The Arabian Sea is a major oceanic nitrogen sink, and its oxygen-deficient zone (ODZ) extends from 150 m to 1200 m water depth. To identify the dominant transformation processes of reactive nitrogen and to quantify the amounts of nitrogen turned over in the different reactions of the nitrogen cycle, we use paired data on stable isotope ratios of nitrogen and oxygen in nitrate and nitrite measured at four near-coastal and five open ocean stations in the Arabian Sea. We find significant nitrate reduction and denitrification between 100 m and 400 m in the open Arabian Sea, which are most intense in the eastern and northern part of the basin, and estimate that about 50% of initial nitrate is being reduced either to dinitrogen gas (denitrification) or to nitrite (nitrate reduction) in the core zone of denitrification. Nitrite accumulates in concentrations above 4 µM in the water column of the eastern and northern Arabian Sea. Large differences in isotopic ratios of nitrate and nitrite and a decoupling of their stable nitrogen and oxygen isotopes can be explained by the reoxidation of nitrite. The observed decoupling of the paired isotopes may be due to the exchange of oxygen of nitrite with oxygen from ambient water. In agreement with model estimates from the literature, about 25% of the nitrate initially reduced to nitrite is returned to the nitrate pool by nitrification in the upper and lower denitrification layer while 40% is denitrified.
 The main reactive nitrogen source to the ocean is diazotrophic N2 fixation, and the major sink was thought to be heterotrophic denitrification [Brandes and Devol, 2002]. During the last years, nitrogen transformation processes known to occur in the sediments were detected in oxygen-deficient zones (ODZs, Figure 1) such as dissimilatory nitrate reduction to ammonium (DNRA) [Lam et al., 2009] or anaerobic ammonium oxidation (anammox), which produces dinitrogen gas from nitrite and ammonium [Kuypers et al., 2003].
 Until recently, it was assumed that the combined oceanic nitrogen losses were larger than the total reactive nitrogen gained by the global ocean from N2 fixation and other external sources [Codispoti, 2007]. This may, however, be due to an underestimation of diazotrophic dinitrogen fixation by the method used previously. New results with a modified methodology supply much higher nitrogen fixation rates and suggest that the nitrogen budget has been balanced for the last 3000 years [Großkopf et al., 2012]. ODZs at midwater depths in the oceans are prominent sinks, where an estimated one third of the loss occurs; the remainder is eliminated in sediments [Brandes and Devol, 2002]. Evidently, most ODZs expanded and deepened in the world's oceans during the last decades [Stramma et al., 2008].
 Whereas anammox appears to be the dominant process of nitrogen loss in the Peru upwelling system [Lam et al., 2009; Thamdrup et al., 2006], the main nitrogen sink in the Arabian Sea is heterotrophic denitrification [Bulow et al., 2010; Ward et al., 2009]. Anammox may be significant in the Oman upwelling region if coupled with DNRA to supply sufficient amounts of ammonium [Jensen et al., 2011]. Nitrite accumulates in the so-called secondary nitrite maximum (SNM) between 150 m and 400 m water depth in the central to northeastern Arabian Sea to concentrations >5 µM [Naqvi, 1991]. This accumulation may be due to the fact that nitrate reduction is more common and carried out by more bacterial species than the reduction of nitrite to dinitrogen gas [Naqvi et al., 2008; Zumft, 1997]. The temporally accumulating nitrite can further be completely denitrified to dinitrogen gas (Figure 1) [Ward et al., 2009]. Alternatively, it is used in the anammox reaction to oxidize ammonium or is reduced to ammonium by DNRA [Jensen et al., 2011]. Nitrite may also become reoxidized to nitrate at the upper or lower fringe of the ODZ at appropriate oxygen concentrations [Anderson et al., 1982]. Other ODZ studies from the Eastern Tropical South Pacific (ETSP) and the Benguela upwelling system detected nitrite oxidation even at low oxygen concentrations (<2.5 µM) [Füssel et al., 2011; Lipschultz et al., 1990]. Dominant processes and rates of N elimination in the ODZ of the Arabian Sea appear to vary on short temporal and spatial scales as 15N-incubation techniques suggest [Jensen et al., 2011; Ward et al., 2009], and the regulating environmental influences on dominance of either denitrification or anammox are unresolved.
 Most incomplete reactions in the nitrogen cycle, such as water column denitrification and probably also anammox, are associated with a kinetic fractionation of stable isotopes 14N and 15N, so that the lighter 14N is preferentially found in the reaction product, and the heavy isotope is enriched in the residual substrate pool [Cline and Kaplan, 1975; Mariotti et al., 1981]. The degree of fractionation is expressed by the isotopic effect (ε) as the difference between the δ values of the source and the product [Mariotti et al., 1981]. Nitrate reduction to nitrite has an isotopic effect of 15ε = 20–30‰ [Altabet et al., 1999; Brandes et al., 1998; Cline and Kaplan, 1975]. Laboratory experiments show that during assimilation of nitrate (15ε = ~5‰) as well as during heterotrophic denitrification, the ratio of N and O enrichment factors 15ε/18ε is ~1 [Granger et al., 2008; Granger et al., 2004]. It can thus be assumed that if only these two processes were responsible for any change in nitrate concentrations, the preformed offset in the original water mass between δ15NNO3 and δ18ONO3 would be maintained. Deepwater nitrate has an average δ15NNO3 value of ~5‰ [Sigman et al., 2005] whereas its δ18ONO3 should be close to 0‰. Both values can deviate locally, and studies have reported average values between 4.8 and 5‰ for δ15NNO3 and between 1.8 and 2.4‰ for δ18ONO3 [Bourbonnais et al., 2009; Sigman et al., 2005].
 Several processes can lead to a decoupling of δ15NNO3 and δ18ONO3. Remineralization and nitrification of organic matter from N2 fixation, for instance, produce nitrate with low δ15NNO3 compared to δ18ONO3 [Sigman et al., 2005]. A second process decoupling δ15NNO3 and δ18ONO3 is the nitrification of ammonium or nitrite in ODZs as it is associated with the uptake of oxygen from either the dissolved oxygen pool or ambient water [Casciotti and McIlvin, 2007; Sigman et al., 2005]. The isotopic effect 15εΝΗ4 associated with ammonia oxidation to nitrite (Figure 1) is between 14 and 19‰ for common oceanic nitrifiers and can be much higher for some species [Casciotti et al., 2003]. Theoretically, half of the oxygen used for ammonia oxidation stems from ambient water, and half is taken from dissolved oxygen, which has a δ18O value of up to 40‰ in ODZs [Bender, 1990; Levine et al., 2009]. The second step of nitrification (nitrite oxidation to nitrate, Figure 1) was found to be associated with a negative kinetic effect on stable nitrogen and oxygen isotopes of ~ −13‰ and ~ −7‰, respectively [Buchwald and Casciotti, 2010; Casciotti, 2009]. The reason is that nitrite takes up water and forms an intermediate with a bound water molecule. The heavier isotope preferably reacts to nitrate, whereas the lighter isotope is more likely to again lose the water molecule [Casciotti, 2009]. According to stoichiometry, one third of the oxygen taken up during ammonia oxidation to nitrate should stem from dissolved oxygen whereas two thirds are taken up from ambient water. However, in reality, the exchange of oxygen with water may result in a dissolved oxygen contribution of less than one sixth [Sigman et al., 2005]. Moreover, any uptake of oxygen from water or the dissolved molecular oxygen pool is associated with considerable positive isotopic effects difficult to distinguish even experimentally [Buchwald and Casciotti, 2010; Casciotti, 2009].
 In addition to the decoupling of δ15NNO3 and δ18ONO3 [Sigman et al., 2005], a strong depletion of nitrite in 15N [Casciotti et al., 2007] suggests that denitrification is not the only significant process in ODZs. This highlights the importance to analytically differentiate between nitrate and nitrite in regions of nitrite accumulation [Casciotti and McIlvin, 2007].
 In this work, we use water column data on oxygen and nutrient concentrations as well as paired isotope measurements of nitrate and nitrite in vertical profiles from the Arabian Sea to identify the predominant processes of nitrogen transformation in the ODZ. Using the fractionation factors, the difference between isotopic ratios of nitrate and nitrite and paired isotope measurements, we attempt to quantitatively estimate the relevance of the various processes of the nitrogen cycle.
1.1 Study Area
 The Arabian Sea is one of the three major sinks of reactive nitrogen in the world's ocean due to widespread reduction of nitrate in its midwater ODZ; the two others are the Eastern Tropical North and South Pacific. High sinking fluxes of organic matter result from monsoonal upwelling of nutrients and conspire with poor ventilation of intermediate waters to cause low oxygen concentrations in the water depth interval from 150 m to >1000 m. Most intense upwelling and primary production occur along the Arabian Peninsula during the SW monsoon [Banzon et al., 2004; Marra and Barber, 2005]. The oxygen deficit is stronger in the NE basin due to poorer ventilation and is spatially decoupled from the western Arabian Sea upwelling area [Naqvi, 1991]. The main water mass in the ODZ is the Indian Central Water (ICW; σθ = 26.7 kg m−3) that combines Subantarctic Mode Water, Antarctic Intermediate Water, and Indonesian Intermediate Water [Böning and Bard, 2009; Morrison, 1997; Morrison et al., 1998]. Already depleted in oxygen, the ICW enters the Arabian Sea in its western part at a core depth of 300 m and continues to lose oxygen while accumulating nutrients from mineralization of sinking organic matter on its way to the eastern and northern parts of the basin. Effective nitrate loss occurs mostly between 150 m and 400 m [Naqvi et al., 2008].
 The inflows of intermediate water masses from the Persian Gulf (PGW) and Red Sea (RSW) [Shailaja et al., 2006] locally modify the water mass structure of the western Arabian Sea. These water masses are surface derived and, thus, oxygen rich and nutrient poor [Morrison et al., 1998]. Their admixture contributes to the nitrate deficit at depth intervals situated at 200–400 m (PGW; σθ = 26.2–26.8 kg m−3) and 500–800 m (RSW; σθ = 27.0–27.4 kg m−3), respectively, and causes better ventilation of the western part of the basin [Mantoura et al., 1993; Prasad et al., 2001].
 O2 concentrations below ~5 µM in the midwater ODZ trigger denitrification [Cline and Richards, 1972; Devol, 1978] which creates a nitrate minimum and high δ15NNO3 values of the residual nitrate [Naqvi et al., 1998]. Upwelling and assimilation of that enriched nitrate transfer the high δ15NNO3 values into biomass, sinking particles of export production and eventually to the sediment record, which evidences large changes in regional denitrification intensity on glacial/interglacial time scales [Altabet et al., 2002; Gaye-Haake et al., 2005; Möbius et al., 2011; Suthhof et al., 2001].
2 Materials and Methods
 Water was sampled at nine stations during the cruise Meteor 74/1b in September/October 2007 (Figure 2 and Table S1 in the supporting information). The open ocean stations had been occupied repeatedly in 1995 to 1997 during the Joint Global Ocean Flux Study (JGOFS) cruises [Codispoti, 2000; Morrison et al., 1998]. Samples for stable nitrate and nitrite isotopes measurements were taken between 0 and 400 m water depth at four stations on the shelf and slope off Oman (#944–#947) in coarse vertical resolution (three to four samples). At five stations in the open Arabian Sea (#949, #950, #953, #955, and #957, Figure 2), the denitrification zone was sampled every 25 m down to 400 m. Sampling intervals increased from 50 to 200 m between 400 m and 1200 m water depth. Ancillary water samples from the denitrification zone were taken for stable isotope analysis of only nitrate (δ15NNO3 and δ18ONO3) by removing nitrite with ascorbate treatment onboard [Granger et al., 2006]. Samples for stable nitrite and nitrate isotopes (δ15NNO3 + NO2 and δ18ONO3 + NO2) were kept frozen until analyses in the home laboratory. Nutrient analyses were carried out onboard for every 25 m at all of these stations.
2.2.1 CTD, Oxygen, and Nutrients
 The conductivity temperature depth profiler (CTD) used was a Sea-bird electronic underwater unit equipped with pressure, temperature, conductivity, oxygen, and fluorescence sensors attached to an Oceanic rosette water sampler with 18 bottles. Raw CTD data were postprocessed with the Sea-bird data processing tool (data conversion, wild edit, split, window filter, and bin average).
 Nutrients (NO3−, NO2−, and PO43−) were measured with a Skalar autoanalyzer directly after sampling onboard R/V Meteor. Oxygen concentrations in discrete samples were measured by using Winkler methods and were further used to calibrate oxygen data obtained from the CTD.
2.2.2 Nitrate and Nitrite Isotopes
 δ15N and δ18O of nitrate and nitrite (δ15NNO3 + NO2 and δ18ONO3 + NO2) were determined using the “denitrifier method” [Casciotti et al., 2002; Sigman et al., 2001]. Based on nitrate and nitrite concentrations, sample volumes calculated to yield 10 nmol N2O were injected into suspensions of Pseudomonas aureofaciens (ATCC #13985) for combined analysis of δ15N and δ18O. The resulting N2O gas in the headspace was purged into a GasBench II (Thermo Finnigan) and analyzed in a Delta Plus XP mass spectrometer. Isotope ratios are reported in per mil using the delta notation
with air N2 and Vienna SMOW as reference for 15N/14N and 18O/16O, respectively. The values were calibrated using International Atomic Energy Agency (IAEA) N3 (δ15NNO3 = +4.7‰ and δ18ONO3 = +25.6‰) and U.S. Geological Survey 34 (δ15NNO3 = −1.8‰ and δ18ONO3 = −27.9‰ [Böhlke et al., 2003]). A further internal potassium nitrate standard was measured twice within each run for quality assurance. Isotope values were corrected using the “bracketing scheme” from Sigman et al. [2009b] for δ18ONO3 and the single point correction referred to IAEA-N3 for δ15NNO3. The standard deviation for IAEA-N3 was 0.2‰ for δ15NNO3 and 0.3‰ for δ18ONO3 which is within the same specification for δ15NNO3 and δ18ONO3 for at least duplicate measurements of the samples.
3.1 Nutrient and Oxygen Concentrations
 The surface mixed layer of 10–50 m is saturated with oxygen (Figures 3a and 3b). Suboxic conditions with oxygen concentrations <5 µM were generally encountered between 100 m and 1025 m water depth, except at nearshore stations #944 and #947 (Figure 3a and Figure S1 in the supporting information). The most intense ODZ was found at station #957 (Figure 3b). Station #950 had the thickest oxygenated surface layer due to the influence of a mesoscale eddy that also caused enhanced mixing of nutrients into surface waters.
 Ratios of nitrate to phosphate decrease in the ODZ as a consequence of denitrification or anammox. Usually, the amount of nitrate removed by denitrification in the ODZ is calculated by using an empirical relationship of nitrate to phosphate molar concentrations of 16:1 in ocean waters [Redfield, 1934]. More specific, however, are regional N:P ratios. In the Arabian Sea, the nitrate deficits are best calculated from a stoichiometric relationship established from Arabian Sea JGOFS data [Codispoti et al., 2001]:
 Ammonium contents are below the detection limit of 20 nM in the ODZ [Lam et al., 2011] and were thus not included in the calculation. The NO3def is most pronounced in the core of the ODZ between 125 m and 400 m with considerable variations among the stations (Figure 3b and Table 1) and vanishes at 1200 m water depth.
Table 1. Depth Ranges of Oxygen Concentrations <5 µM, <1 µM, Nitrite Concentrations >0.5 µM, and a Nitrate Deficit (NO3def) >5 µMa
Depth Range of Oxygen Minimum and Nitrate Deficiency (m)
Total From 100 to 1100 m Water Depth
O2 <5 µM
O2 <1 µM
NO2 >0.5 µM
NO3def >5 µM
NO2 mol m−2
NO3 mol m−2
NO3def mol m−2
aNitrite and nitrate concentrations, nitrogen deficit (NO3def) in mol m−2.
M 74–1 949
M 74–1 950
M 74–1 953
M 74–1 955
M 74–1 957
 Primary nitrite maxima (PNM) occur within the nutricline (at about 10 m at the near-coastal stations and at 50 m at the offshore stations) under oxic conditions (Figures 3a and 3b and Figure S1 in the supporting information). In a global review on PNM occurrences, Lomas and Lipschultz  concluded that PNM most likely originate from the release of nitrite by phytoplankton due to incomplete assimilation of nitrate under conditions of light limitation. A minor amount of nitrite in the PNM may also be an intermediate product of dissimilatory nitrification [Lomas and Lipschultz, 2006]. In accordance with a general link to phytoplankton biomass, the most pronounced PNM of our study occur at those stations where plankton counts suggest the highest primary productivity (Kate Darling, personal communication). These were the nearshore stations #944 and #945, both influenced by active late SW monsoon upwelling, and the offshore station #950, where a mesoscale eddy mixed nutrients into the euphotic zone (Figures 3a and 3b).
 SNM that always coincide with nitrate concentration minima were recognized at all five offshore stations. Nitrite concentrations vary between 1.5 µM (#949) and 5.8 µM (#950) in the core of the denitrification zone (Figures 3a and 3b). The other three offshore stations had a broad nitrite layer with maximum concentrations >4 µM at the top and bottom. The SNM occurred only at two of the near-coastal stations (#945 and #946) and had nitrite concentrations <1.0 µM (Figure 3a).
3.2 δ15N Ratios and δ18O Ratios of Nitrate and Nitrite
 Nitrate and nitrite are both reduced to N2O when analyzing stable N and O isotopes by the denitrifier method so that results represent the stable isotopic N and O ratios of total nitrate plus nitrite (subsequently named δ15NNO3 + NO2 and δ18ONO3 + NO2). Due to different fractionation processes within the nitrogen cycle, nitrate and nitrite can have significantly different stable isotopic ratios [Casciotti, 2009; Casciotti and McIlvin, 2007]. To get an estimate of these differences, δ15NNO3 and δ18ONO3 (after treatment with ascorbate) were determined in two to five samples at each of the five offshore stations.
 Maximum δ15NNO3 + NO2 and δ18ONO3 + NO2 generally correspond to the core depth of nitrite accumulation at 200–300 m water depth (Figure 3b and Figure S1 in the supporting information). Highest values at depths of the nitrite maximum for both δ15NNO3 + NO2 and δ18ONO3 + NO2 were found at stations #953 and #957 (19.2‰ for δ15NNO3 + NO2 and 16.5‰ for δ18ONO3 + NO2, respectively) whereas lowest values (14.1‰ and 15.0‰ for δ15NNO3 + NO2 and δ18ONO3 + NO2, respectively) occur in the western Arabian Sea at station #949.
 Nitrate concentrations <2 µM prevented accurate measurements of nitrate isotope composition in most mixed-layer samples. In the subsurface layer between the ODZ and the mixed layer, we determined δ15NNO3 + NO2 values between 7.4 and 8.1‰ (Figure 3b). Minimum δ18ONO3 + NO2 values of this subsurface layer range between 3.9 and 5.3‰. δ15NNO3 + NO2 and δ18ONO3 + NO2 sharply decrease above and below their maxima in the nitrite accumulation zone (Figure 3b).
 δ15NNO3 and δ18ONO3 are generally low in deepest samples below the ODZ (6.1 to 8.0‰ and 3.6 to 5.7‰, respectively) with average δ15NNO3 of 7.2 ± 0.6‰ and δ18ONO3 of 4.6 ± 0.6‰, respectively (nitrite was not present below 650 m water depth). We use these values as source nitrate stable isotope ratios in the ODZ (ICW) rather than the lower values from other deep oceanic areas, as intermediate water masses entering the Arabian Sea have a preformed nitrate deficit [Mantoura et al., 1993] so that nitrate isotopic values may already be elevated.
 Differences between δ15NNO3 and δ18ONO3 compared with δ15NNO3 + NO2 and δ18ONO3 + NO2 in samples with trace amounts of nitrite are within or very close to the error range of the δ15NNO3 + NO2 and δ18ONO3 + NO2 method. In samples with higher nitrite concentrations, δ15NNO3 and δ18ONO3 are higher by 4–8‰ compared to the δ15NNO3 + NO2 and δ18ONO3 + NO2 (Table 2). Maximum δ15NNO3 and δ18ONO3 values occur in the SNM corresponding to the most pronounced NO3 deficit.
Table 2. Depth in Meter, Measured δ15NNO3 + NO2 and δ18ONO3 + NO2, δ15NNO3 and δ18ONO3, and Calculated δ15NNO2 and δ18ONO2 in Per Mil, Nitrite Concentrations in Micromolara
δ15NNO3 + NO2
δ18ONO3 + NO2
Δ(15,18)NO3 + NO2
aΔδ15N is the difference between stable isotopic values of nitrate and nitrite. Δ(15,18)NO3 + NO2 (Δ(15,18)NO3) is the difference between δ15NNO3 + NO2 and δ18ONO3 + NO2 (δ15NNO3 and δ18ONO3) corrected for the offset between both stable isotopic ratios at 1000–1200 m water depth (see text for explanation). δ15NNO3 + NO2, δ18ONO3 + NO2, δ15NNO3, and δ18ONO3 are shown in italics when δ15NNO2 and δ18ONO2 are not calculated in samples with nitrite <1 µM. Numbers in italics were not included in the average values (Average) and standard deviations (Stdev).
 δ15N and δ18O values of nitrite (δ15NNO2 and δ18ONO2) may be calculated for samples with nitrite concentrations >1 µM according to equations (3) and (4):
 With one exception, calculated δ15NNO2 and δ18ONO2 are negative and range from −11 to −26‰ and from −5 to −22‰, respectively (Table 2). Samples with nitrite concentrations <1 µM were excluded from nitrite isotope calculations because, in these cases, the propagating error becomes much larger than 10‰.
 Nitrite can exchange up to 50% of its oxygen with ambient water in stored samples depending on pH, temperature, and storage time. This exchange can lead to a strong enrichment of δ18ONO2 as oxygen in water has a δ18OH2O of ~0‰, and the isotopic effect of this equilibration is ~11–14‰ [Casciotti et al., 2007]. We thus have to take into account that δ18ONO3 + NO2 was enriched prior to analyses so that calculated δ18ONO2 is too high, and any decoupling between δ15NNO3 + NO2 and δ18ONO3 + NO2 is partly due to this exchange.
4.1 Nitrite Accumulation and Nitrate Deficit
 In essence, the entire depth interval below 100 m and extending to our deepest sample at 1200 m potentially was a sink for nitrate, nitrite, and ammonium. Based solely on oxygen concentrations, heterotrophic denitrification may occur in the interval from 100–175 m down to 625–1025 m (Table 1). Anammox could occur roughly between 100 m and 1200 m at our study sites as oxygen concentrations in that interval are below the anammox threshold of O2 ~10 µM [Jensen et al., 2008] and seem to be limited mainly by the availability of ammonium.
 A plot of phosphate concentrations versus nitrate concentrations (Figure 4) shows that our samples have excess phosphate compared to the global average [Gruber and Sarmiento, 1997] and that most samples are nitrate deficient compared with the regional Redfield ratio adjusted for the preformed nitrate deficit of the Arabian Sea [Codispoti et al., 2001]. The nitrate deficit of virtually all samples illustrates that denitrification has depleted nitrate relative to phosphate in the entire water column down to our deepest samples at 1200 m.
 There are local deviations in the extent of the ODZ and of the nitrate-deficient depth interval: oxygen supply by the PGW entrained into the ODZ water depth interval is very likely responsible for the elevated oxygen concentrations of >10 µM and the suppression of denitrification between 200 m and 350 m water depth at stations #944 and #947 (Figure 3a). O2 concentrations are between 30.5 and 0.7 µM in the PGW [Morrison et al., 1999], and a branch of this water mass has been observed to flow along the coast of Oman and to spread eastward into the central basin [Morrison, 1997; Prasad et al., 2001]. Furthermore, the NO3def is greater at the western and northwestern stations (#949, #950, and #957) than in the eastern part of the Arabian Sea (stations #953 and #955) (Table 1). The main reason for this difference is the larger nitrate deficit in the water masses below 300 m of the western stations, which is probably preformed [Mantoura et al., 1993]. Only a small amount of nitrite builds up at station #949, whereas it comprises more than 20% of the NO3def at station #957 (Table 1). Low nitrite concentrations have previously been reported from the western Arabian Sea, although oxygen concentrations are below the threshold for denitrification [Mantoura et al., 1993]. The virtual absence of nitrite in the western region (in which #949 is situated) may be due to either complete denitrification, which would then increase the NO3def or to nitrification/reoxidation to nitrate, or else to continuous consumption of nitrite by anammox.
4.2 Isotopic Evidence for Heterotrophic Denitrification and Parallel Nitrite Oxidation
 Denitrification is indicated by nitrite accumulation and by maxima of the two stable nitrate isotope ratios between 150 m and 400 m water depth. Midwater denitrification is a process acting on the 18O/16O and 15N/14N mixtures in source nitrate with similar fractionation factors 15ε and 18ε which are between 20 and 30‰ [Altabet et al., 1999; Barford et al., 1999; Granger et al., 2008; Sigman et al., 2009a; Voss et al., 2001]. The “Rayleigh” fractionation model is often used to calculate apparent fractionation factors from δ15NNO3 values [Altabet et al., 1999; Brandes et al., 1998]. This model assumes that the nitrate pool is advected into the ODZ and is not replenishment via isopycnal mixing. The fractionation factors obtained by the Rayleigh model are generally lower than by models taking diffusive replenishment of nutrients into account [Brandes et al., 1998]. Highest isotopic effects are obtained by the so-called “open system model” (Texts S1 and S2 and Figure S2 in the supporting information). When using the Rayleigh fractionation (described by Mariotti et al., 1988), the stable isotope value of the substrate increases according to
with δ15Ninitial being the stable isotope value of the initial product and f being the fraction of the original substrate remaining. The instantaneous product is calculated as
and the accumulated product is calculated as
 The same equations can be used for δ18ONO3 measurements, but they are not applied to δ18ONO3 + NO2 due to the sample storage artifact discussed above. Values for f are calculated according to
when using δ15NNO3 + NO2 and according to
when using δ18ONO3 and δ15NNO3.
 The 15ε and 18ε are obtained from the slope of ln(f) plotted against δ18Osubstrate or δ15Nsubstrate. The ε values obtained in our study are mostly higher than those found in incubation experiments [Barford et al., 1999; Granger et al., 2008] (Table 3). Most differences between δ15NNO3 and calculated δ15NNO2 (Δδ15N; Table 2) are even higher than our calculated 15ε (Table 3). Moreover, the 15ε/18ε of 1.2 is higher than the expected value of 1 (Table 3). Nitrate and nitrite isotopic compositions in the ODZ are thus not consistent with a sole denitrification effect and imply fractionating reactions other than or additional to denitrification as well as reactions that decouple nitrogen and oxygen isotopic compositions in nitrate and nitrite. The only process known to date which could explain both the low nitrite N-isotope values and a decoupling of N and O isotopic fractionation in nitrate is nitrite oxidation. This process shows a rare inverse isotope fractionation, with initial products being isotopically enriched relative to their source signature, depleting the source nitrite of heavy isotope species. A decoupling of oxygen versus nitrogen isotope signatures can occur due to different isotope effects for N and O during nitrite oxidation and due to the incorporation of oxygen from external sources [Buchwald and Casciotti, 2010; Casciotti and McIlvin, 2007; Sigman et al., 2005].
Table 3. Fractionation Factors ε by the Rayleigh Fractionation Model Derived From the Slope of a Plot of ln( f ) Against δ15NNO3 + NO2, δ18ONO3 or δ15NNO3, Respectively (See Text for Explanation of the Rayleigh Model)
Nitrate + Nitrite
 Plots of paired δ15NNO3 + NO2 and δ18ONO3 + NO2 values for depths ≥100 m, as well as δ15NNO3 and δ18ONO3, reveal a deviation from the expected 1:1 line at intermediate values (Figures 5a and 5b).
 A term to express this decoupling of nitrate N and O isotopes was defined by Sigman et al.  as
 For the ratio 15ε/18ε, the isotopic effects of denitrification with 15ε = 18ε are inserted so that 15ε/18ε is about 1, and any deviation of the Δ(15,18) from 0 should reflect processes during which δ15N and δ18O ratios are affected differently. We used the average deepwater nitrate stable isotope ratios from the individual stations, as we have no end member of source waters, and can assume that their stable isotope ratios are elevated above the oceanic average (see above). Most Δ(15,18)NO2 + NO3 and Δ(15,18)NO3 are negative in the interval from 100 m to 1000 m water depths, meaning that O isotope values are relatively more enriched than N isotope values. In the case of Δ(15,18)NO2 + NO3, this is partly an artifact of oxygen exchange of nitrite with water during sample storage, which can lead to elevation of δ18ONO2 toward and above the isotope value of ambient water due to equilibrium fractionation [Casciotti et al., 2007].
 To attain negative Δ(15,18)NO3, an external oxygen source is required. One important source of isotopically enriched oxygen is the exchange of oxygen between 18O-depleted nitrite and ambient water. This is an abiotic process depending on temperature, pH, and the residence time of nitrite, and it leads to a convergence of δ18ONO2 with the δ18O of water [Kool et al., 2007; Snider et al., 2010]. Recent experiments indicate that oxygen equilibration with water can affect 25% of nitrite [Buchwald and Casciotti, 2010; Casciotti et al., 2010]. This relative enrichment of δ18ONO2 in relation to δ15NNO2 can be transferred into the nitrate pool if nitrite is reoxidized.
 Differences in the negative kinetic effects on nitrogen and oxygen isotopes during nitrite oxidation, with N and O isotope effects of ~ −13‰ and ~ −7‰, respectively [Buchwald and Casciotti, 2010; Casciotti, 2009], would further deplete both nitrite isotopes and, additionally, increase δ18ONO2 relative to δ18NNO2.
 In our setting, however, values for Δ(15,18)NO3, where nitrite was removed by ascorbate treatment, are also negative and mostly close to Δ(15,18)NO2 + NO3. We consequently surmise that, barring some uncertainty regarding the abiotic equilibration of nitrite oxygen with ambient water, the two isotopes are truly decoupled (Figure 3b) owed to differences in biological processing. In the center of the nitrite maximum, the Δ(15,18) values are close to 0 suggesting that denitrification is the only significant process affecting Δ(15,18) and nitrification is virtually absent (Figure 3b and Table 2).
 The four stations with significant nitrite accumulation >1 µM in the SNM (Figure 3b) show minimum Δ(15,18)NO3 + NO2 and Δ(15,18)NO3 values at 150 m and 400 m water depth, i.e., above and below the zone with highest isotope values in the core of the denitrification maximum. We interpret this as a nitrification signal, which, as discussed above, differentially modifies nitrite oxygen and nitrogen isotopes, promoting a deviation of Δ(15,18) from zero. This observation agrees with the model of Anderson et al. , who assumed that nitrification occurs adjacent to the zone of denitrification, i.e., between 300 m and 400 m water depth and above 150 m, producing nitrate that diffuses back into the denitrification zone and further fuels denitrification. The tight spatial coupling of nitrification and denitrification may be due to the presence of microenvironments with locally higher oxygen concentrations or due to oxygen diffusion across the oxycline. The lower boundary of the denitrification zone coincides with a small but distinct oxygen peak at 400 m at all stations (Figure 3b), which we attribute to lateral advection of a relatively more oxygenated water mass such as PGW. Furthermore, little oxygen is needed for nitrification to occur: direct measurements of nitrite oxidation rates from the ETSP and the Benguela upwelling system reveal significant nitrite oxidation at O2 concentrations below 2.5 µM [Füssel et al., 2011; Lipschultz et al., 1990]. Nitrite oxidation is independent of prior ammonia oxidation if nitrite is provided by nitrate reduction.
 The production of low Δ(15,18) by nitrification under suboxic conditions helps to interpret the deviating nitrite concentration profiles at stations #949 and #950 (Figure 3b). Nitrite concentration in the SNM at #949 is very low, and the ODZ maximum values of δ15N are significantly lower than at the other locations. But the NO3def is up to 11 µM (Figure S1 in the supporting information), indicating that denitrification has been equally effective as at the other locations. The low nitrite concentrations and relatively depleted δ15NNO3 and δ18ONO3 between 150 m and 500 m water depth indicate that intense nitrification has indeed taken place as suggested by Lam et al.  and that the complete oxidation of depleted nitrite has lowered the δ15NNO3 and δ18ONO3. This unusual pattern may be due to better ventilation of the western part of the basin by PGW or ICW (see above). At station #950, where a mesoscale eddy has eroded the thermocline and mixed oxygen down to 220 m, the layer affected by the eddy is nitrite free and has relatively low isotope values but a pronounced Δ(15,18) minimum as a legacy of nitrification under initially suboxic conditions.
 Based on the concept that low Δ(15,18) thus marks zones of ongoing nitrification, Figure 6 is a schematic plot of variables and processes in the ODZ. Denitrification dominates in the zone between about 200 m and 300 m water depth, where nitrite accumulates and nitrate isotopic ratios attain maxima. This process may also be coupled to the anammox reaction, because ammonium produced by heterotrophic denitrification does not accumulate to any extent. Removal of nitrite by anammox explains the bimodal nitrite peak at locations #953–957, where we observe a relative concentration minimum in the core of nitrite accumulation and a slight isotopic enrichment of nitrate at about 250 m (Table 2). Nitrification is indicated by declining Δ(15,18)NO3 and low δ15NNO2 values in the upper ODZ just below the oxycline and below the core of the denitrification zone. Nitrification probably also adds to the bimodal pattern of nitrite accumulation that has maxima on either side of the denitrification layer. Because nitrite is an intermediate of both nitrification and denitrification, it may accumulate where microenvironments and diffusion allow close coupling of the two processes. Whereas the oxygen concentrations show a steep increase above 100 m, they drop to almost 0 µM below the small oxygen peak at 400 m. We believe that nitrification occurs only in a small layer around 400 m where oxygen remains available. Mixing and diffusion lead to the linear increases of Δ(15,18) above and below the two nitrification layers.
4.3 Box-Model-Derived Mass Balance and Stable Isotopic Ratios in the ODZ
 In order to obtain a rough estimate of the amounts of reactive nitrogen taking part in nitrate reduction, denitrification, and nitrite oxidation, we produced a box model for the upper part of the ODZ (Figure 7 and Text S2 in the supporting information). It encompasses the secondary nitrite maximum and ranges from a water depth of 125 m to 350 m. The areal extent of the secondary nitrate maximum is 1.95 × 1012 m2 [Naqvi, 1987] so that the box volume becomes 4.38 × 1014 m3. The inflow and outflow of water are calculated by dividing the volume of water by the mean residence time of water within the ODZ. As the residence time of ODZ water, we used an estimate of 10 years [Olson et al., 1993]. Nitrate inputs are calculated by multiplying the total inflow with a nitrate concentration of 34 µM; the δ15NNO3 is assumed to be 7‰. The second source of nitrogen is organically bound nitrogen exported from the euphotic zone. There are a variety of equations allowing a calculation of the organic carbon (OC) flux at all water depths if the primary production (Pp) is known. Based on satellite-derived data on primary production and results derived from sediment trap experiments, such an equation was adapted to the condition found in the Arabian Sea (OC = 0.01 Pp2/water depth0.628) [Rixen et al., 2002]. The mean primary production in the Arabian Sea of 180 g C m2 yr−1 is used to calculate the organic carbon flux at the top (125 m) and the bottom (350 m) of the modeled box. The difference between these fluxes is the amount of organic matter remineralized within the box; this number is converted into nitrogen by using the Redfield ratio. Two different remineralization processes are considered: (i) nitrification which is a chain of reactions in the course of which NH4+ is converted via NO2− (ammonia oxidation) to NO3− (nitrite oxidation) and (ii) heterotrophic denitrification which can be described by two reactions which are the NO3− reduction (equation (12)) followed by the NO2− reduction to N2O and N2 (equation (13)):
 The formation of N2 and the outflow of reactive nitrogen are the two sinks balancing the nitrogen inputs. The N2O formation during denitrification is ignored because N2O, in spite of being an important green house gas, is quantitatively of low importance for the nitrogen budget in the ODZ.
 All together, 1000 model years were calculated to get the model into equilibrium whereby the conditions were changed in five steps after steady state was reached for the set conditions (Figure 8 and Text S2 in the supporting information). For the first 200 model years, there was no remineralization of organic nitrogen, and the mean NO3− concentration attained a value of 31 µM, NO2− concentrations were 0 µM, and δ15NNO3 was 7‰ as prescribed. In the second step after the model year 200, the entire organic nitrogen was decomposed and converted into NO3− via complete oxidation of organic nitrogen (complete nitrification). Accordingly, the NO3− concentration increased by approximately 4 µM, and the δ15NNO3 was reduced slightly, as the lighter 14N is preferentially decomposed. An isotopic fraction factor of 15ε = 2‰ [Möbius, 2013] was used as the isotopic effect of ammonification. In a third step, nitrification was reduced, and the remaining organic nitrogen was remineralized by denitrification. Consequently, the NO3− concentration decreased, and the NO2− concentration increased. Assuming a fractionation factor of 15ε = 25‰ for denitrification which is in the range of those determined experimentally and empirically [Altabet et al., 1999; Barford et al., 1999; Granger et al., 2008; Sigman et al., 2009a; Voss et al., 2001], the residual nitrate became heavier and reached values around 20‰ as measured in profile #953 (Table 2). Assuming furthermore that approximately 24% of the produced NO2− is not reduced to N2, the accumulating NO2− attains concentrations of around 5 µM which also agrees quite well with measurements of profile #953. However, the resulting δ15NNO2 is −6‰ which is much higher than the measured δ15NNO2 of ~ −16‰. Assuming, in a fourth step, that the entire available organic nitrogen was utilized by denitrification, the δ15NNO2 further increases. Considering, in the fifth step, that 60% of the produced NO2− is not reduced to N2 and that 40% of the accumulated NO2− is reoxidized to NO3− with an isotopic effect of −13‰ [Buchwald and Casciotti, 2010; Casciotti, 2009], the resulting δ15NNO2 values are similar to the ones measured in profile #953. Thus, a reoxidation of 24% of initial nitrite appears to be the process explaining the low δ15NNO2 values in the upper part of the ODZ.
4.4 Processes in the Lower ODZ
 There is no nitrite accumulation below 400 m at most stations except at #957, although oxygen concentrations are <1 µM down to ~600 m (Figure 3 and Table 1) and would thus promote both denitrification and anammox. Although observations suggest that denitrification occasionally and locally extends down to 600 m [Brand and Griffiths, 2008; Morrison et al., 1999], we observe a gradual decrease of the NO3def, δ15NNO3, and δ18ONO3 suggesting linear mixing of intermediate and deepwater masses. The zone of iron reduction, indicated by maxima of dissolved Fe (II), is equivalent to the nitrite accumulation zone in the Arabian Sea [Moffett et al., 2007]. Manganese [Lewis and Luther, 2000] and iodate reduction [Farrenkopf and Luther, 2002] apparently extend to greater depths than denitrification, so that oxidation of organic matter in the lower ODZ is probably mediated by manganese and iodate reduction. Our oxygen measurements with a conventional electrode on a Seabird CTD are by a factor of 10 higher than oxygen concentrations simultaneously measured with the more sensitive switchable trace amount oxygen (STOX) microsensor [Revsbech et al., 2009] at some stations. Observed thresholds of denitrification or anammox in the oxygen profiles at our stations are thus likely too high. Denitrification is linked to very low oxygen concentrations and depends on sufficient supply of organic substrate. It is possible that denitrification ceases below approximately 400 m in the Arabian Sea, despite low but persistent oxygen concentrations. We attribute this to a limitation by organic substrate caused by rapidly sinking aggregates, reduced suspended matter concentrations, and lower quality of organic matter.
5 Summary and Conclusions
 In a study of oxygen, nutrients, and paired stable isotope ratios of nitrate and nitrite, the major nitrogen transformation processes in the Arabian Sea of the ODZ were investigated in the late SW monsoon of 2007. The near-coastal stations off Oman revealed little nitrite accumulation and sporadic ventilation of the upper ODZ by intrusion of oxygenated waters, probably from the Persian Gulf outflow. All five offshore stations have low oxygen concentrations below 5 µM so that denitrification and anammox are feasible between 100 and 1200 m water depth. However, active denitrification is restricted to the upper ODZ indicated by the accumulation of nitrite and peaks of 15NNO3 + NO2 and δ18ONO3 + NO2. δ15NNO3 and δ18ONO3 measured in selected samples are elevated compared to 15NNO3 + NO2 and δ18ONO3 + NO2 so that calculated δ15NNO2 and δ18ONO2 are negative. As the apparent fractionation factors are higher than those commonly assumed for nitrate reduction and the difference between nitrate and nitrite stable isotopic values are much higher than feasible with nitrate reduction, additional transformation processes of reactive nitrogen are evidently significant in the ODZ of the Arabian Sea. The decoupling of the δ15NNO3 and δ18ONO3, furthermore, suggests that nitrification is an important process. The smaller inverse isotopic effect on oxygen compared to nitrogen during nitrite oxidation, as well as the exchange of oxygen between the strongly depleted oxygen in nitrite with the heavier oxygen of ambient water, is responsible for the relative enrichment of oxygen isotopes compared to nitrogen isotopes. Negative Δ(15,18) values indicate two nitrification layers at 150 m and 400 m, i.e., at the upper and lower margin of the zone of denitrification. We propose that the core of the ODZ is dominated by denitrification, during which dinitrogen gas is released and nitrite is produced. If ammonium is produced by organic matter remineralization, it is removed by anammox bacteria. Bimodal nitrite peaks result not only from nitrite removal but also from coupled denitrification and nitrification at the upper and lower margins of the denitrification zone. Coupled nitrification and denitrification have been proposed by Anderson et al.  with nitrate from nitrification fueling denitrification by diffusion or mixing processes. With a simple box model, the δ15NNO3 and δ15NNO2 found in the ODZ could be reproduced if 40% of the nitrite of nitrate reduction is further reduced to dinitrogen, 24% is reoxidizing to nitrate, and 36% accumulates as nitrite in the ODZ.
 Below 400 m, oxygen concentrations <1 µM prevail, but denitrification occurs only scarcely in the Arabian Sea. The lower margin of the denitrification zone is probably determined by the paucity of organic substrate rather than by low oxygen concentrations. At the lower boundary of the ODZ, other electron acceptors such as iron, manganese, or iodate govern organic matter remineralization.
 We thank Udo Hübner for operating the CTD and water samplers as well as captain Uwe Pahl and crew of R/V Meteor (M 74-1b) for their excellent technical support. We used the program Ocean Data View to make Figures 2, 4, and 5 and Figure S1 in the supporting information and thank Rainer Schlitzer for supplying this open access program. We are grateful to Marcel M.M. Kuypers, Phillis Lam, and Marlene M. Jensen for discussions and kindly providing their ammonium data. Katharina Six and Francesca Guglielmo have helped us with many fruitful discussions and suggestions. Two anonymous reviewers have helped to improve the manuscript with their very constructive comments. Financial support for the Meteor cruise came from the Deutsche Forschungsgemeinschaft (DFG Grant GA 755/4-1).