Transitional ecosystems are among the most degraded ecosystems worldwide, with several groups of organisms investigated for their reliability as biological indicators of human-driven disturbances. Recently non-traditional biochemical biomarkers such as an individual's fatty acids profile have been identified as promising tools for assessing contaminant exposure. In this work, two abundant Atlantic benthic macroalgae (Ulva lactuca and Fucus vesiculosus species) were surveyed in three mudflat areas of the highly urbanized Tejo estuary, with increasing anthropogenic disturbance degrees (Alcochete, Rosário and Seixal mudflats, increasing in contamination by this order) and their fatty acids evaluated as potential biomarkers for exposure to contaminants known to have toxic effects on biota. In terms of contamination the metal pollution index of all the compartments analysed (sediment bioavailable and total metal concentrations and thallus metal concentrations) revealed the same tendencies with lower contamination levels in Alcochete, intermediate in Rosário and high contamination levels in Seixal. In the thallus of U. lactuca thallus could be observed a strong decrease in C18-fatty acids along the contamination gradient, likely due to lipid peroxidation from metal-generated reactive oxygen species. Nevertheless, an increase in stearic and hexadecatrienoic acids in the thallus from the most contaminated site suggested counteractive mechanisms maintain the production of C18-fatty acid pool. A similar response was found in F. vesiculosus but with palmitic acid acting as precursor for the synthesis of stearic acid, allowing the maintenance of oleic and linoleic acids levels in the membranes to counteract oxidative stress. Beyond the physiological interest of these mechanisms, fatty acid profiles were used to develop a novel multivariate pressure index (Multi-PI), that beyond the contaminant concentration would reflect the response of these biomonitor species towards anthropogenic disturbance, through the evaluation of fatty acid profiles, which are also key molecules from a trophic perspective within the estuarine system. The Multi-PI efficiency in responding to different environmental contamination degrees, was substantiated by strong and positive correlations with thallus and sediment contamination. This indicated that fatty acid profiles reflect thallus and benthic habitat contamination and are efficient biomarkers of environmental metal contamination. Therefore, the sessile and abundant nature of benthic macroalgae allied to their fatty acid responses can be leveraged as suitable biomarkers for contaminant monitoring in future impact assessment and ecotoxicology studies.

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