Code release Associated journal article: Mohan, C., Gleeson, T., Famiglietti, J. S., Virkki, V., Kummu, M., Porkka, M., Wang-Erlandsson, L., Huggins, X., Gerten, D., and Jähnig, S. C.: Poor correlation between large-scale environmental flow violations and freshwater biodiversity: implications for water resource management and water planetary boundary, Hydrol. Earth Syst. Sci. Discuss. [preprint], https://doi.org/10.5194/hess-2022-87, in review, 2022. Abstract The freshwater ecosystems all over the world are degrading, such that maintaining environmental flow (EF) in river networks is critical to their preservation. The relationship between streamflow alterations and, respectively, EF violations, and freshwater biodiversity is well established at the scale of stream reaches or small basins (~<100 km²). However, it is unclear if this relationship is robust at larger scales even though there are large-scale initiatives to legalize the EF requirement and EFs have been used in assessing a planetary boundary for freshwater. Therefore, this study intends to evaluate the relationship between EF violation and freshwater biodiversity at large basin scale (median area = 19,600 km2), globally-aggregated scales, and at freshwater ecoregions, and test the prevailing assumption of scalability of this relationship. Four EF violation indices (severity, frequency, the probability to shift to violated state, and probability to stay violated) and nine independent freshwater biodiversity indicators (calculated from observed biota data except one empirically derived from streamflow deviation) were used for correlation analysis. EF violations showed an inverse relationship with the streamflow-derived biodiversity indicator (MSAhy) at the level 5 HydroBASIN scale. However, no statistically significant negative relationship between environmental flows and freshwater biodiversity was found at the global or ecoregion scale except between the streamflow-derived biodiversity indicator (MSAhy) and all EF violation indices. While our results thus suggest that streamflow and EF may not be the main determinants of freshwater biodiversity, they do not preclude the existence of relationships with more holistic EF methods (e.g. including water temperature, water quality, intermittency, connectivity, etc.) or with other biodiversity data or metrics.