Analytical pitfalls when using inhibitors in specific nitrification assays

Environmental context Specific inhibitors of biological reactions in the nitrogen cycle can be used to determine the origin of reactive nitrogen species; these nitrogen species potentially degrade water quality or influence climate. However, inhibitors can potentially interfere with methods for the analysis of stable isotope ratios and concentrations of ammonium, nitrite and nitrate. The effect of this interference on several commonly used methods was investigated. These findings should help avoid the use of inappropriate analytical methods and improve data quality in studies of the nitrogen cycle. Abstract Characterisation of the reaction steps involved in nitrification can help determine the processes that produce potentially harmful environmental pollutants such as nitrite, nitrate and nitrous oxide (N2O). The use of nitrification inhibitors can uncouple the reactions and therefore assist in their mechanistic and isotopic characterisation. However, nitrification inhibitors can interfere with the methods for determining the concentrations and stable isotope ratios of ammonium, nitrite and nitrate. The interference of allylthiourea, hydrazine or sodium chlorate in colorimetric methods and stable isotope measurements were assessed. Ammonium concentrations were measured with the salicylate method. Nitrite and nitrate were measured with the Griess reaction, with nitrate first being reduced to nitrite with vanadium (III) chloride. For the stable isotope analysis, nitrite was reduced to N2O in a 1 : 1 sodium azide and acetic acid buffer solution; preceded, when necessary, by ammonium oxidation to nitrite by hypobromite or nitrate reduction to nitrite on an activated cadmium column. Sodium chlorate did not interfere with any of the analyses and none of the inhibitors interfered with the stable isotope ratios determination of nitrate. Allylthiourea interfered with ammonium and nitrate quantification. Both allylthiourea and hydrazine also clearly interfered in the determination of the nitrogen stable isotope ratio of ammonium, while only allylthiourea interfered in the determination of nitrogen and oxygen stable isotope ratios of nitrite. Although we suggest methods to overcome some of these interferences, our study demonstrated that the analytical methods used in combination with allylthiourea or hydrazine as nitrification inhibitors should be considered with caution when designing experiments.