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Insecticides are designed to target the nervous system of fruit and crop pests (such as the Colorado potato beetle), however they often affect the nervous system of other organisms as well. (Photo: Shutterstock, Sidorov Ruslan)

Insecticides can affect fish behaviour

August 24, 2023 | Annette Ryser

The safety of insecticides is predominantly evaluated based on lethal effects on other animals. However, new findings from Eawag support the notion that also lower concentrations can be problematic, because they affect the nervous systems of fish and cause changes in their behaviour. This can indirectly affect the survival of fish populations and may be one of the reasons for the fish decline that we are observing in Switzerland. Researchers are uncovering the mechanisms of action and suggesting how these effects could be taken into account in the assessment and evaluation processes of insecticides.

Insecticides have a bad reputation. Most of them are designed to target the nervous system of fruit and crop pests (such as the aphid), but they often affect the nervous systems of other organisms as well. These include bees, aquatic insects – and vertebrates, such as fish or even humans. In some cases, this already occurs in very low concentrations. “Many fundamental aspects of the nervous system have changed very little over the course of evolution“, explains Ecotoxicologist Sarah Könemann, who researched the effects of insecticides in her doctoral thesis at the aquatic research institute Eawag and EPFL. For example, some insecticides target certain molecules in the nervous system of insects, but we have almost exactly the same molecules in our nervous system. This is why insecticides can have an effect on humans, too.

In most cases, the concentrations in which insecticides are found in the environment are not high enough to be acutely toxic, i.e. lethal, for vertebrates. ”However, there is a very wide range between a lethal effect and no effect“, explains Könemann. ”This is where I wanted to take a closer look.”

Fish can smell certain insecticides

For this reason, the researcher examined how six common insecticides affect the movement patterns and neuronal activity in the brains of zebrafish larvae. She discovered that these fish perceived the insecticides imidacloprid and diazinon as stress when they were exposed to a high concentration of them in a short time. And: Könemann was able to demonstrate that the fish larvae smelled these insecticides and subsequently avoided the substances. ”At first glance, this seems like a sensible reaction“, says the researcher. ”They flee from the insecticide and thus prevent chronic effects.”

Yet, such a change in behaviour can have negative consequences as well, e.g., if the fish avoid certain habitats as a result. This could lead to them no longer finding sufficient sexual partners or abandoning areas with a particularly rich food supply. “Such effects may therefore be another factor negatively impacting fish populations, which are already challenged by other stressors.”
 

Confocal microscopy image of a zebrafish brain stained with fluorescent dyes visualizing the active (red) and inactive (green) neuronal cells (Photo: Eawag, Sarah Könemann).
Confocal microscopy image of a zebrafish brain stained with fluorescent dyes visualizing the active (red) and inactive (green) neuronal cells (Photo: Eawag, Sarah Könemann).

Behavioural change at the push of a button

In her doctoral thesis, Sarah Könemann also looked at the developing nervous system of zebrafish embryos. “We anticipated that certain insecticides would have a particularly strong influence during the sensitive developmental phase, when the nervous system is forming”, explains the researcher. And indeed, for three quarters of the substances studied, she was able to demonstrate an effect on the behaviour of the fish: Larvae moved less than the control larvae.

Könemann was also able to identify structural changes in the muscle fibres and the peripheral nervous system that could explain the altered movement patterns. But there were also some surprises: Most of these effects (with the exception of those caused by pirimicarb) were reversible when the larvae were no longer exposed to the insecticides. In other words: “We were able to reverse most of the behavioural effects as well as the structural changes within 72 hours.”
 

The insecticide methomyl, for example, led to structural changes in nerve cells of the peripheral nervous system of zebrafish larvae: Compared to control (left), more branches were formed in the nerve tracts on the right. This effect disappeared when the larvae were no longer exposed to the insecticide (Photo: Environ. Sci. Technol. 2022 56 12 8449 8462).
The insecticide methomyl, for example, led to structural changes in nerve cells of the peripheral nervous system of zebrafish larvae: Compared to control (left), more branches were formed in the nerve tracts on the right. This effect disappeared when the larvae were no longer exposed to the insecticide (Photo: Environ. Sci. Technol. 2022 56 12 8449 8462).

Significance for the approval of insecticides

Könemanns investigations also revealed that a group of newer insecticides, which were specifically developed to act on a certain organ that only insects have, did not have far-reaching influence on the behaviour of the fish studied. This is the case for pymetrozine and flonicamid. “This shows us that the impact on vertebrates in the environment can be reduced if the substances are developed in such a way that they more specifically act on the target-organisms”, says Könemann.

She advocates that when evaluating and approving insecticides, one should not only consider whether they will eventually lead to the death of the test animals, but also consider whether their use (even at much lower concentrations) results in behavioural changes. “There is a lot of research going on in the field of ecotoxicology at the moment, especially in the development of tests that can determine the effects of insecticides or other chemicals on the developing nervous system of vertebrates”, says Könemann.

However: “Simply investigating whether a substance affects the behaviour of animals is not enough, in my opinion, to be able to classify the significance of such an effect. I recommend also looking closely at the mechanisms that lead to the behavioural changes and the potential of the organism to recover.”

Award-winning research

It is worth thinking outside the box to gain new insights. In her doctoral thesis, Sarah Könemann applied methods from the neurosciences to an ecotoxicological context and was awarded the Rifcon Early Career Scientist Award from the globally active Society for Environmental Toxicology and Chemistry (SETAC) in 2022 for her detailed and interdisciplinary work.
 

Sarah Könemann (left) and former Eawag Director Janet Hering at the 2022 SETAC Europe meeting in Copenhagen (Photo: Eawag, Barbara Jozef).
Sarah Könemann (left) and former Eawag Director Janet Hering at the 2022 SETAC Europe meeting in Copenhagen
(Photo: Eawag, Barbara Jozef).

Why are problematic insecticides found in water bodies?

In Switzerland, insecticides are approved primarily to be used as plant protection products and biocides. They are mainly used in agriculture, but also in urban areas, the food and feed industry, as well as forestry. They generally enter water bodies through the air and via run-off during rainfall. Although the standards for approving insecticides have been made more stringent in recent years and many substances have been banned, they can still be found in the environment. This is surprising insofar as the most current insecticides degrade quickly in nature. «The fact that we can detect them in our environment in Switzerland (albeit in extremely small quantities of a few pico to nanograms per litre) nevertheless means that they are still being released, despite the bans», says Ecotoxicologist Sarah Könemann. This is made possible by emergency approvals or their use as biocides in stables, which is not explicitly prohibited, unlike the use of the same substance in a field.

Cover picture: Insecticides are designed to target the nervous system of fruit and crop pests (such as the Colorado potato beetle), however they often affect the nervous system of other organisms as well. (Photo: Shutterstock, Sidorov Ruslan)
 

Original publications

Sub-lethal peak exposure to insecticides triggers olfaction-mediated avoidance in zebrafish larvae

In agricultural areas, insecticides inevitably reach water bodies via leaching or run-off. While designed to be neurotoxic to insects, insecticides have adverse effects on a multitude of organisms due to the high conservation of the nervous system among phyla. To estimate the ecological effects of insecticides, it is important to investigate their impact on non-target organisms such as fish. Using zebrafish as the model, we investigated how different classes of insecticides influence fish behavior and uncovered neuronal underpinnings of the associated behavioral changes, providing an unprecedented insight into the perception of these chemicals by fish. We observed that zebrafish larvae avoid diazinon and imidacloprid while showing no response to other insecticides with the same mode of action. Moreover, ablation of olfaction abolished the aversive responses, indicating that fish smelled the insecticides. Assessment of neuronal activity in 289 brain regions showed that hypothalamic areas involved in stress response were among the regions with the largest changes, indicating that the observed behavioral response resembles reactions to stimuli that threaten homeostasis, such as changes in water chemistry. Our results contribute to the understanding of the environmental impact of insecticide exposure and can help refine acute toxicity assessment.
Könemann, S.; Meyer, S.; Betz, A.; Županič, A.; vom Berg, C. (2021) Sub-lethal peak exposure to insecticides triggers olfaction-mediated avoidance in zebrafish larvae, Environmental Science and Technology, 55(17), 11835-11847, doi:10.1021/acs.est.1c01792, Institutional Repository

Zebrafish larvae rapidly recover from locomotor effects and neuromuscular alterations induced by cholinergic insecticides

Owing to the importance of acetylcholine as a neurotransmitter, many insecticides target the cholinergic system. Across phyla, cholinergic signaling is essential for many neuro-developmental processes including axonal pathfinding and synaptogenesis. Consequently, early-life exposure to such insecticides can disturb these processes, resulting in an impaired nervous system. One test frequently used to assess developmental neurotoxicity is the zebrafish light–dark transition test, which measures larval locomotion as a response to light changes. However, it is only poorly understood which structural alterations cause insecticide-induced locomotion defects and how persistent these alterations are. Therefore, this study aimed to link locomotion defects with effects on neuromuscular structures, including motorneurons, synapses, and muscles, and to investigate the longevity of the effects. The cholinergic insecticides diazinon and dimethoate (organophosphates), methomyl and pirimicarb (carbamates), and imidacloprid and thiacloprid (neonicotinoids) were used to induce hypoactivity. Our analyses revealed that some insecticides did not alter any of the structures assessed, while others affected axon branching (methomyl, imidacloprid) or muscle integrity (methomyl, thiacloprid). The majority of effects, even structural, were reversible within 24 to 72 h. Overall, we find that both neurodevelopmental and non-neurodevelopmental effects of different longevity can account for the reduced locomotion. These findings provide unprecedented insights into the underpinnings of insecticide-induced hypoactivity.
Könemann, S.; von Wyl, M.; vom Berg, C. (2022) Zebrafish larvae rapidly recover from locomotor effects and neuromuscular alterations induced by cholinergic insecticides, Environmental Science and Technology, 56(12), 8449-8462, doi:10.1021/acs.est.2c00161, Institutional Repository

Different developmental insecticide exposure windows trigger distinct locomotor phenotypes in the early life stages of zebrafish

Due to their extensive use and high biological activity, insecticides largely contribute to loss of biodiversity and environmental pollution. The regulation of insecticides by authorities is mainly focused on lethal concentrations. However, sub-lethal effects such as alterations in behavior and neurodevelopment can significantly affect the fitness of individual fish and their population dynamics and therefore deserve consideration. Moreover, it is important to understand the impact of exposure timing during development, about which there is currently a lack of relevant knowledge. Here, we investigated whether there are periods during neurodevelopment of fish, which are particularly vulnerable to insecticide exposure. Therefore, we exposed zebrafish embryos to six different insecticides with cholinergic mode of action for 24 h during different periods of neurodevelopment and measured locomotor output using an age-matched behavior assay. We used the organophosphates diazinon and dimethoate, the carbamates pirimicarb and methomyl as well as the neonicotinoids thiacloprid and imidacloprid because they are abundant in the environment and cholinergic signaling plays a major role during key processes of neurodevelopment. We found that early embryonic motor behaviors, as measured by spontaneous tail coiling, increased upon exposure to most insecticides, while later movements, measured through touch-evoked response and a light-dark transition assay, rather decreased for the same insecticides and exposure duration. Moreover, the observed effects were more pronounced when exposure windows were temporally closer to the performing of the respective behavioral assay. However, the measured behavioral effects recovered after a short period, indicating that none of the exposure windows chosen here are particularly critical, but rather that insecticides acutely interfere with neuronal function at all stages as long as they are present. Overall, our results contribute to a better understanding of risks posed by cholinergic insecticides to fish and provide an important basis for the development of safe regulations to improve environmental health.
von Wyl, M.; Könemann, S.; vom Berg, C. (2023) Different developmental insecticide exposure windows trigger distinct locomotor phenotypes in the early life stages of zebrafish, Chemosphere, 317, 137874 (10 pp.), doi:10.1016/j.chemosphere.2023.137874, Institutional Repository

Funding / Partnerships

  • Eawag
  • EPFL
  • University of Zurich
  • Swiss National Science Foundation

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