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Food and Agriculture

Pesticide exposure and impacts on zebrafish development—another read and reason to eat more organic

Photography by Tracey Creed
Words by Isabel Forner

Published August 9 2021

According to the European Commission, a pesticide is “something that prevents, destroys, or controls a harmful organism ('pest') or disease, or protects plants or plant products during production, storage and transport”. Also the term "-cide" comes from the Latin word "to kill”. It is clear that pesticides have contributed to improved crop yields, and to an extent food security since the 1950s, in turn farmers earning potential. It has not come without undesirable outcomes—environmental, ecological and human health impacts. The Food and Agriculture Organization (FAO) estimates that between 20 to 40 per-cent of the annual global crop production is lost due to pests. In the United States, each year, plant diseases cost around US$220 billion, while invasive insects approximately US$70 billion. For that, the use of pesticides became common practice and consequently, their consumption has progressively increased from 1990 (1.55 kg/ha) to 2018 (2.63 kg/ha) with a total of 4 million tons of pesticides applied in 2018.

Pesticide exposure—applications and global overuse

Due to widespread and excessive use of pesticides, pesticide residues can be found in our food, drinking water, air or home dust. For instance, a recent five-year survey in Germany found that pesticides or their metabolites are present in 60 per-cent of the 2.280 sampling points of groundwater. In the Netherlands, 65 per-cent of surface water samples taken in 2013 contained 30 or more insecticides. Consequently, restrictive regulations are being implemented in the use of some agrochemicals, from 1972 for the United States and 1979 in the European Union. Indeed, the number of approved active substances for pesticides has been cut down by 50 per-cent in the European Union in the last few years. Additionally, several policies regulated, for example, the maximum level of pesticides allowed in drinking water or the highest level of pesticide residue that is legally tolerated in food.

Nevertheless, several scientific studies reported the presence of pesticides in human fluids, hair or tissues (for example: here, here, here or here). In humans, pesticides have been linked to several neurologic diseases (i.e. Parkinson, Alzheimer) and a broad range of non-specific symptoms (i.e. headache, dizziness, fatigue, weakness, nausea). We can say that pregnancy is one of the most critical periods because pesticide exposure begins during the gestation by a mother-to-fetus chemical transfer. Transfer of pesticides during pregnancy can have further negative implications for the fetus development and can predispose to adult health problems. Fetuses and newborns are highly vulnerable because their immune system and detoxification mechanisms are not fully developed as they are in adults. Indeed, existing epidemiological evidence linked the exposure of pesticides with adverse pregnancy outcomes in humans (for example: here, here, here or here) and in animal models (for example: here, here, here or here).

Pesticide exposure and neurological disorders in zebrafish

Despite accumulated epidemiological data establishing links between exposure to pesticides and health disorders, experimental research examining offspring neurodevelopment from perinatal (during pregnancy) or postnatal (after pregnancy) pesticide exposure is sparse and not enough. For that, during the last years, our research group in France has been investigating the effect of common pesticides on early neurological development.

To study that, we use a laboratory animal: the zebrafish (Danio rerio). Zebrafish is a small, 3-4 centimeter adult-size, tropical freshwater fish that researchers are using as a model organism to address specific scientific questions. Currently, there are around the world more than 700 labs using this little fish instead of the traditional mammalian models (i.e. mouse, rat, sheep) with a complete on-line wiki. With this teleost, we try to reduce the use of mammals, as well as complex and expensive laboratory setups. Furthermore, zebrafish is a very interesting model because it is translucent during the first days of its life, as you can see in the image below. It means that we can easily study the very early development and the organogenesis—the formation and development of the organs.

Zebrafish is an excellent model to study human diseases, from cardiac regeneration to muscular dystrophy or neurological disorders, as it shares 70% of its genes with us. Its genome has been fully sequenced, allowing us to use several genetic tools, to create transgenic animals and study certain gene functions. As a curiosity, zebrafish have also been sent to the International Space Station to study health issues in microgravity conditions.

Impacts of glyphosate exposure on zebrafish function

For our research, we first focused on the effects of one of the most controversial herbicides, the glyphosate. Glyphosate is the most widely used non-selective herbicide in the world ubiquitously detected in the environment and in human fluids. In addition, glyphosate has been linked to several neurodevelopmental disorders in different animal models (i.e. mouse, rat). From there, we tested different environmental concentrations of glyphosate in zebrafish, specifically during the larvae stage—the first five days of life of the fish, a very sensitive period when the zebrafish is developing its nervous system. After five days, we measured the locomotor activity and observed that the distance, the sense of orientation and the velocity of swimming were significantly lower than for non-exposed fish. In other words, at 1 and 10 mg/L of glyphosate in water, the glyphosate-exposed zebrafish were “lazier”. We can observe an example in the following image, where we have in red the swimming path of the zebrafish. The non-exposed (first line of wells) versus the glyphosate-exposed fish (second to fourth line of wells).

Glyphosate exposure and brain inflammation in zebrafish

In parallel, we performed electrophysiological recordings. Electrophysiology is the branch of neuroscience studying the electrical activity of neurons. So, with the use of micro-electrodes allocated in the head of the zebrafish, we observed abnormal electrical activity in the frontal part of the brain. In other words, neurons from zebrafish exposed to environmental concentrations of glyphosate were sending more “messages” than in a normal situation, consequently, we detected more neuronal activity.

Next, we asked whether this outcome could be followed by brain structural modifications. For that, with transgenic zebrafish (zebrafish with exogenous genes added to the genome to generate fluorescent cells), we detected that certain types of brain cells, called microglial cells, showed morphological modifications. This is remarkable because microglia cells are a hallmark of neuro-inflammation, and such alterations during early development can impair several aspects of the brain development. In the image below, we can see an example of a transgenic zebrafish, with the neurovascular system in green and the microglial cells in red.

Summarizing, only five days of glyphosate exposure at environmental concentrations during the first neuro-developmental steps was able to modify the swimming patterns, alter the electrical activity of neurons, and change the morphology of inflammatory brain cells. If you are interested, you can read the full text here.

Afterwards, in a second study, we decided to expose zebrafish to a mixture of pesticides. Given the high use of agrochemicals, we usually found mixtures of pesticides, rather than one single component, diluted in rivers and streams. We selected a mix of six pesticides commonly used in apple orchards and rarely investigated—captan, ziram, chlorpyrifos, boscalid, thiophanate and thiacloprid. Following the same protocol, zebrafish embryos were exposed for five days to different mixture concentrations. Surprisingly, after five days, concentrations higher than 0.01 mg/L already caused significant body malformations and delayed development, as you can see in the below picture.

Furthermore, differing from the glyphosate results, the behavioural analysis showed an increase in swimming activity, consequently, mixture-zebrafish were more active. Interestingly, when they were exposed to one single component of the mixture, the locomotor response was different, meaning that the impact of a mixture is rather unpredictable, and further studies are needed to understand how mixtures can affect the organism's wellbeing. If you would like to read all the details about this study, you can click here.

Finally, we would like to say that the results you have read here should not be extrapolated to humans or other species (i.e. rodents) yet. This is not always appropriate because, for example, different metabolic pathways exist among animals and humans or different detoxification processes, and no clear-cut approaches exist (at the moment). Human studies have a lot of limitations, therefore, animal toxicology studies like these help us to identify potential biomarkers or early defects from pesticide exposure.

Reducing your pesticide exposure and risk

Over the last decades, statistics reveal a progressive increase in pesticide manufacture and consumption across the world. Studies reporting the presence of pesticides in different environmental matrices and wild species are proven evidence of the environmental problem we are dealing with. As we commented, traces of pesticides have been detected in different human fluids and tissues, and several pieces of research have underscored adverse effects on human health.

A very sensitive window is the gestational period and the first stages of postnatal development. Epidemiological data evidenced that gestational exposure to pesticides is associated with cognitive, behavioural or hormonal disorders, among others, of the offspring. Very recently, the research community has observed that several chemicals, such as pesticides, are able to cause transgenerational effects, meaning that chemical exposure occurring in our grandfathers can have an impact on us or our children.

It is in our hands to change this. Following an organic diet has been associated with reduced levels of pesticides in the U.S. population (see here or here) or France. Moreover, as it is summarised here, organic food has several other health benefits. Unfortunately, land under organic management is still small, for example in the case of the European Union, it represents only 6.2% of the total. In the United States only, there have been more than 500 active pesticide compounds since 1970, and unfortunately, research resources are limited to assess the potential health outcomes for each newly released chemical. The importance of basic research capable of identifying hazardous chemicals is certainly one of the responsibilities that we, as independent researchers, have to address to ensure environmental sustainability and organism wellbeing. Our research is only an example of that. That is why we need your support.

Isabel Forner-Piquer is a researcher at Brunel University London, United Kingdom. Before joining Brunel, she was a postdoctoral researcher at the Institute of Functional Genomics in France. Her research focuses on the study of environmental pollutants and their effects on the endocrine system and neurodevelopment. She holds two Bachelor's degrees—Biology (University of Valencia, Spain) and Marine Science (University of Alicante, Spain) and a PhD degree in Marine Biology and Ecology from the Polytechnic University of Marche (Italy). Awarded numerous grants and accolades, Isabel has published more than twenty scientific articles in peer-reviewed scientific journals.

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