Distinct dynamics in mountain watersheds: Exploring mercury and microplastic pollution-Unraveling the influence of atmospheric deposition, human activities, and hydrology.

The intensification of human activities all around the globe has led to the spread of micropollutants in high-mountain freshwater environments. We therefore aimed to assess the geospatial distribution and determine the potential sources of (total-) mercury (THg) and microplastics (MPs) in mountain freshwater ecosystems. To do so, we analyzed THg and MP concentrations in brown trout, biofilm, and sediments from lotic and lentic ecosystems in the Pyrenees - all subjected to different types of human pressure. Additionally, we assessed the potential impacts of these pollutants on fish, and explored the bioindication capacity of brown trout (Salmo trutta fario) and biofilm regarding THg and MP pollution. For the first time, we measured concentrations of MPs trapped in the matrix of freshwater biofilm. Our results suggest that THg in the Pyrenees might be explained by both legacy (regional) and distant sources, in combination with environmental characteristics such as the presence of peatlands or streamwater physicochemistry, while MPs in fish are linked to recent local pollution sources such as single-use plastics. In contrast, MPs in biofilm matrix and sediments indicate a combination of distant (i.e., atmospheric deposition) and recent local pollution sources. Moreover, hydrodynamics and plastic density likely control MP distribution in rivers. Based on Fulton's condition factor, we also found that higher THg concentrations caused a negative impact on fish health (K < 1), while no impact of MPs could be seen. Therefore, we suggest that brown trout and biofilm can serve as bioindicators of atmospheric deposition of THg in high-altitude lakes and that biofilm is a reliable bioindicator to assess MP pollution in remote environments. Brown trout may also act as a bioindicator of MP pollution, but only efficiently in more polluted areas.

Detection limits are central to improve reporting standards when using Nile red for microplastic quantification.

Beyond simple identification of either the presence or absence of microplastic particles in the environment, quantitative accuracy has been criticised as being neither comparable nor reproducible. This is, in part, due to difficulties in the identification of synthetic particles amidst naturally occurring organic and inorganic components. The fluorescent stain Nile red has been proposed as a tool to overcome this issue, but to date, has been used without consideration of polymer specific fluorescent variability. The aim of this study was to evaluate the efficacy of Nile red for microplastic detection by systematically investigating what drives variations in particle pixel brightness (PPB). The results showed that PPB varied between polymer type, shape, size, colour and by staining procedure. Sand, an inorganic component of the sample matrix does not fluoresce when stained with Nile red. In contrast the organic components, wood and chitin, fluoresce between 1.40 and 12 arbitrary units (a.u.) and 32 and 74 a.u. after Nile red staining, respectively. These data informed the use of a PPB threshold limit of 100 a.u., which improved the detection of EPS, HDPE, PP and PA-6 from the 6 polymers tested and reduced analysis time by 30-58% compared to unstained samples. Conversely, as with traditional illumination, PET and PVC were not accurately estimated using this approach. This study shows that picking a threshold limit is not arbitrary but rather must be informed by polymer specific fluorescent variability and matrix considerations. This is an essential step needed to facilitate comparability and reproducibility between individual studies.