Food Additives and Contaminants , 2000, Vol. 17, No. 8, 679± 687
Cadmium in organs and tissues of horses slaughtered in Italy Massimo Baldini{*, Paolo Stacchini{, Francesco Cubadda{, Roberto Miniero{, Patrizia Parodi{ and Piergiusepp e Facelli{
{ Istituto Superiore di SanitaÁ, L aboratorio Alimenti, V iale Regina Elena 299, 00161 Rome, Italy; { Ministero della SanitaÁ, Piazza Marconi 25, 00144 Rome, Italy
(Received 22 October 1999; revised 3 January 2000; accepted 31 January 2000)
T he cadmium content of muscle, liver, kidney and blood samples from 62 horses slaughtere d in Italy was investigated. Cadmium was determined by graphite furnace atomic absorption spectrometry (GFAAS) after wet digestion of the samples. T he mean and median contents of all samples were (on a fresh weight basis ) 75 and 41 ·g kg ¡1 for muscle, 2.46 and 2.10 mg kg ¡1 for liver, 20.0 and 13.5 mg kg ¡1 for kidney. T he cadmium level in blood samples was always below 6 ·gl ¡1 . T he cadmium concentrations in muscle, liver and kidney were found to be related to the life span of the specimens and increased with age. Females exhibited higher levels than males, but this di erence was signi® cant only in the case of muscle tissue. T he geographica l origin was recognized as the main factor in¯ uencing the cadmium content of the equine specimens analysed. T he di erences between horses coming from the three main breeding countries considered in this study (Poland, L ithuania, Hungary ) were marked and statistically signi® cant. T he average intake of cadmium from equine meat was estimated for the general population and for population groups resident in areas with high consumption of this food item. W hile the cadmium intake from equine meat for the average consumer accounts for about 1% of the total cadmium intake estimated for Italy, in the latter case the enhanced consumption of equine muscle is often accompanied with the consumption of substantial quantities of equine liver and this may lead to high cadmium intakes.
* To whom correspondence should be addressed; e-mail:
[email protected]
Keywords : cadmium, horses, intake, Italy
Introduction Cadmium is a non-essential element with a high potential toxicity for humans. It accumulates predominantly in the kidney, bound to metallothioneines, with a biological half-life of more than 10 years. Excessive long term cadmium exposure may produce irreversible adverse renal e ects (Stoeppler 1991, WHO 1992). This metal is mainly used with its compounds in nickel± cadmium batteries, anticorrosive coating of metals, pigments and stabilizers for plastic, and signi® cant quantities are released by numerous human activities. Ninety per cent of the atmospheric emissions of cadmium comes from anthropogenic sources. Those emissions, together with the release of cadmium into the aquatic and terrestrial environment, may lead to severe local pollution. Moreover, airborne cadmium may be widely spread in the environment due to long range atmospheric transport. The major route of exposure to cadmium for the nonsmoking general population is via food. The most signi® cant sources of cadmium in the diet are cereals and vegetables because of their high consumption rates. Meat and o al (especially liver and kidney) follow in importance. Molluscs and crustaceans may contain high levels of this element (Cicero et al. 1992), but generally constitute only a small part of the diet. Therefore, these commodities have a limited contribution to cadmium dietary intake (Baldini et al. 1987, Coni et al. 1992). The average dietary intake of cadmium in 15 European countries was recently estimated to be in the range 48± 162 mg/person/week, with the exception of one country, Greece, which provided data leading to an estimated intake of 400 mg/person/week (European Commission 1996).
Food Additives and Contaminant s ISSN 0265± 203X print/ISSN 1464± 5122 online # 2000 Taylor & Francis Ltd http://www.tandf.co.uk/journals
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The Joint FAO/WHO Expert Committee on Food Additives (JECFA) established a Provisional Tolerable Weekly Intake (PTWI) for cadmium of 7 mg kg ¡1 bw, corresponding to 490 mg for an adult weighing 70 kg (WHO 1993). In 1993 the International Agency for Research on Cancer (IARC) classi® ed cadmium and its compounds in Group 1 as human carcinogens, based on evidence from human studies, mainly those of lung cancer associated with cadmium inhalation in the workplace, and from animal studies (IARC 1993). Afterwards, the Scienti® c Committee on Food (SCF) of the European Commission reviewed the aspect of carcinogenicit y in order to re-evaluate the toxicology of cadmium from all dietary sources and concluded that a carcinogenic risk from dietary exposure to cadmium cannot be excluded even if it appears to be much lower than that from direct inhalation. Therefore the SCF was unable to establish a safe level for cadmium in food and expressed the opinion that `the present PTWI of 7 mg kg ¡1 bw is still considered to be valid for renal dysfunction but should be reevaluated as soon as further information concerning carcinogenicity or mutagenicity becomes available’ (SCF 1995). In Italy, since 1989, cadmium has been included in the list of the substances monitored by the yearly `National Plan on Residues’ in live animals and their products. The action limits established for cadmium are 0.1 mg kg ¡1 in meat and 1 mg kg ¡1 in o al. With respect to cadmium contamination, equine meat and o al deserve special attention as horses may reach substantially higher levels of this element in their tissues and organs than other slaughter animals (FAO/WHO 1998). Exposure of horses to cadmium is a consequence of their diet and life-span (horses are often slaughtered at a later age than other terrestrial animals). Moreover, in comparison to other mammalian species, horses have kidneys with longer proximal tubules and can, therefore, more e ectively reabsorb and store many toxic metals, including cadmium (Je ery et al. 1989). Therefore, a domestic programme for the monitoring of cadmium levels in organs and tissues of the horses slaughtered in Italy and for the assessment of the human exposure to cadmium due to the consumption of equine products was carried out in 1996. Only a quarter of the horses slaughtered in
Italy are of national origin and very few animals comes from other countries of the European Union. All the other horses (three-quarters of the total) primarily come from Eastern Europe and especially from Poland. Generally, the consumption of equine meat is low in the Italian population but it is much higher among speci® c population groups such as those su ering from anaemia or those who live in areas where equine products are part of the traditional diet. Consequently in the present study we attempted to evaluate the dietary intake of cadmium from equine meat consumption not only for the general population, but also for those speci® c population groups with high consumption levels of equine products.
Materials and methods Sampling Samples of muscle, liver, kidney and blood of 62 horses (33 females, 29 males) slaughtered at the `Centro Carni’ in Rome were collected during four weeks in 1996. Sixty horses were of foreign provenance (38 from Poland, 11 from Lithuania, 10 from Hungary, 1 from Byelorussia ) and two individuals came from Italy (Lazio and Umbria regions). The age and the provenance of the horses were obtained from the data cards provided by the slaughterhouse for each animal. Sampling was conducted according to the guidelines issued by the Food Department of the Istituto Superiore di SanitaÁ in order to mimimize the risk of contamination (Baldini et al. 1996, Coni et al. 1996 ). The 248 collected samples were put in separate plastic bagsÐ each reporting an identi® cation number, the country of origin, the age, the sex and the date of slaughteringÐ and sent to the Food Department where they were kept frozen pending analysis. The potential cadmium release from the plastic bags used for sampling was investigated by exposing them to an acidic extractant (4% acetic acid, contact time 24 h at a temperature of 20 § 28C). In all the cases (the experiment was carried out on ® ve di erent bags) the cadmium concentration in the leachates was below the detection limit.
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Analysis After homogenization of the samples with a laboratory blender, a sub-sample of 1 g was submitted to wet digestion at high pressure by the microwave digestion apparatus Milestone MLS 1200 Mega. All microwave-assisted mineralizations were performed with a mixture of concentrated HNO3/H2 O2 (5 ‡ 2 ml, ultrapure grade). Then, every sample was made up to 25 ml with double distilled water and analysed by graphite furnace atomic absorption spectrometry (GFAAS), using a SIMAA 6000 (Perkin Elmer) instrument in the single element mode equipped with the autosampler AS-72 (Perkin Elmer). All samples were analysed in duplicate. The detection limit (calculated on the basis of sample test portions of 1 g as three times the standard deviation of the mean of blank determinations) was 2 mg kg ¡1 . As regards the quality assurance of the analytical procedure, blanks and certi® ed reference materials (CRMs) were included in each analytical run. The three following BCR CRMs were analysed: 184 (bovine muscle), 185 (bovine liver), 186 (pig kidney). Samples of 0.5 g were used in each case. The results of CRM analysis were satisfactory (found values ˆ 92± 97% of certi® ed values, coe cients of variation ˆ 2± 8%) and are summarized in table 1.
and often did not passed normality tests. In most cases a log-normal distribution appeared valid for describing cadmium concentration in the di erent organs and tissues analysed. Therefore, all data were log-transformed before a two-way analysis of variance (ANOVA) was performed to test di erences related to sex and geographical origin of specimens. After the examination of data scatterplots, the correlation between cadmium concentration and age of specimens was studied on log-transformed data (Pearson product± moment correlation).
Results
The cadmium concentrations in equine muscle, liver and kidney are summarized in tables 2 and 3, where samples are grouped for geographical provenance and age class respectively. As regards blood samples, only four specimens were above the detection limit, with levels ranging from 2 to 6 mg l ¡1 . A detailed report on data concerning muscle and organs follows.
Muscle The mean total content of cadmium in muscle tissue …§ sd† was 75 § 89 mg kg ¡1 . The frequency distribution of cadmium concentrations was positively
Statistical methods Descriptive statistics of the data set were performed with a standard programme and included mean, standard deviation, median, geometric mean, minimum, maximum, skewness. The normality of the frequency distributions of cadmium concentration was checked by means of the Kolmogorov± Smirnov test (with Lilliefors probabilities ) and the Shapiro± Wilks test. All distributions were positively skewed
T able 2. T he cadmium content of equine muscle, liver and kidney in relation to the age of specimens: the ® rst value is the mean, the second is the median and the third is the range (mg kg ¡1 fw). Agea Range
Mean
nb
25c 0.3± 10c > 7c < 2c 8.5d 8.0d 8.2d 3± 20c 7.9d
8 142 13 58 10 65 52 35 209 62
Poland Finland Italy Poland USA Italy Hungary Poland Various origins Various origins
Reference Renon et al. (1980) Salmi and Hirn (1981) Campanini et al. (1982) Decastelli et al. (1991) Decastelli et al. (1991) Ghirarduzzi et al. (1994) Ghirarduzzi et al. (1994) Ghirarduzzi et al. (1994) Weyermann and Luecker (1998) This study
a
Number of samples. In years. Range. d Mean. b c
T able 5.
Cadmium concentrations in equine liver reported by various studies (mg kg ¡1 fw).
Mean
Median
6.58 2.99 2.39 1.90 3.09 2.46
7.62 2.36 Ð Ð Ð 2.10
Range 1.94 ± 0.20 ± 0.16 ± Ð 0.04 ± 0.07 ±
9.31 17.35 8.25 43.50 18.12
na
Ageb
Country
12 142 13 29 854 62
4 2c < 2¡> 25c 0.3± 10c 0.5± 37c Ð 7.9d
Various origins Finland Italy USA Canada Various origins
Reference Renon et al. (1980) Salmi and Hirn (1981) Campanini et al. (1982) Je ery et al. (1989) Salisbury et al. (1991) This study
a
Number of samples. In years. Range. d Mean. b c
fertilizer), have been so far recognized, but there is no information on the presence and respective importance of these sources in Poland. Nevertheless, it is worth noting that Poland has been one of the most important countries for cadmium production (Stoeppler 1991). The mean metal concentrations recorded in equine liver and kidney are comparable with those reported in the literature (tables 5 and 6 ). Generally a few mg kg¡1 are found in liver, while the levels detected in kidney usually lie in the range 20± 30 mg kg ¡1 . Again, the cadmium levels in these organs are related to the age of individuals, but seem to follow a di erent pattern from muscle tissue. In fact, it was reported that cadmium levels in kidney initially increase with age but start to decrease in older horses (Bjorland and Norheim 1981, Salmi and Hirn 1981, Je ery et al. 1989), a trend observed also in humans (WHO 1992). A decrease in liver concentrations of old horses is also
apparent from the data reported by Salmi and Hirn (1981) and Je ery et al. (1989). In the present survey only colts and young adult horses were studied and so a similar trend could not be veri® ed.
Cadmium intake According to the last available data of the National Institute of Nutrition, in Italy the consumption of equine meat in the general population is quite low, averagin g 7.7 g/person/week (Turrini et al. 1991). The average consumption estimated by the Central Institute of Statistics is higher, equal to 25 g/person/ week (ISTAT 1996 ). From these consumption data, using the mean cadmium content in equine muscle found in this study (75 mg kg¡1 ), a weekly cadmium intake of 0.6 ± 1.9 mg from equine meat consumption
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Cadmium in horses slaughtered in Italy
T able 6. Mean 24.1 28.7 48.0 24.8 18.6 25.4 27.7 20.0
Cadmium concentrations in equine kidney reported by various studies (mg kg ¡1 fw). Median 22.1 21.6 Ð Ð Ð 20.6 Ð 13.5
Range 11.7± 32. 1 2.6± 196.3 4119 2.0± 92. 8 Ð 3.4± 117.7 0.01 ± 185.0 1.9± 92. 4
na 12 142 50 13 28 58 854 62
Age b
Country
Reference
4 2c < 2¡> 25c < 2¡> 25c 0.3± 10c 0.5± 37c > 7c Ð 7.9d
Various origins Finland Norway Italy USA Poland Canada Various origins
Renon et al. (1980) Salmi and Hirn (1981) Bjorland and Norheim (1981) Campanini et al. (1982) Je ery et al. (1989) Decastelli et al. (1991) Salisbury et al. (1991) This study
a
Number of samples. In years. Range. d Mean. b c
T able 7. Estimated cadmium intake from equine muscle in relation to di erent consumption patterns observed in some Italian localities (in all cases a Cd concentration of 75 mg kg ¡1 in equine muscle was considered). Consumption pattern 1 2 3 4 a b
Weekly consumption of equine muscle (g)
Cd intake (mg)
% of total intake (national average)a
% of PTWIb
300 675 745 861
22.5 50.6 55.9 64.6
14 32 35 40
5 10 11 13
(Parma) (Barletta) (Barletta) (Barletta)
Total intake (national average ) ˆ 160.3 mg. PTWI ˆ 490 mg (for 70 kg person).
can be calculated for the average consumer. These ® gures account for about 1% of the total weekly cadmium intake (160 mg) estimated for Italy in 1996 (European Commission 1996 ), which in turn is equal to 33% of the FAO/WHO PTWI (490 mg for an adult of 70 kg). The situation is di erent in those geographical areas of Italy where as a consequence of traditional customs the consumption of equine products is much higher than the national average. In 1996 a survey on the consumption of equine muscle and liver was carried out by the Ministry of Health in two of these localities, namely Parma (Emilia Romagna) and Barletta (Puglia ) (Ministero della SanitaÁ, unpublished). At Parma a mean weekly consumption of 300 g of equine muscle and 25 g of equine liver was assessed. At Barletta the mean weekly consumption of equine muscle in those consumers who avoid eating equine liver turned out to be of 675 g. The consumers of both equine meat and equine liver eat on average 745 g/ week of meat and 25 g/week of liver. Finally, in this locality, the heavy consumers of equine products eat a
quantity of equine meat as high as 861 g/week and more than 25 g of liver a week. The cadmium intake from equine muscle consumption in the four above mentioned cases, estimated using the mean cadmium content in equine muscle found in this study, is reported in table 7. The cadmium intake resulting from equine liver consumption has not been calculated. The reason is that, in Italy, only the consumption of liver of domestic horses younger than two years is allowed together with muscle of horses of all ages and origins, while kidney consumption is forbidden (the law is enforced at a slaughterhouse level). The data on cadmium concentrations in liver of domestic colts collected through the present survey are not su cient for an accurate intake calculation. However, both our results and those of other authors (Renon et al. 1980, Salmi and Hirn 1981) show that cadmium levels in the liver of colts up to two years old are one order of magnitude higher than those in the muscle, regardless of the origin of animals. Therefore, the global cadmium intake of the above-mentioned heavy consumer of equine meat may approach the PTWI.
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Conclusions
Acknowledgements
As a whole, the cadmium levels found in the muscle of the specimens selected for this survey are not too high. Both the European Commission (EC) and the FAO/WHO Codex Alimentarius Commission are evaluating a proposed maximum limit (ML) of 0.2 mg kg ¡1 for horse meat (European Commission 1997, FAO/WHO 1998). Ninety-two per cent of the muscle samples analysed showed a cadmium content below this value. Among the ® ve samples above this threshold, four came from Polish horses. Cadmium levels in liver and kidney were considerably higher and greatly exceed the draft MLs currently under evaluation by the EC (0.5± 1 mg kg ¡1 for liver and 1 mg kg¡1 for kidney).
The authors would like to thank the `Centro Carni’ in Rome for providing the equine samples and Barbara Galoppi for her contribution in the analytical determinations.
The cadmium concentrations in equine meat and o al were found to be related to the life-span of the specimens and increased with age. Females exhibited higher levels than males, but this di erence turned out to be signi® cant only in the case of muscle tissue. As suggested by some previous surveys, the horses bred in Poland exhibit higher cadmium levels than those coming from other countries. The cadmium intake from equine meat for the average consumer was calculated and turned out to be negligible, accounting for about 1% of the total cadmium intake estimated for Italy. Conversely, in the case of population groups resident in areas where equine meat is part of the traditional diet, the total cadmium intake may be considerably enhanced as a consequence of high consumption of this food item. Nevertheless, it always remains well below the PTWI and thus, even in this latter case, there is no signi® cant health risk due to equine meat consumption. This conclusion may not hold true if the consumption of equine meat is combined with the consumption of equine liverÐ especially if the consumer is simultaneously exposed to other signi® cant cadmium sourcesÐ depending on the amounts of these food items eaten by the consumer. In this case, an accurate intake calculation was not possible on the basis of the data of the present survey. Hence, it would be advisable to carry out further studies focused on those speci® c consumers, aiming to relate reliable consumption data obtained on a local scale with the actual cadmium concentrations of the equine products consumed.
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