Analysis of 15N-NO3- via anoxic slurries coupled to MIMS analysis: An application to estimate nitrification by burrowing macrofauna

The increasing use of the stable isotope 15N-NO3- for the quantification of ecological processes requires analytical approaches able to distinguish between labelled and unlabeled N forms. We present a method coupling anoxic sediment slurries and membrane inlet mass spectrometry to quantify dissolved 15N-NO3- and 14N-NO3-. The approach is based on the microbial reduction of 14N-NO3- and 15N-NO3- mixed pool, the determination of the produced 29N2 and 30N2, and the calculation of the original 15N-NO3- and 14N-NO3- concentrations. The reduction is carried out in 12 mL exetainers containing 2 mL of sediment and 10 mL of water, under anoxia. To validate this approach, we prepared multiple standard solutions containing 15N-NO3- alone or in combinations with 14N-NO3-, with final concentrations varying from 0.5 to 3000 μM. We recovered nearly 90% of the initial 14N-NO3- or 15N-NO3-, over a wide range of concentrations and isotope ratios in the standards. We applied this method to a 15N-NO3- dilution experiment targeting the measurement of nitrification in sediments with and without the burrower Sparganophilus tamesis. The oligochaete did not stimulate nitrification, likely due to limited ventilation and unfavorable conditions for nitrifiers growth. The proposed method is reliable, fast, and could be applied to multiple ecological studies.