Summary: The need for sensitive biological measures of aquatic ecosystem integrity applicable at large spatial scales has been highlighted by the implementation of the European Water Framework Directive. Using fish communities as indicators of habitat quality in rivers, we developed a multi‐metric index to test our capacity to (i) correctly model a variety of metrics based on assemblage structure and functions, and (ii) discriminate between the effects of natural vs. human‐induced environmental variability at a continental scale. Information was collected for 5252 sites distributed among 1843 European rivers. Data included variables on fish assemblage structure, local environmental variables, sampling strategy and a river basin classification based on native fish fauna similarities accounting for regional effects on local assemblage structure. Fifty‐eight metrics reflecting different aspects of fish assemblage structure and function were selected from the available literature and tested for their potential to indicate habitat degradation. To quantify possible deviation from a ‘reference condition’ for any given site, we first established and validated statistical models describing metric responses to natural environmental variability in the absence of any significant human disturbance. We considered that the residual distributions of these models described the response range of each metric, whatever the natural environmental variability. After testing the sensitivity of these residuals to a gradient of human disturbance, we finally selected 10 metrics that were combined to obtain a European fish assemblage index. We demonstrated that (i) when considering only minimally disturbed sites the index remains invariant, regardless of environmental variability, and (ii) the index shows a significant negative linear response to a gradient of human disturbance. Synthesis and applications. In this reference condition modelling approach, by including a more complete description of environmental variability at both local and regional scales it was possible to develop a novel fish biotic index transferable between catchments at the European scale. The use of functional metrics based on biological attributes of species instead of metrics based on species themselves reduced the index sensitivity to the variability of fish fauna across different biogeographical areas.

Running water ecosystems of Europe are affected by various human pressures. However, little is known about the prevalence, spatial patterns, interactions with natural environment and co‐occurrence of pressures. This study represents the first high‐resolution data analysis of human pressures at the European scale, where important pressure criteria for 9330 sampling sites in 14 European countries were analysed. We identified 15 criteria describing major anthropogenic degradation and combined these into a global pressure index by taking additive effects of multiple pressures into account. Rivers are affected by alterations of water quality (59%), hydrology (41%) and morphology (38%). Connectivity is disrupted at the catchment level in 85% and 35% at the river segment level. Approximately 31% of all sites are affected by one, 29% by two, 28% by three and 12% by four pressure groups; only 21% are unaffected. In total, 47% of the sites are multi‐impacted. Approximately 90% of lowland rivers are impacted by a combination of all four pressure groups.

This work addresses human stressors and their impacts on fish assemblages at pan-European scale by analysing single and multiple stressors and their interactions. Based on an extensive dataset with 3105 fish sampling sites, patterns of stressors, their combination and nature of interactions, i.e. synergistic, antagonistic and additive were investigated. Geographical distribution and patterns of seven human stressor variables, belonging to four stressor groups (hydrological-, morphological-, water quality- and connectivity stressors), were examined, considering both single and multiple stressor combinations. To quantify the stressors' ecological impact, a set of 22 fish metrics for various fish assemblage types (headwaters, medium gradient rivers, lowland rivers and Mediterranean streams) was analysed by comparing their observed and expected response to different stressors, both acting individually and in combination. Overall, investigated fish sampling sites are affected by 15 different stressor combinations, including 4 stressors acting individually and 11 combinations of two or more stressors; up to 4 stressor groups per fish sampling site occur. Stressor-response analysis shows divergent results among different stressor categories, even though a general trend of decreasing ecological integrity with increasing stressor quantity can be observed. Fish metrics based on density of species 'intolerant to water quality degradation' and 'intolerant to oxygen depletion" responded best to single and multiple stressors and their interactions. Interactions of stressors were additive (40%), synergistic (30%) or antagonistic (30%), emphasizing the importance to consider interactions in multi-stressor analyses. While antagonistic effects are only observed in headwaters and medium-gradient rivers, synergistic effects increase from headwaters over medium gradient rivers and Mediterranean streams to large lowland rivers. The knowledge gained in this work provides a basis for advanced investigations in European river basins and helps prioritizing further restoration and management actions.