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Author(s): Harshita Sharma, Anushree Saha, Chhaya Bhatt, Kalpana Wani, Ajay Kumar Sahu, Jyoti Goswami, Arun Kumar Mishra, Manish Kumar Rai*, Joyce Rai

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Address: Department of chemistry, Govt. Nagarjuna P.G. college of science, Raipur (Chhattisgarh), 492007, India
School of Studies in Chemistry, 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:
Sharma et al. (2020). Flotation-Dissolution-Spectrophotometric Determination of Phorate in Various Environmental Samples. Journal of Ravishankar University (Part-B: Science), 33(1), pp. 18-23.


Flotation-Dissolution-Spectrophotometric Determination of Phorate in Various Environmental Samples

Harshita Sharmaa*, Anushree Sahab, Chhaya Bhattb, Kalpana Wanib, Ajay Kumar Sahub, Jyoti Goswamib, Arun Kumar Mishraa, Manish Kumar Raib*, Joyce Raic

aDepartment of chemistry, Govt. Nagarjuna P.G. college of science, Raipur (Chhattisgarh), 492007, India.

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

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

*corresponding author:,

[Received: 25 January 2020; Revised version: 22 August 2020; Accepted: 24 August 2020]        

Abstract: The proposed method is based on flotation–dissolution an easy, impressible, extractive spectrophotometric determination, explained for easy investigation of the organophosphate pesticide phorate (O,O-diethyl S-[ethylthiomethyl] phosphorodithioate) on trace levels. A molybdophospho complex is generated when prorate is treated with ammonium molybdate in acidic medium. As an ion associate complex with methylene blue the complex is present in between of the water and organic layers which is extracted and then dissolved with acetone. The greenish blue complex produced show absorption maxima at 660 nm. Beer’s law range is found to be 0.5 to 16 µg per 10 ml for phorate. The molar absorptivity is 0.989×103 L mol-1 cm-1 and sandell’s sensitivity is 1.00×10-5 µg cm-2. Also calculated the standard deviation and relative standard deviation for the above method were ±0.006 and 1.95% respectively. The method has been applied and checked for the determination of phorate in water, soil and vegetables.

Keywords: Spectrophotometer, Phorate, Organophosphate pesticide, Molybdophospho Complex.


Organophosphorus compounds are possibly the most widely used insecticides worldwide (Borkar et al., 2018; Lakshmajah, 2017; Sharma et al., 2019). Phorate (O,O-diethyl S-[ethylthiomethyl] phosphorodithioate; CAS number 298-02-2) is an organophosphate pesticide that is commonly used to control sucking and biting insects and nematodes on a variety of field crops including corn, cotton, potatoes, sugar beets, and beans. Phorate is highly toxic to birds, fish, and mammals and accidental human exposures, resulting in death in some instances, have been reported. It is globally available for agricultural purposes and is generally applied to the soil as dry granules (Gandhi et al., 2016; Li et al., 2018; Mamta et al., 2019; Moyer et al., 2018). Phorate is a board range pesticide; it has been proved as a most active insecticide against most insect pest species. Phorate has been classified as a most hazardous insecticide according to the world health organization; so its constant usage is a rising alarm. In European communities, it has been banned and while used with limits in the US. Still many agricultural administrations are keenly working to suspend the prohibition on the usage of this enormously lethal insecticide. Phorate has water solubility (50mgl−1), hence percolate through the soil to groundwater. Till now, reported metabolites of phorate are phoratoxon, phoratoxon sulfone, phorate sulfoxide, and phorate sulfone that are more toxic in action. With average LD50 of 2–4 mg kg−1, Phorate is considered as one of the most toxic pesticides (Hazardous Substances Data Base, 1988). Therefore, complete removal of phorate in contaminated soil is the need of the hour (Fu et al., 2019; Jariyal et al., 2018).




Figure 1. Structure of Phorate

Common Name       :          Phorate                    

IUPAC Name          :          O,O-diethyl S-[ethylthiomethyl] phosphorodithioate    

Trade Name             :           Agrimet, Geomet, Granutox, Phorate 10G

Molecular Formula :           C7H17O2PS

Chemical Group      :           Organophosphate

Solubility                 :             Water 0.005% (200C)

LD50                           :             1.1 – 3.2 mg/kg

Melting poin            :          42.9 °C

Experimental Section


For all spectral analysis Systronics Visiscan spectrophotometric model 167 with matched silica cells was used. A Systronics digital pH meter model 335 was used for pH measurements. For centrifugation Remi C-854/4 clinical centrifuge force of 1850 rpm with fixed swing out rotors was used.


Reagents and Chemicals used were of analytical reagent grade or of the best quality available. Double distilled water was used all over the experiment.

Phorate (United phosphorous limited Mumbai): 1mg mL-1 was prepared as stock solution.

Ammonium Molybdate: A 0.05 mol L -1 was prepared in dilute (1.5 mol L -1) Sulfuric acid.

Methylene Blue (sigma-Aldrich chemicals): 4 x 10-5 mole L-1 solution was prepared by dissolving 0.013 g of methylene blue in 100 ml of water.

Oxalic acid: Solution of 0.10 mol L-1 oxalic acid was used.

Butanol: 99% (v/v) solution of butanol was used.

Acetone: 99% (v/v) solution of acetone was used.


Preparation of calibration graph

In 250 ml Erlenmeyer flask, 10ml of aqueous solution consisting 0.5-16 µg per 10 ml of phorate was taken and 1ml of 1.5 mol L -1 sulfuric acid and ammonium molybdate solution 0.6 ml of 0.05 mol L-1 were added respectively. Solution were heated at 80-1000C and then left the solution till it cooled to room temperature. In order to eliminate excess of molybdate, the mixture was treated with 0.5 ml volume of Oxalic acid and the solution was placed into a 250 ml separating funnel. 0.2 ml of methylene blue solution was added into it and then extraction was carried out with 5 ml of butanol. The lower water soluble layer was discarded and the floating ion containing layer with 2 ml of organic phase was transposed to a graduated tube and to which 5 ml of acetone was added and dissolved and then the absorbance was measured at 660 nm against a reagent blank.


The preventative measurements include following: Do not over heat the solution after adding sulphuric acid and ammonium molybdate solution. Secondly, do not use methylene blue solution more than 0.2ml because if the amount of methylene blue solution increased it may affect the analysis and the vegetable samples should be properly centrifuge before experiment.

Results and Discussion

Absorbance Spectra

Maximum absorption shown by the color system at 660 nm because at this wavelength the greenish blue complex produced by proposed method has strongest photon absorption which is evident from Fig. 2. All the spectral determination was carried out against double distilled water as the blank showed negligible absorbance at this wavelength.

Beer’s Law and Sandell’s Sensitivity

The Beer’s law followed at the range of 0.5-16 µg for color system of phorate per 25 ml of final solution at 660 nm.


Molar Absorptivity

The molar absorptivity is calculated 0.989×103 L mol-1 cm-1.

Sandell’s Sensitivity

Sandell’s sensitivity of greenish blue colour dye was found to 1.00×10-5 µg cm-2.


The reproducibility of the method was evaluated by the replicate analysis of 10 µg of phorate 25 ml final solution over a period of 7 days. The Standard deviation and Relative standard deviation are indicated in Table 1. The solution containing 10 µg of phorate was evaluated for reproducibility by performing three replicate analyses for seven days. Later standard deviation was calculated and relative standard deviation ±0.006 and 1.95% respectively. For this wavelength, negligible absorbance was observed for blank reagent.

Table 1. Reproducibility of Method

 No of days

Absorbance (at 660 nm)

















Standard deviation


Relative standard deviation


Molar Absorptivity

0.989×103 L mol-1 cm-1

Sandell’s sensitivity

                1.00×10-5 µg cm-2

Concentration of Phorate = 10 µg/ml

Effect of Ammonium Molybdate concentration

 Effect of Reagent concentration for full color development was observed at favourable condition it was found that 0.6 ml of 0.05 mol L-1 ammonium molybdate solutions was sufficient. The absorbance of the sample solution was found to be increased as the amount of ammonium molybdate was raised.

Effect of reagent concentration

0.2 ml volume of 4 x 10-5 mol L-1 methylene blue solution was found to be enough for complete interaction so that color complex could be formed .If more than 0.2 ml was used it was seen that the blank also produce color.

Effect of Oxalic acid concentration

In order to eliminate excess of molybdate, 0.5 ml volume of oxalic acid solution is quite enough.

Effect of foreign Species

The effects of common foreign species and other pesticides were studied to check the validity of the method. Known amount of pesticides and foreign species were added to a standard solution containing 10 µg of phorate per 10 ml before the determination and the solution was carried out by the above narrated method. It was found that the foreign species are not interfering in the condition as described in the present method, as they degrade and release phosphorous at very higher concentration. The limit acceptable for various other pesticides and ions are shown in Table 2.





Reaction Mechanism


Table 2. Effect of Foreign Species

Foreign species

Tolerance limit in µg/ml













Ba2+, Cl-


Ca2+, SO42-




Pb2+, NO3-


Concentration of Phorate = 10µg/ml


Table 3. Recovery of Phorate in Environmental Samples


Phorate originally found (µg) x


Added (µg)


Total Phorate

Found*(µg) z


(µg) z-x














































*Mean of three replicate analyses     

**Amount of samples 10 ml

***Amount of samples 5 gm


Figure 2. Absorbance curve of colour compound


Figure 3. Calibration curve of the colour compound


Figure 4. Effect of Ammonium Molybdate

Figure 5. Effect of Sulphuric Acid


Determination of phorate in polluted water

Water sample from pond near agricultural field was collected. This sample was extracted with 2 × 25 ml portion of diethyl ether. The ether solution was evaporated to dryness, and the residue was dissolved in 50 ml of ethanol. Aliquots were then analyzed as proposed method.

Determination of phorate in soil sample

Soil sample 5 gm was taken in an Erlenmeyer flask of 250 ml. 20 ml of 0.3% sulfuric acid was added to this flask along with 10 ml of 6% (m/v) hydrogen peroxide and glycerin 0.5 ml. The obtained mixture is boiled at 160-180oC for 20min on a sand – bath, then again add 2 ml of hydrogen peroxide and further boil it for 10 min more. After this bring the mixture to room temperature and add 50 ml of double distilled water.

Determination of phorate in Different Vegetable samples

Different fruits and vegetable samples were weighed, mashed along with acetone and double distilled water (1:1) and then strained from thin cotton cloth. The strained solution is then preceded with centrifugation at 1850 rpm for 10 min. Then 10 ml of sample aliquot were treated with proposed method.                   

Comparison with the earlier reported methods

The proposed method is compared with the other conventional methods reported for the determination of phorate (Table 4). It is evident that the proposed method is highly sensitive and selective.

Table 4. Comparison with other spectrophotometric methods

S. No.

Method / Reagent

λ Max, (nm)

Detection limit




Cu, complex formation


50 µg/ml

Less sensitive and selective

Deshpande et al., 1982


Determination with Chromotrophic acid


11.4 µg/ml

Complicated method involves a number of steps, perbenzoic acid is used which is not easily available

Giang et al., 1960


Detection with ammonium molybdenum and methylene blue


0.5 µg/ml

Uses commonly available reagent, more sensitive and selective

present method


This proposed method is found to be simple, sensitive and rapid spectrophotometric method for the analysis of phorate. Also, it used less toxic substance as reagents for the analysis. The statistical data were calculated and validated. The standard deviation and relative standard deviation obtained for the above method were ±0.006 and 1.95% respectively. Hence, this method can be considered as one of the good alternative to most of the high costing, delicate apparatus which need much more maintenance. It can be very efficiently applied for the determination of phorate in water and vegetable samples.


Borkar, V.; Gokhale, N.; Khobragade, N. and Dhopavkar, R.; “Influence of phorate and carbofuran insecticides on phosphorous availability and its residues in soil and rice”, International Journal of Chemical Studies (2018), 6(1), 238-242.

Deshpande C. M. and Bhende S. S.; Indian J Environ Prot (1982), 2, 73-74.

Fu, J.;  An, X.; Yao, Y.;  Guo, Y.;  Sun, X.; “Electrochemical aptasensor based on one step co-electrodeposition of aptamer and GO-CuNPs nanocomposite for organophosphorus pesticide detection”, Sensors and amp; Actuators: B. Chemical (2019).

Gandhi, K.; Lari, S.; Tripathi, D.; Kanade, G.; “Advanced oxidation processes for the treatment of chlorpyrifos, dimethoate and phorate in aqueous solution”, Journal of Water Reuse and Desalination (2016), 06.1, 195-203.

Giang, P. A. and Schechter M. S.; "Insecticide Residues, Colorimetric Determination of Residues of Phorate and Its Insecticidally Active Metabolites", Journal of Agricultural and Food Chemistry (1960), 8, 51-54.

Jariyala, M.; Jindalb, V.; MandalcK.; Gupta,  V.;  Singh.  B.;“Bioremediation of organophosphorus pesticide phorate in soil by microbial consortia”, Ecotoxicology and Environmental Safety (2018), 159, 310-3016.

Lakshmaiah, G.; “Brain histopathology of the fish Cyprinus carpio exposed to lethal concentrations of an organophosphate insecticide phorate”, International Journal of Advanced Research and Development, (2017), 2(5), 668-672.

Li,  X.;  Shi,  J.;  Chen,  C.;  Li,  W.;  Han,  L.;   Lan,  L.;  Guo,  Y.;  Chang, Y.;  Caia,  J.; and Ding,  Y.; “One-step, visual and sensitive detection of phorate in blood based on a DNA–AgNC aptasensor”, The Royal Society of Chemistry, (2018).

Mamta,; Rao, R.;  Wani, K..; “Status of Organochlorine and Organophosphorus Pesticides in Wetlands and Its Impact on Aquatic Organisms” Environmental Claims Journal (2019), 1-35.

Moyer,  R. A.;  Garry,  K.;  Babin,  M.;  Platoff,  G. E.;  Jett,  D. E.;  Yeung , D. T.; “Kinetic Analysis of Oxime-Assisted Reactivation of Human, Guinea Pig, and Rat Acetylcholinesterase Inhibited by the Organophosphorus Pesticide Metabolite Phorate Oxon (PHO)”, Pesticide Biochemistry and Physiology, (2018).

Sharma, R.; Tiwari, R.; Muralidhar; Maheshwari, S.; Jain, R., Gokhroo, A,; “A Study of Relation of CPK-MB Levels with ECG Parameters in Organophosphorous Poisoning Cases”, Journal of The Association of Physicians of India (2019), 67, 26-19.     

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