Fish Embryonic Testing: Early Detection of Pesticide Toxicity Hazards

The use of pesticides in the agricultural sector continues to rise in line with the increasing demand for high-yield, pest-free crops. However, behind their benefits, pesticides also raise concerns about their impact on human health and other non-target organisms. Therefore, re-evaluating existing pesticides and conducting comprehensive assessments of new formulations are crucial steps.

The main challenge lies in the limitations of conventional toxicity testing methods, which typically involve the use of mammalian animals such as rats or rabbits. In addition to ethical concerns, these methods are considered costly and less physiologically relevant for predicting impacts on humans and ecosystems. In recent years, alternative approaches have been developed to replace or reduce reliance on mammalian test animals. 

One method gaining attention is the Fish Embryonic Test (FET), a toxicity test that uses fish embryos, such as Danio rerio (zebrafish), as a biological model. Fish Embryonic Testing is considered an important part of the early screening process to detect the potential hazards of pesticides available on the market. Amid growing concerns about the environmental and human health impacts of pesticides, FET emerges as an efficient, relevant, and ethically aligned alternative toxicity testing method, in accordance with the principle of reducing animal use in research.

• What is Fish Embryonic Testing?
• Advantages of the Fish Embryonic Test for Pesticide Toxicity Testing

What is Fish Embryonic Testing?

In the context of pesticide research, zebrafish have been used since the 1960s at various developmental stages—embryo, larva, adult fish, as well as cell and tissue levels—to test different types of pesticides from various classes, such as triazines, organophosphates, and pyrethroids. The use of zebrafish models covers a wide range of compounds, including herbicides and insecticides, both as individual chemicals and in combinations, making them an important biological model for assessing the environmental risks of pesticides to aquatic organisms. As a result, the use of fish embryos, particularly zebrafish, in toxicity testing has been further developed into a more standardized method, notably through the Fish Embryo Acute Toxicity Test (FET).

The Fish Embryo Acute Toxicity Test is an acute toxicity testing method that uses fish embryos—primarily from the species Danio rerio (zebrafish)—to evaluate the harmful effects of chemical compounds. The main objective of FET is to assess toxicity at the embryonic stage as an early indicator, which can then be used to plan follow-up testing on juvenile fish, complement other toxicity assessment methods, or even serve as an alternative to standard testing using juvenile fish. The official history of the FET method began when Germany, through its Federal Environment Agency (UBA), proposed its adoption to the OECD (Organisation for Economic Co-operation and Development) working group on September 1, 2006. 

After a review process, the method was approved in May 2013 and officially published as OECD Test Guideline No. 236 on July 26, 2013. This guideline aims to determine how toxic or lethal a chemical substance is to fish embryos. From the beginning, FET was developed as an alternative to conventional toxicity testing methods that used adult fish, such as those outlined in OECD Test Guideline No. 203. The procedure for the Fish Embryo Acute Toxicity Test (FET), as described in OECD TG 236 (2013), requires that newly fertilized zebrafish embryos be exposed to the test chemical as soon as possible—ideally no later than 1.5 hours post-fertilization. 

The exposure lasts for 96 hours (4 days), with the goal of detecting potential acute toxicity at the early stages of embryonic development. During the exposure period, observations are made every 24 hours for four main indicators used to detect mortality or toxic effects. These indicators include: coagulation of the fertilized egg, lack of somite formation (embryonic body segmentation), failure of the embryo's tail to detach from the yolk sac, and absence of a heartbeat. 

If any of these indicators are observed, a toxic effect is considered to have occurred. After the exposure period ends, the acute toxicity level of the test substance is assessed based on the number of embryos exhibiting one or more of these indicators, and from these results, the LC₅₀ value (the concentration that causes death in 50% of embryos) is calculated.

Advantages of the Fish Embryonic Test for Pesticide Toxicity Testing

Toxicity testing using fish embryos, known as the Fish Embryo Acute Toxicity Test (FET), offers numerous advantages that make it well-suited for early screening of hazardous substances in pesticides. One of its main strengths is efficiency. Zebrafish embryos can be tested in large numbers simultaneously using simple tools like 96-well plates, either manually or with automated robotic systems. This approach saves time, cost, and labor compared to toxicity tests on adult fish or mammals.

In addition, zebrafish reproduce quickly and produce large numbers of eggs, ensuring a constant supply of embryos for testing. Thanks to their transparent outer layer, researchers can easily observe malformations or developmental disruptions caused by pesticide exposure. Their small size also enables high-throughput testing and faster results.

Interestingly, in some countries, zebrafish embryos are not legally classified as live animals until they hatch. This makes FET more ethically acceptable, as it is not considered to cause suffering in the same way as testing on higher animals. Scientifically, zebrafish also share a high degree of genetic similarity with humans, about 85% of human drug targets have counterparts in zebrafish—making test results more relevant for predicting the potential risks of chemical substances, including pesticide active ingredients.

With all these advantages, FET has emerged as a promising method for detecting harmful pesticide compounds at early stages of development, before more complex or costly follow-up tests are conducted. Pesticide toxicity testing now relies not only on conventional methods, but also utilizes modern approaches such as the Fish Embryo Acute Toxicity Test (FET) to ensure the safety of active ingredients from the early stages of development. Through IML Research, a trusted laboratory with a team of experts with over 25 years of experience in chemical and pesticide testing.

3 Recommended Pesticide Lab Tests to Pass the Ministry of Agriculture’s Distribution Permit!

You can conduct pesticide testing with accurate, efficient, and ethical results. Ensure that each of your pesticide formulas has undergone laboratory testing according to standards to ensure safety for humans, the environment, and aquatic ecosystems. Consult your pesticide testing needs with IML Research's team of experts via WhatsApp and follow us on Instagram at @imlresearch for the latest information on product laboratory testing!

Author: Dherika
Editor: Sabilla Reza

References:

Braunbeck, T., Kais, B., Eva, L., Jens, O., Katharina, S., Daniel, S., & Ruben, S. (2014). The Fish Embryo Test (FET): Origin, Applications, and Future. Environ Sci Pollut Res. DOI: 10.1007/s11356-014-3814-7.

Glaberman, S., Padilla, S., & Mace, G.B. (2016). Evaluating the Zebrafish Embryo Toxicity Test for Pesticide Hazard Screening. Environmental Toxicology, 9999(9999), 1-6. DOI: 10.1002/etc.3641.

Goncalves, I.F.S., Souza, T.M., Leonardo, R.V., Filipi, C.M., Adailton, P.N., & Davi, F.F. (2020). Toxicity Testing of Pesticides in Zebrafish – A Systematic Review on Chemicals and Associated Toxicological Endpoints. Environmental Science and Pollution Research. https://doi.org/10.1007/s11356-020-07902-5