We report a new method for measurement of the isotopic composition of nitrate (NO3-) at the natural-abundance level in both seawater and freshwater. The method is based on the isotopic analysis of nitrous oxide (N2O) generated from nitrate by denitrifying bacteria that lack N2O-reductase activity. The isotopic composition of both nitrogen and oxygen from nitrate are accessible in this way. In this first of two companion manuscripts, we describe the basic protocol and results for the nitrogen isotopes. The precision of the method is better than 0.2‰ (1 SD) at concentrations of nitrate down to 1 μM, and the nitrogen isotopic differences among various standards and samples are accurately reproduced. For samples with 1 μM nitrate or more, the blank of the method is less than 10% of the signal size, and various approaches may reduce it further.

A Bacterial Method for the Nitrogen Isotopic Analysis of Nitrate in Seawater and Freshwater

Equilibrium and kinetic isotope fractionations during incomplete reactions result in minute differences in the ratio between the two stable N isotopes, 15N and 14N, in various N pools. In ecosystems such variations (usually expressed in per mil [δ15N] deviations from the standard atmospheric N2) depend on isotopic signatures of inputs and outputs, the input–output balance, N transformations and their specific isotope effects, and compartmentation of N within the system. Products along a sequence of reactions, e.g. the N mineralization–N uptake pathway, should, if fractionation factors were equal for the different reactions, become progressively depleted. However, fractionation factors vary. For example, because nitrification discriminates against 15N in the substrate more than does N mineralization, NH4+ can become isotopically heavier than the organic N from which it is derived.Levels of isotopic enrichment depend dynamically on the stoichiometry of reactions, as well as on specific abiotic and biotic conditions. Thus, the δ15N of a specific N pool is not a constant, and δ15N of a N compound added to the system is not a conservative, unchanging tracer. This fact, together with analytical problems of measuring δ15N in small and dynamic pools of N in the soil–plant system, and the complexity of the N cycle itself (for instance the abundance of reversible reactions), limit the possibilities of making inferences based on observations of 15N abundance in one or a few pools of N in a system. Nevertheless, measurements of δ15N might offer the advantage of giving insights into the N cycle without disturbing the system by adding 15N tracer.Such attempts require, however, that the complex factors affecting δ15N in plants be taken into account, viz. (i) the source(s) of N (soil, precipitation, NOx, NH3, N2-fixation), (ii) the depth(s) in soil from which N is taken up, (iii) the form(s) of soil-N used (organic N, NH4+, NO3−), (iv) influences of mycorrhizal symbioses and fractionations during and after N uptake by plants, and (v) interactions between these factors and plant phenology. Because of this complexity, data on δ15N can only be used alone when certain requirements are met, e.g. when a clearly discrete N source in terms of amount and isotopic signature is studied. For example, it is recommended that N in non-N2-fixing species should differ more than 5‰ from N derived by N2-fixation, and that several non-N2-fixing references are used, when data on δ15N are used to estimate N2-fixation in poorly described ecosystems.As well as giving information on N source effects, δ15N can give insights into N cycle rates. For example, high levels of N deposition onto previously N-limited systems leads to increased nitrification, which produces 15N-enriched NH4+ and 15N-depleted NO3−. As many forest plants prefer NH4+ they become enriched in 15N in such circumstances. This change in plant δ15N will subsequently also occur in the soil surface horizon after litter-fall, and might be a useful indicator of N saturation, especially since there is usually an increase in δ15N with depth in soils of N-limited forests.Generally, interpretation of 15N measurements requires additional independent data and modelling, and benefits from a controlled experimental setting. Modelling will be greatly assisted by the development of methods to measure the δ15N of small dynamic pools of N in soils. Direct comparisons with parallel low tracer level 15N studies will be necessary to further develop the interpretation of variations in δ15N in soil–plant systems. Another promising approach is to study ratios of 15N[ratio ]14N together with other pairs of stable isotopes, e.g. 13C[ratio ]12C or 18O[ratio ]16O, in the same ion or molecules. This approach can help to tackle the challenge of distinguishing isotopic source effects from fractionations within the system studied.

Tansley Review No. 95 15N natural abundance in soil-plant systems

We report a novel method for measurement of the oxygen isotopic composition (18O/16O) of nitrate (NO3-) from both seawater and freshwater. The denitrifier method, based on the isotope ratio analysis of nitrous oxide generated from sample nitrate by cultured denitrifying bacteria, has been described elsewhere for its use in nitrogen isotope ratio (15N/14N) analysis of nitrate.1 Here, we address the additional issues associated with 18O/16O analysis of nitrate by this approach, which include (1) the oxygen isotopic difference between the nitrate sample and the N2O analyte due to isotopic fractionation associated with the loss of oxygen atoms from nitrate and (2) the exchange of oxygen atoms with water during the conversion of nitrate to N2O. Experiments with 18O-labeled water indicate that water exchange contributes less than 10%, and frequently less than 3%, of the oxygen atoms in the N2O product for Pseudomonas aureofaciens. In addition, both oxygen isotope fractionation and oxygen atom exchange are consi...

Measurement of the Oxygen Isotopic Composition of Nitrate in Seawater and Freshwater Using the Denitrifier Method

Publisher Summary This chapter focuses on the uses of isotopes to understand water chemistry.I Isotopic compositions generally cannot be interpreted successfully in the absence of other chemical and hydrologic data. The chapter focusses on uses of isotopes in tracing sources and cycling of nitrogen in the water-component of forested catchment, and on dissolved nitrate in shallow waters, nutrient uptake studies in agricultural areas, large-scale tracer experiments, groundwater contamination studies, food-web investigations, and uses of compound-specific stable isotope techniques. Shallow waters moving along a flowpath through a relatively uniform material and reacting with minerals probably do not achieve equilibrium but gradually approach some steady-state composition. The chapter also discusses the use of isotopic techniques to assess impacts of changes in land-management practices and land use on water quality. The analysis of individual molecular components for isotopic composition has much potential as a method for tracing the source, biogeochemistry, and degradation of organic liquids and gases because different materials have characteristic isotope spectrums or biomarkers.

Tracing nitrogen sources and cycling in catchments

Nitrate (NO3 ) concentrations in public water supplies have risen above acceptable levels in many areas of the world, largely as a result of overuse of fertilizers and contamination by human and animal waste. The World Health Organization and the U.S. Environmental Protection Agency have set a limit of 10 mg L nitrate (as N) for drinking water because nitrate poses a health risk, especially for children, who can contract methemoglobinemia (blue-baby syndrome). Nitrate in lower concentrations is non-toxic, but the risks from long-term exposure are unknown, although nitrate is a suspected carcinogen. High concentrations of nitrate in rivers, lakes, and coastal areas can cause eutrophication, often followed by fi sh-kills, due to oxygen depletion. Increased atmospheric loads of anthropogenic nitric and sulfuric acids have caused many sensitive, low-alkalinity streams in North America and Europe to become acidifi ed. Still more streams that are not yet chronically acidic could undergo acidic episodes in response to large rain storms and/or spring snowmelt, seriously damaging sensitive local ecosystems. Future climate changes may exacerbate the situation by affecting biogeochemical controls on the transport of water, nutrients, and other materials from land to freshwater ecosystems. The development of effective management practices to preserve water quality, and remediation plans for sites that are already polluted, requires the identifi cation of actual N sources and an understanding of the processes affecting local nitrate concentrations. In particular, a better understanding of hydrologic fl owpaths and solute sources is required to determine the potential impact of contaminants on water supplies. Determination of the relation between nitrate concentrations in groundwater and surface water and the quantity of nitrate introduced from a particular source is complicated by:

H.-L. Schmidt

Papers

Natürliche Isotopengehalte von Nitrat als Indikatoren für dessen Herkunft