# Evaluation of Sub-Lethal Toxicity of Benzethonium Chloride in _Cyprimus carpio_ [PERSON]
## 4 Discussion  
European Commission has imposed strict criteria for the use of chemicals in products to protect human health and environmental safety. One of these criteria includes the ecotoxicological assessments of acute, sub-acute, and chronic effects of chemicals on various aquatic and terrestrial organisms. Fish are the most sensitive species to pollutants, representing a bioindicator of the water quality and of the status of aquatic ecosystems, being suitable as a biological model in ecotoxicological studies [40]. Aerobic organisms, including the fish, have developed an antioxidant defense system against oxygen radicals derived from the exposed xenobiotics, which can prevent excess oxidation and the damage of cell structures. Among the indicators of oxidative stress, changes in antioxidant enzymes activities, and oxidative damage products, such as protein carbonyls and lipid peroxidation are well-known. Our previous studies on _Cyprinus carpio_ revealed the acute effects of BEC, the LC50 value after 96 h of exposure being calculated at 4.57 mg/L, which indicated acute toxicity on fish according to REACH classification criteria [41]. In this context, our study aimed to reveal the acute toxicity of BEC at a concentration of 1 mg/L in water (simulating the maximum of cationic surfactants detected in surface water) on _Cyprinus carpio_ by assessing the enzymatic and non-enzymatic antioxidative system, the level of lipid peroxidation and histological changes in hepatic tissue. The liver represents the principal system involved in the xenobiotics' metabolism of animals and its antioxidant enzymes are taken into consideration as biomarkers of hepatotoxicity induced by various chemicals. During the xenobiotics' transformation in reactions catalyzed by monooxygenases belonging to CYP 450 families, ROS are generated. The activities of antioxidant enzymes often increase when oxidative stress is generated in fish, in order to contribute to the detoxification of oxygen free radicals and prevent or limit macromolecule damage [42; 43]. However, it was shown that some enzymes can also be partially inactivated by the free radicals; hydrogen peroxide can inactivate SOD [44], superoxide anion and hydrogen peroxide inactivate CAT, and GST is sensitive to products of the Haber-Weiss reaction [45]. Our data showed that CAT and SOD activities decreased following BEC exposure for 96 h (Figure 1). Previously, it was shown that CAT enzymatic activity in _Oncorhynchus mykiss_ liver was significantly reduced under benzalkonium chloride (a cationic surfactant from the same class as BEC) starting from a concentration of 0.18 mg/L [15]. Similar results have been obtained in _Carassius auratus gibelio_ liver in case of exposure to deltamethrin, a decrease in the activities of antioxidant enzymes SOD and CAT as a result of superoxide anions accumulation [46]. However, a sub-lethal exposure for 30 days of freshwater fish _Oreochromis mossambicus_ at concentrations of 1 mg/L of anionic, cationic, or nonionic surfactants has led to severe oxidative stress in the liver, the activities of CAT, SOD, and GPx increasing significantly in the case of all tested surfactants [24]. The difference in fish responses could be explained by the different fish species (subtropical versus tropical fish) and the exposure time, which was longer compared to our experiment. Acute exposure of fish to surfactant induced an antioxidant defense response suggested by the changes of enzyme activities, which was in contrast to a chronic exposure that could induce the expression of an adaptive response. The previous studies showed that the organism's responses could be different and influenced by various factors, such as: fish species and their characteristics, exposure time (acute or chronic), surfactant type and tested concentration, laboratory test conditions (static/semi-static/dynamic test solutions, dilution water characteristics, aeration, and test vessels).