# Evaluation of Sub-Lethal Toxicity of Benzethonium Chloride in _Cyprimus carpio_ [PERSON]
## 4 Discussion
Therefore, the ecotoxicological data and the magnitude of monitoring effects are very varied [1; 3; 5; 8; 9; 14; 16]. GPx reduces the lipid hydroperoxides to hydroxylated lipid derivatives and GST is an enzyme capable of catalyzing multiple reactions in order to detoxify oxidized lipids. The depletion of these two enzymes activities in the liver showed their incapacity to decrease the level of lipid peroxides, explaining the increase of MDA level observed after 96 h of exposure to BEC (Figure 4). The decrease of GPx and GST activity (Figure 2) can be explained by the low level of GSH, the principal non-enzymatic antioxidant of cells that are used substrate by these enzymes. A balance between GSH synthesis and its utilization has to be maintained for proper cell functions. GSH is involved in the diminution ofROS level by acting as an electron-carrier substrate for several enzymes or by reacting directly with radicals in non-enzymatic reactions [47]. The significant decrease of GSH level observed within our study showed the impact of ROS accumulation on cell defense mechanisms. The same response was reported in the carp liver exposed to other pollutants [35]. GRed is involved in the recycling of GSH as this enzyme catalyzes the reaction in which oxidized glutathione returns to its reduced form in the presence of NADPH generated by G6 PDH within the pentose phosphate pathway [48]. A decrease of G6 PDH activity (Figure 3a) determined a decrease in NADPH, which was necessary for GRed to reduce oxidized glutathione. Therefore, the exposure to BEC induced a reduction of all these three antioxidants, GRed, G6 PDH, and GSH. In addition, the increased level of ROS affected the activity of LDH too (Figure 3b). This could be due to an increase of glycolysis rate versus Krebs cycle, consequent to a mitochondrial dysfunction induced by the exposure of fish to this cationic surfactant as previously was noticed [49; 50]. Lipid peroxidation induces oxidation of polyunsaturated phospholipids and changes of peroxidation products, such as MDA, can indirectly reflect cellular membrane injuries in hepatic tissue [51]. In our study, the unchanged level of MDA after 48 h compared to control could be explained by the conjugation of lipid peroxides with GSH catalyzed by GST. The increased level of MDA after 96 h highlighted the idea of ROS generation by BEC presence that interacted with polyunsaturated fatty acids in the context of antioxidant enzyme deficiency, such as GST activity decreased, as it was described also in other previous reports [52]. The present study showed that BEC can induce some unspecific structural changes in liver tissue (Figure 5), identified also for other types of xenobiotics [53; 54]. Generally, the observed changes in the liver tissues are considered compatible with functioning and survival but reported also some moderate effects, such as hepatocytes with cytoplasm vacuolization and an increased macrophage number related also with the decreases of antioxidant enzyme activities, lipid peroxidation being obtained only after 96 h of exposure to BEC. Previous studies on quaternary ammonium compounds, such as benzalkonium chloride and cetyltrimethylammonium bromide, reported oxidative stress, cell apoptosis, necrosis, and cell death, as these pollutants induced overproduction of hydroperoxide and superoxide anion [55]. Similar studies on surfactants' toxicity reported histological changes in the liver of _Cirrhinus mrigala_ associated with SOD and CAT changes [40]. There were reported accumulation of melanomacrophage centers, inflammation, and congestion of hepatocytes [40]. The increase in the density of melanomacrophage aggregates is generally correlated with important liver lesions [53]. Macrophage aggregates are considered areas of inflammation, oxygen-free radical formation, and lipid peroxidation, increasing in fish collected from heavily contaminated waters [56].