**4.1. Chemistry of lead**

Pb is a heavy metal that has malleability, low melting point, low electrical conductivity, and high corrosion resistance. These properties allow its widespread use in the manufacture of blades and pipes of high flexibility and resistance in welds and coatings in the automotive industry; protective plates against ionizing radiation (e.g. X-rays); alloys; coating cables; and paints, dyes, and plastic additives [72]. Usually, inorganic Pb compounds are found as Pb(II) and rarely found as a pure element. Its most common mineral is galena or lead sulfide (PbS). The solubility of Pb compounds is enhanced at lower pH, suggesting that the increased mobility of the Pb is found in ecosystems under stress acidification [73].

#### **4.2. Occurrence in the environment**

Pb is a metal that occurs naturally, making up only about 0.0013% of the earth's crust. However, most Pb concentrations that are found in the environment are the result of human activities such as burning of fossil fuels and mining [74]. Pb can be found in the atmosphere in particulate form, being deposited in water systems, interfering with the characteristics of the water. In other cases, this metal may be found complexed with natural organic compounds [75]. In contact with the ground, Pb can remain for a long time and in various forms (such as insoluble and soluble complexes and colloids) and absorbed by plants, accumulating in the edible parts, causing contamination in humans and animals [74–78].

#### **4.3. Dietary sources of lead**

The WHO and the Expert Committee on Food Additives—"JECFA" initially established a provisional tolerable weekly intake (PTWI) for lead of 50 μg kg−1 body weight for adults. However, after assessing the risk to health, the JECFA later reduced this value to 25 μg kg−1 body weight, equivalent to 3.5 μg kg−1 body weight per day (equal to 1.75 mg week−1 or 1750 μg week−1 for a person weighing 70 kg) [20, 21].

The Expert Committee noted, however, that some foods with high levels of Pb remain commercially available [22]. A reference value for Pb of 0.01 mg L−1 in drinking water was established by the WHO. The concentrations in drinking water are typically below 5 μg L−1, although higher concentrations (above 100 mg L−1) have been reported. The EPA regulations establish limits in the form of maximum contaminant levels (MCLs), and the value for Pb in drinking water is 0.015 mg L−1, even though the EPA has also established a goal for zero Pb in this regard [23].

#### **4.4. Routes of entry into plants, animals, and humans**

The main routes of human exposure to Pb are by ingestion (food, water, and soil), inhalation, and skin [79]. The compounds of tetra-alkyl Pb (Pb tetra acetate, etc.), for example, are rapidly absorbed through the lungs, gastrointestinal tract, and also the skin. Usually, a high level of metal enters the body through the ingestion of contaminated cereals and vegetables [80]. Once absorbed, inside the body Pb is distributed by the blood reaching the soft tissue and then is deposited in the bones and other hard body parts. It is slowly excreted in urine and feces [79].

#### **4.5. Metabolism or transformation in the living system**

In the human body, Pb is not metabolized, but it forms complexes with macromolecules. Pb forms complexes (sulfur groups, –SH) through covalent bonds, causing the intoxication of humans [81]. Pb can disturb the metabolic functions in two ways: (1) it accumulates, thereby disrupting the function of vital organs and glands such as the heart, brain, kidney, bone, liver, so on and (2) it moves the vital nutritional minerals from their original location, hindering their biological function [82].

#### **4.6. Biological functions**

Pb is a toxic metal that would not have known beneficial effects to the body, and its accumu‐ lation over time in the bodies of animals and humans can cause severe illness [83].

#### **4.7. Mechanisms of toxicity of lead**

One of the main reasons by which Pb exerts toxic effect is its ability to substitute diverse cations (calcium, zinc, and magnesium) in their binding sites. Pb has a greater affinity than calcium and zinc ions to protein-binding site because of its larger ionic radius and greater electrone‐ gativity. For example, Pb interacts with oxygen and sulfur binds to sulfhydryl and amide groups of enzymes, altering their configuration and diminishing their activities, and competes with calcium in skeletal tissue and to interact with proteins [84].

In the blood, Pb is distributed to the remaining tissues, where it accumulates; the amount of metal accumulated depends mainly on the vasculature and metabolic characteristics of each tissue [85]. The half-life of Pb is 35 days in the blood and is about 2 years in the brain, and it can last for decades in bone.

Many investigators have demonstrated that Pb affects biomolecules and hence physiological systems. For example, calmodulin is a protein found primarily in the brain and heart. The binding of calcium ions of this protein allows the binding of this protein to cyclic nucleotide phosphodiesterase and adenylate cyclase with subsequent activation. Thus, this protein modulates the levels of AMP and cyclic GMP [86]. Pb is a more potent activator than calcium for calmodulin. According Kern, Pb modifies several signaling cascades and proteins that participate in the vesicular cycle [87]. The alterations caused by the abnormal protein opera‐ tion in second messenger systems and exocytic processes greatly contribute to Pb neurotoxici‐ ty [87].

Pb affects various cellular organelles, for example, mitochondria and endoplasmic reticulum, in different ways. In the mitochondria Pb affects energy metabolism, while in the endoplasmic reticulum Pb increases the cytoplasmic concentration of calcium with a consequent reduction in ion concentration inside this organelle. Many signaling pathways that are within the endoplasmic reticulum are calcium dependent; because the amount is not appropriate, various processes are impaired. Besides, Pb binds to Ape1 nuclease, whose function is to detect and repair DNA damage, inhibiting its operation and allowing the accumulation of mutagenic damages [85–88].

#### **4.8. Incidence of (acute and chronic) toxicity**

Pb is one of the most common environmental contaminants. This element has no known physiological function in the organism, and its damaging effects can affect almost every organ and system in the body [89]. The main way in which Pb enters the body is through the respiratory route (occupational exposure), followed by the digestive route. Organic Pb compounds can penetrate in the body through skin contact and are rapidly absorbed [90, 91].

Exposure to Pb can result in a wide variety of biological effects, depending on the exposure level and duration. The major diseases related to Pb contamination are shown in Table 2. Pb is toxic to various organs and systems, and its effects may vary from enzyme inhibition and anemia to diseases of the nervous, immune, reproductive, and cardiovascular systems, impaired kidney function, and even death.

Studies have suggested an association between Pb exposure and lung cancer and, to a lesser extent, stomach cancer [90]. Pb is hypothesized to be a carcinogen and to enhance the genotoxic effects of other agents. Renal tumors developed in mice that had received high doses of certain Pb compounds and various other animal studies have shown increases in the yield or geno‐ toxicity of known carcinogens. The US Environmental Protection Agency has determined that Pb is a probable human carcinogen [89–91].


**Table 2.** Main health effects related to lead contamination [89–91].

#### **4.9. Comparative analysis of analytical techniques**

The determination of traces of Pb in various food samples is of great importance because Pb is recognized as a cumulative poison in humans and other animals [92]. The determination of Pb requires procedures that are sufficiently sensitive for detection at the pg L−1 level. Tradi‐ tionally, GF-AAS has been applied in such cases, but the direct determination of Pb in complex matrices is usually difficult owing to matrix interference and separation procedures often being required before the sample analysis [93]. The ICP-MS technique is favored because of its low detection limits [93], although many researchers prefer AAS owing to its simpler and less expensive instrumentation. Lead hydride generation and its application to spectrometry analysis have been reviewed by Madrid and Cámara [94]. HG-AAS is a well-developed technique that can be used for the determination of volatile hydride-forming elements such as arsenic, selenium, antimony, and others at trace levels [95]. The advantages of HG-AAS over other atomic absorption spectrometric techniques such as the flame and graphite furnace methods are increase in atomization efficiency and higher selectivity because the analyte is removed from the matrix as a volatile compound and detection limits at the pg L−1 level or lower for the elements cited above. Considering these advantages, this technique could be applied for the determination of Pb, and it is possible to include this element in multi-element analysis schemes involving hydride generation.

The generation of Pb hydride was described by Carrijo et al. [96]. In this study, a flow injection– hydride generation–atomic absorption spectrometry (FI-HG-AAS) system was used for Pb determination. The main characteristics of the flow injection system, that is, high sampling rate and good accuracy, precision, and sensitivity, are maintained.

#### **5. Mercury**

#### **5.1. Chemistry of mercury**

Hg is a metal found in various chemical forms, which can be divided into the following categories: elemental or metallic Hg, inorganic Hg, mainly in the form of mercuric salts (HgCl2 and HgS), and mercuric (Hg2Cl2) and organic Hg, for example, methylmercury and ethylmercury [68, 72].

Metallic mercury (Hg) is in the liquid state at room temperature and easily volatilizes into the atmosphere forming Hg vapors. Hg is a metal with widespread use, especially the production of scientific precision instruments, electrical industry, dentistry (production amalgams), the production of certain types of toys, mining, metal smelting, among others [97].

#### **5.2. Occurrence in the environment**

Hg is a metal found naturally in the earth's crust, occurring in air, soil, and water [98]. It rarely occurs free in nature and is found mainly in cinnabar ore (HgS). It can be found in metal form, as salts of Hg or organic Hg compounds. Once released, Hg remains in the environment among the circulating air, water, sediment, soil, and biota, which assumes various chemical forms.

Most emission to air occurs in the form of elemental Hg, which is very stable and can remain in the atmosphere for months or even years, enabling transport over long distances around the globe [98]. Most of the Hg released by human activities in air is by combustion of fossil fuels, mining, smelting, and combustion waste [99].

The Hg vapor in the atmosphere can be deposited or is converted into the soluble form, returning to the earth's surface in rainwater. From there, two important chemical changes may occur. The metal can be cast again and return the Hg vapor in the atmosphere or may be "methylated" by the microorganisms present in the water sediments, turning into methyl‐ mercury [98]. Furthermore, the Hg can also be released directly in the soil or in water by the application of agricultural fertilizer and disposal of industrial waste water [100].

Atmospheric emissions are the major source of environmental contamination, followed by water pollution and soil contamination, when there is improper disposal of effluents and waste [98].

#### **5.3. Dietary sources of mercury**

Usually, Hg contamination occurs by the presence of this metal in water, soil, air, or food, mostly in the form of methylmercury [100, 101]. The most important source of exposure through diet for the general population is the consumption of fish and other marine organisms. Hg is concentrated in the tissue of fish, becoming increasingly potent in predatory fish and mammals that feed on small fish. The larger carnivorous fish have higher concentrations than smaller ones [99]. The average daily intake of methylmercury (mainly from fish) that can cause demonstrable effects on the health of sensitive individuals is 300 mg day−1 or 4.3 μg Hg day−1 kg body weight−1 [102].

Industrial products can also be contaminated by Hg during the processing steps. Studies have shown the contamination of Hg in breast milk (4–15 μg kg−1) [103], in tea (6 ng g−1) [104], and in products for infant feeding (0.50 μg kg−1) [105].

#### **5.4. Routes of entry into plants, animals, and humans**

The absorption of Hg by humans and animals can be by pulmonary route (inhalation), as well as by gastrointestinal or cutaneous route. In the case of pulmonary route, Hg after inhalation and the presence in the lung is distributed throughout the body, accumulating in various parts of body [106–108]. Soluble compounds are absorbed by mucous membranes following vapor inhalation and by the skin and the sebaceous glands. In the body, organic and inorganic Hg binds GSH [108]. It acts as inactivator because it readily binds to thiol groups of cellular enzymes and disrupts its function by inactivating the metabolism. The non-absorbed Hg is excreted in feces, and absorbed Hg forms are excreted via saliva and skin.

#### **5.5. Metabolism or transformation in the living system**

Hg and its organic compounds in low concentrations cause damage to human health. Their concentrations in surface and ground waters are below 0.5 mg L−1. However, aquatic micro‐ organisms convert the organic Hg into inorganic Hg compounds, which accumulate in the food chain. Methylmercury is the most relevant toxicant [109]. The gastrointestinal tract is the second way (after airway) through which Hg, already now in its organic form, enters the human body through the consumption of fish, shellfish, and other aquatic organisms.

#### **5.6. Biological functions**

Hg has no biological role. All Hg compounds are extremely toxic, particularly methylmer‐ cury [109].

#### **5.7. Mechanisms of toxicity of mercury**

The mechanism of toxicity of Hg is based on its chemical activity and biological features. The main mechanism of toxicity of Hg compounds involves their reactivity with sulfhydryl groups. Once in the cell, both Hg2+ and MeHg form covalent bonds with cysteine residues of proteins and deplete cellular antioxidants [110].

#### **5.8. Incidence of (acute and chronic) toxicity**

Metallic Hg and its organic compounds in very low concentrations cause damage to human health (such as neurotoxic, immunotoxic, and teratogenic properties) and can have high persistence and a high bioconcentration factor (BCF), accumulating in animals, fish, and the environment. Hg poisoning levels and the main symptoms and diseases related to acute and chronic poisoning by Hg are shown in Tables 3 and 4, respectively [111].


**Table 3.** Mercury poisoning levels.



**Table 4.** Main symptoms and diseases related to acute and chronic poisoning by mercury.

#### **5.9. Comparative analysis of analytical techniques**

The determination of Hg in food samples is critical to assess the degree of human exposure, and thus reliable analytical techniques with high sensitivity are required. However, in most situations, the determination of Hg species is not an easy task due to low concentrations in the samples and the characteristic volatility [112, 113]. The volatility of Hg requires special consideration when treating the sample. Food sample preparation using a microwave oven has been widely employed [114].

In the case of the quantification of methylmercury in fish samples and seafood, depending on the nature of the sample and the technique used, an additional pre-concentration step is required. Hg determination has been performed using cold vapor coupled to atomic absorption spectrometry (CV-AAS), cold vapor coupled to atomic fluorescence spectrome‐ try (CV-AFS), inductively coupled plasma optical emission spectrometry (CV-ICP-OES), and inductively coupled plasma mass spectrometry (CV-ICP-MS). Hyphenated techniques involving gas or liquid chromatography separations with detection by element-specific detectors such as ICP-MS and atomic absorption/emission are the most commonly report‐ ed [114].
