Assessment of Fenobucarb and Propoxur insecticides in environmental samples by spectrophotometric and spectrofluorimetric methods

doi:10.21203/rs.3.rs-3400511/v1

Download PDF

Research Article

Assessment of Fenobucarb and Propoxur insecticides in environmental samples by spectrophotometric and spectrofluorimetric methods

https://doi.org/10.21203/rs.3.rs-3400511/v1

This work is licensed under a CC BY 4.0 License

Journal Publication

published 24 Apr, 2024

Read the published version in Chemical Papers →

You are reading this latest preprint version

The widespread use of fenobucarb and propoxur insecticides for agricultural and non-agricultural purposes causes undesirable effects on human health and environment due to their residues in surface and ground water resources. In view of this two new, simple, accurate, sensitive, economical and reproducible optical methods have been investigated for the determination of fenobucarb and propoxur insecticides in pure form, in their commercial formulations and agricultural samples viz. water, grains and vegetables. The methods are based on the alkaline hydrolysis of the insecticides to methyl amine and the reaction of latter to give colored products. In spectrophotometric method the methyl amine formed, in presence of carbon disulfide and Ni(II)acetate gives a yellow colored complex, which was measured at 370 nm. Beer’s law is valid in the concentration range of 0.41–8.29 and 0.42–8.37 µg/ mL for fenobucarb and propoxur respectively. The spectrofluorimetric method is based on the study of yellow colored product, formed by the condensation of methyl amine, with acetylacetone and formaldehyde (Hantzsch reaction) at pH 5.5. The reaction product exhibits fluorescence intensity at an emission wavelength of 470 nm after excitation at a wavelength of 390 nm for both fenobucarb and propoxur insecticides. The method has linearity range 0.14–5.53 and 0.14–5.58 µg/ mL for fenobucarb and propoxur respectively. The different factors which affect the reactions were thoroughly studied and optimized. The proposed methods have been successfully applied for the assay of both insecticides in their commercial formulations and for recovery study from spiked environmental samples. The good results of recoveries of both insecticides demonstrate the accuracy and precision of both methods.

Fenobucarb

propoxur

spectrophotometric method

spectrofluorimetric method

residue analysis

formulation analysis

Carbamate insecticides have become an important class of pesticides because of their wide use in agriculture, in public health, gardening and as therapeutic pharmaceuticals due to their broad spectrum activity and relatively short environmental persistence (Chu and Fan 2009; Dias et al. 2015). These are employed frequently as alternative to organophosphorus insecticides due to greater specificity and lower mammalian toxicity (Vale and Bradberry 2016). Among the carbamate insecticides two N-methylcarbamate pesticides are fenobucarb and propoxur which are used widely due to their vast applications.

Fenobucarb- 2-sec-butylphenyl methylcarbamate, (Scheme 1) is a carbamate insecticide derived from carbamic acid and is used to control biting and sucking insects in rice, cotton and other crops (Zhu et al. 2020: Xia et al. 2019; Chormey 2021; Pham et al. 2021). It effectively controls these insects in paddy fields through inhibiting the activity of acetylcholine esterase. It is considered as Class II toxicity ‘moderately hazardous’ agricultural insecticide (Zhu et al. 2020) according to WHO (World Health Organization). Continuous exposure of fenobucarb may lead to neurotoxicity, skin and eye irritation, inhibition of acetyl cholinesterase, muscle spasm and paralysis of insects, fish and mammals (Zhu et al. 2020: Chormey 2021; Pham et al. 2021).

Propoxur- 2-isopropoxyphenyl methylcarbamate, (Scheme 1) is a non-systemic, broad-spectrum carbamate insecticide with a fast knockdown and long residual effect (Tabibi and Jafari 2006; Patel et al. 2013; Yildiz et al. 2017; Zheng 2017). It is used against a large variety of insects in agriculture, residential and non residential areas, in public health and for in and outdoor spraying (Tabibi and Jafari 2006; Patel et al. 2013; Yildiz et al. 2017). The oral LD₅₀ of propoxur for rats ranged from 40 to 150 mg/kg (Patel et al. 2013). On exposure it leads to inhibition of acetyl cholinesterase, leading to paralysis in insects and mammals, blurred vision, nausea, vomiting, sweating and tachycardia (Tabibi and Jafari 2006; Patel et al. 2013).

Due to solubility of these insecticides in water, fenobucarb 420 mg/L at 20 ^o C and propoxur 1860 mg/ L at 30^o C (Pham et al. 2021; Tabibi and Jafari 2006), they have the potential to enter the surface water through surface run off and also leach down into groundwater. This leads to their residues in different environmental segments and accumulation in humans and other non-target species, which causes potential hazard to human health and environment (Zheng 2017; Ma et al. 2022; Lawrence et al. 2009; Tada et al. 2009). The toxicity of these insecticides makes the monitoring of fenobucarb and propoxur essential for their environmental risk assessment, to decrease potential hazard to the environment. Therefore, it is essential to develop accurate and reliable methods of monitoring their levels for safety purposes.

Many analytical methods have been developed for the analysis of fenobucarb and propoxur and their degradation products. Most of the methods for their analysis are based on chromatographic techniques such as thin layer chromatography (Yeboah and Akpabli 2004), gas chromatography (Chormey 2021; Borahan et al. 2019), liquid chromatography (Zheng 2017; Suma et al. 2005), UPLC–MS (Xia et al. 2019; Mastovska et al. 2010) and HPLC (Yildiz et al. 2017) and optical techniques such as spectrophotometry (Murthy et al. 2012; Kumar et al. 2005; Manjubhashini et al. 2003; Hemasundaram and Naidu 2004), kinetic spectrophotometric method (Chu and Fan 2009) and fluorescence methods (Ni and Cao 2005; Khalaf et al. 1993; Wang and Wang 2005). Optical methods are widely used in all laboratories and find wide applicability due to high cost of highly sophisticated instruments and requirement of skilled technicians. The simplicity, low cost and less time of analysis are some added advantages of optical methods. Most of these spectrophotometric methods of the analysis are based on diazotization reaction with the active reagents, to form coloured compounds with the product formed by alkaline hydrolysis of these insecticides (Murthy et al. 2012; Kumar et al. 2005; Manjubhashini et al. 2003; Hemasundaram and Naidu 2004). However, very less work is done for their analysis by spectrofluorimetric technique. In this paper two simple, efficient, non-extractive and economical optical methods for the determination of fenobucarb and propoxur in bulk and on residues in environmental samples have been developed. In the proposed spectrophotometric method the colored complex Ni(II) methyl dithiocarbamate, formed by the reaction of methyl amine with CS₂ and Ni (II) acetate in an aqueous acetonitrile medium, and its measurement at 370 nm has been made the basis of spectrophotometric analysis. The spectrofluorimetric method involves the measurement of fluorescence intensity of colored product at 470 nm for both insecticides due to condensation of the methyl amine of fenobucarb and propoxur with acetylacetone and formaldehyde in the presence of buffer of pH 5.5.The proposed methods offer high sensitivity, reproducibility and involve a non-extraction procedure. To find the validity of proposed methods these have been validated for the determination of the fenobucarb and propoxur in commercial formulations and on spiked water samples and agricultural products to get reliable residue data.

Apparatus

Perkin Elmer LS 55 Fluorescence Spectrometer (Perkin Elmer, United Kingdom) with 1 cm matched quartz cells was used for fluorescence intensity measurements. All spectral measurements were processed by FL-Win Lab software. The spectrophotometric measurements have been made with a carry100 Bio UV-Vis spectrophotometer (Varian Australia Pvt. Ltd. Mulgrave-Victoria, Australia). A domestic microwave oven, (Samsung electronics, New Delhi, India) was used to carry out hydrolysis. The pH measurements were made on a Cyber Scam 2500 pH meter (Eutech instruments, Singapore). Lab pro 113 15 L water bath was used for heating.

Reagents and samples

The analytical standards of fenobucarb and propoxur procured from Sigma-Aldrich, Bangalore were used. Nickel (II) acetate (Central Drug House, Delhi, India, LR), Acetonitrile, carbon disulfide, sodium bicarbonate, sodium hydroxide, sodium sulfate anhydrous, sodium chloride, glacial acetic acid, (Merck, Mumbai, India), acetylacetone (LR, Laboratory Rasayan) and formaldehyde (AR, HIMEDIA) were used. A Buffer of pH ~ 5.5 (50 mL of 1 N, sodium hydroxide is mixed with 57.4 mL, 1N acetic acid and diluted to 500 mL with distilled water) was used (Lurie 1975). The pH of above buffer was checked and adjusted by a pH meter before use. A reagent solution for spectrofluorimetric analysis was prepared (by mixing 0.8 mL of acetylacetone and 2 mL of formaldehyde in 27.2 mL distilled water) daily. The insecticide formulation "Wilbreak" containing, 50% fenobucarb and a formulation of propoxur "Transportex" containing, 20% active ingredient were procured from the local market.

Preparation of calibration graph for pure compound by Spectrophotometric method

Aliquots 0.1-2 mL of a standard solution of fenobucarb (0.2 mM in 50:50, distilled water and acetonitrile) and 0.1-2 mL of a standard solution of propoxur (0.2 m M in acetonitrile) were taken separately in 10 mL measuring flasks and the volume made to 2 mL with 50:50, distilled water and acetonitrile in fenobucarb and with acetonitrile in propoxur. Each solution was mixed with 1.0 mL, CS₂ (10% in acetonitrile) followed by 1.0 mL aqueous sodium bicarbonate (1M) and then the volume was made to 5 mL with 50:50 distilled water and acetonitrile in fenobucarb and with acetonitrile in propoxur and kept in microwave oven for 60 s (1000 Watts). Then the solution was treated with 1 drop of acetic acid and 1mL, 1 mM nickel (II) acetate solution and the total volume was made to 10.0 mL with 50:50, distilled water and acetonitrile in fenobucarb and with acetonitrile in propoxur. The absorbance of yellow colored solution was measured at 370 nm for both insecticides against a reagent blank. The calibration curve was prepared (Fig. 1) by plotting absorbance values against concentration of the insecticides. The calibration characteristics are given in Table 1.

Table 1

Spectrophotometric and spectrofluorimetric determination of fenobucarb and propoxur as colored Ni(DTC)₂ complex: calibration characteristics.
	FenobucarbPropoxur
Optical characteristics	Corresponding value
λ_max(nm)	370	370
Beer’s law range (µg/ mL)	0.41–8.29	0.42–8.37
Molar absorptivity (L/ mol·cm)	12.08 × 10³	9.48 × 10³
Sandell’s sensitivity (µg/ cm²)	0.0172	0.0221
Stability	2 hr	2 hr
Slope	0.051	0.042
Intercept	0.0349	0.0048
Correlation coefficient (r²)	0.997	0.998
(LOD) (µg/ mL)	0.45	0.42
(LOQ) (µg/ mL)	1.37	1.26

Preparation of calibration graph for pure compound by Spectrofluorimetric method

Aliquots 0.1- 4 mL of a standard solution of fenobucarb (0.1 mM in 50:50, distilled water and acetonitrile) and 0.1- 4 mL of a standard solution of propoxur (0.1 mM in acetonitrile) were taken separately in 25 mL measuring flasks and the volume made to 4 mL with 50:50, distilled water and acetonitrile in fenobucarb and with acetonitrile in propoxur. Each solution was mixed with aqueous sodium hydroxide (1 mL, ~ 0.2 M) and kept in a microwave oven for 90 s. Then the solution was mixed with 3 mL of the buffer of pH ~ 5.5 followed by the addition of 5 mL reagent solution (0.8 mL of acetylacetone and 2 mL of formaldehyde in 27.2 mL distilled water). The mixture was heated in a water bath for 45 min and the final volume of colored solution was made to 15 mL with 50:50, distilled water and acetonitrile in fenobucarb and with acetonitrile in propoxur. The emission intensity of the solution was measured at 470 nm for both insecticides with an excitation at 390 nm. The typical excitation-emission spectrum observed is shown in Fig. 2. The calibration curves were prepared by plotting fluorescence intensity values against the concentration of the insecticides and are shown in (Fig. 2). The calibration characteristics are given in Table 2.

Table 2

Spectrofluorimetric determination of fenobucarb and propoxur: calibration characteristics.

Optical characteristics		Corresponding value
λ _excitation (nm) λ _emission (nm) Linearity range (µg/ mL) Stability Slope Intercept Correlation coefficient (r²) LOD (µg/ mL) LOQ (µg/ mL	390		390
	470		470
	0.14–5.53		0.14–5.58
	3 hr		3 hr
	94.96		86.48
	147.29		224.25
	0.998		0.997
	0.27		0.31
	0.81		0.93

Formulation analysis

One formulation "Wilbreak" containing a 50% (EC) active ingredient of fenobucarb and a formulation of propoxur "Transportex" containing a 20% (EC) active ingredient procured from an authorized pesticide dealer was used. A single large sample of propoxur formulation equivalent to 10 mg active ingredient was dissolved and shaken with 10 mL acetonitrile and filtered. The residue was washed 2–3 times with acetonitrile and the filtrate and washings were diluted to a known volume of 25 mL with acetonitrile. 2.5 mL of this solution is taken and further diluted to 100 mL. A similar procedure is followed for fenobucarb formulation by taking 50:50, distilled water and acetonitrile as solvent. Suitable aliquots of the above solution were taken for analysis and processed in the same manner as described for pure compounds. The assay results are given in Table 3.

Table 3

Assay results of commercial formulation of fenobucarb “Wilbreak” andpropoxur“Transportex” by spectrophotometric and spectrofluorimetric procedure.
			Fenobucarb
Method	Amount taken, µg		Amount found, µg	Recovery (%)^a		Amount found, µg	Recovery (%)^a
Spectrophotometric method		1.0	0.97 ± 0.01	97.4 ± 1.3	0.97 ± 0.01			97.0 ± 1.1
		2.0	1.95 ± 0.02	97.5 ± 1.1	1.95 ± 0.02			97.5 ± 0.9
		4.0	3.93 ± 0.03	98.2 ± 0.7	3.93 ± 0.03			98.2 ± 0.7
		6.0	5.91 ± 0.03	98.5 ± 0.5	5.90 ± 0.03			98.3 ± 0.6
		8.0	7.91 ± 0.04	98.9 ± 0.5	7.89 ± 0.04			98.6 ± 0.5
Spectrofluorimetric method		0.5	0.49 ± 0.01	98.0 ± 1.2	0.48 ± 0.01			96.0 ± 1.0
		2.0	1.94 ± 0.02	97.0 ± 1.1	1.95 ± 0.02			97.5 ± 0.9
		3.0	2.95 ± 0.02	98.3 ± 0.8	2.95 ± 0.03			98.3 ± 0.8
		4.0	3.94 ± 0.03	98.5 ± 0.7	3.94 ± 0.03			98.5 ± 0.7
		5.0	4.93 ± 0.03	98.6 ± 0.6	4.93 ± 0.04			98.6 ± 0.7
^a Values are the mean of five determinations with standard deviation (±)

Determination of fenobucarb and propoxur in grains, vegetable and water samples

Suitable aliquots of a standard solution of fenobucarb and propoxur having a concentration of 10 µg/ mL were added separately to a known weight, 5 gm of grains (rice and wheat), vegetables (pea and cauliflower) and water sample (25 mL). The samples (grains and water) were well mixed and extracted with 2–3 instalments of 5 mL chloroform. The combined extracts were shaken for 5 min and filtered. In the case of vegetables (after thorough mixing, each sample was blended with 50 mL of chloroform in the same containers according to the general procedure of Schenk et al. 2002. The samples were filtered through coarse filter paper (Whatman Grade No. 4) and each filtrate was transferred into 250 mL separating funnels. Sodium chloride (5 gm) was added to each sample, the contents were shaken for one minute and the phases were allowed to separate for 15 min. The lower aqueous phase and any emulsion were discarded. Anhydrous sodium sulphate (4 gm) was added and the funnel was shaken for 30 sec. The dried extract was filtered through coarse filter paper). The solvent from the filtrate (grains, vegetables and water) was removed by heating at 40°C in a water bath. The residue was dissolved in 50:50, distilled water and acetonitrile in fenobucarb and with acetonitrile in propoxur and processed for analysis as above by spectrophotometric and spectrofluorimetric methods. The amounts of fenobucarb and propoxur insecticides were calculated based on calibration graphs and the results of recovery experiments are presented in Tables 4–7.

Table 4

Recovery of fenobucarb from fortified grains, vegetables and water samples by spectrophotometric procedure.
Amount taken, µg	Recovery (%)^a
	Wheat		Rice			Water			Cauliflower		Pea
	Found, µg	Recovery (%)		Found, µg	Recovery (%)		Found, µg	Recovery (%)	Found, µg	Recovery (%)	Found,µg	Recovery (%)
1.0	0.88 ± 0.02	88.0 ± 1.6		0.89 ± 0.02	89.0 ± 1.6		0.95 ± 0.02	95.0 ± 1.6	0.91 ± 0.02	91.0 ± 1.7	0.94 ± 0.02	94.0 ± 1.6
2.0	1.76 ± 0.02	88.0 ± 1.2		1.79 ± 0.03	89.5 ± 1.3		1.90 ± 0.03	95.0 ± 1.4	1.81 ± 0.03	90.0 ± 1.3	1.86 ± 0.02	93.0 ± 1.2
4.0	3.60 ± 0.04	90.0 ± 0.9		3.63 ± 0.03	90.7 ± 0.7		3.81 ± 0.05	95.2 ± 1.2	3.69 ± 0.04	92.2 ± 0.9	3.65 ± 0.05	91.2 ± 0.9
6.0	5.39 ± 0.04	89.8 ± 0.7		5.45 ± 0.03	90.8 ± 0.6		5.71 ± 0.06	95.2 ± 0.9	5.55 ± 0.04	92.5 ± 0.7	5.64 ± 0.04	94.0 ± 0.7
8.0	7.19 ± 0.05	89.9 ± 0.6		7.19 ± 0.05	89.9 ± 0.5		7.66 ± 0.06	95.7 ± 0.8	7.43 ± 0.05	92.9 ± 0.6	7.51 ± 0.05	93.9 ± 0.6
^a Values are the mean of five determinations with standard deviation (±)

Table 5

Recovery of propoxur from fortified grains, vegetables and water samples by spectrophotometric procedure.
Amount taken (µg)	Recovery (%)^a
	Wheat		Rice		Water				Cauliflower		Pea
	Found, µg	Recovery (%)	Found, µg	Recovery (%)			Found, µg	Recovery (%)	Found, µg	Recovery (%)			Found, µg	Recovery (%)
1.0	0.92 ± 0.02	92.0 ± 1.5	0.87 ± 0.02	87.0 ± 1.6		0.92 ± 0.02		90.0 ± 1.5	0.90 ± 0.02	90.0 ± 1.6		0.87 ± 0.01			87.0 ± 1.3
2.0	1.78 ± 0.02	89.0 ± 1.2	1.79 ± 0.02	89.5 ± 1.2		1.87 ± 0.02		92.0 ± 1.0	1.79 ± 0.03	89.5 ± 1.3		1.77 ± 0.02			88.5 ± 0.9
4.0	3.62 ± 0.04	90.5 ± 0.9	3.63 ± 0.03	90.7 ± 0.7		3.75 ± 0.03		93.7 ± 0.7	3.64 ± 0.03	91.0 ± 0.8		3.46 ± 0.03			86.5 ± 0.7
6.0	5.54 ± 0.04	92.3 ± 0.7	5.46 ± 0.04	91.0 ± 0.6		5.66 ± 0.04		94.3 ± 0.6	5.34 ± 0.04	89.0 ± 0.7		5.39 ± 0.03			89.8 ± 0.5
8.0	7.39 ± 0.06	92.4 ± 0.6	7.28 ± 0.04	91.0 ± 0.5		7.57 ± 0.05		94.6 ± 0.5	7.01 ± 0.05	87.6 ± 0.6		7.14 ± 0.04			89.2 ± 0.4
^a Values are the mean of five determinations with standard deviation (±)

Table 6

Recovery of fenobucarbfrom fortified grains, vegetables and water samples by spectrofluorimetric procedure.
Amount taken (µg)	Recovery (%)^a
	Wheat		Rice		Water		Cauliflower			Pea
	Found, µg	Recovery (%)	Found, µg	Recovery (%)	Found, µg	Recovery (%)	Found, µg	Recovery (%)	Found, µg		Recovery (%)
0.5	0.45 ± 0.01	90.0 ± 1.6	0.45 ± 0.01	90.0 ± 1.6	0.48 ± 0.01	96.0 ± 1.8	0.45 ± 0.01	90.0 ± 1.2	0.47 ± 0.01		94.0 ± 1.4
1.0	0.96 ± 0.01	96.0 ± 1.4	0.90 ± 0.01	90.0 ± 1.4	0.98 ± 0.01	98.0 ± 0.9	0.95 ± 0.01	95.0 ± 1.4	0.93 ± 0.02		93.0 ± 1.6
2.0	1.79 ± 0.03	89.5 ± 1.4	1.78 ± 0.02	89.0 ± 1.2	1.90 ± 0.03	95.0 ± 1.6	1.82 ± 0.02	91.0 ± 0.9	1.89 ± 0.03		94.5 ± 1.3
3.0	2.70 ± 0.04	90.0 ± 1.2	2.67 ± 0.03	89.0 ± 0.9	2.86 ± 0.04	95.3 ± 1.4	2.69 ± 0.03	89.7 ± 0.8	2.84 ± 0.03		94.7 ± 1.0
4.0	3.61 ± 0.04	90.2 ± 1.1	3.58 ± 0.03	89.5 ± 0.8	3.85 ± 0.05	96.2 ± 1.2	3.61 ± 0.03	90.2 ± 0.7	3.77 ± 0.04		94.2 ± 0.9
5.0	4.47 ± 0.05	89.4 ± 1.0	4.51 ± 0.04	90.2 ± 0.7	4.78 ± 0.06	95.6 ± 1.1	4.55 ± 0.04	91.0 ± 0.7	4.69 ± 0.04		93.8 ± 0.8
^a Values are the mean of five determinations with standard deviation (±).

Table 7

Recovery of propoxur from fortified grains, vegetables and water samples by spectrofluorimetric procedure.
		Recovery (%)^a
Amount taken (µg)		Wheat		Rice		Water		Cauliflower			Pea
Amount taken (µg)		Found, µg	Recovery (%)	Found, µg	Recovery (%)	Found, µg	Recovery (%)	Found, µg	Recovery (%)	Found, µg		Recovery (%)
0.5	0.46 ± 0.01		92.0 ± 1.4	0.44 ± 0.01	88.0 ± 1.6	0.47 ± 0.01	94.0 ± 1.8	0.46 ± 0.01	92.0 ± 1.2	0.45 ± 0.01		90.0 ± 1.4
1.0	0.92 ± 0.02		92.0 ± 1.8	0.92 ± 0.02	92.0 ± 1.5	0.94 ± 0.02	94.0 ± 1.6	0.89 ± 0.01	89.0 ± 1.3	0.92 ± 0.02		92.0 ± 1.5
2.0	1.80 ± 0.03		90.0 ± 1.3	1.79 ± 0.03	89.5 ± 1.3	1.87 ± 0.03	93.5 ± 1.2	1.82 ± 0.02	91.0 ± 1.0	1.76 ± 0.02		88.0 ± 1.0
3.0	2.71 ± 0.03		90.3 ± 1.0	2.71 ± 0.03	90.3 ± 1.0	2.82 ± 0.03	94.0 ± 1.0	2.74 ± 0.02	91.3 ± 0.7	2.72 ± 0.03		90.7 ± 0.9
4.0	3.65 ± 0.04		91.2 ± 0.9	3.59 ± 0.04	89.7 ± 0.9	3.71 ± 0.04	92.7 ± 0.9	3.65 ± 0.03	91.2 ± 0.7	3.57 ± 0.04		89.2 ± 0.8
5.0	4.59 ± 0.04		91.8 ± 0.8	4.55 ± 0.04	91.0 ± 0.8	4.67 ± 0.04	93.4 ± 0.8	4.44 ± 0.03	88.8 ± 0.6	4.45 ± 0.04		89.4 ± 0.8
^a Values are the mean of five determinations with standard deviation (±)

Interference studies

To the standard solution containing 5 µg of fenobucarb and propoxur, various aliquots of each amines (5 x 10^− 3 M) and pesticides solution (5 x 10^− 4 M) were added separately prior to hydrolysis. Each solution was studied in the same manner as described for both insecticides by spectrophotometric and spectrofluorimetric methods.

Optimization of the reaction conditions for the spectrophotometric method

The optimum conditions for the stability and the development of maximum absorbance of the colored complex by the proposed method were established by varying various parameters such as hydrolysis time, effect of solvents and concentration of CS₂.

Effect of hydrolysis time

The maximum color intensity and stability of the complex formed were obtained at 60 sec by varying the optimum time of hydrolysis in the microwave from 30–120 sec (Fig. 3). Hydrolysis time less than or more than 60 sec gives less intensity of the color.

Effect of solvent

To study the effect of solvents on the stability of complex, different diluting solvents such as water, ethanol, methanol, acetonitrile and acetone were studied. It has been observed that the maximum stability of the complex was obtained by taking 50:50, acetonitrile and water as a solvent for fenobucarb and acetonitrile as a solvent for propoxur insecticide.

Effect of concentration of carbon disulfide

The influence of the concentration of CS₂ on the absorbance of complex was studied by varying different concentrations of CS₂ (2–10% in acetonitrile). It was found that maximum absorbance was obtained at 10%, CS₂ concentration for both insecticides, after which the turbidity of solution increases (Fig. 4).

Quantification

The yellow colored complex formed by the reaction of methyl amine with carbon disulfide and nickel(II) acetate under the standardized experimental conditions is stable for 2 hrs for both insecticides. The Beer’s law in the range of 0.41–8.29 µg/ mL and 0.42–8.37 µg/ mL for fenobucarb and propoxur is obeyed with the limit of detection and limit of quantification 0.45 and 1.37 µg/ mL for fenobucarb and 0.42 and 1.26 µg/ mL for propoxur. The molar absorptivity (ε) and Sandell’s sensitivity values of 12.08 × 10³ L/ mol·cm and 0.0172 µg/ cm² for fenobucarb and 9.48 × 10³ L/ mol·cm and 0.0221 µg/ cm² for propoxur at 370 nm shows the sensitivity of the proposed method. The method is further validated to the analysis of fenobucarb and propoxur insecticides in commercial formulation, grains, vegetables and spiked water samples. The recoveries of the fenobucarb from commercial formulation were 97.4–98.9% of the nominal content with relative standard deviations (RSDs) in the range 0.5–1.3% and 97.0- 98.6% with relative standard deviations (RSDs) in the range 0.5–1.1% for propoxur (Table 3). The recoveries of the fenobucarb insecticide from grains, vegetables and spiked water samples were good, ranging from 88.0–95.7% with RSDs ranging from 0.5–1.7% and 86.5–94.6% for propoxur with RSDs 0.4–1.6% (Tables 4–5).

Proposed mechanism for the spectrophotometric method

The proposed spectrophotometric method is based on the alkaline hydrolysis of fenobucarb and propoxur to methyl amine (Malhotra et al. 2021; Borkovcova et al. 2004), which smoothly and quantitatively reacts with carbon disulfide to form corresponding methyl dithiocarbamate. The latter on reaction with nickel(II) acetate gives a yellow colored complex, nickel(II) methyl dithiocarbamate [Ni(DTC)₂] complex (Scheme 2) in aqueous acetonitrile medium, which is measured under optimized conditions at 370 nm for both insecticides (Fig. 1).The methyl amine formed from the alkaline hydrolysis of fenobucarb and propoxur, reacts smoothly and quantitatively with carbon disulfide (Verma et al. 1986; Sharma et al. 2015) and nickel(II) acetate (in 2:1 molar ratio) forming metallic complex nickel(II) methyl dithiocarbamate, [Ni(DTC)₂] (Fig. 5) is quite well known and thoroughly established in our laboratory (Sharma et al. 2015).

Interference studies

The validity of the proposed method was assessed by investigating the effect of various amines and pesticides in the analysis of a standard solution containing 5µg of fenobucarb and propoxur, separately. It was found that the proposed method is free from interferences due to added common cations and anions, organic nitrogen compounds including tertiary amines, aromatic amines and pesticides. The tolerance limit of added foreign species is given in Table 8.

Table 8

Effect of foreign species on the determination of fenobucarb and propoxur insecticides by spectrophotometric method.
Foreign species	Tolerance limit (µg /mL)	Foreign species	Tolerance limit (µg /mL)
Triethyl amine	350	Benomyl	25
Tributyl amine	300	Captafol	50
Diphenylamine	250	Imazethapyr	200
Dimethylaniline	200	Chloropyrifos	250
*The amount causing an error 2 ± 1.5%

Comparison of proposed spectrophotometric method with other reported methods

A comparison some parameters obtained from the proposed spectrophotometric method for determination of fenobucarb and propoxur has been done with other reported spectrophotometric methods and the results are shown in Table 9. The results indicate that the proposed method is more sensitive, accurate, rapid with instantaneous development of colour with nickel (II) acetate reagent.

Table 9

Effect of foreign species on the determination of fenobucarb and propoxur insecticides by spectrofluorimetric method.
Foreign species	Tolerance limit (µg /mL)	Foreign species	Tolerance limit, (µg /mL)
Triethyl amine	250	Carbendazim	100
Tributyl amine	350	Benomyl	250
Diethylamine	200	Captafol	200
Diphenylamine	200	Imazethapyr	450
Dimethylaniline	250	Thiophanate-methyl	80
Dibutyl amine	20	Chloropyrifos	300
Acephate	150	Mancozeb	200
*The amount causing an error 2 ± 1.5%

Optimization of the reaction conditions for the spectrofluorimetric method

The effect of various parameters such as heating time, hydrolysis time, pH, solvents and reagent concentration on the fluorescence intensity of the formed complex was studied and the reaction conditions are optimized.

Effect of hydrolysis time

The effect of hydrolysis time on the fluorescence intensity was studied by varying the time of hydrolysis from 30–120 sec in microwave. The results indicate that maximum fluorescence intensity was obtained corresponding to 90 sec hydrolysis time (Fig. 6).

Effect of heating time

The reaction time for the complete reaction was studied in water bath by varying heating time from 20–60 min at 55 ^oC. The maximum fluorescence intensity was obtained at 45 min of heating (Fig. 7) after which the fluorescence intensity further decreases.

Effect of pH

The effect of pH on the fluorescence intensity was studied by the use of different acetic- acetate buffer in the range from 3.8–6.3, prepared by mixing certain volumes of 1N acetic acid and 1N sodium hydroxide and diluting the mixture with distilled water [26].The maximum fluorescence intensity has been obtained at pH ~ 5.5 (Fig. 8).

Effect of reagent concentration

The effect of changing the concentration of mixture of acetylacetone and formaldehyde solutions on the fluorescence intensity of fenobucarb and propoxur was studied. The maximum fluorescence intensities were observed upon using 5 mL of reagent solution (prepared by mixing 0.8 mL of acetylacetone and 2 mL of formaldehyde in 27.2 mL distilled water).

Effect of diluting solvents

The stability and intensity of the colored product has been studied by varying diluting solvents such as water, ethanol, acetonitrile, dimethyl formamide, dimethyl sulfoxide and acetone. It is found that acetonitrile in propoxur and 50:50 acetonitrile and water for fenobucarb gives best results as they clears and stabilizes the colored product quantitatively.

Quantification

Under the optimized experimental conditions the proposed spectrofluorimetric method obeys the linearity range of 0.14–5.53 and 0.14–5.58 µg/ mL for fenobucarb and propoxur respectively. The complex formed is stable for 3 hrs for both insecticides. The limit of detection and limit of quantification are 0.27 and 0.81 µg/ mL and 0.31 and 0.93 µg/ mL for fenobucarb and propoxur respectively. The method has been subsequently applied to the determination of fenobucarb and propoxur insecticides in commercial formulation, vegetables and spiked water samples. The recoveries from the commercial formulation were 97.0–98.6% of the nominal content with relative standard deviations (RSDs) in the range 0.6–1.2% and 96.0–98.6% with relative standard deviations (RSDs) in the range 0.7-1.0% for fenobucarb and propoxur respectively (Table 3).The high recoveries of the fenobucarb and propoxur from spiked water, grain and vegetable samples in the range and 89.0–98.0% with RSDs of 0.7–1.8% for the fenobucarb and 88.0–94.0% with RSDs of 0.6–1.8 for propoxur indicates good accuracy and precision of the method (Tables 6 and 7).

Proposed mechanism for the spectrofluorimetric method

The advantage of the reaction of primary amino group (methyl amine, hydrolytic product of fenobucarb and propoxur), with acetylacetone and formaldehyde through Hantzsch reaction has been made the basis of the proposed spectrofluorimetric analysis. The Hantzsch reaction, involves condensation reaction for the synthesis of dihydropyridine and pyridine (Derker and David 1979; Clayden et al. 2016) using acetylacetone as β - diketone and formaldehyde as an aldehyde which forms a colored condensation product (Scheme 3) with methyl amine of fenobucarb and propoxur. The reaction product dihydropyridine derivative, exhibited strong fluorescence at 470 nm after excitation at 390 nm for both insecticides (Fig. 2).

Interference studies

The influence of foreign species on the validity of the proposed spectrofluorimetric method was investigated by adding known aliquots of the diverse foreign species, amines and pesticides to a standard solution containing 5µg of fenobucarb and propoxur separately. Various organic nitrogen compounds including secondary and tertiary amines did not affect determination of fenobucarb and propoxur insecticides by proposed spectrofluorimetric method indicating, the selectivity of method for primary amino group compounds. The tolerance limit of these added foreign species is given in Table 10.

Table 10

Comparison of proposed spectrophotometric method of fenobucarb and propoxur with reported methods.
Spectrophotometric Method	Linear range, µg/ mL	LOD, µg/ mL	LOQ, µg/ mL	Analytical reagent	λ, nm	Samples
[19]	0.5–12	–	–	4- aminopyridine	469	Commercial formulations, water, grains
[1]	0.5–10.0	0.3(PRO) 0.2(BPMC)	–	p-nitroaniline	510	water and fruit
[20]	0.4–10 0.8–10 0.6–10	0.295 0.672 0.426	1.472 3.361 2.131	2,6-dibromo-4-methylbenzenamine 2,6-dibromo-4-nitrobenzenamine 2,4,6-tribromo benzenamine	470 490 478	Water, vegetable, agricultural waste water and soil
[9]	0.8-5.0	–	–	Aniline		Water, fruits, vegetables, and grains
[21]	0.4–10.0 0.4–12.0	– –	– –	2-amino, 4-nitro phenol 2,4-dimethoxy aniline	402 460	Formulations, water and food grains
[22]	0.05–1.2	–	–	3-aminopyridine	463	Water, rice, sugarcane, potato, soil
Proposed method for Fenobucarb and Propoxur	0.41–8.29 0.42–8.37	0.45 0.41	1.37 1.26	Carbon disulphide- nickel(II) acetate	370	Commercial formulations, grains, vegetables and water samples

Comparison of proposed spectrofluorimetric method with other reported methods

Some parameters obtained from the proposed spectrofluorimetric method for the determination of above insecticides, with other reported spectrofluorimetric methods has been done and shown in Table 11. Although very less work by spectrofluorimetric method is reported for these insecticides. It is found that the proposed spectrofluorimetric method has superior sensitivity with the use of low cost reagents.

Table 11

Comparison of proposed spectrofluorimetric method of fenobucarb and propoxur with reported methods.
Spectrofluori metric Method	Linear range	LOD	LOQ	Analytical reagent	λ_excitation(nm) λ_emission (nm)	Samples
[23]	0.04–0.4	5.5 ng L^-1 ^-	–	Britton-Robinson buffer	220–340	Spiked fruit, vegetable samples
[24]	0–4.8 × 10 ^− 6 M	5 × 10 ^− 8 M	–	CTAB micelles	270/295	Bulk
Proposed method for fenobucarb and propoxur	0.14–5.53 µg/ mL 0.14–5.58 µg/ mL	0.27 µg/ mL 0.31 µg/ mL	0.81 µg/ mL 0.93 µg/ mL	Acetylacetone/ formaldehyde	390 470	Formulations, vegetables, grains and water samples

The proposed optical methods are precise, accurate and cost effective for the determination of fenobucarb and propoxur in pure form, commercial formulations and environmental samples. The stability and instant appearance of the colored products, non extractive process, no pre-treatment process and the low consumption of samples and reagents are some added advantages of the proposed methods. The high recoveries of fenobucarb and propoxur insecticides in formulation and residue analysis with low relative standard deviation values indicate good accuracy and precision of the methods.

Statements and Declarations

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Competing Interests

Authors have no financial or non-financial interests to the work submitted for publication. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgment

The authors acknowledge the instrumental facilities provided by the Department of Chemistry, Himachal Pradesh University- Shimla - Himachal Pradesh to carry out this work.

Chu N, Fan S (2009) Sequential injection kinetic spectrophotometric determination of quaternary mixtures of carbamate pesticides in water and fruit samples using artificial neural networks for multivariate calibration. Spectrochim Acta Part A, Mol Biomol Spectrosc 74: 1173- https//doi.org/10.1016/j.saa.2009.09.030
Dias E, Costa FG, Morais S, Pereira ML (2015) A review on the assessment of the potential adverse health impacts of carbamate pesticides. http//dx.doi.org/10.5772/59613
Vale JA, Bradberry SM (2016) Organophosphorus and carbamate insecticides. In: Brent J, Burkhart K, Dargan P, Hatten B, Megarbane B, Palmer R (eds). Critical Care Toxicology, 1^st Springer, Cham. 1 -26. https//doi.org/10.1007/978-3-319-20790-2_52-1
Zhu X, Wu Y, Xia B, Dai M, Huang Y, Yang H, Li C, Li P (2020) Fenobucarb-induced developmental neurotoxicity and mechanisms in zebrafish. Neuro Toxicol 79:11-https//doi.org/10.1016/j.neuro.2020.03.013
Xia W, He Z, Hu K, Gao B, Zhang Z, Wang M, Wang Q (2019)Simultaneous separation and detection chiral fenobucarb enantiomers using UPLC–MS/MS. SN Appl Sci 1: 795- https//doi.org/10.1007/s42452-019-0822-8
Chormey DS (2021) Determination of fenobucarb and chlorbenside in wastewater and lake water samples by Gas Chromatography Mass Spectrometry. European J Sci Technol 27: 518-5 https,//doi.org/10.31590/ejosat.928133
Pham TV, Phuoc VM, Nguyen DV, Koyama J (2021) Treatment efficiency of a combination of alternative technologies in removing pollutants from pesticide containing wastewater. Environ Eng Res 26: 1-9. https,//doi.org/10.4491/eer.2020.263
Tabibi A, Jafari MT (2006) Ultrasonic piezoelectric nebulization of propoxur for the determination by corona discharge ionization ion mobility spectrometry. Anal Sci 36:227- https//doi.org/10.2116/analsci.19P262
Patel V, Rai MK, Rai JK (2013) A new spectrophotometric method for the determination of Baygon in environment and biological samples. Recent Res Sc Technol5: 1- https//updatepublishing.com/journal/index.php/rrst/article/view/1011
Yildiz Y, Jan A, Yildiz B (2017) Determination of 2-Isopropoxyphenyl methyl carbamate propoxur in roach control peanut butter by High Performance Liquid Chromatography. J Environ Anal Chem 4: 1- Doi: 10.4172/2380-2391.1000199.
Zheng W, Park JA, Zhang D, Abd El-Aty AM, Kim SK, Cho SH et al (2017) Determination of fenobucarb residues in animal and aquatic food products using liquid chromatography-tandem mass spectrometry coupled with a QuEChERS extraction method, J Chromatogr B 2017. 1058,1- http//dx.doi.org/10.1016/j.jchromb.2017.05.008
Ma S, Zhang H, Li F, Zhao P, Yin S, Sun J, Xu J, Wang Z, Xu X, Di X (2022) Systemic stereo selectivity study of fenobucarb, environmental behaviors in greenhouse vegetables, fruits, earthworms, and soils and its cytotoxicity. J Agric Food Chem 70: 2127–2135. https//doi.org/10.1021/acs.jafc.1c06420
Lawrence E, Thomas IB, Isioma T (2009) Distribution of propoxur in water, sediment and fish from Warri river niger delta. Nigeria.Turk J Biochem 34: 121–27.
Tada M, Hatakeyama S (2009
) Chronic effects of an insecticide, fenobucarb, on the larvae of two mayflies, Epeoruslatifolium and Baetisthermicus, in Model Streams. Ecotoxicol9: 187–195. https//doi.org/10.1023/A,1008990604166
Yeboah PO, Akpabli C (2004) Comparison of thin layer chromatography (TLC) and gas chromatograph determination of propoxur residues in a cocoa ecosystem. African J Sci Technol 4:24 - DOI: 10.4314/ajst.v4i2.15311
Borahan T, Tekin Z, Zaman BT, Chormey DS, Bakirdere S (2019) An analytical strategy for propoxur determination in raisin samples with matrix matching method after dispersive liquid-liquid microextraction. J Food Compos Anal 84: 103315–19. https,//doi.org/10.1016/j.jfca.2019.103315
Suma R, Sarin RK, Saiprakash PK, Ramakrishna S (2005) Simple liquid chromatographic method for the rapid and simultaneous determination of propoxur and its major metabolite isopropoxy phenol in rat blood and urine using solid-phase extraction. J Anal Toxicol 29: 728- https,//doi.org/10.1093/jat/29.7.728
Mastovska K, Dorweiler KJ, Lehotay SJ, Wegscheid JS, Szpylka KA (2010) Pesticide multiresidue analysis in cereal grains using modified QuEChERS method combined with automated direct sample introduction GC-TOFMS and UPLC-MS/MS techniques. J Agric Food Chem 58: 5959- https//doi.org/10.1021/jf9029892
Murthy PK, Naidu KB, Naidu NV (2012) A simple spectrophotometric method for determination of propoxur using 4- amminopyridine. J Chem Pharm Res 4: 96-
Kumar KS, Suvardhan K, Rekha D, Jayaraj B, Chiranjeevi P(2005) Facile and sensitive spectrophotometric determination of propoxur in formulations and environmental samples. Helv Chim Acta 88: 1022 - https//doi.org/10.1002/hlca.200590072
Manjubhashini AB, Raman GK, Kumar SA (2003) A Spectrophotometric method for the determination of propoxur. Phys Chem Liq 41: 81- https//doi.org/10.1080/0031910021000044483
Hemasundaram A, Naidu NV (2004) Sensitive determination of propoxur by spectrophotometry using 3-aminopyridine. Anal Sci 20: 1707- https,//doi.org/10.2116/analsci.20.1707
Ni Y, Cao D (2005) Simultaneous synchronous fluorescence determination of carbaryl, pPropoxur and carbofuran with multivariate calibration methods. Spectrosc Lett 39: 431- https//doi.org/10.1080/00387010600812640
Khalaf KD, Sancenon J, Delaguardia M (1993) Study of the interaction of propoxur and its metabolites with surfactants. Microchem J 48: 200- https//doi.org/10.1006/mchj.1993.1091
Wang ZD, Wang YT (2005) Study on fluorescence analysis for carbamate pesticides. Spectrosc Spect Anal 25: 1645- http//www.gpxygpfx.com/EN/Y2005/V25/I10/1645
Lurie, Ju,Handbook of Analytical Chemistry, MIR Publishers, Moscow, 1975. p 262.
Schenck FJ, Lehotay SJ, Vega VJ (2002) Comparison of solid-phase extraction sorbents for cleanup in pesticide residue analysis of fresh fruits and vegetables. Sep Sci 25: 883- 90.
Malhotra H, Kaur S, Phale PS (2021) Conserved metabolic and evolutionary themes in microbial degradation of carbamate pesticides. Front Microbiol 12: 1-31. https//doi.org/10.3389/fmicb.2021.648868
Borkovcova I, Janouskova E, Rehurkova I, Ruprich J (2004) Determination of n-methylcarbamates in foods. Cent Eur J Public Health 12: 220-223.

Verma BC, Chauhan S, Sharma N, Sharma U, Sharma DK, Sood A (1986) Analytical applications of the amine-carbon disulphide reaction in acetonitrile. Talanta 33: 703- https//doi.org/10.1016/0039-9140(86)80164-3
Sharma DK, Dharmani T, Sharma N (2015) Extractive spectrophotometric method for the determination of carbaryl in environmental samples. Chem. Soc. Ethiop 29: 173- 186. http//dx.doi.org/10.4314/bcse.v29i2.2
Derker BF, David OW (1979) Comprehensive organic chemistry, the synthesis and reactions of organic compounds. London, Pergamon
Clayden J, Greeves N, Warren S (2016) Oganic Chemistry. Oxford

Schemes 1, 2 and 3 are available in the Supplementary Files section.

Scheme1.jpg
Scheme 1. Chemical structures of fenobucarb (I) and propoxur (II)
Scheme2.jpg
Scheme 2. Proposed mechanism for the spectrophotometric method
Scheme3.jpg
Scheme 3. Proposed mechanism for the spectrofluorimetric method for fenobucarb and propoxur
Graphicalabstract.tif

Download PDF

Journal Publication

published 24 Apr, 2024

Read the published version in Chemical Papers →

Reviewers agreed at journal
16 Nov, 2023
Reviewers invited by journal
25 Oct, 2023
Editor invited by journal
07 Oct, 2023
Editor assigned by journal
05 Oct, 2023
First submitted to journal
30 Sep, 2023

You are reading this latest preprint version

Assessment of Fenobucarb and Propoxur insecticides in environmental samples by spectrophotometric and spectrofluorimetric methods

Status:

Journal Publication

Version 1

Abstract

Figures

INTRODUCTION

Materials and Methods

Apparatus

Reagents and samples

Preparation of calibration graph for pure compound by Spectrophotometric method

Preparation of calibration graph for pure compound by Spectrofluorimetric method

Formulation analysis

Determination of fenobucarb and propoxur in grains, vegetable and water samples

RESULTS AND DISCUSSION

Optimization of the reaction conditions for the spectrophotometric method

Effect of hydrolysis time

Effect of solvent

Effect of concentration of carbon disulfide

Quantification

Proposed mechanism for the spectrophotometric method

Interference studies

Comparison of proposed spectrophotometric method with other reported methods

Optimization of the reaction conditions for the spectrofluorimetric method

Effect of hydrolysis time

Effect of heating time

Effect of pH

Effect of reagent concentration

Effect of diluting solvents

Quantification

Proposed mechanism for the spectrofluorimetric method

Interference studies

Comparison of proposed spectrofluorimetric method with other reported methods

CONCLUSION

Declarations

References

Schemes

Supplementary Files

Status:

Journal Publication

Version 1