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Author(s): Ajay Kumar Sahu*, Manish Kumar Rai*, Joyce Rai, Yaman Kumar Sahu, Deepak Kumar Sahu, Kalpana Wani, Jyoti Goswami and Chhaya Bhatt

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Address: School of Studies in chemistry Pt. Ravishankar Shukla University Raipur (Chhattisgarh), 492010, India
Chhattisgarh Council of Science and Technology, Vigyan Bhawan Vidhan Sabha Road Daldal Seoni, Raipur (Chhattisgarh) 492014, India.

Published In:   Volume - 34,      Issue - 1,     Year - 2021

Cite this article:
Sahu et al. (2021). Assessment of Cymoxanil in Soil, Water and Vegetable Samples. Journal of Ravishankar University (Part-B: Science), 34(1), pp. 80-86.

Journal of Ravishankar University–B, 34 (1), 80-86 (2021)

 Assessment of Cymoxanil in Soil, Water, and Vegetable Samples

Ajay Kumar Sahua*, Manish Kumar Raia *, Joyce Raib, Yaman Kumar Sahua, Deepak Kumar Sahua, Kalpana Wani, Jyoti Goswamia and Chhaya Bhatta

aSchool of Studies in chemistry Pt. Ravishankar Shukla University Raipur (Chhattisgarh), 492010, India.

bChhattisgarh Council of Science and Technology, Vigyan Bhawan Vidhan Sabha Road Daldal Seoni, Raipur (Chhattisgarh) 492014, India.

*Corresponding Author:,

[Received: 20 March 2021; Revised: 25 June 2021; Accepted: 28 June 2021]

Abstract. The present work describes a newly developed method for the spectrophotometric determination of cymoxanil in soil, water and vegetable samples. The detection of the target chemical substance is based on the reaction of cyanide released from the hydrolysed product of cymoxanil with potassium iodide-potassium iodate to form a blue-coloured complex in the presence of starch solution. This complex is water-soluble and shows maximum absorbance at 580 nm. For this complex, Beer's law is obeyed over the concentration range of 2-50 μg mL−1 with molar absorptivity 1.2×105 L mol-1cm-1 and Sandell’s sensitivity 1.0×10-3 µg cm-2. The reproducibility was assessed by carrying out seven days replicate analysis of a solution containing 10 µgmL-1 of cymoxanil in a final solution of a volume of 10 mL. The standard deviation and relative standard deviation for the absorbance value were found to be ± 2.9×10-3 and 1.6% respectively. The proposed method is free from the interference of other toxicants. The analytical parameters were optimized and the method was applied to the determination of cymoxanil in water, soil, and vegetable samples.

Keywords: Spectrophotometric, Cymoxanil, Potassium iodide-potassium iodate and Blue-coloured complex.


Pesticides are used worldwide for the control of insects, microorganisms, fungi, and other harmful pests to protect agricultural products. According to a U.S. Environmental Protection Agency (EPA) report, world pesticide expenditure at the producer level was $55921 million in 2012. (Shin et al 2018). The increasing vegetable intake could signicantly increase pesticide exposure and health risk in humans. The current problems in food safety and phytosanitary barriers demand rigorous control in correctly identifying and quantifying residues of pesticides present in vegetables and fruits as well as the absence of banned pesticides for certain crops (Zhao et al, 2014) India is produced about 109 million tons of vegetables and it is the second-largest producer after China and account for13.4% of world production. Surveys carried out by institutions spread during the country indicate that 50-70% of vegetables are contaminated with pesticide residues (Nirmal et al, 2017).

Cymoxanil-based fungicides have been in use for over 25 years. Today, cymoxanil is registered in over 50 countries and used on over 10 million hectares worldwide for 17 different crops. (Morrica et al, 2004) Cymoxanil, 1-(2-cyano-2-methoxyiminoacetyl)-3-ethyl urea, is a fungicidal cyanooxime effective against plant diseases caused by fungi belonging to the Perenosporales. In practice, it is mainly used against downy mildew of vine (Plasmopara Viticola) and potato blight (Phytophthora infestans). Cymoxanil is applied as a seed treatment to cut potato seed pieces or as a foliar application to the plants to control late blight (Phytophthora infectans). As well as in cucumber, tomatoes, sugar beets and grapes.  So, cymoxanil is a frequently used fungicide and it has been detected in drinking water. (Tellier et al, 2008, Rao et al, 2020, Bavol et al, 2016). Long-term consumption of food containing pesticide residues will inevitably cause great damage to the human body. Excessive intake of cymoxanil by humans will have serious consequences such as the decline of body immunity, the increase of burden on the liver, gastrointestinal diseases, and even cancer. Therefore, there is a crucial need for suitable analytical methods that could quickly detect cymoxanil residue in various foods. (Mi et al, 2021). Because of the highly toxic and widely used cymoxanil, many instrumental systems have been described for the detection or determination of cymoxanil fungicides such as ultrahigh-performance liquid chromatography coupled with electrospray ionization quadrupole Orbitrap high-resolution mass spectrometry (UHPLC/ESI Q-Orbit rap) (Wang et al, 2016), High-performance liquid chromatography (Liu et al, 2016, Balayiannis et al, 2014), Liquid chromatography-tandem mass spectrometry (Holmes et al, 2015), quick, easy, cheap, effective, rugged and safe method (QuEChERS) (Chen et al, 2020). High-performance liquid chromatography equipped utilizing ultraviolet detector lamp (HPLC-UV) (Rao et al. 2020). Some of these techniques suffer from poor sensitivity, analyses are limited to laboratory facilities and expensive due to its analytical cost and instability of colour or longer time required for colour development. To overcome these drawbacks a rapid and sensitive method has been developed for the determination of cymoxanil. UV-Visible spectrophotometry is considered the most convenient analytical technique, because of its inherent simplicity, low cost, and wide accessibility most in laboratories.

In this study, we aimed to establish a simple, sensitive, effective, and accurate method for the determination of cymoxanil. The method is based on the hydrolysis of cymoxanil and bromination reaction followed by reaction with KI and KIO3 and starch. Herein, the determination of cymoxanil in various environmental and vegetable samples using UV-Visible spectrophotometer. Firstly we have collected samples and performed hydrolysis of cymoxanil using sodium hydroxide and further its reaction with bromine water, KI and KIO3, and starch to obtain a blue colour complex.

Material and Methods


UV-Visible absorption spectrum was performed on double beam spectrophotometer make Cary -60 UV-Visible Spectrophotometer (Agilent technologies) with accuracy and quartz glass are used for all spectral measurements with 10 mm matched silica gel. Digital pH meter model pH700 EUTECH was used for pH measurements. A REMI C-854/4 clinical centrifuge having a maximum speed of 3600 rev min-1 and a maximum centrifugal force of 1850 g with fixed swing-out rotors was used for centrifugation.


All reagents used were of Analytical Reagent grade and Double Distilled water was used during the experiment.

Standard solution of cymoxanil- Cymoxanil (Isagro). A stock solution of 1 ppm cymoxanil is prepared in double-distilled water. Working standard solution was prepared by proper dilution of stock.

Sodium hydroxide- Sodium hydroxide (Loba Chemie) aqueous solution of 5 M concentration was prepared.

Bromine Water – A Saturated solution of bromine in water was prepared. This solution was prepared daily.

Formic Acid- 90% solution was prepared.

Potassium iodide-Potassium iodate mixture: prepared by mixing 0.1 mol L-1 Potassium iodide and 0.2 mol L-1Potassium iodate in a 5:1 ratio (Nirmal et al, 2014).

Starch solution- A 100 mg amount of soluble starch was made into a paste with a few drops of hot water and diluted to 100 ml using nearly boiling water. (Patel et al, 2014).

Procedure for determination of cymoxanil in soil, water and vegetable samples

The standard stock solution was prepared in ethanol at the concentration of 100µg mL-1 of cymoxanil. Working standard solutions were prepared by suitable dilution of stock solution in ethanol with different concentration level (2µgmL-1, 5µgmL-1, 10µgmL-1, 15µgmL-1, 20µgmL-1, 25µgmL-1, 30µgmL-1, 35µgmL-1, 40µgmL-1, 45µgmL-1,50µgmL-1) respectively to form calibration curve solution. Recovery was assessed by analysing environmental samples spiked with cymoxanil at a concentration of 40µg mL-1. The method was validated based on previous work regarding the performance of spectrophotometric methods and their explanation of results. The analysis included, develop and validate a method, linear calibration curve, collection of original samples, and preparation of synthetic samples, reproducibility, and recovery. An aliquot of different concentration levels (2µg-50µgmL-1) of cymoxanil solution was placed in a 25 mL calibrated volumetric flask, then 1 mL bromine water was added and shaken gently for 2 minute. After that 0.5 mL of formic was added to remove excess bromine water, and then 1 mL of a mixture of potassium iodide and potassium iodate solution was added to liberate iodine. Then 1 drop of starch solution was added. A blue colour dye was found and their absorption maxima were found at λmax 580 nm. The proposed reaction scheme is shown in (Figure 1).

Sample Preparation

Sample preparation for determination of cymoxanil

Samples were collected from Meghnath farmhouse and Girwar farmhouse situated in village Sukhari which is on the bank of Shivanth River in Rajnandgaon district, Chhattisgarh where cymoxanil was sprayed. Collected samples were chopped into fine pieces and Samples were spiked with a known amount of cymoxanil and kept for 5-6 hours and dipped in ethanol solution for some time. After some time these samples were crushed and shaken for 5-10 minutes and then filtered and centrifuged for 10 minutes. After centrifugation transparent layer was separated and then analysed as given the above-mentioned procedure. (Khatoon et al, 2017).

Results and Discussions

Reaction Mechanism

Cymoxanil was hydrolysed with a basic medium (OH-) and it was dissociated into 1-ethyl-5-methoxyimino-2, 4-dioxoimidazolidine, and cyanide were released (Teller et al, 2008). The bromination of hydrogen cyanide was carried out forming cyanogen bromide (Sahu et al, 2020). The cyanogen bromide was reacted with a mixture of potassium iodide and potassium iodate in presence of formic acid releasing iodine. This iodine further reacts with starch forming a blue coloured complex.


Spectral Characteristics

The absorption spectrum of cymoxanil with starch is observed in the maximum absorbance wavelength peak (λmax) at 580nm as shown in
(Figure 2). All spectral measurements carried out against reagent blank which is double distilled water shows negligible absorption at this wavelength. In the calibration curve, the colour system obeys Beer’s law in the range of 2 to 50µgmL-1 shown in (Figure 3) of cymoxanil in 10mL of final solution at 580nm. The molar absorptivity and Sandell’s sensitivity were found to be 1.25×105 L moL-1 Cm-1 and 1.0×10-3 µg cm-2 % respectively. All parameters and statistical data are shown in (Table I)

Table I.  Optical Characteristics and statistical data of the reaction of cymoxanil with starch reagent



Volume of dye


Λmax (nm)


Beer’s law limit (µgmL-1)


Molar absorptivity (L mol-1 cm-1)


Limit of detection (µgmL-1)


Limit of Quantification (µgmL-1)


Sandell’s sensitivity (µg cm-2)


Standard deviation (µgmL-1)


Relative Standard deviation (%)


Correlation coefficient (R2)








Table II.  Tolerance limit of foreign species and ions on the determination of cymoxanil


Tolerance limit in µgL-1













Fe ++


Zn ++







 It was checked by the repeat analysis of a working standard solution containing a solution of 2µgmL-1 of cymoxanil in 10mL final solution over 7 days traces. The standard deviation and relative standard deviation for the absorbance value were found to be 2.9×10-3 and 1.6% respectively.

Interference studies of foreign species and ions

Different types of foreign ions and species that could be potentially associated with the sample matrix were studied in terms of their effect on the determination of cymoxanil at a concentration of 5.0 µg mL-1. The results showed that most of the foreign ions and species tested did not interfere with their concentrations. The tolerance limit was taken as the concentration causing an error of ±2% in the determination of the pesticide. The tolerance limit for the foreign ions and species studied is shown in (Table II).


The proposed method was significant for the determination of the cymoxanil in soil, water vegetables, and Fruits samples. To ensure the authenticity of the method, different samples of soil, water, vegetables, and fruits were taken. In these samples known amount of cymoxanil was added and examined by the proposed method. (Table III).

Table III. Determination of cymoxanil in different environmental and agricultural samples


Cymoxanil in original found* in µg

Cymoxanil  added in µg/mL

Total Found

Difference (µg/mL)

Recovery (%±RSD)






95.20 ± 0.23






96.50 ± 0.35






97.00 ± 0.20






97.36 ± 0.13






96.40 ± 0.22






96.70 ± 0.25






96.60 ± 0.16






97.10 ± 0.12

*Mean of three replicate analyses.

**Water sample 50mL after treatment 50mL of aliquot was analysed.

***Sample 5g (Taken from the agricultural field, 10mL aliquot was analysed after treatment as described in the currently proposed method).


Stability Studies

Standard stock solutions and working standard solutions were stored in the refrigerator at 4°C and analysed daily for 7 days. No variation in the concentration of both analytes was observed even on the 7th day, which indicates the stability of both analytes.


Based on the literature survey reported analytical methods are expensive and sophisticated. So we have developed a low-cost and simple method for the detection of cymoxanil in environmental and agricultural samples by spectrophotometry. We have studied this method for analysis of cymoxanil in the UV-Visible range and found absorbance maxima at 580nm. The achievement of the proposed method is mainly its simplicity, sensitivity, and higher stability of the coloured system and this gives more advantage of flexibility in performance on any reported instruments. Therefore is proposed method is successfully applied for the determination of cymoxanil in various environmental samples.


Authors are thankful to the Head, School of Studies in Chemistry Pt. Ravishankar Shukla University Raipur and Director-General Chhattisgarh Council of science and technology for providing laboratory facilities and financial assistance.


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