Concentration of potentially toxic elements (PTEs) in milk and its product: a systematic review and meta-analysis and health risk assessment study

ABSTRACT This study evaluated the concentration of potentially toxic elements (PTEs) in milk and its products. The related studies regarding the concentration of PTEs in milk and its products were investigated in international databases such as Scopus, PubMed, and Web of Science. Finally, the health risk assessment was performed using Total Target Hazard Quotient (TTHQ) by the Monte-Carlo Simulation model. According to the results of 30 included articles among 981 retrieved studies, the ranking of metal concentration was Zn > Fe > Se > Cu > As > Ni > Cd > Pb and Zn > Se > Pb > Cu > As > Ni> Fe > Cd in milk and Butter, as well as Fe > Cu > Pb > Se > Zn > Ni> Cd> As and Fe> Zn> Se> Cu > Ni> > As> Cd> Pb in cheese and Yoghurt, respectively. Based on the type of continents, the highest concentrations of As (0.16 µg/kg), Cd (0.49 µg/kg), and Pb (0.49 µg/kg) were found in yoghurt, cheese, and butter, which was related to Western Pacific Region (WPRO) and European Region (EURO), respectively. Regarding trace elements, the highest concentrations of Fe (3.94 µg/kg), Zn (5.90 µg/kg), and Ni (0.23 µg/kg) were observed in yoghurt, butter, and yoghurt belonging to WPRO and Pan Americas Region (PAHO), respectively. In addition, Se (0.78 µg/kg), Cu (0.57 µg/kg), and Se (0.78 µg/kg) represented the highest concentrations in yoghurt and cheese and were associated with WPRO and PAHO, respectively. The non-carcinogenic risk assessment of the PTEs indicated a different risk pattern in various countries, and the TTHQ level in adult groups in all countrieswas lower than 1 except for Italy. Thus, the consumption of milk and its products is safe to the health of consumers.


Introduction
Milk and dairy products are some of the most important food groups that have been widely consumed by various individuals worldwide.Among these dairy products, milk, cheese, yoghurt, and butter are extensively consumed because they are good sources of

Relevant screening, inclusion, and exclusion criteria
The title and abstract of all obtained records were assessed by two authors, FM and AR, independently based on exclusion and inclusion criteria.The kappa statistics (95%) were used to identify the inter-author reliability.Regarding challenging papers, agreement was reached by the third researcher.After removing duplicate articles, the full text of the eligible articles was downloaded.Inclusion criteria in our study were (1) full-text available in the English language (2) detect level of PETs in daily products, (3) cross-sectional research and original, (4) research preferentially conducted online between January 2008 and December 2018.Moreover, only reports published in the English language were evaluated.In this regard, duplicates, qualitative studies, clinical trials, case reports, review articles, and letters to editors were excluded.It also should be noted that articles that did not find raw data, mean values, or standard deviations, authors, journal, year of publication, country of origin and type of milk and its products, and or studies that evaluated the effect of animal feeding, climate change, effect processing to remove of metals in milk and its products, studies that reported fate of metals in milk and its products were excluded.The collected data of each study included the year of study, country of study, type of milk, sample size, average, standard deviation, and concentration range of toxic metals.In order to unify units, all units expressing the concentration of toxic metals, including mg/kg, ppb, and ng/g, was converted to μg/kg.

Risk assessment: estimate non-carcinogenic risk
To determine the, the non-carcinogenic risk of heavy metals intake through the consumption of daily products was applied target hazard quotient (THQ) in accordance with the following equations [16]: Where EDI is estimated daily intake (µg kg-1 bw day −1 ), C is mean level of metal (µg −1 mL); IR, ingestion rate of daily products (mLday −1 ) as shown in the (Table 3); EF, exposure frequency (365 days/year); ED, exposure duration (adults = 30 years) [17], BW is body weight (adults = 70 kg) according to the body weight studies by EPA, ATn (EF×ED) is average time exposure (adults = 10,950 days) [5,18].The average world ingestion rate of milk and dairy products obtained per capita milk consumption in each country [19] .
Target Hazard Quotient (THQ) due to intake metals in dairy products was estimated according to the following equation [20]: Where, EDI indicates daily intake and RfD or TDI is oral reference dose.The TDI values of Pb, and RfD values of Cd, As, Zn, Cu, Fe, Ni were 0.0036, 0.001, 0.003, 0.3,0.04,0.7, and 0.02 mg/kg/day, respectively [18,21] .
Where, TTHQ shows the sum of the each THQ for whole metal analysed in milk and dairy products samples [21].If TTHQ was lower than 1, health hazard was considered acceptable for human health [22].

Data analysis
Meta-analysis technique was applied to assess pooled concentration of metals in daily products by using random effect model.Subgroup analysis was separately preformed according to the type of the metals and continent.Heterogeneity was estimated among the studies by Q and I 2 tests.Cochran Q test (Q statistic, p < 0.10) indicated statistically significant heterogeneity, and I squared indicates heterogeneity among investigations.Statistical analysis was done using second version of the comprehensive Meta-analysis software.Data were analysed by the Stata software (version 14, Stata Corp, College Station, TX, USA) at a significance level of 0.05.

Uncertainty analysis
In order to increase accurate of risk assessment via considering uncertainties, a Monte Carlo simulated (MCS) method was used.To conduct this method, the Oracle Crystal Ball software (version 11.1.2.4.600) was used.In this method, the parameters like the combine concentration of 4 dairy products in each country for each metal (C), total ingestion rate (IR) and body weight (BW) are considered as lognormal distribution [23,24], the number of repetitions was at 10,000 and percentile 95% of THQ and TTHQ was considered cut point of human health risk [25] .

Study characteristics
After an initial literature screening in international databases, including Web of Science, Medline, and Scopus of 981 articles, 430 were removed as duplicates using EndNote citation manager (vX7.4,Thomas Reuters, New York, USA) and 551 articles were remained for further consideration.As shown in (Figure 1), according to the titles of retrieved articles, 319 articles were excluded due to the irrelevant title.Among these articles, 232 articles were selected by reviewing their abstract and then 185 articles were excluded.Next, we could download the full texts of the 47 articles and finally, 30 containing 1874 samples, published from 2008 to 2018, were included in our study for meta-analysis and probabilistic health risk assessment.The studies were done all over the world.The following rank order was obtained for study zones, namely Asia including India (2), Turkey (7), Iran (2), South Korea (2), Lebanon, Saudi Arabia, (15 studies, 50%) >Europe including Serbia, Poland, Romania, Italy (3), Greece, Spain, Croatian, (9 studies, 30%) >, America including Brazil, Argentina (2) (3 studies, 10%) and Africa including Egypt (2) (2 study, 6.6%) .The summary of the selected papers comprising the amounts of Pb, Cd, As, Zn, Cu, Ni and Fe in mother milk in different regions of word is presented in (Table S1-S8).

The concentration of PTEs in milk and dairy products according to the type of metals
Based on results of (Table 1), the ranking of metal concentration was Zn > Fe > Se> Cu > As > Ni> Cd> Pb, Zn > Se > Pb > Cu > As > Ni> Fe > Cd, Fe > Cu > Pb > Se > Zn > Ni> Cd> As, and Fe> Zn> Se> Cu > Ni> > As> Cd> Pb in milk, butter, cheese, and yoghurt, respectively.The results indicated that the highest and the lowest concentrations of As in milk and its products were related to yoghurt and cheese (0.16 and 0.01 μg/kg, respectively).According to findings, the highest concentrations of Cd and Pb were observed in cheese and butter (0.04 and 0.49 μg/kg, respectively) while the lowest As shown, there is a large difference between the concentrations of metals in the milk and dairy product samples in these studies and our study, which can be related to several factors including the physiology or nature of the metal animal body, the source of contamination, the breed of livestock, the agricultural activities (e.g. the types and amounts of the applied fertilisers), and storage conditions and processing technologies [34].The presence of heavy metals in dairy products may be due to the contamination of the primary milk, which may be due to exposure to the environment or the consumption of food and water via livestock [35].The metal contamination of the soil can be transmitted into the plant through the roots of the plant and the consumption of plants by livestock can lead to the contamination of milk and meat of the animals [36].In their study, Singh et al. reported  .The physicochemical properties of metals are considered as the other important factor influencing these differences.Obviously, the higher concentration of Fe, Zn, Cu, and Ni, compared to other investigated elements such as Pb, Cd, and As in many dairy product samples, can be because essential elements have higher transfer factors compared to toxic metals from the soil to plants, thus leading to higher accumulations of these metals in the consuming plants of the animals [37].In addition to the nature of the metal, either natural or anthropogenic origins can increase the content of metals in plants that are used by livestock.In general, excessive use of fertilisers and pesticides in agricultural activities can change the bioavailability of metals in the soil [38].
For example, the presence of Pb, Cd, and As in dairy products can depend on factors such as chemical fertilisers (impurity in phosphate fertilisers), mineral compounds (e.g.limestone and phosphate), and fish meal [39].Furthermore, machinery processing technologies have an important role in the observed differences so that many processes can change the concentration of metals during the production of milk to its products as cheese, butter, and yoghurt.Shabazi et al. reported that high contents of Pb in the cheese samples than milk can be due to the Pb-binding characteristic of casein [26].In another research conducted by Zurera-Cosano et al., the pasteurisation process could increase the Pb concentration by nearly 70% during milk production [40].The findings of another similar study demonstrated that the low concentration of As in cheese, compared to that of other dairy products, may be due to milk processing methods such as the coagulation and dehydration of milk and a need for the final stage of maturation [10].According to a previous study (39), more specifically, the concentration of metals such as Pb and As is high due to their high affinity to casein and fat in milk products while its opposite is true for Cd because this metal has high affinity to bound to components in milk with a relatively low molecular weight (whey proteins, citrates, lactose, and mineral salts).
Likewise, Moran et al. showed that the butter production process from milk reduced the contamination of content metals in high-fat products [41].Eventually, Christophoridis et al. reported that the concentration of metals such as Cu, Ni, Fe, and Zn in milk in addition to temporary milk storage containers and milking equipment can be affected by the method of processing and cooking of milk [10].

Level of metals in milk and its products according to the World Health Organization (WHO) region classification
Based on the data in (  [13].Based on the findings, great differences exist between the concentrations of metals according to different regions.The obtained differences can be related to the characterisation of the geographical origin, the applied plant species and water for animal consumption, weather conditions, industrial and urban activities [37] near heavily traffic roads, factories, mines, highways, usage of chemicals as fertilisers or pesticides, and atmospheric conditions [50].The studies demonstrated that low metal concentrations in some regions may be due to the physicochemical properties of the soil, and the climatic conditions of the region can change the content of metals by making them inaccessible to plants and thus animals [51].The existence of active industries in different countries of the world can affect metal contamination in milk and its products.In a study by Bilandžić et al., atmospheric sediments originated from environmental pollutions by industries could increase Cd content in the milk sample of lactating animals [39].Other studies revealed that the prevalence of contamination to As in milk and milk products is higher in countries where the soil and water are contaminated with As compared to other countries

Health risk assessment
The non-carcinogenic risk assessment of toxic metals based on consumption of the milk and its products in different countries has been shown in (  reported that the average HRI values in adults via consumption of raw and pasteurised cow's milk were within the safe limits (HRI < 1), there was no health risk for the target groups especially adults.Results obtained in regarding risk assessment of metals in different countries indicated different pattern was different, which can be because of the difference in milk and its products consumption content in various countries, milk consumption per capita, concentration of metals in plant and water used for consumption of lactating animals [9,65].The results showed that accounted TTHQ amounts for adult groups in all investigated countries, except for Italy were lower than 1, suggesting that the local inhabitants in all countries except for Italy will not be exposed to a potential health risk from consumption of milk and dairy products, but it can be mentioned that there are also other sources of metal exposures such as dust inhalation, dermal contact and ingestion of other foodstuff and water, which were not included in this study.

Conclusion
This systematic review was performed to evaluate the concentration of the metals in milk and dairy products based on types of metals and WHO regions in the world.Non-carcinogenic risk of toxic metals was also assessed based on the concentration and consumption rate of the milk and dairy products in different countries.The results of 30 papers indicated that the ranking of metals concentration in milk and its products was Zn > Fe > Se> Cu > As > Ni> Cd> Pb in milk, Zn > Se > Pb > Cu > As > Ni> Fe > Cd in Butter, Fe > Cu > Pb > Se > Zn > Ni> Cd> As in Cheese, Fe> Zn> Se> Cu > Ni> > As> Cd> Pb in Yoghurt.In this context, according to continents, the highest concentrations of Fe (17.62 µg/kg), Zn (4.14 µg/kg), Se (1.06 µg/kg), As (0.84 µg/kg), Ni (0.12 µg/kg) and Cd (0.07 µg/kg) was related to (WPRO), while, the highest concentrations of Cu (0.94 µg/kg), Pb (0.42 µg/kg) was belongs to (PAHO) and (EURO), respectively.The plant species, physicochemical characteristics of heavy metals, plant growth conditions and the processing techniques used for the production of milk and dairy products had important roles on the level of metals in these produces.The non-carcinogenic risk assessment of the PTEs in milk and dairy products indicated that there was a different risk pattern in various countries, the TTHQ level in adult groups except for Italy was lower than 1.Therefore it can be concluded that the consumption of milk and dairy products is safe and does not pose risk to the health of consumers.

Figure 1 .
Figure 1.Selection process evidence searches and inclusion.

Table 1 .
Meta-analysis of concentration of toxic metal (PTEs) (µg/kg) in milk and its products based on the type of metals.concentrations of these metals were found in butter and yoghurt (0.01 and 0.01 μg/kg, respectively).Regarding the trace elements, yoghurt and butter represented the highest and lowest concentrations of Fe (3.94 and 0.06 μg/kg, respectively).Moreover, the highest and lowest concentrations of Zn were related to butter and cheese (5.90 and 0.13 μg/kg, respectively).Based on the obtained data, the maximum and minimum concentrations of Ni were observed in yoghurt and milk (0.23 and 0.04 μg/kg, respectively).Likewise, cheese and milk, as well as yoghurt and cheese demonstrated the highest (0.57 and 0.21 μg/kg) and lowest (0.78 and 0.22 μg/kg) concentrations of Cu and Se, respectively.A wide variety of studies have reported different concentrations of metals in milk and its dairy products.In our study, the mean concentrations of Zn, Se, Pb, Cu, As, Ni, Fe, and Cd in milk were 2.76, 1.31, 9.55, 2.17, 0.13, 0.04, 0.03, and 0.02 μg/kg, respectively.Compared to our findings,Shahbazi, et al. reportedthe mean ranges of Pb, Cd, Cu, Zn, and Se as 0.014, 0.001, 0.427, 0.571, and 0.002 mg/kg in the milk samples of Iran, respectively [26].Similarly, Ismail et al. concluded that the mean concentrations of Cd, Pb, Cu, and Ni in the milk samples of Pakistan were 0.001, 0.061, 0.014, 0.738, and 0.028 mg/kg, respectively [27].In another study, Meshref et al. reported that the level of Pb, Cd, Zn, and Cu was in the range of 0.044-0.751,0.008-0.179,0.888-18.316,0.002-1.692,and1.3208-45.6198mg/kginthemilkanddairyproducts of Egypt, respectively [28].In another study, Pavlovic et al. found the mean concentrations of Pb and Cd to be 0.27 and 0.03 mg/ kg in the milk samples of Croatia, respectively[29].In this study, the concentrations of Zn, Se, Pb, Cu, As, Ni, Fe, and Cd in other milk products were investigated in addition to that of the milk sample.According to the results, the mean concentrations of Zn, Se, Pb, Cu, As, Ni, Fe, and Cd were 5.90, 0.49, 0.49, 0.34, 0.13, 0.07, 0.06, and 0.01 (mg/kg), as well as 0.13, 0.22, 0.30, 0.64, 0.01, 0.09, 6.83, and 0.04 (mg/kg) in butter and cheese, and the corresponding levels in yoghurt were 2.58, 0.78, 0.01, 0.57, 0.16, 0.23, 3.94, and 0.04 (mg/kg), respectively.In line with our findings, Rezaei et al. found that the mean concentrations of Cd and Pb were 0.003 and 0.004 mg/kg in yoghurt, along with 0.023 and 0.015 mg/kg in cheese[30].In their study, Cigdem et al. reported Pb concentrations in the studied samples of yoghurt as 0.22 mg/kg[31].Additionally, Starska et al. observed that the mean concentration of Pb, Cd, and As, was 0.017, 0.002, and 0.009 mg/kg in the milk products of Poland, respectively[32].Based on the findings of Yuzbasi et al., the mean concentration of Pb, Cd, Fe, Zn, and Cu in the cheese of Turkey was 0.86, 1.8, 4.2, 0.7, and 0.37 mg/kg respectively[33].Further, Edward et al. found that the mean concentrations of Cd, Pb, and Cu in different types of butter were in the range of 0.09-0.026,0.09-0.014,and0.298-0.364.0 mg/kg, respectively[11].
entry of toxic metals into the milk[12].Likewise, Mohajer et al. indicated that the soil can be contaminated via motorisation, pesticides, and industry and air pollutants.Therefore, plants grown in this soil are contaminated with heavy metals and can enter the body of lactating animals[10] prolonged exposure to soil or water associated with metal sources (e.g.places near contaminant sources and metal scrap) can lead to the

Table 2 .
[54]-analysis of concentration of toxic metal (PTEs) (µg/kg) in milk and its products based on WHO regions.52].For example, Pérez-Carrera et al. found that the rural or suburban areas of Argentina have a groundwater network which is contaminated with remarkable levels of As, therefore, the milk products of this area due to the consumption of the applied drinking water by livestock farms had high levels of contamination with As[53].Similarly, Divrikli et al. indicated that the levels of Cu in Southeastern Anatolia were higher in comparison with all other countries and attributed the high contamination of plants and herbs of this area to Cu[54].Alloway et al. also reported the content and bioavailability of Zn in the applied soil for dairy farming as important factors regarding the presence of this element in milk and its products although the transfer of Zn from the used tools and machinery to food in the manufacture has an important role in the observed differences[55].In another study by Khozam et al., differences in metal concentrations were because of seasonal weather changes and differences in the manufacturing processes of products.Furthermore, they found that higher amounts of Cu in the milk daily of Lebanon, compared to other countries, were related to the use of metallic equipment during manufacturing these products[56].Moreover, Mohajer et al. indicated that the Pb content in Isfahan, Iran was higher than that of other regions due to motorisation (organic derivatives of Pb were widely used as gas additives), industrial causes, fertilisers, pesticides, and air contaminants[57].In similar research in Iran, extremely high levels of Cu were detected in the obtained environmental samples in regions where there were many mining operations[58]. [

Table 3 .
Uncertainty analysis for TTHQ of metals in adult due to consumption of milk and its products in various countries.