A chemical toxicity testing method based on a self-developed pollutant exposure system

Abstract

The present application belongs to the technical field of biological toxicity test of chemicals, and particularly relates to a chemical toxicity test method based on a self-developed pollutant exposure system. The test method is used for simulating the toxicity of chemicals in natural water bodies by means of a self-developed pollutant exposure system, adopts zebrafish as a model organism, establishes a time-concentration linear function of the tested chemicals in a poisoning area, and calculates the toxicity test method of the minimum effect concentration of the tested chemicals by observing the abnormal behavior of zebrafish or analyzing the behavior by coupling a camera tracking technology. The test method is almost a non-destructive experiment, the experimental time is short, most of the zebrafish participating in the experiment can be restored to normal state through appropriate recovery, the time cost is greatly saved, and the test is efficient and has a low mortality rate of test organisms.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological toxicity test of chemicals, and particularly relates to a chemical toxicity test method based on a self-developed pollutant exposure system. BACKGROUND

[0002] Currently, the aquatic biological toxicity test data of chemicals mainly reflect the half lethal concentration (LC 50 ) and the half effect concentration (EC 50 ), and these data often need a long test time (such as 24h, 48h, 96h) and have irreversible damage (such as death) to model organisms; in addition, there are molecular toxicity data such as carcinogenicity, mutagenicity, neurotoxicity, reproductive toxicity and endocrine toxicity, and the test of these toxicity data needs strong professional knowledge and skills, and the instruments and conditions required for the test are relatively expensive, and part of the experiment needs to expose animals and extract target organs and tissues, which also has irreversible damage to model organisms.

[0003] Chemicals enter the environment water through various ways, and the concentration is not constant. The dilution effect can reduce the concentration of chemicals entering the water, which makes the control concentration setting of pollution and leakage lack of basis. If such data is to be obtained, a steady-state exposure experiment with multiple concentration gradients, long time and high mortality is needed, which greatly consumes manpower and material resources. Therefore, an experimental method for gradually and cumulatively exposing natural water to receive and dilute chemicals and biological organisms in the water is needed to guide the management of chemicals.

[0004] Animal behavior experiment is an experimental method for comparing the behavior difference between normal state and abnormal state, which is widely used in toxicology research. Since the behavior needs to be recorded, the amount of animal use and the number of animal death are much lower than those in LC 50 test experiment, and part of the animals can recover to normal through appropriate recovery stage. At the same time, fish can sensitively perceive the changes of water flow and water quality through various sensory organs, and make various behavioral responses to complete the necessary foraging, avoiding danger and courtship behaviors, and the like, and the behavior change is more sensitive than the biochemical biomarker. Moreover, the behavior abnormality can be caused by motor nerve abnormality, which can be caused by neurotoxicity or triggered by endocrine homeostasis imbalance, and the time is much earlier than death, which has early warning function.

[0005] The current aquatic biological toxicity test technology is relatively single, the molecular biology toxicity test technology has high requirement for professional level, and the biological damage and mortality of the test method are also relatively high. SUMMARY

[0006] The technical problem to be solved by the present application is to provide a chemical toxicity testing method based on a self-developed pollutant exposure system, which uses a self-developed pollutant exposure system to simulate the toxicity of chemicals in natural water bodies, uses zebrafish as a model organism, and obtains the concentration that produces long-lasting abnormal behavior without killing zebrafish by observing the abnormal behavior of zebrafish or coupling video tracking technology for behavior analysis, which has the advantages of simple method, short experimental time, low biological mortality, and recoverable test organisms. The technical scheme adopted is:

[0007] A chemical toxicity testing method based on a self-developed pollutant exposure system, which uses a self-developed pollutant exposure system, the system includes a cylinder with an open top, a buffer zone, a contaminated zone and a water outlet zone are arranged in the cylinder in sequence, the buffer zone and the contaminated zone, the contaminated zone and the water outlet zone are separated by a perforated partition, zebrafish are used as model organisms in the exposure system, the testing method specifically includes the following steps:

[0008] (1) Mother liquor preparation, query the LC 50 value of the tested chemical, adjust the initial concentration of the mother liquor according to the LC 50 value of the tested chemical, if the chemical has no obvious effect on the behavior of zebrafish, the concentration can be further increased according to the results of the pre-experiment;

[0009] (2) Establish a time-concentration linear function of the tested chemical in the contaminated zone, which is:

[0010] When t≤40 minutes, C=0.60871t+0.36129;

[0011] When 40 minutes<t≤600 minutes, C=0.04706t+24.10669;

[0012] Wherein, C is the concentration percentage of the chemical relative to the mother liquor, unit is %, t is the water time of the mother liquor;

[0013] (3) Perform a significant behavior test in the exposure system, calculate the concentration of the tested chemical that causes significant changes in the behavior of zebrafish, i.e. the lowest effect concentration;

[0014] (4) Perform behavior analysis on zebrafish in the exposure system by coupling video tracking technology, obtain the lowest effect concentration of the tested chemical;

[0015] Wherein, steps (3) and (4) are optional analysis steps.

[0016] Preferably, the initial concentration of the chemical mother liquor is adjusted according to the LC 50 value of the tested chemical, the adjustment range is 0.5LC 50~2LC 50 .

[0017] Preferably, the distance between the partitions of the self-developed pollutant exposure system is not less than 20 cm, the partitions are punched, the buffer zone, the exposure zone and the effluent zone are provided with an internal circulation system; the buffer zone is provided with an inlet pipe, the effluent zone is provided with an outlet pipe, and the inlet pipe and the outlet pipe are connected to a peristaltic pump to provide power, and the inlet and outlet water flow is set to 0.1-10 L / h.

[0018] As a further preference, the internal circulation system comprises a submersible pump, a circulating water pipe and an aeration assembly, the aeration assembly comprises an aeration pipe and an aeration regulating valve; the circulating flow of the submersible pump is 500-1500 L / h, the submersible pump is placed in the middle of the wall of the effluent zone and maintains a distance of 1-3 mm from the wall, and the pump body and the cylinder body are filled with biochemical cotton to absorb excrement; the circulating water pipe is connected to the outlet of the submersible pump, and the other end is connected to the buffer zone, the outlet end of the circulating water pipe is vertically downward, the outlet is located in the middle of the buffer zone, and a hole is opened 0.5-1 cm above the outlet of the circulating water pipe, the aeration pipe and the aeration regulating valve are inserted, and the aeration regulating valve is placed in the air, the interface is a tubular hard plastic pipe with beveled cutting, and the shorter side faces the outlet of the circulating water pipe to ensure that water flow does not enter the aeration pipe.

[0019] As a further preference, the partitions comprise upper punched partitions, lower punched partitions and full-punched partitions, and the partitions are made of transparent acrylic plates with a 1-3 mm high excrement groove at the bottom.

[0020] As a further preference, the aperture of the holes punched on the partitions is 1-3 mm, and the distance between the holes is set to 1-2 times the aperture.

[0021] Preferably, the time-concentration linear function of the chemical to be tested is obtained by fitting the scatter plot of the pollutant concentration measured at different time points in each region with time, and the fitting method is apparent fitting.

[0022] Preferably, the method for testing the significant behavior is as follows:

[0023] Pure water is pre-loaded in the cylinder body, preheated to 27℃ using a heating rod, sterilized with an ultraviolet lamp and aerated for more than 1 h; adult male zebrafish are placed in the exposure zone for water quality adaptation, and the adaptation stage lasts at least 1 h; after the adaptation period, a blank control experiment lasting 1 h is performed, i.e. the inlet and outlet water peristaltic pumps are started, the inlet peristaltic pump is connected to pure water, and the behavior of the zebrafish during the blank control experiment is observed in detail as a standard behavior control; if it is necessary to observe the toxicity target, a stimulation experiment of the corresponding toxicity target should be additionally performed, such as light stimulation or image stimulation without noise and vibration to measure the visual damage of the chemical to the zebrafish, and the behavioral response of the zebrafish is observed;

[0024] After the end of the blank control experiment, the water inlet pipe is directly connected to the mother liquor of the chemical to be tested, and the poisoning experiment is carried out. During the experiment, the normal behavior and abnormal behavior changes of the zebrafish are observed. The abnormal postures are photographed and recorded. The time of abnormal behavior is recorded.

[0025] If there is no significant abnormal behavior in 9h, the concentration of the chemical to be tested is increased for the second experiment. The concentration of the chemical to be tested can be appropriately increased, for example, by 20% or 50% of the initial concentration, until abnormal behavior occurs.

[0026] If significant abnormal behavior occurs within 9h, the time of abnormal behavior is recorded. According to the time-concentration linear function of the chemical to be tested, the minimum effect concentration is calculated.

[0027] Preferably, the standard / normal behavior includes sinking, floating, cruising, and hitting the wall, but is not limited to these four behaviors. The abnormal behavior includes sudden high-speed movement, long-term abnormal high-speed / stationary state, and swimming posture change. The selection of abnormal behavior should have significant differences to avoid subjective differences of people.

[0028] Preferably, for chemicals to be tested with low toxicity, the behavior changes are analyzed by coupling the camera tracking technology to obtain the non-lethal abnormal behavior concentration, i.e. the minimum effect concentration.

[0029] Preferably, the behavior analysis method of the coupling camera tracking technology is as follows:

[0030] A camera is installed at a suitable height above the center of the cylinder to take pictures of the inside of the cylinder.

[0031] First, a blank control video of 1-2h is taken as a normal state baseline. Then, the chemical mother liquor is introduced into the water. The water is introduced until the behavior of the zebrafish is visibly abnormal or the chemical mother liquor has been introduced for 10h.

[0032] The zebrafish is taken out and placed in sterilized pure water for recovery. The video is analyzed to quantitatively obtain behavior intensity related data such as swimming trajectory, swimming speed, distance and time of the zebrafish. By comparing with the baseline, the long-term abnormal motion state is selected as the abnormal behavior indicator. According to the start time of the abnormal motion state, the minimum effect concentration is calculated.

[0033] Preferably, the resolution of the camera is at least 1288x964, and the frame rate is 30 frames per second. The camera can clearly take pictures of the exposure system.

[0034] Compared with the prior art, the present application has the following advantages:

[0035] The application can realize the simulation of chemical point source emission into larger water area by means of the self-developed pollutant exposure system, gradually increasing exposure from low concentration to high concentration in a gradual and cumulative manner, and dispersing pollutants with high efficiency, and the chemical concentration-time linear function model disclosed in the application can support concentration prediction in the whole period.

[0036] The application uses the self-developed pollutant exposure system for chemical toxicity testing, and the minimum effect concentration of abnormal behavior can reflect the neurotoxicity of chemicals on zebrafish or the characterization of behavior changes caused by endocrine disorders, the experimental method is almost non-destructive, and most of the zebrafish participating in the experiment can be restored to normal state through appropriate recovery, saving the cost of test animals, and since the exposure mode is gradual and cumulative, the response can be obtained faster, and the zebrafish will show more significant abnormal behavior at a concentration lower than the median lethal concentration; and for chemicals with lower toxicity, the behavior change can be analyzed by coupling the camera tracking technology to obtain the non-lethal abnormal behavior concentration, greatly saving the time cost, and the test biological death rate is low. BRIEF DESCRIPTION OF DRAWINGS

[0037] Figure 1 The structure diagram of the self-developed pollutant exposure system used in the application (the camera is omitted);

[0038] Figure 2 The partition diagram of the self-developed pollutant exposure system used in the application;

[0039] Figure 3 The concentration change function equation of the pollutant (to-be-tested chemical) in the three regions of the exposure system;

[0040] Figure 4 The flowchart of significant behavior test in example 1;

[0041] Figure 5 The flowchart of behavior analysis of coupling camera tracking technology in example 2;

[0042] Figure 6 The trajectory diagram of zebrafish in different periods of ZnSO4 water inlet in example 2;

[0043] Figure 7 The motion distance-water inlet time diagram of ZnSO4 water inlet in example 2;

[0044] Figure 8 The motion time-water inlet time diagram of K2Cr2O7 water inlet in example 3;

[0045] Figure 9 The motion time-water inlet time diagram of K2Cr2O7 water inlet in example 3;

[0046] Figure 10 This is a schematic diagram showing the number of times, time, and distance of lead acetate entering the water in Example 4, along with the water inlet time. Detailed Implementation

[0047] The accompanying drawings are for illustrative purposes only; to better illustrate this embodiment, some components in the drawings may be omitted, enlarged, or reduced, and do not represent the actual product dimensions; it should be understood that the orientation or positional relationship indicated by directional terms such as "upper," "lower," "left," "right," "side," "outer," "lateral," "vertical," "horizontal," "top," and "bottom" is usually based on the orientation or positional relationship shown in the accompanying drawings, and is only for the convenience of describing this application and simplifying the description. Unless otherwise stated, these directional terms do not indicate or imply that the device or element referred to must have a specific orientation or be constructed and operated in a specific orientation, and therefore should not be construed as a limitation on the scope of protection of this application; the directional terms "inner" and "outer" refer to the inner and outer contours relative to the outline of each component itself.

[0048] For those skilled in the art, certain well-known structures and their descriptions in the accompanying drawings may be omitted, and therefore should not be construed as limiting the present invention.

[0049] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings.

[0050] Example 1:

[0051] like Figure 1 As shown, this invention uses a self-developed contaminant exposure system, including a top-open tank. Inside the tank, three zones are sequentially arranged: a buffer zone 1, a contamination zone 2, and an outlet zone 3. An inlet baffle 11 separates the buffer zone 1 from the contamination zone 2, and an outlet zone separates the contamination zone 2 from the outlet zone 3. The three zones are separated by baffles. Each baffle has holes with a 2mm diameter to allow water and contaminants to pass through. To improve contaminant diffusion, the distance between holes is set to 1.5 times the hole diameter. All aquatic organisms smaller than this size can be used for experiments. Each baffle is fixed in place using acrylic clips and aquaculture-specific suction cups (i.e., the first fixed suction cup 8). The clips and suction cups should be placed on the buffer zone 1 and the outlet zone side to prevent interference with the normal swimming of the organisms and for photographing.

[0052] The buffer zone 1 is equipped with a sewage inlet pipe 7, the other end of which is connected to a filtered sewage tank that is kept at a constant temperature by a heating rod. The outlet zone 3 is equipped with an outlet pipe 13, the other end of which is connected to a wastewater collection system. Both the sewage inlet pipe 7 and the outlet pipe 13 are connected to peristaltic pumps to provide power.

[0053] The buffer zone 1, contaminated zone 2, and effluent zone 3 are equipped with an internal circulation system.

[0054] The inner circulation system comprises a submersible pump 10, a circulating water pipe 9 and an aeration assembly 4; the submersible pump 10 is a common submersible pump for freshwater fish farming filter system, and the circulating flow is 500-1500 L / h, and the flow size can be set in the range. The submersible pump 10 is arranged in the middle of the cylinder wall of the water outlet area 3, is fixed by using an acrylic fixing buckle and a second fixed suction cup, the pump water outlet is vertically upward, the water inlet section is toward the cylinder wall and keeps a distance of 3 mm from the cylinder wall, and biochemical cotton is filled between the pump body and the cylinder body to absorb excrement.

[0055] The circulating water pipe is connected with the water outlet of the submersible pump, and the other end is connected to the buffer 1, that is, the water outlet of the circulating water pipe 9 is arranged in the buffer area 1. A hole is opened at 1 cm above the water outlet of the circulating water pipe 9, the aeration pipe 5 and the aeration adjusting valve 6 are inserted, and the aeration adjusting valve 6 is arranged in the air, an inclined cutting tubular hard plastic pipe is used at the interface, and the shorter side is toward the water outlet of the circulating water pipe 9 to ensure that the water flow does not enter the aeration pipe 5.

[0056] As shown in Figure 2 As a further preferred, the water inlet partition plate 11 is a full-hole partition plate, and the water outlet partition plate 12 is a lower-hole partition plate.

[0057] The holes on each partition plate can meet different specifications of water distribution systems and pollutant conditions, and the design can also meet the direct exposure to different water depth organisms, for example, the benthic mussel can use the lower hole partition plate.

[0058] The resolution of the camera is 1288x964, and the frame rate is 30 frames per second. The camera can clearly shoot the exposure system. The installation and fixation of the camera use the existing technology, and the installation position is located above the exposure system at a suitable height, so that it can be clearly shot.

[0059] In this embodiment, the cylinder body size is designed as 40x23x25 cm, and the cylinder thickness is 5 mm; each partition plate is vertically inserted into the cylinder body, and the long side of the cylinder is divided into an 8 cm buffer area, a 22 cm exposure area and a 9 cm water outlet area; the sewage inlet pipe and the water outlet pipe are 12 cm away from the bottom of the cylinder; the sewage inlet pipe is 1 cm away from the water outlet end of the circulating water pipe, and the aeration hole is 1 cm away from the water outlet end of the circulating water pipe; the bottom of the submersible pump is 8 cm away from the bottom of the cylinder.

[0060] The partition plate size is 22x24 cm, the bottom manure groove is 2 mm high, the whole plate is punched, the hole diameter is 2 mm, and the thickness is 5 mm.

[0061] The width of the experimental motile organisms is not less than 3 mm, and therefore the bottom manure groove and the aperture are less than 3 mm; the cylinder body is 23 cm wide, and the partition plate is 22 cm wide, which can effectively prevent the motile organisms from leaving the poisoning area from both sides of the partition plate in the presence of manufacturing errors; the height of the partition plate is designed to be slightly lower than the height of the cylinder body, so as to prevent the height of the partition plate from being higher than the cylinder body due to manufacturing errors, which affects the installation of the cover plate in the later stage; the cover plate is an optional item, and the main purpose is to prevent fish from jumping out of the cylinder body.

[0062] Since the fish and shrimp organisms have the behavior of jumping out of the water surface, the water surface needs to be lower than the height of the partition plate / cylinder body to prevent leaving the poisoning area from above or jumping out of the device; in order to meet the vertical swimming distance of aquatic organisms, the water surface height is designed to be 20 cm, the poisoning area size is 22*23*20 cm, and the volume is 10.12 L.

[0063] Linear fitting is realized by origin software, and the fitting method is apparent fitting, and then the time-concentration change function equation of the pollutant (to-be-tested chemical) in the three regions of the exposure system is as shown in Figure 3 The lowest effect concentration corresponding to all abnormal behaviors is obtained by the concentration function of the poisoning area (directly exposed to the water body of zebrafish) and the time of abnormal behavior.

[0064] In this embodiment, the camera is used to record the behavior of zebrafish without analysis, and the abnormal behavior visible to the naked eye is used as the judgment standard, which has a certain indication effect on the minimum effect concentration of high-toxicity chemicals.

[0065] As shown in Figure 4 , phenol is used as a model chemical, and according to the literature, the LC 50 of phenol to adult zebrafish is 144.02 μM, and the phenol stock solution configured based on this concentration is used as the water inlet for pre-experiment, and it is found that it takes a long time for zebrafish to exhibit abnormal behavior, and within 20 minutes in the initial water inlet, no significant avoidance behavior of zebrafish is observed. Therefore, we increase the concentration of the phenol stock solution to 2 mM, and the water inlet flow is set to 1 L / h.

[0066] Pure water is pre-loaded in the cylinder, preheated to 27°C using a heating rod, sterilized by ultraviolet lamp and aerated for 1 h; adult zebrafish (3 months) are placed in the poisoning area for water quality adaptation, and the adaptation stage lasts for 1 h; after the adaptation period, the peristaltic pump is started to connect pure water as the water inlet, and the blank control experiment is performed for 1 h.

[0067] Zebra fish showed stable speed of wall sticking movement in the adaptation stage, indicating that the zebra fish had recognized the range of the experimental water area, and there was almost no state of complete staticity for more than 1 min, and there was normal state of up and down, and the swimming posture was normal, which were all identified as behaviors in the normal state. The exposure of organic matter may cause the zebra fish to be excited and swim fast, swim vertically, swim laterally, struggle and move, and the phenomenon of narcosis, and phenol as a representative of organic matter may have the above behaviors, which need to be paid special attention in observation.

[0068] The water inlet pipe was directly connected to the phenol mother liquor after 1h, and the exposure experiment was carried out. It was observed in the experiment that the zebra fish showed significant wall collision behavior 140 min after the beginning of the exposure, which was an obvious escape behavior, and the behavior lasted for about 1h; the zebra fish began to swim laterally intermittently 220 min after the beginning of the exposure; the zebra fish began to swim laterally continuously and the wall collision behavior decreased 230 min after the beginning of the exposure, and the zebra fish patrolled in the activity area in a relatively stable and continuous swimming state until the end of the experiment 5h after the beginning of the exposure, and then the zebra fish was taken out and placed in a pure water tank for recovery.

[0069] Example 2:

[0070] As shown in Figure 1 The present application uses a self-developed pollutant exposure system, which includes a cylinder with an open top, and the inside of the cylinder is sequentially provided with a buffer zone 1, an exposure zone 2 and a water outlet zone 3. An inlet partition 11 is arranged between the buffer zone 1 and the exposure zone 2, and a water outlet is arranged between the exposure zone 2 and the water outlet zone 3. The three zones are separated by the partitions, and holes are punched on each partition to allow water flow and pollutants to pass through. The hole diameter is 2mm, and the distance between the holes is 1.5 times the hole diameter to improve the diffusion effect of the pollutants. All aquatic organisms with a size less than the size can use the device for experiments. The partition position is fixed by a fixed buckle made of acrylic plate and a special suction cup for breeding (i.e. a first fixed suction cup 8). The buckle and the suction cup should be placed on one side of the buffer zone 1 and the water outlet zone to prevent affecting the normal swimming and shooting of the organisms.

[0071] The buffer zone 1 is provided with a sewage inlet pipe 7, and the other end of the sewage inlet pipe 7 is connected to a sewage tank after filtration and using a heating rod for constant temperature. The water outlet zone 3 is provided with a water outlet pipe 13, and the other end of the water outlet pipe 13 is connected to a wastewater collection system. The sewage inlet pipe 7 and the water outlet pipe 13 are both connected to a peristaltic pump to provide power.

[0072] The buffer zone 1, the exposure zone 2 and the water outlet zone 3 are provided with an internal circulation system.

[0073] The inner circulation system comprises a submersible pump 10, a circulating water pipe 9 and an aeration assembly 4; the submersible pump 10 is a common submersible pump for freshwater fish breeding filter system, and the circulating flow is 500-1500 L / h, and the flow size can be set in the range. The submersible pump 10 is arranged in the middle of the cylinder wall of the water outlet area 3, is fixed by using an acrylic fixing buckle and a second fixing suction cup, the pump water outlet is vertically upward, the water inlet section is towards the cylinder wall and keeps a distance of 3 mm from the cylinder wall, and biochemical cotton is filled between the pump body and the cylinder body to absorb excrement.

[0074] The circulating water pipe is connected with the water outlet of the submersible pump, and the other end is connected to the buffer 1, that is, the water outlet of the circulating water pipe 9 is arranged in the buffer area 1. A hole is opened at 1 cm above the water outlet of the circulating water pipe 9, the aeration pipe 5 and the aeration adjusting valve 6 are inserted, and the aeration adjusting valve 6 is arranged in the air, the interface adopts a tubular hard plastic pipe with beveled cutting, and the shorter side is towards the water outlet of the circulating water pipe 9 to ensure that the water flow does not enter the aeration pipe 5.

[0075] As shown in Figure 2 As a further preferred, the water inlet partition plate 11 is a full-hole partition plate, and the water outlet partition plate 12 is a lower-hole partition plate.

[0076] The holes on each partition plate can meet different specifications of water distribution systems and pollutant conditions, and the design can also meet the direct exposure of different water depth organisms, for example, the benthic mussel can use the lower hole partition plate.

[0077] The resolution of the camera is 1288x964, and the frame rate is 30 frames / s, and the high-definition camera can clearly shoot the exposure system. The installation and fixation of the camera adopt the existing technology, and the installation position is located above the exposure system at a suitable height, so that the camera can clearly shoot.

[0078] In the embodiment, the cylinder body size is designed as 40x23x25 cm, and the cylinder thickness is 5 mm; each partition plate is vertically inserted into the cylinder body, and the long side of the cylinder is divided into an 8 cm buffer area, a 22 cm exposure area and a 9 cm water outlet area; the sewage inlet pipe and the water outlet pipe are 12 cm away from the bottom of the cylinder; the sewage inlet pipe is 1 cm away from the water outlet end of the circulating water pipe, and the aeration hole is 1 cm away from the water outlet end of the circulating water pipe; the bottom of the submersible pump is 8 cm away from the bottom of the cylinder.

[0079] The partition plate size is 22x24 cm, the bottom manure groove is 2 mm high, the whole plate is punched, the hole diameter is 2 mm, and the thickness is 5 mm.

[0080] The width of the experimental motile organisms is not less than 3 mm, so the bottom manure tank and the aperture are less than 3 mm; the cylinder body is 23 cm wide, and the partition is 22 cm wide, which can effectively prevent motile organisms from leaving the dyeing area from both sides of the partition in the presence of manufacturing errors; the height of the partition is designed to be slightly lower than the height of the cylinder body to prevent the height from being higher than the cylinder body due to manufacturing errors, which affects the installation of the cover plate later; the cover plate is an optional item, and the main purpose is to prevent fish from jumping out of the cylinder body.

[0081] Since fish and shrimp organisms have the behavior of jumping out of the water surface, the water surface needs to be lower than the height of the partition / cylinder body to prevent leaving the dyeing area from above or jumping out of the device; in order to meet the vertical swimming distance of aquatic organisms, the water surface height is designed to be 20 cm, the dyeing area size is 22x23x20 cm, and the volume is 10.12 L.

[0082] The time-concentration function equation of the pollutant (to be tested chemical) in the three regions of the exposure system is as shown in Figure 3 The lowest effect concentration corresponding to all abnormal behaviors is obtained by the concentration function of the dyeing area (water body directly exposed to zebrafish) and the time when abnormal behavior occurs.

[0083] As shown in Figure 5 In this embodiment, the zebrafish behavior information and data under ZnSO4 exposure are collected in combination with the zebrafish 2D behavior analysis system of Viewpoint Company, and the specific method is as follows.

[0084] According to the literature, the 96h LC 50 of ZnSO4 on adult zebrafish is 461.54 μM, and the mother liquor concentration is configured to be 450 μM, and the mother liquor of this concentration is used as the water inlet, and the water inlet flow is set to 1 L / h.

[0085] Pure water is pre-loaded in the cylinder, preheated to 27°C using a heating rod, sterilized with a UV lamp and aerated for 1 h; adult zebrafish (3 months) are placed in the dyeing area for water quality adaptation, and the adaptation stage lasts for 1 h; after the adaptation period, the peristaltic pump is started to connect pure water as the water inlet.

[0086] As shown in Figure 6 , the trajectory diagram of zebrafish at different time periods of ZnSO4 water inlet. Open the camera and software of the Viewpoint behavior analysis system, set the ruler, set the minimum detection threshold to 20, the high-speed swimming threshold to 30, and the inactivity threshold to 5, collect data every 1 min and 5 min, and start recording data after removing the background. First, 1 h of pure water inlet is used as a blank experiment, and its behavior data is used as a baseline, and after 1 h, the water inlet pipe is switched to the ZnSO4 mother liquor to start the dyeing experiment. The experiment is carried out until the zebrafish swimming is almost stationary, and the zebrafish is transferred to a pure water cylinder for recovery.

[0087] As shown inFigure 6 It can be seen that in the blank control experiment within 1 h, the zebrafish swimming is concentrated in the center of the video collection area, and there is a small amount of wall sticking swimming. With the pure water entering, the behavior of zebrafish has no significant abnormal change. When ZnSO4 starts to enter, the trajectory range of zebrafish significantly expands, and even the trajectory leaves the collection area and enters the dead angle of collection, which is a stress behavior, but the swimming speed and distribution of zebrafish change little overall. When the experiment starts for 2 h, the motion trajectory changes significantly, the high-speed swimming distance and frequency significantly increase, and the abnormal patrol state behavior trajectory appears. This phenomenon lasts for 1 min, and then the zebrafish starts to move in the center of the video collection area, with slow swimming speed and short swimming distance. This phenomenon lasts until the end of the experiment.

[0088] The moving distances of the three motion states, high-speed moving, low-speed moving and near-stagnant state moving, are counted respectively as shown in Table 1. Figure 7 As shown in Table 1, with the start of ZnSO4 entering, the overall swimming distance of zebrafish gradually decreases at a lower speed, mainly reflected in the decrease of low-speed moving distance. At 120 min, the high-speed moving distance and low-speed moving distance significantly increase, and the near-stagnant state moving distance significantly decreases, indicating that zebrafish has a significant abnormal behavior under external stimulation, but since the duration of this behavior is only 1 min and there is no significant continuity before and after, this phenomenon cannot be ruled out the interference of unknown external factors or individual differences of fish. Subsequently, the overall swimming distance of zebrafish decreases to 1 / 2 of the baseline level after experiencing a slow decrease of about 7 min, but it is worth noting that this decrease is almost only reflected in the low-speed moving distance, and the near-stagnant state moving distance significantly increases, which is consistent with the picture results in the last time of 135-140 min and 155-160 min in Table 1. Figure 6

[0089] Combining the trajectory of zebrafish and the statistical data of zebrafish behavior, we believe that after 127 min of the start of the experiment, that is, 67 min after the start of ZnSO4 entering, zebrafish appears a significant and continuous abnormal behavior of reducing activity. At this time, the concentration of ZnSO4 is 27.26% of the mother liquor, that is, 122.67 μM. This concentration is far less than the 96h LC 50 of ZnSO4 on zebrafish, and at this concentration, zebrafish has no significant damage, but it can be concluded that ZnSO4 at this concentration has an inhibitory effect on the motion behavior of zebrafish. This concentration is far less than the LC 50 of zebrafish, and a significant effect is produced only in 127 min, which confirms that this method saves the experimental time well and is also a non-destructive method for obtaining toxicity data.

[0090] Example 3

[0091] As​Figure 1 As shown, the present application uses a self-developed pollutant exposure system, including a cylinder with an open top, three areas of buffer zone 1, exposure zone 2 and effluent zone 3 are arranged in the cylinder in turn, water inlet partition 11 is arranged between buffer zone 1 and exposure zone 2, and effluent is arranged between exposure zone 2 and effluent zone 3, the three areas are separated by the partition, and holes are punched on each partition to allow water flow and pollutants to pass through, the hole diameter is designed to be 2mm, in order to improve the pollutant diffusion effect, the distance between the holes is set to 1.5 times the hole diameter, and all aquatic organisms with a size less than the size can use the device for experiment. The position of each partition is fixed by using a fixed buckle made of acrylic plate and a special suction cup for breeding (i.e. first fixed suction cup 8), and the buckle and suction cup should be placed on one side of the buffer zone 1 and the effluent zone to prevent affecting the normal swimming and shooting of the organisms.

[0092] The buffer zone 1 is provided with a sewage inlet pipe 7, the other end of the sewage inlet pipe 7 is connected to a sewage tank after filtration and using a heating rod for constant temperature, the effluent zone 3 is provided with an effluent pipe 13, the other end of the effluent pipe 13 is connected to a wastewater collection system. The sewage inlet pipe 7 and the effluent pipe 13 are connected to a peristaltic pump to provide power.

[0093] The buffer zone 1, exposure zone 2 and effluent zone 3 are provided with an internal circulation system.

[0094] The internal circulation system includes a submersible pump 10, a circulating water pipe 9 and an aeration assembly 4; the submersible pump 10 adopts a submersible pump of the same kind of a common freshwater fish breeding filtration system on the market, and the circulating flow is 500-1500L / h, and the flow size can be set within the range. The submersible pump 10 is placed in the middle of the cylinder wall of the effluent zone 3, and is fixed by using an acrylic fixed buckle and a second fixed suction cup, the pump outlet is vertically upward, the water inlet section faces the cylinder wall, and maintains a distance of 3mm with the cylinder wall, and biochemical cotton is filled between the pump body and the cylinder body to absorb excrement.

[0095] The circulating water pipe is connected to the submersible pump outlet, and the other end is connected to the buffer 1, i.e. the circulating water pipe 9 outlet is arranged in the buffer zone 1. A hole is opened at 1cm above the outlet of the circulating water pipe 9, an aeration pipe 5 and an aeration adjusting valve 6 are inserted, and the aeration adjusting valve 6 is placed in the air, an inclined cutting tubular hard plastic pipe is used at the interface, and the shorter side faces the outlet of the circulating water pipe 9 to ensure that the water flow will not enter the aeration pipe 5.

[0096] As shown, Figure 2 As a further preferred, the water inlet partition 11 adopts a full-hole partition, and the effluent partition 12 adopts a lower-hole partition.

[0097] The holes on each partition can meet different specifications of water distribution systems and pollutant conditions, and the design can also meet the direct exposure of different water depth organisms, such as the benthic mussel which can use the lower hole partition.

[0098] The resolution of the camera is 1288x964, and the frame rate is 30 frames per second. The camera can clearly capture the exposure system. The installation and fixation of the camera use existing technology, and the installation position is located above the exposure system at a suitable height to ensure clear shooting.

[0099] In this embodiment, the cylinder size is designed to be 40x23x25cm, and the cylinder thickness is 5mm. Each partition is vertically inserted into the cylinder to divide the long side of the cylinder into an 8cm buffer zone, a 22cm toxic zone, and a 9cm water outlet zone. The sewage inlet pipe and the water outlet pipe are 12cm away from the cylinder bottom. The sewage inlet pipe is 1cm away from the water outlet end of the circulating water pipe, and the aeration hole is 1cm away from the water outlet end of the circulating water pipe. The bottom of the submersible pump is 8cm away from the cylinder bottom.

[0100] The partition size is 22x24cm, and the bottom manure tank is 2mm high. The whole plate is perforated, with a hole diameter of 2mm and a thickness of 5mm.

[0101] The experimental motile organisms are not less than 3mm wide, so the bottom manure tank and the hole diameter are less than 3mm. The cylinder width is 23cm, and the partition width is 22cm. In the presence of manufacturing errors, it can be tightly fitted to effectively prevent organisms with strong motility from leaving the toxic zone from both sides of the partition. The partition height is designed to be slightly lower than the cylinder height to prevent the height from being higher than the cylinder due to manufacturing errors, which may affect the installation of the cover plate later. The cover plate is an optional item, and the main purpose is to prevent fish from jumping out of the cylinder.

[0102] Since fish and shrimp organisms can jump out of the water surface, the water surface needs to be lower than the partition / cylinder height to prevent them from leaving the toxic zone or jumping out of the device from above. To meet the vertical swimming distance of aquatic organisms, the water surface height is designed to be 20cm, the toxic zone size is 22x23x20cm, and the volume is 10.12L.

[0103] The time-concentration function equation of the pollutant (to-be-tested chemical) in the three regions of the exposure system is as shown in the figure. Figure 3 As shown in the figure, the lowest effect concentration corresponding to all abnormal behaviors is obtained by the concentration function of the toxic zone (directly exposed to the water body of zebrafish) and the time when the abnormal behavior occurs.

[0104] In this embodiment, potassium dichromate (K2Cr2O7) is used as the to-be-tested chemical to determine its behavioral toxicity to zebrafish.

[0105] In this embodiment, the circulation flow rate is 1000 L / h, and a heating rod is provided in the buffer zone to maintain the temperature of the water in the cylinder stable, and the temperature is set to 25°C. At the same time, in order to enhance the anti-interference ability of the method, the experimental cylinder is loaded in a soundproof box, infrared rays are emitted through an infrared emitting backboard, and an infrared camera is used to capture video signals. Since the cylinder body is made of glass, the background in the video signal is grayish white (depending on the intensity of the infrared rays), and the zebrafish body does not transmit infrared rays, so it appears black in the video signal. The trajectory is described and quantitatively output by identifying the centroid change of the black pixels.

[0106] The water inflow rate is set to 1 L / h, i.e. 16.7 mL / min, and the zebrafish is placed in the contaminated area after which the video recording starts, and the water inflow of pure water for 1 h is carried out, and K2Cr2O7 is connected at the 60th minute. The initial concentration of K2Cr2O7 is set according to the 96h median lethal concentration (LC 50 ) value (133.5 mg / L) obtained from the literature for zebrafish. The LC 50 indicated in the literature is the concentration of Cr 6+ , which is converted to the concentration of K2Cr2O7 to configure the solution. The K2Cr2O7 solution is watered for 4 h, and after the experiment is finished, the contaminated zebrafish is placed in sterilized aerated pure water for 1 month of recovery before being tested again.

[0107] In this experiment, no obvious abnormal behavior was observed, which was due to the fact that the zebrafish was very active in the initial pure water state, which was related to the exploratory behavior of the zebrafish in a new environment. This behavior is affected by the individual's ability to adapt to the environment. Limited by the hardware limitations of the camera technology, the video image is distorted, with the image on the side close to the camera being larger than the image on the side away from the camera (the image gradually shrinks as the camera moves away radially), and the existing commercial video recognition software is based on the centroid change of the pixels or machine learning to automatically recognize the fish feature parts, and then realize the trajectory tracking. Especially for the reflection produced by the transparent cylinder wall, it may cause label abnormalities, centroid drift and other problems, which still need to be further improved. In this embodiment, the abnormal behavior and the corresponding minimum effect concentration are determined by overall comparison and visual observation, which is more suitable for determining the abnormal behavior of such special individual zebrafish.

[0108] The threshold values for distinguishing each swimming state (high-speed swimming, low-speed swimming, and inactive swimming) are 30 cm and 5 cm, respectively. The time for each swimming state is counted in this way. Each time is 1 minute long, and the behavior analysis is performed for a total of 5 hours. Video recording is achieved through an infrared camera. In this embodiment, the 2D ZebraTower software of the Viewpoint company is used for video analysis. The cylinder wall junction (adhesion and splicing) will appear close to the zebrafish black, which will cause the cylinder wall junction to be identified as "organisms" in the case of a full water area frame analysis area, resulting in the drift of the zebrafish-identified center of mass and the confusion of the trajectory. According to the use method and suggestion provided by the company, only the bottom plane is framed, which results in a monitoring dead angle in the part close to the cylinder wall of each vertical plane. In the normal monitoring state, the sum of the time of each swimming state per minute should be 60 s, and the Figure 8 The part less than 60 s is the monitoring dead angle of the zebrafish swimming near the cylinder wall, resulting in the loss of the trajectory. Previous studies have shown that zebrafish will exhibit exploration behaviors such as diving and swimming around the cylinder wall when entering a new environment. The results of the previous experiments show that different zebrafish individuals exhibit different exploration behavior responses when entering the poisoning device described in this embodiment. Some fish exhibit normal cruising in the cylinder after experiencing a short exploration behavior, and the recorded motion trajectory is soft and evenly distributed. At the same time, there are a small number of individuals that exhibit significant "escape" behavior after entering the poisoning device, that is, they continuously swim around the cylinder wall, the partition, and attempt to swim out of the cylinder. In this embodiment, the abnormal behavior intensity of this small number of individuals is interpreted, and the minimum effective concentration is calculated.

[0109] The first 60 minutes are pure water inflow. The purpose is to adapt the test zebrafish to the water quality and water pump noise of the new environment, and to record the normal behavior data of the zebrafish. As shown in Figure 8 The sum of the time of each swimming state (recordable motion time) in the first 60 minutes is more than 30 s, and only a few time points are less than 30 s. However, due to the large number of data points, it is not intuitive to compare the changes and thus obtain the negative effects of K2Cr2O7. Therefore, the overall comparison is adopted, and the data is integrated into the recordable time every 5 minutes, and then the data comparison is performed. The results are shown in Figure 9 As can be seen from Figure 9 During the pure water inflow stage, the recordable motion time in 45-50 and 50-55 minutes is the shortest, which is 33.9 s and 31.2 s, respectively. The average value of the recordable time starts to be lower than 30 s from 110-115 minutes after K2Cr2O7 starts to flow, and it is restored to 52.6 s from 180-185 minutes. Except that the average value reaches 30.9 s from 120-125 minutes, it is lower than 30 s to different degrees, and Figure 9It can be seen that from 65 to 70 minutes to 110 to 115 minutes, although there is a certain fluctuation, the total recordable movement time shows a downward trend, indicating that in this period, the K2Cr2O7 affected the adhesion movement behavior of the test zebrafish gradually increased with fluctuations.

[0110] The behavior of aquatic organisms affected by toxic substances in previous studies is divided into "adaptation stage, adjustment stage and toxicity stage". In this example, 65 to 115 minutes can be regarded as "adaptation stage", K2Cr2O7 into the water causes the test zebrafish to perceive the abnormal water quality, and trace K2Cr2O7 activates the behavior regulation of the test zebrafish, so that it gradually changes its behavior and adapts to the new water quality; 110 to 185 minutes can be regarded as "adjustment stage", by Figure 8 It can be seen that the concentration of K2Cr2O7 tends to be stable, and enters the Figure 3 slowly rising stage shown in the figure. In this stage, the test zebrafish is affected by external pressure, and the endocrine system adjusts to adapt to the new relative steady state environment. The "toxicity stage" mentioned in previous studies refers to the serious action inhibition of zebrafish, usually manifested as not moving or moving very slowly. In this example, it does not enter this stage. It is worth noting that the behavior data after 185 minutes does not have similar rules as described above, but the total recordable time shows a "first rising, then falling" trend, and is lower than 30s after 260 minutes, lasting about 30 minutes. This stage does not have the characteristics of a new steady state after dyeing.

[0111] Since the average of the recordable movement time every 5 minutes during 110 to 185 minutes is lower than 30s, the behavior data is significantly different from the blank control period, and it has experienced the "adaptation stage" of 65 to 115 minutes, it can be considered that the concentration of 110 minutes is the minimum effective concentration of K2Cr2O7, and the time-concentration linear function is brought in, at this time the concentration of K2Cr2O7 is 26.46% of the mother liquor, that is, 35.32mg / L. This method uses a total of only 5h, which is much lower than the test time of 96h LC 50 , the biological consumption is only 1, if the number of parallel controls is increased, it is also lower than 10, and the test organisms remain alive and active after the recovery period, achieving the purpose of this patent.

[0112] Example 4

[0113] As Figure 1As shown, the present application uses a self-developed pollutant exposure system, including a cylinder with an open top, three areas of buffer zone 1, exposure zone 2 and water outlet zone 3 are arranged in the cylinder in turn, water inlet partition 11 is arranged between buffer zone 1 and exposure zone 2, and water outlet is arranged between exposure zone 2 and water outlet zone 3, the three areas are separated by the partition, and holes are punched on each partition to allow water flow and pollutants to pass through, the hole diameter is designed to be 2mm, in order to improve the pollutant diffusion effect, the distance between the holes is set to 1.5 times the hole diameter, and all aquatic organisms with a size less than the size can use the device for experiment. The position of each partition is fixed by using a fixed buckle made of acrylic plate and a special suction cup for breeding (i.e. first fixed suction cup 8), and the buckle and suction cup should be placed on one side of the buffer zone 1 and the water outlet zone to prevent affecting the normal swimming and shooting of the organisms.

[0114] The buffer zone 1 is provided with a sewage inlet pipe 7, the other end of the sewage inlet pipe 7 is connected to a sewage tank after filtration and using a heating rod for constant temperature, the water outlet zone 3 is provided with a water outlet pipe 13, the other end of the water outlet pipe 13 is connected to a wastewater collection system. The sewage inlet pipe 7 and the water outlet pipe 13 are connected to a peristaltic pump to provide power.

[0115] The buffer zone 1, exposure zone 2 and water outlet zone 3 are provided with an internal circulation system.

[0116] The internal circulation system includes a submersible pump 10, a circulating water pipe 9 and an aeration assembly 4; the submersible pump 10 uses a submersible pump of the same kind of common freshwater fish breeding filtration system on the market, and the circulating flow is 500-1500L / h, and the flow size can be set within the range. The submersible pump 10 is placed in the middle of the cylinder wall of the water outlet zone 3, and is fixed by using an acrylic fixed buckle and a second fixed suction cup, the pump outlet is vertically upward, the water inlet section faces the cylinder wall, and maintains a distance of 3mm with the cylinder wall, and biochemical cotton is filled between the pump body and the cylinder body to absorb excrement.

[0117] The circulating water pipe is connected to the submersible pump outlet, and the other end is connected to the buffer 1, i.e. the circulating water pipe 9 outlet is arranged in the buffer zone 1. A hole is opened at 1cm above the outlet of the circulating water pipe 9, an aeration pipe 5 and an aeration adjusting valve 6 are inserted, and the aeration adjusting valve 6 is placed in the air, an inclined cutting tubular hard plastic pipe is used at the interface, and the shorter side faces the outlet of the circulating water pipe 9 to ensure that the water flow will not enter the aeration pipe 5.

[0118] As shown in Figure 2 As a further preferred, the water inlet partition 11 uses a full-hole partition, and the water outlet partition 12 uses a lower-hole partition.

[0119] The holes on each partition can meet different specifications of water distribution systems and pollutant conditions, and the design can also meet the direct exposure of different water depth organisms, such as the benthic mussel which can use the lower hole partition.

[0120] The resolution of the camera is 1288x964, and the frame rate is 30 frames per second. The camera can clearly capture the exposure system. The installation and fixation of the camera use existing technology, and the installation position is located above the exposure system at a suitable height to ensure clear shooting.

[0121] In this embodiment, the cylinder size is designed to be 40x23x25 cm, and the cylinder thickness is 5 mm; each partition is vertically inserted into the cylinder to divide the long side of the cylinder into an 8 cm buffer zone, a 22 cm poisoning zone, and a 9 cm water outlet zone; the sewage inlet pipe and the water outlet pipe are 12 cm away from the cylinder bottom; the sewage inlet pipe is 1 cm away from the water outlet end of the circulating water pipe, and the aeration hole is 1 cm away from the water outlet end of the circulating water pipe; the bottom of the submersible pump is 8 cm away from the cylinder bottom.

[0122] The partition size is 22x24 cm, the bottom manure tank is 2 mm high, the whole plate is perforated, the hole diameter is 2 mm, and the thickness is 5 mm.

[0123] The width of the experimental motile organisms is not less than 3 mm, so the bottom manure tank and the hole diameter are both less than 3 mm; the cylinder width is 23 cm, the partition width is 22 cm, and in the presence of manufacturing errors, it can be tightly fitted to effectively prevent motile organisms from leaving the poisoning zone from both sides of the partition; the partition height is designed to be slightly lower than the cylinder height to prevent the partition from being higher than the cylinder due to manufacturing errors, which may affect the installation of the cover plate later; the cover plate is an optional item, and the main purpose is to prevent fish from jumping out of the cylinder.

[0124] Since fish and shrimp organisms can jump out of the water surface, the water surface needs to be lower than the height of the partition / cylinder to prevent them from leaving the poisoning zone from above or jumping out of the device; to meet the vertical swimming distance of aquatic organisms, the water surface height is designed to be 20 cm, the poisoning zone size is 22x23x20 cm, and the volume is 10.12 L.

[0125] The time-concentration function equation of the pollutant (to-be-tested chemical) in the three regions of the exposure system is as shown in the figure. Figure 3 The lowest effect concentration corresponding to all abnormal behaviors is obtained by the concentration function of the poisoning zone (directly exposed to the water body of zebrafish) and the time when the abnormal behavior occurs.

[0126] Lead acetate is a chemical with extremely high aquatic organism toxicity. According to literature, the lead ion Pb 2+ The 24h LC 50 of zebrafish is 171 mg / L. Within the allowable range of the method, the initial concentration is appropriately increased to 200 mg / L (1.17 LC 50 ), and this concentration is used as the standard to configure the lead acetate stock solution (313.98 mg / L).

[0127] Figure 10The effects of lead acetate on the number of times, time and distance of zebrafish movement were shown. It was found that Pb 2+ The number of times, time and distance of movement in each stage were affected to different degrees. For the number of times, the pure water infusion in the first 60 minutes did not show a significant regularity, but the overall average was high, especially the number of inactive swimming times was relatively high (77.3 ± 15.6 times) except for the 20th, 21st and 22nd minutes. After lead acetate infusion, the total number of movements decreased significantly at the 93rd minute, the number of inactive swimming times and low-speed swimming times were 5 times, and there was no high-speed swimming time; at the same time, the swimming time graph showed that within 60s, the time in the inactive swimming state was 58.1s, accounting for 96.8% of the analysis time, while before the start of lead acetate infusion, the inactive swimming state accounted for (40.45 ± 9.47)% of the analysis time; the same phenomenon appeared in the swimming distance, the swimming distance at the 93rd minute was 68.2cm, while the average swimming distance from the 60th minute to the 93rd minute was 390.4 ± 65.1cm. At the same time, higher activity appeared at the 91st and 92nd minutes, the swimming distance increased and the high-speed swimming state appeared, all indicating that the zebrafish showed abnormal behavior at this time point. However, due to the short duration (including 2 minutes of recovery to normal state, a total of 5 minutes) and the absence of abnormal data with significant differences from the baseline except for the 93rd minute, it was not possible to directly determine that this concentration was the lowest effect concentration causing abnormal behavior. It is worth noting that at the 100th minute, the recorded movement time was obviously missing (<60s), which indicated that the zebrafish showed wall swimming. Since the zebrafish movement was generally within the analysis area before, the analysis time did not show a lack of time, so the time lack at this time to some extent reflected the behavior of the zebrafish escaping from the area, which lasted until the 152nd minute.

[0128] From 100 to 152 minutes, in addition to the escape behavior, the total movement distance showed a slow downward trend Figure 10The number of movements did not appear to have significant abnormal fluctuations. The decrease in movement distance and escape behavior indicated that the behavior of the test zebrafish was affected by lead acetate, and this period can be considered as an "adaptation stage". From 152 minutes, the number of movements, movement time and movement distance all changed dramatically. First, the number of movements decreased sharply. The total number of movements in the pure water input stage, 60-100 minutes and 100-152 minutes was 158.3±38.1, 177.3±50.8 and 180.3±31.1, respectively, while the total number of movements in 152-187 minutes was 8.2±7.9, which decreased by more than 94.8%. The proportion of inactivity time of 60s increased from (36.5±12.5)%, (43.1±14.4)% and (34.9±13.9)% to (98.1±2.3)%, which increased by more than 127.6%. The total swimming distance decreased from 427.4±118.5cm, 374.9±87.5cm and 367.4±56.4cm to 93.5±19.5cm, which decreased by more than 74.6%. The above statistical data show that the test zebrafish entered an "adjustment stage" from 152 minutes, and the endocrine system and nervous system jointly caused low activity. Although the movement intensity of the zebrafish was weak, it cannot be considered that the zebrafish entered a "toxic stage". The reason is that the behavior intensity of the test zebrafish recovered to some extent from 204 minutes, and the zebrafish jumped out of the water into the water outlet area with a relatively low concentration in order to escape from the contaminated area by 213 minutes, which shows that the test zebrafish did not enter a toxic paralysis state, and the behavior of the test zebrafish recovered to a relatively normal state after being placed in pure water for a short period of time (about 1 day), and no death occurred.

[0129] In summary, it can be considered that the zebrafish produced significant behavioral toxicity effects at 152 minutes, which is reflected in the number of movements, movement time and movement distance, and the behavior intensity changed obviously and consistently. According to the time-concentration linear function, the lead acetate concentration at this time is 28.44% of the mother liquor concentration, which is 89.28mg / L (lead acetate concentration), and Pb 2+ The concentration is 56.87mg / L.

[0130] The whole experiment took 4h, which is much lower than the LC 50 The test time is 24h, and no adverse effects such as death occur to the test organism, which proves that the present application is a non-destructive toxicity test method for obtaining toxicity data.

[0131] Of course, the above description is not a limitation on the present application, and the present application is not limited to the above examples. Changes, modifications, additions or substitutions made by those skilled in the art within the scope of the present application should also be within the scope of the present application.

Claims

1. A chemical toxicity testing method based on a self-developed pollutant exposure system, using a self-developed pollutant exposure system, the system comprising a cylinder with an open top, a buffer zone, a dyeing zone and a water outlet zone being arranged in the cylinder in sequence, the buffer zone and the dyeing zone, the dyeing zone and the water outlet zone being separated by a perforated partition, and using zebrafish as a model organism in the exposure system, characterized in that, The distance between the partitions of the self-developed pollutant exposure system is not less than 20 cm, the partitions are punched, and the buffer zone, the exposure zone and the water outlet zone are provided with an internal circulation system; the buffer zone is provided with an inlet pipe, the water outlet zone is provided with an outlet pipe, the inlet pipe and the outlet pipe are connected to a peristaltic pump to provide power, the inlet and outlet water flow is set to 0.1-10 L / h, the internal circulation system comprises a submersible pump, a circulating water pipe and an aeration assembly, the aeration assembly comprises an aeration pipe and an aeration regulating valve; the circulating flow of the submersible pump is 500-1500 L / h, the submersible pump is arranged in the middle of the cylinder wall of the water outlet zone and is kept at a distance of 1-3 mm from the cylinder wall, and biochemical cotton is filled between the pump body and the cylinder body to absorb excrement; the circulating water pipe is connected to the outlet of the submersible pump, and the other end is connected to the buffer zone; the outlet end of the circulating water pipe is vertically downward, the water outlet is located in the middle of the buffer zone, and a hole is opened 0.5-1 cm above the outlet of the circulating water pipe, the aeration pipe and the aeration regulating valve are inserted, and the aeration regulating valve is arranged in the air, the interface is a tubular hard plastic pipe with beveled cutting, and the shorter side faces the outlet of the circulating water pipe to ensure that the water flow does not enter the aeration pipe; The test method specifically comprises the following steps: (1) Prepare the mother liquor and look up the LC of the chemical to be tested. 50 Value, based on the LC value of the chemical to be tested. 50 The value is adjusted to configure the initial concentration of the mother liquor. If the chemical does not show obvious toxicity to zebrafish behavior, the concentration can be further increased based on the preliminary experimental results. (2) A time-concentration linear function of the test chemical in the exposure zone is established, which is: When t ≤ 40 minutes, C = 0.60871 t + 0.36129; When 40 minutes < t ≤ 600 minutes C = 0.04706 t + 24.10669; wherein, C is the concentration of the chemical in the mother liquor in percent, %, t is the length of time the mother liquor is fed water; (3) A significant behavior test is carried out in the exposure system to calculate the concentration of the test chemical that causes significant changes in zebrafish behavior, i.e. the lowest effect concentration; the method of the significant behavior test is as follows: pure water is pre-loaded in the cylinder body, a heating rod is used to preheat to 27°C, an ultraviolet lamp is sterilized and aerated for more than 1 h; adult male zebrafish are placed in the exposure zone for water quality adaptation, and the adaptation stage lasts at least 1 h; after the adaptation period, a blank control experiment lasting 1 h is carried out, i.e. the inlet and outlet peristaltic pumps are started, the inlet peristaltic pump is connected to pure water, and the behavior of zebrafish during the blank control experiment is observed in detail as a standard behavior control, if it is necessary to observe the toxicity target, a corresponding stimulation experiment of the toxicity target should be additionally carried out; After the blank control experiment, the inlet pipe is directly connected to the test chemical mother liquor for the exposure experiment, and the changes in the routine behavior and abnormal behavior of zebrafish during the experiment are observed, and the abnormal behavior is recorded by taking pictures, and the time of the abnormal behavior is recorded; If there is no significant abnormal behavior within 9 h, the concentration of the test chemical mother liquor is increased for the second experiment, and if significant abnormal behavior occurs within 9 h, the time of the abnormal behavior is recorded, and the lowest effect concentration is calculated according to the time-concentration linear function of the test chemical; The standard behavior or routine behavior includes sinking, floating, cruising and wall collision; the abnormal behavior includes sudden high-speed movement, long-time abnormal high-speed or stationary state and swimming posture change; (4) The behavior of zebrafish in the exposure system is analyzed by coupling the camera tracking technology to obtain the lowest effect concentration of the test chemical; Steps (3) and (4) are selective analysis steps; The starting chemical mother liquor concentration is adjusted according to the LC value of the chemical to be measured, the adjustment range is 0.5 LC ~ 2 LC 50 . 50 50 .​ 2. The method according to claim 1, wherein the method is based on a self-developed pollutant exposure system. The partitions include upper punched partitions, lower punched partitions and full-punched partitions, and the partitions are made of transparent acrylic plates with a 1-3 mm high excrement groove at the bottom.

3. The method according to claim 2, wherein the method is based on a self-developed pollutant exposure system. The aperture of the holes on the partition plate is 1-3 mm, and the distance between the holes is 1-2 times of the aperture.

4. The method according to claim 1, wherein the method is characterized by, The time-concentration linear function of the chemical to be tested is obtained by fitting the scatter plot of the pollutant concentration measured at different time points in each area and time, and the fitting method is apparent fitting.

5. The method according to claim 1, wherein the method is characterized by, For the chemical to be tested with low toxicity, the behavior change is analyzed by coupling the video tracking technology, and the non-lethal abnormal behavior concentration, i.e., the lowest effect concentration, is obtained.

6. The method according to claim 5, wherein the method is based on a self-developed pollutant exposure system. The behavior analysis method of the coupling video tracking technology is as follows: A camera is installed at a suitable height above the center of the cylinder to take pictures of the inside of the cylinder; First, a blank control video of 1-2 hours is taken as the baseline of the normal state, and then the chemical mother liquor is added to the water, and the water is added until the zebrafish behavior is abnormal or the chemical mother liquor has been added to the water for 10 hours; The zebrafish is taken out and put into sterilized pure water for recovery, and the video is analyzed to quantitatively obtain the related data of the swimming trajectory, swimming speed, distance and time of the zebrafish, and by comparing with the baseline, the abnormal motion state for a long time is selected as the abnormal behavior indicator, and according to the starting time of the abnormal motion state, the lowest effect concentration is calculated.

7. The method according to claim 6, wherein the method is a chemical toxicity test method based on a self-developed pollutant exposure system. The resolution of the camera is at least 1288 × 964, and the frame rate is 30 frames per second, so that the camera can clearly take pictures of the exposure system.

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