CN112415182A - Chemical toxicity detection method based on zebra fish behaviors - Google Patents
Chemical toxicity detection method based on zebra fish behaviors Download PDFInfo
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Abstract
The invention provides a chemical toxicity detection method based on zebra fish behaviors, which comprises the following steps: and (3) performing embryonic development detection and larval fish behavior test on the zebra fish through exposure in a chemical solution, thereby detecting the toxicity of the chemical. Compared with molecular experiments, the method has simple technology, easy operation and no need of special training; meanwhile, the fish has optical transparency, is convenient to observe, has large embryo oviposition amount and low cost, has no special requirements on fields, and reduces the application threshold of the invention. The method for rapidly screening the toxicity of chemicals by 'embryo development detection + fry behavior analysis' has wide application prospect.
Description
Technical Field
The invention relates to a chemical toxicity detection method based on zebra fish behaviors.
Background
As a large chemical trade country in China, more than 3000 varieties of chemicals are imported and exported every year, and the amount of the chemicals reaches more than 400 hundred million dollars. Today, it is estimated that exogenous chemicals are more than 1000 thousands, and more than 1000 new chemicals are produced each year. A large amount of chemical substances are released into the environment in various ways, and risks are inevitably caused to the ecological environment and the biological health, so that the research and development of the toxicity test technology of the chemicals in China are particularly important. The apparent toxicity test based on the animal observation level has universality and repeatability, is a widely accepted traditional toxicity test method, is a final embodiment of molecular response on an individual/population level, and is also an important component in a chemical risk control and evaluation system in the 21 st century.
The aquatic organism toxicity test is widely applied to evaluating the water ecological environment safety of chemicals, and the fish embryos/fish larvae are widely applied to toxicity evaluation of endocrine disruptors, pesticides, medicines, personal care products and emerging compounds due to the fact that the fish embryos/fish larvae have optical transparency and high yield and are easy to obtain, and are important data sources and technical supports in aquatic ecological risk evaluation and management. The current toxicity testing technology of zebra fish mainly comprises the following types:
(1) acute toxicity test: setting different concentration gradients of the target compound, carrying out 96-hour acute exposure on zebra fish embryos, counting death number, teratotype number, survival number and the like, and calculating half Lethal Concentration (LC)50) Half maximal Effect Concentration (EC)50) And the like as a basis for judging toxicity.
(2) Developmental toxicity testing: short-term (usually 4-6 days) exposure of zebrafish embryos is performed at sub-lethal concentrations, and embryo development conditions such as hatchability, hatching delay time, heart rate and the like are observed and counted by means of a microscope and other instruments. And (4) performing significant difference analysis on each index and a control group, and judging the toxicity of the compound and the risk to aquatic organisms according to the abnormal condition of embryo development.
(3) Toxicity testing by molecular biology: on the basis of the technology described hereinbefore, the toxicity of the chemicals is evaluated by supplementing the molecular biology technology. This approach allows toxicity assessment to be carried out from individual levels down to gene and protein levels. The method comprises the steps of calculating a malformation index of a chemical by using chemical safety evaluation software, obtaining initial effect concentration of a biological pathway by using a high-flux dose-effect transcription level response test, screening a harmful outcome pathway related to zebra fish developmental malformation from a database, constructing an activation scoring algorithm by combining a result of a targeted high-flux in-vitro test corresponding to a molecular initiation event in the harmful outcome pathway and a test result collected by a document, performing activation scoring on the molecular initiation event, and evaluating the influence of the chemical on the harmful outcome pathway. The whole genome is simplified by screening key genes to form a key gene set representing whole transcriptome information, and the prediction of the embryonic development toxicity of chemicals based on the dose-effect simplified transcriptome can be realized aiming at the transcription expression level of a small amount of key genes. Or by verifying the levels of several mature molecular markers (such as SOD activity, POD activity and the like), judging which biological pathways are toxic.
In addition, behavioral testing of adult zebrafish was also used for compound toxicity assessment. Common techniques include new water tank exploration experiments, individual social behaviors, and crowd experiments.
New water tank exploration experiment: the method comprises the steps of artificially dividing a fish tank into an upper layer, a middle layer and a lower layer or an upper layer and a lower layer, moving the zebra fish exposed by the compound into a water tank, continuously collecting videos about 5 minutes, observing abnormal swimming behaviors of the zebra fish (such as overactivity, spiral or circular swimming, quick convulsion, spastic body contraction, body unbalance, paralysis and the like), and counting the residence time of the zebra fish in the lower layer.
The social behavior of the individual: the water tank is divided into the left half and the right half equally, 1 zebra fish to be detected is placed on one side, and 5 zebra fish treated in the same way are placed on the other side. The middle of the test tube is blocked by a perforated transparent partition plate to ensure that the zebra fish to be tested can see and smell the fish school information (the zebra fish cannot pass through the aperture). And (4) continuously observing for 20 minutes, counting the retention time of the zebra fish to be detected close to the partition plate and the retention time of the zebra fish to be detected close to the partition plate, performing significance difference analysis on the zebra fish and a control group, and judging whether the individual social behaviors of the zebra fish are interfered by the compounds.
Clustering experiments: 5 zebra fish to be tested are placed in a 6L open water tank. After 5 minutes of acclimation, the above was photographed for 10 minutes. Behavior sampling is carried out from video recording, a water tank is divided into n subregions with equal areas, data analysis is carried out once every 30 seconds, the maximum number of zebra fish in a certain subregion is divided by the total number of the subregions occupied by 5 zebra fish, and then the clustering score of each time point can be obtained, and the higher the score is, the more obvious the zebra fish clustering behavior is.
In view of the fact that the toxicity detection speed of the existing chemicals is far from the synthesis speed of new chemicals, the method is urgent, and a living body detection method of 'rapidly responding to the chemicals at an individual level' is established to preliminarily classify and control the chemicals.
Disclosure of Invention
However, the existing chemical toxicity detection methods have a plurality of problems.
The acute toxicity test has the defects of poor environmental relevance, single index, low efficiency, poor safety and the like. The acute toxicity endpoint index is primarily half lethal concentration, LC50However, the design and synthesis of the existing novel chemical substances are converted to low toxicity, and most of the compounds in the environment do not have high acute toxicity, so that toxicity judgment by using endpoints such as lethal teratogenesis generally requires higher exposure concentration, but the actual concentration of the chemical in the environment is far lower than the test concentration, and the biological effect of the compound cannot be effectively represented. In addition, higher exposure concentrations also pose additional risks to the health of the experimenter. "death" is an "end result" of an adverse effect of a foreign compound on an individual level of an organism, as compared to sub-lethal effects such as malformations, dysplasias, behavioral abnormalities, etc., which, although easily observed and convincing, are too single in index and low in sensitivity. Thus leading to an acute toxicity test that is too "conservative" to meet the need for rapid screening of chemical toxicity. With the continuous development of the technology, toxicity screening based on the sub-lethal effect with higher sensitivity has been widely used by researchers.
Although the developmental toxicity test is simple to operate and visual in observation, observation indexes are that before 96h, zebra fish fries from 5 th to 7 th (120h to 168h) can freely move depending on self yolk sac nutrition, and are optimal window periods for detecting the neurodevelopmental toxicity, and after more than 7 days, the zebra fish fries need to be fed with open feed (such as paramecium to) to continue to maintain normal life activities of organisms, and unstable factors for performing the behavior test are increased. Therefore, if the developmental toxicity test and the fry behavior test can be combined, each key stage of the zebra fish from the embryo to the fry development is fully utilized, the advantages of the zebra fish can be exerted to the maximum, and more data support is provided for the chemical toxicity evaluation.
At present, the fish behavior test is less in application and is mostly zebra fish adult fish. Compared with the larval fish, the adult fish has low sensitivity and large water consumption, needs personnel for feeding and has certain requirements on the field. In addition, the existing indexes of qualitative description of zebra fish swimming behavior are that the quantitative data are simple swimming distance, time and the like, and the flux of adult fish testing is low, so that the obtained result has strong subjectivity, great contingency, poor repeatability and lack of persuasion. The brain tissues of fishes, particularly vertebrates, are developed, and if behavioral data of the fishes are deeply mined, abnormal behavioral responses after the fishes are exposed to chemicals can be found, and the interference of the chemicals on the nerve functions can be effectively represented. The incorporation of the behavioral toxicity index can greatly improve the accuracy of the zebra fish embryo/fry testing system. At present, the biggest problem of behavioral testing of zebra fish fries is that no unified standard exists, particularly, the testing time points are selected in a disordered manner and mainly comprise three time points of 120h, 144h and 168h, and if the best testing time is not determined, not only can the toxicity of chemicals be covered, but also the advantages of a zebra fish fry testing model can be weakened.
The invention aims to provide a method for rapidly detecting the toxicity of chemicals, which exposes the chemicals to organisms and living environments thereof practically, expresses the action of the chemicals on the organisms to the maximum extent, and has stronger practicability and practical significance compared with the prediction toxicology based on calculation and models; and the final toxicity data can also be used as a data source for predicting toxicology.
The invention provides a chemical toxicity detection method based on zebra fish behaviors, which is characterized by comprising the following steps: the toxicity of the chemicals is detected by performing embryo development detection and fry behavior test on the zebra fish in a chemical solution.
Wherein the embryo development detection comprises: hatchability, heart rate, body length, full length.
The fry behavior test comprises the following steps: the zebra fish fry is tested on three items of movement, body and direction. The mobile test comprises the following steps: swimming distance, swimming time, speed, acceleration. The physical test includes: jerky time, jerky frequency, active time, active frequency, quiescent time, quiescent frequency, clockwise rotation, counterclockwise rotation. The direction test comprises the following steps: relative angular velocity, absolute angular velocity, relative rotational angle, absolute rotational angle.
The method comprises the following steps:
(1) determining the optimal time point of the zebra fish fry behavior test:
a, continuously culturing zebra fish embryos to obtain larval fish at different development stages;
b, carrying out the incubation of the pore plate before the experiment so as to reduce the experimental error;
c, performing behavior test on the zebra fish fry;
d, determining the optimal time point of the zebra fish fry behavior test;
(2) chemical toxicity comparative exposure experiments:
selecting zebra fish embryos, and exposing the zebra fish embryos to a culture solution containing chemicals to be detected to serve as an experimental group; selecting zebra fish embryo to be exposed in culture solution without chemicals as a control group;
b, culturing the zebra fish embryo at the temperature of 27-29 ℃, and culturing according to the following steps: dark-14: exposure was performed for a 10 hour light cycle;
c, semi-statically exposing for 144-168 hours, updating 80-90% of liquid medicine every 24 hours, and picking out dead embryos in time;
d, performing embryo development detection in the exposure process, namely determining the toxicity index of the embryo at the development stage for 0-96 h;
e. continuously exposing the fry for 144-168 hours, and performing fry behavior test;
(3) and performing data analysis on results of the embryo development detection and the fry behavior test, so as to evaluate the toxicity of the chemicals according to the data analysis results.
Further preferably, when the optimal time point of the zebra fish fry behavior test is determined, continuously culturing zebra fish embryos to obtain 120h, 144h and 168h fry in different development stages;
in a chemical toxicity comparison exposure experiment, semi-static exposure is adopted for 168 hours, 80-90% of liquid medicine is updated every 24 hours, and dead embryos are picked out in time; and the fry are exposed for 168 hours continuously, and fry behavior test is carried out.
The invention detects the toxicity of chemicals based on the sub-lethal level on the whole, the concentration setting interval is smaller than the acute toxicity test, and the health risk to experimenters is low; the method optimizes the zebra fish embryo development toxicity test, the previous development index is single, even the situation that the same development index is counted for multiple times at different time exists, according to the zebra fish embryo development characteristics, several representative and easily observed developmental end points are selected, a zebra fish embryo development index observation axis taking time as a main line is established, namely, the observation index exists every day, and each key period of embryo development is not lacked, so that the development detection is efficient and ordered. Finally, a differential analysis was performed in conjunction with all the data to indicate whether the chemical was developmentally toxic.
In addition, larval fish behavior analysis is an important means of the invention as a rapid screening for chemical toxicity. After the zebra fish fry is exposed to the water containing the drugs, very complex behavioral reaction can be generated, and the zebra fish fry can be used as a sensitive index of toxic exposure. As previously mentioned, current studies are not uniform in the choice of fry behavior test time, and the present invention first makes sensitivity analysis on background fries without chemical exposure. Three time points, for example, fry of 120h, 144h and 168h are subjected to a 50 min-light-dark alternation experiment together, a special instrument is used for collecting data, and the optimal detection time of the zebra fish fry is determined by performing difference analysis on 16 small behavioral indexes of three major items (movement, direction and body). And further, chemical exposure can be carried out on the zebra fish fry, corresponding indexes are analyzed, and interference of the chemicals on the nerve function is represented. The invention realizes high-throughput screening of behavior tests (the fry is placed in a 96-hole plate for data acquisition) by means of special instruments, expands the original 5-6 behavior indexes to 16 behavior indexes, applies the behavior indexes to final toxicity characterization, realizes deep excavation of behavior data, and greatly improves the accuracy of a zebra fish embryo/fry test system.
Finally, although the invention does not relate to molecular biology technology and corresponding indexes, the invention aims to realize the rapid detection and the preliminary screening of the toxicity of the chemicals so as to provide theory and data support for the management of the chemicals, so that the excessive energy does not need to be put on the discussion of a toxicity mechanism; meanwhile, the fish has optical transparency, is convenient to observe, has large embryo oviposition amount and low cost, has no special requirements on fields, and reduces the application threshold of the invention. The method for rapidly screening the toxicity of chemicals by 'embryo development detection + fry behavior analysis' has wide application prospect.
The method of the invention gives full play to the characteristics of fast development and high sensitivity of zebra fish embryos, and not only realizes fast detection, but also can obtain more data in the actual detection of chemical toxicity; meanwhile, biological behavior data acquisition technology is rapidly developed in recent years, monitorable indexes are increasingly diversified, the data volume is far higher than developmental toxicity data, and the data volume is updated to a toxicity detection system in time to provide important evidence support for evaluating the neurotoxicity of the compound.
Drawings
FIG. 1a is a flow chart of a method of detecting the toxicity of a chemical according to the present invention; FIG. 1b shows the specific test items of the larval fish behavior test;
FIG. 2 is a graph showing the "movement" behavior index of zebra fish fries in the "determination of the optimal behavior detection time point" experiment;
FIG. 3 is a "direction" behavior index of zebra fish fries in an experiment of "determination of optimal behavior detection time point";
fig. 4 is a "physical" behavior index of zebra fish fries in an experiment of "determination of optimal behavior detection time point";
FIG. 5 is a zebrafish embryo developmental toxicity index;
FIG. 6 is a "movement" behavior index of the zebra fish fry in the embodiment;
FIG. 7 is a behavior index of the zebra fish fry 'direction' in the example;
FIG. 8 is the "body" behavior index of the zebra fish fry in the example.
Detailed Description
The method for detecting the toxicity of the chemicals is characterized by comprising the following steps: and (3) detecting the toxicity of the chemicals by performing embryo development detection and larva fish behavior test on the zebra fish in the chemical liquid.
Wherein the embryo development detection comprises: hatchability, heart rate, body length, full length.
As shown in fig. 1b, the larval fish behavior test comprises: the zebra fish fry is tested on three items of movement, body and direction. The mobile test comprises the following steps: swimming distance, swimming time, speed, acceleration. The physical test includes: jerky time, jerky frequency, active time, active frequency, quiescent time, quiescent frequency, clockwise rotation, counterclockwise rotation. The direction test comprises the following steps: relative angular velocity, absolute angular velocity, relative rotational angle, absolute rotational angle.
Specifically, the flow chart of the method for detecting the toxicity of chemicals of the present invention is shown in fig. 1 a.
Preferably the method comprises the steps of:
(1) determining the optimal time point of the zebra fish fry behavior test:
a, continuously culturing zebra fish embryos to obtain larval fish at different development stages;
b, carrying out the incubation of the pore plate before the experiment so as to reduce the experimental error;
c, performing behavior test on the zebra fish fry;
d, determining the optimal time point of the zebra fish fry behavior test;
(2) chemical toxicity comparative exposure experiments:
selecting zebra fish embryos, and exposing the zebra fish embryos to a culture solution containing chemicals to be detected to serve as an experimental group; selecting zebra fish embryo to be exposed in culture solution without chemicals as a control group;
b, culturing the zebra fish embryo at the temperature of 27-29 ℃, and culturing according to the following steps: dark-14: exposure was performed for a 10 hour light cycle;
c, semi-statically exposing for 144-168 hours, updating 80-90% of liquid medicine every 24 hours, and picking out dead embryos in time;
d, performing embryo development detection in the exposure process, namely determining the toxicity index of the embryo at the development stage for 0-96 h;
e. continuously exposing the fry for 144-168 hours, and performing fry behavior test;
(3) and performing data analysis on results of the embryo development detection and the fry behavior test, so as to evaluate the toxicity of the chemicals according to the data analysis results.
Wherein, further preferably, when the optimal time point of the zebra fish fry behavior test is determined, the zebra fish embryo is continuously cultured to obtain 120h, 144h and 168h fry in different development stages;
in a chemical toxicity comparison exposure experiment, semi-static exposure is adopted for 168 hours, 80-90% of liquid medicine is updated every 24 hours, and dead embryos are picked out in time; and the fry are exposed for 168 hours continuously, and fry behavior test is carried out.
The method of the invention specifically operates as follows:
1. establishment and optimization of behavior analysis method
1.1 embryo culture
In the experiment, AB-series zebra fishes are used as model organisms and are bred in a recirculating aquaculture system, male and female fishes are separately bred, and the light is 14 h/10 h every day: dark period, cultivation temperature (28 +/-1) DEG C and pH value of 7.0-7.6. In the experiment, high-quality healthy sexual maturity zebra fish is selected, the sexual maturity zebra fish is placed into a mating jar according to the proportion of 1:2 of male and female zebra fish, the light is turned on in the morning on day 2, the zebra fish starts to lay eggs after being stimulated by light, embryos are collected in zebra fish embryo culture solution (ERM, 1L deionized water contains 8g of NaCl,0.4g of KCl and 0.035g of Na2HPO4,0.6g KH2PO4,0.14g CaCl2,0.12g MgSO4,0.35g NaHCO3pH 7.2) and incubated in a thermostated incubator at 28 ℃. Embryo incubation 6-8h later (shield stage), atHealthy developing embryos were selected under microscope.
1.2 well plate incubation
The well plate is incubated prior to the behavioral test because the pores in the inner wall of the well plate may adsorb ions in solution, thereby affecting its actual concentration. First, the well plate was rinsed 2 times with ERM, then 300. mu. LERM was added to each well, the well plate was covered and sealed with wrap, and incubated in a constant temperature light incubator at 28 ℃ for 24 h. Before the experiment, the orifice plate was washed with deionized water 2 times to remove excess liquid, and then placed in a ventilated place to air dry.
1.3 fry behavior testing
The detection process of the zebra fish fry behavior adopts a DanioVision observation system and EthoVision motion trail analysis software matched with the DanioVision observation system. A set number of zebra fish fries (biological replicates per group set according to treatment group number) were selected from each treatment group and transferred to 96-well plates, each well containing 200 μ L of the corresponding exposure solution. And (3) placing the 96-hole plate in a DanioVision observation box, setting the temperature at 28 ℃, performing 50-min light-dark alternate stimulation (10min dark-10 min bright-10 min dark) after adapting for 20min, recording the motion track of the fry during observation, and analyzing each motion index of the fry.
According to the behavior data acquisition instrument, the fry behaviors are divided into three categories of 'movement', 'direction' and 'body'. Wherein, the 'moving' class comprises 4 indexes of swimming distance, swimming time, speed and acceleration; the category of 'direction' comprises 4 indexes of relative angular speed, absolute angular speed, relative rotation angle and absolute rotation angle; the 'body' class index comprises 8 indexes including the time of the manic violence, the frequency of the manic violence, the active time, the active frequency, the rest time, the rest frequency, the number of clockwise rotations and the number of anticlockwise rotations.
1.4 determination of optimal time points for behavior detection
According to a large amount of observation and the characteristics of zebra fish embryo development, the larval fish before 120h has poor movement capability although being hatched, the yolk sac of the larval fish after 168h cannot meet the metabolism of the body, and the larval fish dies if not eaten, and the actual concentration of chemicals is possibly influenced by the bait feeding, thereby interfering the experimental result. Therefore, larval fish behavior detection is focused on days 5,6 and 7, namely 120h, 144h and 168h, and in order to determine the time points of the optimal test, the following experimental procedures are adopted:
randomly selecting 3 sexually mature female zebra fishes and 6 healthy male zebra fishes, respectively placing the zebra fishes and the healthy male zebra fishes in 3 spawning tanks according to the male-female ratio of 1:2 for pairing, continuously spawning for 3 days, respectively setting three time points as groups A (5d), B (6d) and C (7d), carrying out behavior detection on the fry at the three time points together, and selecting well-developed embryos for later use. The embryos are placed in 25mL beakers for culture, 10mL of ERM +10 embryos are placed in each small beaker, and 80% -90% of ERM is updated every 24h, so that sufficient nutrients are provided for the development of the embryos. And meanwhile, dead and abnormal embryos are picked out in time. When the embryos in group C are exposed to day 7 (168h), B, A exposure time periods of the two groups are 6(144h) days and 5 (120h), respectively, 36 larvae are randomly selected from each group to be placed in a 96-well plate (the well plate is incubated for 24h in advance, the specific method is shown as 4.1.2), the motion tracks of the larvae in each group are recorded according to the method of 4.1.3, and the behavior data are exported for significance analysis, so that the most sensitive time point is determined.
1.5 analysis of results
The results are shown in fig. 2-4, fig. 2 is a zebra fish fry "movement" behavior index, fig. 3 is a zebra fish fry "direction" behavior index, and fig. 4 is a zebra fish fry "body" behavior index.
As can be seen from the results in the figure, the fry on the 7 th day is more active than the other two groups, and compared with the 5 th day, 12 indexes in 16 indexes have a significant difference, the difference rate is 75%, and the 7 th day of each index with the significant difference is significantly higher than the 5 th day except the rest time; compared with the 6 th day, 5 of the 16 indexes have obvious difference, the difference rate is 31.25%, although 2 (absolute rotation angle and absolute angular velocity) of the 5 indexes are obviously higher than that of the 7 th day on the 6 th day, the differences of the indexes in the three time periods are comprehensively considered, the fry on the 7 th day is obviously more flexible than those of the fry in the other two time periods and is more sensitive to the indexes, and therefore the 7 th day is determined as the optimal detection time.
2. Application of behavior test in chemical toxicity evaluation
2.1 drug exposure:
200 eggs were selected, and 1 control group and 3 treatment groups (0.01, 0.1, 1 μ M) were provided, each of which was provided with 5 replicates (n-5), each of which was 10 embryos. Zebra fish embryos were cultured in 25mL small beakers at 28 + -1 deg.C and exposed for a 14:10 hour (light: dark) light cycle. Semi-static exposure is adopted for 168h, 80% -90% of liquid medicine is updated every 24h, and dead embryos are picked out in time. And in the exposure process, determining the toxicity index of the embryo at the development stage (0-96h), and counting the hatchability rate 48h, heart rate 72h, body length 96h and total length 96h after exposure. The larval fish were exposed to 168h for behavioral analysis.
2.2 well plate incubation:
the well plates were incubated prior to the behavioral tests because the pores on the inner wall of the well plates adsorbed chemicals, which affected the actual concentration of chemicals. The specific method is shown in 1.2.
2.3 behavioral testing:
the detection process of the zebra fish fry behavior adopts a DanioVision observation system and EthoVision motion trail analysis software matched with the DanioVision observation system. 24 zebra fish fries from each exposure group were transferred to 96-well plates, each containing 200 μ L of the corresponding concentration of exposure solution. The behavior test method and the analysis index were performed according to the method of 4.1.3.
In the embryo exposure process, cell culture with 6 holes, 12 holes or 24 holes can be selected as the exposure container according to actual conditions. When the above well plate is selected as the exposure container, it should be incubated in advance. The specific procedure is similar to the 96-well plate incubation method.
2.4 data analysis:
data analysis was performed in SPSS 22.0. And (4) adopting Kolmogorov-Smirnov to test the normality of the data, and converting the data which does not conform to the normal distribution to conform to the normal distribution. Data homogeneity was checked using Leven's and One-way analysis of variance (One-way ANOVA) was performed using Dunnett's on control and experimental groups, and the experimental data are given as Mean. + -. standard error (Mean. + -. SEM). The histogram is plotted by GraphPad Prism 8.0, with P <0.05 considered a significant difference (, P <0.01 considered a very significant difference (, and P <0.001 considered a very significant difference (, respectively).
Examples
The present invention will be specifically described below by taking 2, 4-di-tert-butylphenol (2,4-DTBP) as an example.
2,4-DTBP as a synthetic phenol antioxidant is widely applied to food packaging, rubber and plastic pipes, but the toxicological effect is not clear. The concentration of 2,4-DTBP was set at 0.01, 0.1 and 1.0 μ M, which is a sublethal level of 2,4-DTBP, while a control group was set, each group being set with 5 biological replicates (n ═ 5) each containing 10 zebrafish embryos. As mentioned above, the development indexes of each type are detected at corresponding time, and the result is shown in FIG. 5, and FIG. 5 is the toxicity index of zebra fish embryo development.
From the results it can be seen that: only 2 of the 4 developmental indicators (FIG. 5-A, FIG. 5-B) showed significant differences compared to the control group, and showed a down-regulation trend. After 48h exposure, there was a significant difference in embryo hatchability at 1.0 μ M, which was only 36.92% of the control group (FIG. 5-A). After 72h exposure, the zebra fish fry heart rate was significantly down-regulated at both 0.1 and 1.0 μ M, with down-regulation amplitudes of 13.51% and 22.97%, respectively (fig. 5-B). And no significant difference is generated in the body length and the full length of the zebra fish at 96 h. Therefore, the development indexes can indicate the development toxicity of the 2,4-DTBP, and have inhibition effects on the hatchability (1.0 mu M) and the heart rate (0.1 mu M and 1.0 mu M) of the fry, but not all the development indexes can generate responses (figure 5-C and figure 5-D), so that the behavior of the fry is detected and the result is significantly analyzed in order to further explore the toxicity of the 2,4-DTBP to the fry.
The results are shown in FIGS. 6 to 8. FIG. 6 is a zebra fish fry 'movement' behavior index; FIG. 7 is a behavior index of zebra fish fry 'direction'; FIG. 8 shows the "body" behavior index of zebra fish fries.
As can be seen from the graph, after 50min of continuous light-dark alternate stimulation, 11 of the 16 indexes generate significant response, the response rate is 68.75%, and the responses are concentrated in zebra fish fries of 0.1 mu M exposure groups, which indicates that the 2,4-DTBP at the concentration generates significant interference on the fry behavior.
Of these, 3 of the 4 indices of the "move" class were significantly up-regulated. Distance, time, and speed were adjusted up 24.83%, 29.37%, and 30.33%, respectively, compared to the control group (fig. 6). As a general measure to provide information on the subject's spontaneous activity, a significant rise in the 3-point index directly indicates that this concentration of 2,4-DTBP may cause the larval fish to be too "excited".
In the "direction" type index, the rotation angle represents the direction change between two consecutive samples, and the absolute relative is only a fraction of the sign. Similarly, the relative angular velocity is used to indicate the change speed of the moving direction, the average value thereof is used to evaluate the turning direction of the entirety of the fry, and the absolute angular velocity indicates the turning amount per unit time. As can be seen from FIG. 4, there was no significant difference in relative angular velocity (FIG. 7-C), indicating that 2,4-DTBP exposure did not interfere with the direction of rotation of the fry; whereas 0.1 μ M exposure resulted in a significant 46.44% decrease in absolute angular velocity of the fry (FIG. 7-A), meaning that 0.1 μ M2, 4-DTBP exposure inhibited the amount of rotation per unit time of the fry.
The physical index is composed of the activity state and the rotation times of the larval fish. The "active state" can be divided into three states, i.e., "violent", "active" and "static", each state having corresponding duration and frequency. As can be seen from fig. 8-a to 8-F, 0.1 μ M, although not affecting the time and frequency of the larval fish in the violent state, significantly increased the time of the larval fish in the active state and, complementarily, significantly decreased the resting time; in addition, the active and quiescent frequencies were significantly up-regulated by 24.68% and 24.92%, respectively. The time of the fish at rest is reduced, but the frequency is increased, which means that the fish fry is frequently switched between rest and activity, and the fish fry is still active again when staying in a rest state, and the process is repeated, so that the fluctuation and the instability of the behavior of the zebra fish fry at 0.1 mu M are reflected. The rotation times of the larval fish in the pore plate area can be divided into anticlockwise rotation and anticlockwise rotation.
As in fig. 8-G, the number of clockwise rotations is not only in the "body" class of metrics, but also the most sensitive one of the 16 metrics (P < 0.001). The number of clockwise rotations at 0.1 μ M exposure rose by 32.89% compared to the control. Similarly, the number of counterclockwise rotations is also adjusted up to 29.71% (fig. 8-H). The turnover times in any direction are obviously increased, and the 2,4-DTBP can not generate obvious interference on the judgment of the direction of the zebra fish fries, and the conjecture also accords with the change of the relative angular speed of the fries.
From the results of significant differences, the response rates of three major categories of indicators, "movement", "direction", and "body" are: 75% (3/4), 50% (2/4), 75% (6/8), the "move" and "body" categories are somewhat more sensitive than the "direction" category.
In fact, in the existing behavior detection method, the swimming distance and speed in the index of the 'moving' type are the two most common indexes, and the two indexes are also found to generate significant difference in the example, so that the stability and the sensitivity of the index are proved.
However, it is only preliminarily proven by these two indexes that the compound interferes with the mobility of the living body, and the change in direction recognition and body state cannot be judged. By increasing the indexes of 'direction' and 'body', we find that the exposure of 2,4-DTBP with 0.1 mu M can inhibit the rotation amount of the larval fish in unit time, but does not interfere the judgment of the direction of the larval fish; the significant increase in activity time and frequency indicates that the exposure of 0.1 μ M2, 4-DTBP causes "hyperactivity" of the fry, and that overexcitement of the fry is precisely the cause of the increase in the fry swimming speed and distance.
Therefore, the invention not only increases a plurality of discrimination indexes, but also increases the effectiveness and persuasion of data, and fully reflects whether the 2,4-DTBP exposure influences the behavior of the larval fish or not.
In conclusion, through the combination of developmental toxicity indexes and behavior tests, the 2,4-DTBP is proved to have developmental toxicity and neurotoxicity to zebra fish embryos/fries. The invention not only utilizes the development sensitivity indexes of the zebra fish from embryo to fry, but also screens out the optimal sensitivity point for the behavior test of the fry, and on the basis, the ethological data is fully mined to form three major indexes of 'movement', 'direction' and 'body', and 16 microspecies indexes, thereby effectively reflecting the neurotoxicity of the fry. Compared with the prior method, the method has the advantages of more data, sufficient evidence and wide coverage, can quickly detect the toxic effect of the chemical with high flux, and provides powerful data support for chemical management.
Claims (8)
1. A chemical toxicity detection method based on zebra fish behaviors is characterized by comprising the following steps: and (3) performing embryonic development detection and larval fish behavior test on the zebra fish through exposure in a chemical solution, thereby detecting the toxicity of the chemical.
2. The method of claim 1, wherein the embryo development testing comprises: hatchability, heart rate, body length, full length.
3. The method of claim 1, wherein the fry behavior test comprises: the zebra fish fry is tested on three items of movement, body and direction.
4. The method of claim 3, wherein the mobile test comprises: swimming distance, swimming time, speed, acceleration.
5. The method of claim 3, wherein the physical testing comprises: jerky time, jerky frequency, active time, active frequency, quiescent time, quiescent frequency, clockwise rotation, counterclockwise rotation.
6. The method of claim 3, wherein the direction test comprises: relative angular velocity, absolute angular velocity, relative rotational angle, absolute rotational angle.
7. Method according to claim 1, characterized in that it comprises the following steps:
(1) determining the optimal time point of the zebra fish fry behavior test:
a, continuously culturing zebra fish embryos to obtain larval fish at different development stages;
b, carrying out the incubation of the pore plate before the experiment so as to reduce the experimental error;
c, performing behavior test on the zebra fish fry;
d, determining the optimal time point of the zebra fish fry behavior test;
(2) chemical toxicity comparative exposure experiments:
selecting zebra fish embryos, and exposing the zebra fish embryos to a culture solution containing chemicals to be detected to serve as an experimental group; selecting zebra fish embryo to be exposed in culture solution without chemicals as a control group;
b, culturing the zebra fish embryo at the temperature of 27-29 ℃, and culturing according to the following steps: dark-14: exposure was performed for a 10 hour light cycle;
c, semi-statically exposing for 144-168 hours, updating 80% -90% of liquid medicine every 24 hours, and picking out dead embryos in time;
d, performing embryo development detection in the exposure process, namely determining the toxicity index of the embryo at the development stage for 0-96 h;
e. continuously exposing the fry for 144-168 hours, and performing fry behavior test;
(3) and performing data analysis on results of the embryo development detection and the fry behavior test, so as to evaluate the toxicity of the chemicals according to the data analysis results.
8. The method of claim 1,
continuously culturing zebra fish embryos to obtain three larval fish of 120h, 144h and 168h in different development stages when the optimal time point of the zebra fish larval fish behavior test is determined;
in a chemical toxicity comparison exposure experiment, semi-static exposure is adopted for 168 hours, 80-90% of liquid medicine is updated every 24 hours, and dead embryos are picked out in time; and the fry are exposed for 168 hours continuously, and fry behavior test is carried out.
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