Method for fluorescence detection of ametryn biotoxicity by using oxyspora clostridium and application

Abstract

The invention belongs to the technical field of organic pesticide biotoxicity detection, and particularly relates to a method for fluorescence detection of ametryn biotoxicity by using oxyspora clostridium and application. According to the method, Anfule is used as a sensitizer, oxyspora clostridium is used as a sensitive indicator organism, and chlorophyll fluorescence kinetic parameters of the oxyspora clostridium are used for detecting the biotoxicity of ametryn. The main components of Anfule are lecithin and vitamin E. The method can rapidly and sensitively detect the ametryn biotoxicity, solves the problems of long determination period and low sensitivity of the ametryn biotoxicity detection method in the prior art, and has important significance for realizing timely early warning and risk assessment of ametryn environmental pollution.

Description

Technical Field

[0001] This invention belongs to the field of organic pesticide biotoxicity detection technology, specifically relating to a method and application of fluorescence detection of atrazine biotoxicity by *Acetobacter spp.* Background Technology

[0002] Ametryn is a triazine selective herbicide with the chemical name N-2-ethylamino-N-4-isopropylamino-6-methylthio-1,3,5-triazine and the molecular formula C9H10⁻¹⁰. 17 Atrazine (N5S) is a systemic herbicide that can be absorbed by the roots, stems, and leaves of plants. It works by inhibiting photosynthesis, thereby eliminating weeds. Atrazine is widely used in crops such as sugarcane, corn, pineapple, and bananas to control annual broadleaf and grassy weeds. It is absorbed through the roots, stems, and leaves and translocated apically throughout the entire plant. However, the extensive use of atrazine not only leads to pesticide residues in crops and soil but also pollutes surface and groundwater through farmland runoff and leaching. Furthermore, it accumulates in the human body through the food chain, causing irritation to the skin, eyes, and respiratory tract, posing a potential threat to human health. Therefore, studying the residue levels of this herbicide in environmental media is of great significance for agricultural product safety and water environment monitoring, and is beneficial for assessing and managing the ecological risks of atrazine in the environment.

[0003] The widespread use of atrazine has caused significant water and soil pollution. Current detection methods for atrazine include immunoassay, spectrophotometry, gas chromatography (GC), high-performance liquid chromatography (HPLC), gas chromatography-mass spectrometry (GC-MS), and liquid chromatography-tandem mass spectrometry (LC-MS / MS), with HPLC, GC-MS, and LC-MS / MS being the most commonly used. However, these methods typically suffer from limitations such as complex pretreatment steps, high consumption of organic solvents, long analysis cycles, high instrument operating costs, and expensive equipment. Furthermore, traditional physicochemical detection methods can only achieve quantitative analysis of the target analyte and cannot comprehensively reflect the biotoxic effects of pollutants on ecosystems. With the development of environmental monitoring needs, the application of biotoxicity detection methods is becoming increasingly widespread.

[0004] Currently, biotoxicity-based detection technologies have become a comprehensive and rapid screening method for assessing herbicides. They can intuitively and comprehensively reflect the toxicity of pollutants to biological populations and their comprehensive impact on the environment, and are gradually developing into an important technical support for pollutant monitoring and early warning systems. Methods for detecting herbicide biotoxicity mainly include the Daphnia acute toxicity method, the fish acute toxicity method, the luminescent bacteria inhibition method, and the algal growth inhibition method. Among these, the algal growth inhibition method uses microalgae as experimental organisms. Due to the advantages of algae—small size, simple structure, wide distribution, rapid reproduction, ease of cultivation, and direct response to toxicity—it exhibits high accuracy and stability. However, this method also has drawbacks such as a relatively long testing cycle and insufficient sensitivity to trace pollutants. Summary of the Invention

[0005] In view of this, the present invention provides a method and application for fluorescence detection of atrazine biotoxicity by *Periflora cephalothorax*. This method can rapidly and sensitively detect the biotoxicity of atrazine, which is of great significance for timely early warning and risk assessment of atrazine environmental pollution.

[0006] To address the above technical problems, this invention provides a method for detecting the biotoxicity of atrazine using fluorescence from *Anatomium spp.* The method uses Anrongle as a sensitizer and *Anatomium spp.* as a sensitive indicator organism, utilizing the chlorophyll fluorescence kinetic parameters of *Anatomium spp.* to detect the biotoxicity of atrazine; the main components of Anrongle are lecithin and vitamin E.

[0007] This invention uses Anrongle as a sensitizer and *Anatomalacia spicata* as a sensitive indicator organism to detect the biotoxicity of atrazine in the presence of Anrongle. The lecithin component in Anrongle forms biological micelles, efficiently encapsulating and solubilizing atrazine molecules, ensuring uniform dispersion in the algal solution and significantly increasing the contact efficiency between atrazine and algal cells. Simultaneously, lecithin reversibly enhances the permeability of algal cell membranes, promoting the transport and accumulation of atrazine towards photosystem II targets. The vitamin E component in Anrongle, by disrupting the algal cell's antioxidant defense system, synergizes with the inhibitory effect of atrazine on photosystem II, exacerbating damage to the photosynthetic electron transport chain and promoting the explosive accumulation of reactive oxygen species, thereby significantly amplifying irreversible damage to the photosynthetic system and improving the sensitivity of atrazine biotoxicity detection.

[0008] In conjunction with the first aspect, the method for detecting the biotoxicity of atrazine by fluorescence in *Phyllostachys nigra* includes the following steps: mixing *Phyllostachys nigra* algal solution, atrazine standard solutions of various concentrations, and Anrongle solution at a volume ratio of 1:1~2:1~2, placing the mixture in the dark for 9~11 min, and measuring the chlorophyll fluorescence kinetic parameters of *Phyllostachys nigra* in the mixture to detect the biotoxicity of atrazine; mixing *Phyllostachys nigra* algal solution, a water sample containing atrazine, and Anrongle solution at a volume ratio of 1:1~2:1~2, placing the mixture in the dark for 10 min, and measuring the chlorophyll fluorescence kinetic parameters of *Phyllostachys nigra* in the mixture to detect the biotoxicity of atrazine; simultaneously, using culture medium instead of atrazine standard solution as a blank control group.

[0009] In conjunction with the first aspect, the OD of the algal solution of the *Nyctaginosa* species... 680 The value is 0.8~1.2, obtained by inoculating *Neptunia capillaris* into the culture medium and culturing it in a constant temperature and light incubator to the logarithmic phase.

[0010] Preferably, the OD of the algal solution of the near-capital-shaped algae is... 680 The value is 1.0, and the cell density range is selected as (1-1.2)×10⁻⁶. 6 The concentration of algal solution corresponding to *Neptune cephalopoda* at 1 ind / mL was used as the concentration of algae for detecting the biotoxicity of atrazine, and was used to determine the biotoxicity of atrazine.

[0011] In conjunction with the first aspect, the culture medium comprises the following components: NaNO3 1.4~1.6 g / L, K2HPO4·3H2O 0.03~0.05 g / L, MgSO4·7H2O 0.07~0.08 g / L, CaCl2·2H2O 0.03~0.04 g / L, citric acid 0.005~0.007 g / L, ferric ammonium citrate 0.005~0.007 g / L, EDTA 0.0005~0.0015 g / L, Na2CO3 0.015~0.025 g / L, H3BO4 0.002~0.003 g / L, MnCl2·H2O The concentrations of the following ingredients are specified: 0.001~0.002 g / L, ZnSO4·7H2O 0.00015~0.00025 g / L, Na2MoO4·2H2O 0.00035~0.00045 g / L, CuSO4·5H2O 0.000075~0.000085 g / L, and Co(NO3)2·6H2O 0.0000045~0.0000055 g / L; the pH of the culture medium is 7.1±0.1.

[0012] Preferably, the culture medium comprises the following components: NaNO3 1.5 g / L, K2HPO4·3H2O 0.04 g / L, MgSO4·7H2O 0.075 g / L, CaCl2·2H2O 0.036 g / L, citric acid 0.006 g / L, ferric ammonium citrate 0.006 g / L, EDTA 0.001 g / L, Na2CO3 0.02 g / L, H3BO4 0.00286 g / L, MnCl2·H2O 0.00181 g / L, ZnSO4·7H2O 0.00022 g / L, Na2MoO4·2H2O 0.00039 g / L, CuSO4·5H2O 0.000079 g / L, Co(NO3)2·6H2O 0.0000049 g / L; the pH of the culture medium is 7.1.

[0013] In conjunction with the first aspect, the culture conditions of the constant temperature and light incubator are as follows: light intensity 2000~2500 lx, temperature 25±2℃, humidity 75±5%RH, light-dark cycle of 12h:12h, and static culture.

[0014] The above-mentioned culture medium and culture conditions enable *Neptune cephalopoda* to grow and reproduce well and rapidly.

[0015] In conjunction with the first aspect, the concentrations of the series of atrazine standard solutions are 0 μg / L, 20 μg / L, 40 μg / L, 60 μg / L, 80 μg / L and 100 μg / L, respectively; the concentration of atrazine in the water sample to be tested is 5~95 μg / L.

[0016] In conjunction with the first aspect, the volume concentration of the Anrongle solution is 0.008~0.012%.

[0017] In conjunction with the first aspect, the chlorophyll fluorescence kinetic parameters include the maximum light energy conversion efficiency of photosystem II, the actual light energy conversion efficiency of photosystem II, the photosynthetic electron transfer efficiency, and the photochemical quenching coefficient.

[0018] The changes in the above chlorophyll fluorescence kinetic parameters can accurately and sensitively reflect the biotoxicity of atrazine, providing a reliable basis for assessing the biotoxicity of atrazine in water bodies contaminated with atrazine.

[0019] In conjunction with the first aspect, the chlorophyll fluorescence kinetic parameters were determined using a water sample chlorophyll fluorometer, with an excitation wavelength of 675~685nm, preferably 680nm.

[0020] The present invention also provides an application of the method for detecting the biotoxicity of atrazine by fluorescence detection of *Acetobacter spp.* in assessing the potential harm of organic pesticides to aquatic ecosystems, early warning of organic pesticide pollution, and precise ecological risk assessment, wherein the organic pesticide is atrazine, atrazine, simazine, cyanazine, or cypermethrin.

[0021] The beneficial effects of this invention are as follows: This invention introduces the pesticide adjuvant "Anrongle" into the detection system for the first time. Utilizing its unique penetration and conduction capabilities, it significantly enhances the accumulation efficiency of the target pollutant atrazine within *Periplaneta macrocarpa* cells, allowing more atrazine molecules to reach its target site—photosystem II—thus successfully amplifying the algal toxicity stress response signal. This invention successfully overcomes the key shortcomings of traditional algal toxicity detection methods, such as insensitive response and long measurement cycles, and constructs a rapid and highly sensitive biotoxicity detection technology. Attached Figure Description

[0022] Figure 1 The curve showing the effect of atrazine on the actual light energy conversion efficiency (Y(II)) of photosystem II of *Neptune cephalopoda* in Example 3 is shown. Figure 2 The curve showing the effect of atrazine on the photosynthetic electron transfer efficiency (ETR) of *Plasmodium septum* in Example 3; Figure 3 The curve showing the effect of atrazine on the photochemical quenching coefficient (qP) of *Plasmodium septemlobus* in Example 3; Figure 4 Example 3 shows the maximum light energy conversion efficiency (F) of atrazine against the photosystem II of *Neptune cephalopoda*. v / F m Influence curve; Figure 5 The curve showing the effect of atrazine on the actual light energy conversion efficiency (Y(II)) of photosystem II of *Plasmodium septemlobus* in Example 4 is shown. Figure 6 The curve showing the effect of atrazine on the photosynthetic electron transfer efficiency (ETR) of *Plasmodium septum* in Example 4; Figure 7 Example 4 shows the effect curve of atrazine on the photochemical quenching coefficient (qP) of *Plasmodium septemlobus*. Figure 8 Example 4 shows the maximum light energy conversion efficiency (F) of atrazine against the photosystem II of *Neptune cephalopoda*. v / F m Influence curve; Figure 9 The curve showing the effect of atrazine on the actual light energy conversion efficiency (Y(II)) of *Phyllostachys nigra* PSII in Example 5; Figure 10The curve showing the effect of atrazine on the photosynthetic electron transport efficiency (ETR) of *Plasmodium septum* in Example 5; Figure 11 The curve showing the effect of atrazine on the photochemical quenching coefficient (qP) of *Acetobacter septemlobus* in Example 5; Figure 12 Example 5 shows the maximum light energy conversion efficiency (F) of atrazine against the photosystem II of *Neptune cephalopoda*. v / F m Influence curve; Figure 13 The curves showing the effect of atrazine on the actual light energy conversion efficiency (Y(II)) of photosystem II of *Hydrocotyle septemlobus*, *Scenedesmus obliquus*, and *Chlorella proteoglycans* in Examples 1 and 1-2 are shown. Figure 14 The curves show the effects of adding Anrongle, sodium dodecylbenzenesulfonate, and Tween-80 to the actual light energy conversion efficiency (Y(II)) of optical system II in Example 1 and Comparative Examples 3-4, respectively. Detailed Implementation

[0023] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0024] It will be understood by those skilled in the art that, unless otherwise defined, all terms used herein (including technical and scientific terms) have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. It should also be understood that terms such as those defined in general dictionaries should be understood to have the meaning consistent with their meaning in the context of the prior art, and should not be interpreted in an idealized or overly formal sense unless specifically defined.

[0025] In recent years, biotoxicity detection methods have been widely used because they can intuitively and comprehensively reflect the ecological risks of pollutants. Among them, the algal growth inhibition method uses microalgae as experimental material and takes advantage of the high sensitivity of algae to herbicides for toxicity assessment. Although this method is accurate and reliable, it suffers from problems such as long measurement cycles and low sensitivity. Specifically, the traditional algal growth inhibition method requires several days to cultivate algae and assess toxicity through changes in biomass. Even rapid detection methods based on fluorescence principles may have high detection limits and insensitive responses when faced with low concentrations of atrazine pollution because the toxic stress signals from algae are weak, making it difficult to achieve early and rapid warnings for the aquatic environment. There are two main reasons for this problem: First, the algal cell wall acts as a natural barrier, limiting the penetration of low concentrations of atrazine molecules into the cell, resulting in weak early toxicity signals that are difficult to capture by traditional methods; second, traditional methods that rely on the algal growth cycle to observe toxic effects cannot achieve rapid response due to the biological processes themselves.

[0026] To address this issue, this invention cleverly introduces the pesticide adjuvant "Anrongle" as a biosensitizer and uses *Plasmodium spiculatum* as a sensitive indicator organism. By addressing various challenges encountered during implementation, a delicate balance is achieved between "sensitization effect" and "biocompatibility." This ensures that Anrongle effectively promotes the penetration of atrazine into the cell wall of *Plasmodium spiculatum*, while simultaneously guaranteeing that it does not cause additional toxic interference to the algal cells, thus preventing distortion of the detection signal.

[0027] Unless otherwise specified, the raw materials, reagents and equipment used in this invention are all conventional commercially available reagents and equipment.

[0028] The algal species used in this invention, *Fachapora cephalothorax* (FACHB-271), was purchased from the Wild Biobank—Freshwater Algae Stock Bank, Chinese Academy of Sciences. *Scenedesmus obliquus* and *Chlorella proteoglycans* were also purchased from the Wild Biobank—Freshwater Algae Stock Bank, Chinese Academy of Sciences. The instruments and models used were: a light incubator (MGC-450BP Shanghai Yiheng), a Water-PAM chlorophyll fluorescence spectrometer (Walz, Germany), a fluorescence microscope (Zeiss, Germany), and a DR6000 UV spectrophotometer. Anronol (effective concentration 3%) was purchased from Nilesco 882 Ltd., South Africa.

[0029] Example 1 This embodiment provides a method for culturing *Aeromonas spp.*: Under aseptic conditions, *Neptunia spicata* was inoculated into Erlenmeyer flasks containing autoclaved medium, the composition of which is shown in Table 1 (the prepared medium needs to be adjusted to pH 7.1 with 1M NaOH or 1M HCl solution). After inoculation, the flasks were placed in a constant temperature and light incubator and cultured until the logarithmic growth phase. The light intensity was set at 2000–2500 lx, the temperature at 25 ± 2℃, the humidity at 75 ± 5% RH, and the light-dark cycle at 12 h:12 h. The flasks were then statically cultured under these conditions, with the flasks shaken 2–3 times daily and the position of the Erlenmeyer flasks changed to ensure uniform light exposure. The flasks were cultured until the logarithmic growth phase was reached and then ready for use.

[0030] Table 1. Specific components of high-temperature and high-pressure sterilized culture medium

[0031] The specific components of the trace element solution used are shown in Table 2: Table 2 Specific components of trace element solution

[0032] Example 2 This embodiment provides a method for preparing the solution used in the fluorescence detection of atrazine biotoxicity by *Achnatherum spicata*: 1. Preparation of *Neptunus cephalosporium* algal solution Select OD 680 A concentration of *N. scutellaria* with a concentration of 1.0 was used as the algal concentration for detecting the biotoxicity of atrazine, at which point the cell density ranged from (1-1.2)×10⁻⁶. 6 The concentration of *Nepaliformis spicata* algal solution in 1 d / mL was used as the algal concentration for detecting atrazine toxicity and was used to determine atrazine toxicity. 2. Preparation of Atrazine Standard Solution To prepare a 20 mg / L atrazine stock solution: Take 1 mL of 100 mg / L atrazine standard solution into a 5 mL volumetric flask, dilute with deionized water to the mark, and obtain the atrazine stock solution. Preparation of 200 μg / L atrazine intermediate solution: Take 1.0 mL of atrazine stock solution into a 100 mL volumetric flask, and dilute to the mark with deionized water to obtain atrazine intermediate solution; Preparation of atrazine standard solutions: Take 1.0, 2.0, 3.0, 4.0 and 5.0 mL of atrazine intermediate solution into five 10 mL volumetric flasks, respectively, and dilute to the mark with deionized water to obtain atrazine standard solutions with concentrations of 20 μg / L, 40 μg / L, 60 μg / L, 80 μg / L and 100 μg / L.

[0033] 3. Preparation of Anrongle Solution Take 1.0 mL of Anrongle stock solution into a beaker, add an appropriate amount of deionized water, mix well with a stirrer, transfer the mixture to a 300 mL volumetric flask, and finally dilute to the mark. Mix well to obtain the Anrongle solution.

[0034] Example 3 This embodiment provides a method for detecting the biotoxicity of atrazine using fluorescence detection of *Periplaneta macrocarpa*, specifically including: Determination of the effect of atrazine on the fluorescence effect of *Acetobacter spp.* without the addition of Anrongle: During the logarithmic growth phase (OD) of *N. occulta* 680 =1.0) Take *Phyllostachys nigra* algal solution, atrazine standard solution of various concentrations, and culture medium, mix them at a volume ratio of 1:1:1 and shake well, and simultaneously set up a blank control group (using culture medium instead of atrazine). After the mixture is incubated in the dark for 10 min, measure the chlorophyll fluorescence kinetic parameters of *Phyllostachys nigra* algal solution using a water sample chlorophyll fluorometer, including the maximum light energy conversion efficiency of photosystem II (F). v / F m The actual light energy conversion efficiency (Y (II)), photosynthetic electron transfer efficiency (ETR), and photochemical quenching coefficient (qP) of photosystem II were determined. Three parallel control experiments were conducted, and the average values ​​were recorded to obtain the changes in each fluorescence parameter in the blank control group and the experimental group, so as to determine the effect of atrazine on each fluorescence parameter of *Acanthopanax senticosus*.

[0035] Determination of the effect of atrazine on the fluorescence effect of *Acetobacter spp.* under the condition of adding Anrongle: Select during the logarithmic growth phase (OD) of atrazine. 680 =1.0) Take the algal solution of *Hylocereus near-capitolatus*, a series of atrazine standard solutions, and Anrongle solution, mix them in a volume ratio of 1:1:1, and shake well. A blank control group (culture medium instead of atrazine) is also set up. After incubating the mixture in the dark for 10 min, the chlorophyll fluorescence kinetic parameters of the algal solution, including the maximum light conversion efficiency (F) of photosystem II, are measured using a water sample chlorophyll fluorometer. v / F m The actual light energy conversion efficiency (Y(II)), photosynthetic electron transfer efficiency (ETR), and photochemical quenching coefficient (qP) of photosystem II were determined. Three parallel control experiments were conducted, and the average values ​​were recorded to obtain the changes in each fluorescence parameter of the blank control group and the experimental group, so as to determine the effect of atrazine on each fluorescence parameter of *Acanthopanax senticosus*. Chlorophyll fluorescence kinetic parameters of photosystem II under different blank control groups and different conditions: actual light conversion efficiency (Y(II)), photosynthetic electron transport efficiency (ETR), photochemical quenching coefficient (qP), and maximum light conversion efficiency (F) of photosystem II. v / Fm The test results are as follows: Figures 1-4 As shown.

[0036] Example 4 This embodiment provides a method for detecting the biotoxicity of atrazine using fluorescence detection of *Periplaneta macrocarpa*, specifically including: Determination of the effect of atrazine on the fluorescence effect of *Acetobacter spp.* without the addition of Anrongle: During the logarithmic growth phase (OD) of *N. occulta* 680 =1.0) Take *Phyllostachys nigra* algal solution, atrazine standard solution of various concentrations, and culture medium, mix them at a volume ratio of 1:1:2 and shake well. A control group (using culture medium instead of atrazine) is also set up. After incubating the mixture in the dark for 10 min, the chlorophyll fluorescence kinetic parameters of the *Phyllostachys nigra* algal solution, including the maximum light conversion efficiency (F) of photosystem II, are measured using a water sample chlorophyll fluorometer. v / F m The actual light energy conversion efficiency (Y (II)), photosynthetic electron transfer efficiency (ETR), and photochemical quenching coefficient (qP) of photosystem II were determined. Three parallel control experiments were conducted, and the average values ​​were recorded to obtain the changes in each fluorescence parameter in the blank control group and the experimental group, so as to determine the effect of atrazine on each fluorescence parameter of *Acanthopanax senticosus*.

[0037] Determination of the effect of atrazine on the fluorescence effect of *Acetobacter spp.* under the condition of adding Anrongle: Select during the logarithmic growth phase (OD) of atrazine. 680 =1.0) Take the algal solution of *Hylocereus near-capitolatus*, a series of atrazine standard solutions, and Anrongle solution, mix them at a volume ratio of 1:1:2 and shake well. A blank control group (culture medium instead of atrazine) is also set up. After incubating the mixture in the dark for 10 min, the chlorophyll fluorescence kinetic parameters of the algal solution, including the maximum light conversion efficiency (F) of photosystem II, are measured using a water sample chlorophyll fluorometer. v / F m The actual light energy conversion efficiency (Y(II)), photosynthetic electron transfer efficiency (ETR), and photochemical quenching coefficient (qP) of photosystem II were determined. Three parallel control experiments were conducted, and the average values ​​were recorded to obtain the changes in each fluorescence parameter of the blank control group and the experimental group, so as to determine the effect of atrazine on each fluorescence parameter of *Acanthopanax senticosus*. Chlorophyll fluorescence kinetic parameters of photosystem II under different blank control groups and different conditions: actual light conversion efficiency (Y(II)), photosynthetic electron transport efficiency (ETR), photochemical quenching coefficient (qP), and maximum light conversion efficiency (F) of photosystem II. v / F m The test results are as follows: Figures 5-8 As shown.

[0038] Example 5 This embodiment provides a method for detecting the biotoxicity of atrazine using fluorescence detection of *Periplaneta macrocarpa*, specifically including: Determination of the effect of atrazine on the fluorescence effect of *Acetobacter spp.* without the addition of Anrongle: During the logarithmic growth phase (OD) of *N. occulta* 680 =1.0) Take *Phyllostachys nigra* algal solution, atrazine standard solution of various concentrations, and culture medium, mix them at a volume ratio of 1:2:1 and shake well. Simultaneously, set up a blank control group (using culture medium instead of atrazine). After incubating the mixture in the dark for 10 min, measure the chlorophyll fluorescence kinetic parameters of the *Phyllostachys nigra* algal solution using a water sample chlorophyll fluorometer, including the maximum light conversion efficiency (F) of photosystem II. v / F m The actual light energy conversion efficiency (Y (II)), photosynthetic electron transfer efficiency (ETR), and photochemical quenching coefficient (qP) of photosystem II were determined. Three parallel control experiments were conducted, and the average values ​​were recorded to obtain the changes in each fluorescence parameter in the blank control group and the experimental group, so as to determine the effect of atrazine on each fluorescence parameter of *Acanthopanax senticosus*.

[0039] Determination of the effect of atrazine on the fluorescence effect of *Acetobacter spp.* under the condition of adding Anrongle: Select during the logarithmic growth phase (OD) of atrazine. 680 =1.0) Take *Achnatherum capitella* algal solution, atrazine standard solution of various concentrations, and Anrongle solution, mix them at a volume ratio of 1:2:1 and shake well. Simultaneously, set up a blank control group (culture medium instead of atrazine). After incubating the mixture in the dark for 10 min, measure the chlorophyll fluorescence kinetic parameters of the algal solution using a water sample chlorophyll fluorometer, including the maximum light conversion efficiency (F) of photosystem II. v / F m The actual light energy conversion efficiency (Y(II)), photosynthetic electron transfer efficiency (ETR), and photochemical quenching coefficient (qP) of photosystem II were determined. Three parallel control experiments were conducted, and the average values ​​were recorded to obtain the changes in each fluorescence parameter of the blank control group and the experimental group, so as to determine the effect of atrazine on each fluorescence parameter of *Acanthopanax senticosus*. Chlorophyll fluorescence kinetic parameters of photosystem II under different blank control groups and different conditions: actual light conversion efficiency (Y(II)), photosynthetic electron transport efficiency (ETR), photochemical quenching coefficient (qP), and maximum light conversion efficiency (F) of photosystem II. v / F m The test results are as follows: Figures 9-12 As shown.

[0040] Depend on Figures 1-12It can be seen that, after adding Anrongle, compared with the same concentration of atrazine, its key fluorescence parameters Y(II), ETR, qP, and F of near-capitaloid photosynthesis were significantly reduced. v / F m All samples showed enhanced inhibition, with the inhibition rate significantly higher than the control group without Anrongle. This indicates that under Anrongle conditions, the inhibitory effect of trace atrazine on the fluorescence parameters of *Anatomium oxysporum* was more pronounced, and Anrongle can significantly improve the sensitivity of *Anatomium oxysporum* in detecting atrazine biotoxicity. Furthermore, the inhibitory effect changed with the ratio of Anrongle to atrazine, especially under trace stress conditions of 20 μg / L atrazine, where the enhancement effect was particularly significant after the addition of Anrongle. These results fully demonstrate that Anrongle can effectively enhance the response intensity and recognition sensitivity of algal biodetection systems to atrazine, providing a reliable basis for improving the performance of detection methods.

[0041] Wherein, inhibition rate (%): ×100%; I 0 represents the chlorophyll fluorescence parameter value of the control group; I i The values ​​represent the chlorophyll fluorescence parameters of the experimental group.

[0042] Comparative Example 1 This comparative example provides a method for detecting the biotoxicity of atrazine using fluorescence from *Anatomium spp.*, with specific steps similar to those in Example 3, except that the *Anatomium spp.* solution in Example 3 is replaced with an equal volume and concentration of *Scenedesmus obliquus* solution.

[0043] Comparative Example 2 This comparative example provides a method for detecting the biotoxicity of atrazine using fluorescence from *Phyllostachys nigra*. The specific steps are similar to those in Example 3, except that the *Phyllostachys nigra* algal solution in Example 3 is replaced with an equal volume and concentration of *Chlorella proteoglycans* algal solution.

[0044] Comparative Example 3 This comparative example provides a method for detecting the biotoxicity of atrazine using fluorescence from *Acetobacter spp.*, with specific steps similar to those in Example 3, except that in Example 3, Anrongle is replaced with an equal amount of sodium dodecylbenzenesulfonate.

[0045] Comparative Example 4 This comparative example provides a method for detecting the biotoxicity of atrazine using fluorescence from *Acetobacter spp.*, with specific steps similar to those in Example 3, except that Anronol in Example 3 is replaced with an equal amount of Tween-80.

[0046] The test results of the actual light energy conversion efficiency (Y(II)) of photosystem II in the algal chlorophyll fluorescence kinetic parameters of Comparative Examples 1-4 and Example 3 are as follows: Figure 14As shown.

[0047] Depend on Figure 13 It can be seen that, compared with *Scenedesmus obliquus* and *Chlorella protozoa* in Comparative Examples 1-2, *Anacentrum spiculatum* used in this invention shows a more significant response to the toxicity of the herbicide atrazine, and a more significant inhibitory effect on the actual light energy conversion efficiency (Y(II)) of photosystem II. This indicates that *Anacentrum spiculatum* has higher sensitivity for atrazine toxicity detection, thus providing a more intuitive and rapid response to the atrazine toxicity test, and significantly shortening the testing cycle. Compared with Comparative Examples 3-4, the inhibition rate of the actual light energy conversion efficiency (Y(II)) of photosystem II in *Anacentrum spiculatum* using the Anrongle adjuvant in this invention is significantly higher than that of sodium dodecylbenzenesulfonate and Tween-80. This shows that Anrongle can greatly enhance the physiological response intensity of *Anacentrum spiculatum* to atrazine stress through a unique synergistic sensitization effect, thereby shifting the traditional detection method from algal growth inhibition endpoint to the early physiological indicator of rapid inhibition of photosystem II, making the testing effect more intuitive and achieving a substantial reduction in the testing cycle.

[0048] In summary, this invention establishes a novel biotoxicity detection method with sensitivity and speed far exceeding traditional methods by selecting specific sensitizing adjuvants and sensitive indicator organisms, providing a powerful technical means for early warning and ecological risk assessment of pesticide pollution such as atrazine.

[0049] The above description is merely a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A method for detecting the biotoxicity of atrazine using fluorescence detection of *Acetobacter spp.*, characterized in that, Using Anrongle as a sensitizer and *Anomala spicata* as a sensitive indicator organism, the biotoxicity of atrazine was detected using the chlorophyll fluorescence kinetic parameters of *Anomala spicata*; the main components of Anrongle are lecithin and vitamin E.

2. The method for detecting the biotoxicity of atrazine by fluorescence detection of *Pericornucus oxysporum* as described in claim 1, characterized in that the steps... include: The algal solution of *N. near-cephalosporium*, a series of atrazine standard solutions and Anrongle solution were mixed evenly in a volume ratio of 1:1~2:1~2, placed in the dark for 10 min, and the chlorophyll fluorescence kinetic parameters of *N. near-cephalosporium* in the mixture were measured to detect the biotoxicity of atrazine. The algal solution of *Neptune cephalopoda*, the water sample containing atrazine, and Anrongle solution were mixed evenly at a volume ratio of 1:1~2:1~2, placed in the dark for 10 min, and the chlorophyll fluorescence kinetic parameters of *Neptune cephalopoda* in the mixture were measured to detect the biotoxicity of atrazine. Meanwhile, culture medium was used instead of atrazine standard solution as a blank control group.

3. The method for detecting the biotoxicity of atrazine by fluorescence detection of *Periflora sphaerocephala* as described in claim 2, characterized in that, The OD of the algal solution of *Neptune cephalopoda* 680 The value is 0.8~1.2, obtained by inoculating *Neptune cephalopoda* into the culture medium and culturing it in a constant temperature and light incubator to the logarithmic phase.

4. The method for detecting the biotoxicity of atrazine by fluorescence detection of *Periflora sphaerocephala* as described in claim 3, characterized in that, The culture medium comprises the following components: NaNO3 1.4~1.6 g / L, K2HPO4·3H2O 0.03~0.05 g / L, MgSO4·7H2O 0.07~0.08 g / L, CaCl2·2H2O 0.03~0.04 g / L, citric acid 0.005~0.007 g / L, ferric ammonium citrate 0.005~0.007 g / L, EDTA 0.0005~0.0015 g / L, Na2CO3 0.015~0.025 g / L, H3BO4 0.002~0.003 g / L, MnCl2·H2O 0.001~0.002 g / L, ZnSO4·7H2O 0.00015~0.00025 g / L, Na2MoO4·2H2O 0.00035~0.00045g / L, CuSO4·5H2O 0.000075~0.000085g / L, Co(NO3)2·6H2O 0.0000045~0.0000055g / L; the pH of the culture medium is 7.1±0.

1.

5. The method for detecting the biotoxicity of atrazine by fluorescence detection of *Periflora sphaerocephala* as described in claim 3, characterized in that, The culture conditions of the constant temperature and light incubator are as follows: light intensity 2000~2500lx, temperature 25±2℃, humidity 75±5%RH, light-dark cycle of 12h:12h, and static culture.

6. The method for detecting the biotoxicity of atrazine by fluorescence detection of *Periflora sphaerocephala* as described in claim 2, characterized in that, The concentrations of the atrazine standard solutions were 0 μg / L, 20 μg / L, 40 μg / L, 60 μg / L, 80 μg / L and 100 μg / L, respectively; the concentration of atrazine in the water sample to be tested was 5~95 μg / L.

7. The method for detecting the biotoxicity of atrazine by fluorescence detection of *Periflora sphaerocephala* as described in claim 2, characterized in that, The volume concentration of the Anrongle solution is 0.008~0.012%.

8. The method for detecting the biotoxicity of atrazine by fluorescence detection of *Periflora sphaerocephala* as described in claim 2, characterized in that, The chlorophyll fluorescence kinetic parameters include the maximum light energy conversion efficiency of photosystem II, the actual light energy conversion efficiency of photosystem II, the photosynthetic electron transport efficiency, and the photochemical quenching coefficient.

9. The method for detecting the biotoxicity of atrazine by fluorescence detection of *Periflora sphaerocephala* as described in claim 8, characterized in that, The chlorophyll fluorescence kinetic parameters were determined using a water sample chlorophyll fluorometer with an excitation wavelength of 675-685 nm.

10. The method for detecting the biotoxicity of atrazine by fluorescence detection of *Anaphalosporium spp.* as described in any one of claims 1 to 9, in assessing the potential harm of organic pesticides to aquatic ecosystems, providing early warning of organic pesticide pollution, and conducting precise ecological risk assessment, is characterized in that... The organic pesticides mentioned are atrazine, simazine, cyanazine, or cypermethrin.

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