RAPID COMMUNICATIONS IN MASS SPECTROMETRY Rapid Commun. Mass Spectrom. 2005; 19: 3298–3306 Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/rcm.2191 A novel dual-isotope labelling method for distinguishing between soil sources of N 2 O N. Wrage 1 * { , J. W. van Groenigen 2 , O. Oenema 2 and E. M. Baggs 1 1 School of Biological Sciences (Plant and Soil Science), University of Aberdeen, Cruickshank Building, St Machar Drive, Aberdeen AB24 3UU, UK 2 Alterra, Wageningen University and Research Centre, P.O. Box 47, 6700 AA Wageningen, The Netherlands Received 14 July 2005; Revised 5 September 2005; Accepted 8 September 2005 We present a novel 18 O- 15 N-enrichment method for the distinction between nitrous oxide (N 2 O) from nitrification, nitrifier denitrification and denitrification based on a method with single- and double- 15 N-labelled ammonium nitrate. We added a new treatment with 18 O-labelled water to quantify N 2 O from nitrifier denitrification. The theory behind this is that ammonia oxidisers use oxygen (O 2 ) from soil air for the oxidation of ammonia (NH 3 ), but use H 2 O for the oxidation of the resulting hydroxylamine (NH 2 OH) to nitrite (NO 2  ). Thus, N 2 O from nitrification would therefore be expected to reflect the 18 O signature of soil O 2 , whereas the 18 O signature of N 2 O from nitrifier denitrification would reflect that of both soil O 2 and H 2 O. It was assumed that (a) there would be no preferential removal of 18 O or 16 O during nitrifier denitrification or denitrification, (b) the 18 O sig- nature of the applied 18 O-labelled water would remain constant over the experimental period, and (c) any O exchange between H 2 18 O and NO 3  would be negligible under the chosen experimental conditions. These assumptions were tested and validated for a silt loam soil at 50% water-filled pore space (WFPS) following application of 400 mg N kg 1 dry soil. We compared the results of our new method with those of a conventional inhibition method using 0.02% v/v acetylene (C 2 H 2 ) and 80% v/v O 2 in helium. Both the 18 O- 15 N-enrichment and inhibitor methods identified nitrifier denitrification to be a major source of N 2 O, accounting for 44 and 40%, respectively, of N 2 O production over 24 h. However, compared to our 18 O- 15 N-method, the inhibitor method over- estimated the contribution from nitrification at the expense of denitrification, probably due to incomplete inhibition of nitrifier denitrification and denitrification by large concentrations of O 2 and a negative effect of C 2 H 2 on denitrification. We consider our new 18 O- 15 N-enrichment method to be more reliable than the use of inhibitors; it enables the distinction between more soil sources of N 2 O than was previously possible and has provided the first direct evidence of the significance of nitrifier denitrification as a source of N 2 O in fertilised arable soil. Copyright # 2005 John Wiley & Sons, Ltd. Emissions of nitrous oxide (N 2 O) are of concern due to the high global warming potential of this gas, its long atmo- spheric lifetime, and its involvement in the destruction of stratospheric ozone. 1 Agricultural soils are the main global source of N 2 O, 2 especially when fertilised. Several different microbial processes produce N 2 O in soil, the most significant of which are thought to be nitrification, nitrifier denitrifica- tion and denitrification. 3 Nitrification and nitrifier denitrification are both carried out by autotrophic nitrifying bacteria. In ammonia oxidation, the first stage of nitrification, ammonia (NH 3 ) is oxidised to nitrite (NO 2  ) and N 2 O can develop as a by-product. As oxygen (O 2 ) is required for this process, it takes place in aerobic microsites of soils. Nitrifier denitrification is a pathway that ammonia oxidisers are thought to turn to under short-term O 2 limitation whereby NO 2  is reduced to molecular nitrogen (N 2 ) via N 2 O. 4 The ability to undertake this process may be a universal trait in the betaproteobacterial ammonia-oxidising bacteria. 5 This reduction is thought to be similar to denitrification, whereby heterotrophic denitrifiers use nitrate (NO 3  ) or NO 2  as an electron acceptor under low O 2 conditions. Although the conditions conducive for nitrification, nitrifier denitrification and denitrification differ, they are thought to take place simultaneously in different microhabitats of the same soil. 6,7 To derive effective management strategies to mitigate N 2 O emissions from soils, the respective contributions of the different microbial processes need to be quantified. To date, any distinctions between N 2 O production from nitrifiers, denitrifiers, and ‘other sources’ encompassing chemodeni- trification, heterotrophic nitrification, dissimilatory nitrate reduction to ammonium (DNRA) or aerobic denitrification, Copyright # 2005 John Wiley & Sons, Ltd. *Correspondence to: N. Wrage, Institute of Agronomy and Plant Breeding, Georg-August-University of Go¨ ttingen, von-Siebold- Str. 8, 37075 Go¨ ttingen, Germany. E-mail: nwrage@gwdg.de { Present address: Institute of Agronomy and Plant Breeding, Georg-August-University of Go¨ ttingen, von-Siebold-Str. 8, 37075 Go¨ ttingen, Germany.