A Comprehensive Review of Pesticide Residues in Peppers

Pesticides are chemicals that are used to control pests such as insects, fungi, and weeds. Pesticide residues can remain on crops after application. Peppers are popular and versatile foods that are valued for their flavor, nutrition, and medicinal properties. The consumption of raw or fresh peppers (bell and chili) can have important health benefits due to their high levels of vitamins, minerals, and antioxidants. Therefore, it is crucial to consider factors such as pesticide use and preparation methods to fully realize these benefits. Ensuring that the levels of pesticide residues in peppers are not harmful to human health requires rigorous and continuous monitoring. Several analytical methods, such as gas chromatography (GC), liquid chromatography (LC), mass spectrometry (MS), infrared spectroscopy (IR), ultraviolet–visible spectroscopy (UV–Vis), and nuclear magnetic resonance spectroscopy (NMR), can detect and quantify pesticide residues in peppers. The choice of analytical method depends on the specific pesticide, that is being tested for and the type of sample being analyzed. The sample preparation method usually involves several processes. This includes extraction, which is used to separate the pesticides from the pepper matrix, and cleanup, which removes any interfering substances that could affect the accuracy of the analysis. Regulatory agencies or food safety organizations typically monitor pesticide residues in peppers by stipulating maximum residue limits (MRLs). Herein, we discuss various sample preparation, cleanup, and analytical techniques, as well as the dissipation patterns and application of monitoring strategies for analyzing pesticides in peppers to help safeguard against potential human health risks. From the authors’ perspective, several challenges and limitations exist in the analytical approach to monitoring pesticide residues in peppers. These include the complexity of the matrix, the limited sensitivity of some analytical methods, cost and time, a lack of standard methods, and limited sample size. Furthermore, developing new analytical methods, using machine learning and artificial intelligence, promoting sustainable and organic growing practices, improving sample preparation methods, and increasing standardization could assist efficiently in analyzing pesticide residues in peppers.


Introduction
Peppers are an important part of many diets worldwide due to their nutritional value. Peppers are a rich source of vitamins and minerals, including vitamins C and A and potassium [1]. They are also high in antioxidants and phytochemicals [2], which are associated with many health benefits, including boosting the immune system [3], The intake of raw and cooked vegetables, such as peppers, is one of the most common pesticide exposure routes [17]. The impact of pesticides on the nutritional value of peppers depends on several factors, including the type and amount of pesticide used, the length of time the pesticides remained on the peppers, and their overall nutritional content. In general, pesticides can potentially reduce the nutritional value of peppers by decreasing the levels of certain nutrients, such as vitamins and minerals [18]. Some pesticides may also have toxic effects on beneficial microorganisms in the soil, which could affect the overall nutrient content of peppers.
The current review discusses different extraction and analytical procedures that are used to determine pesticide residues in peppers. Additionally, dissipation patterns and monitoring strategies are also reviewed. A conclusion and potential future perspectives are proposed based on the authors' viewpoints.

Maximum Residue Limits
Maximum residue limits (MRLs) are regulatory limits that are set for the levels of pesticide that are allowed to remain on food crops, including peppers, after the application of plant production products and preharvest intervals. MRLs are set to protect human health by ensuring that pesticides on food crops are below the levels that could potentially be harmful. MRLs for pesticides can vary between countries, as each country may have different regulations and guidelines for pesticide use. Some countries may have specific MRLs for particular pesticides in peppers, while others may have more general MRLs for specific pesticides in all food crops. MRLs are typically set based on the results of toxicological assessments, which evaluate the potential health risks of different pesticides [19]. MRLs are typically set at levels well below those that could potentially harm human health. It is important to note that MRLs are not intended to measure pesticide safety.
Disagreements over permissible levels between nations could impede trade globally, thus highlighting the urgent need for MRL standardization. The European Union (EU) and the Codex Alimentarius Commission of the Joint Food and Agricultural Organization of the United Nations (FAO)/WHO [20] have established reference MRLs. Each country uses one of two strategies to limit pesticide residues in agricultural commodities: (a) the regulatory monitoring of agricultural raw materials, measuring the residual levels of particular matrices following the MRL [21,22]; or (b) whole diet research, which analyzes the foods people eat to estimate their dietary intake of pesticides [23][24][25]. A viable approach is required to identify and measure residues at a level equal to or lower than the MRL (Table 1) and verify the identification of substances in agricultural products for research and regulatory purposes. The fundamental steps in multi-residue methods (MRMs) and single-residue methods (SRMs) are essentially the same. To monitor or screen different kinds of pesticides in specific products, MRMs are typically used. By contrast, SRMs are often used for substances that cannot be determined by MRM methods and require specific procedures for sample preparation and determination [26][27][28][29]. Consequently, a suitable analytical technique should be established to quantitatively identify the levels of pesticides in peppers for safety and dietary risk assessment.

Sample Pretreatment and Extraction Methods
Analytical methods are vital for estimating MRLs, from sample homogeneity to instrument detection limits. In pesticide research, substantial efforts have been undertaken to create and evaluate analytical techniques and procedures. Suppose the experimental sample is too small to accurately represent the initial batch or unit. In this case, applying sophisticated analytical tools and procedures would be expensive, time-consuming, and inefficient and could provide data that are challenging to understand instead of useful findings [48,49]. Consequently, efficient sample preparation is essential for accurately determining pesticide residues in foods with complex matrices [50].
The distribution of pesticide residues in/on crops is diverse. Thus, the sample needs to be completely homogenized. The matrix components are frequently coextracted with specific pesticides after obtaining a suitably homogeneous sample. Notably, more than 2500 natural compounds are found in paprika [51], which may hide the detection of some pesticide residues. In most conventional methods, the samples are extracted with acetonitrile and/or acetone. NaCl was added to the aqueous phase (either as a saturated solution or in solid form) to broaden the polarity range. Afterward, the extract was partitioned with nonpolar solutions (dichloromethane [DCM] or DCM/petroleum ether) to eliminate water and coextracts (e.g., pigments, phenols, and tannins obtained during liquid-liquid partitioning). The utilization of DCM in the liquid-liquid partitioning process was prohibited in 1980 because of the harmful impacts of chlorinated solvents on the environment and human health [52]. Therefore, many attempts have been made to substitute DCM or remove the liquid-liquid partitioning phase. In this context, a cyclohexane/ethyl acetate combination (1:1, v/v) was employed instead of DCM/petroleum ether (1:1, v/v) during the partitioning step [53,54]. Moreover, a solid-phase extraction (SPE) approach was used in place of liquid-liquid partitioning with DCM. For instance, Luke et al. [55] added fructose, MgSO 4 , and NaCl to the original extract to separate the water from the acetone. To phase-separate mixtures of acetone/water and acetonitrile/water, Schenck et al. [54] used Na 2 SO 4 and MgSO 4 as drying agents. The authors discovered that acetonitrile was more successfully and efficiently separated from the water than acetone and that MgSO 4 was more efficient in removing any remaining water from the organic layer.
Compared to other traditional techniques, the QuEChERS (quick, easy, cheap, effective, rugged, safe) method is widely used because of its many advantages, such as the ease of sample preparation, inexpensiveness, less organic solvent use, high recovery, and accuracy [56,57]. Noh et al. [58] described the QuEChERS technique as a streamlined strategy for analytical chemists to define the concentrations of multiclass and multi-residue pesticides in fruits and vegetables. With this method, MgSO 4 was used in a new way for salting-out extraction and partitioning with acetonitrile, cleaning with dispersive solid-phase extraction (d-SPE), and detection with mass spectrometry (MS). The initial version of the QuEChERS approach demonstrated remarkable performance in detecting hundreds of pesticides in various products. Nevertheless, using the initial approach caused the poor recovery of some pH-dependent pesticides, including pymetrozine, thiabendazole, and imazalil [59]. This approach has undergone some modifications, mainly concerning pH variations and the use of a rather powerful acetate-buffered version, which became the official method of the Association of Official Analytical Chemists (AOAC) [43]. Instead, a citrate-buffered version was adopted as the European Standard (EN) procedure by the European Committee for Standardization (CEN) [60]. Due to frequent modifications of the solvents, salts, buffers, and sorbents used in the QuEChERS analytical approach, the QuEChERS approach is viewed as a sample preparation idea instead of a specific procedure [61]. Modifications are required to avoid pesticide degradation, achieve a reasonable recovery within an acceptable range, and lessen the matrix influence in complex matrices [62].

Challenges in Sample Preparation
Several challenges can arise during the sample preparation process to determine pesticide residues in peppers. Some of these challenges include the following:

•
Sample size: Peppers can vary significantly in size, and can be challenging to accurately sample the fruit in a way that represents the overall population. • Contamination: It is crucial to avoid the contamination of the sample during the preparation process, as this can affect the accuracy of the results.

•
Pesticide distribution: Pesticides may not be uniformly distributed on the surface of the peppers, making it difficult to sample the fruit accurately.
• Extraction efficiency: The efficiency of the extraction process can impact the accuracy of the results, as some pesticides may be more difficult to extract than others. • Matrix effects: The presence of other compounds in the peppers (such as proteins, carbohydrates, and lipids) can interfere with the analysis process and impact the accuracy of the results.
To address these challenges, it is vital to use appropriate sampling techniques and sample preparation methods and to carefully control the conditions of the analysis process to ensure the accuracy and reliability of results.

Cleanup Procedures
Before instrumentation, samples are often purified with sorbents, such as MgSO 4 combined with primary secondary amine (PSA), octadecylsilyl-derivatized silica (C18), and graphitized carbon black (GCB) [60,63]. The limited recovery of C18 in the analysis of nonpolar molecules and the great affinity of the GCB Table for planar analytes are two drawbacks of these often-employed sorbents. Therefore, additional efforts are needed to create novel sorbents or to optimize sorbent combinations to improve the purification effectiveness of matrices.
SPE was created to replace traditional partitioning and reduce the dangerous chlorinated solvents used in the partitioning stage [64]. The technique still needs a sizable glass column with sizable amounts of solvent for washing and elution, even though SPE was used in place of partitioning. Consequently, steps were taken to limit the consumption of solvents. The initial strategy used short florisil columns [64]. Instead of the large classical cartridges, C18 and Florisil column cartridges were also assessed in the cleanup of organo-halogen pesticides in crop matrices, and both cartridges showed acceptable recovery rates [65]. Therefore, SPE cartridges with normal or reversed-phase supports are now commercially available and provide a simple means for sample cleanup without requiring large amounts of solvent. Another extraction and cleanup method, known as matrix solid-phase dispersion (MSPD), was created to overcome the general limitations of liquid-liquid partitioning and SPE columns, including the requirement for many solvents and the emulsification of some fruits and vegetables, which blocks the flow of the analytes [66,67]. The MSPD strategy entails mixing a tiny quantity of the matrix with C18, washing it with a small amount of solvent, and eluting it to extract various chemicals (Barker, 2000a). Nonetheless, because of the minute sample size (0.5 g) used in this method, MSPD did not offer an analytical scope or a process that was adequately broad or straightforward. Anastassiades et al. [53] introduced d-SPE QuEChERS following a similar MSPD strategy [66,68,69]. The sorbent was then combined with an aliquot of the extract instead of the original sample, as in MSPD.

Instrumentation
Gas chromatography (GC), high-performance liquid chromatography (HPLC), and chromatography−mass spectrometry (GC-MS) are among the main methods that are used for pesticide residue detection and metabolite detection [11,28]. These conventional detection methods have good sensitivity, accuracy, precision, and reliability. However, they have some disadvantages, such as cumbersome sample pretreatment steps, the high cost of instruments and equipment requiring professional and technical personnel to operate them, and long detection processes. In this context, Rahman et al. [28] analyzed alachlor residues in the pepper and pepper leaves by GC and verified them through MS with pepper leaf matrix protection. They found that alachlor residues were present in both pepper and pepper leaf samples, with levels exceeding the MRL set by the Malaysian Food Regulation. The authors concluded that the consumption of peppers containing alachlor residues could pose a potential health risk to consumers. The study also highlighted the importance of the regular monitoring of pesticide residues in vegetables and fruits, as well as the need for stricter regulations on the use of pesticides in agriculture.
The main challenges in creating efficient methods for pesticide residue analysis include the low detection thresholds demanded by regulatory agencies, the variance in the polarity, volatility, and solubility of pesticides, and matrix coextraction [70]. Therefore, mass spectrometers, as a universal and more specific type of detector, began to be paired with chromatographic systems to overcome these problems [71]. In addition to improvements in the detection system, improvements in conventional sample preparation have been achieved regarding lowering the use of hazardous organic solvents, time, cost, and labor [72,73]. The QuEChERS sample preparation approach has met global acceptance and has been modified and adapted for various purposes due to its simplicity and flexibility [63,74]. However, this technique was created for gas chromatography−mass selective detection (GC-MSD) or liquid chromatography with tandem mass spectrometry (LC-MS/MS), which demand equipment that is uncommon in laboratories with only the most basic equipment [59,74].
In the quickly expanding food sector, automation in the analytical field is becoming increasingly important. Globally, strict rules and residue monitoring procedures are being created in response to consumer concerns about food safety. Due to the increased sample loads, high-throughput analytical techniques with sufficient precision and accuracy are needed.

Monitoring
The complexity of sample treatment largely depends on the matrix interferences and separation techniques, with GC and HPLC being the most common methods. Peppers typically have higher pesticide residue concentrations than other products because these compounds are constantly applied throughout the growing season. The research conducted in 2017 evaluated the levels of organochlorine pesticides in Nigerian noodles. The findings revealed that the chili peppers used in the noodles contained elevated levels of pesticide residues [41]. Several technologies have contributed to advancing the detection and monitoring of trace pesticide levels, including the GC-electron capture detector (ECD) [24,39,41,46] nitrogen phosphorus detector (NPD) [24,44], flame photometric detector (FPD) [38,43], GC−MS [30,[35][36][37], and gas chromatography−tandem mass spectrometry (GC−MS/MS) due to the high selectivity, separation power, and identification capacity of MS ( Table 2). The latest progress in MRMs associated with GC-MS/MS included the development of an analytical procedure that replaces traditional GC detectors. Nevertheless, due to the inadequate sensitivity for a few compounds, traditional GC detectors are still in use for SRMs [75].

Effect of Household Processing on Pesticide Residue Levels in Peppers
Several household processes can be used to reduce pesticides in fresh peppers, including washing and blanching. These processes can effectively remove or reduce the levels of pesticides on the surface of peppers, but they may not completely eliminate all residues. For instance, washing peppers thoroughly using running water can effectively remove surface contaminants, including pesticides, but it will not remove all pesticides, particularly those that have been absorbed into the pepper tissue [13]. Blanching is a process in which peppers are briefly boiled in water or steam and then cooled in ice water. This process can help loosen the pepper's skin, making it easier to remove. It can also help to reduce the levels of pesticides on the surface of the pepper, as some pesticides may be removed during the boiling process [80]. Again, blanching may not be able to remove all pesticides, particularly those that have been absorbed into the pepper tissue. In this context, Kim et al. [80] evaluated the effects of various household processes, such as washing, blanching, frying, and drying, under different conditions (water volume, blanching time, and temperature) on residual pesticide concentrations. Both washing and blanching (in combination with high water volume and processing time) significantly reduced pesticide residue levels in the leaves and fruit of hot pepper compared with other processes [80]. It is worth considering other conditions/factors, such as selecting peppers that are grown using sustainable and organic practices to further reduce the levels of pesticides in peppers.

Dissipation Patterns and Preharvest Intervals in Peppers
Pesticides that are applied to peppers can be absorbed by the plant while also being present on the surface of the pepper fruit. The rate at which pesticides dissipate or break down can vary depending on several factors, including the type of pesticide, the application rate, the weather, and the application method. Generally, most pesticides will dissipate more quickly in warm, humid conditions and more slowly in cool, dry conditions. Pesticides applied to the surface of the pepper fruit may dissipate more quickly than those absorbed by the plant, as they are more exposed to the environment.
The dissipation behavior of pesticide residues in peppers has been investigated [81][82][83][84][85][86][87]. For instance, Liu et al. [85] reported that the t 1/2 values of metalaxyl in peppers were 3.2-3.9 days at three experimental locations in China. At harvest, pepper samples were found to contain metalaxyl and cymoxanil levels that were well below the MRLs of the EU following the recommended dosage and an interval of 21 days after the last application.
The environmental fate of field-applied synthetic pesticides has been under investigation for several years. Endosulfan 3 EC, a mixture of αand β-stereoisomers, was sprayed on field-grown pepper at the recommended rate of 0.44 kg of active ingredients per acre. Endosulfan sulfate is the major metabolite of endosulfan sulfite, and the β-isomers are relatively more persistent than the α-isomers. In pepper, the α-isomer, which is more toxic to mammals, dissipated faster (t 1/2 = 1.22 day) than the less toxic β-isomer (t 1/2 = 3.0 day). These results confirm the greater loss of the α-isomer than the β-isomer, which can ultimately impact endosulfan dissipation in the environment [82].
The degradation behavior of flonicamid and its metabolites, 4-(trifluoromethyl)nicotinic acid (TFNA) and N-(4-trifluoromethylnicotinoyl) glycine (TFNG), was evaluated in red bell peppers over 90 days under greenhouse conditions, including high temperature, low and high humidity, and in a vinyl house covered with a high-density polyethylene light shade covering film (35% and 75%). For safety reasons, the authors concluded that red bell peppers should be grown under greenhouse conditions because solar radiation increases the rate of flonicamid degradation into its metabolites [88].
It is also possible to reduce the need for pesticides by using integrated pest management techniques, such as introducing natural predators of pests or using physical barriers to prevent pests from accessing plants.
PHIs are the minimum amount of time that must pass between the application of a pesticide and the harvest of a crop. The purpose of PHIs is to allow pesticides to break down or dissipate in the environment and on the surface of the crop to levels that are considered safe for consumption. PHIs vary depending on the specific pesticide used, the type of crop, and the application method. It is important to follow the label instructions for a particular pesticide, as these will include the recommended PHI for the crop in question to ensure that the peppers are safe to consume. It is also worth noting that some pesticides may not be approved for peppers, which means there would be no recommended PHI. It is important to use pesticides only as directed and to follow all label instructions to help ensure the safety of the crop and to protect human health.

Dietary Risk Assessment
The dietary risk assessment of pesticide residues in peppers is an important task that helps determine the potential health effects of consuming peppers treated with pesticides. This assessment typically involves several steps, including: 1.
Identify the pesticides that are commonly used on peppers, as well as their maximum residue levels (MRLs).

2.
Collect data on the levels of pesticide residues found in peppers sold on the market.

3.
Evaluate the potential health risks posed by the consumption of peppers with pesticide residues based on the levels found and the MRLs.
Once the data are collected, they can be used to estimate the average daily intake of each pesticide for different population groups. This can be performed by using data on pepper consumption patterns and the levels of pesticide residues found in peppers. Next, the potential health risks posed by the consumption of peppers with pesticide residues can be evaluated by comparing the estimated daily intake of each pesticide with the appropriate reference doses (RfDs), such as acceptable daily intakes (ADIs) or acute reference doses (ARfDs) [30,89]. These values are established by regulatory agencies, such as the US Environmental Protection Agency (EPA), as a safe level of exposure for the general population. It is worth mentioning that to have a comprehensive view of the impact of pesticide residues in peppers on human health, it is crucial to look not only at the impact of a single pesticide but also at the combined effect of different pesticides that may be present in the pepper [90][91][92]. While the impact of individual pesticides on human health has been extensively studied, the combined effect of multiple pesticides is less understood. However, there is growing evidence to suggest that exposure to multiple pesticides can have additive or synergistic effects on human health and that the cumulative effect of these residues may be greater than the effect of individual pesticides alone. Therefore, it is important to consider the potential combined impact of multiple pesticide residues when evaluating the health risks associated with consuming peppers or other fruits and vegetables. In addition, the levels of the detected pesticide in peppers can be tolerated and do not pose a serious health problem to the community [30,31]. However, it is worth noting that some people may be more sensitive to pesticides than others, such as pregnant women and children [93]. Additionally, long-term exposure to low levels of pesticides may also pose health risks [94]. It is also important to note that the risk assessment process may vary by country, as different countries have different regulations for pesticides, different exposure scenarios, and different methods for assessing risks. It is worth mentioning that regulatory agencies continuously monitor the situation and update their guidelines and regulations as necessary.

The Use of Pepper Leaf Matrix as an Analyte Protectant
The pepper leaf matrix is a complex mixture of compounds that are found in the leaves of pepper plants. It is composed of various organic compounds, such as proteins, carbohydrates, and lipids, as well as inorganic compounds, such as minerals. The specific composition of the pepper leaf matrix depends on the pepper plant variety and the growing conditions. It is possible that the pepper leaf matrix could be used as an analyte protectant during GC analysis [26,28]. Analyte protectants are substances used to stabilize or protect specific molecules or compounds during the analysis process. This can help prevent the degradation or loss of the analyte [26,28], ensuring that accurate and reliable results are obtained.

Challenges and Limitations in Managing Pesticide Residues in Peppers: Author's Perspectives
There are several challenges and limitations when measuring and managing pesticide residues in peppers. Some of the main challenges and limitations include the following:

•
Detection limits: Many pesticides break down or degrade over time, making it difficult to accurately measure their residues in peppers. This can be incredibly challenging when trying to detect low levels of pesticides, as the limits of detection for many analytical methods may be higher than the levels of residues present in the peppers. Pesticide resistance: Some pests and diseases that affect peppers can develop resistance to certain pesticides over time. This can make it more challenging to control these pests and diseases and can lead to the need for more frequent or higher applications of pesticides.
Overall, managing pesticide residues in peppers can be a complex and challenging task. It is important to follow proper pesticide application and management practices to minimize the levels of residues in peppers and to ensure that they meet regulatory limits.

Conclusions and Future Perspectives
Depending on their use, pesticides might have positive and negative effects on peppers. It is important to use pesticides responsibly and to follow all label instructions to minimize any potential negative effects on peppers and other non-target organisms. The analytical approach to monitoring pesticide residues in peppers and their monitoring frequency is important in ensuring the safety of the food supply. Various analytical methods and sample preparation techniques are available, and regulatory agencies and food safety organizations play a crucial role in monitoring pesticide residues in peppers to ensure that they are safe for human consumption. Notably, managing pesticide residues in peppers can be a complex and challenging task. Therefore, it is essential to follow proper pesticide application and management practices to minimize the levels of residues and ensure that they meet regulatory limits. A trend toward using safer and more sustainable pest control methods in pepper production, appropriate sample cleanup methods, techniques designed to remove matrix interferences (such as pigments, lipids, and carbohydrates) and purify the target analytes effectively, and the use of accurate and reliable analytical methods should be considered. It is also important to wash and peel peppers thoroughly before consuming them to reduce the risk of exposure to pesticide residues. Overall, the outcomes of pesticide residue analysis in peppers depend on the specific method used, the type and concentration of pesticides detected, and the regulatory standards that apply.
There are several potential future developments in pesticide residue analysis in peppers that may emerge in the coming years. For instance, more sensitive and accurate methods could be developed to detect the trace levels of new pesticides. As consumer demand for organic produce grows, there may be an increased focus on alternative pest control methods that do not involve synthetic pesticides. This could lead to a decrease in the levels of pesticide residues that are found in peppers. Automating analytical techniques could also become more widespread in the future, improving the efficiency and accuracy of pesticide residue analysis in peppers. MRLs for pesticides in food, including peppers, are periodically reviewed and updated. There is an ongoing debate about what levels of pesticide residues are safe for human consumption and how MRLs should be established. Risk assessment methods are also being developed to help determine the potential health risks associated with different levels of pesticide residues in peppers.