Persistence and safety risk assessment of pyridalyl in cabbage and tomato Essay

Persistence and safety risk assessment of pyridalyl in cabbage and tomato

Abstract

A field experiment had been set up to evaluate persistence behaviour of pyridalyl in tomato, cabbage and field soil over two seasons. An analytical method was developed to analyse pyridalyl residues in different substrates and duly validated based on single laboratory method validation criteria. Pyridalyl residues were detected and quantified using Gas Chromatograph equipped with Electron Capture Detector. The compound showed less persistence in tomato, cabbage and soil. A safe waiting period of 17-18 d after final insecticide application needs to be maintained before harvesting of the crop. Both dietary and soil ecological risk assessment was done and found that the harvested vegetables were toxicologically safe for consumption at recommended dose of pyridalyl. However, there was raised concern for the insecticidal toxicity against the algal population of soil which needs to be reconfirmed in further studies.

Keywords: Pyridalyl. Cabbage. Tomato. Persistence. Risk Assessment

1. INTRODUCTION

Pesticide poisoning is a serious concern in modern agriculture. Although these hazardous compounds seem to be indispensible inputs in crop production; the ill effect of pesticides in human body is now a proven fact. Study of pesticide dissipation in various environmental substrates is very important to judge how long a particular compound can persist in soil, sediments, water etc., and its potency to create toxicity risk. Although, new chemistry molecules have been developed which are less persistent and bio-degradable in nature, there behavior at different environmental conditions needs to be addressed before coming to any conclusion. The present study is about the persistence behavior of a new generation insecticide, known as pyridalyl [2, 6-dichloro-4-(3, 3-dichloroallyloxy) phenyl 3-[5-(trifluoromethyl)-2-pyridyloxy] propyl ether] in important crops like cabbage and tomato in Indian condition.

Cabbage (Brassica oleracea L. var. Capitata), one of the most important vegetable crops, is widely grown and popular in almost all the regions of the country. In India, it contributed 5.5% to the national vegetable production with 90.39 lakh tonnes production and 22.6 MT/ha productivity during 2013-14 (Anonymous 2015). During growing stage it is attacked by different insect pests viz. diamond back moth (Plutella xylostella Linnaeus.), crucifer leaf web worm (Crocidolomia binotalis Zeller), cabbage borer (Hellula undalis Fabricius), cabbage butterfly (Pieris brssicae Linnaeus), cabbage semilooper (Thysanoplusia orichalcea Fabricius), Spodoptera litura which causing severe yield loss to the crop every year. In India, diamondback moth has national importance on cabbage as it causes 50-80% annual loss in the marketable yield (Devjani and Singh 1999; Ayalew 2006) and a loss of US $ 16 million every year (Mohan and Gujar 2003).

Tomato, Lycopersicon esculentum Mill. (Solanaceae) is another popular vegetable for its outstanding antioxidant content. Though, it is extensively grown all over the country with 187.35 lakh tonnes production and shared 11.5% to nation vegetable production but the productivity remained as low as of 21.2 MT/ha during 2013-14 (Anonymous 2015). Insect pests attack is a major constraint for commercial tomato cultivation. The major damage is caused by fruit borer (Helicoverpa armigera), tobacco caterpillar (Spodoptera litura), serpentine leaf miner (Liriomyza trifoli) and white flies (Bemisia tabaci). However, fruit borer (Helicoverpa armigera) is an important pest which causes considerable losses in quantity as well as quality of tomato fruits. It is reported that H. armigera causes yield loss in tomato to the extent of 22–38% (Dhandapani et al. 2003) or one thousand crores rupees per annum (Padmanaban and Arora 2002).

Pyridalyl is a novel insecticide invented and developed by Sumitomo Chemical Co., Ltd. This novel insecticide exerts excellent control against lepidopteron and thysanopteran pests on cotton, vegetable and fruits (Sakamoto et al. 1995; Sakamoto and Umeda 2003). It has dichloropropenyl, phenyl and pyridyl groups in its structure (Fig. 1) but does not share structural similarity with other insecticides. Pyridalyl acts by contact and ingestion and shows cytotoxicity to cells of lepidopteron insects (Anonymous 2009). Its biochemical mode of action is not fully elucidated; hence, it is currently not allocated to any class of insecticides. Many existing resistant as well as susceptible strains of lepidopteron pests can be adequately controlled by pyridalyl. Its efficacy was also reported against populations of Heliothis virescens, Helicoverpa zea (Johnson 2000) and Plutella xylostella (Umeda and Strickland 1999) which are resistant to various currently used insecticides. But before recommending pyridalyl application in cabbage and tomato to control lepidopteran insect-pests in India, it is obvious to find out its persistence in both the crops and soil. The common practice of Indian farmers is to harvest the crop just even after insecticide application for the sake of fetching higher prices in the market. Besides cooked as curry, both tomato and cabbage are consumed as fresh raw salad which might cause risky exposure of pyridalyl to the consumer. Hence, both dietary and soil ecological risk assessment of the insecticide are very much important from consumer and environmental safety point of view. To the best of our knowledge, persistence of pyridalyl in agricultural food crops in Indian condition are limited. Therefore, the present study has been designed to find out the persistence behavior and risk assessment of pyridalyl in both the vegetables over two seasons.

2. MATERIALS AND METHODS

2.1. Chemicals and reagents

Analytical standard of pyridalyl (purity 99.3%) and its formulation pyridalyl 10% EC were obtained from Sumitomo Chemical Co. Ltd., Japan. All the solvents and reagents used in the study were of analytical grade. Stock standard solution (100 mg L-1) of pyridalyl was prepared by dissolving 100 mg analytical standard of pyridalyl in 1 L ethyl acetate. As and when required, working standard was prepared through serial dilution of stock solution.

2.2. Field experiment

Field experiments were conducted during two seasons (I & II; rabi) at Kalyani, West Bengal, India (22˚58´60 N latitude, 88˚28´60 E longitude at an altitude of 9.75 m from mean sea level) to find out the residual fate of pyridalyl in cabbage, tomato and soil. The soil was sandy loam with average pH 6.88, CEC [c mol (p+)/kg] 13.29, organic carbon (g/kg) 5.88. Pyridalyl was applied twice at recommended dose (T1; 75 g a.i. ha-1) and double the recommended dose (T2; 150 g a.i. ha-1) on both the crops at 15 days interval with untreated control (T3; only water spray). The average rainfall (mm) values during tomato field experiment were 56.50 and nil for season I and II respectively and corresponding values during cabbage field experiment were 4.50 and 14.50.

2.3. Sample collection

Cabbage, tomato and soil samples were collected randomly from each plot at 0 (2 h), 1, 3, 7, 10 and 14 d after final insecticide application in each season. Soil samples were collected from 15 cm depth with the help of soil auger. All three samples were also collected at final harvest.

2.4. Extraction and cleanup

Cabbage sample was chopped into small pieces and homogenized (in Polytron homogeizer; PT 3100) for 4-5 min. After that, a representative 20 g sample was placed into 250 mL conical flask and 100 mL mixture of acetone and water (9:1) was added. The flask was shaken for 1 h in a mechanical shaker. Then the sample was filtered through a buchner funnel using 30 mL acetone for two times. The filtrate was concentrated to about 20 mL using rotary vacuum evaporator at 40 0C and transferred to a 1 L separatory funnel. Then 300 mL distilled water along with 50 mL saturated sodium chloride solution were added to the extract. The combined mixture was partitioned thrice with hexane at the volume of 100 mL, 50 mL and 50 mL respectively. After that, the hexane layer was collected over anhydrous sodium sulphate and evaporated to dryness on rotary vacuum evaporator at 40 0C. Finally, the extract was concentrated to 3 mL for column chromatography.

A suitable chromatographic glass column was packed with 10 g florisil in between two layers of anhydrous sodium sulphate (2 g) using packing solvent as hexane. Then the column was eluted with 30 mL mixture of acetone and hexane (30:70). The eluted mixture was evaporated to dryness on a vacuum rotary evaporator at 40 0C and the volume was reconstituted in ethyl acetate (HPLC grade) for analysis in gas chromatography coupled with electron capture detector (ECD). Tomato samples were analyzed following same method as described for cabbage samples.

In case of soil, 50 g representative sample was placed into a 250 mL conical flask and 100 mL mixture of acetone and water (9:1) was added to it and shaken for 1 h on a mechanical shaker. Then soil samples were extracted and cleaned up using same method employed for extraction of cabbage and tomato sample.

2.5. Instrument parameters

Residues of pyridalyl in cabbage, tomato and soil were estimated by Gas Chromatograph (GC) (Model Agilent 6890N, Chemstation software) equipped with Electron Capture Detector (ECD), auto sampler G2614A and HP-5 column (length 32 m, ID 0.32 mm, film thickness 0.25 µm). Sample was injected @ 2 µl with a split ratio of 10:1. Injector and detector temperature were set as 275 0C and 320 0C respectively. Oven temperature was initially set as 240 0C which was increased further @ 10 0C min-1 to 285 0C and held for 7 min. High purity nitrogen was used as carrier gas with a flow rate of 2 ml min-1. Pyridalyl was detected at 5.29±0.02 min in these operating conditions.

2.6. Method validation

2.6.1. Calibration (Linearity)

A calibration curve was formulated by plotting the concentration of analytical standard of pyridalyl (at seven levels of concentration i.e., 0.01, 0.02, 0.03, 0.05, 0.10, 0.20 and 0.50 µg ml-1; X-axis) against the corresponding peak area (mean of six replicates; Y-axis) to judge the linearity of the method.

2.6.2. Recovery study (Trueness)

Untreated (pyridalyl free) samples of tomato, cabbage and soil in six replicates were fortified separately with pyridalyl at three fortification levels, i.e. 0.03, 0.15 and 0.30 µg g-1. The samples were then processed as per same method described in section 2.4. Mean recovery between 70-120% were in well agreement with the acceptance criteria as mentioned by European Union (EU) Guideline (DG-SANTE, 2017).

2.6.3. Repeatability and reproducibility

Repeatability and reproducibility of a method can be evaluated by calculating Relative Standard Deviation (RSD) values. The RSD value is calculated by dividing Standard Deviation (SD) by mean value of the recovery and expressed in percentage. The Repeatability Standard Deviation (RSDr) and Reproducibility Standard Deviation (RSDR) were worked out by analyzing the sample on same day and on three different days respectively. Ten numbers of replicates were being injected in this case.

2.6.5. Matrix effect

Matrix Effect (ME) of the method can be determined by comparing the peak area of the analytical standard in pure solvent (ethyl acetate) with the peak area of the analyte spiked in blank matrix (matrix matched standard) at three fortification levels, i.e. 0.030, 0.150 and 0.300 µg g-1 and expressed in percentage. It can be calculated using the formula: ME = [(peak area of the matrix-matched standard – peak area of the pure analytical standard)/ peak area of the pure analytical standard] X 100. The positive and negative ME values denote matrix-induced enhancement and suppression respectively.

2.6.6. Limit of Detection (LOD) and Limit of Quantification (LOQ)

LOD is the minimum analyte concentration that can be detected in a particular instrument where LOC is the lowest analyte concentration that can be quantified with desired accuracy and precision under certain experimental condition. Both these limits can be determined by using the following formula: LOD = 2 hnCs/hs and LOQ = 6 hnCs/hs (Francotte et al., 1996) where hn = highest deviation of signal from the mean baseline, Cs = injected amount of the analytical standard, hs = peak height of the analytical standard measured from mean baseline to the top of the peak. Likewise, these limits were determined for pyridalyl by injecting different concentration of the analytes following serial dilution.

2.7. Data analysis

Dissipation of pyridalyl in cabbage and tomato followed first order kinetics. The regression equation so derived, can be expressed as: Ct=C0e-kt where Ct stands for residue concentration (μg g−1) at time t (days) after insecticide application, k stands for the dissipation rate constant or the slope of the regression equation and C0 represents initial deposit concentration (μg g−1) at zero time (day of application). The residual half-life (RL50) in days was estimated using the above equation was: RL50 = ln 2/k

Pre-harvest interval (PHI) or safe waiting period is the minimum time (days) interval between pesticide application and crop harvest by which the pesticide residues decreased below the tolerance limit. It was estimated using the following equation: PHI = [ln C0 - ln MRL]/k. MRL of pyridalyl in cabbage and tomato was considered as 0.02 mg kg−1 (FSSAI, 2018).

2.8. Risk assessment

2.8.1. Dietary risk assessment

The Estimated Daily Intake (EDI) of pyridalyl was estimated by multiplying the residue level in cabbage and tomato with average recommended vegetable consumption per day per body weight. The ARfD (Acute Reference Dose) value of pyridalyl is not applicable thereby nullifies the opportunity to assess short term exposure (EU – Pesticide Database, 2013). Long-term dietary Risk Quotient (RQd) was measured by using the following formula: RQd = EDI / (ADI X Average body weight) where ADI is Acceptable Daily Intake expressed in mg kg-1 body weight (bw) day-1. The risk may prevail when RQd is more than 1 and vice versa. ADI value of pyridalyl is considered as 0.03 mg kg-1 bw day-1 (EU – Pesticide Database, 2013). The recommended vegetable consumption of an Indian adult is 300 g d-1 (Krishnaswamy K, 2011) and his average body weight is considered as 55 kg (Mukherjee I & Gopal M, 2000).

2.8.2. Soil ecological risk assessment

Soil ecological Risk Quotient (RQs) was determined based on the available data on algae, earthworm and other arthropods by following the procedure described in Technical Guidance Document on Risk Assessment (European Communities, 2003). Acute 72 h EC50 value for algae, acute 14 d LC50 for earthworm and LR50 value for other arthropods were considered for determining the RQs. Predicted No Effect Concentration (PNEC) values were determined by dividing the corresponding toxicity value with the Assessment factor (AF) i.e. 1000 for this case. Thereafter, RQs values were determined by the following equation: RQs = EC/PNEC (Ccanccapa, 2016), where EC = Effective Concentration of pesticide i.e. pyridalyl for this case in soil. If RQs > 1, then there could be unacceptable risk expected for the presence of pyridalyl in soil ecosystem. Conversely, the risk could be low if the RQs < 0.1 and could be moderate if RQs value remains in between 0.1 to 1.0.

The EC/EDI value was considered as half of the LOQ where the pyridalyl concentration was not detected (ND) i.e. below LOQ for both dietary and soil ecological risk assessment (USEPA, 2000).

3. RESULTS AND DISCUSSION

3.1. Performance of analytical method

The analytical method was validated by meeting up the criteria mentioned in single laboratory method validation approach (DG-SANTE, 2017). Pyridalyl exhibited acceptable linearity with wide range of concentration as plotted in calibration curve (Fig. 1). Data showed in table 1 reveals that performance of the adopted method was highly satisfactory as the average recovery and matrix effect values were within acceptable range irrespective of substrate and level of fortification. High level of precision was observed as both the RSDr and RSDR values were well below the acceptable limit (Accessed on 15th December, 2017.

Dhandapani, N., Umeshchandra, S. R., & Murugan, M. (2003). Biointensive pest management (BIPM) in major vegetable crops: an Indian perspective. Journal of Food Agriculture and Environment, 1, 333–339.

EU- Pesticide Database, (2013) Review report for the active substance pyridalyl, SANCO/12072/2013 rev 4 December 2013 http://ec.europa.eu/food/plant/pesticides/eu-pesticides-database/public/?event=activesubstance.detail&language=EN&selectedID=1801

Francotte, E., Davatz, A., Richert, P., 1996. Development and validation of chiral high performance liquid chromatographic methods for the quantitation of valsartan and of the tosylate of valinebenzyl ester. J. Chromatogr. B. Biomed. Sci. Appl.686, 77e83.

FSSAI. (2018). Food Safety and Standards (Contaminants, toxins and Residues) Third Amendment Regulations, 2018 (REGD. NO. D. L.-33004/99), No. 537] New Delhi, Monday, December 24, 2018/PAUSHA 03, 1940

Isayama, S., Saito, S., Kuroda, K., Umeda, K., & Kasamatsu, K. (2005). Pyridalyl, a novel insecticide: potency and insecticidal selectivity. Archives of Insect Biochemistry and Physiology, 58(4), 226-33.

Johnson, D. R., Lorenz, G. M., Hopkins, J. D., & Page, L. M. (2000). In: Proceedings of the 2000 Cotton Research Meeting and Summaries of Cotton Research in Progress. Derrick, M. Oosterhuis (Eds.). Arkansas Agricultural Experimental Station Fayetteville, Arkansas 72701, 260-265.

Krishnaswamy K. 2011. Dietary Guidelines for Indians—A Manual, 2nd ed. National Institute of Nutrition, Indian Council of Medical Research, Hyderabad, India.

Kumari, B., Madan, V. K., Kumar, R., & Kathpal, T. S. (2002). Monitoring of seasonal vegetables for pesticide residues. Earth and Environmental Science, 74(3), 263–270.

Lewis, K.A., Tzilivakis, J., Warner, D. and Green, A. (2016). An international database for pesticide risk assessments and management. Human and Ecological Risk Assessment: An International Journal, 22(4), 1050-1064.http://dx.doi.org/10.1080/10807039.2015.1133242.

Mohan, M., & Gujar, G. T. (2003). Local variation in susceptibility of the diamondback moth, Plutella xylostella (Linn.) to insecticides and detoxification enzymes. Crop Protection, 22, 495-504.

Mukherjee, I., & Gopal, M. (2000). Environmental behavior and translocation of imidacloprid in eggplant, cabbage and mustard. Pest Management Science, 56, 932-936.

Narasinga Rao, B. S. (2013). Fruits, vegetables, milk and animal foods in balanced Indian diets –a critical appraisal. Bulletin of the Nutrition Foundation of India, 34.

Padmanaban, N., & Arora, R. (2002). Field evaluation of native NPV for the management of tomato fruit borer, Helicoverpa armigera. Pesticide Research Journal, 14, 113–119.

Paramasivam M, Selvi C, Chandrasekaran S. 2014. Persistence and dissipation of flubendiamide and its risk assessment on gherkin (Cucumis anguria L.). Environ Monit Assess 186:4881–4887.

Saini, P., Gopal, M., Kumar, R., Gogoi, R., & Srivastava, C. (2015). Bioefficacy evaluation and dissipation pattern of nanoformulation versus commercial formulation of pyridalyl in tomato (Solanum lycopersicum). Environmental Monitoring and Assessment, 187, 541.

Sakamoto, N., & Umeda, K. (2003). Fine Chemicals, 32 (20), 35-44.

Sakamoto, N., Matsuo, S., Suzuki, M., Hirose, T., Tsushima, K., & Umeda, W. O. (1995). Patent 9611909. Chemical Abstract, 125, 114466.

Sakamoto, N., Saito, S., Hirose, T., Suzuki, M., Matsuo, S., Izumi, K., Nagatomi, T., Ikegami, H., Umeda, K., Tsushima, K., & Matsuo, N. (2004). The discovery of pyridalyl: a novel insecticidal agent for controlling lepidopterous pests. Pest Management Science, 60(1), 25-34.

Svetlana, H., Mária, A., Sherif, B. A. G., & Andrea, P. (2013). Investigation of levels and fate of pyridalyl in fruit and vegetable samples by Fast Gas Chromatography–Mass Spectrometry. Food Analytical Methods, 6(3), 969-977.

Umeda, K., & Strickland, B. (1999). S-1812 Lepidopterous Insect Pest Control in Broccoli Study. 1999 Vegetable Report, College of Agriculture, University of Arizona, index at http://ag.arizona.edu/pubs/crops/az1143/

USEPA. (2000). United States Environmental Protection Agency Office of Pesticide Programs, Washington. Information on Assessing Exposure from Pesticides in Food-A User’s Guide available atAccessed on February 2019

Yoon, J. Y., Park1, J. H., Moon, H. R., Han, G. T., & Lee, K. S. (2013). Residue patterns of indoxacarb and pyridalyl in treated cauliflower. Agricultural Sciences, 4(3), 111-116.

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