Assessing the performance of international laboratories analysing the stable isotope composition (δ15 N, δ18 O, δ17 O) of nitrate in environmental waters.

RATIONALE: Stable-isotope analyses of nitrate (NO3 - ) in various water sources are crucial for understanding nitrogen pollution and its impact on aquatic ecosystems. We evaluated the accuracy and precision of stable-isotope analyses of nitrate conducted by international laboratories. METHODS: Six samples with nitrate (2 mg L-1 NO3 - -N) were sent to 47 laboratories. The NO3 - had a 30-50 ‰ range of δ values for δ15 N, δ18 O and δ17 O. One blind duplicate evaluated reproducibility and the effect of water δ18 O. Laboratories used diverse methods to convert nitrate to N2 O, N2 , CO or O2 for stable-isotopic measurements (microbial, cadmium, titanium and elemental analysis) and isotope-ratio mass spectrometry or laser-based technologies. RESULTS: Thirty-six international laboratories (83 %) reported results; however, 23 % did not analyze the test samples due to technical difficulties. Of the reporting laboratories, 79 % and 84 % produced accurate δ15 N and δ18 O results falling within ±0.8 ‰ and ±1.1 ‰ of the benchmark values, respectively. Three laboratories produced only outliers. The duplicate revealed most laboratories gave internally reproducible results at appropriate analytical precision. For δ17 O, six laboratories reported results, but 67 % could not reproduce results within their claimed analytical measurement precision. One complication is a lack of nitrate reference materials for δ17 O. CONCLUSIONS: Analyst experience contributed to better performance, and underperformance was from compromised standards or inappropriate δ range of working reference materials. The stable isotope community must develop new nitrate reference materials for δ15 N spanning -20 ‰ to +80 ‰ and new materials for δ17 O.

Assessment of rapid low-cost isotope (δ15 N, δ18 O) analyses of nitrate in fruit extracts by Ti(III) reduction to differentiate organic from conventional production.

RATIONALE: The isotopic composition (δ15 N, δ18 O) of nitrate in fruits and vegetables differentiates organic from conventional food production practices. Organic systems do not use synthetic nitrate fertilizers high in 18 O and low in 15 N and thereby help reveal producers' fertilization claims. Isotope analyses of nitrate extracted from fruits and vegetables are done by bacterial reduction which is costly and by specialized laboratories. Rapid, low-cost methods are needed to promulgate nitrate isotope analyses of food products to support organic food product certification and to verify the authenticity of production claims. METHODS: Fresh strawberry samples were obtained from certified organic and conventional growers in Andalucía, Spain. We applied a new, rapid, one-step Ti(III) reduction method to convert the nitrate from strawberry extracts to N2 O gas for headspace isotope analyses using isotope-ratio mass spectrometry. Using the Ti(III) reduction method, 70 samples, controls and references were prepared and analyzed for NO3 - , δ15 N and δ18 O per 48 h. We also analyzed extracts and solids for anions and cations and for bulk δ15 N for multivariate chemometric evaluation. RESULTS: The Ti(III)-based isotope analyses of nitrate in strawberry extracts revealed clear differentiation between organic and conventional production with mean δ18 O and δ15 N values of +18.3 ± 1.2 ‰ and +17.6 ± 1.2 ‰ versus +28.2 ± 4.5 ‰ and +14.9 ± 3.0 ‰, respectively. The δ15 N of strawberry dry mass differed slightly (+3.0 ± 1.4 ‰ versus +4.0 ± 1.4 ‰) between organic and conventional samples, respectively. Chemometric analyses of nitrate isotopes and extract chemistry revealed that the δ18 O of nitrate along with δ15 N and Ca2+ fully differentiated organic from conventional strawberry production. CONCLUSIONS: Our results show the Ti(III) reduction method provides a new low-cost and rapid analytical method to facilitate compound-specific δ15 N and δ18 O isotope analyses of nitrate in selected fruit types, and likely other food products, for the purposes of assessing nitrate fertilization practices of organic versus conventional production claims and to support authenticity investigations.

Progress and challenges in dual- and triple-isotope (δ18 O, δ2 H, Δ17 O) analyses of environmental waters: An international assessment of laboratory performance.

RATIONALE: Stable isotope analyses of environmental waters (δ2 H, δ18 O) are an important assay in hydrology and environmental research with rising interest in δ17 O, which requires ultra-precise assays. We evaluated isotope analyses of six test water samples for 281 laboratory submissions measuring δ2 H and δ18 O along with a subset analyzing δ17 O and Δ17 O by laser spectrometry and isotope ratio mass spectrometry (IRMS). METHODS: Six test waters were distributed to laboratories spanning a wide δ range of natural waters for δ2 H, δ18 O and δ17 O and Δ17 O. One sample was a blind duplicate to test reproducibility and claims of analytical precision. RESULTS: Results showed that ca 83% of the submissions produced acceptable δ18 O and δ2 H results within 0.2‰ (mUr) and 1.6‰ of the benchmark values, respectively. However, 17% of the submissions gave questionable to unacceptable results. A blind duplicate revealed many laboratories reported overly optimistic precision, and many could not replicate within their claimed precision. For Δ17 O, dual-inlet results for IRMS using quantitative O2 conversion were accurate and highly precise, but the results for laser spectrometry ranged by ca 200 per meg (µUr) for each sample, with ca 70% unable to replicate the duplicate to their claimed Δ17 O precision. One complicating factor is the lack of certified primary reference waters for δ17 O. CONCLUSIONS: No single factor or combination of factors was identifiable for poor or good performance, and underperformance came from issues like data normalization including inadequate memory and drift corrections, compromised working reference materials and underperforming instrumentation. We recommend isotope laboratories include high and low δ value controls of known isotope composition in each run. Progress in Δ17 O analyses by laser spectrometry requires extraordinary proof of performance claims and would benefit from the development of adoptable and systematic advanced data processing procedures to correct for memory and drift.

A Ti(III) reduction method for one-step conversion of seawater and freshwater nitrate into N2 O for stable isotopic analysis of 15 N/14 N, 18 O/16 O and 17 O/16 O.

RATIONALE: The nitrogen and oxygen (δ15 N, δ18 O, and δ17 O values) isotopic compositions of nitrate (NO3 - ) are crucial tracers of nutrient nitrogen (N) sources and dynamics in aquatic systems. Current methods such as bacterial denitrification or Cd-azide reduction require laborious multi-step conversions or toxic chemicals to reduce NO3 - to N2 O for 15 N and 18 O isotopic analyses by isotope ratio mass spectrometry (IRMS). Furthermore, the 17 O composition of N2 O cannot be directly disentangled using IRMS because 17 O contributes to mass 45 (15 N). METHODS: We describe a new one-step chemical conversion method that employs Ti(III) chloride to reduce nitrate to N2 O gas in septum sample vials. Sample preparation takes only a few minutes followed by a 24-h reaction producing N2 O gas (65-75% recovery) which partitions into the headspace. The N2 O headspace was measured for 15 N, 18 O and 17 O by IRMS or laser spectrometry. RESULTS: IRMS and laser spectrometric analyses gave accurate and reproducible N and O isotopic results down to 50 ppb (3.5 µM) NO3 -N, similar in precision to the denitrifier and Cd-azide methods. The uncertainties for dissolved nitrate reference materials (USGS32, USGS34, USGS35, IAEA-NO3 ) were ±0.2‰ for δ15 N values and ±0.3‰ for δ18 O values using IRMS. For laser-based N2 O isotope analyses the results were similar, with an δ17 O uncertainty of ±0.9‰ without any need for 15 N correction. CONCLUSIONS: Advantages of the Ti(III) reduction method are simplicity, low cost, and no requirement for toxic chemicals or anaerobic bacterial cultures. Minor corrections may be required to account for sample nitrate concentration variance and potential chemical interferences. The Ti(III) method is easily implemented into laboratories currently using N2 O headspace sampling apparatus. We expect that the Ti(III) method will promulgate the use of N and O isotopes of nitrate in important studies of nutrient dynamics and pollution in a wide range of aquatic ecosystems.

N and O isotope (δ15 Nα , δ15 Nß , δ18 O, δ17 O) analyses of dissolved NO3- and NO2- by the Cd-azide reduction method and N2 O laser spectrometry.

RATIONALE: The nitrogen and oxygen (δ15 N, δ18 O, δ17 O) isotopic compositions of NO3- and NO2- are important tracers of nutrient dynamics in soil, rain, groundwater and oceans. The Cd-azide method was used to convert NO3- or NO2- to N2 O for N and triple-O isotopic analyses by N2 O laser spectrometry. A protocol for laser-based headspace isotope analyses was compared with isotope ratio mass spectrometry. Lasers provide the ability to directly measure 17 O anomalies which can help discern atmospheric N sources. METHODS: δ15 N, δ18 O and δ17 O values were measured on N/O stable isotopic reference materials (IAEA, USGS) by conversion to N2 O using the Cd-azide method and headspace N2 O laser spectrometry. A 15 N tracer test assessed the position-specific routing of N to the α or ß positions in the N2 O molecule. A data processing algorithm was used to correct for isotopic dependencies on N2 O concentration, cavity pressure and water content. RESULTS: NO3- /NO2- nitrogen is routed to the 15 Nα position of N2 O in the azide reaction; hence the δ15 Nα value should be used for N2 O laser spectrometry results. With corrections for cavity pressure, N2 O concentration and water content, the δ15 NαAIR , δ18 OVSMOW and δ17 OVSMOW values (‰) of international reference materials were +4.8 ± 0.1, +25.9 ± 0.3, +12.7 ± 0.2 (IAEA NO3 ), -1.7 ± 0.1, -26.8 ± 0.8, -14.4 ± 1.1 (USGS34) and +2.6 ± 0.1, +57.6 ± 1.2, +51.2 ± 2.0 (USGS35), in agreement with their values and with the isotope ratio mass spectrometry results. The 17 O excess for USGS35 was +21.2 ± 9‰, in good agreement with previous results. CONCLUSIONS: The Cd-azide method yielded excellent results for routine determination of δ15 N, δ18 O and δ17 O values (and the 17 O excess) of nitrate or nitrite by laser spectrometry. Disadvantages are the toxicity of Cd-azide chemicals and the lack of automated sampling devices for N2 O laser spectrometers. The 15 N-enriched tracer test revealed potential for position-specific experimentation of aqueous nutrient dynamics at high 15 N enrichments by laser spectrometry, but exposed the need for memory corrections and improved spectral deconvolution of 17 O.

Automated rapid triple-isotope (δ15N, δ18O, δ17O) analyses of nitrate by Ti(III) reduction and N2O laser spectrometry.

The nitrogen and oxygen (δ15N, δ18O, δ17O) stable isotopic compositions of nitrate (NO3-) are crucial tracers of nutrient N sources and dynamics in aquatic and atmospheric systems. Methods to reduce aqueous NO3- to N2O gas (microbial or Cd method) before 15N and 18O isotope analyses require multi-step conversion or toxic chemicals, and 17O in N2O cannot be disentangled by IRMS due to isobaric interferences. This technical note describes the automation of the stable-isotope analyses of nitrate by coupling the new Ti method with a headspace autosampler and an N2O triple-isotope laser analyzer based on off-axis integrated cavity output spectroscopy. The automation yielded accurate and precise results for routine determinations of δ15N, δ18O, and δ17O values for aqueous nitrate in environmental waters. Systematic corrections were required for cavity pressure, N2O concentration and water vapour content to obtain the highest precision for all three isotopic ratios. For the first time, an automated laser-based system facilitates routine low-cost triple isotope analyses in studies where high-temporal resolution isotope analyses of NO3- are required but have been, until now, cost-prohibitive and time-consuming (e.g. atmospheric N pollution).

Nitrate isotopes reveal N-cycled waters in a spring-fed agricultural catchment.

Nitrate stable isotopes provide information about nitrate contamination and cycling by microbial processes. The Fischa-Dagnitz (Austria) spring and river system in the agricultural catchment of the Vienna basin shows minor annual variance in nitrate concentrations. We measured nitrate isotopes (δ15N, δ18O) in the source spring and river up to the confluence with the Danube River (2019-2020) with chemical and water isotopes to assess mixing and nitrate transformation processes. The Fischa-Dagnitz spring showed almost stable nitrate concentration (3.3 ± 1.0 mg/l as NO3--N) year-round but surprisingly variable δ15N, δ18O-NO3- values ranging from +5.5 to +11.1‰ and from +0.5 to +8.1‰, respectively. The higher nitrate isotope values in summer were attributed to release of older denitrified water from the spring whose isotope signal was dampened downstream by mixing. A mixing model suggested denitrified groundwater contributed > 50 % of spring discharge at baseflow conditions. The isotopic composition of NO3- in the gaining streams was partly controlled by nitrification during autumn and winter months and assimilation during the growing season resulting in low and high δ15N-NO3- values, respectively. NO3- isotope variation helped disentangle denitrified groundwater inputs and biochemical cycling processes despite minor variation of NO3- concentration.