Seasonal variations of nitrous oxide fluxes and soil denitrification rates in subtropical freshwater and brackish tidal marshes of the Min River estuary

STOTEN-24399; No of Pages 10 Science of the Total Environment xxx (2017) xxx–xxx

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Seasonal variations of nitrous oxide fluxes and soil denitrification rates in subtropical freshwater and brackish tidal marshes of the Min River estuary Xuming Wang a,b,c,1, Minjie Hu a,1, Hongchang Ren a, Jiabing Li d, Chuan Tong a,e,⁎, Ronald S. Musenze f,g,⁎ a

School of Geographical Sciences, Fujian Normal University, Fuzhou, China State Key Laboratory of Urban and Regional Ecology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing, China c University of Chinese Academy of Sciences, Beijing, China d School of Environmental Science and Engineering, Fujian Normal University, Fuzhou, China e Key Laboratory of Humid Sub-tropical Eco-geographical Process of Ministry of Education, Fujian Normal University, Fuzhou, China f Air Quality Monitoring Unit, Department of Science, Information Technology and Innovation, Queensland Government, Brisbane, Australia g Department of Civil and Environmental Engineering, College of Engineering, Design, Art and Technology, School of Engineering, Makerere University, Kampala, Uganda b

H I G H L I G H T S

G R A P H I C A L

A B S T R A C T

• N2O fluxes were significantly higher in freshwater marshes than in brackish marshes. • Soil denitrification rates were higher in freshwater than in brackish-water marshes. • Both N2O flux and denitrification rate showed strong seasonal variability. • N2O fluxes had strong negative correlations with salinity in tidal marshes. • Temperature, salinity and ammonia concentration were key controls of denitrification.

a r t i c l e

i n f o

Article history: Received 1 June 2017 Received in revised form 15 October 2017 Accepted 17 October 2017 Available online xxxx Editor: D. Barcelo Keywords: Nitrous oxide emission Denitrification Salinity gradient Brackish tidal marsh Saltwater intrusion Subtropical estuary

a b s t r a c t Estuarine tidal marshes provide favorable conditions for nitrous oxide (N2O) production. Saltwater intrusion caused by sea-level rise would exert complex effects on the production and emission of N2O in estuarine tidal marshes; however, few studies have been conducted on its effects on N2O emissions. Salinity gradients are a common occurrence in estuarine tidal marshes. Studies on production and emission of N2O in tidal marshes with different salinities may elucidate the impact of saltwater intrusion on the emission of greenhouse gases. This study explores the seasonal variations of N2O fluxes and soil denitrification rates in freshwater (Daoqingzhou wetland) and brackish (Shanyutan wetland) tidal marshes dominated by Cyperus malaccensis var. brevifolius (shichito matgrass) in the Min River estuary, southeastern China. N2O fluxes in both marshes showed strong temporal variability. The highest N2O fluxes were observed in the hot and wet summer months, whereas the lowest fluxes were observed in the cold winter and autumn months. N2O fluxes from the freshwater marsh (48.81 ± 9.01 μg m−2 h−1) were significantly higher (p b 0.05) than those from the brackish-water marsh (27.69 ± 4.01 μg m−2 h−1). Soil denitrification rates showed a similar temporal pattern, with the highest rates observed in summer and the lowest in winter. Similarly, soil denitrification rates were significantly higher (p b 0.05) in the freshwater marsh (32.72 ± 19.15 μmol N m−2 h−1) than in the brackish-water marsh (4.97 ± 2.64 μmol N m−2+ h−1). Temperature and the salinity, sulfate (SO2− 4 ), and ammonia nitrogen (NH4 -N) concentrations of the

⁎ Corresponding authors. E-mail addresses: [email protected] (C. Tong), [email protected] (R.S. Musenze). 1 Co first authors who contributed equally to this work.

https://doi.org/10.1016/j.scitotenv.2017.10.175 0048-9697/© 2017 Elsevier B.V. All rights reserved.

Please cite this article as: Wang, X., et al., Seasonal variations of nitrous oxide fluxes and soil denitrification rates in subtropical freshwater and brackish tidal marshes of..., Sci Total Environ (2017), https://doi.org/10.1016/j.scitotenv.2017.10.175

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overlying water were key factors affecting soil denitrification rates. N2O fluxes and soil denitrification rates demonstrated negative correlations with salinity and SO2− 4 concentrations in both marshes. The results indicate that increased seawater intrusion would reduce N2O emissions from estuarine tidal wetlands and exert a negative feedback on the climate system. © 2017 Elsevier B.V. All rights reserved.

1. Introduction Nitrous oxide (N2O) is a potent greenhouse gas with a global warming potential (GWP) 298 times that of carbon dioxide (CO2) over a 100-year time horizon (Denman et al., 2007) and a long atmospheric lifetime (Bauza et al., 2002). Although atmospheric concentrations of N2O are only about 10% of CO2 concentrations, the damage caused to the global environment by N2O emissions is very significant. N2O is also currently recognized as the single most important ozone-depleting substance (Ravishankara et al., 2009). Sea-level rise caused by climate change is of great scientific interest (Wigley and Raper, 1992; Rahmstorf, 2007; Carson et al., 2016). The Fifth Assessment Report (AR5) of the Intergovernmental Panel on Climate Change (IPCC) reported a global average sea level rise of 0.19 m between 1901 and 2010, with saltwater intrusion occurring widely in coastal areas (Myhre et al., 2013). Saltwater intrusion may pose significant ecological threats to fresh- and brackish-water marsh ecosystems (Knowles, 2002; Herbert et al., 2015). Many tidal marshes with various salinity levels develop along estuaries at different tidal extents, influenced by both freshwater river flows and ocean tides. Estuarine tidal marshes, which are gateways of terrestrial nitrogen discharge into the ocean, provide favorable conditions (e.g., alternating dry and wet environments/sediments) for the production and emission of N2O (Galloway et al., 2003; Moseman-Valtierra et al., 2011; Wang et al., 2016). Sediment denitrification, which produces N2O as an intermediate product, is a major pathway of nitrogen removal in tidal marshes (Jordan et al., 2011; Morley et al., 2014). Salinity in tidal marshes is an important environmental factor influencing the generation and emissions of N2O via the sediment denitrification pathway. Salinity affects (1) the enhancement of ammonium release from sediments through physical and chemical processes; (2) the nitrogen release from sediment through physiological processes, and (3) microbial activity (by both nitrifiers and denitrifiers) changing the rate of dissimilatory nitrate reduction to ammonium (Giblin et al., 2010; Wang et al., 2014). However, the effects of salinity on N2O emissions and sediment denitrification are complex. Studies evaluating the effect of salinity changes on N2O emissions in natural environments remain limited, and conflicting data have been reported. Some studies have reported the inhibition of denitrification and a consequent reduction in N2O emissions (Seo et al., 2008; Santoro, 2010; Chauhan et al., 2015; Osborne et al., 2015); however, the conclusions of those studies were inconsistent (Fear et al., 2005; Marton et al., 2012). Previously, little attention was paid to changes in sediment nitrogen transformations and N2O emissions in environments experiencing small salinity changes, such as tidal marshes (Franklin et al., 2016). However, investigation of these changes would add valuable knowledge about climate feedback associated with sea-level rise in the coming decades (Woodroffe and Murray-Wallace, 2012). Simultaneous field measurements of N2O fluxes in both freshwater and brackish-water tidal marshes would be instrumental for enriching the body of knowledge about greenhouse gas emissions in subtropical estuarine systems where data are still scarce (Musenze et al., 2014, 2015). This study presents a quantitative spatial and temporal evaluation of soil denitrification rates and N2O fluxes in both freshwater and brackish-water tidal marshes dominated by Cyperus malaccensis var. brevifolius (shichito matgrass) in the Min River estuary, southeastern China. Shichito matgrass is a common coastal wetlands species,

especially in Fujian and Guangdong provinces. The objectives of this study are to: (1) determine the effects of small increases in salinity on N2O fluxes and denitrification rates in subtropical estuarine tidal marsh ecosystems and (2) establish key environmental controls for N2O emissions in estuarine marshes with narrow salinity gradients in the subtropics. 2. Materials and methods 2.1. Site description This study was conducted in the Min River estuary with measurement sites located in the Daoqingzhou wetland (a freshwater tidal wetland) and the Shanyutan wetland (a brackish-water tidal wetland). The freshwater shichito matgrass marsh site was located in the southwestern part of the Daoqingzhou wetland (25°57′21″N, 119°24′25″E), and the brackish-water shichito matgrass marsh site was located in the middle-western part of the Shanyutan wetland (26°01′46″N, 119°37′31″E) (Fig. 1). The climate of this area is subtropical with hot, humid summers and dry, cold winters. The mean annual temperature of the estuarine region is 19.85 °C, and the mean annual precipitation is 1905 mm. Shichito matgrass is a typical native species in both the Daoqingzhou and Shanyutan wetlands. In the Min River estuary, the tides are considered typical semidiurnal tides; the soil surface is generally submerged for approximately 7 h over a 24 h cycle (Tong et al., 2013). The average salinity of the tidal water (measured from November 2013 to October 2014) was 0.20 ± 0.02‰ and 3.79 ± 1.35‰ in the Daoqingzhou and Shanyutan wetlands, respectively (see Table 2 for variation in seasonal values). There were no significant differences in the heights of the plants or the aboveground and underground biomass between the two study sites (except for the density of the plants measured in a 50 cm × 50 cm area) (see SI Table S1). 2.2. Gas sampling At each site, four sampling plots (four replicates) were established along a transect parallel with the river channel or the coastline at 3-m intervals. Enclosed, opaque static chambers were deployed for the measurement of N2O emitted to the atmosphere. To ensure minimal physical disturbance during chamber deployment and the measurement periods, a wooden boardwalk was constructed to connect all plots. The chambers had a polyvinyl chloride (PVC) bottom collar (35 cm × 35 cm × 30 cm in depth) and an opaque PVC upper chamber (35 cm × 35 cm × 140 cm in height). The bottom collar was permanently inserted into the marsh soil, with 2 cm left protruding above the soil surface. In one side of the top chamber, a gas sampling port lined with rubber cushioning material was installed as an air-tight sealant 90 cm from the ground. The chamber was installed with an electric fan to ensure slow but complete homogeneous air mixing. During the summer field measurement campaign, cotton quilts were used over the chamber tops to ensure effective temperature control. Monthly N2O measurements were made for a full year (November 2013 to October 2014). Samples were collected between 8:00 am and 10:00 am during low or a neap tides each month. Each flux measurement was performed for four samples collected at 10-min intervals. Samples of 50 mL were collected in 100-mL aluminum-foil gas sample bags (Dalian Delin Gas Packing Co., Ltd., China) using 60-mL gas-tight

Please cite this article as: Wang, X., et al., Seasonal variations of nitrous oxide fluxes and soil denitrification rates in subtropical freshwater and brackish tidal marshes of..., Sci Total Environ (2017), https://doi.org/10.1016/j.scitotenv.2017.10.175

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Fig. 1. Location map of the two study sites in the Min River estuary.

syringes (Jiangsu Kangyou Medical Instrument Co., Ltd., China) equipped with three-way locks. 2.3. Estimation of N2O flux N2O concentrations were determined using a gas chromatograph (GC-2014, Shimadzu, Japan) with an electron capture detector (ECD). The injection port, column, and detector temperatures were set at 50, 60, and 320 °C, respectively, with helium as the carrier gas at a flow rate of 30 mL min− 1. Rates of increasing N2O concentrations in the chambers were determined using linear regression, and data were rejected if the R2 value of the regression was ≤ 0.80 (Hirota et al., 2007). N2O fluxes were calculated using the following equation from Wang et al. (2015):
 dc M 273 H F¼ dt V 273 þ T

Waddington, 2008). The 5-cm sections of the samplers protruded above the soil surface and were sealed tightly with covers. All samplers were emptied two days prior to gas sampling. Forty-milliliter pore water samples were collected when gas was sampled, and the physicochemical properties of the pore water were measured in the laboratory: − NH+ 4 -N and NO3 -N concentrations were determined through flow injection analysis (FIA) using a Skalar San++ (Skalar Analytical B·V, Netherlands); Cl− and SO24 − concentrations were determined via ion chromatography (IC) using a Dionex ICS-2100 integrated reagent-free system (Dionex, Sunnyvale, USA); and dissolved organic carbon (DOC) concentration was determined with the high-temperature (680 °C) catalytic oxidation method using a Shimadzu TOC-VCPH analyzer (Shimadzu, Japan). 2.5. Soil denitrification

ð1Þ

2.4. Measurement of environmental variables

2.5.1. Soil cores and overlying water collection Soil cores were collected on the same days as the gas samples in November 2013, February 2014, May 2014, and August 2014. Two undisturbed 15-cm-deep soil cores were collected using two 30-cm-long and 5-cm-inner-diameter PVC corers next to each bottom collar. The bottoms of the corers were plugged tightly with PVC plugs. Beside each soil core sampling site, a 15-cm-deep soil sample was extracted to determine the physicochemical properties of the soil. Overlying water samples were also collected for incubation and analysis.

Various environmental factors were measured, including air temperature, soil temperature, soil electrical conductivity (EC), and soil pH. Air temperature was measured 1.5 m above ground with a Kestrel 3500 hand-held weather meter (Kestrel-3500, USA) and recorded as an average of four consecutive measurements. Soil temperature and EC were measured using a 2265FS EC Meter (Spectrum Technologies Inc., USA), and soil pH was measured with an IQ150 instrument (IQ Scientific Instruments, USA) at the bottom collar at 5-cm depth. Beside each static chamber, a 5-cm-deep soil sample was excavated and a 3mL subsample was taken with a 5-mL syringe to determine the soil bulk density and moisture content. Pore water sampling was done with 5-cm-external-diameter PVC pore water samplers installed at three different depths (5, 15, and 25 cm) near each bottom collar (Ding et al., 2003; Strack and

2.5.2. Incubation and sampling in the laboratory Soil cores and overlying water samples were equilibrated for 12 h in the laboratory to minimize the effects of disturbances during sampling, transportation, and incubation setup. Overlying water samples from the respective study sites were carefully transferred to the corresponding coring tubes. After 2 h, eight soil cores from each study site were divided into two subgroups to conduct the inhibition experiments (Wang et al., 2007). The first subgroup (A) was the “beginning cores”, which were sacrificed at the start of the inhibition experiment. Sixty milliliter subsamples of the overlying water were drawn quickly and injected into the 60-mL headspace vials, pre-equipped with 0.3 mL of 38% formaldehyde, and the lid was tightened. The second subgroup (B) was the “acetylene inhibition samples”. Ten percent of the overlying water in each

where F is the N2O flux (μg m−2 h−1); dc/dt is the slope of the linear regression for the gas concentration gradient with time (μmol mol−1 h−1); M is the molecular mass of N2O (g mol−1); V is the N2O molar volume (L mol−1) under standard conditions; T is the air temperature during sampling (°C); and H is the height of the measurement chamber (m).

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tube was replaced with acetylene-saturated water, which was prepared from the overlying water by replenishing acetylene. Then, 0.2 mL of acetylene-saturated water was injected into the soil through 1-mm holes (filled with silicone rubber) using microsyringes. The holes were pre-drilled at 2-cm vertical intervals in the bottom 15 cm of the PVC coring tubes. The tops of the tubes were tightly sealed upon acetylene injection. Group B cores were put in a cyclotron oscillator (HY-5, Jintan Hongke, China) on mild concussion settings to ensure homogeneity of the overlying water and exchange of materials between the interface while avoiding large disturbances. All incubations were done in the dark for 8 h. After incubation, 60 mL of the overlying water was drawn and quickly injected into 60-mL headspace vials, pre-equipped with 0.3 mL 38% formaldehyde. These vials were then mixed and tightly closed. At the beginning and the end of each inhibition experiment, the temperature and salinity of the extracted overlying water were measured using the 2265FS EC Meter (Spectrum Technologies Inc., USA) and a Salt 6+ salinity meter (Oakton Instruments, IL, USA). 2.5.3. Estimation of dissolved N2O concentration in overlying water The dissolved N2O concentration in the overlying water was measured using static headspace-gas chromatography (Walter et al., 2005). Next, 30 mL of ultra-pure nitrogen was injected into each 60mL headspace vial, and each vial was then filled with the incubated sample of overlying water to exchange 30 mL of overlying water. The 60-mL headspace vials were intensively shaken for 30 min to equilibrate the gas and the liquid phases. Fifteen milliliters of the headspace gas was sampled and injected into 50-mL aluminum foil gas sample bags using 30-mL gas-tight syringes equipped with three-way locks. N2O concentrations of the gas samples (Cg) were measured using the same approach described in Section 2.3. The dissolved N2O concentration in the overlying water was determined according to backward calculation from the headspace gas concentration using Henry's law (e.g. Musenze et al., 2014, 2015, 2016) and the constants of Weiss and Price (1980), based on the overlying water temperature, water salinity, and the headspace gas N2O concentration (Eq. 2 and Eq. 3):

2.5.5. Soil and overlying water characterization To determine the physicochemical properties of the soil, 15-cmdeep soil samples were selected, air-dried, and ground using a ceramic mortar and pestle. Soil EC and pH were measured using a portable conductivity meter (2265FS EC Meter, Spectrum Technologies Inc., USA) and a portable Starter ST300 pH meter (Ohaus, USA), respectively. The soil-water mixture was prepared to a ratio of 2.5:1 (water:soil) and vi− brated for 30 min. NH+ 4 -N and NO3 -N concentrations were determined by measuring the soil leaching liquor (extracted with 2 mol L−1 KCl solution) using flow injection analysis (FIA) (see Section 2.4). Total carbon (TC) and total nitrogen (TN) concentrations of soil samples were determined using a Vario MAX CN element analyzer (Elementar Analysensysteme GmbH, Hanau, Germany). The salinity of the overlying water was measured directly using a Salt 6+ salinity meter (Oakton Instruments, IL, USA). Cl− and SO2− 4 concentrations in the overlying water were determined using the IC meth− od on 0.2-μm filters and syringe-filtered samples. NH+ 4 -N and NO3 -N concentrations were measured using the FIA method described above, whereas DOC was determined using the high-temperature catalytic oxidation method (see Section 2.4). 2.6. Statistical analysis The significance of the observed differences in N2O fluxes and environmental variables between the two sampling sites was determined by comparing the repeated measurement values using analysis of variance (ANOVA). Multiple measurements at a given site over 12 months represented the repeated variables. The differences in denitrification rates and properties of the soil and overlying water at each site in different seasons were examined using least-significant difference (LSD) test in one-way ANOVA. Factors controlling the seasonal variation of N2O fluxes and soil denitrification rates were examined with Pearson correlation analysis. All statistical analyses were performed using the SPSS statistical package (SPSS 17.0 for Windows). The reported statistics were interpreted to be inferential (Musenze et al., 2015). 3. Results

C ¼ C g K 0 þ β=22:4 ln K 0 ¼ A1 þ h A2 ð100=T Þ þ A3 ln ðT=100Þ i þ S B1 þ B2 ðT=100Þ þ B3 ðT=100Þ2

ð2Þ

ð3Þ

where C is the dissolved N2O concentration in the overlying water (μmol L−1); Cg is the N2O concentration in the air (μL L−1); K0 is the solubility of N2O (mol L−1); β is the volume ratio of gas and liquid in the headspace vial (β = 1 in this study); A1, A2, A3, B1, B2, and B3 are constants (− 62.7062, 97.3066, 24.1406, − 0.05842, 0.033193, and − 0.0051313, respectively); T is the thermodynamic temperature of the overlying water (K); and S is the salinity of the overlying water (‰) (Weiss and Price, 1980). 2.5.4. Estimation of soil denitrification rate The soil denitrification rate was calculated using different N2O concentrations in overlying water between group A and group B (Eq. 3) (Yang et al., 2011): RD ¼

C B −C A H w 2 t

ð4Þ

where RD is the soil denitrification rate (μmol N m−2 h−1); CA is the N2O concentration in the overlying water of group A (nmol L−1); CB is the N2O concentration in the overlying water of group B (nmol L−1); Hw is the height of the overlying water in the PVC tube (0.15 m); t is the incubation time (h); and CA and CB are calculated using Eq. 2.

3.1. Monthly and seasonal variations of N2O fluxes N2O fluxes from both sites showed distinct temporal variability (Fig. 2). In the Daoqingzhou wetland, the minimum and maximum N2O fluxes from the shichito matgrass marsh to the atmosphere were 27.11 and 80.30 μg m−2 h−1 in January (winter) and June (summer), respectively. In the Shanyutan wetland, N2O fluxes ranged from 13.22 to 41.54 μg m−2 h−1 throughout the year, as measured in November (autumn) and July (summer), respectively. Overall, the average N2O fluxes were significantly higher (p b 0.05) in Daoqingzhou (48.81 ± 9.01 μg m−2 h−1) than in Shanyutan (27.69 ± 4.01 μg m−2 h−1). For both sites, the maximum average N2O fluxes were observed in the summer (Fig. 2b). 3.2. Environmental variables Details of the monthly variations in air temperature, soil temperature, EC, pH, bulk density, and moisture content at our study sites for the same study period were reported previously (Hu et al., 2016). Briefly, air and soil temperatures in the Daoqingzhou and Shanyutan wetlands demonstrated significant and similar monthly variations. There were no significant differences in air temperatures between the two wetlands, but the average soil temperature was significantly higher (p b 0.05) in Daoqingzhou than in Shanyutan by 0.78 °C. The soil EC in Daoqingzhou and Shanyutan varied between 0.20 and 0.49 mS cm − 1 and 0.80 and 6.62 mS cm− 1 , respectively. The average soil EC in Shanyutan (3.66 ± 0.84 mS cm− 1) was significantly higher (p b 0.05) than that in Daoqingzhou (0.35 ±

Please cite this article as: Wang, X., et al., Seasonal variations of nitrous oxide fluxes and soil denitrification rates in subtropical freshwater and brackish tidal marshes of..., Sci Total Environ (2017), https://doi.org/10.1016/j.scitotenv.2017.10.175

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0.04 mS cm− 1). The soil pH in Daoqingzhou and Shanyutan varied between 4.81 and 6.97 and 5.25 and 7.87, respectively. The average soil pH in Daoqingzhou (5.80 ± 0.41) was significantly lower (p b 0.05) than that in Shanyutan (6.60 ± 0.37). There were no significant differences in the bulk density or moisture content of the soil between the two sites. Monthly variations of pore water concentrations of NH+ 4 -N (Fig. 3a), − NO3 -N (Fig. 3b), Cl− (Fig. 3c), SO24 − (Fig. 3d), and DOC (Fig. 3e), in terms of the mean values at three sampling depths (5, 15, and 25 cm) in the Daoqingzhou and Shanyutan wetlands are shown in Fig. 3. No significant differences were found between the two sites in NH+ 4 -N and − 2− NO− concen3 -N concentrations in pore water. However, Cl and SO4 trations in pore water in Shanyutan were 111 and 24 times greater than the respective concentrations in Daoqingzhou. The two study sites exhibited similar monthly trends for Cl− concentrations. The average DOC concentration in the pore water was significantly lower (p b 0.05) in Daoqingzhou than in Shanyutan for most of the year.

3.3. Influence of environmental variables on N2O fluxes Fig. 2. Monthly and seasonal variations of N2O flux in Daoqingzhou and Shanyutan. Bars show the averages of four months of measurements. Error bars are the standard errors on the mean values (n = 4). “Winter” includes N2O fluxes measured in December 2013, January 2014, and February 2014; “Spring”: March, April, and May 2014; “Summer”: June, July, and August 2014; and “Autumn”: September 2014, October 2014, and November 2013. Different lowercase letters indicate significant differences between seasons at the same sampling site; different capital letters indicate significant differences between sampling sites during the same season. The results of the repeated measurements ANOVA are also reported; asterisks show the significance of factors (site (S) and month (M)) and their interactions: **p b 0.01, *p b 0.05, ns = p N 0.05.

There were strong positive correlations between the N2O flux and air temperature/soil temperature at both sites (see Table S2). The soil EC showed a strong positive correlation with N2O flux in Daoqingzhou, but a negative correlation with N2O flux in Shanyutan. The soil pH and DOC concentrations of the pore water showed strong negative correlations with N2O flux in Daoqingzhou, whereas greater NH+ 4 -N concentration promoted N2O flux significantly. Negative correlation between pore water SO2− concentration and N2O flux was only detected 4 in Shanyutan.

− 2− − Fig. 3. Monthly variations of pore water concentrations of NH+ 4 -N (a), NO3 -N (b), Cl (c), SO4 (d), and DOC (e) in Daoqingzhou and Shanyutan. Data represent the average values of the three sampled depths. Error bars are the standard errors of the average values; n = 4. The results of the repeated measurements ANOVA are also reported; asterisks indicate significance of the factors (site (S) and month (M)) and their interactions: ***p b 0.001, **p b 0.01, *p b 0.05, ns = p N 0.05.

Please cite this article as: Wang, X., et al., Seasonal variations of nitrous oxide fluxes and soil denitrification rates in subtropical freshwater and brackish tidal marshes of..., Sci Total Environ (2017), https://doi.org/10.1016/j.scitotenv.2017.10.175

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and carbon-to-nitrogen ratios (C/N) of the soil were higher (p b 0.05) in the Daoqingzhou wetland. There were significant seasonal variations in the physicochemical properties of the overlying water at both sites (Table 2). Seasonal variability in all physicochemical properties of the overlying water, except for DOC concentration, was higher (p b 0.05) in the Shanyutan wetland. + The salinity and concentrations of Cl−, SO2− 4 , NH4 -N, and DOC of the overlying water were significantly higher (p b 0.05) in Shanyutan than in Daoqingzhou. However, NO− 3 -N concentrations were higher (p b 0.05) in the Daoqingzhou wetland. 3.6. Influence of soil and overlying water on soil denitrification

Fig. 4. Seasonal variations in soil denitrification rates in Daoqingzhou and Shanyutan. Bars represent the averages of monthly values, whereas the error bars are the standard errors of the average monthly values (n = 3). Different lowercase letters indicate significant differences between seasons at the same sampling site; different capital letters indicate significant differences between different sampling sites during the same season.

3.4. Seasonal variations of soil denitrification rates Significant seasonal variations in soil denitrification rates were observed in the Daoqingzhou and Shanyutan wetlands (Fig. 4). In Daoqingzhou, the maximum and minimum soil denitrification rates were 80.91 ± 19.57 and 3.37 ± 1.18 μmol N m−2 h−1 in summer and autumn, respectively. In Shanyutan, the soil denitrification rates ranged from 0.33 ± 0.14 to 11.46 ± 1.64 μmol N m−2 h−1, with the highest denitrification rates detected in spring. Overall, denitrification rates were significantly higher in Daoqingzhou (p b 0.05) than in Shanyutan throughout the study period.

For incubation, the overlying water temperatures in autumn, winter, spring, and summer were 24.0 ± 0.1, 18.4 ± 0.5, 28.0 ± 0.2, and 30.5 ± 0.1 °C, respectively. Linear correlation models were fitted to the relationship between the overlying water temperatures and the soil denitrification rates in both wetlands. There was a significant linear increase in the soil denitrification rate with increased overlying water temperature in both wetlands (n = 16, p b 0.01) (Fig. 5). The relationship between soil physicochemical properties and denitrification rates in the Daoqingzhou and Shanyutan wetlands was inconsistent (Table S3). In the Daoqingzhou wetland, the soil denitrification rate showed negative correlations with EC and C/N and a positive correlation with NH+ 4 -N concentration. In contrast, in the Shanyutan wetland, a negative (and positive) correlation was detected between the denitrification rate and EC (and TN). The relationship between overlying water physicochemical properties and denitrification rates in the Daoqingzhou and Shanyutan wetlands is shown in Table S3. The concentrations of Cl− and SO24 − (NH+ 4 -N) in the overlying water had significantly negative (positive) correlations with soil denitrification rates in both wetlands. However, a strong negative correlation between the denitrification rate and the salinity and DOC concentration (NO− 3 -N concentration) of the overlying water was detected only in Shanyutan (Daoqingzhou). 4. Discussion

3.5. Properties of soil and overlying water

4.1. Nitrous oxide fluxes in estuarine tidal marshes

There were no significant differences in soil pH between the four seasons in Daoqingzhou (Table 1). However, other soil physicochemical properties demonstrated significant seasonal variations in two wetlands. − The seasonal coefficients of variability for EC, NH+ 4 -N, and NO3 -N concentrations were relatively higher than the other physicochemical properties in both wetlands. The EC, pH, and NO− 3 -N concentrations in the soil were significantly higher (p b 0.05) in Shanyutan than in Daoqingzhou. In contrast, the NH+ 4 -N concentrations, TC concentrations,

Both the fresh- and brackish tidal marshes of the Min River estuary were found to be strong sources of atmospheric N2O. Similar results have been previously reported from different climatic zones (Table 3). Estuarine tidal marshes, which are gateways of terrestrial nitrogen discharge into oceans, provide favorable conditions for the production and subsequent emission of N2O, including availability of organic carbon and nitrogen, and alternating dry and wet environments (Galloway et al., 2003; Moseman-Valtierra et al., 2011; Wang et al., 2016). Studies

Table 1 Seasonal variations in soil physicochemical properties in the Daoqingzhou and Shanyutan wetlands of the Min River estuary (numbers are “averages ± standard errors”; n = 4. Nutrient concentrations are expressed per kg of dry soil). Site

Season

EC

pH

(mS cm−1) Daoqingzhou

Shanyutan

Autumn Winter Spring Summer CV Autumn Winter Spring Summer CV

0.26 ± 0.04a B 0.25 ± 0.03a B 0.31 ± 0.05a B 0.16 ± 0.02b B 25.87% 3.13 ± 0.51a A 2.75 ± 0.61a A 1.61 ± 0.48b A 1.14 ± 0.22b A 43.38%

5.02 ± 0.19a B 5.07 ± 0.20a B 4.85 ± 0.08a B 4.96 ± 0.09a B 1.85% 6.54 ± 0.14c A 6.65 ± 0.15bc A 6.89 ± 0.11a A 6.75 ± 0.12ab A 2.23%

NH+ 4 -N

NO− 3 -N

TC

TN

(mg kg−1)

(mg kg−1)

(g kg−1)

(g kg−1)

38.89 ± 4.06b A 42.06 ± 6.20ab A 41.57 ± 5.51ab A 50.04 ± 5.65a A 11.14% 21.20 ± 2.29b B 26.98 ± 4.86ab B 30.28 ± 4.17a B 26.06 ± 6.72ab B 14.38%

0.64 ± 0.20a A 0.16 ± 0.05b B 0.17 ± 0.03b B 0.31 ± 0.18ab A 70.15% 0.52 ± 0.14a A 0.28 ± 0.03bc A 0.26 ± 0.04c A 0.40 ± 0.11b A 32.92%

21.48 ± 0.89a A 18.88 ± 0.69c A 20.92 ± 1.28ab A 19.84 ± 0.81bc A 5.69% 18.70 ± 0.96ab B 17.61 ± 0.67b B 19.03 ± 0.69a B 18.27 ± 1.06ab A 3.33%

1.79 ± 0.07bc A 1.71 ± 0.06c B 1.90 ± 0.12ab A 1.96 ± 0.10a A 6.14% 1.83 ± 0.07b A 1.84 ± 0.06b A 2.00 ± 0.07a A 2.08 ± 0.10a A 6.39%

C/N

12.01 ± 0.20a A 11.07 ± 0.13b A 11.05 ± 0.21b A 10.14 ± 0.17c A 6.87% 10.21 ± 0.23a B 9.60 ± 0.27b B 9.52 ± 0.24b B 8.78 ± 0.17c B 6.17%

Different lowercase letters indicate significant differences between seasons at the same sampling site; different capital letters indicate significant differences between different sampling sites during the same season.

Please cite this article as: Wang, X., et al., Seasonal variations of nitrous oxide fluxes and soil denitrification rates in subtropical freshwater and brackish tidal marshes of..., Sci Total Environ (2017), https://doi.org/10.1016/j.scitotenv.2017.10.175

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Table 2 Seasonal variations in the physicochemical properties of overlying water in the Daoqingzhou and Shanyutan wetlands of the Min River estuary (numbers are “averages ± standard errors”; n = 4). Site

Season

Salinity (‰)

Cl− (mg L−1)

SO2− (mg L−1) 4

−1 NH+ ) 4 -N (mg L

−1 NO− ) 3 -N (mg L

DOC (mg L−1)

Daoqingzhou

Autumn Winter Spring Summer CV Autumn Winter Spring Summer CV

0.21 ± 0.00a B 0.21 ± 0.00a B 0.17 ± 0.01c B 0.19 ± 0.00b B 10.64% 8.10 ± 0.00a A 6.85 ± 0.03b A 1.14 ± 0.00c A 1.03 ± 0.00d A 86.96%

13.02 ± 0.51a B 9.21 ± 0.09b B 2.84 ± 0.20d B 4.28 ± 0.25c B 63.63% 5659.08 ± 40.05a A 4358.99 ± 4.43b A 513.27 ± 1.18c A 396.19 ± 5.46d A 98.21%

17.63 ± 0.06a B 16.19 ± 0.02b B 9.41 ± 0.08c B 8.43 ± 0.10d B 36.13% 771.49 ± 7.58a A 626.49 ± 1.04b A 106.95 ± 0.75c A 27.43 ± 0.78d A 96.84%

0.05 ± 0.00b B 0.19 ± 0.02b B 0.23 ± 0.02ab B 0.24 ± 0.01a B 48.34% 0.80 ± 0.01b A 0.28 ± 0.03c A 1.28 ± 0.02a A 0.83 ± 0.01b A 50.95%

1.87 ± 0.01a A 1.65 ± 0.03b A 0.88 ± 0.01c A 0.82 ± 0.02c A 40.91% 0.72 ± 0.01a B 0.51 ± 0.01c B 0.66 ± 0.01b B 0.08 ± 0.00d B 58.96%

10.68 ± 0.18c B 23.86 ± 0.83b B 28.47 ± 2.91a A 4.64 ± 0.13d B 65.78% 22.79 ± 0.07b A 34.64 ± 3.87a A 12.71 ± 0.29c B 7.59 ± 0.05d A 61.46%

Shanyutan

Different lowercase letters indicate significant differences between seasons at the same sampling site; different capital letters indicate significant differences between different sampling sites during the same season.

on N2O sources/sinks and their key controls are necessary for (1) accurate compilation of N2O emission inventories of tidal marshes, (2) more reliable prediction of N2O emissions in changing climates and environments in the future and (3) better accountability of the global N2O budget. Fluxes of N2O were found to be significantly higher in the freshwater marsh than in the brackish marsh. Although these findings are in agreement with previous studies (Welti et al., 2016; Smith et al., 1983), reported data on the effect of salinity on N2O emissions in estuarine marshes are largely inconsistent (Table 4). The interactions between the drivers of N2O production in different environments are very complex. In the investigated estuarine tidal marshes, the key environmental factors included nitrogen availability, tidal water level, and rooted vegetation depth (Hernandez and Mitsch, 2006; Wang et al., 2007). The results of this study indicate that N2O emissions were higher in the freshwater marsh than in the brackish (oligohaline) marsh (Allen et al., 2011; Chen et al., 2010), even though the range of salinity over the study period was narrow. In general, N2O emission studies in subtropical estuarine areas have shown similar trends (e.g. Musenze et al., 2014; Sturm et al., 2016). Previously, it has been argued that salinity could inhibit N2O production in many ways, including through physical, chemical, and physiological processes (Edmonds et al., 2009; Giblin et al., 2010). This study was not designed to decouple the contributions of the different N2O production processes and mechanisms aside from denitrification rates; however, the results clearly reveal that increased intrusion of saline seawaters into estuarine freshwater wetlands could reduce N2O emissions. This phenomenon would lead to negative feedback on sea-level rise. 4.2. Seasonal variations of nitrous oxide fluxes in estuarine tidal marshes There are various controls on N2O emissions from aquatic systems. Air and soil temperature are key drivers of the temporal dynamics of

N2O flux. Soil temperature controls the decomposition rate of soil organic matter through its effect on microbial activity, enzymes, and metabolism, and consequently regulates the rates of N2O production, consumption, and ultimately emission into the atmosphere. Temperature would also affect the physical properties of soil, as well as the solubility of N2O within both the pore water and the overlying water (Weiss and Price, 1980; Wanninkhof, 1992). Temperature would also affect the water-air gas transfer rate (Guo et al., 2013; Musenze et al., 2014, 2015; Zhu et al., 2014). The significant positive correlation between N2O flux and temperature (Table S2) indicates that temperature is an important control of the seasonal variability of N2O flux. The estimated annual N2O fluxes were 429.6 mg N2O m−2 y−1 and 244.1 mg N2O m−2 y−1 in the Daoqingzhou and Shanyutan wetlands, respectively. During summer, with elevated temperatures (measured average air (soil) temperatures were 35.3 (29.5)°C and 32.0 (29.1)°C in the Daoqingzhou and Shanyutan wetlands, respectively), the summer fluxes accounted for ~33% and ~34% of the annual fluxes in the Daoqingzhou and Shanyutan wetlands, respectively. This finding confirms that N2O emissions in subtropical aquatic systems are normally elevated during the summer months relative to other seasons (Musenze et al., 2014, 2015; Allen et al., 2011). N2O fluxes also increase with sediment and pore water temperatures (Zheng et al., 1997; Li et al., 2010). Zheng et al. (1997) showed that 67% of N2O emissions were released at temperatures of between 15 and 25 °C. Li et al. (2010) showed that temperatures of 25–36 °C were suitable for microbial N2O production (91.3% of total emissions) in a Phragmites australis ecosystem on eastern Chongming Island. However, the observation of elevated emissions in the summer should be interpreted cautiously. Subtropical summers are hot and wet, with increased catchment flows, organic matter, and terrigenous nitrogen deposits, which could sustain increased greenhouse gas production rates for longer periods of time (Musenze et al., 2014, 2015, 2016). These factors could be the reason that aside from elevated summer emissions, seasonal fluxes were similar.

Fig. 5. Relationship between soil denitrification rate and temperature in the Daoqingzhou and Shanyutan wetlands.

Please cite this article as: Wang, X., et al., Seasonal variations of nitrous oxide fluxes and soil denitrification rates in subtropical freshwater and brackish tidal marshes of..., Sci Total Environ (2017), https://doi.org/10.1016/j.scitotenv.2017.10.175

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Table 3 N2O fluxes from estuarine freshwater and brackish tidal marshes in different climate zones worldwide. Location

Climate zone

Vegetation

Habitat

N2O flux (μg m−2 h−1)

References

Min River estuary, China Savannah River, USA

Subtropical Subtropical

Freshwater Freshwater

48.81 ± 9.01 −3.32 ± 5.79

This study Krauss and Whitbeck, 2012

Cooroon Cooroonphah Creek, Australia

Subtropical

Freshwater

59.0 ± 24.3

Welti et al., 2016

Dovey estuary, UK Min River estuary, China Savannah River, USA

Temperate Subtropical Subtropical Tropical Tropical Temperate Temperate Temperate

Freshwater Brackish Brackish Brackish Brackish Brackish Brackish Brackish Brackish

20.2 ± 5.5 27.69 ± 4.01 −1.91 ± 4.05 8.91 ± 4.77 757.24 32.1–533.7 5.2–15.2 4.6 ± 2.5 −30–1

Dausse et al., 2012 This study Krauss and Whitbeck, 2012

Futian, Deep Bay region, China Mai Po, Deep Bay region, China Yellow River estuary, China Dovey estuary, UK Nakaumi lagoon, Japan

Cyperus malaccensis Taxodium dstichum; Nyssa aquatica Gahnia sieberiana; Empodisma minus; Gleichenia spp Agrostis stolonifera Cyperus malaccensis Taxodium dstichum; Nyssa aquatica Kandelia obovata Kandelia obovata Suaeda salsa Festuca rubra Carex rugulosa; Phragmites australis; Solidago altissima

4.3. Soil denitrification rates in estuarine tidal marshes There was strong seasonal variability in sediment denitrification rates in the estuarine tidal marshes. The highest denitrification rates occurred during periods of elevated temperatures in the summer and spring in the Daoqingzhou and Shanyutan wetlands, respectively, whereas the lowest denitrification rates were observed in autumn and winter. The relationship between denitrification rate and overlying water temperature was strong and positive (Fig. 5), with the denitrification rates increasing between the temperature range of ~ 18–30 °C in both tidal marshes. Recently, the optimal temperature for N2O production via the denitrification pathway has been reported as 25 °C in the inter-tidal zone of the Yellow River estuary, China (Sun et al., 2014). Wang et al. (2007) also found temperature to be a key control of sediment denitrification rates in the tidal flat of the Changjiang estuary. The influence of temperature on denitrification rate is likely because of its impact on microbial activity and reaction rates. Under relatively high-temperature conditions, strong microbial activity in sediments leads to higher denitrification potential and higher mineralization. This study found no significant relationship between soil NO− 3 -N content and the denitrification rate. Although this finding could easily be interpreted as indicating that NO− 3 -N concentrations are not a main factor affecting the denitrification rates at the study sites, it could also be an indication that there was an ample supply of NO− 3 -N to meet the nitrogen demands of the denitrifying bacteria, or simply the result of low variability of NO− 3 -N concentration at the study sites over the study period. Therefore, this thrust the other drivers (e.g., temperature) into key roles controlling N2O production via the denitrification pathway.

4.4. Effects of salinity on estuarine soil denitrification rates The results of this study show that the average soil denitrification rates were significantly higher in the freshwater marsh of the Daoqingzhou wetland than in the brackish marsh of the Shanyutan wetland (Fig. 4), although NO− 3 -N concentrations in both wetlands were similar. Salinity (together with EC and Cl− concentrations) of the soil

Chen et al., 2010 Chen et al., 2012 Sun et al., 2017 Dausse et al., 2012 Hirota et al., 2007

and the overlying water were significantly higher in Shanyutan than in Daoqingzhou (Table 1 and Table 2) and had significant negative correlations with soil denitrification rate (Table S3). With all the other presumed critical controls of denitrification and N2O production investigated found to be similar in both wetlands, and only salinity and its precursors found to be significantly higher in Shanyutan than in Daoqingzhou (Table 1 and Table 2), this study demonstrates that salinity was an important controlling parameter responsible for the lower soil denitrification rates in the brackish marsh (Fear et al., 2005; Zimmerman and Benner, 1994; Giblin et al., 2010; Musenze et al., 2014). Salinity values below 6 psu favor retention of NH+ 4 in sediment and higher denitrification rates (Zimmerman and Benner, 1994). This phenomenon is likely the reason for the significant relationship between sediment NH+ 4 -N concentration and denitrification rate in Daoqingzhou, whereas no significant relationship was detected in Shanyutan. Salinity also negatively impacts microbial diversity (Francis et al., 2003; Mosier and Francis, 2008; Moin et al., 2009) and activity (Wang et al., 2010), especially of ammonia-oxidizing bacteria (AOBs). Nonetheless, the influence of salinity is still poorly understood, and conflicting reports suggest that salinity has no influence on denitrification rate (Wang et al., 2007; Fear et al., 2005). Therefore, further studies are still needed for a better understanding of the mechanisms and impacts of salinity on biogenic N2O-producing processes (Musenze et al., 2014, 2015). 4.5. Effects of other environmental factors on denitrification rates and nitrous oxide fluxes Denitrification rates had a strong negative correlation with SO24 − concentrations in both the Daoqingzhou and Shanyutan wetlands (Table S3). This relationship was stronger in Shanyutan than in Daoqingzhou, likely because of the significantly higher SO2− 4 concentrations in the Shanyutan wetland. SO24 − concentrations in freshwater bodies are typically lower than those in saline/sea water (Musenze et al., 2014; Musenze et al., 2016). The higher SO24 − concentrations, the significant negative relationship between SO24 − and denitrification rates, and low N2O saturation levels in the Shanyutan wetland are

Table 4 Comparison of N2O emissions from various estuarine tidal marshes with different salinity levels. Location

Salinity (‰)

N2O flux

References

Barataria Basin, USA Dovey estuary, England Savannah River, USA Cooroon Cooroonphah Creek, Australia Min River estuary, China

Fresh①; Brackish②; Saline③ 1.3①;2.6②; 9.6③; 17.3④ b0.2①; 0.4–2.1②; 1.9–6.8③ Freshwater①; Oligohaline saltwater② 0.20①; 3.79②

①N②N③ ①b②≈③≈④ ①≈②≈③ ①N② ①N②

Smith et al., 1983 Dausse et al., 2012 Krauss and Whitbeck, 2012 Welti et al., 2016 This study

“≈” indicates that there is no significant difference between two sites.

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X. Wang et al. / Science of the Total Environment xxx (2017) xxx–xxx

strong indicators of the negative effect of pore water SO2− concentra4 tions on N2O production and subsequent emissions. Under strong reducing conditions, SO24 − can be reduced to H2S. The H2S thus produced can influence the activities of both nitrifiers and denitrifiers and their effects on N2O production (Giblin et al., 2010). The reduction of SO2− 4 can also enhance dissimilatory nitrate reduction to ammonium (DNRA) (Tiedje, 1988) while inhibiting denitrification (Senga et al., 2006; Larsen et al., 2010), especially at higher metal sulfide concentrations (Brunet and Garcia-Gil, 1996), which directly impacts N2O production and flux. The strong negative relationship of denitrification rates with such SO2− 4 concentrations in overlying water in Daoqingzhou suggests high sensitivity of denitrification to sulfates. Increased sulfate availability in freshwater bodies originating from agricultural croplands and other land uses, together with seawater intrusion into inland freshwater bodies, could significantly affect nitrogen removal processes and N2O emissions. Therefore, in terms of climate feedback, sea level rise, which would lead to increased seawater intrusion into estuarine freshwater tidal wetlands, is likely to lead to a reduction in N2O production in estuarine tidal wetlands. During denitrification, denitrifying bacteria use nitrates as electron acceptors in the absence of oxygen for their respiration (respiratory denitrification). In the process of nitrate reduction, N2O is normally produced as an intermediate that may either be emitted or further reduced under strong reducing conditions to the end product, N2 gas. Therefore, denitrification is strongly dependent on nitrate concentration, and in most cases, nitrates would be a limiting factor of denitrification (Moseman-Valtierra et al., 2011). In contrast, our data indicate that sediment nitrate concentrations had no significant relationship with denitrification rates, but were strongly correlated with the NH+ 4 concentrations of overlying water. In a well-oxygenated water column, denitrification mainly occurs in the sediment (Musenze et al., 2014; Musenze et al., 2015). The overlying water column would be dominated by nitrification because of the availability of both oxygen and NH+ 4 . It is likely that most nitrification occurs in the uppermost few centimeters of the sediments under reducing conditions because of the sensitivity of nitrifiers to oxygen (Knowles, 1982). Therefore, the availability of − NH+ 4 is important for this step. This process is the main source of NO3 for denitrifiers (Seitzinger, 1988; LaMontagne et al., 2003), and is therefore the reason for their low dependence on sediment NO− 3 levels. The most conducive conditions for biogenic greenhouse gas production are at the oxycline (Hynes and Knowles, 1984; Pina-Ochoa and Álvarez-Cobelas, 2006; Musenze et al., 2016), where NO− 3 would be readily available for denitrifiers. These conditions would be synonymous with coupled nitrification and denitrification, which is often mistaken for the nitrifier-denitrifier pathway, the N2O production potential of which remains poorly understood (Webster and Hopkins, 1996; Wrage et al., 2001). Soil organic carbon, an electron donor and energy source for denitrifiers, could significantly affect soil denitrification rates. In general, soil C/ N is used as an indicator of soil organic carbon availability. Lower C/N indicates a higher decomposition rate of soil organic carbon (Guo et al., 2012). The decomposition of organic matter consumes a large amount of oxygen, which creates an anaerobic environment favorable for denitrification. In this study, soil denitrification rates showed significant negative correlations with C/N (Table S3), which suggests that high organic carbon availability exerts a strong oxygen demand that would leave insufficient oxygen for nitrification and eventually support denitrification. Alternatively, the high organic carbon availability in relation to the nitrogen levels could trigger DNRA (Liu et al., 2016), especially in the Shanyutan wetland, since DNRA is favored by the labile C/NO− 3 levels (Silver et al., 2001). 5. Conclusions Both freshwater and brackish tidal marshes of the Min River estuary are strong emitters of atmospheric N2O with significant temporal

9

variability. N2O emissions were significantly higher in the hot and wet summer season and lower in the cold autumn and winter seasons. N2O emissions from the freshwater marsh were significantly higher than those from the brackish marsh. Soil denitrification rates in the freshwater and brackish tidal marshes showed strong seasonal variability; the maximum (minimum) values were observed in summer (autumn) and spring (winter), respectively. Denitrification was strongly dependent on soil temperature. Soil denitrification rates in the brackish marsh were significantly lower than those in the freshwater marsh. N2O fluxes and soil denitrification rates demonstrated negative correlation with salinity and sulfate (SO2− 4 ) concentrations in both the freshwater and brackish tidal marshes. Increased intrusion of saline seawater into subtropical tidal estuarine freshwater aquatic systems (e.g., wetlands) could reduce N2O emissions, and exert negative feedback with respect to climate change. Acknowledgments This study was supported by the National Natural Science Foundation of China (41371127), the Program for Innovative Research Teams of Fujian Normal University (IRTL1205), and the Key Sciences and Technology Project of Fujian Province (2014R1034-1). We thank Wenlong Zhang, Ping Yang, Weiqi Wang and Jiafang Huang for their help with field sampling. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi. org/10.1016/j.scitotenv.2017.10.175. References Allen, D., Dalal, R.C., Rennenberg, H., Schmidt, S., 2011. Seasonal variation in nitrous oxide and methane emissions from subtropical estuary and coastal mangrove sediments, Australia. Plant Biol. 13 (1), 126–133. Bauza, J.F., Morell, J.M., Corredor, J.E., 2002. Biogeochemistry of nitrous oxide production in the red mangrove (Rhizophora mangle) forest sediments. Estuar. Coast. Shelf Sci. 55 (5), 697–704. Brunet, R.C., Garcia-Gil, L.J., 1996. Sulfide-induced dissimilatory nitrate reduction to ammonia in anaerobic freshwater sediments. FEMS Microbiol. Ecol. 21, 131–138. Carson, M., Köhl, A., Stammer, D., Slangen, A.B.A., Katsman, C.A., van de Wal, R.S.W., Church, J., White, N., 2016. Coastal sea level changes, observed and projected during the 20th and 21st century. Clim. Chang. 134, 269–281. Chauhan, R., Datta, A., Ramanathan, A.L., Adhya, T.K., 2015. Factors influencing spatiotemporal variation of methane and nitrous oxide emission from a tropical mangrove of eastern coast of India. Atmos. Environ. 107, 95–106. Chen, G.C., Tam, N.F.Y., Ye, Y., 2010. Summer fluxes of atmospheric greenhouse gases N2O, CH4 and CO2 from mangrove soil in South China. Sci. Total Environ. 408, 2761–2767. Chen, G.C., Tam, N.F.Y., Ye, Y., 2012. Spatial and seasonal variations of atmospheric N2O and CO2 fluxes from a subtropical mangrove swamp and their relationships with soil characteristics. Soil Biol. Biochem. 48, 175–181. Dausse, A., Garbutt, A., Norman, L., Papadimitriou, S., Jones, L.M., Robins, P.E., Thomas, D.N., 2012. Biogeochemical functioning of grazed estuarine tidal marshes along a salinity gradient. Estuar. Coast. Shelf Sci. 100, 83–92. Denman, K.L., Brasseur, G., Chidthaisong, A., Ciais, P., Cox, P.M., Dickinson, R.E., Hauglustaine, D., Heinze, C., Holland, E., Jacob, D., Lohmann, U., Ramachandran, S., da Silva Dias, P.L., Wofsy, S.C., Zhang, X., 2007. Couplings between changes in the climate system and biogeochemistry. In: Solomon, S., Qin, D., Manning, M., Chen, Z., Marquis, M., Averyt, K.B., Tignor, M., Miller, H.L. (Eds.), Climate Change 2007: the Physical Science Basis. Contribution of Working Group I to the Fourth Assessment Report of the Intergovernmental Panel on Climate Change. Cambridge University Press, Cambridge, United Kingdom and New York, NY, USA. Ding, W.X., Cai, Z.C., Tsuruta, H., Li, X., 2003. Key factors affecting spatial variation of methane emissions from freshwater marshes. Chemosphere 51, 167–173. Edmonds, J.W., Weston, N.B., Joye, S.B., Mou, X., Moran, M.A., 2009. Microbial community response to seawater amendment in low-salinity tidal sediments. Microb. Ecol. 58, 558–568. Fear, J.M., Thompson, S.P., Gallo, T.E., Pearl, H.W., 2005. Denitrification rates measured along a salinity gradient in the eutrophic Neuse River Estuary, North Carolina, USA. Estuaries 28 (4), 608–619. Francis, C.A., O'Mullan, G.D., Ward, B.B., 2003. Diversity of ammonia monooxygenase (amoA) genes across environmental gradients in Chesapeake Bay sediments. Geobiology 1, 129–140. Franklin, R.B., Morrissey, E.M., Morina, J.C., 2016. Changes in abundance and community structure of nitrate-reducing bacteria along a salinity gradient in tidal wetlands. Pedobiol. J. Soil Biol. https://doi.org/10.1016/j.pedobi.2016.12.002.

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Please cite this article as: Wang, X., et al., Seasonal variations of nitrous oxide fluxes and soil denitrification rates in subtropical freshwater and brackish tidal marshes of..., Sci Total Environ (2017), https://doi.org/10.1016/j.scitotenv.2017.10.175