Conference Agenda

Recent progress in the field of laser spectroscopy has transformed the measurement of atmospheric greenhouse gases, enabling in-situ field measurements of carbon dioxide (CO₂) and methane (CH₄) stable isotope ratios. These new measurement capabilities have generated an urgent demand for commutable isotopic gas reference materials for CO₂ and CH₄ at ambient amount fractions. However, achieving the level of uncertainty required for such reference materials remains challenging. Additionally, reliance on individual calibration procedures by end-users has resulted in inconsistent data, hindering comparability across datasets.

Addressing these challenges requires the development of new isotopic reference materials, improved validation protocols, and standardised calibration guidelines. This effort is essential to ensure traceability for field-deployable spectroscopic methods and traditional offline flask sampling techniques using mass spectrometry.

In response to the growing need for improved metrological support, a joint CCQM GAWG/IRWG Isotope Ratio Task Group was established in April 2023. This group coordinates efforts among National Metrology Institutes (NMIs), Designated Institutes (DIs), and intergovernmental organisations to develop a robust infrastructure for traceable isotopic reference materials and calibration guidelines, ensuring accurate measurement of stable isotope ratios for atmospheric greenhouse gases.

As part of its foundational work, the task group recently submitted a comprehensive paper outlining key recommendations for advancing the measurement of CH₄ stable isotope ratios. The paper also reviews Calibration and Measurement Capabilities (CMCs) to help NMIs and DIs develop their capabilities in support of the atmospheric measurement community.

This presentation will summarise the task group’s objectives, progress, and key recommendations. Additionally, it will provide preliminary results from a global survey conducted in early 2025, mapping current capabilities for the measurement of the stable isotope ratio of CH₄.

Within the German Isotope Network (GIN) five laboratory inter-comparison tests were conducted between 2018 and 2024 with between 8 and 17 participating laboratories. For the most recent comparison GIN5 in 2024, three objectives were to be met: First, participants measured ten unknown water samples using their established procedures and working standards akin to earlier comparisons. The second task was to measure the same ten unknown water samples but using unified laboratory working standards that were distributed to all laboratories together with the unknown samples. Third, all laboratories were asked to measure a unified control standard as often as possible during their daily measurements to compare laboratory accuracies for long-term measurements of a quality check sample.

Thirteen out of seventeen laboratories returned results in time for the GIN5 comparison. The results clearly indicate a better overall accuracy for each of the ten unknown water samples measured during the test when all laboratories use the same working standards. For all five inter-comparison tests, we discuss the accuracy and comparability of lab results when independently calibrated working standards were used. In addition to well established international inter-comparison tests (e.g., WICO by IAEA), we highlight and discuss potentials and challenges for inter-comparison tests that are organized and conducted on a national level.

The efficient catalytic transformation of greenhouse C₁ molecules (e.g., CO₂ and CH₄) into valuable chemical building blocks is critical for the sustainable energy transition. Advancing catalytic materials and processes requires a fundamental understanding of reaction mechanisms, and stable isotope techniques are powerful tools that provide molecular-level insights into catalytic reactions.

In this contribution, I will highlight isotope-based methodologies for probing the molecular details of CO₂ hydrogenation and CH₄ aromatization. I will describe the application of steady-state isotope transient kinetic analysis (SSITKA) and pulsed isotope transient techniques to investigate the reactivity of surface species and their interactions with gas-phase molecules. Furthermore, I will discuss how these isotope labeling methods can be combined with isotope-sensitive spectroscopic techniques, including infrared (IR) and nuclear magnetic resonance (NMR) spectroscopy, to gain deeper mechanistic insights.

First, I will focus on how a combination of operando diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS) and ¹²C/¹³C SSITKA provided a mechanistic understanding of the unusual particle size effects in Co- and Ni-based catalysts for CO₂ hydrogenation to hydrocarbons [1,2]. Second, I will discuss our studies on CH₄ aromatization, where pulsed ¹²C/¹³C isotope transient techniques and ¹³C/¹H NMR spectroscopy revealed an unexpected hydrocarbon pool mechanism. In this process, benzene forms through secondary reactions between confined polyaromatic carbon species and the primary products of methane activation. [3,4,5].

References

[1] J.F.M. Simons, N. Kosinov, E.J.M. Hensen, et al. J. Am. Chem. Soc. 145 (2023) 20289–20301.

[2] J.J.C. Struijs, N. Kosinov, et al. Angew. Chemie Int. Ed. 62 (2023) e202214864.

[3] N. Kosinov, E.J.M. Hensen, et al. Angew. Chemie Int. Ed. 57 (2018) 1016–1020.

[4] N. Kosinov, E.J.M. Hensen, et al. Angew. Chemie Int. Ed. 58 (2019) 7068–7072.

[5] H. Zhang, N. Kosinov, et al. Angew. Chemie Int. Ed. 62 (2023) e202306196.

The Stable Isotope Mass Spectrometry Laboratory (SIRMS Lab) at the University of Southampton is a state-of-the-art research, teaching and analytical facility dedicated to high-precision light stable isotope analysis across a diverse range of scientific disciplines. Located within the Ocean and Earth Science department of the University of Southampton at the National Oceanography Centre Southampton, Waterfront Campus, the lab supports academic research, external collaboration and commercial sample analysis, whilst offering expert guidance on sample preparation, analysis, and data interpretation.

Equipped with several advanced gas source isotope ratio mass spectrometers (IRMS) and peripheral devices operating in both dual inlet and continuous flow modes, the SIRMS lab specializes in analysing light stable isotopes such as δ²H, δ¹³C, δ¹⁵N, δ¹⁸O, and δ³⁴S across a wide array of organic and inorganic materials. This analytical capability supports research in environmental sciences, oceanography, geology, archaeology, biology, and climate science.
Researchers benefit from tailored support by dedicated personnel for sample preparation, instrument maintenance, method development, quality assurance and sample analysis, ensuring high-quality data and robust interpretations. The lab staff is actively involved in training and mentoring students and postdoctoral researchers, fostering the next generation of scientists in stable isotope techniques.
Here we present an overview of the lab and its capabilities, introduce our new instrumentation with examples of recent research applications, demonstrating its role in advancing scientific understanding across multiple fields.

The online combustion of analytes between gas chromatography and isotope ratio mass spectrometry (GC-C-IRMS) has enabled compound-specific isotope analysis (CSIA) for various applications, such as assessment of environmental contaminants or doping in sports. However, CSIA is challenged by the need for complete peak separation and for best sensitivity. Comprehensive gas chromatography could deliver a breakthrough, but hinges on the development of robust miniaturized online combustion tubes that offer sufficient oxidation capacity and catalytic surface area to accomplish complete analyte conversion to CO₂, while being narrow enough to preserve narrow analyte peak shapes within the continuous flow carrier stream. The current step change when He carrier gas passes from GC capillary columns (inner diameter, i.d.: 0.22–0.32 mm) to commercial combustion tubes (i.d.: 0.5 mm) generates substantial peak broadening. Even smaller GC capillaries are needed, however, to support GCxGC applications and to improve sensitivity by reducing flows and, therefore, minimizing losses in an open split before IRMS. Since commercial reactor tubes are not compatible with fast GC-C-IRMS and GCxGC-C-IRMS, efforts have been directed at developing alternative miniaturized reactor tubes.

To pioneer the necessary dramatic reduction of reactor tube size, a Ni wall-coated catalytic microreactor is constructed by coating different capillaries (Alumina, Quartz and Fused silica) by electroless plating. An 18 cm long Ni layer is coated in the middle inner channel to fit the furnace hot zone. This approach has the benefit of simplicity, while allowing for the fabrication of narrow bore capillary reactors (inner diameter < 0.2 mm), which is not achievable with hand-loading of metal wires. The weight of the coating is determined by ICP-MS, while the layer thickness is measured by SEM and EDX. Precise and accurate data were obtained for Caffeine on quartz (o.d. 1.5 mm, i.d. 0.32 mm), with an overall mean Δδ¹³C of -0.16‰ and a standard deviation of ±0.12‰.

We present a compact laser based analyzer for the measurement of ¹³C/¹²C and ¹⁸O/¹⁶O isotope ratios in CO₂ samples. The analyzer is based on the tunable diode laser spectroscopy method, utilizing interband cascade lasers in the mid-infrared region. The instrument is optimized for the analysis of sample concentrations ranging from 1-100% CO₂ with precision better than 0.1‰. With the combination of small sample cell to efficient sample gas handling, the sample consumption per analysis is below 10 mL and measurement time down to few seconds. The analyzer weights only 8kg and has a compact frame of 30cm x 20cm x 10 cm and battery powered operation capability. In this work, we present validation of the accuracy and precision of the instrument for isotope ratio determination for different measurement times and total sample consumption. The instrument is also used for measuring samples from CO₂ leak sources in European union METCCUS project, that supports carbon capture utilization and storage.

The CEREGE, a research institute located in the southeast of France, acquired a Thermo Kiel IV & 253+ system in 2023, dedicated to the measurement of clumped isotopes. Notably, this was the first installation of such a system operating with Qtegra ISDS as its piloting software.

Following initial months of adjustments, interesting challenges, and fine-tuning, the system has begun to yield reliable data as of 2024. However, operating such highly sensitive equipment in a region subjected to acute temperature variations —with very hot summers and cold winter nights transitioning to warmer daytime temperatures—presents significant challenges that necessitate continuous adjustments and monitoring.

In our presentation, we will demonstrate how operating the system in Long Integration Dual Inlet “Bracketed” (LIDI 2) mode, compared to the standard LIDI and Change Over modes, can help mitigate the adverse effects of room temperature fluctuations. On the other hand, we will also shed light on the drawbacks and limitations of such mode and the reasons behind them.

We will also aim at sharing practical insights and strategies gathered from both existing literature and our hands-on laboratory experience through round-the-clock monitoring, aimed at minimizing fluctuations and improving data consistency.

Establishing compatibility among laboratories measuring high-precision stable isotopes of atmospheric methane (CH₄) is challenging because the reference materials differ from the measured samples. Significant offsets are common as each laboratory has a different tie to the VPDB or SMO-SLAP scales. Likewise, for clumped isotopes, since the measurements are not done against a standard, but against the isotopologues' respective stochastic distribution to calculate the anomaly of the clumping, laboratories have to periodically compare cylinders, measurement procedures, and extraction protocols. Inter-laboratory comparison of CH₄ isotope measurements, both stable and clumped, is essential so that the data from different laboratories can be compared and consolidated for scientific interpretation. This process is referred to as harmonisation of isotopic measurements.

To achieve this, we adopt methods to quantify and adjust inter-laboratory scales. For stable isotopes, we compare routine atmospheric measurements conducted by these laboratories at high-latitude stations in the Northern and Southern Hemispheres, where we assume that the air masses are sufficiently homogenized for direct comparison. The long-term mean offsets are verified against various inter-laboratory round-robin exercises and align well with the findings of Umezawa et al. (2018). For clumped isotopes, we compare the measurement procedures and results for both mass spectrometry and laser spectroscopy methods by circulating pure CH₄ samples among participating laboratories. Although the clumping anomaly data cannot be verified against a standard scale, they do agree well among the laboratories and with the theoretical thermal equilibration scale of CH₄ isotopologues. We present the results from both exercises and report noteworthy agreement among laboratories, especially given the substantial effort, complexity, and logistical challenges involved in conducting CH₄ isotope measurements. Continuous intercomparison and harmonisation efforts are crucial for maintaining high precision and consistency in isotopic measurements, ultimately improving our understanding of methane's role in the global carbon cycle and its impact on climate change.

Reference materials (RMs) are essential to allow traceable measurements of isotope delta values to be made in laboratories around the world. Often, new isotope delta RMs have been characterized by inter-laboratory studies. These involve several laboratories each making measurements on the candidate RM using their own measurement protocol and data processing procedures. This provides additional confidence in the assigned isotope delta values but makes the estimation of the single assigned value and associated uncertainty more challenging. Some published approaches have been criticized for providing uncertainties that are too small with a potential cause being inadequate separation of random (e.g. precision of measurement results) and systematic (e.g. assigned values of existing RMs used for calibration) sources of uncertainty [1].

In this work, we use a dataset from the interlaboratory characterization of various nitrous oxide candidate RMs [2] to attempt to address these shortcomings. Each participating laboratory was completely free to apply the measurement methods they normally would and to process the data following their usual procedures. These included both elemental analyzer (EA) and dual-inlet (DI) isotope ratio mass spectrometry (IRMS) as well as optical isotope ratio spectroscopy (OIRS).

Participating laboratories provided uncalibrated data, so the final step of calibrating measured isotope delta values to the international reporting scale could be done centrally while also propagating uncertainties. This approach allowed complete separation of random from systematic sources of uncertainty through each calculation stage. It also allowed correlations among laboratories arising from the use of the same RMs for calibration to be accounted for. Several fundamental features of single- and two-point calibration of isotope delta by DI- and EA-IRMS and resulting measurement uncertainty for both individual laboratories and for combinations of inter-laboratory data will be presented.

[1] https://doi.org/10.1007/s00769-022-01527-6

[2] https://doi.org/10.1002/rcm.9296

Rapidly expanding biogeochemical applications based on compound specific isotope ratios require instrumentation versatility to meet different analytical challenges. Here we present features and benefits of using the following GC injection techniques: on-column injection, Large Volume Injection (LVI) Programmed Temperature Vaporization (PTV) technique, Static Headspace Sampling (SHS) injection and conventional Split/Splitless injection. We will demonstrate capability of Thermo Scientific™ GC IsoLink™ II IRMS System to support these injection techniques to properly transfer a representative portion of the sample to the analytical column while avoiding discrimination and isotopic effects.

On-column injection is applied for analysis of thermally labile or unstable compounds, as well as for samples with large analyte-boiling-point differences. It can be advantageous in a wide area of applications, i.e. for investigations of alkenones and alkanes from soils and sediments. We will present an optimized GC-IRMS analytical setup for stable carbon isotope ratios analysis of saturated hydrocarbons.

The LVI PTV is an injection technique which allows the introduction of larger volumes of samples in the GC injector which can be particularly useful for analysis of organic pollutants present in very small quantities. Here we present an optimized methodology for analysis of very small amounts of saturated hydrocarbons.

The SHS injection via split/splitless injector eliminates the need for direct liquid sample injection, reducing column contamination and improving analyte separation and reproducibility of isotope data. Here we demonstrate excellent precision and accuracy for GC-C-IRMS analysis of VOCs by using an optimized method for SHS, including improved sensitivity and lower detection limits.

Finally, we also present an optimized workflow for the analysis of PAHs by GC-IRMS with conventional Splitless injection, including characterization of PAHs standards and data evaluation.

Hydroxylamine (NH₂OH) is a short-lived compound of the marine N cycle (Ward, 2008) and is formed as an intermediate during the nitrification (Yoshida and Alexander, 1964). Greenhouse gas N₂O is produced from hydroxylamine in a side reaction during ammonium oxidation in the water. The detection of dissolved hydroxylamine and its stable isotopes in oceanic waters is a key to additional insights in the mechanisms of the marine N cycle and especially the production mechanisms of N₂O since it could serve as a tracer for the occurrence of bacterial nitrification in the water column. In 2011, Casciotti et al suggested that the measurement of δ¹⁸O in NH₂OH is necessary to understand the isotope effect for O incorporation by certain bacteria.

Butler and Gordon (1986) developed a FAS conversion method which is based on the oxidation of NH₂OH to N₂O using iron (III) as oxidation agent and subsequent quantitative analysis of the resulting N₂O. It is the only available method for this measurement at nanomolar level. Based on this we have developed a new fully automated method by using McIlvin and Casciotti (2010) approach. We have successfully measured the concentration and stable isotope signatures of NH₂OH for both marine and freshwater samples collected from the Otago Harbor and Leith river respectively. We have attained the best conversion ratio of NH₂OH for the natural waters withing 4-10 hours. The recovery rate was 32- 44% between standards of 5-50 nM. This is the first attempt to measure the δ¹⁸O and δ¹⁵N signature of NH₂OH and these results will give more insights in to the nitrification pathways in the oceanic waters during N₂O formation.

In soils, inositol hexaphosphate (phytate) is a recalcitrant form of organic phosphorus (OP). Hence, the ability to quantify phytate turnover in soils and to identify the factors that control it, is crucial for understanding phosphorus (P) cycling in terrestrial ecosystems. However, the lack of multiple stable P isotopes has hindered investigations of phytate dynamics under natural conditions over extended periods. To address this, we propose a novel technique for determining the carbon isotopic composition (δ¹³C) of inositol in phytate at a compound-specific level. For this purpose, phytate was extracted from soil, and purified via ion exchange chromatography, followed by dephosphorylation, derivatization, and analysis using GC-MS and GC-C-IRMS. Pure compounds were also analyzed to assess protocol efficiency, identify isotopic fractionations, and apply isotopic corrections due to derivatization. Phytate extracted from soil samples was identified using GC-MS chromatograms. Replicate analyses of the pure compounds showed that the protocol is highly reproducible. The proposed method was able to identify, quantify, and measure the δ¹³C values of inositol in phytate separately from other sugar molecules such as glucose and fructose. The δ¹³C values showed high reproducibility, with values varying by less than 0.5‰, and with no detectable isotopic fractionation during sample preparation. The δ¹³C values of phytate in soil samples reflected the dominant vegetation type (C₃ or C₄) at the study site. This study introduces a novel approach to measuring the δ¹³C values of inositol in phytate from environmental samples, offering new opportunities for investigating and quantifying OP dynamics using stable carbon isotopes.

Laser based analyzers are widely used in ecohydrology to analyze plant water isotopic compositions (δ¹⁸O and δ²H). The suitability of three different water extraction and isotope equilibration techniques was compared. We examined whether co-extracted organic contaminants (VOCs) affect laser-based isotope measurements and used the instrument’s spectral parameters to post-correct for interfering VOCs.

Cryogenic vacuum extraction, vapor headspace-equilibration in bags and vapor equilibration in-situ probes were used to extract liquid water or water vapor for laser-based isotope analysis (CRDS). Isotope data were calibrated by standards for each method separately. Spectral parameters of the instrument, appropriate to identify spectral interferences with MeOH and CH₄ were identified and used for post-correction. Differences between the three methods and between the origins of the vegetables were identified by statistical tests.

VOCs were found in various amounts for the three different methods. They were co-extracted or co-equilibrated during the different extraction/equilibration methods. Correlation coefficients of isotope data and ‘CH4’ (spectral parameter) were 0.99 or better, however slopes for δ¹⁸O were similar on different instrument types but different for δ²H. Our correction approach improved results and inter-comparability of the methods considerably without knowing the chemical composition of the plant sap.

All three methods were sensitive enough to distinguish and resolve differences in natural abundance. Data quality was improved by the ‘CH4-correction’ approach but could probably be optimized by a plant species-specific correction. Standardized tools for contaminant removal or post-correction applications from manufacturers, in particular for vapor-mode analysis are still needed.

Keywords
water extraction, volatile organic compound, plant water stable isotopes, laser-based isotope analysis (CRDS), spectral interference, organics correction

References
Herbstritt B, Wengeler L, Orlowski N.: Coping with spectral interferences when measuring water
stable isotopes of vegetables. Rapid Commun Mass Spectrom. 2024;38(22):e9907.
doi:10.1002/rcm.9907

Stable sulfur isotopes are crucial for studying geological, environmental, and biological processes, offering key insights into the sulfur cycle and related systems. This work presents a novel method for precise sulfur isotope ratio determination using an elemental analyzer coupled with multi-collector inductively coupled plasma mass spectrometry (EA-MC-ICPMS). The method enables accurate analysis of ³⁴S/³²S and ³³S/³²S ratios, providing a reliable tool for sulfur isotope research.

Validation with international reference materials, including Ag₂S and BaSO₄, demonstrated that δ³⁴S values agreed with reported values within ±0.3‰. The method also achieved a high analytical precision (σ), typically better than 0.3‰ for both δ³³S and δ³⁴S, highlighting its robustness and reliability.

This versatile approach supports the analysis of bulk sulfur samples and addresses complex research questions. It is particularly suited for investigating mass-independent isotope effects in sulfur-containing organic compounds, which help uncover unique chemical pathways and reaction mechanisms. Additionally, it enables identification of sulfur sources and the tracking of biological and chemical transformations in environmental systems.

The EA-MC-ICPMS method delivers accurate and precise δ³⁴S and δ³³S data, making it a valuable tool for geochemical, environmental, and biogeochemical studies. Its capability to analyze isotopic variations in natural and anthropogenic systems ensures its relevance for advancing sulfur isotope research and addressing emerging scientific challenges.

Chamber-based measurements of soil CO₂ flux have long been a key tool in understanding the role of soil in ecosystem carbon cycling. However, bulk CO₂ flux measurements are limited in their ability to inform understanding of the processes contributing to observed fluxes. Estimation of the stable carbon isotope ratio (δ¹³C) of soil-respired CO₂ provides a more comprehensive measurement which can help disentangle the drivers of CO₂ fluxes, to gain a fuller picture of soil carbon dynamics.

Soil CO₂ flux has commonly been calculated using coupled chamber-infrared gas analyser (IRGA) systems. Enhanced cavity absorption analysers are increasingly used to measure CO₂ isotopologues and δ¹³C. However, combining this with chamber flux measurement to estimate soil-respired δ¹³C has traditionally been challenging to perform in situ. When measuring low abundance gases, such as ¹³CO₂, one limitation is the magnitude of H₂O vapour transients during the measurement period. This is because the necessary H₂O corrections implemented in commercial gas analysers introduce potentially large levels of uncertainty. An additional source of uncertainty stems from the tendency in commercial analysers for the reported δ¹³C value to exhibit a non-negligible CO₂ concentration dependency.

This work presents a detailed sensitivity analysis of the uncertainty in estimated soil δ¹³C which can be introduced by both issues, revealing potential for a large magnitude of error in δ¹³C estimates if H₂O and instrument effects are not minimized or properly accounted for. As well, a measurement system is presented which employs a novel, completely passive H₂O vapour equilibration system and simple, automated calibration apparatus which simultaneously reduces implementation complexity and improves the quality of field chamber-based estimates of the δ¹³C of soil-respired CO₂.

Laser-based water stable isotope analyzers have become increasingly popular in the last one and a half decades. Their direct and continuous measurement capabilities make them perfectly suitable for, e.g., in situ isotope assays in environmental settings. However, field-deployed analyzers may be subjected to inconsistent background gas matrices between individual samples and compared to co-measured calibration standards. Therefore, we tested the gas matrix effects by varying the carrier gas proportions of nitrogen (N₂), oxygen (O₂) and carbon dioxide (CO₂) in naturally occurring ranges on three different generations of analyzers employing cavity ring-down spectrometry (Picarro L21xx series). The observed magnitudes of gas matrix effects exceeded commonly accepted measurement uncertainty by two orders of magnitude on all analyzers tested but with very contrasting patterns being displayed by the different generations. For example, oxygen isotope readings increased on all instruments as the carrier gas was transitioned from air to a mixture of 80% N₂ and 20% CO₂, which may be encountered on poorly aerated, organic-rich or contaminated sites. For the same transition, hydrogen isotope readings decreased on the L2120-i while they increased on all other instruments tested. Besides, we also checked for potential drift over the course of eight years on the oldest generation tested (L2120-i). Finally, we determined the consistency of the gas matrix effect on three different analyzers of the same generation (L2130-i). We present a post-measurement routine how the effects can be reliably corrected for and how isotope data would be misinterpreted in dual isotope space when the suggested correction is omitted.

Nitrous oxide (N₂O) is a potent greenhouse gas that contributes to global warming and ozone depletion. Precise measurements of its stable isotopes are essential for understanding nitrogen cycling across soil, ocean, atmospheric, and wastewater systems. In response to the growing demand for high-precision N₂O isotope analysis, Picarro introduces the PI5131-i isotopic and gas concentration analyzer. This instrument enables simultaneous measurements of site-specific isotopic signatures (δ¹⁵N_α, δ¹⁵N_β) along with bulk δ¹⁵N and δ¹⁸O. It is based on the renowned mid-infrared, laser-based cavity ring-down spectrometry (CRDS) technology, but with significant software and hardware enhancements, ensuring a robust and stable measurement platform. Here, we present the analyzer’s capabilities and performance metrics for continuous N₂O isotope measurements, including precision and long-term stability.

Compound-specific stable isotope analysis (CSIA) is a powerful tool for investigating sources and transformation processes of micropollutants and verifying food authenticity. Using liquid chromatography coupled to an isotope ratio mass spectrometer (LC-IRMS), stable carbon isotope signatures in aqueous samples can be measured. However, the LC-IRMS interface requires wet chemical oxidation, where all oxidizable carbon is converted to carbon dioxide (CO₂). This necessitates avoiding organic eluents, buffers, or modifiers to prevent falsified isotope signatures. Consequently, chromatographic separations that rely on organic additives—widely used in conventional LC methods—are incompatible with this technique. This restricts method development to inorganic buffers and temperature adjustments, which can be labor-intensive, particularly for complex samples.

We present a novel approach integrating two-dimensional liquid chromatography (2D-LC) with LC-IRMS to address these challenges. This advanced coupling enables the use of established LC methods involving organic additives in the first dimension. Through heart-cut modulation, the analyte of interest is selectively transferred to the second dimension, where organic solvents are separated prior to oxidation.

Our results demonstrate how this innovative 2D-LC-IRMS system overcomes the limitations of traditional aqueous LC-IRMS methods. We will showcase its ability to handle complex sample matrices and resolve issues like analyte coelution, expanding the range of CSIA applications. This technique not only simplifies method development but also enhances the analytical potential of LC-IRMS in addressing critical environmental and authenticity challenges. Attendees will gain valuable insights into the system’s development, implementation, and broader implications for the future of CSIA.

Landuse and climate change alter hydrological processes and affect drinking water re sources. Practical tools for understanding and quantifying these processes becomes increasingly important, for example to sustainably manage groundwater reservoirs. Analyses of the water isotopes deuterium (δ²H), oxygen (δ¹⁸O), and tritium (³H) provide useful tools, which can be applied to determine groundwater ages, assess bank filtration quantities or to identify mixings of groundwater aquifers.

The objective of the IsoGW-project (2023-2026) is to create nation-wide interpolated isotope maps (i.e., isoscapes) of δ²H, δ¹⁸O and of ³H concentrations in German groundwaters. By establishing such a service for the first time, Germany is following its European partners, which have already published some preliminary work on the matter. Data has been collected from all 16 german federal states authorities, additional samplings, partner laboratories and literature. With more than 4 000 groundwater sampling points and additional surface water and precipitation stations, our work relies on a dataset with an exceptionally high density. Priorities of the data analysis and management include filtering the data to focus on the upper unconfined aquifer and the extension of the database with additional information. These are needed as covariables for the interpolation of the stable isotopes. At the same time, different methods accounting for the ³H half-life of 12.3 years are compared. Overall, we are confident that this isoscapes and the data publication coming along with it will enable systematic large-scale assessments of the water cycle and provide an important work basis for local studies and sustainable groundwater management.

A quantitative assessment of water fluxes and dynamics is crucial for a sustainable use of groundwater ressources. Aside from other key features of the soil water budget the determination of seepage water is of high relevance. Isotope-based methods can be used for this purpose, yet these approaches often apply simplified assumptions regarding water movement in the soil. To which extent, or under which conditions this yields imprecise results requires further reseach, especially in the context of changing system properties and/or changing environmental conditions. Our contribution addresses the impact of soil physical parameters on observed isotope singnatures in seepage water and the resulting estimates of groundwater recharge.

Subterranean estuaries (STE) connect the water and some element cycles on land with the marine coastal area and represent areas of steep biogeochemical gradients that affect, for example, the transport and transformation of carbon and sulfur species. The availability of dissolved sulphate is of particular importance for carbon mineralization. Interactions with the mineral solid phases in the aquifer and sediment contribute to the hydrochemical modulation of the solutions finally leaving the sediment, and minerals act as sources/sinks for elements upon biogeochemical cycling. DynaDeep deals with the processes in a STE characterised by permeable sands in the north of Spiekeroog Island. The beach system, in contact with the southern North Sea, represents a unique and highly dynamic system, previously unexplored in a regional and global context, which enables the interactions between fresh and salt water with a view to current and future system changes. Boreholes and multi-level wells allow seasonal observations of the composition in pore waters as well as sediment sampling. This study presents the results of multi-isotope (COSH) studies on water, dissolved C species and sulphate as well as iron sulphides, combined with the hydrochemical gradients and phase analysis of sedimentary minerals. The water isotope ratios characterise the mixing with fresh water. The C isotope signatures in the DIC identify the degradation of organic matter, but also the corrosion of marine carbonates as sources. The isotope signatures of dissolved sulphate and iron sulphides indicate the origin of reduced phases from microbial sulphate reduction, and potential sulphide oxidation near the surface or an addition of non-marine sulphate sources with decreasing salinities. Micro and phase analytical investigations indicate the presence of calcite, apatite, and barite in the sediments, all probably of in-situ and/or detrital origin. Dolomite, which may act as further alkalinity source is of detrital origin.

The objective of the IsoGW-project funded by the German LURCH initiative is to create a nation-wide interpolated isotope map (i.e. isoscape) of stable water isotope ratios (δ²H and δ¹⁸O) and of tritium (³H) concentrations in the upper unconfined aquifer in Germany. This groundwater dataset is completed with isotope data from precipitation and surface waters, allowing systematical and large-scale assessments of various compartments of the water cycle. δ²H, δ¹⁸O and 3H analyses can also be applied locally to quantify contributions of river water and groundwater where river bank filtration occurs.

Here, three pilotsites have been selected across Germany to investigate the surface water/groundwater interactions in regions where drinking water is gained from river bank filtration. These sites cover different conditions in terms of topography, urban influence, hydrogeology and groundwater extraction. Data was collected via bimonthly samplings over one year at each site. The analyses combine stable isotope measurements with main ions and urban settlement indicators such as gadolinium. Results show good applicability of these isotopes to quantify mixing processes between groundwater and freshly infiltrated river water. More diverging results are obtained when quantifying the travel velocity of the groundwater. Overall, the data obtained so far provided valuable insights in the mixing processes thus supporting that isotopes are good for such assessments, but ideally would necessitate longer time-series to improve accuracy.

Nitrogen in soil is strongly influenced by anthropogenic activities, such as fertilization and atmospheric nitrogen deposition. Recent studies, however, show the possible importance of geogenic organic matter. Geogenic organic matter (also known as fossil, rock or petrogenic organic matter) consists of plant and microbial remains preserved in rocks. While typically inert due to deep isolation, it can be mobilized by human activities such as mining and construction or naturally by weathering. This makes it an active but understudied component of soil systems that enters the decomposer food chain.

Geogenic nitrogen can substantially affect nitrogen availability, which is a key factor shaping terrestrial ecosystems' responses to rising atmospheric CO2 levels. Quantifying this flux is essential for accurately modeling biogeochemical cycles and better understanding terrestrial ecosystem dynamics.

The study is set at post-mining sites in the NW Czech Republic, utilizing a unique chronosequence of spoil heaps with varying ages, developmental stages, and two types of reclamation approaches characterized by different vegetation. This setting allows us to investigate the integration of geogenic organic matter into soil processes and its utilization by microorganisms across the site development. By extracting amino sugars and determining their natural abundance nitrogen isotope ratio, we will gain insights into microbial assimilation of geogenic nitrogen as it is isotopically enriched compared to recent nitrogen sources. We will use a mixing model to assess the contribution of geogenic organic matter and recent nitrogen sources to microbial uptake.

We aim to quantify quantify the amount of geogenic nitrogen assimilated by soil microorganisms, as determined by the isotopic composition of nitrogen in their amino sugars, across the chronosequence and assess how the developmental stage of the sites and the type of reclamation influence this assimilation.

We measured δ¹³C and δ¹⁵N values of plants from herbarium specimen collected between 1870 and 1930, as well as contemporary specimen, collected from the same area or possible similar ecosystems. The specimen represented dry grassland species and wetland species. We found a decrease in δ¹³C values of wetland species which exceeds the shift we could expect to be caused by the Suess effect, which indicates changing water regimes. Our results show an increase by 1.29‰ in δ¹⁵N in wetland plants, and a decrease by 3.03‰ in dry grassland species. This suggests, that wetlands are mainly impacted by organic fertilizer and sewage discharge into aquatic ecosystems, while grasslands have a strong sign of anthropogenic nitrogen deposition and/or artificial fertilizer. However, we found no increase in total nitrogen content in plant biomass. We found no correlation between the size of the shift in δ¹⁵N and the use of artificial fertilizer per hectare in the adjacent area or the cover of agricultural land.

Herbarium specimen are a unique proxy for long-term environmental change. In our case, different nitrogen sources have opposing isotopic footprints. This allows the identification of the dominating source. But in most ecosystems nitrogen input is a mixture of different sources, it seems impossible to calculate the contribution of each source, for example using mixing models.

Mixed and coniferous forests are complex ecosystems with understory vegetation playing a vital yet often overlooked role in their functioning. Among the diverse understory plants are evergreen lycophytes such as Lycopodium annotinum L. (interrupted clubmoss), a protected species in the EU. Assessing long-term ecophysiological processes in this evergreen species is crucial to understand its interactions with its environment.
This study explores seasonal and organ-specific ecophysiological shifts in L. annotinum by using stable nitrogen isotopes (¹⁵N/¹⁴N) as integrators of ecophysiological processes. We hypothesized that ¹⁵N/¹⁴N ratios and nitrogen content (N%) in L. annotinum should vary across seasons and organs while being related with local plant diversity.
Plant specimens were collected in 2021-2022 during different seasons in Lithuania. Plant material was dried, separated into different organs (roots, plagiotropic stems and their leaves, orthotropic stems and their leaves), and ground into powder-like material. ¹⁵N/¹⁴N ratios and N% in bulk biomass were measured by employing isotope ratio mass spectrometry coupled with elemental analysis (EA-IRMS). ¹⁵N/¹⁴N ratios were expressed as δ¹⁵N (‰) in relation with N-AIR.
The observed seasonal and organ-specific differences in nitrogen isotopic composition highlight adaptive ecophysiological responses of L. annotinum. Our results revealed significant seasonal shifts in ¹⁵N/¹⁴N ratios and N% of L. annotinum. Moreover, ¹⁵N/¹⁴N ratios and N% differed among different organs in L. annotinum. On the other hand, no significant relationships between neighbouring plant diversity and ¹⁵N/¹⁴N ratios and N% in L. annotinum were found, suggesting that nitrogen uptake in these plants might be independent from local plant diversity patterns. Our study contributes to understanding nitrogen dynamics in evergreen understory species and provides a foundation for further investigations into the nitrogen balance of L. annotinum.

Harbour porpoise (Phocoena phocoena) populations reside in one of the most heavily used shelf seas worldwide, the North Sea. In the last 200 years impacts occurred from hunting, fishing, shipping, chemical pollution, man-made underwater noise, eutrophication and climate change. Combined impacts caused a massive retreat of the population from the SE North Sea in the 1950’s as well as a remarkable recovery through the late 1990’s to early 2000’s. Both trends cannot be clearly explained using available records, but food base alterations are a likely steering factor. To better understand North Sea harbour porpoise population dynamics, methods assessing long-term ecosystem health are needed, but gauging human impact on the ecosystem requires long-term records of ecosystem function that are extremely difficult to assemble. Here we show that molecular-level isotope measurements of amino acid carbon and nitrogen from harbour porpoise collagen allow for reconstruction of ecosystem status across ~170 years in the North Sea. We found increased reliance on high trophic position (TP) prey items (e.g. gadoids) from 1939 to 1969 which shifted towards lower TP demersal prey (e.g. gobies, sandeel) in 1970-1990. The trend of downward TP predation continued with reliance on smaller, abundant pelagic prey (e.g. herring, sprat, squid) through the 2010’s as well as clear shift from dinoflagellate towards diatom support of the North Sea food web since 2008. These trends reflect a changing food base due to the combination of fisheries’ pressure as well as climate warming in the North Sea and align with long-term regime shifts identified from fishery and plankton community records from the last 60 years. Additionally, porpoise feeding dynamics revealed a sex-based shift towards overlap of amino acid C records indicating development of competition for similar prey items that may explain increased stranding of juvenile males observed on the Dutch coast since 2005.

Orestias chungarensis is a small-bodied fish whose global distribution is limited to a single high-altitude (4520 m) Andean lake (Chungará) located in the Altiplano of northern Chile. Until the late 20^th century O. chungarensis was the only fish species inhabiting the lake. The introduction of rainbow trout at this time led to the loss of Orestias from the River Chungará. Although of elevated conservation concern, little is known regarding Orestias ecology: the few studies conducted have relied on individuals captured from the shallow littoral. Here we analysed multi-tissue stable isotopes (δ¹³C, δ¹⁵N, δ³⁴S) and stomach contents to study the trophic ecology of Orestias captured in different lake habitats.

Stable isotope values showed the existence of two putative groups of Orestias. A discriminant function analysis function supports the separation of two groups with a classification success of 98%. The Orestias included individuals belonging to a putative pelagic group (¹³C-depleted, ¹⁵N-enriched, ³⁴S-depleted) and a benthic /littoral group (¹³C-enriched, ¹⁵N-depleted and ³⁴ S-enriched). Mixing models analysis showed the main contribution to the assimilated diet of the pelagic group Orestias in a short and longer time was pelagic zooplankton and benthic/littoral group amphipods and zooplankton from littoral indicating different foraging habitats.

Our stomach contents results showed that Orestias feed mainly on benthic macroinvertebrates (amphipods, chironomids, and gastropods.). However, the isotopic variation shown by O. chungarensis in Lake Chungará suggests that individuals forage across different habitats over time. Given the remarkable plasticity found in the genus, may reflect the existence of a previously unrecognized ecotype.

Phytoplankton play a key role in biogeochemical cycles, impacting atmospheric and aquatic chemistry, food webs, and water quality. However, it remains challenging to reconstruct past changes in algal community composition, as existing proxies are suitable only for a subset of taxa and/or influenced by degradation. Here, we demonstrate the potential of compound-specific hydrogen isotope ratios (δ²H values) of common algal lipids (fatty acids, phytol, and phytosterols) to serve as (paleo)ecological indicators. We present data from (1) batch cultures of 20 species of algae, (2) large volume mesocosms that were manipulated with nutrient loading and the presence/absence of keystone species, and (3) a one-year time series from Rotsee, a small, eutrophic lake.

In our culturing data, water δ²H values were constant but lipid δ²H values ranged from -455 ‰ to -52 ‰, incorporating variability associated with chemical compound classes and taxonomic groups. Hydrogen isotope offsets among lipids of different compound classes (expressed as δ²H_{Lipid1/Lipid2} values) are independent of source water δ²H values and covary with changes in algal community composition. In our mesocosms and throughout the year in Rotsee, increases in the relative abundance of cyanobacteria and/or green algae relative to other eukaryotes are associated with high δ²H_C16:0/Phytol values. High δ²H_C16:0/Sterol values indicate increased abundance of green algae and dinoflagellates relative to diatoms, while high δ²H_{Sterol/Phytol} values indicate increases in diatom abundance relative to other eukaryotic algae.

We suggest that measuring δ²H values of multiple common lipids from sedimentary records and calculating δ²H_{Lipid1/Lipid2} values can resolve changes in algal community composition from changes in source water isotopes. With an appropriate availability of sedimentary lipids, ideally including source-specific biomarkers limited to a single taxonomic group, this approach permits the reconstruction of both taxonomic variability and hydroclimate from diverse aquatic systems.

Photosynthetic Sacoglossa sea slugs exhibit the remarkable ability to incorporate chloroplasts stolen from their algal prey—a process known as kleptoplasty. These algae organelles are then stored in the cells of the digestive diverticula, where, in some species, they can remain functional up to several months. The longevity of these kleptoplasts varies depending on the sea slug species and the algal donor. However, the extent to which the origin of the chloroplasts influences their contribution to the animal's metabolism remains unclear.

In this study, we incubated three species of sea slugs (Elysia viridis, Elysia timida and Elysia crispata) in seawater enriched with ¹³C-bicarbonate and ¹⁵N-ammonium under both light and dark conditions. The isotopic composition analysis using Isotope Ratio Mass Spectrometry (IRMS) allowed us to assess the incorporation of carbon and nitrogen, mediated by the stolen chloroplasts, into the animal tissues. The impact of the algal chloroplast donor (Bryopsis sp., Acetabularia acetabulum) on carbon fixation and nitrogen assimilation will be discussed, with evidence highlighting the significant role of chloroplast functionality in the metabolism of Sacoglossan sea slugs.

Anisakis simplex larvae, commonly found in marine fish, cause anisakiasis in humans, resulting in gastric to gastro-allergic symptoms. Despite known health risks, the impact of Anisakidae larvae on fish hosts is less understood. This study aimed to investigate this interaction by assessing the feeding strategy of A. simplex. Anisakis larvae were isolated from North Sea Merluccius merluccius tissues (stomach, body cavity, liver and muscle) and were analysed for carbon (δ¹³C) and nitrogen (δ¹⁵N) isotope values. Significant differences in δ¹³C values were found among host tissues, with the liver differing from muscle and stomach tissues. In contrast, no differences were noted for the associated parasites. Additionally, δ¹⁵N values indicated that the host occupied a significantly higher relative trophic position than its parasite. This suggests a lack of direct nutrient transfer from host to parasite, as the parasite would typically exhibit higher stable isotope values than the tissue they feed on. Therefore, A. simplex's stable isotope values might reflect those of its previous host (crustacean and/or small fish), providing insights into diet and movement of the paratenic M. merluccius host. Further research is needed to confirm these findings across different fish species and to explore A. simplex as a proxy for trophic ecology.

Norway spruce (Picea abies (L.) Karst) is a widespread tree species in Austria. Due to its economic importance and planting recommendations in the past, it can now also be found on sites that are close to the limits of suitability. Changing climate conditions affect plant growth and plant performance. Prolonged droughts and rising temperatures reduce the plant’s resistance to pest insects but mild winters result in high insect populations. In recent years we see more regions where water availability is the limiting factor due to more frequent and longer droughts or changing precipitation patterns. Soil has a certain buffer function but only the water pools of the upper layers are accessible for spruce trees as they have shallow roots. Economic losses due to massive pests are becoming more common. To compare Norway spruce’s strategies and resilience capacity we investigated sites all over Austria.

Stable isotope (¹³C,¹⁸O) data in tree rings is a very effective tool to investigate physiological responses to environmental and geomorphological conditions, specifically soil water availability and usage. It acts as an archive that provides insights into individual tree water status, temperature and source water use. We sampled 28 spruce plots all over Austria with 14 different soil types and linked the isotope data of selected dry and wet years with temperature and evaporation data as well as the precipitation amount during the growing season. We assessed radial growth through tree-ring width and intrinsic water-use efficiency (iWUE) for the individual site-specific conditions. This information should support forest management strategies to integrate specific environmental conditions, including soil properties and water status, to effectively mitigate impact of climate extremes and adapt our forests for future scenarios.

Hillside slopes within the Mojave Desert are typically rocky with limited soil development. Here low soil nitrogen (N) contents typify soils between isolated shrubs, with higher d¹⁵N values (~7‰) as denitrification processes exceed N₂ fixation by free-living bacteria. Yet, under shrubs nitrogen contents are higher, creating a phenomenon known as “islands of fertility”. Adult leaf d¹⁵N values are lower (2-3‰). Here we describe leaf d¹⁵N values in seedling, maturing, and adult Encelia farinosa, a common, drought-deciduous Mojave Desert shrub lacking any association with symbiotic N₂-fixing bacteria. As seedlings develop in bare soils and develop canopies over time, leaf d¹⁵N values progressively decrease. It is hypothesized that leaf litter falling below shrubs decomposes, providing a carbon source to support free-living N₂ fixing bacteria and enrich shrub soils in N.

The so-called crucial field is an agricultural test project west of Copenhagen since 2001, where various fertilizers (such as cattle manure, mineral fertilizer and organic household waste) have been applied on different sub-plots to study their long-term effects. Moreover, some sub-plots have been 'retired' and received only minimal fertilization after 2012. To investigate the numerous effects on C and N isotopes, I am analyzing stored fertilizer, soil and grain samples. The main points of investigation are

1. how the isotopic composition of the soil and the grain changes depending on the fertilizer treatment over time (caused by the fertilizer delta values or alternated soil processes due to fertilization);
2. how the isotopic delta values of the fertilizers themselves have changed since the beginning of the experiment and
3. how long the soil isotopic delta values take to reach pre-experimental values after 'retirement'.

In addition, it is intended to do GHG-flux measurements to investigate emissions of the sub-plots and examine whether high emissions can be linked to high delta values in the soil.

Earth atmospheric dioxygen is mainly produced by biosphere photosynthesis, biosphere respiration being one of its main consumers. Atmospheric O₂ evolution is thus linked to global biosphere productivity.

In ice cores we extract air from bubbles to study past atmosphere composition. However, as O₂ concentration in air bubbles is affected by close-off processes, it is difficult to reconstruct its past atmospheric variations. In turn, O₂ isotopic composition (δ¹⁸O and δ ¹⁷O), is also modified by biological processes, but less influenced by close-off processes so this tracer should provide useful information on past biosphere activity.

Quantitative interpretation of O₂ past isotopic composition relies on robust estimate of oxygen fractionation coefficients associated with photosynthesis and respiration. In the past decades, some determinations of these biological fractionation coefficients were performed in uncontrolled large-scale environments or at the micro-organisms scale in conditions very different from the natural environment. There are thus inconsistencies in previous determinations of the O₂ fractionation coefficients limiting the interpretation of δ¹⁸O and δ ¹⁷O of O₂.

In order to come up with coherent estimates of oxygen fractionation coefficients during biological processes, we developed closed biological chambers as a biosphere replica, with controlled environment parameters (light, temperature, CO₂ concentration), which were used in combination with a newly designed optical spectrometer for continuous measurements of O₂ concentration and of its isotopic composition.

In this presentation, we show the design and realisation of our aquatic biological chambers as well as the associated development of the multiplexing system to be able to run parallel experiments with the same environmental conditions. Then, we show the results obtained for light and dark periods, and the corresponding fractionation coefficients calculated for photosynthesis and respiration. Finally, we use the newly determined fractionation coefficients to improve interpretation of O₂ δ¹⁸O records in ice cores air bubbles.

Root carbon has been shown to be one of the most dominant forms of soils carbon inputs in agricultural systems. New paradigms in soil organic matter theory suggest the role of root derived soil carbon in its influence on carbon storage and decomposition may have been overlooked.

Mixed species systems are currently gaining traction Europe providing opportunities for sustainable intensification of agriculture and other ecosystem-service co-benefits. In Mix&Max-Root-C we aimed to gain a management-oriented understanding of the effect of mixed-species root systems on carbon flows and organic matter accumulation across a European gradient

We conducted pan-European in-situ field experiments to measure decomposition rates and the fate of carbon using labelled litter. Treatments included:((i) monoculture (1 species), (ii) low diversity (2-4 different plant species in the mix culture) and (iii) high diversity (≥ 5 different plant species in the mix culture)). The goal was to determine the impact of increased plant diversity organic matter breakdown to develop a trans-European decomposition index. Using a hub spoke design, a common ¹³C-labelled material was supplied to each participant and was mixed in a similar manner with the soil from the local treatments, packed into mesh bags and buried in the treatment plots. This was done at the start of the growing season and excavated at the end of the growing season and returned to Tulln for a suite of carbon pool analysis.

The experiment, covered eight sites, diverse climates and cropping systems. We tested the null hypothesis that increased plant diversity does not increase the decomposition rate in the field. Initial results suggest that decomposition rates were 40-65% across sites and that diverse cover-cropping mixtures did not lead to lower decomposition rates. These data and results will be used to guide model predictions.

Keywords* Mixed cropping, Diversity, ¹³C labelled, Maize litter, Monoculture, Carbon

The priming effect (PE) refers to the enhanced remineralization of recalcitrant organic carbon (OC) driven by the respiration of labile OC, potentially increasing CO₂ fluxes from aquatic ecosystems. Investigating PE dynamics requires precise methods to trace the fate of organic matter sources and their transformation within sedimentary environments. In this study, we apply compound-specific stable isotope analysis (CSIA) within an isotopic mixing model framework to quantify the contribution of priming to the degradation of hydrocarbons (C₁₅–C₃₀) in coastal sediments. By integrating stable isotope data with concentration profiling, we assess how the remineralization of recalcitrant OC is influenced by fresh organic inputs. To achieve this, we conducted controlled microcosm experiments using coastal sediments (δ¹³C_bulk = −25.26 ± 0.06 ‰, 1.63 ± 0.07% OC) spiked with isotopically distinct marine and terrestrial OC sources such as Nannochloropsis phytoplankton (δ¹³C = −43.18 ± 0.31 ‰) and C₄ corn leaves (δ¹³C = −13.90 ± 0.09 ‰). The temporal variations in isotopic composition of respired OC were tracked by difference through CSIA at multiple time points across 30 microcosms, allowing us to resolve source-specific degradation pathways. Our results demonstrate that stable isotope values effectively capture the acceleration of recalcitrant OC remineralization following the addition of fresh, labile OC. The protein/peptide-rich Nannochloropsis and polysaccharide-rich corn leaves both enhanced OC degradation compared to sediment incubated alone under identical conditions. Additionally, CSIA revealed a decline in fractionation over time, indicating stabilization of the degradation process within the sedimentary matrix. These findings highlight the power of CSIA in isotopic mixing models as a robust tool for quantifying OC transformations and source contributions in complex sedimentary systems. Understanding these dynamics is crucial for refining global carbon cycle models, particularly in the context of increasing atmospheric CO₂ levels, eutrophication, and enhanced terrestrial OC fluxes to marine environments.

The biofilter, represented by a microbially active biochar filter, has been explored as a potential tool for groundwater remediation, offering the prospect of self-renewal of the sorbent's sorption capacity through the microbial degradation of introduced contaminants, as well as the utilisation of eco-friendly filter materials, such as biochar.

Utilising tetrachloroethene (PCE, C₂Cl₄), a prominent groundwater contaminant, as a model substance, a comprehensive experimental approach was adopted, encompassing batch and column studies in the laboratory. These experiments involved the use of different biochars and activated carbon in conjunction with two organohalide respiring microbial communities. The fate and behaviour of PCE and its daughter products were monitored using a combination of compound specific isotope analysis (CSIA) and sorption/desorption analysis. Additionally, molecular biological and biochemical methods were employed to analyse the composition and proliferation of microbes.

The results obtained provided compelling evidence for the complete dechlorination of PCE and its daughter products to ethene. Furthermore, the results indicated that microorganisms may proliferate more readily on biochar compared to activated carbon. In essence, a functional biofilter requires both an adaptable microbial community and a corresponding filter medium. The outcome of a recently completed project will be presented, along with the plans for a follow-up project that is currently in its initial stages. The objective of this subsequent project is to enhance the biofilter approach to a technical level and to conduct a pilot study in a field setting.

Soil N₂O emissions are laborious and difficult to quantify and upscale to larger areas due to high spatiotemporal variations. Nitrogen isotopes are gaining increasing attention as a potential tool for improving landscape-scale assessments of N₂O emissions. This is because biochemical processes in the nitrogen (N) cycle discriminate against the heavier N isotope, ¹⁵N, resulting in distinct isotopic signatures in the product and residual substrate. Maps of the spatial distribution of isotopes, isoscapes, offer a promising approach for identifying spatial variability in N processes. However, integrating soil N isotopes into ecosystem models requires better understanding of the drivers behind their spatial variation.

Moist depressions in crop fields are known to be N₂O hotspots and represent key sites for exploring soil ¹⁵N patterns associated with N₂O emissions. This study focused on spatial patterns of soil N isotopes across two rolling fields in Zealand, Denmark. A total of 148 topsoil samples (0-10 cm) were collected along multiple topographic transects and analyzed for soil N and carbon content and isotopic composition, with soil texture (at selected locations). Correlations between parameters were assessed using Spearman’s Rank Correlation Coefficient.

Emitted N₂O is expected to be depleted in ¹⁵N relative to its source substrate. Based on this, we hypothesized that soil N in N₂O hotspots (moist depressions) would show higher ¹⁵N enrichment compared to adjacent soils, due to greater losses of ¹⁵N depleted N. Contrary to this hypothesis, results showed a significant positive correlation between soil δ¹⁵N and elevation, with the lowest δ¹⁵N values observed in depressions. This suggests that processes other than N₂O emissions play an important role in shaping the isotopic patterns. The study revealed substantial spatial variability in soil δ¹⁵N (3.8-9.8‰) underscoring the importance of sample location in determining isotope fractionation patterns and highlighting the need for further investigation to refine application of isoscapes.

The present study focuses on how anthropogenic activities affected the carbon cycle and biological productivity in oligotrophic lake during the last 130 years. We analysed stable carbon isotopes and radiocarbon (¹⁴C) distribution in two organic sediment fractions: alkali-soluble and alkali-insoluble was performed in lake sediments. Additionally, diatom analysis and organic matter content in sediments was performed. Over 130 years, the reservoir age (RA) in both organic sediment fractions in this ecosystem changed by 872.4 ± 80 years. In this lake ecosystem, during periods when the water level was maintained more or less constant (1885-1932 and after 1985), the ¹⁴C specific activity values in both sediment fractions remained very similar, varying in the range of 1 pMC. Any changes in the plankton community that affect the stable carbon isotope ratio in the alkali-soluble fraction did not affect the redistribution of ¹⁴C between both organic sediment fractions. Differences in RA up to 600 years changes between fractions were associated with the input of allochthonous substances into the lake.

Clumped isotopes of methane (CH₄), specifically ∆¹³CH₃D and ∆¹²CH₂D₂, provide additional information to constrain its sources and sink processes. These isotopes complement interpretations of CH₄ provenance based on bulk isotopes. However, interpreting the origin of CH₄ using isotopes becomes challenging when the isotopic signature is altered by post-generation processes. In this study, we measured, for the first time, the bulk and clumped isotopic composition of sub-glacial CH₄ samples. These samples were collected from the air-filled headspace of the glacier portal (ice cave) at the edge of the Isunnguata Sermia glacier (ISG), located at the western margin of the Greenland ice sheet (GrIS). Our goal was to identify the processes underlying the sub-glacial production and potential processing of CH₄. The ∆¹³CH₃D and ∆¹²CH₂D₂ values of the samples measured in this study are 3.7 ± 0.3 ‰ and 39.7 ± 2.0 ‰, respectively (95 % confidence interval). The ∆¹²CH₂D₂ values are close to those of atmospheric CH₄. The elevated ∆¹²CH₂D₂ values can be attributed to the alteration of the source’s isotope signal by aerobic oxidation. This conclusion is supported by previous studies at this site, which reported the presence of methanotrophic bacteria and dissolved oxygen close to saturation in the meltwater. Our results confirm that the correlation between ∆¹³CH₃D and ∆¹²CH₂D₂ is a useful tool for deciphering oxidation pathways. Our results support the inference that aerobic CH₄ oxidation can strongly modify the ∆¹²CH₂D₂ isotope signal, which must be considered when determining the source signatures of environmental samples.

Information on fungal contribution to nitrous oxide (N₂O) emission from denitrification in soil is scarce. However, some fungal species were identified to produce substantial amounts of N₂O in controlled experiments and several soil incubation experiments even suggest that fungi may dominate N₂O emissions compared to bacteria. The present study aimed to answer the question, whether it is possible to identify soils with substantial contribution of fungal denitrification on N₂O emissions.

Repacked samples of eight arable and grassland soils and one artificial compost from different locations were incubated in a fully automated incubation system to investigate N₂O emissions and its origin. Two treatments without and with homogeneous straw incorporation were compared to vary the carbon availability and sources. Nitrate was added to allow denitrification. Three phases were established for a period of one week each, with varying experimental conditions. Initially, the water saturation was adjusted to 60% of water filled pore space (WFPS) with oxic conditions, followed by a second oxic phase with water saturation raised to 80% WFPS. During a third phase the oxygen was removed from the system. CO₂ and N₂O fluxes were measured continuously and gas samples for isotopic analysis of N₂O were collected manually at selected time points.

CO₂ fluxes decreased with the onset of a new phase, while CO₂ fluxes of treatments with straw exceeded the ones without straw. In contrast to that, N₂O fluxes increased from phase 1 to 3. Applying the FRAME model, which is used for N₂O source partitioning based on isotopic data taking into account mixing and fractionation effects, revealed bacterial denitrification or nitrifier denitrification being the dominant N₂O producing pathways. Thus, fungal denitrification played a minor role in N₂O production, although isotopic data of some soils indicated a fungal contribution by denitrification up to 0.4 %.

Rogoznica Lake – Dragon Eye (RL) is a highly eutrophic, stratified euxinic marine system on the Adriatic coast, significantly affected by environmental changes, including water column warming, deoxygenation, accumulation of toxic sulfides and ammonia, and an increased frequency of anoxic holomictic events. These factors strongly influence the dynamics and properties of organic matter (OM).

Long-term data on OM reveal the accumulation of particulate (POC) and dissolved (DOC) organic carbon, particularly in the anoxic hypolimnion, with DOC ranging from 0.809 to 7.16 mg L⁻¹ and POC from 0.572 to 10.5 mg L⁻¹. Qualitative changes in OM are assessed using DOC-normalized surfactant activity (NSA = SAS/DOC), determined by monitoring DOC levels and its surface activity, i.e., surface-active substances (SAS). Further characterization of OM is conducted through the analysis of stable isotopes of light elements (¹³C/¹²C, ¹⁵N/¹⁴N, ³⁴S/³²S) and the C:N ratio in the POC fraction, providing insights into phytoplankton community structure, OM sources and origins, and its role in biogeochemical cycles. These analyses are performed using isotope-ratio mass spectrometry (IRMS).

Preliminary seasonal isotope data (δ¹³C, δ¹⁵N, δ³⁴S) from the RL water column reveal values ranging from -20.89‰ to -32.30‰ for δ¹³C, -8.07‰ to 7.24‰ for δ¹⁵N, and -10.83‰ to 21.74‰ for δ³⁴S, with noticeable seasonal shifts along the water column driven by variable physico-chemical parameters (salinity, oxygen saturation, atmospheric deposition). The position of the chemocline, which separates the surface oxic and bottom anoxic water layers, is distinctly observable in the δ³⁴S values, showing pronounced seasonality.

These findings indicate that OM in the RL water column is predominantly autochthonous, largely derived from phytoplankton activity, with occasional allochthonous inputs, as suggested by the C:N ratio (ranging from 1.09 to 6.51), contributing to eutrophication. During holomictic events, when the water column becomes entirely mixed and anoxic, isotope ratios suggest a significant presence of bacterially-produced OM.

Biochar as an alternative filter to activated carbon (AC) was tested for the removal of tetrachloroethene (PCE) from contaminated groundwater by means of sorption and biodegradation by organo-halide respiring bacteria in batch and column experiments. Besides quantifying PCE degradation and identifying the relevant bacteria, microbial biomass and its carbon isotope composition were determined using microbial phospholipid fatty acids (¹³C-PLFA) analysis in order to allow a quantitative and functional observation of the microbial community.

PLFA analysis revealed that microorganisms and also those groups that can be assigned to the PCE-degrading organisms preferentially colonize biochar, while AC is avoided possibly due to the higher PCE sorption capacity of the activated charcoal and hence lower bioavailability of PCE. If pure PCE was added to AC to increase its concentration in the liquid phase, the microbial colonization of the AC still remained low, while the amount of floating microbial biomass increased. The carbon isotope value of the microorganisms (¹³C PLFA) indicated the use of alternative carbon sources besides added lactate and acetate and/or the presence of strongly isotope-fractionating biochemical processes. This effect was especially strong on and under the presence of biochar and in gram positive bacteria. Besides incorporation of biochar, also the use of ethene (PCE degradation product) and methane (derived from methanogenesis) might be considered as carbon source.

Carbon cycling in this specific environment still need to be verified by further research. Nonetheless, we can conclude that it is advisable to choose the filter material not only on the basis of the sorption capacity, but above all on the synergy effects that leads to a permanently active microbial community and an extension of the filter life due to the continuous and complete degradation.

Mineral formation from hard water creeks is sensitive to variations of physico-chemical boundary conditions, including climate change. Groundwaters saturated in CaCO₃ and supersaturated in CO₂ emerge from springs, degas CO₂, and after exceeding a critical supersaturation carbonate starts to precipitate. Lithology-dependent geogenic and anthropogenic loads with sulfate are observed, too. Trace element and stable isotope partitioning upon crystallization are controlled by non-equilibrium processes. Both may be used to estimate element sources, subterrestrial weathering, and surface precipitation, and degassing rates. The fresh waters will discharge to rivers or coastal waters and impact their buffer capacity with consequences for green-house gas levels and surface water acidification.

Examples for hard water creeks in the temperate climate zone showing recent active sinter formation were chosen from Mühlengrund and Kollicker Bach (Rügen), Nohn (Eifel), and Westerhof (Harz foreland). Westerhof has a well-documented long research history dating back to the 60s that allowing for anthropogenic impact identification.

The hydro- and stable isotope (H, C, O, S) geochemistry of dissolved and solid phases were analyzed. Carbonate precipitation rates are estimated from trace element distribution.

The systems are characterized by a two-stage development of the surface waters: An induction period with sol CO₂ degassing, and a stage where calcite formation drives further degassing. The liberation of CO₂ is associated with an enrichment of the heavy carbon isotope in the remaining dissolved inorganic carbon. Trace element discrimination between the aqueous solutions and fresh calcite precipitates along the flow path are compared of distribution coefficients derived in previous experimental studies and seasonal and spatial resolved field calcite-precipitation rates. Stable isotope signatures (H, C, O, S) allow water and element source identification and mechanistic interpretation of the processes controlling the carbonate system. Carbonate-associated-sulfate in sinter is a new proxy that allows for a source characterization of sulfate in fresh water carbonates.

Coastal ecosystems are dynamic regions rich in diverse biological and geochemical interactions. However, major gaps exist in our knowledge of these biogeochemical processes and the factors regulating their relative importance. The study of nitrogen and carbon cycling in aquatic systems is important for understanding these biogeochemical processes and the impact of human and natural inputs to the ecosystems. Nitrous oxide (N₂O) and methane (CH₄) have important roles in the nitrogen and carbon biogeochemical processes as they are produced or externally introduced and then cycled within coastal and ocean environments. The gaps in our understanding of the distribution and dynamics of the underlying processes controlling these N and C cycles can be filled with the development and deployment of high-resolution spatial-temporal measurement methods.

We have developed a real-time, in situ system to quantify dissolved greenhouse gases (N₂O and CH₄ and their isotopologues) in aquatic ecosystems including coastal wetlands. This measurement system consists of i) an array of permeable, hydrophobic probes to passively sample dissolved gases; ii) a collection protocol for efficiently transferring dissolved gases without isotopic fractionation; and iii) an interface of the probe array and sampling system with an Aerodyne tunable infrared laser direct absorption spectrometer (TILDAS). The TILDAS provides real time determination of concentration and isotopic abundances of N₂O and CH₄.

In laboratory studies, we have compared dissolved gases extracted from a variety of collected water samples including different tap water sources, ocean water, and wetland “swamp” water. A field deployment of the system is planned for Spring 2025 at a coastal site on Cape Cod, MA with an array of 16 probes to be deployed in the coastal ecosystem. Dissolved N₂O and CH₄ concentrations and isotopic signatures will be measured continuously over several weeks at the site. Preliminary results and the biogeochemical implications will be discussed.

Sulfamethoxazole (SMX) is a frequently detected sulfonamide antibiotic in surface and groundwater, raising environmental concerns about its fate. Oxidative treatments, such as persulfate application, are commonly used for micropollutant removal. To investigate and differentiate SMX transformation by various radicals from other SMX dissipation processes, SMX transformation experiments were conducted using heat-activated persulfate at pH 3, 7, and 10. SMX hydroxylamine (TP269a) and TP178 were identified as the dominant transformation products across all pH levels. The exclusive formation of 4-nitroso-SMX, 4-nitro-SMX, and TP518 at pH 3 highlighted the role of SO₄^•– in attacking the amino group. At pH 7 and pH 10, 3A5MI emerged as the dominant TP. Normal carbon isotope fractionation (Δδ¹³C from 1.9‰ to 2.3‰), with consistent isotopic values across pH levels, was attributed to the formation of TP178, which involves C-S bond cleavage. An inverse nitrogen isotope fractionation at pH 3 (ε_N = +0.68 ± 0.11‰) was linked to SO₄^•–-induced single-electron transfer, leading to the formation of N-centered SMX radicals. Conversely, normal nitrogen isotope fractionation at pH 10 (ε_N = −0.27 ± 0.04‰), was associated with multiple bond cleavages, including N-H bond cleavage initiated by H abstraction through HO• and N-S bond cleavage leading to the formation of 3A5MI. The inverse nitrogen isotope fractionation observed at pH 7 indicated that the dominant pathway involved SO₄^•– reactions, accounting for 76% of the overall transformation. Overall, the results highlight the potential of CSIA to elucidate SMX persulfate oxidation pathways and evaluating the natural attenuation of SMX through radical reactions in aquatic systems.

Quantification of N₂ and N₂O fluxes from agricultural ecosystems is needed to investigate gaseous fertilizer losses and greenhouse gas fluxes. The non-random distribution of N₂ and N₂O isotopologues evolved from ¹⁵N-labelled N pools is used to quantify the ¹⁵N enrichment of the N pool producing N₂ and N₂O (ap values) and the pool-derived fluxes (fp values). Accuracy and precision for ap and fp depends on the flux strength and the limit of detection (LOD) of the IRMS approach, i.e. LOD for ²⁹R and ³⁰R for N₂ and ⁴⁵R and ⁴⁶R for N₂O. For robust evaluation of accuracy, standard gases are needed containing defined contents of single and double-substituted N₂ and N₂O.

Sensitivity for N₂ fluxes has been improved by establishing N₂-depleted atmospheres in the lab and in the field. Combining ¹⁵N tracing and natural abundance approaches can be used to distinguish heterotrophic denitrification and nitrifier denitrification, but this requires precise determination of fp, which can be subject to bias by inaccurate instrumental calibration.

We developed and analyzed multiple isotopic standard gases containing defined concentrations of single and double substituted N₂ and N₂O. N₂ background concentrations were either atmospheric or similar to the N₂ -depleted atmospheres (0.5 or 2% N₂) of respective experiments. Stock mixtures were designed as part of the DASIM project where premixtures were produced by Thünen and stock mixtures manufactured by Westfalen AG, Hörstel, Germany. Moreover, stock mixtures for N₂O were further diluted with Helium and unlabeled N₂O to obtain defined mixtures close to the LOD of the IRMS. To optimize drift correction of the IRMS, we extended the system to enable alternating sample and standard gas injections.

We will explain the production of the manufactured standard gases and show first results of analysis in comparison with ideal values.

Carbon, nitrogen, and sulfur isotope signatures of bone collagen and dentine have been used as powerful tools to interpret diet and population mobility in archeological studies [1]. However, owing to extremely low S concentration (typically <0.3 wt%) and limited sample size of archeological samples, precise δ³⁴S measurements can be difficult, and simultaneous δ¹⁵N, δ¹³C and δ³⁴S analyses are even more challenging.

Thermo Scientific™ EA IsoLink™ IRMS system provides a solution to this challenge with enhanced hardware components and an optimized analytical method that improve S sensitivity by 3 times and suppress S memory effect, thus achieving high precision S isotope data with only 3-5 μg S per analysis.

Here we present sequential δ¹⁵N, δ¹³C and δ³⁴S measurements of well-certified collagen reference materials USGS88 and USGS89, and in-house standards Bovine Gelatine, Porcine Gelatine and Fish Gelatine using this optimized setup to assess SO₂ memory effect and the performance of NCS isotope measurements.

Replicates of these collagen standards with distinct δ³⁴S (~1.5mg, equivalent to 4-7 μg S) were run in sequences with one or two empty runs added in between as a cleaning procedure. Data showed great reproducibility with an uncertainty (1SD) of ≤ 0.3 ‰ on δ³⁴S and minimal SO₂ carry-over.

The quality control standard bovine gelatine was separately measured with USGS88 and USGS89 to check the sequential NCS isotope measurements. Bovine gelatine analyses (~1.5 mg, equivalent to 248 μg N, 669 μg C, 4 μg S) in two sequences gave averages values of δ¹⁵N_AIR = 7.3 ± 0.2 ‰, δ¹³C_VPDB = -17.1 ± 0.1 ‰ and δ³⁴S_VCDT = 6.8 ± 0.1 ‰ (n=27), after scale correction using USGS88 and USGS89. The measurement precision and accuracy on δ¹⁵N, δ¹³C and δ³⁴S of bovine gelatine are well within the expected values [1].

[1] Sayle et al., RCM 33 (2019) 1258-1266.