Common Fungicide Cocktail Silently Rewires Fish Hearts, Nerves and Social Behavior

A commercial fungicide mixture sprayed on crops across the globe is doing far more damage to fish than simply killing them. New research on zebrafish reveals that exposure to an azoxystrobin and tebuconazole premix, at concentrations actually detected in rivers and permitted in drinking water, disrupts survival, slows the embryonic nervous system, scrambles heart rhythms, and fundamentally alters how young fish interact with their shoal mates. The findings, published in the journal Ecotoxicology, add to a growing body of evidence that pesticide mixtures—products deliberately formulated as chemical cocktails—pose ecological risks that conventional single-substance safety testing largely misses.

The study, led by Aline Pompermaier of the Federal University of Fronteira Sul in Brazil with collaborators at Purdue University, focused on a commercial premix fungicide containing two active ingredients: azoxystrobin, a strobilurin compound that blocks mitochondrial respiration in fungi, and tebuconazole, a triazole that inhibits sterol biosynthesis in fungal cell membranes. These dual mechanisms, belonging to Fungicide Resistance Action Committee Groups C3 and G1, make the product highly effective against a broad range of crop diseases, from soybean rust to wheat stripe rust. It is registered for use on more than two dozen crops, including cotton, rice, corn, coffee, sugarcane, and grapes, and is sold in countries including Brazil, Australia, Argentina, Canada, Spain, and Germany.

What makes the new work particularly significant is that the researchers tested the product exactly as it is sold—as a ready-made formulation containing 120 grams per liter of azoxystrobin, 200 grams per liter of tebuconazole, and a substantial fraction of undisclosed excipients. Most previous ecotoxicology studies have either tested single active ingredients or combined pure compounds in the laboratory. Commercial premixes, by contrast, enter waterways as complete formulations, and the inert ingredients they contain can modify how the active substances are absorbed and how toxic they become. The authors argue this distinction matters, and their results suggest it does.

The exposure regime was designed around environmental reality. Zebrafish embryos were placed in solutions containing 30, 100, or 180 micrograms per liter of the fungicide, with concentrations benchmarked against the highest dose of the mixture component, tebuconazole. The two lower levels correspond to tebuconazole concentrations actually measured in surface and groundwaters in Brazil and elsewhere, while the highest matches Brazil’s maximum allowable limit for tebuconazole in drinking water. Notably, all three levels fall well below concentrations of azoxystrobin recorded in some Brazilian agricultural watersheds, where average detections have reached 1,055 micrograms per liter and peaks of 6,672 micrograms per liter—values that dwarf the experimental doses.

Embryos were exposed from 3 to 120 hours post-fertilization, a window covering the critical organogenesis phase when the nervous system, heart, and other organs take shape. The zebrafish is an ideal model for such work: its embryos are transparent, develop externally, and within days display species-specific behaviors that can be quantified with simple video recording. The team monitored 110 embryos per group for survival and hatching, tracked spontaneous movement in 20 embryos per group under a stereomicroscope at 24 hours post-fertilization, and counted heartbeats at 72 hours. Later, they filmed shoaling behavior at five days and tested antipredator responses at seven days using a moving red sphere displayed on a monitor beneath the test plates.

The survival results were stark. Kaplan-Meier analysis revealed that larvae exposed to 100 and 180 micrograms per liter died at significantly higher rates than controls. In the 100 microgram per liter group, the hazard ratio indicated a 70 percent lower probability of survival compared with unexposed animals; at 180 micrograms per liter, survival probability dropped by 63 percent. These are not laboratory extremes—they are concentrations of tebuconazole documented in real waterways and legally permitted in Brazilian tap water. Hatching, remarkably, was unaffected, meaning the fungicide killed embryos after they emerged rather than preventing emergence itself. The authors point out that azoxystrobin, unlike tebuconazole, has no established regulatory limit in drinking water in Brazil, a regulatory gap they describe as a genuine threat to aquatic life.

Equally troubling were the sublethal effects on developing physiology. At the highest concentration, embryos showed significantly reduced spontaneous movement, the early wriggling behavior controlled by the nascent nervous system. Because this reflexive coiling is orchestrated by developing neural circuits, its suppression is widely interpreted as an early warning sign of neurotoxicity or broader developmental impairment. Heart rate told a stranger story: exposure to 30 micrograms per liter slowed larvae heartbeats, while 100 micrograms per liter sped them up. This non-monotonic, potentially biphasic pattern mirrors hormetic responses documented in fish embryos exposed to other contaminants, and the authors caution that the underlying mechanism—whether adaptive compensation, direct cardiotoxicity, or oxidative stress—remains unresolved and warrants dedicated investigation.

The behavioral findings may be the most ecologically provocative. In the shoaling test, larvae exposed to 100 and 180 micrograms per liter bunched significantly closer together than controls, with reduced average distance between individuals. Zebrafish are intensely social animals, and tight shoaling is their primary defense against predators. But abnormally heightened cohesion can signal an anxiety-like state—a baseline perception of danger that persists even when no threat exists. The researchers suggest the exposed larvae behaved as though a predator were always present. Intriguingly, when the team later confronted the animals with an actual simulated threat in the aversive stimulus test, the exposed larvae responded normally, dodging away from the moving sphere just like controls. This dissociation is telling: it indicates the fungicide did not paralyze the animals or destroy their motor capacity, but rather shifted their default behavioral state, plausibly through dysregulation of cholinergic neurotransmitter pathways that govern anxiety and sociability.

The mechanistic picture drawn from prior literature is consistent with this interpretation. Azoxystrobin has been shown to disturb acetylcholinesterase activity, elevate reactive oxygen species, and disrupt dopaminergic signaling in zebrafish brains, while tebuconazole is known to inhibit acetylcholinesterase in larvae, induce oxidative stress in carp and zebrafish, and cause neurochemical and behavioral alterations. A mixture combining a mitochondrial respiratory inhibitor with a sterol biosynthesis inhibitor may therefore impose a dual burden—neural and oxidative—on embryos whose defenses are still under construction, potentially overwhelming redox homeostasis and cardiac regulation simultaneously. The authors are careful to note that they did not measure biochemical biomarkers directly, framing these pathways as plausible hypotheses grounded in the literature rather than confirmed mechanisms, and they call for follow-up work measuring acetylcholinesterase activity, antioxidant enzymes, lipid peroxidation, and gene expression.

The ecological and regulatory implications are difficult to overstate. A fish larva locked in a perpetual state of vigilance burns energy that should fuel growth and reproduction, and an abnormally cohesive shoal may become easier for predators to spot or less efficient at finding food. Reduced larval survival at environmentally realistic concentrations, meanwhile, can ripple through food webs and disrupt trophic cascades. The study’s central message is that environmental risk assessment must evolve beyond testing isolated active ingredients and counting corpses. Sublethal endpoints—heart function, embryonic movement, social behavior—capture harms that mortality statistics miss entirely, and commercially formulated mixtures must be evaluated as the integrated chemical systems they are. As the authors conclude, premix fungicides entering the environment as ready-made cocktails may act additively or synergistically on aquatic fauna, and regulatory frameworks that ignore this reality leave ecosystems—and potentially drinking water supplies—protected by standards built for a simpler chemical age.

Subject of Research: Toxicological effects of a commercial azoxystrobin-tebuconazole fungicide mixture on zebrafish physiology and behavior

Article Title: Beyond mortality: A fungicide mixture alters physiology and causes impaired behavior in zebrafish

Article References: Beyond mortality: A fungicide mixture alters physiology and causes impaired behavior in zebrafish. (n.d.). https://doi.org/10.1007/s10646-026-03147-z

Image Credits: AI Generated

DOI: 10.1007/s10646-026-03147-z

Keywords: zebrafish, fungicide mixture, azoxystrobin, tebuconazole, ecotoxicology, pesticides, shoaling behavior, developmental toxicity, heart rate, neurotoxicity, water contamination, environmental risk assessment