Herbicides are the most widely used agrochemicals worldwide, accounting for approximately 47.5 % of the total utilized in agriculture (Rajmohan et al., 2020). Glyphosate (N-(phosphonomethyl) glycine) is a synthetic, broad-spectrum herbicide commonly applied to control various weed species in crops such as rice and soybean. It belongs to the organophosphates group and works by inhibiting 5-enolpyruvylshikimate-3-phosphate synthase (EPSP) (Kanissery et al., 2019) an enzyme essential for the synthesis of aromatic amino acids in plants.

Structurally, glyphosate has a central carbon atom (C) that acts as the backbone of its molecular framework. This carbon atom is attached to three different groups: an amino group (NH2), a carboxylic acid group (COOH), and a phosphoric acid group (PO3H2). The amino group is crucial for glyphosate's herbicidal activity, facilitating its interaction with EPSP. The carboxylic acid group increases its water solubility, while the phosphoric acid group strongly binds to metal ions like manganese and zinc, essential cofactors for EPSP enzyme activity, disrupting the enzymatic processes critical for plant growth (Gill et al., 2018; Heineke et al., 1994).

Although glyphosate targets plants, it has been associated with alterations in acetylcholinesterase (AChE) activity (Lopes et al., 2022; Robb and Baker, 2023). The AChE enzyme degrades acetylcholine (ACh) into choline and acetate present in the synaptic clefs, playing a vital role in the cholinergic nervous system of insects (Lang et al., 2012), crustaceans (Pala, 2019), fish (Lopes et al., 2022), rodents (Wang et al., 2021) and humans (Roseiro et al., 2012). Specifically, human acetylcholinesterase (hs-AChE) contains a main binding site, composed of a catalytic triad of serine (Ser203), histidine (His447), and glutamate (Glu334), that catalyzes the hydrolysis of acetylcholine. Additionally, hs-AChE features a negatively charged anionic choline-binding pocket that interacts with the positively charged quaternary amine (N+) of ACh (C7H16NO2+), as well as with cationic substrates and inhibitors (Bourne et al., 2003; Dvir et al., 2010; Ordentlich et al., 1998; Sussman et al., 1991). Besides, experimental evidence indicates that organophosphates can inhibit hs-AChE activity through a nucleophilic attack on the oxygen atoms of regulatory serine residues in its catalytic site (Čolović et al., 2013).

The interference of glyphosate and glyphosate-based herbicides (GBH) with the cholinergic system of fish has been previously demonstrated. In vitro studies with fish brain and muscle show inhibition of AChE activity (Sandrini et al., 2013). In vivo exposure to glyphosate and to GBH reduces cholinesterase activity (Modesto and Martinez, 2010; Samanta et al., 2014; Sánchez et al., 2017), and induces AChE mRNA levels in fish (Lopes et al., 2017). However in 2023, Moraes et al. (2023) have observed an increase in AChE activity in fish exposed to environmentally relevant concentrations of glyphosate.

Amongst various fish species, the zebrafish (Danio rerio) stands out as a well-studied model for investigating the effects of glyphosate or GBH exposure on AChE activity. Zebrafish species are well characterized regarding brain organization, cell morphology, neuromodulator systems, and chemical sensitivity (Kalueff et al., 2014). Yet, to the best of our knowledge, studies demonstrating the interaction between glyphosate and AChE at the intermolecular level in any animal species have not been carried out.

The present study aims to address such knowledge gap by employing a novel approach that combines computational ecotoxicology and experimental techniques. Firstly, molecular docking simulations are used to predict how glyphosate interacts with zf-AChE at the molecular level. These computational findings are then validated using spectrofluorimetric techniques such as UV–vis and fluorescence. This validation provides mechanistic insights into the experimental concentrations that could potentially induce structural perturbations in the enzyme (González-Durruthy et al., 2022; Rial et al., 2022). This combined approach offers valuable insights into the potential effects of glyphosate on the zebrafish nervous system and opens new avenues for research in ecotoxicology.