Priority Environmental Chemical Contaminants in Meat

Gianfranco Brambilla, Annalaura Iamiceli, and Alessandro di Domenico

Introduction

Generally, foods of animal origin play an important role in determining the exposure of human beings to contaminants of both biological and chemical origins (Ropkins & Beck, 2002; Lievaart et al., 2005). A potentially large number of chemicals could be considered, several of them deserving a particular attention due to their occurrence (contaminations levels and frequencies) and intake scenarios reflecting the differences existing in the economical, environmental, social and ecological contexts in which the ''from-farm-to-fork'' activities related to meat production are carried out (FAO - Food and Agriculture Organization, 2008).

For the reasons reported above, the prioritization of contaminants of potential relevance to meat safety should be adequately framed within a risk analysis, a scientific process targeted on public health protection, aiming to estimate how much of a contaminant consumers in general and/or population-sensitive groups (such as children or elderly people) may be exposed to without (appreciable) risk. Health risk assessment is commonly divided into four steps providing answers to the following questions: (1) hazard identification - what can go wrong? (2) hazard characterization - what are the consequences? (3) exposure assessment - how can it happen? (4) risk estimation - what is the likelihood the adverse effect would happen (Gaylor et al., 1997; FAO, 1997)?

Prioritization of Chemical Contaminants in Meat

When talking about meat, we should consider for contaminants prioritization with respect to food safety the following factors: the chemical potential to bioaccumulate in food-producing animals; the relevance for humans of the

Istituto Superiore di Sanita, Toxicological Chemistry Unit, Viale Regina Elena 299, I-00161 Rome, Italy e-mail: [email protected]

F. Toldra (ed.), Safety of Meat and Processed Meat,

Food Microbiology and Food Safety, DOI 10.1007/978-0-387-89026-5_15,

© Springer Science+Business Media, LLC 2009

intakes via food of animal origin; and the ability of some chemicals to cause severe toxicological effects as a consequence of long-term exposures (UNEP -United Nations Environmental Program).

Many of the toxic substances sharing the aforesaid features have been already framed within the Stockholm Convention (2001) and conventionally identified as persistent organic pollutants (POPs). By definition, POPs are "chemical substances that persist in the environment, bioaccumulate through the food web, and pose a risk of causing adverse effects to human health and the environment" (the Stockholm Convention, 2001).

The environmental persistence of POPs, generally correlated to their chemical stability that makes these substances highly resistant to biological and chemical degradation, represents the main factor relating such chemicals to long-time exposure. Bioaccumulation magnitude depends on several factors, one being the solubility of the substance in lipids (Bernes, 1998). This feature strengthens the tendency of POPs to be concentrated in the fatty tissue of a living organism and they are found in food-producing animals at higher concentrations than those present in the environment and/or feedingstuffs (Hoogenboom, 2004).

Potential adverse effects on the environment and human health caused by exposure to POPs are of considerable concerns for governments, non-governmental organizations and the scientific community.

The worldwide dimension and relevance of POPs in meat production is enhanced not only by their environmental persistence and the capacity of covering long distances away from the point of release but also, within the context of this paper, by the potential to reach Consumers of different countries, via the world trade of feeds and food of animal origin of unreliable quality.

Concerted international measures were adopted to efficiently control the environmental release of POPs: the Stockholm Convention opened for signatures in 2001 and entered into force in May 2004 (EU Council, 2006); it provides an international framework, based on the precautionary principle, that seeks to guarantee the elimination of POPs or the reduction of their production and use.

The substances actually under the Convention include eight individual orga-nochlorine pesticides, hexachlorobenzene, polychlorobyphenyls (PCBs), poly-chlorodibenzodioxins (PCDDs) and polychlorodibenzofurans (PCDFs), all present in Annex A (substances to be eliminated); in Annex B, the substances whose production and use are restricted are reported, whereas Annex C comprises substances unintentionally produced whose releases have to be reduced and finally eliminated.

At present a second group of chemicals (candidate POPs) is under consideration for inclusion in the Convention on the basis of their risk profile prepared by the POP Review Committee; a third group (proposed POPs) has been proposed for risk evaluation to the Review Committee. In Table 15.1 the compounds belonging to the three different groups are listed; Table 15.2 provides hints on

Table 15.1 Priority, candidate and proposed-for-inclusion POPs within the Stockholm Convention

Priority POPsa

Aldrin Chlordane DDT (p,p'-DDT) Dieldrin

Endrin Heptachlor

Hexachlorobenzene (HCB) Mirex

Polychlorobiphenyls (PCBs) Polychlorodibenzodioxins (PCDDs) Polychlorodibenzofurans (PCDFs) Toxaphene

Candidate POPsb

Chlordecone Hexabromobiphenyl

Lindane (gamma-HCH) Pentabromodiphenyl ether

Perfluorooctane sulfonate (PFOS)

Proposed for inclusion POPsb

alpha-HCH beta-HCH

Octabromodiphenyl ether Pentachlorobenzene

Short-chained chlorinated paraffins

a All compounds are targeted for ''elimination'', with the exception of DDT (''restricted use'') and PCDDs and PCDFs (''unintentional production''). In most cases, production and/or use are subject to specific exemptions, likely reflecting local requirements. b Updating of Annex A, B or C of the Convention by the POPs Review Committee. Third meeting of the POPs Review Committee (POPRC-3) 19-23 November 2007, Geneva, Switzerland.

a All compounds are targeted for ''elimination'', with the exception of DDT (''restricted use'') and PCDDs and PCDFs (''unintentional production''). In most cases, production and/or use are subject to specific exemptions, likely reflecting local requirements. b Updating of Annex A, B or C of the Convention by the POPs Review Committee. Third meeting of the POPs Review Committee (POPRC-3) 19-23 November 2007, Geneva, Switzerland.

their main physical-chemical and toxicological properties. The inclusion of organic chemicals in the frame of the Convention presumes that the above-mentioned science-based requirements (persistence, toxicity, bioaccumulation, long-range transport) should be met.

Factors Influencing the Exposure of Meat Animals to Chemical Contaminants

Meat production scenarios are driven by a variety of variable factors linked to each whose final result could enhance or reduce the risk of exposure and bioaccumulation of those contaminants of priority relevance in food-producing animals.

Schematically they can be summarized as follows: (a) the globalization of the markets of feeds and foods produced in countries that may be acknowledged for differences in environmental risk (Reardon & Barrett, 2002) (Fig. 15.1); (b) the proposition of feed materials innovative for origin and provenience (Brambilla & De Filippis, 2005; Sapkota, Lefferts, McKenzie, & Walker, 2007), to improve the meat nutritional quality (less fat with a modified composition in favour of (poly)unsaturated fatty acids with respect to cholesterol and saturated fats) (Givens, 2005), that could determine a lower dilution in the fat mass of lipophylic contaminants; (c) a cost increase of some agriculture practices that determine to reconsider the use of wastes (i.e. the turnover from more expensive chemically synthesized fertilizers to

Table 15.2 Synopsis of the relevant chemical, physical and toxicological features of consolidated, Candida ted and proposed POPs (WHO, 2006; Stow, 2005; IARC)

Compound

Structure

Molecular weight

LogKow

Half-life in soil (years)

Non-carcinogenic chronic toxicity

Guidance values

IARC

group

Aldrin

Reproductive and developmental toxicity, neurotoxicity

WHO pTDIO.l ng/kg h.w. per day cumulative with Dieldrin ASTDR MRL -0.03 ng/kg/day USEPA RID - 0.03 \igj kg/day

Chlordane (CAS 57-74-9)

Reproductive and developmental toxicity, neurotoxicity

6.19

Reproductive and developmental toxicity, neurotoxicity

kg/day Health Canada pTDI -20 fig/kg/day

Dieldrin (CAS 60-57-1)

Reproductive and developmental toxicity, neurotoxicity

WHO pTDI 0.1 ng/kg h.w. per day cumulative with Dieldrin ASTDR MRL -0.03 ng/kg/day USEPA RID - 0.03 \ig/ kg/ day_

Table 15.2 (continued)

Compound

Structure

Molecular weight

LogKow

Half-life in soil (years)

Non-carcinogenic chronic toxicity

Guidance values

IARC

group

322-460

10-12"

PCDFsef

306-444

Reproductive and developmental toxicity, immuno- and neurotoxicity

Reproductive and developmental toxicity, immuno- and neurotoxicity

Reproductive and developmental toxicity, neurotoxicity

WHO TDI - 1-4 pg TEQ/kg/day (cumulative with PCDF and DL-PCBS) ASTDR MRL - 1 pg TEQ/kg/day

WHO TDI - 1-4 pg TEQ/kg/day (cumulative with PCDF and DL-PCBS) ASTDR MRL - 1 pg TEQ/kg/day

WHO pTDI -

3"

Hexachlorobenzene ÇI

cr y CI CI

Reproductive and WHO ADI -0.17 ng/ 2 B

developmental kg/day toxicity, neurotoxicity

Table 15.2 (continued)

Compound

Structure

Molecular weight

LogKow

Half-life in soil (years)

Non-carcinogenic IARC

chronic toxicity Guidance values group

Heptachlor (CAS 76-44-8)

Hepatic, reproductive and developmental toxicity

Mirex (CAS 2385-85-5)

Mirex (CAS 2385-85-5)

5.28

Reproductive a nd developmental toxicity, immuno- and neurotoxicity

kg/day

Health Canada pTDI - 0.07 fig/kg/day

PCBss

1S9-499

Reproductive and developmental toxicity

ASTDR MRL -

0.02 ng/kg/day USEPA RfD - 0.02 \igj kg/day Health Canada pTDI -

1 ng/kg/day

Toxaphene (CAS 8001-35-2)

Toxaphene (CAS 8001-35-2)

0.3-12 years

Reproductive and developmental toxicity, immuno- and neurotoxicity

Health Canada pTDI 2B

Table 15.2 (continued)

Compound Structure

Molecular weight

LogKow

Half-life in soil (years)

Non-carcinogenic IARC

chronic toxicity Guidance values group

Chlordecone (CAS 143-50-0)

Chlordecone (CAS 143-50-0)

490.6

Hepatic, reproductive and developmental toxicity

Hexabro-

mobiphenylh Bf e

627.58 6.39

Hepatic, reproductive, immuno- and thyroid toxicity

Lindane (gamma-hexachloro-cyclohexane) (CAS 58-89-9)

290.83 3.8

Reproductive and developmental toxicity, neurotoxicity

WHO tADI y-HCH

PFOS1

538 Not measurable

Hepatic, reproductive and immuno (thymus) toxicity

Penta-BDE

(commercial mixture)

Hepatic, reproductive and thyroid toxicity

Table 15.2 (continued)

Compound

Structure

Molecular weight

LogKow

Half-life in soil (years)

Non-carcinogenic chronic toxicity

Guidance values

IARC

group

Pentachloro-

benzene (CAS 608-93-5)

250.32

Hepatic, nephric, hematological and developmental toxicity

Octa-BDE

Octa-BDE

mixture) (CAS 32536-52-0)

801.38

6.29

mixture) (CAS 32536-52-0)

Fetotoxicity, delayed fetal skeletal ossification, hepatic and thyroid toxicity

SCCPsm (CAS 85535-84-8)

320-500

>365

Hepatic toxicity

fl/pAo-HCH"

290.S3

48-125

Neuro-, hepato-, immuno-toxicity

ASTDR MRL ct-HCH -

S ng/kg/day

Table 15.2 (continued)

Compound

Structure

Molecular weight

LogKow

Half-life in soil (years)

Non-carcinogenic chronic toxicity

Guidance values

IARC

290.83

91-122

Hepatic reproductive toxicity

a 1,1 ,l-trichloro-2,2-bis(4-chlorophenyl)ethane. b Polychlorinated dibenzo-/?-dioxins.

c Data refer to the seven 2,3,7,8-chlorosubstituted toxic congeners only. d Data refer to 2,3,7,8-T4CDD. e Polychlorinated dibenzofurans.

f Data refer to the ten 2,3,7,8-chlorosubstituted toxic congeners only. g Polychlorinated biphenyls. h Only one isomeric structure is shown. 1 Perfluorooctane sulfonate (the potassium salt is shown).

k Pentabromodiphenyl ether. The commercial mixture contains penta- through heptabromo-substituted homologues. 1 Octabromodiphenyl ether. The commercial mixture contains penta- through decabromo-substituted homologues. 111 Short-chained chlorinated paraffins. n alpha-Hexachlorocyclohexane. ° beta-Hexachlorocyclohexane.

ASTDR, Agency for Toxic Substances and Disease Registry. MRL, minimum risk levels for chronic exposure.

USEPA RfD, United States Environmental Protection Agency Reference doses. WHO tADI, World Health Organization's temporarily acceptable daily intake. WHO (TDI/pTDI), World Health Organization's (provisional) tolerable daily intake. Health Canada (pTDI) provisional daily intakes.

IARC, International Agency of Research on Cancer; 1: The agent is carcinogenic to humans; 2A: The agent is probably carcinogenic to humans; 2B: The agent is possibly carcinogenic to humans; 3: The agent is not classifiable as to its carcinogenicity to humans; 4: The agent is probably not carcinogenic to humans.

Fig. 15.1 Changes in meat (above) and feedingstuffs (below) imports from third countries to the European Union, 1 in 1994 and 2004 (Eurostat, 2006)

cheaper potentially contaminated sludges (Fiedler, Hutzinger, Welsch-Pausch, & Schmiedinger, 2000; Schoof & Houkal, 2005); (d) new trends in animal managements, towards less intensive farming systems, and an improved welfare, that could lead to an increased exposure to environmental contaminants (i.e. through forages and soils) with respect to that coming from feedingstuffs placed on the market (Schierea, Ibrahim, & van Keulenc, 2002) (FAO, 2000); (e) climate changes and new evaluations of the risk/ benefit ratio in the use of pesticides, such as dichlorodiphenyltrichloroethane (DDT) to prevent arthropod-borne transmissible diseases both to animals and to humans (FAO, 2008; WHO, 2007); (f) not sufficiently implemented protective farming practices with respect to possible backyard emissions due to the improper disposal of agriculture wastes (Codex Alimentarius, 2006; United States Environmental Protection Agency - US EPA, 2008). In Tables 15.3 and 15.4 an inventory of the ports of entry for "dioxins" and other POPs in the food chain are, respectively, reported (Brambilla, Iamiceli, Ferri, & di Domenico, 2008).

Table 15.3 Inventory of potential sources of exposure to PCDDs, PCDFs and PCBs in farmed animals

Item

Origin of contamination

1. Anticaking agents (clay)

2. Mineral components

3. Zinc and copper oxide

4. Calcium phosphate

5. Rendered oils for animal nutrition

6. Fish oils and meals

1. Drying of feed material

2. Crop harvesting

3. Atmospheric ''fall-out''

1. Grazing fields

2. Water

1. Stable

2. Bedding

3. Anti-slippery floors

Feedingstuffs (in market)

• Waste from high-temperature furnaces

• Ashes from rotary kilns

• Hydrolysis of bones and shells with hydrochloric acid used in PVC production

• Contamination with heat-stressed or aged PCB-containing dielectric fluids

• Contamination from exhausted gasoline or combustion oil residues in barrels

• Contaminated aquatic webs

Forages (in farm)

• Open flame devices

• Crop exposure to contaminated soil

• Improper disposal (burning) of agricultural waste

Grasslands and water

• Use of sewage sludge as fertilizers

• Use of incineration ashes as mineral fertilizers

• Vicinity to chloro-pesticide plants/stores

• ''Fall-out'' from local regular emissions (metal smelters, plastic material manufacturers, etc.)

• Destruction of animal carcasses

• Improper disposal (burying) of agricultural and veterinary wastes

• Vicinity to paper mills, to chlorinated pesticide factories; releases from pesticide stores

Farm work and indoor areas

• Leakage from old electrical apparatuses

• Timber treated with antifouling agents (copper chromium arsenate (CCA), pentachlorophenol (PCP), etc.)

• CCA- and PCP-contaminated shavings and dust

• Recycled PVC, rubber and plastic materials

From Residues Monitoring Plans to Intake Assessment

Although many POPs are already strictly regulated or are no longer in production, as the food of animal origin still represents the main source of exposure, the measurement of POPs in food and, in particular, in products of animal origin is particularly relevant for the protection of human health and for the consumers' perception about food safety. Appropriate monitoring plans and maximum residue limits (MRLs) for some POPs (organochlorine pesticides) in a variety of food commodities were established by European Union (EU) and

Table 15.4 Potential sources of exposure to candidate POPs in meat animals (UNEP, 2002)

Compound

Exposure source

1. Chlordecone

2. Dicofol

3. Endosulfan

4. Hexabromobiphenyl

5. Hexachlorocyclo-hexanes (alpha-, beta-gamma-HCH)

6. Pentachlorobenzene

7. Perfluoro-organic compounds (PFCs, PFOS, PFOA, etc.)

8. Polybromodiphenyl ethers (PBDEs)

9. Short-chained chlorinated paraffins (SCCPs)

10. Polychloronaphtha-lenes (PCNs)

Insecticide, fungicide and degradation product of insecticide mirex Acaricide structurally similar to DDT, used on grapes, beans, cotton, pumpkin, ornamental plants, melon and watermelon Insecticide and acaricide (against mites) used in horticulture, crop industry and cotton Flame retardant in thermoplastics for industrial and electrical products

• Insecticide for treatment of seeds, lice and scabies

• Used on soils intended for sugarbeet cultivation

Obsolete pesticide, flame retardant and intermediate to make fungicides Stain resistance treatments for fabrics/paper, coatings for metal surfaces including nonstick cookware and electronics components, fire fighting foams

Flame retardants in plastics for TV sets and computers, in carpets, car interiors and polyurethane foams for furniture and bedding High-temperature lubricants, plasticizers, flame retardants and additives in adhesives, paints, rubbers and sealants Cable insulation, wood preservative, engine oil additive, capacitor fluids, dye intermediate and flame retardants

• Contamination of crops and seeds

• Contamination of forages

• Forages (feed) contamination

• Top soil treated with sewage sludge

• Contact materials

• Feed contamination

• Soil and water contamination

• Closeness to product stock piles

• Topsoil treated with sewage sludge

• Litter from recycled paper

• Contact materials (rubbers, carpets)

• Topsoil treated with sewage sludge

• Topsoil treated with sewage sludge

• Contact material (bedding)

• Topsoil treated with sewage sludge non-EU countries, thus making mandatory the development of sensitive methods to analyse these pollutants in food, along with the establishment of correct sampling procedures and sample pre-treatments (Food and Agriculture Organization/World Health Organization - FAO/WHO, 2001; EU Regulation, 2005).

More recently, international and national bodies have focused their attention on alimentary exposure (intake) assessment (WHO, 2000; 2002), trying to verify to what extent the regulatory actions are effective in keeping consumers and sensitive population groups from unacceptable levels of exposures. Schematically, such an assessment may be carried out following two different approaches. The first is a so-called indirect approach or dietary modelling: dietary modelling is the process of combining country-based food consumption data (European Food Safety Authority - EFSA, 2008) and chemical concentration data in foods (i.e. those data coming from residue monitoring plans) to estimate the intake of the selected contaminants. Intake estimates are then compared to reference health standards for life-long exposures, usually expressed as "tolerable daily, weekly or monthly intakes'' (TDI, TWI and TMI) to estimate the risk to population health (WHO, 1999). These estimates may be carried out on a deterministic (average or worst-case exposures) or a (semi-) probabilistic basis, taking into account the distribution curves of the data and the relative percentiles (Kroes et al., 2002). However, they are affected to different extents by uncertainties that suggest a prudent use of the exposure outcomes.

Uncertainties may arise from both the quality and the representativeness of the data, such as

- sampling strategy focused on meat batch and consignment and not on the food really eaten by consumers;

- data not consistent enough to describe the distribution of contamination for each category of food considered in the consumption database;

- food of animal origin with different fat content grouped in the same class of food item (i.e. meat and meat products);

- analytical methods basically focused on contamination values only around the MRL range, sufficient to give a compliance/non-compliance evaluation, but not validated for determination over the entire range of possible contamination levels.

To overcome the aforesaid biases in dietary modelling, some countries have planned specific studies (the so-called total diet studies (TDS) or "market basket studies'') that involve the purchasing of food samples at retail level, according to their representativeness in the food diet of a selected population and their preparation according to national household procedures. The foods so prepared are then aggregated for macro categories (fish and fishery products, milk and dairy products, egg and egg-based products, meat) and the resulting composite samples analysed for the contaminants of interest (WHO, 2002).

The second (direct) approach is represented by the ''duplicate diet'' (DD) methodology (Thomas et al., 1997); selected population groups as representative of country food habits are asked to duplicate their meals within a fixed time frame (e.g. 1 week), meals that will be analysed. Such direct approach provides at the same time accurate information on both dietary habits and contaminant intakes Moreover, it takes into account possible factors that may influence the contaminant content, such as meat cooking process (e.g. frying, boiling, roasting, steaming) (Noel, Leblanc, & Gueerin, 2003). However, the necessity of an active check of consumers may affect the representativeness of the outcomes due to the rather limited number of observations usually carried out in such studies.

In the following sections, the relevant information about chemistry, occurrence, exposure and analysis of the main categories of priority chemical contaminants in meat will be provided.

Organochlorine Pesticides

The family of organochlorine pesticides groups a wide range of organic chemicals containing chlorine atoms, used in agriculture and public health to effectively control pest. Although most of them were during the 1970 s and 1980 s, they are still found in the environment (Rhind, 2002; Konstantinou, Hela, & Albanis, 2006) and in biological matrices (Torres et al., 2006; Meeker, Altshul, & Hauser, 2007). Due to their chemical-physical properties as POPs, food is considered to represent a long-term source of exposure. Current EU MRLs established for the organochlorine pesticides of interest in animal products are set between 0.02 and 1 mg/kg fat. An inventory of the regulatory limits in different countries according to the animal species is reported in Table 15.5. The official occurrence data in EU meat are available in the binary form "compliant/non-compliant". However, in 2004, of 436 samples targeted on different types of meat, a few non-compliant outcomes were found in cattle for gamma-hexachlorocyclohexane (HCH) (lindane) residues (three results), pigs (one for beta-HCH and one for DDT, as the sum of its isomers and related compounds) and sheep (two for beta-HCH) (EU Commission, 2004). At international level, MRLs in meat and meat products recommended by Food and Agriculture Organization (FAO) jointly with World Health Organization (WHO) vary from 0.05 to 3 mg/kg fat. In the United States, legislation was enacted in 1996 with the Food Quality Protection Act, including stricter safety standards, especially for infants and children, and a complete reassessment of all existing pesticide tolerances. For the pesticides of our concern, US residue limits are established between 0.1 and 7 mg/kg fat. The US Pesticide Monitoring Program does not focus on meat, but uses primarily cow milk and eggs as the more relevant animal food source of exposure and biomarkers.

A Swedish market basket survey (Darnerud et al., 2006) supports the evidence that meat products do not represent a relevant source for organochlorine pesticide intake; for DDT (sum of p,p -DDE, p,p0-DDD, p,p -DDT and p-DDT), meat contribution to intake is on average 83 ng on a total dietary intake of 524 ng (15% of contribution), whereas for the three HCH and for the four chlordane congeners the ratio (in nanograms) is 9.5/81 and 6.2/115, corresponding to 12 and 5% contributions, respectively.

Multi-residue procedures and highly sensitive methods are currently a requirement in organochlorine pesticide analysis in products of animal origin. Hercegova,

Table 15.5 Regulatory limits (MRLs) in |ig/g fat for residues of POP pesticides in meat in EU and several non-EU countries

POP

Australia

EUa Meat

Offals

Fat

FAOb

Japan

Korea

United Kingdom

United States

Aldrin

0.2

0.2

0.2

0.2

0.2

0.2

0.2

0.2

0.3

Chlordane

0.2

0.05

0.05

0.05

0.05

0.08

0.5

0.05

0.3

DDT

5

1

1

1

5

1

5

1

5

Dicofol

-

0.05-0.5

0.05

0.05

3

0.3-0.08

-

0.05-0.5

-

Dieldrin

0.2

0.2

0.2

0.2

0.2

0.2

-

0.2

0.3

Endrin

-

0.05

0.05

0.05

-

0.05

0.1

0.05

0.3

HCB

1

0.2

0.2

0.2

-

0.2

-

0.2

0.5

HCH

-

0.3

0.3

0.3

-

-

-

0.1-0.2

-

Heptachlor

0.2

0.2

0.2

0.2

0.2

0.2

-

0.2

0.2

Lindane0

2

0.02

0.02

0.02

0.1

0.02

2

0.02

4-7

Mirex

-

-

-

-

-

-

-

-

0.1

Toxaphene

-

0.05

0.05

0.05

-

-

-

0.05

-

a Legislative reference (harmonization in progress within the framework of Regulation 2005/396/EC). For aldrin, chlordane, DDT, dieldrin, endrin, HCB, HCH, heptachlor: Directive 1993/57/EEC. For dicofol: Directives 1993/57/EEC and 2000/24/EC. For lindane: Directive 2002/66/EC. For toxaphene: Directive 2004/61/EC. b Together with WHO. c gamma-HCa.

a Legislative reference (harmonization in progress within the framework of Regulation 2005/396/EC). For aldrin, chlordane, DDT, dieldrin, endrin, HCB, HCH, heptachlor: Directive 1993/57/EEC. For dicofol: Directives 1993/57/EEC and 2000/24/EC. For lindane: Directive 2002/66/EC. For toxaphene: Directive 2004/61/EC. b Together with WHO. c gamma-HCa.

Dômôtôrovâ, and Matisova (2007) have recently reviewed available methods and drawn the following decalogue: (a) possibility to determine a number of pesticides as high as possible in a single analysis; (b) high recoveries; (c) high selectivity obtained by means of an effective removal of potential interferences from the sample; (d) high sensitivity; (e) high precision; (f) good ruggedness; (g) low cost; (h) high speed; (i) use of less harmful solvents and in low amounts.

Multi-residue methods developed for organochlorine pesticides follow the general scheme shown in Fig. 15.2. After a sample pre-treatment

SAMPLE

Pre-treatment

(grinding, homogenization, addition of a drying agent, addition of the Internal Standard(s)

Extraction

(Soxhlet, high speed extraction, SFE, PLE, MAE, MSPD, etc.)

Extraction

(Soxhlet, high speed extraction, SFE, PLE, MAE, MSPD, etc.)

Removal of lipids

Removal of lipids

Disruptive methods

(treatment with concentrated sulfuric acid or alkaline treatment)

Non-disruptive methods

(GPC, liquid-liquid partitioning, adsorption chromatography, solid phase extraction,... )

Non-disruptive methods

(GPC, liquid-liquid partitioning, adsorption chromatography, solid phase extraction,... )

Fractioning

(adsorption chromatography)

Fractioning

(adsorption chromatography)

Instrumental analysis

Fig. 15.2 General flow chart for the analysis of organochlorine pesticides, PCDDs, PCDFs, PCBs and PBDEs

(homogenization and drying), the analytes and fat are co-extracted. Lipids are separated from analytes using different non-destructive procedures, such as liquid-liquid partitioning and gel permeation chromatography (GPC). Adsorbent phases Florisil® (US Silica Company, Berkeley Springs), alumina or silica gel are then used for the clean-up step. Instrumental determination is performed by high-resolution gas chromatography (HRGC) coupled with electron capture detection (ECD) or with mass spectrometry (MS). The principal procedures currently used for the analysis of organochlorine pesticides in meat samples were reviewed by the Codex Committee on Pesticide Residues (Codex, 2003) and included in the Official Methods of Analysis of the Association of Official Analytical Chemists (AOAC International, 2005) and in the Pesticide Analytical Manual of the Food and Drug Administration (US FDA, 1994).

Non-dioxin-Like Polychlorobiphenyls (NDL-PCBs)

Polychlorinated biphenyls (PCBs) are a family of 209 chlorinated compounds, no longer produced and used in Europe, United States and Canada, but still commercially available under various trade names in other parts of the world. PCBs are substantially insoluble in water, very lipophilic and highly persistent (Table 15.2). For their chemical-physical stability and dielectric properties, they were used worldwide as dielectric fluids in transformer and capacitor oils, as hydraulic and heat exchange fluids and as lubricating and cutting oils. Non-dioxin-like (NDL)-PCBs have a different toxicological profile from PCDD, PCDF and dioxin-like (DL)-PCBs.

In experimental animal studies carried out with individual NDL-PCB congeners (PCB 28,128 and 153 at a dose range of 30-40 mg/kg-bw per day) showed liver and thyroid toxicity to be the most sensitive toxicological end-points. However, because in field conditions a contamination with both NDL-PCB and DL-PCBs could occur, possible confounding effects can be determined by the simultaneous presence of DL and NDL congeners, owing to toxicological end-points being almost the same. In a recent opinion EFSA (2005) concluded that no health-based guidance value for humans could be established for NDL-PCB; however, regulatory levels as indicators of an overall good farming practices are suggested.

NDL-PCBs bioaccumulate in meat, liver and particularly in fat tissues of farmed animals and have been included in the EU national residue monitoring plans in food of animal origin since 1990. Data usually refer to the following six (S6) NDL-PCB congeners (PCB 28, 52, 101, 138, 153, 180) as indicator or marker PCBs. The six individual congeners were not selected from a toxicolo-gical point of view, but considered to be representative for the different PCB patterns in various sample types. The S6 NDL-PCB represents about 50% of total NDL-PCB in food. In some countries, the dioxin-like PCB 118 has been added to the former six, to form a group of seven (S7) indicator PCBs. Lacking toxicology and health-based guidance values, the EU Commission may set a MRL for the S6 (NDL-PCBs) in meat products, considering its occurrence recorded from national residue control plans. A 99th percentiles around 48 and 28 ng/g fat have been recently reported for ruminant and pig meats, respectively (European Commission, DG Sanco working document, January 2008).

More than 90% of the NDL-PCB exposure in the general population is via food. Average dietary daily intakes of total NDL-PCB were estimated to be in the range of 10-45 ng/kg b.w. per day, excluding breastfed infants; children had exposure levels 2.5-fold higher than adults due to a larger intake of milk and dairy products. Moreover, some European (sub)-populations and other specific groups consuming fish-based diets may be exposed to twofold higher intakes than the average population. This determines the efforts to lower the levels of NDL-PCB in foods (EFSA, 2005).

An inventory of intake estimates of NDL-PCBs was carried out by EFSA (2005) for three different European countries, by considering the average occurrence in food items, their average fat contents and the average food consumption (Table 15.6). In the Netherlands (Baars et al., 2004), the estimated median life-long-averaged intake of the S6 NDL-PCBs was 5.6 ng/kg b.w. per day, with a meat product contribution of 27%. These data indicated, however, a diminishing trend in the concentration of the contaminants in selected food items with respect to previous national studies. The same diminishing trend was reported by Fattore, Fanelli, Dellatte, Turrini, and di Domenico, (2008) for the S6 NDL-PCBs dietary intake in the Italian population: the mean exposure resulted in 10.9 ng/kg b.w. in adults (13-94 years old). Fish and fishery products and milk and dairy products were the major contributors to the total dietary intake, with meat contributing at 10%.

As reported above, most analytical studies on NDL-PCBs are limited to the determination of a small number of congeners (PCBs 28, 52, 101, 138, 153 and 180) as indicators of the presence of all the chemical class. The analytical procedures for NDL-PCBs have recently been reviewed by Ahmed (2001), for PCB analysis in food. With respect to the analytical determination of "dioxins'' (PCDDs and PCDFs) and DL-PCBs, differences may be observed in the chromatographic and detection systems utilized (Fig. 15.2). The general scheme consists on an organic solvent extraction; then lipids are removed by gel permeation chromatography (GPC) or treatment with sulfuric acid and co-extracted substances are eliminated by adsorption chromatography. A comprehensive review on developments in the high-resolution gas chromatography (HRGC) of PCBs is given by Cochran and Frame (1999), who evaluated a variety of stationary phases commonly used for PCB analysis. The 5%-phenyl type column has substantially become the standard for PCB analysis. The final determination is performed by HRGC-ECD (electronic capture detector) or, preferably, by HRGC-LRMS (low-resolution mass spectrometry). The internal standard (IS) technique is generally adopted in accord with the US EPA Methods 1668 (US EPA, 1999).

Table 15.6 Estimates of the dietary exposures for the average consumer to NDL-PCBs of three EU countries (EFSA, 2005)

Sum PCBs Consumption Consumption Consumption Exposure Exposure Exposure

Table 15.6 Estimates of the dietary exposures for the average consumer to NDL-PCBs of three EU countries (EFSA, 2005)

Sum PCBs Consumption Consumption Consumption Exposure Exposure Exposure

Food groups

(mean) ng/g

Italy

France

Sweden

Italy

France

Sweden

Cereals and cereals products

0.0213

270

218

292

6

5

6

Fruits and vegetables21

0.0495

498

313

387

25

15

19

Eggsb

0.73

18

17

15

13

12

11

Fats and oils0

5.05

38

18

24

192

91

121

Meat and meat products'3

1.52

134

117

143

204

178

218

Offalsc

0.74

3

3

7

2

2

5

Fish and fish products

12.50

43

32

35

538

400

438

Milk

0.17

124

106

343

21

18

59

Cheese and dairyf

0.98

87

100

45

86

98

44

Total (ng/day)

1086

821

921

Total (ng/kg b.w. per day

18.1

13.7

15.4

a Including potatoes. b Assuming 11.1 g fat for 100 g product. c An average value for vegetal oil was used. d Assuming 12 g fat for 100 g meat. c Assuming 10.9 g fat for 100 g liver. f Assuming 1.6 fat for 100 ml milk.

Polychlorodibenzodioxins, Polychlorodibenzofurans and Dioxin-Like PCB (PCDDs, PCDFs and DL-PCBs)

PCDDs and PCDFs (altogether also commonly known as "dioxins") are not produced intentionally; in fact their formation and release into the environment occur primarily in thermal or combustion processes or as unwanted byproducts of industrial processes involving chlorine. They form two groups of tricyclic aromatic compounds containing between one and eight chlorine atoms, thus resulting in 210 congeners (75 PCDDs and 135 PCDFs), different in the number and/or position of chlorine atoms; only the 17 congeners with chlorines at positions 2, 3, 7 and 8 are of toxicological interest. "Toxicity equivalency factors'' (TEFs) have been proposed by different regulatory bodies (for instance, US EPA, NATO, WHO) since 1970 and applied to evaluate the cumulative effect of the toxicity on mammals or environmental species. In 1997 the World Health Organization (WHO) adopted the (WHO-TEF) based approach also for food and feedingstuffs to each of the 17 congeners: the highest TEF of 1 was assigned to 2,3,7,8-T4CDD (as the most toxic congener) (Van den Berg et al., 1998): an update of the aforesaid WHO-TEFs was carried out in 2005 (van den Berg et al., 2006).

Due to the shared mode of action, WHO-TEFs have also been assigned to 11 PCB congeners, named as dioxin-like PCBs. In Table 15.7, the former consensus-based 1997 and the new adopted 2005 TEFs for all the PCDD, PCDF and PCB congeners with a dioxin-like activity are reported.

Humans are exposed to PCDDs, PCDFs and DL-PCBs mainly through the diet. The contribution of foods of animal origin (i.e. meat and fish and their products) may be higher than 90% of the total exposure to the aforesaid contaminants (Fattore, Fanelli, Turrini, & di Domenico, 2006; Tard, Gallotti, Leblanc, & Volatier, 2007). In order to reduce human exposure and protect consumer health, the EU has progressively issued regulatory measures setting maximum levels (MLs) and action levels (ALs) for PCDDs, PCDFs and DL-PCBs in food (Table 15.8). For example, a ML of 3.0 pgWHO-TE/g fat was established for PCDDs and PCDFs in bovine and sheep meat corresponding to a ML of 4.5 pgWHO-TE/g fat when DL-PCBs are considered (EU Regulation 1881, 2006); in pork meat, the corresponding ML values are 1.0 and 1.5 pgWHO-TE/g fat. When contaminant concentrations are greater than ALs but not MLs, the meat is not withdrawn from the market, but it is mandatory to trace back the source of exposure(s) that may have determined the unwanted contamination level higher than the average, i.e. through a feed.

Many data are presently available for their occurrence in carcasses from pigs and beefs. A former US EPA, United States Department of Agriculture (USDA) and Food and Drug Administration (FDA) survey carried out in 1994-1996 on 56 carcasses of pigs and 51 carcasses of steers and heifers found a mean 1.44 (median, 1.19) pgWHO-TE/g fat for PCDDs and PCDFs, and a mean 1.47 (median, 1.22) pgWHO-TE/g fat for PCDDs, PCDFs and DL-PCBs

Table 15.7 Comparison of the 1998 and 2005 TEFs for PCDDs, PCDFs and DL-PCBs; values in italics indicate a change in TEF value (Van den Berg et al., 2005)

Compound_1998 WHO-TEFs_2005 WHO-TEFs

Chlorinated dibenzodioxins

1.2.3.7.8.9-HxCDD 0.1 0.1 1,2,3,4,6,7,8-HpCDD 0.01 0.01 OCDD 0.0001 0.0003

Chlorinated dibenzofurans

1.2.3.7.8.9-HxCDF 0.1 0.1 2,3,4,6,7,8-HxCDF 0.1 0.1

Non-ortho-substituted PCBs

PCB 77 0.0001 0.0001

PCB 81 0.0001 0.0003

PCB 169 0.01 0.03

Mono-ortho-substituted PCBs

PCB 105 0.0001 0.00003

PCB 114 0.0005 0.00003

PCB 118 0.0001 0.00003

PCB 123 0.0001 0.00003

PCB 156 0.0005 0.00003

PCB 157 0.0005 0.00003

PCB 167 0.00001 0.00003

PCB 189 0.0001 0.00003

together. Later data, referred to the 2002-2003 period, indicated a mean 0.24 (median, 0.15) pgWHO-TE/g fat for PCDDs and PCDFs in 136 pig carcasses and a mean 0.28 (median, 0.18) pgWHO-TE/g fat when DL PCB contributions are included. The mean level of PCDDs and PCDFs in heifer and steer carcasses (N = 139) was 0.79 (median, 0.73) pgWHO-TE/g lb, whereas the sum of PCDDs, PCDFs and DL-PCBs was found to be 0.93 (median, 0.56) pgWHO-TE/g fat (US FDA, 2006). An USDA market basket study (Huwe & Larsen, 2005) reported a PCDD and PCDF mean contaminations of 0.64,1.54 and 0.37 pgWHO-TE/g fat for beef, hamburgers and pig meat, respectively; the

Table 15.8 MLs and ALs for meat and meat products according to Regulation 1881/2006/EC and Recommendation 2006/88/EC. Levels concern PCDDs plus PCDFs (I), DL-PCBs (II) or their sum (I + II), to be compared with upper bound analytical outcomes

Food or feed item

ML(I)

ML(I + II)

AL(I)

AL(II)

EU acceptance levels for meat and meat productsa

Ruminants (bovine, ovine)

3.0

4.5

1.5

1.0

Poultry and farmed game

2.0

4.0

1.5

1.5

Pork

1.0

1.5

0.6

0.5

Liver and liver products

6.0

12.0

4.0

4.0

a Values in pgWHO-TE/g fat (fat, >1%). The same levels apply to the fats derived from ruminants, poultry and pork.

a Values in pgWHO-TE/g fat (fat, >1%). The same levels apply to the fats derived from ruminants, poultry and pork.

corresponding DL-PCB mean concentrations were 0.11, 0.15 and 0.05 pgWHO-TE/g fat, respectively.

In 2000, the EU Scientific Committee on Food made an inventory on 138 meat products within the EU. In ruminants, the mean concentration of PCDDs and PCDFs was 0.74 pgWHO-TE/g fat and that of DL-PCBs 0.72 pgWHO-TE/g fat. In pigs, PCDDs and PCDFs were found at a mean 0.85 pgWHO-TE/g fat, whereas DL-PCB concentration was 0.40 pgWHO-TE/g fat (EC/EU SCF, 2000). New data coming from implementing EU national residue plans followed in 2004: the mean concentrations in ruminants for PCDDs and PCDFs alone, or together with DL-PCBs, were 0.46 and 0.80 pgWHO-TE/g fat, respectively. The corresponding values of 0.21 and 0.23 pgWHO-TE/g fat were reported for pigs (Gallani & Boix, 2004). Compared with the older data, the most recent monitoring results show a decrease of PCDD and PCDF levels, in line with the observed general trend (US EPA, 2006).

According to results of a national PCDD, PCDF and DL-PCB monitoring program, Australian Government (2005) reported total mean contaminations of 0.845, 0.575 and 0.803 pgWHO-TE/g fat in beef, pigs and lamb, respectively.

For the exposure assessment, guidance values as TDI, TWI and TMI have been set by different international bodies (Table 15.9). In most countries, the estimated mean intakes in adults seem close to such values, thus indicating a not

Table 15.9 Guidelinesa for total human exposure to PCDDs, PCDFs and DL-PCBs. Values expressed in pgWHO-TE/kg body weight

Organization or country

TDIb

TWIb

TMIb

EU (2001)

2

14

60

WHO (1998)

1-4

7-28

30-120

JEFCA (2001)

2.3

16.3

70

NL (2000)

1

7

30

Japan (2000)

4

28

120

Australia (2002)

2.3

16.3

70

a Original guidelines in bold; values extrapolated by these authors in italics. b TDI, TWI, TMI: tolerable daily, weekly or monthly intake.

a Original guidelines in bold; values extrapolated by these authors in italics. b TDI, TWI, TMI: tolerable daily, weekly or monthly intake.

negligible part of population may be over-exposed. However, a diminishing trend is noted with respect to previous studies, as already observed for NDL-PCBs. Among food items, meat consumption represents the third contribution in order of relevance to intake, after fish and fishery products and milk and dairy products, with some possible relevant differences according to the country or local food habits (Table 15.10).

The necessity to evaluate the cumulative presence of PCDDs, PCDFs and DL-PCBs along with their possible low contamination levels (in the order of pg/ g WHO-TE) makes the analytical approach rather complex, time consuming and rather expensive. Reference methods have been elaborated by the US Environmental Protection Agency for the determination of the PCDD and PCDF toxic congeners (US EPA, 1994) and for DL-PCB congeners (US EPA, 1999) by HRGC-HRMS. The basic requirements for the EU official analytical methods to determine PCDD, PCDF and DL-PCB levels in foodstuffs are reported in Regulation 1883/2006 EC (2006). A critical review of the various methods used to analyse DL-PCBs is given by Iamiceli, Fochi, Bram-billa, and di Domenico (2008). Many analytical methods follow the following

Table 15.10 Contribution of meat consumption to the mean total intake of PCDDs, PCDFs and DL-PCBs in different studies

Average

meat

Country

Estimated

contribution

(town)

Year

Congeners

intake

(%)

References

Japan

2004

PCDDs, PCDFs

1.55 pgWHO-

11

Sasamoto

(Tokyo)

and DL-PCBs

TE/kg b.w. per day

et al. (2006)

Finland

2004

PCDDs, PCDFs and DL-PCBs

115 pg 1.5 pgWHO-TE/kg b.w.

Kiviranta et al. (2004)

Australia

2004

PCDDs, PCDFs and DL-PCBs

3.7-15.6 pgWHO-TE/kg b.w. per month

4***

Australian Government (2005)

United

2004

PCDDs and

9.6 pgWHO-

47

EPA (2006)

States

PCDFs

TE/kg b.w. per month*

Sweden

2006

PCDDs, PCDFs and DL-PCBs

1.30 pgWHO-TE/kg/b.w. per day**

16

Darnerud et al. (2006)

Italy

2006

PCDDs, PCDFs and DL-PCBs

2.28 pgWHO-TE/kg b.w.

11

Fattore et al. (2006)

France

2005

PCDDs, PCDFs and DL-PCBs

1.8 pgWHO-TE/kg b.w.

10

Tard et al. (2007)

* Lower bound approach, all groups. ** Medium bound approach. *** Including eggs.

* Lower bound approach, all groups. ** Medium bound approach. *** Including eggs.

general scheme: known quantities of isotopically labelled analytes are added to the samples at the earliest possible stage of extraction to provide proper correction for analyte losses, then the test sample has to be homogenized and dehydrated as described for organochlorine pesticide analysis. The analytes of interest are extracted with a suitable organic solvent and the extract is purified by the use of sulfuric acid, as far as all the analytes of interest are resistant to acid treatment and this step allows a selective destruction of most of the interfering substances (i.e. fats) co-extracted with the target compounds. Due to the difference in concentrations between planar (PCDDs, PCDFs and non-ortho DL-PCBs) and non-planar analytes (mono-ortho DL-PCBs) and the presence of other co-extractive compounds resistant to clean-up procedure (i.e. chlorinated pesticides), fractioning steps are generally included during purification before instrumental analysis by HRGC-HRMS.

Polybrominated Diphenyl Ethers (PBDEs)

PBDEs are a group of 209 congeners, differing in the number of bromine atoms and in their position on two phenyl rings linked by an oxygen. Their nomenclature is identical to that of PCBs. PBDEs were first introduced into the market in the 1960s and used as flame retardants to improve fire safety in various consumer products and in electronics. There are three types of commercial PBDE products, referred to as pentabromo- (Penta-BDE), octabromo- (Octa-BDE) and decabromodiphenyl ether (Deca-BDE), each product being a mixture of various PBDE congeners (Alaee, Arias, Sjodin, & Bergman, 2003). These chemicals are persistent and lipophilic, thus resulting in bioaccumulation in fatty tissues of organisms and in enrichment through the food chain (Law et al., 2003). The EU has prohibited the uses of Penta- and Octa-BDE (EU Directive, 2002), but these substances are still on the market in many regions of the world. In any case, a substantial reservoir of PBDEs exists in products that could release them to the environment.

Despite the fact that dietary intake is probably the main route of exposure to PBDEs for the general population (Schuhmacher, Kiviranta, Vartiainen, & Domingo, 2007; Schecter, Papke, Tung, Staskal, & Birnbaum, 2004), no MLs for PBDEs in food have been set by the EU yet. Tolerable daily intakes, due to the scarcity of data on human beings, have not been established yet.

Because they are not framed within national monitoring programs of contaminant residues in foods, PBDE occurrence data are basically recovered from intakes studies and generally are referred to the following congeners 28, 47, 99, 100, 153, 154, 183 and 209, as the most recurrent.

In the Netherlands, a granted national project revealed that oils and fats accounted for 25% of the total PBDE exposure, while milk, fish and meat contributed for 19, 13 and 11% of the intake. Average levels found in meat products, expressed as ng/g product (medium bound approach), were 0.152 for beef (16% fat) and 0.273 for pork (26% fat) (de Mul et al., 2005); when converted on a fat basis, mean values resulted in 0.950 and 1.0 ng/g lipid base (lb) for beef and pig, respectively. An USDA (2005) study based on food consumption data recorded contamination values in pig meat for the selected eight PBDEs of 2.6 ng/g fat on average, with a rather wide range spanning from 0.190 to 16.3 ng/g fat. Such a range may indicate the presence of occasional sources capable of determining high levels of contamination. By contrast in beef, against a reported mean contamination of 0.250 ng/g fat, levels ranged from the detection limit to 0.880 ng/g fat (Huwe et al., 2005).

A total diet study recently carried out by the United Kingdom Food Safety Agency (UK FSA) estimated an upper bound dietary intake in adults of five PBDEs (PBDE 47, PBDE 99, PBDE 100, PBDE 153, PBDE 209) of 5.9 ng/kg b.w. per day, where meat products accounted for 68% contribution (FSA, 2006a). In Sweden (Darnerud et al., 2006), for an estimated intake of 50.9 ng/ person/day (0.69 ng/kg b.w.), meat products contributed up to 14%. In Finland (Kiviranta, Ovaskainen, & Vartiainen, 2004), PBDE (six congeners) intake was estimated in 44 ng/day per person, where meat category (including eggs) represented 4% of the total.

As in the case of NDL-PCBs, PBDE determination in meat is limited to the of a small number of congeners used as indicators. The EFSA Scientific Panel on Contaminants in the Food Chain has recently recommended the inclusion of the following congeners in a European monitoring programme: PBDE 28, 47, 99, 100, 153, 154, 183 and 209 (EFSA, 2006) as the most frequently congeners found in food. Covaci, Voorspoels, and de Boer (2003) and Covaci et al. (2007) have recently reviewed the advances in the analysis of brominated flame retardants that in principle do not greatly differ from the approaches used for PCBs and "dioxi

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