Pesticide Determination in Fresh Coconut Water (Cocos nucifera Linn.) by GC-MS Using Microwave-Assisted Liquid-Liquid Extraction

Fresh coconut (Cocos nucifera) water, a popular beverage in tropical countries, is often exposed to pesticide contamination due to agricultural practices. Thus, this work proposed the simultaneous determination of 36 pesticides in fresh coconut water samples using liquid-liquid extraction, drying assisted by a household microwave oven, and gas chromatography-mass spectrometry (GC-MS) analysis. Limits of detection varied from 5.4 (disulfoton) to 9.6 ng L-1 (parathion-methyl). Nine pesticide residues were detected (dimethoate, γ-HCH, β-HCH, 4,4’-dichlorodiphenyldichloroethylene, endosulfan II, 4,4’-dichlorodiphenyldichloroethane, 4,4’-dichlorodiphenyltrichloroethane, endrin ketone, and methoxychlor) with concentrations from < limit of quantification to 5,768.34 ± 1.64 ng L-1. Dimethoate was quantified in all samples, indicating its recent use. The proposed methodology offers several advantages, including the use of small sample volumes and solvent extraction, which eliminates the need for costly cartridges and reduces waste production. Additionally, the obtained results can be valuable for regulatory agencies, aiding in the mitigation of environmental damage and the protection of human health.


Introduction
The coconut (Cocos nucifera Linn.) is an economically recognized plant in the food and cosmetic industry due to its use in a wide variety of by-products including pulp, water, oil, and coconut milk. 1 Coconut water, taken directly from the inner part of the fruit, is widely consumed in tropical countries because it presents attractive sensorial characteristics such as sweet taste and refreshing sensation. 2,3Also, it is rich in various functional bioactive compounds, including vitamins B and C, minerals, and enzymes, related to anti-inflammatory and antioxidant activities. 4oconut crop is an important agricultural activity for the Brazilian economy, amounting to approximately R$ 1.3 billion in 2021.The country produced 1.64 million metric tons, being the Northeast region responsible for more than 70% of the national production. 5In this scenario, Bahia State stands out with 552.5 thousand tons (i.e., 30.3%). 1 Frequently, pests attack coconut palms and reduce its productivity and lifespan.Then, pesticides are sprayed directly on foliage or injected into the stem and root systems to prevent or revert the damages, and consequently minimize losses in the coconut production. 6The United States Environmental Protection Agency (US-EPA) defines pesticide as any substance or mixture of substances intended for preventing, destroying, repelling, or mitigating pests. 7Therefore, coconut water may be contaminated by residues of pesticides used during the planting, handling, and/or harvesting processes. 8ome pesticides are bioaccumulative and can also biomagnify, resulting in their residues persisting in crops and the environment.This not only impacts the quality of water, soil, and atmosphere but also poses negative health risks to humans and animals. 9Exposure to pesticide residues may cause increased susceptibility to endocrine disrupting effects, 10 teratogenic fetal abnormalities, 11 occurrences of neurological disorders, 12 psychiatric disorders, 13 and neoplasms. 14he Food and Agriculture Organization of the United Nations (FAO) reports the worldwide use of pesticides in agriculture at 2.7 million tonnes (Mt) of active ingredients, while the total trade of formulated products attained approximately 7.2 Mt in 2020, reaching USD 41.1 billion. 15n 2017, Bahia State used 26.3 thousand tons of active ingredients of pesticides, representing 4.87% of the Brazilian consumption, i.e., the eighth largest consumer. 16espite this, Brazilian and international laws do not establish maximum levels for these chemical species in this beverage, although studies confirmed the presence of these contaminants in coconuts produced and marketed in Brazil. 88][19][20][21][22][23] To the best of our knowledge, no works involving the use of a household microwave (which is a cheap and accessible device) for the drying step of thirty-six pesticides in coconut water has been developed.Therefore, this work proposes the simultaneous GC-MS determination of thirty-six pesticides (from the organochlorine, organophosphate, pyrethroid, carbamate, thiocarbamate, and strobilurin classes) in commercial fresh coconut water samples using a simple, fast, and efficient liquid-liquid extraction (LLE) methodology that employs small volumes of samples and solvents, and a drying step assisted by a household microwave oven.
The stock standard solution of the thirty-six analytes were also prepared in hexane:toluene (1:1) at a concentration of 1,000 μg L -1 .Then, working solutions were prepared by appropriated dilution of the stock standard solution using the same solvent.
The "green dwarf coconut" variety was selected for validation and method applicability.A total of thirty coconuts were acquired from two farms that supply the product to local markets in Barreiras city, Bahia State, Brazil.All samples were within the 6 to 8 months of maturity.Fresh coconut water samples were then stored in amber glass bottles (100 mL) previously cleaned, and finally frozen at -20 °C until analysis.

Sample preparation
Initially, the efficiency of the household microwave oven for the drying step was evaluated.For this, standard solution was dried in different ways: (i) 15 min of heating; (ii) 5 min of heating and 5 min of cooling; and (iii) 3 min heating and 3 min of cooling.After total drying, they were resuspended and injected into the GC-MS.The drying step using a household microwave oven was developed following a previous work. 22A household microwave oven (Consul, model CMS25ABHNA, 2450 MHz, 1.2 KW) was used.
Based on the best results obtained for the drying step, the experimental conditions for the extraction method were optimized by varying the solvents (hexane and hexane:toluene), the proportion of the hexane:toluene mixtures (1:1 and 7:3), stirring time (20 and 30 min), and stirring speed (200, 600 and 800 rpm).Six tests were then carried out: (i) toluene, 20 min of stirring, and speed of 200 rpm; (ii) toluene, 30 min of stirring, and speed of 200 rpm; (iii) toluene, 30 min of stirring, and speed of 600 rpm; (iv) toluene, 30 min of stirring, and speed of 800 rpm; (v) hexane:toluene (1:1), stirring for 30 min, speed 800 rpm; (vi) hexane:toluene (7:3), 30 min of stirring, and speed of 800 rpm.Magnetic stirring was performed using a Fisatom stirrer (model 753A, São Paulo, Brazil).All tests were performed with spiked samples of fresh coconut water at 1,000 ng L -1 .After total drying, they were resuspended and analyzed by GC-MS, followed by the analysis of the standard solution at equivalent concentration.
After optimization, thirty-six pesticides were simultaneously extracted using 10 mL of fresh coconut water and 500 μL of hexane:toluene (7:3) solution into a glass vial.The mixture was then subjected to magnetic stirring for 30 min (800 rpm).The supernatant (400 μL) was collected in an amber vial and dried in a household microwave oven at the minimum power (70 W), with intervals of 3 min between the heating and cooling steps.The cooling step was performed by removing the amber vial from the household microwave and left at room temperature.After complete drying, the samples were resuspended in 200 μL of hexane:toluene (7:3) solution and then subjected to GC-MS analysis.
For the pesticide analysis, the GC oven was initially heated to 60 °C (t = 0 min).After 1 min at this temperature, the oven was heated to 200 °C at a rate of 25 °C min -1 .Then, it was heated to 230 °C at 5 °C min -1 , and finally to 300 °C, maintaining for 1.5 min.The injector was set at 300 °C in the splitless mode and the injection volume was 1.0 μL.The temperatures of the ion source and interface were set at 300 °C.The mass spectrometer was operated in the electron ionization mode at ionization energy of 1.1 kV.
The analytical method was developed in the scan mode, using a standard solution at 100 μg L -1 .Selected ion monitoring (SIM) was used for the quantification of pesticides.The most intense and specific ions were chosen for each analyte (one quantification ion and up to three confirmation ions).The ion ratios from sample extracts were within ± 30% (relative) of the average of the calibration standards. 24The quantification and confirmation ions were confirmed using the National Institute of Standards and Technology (NIST) database, 25 as indicated in Table 1.All analyzes were carried out in triplicate.

Method validation
The parameters used for the method validation followed the recommendations of the Codex Alimentarius, 24 guideline CXG 90-2017: selectivity, calibration, linearity, limit of detection (LOD), limit of quantification (LOQ), intra-and inter-day precision, matrix effect, accuracy (recovery test), and application of the method to real samples of fresh coconut water.
The calibration was performed by constructing analytical curves in the form y = ax + b, where y is the area of the peaks, a is the angular coefficient, x is the concentration value, and b is the linear coefficient of regression line.Coefficient of determination (R 2 ) greater than 0.99 was considered as linearity criterion in the regression of the calibration curves constructed using seven different concentration levels in the range from 10 to 400 ng L -1 .The LOD and LOQ were calculated as (3×S b )/a and (10×S b )/a, respectively, where S b is the standard deviation of the linear coefficient and a is the angular coefficient of the calibration curve.The matrix effect was investigated by comparing the slope of the analytical curve obtained from the analyte standards with the slopes of the analytical curves obtained from the spiked samples.Precision and accuracy were estimated by analyzing fresh coconut water samples spiked with the standard work solution containing all the pesticides, to achieve final concentrations of 32, 200, and 350 ng L -1 , in triplicate.Precision was assessed by the coefficient of variation (CV) of nine injections performed at three different times (morning, afternoon, and night) on the same day (intra-day precision) and at the same time by seven consecutive days (inter-day precision).Accuracy was assessed by conducting analyte recovery tests, which involve adding a known quantity of the analyte (32, 200, and 350 ng L -1 ) to a sample and subsequently determining the percentage of the added amount that was detected.After validation, the analytical methodology was applied to determine the concentration of thirty-six pesticides in fresh coconut water samples.

Validation method and optimization of extraction conditions
The selectivity of the method was evaluated by comparing the chromatograms resulting from the analysis of the standard solution containing the pesticides at 50 μg L -1 (Figure 1a), extraction of a spiked sample (Figure 1b), and extraction of a non-spiked sample (Figure 1c).These chromatograms were obtained in full scan mode, so at the beginning, it is possible to observe the elution of several compounds.The scan mode allowed for a comprehensive visualization of the compounds present in the matrix.However, confirmation that only the analytes eluted was achieved through validation tests (matrix effect, accuracy) in the SIM mode.Since a blank sample of fresh coconut water was not available, the selectivity test was performed on a real sample that contained pesticides.The selectivity was confirmed through the recovery test, where the area values of the pesticides identified in Figure 1c (non-spiked sample) were compared to the differences observed in Figure 1b (spiked sample) and Figure 1a (analytical standard).
Table 2 shows the analytical validation parameters for the simultaneous determination of thirty-six pesticides in fresh coconut water samples.As observed, the values of R 2 were from 0.9990 to 0.9997 for all pesticides.For the analysis of the commercial fresh coconut water samples, dilutions of the extracts were performed for all concentrations above the working linear range.LOD values varied from 5.4 ng L -1 for disulfoton to 9.6 ng L -1 for parathion-methyl, while LOQ values varied from 17.8 to 31.8 ng L -1 for the same analytes, respectively.For all the analytes, the intra-day precision presented CV values lower than 10%, being the highest one of 7.86% for dimethoate at 350 ng L -1 .The inter-day precision, in turn, had the highest CV value of 21.12% for methyl parathion at 32 ng L -1 .Both intra-and inter-day precision values were in accordance with the Official Methods of Analysis of AOAC International, which precision varies according to the analyte concentration.At concentrations below or equal to 1,000 ng L -1 , the CV should be within 30%. 26he accuracy was assessed through a recovery study, which considered the efficiency of the extraction and drying steps.When comparing the results, extraction with toluene for 30 min at 800 rpm, with 3 min of heating followed by  3 min of cooling, showed better result.This approach was chosen because the amber glass vial containing the standard solution did not reach a high enough temperature to degrade or completely evaporate the analytes.In other words, there was no significant difference (at a significance level of 0.05) between the peak areas of the standard solution after microwave-assisted drying and a standard solution at the same concentration.Figure 2 shows the recovery results obtained for the optimization of the extraction method using the six different experimental conditions.Comparing the results of tests (i) to (iv), where the samples were extracted using only toluene, it is possible to conclude that the longer the extraction time and the higher the agitation speed, the better the recovery values for the studied analytes.Moreover, it was verified that the analytes with lower polarity presented low recovery values.Thus, solvent mixtures with different dipole moment (μ) (hexane (μ = 0.00) and toluene (μ = 0.360)) were studied to determine the best proportion that allows an efficient recovery for all thirty-six pesticides.
After optimization, the best condition for simultaneous extraction of the studied analytes was add 500 μL of hexane:toluene (7:3) to 10 mL of sample, under magnetic stirring at 800 rpm for 30 min and drying in household microwave oven with intervals of 3 min between heating and cooling steps.Under this condition, recoveries of the pesticides (Table 2) ranged from 72.9% (fenthion) to 110% (parathion-methyl) at 32 ng L -1 , from 75.5% (endosulfan II) to 117.9% (endrin ketone) at 200 ng L -1 , and from 84.1% (endosulfan II) to 113.5% (endrin ketone) at 350 ng L -1 .All these values are in accordance to the Codex Alimentarius guideline, 24 which establishes recovery values in the range 70-120% with CV ≤ 20% in complex matrices.
For the matrix effect study, the parallelism test showed p-values ranging from 0.09 to 0.95 (p > 0.05).In this sense, the null hypothesis that the slope coefficients are equal is valid for the thirty-six analytes at a significance level of 0.05, i.e., there are no interferences in the analytical instrumental response related to the various components of the matrix.
Regarding the literature, the results obtained in this work for LOQ were significantly lower than those reported in previous studies employing different extraction and detection methods (Table 3).For the accuracy, despite some studies 23 presented better recovery results, they made use of expensive and laborious extraction techniques such as solid-phase microextraction (SPME) and matrix solidphase dispersion (MSPD).Therefore, the present work stands out because it used small solvent volumes, besides replaced gas purge with drying in a household microwave oven, leading to a noteworthy analytical method able to determine simultaneously thirty-six pesticides of different chemical classes at very small concentrations in a complex matrix such as coconut water.
Application in real samples of fresh coconut water After validation and optimization of the extraction method, the thirty-six pesticides studied were determined in thirty samples of fresh coconut water.Although most of the pesticides have been or are still used in the crops of the studied region, twenty-seven from the thirty-six pesticides were not identified in any of the samples of fresh coconut water analyzed.The concentration of the nine pesticides found in commercial fresh coconut water samples are shown in Table 4.
In fact, only γ -HCH and dimethoate were found in all samples, although only dimethoate (68.90 ± 0.92-5,768.34± 1.64 ng L -1 ) was quantified.Analytes such as 4,4'-DDE, detected in sixteen samples, and endosulfan II, detected in twenty-two samples, presented concentrations below their respective LOQ values, while β-HCH, 4,4'-DDD, 4,4'-DDT, endrin ketone, and methoxychlor had lower detection frequencies.It is worth noting that the use of dimethoate in coconut crop is a common practice in Brazil, which is described by the Brazilian Agricultural Research Corporation (EMBRAPA) as an agent used to mitigate pest action in this crop. 27t must also be highlighted that dimethoate is resistant to photodegradation and has a half-life lower than two months. 28,29The results obtained in this work may be associated to recent contamination by dimethoate in the cultivation areas, either by direct application to the coconut crop or indirectly through the mass transport from cultivated areas of other crops.This prohibition is also applied to aldrin, dieldrin, endrin, and heptachlor.The presence of γ-HCH in analyzed samples indicates a recent exposure, since its halflife is just a few days. 32Although the absence of insecticidal activity of its alpha, beta, gamma, delta, and epsilon isomers, they have half-lives of up to nine years and also induce harmful effects on human health. 33These compounds can act as endocrine disruptors, causing changes in thyroid hormone levels during pregnancy, 34 increasing the susceptibility to Parkinson's disease, 35 and present carcinogenic activity. 36n Brazil, the United States, and the European Union, there is no specific law for the presence of pesticide residues in fresh coconut water.Thus, the results obtained in this study were compared with the established standards for drinking water.We assumed that the fresh coconut water is mainly ingested from the fruit directly, without any industrial processing.Among the analytes found in the studied samples, the Brazilian regulation establishes the maximum levels only for 4,4'-DDE, 4,4'-DDD, and 4,4'-DDT pesticides, whose the sum of their concentrations must be lower than 1,000 ng L -1 . 37n the United States, there is a maximum level only for the γ and β-HCH at 200 ng L -1 . 38In European Union, it is established a maximum concentration at 100 ng L -1 for pesticides regardless of the type, and 500 ng L -1 for the total pesticide sum. 39Therefore, the results obtained in this work were below the limits established by Brazilian and US regulations, but higher than those ones adopted by the European regulation.In addition, the levels of dimethoate alone were higher than the limit allowed for the sum of all pesticides, considering the European regulation.
It is important to highlight that the results obtained in this work contribute to further knowledge about the distribution of the studied pesticides in environmental matrices in the Western region of Bahia, Brazil, such as surface and bottom sediments. 40,41

Conclusions
This study demonstrated the development and validation of an efficient analytical method for the simultaneous determination of thirty-six pesticides belonging to the chemical classes of organochlorines, organophosphates, pyrethroids, carbamates, thiocarbamate, and strobilurin in fresh coconut water samples using GC-MS.The main advantage of the proposed methodology is the use of a small solvent volume through a liquid-liquid extraction method, and drying of the sample extracts assisted by a household microwave oven.This reduces waste production, besides avoiding the use of expensive extraction cartridges.The application of the method in real samples of fresh coconut water collected in two locations revealed high concentrations of dimethoate, which may be related to the contamination of the irrigation water from the two rivers in the region.Consequently, the proposed methodology has the potential to provide data to pesticide regulatory agencies, and also contribute for mitigating actions to environmental damages and prevention to human health.