Sedimentary nitrogen dynamics in a coastal reef area with relatively high nitrogen concentration

1. Introduction

Coral reefs have the most abundant marine biological resources, and thus, the serious degradation of coral reefs around the world has attracted extensive attention (Eakin et al., 2019; Hughes et al., 2003). Although coral thermal bleaching caused by climate warming is considered to be the main factor of coral reef degradation worldwide (Hughes et al., 2017), coral bleaching caused by excess nitrogen (N) generated and discharged by anthropogenic activities in coral reefs at relatively high latitudes should not be ignored (Morris et al., 2019; Wiedenmann et al., 2013; Rosset et al., 2017).

Due to the influence of anthropogenic activities, the nutrient structure in coastal waters of China often has an excess of N (Guo et al., 2019; Ning et al., 2020; Xie et al., 2021), whereas the nutrient exchanges between the seawater and sediment interface play an important role in regulating the N concentration and nutrient structure in shallow reefs (Ning et al., 2019; Rasheed et al., 2003; Eyre et al., 2008). Carbonate sands are the dominant sediments in reef environments, and their porous structure leads to greater porosity and permeability and a microbial abundance of carbonate sand compared to silicate sand (Wild et al., 2005; Rasheed et al., 2003). Thus, the N dynamics of these two sedimentary types are supposed to be different (Kessler et al., 2014; Cook et al., 2017). However, studies on N dynamics in carbonate sands are very rare, and the regulation mechanisms of the sedimentary N dynamics in coral reefs are unclear (Erler et al., 2014; Robertson et al., 2019).

Weizhou Island reef is located in a relatively high latitude area in the northern South China Sea (Fig. 1) and is often threatened by eutrophication attributed to anthropogenic activities (Yu et al., 2019). Anthropogenic activities have altered the global N cycle (Yang and Gruber, 2016; Wang et al., 2019), but their perturbation of N dynamics in reef sediments is not clear, so the N cycle in coral reefs could not be clearly understood. In this study, rising temperatures indicate climate warming, and enrichments of ${\rm{NO}}_3^- $ in seawater and total organic carbon (TOC) in sediments are the alternative indicators of anthropogenic activities. We hypothesize that the sedimentary N flux in coral reefs would be stimulated by climate warming and anthropogenic activities. In order to test this hypothesis, we conducted repacked flow-through reactor experiments to investigate the fluxes of different N forms at the interface of sediment and seawater at Weizhou Island, and analyzed their regulation mechanism by environmental factors, including temperature, ${\rm{NO}}_3^- $ concentration, the content of TOC in sediments, porewater advection rates, and flow path lengths. With these comprehensive data, we were able to unveil the N dynamics in reef sediments under the influences of climate warming and anthropogenic activities.

Figure  1.  Sampling stations in the tidal flats (the black areas) around Weizhou Island. The dark grey areas are coral covering areas. I1−I5 are the station IDs.

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2. Materials and methods

2.1 Study site and sampling

Weizhou Island is a volcanic island located in the Beibu Gulf, and its relatively high latitude provides an ideal habitat for coral growth. Carbonate sands are the dominant sediments in reef environments, and coupled with the input of terrigenous weathering products, admixed terrigenous silicate and reef-derived carbonate components can be found in the tidal flats of Weizhou Island. During September 2019 at stations in the tidal flats (Fig. 1), seawater samples were collected by submersing a 10 L polyethylene bucket, and sediments (0–10 cm) were collected by pushing Plexiglas tubes (inner diameter of 10 cm) into the sediment. At the same time, 100 mL of surface seawater samples for nutrient analysis were filtered with 0.45 μm pore-size syringe polyethersulfone filters, and Rhizon soil moisture samplers (19.21.23F Rhizon CSS) were pushed into the sediment in the field to collect and filter porewater samples. The blanks of ${\rm{NH}}_4^+ $, ${\rm{NO}}_2^- $, and ${\rm{NO}}_3^- $ were 0.10 μmol/L, <0.01 μmol/L, and 0.01 μmol/L for syringe filters and 0.08 μmol/L, 0.02 μmol/L, and 0.03 μmol/L for Rhizon samplers, respectively. Both surface seawater and porewater samples were frozen at −20℃ until later analysis of nutrients. In addition, the sediment samples for the analysis of N content were placed in Ziploc plastic bags and then frozen at −20℃.

2.2 Flow-through reactor experiments

The flow-through reactor (FTR) experiment was implemented according to Ning et al. (2020) using Plexiglas columns (with an inner diameter of 4 cm) and lids designed by Rao et al. (2007), in which radial grooves were milled around the inflow and outflow ports. Briefly, homogenized sediment was packed into each Plexiglas column, and seawater was pumped from the bottom up through the sediments from a large carboy with continuous air flushing. During incubations, the sediment columns were immersed in a tank filled with seawater (an external temperature controller was used to control the ambient temperature). Flux measurements began 6 h after the start of the column percolation to acclimatize under the laboratory conditions. The measurements were conducted twice, with the intervals of 1 h, and the duration of measurements was within 2 h. At each sampling time, seawater samples for nutrient analysis were collected directly from the outflow, and the inflow seawater samples were collected from the source using 60 mL syringes. Additionally, an aliquot of seawater for N₂ measurement was sampled and transferred to a 12 mL Exetainer vial (Labco Ltd.), and the dissolved oxygen (DO) of inflow and outflow seawater was measured using a multiparameter probe (YSI ProDSS). The FTR experiments were carried out in duplicate.

Five experiments were performed covering the conditions summarized in Table 1. In the first experiment, sediments and seawater collected from each station were incubated to investigate the sedimentary N dynamics. In the second experiment, all the sediments collected from different stations were mixed and repacked in FTRs, and different ${{\rm {NO}}_3^-} $ doses were added into the inflow seawater with the final ${{\rm {NO}}_3^-} $ concentrations of 1 μmol/L, 4 μmol/L, 10 μmol/L, 30 μmol/L, and 45 μmol/L, respectively. In the third experiment, sediments were mixed with different proportions of freeze-dried phytoplankton to obtain gradient TOC content of 0.05%, 0.14%, and 0.22%, and other variables were kept constant. The porewater flow rate in the former three experiments was maintained at 1 mL/min (equivalent to 48 L/(m²·h)), with a residence time of 1 h. In the fourth experiment, mixed sediments were used as in the second experiment, and the seawater was pumped through the FTRs at variable flow rates (0.5 mL/min, 1 mL/min, 2 mL/min, and 3 mL/min); meanwhile, four column lengths (5 cm, 10 cm, 15 cm, and 20 cm) were used to assess how flow path lengths influence nutrient fluxes. In the fifth experiment, sediments were incubated at a temperature of 20℃, 26℃, and 32℃, respectively.

Table  1.  Summary of conditions in flow-through reactor experiment

Experiment	Temperature/℃	${\rm{NO}}_3^- $/(μmol·L–1)	TOC content	Advection rate/(mL·min–1)	Column length/cm
1− Station	26	see Table 2	0.03%±0.01%	1	10
2− ${\rm{NO} }_3^- $ concentration	26	1, 4, 10, 30, 45	0.05%	1	10
3− TOC concentration	26	25±5	0.05%, 0.14%, 0.22%	1	10
4− Advection rate and flow path length	26	45±5	0.05%	0.5, 1, 2, 3	5, 10, 15, 20
5− Temperature	20, 26, 32	10±5	0.05%	1	10
Note: The variable parameters are indicated in bold.

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2.3 Analytical methods

Each frozen sediment sample was freeze-dried, and the total N (TN) content in sediment was determined using a CHNOS Elemental Analyzer (Vario EL III, Elemental Analyzer). The precision for TN had a <6% coefficient of variation (CV). Nutrient concentrations in seawater were determined using an autoanalyzer (QuAAtro, SEAL Analytical). The measurement precisions for the ${\rm{NO}}_3^- $, ${\rm{NO}}_2^- $, ${\rm{NH}}_4^+ $, dissolved inorganic phosphorus (DIP), and total dissolved nitrogen (TDN) analyses had a <6% CV. The dissolved organic nitrogen (DON) concentration was obtained from the difference between the TDN concentration and the dissolved inorganic nitrogen (DIN includes ${\rm{NO}}_3^- $, ${\rm{NO}}_2^- $ and ${\rm{NH}}_4^+ $) concentration. The dissolved N₂ was measured by membrane inlet mass spectrometry.

2.4 Calculations and statistical analyses

Fluxes (F, mmol/(m²·h)) were calculated from the differences in the concentrations of ${\rm{NO}}_3^- $, ${\rm{NO}}_2^- $, ${\rm{NH}}_4^+ $, DIP, DO, and N₂ between the influent and effluent seawater, flow rate, and cross-sectional area of the sediment column according to Eq. (1). Positive values represent an efflux out of the sediment, whereas negative values represent an influx into the sediment.

where, C_in and C_out are the concentrations of ${\rm{NO}}_3^- $, ${\rm{NO}}_2^- $, ${\rm{NH}}_4^+ $, DIP, DO, and N₂ in the influent and effluent seawater, respectively. R is the flow rate, and S is the cross-sectional area of the sediment column (12.56 cm²).

Pearson’s correlation analysis with a two-tailed test of significance was used to evaluate the relationship between the parameters. Statistical analyses were carried out using the Statistical Package for the Social Sciences (SPSS) software (version 22.0), and the statistical significance was judged at the criterion of p<0.05.

3. Results

3.1 N and P contents in the surface seawater and sediment around the Weizhou Island during September 2019

The average concentrations of ${\rm{NH}}_4^+ $, ${\rm{NO}}_2^- $, and ${\rm{NO}}_3^- $ in the surface seawater of Weizhou Island were (1.19±0.52) μmol/L, (0.35±0.06) μmol/L, and (6.01±3.56) μmol/L, respectively (Table 2). ${\rm{NO}}_3^- $ dominated the DIN, and the concentration of DIN was 27−399 times that of DIP ((0.15±0.16) μmol/L). The nutrient concentrations in porewater were several times or even dozens of times higher than those in surface seawater, but the concentrations were significantly different among different stations (Table 2). The proportion of ${\rm{NH}}_4^+ $ in DIN in porewater was significantly higher than that in surface seawater, especially at Stations I3 and I5, in which ${\rm{NH}}_4^+ $ absolutely dominated the DIN. The DIP concentration in porewater ranged from 0.46 μmol/L to 0.69 μmol/L, and the ratio of DIN:DIP was 24−418. The TN content in the sediments of Weizhou Island was as low as 1.25−4.28 μmol/g.

Table  2.  Nutrient concentrations in overlying seawater and porewater in sediments, and total nitrogen (TN) content in bulk sediments

Station	Seawater					Porewater					Sediment
	${{\rm {NH}}_4^+} $/ (μmol·L−1)	${{\rm {NO}}_2^-} $/ (μmol·L−1)	${\rm{NO}}_3^- $/ (μmol·L−1)	DIP/ (μmol·L−1)		${\rm{NH}}_4^+ $/ (μmol·L−1)	${\rm{NO}}_2^- $/ (μmol·L−1)	${\rm{NO}}_3^- $/ (μmol·L−1)	DIP/ (μmol·L−1)		TN (dry weight)/ (μmol·g−1)
I1	0.70	0.33	4.36	0.02		6.72	3.16	6.76	0.46		2.90
I2	0.81	0.33	9.95	0.08		8.64	2.54	214.03	0.54		3.34
I3	1.14	0.30	1.44	0.08		113.28	0.81	0.43	0.54		4.28
I4	1.27	0.32	5.04	0.15		14.75	3.94	48.80	0.50		1.25
I5	2.02	0.46	9.28	0.43		156.03	1.29	0.56	0.69		2.47

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3.2 The first FTR experiment: clear release of N₂ of sediment of all sites

The ${\rm{NH}}_4^+ $ was released from the sediment into the seawater at rate of 0.01–0.04 mmol/(m²·h), but was transferred from the seawater into the sediment at Station I5; ${\rm{NO}}_2^- $ and ${\rm{NO}}_3^- $ were transferred from the seawater into the sediments at the rate of 0.01–0.02 mmol/(m²·h) and 0.01–0.25 mmol/(m²·h), respectively (Fig. 2). Meanwhile, the N₂ flux (0.11–0.25 mmol/(m²·h)) is equivalent to or even greater than the DIN efflux (Fig. 2).

Figure  2.  Fluxes of different N forms at the seawater-sediment interface of different stations at Weizhou Island during September 2019.

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There were significant differences in the N flux among stations at Weizhou Island, not only in the order of magnitude but also in the flux direction (Fig. 2). For example, the N flux at Station I5 was differed significantly from that at other stations, and the influx of ${\rm{NH}}_4^+ $ and the efflux of ${\rm{NO}}_3^- $ were observed at this station, reflecting the strong nitrification occurring in the sediments of this station. The N₂ flux was positively correlated with the DIN flux, but the N₂ flux was negatively correlated with the ${\rm{NO}}_3^- $ concentration in the seawater (Table 3). Although complicated, DON flux (−0.18–0.39 mmol/(m²·h)) was related to the dissolution and adsorption of particulate organic N, exhibiting significant spatial differences in magnitude and direction.

Table  3.  Correlation analysis results for ${\rm{NO}}_3^- $ and DIP concentrations in seawater and the TN in sediments and fluxes of DO, N₂, DIN, and DON obtained from the first FTR experiment

	${\rm{NO}}_3^- $ concentration	DIP concentration	TN concentration	DO flux	N2 flux	DIN flux
DIP concentration	0.23
TN concentration	0.22	−0.02
DO flux	−0.05	0.13	−0.45*
N2 flux	−0.72*	0.01	0.05	−0.04
DIN flux	−0.65*	0.34	−0.06	0.18	0.47*
DON flux	0.34	0.92*	0.00	0.17	−0.20	0.34
Note: * correlation is significant at the 0.05 level, n=20.

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3.3 The second FTR experiment: the increasing ${{{\boldsymbol{NO}}_3^-}}$ concentration corresponded to the increasing ${{{\boldsymbol{NO}}_3^-}}$ influx into the sediment

The second FTR experiment with different ${\rm{NO}}_3^- $ concentrations of 1 μmol/L, 4 μmol/L, 10 μmol/L, 30 μmol/L, and 45 μmol/L was implemented to access the effect of the increasing ${\rm{NO}}_3^- $ on the N flux at the seawater–sediment interface of Weizhou Island. When the concentration of ${\rm{NO}}_3^- $ in seawater was lower than (2.4±4.3) μmol/L, ${\rm{NO}}_3^- $ was released from the sediment to the seawater at a rate of >0.19 mmol/(m²·h), but when the concentration was higher than this threshold, ${\rm{NO}}_3^- $ was transferred from the seawater to the sediment, and the influx of ${\rm{NO}}_3^- $ increased with the increasing concentration of ${\rm{NO}}_3^- $ (Fig. 3).

Figure  3.  Relationship between ${\rm{NO}}_3^- $ flux and ${\rm{NO}}_3^- $ concentration in the influent seawater of flow-through reactor. Dashed lines represent 95% confidence intervals.

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3.4 The third FTR experiment: the increasing TOC corresponded to the increase of ${{{\boldsymbol{NH}}}}_4^+$ and DON output, and ${\boldsymbol{NO}}_3^-$ input from the sediment to the seawater

The release rates of ${\rm{NH}}_4^+ $ and DON from the sediment to the seawater gradually increased with the increase of TOC content, and the consumption rate of ${\rm{NO}}_3^- $ in sediment also increased at the same time (Fig. 4). However, the ${\rm{NH}}_4^+ $ flux (<0.008 mmol/(m²·h)) was negligible in comparison with ${\rm{NO}}_3^- $ flux (−0.88 mmol/(m²·h) to −0.70 mmol/(m²·h)) and DON flux (0.02–1.04 mmol/(m²·h)).

Figure  4.  Fluxes of DON (a), ${\rm{NO}}_3^- $ (b), and ${\rm{NH}}_4^+ $(c) under variable TOC contents.

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3.5 The fourth FTR experiment: a selective increase of ${{{\boldsymbol{NO}}_3^-}} $ and DON fluxes at the intermediate advection rate and flow length

The peak of ${\rm{NO}}_3^- $ influx was observed at the intermediate advection rate (96 L/(m²·h)), and ${\rm{NO}}_3^- $ also peaked at the intermediate flow path length (10 cm) under intermediate advection (Fig. 5a). Meanwhile, because the TOC contents in sediments were low, the ${\rm{NH}}_4^+ $ released by mineralization was insignificant under variable advection rates and flow path lengths (Fig. 5b). Similar to ${\rm{NO}}_3^- $ flux, DON efflux peaked at the intermediate advection rate (96 L/(m²·h)) and flow path length (10 cm; Fig. 5c).

Figure  5.  Fluxes (isoline, mmol/(m²·h)) of ${\rm{NO}}_3^- $ (a), ${\rm{NH}}_4^+ $ (b), and DON (c) under different advection conditions.

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3.6 The fifth FTR experiment: temperature significantly affected sedimentary N dynamics at Weizhou Island

The flux of various forms of N increased with the increase of temperature from 20℃ to 32℃ (Fig. 6), indicating that the increase of temperature promoted the activity of the bacteria involved in N cycling in sediments. However, the DON flux decreased significantly to (0.13±0.14) mmol/(m²·h) at 26℃ (Fig. 6a), indicating that there was a process consuming DON, such as the degradation of DON to ${\rm{NH}}_4^+ $. Indeed, the highest ${\rm{NH}}_4^+ $ efflux was observed at the same temperature ((0.07±0.02) mmol/(m²·h); Fig. 6b). When the temperature rose to 32℃, only DON released by the dissolution of refractory organic matter was stimulated, probably due to the low proportion of degradable organic matter in total organic matter content. Therefore, the DON flux significantly increased to (1.23±0.02) mmol/(m²·h), but the ${\rm{NH}}_4^+ $ flux did not increase significantly (Fig. 6).

Figure  6.  Fluxes of DON (a), ${\rm{NH}}_4^+ $ (b), and ${\rm{NO}}_3^- $ (c) under variable temperature conditions.

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At the temperature of 20℃, ${\rm{NO}}_3^- $ was released from sediment to seawater at a flux of (0.05±0.22) mmol/(m²·h), but when the temperature increased to 26℃, ${\rm{NO}}_3^- $ was transferred from seawater to sediment ((0.25±0.12) mmol/(m²·h)), and the magnitude of its influx increased with the increase of temperature (Fig. 6c).

4. Discussion

4.1 High potential rates of sedimentary N transformation at Weizhou Island during September 2019

Clear release of N₂ from sediments was observed at all sites (Fig. 2), and the sedimentary N removal pathway would deplete the ${\rm{NO}}_3^- $ in water column within a couple of days. Indeed, it seems the sedimentary N flux measured here were quite high in comparison with those in other coral reefs measured by benthic chamber or intact core incubation (Eyre et al., 2008). Previous studies have assessed the limitation of FTRs, because fixed porewater advection rate, flow paths or compositions of inflow seawater are far from the natural conditions (Santos et al., 2012; Ning et al., 2019). Thus, these fluxes measured by FTRs may represent potential rates for the Weizhou Island sediments. Furthermore, benthic categories of coral reefs include corals, turf algae, biogenic rock, etc., besides sediments, and N₂ flux varied significantly at different categories (El-Khaled et al., 2021). Thus, we should avoid direct extrapolations from the laboratory results to field conditions.

The potential rate of ${\rm{NH}}_4^+ $ relese from the Weizhou Island sediments (0.01–0.04 mmol/(m²·h), Fig. 2) was much lower than that of the Great Barrier Reef (about 0.5 mmol/(m²·h); Santos et al., 2012). One explanation is because the TN content in the sediments of Weizhou Island (1.25−4.28 μmol/g) was lower than that in the sediments of the Great Barrier Reef (19−44 μmol/g; Alongi et al., 2008). Indeed, DO flux (an indicator of mineralization rate) was closely related to the TN content in the sediments (Table 3). An alternative explanation is that most of the ${\rm{NH}}_4^+ $ released by mineralization of organic matter was adsorbed within porous carbonate sand grains or was consumed by nitrifying bacteria (Jäntti et al., 2011).

The N₂ flux here are supposed to be the potential rate of denitrification, because the N₂ fixation (N₂ consumption) and anammox (N₂ production) in the Weizhou Island sediments was negligible (Ning et al., 2022). In general, coupled nitrification-denitrification is active in permeable sand sediments (Marchant et al., 2016), which explains why N₂ flux was positively correlated with DIN flux (Table 3). The sufficient DO in the surface permeable sediments enhances the rates of nitrification and its coupled denitrification (Rysgaard et al., 1994). In similar, the N₂ flux was negatively correlated with the ${\rm{NO}}_3^- $ concentration in the seawater (Table 3), indicating that denitrification in the sediment of Weizhou Island depended on the ${\rm{NO}}_3^- $ provided by nitrification rather than the ${\rm{NO}}_3^- $ in the overlying seawater. However, the availability of organic matter and its degradation product (i.e., ${\rm{NH}}_4^+ $) is a limiting factor of nitrification (Jäntti et al., 2011; Rysgaard et al., 1994), which explains why ${\rm{NH}}_4^+ $ was transferred from the seawater into the sediment at Station I5 (Fig. 2).

4.2 Inorganic N retaining within the sediments versus losing as N₂ products

FTR experiments allow the manipulation of experimental conditions to gain insights into the mechanisms controlling the N dynamics in permeable sediments (Santos et al., 2012). Through changing flow rates and flow length, a peak of ${\rm{NO}}_3^- $ influx was also observed at the intermediate advection rate (96 L/(m²·h); Fig. 5a) because the intermediate advection rate enhances the development of the microenvironment (i.e., steep DO gradients) within porous carbonate sediments, perhaps providing optimal conditions for the dissimilatory reduction of the external ${\rm{NO}}_3^- $ transmitted to the microenvironment (Santos et al., 2012). Furthermore, ${\rm{NO}}_3^- $ influx also peaked at the intermediate flow path length (10 cm) under intermediate advection (Fig. 5a), indicating that there may be an oxidation process in the deeper layer of sediment (>10 cm), which results in the reproduction of ${\rm{NO}}_3^- $ (Zhang and Furman, 2021). If ${\rm{NO}}_3^- $ was reduced to N₂ by denitrifiers, ${\rm{NO}}_3^- $ reproduction would not occur; thus, the possible explanation is that ${\rm{NO}}_3^- $ was mainly reduced to ${\rm{NH}}_4^+ $ by dissimilatory nitrate reduction to ammonium (DNRA) bacteria, and when these ${\rm{NH}}_4^+ $ accumulated to a certain concentration within the anoxic microenvironment, they were transferred to the external oxic environment where they were reoxidized to ${\rm{NO}}_3^- $. However, the ${\rm{NH}}_4^+ $ flux was insignificant in comparison with those of other N forms (Fig. 5b), perhaps because of the adsorption of ${\rm{NH}}_4^+ $ by sediment pores (Mackin and Aller, 1984).

In short, a selective increase of ${\rm{NO}}_3^- $ flux at given advection rate and flow length showed that more inorganic N was retained within the sediments than lost as N₂ products. Meanwhile, DON can be released during growth of denitrification or DNRA bacteria (Kawasaki and Benner, 2006), resulting in greatest DON efflux at this situation (Fig. 5c). Thus, the interaction between currents and seabed topography makes permeable carbonate sands acting as reservoir of N. This implies that permeable carbonate sands may help to buffer coral reefs during periods of eutrophication or extreme oligotrophy (Erler et al., 2014).

4.3 Implication for the N dynamics in reef sediments under the influences of climate warming and anthropogenic activities

The increasing anthropogenic pollution has resulted in an increasing trend in ${\rm{NO}}_3^- $ concentration (Yu et al., 2019; Pan et al., 2021). Thus, N-excess conditions occurred at Weizhou Island, which was consistent with other reefs affected by anthropogenic activities (Lapointe et al., 2019; Guo et al., 2019). The input of anthropogenic ${\rm{NO}}_3^- $ may be transferred into the sediment, and the influx of ${\rm{NO}}_3^- $ increased with the increasing ${\rm{NO}}_3^- $ concentration in seawater (Fig. 3). This is consistent with the finding in a intertidal flat (Jiang et al., 2021), indicating that water ${\rm{NO}}_3^- $ directly regulated the N flux between seawater and permeable sediments (Wang et al., 2021). However, denitrification did not benefit from the increase of ${\rm{NO}}_3^- $, indicated by the negative relationship between N₂ flux and ${\rm{NO}}_3^- $ concentration in seawater (Table 3). Thus, the anthropogenic ${\rm{NO}}_3^- $ would be retained in the sediment as adsorbed ${\rm{NH}}_4^+ $ following DNRA.

Anthropogenic sediment loads could impact on coral bleaching (Baird et al., 2021) and lead to the increase of DON released from the sediment (Fig. 4a), which enhances the risk of eutrophication in N-excess areas (Lu et al., 2020) such as Weizhou Island. Meanwhile, the increase of TOC content promoted the consumption of ${\rm{NO}}_3^- $ in sediments (Fig. 4b), because the availability of organic matter limits the potential denitrification rate of heterotrophic denitrifiers (Deek et al., 2012).

Temperature is a key factor affecting benthic N flux (Tan et al., 2020; Canion et al., 2014; Muta et al., 2020). The optimal activity temperature of denitrifying bacteria is greater than 25℃ (Tan et al., 2020), which can explain the increase of ${\rm{NO}}_3^- $ consumption in sediments due to temperature rise. However, DNRA bacteria are also thermophilic (Rahman et al., 2019). With the impacts of anthropogenic activities and global warming, further studies using isotope tracing technology and gene sequencing are still needed to assess whether the dissimilatory reduction of ${\rm{NO}}_3^- $ in carbonate sands is mainly regeneration by DNRA or removal by denitrification. However, the increase of DON flux was much greater than that of ${\rm{NO}}_3^- $ flux, and thus, the impacts of anthropogenic activities and climate warming will exacerbate the N-excess condition in Weizhou Island reefs.

5. Conclusions

We conducted a series of FTR experiments to investigate the mechanisms controlling the N dynamics in permeable carbonate sands. The results indicated that permeable carbonate sands act as reservoirs of N under the influence of advective flow, but the sedimentary N dynamics had limited contributions to the N removal at Weizhou Island reefs under N-excess conditions. In the future, the escalating anthropogenic activities (enrichments of ${\rm{NO}}_3^- $ in seawater and TOC in sediments) and climate change (increasing temperatures) will exacerbate the N-excess condition in Weizhou Island reefs. However, further studies on N dynamics in benthic categories of coral reefs include corals, turf algae, sediments, biogenic rock using isotope tracing technology are still needed to reveal the N cycling in coral reefs.