Cattle tissues as a source of cadmium for consumers

The aim of this study was to re-evaluate the analytical results and time trends of the content of cadmium in muscle, liver, and kidney samples of young bovine animals and cows slaughtered in the Czech Republic during the years 1993–2010, and to estimate the total dietary exposure for the entire population aged 4–90 years and specifically, for children at the age of four to six years. Analyses showed that the arithmetic means of cadmium concentration in cow kidney and liver are higher than in young bovine (P < 0.0001), but not in muscle (P = 0.227). The mean cadmium concentration in cow kidney (0.595 mg·kg-1) was twice as high as in young bovine kidney (0.285 mg·kg-1) for the entire studied period. The mean concentration of cadmium in cow liver (0.113 mg·kg-1) was higher compared to young bovine liver (0.078 mg·kg-1). The mean concentration of cadmium in cow muscle (0.008 mg·kg-1) was comparable with young bovine muscle (0.006 mg·kg-1). The evaluation of time trends showed increasing concentrations of cadmium for cow (increasing slaughtering age) and young bovine kidney and decreasing concentrations for cow and young bovine muscle, but no significant change for liver. The estimated Cd burden of the entire population is worthy of attention (10% were over the tolerable dose of 2.5 μg·kg-1 body weight and week). For more than 50% of four to six-year-old children the burden exceeded the tolerable weekly intake. The contribution of food belonging to the group of meat and meat products, including cattle tissues, to usual cadmium exposure is only 5%. Nevertheless, the food safety recommendation is not to consume cow kidney as food. Food, kidney, contamination, heavy metals, trend, exposure Cadmium (Cd) is a toxic metal occurring in the environment from natural sources and also as a consequence of anthropogenic activities related to industry and agriculture. Many legislative control measures have been taken to decrease the contamination of foodstuff in Europe since the 1970s and this regulatory effort continues until the present time when new emerging toxicological data were collected (EFSA 2011). For a non-smoking person who is not occupationally exposed to Cd, foodstuffs represent the source of up to 90% of his or her total intake of Cd (EFSA 2009). Smokers have × 4–5 higher Cd concentrations in the blood and × 2–3 higher Cd contents in the kidneys (Dallongewille et al. 1998). The absorption of Cd through the human digestive tract is relatively low (3–5%). Cadmium accumulated in the kidneys and liver has a very long half-time of excretion from the human body, in the range of 10–30 years (Kjellström and Nordberg 1978; EFSA 2009). Cadmium is primarily toxic for the kidneys. Exposure to Cd is associated with many other effects such as nephrotoxic, neurotoxic, carcinogenic, genotoxic, and teratogenic effects, the development of osteoporosis or osteomalacia, damage to the endocrine system and reproductive functions (EFSA 2009). It is also classified as a group 1 human carcinogen (IARC 2015). In 2009, EFSA re-evaluated the existing Tolerable Weekly Intake (TWI) of Cd and set it to 2.5 μg·kg-1 body weight (EFSA 2009, 2011). Regulation (EC) No. 1881/2006 establishes the maximum limit (ML) for Cd content in meat (excluding offal) of ACTA VET. BRNO 2015, 84: 289–295; doi:10.2754/avb201584030289 Address for correspondence: prof. MVDr. Jiří Ruprich, Ph.D. National Institute of Public Health Palackého tř. 3a, 612 42 Brno, Czech Republic Phone: +420 541 211 764; +420 602 245 606 E-mail: jruprich@chpr.szu.cz http://actavet.vfu.cz/ bovine animals, sheep, porcine animals and poultry as follows: 0.050 mg·kg-1 wet weight; for liver: 0.50 mg·kg-1 wet weight; and for kidney: 1.0 mg·kg-1 wet weight. The aim of this study was to re-evaluate the recent analytical results and time trends of the content of Cd in muscle, liver and kidney samples of young bovine animals and cows slaughtered in the Czech Republic during the years of 1993–2010, and to estimate consequences for the total dietary exposure of the whole population of consumers and specifically, for younger children. Materials and Methods Sampling of cattle tissues and market food samples Samples of bovine meat and offal were collected by the Czech State Veterinary Administration (SVA), within the implementation of the national plan for the monitoring of residues and contaminants in accordance with the Council Directive 96/23/EC (1996) and Commission Decision 97/747/EC (1997) in the years 1993–2010. The sampling methodology was previously described by Drápal et al. (2012). Market food samples representing a common Czech diet were collected in all country regions as part of the national Total Diet Study during the years 2004–2011. The sampling methodology was described by Ruprich (2014). Analytical methods Samples of cattle tissues were analysed by SVA laboratories. Homogenized samples of biological material were mineralized in a microwave device (Ethos plus, Milestone) after the addition of concentrated nitric acid and hydrogen peroxide. Determination of cadmium was carried out using graphite furnace atomic absorption spectrometry with Zeeman correction (GF-AAS) (Varian 1985, 1988), and in some cases by using inductively coupled plasma mass spectrometry (ICP-MS) (Varian 2004). The levels of quantification (LOQ) were 0.01 mg·kg-1 for liver and kidney, and 0.005 mg·kg-1 for muscle. The results were provided in the Standard sample description for food and feed format developed by EFSA (2010). Analyses of market food at the laboratories of the National Institute of Public Health used the microwave wet digestion in a closed system (Ethos plus, Milestone) using nitric acid and hydrogen peroxide. Then, evaporation (Start D, Milestone) was used too, for mineralization of food samples and GF-AAS with Zeeman correction for determination of Cd (Perkin-Elmer 1991, 1999). The range of LOQs was 0.0001–0.002 mg·kg-1 of various food samples. All methods employed were accredited according to the European Standard EN ISO/IEC 17025. Statistical analyses Statistical evaluation of trends was performed using the linear regression analysis. T-test for independent samples was used to analyse the difference between observed concentrations of Cd for both cattle categories. A probability lower than 0.05 (P < 0.05) was considered as significant. Regression analyses and t-test were performed using the STATISTICA 12 software (StatSoft 2013). Exposure assessment Exposure assessment was done by the Monte Carlo Risk Assessment modelling software (MCRA), version 8.0 (RIVM, NL). Food consumption datasets, used from the Individual Food Consumption national study SISP04 (Ruprich et al. 2006), were reorganized into 20 Level 1 food groups, according to the FoodEx1 EFSA hierarchic classification system. Food concentration data (3520 analytical results) for Cd came from the national Total Diet Study (Ruprich 2014). The data represent dietary sources from analyses performed during the time period of 2004–2011. For evaluation of total exposure doses of Cd the following MCRA calculation parameters were used: Logistic-Normal Normal (LNN) exposure model, uncertainty analysis based on 10000 MC simulations, 100 resampling cycles, replacement of values < LOQ by 50% of LOQ (middle bound).

Cadmium (Cd) is a toxic metal occurring in the environment from natural sources and also as a consequence of anthropogenic activities related to industry and agriculture.Many legislative control measures have been taken to decrease the contamination of foodstuff in Europe since the 1970s and this regulatory effort continues until the present time when new emerging toxicological data were collected (EFSA 2011).For a non-smoking person who is not occupationally exposed to Cd, foodstuffs represent the source of up to 90% of his or her total intake of Cd (EFSA 2009).Smokers have × 4-5 higher Cd concentrations in the blood and × 2-3 higher Cd contents in the kidneys (Dallongewille et al. 1998).The absorption of Cd through the human digestive tract is relatively low (3-5%).Cadmium accumulated in the kidneys and liver has a very long half-time of excretion from the human body, in the range of 10-30 years (Kjellström and Nordberg 1978;EFSA 2009).Cadmium is primarily toxic for the kidneys.Exposure to Cd is associated with many other effects such as nephrotoxic, neurotoxic, carcinogenic, genotoxic, and teratogenic effects, the development of osteoporosis or osteomalacia, damage to the endocrine system and reproductive functions (EFSA 2009).It is also classified as a group 1 human carcinogen (IARC 2015).In 2009, EFSA re-evaluated the existing Tolerable Weekly Intake (TWI) of Cd and set it to 2.5 μg•kg -1 body weight (EFSA 2009(EFSA , 2011)).Regulation (EC) No. 1881/2006 establishes the maximum limit (ML) for Cd content in meat (excluding offal) of bovine animals, sheep, porcine animals and poultry as follows: 0.050 mg•kg -1 wet weight; for liver: 0.50 mg•kg -1 wet weight; and for kidney: 1.0 mg•kg -1 wet weight.
The aim of this study was to re-evaluate the recent analytical results and time trends of the content of Cd in muscle, liver and kidney samples of young bovine animals and cows slaughtered in the Czech Republic during the years of 1993-2010, and to estimate consequences for the total dietary exposure of the whole population of consumers and specifically, for younger children.

Sampling of cattle tissues and market food samples
Samples of bovine meat and offal were collected by the Czech State Veterinary Administration (SVA), within the implementation of the national plan for the monitoring of residues and contaminants in accordance with the Council Directive 96/23/EC (1996) and Commission Decision 97/747/EC (1997) in the years 1993-2010.The sampling methodology was previously described by Drápal et al. (2012).
Market food samples representing a common Czech diet were collected in all country regions as part of the national Total Diet Study during the years 2004-2011.The sampling methodology was described by Ruprich (2014).

Analytical methods
Samples of cattle tissues were analysed by SVA laboratories.Homogenized samples of biological material were mineralized in a microwave device (Ethos plus, Milestone) after the addition of concentrated nitric acid and hydrogen peroxide.Determination of cadmium was carried out using graphite furnace atomic absorption spectrometry with Zeeman correction (GF-AAS) (Varian 1985(Varian , 1988)), and in some cases by using inductively coupled plasma mass spectrometry (ICP-MS) (Varian 2004).The levels of quantification (LOQ) were 0.01 mg•kg -1 for liver and kidney, and 0.005 mg•kg -1 for muscle.The results were provided in the Standard sample description for food and feed format developed by EFSA (2010).
Analyses of market food at the laboratories of the National Institute of Public Health used the microwave wet digestion in a closed system (Ethos plus, Milestone) using nitric acid and hydrogen peroxide.Then, evaporation (Start D, Milestone) was used too, for mineralization of food samples and GF-AAS with Zeeman correction for determination of Cd (Perkin-Elmer 1991, 1999).The range of LOQs was 0.0001-0.002mg•kg -1 of various food samples.All methods employed were accredited according to the European Standard EN ISO/IEC 17025.

Statistical analyses
Statistical evaluation of trends was performed using the linear regression analysis.T-test for independent samples was used to analyse the difference between observed concentrations of Cd for both cattle categories.A probability lower than 0.05 (P < 0.05) was considered as significant.Regression analyses and t-test were performed using the STATISTICA 12 software (StatSoft 2013).

Exposure assessment
Exposure assessment was done by the Monte Carlo Risk Assessment modelling software (MCRA), version 8.0 (RIVM, NL).Food consumption datasets, used from the Individual Food Consumption national study SISP04 (Ruprich et al. 2006), were reorganized into 20 Level 1 food groups, according to the FoodEx1 EFSA hierarchic classification system.Food concentration data (3520 analytical results) for Cd came from the national Total Diet Study (Ruprich 2014).The data represent dietary sources from analyses performed during the time period of 2004-2011.For evaluation of total exposure doses of Cd the following MCRA calculation parameters were used: Logistic-Normal Normal (LNN) exposure model, uncertainty analysis based on 10000 MC simulations, 100 resampling cycles, replacement of values < LOQ by 50% of LOQ (middle bound).

Results
The analytical results from SVA (9729 individual samples) were organized according to tissue, year of collection, and category of slaughtered animals.The means and range of cadmium content in bovine tissues (mg•kg -1 wet weight) in the Czech Republic during the years 1993-2010 are summarized in Table 1.Statistical testing (t-test) showed significant differences between the observed mean concentrations for both studied cattle categories for samples of kidney (P = 0.000004) and liver (P < 0.000001), but not for muscle (P = 0.2265).Evaluation of trends showed increasing concentrations of Cd in cow and young bovine kidney.The result of the analysis is presented in Fig. 1.The model for cow kidney (y = -3.3274+ 0.022*x) explains 81% of the data variability.The models for young bovine kidney (y = -4.8013+ 0.0025*x) explain 40% of the data variability.Evaluation of trends for cow and young bovine muscle is presented in Fig. 2. The model for cow muscle (y = 0.687 -0.0003*x) accounts for 49% of the data variability.The models for young bovine muscle (y = 0.8935 -0.0004*x) explain 89% of the data variability.The data point for cows and year 2007/2008 was considered as an outlier.Linear regression showed that mean concentrations of Cd in cow and young bovine liver are not dependent on time.
The distribution of total Cd dietary exposure doses were studied for the entire Czech population (aged 4-90) and specifically, for younger children (four to six years of age).four to six years of age this burden is even more critical.More than 50% of children are exposed to doses equal to TWI, 5% of children are exposed to doses at 190% TWI EFSA.The three most significant sources of usual exposure of children are (1) grains and grainbased products (38%), (2) vegetables and vegetable products (14%), and (3) starchy roots and tubers (14%).The contribution of meat and meat products (including offal) is only about 4%, while e.g.milk and dairy products could potentially represent more than 8% of usual exposure.This analysis confirmed the assumption that although the Cd concentration in some cattle tissues is growing up, total usual exposure from these tissues is still relatively low.However, due to emerging total exposure doses, the food safety recommendation is not to use cow kidney as food, especially for younger children.Even as small a portion as 70 g of cow kidney consumed within one week, with about 0.8 mg Cd•kg -1 , can pose a certain health risk.

Fig. 1 .
Fig. 1.Trends in the observed mean Cd concentrations in mg•kg -1 wet weight in cow and young bovine kidney during the time period of 1993-2010.The figure shows regression lines with 95% confidence interval (CI) of estimated models.

Fig. 2 .
Fig. 2. Trends in the observed mean Cd concentrations in mg•kg -1 wet weight in cow and young bovine muscle during the time period of 1993-2010.The figure shows regression lines with 95% confidence interval (CI) of estimated models.The point from year 2007/2008 in dataset for cows can be considered as an outlier.

Table 1 .
Table2presents mean exposure doses of Cd estimated for the Czech population aged 4-90 and for four to six-year-old children and uncertainty bounds (2.5-97.5 percent) of exposure estimates.The cadmium content in bovine tissues in the Czech Republic in the years 1993-2010 (mg•kg -1 wet weight) (for calculation of mean and range values < LOQ were replaced by 50% of LOQ value = middle bound values)

Table 2 .
Exposure doses of Cd estimated for the Czech population 4-90 years old and young children four to six years old, and uncertainty bounds (2.5-97.5 percent).TWI EFSA (2011) is 2.5 μg•kg -1 of body weight and week.