## 3 Results and Discussion
### Oxidative Stress Indices  
The potentially negative impacts of zebrafish subchronic exposure to tramadol hydrochloride were also studied by conducting an analysis of selected oxidative stress indices. Most often, induction of oxidative stress in aquatic animals is assessed by using changes in various enzyme activities, which play crucial roles in antioxidant defense. The role of the first-line defense enzyme antioxidants is primarily provided by superoxide dismutase, CAT and GPx, which suppress or prevent the formation of free radicals or reactive species in cells. In the next defense line, the oxidized form of glutathione, created as a result of the reaction catalyzed by GPx, is regenerated to the reduced form of glutathione by GR [41; 42; 43]. An important enzyme in antioxidant protection is also multifunctional phase II biotransformation enzyme - GST, which is involved in the xenobiotic detoxification and in elimination of reactive oxygen species, as well as products of lipoperoxidation [44]. One of the significant manifestations of antioxidant defense disruption is lipid peroxidation, which is often used as a useful oxidative stress indicator [40]. In our study, changes in various antioxidant enzyme activities (CAT, GR, GPx and GST) and lipid peroxidation were evaluated. Obtained results are given in Figures 2-6. In our study, fish exposed to tramadol hydrochloride did not show any considerable changes in GR, GPx or CAT activities between control and experimental groups. Surprisingly, a significant increase in GST activity was recorded only at the concentration of 2 ug/L (\(p<0.01\)), compared to the control group. No changes were recorded in higher concentrations. An increase in GST activity, mainly in low environmentally relevant concentrations, was also found by [PERSON] et al. [45]. They studied the effect of salicylic acid on zebrafish, after 28 days  
Figure 1: Abnormal glycogen accumulation in liver of _Danio rerio_ after exposure to 600 ug/L of tramadol hydrochloride (hematoxylin and eosin, 400x). of exposure. An increase in activity of the detoxification enzyme GST probably indicates an activation of antioxidant system against xenobiotic. A similar study was performed by [PERSON] et al. [27]. They examined effects of tramadol hydrochloride on common carp (_Cyprinus carpio_) embryos and larvae. No changes in GR and GST activities after tramadol hydrochloride exposure were reported. In contrast, they observed a significant reduction in GPx activity at the highest concentrations of 100 and 200 \(\upmu\)g/L of tramadol hydrochloride. Furthermore, [PERSON] et al. [12] did not find differences in GR activity after exposure of a mixture of tramadol hydrochloride and naproxen sodium in common carp. They noticed a significant drop in GST activity, but only at a concentration of 100 \(\upmu\)g/L. Likewise, they recorded a decrease in GPx activity among various groups exposed to a mixture of tramadol hydrochloride and naproxen sodium (50, 100 and 200 \(\upmu\)g/L). Figure 3: Analysis of glutathione peroxidase activity (GPx) Data are expressed as mean \(\pm\) standard deviation. No significant differences (\(p>0.05\)) were found between control and experimental groups. Figure 2: Analysis of catalase (CAT). Data are expressed as mean \(\pm\) standard deviation. No significant differences (\(p>0.05\)) were found between control and experimental groups. The results of our study indicate a significant increase in lipid peroxidation after exposure to tramadol hydrochloride. In particular, TBARS levels were increased among groups in comparison to the control, but a statistically significant (\(p<0.01\)) difference was observed only in concentrations of 20 and 200 \(\mathrm{\SIUnitSymbolMicro g}\)/L of tramadol hydrochloride. Surprisingly, TBARS values were not significantly increased in the highest tested concentration, i.e., 600 \(\mathrm{\SIUnitSymbolMicro g}\)/L. The explanation for these results might be, as suggested by [PERSON] et al.