Polycyclic Aromatic Hydrocarbons

Polycyclic Aromatic Hydrocarbons

Polycyclic Aromatic Hydrocarbons G Purcaro, S Moret, and LS Conte, Department of Food Science, Udine, Italy ã 2016 Elsevier Ltd. All rights reserved. ...

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Polycyclic Aromatic Hydrocarbons G Purcaro, S Moret, and LS Conte, Department of Food Science, Udine, Italy ã 2016 Elsevier Ltd. All rights reserved.

General Overview Polycyclic aromatic hydrocarbons (PAHs) are a large group of carcinogenic organic compounds, which originated from incomplete combustion or pyrolysis of organic matter. They are formed by two to six fused aromatic rings (linear, cluster, or angular arrangement), and they solely consist of carbon and hydrogen. They can present alkyl substituents, depending on the generating process. Although the International Union of Pure and Applied Chemistry defined a systematic nomenclature for these compounds, many of them have maintained their common name, reflecting the initial isolation from coal tar (e.g., naphthalene and pyrene), their color (e.g., fluoranthene and chrysene; the latter erroneously, because contaminated with naphthacene, which is orange, when first isolated), and their shape (e.g., coronene and ovalene). The numbering and naming of PAHs are based on a few basic rules: Rings are drawn with two vertical sides (when possible), with as many rings as possible in a line and the rest of the structure sitting in the top right quadrant; the numbering starts from the first carbon atom not engaged in ring fusion in the right-hand ring and proceeds clockwise; the name of a fused-ring system is made up of a prefix of the fixed part (e.g., benzo) followed by an italic letter denoting the bond of the base (e.g., a refer to the 1,2bond) and the name of the parent compound follows (e.g., benzo[a]anthracene reported in Figure 1). A list of the most analyzed ones is reported in Table 1, along with their chemical structure, molecular formula, molecular weight (mw), solubility in water at 25  C, partition coefficient octanol–water (log Kow), and abbreviation (that will be used all over the chapter). PAHs are solid at ambient temperature and are the least volatile of the hydrocarbons (the boiling points of the PAH are markedly higher than those of the n-alkanes of the same carbon number). Nonetheless, light PAHs (2–3 rings) are present in the environment mainly in the vapor form, while heavier PAHs (more than five rings) are predominantly adsorbed on organic particulate matter, usually on small particles (<2.5 mm); four-ring compounds have an intermediate behavior. The high mw and the absence of polar substituent groups make these compounds very insoluble in water, but the presence of detergent or organic matter causing emulsions in water, or PAHs adsorbed on suspended particles, can increase the content of PAHs in wastewater or in natural waters. Although PAHs are chemically stable and poorly degraded by hydrolysis, they are susceptible to oxidation and photodegradation, and this behavior is particularly relevant to possible losses during analysis.

Legislation In the European Union, the necessity of a harmonized European legislation appeared evident after 2001, when a batch of

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pomace oil entering the Czech Republic was found highly contaminated. Following this event, some states fixed their own limits for PAH contamination in edible oil, mainly considering the 16 PAHs highlighted by the US Environmental Protection Agency (EPA) (called 16 EPA PAHs). These different limits among the European Member states caused several commercial problems. In 2002, the Scientific Committee on Food (SCF) published an opinion where 15 PAHs, over 33 assessed for toxicity information, were identified as both genotoxic and carcinogenic. Furthermore, BaP was pinpointed as a suitable marker for PAH contamination in food. Subsequent to the opinion, the European Commission (EC) introduced a new regulation (Reg. 208/2005, shortly followed by Reg. 1881/2006) harmonizing the PAH legislation among all the member states and fixing a limit for the presence of BaP only, used as a marker of PAH contamination. The EC also published a recommendation (Rec. 2005/108/EC), requiring all the member states to investigate the presence of the 15 PAHs highlighted by the SCF plus the one (BcF) considered by the Joint FAO/WHO Expert Committee on Food Additives (JECFA) (called the 16 EU PAHs) to review the limits already set and to add new food classes by 1 April 2007. No new limits were set in 2007, but the European Food Safety Authority (EFSA) raised doubts on the suitability of BaP as a marker. In 2008, it was definitely concluded that BaP was not appropriate, while the sum of eight heavy PAHs (PAH8) (BaA, Ch, BbF, BkF, BaP, DBahA, BghiP, and IP) or of a subgroup of four (PAH4) (BaA, Ch, BbF, and BaP) was suggested. Following the EFSA opinion, the EC enacted a new regulation in 2011 (Reg 835/2011) fixing new limits considering both the BaP only and the PAH4. Additional food classes compared to the previous legislation were added, but the limits for these newly introduced classes came into force later than the previously regulated classes, giving more time to adapt the production technologies. The complete overview of the considered food classes and respective limits are shown in Table 2. Meanwhile, the EC, not dictating any official method, fixed the performance criteria required by any analytic method applied for PAH analysis (Reg. 836/2011), extending the parameters set only for BaP in previous regulations (Reg. 333/2007 and Dir. 2005/10/EC), to all the analytes included in the PAH4 sum (Table 3). Finally, if any of the four PAHs is at the limit of quantification (LOQ), it would be considered as zero in the summing of the four PAHs. Outside Europe, regulations are much less restrictive. In the United States, even though EPA has always paid a great attention to PAHs as environmental pollutants, no standards governing the PAH content of foodstuffs have been established by the US Food and Drug Administration (FDA). The only exception regards the level of PAHs in seafoods after the Deepwater Horizon oil spill, which occurred in the Gulf of Mexico in 2010. In response to this event, the FDA developed risk criteria and established threshold for allowable levels (levels of concern) for

Encyclopedia of Food and Health

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Polycyclic Aromatic Hydrocarbons

407

2 3

1 11

12

10

4

a

9

5 8

7

6

Figure 1 Numbering of benzo[a]anthracene. Table 1 Compound name, abbreviation, molecular formula and weight (mw), solubility at 25  C, log Kow, and structure of some of the most studied PAHs Compounds (abbreviation)

Molecular formula (mw)

Solubility at 25  C (mg l 1)

Log Kow

Naphthalene (Na)

C10H8 (128)

12 500–34 000

3.37

Acenaphthene (Ac)

C12H10 (154)

Acenaphthylene (Ap)

C12H8 (152)

3420

4.07

Fluorene (F)

C13H10 (166)

800

4.18

Phenanthrene (Pa)

C14H10 (178)

435

4.46

Anthracene (A)

C14H10 (178)

59

4.5

Fluoranthene (Fl)

C16H10 (202)

260

4.90

Pyrene (P)

C16H10 (202)

133

4.88

Benzo[c]fluorene (BcF)

C17H12 (216)

103

5.19

Cyclopenta[c,d]pyrene (CPP)

C18H10 (226)

33.52

5.70

Structure

3.98

(Continued)

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Table 1

Polycyclic Aromatic Hydrocarbons

(Continued)

Compounds (abbreviation)

Molecular formula (mw)

Solubility at 25  C (mg l 1)

Log Kow

Benzo[a]anthracene (BaA)

C18H12 (228)

11.0

5.63

Chrysene (Ch)

C18H12 (228)

1.9

5.63

5-Methylchrysene (5MeCh)

C19H14 (242)

65

6.07

Benzo[j]fluoranthene (BjF)

C20H12 (252)

2.4

6.21

Benzo[b]fluoranthene (BbF)

C20H12 (252)

2.4

6.04

Benzo[k]fluoranthene (BkF)

C20H12 (252)

0.8

6.11

Benzo[a]pyrene (BaP)

C20H12 (252)

3.8

6.06

Dibenzo[a,h]anthracene (DBahA)

C22H14 (278)

0.4

6.86

Benzo[g,h,i]perylene (BghiP)

C22H12 (276)

0.3

6.78

Indeno[1,2,3-cd]pyrene (IP)

C22H12 (276)

-

6.58

Structure

(Continued)

Polycyclic Aromatic Hydrocarbons

Table 1

(Continued)

Compounds (abbreviation)

Molecular formula (mw)

Solubility at 25  C (mg l 1)

Log Kow

Dibenzo[a,l]pyrene (DBalP)

C24H14 (302)

0.24

7.71

Dibenzo[a,e]pyrene (DBaeP)

C24H14 (302)

0.24

7.71

Dibenzo[a,i]pyrene (DBaiP)

C24H14 (302)

0.5

7.28

Dibenzo[a,h]pyrene (DBahP)

C24H14 (302)

-

6.26

Table 2

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Structure

Limits fixed by EC Regulation 835/2011 for BaP and the sum of PAH4 (BaA, Ch, BbF, and BaP) Maximum levels (mg kg 1)

Foodstuffs

BaP

Sum of PAH4

Oils and fats intended for direct human consumption or use as ingredient in food (excluded cocoa butter and coconut oil) Coconut oil for direct human consumption or use as ingredient in food Cocoa beans and derived products

2 2 5

Processed cereal-based foods and baby foods for infants and young children Infant and follow-on formulae, including infant and follow-on milk Dietary foods for special medical purposes, intended specifically for infants Smoked sprats and canned smoked sprats Bivalve mollusks (fresh, chilled, or frozen) Heat treated meat and meat products sold to the final consumer Smoked bivalve mollusks Muscle meat of smoked fish and smoked meat and meat products

1

10 20 35a 30b 1

5

30

6 5c 2d

35 30c 12d

a

Until 31/3/2015. From 1/4/2015. c Until 31/8/2014. d From 1/9/2014. Adapted from EC Regulation n 835/2011. b

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Polycyclic Aromatic Hydrocarbons

Table 3 Performance criteria required for analytic methods by the EC Regulation 836/2011 Parameter

Value

Applicability Specificity

Foodstuffs listed in Reg 1881/2006 Free from matrix or spectral interferences, verification of positive detection 0.3 mg kg 1 for each PAH 0.9 mg kg 1 for each PAH HORRATr or HORRATR < 2 in ring test

LOD LOQ Precision (repeatability and reproducibility) Recovery

50–120%

Adapted from EC Regulation n 836/2011.

shrimp, crabs, oyster, and finfish. Levels of concerns were calculated for 13 PAHs and their alkylated homologues selected among the most studied PAHs in petroleum mixture, namely, Na (including C1, C2, C3, and C4 homologues), F (including C1, C2, and C3), A and Pa (including C1, C2, C3, and C4), P, Fl, Ch, BkF, BbF, BaA, BaP, DBahA, and IP. A daily consumption of 13 g day 1 for shrimp and crab, 12 g day 1 for oyster, and 49 g day 1 for finfish was considered for a reference person of 80 kg (http://www.fda.gov/food/ucm217601.htm). Since 2001, the Canadian legislation has settled a limit of 3 mg kg 1 for the sum of heavy PAHs (BaP, DBahA, BaA, BbF, BkF, IP, Ch, and BghiP) calculated on the basis of the toxic equivalence to BaP only in pomace oil. The toxic equivalence factor approach considers the risk of any single PAH compared to that of BaP. This approach assumes the additivity of the risks, but some studies showed that PAH mixtures can present not only additive but also synergistic and/or antagonistic effects. On the other hand, the toxicity of most environmentally relevant PAHs has not yet been quantified; therefore, the risk may be underestimated. The Chinese government set a maximum level for the only BaP content in different classes of product, namely, 5 mg kg 1 for cereal and cereal products, meat and meat products, and fish and fishery products both smoked and roasted and 10 mg kg 1 for fats and oils (GB2762-2012). In Australia and New Zealand, no regulatory limits have been set for PAHs, but the general approach is to minimize the exposure following the ALARA (as low as reasonably achievable) principle and applying good operational practices.

Source and Occurrence in Food PAHs are formed from both natural and anthropogenic sources. The former includes natural combustion, such as forest fires and volcanoes, and long-term degradation followed by synthesis (fossil fuels contain extremely complex mixtures of these compounds, with a prevalence of highly alkylated ones). The anthropogenic sources are mainly related to incomplete combustion of organic material (forming mainly parent PAHs). The formation mechanism occurs primarily by partial cracking of organic compounds to smaller unstable fragments at high temperatures (pyrolysis), mostly radicals, which recombine to give relatively stable PAHs (pyrosynthesis). The main

rate of formation occurs linearly between 500 and 750  C, whereas above 3000  C, they are not formed. The ratio between alkylated PAHs and parent PAHs can be an indication of a source of contamination. Foods can be contaminated by PAHs through two main routes, namely, environmental pollution and lack of good manufacturing practices during food production. The two sources are discussed separately in the following sections.

Environmental Sources Natural sources of PAH emissions in the environment are forest fires, volcanoes, hydrothermal process, oil seepage, and carbonization. Nevertheless, they are present in the environment mainly due to anthropogenic sources, such as exhaust from motor vehicles; domestic combustion of wood, oil, gas, and charcoal; industrial biomass combustion; open burning of agricultural debris; tobacco smoke, laying tar, coke oven, and roofing tar emission; asphalt roads; and coal, coal tar, and hazardous waste sites. Once released to the atmosphere, PAHs may be transported quite far from the emission source and can be removed from the atmosphere by wet and dry deposition onto soil, water, and vegetation. In the air, PAHs are subjected to reactions with oxidant gases, such as NO2, O3, and SO3, and are photooxidized. Many PAHs, such as BaP, are rapidly destroyed by UV light. After deposition, PAHs may undergo different fates: In surface water, they can be volatilized, photolyzed, biodegraded, and bound to suspended particles or sediments or accumulate in aquatic organisms; in soil, they can be volatilized or undergo biotic or abiotic degradation, mainly photolysis and oxidation. PAHs in soil can also enter groundwater and be transported within an aquifer. The relative solubility in water and organic solvents (characterized by the log Kow value) affects transport and distribution of PAHs between different environmental compartments and their uptake and accumulation by living organisms. The chemical reactivity influences their adsorption to organic matter or their degradation. The PAH mixture profile, which may be found in foods and feeds contaminated by environmental sources, can be very different from the original source, due to the different properties within the large group of PAHs. Due to the lipophilic characteristics of PAHs, high-watercontent plant tissues have a limited amount of these contaminants, but the waxy surfaces of vegetables and fruits can concentrate PAHs through surface adsorption. The superficial contamination of vegetables in urban areas can be up to 100 times higher than in rural areas. Some authors found that the amount of BaP in fruits from rural areas ranged between 0.19 and 0.34 mg kg 1, while in urban areas, it ranged between 30 and 60 mg kg 1. Up to 50% of these kinds of contamination can be removed by careful washing of the surface of vegetables and fruits.

Technological Processing Heat-processing procedures during food preparation, in particular grilling, roasting, and smoking, involving direct contact with combustion gases, are among the major sources of PAH

Polycyclic Aromatic Hydrocarbons

contamination in food. The level of contamination in smoked products is related to the time and temperature of processing, humidity, types of control and smoke used, and the design and types of kilns. Modern kilns have strict electronic control of the entire process. Different types of wood or woodchips used to generate smoke produce different amounts of PAHs, with the highest concentration found when spruce, hazel tree, plum tree, and aspen are used, whereas they are lowest when apple tree, alder, and maple are burned. Over the last decades, commercial smoked food production has generally replaced the traditional smoking process with the use of liquid smoke flavoring. The latter derives from smoke subjected to fractionation and purification; thus, the content of PAHs on the final product is greatly reduced and strictly controlled. It has been demonstrated that in canned smoked fish, over 70% of the entire amount of BaP migrates from the fish to the oil fraction. Roasting may also lead to direct or indirect contamination, but to a small extent, considering that the temperature reached is relatively low for PAHs formation (up to 260  C). It has been proven that strong coffee bean roasting conditions lead to significant levels of P, Ch, and BaA but low BaP (approximately 0.2 mg kg 1). However, the transfer from the coffee bean to the coffee brew was reported low (in general lower than 140 ng l 1) and obviously related to the water solubility of the specific PAH. Differently from industrial process, considerable variability was found in the PAH level in domestic grilled products, depending on the heat source, the type and geometry of the grill, the composition of the food grilled, and the cooking time. However, since PAHs arose mainly from the pyrolysis of fat dropped into the flame, as long as the food is protected from direct contact with flames, contamination can be controlled. Another important source of contamination of PAHs is related to the mineral oil contamination, which is composed of two main groups of compounds, mineral oil saturated hydrocarbons (MOSH) and (mainly alkylated), mineral oil aromatic hydrocarbons (MOAH). Mineral oils, previously purified of the aromatic fraction (food-grade quality), are widely employed in the food industries as releasing agents, antidust agents, etc. However, accidental or intentional contamination with nonfood-grade mineral oil may occur. The main sources of the latter are jute bags, recycled cardboard, plastics, lubricating oils, printing inks, and environmental contamination.

Occurrence and Dietary Intake Several reports have been published concerning surveys of PAH contamination in foodstuffs. The most recent one is the report published by EFSA in 2008 on the data collected from the European member states on request of the EC (Rec. 2005/ 108/EC). Figure 2 outlines a summary graphic on the BaP content (average and the maximum amount found) in different Codex food categories, extrapolated from the most relevant data in the report (considering only the food categories in which more than ten samples were analyzed). Among the main categories, the most contaminated ones, on average, were coffee and tea, food supplements, herbs and spices, fresh and treated mollusks, and fats and oils. There is no specification of the type of lipids included in the two distinct

411

categories reported, namely, fats and oils and vegetable oil. However, in a previous report published in 2004 by the EC, olive pomace oil, grape seed oil, and seed oils resulted much more contaminated compared to virgin olive oil, due mainly to the drying process carried out before oil extraction and the raw material handling. The refining process reduces the amount of PAHs; specifically deodorization removes light PAHs, while bleaching in the presence of activated charcoal reduces heavy PAHs. A high level of contamination can be found in smoked meat and fish, which is related to the method of smoking. The liquid smoked products are practically noncontaminated, while the traditionally smoked products can present over 5 mg kg 1 of BaP. Seawater may be highly contaminated with PAHs due to oil spills, industrial and urban effluents, and leaking from creosote wharfs and pilings. However, bluefish species, such as tuna, mackerel, and salmon, generally present lower contamination than mollusks. Indeed, fish, in contrast with bivalves, oxidize and metabolize PAHs to water-soluble compounds, which are gradually excreted. Since bivalves filter large volumes of water, their levels of BaP can be significantly high (several hundreds of ng g 1). There are not many studies on the dietary intake of PAHs, and it is difficult to generalize since it is strictly related to the availability of food consumption surveys and their criteria and the PAH occurrence data in the national diet. In 2005, the EFSA claimed the necessity of a common database on food consumption to improve the consistency and reliability of exposure assessment carried out by the various EFSA Panels. As a result, in 2007, the development of the Concise European Food Consumption Database started, which was published in 2008. Upon processing of such data, cereals and seafoods were the main contributors to the dietary intake, mainly due to high consumption, rather than a high contamination level of these products. In particular, cocoa butter and pomace oil were excluded from the food categories examined, since they were not consumed as such; therefore, the fats and oils category, which was among the main sources in a previous survey by the JECFA, was surpassed by seafood. Notwithstanding the general low consumption, some foodstuffs present very high levels of contamination and may affect the overall exposure in particular cases. Some food supplements (such as fish oil, ginseng, bee products, and plant extract), whose consumption is very variable depending on their intended use, were found highly contaminated (up to 270 mg kg 1). Equally, some spices were found with very high level of PAHs (up to 75 mg kg 1, with an average of 4.75 mg kg 1). Considering all the European Countries, an average daily intake can be estimated in the 185–255 ng day 1 range for BaP and in the 936–1449 ng day 1 and in the 1415–2136 ng day 1 range for PAH4 and PAH8, respectively.

Toxicity and Risk Assessment PAHs, once absorbed in the organism, become toxic during the attempt to eliminate them through feces or urine. In fact, some intermediates of the metabolic pathway are very reactive,

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Polycyclic Aromatic Hydrocarbons

Mean

Max 101

Coffee, tea (n=99)

270

food supplements (n=325) Infant formulae (n=35)

75

Herbs, spices (n=77)

101

Smoked, dried, fermented mollusc¼

127

fresh mollusc (n=383) fresh fish (n=161)

48.8

heat-treated meat (n=400) fermented meat (n=49) cured meat (n=161) processed meat (n=73) breads and rolls (n=12) breakfast cereals (n=25) grain whole, flakes (n=21) Cocoa, chocolate (n=225) dried vegetables (n=90) vegetables (n=55) dried fruit (n=256) Fresh fruit (n=16) emulsion (n=20) margarine (n=37) butter (n=17)

61

vegetable oils (n=2100) fats and oils (n=25) cheese (n=42) 0

10

20

30

40

50

mg kg–1 Figure 2 A summary graphic on the BaP content (average and the maximum amount found, mg kg 1) in different Codex Food Categories, extrapolated from the most relevant data reported in the report published by EFSA in 2008. Reproduced from European Food Safety Authority (EFSA) (2008). Polycyclic aromatic hydrocarbons in food, scientific opinion of the panel on contaminants in the food chain (Question N EFSA-Q-2007-136), The EFSA Journal 724, 1–114.

forming adducts with DNA, while the final product can be used as an exposure biomarker.

Absorption There are three main routes of absorption of PAHs in humans: inhalation, skin contact, and ingestion. The rate of absorption by the lungs depends on the structure of the PAH, the size, and the chemical nature of the particles where PAHs are adsorbed. The absorption through skin contact is characteristic of occupational exposure, like coal cooking, petroleum refining, and road paving, and it involves both diffusional and metabolic processes. The main route that will be discussed hereafter is

ingestion of PAHs occurring through food and water. Some authors have suggested two phases involved in the absorption process: uptake by the mucosa, followed by diffusion through the intestinal wall. The composition of the diet affects PAH absorption. In fact, it has been shown that lipophilic foods increase absorption of 14C-BaP, while cellulose, lignin, bread, rice flakes, and potato flakes suppress it in Wistar rats. Due to the lack of functional groups, these compounds can easily pass through lipoprotein membranes and reach all internal organs. Several authors have shown that detectable levels of PAHs can be identified in almost all internal organs. Organs rich in adipose tissue can store hydrocarbons and gradually release them, while the gastrointestinal tract contains high

Polycyclic Aromatic Hydrocarbons

S,R-oxide (K-region)

413

R,R-diol EPHX1 R

S

R

O GST

OH

UGT

OH

GSH adducts

Sulfo- or glucuronic acid conjugates

UGT SULT

polynuclear quinones

phenol

R

quinols (hydroquinone)

UGT SULT

UGT SULT UGT SULT B[a]P

catechol (hydroquinone)

phenol

Sulfo- or glucuronic acid conjugates

O O ortho-quinone

OH AKR

O EPHX1 S

R,S-oxide (non-K-region)

O

CYP R

R

R

HO R,R-diol

S S

R

anti-(R,S,S,R)diol-epoxide

R

HO OH

OH UGT

GST

Glucuronic acid conjugates

GSH adducts

Figure 3 Scheme of benzo[a]pyrene metabolism. Reproduced from Luch A., Baird W.M. (2010). Carcinogenic polycyclic aromatic hydrocarbons. In McQueen C. A. (ed.) Comprehensive Toxicology (2nd ed.), pp. 85–123. Elsevier.

levels of PAHs and their metabolites, independently of the route of absorption.

Metabolism and Excretion The metabolism of PAHs, using BaP as an example, is schematized in Figure 3. The general scheme of the PAH metabolism involves oxidation to a range of primary (epoxides, phenols, and dihydrodiols) and secondary (diol epoxides, tetrahydrotetrols, and phenol epoxides) metabolites, followed by conjugation with glutathione, glucuronic acid, or sulfate. A large number of inducible enzymes are involved in these stages (microsomal cytochrome P450-dependent monooxygenase, glutathione transferases, dehydrogenases, hydrolases, peroxidases, and oxidoreductases). The most important metabolite of BaP is benzo [a]pyrene-7,8-diol-9,10-epoxide, which presents the highest tumor-inducing activity, because it forms adducts with proteins or DNA. The main site for bonding is the amino group of guanine or adenine (Figure 4). If the adduct PAH–DNA is not repaired, it can start a carcinogenic process. The major metabolites of BaP excreted through different routes are 3-hydroxy- and 9-hydroxy-BaP, part of them conjugated to sulfate or glucuronic acid. Most of the metabolites are excreted through feces, while a smaller proportion by urine. Excretion of metabolites through

milk may occur as well, but to a lower extent. The proportion among the excretion routes varies with the PAH congener. Although the use of several biomarkers in urine, such as 1-hydroxypyrene, hydroxynaphthalene, and hydroxyphenanthrene, is widely accepted to estimate the PAH exposure in workplace and in smokers, there are still few studies on dietary exposure evaluation. Several methods have been developed to assess human exposure through urinary biomarkers, such as 1-hydroxypyrene, a metabolite of pyrene. However, the relative amount of P and BaP may vary considerably between different sources of contamination, and proofs of correlation are still weak. Quantification of adducts of PAHs with DNA in peripheral lymphocytes, and other tissues and with proteins, such as albumin, has also been used as an indicator of the dose of the reactive metabolite.

Toxicity Evaluation and Risk Assessment Most of the toxicity studies on PAHs have been carried out on dermal, subcutaneous, and inhalation exposure, while only limited studies have dealt with oral administration. Table 4 reports the evaluation of toxicity (genotoxicity and carcinogenicity) according to the International Programmme on Chemical Safety (IPCS) of the WHO, the International Agency for Research on Cancer (IARC), and the SCF. Fourteen of the 33 PAHs considered by the study of the SCF published in 2002 showed clear evidence of both genotoxicity

414

Polycyclic Aromatic Hydrocarbons

CYP

O

BaP

CYP

EH

7S,8S-diol,

7R,8R-diol > 95%

HO

7S,8R-epoxide

O

7R,8S-epoxide > 95%

EH

HO OH

OH O

O

O

CYP

HO

HO

HO OH

OH I

DNA dNu

dNu

DNA

HO

HO dNu

HO OH 1 trans-

OH 2 cis-

dNu

HO +

HO

HO

HO

trans-

OH 3

OH 4

trans-

cis-

dNu

HO OH 5

HO

DNA

dNu HO +

+ HO

IV 7S,8R,9R,10S-

(+) syn 97 : 3 (-) anti

DNA HO

OH

7S,8R,9S,10R-

(-) syn 14 : 86 (+) anti

dNu

O

HO

OH III

II 7R,8S,9S,10Rdiol-epoxide

7R,8S,9R,10S-

CYP

dNu = Deoxynucleotide

dNu

HO OH 6 cis-

HO +

HO

HO OH 7 trans-

OH 8 cis-

Figure 4 Formation of BaP-DNA adduct: configurational isomers of BaP-7,8-diol-9,10-epoxide and DNA adducts. Reproduced from Xue W., Warshawsky D. (2005). Metabolic activation of polycyclic and heterocyclic aromatic hydrocarbons and DNA damage: a review. Toxicology and Applied Pharmacology 206(1), 73–93.

and carcinogenicity, according to both SCF and IPCS. The PAHs are BaA, BbF, BjF, BkF, BaP, Ch, CPP, DBahA, DBaeP, DBahP, DBaiP, DBalP, IP, and 5-MeCh. BghiP is considered genotoxic, but not carcinogenic due to the limited data. According to IPCS, the carcinogenicity of Ac, Ap, benzo[g,h,i] fluoranthene, benzo[a]fluorene, benzo[b]fluorene, benzo[e] pyrene, coronene, Na, Pa, and P was considered questionable. Among these, Na was considered to be noncarcinogenic due to its negative genotoxicity. Finally, A, benzo[g,h,i]fluoranthene, F, 1-methylphenanthrene, perylene, and triphenylene were evaluated as noncarcinogenic. According to the experiments carried out using the same route of exposure, the most potent carcinogenic PAHs seem to be DBahA, DBahP, DBalP, BaP, BbF, and 5-MeCh. BjF and BkF are of moderate potency, while BaA and Ch are relatively weak. Tumor formation is related to the route of administration but not restricted to the site of application, that is, oral administration can induce tumor of the gastrointestinal tract, dermal application can induce mainly skin tumors, and inhalation can induce mainly lung tumors; however, liver and mammary gland tumors may be developed as well. The risk assessment process consists of several steps including hazard identification, hazard characterization, exposure assessment, and risk characterization.

For most toxic process, there is a critical level of interaction, below which the homeostatic mechanisms would be able to avoid any structural or functional changes. Nevertheless, when genotoxic substances interacting with DNA, directly or after metabolic transformations, are considered, the absence of a threshold in their mechanism of action is generally assumed. This means that there is no dose without a potential effect. The general approach to minimize the exposure to these xenobiotics is expressed in the ALARA (as low as reasonably achievable) principle. However, this principle is not helpful to the risk management, which should establish a reasonable level of risk. To evaluate the cancer risk of any xenobiotic, epidemiological data are the more informative and reliable way. However, when the latter are not available, animal assays are used to determine the risk. These experiments are carried out by exposing animals to high levels of toxic compounds in order to compensate for the low number of experimental groups and to detect significant tumor incidence in a reasonable time frame. Therefore, the extrapolation of low levels of exposition to which a human population may be exposed is necessary and very delicate. A wide range of models can be used to extrapolate these data, but unfortunately, each leads to a very different conclusion. In 2005, the SCF suggested the possibility to use the margin of exposure (MoE) approach. It is similar to the

Polycyclic Aromatic Hydrocarbons

Table 4

415

Evaluation of genotoxicity and carcinogenicity of several PAHs according to different references

Compounds (abbreviation)

Genotoxicity (SCF, 2002)a

Genotoxicity (IPCS, 1998)b

Carcinogenicity (IPCS, 1998)b

Naphthalene (Na) Acenaphthene (Ac) Acenaphthylene (Ap) Fluorene (F) Phenanthrene (Pa) Anthracene (A) Fluoranthene (Fl) Pyrene (P) Cyclopenta[c,d]pyrene (CPP) Benzo[a]anthracene (BaA) Chrysene (Ch) 5-methylchrysene (5MeCh) Benzo[j]fluoranthene (BjF) Benzo[b]fluoranthene (BbF) Benzo[k]fluoranthene (BkF) Benzo[a]pyrene (BaP) Dibenzo[a,h]anthracene (DBahA) Benzo[g,h,i]perylene (BghiP) Indeno[1,2,3-cd]pyrene (IP) Dibenzo[a,l]pyrene (DBalP) Dibenzo[a,e]pyrene (DBaeP) Dibenzo[a,i]pyrene (DBaiP) Dibenzo[a,h]pyrene (DBahP)

Probably not genotoxic Inadequate data Inadequate data Inadequate data Equivocal Not genotoxic Equivocal Not genotoxic Genotoxic Genotoxic Genotoxic Genotoxic Genotoxic Genotoxic Genotoxic Genotoxic Genotoxic

Negative Inconsistent, limited database Inconsistent, limited database Negative, with few exception Inconsistent Negative, with a few exceptions Positive Inconsistent Positive Positive Positive Positive Positive Positive Positive Positive Positive

Questionable Questionable No studies Negative Questionable Negative Positive, limited database Questionable Positive Positive Positive Positive Positive Positive Positive Positive Positive

Genotoxic Genotoxic Genotoxic Genotoxic Genotoxic Genotoxic

Positive Positive Positive, limited database Positive Positive Positive, limited database

Negative, limited database Positive Positive Positive Positive Positive

Carcinogenicity (IARC, 1987)c

3

3 3 2A 3 2B 2B 2B 2B 2A 2A 3 2B 2B 2B 2B 2B

a

SCF, Scientific Committee on Food (2002). Opinion of the Scientific Committee on Food on the risks to human health of polycyclic aromatic hydrocarbons in food. 4 December 2002. European Commission, Brussels. b IPCS, International Programme on Chemical Safety (1998). Selected non-heterocyclic polycyclic aromatic hydrocarbons. Environmental health criteria 202. Geneva: International Programme on Chemical Safety, World Health Organization. c IARC, International Agency for Research on Cancer (1987). Overall evaluation of carcinogenicity: an updating of IARC Monographs Volumes 1 to 42. IARC monographs on the evaluation of carcinogenic risks to humans. Supplement 7. Lyon: International Agency for Research on Cancer, World Health Organization.

margin of safety (MoS), where the no observed adverse effect level is compared to human exposition to the xenobiotic. However, the MoS approach is not appropriate for carcinogenic and genotoxic substances, where there is not a level without effect, while the MoE is. The MoE is the ratio between a defined point on the dose – response curve for the adverse effects and the human intake. To establish the appropriate point on the dose – response curve the SCF considered the benchmark dose (BMD), which is calculated on the best-fitting curve to the experimental data. In particular, it has been recommended to use the BMDL10 (BMD lower confidence limit 10%), which is the lower limit of the interval of confidence at 95% of the BMD, because this is the lowest effect measurable as statistically significant. Furthermore, considering that at low doses the carcinogenic process seems to be nonlinear, and the assays are carried out on animals, other two factors of 100-fold have been added to include the inter- and intraspecies variability and the uncertainty of the carcinogenic process. Therefore, an MoE of 10 000 or higher would generally be of low concern for the public health. Comparable approaches have been used by Health Canada for Priority Substances under the Canadian Environmental Protection Act, the National Health and Medical Research

Council in Australia and New Zealand for the Toxicity Assessment for Carcinogenic Soil Contaminants, and the JECFA.

Analytic Determination Sample Preparation Analytic methods have to be carefully optimized and be very sensitive and selective, minimizing handling to avoid spurious contributions from solvents and glassware and manipulation. Plastic materials have to be avoided since PAHs can be adsorbed (especially in polyethylene); thus, inert materials, such as aluminum, glass, and stainless steel, previously rinsed with high purity acetone or hexane, should be used. Extracts have to be protected from light by using amber or foil-wrapped vials to avoid photooxidation of analytes, and drastic evaporation should be avoided to minimize loss of more volatile compounds. The use of a keeper, such as toluene, may reduce such a loss. For PAH determination in complex samples, the first step is to extract the lipid fraction to which PAHs are associated. Traditionally, Soxhlet extraction or saponification using alcoholic KOH, followed by a purification step performed by gel

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permeation or, recently, by solid phase extraction (SPE), was employed. Over the last decades, more efficient and/or rapid techniques, which minimize sample manipulation and solvent consumption, have been largely employed for PAHs analysis. Many papers report the use of ultrasound-assisted extraction, a cheap and easy technique, which, if properly optimized, can be a satisfactory alternative to Soxhlet and saponification extraction, depending on the nature of the contamination and the complexity of the matrix. Generally, the sample has to be lyophilized to optimize the extraction yield. More innovative techniques have been used exploiting the effect of pressure and temperature to improve the extraction efficiency and rapidity, such as pressurized liquid extraction (PLE), microwave-assisted extraction (MAE), and supercritical fluid extraction (SFE) using CO2. To obtain a satisfactory extraction yield over an extended range of PAHs by SFE, the addition of a modifier (e.g., methanol) was reported, leading to a necessity of a further purification step. More effective results were obtained by mixing a sorbent (e.g., C18 beads) with the ground sample directly on the extraction thimble (Figure 5). Applying classical PLE extraction (with the extraction vessel packed with the ground sample and inert material), a further cleanup step is required to purify the analytes from the coextracted lipids. Many works proposed the use of a sorbent directly mixed in the vessel to perform a single extraction/ purification step. In general, activated silica (over Florisil®, sulfuric acid-impregnated silica, and neutral, basic, and acidic alumina) gave better results. A different approach to reduce coextracted lipid matter can be applied in the MAE technique. A simultaneous saponification and extraction method can be performed in the same extraction vessel. The approach succeeded for simple samples, such as vegetables and propolis, but still a mild purification was needed when complex samples, such as fish and meat, were analyzed. SPE is the most applied approach for purification, not only for lipid extracts but also for fatty and nonfatty liquid foods,

Figure 5 GC-MS chromatograms of a spiked crab tissue extracted by SFE. (a) Conventional SFE; (b) SFE plus C18 as sorbent. Peaks: (1) Pa. (2) A, (3) Fl, (4) P, (5) BaA, (6) Ch, (7) BbF, (8) BeP, (9) BaP, (10) perylene; letters are natural lipids. Reprinted with permission from Ali M.Y., Cole R.B.(1998). Analytical Chemistry 70, 3242–3248.

such as edible oil, milk, water, and beverages (wine, beer, and spirits). Many different sorbent phases have been employed to exploit different retention mechanisms according to the nature of the sample. Among the traditional stationary phases, silica, C18, C8, Florisil®, styrene–divinylbenzene, or mixture of these phases has been the most employed, giving satisfactory purification and recovery results. Recently, the applications of novel sorbent phases for PAHs analysis have been published, such as molecular imprinted polymers, sulfonated graphene sheets, and carbon nanotubes. Solid-phase microextraction (SPME) has been largely employed thanks to its peculiar characteristics. In fact, it is easy to use, rapid, and practically solvent-free. It can be used both in the head-space (HS) and in direct immersion (DI) mode by carefully selecting the fiber coating. HS-SPME is obviously more selective for more volatile PAHs (2–4 rings compounds) rather than a heavy one. However, even in the DI mode, the selectivity does not improve much when a polydimethylsiloxane (PDMS) fiber is employed for analyzing PAHs in water (Figure 6). Sensitivity can be much improved using a stir bar sorptive extraction approach, which exploits the same principle as SPME, employing a larger amount of coating on a magnetic stir bar, but the discrimination between light and heavy PAHs persists. Interesting results were obtained using a noncommon fiber coating, namely, Carbopack Z/PDMS, which has a high affinity for heavy PAHs, while very low, almost none, for light PAHs. The primary mechanism, when dipped in a nonpolar solvent, is the p–p interaction between the carbon surface and the planar aromatic compounds. Such a fiber was used directly immersed in a diluted oil solution, obtaining very good limits of detection and repeatability. Sensitivity can be further improved by performing a rapid liquid–liquid extraction of the edible oil to reduce the bulk of triglycerides, thus improving the signal-tonoise ratio in the gas chromatographic (GC) analysis.

Chromatographic Techniques Both liquid chromatographic (LC) and GC techniques can be used to analyze PAHs after sample purification. The LC systems are usually equipped with fluorimetric detectors (FLD), which give very high selectivity and sensitivity, although in recent years, the use of the mass spectrometer (MS) has been increased, obtaining sensitivity comparable to FLD. Despite the significant lower sensitivity, UV detector is still used in several works. LC equipped with dedicated C18 columns were widely employed to easily resolve the 16 EPA PAHs, but since the focus has moved to the 16 EU PAHs, a proper separation of all the isomers has become more cumbersome. Some critical pairs, such as P and BcF, BjF and BeP, and DBahP and BghiP, are not resolved even using a different step elution gradient; thus, more FLD detectors in series are employed by setting different wavelengths for the eluted PAHs in each FLD, thus exploiting the selectivity of the detector. Furthermore, the use of a UV detector is required to detect CPP, which does not give rise to fluorescence. GC-MS is a valid alternative to circumvent the absence or the low intensity of the fluorescence signal, as is the case of CPP and alkylated PAHs. Sensitivity of the pg order can be reached using the single ion monitoring (SIM) mode in

Polycyclic Aromatic Hydrocarbons

417

1.2 × 106 100-µm PDMS

1.0 × 106

30-µm PDMS 7-µm PDMS 85-µm PA

Peak area

8.0 × 105

Carboxen/PDMS

6.0 × 105 4.0 × 105

2.0 × 105

0.0

p Y P Na AcP Ac

u

Fl

e

Ph

t

An

FL

r

Py

A

Ba

r

Ch

FL kFL BaP B

Bb

P

In

A iP DB Bgh

2.4 × 106 SPME(25⬚ C)

2.1 × 106

HSSPME(80⬚ C) HSSPME(60⬚ C)

Peak Area

1.8 × 106

HSSPME(25⬚ C)

1.5 × 106 1.2 × 106 9.0 × 105 6.0 × 105 3.0 × 105 0.0

P Y P Na AcP Ac

u

Fl

e

Ph

t

An

FL

r

Py

A

Ba

r

Ch

FL kFL BaP B

Bb

P

In

A iP DB Bgh

Figure 6 Profile of PAH uptake obtained using different fiber coating in SPME in (a) head-space mode and (b) comparison between direct immersion and head-space at different temperature. Reprinted with permission from Doong R., Chang S., Sun Y. (2000). Journal of Chromatography A 879, 177–188.

the MS detector. The US EPA did not consider the GC reliable enough due to several coelution problems, thus suggesting the use of LC, but it has been largely proved that a careful selection of the stationary phase and column dimensions gives an optimal resolution of critical pairs. Good results can be obtained using a 50% phenyl column. The selection of the injection mode (splitless, PTV, or on-column), along with the proper injection liner (multibaffle, packed, etc.), is fundamental in avoiding discrimination, especially of the lighter PAHs.

Hyphenated Chromatographic Techniques An interesting approach to reduce sample manipulation and thus the risk of cross contamination is the application of

hyphenated techniques. The most exploited technique is the coupling of an LC step (equipped with one or two LC columns) for purification purposes, followed by GC analysis for the final determination. This technique has been mainly applied to analyze alkylated PAHs. The analysis of parent PAHs can be performed using a silica column in the LC purification, retaining triglycerides, which are then removed by backflushing the column, while PAHs are eluted and analyzed in GC-MS, obtaining a limit of detection of about 0.5 mg kg 1 in SIM mode. Coupling two LC columns of different selectivity prior to GC analysis made it possible to analyze alkylated PAHs in edible oil. A first silica column removed triglycerides, while a second amino one allowed to fractionate PAHs according to the ring number.

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Polycyclic Aromatic Hydrocarbons

Several applications coupled two LC systems or similar chromatographic systems, such as the donor-acceptor complex chromatography (DACC). The working principle of DACC is based on the p–p interaction between PAHs and the sorbent, which was exploited to retain PAHs while removing the matrix interferences, and then PAHs were backflushed directly onto the LC analytic column to be analyzed. A silica column was also applied as preparative chromatography prior to a C18 column for PAH separation. Although comprehensive multidimensional GC (GC  GC) cannot disregard a preparation step, the increased separation power, obtained by coupling two columns with different selectivity, can enable the isolation of matrix interferences; thus, in several cases, a less intensive cleanup step can be performed. GC  GC has been often applied to characterize alkylated PAH contamination, rather than to analyze parent PAHs, even if some few papers have been published on this.

Conclusion and Future Perspective PAHs are a class of compounds that have been under investigation for a long time. However, further investigation is still needed, in particular considering the high toxicity of such compounds, along with the possible occurrence in rather complex mixtures. In particular, the EFSA is still requiring data on oral carcinogenicity of mixtures relevant for dietary exposure and occurrence data for less considered and often less abundant compounds, such as BcF. Therefore, to satisfy such requirements, improved analytic methods, in terms of both selectivity and sensitivity, are required to study the occurrence of PAHs in different food matrices and of metabolites in biological fluids. Miniaturized and hyphenated techniques will lead the development of further methods in the next future following the general trend of reducing solvent and time consumption and reducing sample manipulation avoiding artifacts formation. An interesting future challenge will be to extend the study to the reliable identification and quantification of not only parent PAHs but also the alkylated ones, for which a detailed toxicological evaluation is still lacking.

See also: Carcinogenic: Carcinogenic Substances in Food; Carcinogens: Identification of Carcinogens; Chromatography: Combined Chromatography and Mass Spectrometry; Chromatography: Focus on Multidimensional GC; Chromatography: High-Performance Liquid Chromatography; Codex Alimentarius; Risk Assessment of Foods and Chemicals in Foods.

Further Reading Canada (2001). Canadian Food Inspection Agency, Industry Advisory, 17 September. 2001. EFSA, European Food Safety Authority (2008b). European Food Safety Authority, A report from the Unit Collection and Exposure on a Request from the European Commission, first issued on 29 June 2007, revised on 31 July 2008. EFSA/DATEX/ 002. EFSA, European Food Safety Authority (2005) Opinion of the scientific committee on a request from EFSA related to a harmonised approach for risk assessment of substances which are both genotoxic and carcinogenic (Request No EFSA-Q-2004020). The EFSA Journal 282: 1–31. EFSA, European Food Safety Authority (2008) Polycyclic aromatic hydrocarbons in food, scientific opinion of the panel on contaminants in the food chain (Question N EFSA-Q-2007-136). The EFSA Journal 724: 1–114. IARC, International Agency for Research on Cancer (1987) Overall evaluation of carcinogenicity: an updating of IARC monographs volumes 1 to 42. In: IARC monographs on the evaluation of carcinogenic risks to humans. Lyon: World Health Organization Supplement 7. IPCS, International Programme on Chemical Safety (1998) Selected non-heterocyclic polycyclic aromatic hydrocarbons. Environmental health criteria 202. Geneva: World Health Organization. JECFA (2006). Summary and conclusions of the sixty-fourth meeting of the Joint FAO/ WHO Expert Committee on Food Additives (JECFA), Expert Committee on Food Additives, Food and Agriculture Organization (FAO), Rome/World Health Organization (WHO), Geneva. Lee ML, Novotny MV, and Bartle KD (1981) Analytical chemistry of polycyclic aromatic compounds. New York: Academic Press. Luch A and Baird WM (2010) Carcinogenic polycyclic aromatic hydrocarbons. Comprehensive Toxicology 14: 85–123. Moret S, Purcaro G, Marega M, and Conte LS (2012) Sample preparation techniques for the determination of some food contaminants (polycyclic aromatic hydrocarbons, mineral oils and phthalates). In: Pawliszyn J (ed.) Comprehensive sampling and sample preparation, vol. 2, pp. 313–356. Oxford: Elsevier, Academic Press. SCF, Scientific Committee on Food (2002) Opinion of the Scientific Committee on Food on the risks to human health of polycyclic aromatic hydrocarbons in food. Brussels: European Commission. Xue W and Warshawsky D (2005) Metabolic activation of polycyclic and heterocyclic aromatic hydrocarbons and DNA damage: a review. Toxicology and Applied Pharmacology 206(1): 73–93.