Investigating the effect of El Niño on nitrous oxide distribution in the eastern tropical South Pacific

Abstract. The open ocean is a major source of nitrous oxide
(N2O), an atmospheric trace gas attributable to global warming and
ozone depletion. Intense sea-to-air N2O fluxes occur in major oceanic
upwelling regions such as the eastern tropical South Pacific (ETSP). The
ETSP is influenced by the El Niño–Southern Oscillation that leads to
inter-annual variations in physical, chemical, and biological properties in
the water column. In October 2015, a strong El Niño event was developing
in the ETSP; we conduct field observations to investigate (1) the N2O
production pathways and associated biogeochemical properties and (2) the
effects of El Niño on water column N2O distributions and fluxes
using data from previous non-El Niño years. Analysis of N2O natural
abundance isotopomers suggested that nitrification and partial
denitrification (nitrate and nitrite reduction to N2O) were occurring
in the near-surface waters; indicating that both pathways contributed to
N2O effluxes. Higher-than-normal sea surface temperatures were
associated with a deepening of the oxycline and the oxygen minimum layer.
Within the shelf region, surface N2O supersaturation was nearly an
order of magnitude lower than that of non-El Niño years. Therefore, a
significant reduction of N2O efflux (75 %–95 %) in the ETSP
occurred during the 2015 El Niño. At both offshore and coastal stations,
the N2O concentration profiles during El Niño showed moderate
N2O concentration gradients, and the peak N2O concentrations
occurred at deeper depths during El Niño years; this was likely the
result of suppressed upwelling retaining N2O in subsurface waters. At
multiple stations, water-column inventories of N2O within the top 1000 m were up to 160 % higher than those measured in non-El Niño years,
indicating that subsurface N2O during El Niño could be a reservoir
for intense N2O effluxes when normal upwelling is resumed after El
Niño.


2 phases, El Niño, La Niña and neutral. During El Niño / La Niña years, higher / lower sea surface temperature and deepening / shoaling of the thermocline occur in the Eastern tropical South Pacific (ETSP) (Barber and Chavez, 1983). During El Niño years, upwelling is suppressed in the ETSP, leading to a reduction of upward nutrient fluxes to the surface waters and decreased primary production (Chavez et al., 2003;Ñiquen and Bouchon, 2004).
The ETSP is an oceanic region with intense sea-to-air flux of nitrous oxide (N2O), a strong greenhouse gas and a potent 5 ozone depleting agent (Ravishankara et al., 2009). Diverse microbial processes involved in the production and consumption of N2O occur in the ETSP, a major oceanic oxygen minimum zones (OMZs) having wide range of O2 concentrations spanning sub-nanomolar level at intermediate depths (Revsbech et al., 2009) to atmospheric saturation at the surface. In the presence of oxygen, N2O is a by-product during the first step of nitrification, i.e. ammonium (NH4 + ) oxidation to nitrite (NO2 -) (Anderson, 1964). Under suboxic and anoxic conditions, N2O is produced via partial denitrification, i.e. NO2 -10 reduction and nitrate (NO3 -) reduction (Codispoti and Christensen, 1985). The dominant biological sink of N2O in the ocean is the last step of denitrification where N2O is reduced to N2 under anoxic conditions (Babbin et al., 2015). Recent investigations suggest that N2O uptake by diazotrophs is a possible N2O sink at the surface waters (Farí as et al., 2013;Cornejo et al., 2015). Its environmental significance awaits further exploration.
Recent modelling efforts have highlighted that the ENSO events could prominently change biogeochemical processes 15 related to nitrogen cycling (Carrasco et al., 2017;Mogollón and Calil, 2017;Yang et al., 2017). During El Niño events in the 1980's, oceanic N2O fluxes decreased (Cline et al., 1987;Butler et al., 1989), which could be related to changes O2 and organic matter availabilities that are critical environmental factors regulating N2O production (Elkins et al., 1978;Farí as et al., 2009;Aré valo-Martí nez et al., 2015;Kock et al., 2016). Here we report water column nitrogen biogeochemistry, N2O distribution and natural abundance isotopes during October 2015 when a strong El Niño event (recurrence interval > 10 20 years) was developing Santoso et al., 2017). Stable isotope analyses ( 15 N vs. 14 N and 18 O vs. 16 O) were used to determine the pathways of N2O production and consumption as outlined previously (Yamagishi et al., 2007;Grundle et al., 2017). Recent publications of time-series studies focusing on biogeochemical variations in the ETSP (Gutié rrez et al., 2008;Farí as et al., 2015;Graco et al., 2017) and the marine N2O database  allow us to present the effects of a strong El Niño event on water column hydrography and N2O distributions. 25

Field sampling and laboratory measurements
The progress and the strength of El Niño was quantified by the Ocean Niño Index (ONI, Figure 1), defined as the running 3-month average sea surface temperature anomaly for the Niño 3.4 region in the east-central tropical Pacific (5˚S -5˚N, 120˚W -170˚W). The 2015-16 El Niño was an "extreme El Niño event" indicated by ONI ≥ 0.5 °C. The ASTRA- OMZ 30 SO243 cruise on board the R/V Sonne took place between the 5 th and 22 nd October 2015 from Guayaquil, Ecuador to Biogeosciences Discuss., https://doi.org /10.5194/bg-2018-453 Manuscript under review for journal Biogeosciences Discussion started: 6 November 2018 c Author(s) 2018. CC BY 4.0 License.
3 Antofagasta, Chile (Figure 2a). In October 2015, the El Niño was still developing with ONI = 2.1°C, comparable to other strong El Niño events in 1972-73, 1982-83, 1997-98 (Stramma et al., 2016. according to their respective water depth: The coastal stations are shallower than 250 m whereas the offshore stations are > 3000 m in depth. Water samples were taken from a 24 x 10L bottle CTD-rosette system. At every station, CTD-Niskin 5 bottles collected water samples at approximately 10 -20 depths spanning the observed oxygen concentration range. The CTD system was equipped with two independent sets of sensors for temperature, conductivity (salinity) and oxygen measurements. Calibration for temperature, salinity and oxygen measurements were reported previously, with standard deviations of 0.002°C, 0.0011 PSU, and 0.8 μmol L -1 [O2], respectively . The detection limit of dissolved oxygen was ~ 3 μmol L -1 ; the ODZ was operationally defined as water parcels with [O2] < 5 μmol L -1 , and the 10 boundary of oxygenated layer was defined as [O2] = 20 μmol L -1 . Dissolved NO3and NO2concentrations were measured at sea with an auto-analyzer (QuAAtro, Seal Analytical, Germany). The detection limit for NO3and NO2was 0.1 and 0.02 μmol L -1 , respectively. For N2O concentration measurements, triplicate samples were collected in 20 mL glass vials and were crimp-sealed with butyl stoppers and aluminum caps. Immediately following this, a 10 mL helium headspace was created and 50 μL of saturated mercuric chloride (HgCl2) solution was added. After an equilibration period of at least 2 hours the 15 headspace sample (10 mL) was measured by a gas chromatograph equipped with an electron capture detector (GC/ECD).
The GC was calibrated on a daily basis using dilutions of two standard gas mixtures. The detailed GC/ECD setup and calculation of N2O concentration were reported previously (Walter et al., 2006;Kock et al., 2016).
Samples for natural abundance N2O isotopes were collected and preserved with 100 μL of saturated HgCl2 in 160 mL glass serum bottles with butyl stoppers and aluminum seals. Isotopic measurements of N2O were carried out at the 20 University of Massachusetts Dartmouth following procedures previously reported (Grundle et al., 2017). In brief, dissolved N2O was extracted by an automated purge-and-trap system and concentrated with liquid nitrogen. Interfering molecules such as H2O and CO2 were isolated from N2O to increase measurement precision. A multi-collector isotope ratio mass spectrometer detected NO + fragment of N2O (mass 30, 31, for δ 15 Nα) and intact N2O molecule (mass 44, 45 and 46, for δ 15 Nbulk and δ 18 O). 25

Data Analysis
Water column N2O saturation was quantified by the N2O excess (ΔN2O), defined as the concentration difference between measured and equilibrium values: The N2O equilibrium concentration was calculated according to Weiss and Price (1980) with in-situ temperature, salinity and 30 the atmospheric N2O dry mole fraction in the year of 2015, 328 ppb at 1 atmospheric pressure (Blasing, 2016).

4
The N2O efflux from the ocean to the atmosphere was calculated as the product of N2O excess and gas transfer coefficient (kw, cm hr -1 ) that was derived according to empirical relationship proposed by Wanninkhof (2014): where U10 denotes wind speed (m s -1 ) at 10 m above sea surface, Sc denotes the Schmidt number for N2O under in-situ temperature (Wanninkhof, 2014). 5 The notations for N2O isotopomer ratios (δX) are defined as the relative difference between isotopic ratio (R) of sample and reference material: where X denotes 15 15 15 2 NN SP     (5) The value of δX is expressed as permil (‰) deviation relative to a set of reference materials: atmospheric N2 for δ 15 Nbulk δ 15 Nα and δ 15 Nβ (Mohn et al., 2014), and Vienna standard mean ocean water (VSMOW) for δ 18 O. 15

Additional datasets
The twice-weekly, 50-km resolution of sea surface temperature anomaly from NOAA's Satellite Coral Bleaching Monitoring Datasets (https://coralreefwatch.noaa.gov/satellite/ methodology/methodology.php) were used to quantify the sea-surface temperature difference of the ETSP during October 2015 relative to 1985 -1993. For N2O flux calculations, instantaneous wind speed data at each of our sampling locations were acquired from shipboard metrological measurements. 20 Seawater N2O and oxygen concentrations from previous sampling campaigns in the ETSP were extracted from the MEMENTO database . Specifically, data from the following cruises were used for comparison

Hydrography, distribution of oxygen and inorganic nitrogen
An extreme El Niño event during 2015-16 impacted the ETSP with a relatively high sea surface temperature anomaly, especially at the equatorial region (2°S -2°N and 80 -90°W) where the highest anomaly between 3 and 5°C was observed at offshore waters ( Figure 2a). The El Niño-induced warming effect decreased southwards. Between 5°S and 12°S, the 5 temperature anomaly was 2 -3°C. South of 12°S the anomaly was generally < 1°C. The shelf areas (7°S -14°S) had a progressively lower temperature anomaly southwards; > 1.5 °C and < 1 °C north and south of 12°S, respectively.

Discussion
The ETSP is one of the world's major OMZs having active N2O production and intense efflux to the atmosphere (Aré valo-Martí nez et al., 2015; Kock et al., 2016). The gradient spanning from fully oxygenated conditions to anoxia creates suitable conditions for N2O production and consumption, which causes the co-existence of water column N2O supersaturation and undersaturation (Codispoti and Christensen, 1985). To identify the N2O cycling pathways, we input N2O 5 isotopomer measurements into a simple mass balance model (section 4.1). Quantitative relationships linking O2, NO3and N2O were examined to characterize the effect of oxygenation on N2O production from NH4 + oxidation (section 4.2).
Previously measured N2O concentrations from the ETSP (MEMENTO database, Kock and Bange (2015)) were compared to data from this study to investigate the difference in water column N2O distribution and effluxes between El Niño and non-El Niño years (section 4.3), which would better constrain the natural variability of N2O cycling in the ETSP. 10

N2O cycling pathways inferred from natural abundance isotopic and isotopomeric signatures
The analyses of natural abundance isotopomers quantify the substitutions of nitrogen and oxygen isotopes ( 15 N, 14 N, 18 O and 16 O) occurring on the linear asymmetric N2O molecule (Yoshida and Toyoda, 2000), and can be used to identify potential production and consumption pathways (Yamagishi et al., 2007;Grundle et al., 2017). The production of N2O in an 15 isolated water body follows mass conservation of the respective isotopes. The mass balance model proposed by Fujii et al. (2013) quantified the isotopic signature of N2O produced within the water mass (δproduced) by the linear regression of the inverse N2O concentration (1/[N2O]measured) and the respective isotope values (δobserved): where [N2O]initial and δinitial refer to source water N2O concentration and isotopic signature, respectively. It has been shown 20 that SP is indicative of N2O production pathways, because SP is independent of isotopic values of N2O production substrates; generally, N2O produced via NH4 + oxidation and partial denitrification have distinctive SP values of 30 ± 5 ‰ and 0 ± 5 ‰, respectively (Toyoda et al., 2011). Thus, N2O production processes can be qualitatively characterized by means of SPproduced. We further identified four water bodies (coastal and offshore stations combined) from shallow to deeper depths with distinctive features such as O2, NO2concentrations and depths (Table 1) to discuss N2O cycling pathways as follows. 25 (1) Upper oxycline and surface ( Figure S1a): [O2] > 20 μmol L -1 . All the samples were from < 200 m (data not shown), N2O production from this water body could actively contribute to atmospheric efflux. The samples had variable SP values (-9 -34 ‰); some coastal samples had the lowest water column SP values ever reported (-9 ‰, Figure 4g). The low SPproduced (6.4±1.9) indicates that both nitrification and denitrification were sources of N2O to the upper oxycline, with the majority appearing to come from denitrification. Given that the O2 concentrations were too high for denitrification to proceed locally 30 in the upper oxycline and the surface (Codispoti and Christensen, 1985), the SP signature in this water body was a mixture of (2) N2O peak ( Figure S1b): [O2] < 20 μmol L -1 and [NO2 -] < 1 μmol L -1 . Generally the samples were from N2O concentration maxima near the upper boundary of the ODZ. The SPproduced is relatively low (8.3±3.0 ‰) at this suboxic water 5 body ([O2] < 20 μmol L -1 ), which allowed N2O production from denitrification while inhibited N2O consumption (Bonin et al., 1989;Farí as et al., 2009). With the SPproduced, we conclude that water column N2O maximum was mainly attributed to partial denitrification (i.e. NO2and NO3reduction), with minor contribution from nitrification. This is consistent with previous 15 N tracer incubation experiments demonstrating the coincidence between local N2O concentration maximum and high rates of N2O production from NO2and NO3reduction that are 10 -100 fold higher than the rate of N2O production 10 from NH4 + oxidation (Ji et al., 2015).
(3) Oxygen deficient zone ( Figure S1c): [O2] < 5 μmol L -1 and [NO2 -] > 1 μmol L -1 . Accumulation of NO2within the anoxic layer is prominent feature of ODZ, where N2O consumption occurs (Codispoti and Christensen, 1985). The isotopic signature of "produced N2O" had distinctively high δ 15 Nbulk (8.5 ‰), and δ 18 O (71 ‰, Table 1 and Figure S2), and this is indicative of N2O reduction to N2 which results in an isotope enrichment of the remaining N2O pool in the process of N-O 15 bond breakage (Toyoda et al., 2017). The SP signature was also high (39.9 ‰). While NH4 + oxidation can produce N2O with similar SP values, we rule this out given the low O2 concentrations which were observed (Peng et al., 2016). instead, similar to the high δ 15 Nbulk and δ 18 O values which were observed, we suggest that the high SP values which were recorded in the ODZ, where N2O undersaturation occurred, were also a result of N2O consumption, as reduction of N2O can also result in high SP values (Popp et al., 2002;Well et al., 2005;Mothet et al., 2013). Based on the observed δ 15 Nbulk, δ 18 O and SP values 20 of N2O, we conclude that N2O consumption was the predominant N2O cycling pathway in the water body with [O2] < 5 μmol L -1 and [NO2 -] > 1 μmol L -1 in the ETSP.
(4) Intermediate waters ( Figure S1d): Samples from depths 500 -1000 m. Generally, the N2O concentrations peaks below the oxygen minimum layer at the offshore waters can be found in this water body (Figure 4a). From the linear regression, the SPproduced is 15.6±4.1 ‰. The samples had [O2] = 5 -70 μmol L -1 , suitable for N2O production from both 25 nitrification and denitrification. Downward mixing and diffusion from ODZ is unlikely because the ETSP is a major upwelling region and ODZ samples had high SP values (see next paragraph). We conclude that localized N2O production from nitrification and denitrification are important pathways in this region of the water column, and probably contributed to production was not derived due to the lack of complimentary dataset (i.e. nitrate and nitrite isotopes) and thus we are not able to investigate the change of N2O production rates during nitrification and denitrification that are affected by El Niñoinduced lower export production (in comparison to non-El Niño years, Espinoza-Morriberón et al. (2017)).

The effect of O2 on N2O production from NH4 + oxidation 5
The oxygenated surface mixed layer is constantly in direct contact with the atmosphere. Thus, N2O production via NH4 + oxidation is important to oceanic N2O fluxes. During NH4 + oxidation to NO2 -, the effectiveness of N2O production in oxygenated waters can be quantified with the N2O yield, which is defined as the molar nitrogen ratio of N2O produced and NH4 + oxidized. In oxygenated waters, the near absence of NH4 + and NO2suggest the amount of NH4 + oxidized produces equal amounts of NO3within measurement error. Rees et al. (2011) and Grundle et al. (2012)  observed. Here, to avoid sampling the ODZ where suboxic condition stimulates N2O production from partial denitrification, 15 only data from the upper oxycline were used to perform linear regression. The slope of the regression at [NO3 -] < 20 μmol L -1 (corresponding to [O2] > 100 μmol L -1 ) is 0.85 ± 0.11, indicating that 0.085 ± 0.011 nmol of N2O is produced for every μmol of NO3produced (or NH4 + oxidized), equating to a molar nitrogen yield (mol N2O-N produced / mol NO3produced) of 0.17 ± 0.02 %. At [NO3 -] > 20 μmol L -1 (corresponding to [O2] < 100 μmol L -1 ) the yield increases to 0.85 ± 0.13 %.
These N2O yield estimates are generally comparable to previously reported values (0.04 -1.6 %) in the ETSP (Elkins 20 et al., 1978;Ji et al., 2015), and indicating that potential N2O production from NH4 + oxidation decreases with water column oxygenation due to intrusion of oxygen-rich water masses (Llanillo et al., 2013;Graco et al., 2017). As discussed earlier, the oxycline samples were probably influenced by mixing of suboxic water with active denitrification producing high N2O concentrations and low NO3concentrations; the N2O yield estimates here are thus spatially and temporally integrated. As a comparison, 15 N tracer incubation method directly measured 0.04 % N2O yield during NH4 + oxidation at [O2] > 100 μmol L -25 1 (Ji et al., 2015).

N2O distribution and fluxes during El Niño
Excess N2O (ΔN2O) in surface waters is one of the principal factors regulating N2O fluxes. To evaluate the effect of strong El Niño events on oceanic N2O fluxes, we compare surface and water column ΔN2O concentrations in shelf waters (< 30 300 m depth) along 8 -16 °S during El Niño (October 2015) and "neutral" conditions (December 2012, from Kock et al. (2016)). Both data sets revealed that higher surface ΔN2O concentrations and thus higher potential N2O efflux occurred at Suppressed upwelling or increased downwelling during El Niño events, as observed in both observational and model studies (Llanillo et al., 2013;Graco et al., 2017;Mogollón and Calil, 2017), can directly and indirectly affect N2O fluxes to the atmosphere: First, reduced upward transport of subsurface N2O-rich water not only decreased surface ΔN2O, but also increased subsurface ΔN2O, which is illustrated by the comparative observation of higher subsurface ΔN2O concentrations in coastal waters in October 2015 (Figure 7b, 7c) than those in December 2012 (Figure 7e, 7f). Second, because the oxygen 10 sensitivity of the denitrification sequence increases with each step (Körner and Zumft, 1989) The decrease of surface ΔN2O concentration during El Niño was associated with an increase of subsurface N2O 20 concentrations. Water column ΔN2O concentration profiles at expanded temporal and spatial coverage (see Figure 2a for station coordinates) were compared within the same season between El Niño and non-El Niño years (Figure 8). We included data from January 2015, which had the highest ONI during austral summer than any other years. Generally, subsurface ΔN2O concentration peaks were observed at deeper depths during 2015. Offshore stations had higher subsurface peak ΔN2O concentrations during El Niño (Figure 8a, 8b), except at station C where the peak concentration during October 2015 was 25 comparable to that of December 2012 (Figure 8c). At coastal stations D and E, higher ΔN2O concentrations were found below 50 m but peak ΔN2O concentrations were lower during El Niño years (Figure 8d, 8e). In the southernmost coastal station F, the peak ΔN2O concentration was higher in 2015 than that of 1985; both were found at similar depths at ~ 60 m.
The increase of subsurface N2O concentrations during El Niño resulted in OMZ water column retaining larger amount of N2O, as shown by higher depth-integrated N2O concentrations during El Niño years than normal years in both coastal and 30 offshore waters (Figure 9).
In all, the apparent decrease in N2O efflux during the El Niño event in the tropical Pacific, as shown in this study and others (Cline et al., 1987;Butler et al., 1989) is the result of complex physical and biochemical changes. The above comparative analyses are simple due to limited data availability. Consequently, these following aspects are yet to be Biogeosciences Discuss., https://doi.org /10.5194/bg-2018-453 Manuscript under review for journal Biogeosciences Discussion started: 6 November 2018 c Author(s) 2018. CC BY 4.0 License. resolved: (1) It is unclear how offshore N2O fluxes vary from "neutral" to El Niño years. Current ΔN2O profiles show higher surface ΔN2O concentrations at station A and B in 2015 (Figure 8a and 8b), whereas the surface ΔN2O was lower in 2015 at station C (Figure 8c). A zonal (east-west) section near 12°S showed slightly higher offshore surface ΔN2O in 2015 (~ 5 nmol L -1 , Figure 7c) than in 2012 (~ 1 nmol L -1 , Figure 7f). The decrease in coastal N2O fluxes during El Niño could be compensated by increase in offshore fluxes. (2) The southward relocation of high surface ΔN2O from neutral to El Niño 5 years (Figure 7a and 7d) possibly results in higher surface ΔN2O hence higher N2O flux in southern region of ETSP (e.g. south of 16°S, Figure 8f). (3) It is possible that once the normal upwelling is resumed after the El Niño event, N2O produced and retained in the subsurface layer in coastal and offshore waters could be a potential reservoir contributing to high N2O fluxes. (4) The co-occurrence of El Niño and mesoscale eddy formation along the Peruvian coast will have complicated effects on N2O fluxes, which remains unexplored. 10

Conclusions
The eastern tropical South Pacific Ocean is a major upwelling region that is ideal to study the effect of strong El Niño events on N2O efflux and associated water column biogeochemistry. During a developing strong El Niño event in October 2015, a more pronounced warming effect occurred at lower latitudes in the ETSP. In comparison to conditions in December 2012 (non-El Niño), the depths of the oxygen deficient zone's upper boundary at lower latitudes were deeper in October 15 2015, coinciding with lower peak N2O concentrations. Shelf N2O effluxes were significantly lower during 2015 El Niño as a result of lower surface levels of N2O supersaturation. However, a change of upwelling pattern appeared to cause higher subsurface N2O concentrations and doubled the depth-integrated N2O concentrations during El Niño than in other non-El Niño years. Natural abundance isotopic and isotopomer analysis indicated that both nitrification and denitrification are important pathways for N2O production, and denitrification-derived N2O probably contributes to the efflux to the 20 atmosphere. Decreased N2O efflux and subsurface accumulation during strong El Niño events is likely the result of suppressed upwelling and water column oxygenation. However, the complex spatial and temporal patterns of El Niñoinduced N2O distribution in ETSP remain to be explored.        Table S1 for references.

Data availability
Raw data presented in this manuscript can be found in the Supplementary material.