Perspective: Hidden biases in isotope delta results and the need for comprehensive reporting.

Measurements of stable-isotope composition on an isotope-delta scale can be subject to bias between laboratories or over time within a single laboratory. This bias can arise not just from differences in method protocol but also from changes in reporting guidelines, or even to the isotope-delta scales themselves. Without a clear description of method protocols, including all sample preparation steps, instrumental parameters and settings, data processing including calibration of results and estimation of measurement uncertainty, the traceability and comparability of isotope-delta values cannot be assured as bias(es) may remain hidden. To address this need, there are now clear guidelines published by IUPAC for reporting isotope-delta values for the "light" elements hydrogen, carbon, nitrogen, oxygen and sulfur.1 We recommend that authors and reviewers adhere to those guidelines when preparing and reviewing future publications.

Gamma-irradiation of common biological samples for stable carbon and nitrogen isotope and elemental analyses.

RATIONALE: Around the world biosecurity measures are being strengthened to prevent the spread of pests and diseases across national and international borders. Quarantine protocols that involve sample sterilisation have potential effects on sample integrity. The consequences of sterilisation methods such as gamma (γ)-irradiation on the elemental and chemical properties of biological samples have not been widely examined. METHODS: We tested the effect of γ-irradiation (50 kGy) on the stable carbon and nitrogen isotope compositions (δ13 C and δ15 N values) and elemental concentrations (C % and N %) of common biological samples (fish, plants and bulk soils). The analysis used a continuous flow system consisting of a Delta V Plus isotope ratio mass spectrometer connected with a Thermo Flash 1112 elemental analyser via a ConFlo IV interface. Results were compared using two one-sided tests (TOST) to test for statistical similarity between paired samples. RESULTS: There was no change in the δ15 N values or N % of γ-irradiated samples, and only small changes to the δ13 C values of consumers (range: 0.01‰ to 0.04‰), producers (-0.02‰ to 0.04‰) and sediments (0‰ to 0.07‰). The magnitude of change in δ13 C values was greatest at low carbon concentrations but appeared negligible when measured against replicated sample analysis and the combined analytical uncertainty (i.e., 0.10‰). The C % values of irradiated samples were higher for consumers (0.23%) and lower for producers and sediments (0.04% and 0.05%, respectively) which may have implications for certain types of biological material. CONCLUSIONS: Routine γ-irradiation has little effect on the stable carbon and nitrogen isotope compositions of common biological samples and marginal effects on carbon elemental concentrations. This is unlikely to warrant concerns since the observed difference is typically of a magnitude lower than other sources of potential uncertainty.

New Ag3 PO4 comparison material for stable oxygen isotope analysis.

RATIONALE: A silver phosphate reference material (Ag3 PO4 ) for the measurement of stable oxygen isotope compositions is much needed; however, it is not available from the authorities distributing reference materials. This study aims to fill this gap by calibrating a new Ag3 PO4 stable isotope comparison material produced by the University of Natural Resources and Life Sciences (BOKU). METHODS: Aliquots of Ag3 PO4 were distributed to four laboratories who frequently measure the δ18 O value in Ag3 PO4 ; the University of Natural Resources and Life Sciences (BOKU), the University of Western Australia (UWA), the University of Helsinki (UH), and the Helmholtz Centre for Environmental Research (UFZ). The instruments used to perform the measurements were high-temperature conversion elemental analysers coupled with continuous flow isotope ratio mass spectrometers. The working gas δ18 O value was set to 0‰ and the normalization was done by a three-point linear regression using the reference materials IAEA-601, IAEA-602, and NBS127. RESULTS: The mean δ18 O value of the new BOKU Ag3 PO4 comparison material on the VSMOW-SLAP scale is 13.71‰ and the combined uncertainty is estimated as ±0.34‰. This estimated uncertainty is within the range typical for comparison materials of phosphates and sulphates. Consistent results from the different laboratories probably derived from similar instrumentation, and use of the same reference materials and normalization procedure. The matrix effect of the different reference materials used in this study was deemed negligible. CONCLUSIONS: The BOKU Ag3 PO4 can be used as an alternative comparison material for stable oxygen isotope analysis and is available for stable isotope research laboratories to facilitate calibration.

Recalculation of stable isotope expressions for HCNOS: EasyIsoCalculator.

RATIONALE: The stable HCNOS isotope compositions can be reported as (a) the isotope ratio of two stable isotopes (R); (b) the isotope delta value (δ); and (c) the atom fraction of the isotopes (x). Recalculations between these different expressions are needed frequently and require the use of the absolute isotope ratio for the zero points of the stable isotope delta scales (Rstd). The inconsistent use of Rstd values may lead to a discrepancy in recalculated results. METHODS: We summarised the recalculation procedures between different expressions of the stable isotope compositions and introduced a user-friendly EasyIsoCalculator that allows the recalculation between the main expressions of isotope compositions. We mathematically and empirically evaluated the possible inconsistencies in reporting of the stable isotope data due to the use of different Rstd and different normalisation methods. RESULTS: The recalculation between δ-values and other expressions of the stable isotope compositions always involves the use of Rstd. The choice of Rstd will have a significant influence on the recalculated values. The use of different Rstd values has a significant influence also on the normalisation of raw values but only when the normalisation is conducted versus the working standard gas value, causing discrepancy, e.g. for δ(13C/12C) up to ~ 0.3 ‰. CONCLUSIONS: Differences in the selection of Rstd value may lead to significant differences among different laboratories. The uncertainty in the calculations originates primarily from the uncertainty in the Rstd determination; however, it is lower than the discrepancy arising from the inconsistent use of Rstd. Consistent use of the same Rstd values is required to eliminate the unnecessary discrepancy if different data sets are recalculated from delta value to other expressions.

Absolute isotope ratios defining isotope scales used in isotope ratio mass spectrometers and optical isotope instruments.

RATIONALE: The isotope delta is calculated from the isotope ratio of a sample and the absolute isotope ratio of the zero reference point defining each stable isotope international scale (Rstd ). Therefore, Rstd requires accurate determination. However, the literature contains a large number of Rstd values, and selection of different Rstd may lead to inconsistency in reporting and recalculating stable isotope results. METHODS: We reviewed Rstd used in the proprietary software provided by the manufacturers of stable isotope instruments commonly employed for analyses of stable HCNOS compositions. We compared the Rstd values and assessed the potential implications of using different Rstd and the normalization versus tank working gas standard for consistency in reporting stable isotope results. RESULTS: Different Rstd values are used by different manufacturers of stable isotope analytical instruments. For R(2 H/1 H)VSMOW two different but very similar values are used, 0.00015575 and 0.00015576; for R(13 C/12 C)VPDB three different values are used, 0.0111802, 0.0112372 and 0.01118028; and for R(15 N/14 N)Air-N2 two values, 0.0036782 and 0.0036765, are used. All manufacturers are using the same value for R(18 O/16 O)VSMOW , 0.00200520, but three different values for R(18 O/16 O)VPDB , 0.002067200, 0.00208835 and 0.002088349. For R(34 S/32 S)VCDT four different Rstd are used, 0.0441509, 0.0441626, 0.044162589 and 0.0441520597. CONCLUSIONS: The use of different Rstd values may lead to differences in the isotope delta values obtained if the normalization versus working standard gas is applied. For the range of Rstd used in proprietary software, the potential differences are lowest for oxygen (< 0.002 ‰) and nitrogen (< 0.001 ‰), and highest for carbon (0.107 to 0.112 ‰) and sulfur (0.023 ‰). Evaluation of the existing Rstd values and recommendations for the best estimates are highly desirable to ensure worldwide consistency in stable isotope data reporting.

Comparisons of stable isotope composition among tissues of green turtles.

Ecologists often need to make choices about what body parts (tissues or organs) of an animal to sample. The decision is typically guided by the need to treat animals as humanely as possible, as well as the information that different body parts can provide. When using stable isotopes, decisions are also influenced by whether specimens would require preservation, and whether they have properties (such as high lipid concentrations) that would influence measurements. Sometimes we cannot use a preferred tissue (for example, because of ethical or logistical constraints), and in such cases an ability to reliably predict stable isotope composition for one tissue from data yielded by another would be useful. METHODS: In this study we analysed multiple tissues (skin, whole blood, red blood cells, plasma and nail) from green turtles (Chelonia mydas) to evaluate variation in C:N ratios, and test hypotheses about the intercept and slope of regressions of stable carbon and nitrogen isotope compositions among tissues. RESULTS: Regression models revealed that linear relationships were present for most comparisons, except those involving the δ13 C of skin, and the slopes (ß1 ) of most regressions were different from unity. The C:N ratios of skin were significantly higher and more variable than those of other tissues. The δ13 C and δ15 N of nail were highly correlated with those of the whole blood, red blood cells and plasma. Nail and red blood cells showed low variation in C:N. CONCLUSIONS: The patterns in slopes of regressions indicate that comparisons of measurements yielded by different tissues of wild animals are complicated by the fact that the tissues are unlikely to be in isotopic equilibrium with their diet. Of the tissues used in this study, nail is simple to collect, requires minimal disturbance to the animal and no special preservation; these traits should make it attractive to turtle ecologists, but more information is needed on aspects such as growth rates.

Reference materials selection for the stable carbon isotope analysis of dissolved carbon using a wet oxidation system.

RATIONALE: Wet chemical oxidation combined with isotope ratio mass spectrometry has become a routine technique for analyzing the stable carbon isotope composition of dissolved organic (DOC) and inorganic (DIC) carbon. However, methodological inconsistencies between laboratories in using different reference materials lead to a discrepancy in results. We experimentally tested the precision and accuracy of the analysis of commonly available international reference materials and other chemicals potentially suitable for laboratory standards. METHODS: The solid international reference materials and other simple chemicals were used to prepare water solutions. A range of carbon concentrations was chosen to optimize tests for (1) precision and accuracy, (2) linearity, (3) detection limits, (4) memory effects, and (5) efficiency of DIC removal from a DOC/DIC mixtures. Samples were analyzed using an LC-IsoLink coupled with a Delta V Plus isotope ratio mass spectrometer (Thermo Fisher Scientific). RESULTS: The analytical setup had a negligible memory effect, good reproducibility (<0.21‰) and accuracy (maximum difference from the true values <0.35‰) for the analyzed organic compounds if approximately ≥9 × 10-09 moles of dissolved carbon was injected into the system (~11 mg C L-1 if a 10-µL loop was used). Analyses of sodium bicarbonate or calcium carbonate solutions had a two-fold lower accuracy despite maintaining a high precision. CONCLUSIONS: Aqueous solutions of international reference materials such as L-glutamic acids (USGS40, USGS41), benzoic acid (IAEA-601) and sucrose (IAEA-CH-6) can be successfully used for direct normalization of results to the VPDB scale. By contrast, analyses of caffeine and urea returned very reproducible but highly inaccurate results and these materials are not recommended for standards.

Stable isotope analysis of saline water samples on a cavity ring-down spectroscopy instrument.

The analysis of the stable hydrogen and oxygen isotope composition of water using cavity ring-down spectroscopy (CRDS) instruments utilizing infrared absorption spectroscopy have been comprehensively tested. However, potential limitations of infrared spectroscopy for the analysis of highly saline water have not yet been evaluated. In this study, we assessed uncertainty arising from elevated salt concentrations in water analyzed on a CRDS instrument and the necessity of a correction procedure. We prepared various solutions of mixed salts and separate solutions with individual salts (NaCl, KCl, MgCl2, and CaCl2) using deionized water with a known stable isotope composition. Most of the individual salt and salt mixture solutions (some up to 340 g L(-1)) had δ-values within the range usual for CRDS analytical uncertainty (0.1‰ for δ (18)O and 1.0‰ for δ (2)H). Results were not compromised even when the total load of salt in the vaporizer reached ∼38.5 mg (equivalent to build up after running ∼100 ocean water samples). Therefore, highly saline mixtures can be successfully analyzed using CRDS, except highly concentrated MgCl2 solutions, without the need for an additional correction if the vaporizer is frequently cleaned and MgCl2 concentration in water is relatively low.

Rain use efficiency gradients across Australian ecosystems.

Rain use efficiency (RUE) quantifies the ecosystem's capacity to use precipitation water to assimilate atmospheric CO2. The spatial distribution of RUE and its drivers across the Australian continent is largely unknown. This knowledge gap limits our understanding of the possible contribution of Australian ecosystems to global carbon assimilation. This study investigates the spatial distribution of RUE across diverse terrestrial ecosystems in Australia. The results show that RUE ranges from 0.43 (1st percentile) to 3.10 (99th percentile) g C m-2 mm-1 with a continental mean of 1.19 g C m-2 mm-1. About 68 % of the spatiotemporal variability of RUE can be explained by a multiple linear regression model primarily contributed by climatic predictors. Benchmarked by the model estimation, drainage-diverging/converging landscapes tend to have reduced/increased RUE. The model also revealed the impact of increasing atmospheric CO2 concentration on RUE. The continental mean RUE would increase by between 29.3 and 64.8 % by the end of this century under the SSP5-8.5 scenario in which the CO2 concentration is projected to double from the present level. This increase in projected RUE is attributed to the assumed greening effect of increasing CO2 concentration, which does not consider the saturation of CO2 fertilisation effect and the warming effect on increasing wildfire occurrence. Under the SSP1-2.6 scenario, RUE would decrease by about 7 %. This study provides baseline RUEs of various ecosystems in Australia for investigating the impacts of human interferences and climate change on the capacity of Australian vegetation to assimilate atmospheric CO2 under given precipitation.

Tracing sulfate sources in a tropical agricultural catchment with a stable isotope Bayesian mixing model.

Sulfate (SO42-) is an essential anion in drinking water and a vital macronutrient for plant growth. However, elevated sulfate levels can impact ecosystem or human health and could be an important indicator of acid rock drainage or pollution. Therefore, monitoring SO42- sources and transport is important for water quality assessments. This study focused on exploring the sources and transformations of SO42- as well as estimating the proportional contribution of the potential SO42- pollutant sources to groundwater and surface water in a tropical river basin, the Densu River Basin. The study used major ions combined with stable sulfur and oxygen isotope compositions and a Bayesian isotope mixing model, MixSIAR. The major ion characteristics indicate that SO42- concentrations remain stable throughout the rainy and dry seasons but originate from diverse sources. The multi-isotope model (δ34SSO4, δ18OSO4) identified four potential SO42- sources: detergent, precipitation, sewage, and sulfate fertilizer. However, the δ34SSO4 and δ18OSO4 values of the fertilizer source signatures overlapped with those of precipitation and sewage. Nevertheless, the contributions from each source were disentangled using the MixSIAR model, which revealed sewage as the most dominant SO42- pollutant in the Densu Basin, accounting for ~47 % of sulfate in groundwater and ~ 56 % of sulfate in surface water. Sulfate fertilizer (~33 %) was the second most important source after sewage for groundwater, while detergent (~23 %) was the second most important source for surface water. The redox processes of bacterial sulfate reduction and sulfide oxidation were determined to have a minimal impact on the sulfur isotope fractionation within the basin. This study highlights the benefits of combining major ions, sulfur isotopes and the MixSIAR model for identifying sources of sulfate. This approach accounts for uncertainties in source contributions which allows for more robust and reliable apportionment of sulfate sources. The study emphasizes the need for effective waste management and pollution control measures to protect water quality and provides vital guidelines on how to partition sulfate sources on a large catchment scale and evidence for making pollution management decisions on water resources.

Molecular mechanisms of microbiome modulation by the eukaryotic secondary metabolite azelaic acid.

Photosynthetic eukaryotes, such as microalgae and plants, foster fundamentally important relationships with their microbiome based on the reciprocal exchange of chemical currencies. Among these, the dicarboxylate metabolite azelaic acid (Aze) appears to play an important, but heterogeneous, role in modulating these microbiomes, as it is used as a carbon source for some heterotrophs but is toxic to others. However, the ability of Aze to promote or inhibit growth, as well as its uptake and assimilation mechanisms into bacterial cells are mostly unknown. Here, we use transcriptomics, transcriptional factor coexpression networks, uptake experiments, and metabolomics to unravel the uptake, catabolism, and toxicity of Aze on two microalgal-associated bacteria, Phycobacter and Alteromonas, whose growth is promoted or inhibited by Aze, respectively. We identify the first putative Aze transporter in bacteria, a 'C4-TRAP transporter', and show that Aze is assimilated through fatty acid degradation, with further catabolism occurring through the glyoxylate and butanoate metabolism pathways when used as a carbon source. Phycobacter took up Aze at an initial uptake rate of 3.8×10-9 nmol/cell/hr and utilized it as a carbon source in concentrations ranging from 10 µM to 1 mM, suggesting a broad range of acclimation to Aze availability. For growth-impeded bacteria, we infer that Aze inhibits the ribosome and/or protein synthesis and that a suite of efflux pumps is utilized to shuttle Aze outside the cytoplasm. We demonstrate that seawater amended with Aze becomes enriched in bacterial families that can catabolize Aze, which appears to be a different mechanism from that in soil, where modulation by the host plant is required. This study enhances our understanding of carbon cycling in the oceans and how microscale chemical interactions can structure marine microbial populations. In addition, our findings unravel the role of a key chemical currency in the modulation of eukaryote-microbiome interactions across diverse ecosystems.

Chemical tools for the Gid4 subunit of the human E3 ligase C-terminal to LisH (CTLH) degradation complex.

We have developed a novel chemical handle (PFI-E3H1) and a chemical probe (PFI-7) as ligands for the Gid4 subunit of the human E3 ligase CTLH degradation complex. Through an efficient initial hit-ID campaign, structure-based drug design (SBDD) and leveraging the sizeable Pfizer compound library, we identified a 500 nM ligand for this E3 ligase through file screening alone. Further exploration identified a vector that is tolerant to addition of a linker for future chimeric molecule design. The chemotype was subsequently optimized to sub-100 nM Gid4 binding affinity for a chemical probe. These novel tools, alongside the suitable negative control also identified, should enable the interrogation of this complex human E3 ligase macromolecular assembly.

Normalization procedures and reference material selection in stable HCNOS isotope analyses: an overview.

The uncertainties of stable isotope results depend not only on the technical aspects of measurements, but also on how raw data are normalized to one of the international isotope scales. The inconsistency in the normalization methods used and in the selection of standards may lead to substantial differences in the results obtained. Therefore, unification of the data processing protocols employed is highly desirable. The best performing methods are two-point or multipoint normalization methods based on linear regression. Linear regression is most robust when based on standards that cover the entire range of δ values typically observed in nature, regardless of the δ values of the samples analysed. The uncertainty can be reduced by 50 % if measurements of two different standards are performed four times, or measurements of four standards are performed twice, with each batch of samples. Chemical matrix matching between standards and samples seems to be critical for δ (18)O of nitrate or δ (2)H of hair samples (thermal conversion/elemental analyser), for example; however, it is not necessarily always critical for all types of samples and techniques (e.g. not for most δ (15)N and δ (13)C elemental analyser analyses). To ensure that all published data can be recalculated, if δ values of standards or the isotope scales are to be updated, the details of the normalization technique and the δ values of the standards used should always be clearly reported.

The measurement of ambient air moisture stable isotope composition for the accurate estimation of evaporative losses.

Accurate estimation of evaporative losses from a water body, using the Craig-Gordon model and the stable hydrogen and oxygen isotope composition of water, requires knowledge of the stable isotope composition of ambient air moisture. This is rarely measured in the field, and it is usually estimated assuming that recent rainfall remains in isotopic equilibrium with atmospheric moisture. However, the ambient air moisture stable isotope composition may vary significantly at different heights above the water body. In this study, we set up outdoor pan evaporation experiments and simultaneously measured the stable isotope composition of ambient moisture in the atmosphere at three different heights. Using these measurements, we calculated evaporative losses, compared them with the observed losses in the pan, and assessed the uncertainty introduced by differences in ambient moisture measurements. Three main steps in the experimental method:•Daily water sampling from the evaporation pan for analysis of stable hydrogen and oxygen isotope compositions.•Recording the stable isotope composition of ambient air moisture at three different heights using the Picarro L2130-i system over a period of experiments.•Calculating evaporative losses from the pan using the Craig-Gordon model and ambient air stable isotope composition measured at three different levels and comparing to the observed losses.

Corrigendum to: The measurement of ambient air moisture stable isotope composition for the accurate estimation of evaporative losses. MethodsX 11 (2023) 102265.

[This corrects the article DOI: 10.1016/j.mex.2023.102265.].

Disentangling nitrate pollution sources and apportionment in a tropical agricultural ecosystem using a multi-stable isotope model.

Fertilizers increase agricultural productivity and farmers' income. However, intensive agriculture frequently overuses fertilizers, which in turn can contaminate surface and groundwater. In this study, hydrochemical and multi-isotope (δ15NNO3, δ18ONO3 and δ18OH2O) data have been combined to identify nitrate pollution sources in Ghana's Densu River Basin, trace the Nitrogen (N) biogeochemical processes in the basin and apportion the contribution of each pollution source. Surface water NO3- ranged from 0.3 to 10.6 mg/L (as N), while groundwater NO3- ranged from 0.9 to 34 mg/L. Hierarchical cluster analysis classified the water samples into three spatial categories: upstream, midstream, and downstream, reflecting river and land use patterns. The multi-isotope model considered five primary NO3- sources: atmospheric deposition, manure/sewage, NH4+ in fertilizers, other NO3- based fertilizers and soil N. Nitrification was identified as the major biogeochemical process upstream, whereas mixing of sources and denitrification dominate the midstream to downstream sections of the basin. Nitrate source apportioning using a MixSIAR model reveal that N fertilizers (40 %) and soil N (34 %) contribute the most to nitrate pollution upstream of the river. From the midstream to downstream sections, manure/sewage (43 %) become the dominant nitrate source, reflecting the transition from agriculture to peri-urban and urban land use. This study has shown that soil erosion and runoff contribute to nitrate pollution in the Densu River, at levels comparable to N fertilizers, and groundwater across the basin is impacted mainly by manure/sewage. The multi-isotope analyses allowed the partitioning of N sources in other ways not possible using only classical hydrochemical methods.

Stable Isotope Provenance of Unidentified Deceased Migrants-A Pilot Study.

In the global migration crisis, one of the challenges in the effort to identify deceased migrants is establishing their region of origin, which facilitates the search for ante mortem data to be compared with the post mortem information. This pilot study explores the potential of using stable isotope analysis to distinguish between individuals coming from West Africa and the Horn of Africa. Six individuals (four of known origin and two of unknown origin) were sampled. δ13CVPDB(keratin), δ15NVPDB(keratin) and δ18OVSMOW(keratin) of hair were analysed using Elemental Analyzers coupled with Isotope Ratio Mass Spectrometry (IRMS). δ18OVSMOW(carbonate) and δ13CVPDB(carbonate) of bone were analysed using GasBench II with IRMS, while 87Sr/86Sr composition was determined in bone and dental enamel using laser ablation multi-collector inductively coupled plasma mass spectrometry. The stable isotope compositions of the individual from the Horn of Africa differed from the other individuals. The differences found between 87Sr/86Sr of enamel and bone and between δ18O and δ13C in bone and hair reflect changes in sources of food and water in accordance with regionally typical migration journeys. The analysis of multiple stable isotopes delivered promising results, allowing us to narrow down the region of origin of deceased migrants and corroborate the information about the migration journey.

FRAME-Monte Carlo model for evaluation of the stable isotope mixing and fractionation.

Bayesian stable isotope mixing models are widely used in geochemical and ecological studies for partitioning sources that contribute to various mixtures. However, none of the existing tools allows accounting for the influence of processes other than mixing, especially stable isotope fractionation. Bridging this gap, new software for the stable isotope Fractionation And Mixing Evaluation (FRAME) has been developed with a user-friendly graphical interface (malewick.github.io/frame). This calculation tool allows simultaneous sources partitioning and fractionation progress determination based on the stable isotope composition of sources/substrates and mixture/products. The mathematical algorithm applies the Markov-Chain Monte Carlo model to estimate the contribution of individual sources and processes, as well as the probability distributions of the calculated results. The performance of FRAME was comprehensively tested and practical applications of this modelling tool are presented with simple theoretical examples and stable isotope case studies for nitrates, nitrites, water and nitrous oxide. The open mathematical design, featuring custom distributions of source isotope signatures, allows for the implementation of additional processes that alternate the characteristics of the final mixture and its application for various range of studies.

A strategy for selection of reference materials in stable oxygen isotope analyses of solid materials.

The propagation of uncertainties associated with the stable oxygen isotope reference materials through a multi-point normalisation procedure was evaluated in this study using Monte Carlo (MC) simulation. We quantified the normalisation error for a particular selection of reference materials and their number of replicates, when the choice of standards is restricted to either nitrates, sulphates or organic reference materials alone, and in comparison with when this restriction was relaxed. A lower uncertainty in stable oxygen isotope analyses of solid materials performed using High-Temperature Pyrolysis (HTP) can be readily achieved through an optimal selection of reference materials. Among the currently available certified reference materials the best performing pairs minimising the normalisation errors are USGS35 and USGS34 for nitrates; IAEA-SO-6 and IAEA-SO-5 for sulphates; and IAEA-601 and IAEA-602 for organic materials. The normalisation error can be reduced further--by approximately half--if each of these two analysed reference materials is replicated four times. The overall optimal selection among all nine considered reference materials is the IAEA-602 and IAEA-SO-6 pair. If each of these two reference materials is replicated four times the maximum predicted normalisation error will equal 0.22‰, the minimum normalisation error 0.12‰, and the mean normalisation error 0.15‰ over the natural range of δ(18)O variability. We argue that the proposed approach provides useful insights into reference material selection and in assessing the propagation of analytical error through normalisation procedures in stable oxygen isotope studies.