Anthropogenic nutrient loads and season variability drive high atmospheric N2O fluxes in a fragmented mangrove system

Fragmented mangroves are generally ignored in N2O flux studies. Here we report observations over the course of a year from the Mangalavanam coastal wetland in Southern India. The wetland is a fragmented mangrove stand close to a large urban centre with high anthropogenic nitrogen inputs. The study found the wetland was a net source of N2O to the atmosphere with fluxes ranging between 17.5 to 117.9 µmol m−2 day−1 which equated to high N2O saturations of between 697 and 1794%. The average dissolved inorganic nitrogen inputs (80.1 ± 18.1 µmol L−1) and N2O emissions (59.2 ± 30.0 µmol m−2 day−1) were highest during the monsoon season when the rainfall and associated river water inputs and terrestrial runoff were highest. The variation in N2O dynamics was shown to be driven by the changes in rainfall, water column depth, salinity, dissolved oxygen, carbon, and substrate nitrogen. The study suggests that fragmented/minor mangrove ecosystems subject to high human nutrient inputs may be a significant component of the global N2O budget.

www.nature.com/scientificreports/ Along the Indian coast (7,516.6 km long 7 ), mangrove cover an area of 497,500 ha 8 . The India state Kerala in southern India, was once rich in mangrove habitats, but the area of intact mangrove has drastically declined due to anthropogenic pressures such as urbanisation, burgeoning power projects and rapid industrialisation. It is estimated that the Ernakulum district in Kerala has 943 mangrove stands of which 880 (~ 93%) of them have an area less than 1 hectare. The contribution of these smaller mangrove systems is often ignored and their role in global nutrient cycling has yet to be adequately determined. As most of these mangrove stands are generally observed near the vicinity of human settlements, they can be critically influenced by the N loading there. Recent global studies suggest that aquatic environments influenced by intensive agricultural practices and industrial activities can act as source of N 2 O to the atmosphere [9][10][11] and this predicted rise in terrestrial N concentrations can potentially triple the global N 2 O emissions from coastal wetlands 1 . However, Indian mangrove stands have been mostly ignored from the N input and output studies due to their relatively smaller area and footprint. The few studies that have taken place in Indian mangroves suggest that they are highly influenced by anthropogenic nutrient inputs and are significant sources of atmospheric N 2 O [12][13][14] . In light of the increasing anthropogenic N inputs, it is important to monitor and assess the potential of fragmented/minor mangrove stands for their contribution of N 2 O to the atmosphere.
In order to understand the contribution of fragmented/minor mangrove stands to atmospheric N 2 O emissions, this study aimed to assess the Mangalavanam Coastal Wetland (MCW) a fragmented minor mangrove stand adjacent to the city of Kochi, India over a one year period incorporating a range of distinct seasons. The N 2 O flux rates were calculated using three different k models [15][16][17] to reduce the uncertainty in calculated N 2 O flux rates. The study also tested the relation between N substrates and N 2 O fluxes with temperature, pH, DO, and C loading of the wetland to determine the drivers of N 2 O dynamics. Study area. The MCW is a semi-closed fragmented mangrove stand in the Ernakulam district along the south-west coast of India at 9° 59′ 23.83″ N and 76° 16′ 26.74″ E (Fig. 1). It receives tidal input through a narrow channel/feeder canal from the adjacent nutrient rich Cochin Estuarine System (CES), where eutrophication is of great concern 18 . This tidal wetland has a wet and maritime tropical climate with three distinct seasons: premonsoon (February to May), southwest monsoon (June to September, from here on called monsoon) and northeast monsoon (October to January) or post-monsoon. The wetland is relatively shallow (< 0.5 m) and is largely inundated during high tide flooding. The dominant mangrove species present in the MCW are Rhizophora mucronata, Avicennia officinalis and Acanthus ilicifolius. Mangrove associates such as Acrostichum aureum, Derris trifoliate and Morinda citrifolia are also common. Kochi is the most densely inhabited city in Kerala with an  Carbon and nitrogen measurements. The total nitrogen (TN) concentration in the water column ranged from 172.1 to 877.1 µmol L −1 , with an annual mean of 399.4 ± 244.8 µmol L −1 and in the sediments it ranged from 158.6 to 301.4 µmol g −1 ( Table 1). The mean TN in the water column was highest during the monsoon season and the lowest during post-monsoon season, while in sediments the maximum concentration was observed during the post-monsoon season and the minimum during the pre-monsoon season. Dissolved nitrogen (DN) contributed 66 to 88% of the observed TN with an annual mean of 322.1 ± 182.0 µmol L −1 and varied significantly on temporal basis (f = 9.16, ρ = 0.002). Dissolved inorganic nitrogen constituted 12-41% of the DN concentrations with an annual mean of 60.7 ± 18.1 µmol L −1 , while DON was 58-88% of the DN concentration with an annual mean of 248.6 ± 168.9 µmol L −1 . The DIN and DON concentrations in the water column varied significantly on seasonal basis (f = 17.15, ρ ˂ 0.001 and f = 8.26, ρ = 0.003, respectively) with higher DIN and DON in the monsoon season. NO 3 was the major constituent of DIN, with a mean of 62 ± 8% of DIN. NO 3 was followed by NH 4 and NO 2 , constituting about 36 ± 8% and 2 ± 1%, of the DIN respectively. There was a significant difference in water column NH 4 (f = 8.89, ρ = 0.002), NO 2 (f = 15.47, ρ < 0.001) and NO 3 (f = 11.89, ρ = 0.001) seasonally. Particulate nitrogen (PN) in the water column ranged from 20.1 µmol L −1 to 232.9 µmol L −1 during the study period and varied significantly between seasons (f = 8.76, ρ = 0.002). The total carbon (TC) concentration in the water column ranged from 2473.3 to 7391.7 µmol L −1 (average 3916.3 ± 1453.0 µmol L −1 ) with total organic carbon (TOC) contributing 33 ± 10% of the observed TC concentrations (range from 484.2 to 3792.5 µmol L −1 ) which varied significantly on seasonal basis (f = 4.63, ρ = 0.02). In the sediment, TOC concentrations ranged between 2877.5 µmol g −1 to 9911.7 µmol g −1 over the study period. In the water column, the mean TOC concentration was highest during the monsoon season and lowest during the post-monsoon season, while in sediments, the maximum concentration was observed during the post-monsoon season (6230.3 ± 2292.4 µmol g −1 ) and the minimum concentration was observed during the pre-monsoon season (4000.8 ± 1323.8 µmol g −1 ). The total inorganic carbon was the major constituent of TC in the water column, with an annual mean of 2556.4 ± 771.6 µmol L −1 , ranged from 1578.3 µmol L −1 in July to 4605.0 µmol L −1 in November and varied significantly between seasons (f = 7.57, ρ = 0.003).
Nitrous oxide dynamics. The dissolved N 2 O concentration in the water column of the MCW ranged from 50.3 nM in November to 131.5 nM in June (Fig. 3, Table 1). The mean percentage saturation of N 2 O was 1088 ± 252% and ranged from 696 to 1794%, with relatively little seasonal variations. Using Wanninkhof 15

Discussion
This study found that the MCW fragmented mangrove stand is a C and N rich environment which favours substantial N 2 O production and its release into the atmosphere. The N pool and the atmospheric flux of N 2 O in the MCW (39.1 ± 22.6 μmol m −2 day −1 ) was found to be higher than that reported in the adjacent CES (11.4 ± 6.9 μmol m −2 day −1 ) where there are significant anthropogenic N inputs 19 . In the Bhitarkanika mangrove on the east coast of India 12 , high N 2 O emissions were observed (5-113.38 μmol m −2 day −1 ) driven by high anthropogenic nutrient loading. In other Indian mangroves-Muthupet mangrove in southern India, comparatively low N 2 O emissions (9.3-18.18 μmol m −2 day −1 ) were reported even though the mangrove is subjected to a range of anthropogenic inputs including aquaculture (shrimp-farming effluent) and seasonal agricultural run-off 13  www.nature.com/scientificreports/ observations were also in the range of previous studies from the large eutrophic Pearl River Estuary in China (37 ± 15 μmol m −2 day −1 ) which receives extensive urban, industrial and agricultural run-off 10 .
In a range of pristine mangroves in Australia 20 , the uptake of N 2 O has been reported with an uptake rate of 1.52 ± 0.17 μmol m −2 day −1 . However, the relative pristine mangrove site selected in our study (Site 2) was supersaturated with dissolved N 2 O (1122.8 ± 266.9%) and acted as atmospheric source of N 2 O (41.0 ± 26.1 μmol m −2 day −1 ). A recent study on N 2 O dynamics from four southern hemisphere subtropical www.nature.com/scientificreports/ estuaries 11 (an urbanised estuary, mixed, urban and agricultural estuary and pristine estuary) reported that the N 2 O saturation ranged from 77.7 to 381.5% with highest saturations in the pristine estuary and lowest in the agricultural estuary, which was much lower than the range seen in this study (696.7-1794.3%). The study also reported that estuaries in the two urbanised catchments had the highest mean N 2 O flux rates during summer (18.8 ± 11.6 μmol m −2 day −1 and 18.7 ± 12.8 μmol m −2 day −1 , respectively) with the pristine estuary having a summer flux rate of 5.1 ± 6.9 μmol m −2 day −111 . Other studies from estuaries in urbanised catchments have reported low mean N 2 O flux rates, with 4.0 μmol m −2 day −1 reported in the Yarra River in Melbourne 21 , and 7.3 μmol m −2 day −1 in the Brisbane River 22 . The high flux rates observed here are likely a result of high rainfall in the tropics driving higher freshwater inputs, terrestrial runoff and the enhanced anthropogenic N enrichment compared to other estuaries cited above. The pristine site (Site 2) also received tidal inputs from the nutrient enriched CES which would lead to higher N 2 O concentrations in the water column compared to Site 1 that was heavily influenced by upstream estuarine inputs. Also, the water column of Site 1 drains out completely into the CES during low tides; however the water column depth of Site 2 was less affected during low tides which likely reduced the export of N to adjacent waters. Significant uncertainties exist in global N 2 O budget due in part to the differences in methods used in calculating flux rates. Two meta-analyses published in the past 20 years highlight that the lack of direct measurements of k significantly hampers the ability to parameterize air-water gas exchange in estuaries and that care should be given when choosing values of k, with respect to location-dependent controls on gas exchange 17,23 . Here we offer a comparison of the different flux rates calculated using three different and commonly used k value models (Table 1) and the relationship of various measured parameters to N 2 O flux rates calculated using three models ( Table 2). The parameterization of Raymond and Cole 17 is widely used for rivers and streams, where the turbulence reaching the air-water interface is chiefly generated by friction with the bottom. The parameterization of Wanninkhof and McGillis 16 is best used for open water bodies but can underestimate N 2 O flux rates where wind speeds are low such as sheltered mangrove systems. The k value model of Wanninkhof 15 is also commonly used and uses the Schmidt number of the water related to viscosity and with its exponent reflecting the surface layer's rate of turbulent renewal. As water column depth, wind speed, and current velocity act as main drivers of these k models and influences turbulence at the air-water interface, in shallow flowing ecosystems 24,25 , such as in mangroves, we felt the k value model of Wanninkhof 15 best fits for the study. www.nature.com/scientificreports/ The study observed a relationship between N 2 O dynamics and the availability of nitrogen species ( Table 2). Concentrations of TN, DN, DON, NO 3 , NH 4 and NO 2 in the study area were high and at the high end of the range of concentrations seen in other highly polluted estuaries 26 . MCW is located in the heart of the densely populated Kochi city and has several sewage inlets from surrounding human settlements. This significantly increases the availability of N in both sediments and the water column leading to higher N 2 O production in the study area. Recent studies have shown that sewage influxes increase N 2 O production 19,22 . The release of nitrogenous compounds through the excreta of water-birds can also cause N enrichment in mangroves 26 and the abundant water bird population of MCW would also lead to higher nitrogen loads in the study sites.
The MCW also receives significant N inputs through the narrow channel from CES that receives substantial fresh water discharge (approx. 1.2 × 10 10 m 3 year −1 ) from the six major rivers in the area (Pamba, Manimala, Achancoil, Meenchil, Periyar and Muvattupuzha Rivers) during the monsoon season 27,28 and also receives inputs  (Table 2). Similar observations of increased N loads and associated N 2 O fluxes due to rainfall have been reported in the eutrophic Taihu Lake in China 31 . The high resident time of the water column in the study lead to greater accumulation of N in the water column and longer processing times of N before being exported which likely influence N 2 O production and flux rates in the study area. High N and organic carbon concentrations have been shown to accelerate the nitrification-denitrification processes 31-33 and lead to higher N 2 O production although C and N concentrations did not show a clear relationship to dissolved N 2 O concentration in the MCW. Non-significant relationships between dissolved N 2 O concentration and DIN have been observed in other recent studies [34][35][36] . However, during this study significant relationships were observed between TC, TIC, TN, DN, DIN, NO 3 , NH 4 , DON, PN and N 2 O flux calculated using Wanninkhof 15 (Table 2). This indicates a consistent relationship existed between N, C inputs and N 2 O outputs. Significant relationships between DIN inputs and N 2 O fluxes have also been shown in the nearby CES in India 19 , rivers 37 and estuaries globally 38 . Positive relationships were also observed between N 2 O and NH 4 and NO 3 + NO 2 during the summer dry season, while during winter, N 2 O saturation was strongly correlated to NO 3 + NO 2 but not with NH 4 11 . The significant positive correlation between NO 3 and NH 4 concentrations to N 2 O flux calculated using Wanninkhof 15 and strong negative correlation between DO and NH 4 (r = − 0.558, ρ = 0.005), NO 2 (r = − 0.586, ρ = 0.003) and NO 3 (r = − 0.788, ρ ˂ 0.001) suggest high N 2 O flux rates produced through nitrification. However, lower DO during the monsoon months along with higher availability of NO 3 in the presence of high organic carbon loading likely promotes denitrification increasing N 2 O production and its fluxes. Several studies suggest that estuaries receiving higher N inputs generally show oxygen depletion, which in turn triggers denitrification 34,39,40 . Increasing anthropogenic nitrogen inputs to the mangrove sediments along with periodic tidal flooding can generate anoxic conditions in the sediment; thereby further enhancing denitrification 41 . This process along with nitrification could significantly contribute to nutrient turnover and N 2 O production in mangroves. However, the magnitude at which a wetland can act as source of N 2 O cannot be explained solely based on N loads as the magnitude of N 2 O flux depends on both denitrification-nitrification rates (how efficiently N is cycled) and N 2 O reduction rates (how efficiently N 2 O is reduced to N 2 ) 31 .
The highest dissolved N 2 O concentrations and saturations were observed during the pre-monsoon season when temperatures were highest, while the N 2 O flux rates were highest during monsoon season. However, there was no significant relationship between water column and sediment temperatures and N 2 O concentrations, saturations and flux rates. Recent studies suggest that higher temperatures during summer seasons (pre-monsoon) can enhance microbial activity favouring N 2 O production 9 , while the reduced freshwater discharge during summers can increase the resident time of the water column, leading to greater accumulation of N 2 O in water and reducing the N 2 O loss to the atmosphere due to the decrease in gas transfer velocity 42 .
The shallow depth profile of the MCW likely promoted easier diffusion of oxygen molecules into the water column and to the sediments, favouring nitrification. This can drive higher dissolved N 2 O concentrations and  www.nature.com/scientificreports/ its saturation during the pre-monsoon than the monsoon and post-monsoon seasons. A recent report suggests that the shallow bathymetry of the CES favoured easier exchange of N 2 O that was produced in the estuarine sediment and water column to the atmosphere 19 . A positive correlation between water column depth and N 2 O flux was observed using all models ( Table 2). The deepest water column depth was mainly observed during monsoon season when the C and N loads were highest; there was low DO saturation and wind speeds were highest. However the mean water column depth during the monsoon season remained shallow (less than 0.5 m) even though it had the highest range in depths (0.3-0.5 m). Combined with the higher wind speeds during the monsoon season, this resulted in higher N 2 O fluxes rates. The significant negative correlation between salinity and N 2 O flux calculated using Wanninkhof 15 was likely driven by either nutrient rich seawater returned from the CES or the reduced solubility of gases in saline conditions. During the study, DON was the major constituent of the N and signified that the N enrichment in the study area was mainly through organic matter inputs or oceanic inputs where DON is the most prevalent form of N. As the major source of organic carbon in the study area was likely from mangrove litter which is generally nitrogen deficient 43,44 , the positive correlation of TC to DON in the water column (r = 0.870, ρ < 0.001) suggests that the study area receives significant amount of organic inputs from allochthonous sources, particularly with high N concentrations. The significant positive correlation between TC and TIC in the water column and N 2 O flux indicates that the N 2 O production and higher fluxes occurs in N rich environments with regard to C availability. However, the study failed to explain the relationship of TOC to N 2 O production and fluxes. A recent study in the tropical estuaries of north-western Borneo 45 also failed to explain the significant correlation between DOC and N 2 O concentrations. Although temperature and pH are important factors affecting N 2 O production, no clear relationship was found between these variables and dissolved N 2 O concentrations and fluxes. Several studies also suggest that the influence of water temperature on the denitrification rate may vary between regions and seasons 31,46,47 .
As the MCW is a fragmented mangrove, it's atmospheric contribution of N 2 O calculated using Wanninkhof 15 is quite small (ranging from 1.1 ± 0.2 × 10 6 μmol day −1 during post-monsoon season to 2.4 ± 1.2 × 10 6 μmol day −1 during monsoon season) and on its own may not be significant. However considering the numerous smaller mangrove stands that exist all over the tropics, fragmented mangroves may represent a significant source of N 2 O to the atmosphere. For example, the Ernakulam district in the state Kerala alone has about 933 minor mangrove stands, covering a total area of 206 hectares. This suggests that fragmented/minor mangrove stands may have an as yet unquantified but significant influence on atmospheric N 2 O budgets and atmospheric warming into the future.

Conclusion
The net N 2 O flux from MCW suggest that it is a source of atmospheric N 2 O, however due to its small area of coverage its contribution to atmospheric N 2 O is minor. The high precipitation rates in the study, through terrestrial runoff and river water discharge and high N inputs, influenced N 2 O flux rates particularly during monsoon season. The study suggests that when taken as whole, fragmented/minor mangroves which are abundant in tropical regions, need to be critically assessed and protected from further anthropogenic loading. Further studies in a range of different geologic and hydrologic conditions will help to include this potentially significant ecosystem type in global N 2 O budgets. The study highlights that N 2 O flux rates were dependant on the availability of DIN as well as salinity, DO, estuarine depth, rainfall, wind speed and availability of carbon.

Materials and methods
Field work was carried out over a period of 12 months from June 2014 to May 2015 with observations made during the morning hours on a monthly basis. Rainfall data was obtained from the Indian Meteorological Department. The depth of the wetland was measured by lowering a graduated weighted rope until it touched the top of the sediments. Transparency was measured using a 20 cm diameter Secchi disc 48 . Triplicates of nutrient samples were collected from surface waters at each site. Temperature, salinity and pH were measured using an Eutech water quality analyser (CyberScan Series SCD 650). Water samples for DO were taken in 60 ml glass bottles fixed with 0.5 ml each of Winkler reagents and titrated against sodium thiosulphate using visual endpoint detection 49 . Apparent oxygen utilisation (AOU) was calculated as outlined by Garcia and Gordon 50 , using the measured DO concentration. Dissolved inorganic nitrogen (DIN, which includes NH 4 , NO 2 , and NO 3 ) were analysed spectrophotometrically following standard procedures 51 .
Water samples for total organic carbon (TOC) and total inorganic carbon (TIC), total nitrogen (TN), dissolved nitrogen (DN) and dissolved N 2 O were collected in 120 ml glass bottles. The samples were fixed using saturated mercuric chloride (0.6 ml/120 ml) to arrest microbial activity 52 . An aliquot of each sample was filtered through a 25 mm GF/F filter and the filtrate was collected for the measurement of DN, while the remainder of the sample was analysed for TOC, TIC and TN by wet combustion method using a TOC elemental analyser (Multi N/C 2100 S Analytik jena). Particulate nitrogen (PN) and dissolved organic nitrogen (DON) concentrations in the water column were calculated by subtracting DN from TN and DIN from DN, respectively. Sediment samples were obtained using a van-Veen grab sampler (area 0.04 m 2 ) with a glass corer (3 cm diameter) inserted into each grab sample. As the surface sediment contained mangrove litter, samples were collected at a depth of 2 cm from the surface of the sediment and sieved (usually < 2 mm), dried at 60 °C for 24-72 h and ground to a fine powder. An aliquot of the dried sediment sample was acidified using 1 M hydrochloric acid to remove the inorganic carbon present in the sample 53 . The acidified samples were then washed with distilled water, dried and ground to powder. The samples were then analysed for TOC using dry combustion method (TOC elemental analyser Multi N/C 2100 S, Analytik jena). The other part of the sample that was not treated with acid was used to measure TN in the sediment using Pyro-cube IRMS.
Dissolved N 2 O was determined by the multiple phase equilibration technique 54 .

Data availability
Most of the data generated during the current study is graphically represented in the manuscript. The datasets generated during and/or analysed during the current study are also available from the corresponding author on reasonable request. www.nature.com/scientificreports/