## 2 Materials and Methods
### Biochemical Analysis  
At the end of the stress test, nine fish from each treatment group were euthanized by freezing, and three tissues were collected: gills, liver, and muscle from each fish, and preserved in an ultrafreezer at \(-\)80 \({}^{\circ}\)C until homogenization and analysis. Tissues were homogenized (1:5, _w/v_) in Tris-HCl buffer (100 mM, pH 7.75) with EDTA (2 mM) and Mg\({}^{2+}\) (5 mM). The homogenate was centrifuged at 20,000\(\times\)\(g\) (14,010 rpm) for 10 min at 4.0 \({}^{\circ}\)C and used for the following analyses. The total protein content was determined using the Biuret method.  
The total antioxidant capacity against peroxyl radicals (ACAP) was determined according to the methodology of [PERSON] et al. [38], with the detection of reactive oxygen species. The reading was carried out on a microplate containing 10 \(\mu\)L of tissue extract, 7.5 \(\mu\)L of ABAP solution (2,2-azobis-2-methylpropionamidine dihydrochloride) at a final concentration of 20 \(\mu\)M, and finally 10 \(\mu\)L H2 DCF-DA (2\({}^{\prime}\), 7\({}^{\prime}\) dichlorfluorescein diacetate) at a concentration of 40 \(\mu\)M. The plate was read using a fluorimeter (Victor 2, Perkin Elmer) at a wavelength of 485/520 nm and maintained at 37 \({}^{\circ}\)C. The result was interpreted as follows: the smaller the area, the greater the antioxidant capacity.  
Lipid peroxidation levels were determined by quantifying thiobarbituric acid reactive substances (TBARS), as proposed by [PERSON] and [PERSON] [39]. For this, 10 \(\mu\)L of homogenate was used, and 20 \(\mu\)L of butylated hydroxytoluene (BHT, 67 \(\mu\)M) solution, 150 \(\mu\)L of 20% acetic acid, 150 \(\mu\)L of 0.8% TBA solution, 50 \(\mu\)L of milli Q water, and 20 \(\mu\)L of 8.1% sodium dodecyl sulfate were added. The samples were then placed in a water bath at 95 \({}^{\circ}\)C for 30 min, followed by a 10 min rest at room temperature. Milli-Q water (100 \(\upmu\)L of milli Q water) and n-butanol (500 \(\upmu\)L of n-butanol) were added, and the samples were vortexed and centrifuged at 3000 rpm for 10 min at 15 \({}^{\circ}\)C to separate the phases. Then, 150 \(\upmu\)L of the supernatant was removed and placed in a microplate, and fluorescence was measured at 553 nm for emission and 515 nm for excitation using a fluorimeter (Victor 2, Perkin Elmer). The results are expressed as MDA per milligram of tissue.  
Glutathione-S-transferase (GST) activity was determined following the methodology described by [PERSON] et al. [40]. Following the procedure of incorporating 15 \(\upmu\)L of homogenized sample, 10 \(\upmu\)L of GSH 25 Mm, 80 \(\upmu\)L of CDNB, and a potassium phosphate reaction medium heated to 25 \({}^{\circ}\)C in a water bath. The absorbance of the samples was read using a spectrophotometer (BiotelELx 800, DeMorellis, Osasco, SP, Brazil) at 340 nm. The concentration of reduced glutathione (GSH) was measured according to the methodology proposed by [PERSON] and [PERSON] [41]. After incubating 240 \(\upmu\)L of sample extract with 28 \(\upmu\)L of trichloroacetic acid at a final concentration of 5% (TCA), centrifugation was performed for 10 min at 20,000 g at 4 \({}^{\circ}\)C. After centrifugation, 100 \(\upmu\)L of the supernatant was removed to mount the plate, 200 \(\upmu\)L of 0.4 M Tris-Base pH 8.9, and 10 \(\upmu\)L of 5, 5\({}^{\prime}\)-dithiobis (2-nitrobenzoic acid) (DTNB) were added. The plate was incubated at room temperature for 15 min and then read using a spectrophotometer (BiotelELx 800) at an absorbance of 405 nm. The results are expressed as \(\upmu\)mol of GSH per mg of protein.  
To measure the sulfhydryl group (P-SH) [41], the pellet formed in the previous analysis was resuspended in 240 \(\upmu\)L of homogenization buffer. Then, 20 \(\upmu\)L of the extract obtained was added to a microplate along with 160 \(\upmu\)L of 0.2 M Tris-Base (pH 8.2) and 10 \(\upmu\)L of DTNB. The plate was read using a spectrophotometer (BiotelELx 800) at an absorbance of 405 nm after an incubation time of 15 min.