# Illegal Nitrite Treatment of Red Tuna and Prolonged Storage: What About Other Food Safety Risks?
## 3 Results and Discussion  
Three samplings in total were carried out, comparing the results obtained for fresh samples with those obtained for treated samples after 5 days of storage under refrigeration. Specifically, regarding chemical analysis, the presence and concentration of HIM, TVBN, biogenic amines, and nitrites/nitrates was evaluated, while microbiological investigationswere focused on assessing total microbial count at 30 \({}^{\circ}\)C (_Enterobacteriaceae_, _Vibrionaceae_, coagulase-positive staphylococci, _Salmonella_ and _Escherichia coli_). All samples were preliminarily checked for the detection of two food additives which can influence these parameters: sulfites and ascorbic acid. The absence of sulfites was determined in all samplings, while ascorbic acid was quantified at a mean concentration of 245.5 mg/kg, below the 300 mg/kg limit specified by EU Regulation No. 1333/2008. Obviously, all samples were also analyzed for quantifying the nitrite concentration both in treated samples and those not treated. In Figure 2, some chromatogram examples are shown. Histamine analysis is needed to assess the spoilage of fish products, in particular for fresh fish where the eventual use of illegal additives, such as nitrites, can alter sensory evaluations. HIM is used as a useful parameter of food quality, relating to freshness degree. A content lower than 10 mg/kg ensures a good quality level, while concentrations up to 30 mg/kg and 50 mg/kg are indexes of significant deterioration and definite decomposition, respectively [20]. The European Commission fixed some safety requirements for HIM presence in seafood [35]. More in depth, for fish species associated with high levels of histidine (including Scombridae, like tuna), nine samples have to be taken from each batch for analysis; the mean level must not exceed 100 mg/kg, no sample may have a level exceeding 200 mg/kg, and two samples may have a concentration higher than 100 mg/kg but lower than 200 mg/kg. Finally, the fishery products which have undergone enzyme ripening or treatment in brine may have HIM levels not higher than twice the limits listed above. Figure 3 shows an example of HIM analysis performed using HPLC/FLD after on-line post-column chemical derivatization of the samples. Figure 2: Chromatogram examples of red tuna analysis. Quantified ascorbic acid: 300.9 mg/kg (**A**); quantified nitrites: 130.8 mg/kg (**B**). Other biogenic amines analyzed during this part of the study are shown in Figure 4, together with an example of a chromatogram obtained by ion chromatography. The levels of these compounds contribute to the assessment of food quality by calculating the Biogenic Amines Index (BAI):  
BAI = (cadaverine + putrescine + histamine)/(1 + spermine + spermidine)  
A product can be described as fresh for BAI values between 0 and 1 mg/kg; for BAI values between 1 and 10 mg/kg, the product is initially altered, and it is decomposing for BAI values higher than 10 mg/kg. The overall results obtained from these three samplings are reported in Tables 1 and 2. Figure 4: Biogenic amines: (a) Trimethylamine; (b) cadaverine; (c) putrescine; (d) spermidine; (e) spermine (**A**). Chromatogram examples of a standard solution at a concentration of 5 mg/L of trimethylamine, spermidine, and spermine, and 1.25 mg/L of putrescine and cadaverine, obtained by ion chromatography (**B**). Figure 3: Chromatographic analysis of two tuna samples: (a) histamine-free sample (blank); (b) sample spiked with HIM at 100 mg/kg. It was noted that the residual concentration of nitrite in the samples, given the same treatment, is highly variable. This can be due to the different dimensions, shape, and thickness of the red tuna slices injected with nitrite solution and related solution release during refrigerated storage. In particular, the concentration detected at the end of the first sampling was almost twice that quantified on the second sampling (equal to 152.1 mg/kg vs. 84.0 mg/kg), and even greater is that found in the third sampling, 200.9 mg/kg.