Analysis of Endocrine Disrupting Compounds in Food

By Leo M. L. Nollet

Analysis of Endocrine Disrupting Compounds in Food provides a unique and comprehensive professional reference source covering most of the recent analytical methodology of endocrine disrupting compounds in food. Editor Nollet and his broad team of international contributors address the most recent advances in analysis of endocrine disrupting chemicals in food. While covering conventional (typically lab-based) methods of analysis, the book focuses on leading-edge technologies that recently have been introduced. The book looks at areas such as food quality assurance and safety. Issues such as persistent organic pollutants, monitoring pesticide and herbicide residues in food, determining heavy and other metals in food and discussing the impacts of dioxins, PCBs, PCDFs and many other suspected chemicals are covered.

The book discusses the relationship between chemical compounds and hormone activity. What are the health impacts of different chemical compounds for men and animals? How are these compounds entering in foodstuffs? Analysis of Endocrine Disrupting Compounds in Food offers the food professional what its title promises – a compendium of sample preparation and analysis techniques of possible endocrine disrupting compounds in food.

Special Features:

• Uniquely concentrates on analysis and detection methods of EDCs in foodstuffs
• Extensive coverage of the main types of globally available analytical techniques and methodologies
• Fully detailed properties, sample procedures, and analysis steps for each EDC
• Renowned editor Leo Nollet leads a broad team of international experts

• Language: English
• Publisher: Wiley
• Release date: Jun 9, 2011
• ISBN: 9780470961841

Book preview

Chapter 1

Endocrine Disrupting Chemicals. What? Where?

Guang-Guo Ying

Introduction

There is a concern that some natural and synthetic chemicals can interfere with the normal functioning of endocrine systems, thus affecting reproduction and development in wildlife and humans. These chemicals are called endocrine disruptors or endocrinedisrupting chemicals (EDCs). Although endocrine disruption has been known since the 1930s (Dodds et al. 1938), this issue has regained attention and generated immense scientific and public interest since 1992 (Colborn and Clement 1992), and especially since the publication of the book Our Stolen Future (Colborn et al. 1996). The chemicals identified or suspected as being endocrine disruptors in the literature include pesticides (e.g., dichlorodiphenyltrichloroethane [DDT], dichlorodiphenyldichloroethylene [DDE], dieldrin, endosulfan), pharmaceuticals (e.g., diethylstilbestrol [DES]) and industrial chemicals or pollutants (e.g., polychlorinated biphenyls [PCBs], dioxins, bisphenol A) (Table 1.1). Since then, many studies have been carried out on endocrine disruption. Some reproductive problems in wildlife and humans have been linked to exposure to these chemicals. Wildlife and humans are exposed daily to these pervasive chemicals that have already caused numerous adverse effects in wildlife and are most likely affecting humans as well.

There is compelling evidence regarding the effects of exposure to EDCs on wildlife (Damstra et al. 2002 ). These include imposex of mollusks by organotin compounds (Alzieu 2000; Gibbs et al. 1990; Horiguchi et al. 1994); developmental abnormalities, demasculinization and feminization of alligators in Florida by organochlorines (Guillette et al. 1994, 2000); and feminization of fish by wastewater effluent from sewage treatment plants and paper mills (Table 1.2 ) (Jobling et al. 1 998; Bortone et al. 1989). There is also evidence that human testicular and breast cancer rates have increased during the last four decades, especially in developed countries (Brown et al. 1986; Hakulinen et al.

1986; Adami et al. 1994; Feuer 1995; Moller 1993; Ries et al. 1991; Wolff et al. 1993). However, except in a few cases (e.g., DES), a causal relation between exposure to chemicals and adverse health effects in humans has not been firmly established. Owing to the scientific evidence and public concern about potential effects on humans and wildlife, the U.S. Congress made amendments to the Safe Drinking Water Act (SDWA) in 1996 and required the U.S. Environmental Protection Agency (U.S. EPA) to develop a screening program for endocrine disruptors (Fenner-Crisp et al. 2000). In April 2000, a meeting of the environment ministers of the G8 group of industrialized countries listed EDCs as one of the high priorities and called for a furtherance of knowledge acquisition on EDCs through jointly planned and implemented projects and international information sharing (Loder 2000). Surveys of some new emerging endocrine disrupting chemicals (e.g., nonylphenol and steroids) in major rivers of some countries have been undertaken (e.g., Naylor et al. 1992; Blackburn et al. 1999; Ahel et al. 2000; Tabata et al. 2001; Kolpin et al. 2002). The U.S. EPA and the Organization of Economic and Cooperative Development (OECD) have invested considerable resources to develop tiered procedures for the testing and assessment of EDCs (Fenner-Crisp et al. 2000; Huet 2000; Parrott et al. 2001). The U.S. EPA planned to screen 15,000 chemicals for their possible effects as endocrine disruptors in animals and humans (Macilwain 1998).

Table 1.1. List of suspected/known EDCs.

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This chapter will give some background information about the endocrine disruption issue, EDCs in food, and potential effects associated with exposure to EDCs.

Table 1.2. Effects associated with exposure to EDCs in wildlife (representative examples).

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Endocrine-disruption chemicals

Endocrine system

An endocrine system is found in nearly all animals, including mammals, nonmammalian vertebrates (e.g., fish, amphibians, reptiles and birds) and invertebrates (e.g., snails, lobsters, insects, and other species). Along with the nervous system, the endocrine system is one of the two communication systems that regulate all responses and functions of the body. The endocrine system consists of glands and the hormones they produce that guide the development, growth, reproduction, and behavior of humans and animals. The major endocrine glands of the body include the pituitary, thyroid, parathyroids, adrenals, pancreas, pineal gland, and gonads (ovaries in females and testes in males). Hormones are biochemicals that are produced by endocrine glands in one part of the body, travel through the bloodstream, and cause responses in other parts of the body. They act as chemical messengers and interact with specific receptors in cells to trigger responses and prompt normal biological functions such as growth, reproduction, and development.

Hormones generally fall into four main categories: (1) amino acid derivatives, (2) proteins, (3) steroids, and (4) eicosanoids (Lister and van der Kraak 2001 ). The unifying nature of hormone action is the presence of receptors on target cells, which bind a specific hormone with high affinity and stereospecificity. Steroid and thyroid hormones act by entering target cells and stimulating specific genes. All other hormones bind to receptors on the cell surface and activate second-messenger molecules within the target cells (Raven and Johnson 1999). The body has hundreds of different kinds of receptors; each one is designed to receive a particular kind of chemical signal. The hormone and its receptor have a lock-and-key relationship (Figure 1.1). When a hormone encounters its receptor, they grab hold, engaging in a molecular embrace known as binding . Once joined, the hormone molecule and its receptor trigger the production of particular proteins that turn on the biological activity associated with the hormone. The actions of hormones have two types: organizational and activational (Lister and van der Kraak 2001). The first type of action occurs during critical periods of development and induces permanent effects such as the actions of sex steroids. The second type of action causes only transient changes in a myriad of cellular processes such as the effects of glucagon and insulin on glucose homeostasis. Organizational actions are more important in terms of effect with respect to environmental contaminants (Guillette et al. 1996). Timing of hormone release is often critical for normal function, especially during fetal development (Palanza et al. 1999 ).

Figure 1.1. Functioning of hormone system. VTG, vitellogenin; ER, estrogen receptor; Hsp90, heat shock protein 90 kDa; ERE, estrogen response element.

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Endocrine disruption

The Society of Environmental Toxicology and Chemistry (SETAC) defined endocrine disruption as follows: Synthetic, and naturally occurring, chemical substances in the environment are disrupting the normal functions of the endocrine system and its hormones in humans and wildlife (SETAC 2000). This hypothesis has received much attention in recent years because there is increasing evidence that some chemicals in our environment disrupt the endocrine systems in wildlife as well as humans.

There are several ways that chemicals can interfere with the endocrine system (Sonnenschein and Soto 1998). They can mimic or block natural hormones and alter hormonal levels, thus affect the functions that these hormones control. Less direct disruption involves alteration of the body’ s ability to produce hormones, interference with the ways hormones travel through the body, and changes in numbers of receptors. Regardless of the situation, having too much or too little of the hormones it needs may cause the endocrine system to function inappropriately. Very subtle disruptions of the endocrine system can result in changes in growth, development, or behavior that can affect the organism itself or the next generation (Guillette et al. 1 996; vom Saal et al. 1997; Palanza et al. 1 999).

Hormones play a crucial role in the proper development of the growing fetus. Embryos and fetuses are especially sensitive at particular times to low doses of endocrine disruptors (Guillette et al. 1 996; vom Saal et al. 1997; Palanza et al. 1999 ). Substances that have no effect in an adult can become poisonous in the developing embryo. The timing of exposure may be more important than the dose of the substance. The ultimate effects of endocrine disruption might not be seen until later in life or even until the next generation (Colborn et al. 1996; U.S. EPA 1997).

Endocrine disruptors

Endocrine disruptors have received growing attention in public media and the scientific community due to their potential impacts on humans and wildlife. There are several definitions used by scientists and policy makers. In the Organization of Economic and Cooperative Development (OECD), an endocrine-disrupting chemical has been defined as an exogenous substance or mixture that alters the function(s) of the endocrine systems and consequently causes adverse health effects in an intact organism or its progeny or (sub) populations (Lister and Van Der Kraak 2001). An environmental endocrine disruptor was also broadly defined by the U.S. EPA as an exogenous agent that interferes with the production, release, transport, metabolism, binding, action, or elimination of natural hormones in the body responsible for maintenance of homeostasis and the regulation of developmental processes (Kavlock et al. 1996). A potential endocrine disruptor can be simply defined as a substance that possesses properties that might be expected to lead to endocrine disruption in an intact organism.

An extensive list of the chemicals (Table 1.1) (Colborn et al. 1996; Guillette et al. 1996; Sonnenschein and Soto 1998; U.S. EPA 1997 ; Depledge and Billninghurst 1999 ) that have been found to be or are suspected to be capable of disrupting the endocrine systems include many pesticides that are designed to be bioactive (e.g., DDT, vinclozolin, tributyltin [TBT], atrazine), persistent organochlorines (e.g., PCBs, dioxins and furans), alkyl phenols (e.g. nonylphenol and octylphenol), heavy metals (e.g., cadmium, lead, mercury), phytoestrogens (e.g., isoflavoids, lignans, β-sitosterol), and synthetic and natural hormones (e.g., β-estradiol, ethinyl estradiol). Many of these compounds have little in common structurally or in terms of their chemical properties, but they evoke agonist or antagonist responses, possibly through comparable mechanisms of action. These chemicals are released from a wide variety of sources such as intensive agriculture, industrial wastes, mining activity, domestic sewage, and landfills. Suspected EDCs can be found in every division of our environment (air, water, soil, sediment, and biota), in industrial products and household items, and even in the food we eat. They are often found in mixtures, such as effluents from sewage treatment plants, paper mills, and textile factories. It is not clear whether the components in a mixture act additively, synergistically, or antagonistically.

EDCs can be classified into the following categories:

1. Environmental estrogens, for example, methoxychlor, bisphenol A

2. Environmental antiestrogens, for example, dioxin, endosulfan

3. Environmental antiandrogens, for example, vinclozolin, DDE, kraft mill effluent

4. Toxicants that reduce steroid hormone levels, for example, fenarimol and other fungicides, endosulfan

5. Toxicants that affect reproduction primarily through effects on the central nervous system (CNS), for example, dithiocarbamate

6. Toxicants that affect hormone status, for example, cadmium, benzidine-based dyes (Depledge and Billinghurst 1999)

The chemistry of the potential endocrine species far from their original source. In disruptors varies greatly, as does potency, that is, the effectiveness in binding and turning on the response. Most endocrine disruptors have very low potency because their chemistry is significantly different from the hormones they mimic. In addition to potency, the potential for a hormonelike effect depends on dose. For most of the endocrine disruptors, the dose-response relationship has not yet been established, especially at the low-dose range, and this may differ from species to species.

The risk of endocrine disruptors to humans and wildlife also depends on their behavior and fate in the environment. Chemicals behave differently in different media. For example, nonylphenol had a dissipation half life of ≤1 .2 days in the water column, 28 104 days in sediment, and 8–13 days on macrophytes in an experimental littoral ecosystem (Liber et al. 1999 ). Some EDCs (e.g., DDT and PCBs) are ubiquitous and persistent in the environment (Atlas and Giam 1981). They accumulate in the fatty tissue of organisms and increase in concentration as they move up through the food web (biomagnification). Because of their persistence and mobility, they accumulate in organisms and harm species far from their original source. In order to assess the risks, it is necessary to carry out monitoring of those chemicals possessing endocrine-disrupting characteristics in environmental media and foods we eat.

Human exposure of e ndocrinedisrupting c hemicals

Endocrine-disrupting chemicals need to enter an organism before they can disrupt its endocrine system. Humans can be exposed in a variety of ways (Figure 1.2 ): the food we eat, the air we breathe and the particles or vapors it contains, the pharmaceuticals we ingest for medical reasons, the water we drink, the soil we accidentally or intentionally eat, and in utero exposure from the mother’ s body burden (Crisp et al. 1998). For fat-soluble chemicals such as PCBs, for example, food is the major source for people. Dairy products, meat, and processed foods are all major contributors. Breast milk is also a contributor (Table 1.3). These fat-soluble chemicals remain in the body for a long time, and their accumulation early in life contributes significantly (approximately 15%) to the adult body burden (Patandin et al. 1999).

Figure 1.2. Routes of human exposure to chemicals.

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Table 1.3. Comparison of levels of selected organochlorines ( μ g/g fat) in breast milk of women from Australia and other countries.

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a Concentration range and mean in parentheses.

HCH, hexachlorocyclohexane isomers (α, β-HCH); HCB, hexachlorobenze; Σ DDT, dichlorodiphenyltrichloroethane (DDT and its metabolite DDE); PCBs, polychlorinated biphenyls.

Various endocrine-disrupting chemicals have been detected in foods, including persistent organic pollutants such as polychlorinated biphenyls and organochlorine pesticides, and emerging contaminants such as 4-nonylphenols, sunscreen agents, and bisphenol A (Tables 1.3 and 1.4). Some contaminants such as 4-nonylphenols have been found to be ubiquitous in various foodstuffs (Guenther et al. 2002 ). These chemicals may accumulate in the human body and cause endocrine-disrupting effects.

Table 1.4. Concentrations of some EDCs in foods.

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aMinimum to maximum (mean).

4-NP, 4-nonylphenols; BPA, bisphenolA; DEHP, di-2-ethylhexyl phthalate; PCBs, polychlorinated biphenyls; 4-MBC,

4-methylbenzylidene; OC, octyocrylene; nd, no data.

Effects a ssociated with e xposure to e ndocrine-disrupting c hemicals

Wildlife

A variety of reproductive and developmental effects in wildlife have been attributed to exposure to EDCs (see Table 1.2). This includes changes of sex, population decline, increase in cancers, reduced reproductive function and disorders, disrupted immune and nervous systems, as well as abnormal behavior (U.S. EPA 1997; Jimenez 1997; Depledge and Billinghurst 1999; Damstra et al. 2002). These adverse effects have been reported in various species including invertebrates, fish, reptiles, birds, and mammals.

Marine invertebrates

The best-documented cases of endocrine disruption in invertebrates are in mollusks exposed to organotin compounds (e.g., tributyltin [TBT]) contained in antifouling paints. Since 1971, many studies have reported detrimental effects of TBT on biota including high larval mortality and severe malformations of shells in oysters (Alzieu et al. 1986; Alzieu 1991); imposex in gastropods (Gibbs et al. 1 990; Horiguchi et al. 1994) and dogwhelks (Bryan et al. 1986); growth retardation in mussels (Salazar and Salazar 1991) and microalgae (Beaumont and Newman Munkittrick et al. 1991). Organochlorines 1986 ); and deformities in fiddler crabs (Weis et al. 1987 ). These effects have been observed at exposure concentrations as low as 1 ng/L (Gibbs et al. 1988;Alzieu 2000).The imposex phenomenon is currently the only example of chemical-mediated endocrine disruption that has resulted in an effect at the population level. There have also been reports on the endocrine disruption in crustaceans by trace levels of metals (Cd, Zn) and polychlorinated biphenyls (PCBs) (Bodar et al. 1990; Kristoforova et al. 1984).

Fish

Endocrine disruption in fish has been widely studied, and the observed reproductive effects include feminization, vitellogenisis, impaired reproductive performance, altered thyroid function, decreased fertility, and decreases in population through exposure to pesticides, PCBs, polyaromatic hydrocarbons (PAHs), nonylphenols, hormone steroids, and phytosterols (U.S. EPA 1997). Widespread sexual disruption in wild fish in the United Kingdom has been found due to exposure to the discharges from sewage treatment plants that contain estrogenic chemicals such as nonylphenol and hormone steroids (Jobling et al. 1 998; Sumpter 1998; Tyler and Routledge 1998). In fish, the threshold concentrations for an estrogenic response to a 3-week exposure to 17 α-ethynylestradiol, 17 β-estradiol, nonylphenol, and octylphenol were 0.1ng/L, 1ng/L, 10μg/L, and 3μg/L, respectively (Purdom et al. 1994; Routledge et al. 1 998; Jobling et al. 1995). The levels of these EDCs in some English rivers are well above the threshold concentrations (Tyler and Routledge 1998). A number of abnormalities, including reductions in gonadal size, delayed sexual maturation, and reduced expression of secondary sexual characteristics, have been observed in white sucker exposed to bleached kraft mill effluent in Canada (McMaster et al. 1991, 1992; have been implicated in a number of developmental and reproductive abnormalities and altered thyroid function in fish in the Great Lakes of North America (U.S. EPA 1997; Leatherland 1992).

Reptiles

Alligators in Lake Apopka in Florida were extensively studied and are the subject of a well-documented case of endocrine disruption. The population of alligators declined in the 1980s following a major spill of dicofol. The lake has also received other pesticides from nearby agricultural land (Guillette et al. 2000 ). Abnormalities ranging from low hatchability of eggs to morphological and physiological deficits in both males and females have been reported (Guillette et al. 1994). These were attributed to endocrine disruption caused by embryonic exposure to elevated tissue concentrations of DDT and its metabolites or other pesticides, as well as an embryotoxic effect of high contaminant concentrations in the eggs (Guillette et al. 2000 ).

Birds

The phenomena of eggshell thinning, altered thyroid function, and supernormal clutches (female-female pairing) in wild birds have been attributed to endocrine disruption (U.S. EPA 1997; Fry and Toone 1981). Thin eggshells of birds have been widely reported in the past and were caused by DDE (Dawson 2000). Fry and Toone (1981) suggested that DDT-induced feminization of male gull embryos may be responsible for the biased sex ratio. This resulted in greater numbers of supernormal clutches in western gulls on Santa Barbara Island, California. Low hatching success rates and abnormal thyroid in fish-eating birds (gulls, bald eagles) in the Great Lakes were also linked to organochlorines (DDT, PCBs) (U.S. EPA 1997).

Mammals

Reproductive problems have been found in male Florida panthers, grey seals and common seals and attributed to pollution by PCBs and other organochlorine chemicals (Reijinders 1986; Facemire et al. 1995; U.S. EPA 1997). Poor reproduction of common seals in the western part of the Wadden Sea, the Netherlands, was reported and believed to be caused by pollutants (e.g., PCBs) carried from the Rhine River (Reijinder 1986).

Humans

Endocrine disruptors have been linked to a range of human health problems, including increased incidence of testicular, prostate, and female breast cancer; decreased semen quality and counts; increased frequency of cryptorchidism and hypospadias; increased incidence of polycystic ovaries in women; endometriosis; altered physical and mental development (IEH 1995 ; Colborn et al. 1996 ; U.S. EPA 1997; Foster 2001). With few exceptions (e.g., DES), a direct causal relationship between exposure to a specific chemical and an adverse effect on human health via an endocrine disruption mechanism has not been established, although some laboratory studies on animals showed that some chemicals might be responsible for the reported reproductive, developmental, and carcinogenic effects in humans.

The best-known example of endocrine disruption in humans is the drug DES, a synthetic estrogen that was prescribed to many women in an effort to prevent miscarriage between the late 1940s and early 1970s. Not only did DES not prevent miscarriage, it also had many harmful effects on the children of many of these women. The effects of DES in the offspring of these women were not only cancers, but also birth defects of the uterus and ovaries, reproductive organ dysfunction, and immune suppression (IEH 1995 ; Colborn et al. 1 996; U.S. EPA 1997).

Observations in humans from exposure to contaminated cooking oil in two incidents in Asia provide another good example of how EDCs affect human health (Rogan 1982; Yu et al. 1994 ; Guo et al. 2000 ; Aoki et al. 2001 ). In 1968 in Northern Kyushu in Japan, about 2000 people were poisoned by PCBs and polychlorinated dibenzofurans (PCDFs) that contaminated rice oil. Their condition was named Yusho disease . A similar poisoning by PCBs in Taiwan in 1979 was named Yu Cheng disease . The symptoms of these two diseases in exposed people were dermal and ocular lesions, irregular menstrual cycles, and altered immune responses. Further studies showed that children born to mothers after the event had impaired cognitive development, intrauterine growth retardation, and dysmorphic and hyperpigmented skin and nails. Prenatally exposed boys had sperm with abnormal morphology, reduced motility, and reduced strength.

Recent studies have shown the possible impacts of endocrine disruptors on intelligence and behavior of humans (Laessig et al. 1999 ; Porter et al. 1999 ). During the 9 months between conception and birth, the fetal brain is developing and guided by natural chemical signals, including hormone systems; therefore, it is vulnerable to endocrine disruption. Jacobson and Jacobson (1996) found significant learning and attention problems in children of women who had eaten contaminated fish from Lake Michigan in the 6 years prior to pregnancy. The fish from Lake Michigan contained significant levels of PCBs and other contaminants, which had effects on their children. In a similar study by Lonkey et al. (1996), measurable neurobehavioral deficits in the newborn children of women who had eaten the equivalent of 40 pounds of Lake Ontario salmon in a lifetime were observed.

The cases discussed above have shown that adverse effects on human health are possible through exposure to some endocrine disruptors. The released Global Assessment of the State of the Science of Endocrine Disruptors by Damstra et al. (2002) concluded that studies examining EDC-induced effects in humans have yielded inconsistent and inconclusive results, which is responsible for the overall data being classified as ‘weak’. This classification is not meant to downplay the potential effects of EDCs; rather, it highlights the need for more rigorous studies. The only evidence showing that humans are susceptible to EDCs is currently provided by studies of high exposure levels. More evidence has recently emerged that exposure to EDCs such as phthalates, PCBs, and mercury can disrupt reproductive and developmental processes (Barrett 2005; Hood 2005). However, many uncertainties about endocrine disruptors are of concern. For example, how great is the effect of EDCs on human health, especially at low exposure levels? What are the unobserved effects of the EDCs? The presently available evidence warrants further research.

Summary

Many chemicals can potentially disrupt normal function of reproductive systems in wildlife and humans. These endocrine disruptors could enter food chains through various pathways. Based on the precautionary principle, it is necessary to monitor these chemicals in order to protect human health. Due to the complex nature of food matrices, it is crucial to have sensitive and selective analytical methods for the determination of EDCs in food. We will discuss in detail the extraction and instrumental analysis in the following chapters.

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Chapter 2

Analysis of PCBs in Food

Manuela Melis and Ettore Zuccato

Introduction

Polychlorinated biphenyls (PCBs) are apolar and nonflammable industrial fluids characterized by high electrical isolation properties and good thermal and chemical stability. These chemicals were intentionally produced as technical mixtures and are widely used in industrial and commercial applications as dielectric fluids, organic diluents, plasticizers, adhesives, and flame retardants, especially during the 1970s. Even though their production and use has been banned for a few decades in the United States and Europe, they are still widespread pollutants in air, soil, sediments, and biota, especially in industrialized regions. In addition, PCBs can be unintentionally produced as by-products of some chemical processes or degradation of chlorinated organic compounds. Chemically, the term PCBs includes a family of 209 different congeners that can be divided into two principal groups according to their toxicological properties. The group of dioxin-like PCBs (DL-PCBs), including non-ortho and mono ortho PCBs and consisting of 12 congeners, shows toxicological properties similar to those of dioxins. The other group, non-dioxin like PCBs (NDL-PCBs), includes the most abundant congeners in the environment and human tissue; those toxicological characteristics have not been completely clarified. PCBs are lipophilic and bioaccumulate in the food chain. The diet, in particular the consumption of fish and animal products, is the major route of exposure for humans. After intake, due to their hydrophobic characteristics and resistance to metabolic degradation, these substances can accumulate in fatty tissues, where they can exist as mixtures of different congeners with half-lives of several years. Because DL-PCBs can adopt a coplanar conformation and become isosteric with dioxins, these congeners can bind the aryl hydrocarbon receptor (AhR) and have toxicological characteristics similar to those of dioxins (Barouki et al. 2007). NDL-PCBs, indeed, show a different toxicological profile, and some studies suggest that these congeners can reduce dopamine neurotransmitter levels, and as a result, interfere with calcium homeostasis in neurons (Brown et al. 1998; Seegal 1998; Tilson et al. 1998).

Analysis of PCB s in f ood

Because of their physicochemical properties, PCBs can persist in the environment and bioaccumulate in foods. The main source of nonoccupational exposure to these substances for humans is the diet, and this makes it important to analyze food items to estimate the real exposure of the population to PCBs. These compounds are lipophilic and apolar. As a consequence, they have low water solubility, and their highest concentrations are found in fatty foods such as fish and meat rather than in vegetables, cereals, and fruits. Owing to the variety and the complexity of the sample matrix and the possibility of crosscontamination, the analysis of PCBs in foods is difficult (Carabias-Martínez et al. 2005). For instance, because in the 1970s-1980s the use of PCBs in industrial fields was common, these substances may be present in laboratories built during these years. In some cases, important sources of contamination were found in some electronic equipment placed next to the laboratories (Weistrand et al. 1992). After that time, to prevent significant contamination, samples were not allowed to be exposed to the laboratory air for extended periods (Alcock et al. 1994), and some researchers suggested that the air of the laboratory be prefiltered through HEPA and carbon filters (Muir and Sverko 2006). Moreover, plastic materials should be avoided during storage, extraction, and cleanup of samples because such materials may contain plasticizers such as phthalates that might interfere with the target compound in the analysis (de Boer 1999).

The procedure for PCB analysis in food consists of several different steps: matrix preparation, extraction from the matrix (often together with other lipophilic matrix components), cleanup or purification, and instrumental determination (Bucholski et al. 1996; Letellier and Budzinski 1999).

Sample pretreatment

European Commission (2006) directive 2006/1883/EC of 19 December 2006 established that samples must be stored and transported in glass, aluminum, polypropylene, or polyethylene containers, and storage and transportation must be performed in a way that maintains the integrity of the food sample. A basic requirement for a laboratory dedicated to PCB analysis is the availability of sufficient freezer and refrigerator capacity for sample storage and archiving (Muir and Sverko 2006 ). Samples of tissues are, in fact, usually preserved by freezing after dissection in small pieces (Hess et al. 1995 ) and homogenization or grinding to cause rupture of membranes (Sparr Eskillson and Bj ö rklund 2000 ). Storage of wet samples at −25°C is a safe procedure for fish for a period of up to 2 years, and storage of freeze-dried fish samples in a dark place at ambient temperature is also possible. For storage periods longer than 2 years, it is recommended that the samples be stored at −7 0 ° C or lower (de Boer 1999); however, dried products are more stable and can be more conveniently stored than wet food samples (Fernandes et al. 2004 ). Drying with anhydrous sodium sulfate is convenient, particularly for samples with smaller sizes and more lipid-rich matrices, such as oily fish (Fernandes et al. 2004 ). Drying is particularly important when using nonpolar solvents for solid sample extraction, and the use of sodium sulfate, diatomaceous earth, or cellulose can improve the efficiency of extraction that could be compromised by moisture (Ridgway et al. 2007). Witczak and Chlewinska (2008) have compared the effect of freeze-drying and anhydrous sodium sulfate in the muscles of selected fish species. In fish tissues, freezedrying caused the loss of PCBs during sublimation, especially lighter PCBs (Juan et al. 1999 ), but the difference in recovery between this method and drying with anhydrous sodium sulfate was not statistically significant. Despite smaller recoveries, freezedrying is recommended for extraction of fish tissues because it saves on solvent and the extracts are easier to prepare. Animal or fish tissue handling can cause problems, however; drying before extraction may not always be practical because these samples can be characterized by high proportions of fats or because cell collapse can cause retaining of the analytes. Tissue matrices, in particular, tend to clump, preventing the extraction by solvent (Ridgway et al. 2007). Dispersion of tissue with solid support can be used to avoid aggregation of sample particles and to enhance solvent extraction (Ridgway et al. 2 007).

Vegetables usually have high water content and should be dehydrated prior to the analysis unless extracted immediately after sampling. Vegetable matter is therefore crushed, chopped, and freeze-dried, or gently dried at 40°-50°C prior to storage and analysis (Liem 1999a ). Analysis of solid and semisolid foods are at a disadvantage because liquid samples require fewer pretreatment steps (Carabias-Martínez et al. 2005). Liquid samples are usually freeze-dried for storage (Thomsen et al. 2002). In general, analysis of halogenated contaminants in water is recommended immediately after sampling (de Boer 1999).

Extraction

The extraction step serves to isolate analytes from potentially interfering sample components while getting these analytes into a form suitable for analysis (Raynie 2006 ). Extraction is a fundamental step that prepares food samples for instrumental analysis and consists of a transfer by partition of the analytes from food matrices to an extraction matrix with a simultaneous elimination of interfering substances. PCBs are lipophilic, thus extraction methods are based on the isolation of the lipid fraction from the sample matrix. Methods for the isolation of the fat fraction from individual food differ depending on the type of sample (Liem 1999a). Butter, fats, and oils do not normally require extraction procedures (Cencič Kodba and Brodnjak Von čina 2007). Sample weight used for the extraction must be sufficient to fulfill the requirements with respect to sensitivity. There are many satisfactory specific sample extraction procedures that may be used for the products under consideration. Every laboratory may use their preferred procedure as long as it is validated according to internationally accepted guidelines (EU Commission Directive 2006/1883/EC ). Below, we describe the most common techniques used for PCB extraction from foods (see also Table 2.1, which summarizes the principal characteristics of each technique).

Liquid-liquid e xtraction ( LLE)

Liquid-liquid extraction (LLE) is easy and popular, and it is one of the most common methods of extraction, especially for organic analytes from liquid matrices. Analytes in solution or liquid samples can be extracted by direct partitioning with immiscible solvents mixed by shaking. Choosing the proper solvent polarity for solubilizing the analytes is fundamental, and generally a combination of nonpolar solvents with solvents of various polarity is used. As an example, to extract the lipid fraction of butter for PCB analysis, Ramos et al.( 1999 ) used a mixture of acetone/ hexane (2:1 v/v), and Battu et al.(2004) used acetonitrile saturated with hexane, then partitioned the acetonitrile fraction with dichloromethane. However, this technique can also be adopted to extract solid samples after homogenization. For instance Mondon et al. (2001) used acetone/hexane (1:1v/v) to extract PCBs from 2 g of sand flathead and Pacific oysters after homogenization. Johansen et al. (1993) extracted 10-20g of crab tissue with cyclohexane, acetone, and water, and Matthews et al. (2008) used two different methods, dichloromethane (DCM)/cyclohexane (1:1v/v) and hexane/acetone (1:1v/v), to extract PCBs from skin and muscle of edible fish, crustaceans, and shellfish from Queensland, Australia. Zuccato et al. (2004) extracted 25 g of homogenized salmon with 100 mL of 25% hydrochloric acid and 200 mL of 1 : 1 DCM/hexane to study PCB concentrations in salmon samples from Europe. Zuccato and colleagues (2008) used acetone to extract cabbage and concentrated sulfuric acid and DCM/hexane 1 : 1 to extract butter to study PCBs in foods from Belgium, Italy, Portugal, and Spain.

This technique is easy but presents several disadvantages, such as the use of large volumes of solvents that have to be evaporated after extraction. It is expensive and time consuming and requires the use of highly flammable and toxic solvents. Afterward, it is important to note that in order to apply LLE, the size of the sample should be relatively large (Hess et al. 1995 ; Bucholski et al. 1 996; Carro et al. 2002; Ahmed 2003; Focant et al. 2004a; Ridgway et al. 2007; Beyer and Biziuk 2008).

Table 2.1.Summary of the extraction techniques used for PCB analysis.

c02_image001.jpg

L-L, liquid-liquid extraction; SPE, solid-phase extraction; MSPD, matrix solid-phase dispersion; ASE, accelerated solvent extraction; MAE, Microwave-assisted extraction; SFE, supercritical fluid extraction

Soxhlet extraction

The traditional extraction method for the determination of a wide variety of compounds in food samples is Soxhlet extraction (Carabias-Martínez et al. 2005). Before extraction, animal and fish tissues are macerated and then ground with sodium sulfate and silica; this first passage can reduce the water content, enhancing the extraction efficiency (Hess et al. 1995). In a conventional Soxhlet apparatus, up to 10 g of sample is placed in a thimble holder, which is gradually filled with 50-200 mL of condensed fresh solvent from a distillation flask. When the liquid reaches the overflow level, a siphon aspirates the solute from the thimble holder and unloads it back into the distillation flask, carrying the extracted analytes into bulk liquid. This operation is repeated until extraction is complete (Dean and Xiong 2000 ; Ridgway et al. 2007 ). No filtration is required after a leaching step (Luque de Castro and Gárcia-Aruso 1998; Letelier and Budzinski 1999; Carro et al. 2002). Nonpolar solvents, such as n-hexane, have been frequently used for PCB analysis (Hess et al. 1995). Nonpolar solvents are too immiscible with water to penetrate the wet material however, thus a medium polarity or a binary solvents mixture is recommended, such as DCM or hexane/acetone (1:1v/v) (de Boer 1988 ; Folch et al. 1996 ). During the last decade, Soxhlet extraction has been used for determination of PCBs in fish, mammals, and mussel tissues (Vives and Grimalt 2002; Manirakiza et al. 2002; Corsolini et al. 2002; Kirivanta et al. 2003; Malavia et al. 2004; Asmund et al. 2004; Isosaari et al. 2006; Pan et al. 2007 ; Koistinen et al. 2008 ; Witczak and Chlewinska 2008 ). Zuccato et al. (1999) used Soxhlet to extract (for 8 hours with hexane/ acetone, 9:1v/v) 5g of lyophilized sample of mixed meat, dairy products, fish, oils, green and other leafy vegetables, cereals, sweets and sweeteners, fruits, alcoholic and nonalcoholic beverages, in order to study the source and toxicity of PCBs in the Italian diet. Focant et al. (2002) extracted 10 g of powdered milk with Soxhlet using pentane/DCM (1:1v/v) as the solvent mixture. Zhao et al. (2009) have homogenated and individually dried vegetables, rice, pulses, pork, and chicken and fish muscle samples that were then Soxhlet extracted with hexane/acetone (3:1v/v). Grassi et al. (2008) have used Soxhlet to extract vegetables with a solution of hexane/ acetone (9 : 1 v/v) for 24 hours. Huckins et al. (1990) have proposed the use of semipermeable membrane during extraction for separating the component fraction from the lipid matrix of foodstuffs. During the extraction, PCBs are allowed to cross the semipermeable membrane of a polyethylene bag, and the lipid matrix is retained; extraction time is shorter than normal, and it is sufficient for a quantitative recovery of all the components of interest.

Soxhlet extraction was born from the need to reduce the consumption of solvents, and it is faster and less expensive than LLE. Despite these advantages, it is necessary to evaporate the solvents to concentrate the samples, and a single sample run requires many hours to be completed (Liem 1999b ; Abrha and Raghavan 2000; Buldini et al. 2002; Beyer and Biziuk 2008). Moreover, the temperature of the system remains high because samples are extracted by keeping the solvent at the boiling point for a long time, thus the possibility of thermal decomposition of thermolabile analytes cannot be excluded.

Solid-phase extraction

Solid-phase extraction (SPE) is widely accepted as an alternative to laborious and time-consuming liquid-liquid methods for extraction of nonvolatile organic compounds such as PCBs from liquid matrices. SPE can be used directly as an extraction technique for liquid matrices or as a cleanup method after solvent extraction (Hennion 1999; Ahmed 2001; Focant et al. 2004a; Gilbert-López et al. 2009).

SPE is based on the use of disposable cartridges to trap the analytes and separate them from the matrix. The use of SPE requires that the sample must be homogeneous and in a liquid state prior to addition to a SPE column or disk device. The presence of particulate can complicate the use of SPE, impeding and blocking the flow and creating variability in the particulate content of samples, possibly leading to variability of recoveries (Barker 2000a). The extraction is performed in four steps: conditioning of the SPE column (the functional groups of the sorbent are solvated in order to activate them to permit the interaction with analytes), retention (solutions are passed through the column and the analytes are bound to the bed surface), selective washing (to eliminate the undesired molecules), and elution (the analytes are desorbed and collected for analysis).

When a sample solution passes through the activated sorbent bed, analytes are concentrated on the surface while other components pass through the bed. Selection of the appropriate SPE sorbent depends on the mechanism of interaction between the sorbent and the analytes of interest. Many types of sorbent, such as alumina, magnesium silicate, and graphitized carbon are available. Silica is widely used because it is reactive enough to permit its surface to be modified by chemical reaction, yet stable enough to allow its use with a wide range of solvents (Buldini et al. 2 002). Nonpolar sorbents can be made of functional groups (C8, C18, cyano, amino, sulfunic acid, etc.) bound to the surface of silica to alter its retention properties or they may be made of functionalized styrenedivinylbenzene synthetic polymers (Focant et al. 2 004a). In particular, C18 cartridges have nonpolar characteristics that retain organochlorine compounds and have a size exclusion function suitable to eliminate several macromolecules coextracted with PCBs. For these reasons C 18 cartridges can be used either for extraction or for sample cleanup (Covaci and Schepens 2001). For instance, C18 cartridges have been used for the analysis of PCBs in milk (Pic ó et al. 1995; Ahmed 2003; Centi et al. 2007 ), and Dong et al. (2000) and Sun et al. (2002) have used these cartridges to analyze PCBs in water samples from the Yangtze River in China.

Diatomaceous earth (diatomite, Extrelut), a form of silica composed of the siliceous shells of unicellular aquatic plants, has also been used frequently for extraction of PCBs. SPE decreases the sample-preparation time and the consumption of solvents, permits the simultaneous removal of several interfering substances, and allows multiple samples to be treated in parallel using small volumes of solvents. SPE is an advantageous technique because there is no emulsion formation and no requirement for repeated extraction or centrifugation steps. However, the presence of particulate in unfiltered samples might cause clogging of the SPE cartridge, which may lead to longer extraction time (Hennion 1999 ; Ahmed 2001; Björklund et al. 2002; Buldini et al. 2 002). Moreover, during extraction by a SPE method, part of the lipids may remain in the cartridge, even after elution with nonpolar solvents (Focant et al. 2004a ). To solve this problem Sj ö din et al. (2004) used a solid phase dispersion of diatomaceous earth in the SPE cartridge to determine PCBs. This permitted the extraction of milk without the inconvenience of loosing part of the lipids.

Matrix solid-phase dispersion

Matrix solid-phase dispersion (MSPD), a tissue disruption/extraction method, uses a mortar and pestle to blend octadecylsilylderivatized silica (C18) or other chemically modified solid supports with the tissue sample (Crouch and Barker 1997). For extraction of analytes from animal tissues,