River-length patterns in the chemistry and biology of the Charlton Brook, an unclassified watercourse in Sheffield, England, have been examined. Five sampling sites for macroinvertebrates and pollutant analysis were used, in conjunction with Environment Agency's General Quality Assessment (GQA) methodologies and hydraulic analysis of the catchment. Sites were strategically located to account for the tributaries and the brook downstream of their confluence, to assess the potential impact from surface water outfalls (SWOs). Variations in GQA parameters indicate a significant drop in quality downstream of the SWOs that discharge to the study watercourse, with a marked drop in biological diversity noted at the onset of urbanisation. The decline in biological quality however is greater than that suggested by physicochemical analysis alone. There was a significant inverse relationship between impermeable area and biological diversity. Analysis of polycyclic aromatic hydrocarbons (PAHs) and trace metals in sediment from the watercourse showed significant yet irregular variations between sites. The potential toxicity of instream metal concentrations was determined using cumulative criterion unit (CCU) scores, which highlighted cadmium, copper and lead as the major sources of potential chronic instream toxicity. The threshold for likely harm to aquatic life is exceeded at all sites. In the absence of different physical characteristics, comparisons of the chemical and biological data indicate that the benthic macroinvertebrate population of such watercourses are adversely affected by the stormwater inputs.