Nitrate (NO₃) in Water Sample Preparation

Nitrate in water samples for bacterial denitrifier preparation [1, 2] by the SIF should be filtered (0.1 to 0.2 micron pore size), stored frozen, and shipped frozen. Do not use methods of preservation aside from filtration and freezing. Poisons, high alkalinity, and high acidity can adversely affect the denitrifying bacteria.

Collect samples in 30-60 mL nitrate-free, wide-mouthed, screw-top containers made of either polyethylene or polycarbonate. Please avoid polypropylene and polystyrene, which are prone to cracking at low temperatures. Thoroughly flush all supplies, including filters, with sample prior to use.

A minimum of 10 mL of water sample is required, with 15-30 mL preferred. Samples requiring extensive handling by the SIF (e.g., filtering) will be charged incidental fees per sample.

As the bacterial denitrifier method does not discriminate between nitrate (NO₃^-) and nitrite (NO₂^-), samples should be treated for nitrite removal [3] prior to submission to the SIF.

Accurate nitrate concentrations are required for all samples. Please specify if your concentrations are NO₃^- vs. NO₃^--N, μM vs. mg/L, etc. If this information is not available, please allow the SIF to refer you to an analytical facility that can determine the concentration for you.

Required concentration range is 4 μM to 7000 μM nitrate. Samples above 7000 μM will be diluted by SIF for an incidental fee. Samples with missing or inaccurate nitrate concentrations will be charged for reruns and take longer to process.

Special Sample Types
For enriched samples / tracer experiments, please contact the SIF prior to your experiment.

The SIF no longer accepts customer-prepared nitrate in water samples. Due to the nature of bacterial denitrification preparation and analysis, we cannot offer appropriate quality assurance unless we prepare the samples (and standards) ourselves.

We do accept KCl soil extracts[4], TDN digests[5], and NH₄⁺ digests[6]. Please contact us for details and special requirements.

For ammonium, most of our clients diffuse from solution onto acidified discs[7]. The resulting disc is then submitted and analyzed as a solid sample.

Supplies

 

Freezing and Shipping Samples
Please use only polyethylene or polycarbonate containers. At low temperatures, both polypropylene and polystyrene become brittle and are prone to cracking. Leave enough headspace to account for expansion during freezing, and pre-chill your samples in a refrigerator for the first 12-24 hours prior to freezing.

Once frozen, carefully pack small sets of samples in ziplock bags or small boxes with dividers. Pack these sets in a larger hard-walled cooler or Styrofoam insulated shipping kit with packing peanuts or bubble wrap. Make sure the samples are well cushioned on all sides, as loose bottles can break during shipping. Do not use foam-only coolers or cardboard-only boxes for the exterior shipping container; such containers will disintegrate due to condensation and hard impacts when tossed by couriers.

Keep the entire shipment frozen until time to ship. Pre-chilling your cooler will extend refrigeration time. We recommend blue ice blocks, cold packs, or ice blankets for packages sent by overnight or 1-day delivery. Dry ice is advisable for delivery times exceeding 2 days. Pack about 15-20 pounds of dry ice for the first 24 hours of transit, plus an additional 5-10 pounds of dry ice for each additional day of transit. Fill any remaining void space in the cooler with newspaper or other filler to slow sublimation and protect against damage.

Ship your samples by the fastest available method to arrive at the SIF midweek (Tuesday through Thursday). If you would like your cooler returned, please include a prepaid shipping label and note the request on your Analysis Order Form.

We recommend saving a duplicate set of samples in case of sample loss, whether due to shipping loss or cracked vials.

Please complete and submit the online Analysis Order Form and Sample List for your samples. Include nitrate concentrations for all samples for bacterial denitrification preparation by the SIF. Samples with missing or inaccurate nitrate concentrations will be charged for reruns and take longer to process. Be sure to include a printed copy of both forms with your samples. The SIF uses these forms to track your samples and to contact you regarding sample receipt, data, and invoicing. For mixed analysis requests, you must complete a separate set of forms for each type of analysis.

Sample Storage and Retention by the SIF
The SIF will store your samples in the freezer. Due to limited freezer space, please ship your samples to arrive only 1 to 3 weeks prior to your appointment. The SIF will send an email reminder approximately 4 to 6 weeks prior to your appointment. After analysis and reruns are complete, samples will be stored by SIF for 2 months and then discarded. Please review your results during this 2-month window.

References
[1] D. M. Sigman, K. L. Casciotti, M. Andreani, C. Barford, M. Galanter, and J. K. Böhlke. 2001. A bacterial method for the nitrogen isotopic analysis of nitrate in seawater and freshwater. Anal. Chem. 73: 4145-4153. DOI: 10.1021/ac010088e

[2] K. L. Casciotti, D. M. Sigman, M. Galanter Hastings, J. K. Böhlke, and A. Hilkert. 2002. Measurement of the oxygen isotopic composition of nitrate in seawater and freshwater using the denitrifier method. Anal. Chem. 74: 4905-4912. DOI: 10.1021/ac020113w

[3] J. Granger and D. M. Sigman. 2009. Removal of nitrite with sulfamic acid for nitrate N and O isotope analysis with the denitrifier method. Rapid Comm. Mass Spectrom. 23: 3753-3762. DOI: 10.1002/rcm.4307

[4] L. Rock and B. H. Ellert. 2007. Nitrogen-15 and oxygen-18 natural abundance of potassium chloride extractable soil nitrate using the denitrifier method. Soil Science Society of America Journal, 71(2), 355-361. DOI: 10.2136/sssaj2006.0266

[5] A. N. Knapp and D. M. Sigman. 2005. N isotopic composition of dissolved organic nitrogen and nitrate at the Bermuda Atlantic time-series study site. Global Biogeochem. Cycles, 19, GB1018. DOI: 10.1029/2004GB002320

[6] J. D. Felix, E. M. Elliott, T. J. Gish, L. L. McConnell, and S. L. Shaw. 2013. Characterizing the isotopic composition of atmospheric ammonia emission sources using passive samplers and a combined oxidation-bacterial denitrifier approach. Rapid Commun. Mass Spectrom. 27, 2239–2246. DOI: 10.1002/rcm.6679

[7] R. M. Holmes, J. W. McClelland, D. M. Sigman, B. Fry, and B. J. Peterson. 1998. Measuring ¹⁵N-NH₄⁺ in marine, estuarine, and fresh waters: An adaptation of the ammonia diffusion method for samples with low ammonium concentrations. Mar. Chem. 60: 235-243.