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

[18]. Mass fish mortalities are increasingly occurring in freshwater and marine habitats after unusually heat waves and hot weather. This has been described as the greatest challenge of aquatic ectothermic fauna due to the elevated frequency, intensity and duration of heat waves. The mechanism underlying all these claims is not yet fully understood. This is only possible by monitoring individual responses and physiological functions. Therefore, experimental biology is particularly important in understanding the effects of global warming on fish [19, 20]. As a result of the sensitivity of physiological parameters and to adapt to temperature changes, fish alter their endocrine processes, metabolic rates, immune and antioxidant systems [7, 9]. Based on the above strategies, in this article, we investigated the complex interaction between global warming and the physiology of freshwater aquatic organisms. In relation to the general animal physiology and oxidant levels of anthropogenic temperature increase, aquatic organisms, in particular freshwater and cold-water fish, rainbow trout (Oncorhynchus mykiss) are selected to study and discuss multiple biomarker responses of increasing water tempera- tures from a broad perspective. 2. Material and methods Certified juvenile rainbow trout (O. mykiss) were obtained from Atatu¨rk University Fisheries Faculty Inland Water Fish Application and Research Center (B.25.42.0028, 01.01.2013 by Agri- culture Ministry, Turkiye) with an average weight of 20 ±0.8 g. After a 14-day acclimation

period, 480 rainbow trout were treated as indicated below. The feeding was done twice a day with commercial trout pellets with the rate of 1.5% of body weight during the acclimatization periods The trail design was organized as 30 fish x 4 group with 4 replications. Fish were ran- domly distributed to fiberglass tanks according to the planned temperature applications. Experiment methodology, design protocol, and fish welfare were approved by Atatu¨rk Univer- sity Faculty of Veterinary Medicine, Experimental Animal Use, and Ethics Committee (approved in 28.12.2021, Acceptance No:11). The stock ratio in these tanks (as 20 kg/ m3) was determined with the aim of reaching the final weight of 250±17 g. For this purpose, the limits on which water temperatures in the cur- rent working environment can be manipulated have been studied. At the determined tempera- tures (12±0.3˚C, B: 15±0.3˚C, C: 18±0.3˚C, and D: 21±0.3˚C), O2 level for fish between temperatures was adjusted by increasing the amount of water for survival comfort. Thanks to this setup, O2 level was kept above 7.5–8 mg L-1 in all groups. For reaching to 250 g final live weight day counts according to the groups were as A: 12±0.3˚C (315 days), B: 15±0.3˚C (300 days), C: 18±0.3˚C (310 days), and D: 21±0.3˚C (310 days). At the end of the time when reached to the determined weight at 4 different temperatures [A: 12±0.3˚C, B: 15±0.3 ˚C, C:18 ±0.3 ˚C, and D: 21±0.3 ˚C] under controlled conditions, random samplings were made from the fish belonging to each temperature group. In order to determine the stress physiology and

antioxidant status, the blood, liver and muscle tissue samples of fish (n = 15) from each tank- were taken. 2.1 Determination of blood physiology and antioxidant status a. Determination of hematologic indexes. The rainbow trout were transferred from the tanks to an aerated clove oil anesthetic bath (60 mg/L). The samples were collected from the caudal vein with syringe. Blood samples were analyzed with PROKAN-6800VET fully auto- matic blood counting device. For each sample, leukocyte (WBC), hematocrit (HCT), hemoglo- bin (HGB), erythrocyte (RBC), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC) values were measured. The working principle of the blood counting device is based on the counting and PLOS ONE Effect of temperature on fish physiology and biochemistry PLOS ONE | https://doi.org/10.1371/journal.pone.0294656 November 30, 2023 3 / 13