Summer-time denitrification and nitrous oxide exchange in the intertidal zone of the Yangtze Estuary

Estuarine, Coastal and Shelf Science 73 (2007) 43e53 www.elsevier.com/locate/ecss

Summer-time denitrification and nitrous oxide exchange in the intertidal zone of the Yangtze Estuary Dongqi Wang a,b, Zhenlou Chen a,*, Jun Wang a, Shiyuan Xu a, Hongxia Yang a, Hua Chen a, Longyuan Yang c, Lingzhen Hu a a

School of Resources and Environment Science, East China Normal University, Shanghai 200062, China b School of Life Science, East China Normal University, Shanghai 200062, China c Nanjing Institute of Geography & Limnology, China Academy of Science, Nanjing 210008, China Received 14 August 2006; accepted 8 November 2006 Available online 17 April 2007

Abstract Sediment denitrification rates and nitrous oxide (N2O) exchange fluxes in the Yangtze estuary intertidal zone (mudflats and salt marshes) were measured in summer-time using an acetylene inhibition technique and an in situ static closed chamber method. N2O natural production rates ranged between 0.10 mmol N m2 h1 and 8.50 mmol N m2 h1, and the denitrification rates ranged from 18.71 mmol N m2 h1 to 35.87 mmol N m2 h1. The N2O natural production rates in overlying water were low, with intertidal sediment being the source of overlying water N2O during the submerged period. Data analysis indicated that most sediment N2O was not derived from denitrification, but from several other nitrogen-cycling processes. During the low tide, the middle tidal marsh was the source of atmosphere N2O (exchange fluxes changed between 11.03 mmol N m2 h1 and 13.17 mmol N m2 h1). 5 cm and 10 cm depth ground temperatures were significant factors controlling the emission flux. At the low tidal flat, N2O emission flux rates ranged from 5.75 mmol N m2 h1 to 0.49 mmol N m2 h1 at the sedimentatmosphere interface. Overall, the middle tidal marsh was the source of atmospheric N2O, while the low tidal flat was a sink for atmospheric N2O. Plants of the intertidal zone (Scirpus mariqueter and benthic algae) were significant factors controlling N2O exchange flux. Photosynthesis of intertidal zone plants inhibited the emission of N2O and induced consumption, while plant respiration may enhance N2O emission from the intertidal zone. N2O emission and consumption at the intertidal zone-atmosphere interface correlated positively with the emission and consumption of CO2. Ó 2007 Elsevier Ltd. All rights reserved. Keywords: Yangtze Estuary; intertidal zone; denitrification; N2O; nitrous oxide exchange

1. Introduction The nitrogen load imported to estuarine and coastal areas has increased year by year with the increase of waste from human activity, agricultural runoff and industrial production. The average total nitrogen transported to the sea by rivers worldwide has reached about 40.6  1012 T N yr1 (Nedwell et al., 1999). Near-shore water eutrophication is a severe global environmental problem. Long-term data indicates that * Corresponding author. E-mail address: [email protected] (Z. Chen). 0272-7714/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.ecss.2006.11.002

nitrogen pollution results in rapid reduction of biological diversity in grass ecosystems and the extinction of natural ecological systems (Stevens et al., 2004; Thomas et al., 2004). Estuarine wetlands serve as a natural barrier in purifying land generated pollutants and in attenuating the load from the land to the sea (Nedwell and Trimmer, 1996). Wetlands play a key role in controlling marine eutrophication and the recycling of global nitrogen (Jickells, 1998). Estuarine sediment can eliminate 10e60% of the land generated nitrogen load (Nixon et al., 1995; Nedwell, 1996; Nedwell and Trimmer, 1996; Ogilvie et al., 1997; Stockenberg and Johnstone, 1997; Barnes and Owens, 1998; Berelson et al., 1998;

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Beusekom and Jonge, 1998; Trimmer et al., 1998), About one half of the total nitrogen load received by estuarine areas globally is removed by denitrification (Nixon et al., 1996). Under anoxic conditions, denitrifying bacteria switch to anaerobic respiration, using NO 3 as an electron acceptor instead of O2. NO 3 is reduced to N2 and some N2O, N2 and N2O are released into the atmosphere. N2 is an inert gas and abundant in the earth’s atmosphere, because denitrification converts NO 3 into gas, chronic N inputs will not cause this sink to become saturated with nitrogen (Martin et al., 1999), so denitrification is considered as a desirable means of removing nitrate from polluted water and water environments suffering from eutrophication. N2O is a major atmospheric greenhouse gases whose abundance in the troposphere has increased from a pre-industrial level of around 288 ppbv to 310 ppbv (ppbv ¼ 1012 volume concentration; Bauza et al., 2002). The ability of N2O to absorb solar radiation and induce global warming is about 190 to 300 times than that of carbon dioxide (CO2) and 4 to 21 times than that of methane (CH4). It has a lifespan in the atmosphere of about 150 years (Qi and Dong, 1999; Bauza et al., 2002), and thus a large greenhouse warming potential (Rodhe, 1990). It also contributes to ozone destruction resulting in stratospheric ozone depletion (Groffman et al., 2000). Thus increasing troposphere N2O concentration and the potential effect on global climate change requires serious attention. Increases in nitrogen loadings to estuarine and coastal environments stimulate microbial processes and associated N2O emissions (Bange et al., 1996; Seitzinger and Kroeze, 1998). Estuarine and coastal zones could account for about one half of the total ocean N2O emissions (Bange et al., 1996). Many recent research programs have focused on the production and emission of N2O in these areas (Barnes and Owens, 1998; Robinson et al., 1998; Groffman et al., 2000; Usui et al., 2001; Dong et al., 2002, 2004; Kenny et al., 2004; Punshon and Moore, 2004). In the past fifty years, Yangtze (Changjiang) river nitrate-N concentration have increased almost 14-fold for 1968 to 1997 (Yan et al., 2003), because of the effect of human activity (fertilizer application, waste water and sewage drainage, soil erosion, etc.). In the Yangtze estuary, nitrate concentration has increased from 16 mmol N l1 in 1961 (Gu et al., 1981) to about 65 mmol N l1 in 1980e 1981 (Edmond et al., 1985). In the past two decades, although nitrate concentration fluctuated, overall it was increasing continually (Shen et al., 2003) and reached about 75 mmol N l1 in 1998e1999 (Shen, 2000; Fu and Shen, 2002). Because of the drainage along the estuarine and coastal shore-line, the average tidal-flat water nitrate concentration is approximately 130 mmol N l1 (Chen et al., 2005). With the increasing nitrate concentration, denitrification progress will be accelerated and nitrous oxide production and emission would become prominent. As one of the biggest estuaries with a high water nitrate concentration, nitrogen cycling in this area, especially concerning, denitrification and nitrous oxide emission, would play an important part in global estuarine coastal zone nitrogen cycling. Only a few studies on sediment denitrification and N2O production and emission have been reported for

estuarine coastal zones in China (Xu et al., 2005; Wang et al., 2006). This study focuses on nitrogen cycling in the Yangtze estuary intertidal zones (marshes and flats). Intertidal zone sediment denitrification and intertidal zone-atmosphere interface N2O emission and consumption were investigated during the summer, using simulation incubation, acetylene inhibition technology and the in situ static sampling chamber. The data should provide better understanding of nitrogen cycling in the Yangtze estuarine coastal zone. 2. Materials and methods 2.1. Physical setting The Yangtze River (Changjiang River) is the longest river in China, and the third longest (6300 km) in the world. The water discharge is the fifth largest in the world, and its sediment load is the fourth largest (Hori et al., 2001). It carries more than 4.86  108 metric tonnes of sediment to the coast annually via its tremendous discharge, 9.0  1011 m3 water (Chen and Zhong, 1998). About 50% of the sediment is deposited in its estuary (Milliman et al., 1985; Chen et al., 1988). Because of the abundant sediment supply, the delta front develops seawards rapidly with the intertidal mudflats growing at an average rate of tens to hundreds meters per year in recent decades (Yang et al., 2001). Land reclamation of the intertidal mudflats reflects increasing land use in Shanghai, and in recent years, many parts of the Yangtze estuary intertidal flats have disappeared because of exploitation and reclamation. There remains about 800 km2 of active intertidal mudflats fringing the delta (Fan et al., 2006). The Yangtze estuary is in a typical semi-tropical monsoon area, mild and wet, with four distinct seasons. The yearly average temperature is 16  C, the long-term average annual maximum water temperature is 28  C (August), and the minimum water temperature is 6.7  C (February) (Shen, 2001). Rainfall is abundant with an average annual rainfall of 1144 mm (Xu, 1997). Intertidal zone sediment is composed mainly of fine particles. Natural geomorphology and depositing zones are distinct. High, middle, and low tidal flats develop from the land to the sea. The high tidal flat (marsh) sediment usually has the finest texture and contains black-gray silty clay and clayey silt. The reed (Phragmites communis) and the bulrush (Scirpus mariqueter) thrive on the flat, with continuous distribution across the ground. Middle tidal flat (marsh) sediment is composed of gray silty clay and silt, and the bulrush (S. mariqueter) is the dominant plant in this region. Low tidal flat (bare flat) sediments are relatively coarse, with silt and fine sandy silt as major constituents (Chen et al., 2001). 2.2. Sampling Six sampling locations were selected from the north bank to the south bank of Yangtze estuary. Those locations were Yinyang (YY), Chongming island (CM), Gulu (GL), Bailonggang (BLG), Chaoyang (CY) and Laogang tidal flat (LG) (see Fig. 1). Two sampling sites were located at CM, CY and LG,

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Fig. 1. Map of sampling locations.

respectively. One site was on the salt marsh and the other on the mudflat. There were only mudflat sampling sites at YY, GL and BLG because of the salt marsh had disappeared in reclamation. 2.2.1. Sediment cores and water collecting Nine small intact sediment cores (w10 cm) were collected during low tide at every sampling sites, using 24 cm long  3.5 cm i.d. (internal diameter) Perspex tubes, each equipped with diametrically opposed sets of 1.5 mm diameter silicon rubber-filled injection ports, and sealed with rubber bungs. These ports were aligned vertically at 0.5 cm intervals, facilitating injection of acetylene. 20 l of overlying seawater was collected using plastic buckets at each sampling site for inhibition incubation in the laboratory. Another sediment core was collected using a PVC tube (20  8 cm i.d.) at each site for sediment characterization analysis. Samples were carried to the laboratory after collection. 2.2.2. Air samples collecting CM was selected as the representative sampling location to measure the N2O exchange fluxes. Triple dark (opaque) and white (transparent) air sample collection chambers (50 cm net height  30 cm i.d.) were used to collect air exchange samples at the intertidal zone-atmosphere interface during the low tide. The dark chambers were constructed from a 0.4 mm iron sheet, covered by an insulating layer and

aluminum foil, to insulate and reduce heat transmission and reflect light. The white chambers were made of a 3 mm thickness Perspex cylinder. An electric fan was fixed in the chamber to mix air, a thermometer was installed to measure temperature (temperature inside the chamber changed from 29.5  C in the morning to 37.0  C in the afternoon in dark chambers and from 36.0  C to 44.2  C in white chambers), and a balance pipe was used to equalize the air pressure between the inside and the outside of the chamber (Khalil et al., 1990). Every chamber had a sampling pipe on top with a three-way air-tight valve. All the connections were made ‘‘air tight’’ and sealed by silicon rubber. Each air chamber was set on the ground at the middle tidal marsh and low tidal flat during low tide. Immediately after installing the chamber and again after 30 min, a 180 ml gas sample was drawn out from each chamber by a syringe with a three-way air-tight valve, and then was injected into special gas sampling bag, which is inert to greenhouse gases (Xu, 1999), for transport back to the laboratory and analysis by gas chromatography (GC). At the end of each time collection, chambers were opened until the next sampling. Air samples were collected every 1.5 h during the emergence (low tide) time in the sampling day. The day after air sampling on the marsh, six plots of the marsh plant Scirpus mariqueter, about 40  40 cm, were carefully cut and removed leaving just 1e2 cm stubble without disturbing the surface sediment, to measure the marsh

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sediment-atmosphere interface N2O exchange and compute the effect of S. mariqueter. Light luminosity (test by TES1332 photometer), air temperature, ground surface temperature and the ground temperature at both 5 cm depth and 10 cm depth (test by kerosene thermometer) were recorded synchronously with sampling. Air samples were sent to Taihu Lake Ecosystem Research Station, Nanjing Institute of Geography & Limnology, China Academy of Science (NIGLAS) after collection and analyzed within 3 days. 2.3. Incubation and sampling in the laboratory Acetylene can inhibit the process of microbes reducing N2O to N2. The Acetylene Inhibition Technique (AIT) was developed based on this principle (Yoshinari et al., 1977; Sørensen, 1978). This method provides a simple and precise way to measure denitrification rates. But Acetylene could blocks both denitrification and nitrification and it has incomplete inhibition under low nitrate concentrations (<10 mmol N l1) (Knowles, 1990), therefore, this method tends to underestimate denitrification in systems where nitrate pools are small or if the nitrification and denitrification processes are coupled. Tidal flat overlying water nitrate concentrations are higher in the Yangtze estuary (most >100 mmol N l1, only at CM, it >50 mmol N l1). Denitrification only removed about 5 w 10% NO 3 of water body during our three hour incubations. The NO 3 concentration in overlying water did not decrease greatly so the sediment denitrification would be supported by nitrate from the overlying water. This result suggested that the acetylene inhibition should work in this system. In view of the high dissolved oxygen (DO) concentration in overlying water (>6.9 mg l1 at all of the sites), nitrification would take place in the surface layer of the sediment because of DO penetration although nitrification was not measured in this research, so coupled nitrification-denitrification cannot be measured by AIT because acetylene inhibits nitrification progress. The data was only the NO 3 denitrification rate and did not include the coupled nitrification-denitrification rate; the AIT method might thus underestimate the rate of denitrification in this system. 2.3.1. Sediment denitrification and N2O production Denitrification and N2O production rates were measured as described by Ogilvie et al. (1997). Sediment cores were placed in custom-built, black glass, incubation tanks at the laboratory. Overlying seawater was carefully added to the tank, avoiding disturbance of the sediment surface greatly. An electromagnetic pump was used to pump the air into every sediment core tube and the tank. Sediment cores were equilibrated for 12 h in the black tank to minimize the effect of the disturbances during the sampling, transporting and incubation instrument setting. After equilibration, nine sediment cores from each site were divided into three subset groups. The first group was the ‘‘beginning cores’’, sacrificed at the beginning of the inhibition experiment. The top 2 cm sediment of the core was gently stirred

in order to homogenize the dissolved N2O pools in the sediment and the water column. The 20 ml sub-samples of the slurry were withdrawn quickly and injected into 40 ml air-tight vials, preequipped with 100 ml 38% formaldehyde, and the lid was tightened quickly. The second group was the ‘‘natural samples.’’ Cores were left untreated, at the beginning of the inhibition experiment, adding the air-tight rubber stopper quickly, and placed in the blank tank. The third group was the ‘‘acetylene inhibition samples’’. Ten ml acetylene-saturated seawater, made up from the overlying water, was added to each tube and 1 ml acetylene-saturated seawater was injected in to the top 2 cm of sediment via the silicon rubber-filled injection ports at 90 to give a final acetylene partial pressure of 10 kPa. The rubber stopper was inserted quickly, and cores were placed and incubated in black tank with the natural samples. After 3 h incubation, sub-samples were collected as ‘‘beginning cores.’’ Laboratory air was sampled at the same time as the core samples to test the background value of laboratory air. 2.3.2. Water column N2O production and consumption Nine identical blank tubes were placed in every site incubation tank, along with the sediment cores, to measure possible denitrification and N2O production rates in the water column. Sub-samples were collected as sediment cores, but 20 ml of acetylene-saturated seawater was added into the acetylene inhibition cores since water volume was about twice that of the sediment core because blank core tube contained no sediment. 20 ml water samples of sea water were collected at the beginning and the end of each inhibition, filtrated by a 0.45 mm filter, and stored at 0e4  C in a refrigerator for the analysis of  þ dissolved inorganic nitrogen (DIN ¼ NO 3 þ NO2 þ NH4 ), two drop saturated HgCl2 was added to kill the bacteria. Water samples were analyzed within 3 days. 2.4. Analysis Water samples were analyzed for the DIN. NO 3 was measured using colorimetry after Zn-Cd reduction, NO 2 using the sulfanilamide and N-(1-naphthyl) ethylene diamine dihydrochloride colorimetry, NHþ 4 using sodium hypobromite oxidation colorimetry (Han, 1986). The sea water salinity and dissolved oxygen (DO) were measured by YSI-30 and CENTRY M-2 portable analyzer respectively with temperature parameter record; N2O was analyzed by a gas chromatograph, an HP5890II, equipped with an electron capture detector (ECD) for the detection of N2O. The chromatograph and analyzing conditions were as follows: #1, #2 chromatograph columns, #3: SS-1 m  2 mm packed with Poropak Q (80/100), #4: SS-3 m  2 mm packed with Poropak Q (80/100); High purity N2 as the carrier gas for ECD (30 cm3 min1); the ECD temperature was 300  C; the column temperature was 55  C. The organic carbon content of the sediment was analyzed by the K2CrO7-FeSO4 $ 7H2O method (Bao, 2000) and the chlorophyll a content via the cool-acetone method (SEPAC, 2002). SPSS was used for correlation analysis between N2O fluxes and environmental factors.

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3. Results

Table 2 N2O concentration change in overlying water. *Mean  SE, n ¼ 3

3.1. Environmental factors

Sampling location

Blank water (nmol l1 h1)

Inhibition blank water (nmol l1 h1)

The environmental factors at the six sampling locations are shown in Table 1. In summer, Yangtze estuarine tidal flat sea water temperature is high because the shallow water. South channel is the main discharge channel; water salinity at north bank location (YY) is higher than island and south bank locations and the salinity increased from inner estuary to seaward (from GL to BLG, CY, and LG). Because of wind disturbing, DO is high in water column. Because of the drainage along the estuarine, coastal bank line Water DIN concentration in tidal flat overlying water is higher than the data in estuary (Chen et al., 2005), which reported by Shen (2000), Fu and Shen (2002) and Shen et al. (2003), beside of at CM, which is located at estuarine island and affect little by sewerage. Sediment organic carbon content is lower at CY and higher at GL according to the topography and water dynamic difference.

CM BLG CY LG

0.60  0.62 0.26  0.25 0.29  0.13 0.34  0.17

0.07  0.48 1.14  0.04 0.85  0.14 1.08  0.08

BLG-L, CY-L and LG-L) (see Fig. 2). This result may be related to the higher water temperature and DIN content at YY (see Table 2). Between the different tidal flat physiognomy zones, the low tidal flat sediment (CM-L, CY-L, LG-L) denitrification rates were lower than those of the middle tidal marshes sediment (CM-M, CY-M, LG-M) (see Fig. 2). The natural production rates of N2O in sediment ranged from 0.10 to 8.50 mmol N m2 h1, GL-L and BLG-L location sediments were the stronger N2O emission sources (see Fig. 2). 3.4. Marsh zone sediment-atmospheric interface exchange of N2O during emergence

3.2. Water column N2O production and consumption In CM tidal flat overlying water, N2O production and consumption rates were very low (see Table 2); changes in N2O concentration ranged from 0.34 to 0.60 nmol N l1 h1. Considering the volume (0.096 l) and cross sectional area (9.62  104 m2) of the incubation core, this process had little effect on the N2O sediment-water exchange (from 0.034 to 0.060 mmol N m2 h1). 3.3. Denitrification in sediments and sediment-water interface exchanges of N2O during submergence Denitrification rates in the Yangtze estuary tidal sediment were between 18.71 and 35.87 mmol N m2 h1 during the submergence of the tide. Those were same magnitude but lower than the denitrification in North Atlantic continental shelf sediments within the same latitude range (20 N w 40 N, average denitrification rate was 54.17 mmol N m2 h1, Seitzinger and Giblin, 1996). Spatial differences in distribution of Yangtze estuary intertidal zone sediment denitrification rates were not very large. The denitrification rate at the north bank YY low tidal flat site (YY-L) was higher than that at the CM and south bank locations low tidal flat (CM-L, GL-L,

3.4.1. Marsh zone sediment-atmospheric N2O emission and consumption when vegetation was cut CM marsh sediment (CM-MS) was a source of atmospheric N2O (see Fig. 3). CM-MS in the dark chambers without vegetation (CM-MSD) was a source of atmospheric N2O during the first sampling. However, CM-MS in the white chambers (CM-MSW) consumed some atmospheric N2O. This interface consumption became faint in the second time sampling, indicating that consumption and emission were almost balanced. The N2O emissions increased gradually in both the white chambers and the dark chambers after third sampling, probably due to increasing ground temperature. The difference between the emission flux in the white chambers and in the dark chambers may have resulted from algal photosynthesis and respiration on the tidal flat sediment surface (chlorophyll a content of CM-MS: 5.4  0.89 mg g1, n ¼ 6) and temperature differences. 3.4.2. Marsh zone-atmosphere interface N2O emission and consumption CM marsh (CM-M) in dark chambers (CM-MD) was the source of atmospheric N2O, N2O emission flux ranged

Table 1 Environment factors at sampling locations. *Mean  SE, n ¼ 3. **Value of top 3 cm sediment Sampling location

Water temperature ( C)

Salinity

DO (mg l1)

NO 3* (mmol l1)

NHþ 4* (mmol l1)

YY CM GL BLG CY LG

29.0 28.5 30.0 23.8 23.9 24.0

16.5 0.2 0.1 0.2 3.8 4.5

8.4 7.4 7.6 7.0 6.9 7.3

184.8  0.0 56.55  5.7 147.7  0.0 103.6  2.5 176.0  27.2 125.1  10.5

14.3  0.0 3.9  1.6 10.2  0.0 7.8  4.1 7.3  1.4 18.8  5.4

NO 2* (mmol l1)

Organic carbon (&)** Middle tidal flat

Low tidal flat

1.0  0.1

3.3

1.1

2.5  0.0 1.3  0.1 3.0  0.2

1.1 7.1

5.1 1.4 6.1

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Fig. 2. Denitrification and natural N2O production rates in sediment during submergence.

between 9.22 and 13.17 mmol N m2 h1. In white chambers (CM-MW), marsh consumed N2O, consumption fluxes decreased gradually from morning (11.03 mmol N m2 h1) to noon (2.89 mmol N m2 h1). In the afternoon, atmospheric N2O was emitted (flux between 0.42 and 0.51 mmol N m2 h1). The exchange direction difference in dark chambers and white chambers indicated that the photosynthesis of Scirpus mariqueter restrained the production of N2O in sediment, and even induce the extra consumption. But N2O production and emission rate would be enhanced, because the ground temperature increased as the day progressed and may have counteracted the effect of S. mariqueter; the N2O consumption decreased in white chambers from morning to noon (see Fig. 4). The emergence of the tidal flat surface allowed oxygen to penetrate into the sediment and accelerate the nitrification process in the sub-surface layer of the sediment to produce N2O

during low tide, although the sediment remained saturated or near saturated with water. At the same time, nitrification may have provided more NO 3 for denitrification in the anoxic layer under the sediment surface layer, thus producing more N2O. Emergence during low tide made the Scirpus mariqueter zone a strong emission source of atmospheric N2O, and the emission flux was one to two orders greater than that during submergence.

3.5. Low tidal flat-atmosphere interface N2O emission and consumption during emergence Because the emergence time at low tide is shorter in CM low tidal flat (CM-L) than in the middle tidal marsh, air samples at this site were collected four times from early morning to noon, at one and a half hour intervals.

Fig. 3. CM marsh sediment-atmosphere interface N2O exchange without vegetation.

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Fig. 4. CM marsh-atmosphere interface N2O exchange.

During the tidal cycle, re-suspension damaged benthic algae growth on the low tidal flat sediment surface, but the living benthic algae remaining on the sediment surface was sufficient for investigation (chlorophyll a content of CM-L surface sediment: 2.0  0.53 mg g1, n ¼ 6). The tidal flat-atmosphere interface exchange of N2O was weak (flux between 1.31 mmol N m2 h1 and 0.49 mmol N m2 h1) in CM-L dark chambers (CM-LD); there is a little emission at the first and faint consumption in the other three samplings (see Fig. 5). In CM-L white chambers (CM-LW), algae photosynthesis inhibited N2O emission and induced the low tidal flat to consume atmospheric N2O (see Fig. 5). The flats absorbed atmospheric N2O, with consumptive fluxes between 5.75 mmol N m2 h1 and 1.47 mmol N m2 h1. 4. Discussion 4.1. Denitrification and N2O production rate in Yangtze estuarine tidal flat sediments Environment factors have an effect on the denitrification process (Martin et al., 1999). The highest denitrification rates

Fig. 5. CM low tidal flat-atmosphere interface N2O exchange.

in USA Texas bay sediment appeared in the upper estuary in summer. Temperature, organic carbon (OC) content and salinity were the three most important factors, explaining 52%, 28% and 15% of the variation, respectively (Zimmerman and Benner, 1994). The character of the sediment was also an important factor; the coarser sediment may have enhanced the diffusion of overlying water into the sediment, and provided NO 3 for denitrification (Casey et al., 2004). Likewise, abundant OC in the sediment would benefit denitrification (Saunders and Kalff, 2001). The Yangtze estuary is a typical semi-tropical monsoon estuary where environmental factors change greatly in different seasons. It is certain that denitrification would have great seasonal changes as well. In this summer study, we still could not get any significant correlation between the denitrification rates and environment factors. The temporal and spatial changes in Yangtze estuary and the effect of them on the denitrification process need research over the whole year. In most estuarine and coastal area, where the nitrification in sediment is weak, sediment denitrification is supported by the overlying water NO 3 , which diffuses into sediment through sediment-water interface. NO 3 concentration of water column was the primary factor controlling estuarine sediment denitrification, and sediment denitrification rates appeared to obey saturation kinetics, becoming NO 3 saturated at water column 1 NO concentrations >200 mmol l (Ogilvie et al., 1997). In 3 summer, average denitrification rates in the Yangtze estuary tidal sediment was 27.42 mmol N m2 h1. It is lower than those in estuaries with high NO 3 concentrations (most were >400 mmol l1) (Ogilvie et al., 1997; Barnes and Owens, 1998; Trimmer et al., 1998), and higher than those in the re1 gions with low NO 3 concentrations (most <30 mmol l ) (Conley et al., 1997; Wang et al., 2003). The middle tidal marshes and low tidal flats water had the same environment factors, such as water temperature, salinity and DO, during the incubations. The difference in sediment characteristics between the marshes’ and flats’ sediment, such as particle size character, material content and so on,

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may be the primary reasons of denitrification rate differences between the two tidal flats. Denitrifying bacteria in wetland sediments to be more abundant in the spring/summer compared to the fall/winter. Degradation organic carbon promoted the higher denitrification rates (Sirivedhin and Gray, 2006). In addition, sediment nitrification rates accelerated under higher summer temperatures, and may have provided adequate NO 3 to support the denitrification rates (Jensen et al., 1993; Nowicki, 1994). Higher organic carbon content may have enhanced sediment denitrification rates in the middle tidal marsh. Denitrification in the fine-textured soils presented significantly higher rates than coarse ones (Pinay et al., 2000). Sediment denitrification rates did not correlate significantly to these main environment factors. In Yangtze estuary intertidal zones, environmental factors change greatly and have a complex effect on microbial activity. GL-L and BLG-L location sediments were the stronger N2O emission sources being markedly different to the other locations (see Fig. 2). GL and BLG are near the drainage of Shanghai City and the particles of drainage water settle on the GL and BLG tidal flat. The microbial community in these particles may be different from that in sediments from other locations and produce more N2O during nitrogen cycling. Sediment denitrification did not have a significant correlation to natural N2O production at all sampling site. The magnitude of the natural N2O production rate followed denitrification rate and indicating that the N2O came from the denitrification and not nitrification Ogilvie et al. (1997). Not considering the dissimilation of nitrate, N2O was about 2% of the denitrification production (Nedwell, 1996; Ogilvie et al., 1997). In our research the GL-L and BLG-L sediment natural N2O production rate reached more than 30%, and at YY and LG, it also exceeded 2%. So most natural sediment N2O production might come from nitrification rather than denitrification in the Yangtze estuarine intertidal zone where the DO condition is ideal for nitrification.

4.2. The correlation between environment factors and N2O exchange fluxes Environmental factors obviously changed during air sample collection throughout the day, These factors include sunlight luminous flux (SLF), air temperature (AT), sampling chamber temperature (SCT), surface ground temperature (SGT), ground temperature at 5 cm depth (5 cm GT) and ground temperature at 10 cm depth (10 cm GT). Correlation analysis between these factors and N2O emission and consumption indicated that at depths of 5 cm and 10 cm the ground temperature had a significant positive correlation to N2O emission, except in the dark chamber having Scirpus mariqueter, in which correlation test was not significant but the correlation was higher than 0.5 (see Table 3). N2O diffusion velocity increased at higher temperatures. The activity of microbes in the sediment is also enhanced with increasing temperature. We conclude that N2O emission flux was positively correlated with ground temperature.

4.3. The correlation between N2O fluxes and CO2 fluxes At the middle tidal marsh (Scirpus mariqueter community), seven quadrats (0.5  0.5 m2) were selected randomly and investigated for the height and abundance of S. mariqueter. After air sample collection at the marsh and flat, a 1 w 2 mm thick layer was shaved from the top of the sediment in the air chamber and used to measure the sediment chlorophyll a content, to calculate the benthonic algae abundance on the sediment surface. The abundance of S. mariqueter was 3136  500 m2 (n ¼ 7) and the height of it was 51.4  3.3 cm (n ¼ 7). Chlorophyll a content in CM-M surface sediment was 5.4  0.89 mg g1 of wet sediment (n ¼ 6) and 2.0  0.53 mg g1 of wet sediment (n ¼ 6) at CM-L. The CO2 emission and consumption flux data for Fig. 6 was collected from the interrelated research work taken simultaneously at the same sampling sites (Yang et al., 2006). The CM intertidal zone-atmosphere interface N2O emission and consumption correlated significantly to the photosynthesis and respiration of intertidal plants (Scirpus mariqueter and benthonic algae) and the respiration of tidal flat sediment (Pearson correlation, R ¼ 0.956; 2-tailed test was highly significant, p z 0.000). The photosynthesis and respiration of tidal flat plants (Scirpus mariqueter and benthonic algae) was one of the main factors controlling N2O emission from sediment. When photosynthesis was stronger, more oxygen was released from the sediment surface algae or the roots of the S. mariqueter, a process which may have restrained the denitrification process. The microbial activity, reducing the N2O into N2, may have been restrained because of oxygen exudation from the roots. The N2O microbe metabolism in the sediment may have absorbed atmospheric N2O and caused the intertidal zone to become a sink for atmospheric N2O. Previous research indicates that some plants absorb or emit N2O via their photosynthesis and respiration (Chen et al., 2003), but more research is needed to prove whether S. mariqueter and algae have an N2O metabolism process. In the dark air chambers, plant respiration made the sediment more anoxic, which may have enhanced the denitrification process. CO2 and N2O emissions increased together. At CM marsh, plant photosynthesis decreased N2O emission flux at 14.0 mmol N m2 h1 on average. N2O emission flux induced by respiration was about 6.18 mmol N m2 h1 on average. At CM flat, sediment surface algae photosynthesis induced the consumption flux from 1.96 mmol N m2 h1 to 4.44 mmol N m2 h1.

4.4. Conclusions In summer, N2O production rates were low in the tidal water body in Yangtze estuary, ranging from 0.34 nmol N l1 h1 to 0.60 nmol N l1 h1. During submergence, the denitrification rate in Yangtze estuarine tidal flat sediment was from 18.71 mmol N m2 h1 to 35.87 mmol N m2 h1; and the intertidal zone sediment was the source of overlying water N2O

D. Wang et al. / Estuarine, Coastal and Shelf Science 73 (2007) 43e53

51

Table 3 Correlation between N2O exchange fluxes and environment factors. *Correlation is significant at the 0.05 level. **Correlation is significant at the 0.01 level SLF (LUX) Having plant

CM-MD CM-MW

Without plant

CM-MD CM-MW

Samples Number

Correlation Sig. (2-tailed) Correlation Sig. (2-tailed) Correlation Sig. (2-tailed) Correlation Sig. (2-tailed) N

0.535 0.172

0.312 0.452 8

AT ( C)

SCT ( C)

SGT ( C)

5 cm GT ( C)

10 cm GT ( C)

0.487 0.221 0.351 0.393 0.683 0.062 0.870** 0.005 8

0.683 0.062 0.819* 0.013 0.535 0.172 0.705 0.051 8

0.470 0.240 0.883** 0.004 0.704 0.051 0.808* 0.015 8

0.556 0.152 0.941** 0.000 0.848** 0.008 0.927** 0.001 8

0.574 0.137 0.922** 0.001 0.953** 0.000 0.993** 0.000 8

(natural production rate was between 0.10 mmol N m2 h1 and 8.50 mmol N m2 h1). During the low tide, intertidal marsh became an atmospheric N2O emission source in dark chambers (fluxes were between 9.22 mmol N m2 h1 and 13.17 mmol N m2 h1), and a sink in white chambers from early morning to noon (fluxes were between 11.03 mmol N m2 h1 and 2.89 mmol N m2 h1). But in the afternoon, the marsh-atmosphere exchange was nearly balanced (fluxes were between 0.42 mmol N m2 h1 and 0.51 mmol N m2 h1). On the whole, CM intertidal marsh was an emission source of atmospheric N2O when it emerged. In the dark chambers, low tidal flat N2O exchange at the flatatmosphere interface was between 1.31 mmol N m2 h1 and 0.49 mmol N m2 h1 during emergence. In the white chambers, low tidal flats absorbed atmospheric N2O (fluxes ranged from 5.75 mmol N m2 h1 to 1.47 mmol N m2 h1). CM low tidal flat was a sink for atmospheric N2O during the low tide. The photosynthesis of CM intertidal plants may decrease N2O emission, and even induce consumption, and their respiration could increase N2O emissions. Plants were the main factors controlling N2O emission or consumption. Intertidal

zone-atmosphere interface N2O emission and consumption flux were positively correlated to CO2 emission and consumption.

Acknowledgements This study was supported by the National Natural Science Foundation of China (Grant No. 40173030, 40571006, 40131020), the Science & Technology Department of Shanghai (Grant No. 05DZ12007, 05JC14059), and the PhD Program Scholarship Fund of ECNU 2005. The authors would like to thank the Nanjing Institute of Geography & Limnology of the China Academy of Science (NIGLAS) and the Taihu Lake Ecosystem Research Station of NIGLAS, plus all of the laboratory persons providing their assistance in samples analysis. The authors also would like to thank Dr. Wayne S. Gardner for his helpful comment and linguistic and editorial correction and two anonymous reviewers aided in the development and improvement of this paper.

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Fig. 6. Correlation between N2O and CO2 exchange fluxes at the tidal flatatmosphere interface.

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