Beyond Pesticides, June 6, 2024

Researchers link pesticide exposure to behavioral effects on zebrafish (Danio rerio) larvae, signaling a larger issue for overall population and ecosystem effects. In a study published in Biomedicines, the authors conduct a multi-behavioral evaluation of the effects of three pesticides, both individually and as mixtures, on larvae.

As the authors state, “Even at low concentrations, pesticides can negatively affect organisms, altering important behaviors that can have repercussions at the population level.” By analyzing effects on individual zebrafish with single compounds and mixtures, this study shows the dangers of pesticides in aquatic systems regarding synergy (a greater combined effect when substances mix) and the ripples created throughout entire ecosystems.

Researchers from the Department of Morphology and Animal Physiology, as well as the Department of Physics, from the Rural Federal University of Pernambuco in Brazil collaborated with the Department of Biology at Indiana University in Indianapolis to perform the study. The researchers exposed zebrafish larvae to carbendazim, fipronil, and sulfentrazone to determine any behavioral effects on anxiety, fear, and spatial/social interaction for each compound separately and in combination.

Each compound and mixture were applied to embryo medium, exposing fertilized zebrafish eggs. The embryos of zebrafish hatch, or become larvae, within 48-72 hours of fertilization. For this study, the zebrafish were kept in the solutions for six days post-fertilization during which their behavior was monitored and recorded. The solutions, containing just embryo medium (controls) or the embryo medium and pesticide(s) (test groups), were refreshed daily to prevent degradation.

As the study finds, “Pesticide concentrations were based on values equal to or lower than the maximum residue limits (MRL) of these pesticides in foods allowed by the Brazilian Health Regulatory Agency,” which establishes real-world environmental exposure levels. In most ecotoxicology studies, lethal exposure is the primary focus, limiting the ability to determine effects for sublethal concentrations. These lower levels are crucial in understanding environmental impact because, as the authors state, “Pesticides are commonly found in the aquatic environment at low concentrations, which accumulate, and effects are amplified through the food chain.”

The behaviors of the zebrafish monitored include swimming patterns within the chamber; swimming behavior when touched; direction and distance of travel; and spatial interaction and social/escape responses to stimuli. These behaviors are important for the survival of zebrafish and many other species. As the study concludes, “Changes in swimming behavior can have important consequences for feeding behaviors (prey capture) and susceptibility to predation (predator escape), among others.”

Carbendazim, fipronil, and sulfentrazone were chosen as pesticides to test as they are all commonly used in crop production and have been detected in aquatic ecosystems and food products. There is also prior research linking each with effects on zebrafish and similar species. Carbendazim is a fungicide that causes disorganized swimming patterns, while fipronil is an insecticide that has been found to adversely affect the nervous system with test populations exhibiting convulsions and muscle spasms. Sulfentrazone is an herbicide with the least knowledge on its neurotoxicity, but it acts like an endocrine disruptor and affects zebrafish heart development.

Zebrafish are commonly used in ecotoxicology studies, especially as a model in behavioral studies “not only because of their morphological and genetic conservation with humans (60 to 80% homology), but also behavioral similarities, exhibiting a wide range of complex behaviors including social interactions, anxiety, learning, memory, and avoidance behaviors that may be useful for modeling neurological and psychiatric diseases,” the researchers say. Additional studies on zebrafish have found organ damage and transgenerational toxic effects with pesticide exposure.

Within this study, the authors report, “Significant changes were observed in the performance of larvae exposed to all compounds and their mixtures… Among the single pesticides, exposure to S[ulfentrazone] produced the most behavioral alterations, followed by F[ipronil] and C[arbendazim], respectively. A synergistic effect between the compounds was observed in the C[arbendazim] + F[ipronil] group, which showed more behavioral effects than the groups exposed to pesticides individually.”

Specifically, behavioral changes between the control groups and exposed groups are seen in the larvae and their responses to stimuli, movement (speed, trajectory, and distance traveled), and visual abilities. Immobility is one of the results of pesticide exposure, which is an extreme effect in terms of movement being impaired.

In the study’s discussion, the authors state, “The changes in the behavior result from one or a combination of molecular, biochemical, and physiological changes. The group exposed to sulfentrazone was the one that showed the highest number of behavioral changes. Showed significant differences in mean speed, distance traveled, and presented an abnormal trajectory.” This effect on muscle mechanics within the larvae can lead to lower rates of survival in the species.

The authors continue, writing, “Larvae exposed to fipronil showed a significant increase in immobile behavior during the exploratory activity test and showed an abnormal trajectory in the test.” Fipronil causes larvae to lose their mobility, which makes them more vulnerable to predators. Carbendazim is the least toxic of the three single compounds in the study, but zebrafish still exhibit behavioral effects in abnormal trajectory and impairment of their visual systems with exposure.

Being able to see and respond to visual stimuli is essential for fish, who need to process their surroundings to navigate their habitat, find food, and avoid predators. “About escape from the visual stimulus, larvae exposed to the single pesticides and their mixtures stayed significantly longer in the lower part of the well (area with stimulus) and did not show evasive behavior,” the study states. Since many stimuli in their natural environment can represent a threat, not being able to process and respond to potential predators can lead to death for impaired larvae.

The synergistic responses in zebrafish, such as with the reduction in speed, distance traveled, and optomotor response, show that mixtures of chemicals can increase effects on organisms. The study concludes, “These results highlight the complexity of pesticide mixture toxicity and the sensitivity of behavioral tests, which can be used as initial indicators of environmental stress. Experiments testing pesticide mixtures will help us understand their additive and synergistic environmental exposure effects.” Scientists and advocates have urged prioritizing further studies on the effect of mixtures on organisms, given that exposure in the environment is not limited to a single pesticide at a time.

This study helps show that pesticide exposure goes beyond human health issues to other vertebrates, invertebrates, and ecosystems in general. Pesticide use negatively impacts nontarget organisms and can “reduce the fitness of an individual, leading to population decline and serious effects on the ecosystem.” By using petrochemical pesticides and fertilizers, impacts can reach a multitude of organisms through the soil, water, and air that can lead to declines in populations that spread throughout the food web.

With alternatives available, petrochemical pesticides and fertilizers are causing unnecessary and avoidable effects on health and the environment. Organic agriculture offers a holistic approach to sustainable crop production that improves soil quality, increases biodiversity, minimizes water pollution, and more.  

Stay informed about pesticides and their negative effects with the Gateway on Pesticide Hazards and Safe Pest Management and through the Daily News Blog, which highlights the ever-growing body of science that supports the need for organic alternatives. Spring Into Action with a pesticide-free garden and make The Safer Choice to avoid hazardous home, garden, community, and food use pesticides.    

All unattributed positions and opinions in this piece are those of Beyond Pesticides.

Source:

Gomes, S. da S. et al. (2024) Behavioral effects of the mixture and the single compounds carbendazim, fipronil, and sulfentrazone on zebrafish (danio rerio) larvae, Biomedicines. Available: https://www.mdpi.com/2227-9059/12/6/1176.