Methylene Blue Is a Widely Used Antifungal Agent That Confounds Behavioral Toxicity Assays in Larval Zebrafish

Zebrafish are widely used as model organisms in biological research with embryos typically reared in media supplemented with methylene blue (MB) as an antifungal agent. Many animal care guidelines recommend the use of MB during early development stages. However, this practice overlooks MB’s known effects as a monoamine oxidase inhibitor and antidepressant. This study demonstrates that at recommended husbandry concentrations, MB significantly reduces zebrafish locomotion in a 24 h behavior assay, a finding consistent across strains and laboratories. Gene expression profiling and pharmacological experiments using the MAO-inhibitor deprenyl suggest that MB induces hypolocomotion by increasing the serotonergic tone. Importantly, MB use in standard embryo medium masks known hypolocomotor responses to fluoxetine, a common aquatic contaminant and selective serotonin-reuptake inhibitor. These findings have significant implications for the increasing use of larval zebrafish in high-throughput neurotoxicity assessments and highlight the need to reconsider the use of MB in zebrafish research. The study emphasizes the importance of eliminating potential confounds in husbandry practices and improving experimental protocol reporting to enhance reproducibility in zebrafish-based (eco)toxicity testing.