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Author(s): Chhaya Bhatt*, Deepak Kumar Sahua, Thakur Vikram Singh, Kalpana Wani, Jyoti Goswami, Ajay Kumar Sahu, Harshita Sharma, Geetanjali Deshlehre, Manish Kumar Rai*, Joyce Rai.

Email(s): , mkjkchem@gmail

Address: School of Studies in Chemistry, Pt. Ravishankar Shukla University, Raipur (Chhattisgarh), 492010, India
School of Studies in Environmental Science, Pt. Ravishankar Shukla University, Raipur (Chhattisgarh), 492010, India
Chhattisgarh Council of Science and Technology,MIG-25, Indrawati Colony,Raipur (Chhattisgarh),492007, India.

Published In:   Volume - 33,      Issue - 1,     Year - 2020

Cite this article:
Bhatt et al. (2020). Spectrophotometric Determination of Phenthoate in Vegetables and Fruit Samples of Kabirdham (Chhattisgarh). Journal of Ravishankar University (Part-B: Science), 33(1), pp. 08-17.

Journal of Ravishankar University–B, 33 (1), 08-17 (2020)

Spectrophotometric Determination of Phenthoate in Vegetables and Fruit Samples of Kabirdham (Chhattisgarh)

Chhaya Bhatta*, Deepak Kumar Sahua,c, Thakur Vikram Singhb, Kalpana Wania, Jyoti Goswamia, Ajay Kumar Sahua, Harshita Sharmaa, Geetanjali Deshlehrea, Manish Kumar Raia*, Joyce Raic

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

bSchool of Studies in Environmental Science, Pt. Ravishankar Shukla University, Raipur (Chhattisgarh), 492010, India.

cChhattisgarh Council of Science and Technology,MIG-25, Indrawati Colony,Raipur (Chhattisgarh),492007, India.

*Corresponding Author: mkjkchem@gmail,

[Received: 27 November 2019; Revised version: 07 July 2020; Accepted: 09 July 2020]

Abstract. A large variety of pesticides have been used in the agriculturearea to raise the quality, extend and yield storage life of crops.However, the use of pesticides has been greater than before now a day dueto the growing rapid urbanization and population. The nonstop uses ofthese pesticides have resulted in pollution of the environment and alsocaused risk to human health. For the rapid detection of selective phenthoate pesticides, we developed a simple and highly sensitive spectrophotometric method based on azo-coupling reaction. After a simple pretreatment of phenthoatewith a diazotized solution,the resulting is rapidly undergoing azo-coupling reaction with 4-aminoazobenzene with adramatic color change only in few minutes.Finally, we successfully applied the concentration of phenthoate pesticides on vegetable, fruit, water, and soil samples. In this method, we found the absorption maxima of the orange dye compound was measuredat480nm.ThecolorsystemobeysBeer’slawinthefollowingworkingrangeof5µg to 40 µg in a final solution of 25 mL. The molar absorptivity, Sandell’s sensitivity, standard deviation, and the relative standard deviation was found to be 1.083X107L mol-1 cm-1, 0.99X10-5µg cm-2, ±0.003 and 0.89% respectively. This method is simple, sensitive, andfree from interferences of other pesticides and diverse ions. Other pesticides do not interfere with the proposed method. The method is highly reproducible and has been successfully applied for the determination of phenthoate insecticides in vegetables and fruit samples.

Keywords: Spectrophotometer, Phenthoate, Diazotization, vegetables, and fruit Samples.


Pesticides are substances, or a mixture of substances that are widely used on crops and vegetables it increases agricultural productivity because of their effectiveness, low cost, and acute toxicity to the target organism. (Khedr et al., 2019). Pesticides are used to prevent, repel, or kill organisms and substances that are harmful to crop growth. (Wu et al., 2018). Pesticides and herbicides are divided into four sections with a focus on toxicology, ecology, risk assessment, modeling, and treatment strategies (Choudri et al., 2018).Pesticides may simultaneously be harmful both for the environment (e.g., accumulating in ecosystems) and for human health(N.M. Bhojane et al.,2018). In this second task, they can give adverse effects, including cancer, acute and chronic injury to the nervous system, lung damage, reproductive dysfunction, and possibly dysfunction of the endocrine and immune systems(Christopher DN Paul CE et al.,2013). One of the main pesticide sources is the diet. Indirectly, the consumer assimilates small pesticide quantities (residues) from different foods such as meat, fruit, and vegetables (Notardonato et al., 2018).

Phenthoate is a systemic organophosphate insecticide thatis ethylmandelate inwhich the hydroxy group has beenreplaced by a (dimethoxyphosphorothioyl) sulfanediylgroup(KarraSomaiah et al., 2015). Itis used against Lepidoptera, jassids, aphids, soft scales, mosquitoes, blowflies, houseflies anda phenthoate used to control common insects in Rice, Vegetables including brassicas, eggplant, peppers, radish, turnips, carrot, Soybean, Potatoes, Tea, Citrus(Jian Wanget al., 2012).It has a role as an acetylcholinesterase inhibitor, an acaricide, and an agrochemical, It is used for termite control in construction, forestry and fieldcrops(Kaufmann, A et al.,2012). It is widely used by farmers to control insect pests of vegetables(KarraSomaiah et al., 2015). Historically pesticides were known as economic poison (Reddy et al., 2017). The acute oral LD50 for rats proposed for phenthoate is 270 mg/kg. The literature revealed many efforts made to determine phenthoate in various environmental samples.

These methods include high-performance liquid chromatography, capillary electrophoresis, liquid chromatography/time-of-flightmassspectrometry,andultra-performanceliquidchromatographycoupledtotandemmass spectrometry (González et al., 2008). Analytical methods for the analysis of pesticide residues in grapes and by-products in the last decade. The most widely used detection technique for the determination of pesticides in grapes is mass spectrometry combined with gas and/or liquid chromatography (Grimalt et al.,2016).Most of these pesticides are not completely degraded after application, and their metabolism as well as some un-changed forms entire the ecosystem (Zhang et al., 2017). Techniques applied fororganophosphoruspesticides analyses are mostly chromatographic (gas chromatography and liquid chromatography), electrochemical, immunochemical, and biosensors based ones (Stoytcheva et al., 2014). Gas Chromatographic Analysis of Extracts from Sediment and Water Samples (Okoya et al.,2013).

A new data analysis method was developed to determine the concentration of Carbofuran pesticides by cyclic voltammetry (Chen andChen, 2013).Despite their versatile uses, these techniques have a lot of drawbacks such as tedious procedure, non-linearity of the calibration curve, and matrix interference, also these techniques required a large number of solvents for the extractionand also some limitations in term of high-cost instruments used in routine analysis and matrix effects. In the proposed work first time a spectrophotometric method is developed for the determination of phenthoate in various environmental samples as the literature review suggested no single spectrophotometric method is reported for estimation of phenthoate. This contamination poses a great risk to human health since residues in food and drinking water can enter the body and accumulate in organs and tissues. (Alkan et al., 2018). Due tothe common availability of the instrumentation, simplicity of the procedure, speed, precision, and accuracy, spectrophotometric methods enjoy wide popularity. Also, they are more economical and simpler compared to other analytical methods. So, the present communication involves developed a spectrophotometric method based on diazotization with diazotized 4- aminoazobenzene and determined after extraction of phenthoate insecticides in Environmental and agriculturalSamples.

Structural Formula:


Figure 1. C12H17O4PS2


        Molecularweight                          :              320.358 g mol-1

        Appearance                                  :               Reddish yellow oily liquid

Meltingpoint                                :               186-187℃ at 5mm Hg

Boilingpoint                                  :               The compound decomposes at normal pressure before reaching the boiling point.

Partitioncoefficient                     :               Octanol//water log P = 3.69, 3.82at

                two concentrations


        Water solubility                            :             11 mg/L (20℃)

Sampling Location Site

Kabirdham, earlier known as Kawardha, is famous for historic Bhoramdeo temple located between 21.32' to 22.28' N latitude and 80.48' to 81.48' E longitude covering an area of 4,447.5 km2 (1,717.2 sq mi).The samples were taken from farmhouse, and various vegetable and fruit markets. The location of the study area has been shown in Figure 2.

Figure 2. location site

Materials and Method

Apparatus:SystronicsVisiscan Spectrophotometric model 167 with matched silica cells was used for all spectral measurements. A Systronics digital pH meter model 335 was used for pH measurements. A Remi C-854/4 clinical centrifuge force of 1850 rpm with fixed swing-out rotors was used for centrifugation. All reagents used were of Sigma –Aldrich, Pvt. Ltd. Double distilled water was used throughout. Calibrated glassware was utilized after getting cleaned and marinating in the acidified solution of potassium dichromate and rinsing two times with double distilledwater.

Reagents:Phenthoate (Coromandel international limited): A stock solution of 1 mg/mL was prepared in double-distilled water Working standard solutions were prepared by appropriate dilution of the stock standard solution with double distilled water.

Sodium Nitrite (Reidel Chemicals, India): A 1% m/v solution was prepared in double-distilled water.

HCl: 0.5 m/L solution was prepared in double-distilled water.

4-aminoazobenzene: 1% solution of 4 aminoazobenzene was prepared with 25% of ethanol.

Diazotized solution: To 1 mL of 4- aminoazobenzene, 1 mL of 1% sodium nitrite in hydrochloric acid was added and the solution was kept in a brown bottle at 0-5℃. This was stable for 4 h when kept in cold.


Reaction mechanism of the proposed method



Figure 3. Reaction mechanism for the determination of phenthoate




An aliquot of test solution containing 5 to 40 µg ofphenthoate was taken in a 25 mL calibrated flask, then 1 mL of diazotized solution was added and shaken thoroughly and kept at 0-5℃ for 10 minutes for full-color development and orange color was obtained. The solution was then diluted to the mark with water and absorbance was measured 480 nm against a reagentblank.

Results and Discussion

Spectral Characteristics: The 4-aminoazobenzene reagent dye formed in the proposed reaction shows maximum absorption at 480 nm (Figure 4). All spectral measurements were carried out against double distilled water as the reagent blank showed negligible absorption at this wavelength. The color system obeys Beer’s law in the range of 5 to 40 µg of phenthoate per 25 mL of the final solution at 480 nm (Figure5).

Figure 4. Absorbance Curve of the phenthoate                 Figure 5. Calibration curve for the determination of phenthoate

      Table 1. Reproducibility of Method

No. of Days

Absorbance at 480nm

















Standard deviation


Relative Standard Deviation


*Concentration of Phenthoate used was 10µg /25mL

Method validation

Linearity: For this experiment, the linear graph plotted between absorbance and concentration of phenthoate in the range of 5 to 40 µg in 25 ml volume and we obtained a linear graph. The graph was plotted between a small intercept and a high correlation coefficient of a regression equation (y=mx+c). In the study, the molar absorptivity was also calculated as shown in Table 2.

Accuracy and precision: The precision of the method was checked by the three replicate analysis of a working standard solution containing 10µg of phenthoate in 25 mL final solution over a period of 7 days. recovery range was found in the range of 85-98%. The standard deviation and relative standard deviation were found to be ± 0.003 and 0.89% respectively

Robustness and ruggedness: Robustness and ruggedness were evaluated for the new method. Sometimes parameters like pH, temperature, and time were changed. In this method one parameter altered but another was constant we calculated recovery each time and observed that the change of parameter does not affect reliving values shown in Table 3.

Lower Limit of Detection (LOD): Limit of detection is the lowest concentration of pesticide in a sample, which is detected, but it is not necessary to this value is quantified,under the condition of analysis.

LOD = 3.3 σ/Slope Where: σ = the standard deviation of the answer at low concentrations

Slope = the slope of the calibration curve (Saadati et al., 2013; Kumari et al., 2010).


Limit of Quantitation (LOQ): Limit of quantitation is the lowest concentration of a pesticide in a sample which is determined with acceptable precision and accuracy under the condition of analysis.

LOQ = 10 σ/Slope Where: σ = the standard deviation of the answer at low concentrations.

Slope = the slope of the calibration curve (Shrivastava et al., 2011).

Tolerance Limit: Tolerance of pesticide is a maximum quantity of pesticide residue that can be present in food. Tolerance is expressed in parts per million (Kumari et al.,2010).

        Table 2. Optical characteristics and Statistical Data of theRegression Equations for the Reaction



Values for the reaction








Beer’s law limit

5 to 40 µg in 25 mL


Molar absorptivity

1.083 X107L mol-1 cm-1


Sandell’s sensitivity

0.99X 10-5µg cm-2


Standard deviation



Relative standard deviation



Correlation coefficient(R2)



Regression equation









Limit of Quantification(LOQ)

3.0 µg/mL


Limit of Detection (LOD)



Lethal Dose (LD50)


Salvaneschi, 1968;He&Gera,1978a; Toyoshima et al, 1978; Trabucchi, 1965; Pellegrini & Santi,1972.


Effect of different reaction conditions

Effect of temperature: The effect of temperature on the reaction was studied by the varying temperature range from 0℃-10℃ for the phenthoate and found that the 0℃-5℃ temperature was sufficient for the complete reaction.With an increase or decrease in temperature, the absorption values also increased or decreased.

Effect of pH: The effect of pH on the color reaction was studied and it was found that constant absorbance values were obtained at a pH range of ~ 2– 4.0 and no buffer solution was required to stabilize the color. The colored species remain stable for more than 7 hr. under optimum condition (Figure-7).

Effect of reagent: when p-amino azobenzene reacts with sodium nitrite and HCL than form diazonium salt.when the amount of diazonium salt was increased or decreased the absorbance value also changed. when the concentration of p-amino azobenzene was altered, firstly absorbance value was increased by increasing the concentration of p-amino azobenzene,and after that absorbance value was observed to be decreasing rapidly with increasing concentration (Figure-6).


Figure 6. Effect of reagent                                                 Figure 7. Effect of pH



Effect of foreign species: The effect of common foreign species and pesticides was studied to assess the validity of the method. A known amount of metal ions and pesticides were added to the standard 10 μg/25 mL of phenthoate. The method was found to be free from interferences of most of the foreign species and pesticide(Table03).

Effect of interference: We studied different vegetable samples and we found that different types of compounds are already present in these vegetables. We added different pesticides and foreign species solution containing 10 µg of phenthoate in 25ml of the final solution and developed a method for the analysis by the proposed methods and we found that there was no interference of those compounds in the method.

        Table 3. Effect of Foreign Species


Foreign Species


Tolerance limit* µg in 25 mL






















 *Concentration of Phenthoate 10 µg / 25 mL.

*The amount causing an error of ±2% in absorbance value. 

    Table 4. Recovery of Phenthoate in Various Environmental and Agricultural Samples


Phenthoate originally found

(µg mL-1)a

Phenthoate added

(µg mL-1)b

Total Phenthoate found after standard additionc












92 ± 0.37














85 ± 0.27







93 ±0.81













86 ±0.36






93 ±0.56






87 ±0.19






98 ±0.16

Mean of three replicate analyses

*Water sample 5 mL; ** Amount of Sample 5 g.


The proposed method was applied satisfactorily, for the analysis of phenthoate in various samples i.e. polluted water, vegetables, fruits. The amount of phenthoate found in various samples i.e. water, rice, soil, green beans, potatoes, cauliflower, tomato, orange was purchased from the market and farmhouse respectively.The results are analyzed in Table-04.

Analysis of phenthoate in polluted water:  Water samples from rivers receiving runoff from various agricultural fields, where phenthoate was sprayed are collected. Then these samples are filtered through Whatman No. 40 filter paper. Now the water is evaporated to dryness and the residue was dissolved in 10mL of double-distilled water. Aliquot of water samples were taken in a 25 mL graduatedtube.

Analysis of Phenthoate in Different Fruit, Vegetables, and soil: Various samples of vegetables, fruits, and soil each 5.0 gm were collected from agricultural field and market, where phenthoate had been sprayed as an insecticide. The samples were macerated with 50 mL double distilled water filtered through a Whatman filter paper No. 40 and the filtrate were centrifuged at 1850 rpm for 10 minutes. In the case of vegetables and fruits, the filtrate was quantitatively transferred into a 50 mL calibrated flask and made up to the mark with double distilled water. 10 mLaliquots were taken in a beaker. Then 1.0 mL diazotized 4-aminoazobenzene was added.Shakenthoroughlyandkeptat0-5oCfor10minutesforfull-colordevelopment.

Comparison with other analytical methods:

A newly proposed method for the determination of phenthoate with P-amino azobenzene is described. The proposed method could be used as a simple and cheap method as compared to other expensive techniques. The present method is cheap and easy to handle. In this method reagents used are easily available and are cheap reagents. A comparison of the proposed method with other analytical methodsis shown in (Table 5).

   Table 5. Comparison with Various Spectrophotometric Methods for Determination of Phenthoate




Beer’s Law rangedetection limit




Leuco crystal violet  




Less sensitive

[Pradhan et al.2007]


Anthranilic acid



Less sensitive

[Amin et al.2015]


4-amino antipyrine


2-20 μg/10mL

Toxic reagent

[Amin et al.2015]





5-40 μg/10mL

Less sensitive

[Amin et al.2015]


Leuco malachite green


10-50 μg/10mL

Less sensitive

[Nirmal et al. 2015]



(proposed method)







Fruits and vegetables are the food of alternative for practically all people due to their healtheffects and cost compared to different methods, so vegetables are essential for the people in dailylife which increases the risk of different pesticide residues toxicity.The proposed method is rapid, simple, sensitive and reagent described here is sensitive and selective for analysis of phenthoate in various vegetables, fruits, soil, and water samples. The proposed method was best as compared to other methods. In this spectrophotometer method interference of any foreign species are negligible. It can successfully apply for the determination of phenthoate in water, soil, vegetable sample. Check the recoveries, a known amount of phenthoate was added to various samples and then analyzedby the proposed method.Thecalculated LOD and LOQ values for every constituent make sure that the developed methods arevery responsive and sensitive to their small doses. Being simpler, good applicability, and moreeconomic than the reported other expansive techniques, the introduced methods can be enhancedselections over the reported techniques. That method uses very complicated, costly, andprocessing steps apparatus.


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 Raipurfor providing lab facility. financial support of this work by the DST-FIST [NO.SR/FST/CSI-259/2014(C)] is gratefully acknowledged.


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