2. Materials and Methods
2.1. Experimental conditions
The experimental fish used in this experiment were provided by Shandong Oriental Ocean Sci-Tech Co., Ltd. (Shandong, China). The experimental area was equipped with 15 RAS, each comprising an aquaculture tank (3 m3), foam separator, waste collector, storage tank (1.5 m3), biological filter, dissolved O­2 source, circulating pump, and an integrated control box. The fish were domesticated for two weeks before the experiment. After acclimation, the fish were divided into five groups (three replicates in each group, each containing 3 samples). Following our experimental scheme and previous studies (Fivelstad et al . 2017; Khan 2018; Mota et al . 2019; Stiller et al . 2015), we used the following CO2 concentrations: 0 mg/L (control), and 8, 16, 24, and 32 mg/L. Fish treatment was approved by the Animal Protection and Utilization Committee of the Institute of Oceanography, Chinese Academy of Sciences.
Throughout the experiment, dissolved O2 saturation was 90% to 100%, temperature was 14.6 ± 0.1 °C, and salinity was 33 ± 1.0 ‰. The mean body weight of the experimental juveniles was 163.63 ± 1.92 g. Fish were initially stocked at a density of 2.35 kg/m3. Natural light was provided for 10–14 h per day. Fish were fed twice daily at 0.8% of body mass with a commercial fish feed (52% crude protein, 12% crude fat, 16.0% crude ash, 3.0% crude fiber, 12% water, 5% Ca, ≥2.3% P, ≥2.3% lysine, and ≤3.8% NaCl).