Comprehensive toxicity assessment of anaerobic digestates from livestock manure in <i>Rhinella arenarum</i> larvae

The use of anaerobic digestates from livestock manure as biofertilizers is a sustainable practice within the framework of the circular economy. However, their potential toxicity to aquatic ecosystems is insufficiently understood. This study assesses the toxicity of three liquid digestates (LDs) derived from pig (DP), dairy (DD), and cattle (DC) manure by means of standardised bioassays with Rhinella arenarum larvae. Physicochemical and microbiological characterisation was conducted and compared with regulatory standards. Biomarkers of oxidative stress (catalase and glutathione-S-transferase activities, and glutathione content), anticholinesterasic activity (acetylcholinesterase and butyrylcholinesterase activities), and genotoxicity (micronucleus frequency) were also performed. The LDs exhibited high levels of organic matter, carbon, and nutrients, confirming their potential as agricultural amendments. Elevated concentrations of ammonium, metals (Cu and Zn), and electrical conductivity were also detected. The DP (LC50-504 h = 0.338%) and DC (LC50-504 h = 0.347%) showed high chronic toxicity, whereas DD (LC50-504 h = 1.18%) was significantly less toxic. All LDs altered oxidative stress biomarkers, induced anticholinesterasic effects, and caused genotoxicity at sublethal concentrations. These findings highlight the importance of amphibian bioassays in the ecotoxicological evaluation of anaerobic digestates prior to their environmental application.