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Trace element levels in raw milk from northern and southern regions of Croatia
Food Chemistry 127 (2011) 63–66
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Trace element levels in raw milk from northern and southern regions of Croatia Nina Bilandzˇic´ a,⇑, Maja Ðokic´ a, Marija Sedak a, Bozˇica Solomun a, Ivana Varenina a, Zorka Knezˇevic´ a, Miroslav Benic´ b a b
Laboratory for Residue Control, Department for Veterinary Public Health, Croatian Veterinary Institute, Savska cesta 143, HR-10000 Zagreb, Croatia Laboratory for Mastitis and Raw Milk Quality, Department for Bacteriology and Parasitology, Croatian Veterinary Institute, Savska cesta 143, HR-10000 Zagreb, Croatia
a r t i c l e
i n f o
Article history: Received 12 July 2010 Received in revised form 7 December 2010 Accepted 19 December 2010 Available online 23 December 2010 Keywords: Milk Croatia Trace metals Atomic absorption spectroscopy
a b s t r a c t A total of 157 raw milk samples were collected from tankers arriving at processing facilities from rural areas in northern and southern regions of Croatia during 2009 and 2010. Concentrations of As, Cd, Cu, Hg and Pb in the samples were analysed by graphite furnace-atomic absorption spectroscopy. Mean Pb concentrations in northern and southern regions were 58.7 and 36.2 lg l 1, respectively, and both exceeded the maximum recommended level. Arsenic concentrations ranged from 1 to 283 lg l 1 in the southern and to 1019 lg l 1 in the northern regions. Mean Cd and Hg levels were: 1.76 and 1.59 in the northern and 3.4 and 7.1 lg l 1 in the southern region. Significantly higher Cd and Hg levels were observed in the southern than in the northern region (p < 0.001, both). Similar mean Cu levels were found in both regions: 931.9 in the north and 848.4 lg l 1 in the south. The results indicate that particular attention should be paid to Pb residues. In future studies, a greater number of milk samples and grass samples from pastures from different regions of Croatia should be controlled to confirm the absence of possible toxicological risks. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction Various industrial environmental contaminations of soil, atmosphere and waters, foods and plants with toxic metals cause their incorporation into the food chain. In regions with metallurgic, petrochemical and fertiliser industries, high levels of As, Cd, Cu, Hg and Pb have been determined in the air (Pacyna & Winchester, 1990; Sanchez de la Campa et al., 2008). Fodder grown on contaminated soils with lead and cadmium will accumulate these metals, and high levels were observed in both fodder and soil samples at polluted localities (Patra, Swarup, Sharma, & Naresh, 2007; Patra et al., 2008; Swarup, Patra, Naresh, Kumar, & Shekhar, 2005). Elevated blood and milk lead and cadmium levels have been reported in lactating cows reared near these localities. Due to their potential toxicity and accumulation, chronic lower level intakes of cadmium and lead have adverse affects on human health and also on domestic and wild animal health (Ikeda et al., 2000; Satarug et al., 2003). Milk is an essential dietary component for infants, as its minerals and proteins are essential for the growth and maintenance of humans and animals. It is important that milk is free of noxious xenobiotic substances such as, medicines, dioxins, pesticides, chemicals, toxic metals and all other kind of environmental contaminants. It is, therefore, imperative that the presence of heavy metals in milk be controlled in accordance with the defined maximum residue levels set by the EU, or to compare data with ⇑ Corresponding author. Tel.: +385 1 612 3601; fax: +385 1 612 3636. E-mail address: [email protected] (N. Bilandzˇic´). 0308-8146/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2010.12.084
the literature. Heavy metal contamination in milk has been reported in different countries and regions (Baldini, Coni, Stacchini, & Stacchini, 1990; Caggiano et al., 2005; Licata et al., 2004; Patra et al., 2008; Simsek, Gultekin, Oksuz, & Kurultay, 2000; Soares et al., 2010; Tajkarimi et al., 2008). There is no increased industrialisation in Croatia. However, due to increased urban, agricultural and industrial emissions of metals reported in other countries (Hellström, Persson, Brudin, Öborn, & Järup, 2007; Patra et al., 2008; Reglero, Monsalve-González, Taggart, & Mateo, 2008; Swarup et al., 2007), it thus becomes necessary to assess the levels of heavy metal concentrations in animal tissues and milk. There is little data on toxic metal concentrations in raw milk in Croatia. There are even fewer reports comparing metal contamination levels in milk originating from different regions in Croatia. The northern regions are more populated and urbanised than southern regions near the Adriatic Sea, which are characterised by varying ecological features. Therefore, the present study was proposed to assess the concentrations of As, Cd, Cu, Hg and Pb residues in cattle milk from dairy farms from different areas in Croatia for the purpose of safeguarding human and animal health. 2. Materials and methods 2.1. Sample collection A total of 157 raw milk samples (500 ml) were collected from tankers arriving at processing facilities from rural areas from southern and northern counties of Croatia during 2009 and 2010.
N. Bilandzˇic´ et al. / Food Chemistry 127 (2011) 63–66
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Upon collection, all the samples were placed in clean, acid-washed polyethylene bottles, labelled and stored at 18 °C until analysis. 2.2. Reagents and standards Reagents HNO3 and HCI used were of analytical-reagent grade (Analytical Grade, Kemika, Croatia). Double deionised water (Milli-Q Millipore, 18.2 MX cm resistivity) was used for all dilutions. Plastic and glassware were cleaned by soaking in diluted HNO3 (1:9, v/v) and by subsequent rinsing with double deionised water and drying prior to use. Calibrations were prepared with elemental standard solutions of 1 g l 1 of each element supplied by Perkin Elmer. Stock solution was diluted in HNO3 (0.2%). In the preparation of Hg working standards, 1 ml of HNO3 conc., 0.1 ml 10% K2Cr2O7 and 0.1 ml HCl conc. were added to all working standards and prepared in brown glass volumetric flasks. As matrix modifiers in each atomisation for As, Cd, Cu and Pb, 0.005 mg Pd(NO3)2 and 0.003 mg Mg(NO3)2 (Perkin Elmer, USA) were used. 2.3. Sample preparation Samples (0.5 ml) were accurately weighed in a PFA digestion vessel, before the addition of 1 ml of double deionised water and 6 ml HNO3 (65% v/v). A blank digest was carried out in the same way. The Multiwave 3000 microwave closed system (Anton Paar, Germany) was used for sample digestion. The digestion programme began at a power of 800 W then ramped for 15 min, after which samples were held at 800 W for 15 min. The second step began at a power of 0 W and held for 15 min. Each digested sample was diluted to a final volume of 50 ml with double deionised water. Detection limits were determined as the concentration corresponding to three times the standard deviation of ten blanks. All specimens were run in batches that included blanks, a standard calibration curve, two spiked specimens and one duplicate. The quality of data was checked by the analysis of recovery rate using certified reference materials: mussel tissue (ERM-CE278, IRMM, Belgium) and skim milk powder (BCR-151, IRMM, Belgium). 2.4. Analysis of metals The analyses of As, Cd, Cu and Pb were conducted using a graphite spectrometer with an AAnalyst 800 (Perkin Elmer, USA) equipped with an AS 800 autosampler (Perkin Elmer, USA). For
graphite furnace measurements, argon was used as the inert gas. Pyrolytically coated graphite tubes with a platform were used. Measurements (integrated absorbance peak areas) were carried out by using single element hollow lamps. Mercury in milk samples were quantified without acid digestion using the AMA-254 (Advanced Mercury Analyzer, Leco, Poland), which employs direct combustion of the sample in an oxygen-rich atmosphere. The instrumental settings and optimising temperature programmes of the spectrometer and mercury analyser are summarised in Table 1. 2.5. Data analysis Concentrations were expressed as mean ± standard deviation, minimum and maximum values. The data was analysed using one-way analysis of variance (ANOVA) to examine statistical significance of differences in the mean concentration of As, Cd, Cu, Hg and Pb, determined in milk samples. A probability level of p 6 0.05 was considered statistically significant. All calculations and statistical analysis were performed with Statistica 6.1 (StatSoftÒ Inc., USA). 3. Results and discussion The accuracy of results was checked by analysing standard reference materials (Table 2) and showed good accuracy, with recovery rates for metals of 96.4–99.3% for muscle tissue (ERM-CE278, IRMM, Belgium) and 96.8–98.9% for skim milk powder (BCR-151, IRMM, Belgium). The limits of detection (LODs, lg l 1) in milk were found to be: As 5.0, Cd 0.4, Cu 0.5, Hg 0.5 and Pb 4.0. The concentrations of five metals in milk samples from northern and southern regions of Croatia are reported in Tables 3. The occurrence of trace elements in different concentration ranges in milk samples are presented in Table 4. Maximum residue levels for trace elements in milk, with the exception of arsenic (100 lg kg 1 w/w) and lead (20 lg kg 1 w/ w; European Commission, 2006), have not yet been established. In the present study, the particular concern is that measured mean lead concentrations exceed the maximum residue levels in both regions (58.7 and 36.2 lg l 1) of Croatia. Levels above 20 lg l 1 were measured in 35.5% of samples from the north and 28.3% of samples from the south regions. There were no significant differences in lead levels between the two regions. Furthermore, observed concentrations were similar to levels found in milk samples in
Table 1 Instrumental parameters for atomic absorption spectrometry and mercury analyser and graphite furnace programme (temperature and time) for Pb, Cd, Cu, As and Hg determination in raw milk samples. Conditions for graphite furnace-atomic absorption spectrometry Lead
Cadmium
Copper
Arsenic
Wavelength (nm) Argon flow (ml min 1) Sample volume (ll) Modifier volume (ll)
228.8 250 20 5
324.8 250 20 5
193.7 250 20 5
110 (1, 30) 130 (15, 30) 700 (10, 20) 1550 (0, 5) 2450 (1, 3)
110 (1, 30) 130 (15, 30) 1200 (10, 20) 2000 (0, 5) 2450 (1, 3)
110 (1, 30) 130 (15, 30) 1600 (10, 20) 2000 (0, 5) 2450 (1, 3)
283.3 250 20 5
Heating programme temperature °C (ramp time (s), hold time (s)) Drying 1 110 (1, 30) Drying 2 130 (15, 30) Ashing 900 (10, 20) Atomisation 1850 (0, 5) Cleaning 2450 (1, 3) Conditions for determination on mercury analyser Wavelength (nm) Drying time (s) Decomposition time (s) Wait time (s) Weight (mg)/volume of sample (ml) Working range (ng)
253.65 60 150 45 100/100 0.05–600
N. Bilandzˇic´ et al. / Food Chemistry 127 (2011) 63–66 Table 2 Trace metal concentrations in two certified references materials. Certified references material
Element Certified value (lg kg 1)
BCR-151 (skim milk powder)
Cd Cu Hg Pb As Cd Cu Hg Pb
ERM-CE278 (mussel tissue)
Measured value (lg kg 1) (n = 5)
101 ± 8 99.5 ± 3.61 5.23 ± 0.08 5.11 ± 0.312 101 ± 10 97.8 ± 4.21 2.002 ± 0.026 1.98 ± 0.198 6.07 5.87 ± 0.211 0.348 0.341 ± 0.051 9.45 9.38 ± 0.231 0.196 0.189 ± 0.012 2.00 1.95 ± 0.211
Recovery (%)
98.5 97.7 96.8 98.9 96.6 97.9 99.3 96.4 97.5
Table 3 Concentrations of trace elements in raw cow’s milk samples from southern and northern regions of Croatia. Element
Region
Number of samples
Mean (lg l 1)
Range (min–max) (lg l 1)
SD
As
Northern Southern Northern Southern Northern Southern Northern Southern Northern Southern
90 67 90 67 90 67 90 67 90 67
18.5 43.5 1.76* 3.40* 931.9 848.4 1.59* 7.10* 58.7 36.2
1.0–283.0 1.0–1019.0 1.0–11.0 1.0–20.0 2.0–17,077 1.0–3773.0 1.0–9.0 1.0–90.0 1.0–370.0 1.0–476.0
38.9 131.6 1.94 3.89 2448.0 1190.1 1.45 16.6 82.9 72.3
Cd Cu Hg Pb
*
Significant difference between two regions: p < 0.001.
Table 4 Frequency distribution of trace elements in milk samples in two regions in Croatia. Element
As Cd Cu Hg Pb
Concentration range (lg l 1)
Number of samples in range Northern region (n = 90)
Southern region (n = 67)
1–100 >100 1–5 >5 10–100 >100 1–5 >5 10–20 >20
88 2 84 6 72 18 87 3 58 32
61 6 54 13 49 18 55 12 48 19
industrial (0.049 mg kg 1) and traffic intensive regions (0.032 mg kg 1) from Turkey (Simsek et al., 2000). However, concentrations found were higher than those found in milk from different regions of Iran in the range from 1 to 46 lg l 1, with average value of 7.9 lg l 1 (Tajkarimi et al., 2008), southern Poland (10–18 lg kg l 1; Krelowska-Kulas, Kedzior, & Popek, 1999), Italy (0.2–1.19 lg kg 1; Caggiano et al., 2005; Licata et al., 2004), Spain (1.8 lg kg l 1 and 5.23 lg l 1; Martino, Sanchez, & Sanz-Medel, 2001; Sola-Larranaga & Navarro-Blasco, 2009) and South Africa (8.00–19.7 lg kg l 1; Ataro, McCrindle, Botha, McCrindle, & Ndibewu, 2008). Previous studies suggest the importance of assessing lead in animals reared in the vicinity of polluted areas as its presence in milk is due to various factors such as, different industrial activities, climatic factors, contaminated agricultural water for irrigation, accumulation along roads and motorways, and the use of pesticide compounds (Licata et al., 2004; Tajkarimi et al., 2008). Higher lead levels in milk were reported in an unpolluted area (0.25 mg l 1) and around plant and smelter areas in India
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(0.65–0.85 mg l 1; Patra et al., 2008), and also in Brazil (0.23 mg l 1; Soares et al., 2010) and Pakistan (21.78 and 15.96 mg l 1; Javed et al., 2009). Arsenic has been the only human carcinogen with registered evidence of carcinogenic risk by both inhalation and ingestion (Bhattacharya et al., 2007) and has been connected with certain types of cancer, including lung, liver, skin and bladder cancer in humans (Kapaj, Peterson, Liber, & Bhattacharya, 2006). It is also directly toxic and is accumulative. In the present study, arsenic concentrations ranged from minimum values of 1 to 283 lg l 1 in the south and 1019 lg l 1 in the north region and there were no significant differences in arsenic levels between the regions. Arsenic concentrations exceeding the maximum residue levels were found in only 2% of samples from the northern and 9% of samples from the southern region. However, mean arsenic levels obtained (18.5 and 43.5 lg l 1) were lower than results reported in milk from Calabria, Italy (0.242–0.684 mg kg 1; Licata et al., 2004). The authors indicated that observed high arsenic levels could be due to the use of pesticides and environmental disinfectants (arsenical compounds) used in observed farmland areas (Licata et al., 2004). Significant differences in arsenic levels between traffic and industrial regions (0.05 and 0.04 mg kg 1) and rural regions (0.0002 mg kg 1) were reported in Turkey (Simsek et al., 2000). Cadmium concentrations in milk vary greatly depending on the type of food and cadmium load in the food production environment (Olsson et al., 2002). A recent study suggested that the main inputs of cadmium to animal feed in farmed animals are feeding crops, trace element premixes, fish meal, and minerals such as, limestone and phosphate (Tu, Han, Xu, Wang, & Li, 2007). Cadmium concentrations measured in the present study ranged from 1 to 20 lg l 1, and more than 80% of samples were below 5 lg l 1 in both regions. Mean cadmium levels were significantly higher in the south than the north region of Croatia (p < 0.001). However, cadmium concentrations measured in milk in different countries were: below detection level of 0.006 lg kg 1 in different farms from South Africa (Ataro et al., 2008), 1–6 lg kg 1 in southern Poland (Krelowska-Kulas et al., 1999), 0.01–22.80 lg kg 1 in Calabria, Italy (Licata et al., 2004), and 0.47 lg kg 1 and 0.40 lg l 1 in Spain (Martino et al., 2001; Sola-Larran´aga & Navarro-Blasco, 2009). In contrast, levels found in the present study were lower in comparison with milk from unpolluted areas (0.033 mg l 1) and regions around plants and smelters (0.057–0.265 mg l 1) in India (Patra et al., 2008) and levels of 0.089 and 0.062 mg l 1 in Pakistan (Javed et al., 2009). There are limited data on mercury residues in milk in comparison with other trace metals, especially lead and cadmium. The most important anthropogenic sources of mercury pollution in the environment are mining and combustion, agricultural materials, industrial and urban discharges (Zhang & Wong, 2007). Mercury is one of the most toxic heavy metals in our environment and it is a well-known fact that the general population is most commonly exposed to mercury by eating fish and marine mammals that may contain methyl–mercury in tissues (Castro-Gonzáleza & Méndez-Armenta, 2008). It has been suggested that fungicidal treatment with organomercurial seed dressings have traditionally been a major source of mercury for farm animals, whilst the recent practice of adding fish meal to feed is the main source of mercury for livestock (López-Alonso et al., 2007). Mercury concentrations measured in the present study ranged from 1 to 9 lg l 1 in the north and 1 to 90 lg l 1 in the south regions. Levels below 5 lg l 1 were measured in 97% of samples from the north and 82.1% of samples from the south regions. However, mercury levels measured in the south region were significantly higher than those from the north region (p < 0.001). Values observed were higher than those measured (<0.005 mg kg 1) in industrial and rural regions in Turkey (Simsek et al., 2000). However, the levels of mercury reported here are similar to concentra-
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tions found on ovine farms in southern Italy (0.0025 mg kg 1; Caggiano et al., 2005) and milk powder from Brazil (0.0069 mg kg 1; Nardi et al., 2009). In this study, copper levels were also measured. Copper is an essential element required in the diet due to its role in vital oxidation–reduction reactions. At supraoptimal concentrations, copper may generate toxic effects such as, dermatitis, liver cirrhosis and neurological disorders (Storelli, Barone, Garofalo, & Marcotrigiano, 2007). Copper deficiency is a common nutritional problem in ruminants, though Cu excess is also commonly encountered, especially in sheep (Puls, 1994). It is considered that Cu concentrations between 0.1 and 0.9 mg l 1 are the ‘‘normal’’ range in milk (Puls, 1994). Mean copper concentrations presented were 931.9 lg l 1 in the north and 848.4 lg l 1 in the south region. Significant differences in copper levels between regions were not observed. Levels obtained were in line with copper concentrations in industrial (0.96 mg kg 1) and heavy traffic regions (0.58 mg kg 1) reported in Turkey (Simsek et al., 2000). On the other hand, copper levels were higher than those reported in southern Poland (0.2– 0.3 mg kg 1; Krelowska-Kulas et al., 1999), Spain (60 lg kg 1 and 51.8 lg l 1; Martino et al., 2001; Sola-Larranaga & Navarro-Blasco, 2009), Calabria in Italy (0.14–737.58 lg kg 1; Licata et al., 2004) and unpolluted areas of India (0.101 mg l 1; Patra et al., 2008). 4. Conclusions In conclusion, this study presents the first regional study in Croatia and a significant affect of region was noted for cadmium and mercury concentrations in milk samples. Significantly higher cadmium and mercury levels were observed in the southern than in the northern region. Also, of particular concern is the measurement of lead levels above the maximum residue levels in both the regions studied and very high levels of cadmium, arsenic and mercury were measured in some milk samples. This demonstrates that particular attention should be paid to toxic lead residues and a greater number of milk samples and grass samples from pastures in different regions of Croatia should be controlled in future studies to confirm the absence of possible toxicological risks. Acknowledgements The authors are thankful to all those who contributed to the study. Special thanks go to Marijana Fluka and Mirjana Hren for sample preparation. This study was supported with a grant from the Ministry of Agriculture, Fisheries and Rural Development Veterinary Directorate, Croatia. References Ataro, A., McCrindle, R. I., Botha, B. M., McCrindle, C. M. E., & Ndibewu, P. P. (2008). Quantification of trace elements in raw cow’s milk by inductively coupled plasma mass spectrometry (ICP-MS). Food Chemistry, 111, 243–248. Baldini, M., Coni, E., Stacchini, A., & Stacchini, P. (1990). Presence and assessment of xenobiotic substances in milk and dairy products. Annali dell’Istituto Superiore di Sanità, 26, 167–176. Bhattacharya, P., Welch, A. H., Stollenwerk, K. G., McLaughlin, M. J., Bundschuh, J., & Panaullah, G. (2007). Arsenic in the environment: Biology and Chemistry. The Science of the Total Environment, 379, 109–120. Caggiano, R., Sabia, S., D’Emilio, M., Macchiato, M., Anastasio, A., Ragosta, M., et al. (2005). Metal levels in fodder, milk, dairy products, and tissues sampled in ovine farms of Southern Italy. Environmental Research, 99, 48–57. European Commission. (2006). European commission, regulation (EC) no. 1881 (2006): 19 of December 2006, setting maximum levels for certain contaminants in foodstuffs. Official Journal, L364, 5–24. Castro-Gonzáleza, M. I., & Méndez-Armenta, M. (2008). Heavy metals: Implications associated to fish consumption. Environmental Toxicology and Pharmacology, 26, 263–271.
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Trace element levels in raw milk from northern and southern regions of Croatia Nina Bilandzˇic´ a,⇑, Maja Ðokic´ a, Marija Sedak a, Bozˇica Solomun a, Ivana Varenina a, Zorka Knezˇevic´ a, Miroslav Benic´ b a b
Laboratory for Residue Control, Department for Veterinary Public Health, Croatian Veterinary Institute, Savska cesta 143, HR-10000 Zagreb, Croatia Laboratory for Mastitis and Raw Milk Quality, Department for Bacteriology and Parasitology, Croatian Veterinary Institute, Savska cesta 143, HR-10000 Zagreb, Croatia
a r t i c l e
i n f o
Article history: Received 12 July 2010 Received in revised form 7 December 2010 Accepted 19 December 2010 Available online 23 December 2010 Keywords: Milk Croatia Trace metals Atomic absorption spectroscopy
a b s t r a c t A total of 157 raw milk samples were collected from tankers arriving at processing facilities from rural areas in northern and southern regions of Croatia during 2009 and 2010. Concentrations of As, Cd, Cu, Hg and Pb in the samples were analysed by graphite furnace-atomic absorption spectroscopy. Mean Pb concentrations in northern and southern regions were 58.7 and 36.2 lg l 1, respectively, and both exceeded the maximum recommended level. Arsenic concentrations ranged from 1 to 283 lg l 1 in the southern and to 1019 lg l 1 in the northern regions. Mean Cd and Hg levels were: 1.76 and 1.59 in the northern and 3.4 and 7.1 lg l 1 in the southern region. Significantly higher Cd and Hg levels were observed in the southern than in the northern region (p < 0.001, both). Similar mean Cu levels were found in both regions: 931.9 in the north and 848.4 lg l 1 in the south. The results indicate that particular attention should be paid to Pb residues. In future studies, a greater number of milk samples and grass samples from pastures from different regions of Croatia should be controlled to confirm the absence of possible toxicological risks. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction Various industrial environmental contaminations of soil, atmosphere and waters, foods and plants with toxic metals cause their incorporation into the food chain. In regions with metallurgic, petrochemical and fertiliser industries, high levels of As, Cd, Cu, Hg and Pb have been determined in the air (Pacyna & Winchester, 1990; Sanchez de la Campa et al., 2008). Fodder grown on contaminated soils with lead and cadmium will accumulate these metals, and high levels were observed in both fodder and soil samples at polluted localities (Patra, Swarup, Sharma, & Naresh, 2007; Patra et al., 2008; Swarup, Patra, Naresh, Kumar, & Shekhar, 2005). Elevated blood and milk lead and cadmium levels have been reported in lactating cows reared near these localities. Due to their potential toxicity and accumulation, chronic lower level intakes of cadmium and lead have adverse affects on human health and also on domestic and wild animal health (Ikeda et al., 2000; Satarug et al., 2003). Milk is an essential dietary component for infants, as its minerals and proteins are essential for the growth and maintenance of humans and animals. It is important that milk is free of noxious xenobiotic substances such as, medicines, dioxins, pesticides, chemicals, toxic metals and all other kind of environmental contaminants. It is, therefore, imperative that the presence of heavy metals in milk be controlled in accordance with the defined maximum residue levels set by the EU, or to compare data with ⇑ Corresponding author. Tel.: +385 1 612 3601; fax: +385 1 612 3636. E-mail address: [email protected] (N. Bilandzˇic´). 0308-8146/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2010.12.084
the literature. Heavy metal contamination in milk has been reported in different countries and regions (Baldini, Coni, Stacchini, & Stacchini, 1990; Caggiano et al., 2005; Licata et al., 2004; Patra et al., 2008; Simsek, Gultekin, Oksuz, & Kurultay, 2000; Soares et al., 2010; Tajkarimi et al., 2008). There is no increased industrialisation in Croatia. However, due to increased urban, agricultural and industrial emissions of metals reported in other countries (Hellström, Persson, Brudin, Öborn, & Järup, 2007; Patra et al., 2008; Reglero, Monsalve-González, Taggart, & Mateo, 2008; Swarup et al., 2007), it thus becomes necessary to assess the levels of heavy metal concentrations in animal tissues and milk. There is little data on toxic metal concentrations in raw milk in Croatia. There are even fewer reports comparing metal contamination levels in milk originating from different regions in Croatia. The northern regions are more populated and urbanised than southern regions near the Adriatic Sea, which are characterised by varying ecological features. Therefore, the present study was proposed to assess the concentrations of As, Cd, Cu, Hg and Pb residues in cattle milk from dairy farms from different areas in Croatia for the purpose of safeguarding human and animal health. 2. Materials and methods 2.1. Sample collection A total of 157 raw milk samples (500 ml) were collected from tankers arriving at processing facilities from rural areas from southern and northern counties of Croatia during 2009 and 2010.
N. Bilandzˇic´ et al. / Food Chemistry 127 (2011) 63–66
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Upon collection, all the samples were placed in clean, acid-washed polyethylene bottles, labelled and stored at 18 °C until analysis. 2.2. Reagents and standards Reagents HNO3 and HCI used were of analytical-reagent grade (Analytical Grade, Kemika, Croatia). Double deionised water (Milli-Q Millipore, 18.2 MX cm resistivity) was used for all dilutions. Plastic and glassware were cleaned by soaking in diluted HNO3 (1:9, v/v) and by subsequent rinsing with double deionised water and drying prior to use. Calibrations were prepared with elemental standard solutions of 1 g l 1 of each element supplied by Perkin Elmer. Stock solution was diluted in HNO3 (0.2%). In the preparation of Hg working standards, 1 ml of HNO3 conc., 0.1 ml 10% K2Cr2O7 and 0.1 ml HCl conc. were added to all working standards and prepared in brown glass volumetric flasks. As matrix modifiers in each atomisation for As, Cd, Cu and Pb, 0.005 mg Pd(NO3)2 and 0.003 mg Mg(NO3)2 (Perkin Elmer, USA) were used. 2.3. Sample preparation Samples (0.5 ml) were accurately weighed in a PFA digestion vessel, before the addition of 1 ml of double deionised water and 6 ml HNO3 (65% v/v). A blank digest was carried out in the same way. The Multiwave 3000 microwave closed system (Anton Paar, Germany) was used for sample digestion. The digestion programme began at a power of 800 W then ramped for 15 min, after which samples were held at 800 W for 15 min. The second step began at a power of 0 W and held for 15 min. Each digested sample was diluted to a final volume of 50 ml with double deionised water. Detection limits were determined as the concentration corresponding to three times the standard deviation of ten blanks. All specimens were run in batches that included blanks, a standard calibration curve, two spiked specimens and one duplicate. The quality of data was checked by the analysis of recovery rate using certified reference materials: mussel tissue (ERM-CE278, IRMM, Belgium) and skim milk powder (BCR-151, IRMM, Belgium). 2.4. Analysis of metals The analyses of As, Cd, Cu and Pb were conducted using a graphite spectrometer with an AAnalyst 800 (Perkin Elmer, USA) equipped with an AS 800 autosampler (Perkin Elmer, USA). For
graphite furnace measurements, argon was used as the inert gas. Pyrolytically coated graphite tubes with a platform were used. Measurements (integrated absorbance peak areas) were carried out by using single element hollow lamps. Mercury in milk samples were quantified without acid digestion using the AMA-254 (Advanced Mercury Analyzer, Leco, Poland), which employs direct combustion of the sample in an oxygen-rich atmosphere. The instrumental settings and optimising temperature programmes of the spectrometer and mercury analyser are summarised in Table 1. 2.5. Data analysis Concentrations were expressed as mean ± standard deviation, minimum and maximum values. The data was analysed using one-way analysis of variance (ANOVA) to examine statistical significance of differences in the mean concentration of As, Cd, Cu, Hg and Pb, determined in milk samples. A probability level of p 6 0.05 was considered statistically significant. All calculations and statistical analysis were performed with Statistica 6.1 (StatSoftÒ Inc., USA). 3. Results and discussion The accuracy of results was checked by analysing standard reference materials (Table 2) and showed good accuracy, with recovery rates for metals of 96.4–99.3% for muscle tissue (ERM-CE278, IRMM, Belgium) and 96.8–98.9% for skim milk powder (BCR-151, IRMM, Belgium). The limits of detection (LODs, lg l 1) in milk were found to be: As 5.0, Cd 0.4, Cu 0.5, Hg 0.5 and Pb 4.0. The concentrations of five metals in milk samples from northern and southern regions of Croatia are reported in Tables 3. The occurrence of trace elements in different concentration ranges in milk samples are presented in Table 4. Maximum residue levels for trace elements in milk, with the exception of arsenic (100 lg kg 1 w/w) and lead (20 lg kg 1 w/ w; European Commission, 2006), have not yet been established. In the present study, the particular concern is that measured mean lead concentrations exceed the maximum residue levels in both regions (58.7 and 36.2 lg l 1) of Croatia. Levels above 20 lg l 1 were measured in 35.5% of samples from the north and 28.3% of samples from the south regions. There were no significant differences in lead levels between the two regions. Furthermore, observed concentrations were similar to levels found in milk samples in
Table 1 Instrumental parameters for atomic absorption spectrometry and mercury analyser and graphite furnace programme (temperature and time) for Pb, Cd, Cu, As and Hg determination in raw milk samples. Conditions for graphite furnace-atomic absorption spectrometry Lead
Cadmium
Copper
Arsenic
Wavelength (nm) Argon flow (ml min 1) Sample volume (ll) Modifier volume (ll)
228.8 250 20 5
324.8 250 20 5
193.7 250 20 5
110 (1, 30) 130 (15, 30) 700 (10, 20) 1550 (0, 5) 2450 (1, 3)
110 (1, 30) 130 (15, 30) 1200 (10, 20) 2000 (0, 5) 2450 (1, 3)
110 (1, 30) 130 (15, 30) 1600 (10, 20) 2000 (0, 5) 2450 (1, 3)
283.3 250 20 5
Heating programme temperature °C (ramp time (s), hold time (s)) Drying 1 110 (1, 30) Drying 2 130 (15, 30) Ashing 900 (10, 20) Atomisation 1850 (0, 5) Cleaning 2450 (1, 3) Conditions for determination on mercury analyser Wavelength (nm) Drying time (s) Decomposition time (s) Wait time (s) Weight (mg)/volume of sample (ml) Working range (ng)
253.65 60 150 45 100/100 0.05–600
N. Bilandzˇic´ et al. / Food Chemistry 127 (2011) 63–66 Table 2 Trace metal concentrations in two certified references materials. Certified references material
Element Certified value (lg kg 1)
BCR-151 (skim milk powder)
Cd Cu Hg Pb As Cd Cu Hg Pb
ERM-CE278 (mussel tissue)
Measured value (lg kg 1) (n = 5)
101 ± 8 99.5 ± 3.61 5.23 ± 0.08 5.11 ± 0.312 101 ± 10 97.8 ± 4.21 2.002 ± 0.026 1.98 ± 0.198 6.07 5.87 ± 0.211 0.348 0.341 ± 0.051 9.45 9.38 ± 0.231 0.196 0.189 ± 0.012 2.00 1.95 ± 0.211
Recovery (%)
98.5 97.7 96.8 98.9 96.6 97.9 99.3 96.4 97.5
Table 3 Concentrations of trace elements in raw cow’s milk samples from southern and northern regions of Croatia. Element
Region
Number of samples
Mean (lg l 1)
Range (min–max) (lg l 1)
SD
As
Northern Southern Northern Southern Northern Southern Northern Southern Northern Southern
90 67 90 67 90 67 90 67 90 67
18.5 43.5 1.76* 3.40* 931.9 848.4 1.59* 7.10* 58.7 36.2
1.0–283.0 1.0–1019.0 1.0–11.0 1.0–20.0 2.0–17,077 1.0–3773.0 1.0–9.0 1.0–90.0 1.0–370.0 1.0–476.0
38.9 131.6 1.94 3.89 2448.0 1190.1 1.45 16.6 82.9 72.3
Cd Cu Hg Pb
*
Significant difference between two regions: p < 0.001.
Table 4 Frequency distribution of trace elements in milk samples in two regions in Croatia. Element
As Cd Cu Hg Pb
Concentration range (lg l 1)
Number of samples in range Northern region (n = 90)
Southern region (n = 67)
1–100 >100 1–5 >5 10–100 >100 1–5 >5 10–20 >20
88 2 84 6 72 18 87 3 58 32
61 6 54 13 49 18 55 12 48 19
industrial (0.049 mg kg 1) and traffic intensive regions (0.032 mg kg 1) from Turkey (Simsek et al., 2000). However, concentrations found were higher than those found in milk from different regions of Iran in the range from 1 to 46 lg l 1, with average value of 7.9 lg l 1 (Tajkarimi et al., 2008), southern Poland (10–18 lg kg l 1; Krelowska-Kulas, Kedzior, & Popek, 1999), Italy (0.2–1.19 lg kg 1; Caggiano et al., 2005; Licata et al., 2004), Spain (1.8 lg kg l 1 and 5.23 lg l 1; Martino, Sanchez, & Sanz-Medel, 2001; Sola-Larranaga & Navarro-Blasco, 2009) and South Africa (8.00–19.7 lg kg l 1; Ataro, McCrindle, Botha, McCrindle, & Ndibewu, 2008). Previous studies suggest the importance of assessing lead in animals reared in the vicinity of polluted areas as its presence in milk is due to various factors such as, different industrial activities, climatic factors, contaminated agricultural water for irrigation, accumulation along roads and motorways, and the use of pesticide compounds (Licata et al., 2004; Tajkarimi et al., 2008). Higher lead levels in milk were reported in an unpolluted area (0.25 mg l 1) and around plant and smelter areas in India
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(0.65–0.85 mg l 1; Patra et al., 2008), and also in Brazil (0.23 mg l 1; Soares et al., 2010) and Pakistan (21.78 and 15.96 mg l 1; Javed et al., 2009). Arsenic has been the only human carcinogen with registered evidence of carcinogenic risk by both inhalation and ingestion (Bhattacharya et al., 2007) and has been connected with certain types of cancer, including lung, liver, skin and bladder cancer in humans (Kapaj, Peterson, Liber, & Bhattacharya, 2006). It is also directly toxic and is accumulative. In the present study, arsenic concentrations ranged from minimum values of 1 to 283 lg l 1 in the south and 1019 lg l 1 in the north region and there were no significant differences in arsenic levels between the regions. Arsenic concentrations exceeding the maximum residue levels were found in only 2% of samples from the northern and 9% of samples from the southern region. However, mean arsenic levels obtained (18.5 and 43.5 lg l 1) were lower than results reported in milk from Calabria, Italy (0.242–0.684 mg kg 1; Licata et al., 2004). The authors indicated that observed high arsenic levels could be due to the use of pesticides and environmental disinfectants (arsenical compounds) used in observed farmland areas (Licata et al., 2004). Significant differences in arsenic levels between traffic and industrial regions (0.05 and 0.04 mg kg 1) and rural regions (0.0002 mg kg 1) were reported in Turkey (Simsek et al., 2000). Cadmium concentrations in milk vary greatly depending on the type of food and cadmium load in the food production environment (Olsson et al., 2002). A recent study suggested that the main inputs of cadmium to animal feed in farmed animals are feeding crops, trace element premixes, fish meal, and minerals such as, limestone and phosphate (Tu, Han, Xu, Wang, & Li, 2007). Cadmium concentrations measured in the present study ranged from 1 to 20 lg l 1, and more than 80% of samples were below 5 lg l 1 in both regions. Mean cadmium levels were significantly higher in the south than the north region of Croatia (p < 0.001). However, cadmium concentrations measured in milk in different countries were: below detection level of 0.006 lg kg 1 in different farms from South Africa (Ataro et al., 2008), 1–6 lg kg 1 in southern Poland (Krelowska-Kulas et al., 1999), 0.01–22.80 lg kg 1 in Calabria, Italy (Licata et al., 2004), and 0.47 lg kg 1 and 0.40 lg l 1 in Spain (Martino et al., 2001; Sola-Larran´aga & Navarro-Blasco, 2009). In contrast, levels found in the present study were lower in comparison with milk from unpolluted areas (0.033 mg l 1) and regions around plants and smelters (0.057–0.265 mg l 1) in India (Patra et al., 2008) and levels of 0.089 and 0.062 mg l 1 in Pakistan (Javed et al., 2009). There are limited data on mercury residues in milk in comparison with other trace metals, especially lead and cadmium. The most important anthropogenic sources of mercury pollution in the environment are mining and combustion, agricultural materials, industrial and urban discharges (Zhang & Wong, 2007). Mercury is one of the most toxic heavy metals in our environment and it is a well-known fact that the general population is most commonly exposed to mercury by eating fish and marine mammals that may contain methyl–mercury in tissues (Castro-Gonzáleza & Méndez-Armenta, 2008). It has been suggested that fungicidal treatment with organomercurial seed dressings have traditionally been a major source of mercury for farm animals, whilst the recent practice of adding fish meal to feed is the main source of mercury for livestock (López-Alonso et al., 2007). Mercury concentrations measured in the present study ranged from 1 to 9 lg l 1 in the north and 1 to 90 lg l 1 in the south regions. Levels below 5 lg l 1 were measured in 97% of samples from the north and 82.1% of samples from the south regions. However, mercury levels measured in the south region were significantly higher than those from the north region (p < 0.001). Values observed were higher than those measured (<0.005 mg kg 1) in industrial and rural regions in Turkey (Simsek et al., 2000). However, the levels of mercury reported here are similar to concentra-
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tions found on ovine farms in southern Italy (0.0025 mg kg 1; Caggiano et al., 2005) and milk powder from Brazil (0.0069 mg kg 1; Nardi et al., 2009). In this study, copper levels were also measured. Copper is an essential element required in the diet due to its role in vital oxidation–reduction reactions. At supraoptimal concentrations, copper may generate toxic effects such as, dermatitis, liver cirrhosis and neurological disorders (Storelli, Barone, Garofalo, & Marcotrigiano, 2007). Copper deficiency is a common nutritional problem in ruminants, though Cu excess is also commonly encountered, especially in sheep (Puls, 1994). It is considered that Cu concentrations between 0.1 and 0.9 mg l 1 are the ‘‘normal’’ range in milk (Puls, 1994). Mean copper concentrations presented were 931.9 lg l 1 in the north and 848.4 lg l 1 in the south region. Significant differences in copper levels between regions were not observed. Levels obtained were in line with copper concentrations in industrial (0.96 mg kg 1) and heavy traffic regions (0.58 mg kg 1) reported in Turkey (Simsek et al., 2000). On the other hand, copper levels were higher than those reported in southern Poland (0.2– 0.3 mg kg 1; Krelowska-Kulas et al., 1999), Spain (60 lg kg 1 and 51.8 lg l 1; Martino et al., 2001; Sola-Larranaga & Navarro-Blasco, 2009), Calabria in Italy (0.14–737.58 lg kg 1; Licata et al., 2004) and unpolluted areas of India (0.101 mg l 1; Patra et al., 2008). 4. Conclusions In conclusion, this study presents the first regional study in Croatia and a significant affect of region was noted for cadmium and mercury concentrations in milk samples. Significantly higher cadmium and mercury levels were observed in the southern than in the northern region. Also, of particular concern is the measurement of lead levels above the maximum residue levels in both the regions studied and very high levels of cadmium, arsenic and mercury were measured in some milk samples. This demonstrates that particular attention should be paid to toxic lead residues and a greater number of milk samples and grass samples from pastures in different regions of Croatia should be controlled in future studies to confirm the absence of possible toxicological risks. Acknowledgements The authors are thankful to all those who contributed to the study. Special thanks go to Marijana Fluka and Mirjana Hren for sample preparation. This study was supported with a grant from the Ministry of Agriculture, Fisheries and Rural Development Veterinary Directorate, Croatia. References Ataro, A., McCrindle, R. I., Botha, B. M., McCrindle, C. M. E., & Ndibewu, P. P. (2008). Quantification of trace elements in raw cow’s milk by inductively coupled plasma mass spectrometry (ICP-MS). Food Chemistry, 111, 243–248. Baldini, M., Coni, E., Stacchini, A., & Stacchini, P. (1990). Presence and assessment of xenobiotic substances in milk and dairy products. Annali dell’Istituto Superiore di Sanità, 26, 167–176. Bhattacharya, P., Welch, A. H., Stollenwerk, K. G., McLaughlin, M. J., Bundschuh, J., & Panaullah, G. (2007). Arsenic in the environment: Biology and Chemistry. The Science of the Total Environment, 379, 109–120. Caggiano, R., Sabia, S., D’Emilio, M., Macchiato, M., Anastasio, A., Ragosta, M., et al. (2005). Metal levels in fodder, milk, dairy products, and tissues sampled in ovine farms of Southern Italy. Environmental Research, 99, 48–57. European Commission. (2006). European commission, regulation (EC) no. 1881 (2006): 19 of December 2006, setting maximum levels for certain contaminants in foodstuffs. Official Journal, L364, 5–24. Castro-Gonzáleza, M. I., & Méndez-Armenta, M. (2008). Heavy metals: Implications associated to fish consumption. Environmental Toxicology and Pharmacology, 26, 263–271.
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