Effect of climate change on hematotoxicity/hepatoxicity oxidative stress, Oncorhynchus mykiss, under controlled conditions

Total antioxidant status (TAS). Was measured using the method developed by Erel [29] on an auto-analyzer. In this method, while measuring the TAS level, the Fe2+-o-dianisi- dine complex forms the OH radical by forming a Fenton-type reaction with hydrogen per- oxide. This strong ROS is reduced and reacts with the colorless o-dianisidine molecule at low pH and brown-yellow colored dianisidyl radicals are formed. Dianiside radicals par- ticipate in further oxidation reactions, enhancing color formation. However, the samples’ antioxidant compounds depress these oxidation-reduction reactions and diminish the color formation. TAS results were determined by reading this decrease spectrophotometrically. Results areexpressed as mmol Trolox Equiv/L. (TOS) levels were measured with automatic colorimetric method according to [29]. In this method, the sample’ oxidants oxidize the fer- rous ion-o-dianisidine complex to ferricion. The glycerol in the environment accelerates this reaction approximately three times. In acidic medium, ferric ions form a colored complex with “xylenol orange”. The intensity of this color in relation to the amount of the samples’ oxi- dants was measured spectrophotometrically in the autoanalyzer and TOS values were obtained. The results were recorded as μmol H2O2 Equiv/L. Oxidative stress index (OSI) was calculated as: OSI = TOS/TASx10, according to [29]. Measurement of erythrocyte and tissue supernatant myeloperoxidase (MPO) enzyme activity. Was performed according to Bradley et al. [30]. This method is based on the reduction of

H2O2 oxidized by MPO with O-dianisidine hydrochloride and measuring the absorbance of this reduced product at 460 nm [30, 31]. Measurement of lipid peroxidation level. The level of malondialdehyde (MDA), which is a product of lipid peroxidation, was determined [23]. The tubes were mixed for a short time by taking 200 μl from the prepared homogenate and adding 800 μl phosphate buffer and 25 μl of a synthetic antioxidant (BHT) and 500 μl of 30% TCA. It was then incubated at -20 ˚C for 2 hours. After incubation, the samples were centrifuged (15 minutes 2000 rpm) and 1 ml of the supernatant was taken and transferred to another Eppendorf tube, 75 μl EDTA-Na2H2O, 250 μl TBA were added and after a light vortexing process, they were kept in a water bath (90 ˚C) for 15 minutes. MDA levels were calculated by reading the samples cooled to room tem- perature at 532 nm [23, 31]. 2.2 Statistical analyses At the end of the study, the data of the groups were evaluated using the SPSS (version 13) sta- tistical package program. Duncan’s multiple comparison test was used to identify different groups following analysis of variance. The raw values from all analyzes were presented as the mean ± standard error of the groups. The statistical significance level was taken as p <0.05 in the calculations. 3. Results It was found that different temperature applications caused alterations in rainbow trout blood samples and these changes were significant at p<0.05 level (Fig 1). RBC, Plt, Hct and Hg levels decreased due to temperature increase, while increases in WBC level were determined. RBC,

Plt, Hct and Hg levels decreased by 25%, 40%, 20% and 23%, respectively, compared to the minimum value of optimum temperature group (Group A 12 ˚C) and WBC increased by 40% compared to the hematological index values determined in the group other than the optimum temperature values (D group 21 ˚C). To understand how thermal stress affects the metabolism of this species and to assess the effect of changing temperature on blood and liver, we measured oxidative markers in these PLOS ONE Effect of temperature on fish physiology and biochemistry PLOS ONE | https://doi.org/10.1371/journal.pone.0294656 November 30, 2023 5 / 13