Peek-A-Boo Test: A Simple Test for Assessing the Effect of Anxiolytics on Fish Behavior

The potential of pharmaceuticals and personal care products to alter the behavior of aquatic organisms is a growing concern. To assess the actual effect of these substances on aquatic organisms, a simple but effective behavioral test is required. We devised a simple behavioral (Peek-A-Boo) test to assess the effect of anxiolytics on the behavior of a model fish (medaka, Oryzias latipes). In the Peek-A-Boo test, we investigated the response of medaka to an image of a predator fish (donko fish, Odontobutis obscura). The test revealed that the time taken for test medaka exposed to diazepam (0.8, 4, 20, or 100 µg/L) to approach the image was shorter by a factor of 0.22 to 0.65, and the time spent in the area close to the image was longer by a factor of 1.8 to 2.7 than in the solvent control group for all diazepam exposure groups (p < 0.05). Hence, we confirmed that the test could detect changes in medaka behavior caused by diazepam with high sensitivity. The Peek-A-Boo test we devised is a simple behavioral test with high sensitivity for fish behavioral alteration. Environ Toxicol Chem 2023;42:2358-2363. © 2023 SETAC.