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Assessing spatial variation in the chemical sensitivity of natural assemblages will enhance ecological relevance and reduce uncertainty in ecological risk assessments and the derivation of environmental quality standards (EQSs). However, the majority of species in natural communities have not undergone toxicity testing for any chemical, which poses a major challenge when assessing their sensitivity. We investigated spatial variation and patterns in the sensitivity of 4084 freshwater macroinvertebrate assemblages across England to 5 general-acting chemicals (heavy metals) and 13 specifically acting chemicals (insecticides) using a novel hierarchical species sensitivity distribution method based on taxonomic relatedness. Furthermore, we explored how river typology relates to spatial variation in assemblage sensitivity to chemicals and the potential impacts of such variation on current EQSs. Our findings revealed that, whereas assemblages with similar taxonomic compositions exhibit comparable sensitivity distributions, assemblages with different taxonomic compositions could have very similar or very different sensitivity distributions. The variation in assemblage sensitivity was greater for specifically acting chemicals than for general-acting chemicals and exhibited spatial clustering patterns. These spatial clustering patterns varied depending on the chemical, and the regions where assemblages were most sensitive to metals were generally not the same as the regions where assemblages were most sensitive to insecticides. Spatial variation in assemblage sensitivity was related to river typology with sensitive assemblages being more common than expected in lowland calcareous (or mixed geology) rivers within very small to small catchments. Comparing spatial variation in assemblage-specific chemical sensitivity to EQSs, we found that the operational EQSs in England would protect most study assemblages (i.e., > 99.5 %), although a small proportion of assemblages may face potential risks associated with azinphos-methyl, copper, and malathion. In many cases the EQSs were very precautionary, potentially requiring expensive control measures or restricting beneficial chemical use with no additional environmental benefit. The development of spatially defined EQSs, possibly based on river types, could be developed to target areas that require the highest level of protection and thus strike a balance between the benefits of chemical use and environmental protection., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Ltd.)