Tricaine methane-sulfonate (MS-222) is one of the most widely used anaesthetics for poikilotherms worldwide. This paper outlines its anaesthetic efficacy and dosage in fish and legislation for its use, fish stress responses to MS-222 anaesthesia and its effect on fish physiology and blood properties, pharmacokinetics, genotoxicity, immune response, potential interference with fish hepatic cytochrome P450 spectra, and its impact on nerve sensitivity. Key questions arising from the available data are analysed, such as regulatory constraints on its use, the need for the standardization of buffering protocols, and interdependencies of the factors impacting the specific applicative efficacy of MS-222. Current research has provided an abundance of data on MS-222 use in fish, although the applications within these studies are often impractical at the farming level. Specific emphasis is therefore placed on highlighting application strategies on a practical basis, presenting potential future research on topics that require in-depth analysis (preparation and storage of anaesthetic solutions, pre-anaesthetic sedation and stress reduction, cortisol response in aquarium fish, toxicity of MS-222 metabolites, and possible immunodepressive properties). Additionally, both from a scientific and practical perspective, it is necessary to have a better understanding of safety margins, induction, immersion and recovery times for many (marine and freshwater, farmed and ornamental) fish species in order to achieve optimal utilization.

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a b s t r a c tA two-season investigation of the wastewater treatment plant (WWTP) effluent, of related waters, sludge and fish across a wide area and 11 stations, with emphasis on Aeromonas spp. was conducted. Aeromonas veronii was the prevailing aeromonad isolated by MALDI TOF MS in the summer period. A rise of Aeromonas hydrophila was observed in summer in raw sewage, treated wastewater and effluent-carrying canal. The ratio of aeromonad species retrieved from fish tissues did not correspond with the water and sludge findings, as in spring in the effluent-carrying canal fish carried Aeromonas salmonicida ssp. salmonicida and Aeromonas bestiarum, while in summer mainly A. veronii and Acinetobacter johnsonii were isolated from fish tissues in the same location. No correlation was established between fecal coliforms/ enterococci and aeromonad occurrence. All retrieved Aeromonas species demonstrated a distinct spectral pattern, with peaks showing unique mass distribution ranging from 4000 to 10,000 Da. Hierarchical clustering separated aeromonads of all isolated species and clustered closely related strains together. Resistance was determined towards amoxicillin, and frequently towards sulfamethoxazole and erythromycin. In summer, a high proportion of water and sludge Aeromonas species demonstrated multiple resistance patterns towards five or more antimicrobials. The quinolone resistance of water aeromonads was mostly related to A. veronii. There are potential health concerns regarding aeromonad exposure amongst recreational fishermen who come into contact with fish inhabiting waters downstream from the WWTP, and WWTP workers who are occupationally exposed to wastewaters and their aerosols.