and the PRE (17%) [2], suggesting that the important nitrate sources from atmospheric deposition in the NSCS outside the PRE areas. The results indicated that the contribution of nitrate from nitrification, nitrogen fixation and the Kuroshio Current water was 68%. According to the difference in isotope fingerprint characteristics, we can use δ¹⁵N-NO₃^- to further calculate the contribution of Kuroshio intrusion, nitrification and nitrogen fixation. The δ¹⁵N-NO₃^- values of nitrification and nitrogen fixation are close to 0‰, and the value of the Kuroshio Current is approximately 5.6‰ [44, 45]. We use the sample model as follow: (12) where Y represents the δ¹⁵N-NO₃^- measured value, X represents the δ¹⁵N-NO₃^- value for nitrification and nitrogen fixation (0‰), Z represents the δ¹⁵N-NO₃^- value of the Kuroshio Current (5.6‰), and a represents the contribution rate of nitrification and nitrogen fixation. The results indicated that the contribution of nitrate sources from nitrification and nitrogen fixation ranged from 0% to 38% (average of 8%) and that from the Kuroshio Current water ranged from 33% to 72% (average of 60%). The proportion of nitrate sources in the outer area was summarized in Table 2. Among the three influencing factors of nitrate concentration, the Kuroshio intrusion was the dominant factor that controlled the nitrate concentration in the outer areas of NSCS. This phenomenon may be responsible for the lower nutrients in the areas.

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Table 2. The proportion of nitrate sources in the outer area.

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Complex processes for high nitrate and isotopic values in bottom waters

In bottom waters, particularly in coastal areas near the PRE (Transect A and B), higher nutrients and δ¹⁵N-NO₃^- values were also observed, while the δ¹⁸O-NO₃^- values exhibited lower values compared with the upper waters (Fig 4). Due to the strong thermocline and halocline during the summer, nitrification may be responsible for the higher concentrations of NO₃^- and NO₂^- in bottom waters. However, the higher isotopic values of nitrate observed in the bottom waters indicated that other processes may have also occurred, such as assimilation and denitrification. Denitrification or nitrate assimilation by phytoplankton leads to an increase in δ¹⁵N-NO₃^- and δ¹⁸O-NO₃^- values because lighter isotopes ¹⁴N and ¹⁸O are preferentially metabolized by microorganisms or phytoplankton. This makes the remaining nitrate pools simultaneously enriched in δ¹⁵N and δ¹⁸O at a ratio of 1:1, while nitrification leads to the decoupling of δ¹⁵N-NO₃^- and δ¹⁸O-NO₃^- values, as a decrease in δ¹⁸O-NO₃^- is smaller than that in δ¹⁵N-NO₃^- [17, 34]. However, denitrification seems unlikely to have occurred due to a relatively high DO level (4.25 ~ 7.03 mg L^-1, average of 5.41 mg L^-1) observed in the bottom waters. Thus, phytoplankton assimilation primarily contributed to the enrichment of δ¹⁵N-NO₃^- and δ¹⁸O-NO₃^- values in the bottom waters of the NSCS. As shown in Fig 7, except for Transect E, most of the δ¹⁵N-NO₃^- and δ¹⁸O-NO₃^- values in the bottom waters fell within the theoretical value (1:1). In addition, an extremely high level of Chl-a was found in the middle and bottom waters of the coastal areas near the PRE (Transect A and B, Fig 2), which further confirmed that phytoplankton assimilation occurred in these areas. However, the δ¹⁵N-NO₃^- and δ¹⁸O-NO₃^- values slightly deviated from the assimilation line, which could be influenced by nitrification and other sources. For example, significantly higher δ¹⁸O-NO₃^- values occurred in Transect E, which may be influenced by atmospheric deposition. Atmospheric deposition would be first deposited in the upper waters of the ocean and then mixed in the water column and transported with ocean currents. In addition, the NH₄⁺ concentrations (ranged from 0.46 to 3.42 μmol L^-1, average of 1.18 μmol L^-1) in the bottom water was significantly lower than NO₃^- concentration (ranged from 0.08 to 4.42 μmol L^-1, average of 1.94 μmol L^-1), this further indicated that nitrification indeed occurred at the bottom water.

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Fig 7. The range of δ¹⁵N-NO3- and δ¹⁸O-NO3- values measured in the bottom waters of the NSCS.

https://doi.org/10.1371/journal.pone.0209287.g007

Conclusions

Nitrate dual stable isotopes in the water column of the NSCS were investigated to provide information about nitrogen sources and cycles. A higher NO₃^- concentration and δ¹⁵N-NO₃^- value were observed in the upper waters of the coastal areas near the PRE, which were mainly influenced by manure and sewage (63%), atmospheric deposition (19%), soil organic nitrogen (12%) and reduced N fertilizer (6%). For the upper waters of the outer areas, low NO₃^- concentrations and δ¹⁵N-NO₃^- values but high δ¹⁸O-NO₃^- values were observed, which were mainly influenced by Kuroshio intrusion (61%), atmospheric deposition (31%) and nitrogen fixation/nitrification (8%). The results indicated that the important nitrate source from atmospheric deposition in the outer areas. Complex processes were found in the bottom waters, and the nitrification and phytoplankton assimilation may be responsible for the higher nitrate and δ¹⁵N-NO₃^- values. Our study, therefore, manifests the combination of isotopic and nitrate data to help illustrate the spatial variation in nitrate sources and the complex nitrogen cycles in the NSCS.

Acknowledgments

The authors would like to thank Xijie Yin for the technical support from the Third Institute of Oceanography at the State Oceanic Administration.

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Figures

Abstract

Introduction

Materials and methods

Field sampling

Chemical analyses

Data analysis

SIAR mixing model

Results

Physiochemical parameters

Nutrient concentrations

Isotopic compositions of nitrate

Discussion

Sources of nitrate in the upper waters of the coastal areas near the PRE

Impact of the Kuroshio intrusion on nitrate in the upper and mid-waters in the outer areas

Complex processes for high nitrate and isotopic values in bottom waters

Conclusions

Acknowledgments

References