## 4 Discussion
### HIM Levels in Fish Samples
#### 4.1.1 Fresh, Frozen, and Defrosted Fish  
As shown in Figure 2, the fresh, frozen, and defrosted fish category, mainly represented by tuna, anchovies, and mackerel, showed a particular situation: the highest number of samples with concentrations below 10 mg kg\({}^{-1}\). At the same time, the highest number of \"non-compliant\" samples was found in this category. The non-compliances concerned nine frozen and defrosted tuna samples and two anchovie samples with concentration values from 328 to 5542 mg kg\({}^{-1}\), much higher than the limits set in the Regulation (EC) No. 2073/2005 (Table 1). Critical points such as inadequate frosting/defrosting cycles, incorrect treatment before consumption of the defrosted tuna, and inappropriate packaging or storage conditions at the retail market are, presumably, at the basis of these findings in agreement with those reported by [PERSON] et al. and [PERSON] et al. [40; 41] Furthermore, fraudulent practices in the treatment of raw tuna with red extracts containing nitrates and/or high levels of antioxidants or with carbon monoxide, as reported in the last years [20], may be other causes of the HIM presence. As shown in Table 1, in the fresh, frozen and defrosted fish category, tuna showed the highest HIM concentrations, in accordance with most of the studies reported in the literature [38; 42; 43; 44], confirming that tuna is more susceptible to HIM development than mackerel, anchovies, and sardines because of its high content of free histidine and its composition and presence of high levels of bacterial flora. As reported in Table 1 for fresh anchovy samples, an HIM content in the range 2.57-559 mg kg\({}^{-1}\) was found, with a mean concentration of 19.6 mg kg\({}^{-1}\). This value was lower than that of 41.1 mg kg\({}^{-1}\) reported by [PERSON] et al. [45] In the same study, the authors found a mean HIM concentration of 39.3 mg kg\({}^{-1}\) on 30 frozen mackerel lots. This value was higher than that of 7.8 mg kg\({}^{-1}\) detected, in the present survey, in fresh/frozen mackerel samples that showed also HIM levels ranging to 2.57 from 68.4 mg kg\({}^{-1}\). The HIM levels found in mackerel were also much lower than those reported by [PERSON] et al. in Indian mackerels (_Rastrelliger kanagurta_) of Pakistan (144.72 \(\pm\) 2.47 mg kg\({}^{-1}\)) [46]. Finally, our results are quite similar to those reported by [PERSON] et al. for Mediterranean mackerel sampled in the years 2010-2012 but were much lower with respect to the HIM values found in the same kind of product analyzed in 2014 [38].  
HIM concentrations less than 10 mg kg\({}^{-1}\) were detected in non-scombroid fish such as salmon and other species for which no limits were set in the Regulation (EC) No. 2073/2005. These findings are consistent with the literature data [47] even though salmon consumption is reported as a cause of intoxication [48; 49] and recent studies showed the presence of HIM in non-scombroid species such as mahi-mahi and swordfish in fillets [50; 51].  
Two samples of tuna slices stored in the freezer, taken from a local restaurant and from a company canteen, collected after allergic reaction cases, showed HIM concentration levels of 4895 mg kg\({}^{-1}\) and 5542 mg kg\({}^{-1}\), respectively. Restaurants, company canteens and sandwich shops are often reported as sources of scombroid poisoning cases [52]. Apart from frozen or defrosted tuna, used in restaurants for meal preparation but previously stored in inappropriate conditions and not consumed within 24 h, canned tuna is also used as an ingredient in salad, sandwic and pizza, and when opened a long time before consumption, can be subject to bacterial contamination and HIM production as reported by [PERSON] et al. [53].