Eutrophication is a threat to wetlands worldwide. Elevated phosphorus concentration is often the main driver of loss of biodiversity and ecosystem function, but effects of phosphorus and nitrogen have proved largely inseparable, because they vary colinearly. Charophytes, aquatic algae that are very close to the evolutionary link with the land–plant lineage, provide a sensitive system for disentangling complex pollutant threats. Here, we investigated aquatic vegetation and water quality at the principal sites for charophyte biodiversity in the UK and used hierarchical partitioning to discriminate independent effects of pollutants on their occurrence. A laboratory experiment examined the growth responses of a representative species (Chara globularis) to nitrate. Nitrate-N exerted the greatest detrimental effect on charophyte occurrence in the field. Furthermore, growth of C. globularis in the laboratory was inhibited above very low concentrations. Smaller independent effects of copper (Cu), cadmium (Cd), cobalt (Co), phosphate-P, nickel (Ni), boron (B) and manganese (Mn) on charophyte occurrence were discriminated. It is possible to separate the deleterious effects of phosphorus and nitrogen on aquatic organisms in the field. Nitrate is a critical factor and a mean annual average concentration limit of c. 2 mg l-1 nitrate-N is necessary to protect charophytes and their services within wetland ecosystems.