Mari Reinik

Terje Tamme

Mati Roasto

Kadrin Juhkam

Toomas Tenno

A Kiis

Polycyclic aromatic hydrocarbons

Keywords: polycyclic aromatic hydrocarbons, benzo(a)pyrene, meat products, intake

incomplete combustion processes or in high-temperature pyrolysis of coal, oil and other organic materials. The most important sources of PAHs have been identified as coke ovens in the production of aluminium, iron, and steel; heating in power plants and residences; cooking; motor vehicle traffic; environmental tobacco smoke; and the incineration of waste material (WHO 1998). Over 100 PAHs have been identified in the environment as pollutants occuring as complex mixtures (Mottier et al. 2000).

Many of PAHs have been demonstrated to be carcinogenic for experimental animals and they are also supposed to have a significant impact on the disease burden caused by various cancers in human population [START_REF] Phillips | Polycyclic aromatic hydrocarbons in the diet[END_REF]. PAHs highlighted to be genotoxic and carcinogenic by the Scientific Committee on Food (SCF) are the following: benz(a)anthracene, benzo(b)fluoranthene, benzo(j)fluoranthene, benzo(k)fluoranthene, benzo(g,h,i)perylene, benzo(a)pyrene, chrysene, cyclopenta(c,d)pyrene, dibenz(a,h)anthracene, dibenzo(a,e)pyrene, dibenzo(a,h)pyrene, dibenzo(a,i)pyrene, dibenzo(a,l)pyrene, indeno(1,2,3-cd)pyrene,

5-methylchrysene (European Commission 2005a). Joint Expert Committee on Food

Additives (JECFA) has added 16th analyte to the list -7H-benzo-(c)fluorene (JECFA 2005). European Commission has required further investigation of the levels of benzo(a)pyrene and other PAHs, in particular those highlighted to be carcinogenic by benzo(k)fluoranthene, dibenz(a,h)anthracene, dibenzo(a,e)pyrene, dibenzo(a,i)pyrene, dibenzo(a,l)pyrene dibenzo(a,l)pyrene, indeno(1,2,3-cd)pyrene and 5-methylchrysene, as reasonably anticipated to be human carcinogens (IARC 1973(IARC , 1983(IARC , 1987)). In mammalian cells PAHs undergo metabolic activation to diol epoxides that bind covalently to cellular macromolecules, including DNA, thereby causing errors in DNA replication and mutations that initiate the carcinogenic process [START_REF] Janoszka | Organic Compounds Formed in Thermally Treated High-protein Food. Part I: Polycyclic Aromatic Hydrocarbons[END_REF].

Diet is the primary source of human exposure to PAHs contributing to more than 90% of total exposure to PAHs (WHO 1998, SCF 2002). PAHs may be present as contaminants in various food items and beverages including drinking water [START_REF] Chen | Determination of polycyclic aromatic hydrocarbons in water by solidphase microextraction and liquid chromatography[END_REF][START_REF] King | Determination of polycyclic aromatic hydrocarbons in water by soli-phase microextraction-gas chromatography-mass spectrometry[END_REF]), vegetables, fruits, cereals, oils (Dennis et al.1991, Moret and Conte 2000, Moret and Conte 2002[START_REF] Camargo | Polycyclic aromatic hydrocarbons in Brazilian vegetables and fruits[END_REF], Tao et al. 2006, Voutsa and Samara 1998), fish (Šimko 1991, Lodovici et al. 1995, Moret and Conte 2000, Yurchenko and Mölder 2005[START_REF] Karl | Determination of polycyclic aromatic hydrocarbons in smoked fishery products from different smoking kilns[END_REF], Stołyhwo and Sikorski 2005) and meat [START_REF] Kazerouni | Analysis of 200 food items for benzo(a)pyrene and estimation of its intake in an epidemiologic study[END_REF], Elhassaneen 2004, Šimko 2002, Mottier 2000[START_REF] Janoszka | Organic Compounds Formed in Thermally Treated High-protein Food. Part I: Polycyclic Aromatic Hydrocarbons[END_REF]). The possible sources of PAHs in food are environmental contamination from the atmosphere, soil or water, contamination from packaging materials and thermal treatment which is used for the preparation and Food processing procedures, such as smoking, drying and cooking, are generally supposed to be the major sources of PAH contamination of food. Formation of PAHs in meat products is affected by several factors: the methods used for preparation of food (grilling, frying, roasting etc.), temperature and time of cooking, distance from the heat source and drainage of fat (SCF 2002[START_REF] Kazerouni | Analysis of 200 food items for benzo(a)pyrene and estimation of its intake in an epidemiologic study[END_REF]. Several mechanisms of formation of PAHs have been proposed, such as melted fat that undergoes pyrolysis when dripping onto the heat source and pyrolysis of the meat due to the high temperature [START_REF] Lijinsky | The formation and occurrence of polynuclear aromatic hydrocarbons associated with food[END_REF]. Because PAH formation occurs on or near the surface of meats, food cooked without being exposed to smoke does not show significant levels of PAH. The PAH concentrations in smoked food depend on several variables in the smoking process, including the type of smoke generator and wood, combustion temperature and degree of smoking (Moret et al. 1997). Lowering the temperature of smoke formation to 300-400 °C combined with the use of filters, the PAH content of smoke can be decreased by about 90% (Sikorski 2005).

Benzo(a)pyrene (BaP) is the most widely known and studied of the PAHs due to its importance as one of the most potent animal carcinogenic PAH, its relatively easy analysis and presence in wide variety of food items. BaP seems to be a good marker for other PAHs in food items, the correlation coefficient between the total PAH and the BaP concentration has been detected to be 0.87 and between the carcinogenic PAHs and BaP 0.98 [START_REF] Kazerouni | Analysis of 200 food items for benzo(a)pyrene and estimation of its intake in an epidemiologic study[END_REF]. Maximum acceptable concentration of 5 (Kazerouni et al. 2001, Moret andConte 2000), high performance liquid chromatography [START_REF] Chen | Evaluation of analysis of polycyclic aromatic hydrocarbons in meat products by liquid chromatography[END_REF][START_REF] Chiu | Comparison of GC/MS and HPLC for overcoming matrix interferences in the analysis of PAHs in smoked food[END_REF][START_REF] Camargo | Polycyclic aromatic hydrocarbons in Brazilian vegetables and fruits[END_REF][START_REF] De Boer | Developments in the use of chromatographic techniques in marine laboratories for the determination of halogenated contaminants and polycyclic aromatic hydrocarbons[END_REF], gas chromatography with mass-selective detector (Mottier et al. 2000, Yurchenko and Mölder 2005[START_REF] Jira | A GC/MS method for the determination of carcinogenic polycyclic aromatic hydrocarbons (PAH) in smoked meat products and liquid smokes[END_REF]) or flame ionization detector (Moret and Conte 2000).

Intake of PAHs has been estimated in some European countries: in Austria, Italy, The Netherlands and UK, furthermore the estimated BaP intake is available for Germany, Sweden and USA (SCF 2002). According to several studies the major dietary contributors are cereals, oils and vegetables although PAH levels in cereals are often low [START_REF] Dennis | Analysis of polycyclic aromatic hydrocarbons in the UK total diet[END_REF], de Vos et al.1990, Tao et al. 2006). Grilled or barbequed meat caused a significant proportion (21%) of the average total daily BaP intake in USA according to the data obtained by [START_REF] Kazerouni | Analysis of 200 food items for benzo(a)pyrene and estimation of its intake in an epidemiologic study[END_REF].

Various food items and products in Estonia have been analysed for BaP content starting from year 1999 and for PAH content from 2003 in the framework of the Estonian food safety monitoring programme, initiated in 1998 [START_REF] Reinik | Estonian National Monitoring programme of Food Additives[END_REF].

The aim of the present study was to determine the levels of PAHs in commercial meat products and grilled meat prepared by traditional Estonian habits. Furthermore, the aim here was to estimate the human exposure to PAHs and BaP through various meat products. As grilling, smoking and barbequing of meat are widely used in Estonia, the assessment of dietary risks connected with the intake of PAHs is essential.

Materials and methods

Meat samples

Altogether Various types of grilling equipment are used for home-grilling. In present study two commonly used grilling methods were used: traditional wood burning grill and small disposable charcoal grill unit. In the traditional wood-burning grill the distance between burning coals and meat is approximately 20 cm and the temperature at the surface of the meat is between 180-240 °C. In recent years disposable charcoal grills have gained popularity for their convenience and shorter preparation time. Compared to traditional grilling the heat source is much closer to the grilled product in these disposable grills. 14 samples of meat products were grilled using both methods and analyzed for the content of 12 PAHs. ionization mass spectrometry was performed with ammonia reagent gas. The method performance parameters are given in Table I.

Both methods are accredited by Estonian Accreditation Centre. This laboratory has successfully participated in international proficiency tests using both methods of analysis. A reference material, smoked fish paste containing 16 PAHs (prepared by Estonian Institute of Chemical Physics and Biolophysics), was analysed in each series of analyses, the results for HPLC and GC/MS analysis of benzo(a)pyrene are coincident in accepted range.

[Insert Table I about

Results and discussion

BaP in commercial meat products

During the years 2001-2005 the range and average concentrations of BaP in 322 samples from various industrial and commercially available meat products were determined (Table II). BaP contents in analyzed meat products were at range of <0.3

(below the determination limit) and 31.2 µg kg -1 . In 64% of the analyzed samples BaP concentrations remained below the limit of determination (<0,3 µg kg -1 ). The maximum acceptable concentration of 5µg kg -1 for BaP in smoked meat products (European Commission 2005b) was exceeded in 11 samples. High PAH concentration containing products were home-made ham, smoked meat, smoked chicken and smoked sausages with concentrations of 30.6, 31.2, 15.0 and 20.0 µg kg -1 , respectively.

[Insert Table II about here]

In one earlier study concentrations of BaP have been found in the range of 0.03-100 µg kg -1 in smoked meat products (Šimko 2002). The highest concentrations were detected in smoked sausages and smoked ham. In cooked meat products in Poland 0.11-3.93 µg kg -1 of BaP have been detected [START_REF] Janoszka | Organic Compounds Formed in Thermally Treated High-protein Food. Part I: Polycyclic Aromatic Hydrocarbons[END_REF]. III). Eleven of those, except benzo(g,h,i)perylene, are categorized by IARC as probable or possible carcinogens (IARC 1973(IARC , 1983(IARC , 1987)).

[Insert Table III about here] Benzo(a)anthracene was detected in 15 samples, BaP in 13 samples.

Benzo(a)anthracene and chrysene were found in highest concentrations. 5methylchrysene, dibenzo(a,h)anthracene, dibenzo(a,l)pyrene, dibenzo(a,e)pyrene concentrations remained below the determination limit in all samples. PAHs were not found in 4 samples. There are several factors influencing the final PAH content in the products in smoking and grilling process: type of wood and smoke generator, smoke temperature, duration of smoking or grilling, availability of oxygen and oven dimensions. As most of the cooked sausages undergo only a slight smoking process, BaP and PAH concentrations close to or under the determination limits were observed.

Our results are consistent with the study by [START_REF] Phillips | Polycyclic aromatic hydrocarbons in the diet[END_REF] where the concentration range of total PAHs in smoked meat was between 2.6 and 29.8 µg kg Home-prepared meat products, especially those which were prepared using a disposable grill, contained higher concentrations of BaP and PAHs compared to commercial products. The mean concentration of BaP in industrial and disposablegrill meat was 0.7 µg kg -1 and 1.0 µg kg -1 , respectively. The average levels of PAHs in home-grilled meat were higher compared to industrial smoked products, mean contents being correspondingly 12.5 µg kg -1 and 7.5µg kg -1 .

Elhassaneen ( 2004) determined 11 PAHs in charcoal-broiled beefburgers. The range of PAHs was between 0.31-14.95 µg kg -1 and most frequently pyrene and benzo(a)pyrene were detected. The range of BaP concentration was 0.99-4.8 µg kg -1 .

Contents of 16 PAHs in seven different barbequed meat sausages were determined in a Swiss study. The highest concentrations were found for phenanthrene and naphtalene, BaP contents were between "not detected" and 2.81 µg kg -1 (Mottier et al.

2000).

The highest concentrations of PAHs reported in meat have been detected in food cooked over open flames. For example, in barbecued meat, the total PAHs were found to be present at levels up to 164 µg kg -1 ; with BaP being present at levels as high as 30 µg kg -1 [START_REF] Panalaks | Determination and identification of polycyclic aromatic hydrocarbons in smoked and charcoal-broiled food products by high pressure liquid chromatography[END_REF]). The results for the total sum of PAHs are difficult to compare between the studies published earlier by other authors as there is variation in analyzed compounds and several non-carcinogenic PAHs have been included in earlier works.

Intake of BaP and PAHs by children from the consumption of meat products

The questionnaire concerning the daily consumption of meat products children's parents were asked to complete during one observation week. Data from 346 children at the age of 1 to 16 years were obtained [START_REF] Reinik | Nitrites, nitrates and N-nitrosoamines in Estonian cured meat products: Intake by Estonian children and adolescents[END_REF]). In the PAH and BaP intake estimation an individual approach was applied: using the mean concentrations of BaP (Table II) and PAHs detected in meat products (Table III andIV V.

[Insert Tables V-VI about here]

The mean BaP intake by children was found to be 14 ng per day, maximum reaching to 140 ng per day. Intake per kg of body weight (bw) is higher in younger age groups

(1-9 years), maximum intake is more than 10-fold compared to the average value of 0.46 ng per day per kg of bw. The average intake of PAHs from meat products was found to be 192 ng per day (Table VI), whereas maximum intake was estimated to be up to 1575 ng per day. BaP comprises 7.3% of total PAH intake. Both for BaP and PAH the highest intake is obtained from smoked meat products followed by cooked sausages and grilled products.

Dietary intake of BaP by schoolchildren and toddlers estimated in a UK survey COT (SCF 2002) was in the range of 1.4-3.8 ng kg -1 bw per day. Similarly to our work the youngest age groups were found to have the highest exposure for both BaP and PAH per kg of bw, about 2.4-fold higher intake than for adults (SCF 2002). to the large extent. In USA and in some European countries the estimated BaP intake from food is reported to be between 0.1-1.6 µg per day (Guillen and Sopelana 2003), according to surveys conducted in six EU countries the mean or national-averaged intake of BaP for an adult person was estimated to be between 0.05 and 0.29 µg per day (SCF 2002) and the total intake of 19 PAHs was estimated to be 8.8 µg per day in Finland (SCOOP report 2004). Large variation in estimated intake values is probably due to the food groups and items considered, differences in analytical methods and to statistical analysis of concentration values below the detection and determination limits. The estimated contribution of meat products to the overall intake of PAHs also differs between countries from very low for UK to 21 % in USA and 27 % in France resulting in the second contributing food group after bread and cereals (SCF 2002, SCOOP report 2004).

Intake of BaP and total

As smoking and grilling are prevalent meat preparation methods in Estonia, the impact of meat products is assumed to be significant in the overall PAH intake. The mean intake values obtained in this study were lower compared to the estimates by other countries, however the results showed that for high consumers intake may be significant and exceed average value up to 10 times.

Conclusions

The content of benzo(a)pyrene in 322 meat products was determined by an HPLC method. BaP contents in meat products were in the range of <0.3 and 31.2 µg kg -1 , with a mean concentration of 0.8 µg kg -1 . The maximum acceptable concentration of 5 µg kg -1 for BaP in smoked meat products was exceeded in 11 samples, mainly home-made ham, smoked meat and smoked chicken. Concentrations of 12 PAHs were determined in 22 commercial smoked and 14 home-grilled meat products. For smoked products the highest PAH concentrations were detected to be 19 µg kg -1 , whereas the average concentration was 7.5 µg kg -1 .

The intake of BaP and PAHs by Estonian children from meat products was estimated on the basis of the data obtained from the individual food consumption questionnaires. The mean BaP intake was found to be 14 ng per day, maximum intake being more than 10 times higher. The average intake of PAHs by children from meat products was found to be 192 ng per day, while maximum intake values were estimated to be up to 1575 ng per day.

The mean intake by the general population from the consumption of meat products was found to be 29 ng of BaP per day and 346 ng of total PAH per day. For consumption of 100 g of smoked or grilled meat products per day the mean daily intake was estimated to be 87 ng for BaP and 942 ng for PAHs. The PAH intake from the consumption of meat products per kg of body weight is highest among children in a age group of 1-9 years; the mean daily intake exceeding by 1.5 times, and the maximum by 10-fold the average intake of the whole population (5.3 ng kg -1 bw per day).

Considering the toxicity of PAHs and high consumption of grilled and smoked meat products in Estonia, the data reported on the levels of carcinogenic PAHs in food and information on daily intake by children and the whole population is clearly significant. Σ Benzo(b),(k),(j) fluoranthene <0.9-3.8 1.3 <0.9-2.4 0.9 <0.9-1.2 0.7

F

Benzo(g,h,i)perylene Dibenzo(a,e)pyrene <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 Dibenzo(a,l)pyrene <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 Indeno(1,2,3)pyrene 0.5-1.7 0.9 1.7-2.0 1.9 0.5 0.5 0.5 0.5 0.5-1.5 1.0 0.5-

  (PAHs) include several potent carcinogenic compounds consisting of two or more fused aromatic rings. PAHs are formed during

  SCF, in food(European Commission 2005a, Wenzl et al. 2006). The International Agency of Research on Cancer (IARC) has categorized 15 PAHs, including benzo(a)pyrene, benz(a)anthrazene, benzo(b)fluoranthene, benzo(j)fluoranthene,

  manufacturing of food(Guillen 1994). PAHs are lipophilic and have very low aqueous solubility so they accumulate in lipid tissue of plants and animals. The levels of PAHs found in unprocessed foods reflect the background contamination which originates from long distance airborne transportation of contaminated particles and natural emissions.

  Analyses of the PAHs in meat samples were carried out at Tartu Laboratory of Estonian Health Protection Inspectorate. The gas chromatographic method with massselective detector (GC/MS) was used to determine the following PAHs: benz(a)anthracene, sum of benzo(b)-, benzo(j)-and benzo(k)fluoranthene, performance liquid chromatography (HPLC) with fluorescence detection was used for samples in which only benzo(a)pyrene content was measured.For sample preparation the method described by[START_REF] Ojaveer | Levels of polycyclic aromatic hydrocarbons in eelpout, zoarces viviparous, in Estonian marine waters[END_REF] with some modifications was used. The sample was homogenized and hydrolyzed in concentrated hydrochloric acid overnight. Dichloromethane/hexane solution 25:75 was added. The solution was filtered through the layer of Na 2 SO 4 and NaHCO 3 1:1.The eluate was evaporated to dryness, ethyl acetate/cyclohexane 50:50 solution was added. Portion of the extract was injected into a gel chromatograph. The fraction containing PAHs was collected and evaporated to dryness. The residue was dissolved in methanol.For benzo(a)pyrene analysis an Hewlett Packard 1100 HPLC equipment with Agilent 1100 fluorescence detector was used. The limit of determination was 0,3 µg kg -1 and recovery was 80 %. A Hewlett Packard Model 6890 gas chromatograph equipped with mass selective detector and chromatographic column HP-5 (30m x 0.25 mm i.d. silica capillary column with film thickness of 0.25 µm) was used for the analysis of PAHs. The temperature was programmed from 80 to 300 °C. Positive-ion chemical

  here] Method for intake estimation To obtain data about the consumption of cured meat products by children in Estonia a questionnaire developed by the scientists of the Food Hygiene and Control Department of Estonian University of Life Sciences was used. Questionnaires were distributed to schools, kindergartens and family doctor centres and asked to fill in by the children's parents. The main aim was to estimate the daily consumption of meat products by children. Data on the number and age of children in the family, consumption of meat products per child, preference for domestic and imported products and attitude towards food additives were included in the questionnaire. The data from 346 children were received in 2000 -2003. For the statistical analysis of data the responses were grouped in five groups by age of the children. According to registered data the maximum and average amounts of consumed meat products in grams in various age groups were calculated.

  Food consumption data from Estonian Board of Statistics were used for the estimation of average intake of PAHs and BaP in the general population in Estonia. Furthermore, an estimation was made for PAH and BaP intake through theoretical consumption of 100 g of each meat product per day. The national food consumption data were collected in2001 -2004.

  PAHs in commercial smoked meat products 22 samples of meat products were analyzed for the concentrations of 12 PAHs in 2005 (Table

  Benzo(a)pyrene, benzo(a)anthracene, sum of benzo(b),(k),(j)fluorenes, chrysene and benzo(g,h,i)perylene were detected in all samples. The highest concentrations were determined for chrysene, sum of benzo(b),(k),(j)fluorene and benzo(a)anthracene, maximum values being 6.2, 5.1 and 3.0 µg kg -1 , respectively. The results in tableIVindicate a certain relation between the amount of benzo(a)pyrene and total amount of PAHs. According to our results BaP comprises in general 6.3 % of the total sum of the PAHs in home-grilled meat products.

  for consumption of cooked sausages, smoked sausages, ham and grilled meat products by each respondent child. The intake of BaP by children, for the period of 2001-2005 is represented in ng per day in Table

  Several methods for analysis have been used for the determination of PAHs in food.

	PAHs have been extracted from homogenized food samples using various techniques
	such as liquid-liquid extraction (Rojo Camargo and Toledo 2003) or supercritical
	fluid extraction (Järvenpää et al. 1996, Lage Yusty and Daviña 2005). Solid phase
	extraction (Nazarkina et al. 2001) and gel permeation chromatography (Yurchenko
	and Mölder 2005) have been used for sample cleanup. The determination of PAHs in
	F food has been carried out by various analytical methods, including thin layer o r chromatography
	P
	e
	e r
	R
	e
	v i e
	w
	O n l
	y
	µg kg -1 has been established for BaP in smoked meats and smoked meat products
	(European Commission 2005b).

  -1 , and highest concentration of carcinogenic PAHs was 16.0 µg kg -1 . Reports of previous works(Šimko, 2002) indicate that BaP concentrations are between 1% and 20% of the total carcinogenic PAHs. According to our results BaP comprises in average 6.1 % of the total sum of PAHs in commercial smoked meat products.PAHs in home-made grilled meat productsThe contents of PAHs in grilled products are shown in TableIV. Using of disposable grill resulted in higher (maximum 1.6 times) concentrations of PAHs compared to traditional grilling.[Insert TableIV about here]Total amounts of 12 PAHs was higher in grilled pork than in grilled chicken or sausages. The difference occurs probably due to variation in fat content. When fatty meat is grilled, more fat drops onto the coals and due to the high temperature, PAHs are formed. In consequence, smoke carries the PAHs onto the surface of meat products. Lower concentrations of PAHs in sausages can be explained by shorter grilling time.

	F o r
	P
	e
	e r
	R
	e
	v i e
	w
	O n l
	y

  intake for the general population. Data include the size of the population and average amounts of monthly consumed meat products in Estonia in2001-2004. The lowest concentrations of BaP were detected in cooked sausages and beef hamburgers, 0.15 µg kg -1 as a mean value. However, cooked sausages are the most frequently used meat product in general population with the average consumption of 35 g per day. The mean intake of BaP from consuming cooked sausages was estimated to be 5.3 ng per day in our study. The highest concentrations of BaP were determined in smoked and grilled meat products. From before-mentioned product groups smoked sausages give the highest intake of BaP; 11 ng per day. The average consumption of grilled, smoked and other cured meat products is 65 g per day in

	F o r F o r
	P P
	e e
	e r e r
	R R
	e e
	v i e w v i e According to mean concentrations of PAHs found in present study and taking into [Insert Table VII about here] w
	O n l y account average daily consumption and the theoretical consumption of 100 g of meat product, average daily PAH intake was estimated. Highest concentrations of PAHs O n l were detected in grilled meat, concentration of 16 µg kg -1 . Consuming 26 g of smoked y
	and grilled meat products per day as average in Estonia, causes a 234 ng daily PAH
	intake (3.6 ng kg -1 bw per day).

PAHs by Estonian population from the consumption of meat products Dietary intake of food contaminants depends on both the nutritional habits of the examined population group and the concentrations of contaminants in food. Food consumption data from the Board of Statistics were used in the calculation of average general Estonian population (Table

VII

) which generates BaP intake of 29 ng per day (0.45 ng kg -1 bw per day). As food consumption database indicates only average consumption, the real intake values for persons who eat high quantities of meat products frequently can be much higher.

As different methods have been used for the estimation of intake of food contaminants and extensive variation of diets, results obtained in earlier studies differ

  ElhassaneenYA. 2004. The effects of charcoal-broiled meat consumption on antioxidant defense system of erythrocytes and antioxidant vitamins in plasma.Voutsa D, Samara C. 1998. Dietary intake of trace elements and polycyclic aromatic hydrocarbons via vegetables grown in an industrial Greek area. The Science of the Table III PAH concentrations (µg kg -1 ) in 22 commercial smoked meat products in Estonia 2005.

	Lodovici M, Dolara P, Casalini, C, Clapellano S, Testolin G. 1995. Polycyclic health of polycyclic aromatic hydrocarbons in food. Brussels Table I GC/MS method performance characteristics Table IIBenzo(a)pyrene concentrations (µg kg -1 ) in 322 commercial meat products in Estonia
	2001-2005.
	aromatic hydrocarbon contamination in the Italian diet. Food Additives and Contaminants, 12:703-713. http://europa.eu.int/comm/food/fs/sc/scf/index_en.html Total Environment 218:203-216. PAHs Retention time (min) Detection limit (µg kg -1 ) Determination limit (µg kg -1 ) Ham Smoked sausage Smoked chicken Recovery Concentration Median Average n=6 n=13 n=3 PAH % Benzo(a)anthracene 12.25 0.2 0.7 101 Product group No. of samples range (µg kg -1 ) concentration (µg kg -1 ) concentration range mean range mean range mean (µg kg -1 ) Benzo(a)anthracene <0.7-3.2 1.6 <0.7-5.6 1.6 1.0-2.2 1.7
	Nutrition Research, 24: 435-446. Sikorski E. 2005. The effect of processing on the nutritional value and toxicity of Σ Benzo(b),(k),(j) fluoranthene 17.09 0.3 0.9 Smoked sausage 115 <0.3-20 <0.3 1.1 Ham 116 <0.3-30.6 <0.3 0.8	97
	Moret S, Piani B, Bortolomeazzi R, Conte LS. 1997. HPLC determination of foods. In: Dabrowski M, Sikorski ZE, editors. Toxins in food. USA: CRC press Wenzl T, Simon R, Kleiner J, Anklam E. 2006. Analytical methods for polycyclic Benzo(g,h,i)perylene 25.73 0.2 0.5 Smoked meat 20 <0.3-31.2 0.5 0.7	87
	Benzo(a)pyrene Smoked chicken	18.75 29	0.1 <0.3-15.0	0.3	0.3	1.3	90
	European Commission. 2005a. Commission Recommendation 2005/108/EC of 4 polyaromatic hyrdocarbons in olive oils. Zeitschrift für Lebensmittel-Untersuchung p.285. aromatic hydrocarbons (PAHs) in food and the environment needed for new Chrysene 12.40 0.2 0.7 Grilled chicken 6 <0.3-1.0 0.6 0.5	97
	o r F o r February 2005 on the further investigation into the levels of polycyclic aromatic hydrocarbons in certain foods. Official Journal of the European Union L34: 43-45. F o r F o r F o r und -Forshung A, 205:116-120. F o r F o r Šimko P. 2002. Determination of polycyclic aromatic hydrocarbons in smoked meat F o r legislation in the European Union. Trends in Analytical Chemistry 25:716-725. F o r Dibenzo(a,h)anthracene 24.83 0.2 0.7 Dibenzo(a,e)pyrene 32.57 0.2 0.5 Dibenzo(a,l)pyrene 30.74 0.2 0.5 Indeno(1,2,3)pyrene 24.57 0.3 1.0 5-methylchrysene 14.21 0.2 0.7 F o r Grilled sausage 10 <0.3-1.2 0.7 0.6 Grilled meat 6 0.7-1.8 1.0 1.1 Cooked sausage 16 <0.3-1.0 <0.3 0.15 Others 4 <0.3 <0.3 F o r	99 99 85 92 98
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	R R R R R determination in vegetable oil. Journal of Separation Science, 25:96-100. R R Chemistry, 40:293-300. R hydrocarbons in smoked fish by gas chromatography mass spectrometry with positive R R R
	e v i e w e v i e w Guillen MD. 1994. Polycyclic aromatic compounds: extraction and determination in food. Food Additives and Contaminants, 11:669-684. Guillén MD, Sopelana P. 2003. Polycyclic Aromatic Hydrocarbons in Diverse Foods. e v i e w e v i e w e v i e w Mottier P, Parisod V, Turesky R.J. 2000. Quantitative Determination of Polycyclic Aromatic Hydrocarbons in Barbecued Meat Sausages by Gas Chromatography Coupled to Mass Spectometry. Journal of Agricultural Food Chemistry, 48:1160-e v i e w e v i e w Stołyhwo A, Sikorski ZE. 2005. Polycyclic aromatic hydrocarbons in smoked fish -e v i e e e e ion chemical ionization. Journal of Food Composition and Analysis 18:857-869. v i e v i e v i e a critical review. Food Chemistry, 91:303-311. w w w w
	O n l y O n l y In: D'Mello JPF, editor. Food Safety. Contaminants and Toxins. Edinburgh, UK: CABI. p. 175. O n l y O n l y O n l y 1166. Nazarkina SG, Bulanova AV, Larionov OG. 2001. Solid-phase extraction of O n l y O n l y Tao S, Jiao XC, Chen SH, Liu WX., Coveney Jr RM., Zhu LZ, Luo YM.. 2006. Accumulation and distribution of polycyclic aromatic hydrocarbons in rice (Oryza O n l O n l O n l O n l sativa). Environmental Pollution, 140:406-415. y y y y
	IARC. 1973. Some polycyclic aromatic hydrocarbons and heterocyclic compounds. polycyclic aromatic hydrocarbons using polymer sorbents. Zhurnal analiticheskoi
	In: IARC monographs on the evaluation of carcinogenic risk of chemicals to humans. Khimii, 56:394-397. de Vos RH, van Dokkum W, Schouten A, de Jomg-Berkhout P. 1990. Polycyclic
	Vol.3. Lyon, France: IARC, 271 pp. aromatic hydrocarbons in Dutch total diet samples 1984-1986. Food Chemistry and
	Toxicology, 28:263-268.

  Table IV PAH concentrations (µg kg -1 ) in 14 Estonian home-grilled meat products.

			Grilled pork		Grilled sausage		Grilled chicken
		Wood burning	Disposable	Wood burning	Disposable	Wood burning	Disposable
	PAHs	grill		charcoal grill	grill		charcoal grill	grill		charcoal grill
		n=3		n=3		n=2		n=2		n=2		n=2
		range mean range mean range mean range mean range mean range mean
	Benzo(a)anthracene 1.6-2.5	1.9	3.0	3.0	1.4-2.1	1.8	1.0-1.7	1.4	0.7-1.2	1.0	1.5-1.7	1.6
	Σ Benzo(b),(k),(j)-fluoranthene	1.7-3.8	3.0	4.3-5.1	4.6	2.6-2.7	2.6	2.7-3.5	3.1	2.2-2.6	2.4	3.0-3.6	3.3
	Benzo(g,h,i)perylene 0.5-1.5	1.1	1.8-2.3	2.1	1.3-1.4	1.4	1.4-1.5	1.4	1.2-1.4	1.3	1.5-1.8	1.7
	Benzo(a)pyrene	0.7-1.0	0.8	1.0-1.8	1.4	0.6-0.8	0.7	0.5	0.5	0.3-0.5	0.4	0.8-1.0	0.9
	Chrysene	2.4-6.2	3.7	4.7-6.2	5.6	1.5-2.3	1.9	1.5-3.5	2.5	0.9-1.6	1.3	2.2-3.1	2.7
	Dibenzo(a,h)-anthracene	<0.7		<0.7		<0.7		<0.7		<0.7		<0.7

Table V .

 V Estimated daily intake of benzo(a)pyrene (ng per day and ng kg -1 body weight per day) from the consumption of meat products by Estonian children.

	Page 31 of 33	Food Additives and Contaminants
	F o r F o r F o r	1.8	1.2
	5-methylchrysene Σ 12all PAHs Concentrations below determination limit (LOQ) were counted as LOQ/2 to calculate mean levels <0.7 0.4-0.7 0.6 <0.7 0.4 0.4 <0.7 0.4-0.7 13 20 10 11 8.6 P P P e e e of PAHs. e r e r e r	0.6 13
		R R R
		e e e
		v i e v i e v i e
		w w w
		O n l O n l O n l
		y y y

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Table VII .

 VII Mean daily intake of benzo(a)pyrene and PAHs from the consumption of meat products in general population in Estonia calculated on the basis of the food consumption survey from the National Board of Statistics and the theoretical consumption of 100g of each product per day.

								Page 34 of 33
	Product group	Average consumption (g per day)	Mean BaP content (µg kg -1 )	Mean Σ12 PAHs content (µg kg -1 )	Mean BaP intake (ng per day) At average Through daily daily consumption consumption of 100g	Mean total PAH intake (ng per day) At average Through daily daily consumption consumption of 100g
	Cooked sausage	35	0.15	3.2	5.3	15	112	320
	Smoked sausage	10	1.1	7.3	11	110	75	730
	Ham	2.0	0.8	8.0	1.6	80	16	800
	Smoked meat	6.7	0.7	8.0	4.7	70	54	800
	Smoked chicken	1.0	1.3	5.7	1.3	130	5.7	570
	Grilled chicken	1.7	0.5	11	0.85	50	18	1100
	Grilled sausage	2.0	0.6	10	1.2	60	21	1000
	Grilled meat	2.7	1.1	16	3.0	110	44	1600
	Others	2.9	0.15	-	0.45	15	-	-
	Total	64			29		346

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Food Additives and Contaminants

http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.ukFood Additives and Contaminants


Marta López Alonso

Marta Miranda

Cristina Castillo

Joaquín Hernández

Marco García

José Luis Benedito

Toxic and essential metals in liver, kidney and muscle of pigs at

Keywords: toxic and trace metals, liver, kidney, muscle, pig

Introduction

One of the most important aspects of environmental pollution for humans is the intake of toxic elements in the diet. Although contamination of food by toxic metals cannot be entirely avoided given the prevalence of these pollutants in the environment, there is a clear need for such contamination to be minimized (SCAN, 2003).

Meat and meat products form an important part of the human diet. Although toxic metal contents in muscle are generally low, offal products such as liver and kidney often accumulate higher metal concentrations than most other foods. Maximum admissible levels have been established by the European Union for meat and meat products (European Commission, 2001), and monitoring programmes have been carried out in many countries with the aim of avoiding the distribution of foodstuffs that could pose a risk to human health if consumed (Vos et al., 1986;[START_REF] Jorhem | Lead, cadmium, arsenic and mercury in meat, liver and kidney of Swedish pigs and cattle in 1984-88[END_REF][START_REF] Salisbury | Multielement concentrations in liver and kidney tissues from five species of Canadian slaughter animals[END_REF][START_REF] Tahvonen | Lead and cadmium contents in pork, beef and chicken and in pig and cow liver in Finland during 1991[END_REF]Doganov, 1996). These monitoring programmes have been accompanied by legislation and other measures to reduce metal content in animal feed, with the aim of reducing metal residues in meat, as well as toxic effects on livestock (EFSA, 2004a,b): examples include the great reduction in environmental lead levels as a consequence of lead reduction in petrol, or the introduction of raw materials with low cadmium content in fertilizer manufacturing [START_REF] Jorhem | Lead, cadmium, arsenic and mercury in meat, liver and kidney of Swedish pigs and cattle in 1984-88[END_REF][START_REF] Niemi | The lead, cadmium and mercury concentrations in muscle, liver and kidney from Finnish pigs and cattle during 1987-1988[END_REF][START_REF] Tahvonen | Lead and cadmium contents in pork, beef and chicken and in pig and cow liver in Finland during 1991[END_REF].

In extensive production systems, most of animal feeds are locally produced, and animal metal exposure is directly related to environmental contamination in the area; in fact, livestock have proved to be good biomonitor species for metal contamination (Petersson-Grawe et al., 1997;[START_REF] López-Alonso | Cattle as biomonitors of soil arsenic, copper and zinc concentrations in Galicia (NW Spain)[END_REF]. By contrast, in intensive production systems (mainly used for non-ruminant livestock, including pigs, poultry, and fish), animals are typically fed compounded feed, consisting of a mixture of individual non-locally produced feed components (generally imported from various countries), to which additives and/or mineral supplements with considerable amounts of toxic metals are added (EFSA, 2004a).

In a previous study in Galicia, a rural region in NW Spain, we determined toxic and essential metal levels in cattle, the main livestock species in this region, predominantly fed locally grown fodder (Lopez-Alonso et al., 2000). This study found that metal levels in cattle muscle, liver and kidney were generally low, similar to those recently reported in other regions, and only in rare cases exceeding maximum acceptable levels as specified by the European Union. However, information on metal concentrations in other major types of meat from intensive production systems in NW Spain, such as for example pork, is not available. The aims of the present study were (i) to evaluate metal concentrations in swine meat and offal from intensive farming in Galicia, (ii) to compare these with reported metal levels in pigs in other countries and in cattle in this region, and (iii) to relate the observed concentrations to maximum acceptable concentrations.

Material and methods

Sampling

Samples (n=63 pigs) of liver, kidney and muscle were taken from randomly selected healthy animals from the slaughter lines of major abattoirs in NW Spain during 2004. Samples of at least 200 g of liver (right lobe) and diaphragm, and a whole kidney, were taken from each animal. All samples were packed individually in plastic bags and immediately transported to the laboratory and stored at -18ºC until analysis.

Chemical analysis

For liver and muscle analyses, approximately 2 g sub-samples were excised from semithawed tissues and digested in 5 ml of concentrated nitric acid (Suprapur grade, Merk) and 2 ml of 30% w/v hydrogen peroxide in a microwave digestion system (Milestone, Ethos Plus). Because of the different metal contents in the cortex and medulla, the kidney samples were homogenized before digestion to ensure that the subsample analysed was representative of the whole organ. Digested samples were transferred to polypropylene sample tubes and diluted to 25 ml with Ultrapure water (18 MΩ.cm specific resistivity, Millipore Milli-Q system). Metals at high concentrations in the digest (Cu, Zn, Fe, Mn, Se and Mo) were determined by inductively coupled plasma atomic emission spectrometry (ICP-OES, Perkin Elmer Optima 4300 DV) while metals at low concentrations (Cd, Pb, As, Hg, Co, Cr and Ni) were determined by inductively coupled plasma mass spectrometry (ICP-MS, VGElemental PlasmaQuad II-SOption).

Quality control

An analytical quality control programme was employed. Blank absorbance values were monitored throughout the study and were subtracted from the readings before the results I). The limits of quantification, expressed as concentration in tissue, were calculated on the basis of mean sample weights and volumes analysed. Analytical recoveries were determined from a certified reference material (Pig Kidney CRM 186; BCR Reference Materials) analysed together with the samples. The results are given in Table I and the metal concentrations analysed were in close agreement with the reference values. No information is given regarding Co and Mo in the CRM, and analytical recoveries were determined using spiked samples at a level giving absorbance values that were generally 2-10 times higher than the normal levels in the various tissues: recoveries were 91 and 97% respectively. The precisions of the analytical method and the overall procedure were evaluated as the relative standard deviations of 12 absorbance readings from the same digest and single absorbance readings from 12 digests of the same sample, respectively, these values being 0.63-7.91% for the analytical method and 3.02-11.3% for the overall procedure.

F

[Insert Table I about here] For statistical purposes, non-detectable concentrations were assigned a value of half of the detection limit.

Results and discussion

Descriptive statistics for toxic and essential metal levels in liver, kidney and muscle samples are presented in Tables II andIII Data on toxic and essential metal levels reported for pigs from other countries are presented in Tables IV-XV. Comparisons of data between studies must be done with caution. For example, average metal concentrations in tissues, especially in liver and kidney, depend in part on the age, sex and breed of the animals, which are usually not reported. In addition, for metals at very low concentrations with many samples very close to or below the quantification limit (especially toxic metals in muscle), the reported mean values are highly dependent on the limits of detection of the technique (which often vary between studies by more than one order of magnitude), the value assigned to non-detected concentrations (zero, half the limit of detection, or the limit of detection) and their inclusion or not in the calculation of mean values, and even the way in which the data are presented (arithmetic or geometric means, medians or ranges).

Cadmium

Cadmium concentrations in the samples analysed in this study ranged from 0.002 to 0.669 mg/kg fresh weight (Table II). Mean cadmium concentrations were significantly higher in the kidney (0.308±0.103 mg/kg) than in the liver (0.073±0.019 mg/kg), and mean concentrations in both were higher than in muscle (0.009±0.007 mg/kg).

[Insert Table IV about here]

Cadmium is possibly the most important toxic element in animal products, because of its accumulation in offal, and for this reason fairly extensive data is available in the literature (Table IV). In general, cadmium residues in pig tissues in our study are very 7 similar to values reported for pigs slaughtered at similar ages (5-8 months) in other countries. The Scandinavian countries are the exception, with cadmium concentrations about half those reported elsewhere, possibly reflecting the great effort made by these countries in recent decades to reduce cadmium levels in feedstuffs, especially phosphate mineral supplements and fertilizers (Vos et al., 1986;[START_REF] Niemi | The lead, cadmium and mercury concentrations in muscle, liver and kidney from Finnish pigs and cattle during 1987-1988[END_REF][START_REF] Tahvonen | Lead and cadmium contents in pork, beef and chicken and in pig and cow liver in Finland during 1991[END_REF].

Cadmium concentrations in our pig samples were higher than those seen in young cattle (slaughtered at 6-10 months) in the same region (liver 0.032, kidney 0.070, muscle 0.001 mg/kg fresh weight; [START_REF] López-Alonso | Toxic and trace elements in liver, kidney and meat from cattle slaughtered in Galicia (NW Spain)[END_REF]. In contrats, results of previous comparative studies of both livestock species in other countries have found higher cadmium concentrations in cattle than in pigs [START_REF] Jorhem | Lead, cadmium, arsenic and mercury in meat, liver and kidney of Swedish pigs and cattle in 1984-88[END_REF][START_REF] Niemi | The lead, cadmium and mercury concentrations in muscle, liver and kidney from Finnish pigs and cattle during 1987-1988[END_REF][START_REF] Tahvonen | Lead and cadmium contents in pork, beef and chicken and in pig and cow liver in Finland during 1991[END_REF], which has generally been attributed to differences in age at slaughter: around 6 months for pigs and generally more than two years for cattle.

We do not have sufficiently precise information about cadmium concentrations in feed in cattle and pigs in NW Spain to evaluate whether the observed differences between species are linked to differences in cadmium content in the diet or to physiological differences in metal accumulation. Cadmium contamination levels seem to be very low in Galicia [START_REF] Paz-Gonzalez | Total and extractable nickel and cadmium contents in natural soils[END_REF] (EFSA, 2004a) are similar for ruminants (mean 11, range 0.03-0.85 mg/kg dry matter) and pigs (means for different ages 0.07-0.16, range 0.01-0.50 mg/kg dry matter). It is possible that the higher cadmium levels in pigs could be related in part to the widespread practice in intensive production systems of giving copper and zinc supplements [START_REF] Rambeck | The effect of high dietary copper supplements on cadmium residues in pigs[END_REF]. Experimental studies have demonstrated that in pigs receiving a conventional intensive diet, cadmium concentrations in the liver and kidney were twice as high as in animals that received high copper supplements [START_REF] Rothe | Dietary factors influencing cadmium retention[END_REF] compared to the control animals. In connection with this, the European Food Safety Authority (EFSA, 2004a) has pointed out that high copper supplementation of feeds for pigs increases the risk of undesirable cadmium accumulation in the liver and kidney.

Lead

Lead concentrations in the tissues of Galician pigs were very low and varied between non-detected and 0.046 mg/kg fresh weight (Table II). The kidney was the tissue with the highest lead concentrations (0.008±0.006 mg/kg), followed by the liver (0.004±0.002 mg/kg), whereas in muscle lead concentrations were undetectable in most samples (74%).

[Insert Table V about here]

Lead concentrations have been also widely monitored in meat and meat products.

Unlike cadmium, tissue levels of which are very similar between studies, lead concentrations vary by more than 100-fold (Table V). Lead concentrations in our Galician pig tissues were at the lower end of the ranges reported in the literature, similar to those recently reported from Finland and Sweden, countries where there were early measures for reducing the use of leaded petrol, the main source of lead pollution to the atmosphere [START_REF] Jorhem | Lead, cadmium, arsenic and mercury in meat, liver and kidney of Swedish pigs and cattle in 1984-88[END_REF][START_REF] Tahvonen | Lead and cadmium contents in pork, beef and chicken and in pig and cow liver in Finland during 1991[END_REF]. These measures have possibly contributed to significantly reduced lead concentrations in animal products in recent years in many countries (see Table V), as well as in other foods [START_REF] Bellés | Reduction of lead concentrations in vegetables grown in Tarragona province, Spain, as a consequence of reduction of lead in gasoline[END_REF]. But differences in lead concentrations between studies may also be related in part to the analytical techniques: detection of lead in animal products is problematic (especially in muscle) and concentrations may be overestimated when they are close to the detection limit (Falandysz, 1993b). In fact, in the opinion of [START_REF] Jorhem | Levels of certain trace elements in beef and pork imported to Sweden[END_REF] a major reason for the apparent widespread decrease in lead levels in muscle in recent years may be improvement in analytical methods.

Lead residues in Galician pigs were lower than those previously found in calves from this region (liver 0.053, kidney 0.052, muscle 0.009 mg/kg fresh weight; [START_REF] López-Alonso | Toxic and trace elements in liver, kidney and meat from cattle slaughtered in Galicia (NW Spain)[END_REF]. Similar results have been obtained in other pork-beef compatative studies in Finland [START_REF] Tahvonen | Lead and cadmium contents in pork, beef and chicken and in pig and cow liver in Finland during 1991[END_REF], Sweden [START_REF] Jorhem | Lead, cadmium, arsenic and mercury in meat, liver and kidney of Swedish pigs and cattle in 1984-88[END_REF] and Slovenia (Doganov, 1996). Since lead does not accumulate with age in the tissues analysed in this study, these interspecies differences in lead accumulation could be associated with differences in dietary lead content (lead concentrations in forage crops are generally higher than in concentrate feed materials; EFSA, 2004b), as well as interspecies differences in lead accumulation and tolerance: pigs seem to be more tolerant of lead poisoning than most other species, including ruminants (EFSA, 2004b). Arsenic concentrations in the tissues of pigs in this study varied between non-detected and 0.026 mg/kg fresh weight. Appreciable arsenic concentrations were found only in the liver (0.013±0.003 mg/kg) and kidney (0.011±0.008 mg/kg), whereas in muscle arsenic concentrations were undetectable in most samples (98%).

[Insert Table VI about here]

The arsenic concentrations in our pigs are in general in good agreement with those reported in other countries (Table VI); though in Canada [START_REF] Salisbury | Multielement concentrations in liver and kidney tissues from five species of Canadian slaughter animals[END_REF] and the USA [START_REF] Doyle | Toxic and essential trace-elements in meat -Review[END_REF] arsenic residues were about one order of magnitude higher than in other countries, which has been associated with the use of organoarsenicals such as arsanilic and phenilarsonic acids as growth promotants in pigs [START_REF] Salisbury | Multielement concentrations in liver and kidney tissues from five species of Canadian slaughter animals[END_REF]. However, nowadays these arsenic compounds are not allowed and arsenic concentrations in pig feeds depend mainly on the content of products derived from fish and other marine organisms [START_REF] Jorhem | Lead, cadmium, arsenic and mercury in meat, liver and kidney of Swedish pigs and cattle in 1984-88[END_REF]EFSA, 2005). Arsenic residues in our Galician pigs were lower than those previously found in calves in this region (liver 0.043, kidney 0.055, muscle 0.004 mg/kg fresh weight; [START_REF] López-Alonso | Toxic and trace elements in liver, kidney and meat from cattle slaughtered in Galicia (NW Spain)[END_REF]. In general arsenic concentrations reported in cattle products have been very low (see [START_REF] López-Alonso | Toxic and trace elements in liver, kidney and meat from cattle slaughtered in Galicia (NW Spain)[END_REF] for review) and similar to or even lower than those reported for pigs from the same countries [START_REF] Jorhem | Lead, cadmium, arsenic and mercury in meat, liver and kidney of Swedish pigs and cattle in 1984-88[END_REF][START_REF] Salisbury | Multielement concentrations in liver and kidney tissues from five species of Canadian slaughter animals[END_REF][START_REF] Kluge-Berge | Lead, cadmium, mercury and arsenic in Norwegian cattle and pigs[END_REF]; this is possibly because arsenic concentration in forages is low compared to commercial compound feeds (EFSA, 2005). The relatively 11 high mean arsenic concentrations found in the liver and kidney of Galician cattle were due to a small proportion of animals having relatively high arsenic burdens (maximum of 0.474 mg/kg fresh weight) which could be associated with relatively elevated levels of arsenic in some soils in Galicia; in fact, a positive association between arsenic content in the soil and arsenic residues in the liver and kidney of cattle has been demonstrated [START_REF] López-Alonso | Cattle as biomonitors of soil arsenic, copper and zinc concentrations in Galicia (NW Spain)[END_REF].

Mercury

Mercury levels were very low in pig meat and offal in this study: undetectable concentrations were found in 86, 68 and 97% of samples for liver, kidney and muscle respectively, while the remaining samples were very close to the detection limit (maximum value: 0.008 mg/kg fresh weight) (Table II).

[Insert Table VII about here]

Mercury levels in pigs in our study were in good agreement with data reported in the literature (Table VII), in which a tendency for declining mercury content in meat products has been observed in recent decades, largely reflecting prohibition of the use of organomercurials for dressing seed, and the reduced use of fish products in pig feed [START_REF] Jorhem | Lead, cadmium, arsenic and mercury in meat, liver and kidney of Swedish pigs and cattle in 1984-88[END_REF]Falandysz, 1993a).

Mercury levels in the liver (0.0008 mg/kg fresh weight) and muscle (0.0004 mg/kg) of Galician calves were even lower than in the pigs analysed in this study, whereas in the kidney of calves mercury concentrations were higher (0.012 mg/kg) (López-Alonso et , 2003a). In a previous pork-beef comparative study, mercury concentrations were in general higher in pork samples [START_REF] Jorhem | Lead, cadmium, arsenic and mercury in meat, liver and kidney of Swedish pigs and cattle in 1984-88[END_REF], which was attributed to the higher mercury content of compound feeds for pigs. In Galicia, however, the mercury exposure of cattle has been shown to be associated with atmospheric mercury emissions from coal-fired power plants, which explains most of the inter-individual variability in kidney mercury levels in animals grazing in the vicinity of these plants (López-Alonso et al., 2003b).

Essential metals

Concentrations of the main essential metals (copper, zinc, iron, selenium and manganese) in the liver and kidney of our Galician pigs (Table III) were within the adequate-safe ranges for this animal species as proposed by [START_REF] Puls | Mineral levels in Animal Health[END_REF], and in general are in good agreement with those reported in the literature (Tables VIII-XV), which indicates that levels of these elements in current pig diets are appropiate and/or that pigs have effective homeostatic mechanisms that maintain tissue metal concentrations within acceptable ranges. Cobalt concentrations in the liver and kidney of pigs in our study were below those considered adequate for this animal species (liver 1.0-2.0, kidney 0.40 mg/kg dry weight; [START_REF] Puls | Mineral levels in Animal Health[END_REF], but in good agreement with those reported in previous studies (Table XIII). Other essential metals that have been less extensively studied in animal products are nickel and chromium. Although their essentiality has been demonstrated in experimental studies, adequate levels in pigs and other livestock have not been well established; in addition, nickel and chromium concentrations in pig tissues have varied widely from study to study (Tables XIV-XV), possibly reflecting variation in the limits of detection of the method. It is therefore not possible to draw [Insert Tables VIII to XV about here] It is worth noting that mean concentrations of most of the essential metals in muscle of pigs in this study are higher than reported in previous monitoring studies. This is especially true for mean copper, manganese and selenium concentrations, which were 5to 10-fold higher than reported from other countries. These differences may be due to differences between studies in the type of muscle analysed: diaphragm contains significantly more copper than pectoral muscle in cattle [START_REF] López-Alonso | Toxic and trace elements in liver, kidney and meat from cattle slaughtered in Galicia (NW Spain)[END_REF], and it is possible that the concentrations of other metals can also be different.

Unfortunately, information about the muscle sampled is rarely reported.

Comparison of essential metal concentrations between Galician cattle and pigs is not meaningful since in animals receiving adequate diets essential metal concentrations in tissues depend basically on homeostatic mechanisms, with the result that tissue concentrations of these elements are characteristic for each animal species. However, from a nutritional point of view, it is worth noting that the main difference between the two species in trace metal content is in copper concentrations in the liver. Cattle, and ruminants in general, show a marked capacity to accumulate copper in the liver when this element is supplied at levels above physiological requirements, and in fact a [START_REF] López-Alonso | Toxic and trace elements in liver, kidney and meat from cattle slaughtered in Galicia (NW Spain)[END_REF]. In contrast, pigs do not accumulate copper in the liver and mean concentrations are less than 25% of those seen in cattle, even though pigs in intensive systems receive high copper supplementation as grown promoters. As in other pork-beef comparative studies [START_REF] Jorhem | The levels of zinc, copper, manganese, selenium, chromium, nickel cobalt and aluminium in the meat, liver and kidney of Swedish pigs and cattle[END_REF], liver from NW Spain cattle [START_REF] López-Alonso | Interactions between toxic (As, Cd, Hg And Pb) and nutritional essential (Ca, Co, Cr, Cu, Fe, Mn, Mo, Ni, Se, Zn) elements in the tissues of cattle from NW Spain[END_REF]) contained approximately 4 times as much cobalt as pig's liver.

Maximum admissible levels in animal products

The European Commission has established maximum admissible levels for cadmium in liver, kidney and muscle of 0.5, 1 and 0.050 mg/kg fresh weight respectively (European Commission, 2001); none of the pigs sampled in this study exceeded these levels in any of the tissues analysed. Although cadmium accumulation rates are high in pigs compared to other livestock, especially when receiving copper supplementation [START_REF] Rambeck | The effect of high dietary copper supplements on cadmium residues in pigs[END_REF], the fact that these animals are slaughtered very young generally means that cadmium levels in offal do not exceed the maximum limits.

For lead the maximum admissible levels are 0.5, 0.5 and 0.1 mg/kg fresh weigh for liver, kidney and muscle respectively (European Commission, 2001). As with cadmium, none of the sampled animals exceeded these limits: maximum values in our pigs were about one order of magnitude lower. For the other toxic metals, arsenic and mercury, the European Commission has not established statutory limits for meat products. Mercury levels in our samples very low (about 1000-fold lower than allowed in fish; European Commission, 2001).

Conclusions

The concentrations of the toxic metals cadmium, lead, arsenic and mercury in liver, kidney and muscle of pigs raised in NW Spain are low, and maximum admissible concentrations established by the European Union were not exceeded in any sample. In general, toxic metal concentrations in our Galician pig samples were similar to those reported in other studies from other countries in recent years years, which in general have shown a declining trend. In addition, the lower variability between studies in toxic metal levels in pigs compared to cattle possibly indicates that pig diets are rather standard in their toxic metal contents, and fairly independent of local pollution levels. In the case of the essential metals analysed, their concentrations in pigs in our study are within the adequate-safe ranges for this animal species, and in general are in good agreement with those reported previously in the literature.

  pigs at slaughter is normally around 6 months, when they weigh approximately 120 kg.

  The limit of detection in the acid digest was set at three times the standard deviation of the reagent blanks (Table

  respectively.

[

  Insert TablesII and III about here]

  the levels of these metals in Galician pigs versus pigs from other regions. Finally, we are not aware of any previous studies of molybdenum concentrations in pigs, and the adequate-safe range has not yet been established for this animal species.

  of calves in Galicia have hepatic copper concentrations above normal levels

  Teeuwen JJMH, Van Delft W. 1986. Arsenic, cadmium, lead and mercury in meat, livers and kidneys of swine slaughtered in the Netherlands during the period 1980-1985. Zeitschrift für Lebensmittel Untersuchung und Forschung,

Table I .

 I Detection limits (µg/l) and results of analysis of the certified reference material (Pig Kidney CRM 186) expressed as mg/kg.

	Element Detection limit Certified Reference Material
				Certified levels *	Analysed levels
				(mean±95%CI)	(mean±95%CI)
	As	0.5		0.063±0.009	< ld
	Cd Co Cr Cu Fe	0.04 0.02 1.4 2.5 3.0 F o r	2.71±0.15 ---(58-142) 31.9±0.4 299±10	2.47±0.10 0.287±0.032 198±43 29.6±0.3 284±10
	Hg Mn	0.15 0.9	P	1.97±0.04 8.5±0.3	1.76±0.05 7.94±0.29
	Mo Ni Pb Se	3.8 0.62 0.26 13	---(0.420) e 0.306±0.011 10.3±0.5 e r	3.54±0.10 0.478±0.051 0.329±0.053 11.0±0.5
	Zn	2.8		128±3 R	124±3
				e
				v i e
				w
				O n l
					y
					24

Table II .

 II Concentrations of the toxic metals cadmium (Cd), lead (Pb), arsenic (As) and mercury (Hg) in liver, kidney and muscle (mg/kg fresh weight) in Galician pigs.

		Tissue	N (<ld)	Mean±DS	Median	90 th percentile	Range
	Cd	liver	63	0.073±0.019	0.068	0.100	0.043-0.136
		kidney	62	0.308±0.103	0.292	0.446	0.172-0.669
		muscle	62	0.009±0.007	0.007	0.015	0.002-0.038
	Pb As	liver kidney muscle liver kidney F o 63 (16) 62 (1) 62 (46) 63 62 (26) r	0.004±0.002 0.008±0.006 0.003±0.003 0.013±0.003 0.011±0.008	0.004 0.007 0.002 0.013 0.013	0.007 0.011 0.006 0.017 0.022	ND-0.012 ND-0.046 ND-0.018 0.009-0.023 ND-0.026
	Hg	muscle liver kidney muscle	62 (61) 63 (54) 62 (42) P 62 (60) e 0.003±0.001 0.001±0.001 0.002±0.002 0.001±0.000	0.003 0.001 0.001 0.001	0.003 0.004 0.006 0.001	ND-0.013 ND-0.005 ND-0.008 ND-0.004
	<ld: below the detection limit; ND: not detected. e r
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Table III .

 III Concentrations of the essential metals copper (Cu), zinc (Zn), iron (Fe),

	selenium (Se), manganese (Mn), cobalt (Co), chromium (Cr), nickel (Ni) and
	molybdenum (Mo) in liver, kidney and muscle (mg/kg fresh weight) in Galician pigs.
		Tissue	N(<ld)	Mean±SD	Median	90 th percentile	Range
	Cu	liver	63	14.9±12.6	11.3		25.3	5.7-87.2
		kidney	62	5.63±1.44	5.32		7.40	3.42-9.78
	Zn	muscle liver kidney muscle F o 62 63 62 62 r	6.85±1.48 81.3±19.7 28.9±4.5 42.5±6.9	6.50 78.5 28.0 41.4		9.20 107 35.6 53.1	4.60-10.24 47.8-137 20.5-41.9 32.0-60.7
	Fe Se Mn	liver kidney muscle liver kidney muscle liver	63 62 62 P 63 e 195±71 51.6±13.6 26.5±5.4 1.17±0.13 62 2.51±0.33 62 0.656±0.134 63 3.32±0.44 e r	190 51.7 26.3 1.16 2.52 0.653 3.33		278 68.0 33.7 1.33 2.93 0.843 3.81	61-430 25.2-95.3 18.6-38.9 0.890-1.44 1.82-3.16 0.370-0.979 2.30-4.38
		kidney muscle	62 62	1.56±0.20 1.01±0.23 R	1.55 0.96		1.83 1.30	1.16-2.07 0.65-1.59
	Co Cr Ni	liver kidney muscle liver kidney muscle liver	63 62 62 63 62 62 63 (28)	0.023±0.004 0.027±0.010 e 0.003±0.001 0.120±0.028 0.077±0.025 v 0.023 0.024 0.003 0.115 0.071 i e 0.131±0.054 0.116 0.009±0.007 0.008 w	0.042 0.003 0.041 0.139 0.091 0.165 0.017	0.014-0.032 0.012-0.064 0.002-0.013 0.078-0.284 0.052-0.215 0.070-0.441 ND-0.031
	kidney muscle liver kidney muscle <ld: below the detection limit; ND: not detected. 62 (14) 0.027±0.025 62 (1) 0.026±0.012 Mo 63 1.62±0.23 62 0.683±0.079 62 0.140±0.022	0.024 0.022 1.65 0.680 0.141	0.044 0.041 1.81 0.792 0.164 O n l y 0.519-0.837 ND-0.161 ND-0.079 0.810-1.99 0.097-0.203

Table IV .

 IV Published data on cadmium concentrations in pig liver, kidney and muscle.Unless otherwise specified, values shown are average concentrations (mg/kg fresh weight). The numbers of samples analysed are in parentheses. not calculated, because more than 70% of the values were below the detection limit; b calculated per wet weight based on dry matter contents of 26% for meat, 29.5% for liver and 20% for kidney; c median value; d adults; e age 8 months; f age 3-24 months; g age more than 2 years; h age around 6 months; i age 5-7 months; j market hogs.

	Liver	Kidney	Muscle	Country	Reference
	_	0.6 (315)	_	Australia d	Morcombe et al. (1992)
	n.c. (662) a	0.26 (607)	_	Canada	Korsrud et al. (1985)
	0.09 (2062)	0.46 (2061)	_	Canada	Salisbury et al. (1991)
	0.0325 (13) 0.0314 (41) 0.070 (4) 0.028 (218) 0.00619 (258) b _ 0.1471 (14) 0.465 (42) 0.180 (4) 0.17 (217) 0.069 0.379 0.069 (80) c 0.378 (80) c F o r	0.0079 (14) 0.00907 (42) 0.005 (20) 0.0015 (121) <0.00039 (241) b Finland Czech Republic e Raszyk et al. (1996) Czech Republic e Ulrich et al. (2001) Finland Nuurtamo et al. (1980) Finland Niemi et al. (1991) Tahvonen and Kumpulainen (1994) 0.004 Germany Potthast (1993) 0.003 (80) c Germany Hecht (1983)
	0.012 (200) 0.199 (30) 0.075 (131) _ 0.052 (806) 0.041 (1509) 0.09 (73) 0.019 (426)	0.066 (200) 0.666 (30) 0.429 (203) P 0.011 (200) 0.048 (30) 0.004 (203) 0.09 (567) _ 0.28 (805) 0.017 (926) e 0.24 (1516) 0.005 (658) 0.39 (272) 0.009 (84) 0.11 (626) 0.001 (426) e r	Hungary Italy The Netherlands f Vos et al. (1986) Gyõri et al. (2005) Amodio-Cocchieri and Fiore (1987) Norway Kluge-Berge et al. (1992) Poland Falandysz and Lorenc-Biala (1989) Poland Falandysz (1993a) Slovenia g Doganoc (1996) Sweden h Jorhem et al. (1991)
	_ 0.05 (13) 0.14 (326) 0.21 (282)	0.11 (1051) 0.1 (13) 0.30 (321) 0.65 (281)	_ _ n.c. (326) a n.c. (281) a R Sweden i UK USA j USA d e	Pettersson-Grawé et al. (1997) MAFF (1998) Coleman et al. (1992) Coleman et al. (1992)
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Table V .

 V Published data on lead concentrations in pig liver, kidney and muscle. Unless otherwise specified, values shown are average concentrations (mg/kg fresh weight). The numbers of samples analysed are in parentheses. not calculated, because more than 70% of the values were below the detection limit; b calculated per wet weight based on dry matter contents of 26% for meat, 29.5% for liver and 20% for kidney; c median value; d age 8 months; e age 3-24 months; f age more than 2 years; g age around 6 months; h market hogs; i adults.

	Liver	Kidney	Muscle	Country	Reference
	n.c. (662) a	n.c. (607) a	_	Canada	Korsrud et al. (1985)
	0.07 (2062)	0.07 (2061)	_	Canada	Salisbury et al. (1991)
	0.137 (41)	0.1515 (42)	0.099 (42)	Czech Republic d	Ulrich et al. (2001)
	0.1415 (13) <0.020 (4) 0.038 (206) 0.00324 (258) b _ 0.1250 (14) 0.020 (4) 0.04 (205) 0.055 0.023 0.055 (80) c 0.029 (80) c 0.075 (200) 0.070 (200) F o r	0.1236 (14) 0.020 (20) 0.015 (187) 0.00234 (241) b Finland Czech Republic d Finland Finland 0.033 Germany 0.033 (80) c Germany 0.079 (200) Hungary	Raszyk et al. (1996) Nuurtamo et al. (1980) Niemi et al. (1991) Tahvonen and Kumpulainen (1994) Potthast (1993) Hetch (1983) Gyõri et al. (2005)
	0.357 (30) 0.05 (131) _ 0.16 (804) 0.094 (1509) 0.06 (72) 0.019 (426) 0.3 (13)	0.511 (30) 0.13 (203) 0.00 (567) P 0.19 (30) 0.03 (204) _ 0.2 (805) 0.092 (926) 0.12 (1516) 0.02 (334) e 0.06 (274) <0.05 (83) 0.016 (626) <0.005 (426) 0.09 (13) _ e r	Italy The Netherlands e Vos et al. (1986) Amodio-Cocchieri and Fiore (1987) Norway Kluge-Berge et al. (1992) Poland Falandysz and Lorenc-Biala (1989) Poland Falandysz (1993a) Slovenia f Doganoc (1996) Sweden g Jorhem et al. (1991) UK MAAF (1998)
	n.c. (318) a n.c. (280) a	n.c. (312) a n.c. (279) a	n.c. (318) a <0.5 (279) R USA h USA i	Coleman et al. (1992) Coleman et al. (1992)
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Table VI .

 VI Published data on arsenic concentrations in pig liver, kidney and muscle.Unless otherwise specified, values shown are average concentrations (mg/kg fresh weight). The numbers of samples analysed are in parentheses. not calculated, because more than 70% of the values were below the detection limit; b age 3-24 months; c age around 6 months.

	Liver	Kidney	Muscle	Country	Reference
	n.c. (662) a	n.c. (607) a	_	Canada	Korsrud et al. (1985)
	0.26 (2062)	0.17 (2061)	_	Canada	Salisbury et al. (1991)
	<0.02 (1) 0.005 (62) 0.026 (385) 0.006 (103) 0.01 (563) 0.023 (625) 0.19	<0.02 (1) 0.008 (65) 0.037 (387) 0.005 (174) _ 0.019 (338) 0.13 F o r	<0.02 (20) 0.003 (65) <0.005 (48) 0.002 (183) _ 0.024 (338) 0.01	Finland Germany Germany The Netherlands b Vos et al. (1986) Nuurtamo et al. (1980) Holm (1978) Crossmann (1981) Norway Kluge-Berge et al. (1992) Sweden c Jorhem et al. (1991) USA Doyle and Spaulding (1978)
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Table VII .

 VII Published data on mercury concentrations in pig liver, kidney and muscle.Unless otherwise specified, values shown are average concentrations (mg/kg fresh weight). The numbers of samples analysed are in parentheses. not calculated, because more than 70% of the values were below the detection limit; b age 8 months; c age 3-24 months; d age around 6 months.

	Liver	Kidney	Muscle	Country	Reference
	n.c. (662) a	n.c. (607) a	_	Canada	Korsrud et al. (1985)
	0.01 (2062)	0.02 (2061)	_	Canada	Salisbury et al. (1991)
	0.0149 (41)	0.026 (42)	0.00504 (42)	Czech Republic b	Ulrich et al. (2001)
	0.01 (13) 0.011 (4) 0.012 (183) 0.002 (131) _ 0.005 (181) 0.0037 (1516) 0.007 (1516) 0.0138 (14) 0.013 (4) 0.014 (95) 0.007 (200) 0.01 (567) 0.008 (180) F o r	0.0088 (14) <0.002 (20) 0.011 (163) 0.002 (204) _ 0.003 (181) 0.0019 (1513)	Czech Republic b Finland Finland The Netherlands c Vos et al. (1986) Raszyk et al. (1996) Nuurtamo et al. (1980) Niemi et al. (1991) Norway Kluge-Berge et al. (1992) Poland Falandysz and Gajda (1988) Poland Falandysz (1993a)
	0.015 (390) 0.02 (156) 0.01 (136)	0.019 (626) _ 0.02 (136) P 0.009 (390) 0.011 (156) 0.003 (136) e	Sweden d USA USA	Jorhem et al. (1991) Sell et al. (1975) Doyle and Spaulding (1978)
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Table VIII .

 VIII Published data on copper concentrations in pig liver, kidney and muscle.Values shown are average concentrations (mg/kg fresh weight). The numbers of samples analysed are in parentheses.

	Liver	Kidney	Muscle	Country	Reference
	12.0 (662)	7.3 (607)	_	Canada	Korsrud et al. (1985)
	12.2 (2062)	7.58 (2061)	_	Canada	Salisbury et al. (1991)
	16 (4)	6.0 (4)	0.87 (20)	Finland	Nuurtamo et al. (1980)
	_ _ 6.2 (804) 8.5 (661) 9.0 (126) 60.5 (13) 11.1 (326)	_ 7.9 (71) 6.2 (805) 8.4 (663) 6.1 (75) 3.57 (13) 6.65 (321) F o r	0.5 _ 0.7 (926) 1.1 (658) 0.9 (126) _ 1.16 (326)	Italy The Netherlands a Ellen et al. (1989) Lombardi-Boccia et al. (2005) Poland Falandysz & Lorenc-Biala (1989) Poland Falandysz (1993a) Sweden b Jorhem et al. (1989) UK MAFF (1998) USA c Coleman et al. (1992)
	18.3 (282) a age 4-24 months (mean 7 months); b age around 6 months; c market hogs; d adults 6.73 (281) 0.93 (281) USA d Coleman et al. (1992) P
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Table IX .

 IX Published data on zinc concentrations in pig liver, kidney and muscle. Values shown are average concentrations (mg/kg fresh weight). The numbers of samples analysed are in parentheses.

	Liver	Kidney	Muscle	Country	Reference
	61.6 (662)	25.9 (607)	_	Canada	Korsrud et al. (1985)
	65.6 (2062) 90 (4) _ _ 39 (804) 50 (661)	27.4 (2061) 26 (4) _ 22.7 (71) 39 (804) 30 (663) F o r	_ 22 (20) 15.5 _ 20 (926) 26 (658)	Canada Finland Italy The Netherlands a Ellen et al. (1989) Salisbury et al. (1991) Nuurtamo et al. (1980) Lombardi-Boccia et al. (2005) Poland Falandysz & Lorenc-Biala (1989) Poland Falandysz (1993a)
	74 (126) 39.8 (13) 66.9 (326) 63.7 (282) a age 4-24 months (mean 7 months); b age around 6 months; c market hogs; d adults 22 (75) 24 (126) Sweden b Jorhem et al. (1989) 18.8 (13) _ UK MAFF (1998) 25.0 (321) 24.0 (326) USA c Coleman et al. (1992) 24.7 (281) 33.8 (281) USA d P Coleman et al. (1992) e
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Table X .

 X Published data on iron concentrations in pig liver, kidney and muscle. Values shown are average concentrations (mg/kg fresh weight). The numbers of samples analysed are in parentheses.

	Liver	Kidney	Muscle	Country	Reference
	_	_	4.18 (104)	Germany	Reichardt et al. (2002)
	_	_	4.2	Italy	Lombardi-Boccia et al. (2005)
	56 (804)	57 (805)	14 (926)	Poland	Falandysz & Lorenc-Biala (1989)
	54 (661) 123 (15) a 191.0 (326) 363.0 (282) a median value; b market hogs; c adults 63 (663) 13 (658) _ 21 (15) a 46.5 (320) 10.2 (326) 79.0 (281) 16.5 (281) F o r	Poland Thailand USA b USA c	Falandysz (1993a) Kongkachuichai et al. (2002) Coleman et al. (1992) Coleman et al. (1992)
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Table XI .

 XI Published data on selenium concentrations in pig liver, kidney and muscle.Values shown are average concentrations (mg/kg fresh weight). The numbers of samples analysed are in parentheses.

	Liver	Kidney	Muscle	Country	Reference
	0.470 (4)	1.710 (4)	0.070 (20)	Finland	Nuurtamo et al. (1980)
	0.56 (48)	2.41 (48)	0.08 (48)	Finland	Salmi and Hirn (1984)
	_	_	0.16 (50)	Norway	Froslie et al. (1985)
	0.51 (48) 0.50 (72) a age around 6 months 2.34 (48) 1.9 (72) F o r	0.14 (48) 0.094 (72)	Sweden Sweden a	Kolar (1983) Jorhem et al. (1989)
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Table XII .

 XII Published data on manganese concentrations in pig liver, kidney and muscle. Values shown are average concentrations (mg/kg fresh weight). The numbers of samples analysed are in parentheses. age 4-24 months (mean 7 months); b age around 6 months; c market hogs; d adults

	Liver	Kidney	Muscle	Country	Reference
	3.9 (4)	1.5 (4)	0.13 (20)	Finland	Nuurtamo et al. (1980)
	_	1.45 (71)	_	The Netherlands a Ellen et al. (1989)
	1.1 (804)	1.1 (805)	0.11 (926)	Poland	Falandysz and Lorenc-Biala (1989)
	1.3 (663) 3.0 (46) 4.20 (324) 2.37 (281)	1.3 (663) 1.5 (46) 1.40 (318) 1.22 (280) F o r	0.11 (658) 0.12 (45) 0.13 (324) 0.14 (280)	Poland Sweden b USA c USA d	Falandysz (1993a) Jorhem et al. (1989) Coleman et al. (1992) Coleman et al. (1992)
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Table XIII .

 XIII Published data on cobalt concentrations in pig liver, kidney and muscle.Values shown are average concentrations (mg/kg fresh weight). The numbers of samples analysed are in parentheses. not calculated because of large number of data below the detection limit ; b age around 6 months; c market hogs; d adults

	Liver	Kidney	Muscle	Country	Reference
	0.010 (4)	0.070 (4)	<0.010 (20) Finland	Nuurtamo et al. (1980)
	0.010 (36)	0.004 (36)	0.001 (36)	Sweden b	Jorhem et al. (1989)
	n.c. (324) a	n.c. (318) a	n.c. (324) a	USA c	Coleman et al. (1992)
	n.c. (281) a	n.c. (280) a	n.c. (280) a	USA d	Coleman et al. (1992)

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Table XIV .

 XIV Published data on chromium concentrations in pig liver, kidney and muscle. Values shown are average concentrations (mg/kg fresh weight). The numbers of samples analysed are in parentheses. not calculated because of large number of data below the detection limit; b calculated per wet weight based on dry matter content of 26% for meat, 29.5% for liver and 20% for kidney; c age 4-24 months (mean 7 months); d age around 6 months; e sows.

	Liver	Kidney	Muscle	Country	Reference
	0.010 (4)	0.010 (4)	0.040 (20)	Finland	Nuurtamo et al. (1980)
	_	n.c. (71) a	_	The Netherlands c	Ellen et al. (1989)
	<0.010 (71)	<0.010 (71)	0.014 (71)	Sweden d	Jorhem et al. (1989)
	0.006726 (4) b F 0.00716 (5) b _ o r	USA e	Lindemann et al. (2004)
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Table XV .

 XV Published data on nickel concentrations in pig liver, kidney and muscle.Values shown are average concentrations (mg/kg fresh weight). The numbers of samples analysed are in parentheses. not calculated because of large number of data below the detection limit; b age 4-24 months (mean 7 months); c age around 6 months; d market hogs; e adults.

	Page
	Liver	Kidney	Muscle	Country	Reference
	<0.020 (4)	<0.020 (4)	<0.020 (20)	Finland	Nuurtamo et al. (1980)
	_	0.045 (71)	_	The Netherlands b Ellen et al. (1989)
	0.011 (34)	0.011 (34)	<0.010 (34)	Sweden c	Jorhem et al. (1989)
	2.14 (326)	0.57 (320)	n.c. (326) a	USA d	Coleman et al. (1992)
	0.26 (282)	0.29 (281)	n.c. (281) a	USA e	Coleman et al. (1992)

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