Small Molecule Screening and Toxicity Testing in Early-stage Zebrafish Larvae

Zebrafish have become a prominent model organism for human translational research, toxicity testing of small molecules and environmental pollutants, therapeutic drug discovery, and other biomedical research applications. The presented protocol aims to provide a clear guide for drug screening and toxicity testing in early-stage zebrafish larvae (aged 3-7 days post fertilization, dpf). Several methodologies describing toxicity testing in zebrafish larvae have been published, albeit lacking a standardized protocol for exposure durations, rate of media changes, and morphology scoring, amongst others. We experimentally determined the ideal larvae ages for conducting the experiments, the plate sizes that work best for those ages, the rate of media changes that provide reliable results while limiting wastage of test compound, and a morphology scoring system that is easily quantifiable in a statistical manner. The manuscript describes in detail a protocol for toxicity testing in zebrafish larvae aged between 3 to 7 dpf, aiming to target the gap in drug toxicity testing and taking the first step towards a standardized method similar to the Fish Embryo Acute Toxicity test.