Concentration of mineral and heavy metals in raw mare (horse) milk consumed in Yazd, Iran: A risk assessment study

Abstract Background In recent years, the mare's milk has been introduced as a rich source of nutrients with hypoallergic characteristics which is widely used for Iranian infants. Objectives The present study aimed to investigate the heavy metal concentration of mare's milk and its consumption risk assessment. Methods About 88 mare's milk was collected from Yazd, the centre of Iran, during the summer of 2020. The raw mare's milk was digested and analysed for mineral and heavy metal content (As, Ca, Cd, Co, Cu, Fe, Mg, Mn, Ni, P, Pb and Zn) by ICP‐OES. To estimate the health hazard for consumers the Estimated Daily Intake (EDI), Hazard Quotient (HQ) and Hazard Index (HI) of heavy metals were determined. Results The Ca ranged from 260.52 to 201.43 mg/L, which was the highest mineral in mare's milk followed by P and Mg. By increasing the age, P and Ca content was increased. The obtained ranges of Cu, Co, Fe, Mn and Zn were 72.12–75.11, 1.12–9.3, 180.69–230.21, 31.24–47.13 and 1060–1200 μg/L, respectively. The Cd and Arsenic content of mares' 8–11 years of age had higher concentrations. The highest Pb content was reported in mares 4–7 years old (10 μg/L). Although, Pb, Cd and As content of the mare's milk was evaluated lower than the permissible limit. Also, the HQ value was As > Cd > Pb > Zn > Ni > Cu for infants, toddlers and adults. The HI of mare's milk was 0.16, 0.15 and 0.022 for infants, toddlers and adults, respectively. Conclusions Mare's milk could be an effective nutrition source for infants and children suffering from milk protein allergies.


Sample collection
The milk samples were collected from 88 lactating mares and their health condition was confirmed by a veterinarian. The mares were chosen due to their milk consumption by people from active milk production centres in Yazd, Iran. Samples were collected from the 15th day of April to the end of September. The criteria for sample collection were as follows: (1) consumption of mare's milk by people, (2) confirmation of the health condition of mares by the veterinarian. Then, mares were categorised into three groups: 4-7 years old (42 mares), [8][9][10][11] years old (25 mares) and >12 years old (21 mares). The mare's milk was collected into sterile polypropylene tubes and transferred to the laboratory under a cold chain and stored at −20 • C until further analysis (Paksoy et al., 2018). Prior to digestion, samples were thawed at ambient temperature.

Wet digestion
The wet digestion procedure was used to digest raw milk samples. A total of 2 mL of raw mare's milk were digested in presence of 2 mL HNO 3 (65%) and 1 mL H 2 O 2 (30%) and heated at 90 • C for 2-3 h. The cooled samples were filtered through a 0.45 μm filter membrane and diluted to 10 mL of distilled deionised water. The blank sample was prepared in the same procedure (Beikzadeh et al., 2019).

ICP-OES analysis
Heavy metal analyses were carried out with an Inductively Coupled

Method validation
The validation and accuracy of the method were verified by the determination of target elements in certified reference material (CRM, Multi-element standard TraceCERT® in 10% nitric acid). All tests were done in triplicate.

Heavy metal risk assessment
The exposure dose of the heavy metals, Estimated Daily Intake or EDI, was evaluated according to the following equation (Kiani et al., 2021).
where C is the concentration of heavy metal in the mare's milk sample (the mean concentration of all groups was considered as the final concentration); IR is the consumption rate of milk (160 g/day for 6to 12-month-old infant, 200 g/day for 1-to 2-year-old toddler and 164.38 g/day for adults) by different age groups; and W is the reference body weight for different consumer age groups (9.3 kg for 6-to 12-month-old infants, 12.2 for 1-to 2-year old toddlers and 70 kg for adults) (Kiani et al., 2021). The Estimated Weekly Intake (EWI) was evaluated by multiplying EDI by 7.
By estimating the EDI, the Hazard Quotient (HQ) of heavy metals for determination of noncarcinogenic health risk of mare's milk consumption was assessed (Equation 2).

Data analysis
The statistical analysis was done with SPSS 22.0 software package. The Kruskal-Wallis comparison tests were used for normal nondistributive variables. The differences in metal concentration were analysed by one-way analysis of variance (ANOVA). The significant differences (p < 0.05) between the means were determined by Tukey's multiplerange test. The metal concentration was reported as means ± standard deviation.

Result and discussion
Recently there has been growing interest in the consumption of mare's milk in infants and sensitive people due to its functional properties (Malacarne et al., 2002). It has been established that the nutritional composition of mare's milk is similar to human milk. Therefore, mare's milk could be a rich nutritional source for infants and even sensitive and old people (Nayak et al., 2020). Although the hypoallergic and   (Homayonibezi et al., 2021). Likewise, the contamination of water and forage resources of livestock by heavy metals can lead to the accumulation and secretion of heavy metals into animal products including milk and meat (Homayonibezi et al., 2021). To the best of our knowledge, there are limited studies on the heavy metal and mineral content of mare's milk consumed in Iran. Due to the consumption of mare's milk by infants and immunosuppressive people, the concern of heavy metal contamination would be elevated. Therefore, this study was  (<0.01 mg/kg). The limit of nickel in milk is prescribed as 0.4 mg/kg (Homayonibezi et al., 2021), but all milk samples in the current study had lower Ni content.
In overall, the mineral composition of mare's milk was shown lower than in many studies. This also could be attributed to the age of the mares, which participated in the current study, which was higher than 4 years old. The variation in metal composition of dairy products is affected by the stage of lactation, species and genetics, age, mammary gland health status, milk protein content, nutritional status of animal, environmental factors, sample size and analytical method accuracy (Fantuz et al., 2015;Fantuz et al., 2012;Homayonibezi et al., 2021;Pietrzak-Fiećko & Kamelska-Sadowska, 2020).  Sadowska, 2020). In addition, the main way of Pb elimination is through urine and faeces, small amounts could be released in milk (Longodor et al., 2018). There are some reports that supplementation of animal diets with Ca could limit the absorption of Cd and Pb (Fantuz et al., 2015). The lack of information about heavy metal distribution in mare's feedstuff and diet as well as mare's blood was a limitation of the current study. Nevertheless, the mammary gland plays an important role in the regulation of metal secretion in milk (Fantuz et al., 2015). Miclean et al. (2019) confirmed that the main factor in metal contamination of food resulted from contamination by soil. Researchers have illustrated the contamination of soil due to human activities, could lead to the high variability of metal content in different studies (Miclean et al., 2019).
Apart from water and forage, ingestion of contaminated fodder with soil and supplementary minerals in diets could trigger metal contamination (Miclean et al., 2019). It was shown that the metal content of milk could be affected by the season of the year. The high metal contamination of collected milk in winter is reported owing to high rainfall and contamination of soil and food crops consequence of waste wash down (Shahbazi et al., 2016).