**7. Methods to detect pesticides**

Pyrethrins and Pyrethroids act on sodium channels in the nervous system of numerous phyla, such as arthropods and chordates [87]. Pyrethrins and Pyrethroids present low acute toxicity to mammals and birds and constitute one of the safest insecticides to man. However, at low concentrations these chemicals are acutely toxic to a wide range of aquatic organisms and

Pyrethrins are natural compounds extracted from chrysanthemum flowers; pyrethroids are synthetic compounds whose structure resembles the structure of pyrethrins [87]. Light degrades these chemicals. Modification of pyrethroids over the years has enhanced their insecticidal activity and persistence in the environment [109]. Compared with pyrethrins, pyrethroids are more stable under light [108], which incurs increased environmental risks associated with their use [5]. Pyrethrins and Pyrethroids display high selectivity and easy degradability in the environment as compared with other pesticides, been a favored replace‐

Pyrethroids strongly adsorb to soil particles, but they can move in runoff with soil particles and reach sediments, consequently entering aquatic ecosystems and affecting aquatic organ‐ isms like invertebrates and fish [108]. Fish are highly sensitive to pyrethrin and pyrethroid products, and contamination of lakes, streams, ponds, or any aquatic habitat is a concern [109]. Moreover, some formulations contain additional insecticides, insect repellents, and solvents

Triazines basically consist of herbicide compounds, are relatively persistent and migrate easily through the soil into surface and ground waters [111]. In soil, they undergo degradation mainly in a microbial action, but the role of photodegradation is still significant [112]. Residues of triazines have emerged in soil, surface waters, and groundwater in areas where the application

Herbicides are often benign with regard to impacts on animals; however, these compounds can have toxic effects at concentrations found in the environment [5]. Furthermore, indiscriminate use of this herbicide, careless handling, accidental spillage, or discharge of untreated effluents into natural water ways can harm the fish population and other aquatic organisms and may contribute to long-term effects in the environment. Atrazine, a triazine herbicide, is one of the most often detected pesticides in streams, rivers,

Phenoxy derivatives basically consist of compounds with herbicide action. They are soluble in water and can pollute surface and ground waters. Phenoxy derivatives display moderate toxicity, but some chlorinated metabolites can be toxic to human and aquatic organisms [114]. In addition, the metabolites may have mutagenic and carcinogenic properties. 2,4-D and MCPA, which are also phenoxy herbicides, can undergo degradation by biotic and abiotic mechanisms. However, these processes may not suffice to reduce the concentrations of

Regarding dipyridyl derivatives, the best-known compounds are diquat and paraquat, developed as herbicides and desiccants. Diquat is water soluble and persistent in the aquatic system. However, it can bind to soil, which reduces its mobility in the environment. Although

such as alcohol and petroleum, which increase pesticide toxicity [109].

insects [108].

218 Toxicology Studies - Cells, Drugs and Environment

ment for organophosphorus compounds [110].

of agrochemicals has taken place [111].

ponds, reservoirs, and groundwater [113].

chlorinated phenoxy derivatives on many sites [115].

A wide range of methodologies exist to identify possible exposure to pesticides. When identification is necessary due to poisoning of a patient attended in the clinic, the general procedures include anamnesis, physical examination, evaluation of clinical signs, and diag‐ nostic and toxicological analysis. If the investigation aims to qualify and/or quantify a possible pesticide, it is generally necessary to collect a sample and analyze it for the presence of pesticides and/or metabolites in biological samples (blood, liver, stomach contents) and/or the environment (air, water, ground). Selection of the test will depend on the purpose of the analysis. It is also essential to consider the financial costs of a method. Simpler tests are still important, – apart from been inexpensive, many offer high sensitivity, specificity, precision, and accuracy, all of which are factors that are crucial for reliable analysis [123, 124].

Prior to analyzes pesticides samples analysts have to go through similar steps: definitions of the analytical problem (target analyte and its properties), choice of detection methods (immu‐ noassays spectrometry), sampling (how to collect and store the sample), sample preparation (solubilization, extraction, concentration, and separation), calibration (qualification and/or quantification of the analyte), calculation and evaluation of the results, and actions to complete the analysis [125].

Sample storage for long periods should ensure that no sample degradation or external contamination occurs. Well-sealed containers stored under refrigeration and protected from light are mandatory. To avoid any type of external interference during analysis, none of the employed materials should modify or degrade the pesticide in the sample. The analysis of pesticides, mainly in water, ambient air, and soil sediments, often requires a purification step to clean the sample and pre-concentrate the analytes, to improve the quality of the analytical results. The extraction process is a key analytical step – it extracts the desirable compounds for further separation and characterization. Liquid-liquid extraction, and pre-concentration procedures, such as solid-phase extraction and solid-phase microextraction, are the most commonly used methods, but other extraction methods are also applicable depending on the objective [126]. Extraction of residues from the sample matrix demands appropriate solvents for maximum extraction efficiency and minimal co-extraction of interfering substances. The extraction solvents must be highly pure. Blank tests help to prove that the matrix does not interfere in the analyzes. After extraction, a purification step removes the interfering substance with minimal loss of the analyte. The final solution should include an appropriate solvent for analyte determination by the selected method [127].

Below is a didactic description of the main separations and detection methods.

#### **7.1. Physicochemical methods**

Gas chromatography (GC), Liquid Chromatography (LC), and Capillary Electrophoresis (CE) constitute physicochemical separation methods.

When the analyzed pesticide is volatile or semi-volatile, GC still is the method of choice: it offers higher resolution and lower detection limits. GC is usually associated with multiple detectors whose choice will depend on the characteristics of the target analytes. GC is based on sample volatilization and introduction into a chromatographic column coated or packed with a solid or liquid stationary phase. A gaseous mobile phase elutes the analyte; this phase is inert, and does not interact with the analyte. The carrier gases should be pure and chemically inert, too, and the choice will depend on the detector. The commonest carrier gases are helium, argon, nitrogen, carbon dioxide, and hydrogen [128].

LC has emerged as a great separation tool. It allows for effective separation of nonvolatile and thermally unstable pesticides that are incompatible with GC. During LC, extracts pass through multiple adsorbent columns that can discriminate between the components of the matrix and target analyte. The degree of selectivity will vary according to the adsorbent present in the column (alumina, silica gel, or Florisil), mesh size, and activity levels. Columns can be used separately or in combination [129].

CE is a powerful tool to separate and identify a wide range of molecules. EC provides high resolution, and large separation efficiency. It requires small sample size and low solvent consumption analyzes is faster and operational coats are low [130].

An ideal detector should ensure adequate sensitivity, good stability and reproducibility, and linear response to various concentrations of the analytes. It should also operate in a wide range of temperature, have reduced response time (independent of the flow), and be easy to handle. The detector response should be equivalent for all the analytes or selective to certain classes of compounds. Ultimately, the detector should not destroy the sample. Unfortunately, a switch that exhibits all these characteristics does not exist, so it is necessary to select the detector according to the desired goal [128].

Several types of detectors are commercially available. They can come coupled to the separation device. These detectors use photometric or fluorimetric methods, thermal conductivity, diode array detection, electrons capture, atomic absorption, or pesticides mass/charge evaluation. The latter method is currently in evidence due because it is highly sensitive, offers autonomy, and performs a variety of functions. Electron capture and mass spectrometry are the most often used to detect pesticides.

The electron capture detector (ECD) is usually employed to search for organic pesticides, because it is highly sensitive and selective toward molecules containing electronegative functional groups. It also detects masses in the order of pictograms and can analyze traces of pesticides. However, ED cannot detect compounds with low electron affinity. Its excellent properties are useful for analysis of pesticides in both the environmental area and hospitals. A detector called µECD is also available in the market. It is advantageous over ECD in term of sensitivity, stability, and robustness [131].

Mass spectrometry (MS) is based on the ionization, acceleration, and separation of the generated molecules and ions according their mass/charge (m/z) ratio. This Provides a typical spectrum that gives the relative mass abundance of the different ionic species as a function of m/z so, which permits unambiguous identification of molecules. Mass spectrometry is a confirmation technique that is less subject to misunderstanding. Nevertheless, it has a drawback – it destroys the analyte [132].

As mentioned previously, the choice of method will depend on the case. LC-MS and GC-MS are the methods that generally separate and detect pesticides most suitable. These methods play a very important role in the analysis of pesticides and related compounds and are applicable in several areas like environmental analysis, food safety, and occupational toxicol‐ ogy, among others. Because they can serve various purposes, these methods also help to detect compounds in different samples, such as water, soil, sediment, sludge, vegetables and fruits, and animals and humans tissues and fluids [124, 126]. Obviously, method will based on the needs and characteristics of the target pesticide, and each sample will have their own features, which will depend on their physicochemical properties.

#### **7.2. Biological**

Sample storage for long periods should ensure that no sample degradation or external contamination occurs. Well-sealed containers stored under refrigeration and protected from light are mandatory. To avoid any type of external interference during analysis, none of the employed materials should modify or degrade the pesticide in the sample. The analysis of pesticides, mainly in water, ambient air, and soil sediments, often requires a purification step to clean the sample and pre-concentrate the analytes, to improve the quality of the analytical results. The extraction process is a key analytical step – it extracts the desirable compounds for further separation and characterization. Liquid-liquid extraction, and pre-concentration procedures, such as solid-phase extraction and solid-phase microextraction, are the most commonly used methods, but other extraction methods are also applicable depending on the objective [126]. Extraction of residues from the sample matrix demands appropriate solvents for maximum extraction efficiency and minimal co-extraction of interfering substances. The extraction solvents must be highly pure. Blank tests help to prove that the matrix does not interfere in the analyzes. After extraction, a purification step removes the interfering substance with minimal loss of the analyte. The final solution should include an appropriate solvent for

analyte determination by the selected method [127].

constitute physicochemical separation methods.

argon, nitrogen, carbon dioxide, and hydrogen [128].

consumption analyzes is faster and operational coats are low [130].

**7.1. Physicochemical methods**

220 Toxicology Studies - Cells, Drugs and Environment

separately or in combination [129].

Below is a didactic description of the main separations and detection methods.

Gas chromatography (GC), Liquid Chromatography (LC), and Capillary Electrophoresis (CE)

When the analyzed pesticide is volatile or semi-volatile, GC still is the method of choice: it offers higher resolution and lower detection limits. GC is usually associated with multiple detectors whose choice will depend on the characteristics of the target analytes. GC is based on sample volatilization and introduction into a chromatographic column coated or packed with a solid or liquid stationary phase. A gaseous mobile phase elutes the analyte; this phase is inert, and does not interact with the analyte. The carrier gases should be pure and chemically inert, too, and the choice will depend on the detector. The commonest carrier gases are helium,

LC has emerged as a great separation tool. It allows for effective separation of nonvolatile and thermally unstable pesticides that are incompatible with GC. During LC, extracts pass through multiple adsorbent columns that can discriminate between the components of the matrix and target analyte. The degree of selectivity will vary according to the adsorbent present in the column (alumina, silica gel, or Florisil), mesh size, and activity levels. Columns can be used

CE is a powerful tool to separate and identify a wide range of molecules. EC provides high resolution, and large separation efficiency. It requires small sample size and low solvent

An ideal detector should ensure adequate sensitivity, good stability and reproducibility, and linear response to various concentrations of the analytes. It should also operate in a wide range of temperature, have reduced response time (independent of the flow), and be easy to handle. Chemical analysis of isolated compounds is commonly used to monitor environmental pollution, but such analyses can be limited and expensive and cannot indicate the biological effects. In contrast, biological tests indicate the toxicity of a ride range of compounds or environmental samples, and are therefore essential to determine the environmental impacts of the presence of these chemicals [133]. Immunoassays and biosensors are methods related to the biological factor. Immunoassays are a powerful tool in clinical laboratories and one of the most widely applied analytical techniques. The reagents kits and the equipment necessary to perform immunoassays are commer‐ cially available and rely on fluorescent, chemiluminescent or other detection methods. Immunoassays can detect a wide range of compounds including drugs, proteins, and hormones; they can also identify and quantify the presence of pesticides residues in various samples such as natural water, food, and blood, among others [129].

Regarding biosensors, organisms such as *Drosophila melanogaster* fly species may aid the detection of pesticides in food samples and other matrixes such as water, soil, plants, and animal tissue. This test model is advantageous, because these insects have low tolerance to toxic substances with insecticidal character, besides being experimental models of easy creation, manipulation, and maintenance. In addition, they require few financial resources and can remain under laboratory conditions. However, this method only serves to detect the presence of pesticides, but it cannot identify the detected compound. Therefore, after using this probe, the analyst has to employ a chromatographic, for example, to identify the group of pesticides in that sample [123].
