*2.2. Analytical Method*

At 0, 6, 24, and 48 h, three fish from each tank were randomly collected for enzyme activity measurement. The fish were anesthetized with overdose AQUI-S (100 mg L−1, AQUI-S New Zealand Ltd., Lower Hutt, New Zealand) and dissected on an ice tray. The liver, gill, and muscle samples were weighed and immediately stored in liquid nitrogen. For each assay, an individual sample was partially thawed, weighed, and homogenized using a glass homogenizer on ice in 0.2 M NaCl. The suspensions were centrifuged at 2500 r·min−<sup>1</sup> for 10 min at 2 ◦C. Then, the supernatant was incubated in the enzyme substrate and read on a spectrophotometer (Synergy H1, BioTek Instruments, Winooski, VT, USA). All of the measurements were conducted in triplicate.

The protein content was determined by the Coomassie Brilliant Blue method with bovine serum protein as the standard used the protein quantitative kit (catalog number A045-4, Nanjing, China), incubated at 37 ◦C for 30 min at 562 nm wavelength, and the protein concentration measured by microplate colorimetry. The SOD test kit (catalog number A001-3, Nanjing, China) was used to measure the activity of the SOD in the animal tissue samples. The activity of the SOD was determined by the xanthine oxidase method. The absorbance value was measured at the wavelength of 550 nm by colorimetry to calculate its activity. The activity unit was defined as follows: when the SOD inhibition rate reached 50% per milligram of tissue protein in 1 mL of the reaction solution, the corresponding amount of SOD was 1 SOD activity unit (U·mgprot<sup>−</sup>1). The GSH-Px activity in tissues can

be measured with a GSH-Px determination kit (catalog number A005-1, Nanjing, China). The GSH-Px activity was expressed by the consumption rate of GSH in the enzymatic reaction, while the more stable yellow substance formed by GSH and dithiodinitrobenzoic acid can be determined by colorimetry to calculate the GSH-Px activity. Through the colorimetric method, a 1 cm optical path cuvette was used at 412 nm wavelength, the distilled water was adjusted to zero, the absorbance value was measured, and its activity was calculated. The activity unit U represents that the GSH concentration in the reaction system is reduced by 1% per milligram of protein per minute by deducting non-enzymatic reaction <sup>μ</sup>mol·L−1. The MDA determination kit (catalog number A003-1, Nanjing, China) was used to measure the content of MDA in the animal tissues. The MDA was condensed with thiobarbituric acid to form a red substance, and MDA was determined by colorimetry at 532 nm. The method according to Gan L as well as other methods were used with slight modifications [23,24].
