Fish embryo test plate and method for evaluating neurodevelopmental toxicity using the same

Abstract

This invention provides a plate for evaluating toxic substances using fish embryos, a screening method for neurodevelopmental toxic substances using the same, and an apparatus and kit for evaluating neurodevelopmental toxic substances including the plate. [Solution] A plate for evaluating toxic substances using fish embryos is provided, comprising n outer wells and n inner wells located inside the outer wells, where n is an integer between 1 and 96, and the height of the inner wells is lower than the height of the outer wells. The plate and screening method provide an alternative test method to animal experiments as a kinetic test method that utilizes the characteristics of the swim bladder formation process in fish embryos, and at the same time, have the advantage of being able to effectively evaluate toxicity even with a small amount of sample.

Description

【Technical Field】 【0001】 The present disclosure relates to a plate for non-animal toxicity substance evaluation using fish embryos and a method for evaluating neurodevelopmental toxicity using the same. 【Background Art】 【0002】 Conventional neurodevelopmental toxicity evaluations have been utilized for hazard assessments by referring to the OECD Test Guideline (TG) 426: Developmental Neurotoxicity Study based on mammals such as rodents. OECD TG426 evaluates changes in the nervous system in postpartum mothers and the effects on general nervous system development, such as the behavior, motility, learning, and memory of the offspring born. 【0003】 Recently, the demand for the spread of ethical awareness towards animals and the reduction of animal tests has increased, leading to a growing need for the introduction of alternative animals for the hazard assessment of chemicals and the production and verification of alternative test materials using them. Such trends are steadily being discussed in the international community, and in neurodevelopmental toxicity evaluations, the development and application of various alternative test methods are actively underway. The National Institute of Health (NIH) in the United States has published a list of alternative test methods accepted by the U.S. government by referring to the guidelines presented by various organizations such as the Organization for Economic Co-operation and Development (OECD), the U.S. Food and Drug Administration (FDA), and the U.S. Environmental Protection Agency (EPA). Thus, alternative test methods using non-animal fish embryos and the like have been proposed for the replacement of mammals such as rodents, which are widely used in toxicity tests. 【0004】 For example, the 2013 OECD guidelines for test animals included "TG236: Fish Embryo Acute Toxicity (FET) Test," a method for evaluating acute toxicity in fish. The FET test involves exposing zebrafish embryos to a chemical substance for 96 hours. If one or more of the following are observed, the chemical substance and its concentration are judged to be acutely toxic, and a LC (Low-Coagulation) test is performed. 50 Calculate the result (reference [OECD, Test No. "236: Fish embryo acute toxicity (FET) test." OECD Guidelines for the Testing of Chemicals, Section 2 2013:1-22]). 【0005】 In humans, neurodevelopmental abnormalities can lead to various postnatal neurological disorders, as well as prenatal miscarriages and stillbirths. The first cry of a newborn immediately after birth enables independent lung respiration, and failure to do so is a major cause of stillbirth. Animal respiration is maintained by the complex regulatory action of the nervous and muscular systems. The swim bladder of fish is evolutionarily homologous to the lungs of mammals, and its formation and function also develop through the complex reciprocal regulatory action of the autonomic nervous and muscular systems during development. The inventors of this invention sought to utilize these complex stages of swim bladder development in fish as an indicator for evaluating neurodevelopmental toxicity. [Prior art documents] [Non-patent literature] 【0006】 OECD, Test No. "236: Fish embryo acute toxicity (FET) test." OECD Guidelines for the Testing of Chemicals, Section 2 (2013): 1-22. [Overview of the Initiative] [Problems that the invention aims to solve] 【0007】 This disclosure aims to provide a plate for evaluating and screening neurodevelopmental toxic substances using fish embryos. 【0008】 Furthermore, this disclosure aims to provide a screening method for evaluating neurodevelopmental toxicity using fish embryos, including the aforementioned plate. 【0009】 Furthermore, this disclosure aims to provide a device for evaluating toxic substances using fish embryos, including the aforementioned plate. 【0010】 Furthermore, this disclosure aims to provide a kit for evaluating neurodevelopmental toxicity using fish embryos, including the aforementioned plate. [Means for solving the problem] 【0011】 A plate for evaluating toxic substances using fish embryos according to one embodiment includes n outer wells and n inner wells located inside the outer wells, where n is an integer between 1 and 96, and the height of the inner wells is lower than the height of the outer wells. 【0012】 The internal well may be non-permeable. 【0013】 The material of the plate may be one of the following: polystyrene (PS), polycarbonate (PC), polypropylene (PP), polymethyl methacrylate (PMMA), polyethylene terephthalate (PET), polylactic acid (PLA), polyvinyl chloride, or a combination thereof. 【0014】 The plate is filled with a liquid selected from water and a test solution in an external well and an internal well, and the height of the liquid is lower than the height of the external well and higher than the height of the internal well. 【0015】 The height of the internal well is from about 5 mm to about 21 mm, and the difference (Δh) between the height of the internal well and the height of the liquid satisfies the relationship of the following formula (1). [Formula 1] 【0016】 3 mm ≤ height of liquid - height of internal well (Δh) ≤ 10 mm 【0017】 The height ratio of the internal well to the external well may be from about 1:3 to about 1:1.2. 【0018】 The shape of the external well or the internal well may be a prism or a cylinder, respectively. 【0019】 The diameter of the external well is from about 10 mm to about 40 mm and satisfies the relationship of the following formula (2). [Formula 2] 【0020】 1.1 ≤ diameter of external well / diameter of internal well ≤ 4 【0021】 [[ID=3३]] The embryo of the fish may be an embryo of a zebrafish. 【0022】 The plate may be a plate for screening a neurodevelopmental toxicant using an embryo of a fish. 【0023】 A method according to an embodiment is a method for screening neurodevelopmental toxicants using fish embryos, and the method includes: a) providing the plate; b) filling water or a test solution in each of the plates such that the height is higher than the height of the inner wells and lower than the height of the outer wells, and positioning fish embryos in the inner wells; c) when water is filled in step b), adding a sample to the inner wells of the plate; and d) checking whether the fry formed from the fish embryos move between the inner wells and the outer wells. 【0024】 The fish embryo may be a zebrafish embryo. 【0025】 The fish embryos in step b) are fish embryos from immediately after fertilization to about 48 hours. 【0026】 Step d) is performed 100 to 150 hours after the modification of the fish embryos. 【0027】 Step d) may include counting the number of fry that failed to move between the inner wells and the outer wells and the number of fry that successfully moved between the inner wells and the outer wells, respectively. 【0028】 Among the fry that successfully moved between the inner wells and the outer wells, if the swim bladder of the fry inflated into a round, balloon-like shape, it can be determined that the inflation of the swim bladder was successful. 【0029】 The screening method may further include: e) when the number of fry with successful swim bladder inflation is 50% or less of the total number of embryos, determining that the sample is a toxicant. 【0030】 An apparatus according to an embodiment is an apparatus for evaluating toxicants using fish embryos, including the plate and an image reader that performs the above counting through automated image analysis. 【0031】 The aforementioned fish embryo may be a zebrafish embryo. 【0032】 One embodiment of the kit is a kit for evaluating neurodevelopmental toxic substances using fish embryos, comprising the plate and instructions for using the plate in a neurodevelopmental toxic substance screening method using fish embryos. 【0033】 The aforementioned fish embryo may be a zebrafish embryo. [Effects of the Invention] 【0034】 The plate described herein, by including an internal well, allows for the evaluation of swim bladder expansion behavior in fish embryos, distinguishing it from the reduction in migratory ability caused by general developmental toxicity. Therefore, the plate described herein can be used to assess the presence or absence of neurodevelopmental toxicity from chemicals, etc. The screening method using the above plate has a sensitivity approximately 10 times higher than conventional methods, and the presence or absence of neurodevelopmental toxicity can be determined even with a small amount of sample. [Brief explanation of the drawing] 【0035】 [Figure 1] A schematic diagram of the swim-up and swim bladder inflation behavior of a zebrafish is shown. The explanation for each stage is as follows: <1> Zebrafish move to the water's edge and attach themselves to walls using the adhesive on their jaws. <2> They swim up along the wall in the opposite direction of gravity, then attach their jaws to the wall again to secure themselves. <3> The process until it reaches the water surface <2> After repeating this process, the swim bladder expands. <4> After adjusting the expansion of the swim bladder to an appropriate size and ensuring neutral buoyancy, free swimming and independent behavior of the individual animal eventually become possible. 【0036】 [Figure 2] This demonstrates the measurement of zebrafish swim-up behavior using conventional glass test tubes. 【0037】 [Figure 3]The initial schematic diagrams of plates manufactured by attaching inner wells (IWs) to 2-well plates and 6-well plates are shown. 【0038】 [Figure 4] This shows a plate for evaluating toxic substances using fish embryos. Plate 1 consists of 100 wells. Figure 4 shows an example with 24 wells, but the number of wells is not limited. 【0039】 [Figure 5] Figure 4 is an enlarged perspective view of well 100. Well 100 consists of an outer well 110 and an inner well 120. 【0040】 [Figure 6] Figure 4 is an enlarged cross-sectional view of well 100. Do is the diameter of the outer well 110, and Di is the diameter of the inner well 120. Lo is the height of the outer well 110, and Li is the height of the inner well 120. 【0041】 [Figure 7] An exemplary embodiment of the plate is shown. One exemplary embodiment of the plate may be a 24-well plate, with an internal well height of 9 mm, a water level height of 12 mm, and 2 mL of the test solution being used. 【0042】 [Figure 8] An exemplary embodiment of the plate is shown. One exemplary embodiment of the plate may be a 24-well plate, in which the height of the internal wells may be 20 mm, the water level may be 24 mm, and the height of the external wells may be 29-30 mm. 【0043】 [Figure 9] An exemplary embodiment of the plate is shown. The plate may be a 24-well plate and further includes a cover that covers the upper part of the plate. The numbers shown in Figure 9 indicate exemplary specifications, and the unit of the numbers is mm. 【0044】 [Figure 10] The results of the negative and positive control groups tested using plates are shown. In the diagram, <1> If the movement from the inner well to the outer well fails, <2> If the swim bladder successfully moves from the inner well to the outer well, but fails to expand, <3> This indicates a case where the fish successfully moved from the inner well to the outer well and its swim bladder expanded. The left panel of Figure 10 shows the results of the test where the negative control group was treated with egg water. The right panel of Figure 10 shows the results when the negative control group was treated with egg water and when the positive control group was treated with the anesthetic tricaine. 【0045】 [Figure 11] An example of a design for a neurodevelopmental toxicity screening method is shown. Here, 1-3 represent test groups under three different concentration conditions for a specific drug, nC represents the negative control group (e.g., rearing water), pC represents the positive control group (e.g., tricaine 40 mg / L), and sC represents the solvent control group. 【0046】 [Figure 12] This shows examples of successful or unsuccessful swim bladder expansion in zebrafish embryos. When swim bladder expansion is normal (i.e., successful), the a / b value is approximately 0.5 or greater, and when swim bladder expansion is abnormal (i.e., unsuccessful), the a / b value is approximately less than 0.5. 【0047】 [Figure 13] The CAD drawings of plate 5 of Embodiment 1-4 and a prototype manufactured using 3D printing technology are shown. 【0048】 [Figure 14] The results of neurodevelopmental toxicity assessments using plates for 30 different chemical substances are presented. [Modes for carrying out the invention] 【0049】 The embodiments will be described in detail below with reference to the attached drawings. However, various modifications may be made to the embodiments, and the scope of the patent application will not be limited or restricted by such embodiments. All modifications, equivalents, or substitutes to the embodiments should be understood to be included within the scope of the patent. 【0050】 The terms used in the embodiments are for illustrative purposes only and should not be construed as limiting. Singular expressions include plural expressions unless, in context, they have a clearly different meaning. In this specification, terms such as “includes” or “has” indicate the presence of features, figures, steps, actions, components, parts, or combinations thereof described in the specification, and should not be understood as preemptively excluding the possibility of the presence or addition of one or more other features, figures, steps, actions, components, parts, or combinations thereof. 【0051】 Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as those commonly understood by a person of ordinary skill in the art to which this embodiment belongs. Commonly used predefined terms should be interpreted as having the meaning consistent with their meaning in the context of the relevant art, and not as ideal or overly formal unless expressly defined herein. 【0052】 Furthermore, when explaining with reference to the attached drawings, the same components will be assigned the same reference numerals regardless of the reference numerals used in the drawings, and redundant explanations will be omitted. In the description of embodiments, if a specific explanation of related prior art is deemed to unnecessarily obscure the gist of the embodiment, such detailed explanation will be omitted. 【0053】 Furthermore, in describing the components in the embodiments, terms such as 1st, 2nd, A, B, (a), (b), etc., may be used. These terms are used to distinguish a component from other components, and the terms do not limit the essence, order, or sequence of the component. 【0054】 Components that have functions common to components included in any of the embodiments will be described using the same name in the other embodiments. Unless otherwise stated, the description given in one embodiment can be applied to the other embodiments, and specific descriptions will be omitted to the extent that they overlap. 【0055】 In this specification, the term "about" is understood to indicate a range of numbers that a person skilled in the art should consider equivalent to the stated value in terms of achieving the same function or result. The term "about" refers to ±20% of the given number, generally ±10%, often ±5%, and less precisely ±2% of the given number. In some forms, the term "about" means the given number itself. 【0056】 All numerical ranges presented throughout this specification include their upper and lower limits, and all narrower numerical ranges belonging to such ranges, all of which are deemed to be clearly and specifically described herein. 【0057】 In this specification, even without explicit indication of singular or plural forms such as "one," "one," or "~ra" for a noun, a noun always means "at least one" or "one or more," and includes plural forms of nouns. 【0058】 The present invention will be described in detail below. 【0059】 The swim bladder of fish is an organ that regulates buoyancy and is essential for free swimming and survival. The development of the swim bladder in fish requires the injection of an appropriate amount of air from the outside for normal function, along with the formation of the organ. For example, in the case of zebrafish embryos, normal motor skills toward the water surface are required to come into contact with external air. In particular, the normal development of the entire nervous system, including complex stages of sensory and motor systems, is required, including upward movement based on gravity detection, movement to crawl up walls, spatial awareness of the water surface, inhalation of external air bubbles, sensory regulation to adjust air bubbles to the appropriate size, and ultimately, movement for free swimming (see Figure 1). Embryos that fail to develop their swim bladder, inhale external air droplets, and expand their swim bladder will fail to achieve neutral buoyancy, experience difficulty in foraging for food through free swimming, and consequently, cannot survive. Therefore, the normal developmental process of the swim bladder can be used as an indicator for evaluating neurodevelopmental toxicity and the effectiveness of related nervous system modulochemicals. 【0060】 In the case of zebrafish embryos, swim bladder expansion occurs during the normal developmental process 4-5 days after fertilization. Various nervous system functions, including dopamine and neuropeptide Y, directly related to this process, regulate buoyancy, enabling free swimming and feeding, thus allowing the individual to survive (see references [Robertson, GN, et al. "Development of the swimbladder and its innervation in the zebrafish," Danio rerio. "Journal of Morphology 268.11 2007:967-985."] and [Smith, Frank M., and Roger P. Croll. "Autonomic control of the swimbladder." "Autonomic Neuroscience 165.1 2011:140-148"]). 【0061】 The swim bladder is evolutionarily homologous to the mammalian lung. In humans, lung respiration begins with the first cry of a newborn immediately after birth, and this process, like in fish, is regulated by the normal activity of the nervous and muscular systems. In relation to this, the ultimate consequences of neurodevelopmental abnormalities in humans include various neurological diseases, as well as miscarriage and stillbirth. Since the formation and function of the swim bladder also develop through the complex interaction and regulation of the autonomic nervous and muscular systems during development, it can be used as a primary indicator for evaluating neurodevelopmental toxicity. The inventors of this invention have attempted to utilize the development of the swim bladder in fish as an indicator for evaluating neurodevelopmental toxicity by taking advantage of these characteristics. 【0062】 In the conventional OECD method for evaluating acute toxicity in fish (FET test), zebrafish embryos are exposed to a chemical substance for 96 hours. If one or more of the following are observed, the chemical substance and its concentration are judged to be acutely toxic, and the LC50 is calculated (Reference: [OECD, Test No. "236: Fish embryo acute toxicity (FET) test. "OECD Guidelines for the Testing of Chemicals, Section 2 2013:1-22"]). In other words, there is no conventional method for screening neurodevelopmental toxic substances based on the presence or absence of swim bladder expansion or development, and this method was newly developed by the present inventor. 【0063】 The FET test uses indicators of toxicity such as (i) coagulation of fertilized eggs due to cell growth toxicity, (ii) stagnation of somite formation and (iii) failure of the tailbud to separate from the yolk sac due to cell differentiation toxicity, and (iv) stagnation of heartbeat due to myocardial motion dysregulation. Occasionally, one or more of the above (i) to (iv) may have a secondary effect on swim bladder development, but this is distinct from the complex nervous and muscular regulatory effects of swim bladder expansion. Here, the inventors added a structure called an internal well, allowing them to observe the swim bladder expansion stage of fish embryos in three-dimensional movement (i.e., up, down, left, and right), which is clearly distinct from structural and morphological developmental toxicity due to early cytotoxicity. Furthermore, if the embryo successfully moves from the internal well to the external well (i.e., escapes the internal well), it can be determined that there is no problem with the embryo's basic locomotion ability, thus distinguishing indicators of the complex swim bladder expansion process from results due to simple locomotion ability. 【0064】 This disclosure relates to a plate for evaluating toxic substances using fish embryos, wherein the plate includes n outer wells and n inner wells located inside the outer wells, where n is an integer between 1 and 96, and the height of the inner wells is lower than the height of the outer wells. 【0065】 The term "fish" refers to vertebrates that live in water and breathe with gills. For example, "fish" includes, but is not limited to, OECD standard test species such as zebrafish (danio rerio), fathead minnow (pimephales promelas), carp (Cyprinus caripio), southern medaka (oryzias latipes), guppy (poecilia reticulata), bluegill (Lepomis macrochirus), rainbow trout (Oncorhynchus mykiss), three-spined stickleback (gasterosteus aculeatus), sheephead minnow (cyprinodont variegatus), Western sea bass (dicentrarchus labrax), or red sea bream (pagrus major). The fish may be small fish including, but not limited to, zebrafish (Danio rerio), medaka (Oryzias latipes), Danionella cerebrum, and minnow (Phoxinus phoxinus). Preferably, the fish may be zebrafish. As a small fish, the zebrafish is a vertebrate model similar to humans, and hundreds or more embryos can be produced at once through in vitro fertilization, making it suitable for large-scale screening studies of pharmaceuticals or chemicals. In addition, the embryos are transparent, larger in size than mammals, and develop rapidly, making it easy to observe tissue development and function. As a current international standard, zebrafish embryos up to 5 days post-fertilization are not considered animals and can be freely used in tests, and the OECD TG 236 (Fish acute embryo test, FET) also utilizes zebrafish embryos up to 96 hours. Furthermore, the European Union (EU), in its Directive 2010 / 63 / EU on the protection of animals used for scientific purposes, includes only larval fish forms that are capable of independent feeding as subject to its application. 【0066】 Fish embryos transform into larvae upon hatching or the onset of exogenous feeding. From this point until metamorphosis leads to juvenile fish, they are called "larvae," and after sexual maturity, they are classified as "adults." In the case of zebrafish, hatching generally occurs between 48 and 72 hours after fertilization. Typically, zebrafish embryos are considered "larvae" after 72 hours, regardless of whether they hatch or not. They then undergo metamorphosis to become juvenile fish around 30 days later, and become sexually mature after 3-4 months (see reference [Ali, Shaukat, et al. "Zebrafish embryos and larvae: a new generation of disease models and drug screens. "Birth Defects Research Part C: Embryo Today: Reviews 93.2 2011: 115-133"]). 【0067】 As used herein, the term "fish embryo" broadly encompasses the period from immediately after fertilization to approximately 72 hours later, and further includes the early larval stage up to approximately 144 hours after fertilization. On the other hand, in this agreement, "fish embryo" refers to the period from immediately after fertilization to approximately 72 hours later, and "larva" refers to individuals from approximately 72 hours after fertilization to 30 days of age. Unless otherwise specified herein, the term "fish embryo" shall be used in the broad sense described above. 【0068】 The term "plate" refers to a thin, flat, plate-like instrument primarily used for testing and research purposes. The plate consists of n wells, where n is an integer greater than or equal to 1, preferably an integer between 1 and 96, more preferably 6, 12, 24, 48, or 96. The number of wells is determined based on the experimenter's convenience or the dilution ratio of the drug to be screened. The plate may further include a cover over the upper section of the plate (Figure 9). 【0069】 For illustrative purposes, referring to Figures 4-6, plate 1 may be a 24-well plate, in which case the plate contains 24 wells 100. The wells 100 consist of an outer well 110 and an inner well 120 located inside the outer well. 【0070】 The internal well is lower in height than the external well. Because the height Li of the internal well is lower than the height Lo of the external well, when the well 100 is filled with liquid, the liquid surface is positioned above the height of the internal well but lower than the height of the external well. Therefore, it is possible to observe whether or not fish embryos move from the internal well to the external well. The internal well may be permeable, semi-permeable, or impermeable. Preferably, the internal well may be impermeable. The internal well can be permeable to such an extent that fish cannot pass through it, as long as it does not allow fish to pass through the walls of the internal well and move to the external well. In other words, there is no limit to the degree of permeability as long as fish cannot pass through the walls of the internal well and move to the external well. 【0071】 The material of the plate may be one of the following: polystyrene (PS), polycarbonate (PC), polypropylene (PP), polymethyl methacrylate (PMMA), polyethylene terephthalate (PET), polylactic acid (PLA), polyvinyl chloride, or a combination thereof. Preferably, it may be polystyrene, but is not limited thereto. 【0072】 The height of the internal well may be approximately 5 mm or more. The height of the internal well may be approximately 5 mm, approximately 6 mm, approximately 7 mm, approximately 8 mm, approximately 9 mm, approximately 10 mm, approximately 11 mm, approximately 12 mm, approximately 13 mm, approximately 14 mm, approximately 15 mm, approximately 16 mm, approximately 17 mm, approximately 18 mm, approximately 19 mm, approximately 20 mm, or approximately 21 mm or more. Preferably, the height of the internal well may be approximately 19 mm to approximately 21 mm, but is not limited thereto. If the height of the internal well is less than approximately 5 mm, the fish embryo can escape the internal well regardless of whether or not the swim bladder is inflated, so it is preferable that the height of the internal well be approximately 5 mm or more. The height of the internal well can be appropriately selected considering the size of the fish embryo. 【0073】 The height of the outer well may be approximately 12 mm or more. The height of the outer well may be approximately 12 mm, approximately 13 mm, approximately 14 mm, approximately 15 mm, approximately 16 mm, approximately 17 mm, approximately 18 mm, approximately 19 mm, approximately 20 mm, approximately 21 mm, approximately 22 mm, approximately 23 mm, approximately 24 mm, approximately 25 mm, approximately 26 mm, approximately 27 mm, approximately 28 mm, approximately 29 mm, approximately 30 mm, or approximately 31 mm or more. Preferably, the height of the outer well may be approximately 28 mm to approximately 31 mm, but is not limited thereto. The height of the outer well can be appropriately selected considering the height of the inner well and the size of the fish embryo, etc. 【0074】 The plate is configured such that, by filling the outer and inner wells with a liquid selected from water and a test solution, the height of the liquid is lower than the height of the outer well and higher than the height of the inner well. The difference (Δh) between the height of the inner well and the height of the liquid satisfies the relationship given by equation (1) below. [Formula 1] 【0075】 3mm ≤ liquid height - internal well height (Δh) ≤ 10mm 【0076】 The height ratio of the internal well to the external well may be about 1:3 to about 1:1.2. The height ratio of the internal well to the external well may be about 1:3, about 1:2.9, about 1:2.8, about 1:2.7, about 1:2.6, about 1:2.5, about 1:2.4, about 1:2.3, about 1:2.2, about 1:2.1, about 1:2, about 1:1.9, about 1:1.8, about 1:1.7, about 1:1.6, about 1:1.5, about 1:1.4, about 1:1.3, or about 1:1.2. Preferably, the height ratio of the internal well to the external well may be about 1:1.4 to about 1:1.6, but is not limited thereto. 【0077】 The shape of the outer well or inner well may be a prism or a cylinder, respectively. The shape of the outer well or inner well may be a triangular prism, a square prism, a pentagonal prism, a hexagonal prism, a heptagonal prism, an octagonal prism, a nonagonal prism, a decagonal prism, or a cylinder. Preferably, the shape of the outer well and inner well may be a cylinder, but is not limited to these. 【0078】 The diameter of the outer well may be about 10 mm to about 40 mm. Preferably, the diameter of the outer well may be about 15 mm to about 20 mm, but is not limited thereto. The diameter of the inner well may be about 5 mm to about 35 mm. Preferably, the diameter of the inner well may be about 5 mm to about 10 mm, but is not limited thereto. If the shape of the outer well or inner well is a prism, the diameter is defined as the length of the circle that circumscribes the interior of the polygon that is the base of the prism. 【0079】 The diameter ratio of the inner well to the outer well is approximately 1:1.1 to approximately 1:4. The diameter ratio of the inner well to the outer well may be approximately 1:1.1, approximately 1:1.2, approximately 1:1.4, approximately 1:1.6, approximately 1:1.8, approximately 1:2, approximately 1:2.2, approximately 1:2.4, approximately 1:2.6, approximately 1:2.8, approximately 1:3, approximately 1:3.2, approximately 1:3.4, approximately 1:3.6, approximately 1:3.8, or approximately 1:4. Preferably, the diameter ratio of the inner well to the outer well may be approximately 1:2.4 to approximately 1:3.2, but is not limited thereto. The diameter of the outer well and the diameter of the inner well satisfy the relationship given by equation (2) below. [Formula 2] 【0080】 1.1 ≤ outer well diameter / inner well diameter ≤ 4 【0081】 The plate is further filled with a liquid selected from water or a test solution. The water or test solution is filled to a height higher than the height of the internal wells and lower than the height of the external wells. The plate may be filled with egg water instead of water. The test solution may contain or be a solution in which the chemical substance or sample to be tested is dissolved. The test solution may be a solution corresponding to a negative control group or a positive control group. Fish embryos are placed in the internal wells of the plate. The fish and fish embryos are as described above. 【0082】 The aforementioned plate is for screening neurodevelopmental toxic substances using fish embryos. Neurodevelopmental toxic substance screening means evaluating the effects of chemicals, drugs, or samples on the development of the nervous system. As mentioned above, the swelling or development of the swim bladder in fish embryos is closely related to neurodevelopment. Therefore, by placing fish in the internal wells of the plate, treating them with a test solution or sample, and evaluating whether or not the fish's swim bladder swells or develops, neurodevelopmental toxic substances can be screened. 【0083】 This disclosure relates to a method for screening neurodevelopmental toxic substances using fish embryos, the method comprising: a) providing the plates; b) filling each of the plates with water or a test solution to a height greater than the height of the internal wells and less than the height of the external wells, and positioning the fish embryos in the internal wells; c) if water is filled in step b), introducing a sample into the internal wells of the plates; and d) checking for any movement of the internal and external wells of the larvae formed from the fish embryos. 【0084】 The aforementioned fish embryos may be, but are not limited to, embryos of OECD standard test species, including, for example, zebrafish (danio rerio), fathead minnow (pimephales promelas), carp (Cyprinus caripio), southern medaka (oryzias latipes), guppy (poecilia reticulata), bluegill (Lepomis macrochirus), rainbow trout (Oncorhynchus mykiss), three-spined stickleback (gasterosteus aculeatus), sheephead minnow (cyprinodont variegatus), Western sea bass (dicentrarchus labrax), or red sea bream (pagrus major). The aforementioned fish embryos may be, but are not limited to, those of small fish, including zebrafish (Danio rerio), medaka (Oryzias latipes), Danionella cerebrum, and minnow (Phoxinus phoxinus). Preferably, the fish embryos may be zebrafish embryos. As mentioned above, zebrafish are a human-like vertebrate model, capable of producing hundreds or more embryos at once through in vitro fertilization, and are suitable for large-scale screening studies of pharmaceuticals or chemical substances. Furthermore, zebrafish embryos are transparent, larger in size than mammalian embryos, develop rapidly, and allow for easy observation of tissue development and function. 【0085】 Step b) is to fill each plate with water or test solution so that it is higher than the height of the internal wells and lower than the height of the external wells, and to position the fish embryos in the internal wells. The water may be egg water. For example, egg water can be prepared by dissolving 6 g of sea salt in 1 L of tertiary distilled water, diluting the concentrated solution to 1 / 100 to a final concentration of 60 ug / ml. The test solution may contain the chemical substance or sample to be tested, or a solution in which the chemical substance or sample is dissolved. The test solution may be a solution corresponding to a negative control group or a positive control group. 【0086】 Step c) of adding samples to the internal wells of the plate is a step of adding samples at different concentrations to each well of the plate in order to evaluate the presence or absence of neurodevelopmental toxicity. For example, as shown in Figure 11, along with a solvent control group, a negative control group, and a positive control group, samples can be added to each well (internal well, or both internal and external wells) at three different concentrations. For example, samples can be added at concentrations of approximately 25 mg / L, 12.5 mg / L, and 6.25 mg / L, or at concentrations of approximately 100 mg / L, 50 mg / L, 25 mg / L, and 12.5 mg / L, or at concentrations of approximately 50 mg / L, 25 mg / L, 12.5 mg / L, and 6.25 mg / L. For example, the sample concentrations may be approximately 2-fold dilution, 3-fold dilution, 4-fold dilution, 10-fold dilution, or 100-fold dilution. In the step of introducing the sample, the method of introducing the sample is not particularly limited, and any method commonly used in the art can be used, taking into consideration the chemical properties of the sample, the expected toxicity range, etc. 【0087】 In steps b) and c) above, the plate may be filled with water to exceed the height of the internal wells, the fish embryos may be placed in the internal wells, and then the sample may be added to the internal wells of the plate. Here, the sample may be a liquid sample or a solid sample such as a powder. Alternatively, in steps b) and c) above, the plate may be filled with a test solution to exceed the height of the internal wells, and the fish embryos may be placed in the internal wells. The test solution may contain the chemical substance or sample to be tested, or a solution in which the chemical substance or sample is dissolved. The test solution may be a solution corresponding to a negative control group or a positive control group. That is, in steps b) and c) the sample may be added after the plate has been filled with water, or the test solution in which the sample is dissolved in water may be immediately filled into the plate. 【0088】 Step d) includes counting the number of larvae that failed to move between the inner well and the outer well, and the number of larvae that succeeded in moving between the inner well and the outer well. In one embodiment, among the larvae that succeeded in moving between the inner well and the outer well, if the swim bladder of the larvae inflates into a round, balloon-like shape, it is determined that the swim bladder has successfully inflated. 【0089】 Alternatively, the screening method, in step d), <1> The number of fish embryos that failed to move from the inner well to the outer well. <2> The number of fish embryos that successfully moved from the inner well to the outer well but failed to expand their swim bladder, and <3> The method further includes the step of counting and quantifying the number of fish embryos that successfully moved from an inner well to an outer well and successfully expanded their swim bladder. Instead of counting the number of fish embryos, the number of wells in which the fish embryos were located may be counted. However, the method is not limited to this, and one fish embryo may be located in each well, in which case the number of wells counted and the number of fish embryos will be the same. 【0090】 The aforementioned screening method has the above-mentioned quantitative data, EC 50 This further includes the step of finding the value. For example, EC 50 When calculating the half maximal effective concentration, if "effect" is defined as "failure to form swim bladder," then here, EC 50 The value represents the case where 50% of the individuals failed to form a swim bladder. The total number of individuals used in the tests for each concentration and the above <1> The number of fish embryos that failed to move from the inner well to the outer well, and <2> The total number of fish embryos that successfully moved from the inner well to the outer well but failed to expand their swim bladder was obtained using the Probit regression method, one of the regression analysis methods, with the statistical program SPSS (IBM). 50 The value can be calculated. However, EC 50 There are no particular restrictions on the method used to determine the value; any method commonly used in this art can be used. 【0091】 The step of evaluating the formation of the swim bladder in the fish embryo can be used to assess the success or failure of swim bladder expansion through microscopic observation. Success is indicated by the swim bladder of the fish embryo expanding into a round, balloon-like shape, while failure is indicated by the swim bladder of the fish embryo being flat (see Figure 12). The term "successful swim bladder expansion" may be used interchangeably with "normal swim bladder expansion," and the term "failure of swim bladder expansion" may be used interchangeably with "stagnant swim bladder expansion" or "abnormal swim bladder expansion." 【0092】 The aforementioned microscopic observation is performed using a stereoscopic microscope, but is not limited to this. Any microscope capable of observing swim bladder expansion is acceptable. Furthermore, step d) is performed using an automated observation device. Examples of such automated devices include DanioVision (Noldus) and Zebralab (Viewpoint). 【0093】 When the swim bladder expands into a round, balloon-like shape, the shortened length (vertical length) of the swim bladder is denoted as "a," and the length of the long axis (horizontal length) of the swim bladder is denoted as "b," meaning that the value of a / b is approximately 0.5 or greater (left-hand diagram in Figure 12). In one embodiment, if the fish embryo is a zebrafish embryo, the a value may be approximately 213 ± 5 μm, and the b value may be approximately 385 ± 11 μm. The size of fish embryos can vary even within the same species, and a person skilled in the art can easily determine whether the swim bladder of a fish embryo has expanded into a round, balloon-like shape, i.e., whether the expansion was successful. Therefore, the success of the swim bladder expansion is not limited to the numerical range presented above. 【0094】 When the swim bladder is flat, it means that the value of a / b is less than approximately 0.5, where "a" is the length of the short axis (vertical length) of the swim bladder and "b" is the length of the long axis (horizontal length) of the swim bladder (right-hand diagram in Figure 12). In one embodiment, when the fish embryo is a zebrafish embryo, the value of a may be approximately 83 ± 7 μm and the value of b may be approximately 344 ± 18 μm. However, the size of fish embryos can differ even within the same species, and a person skilled in the art can easily determine if the swim bladder of a fish embryo is flat, i.e., if it has failed to expand. Therefore, the presence or absence of swim bladder expansion failure is not limited to the numerical range presented above. 【0095】 The fish embryos in step b) above may be fish embryos from immediately after fertilization to about 48 hours later. In one embodiment, the fish embryos in step b) above may be approximately 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 25 hours, 26 hours, 27 hours, 28 hours, 29 hours, and 30 hours after fertilization. Fish embryos may be present in intervals of approximately 31 hours, 32 hours, 33 hours, 34 hours, 35 hours, 36 hours, 37 hours, 38 hours, 39 hours, 40 hours, 41 hours, 42 hours, 43 hours, 44 hours, 45 hours, 46 hours, 47 hours, 48 ​​hours, 49 hours, 50 hours, 51 hours, 52 hours, 53 hours, 54 hours, 55 hours, 56 hours, 57 hours, or up to 58 hours. 【0096】 Step d) above is performed 100 to 150 hours after fertilization of the fish embryo. Step d) above is performed approximately 100 hours, 101 hours, 102 hours, 103 hours, 104 hours, 105 hours, 106 hours, 107 hours, 108 hours, 109 hours, 110 hours, 111 hours, 112 hours, 113 hours, 114 hours, 115 hours, 116 hours, 117 hours, 118 hours, 119 hours, 120 hours, 121 hours after fertilization of the fish embryo. The process may be performed at approximately 122 hours, 123 hours, 124 hours, 125 hours, 126 hours, 127 hours, 128 hours, 129 hours, 130 hours, 131 hours, 132 hours, 133 hours, 134 hours, 135 hours, 136 hours, 137 hours, 138 hours, 139 hours, 140 hours, 141 hours, 142 hours, 143 hours, and 144 hours. Step d) may be performed approximately 36 hours after the sample input in step c). 【0097】 The procedure may further include a step of sealing the plate after step c) above. If a plate cover is present, the step of sealing the plate is a step of covering the plate with the cover. The step of sealing the plate is for preventing the evaporation of water or the test solution, and there are no particular limitations thereto. 【0098】 This disclosure relates to a toxic substance evaluation apparatus using fish embryos, comprising a plate and an image reader that counts the number of larvae that failed to move between the inner and outer wells and the number of larvae that succeeded in moving between the inner and outer wells through automated image analysis. 【0099】 The aforementioned plates and fish embryos are as described above. 【0100】 This disclosure relates to a kit for evaluating neurodevelopmental toxic substances using fish embryos, comprising the plate and instructions for using the plate in a neurodevelopmental toxic substance screening method using fish embryos. 【0101】 The aforementioned plates and fish embryos are as described above. 【0102】 The aforementioned instructions relate to a method for using the plate in a neurodevelopmental toxicology screening method using fish embryos. The instructions include instructions for using, storing, and handling the kit, the function of each component of the kit, and how to interpret the results. The instructions include explanations for each stage of the screening method described above. 【0103】 The contents of the above disclosure will be described in more detail in the following embodiments. 【0104】 Embodiment 1: Plate fabrication for evaluating the formation and expansion of the swim bladder of a zebrafish. 【0105】 Embodiment 1-1: Manufacturing and evaluation of plate 1 【0106】 As an initial model for observing the swim-up behavior of zebrafish, we used a method of arranging various test tubes as shown in Figure 2. By using several test tubes to simultaneously test numerous embryos, it becomes possible to treat and test various test substances. 【0107】 Embodiment 1-2: Manufacturing and evaluation of plates 2 and 3 【0108】 Plates 2 and 3 were prepared to evaluate the formation and expansion of the swim bladder in zebrafish (see Figure 3). Plate 2 was prepared by attaching a 2 mL tube (Axygen, microtube (Clear 2.0 ml), Cat. No. 27-00619-01, Model: MCT-200-C) to a 0.5 cm high cylinder in a 12-well plate (SPL Life Science, South Korea, Cat. No. 30012). Plate 3 was prepared by attaching a 15 mL tube (SPL Life Science, South Korea, Cat. No. 50015) to a 1 cm high cylinder in a 6-well plate (SPL Life Science, South Korea, Cat. No. 30006). In plates 2 and 3, the required amounts of test material for each test were 2.5 mL and 11 mL, respectively. 【0109】 In the case of plate 2, the height of the internal wells was extremely low, which meant that the embryos could escape without performing a swim-up action. In the case of plate 3, only 6 zebrafish embryos could be tested per plate, which resulted in excessive consumption of the test material. 【0110】 Embodiment 1-3: Manufacturing and evaluation of plate 4 【0111】 A 24-well plate (SPL Life Science, South Korea, Cat. No. 30024) was used for the experiment by attaching a 9mm high internal well to each well, creating an internal space large enough to accommodate one zebrafish embryo (see Figure 7). The 24-well plate has wells arranged in a 4x6 configuration. Each well has an internal well that forms a concentric circle with the external well, but is characterized by having an internal well to identify the effects of reduced zebrafish mobility and an external well that serves as the waterside space where swim bladder expansion occurs. 【0112】 If zebrafish embryos 5 days after fertilization cannot pass through the internal well with a specific drug and treatment concentration, it indicates that they failed in the swim-up behavior step, which is the behavior of searching for the aquatic air layer before swim bladder inflation, and therefore the drug and treatment concentration in question does not have a discernible effect on swim bladder inflation. Conversely, if zebrafish embryos 5 days after fertilization that have passed through the internal well with a specific drug and treatment concentration exhibit abnormal swim bladder inflation (for example, if the embryo cannot swim normally and sinks to the bottom or lies on its side, this is considered an abnormality in swim bladder inflation), then the effect on swim bladder inflation unrelated to the absence of mobility can be accurately identified. 【0113】 In summary, this plate-based neurodevelopmental toxicology screening allows for the identification of neurodevelopmental toxicity related to swim bladder expansion behavior, while excluding toxicity related to the mobility of specific drugs under various concentration conditions. Examples of tests using this plate are described in detail in Embodiment 2. 【0114】 Embodiments 1-4: Manufacturing and evaluation of plate 5 【0115】 In the case of plate 4, the following problems were identified. Firstly, the height of the internal well (9 mm) was too low to determine the minimum threshold for distinguishing motility, as the embryo could escape from the internal well regardless of its swimming behavior when stimulated. Secondly, the low height of the internal well meant that even slight shaking during the test could cause the embryo to detach from the internal well. 【0116】 To address the above issues, the height of the internal wells was increased from 9 mm to 20 mm, and the height of the external wells was adjusted to 30 mm so that all internal wells were submerged. The adjusted internal well height (20 mm) was suitable for evaluating mobility, as it prevented the embryo from moving from the bottom in a single attempt (trusting bout) and induced swim-up behavior. Furthermore, the detachment of embryos from the internal wells due to shaking was significantly reduced. 【0117】 To confirm the reproducibility of the experiment with Plate 5, a prototype was manufactured using 3D printing technology with PLA material and tested with zebrafish embryos treated with culture water (negative control group) and tricaine (MS-222) (positive control group) (Figure 13). The results were the same as those of the negative and positive control groups in Plate 4, and at the same time, the ease of control for the experimenter was improved by reducing the detachment of embryos from the internal wells due to experimenter error. 【0118】 Embodiment 2: Evaluation of neurodevelopmental toxicity in zebrafish using Plate 4 【0119】 Embodiment 2-1: Preliminary study of the positive control group 【0120】 First, before evaluating the neurodevelopmental toxicity of zebrafish, a preliminary test was conducted using the anesthetic tricaine (MS-222) as a positive control group. Four plates were used, with each plate treated with tricaine at concentrations of 80 mg / L, 40 mg / L, and 20 mg / L, and egg water as the negative control group. For example, the egg water used was prepared by dissolving 6 g of sea salt (Instant Ocean, Aquarium Systems product) in 1 L of tertiary distilled water, diluting the concentrated solution to 1 / 100, and finally achieving a concentration of 60 ug / ml. 【0121】 The results are shown in Table 1. In Table 1, <1> If the movement from the inner well to the outer well fails, <2> If the swim bladder successfully moves from the inner well to the outer well, but fails to expand, <3> This indicates a successful migration from the inner well to the outer well, as well as successful expansion of the swim bladder. The test results showed that using 40 mg / L of tricaine as a positive control group was appropriate. 【0122】 Table 1 [Table 1-1] 【0123】 Embodiment 2-2: Alternative Test Method for Swimsuit Development (First Cry Assay) - Neurodevelopmental Toxicity Evaluation of 30 Chemical Substances Using Plate 4 【0124】 Zebrafish develop extremely quickly, with most of their tissues already formed and functioning by the first day after fertilization. Furthermore, zebrafish embryos can be used to observe changes in neurobehavior due to short-term exposure to chemicals (see reference [Kim, Seong Soon, et al. "Neurochemical effects of 4-(2Chloro-4-fluorobenzyl)-3-(2-thienyl)-1,2,4-oxadiazol-5 4H-one in the pentylenetetrazole (PTZ)-induced epileptic seizure zebrafish model. "International journal of molecular sciences 22.3 2021:1285."]). To evaluate only neuro-specific effects while excluding other secondary effects such as initial cytotoxicity from the test substance, normal individuals with no developmental problems at 24 or 48 hours post-fertilization were selected and dispensed into the inside of each inner well of plate 4. Next, 4 mL of the test solution was treated into each well, with the water level approximately 24 mm. Finally, the presence or absence of swim bladder swelling was evaluated on day 5 post-fertilization, which was 3 days after exposure to the test substance. 【0125】 The presence or absence of swim bladder expansion was quantified by classifying the results according to the following criteria. <1> Failure to move from the inner well to the outer well. <2> Successful movement from the inner well to the outer well and failure of swim bladder expansion, and <3> Successful migration from the inner well to the outer well and successful expansion of the swim bladder. 【0126】 Five days after fertilization, zebrafish instinctively move to the aquatic environment and inflate their swim bladder. Therefore, they do not move from the outer well to the inner well until they achieve neutral buoyancy through swim bladder inflation, maintaining their swim-up behavior. In controlled experiments, when swim bladder inflation is complete, embryos located in the inner well swallow air bubbles through the water surface of the outer well and then move to the inner well, which does not indicate an abnormality in mobility. If an embryo located in the inner well fails to inflate its swim bladder, it is judged to be an abnormality in mobility. If an embryo located in the outer well fails to inflate its swim bladder, it is judged to be an abnormality in swim bladder inflation. 【0127】 For each of the 30 chemical substances <1> , <2> , <3> The number of embryos corresponding to each category is as follows: The total number of embryos used in the tests for each concentration and <1> and <2> The total number of fish embryos corresponding to the specified species was used, and the EC50 value was calculated using the Probit regression method, a type of regression analysis, with the statistical program SPSS (IBM). Here, the EC50 value represents the concentration at which 50% of the embryos fail to form a swim bladder. 【0128】 (1) 4,4'-Oxydianiline 【0129】 [Table 1-2] 【0130】 (2) 1,3-Benzenediamine 【0131】 [Table 1-3] 【0132】 (3) N-methyl-2-pyrrolidone 【0133】 [Table 1-4] 【0134】 (4) 4-Methylbenzenamine 【0135】 [Table 1-5] 【0136】 (5) 4,4'-Diaminobiphenylmethane 【0137】 [Table 1-6] 【0138】 (6) 1,4-Benzenediamine 【0139】 [Table 1-7] 【0140】 (7) Triethylene glycol dimethyl ether 【0141】 [Table 1-8] 【0142】 (8) Triethylene glycol monobutyl ether 【0143】 [Table 1-9] 【0144】 (9) Ethylene glycol monophenyl ether 【0145】 [Table 1-10] 【0146】 (10) Nicotinamide 【0147】 [Table 1-11] 【0148】 (11) Catechol 【0149】 [Table 1-12] 【0150】 (12) Diethylene glycol 【0151】 [Table 1-13] 【0152】 (13) Diethylene glycol monobutyl monobutyl ether acetate 【0153】 [Table 1-14] 【0154】 (14) Triethylene glycol diacetate 【0155】 [Table 1-15] 【0156】 (15) Caffeine 【0157】 [Table 1-16] 【0158】 (16) Tetraethylene glycol monobutyl ether 【0159】 [Table 1-17] 【0160】 (17) Ethanol 【0161】 [Table 1-18] 【0162】 (18) Phenol 【0163】 [Table 1-19] 【0164】 (19) Tetrachloroethylene 【0165】 [Table 1-20] 【0166】 (20) p-tert-butylphenol 【0167】 [Table 1-21] 【0168】 (21) Nitrobenzene 【0169】 [Table 1-22] 【0170】 (22) Isopropyl alcohol 【0171】 [Table 1-23] 【0172】 (23) 1,1,1-Trichloroethane 【0173】 [Table 1-24] 【0174】 (24) Trichloroethylene 【0175】 [Table 1-25] 【0176】 (25) 1,2-Dichloroethane 【0177】 [Table 1-26] 【0178】 (26) t-butyl hydroquinone 【0179】 [Table 1-27] 【0180】 (27) Diethylenetriaminepentaacetic acid 【0181】 [Table 1-28] 【0182】 (28) Ethylenediaminetetraacetic acid 【0183】 [Table 1-29] 【0184】 (29) Nickel(II) chloride 【0185】 [Table 1-30] 【0186】 (30) 9-cis-retinoic acid 【0187】 [Table 1-31] 【0188】 The analysis results for 30 chemical substances were compared with the results of the OECD's Acute Fetal Toxicity Test (FET test) and are shown in Table 2 and Figure 14 below. As a result, 24 substances (numbers 1, 2, 3, 4, 5, 6, 8, 9, 11, 12, 13, 14, 15, 16, 17, 18, 20, 21, 24, 26, 27, 228, 29, and 30 in Table 2 below) showed toxic effects on the swim bladder formation process. In particular, for numbers 6, 11, 13, 15, 20, and 29 in Table 2 below, the LC tests performed using the Acute Fetal Toxicity Test (FET) were not effective. 50 Compared to the value, the amount of EC is up to 10 times less. 50 It was confirmed that it possesses (Table 2 and Figure 14). This indicates that the swim bladder developmental effect alternative test method of the present invention can efficiently confirm neurodevelopmental toxicity with sensitivity up to approximately 10 times greater than conventional acute toxicity test methods for fish. 【0189】 Table 2 [Table 2] 【0190】 Although embodiments of the present invention have been described in detail above with reference to the drawings, the present invention is not limited to the embodiments described above, and a person with ordinary skill in the art can apply various technical modifications and variations based on the above. For example, the described techniques may be performed in a different order than described, and / or the components of the described systems, structures, apparatus, etc. may be combined or combined in a different manner than described, or substituted or replaced by other components or equivalents, and still achieve appropriate results. Accordingly, other realizations, other embodiments, other aspects, and equivalents to the claims also fall within the scope of the claims described later. 【0191】 This disclosure provides the following aspects: 【0192】 Side view 1. A plate for evaluating toxic substances using fish embryos, 【0193】 The plate includes n external wells and n internal wells located inside the external wells, where n is an integer between 1 and 96. 【0194】 A plate for evaluating toxic substances using fish embryos, wherein the height of the internal wells is lower than the height of the external wells. 【0195】 Side 2. In side 1, the internal wells are non-permeable, in the plate. 【0196】 Side 3. In any one of the aforementioned sides, the plate is a plate in which the outer well and the inner well are filled with a liquid selected from water and a test solution, such that the height of the liquid is lower than the height of the outer well and higher than the height of the inner well. 【0197】 Side 4. On any one of the aforementioned sides, the height of the internal well is approximately 5 mm to approximately 21 mm, and the difference (Δh) between the height of the internal well and the height of the liquid satisfies the relationship given by equation (1) below, a plate. [Formula 1] 【0198】 3mm ≤ liquid height - internal well height (Δh) ≤ 10mm 【0199】 Side 5. A plate in which, on any one of the aforementioned sides, the height ratio of the internal wells to the external wells is approximately 1:3 to approximately 1:1.2. 【0200】 Side 6. A plate in which, on any one of the aforementioned sides, the shape of the external well or internal well is a prism or a cylinder, respectively. 【0201】 Side 7. A plate in which, on any one of the aforementioned sides, the diameter of the external well is approximately 10 mm to approximately 40 mm, and the relationship in equation (2) below is satisfied. [Formula 2] 【0202】 1.1 ≤ outer well diameter / inner well diameter ≤ 4 【0203】 Side 8. On any one of the aforementioned sides, the fish embryo is a zebrafish embryo, plate. 【0204】 Side 9. In any of the aforementioned sides, the plate is for screening for neurodevelopmental toxic substances using fish embryos. 【0205】 Aspect 10. A method for screening neurodevelopmental toxic substances using fish embryos, wherein the method is: 【0206】 a) A step of providing one of the plates from sides 1 to 9, 【0207】 b) Fill each of the plates with water or a test solution to a height higher than the height of the internal wells and lower than the height of the external wells, and position the fish embryos in the internal wells. 【0208】 c) If water is filled in step b) above, the step of putting the sample into the internal well of the plate and 【0209】 d) A screening method comprising the step of checking whether or not there is movement between the internal and external wells of larvae formed from the embryo of the fish. 【0210】 Aspect 11. A screening method in which, in Aspect 10, the fish embryo is a zebrafish embryo. 【0211】 Aspect 12. A screening method in which, in Aspect 10, the fish embryos in step b) are fish embryos from immediately after fertilization to approximately 48 hours later. 【0212】 Side 13. In Side 10, step d) is a screening method performed 100 to 150 hours after fertilization of fish embryos. 【0213】 Aspect 14. A screening method in which step d) in Aspect 10 includes counting the number of larvae that failed to move between the inner well and the outer well, and the number of larvae that succeeded in moving between the inner well and the outer well. 【0214】 A screening method in which, in any one of the sides 10 to 14, if the swim bladder of a larva that has successfully moved between the internal well and the external well has inflated into a round balloon shape, it is determined that the swim bladder has successfully inflated. 【0215】 Aspect 16. In any one of Aspects 10 to 15, the screening method further comprises the step of determining that the sample is a toxic substance if the number of larvae that have successfully expanded their swim bladder is 50% or less of the total number of embryos. 【0216】 Apparatus for evaluating toxic substances using fish embryos, comprising a plate described on one of sides 1 to 9, and an image reader that performs a 14th count through automated image analysis. 【0217】 A device in which, in side view 18 and side view 17, the fish embryo is a zebrafish embryo. 【0218】 Side 19. The plate described on any one of sides 1 to 9 and 【0219】 Instructions for using the aforementioned plate in a screening method for neurodevelopmental toxic substances using fish embryos. 【0220】 A kit for evaluating neurodevelopmental toxicities using fish embryos, including [specific example of a product]. 【0221】 In side view 20 and side view 19, the fish embryo is a zebrafish embryo, kit. [Explanation of Symbols] 【0222】 1: Plate 100: Well 110: External well 120: Internal well Do: Diameter of the outer well Di: Diameter of the internal well Lo: Height of the external well Li: Height of the internal well

Claims

[Claim 1] A plate for evaluating toxic substances using fish embryos, The plate includes n external wells and n internal wells located inside the external wells, where n is an integer between 1 and 96. The aforementioned plate for evaluating toxic substances using fish embryos has an internal well height lower than the external well height. [Claim 2] The plate according to claim 1, wherein the internal well is non-permeable. [Claim 3] The plate according to claim 1, wherein the plate has an outer well and an inner well filled with a liquid selected from water and a test solution, the height of the liquid being lower than the height of the outer well and higher than the height of the inner well. [Claim 4] The plate according to claim 3, wherein the height of the internal well is approximately 5 mm to approximately 21 mm, and the difference (Δh) between the height of the internal well and the height of the liquid satisfies the relationship shown in equation (1) below. [Formula 1] 3 mm ≤ liquid height - internal well height (Δh) ≤ 10 mm [Claim 5] The plate according to claim 1, wherein the height ratio of the internal well to the external well is approximately 1:3 to approximately 1:1.

2. [Claim 6] The plate according to claim 1, wherein the shape of the external well or internal well is a prism or a cylinder, respectively. [Claim 7] The plate according to claim 1, wherein the diameter of the external well is approximately 10 mm to approximately 40 mm and satisfies the relationship shown in equation (2) below. [Formula 2] 1.1 ≤ outer well diameter / inner well diameter ≤ 4 [Claim 8] The plate according to claim 1, wherein the fish embryo is a zebrafish embryo. [Claim 9] The plate according to claim 1, wherein the plate is for screening neurodevelopmental toxic substances using fish embryos. [Claim 10] A screening method for neurodevelopmental toxic substances using fish embryos, The aforementioned method, a) the step of providing the plate according to any one of claims 1 to 9, b) Filling each of the plates with water or a test solution to a height higher than the height of the internal wells and lower than the height of the external wells, and positioning the fish embryos in the internal wells, c) If water is filled in step b) above, the step of introducing the sample into the internal well of the plate, d) A step of confirming whether or not there is movement between the inner well and the outer well of the larvae formed from the fish embryo, A screening method that includes this. [Claim 11] The screening method according to claim 10, wherein the fish embryo is a zebrafish embryo. [Claim 12] The screening method according to claim 10, wherein the fish embryo in step b) is a fish embryo from immediately after fertilization to about 48 hours later. [Claim 13] The screening method according to claim 10, wherein step d) is performed 100 to 150 hours after fertilization of the fish embryo. [Claim 14] The screening method according to claim 10, wherein step d) includes counting the number of larvae that failed to move between the inner well and the outer well, and the number of larvae that succeeded in moving between the inner well and the outer well. [Claim 15] Among the larvae that successfully moved between the inner well and the outer well, The screening method according to claim 14, wherein if the swim bladder of a larval fish inflates into a round, balloon-like shape, it is determined that the swim bladder has successfully inflated. [Claim 16] The aforementioned screening method is e) The screening method according to claim 10, further comprising the step of determining that the sample is a toxic substance if the number of larvae that have successfully expanded their swim bladder is 50% or less of the total number of embryos. [Claim 17] A plate according to any one of claims 1 to 9, An image reader that performs the count described in claim 14 through automated image analysis, A device for evaluating toxic substances using fish embryos, including [specific example]. [Claim 18] The apparatus according to claim 17, wherein the fish embryo is a zebrafish embryo. [Claim 19] A plate according to any one of claims 1 to 9, Instructions for using a plate described in any one of claims 1 to 9 in a method for screening neurodevelopmental toxic substances using fish embryos, A kit for evaluating neurodevelopmental toxicities using fish embryos, including [specific example of a product]. [Claim 20] The kit according to claim 19, wherein the fish embryo is a zebrafish embryo.

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