Green Spectrophotometric Determination of Organophosphate in Selected Fruits and Vegetables

A simple, sensitive, precise, and environmentally safe spectrophotometry method was developed and validated for the determination of organophosphate in various fruits and vegetables using a UV-Visible spectrophotometer using a magnesia mixture. The volume of reagent used for analysis and the stability of the color complex were also optimized. The drug showed a stable white color complex at 420 nm. The greenness of the methods was estimated using an ecoscale (84), the Green Analytical Procedure Index, and AGREE (0.89), which were found to be excellent green method based on spectrophotometric determination. The method was validated using ICH guidelines and has acceptable values for linearity (0.5–2.5 mg/ml), accuracy (98.5–102.5%), precision, robustness, limit of detection (0.16 mg), and limit of quantification (0.486 mg). The concentration of the organophosphate in the analyzed sample was in the range of 0.003 to 2.45 mg. Altogether, the proposed green analytical method was found to be a simple, selective, sensitive, accurate, and ecofriendly method for the analysis of organophosphate in various fruits and vegetables.


Introduction
Pesticides are chemical substances or mixture of chemicals meant for preventing, repelling, destroying, or controlling any pest like insects or other organisms. As per WHO 2008, pesticide poisoning is any pesticide-related injury or health efect including systemic and nonsystemic efect, resulting from suspected or confrmed exposure to a pesticide [1]. Tese are synthetic molecules aimed at being toxic towards fungi, plants, or animals that are detrimental to cultures. Fungicides, herbicides, and insecticides have been developed in order to control as specifcally as possible these pests in order to protect cultures. Nevertheless, these pesticides can be toxic to human and wild fauna [2]. Vegetable cultivation attracts high rates of the application of pesticides and farmers in many developing countries use many acutely toxic insecticides on those crops. Apart from application of pesticides exposures can also occur for farmers involved in the harvesting process or who enter the sprayed feld too soon after spraying [3]. Farmers, particularly in areas of commercial vegetable production, are primarily relying on chemical pest control methods. Te frequency of pesticides used is 3 times per week. Around 97.6% of subjects used incomplete personal protective equipment [4].
Most of the Nepalese population lives in rural areas, and nearly 80% of the country's people are involved in agriculture which is an important factor for the national economy. Cultivation of vegetables in the agricultural lands of Nepal normally generates high earnings per unit area in comparison to the crops although it is cultivated in a wide area of Nepalese agricultural lands. Te number of smallscale farmers growing vegetables for the domestic market in Nepal is increasing. Pesticides are widely used in Nepal to control various pests and disease in agriculture as well as livestock production. Te increasing demands of food and vegetables have led to an increased use of pesticides. Among the pesticides used in Nepal, organophosphate compounds are the most commonly used [3].
In the 1930s, organophosphates were used as insecticides, but the German military developed these substances as neurotoxins in the Second World War [5]. Organophosphates (OP) are chemical substances produced by the process of esterifcation between phosphoric acid and alcohol. Tese chemicals are the main components of herbicides, pesticides, and insecticides [6]. Organophosphate poisoning can be acute or chronic. Te symptoms of acute toxicity are hypersecretion, bronchoconstriction, myosis, diarrhea, bradycardia, central nervous system (CNS) depression, seizure, cyanosis, and coma [7]. Te organophosphate poisoning was experienced by farmers through inhalation and dermal contact. Te severity depends on pesticide type, dose, and duration of application and frequency of application. Te intensity of organophosphate poisoning was infuenced by the area of pesticide application, climate skill of the application, and personal [8]. Te mechanism of organophosphate`s toxicity is via inhibiting the acetylcholinesterase (AchE), is an enzyme that degrades the neurotransmitter acetylcholine to choline and acetic acid. Te inhibition of AchE will cause the increase in acetylcholine concentration in the synapse. Tis mechanism will cause some nicotinic and muscarinic symptoms and central and peripheral nervous system toxicity [9].
Due to the excess demand of numbers of fruits and vegetables, Nepal depends a lot in India which supplies these food stufs from a long decade. Tere were few news stories in the newspaper, TV, and so on. related to use of excess pesticides in fruits and vegetables which are transported in border area of Nepal from India. Hence, this study will defnitely provide the scientifc statement regarding this issue. Among various fruits available in the markets, the selection of fruits i.e. mango and watermelon is to rationalize the study as these fruits are mostly available and consumed fruits in our Nepalese society. Tese are consumed in every family visit and devour by children, so studying these can make our research more relevant. Te reason for choosing other fruits like kiwi is the popularity in the current context of Nepal. Kiwi and lemon are now famous as a source of Vitamin C, support heart health and the digestive system, protect anemia, reduce cancer risk, and to boost immune system. Tere is a rising demand for organic agricultural products due to consumer concern about the strong contamination of vegetables from the applied pesticides. Terefore, it is essential to identify the presence of pesticide residue in vegetables so that consumers could be protected. In this regard, this study is going to be initiated to assess the present status of organophosphate pesticide residue in eggplant, tomato, cucumber, cabbage, ladyfnger, cowpea, pointed gourd, and bean.
Various research studies have conducted for the determination of the organophosphate in foods using methods such as P-NMR [10], conductometry [11], and GC-MS/ GC-NPD [12], where most of the methods use HPLC [13][14][15][16] or liquid-mass spectrometry [17]. Since these methods are highly sophisticated, expensive, and use a large amount of organic solvents along with a high amount of instrumental energy, so a developing country such as Nepal could not aford the availability of such machines in every local area. Te use of less sophisticated analytical methods like UV-Visible spectrophotometer with more concern on greenness profle, if provides the similar results like abovementioned methods, this will be an extremely economical method of detection. Hence, a simple, green economic UV-Visible spectrophotometric method was developed and validated for the analysis of fruits and vegetables.

Equipment and Software
Required. LT-2100 double beam UV-visual spectrophotometer with a 10 mm quartz cuvette was used to record the absorbance. Te greenness of the method was established using GAPI chart version 1.0.0.

Collection of Samples.
Te fruits and vegetables samples were collected from the local market (Chitwan) as well as from Raxaul, India, and proceed for extraction.

Extraction.
A stainless steel knife was used to cut fruits and vegetable samples, sliced and diced them, and 1 gm sample was placed inside a glass test tube, as shown in the Figure 1. Te extraction was carried as per Knowledge-Based Integrated Sustainable Agriculture and Nutrition (KISAN) II Project [18] using 2 ml of 95% alcohol (ethanol) and bromine water solution. Finally, the extracted solution was poured into a clean test tube prior to proceeding for test.

Mechanism of Color Complex Formation.
Magnesia mixture was prepared by adding 2 gm magnesium chloride solution to 1 gm ammonium chloride and adding about 30 drops of ammonium hydroxide to above-mentioned solution after boiling and cooling it till the strong smell of ammonia is obtained. Tus, prepared solution is allowed to react with the sample extract to obtain white color of the magnesium phosphate and detected in UV-Visible spectrophotometer. Te intensity of the white precipitate depends on the amount of magnesium phosphate, which in turn depends on the phosphate present in fruit and vegetable samples. Tis is the basis of organophosphate pesticide analysis.

Sample Preparation.
From each of the extracts, 1 ml sample was taken and added with the 2 ml of magnesia mixture in 10 ml of volumetric fask. Te fnal volume is made up by distilled water for both Indian and local samples.

Selection of Wavelength
Maxima. Disodium orthophosphate was taken as the standard for this analysis. It was allowed to react with the magnesia mixture to form a white color complex. Te absorbance was observed at the maximum wavelength 420 nm after varying wavelength from 200 to 800 nm.

Optimization of Volume of Reagent.
Te optimization of the reagent was frst established by varying volume of the reagent (1 ml to 3 ml), where the maximum absorbance at 2 ml was found. Hence, it was selected after optimizing volume of reagent, as shown in Table 1.

Stability of Colored Complex.
After optimizing the volume of the reagent it was subjected for the stability test. Te optimum time for completion of the reaction between disodium orthophosphate and magnesia mixture to obtain the white color was 1 min, and the complex was stable for 1 hour. Ten the absorbance was measured, and it was quite stable with precise measurement which is shown in Table 2.

Preparation of Calibration
Curve. 0.5 gm of disodium orthophosphate was weighed and dissolved in distilled water and made up to 100 ml to prepare a 5 mg concentration of solution. Te secondary stock solution was prepared at 0.5 mg, 1 mg, 1.5 mg, 2 mg, and 2.5 mg concentrations, respectively, after the addition of magnesia mixture and dilution with water. Finally, absorbance was observed in a UV spectrophotometer at a wavelength of 240 nm after obtaining it as the maximum wavelength.

Determination of Concentration of Organophosphate in Samples
3.5.1. Analysis in Vegetables. All randomly selected samples were collected and extraction of it was carried out. 1 ml of the extract was taken and diluted to 10 ml volumetric fask with 2 ml of magnesia mixture and 7 ml of water. Te mixture was observed in a UV-Visible spectrophotometer and the absorbance obtained was recorded. Finally, the organophosphate concentration on each vegetable was calculated and compared, as shown in Figure 2 and a statistical analysis was established (Table 3).

Analysis in Fruits.
Due to the unavailability of local seasonal fruit samples during the time of research, only Indian fruit samples were used for the determination of organophosphate, as shown in Figure 3.

Validation. Te method was validated as per International Conference on Harmonization (ICH)
Guidelines [20].
3.6.1. Linearity. Te absorbance of complex was analyzed using UV-Visible spectrophotometer. Te linearity graph is shown as per Figure 4.

Limit of Detection and Quantifcation.
Te limit of detection (LOD) and limit of quantifcation (LOQ) for the procedure were performed and the data were obtained as 0.160 mg and 0.486 mg, respectively.          4 International Journal of Analytical Chemistry

Robustness
(1) Variation of Wavelength. Te robustness of the sample was carried out by the variation of wavelength at 239 and 241 nm (Table 6). Te concentration of the sample selected was 1 mg/ml. Te % RSD should be less than 2.5%, which showed that variation in the wavelength showed the method to be not robust.
(2) Variation of Reagent Volume. Te robustness of the sample was carried out by variation of reagent volume i.e. 1.8 ml and 2.2 ml, as shown in Table 7. Te concentration of    International Journal of Analytical Chemistry    Additional mark: quantifcation ring in the middle of GAPI: procedure for quantifcation. 6 International Journal of Analytical Chemistry the sample selected was 1 mg/ml. Te % RSD should be less than 2.5%, which showed that variation in the reagent volume showed the method to be not robust.

Assessment Using Analytical
Ecoscale. An excellent semiquantitative method applied to assess the greenness profle of the analytical methods is the analytical ecoscale [21,22]. Based on the penalty points, the total score of the method is calculated. Te ideal green analytical method is with an ecoscale score of 100.75 and 50 are the green methods which are named as excellent and fair green analytical methods, respectively. If the penalty point is less than 50, it is called the defcient green method. Te ecoscale score of the proposed green analytical method is 84, as shown in Table 8.

Assessment Using Green Analytical Procedure Index (GAPI).
Te qualitative method meant to measure the greenness is GAPI, which calculate greenness based on the stages involved in an analytical method [23,24]. Te two main stages of GAPI are sample preparation and instrumental assessment. A pictogram of fve pentagrams is a visual output in GAPI and is used to evaluate and quantify the low, medium, and high environmental impact involved for each step of the methodology which can be analyzed by the green, yellow, or red color in each pentagram. Te application of GAPI in the proposed method is given in Table 9, and the pictogram is represented in Figure 5, which shows the method, has satisfed most of the criteria and confrms the proposed method as ecofriendly. (AGREE). AGREE depends on 12 parameters equal to the 12 principles of Green Analytical Chemistry. Each principle or parameter contains a score range 0-1, which is calculated based on the hazardous to a particular principal of greenness. It looks like a classical clock shape consisting of numbers 1-12 on the edge of the circle, representing the philosophy of 12 principles [25]. As shown in Figure 6 with 0.89, the proposed method indicates the method was greenest in all aspects of green principles.

Conclusion
A novel green spectrophotometric method was designed using a magnesia mixture for the analysis of organophosphates in fruits and vegetables. Direct analysis of organophosphates was not possible due to a lack of chromophore which was resolved by a simple derivatization method using magnesia mixture. Validation of the proposed method was carried out as per ICH guidelines, allowing application of the proposed method in the determination of the fruits and vegetables samples. Ecoscale, GAPI, and AGREE assessment methods also afrmed the ecosafety of the developed spectrophotometric method and can be adapted to the established quality of other fruits and vegetables for organophosphate analysis.

Data Availability
Te data used to support the fndings of this study are included within the article.

Conflicts of Interest
Te authors declare that there are no conficts of interest regarding the publication of this article.