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A conference abstract from NIRItalia online 2021 in English and Italian., This study is supported by the Italian Ministry of Health under Grant nr. IZSPLV 02-18 - RC.

Under Council Regulations, authentication of fish as fresh or frozen-thawed is compulsory because of the widespread fraudulent practice of retailing fish products as fresh, when they have actually been frozen. Moreover, in order to satisfy the health requirements of EC Regulations 853/2004 and 1276/2011, the fish intended for raw consumption has to be deep-frozen before usage, to protect consumers against Anisakiasis.. The aim of this study was to apply histological analysis as a method for distinguishing between fresh and frozen-thawed fish, with a view to evaluate its potential use as a routine screening technique in compliance with the requirements of EC Regulation No. 882/2004 on official food and feed controls. In a first experiment, method performance (i.e., accuracy and precision) was evaluated on tissue samples from three common Mediterranean fish species; the evaluation was subsequently extended to include samples from 35 different fish species in a second experiment to test for method robustness. Specifically, method accuracy was tested by comparing histological results against a gold standard obtained from the analysis of frozen and unfrozen fish samples prepared for the study; method precision was evaluated according to inter-rater agreement (i.e., three laboratories with expertise in histopathology in the first experiment, and three expert analysts in the second experiment) by estimating Cohen's kappa (and corresponding 95% confidence intervals) for each pair of labs and experts and combined Cohen?s kappa for all three experts and labs. The observed interrater agreement among the three labs and the three experts showed good method accuracy and precision (high sensitivity and specificity), as well as good method reproducibility. Our results suggest that, besides being a rapid and simple tool, histology is a highly accurate method for distinguishing between fresh and frozen-thawed fish, irrespective of the fish species analysed. Furthermore in a third experiment ten samples obtained both from red and white-meat fish, based on real production of the food business, were designated to evaluate the performance of the histological method in correctly classifying the fish as fresh/frozen as well as the microbiological and chemical safety issues possibly related to the fishery products. All samples were collected and were subjected to histological, microbiological and chemical analyses before and after freezing. For two samples species identification was needed. Based on the histological method, one out of ten fish, all claimed to be fresh by the supplier, was not; all the remaining nine samples following freezing could be characterized microscopically as frozen; microbiological parameters resulted normal, while flesh from a tuna fish (Euthynnus alletteratus) contained mercury residues three times the legally permitted level.