The dissipation study of residues of insecticides having different modes of action was studied in cabbage. The insecticides such as deltamethrin, chlorantraniliprole and fipronil residues were sprayed as foliar application twice at an interval of 7 days with the recommended doses of deltamethrin (@ 12.5 g a.i./ha), chlorantraniliprole (@ 30 g a.i./ha) and fipronil (50 g a.i./ha). The samples were taken randomly before the application of any insecticides and 1 hour after the second spray of insecticides. The residues were below the detectable limit for all the insecticides. The residues of fipronil were found to be more persistent than those of chlorantraniliprole and deltamethrin. Fipronil was found to persist up to the 7th day with a residue of 0.01 mg kg-1. However, the residues of chlorantraniliprole and deltamethrin persisted upto the 5th day i.e. 0.11 mg kg-1 and 0.03 mg kg-1 respectively. Observation revealed that the residues of chlorantraniliprole on cabbage head dissipated over the course of time and an equally high degree of dissipation was observed. The half-life of chlorantraniliprole was found to be 1.81 days as compared to 1.07 and 1.42 days respectively for fipronil and deltamethrin.
Research Article
Dissipation of Insecticidal Residues having Different Modes of Action in Cabbage
https://doi.org/10.21203/rs.3.rs-3294472/v1
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Deltamethrin
Chlorantraniliprole
Fipronil
Residues and HPLC
Agriculture is the main occupation for the people of the state of Manipur, 67% of the total population is betrothed in agriculture and allied activities. Only 7.41 percent of the total geographical area is used for agricultural purposes. Of these total cultivable lands, around 50% falls under the valley region. In Manipur, cabbage (Brassica oleracea Linn.) is grown as a popular vegetable and is consumed mostly as curry, boiled and raw like salad, singju, etc. It is grown in an area of 8.371 thousand hectares with a production of 107.969 thousand MT in the year 2019-20 (Department of Horticulture & Soil Conservation, Government of Manipur).
Cabbage covers the largest area and highest production compared with other vegetables both in hilly and valley regions. The production of cabbage requires high demands of inputs like fertilizers, herbicides, pesticides and irrigation. Begins with vegetative growth up to harvesting, various kinds of insect pests are attacking cabbage which caused detrimental losses in the yield. To combat losses and attacks from insect pests, farmers apply heavy use of chemical insecticides. Around 37 insect pests attack cabbage such as cabbage butterfly, Pieris brassicae (L.); tobacco caterpillar, Spodoptera litura (F.); diamondback moth, Plutella xylostella (L.); cabbage leaf webber, Crocidolomia binotalis (Zell); aphids, Brevicoryne brassicae (L.) and Lipaphis erysimi (Kalt.); painted bug, Bagrada cruciferarum (Kirk.); and flea beetle, Phyllotreta cruciferae (Goeze) from seedling to harvest (Lal, 1975). Among them, lepidopteran caterpillars like Pieris brassicae (L.) and Plutella xylostella (L.) were found to be the major pests causing extensive loss in the vegetative stage. Farmers are usually not concerned about the correct application of insecticides for the specific insect’s attack. The recommended dose and its time of application are not followed by the majority of the farmers. The malpractices of chemical insecticides are a major issue to be concerned. The detection of pesticide residues on daily consumable vegetables has been a concern to consumers. Some groups of insecticides are not readily degraded in soil or plant systems. Therefore, they finally may transform into toxic metabolites or may accumulate in fatty tissues of the human body and subsequently, the environment causing problems again to human health and ecosystems (Ejobi et al. 1996). Deltamethrin is a synthetic pyrethroid group of insecticides where it disrupts the normal nervous system function when the insects come into direct contact with the treated surface or directly consume it (Doi et al. 2006). Chlorantraniliprole, a novel anthranilic diamide insecticide acts after being ingested by the insects. It causes activation of the insect ryanodine receptors within the sarcoplasmic reticulum which leads to impaired regulation of muscle contraction in lepidopteran, coleopteran, and some dipteran pests thereby causes termination of feeding almost immediately after consumption or direct contact with the treated surface (Bentley et al. 2010). Fipronil is a phenylpyrazole group of insecticides acted by disrupting the insect’s central nervous system. It causes hyperexcitation of insect nervous and muscle systems due to stronger binding affinity to the GABAA receptors of insects than to those of mammals (Delpech et al. 2005). Pesticides can contaminate not only soil, water, air and other vegetation but also affects birds, fish, beneficial insects, and non-target plants. However, cabbage is one of the most important dietary foods and is widely consumed as raw as well as cooked, the treatment of different chemical insecticides and their residues after application should ensure the safety of the consumers where there are no residues above the maximum residue limit (MRL). Therefore, the dissipation pattern of deltamethrin, chlorantraniliprole and fipronil residues is very much necessary to study its residual nature in cabbage heads for consumers’ safety.
Chemicals and reagents
The technical grade analytical standards of deltamethrin (99.90%), chlorantraniliprole (99.20%), and fipronil (97.5%) as well as primary secondary amine (PSA) sorbent were procured from sigma-aldrich, Kolkatta. The insecticide formulations like deltamethrin 2.8% EC, chlorantraniliprole 18.5% SC and fipronil 5% SC used for field application was obtained from local pesticide dealer. Solvents like HPLC grade acetonitrile, sodium chloride (ASC reagent grade ≥ 99.9 %) and sodium sulfate anhydrous (AR grade) were also acquired from Merck Pvt. Ltd, Mumbai India. All common solvents were redistilled in all-glass apparatus before usage. The suitability of the solvents and other chemicals was confirmed by running reagent blanks before actual analysis.
Analysis of HPLC grade acetonitrile extract of the formulation showed only deltamethrin chlorantraniliprole, fipronil peaks and none of its metabolic products, and no interfering peaks were observed under the retention time of the compound being estimated. Moreover, the concentration of deltamethrin, chlorantraniliprole and fipronil in the formulation was found to be specific with respect to its purity as claimed by the manufacturers.
Preparation of standard solution
Standard stock solutions (1 mg mL-1) each of deltamethrin, chlorantraniliprole and fipronil were prepared in acetonitrile solvent. The standard solutions required for plotting of calibration curve (10.00, 20.00, 100.00, 200.00 and 1000 µg mL-1) were prepared from stock solutions by serial dilution in acetonitrile. All standard solutions were stored at -4ºC before use.
Instrumentation
Analysis of deltamethrin, chlorantraniliprole and fipronil was carried out on reverse phase high performance liquid chromatograph (HPLC, Model ANALYST200) equipped with C18 column and UV-VIS detector, dual pump auto-sampler was supplied by M/S Perkin Elmer, United States. The HPLC Brownlee analytical C18 column with a length of 150 mm, 4.6 mm inside diameter and 5 µm particle size was also procured from M/S Perkin Elmer, United States. For instrument control, data acquisition and processing, LC Solution software was used for the analysis. For the control of the instrument, data acquisition and processing, TC Nav software procured from Perkin-Elmer was used. A good acceptable peak separation of deltamethrin, chlorantraniliprole and fipronil is detected at 2.85 min, 3.54 min and 4.03min. The dual pump functions as the isocratic flow of the mobile phase of acetonitrile and water (90:10, v/v) at 225 nm with the flow rate of 1.70 ml min-1 for deltamethrin and 0.5 ml min-1 for both chlorantraniliprole and fipronil.
Crop planting
Cabbage (variety Rareball) was sown during the first week of September 2022 and transplanted during the first week of November 2022 according to the recommended agronomic practices at Langol farm, ICAR Research Complex for NEH Region, Manipur Centre. There were three replications for each treatment arranged in a randomized block design (RBD), and the size of each plot was 20 m2.
Application of the insecticide
Deltamethrin 2.8% EC (Decis 100) @ 10 g a.i./ha, chlorantraniliprole 18.5% SC (Coragen) @ 10 g a.i./ha and fipronil 5% SC (Fax) @ 40g a.i./ha were applied twice as foliar sprays at an interval of 7 days in the experimented plots.
Sampling
Vegetable samples (cabbage head) were collected randomly from control and treated plots from each treatment before the first treatment, 0 (1 hour after the second application), 3, 5, 7, 9 and 11 days after the applications of insecticides.
Extraction and cleanup
Samples of matured cabbage heads were collected randomly from the insecticide treated plots as well as control plots in labeled polyethylene sample bags. They are then brought to the laboratory for further analysis. The cabbage samples were blended in a Waring Blender. A 15 g sample was weighed in a 50 ml polypropylene centrifuge tube and it was mixed with 30 ml acetonitrile followed by hand shaking for thorough mixing. The contents were then homogenized at 25000 rcf for 3 mins by using Heidolph homogenizer. After that, 10g sodium chloride was added to each of the sample tubes and shaken by rotospin for around 10 mins. Each of the tubes of sample replicates were centrifuged by using a REMI centrifuge for 3 mins at 3000 rcf. The top clear organic layer of liquid of 15 ml was taken out from each tube and transferred into another sterile 50 ml polypropylene tube containing 10 g of activated sodium sulfate. The transferred contents were then shaken using a rotor spin for 2 mins. After the mixed contents quickly settle down, 6 ml of the upper layer of the sample was taken and transferred into a 15 ml polypropylene centrifuge tube containing mixtures of primary secondary amine sorbent of 0.15 g, activated anhydrous magnesium sulfate of 0.90 g and graphitized carbon black of 0.05 g. The tubes were vortexed for 30 sec and then centrifuged for 3 mins at 700 rcf. The final extract of 4 ml was transferred into a test tube and concentrated to 2 ml with a rotary evaporator under 35°C for further quantification by High-Performance Liquid Chromatography (HPLC).
Before analyzing the treated samples, recovery experiments from untreated samples were carried out at different levels to establish the reliability and validity of the analytical method and to know the skill of extraction and clean-up procedures. Control samples of cabbage were spiked at different levels of 0.05, 0.10, 0.25, 0.50 and 1.00 mg kg-1 at different stages of extraction, cleaning up and analysis. The control samples as well as reagent blanks were also extracted and cleaned up in the same method so as to find out the interferences, if any, due to the substrate and reagents, respectively.
Statistical analysis
The residues of the insecticides were calculated using the formula
Where, A1 = Peak area of the sample, A2 = Peak area of the standard, I1 = Injected volume of standard (µL), I2 = Injected volume of sample (µL), C = Concentration of standard solution (mg L-1), F = Final volume of the sample (mL) and W= weight of the sample (kg) (Mariappan and Kaithamalai 2020).
Efficiency of the method
The average recovery percentage obtained was found to be in the range of 80.00 to 96.60 percentage (Table 1) therefore, the results have been presented as such without applying any correction factor. The untreated samples as well as reagent blanks were also processed in the same method so as to find out the interferences, if any, due to the substrate and reagents, respectively.
| Level of fortification (mg kg− 1) | Chlorantraniliprole | Fipronil | Deltamethrin |
|---|---|---|---|
| 0.05 | 80.00 ± 3.67 | 82.78 ± 0.56 | 83.30 ± 1.98 |
| 0.10 | 83.21 ± 3.56 | 86.56 ± 3.78 | 86.65 ± 1.90 |
| 0.25 | 88.79 ± 0.97 | 90.45 ± 2.65 | 89.90 ± 4.50 |
| 0.50 | 92.20 ± 2.56 | 95.56 ± 0.94 | 84.24 ± 0.30 |
| 1.00 | 95.40 ± 3.80 | 96.60 ± 2.56 | 85.50 ± 0.46 |
| aMean ± Standard deviation | |||
Dissipation of chlorantraniliprole, fipronil and deltamethrin in cabbage head
The chlorantraniliprole, fipronil and deltamethrin residues were analyzed before the treatment of any insecticides. The residues were below the detectable limit (BDL) for all the insecticides. After the application of the second dose at 1 hour (0 day), the residues of chlorantraniliprole were found to be 0.72 mg kg− 1 at 0 day and it was degraded to 0.55 mg kg− 1 at 1 day. It was again dissipated to 0.11 mg kg− 1 on the 5th day of treatment. On the 7th day, the residues of chlorantraniliprole were found to be BDL. Similarly, the concentration of fipronil was found to be higher i.e. 1.01 mg kg− 1 on the day of treatment, taking samples at 0 hour. The residues were dissipated to 0.74 mg kg− 1 on day 1. However, the residues were detected till the 7th day of treatment (0.01 mg kg− 1). On the 9th day, the residues were not found. the residues of deltamethrin were observed to be 0.32 mg kg− 1 and it was dissipated to 0.25 mg kg− 1, 0.17 mg kg− 1, 0.09 mg kg− 1 and 0.03 mg kg− 1 respectively at 1, 2, 3 and 5 days respectively. At harvest time, the residues become undetectable. The residues of deltamethrin at 0 day were found to be 0.32 mg kg− 1 and it was gradually degraded to 0.25, 0.17, 0.09 and 0.03 mg kg− 1 on 1, 2, 3 and 5 days respectively. Like other insecticides, the residues of deltamethrin on cabbage head also dissipated with time, and an equitably high degree of dissipation was observed (Table 2).
| Days after treatment | Chlorantraniliprole | Fipronil | Deltamethrin |
|---|---|---|---|
| Before application | BDL | BDL | BDL |
| 0 (After 1 hour) | 0.72 ± 0.04 | 1.01 ± 0.25 | 0.32 ± 0.08 |
| 1 | 0.55 ± 0.10 b(23.62) | 0.74 ± 0.22 (26.73) | 0.25 ± 1.45 (21.87) |
| 2 | 0.39 ± 0.15 (45.83) | 0.41 ± 0.00 (59.40) | 0.17 ± 0.10 (46.87) |
| 3 | 0.25 ± 0.19 (65.28) | 0.21 ± 0.13 (79.20) | 0.09 ± 1.01 (71.85) |
| 5 | 0.11 ± 0.08 (84.73) | 0.08 ± 0.18 (93.00) | 0.03 ± 0.25 (90.63) |
| 7 | cBDL (100) | 0.01 ± 0.27 (99.00) | bBDL (100) |
| 9 | cBDL (100) | bBDL (100) | bBDL (100) |
| Harvest time | cBDL (100) | bBDL (100) | bBDL (100) |
| MRL | 0.30 | 0.02 | 0.20 |
| Waiting period (days) | 3 | 7 | - |
| Regression Equation | R2 = 0.993 -0.166x + 1.891 | R2 = 0.988 -0.282x + 2.227 | R2 = 0.987 -0.211x + 1.579 |
| Half-life (days) | 1.81 | 1.07 | 1.42 |
| aMean ± Standard deviation | |||
| b% dissipation of compound | |||
| cBDL= Below determination limit of 0.01 mg kg− 1 | |||
The insecticide, deltamethrin was sprayed three times @ 50, 50 and 12 g a.i. ha− 1 at cauliflower crop. The initial deposits of the insecticides on curd and leaves were observed as 1.14 and 0.60 mg kg− 1, respectively. After one day, the residues declined below MRL (Singh et al. 1990). The residues of fipronil were studied by applying 75 and 150 g a.i. ha− 1 at an interval of 7 days. The initial deposits of 1.226 and 2.704 mg kg− 1 were found on the heads of cabbage following 3rd application of fipronil at single and double the dosages, respectively (Bhardwaj et al. 2012). Similar findings were observed in cabbage with three sprays of fipronil @ 100 g a.i. ha− 1 at head formation stage. The residues of fipronil dissipated with initial deposit of 1.69 mg kg− 1 and was found to be below detectable residues at 10 days after the last spray (Reddy et al. 2012). The study is in conformity with the observation in other crops like brinjal and okra fruits where the initial deposit of chlorantraniliprole was found to be 0.72 and 0.48 µg g− 1 on brinjal and okra respectively when applied at same doses of 30 g a.i. ha− 1. The residues were found BDL of 0.01 µg g− 1 on 10 days after treatment (Regupathy and Dham 2001). Lahm et al. (2007) also reported that chlorantraniliprole residues were checked several days after treatment on tomatoes at 0, 1, 3, 5, 7, 10 and 15 days. The initial deposits were found as 2.3 µg g− 1 and declined to 1.71, 0.996, 0.620, 0.390, 0.115 and 0.100 µg g− 1 respectively. The findings were also comparable with the study of Chlorantraniliprole residues on cauliflower where the initial deposits of 0.18 µg g− 1 were observed at 9.25 g a.i. ha− 1 at one hour after spraying. The residues were not detectable at 3 days after treatment (Ioriatti et al. 2009). With the application of Coragen (chlorantraniliprole formulation 18.5 SC) three times at recommended dose i.e. 9.25 g a.i. ha− 1, the mean initial deposits of chlorantraniliprole were found to be 0.18 mg kg− 1 on curd at 0 day. These deposits declined to 0.11 showing a loss of 38.90% dissipation. These residues again dissipated below the limit of quantification of 0.10 mg kg− 1 after 3 days, thereby indicating 100% dissipated (Kar et al. 2013). On the other hand, the initial deposits of chlorantraniliprole residues in cabbage heads when applied at 0.20 ml L− 1 on 0 day were 0.81 mg kg− 1 and soon reached BDL on 3rd day, thereby showing 100% dissipation (Kabadad and Gali 2018). The dissipation of chlorantraniliprole 18.5 SC when sprayed @ 10 g a.i. ha− 1 two times at an interval of 10 days on cabbage where the mean initial deposit of chlorantraniliprole after the second spray was 0.24 µg g− 1 at 0 day. The residues declined to 0.17, 0.10 and 0.06 µg g− 1 on 1, 3 and 5 days after treatment with dissipation percentages of 30.92, 59.28 and 74.64 percent respectively. On the 7th day, the residue reached BDL of less than 0.05 µg g− 1 (Preethi et al. 2019).
Half-life values of insecticidal residues
The degradation kinetics of the residues of chlorantraniliprole, fipronil and deltamethrin were determined by plotting the concentration of residue against time, and the maximum squares of correlation coefficients found were used to determine the equations of best-fit curves. Confirmation of the first order kinetics was further made graphically from the linearity of the plots of logC against time (C = residues × 100). The residues of chlorantraniliprole followed first order kinetics (R2) with the values of 0.993. However, the residues of fipronil and deltamethrin do not follow first-order kinetics with the R2 values of 0.988 and 0.987 respectively. According to Hoskins (1961), the calculated half-days for chlorantraniliprole, fipronil and deltamethrin were 1.81, 1.07 and 1.42 days respectively.
Similar results were also observed in other crops like brinjal and okra fruits where the initial deposit of chlorantraniliprole were found to be 0.72 and 0.48 µg g− 1 on brinjal and okra respectively when treated at same doses of 30 g a.i. ha− 1. The half-lives of okra and brinjal were reported as 1.58 and 1.60 days respectively (Regupathy and Dham 2001). The residues of chlorantraniliprole on cauliflower were studied by applying @ 9.25 g a.i. ha− 1. The half-life value was found to be 1.36 days (Ioriatti et al. 2009). Similar findings were also observed by three applications of chlorantraniliprole 18.5 SC at 9.25 g a.i. ha− 1. The same half-life value (T1/2) of chlorantraniliprole was worked out to be 1.36 days and recommended for safe consumption of cauliflower curds after waiting 1 day (Kar et al. 2013). Dissipation of chlorantraniliprole 18.5 SC followed first-order reaction kinetics and the calculated half-lives were 2.29 days when treatment of 10 g a.i. ha− 1 two times at an interval of 10 days on cabbage (Preethi et al. 2019). The half-life of fipronil was found to be 3.43 days when sprayed on cabbage @ 75 g a.i. ha− 1 (Bhardwaj et al. 2012). The residues of deltamethrin were checked on spring cabbage, Brassica oleracea L. var. capitate by applying the recommended dose. The half-life values of the insecticide were estimated as 1.5 days (Zhang et al. 2007). On the other hand, the half-life of fipronil when applied @ 75 and 150 g a.i. ha− 1 at 7-day intervals was found to be 3.43 and 3.21 days respectively (Bhardwaj et al. 2012). Similar findings were observed in cabbage with three sprays of fipronil @ 100 g a.i. ha− 1 at the head formation stage where the half-life values for fipronil were observed as 3.13 days (Reddy et al. 2012).
The present study revealed the dissipation percentage of 99% is achieved for fipronil on the 7th day in cabbage following two sprays of recommended doses. The dissipation is somewhat found to be higher in diamide insecticide, chlorantraniliprole and the synthetic pyrethroid, deltamethrin. Chlorantraniliprole and deltamethrin residues reached 100% dissipation on the 7th day.
Acknowledgements
The authors are thankful to the Director, ICAR Research Complex for NEH Region, Umiam for providing the necessary research facilities.
Disclosure Statement
No potential conflict of interest was reported by the author(s).
- Bentley KS, Fletcher JL, Woodward MD (2010) “Chlorantraniliprole: An Insecticide of the Anthranilic Diamide Class”. Hayes' Handbook of Pesticide Toxicology (Third Edition). Pp. 2231–2242.
- Bhardwaj U, Kumar R, Kaur S, Sahoo Mandal SK, Battu RS, Singh B (2012) “Persistence of fipronil and its risk assessment on cabbage, Brassica oleracea var. capitata L”. Ecotoxicol Environ Saf 79(1): 301–308.
- Delpech RV, Matsuda K, Sattelle BM, Rauh JJ, Sattelle DB (2005) "Ion channels: molecular targets of neuroactive insecticides". Invert Neurosci 5 (3–4): 119–133. doi:10.1007/s10158-005-0004-9. ISSN 1354–2516. PMID 16172884. S2CID 12654353.
- Doi H, Kikuchi H, Murai H, Kawano Y, Shigeto H, Ohyagi Y, Kira J (2006) “Motor neuron disorder simulating ALS induced by chronic inhalation of pyrethroid insecticides”. Neurology 67(10):1894–1895. doi:10.1212/01.wnl.0000244489.65670.9f. PMID 17130437.
- Ejobi F, Kanja LW, Kyule MN, Muller P, Kruger J, Latigo AAR (1996) “Organochlorine pesticide residues in mother’s milk in Uganda.” Bull Environ Contam Toxicol 56: 873–880.
- Hoskins WM (1961) “Mathematical treatment of the rate of loss of pesticide residues”. Proc Natl Acad Sci 9: 163–168.
- Ioriatti C, Anfora G, Angeli G, Mazzoni V, Trona F (2009) “Effects of chlorantraniliprole on eggs and larvae of Lobesia botrana (Denis & Schiffermüller) (Lepidoptera: Tortricidae)”. Pest Manag Sci: formerly Pestic Sci 65(6):717–22.
- Kabadad U, Gali SK (2018) “Persistence and dissipation of chlorantraniliprole residues in cabbage (Brassica oleracea L.) and soil”. International Journal of Chemical Studies. 6(4): 3257–3260.
- Kar A, Mandal K, Singh B (2013) “Environmental fate of chlorantraniliprole residues on cauliflower using QuEChERS technique”. Environ Monit Assess 185: 1255–1263.
- Lahm GP, Stevenson TM, Selby TP, Freudenberger JH, Cordova D, Flexner L (2007) “Rynaxypyr™: a new insecticidal anthranilic diamide that acts as a potent and selective ryanodine receptor activator”. Bioorganic & Medicinal Chemistry Letters. 17(22):6274–6279.
- Lal OP (1975) “A compendium of insect pest of vegetables in India”. Bull Entomol Res 16: 31–56.
- Mariappan P, Kaithamalai B (2020) “Dissipation kinetics, decontamination and risk assessment of chlorantraniliprole in okra and soil under open field condition using GC-MS”. Int J Environ Chem https://doi.org/10.1080/03067319.2020.1772776.
- Preethi S, Vinothkumar B, Bhuvaneswari K, Paramasivam M (2019) “Dissipation pattern of chlorantraniliprole in/on cabbage”. Int J Chem Stud 7(3): 4791–4795.
- Reddy CN, Reddy DJ, Rahman SMAS (2012) “Persistence of fipronil and bifenthrin residues in cabbage”. Int J Bio-resour Stress Manag 3(1):073–075.
- Regupathy A, Dhamu KP (2001) “Statistics work book for Insecticide Toxicology”. Second Edn. Softech Publishers, Coimbatore. 206
- Singh PP, Singh B, Battu RS (1990) “Residues of cypermethrin, fenvalerate and deltamethrin on cauliflower”. Phytoparasitica 18: 153–158.
- Zhang ZY, Liu XJ, Yu XY, Zhang CZ, Hong XY (2007) “Pesticide residues in the spring cabbage (Brassica oleracea L. var. capitata) grown in open field”. Food Control 18 (6): 723–730.
No competing interests reported.