First steps in platform development for toxicology trials with Danio rerio

A dose of 0.32 µmol/L Pendimethalin was applied following OECD guideline 236 over 144 hours starting 2 – 3 hours post fertilization. For acute fish toxicity 50 - 70 embryos were preserved for each sample (effect group n = 4 , control group n = 8) in liquid nitrogen, followed by homogenization. About two milligrams of homogenate was used for non-targeted metabolite analysis by GC-MS.

Principle component analysis shown as biplot. The PCA was calculated with an targeted list of manually inspect compounds n = 20 (no missing value). PCA: Scale pareto (center), method svd, ellipse based on Hotelling's T-squared distribution.

left: Overview of the TIC. Overlay of wild type (green) and effect group (red).
right: Semiquantitative determination of cholesterol in scan mode, quantification ion m/z 129.

Boxplot of phosphoric acid (left) and cholesterol (right). Phosphoric acid and cholesterol are involved in many pathways, beside that phosphoric acid is a common functional group for endogenous metabolites as phosphate. However, an enhancement in pathways related to energy consumption for the treated group could be associated with stress induced by Pendimethalin.

Metabolomics can be used as an analytical lens into the biochemical state of a given organism. Primary metabolite profiling for toxicology trials provides an orthogonal view on traditional toxicology methods. GC-MS data could be combined with lipid and secondary metabolite LC-MS analysis. GC-MS analysis is a robust, reliable and standardized analytical method, which could be used in automatization and in high-throughput mode.