A Ti(III) reduction method for one-step conversion of seawater and freshwater nitrate into N2O for stable isotopic analysis of 15N/14N, 18O/16O and 17O/16O

Rationale The nitrogen and oxygen (delta N-15, delta O-18, and delta O-17 values) isotopic compositions of nitrate (NO3-) are crucial tracers of nutrient nitrogen (N) sources and dynamics in aquatic systems. Current methods such as bacterial denitrification or Cd-azide reduction require laborious multi-step conversions or toxic chemicals to reduce NO3- to N2O for N-15 and O-18 isotopic analyses by isotope ratio mass spectrometry (IRMS). Furthermore, the O-17 composition of N2O cannot be directly disentangled using IRMS because O-17 contributes to mass 45 (N-15). Methods We describe a new one-step chemical conversion method that employs Ti(III) chloride to reduce nitrate to N2O gas in septum sample vials. Sample preparation takes only a few minutes followed by a 24-h reaction producing N2O gas (65-75% recovery) which partitions into the headspace. The N2O headspace was measured for N-15, O-18 and O-17 by IRMS or laser spectrometry. Results IRMS and laser spectrometric analyses gave accurate and reproducible N and O isotopic results down to 50 ppb (3.5 mu M) NO3-N, similar in precision to the denitrifier and Cd-azide methods. The uncertainties for dissolved nitrate reference materials (USGS32, USGS34, USGS35, IAEA-NO3) were +/- 0.2 parts per thousand for delta N-15 values and +/- 0.3 parts per thousand for delta O-18 values using IRMS. For laser-based N2O isotope analyses the results were similar, with an delta O-17 uncertainty of +/- 0.9 parts per thousand without any need for N-15 correction. Conclusions Advantages of the Ti(III) reduction method are simplicity, low cost, and no requirement for toxic chemicals or anaerobic bacterial cultures. Minor corrections may be required to account for sample nitrate concentration variance and potential chemical interferences. The Ti(III) method is easily implemented into laboratories currently using N2O headspace sampling apparatus. We expect that the Ti(III) method will promulgate the use of N and O isotopes of nitrate in important studies of nutrient dynamics and pollution in a wide range of aquatic ecosystems.