Developmental and behavioral disruption in zebrafish larvae by mixtures of anatoxin-a and neonicotinoids

Harmful algal blooms caused by cyanobacteria have intensified globally due to eutrophication and climate change, posing increasing risks to aquatic ecosystems and public health. During blooms, cyanobacteria can release potent neurotoxins such as anatoxin-a, a nicotinic acetylcholine receptor (nAChR) agonist that causes rapid neurotoxicity. Likewise, neonicotinoid insecticides, including acetamiprid and thiacloprid, also target nAChRs and frequently contaminate aquatic environments. Despite their shared mode of action and environmental co-occurrence, their combined toxicity remains poorly understood. This study evaluated the individual and combined effects of (±)-anatoxin-a, acetamiprid, and thiacloprid on zebrafish (Danio rerio) larvae using the Fish Embryo Toxicity (FET) test. Sublethal endpoints included morphology, morphometrics, cardiac function, locomotor behavior, and skeletal muscle integrity. The 120-h EC₅₀ values were 1.07 mg/L for (±)-anatoxin-a, 51.04 mg/L for acetamiprid, and 36.55 mg/L for thiacloprid, confirming the markedly higher potency of the cyanotoxin. Individual exposures caused impaired locomotor activity, uncontrolled movements and trembling, pericardial enlargement, reduced heart rate, developmental alterations including reduced body length, eye size and swim bladder inflation, and disruption of muscle organization characterized by increased somite angle and reduced birefringence. Binary and tertiary mixtures frequently produced synergistic interactions, particularly for behavioral, cardiac, and muscle-related endpoints, with several combinations inducing toxicity beyond that predicted by concentration addition. Notably, mixtures containing environmentally relevant concentrations of anatoxin-a elicited significant behavioral, cardiotoxic, and neuromuscular impairments. These findings demonstrate that co-exposure to natural and synthetic nAChR agonists can substantially enhance developmental toxicity and highlight the need to incorporate realistic mixture scenarios into environmental risk assessment.