14-day exercise behavior records of rainbow trout in the control group and the microplastic exposure group

After the official experiment commenced, we removed one fish each from the control and treatment groups on days 0 and 14, respectively, and placed them in a water tank (810×605×590 mm). The tank was filled to a water depth of 400 mm, and the water quality parameters (temperature, pH, DO) were maintained consistent with the rearing conditions. Behavioral recordings were conducted under uniform overhead white light illumination at an intensity of 300 ± 20 lux, measured at the water surface. Throughout the experiment, we ensured that the indoor environment remained quiet and undisturbed, while positioning the camera at an appropriate location. After allowing the camera to acclimate for 10 minutes, we recorded the movement trajectory of the rainbow trout over the subsequent 15 minutes. The experiment was conducted with three biological replicates. For each replicate, one healthy individual was randomly selected from an independent rearing tank containing 20 rainbow trout. Each tank was considered one biological replicate, with three tanks each for the control group and the exposure group. The 15-minute video recordings were processed to quantify locomotor activity. Briefly, video files were converted to image sequences for trajectory analysis using ImageJ software (version 1.8.0-172). The total swimming distance and average speed for each fish were calculated based on the tracked movement trajectories.