A colorimetric method for rapid detection of chlorothalonil in fruits and vegetables

Abstract

The application discloses a colorimetric method for rapidly detecting chlorothalonil in fruits and vegetables, and comprises the following steps: sample extraction: adding a fruit and vegetable sample into water to scrub the fruit and vegetable skin, so as to obtain a to-be-tested solution; detection: adding chlorothalonil detection colorimetric reagent into the to-be-tested solution, heating, then adding an adsorbent, oscillating, standing, pouring off supernatant, and reserving the precipitate; colorimetry: observing the color of the precipitate, comparing with a chlorothalonil standard colorimetric card, and determining the residual amount of chlorothalonil in the fruit and vegetable sample; the chlorothalonil detection colorimetric reagent is a mixture containing tetrabutylammonium hydroxide, o-dinitrobenzene and p-nitrobenzaldehyde; and the adsorbent is modified chitosan. The hydrophobic product generated by the chemical color reaction of chlorothalonil and an indicator is uniformly adsorbed by the long-chain modified chitosan, and whether the content of chlorothalonil in the fruit and vegetable sample exceeds the standard can be rapidly determined by comparison with the chlorothalonil colorimetric card.

Description

Technical Field

[0001] This application relates to a colorimetric method for rapid detection of chlorothalonil in fruits and vegetables, belonging to the field of rapid pesticide detection technology. Background Technology

[0002] Chlorothalonil is a broad-spectrum protective fungicide widely used to control various pests on fruit trees and vegetable leaves. It is also used as an industrial antifungal agent and fruit preservative. Statistics show that the demand for chlorothalonil has been increasing year by year. In 2016, the global demand for chlorothalonil was 49,000 tons, with a compound annual growth rate of 6.1%. It is projected that global consumption of chlorothalonil will increase to 72,000 tons by 2025.

[0003] Chlorothalonil lacks systemic activity and adheres well to plant surfaces after spraying, making it difficult to wash away by rain. Therefore, its efficacy is relatively long, with a residual effect typically lasting 7-10 days. Toxicological studies have shown that chlorothalonil can induce cell mutations and, under certain dosage conditions, induce sister chromatid exchange in mouse bone marrow lymphocytes. Chlorothalonil is also highly toxic to fish and amphibians, being a chemical that can cause genetic mutations in aquatic organisms, amphibians, and plant cells, and has a certain sensitizing effect on humans. In 2017, the International Agency for Research on Cancer (IARC) of the World Health Organization preliminarily classified chlorothalonil as a Group 2B carcinogen.

[0004] Chlorothalonil residues in fruits and vegetables pose a threat to human health, making detection necessary. Currently established detection methods primarily rely on large-scale instruments, including gas chromatography, high-performance liquid chromatography, and gas chromatography-mass spectrometry. However, these methods often require cumbersome sample pretreatment, are highly technical, costly, and time-consuming. Therefore, developing a rapid, intuitive, and cost-effective method for detecting chlorothalonil pesticide residues in fruit and vegetable samples is essential. Summary of the Invention

[0005] The purpose of this invention is to provide a method for rapid detection of chlorothalonil based on colorimetric principles. This method has good stability and high sensitivity, and can meet the needs of rapid on-site detection of chlorothalonil pesticides in fruit and vegetable samples.

[0006] According to one aspect of this application, a colorimetric method for rapid detection of chlorothalonil in fruits and vegetables is provided, the colorimetric method comprising at least the following steps:

[0007] (1) Sample extraction: Add water to the fruit and vegetable samples and rub the fruit and vegetable skin to obtain the test solution;

[0008] (2) Detection: Add chlorothalonil colorimetric reagent to the test solution, mix, heat, add adsorbent, shake, let stand, discard the supernatant, and retain the precipitate;

[0009] (3) Colorimetric analysis: Observe the color of the precipitate and compare it with the standard colorimetric card of chlorothalonil to determine the residual amount of chlorothalonil in the fruit and vegetable samples.

[0010] The chlorothalonil colorimetric reagent is a mixture containing tetrabutylammonium hydroxide, o-dinitrobenzene, and p-nitrobenzaldehyde;

[0011] The adsorbent is modified chitosan.

[0012] Optionally, the method for preparing the modified chitosan includes: reacting a mixture containing chitosan, ethylene glycol diglycidyl ether, sodium dodecyl sulfonate, and tetradecyl dimethyl tertiary amine, centrifuging, and obtaining the modified chitosan.

[0013] Optionally, the mass ratio of chitosan, ethylene glycol diglycidyl ether, sodium dodecyl sulfonate, and tetradecyl dimethyl tertiary amine is 1:1 to 3:0.05 to 0.2:2 to 4.

[0014] Optionally, the mixture may further contain a solvent, wherein the solvent is ethanol.

[0015] Optionally, the pH value of the mixture is 11 to 12.

[0016] Optionally, the reaction temperature is 60–80°C and the reaction time is 1–2 hours.

[0017] Optionally, the reaction temperature is selected from any value of 60℃, 65℃, 70℃, 75℃, 80℃ or a range between any two of the above points.

[0018] Optionally, the reaction time is selected from any value of 1 hour, 1.5 hours, 2 hours, or a range between any two of the above points.

[0019] Optionally, the centrifugation speed is 8000 r / min to 10000 r / min, and the centrifugation time is 5 min to 8 min.

[0020] Optionally, the centrifugal speed is selected from any value among 8000 r / min, 9000 r / min, and 10000 r / min, or a range between any two of the above points.

[0021] Optionally, the centrifugation time is selected from any value among 5 min, 6 min, 7 min, and 8 min, or a range between any two of the above points.

[0022] Optionally, the preparation method of the chlorothalonil standard colorimetric card includes: preparing chlorothalonil standard working solutions of different concentrations, adding chlorothalonil detection colorimetric reagent to the chlorothalonil standard working solutions respectively, heating, adding adsorbent and mixing, shaking and standing, discarding the supernatant, and then recording and processing the precipitate using instruments and equipment to obtain the chlorothalonil standard colorimetric card under the corresponding conditions.

[0023] Optionally, in the chlorothalonil colorimetric reagent, the molar ratio of tetrabutylammonium hydroxide, o-dinitrobenzene, and p-nitrobenzaldehyde is 10-20:1-2:1-2.

[0024] Optionally, in step (1), the fruit and vegetable samples have the same weight as the water.

[0025] Optionally, in step (2), the volume ratio of the test solution to the chlorothalonil colorimetric reagent is 1:1 to 1.2.

[0026] Optionally, in step (2), the heating temperature is 45-60°C and the heating time is 10-15 minutes.

[0027] Optionally, the heating temperature is selected from any value among 45°C, 50°C, 55°C, and 60°C, or a range between any two of the above points.

[0028] Optionally, the heating time is selected from any value among 10 min, 11 min, 12 min, 13 min, 14 min, and 15 min, or a range between any two of the above points.

[0029] Optionally, in step (2), the oscillation time is 1 min to 2 min, and the resting time is 5 min to 10 min.

[0030] Optionally, the oscillation time is selected from any value among 1 min, 1.5 min, 2 min, or a range between any two of the above points.

[0031] Optionally, the settling time is selected from any value of 5 min, 6 min, 7 min, 8 min, 9 min, 10 min or a range between any two of the above points.

[0032] One optional method for rapid detection of chlorothalonil in fruits and vegetables based on colorimetric principles is as follows:

[0033] (1) Pretreatment: Weigh the fruits and vegetables, and wash the skin of the fruits and vegetables with an equal weight of pure water to obtain the test solution;

[0034] (2) Detection: Take 1 mL of the test solution and put it into a transparent centrifuge tube. Add reaction reagents A, B and C to it respectively, mix well, react at 45-60℃ for 10 min, then add 50 mg of product adsorbent material D, shake for 1 min, let stand for 5 min, pour off the supernatant and keep the precipitate.

[0035] (3) Colorimetric analysis: Observe the color of the precipitate and determine the residual amount of chlorothalonil in the fruit and vegetable samples by referring to the standard colorimetric card of chlorothalonil.

[0036] The names, solvents, concentrations, and amounts of the reaction reagents A, B, and C are shown in the table below:

[0037] Reagent Name Tetrabutylammonium hydroxide o-Dinitrobenzene p-Nitrobenzaldehyde solvent methanol ethanol ethanol reagent concentration 100~150g / L 6~10g / L 6~10g / L reagent dosage 50 microliters 500 microliters 500 microliters

[0038] Because the products generated by the reaction of reagents A, B, and C with chlorothalonil are lipid-soluble, the centrifuge tubes exhibit a heterogeneous system after the reaction, making direct absorbance comparison impossible. To accurately quantify the detection results, uniform adsorption of the colored products is necessary; therefore, product adsorbent D is synthesized.

[0039] The product adsorbent D is modified chitosan, and its preparation method is as follows: Under alkaline conditions, 2g of chitosan, (3-4)mL of ethylene glycol diglycidyl ether, (0.1-0.3)g of sodium dodecyl sulfonate, and (4-6)g of tetradecyl dimethyl tertiary amine are added to 100mL of 50% (v / v) ethanol aqueous solution. The mixture is reacted in a water bath at 60-80℃ for 1-2 hours, centrifuged at 8000r / min for 5min, and the precipitate is collected. The precipitate is washed three times with ethanol and then vacuum dried to obtain modified chitosan with long fatty chains. The pH range of the alkaline conditions is 11-12.

[0040] Optionally, the method for preparing the chlorothalonil standard colorimetric card is as follows:

[0041] Take 1 mL of chlorothalonil standard solutions with concentrations of 0, 1, 2, and 4 mg / L respectively, add reaction reagents A, B, and C respectively, mix well, react at 45–60℃ for 10 min, then add 50 mg of product adsorbent D, shake for 1 min, let stand for 5 min, discard the supernatant, record the precipitate using an imaging device, process the record using Photoshop software, and obtain the chlorothalonil standard colorimetric card under the corresponding conditions.

[0042] The beneficial effects that this application can produce include:

[0043] The colorimetric rapid detection method for chlorothalonil of the present invention is simple to operate, has good stability and high sensitivity, and the detection limit can be as low as 1 mg / Kg. The entire detection process can obtain the detection result in less than 20 minutes, making it suitable for rapid on-site detection of this pesticide.

[0044] The present invention provides a rapid colorimetric detection method for chlorothalonil using product adsorption. The prepared product adsorption material is chitosan with long fatty chains on its surface, which can uniformly adsorb the colored insoluble product generated by the reaction of chlorothalonil and reagents. The color of the material after adsorption is recorded, which solves the problem that chlorothalonil cannot be measured by conventional colorimetric methods because the reaction between chlorothalonil and reagents results in a heterogeneous system.

[0045] This invention utilizes long-chain modified chitosan to uniformly adsorb the hydrophobic product generated by the chemical colorimetric reaction between chlorothalonil and an indicator. By comparing with a chlorothalonil colorimetric card, it can quickly determine whether the chlorothalonil pesticide content in fruit and vegetable samples exceeds the standard. Attached Figure Description

[0046] Figure 1 This is the standard colorimetric card for chlorothalonil used in this application. Detailed Implementation

[0047] The present application is described in detail below with reference to the embodiments, but the present application is not limited to these embodiments.

[0048] Unless otherwise specified, all raw materials used in the embodiments of this application were purchased commercially. The imaging device was a flatbed scanner, model Epson V200, with a resolution set to 300 dpi.

[0049] Example 1

[0050] Preparation method of adsorbent (product adsorbent material D): The product adsorbent material D is modified chitosan, and its preparation method is as follows: 2g chitosan, 3mL ethylene glycol diglycidyl ether, 0.2g sodium dodecyl sulfonate, and 5g tetradecyl dimethyl tertiary amine are respectively added to 100mL of 50% ethanol aqueous solution, the pH is adjusted to 11, and the reaction is carried out in a water bath at 80℃ for 1.5 hours. After centrifugation at 8000r / min for 5min, the precipitate is collected, and the precipitate is washed three times repeatedly with ethanol. After vacuum drying, modified chitosan with long fatty chains is obtained.

[0051] Example 2

[0052] Preparation method of adsorbent (product adsorbent material D): The product adsorbent material D is modified chitosan, and its preparation method is as follows: Under alkaline conditions, 2g chitosan, 4mL ethylene glycol diglycidyl ether, 0.3g sodium dodecyl sulfonate, and 4g tetradecyl dimethyl tertiary amine are respectively added to 100mL of 50% ethanol aqueous solution, the pH is adjusted to 12, the reaction is carried out in a water bath at 70℃ for 2 hours, centrifuged at 8000r / min for 5min, the precipitate is collected, the precipitate is washed three times repeatedly with ethanol, and after vacuum drying, modified chitosan with long fatty chains is obtained.

[0053] Example 3

[0054] Preparation of chlorothalonil standard colorimetric cards: Take 1 mL of chlorothalonil standard solutions with concentrations of 0, 1, 2, and 4 mg / L, respectively, and add chlorothalonil detection colorimetric reagents (reaction reagents A, B, and C) to each solution. The names, solvents, concentrations, and amounts of these three reagents are shown in Table 1 below.

[0055] Table 1: Colorimetric Reagents for Chlorothalonil Detection

[0056] Reagent Name Tetrabutylammonium hydroxide o-Dinitrobenzene p-Nitrobenzaldehyde solvent methanol ethanol ethanol reagent concentration 120g / L 6g / L 8g / L reagent dosage 50 microliters 500 microliters 500 microliters

[0057] After mixing the above solution, react it at 50°C for 10 min. Then, add 50 mg of the adsorbent (product adsorbent material D) prepared in Example 1, shake for 1 min, let stand for 5 min, discard the supernatant, record the precipitate using an imaging device, and process the record using Photoshop software to obtain the chlorothalonil standard colorimetric card under the corresponding conditions. Figure 1 ).from Figure 1 It can be seen that as the concentration of chlorothalonil standard increases, the color of the adsorbent gradually deepens.

[0058] Example 4

[0059] Detection of chlorothalonil residues in spiked eggplant:

[0060] (1) Pretreatment: Weigh 86g of spiked eggplant and wash the eggplant skin with 86mL of purified water to obtain the test solution;

[0061] (2) Detection: Take 1 mL of the test solution and put it into a transparent centrifuge tube. Add chlorothalonil detection colorimetric reagent (reaction reagents A, B, and C) to it respectively, mix well, react at 50°C for 10 min, then add 50 mg of the product adsorbent material D prepared in Example 1, shake for 1 min, let stand for 5 min, pour off the supernatant, and retain the precipitate.

[0062] The names, solvents, concentrations, and amounts of the colorimetric reagents for chlorothalonil detection (reaction reagents A, B, and C) are shown in Table 2 below:

[0063] Table 2: Colorimetric reagents for chlorothalonil detection

[0064] Reagent Name Tetrabutylammonium hydroxide o-Dinitrobenzene p-Nitrobenzaldehyde solvent methanol ethanol ethanol reagent concentration 120g / L 6g / L 8g / L reagent dosage 50 microliters 500 microliters 500 microliters

[0065] (3) Colorimetric analysis: Observe the color of the precipitate and determine the residual amount of chlorothalonil in the eggplant by comparing it with the standard colorimetric card of chlorothalonil. The residual amount of chlorothalonil is 3 mg / Kg.

[0066] Example 5

[0067] Detection of chlorothalonil residues in spiked apples:

[0068] (1) Pretreatment: Weigh 153g of spiked apples and wash the apple skin with 153mL of purified water to obtain the test solution;

[0069] (2) Detection: Take 1 mL of the test solution and put it into a transparent centrifuge tube. Add chlorothalonil detection colorimetric reagent (reaction reagents A, B, and C) to it respectively, mix well, react at 50°C for 10 min, then add 50 mg of the product adsorbent material D prepared in Example 1, shake for 1 min, let stand for 5 min, pour off the supernatant, and retain the precipitate.

[0070] The names, solvents, concentrations, and amounts of the colorimetric reagents (reaction reagents A, B, and C) for chlorothalonil detection are shown in Table 3 below:

[0071] Table 3: Colorimetric Reagents for Chlorothalonil Detection

[0072] Reagent Name Tetrabutylammonium hydroxide o-Dinitrobenzene p-Nitrobenzaldehyde solvent methanol ethanol ethanol reagent concentration 120g / L 6g / L 8g / L reagent dosage 50 microliters 500 microliters 500 microliters

[0073] (3) Colorimetric analysis: Observe the color of the precipitate and determine the residual amount of chlorothalonil in the apple by comparing it with the standard colorimetric card of chlorothalonil. The residual amount of chlorothalonil is 2 mg / Kg.

[0074] The above description is merely a few embodiments of this application and is not intended to limit this application in any way. Although this application discloses preferred embodiments as described above, it is not intended to limit this application. Any changes or modifications made by those skilled in the art without departing from the scope of the technical solution of this application using the disclosed technical content are equivalent to equivalent implementation cases and fall within the scope of the technical solution.

Claims

1. A colorimetric method for rapid detection of chlorothalonil in fruits and vegetables, characterized in that, The colorimetric method includes at least the following steps: (1) Sample extraction: Add water to the fruit and vegetable samples and rub the fruit and vegetable skin to obtain the test solution; (2) Detection: Add chlorothalonil colorimetric reagent to the test solution, mix, heat, add adsorbent, shake, let stand, discard the supernatant, and retain the precipitate; (3) Colorimetric analysis: Observe the color of the precipitate and compare it with the standard colorimetric card of chlorothalonil to determine the residual amount of chlorothalonil in the fruit and vegetable samples; The chlorothalonil colorimetric reagent is a mixture containing tetrabutylammonium hydroxide, o-dinitrobenzene, and p-nitrobenzaldehyde; The adsorbent is modified chitosan; The method for preparing the modified chitosan includes: reacting a mixture containing chitosan, ethylene glycol diglycidyl ether, sodium dodecyl sulfonate, and tetradecyl dimethyl tertiary amine, centrifuging, and obtaining the modified chitosan.

2. The colorimetric method according to claim 1, characterized in that, The mass ratio of chitosan, ethylene glycol diglycidyl ether, sodium dodecyl sulfonate, and tetradecyl dimethyl tertiary amine is 1:1~3:0.05~0.2:2~4; The mixture also contains a solvent, which is ethanol; The pH value of the mixture is 11-12.

3. The colorimetric method according to claim 1, characterized in that, The reaction temperature is 60~80 ℃, and the reaction time is 1~2 hours; The centrifugation speed is 8000 r / min to 10000 r / min, and the centrifugation time is 5 min to 8 min.

4. The colorimetric method according to claim 1, characterized in that, The preparation method of the chlorothalonil standard colorimetric card includes: preparing chlorothalonil standard working solutions of different concentrations, adding chlorothalonil detection colorimetric reagent to the chlorothalonil standard working solutions respectively, heating, adding adsorbent and mixing, shaking and standing, discarding the supernatant, and then recording and processing the precipitate through instruments and equipment to obtain the chlorothalonil standard colorimetric card under the corresponding conditions.

5. The colorimetric method according to claim 1, characterized in that, In the chlorothalonil colorimetric reagent, the molar ratio of tetrabutylammonium hydroxide, o-dinitrobenzene, and p-nitrobenzaldehyde is 10~20:1~2:1~2.

6. The colorimetric method according to claim 1, characterized in that, In step (1), the fruit and vegetable samples have the same weight as the water.

7. The colorimetric method according to claim 1, characterized in that, In step (2), the volume ratio of the test solution to the chlorothalonil colorimetric reagent is 1:1~1.

2.

8. The colorimetric method according to claim 1, characterized in that, In step (2), the heating temperature is 45~60 ℃ and the heating time is 10 min~15 min.

9. The colorimetric method according to claim 1, characterized in that, In step (2), the oscillation time is 1 min to 2 min, and the resting time is 5 min to 10 min.

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