Evaluating sub-lethal stress from Roundup® exposure in Artemia franciscana using 1H NMR and GC-MS

Global salinization trends present an urgent need for methods to monitor aquatic ecosystem health and characterize known and emerging stressors for water bodies that are becoming increasingly saline. Environmental metabolomics methods that combine quantitative measurements of metabolite levels and multivariate statistical analysis are powerful tools for ascertaining biological impacts and identifying potential biomarkers of exposure. We propose the use of the saltwater aquatic crustacean, Anemia franciscana, as a model organism for environmental metabolomics in saltwater ecosystems. Artemia are a good choice for ecotoxicity assays and metabolomics analysis because they have a short life cycle, their hemolymph is rich in metabolites and they tolerate a wide salinity range. In this work we explore the potential of Anemia franciscana for environmental metabolomics through exposure to the broad-spectrum herbicide, glyphosate. The LC50 for a 48 h exposure of Roundup (R) was determined to be 237 +/- 23 ppm glyphosate in the Roundup (R) formulation. Anemia cysts were hatched and exposed to sub-lethal glyphosate concentrations of 1.00, 10.0, 50.0, or 100 ppm glyphosate in Roundup (R). We profiled 48 h old Anemia extracts using H-1 NMR and GC-MS. Dose-dependent metabolic perturbation was evident for several metabolites using univariate and multivariate analyses. Metabolites significantly affected by Roundup (R) exposure included aspartate, formate, betaine, glucose, tyrosine, phenylalanine, gadusol, and isopropylamine. Biochemical pathway analysis with the KEGG database suggests impairment of carbohydrate and energy metabolism, folate-mediated one-carbon metabolism, Artemia molting and development, and microbial metabolism.