Food Safety Assessment and Residue Burden Profiling of Pesticides, Aflatoxins, and Ochratoxin-A in Three Commercial Chilli Varieties

Chilli is among the most popularly consumed and commercially exported spices from India; nevertheless, food safety investigations involving the concurrent estimation of pesticide residues, aflatoxins, and ochratoxin-A in different chilli commercial cultivars have been poorly studied. As such, this study sought to determine the food safety implications and residue burden of pesticides, aflatoxins, and ochratoxin-A in three chilli commercial cultivars, including Teja, Byadgi, and Guntur Sannam. Multi-residue determination for pesticides was done using both LC-MS and GC-MS, whereas mycotoxins were estimated using an HPLC-FLD method after immunoaffinity column clean-up. A total of 28 pesticide residues and 4 mycotoxin parameters were studied in a residue burden model and Kruskal-Wallis statistics. Complete detection of all pesticides and mycotoxins was observed in all varieties, suggesting extensive chemical and fungal contamination of chilli. Among the cultivars, Teja had the highest total pesticide burden of 3.6834 mg/kg and mean pesticide content of 0.1316 mg/kg, mainly attributed to acetamiprid, prochloraz, imidacloprid, thiamethoxam, and clothianidin. Conversely, Guntur Sannam demonstrated a high mycotoxin burden of 32.3801 µg/kg with a mean of 8.0950 µg/kg, signifying a serious aflatoxin and ochratoxin-A contamination problem. However, the Byadgi cultivar showed relatively low pesticide burden of 1.9521 mg/kg and a mycotoxin burden of 5.4957 µg/kg. In total, nine pesticide residues and three mycotoxins were significantly associated across varieties (p<0.05) via Kruskal-Wallis analysis.

Chilli varieties; Pesticide residues; Aflatoxins; Ochratoxin-A; Food safety; Residue burden

1. Spices Board of India, *Annual Report 2023–24*. Cochin, India: Ministry of Commerce and Industry, Government of India, 2024. [Google Scholar] [Crossref]

2. European Commission, *Rapid Alert System for Food and Feed (RASFF) Annual Report 2023*. Brussels, Belgium: Directorate-General for Health and Food Safety, 2024. [Google Scholar] [Crossref]

3. World Health Organization, *The WHO Recommended Classification of Pesticides by Hazard and Guidelines to Classification*, 2019 ed. Geneva, Switzerland: World Health Organization, 2020. [Google Scholar] [Crossref]

4. European Food Safety Authority (EFSA), “The 2021 European Union report on pesticide residues in food,” *EFSA Journal*, vol. 21, no. 4, Art. no. e07939, 2023. [Google Scholar] [Crossref]

5. International Agency for Research on Cancer (IARC), *Some Traditional Herbal Medicines, Some Mycotoxins, Naphthalene and Styrene*, IARC Monographs on the Evaluation of Carcinogenic Risks to Humans, vol. 82. Lyon, France: IARC, 2002. [Google Scholar] [Crossref]

6. European Commission, “Commission Regulation (EU) 2023/915 of 25 April 2023 on maximum levels for certain contaminants in food,” *Official Journal of the European Union*, L 119, May 5, 2023. [Google Scholar] [Crossref]

7. Food Safety and Standards Authority of India (FSSAI), *Food Safety and Standards (Contaminants, Toxins and Residues) Regulations, 2011*, as amended. New Delhi, India: FSSAI, 2023. [Google Scholar] [Crossref]

8. Joint FAO/WHO Meeting on Pesticide Residues (JMPR), *Pesticide Residues in Food—Report of the Joint FAO/WHO Meeting*, FAO Plant Production and Protection Paper. Rome, Italy: Food and Agriculture Organization of the United Nations, 2022. [Google Scholar] [Crossref]

10. DIN EN 14132:2008, *Foodstuffs—Determination of Aflatoxin B1, B2, G1 and G2 in Hazelnuts, Peanuts, Pistachios, Figs and Paprika Powder—High-Performance Liquid Chromatographic Method with Post-Column Derivatization and Immunoaffinity Column Clean-Up*. Berlin, Germany: German Institute for Standardization, 2008. [Google Scholar] [Crossref]

11. K. R. N. Reddy, B. Salleh, B. Saad, H. K. Abbas, C. A. Abel, and W. T. Shier, “An overview of mycotoxin contamination in foods and its implications for human health,” *Toxin Reviews*, vol. 29, no. 1, pp. 3–26, 2010. [Google Scholar] [Crossref]

12. M. R. Jang, E. H. Kim, J. M. Shin, Y. H. Park, H. W. Park, J. K. Kim, M. S. Hong, I. S. Yu, and Y. S. Shin, “Evaluation of residual pesticides in dried chili peppers and chili powders using LC-MS/MS,” *Journal of Food Hygiene and Safety*, vol. 36, no. 1, pp. 9–16, 2021, doi: 10.13103/JFHS.2021.36.1.9. [Google Scholar] [Crossref]

13. D. Lee, H. Kim, J. Lee, S. Kim, and J. H. Shim, “Analysis of 207 residual pesticides in hot pepper powder using LC-MS/MS and GC-MS/MS,” *Food Science and Biotechnology*, 2023. [Google Scholar] [Crossref]

14. M. Dhanshetty, S. Banerjee, A. K. Singh, and D. K. Sharma, “Analysis of aflatoxins and ochratoxin A in chilli powder using ultrahigh performance liquid chromatography with fluorescence detection and tandem mass spectrometry,” *Journal of Food Composition and Analysis*, 2022. [Google Scholar] [Crossref]

15. Z. Zareshahrabadi, R. Bahmyari, H. Nouraei, H. Khodadadi, P. Mehryar, F. Asadian, and K. Zomorodian, “Detection of aflatoxin and ochratoxin A in spices by high-performance liquid chromatography,” *Journal of Food Quality*, vol. 2020, Art. no. 8858889, 2020, doi: 10.1155/2020/8858889. [Google Scholar] [Crossref]

16. D. Pickova, J. Toman, V. Ostry, and F. Malir, “Natural occurrence of ochratoxin A in spices marketed in the Czech Republic during 2019–2020,” *Foods*, vol. 10, no. 12, Art. no. 2984, 2021, doi: 10.3390/foods10122984. [Google Scholar] [Crossref]

17. M. Anastassiades, S. J. Lehotay, D. Štajnbaher, and F. J. Schenck, “Fast and easy multiresidue method employing acetonitrile extraction/partitioning and dispersive solid-phase extraction for the determination of pesticide residues in produce,” *Journal of AOAC International*, vol. 86, no. 2, pp. 412–431, 2003. [Google Scholar] [Crossref]

18. S. J. Lehotay, “Determination of pesticide residues in foods by acetonitrile extraction and partitioning with magnesium sulfate: Collaborative study,” *Journal of AOAC International*, vol. 90, no. 2, pp. 485–520, 2007, doi: 10.1093/jaoac/90.2.485. [Google Scholar] [Crossref]

19. European Commission, “Analytical quality control and method validation procedures for pesticide residues analysis in food and feed,” Document No. SANTE/11312/2021, 2021. [Google Scholar] [Crossref]

20. J. H. Shim, D. Lee, H. Kim, and J. Lee, “A comprehensive review of pesticide residues in peppers,” *Foods*, vol. 12, no. 6, Art. no. 1264, 2023. [Google Scholar] [Crossref]

21. European Parliament and Council of the European Union, “Regulation (EC) No. 396/2005 of 23 February 2005 on maximum residue levels of pesticides in or on food and feed of plant and animal origin,” *Official Journal of the European Union*, 2005. [Google Scholar] [Crossref]

22. AOAC International, *Official Methods of Analysis of AOAC International*, 21st ed. Rockville, MD, USA: AOAC International, 2019. [Google Scholar] [Crossref]

23. ISO/IEC 17025:2017, *General Requirements for the Competence of Testing and Calibration Laboratories*. Geneva, Switzerland: International Organization for Standardization, 2017. [Google Scholar] [Crossref]

24. European Parliament and Council of the European Union, “Regulation (EC) No. 882/2004 of 29 April 2004 on official controls performed to ensure the verification of compliance with feed and food law, animal health and animal welfare rules,” *Official Journal of the European Union*, L 165, Apr. 30, 2004. [Google Scholar] [Crossref]