# Methods Manual for Salt Lake Studies/Nitrogen

Authors: PSJ Coleman,

### Nitrogen overview

Nitrogen species are analysed as indicators of the health of salt lakes, providing information about the likelihood that the water body will support microalgal blooms. Nitrogen is a very labile element and is found in many forms, from simple oxidized inorganic nitrogen to complex proteins.
Considerable confusion can arise about what species of N are being tested and what units are being reported in nitrogen analysis. For example, the common ‘nitrate tests’ available in zinc-reduction field kits actually measure oxidized N in the form of nitrite, after the zinc reduces a known percentage of the nitrate present to nitrite, then the method calculates the quantity of nitrate, or of nitrogen, depending on the kit (Eaton et al, 1995). Unfortunately, any nitrite present produces a high reading, which may be unquantifiable using colour chart type tests in the field.
The choice of analyses depends on what you wish to understand about the salt lake you are studying, and whether you will be conducting the analyses in the field or in a laboratory.
Total oxidised nitrogen (N$_{ox}$) analysis measures the two types of nitrogen (nitrite and nitrate) that are immediately available to plants in brine. Because of their immediate availability to plant life, testing for these species provides a useful indicator of a waterbody's ability to support a bloom. Separate nitrate (NO$_3$) and nitrite (NO$_2$) test methods are also available that are suitable for either field or laboratory use.
Ammonium (NH$_4$) is a common nitrogenous pollutant that is toxic to many species in excess amounts. For fieldwork, it is possible to measure ammonium at the site, as the method does not require digestion.
Total nitrogen (TN) is the most useful routine laboratory measure, as N is very labile and changes rapidly, both within the waterbody and in the removed sample. The full suite of nitrogen testing should be conducted where it is important to understand speciation within the waterbody. Should total nitrogen be required, use an appropriate method and analyse in the laboratory, as a high temperature digestion is required. Total nitrogen measures a combination of Kjeldahl (organic) nitrogen, oxidised nitrogen and ammonia. Subtracting oxidised nitrogen from total nitrogen provides a measure known as TKN (Total Kjeldahl Nitrogen) which is a sum of the organic N and ammonium components.
When you wish to discover whether your salt lake is nitrogen or phosphorus limited, you need to measure an appropriate range of species. It is common to compare Total N to Total P (TN:TP) or to compare dissolved inorganic N to dissolved inorganic P (DIN:DIP). The nitrogen tests required for DIN are the measures of total oxidised N (NO$_3$ and NO$_2$) and ammonium (NH$_4$).

### Sample handling

Nitrogen may be fixed from the air by bacteria within the sample. Always fill sample jar to the top if it will be a while before testing. Keep samples cool and test on the day of collection.

### Notes about oxidised nitrogen analysis

The two most common methods for the analysis of nitrate in saline waters are the cadmium reduction colorimetric method and the zinc powder reduction colorimetric method. Both methods are affected by the salinity of the sample.
Thomas and Chamberlin (1953) propose the use of a zinc powder reductant to avoid salt effects. Despite being adequate in salinities around that of sea-water, the method is impacted, physically, at higher salinities. High salt levels interfere with flocculation of the zinc reductant used in many field test kits, so dilution or filtration may be necessary. Most field test kits rely on gravity to 'floc' the zinc, and in dense brines the fine powder may not settle completely. Any zinc remaining in the sample will prevent the azo dye from developing its full colour causing underestimation of the oxidized nitrogen concentration, so the zinc must be removed completely. Diluting slightly hypersaline samples with distilled water(minimally) is one way to correct this. However dilution reduces the sensitivity of the method, so always use the minimal dilution possible. It is good practice not to exceed a 5 times dilution. Carefully quantify your dilution so that you can correct your results. Remember that the chance of error is lessened if you dilute a large ampunt of sample and then use an aliquot, rather than trying to measure tiny amounts of sample and diluent.
An alternative to dilution is to use a nitrate-free membrane filter (e.g. Supor) attached to a syringe to separate the reduced sample from the zinc powder. Make sure you do not use cellulose nitrate membrane filters!
Ghassemzadeh et al (1997) examined salt effects on the cadmium column reduction colorimetric method. They discovered a significant salt effect on this method. The authors felt that dilution was not always appropriate in highly saline samples, as the concentration of nitrate in the natural brines they were testing were very low, thus becoming undetectable with dilution. The authors suggested that using spiked samples of varying salinities of brine from specific salt lakes would allow researchers to develop a salt effect correction formula that was specific to each lake they were studying. The authors provided a salt effect correction for hypesaline water derived from seawater. Such brines are common in coastal lagoons, salinas and commercial saltworks.
% salt error = -0.0007salinity$^2$ + 0.3322salinity + 1.1095
The r$^2$ of the equation is 0.9638

### Notes about ammonium analysis

Check the selected test method's sensitivity to salt and use an appropriate dilution, based on the salinity of your sample. Overly saline samples may affect the colour development of many ammonium test methods and this may be specified in the reagent manufacturer's notes. If no information on salinity effects has been provided, using a bracketed series of dilutions or a spiked sample will provide you with the information you need to determine what dilutions are appropriate and what colour changes indicate an overly saline sample.