ORBITRAP-MS FOR δ¹⁵N NITRATE MEASUREMENT: INVESTIGATING NITROGEN DYNAMICS IN THE RIO NEGRO WATERS

Introduction

Nitrogen isotopes, particularly δ¹⁵N, are valuable indicators of biogeochemical processes in natural aquatic environments, offering insights into the sources and transformations of nitrogen compounds (Denk et al., 2017). In river systems like the Amazon basin, nitrogen cycling is driven by a complex interplay of biological and physical processes, including microbial activity, organic dissolved matter, and hydrological variations. The Amazon basin is an ideal natural laboratory for investigating these processes due to its vast scale, high biodiversity, and dynamic hydrology (Choisnard et al., 2024). Despite its significance, high-resolution isotopic studies of nitrogen in the Amazon’s aquatic systems remain scarce. Traditional isotopic studies are conducted using the isotope ratio mass spectrometry (IRMS), however, requires analyte the conversion into simple molecular gases, limiting site-specific analysis on complex compounds. Orbitrap mass spectrometry overcomes this by directly measuring isotope ratios in complex compounds, preserving position-specific data and offering high-resolution mass measurements (Eiler et al., 2017).

This study aimed to evaluate the nitrogen isotopic composition of nitrate (δ¹⁵N) in water samples collected from the Amazon basin using Orbitrap mass spectrometry. The primary objective was to establish a rapid, sensitive, and reliable analytical protocol for δ¹⁵N determination, minimizing sample preparation and enabling detailed assessment of nitrogen cycling in this critical ecosystem. The findings contribute to the growing understanding of nitrogen dynamics in tropical aquatic systems and provide a foundation for future geochemical and environmental monitoring efforts.

Experimental

Sampling was conducted in the Rio Negro region, Amazonas, at specific points from P1 to P9 (Figure 1), to assess the nitrogen isotopic composition (δ¹⁵N) of nitrate in the collected water samples. The samples were initially subjected to SpeedVac drying to concentrate dissolved nitrate, and the resulting dry residue was resuspended in methanol for isotopic analysis.

Analytical measurements were performed using an Orbitrap Exploris 240 MS mass spectrometer, operated in dual inlet configuration. This mode allows alternation between sample and reference standards, enhancing measurement accuracy. The reference standard used was USGS 32 nitrate with a δ¹⁵N value of +180‰. The system was set to operate in negative ion mode, targeting the nitrate ion (m/z 61.2-64.5) as the analyte of interest. The flow rate was adjusted to 5 µL/min.

Injection sequences were programmed in alternating blocks of seven injections for reference and sample solutions, ensuring robust precision and reproducibility by accounting for instrumental drift and variability. This procedure was adopted to optimize the analysis, ensuring correction for any data fluctuations and maintaining consistency in measurements throughout the experiment.

Data processing was conducted using IsotoPy, a custom Python-based software developed by our team, specifically designed to enhance the precision of isotope ratio measurements by correcting for instrumental biases and drift.

Results and Discussion

The nitrogen isotopic compositions (δ¹⁵N) of nitrate for the water samples collected from nine sampling points (P1 to P9) along the Rio Negro exhibited significant variability, ranging from +22‰ to +48‰ (Figure 2). These findings suggest a complex interplay of environmental and analytical factors influencing the δ¹⁵N values in the region.

The observed spatial variability in δ¹⁵N values across sampling sites likely reflects heterogeneity in local microbial activity, redox conditions, and hydrological dynamics. Areas with higher organic matter content, lower oxygen availability, or slower water flow tend to promote microbial processes that fractionate nitrogen isotopes more strongly, resulting in higher δ¹⁵N values (SHE et al., 2024). Seasonal changes in the Amazon basin, such as flooding events, could further modulate these isotopic patterns by altering nutrient availability and redox gradients. Additionally, δ¹⁵N values can also be influenced by the presence of specific nitrate sources with higher δ¹⁵N values, such as anthropogenic nitrogen (e.g., fertilizers, sewage). These sources, when present in the river system, could significantly elevate the δ¹⁵N in the water samples, leading to the higher values observed at some sampling points (Chen et al., 2024).

On the other hand, one important aspect to consider is the matrix effect, as the samples were not subjected to any purification process prior to analysis. The absence of purification means that there could have been interference from other nitrogenous compounds, such as ammonia, nitrites, or organic matter dissolved in the water. These compounds may have affected the accurate measurement of δ¹⁵N by introducing additional sources of nitrogen or altering the isotopic composition of the nitrate. This could have led to the observed enrichment of δ¹⁵N in some of the samples, especially if contamination occurred from sources such as fertilizers or wastewater, which are typically enriched in ¹⁵N.

Despite the potential interference from matrix effects and the absence of sample purification, the δ¹⁵N values observed still allowed for the differentiation of the sampling points. The significant variation in isotopic ratios across the different points demonstrates the ability to distinguish between locations based on their isotopic signatures. This suggests that, even with the analytical limitations, the data still reflect spatial differences in nitrogen cycling within the Rio Negro River system.

Given the potential for interference from matrix effects and the lack of purification, further studies with purified samples and more rigorous control over analytical procedures would be necessary to more accurately assess the δ¹⁵N values in the Rio Negro and determine the sources of enriched nitrate.

Conclusions

This study enhances our understanding of nitrogen cycling in the Amazon basin by analyzing δ¹⁵N values of nitrate in the Rio Negro. The findings suggest that nitrogen dynamics are influenced by both natural processes, such as microbial activity and hydrological conditions, and anthropogenic sources like fertilizers. Despite potential interference from matrix effects and lack of sample purification, distinct isotopic signatures were observed across sampling points, reflecting spatial variability in nitrogen sources. These results underscore the complexity of nitrogen cycling in tropical ecosystems and highlight the need for further research with purified samples to refine measurements and better assess environmental changes in the region.

Acknowledgements

The authors acknowledge the financial support from Petróleo Brasileiro SA-Petrobras, CAPES, and CNPq.