## 1 Introduction  
Fish are high-trophic-level organisms among various aquatic eco-receptors. As key receptors in aquatic systems, it is necessary to protect them from chemical or contaminant pollution. Water quality standards (WQS) are derived by considering both the level of contaminant pollution in the environment and its toxicity to aquatic eco-receptors [1, 2, 3, 4, 5]. The United States, Canada, the European Commission, and the governments of Australia and New Zealand derive the WQS for contaminants to protect aquatic eco-receptors, including fish [1, 2, 3, 4]. Moreover, the WQS in Japan primarily aims to protect fish, leading to a high demand for chemical toxicity data on fish [5]. These demands have resulted in conducting animal tests on fish to evaluate the toxicity of specific chemicals. From the perspective of animal care and use ethics, researchers must strive to maintain the welfare of experimental animals, including vertebrates such as fish and amphibians in aquatic environments, under the 3R principles (reduction, refinement, and replacement) [6, 7]. Direct pain or unnecessary harm to test organisms are not permitted, and this has driven the development of alternative testing methods; one such alternative tool is the fish embryo assay, which uses organisms that have not yet developed a nervous system. Since fish embryos have primitive nervous systems and do not experience discomfort or pain, they are considered replacements for organisms used in traditional toxicity testing [6, 7, 8]. According to European Directive 2010/63/EU, fish embryos and larvae, which are not capable of independent feeding, are excluded from the group of protected animals [8]. In accordance with the fish embryo test guidelines [9; 10; 11; 12; 13; 14; 15; 16], various fish species are used for assays, including _Catostomus commercially_, _Cyprinodon variegatus_, _Cyprinus carpio_, _Danio rerio_, _Esox lucius_, _Ictalurus punctatus_, _Lepomis macrochirus_, _Misgurus anguillicaudatus_, _Oncorhynchus kisutch_, _Oncorhynchus mykiss_, _Oncorhynchus tshauytscha_, _Opsanus beta_, _Oryzias latipes_, _Oryza sinensis_, _Pimephales promelas_, _Salmo salar_, _Salmo trutta_, _Salvelinus fontinalis_, and _Salvelinus namaycush_. By screening previous studies for \"each species name\", \"fish\" AND \"toxicity\", or \"fish embryo\" AND \"toxicity\" in Google Scholar, the research ratios of \"fish embryo\" in \"fish\" toxicity research were shown to be 8.30% for zebrafish and 7.48% for _O. latipes_, respectively (Figure 1). These two fish species have high proportions of fish embryo studies. Furthermore, these two main fish species are included in the test guidelines of individual countries and international organizations to assess the toxicity of chemicals and contaminants for deriving water quality criteria, which helps to protect aquatic ecosystems. Focused on both fish species (zebrafish and _O. latipes_), this study examined the overall development of each species from birth to adulthood in laboratory conditions. We also reviewed the test guidelines related to fish embryos for specific nations and international organizations, and analyzed previous research on both acute and embryonic experiments to understand how toxicity levels change. The ultimate purpose of this study is to provide suggestions for suitable exposure duration to determine acute and chronic toxicity using fish embryo assays. Ultimately, we aim to promote the fish embryo assay as an alternative tool for adult fish tests. Figure 1: Research trends of fish toxicity tests using 19 fish species. (**A**) Numbers of studies found in Google Scholar research engine, and three most frequently used fish species were _’Cyprinus carpio’_, _’Danio rerio_ (zebrafish)’, and _’Oncorhynchus mykiss’_. (**B**) Relative ratios of fish embryo test in fish toxicity tests in 19 test species implied that _Cypzias latipes_ showed highest rates of fish embryo assay compared to other test species. Blue bars indicate the research ratio for _D.