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The widespread use of Carbon nanotubes (CNTs) has been increasing exponentially, leading to a significant potential release into the environment. Nevertheless, the toxic effects of CNTs in natural aquatic systems are related to their ability to interact with abiotic compounds. Considering that salinity variations are one of the main challenges in the environment and thus may influence the behavior and toxicity of CNTs, a laboratory experiment was performed exposing the tube-building polychaete Diopatra neapolitana (Delle Chiaje 1841) for 28 days to pristine multi-walled carbon nanotube (MWCNTs) and carboxylated MWCNTs, maintained at control salinity 28 and low salinity 21. An innovative approach based on thermogravimetric analysis (TGA) was adopted for the first time to assess the presence of MWCNTs aggregates in the organisms. Both CNTs generated toxic impacts in terms of regenerative capacity, energy reserves and metabolic capacity as well as oxidative and neuro status, however greater toxic impacts were observed in polychaetes exposed to carboxylated MWCNTs. Moreover, both CNTs maintained under control salinity (28) generated higher toxic impacts in the polychaetes compared to individuals maintained under low salinity (21), indicating that exposed polychaetes tend to be more sensitive to the alteration induced by salinity variations on the chemical behavior of both MWCNTs in comparison to salt stress.