Accumulation of heterocyclic nitrogen in humi®ed organic matter: a 15 N-NMR study of lowland rice soils N.M AHIEU a ,D.C.O LK b &E.W.R ANDALL a a Department of Chemistry, Queen Mary & West®eld College, London E1 4NS, UK, and b International Rice Research Institute, MCPO Box 3127, 1271 Makati City, Philippines Summary Recent intensi®cation of cropping and the attendant longer submergence of the soil for lowland rice in tropical Asia appear to have altered the nature of the soil organic matter, and perhaps also nutrient cycling. To identify the dominant forms of organic nitrogen in the soils we extracted the labile mobile humic acid (MHA) and the more recalcitrant calcium humate (CaHA) fractions from soils under several long-term ®eld experiments in the Philippines and analysed them by 15 N-nuclear magnetic resonance spectroscopy. Amide N dominated the spectra of all humic acid (HA) samples (60±80% of total peak area). Its proportion of total spectral area increased with increasing intensity of cropping and length of time during which the soil was ¯ooded and was greater in the MHA fraction than in the CaHA fraction. Simultaneously the spectral proportion of free amino N and other chemical shift regions decreased slightly with increasing length of submergence. Heterocyclic N was detected at modest proportions (7± 22%) and was more prevalent in more humi®ed samples, especially in the CaHA of aerated soils. Correlations of spectral proportions of heterocyclic N with other properties of the HA, reported elsewhere, were highly signi®cant. Correlations were positive with visible light absorption (r =0.86)and concentration of free radicals (r =0.85), both of which are indices of humi®cation, and negative with concentration of H (r =±0.86), a negative index of humi®cation. Correlations of spectral proportions of amide N with these properties were also highly signi®cant but in each case of opposite sign to that of heterocyclic N. Proportions of heterocyclic N declined with increasing duration of submergence. The resultssuggestthat(i) 15 N-NMRcanreproduciblymeasuresomeportionofheterocyclicN,(ii)formation of heterocyclic N is associated solely with gradual humi®cation occurring over many years, and (iii) the abundant phenols in the submerged rice soils did not promote formation of heterocyclic N, and hence someotherprocessisresponsibleforasubstantialdecreaseintheavailabilityofnativeNassociatedwith intensive rice cropping. Introduction Long-term trends in yields for double- and triple-cropped ®eld experiments in tropical lowland rice indicate a gradual decrease in the availability of native nitrogen in the soil after the intensi®cation of cropping and the associated submergence of the soil. However, total soil N has remained constant, suggesting some change in the organic nature of soil organic matter(SOM)(Cassman etal.,1995).Thisstudycontinuesour earlierinvestigationsintothechemicalnatureofSOMinthese circumstances. Using nuclear magnetic resonance (NMR) spectroscopy, we established the effects of long-term intensive rice cropping on forms of carbon (Olk etal., 1996, 1998) and phosphorus (Mahieu etal.,2000)inthesoil.Inshort,extractedhumicacid (HA) fractions were found to contain less humi®ed materials (e.g. phenolic C and diester P) with increasing intensity of irrigated rice cropping. Other analyses of the HA also indicated their less humi®ed nature with increasing intensity of rice cropping: their C:N ratio increased and absorption of visible light decreased (Olk etal., 1996), their concentrations of H, sulphur, and amide and amino, hydroxyl, and methoxy groups increased, while the wavelengths of ¯uorescence emission maximum and concentrations of free radicals measured by electron spin resonance spectroscopy and of carboxyl groups decreased (Olk etal., 2000). In this study we follow the conventional de®nition of humi®cation as a gradual reworking of the organic matter through oxidative and hydrolytic reactions mediated by soil microorganisms and possibly also through various abiotic reactions such that labile compounds are depleted over time in favour of more recalcitrant compounds (Stevenson, 1994; Zech etal., 1997). Correspondence: N. Mahieu. E-mail: n.mahieu@qmw.ac.uk Received 22 December 1998; revised version accepted 21 March 2000 European Journal of Soil Science, September 2000, 51, 379±389 # 2000 Blackwell Science Ltd 379