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Food Forensics Cases Related to Food Bioterrorism/Food Bio‐Weapons and Food Poisoning Agents: Agrochemical Food Poisoning Agents

Food bioterrorism refers to any deliberate/intentional practice or actions that may lead to the presence of harmful substances such as poisons in food that may affect the consumer negatively by causing illness, disease, sickness, physical body deformation, or death. Substances that may poison foods may be in the form of chemical substances such as agrochemicals/pesticides/insecticides, antibiotics, metals (e.g. cadmium, arsenic, lead, mercury, radionuclides, etc.), they may also be in the form of biological agents such as biological substances caused by naturally occurring toxins that are present in food such as algal toxins, red kidney beans, or green potatoes, and they may be microbially derived poisons caused by pathogenic microorganisms deliberately introduced into foods. In this category, there may be microbial toxic secondary metabolites (e.g. mycotoxins, cyanotoxins, botulinum, tetanus, anthrax, H5N1 virus poison, H1N1 virus poison, etc.) in foods, or microbes or their spores introduced into foods.

Introduction

Conflicts within and between societies have been characteristic of our planet since time immemorial. Political, social, and economic factors have been at the center of many if not all of the major frictions that have led to the loss of peace, and in many cases loss of life. One of the easy target weapons for inflicting damage or even eliminating those who are on the opposite side of the conflict, has been in the area of food and water (bioterrorism), through our basic needs such as for food that is required by all human beings on a daily basis. The deliberate or intentional unlawful act of inflicting damage to or annihilation of human life and well‐being or livestock, crops, social, or economic installations, etc., that involves the use of biological agents such as microorganisms or secondary metabolites derived from microbes, is known as bioterrorism (Centers for Disease Control and Prevention (2008); Frerichs et al., 2004; Wein and Liu, 2005).

There are several food production, processing, or distribution stages that could be a target in food bioterrorism. They include the whole production and supply chain (from farm‐to‐table food continuum), for example crops that are still in the field, livestock, food products that have entered the processing stage, supply and distribution chain, wholesale/retail shops, storage facilities, transportation, food laboratories, and agriculture research infrastructure (Mackby, 2006). Bioterrorism agents can inflict serious health problems, illness, sickness, and disease ranging from severe acute respiratory syndrome (SARS), foot‐and‐mouth disease, botulism, tetanus, mad cow disease, central nervous system collapse, monkey pox, pulmonary related diseases, avian influenza, etc.

Chemical Food Poisoning Agents: Agrochemicals in Foods

The main fact about agrochemical toxicity is that all agrochemicals are usually, but not always, poisonous to humans. Although they are termed agrochemicals, pesticides, insecticides, fungicides, herbicides, rodenticides, algicides, bactericides, etc., they are used for both agricultural‐related applications and also non‐agricultural‐related application. The main applications of these chemicals in agriculture involve the prevention, destruction, repulsion, and mitigation of pests, such as insects, rodents, or other destructive animals, weeds and harmful microbes including fungi, bacteria, and viruses, that can cause or act as disease vectors (Laws and Hayes, 1991). Non‐agricultural use of agrochemicals includes their application in the control and reduction of food‐borne and vector‐borne diseases, such as mosquitoes (Centers for Disease Control and Prevention (Disease Information), 2002; Gubler, 1998).

Agrochemicals and their residues can be transferred to humans in several possible ways, including drinking water that is contaminated with agrochemicals, eating meat from animals that have previously been fed on feed contaminated with agrochemicals, or animals previously subjected to medicated premixes, consumption of eggs, milk, honey, vegetables, and fruits.

The agrochemical classes and their respective metabolites that have been reported in human biological matrices for forensic poisoning‐related cases include organophosphate agrochemicals and their respective metabolites (Hardt and Angerer, 2000; Lacassie et al., 2001; Pitarch et al., 2003); organochlorine agrochemicals (Barr et al., 2003; Burke et al., 2003; Burse et al., 2000; Campoy et al., 2001; Conka et al., 2005; Hong et al., 2002; Lacassie et al., 2001; Sundberg et al., 2006); synthetic pyrethroid‐based insecticides (Arrebola et al., 1999; Lacassie et al., 2001; Leng and Gries, 2005; Ramesh and Ravi, 2004; Schettgen et al., 2002); triazines (Barr et al., 2002; Lacassie et al., 2001; Norrgran et al., 2006); chloroacetanilides (Bradman et al., 2003; Whyatt et al., 2003); phenoxyacetic acid herbicides (Hill et al. 1995); chlorophenols (Bravo et al., 2005); and neurotoxic carbamates (Meeker et al., 2004).

Sampling of Target Specimens

Human specimens that are normally collected for the analysis of agrochemical poisoning include hair, fluids (urine, sweat, saliva, blood, intestinal fluids, milk, human hair, finger nails, amniotic fluids, semen, etc.). Of the fluid specimens, urine is the most commonly used, due to the fact that it is easily obtained in abundance. However, there are some drawbacks that are associated with urine sampling, which are due to temporal variability of the volume, and also the variability of the levels of endogenous and exogenous compositions. Some reports have suggested that urine sampling should be done first thing in the morning to ensure the stability of the acceptable representative as opposed to sampling at other times of the day (Kapka‐Skrzypczak et al., 2011). Moreover, it is required that normalization using specific standards such as creatine should be included to account for cases of either over‐dilution or over‐concentration of the samples that have been collected on the spot (Arcury et al., 2009).

Milk is known to be a very complex matrix due to the presence of numerous biomolecules such as fats and lipids, proteins, carbohydrates, etc., which may have strong affinity to the agrochemical residues that may be present, making it difficult to extract and quantify them properly.

Saliva sampling is easy but the analyst must be knowledgeable enough to be able to present acceptably reliable measurements related to an internal dose from a saliva specimen that has been instantly collected (spot saliva sample) and must have a thorough knowledge of the stability (half‐life), pharmacokinetics data of the agrochemical molecules being investigated, and the distribution ratio of the same agrochemical between the saliva and blood (Barr et al., 2006; Timchalk et al., 2004). Some researchers have suggested that the distribution ratio of agrochemicals in saliva is outweighed by that found in the blood and this ratio is governed by the type and chemistry of the protein binding to the agrochemical species (Kapka‐Skrzypczak et al., 2011). Sweat has also been reported to be a possible specimen for the analysis of pesticide poisoning (Rosenberg et al., 1985).

Hair specimens have been used as an indicator of long‐term agrochemical exposure/poisoning because the drugs remain longer in the hair. Hair strands from the suspected victims have to be decontaminated from cosmetics and facial secretions using appropriate aqueous‐organic reagents (e.g. surfactants such Triton‐X 100, deionized water, acetone), cut into small pieces and incubated at temperatures between 45 and 75 °C (depending on the type of suspected agrochemicals). Then hair samples are dissolved in methanol to extract agrochemical residues before injecting into an analytical instrument such as GC‐FID/NPD/FPD/MS for detection and quantification (Cirimele et al., 1999; Oluremi et al., 2011a,b).

However, despite the fact that a hair specimen gives a relatively good biological measure of exposure or poisoning due to many persistent organic pollutants (POPs), including some of the agrochemicals, there are some limitations that are associated with the difficulties in distinguishing between exogenous and endogenous contamination. Another bottleneck related to the use of hair specimens in the analysis of agrochemicals and other POPs is lack of availability of standardized methods for hair sampling, decontamination (washing), and analysis, as well as the inadequacy of useful information that is available in the literature regarding the correlation and distribution ratio between concentrations of the POPs, such as agrochemicals in hair and other body matrices such as blood, adipose tissue, etc. (Altshu et al., 2004).

Finger nails are also used as specimens in food poisoning cases, as they can retain residues of metabolites for a considerable period of time (Dablool, 2014).

Blood samples are also useful in providing evidence in cases of agrochemical poisoning. For example, for poisoning with organophosphates or carbamates, blood samples can provide a good measure of the depression associated with plasma butyrylcholinesterase (pseudocholinesterase) as well as that of red blood cell (RBC) AChE levels as caused by the absorption of organophosphate in the blood (Eddleston et al., 2008).

Other specimen that are normally collected to examine microbial food poisoning include throat swabs, nasal swabs, nail swabs, and stool samples and after collection the culturing of the microbes follows and they can be identified using molecular biological techniques.

The umbilical cord has been mentioned as a suitable sampling organ for analysis of agrochemical poisoning (Burse et al., 2000).

Agrochemical Poisoning Metabolite Indicators and Markers: Indicator Molecules and Markers Found in Urine Samples After Poisoning

In most cases, when there is poisoning due to chemicals, it is difficult to track parent molecules in any of the body matrices for several reasons, including the fact that they become metabolized in the body system and change their chemistry altogether.

In general, the trend and series of events that follow just after the poisonous or toxic molecule comes into contact with and/or if it gets into the body’s system, a toxicokinetic process commences, which involves absorption processes, distribution, metabolism, and elimination through excretion routes (NB: the rate and time it takes for agrochemicals or their metabolites to be eliminated differs within and between agrochemicals from various matrices, urine, blood/blood components, etc., with some being rapid while others have slow elimination rates depending on their respective half‐life (Angerer et al., 2007)). These processes trigger and result in the body metabolic pathways producing certain specific molecules (biomarkers/indicators) in response to the presence of these toxic molecules. Each of the poisonous/toxic molecules results in a particular set of markers/indicator molecules, which can be identified and measured from each of the processes, whether it is during absorption, distribution, metabolism, or elimination. Therefore toxicity due to poisoning molecules takes into account the detection and measurement of these specific biomarkers or indicator molecules that may include parent molecules (in case they are present in the matrix chosen for analysis), the specific metabolites in any of the processes or the reaction products present in the sampled specimen’s matrix such as urine, hair, tissues, blood, and/or blood‐derived components, nails, exhaled air, etc. (Barr et al., 2006; Ngo et al., 2010). In some cases, such as the phenomena that happen with organophosphorus agrochemicals that are characterized with having common urinary metabolites but which have a tendency to mask the signals of their respective parent molecules in the victim’s body system, it is thus expedient to deal with the measurements of mainly metabolites rather than the parent molecules (Albertini et al., 2006; Barr et al., 2006; Kissel et al., 2005).

Analysis of Agrochemical Residues and their Metabolites in Biologic Samples in Individuals Affected by Food Poisoning

There are three major classes of organophosphorus agrochemicals including: i) phosphoroamidothiolates; ii) phosphorodithioates; and iii) phosphothionates (Chambers, 1992). Within these three classes there are numerous individual organophosphate compounds. The analytical strategy for the analysis includes the measurement of both the intact agrochemical molecules and/or their metabolites in various biologic matrices such as serum, breast milk, stool, meconium (the earliest stool of an infant), urine, etc. The analytical procedures for the agrochemical parent molecules and their respective metabolites in all of these biological matrices involve the application of effective sample extraction and sample purification procedures, as well as very sensitive and selective detection methods.

Biomarkers in Agrochemical Poisoning

There are different types of biomarkers that can be used to provide indication of the presence of a foreign substance inside living systems, which triggers the body’s system of an organism to respond by either upregulating or downregulating certain biomolecules (biomarkers) specific to that particular foreign substance (WHO, 1993). The upregulation and/or downregulation of these biomolecules shows that biomarkers are genetically controlled and are also useful in predicting parameters or species that will either increase or decrease as a result of poisoning (Hernandez et al., 2003). Biomarkers are useful in forensic cases because their detection provides evidence of not only poisoning but which poisons were actually used (WHO, 1993).

Specific biomarkers in agrochemical poisoning cases arise during the metabolism of the agrochemical once ingested, which then proceeds through bioactivation steps before the detoxification procedures begin. It should be noted that the bioactivation and detoxification mechanisms can be influenced by genetic polymorphisms through the catalytic actions of a number of enzymes that are coded by various genetic factors. For this reason, all these biotransformation pathways and processes may significantly influence the toxicity of different agrochemicals to humans (Costa et al., 2005; Rose et al., 2005).

These specific responses by the body after poisoning, which result in the formation of different types of biomarkers, are caused by several polymorphic principal enzymes that may belong to a number of enzyme classes and in some cases a synergetic combination of these enzymes from different classes, especially in the processes involved in bioactivation (Hernandez et al., 2003). On the other hand, the process of elimination of agrochemical intact molecules and their metabolites from the body is genetically controlled, such that some individuals with weaker versions of polymorphic genes may experience elevated levels of bioactivation that may be associated with slow detoxification of poisoning agents and these individuals are likely to suffer severe poisoning consequences (Bolognesi, 2003). The metabolic process of elimination of poisons emanating from agrochemical poisoning involves glutathione S‐transferase enzymes, cytochrome P450 enzymes, and also esterase enzymes. The glutathione S‐transferase enzymes are useful in identifying genetic polymorphisms that cause glutathione conjugation due to agrochemical or other chemical poisoning (Bolognesi, 2003; Schroeder, 2005). These enzymatic controlled polymorphisms play different roles, for example, glutathione S‐transferase polymorphism 1 (GSTP1) has been reported to play a role in influencing both substrate selectivity and stability, also the rate at which metabolites are formed or the rate at which metabolites and other toxic species are eliminated (Liu et al., 2006).

The enzymes that belong to the family of cytochrome P450 (CYP450) are important in the metabolism of agrochemicals, such as carbamates, nicotinoid class of agrochemicals, etc., as they are responsible for catalyzing various metabolic reactions and processes and their variation profiles in the body may be a useful indication of agrochemical poisoning (Hodgson, 2003; Mutch and Williams, 2006). The catalytic mechanism of action of CYP450 enzymes (e.g. CYP2C8, CYP3A4, CYP3A5, CYP2D6, CYP1A2) involves oxidative desulfuration of the agrochemical molecules (mainly the anticholinerase ones such as carbamates and organothiophosphates to the oxon derivatives of the respective agrochemicals). These enzymes (e.g. CYP1A2, CYP2, and CYP3) are also important in catalyzing both the bioactivation and elimination processes of anticholinerase agrochemicals (Costa et al., 2005; Nebert, 2005).

Another class of enzyme that is important in catalyzing bioactivation and detoxification of agrochemicals and their metabolites is esterase enzymes, which include the serine hydrolase class of enzymes (mainly carboxylesterase enzymes), paraoxonase enzymes, and cholinesterase enzymes.

The carboxylase enzymes (produced in the liver) are mainly responsible for the hydrolysis of ester‐containing agrochemicals, as well as the detoxification of anticholinesterase agrochemicals (Ross et al., 2006; Wu et al., 2007; Zhou et al., 2007). In the case of paraoxonase (PON), it also includes several classes such as classes 1, 2, and 3, such that there are PON1, PON2, and PON3, mainly responsible for the metabolism of oxygen containing agrochemicals (Hernandez et al., 2004). The esterases normally target the anticholinesterase agrochemicals, mainly the organophosphates.

Organophosphate and Carbamate Agrochemicals: Mode of Action, Toxicity, Metabolism, and Biomarkers

The organophosphate class of agrochemicals includes a number of other sub‐classes (with nerve poisoning moiety), as shown in Figure 4.1a to g.

Figure 4.1 General chemical structures of organophosphate agrochemical sub‐classes: (a) phosphorothioate; (b) sodium‐di‐sec‐butyl‐phosphorodithioate; (c) phosphorothioic acid, methyl, 0‐ethyl; (d) phosphates; (e) phosphonates; (f) phosphoramidite; and (g) O,S‐dimethyl phosphoramidothioate.

The raw materials that are normally used in the preparation of organophosphate agrochemicals, are mainly phosphorylating compounds that originate from various sources such as diisopropylfluorophosphate, sarin, etc. These substances are well known for their powerful effects as nerve agents. In cases of poisoning, organophosphates normally tend to phosphorylate specific targets in the body of the victim, mainly an enzyme acetylcholinesterase that plays an important role in facilitating the hydrolysis of acetylcholine (a neurotransmitter), found in the synaptic membrane in the central nervous system to form choline and acetic acid. Other targets include proteins and enzymes such as neuropathy target esterases (NTE), nicotinic acid, and muscarinic acetylcholine receptors (Eldefrawi et al., 1992).

The action of organophosphates in the body system prevents the hydrolysis of acetylcholine, thus causing failure in the binding of the ion channel of the nicotinic acetylcholine. Due to this, these ion channels will remain open permanently and this will allow sodium ions to pass through, continuously causing a number of fatal events including membrane depolarization, convulsions which may be fatal to the victim, and also non‐stop neuronal firing that may also be fatal. Cases where organophosphates bind NTE will lead to disorders related to progressive neuronal degeneration syndrome, also known as organophosphate‐induced delayed neuropathy (OIDN), which results in symptoms such as ataxia.

The reaction of organophosphates with acetylcholinesterase proceeds via mechanisms that involve nucleophilic reactions of the serine hydroxyl group, which is the active site on the phosphorus atoms that belong to phosphate ester functionality (Schemes 4.1–4.3). The nucleophilic attack is thus responsible for the cleavage of bonds such as P–O and P–S. After these processes follows a step that involves phosphorylation of the amino acid serine followed by the release of the leaving group. Serine may also undergo acetylation using acetylcholine as the process of acetylcholine hydrolysis proceeds.

Scheme 4.1 Proposed reactions of organophosphates and acetylcholinesterase (Raushel, 2011).

Scheme 4.2 Proposed acetylcholinesterase mechanism of action.

Scheme 4.3a Proposed interaction of acetylcholine with acetyl cholinesterase.

Scheme 4.3b Proposed interaction of acetylcholine with carbaryl.

Scheme 4.3c Proposed interaction of acetylcholine with chlorpyrifos.

Organophosphate Agrochemicals: Malathion

Malathion (carbophos) (C₁₀H₁₉O₆PS₂; molar mass: 330.358 g/mol; density: 1.23 g/mL; chemical name diethyl‐2‐[(dimethoxyphorothothionyl) sulfanyl butanedionate) is an organophosphate pesticide with a water solubility value of 145 mg/L (at 25 °C) and logK_OW value of 2.75. The chemical structure for malathion is depicted in Figure 4.2.

Figure 4.2 Chemical structure of malathion.

Malathion is known to produce a number of metabolic compounds in metabolically active plant tissues, fish, animals, and soils (Mostaf et al., 1974).

The metabolites of malathion formed in fish are mainly monoacids and dicarboxylic acids (Cook and Moore, 1976). In humans, the principal metabolites include malathion monoacids, malathion dicarboxylic acids, and malaoxon, which is among several minor metabolites. Other metabolites of malathion include O,O‐dimethylphosphorodithionate, O,O‐dimethylphosphorothionate, dimethylphosphate, and monomethyl phosphate. Detailed metabolic pathways for malathion are presented in Figure 4.3a–d.

Figure 4.3a(i) Proposed metabolic pathways of malathion in humans: Formation of specific malathion/malaoxon biomarkers in urine (MCA and MDA).

Figure 4.3a(ii) Proposed metabolic pathways of malathion in humans: Formation of non‐specific malathion biomarkers in urine (DMDTP and DMTP).

Figure 4.3b Proposed metabolism of malathion in fish/aquatic organisms.

Figure 4.3c Proposed formation mechanism of malathion metabolic biomarkers in plants (e.g. plant vegetables, crop plants).

Figure 4.3d Proposed metabolites of malathion in water.

Organophosphate Agrochemicals: Parathion and Methyl Parathion

Parathion (folidol) (O,O‐Diethyl O‐(4‐nitrophenyl) phosphorothioate) has a water solubility of 11 mg/L (at 20 °C), logK_OW value of 3.83, chemical formula: C₁₀H₁₄NO₅PS, and molar mass: 291.26 g · mol⁻¹. The chemical structure of parathion is depicted in Figure 4.4.

Figure 4.4 Chemical structure of parathion.

In humans and other animals, the metabolism of parathion proceeds via the route of the de‐esterification process that generates 4‐nitrophenol and de‐ethlation, and oxidation that produces paraoxon products. In the case of orally ingested pathion, the detoxification process proceeds via dearylation, which generates 4‐nitrophenol and O,O‐diethylphosphorothionate. The dearylation is an enzymatic mediated reaction where NADPH‐dependent microsomal oxidases occur as well as glutathione‐S‐aryl transferases (Hollingworth et al., 1973).

Anticholinesterase paraoxon as well as sulfates are other metabolites generated through the oxidative desulfuration process, which is mediated by cytochrome P450 enzymes. Paraoxon metabolites also undergo hydrolytic biotransformation, which catalyzes esterases to generate 4‐nitrophenol and diethyl phosphate. Another biotransformation reaction of paraoxon produces desethyl paraoxon, a product which is excreted in urine, and is catalyzed by glutathione–S‐alkyl transferases. Other parathion metabolites that are excreted in urine include monoethyl phosphate, which is itself a by‐product of desethyl paraoxon hydrolysis and also an inorganic phosphate.

By‐products of 4‐nitrophenol metabolism proceed via the route of glucuronide conjugation and form major metabolites in urine. In some cases, only glucuronide conjugation products without other unconjugated metabolites are the ones excreted in urine. Parathion, paraoxon, and ethyl paraoxon (EPN) can also be reduced by in vitro enzymes such as NADPH and NAD to form several products, including amino paraoxon and amino ethyl paraoxon, 4‐aminophenol and these are excreted in urine as one of the major metabolite products. The detailed metabolic pathways for parathion are depicted in Figure 4.5a–f.

Figure 4.5 (a) Proposed metabolic pathways for parathion in animals; (b) Proposed photolysis metabolic pathways for parathion.

Parathion‐methyl, on the other hand, has a logK_OW value of 3.0 (thus relatively less hydrophobic as compared to parathion) and water solubility of 55 mg/L (20 °C) (thus more soluble in water as compared to parathion.

The major biotransformation pathways for parathion‐methyl proceed via desulfuration, a process that leads to the generation of paraoxon methyl (an oxon derivative). Another biotransformation route for parathion methyl proceeds via hydrolysis, which results in the generation of products such as dimethyl phosphate, dimethylphosphorothionate, and 4‐nitrophenol. Other metabolites include those which are obtained via the reduction of the nitro functional group and also via desmethylation to produce the desmethyl‐paraoxon methyl. The process of desmethylation takes place in the liver and is catalyzed by glutathione‐S‐methyl transferase. The metabolic pathways in phase two metabolism proceed mainly via conjugation of 4‐nitrophenol and then these conjugated metabolites are eliminated through excretion. Figure 4.6a–f depicts the detailed metabolic pathway of parathion methyl.

Figure 4.6 (a) Proposed metabolic pathway for parathion‐methyl; (b) Proposed acidic hydrolysis metabolic pathway for parathion‐methyl; (c) Proposed alkaline hydrolysis metabolic pathway for parathion‐methyl; (d) Proposed metabolic pathway for parathion‐methyl in goats.

Organophosphate Agrochemicals: Pirimiphos‐methyl

Pirimiphos‐methyl (Actellic) (O‐[2‐(diethylamino)‐6‐methylpyrimidin‐4‐yl] O,O‐dimethyl phosphorothioate), has a molecular formula of C₁₁H₂₀N₃O₃PS (Mwt 305.334); is an organophosphate pesticide with log K_OW value of 4.2; and water solubility of 9.9 mg/L (at 30 °C and pH 5.2). Its chemical structure is presented in Figure 4.7.

Figure 4.7 Chemical structure of pirimiphos‐methyl.

The metabolism of pirimiphos‐methyl begins with its hydrolysis to produce 2‐diethylamino‐6‐methyl‐pyrimidin‐4‐ol and the hydrolytic by‐product may either undergo conjugation or become N‐de‐ethylated. The hydrolysis can either be acidic or alkaline, which generates pyrimidinol as the major metabolic product (Figure 4.8a,b).

Figure 4.8a Proposed metabolic pathways of pirimiphos‐methyl in animals.

Figure 4.8b Proposed metabolic pathways of pirimiphos‐methyl in plants.

Organophosphate Agrochemicals: Diazinon

Diazinon (Figure 4.9) (O,O‐diethyl O‐[4‐methyl‐6‐(propan‐2‐yl)pyrimidin‐2‐yl] phosphorothioate; chemical formula C₁₂H₂₁N₂O₃PS; and Mwt = 304.34 g · mol⁻¹), is another organophosphate agrochemical. It has a logK_OW value of 3.3 and water solubility of 60 mg/L (20 °C). The chemical structure is depicted in Figure 4.9.

Figure 4.9 Chemical structure of diazinon.

The metabolic pathways for diazinon proceed via the cleavage of the P‐O‐pyrimidine linkage to form 3‐isopropyl‐4‐methyl‐6‐hydroxypyrimidine. Other metabolic products such as diazoxon, in which the pyrimidinyl group has been lost, are obtained through the process that involves the oxidative desulfuration of the thiono functional group. This process is mediated by microsomal mixed function oxidases. The diazoxon then becomes hydrolysed by the action of either A‐esterases or enzymatic oxidative processes mediated by mixed function oxidases to generate diethyl phosphorothioate. In addition to these, other metabolites are formed through hydroxylation processes of the alkyl groups, mainly the methyl and isopropyl groups present on the pyrimidine ring. Figure 4.10a–d depicts the metabolic pathways of diazinon.

Figure 4.10a Proposed metabolic pathways for diazinon in plants.

Figure 4.10b Proposed hydrolysis metabolic pathways for diazinon.

Figure 4.10c Proposed photolysis metabolic pathways for diazinon.

Figure 4.10d Proposed metabolic pathways for diazinon in animals.

Organophosphate Agrochemicals: Dichlorvos

Dichlorvos (2,2‐dichlorovinyl dimethyl phosphate, DDVP; molar mass: 220.98 g/mol; logK_OW value of 1.9; and water solubility of 18.0 mg/L (25 °C)), is an organophosphate with the chemical structure shown in Figure 4.11.

Figure 4.11 Chemical structure of dichlorvos.

The biotransformation of dichlorvos proceeds via enzymatically catalyzed hydrolytic processes, which then generate dimethyl phosphate and dichloroacetaldehyde as initial products. Dichloroacetaldehyde can then proceed through more steps of metabolic reactions to yield 2,2‐dichloroethanol, dechlorination of dichloroacetaldehyde, to form glycolic acid. Other metabolites that are of importance include methyl glutathione and desmethyldichlorvos, which are produced via the demethylation processes that are mediated by glutathione‐S‐methyl transferases.

Organophosphate Agrochemicals: Coumaphos

Coumaphos organophosphate (O,O‐diethyl O‐3‐chloro‐4‐methyl‐2‐oxo‐2H‐chromen‐7‐yl phosphorothioate) has a molecular formula C₁₄H₁₆C_lO₅PS; Mwt 362.77 g/mol; logK_OW value of 4.13; and water solubility of 1.3 mg/L (20 °C). Its chemical structure is depicted in Figure 4.12.

Figure 4.12 Chemical structure of coumaphos.

The metabolites related to coumaphos are normally produced through a number of processes such as hydrolysis, oxidation, and oxidative desulfuration, which generate coumaphos‐oxon prior to hydrolysis. Other metabolic products are formed through dechlorination, as well as those formed after the lactone group of the coumarin ring has been opened. The metabolic pathways for coumaphos are depicted in Figure 4.13a–c.

Figure 4.13a Proposed metabolic pathways for coumaphos in animals.

Figure 4.13b Proposed hydrolysis metabolic pathways for coumaphos.

Figure 4.13c Proposed photolysis metabolic pathways for coumaphos.

Organophosphate Agrochemicals: Chlorpyrifos

Chlorpyrifos (O,O‐diethyl O‐3,5,6‐trichloropyridin‐2‐yl phosphorothioate) has a molecular formula C₉H₁₁C_l3NO₃PS; Mwt = 350.59 g/mol; logK_OW value of 4.7; and water solubility of 1.4 mg/L (25 °C). The structure of chlorpyrifos is depicted in Figure 4.14.

Figure 4.14 Chemical structure of chlorpyrifos.

The metabolites in cases of chlorpyrifos poisoning are generated through a number of metabolic pathways, including oxidative dealkylation or hydrolysis. The oxidation of chlorpyrifos normally proceeds via acidic, neutral, and alkaline media. The products of hydrolysis include diethyl phosphorothioate and 3,5,6‐trichloro‐2‐pyridinol. The 3,5,6‐trichloro‐2‐pyridinol is then conjugated to form either glycoside or glucuronide derivatives. Chlorpyrifos‐oxon is another metabolite that is generated; however, it is rapidly hydrolysed, thus normally not detected. The metabolic pathways are shown in Figure 4.15a–d.

Figure 4.15a Proposed metabolic pathways for chlorpyrifos in humans.

Figure 4.15b Proposed metabolic pathways for chlorpyrifos in plants.

Figure 4.15c Proposed metabolic pathways for chlorpyrifos in goats.

Figure 4.15d Proposed metabolic pathways for chlorpyrifos in fish.

Organophosphate Agrochemicals: Chlorpyrifos‐methyl

Chlorpyrifos‐methyl (O,O‐dimethyl O‐3,5,6‐trichloro‐2‐pyridyl phosphorothioate) with a molecular formula of C₇H₇C_l3NO₃PS; Mwt = 322.5 g/mol; water solubility of 2.6 mg/L (25 °C); and logK_OW value of 4.26, is an organophosphosphate pesticide with the chemical structure as depicted in Figure 4.16.

Figure 4.16 Chemical structure of chlorpyrifos‐methyl.

Unlike the metabolic pathways in chlorpyrifos, in chlopyrifos‐methyl, the most important metabolic route for the degradation of the compound involves the demethylation process, which is catalyzed by glutathione‐S‐alkyl transferase to generate demethylchlorpyrifos‐methyl. On the other hand, the major elimination route of chlorpyrifos‐methyl metabolites from the body is mediated by the NADP‐dependent oxidative dearylation process, which yields a conjugated product 3,5,6‐trichloro‐2‐pyridinol. Like chlorpyrifos, the oxon derivative is rapidly hydrolysed and therefore difficult to detect. The metabolic pathways for chlorpyrifos‐methyl are presented in Figure 4.17a–c.

Figure 4.17a Proposed metabolic pathways for chlorpyrifos‐methyl in animals.

Figure 4.17b Proposed metabolic pathways for chlorpyrifos‐methyl in plants.

Figure 4.17c Proposed hydrolysis metabolic pathways for chlorpyrifos‐methyl.

Organophosphate Agrochemicals: Azinphos‐ethyl

Azinphos‐ethyl (3‐(diethoxyphosphinothioylsulfanylmethyl)‐1,2,3‐benzotriazin‐4‐one) with a chemical formula of C₁₂H₁₆N₃O₃PS₂, Mwt = 345.37 g/mol; logK_OW value of 3.18; and water solubility of 4.5 mg/L (20 °C), is an organophosphate agrochemical with the chemical structure depicted in Figure 4.18.

Figure 4.18 Chemical structure of azinphos‐ethyl.

In poisoning incidences that involve azinphos‐ethyl, metabolites are produced via both acidic and alkaline hydrolysis processes. Azinphos is known to be unstable under neutral conditions. The details of the metabolic pathway are depicted in Figure 4.19a,b.

Figure 4.19a Proposed metabolic pathways for azinphos‐ethyl in animals.

Figure 4.19b Proposed metabolic pathways for azinphos‐ethyl in plants.

Organosphosphate Agrochemicals: Azinphos‐methyl

Azinphos‐methyl (O,O‐dimethyl S‐[(4‐oxo‐1,2,3‐benzotriazin‐3(4H)‐yl)methyl]dithiophosphate) with molecular formula C₁₀PN₃H₁₂S₂O₃; Mwt of 317.324 g/mol; logK_OW value of 2.96; water solubility of 28 mg/L (20 °C), has its chemical structure given in Figure 4.20.

Figure 4.20 Chemical structure of azinphos‐methyl.

In azinphos‐methyl poisoning incidences, the metabolites that are generated include 3‐(thiomethyl)benzazimide, which is further biotransformed through dimerization processes on the one hand and also by another route, the metabolites may proceed either via the reduction of the thiol group or via 3‐demethylation or ring opening that generates disulfide metabolites such as 3‐methylbenzazimide, benzazimide, and anthranilic acid. Conjugated metabolites are also formed. Figure 4.21a–d details the metabolic pathways for azinphos‐methyl.

Figure 4.21a Proposed photolysis metabolic pathways for azinphos‐methyl.

Figure 4.21b Proposed metabolic pathways for azinphos‐methyl in animals.

Figure 4.21c Proposed metabolic pathways for azinphos‐methyl in plants.

Figure 4.21d Proposed thermal degradation metabolic pathways for azinphos‐methyl.

Generally, different classes and individual organophosphates as well as carbamates tend to inhibit acetylcholinesterase (Costa et al., 2005).

However, there is a common factor in that all classes and individual organophosphates and carbamates poisonings result in toxic effects to the victims when they inhibit cholinesterase enzymes in the nervous system, because these agrochemicals cause cholinesterase enzymes to be phosphorylated, thus blocking their ability to break down acetylcholine into choline and acetic acid (Costa et al., 2005). The high levels of toxicity due to the organophosphates and their metabolites in the body can thus be indicated by a measure in the reduction in the level of a number of enzymes, including that of cholinesterase enzyme activity in blood as worked out from the difference between the pre‐dose and post‐dose poisoning (He et al., 2002). The measure of cholinesterase to indicate poisoning due to organophosphates may become unreliable in cases of low level poisoning, due to lack of selectivity and sensitivity and it also requires that the experiment incorporates a control experiment for the sake of establishing the baseline activity (Margariti et al., 2007).

Apart from acetylcholinesterase (AChE), measurement of other blood cholinesterase enzymes such as butyrylcholinesterase (BChE) activities have also been used as primary biomarkers for poisoning cases related to organophosphate and/or carbamate agrochemicals (Simoniello et al., 2010; Stefanidou et al., 2009). It should be noted that acetylcholinesterase is an enzyme that performs the hydrolytic cleavage of acetylcholine, which is a mediator molecule that facilitates the physiological transmission of nerve action potential in the nervous system (Araoud, 2011).

Butyrylcholinesterase (BChE) is a plasmic pseudo‐cholinesterase enzyme that is found in plasma (Costa et al., 2005). Unlike acetylcholinestearse, the decrease in the activity of BChE in the blood is not necessarily an indication of poisoning that is associated with anti‐cholinergic substances such as carbamates or organophosphorus compounds, but BChE is normally used as a predictive biomarker for anti‐cholinesterase agents such as organophosphates and carbamate possible poisoning cases (Ranjbar et al., 2002; Rastogi et al., 2008). However, according to some reports, the poisoning due to certain organophosphate compounds such as diazinon, malathion, dichlorvos, etc. can have plasma cholinesterase activity depression effects as a more sensitive indicator than it does for the other organophosphates (Costa et al., 2005). This can explain why the extent of inhibition of both AChE and BChE tends to vary significantly within different classes and individual compounds of organophosphates and carbamates, though the measurements of the depression of AChE activity has been universally accepted as a better indication of chronic poisoning exposure to organophosphorus compounds than BChE measurements (Kamel and Hoppin, 2004).

Apart from the toxicity that can be monitored by measuring the depression of cholinesterase and which causes cholinergenic symptoms associated with illness, organophosphorus poisoning is also known to cause another form of neurotoxicity, a central peripheral distal sensory‐motor axonopathy, which is known as organophosphate‐induced delayed‐polyneuropathy (OPIDP). This neurotoxicity disease is not associated with any depression of cholinesterase enzymes such as AChE, but with phosphorylation of an esterase enzyme known as neuropathy target esterase (NTE) that is present in the nervous system, blood lymphocytes, and platelets and liver (Maroni et al., 2000; Costa et al., 2005). The chain of biochemical reactions during organophosphorus compound poisoning begins with phosphorylation and this reaction is followed by the transformation of the phosphorylated target and is highly dependent on the nature and functional groups present in organophosphorus compounds, because it can only take place with phosphate, phosphonates, and phosphoramidates compounds. This transformation reaction cannot take place for sulfonates and carbamates (Maroni et al., 2000; Costa et al., 2005). Therefore measuring levels of NTE in lymphocytes may be a good biomonitoring indicative predictive marker, especially for the organophosphorus compounds that are associated with causing delayed polyneuropathy (Costa and Manzo, 1995).

Another useful biomarker for poisoning that is related to anticholinesterase agrochemicals is β‐glucuronidase activity measurements (Ueyama et al., 2010). Poisoning due to either organophosphorus or carbamate agrochemicals is normally followed by the cleavage of an enzyme known as the egasyn‐glucuronidase complex, which results in an increase in β‐glucuronidase activity in the plasma, especially in cases of acute organophosphate poisoning (Hernandez et al., 2004).

Genotoxicity (DNA damage) effects caused by agrochemicals (e.g. organochlorines, herbicides, etc.) poisoning has been reported widely and is normally monitored by using cytogenetic markers that include the measurements of chromosomal aberrations (CA), sister chromatid exchange (SCE), and micronuclei (MN) (Bolognesi, 2003; Das et al., 2007). The techniques that are normally used to measure changes in the measurements for these markers are mainly either single cell gel electrophoresis (SCGE) or Comet assay, which are capable of determining the degree of DNA or chromosome damage in the victim’s blood cells, such as leukocytes, lymphocytes, etc. (Angerer et al., 2007).

Some agrochemical poisoning such as that due to paraquat is known to influence changes in the levels of an erythrocyte enzyme known as erythrocyte δ‐aminolevulinic acid dehydratase (ALA‐D) (Hernandez et al., 2005). Scientific reports have indicated that poisoning due to some agrochemicals causes significant depression of ALA‐D due to a number of possible reasons, including (i) the generation of oxidative stress; (ii) non‐competitive binding of agrochemical molecules to the enzyme; and (iii) modification of the vicinal sulfhydryl of ALA‐D after poisoning, which might trigger certain phenomena that may induce the inhibition of enzyme activity (Noriega et al., 2002; Hernandez et al., 2005). Variations in the mean corpuscular hemoglobin concentration (MCHC) and variation in the mean platelet volume (MPV) have been used as biomarkers and indicator parameters in cases involving poisoning due to agrochemicals such as organophosphates, organochlorines, and others (Parron et al., 1996). In some cases, there is abnormality in the functioning of some organs such as the liver (where there are unusual variations in liver enzyme activities, e.g. in serum alanine aminotransferase (ALT), as well as that of aspartate aminotransferase (AST) (Anwar, 1997)) or kidneys (e.g. nephrotic changes as exemplified by increased levels of serum creatine, as well as that of blood urea (Hernandez et al., 2006)).

In some cases, poisoning due to agrochemicals tends to disrupt the metabolism of certain amino acids such as tryptophan causing hyperglycemia, and also alters the activity of ALT, AST, gamma glutamyl transferase (GGT), and lactate dehydrogenase (LDH) enzymes (Tsatsakis et al., 2009). Therefore, levels of these enzymes can be used as biomarkers of agrochemical poisoning. Moreover, enhancement of the concentration of some molecules such as triglycerides, GGT activity, inorganic phosphorus, and creatine kinase (CK), can be used as an indication of poisoning due to agrochemicals that possess anticholinesterase properties (Parron et al., 1996).

However, the measurement of parent molecules of organophosphate agrochemicals in the biological matrices is in most cases not possible, because they are rapidly metabolized by the body system and may not appear in blood or urine. Immediately when they enter the body, they become converted to oxo‐phosphate metabolites by the action of enzymes and these oxo‐phosphates are then subjected to a reaction with cholinesterase enzymes (Barr et al., 2004). Further reactions of oxo‐phosphates may take place and such reactions include enzyme/spontaneous hydrolysis, which may result in the generation of specific metabolites and non‐specific metabolites, mainly dialkyl phosphates (DAP), which are common metabolites of organophosphate agrochemicals.

Examples of specific organophosphate metabolites include para‐nitrophenol (PNP), which is associated with poisoning due mainly to methyl parathion, but also other similar compounds such as ethyl parathion and nitrobenzene (Barr and Needham, 2002; Esteban et al., 1996). Other specific organophosphate metabolites include 2‐isopropyl‐4‐methyl‐6‐hydroxypyrimidine (IMPY), which is a specific metabolite for diazinon poisoning; malathion dicarboxylic acid (MDA), a specific metabolite for malathion poisoning; para‐nitrophenol (PNP), a specific metabolite for parathion and methyl parathion poisoning; 3,5,6‐trichloro‐2‐pyridinol (TCPY), a specific metabolite for chlorpyrifos; and chlorpyrifos methyl poisoning (Margariti et al., 2007).

In some cases, it is possible to have a situation where organophosphates do not react to generate oxo‐phosphate metabolites and the unconverted organophosphates become hydrolysed to generate their respective specific metabolites as well as dialkylthionate metabolites, such as dialkylthiophosphate, glucono‐dialkylthiophosphate, sulpho‐dialkylthiophosphate metabolites, and/or dialkyldithiophosphate, glucono‐dialkyldithiophosphate, and sulpho‐dialkyldithiophosphate metabolites, which are normally excreted through urine and can thus be regarded as useful biomarkers for poisoning by organophosphate agrochemicals (Barr et al., 2004).

The most useful urinary dialkyl phosphate metabolites that can be used as biomarkers or indicators of organophosphate poisoning include dimethylphosphate (which is an indication of poisoning by azinphos‐methyl, dichlorvos, dicrotophos, dimethoate, fenitrothion, fenthion, malathion, methyl parathion, trichlorfon); diethylphosphate (indicator for poisoning due to chlorpyrifos, coumaphos, diazinon, disulfoton, ethion, parathion, phorate); dimethylthiophosphate (poisoning due to azinphos‐methyl, dimethoate, fenchlorphos, fenitrothion, fenthion, malathion, methyl parathion); dimethyldithiophosphate (poisoning due to azinphos‐methyl, dimethoate, malathion); diethylthiophosphate (poisoning due to chlorpyrifos, coumaphos, diazinon, disulfoton, ethion, parathion, phorate); and diethyldithiophosphate (poisoning due to disulfoton, phorate) (Margariti et al., 2007).

Although these dialkyl phosphate metabolites can be detected in urine, they will only serve as an indication of organophosphate poisoning and not of a specific organophosphate compound. These dialkyphosphates are normally excreted in urine in the form of sodium or potassium salts.

The potency and affinity of organophosphate compounds or their metabolites differ from one compound to another, or may differ between a parent compound and its metabolite. For example, chlorpyrifos, a phosphorothionate organophosphorus compound, may exhibit weak inhibition for acetylcholinesterase as a parent compound, but the metabolic reactions through desulfation activation result in an oxygenated oxo moiety derivative known as chlorpyrifos‐oxon, which becomes more potent as compared to the parent compound, due to the fact that the chlorpyrifos‐oxon displays a stronger affinity as well as potency in terms of phosphorylating the hydroxyl groups of the amino acid serine present on the active sites of the enzyme acetylcholinesterase (Timchalk, 2010).

The toxic potency of organophosphorus agrochemicals and their metabolites is governed by a number of factors, including the amount of the organophosphate ingested or of the metabolite formed, and the kinetics of their bioactivation/bioelimination (Calabrese, 1990). The mediation of the metabolic bioactivation of all these organophosphorus compounds is catalyzed by cytochrome P450 oxidases and the reactions take place mainly in the liver and in some cases in other organs such as the brain (Chambers and Chambers, 1989).

Carbamate Agrochemicals: Mode of Action, Toxicity, and Metabolism

Carbamates are derivatives of carbamic acid, NH₂COOH, which share a similar mode of action as organophosphates, whereby they both exert an anticholinesterase action on the nervous system. Unlike organophosphates, the inhibitory effect to carbon cholinesterase due to carbamate poisoning is short‐lived and so is the reverse of the effect of carbamate levels on the cholinesterase enzyme in the blood, such that in most cases the level may even appear normal. Apart from their agricultural applications to control plant and crop disease, they are also used in household fumigation in the forms of spray or baits (control of pests). Examples of carbamate agrochemicals include carbaryl, propoxur, bendiocarb, methomyl aldicarb, carbofuran, methiocarb, and mexacarbate.

In cases of carbamate poisoning, the level of cholinesterase activity is normally measured in blood samples and because the reduction in cholinesterase is short‐lived, it is imperative that the sampling of whole blood specimens be done immediately after the poisoning incident. Since the rapidly metabolized carbamates, together with their metabolites are excreted through urine, then urine can be another suitable biological matrix where carbamates and their metabolites can be measured.

The Biotransformation of Carbamate Agrochemicals

There are several chemical reactions that facilitate the biotransformation of carbamates once poisoning has taken place and include oxidative reactions, ester hydrolysis reactions, and also conjugate reactions (Ecobichon, 1994, 2001). The degree of metabolism for different carbamate compounds is governed by the chemistry of the carbamate molecules themselves, mainly the nature of substituent groups as well as their positions where they attach on either side of oxygen atom or nitrogen atom (Ecobichon, 2001; Timchalk, 2010).

The pathways of bioelimination reactions of carbamates and their respective metabolites normally involve hydrolysis processes, which are generally catalyzed by non‐specific esterases. These hydrolytic processes result in a number of products such as alpha‐naphthol and methyl‐carbamic acid. The formed methyl‐carbamic acid is very unstable, as it rapidly decomposes into carbon dioxide and monomethylamine. The alpha‐naphthol tends to undergo conjugation reactions with other compounds such as glucuronide or some sulfate compounds, which are excreted together in urine (Chin et al., 1979a). There are other non‐enzymic mediated metabolites that are associated with carbamate poisoning, which include oxidative metabolites that contain ring structures or ring structures with side chain hydroxylations that are also excreted in urine (Chin et al., 1979b).

Poisoning Biomarkers/Indicator Compounds, Metabolites, and Monitoring Matrices

Both carbamate parent compounds and their respective metabolites are normally monitored in poisoning cases in target biomatrices. For example, aldicarb‐sulfone is monitored in urine in aldicarb‐related poisoning cases, while carbaryl is monitored in blood, and its metabolite (1‐naphthol) is monitored in urine. Methonyl is monitored as a parent compound in blood, and pyrimicarb’s metabolites, mainly 2‐dimethyl‐4‐hydroxy‐5,6‐dimethylpyrimidine and 2‐methyl‐4‐hydroxy‐5,6‐dimethylpyrimidine, are normally monitored in urine. Propoxur carbamate is monitored as a parent compound in blood, while its metabolite (2‐isopropoxyphenol) is monitored in urine. Poisoning due to carbofuran and carbosulfan is monitored in appropriate biomatrices using their metabolites, mainly carbofuranphenol and carbosulfanphenol.

Organohalogen Agrochemicals Poisoning: Indicator Compounds and Monitoring Biomatrices

Agrochemicals that belong to the class of organohalogens that include polychlorinated biphenyls (PCBs) and pesticides such as dichlorodiphenyl tetrachloroethane (DDT), metabolites of DDT, such as DDD and DDE, hexachlorocyclohexane (HCH), dieldrin, hexachlorobenzene (HCB), chlordanes, endosulfan, dioxins, polybrominated diphenyl ethers (PBDE), etc. are known to have high lipophilicity property, which enables them to cross the lipophilic cell membranes of animal cells by the process of diffusion. These molecules have been found to be contaminating foodstuffs such as fruits, vegetables, honey, cow’s milk, human milk, fat tissues of animals, etc. The consumption of food items that are contaminated with these persistent molecules can lead to poisoning. The poisoning effects are aggravated by the fact that these molecules tend to bioaccumulate and bioconcentrate in the body system. There have been many deliberate poisoning cases that have resulted in health problems of varying magnitude to the victims, due to the fact that their fate when they are in an individual’s body system varies significantly, depending on the nature of either degradation taking place by metabolism or by the processes in various tissues such as lipid tissue where these molecules bioaccumulate (Letcher et al., 2000).

Organohalogen agrochemicals and their metabolites are known for their persistence as well as their potential adverse long‐term effects to human health in cases of poisoning. This makes it plausible to monitor their respective parent/intact compounds in addition to their metabolites in various matrices, mainly serum, breast milk, meconium, and umbilical cord (Margariti et al., 2007). For example, dichlorodiphenyltrichloroethane (p,p’‐DDT) is normally monitored both as an intact molecule and also in its metabolite forms such as dichlorodiphenyldichloroethylene (p,p’‐DDE). Due to their hydrophobicity, monitoring of the intact molecules and/or their respective metabolites in these biological matrices requires the experimental design to employ effective sample extraction and sample purification methods and very sensitive detection methods (Margariti et al., 2007).

Metabolism of Organohalogen Agrochemicals and their Respective Metabolites

Metabolism of organohalogens in the human/animal body system is known to be dependent on the presence and action of a number of microsomal enzymes. The bioactivity and induction of these microsomal enzymes can be indicated by the presence of 6‐β‐hydroxycortisol and D‐glucaric acid excretion in biomatrices such as urine from the victims. The challenge of using such enzymatic induction biomarkers as an indication of poisoning due to organochlorine lies in the fact that poisoning due to some other chemicals such as tranquilizers (e.g. barbiturates), alcohol, etc. are also known to result in the same induction biomarkers. For this reason, identification of intact organohalogens and/or their metabolites in biomatrices such as blood and its components or urine is always highly recommended.

Each intact organohalogen molecule is associated with certain metabolites that can be traced in specific biometrics. For example, hexachlorobenzene poisoning in humans is associated with the porphyria metabolite biomarker that is eliminated from the body via urine (Maroni et al., 2000).

Other organohalogen compounds, such as dichlorodiphenyl trichloroethane (DDT), has mainly 4.4’‐dichlorodiphenyldichloroethane as the main metabolite biomarker, which together with its parent/intact molecule (DDT), are analyzed in either urine, breast milk, blood, or adipose tissues. Heptachlor‐epoxide is a biomarker metabolite associated with heptachlor poisoning and both parent and metabolites of this compound are normally analyzed in milk, urine, serum, and adipose tissues. Endrin and its metabolite biomarker (anti‐12‐hydroxy‐endrine) are normally analyzed in the urine matrix, while aldrin poisoning yields dieldrin as a metabolite biomarker and both the metabolite and parent compound are analyzed in biomatrices such as serum, milk, and fat tissues. Organohalogen agrochemicals such as α‐ and β‐endosulfan (analyzed in serum), lindane, vinclozolin, and β‐HCH are analyzed in serum and breast milk. Metolachlor and its metabolite metolachlor mercapturate are also analyzed in the same biomatrices.

Generally, all other toxic organohalogens such as dichlorobenzene (with its metabolite 2,4‐dichlorophenol); p‐dichlorobenzene (with its metabolite 2,5‐dichlorophenol); chlorinated benzene (with its metabolites 2,4,5‐trichlorophenol and 2,4,6‐trichlorophenol); dioxins; polybrominated diphenyl ethers; and brominated flame retardants etc., are analyzed as both intact molecules and their respective metabolites in biomatrices such as urine, serum, and milk.

Pyrethroid Agrochemicals

Research has shown that in mammals, pyrethroid insecticides are normally rapidly metabolized into their respective carboxylic acids through a mechanism that involves hydrolytic cleavage of their ester bonds, then by oxidation processes such as glucuronidization (Figure 4.22). They are normally excreted through urine as conjugate metabolites (Dorman and Beasley, 1991; Soderlund et al., 2002). Other biological matrices such as blood and its components (i.e. serum) are normally not preferred as important biomatrices where pyrethroid metabolites can be found simply because the metabolic process is rapid metabolism and therefore levels of either intact molecules or their respective metabolites are at any time low as compared to that which may be found in urine (Margariti et al., 2007).

Figure 4.22 Proposed urinary metabolites of permethrin in mammals (Crawford et al., 1981; Miyamoto et al., 1988).

Pyrethroid insecticide intact compounds such as cyfluthrin, cypermethrin, and permethrin are normally monitored in their metabolite forms, which include cis‐ and trans‐3‐(2,2‐dichlorovinyl)‐2,2‐dimethylcyclopropane‐1‐carboxylic acids (cis‐/trans‐DCCA), while other pyrethroid herbicides such as deltamethrin are monitored as cis‐3‐(2,2‐dibromovinyl)‐2,2‐dimethylcyclopropane‐1‐carboxylic acid (DBCA) metabolites. Cyfluthrin is monitored as 4‐fluoro‐3‐phenoxybenzoic acid (4F3PBA), while a number of synthetic pyrethroids such as cypermethrin, deltamethrin, and permethrin are usually monitored in urine or milk in the form of their common metabolite 3‐phenoxybenzoic acid (3PBA) (Margariti et al., 2007).

Herbicides Poisoning

Scientific findings have revealed that phenoxy herbicides hardly undergo biotransformation in mammals and therefore monitoring of these compounds in cases of poisoning involves mainly parent/unmodified compounds, mainly in urine (Garry et al., 2001). Chloroacetanilide herbicides such as acetochlor are monitored in the form of the metabolites, mainly acetochlor mercapturate, while alachlor is monitored as alachlor mercapturate and metolachlor as mercapturate metolachlor. Atrazine herbicides are normally monitored in their atrazine mercapturate (AM) forms (Margariti et al., 2007).

Detection Methods for Food Forensic Specimens Suspected of Contamination with Agrochemical Molecules and/or their Metabolites in Various Biomatrices

From the above discussion, it follows that the most likely specimens that may be presented as evidence in forensic investigation due to food poisoning by agrochemical molecules or others will be biological fluids (urine, breast milk, blood and its components such as serum, saliva, and vomit), hair, nails, meconium, stool, etc.

The majority of these biomatrices are complex and interact with the analytes in various ways and to a various degree of bonding strengths. This therefore requires very selective sample preparation methods to isolate analytes of interest before subjecting them to analytical instruments for determination. A number of sample preparation methods, separation, and detection techniques have been reported for agrochemical molecules and their respective residues in various biomatrices, as outlined below.

Analytical Methods of Organophosphate Agrochemicals and their Residues in Human Urine Specimens

The urine matrix is one of the most used specimens in the determination of organophosphate agrochemical molecules and their metabolites. Different sample preparation approaches with different separation and detection techniques have been reported. For example, Barr et al. (2002) determined methyl parathion and its metabolite p‐nitrophenol where they reported sample preparation methods that involved enzyme hydrolysis followed by solvent extraction and then concentrated the sample by drying using anhydrous sodium sulfate (Barr et al., 2002). Enzyme hydrolysis in combination with solid phase extraction was also reported by Olsson et al. (2003) in the analysis of certain organophosphate biomarkers in urine before detection using high resolution LC‐MS/MS (Olsson et al., 2003).

Liquid–liquid extraction (solvent extraction) has been used alone and also in combination with solid phase extraction (florisil/PSA cartridge sorbent) as a sample preparation for dialkylphosphates and their metabolites (mainly dimethylphosphate, dimethylthiophosphate, dimethyldithiophosphate, diethylphosphate diethylthiophosphate, and diethyldithiophosphate) in urine samples (Dulaurent et al., 2006; Hardt and Angerer, 2000; Ueyama et al., 2006). Where the detection method involved GC‐EI‐MS, the derivatization step was also involved to ensure that the derivative was volatile enough for GC‐MS analysis.

In certain other reports, organophosphates (dimethylthiophosphate, diethylphosphate, diethylthiophosphate, and diethyldithiophosphate) have been analyzed using direct injection onto high resolution LC‐MS/MS (Hernandez et al., 2002, 2004). Mainly dimethylphosphate and dimethylthiophosphate, and other compounds were analyzed. In other reports, azeotropic distillation, chloropropylation, and concentration of dialkyl organophosphates and their metabolites (mainly dimethylphosphate, dimethylthiophosphate, dimethyldithiophosphate, diethylphosphate diethylthiophosphate, and diethyldithiophosphate) were used as sample preparation methods before determination using high resolution GC‐MS/MS, where the use of isotopic internal standards were employed in quantitation steps (Bravo et al., 2002). Lyophilization in combination with solvent extraction using methyl cyanide (acetonitrile) and diethyl ether, followed by chloropropylation concentration was the method for sample preparation before detecting dimethylphosphate, dimethylthiophosphate, dimethyldithiophosphate, diethylphosphate diethylthiophosphate, and diethyldithiophosphate, using high resolution GC‐MS/MS, which was operated in the positive chemical ionization mode (Bravo et al., 2004).

Analytical Methods of Organophosphate Agrochemicals and their Residues in Human Serum Specimens

Protein precipitation has been reported as a sample preparation method in the analysis of chlorpyrifos and its major metabolite (3,5,6‐trichloro‐2‐pyridinol) in human serum and urine, where high resolution LC‐MS/MS was used for separation and detection of these molecules (Sancho et al., 2000). Solid phase extraction has been used as a sample preparation in the multiresidue analysis that involved organochlorine and organophosphates in human serum, where GC‐MS was used for separation and detection purposes (Pitarch et al., 2003).

Analytical Methods of Organophosphate Agrochemicals and their Residues in other Biomatrices Specimens

Other biomatrices where organophosphates (dimethylphosphate, dimethylthiophosphate, dimethyldithiophosphate, diethylphosphate diethylthiophosphate, and diethyldithiophosphate) are analyzed, include meconium and amniotic fluid. For meconium, the sample preparation method involving a combination of lyophilization, and solvent extraction using methanol and chloropropylation was reported by Whyatt and Barr (2001), where they measured metabolites of certain organophosphates in postpartum meconium. The use of amniotic fluid in the analysis of biomarkers associated with organophosphorus agrochemical poisoning was reported by Bradman et al. (2003), where the sample preparation method of choice for dimethylphosphate, dimethylthiophosphate, dimethyldithiophosphate, diethylphosphate, diethylthiophosphate, and diethyldithiophosphate involved azeotropic distillation followed by chloropropylation and concentration prior to detection using high resolution GC‐MS/MS that was operated in the positive chemical ionization mode.

Analytical Methods of Organochlorine Agrochemicals and their Residues in Human‐related Biomatrices

The majority (if not all) of the organochlorine agrochemicals and their respective metabolites are very hydrophobic and characterized with long half‐lives in the biomatrices. This suggests that some of the biomatrices such as urine will be of little importance when considering specimen collection for analyses intended to find evidence of poisoning. The method of organochlorine detection has mainly been either GC‐MS or GC‐ECD.

Analytical Methods of Organochlorine Agrochemicals and their Residues in Human Milk Specimens

The sample preparation approaches for organochlorine and their respective metabolites (mainly dichlorodiphenyldichloroethylene (p,p‐DDE), o,p‐dichlorodiphenyltrichloroethane (o,p‐DDT), dichlorodiphenyltrichloroethane (p,p‐DDT), mirex, and dieldrin in milk) involve a combination of solvent extraction (using appropriate solvents for appropriate compounds) and solid phase extraction (mainly florisil sorbent) (Burke et al., 2003; Campoy et al., 2001).

Analytical Methods of Organochlorine Agrochemicals and their Residues in Human Serum Specimens

A sample preparation method utilizing a combination of solid phase extraction and solvent extraction (methanol:dichloromethane) and clean‐up using silica and gel permeation chromatography for a multiresidue analysis of a mixture of PCBs and chlorinated pesticide molecules (mainly dichlorodiphenyldichloroethylene (p,p‐DDE); o,p‐dichlorodiphenyltrichloroethane (o,p‐DDT); dichlorodiphenyltrichloroethane (p,p‐DDT); mirex; dieldrin; heptachlor epoxide; oxychlor; trans‐nonachlor; hexachlorobenzene (HCB); gamma‐hexachlorocyclohexane (lindane, γ‐HCH); and beta‐hexachlorocyclohexane (β‐HCH)) in serum has been reported by Barr et al. (2003). A combination of lyophilization and acceleration solvent extraction with clean‐up using gel permeation for PCBs and agrochemicals and their metabolites (dichlorodiphenyldichloroethylene (p,p‐DDE); o,p‐dichlorodiphenyltrichloroethane (o,p‐DDT); dichlorodiphenyltrichloroethane (p,p‐DDT); mirex; dieldrin; heptachlor epoxide; oxychlor; trans‐nonachlor; hexachlorobenzene (HCB); gamma‐hexachlorocyclohexane (lindane, γ‐HCH); and beta‐hexachlorocyclohexane (β‐HCH)) in serum was reported by Barr et al. (2003).

Čonka et al. (2005) used solid phase extraction (SPE) and liquid–liquid extraction (dichloromethane:hexane) and clean‐up with florisil/silica gel column as sample preparation methods before GC‐ECD detection of a mixture of PCBs and organochlorines (mainly dichlorodiphenyldichloroethylene (p,p‐DDE); dichlorodiphenyltrichloroethane (p,p‐DDT); hexachlorobenzene (HCB); gamma‐hexachlorocyclohexane (lindane, γ‐HCH); beta‐hexachlorocyclohexane (β‐HCH); and alpha‐hexachlorocyclohexane (α‐HCH)) (Čonka et al. 2005).

Lacassie et al. (2001) employed solid phase extraction for serum specimen and eluted the sorbed PCBs and organochlorines (dichlorodiphenyldichloroethylene (p,p‐DDE); dichlorodiphenyltrichloroethane (p,p‐DDT); dieldrin; hexachlorobenzene (HCB); gamma‐hexachlorocyclohexane (lindane, g‐HCH); a‐endosulfan; b‐endosulfan; and aldrin) using ethyl acetate prior to GC‐EI‐MS, while Sundberg et al. (2006) eluted the same classes of chlorinated compounds (mainly dichlorodiphenyldichloroethylene (p,p‐DDE); hexachlorobenzene (HCB); aldrin; chlordane; endrin; and dichlorodiphenyldichloroethane (p,p‐DDD)) using methylene chloride prior to GC‐ECD detection.

Analytical Methods of Organochlorine Agrochemicals and their Residues in Meconium Specimens

A method involving solvent extraction of the organochlorine metabolite, dichlorodiphenyldichloroethylene (p,p‐DDE), followed by filtration and concentration prior to GC‐EI‐MS has been reported by Hong et al. (2002).

Analytical Methods of Multiresidue Analysis of Miscellaneous Agrochemicals and their Metabolites in Various Biomatrices

Methods for the analysis of different classes of herbicides, insecticides, fungicides, molluscicides, rodenticides, etc. in various biomatrices have been developed. For example, Baker et al. (2000) and Bradman et al. (2003) reported the analysis of pyrethroid metabolites (2,4‐dichlorophenoxyacetic acid (2,4‐D); atrazine mercapturate (AM); malathion dicarboxylic acid (MDA, malathion metabolite); and 2‐isopropyl‐4‐methyl‐6‐hydroxypyrimidine (IMPY, diazinon metabolite)) in urine and amniotic fluid biomatrices respectively. In both reports, the sample preparation method involved enzyme hydrolysis and solvent extraction, while the detection was by LC‐APCI‐MS/MS.

Corrion et al. (2005) and Bielawski et al. (2005) reported the analysis of pyrethroid metabolites (pyrethroids, 3,5,6‐trichloro‐2‐pyridinol (TCPY) and methyl/ethyl chlorpyrifos metabolite), organophosphates, carbamates and/or metabolites, chloroacetanilides, organochlorine pesticides, and malathion monocarboxylic acid (MMA, malathion metabolite)) in blood and meconium biomatrices respectively. In the report by Corrion et al. (2005), the sample preparation method involved the use of solvent extraction and derivation prior to GC‐EI‐MS, while Bielawski et al. (2005) used the solid phase extraction method and the sample was analyzed using GC‐MS. In another report by Barr et al. (2002), a multiresidue mixture comprised of pyrethroids (N,N‐diethyl‐m‐toluamide (DEET), atrazine, organophosphates, carbamates and/or metabolites, and chloroacetanilides in serum and plasma biomatrices). Solid phase extraction sample preparation method used methyl chloride as the elution solvent prior to detection using high resolution GC‐MS.

The analysis of 2,4‐dichlorophenoxyacetic acid (2,4‐D), 2,4,5‐trichlorophenoxyacetic acid (2,4,5‐T), acetochlor mercapturate, alachlor mercapturate, metolachlor mercapturate, and atrazine mercapturate (AM) in urine was reported by Norrgram et al. (2006), where solid phase extraction with methanol elution was employed before detection by high resolution LC‐APCI‐MS/MS.

Generally, from the majority of the reports, enzyme hydrolysis, acid/base hydrolysis, solvent extraction, or solid phase extraction have been widely used as sample preparation methods and detection with either GC‐MS or LC‐MS have been the principal techniques.

Conclusions

The knowledge of the pathways of various agrochemical residues after consuming foods that are contaminated with these molecules is crucial for a number of reasons, as it will guide the analyst as to which are the best sample specimens to collect. It will also be helpful for the analyst to estimate the time needed from poisoning to analysis and which metabolites to expect from the various possible specimens (urine, milk, blood, serum, etc.). In addition to this, the choice and knowledge of the analytical instrument to use for analysis is important, as it will result in the correct analysis of the residues, both qualitatively and quantitatively.

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