Methane Emissions from Surface of Mangrove River on Hainan Island, China

: The surfaces of rivers are considered important sources of atmospheric methane (CH 4 ), however research on this topic is still constrained, especially in freshwater rivers and with the consideration of spatial heterogeneity. Three regions (upper reaches, midstream and downstream) were selected to examine the CH 4 ﬂuxes from a freshwater river surface in a mangrove forest wetland from 2012 to 2013, using ﬂoating chambers. Results showed that the CH 4 ﬂuxes varied signiﬁcantly among the three regions, with the lowest ﬂuxes at downstream (0.50 ± 0.20 mg m − 2 h − 1 ), and highest at upper reaches (1.19 ± 0.36 mg m − 2 h − 1 ). The average emission rate at midstream was 0.95 ± 0.37 mg m − 2 h − 1 . The methane ﬂux also varied with seasons, with higher ﬂux in rain-abundant seasons. On average, the CH 4 ﬂux in our research river was 0.88 ± 0.31 mg m − 2 h − 1 , which was less than other tropical rivers. In addition, we found that the CH 4 ﬂux was signiﬁcantly correlated with the water characteristics of temperature and atmospheric pressure. Thereby, this study quantiﬁed the methane emission from a freshwater river surface in a tropical mangrove forest, enriching the existing knowledge of river surface CH 4 ﬂux. investigate the seasonal and spatial dynamics of CH 4 ﬂux from a typical river in mangrove wetland; (2) to examine the inﬂuencing factors of CH 4 ﬂux.


Introduction
Methane (CH 4 ) is an important greenhouse gas, and is the second-most abundant after CO 2 [1]. Previous research has shown that rivers could be important sources of atmospheric methane (CH 4 ), but this remains uncertain due to a lack of global studies [2,3]. Global water CH 4 emissions into the atmosphere were estimated at about 1.2-2.1 Pg C ·year −1 [4], of which more than 20% originated from freshwaters [5,6]. Research has also estimated that the CH 4 emission from rivers is about 1.5 Tg CH4 ·year −1 [4]. Despite their small area, rivers play an important role in the global methane budget [4]. CH 4 is produced in anaerobic environments and the production process can be divided into the following three steps: (1) complex organic matter is transformed into monosaccharides by hydrolytic enzymes (synthesized by hydrolytic fermentation bacteria), and further fermented to synthesize fatty acids, carbon dioxide and hydrogen; (2) fatty acids, carbon dioxide and hydrogen are oxidized to acetic acid, CO 2 , and H 2 ; (3) acetic acids are oxidated to CH 4 , or the CO 2 is reduced by H 2 to produce CH 4 by acetoclastic methanogenesis (AM) and hydrogenotrophic methanogenesis (HM), respectively [7][8][9]. According to the pathway of CH 4 formation in step (3), CH 4 formation always divides into two pathways: (1) fermentation of acetate by AM; (2) acetate oxidation during which CO 2 is reduced with H 2 by HM. The dominant pathway of CH 4 production is mainly determined by the substance supplying the methanogens [10].
In general, tropical regions were shown to have the highest CH 4 emissions [11]. High content of organic matter, strong heat and strictly anaerobic environments were favorable

Study Site
The river we studied was located in Dongzhaigang National Nature Reserve (19 • 57 -20 •  rivers in these regions also have a large amount of carbon substrates [10] which supply abundant substance for methanogens, leading to a higher CH4 emission. Large amounts of CH4 emission from waters and rivers, especially through the water-gas interface, is an important source of CH4 emissions [12].
Rivers in mangrove wetlands are well adapted to the transition zone between land and sea and the river water body has a natural salinity difference [13,14]. At present, there are many studies on swamps and lakes, and relatively few studies on rivers. Some studies have shown that freshwater rivers, though small in proportion to entire wetlands, contribute more than their share of methane emissions [15,16].
At present the study of river methane emissions occurs mainly in the Amazon in South America, the Congo River basin in West Africa, the Mississippi River basin in North America, Costa Rica in Central America, and in some of Europe's rivers [3,11,[17][18][19][20][21][22]. In China, the research surrounding river CH4 flux is mainly concentrated in the Pearl River watershed, the Yangtze River basin, and in the Yellow River [23][24][25][26].
Rivers are an important source of atmospheric CH4 emission, especially in tropical areas [27]. We speculate that river-flow through the mangrove wetland may have a high CH4 emission rate due to the high organic carbon [13,14] and varied salinity, which could inhibit the activity of methanogens and their reduction in CH4 emission [15,28].
In tropical areas, rivers are characterized by fast substance-exchange such as available carbon, higher rainfall and higher oxygen levels [29]. These characteristics have led to the CH4 emissions in this region being more complex than those in temperate areas. Natural mangrove wetlands are the largest and earliest nature reserves in China. With the characteristics of tropical rivers, the rivers distributed throughout natural mangrove wetlands are also contaminated by human waste, which further increases the organic carbon in the water. Until now, the CH4 flux in such rivers has remained unknown, especially with consideration of spatial-temporal heterogeneity. Thus, the objectives of this study are: (1) to investigate the seasonal and spatial dynamics of CH4 flux from a typical river in mangrove wetland; (2) to examine the influencing factors of CH4 flux.

Study Site
The river we studied was located in Dongzhaigang National Nature Reserve (19°57′-20°01′ N, 110°32′-110°37′ E), in the north east of Hainan province ( Figure 1). Dongzhaigang National Nature Reserve is the first-founded (1980) and the largest mangrove forest reserve in China. It accounts for 44.51% of the mangrove forests of Hainan Island and holds the most abundant mangrove species.    Figure 1). The UP region is distal to the estuary, is rarely influenced by tides and its salinity is lowest. The DOWN region is near the estuary, is severely influenced by tides, and its salinity is highest. The MID region is the transition area between UP and DOWN. The hydrological environments of the three regions also differed.

Gas Sampling
Three plots at each region of the river were established to measure the CH 4 flux of the water surface, using vented closed chambers (26 cm in diameter, 46 cm in height) which consisted of two parts. One was made out of a cylindrical polycarbonate (PC) pipe, and the other was made out of a gas filled floating chamber (Figure 2c,d) [37,38]. Three plots were selected with a distance of about 20 m, and the distance to riverbank was 4 m. The CH 4 was measured monthly from February 2012 to December 2013. The gas was sampled from 8:30 a.m. to 11:30 p.m., with 10 mL disposable vacuum tubes at 10 min intervals over a 30 min period after floating on the water. The air temperature inside the chamber was measured at 0 and 30 min. The concentration of CH 4 in the samples was determined by a gas chromatograph (Agilent 7890A, Agilent Co., Santa Clara, CA, USA) equipped with a flame ionization detector (FID), which operated at 350 • C. The Nickel conversion furnace temperature was maintained at 350 • C. The column temperature was maintained at 250 • C and the carrier gas was pure nitrogen at a flow rate of 20 mL min −1 , the combustion gas was air at a flow rate of 500 mL min −1 , the combustion supporting gas was pure hydrogen with a flow rate of 50 mL min −1 .
The flux (mg CH 4 m −2 h −1 ) of CH 4 was calculated as: is the rate of concentration change; M (mg mol −1 ) is the molar mass of CH 4 ; P (Pa) is the atmospheric pressure of the sampling site; T (K) is the absolute temperature at the sampling time; V 0 (m 3 ), P 0 (Pa), T 0 (k) are the molar volume, atmospheric pressure, and absolute temperature, respectively, under the standard condition; H (m) is the chamber height over the water surface. The flux (mg CH4 m −2 h −1 ) of CH4 was calculated as: where dC/dt (mol·h −1 ) is the rate of concentration change; M (mg mol −1 ) is the mola of CH4; P (Pa) is the atmospheric pressure of the sampling site; T (K) is the absolut perature at the sampling time; V0 (m 3 ), P0 (Pa), T0 (k) are the molar volume, atmos pressure, and absolute temperature, respectively, under the standard condition; H the chamber height over the water surface.

Water Sampling and Analysis
Water was collected in 10 mL glass bottles monthly and randomly at our sam site, and was transported to the lab at 4 °C and analyzed within one week. The cha istics of water temperature, salinity, pH, oxidation-reduction potential (ORP) an ductivity were measured during sampling using a portable digital meter (YSI P Portable multi-parameter water quality analyzer, USA). The water and air tempe were also measured using a handheld digital thermometer, and air pressure was ured monthly. In the lab, water DOC, NO 4 -P concentration analyzed using a continuous flow analytical system (SKALAR San ++ , SKALAR Co., The Netherlands). Before DOC measurement, the sampled water was filtered thro 0.45 μm cellulose acetate membrane filter, and before other index measurements th pled water was filtered through filter paper to eliminate the impurities.

Water Sampling and Analysis
Water was collected in 10 mL glass bottles monthly and randomly at our sampling site, and was transported to the lab at 4 • C and analyzed within one week. The characteristics of water temperature, salinity, pH, oxidation-reduction potential (ORP) and conductivity were measured during sampling using a portable digital meter (YSI ProPlus Portable multi-parameter water quality analyzer, USA). The water and air temperature were also measured using a handheld digital thermometer, and air pressure was measured monthly. In the lab, water DOC, NO − 3 -N, NH + 4 -N and PO 3− 4 -P concentrations were analyzed using a continuous flow analytical system (SKALAR San ++ , SKALAR Co., Breda, The Netherlands). Before DOC measurement, the sampled water was filtered through a 0.45 µm cellulose acetate membrane filter, and before other index measurements the sampled water was filtered through filter paper to eliminate the impurities.

Statistical Analysis
The difference in CH 4 emissions from different regions of the river was analyzed by one-way analysis of variance (ANOVA). ANOVA was also used to compare the differences in water temperature, salinity, pH, oxidation-reduction potential, conductivity, DOC, NO − 3 -N, NH + 4 -N and PO 3− 4 -P among the three regions. The relationships between CH 4 flux and CH 4 concentration in water, air and water temperatures and DOC concentration were detected by regression analysis. All data were analyzed using SPSS 22.0 for Windows (SPSS Inc., Chicago, IL, USA) and all graphs were drawn using Origin Pro 8.1.

Water Characteristics and Environmental Factors
Of the environmental factors, water salinity and conductivity significantly increased from UP to DOWN (p < 0.05 for both). The same was true for pH and PO 3− 4 -P (p < 0.05 for both). Other characteristics did not show any significant difference among the three regions  (Table 1). In addition, salinity, water temperature, conductivity, ORP, and air temperature also varied seasonally (Figure 3a-g).

CH4 Fluxes
The CH4 fluxes of the three regions varied by month (Figure 3). At the UP region, higher CH4 flux was observed during June to September 2012 and May and August 2013. At the MID region, higher CH4 flux was observed during Feb. to Apr. and June to Aug. 2013. At the DOWN region, higher CH4 flux was observed during the later months of each

CH 4 Fluxes
The CH 4 fluxes of the three regions varied by month (Figure 3). At the UP region, higher CH 4 flux was observed during June to September 2012 and May and August 2013. At the MID region, higher CH 4 flux was observed during Feb. to Apr. and June to Aug. 2013. At the DOWN region, higher CH 4 flux was observed during the later months of each year. In summary, higher CH 4 fluxes were observed in rain-abundant seasons at the UP and MID regions and later in the rainy season at the DOWN region.
Of the three regions, the UP region had significantly higher CH 4 fluxes than the MID and DOWN regions in 2012 (p < 0.01). Additionally, the UP and MID regions had relatively higher CH 4

Relationship between Methane Fluxes and Factors
Correlation tests were conducted among CH4 fluxes and water environment factors. The results showed that the significantly correlated variables were different among the three regions. At the UP region, water temperature was significantly positively correlated with CH4 fluxes and atmospheric pressure was significantly negatively correlated with CH4 fluxes. The MID and DOWN regions did show any significant correlations with water variables ( Table 2).

Relationship between Methane Fluxes and Factors
Correlation tests were conducted among CH4 fluxes and water environment factors. The results showed that the significantly correlated variables were different among the three regions. At the UP region, water temperature was significantly positively correlated with CH4 fluxes and atmospheric pressure was significantly negatively correlated with CH4 fluxes. The MID and DOWN regions did show any significant correlations with water variables (Table 2).

Relationship between Methane Fluxes and Factors
Correlation tests were conducted among CH 4 fluxes and water environment factors. The results showed that the significantly correlated variables were different among the three regions. At the UP region, water temperature was significantly positively correlated with CH 4 fluxes and atmospheric pressure was significantly negatively correlated with CH 4 fluxes. The MID and DOWN regions did show any significant correlations with water variables (Table 2).

Comparisons with Other Studies
The CH 4 flux in our study ranged from 0.47 to 1.30 mg CH 4 m −2 h −1 , which was severely lower than in other research [3,[39][40][41] (Table 3). The possible reason was that we neglected the pathway of ebullition of CH 4 emission, resulting in relatively lower CH 4 flux. With deeper research about CH 4 flux in waters and lakes, results have found that ebullition is one of the dominant pathways in open water CH 4 flux [4]. In general, the CH 4 flux in tropical and subtropical open water/water bodies was relatively higher, such as in the open water in India and South America [40,41]. Polluted water CH 4 fluxes were also higher than other water bodies due to their relatively high organic carbon, such as one open water-source in India [3]. On the contrary, the CH 4 flux in water bodies with lower organic carbon in sediments was lower than natural water bodies [42]. In addition, macrophytes were also one of critical factors enhancing water bodies CH 4 flux, as was observed in South America [40]. Macrophytes could supply an abundance of organic matter into waters, which is the original substance during CH 4 production, and may therefore lead to higher CH 4 flux [43].

Spatial and Seasonal Variations of CH 4 Fluxes
Without four seasons, time frames in our research were divided into rainy season (end of March to the beginning of November)) and dry season (beginning of November to the end of March). Some research has showed that heavy rain enhances CH 4 emission in North India [42] with obviously seasonal dynamics. Some research has showed that the CH 4 fluxes did not vary with seasons, such as in Amazon River basin [45]. Research in Pantanal also showed that the emission of CH 4 was slightly higher during the rainy season than the dry season [40]. The seasonal dynamic of CH 4 fluxes in tropical rivers is still controversial. In our research, we found that the CH 4 fluxes were higher in the rainy season than in the dry season.
The CH 4 fluxes in our research also had significant spatial variability, with the highest flux at the UP region in 2012, and relatively higher flux at the UP and MID regions in 2013. Taken together, a relatively higher emission rate was observed at the UP region. The abnormal or extreme value of the middle reaches is mainly due to the presence of the town and severe human activity.

Effect of Environmental Factors on CH 4 Fluxes
The heterogeneity of CH 4 throughout seasons and sites mainly resulted from the variation of water characteristics [17]. Of the water characteristics, salinity is one of the most critical factors. Numerous studies have shown that higher salinity consistently reduces CH 4 emission in waters by inhibiting methanogen activities [46]. The inhibiting effect of salinity on CH 4 emission could explain why CH 4 flux was higher in the rainy season. In the rainy season, an abundance of water reduces water salinity and its inhibiting effect on methanogens, together resulting in a higher CH 4 flux [13,38]. The inhibiting effect of salinity on methanogens also could explain the higher CH 4 flux in the UP region. The UP region, which is far from the bay and was little-affected by tides, has a relatively low salinity and higher CH 4 flux.
Water temperature and air pressure were also critical factors in influencing CH 4 flux, as observed in our study. Research has showed that water temperature affects CH 4 flux through regulating methanogen activity. Atmospheric pressure is always negatively correlated with CH 4 flux, for higher air pressure inhibits the emission of CH 4 from water to air. Higher air pressure also reduces the transportation of bubbles to air, reducing CH 4 flux. At our research site, extreme weather such as typhoons always causes low pressure and an increase in CH 4 flux.

Conclusions
This research quantified the CH 4 flux of a tropical river in a mangrove wetland. The results showed that CH 4 flux varied significantly during rainy and dry seasons, and varied among regions of the river. In our river, the UP region always showed higher CH 4 flux than the MIN and DOWN regions due to higher salinity which inhibits the activity of methanogens and reduces CH 4 production. Water temperature also affected CH 4 flux through regulating methanogen activity. Air pressure physically regulated CH 4 flux through control of the transportation of CH 4 from water to air. Our research could enrich the available data on river CH 4 flux in tropical mangrove wetlands.