Goncalves_2026_Environ.Res__124596

The enzyme acetylcholinesterase (AChE) is one of the primary biomarkers used in the biomonitoring of aquatic ecosystems. However, traditional colorimetric methods exhibit selectivity limitations in complex matrices, such as mollusk, fish, and amphibian tissues. This study presents a rapid and selective HPLC-UV method for the determination of AChE activity, based on the quantification of the enzymatic conversion of 1-naphthyl acetate to 1-naphthol, using a novel extraction protocol designed to minimize matrix interferences. The method was validated following the ICH (International Council for Harmonisation) guidelines, demonstrating adequate selectivity, linearity, precision, accuracy, limit of detection (LOD), and limit of quantification (LOQ). We applied the proposed method to biological samples of three aquatic species from different regions with distinct degrees of anthropogenic impact. Field application revealed lower AChE activity in samples from contaminated sites (0.00 to 4.44 AChE units/mL) compared to the control (6.11 AChE units/mL), suggesting that the proposed method is suitable for aquatic contamination monitoring. To further validate our methodology, we determined trace metal concentrations (copper, chromium, nickel, and manganese) in biological samples and investigated the association between metal concentrations and AChE activity. The results revealed a clear association (negative correlation) between acetylcholinesterase enzymatic activity and metal concentrations. Thus, the proposed approach successfully identified that AChE activity decreased in regions most impacted by metals, indicating neurotoxic effects.