Corso di Laurea Magistrale in Farmacia
TESI DI LAUREA
Links between the Persistent
Organic Pollutants and the components of the
Metabolic Syndrome in a Lebanese Population
Supervisors
Candidate
Prof. Vincenzo Calderone
Fabienne Frenn
Prof. Joseph Matta
2 TABLE OF CONTENTS
ABSTRACT ... 4
AIM OF THE STUDY ... 5
CHAPTER 1. INTRODUCTION ... 6
CHAPTER 2. METABOLIC SYNDROME ... 10
2.1. Hyperglycemia ... 12
2.2. Dyslipidemia ... 14
2.3. Obesity ... 14
2.4. Hypertension ... 14
2.5. Cardiovascular Diseases ... 16
CHAPTER 3. PERSISTENT ORGANIC POLLUTANTS ... 17
3.1. Polychlorinated Biphenyls... 17 3.2. Chlordane ... 18 3.3. DDT ... 18 3.4. Dieldrine ... 19 3.5. Dioxins... 19 3.6. Endrin ... 19 3.7. Furans ... 20 3.8. Heptachlor... 20 3.9. Hexachlorobenzene (HCB) ... 21 3.10. Mirex ... 21 3.11. Toxaphene ... 21
3
3.12. Aldrin ... 22
CHAPTER 4. POLYCHLORINATED BIPHENYLS (PCBS) ... 23
4.1. Identification ... 23
4.2. Chemical and Physical Properties ... 27
4.3. Environmental Occurrence and Exposure ... 31
4.4. Population Biomonitoring ... 35
4.5. Regulations and Guidelines ... 38
CHAPTER 5. MATERIALS AND METHODS ... 39
5.1. Study Design and Population ... 39
5.2. Confidentiality and Ethical Considerations ... 40
5.3. Blood Sampling and Storage ... 41
5.4. STATISTICAL ANALYSIS ... 41
CHAPTER 6. RESULTS AND DISCUSSION ... 43
6.1. Characteristics of the Studied Population... 43
6.2. PCB Concentrations in the Serum ... 46
6.3. Comparison of PCB Levels with International Values ... 51
6.4. PCBS and Critical Limits ... 55
6.5. Correlation of PCB Levels ... 56
6.6. Correlations between pairs of PCBS ... 61
CONCLUSION ... 62
4
ABSTRACT
Polychlorinated biphenyls (PCBs) are persistent organic pollutants that are still routinely detected 30 years after their restriction in many countries. PCBs have been associated with several non-communicable diseases. They are best measured via “Human Biomonitoring” (HBM). The concentrations of six indicator PCBs (PCBs 28, 52, 101, 138, 153 and 180) were measured in the serum samples of 316 Lebanese population, using gas chromatography coupled to an iron trap mass spectrometer detector. PCBs were detected in 56.3 to 59.2% of the serum samples. The sum of PCB (∑PCB) levels ranged from < LOD to 338.84 ng/g lipids, with a geometric mean level of 10.34 ± 0.98 ng/g lipids. The major contributor to the ∑PCBs was PCB 180. In the present study, the levels were in general lower than the values observed in several Western and European countries. No association was found between age and concentration of any of the PCBs. In terms of risk for health, the highest levels were lower than critical limits as such as HBM I and II values. We observed an inverted U-shaped association between levels of serum PCBs and the risk of overweight/obesity. Regarding potential food contributors, we found no relation between PCB levels and fish consumption and a moderate relation with dairy products consumption (moderate consumers of dairy products had higher PCB levels compared to lower consumers (16.92±0.1/6.92±0.12; p=0.025). The present study is the first to provide information regarding PCB levels in a Lebanese population. Larger studies are required in order to estimate the PCB exposure parameters of the Lebanese population.
5 AIM OF THE STUDY
This is the first study to monitor levels of serum PCBs in a Lebanese population sample, which allows us to draw a first conclusion on the exposure to PCBs, as well as the influence of age, diet and geographical vicinity of the sources of pollution.
6
CHAPTER 1
INTRODUCTION
Persistent Organic Pollutants (POPs) are organic compounds that are resistant to environmental degradation, hence, they persist in the environment, bio-accumulate in human and animal tissue, and bio-magnify in food chains like milk, fish and eggs (WHO, 2007). The United Nations Environment Program (UNEP) Governing Council originally created a list of 12 POPs – known as the “dirty dozen.” These were Aldrin, Chlordane, Dichlorodiphenyltrichloroethane (DDT), Dieldrin, Endrin, Heptachlor, Hexachlorobenzene, Mirex, PolyChlorinated Biphenyls (PCBs), PolyChlorinated Dibenzo-p-Dioxins (PCDD or Dioxins), PolyChlorinated DibenzoFurans (PCDF or Furans), and Toxaphene. In recent years, this list has been expanded to include Polycyclic Aromatic Hydrocarbons (PAHs), PolyBrominated Diphenyl Ethers (PBDE), and TriButylTin (TBT) (Ashraf, 2015). The groups of compounds that make up POPs are also classed as Persistent, Bioaccumulative, and Toxic (PBTs) or Toxic Organic Micro Pollutants (TOMPs). These terms are essentially synonyms for POPs (Crinnion, 2011). Figure 1 shows the different groups of POPs: intentionally and unintentionally produced (Ashraf, 2015).
7 There are many thousands of POP chemicals, often coming from certain series or families of chemicals (for example there are theoretically 209 different polychlorinated biphenyls, differing from each other by level of chlorination and substitution position). POPs are persistent in the environment, having long half-lives in soils, sediments, air or biota. There seems to be no consensus of opinion about how long the half-life in a given media should be for the term “persistent” to be conferred; however, in practice a POP could have a half-life of years or decades in soil/sediment and several days in the atmosphere (Jones & de Voogt, 1999). A recent approach has considered defining “persistence in the environment” operationally from a model calculated overall residence time at steady state in a multimedia environment (Webster, Mackay, & Wania, 1998). POPs are typically “water-hating” and `fat-loving' chemicals, i.e. hydrophobic and lipophilic. In aquatic systems and soils, they partition strongly to solids, notably organic matter, avoiding the aqueous phase (Shree, 2017). They also partition into lipids in organisms rather than entering the aqueous milieu of cells and become stored in fatty tissue. POPs have a slow metabolism and may therefore accumulate in food chains (Mrema et al., 2013). Importantly, POPs have the propensity to enter the gas phase under environmental temperatures (Jones & de Voogt, 1999) . Hence, they may volatilize from soils, vegetation and water bodies into the atmosphere and because of their resistance to breakdown reactions in air, travel long distances before being re-deposited. The cycle of volatilization and deposition may be repeated many times, with the result that POPs could accumulate in a much further area from where they were used or emitted. In the atmosphere itself, POPs can partition between particles and aerosols depending on ambient temperature and the physico-chemical properties of the compound itself (Jones & de Voogt, 1999). As for the origin of the POPs, some are accidental byproducts of combustion or the industrial synthesis of other chemicals. Many have been synthesized for industrial uses (eg. PCBs, chlorinated paraffins, PBDEs) or as agrochemicals (e.g. DDT, Lindane, chlordane). Examples of more polar POPs are phenols (e.g. polyethoxylated alkylphenols which are nonionic surfactants), and chlorinated phenols. The POPs properties include aqueous solubility, vapor pressure, partition coefficients between water/solid, air/solid or air/liquid, and half-lives in air, soil and water. These properties have been compiled for many POPs in various databases, e.g. those of Mackay
8 and co-workers (Mackay et al. 1992), the United States Environmental Protection Agency (US EPA) and the Dutch AQUAPOL system (Krop, Velzen, Voogt, Govers, & Heijden, 1995), either from direct measurements made in the laboratory or calculated from theory. However, there are often wide variations in the values reported and their quality, conferring uncertainty on the precise behavior of the POPs, and these databases are constantly being revised and improved. Due to POPs effects and their correlation with the metabolic syndrome, this study focused on links between the POPs, specifically PCBs, and the components of metabolic syndrome in a Lebanese population. In addition, a strong statistical association was found between the presence of metabolic syndrome and higher lipid-adjusted toxic equivalents (TEQs) of dioxin and dioxin-like POPs (e.g., polychlorinated dibenzo-p-dioxins [PCDDs or dioxins], polychlorinated dibenzofurans [PCDFs or furans], and dioxin-like polychlorinated biphenyls [PCBs]). Expert groups have developed several sets of clinical criteria for the diagnosis of the syndrome. The prevalence of metabolic syndrome is increasing globally as a result of changes in lifestyle, available nutrition, and the exposure to stress (Kolb & Martin, 2017). Early detection prevents or delays the progression of MetS to the more serious conditions. Due to the fact that the prevalence of all forms of diabetes is increasing in Lebanon (Noureddine, Nakhoul, Galal, Soubra, & Saleh, 2014), it is imperative to investigate the prevalence of MetS, and initiate local programs for management. No properly conducted survey was carried out in this mentioned region and especially in Lebanon. Therefore, we aimed to investigate the prevalence of MetS in apparently healthy individuals living in different regions in Lebanon.
Human exposure to persistent environmental chemicals is best determined via Human Body Model (HBM) which detects levels of pollutants in human matrices such as blood, serum, urine and others, Combining biomonitoring to health risk assessment is of particular importance when it comes to pollutants that accumulate in the body for long periods of time (WHO, 2015). Several HBM programs have been launched in many westerncountries worldwide, in order to assess the nationwide concentration of specific persistent pollutants, notably the “German Environment Surveys”, The “French Nutrition and Health Survey” (ENNS), the “United States National Health and Nutrition Examination Survey” (NHANES), the “Canadian Health Measures Survey” (CHMS) and the
9 “BIOAMBIENT.ES” in Spain, among others (Choi, Mørck, Polcher, LE., & Joas, 2015). In HBM studies, the PCBs amongst the POPs are most commonly monitored in blood, due to the blood’s standardized sampling procedures, the familiarity of this intervention, as well as the pollutants’ equilibrium with the human organs (WHO, 2015). In fact, the Stockholm Convention identified PCBs among the initial twelve persistent organic pollutants (POPs) in 2001. The Community Bureau of Reference of the European Commission selected 7 congeners among the 209, to be primarily monitored in environmental and biological matrices, known as “indicator PCBs” (INERIS, 2011). Given the highly toxic nature of the POPs, and knowing that they haven’t been previously monitored in Lebanon, the aim of the present study was to determine, for the first time, the serum levels of 6 indicator PCBs (28, 52, 101, 118, 138, 153, 180) in a sample of a Lebanese population and to consider these levels as first values of reference. Furthermore, possible difference in PCB levels by gender, age and Body Mass Index (BMI) were also evaluated. The results obtained were compared with data from other countries.
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CHAPTER 2
METABOLIC SYNDROME
The “metabolic syndrome” (MetS) is a clustering of components that reflect overnutrition, sedentary lifestyles, and resultant excess adiposity, and includes the clustering of abdominal obesity, insulin resistance, dyslipidemia, and elevated blood pressure (Cornier et al., 2008). PCBs have been linked to the development or exacerbation of cardiovascular disease. Causally, laboratory and epidemiological studies have shown that PCBs can lead to obesity, diabetes, and lipid abnormalities; all of which are risk factors for developing cardiovascular disease (Perkins, Petriello, Newsome, & Hennig, 2016).
11 A diagnosis of the MetS is made when any 3 of the 5 following risk factors are present (Table 1) : enlarged waist circumference with population-specific and country-specific criteria; elevated triglycerides, defined as ≥ 150 mg/dL, decreased HDL-C, defined as < 40 mg/dL in men and < 50 mg/dL in women, elevated blood pressure, defined as systolic blood pressure ≥ 130 mm Hg or diastolic blood pressure ≥ 85 mm Hg and elevated fasting glucose, defined as blood glucose > 100 mg/dL, with the inclusion of patients taking medication to manage hypertriglyceridemia, low HDL-C, hypertension. and hyperglycemia (Lam & LeRoith 2019).
Table 1: Criteria for Diagnosis of the Metabolic Syndrome
Measure Categorical Cut-Points
Waist circumference Population and country specific definitions
Triglycerides * ≥ 150 mg/dL
High Density Lipoprotein Cholesterol (HDL-C)*
Men < 40 mg/dL Women < 50 mg/dL
Blood Pressure* ≥ 130/ ≥8
Fasting Glucose* ≥ 100 mg/dL
Table 1: Metabolic Syndrome (Lam & LeRoith 2019)
*Drug treatment for elevated triglycerides, low HDL-C, elevated blood pressure or elevated glucose are alternate indicators
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2.1-Hyperglycemia
Hyperglycemia is characterized by an elevated concentration of plasma glucose. Two patterns of higher glucose define prediabetes: a fasting glucose in the range of 100_125 mg/dL or a 2-h postprandial level of 140– 199 mg/dL. Categorical diabetes is defined as a fasting glucose ≥126 mg/dL or a postprandial level ≥200 mg/dL. Microvascular diseases, such as kidney diseases and retinopathy, are the primary clinical outcome of hyperglycemia. Type 2 diabetes is in fact the primary cause of chronic kidney disease (Grundy, 2016). It is a physiopathology caused by hyperglycemia due to an absolute or relative deficit in insulin production. Chronic hyperglycemia can lead up to organ failures and dysfunctions such as the kidneys, heart and blood vessels (Alam, Asghar, Azmi, & Malik, 2014).
Insulin is a polypeptide hormone secreted by the beta cells of the pancreatic islets of Langherhans, and acts via glycoprotein receptors located on target tissues of the liver, skeletal muscles and adipocytes. Insulin binds to its receptor allowing transmittance of signals across the plasma membrane. These signals lead up to the activation of different reactions such as kinase mediated reactions and phosphorylation. The end result of the chain reactions is the activation of glycogen synthesis, storage of glucose as glycogen (with the help of glucose transporter protein GLUT4), inhibition of gluconeogenesis and glycogenolysis (McCracken, Monaghan, & Sreenivasan, 2018).
Insulin resistance occurs whenever there is a reduced responsiveness to normal insulin levels. At an early stage, the beta cells of the pancreatic islets of Langerhans secret an excessive amount of insulin as a compensatory mechanism to maintain normal levels of glycaemia. This mechanism, however, will decompensate eventually resulting in the reduction of glycogen synthesis, glucose transport and effectiveness of the insulin signaling pathways (McCracken et al., 2018). Phenotypes who are at most risk of developing insulin resistance have a poor diet habit and are most likely to be obese; this fatty acid excess is cause by an inappropriate lipolysis. Nevertheless, this does not exclude phenotypes who maintain a balanced diet. The targeted tissues, as we mentioned above, are the liver, skeletal muscles and adipocytes.
Persistent organic pollutants (POPs) are lipophilic compounds, they tend to accumulate mainly in adipose tissues. An exposure to chlorinated organic pollutants increases the risk
13 developing type 2 diabetes and can no longer be ignored as POPs have influenced the pathogenesis of type 2 diabetes (D. H. Lee, Porta, Jacobs, & Vandenberg, 2014). Figure 3 shows that excess energy intake and physical inactivity lead to obesity, and as a result an increase in mitochondrial dysfunction, excessive fat in the liver, muscle and pancreas and finally inflammation in the adipose tissue. These “traditional risk factors” cause insulin resistance. The chronic presence of POPs mixtures in adipose tissue are classified as “new risk factors” and have a direct effect on obesity and its consequent results (D. H. Lee et al., 2014).
Figure 3. Traditional Risk Factors and New Risk Factors on development of Type 2 Diabetes (D. H. Lee et
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2.2-Dyslipidemia
Dyslipidemia, defined as elevated total or low-density lipoprotein (LDL) cholesterol levels, or low levels of high-density lipoprotein (HDL) cholesterol, is an important risk factor for coronary heart disease (CHD) and stroke (Fodor, 2011). There is a small volume of epidemiological literature directly examining a potential causal relationship between dyslipidemia and PCB exposure is certainly a limiting factor. The difficulty of finding literature examining this is further compounded by the fact that serum lipid measurements are often published within much larger analyses and are often not a point of emphasis, meaning that searching for appropriate PCB literature is complicated. Despite these limitations, though, there is epidemiological evidence suggesting PCB exposure may contribute to dyslipidemia (Goncharov et al., 2008) , (Lee et al. 2011), (Uemura et al., 2009).
2.3-Obesity
While atherosclerosis and hypertension are commonly associated with the development of CVD, obesity serves as a primary modulator of these conditions and has been implicated heavily in CVD development (Kenchaiah et al., 2002) ; (Poirier et al., 2006) ; (Van Gaal, Mertens, & De Block, 2006). A limited amount of literature has focused on PCB exposure and its implications on the etiology of obesity development, with primary correlations drawn from laboratory studies (Arsenescu, Arsenescu, King, Swanson, & Cassis, 2008) ; (Ferrante et al., 2014) ; (Kim et al., 2012) ; (Myre & Imbeault, 2014). Epidemiological studies into the association of PCB exposure and the development of obesity primarily have relied upon the comparison of serum PCB levels and body mass index (BMI) or birth weight, although studies have yielded mixed results (Perkins et al., 2016).
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2.4-Hypertension
While the NHANES data set has provided a wealth of information, a variety of epidemiological studies involving different cohorts have also found similar associations between PCB exposure and an increased risk of hypertension (Perkins et al., 2016). Two studies of the Anniston, Alabama cohort, a group of 758 participants residing near the original Monsanto Corporation PCB manufacturing site, showed a correlation between rates of hypertension and serum PCB concentration (Goncharov, Bloom, Pavuk, Birman, & Carpenter, 2010) and that other than age, total serum PCBs were the strongest determinant of blood pressure level in 394 participants (Goncharov, Pavuk, Foushee, Carpenter, & Consortium, 2011). Additionally, a study of 1,374 Japanese residents is consistent with these findings with their own data indicating that serum levels of dioxin-like PCBs are directly correlated with high blood pressure (Uemura et al. 2009). Studies analyzing the correlation between PCB exposure and rates of hypertension have been performed on a wide array of sample sizes (Akagi & Okumura, 1985) , (Goncharov et al., 2011) , (Peters, Fabian, & Levy, 2014), (Stehr-Green, Ross, Liddle, Welty, & Steele, 1986). Of all the epidemiological studies examined, all but one of the studies found a positive relationship. An important observation is that the studies that found a positive relationship had higher sample sizes, ranging from 394 to 12,200 participants, than the study in disagreement, which had a sample size of 59 participants (Akagi & Okumura, 1985), suggesting sample size may have influenced these findings.
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2.5-Cardiovascular Diseases
Cardiovascular disease (CVD) is a non-communicable disease that encompasses a group of disorders of the heart and blood vessels including coronary heart disease, cerebrovascular disease, peripheral arterial disease, rheumatic heart disease, congenital heart disease, deep vein thrombosis, and pulmonary embolism (Labarthe, 2011). The World Health Organization estimates that 17.3 million people died from CVD in 2008 alone, accounting for 30% of global deaths and serving as the number one cause of death globally who (WORLD HEALTH ORGANISATION, 2011). Several risk factors and associated diseases including type 2 diabetes, hypertension, obesity, sedentary lifestyle, and over nutrition can contribute to the pathology of CVD. Furthermore, environmental pollutants, such as PCBs, can thus contribute to CVD directly or indirectly via promotion of these and other risk factors and associated diseases. Considering the significant burden that CVDs have on global mortality, it is imperative to explore and assess factors that may promote or exacerbate the pathogenesis of these diseases world (WORLD HEALTH ORGANISATION, 2011).
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CHAPTER 3
PERSISTENT ORGANIC POLLUTANTS
The Stockholm Convention has enlisted the 12 most life-threatening and harmful POPs in a list called “the dirty-dozen”, having effects not only on humans but on the ecosystem as well. These 12 POPs have been placed under 3 categories:
• PESTICIDES: aldrin, chlordane, DDT, dieldrin, endrin, heptachlor, hexachlorobenzene, mirex, toxaphene
• INDUSTRIAL CHEMICALS: hexachlorobenzene, polychlorinated biphenyls
(PCBs)
• BY-PRODUCTS: hexachlorobenzene; polychlorinated dibenzo-p-dioxins and polychlorinated dibenzofurans (PCDD/PCDF), and PCBs.
(UNEP, 2008)
3.1- Polychlorinated Biphenyls (PCBs)
These compounds are used in industry as heat exchange fluids, in electric transformers and capacitors, and as additives in paint, carbonless copy paper, and plastics. Of the 209 different types of PCBs, 13 exhibit a dioxin-like toxicity. Their persistence in the environment corresponds to the degree of chlorination, and half-lives can vary from 10 days to one-and-a-half years. PCBs are toxic to fish, killing them at higher doses and causing spawning failures at lower doses. Research also links PCBs to reproductive failure and suppression of the immune system in various wild animals, such as seals and mink. Large numbers of people have been exposed to PCBs through food contamination. Consumption of PCB-contaminated rice oil in Japan in 1968 and in Taiwan in 1979 caused pigmentation of nails and mucous membranes and swelling of the eyelids, along with fatigue, nausea, and vomiting. Due to the persistence of PCBs in their mothers' bodies,
18 children born up to seven years after the Taiwan incident showed developmental delays and behavioral problems. Similarly, children of mothers who ate large amounts of contaminated fish from Lake Michigan showed poorer short-term memory function. PCBs also suppress the human immune system and are listed as probable human carcinogens. (UNEP, 1998)
3.2-Chlordane
Used extensively to control termites and as a broad-spectrum insecticide on a range of agricultural crops, chlordane persists in the soil for a long time and has a reported half-life of one year. Chlordane may affect the human immune system and is classified as a possible human carcinogen. It is believed that human exposure occurs mainly through the air, and chlordane has been detected in the indoor air of residences in the US and Japan. Chlordane has been severely restricted and banned in different countries around the world. (UNEP, 1998)
3.3-DDT
DDT was widely used during World War II to protect soldiers and civilians from malaria, typhus, and other diseases spread by insects. After the war, DDT continued to be used to control disease, and it was sprayed on a variety of agricultural crops, especially cotton. DDT continues to be applied against mosquitoes in several countries to control malaria. Its stability, its persistence (as much as 50% can remain in the soil 10-15 years after application), and its widespread use have meant that DDT residues can be found everywhere; residual DDT has even been detected in the Arctic. 34 countries have banned DDT, while 34 others severely restrict its use. Nonetheless, it has been detected in food from all over the world. Although residues in domestic animals have declined steadily over the last two decades, food-borne DDT remains the greatest source of exposure for the general population. The short-term acute effects of DDT on humans are limited, but long-term exposures have been associated with chronic health effects. DDT has been detected in breast milk, raising serious concerns about infant health. (UNEP, 1998)
19
3.4-Dieldrin
Used principally to control termites and textile pests, dieldrin has also been used to control insect-borne diseases and insects living in agricultural soils. Its half-life in soil is approximately five years. The pesticide aldrin rapidly converts to dieldrin, so concentrations of dieldrin in the environment are higher than dieldrin use alone would indicate. Dieldrin is highly toxic to fish and other aquatic animals, particularly frogs, whose embryos can develop spinal deformities after exposure to low levels. Dieldrin residues have been found in air, water, soil, fish, birds, and mammals, including humans. Food represents the primary source of exposure to the general population. For example, dieldrin was the second most common pesticide detected in a US survey of pasteurized milk.
(UNEP, 1998)
3.5-Dioxins
These chemicals are produced unintentionally due to incomplete combustion, as well during the manufacture of pesticides and other chlorinated substances. They are emitted mostly from the burning of hospital waste, municipal waste, and hazardous waste, and also from automobile emissions, peat, coal, and wood. There are 75 different dioxins, of which seven are considered to be of concern. One type of dioxin was found to be present in the soil 10 - 12 years after the first exposure. Dioxins have been associated with a number of adverse effects in humans, including immune and enzyme disorders and chloracne, and they are classified as possible human carcinogens. Laboratory animals given dioxins suffered a variety of effects, including an increase in birth defects and stillbirths. Fish exposed to these substances died shortly after the exposure ended. Food (particularly from animals) is the major source of exposure for humans. (UNEP, 1998)
3.6-Endrin
This insecticide is sprayed on the leaves of crops such as cotton and grains. It is also used to control rodents such as mice and voles. Animals can metabolize endrin, so it does not accumulate in their fatty tissue to the extent that structurally similar chemicals do. It
20 has a long half-life, however, persisting in the soil for up to 12 years. In addition, endrin is highly toxic to fish. When exposed to high levels of endrin in the water, sheepshead minnows hatched early and died by the ninth day of their exposure. The primary route of exposure for the general human population is through food, although current dietary intake estimates are below the limits deemed safe by world health authorities. (UNEP, 1998)
3.7-Furans
These compounds are produced unintentionally from many of the same processes that produce dioxins, and also during the production of PCBs (see below). They have been detected in emissions from waste incinerators and automobiles. Furans are structurally similar to dioxins and share many of their toxic effects. There are 135 different types, and their toxicity varies. Furans persist in the environment for long periods, and are classified as possible human carcinogens. Food, particularly animal products, is the major source of exposure for humans. Furans have also been detected in breast-fed infants. (UNEP, 1998)
3.8-Heptachlor
Primarily used to kill soil insects and termites, heptachlor has also been used more widely to kill cotton insects, grasshoppers, other crop pests, and malaria-carrying mosquitoes. It is believed to be responsible for the decline of several wild bird populations, including Canadian Geese and American Kestrels in the Columbia River basin in the US. The geese died after eating seeds treated with levels of heptachlor lower than the usage levels recommended by the manufacturer, indicating that even responsible use of heptachlor may kill wildlife. Laboratory tests have also shown high doses of heptachlor to be fatal to mink, rats, and rabbits, with lower doses causing adverse behavioral changes and reduced reproductive success. Heptachlor is classified as a possible human carcinogen, and some two dozen countries have either banned it or severely restricted its use. Food is the major source of exposure for humans, and residues have been detected in the blood of
cattle from the US and from Australia.
21
3.9-Hexachlorobenzene
(HCB)
First introduced in 1945 to treat seeds, HCB kills fungi that affect food crops. It was widely used to control wheat bunt. It is also a byproduct of the manufacture of certain industrial chemicals and exists as an impurity in several pesticide formulations. When people in eastern Turkey ate HCB-treated seed grain between 1954 and 1959, they developed a variety of symptoms, including photosensitive skin lesions, colic, and debilitation; several thousand developed a metabolic disorder called porphyria turcica, and 14% died. Mothers also passed HCB to their infants through the placenta and through breast milk. In high doses, HCB is lethal to some animals and, at lower levels, adversely affects their reproductive success. HCB has been found in food of all types. A study of Spanish meat found HCB present in all samples. In India, the estimated average daily intake of HCB is 0.13 micrograms per kilogram of body weight. (UNEP, 1998)
3.10-Mirex
This insecticide is used mainly to combat fire ants, and it has been used against other types of ants and termites. It has also been used as a fire retardant in plastics, rubber, and electrical goods. Direct exposure to mirex does not appear to cause injury to humans, but studies on laboratory animals have caused it to be classified as a possible human carcinogen. In studies mirex proved toxic to several plant species and to fish and crustaceans. It is considered to be one of the most stable and persistent pesticides, with a half life of up to 10 years. The main route of human exposure to mirex is through food,
particularly meat, fish, and wild game.
(UNEP, 1998)
3.11-Toxaphene
This insecticide is used on cotton, cereal grains, fruits, nuts, and vegetables. It has also been used to control ticks and mites in livestock. Toxaphene was the most widely used pesticide in the US in 1975. Up to 50% of a toxaphene release can persist in the soil for up to 12 years. For humans, the most likely source of toxaphene exposure is food. While the toxicity to humans of direct exposure is not high, toxaphene has been listed as a possible
22 human carcinogen due to its effects on laboratory animals. It is highly toxic to fish; brook trout exposed to Toxaphene for 90 days experienced a 46% reduction in weight and reduced egg viability, and long-term exposure to levels of 0.5 micrograms per liter of water reduced egg viability to zero. Thirty-seven countries have banned toxaphene, and 11 others have severely restricted its use. (UNEP, 1998)
3.12-Aldrin
Aldrin is a pesticide applied to soils to kill termites, grasshoppers, corn rootworm, and other insect pests. It can also kill birds, fish, and humans, where the fatal dose for an adult male is estimated to be about five grams. Humans are mostly exposed to Aldrin through dairy products and animal meats. According to an Indian study, the quantity of Aldrin and its byproduct Dieldrin that is consumed on a daily basis is 19 micrograms per person. The use of Aldrin has been severely restricted and banned in different countries around the world.
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CHAPTER 4
POLYCHLORINATED BIPHENYLS
4.1-Identification
Polychlorinated biphenyls or PCBs are part of the large family of Persistent Organic Pollutants (POPs). They are aromatic compounds formed by two phenyl rings, which most hydrogens atoms are substituted by chlorine atoms. PCBs have a chemical formula of C12H(10-m-n)Cl(m+n) where the sum of m and n is the number of the substituted chlorine atoms present in the molecule (IARC, 2016).
There are 209 different PCBs that are also called congeners. They are numbered according to a strategy made by Ballschmiter & Zell called “BZ number” that attributes a number from 1 to 209 to each congener; the BZ number correlates the structural arrangement of the PCB congener and ascending order of number of chlorine substitutions within each sequential homologue (Ballschmiter & Zell, 1980). This shorthand nomenclature has become quite popular and is convenient for many uses, although it is important to note that it obscures the chemical identity of the congener and does not strictly follow the IUPAC rules (IARC, 2016). A number assigned by the Chemical Abstracts Service and maintained in the CAS Registry database (American Chemical Society, 1999). The registry number is
24 Table 2 shows the correspondence between the BZ number and the position of the chlorine atoms on the biphenyl rings of the PCB congeners.
Table 2 Polychlorinated Biphenyls and Polybrominated Biphenyls (IARC, 2016)
The chemical structure of a congener has different ortho, meta and/or para substitutions of the chlorine atoms. The ortho substitutions are found in positions 2, 2’, 6, and 6’ of the two benzene rings. The meta substitutions are found in positions 3, 3’, 5 and 5 and finally the para substitutions can be found in positions 4 and 4’. Their configurations can be planar, in which the two benzene rings are on the same plane, or non-planar in which the benzene
25 rings are perpendicular to each other, forming a 90◦ angle (Faroon, Jones, & de Rosa, 2000). The replacement of the hydrogen atom by the chlorine atom in the ortho position causes the molecule to rotate and thus becomes non-planar. This is due to the fact that the chlorine atom is bigger and bulkier than the hydrogen atom. In case of non-ortho substitutions or a mono-ortho substitution the congeners may assume a planar or co-planar configuration (MD Erickson, 1997).
Figure 4. shows 3 examples of substitutions on the biphenyl rings:
Figure 4. Polychlorinated biphenyls and links to cardiovascular disease (Perkins et al., 2016): A. General structure of a polychlorinated biphenyl with relevant nomenclature
highlighted B. Structure of 3,3′,4,4′,5-Pentachlorobiphenyl (PCB 126), a coplanar,
non-ortho substituted PCB C. Structure of 2,2′,4,4′,5,5′-Hexachlorobiphenyl (PCB 153), a
26 Figure 5. 3D structure of some PCB congeners.
Figure 5. Polychlorinated Biphenyls and Polybrominated Biphenyls (IARC, 2016)
In the biphenyl molecule, the two aromatic rings can rotate about the connecting single 1,1′-bond. As with all molecules, there is a low-energy preferred conformation. With PCBs, this conformation is dependent on the degree of chlorine substitution, since chlorine is larger than hydrogen and creates more steric hindrance to the rotation (MD. Erickson, 2001). According to studies made by the Stockholm Convention on Persistent Organic Pollutants (POPs), the most concentrated PCBs found in the environment, in food, in human fluids and tissues are the following: 28, 52, 101, 138, PCB-153, PCB-180 (Convention, 2008).
27
4.2-Chemical and Physical Properties
PCBs are classified as pure congeners or commercial products. The pure congeners are transparent or slightly yellowish crystalline compounds with no evident odor or smell. The commercial products, however, are viscous liquid mixtures. Their viscosity depends on the quantity of substituted chlorine atoms. Their color ranges from light yellow to dark
brown (IARC, 2016).
One of the most important properties of PCBs is the fact that they are inert compounds, meaning that they are resistant to acidic, alkaline and oxidant environments. They are thermally stable to a certain extent; this is due to the fact that they can be destroyed by chemical and biochemical processes under certain or severe conditions (IARC, 2016) PCBs are nonpolar compounds, which makes them slightly soluble to insoluble in water. The degree of chlorination plays an important role in the solubility of the PCBs: the solubility decreases with increased chlorination. The range of solubility varies from 0.0012 to 4830 μg/L. Ortho-substituted congeners however have a higher solubility in water; it is due to hydrogen bonds. PCB congeners are soluble in nonpolar organic solvents and biological liquids. The degree of chlorination plays an important role as well in determining the melting and boiling points of the PCBs. Their melting points range from 25℃ to 309℃ and their boiling points range from 205℃ to 450℃. The more chlorinated the PCB congener is, the higher the melting and boiling point is. The octanol/water partition coefficient (Kow) is also a parameter to be considered in determining the properties and aspects of PCBs (IARC, 2016). Kow is defined as the ratio of a chemical’s concentration in the octanol phase to its concentration in the aqueous phase in a two phase octanol/water system. Figure 6 shows the correlation between the BZ numbers and the log Kow ; they are directly proportional, meaning that the more lipophilic the compound, the more it drifts away from the aqueous phase (MD. Erickson, 2001).
28 Figure 6. correlation between Octanol/water partition coefficient (Kow) and BZ number.
Figure 6. BZ= Ballschmiter and Zell number. Polychlorinated biphenyls and Polybrominated biphenyls
(IARC, 2016) .
PCBs are characterized by Henry’s law constants which is a measure of the equilibrium distribution coefficient between air and water, that tend to decrease with a higher degree of chlorination. Less chlorinated PCB congeners have a considerably higher vapour pressure (1–2 Pa at 25 °C for monochlorobiphenyls) than the more highly chlorinated congeners (1.4 × 10−6 Pa for decachlorobiphenyl) (Shiu & Mackay, 1986). The vapour pressure of PCBs is higher when the congener is less chlorinated ; it is determined by Henry’s law constants (a measure of the equilibrium distribution coefficient between air and water)
29 (Shiu & Mackay, 1986). PCBs are highly combustible but safe compounds the products of their combustion are hazardous materials that are life threatening. They include polychlorinated dibenzodioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs) (IARC, 2016).
Table 3. Characteristics of the studied PCB congeners: BZ number, CAS registry number and log Kow of he studied PCB congeners.
BZ number CAS number log Kow
28 7012-37-5 5.67 52 35693-99-3 5.84 101 37680-73-2 6.38 118 31508-00-6 6.74 138 35065-28-2 6.83 153 35065-27-1 6.92 180 35065-29-3 7.36
Table 3. BZ= BZ number (Ballschmitter and Zell, 1980), CAS= Chemical Abstract Service, Kow =
30 Figure 7. shows the model for the behavior of persistent organic pollutants in the air-plant-soil system (Ashraf, 2015).
31
4.3-Environmental Occurrence and Exposure
PCBs are found worldwide at measurable levels in all environmental media (soils and sediments, water, air), in wildlife, and also probably in the body of every human. Human exposure to PCBs occurs mostly via ingestion of contaminated food, but also via inhalation and dermal absorption (IARC, 2016). Soils are natural sinks for persistent and lipophilic compounds such as PCBs; PCBs are absorbed by the organic carbon of the soil, and once absorbed they are relatively persistent (Buckley-Golder, 1999). PCBs enter the soil via different pathways: industrial releases from manufacture, use and disposal, accidental releases, atmospheric deposition, application of sewage sludge, and erosion and leachate from nearby contaminated areas. PCBs in organic liquids may be dissolved by soils and then migrate with the solvent. The congener patterns of PCBs in soils and sediments change over time as a result of the activity of aerobic bacteria (that degrade less chlorinated congeners) and anaerobic bacteria (that can cause partial dechlorination of more highly chlorinated congeners) (Hardell, Tilander, Welfinger-Smith, Burger, & Carpenter, 2010). The patterns found in environmental biota are often referred to as “weathered,” since they result from alterations in the composition of a mixture (e.g. resulting from bio accumulative and metabolic processes in higher biological organisms and through bacterial action, exposure to ultraviolet radiation, etc.). “Weathering” processes result in PCB patterns with either a higher chlorinated fraction or congeners with higher bioaccumulative properties compared with the commercial products. “Weathering” must be considered when assessing PCB-associated risks based on studies with experimental animals exposed to commercial PCB products.Water is a major pathway for migration of PCBs, both in solution and particulate-bound, although PCBs are lipophilic and generally not very soluble in water. Less chlorinated PCB congeners have greater solubility than more highly chlorinated congeners (IARC, 2016).
Air is another major pathway for PCB migration. PCBs are semivolatile compounds and, as with water solubility, less chlorinated congeners are more volatile than more highly chlorinated ones (Totten, Stenchikov, Gigliotti, Lahoti, & Eisenreich, 2006). There is extensive evidence that PCBs in aquatic systems exchange with PCBs in air (Bamford,
32 Ko, & Baker, 2002). Air transport of PCBs can occur in either the vapour phase or particulate-bound, thus contributing to global pollution and PCB contamination of remote regions of the earth. PCBs in air come from several direct or indirect sources, including industrial facilities, military sites, contaminated bodies of water, landfills and hazardous waste sites, electric arc furnaces, incineration and other forms of combustion, sewage sludge applied to agricultural lands, and construction materials, including in paints (Hu & Hornbuckle, 2010), caulking, light ballasts, floor sealants, and adhesives and plasticizers in older buildings (Wallace, Basu, & Hites, 1996). PCBs from soil, sediment, air and water enter the food-chain by uptake and bioaccumulation in plants and animal fats. There is significant biological magnification of PCB concentration as PCBs move up the food-chain. PCB concentrations vary depending on the degree of bioaccumulation, and are usually highest in carnivorous fish coming from contaminated waters. PCBs are found in the fat of all meat animals, in all dairy products containing fat, and in eggs (Registry & (ATSDR), 2000); (Institute of Medicine, 2003), sometimes at high concentrations due to local contamination of grasses, and feeding practices in some countries. Also, it is not uncommon to feed domestic animals with fish meal or oil, or waste animal fats, which results in recycling of PCBs (Institute of Medicine, 2003). For example, farmed salmon fed with concentrated fish meal or fish oil containing significant amounts of PCBs showed elevated concentrations of PCBs (Hites et al., 2004). PCBs found in food are typically of higher chlorination, since they are less volatile and more biologically persistent in plants and animals than the lower congeners. Another important route of exposure to PCBs is inhalation; however, it is difficult to determine the relative contribution of inhalation compared with ingestion. (Harrad, Hazrati, & Ibarra, 2006) have suggested that inhalation may account for 4–63% (median, 15%) of overall exposure in humans. PCBs may be attached to indoor dust, which can be either ingested or inhaled. Individuals who spend significant periods of time in the presence of either outdoor or indoor vapour-phase PCBs will have continuous exposure that is not reflected in measurements of “total” PCBs, because the less chlorinated congeners are more rapidly metabolized and excreted by the human body, as shown in Figure 8 (Johansson, Hanberg, & Wingfors, 2003). Concentrations of different PCB congeners were measured in blood from individuals living in houses where PCB-containing sealant was used. Concentrations of most congeners were only slightly elevated (1.2 to 3.2 times), but the two congeners with a low level of chlorination (PCB-28 and PCB-66) were detected at much higher concentrations (30 and 9 times, respectively) in contaminated flats than in control flats.
33 Figure 8. Blood PCB concentrations in individuals living in PCB-contaminated flats relative to individuals living in control flats
Figure 8. PCB in building sealant is influencing PCB levels in blood of residents (Johansson et al., 2003)
Dermal absorption of PCBs may occur primarily in the occupational setting, but also through contact with contaminated sediments or other applications to the skin (Wester, Maibach, Sedik, Melendres, & Wade, 1993; Wester, Mobayen, & Maibach, 1987). Less chlorinated congeners are more rapidly absorbed through the skin than more highly chlorinated congeners (Garner & Matthews, 1998). Congener patterns in the general human population are always different from any pattern found in commercial PCB products (Patterson et al., 2009). The factors that may explain this are:
34
• The general public is exposed to multiple sources of PCBs, only rarely to a single commercial product.
• There may be more than one route of exposure for almost all matrices/animals/humans.
• Dechlorination occurs to varying degrees in sediments, soils, water and air. Commercial PCB products will volatize to some degree, and in doing so, will lose less chlorinated congeners.
• PCBs ingested by fish and animals will be metabolized (to less chlorinated and hydroxylated congeners) to different degrees. Thus most food stuffs will demonstrate a shift in the congener profile compared to the commercial product.
• When inhalation is the major route of exposure, there is selective exposure to the more volatile, less chlorinated and less persistent congeners.
• Genetic differences among individuals may confer differences in metabolic activity and selective metabolism of different congeners.
35
4.4-Population Biomonitoring:
The presence of PCBs in serum or blood may reflect exposure from any source (Dewailly, Flaugnatti, Haguenoer, & al, 1988). Results from different studies in humans have indicated that measurements of PCBs in serum generally reflect cumulative past exposure. Many PCB congeners can remain in the body for years after exposure, although some of the less chlorinated congeners are more volatile and consequently show shorter residence times.
4.4.1- USA
Hopf et al. provided an extensive review of reports on background levels of PCBs in the USA population (Hopf, Ruder, & Succop, 2009). The NHANES survey over the period 2002–2004 reported increasing concentrations of PCBs with age, and concentrations were higher in men than in women, and higher in African-Americans and Caucasians than in Mexican-Americans (Patterson et al., 2009).
4.4.2-EUROPE
Several European studies on human biomonitoring have reported blood PCB
concentrations in adults or children. Past environmental contamination in industrial areas has polluted surrounding soils and forage, leading in turn to high blood PCB concentrations in the adult population. Age-related accumulation of PCBs has been observed in these (Patterson et al., 1994) (Apostoli et al., 2005) (Park, Lee, Kang, & Chang, 2007), and may be partially explained by historical high levels of exposure in the 1970s.
In Germany, Environmental Surveys (GerES) were carried out in 1998 becker (Becker et al., 2002) and during 2003–2006 becker (Becker et al., 2009). GerES data show mean blood concentrations for the sum of PCBs of 1.3–1.7 µg/L in 1998 and of 286 ng/L in the more recent survey, with strong difference (factor of 5.6) between age groups 18–25 and 66–69 years.
36 In Belgium in 2007–2011 (Schoeters, Colles, Den Hond, & al., 2011), the Flemish Human Environmental Survey reported average blood PCB concentrations of 333 ng/g lipid. Average concentrations in the United Kingdom in 2003 were 170 ng/g lipid (Thomas, Wilkinson, Hodson, & Jones, 2006) . In Spain in 2004–2008, concentrations of the most common PCBs were in the range of 21.8 to 38.9 ng/g lipid (Ibarluzea et al., 2011). In France, blood analysis in the general adult population was first carried out in 1986 dewailly (Dewailly et al., 1988) and then in 2006–2007 (French Nutrition and Health survey (Fréry et al., 2013). The reported blood PCB concentrations in populations in industrial polluted areas such as Italy turci (Turci et al., 2004); (Apostoli et al., 2005); (Turrio-Baldassarri et al., 2008) and Slovakia (Jursa, Chovancová, Petrík, & Loksa, 2006) were high compared with those in non-occupationally exposed populations such as in Sweden (Salihovic et al., 2012).
The most frequently detected di-ortho-chlorine-substituted PCBs in population studies are PCB-138, PCB-153, and PCB-180 (Glynn et al., 2000), accounting for 65–78% of the measured sum of total (Needham et al., 2005). The seven PCB indicator congeners (118, 138, 153, 156, 170, 180, and 194) contributed to 99% of the total PCB levels. In several countries in the European Union, a clear decrease in blood concentrations of PCBs has been observed in the last two decades. Overall, mean whole blood concentrations of PCB-138, PCB-153, and PCB-180 appear to have decreased by approximately 80% in 20 years (Link et al., 2005) ; (Hagmar et al., 2006) ; (Agudo et al., 2009) ; (AMAP, 2009).
4.4.3- AFRICA
Rollin et al. (Röllin, Sandanger, Hansen, Channa, & Odland, 2009) reported overall low blood concentrations of PCBs (99, 118, 138, 153, 170, 180 and 187) in delivering mothers from seven geographical regions in South Africa. Large regional differences were observed, with women from rural areas having the lowest levels of PCBs. PCB-138 and PCB-153 were found in the blood of mothers from all of the 61 sites studied at geometric mean concentrations of 3.56 and 3.2 ng/g lipid, respectively. (Ahmed, Loutfy, & El Shiekh, 2002)reported the sum concentration of 29 congeners in blood from Egyptian women to be 61.9 ng/g. Weiss et al. (Weiss et al., 2006) reported concentrations of
PCB-37 153 in infertile women in the United Republic of Tanzania to be 0.17 µg/kg. PCB concentrations were significantly higher in men than in women (Ben Hassine et al., 2014).
38
4.5-Regulations and Guidelines
For Parties to the Stockholm Convention on Persistent Organic Pollutants (POPs) (UNEP, 2001) presently 179 Member States, the production of PCBs is totally prohibited, although the presence of PCBs in equipment is allowed to continue until 2025. The environmentally sound management of waste containing or contaminated with PCBs at a content above 0.005% must be achieved by 2028 (IARC, 2016). Annex I of the Basel Convention on the Transboundary Movements of Hazardous Wastes and Their Disposal (UNEP, 2011) defines a category of hazardous waste specific to PCBs: “Y10 waste substances and articles containing or contaminated with PCBs and/or polychlorinated terphenyls (PCTs) and/or polybrominated biphenyls (PBBs).” Additionally, Annex VIII defines as “hazardous” any electrical waste containing or contaminated with PCBs at a concentration greater than 50 mg/kg. The Basel Convention is legally binding for 179 countries (status in 2013). The Codex Alimentarius Commission, recognizing the importance of prevention of human exposure through source-directed measures (i.e. strict control of industrial and agricultural processes that may generate and release PCDDs, PCDFs, and PCBs), adopted the Code of Practice Concerning Source Directed Measures to Reduce Contamination of Food with Chemicals (Codex Alimentarius, 2001) and the Code of Practice for the Prevention and Reduction of Dioxin and Dioxin-like PCB Contamination in Foods and Feeds (Codex Alimentarius, 2006). No limits in foodstuffs were included, but management options were
recommended (IARC, 2016).
Air quality guidelines for PCBs have not been established, because exposure by direct inhalation generally constitutes only a small proportion of total exposure, in the order of 1– 2% of the daily intake from food. Although this air concentration is only a minor contributor to direct human exposure, it is a major contributor to contamination of the food-chain world (WORLD HEALTH ORGANISATION, 2000).
39
CHAPTER 5
MATERIALS AND METHODS
5.1-Study Design and Population
The study design was a cross-sectional survey made at the Saint Joseph University. A list of currently enrolled students and employees was obtained from the university’s records and registration department. 316 students and employees were randomly selected for recruitment using an automated random selection procedure. To be eligible for the study, the participants had to be Lebanese, aged between 17 and 65 years and should have spent the last 10 years in Lebanon. Each participant completed a “face-to-face” questionnaire comprising socio-demographic information, details about their housing locations, smoking and dietary habits. In order to determine the geographical vicinity to the sources of pollution, the distance in kilometers (km) from each participant’s housing location to the nearest source of pollution was determined. Participants were therefore categorized by living within or out of 5 km range from known sources of exposure. A previously validated food-frequency questionnaire (FFQ) was administered to the participants in order to assess the intake of 14 major food categories, over the last year, including culturally adapted food items. These categories comprised: cereals and grains, dairy products, fruits, vegetables, legumes, meats including fish and shellfish, fast-food, nuts and seeds, oils and fats, salty snacks, sweets and beverages. The frequency of consumption of each food item was noted per day, week, month or year, in addition to the number of portions consumed each time. The portion sizes of food were estimated using measuring cups and food models (Biró, Hulshof, Ovesen, & Amorim Cruz, 2002)
40
5.2- Confidentiality and Ethical Considerations
This study was approved by the Ethics Committee of “Saint Joseph University of Beirut” and “Industrial Research Institute” (IRI). Participants were fully informed about the purpose and procedures of the study before reading and signing the informed consent form. The confidentiality of the results was maintained.
5.3-Blood Sampling and Storage
Blood serum was collected from fasting subjects in 5ml vacutainer tubes (BD vacutainer, Plymouth, United Kingdom) without anticoagulants and were centrifuged at 3,500rpm for 15 minutes. The serum was divided in 2 aliquots: the first one was analyzed for total lipids using an automatic biochemistry analyzer (HumaStar) and the second one was stored in “eppendorf” tubes at -80 Celsius degrees to be tested for PCBs at the Lebanese laboratories “Industrial Research Institute” (IRI), using gas chromatography coupled to an ion trap mass spectrometer detector.
5.3.1 - SAMPLE PREPARATION
The human serum samples (10 ml) were homogenized manually by shaking for 1 min; 5 ml of human serum was centrifuged. 100 µL Internal Standard (PCB103 + PCB193) (125 ng/mL in isooctane) was added and the samples were sonicated for 20 min. Then, the samples were stored overnight at 4°C before adding 1.5 mL formic acid and 2 mL demineralized water.
5.3.2 - SAMPLE EXTRACTION
The SPE cartridge was washed and conditioned with 5 ml DCM (dichloromethane) and 5 ml methanol-water with a flow of 1.5 mL/min; the sample extract was added to the SPE cartridge with a flow of 0.4 mL/min; Then, the cartridge was dried under a nitrogen stream at 20 psi for 10 min, followed by centrifugation (15 min, 4000 rpm); the PCBs were
41 eluted with 5 ml hexane followed by 3 ml DCM and the extracts were concentrated to approximately 1 ml in a water bath (≤40ºC) under a nitrogen flow.
5.3.3 – SAMPLE PURIFICATION
The samples were cleaned up over an Al2O3 column, fractioned over a 1.5% (w/w) deactivated silica column and then purified over an acidic silica column (40% H2SO4 -silica).
5.3.4 – INSTRUMENTAL ANALYSIS
Analysis of PCBs was performed using a gas chromatograph (Agilent Technologies) equipped with a split/splitless injector port and an electron captor detector. The injector was operated in the splitless mode (1.5 min). Helium (He) served as a carrier gas with a flow-rate of 1 ml/min. Chromatographic separation was accomplished with a CP-SIL 8 CB (Agilent Chrompack CP8753), (60 m x 0.25 mm, i.d.; 0.25 µm film thickness) capillary column. The temperature program used was 90ºC initial for 3 min, increased at 30ºC/min to 200ºC for 15 min, then at 5 ºC/min to 265ºC for 5 min and finally at 3ºC/min to 27 ºC for 15 min. The detector was with an N2 make-up gas. A CP-SIL 19 CB (Agilent Chrompack CP8722), (60 m x 0.25 mm, i.d.; 0.25 µm film thickness) capillary column was used as second column to confirm the identifications of PCBs in human serum. The corresponding limit of detection (LOD) was 1.4 ng/l.
5.4 – Statistical Analysis
Given that more than 30% of the values were not detected, a multiple imputation (MI) analysis was carried out. All missing data were created by imputing random values after generating multiple linear regression and creating standardized β which provides M estimators of the parameters of interest. The independent predictors used were age, BMI, fish, shellfish and dairy products consumption. The upper bound was fixed and known in advance (=1.4 ng/l). PCBs with concentrations below the LOD were also reported as LOD/2 and the results were compared to those obtained from the MI method. Resulting
42 PCBs concentrations were adjusted for total lipids. Continuous variables were determined as means and standard deviations while categorical variables were determined as frequencies and percentages. Geometric means (GM) (log10 and square root of quantitative variable) was used in case of non normality of distribution. Bivariate analysis (spearman test) was carried out to determine linear correlation between continuous variables.
43
CHAPTER 6
RESULTS AND DISCUSSIONS
6.1-Characteristics of the Studied Population
Demographic and anthropometric characteristics of the participants are summarized in Table 4. The study sample was 42.7 % men and 57.3 % women. Mean age of the participants was 25.58 years. 97.5% of our participants reside in Beirut and Mount Lebanon. Fifty percent live within 5 km from an ongoing or accidental source of exposure. The Body Mass Index (BMI) of the participants varied between 16.9 and 45.93 kg/m2 with a mean value of 25.37 kg/m2 for men and 23 kg/m2 for women. The mean percentage of body fat was 20.94% in men and 30.66% in women. 60.1% were nonsmokers, while 39.9% had ever smoked.
44
Table 4. Characteristics of the study population (n = 316)
Variables N % Gender • Male • Female 135 181 42.70 57.30 Age (mean in years ± SD) 25.58 ± 0.15
• Men • Women 25.19 ± 0.13 25.88 ± 0.16 Governorate of residence • Beirut • Mount Lebanon • North • Bekaa • South • Nabatieh 110 198 3 1 2 2 34.8 62.7 0.9 0.3 0.6 0.6 Geographical vicinity to source
of exposure ≤ 5 km > 5 km 160 156 50.63 49.36 BMI (mean in Kg/m2 ± SD) 24.01 ± 4.32 • Men • Women 25.37 ± 4.26 23.00 ± 4.10 BMI classification (Kg/m2) • Underweight (< 18.5) • Normal weight (18.5-25) • Overweight (25-30) • Obese (> 30) 21 177 92 26 6.60 56.00 29.10 8.20 Percentage of body fat (mean ±
SD) • Men • Women 20.94 ± 8.19 30.66 ± 8.68
45
Percentage of body fat (%)
< 30 ♀ and 25 ≥ 30 ♀ and 25 ♂ 173 130 57.10 42.90 Smoking • Non smoker • Previous smoker • Current smoker 190 19 107 60.10 6.00 33.90
Fish and shellfish consumption 1 portion per week 2 - 4 portions per week
≤ 5 portions per week
49 112 144 16.10 36.70 47.20
Dairy products consumption < 2 portions per day 2 - 3 portions per day
> 3 portions per day
85 99 120 28.00 32.60 39.50
All quantitative variables were normalized by calculating the geometric mean ± SD. Elevated percentage of body fat is defined by a percentage of fat ≥ 25% for men and 30% for women (Lysen and Israel, 2012)
46
6.2-Polychlorinated Biphenyls Concentrations in the Serum Samples
In order to determine which value to attribute to the PCB concentrations below the LOD, results obtained from multiple imputation and LOD/2 methods were compared (Table 5). Given that no differences were found, LOD/2 was used as a replacement for all missing values obtained while doing our analytical analysis for PCBs.
Table 5. Geometric means of the PCBs obtained with MI and LOD/2 methods respectively PCB 28 PCB 52 PCB 101 PCB 138 PCB 153 PCB 180 ∑PCB MULTIPLE IMPUTATIONS (MI) 0.96 0.28 0.55 1.92 3.01 3.49 10.22 ND = LOD/2 0.86 0.34 0.58 1.88 2.95 3.72 10.34
LOD= Limit of detection ND= Not Detected
Missing data are unavoidable in epidemiological and clinical research but their potential to undermine the validity of research results has often been overlooked in the medical literature. This is partly because statistical methods that can tackle problems arising from missing data have, until recently, not been readily accessible to medical researchers. However, multiple imputation, a relatively flexible, general purpose approach to dealing with missing data—is now available in standard statistical software (Roystone,P. ; Roystone, P.) ; (Online.) ; (Institute) making it possible to handle missing data semi routinely. Results based on this computationally intensive method are increasingly reported.
47
Figure 9. Multiple Imputation
Table 6 shows the concentrations of PCB in human serum as well as the percentage of measurements above the LOD for each congener. Since PCB concentrations depend on the amount of lipid in the sample, the results are expressed as ng/g serum lipid. The mean level of lipid in serum was 523.6 ± 120.5 mg/dl. The concentrations of individual serum PCB congeners were above the LOD in 56.3 to 59.2% of the cases. PCB congeners 138, 153 and 180 predominated and accounted for about 15.7, 25.2 and 34.1% of the amount of the indicator congeners analyzed. This is in agreement with previous findings (Ben Hassine et al., 2014) ; (Černá et al., 2008) ; (Hirai, Fujimine, Watanabe, Nakano, & T., 2005) ; (Huetos et al., 2014) ; (Kalantzi et al., 2011) ; (Luis A Henríquez-Hernández & O.P.L., 2011); (Pavuk et al., 2014); (Ulutaş et al., 2015) ; (Zani et al., 2013). The main contributor was PCB 180, similarly to the BIOAMBIENT study (Huetos et al., 2014), whereas the main contributor found in other studies was PCB 153 (Ben Hassine et al., 2014; Kalantzi et al., 2011; Pavuk et al., 2014; Ulutaş et al., 2015). In fact, the higher the chlorination is, the more a congener is persistent and resistant to biodegradation. As shown in Table 7, PCB
48 180 has the highest number of chlorine atoms with 7 atoms , while PCB 138 and 153 have 6 atoms, PCB 101 has 5, PCB 52 has 4 and PCB 28 has 2 (Agence de l’eau Seine-Normandie & Chevreuil, 2009). The lower chlorinated congeners are more rapidly metabolized by the human body, while the higher chlorinated congeners tend to bioaccumulate in serum lipids and in the adipose tissue (Grimm et al., 2015). PCB 25, 52 and 101 can reach half-lives of 5 years in the human body. In contrast, the higher chlorinated PCBs can persist for 10 to 47 years (Agency for Toxic Substances and Disease Registry (ATSDR), Public Health Service, 2000; European Food Safety Authority (EFSA), 2005; Ritter et al., 2010). This explains the high contribution of PCB 138, 153 and 180 (75%) to the total PCB level. Another contributor is the source of contamination. Historically, technical mixtures of PCBs were manufactured under several trademarks, among which the most widely used was Aroclor (Monsanto Company, United States). Several types of Aroclor mixtures exist depending on the composition in chlorine in PCBs (Agence de l’eau Seine-Normandie et al., 2009; Agency for Toxic Substances and Disease Registry (ATSDR), U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES, Public Health Service, 2000). In particular, Aroclor 1260 is richer in PCB 180 than in PCB 153, whereas Aroclor 1254a contains more PCB 153 than PCB 180 (U.S. Environmental Protection Agency, 2006). Clophen A-60 (Germany) and Phenoclor DP-6 (France) are similar in composition to Aroclor 1260 whereas Kanechlor 500 (Japan) is similar to Aroclor 1254 PCBs. When used as coolants in the electrical devices, Aroclor 1254 was used to fill transformers and capacitors while Aroclor 1260 was used to fill only transformers (Agence de l’eau Seine-Normandie et al., 2009; Agency for Toxic Substances and Disease Registry (ATSDR), U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES, Public Health Service, 2000; INERIS, 2011). Therefore, the higher contribution of PCB 180 in the present study to the detriment of PCB 153 could be due to the contamination in PCBs by transformers in the power sector.
49 Table 6. PCB concentrations (in nanograms per gram lipid) found in human serum samples (n =
316)
% >LOD % >LOD Min Q2 Q4 Max
Men Women PCB 28 56.30 55.60 56.90 < LOD 2.17 5.60 18.19 PCB 52 58.20 58.50 58.00 < LOD 0.34 1.49 17.38 PCB 101 57.60 57.80 57.50 < LOD 0.60 3.56 17.38 PCB 138 59.20 58.50 59.70 < LOD 8.17 16.67 50.12 PCB 153 58.20 58.50 58.00 < LOD 16.42 45.89 87.09 PCB 180 58.20 58.50 58.00 < LOD 24.06 66.76 169.82 ∑PCB138,1 53,180 - - - < LOD 52.57 127.97 302.00 ∑PCB - - - < LOD 57.90 134.58 338.84
LOD= Limit of detection; Q2= second quartile or the 50th percentile, also known as the median ; Q4= fourth
quartile or 95th percentile
The sum PCBs concentrations defined as the sum of all detected and quantified congeners ranged from below the Limit of Detection (< LOD) to 338.8 ng/g lipid with geometric mean (± SD). A percentile is a measure used in statistics indicating the value below which a given percentage of observations in a group of observations falls. For example, the 50th percentile is the value below which 50% of the observations may be found. The 25th percentile is also known as the first quartile (Q1), the 50th percentile as the median or second quartile (Q2), the 75th percentile as the third quartile (Q3) and the 95th percentile as the fourth quartile (Q4).
50 Table 7: Identification of PCB congeners
PCB-180 2,2',3,4,4',5,5'-Heptachlorobiphenyl PCB-153 2,2',4,4',5,5'-Hexachlorobiphenyl PCB-138 2,2',3,4,4',5'-Hexachlorobiphenyl PCB-101 2,2',4,5,5'-Pentachlorobiphenyl PCB- 52 2,2',5,5'-Tetrachlorobiphenyl PCB-28 2,4,4'-Trichlorobiphenyl
