Hey Alex,

This protocol from the Lotic Intersite N Experiment (https://lter.kbs.msu.edu/docs/linx/linx2/linx2_protocols_15n_experiment.pdf) outlines a method for measuring 15NO3 in freshwater samples.

Here’s the paper they adapted it from: https://sigman.princeton.edu/sites/default/files/pdfs/SigmanMC1997.pdf

Hey Alex;

This is a pretty complex technique, as you see from the previous answer. In a nutshell, the most common approach uses a specialized microbial culture to partially denitrify the nitrate to nitrous oxide (which is volatile) and then measure d15N in the nitrous oxide via IRMS. The value is subsequently corrected for fractionation due to the microbial conversion. The good news is that the USGS Reston Isotope Laboratory (RSIL) will run this analysis for you at a very reasonable cost. I believe the samples only need to be analyzed for nitrate (you need to provide the concentration to them) and filtered through a 0.2 um filter - but double check that. The website is as follows. https://isotopes.usgs.gov/lab/services.html.

Barium Protocol: Ba (NO3)2 for measuring 15N-NO3

Materials and products: - 50 mL Centrifuge Tubes Resistant to Acetone -Centrifuge - stirrer - Freeze - NaI to 1M - BaI2 to 0.1M - Acetone - n-Hexane - Pipette - Heating at 60 ° C (oven) - Tin Capsules for Combustion - Desiccator to dry

Protocol:

- Dry lyophilization of 100 ml of water - introduced into conical tubes resistant to acetone - Addition of 350μL-500μL of NaI - Vortex shaker 2 min - Addition of 1 mL of acetone in each tube + agitation - Addition of 20 mL of acetone and 10 mL of hexane + stirring - Refrigeration overnight at 4 °C - Centrifugation 5 min (RCF = 6240g) or decantation - Pipette the supernatant liquid into a new tube - Addition of 750μL of BaI2 - Refrigeration at 4 ° C of 12h - Centrifugation and decantation of Ba (NO3) 2 - Ba (NO3) 2 oven dried at 60 °C - Put 0.5 mg in tin capsule for EA-IRMS isotopic analysis in 15N-NO3.