A multidisciplinary approach to risk assessment enables the identification of deteriorated sections within a major...

To date, few studies have assessed the quality of the Guadalquivir River, the main hydrographic basin of the Southern Iberian Peninsula. Here, we present the first comprehensive study to integrate physico-chemical characterisation, bacterial community assessment, and quality indices along its entire course. The Water Quality Index showed a gradual, progressive deterioration in water quality downstream, with 10% of the stations showing fair quality, signs of eutrophication (∼400 mg/m³ chlorophyll), and microbiological contamination (Escherichia coli, up to 3000 CFU/100 mL). The lower-middle section showed significant changes in the levels of organic carbon, metals, suspended solids and turbidity, as well as high nitrate (up to 19 mg L^-1) and ammonia (up to 1.4 mg L^-1) levels influenced by agricultural activities. Water quality may also be negatively influenced by the impact of tributaries. Since microorganisms are ubiquitous and play indispensable environmental roles, changes in water microbiome composition, structure and functionality were evaluated. Metabarcoding analysis revealed significant alterations, particularly in the upper reaches, despite the prevalence of parameters associated with good quality. In this section, a considerable decrease in α-diversity, very high values of microgAMBI ecological index, and potential functional changes revealed signs of environmental degradation. In addition, the predicted high prevalence of "ABC transporters" and the "two-component system" suggests a response to adverse conditions, driven by a small number of tolerant opportunistic microorganisms, notably the family Comamonadaceae. This area, characterised by extensive olive monoculture, has traditionally been exposed to contamination from biocides. In practical terms, this research confirmed that although widely recognised indices may report high overall water quality, complementary microbiome analyses might help reveal undetected hazards affecting the whole ecosystem. This multidisciplinary study could be used as a model for designing risk maps that identify "hotspots" for action to improve the management of essential water resources.