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: mkjkchem@gmail.com,
harshitasharmachem23@gmail.com
[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.
Introduction
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
Apparatus
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.
Chemicals
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.
Procedure
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.
Precautions
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.
Reproducibility
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)
|
1
|
0.375
|
2
|
0.376
|
3
|
0.380
|
4
|
0.372
|
5
|
0.384
|
6
|
0.387
|
7
|
0.389
|
Mean
|
0.380
|
Standard deviation
|
±0.006
|
Relative standard deviation
|
1.95%
|
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
|
Lamda-cyhalothrin
|
1000
|
Ethion
|
900
|
Metsulfuron
|
1500
|
Propanofos
|
1200
|
Fenvalrate
|
1000
|
Al3+
|
800
|
Ba2+, Cl-
|
950
|
Ca2+, SO42-
|
900
|
Zn2+
|
500
|
Pb2+, NO3-
|
600
|
Concentration
of Phorate = 10µg/ml
Table
3.
Recovery of Phorate in Environmental Samples
|
Samples
|
Phorate
originally found (µg) x
|
Phorate
Added
(µg)
y
|
Total
Phorate
Found*(µg) z
|
Difference/
(µg) z-x
|
Recovery/(%)
(z-x)×100/y
|
Water**
|
0.29
|
10
|
9.87
|
9.58
|
95.8±0.223
|
Soil***
|
0.65
|
10
|
10.25
|
9.6
|
96.7±0.221
|
Cucumber***
|
0.41
|
10
|
10.28
|
9.87
|
98.7±0.588
|
Potato***
|
0.12
|
10
|
9.85
|
9.73
|
97.3±0.488
|
Tomato***
|
0.57
|
10
|
10.42
|
9.85
|
98.5±0.545
|
Cauliflower***
|
0.31
|
10
|
10.20
|
9.89
|
98.9±0.210
|
Brinjal***
|
0.52
|
10
|
10.30
|
9.78
|
97.8±0.513
|
*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
|
Applications
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
|
Remarks
|
Reference
|
1
|
Cu, complex formation
|
420
|
50 µg/ml
|
Less sensitive
and selective
|
Deshpande et
al., 1982
|
2
|
Determination with Chromotrophic acid
|
570
|
11.4 µg/ml
|
Complicated
method involves a number of steps, perbenzoic acid is used which is not
easily available
|
Giang et al.,
1960
|
3
|
Detection with ammonium molybdenum and
methylene blue
|
660
|
0.5 µg/ml
|
Uses commonly
available reagent, more sensitive and selective
|
present method
|
Conclusion
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.
References
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.