EFFECT OF METHYLENE BLUE ON DEVELOPING ZEBRAFISH EMBRYOS Danio rerio

Our laboratory routinely conducts zebrafish (Danio rerio) developmental studies on 100s of chemicals, it is therefore imperative that the chemicals we use for animal husbandry are safe. It was brought to our attention that methylene blue, an antifungal agent commonly used in zebrafish embryo rearing, has been reported to affect swim bladder inflation (angelfish), increased fetal death (rats and humans), and produce hyperbilirubinemia, hemolytic anemia, and intestinal atresia (humans). Therefore, we conducted a developmental toxicity and neurotoxicity assessment of methylene blue in zebrafish using concentrations commonly employed by zebrafish researchers.

A 6 day developmental/neurodevelopmental assay was conducted using zebrafish embryos/larvae. At 0 days post fertilization (dpf), methylene blue exposure began in 96 well plates, which were kept in a 26^°C incubator on a 14:10 (light:dark) light cycle.  Embryos were dosed once daily on 0-4 dpf with 0.6, 1.6, 5.0 or 10.0 µM methylene blue (MEB); 1 µM chlorpyrifos (CPF) was used as a positive control (n=24 per chemical per dose).  At 5 dpf embryos were removed from the chemical, and placed in water, and then tested on 6 dpf.  

Behavioral testing consisted of assessing the locomotor activity of individual larval zebrafish in a 96-well plate, under a light:dark test paradigm: 20 minute acclimation in the dark, followed by 40 minutes of light (5 lux) and then 40 minutes of dark.   Embryos were examined daily on 0-5 dpf and after locomotor testing on 6 dpf for death, hatching, swim bladder inflation, and morphological appearance.

Although the CPF treated larvae showed the expected decrement in locomotor activity (Figure B1 inset), no differences were seen between MEB exposed and control larval locomotor activity (Figure A1) or any of the developmental aspects (death, hatching rate, swim bladder inflation or dysmorphology).  These data indicate that MEB is not developmentally or neurodevelopmentally toxic to larval zebrafish at commonly used concentrations.  This abstract does not necessarily reflect U.S. EPA policy.