A quantitative analysis method for herbicide toxicity based on the full curve characteristics of microalgae OJIP

Abstract

The present invention discloses a method for quantitatively analyzing the toxicity of herbicides based on the full curve characteristics of microalgae OJIP, specifically including the following steps: Step 1, conduct exposure experiments of different concentrations of herbicides on microalgae; Step 2, measure the rapid chlorophyll fluorescence induction kinetics OJIP curve of the microalgae sample to be tested; Step 3, extract the fluorescence intensity information Fo, F J , F I , F P of the O item, J item, I item and P item in the OJIP curve of the microalgae sample to be tested; Step 4, calculate the change information Fv of the fluorescence intensity between the J item and the O item in the OJIP curve (J‑O) , the change information Fv of the fluorescence intensity between the I item and the J item (I‑J) , the change information Fv of the fluorescence intensity between the P item and the I item (P‑I) ; Step 5, construct a herbicide toxicity response index RI based on the full curve characteristics of the OJIP curve; Step 6, establish a dose-effect quantitative relationship curve between the herbicide concentration and the inhibition rate of the toxicity response index RI; Step 7: Calculate the 50% effect concentration value of the herbicide to characterize the toxicity of the herbicide.

Description

Technical Field

[0001] The invention belongs to the technical field of water quality detection, and in particular relates to a method for quantitatively analyzing herbicide toxicity based on the full curve characteristics of microalgae OJIP. Background Art

[0002] As a major agricultural country, the continuous development of modern agriculture in my country is inseparable from the extensive use of pesticides. Among them, herbicides, as a very important member of chemical pesticides, play an important role in quickly and efficiently preventing and controlling farmland weeds and improving the quality and yield of crops. Therefore, the production and use of herbicides in my country are also increasing. For example, in 2015, the total output of pesticides in my country was 1.328 million tons, of which the total output of herbicides was 827,000 tons, accounting for 62.35% of the total pesticide output (Shen Yan et al. Anhui Agricultural Science, 2019, 47(19):148-151.); by 2020, the total output of herbicide technical in my country has reached 1.004 million tons, a year-on-year increase of 7.38% (National Bureau of Statistics). However, during the application of herbicides, only a small amount of herbicides can eliminate weeds, while nearly 60% to 80% of herbicides will enter the water ecosystem through spray drift, soil leaching, rainfall erosion, surface runoff and other pathways. Because herbicides have a disinfecting and toxic effect on some plants, herbicides entering water bodies will not only pollute the water environment, but also have potential toxic effects on non-target organisms. Therefore, the large-scale application of herbicides can avoid weed damage in farmland and increase crop yields, but it will also bring a series of environmental safety issues.

[0003] In aquatic ecosystems, microalgae, as a type of aquatic plants, are at the forefront of the aquatic food chain and are the most important primary producers and energy converters in aquatic ecosystems. Microalgae can provide material and energy sources for higher aquatic organisms such as planktonic invertebrates and fish through photosynthesis. Therefore, microalgae play a vital role in aquatic ecosystems, and their photosynthesis plays an important role in maintaining the stability of the structure and function of aquatic ecosystems. However, due to the small size of microalgae cells, algae cells can directly contact herbicides and are extremely sensitive to herbicide toxicity. Therefore, in aquatic ecosystems, microalgae are the most susceptible to the poisoning of residual herbicides in water. Photosynthesis, as an extremely important physiological process of microalgae, is easily affected by herbicides in water bodies. Many studies have shown that herbicides can inhibit the photosynthesis of microalgae by inhibiting the absorption of light energy, the conversion of excitation energy and the transfer of photosynthetic electrons, which will seriously affect and threaten the normal physiological and growth processes of microalgae and even the normal structure and function of aquatic ecosystems. Therefore, accurate and quantitative detection of the toxicity of herbicides in water based on the photosynthetic inhibition effect of microalgae is of great significance for ensuring the safety of water environment quality and accurately assessing the risks of aquatic ecological environment.

[0004] As a simple, rapid, and non-destructive in vivo fluorescence detection technology, chlorophyll fluorescence induction kinetics has become an important technology for studying the photosynthetic process of living plants and for rapid analysis of photosynthetic status because it can quickly and easily obtain the rapid chlorophyll fluorescence induction kinetic curve (OJIP curve) and various photosynthetic fluorescence parameters that characterize the photosynthetic status of plants. Based on this, chlorophyll fluorescence induction kinetics has also become a very useful tool for the toxicity detection of water pollutants such as herbicides based on the photosynthetic inhibition effect of microalgae. However, at present, the application of chlorophyll fluorescence induction kinetics technology in herbicide toxicity analysis and detection is mainly based on Fo (initial fluorescence of OJIP curve), Fm (maximum fluorescence of OJIP curve), Fv (the change between maximum fluorescence Fm and initial fluorescence Fo in OJIP curve), Fv / Fm (maximum photochemical quantum yield of photosystem II), Fv / Fo (potential photochemical efficiency of photosystem II) (Wang Z, Sun X, Ru S, et al. Science of the Total Environment, 2022, 807: 150609; Yang L, Li H, Zhang Y. Environment International, 2019, 133: 105175; Majewska M, Harshkova D, M, et al. Chemosphere, 2018, 209: 989-997; Deblois CP, Dufresne K, Juneau P. Aquatic Toxicology, 2013, 126: 77-84.) These commonly used photosynthetic fluorescence parameters are used as response indicators of herbicide toxicity to evaluate the degree of influence of herbicides on microalgae photosynthesis, and then judge the toxicity of herbicides. However, for chlorophyll fluorescence induction kinetics technology, the measured OJIP curve is the most direct and intuitive information to fully reflect the photosynthetic state of plants. The overall information of the OJIP curve is the key to truly and accurately reflect the degree of influence of herbicides on microalgae photosynthesis and judge the toxicity of pollutants. In addition, Chen et al. also showed that under herbicide stress, the fluorescence intensity of the J item in the algae OJIP curve increased significantly, resulting in a significant difference in the entire OJIP curve compared with the blank control sample (Chen S, Xu X, Dai X, et al. Biochimica et Biophysica Acta-Bioenergetics, 2007, 1767: 306-318.). However, the photosynthetic fluorescence parameters Fo, Fm, Fv, Fv / Fm or Fv / Fo only correspond to single-point or two-point information (such as maximum fluorescence intensity information or minimum fluorescence intensity information) in the microalgae OJIP curve. Using these commonly used photosynthetic fluorescence parameters as response indicators of herbicide toxicity cannot fully and accurately reflect the toxic effects of herbicides on the degree of damage to the photosynthetic reaction center of microalgae and the degree of inhibition of photosynthetic energy flow and electron transfer processes. Therefore, the current use of these commonly used photosynthetic fluorescence parameters as toxicity response indicators makes chlorophyll fluorescence induction kinetics technology have certain limitations in the accurate quantitative analysis and detection of herbicide toxicity, and often cannot truly reflect the toxicity of the herbicide.

[0005] Therefore, in response to the above problems, a quantitative analysis method for herbicide toxicity based on the full curve characteristics of microalgae OJIP was established, which is of great importance and significance for accurately judging the toxicity of herbicides residual in water and accurately evaluating the risks of herbicides to aquatic ecosystems. Summary of the invention

[0006] In view of the problems existing in the above-mentioned technology, the purpose of the present invention is to provide a method for quantitative analysis of herbicide toxicity based on the full curve characteristics of microalgae OJIP. The method uses microalgae, which are the main primary producers in aquatic ecosystems, as test organisms. Based on the inhibitory effect of herbicide toxicity on microalgae photosynthesis, the full curve characteristics of the OJIP curve characterizing the photosynthetic state of microalgae obtained by chlorophyll fluorescence induced kinetics technology are used to construct a herbicide toxicity response index, so as to achieve a comprehensive reflection of the impact of herbicide toxicity on microalgae photosynthesis. By establishing a dose-effect quantitative relationship curve between the inhibition rate of the toxicity response index and the herbicide concentration under the stress of different concentrations of herbicides, the 50% effect concentration value of the herbicide is obtained, so as to accurately judge the toxicity of different herbicides, thereby solving the problem in the prior art that the toxicity of herbicides is judged as a one-sided and inaccurate response index using only photosynthetic fluorescence parameters such as Fv / Fm obtained from single-point or two-point information in the OJIP curve as response indicators.

[0007] The technical solution of the present invention is: a method for quantitative analysis of herbicide toxicity based on the full curve characteristics of microalgae OJIP, which specifically includes the following steps:

[0008] Step 1, conducting an exposure experiment of microalgae to different concentrations of herbicides;

[0009] Step 2, measuring the rapid chlorophyll fluorescence induction kinetics OJIP curve of the microalgae sample to be tested;

[0010] Step 3: Extract the fluorescence intensity information Fo, F of the O, J, I and P items in the OJIP curve of the microalgae sample to be tested. J 、F I 、F P ;

[0011] Step 4: Calculate the fluorescence intensity change information Fv between the J term and the O term in the OJIP curve (J-O) , the information of the change of fluorescence intensity between item I and item J Fv (I-J) , the fluorescence intensity change information Fv between P and I (P-I) ;

[0012] Step 5, constructing the herbicide toxicity response index RI based on the full curve characteristics of the OJIP curve;

[0013] Step 6, establishing a dose-effect quantitative relationship curve between the herbicide concentration and the toxic response index RI inhibition rate;

[0014] Step 7, calculating the 50% effect concentration value of the herbicide to characterize the toxicity of the herbicide.

[0015] Beneficial effects:

[0016] The present invention establishes a method for quantitative analysis of herbicide toxicity based on the full curve characteristics of microalgae OJIP. The method is based on the microalgae rapid chlorophyll fluorescence induced kinetics OJIP curve, and uses Fo, Fv, and Fv representing the full curve characteristics of the OJIP curve. (J-O) 、Fv (I-J) 、Fv (P-I) The key elements are variables that jointly construct the response index of herbicide toxicity, thereby establishing a dose-effect quantitative relationship curve between the herbicide concentration and the inhibition rate of the toxicity response index, and obtaining the 50% effect concentration value of the herbicide through the curve, so as to achieve accurate quantitative analysis and detection of herbicide toxicity based on the photosynthetic inhibition effect of microalgae. The method established by the invention effectively solves the problem in the prior art that the single point or two-point information in the OJIP curve as the toxicity response index cannot fully and accurately reflect the toxicity of the herbicide, and the method is simple and fast, which can meet the needs of rapid on-site detection of herbicide toxicity and provide a methodological basis for water quality safety management and water ecological risk assessment. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 : Flow chart of the quantitative analysis method of herbicide toxicity based on the full curve characteristics of microalgae OJIP of the present invention;

[0018] Figure 2 : OJIP curves of Chlorella pyrenoidosa samples and their blank control samples under different concentrations of atrazine stress;

[0019] Figure 3 : OJIP curves of Chlorella pyrenoidosa samples and their blank control samples under different concentrations of terbuthylazine stress;

[0020] Figure 4 : The dose-effect quantitative relationship curve between atrazine concentration and toxic response index RI inhibition rate;

[0021] Figure 5 : The dose-effect quantitative relationship curve between terbuthylazine concentration and toxic response index RI inhibition rate. DETAILED DESCRIPTION

[0022] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the protection scope of the present invention.

[0023] According to an embodiment of the present invention, a method for quantitative analysis of herbicide toxicity based on the full curve characteristics of microalgae OJIP is proposed, which is characterized by comprising the following steps:

[0024] Step 1: Conduct an exposure experiment of different concentrations of herbicides on microalgae: In the exposure experiment, the same volume V 1 A series of herbicides with different concentrations were added to a series of equal volumes of V 2 The microalgae samples under different concentrations of herbicide stress were obtained from the microalgae suspension of volume V 1 Add deionized water to a volume of V 2 A blank control sample of microalgae under non-herbicide stress was obtained from the microalgae suspension of 10 μg / ml. For each microalgae sample under herbicide stress and the blank control sample, the final volume of the sample and the microalgae cell density in the sample were the same.

[0025] Step 2: Measuring the rapid chlorophyll fluorescence induction kinetics OJIP curve of the microalgae sample to be tested: When the exposure time is t, the rapid chlorophyll fluorescence induction kinetics OJIP curves of the microalgae samples under the stress of different concentrations of herbicides and the blank control sample are measured respectively to obtain the rapid chlorophyll fluorescence induction kinetics OJIP curves of the chlorophyll fluorescence intensity of the microalgae samples under the stress of different concentrations of herbicides changing with time and the rapid chlorophyll fluorescence induction kinetics OJIP curves of the chlorophyll fluorescence intensity of the blank control sample changing with time.

[0026] Step 3: Extract the fluorescence intensity information Fo, F of the O, J, I and P items in the OJIP curve of the microalgae sample to be tested J 、F I 、F P :The OJIP curve of the rapid chlorophyll fluorescence induction kinetics of microalgae shows four obvious fluorescence transient states: O term, J term, I term and P term. Among them, the O term is the initial term of the microalgae OJIP curve, that is, the point in the OJIP curve corresponding to the initial recording of the fluorescence intensity. This point is the lowest point of the fluorescence intensity in the OJIP curve, and the corresponding fluorescence intensity is Fo, that is, Fo is the fluorescence intensity corresponding to the first time point 20μs in the measured OJIP curve; the J term is the first platform that appears at 2ms in the process of the fluorescence intensity gradually increasing over time in the microalgae OJIP curve, and the corresponding fluorescence intensity F J is the fluorescence intensity value at 2ms in the OJIP curve; I is the second platform that appears at 30ms in the process of the fluorescence intensity gradually increasing over time in the microalgae OJIP curve, and the corresponding fluorescence intensity F I is the fluorescence intensity value at 30ms in the OJIP curve; P is the point corresponding to the maximum fluorescence intensity value in the microalgae OJIP curve, and the corresponding fluorescence intensity value F P is the maximum fluorescence intensity value of the OJIP curve.

[0027] Step 4: Calculate the fluorescence intensity change information Fv between the J term and the O term in the OJIP curve(J-O) , the information of the change of fluorescence intensity between item I and item J Fv (I-J) , the fluorescence intensity change information Fv between P and I (P-I) :According to the Fo and F in the OJIP curve of each microalgae sample J 、F I 、F P The four fluorescence intensity information are calculated according to the following formulas (1), (2) and (3) respectively: the change information Fv of the fluorescence intensity between the J item and the O item in the corresponding OJIP curve (J-O) , the information of the change of fluorescence intensity between item I and item J Fv (I-J) , the fluorescence intensity change information Fv between P and I (P-I) :

[0028] F (J-O) =F J -F O (1)

[0029] F (I-J) =F I -F J (2)

[0030] F (P-I) =F P -F I (3)

[0031] Step 5: Construct the herbicide toxicity response index RI based on the full curve characteristics of the OJIP curve: O 、Fv (J-O) 、Fv (I-J) 、Fv (P-I) These key elements representing the characteristics of the full curve of the OJIP curve are variables. (J-O) 、Fv (I-J) 、Fv (P-I) According to the changing law of herbicide concentration, the response index RI that characterizes the toxicity of herbicides is constructed using formula (4):

[0032]

[0033] Step 6: Establish a dose-effect quantitative relationship curve between herbicide concentration and toxicity response index RI inhibition rate: Calculate the inhibition rate Ir of the microalgae toxicity response index RI under different concentrations of herbicide stress according to the following formula (5):

[0034]

[0035] In the formula, RI 0is the toxicity response index value of the blank control sample under non-stress, RI is the toxicity response index value of the microalgae sample under herbicide stress, and Ir is the toxicity response index RI of the microalgae under herbicide stress relative to the toxicity response index RI of the blank control sample 0 The Logistic model was used to establish the dose-effect quantitative relationship curve between the herbicide concentration and the toxic response index RI inhibition rate under different herbicide concentrations.

[0036] Step 7: Calculate the 50% effect concentration value of the herbicide to characterize the toxicity of the herbicide: Based on the established dose-effect quantitative relationship curve between the herbicide concentration and the toxicity response index RI inhibition rate, calculate the herbicide concentration value corresponding to the toxicity response index RI inhibition rate of 50%. This concentration value is the 50% effect concentration value of the herbicide, and this value is used to characterize the toxicity of the corresponding herbicide.

[0037] According to a specific embodiment, the herbicides atrazine and terbuthylazine are used as the analysis objects, and the green algae Chlorella pyrenoidosa in freshwater microalgae is used as the test organism to carry out the quantitative toxicity analysis of the two herbicides atrazine and terbuthylazine based on the full curve characteristics of the microalga Chlorella pyrenoidosa OJIP. Figure 1 As shown, the specific steps include:

[0038] Step 1: Conduct an exposure experiment of different concentrations of herbicides on microalgae: In the exposure experiment, the same volume V 1 A series of herbicides with different concentrations were added to a series of equal volumes of V 2 The microalgae samples under different concentrations of herbicide stress were obtained from the microalgae suspension of volume V 1 Add deionized water to a volume of V 2 A blank control sample of microalgae under non-herbicide stress was obtained from the microalgae suspension of 10 μg / ml. For each microalgae sample under herbicide stress and the blank control sample, the final volume of the sample and the microalgae cell density in the sample were the same.

[0039] In this example, an exposure experiment of different concentrations of atrazine herbicide and different concentrations of terbuthylazine herbicide on microalgae Chlorella vulgaris was carried out: in the exposure experiment, a series of atrazine solutions and terbuthylazine solutions with different concentrations in volume of 1 mL were added to a series of 49 mL Chlorella vulgaris suspensions, respectively, to obtain Chlorella vulgaris samples under different concentrations of atrazine and terbuthylazine stress, wherein the concentrations of atrazine in the Chlorella vulgaris samples under atrazine stress were 1, 2, 5, 7.5, 10, 20, 30, 40, and 60 μg / L, respectively, and the concentrations of terbuthylazine in the Chlorella vulgaris samples under terbuthylazine stress were 0.5, 1, 2, 5, 10, 20, 30, 40, and 60 μg / L, respectively; similarly, a volume of 1 mL of deionized water was added to a volume of 49 mL of Chlorella vulgaris suspension to obtain a blank control sample of Chlorella vulgaris under non-herbicide stress. For each sample of Chlorella vulgaris under herbicide stress and the blank control sample, the final volume of the sample was 50 mL, and the cell density of the microalgae in the sample was 5×10 5 cells / mL.

[0040] Step 2: Measuring the rapid chlorophyll fluorescence induction kinetics OJIP curve of the microalgae sample to be tested: When the exposure time is t, the rapid chlorophyll fluorescence induction kinetics OJIP curves of the microalgae samples under the stress of different concentrations of herbicides and the blank control sample are measured respectively to obtain the rapid chlorophyll fluorescence induction kinetics OJIP curves of the chlorophyll fluorescence intensity of the microalgae samples under the stress of different concentrations of herbicides changing with time and the rapid chlorophyll fluorescence induction kinetics OJIP curves of the chlorophyll fluorescence intensity of the blank control sample changing with time.

[0041] In this embodiment, when the exposure time is 1 h, the AquaPenAP110 / C handheld algae fluorescence measuring instrument (PSI, Czech Republic) is used to measure the rapid chlorophyll fluorescence induction kinetics OJIP curves of the pyrenoid Chlorella samples under different concentrations of atrazine and different concentrations of terbuthylazine stress and the blank control samples. The measured rapid chlorophyll fluorescence induction kinetics OJIP curves of the pyrenoid Chlorella samples under different concentrations of atrazine stress and the blank control samples are shown in Figure 2. Figure 2 As shown in the figure, the OJIP curves of the fast chlorophyll fluorescence induction kinetics of the Chlorella vulgaris samples and the blank control samples under different concentrations of terbuthylazine stress are shown in Figure 3 shown.

[0042] Step 3: Extract the fluorescence intensity information Fo, F of the O, J, I and P items in the OJIP curve of the microalgae sample to be tested J 、F I 、F P:The OJIP curve of the rapid chlorophyll fluorescence induction kinetics of microalgae shows four obvious fluorescence transient states: O term, J term, I term and P term. Among them, the O term is the initial term of the microalgae OJIP curve, that is, the point in the OJIP curve corresponding to the initial recording of the fluorescence intensity. This point is the lowest point of the fluorescence intensity in the OJIP curve, and the corresponding fluorescence intensity is Fo, that is, Fo is the fluorescence intensity corresponding to the first time point 20μs in the measured OJIP curve; the J term is the first platform that appears at 2ms in the process of the fluorescence intensity gradually increasing over time in the microalgae OJIP curve, and the corresponding fluorescence intensity F J is the fluorescence intensity value at 2ms in the OJIP curve; I is the second platform that appears at 30ms in the process of the fluorescence intensity gradually increasing over time in the microalgae OJIP curve, and the corresponding fluorescence intensity F I is the fluorescence intensity value at 30ms in the OJIP curve; P is the point corresponding to the maximum fluorescence intensity value in the microalgae OJIP curve, and the corresponding fluorescence intensity value F P is the maximum fluorescence intensity value of the OJIP curve.

[0043] In this example, the fluorescence intensity information Fo, F of the O, J, I and P items in the OJIP curves of the Chlorella pyrenoidosa samples and their blank control samples under different concentrations of atrazine stress were J 、F I 、F P As shown in Table 1, the fluorescence intensity information F of O, J, I and P in the OJIP curve of Chlorella pyrenoidosa samples and their blank control samples under different concentrations of terbuthylazine stress O 、F J 、F I 、F P As shown in Table 2 below.

[0044] Table 1 F in OJIP curves of Chlorella pyrenoidosa samples under different concentrations of atrazine stress O 、F J 、F I 、F P Fluorescence intensity

[0045]

[0046] Table 2 F in OJIP curve of Chlorella pyrenoidosa samples under different concentrations of terbuthylazine stress O 、F J 、F I 、F P Fluorescence intensity

[0047]

[0048] Step 4: Calculate the fluorescence intensity change information Fv between the J term and the O term in the OJIP curve (J-O) , the information of the change of fluorescence intensity between item I and item J Fv (I-J) , the fluorescence intensity change information Fv between P and I (P-I) Fluorescence information: According to the F in the OJIP curve of each microalgae sample O 、F J 、F I 、F P The four fluorescence intensity information are calculated according to the following formulas (1), (2) and (3) respectively: the change information Fv of the fluorescence intensity between the J item and the O item in the corresponding OJIP curve (J-O) , the information of the change of fluorescence intensity between item I and item J Fv (I-J) , the fluorescence intensity change information Fv between P and I (P-I) :

[0049] F (J-O) =F J -F O (1)

[0050] F (I-J) =F I -F J (2)

[0051] F (P-I) =F P -F I (3)

[0052] In this embodiment, according to F in the OJIP curve O 、F J 、F I 、F P Four fluorescence intensity information and formulas (1), (2), (3) calculated the Fv in the OJIP curve of Chlorella vulgaris samples and blank control samples under different concentrations of atrazine stress (J-O) 、Fv (I-J) 、Fv (P-I) The fluorescence information is shown in Table 3. The Fv in the OJIP curve of Chlorella vulgaris samples and blank control samples under different concentrations of terbuthylazine stress (J-O) 、Fv (I-J) 、Fv (P-I) The fluorescence information is shown in Table 4.

[0053] Table 3 Fv in OJIP curves of Chlorella pyrenoidosa samples under different concentrations of atrazine stress (J-O) 、Fv (I-J) 、Fv (P-I) Fluorescence information

[0054]

[0055] Table 4 Fv in OJIP curves of Chlorella pyrenoidosa samples under different concentrations of terbuthylazine stress (J-O) 、Fv (I-J) 、Fv (P-I) Fluorescence information

[0056]

[0057]

[0058] Step 5: Construct the herbicide toxicity response index RI based on the full curve characteristics of the OJIP curve: Fo, Fv (J-O) 、Fv (I-J) 、Fv (P-I) These key elements representing the characteristics of the full curve of the OJIP curve are variables. (J-O) 、Fv (I-J) 、Fv (P-I) According to the changing law of herbicide concentration, the response index RI that characterizes the toxicity of herbicides is constructed using formula (4):

[0059]

[0060] In this embodiment, the toxicity response index RI of Chlorella pyrenoidosa and its blank control samples under different concentrations of atrazine stress calculated by formula (4) is shown in Table 5, and the toxicity response index RI of Chlorella pyrenoidosa and its blank control samples under different concentrations of terbuthylazine stress is shown in Table 6.

[0061] Table 5 Toxicity response indexes of Chlorella pyrenoidosa under different concentrations of atrazine stress

[0062]

[0063] Table 6 Toxicity response indexes of Chlorella pyrenoidosa under different concentrations of terbuthylazine stress

[0064]

[0065] Step 6: Establish a dose-effect quantitative relationship curve between herbicide concentration and toxicity response index RI inhibition rate: Calculate the inhibition rate Ir of the microalgae toxicity response index RI under different concentrations of herbicide stress according to the following formula (5):

[0066]

[0067] In the formula, RI 0is the toxicity response index value of the blank control sample under non-stress, RI is the toxicity response index value of the microalgae sample under herbicide stress, and Ir is the toxicity response index RI of the microalgae under herbicide stress relative to the toxicity response index RI of the blank control sample 0 The Logistic model was used to establish the dose-effect quantitative relationship curve between the herbicide concentration and the toxic response index RI inhibition rate under different herbicide concentrations.

[0068] In this embodiment, the inhibition rate of the toxicity response index RI of Chlorella pyrenoidosa under the stress of different concentrations of atrazine was calculated according to formula (5), and the inhibition rate of the toxicity response index RI of Chlorella pyrenoidosa under the stress of different concentrations of terbuthylazine was calculated. The dose-effect quantitative relationship curve between the atrazine concentration and the toxicity response index RI inhibition rate under the stress of different concentrations of atrazine herbicide was established by using the Logistic model. The results are as follows: Figure 4 As shown in Figure 2, the Logistic model was also used to establish the dose-effect quantitative relationship curve between the concentration of terbuthylazine and the toxic response index RI inhibition rate under the stress of different concentrations of terbuthylazine herbicide. The results are shown in Figure 2. Figure 5 shown.

[0069] Step 7: Calculate the 50% effect concentration value of the herbicide to characterize the toxicity of the herbicide: Based on the established dose-effect quantitative relationship curve between the herbicide concentration and the toxicity response index RI inhibition rate, calculate the herbicide concentration value corresponding to the toxicity response index RI inhibition rate of 50%. This concentration value is the 50% effect concentration value of the herbicide, and this value is used to characterize the toxicity of the corresponding herbicide.

[0070] In this embodiment, according to the established dose-effect quantitative relationship curve between atrazine concentration and toxicity response index RI inhibition rate, the calculated 50% effect concentration of atrazine herbicide when the toxicity response index RI inhibition rate is 50% is 11.743 μg / L, and according to the established dose-effect quantitative relationship curve between terbuthylazine concentration and toxicity response index RI inhibition rate, the calculated 50% effect concentration of terbuthylazine herbicide when the toxicity response index RI inhibition rate is 50% is 11.098 μg / L. Therefore, by comparison, for Chlorella pyrenoidosa, the toxicity intensity of terbuthylazine herbicide is higher than that of atrazine herbicide.

Claims

1. A quantitative analysis method for herbicide toxicity based on the full curve characteristics of microalgae OJIP, characterized in that: The specific steps include: Step 1, conducting an exposure experiment of microalgae to different concentrations of herbicides; Step 2, measuring the rapid chlorophyll fluorescence induction kinetics OJIP curve of the microalgae sample to be tested; Step 3: Extract the fluorescence intensity information Fo, F of the O, J, I and P items in the OJIP curve of the microalgae sample to be tested. J 、F I 、F P ; Step 4: Calculate the fluorescence intensity change information Fv between the J term and the O term in the OJIP curve (J-O) , the fluorescence intensity change information Fv between item I and item J (I-J) , the fluorescence intensity change information Fv between P and I (P-I) ; Step 5, constructing the herbicide toxicity response index RI based on the full curve characteristics of the OJIP curve; Step 6, establishing a dose-effect quantitative relationship curve between the herbicide concentration and the toxic response index RI inhibition rate; Step 7, calculating the 50% effect concentration value of the herbicide to characterize the toxicity of the herbicide; The step 4 specifically includes: According to the Fo and F in the OJIP curve of each microalgae sample J 、F I 、F P The four fluorescence intensity information are calculated according to the following formulas (1), (2), and (3) to calculate the fluorescence intensity change information Fv between the J item and the O item in the corresponding OJIP curve: (J-O) , the fluorescence intensity change information Fv between item I and item J (I-J) , the fluorescence intensity change information Fv between P and I (P-I) : (1) (2) (3) The step 5 specifically includes: Fo、Fv (J-O) 、Fv (I-J) 、Fv (P-I) These key elements representing the characteristics of the full curve of the OJIP curve are variables. (J-O) 、Fv (I-J) 、Fv (P-I) According to the changing law of herbicide concentration, the response index RI that characterizes the toxicity of herbicides is constructed using formula (4): (4) The step 6 specifically includes: The inhibition rate Ir of the microalgae toxicity response index RI under different concentrations of herbicide stress was calculated according to the following formula (5): (5) Wherein, RI0 is the toxicity response index value of the blank control sample under non-stress, RI is the toxicity response index value of the microalgae sample under herbicide stress, Ir is the inhibition rate of the microalgae toxicity response index RI under herbicide stress relative to the toxicity response index RI0 of the blank control sample. The Logistic model was used to establish the dose-effect quantitative relationship curve between the herbicide concentration and the toxicity response index RI inhibition rate under different herbicide concentrations.

2. A method for quantitative analysis of herbicide toxicity based on the full curve characteristics of microalgae OJIP according to claim 1, characterized in that: The step 1 is to conduct an exposure experiment of microalgae to herbicides of different concentrations, specifically comprising: in the exposure experiment, a series of herbicides of the same volume V1 but different concentrations are respectively added to a series of equal volumes of microalgae suspensions of volume V2 to obtain microalgae samples under the stress of herbicides of different concentrations; similarly, deionized water of volume V1 is added to the microalgae suspension of volume V2 to obtain a blank control sample of microalgae under non-herbicide stress.

3. The method for quantitative analysis of herbicide toxicity based on the full curve characteristics of microalgae OJIP according to claim 1, characterized in that: The step 2 specifically includes: When the exposure time is t, the rapid chlorophyll fluorescence induction kinetics (OJIP) curves of the microalgae samples under different concentrations of herbicide stress and the blank control samples are measured respectively to obtain the rapid chlorophyll fluorescence induction kinetics (OJIP) curves of the chlorophyll fluorescence intensity of the microalgae samples under different concentrations of herbicide stress changing with time and the rapid chlorophyll fluorescence induction kinetics (OJIP) curves of the chlorophyll fluorescence intensity of the blank control samples changing with time.

4. The method for quantitative analysis of herbicide toxicity based on the full curve characteristics of microalgae OJIP according to claim 1, characterized in that: The step 3 specifically includes: The OJIP curve of the microalgae rapid chlorophyll fluorescence induced dynamics shows four obvious fluorescence transients, namely, the O term, J term, I term and P term. The O term is the initial term of the microalgae OJIP curve, that is, the point in the OJIP curve corresponding to the initial recording of the fluorescence intensity. This point is the lowest point of the fluorescence intensity in the OJIP curve, and the corresponding fluorescence intensity is Fo, that is, Fo is the fluorescence intensity corresponding to the first time point 20μs in the measured OJIP curve; the J term is the first platform that appears at 2ms in the process of the fluorescence intensity gradually increasing over time in the microalgae OJIP curve, and the corresponding fluorescence intensity F J is the fluorescence intensity value at 2ms in the OJIP curve; I is the second platform that appears at 30ms in the process of the fluorescence intensity gradually increasing over time in the microalgae OJIP curve, and the corresponding fluorescence intensity F I is the fluorescence intensity value at 30ms in the OJIP curve; P is the point corresponding to the maximum fluorescence intensity value in the microalgae OJIP curve, and the corresponding fluorescence intensity value F P is the maximum fluorescence intensity value of the OJIP curve.

5. The method for quantitative analysis of herbicide toxicity based on the full curve characteristics of microalgae OJIP according to claim 1, characterized in that: The step 7 specifically includes: Based on the established dose-effect quantitative relationship curve between herbicide concentration and toxicity response index RI inhibition rate, the herbicide concentration value corresponding to a toxicity response index RI inhibition rate of 50% is calculated. This concentration value is the 50% effect concentration value of the herbicide, and this value is used to characterize the toxicity of the corresponding herbicide.

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