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Residues and Safety Evaluation of Etoxazole, Bifenazate and Its Metabolite Bifenazate-diazene in Citrus Under Open-Field Conditions


The residues of bifenazate (sum of bifenazate and bifenazate-diazene) and etoxazole in whole citrus and pulp collected from twelve regions of China were monitored and their chronic dietary risk to consumer were also evaluated. The citrus samples were extracted by a QuEChERS (quick, easy, cheap, effective, rugged, and safe) method, and analyzed by high performance liquid chromatography-tandem mass spectrometry (HPLC–MS/MS). The average recoveries of target compounds were ranged from 83 to 100% with relative standard deviations (RSDs) of 0.59–11.8%. The limits of quantification (LOQs) for three analytes were 0.01 mg/kg. At the interval to harvest of 20 and 30 days, the residues of total bifenazate and etoxazole were from below 0.02 to 0.26 mg/kg and from below 0.01 to 0.30 mg/kg in citrus samples. The chronic risk quotients (RQs) were below 100%, indicating no unacceptable risk to consumers.

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  1. Anastassiades M, Lehotay SJ, Stajnbaher D, Schenck FJ (2003) Fast and easy multiresidue method employing acetonitrile extraction/partitioning and dispersive solid-phase extraction for the determination of pesticide residues in produce. J AOAC Int 86(2):412–431.

    CAS  Article  Google Scholar 

  2. Chen H, Li W, Guo L, Weng H, Wei Y, Guo Q (2019) Residue, dissipation, and safety evaluation of etoxazole and pyridaben in Goji berry under open-field conditions in the China’s Qinghai-Tibet Plateau. Environ Monit Assess 191(8):517.

    CAS  Article  Google Scholar 

  3. CNS (2012) The chinese dietary guidelines. People’s Publishing House, Beijing

    Google Scholar 

  4. Dekeyser MA (2005) Acaricide mode of action. Pest Manag Sci 61(2):103–110.

    CAS  Article  Google Scholar 

  5. European Food Safety, Authority (2012) Reasoned opinion on the review of the existing maximum residue levels (MRLs) for etoxazole according to Article 12 of Regulation (EC) No 396/2005. EFSA J.

    Article  Google Scholar 

  6. European Food Safety Authority, Anastassiadou M, Brancato A, Carrasco Cabrera L, Ferreira L, Greco L, Jarrah S, Kazocina A, Leuschner R, Magrans JO, Miron I, Nave S, Pedersen R, Reich H, Rojas A, Sacchi A, Santos M, Stanek A, Theobald A, Vagenende B, Verani A (2019) Modification of the existing maximum residue level for bifenazate in elderberries. EFSA J 17(11):e05878.

    Article  Google Scholar 

  7. European Food Safety Authority, Brancato A, Brocca D, De Lentdecker C, Erdos Z, Ferreira L, Greco L, Janossy J, Jarrah S, Kardassi D, Leuschner R, Lythgo C, Medina P, Miron I, Molnar T, Nougadere A, Pedersen R, Reich H, Sacchi A, Santos M, Stanek A, Sturma J, Tarazona J, Theobald A, Vagenende B, Verani A, Villamar-Bouza L (2017) Modification of the existing maximum residue level for bifenazate in soya bean. EFSA J 15(9):e04983.

    CAS  Article  Google Scholar 

  8. FAO. (2006a) Pesticide residues in food 2006. Accessed 12 October 2006

  9. FAO. (2006b) Updating the principles and methods of risk assessment: MRLs for pesticides and veterinary drugs (Rome). Accessed 23 January 2006

  10. FAO. (2010a) BIFENAZATE (219). Accessed 09 July 2010

  11. FAO. (2010b) ETOXAZOLE (241). Accessed 09 July 2010

  12. Gómez-Almenar MC, García-Mesa JA (2015) Determination of pesticide residues in olives by liquid extraction surface analysis followed by liquid chromatography/tandem mass spectrometry. Grasas Aceites.

    Article  Google Scholar 

  13. Ham J, You S, Lim W, Song G (2020) Etoxazole induces testicular malfunction in mice by dysregulating mitochondrial function and calcium homeostasis. Environ Pollut 263(Pt A):114573.

    CAS  Article  Google Scholar 

  14. Hayward DG, Wong JW, Park HY (2015) Determinations for pesticides on black, green, oolong, and white teas by gas chromatography triple-quadrupole mass spectrometry. J Agric Food Chem 63(37):8116–8124.

    CAS  Article  Google Scholar 

  15. Hiragaki S, Kobayashi T, Ochiai N, Toshima K, Dekeyser MA, Matsuda K, Takeda M (2012) A novel action of highly specific acaricide; bifenazate as a synergist for a GABA-gated chloride channel of Tetranychus urticae [Acari: Tetranychidae]. Neurotoxicology 33(3):307–313.

    CAS  Article  Google Scholar 

  16. IUPAC. (2021) Pesticides properties database. Accessed 28 April 2021

  17. Lin H, Liu L, Zhang Y, Shao H, Li H, Li N, Zou P, Lu N, Guo Y (2020) Residue behavior and dietary risk assessment of spinetoram (XDE-175-J/L) and its two metabolites in cauliflower using QuEChERS method coupled with UPLC-MS/MS. Ecotoxicol Environ Saf 202:110942.

    CAS  Article  Google Scholar 

  18. Liu S, Kou H, Mu B, Wang J, Zhang Z (2019) Dietary risk evaluation of tetraconazole and bifenazate residues in fresh strawberry from protected field in North China. Regul Toxicol Pharmacol 106:1–6.

    CAS  Article  Google Scholar 

  19. Ma J, Huang Y, Peng Y, Xu Z, Wang Z, Chen X, Xie S, Jiang P, Zhong K, Lu H (2021) Bifenazate exposure induces cardiotoxicity in zebrafish embryos. Environ Pollut 274:116539.

    CAS  Article  Google Scholar 

  20. Malhat F, Hassan A (2011) Level and fate of etoxazole in green bean (Phaseolus vulgaris). Bull Environ Contam Toxicol 87(2):190–193.

    CAS  Article  Google Scholar 

  21. Ochiai N, Mizuno M, Mimori N, Miyake T, Dekeyser M, Canlas LJ, Takeda M (2007) Toxicity of bifenazate and its principal active metabolite, diazene, to Tetranychus urticae and Panonychus citri and their relative toxicity to the predaceous mites, Phytoseiulus persimilis and Neoseiulus californicus. Exp Appl Acarol 43(3):181–197.

    CAS  Article  Google Scholar 

  22. Park W, Lim W, Park S, Whang KY, Song G (2020) Exposure to etoxazole induces mitochondria-mediated apoptosis in porcine trophectoderm and uterine luminal epithelial cells. Environ Pollut 257:113480.

    CAS  Article  Google Scholar 

  23. Peng Y, Li M, Huang Y, Cheng B, Cao Z, Liao X, Xiong G, Liu F, Hu C, Lu H (2021) Bifenazate induces developmental and immunotoxicity in zebrafish. Chemosphere 271:129457.

    CAS  Article  Google Scholar 

  24. Perestrelo R, Silva P, Porto-Figueira P, Pereira JAM, Silva C, Medina S, Camara JS (2019) QuEChERS—fundamentals, relevant improvements, applications and future trends. Anal Chim Acta 1070:1–28.

    CAS  Article  Google Scholar 

  25. Rejczak T, Tuzimski T (2017) QuEChERS-based extraction with dispersive solid phase extraction clean-up using PSA and ZrO2-based sorbents for determination of pesticides in bovine milk samples by HPLC-DAD. Food Chem 217:225–233.

    CAS  Article  Google Scholar 

  26. Rencuzogullari E, Ila HB, Kayraldiz A, Arslan M, Diler SB, Topaktas M (2004) The genotoxic effect of the new acaricide etoxazole. Russ J Genet 40(11):1300–1304.

    Article  Google Scholar 

  27. Saber AN, Malhat F, Anagnostopoulos C, Kasiotis KM (2020) Evaluation of dissipation, unit–unit-variability and terminal residue of etoxazole residues in strawberries from two different parts in Egypt. Journal of Consumer Protection and Food Safety 15(3):229–236.

    CAS  Article  Google Scholar 

  28. SANTE/11945/2015. (2015) Analytical quality control and method validation procedures for pesticide residues and analysis in food and feed. Accessed 1 January 2016

  29. Satheshkumar A, Senthurpandian VK, Shanmugaselvan VA (2014) Dissipation kinetics of bifenazate in tea under tropical conditions. Food Chem 145:1092–1096.

    CAS  Article  Google Scholar 

  30. Sharma KK, Tripathy V, Mohapatra S, Matadha NY, Pathan ARK, Sharma BN, Dubey JK, Katna S, George T, Tayade A, Sharma K, Gupta R, Walia S (2021) Dissipation kinetics and consumer risk assessment of novaluron + lambda-cyhalothrin co-formulation in cabbage. Ecotoxicol Environ Saf 208:111494.

    CAS  Article  Google Scholar 

  31. Sun D, Pang J, Fang Q, Zhou Z, Jiao B (2016) Stereoselective toxicity of etoxazole to MCF-7 cells and its dissipation behavior in citrus and soil. Environ Sci Pollut Res Int 23(24):24731–24738.

    CAS  Article  Google Scholar 

  32. Thekkumpurath AS, Girame R, Hingmire S, Jadhav M, Jain P (2020) Residue dissipation, evaluation of processing factor and safety assessment of hexythiazox and bifenazate residues during drying of grape to raisin. Environ Sci Pollut Res Int 27(33):41816–41823.

    CAS  Article  Google Scholar 

  33. Uner N, Oruc E, Sevgiler Y (2005) Oxidative stress-related and ATPase effects of etoxazole in different tissues of Oreochromisniloticus. Environ Toxicol Pharmacol 20(1):99–106.

    CAS  Article  Google Scholar 

  34. Wang Z, Pang J, Liao C, Zhang Q, Sun D (2020) Determination of etoxazole in different parts of citrus fruit and its potential dietary exposure risk assessment. Chemosphere.

    Article  Google Scholar 

  35. Yao Z, Li Z, Zhuang S, Li X, Xu M, Lin M, Wang Q, Zhang H (2015) Enantioselective determination of acaricide etoxazole in orange pulp, peel, and whole orange by chiral liquid chromatography with tandem mass spectrometry. J Sep Sci 38(4):599–604.

    CAS  Article  Google Scholar 

  36. Zheng X, Liu C, Hu J (2020) Residues and dietary risk assessments of 2,4-D Isooctyl ester, metribuzin, acetochlor, and 2-Ethyl-6-methylaniline in corn or soybean fields. J Agric Food Chem 68(15):4315–4324.

    CAS  Article  Google Scholar 

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This work was supported by financial support from National Natural Science Foundation of China (Project No. 21677009).

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Correspondence to Jiye Hu.

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Hou, H., Yu, X., Dong, B. et al. Residues and Safety Evaluation of Etoxazole, Bifenazate and Its Metabolite Bifenazate-diazene in Citrus Under Open-Field Conditions. Bull Environ Contam Toxicol 107, 281–288 (2021).

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  • Bifenazate
  • Bifenazate-diazene
  • Etoxazole
  • Pesticide residue
  • Risk assessment