sm81n3-4637

Spatial variability of nitrous oxide in the Minho and Lima estuaries (Portugal)

Célia Gonçalves, Maria José Brogueira

Instituto Português do Mar e da Atmosfera, IPMA Rua Alfredo Magalhães Ramalho, 6, 1495-006, Lisboa, Portugal.
(CG) (Corresponding author) E-mail: celia.pgoncalves@gmail.com. ORCID iD: http://orcid.org/0000-0001-5524-1256
(MJB) E-mail: mjbrogueira@gmail.com. ORCID iD: http://orcid.org/0000-0003-3692-4099

Summary: Nitrous oxide (N2O) is a potent long-lived greenhouse gas and estuaries represent potentially important sources of this biogas to the atmosphere. In this work, we analyse the first N2O data obtained in the Minho and Lima estuaries, and the processes and environmental factors that may regulate its production in these systems. In September 2006, N2O attained values of up to 20.0 nmol L–1 in the upper reaches of the Lima estuary and the river was, apparently, the main source of biogas to the system. In Minho N2O reached a maximum of 14.4 nmol L–1 and nitrification appeared to contribute to the enhancement of N2O. In the upper estuary, the relatively high concentrations of nitrification substrate NH4+, the positive correlations found between N2O level above atmospheric equilibrium (ΔN2O) and apparent oxygen utilization and NO2, and the negative correlations between ΔN2O and NH4+ and pH can be interpreted as in situ N2O production through pelagic nitrification. Principal component analysis gave evidence of considerable differences between upper estuaries, particularly in terms of higher N2O in Lima and NH4+ in Minho. Surface waters of both estuaries were always N2O-supersaturated (101-227%) and estimated N2O emissions from Minho and Lima were 0.28 Mg N2O-N yr–1 and 0.96 Mg N2O-N yr–1, respectively, which represent a reduced fraction of N2O global emission from European estuaries.

Keywords: N2O; greenhouse gas; fluxes; emission; Portuguese estuaries.

Variabilidad espacial del óxido nitroso en los estuarios del Miño y Lima (Portugal)

Resumen: El óxido nitroso es un poderoso gas de efecto invernadero y los estuarios representan potenciales fuentes de este biogás a la atmósfera. En este trabajo se analizan los primeros datos de N2O obtenidos en los estuarios de Miño y Lima, así como los procesos y factores ambientales que pueden regular su producción en estos sistemas. En septiembre de 2006, N2O alcanzó en el tramo superior del Lima valores de hasta 20,0 nmol L–1 y el río Lima fue, aparentemente, la principal fuente de biogás para el sistema. En el Miño N2O alcanzó una concentración máxima de 14,4 nmol L–1 y la nitrificación parece contribuir al aumento de N2O. En el tramo superior del Miño las concentraciones relativamente altas del substrato de nitrificación NH4+ y las correlaciones positivas encontradas entre el exceso de N2O (ΔN2O) y el consumo aparente de oxígeno (AOU) y NO2, y las correlaciones negativas entre ΔN2O, NH4+ y pH pueden interpretarse como una producción in situ de N2O debido a la nitrificación pelágica. El Análisis de Componentes Principales evidenció diferencias considerables entre la cabecera de los estuarios particularmente en términos de mayor concentración de N2O en el Lima y NH4+ en el Miño. Las aguas superficiales del Miño y del Lima presentaban sobresaturación de N2O (101-227%) y las emisiones estimadas de N2O fueron de 0,28 Mg de N2O-N año–1 y 0,96 Mg de N2O-N año–1, respectivamente, que representan una fracción reducida de las emisiones totales de N2O procedentes de los estuarios europeos.

Palabras clave: N2O; gas de efecto invernadero; flujos; emisión; estuarios portugueses.

Citation/Como citar este artículo: Gonçalves C., Brogueira M.J. 2017. Spatial variability of nitrous oxide in the Minho and Lima estuaries (Portugal). Sci. Mar. 81(3): 317-326. doi: http://dx.doi.org/10.3989/scimar.04637.26A

Editor: X.A. Álvarez-Salgado.

Received: March 21, 2017. Accepted: June 14, 2017. Published: September 4, 2017.

Copyright: © 2017 CSIC. This is an open-access article distributed under the terms of the Creative Commons Attribution (CC-by) Spain 3.0 License.

Contents

Summary
Resumen
Introduction
Materials and methods
Results
Discussion
Acknowledgements
References

IntroductionTop

In the last few decades, the study of N2O has acquired greater importance due to its contribution to global climate change. N2O is an important long-lived greenhouse gas in terms of radiative forcing (0.17±0.03 W m–2) (Myhre et al. 2013Myhre G., Shindell D., Bréon F.M., et al. 2013. Anthropogenic and Natural Radiative Forcing. In: Stocker T.F., Qin D., Plattner G.K., et al. (eds), Climate Change 2013: The Physical Science Basis. Contribution of Working Group I to the Fifth Assessment Report of the Intergovernmental Panel on Climate Change. Cambridge University Press, pp. 659-740.) and represents the major anthropogenic contributor to stratospheric ozone destruction. It has a long atmospheric lifetime of 131±10 years (Prather et al. 2012Prather M.J., Holmes C.D., Hsu J. 2012. Reactive greenhouse gas scenarios: Systematic exploration of uncertainties and the role of atmospheric chemistry. Geophys. Res. Lett. 39: L09803. ) and its global warming potential is 310 times greater than that of carbon dioxide, in a time horizon of 100 years. In 2011 atmospheric N2O levels (324.2±0.1 ppb) exceeded the pre-industrial levels (270±7 ppb) by about 20% (Myhre et al. 2013Myhre G., Shindell D., Bréon F.M., et al. 2013. Anthropogenic and Natural Radiative Forcing. In: Stocker T.F., Qin D., Plattner G.K., et al. (eds), Climate Change 2013: The Physical Science Basis. Contribution of Working Group I to the Fifth Assessment Report of the Intergovernmental Panel on Climate Change. Cambridge University Press, pp. 659-740.), largely due to increased agricultural activity and industry.

Estuaries have been considered significant N2O contributors to the atmosphere as a consequence of their high productivity and anthropogenic nitrogen loadings. N2O is mainly formed during the first step of nitrification, the aerobic oxidation of ammonium (NH4+) to nitrite (NO2), mediated by ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA), and microbiological denitrification, the biological reduction of nitrate (NO3) to N2O, and, in turn, nitrogen gas (N2). Nitrification and denitrification often occur simultaneously in aquatic ecosystems and their relative contribution to total N2O production is difficult to disentangle. As nitrification is an aerobic process in well-oxygenated estuarine systems, the water column mostly contributes N2O through nitrification production (de Wilde and de Bie 2000de Wilde H.P.J., de Bie M.J.M. 2000. Nitrous oxide in the Schelde estuary: Production by nitrification and emission to the atmosphere. Mar. Chem. 69: 203-216., Barnes and Upstill-Goddard 2011Barnes J., Upstill-Goddard R.C. 2011. N2O seasonal distributions and air-sea exchange in UK estuaries: Implications for the tropospheric N2O source from European coastal waters. J. Geophys. Res. B. 116: G01006.). Denitrification is usually limited to zones under hypoxic conditions (DO <2 ml L–1), although some denitrification may occur even in relatively oxygenated waters (de Bie et al. 2002de Bie M.J.M., Middelburg J.J. Starink M., et al. 2002. Factors controlling nitrous oxide at the microbial community and estuarine scale. Mar. Ecol. Prog. Ser. 240: 1-9. ).

Both nitrification and denitrification are sensitive to the ongoing environmental changes and any natural or anthropogenic-induced shifts in the N availability have the potential to alter nitrogen cycling in coastal environments and affect N2O formation and release to the atmosphere (Bange et al. 2010Bange H.W., Freing A., Kock A., et al. 2010. Marine pathways to nitrous oxide. In: Smith K.A. (ed.), Nitrous oxide and climate change. Earthscan, London. U.K. pp. 36-62.). The extent of the denitrification is also strongly controlled by temperature, nitrate concentrations and the availability of organic carbon (e.g. Dong and Nedwell 2006Dong L.F., Nedwell D.B. 2006. Sources of nitrogen used for denitrification and nitrous oxide formation in sediments of the hypernutrified Colne, the nutrified Humber and oligotrophic Conwy Estuaries, United Kingdom. Limnol. Oceanogr. 51(1, part 2): 545-557.). In addition to the supply of oxygen and ammonia, which are the main controls on nitrification, other environmental variables may affect this biological process: temperature (Dai et al. 2008Dai M., Wang L., Guo X., et al. 2008. Nitrification and inorganic nitrogen distribution in a large perturbed river/estuarine system: the Pearl River Estuary, China. Biogeosci. 5: 1227-1244.), salinity (Bollmann and Laanbroek 2002Bollmann A., Laanbroek H.J. 2002. Influence of oxygen partial pressure and salinity on the community composition of ammonia-oxidizing bacteria in the Schelde estuary. Aquat. Microb. Ecol. 28: 239-247. ) and pH (Strauss et al. 2002Strauss E.A., Mitchell N.L., Lamberti G.A. 2002. Factors regulating nitrification in aquatic sediments: effects of organic carbon, nitrogen availability, and pH. Can. J. Fish. Aquat. Sci. 59: 554-563. ).

Estimates of N2O release from estuaries to the global inventory reveal wide uncertainties due to the large variability in N2O data (Bange et al. 2010Bange H.W., Freing A., Kock A., et al. 2010. Marine pathways to nitrous oxide. In: Smith K.A. (ed.), Nitrous oxide and climate change. Earthscan, London. U.K. pp. 36-62., Barnes and Upstill-Goddard 2011Barnes J., Upstill-Goddard R.C. 2011. N2O seasonal distributions and air-sea exchange in UK estuaries: Implications for the tropospheric N2O source from European coastal waters. J. Geophys. Res. B. 116: G01006.). However, considerable efforts have been made in the last few decades to better understand the nitrogen cycle, the dynamics of N2O production and the quantification of the respective emission from European estuaries. More recently Murray et al. (2015)Murray R.H., Erler D.V., Eyre B.D. 2015. Nitrous oxide fluxes in estuarine environments: response to global change. Global Change Biol. 21: 3219-3245. reviewed N2O global fluxes from estuarine environments and reported a variation of 0.17-0.95 Tg N2O-N yr–1.

Studies on the N2O dynamics and fluxes have been carried out in the Portuguese Tagus, Sado and Douro estuaries (e.g. Gonçalves et al. 2010Gonçalves C., Brogueira M.J., Camões M.F. 2010. Seasonal and tidal influence on the variability of nitrous oxide in the Tagus estuary, Portugal. Sci. Mar. 74S1: 57-66. , 2015Gonçalves C., Brogueira M.J, Nogueira M. 2015. Tidal and spatial variability of nitrous oxide (N2O) in Sado estuary (Portugal). Est. Coast. Shelf Sci. 167: 466-474, Teixeira et al. 2013Teixeira C., Magalhães C., Joye S.B., et al. 2013. The role of salinity in shaping dissolved inorganic nitrogen and N2O dynamics in estuarine sediment-water interface. Mar. Pollut. Bull. 66: 225-229. ). However, no data on N2O levels and fluxes are available for the Minho and Lima estuaries. In this work, we (1) report spatial variability of N2O concentration in these systems, (2) assess the contribution of different N2O sources, (3) evaluate the role of environmental properties on the increment of N2O fluxes, and (4) estimate N2O emission in a perspective of global N2O estuarine emissions.

MATERIALS AND METHODSTop

Area description

The Minho and Lima estuaries, both situated in the northern part of Portugal (Fig. 1), differ essentially in terms of river discharge, anthropogenic pressures and morphology (Table 1).

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Fig. 1. – Map of the Minho and Lima estuaries. Dots and numbers represent sampling sites.

Table 1. – Main physical and hydrological characteristics of the Minho and Lima estuaries (Ferreira et al. 2005Ferreira J.G., Nobre A.M., Simas T.C., et al. 2005. Monitoring plan for water quality and ecology for Portuguese traditional and coastal waters: Development of guidelines for the application of the European Union Water Framework Directive. Editions INAG-Instituto da Água IMAR-Institute of Marine Research. Lisbon, Portugal, 142 pp.).

Estuary Area (km2) Mean depth (m) Mean water volume (×106 m3) Mean residence time (days) Mean tidal range (m) Mean annual freshwater input
(m3 s–1)
Watershed population density
(hab km–2)
N load (t yr–1)
Minho 23 2.6 70 1.5 3 300 112 20051
Lima 5 4.0 19 1 2 54 87 1078

Minho estuary

The Minho estuary is situated in the border region between Portugal (Minho region) and Spain (Galicia region). The estuary has an area of approximately 23 km2, a mean depth of 2.6 m, and a maximum width of about 2 km at the confluence of the Coura River, at Caminha. The estuary is mesotidal, with a mean tidal range of 3 m, and its dynamic tidal effects extends 35 km upstream (Bettencourt et al. 2003Bettencourt A., Ramos L., Gomes V., et al. 2003. Estuários Portugueses. Editions INAG - Ministério das Cidades, Ordenamento do Território e Ambiente, Lisboa, 311 pp.).

The annual mean discharge of the Minho River is 300 m3 s–1 (Table 1), ranging between approximately 100 m3 s–1 in August and 800 m3 s–1 in February, and the water residence time is 1.5 days (Sousa et al. 2013Sousa C., Vaz M.N., Alvarez I., et al. 2013. Effect of Minho estuarine plume on Rias Baixas: numerical modeling approach. J. Coast. Res. Spec. 65: 2059-2065.). The estuary has high ecological value, mainly due to the large diversity of habitats and biodiversity and great socio-economic importance from tourism, fishing and agriculture. However, increasing pressure has been detected over recent years, involving in particular the pollution of surface water from both specific and diffuse sources, morphological alterations, changes of land use in the drainage basin and other impacts from human activity, such as aquaculture, textile, rubber and plastic processing industries (Ferreira et al. 2005Ferreira J.G., Nobre A.M., Simas T.C., et al. 2005. Monitoring plan for water quality and ecology for Portuguese traditional and coastal waters: Development of guidelines for the application of the European Union Water Framework Directive. Editions INAG-Instituto da Água IMAR-Institute of Marine Research. Lisbon, Portugal, 142 pp.). The Minho and Coura rivers are the main nitrogen sources to the estuary, contributing 13000 t N yr–1 and 7000 t N yr–1, respectively, and effluents from domestic origin contribute 51 t N yr–1 (Ferreira et al. 2005Ferreira J.G., Nobre A.M., Simas T.C., et al. 2005. Monitoring plan for water quality and ecology for Portuguese traditional and coastal waters: Development of guidelines for the application of the European Union Water Framework Directive. Editions INAG-Instituto da Água IMAR-Institute of Marine Research. Lisbon, Portugal, 142 pp.) (Table 1).

Lima estuary

The Lima estuary, located south of Minho, has an area of approximately 5 km2 and a mean depth of 4 m (Table 1). It has a semidiurnal mesotidal regime and the tidal effect extends 20 km upstream (Ramos et al. 2006Ramos S., Cowen R.K., Ré P., et al. 2006. Temporal and spatial distributions of larval fish assemblages in the Lima estuary (Portugal). Est. Coast. Shelf Sci. 66: 303-314. ). The upper estuary is constituted by a narrow channel with some intertidal areas and undisturbed banks. The middle estuary is a shallow salt marsh zone, and the lower estuary consists of a wider and shallow basin that communicates with the sea via a deep, narrow channel (with a typical depth of about 10 m). The estuary mouth is artificially obstructed by a 2-km-long jetty. The Lima River has an annual freshwater discharge of 54 m3 s–1, and the mean water residence time in the estuary is one day (Table 1).

The lower estuary is a highly urbanized zone and has been subjected to several anthropogenic impacts resulting from harbour activities (Viana do Castelo). However, nutrient loadings also originate from diffuse sources, largely agriculture (0.51 t N yr–1), although the main N source to the estuary is the Lima River (1077 t N yr1) (Ferreira et al. 2005Ferreira J.G., Nobre A.M., Simas T.C., et al. 2005. Monitoring plan for water quality and ecology for Portuguese traditional and coastal waters: Development of guidelines for the application of the European Union Water Framework Directive. Editions INAG-Instituto da Água IMAR-Institute of Marine Research. Lisbon, Portugal, 142 pp.). This N load is, however, approximately 19 times lower than the load to the Minho estuary (Table 1).

Sampling

Water sampling was undertaken in September 2006 during ebb tide (at spring tide), at nine stations located along a main transect of both estuaries, from the upstream limit of tidal influence to the estuary mouth, covering a full range of salinity of 0-30 corresponding to a distance of 26.5 km in Minho and 15 km in Lima estuary (Fig. 1).

Surface water (0.2 m depth) was collected using 2-L Niskin bottles (General Oceanics) for analysis of salinity (S), temperature (T), pH, dissolved inorganic nitrogen (nitrate NO3, nitrite NO2 and ammonium NH4+), dissolved oxygen (DO) and nitrous oxide (N2O). The hydrological characteristics of the Minho and Lima estuaries and the meteorological conditions observed during the sampling period are presented in Table 2.

Table 2. – Hydrological characteristics of the Minho and Lima estuaries and meteorological conditions observed in September 2006. Q, daily mean flow of the Minho and Lima rivers measured at Foz Mouro and Ponte da Barca, respectively (SNIRH 2013); u10, daily wind speed normalized to 10 m height.

Estuary Sampling date Tidal amplitude (m) Q daily mean (m3 s–1) u10 (m s–1)
Minho 12 Sep 0.6-3.3 90.7 2.5-6.2
Lima 13 Sep 0.9-3.1 8.1 1.3-4.1

Analytical procedure

Water temperature (T) was measured in situ with a Seabird SBE19/CTD probe with an accuracy of 0.01°C.

Salinity (S) measurements were carried out using a temperature-controlled Guideline Salinometer (Portasal 8410A), and accuracy was 0.03 salinity. Equipment was calibrated with a certified IAPSO Standard Seawater reference.

Meteorological parameters (air temperature, pressure, and wind speed and direction) were determined using a portable meteorological station (Campbell Scientific CR510). Measurements represent the average of physical parameters taken using a sampling time of 5 seconds and a storage time of 1 minute. Wind speed 10-minute average was determined for each sampling station and converted to wind speed values at 10 m height (u10) using a logarithmic correction (Hartman and Hammond 1985Hartman B., Hammond D. 1985. Gas exchange in San Francisco Bay. Hydrobiology 129: 59-68.).

The Minho and Lima River discharges were calculated as an average of the flow 10 days before sampling, at the hydrometric stations of Foz de Mouro and Ponte da Barca, respectively (SNIRH 2013SNIRH (Sistema Nacional de Informação de Recursos Hídricos). 2013. Instituto da Água IP.).

Dissolved oxygen (DO, μmol L–1) was measured using whole-bottle Winkler’s titration method (Aminot and Chaussepied 1983Aminot A., Chaussepied M. 1983. Manuel des analyses chimiques en milieu marin. Centre National pour l’Exploitation des Océans. CNEXO, Brest, France, 395 pp.). A Methrom titrator was used to dispense small amounts of thiosulphate, and starch endpoint was detected visually. Precision of the method was in the range of 0.08% to 0.25%. DO saturation, expressed in percentage (%), was determined as the ratio of the oxygen concentration determined and the equilibrium values of DO calculated with the Weiss (1970)Weiss R.F. 1970. The solubility of nitrogen, oxygen, and argon in water and seawater. Deep-Sea Res. Oceanogr. Abstr. 17: 721-735. equation. Apparent oxygen utilization (AOU, μmol L–1) was calculated as the difference between the saturation oxygen concentration and the dissolved oxygen concentration measured in the sample.

Water samples for determination of dissolved inorganic nitrogen were filtered through acetate cellulose filters (pore size 0.45 μm) and stored at –20ºC until analysis. Analyses were carried out using a Traacs autoanalyser following colorimetric techniques outlined by the manufacturer. Estimated precision was ±0.8% for nitrate and nitrite (NO3 and NO2) and ±2.0% for ammonium (NH4+), at mid-scale concentrations. Accuracy of nutrient measurements was maintained by using CSK Standards (WAKO, Japan).

pH measurement was carried out immediately after collecting water samples using a Metrohm 704 pH-meter and a combined electrode (Metrohm), standardized against NBS buffers (6.865 and 9.180 pH). Precision of pH measurements was ±0.01.

Water samples for determination of N2O were collected in triplicate in 20-mL glass headspace vials and poisoned with saturated aqueous mercury chloride (HgCl2) to stop biological activity. The vials were stored upside down, in the dark, at 4°C in the refrigerator until analysis, performed within 10 days. Dissolved N2O was determined by a headspace equilibration technique coupled with gas chromatographic analysis (GC-3800, Varian). Briefly, 20 mL of sample was equilibrated with 5 mL of highly purified helium (purity =99.9999%) in a headspace CombiPAL autosampler. Gas chromatographic separation was carried out using a stainless steel column packed with 80/100 (mesh) Porapak. Oven and detector temperature was set at 50°C and 320°C, respectively, and high purity nitrogen (99.9999%) was used as the carrier gas (flow rate 30 mL min–1). To remove water vapour and carbon dioxide, absorbent columns packed respectively with Mg(ClO4)2 and Carbosorb were located in the carrier gas line between the sample loop and the separation column. N2O peak was detected with a 63Ni electron capture detector (ECD). Calibration of ECD response was performed using standard gas mixtures with 400, 780 and 1980 ppb N2O in synthetic air (Air Liquide), and method precision was 2.6% (30 replicate measurements using samples containing 10 nmol L1 of N2O). In situ concentration of N2O (C, nmol L–1) was calculated from the concentrations measured in the headspace according to the solubility equation of Weiss and Price (1980)Weiss R.F., Price B.A. 1980. Nitrous oxide in water and seawater. Mar. Chem. 8: 347-359. :

C = β (TS) x′P

where x′ is the measured N2O dry mole fraction, P is the atmospheric pressure, and β is the solubility coefficient, which is a function of the water temperature (T) and salinity (S). N2O equilibrium concentrations were calculated assuming an atmospheric N2O mixing ratio of 320.1 ppb (WMO 2006WMO (World Meteorological Organization). 2006. World Meteorological Organization greenhouse gas bulletin: The State of Greenhouse Gases in the Atmosphere Using Global Observations through 2006. Bulletin No. 3. Global Atmosphere Watch, Geneva, Switzerland, 4 pp.).

N2O saturation, expressed in percentage (%), was determined as the ratio between the measured dissolved N2O concentration and the equilibrium concentration. The N2O water-air flux (FN2O) was estimated according to the following equation:

FN2O = kN2O ΔN2O

where ΔN2O, the excess of N2O, is the difference between the measured concentration and the equilibrium concentration with the atmosphere in the estuarine water at the local temperature and salinity; and kN2O (cm h–1) is the N2O transfer velocity, which is expressed as a function of the wind speed and the Schmidt number (Sc). Since no direct measurements of kN2O were made in the Minho and Lima estuaries, both the k-wind relationships of Carini et al. (1996)Carini S., Weston N., Hopkinson C., et al. 1996. Gas exchange rates in the Parker River estuary, Massachusetts. Biol. Bull. 191(2): 333-334. (hereinafter referred to as C96) and Raymond and Cole (2001)Raymond P.A., Cole J.J. 2001. Gas exchange in rivers and estuaries: choosing a gas transfer velocity. Estuaries 24: 312-317. (hereinafter referred to as RC01) were, respectively, used to compute k:

kC96 = 0.045 + 2.0277u10

kRC01 = 1.91 e0.35u10

The gas transfer velocities and air-sea fluxes were estimated using in situ wind speeds normalized to 10 m height (u10). The k coefficients were corrected for in situ temperature using the following relationship:

kN2O /k600 = (ScN2O /600)–0.5

where ScN2O is the Schmidt number for N2O calculated according to the equation of Wanninkhof (1992)Wanninkhof R. 1992. Relationship between wind speed and gas exchange over the ocean. J. Geophys. Res. C 97(C5): 7373-7382.:

Sc = 2301.1 – 151.1 T + 4.7364 T2 + 0.057431 T3

where T is the temperature (ºC).

Statistical analysis

An unpaired t-test was used to identify statistical differences in levels of N2O and other environmental variables between and along estuaries.

Pearson’s correlation analyses were performed to evaluate the existence of relationships between ΔN2O and the variables NH4+, AOU, NO2 and pH assumed to be connected with production pathways of this biogas.

The multivariate techniques principal component analysis (PCA) and cluster analysis were applied to environmental data in order to identify and compare inter-relationships between these variables in both estuaries. Data were log(x+1) transformed and handled using correlation-based PCA on the basis of standard Euclidean distance between samples to define their dissimilarity. PRIMER (version 6) was employed for the multivariate analysis.

Figures were created in Golden Software Grapher program (version 9.6.1001).

RESULTSTop

N2O levels and fluxes

Concentrations of N2O and the studied environmental parameters plotted against salinity along both Minho and Lima estuaries are shown in Figure 2.

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Fig. 2. – N2O and other environmental parameters against salinity in the Minho (black circles) and Lima (open squares) estuaries (error bars=±1 standard deviation, sd). Conservative lines (solid line, Minho; dashed line, Lima) are shown when appropriate.

Distribution of N2O exhibits a declining tendency towards the mouths of both estuaries. In general, concentrations were higher in Lima than in Minho (Fig. 2A). In Lima values ranged from 10.1 to 20.0 nmol L1 and were below the conservative mixing line. The maximum value was reached in the upper estuary, suggesting that the Lima River was the main source of N2O to the estuarine system. From salinity 0 to 4 a sharp decrease was observed, suggesting a greater N2O loss in the zone, presumably through water-air gas exchange. Downstream of salinity 4, N2O decreased more slightly and the mixing with the N2O-poorer seawater is well perceptible. In the Minho estuary, concentrations varied from 8.6 to 14.4 nmol L1 and by contrast with those observed in Lima were above the conservative mixing line, pointing to the existence of N2O sources within the estuary. A major concentration increase from 10.5 to 14.4 nmol L1 was detected between 0 and 2.5 salinity, suggesting the existence of internal N2O sources in this zone of the estuary, presumably from manufacturing industries located nearby. N2O saturation values ranged from 101% to 166% along the Minho estuary and from 113% to 227% along the Lima estuary (see Table 4), indicating that both estuaries are potential N2O sources to the atmosphere.

Surface waters of both estuaries were well oxygenated during our study period. Concentrations of DO were higher than 280 µmol L–1 in the upper Lima estuary at salinity 0.2, decreasing to 240 µmol L–1 at salinity 2.8 (Fig. 2C). Seawards of this salinity, an increasing tendency was observed and a concentration of 255 µmol L–1 was also reached in the vicinity of the estuary mouth (salinity 30.2). In the Minho estuary, a decrease in DO was also detected in the upper estuary and the concentration dropped from 243 µmol L–1 at 0.1 salinity to 199 µmol L–1 at 2.3 salinity. Afterwards, a sharp increase in DO was measured along the estuary and a maximum value of 264 µmol L–1 was reached at the estuary mouth (salinity 29.7). DO-enriched seawater probably accounted for the similar increasing trend seaward in both estuaries. Saturation values were higher than 70% in the Minho estuary and 90% in the Lima estuary.

In the Minho estuary pH increased from 7.3 in the more river-influenced zone to 8.1 at the estuary mouth (Fig. 2D). In the Lima estuary pH showed a larger range of values, increasing from 6.7 in the most river-influenced site to 8.0 at the estuary mouth.

NO3 was the dominant species of inorganic nitrogen in both estuaries, reaching a similar maximum concentration in the river input (47.6 µmol L–1 in Minho and 44.4 µmol L–1 in Lima) (Fig. 2E). Values decreased seawards and in general followed the theoretical conservative mixing line. Along the Minho estuary both NO2 and NH4+ exhibited an irregular behaviour, though the system seemed to function as an NO2 source and an NH4+ sink (Fig. 2F, G). Between salinity 0 and ~5-7 the decline in NH4+ (~4.0 to 0.5 µM L–1) was simultaneous with an increase in NO2 (~0.6 to 1.0 µM L–1) and N2O (~10 to 13-14 nmol L–1), suggesting the occurrence of nitrification.

Figure 3 displays relationships between ΔN2O and AOU, NH4+, NO2, NO3 and pH in the Minho estuary. In the mentioned salinity zone (0 and ~5-7) a significant positive correlation was found between ΔN2O and AOU (R2=0.75). This indicates the occurrence of nitrification as a source of N2O and the respective slope provides an estimate of the biological N2O yield per mole O2 consumed (Yoshinari 1976). Further, the simultaneous (negative) correlations between ΔN2O and the primary substrate for nitrification, NH4+ (R2=0.40), and pH (R2=0.80), and the positive correlation between ΔN2O and the byproduct of nitrification, NO2 (R2=0.41), are consistent with the predominance of nitrification as a mechanism of N2O production in the upper part of the Minho estuary. No correlation was found with NO3, whose high concentrations were mostly riverine derived.

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Fig. 3. – Relationships between ΔN2O and environmental parameters in the Minho estuary. Lower salinity sites (0-10 salinity) are represented by black circles and higher salinity sites (15-30 salinity) by open circles. R2, correlation coefficient.

In the Lima estuary no relationships were found, suggesting the occurrence of nitrification (Fig. 4).

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Fig. 4. – Relationships between ΔN2O and environmental parameters in the Lima estuary.

The application of PCA to the studied environmental variables in the Minho and Lima estuaries allowed us to identify two main composite variables, PC1 and PC2 (eigenvalues >1.0), which explain 82% of the variance (Table 3) and represent a good description of the environmental structure across the estuarine sampled sites.

Table 3. – Results of principal component analysis showing loadings of variables for the first two principal components for the Minho and Lima estuaries.

Environmental variable PC1 PC2 PC3
Eigenvalues 4.27 2.17 0.74
% Variation 53.4 27.1 9.7
Cum. % Variation 53.4 80.6 90.2

PC1 explained 53% of variance and had the highest positive loading for NO3, T and N2O and a negative loading for S and pH (Fig. 5). This component represents the separation of major river-influenced stations from major marine-influenced ones in both estuaries. PC2 explained 27% of the variance and correlated positively with DO and negatively with NO2and NH4+. This component appears to represent relevant parameters to N dynamics, particularly in Minho estuary. In fact, projection of stations along PC2 reveals a clear separation of sites from the upper Lima estuary (L1-L5) (Fig. 5), mostly associated with higher values of N2O, and sites from the upper Minho (M1-M5) more associated with higher NO2 and NH4+, apparently from river origin, as the N load from the Minho River is considerable at this point (Table 1). It was also observed that the stations from both middle/lower estuaries did not differ in terms of studied environmental variables.

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Fig. 5. – PCA ordination of variables loadings (A) and scores of sampling stations (B) in the Minho and Lima estuaries.

N2O water-air fluxes are shown in Figure 6. Positive values prevailed at all stations but decreased, in general, between the upper and lower zone of the estuaries. This tendency was more pronounced in the Lima estuary, where higher N2O fluxes in the river-influenced area were about twice (~12.0 μmol m2 d1; C96) (Fig. 6B) those observed in the upper part of the Minho estuary (~6.0 μmol m2 d1; C96) (Fig. 6A). The higher fluxes in the Lima estuary were mainly associated with the higher levels of N2O observed in the upper estuary area (St.1 to St.3; Fig. 2), indicating that the low salinity zone (0-5) is an important source of N2O to the atmosphere.

sm4637fig6.jpg

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Fig. 6. – Water-air N2O fluxes (bar charts) along the Minho (A) and Lima (B) estuaries. Wind speed (u10) is represented by solid line (Error bars =±1 sd).

Using the two different parameterizations (C96, RC01) to calculate the gas transfer coefficients, the averaged N2O water-air fluxes from Minho estuary to the atmosphere ranged between 4.0±3.3 µmol m2 d1 (RC01) and 4.1±2.8 µmol m2 d1 (C96), corresponding to a mean N2O concentration of 11.3±1.3 nmol L1 (132±22% saturation) and a mean wind speed of 3.9±0.1 m s1. Slightly higher N2O water-air fluxes were found in the Lima estuary, with averaged values ranging between 4.7±1.9 µmol m2 d1 (RC01) and 5.0±2.0 µmol m2 d1 (C96), corresponding to a higher mean N2O concentration of 13.7±1.6 nmol L1 (153±26% saturation) and a lower mean wind speed level (2.4±0.1 m s1).

N2O fluxes from the Minho and Lima estuaries were regressed versus the first two PCs’ ordination of station scores to test their ability to predict the fluxes. We found out that only PC1 showed to be correlated with N2O flux, with a strong positive correlation (R2=0.61) (Fig. 7). N2O flux also increased along a gradient of increasing NO3, T and N2O. These results suggest that future global changes in these parameters will result in an increase of N2O flux in these estuarine systems.

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Fig. 7. – Relationship between log-transformed values of N2O fluxes and the first principal component in the Minho and Lima estuaries. R2, correlation coefficient. Variable trends are indicated along the top of the figure.

DiscussionTop

The present study reveals that the Minho and Lima estuaries, particularly the upper reaches, behave differently regarding N2O levels, sources and fluxes. N2O distribution exhibits a pronounced spatial variability in both estuaries but in the Lima estuary concentrations are higher than in Minho. The Lima River was the main N2O contributor to the Lima estuary, whereas the occurrence of nitrification seems to represent an additional N2O source within the Minho estuary. As NH4+ is a primary substrate for nitrification, low NH4+ concentration may limit nitrification. It has been suggested that the AOA and AOB niches are defined by ammonium concentrations (Martens-Habbena et al. 2009Martens-Habbena W., Berube P.M., Urakawa H., et al. 2009. Ammonia oxidation kinetics determine niche separation of nitrifying Archaea and Bacteria. Nature 461: 976-981.), with AOA dominating in ammonia-limited acid, whereas AOB have a tolerance of high ammonia concentrations.

Though no information on benthic AOA and AOB communities along the Minho and Lima estuaries is available, NH4+ concentration in the upper Minho estuary (maximum 4.4 μmol L–1) seems more suitable for the occurrence of nitrification than in the Lima estuary (maximum 1.8 μmol L–1). Nitrification reactions typically happen within a DO range of 15.6-78.0 µmol L–1, and the Minho and Lima surface waters were well above these concentrations, leading to nitrification conditions. However, only in the upper Minho do correlations found between ΔN2O and AOU, NH4+ and NO2 suggest that nitrification may have been acting as an NH4+ sink and a source of N2O. The calculated biological N2O yield (0.060 nmol per μmol O2 consumed) falls within the range observed in marine systems and in particular in the Atlantic off the Iberian coast (Nevison et al. 2003Nevison C.D., Butler J.H., Elkins J.W. 2003. Global distribution of N2O and the ΔN2O-AOU yield in the subsurface ocean. Global Biogeochem Cycles 17: 1119. ). The effect of salinity on nitrification is well documented and in many estuarine systems nitrification rates are highest at lower and intermediate salinities (Bianchi et al. 1999Bianchi M., Feliatra F., Lefevre D. 1999. Regulation of nitrification in the land-ocean contact area of the Rhône River plume (NW Mediterranean). Aquat. Microb. Ecol. 18: 301-312., Teixeira et al. 2013Teixeira C., Magalhães C., Joye S.B., et al. 2013. The role of salinity in shaping dissolved inorganic nitrogen and N2O dynamics in estuarine sediment-water interface. Mar. Pollut. Bull. 66: 225-229.). Our results from the upper Minho are in accordance with these findings, as the potential nitrification occurred at low salinity (between ~2 and ~10). The community composition of nitrifying microbes is very dependent on salinity, but a combination of other environmental factors may shape AOB diversity along an estuary (Mosier et al. 2008Mosier A.C., Francis C.A. 2008. Relative abundance and diversity of ammonia-oxidizing archaea and bacteria in the San Francisco Bay estuary. Environ. Microbiol. 10: 3002-3016.).

pH may regulate nitrification, and Wild et al. (1971)Wild H.E., Sawyer C.N., McMahon T.C. 1971. Factors affecting nitrification kinetics. J. Water Pollut. Control Fed. 43: 1845-1854. found an ideal pH range for nitrification between 7.5 and 8.5. As nitrifiers are known to decrease pH, the sharp negative correlation found between pH and ΔN2O in the pH range 7.2-7.4 in the upper Minho estuary (Fig. 3E) may be a direct result of nitrification. N2O saturation values ranging from 101% to 166% in the Minho estuary and 113% to 227% in the Lima estuary indicate that both estuaries behave as a potential N2O source to the atmosphere. Positive N2O water-air fluxes prevailed in all sampling stations, decreasing in general from upper to lower estuaries. However, this tendency was more pronounced in the Lima estuary, where higher N2O fluxes in the river-influenced area were about twice (~12.0 μmol m2 d1; C96) those observed in the upper part of the Minho estuary (~6.0 μmol m2 d1; C96). It is likely that a greater turbulence of Minho upper estuary waters leads to a more rapid degassing of N2O to the atmosphere in this part of the system.

Estimated N2O fluxes were similar to those reported from the Portuguese Tagus and Sado estuaries (Gonçalves et al. 2010Gonçalves C., Brogueira M.J., Camões M.F. 2010. Seasonal and tidal influence on the variability of nitrous oxide in the Tagus estuary, Portugal. Sci. Mar. 74S1: 57-66. , 2015Gonçalves C., Brogueira M.J, Nogueira M. 2015. Tidal and spatial variability of nitrous oxide (N2O) in Sado estuary (Portugal). Est. Coast. Shelf Sci. 167: 466-474. ) (Table 4) but much lower than that of the Douro estuary (Barnes and Upstill-Goddard 2011Barnes J., Upstill-Goddard R.C. 2011. N2O seasonal distributions and air-sea exchange in UK estuaries: Implications for the tropospheric N2O source from European coastal waters. J. Geophys. Res. B. 116: G01006.). These fluxes were also much lower than those reported from the Scheldt estuary in Belgium, the Ems estuary in Germany, the Humber estuary in the UK, the Guadalete estuary in Spain and the Pearl River estuary in China (Table 4).

Table 4. – N2O saturation, water-air fluxes and emissions for several world estuaries. Estimated using the relationships of (a) Clark et al. (1995)Clark J.F., Schlosser P., Simpson K.J, et al. 1995. Relationship between gas transfer velocities and wind speeds in the tidal Hudson River determined by the dual tracer technique. In: Jhane B., Monahan E. (eds), Air-Water Gas Transfer. AEON Verlag and Studio, Germany, pp. 785-800.; (b) Carini et al. (1996)Carini S., Weston N., Hopkinson C., et al. 1996. Gas exchange rates in the Parker River estuary, Massachusetts. Biol. Bull. 191(2): 333-334. ; (c) Raymond and Cole (2001)Raymond P.A., Cole J.J. 2001. Gas exchange in rivers and estuaries: choosing a gas transfer velocity. Estuaries 24: 312-317.; (e) unpublished data.

Estuaries Area (km2) Nitrous Oxide (N2O) Sampling survey Reference
Saturation (%) Water-air fluxes (µmol m2 d1) Emission (Mg N2O-N yr–1)
Loire, France 41 84 - 271 14.3 (a) 6.1 Single de Wilde and de Bie (2000)de Wilde H.P.J., de Bie M.J.M. 2000. Nitrous oxide in the Schelde estuary: Production by nitrification and emission to the atmosphere. Mar. Chem. 69: 203-216.
Scheldt, Belgium 269 710 66.6 (a) 1.8×102 Seasonal de Wilde and de Bie (2000)de Wilde H.P.J., de Bie M.J.M. 2000. Nitrous oxide in the Schelde estuary: Production by nitrification and emission to the atmosphere. Mar. Chem. 69: 203-216.
Tagus, Portugal 320 101 - 147 –1.0 - 10.4 (a) 12.8 - 16.0 (e) Seasonal Gonçalves et al. (2010)Gonçalves C., Brogueira M.J., Camões M.F. 2010. Seasonal and tidal influence on the variability of nitrous oxide in the Tagus estuary, Portugal. Sci. Mar. 74S1: 57-66.
Humber, UK 303.6 100 - 4250 76.6 (a) 2.5×102 Seasonal Barnes and Upstill-Goddard (2011)Barnes J., Upstill-Goddard R.C. 2011. N2O seasonal distributions and air-sea exchange in UK estuaries: Implications for the tropospheric N2O source from European coastal waters. J. Geophys. Res. B. 116: G01006.
Tay, UK 121.3 100 - 118 2.5 (a) 2.5 Single Barnes and Upstill-Goddard (2011)Barnes J., Upstill-Goddard R.C. 2011. N2O seasonal distributions and air-sea exchange in UK estuaries: Implications for the tropospheric N2O source from European coastal waters. J. Geophys. Res. B. 116: G01006.
Tyne, UK 7.9 98 - 280 7.5 0.37 Seasonal Barnes and Upstill-Goddard (2011)Barnes J., Upstill-Goddard R.C. 2011. N2O seasonal distributions and air-sea exchange in UK estuaries: Implications for the tropospheric N2O source from European coastal waters. J. Geophys. Res. B. 116: G01006.
Ems, Germany 162 181 - 1794 76.7 (a) 1.3×102 Single Barnes and Upstill-Goddard (2011)Barnes J., Upstill-Goddard R.C. 2011. N2O seasonal distributions and air-sea exchange in UK estuaries: Implications for the tropospheric N2O source from European coastal waters. J. Geophys. Res. B. 116: G01006.
Gironde, France 442 120 - 463 25.5 (a) 1.2×102 Seasonal Barnes and Upstill-Goddard (2011)Barnes J., Upstill-Goddard R.C. 2011. N2O seasonal distributions and air-sea exchange in UK estuaries: Implications for the tropospheric N2O source from European coastal waters. J. Geophys. Res. B. 116: G01006.
Douro, Portugal 2.4 280 - 650 74.7 (a) 1.9 Single Barnes and Upstill-Goddard (2011)Barnes J., Upstill-Goddard R.C. 2011. N2O seasonal distributions and air-sea exchange in UK estuaries: Implications for the tropospheric N2O source from European coastal waters. J. Geophys. Res. B. 116: G01006.
Sado, Portugal 180 91 - 162 –2.2 (b)- 3.8 (c) 3.7 - 4.4 Single Gonçalves et al. (2015)Gonçalves C., Brogueira M.J, Nogueira M. 2015. Tidal and spatial variability of nitrous oxide (N2O) in Sado estuary (Portugal). Est. Coast. Shelf Sci. 167: 466-474.
Guadalete, Spain - 96 - 2174 −0.1 - 313.2 - Seasonal Burgos et al. (2015)Burgos M., Sierra A., Ortega T., et al. 2015. Anthropogenic effects on greenhouse gas (CH4 and N2O) emissions in the Guadalete River Estuary (SW Spain). Sci. Total Environ. 503-504: 179-189.
Pearl River, China 2789 101 - 3800 0.1 - 733 1.35×103 Seasonal Lin et al. (2016)Lin H., Dai M., Kao S.J. et al. 2016. Spatio temporal variability of nitrous oxide in a large eutrophic estuarine system: The Pearl River Estuary, China. Mar. Chem. 182: 14-24.
Minho, Portugal 23 101 - 166 4.0 (c) - 4.1 (b) 0.94 - 0.96 Single This study
Lima, Portugal 5.4 113 - 227 4.7 (c) - 5.0 (b) 0.26 - 0.28 Single This study

Though N2O water-air fluxes were obtained during a single sampling, aware of the seasonal variability that characterizes these estuarine systems, we estimated the annual contribution of Minho and Lima estuaries to the global N2O emissions. Taking into account estuarine areas (23 km2 for Minho and 5.4 km2 for Lima) and the estimated mean N2O fluxes, we extrapolated an emission of 0.94-0.96 Mg N2O-N yr–1 (estimated using RC01 and C96, respectively) for the Minho estuary and 0.26-0.28 Mg N2O-N yr–1 (RC01 and C96, respectively) for the Lima estuary.

On a global perspective, estimated N2O emissions from the Minho and Lima estuaries (<1.0 Mg N2O-N yr–1) represent a reduced fraction (<0.02%) of emissions from European estuaries (6.8 Gg N2O yr1, Barnes and Upstill-Goddard 2011Barnes J., Upstill-Goddard R.C. 2011. N2O seasonal distributions and air-sea exchange in UK estuaries: Implications for the tropospheric N2O source from European coastal waters. J. Geophys. Res. B. 116: G01006.). Nevertheless, being aware of our unique seasonal sampling, and particularly the higher values of N2O emissions measured in winter spring and in other Portuguese estuaries (Gonçalves et al. 2010Gonçalves C., Brogueira M.J., Camões M.F. 2010. Seasonal and tidal influence on the variability of nitrous oxide in the Tagus estuary, Portugal. Sci. Mar. 74S1: 57-66. ), more studies assessing the seasonal variability of N2O emissions in our systems are needed.

ACKNOWLEDGEMENTSTop

Acknowledgements are due to colleagues from the IPMA Oceanography Laboratory for their assistance in sampling, technical and analytical procedures. The authors also want to thank the Instituto Hidrográfico for their assistance during sampling. The research was supported by the PoPesca MARE project (22-05-01-FDR-001) and by the FCT-Portuguese Foundation of Science and Technology (POCI 2010 and FSE) through grant SFRH/BD/28569/2006.

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