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    • 专利摘要:
    The invention discloses a joint toxicity test method for cadmium and pesticide atrazine and chlorpyrifos to earthworms, and provides a scientific basis for the ecological risk of compound pollution. The filter paper method and artificial soil method are used to evaluate the single and joint toxicity of cadmium, pesticide atrazine and chlorpyrifos to earthworm. The joint toxic effects of equal toxicity and equal concentration between pollutants were evaluated according to the Marking additive index method. The results of a single toxicity study showed that when tested by the filter paper method, the cadmium, atrazine, and chlorpyrifos to earthworm at 48 h LC50 values are 2269 (176. 9 to 293. 7), 156.6 (119. 5 to 201. 6.), and 1015.9 (756. 1 to 1334. 4) mg L'l", respectively, using artificial soil test method, the cadmium, atrazine, and chlorpyrifos to earthworm 14 d LCso values are 1428.4 (132.9 to 1599. 1), 180.4 (158. 4 to 204.5), and 384.9 (353.5 to 444.3) mg kg'l, respectively. Results of joint toxicity show in both test systems that chlorpyrifos and cadmium having a toxicity ratio of l: 1 and a concentration ratio of l: 1 are mainly antagonistic, and the joint toxicity of pollutants to earthworms is more complicated, which is closely related to pollutant types, concentration ratios, test systems and functions.
    公开号:NL2025052A
    申请号:NL2025052
    申请日:2020-03-05
    公开日:2020-09-11
    发明作者:Wang Yanhua；Yang Guiling；Zhang Changpeng；Li Xinfang；Xu Zhenlan；Wang Qiang
    申请人:Zhejiang Acad Agricultural Sci；
    IPC主号:
    专利说明:

    TEST METHOD FOR JOINT TOXICITY OF CADMIUMWITH PESTICIDE ATRAZINE AND CHLORPYRIFOS TOEARTHWORMS
    TECHNICAL FIELD The invention belongs to the field of the research of pesticides atrazine and chlorpyrifos to earthworms, and in particular relates to a method for testing the joint toxicity of cadmium and pesticides atrazine and chlorpyrifos to earthworms.
    BACKGROUD For a long time, toxicology research has focused on the environmental toxicity of a single chemical. Many standards, such as safe concentration standards, allowable discharge standards for wastewater, soil environmental quality standards, and surface water environmental quality standards and so on, are established based on the toxicity of a single chemical. However, the pollutants in the environment are diversified and complicated, and mainly exist in the form of composite or mixed of organic and inorganic pollutants. The related evaluation standards established by the single effect in the past cannot truly reflect the environmental quality requirements. Many environmental toxicological effects cannot be explained by the action mechanism of a single pollutant. Studies have shown that even if the concentration of each chemical is lower than the no observed effect concentration NOEC, their mixture may produce significant toxic effect on the organism. Therefore, it is not enough to rely on the ecotoxicity of a single chemical to assess environmental quality. In recent years, although scholars at home and abroad have successively carried out research on the interactions between multiple heavy metals and multiple organic pollutants and their ecotoxicity, and have also achieved very important research results, in general, environmental composite pollution and the study of its toxicological effects is still in its initiating and needs to be further improved and developed. With the rapid development of the pesticide industry and its widespread use, its pollution effects have also spread to the entire ecosystem. Studies have shown that 20% to 70% of herbicides will remain in the soil for a long time, and 80% to 90% of pesticides will enter rivers, lakes and soil through rainfall, sedimentation and runoff, that is to say, the combined pollution of two or more pesticide residues in the soil environment is widespread. Atrazine is currently the most used triazine herbicide in the world. It is long lasting in the environment. It has been reported that the agent is frequently detected in soil, groundwater and surface water. Chlorpyrifos is the most used organophosphorus pesticide in China. It is relatively stable in soiland water and can be used in a variety of organisms. Concentrated in the organisms, and even cause serious adverse effects on soil organisms and aquatic organisms through the food chain. Cadmium is a heavy metal that is highly toxic to living organisms. It is non-degradable and accumulative, widely used in electroplating, pigment, paint, printing and dyeing and other industrial fields. With the rapid development of the economy, a large amount of cadmium enters the soil, water, sediments and the atmosphere with sewage, garbage and industrial wastes, causing pollution. Therefore, the threat of combined pollution of cadmium and pesticides exists in farmland ecosystems, although domestic and foreign scholars have done a lot of research on the toxic effects and mechanism of atrazine, chlorpyrifos and cadmium single pollution, the research on the toxic effects of cadmium and pesticide combined pollution is not enough. Earthworm is a kind of soil animal with the largest terrestrial animal biomass. It plays an important role in improving the physical properties of the soil and the nutrition cycle of plants. Studies on the ecological toxicity of earthworms can not only reflect the toxic effects of pollutants, but also reflect the pollution of soil. It can also provide a safety threshold to protect the entire soil fauna. Therefore, this study uses earthworm as model organism, and uses the compound chlorpyrifos, atrazine and heavy metal cadmium as the research object to investigate combined toxic effects on earthworms when substances coexist, aiming to obtain basic data on the joint toxicity of these pollutants joint pollution, with a view to providing basic data for the correct evaluation of the biological effects of pollutants on earthworms, and provide scientific evidence for the protection of soil animals and environmental risk.
    SUMMARY The purpose of the present invention is to provide the joint toxicity test method of cadmium and the pesticide atrazine and chlorpyrifos to earthworms in order to solve the above problems, and solve the problems of low biological utilization rate and poor absorption rate of the existing joint toxicity test methods. In order to solve the above problems, the present invention provides a technical solution: A joint toxicity test method for cadmium and pesticide atrazine and chlorpyrifos to earthworms, characterized in that, comprising of experimental materials, experimental methods and data processing. Preferably, the experimental material comprises a test organism and a test chemical: The test organism Eisenia fetida, provided by the earthworm farm of the College of Animal Science, Zhejiang University, was first pre-cultured for a period of time, and then selectinghealthy adult earthworm for 2 months and older, weighing 350 ~ 500mg, with obvious clitellum and same size for toxicity test; Before the toxicity test, checking the sensitivity of the earthworms with chloroacetamide according to the method of the OECD TG 207 guideline, selecting the chloroacetamide on the earthworm with 14 d-LCso value at 10mg / kg, dw-50mg / kg for toxicity test; The tested chemicals comprises 92% atrazine technical product, 95% chlorpyrifos technical product and CdCl, « 2.5H;0 (99%, AR). Preferably, the experimental method comprises a single toxicity test and a joint toxicity evaluation method;
    The single toxicity test use filter paper method and artificial soil method,
    Filter paper method: refer to the method of OECD TG 207, and improve on this basis. (D Adding a small amount of deionized water to a flat bottom beaker (9cm =12cm in height) with a layer of filter paper at the bottom to just immerse the filter paper, place the earthworm on the filter paper, seal it with a plastic film, pierce the plastic film with a dissecting needle, and placethe beaker in a thermostat with a temperature of (20 + 1) °C and a humidity of 80% to 85%, clear the intestine for 1 d; ©) Putting a layer of filter paper with a diameter of 11cm in a 9cm diameter petri dish (the filter paper covers the edge of the petri dish), and determining the effective concentration range of the pesticides through preliminary experiments.
    Diluting the test pesticide with acetone into 5 to 7 series of gradient concentrations in equal steps; Taking 2mL of a certain concentration of the pesticide solution on filter paper; Each concentration is set to 30 replicates, with a solvent control group and a blank control group; Placing the petri dishes in the fume hood for 1 d: After the acetone has completely evaporated, adding 2mL of distilled water to moisten the filter paper; Rinsing the earthworms after deionization with deionized water, absorbing excess water with filter paper, and placing them in a petri dish; Putting 1earthworm in each petri dish as one repeat, sealing with plastic film, puncturing the plastic film with a dissecting needle, and placing it in an incubator with a temperature of (20 + 1) °C and a humidity of 80% to 85% in the dark, and counting each at 24 h and 48 h, recording the number of deaths and symptoms of poisoning, and considering the anterior tail of the earthworm to not respond to mechanical stimulation as death;
    Artificial soil method: © Artificial soil is prepared according to the standard method proposed in OECD TG 207; @ The test pesticide is dissolved in acetone and mixed in 10 g quartz sand (when some of the original drugs have low solubility in acetone, adding additive 1% TritonX-100), after acetone is completely evaporated, mixing with 490 g artificial soil, addingdistilled water to make the soil water content reach about 35%, dissolving CdCl, + 2.5H:0 directly in water; Putting the artificial soil into a 500 mL glass standard bottle, and then putting 10 earthworms domesticated for 24 hours in artificial soil without pesticide treatment into the glass standard bottle as a repeat, puncturing the plastic film with a dissecting needle and sealing the glass standard bottle mouth, and then placing the glass standard bottle in an incubator with a temperature of (20 + 1) °C and a humidity of 80% to 85 %, with continuous light culture (light intensity 400-800 lux); According to the results of the preliminary experiment, each drug is set to 5 to 7 concentrations according to a certain level difference, each concentration is set to 3 replicates, with a blank control group, a solvent and an adjuvant control without the drug; Counting on the 7 th and 14 th days of the test and record deaths and symptoms of poisoning, the front tail of earthworms not responding to mechanical stimulation are considered dead; The joint toxicity evaluation method is based on the toxicity data of a single pollutant to earthworms, taking 48 h-LCso (filter paper method) and 14 d-LCse (artificial soil method) as criterion, setting six different test concentrations according to the mixing ratio of equal toxicity and equal concentration at equal logarithmic intervals (refer to the design of the more toxic substance), the test method and the calculation method of the LCs value at each time are the same as the single toxicity test; Evaluating the joint toxicity of chlorpyrifos, atrazine and heavy metal cadmium, and the sum of biological toxicity S was obtained using the following formula: S = Am / Ai + Bm /Bi + Cm / Ci, where Am, Bm and Cm are the toxicity of each poison in the mixture (LC), Ai, Bi and Ci are the toxicity when the poisons A, B and C act separately (LCso); S is converted into an additive index AI (Additive Index), when S <1, AI =(1/S) -1.0; when S > 1, AI = 1.0-S; finally, use Al to evaluate the compound effect of the chemical, when -0.2 < AT < 0.25, it is an addition; when AI > 0.25, it 1s greater than the additive effect, that is, synergism; When AI < -0.2, itis less than the additive effect, that is, antagonism.
    Preferably the data processing calculates the LCso value of the pesticide to the earthworm and its 95% confidence limit based on the number of deaths and the time of death of the earthworm using the probability value analysis method, and uses whether the 95% confidence limit of the LCso value has an overlap as criteria to determine the different agents for whether toxicity differences are significant.
    The beneficial effect of the present invention is that the present invention relates to a joint toxicity test method of cadmium, the pesticide atrazine and chlorpyrifos to earthworms. Atrazine has been identified as an endocrine disruptor, which can produce a cytotoxic effect and can cause DNA damage in biological cells. It is genotoxic and may be carcinogenic to thehuman body. In the filter paper method, pesticides adhere to the surface of the filter paper and have high bioavailability. In addition, earthworms directly contact with pollutants in vitro, resulting in large absorption and fast absorption rate. 5 BRIEF DESCRIPTION OF THE DRAWINGS For ease of explanation, the present invention is described in detail by the following specific implementations and drawings.
    Figure 1 is a schematic flow chart of the experimental steps of the present invention.
    DESCRIPTION OF THE EMBODIMENTS As shown in FIG 1, this specific embodiment employs the following technical schemes: A joint toxicity test method for cadmium and pesticide atrazine and chlorpyrifos to earthworms, characterized in that, comprising of experimental materials, experimental methods and data processing, Wherein, the experimental material comprises a test organism and a test chemical: The test organism Fisenia fetida, provided by the earthworm farm of the College of Animal Science, Zhejiang University, was first pre-cultured for a period of time, and then selecting healthy adult earthworm for 2 months and older, weighing 350 to 500 mg, with obvious clitellum and same size for toxicity test; Before the toxicity test, checking the sensitivity of the earthworms with chloroacetamide according to the method of the OECD TG 207 guideline, selecting the chloroacetamide on the earthworm with 14 d-LCso value at 10mg / kg, dw-50mg / kg, dw for toxicity test; The tested chemicals comprises 92% atrazine technical product, 95% chlorpyrifos technical product and CdCl, « 2.5H;0 (99%, AR).
    Wherein the experimental method comprises a single toxicity test and a joint toxicity evaluation method; The single toxicity test use filter paper method and artificial soil method; Filter paper method: refer to the method of OECD TG 207, and improve on this basis. (1 Adding a small amount of deionized water to a flat bottom beaker (9cm x12em in height) with a layer of filter paper at the bottom to just immerse the filter paper, place the earthworm on the filter paper, seal it with a plastic film, pierce the plastic film with a dissecting needle, and place the beaker in a thermostat with a temperature of (20 + 1) °C and a humidity of 80% to 85%,
    clear the intestine for 1 d; ©) Putting a layer of filter paper with a diameter of 11cm in a 9cm diameter petri dish (the filter paper covers the edge of the petri dish), and determining the effective concentration range of the pesticides through preliminary experiments. Diluting the test pesticide with acetone into 5 to 7 series of gradient concentrations in equal steps; Taking 2 mL of a certain concentration of the pesticide solution on filter paper; Each concentration is set to 30 replicates, with a solvent control group and a blank control group; Placing the petri dishes in the fume hood for 1 d; After the acetone has completely evaporated, adding 2mL of distilled water to moisten the filter paper; Rinsing the earthworms after deionization with deionized water, absorbing excess water with filter paper, and placing them in a petri dish.
    Putting 1 earthworm in each petri dish as one repeat, sealing with plastic film, puncturing the plastic film with a dissecting needle, and placing it in an incubator with a temperature of (20 + 1) °C and a humidity of 80% to 85%in the dark, and counting each at 24 h and 48 h, recording the number of deaths and symptoms of poisoning, and considering the anterior tail of the earthworm to not respond to mechanical stimulation as death; Artificial soil method: © Artificial soil is prepared according to the standard method proposed in OECD TG 207; @ The test pesticide is dissolved in acetone and mixed in 10 g quartz sand (when some of the original drugs have low solubility in acetone, adding additive 1% TritonX-100), after acetone is completely evaporated, mixing with 490 g artificial soil, adding distilled water to make the soil water content reach about 35%, dissolving CdCl; « 2. SH.O directly in water; Putting the artificial soil into a 500 mL glass standard bottle, and then putting 10 earthworms domesticated for 24 hours in artificial soil without pesticide treatment into the glass standard bottle as a repeat, puncturing the plastic film with a dissecting needle and sealing the glass standard bottle mouth, and then placing the glass standard bottle in an incubator with a temperature of (20 + 1) °C and a humidity of 80% to 85 %, with continuous light culture (light intensity 400-800lux); According to the results of the preliminary experiment, each drug is set to 5 to 7 concentrations according to a certain level difference, each concentration is set to 3 replicates, with a blank control group, a solvent and an adjuvant control without the drug; Counting on the 7 th and 14 th days of the test and record deaths and symptoms of poisoning, the front tail of earthworms not responding to mechanical stimulation are considered dead; The joint toxicity evaluation method 1s based on the toxicity data of a single pollutant to earthworms, taking 48 h-LCso (filter paper method) and 14 d-LCsp (artificial soil method) as criterion, setting six different test concentrations according to the mixing ratio of equal toxicity and equal concentration at equal logarithmic intervals (refer to the design of the more toxic substance), the test method and the calculation method of the LCso value at each time are thesame as the single toxicity test; Evaluating the joint toxicity of chlorpyrifos, atrazine and heavy metal cadmium, and the sum of biological toxicity S was obtained using the following formula: S=Am/Ai+Bm/Bi+ Cm/ Ci, where Am, Bm and Cm are the toxicity of each poison in the mixture (LCsp), A1, Bi and Ci are the toxicity when the poisons A, B and C act separately (LCso); Sis converted into an additive index AI (Additive Index), when S <1, AT =(1/S) -1.0; when S > 1, AI = 1.0-S; finally, use AI to evaluate the compound effect of the chemical, when -0.2 < Al < 0.25, it is an addition; when AI > 0.25, it is greater than the additive effect, that is, synergism; When AI < -0.2, it is less than the additive effect, that is, antagonism.
    Wherein the data processing calculates the LCsp value of the pesticide to the earthworm and its 95% confidence limit based on the number of deaths and the time of death of the earthworm using the probability value analysis method, and uses whether the 95% confidence limit of the LCso value has an overlap as criteria to determine the different agents for whether toxicity differences are significant.
    Experimental results: Filter paper method measurement results: Atrazine and chlorpyrifos were formulated with a toxicity ratio of 1: 1, and the additive indexes were 0.94 and 1.36 when the exposure time was 24 h and 48 h, respectively.
    The joint effects were synergistic, and with the increase of the exposure time, the synergistic effect is enhanced; when the two are joint at a concentration ratio of 1: 1 and the exposure time is 24 h and 48 h, the additive index is -2.04 and -0.18, respectively, and the joint effect results are antagonism and additive effect (Table 1), Atrazine and cadmium are formulated with a toxicity ratio of 1: 1, and the additive indexes are -0.82 and -1.78 when the exposure time is 24 h and 48 h, respectively.
    The joint effect results are antagonism, and with the prolongation of exposure time the antagonism effect is enhanced; when the two are joint at a concentration ratio of 1: 1 and the exposure time is 24 h and 48 h, the additive indexes are -0.62 and -0.666, respectively, and the joint effect are shown as antagonism and additive effect (Table 2). Similar to the joint effect of atrazine and cadmium, chlorpyrifos and cadmium are formulated with a toxicity ratio of 1: 1, and the joint indexes at exposure time of 24 h and 48 h are -0.28 and -1.02, respectively, and the joint effects are shown as -0.34 and -0.6, and the joint effect is shown as antagonism.
    The antagonism effect increased with the prolongation of exposure time; The results were shown as antagonism and additive effects (Table 3). Atrazine, chlorpyrifos and cadmium were formulated with a toxicity ratio of 1: 1: 1. When the exposure time is 24 h and 48 h, the additive indexes are 0.75 and -0.28, respectively, and the joint effects are synergy and tangerine resistance.
    At 48 h, the additive indices were 0.04 and 0.67, respectively, and the joint results were additive and synergistic (Table 4).
    Table 1 Joint toxicity of atrazine and chlorpyrifos to earthworms measured by filter paper method Table 2 Joint toxicity of atrazine and Cd to earthworms measured by filter paper method Table 3 Joint toxicity of chlorpyrifos and Cd to earthworms measured by filter paper method Table 4 Joint toxicity of atrazine, chlorpyrifos and Cd to earthworms measured by filter paper method In general, the joint effects of atrazine, chlorpyrifos and cadmium were mainly antagonism at a toxicity ratio of 1: 1 and a concentration ratio of 1: 1, while the joint effects of atrazine and chlorpyrifos and the concentration ratio were close correlation.
    It is mainly synergistic when the toxicity Is 1: 1, and it is more important than orange resistance when the concentration is 1: 1. The joint effect of atrazine, chlorpyrifos and cadmium is more complex when they are co-existing, and are closely related to the exposure time and the ratio of the pollutants.
    The toxicity is synergistic at 1: 1: 1 and 24 h after exposure, but with the exposure time prolonged, it is an antagonism effect when it is exposed to 48 h; when the concentration ratio is 1: 1: 1, it is an additive effect when it is exposed for 24 h, but as the exposure time 1s prolonged, it is a synergistic effect when it 1s exposed to 48 h.
    Table 1 Joint toxicity of atrazine and chlorpyrifos to earthworms with filter paper . Exposure LC sy (95% fiducial limit) mg Lt Additive time Atrazine Chlorpyrifos index ee . 24 h 203.4(127.2~706.2) 1324.1(827.7~4597.2) 0.94 Toxicity ratio of 1:1 48h 33.10(19.12~41.59) 215.5(124.4~270.8) 1.36 Concentration ratio of 1:1 24h 1948.5(1359.6~4357.6) 1948.5(1359.6~4357.6) -2.04 48h 159.9(122.3-256.9) 159.9(122.3~256.9) -0.18table 2 Joint toxicity of atrazine and Cd to earthworms with filter paper method . Exposure LCs (95% fiducial limit) mg L*! Additive Ratio . Ty TT . time Atrazine Cd index Toxicity ratio of 1:1 24h 531.7(263.3~12554 4) 769.1(380.8~1815.8) -0.82 AE ’ 48 h 218.1(153.7~445.2) 315.4(222.3~643.9) -1.78 Concentration ratio of 1:1 24 h 579.9(340.2~2508.7) 579.9(340.2~2508.7) -0.62 Table 3 Joint toxicity of chlorpyrifos and Cd to earthworms with filter paper method Ratio Exposure LCs (95% fiducial limit) mg L~1 Additive © time Chlorpyrifos Cd index SRT / 24h 2623.8(1509.8~16106.3) 584.1(335.9~3585.3) -0.28 Toxicity ratio of 1:1 48 h 1025.6(737.6~1672.8) 228.3(164.2~372.4) -1.02 Concentration ratio of 1-1 24 h 850.7(421.3~2008.6) 850.7(421.3~2008.6) -0.34 Table 4 Joint toxicity of atrazine, chlorpyrifos and Cd to earthworms with filter paper method
    ~ . < -1 Ratio Exposure ee ee Additive index 2 57 145 313 Tomes Mb ELSES mon ] 9 2930 ; Test results by artificial soil method: Atrazine and chlorpyrifos were formulated with a toxicity ratio of 1: 1, and the additive indexes were -1.34 and -0.54 when the exposure time was 7 d and 14 d, respectively.
    The prolonged exposure time weakened the antagonism effect; when the two were used at a concentration ratio of 1: 1 and the exposure time was 7 d and 14 d, the additive indexes were -0.57 and -0.33, respectively.
    And, with the increase of exposure time, antagonism also weakened (Table 5). Atrazine and cadmium have a toxicity ratio of 1: 1, and the additive index is 0.21 and 0.51 when the exposure time 1s 7 d and 14 d, respectively.
    The joint effect results are additive K synergistic, respectively.
    The concentration ratio was 1: 1, and the additive index was 1.26 and 1.60 when the exposure time was 7 d and 14 d, respectively.
    The joint effects were synergistic, and the synergistic effect increased with the extension of exposure time (Table 6). Chlorpyrifos and cadmium are formulated with a toxicity ratio of 1: 1, and the additive index is -0.66 and -0.33 when the exposure time is 7 d and 14 d, respectively.
    The joint effect results are antagonism, and the prolongated exposure time weakens the antagonism; When the two were formulated at a concentration ratio of 1: 1 and the exposure time was 7 d and 14 d, the additive index was -0.79 and 0.10, respectively, and the joint effect was shown as antagonism and additive effect (Table 7). Atrazine, chlorpyrifos, and cadmium are formulated with a toxicity ratio of 1: 1: 1, and the additive indices are -1.91 and -1.05 when the exposure time 1s 7 d and 14 d, respectively. the Prolongated exposure time weakens the antagonism; When the three were joint at a concentration ratio of 1: 1: 1 and the exposure times were 7 d and 14 d, the additive indices were 2.41 and 3.18, respectively.
    The joint effects were all synergistic, and the synergistic effects increased with the prolongation of the exposure time (Table 8). Table 5 Joint toxicity of atrazine and chlorpyrifos to earthworms measured by artificial soil method Table 6 Joint toxicity of atrazine and Cd to earthworms measured by artificial soil method Table 7 Joint toxicity of chlorpyrifos and Cd to earthworms measured by artificial soil method Table 8 Joint toxicity of atrazine, chlorpyrifos, and Cd to earthworms measured by artificial soil method In general, the joint effect of atrazine and cadmium is mainly synergistic, while the jointeffects of chlorpyrifos, cadmium and atrazine are mainly antagonism; The joint toxicity when atrazine, chlorpyrifos and cadmium coexist is related to the concentration ratio, and the antagonism is the main effect when the toxicity ratio is 1: 1: 1, while the synergy is the main effect when the concentration ratio 1s 1: 1: 1. Table 5 Joint toxicity of atrazine and chlorpyrifos to earthworms with artificial soil es method i . Exposur LCso (95% fiducial limit) mg kg! Additive Ratio . Se RR e time Atrazine Chlorpyrifos index Toxicity ratio of 1:1 7d 244.3(196.1~287.2) 482.4(380.2~572.4) -1.34 14 d 140.8(82.51~185.6) 292.4(171.4~385.6) -0.54 Concentration ratio of 1:1 7d 216.1(174.0~250.6) 216.1(174.0~250.6) -0,57 14d 163.6(117.3~198.8) 163.6(117.3~198.8) -0.33 Table 6 Joint toxicity of atrazine and Cd to earthworms with artificial soil method . Exposure LC (95% fiducial limit) mg kg’! Additive time Atrazine Cd index . . 7d 82.79(67.75~94.52) 650.5(532.3~742.6) 021 Toxicity ra f1:1 Ord Nem oxicily ratio o 14d S9.86(43.29~72.89) 470.3(340.2~572.8) 0.51 . . 7d 80.17(65.14~93.67) 80.17(65.14~93.67) 1.26 Concentration ratio of 1:1 = i css ln SL IOES 3CTHIE) | GLSOUSICTANG) | LEO Table 7 Joint toxicity of chlorpyrifos and Cd to earthworms with artificial soil ee method i . Exposure LCs (95% fiducial limit) mg kg! Additive Ratio . ee Re Res time Chlorpyrifos Cd index Toxicity ratio of 1:1 7d 347.5(290.1~470.8) 1287.2(1074.7~1744.3) -0.66 vs / 14d 255.7(212.7~317.5) 947.3(787.9~1176.2) -0.33 . 7d 593.9(459.7-954.]} 593.9(459.7~954.1) -0.79 Concentration ratio of 1:1 14d 275.1(231.8-325.5) 275.1(231.8~325.5) 0.10 Table 8 Joint toxicity of atrazine, chlorpyrifos and Cd to earthworms with artificial Lo soil method | Ratio Exposure ~~ LCsymgkg! Additive * time Atrazine Chlorpyrifos Cd index ne . 7d 193.8 411.1 1522.9 -1.91 Toxicity ratio of 1:1 ICI 14d 123.6 262.2 971.4 -1.05 . . 7d 37.11 37.11 37.11 241 n Ki [e : Ad 2708 27.08 2708 dl The joint effect of chlorpyrifos and cadmium determined by artificial soil method is similar to the filter paper method, and the main effect is antagonism.
    The artificial soil method is mainly used to determine the joint effect of atrazine and cadmium.
    It is mainly antagonism when employs filter paper method.
    The atrazine and chlorpyrifos are mainly antagonism when employ artificial soil method.
    The joint effect of the two pesticides is more complicated when tested by the filter paper method.
    When the toxicity ratio is 1: 1, the joint effect is mainly synergistic, and when the concentration ratio is 1: 1, it 1s more the antagonism.
    The joint effect of chlorpyrifos, atrazine and Cadmium coexisting 1s more complicated and is closely related to the test system, concentration ratio and exposure time.
    In the filter paper method, pesticidesadhere to the surface of the filter paper, which has high bioavailability, and the earthworms are in direct contact with the pollutants outside the body, resulting in large absorption and fast absorption rate. In the artificial soil method, the pollutants will be absorbed by soil colloid and organic matter, and degraded by biological, light and chemical oxidation, which greatly reduces the bioavailability of the test substance in the soil, thus reducing the absorption rate of earthworm to pollutants. The difference in the toxicity of different types of pollutants to earthworms may be due to its different chemical structure, behavior in the environment and bioavailability, which results in different mechanisms of toxicity to earthworms, Atrazine has been identified as an endocrine disruptor, which can produce cytotoxic effects, can cause DNA damage in biological cells, is genotoxic, and may be carcinogenic to humans. The mechanism of chlorpyrifos is to inhibit acetylcholinesterase and the effect is irreversible. Cadmium is a poison that causes rapid damage and is easy to accumulate in various organs. It can cause oxidative damage to the body, DAN single-strand breaks, and damage the DNA repair system, leading to apoptosis.
    The impact of pollutants on the ecosystem, no matter how complicated and serious the final consequences, initiate at the molecular level of the individual organism, and the physiological response of the organism at the sublethal concentration of pollutant species is mainly expressed at the molecular level. The joint toxicological effects of pollutants on earthworms are mainly carried out from the perspective of acute toxicology. The analysis based on these data can only be used as the first step of toxicological risk analysis, and cannot truly reflect the long-term and potential threats of toxicants to soil biology. Therefore, it is necessary to continue to study the joint toxicity of pollutants at the cell and molecular level to reveal the mechanism of joint toxicity. In addition, from an ecological point of view, earthworms are at the bottom of the terrestrial ecological food chain and have a concentrated effect on most pollutants.
    These concentrated pollutants may not cause serious damage to the earthworms, but they may affect senior organisms in the food chain. In order to better protect the soil organisms and the ecological environment of the soil environment, the acute and chronic toxicity of a variety of soil organisms, including jumping insects, needs to be studied to enrich the basic test data of pollutants on soil organism’s toxicology. Research on the biological transmission and bioaccumulation of pollutants in the soil ecosystem through the food chain, food webs needs to be further developed to provide a reference for the formulation of standards for the safe concentration of pollutants on soil organisms.
    Different types of tested organisms respond to the same joint pollutants in different types (possibly due to the different enzyme structures in different organisms). It is not sufficient to use only one or a certain type of organism as the indicator for the risk assessment of joint toxicity. It
    Is necessary to conduct a composite evaluation of multiple indicator organisms and even the entire biological community in order to more accurately determine the impact of composite pollutants on the entire ecosystem. In addition, previous studies have shown that in addition to the joint physical and chemical properties of pollutants in the process of joint pollution, the joint relationship between the mass and concentration of pollutants plays a more direct and important role, consistent with the results of this study. This shows that different combinations of mass concentration may have different joint toxicity mechanisms. In this study, only the ratio of concentration ratio of 1: 1 and toxicity ratio of 1: 1 between pollutants was carried out. In order to fully reflect the interaction law of pollutants, research on multiple mass concentration ratios should be carried out as much as possible.
    At present, the joint toxicity mechanisms related to complex pollution mainly include several theories of competition for binding sites, affecting biological enzyme activities, interfering with normal physiological processes of organisms, changing the structure and function of biological cells, chelation and precipitation, and interfering with the structure and function of biological macromolecules. The joint toxicity of atrazine and chlorpyrifos to earthworms measured by artificial soil method in this research is mainly antagonism, whether it Is concentration ratio of 1: 1 or concentration ratio of I: 1, and may be related with the above-mentioned mechanism of joint toxicity, some scholars believe that the mechanism of pollutant synergy is mainly that the structure of the cell membrane can be changed when the pollutants coexist, so that the permeability of the cell membrane is increased. In this study, the joint toxicity of atrazine and cadmium to earthworms measured by artificial soil method was synergistic in both toxicity ratio of 1: 1 and concentration ratio of 1: 1, and the synergy increased with the extension of the experiment. It is possible that with the increase of exposure time, the permeability of earthworm cell membranes will increase, thereby increasing the absorption of pollutants by organisms, enhancing the biological toxicity of the mixed system, and producing a synergistic effect.
    The change of toxicity when pollutants coexist is a comprehensive reflection of their processes in vivo. The interaction of pollutants in the organism may occur in the process of ingestion, absorption, distribution, metabolism, transformation, and excretion of pollutants to change the processes in their respective bodies, or produce related biological effects when they act on the same target organ. That is to say, the joint action effect can occur by affecting the respective toxicological metabolic kinetics and toxicological kinetics. The metabolic transformation of pollutants in the organism makes the study of compound pollution more complicated. For example, when the original compound and its metabolites are toxic, but targeting different target organs or toxicity endpoints, it is difficult to determine the jointtoxicity. Therefore, the interaction of compounds in the body and the absorption, distribution, metabolic transformation and toxic effects are more meaningful, which is of great significance for formulating relevant environmental quality standards, exploring their joint toxicity mechanism and taking preventive measures.
    The basic principles and main features of the present invention and the advantages of the present invention have been shown and described above. Those skilled in the art should understand that the present invention is not limited by the above embodiments. What is described in the above embodiments and the description is only to clarify the principle of the present invention, various modifications and improvements can be made without departing from the spirit and scope of the present invention. These modifications and improvements shall all fall within the scope of the claimed invention. The claimed scope of the invention is defined by the appended claims and their equivalents.
    权利要求:
    Claims (3)
    [1]
    1. Joint toxicity testing method for cadmium and the pesticides atrazine and chlorpyrifos for earthworms, characterized in that the method comprises experimental materials, experimental methods and data processing.
    [2]
    Joint test method for cadmium and the pesticides atrazine and chlorpyrifos for earthworms according to claim 1, characterized in that the experimental materials comprise a test organism and test chemical: - the test organism Eisenia fetida, provided by the earthworm farm of the College of Animal Science, Zhejiang University , is first pre-cultured for a period of time and then healthy adult earthworms of 2 months and older, weighing 350-500 mg and with clear clitellum and equal size, are selected for the toxicity test; - before the toxicity test, the susceptibility of the earthworms to chloroacetamide according to the method of the OECD TG 207 guideline, where earthworms with a 14d-LCs value of 10 mg / kg, dw (dry weight}) - 50 mg / kg, dw (dry weight) are selected for the toxicity test, - the test chemicals include 92% atrazine technical product, 95% chlorpyrifos technical product and CdCl; : 2.5 HO (99%, AR (analytical reagent)).
    [3]
    Joint test method for cadmium and the pesticides atrazine and chlorpyrifos for earthworms according to claim 1, characterized in that the experimental method comprises a single toxicity test and a joint toxicity evaluation method: - the single toxicity test uses a filter paper method and an artificial soil method; |) the filter paper method: the method of OECD TG 207 is consulted and improved on the following basis, i) a small amount of deionized water is added to a flat bottom beaker (9 cm x 12 cm high) with a layer of filter paper on the bottom to properly immerse the filter paper, the earthworm is placed on the filter paper, it is closed with a plastic film, the plastic film is pierced with a dissociation needle, and the beaker is placed in a thermostat with a temperature of 20 + 1 ° C and a humidity of 80% to 85%, the bowel is cleaned for 1 day;
    ii) a layer of filter paper with a diameter of 11 cm is placed in a petri dish with a diameter of 9 cm (the filter paper covers the edge of the petri dish), and the effective concentration range of the pesticides is determined by preliminary experiments, the test pesticide is measured with acetone diluted in equal steps in 5 to 7 series of gradient concentrations; 2 ml is taken of a certain concentration of the pesticide solution on filter paper; each concentration is set to 30 replicates, with a solvent control group and a blank control group; the Petri dishes are placed in a fume hood for 1 day; after the acetone has completely evaporated, 2 ml of distilled water is added to moisten the filter paper; the earthworms are rinsed with deionized water after deionization, excess water is absorbed with filter paper and placed in a petri dish; in each petri dish 1 earthworm is put as a repeat, closed with plastic film, and the plastic film is pierced with a dissection needle and placed in an incubator at 20 + 1 ° C and a humidity of 80% to 85% in the dark and counted every 24 hours and 48 hours, recording the number of deaths and symptoms of poisoning and failure of the earthworm's anterior tail to respond to mechanical stimulation is considered dead; II} the artificial soil method: artificial soil is prepared according to the standard method proposed in OECD TG 207; The test pesticide is dissolved in acetone and mixed in 10 g of quartz sand (when some of the original agents have a low solubility in acetone, 1% TritonX-100 is added}, after the acetone is completely evaporated, is mixed with 490 g of artificial soil, distilled water is added to make the groundwater content reach about 35%, CdCl;: 2.5 HzO is dissolved directly in water; the artificial soil is put into a standard 500ml glass bottle, then 10 earthworms are left to dry for 24 hours have been domesticated in artificial soil without pesticide treatment placed in the standard glass bottle as repeat,
    the plastic film is pierced with a dissection needle, and the mouth of the standard glass bottle is closed, then the standard glass bottle is placed in an incubator with a temperature of 20 + 1 ° C and a humidity of 80% to 85%, with continuous light culture (light intensity 400-800 lux); according to the results of the preliminary experiment, each agent is set at 5 to 7 concentrations according to a certain level difference, each concentration is set at 3 replicates, with a blank control group, a solvent and an adjuvant control without the agent; on the 7th and 14th days of the test and deaths and symptoms of poisoning are counted and recorded, and failure of the anterior tail of the earthworm to mechanical stimulation is considered dead; - the joint toxicity evaluation method is based on the toxicity data of a single pollutant to earthworms, using 48 h-LC50 (filter paper method) and 14 d-LC: o (artificial soil method) as the criterion, setting six different test concentrations based on the mixing ratio of equal toxicity and equal concentration at equal logarithmic intervals (referring to the design of the more toxic substance), the test method and the method of calculation of the LCse value are the same as the single toxicity test; evaluation of the joint toxicity of chlorpyrifos, atrazine and heavy metal cadmium, and the sum of the biological toxicity S is obtained using the following formula: S = Am / Ai + Bm / Bi + Cm / Ci, where Am, Bm and Cm are the toxicity of each poison in the mixture (LC 50). A 1, Bi and C 1 are the toxicity when the toxins A, B and C act alone (LC 50); S is converted to an additive index Al (Additive Index) when S <1, Al = (1/5) -1.0; when S> 1, Al = 1.0-S; finally, AI is used to evaluate the composite effect of the chemicals, when -0.2 <Al <0.25 it is an additive effect; when Al = 0.25, it is greater than the additive effect, i.e. synergism; when Al <-0.2, it is less than the additive effect, i.e. antagonism.
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    同族专利:
    公开号 | 公开日
    CN109953001A|2019-07-02|
    NL2025052B1|2021-09-23|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    KR101920048B1|2016-10-17|2018-11-19|건국대학교 산학협력단|Earthworm Animal model for Diagnosis of skin toxicity and diagnosis method using the same|
    法律状态:
    优先权:
    申请号 | 申请日 | 专利标题
    CN201910169542.4A|CN109953001A|2019-03-06|2019-03-06|Cadmium and Pesticide Atrazine, chlopyrifos are to the joint toxicity test method of earthworm|
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