Biogeochemistry of nitrous oxide in groundwater in a forested ecosystem elucidated by nitrous oxide isotopomer measurements

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Geochimica et Cosmochimica Acta 73 (2009) 3115–3133 www.elsevier.com/locate/gca

Biogeochemistry of nitrous oxide in groundwater in a forested ecosystem elucidated by nitrous oxide isotopomer measurements K. Koba a,b,c,*, K. Osaka d,h, Y. Tobari a,b, S. Toyoda b,e, N. Ohte d,i, M. Katsuyama d,j, N. Suzuki b,e,k, M. Itoh d,h, H. Yamagishi a,b,l, M. Kawasaki d,m, S.J. Kim d,n, N. Yoshida a,b,e,f, T. Nakajima g a

Department of Environmental Science and Technology, Tokyo Institute of Technology, Yokohama 226-8502, Japan b SORST Project, Japan Science and Technology Corporation, Saitama 332-0012, Japan c Faculty of Agriculture, Tokyo University of Agriculture and Technology, Saiwai-cho, 3-5-8, Fuchu-city, Tokyo 183-8509, Japan d Department of Environmental Science and Technology, Kyoto University, Kyoto 606-8502, Japan e Department of Environmental Chemistry and Engineering, Tokyo Institute of Technology, Yokohama 226-8502, Japan f Frontier Collaborative Research Center, Tokyo Institute of Technology,Yokohama 226-8502, Japan g Lake Biwa Environmental Research Institute, Shiga 520-0022, Japan h National Institute for Agro-Environmental Sciences, Tsukuba 305-8604, Japan i Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo 113-8657, Japan j Research Institute for Human and Nature, Kyoto 603-8047, Japan k Kitami Institute of Technology, Hokkaido 090-8507, Japan l National Institute for Environmental Studies, Ibaraki 305-8506, Japan m Suntory, Ltd., Osaka 618-8503, Japan n Global Environment Laboratory, Yonsei University, Seoul 120-749, South Korea Received 21 September 2008; accepted in revised form 13 March 2009; available online 2 April 2009

Abstract The biological and physical controls on microbial processes that produce and consume N2O in soils are highly complex. Isotopomer ratios of N2O, with abundance of 14N15N16O, 15N14N16O, and 14N14N18O relative to 14N14N16O, are promising for elucidation of N2O biogeochemistry in an intact ecosystem. Site preference, the nitrogen isotope ratio of the central nitrogen atom minus that of the terminal nitrogen atom, is useful to distinguish between N2O via hydroxylamine oxidation and N2O via nitrite reduction. We applied this isotopomer analysis to a groundwater system in a temperate coniferous-forested ecosystem. Results of a previous study at this location showed that the N2O concentration in groundwater varied greatly according to groundwater chemistry, i.e. NO3, DOC, and DO, although apportionment of N2O production to nitrification or denitrification was ambiguous. Our isotopic analysis (d15N and d18O) of NO3 and N2O implies that denitrification is the dominant production process of N2O, but definitive information is not derived from d15N and d18O analysis because of large variations in isotopic fractionations during production and consumption of N2O. However, the N2O site preference and the difference in d15N between NO3 and N2O indicate that nitrification contributes to total N2O production and that most measured N2O has been subjected to further N2O reduction to N2. The implications of N2O biogeochemistry derived from isotope and isotopomer data differ entirely from those derived from conventional concentration data of DO, NO3, and N2O. That difference underscores the need to reconsider our understanding of the N cycle in the oxic–anoxic interface. Ó 2009 Elsevier Ltd. All rights reserved. *

Corresponding author. Fax: +81 42 367 5900. E-mail address: [email protected] (K. Koba).

0016-7037/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.gca.2009.03.022

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1. INTRODUCTION Nitrous oxide (N2O) is an atmospheric trace gas that contributes to global warming and stratospheric ozone depletion. A major source of N2O in terrestrial ecosystems is microbial activity: nitrification and denitrification. Despite extensive studies of such microbial activity related to N2O, estimation of N2O emissions from terrestrial ecosystems, especially from soils, includes large uncertainties because of the high spatial and temporal variation of microbial activities in soils, which results in poorly constrained global N2O budgets (Forster et al., 2007). Autotrophic nitrifiers carry out nitrification in soils in oxic environments with ammonia as the substrate; N2O is produced as a by-product of this process. Regarding denitrification, N2O is produced as an intermediate product and is further reduced to dinitrogen gas (N2O reduction). This process is performed by heterotrophic denitrifiers under anoxic conditions with nitrate (NO3) as substrate (hereinafter, we use the term ‘‘denitrification” as NO3 and nitrite (NO2) reduction to N2O). Evaluating the balance of nitrification, denitrification, and N2O reduction in soils remains difficult despite remarkable differences in oxygen and carbon demands between nitrification and denitrification. High spatiotemporal heterogeneity of the soil environments in terms of porosity, moisture contents, and water flow paths prevents a complete understanding of N2O dynamics in soil. This lack of knowledge related to N2O makes it difficult to model N2O emissions from terrestrial ecosystems. Herein, we address the biogeochemistry of N2O in groundwater in a small coniferous forest ecosystem. A prior study revealed high spatial variation of N2O concentrations in this small watershed, but the relative importance of microbial production pathways to N2O production was ambiguous based on correlation analysis with concentration data of dissolved oxygen (DO), dissolved organic carbon (DOC), NO3, and N2O (Osaka et al., 2006). For separation of nitrification, denitrification, and N2O reduction, inhibitors such as acetylene (Davidson et al., 1993) and isotopically enriched substrates (Panek et al., 2000; Wrage et al., 2005; Menyailo and Hungate, 2006a) have been used intensively (see the review of Groffman et al. (2006) for details). Although such applications of inhibitors and isotope tracers can provide quantitative information related to production and consumption processes, these approaches also present important limitations such as unintended effects on microbial activities caused by inhibitors, and so-called priming effects resulting from addition of isotopically enriched substrates (Kuzyakov et al., 2000). As another promising approach, changes in the natural abundances of N and O isotopes (expressed as d15N and d18O, respectively) have been used to characterize N2O in intact ecosystems (Yoshida et al., 1989; Kim and Craig, 1993; Dore et al., 1998; Naqvi et al., 1998). Although isotopic signatures of inorganic N species tend to be variable because of the high turnover of N in terrestrial ecosystems, variations in their d15N and d18O can, in fact, provide important information about ecosystem functions (Ostrom et al., 2002) without disturbing the environment through

addition of inhibitors and tracer-labeled compounds. Large isotope fractionation during nitrification (Mariotti et al., 1981) can induce production of 15N-depleted N2O (Yoshida, 1988); this unique signature has been used as evidence of N2O produced by nitrification. Ueda et al. (1991) found 15N-depleted N2O dissolved in groundwater, which suggests that N2O in the groundwater was produced through nitrification. More recently, Priscu et al. (2008) observed extremely 15N-depleted N2O (80&) in a perennially ice-covered Antarctic lake, where nitrification is responsible for N2O production (Priscu, 1997). For denitrification in the soil, Perez et al. (2000) compared d15N of N2O to d15N of substrates (NO3 and total soil N), reporting that high d15N of N2O compared with that of substrates was attributed to N2O reduction. Furthermore, the degree of N2O reduction regulated the emission rates and isotopic signature of N2O emitted from agricultural fields (Perez et al., 2000). Although the use of d15N and d18O of N2O is advantageous because it is non-invasive, interpretation of its results is limited and source attributions using this approach are inherently inconclusive, often only giving an indication of whether a process is occurring (Baggs, 2008). The lack of quantification for kinetic isotope effects during N2O production and consumption is, in part, responsible for the limited use of d15N and d18O of N2O. Recently, analyses of intramolecular distribution of 15N in N2O have been used to characterize N2O (Brenninkmeijer and Ro¨ckmann, 1999; Toyoda and Yoshida, 1999; Stein and Yung, 2003). Sutka et al. (2006) demonstrated that quantification of the relative abundances of 15N in the central (a) and terminal (b) N atoms of the N2O molecule can be a promising tool for separating nitrification from denitrification. In the present study, we specifically address the biogeochemistry of N2O in groundwater in a small forested ecosystem using d15N and d18O of N2O and NO3, in addition to the intramolecular distribution of 15N in N2O (N2O isotopomer analysis). Our testing hypotheses are that denitrification is the main production process in the groundwater in temperate, forested headwaters, and that dissolved N2O is strongly reduced to N2, causing very high variability in N2O concentrations in groundwater (Osaka et al., 2006). 2. MATERIALS AND METHODS 2.1. Study site This study was conducted in an unchanneled headwater catchment (Matsuzawa catchment, 0.68 ha) at the Kiryu Experimental Watershed (KEW, 5.99 ha) located at 34°580 N, 136°000 E in central Japan (Katsuyama et al., 2001). The average annual precipitation and air temperature during 1999–2003 were, respectively, 1607 mm and 13.5 °C. Fig. 1a shows a topographical map of the Matsuzawa catchment. The locations and depths of observation wells are shown, respectively, in Table 1 and Fig. 1a. Katsuyama et al. (2008) reported denitrification activities of surface soils collected from the area near SG1 (254–505 nmol N/g

Biogeochemistry of N2O in groundwater elucidated by N2O isotopomer measurements

3117

(b) (a) TGB rface

Soil su

ck

Bedro

SG34-585

(c)

Soil

ce

surfa

SG1-75 SG1-130 SG2-177 SG1-250

Springwater 75 cm 130 cm 177 cm 250 cm

Bedrock

Fig. 1. Location of sampling points in KEW and a schematic depiction of the well location. (a) Topographical map of the Matsuzawa catchment. The contour interval is 1 m; the shaded area represents the groundwater body. Two dotted arrows indicate the directions of the cross-sections for (b) and (c). (b) Cross-section for TGB. The solid line with $ shows the normal groundwater level. The TGB is located at the edge of groundwater body. (c) Cross-section for SG34-585, SG1 (SG1-75, -130 and -250), SG2-177 and spring water. The solid line with $ shows the normal groundwater level. SG1-75, -130 and -250 are installed in the same area (within 1 m  1 m).

Table 1 Details of observation wells. Well name

Type

Well depth (m)

Soil depth (m)

MRTa (month)

DOb

NO3b

DOCb

TGB SG1-75 SG34-585 SG1-130 SG2-177 SG1-250

Transitional Saturated, shallow Saturated, deep Saturated, deep Saturated, deep Saturated, deep

0.82 0.75 5.85 1.30 1.77 2.50

0.32 4.70 – 4.70 4.46 4.70

2.6 6.8 – – – –

0.51* n.s. n.s. 0.65** n.s. 0.32*

n.s. 0.27* 0.41* n.s. n.s. n.s.

n.s. n.s. 0.30* n.s. n.s. n.s.

Spring water

–

–

–

7.7

n.s.

n.s.

n.s.

a b

Mean residence time estimated by Kabeya et al. (2007). Regression coefficient (R2) against dissolved N2O concentrations; *P<0.05; **P<0.005; n.s., not significant.

dry soil/h) and the groundwater collected from SG1-250 (from 1.4  104 to 1.7  103 nmol N2/ml/h). In addition, Koba et al. (1997) examined the concentration and d15N of NO3 in soil solutions collected in the SG1 area and found the occurrence of denitrification with response to the precipitation. Consequently, denitrification is considered the main process for N2O production in this small watershed. We collected groundwater samples during 2002–2005 (almost monthly during October 2003–April 2005, 24 times in total, 121 samples). Five wells (SG1-75, -130, -250 in the SG1 area, SG2-177 at the SG2 area, and SG34-585 at the SG34 area in Fig. 1a) are located in the saturated zone (SZ), and TGB is located in the transitional zone (TZ). Groundwater collected from SG1-75 is shallow groundwater in SZ, whereas other wells (SG34-585, SG1-130, SG2-

177, and SG1-250) are deep groundwater in SZ (Table 1 and Fig. 1b and c). We collected groundwater from these six wells and spring water (Table 1) for each sampling date, although the number of collected samples varied according to the occurrence of groundwater (Katsuyama et al., 2001). Analyses of groundwater of different types might provide a wide spectrum of water chemistries, enabling us to interpret the biogeochemical processes occurring in the whole KEW groundwater body. Groundwater samples were collected from the bottoms of the wells using a silicone tube attached to a plastic syringe. After rinsing the tubing and syringe, groundwater was collected slowly into the syringe with special care to remain the water bubble-free. Samples for dissolved N2O analysis were stored with 200 ml glass vials (Kimble/Kontes, 61000G-200) preserved by addition of

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2 ml of saturated HgCl2, and sealed using a butyl rubber stopper and an aluminum seal. 2.2. Chemical analysis Details of chemical analysis (DO using the Winkler method; DOC using a Shimadzu TOC-5000; and nutrient concentrations by HPLC) of water samples are described in Osaka et al. (2006). The N2O analyses were performed using an on-line analytical system comprising a gas extraction chamber (Koshin Rika, Tokyo), a stainless-steel gas transfer line, pre-concentration traps, chemical traps for removal of H2O and CO2, and a gas chromatograph (GC)/ isotope-ratio monitoring mass spectrometer (MAT 252; Thermo Fisher Scientific, Toyoda et al. (2008)). The notation of the isotopomer ratios is the following: d15 Ni ¼ ð15 Risample =15 Rstd  1Þ  1000 ð&Þ

ði ¼ a; b; or bulkÞ

The d15N and d18O of NO3 in the selected number of water samples (filtered by 0.45 lm syringe filter) were measured after Sigman et al. (2001) and Casciotti et al. (2002). Isotopic data of NO3 were calibrated using USGS32, USGS34, USGS35, and IAEA-NO3 (Bo¨hlke et al., 2003). The d15N and d18O of NO3 were reported, respectively, relative to atmospheric N2 and V-SMOW. The precision (obtained by repeated analysis of calibrated in-house AgNO3) was typically better than 0.2& for d15N and 0.5& for d18O. 2.3. Statistical analysis Statistical analyses were conducted using a statistical software package (R; R Development Core Team, 2008) with one-way analysis of variance (ANOVA, Tukey posthoc test) and Pearson’s product-moment correlation: P-values <0.05 were inferred as statistically significant.

d18 O ¼ ð18 Rsample =18 Rstd  1Þ  1000 ð&Þ

2.4. Interpretation of isotopic signature of N2O dynamics

Therein, 15Ra and 15Rb, respectively, represent the 15N/14N ratios of a and b N atoms; 15Rbulk and 18R, respectively, denote average isotope ratios for 15N/14N and 18O/16O. Subscripts ‘‘sample” and ‘‘std”, respectively, signify isotope ratios for the sample and the standard, atmospheric N2 for N and Vienna Standard Mean Ocean Water (VSMOW) for O. We also define the 15N site preference (hereinafter, SP) as an illustrative parameter of the intramolecular distribution of 15N (Toyoda and Yoshida, 1999):

Although the 15N and 18O natural abundance method can provide valuable information related to processes occurring in intact ecosystems, several precautions must be taken for better application of this method. First, isotopic signatures of substrates (NH4+ and NO3) can vary greatly in soils (Koba et al., 1998). Consequently, measurement of the isotopic signature of substrates such as NH4+ and NO3 is necessary to constrain the N2O biogeochemistry (Perez et al., 2000; Po¨rtl et al., 2007). Secondly, the isotopic fractionation factors we use to interpret N2O biogeochemistry have considerably wide ranges (e.g. Bryan et al., 1983; Casciotti et al., 2003). Therefore, to draw conservative conclusions about N2O biogeochemistry, we consider such large ranges of isotopic fractionations in each process together with isotopic signature of the substrates. Isotopic fractionation is often described using a difference in the isotope values between the product and substrate (i.e. net isotope effect) because most biological reactions include multiple fractionating steps (JinuntuyaNortman et al., 2008). This factor is useful as an alternative to the kinetic isotope effect (referred as e; Appendix A when the reactant concentration is large and when e is small relative to 1000 (Mariotti et al., 1981; Toyoda et al., 2005). We applied this net isotope effect to apportion N2O production (Fig. 2a). In addition, Sutka et al. (2006) clarified that the SP is large (33&) for N2O produced by hydroxylamine oxidation (considered as nitrification in this study), although SP is small (0&) for N2O produced by NO2 reduction (denitrification by heterotrophic denitrifier and nitrifier-denitrification by autotrophic ammonia-oxidizing bacteria). This SP is likely to be constant with different substrate concentrations (Toyoda et al., 2005; Sutka et al., 2006) and with different microbes of different functional groups (e.g. ammonia-oxidizing bacteria and methane-oxidizing bacteria for hydroxylamine oxidation; Sutka et al., 2004, 2006). We apply 33& of SP for nitrification and 0& of SP for denitrification (Fig. 2a).

15

N  site preference ðSPÞ ¼ d15 Na  d15 Nb :

Site-specific N isotope analysis in N2O was conducted using ion detectors modified for mass analysis of the N2O fragment ions (NO+), which contained N atoms in the a position of the N2O molecules, whereas bulk (average) N and O isotope ratios were determined from molecular ions (N2O+) (Toyoda and Yoshida, 1999). An aliquot of water sample (1–100 ml) containing 1–5 nmol of N2O was measured gravimetrically and introduced to the gas extraction chamber with carrier gas (helium). The dissolved N2O was purged from the water sample using helium gas for 20 min with a 70 ml/min flow rate. The stripped N2O was then passed through water/CO2 traps, cryofocused by liquid N, then introduced to GC. Trace amounts of CO2 were separated from N2O by GC; then the purified N2O was introduced into a mass spectrometer. The measurement precision was typically better than 0.2& for d15Nbulk and better than 0.5& for d18O, d15Na and d15Nb. The calibrated reference N2O gas (8.82 ppm) introduced into the sample loop (5.09 ml) was first measured manometrically for its amount. The reference gas in the sample loop was introduced into the degassed de-ionized water (about 100 ml) in the gas extraction chamber. Then the dissolved reference N2O gas was measured exactly as the sample had been. The peak area of the reference gas was used to obtain the conversion factor between the peak area (mass 44 or 30) and the amount of N2O (nmol). Then we used this conversion factor to obtain the concentration of dissolved N2O for each sample.

Biogeochemistry of N2O in groundwater elucidated by N2O isotopomer measurements

a

Site Preference/‰

Sutka et al. (2006)

33

Yoshida (1988)

Nitrification

Toyoda et al. (2006)

0

Denitrification

Brandes and Devol (1997)

0

39 47

3

b

2

Nitrification + Denitrification + N2O reduction

60 50

68

Δδ15NNO - – N O /‰

Denitrification + N2O reduction

Site Preference/‰

40

20

Nitrification

É

30 É

10

TGB SG1-75

0

SG34-585

Denitrification

SG1-130 SG2-177 SG1-250

-10

Springwater

-20 -30 -20 -10

0

10

40 50

20 30

60

70

Δδ15NNO - – N O /‰ 3

2

Fig. 2. Proposed isotopomer maps to interpret the biogeochemistry of N2O. (a) Schematic Dd15N–SP map. The open squares with ‘‘denitrification” and ‘‘nitrification”, respectively, denote the range of the data for N2O produced by denitrification and nitrification. Dd15N signifies the differences between substrate (NO3 and NH4+) and product (N2O) in d15N values. The shaded area represents mixing of N2O produced by nitrification and denitrification. The N2O reduction increases SP and decreases Dd15N. The slopes (SP/ Dd15N) of vector of the N2O reduction (open arrow) can be assigned as 1/1.2 from Table 2. (b) Dd15N–SP map for KEW groundwater. Most data are shown outside the shaded area, indicating the N2O reduction in KEW groundwater. The dotted arrows are the vectors of N2O reduction, and the data in the dotted circle are not explainable by denitrification plus N2O reduction, which suggests the contribution of nitrification for these data.

The net N isotope effect (Dd15N) during denitrification varies greatly. As large values of Dd15N, which are defined as Dd15N = d15N of NO3 (or NO2) minus d15N of N2O, Sutka et al. (2004) reported 35.1& for NO2 reduction by Nitrosomonas europea. Sutka et al. (2006) also reported large Dd15N (36.7&) during NO2 reduction by Pseudomonas aureofaciens. In addition, Toyoda et al. (2005) found a large Dd15N value (39&) for P. fluorescens. Therefore, we apply 39& as the largest value for Dd15N of N2O by denitrification (Fig. 2a). For the smallest value of Dd15N, we

3119

should apply 0& for safety because a small to zero isotopic fractionation can be performed under substrate-limited conditions such as denitrification in sediments (Bryan et al., 1983; Brandes and Devol, 1997). For N2O production during nitrification, Dd15N, i.e. d15N of NH4+ minus d15N of N2O, has much larger values than denitrification because N2O is a by-product of nitrification. Yoshida (1988) reported as large a value as 68& with N. europea; this large Dd15N value is partly confirmed by Sutka et al. (2006), who reported 46.9& with N. europea. Therefore, we apply these two values in Fig. 2a. In this groundwater where the complete conversion of NH4+ to NO3 is highly likely based on the low NH4+ concentration (see Section 3.5), we can assume that d15N of NH4+ and NO3 are equal, which allows us to put the whole data into a single SP–Dd15N scheme (Fig. 2a). For N2O reduction, recent studies have revealed constant co-variations for d15N, d18O, and SP (Table 2). These co-variations are useful to interpret N2O biogeochemistry (Fig. 2a). We assume that, as groundwater becomes anoxic, NO3 starts to be reduced to N2O and that this N2O produced via denitrification mixes with N2O already produced by nitrification in the groundwater. This mixed N2O can have isotopic signatures falling in the shaded area shown in Fig. 2a; this N2O can also be reduced when the groundwater becomes more anoxic. In N2O reduction, SP is expected to increase along with the decrease in Dd15N with the constant co-variations (Fig. 2a) presented in Table 2. The diagrams are modified from Yamagishi et al. (2007). In their study, a similar diagram presented their data of oceanic N2O to elucidate the relative contributions of nitrification, denitrification, and N2O reduction. The diagram of Dd18O and SP is also possible (Appendix A), although a lack of information related to the O isotopic fractionation during N2O produced by denitrification and nitrification prevents us from the effective use of this diagram. 3. RESULTS AND DISCUSSION 3.1. Spatial variations in DO, NO3, DOC, and N2O concentrations Water chemistries of groundwater and spring water are presented in Table 3. Large variations in N2O concentrations were found (4.7–1585.4 nM). TGB had the highest N2O concentrations (Table 3) and occasionally showed high N2O concentrations of greater than 1000 nM (19.4– 1585.4 lM). The NO3 concentration was 2.8–73.2 lM; SG34-585 had the lowest (2.8–26.1 lM) and SG2-177 had the highest NO3 concentrations (34.7–53.9 lM). Neither NH4+ nor NO2 was detected, suggesting complete consumption of NH4+ by nitrification. The DOC concentration was 2.4–604.4 lM; TGB showed the highest DOC concentrations (147.3–604.4 lM). The DO concentration was 3.2–420.3 lM; particularly, SG34-585 had the lowest (3.2– 118.8 lM) and spring water had the highest (159.6– 420.3 lM) DO concentrations. The high variation in concentrations of TGB (Table 3) is typical for groundwater in TZ (Katsuyama et al., 2001).

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Table 2 Covariation in d15N and d18O during N2O reduction. Ratio of isotopic fractionation factors 18

15

bulk

18

15

e O/e N

e O/eSP

e N

2.7 2.3 2.6 2.7 2.4 2.4 2.5 2.0 2.0

2.2 2.2 1.9 4.9 – – – – –

1.3 1.0 0.7 1.9 – – – – –

Average 2.4

2.8

1.2

Condition/sample

Reference

Pseudomonas stutzeri Pseudomonas denitrificans ETNP (calculated) Soil incubation Soil incubation Soil incubation Soil incubation Soil incubation Soil incubation

Ostrom et al. (2007) Ostrom et al. (2007) Yamagishi et al. (2007) Jinuntuya-Nortman et al. (2008) Ostrom et al. (2007) Vieten et al. (2007) Menyailo and Hungate (2006b) Mandernack et al. (2000) Webster and Hopkins (1996)

bulk

/eSP

Significant (negative) correlations were found between DO and N2O for TGB, SG1-130, and SG1-250, indicating that denitrification predominates in these groundwaters (Table 1). Positive correlations between NO3 and N2O were observed for SG1-75 and SG34-585, which suggests nitrification in these two groundwaters (Table 1). Moreover, SG34-585 groundwater shows a positive correlation between DOC and N2O (Table 1), implying the occurrence of denitrification and that denitrification is somewhat carbon-limited in this groundwater. Consequently, both nitrification and denitrification can be responsible for the N2O production in SG34-585 by correlations of measured concentrations (Table 1). No significant correlations between N2O versus DO, DOC, and NO3 were observed for SG2-177 and spring water (Table 1). 3.2. Spatial variations in NO3 isotope ratios The range of d15N of NO3 was from 2.1 to +6.6&; the d18O range of NO3 was 7.1 to +11.9& (Table 3). SG1-250 had the highest average value of d15N (ranging 2.8–5.8&) and SG2-177 had the highest d18O value (ranging 1.5–7.7&). The d15N of NO3 correlated negatively with NO3 concentration (R2 = 0.67, P < 0.05, n = 8, Fig. 3a) and with the logarithm of NO3 (R2 = 0.66, P < 0.05, n = 8, Fig 3b) for SG1-75, indicating the occurrence of denitrification. No significant correlation was found for other sampling points. The O isotope effect associated with denitrification covaries with the N isotope effect, such that the d18O and d15N of NO3 change concomitantly with a consistent ratio (e18O/e15N) of about 1 (Granger et al., 2006, 2008) or 0.5 (Lehmann et al., 2003; Granger et al., 2008). In KEW groundwater, however, no such concomitant increase in d15N and d18O was found, except for SG2-177 (R2 = 0.57, P < 0.05, n = 8, d18O = 5.9  d15N  6.3). Thus, the occurrence of denitrification in SG1-75 was not supported by d15N–d18O relationship. Even in the case of SG2-177, there was no significant correlation between d15N and the NO3 concentration (Fig. 3a). In addition, the slope for the regression line (5.9) for SG2-177 differed from the expected 0.5–1.0 denitrification slope. Thus, the occurrence of denitrification in SG2-177 was not fully supported, either.

Moreover, no significant correlation between d18O and NO3 concentration was observed for any sampling points. Thus, the lack of significant relationships among d15N, d18O and concentration of NO3 that support the occurrence of denitrification implied either that denitrification was not important or mixing of nitrification-dominated water and denitrification-dominated water was effective to mask the unique isotopic signature of denitrification in this groundwater. 3.3. Isotope data of N2O In spite of these narrow ranges of d15N of NO3, N2O exhibited wide ranges in d15Nbulk (24.4 to +5.6&) and d18O (28.9–53.6&). The d15Nbulk was lower than the atmospheric value (6.5 ± 0.5&; Yoshida and Toyoda, 2000). SG1-250 had the highest d15Nbulk values (ranging 10.2 to +5.3&). The range of d18O of N2O overlapped the atmospheric value (43.7 ± 0.9&; Yoshida and Toyoda, 2000). The SG34-585 sample had the lowest d18O of N2O (ranging 28.9–45.5&), whereas SG2-177 showed the highest values among the samples (ranging 45.0–53.6&). The N2O concentrations decreased concomitantly with increased d15Nbulk (Fig. 4) and there were significant correlations for TGB (R2 = 0.39, P < 0.05, n = 11), SG1-75 (R2 = 0.66, P < 0.0005, n = 15), SG1-250 (R2 = 0.38, P < 0.005, n = 24), spring water (R2 = 0.60, P < 0.0001, n = 20). Assuming N2O reduction, the apparent isotopic fractionation factor can be estimated using Rayleigh equation (Eq. (2) in Appendix A) as 4.2& for TGB (R2 = 0.78, P < 0.0001, n = 11), 9.5& for SG1-75 (R2 = 0.63, P < 0.0001, n = 15), 4.1& for SG1-250 (R2 = 0.56, P < 0.0001, n = 24), and 5.6& for springwater (R2 = 0.62, P < 0.0001, n = 20). The application of Rayleigh equation for N2O reduction requires an assumption that d15Nbulk of source N2O is constant, which was not possibly supported because both nitrification and denitrification are expected to be N2O sources, each of which can leave different isotopic signatures. Furthermore, d18O of N2O showed no significant correlation with either N2O concentrations or log N2O for any sampling points. Moreover, as in the case of NO3, the O isotope effect associated with N2O reduction is expected to co-vary with the N isotope effect such

Table 3 Groundwater Chemistry in KEW. All

TGB

SG1-75

SG34-585

SG1-130

SG2-177

SG1-250

Spring water

Average s.d. n Stats*

77.6 195.6 121

354.7 592 11 b

60.9 34.8 15 a

74.3 71.5 13 a

38.2 24.8 24 a

44.2 22.3 14 a

40.9 35.8 24 a

54.8 26.6 20 a

NO3 (lM)

Average s.d. n Stats*

34.9 14.9 111

39.0 17.4 11 bc

38.9 11.1 15 bc

10.5 7.2 12 a

38.8 11.3 20 bc

45.1 5.8 13 c

29.3 12.7 21 b

39.8 12.1 19 bc

DOC (lM)

Average s.d. n Stats*

72.2 87.2 108

281.2 143 11 b

59.1 29.5 14 a

50.0 30.5 13 a

40.7 18.6 20 a

30.7 20.9 12 a

50.9 32.8 18 a

56.3 26.5 20 a

DO (lM)

Average s.d. n Stats*

200.1 89.6 106

188.6 99.1 11 bc

248.8 50.1 14 cd

68.7 29.5 13 a

235.6 48.9 18 bcd

165.7 73.8 14 b

183.2 76.8 18 bc

272.1 74.0 18 d

d15N of NO3 (&)

Average s.d. n Stats*

2.0 1.7 58

0.7 3.0 6 a

0.9 1.0 8 a

2.3 2.6 8 ab

2.2 1.0 10 ab

1.8 0.3 8 ab

3.6 0.9 9 b

1.7 0.5 9 ab

d18O of NO3 (&)

Average s.d. n Stats*

0.0 4.1 58

0.9 8.0 6 ab

0.5 2.5 8 ab

0.1 3.0 8 ab

3.6 2.9 10 a

4.4 2.3 8 b

1.7 1.9 9 a

0.4 3.1 9 ab

d15Nbulk of N2O (&)

Average s.d. n Stats*

4.4 6.0 121

8.5 7.7 11 a

7.9 6.9 15 a

5.7 7.9 13 a

0.8 3.7 24 ab

4.1 1.7 14 ab

0.3 3.9 24 b

8.1 3.3 20 a

d18O of N2O (&)

Average s.d. n Stats*

44.2 3.8 121

46.3 3.4 11 bc

43.2 2.6 15 b

38.8 5.4 13 a

43.4 2.1 24 b

48.6 2.2 14 c

45.0 2.3 24 b

44.4 2.3 20 b

Site preference (&)

Average s.d. n Stats*

21.8 8.1 121

23.1 8.7 11 bc

19.0 4.0 15 b

9.8 8.6 13 a

19.6 6.3 24 b

30.0 2.8 14 c

24.6 7.3 24 bc

24.3 4.9 20 bc

*

3121

Statistically significant differences between sampling points are indicated by different letters.

Biogeochemistry of N2O in groundwater elucidated by N2O isotopomer measurements

N2O (nM)

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K. Koba et al. / Geochimica et Cosmochimica Acta 73 (2009) 3115–3133 7 6

δ15N off NO3- /‰ ‰

5

7

δ15N of NO O3- /‰ ‰

6

4 3 2 1 0 -1

5

-2

4

-3

(b) 0

1

2

3

4

5

6

Log NO3-

3 2

TGB SG1-75 SG34-585 SG1-130 SG2-177 SG1-250 Springwater

1 0

-1 -2

(a)

-3 0

10

20

30

40

50

60

70

NO3- (μM) Fig. 3. d15N of NO3. (a) The relation between concentration and d15N of NO3. The negative significant correlation between d15N and concentration of NO3 (R2 = 0.67, P < 0.05, n = 8) for SG1-75 was shown. (b) The relation between the logarithm of NO3 and d15N of NO3. A significant relationship was found for SG1-75 (R2 = 0.66, P < 0.05, n = 8) and the apparent isotopic fractionation factor e (see Appendix A) was calculated as 2.5&.

10 5

TGB SG1 75 SG1-75 SG34-585 SG1-130 SG2-177 SG1-250 Springwater

0

δ15Nbulk/‰ ‰

tion of N2O cannot fully support the simple interpretation that N2O reduction following denitrification controlled the N2O biogeochemistry in this groundwater. As indicated by NO3 isotopes (Section 3.2), the mixing of nitrificationdominated water and denitrification-dominated water would mask the isotopic signature of denitrification in N2O.

-5

-10

3.4. Site preference of N2O

-15 -20 -25

0

100

200

300 400 N2O (nM)

1500

1600

Fig. 4. Concentration and d15Nbulk of N2O. For TGB, SG1-75, SG1-250 and spring water, there were significant correlation between concentration and d15Nbulk of N2O (see text).

that the d18O and d15N of N2O change concomitantly with a consistent ratio (e18O/e15N) of about 2.4 (Table 2), which was not observed in the KEW groundwater system. Among different sampling points, only SG34-585, for which no significant correlation between d15Nbulk and either N2O concentration or log N2O was found, showed significant correlation between d15Nbulk and d18O (R2 = 0.81, P < 0.0001, n = 13), but the ratio of d18O/d15N was 0.61, different from 2.4 (Table 2). Consequently, although N2O reduction was considered to play an important role, the lack of relationships among d15Nbulk, d18O and concentra-

SP of N2O had a wide range (1.9–39.6&); SG34-585 showed the lowest SP values (Table 3). The strong positive relation with all individual data between SP and d18O (Fig. 5; d18O = 0.32  SP + 37.3, R2 = 0.47, P < 0.0001, n = 121) demonstrates that the stability of the Na-O bond is responsible for fractionation of N and O isotopes, as proposed in previous studies (Yoshida and Toyoda, 2000; Popp et al., 2002; Toyoda et al., 2002; Schmidt et al., 2004; Well et al., 2005). The same correlations were found for TGB (slope = 0.33, R2 = 0.71, P < 0.005, n = 11), SG34-585 (slope = 0.37, R2 = 0.34, P < 0.05, n = 13), SG1-130 (slope = 0.20, R2 = 0.38, P < 0.005, n = 24) and springwater (slope = 0.28, R2 = 0.37, P < 0.005, n = 20). Well et al. (2005) reported a similar relation (d18O = 0.77  SP + 10.3) in the groundwater. Although this relation implies that N2O reduction controls SP and d18O and that N2O reduction plays an important role in shaping N2O isotopomer signatures, the slope of the regression line (d18O/SP = 0.2–0.37) differs remarkably from the previously reported value (2.8; Table 2). Denitrification is thought to produce N2O with the constant SP of 0& (Sutka et al., 2004, 2006; Toyoda et al.,

Biogeochemistry of N2O in groundwater elucidated by N2O isotopomer measurements

denitrifier (Casciotti et al., 2002; Toyoda et al., 2005), presumably because of branching isotopic effect during denitrification (O atoms in NO3 should be transferred to the subsequent N oxide pool or lost as H2O: Casciotti et al., 2007). Concentration of NH4+ in KEW groundwater was undetectable. For that reason, it was impossible to measure the d15N of NH4+. However, this near-zero NH4+ concentration enables us to assume that NH4+ has, originally, the same d15N as that of NO3 because of complete conversion of NH4+ to NO3 in KEW groundwater. Although some of NH4+ should be converted into N2O, the amount of this N loss as N2O was negligible because of much larger pool size of NO3 and assumed original NH4+ level (millimole level) than N2O pool size (nanomole level). Assuming that NH4+ has d15N of 2 to +7& (the same range as that of NO3), the N2O produced via nitrification is expected to have d15Nbulk of 40 to 70& (40 = 7 + (47) after Sutka et al. (2006) for the highest value of 47&, and 70 = 2 + (68) after Yoshida (1988) for the lowest value of 68&; Fig. 6). The d18O range of N2O produced by nitrification is defined according to d18O of H2O (8.4& after Kabeya et al., 2007) and DO (23.5& by Kroopnick and Craig, 1972), although DO consumption can increase the d18O of residual DO (Nakayama et al., 2007). For d15N of N2O produced by denitrification, its maximum value is assigned as equal to that of NO3 (6.6&) with no isotopic fractionation during N2O production, although the minimum value is set as 41&, as calculated with the observed minimum d15N of NO3 (2&) and isotopic fractionation factor (39& after Toyoda et al., 2005). For N2O produced by denitrification, the d18O range is assigned from 8& (complete incorporation of oxygen atom from H2O; Ye et al., 1991) to 57& (65& after Toyoda et al., 2005, minus 8& of H2O d18O). The lowest d15Nbulk and d18O of N2O observed in KEW groundwater is 24.4& and 28.9&, respectively, which are both much higher than this nitrification range (area with

55

δ18O of N2O O/‰

50 45 40 35

TGB SG1-75 SG34-585 SG1-130 SG2 177 SG2-177 SG1-250 Springwater

30 25 0

5

10

15 20 25 30 Site Preference/‰

35

40

Fig. 5. Relation between d18O and SP of N2O.

2005). Therefore, the combination of denitrification plus N2O reduction can lead the pseudo-Rayleigh relation between SP and the concentration of N2O. However, no significant correlation between SP and N2O concentrations was observed, which also reflects that SP is not determined simply by denitrification and N2O reduction. Therefore, the lack of significant correlation between SP and N2O concentration cannot support our hypothesis as well as in the case for NO3 (Section 3.2) and N2O isotopes (Section 3.3) that N2O reduction following denitrification is controlling N2O biogeochemistry in the KEW groundwater system. 3.5. A d15N–d18O map of N2O and NO3 in groundwater Fig. 6 depicts a d15N–d18O map of N2O and NO3. It is the first evidence, to our knowledge, that N2O is more enriched in 18O than NO3 in the intact ecosystem, as observed from laboratory experiments using pure-cultured

80

TGB SG1-75

3123

SG34-585 SG1-130

SG2-177 SG1-250

Springwater

70 60

δ18O/‰ ‰

50 40 N2O

30

O2

20

Nitrification

10 NO3-

0

- 10 -20 -70

Denitrification H2O

-60

-50

-40 40

-30 30

-20 20

-10 10

0

10

20

δ15N/‰ Fig. 6. d15N–d18O map of N2O and NO3. The expected d15N and d18O ranges for N2O produced by denitrification and nitrification are depicted, respectively, as areas with solid lines and dotted lines. The N2O reduction is expected to increase both d15N and d18O of residual N2O with the constant ratio of (e18O/e15N) of 2.4 (Table 2), as represented in the map by solid and broken arrows. Details are given in the text.

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K. Koba et al. / Geochimica et Cosmochimica Acta 73 (2009) 3115–3133

dotted line: Fig. 6), suggesting that nitrification cannot be the exclusive source of N2O. The d15Nbulk and d18O fell into the denitrification region in this map (area with solid line: Fig. 6), strongly indicating that N2O in KEW groundwater is produced via denitrification. Although N2O produced via nitrification can become enriched both in 15N and 18O through N2O reduction, the vectors of the N2O reduction in this d15N–d18O map (dotted arrows with the slope of 2.4 in Fig. 6) suggest that the reduction of N2O produced by nitrification cannot explain most of the isotopic signature of N2O. It is also highly likely that N2O reduction follows the occurrence of denitrification because N2O reduction is more sensitive to O2 than NO3 reduction (Betlach and Tiedje, 1981). Thus, N2O reduction without the occurrence of denitrification is unlikely. Although nitrification is unlikely to be the exclusive source of N2O in KEW groundwater, the observed isotopic range of N2O in this map does not contradict the possibility of mixing of N2O produced via nitrification and denitrification, with subsequent N2O reduction, which was implied by isotopic signatures of NO3 and N2O and SP of N2O.

Although the estimation is semi-quantitative because of uncertainty related to isotopic fractionation factors, we can calculate the contribution of nitrification to total N2O production and degree of N2O reduction (see Appendix B for calculations) from Fig. 2b using an isotope fractionation factor during N2O reduction in water-saturated soil (4.5&) reported by Jinuntuya-Nortman et al. (2008). We report the minimum contribution of nitrification to emphasize the importance of nitrification in this denitrifying groundwater system (Table 4). The average minimum contribution of nitrification to total N2O production was 3.2% (ranging 0–23%), and the average reduction of N2O was 94.3% (ranging 34–99%). This high contribution of denitrification together with strong N2O reduction was common for all groundwater (Table 4). Consequently, it is concluded that denitrification is the main process for N2O production, and that the produced N2O is strongly reduced to N2 in KEW groundwater. 4. CONCLUSION

15

3.6. SP and Dd N of N2O The Dd15N–SP map indicates that all individual data can be interpreted as resulting from the mixing of nitrification N2O and denitrification N2O followed by N2O reduction (Fig. 2b). The combination of denitrification plus N2O reduction, which are considered dominant processes in KEW groundwater from the d15N–d18O map (Fig. 6), cannot explain 20 of the 58 data located in the dotted circle above the N2O reduction line running from the denitrification region (Fig. 2b). For these data, at least, the contribution of nitrification must be incorporated. Moreover, the fact that most data fall outside the mixing zone of nitrification and denitrification (shaded area in Fig. 2b) strongly suggests that most N2O measured in this study underwent partial N2O reduction. Consequently, N2O concentration data cannot readily provide conclusive information of N2O production processes, which would explain why the categorization based on the correlation analysis (Table 1) was not supported by NO3 and N2O isotopes and SP of N2O.

Results of this study underscore the usefulness of isotope and isotopomer measurements of N2O and NO3 to investigate N2O biogeochemistry. Interpretations of concentration and conventional isotopic data in this groundwater contradicted each other and it was not straightforward to derive the information on the balance among nitrification, denitrification and N2O reduction. Although further studies of N2O biogeochemistry and accumulation of data of isotopic fractionations are necessary to support a quantitative discussion, our conservative estimate based on SP and Dd15N revealed that the contribution of nitrification to N2O production is considerable, and that N2O is reduced strongly in KEW groundwater. It must be emphasized here that the contribution of nitrification and N2O reduction elucidated by isotopomer data cannot be derived directly from concentrations of N2O, DO, DOC, or NO3, or from conventional isotope data (d15N and d18O of NO3, and N2O), which are often used to interpret N2O dynamics.

Table 4 Balance of N2O production and consumption processes in KEW groundwater. Sample

Contribution to total N2O production (%)

N2O reduction (%)

SE

n

4.1 2.8 0.2 0.3 0.9 2.5 0.8

97.6 98.6 68.5 95.6 99.9 99.3 99.4

1.2 0.3 8.3 2.1 0.0 0.3 0.3

6 8 8 10 8 9 9

0.8

94.3

1.8

58

Nitrification

Denitrification

SE

TGB SG1-75 SG34-585 SG1-130 SG2-177 SG1-250 Spring water

8.8 3.5 0.2 0.3 5.4 4.9 1.2

91.2 96.5 99.8 99.7 94.6 95.1 98.8

All data

3.2

96.8

*SE is the standard error of the mean; n is the number of samples analyzed.

Biogeochemistry of N2O in groundwater elucidated by N2O isotopomer measurements ACKNOWLEDGEMENTS

3125

a Casciotti (2002)

Site Preference/‰

We thank Drs. K. Yamada, Y. Ueno, A. Makabe and other members in Yoshida’s laboratory in TITECH and the Lab. of Forest Hydrology in Kyoto University. We also thank Dr. J.A. Brandes and three anonymous reviewers for valuable comments on this manuscript. This work was partly supported by the Solution Oriented Research for Science and Technology (SORST) project and the Core Research for Evolutional Science and Technology (CREST) from Japan Science and Technology Agency, and the Grants-in-Aid for Creative Scientific Research (Nos. 18780112 and 20780113) and the Program to Create an Independent Research Environment for Young Researchers from the Ministry of Education, Culture, Sports, Science and Technology, Japan.

33

Nitrification

Ye et al. (1991)

Toyoda et al. (2005)

0

Denitrification

H2O, NO3O2 ?

-65

-32

0

18O (substrate – N2O) /‰

APPENDIX A. INTERPRETATION OF THE ISOTOPIC SIGNATURE OF N2O DYNAMICS

b

60 50

Nitrification + Denitrification + N2O reduction

Denitrification + N2O reduction

40

Site Preference/‰

Isotopic fractionation is expressed in terms of the kinetic isotope effect, e15N and e18O, where e = (1  (KL/ KH))  1000 (&) and where KL and KH, respectively, signify the reaction rates of light and heavy isotopes. Isotopic fractionation for many microbial reactions has been described using a Rayleigh distillation equation in which the isotopic composition of the residual substrate of a reaction (ds) is related to that of the initial substrate (ds0), e, and the ratio of the observed to initial concentration (C/C0) (Mariotti et al., 1981): ds  ds0 ¼ e  lnðC=C 0 Þ ð1Þ

Nitrification

30 20 10 0

The modified Eq. (1) is often used to calculate e (Mariotti et al., 1988; Koba et al., 1997):

-10

ð2Þ

Eq. (2) is applicable if the fundamental assumptions inherent in the use of the Rayleigh equation, mainly a unidirectional reaction within a closed system, are not severely compromised. The Rayleigh equation has been applied for many groundwater environments (Mariotti et al., 1988; Ostrom et al., 2002). In addition to the kinetic isotope effect, isotopic discrimination is often described using a difference in the isotope values between the product and substrate (i.e. net isotope effect) because most biological reactions include multiple fractionating steps (Jinuntuya-Nortman et al., 2008). This factor, expressed as Dd15N or Dd18O in this text, is useful as an alternative to the kinetic isotope effect when the reactant concentration is large and when e is small relative to 1000 (Mariotti et al., 1981; Toyoda et al., 2005). The N2O concentration is much lower (nanomolar level) than the NO3 concentration (micromolar level) in KEW groundwater. Consequently, it can be assumed that the reactant concentration is large. Furthermore, the e values for the N2O production during nitrification and denitrification are usually much smaller than 1000. Consequently, we can apply this net isotope effect to the apportioned production of N2O to nitrification and denitrification (Fig. 2a and b and Fig. A1a and b). Unfortunately, a lack of information related to oxygen isotopic fractionation during N2O produced by denitrification and nitrification prevents us from good use of this

-20 -70

-60

TGB

SG34-585

SG2-177

SG1-75

SG1-130

SG1-250

-50

-40

-30

Springwater

-20

-10

0

Δδ18ONO - – N O /‰ 3

2

Fig. A1. Dd18O and N2O isotopomer in KEW groundwater: (a) schematic Dd18O–SP map, (b) Dd18O–SP map for KEW groundwater. Details are given in the text in Appendix B.

A

A(XA, YA) Nitrification area

Site Preference/‰

ds ¼ e  lnðCÞ þ Constant

Denitrification

Slope = - 1/1.2 or -1/2.1

33

Nmax A’ A’(XA’, YA’) B(XB, YB)

Amin (XAmin, YAmin)

B

0 Denitrification area

Bmin (XBmin, 0)

Dmax B’min (XB’min, YB’min)

Dmin

Dmax Nmin

Δδ15N

or

Nmax

Δδ18O/‰

Fig. A2. Calculation of minimum contribution of nitrification to total N2O production. Details are described in the text in Appendix B.

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K. Koba et al. / Geochimica et Cosmochimica Acta 73 (2009) 3115–3133

C Concentrati ion C Concentrati ion

N2O or isoto ope ratio

Isotope e ratio

b

60 50 40 30 20 10 0 -10 -20 -30 -40 40 1600 1400 1200 1000 800 600 400 200 0 700 600 500 400 300 200 100 0

TGB

50 40 SG1-75 30 20 10 0 -10 -20 -30 -40 40 140 120 100 80 60 40 20 0 -20 400 350 300 250 200 150 100 50 0

δ15Nbulk/‰ δ15Nα/‰ δ15Nβ/‰ SP/‰ δ18O/‰

12 10 8 6 4 2 0 -2 -4 -6 6 -8

Isottope ratio

N2O

Isotope e ratio

a

N2O ((nM)) δ15N of NO3-/‰ δ18O of NO3-/‰

NO3 (μM) DO (μM) DOC (μM)

δ15Nbulk/‰ δ15Nα/‰ δ15Nβ/‰ SP/‰ δ18O/‰

N2O ((nM)) δ15N of NO3-/‰ δ18O of NO3-/‰

NO3 (μM) DO (μM) DOC (μM)

Fig. A3. Seasonal variations in water chemistry: (a) TGB, (b) SG1-85, (c) SG34-575, (d) SG1-130, (e) SG2-177, (f) SG1-250, and (g) spring water.

scheme (Fig. A1a). We can assume that d18O of NO3 can become as low as that of H2O (Casciotti et al., 2002; Osaka, 2007). If that were so, our Dd18ONO3 –N2 O for denitrification

(d18O of NO3 minus d18O of N2O) would be equal to d18O of H2O minus d18O of N2O (Dd18OH2 O–N2 O ). The maximum Dd18ONO3 –N2 O can be 0& because some denitrifiers can ex-

Biogeochemistry of N2O in groundwater elucidated by N2O isotopomer measurements

N2O or isoto ope ratio

50 40 SG34-585 30 20 10 0 -10 -20 -30 30 230

130

C Concentrati on

Isotope e ratio

c

-20 140 120 100 80 60 40 20 0

Co oncentration n

N2O or isotop pe ratio

Isotope rratio

d

3127

δ15Nbulk/‰ δ15Nα/‰ δ15Nβ/‰ SP/‰ δ18O/‰

180 N2O ((nM)) δ15N of NO3-/‰

80

δ18O of NO3-/‰

30

50 40 30 20 10 0 -10 -20 -30 120 100 80 60 40 20 0 -20 350 300 250 200 150 100 50 0

NO3 (μM) DO (μM) DOC (μM)

δ15Nbulk/‰ δ15Nα/‰ δ15Nβ/‰ SP/‰ δ18O/‰

SG1-130 N2O (nM) δ15N of NO3-/‰ δ18O of NO3-/‰

NO3 (μM) DO (μM) DOC (μM)

Fig. A3 (continued)

change O atoms completely between NO2 and H2O during denitrification (Ye et al., 1991; Casciotti et al., 2002). Toyoda et al. (2005) reported Dd18OH2 O–N2 O of 29.5 to

65.2&. More studies must be done to constrain this parameter. However, we assign the Dd18ONO3 –N2 O range as 0 to 65& (which is equal to the range of Dd18OOH2 O–N2 O

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K. Koba et al. / Geochimica et Cosmochimica Acta 73 (2009) 3115–3133

Co oncentration

N2O or isotop pe ratio

Isotope rratio

e

60 50 40 30 20 10 0 -10 20 -20 -30 100 90 80 70 60 50 40 30 20 10 0 300

SG2-177

δ15Nbulk/‰ δ15Nα/‰ δ15Nβ/‰ SP/‰ δ18O/‰

N2O ((nM)) δ15N of NO3-/‰ δ18O of NO3-/‰

250 200

NO3 (μM)

150

DO (μM)

100

DOC (μM)

50 0

Co oncentration

N2O or isotop pe ratio

Isotope rratio

f

50 40 30 20 10 0 -10 -20 -30 160 140 120 100 80 60 40 20 0 -20 300

δ15Nbulk/‰ δ15Nα/‰ δ15Nβ/‰ SP/‰ δ18O/‰

SG1-250 N2O ((nM)) δ15N of NO3-/‰ δ18O of NO3-/‰

250 200

NO3 (μM)

150

DO (μM)

100

DOC (μM)

50 0

Fig. A3 (continued)

used in this study). In fact, Dd18ONO3 –N2 O during nitrification is expected to have a range defined by d18O of H2O

(8.4& after Kabeya et al., 2007) and that of hydroxylamine theoretically with an assumption that no apparent

Biogeochemistry of N2O in groundwater elucidated by N2O isotopomer measurements

C Concentrat ion

N2O or isoto ope ratio

Isotope e ratio

g

60 50 40 30 20 10 0 -10 -20 -30 30 140 120 100 80 60 40 20 0 -20 450 400 350 300 250 200 150 100 50 0

3129

15Nbulk/‰ 15Nα/‰ 15Nβ/‰

SP/‰ 18O/‰

Spring water

N2O ((nM)) 15N

of NO3-/‰

18O

of NO3-/‰

NO3 (μM) DO (μM) DOC (μM)

Fig. A3 (continued)

isotopic fractionation exists during oxygen incorporation from H2O and DO to N2O. The d18O of hydroxylamine is expected to be equal to that of DO, which is then assumed as 23.5&, as in work by Kroopnick and Craig (1972), although consumption of DO in groundwater increases the d18O of DO. Casciotti (2002) reported 16.7 to 31.7& as Dd18OH2 O–N2 O in an incubation study. Therefore, we apply 32& as a lowest Dd18OH2 O–N2 O for nitrification. For safety, we apply 0& as the maximum Dd18OH2 O–N2 O for nitrification, signifying that N2O and H2O have the same d18O in the case with strong incorporation of oxygen from H2O to N2O. The assignment of this maximum value for d18O might be too conservative because N2O produced by nitrification (hydroxylamine oxidation in this study) is believed to come from hydroxylamine; there might be no chance for O atoms of N2O to exchange with the O atoms of H2O. Such low d18O of N2O has not been observed often in nitrification (cf. 10& of d18O of N2O by Webster and Hopkins, 1996). The assumption that d18O of H2O is the common baseline for both Dd18O for nitrification and Dd18O for denitrification allows us to put the whole data into a single Dd18OH2 O–N2 O –SP diagram. Despite the large uncertainties described above, the Dd18O–SP map exhibits the same trend as Dd15N–SP: of 58 data, only 26 can be interpreted as the result of denitrification plus N2O reduction (Fig. A1b), which also indicates the importance of nitrification as an N2O production process in KEW groundwater.

Appendix B. Calculation of the minimum contribution of nitrification to total N2O production from Fig. 2a Calculation to estimate the maximum and minimum contributions of nitrification to total N2O production relies on the fact that maximum Dd15N for denitrification is less than that for nitrification (Fig. 2a) and on the slope of the N2O reduction vector in this map (1/1.2; Table 2). Consequently, this calculation presented here must be modified when the range of Dd15N and slope are updated for nitrification and denitrification. The N2O isotopomer data are the consequence of mixing of nitrification and denitrification, and of N2O reduction following mixing. It is necessary to calculate SP and Dd15N values of N2O, which are not reduced yet (i.e. all data points are expected to have fallen into the shaded area in the map if N2O reduction did not occur) in the SP–Dd15N map (Fig. A2). If the measured data A originate from A0 , which is not reduced (merely mixed with nitrification N2O and denitrification N2O) and its SP is YA0 , then the relative contribution of nitrification (N2Onit) is calculable simply as N2 Onit ¼ 1  ðð33  Y A Þ=33Þ ¼ Y A =33:

ð3Þ

Although we cannot know the actual YA0 , it is possible to calculate the minimum YA0 . Therefore, we can calculate the minimum contribution of nitrification. The minimum YA0 (YAmin) is calculated as the y-coordinate of the intersection point (Amin) of a line running through data A with a

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K. Koba et al. / Geochimica et Cosmochimica Acta 73 (2009) 3115–3133 8

8

6

6

4

4

2

2

0 8

0 8

6

6

4

4

2

2

0 8

0 8

0 8

6

6

6

6 4

TGB

2 0 8

0

200

400

600

800

1000

1200

1400

1600

6

SG1-75

4 2

SG34-585

4

4

2

2

2

0 8

0 8

0 8

6

6

4

4

2

2

0 8

0 8

0 8

6

6

6

4

4

4

6

SG1-130

4 2

SG2-177

4

2

2

0 8

0 8

0 8

6

6

6

2

SG1-250

4

2

2

0 8

0 8

0 8

6

6

Spring water

4

4

4

2

2

2

0

0

0

0

20

40

60

80

100

120

140

160

180

0

200

10

20

30

40

50

60

70

80

90

TGB

4 2 0 6

3 2

3

SG1-75

2 1 0 4

0 6

SG34-585

2

3 2 1 0 4

0 6

3

SG1-130

2

2 1 0 4

0 6

3

4

SG2-177

2

2 1 0 4

0 6

SG1-250

4 2

3 2 1 0 4

0 6

Spring water

4 2

3 2 1 0

0 -5 -4 -3 -2 -1

0

1

2

3

4

5

6

10 5 0 15 10 5 0 15 10 5 0 15 10 5 0 15 10 5 0

50

100

7

8

δ15N of NO3-/‰

9 10 11

150

200

250

300

350

400

450

500

-15 -13 -11 -9

-7

-5

-3

-1

1

3

5

7

0

50 100 150 200 250 300 350 400 450 500 550 600 650 700

DO (μM)

0 4

4

5 0 15

DOC (μM)

12 10 8 6 4 2 0 12 10 8 6 4 2 0 12 10 8 6 4 2 0 12 10 8 6 4 2 0 12 10 8 6 4 2 0 12 10 8 6 4 2 0 12 10 8 6 4 2 0

1

4

10

0

100

4

6

2

5 0 15

NO3- (μM)

N2O (nM)

4

10

4

2

6

Frequency

4

15

9

11 13 15

δ18O of NO3-/‰

12 10 8 6 4 2 0 12 10 8 6 4 2 0 12 10 8 6 4 2 0 12 10 8 6 4 2 0 12 10 8 6 4 2 0 12 10 8 6 4 2 0 12 10 8 6 4 2 0

-25 -23 -21 -19 -17 -15 -13 -11 -9 -7 -5 -3 -1 1

δ N 15

bulk

3

5

7

9 11

6 4 2 0 6 4 2 0 6 4 2 0 6 4 2 0 6 4 2 0 6 4 2 0 6 4 2 0 25 27 29 31 33 35 37 39 41 43 45 47 49 51 53 55

δ18O of N2O/‰

/‰

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40

Site Preference/‰

Fig. A4. Distribution of the groundwater chemistries of KEW groundwater. The N2O concentration in TGB was much higher than that in other groundwater samples.

Y Amin  Y A ¼ 4:5  lnð½N2 OA =½N2 OA0

ð4Þ

Therein, [N2O]A and [N2O]A0 are, respectively, the N2O concentration of data A and the expected N2O concentration of data A prior to the N2O reduction. Eq. (4) provides the degree of N2O reduction (N2O]A/[N2O]A0). For data B, the [N2O]A/[N2O]A0 is calculable in the same manner as that used for data A. 0  Y B ¼ 4:5  lnð½N2 OA =½N2 OA0

ð5Þ

12 10

TGB SG1-75 SG34-585 SG1-130 SG2-177 SG1-250 Springwater

8 6

-

δ18O of NO O 3 /‰

slope of 1/1.2 and a line connecting Nmax (Nmax, 33) and Dmax (Dmax, 0). For data whose calculated y-coordinate of the intersection point is less than zero (i.e. YB0 min for data B (XB, YB) in Fig. A1), the minimum N2Onit is assigned as zero (i.e. YBmin = 0) (see Figs. A3–A6). We can also calculate the degree of N2O reduction using data that are used for minimum N2Onit. According to results presented by Jinuntuya-Nortman et al. (2008), the fractionation factor of SP during N2O for water-saturated soil is 4.5&. Therefore, we can apply this factor to the Rayleigh equation (Eq. (1) in Appendix A) as to data A.

4 2 0 -2 -4 4 -6 -8 8 -3

-2

-1

0

1

2

3

4

5

6

7

δ15N of NO3-/‰

Fig. A5. d15N and d18O map of NO3. Denitrification is expected to increase both d15N and d18O with a consistent ratio (e18O:e15N) of ca. 1:1. However, no such relation was found. There was a significant relationship between d15N and d18O for SG2-177 (R2 = 0.57, P < 0.05, n = 8, d18O = 5.9  d15N  6.3), although this slope was far from the from the expected denitrification slope.

Biogeochemistry of N2O in groundwater elucidated by N2O isotopomer measurements 55

δ18O of N2O O/‰

50 45 40 35 30

TGB SG1-75 SG34-585 SG1-130 SG2-177 SG1-250 Springwater

25 20 -25

-20

-15

-10 -5 δ15Nbulk/‰

0

5

10

Fig. A6. d15Nbulk and d18O map of N2O. Denitrification is expected to increase both d15Nbulk and d18O with a consistent ratio (e18O:e15N) of about 2.4:1 (Table 2). However, no such relation was found. SG34-585 showed a significant relationship between d15Nbulk and d18O (R2 = 0.81, P < 0.0001, n = 13), although the ratio of d18O/d15N was 0.61, far from 2.4.

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