Seasonal and tidal influence on the variability of nitrous oxide in the Tagus estuary , Portugal

In order to evaluate seasonal and tidal influence on the variability of dissolved nitrous oxide (N2O) in the Tagus estuary, Portugal, water sampling was carried out along the salinity gradient (May and November 2006) and during several tidal cycles (February and April 2007) at a fixed site. N2O and other relevant environmental parameters, temperature, salinity, dissolved oxygen and inorganic nitrogen forms (nitrate, nitrite and ammonium) were measured. Dissolved N2O concentrations showed strong tidal and seasonal variability, with the highest values occurring in February 2007 (13.7 nM, spring tide) and November 2006 (18.4 nM, upper estuary), apparently related to major Tagus river discharge. The existence of N2O sources was noticed in middle estuary. During spring tide, the input from external sources may be augmented by water column nitrification, making this process a contributor to the enhancement of N2O concentration in the estuary. Estimated N2O air-sea fluxes to the atmosphere reached a maximum value of ~10.4 μmol m -2 d-1 in February 2007 during spring tide and in May 2006 in the upper and lower (left bank) estuary. Although the Tagus estuary behaves predominantly as a source of atmospheric N2O, it appears to be a weaker source than other, more eutrophic estuaries.


INTRODUCTION
In the last few decades, the study of nitrous oxide (N 2 O) has acquired greater importance due to its role in the Earth's climate.N 2 O acts as a greenhouse gas, with a global warming potential per molecule more than 200 times that of carbon dioxide (IPCC, 2007).Stratospheric N 2 O is the major source of NO radicals, which are involved in the depletion of the ozone layer in the stratosphere (Cicerone, 1987).Despite the substantial advances in research on this biogas, uncertainty still remains concerning N 2 O balances and production and consumption mechanisms (IPCC, 2001).The ocean's contribution to global emissions of atmospheric N 2 O was estimated to be about 30%, of which 60% comes from highly productive regions, such as estuaries and coastal areas (Bange et al., 1996;Seitzinger et al., 2000).The production of N 2 O is linked to the microbial turnover of inorganic nitrogen by nitrifying and denitrifying organisms (Ritchie and Nicholas, 1972;Poth and Focht, 1985;Wrage et al., 2001).During nitrification, the aerobic two-step process in which ammonium is oxidized to nitrate N 2 O is assumed to be a by-product, although the exact metabolism is still under discussion (Ostrom et al., 2000).In suboxic habitats, nitrate can be reduced by denitrification to molecular nitrogen, with N 2 O as an intermediate (Cohen and Gordon, 1978).
As N 2 O production depends strongly on environmental conditions (Naqvi et al., 2000), any natural or anthropogenic-induced shifts in the aquatic system will affect the formation and subsequent release of N 2 O to the atmosphere.Nitrogen load is a main factor controlling N 2 O emissions from estuarine sediments (Seitzinger and Nixon, 1985;Middelburg et al., 1995).Seitzinger et al. (2000) and Cabrita and Brotas (2000) found denitrification to be an important process of N 2 O formation in intertidal sediments of the Tagus estuary, which receives relatively large inorganic nitrogen inputs from treated and untreated wastewater systems.
Oxygen is known to be another key factor controlling both N 2 O production in aquatic ecosystems and the temporal variability of ammonium.It is therefore important to understand the main factors regulating the production and variability of N 2 O within individual systems.In the present work we study the seasonal and tidal variability of N 2 O levels and fluxes, and their relationship with inorganic nitrogen species and other environmental variables in the Tagus estuary.

Description of the study area
The Tagus estuary, one of the largest in Western Europe, is located in the most populated area of Portugal that includes the capital, Lisbon (Fig. 1), and occupies an area of about 320 km 2 (Ferreira et al., 2003).The main freshwater source to the estuary is the River Tagus, which produces an annual mean discharge of approximately 400 m 3 s -1 (Bettencourt et al., 2003).There is a pronounced dry/wet season discharge signal (~30 m 3 s -1 in a dry summer and 2000 m 3 s -1 in a wet winter) (SNIRH, 2006), so the residence time of freshwater in the estuary is highly variable, ranging from approximately 6 to 65 days (Martins et al., 1984).Though organic loadings to the estuary have been reduced in recent years, partially treated or untreated effluents from greater Lisbon, particularly from the southern shoreline, still contribute approximately 30% of total organic nitrogen and 10% of nitrate (IST/ Maretec, 2002).Diffuse nutrient loads due to the leaching of agricultural land also exist, but they are negligible compared with the contribution of the agricultural load carried by the Tagus river.This mesotidal estuary has three morphologically distinct regions corresponding to different habitats: the upstream, middle and downstream sections (Brogueira and Cabeçadas, 2006).The large and shallow upper part of the estuary has an average depth of 2 m and includes extensive mudflats and salt marshes, the middle part is deeper and has an average depth of 7 m, and the lower part has maximum depths of 46 m.The combined effects of relatively shallow depths and strong tidal currents make the Tagus estuary a generally well-mixed system.Tides are semi-diurnal with amplitudes ranging from about 1 m at neap tides to about 4 m at spring tides.The Tagus estuary shows an asymmetric behaviour of flood and ebb, which leads to stronger velocities during ebbs (Fortunato et al., 1999).The wind also plays an important role in estuarine circulation, blowing predominantly from south and southwest in winter, rotating progressively to the northwest and north in spring and maintaining this direction in summer (Braunschweig et al., 2003).

Sampling and analytical methods
Surface water (0.2 m depth) samples (duplicate) were collected along the salinity gradient, during ebb, in May and in November 2006, corresponding to low and high water river flow, 46 m 3 s -1 and 768 m 3 s -1 , respectively (SNIRH, 2007).In May, sampling was carried out all along the estuary, including the Barreiro/ Montijo area (Fig. 1B), while in November samples were taken along a main transect following the right bank, as shown in Figure 1C.
In 2007, sampling was performed at a fixed site (38.8ºN,9.2ºW) (Fig. 1A) during February and April at spring and neap tides.Surface water samples (triplicate) were taken every hour covering one tidal cycle (approximately 13 h).Sampling dates, tidal conditions, Tagus river flow and wind speed are detailed in Table 1.
Water samples for analysis of physical (salinity) and chemical parameters (dissolved nitrate, nitrite, ammonium, oxygen and nitrous oxide) and chlorophyll a were collected with 2-L Niskin bottles.
Salinity (S) measurements were carried out using a temperature-controlled conductive salinometer (Guideline).Temperature (T) was determined in situ with an Aanderaa probe (CTD sensor).Meteorological parameters were measured using a portable meteorological station (Campbell Scientific CR 510).Measured wind speeds were converted to wind at 10 m (U 10 ) using a logarithmic correction (Pond, 1975;Hartman and Hamond, 1985).Monthly means of the Tagus river outflow at the hydrometric station in Almourol (39.8ºN, 8.4ºW), located upstream of the Tagus estuary, were obtained from SNIRH ( 2007) and are available online at www.snirh.pt.
Dissolved oxygen (referred herein as O 2 ) was determined according to the Winkler method, as described by Aminot and Chaussepied (1983).The precision of this method was between 0.08% and 0.25%.
Water samples for determination of dissolved nutrients were filtered through acetate cellulose filters (pore size 0.45 µm) and frozen until analysis.Nutrient analyses were carried out using a TRAACS autoanalyzer following colorimetric techniques outlined by the manufacturer.Estimated precision (10 replicates) is as follows: ±2.6% for nitrate and nitrite (referred to as NO 3 -and NO 2 -, respectively) and ±2.0% for ammonium (referred to as NH 4 + ), at mid-scale concentrations.Accuracy of nutrient measurements was maintained by using CSK Standards (WAKO, Japan).
For determination of nitrous oxide (N 2 O), bubble free sub-samples were taken (three replicates) into 20-ml glass vials, immediately poisoned with 40 µl of saturated aqueous mercury chloride (HgCl 2 ) to prevent microbial activity, and sealed with gastight caps.The vials were stored upside down, in the dark, at 4ºC and analyzed within 10 days after collection.Dissolved N 2 O concentration was determined by gas chromatography using a GC -ECD (GC Varian CP3800) headspace technique.In this procedure 5 ml of the sample were replaced by helium and equilibrium was performed in a headspace CombiPAL autosampler.Gas chromatographic separation was carried out using a stainless steel column packed with 80/100 (mesh) Porapak and detected by a 63 Ni electron capture detector (ECD).Calibration of ECD response was done using standard N 2 O gas mixtures in synthetic air (Air Líquide) and the precision of the method was 3%.N 2 O concentrations in the water samples were calculated from the concentrations meas-  ured in the headspace according to the solubility equation of Weiss and Price (1980).The equilibrium N 2 O concentrations were calculated from these values and from atmospheric N 2 O concentrations.The N 2 O fluxes across the air-water interface were estimated using the Broecker and Peng (1974) equation and the relation of Clark et al. (1995), since it contains a non-zero intercept, which accounts for gas exchange resulting from tidally induced turbulence in the estuary.
Chlorophyll a (referred to as Chl a) was analyzed by fluorometry (Hitachi F-7000 fluorescence spectrophotometer) in 90% acetone extracts after filtration (Whatman GF/F filters) following Lorenzen's (1967) method.The precision of this method was between 1.5% and 1.8%.

Spatial variability
The spatial distributions of S, T, O 2 , NO 3 -+NO 2 -, NH 4 + and N 2 O in May and November 2006 are shown in Figure 2. In November (Fig. 2B), under high Tagus river flow (768 m 3 s -1 ) salinity values from ~0 to ~20 extended to the middle estuary, while in May (Fig. 2A), under low Tagus river flow (46 m 3 s -1 ), this range was just limited to the upper estuary.Temperature, also differed greatly from November to May, when values higher than 18ºC were measured all over the main body of the estuary.O 2 concentrations were above 4.5 ml L -1 along the estuary in both periods, and maximum concentrations (7.1 ml L -1 ) were found in May in the upper estuary, simultaneously with higher chl a levels (~40 mg m -3 ) (not shown).
Maximum concentrations of NO 3 -+ NO 2 -were observed in the upper estuary (Fig. 2) in November (~85 µM) and in May (~38 µM), indicating the major input from Tagus river.The existence of local inputs along the system is perceptible through the mixing diagrams (Fig. 3), which show the non-conservative behaviour of this nutrient in both sampling periods.The highest concentrations of NH 4 + were observed in May (Fig. 2A) when a gradual increase was observed from the upper to the lower estuary.In the vicinity of Barreiro/ Montijo (stations 13, 14 and 15), NH 4 + values of up to 26 µM were attained simultaneously with lower O 2 values (4.6 ml L -1 ).The mixing diagrams (Fig. 3) show the non-conservative behaviour of NH 4 + in both sampling periods, with the system acting as an NH 4 + sink in May (Fig. 3A) except for the mentioned area (stations 13, 14, 15) and as an NH 4 + source in November (Fig. 3B).Regarding the N 2 O distributions, higher values were observed in November (Fig. 2B) in the upper estuary, where values of up to ~18.0 nM were attained, decreasing towards the lower estuary.Apparently, the River Tagus river was also the main N 2 O source in this high river flow period.Further, the concave curve of the N 2 O profile along salinity (Fig. 3B) indicates that N 2 O in the estuary decreases faster than would be expected from conservative mixing with the N 2 O-poor coastal water.In May N 2 O concentrations varied only between 10.2 nM and 12.6 nM, with the highest values being observed in the middle estuary (Fig. 2A).In this area, the existence of N 2 O local inputs and/or production is also noticeable through the mixing diagram (Fig. 3A).N 2 O concentrations were above atmospheric equilibrium throughout the entire salinity gradient in both sampling periods (Table 2), indicating that the estuary acts as a potential source of this gas to the atmosphere.

Tidal variability
Data obtained during tidal cycles are summarized in box plots (Fig. 4).It can be noticed that outliers exist only in April for T, at both spring and neap tides and for S, at neap tide.S, NO 3 -and N 2 O exhibited a larger spread in February, while the reverse was true for Chl a which had much lower values in February.Variables T, S, NO 3 -, NO 2 -and N 2 O as well, exhibit a good separation between February and April.S, dissolved nutrients (NO 3 -, NO 2 -and NH 4 + ), N 2 O and Chl a revealed a typical seasonal variability.Higher T, S and Chl a values were attained in April, while dissolved nutrients and N 2 O showed higher values in February, when higher external nutrient loading occurred.Furthermore, in February NO 3 -, NH 4 + and N 2 O exhibited a good distinction between neap and spring tides.In April a separation between tides is also seen in O 2 , NH 4 + and Chl a. Trends of N 2 O are compared with those of the most relevant parameters assumed to be directly connected with the production pathways of this biogas (NH 4 + , O 2 , NO 2 -) (Figs. 5, 6).In February during spring tide (Fig. 5A), when higher levels of N 2 O were detected, the simultaneous rise in N 2 O and NO 2 -concentrations and the fall in NH 4 + and O 2 concentrations occurred at slack water, both at low tide (LT) and at high tide (HT).Negative correlations were found between N 2 O and NH 4 + (N=6, R 2 =0.80,P<0.02) and between N 2 O and O 2 (N=5, R 2 =0.80,P<0.04), which suggests in situ N 2 O production through the nitrification process.At neap tide (Fig. 5B), the increase in N 2 O concentrations also detected near slack water (HT) was, however, accompanied by simultaneous increases in NH 4 + , O 2 and NO 2 -, which does not suggest the occurrence of nitrification under this tidal condition.In April, N 2 O variability was also much lower during spring tide (Fig. 6A) than at neap tide (Fig. 6B), although higher N 2 O values were also observed at slack water (either at HT or at LT). Surface waters were always supersaturated with N 2 O and a maximum mean N 2 O saturation value of 135% was attained in February during spring tide (Table 2).10.4 µmol m -2 d -1 ) were observed in May (Fig. 7A) in both the upper and lower (left bank) estuary, when wind speed attained the maximum values (~8 m s -1 ).In November (Fig. 7B), under lower wind speeds (≤3.5 m s -1 ), N 2 O air-sea fluxes varied only between 1.5 and 6.8 µmol m -2 d -1 , despite the highest N 2 O concentration measured in the upper estuary.Fluxes compare with those calculated from intertidal sediments of the lower Tagus estuary by Cabrita and Brotas (2000), who also detected seasonal variability.
N 2 O air-sea fluxes estimated during tidal cycles in February and April 2007 are shown in Figure 8.Much higher N 2 O exchange values were recorded in February during spring tide, when higher N 2 O concentrations and simultaneous high river runoff and wind speeds were recorded.At spring tide a progressive increase in N 2 O flux was estimated during the flood and maximum value of 10.4 µmol m -2 d -1 was reached.At neap tide values did not exceed 4.1 µmol m -2 d -1 and the variability of N 2 O fluxes during the tidal cycle was not so pronounced.In April during spring tide, much lower and even some negative fluxes were estimated.During the neap tidal cycle, wind speed never exceeded 0.6 m s -1 and estimated fluxes were always positive though they did not exceed 1.2 µmol m -2 d -1 .

DISCUSSION
Our results provide evidence of pronounced seasonal and tidal variability of N 2 O levels in the Tagus  ), apparently associated with a major Tagus river discharge observed in these periods.Lower water temperatures may also have favoured higher N 2 O solubility in water in autumn/winter.The non-conservative N 2 O behaviour along the estuary indicates that the middle estuary was acting as a sink.As the entire Tagus estuary is well oxygenated (>4.5 ml L -1 ), the N 2 O decrease seems to result mainly from gas emission to the atmosphere, as it is unlikely that N 2 O is consumed at oxygen concentrations above 2 µmol dm -3 (Codispoti and Christensen, 1985).During spring (May 2006), when the River Tagus flow was more reduced, N 2 O concentrations were much lower, even in the upper es-  tuary.Nevertheless, internal sources/production were apparently acting in the middle/lower estuary.From relevant parameters connected with the production of N 2 O, the occurrence of nitrification could not be deduced.Other additional N 2 O sources are probably influencing N 2 O production in the estuary.According to Cabrita and Brotas (2000), sediment denitrification in the lower Tagus estuary constitutes an N 2 O source, which may influence N 2 O concentrations in specific areas of this estuary.Furthermore, the existence of inputs from point sources (untreated and partially treated sewage and industrial effluents) in the middle estuary may account for higher N 2 O levels.N 2 O variability along tidal cycles was consistently higher at neap tides, though maximum values were reached in winter both at spring and neap tides.Apparently, N 2 O production through nitrification may represent an additional N 2 O source in the estuary.In fact, the correlations found between N 2 O concentrations and NO 2 -(positive) and O 2 and NH 4 + (negative) close to slack water can be interpreted as resulting from in situ N 2 O production by nitrification, probably favoured by larger amounts of particles during spring tides and higher residence time.This process, whose primary substrate NH 4 + maintained relatively high levels in the estuary in winter, is likely favoured by the presence of a larger amount of particles and higher residence time at slack water.It is known that most nitrifiers are attached to particles (Stehr et al., 1995) and their accumulation contributes to the enhancement of nitrifying population growth and nitrifying action in many estuaries (Abril et al., 2000;Brion et al., 2000).Also, near slack water less atmospheric exchange favours the enhancement of N 2 O levels in seawater.
Similarly to fluctuation in N 2 O levels, N 2 O fluxes in the Tagus estuary varied seasonally, along the salinity gradient, and also with the tidal state.The highest N 2 O air-sea fluxes along the estuary (10.4 µmol m -2 d -1 ) were measured in May in the upper and lower (left bank) estuary, despite the higher N 2 O levels found in November.The rate of gas exchange with the atmosphere is largely determined by wind speed (Liss and Merlivat, 1986;Wanninkhof, 1992) and approximately proportional to the square of the wind speed (Clark et al., 1995).Actually, wind speed recorded in May was 8 m s -1 while in November it did not exceed 3.5 m s -1 .
In relation to N 2 O emissions during tidal cycles, the enhancement of N 2 O fluxes at spring tide, during flood, observed in February seems to be caused not only by wind stress, but also by strong water turbulence induced by simultaneous effects of tidal force and higher river flow.Although this estuary behaves predominantly as a source of atmospheric N 2 O, in particular during the winter period, fluxes are low and the area may even function as a potential sink of atmospheric N 2 O at times, suggesting that this system is a weaker source of atmospheric N 2 O than other more eutrophic estuaries.

Fig. 1 .
Fig. 1. -Map of Tagus estuary showing the location of sampling sites in A) 2007 (fixed station) ; B) May 2006 and C) November 2006.

Fig. 2 .
Fig. 2. -Spatial distribution of physical (Salinity (S) and Temperature (T)) and chemical parameters (dissolved oxygen (O 2 ), nutrients (NO 3 - Air-sea N 2 O fluxes N 2 O air-sea fluxes estimated in the Tagus estuary in May and November 2006 are shown in Figure 7. Similar patterns were observed along the right bank of the estuary (St. 1 to St. 10/St.8, in May/November, respectively) in both sampling periods, with a progressive decrease towards the lower estuary.The highest N 2 O air-sea fluxes (

Fig. 7 .
Fig. 7. -Air-sea N 2 O fluxes (bar charts) in (A) May and (B) November 2006.Wind speed is represented by solid lines.The river flow (Q) is also indicated.

Fig. 8 .
Fig. 8. -Air-sea N 2 O fluxes (bar charts) in February and April 2007 during (A) spring tide and (B) neap tide.Wind speed is represented by solid lines and tidal amplitude by dotted lines.The river flow (Q) is also indicated.

Table 1 .
-Surface temperature (T), Tidal amplitude, river flow rate (Q) and wind speed (U10), along the Tagus estuary(May and November 2006)and at the fixed station during tidal cycles(February  and April 2007).
bMean wind velocity (10 min average) at the observation site.

Table 2 .
-Mean N 2 O concentrations and percent saturation (± standard deviation, SD) along the Tagus estuary (May and November 2006) and at the fixed station during tidal cycles (February and April 2007).
2 O) in (A) May and (B) November 2006.Numbers correspond to sampling sites according to map in Figure 1.