Geochemical and environmental isotope techniques are successfully applied to understand the origin and evolution of groundwater in aquifers, with the aim of producing consistent conceptual models. In the present study, δ18O, δ2H, and δ13C stable isotope ratios and 14C and 3H levels were used for this purpose. One of the most important aquifer systems in South America, the Amazon Aquifer System (AAS), remains almost entirely unstudied in this aspect. The combination of hydrochemical and isotopic tracers was applied in a pioneer study to foster the understanding of the driving hydrogeological processes in the southwestern portion of the AAS, in the Acre Sedimentary Basin (ASB), northwestern Brazil. The study area comprises three aquifers: from top to bottom of the system, the Quaternary Aquifer, the Içá–Solimões Aquifer (Paleogene/Neogene), and the Tikuna Aquifer (Cretaceous). The aquifers have distinct hydrochemical characteristics, acidic pH and Na–HCO3 waters in the Quaternary Aquifer, pH ~8 and Ca–HCO3 and Na–HCO3 waters in the Içá–Solimões Aquifer, and neutral pH and Ca–HCO3 waters in the lower Tikuna Aquifer system. Ion exchange processes predominate in all three aquifers, but carbonate dissolution also occurs in some parts of the Içá–Solimões Aquifer. The Içá–Solimões Aquifer shows a geochemical evolution along the regional flow from southwest to northeast. δ18O and δ2H measurements of the groundwater align with the global meteoric water line, without a significant evaporative effect or dispersion. Groundwater from the Içá–Solimões and Tikuna Aquifers is more depleted than the weighted rainfall average (δ2H = −27.9‰ and δ18O = −5.35‰, respectively). The deuterium excess of the local rainfall is approximately +14‰. Residence times, estimated by 14C, range between 13 and 18 ka in the Içá–Solimões Aquifer and reach approximately 6 ka in the Tikuna Aquifer. In addition, tritium concentrations lower than 0.6 TU pointed to the absence of recent waters in the studied aquifers. Local structural and stratigraphic features in the Serra do Divisor area, where asymmetric folding and faulting occur, affect the functioning and features of these aquifer systems, originating from the erosive exposure of deeper and older layers to recent recharge. Thus, geologically older and subjacent aquifer units (Tikuna Aquifer) hold more recently circulating waters than the younger and shallower ones (Içá–Solimões Aquifer).
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