The recovery of European freshwater biodiversity has come to a halt.

Owing to a long history of anthropogenic pressures, freshwater ecosystems are among the most vulnerable to biodiversity loss1. Mitigation measures, including wastewater treatment and hydromorphological restoration, have aimed to improve environmental quality and foster the recovery of freshwater biodiversity2. Here, using 1,816 time series of freshwater invertebrate communities collected across 22 European countries between 1968 and 2020, we quantified temporal trends in taxonomic and functional diversity and their responses to environmental pressures and gradients. We observed overall increases in taxon richness (0.73% per year), functional richness (2.4% per year) and abundance (1.17% per year). However, these increases primarily occurred before the 2010s, and have since plateaued. Freshwater communities downstream of dams, urban areas and cropland were less likely to experience recovery. Communities at sites with faster rates of warming had fewer gains in taxon richness, functional richness and abundance. Although biodiversity gains in the 1990s and 2000s probably reflect the effectiveness of water-quality improvements and restoration projects, the decelerating trajectory in the 2010s suggests that the current measures offer diminishing returns. Given new and persistent pressures on freshwater ecosystems, including emerging pollutants, climate change and the spread of invasive species, we call for additional mitigation to revive the recovery of freshwater biodiversity.

Scrutinizing the Role of Venoarterial Extracorporeal Membrane Oxygenation: Has Clinical Practice Outpaced the Evidence?

The use of venoarterial extracorporeal membrane oxygenation (VA-ECMO) for temporary mechanical circulatory support in various clinical scenarios has been increasing consistently, despite the lack of sufficient evidence regarding its benefit and safety from adequately powered randomized controlled trials. Although the ARREST trial (Advanced Reperfusion Strategies for Patients with Out-of-Hospital Cardiac Arrest and Refractory Ventricular Fibrillation) and a secondary analysis of the PRAGUE OHCA trial (Prague Out-of-Hospital Cardiac Arrest) provided some evidence in favor of VA-ECMO in the setting of out-of-hospital cardiac arrest, the INCEPTION trial (Early Initiation of Extracorporeal Life Support in Refractory Out-of-Hospital Cardiac Arrest) has not found a relevant improvement of short-term mortality with extracorporeal cardiopulmonary resuscitation. In addition, the results of the recently published ECLS-SHOCK trial (Extracorporeal Life Support in Cardiogenic Shock) and ECMO-CS trial (Extracorporeal Membrane Oxygenation in the Therapy of Cardiogenic Shock) discourage the routine use of VA-ECMO in patients with infarct-related cardiogenic shock. Ongoing clinical trials (ANCHOR [Assessment of ECMO in Acute Myocardial Infarction Cardiogenic Shock, NCT04184635], REVERSE [Impella CP With VA ECMO for Cardiogenic Shock, NCT03431467], UNLOAD ECMO [Left Ventricular Unloading to Improve Outcome in Cardiogenic Shock Patients on VA-ECMO, NCT05577195], PIONEER [Hemodynamic Support With ECMO and IABP in Elective Complex High-risk PCI, NCT04045873]) may clarify the usefulness of VA-ECMO in specific patient subpopulations and the efficacy of combined mechanical circulatory support strategies. Pending further data to refine patient selection and management recommendations for VA-ECMO, it remains uncertain whether the present usage of this device improves outcomes.

Freshwater-related species richness in Natura 2000 sites strongly depends on the surrounding land use besides local habitat conditions.

The Birds and Habitats Directive are the cornerstones of Europe's nature conservation policy and the resulting Natura 2000 (N2k) sites form the largest coordinated network of protected areas in the world. Despite the ambitious targets of these directives and decades of efforts, biodiversity, especially of freshwater-related species, continues to decline in Europe. While multiple stressors at larger spatial scales are known to limit the effect of river restoration projects, the importance of surrounding land use outside the N2k sites for freshwater-related species richness inside N2k sites has rarely been studied. Conditional inference forests were used to assess the importance of land use in the surrounding and upstream of the German N2k sites compared to local habitat conditions inside. Freshwater-related species richness depended on land use in the surrounding besides local habitat conditions. Results indicated that this was especially true for birds in small N2k sites embedded in a wet, diverse, and patchy landscape and for non-birds due to the provision of additional habitats outside the N2k sites. Given that most N2k sites in Europe are rather small, the surrounding habitat conditions and land use potentially influences and affects freshwater-related species in many N2k sites across Europe. The additional conservation and restoration areas to be designated under the EU Biodiversity Strategy and upcoming EU restoration law should either be large enough or surrounded by extensive land use to optimize their effect on freshwater-related species.

Of causes and symptoms: using monitoring data and expert knowledge to diagnose the causes of stream degradation.

Ecological status assessment under the European Water Framework Directive (WFD) often integrates the impact of multiple stressors into a single index value. This hampers the identification of individual stressors being responsible for status deterioration. As a consequence, management measures are often disentangled from assessment results. To close this gap and to support river basin managers in the diagnosis of stressors, we linked numerous macroinvertebrate assessment metrics and one diatom index with potential causes of ecological deterioration through Bayesian belief networks (BBNs). The BBNs were informed by WFD monitoring data as well as regular consultation with experts and allow to estimate the probabilities of individual degradation causes based upon a selection of biological metrics. Macroinvertebrate metrics were shown to be stronger linked to hydromorphological conditions and land use than to water quality-related parameters (e.g., thermal and nutrient pollution). The modeled probabilities also allow to order the potential causes of degradation hierarchically. The comparison of assessment metrics showed that compositional and trait-based community metrics performed equally well in the diagnosis. The testing of the BBNs by experts resulted in an agreement between model output and expert opinion of 17-92% for individual stressors. Overall, the expert-based validation confirmed a good diagnostic potential of the BBNs; on average 80% of the diagnosed causes were in agreement with expert judgement. We conclude that diagnostic BBNs can assist the identification of causes of stream and river degradation and thereby inform the derivation of appropriate management decisions.

A global agenda for advancing freshwater biodiversity research.

Global freshwater biodiversity is declining dramatically, and meeting the challenges of this crisis requires bold goals and the mobilisation of substantial resources. While the reasons are varied, investments in both research and conservation of freshwater biodiversity lag far behind those in the terrestrial and marine realms. Inspired by a global consultation, we identify 15 pressing priority needs, grouped into five research areas, in an effort to support informed stewardship of freshwater biodiversity. The proposed agenda aims to advance freshwater biodiversity research globally as a critical step in improving coordinated actions towards its sustainable management and conservation.

Multiple stressors determine river ecological status at the European scale: Towards an integrated understanding of river status deterioration.

The biota of European rivers are affected by a wide range of stressors impairing water quality and hydro-morphology. Only about 40% of Europe's rivers reach 'good ecological status', a target set by the European Water Framework Directive (WFD) and indicated by the biota. It is yet unknown how the different stressors in concert impact ecological status and how the relationship between stressors and status differs between river types. We linked the intensity of seven stressors to recently measured ecological status data for more than 50,000 sub-catchment units (covering almost 80% of Europe's surface area), which were distributed among 12 broad river types. Stressor data were either derived from remote sensing data (extent of urban and agricultural land use in the riparian zone) or modelled (alteration of mean annual flow and of base flow, total phosphorous load, total nitrogen load and mixture toxic pressure, a composite metric for toxic substances), while data on ecological status were taken from national statutory reporting of the second WFD River Basin Management Plans for the years 2010-2015. We used Boosted Regression Trees to link ecological status to stressor intensities. The stressors explained on average 61% of deviance in ecological status for the 12 individual river types, with all seven stressors contributing considerably to this explanation. On average, 39.4% of the deviance was explained by altered hydro-morphology (morphology: 23.2%; hydrology: 16.2%), 34.4% by nutrient enrichment and 26.2% by toxic substances. More than half of the total deviance was explained by stressor interaction, with nutrient enrichment and toxic substances interacting most frequently and strongly. Our results underline that the biota of all European river types are determined by co-occurring and interacting multiple stressors, lending support to the conclusion that fundamental management strategies at the catchment scale are required to reach the ambitious objective of good ecological status of surface waters.

Trait-based ecology at large scales: Assessing functional trait correlations, phylogenetic constraints and spatial variability using open data.

The growing use of functional traits in ecological research has brought new insights into biodiversity responses to global environmental change. However, further progress depends on overcoming three major challenges involving (a) statistical correlations between traits, (b) phylogenetic constraints on the combination of traits possessed by any single species, and (c) spatial effects on trait structure and trait-environment relationships. Here, we introduce a new framework for quantifying trait correlations, phylogenetic constraints and spatial variability at large scales by combining openly available species' trait, occurrence and phylogenetic data with gridded, high-resolution environmental layers and computational modelling. Our approach is suitable for use among a wide range of taxonomic groups inhabiting terrestrial, marine and freshwater habitats. We demonstrate its application using freshwater macroinvertebrate data from 35 countries in Europe. We identified a subset of available macroinvertebrate traits, corresponding to a life-history model with axes of resistance, resilience and resource use, as relatively unaffected by correlations and phylogenetic constraints. Trait structure responded more consistently to environmental variation than taxonomic structure, regardless of location. A re-analysis of existing data on macroinvertebrate communities of European alpine streams supported this conclusion, and demonstrated that occurrence-based functional diversity indices are highly sensitive to the traits included in their calculation. Overall, our findings suggest that the search for quantitative trait-environment relationships using single traits or simple combinations of multiple traits is unlikely to be productive. Instead, there is a need to embrace the value of conceptual frameworks linking community responses to environmental change via traits which correspond to the axes of life-history models. Through a novel integration of tools and databases, our flexible framework can address this need.

Diverging response patterns of terrestrial and aquatic species to hydromorphological restoration.

Although experiences with ecological restoration continue to accumulate, the effectiveness of restoration for biota remains debated. We complemented a traditional taxonomic analysis approach with information on 56 species traits to uncover the responses of 3 aquatic (fish, macroinvertebrates, macrophytes) and 2 terrestrial (carabid beetles, floodplain vegetation) biotic groups to 43 hydromorphological river restoration projects in Germany. All taxonomic groups responded positively to restoration, as shown by increased taxonomic richness (10-164%) and trait diversity (habitat, dispersal and mobility, size, form, life history, and feeding groups) (15-120%). Responses, however, were stronger for terrestrial than aquatic biota, and, contrary to our expectation, taxonomic responses were stronger than those of traits. Nevertheless, trait analysis provided mechanistic insights into the drivers of community change following restoration. Trait analysis for terrestrial biota indicated restoration success was likely enhanced by lateral connectivity and reestablishment of dynamic processes in the floodplain. The weaker response of aquatic biota suggests recovery was hindered by the persistence of stressors in the aquatic environment, such as degraded water quality, dispersal constraints, and insufficient hydromorphological change. Therefore, river restoration requires combined local- and regional-scale approaches to maximize the response of both aquatic and terrestrial organisms. Due to the contrasting responses of aquatic and terrestrial biota, the planning and assessment of river restoration outcomes should consider effects on both components of riverine landscapes.

Effects of agriculture on river biota differ between crop types and organism groups.

While the general effects of agricultural land use on riverine biota are well documented, the differential effects of specific crop types on different riverine organism groups, remain largely unexplored. Here we used recently published land use data distinguishing between specific crop types and a Germany-wide dataset of 7748 sites on the ecological status of macroinvertebrates, macrophytes and diatoms and applied generalized linear mixed models to unravel the associations between land use types, crop types, and the ecological status. For all organism groups, associations of specific crop types with biota were stronger than those of urban land use. For macroinvertebrates and macrophytes, strong negative associations were found for pesticide intensive permanent crops, while intensively fertilized crops (maize, intensive cereals) affected diatoms most. These differential associations highlight the importance of distinguishing between crop types and organism groups and the urgency to buffer rivers against agricultural stressors at the catchment scales and to expand sustainably managed agriculture.

How tolerances, competition and dispersal shape benthic invertebrate colonisation in restored urban streams.

Biotic communities often respond poorly to river restoration activities and the drivers of community recovery after restoration are not fully understood. According to the Asymmetric Response Concept (ARC), dispersal capacity, species tolerances to stressors, and biotic interactions are three key drivers influencing community recovery of restored streams. However, the ARC remains to be tested. Here we used a dataset on benthic invertebrate communities of eleven restored stream sections in a former open sewer system that were sampled yearly over a period of eleven years. We applied four indices that reflect tolerance against chloride and organic pollution, the community's dispersal capacity and strength of competition to the benthic invertebrate taxa lists of each year and site. Subsequently, we used generalised linear mixed models to analyse the change of these indices over time since restoration. Dispersal capacity was high directly after restoration but continuously decreased over time. The initial communities thus consisted of good dispersers and were later joined by more slowly dispersing taxa. The tolerance to organic pollution also decreased over time, reflecting continuous improvement of water quality and an associated increase of sensitive species. On the contrary, chloride tolerances did not change, which could indicate a stable chloride level throughout the sampling period. Lastly, competition within the communities, reflected by interspecific trait niche overlap, increased with time since restoration. We show that recovery follows a specific pattern that is comparable between sites. Benthic communities change from tolerant, fast dispersing generalists to more sensitive, slowly dispersing specialists exposed to stronger competition. Our results lay support to the ARC (increasing role of competition, decreasing role of dispersal) but also underline that certain tolerances may still shape communities a decade after restoration. Disentangling the drivers of macroinvertebrate colonisation can help managers to better understand recovery trajectories and to define more realistic restoration targets.

River ecological status is shaped by agricultural land use intensity across Europe.

Agriculture impacts the ecological status of freshwaters through multiple pressures such as diffuse pollution, water abstraction, and hydromorphological alteration, strongly impairing riverine biodiversity. The agricultural effects, however, likely differ between agricultural types and practices. In Europe, agricultural types show distinct spatial patterns related to intensity, biophysical conditions, and socioeconomic history, which have been operationalised by various landscape typologies. Our study aimed at analysing whether incorporating agricultural intensity enhances the correlation between agricultural land use and the ecological status. For this, we aggregated the continent's agricultural activities into 20 Areas of Farming-induced Freshwater Pressures (AFFP), specifying individual pressure profiles regarding nutrient enrichment, pesticides, water abstraction, and agricultural land use in the riparian zone to establish an agricultural intensity index and related this intensity index to the river ecological status. Using the agricultural intensity index, nearly doubled the correlative strength between agriculture and the ecological status of rivers as compared to the share of agriculture in the sub-catchment (based on the analysis of more than 50,000 sub-catchment units). Strongest agricultural pressures were found for high intensity cropland in the Mediterranean and Temperate regions, while extensive grassland, fallow farmland and livestock farming in the Northern and Highland regions, as well as low intensity mosaic farming, featured lowest pressures. The results provide advice for pan-European management of freshwater ecosystems and highlight the urgent need for more sustainable agriculture. Consequently, they can also be used as a basis for European Union-wide and global policies to halt biodiversity decline, such as the post-2027 renewal of the Common Agricultural Policy.

Differential associations of five riverine organism groups with multiple stressors.

The decline of river and stream biodiversity results from multiple simultaneous occuring stressors, yet few studies explore responses explore responses across various taxonomic groups at the same locations. In this study, we address this shortcoming by using a coherent data set to study the association of nine commonly occurring stressors (five chemical, one morphological and three hydraulic) with five taxonomic groups (bacteria, fungi, diatoms, macro-invertebrates and fish). According to studies on single taxonomic groups, we hypothesise that gradients of chemical stressors structure community composition of all taxonomic groups, while gradients of hydraulic and morphological stressors are mainly related to larger organisms such as benthic macro-invertebrates and fish. Organisms were sampled over two years at 20 sites in two catchments: a recently restored urban lowland catchment (Boye) and a moderately disturbed rural mountainous catchment (Kinzig). Dissimilarity matrices were computed for each taxonomic group within a catchment. Taxonomic dissimilarities between sites were linked to stressor dissimilarities using multivariable Generalized Linear Mixed Models. Stressor gradients were longer in the Boye, but did in contrast to the Kinzig not cover low stress intensities. Accordingly, responses of the taxonomic groups were stronger in the Kinzig catchment than in the recently restored Boye catchment. The discrepancy between catchments underlines that associations to stressors strongly depend on which part of the stressor gradient is covered in a catchment. All taxonomic groups were related to conductivity. Bacteria, fungi and macro-invertebrates change with dissolved oxygen, and bacteria and fungi with total nitrogen. Morphological and hydraulic stressors had minor correlations with bacteria, fungi and diatoms, while macro-invertebrates were strongly related to fine sediment and discharge, and fish to high flow peaks. The results partly support our hypotheses about the differential associations of the different taxonomic groups with the stressors.

Multi-decadal improvements in the ecological quality of European rivers are not consistently reflected in biodiversity metrics.

Humans impact terrestrial, marine and freshwater ecosystems, yet many broad-scale studies have found no systematic, negative biodiversity changes (for example, decreasing abundance or taxon richness). Here we show that mixed biodiversity responses may arise because community metrics show variable responses to anthropogenic impacts across broad spatial scales. We first quantified temporal trends in anthropogenic impacts for 1,365 riverine invertebrate communities from 23 European countries, based on similarity to least-impacted reference communities. Reference comparisons provide necessary, but often missing, baselines for evaluating whether communities are negatively impacted or have improved (less or more similar, respectively). We then determined whether changing impacts were consistently reflected in metrics of community abundance, taxon richness, evenness and composition. Invertebrate communities improved, that is, became more similar to reference conditions, from 1992 until the 2010s, after which improvements plateaued. Improvements were generally reflected by higher taxon richness, providing evidence that certain community metrics can broadly indicate anthropogenic impacts. However, richness responses were highly variable among sites, and we found no consistent responses in community abundance, evenness or composition. These findings suggest that, without sufficient data and careful metric selection, many common community metrics cannot reliably reflect anthropogenic impacts, helping explain the prevalence of mixed biodiversity trends.

Stressors affecting the ecological status of temporary rivers in the Mediterranean region.

Temporary rivers are widespread in the Mediterranean region and impose a challenge for the implementation of the Water Framework Directive (WFD) and other environmental regulations. Surprisingly, an overarching analysis of their ecological status and the stressors affecting them is yet missing. We compiled data on the ecological status of 1504 temporary rivers in seven European Mediterranean region countries and related their ecological status (1) to publicly available data on pressures from the European WISE-WFD dataset, and (2) to seven more specific stressors modelled on a sub-catchment scale. More than 50 % of the temporary water bodies in the Mediterranean countries reached good or even high ecological status. In general, status classes derived from phytobenthos and macrophyte assessment were higher than those derived from the assessment of benthic invertebrates or fish. Of the more generally defined pressures reported to the WISE-WFD database, the most relevant for temporary rivers were 'diffuse agricultural' and 'point urban waste water'. Of the modelled more specific stressors, agricultural land use best explained overall ecological status, followed by total nitrogen load, and urban land use, while toxic substances, total phosphorus load and hydrological stressors were less relevant. However, stressors differed in relevance, with total nitrogen being most important for macrophytes, and agricultural land use for phytobenthos, benthic invertebrates and fish. For macrophytes, ecological quality increased with stressor intensity. The results underline the overarching effect of land use intensity for the ecological status of temporary water bodies. However, assessment results do not sufficiently reflect hydrological stress, most likely as the biological indicators used to evaluate these systems were designed for perennial water bodies and thus mainly target land use and nutrient impacts. We conclude that biomonitoring systems need to be updated or newly developed to better account for the specific situation of temporary water bodies.

The Asymmetric Response Concept explains ecological consequences of multiple stressor exposure and release.

Our capacity to predict trajectories of ecosystem degradation and recovery is limited, especially when impairments are caused by multiple stressors. Recovery may be fast or slow and either complete or partial, sometimes result in novel ecosystem states or even fail completely. Here, we introduce the Asymmetric Response Concept (ARC) that provides a basis for exploring and predicting the pace and magnitude of ecological responses to, and release from, multiple stressors. The ARC holds that three key mechanisms govern population, community and ecosystem trajectories. Stress tolerance is the main mechanism determining responses to increasing stressor intensity, whereas dispersal and biotic interactions predominantly govern responses to the release from stressors. The shifting importance of these mechanisms creates asymmetries between the ecological trajectories that follow increasing and decreasing stressor intensities. This recognition helps to understand multiple stressor impacts and to predict which measures will restore communities that are resistant to restoration.

Environmental ranges discriminating between macrophytes groups in European rivers.

Riverine macrophytes form distinct species groups. Their occurrence is determined by environmental gradients, e.g. in terms of physico-chemistry and hydromorphology. However, the ranges of environmental variables discriminating between species groups ("discriminatory ranges") have rarely been quantified and mainly been based on expert judgement, thus limiting options for predicting and assessing ecosystem characteristics. We used a pan-European dataset of riverine macrophyte surveys obtained from 22 countries including data on total phosphorus, nitrate, alkalinity, flow velocity, depth, width and substrate type. Four macrophyte species groups were identified by cluster analysis based on species' co-occurrences. These comprised Group 1) mosses, such as Amblystegium fluviatile and Fontinalis antipyretica, Group 2) shorter and pioneer species such as Callitriche spp., Group 3) emergent and floating species such as Sagittaria sagittifolia and Lemna spp., and Group 4) eutraphent species such as Myriophyllum spicatum and Stuckenia pectinata. With Random Forest models, the ranges of environmental variables discriminating between these groups were estimated as follows: 100-150 µg L-1 total phosphorus, 0.5-20 mg L-1 nitrate, 1-2 meq L-1 alkalinity, 0.05-0.70 m s-1 flow velocity, 0.3-1.0 m depth and 20-80 m width. Mosses were strongly related to coarse substrate, while vascular plants were related to finer sediment. The four macrophyte groups and the discriminatory ranges of environmental variables fit well with those described in literature, but have now for the first time been quantitatively approximated with a large dataset, suggesting generalizable patterns applicable at regional and local scales.

A metric-based analysis on the effects of riparian and catchment landuse on macroinvertebrates.

Woody riparian vegetation along rivers and streams provides multiple functions beneficial for aquatic macroinvertebrate communities. They retain fine sediments, nutrients and pesticides, improve channel hydromorphology, control water temperature and primary production through shading and provide leaves, twigs and large wood. In a recent conceptual model (Feld et al., 2018), woody riparian functions were considered either independent from large-scale landuse stressors (e.g. shading, input of organic matter), or dependent on landuse at larger spatial scales (e.g. fine sediment, nutrient and pesticide retention). We tested this concept using high-resolution data on woody riparian vegetation cover and empirical data from 1017 macroinvertebrate sampling sites in German lowland and mountain streams. Macroinvertebrate metrics indicative for individual functions were used as response variables in structural equation models (SEM), representing the hierarchical structure between the different considered stressors at different spatial scales: catchment, upstream riparian, local riparian and local landuse cover along with hydromorphology and water quality. The analysis only partly confirmed the conceptual model: Biotic integrity and water quality were strongly related to large-scale stressors as expected (absolute total effect 0.345-0.541), but against expectations, fine sediments retention, considered scale-dependent in the conceptual model, was poorly explained by large-scale stressors (absolute total effect 0.027-0.231). While most functions considered independent from large-scale landuse were partly explained by riparian landuse cover (absolute total effect 0.023-0.091) they also were nonetheless affected by catchment landuse cover (absolute total effect 0.017-0.390). While many empirical case studies at smaller spatial scales clearly document the positive effects of restoring woody riparian vegetation, our results suggest that most effects of riparian landuse cover are possibly superimposed by larger-scale stressors. This does not negate localized effects of woody riparian vegetation but helps contextualize limitations to successful restoration measures targeting the macroinvertebrate community.

GPR55 in B cells limits atherosclerosis development and regulates plasma cell maturation.

Dissecting the pathways regulating the adaptive immune response in atherosclerosis is of particular therapeutic interest. Here we report that the lipid G-protein coupled receptor GPR55 is highly expressed by splenic plasma cells (PC), upregulated in mouse spleens during atherogenesis and human unstable or ruptured compared to stable plaques. Gpr55-deficient mice developed larger atherosclerotic plaques with increased necrotic core size compared to their corresponding controls. Lack of GPR55 hyperactivated B cells, disturbed PC maturation and resulted in immunoglobulin (Ig)G overproduction. B cell-specific Gpr55 depletion or adoptive transfer of Gpr55-deficient B cells was sufficient to promote plaque development and elevated IgG titers. In vitro, the endogenous GPR55 ligand lysophsophatidylinositol (LPI) enhanced PC proliferation, whereas GPR55 antagonism blocked PC maturation and increased their mitochondrial content. Collectively, these discoveries provide previously undefined evidence for GPR55 in B cells as a key modulator of the adaptive immune response in atherosclerosis.

Perceived multiple stressor effects depend on sample size and stressor gradient length.

Multiple stressors are continuously deteriorating surface waters worldwide, posing many challenges for their conservation and restoration. Combined effect types of multiple stressors range from single-stressor dominance to complex interactions. Identifying prevalent combined effect types is critical for environmental management, as it helps to prioritise key stressors for mitigation. However, it remains unclear whether observed single and combined stressor effects reflect true ecological processes unbiased by sample size and length of stressor gradients. Therefore, we examined the role of sample size and stressor gradient lengths in 158 paired-stressor response cases with over 120,000 samples from rivers, lakes, transitional and marine ecosystems around the world. For each case, we split the overall stressor gradient into two partial gradients (lower and upper) and investigated associated changes in single and combined stressor effects. Sample size influenced the identified combined effect types, and stressor interactions were less likely for cases with fewer samples. After splitting gradients, 40 % of cases showed a change in combined effect type, 30 % no change, and 31 % showed a loss in stressor effects. These findings suggest that identified combined effect types may often be statistical artefacts rather than representing ecological processes. In 58 % of cases, we observed changes in stressor effect directions after the gradient split, suggesting unimodal stressor effects. In general, such non-linear responses were more pronounced for organisms at higher trophic levels. We conclude that observed multiple stressor effects are not solely determined by ecological processes, but also strongly depend on sampling design. Observed effects are likely to change when sample size and/or gradient length are modified. Our study highlights the need for improved monitoring programmes with sufficient sample size and stressor gradient coverage. Our findings emphasize the importance of adaptive management, as stress reduction measures or further ecosystem degradation may change multiple stressor-effect relationships, which will then require associated changes in management strategies.