2.3. Experimental Design

The fish were randomly transferred from the housing aquarium to the experimental tanks, with a capacity of 5 L each, and left to adjust to the new space. Four experimental groups were divided from each other as follows: untreated, 0.24 μg L⁻¹ vit. B₁₂, 600 μg L⁻¹ FIP + PYR, and 0.24 μg L⁻¹ vit. B₁₂ + 600 μg L⁻¹ FIP + PYR. Zebrafish juveniles were exposed to a single dose of the pesticide mixture, while vit. B₁₂ was administered for a period of two weeks in order to simulate a real-life situation.

Following the accommodation period, the initial behavior of each fish was evaluated through the locomotor activity and social interaction tests.

A newly prepared pesticide solution was dissolved directly into the experimental tanks for the mixture and mixture + vit. groups. As regards vitamin administration, this was performed by placing each fish in a plastic vial filled with vit. B₁₂ solution and leaving it in for 30 min. The control and mixture groups simulated exposure to the vitamin. Each fish was tested in the T-maze to collect data on locomotor activity and sociability as in the initial assessment of behavior. These tests were repeated daily for two weeks. Data were acquired every day using a camera situated above the maze, connected to a computer with EthoVision XT 11.5 software (Noldus Information Technology, Wageningen, Holland), with which all the behavioral parameters were calculated. In the end, fish were killed by immersion in ice-cold water for a minimum of 5 min after opercular motion had stopped and then stored in the freezer at −80 °C for oxidative stress analysis. The study included two more replicates. A schematic representation of the entire study is presented in Figure 1.

The experimental design of the study for evaluating the impact of vitamin B₁₂ and pesticide mixture on zebrafish.