Assessing the effects of fresh, air-drying and freezing preservation methods on enzymatic activities from fluvial ecosystems

Extracellular enzymes play a major role in ecosystem functioning and global biogeochemical cycles, driving the degradation, transformation, and mineralisation of both organic and inorganic compounds. Enzyme activities are commonly analysed in fluvial ecosystems using fresh samples shortly after collection. Yet, how long-term preservation methods, such as freezing and air-drying storage, influence enzyme activity and the interpretation of ecological patterns remains poorly understood in fluvial ecosystems. This knowledge is essential as the current ‘gold standard’ using fresh samples restricts the spatial scope and sample size of freshwater studies, complicating large-scale or global assessments. Here, we investigated the effects of fresh, freezing and air-drying preservation methods on the activities of four enzymes representing nitrogen, carbon, and phosphorus cycling in fluvial ecosystems during both wet and dry phases. To do this, we sampled 10 streams with contrasting water availability in southern Spain: five during their aquatic phase and five during their terrestrial/dry phase. Five replicate samples of streambed sediment were collected per stream, with each sample analysed for four enzymes (β-glucosidase, β-D-cellobiosidase, N-acetyl-β-glucosaminidase and phosphatase) after undergoing one of three storage methods: fresh, frozen (−20°C), and air-dried at room temperature (20°C–25°C). We then quantified the bias of each preservation method relative to freshly analysed samples under both wet and dry conditions, and whether preservation influenced the detection of hydrological regime-related differences in enzymatic activities. We further assessed whether enzyme activity values estimated from air-dried and frozen samples could accurately reflect those measured in fresh samples. Using fresh sediment analyses as a benchmark, our results showed that air-drying introduced less bias than freezing when estimating extracellular enzyme activity in streambed sediments across both wet and dry phases. Despite these biases, fresh, air-dried and frozen samples revealed similar enzymatic response patterns to hydrological variation. Specifically, β-glucosidase and β-D-cellobiosidase activities increased under dry conditions, while N-acetyl-β-glucosaminidase and phosphatase activities remained stable across hydrological phases. Enzyme activities measured in air-dried and frozen samples generally showed moderate to high explanatory power for those measured in fresh sediments, except for phosphatase in air-dried samples. Our study demonstrates that, despite producing different biases, both air-drying and freezing treatments are generally able to accurately predict enzyme activity in fresh sediments and consistently capture variations between contrasting abiotic conditions. These findings provide novel evidence supporting the use of air-drying and freezing as viable methods for preserving streambed sediment samples. These methods open up new opportunities for broader, more comprehensive large-scale studies on fluvial ecosystems in the context of global change.