# Illegal Nitrite Treatment of Red Tuna and Prolonged Storage: What About Other Food Safety Risks?
## 3 Results and Discussion
The greatest variations in the parameters under investigation were again observed in the second sampling, in which case the levels of total bacterial count and Enterobacteriaceae were as much as 10 times higher for the treated sample. The absence of all pathogens was confirmed and there was no development of pathogenic microorganisms such as coagul-positive staphylococci, _Salmonella_ spp., _Escherichia coli_, or _Vibrio_ spp. in these samples. The results of the third sampling show high levels of total bacterial count and Enterobacteriaceae that were not reduced by adding the treatment to the samples. As appreciable from Table 3, where the mean values calculated considering all tests performed on fresh samples were compared to those obtained on nitrite-treated samples after 5 days of storage at 4 \({}^{\circ}\)C, although the latter results were higher, a statistically significant difference (\(p<0.05\)) was verified only for the histamine and TVBN concentrations. Anyway, although statistically significant, this increase does not represent a food safety concern, since the final values are still largely below the legal limits in force at the European level. Further studies in order to clarify the effect of nitrite treatment on red tuna samples in the presence of pathogen contamination were also investigated (Table 4). During these trials, the survival and the possible growth of _Salmonella_ spp. and _M. morganii_ were evaluated in tuna samples during storage of samples treated with nitrite and those not treated. As observed in Table 4, while the increase of contamination from 4 \(\times\) 10 CFU/g to 1.5 \(\times\) 10\({}^{2}\) and from 4.5 \(\times\) 10\({}^{6}\) CFU/g to 2.7 \(\times\) 10\({}^{7}\) CFU/g was verified for _Salmonella_ and _Morganella morganii_, respectively, the treatment with nitrite caused the decrease in both species' levels. More in depth, _Salmonella_ was inhibited, and it was no longer detected after 5 days of storage, where the initial contamination level was 10\({}^{2}\) CFU/g. The decrease in both species' contamination level from 4 \(\times\) 10 CFU/g to 2 \(\times\) 10 CFU/g, and from 1.7 \(\times\) 10\({}^{6}\) CFU/g to 9.0 \(\times\) 10\({}^{5}\) CFU/g was verified for _Salmonella_ and _Morganella morganii_, respectively. This means that the \"stabilizing\" effect of nitrite against microbial growth ensures that a possible contamination of tuna samples and the consequent prolonged storage after nitrite treatment do not pose a health risk due to the growth of _Salmonella_ and/or _Morganella morganii_. This finding was confirmed after chemical analysis of such samples voluntarily contaminated with _Morganella morganii_. Indeed, no HIM and other biogenic amines were detected in the fresh samples. After 5 days of storage, the final HIM concentration quantified in the non-treated sample was equal to 32.0 mg/kg, while a value of 11.0 mg/kg was quantified in the treated sample. Accordingly, the BAI calculated in the treated sample (0.7) result was lower than that of the non-treated sample (1.6). \begin{table}
\begin{tabular}{c c c} \hline \multirow{2}{*}{**Parameter**} & **T\({}_{0}\) (Control)** & **Nitrite-Treated Sample t\({}_{5\text{gg}}\)** \\  & **Mean Value \({}^{*}\)** & **Mean Value \({}^{*}\)** \\ \hline Histamine (mg/kg) & 2.8 \({}^{\text{a}}\) & 16.8 \({}^{\text{b}}\) \\ TVBN (mg/100 g) & 0.0 \({}^{\text{a}}\) & 17.1 \({}^{\text{b}}\) \\ BAI\({}_{\text{index}}\) & 0.0 \({}^{\text{a}}\) & 6.1 \({}^{\text{a}}\) \\ Total microbial count & 2.4 \(\times\) 10\({}^{6}\) \({}^{\text{a}}\) & 3.5 \(\times\) 10\({}^{7}\) \({}^{\text{a}}\) \\ _Enterobacteriaceae_ (CFU/g) & 1.0 \(\times\) 10\({}^{5}\) \({}^{\text{a}}\) & 1.7 \(\times\) 10\({}^{6}\) \({}^{\text{a}}\) \\ \hline \end{tabular} \({}^{*}\)n = 6 replicates; Values with different superscript \({}^{\text{ab}}\) statistically differ (\(p<0.05\)).  
\end{table} 
Table 3: Statistical analysis.