Transcriptomic and behavioral analyses reveal unique target tissues and molecular pathways associated with embryonic exposure to low level glyphosate and metal mixtures in zebrafish.

Investigation of developmental molecular events following exposure to environmentally relevant agrochemical mixtures is critical to predicting their potential long-term ecological and human health risks. Here, we sought to uncover transcriptomic changes during zebrafish (Danio rerio) embryonic development following exposure to glyphosate and co-exposure to metals. Glyphosate is widely used globally with an allowable drinking water limit of 700 ppb. We examined effects of glyphosate (10 ppb) alone and when co-exposed to a metal mixture containing low levels of arsenic (4 ppb), lead (5 ppb), cadmium (2 ppb), and vanadium (15 ppb). This mixture was derived based on behavioral and morphological toxicity findings and environmentally relevant concentrations found in agricultural regions where glyphosate and metals are ubiquitously present. Gene expression patterns coupled to a single-cell transcriptomic dataset revealed that developmental exposure (28-72 h post fertilization) to glyphosate dysregulates expression of developmental genes specific to the central nervous system. Subsequent studies indicated significant suppression of larval zebrafish movement with 10 ppb glyphosate exposure. Studies with glyphosate + metals mixture and metals mixture alone showed unique developmental transcriptomic patterns and behavioral changes compared to glyphosate exposure alone. However, some outcomes (e.g., changes in expression of genes involved in epigenetic regulation and extracellular matrix patterning) were common across all three exposures compared to the control. Notably, glyphosate + metals co-exposure distinctly suppresses lysosomal transcripts and targets renal developmental genes. While further studies are required to uncover the precise nature of the interactions between glyphosate and metals, our study shows that glyphosate at very low levels is a behavioral and neurotoxicant that changes when metals are present. Given this herbicide affects distinctive physiological processes, including renal development and lysosomal dysregulation when co-exposed with metals, we conclude that environmental cation levels should be considered in glyphosate toxicity and risk assessment.