Eur Food Res Technol (2001) 212 : 426430

Q Springer-Verlag 2001

ORIGINAL PAPER

Marta Miranda 7 Marta Lpez Alonso 7 Cristina Castillo Joaqun Hernndez 7 Jos Luis Benedito

Cadmium levels in liver, kidney and meat in calves from Asturias (North Spain)

Received: 30 May 2000 / Revised version: 27 July 2000

Abstract The aim of the present study was to investigate the levels of cadmium in calves from Asturias and to compare them with the levels reported in other countries and with the maximum tolerance levels in products for human consumption. The Principado de Asturias contains a large mining area and, for that reason, may deserve special attention with regard to possible heavy metal contamination. Samples of liver, kidney and meat from 312 animals aged 612 months were collected from the whole region. Samples were acid digested and cadmium concentrations were determined by graphite furnace atomic absorption spectrophotometry (GFAAS). A strict analytical quality control was carried out during the study. The average wet weight concentrations in liver, kidney and meat were respectively 30.7 mg/kg, 161 mg/kg and 2.03 mg/kg and the levels found in the different tissues were comparable to those reported from other investigations carried out in different countries in recent years. Only 2.9% of kidneys exceeded the tolerance limits established in the strictest legislation, whereas no samples of liver and meat exceeded the limits. Keywords Cadmium 7 Calves 7 Liver 7 Kidney 7 Meat 7 Asturias 7 Spain

Introduction
One of the most important aspects of environmental pollution to humans is that a significant intake of heavy metals occurs through diet. Food-producing animals are a rich source of food, but they are also a source of

M. Miranda (Y) 7 M.L. Alonso 7 C. Castillo 7 J. Hernndez J.L. Benedito Departamento de Patoloxa Animal, Facultade de Veterinaria, Universidade de Santiago de Compostela. 27002 Lugo, Spain e-mail: mmiranda6lugo.usc.es

various contaminants. Since diet is an important source of heavy metals for humans, the World Health Organisation has indicated the necessity of reducing to a minimum the content of toxic metals in food [10]. Cadmium is recognised as one of the most toxic elements to man and animals. This metal is naturally present in all parts of the environment, including sea and fresh water, soils, sediments and the air [28]. It is used in many industrial processes and is also a contaminant in some fertilizers, especially in partially acidulated phosphate fertilizers [24] and in urban sewage sludges used to fertilize pastures or crops [9]. This widespread distribution and the industrial fallout have resulted in all food being exposed to, and containing, cadmium. Currently, programs monitoring heavy metal levels in animal products, such as meat, liver and kidney, are conducted with a view to determining whether there are residues and to prevent the distribution of products that pose a risk to human health. In recent years a large number of countries have carried out a control programme to monitor the levels of heavy metals in bovine tissues: Germany [20, 21], Austria [15], Italy [1], The Netherlands [31], Norway [14], Sweden [1113], Finland [26, 30], Poland [5], Slovakia [17, 18], Slovenia [3], Czech Republic [27], Australia [16, 19, 22], Brazil [2] and Canada [29], but not Spain. Here the first studies in monitoring the levels of heavy metals in bovine tissues are more recent [23, 25]. Asturias is a region where the number of animals of the bovine species and the production of meat are economically very important. As cadmium is considered one of the most dangerous trace metals in the food, we thought it could be interesting to determine the levels of cadmium in bovine products for human consumption. Now in Spain, there is no legislation regarding maximum levels of cadmium in animal products and we thought that this study could contribute to the preparation of a guide to recommended values or to the laying down of admissible maximum values. The aim of the present study has been to investigate the levels of cadmium in liver, kidney and meat in

427

calves from Asturias, and to compare them with levels and legislation in different countries.

Material and methods

Sampling. The samples were taken in all cattle slaughterhouses in Asturias between November 1996 and July 1997. The age of animals was 612 months. A total of 312 animals and 936 samples were analysed. Samples of liver, kidney and muscle were taken from the same animal. Approximately 200 g of liver, kidney and muscle were taken from each animal. The liver samples were taken from the caudate lobule, the kidney from the anterior half of the right kidney and the muscle from the diaphragm. The samples were packed in polyethylene bags and deep frozen during transport to the laboratory. At the laboratory the samples were freed of fat, major blood vessels and tendons, and homogenised. Each sample was divided into three sub-samples (10 g approximately) and identified with an animal code. The samples were stored at 18 7C until analysis. Digestions. All samples (1 g approximately) were weighed in glass digestion tubes in a precision balance. All samples were thereafter dried in a furnace at 85 7C to obtain a constant weight (24 h approximately). Then 2 ml of concentrated high purity HNO3 (Suprapur grade, Merck) was added to samples to obtain a cold digestion, for 24 h. After that, samples were placed in a thermostatic block at 120B2 7C for 60 min and then 2 ml of hydrogen peroxide 30% w/v was added to complete the digestion of organic matter. The solutions were then diluted with ultrapure water to 20 ml for liver, 15 ml for kidney and 10 ml for muscle. Glassware was usually washed and soaked in 10% diluted HNO3 for at least 24 h and then well washed (3 times) with double-distilled water before use. Determinations. The determinations were carried out in an atomic absorption spectrophotometer using the Zeeman effect (HITACHI Z-8100) with graphite furnace. Cadmium was determined by means of a graphite furnace. The drying step was carried out at temperatures that ensure smooth drying. The atomisation temperature used was 2000 7C and the wavelength was at 228.8 nm. Ammonium di-hydrogen phosphate (NH4H2PO4) was tested as matrix modifier but we did not observe significant absorbency differences, so that no matrix modifier was used. For the purpose of detecting interference we tested the method of standard addition, and the results show that there is no interference, so cadmium was analysed using a calibration curve. Quality control. A strict analytical quality control programme was employed during the study. The results of the analytical qual-

ity control programme are presented in Table 1. In every series of 24 samples, 2 blanks and 1 sample of international reference material (Pig Kidney CRM 186, from the Community Bureau of Reference, Commission of the European Community) were included. The results of these analyses were in close agreement with the reference values. Recovery tests were made during the analysis of each sample type. The samples were spiked with three different concentrations of the elements; the level of the additions was adjusted to give a signal that was generally 2- to 10-times greater than the normal levels in the different tissues. The blank levels were monitored throughout the investigation period and were deducted from the reading before the results were calculated. The limit of determination was set at three times the standard deviation of the mean blank. The detection limit of samples was calculated according to final dilution and weight. The precision of the analytical method was evaluated by measuring the absorbance signals in the same digested sample 12 times. To assess the precision of the overall procedure, readings of 12 different digested aliquots of the same organ were performed. The relative standard deviations (RSD) are very acceptable values.

Results and discussion

The results obtained in this investigation are shown in Table 2. The results obtained are presented as mean, range and median values. Although the trace elements often did not show a normal distribution, it is necessary to present the mean values in order to compare results, as in most papers the results are presented in this way. The results are presented in mg/kg, although in Table 3, where the results of the present study are compared with some literature data, results are presented in mg/ kg for the purpose of comparison. When the element concentration was less than the detection limit, a value of half the detection limit was assigned for the purpose of calculating the means. For liver, kidney and meat, mean values of 30.7 mg/ kg, 161 mg/kg and 2.03 mg/kg wet weight, respectively, were obtained. For liver and kidney all samples were above the detection limit, whereas 36% of meat samples were below this limit. The data shows that the kidney was the main organ involved in cadmium accumulation, followed by the liver and finally the muscle, the difference between organs being statistically significant

Table 1 Results of the analytical quality control program used in determination of cadmium in liver, kidney and meat Liver Accuracy (Pig Kidney CRM 186) Determined mean (mg/kg)BSD (np24) Certified mean (mg/kg)BSD Recovery % Recovery test (np21) Blank MeanBSD (np12) (mg/l) Detection limit Acid digestion (mg/l) 3!SD of blank In samples (mg/kg) Precision (RSD %) Analytical method (np20) Overall procedure (np20) 0.071B0.038 0.11 1.95 4.72 5.82 1.43 4.39 6.41 0.96 5.95 8.47 2601B612 2710B150 96.0 93.3B4.13 96.5B3.18 95.0B5.31 Kidney Meat

428 Table 2 Cadmium levels in liver, kidney and meat (mg/kg wet weight) in calves from Asturias Liver Number of samples ( a) MeanBSE b Range Median 312 (0) 30.7B1.24 3.39221 27.0 Kidney 312 (0) 161B7.03 4.2717 124 Meat 312 (119) 2.03B0.154 ND20.7 c 1.34

a Numbers in parenthesis denote number of samples below the limit of detection b SEpstandard error c ND: Below the limit of detection

(p~0.001). The long half-life and the low elimination of the cadmium explain the predominance of the renal and hepatic levels over muscle [7, 8]. We found a significant correlation (rp0.733, p~0.001) between the cadmium concentrations in livers and kidneys; a similar correlation was reported by Vos et al. [31] and Langlands et al. [22]. This relationship of hepatic and renal cadmium in this study is indicative of prolonged exposure over time in low dosages [7, 8]. Cadmium concentrations in animal tissues, especially livers and kidneys, are strongly related to the cadmium levels in animal feedstuffs [32]. Cadmium accumulation in meat was very low; Salisbury et al. [29] suggest that only animals ingesting very high amounts of cadmium will have significant residues in muscle. In Table 3 some literature data on the cadmium levels in liver, kidney and meat of cattle in different countries are given. Cadmium levels found in liver were similar to those reported in Canada [29], Germany [20], and Galicia (Spain) [23], in these cases data corre-

sponding to young animals (Table 3). However, levels were lower than those described in other countries, where data refers to cattle. Kidney levels were also lower than those reported in most of countries, even in studies of young animals [20, 29]. They were only higher than those reported in Galicia (Spain) [23]. This difference could be explained by considering that Asturias is a more industrialised region than Galicia, and this difference is only present in the kidney because it is the main organ for cadmium accumulation. It is difficult to compare the results for liver and kidney from different investigations since cadmium accumulates with age [6, 22, 30, 31], and generally the age of the slaughtered animals is not always known or reported, or is not reported precisely enough. The reason why cadmium levels in liver and kidney in this investigation were lower than the majority reported in the literature can be due to the animals being young (612 months). This is because in Asturias, like other regions of Spain, cattle are slaughtered at this age, whereas in other countries they are slaughtered after at least two years. In Spain we could not evaluate the trend of cadmium accumulation during recent years because there are no previous studies, and the levels could probably be lower now, as the use of cadmium is more controlled. In fact, Niemi et al. [26] and Tahvonen and Kumpulainen [30] have suggested that lower cadmium concentrations in fertilizers and improvement in public waste management and manufacturing methods may have resulted in a reduction in cadmium emissions into the environment. Levels found in muscle were similar to those described in Australia [19], Germany [20], Finland [26], Sweden [12] and Galicia (Spain) [23]. The levels of cadmium in muscle are very low and similar in the different studies (Table 3).

Table 3 Data from recent literature on the levels of cadmium in liver, kidney and meat of cattle from various countries. Average concentrations (in mg/kg wet weight) are given. The number of samples analysed is in brackets Liver 0.031 (312) 0.06 (179) 0.176 (1100) 0.12 (8635) 0.04 (210) 0.105 (146) 0.119 (30) 0.034 (87) 0.05 (61) 0.09 (69) 0.061 (113) 0.052 (350) 0.070 0.120 0.077 0.316 0.032
a b

Kidney 0.161 (312) 0.37 (174) 0.650 (1227) 0.52 (8635) 0.26 (209) 0.522 (210) 0.342 (30) 0.222 (87) 0.51 (90) 0.23 (256) 0.37 (331) 0.350 (98) 0.21 (578) 0.39 (68) 0.610 (291) 0.298 (21) 0.259 (6) 0.071 (427)

Meat 0.002 (312) 0.001 (181) 0.029 (1812) 0.0009 (262) 0.004 (215) 0.038 (30) 0.001 (87) 0.004 (87) 0.001 (113) ND (138) ND (80) 0.001 (34) 0.006 (92) 0.010 (21) 0.023 (6) 0.001 (438)
c

Country Asturias (Spain) Australia Australia Australia c Canada a The Netherlands Italy Germany b Austria Brazil Slovenia Finland Finland Norway c Sweden Poland Czech Republic Slovakia Galicia (Spain) b Median value NDpnon detected

Year 1983 1988 1998 1991 1987 1987 1988 1993 1994 1996 1991 1994 1992 1991 1993 1998 1995 1999

Reference This study [19] [22] [16] [29] [31] [1] [20] [15] [2] [3] [26] [30] [14] [12] [5] [27] [17] [23]

(33) (290) (21) (6) (437)

Veal Calves

429 Table 4 Maximum permissible levels in cattle products for human consumption from various countries Country Australia Canada The Netherlands Germany Germany Brazil Slovenia Slovakia European Community Liver 1.25 1 1 0.5 1 1 0.5 0.5 0.51 Kidney Meat mg/kg wet weight 2.5 1 3 0.5 1 1 1 1 1 0.1 0.05 0.050.1 0.2 1 0.05 0.1 0.2 Legislations Maximum acceptable concentrations. National Health and Medical Research Council Action levels. Agri-Food Safety Division of Agriculture Canada Provisional legal limits (maximum acceptable concentrations) ZEBS Guide value recommendations FIHV permissible maximum values National programme of biological residues in meat Allowed maximum tolerance levels. Uradni list SFRJ 1987 Permissible hygienic limits. Ministry of Health of the Slovak Socialist Republic Commission Regulation 194/97 setting maximum limits for certain contaminants in foodstuffs References [22] [29] [31] [20] [20] [2] [3] [17] [4]

Most countries have carried out surveys of toxic elements in animal tissues and the results have allowed them to determine the levels of toxic metals and to establish the maximum tolerable levels in products for human consumption. Nowadays in Spain there are no limits of acceptability for toxic metals in animal tissues. Consequently, in this study cadmium concentrations detected in calves from Asturias have been compared with the maximum levels established in other countries and with the Regulation proposed by the European Commission (Table 4). Cadmium is one of the first toxic metals for which maximum acceptable concentrations have been set. Regulatory limits for cadmium in liver and kidney vary among countries (Table 4). The highest regulatory limit for liver and kidney is from Australia [22], with 1.25 mg/kg and 2.5 mg/kg wet weight respectively; no sample had levels above these limits. In contrast, the German ZEBS guide values [20] are the lowest for both tissues, with 0.5 mg/kg wet weight; 2.9% (9) of kidneys exceeded this level. However, the acceptable maximum concentration for kidney in most legislations, including the Commission Regulation N0 194/97 of the European Community [4], is 1 mg/kg (Table 4); no sample of kidney had cadmium concentrations above this level. In most countries 115% of kidney samples have cadmium concentrations above 1 mg/kg and a few even have liver cadmium concentrations above this level. Cadmium concentrations in meat are generally very low, rarely exceeding the tolerance limits. Similarly to other countries, no samples of calves from Asturias had muscle cadmium concentrations above the acceptable maximum concentration.

In conclusion, we could say that there was no evidence of accumulation of toxic levels of cadmium. There also did not seem to be any need to recommend restrictions in the human consumption of meat, liver and kidneys from calves in this region. This study provided baseline data on cadmium concentrations for calves in this region of Spain and allows guide value recommendations to be made for cadmium in calves.
Acknowledgements This work was supported by the Xunta de Galicia (Spain), grant no. XUGA 26103B98. Marta Miranda was the recipient of a research fellowship from the Xunta de Galicia (Spain).

References
1. Amodio-Cocchieri R, Fiore P (1987) Bull Environ Contam Toxicol 39 : 460464 2. Aranha S, Nishikawa AM, Taka T, Salioni EMC (1994) Rev Inst Adolfo Lutz 51(1) : 1620 3. Doganoc DZ (1996) Food Addit Contam 13(2) : 237241 4. European Commission (1997) Commission Regulation (EC) No. 194/97 of 31 January 1997 setting maximum limits for certain contaminants in foodstuff 5. Falandysz J (1993) Sci Total Environ 136 : 177191 6. Frank R, Suda P, Luyken H (1989) Bull Environ Contam Toxicol 43 : 737741 7. Friberg L, Kjellstrm T, Nordberg G, Piscator M (1979) Cadmium. In: Friberg L, Nordberg GF, Vouk VB (eds) Handbook on the toxicology of metals, 1st edn. Elsevier/North Holland Biomedical Press, pp 355381 8. Garca-Fernndez AJ, Snchez-Garca JA, Gmez-Zapata M, Luna A (1996) Arch Environ Contam Toxicol 30 : 252258 9. Jing J, Logan TJ (1992) J Environ Qual 21 : 7381 10. Joint Expert Committee of the FAO/WHO (1972) 16th Report of the Joint FAO/WHO Expert Committee on Food and Additives. WHO, Geneva, Switzerland

430 11. Jorhem L, Sundstrom B, Astrand C, Haegglund G (1989) Z Lebensm Unters Forsch 188 : 3944 12. Jorhem L, Slorach S, Sundstrom B, Ohlin B (1991) Food Addit Contam 8(2) : 201212 13. Jorhem L, Sundstrom B, Engman J, Astrand C, Olsson I (1996) Food Addit Contam 13(7) : 737745 14. Kluge-Berge S, Skjerve E, Sivertse T, Godal A (1992) In: Proceedings of 3rd World Congress, Foodborne Infections and Intoxications Berlin, vol II, pp 745748 15. Kfer J, Fuchs K (1993) Wien Tierrztl Mschr 80 : 264267 16. Koh TS, Bansemer PC, Frensham AB (1998) Aust J Exp Agric 38 : 535540 17. Kottferov J, Kornekov B (1995) Arch Environ Contam Toxicol 29 : 400405 18. Kottferov J, Kornekov B (1997) Arch Tierz 40(4) : 309316 19. Kramer HL, Steiner JW, Vallely PJ (1983) Bull Environ Contam Toxicol 30 : 588594 20. Kreuzer W, Rosopulo A, Sell D, Frangenberg J, Koberstein S (1988) Fleischwirtsch 68(1) : 101108 21. Kreuzer W, Gabriel W, Lucker E, Zerzawy H, Rosopulo A (1991) Fleischwirtsch 71(3) : 339344 22. Langlands JP, Donald GE, Bowles JE (1988) Aust J Exp Agric 28 : 291297 23. Lpez Alonso M (1999) Principales elementos contaminantes en ganado vacuno de Galicia. Thesis, University of Santiago de Compostela 24. McLaughlin MJ, Simpson PG, Fleming N, Stevens DP, Cozens G, Smart MK (1997) Aust J Exp Agric 37(8) : 10191026 25. Miranda M (1999) Valoracin de los principales elementos txicos en ganado vacuno de Asturias. Thesis, University of Santiago de Compostela 26. Niemi A, Venlinen ER, Hirvi T, Hirn J, Karppanen E (1991) Z Lebensm Unters Forsch 192 : 427429 27. Raszyk J, Gajduskov V, Ulrich R, Jarosov A, Npravnk A, Salava J (1998) Vet Med Czech 43(8) : 233238 28. Roberts AHC, Longhurst RD, Brown MW (1994) N Z J Agric Res 37 : 119129 29. Salisbury CDC, Chan W, Saschenbrecker P (1991) J Assoc Off Anal Chem 74(4) : 587591 30. Tahvonen R, Kumpulainen J (1994) Food Addit Contam 11(4) : 415426 31. Vos G, Hovens JPC, van Delft W (1987) Food Addit Contam 4(1) : 7388 32. Vreman K, van der Veen NG, van der Molen EJ, de Ruig WG (1986) Netherlands J Agric Sci 34(2) : 129144