This study combines a traditional chemical characterization with a simultaneous biological evaluation through histological, immunohistochemical, and enzymatic observations to assess the efficiency and sustainability of soil washing on Hg-contaminated sediment in terms of the bioavailability of the contaminant before and after the treatment, as well as the potential drawbacks of the treatment that are not revealed by a simple chemical characterization of treated sediments on its own. Different extracting agents, that is, ethylenedinitrilotetraacetic acid (EDTA), ethylenediaminedisuccinic acid (EDDS), sodium thiosulfate, potassium iodide (KI), and iodine (I2), have been compared in this work to evaluate their efficiency in the removal of Hg from contaminated sediments. Speciation analysis was applied to assess the mobility of Hg from different fractions of aged sediments. Biological evaluation was carried out through the use of large mesocosms and Mytilus galloprovincialis as biological sentinels. Results from bench scale tests have shown Hg removal of up to 93% by means of the multi-step KI/I2 washing process of the sediment. Results from histological, immunohistochemical, and enzymatic analysis have shown significant differences in the degree of alteration of biological tissues and their functional integrity between organisms in contact with contaminated and restored sediments. The reduction in 5-HT3R immunopositivity in the mesocosm with treated sediments suggests a tendency for mussels to recover a healthy condition. This result was also confirmed by the measurement of the enzymatic activity of AChE in mussel gills, which was significantly reduced in organisms from the mesocosm with polluted sediments compared with those from the one with restored sediments. The proposed approach could help stakeholders all over the world select, at an early stage, the most efficient cleaning action from a more holistic perspective, including not only pollutant concentration and economic reduction but also a direct assessment of the ultimate impact of the selected process on the biological system. © 2023 by the authors.

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