doi: 10.1111/j.1471-0307.2012.00846.x ORIGINAL RESEARCH Heavy metal concentrations in raw milk collected from different regions of Samsun, Turkey HASAN TEMIZ* and ARZU SOYLU Department of Food Engineering, Engineering Faculty, University of Ondokuz Mayis, TR-55139 Samsun, Turkey In this study, 144 raw milk samples were analysed for heavy metal contamination derived from emissions from industrial operations in Tekkekoy, Samsun, Turkey. Cu, Fe, Zn, Cr, Ni, Cd, As and Pb levels in samples were determined by inductively coupled plasma mass spectrometry (ICP-MS). The average amounts of copper, chromium, iron, zinc, nickel, cadmium, arsenic and lead were determined as 1.130, 0.441, 12.920, 0.032, 0.483, 0.006, 0.003 and 0.004 mg ⁄ kg, respectively. It was determined that the summer period has the highest levels for copper, lead and cadmium. The highest contents of arsenic and copper were found at the two industrial regions that were close to Black Sea, described as ‘1st’ and ‘2nd’ region. Whereas the 3rd and 4th regions that were far from an industrial zone and also from the Black Sea, nickel, lead and chromium had the highest levels. Keywords Heavy metals, ICP- MS, Milk, Milk contaminants. INTRODUCTION *Author for correspondence. E-mail: hasant@omu.edu.tr 2012 Society of Dairy Technology Rapid urbanisation and industrial development have caused environmental pollution all over the world. As a result, many dangerous elements or compounds, such as dioxins, pesticides, metals and metalloids, accumulate along the food chain. Their concentrations in the environment grow with the increase in urban, agricultural and industrial emissions (Rubio et al. 1998; Zheng et al. 2007). Heavy metals are invaluable and unavoidable components of our environment (Vidovic et al. 2005). They are in varying quantities throughout the geosphere and are being cycled continuously through different components of the ecosphere. The amounts of different heavy metals in ambient atmospheres have been increasing with advances of human civilisation and are likely to increase further with increasing exploitation of geological resources, such as mining and fossil fuel development. An additional concern with the metals is their concentration in domestic and industrial waste products, because the elements are indestructible (Sharma et al. 1982). Heavy metals may enter to the human body through inhalation of dust, consumption of contaminated drinking water, direct ingestion of soil and consumption of food plants grown in metal-contaminated soil (Chary et al. 2008). Vol 65 International Journal of Dairy Technology Milk and most of the dairy products, which are important parts of human’s diet, are likely to be exposed to heavy metal contamination from ingestion of feed during the lactation period (Simsek et al. 2000). Some heavy metals having toxic effects on human and animal health can accumulate in these products (Dahiya et al. 2005). The toxicity induced by excessive levels of these elements, such as chromium (Cr), lead (Pb) and mercury (Hg), is well known (Anastasio et al. 2006). Heavy metal residues in milk are of particular concern because milk is largely consumed by infants and children. The toxic metal content of milk and dairy products depend on several factors, in particular environmental conditions, the manufacturing process and the possible contamination during several steps of the manufacturing processes. Tekkekoy region is located at eastern part of Samsun Province of Turkey (Figure 1) and has different pollution parameters (high volume of traffic, industrial plants, small-medium towns, intensive agricultural activities). The Gelemen Farm that serves as a source of food for the city is located in Tekkekoy. In this region, there are some researches about heavy metal concentration at soil and vegetables samples, but there is no data about milk and dairy products. The aim of this work was to determine 1 Vol 65 Table 1 Monthly wind situation, Samsuna Study Area Figure 1 Map of Samsun. Months Wind speeds (m ⁄ s) Prevailing direction Maximum speed (m ⁄ s) Strongest direction January February March April May June July August September October November December Yearly 2.3 2.2 2.1 1.4 1.2 1.9 2.3 2.0 1.8 1.4 2.2 2.0 1.9 SSE SSE SSE NNE NNW NNW WNW NNE SW SW WSW WSW NNW 15.1 16.5 19.7 10.7 8.5 10.7 11.1 12.5 12.2 15.6 18.2 14.7 19.7 SSE S S SW WNW WNW WNW N NW NNW SSW SW S a the concentration of some heavy metals in milk, collected from several farms in the Tekkekoy region. Source: Anonymous 2006 (N, north; S, south; SW, south-west; SSE, south-south-east; NNE, north-north-east; NNW, north-north-west; WSW, west-south-west; WNW, west-north-west; NW, north-west; SSW, south-south-west). MATERIALS AND METHODS Site characteristics The study area is located in northern Turkey, at the coast of Black Sea, 4121¢ N latitude and 3615¢ E longitude and surrounded by Carsamba Plain in the east and the south-east. There are small-scale industrial operations and small settlements located in the centre of Samsun Province. 2.km Climate The minimum temperature was measured in January as 2 C, and the maximum temperature was measured in August as 29.9 C. The mean temperature is 14.5 C. The mean annual rainfall is 714.7 mm. Even though changes in wind-direction occur every month, the prevailing wind is from the north and west, as shown in Table 1 (Anonymous 2006). Sampling Milk sampling was carried out at 24 different locations along the south–east of Samsun. The location map of the Tekkekoy with 24 sampling sites is shown in Figure 2. The sampling area started from the seaside and going up to 30 km north. Sampling areas have been selected in relation to the distance from the Black Sea because the industrial zones were located through the seaside. These areas were characterised by different elevations and ecological features. Therefore, the region was divided into four different spots according to their distance: 1st region up to 2 km from seaside, 2nd region between 2 and 5 km, 3rd region between 5 and 15 km and 4th region between 15 and 30 km. The 1st and 2nd regions were close to industrial area and have a low altitude. The 3rd and 4th regions were far from the industrial area according to the other regions and have a high altitude. Between the first and second regions, there is a 2 5.km 15.km 30.km Figure 2 Map of Tekkekoy. highway having a high traffic volume. The heavy metal emissions of some domestic large industrial corporations located at study area are shown in Table 2. 2012 Society of Dairy Technology Vol 65 Table 2 Heavy metal emissions of the industrial branches of Tekkekoy region, Samsuna Metals Petro chemistry Choler-alkali production Fertilizer industry Iron–steel industry Power plant Cd Cr Cu Hg Pb Ni Sn Zn + + ) + + ) + + + + ) + + ) + + + + + + + + ) + + + + + + + + + + + + + + + + + a Source: Kahvecioglu et al. (2009). Sampling was carried out with three replications in two periods: summer (May, July and September of 2010) and winter (November of 2009, January and March of 2010). All the samples were collected according to procedures excluding metallic containers, mechanical milkers, etc. to minimise possible external contaminations. For each sampling location, 10 milk samples were collected from different farms and they were mixed to obtain laboratory sample. So, in all sampling period, 240 milk samples were collected, and totally 1640 milk samples were collected to reach homogenous sample that represents each area. Milk samples were collected from the cattle grazing in pasture land between 6:00 to 10:00 a.m. in clean polyethylene bottles. Each milk sample was stored at )18 C until analysis. Sample preparation Test portions were dried in an oven at 95 C and then ashed at 450 C under a gradual increase (<50 C ⁄ h). 5 mL 6 M HCl (1 + 1) was added to ashed samples, and then the solution was evaporated to dryness. The residue was dissolved in 0.1 M HNO3, and the analyses were performed using ICP-MS (Jorhem 2000). Inductively coupled plasma mass spectrometry (ICP-MS) measurements Inductively coupled plasma mass spectrometry analyses were performed in the Environmental Geochemistry laboratory at the Mersin University Geological Engineering Department, Mersin, Turkey. Concentrations of eight elements (Cu, Fe, Zn, Cr, Ni, Cd, As and Pb) in the solution extracts were determined in triplicate by Agilent 7500ce ICP-MS (Tokyo, Japan) equipped with a collusion ⁄ reaction cell in the form of octopole reaction system (ORS). The argon gas utilised was of spectral purity (99.998%). The external standard calibration method was applied to all determinations, using Li, Sc, Ge, Y, In, Tb and Bi internal standard mix (in 2% HNO3 matrix). NIST single-element reference standards are used to construct five-point calibration curves. At regular intervals during analysis, calibration 2012 Society of Dairy Technology standards were analysed as samples to monitor instrument drift. Furthermore, extractant and ultra pure water blanks were frequently analysed alongside samples to check for contamination. Blanks were prepared by completion of the full analytical procedure without samples. The analytical accuracy was checked from replicate measurement of several samples and by measuring certified reference materials. The relative error is <±5% for all analysed elements (Guler and Alparslan 2009). Statistical analysis The data obtained from three replicates were analysed by ANOVA using the SPSS statistical package program, and the differences between the means were compared using the Duncan’s multiple range test at the significance level of 0.05. RESULTS AND DISCUSSIONS Copper Copper contents of the raw milk samples from different regions are shown in Table 3. According to analysis of variance, the differences between both region and period were statistically significant (P < 0.05). The copper values obtained in this study ranged between 0.618 and 1.889 mg ⁄ kg. The highest copper value was obtained from samples collected from 2nd region at the summer period. The copper value detected from summer period was higher than those of winter period. Herbicide used in agriculture might increase the amount of copper in the milk. So, the copper amount of milk could be increased by both industrial emissions and herbicides at the summer season. The results for the copper concentrations of milk were higher than that reported by Simsek et al. (2000) and by Licata et al. (2004) but lower than that reported by Temurci and Guner (2006). The World Health Organization (FAO ⁄ WHO, 1982) and Food and Nutrition Board (2001) have reported some critical levels for metals that are shown at Table 4 as provisional maximum tolerable daily intake (PMTDI) level, provisional tolerable weekly intake level (PTWI), dietary reference intakes (DRI) and an upper level (UL). While the copper values obtained in this study compared with PMTDI, DRI and UL values, it seems that the results are between the published values, but milk is not the only source of this metal. So, it can be concluded that consuming large amounts of milk obtained from the region may cause toxic effects in humans. Iron The iron concentrations observed in this study varied between 0.12 and 0.64 mg ⁄ kg (Table 3). No statistical difference (P > 0.05) was detected between both region and periods. The iron results of this study were lower than the values reported by Simsek et al. (2000) and by Temurci and Guner (2006). While the copper results obtained in this study are compared with PMTDI, DRI (FAO/WHO, 1983) and UL values (Table 4), it seems that the results are lower than the established values. The 3 ± ± ± ± ± ± ± ± 0.47 0.92 0.04 12.58 0.00 0.01 0.64 0.03 0.62 0.59c 0.03bc 2.96 0.00b 0.00ab 0.55b 0.11a ± ± ± ± ± ± ± ± 0.32 0.63 0.03 12.25 0.00 0.01 0.49 0.07 0.55 1.17 0.05 11.59 0.00 0.01 1.13 0.02 0.44 1.25 0.03 13.80 0.00 0.00 0.44 0.02 ± ± ± ± ± ± ± ± 5–15 km 2–5 km 15–30 km Copper (mg ⁄ kg) DRI UL PMTDI Nickel (mg ⁄ kg) UL Lead (mg ⁄ kg) PTWI Zinc (mg ⁄ kg) DRI PMTDI ± 1.00 ± 0.66abc ± 0.03c ± 1.70 ± 0.00 ±.0.00bc ± 0.45b ± 0.02b 1.53 0.42c 0.02abc 4.56 0.00b 0.00ab 0.29b 0.01b ± ± ± ± ± ± ± ± 0–2 km 0.60 0.62 0.04 12.70 0.00 0.01 0.35 0.01 ± ± ± ± ± ± ± ± Letters indicate difference among period (P < 0.05). Letters indicate difference among region (P < 0.05). * All values are mean ± SD of triplicate. abc AB Average 0.40 1.36 0.05 13.26 0.00 0.01 0.33 0.03 0.46 1.02abc 0.04abc 2.53 0.00ab 0.00bc 0.10b 0.07ab ± ± ± ± ± ± ± ± 0.26 1.22 0.04 14.91 0.00 0.01 0.33 0.05 15–30 km 0.3–1 0.025 8–11 1 Iron (mg ⁄ kg) DRI 8–18 UL 40–45 PMTDI 0.8 Chromium (mg ⁄ kg) DRI 0.015–0–030 Cadmium (mg ⁄ kg) PTWI 0.007 Arsenic (mg ⁄ kg) PTWI 0.015 average iron concentration of milk was reported as 1.4 mg ⁄ kg by Metin (2010). The average iron concentration of samples in this study was found to be lower than that reported by Metin (2010). According to results, the milk obtained from research area supplies 3.17% of daily iron requirement of an individual. This rate is consistent with the rate given by Metin (2010). ± 0.87 ± 1.17ab ± 0.004a ± 5.16 ± 0.00ab ± 0.00bc ± 0.17b ± 0.01b 0.64 1.42 0.07 13.48 0.00 0.00 0.31 0.02 5–15 km 0.95 1.43a 0.05ab 5.40 0.00b 0.00c 0.23b 0.03ab ± ± ± ± ± ± ± ± 0.62 1.89 0.06 11.96 0.00 0.00 0.33 0.05 2–5 km 0.25 0.79bc 0.01abc 4.12 0.01a 0.00ab 0.01b 0.02b ± ± ± ± ± ± ± ± 0–2 km 0.12 0.89 0.05 12.70 0.01 0.01 0.34 0.02 Iron Copper Lead Zinc Arsenic Cadmium Nickel Chromium Summer period Table 3 Mean values of heavy metal concentrations measured in regions and periods (mg ⁄ kg)* 4 0.7–0.9 3–10 0.05–0.5 DRI, Dietary reference intakes; UL, upper level; PMTDI, provisional maximum tolerable daily intake, PTWI, provisional tolerable weekly intake level. 0.72 1.16A 0.04A 4.49 0.00 0.00B 0.16B 0.04 Winter period Table 4 Some critical levels of observed metals, published by WHO ⁄ FAO and Food and Nutrition Board 0.77 1.04bc 0.07abc 2.25 0.00ab 0.01a 1.84a 0.02b Average 1.02 0.76B 0.04B 3.10 0.00 0.01A 1.04A 0.06 Vol 65 Zinc The average zinc values of the raw milk samples were between 11.597 and 14.907 mg ⁄ kg (Table 3). According to the statistical analysis, the differences between the regions and periods were not statistically significant (P > 0.05). The results obtained in this study were higher than those reported by Simsek et al. (2000) and by Licata et al. (2004) and lower than those reported by Baranowska et al. (2005). The study carried out by Vidovic et al. (2005) shows that 58% decrease in the rate of zinc in the atmospheric deposits caused 17% decrease in the rate of zinc in milk. Compared to the zinc results obtained in this study with established levels at Table 4, it might be speculated that the milk itself could provide the requirement zinc of an individual. The amount of zinc in milk was reported as 3.5 mg ⁄ kg by Metin (2010). The results obtained from research area were four times higher than that reported by Metin. In this case, zinc amount of milk was determined over the toxic limits. Chromium The chromium values obtained in this study varied between 0.013 and 0.075 mg ⁄ kg (Table 3). The differences among the regions were statistically significant (P < 0.05), and the highest chromium value was detected in 4th region during winter period. Compared to the previous results, the chromium results of this research were lower than the values detected for some region of Ankara, Turkey, by Temurci and Guner (2006) and for two regions of southern Italy by Anastasio et al. (2006). 2012 Society of Dairy Technology Vol 65 But higher values were detected in raw milk which was collected from different regions of Calabria, by Licata et al. (2004) and Southern Italy by Caggiano et al. (2005). Chromium is used in the leather industry, in inks and in processing of steel. The sampling sites in the Tekkekoy region, there are some of those types of industries. The results obtained in the study were higher than DRI value given at Table 4. Metin (2010) reported the amount of chromium in milk as 0.015 mg ⁄ kg and that were lower than those of study area. Nickel Nickel contents of the raw milk samples from different regions are shown in Table 3. According to analysis of variance, the differences among both region and period were statistically significant (P < 0.05). The nickel values obtained in this study were ranged between 0.312 and 1.127 mg ⁄ kg. Figure 3 presents mean values of nickel associated with regions and period interactions. As shown in Figure 3, the highest nickel value was detected at 3rd region during winter period. Temurci and Guner (2006) studied on heavy metal contents of milk and cheese in Ankara, Turkey. In this research, the nickel contents of milk were determined as 2.754 mg ⁄ kg, which were higher than that observation. According to the Table 4, the amount of nickel at this research was higher from UL values at some regions. However, the Turkish Codex (Anonymous 2002) had published the maximum amount of nickel obtained from milk as 0.1–0.2 mg ⁄ kg. So, the values for nickel concentrations in milk samples observed in this research were higher than those reported in Turkish Codex and threaten human health. Figure 4 Interactions of period and region for cadmium. concentration observed in the study, and the lowest concentration was determined in samples of 2nd region, collected in summer period (Figure 4). The values were lower than those reported by Rubio et al. (1998), Anastasio et al. (2006), Caggiano et al. (2005) and Temurci and Guner (2006), but higher than those reported by Licata et al. (2004) and Baranowska et al. (2005). The cadmium results of this research are closely related to the values reported by Vidovic et al. (2005). There are no data on Turkish Codex about the cadmium levels of milk, but for the other foodstuff, the cadmium levels ranged from 0.005 to 1.0 mg ⁄ kg (Anonymous 2008). The results of the study were less than PTWI values (FAO/WHO, 2006) (Table 4). So, cadmium levels could not lead to any health risk at the researched area according to results of this study. Cadmium The concentrations of cadmium in analysed samples are reported in Table 3. The cadmium values were changed between 0.001 and 0.013 mg ⁄ kg. According to analysis of variance, the differences among regions and periods were found to be significant (P < 0.05). Besides, a significant region–period interaction was detected (P < 0.05). The samples collected in 3rd region during winter period produced the highest cadmium Arsenic Arsenic contents of the raw milk samples from different regions are shown in Table 3. The arsenic values varied between 0.001 and 0.007 mg ⁄ kg. No statistical significance was detected between periods, but the difference in the content of arsenic in milks, sampled from different regions, was found to be significant (P < 0.05). Our results of arsenic concentrations were in accordance with those of Simsek et al. (2000), while Licata et al. (2004) reported higher values. According to PTWI value (FAO/WHO, 1989) (Table 4), the amount of arsenic in milk samples of the present study had no health concern. Figure 3 Interactions of period and region for nickel. Lead The lead concentrations observed in this study were 0.028– 0.068 mg ⁄ kg (Table 3). As shown in Table 3, lead levels in two sets of samples, obtained from 2nd and 3rd regions, were higher than the levels of other regions. It is clear that the lead level was variable in Turkey, especially in these areas. While the highest lead concentration was detected in the samples from 3rd region during summer period, the lowest lead concentration was detected in samples from 2nd region during winter period. The differences among the regions were statistically significant (P < 0.05), and the highest lead concentration was detected at samples collected during summer period. The lead contents of milk from regions were higher than the limits of Turkish Codex, 0.02 mg ⁄ kg (Anonymous 2008). The lead results of 2012 Society of Dairy Technology 5 Vol 65 this research were closely related to the values reported by Simsek et al. (2000) and lower than those reported for the milk samples collected from in two regions of southern of Italy by Anastasio et al. (2006), different regions of India by Singh et al. (1997) and different regions of Poland by Baranowska et al. (2005), but were higher than those reported for milk samples collected from different regions of Iran by Tajkarimi et al. (2008), different regions of Calabria, Italy, by Licata et al. (2004) and the industrial area of Huludao city, China, by Zheng et al. (2007). One of the possible reasons for the increase in lead concentrations of milk among countries may be the wide use of leaded gasoline during recent decades. The PTWI value (FAO/WHO, 2000) of lead was reported as 0.025 mg ⁄ kg (Table 4). For a person of an average of 60-kg body weight, this would mean 0.214 mg lead ⁄ day. The amount of lead in milk samples obtained from the research was higher than those reported in both the Turkish Codex and FAO ⁄ WHO lead residue level. It is recommended that local and national authorities should make farmers aware of appropriate practices for preventing heavy metal contaminations of farmlands. CONCLUSION The results of this study show that the raw milk samples collected in summer period were highest for copper, lead and cadmium, also higher than those of the winter period. The results indicated that raw milk samples obtained from those regions may not be safe for consumers in terms of copper, lead, zinc, nickel and chromium metals. The environmental pollution at study area was attributed to adjacent heavy industry. Further studies are necessary to evaluate the content of heavy metals on a greater number of milk samples from various dairy in Tekkekoy and to confirm the absence of possible toxicological risks in this region. ACKNOWLEDGEMENTS This research was supported with PYO.MUH. 1904.09.007 Project Number by the University of 19 Mayis, Samsun, Turkey. REFERENCES Anastasio B A, Caggiano R, Macchiato M, Paolo C, Ragosta M, Salvatore P and Cortesi M L (2006) Heavy metal concentrations in dairy products from sheep milk collected in two regions of southern Italy. Acta Veteinaria Scandinavica 47 69–74. Anonymous (2002) Gida maddelerinde belirli bulasanlarin maksimum seviyelerinin belirlenmesi hakkinda teblig, 23.04.2002. Turk Gida kodeksi Yonetmeligi. Anonymous (2006) Samsun Valiligi ll Cevre ve Orman Mudurlugu, Samsun il cevre durum raporu. 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