Determination of the Level of Heavy Metals in Cow’s Milk Collected from Butajirra and Meskan Districts, South Central Ethiopia.


 BackgroundMilk is a complete food useful to promote growth and development of the infant mammals as it contains vital nutrients including proteins, essential fats, vitamins, and minerals, in a balanced proportion. Milk can also contain chemical hazards and contaminants, such as heavy metals which can be a risk for health. This study was aimed at determining the level of the heavy metals in cow’s milk collected from Butajirra and Meskan districts, south Ethiopia. Cows’ milk was collected from 193 healthy and lactating cows. Samples were digested by optimized microwave digestion method using HNO 3 and H 2 O 2 . Analysis was done using ICP-OES for Cd, Cr, Cu, Fe, Mn, Ni, Pb, and Zn. MP-AES was used for Ca, Mg, K and Na. ResultsNi was not detected in all the milk samples. The concentrations of metals in the studied milk samples were, Cd (0.0 – 0.03), Cr (0.0 – 0.4), Cu (0.03 – 1.1), Fe (0.0– 1.9), Mn (0.0– 0.7), Pb (0.0– 12.3), Zn (0.0–8.2), Ca (380.1– 532.4), Mg (159.6– 397.9), K (1114.2–1685.8) and Na (495.9–1298.3) ppm. These values were compared with guide lines of different international organizations for food and other available literatures. Cd, Cr, Cu, Mn, Pb, Zn and Mg were found over the permissible limits. ConclusionSpecial attention should be given to the level of heavy metals in cows’ milk, since they are difficult to remove from the body and are dangerous to health once they accumulate to a level greater than their limit.

of them (Zn and Fe) are essential trace elements for living organisms when consumed at low concentration. However, they become toxic at high concentration. Most of these metal ions (Cd, Cu, Zn, Cr and Fe) are released from the industries in simple cationic forms (Volesky and May-Phillips, 1995). Now days, the contamination of milk with toxic elements is a global concern (Arianejad et al., 2015).This is because of the increasing industrial, vehicle and agricultural processes which can result in increased concentration of toxic heavy metals in the air, water, plants and soil (Yahaya et al., 2010).
Heavy metals such as Cd and Pb are common air pollutants and are emitted into the air as a result of various industrial activities. Although the atmospheric levels are low, they contribute to the deposition and build-up in soils. Various industrial environmental contamination of soil, waters, foods and plants with these metals cause their incorporation into the food chain and impose threat to human and animal health (Bilandžić et al., 2011).
The bioaccumulation nature of heavy metals in the environment makes them very dangerous for living organisms. Heavy metals cannot be degraded and are very toxic even at low concentration.
When living organisms are exposed to these compounds from food chain such as milk, they are accumulated and stored at a rate faster than their metabolism and excretion (Tsoumbaris and Papadopoulou, 1994).
These heavy metals are taken in by plants and consequently accumulate in their tissues. The accumulation of toxic heavy metals leads to metabolic disorders and serious health problems such as weakness, heart failure, cancer, and kidney problem (Licata et al., 2004).
The toxic effect of heavy metals depends on the total amount absorbed, route of exposure, duration of exposure and age of the person. For example, young children are more susceptible to the effects of Pb exposure because they absorb several times more of the percent ingested and yet to develop excretory system compared with adults (Tungegova et al., 2016).
There is evidence that milk and other dairy products might contain varying amounts of different toxic contaminants. Causes for the presence of heavy metals in cow's milk are due to exposure of the cows to contaminated feed sources like grass, drinking water, dust that settle on the grass, pharmaceutical medicines and their bioaccumulation process which potentially influence human health (Singh et al., 2010, Amponsah, 2014 Therefore, information on the level of heavy metals in cow's milk is important in assessing risk to human health. Currently, many analytical methods are being developed and validated to perform accurate measurements of pollutants such as heavy metals in environmental matrices in order to assess compliance with national and international legislations. Those legislations limit the allowable concentration of pollutants in the atmosphere to ultra-trace levels. When analytical methods are performed, validation is often supported by primary standards and certified reference materials at low concentrations, developed for the purpose of accurate calibration, with a traceable uncertainty statement (Brown et al., 2005).
The heavy metal concentration values need to be compared with recommended dietary allowance (RDA) values. It is also advisable to compare with the corresponding values for milk in different countries as reported in literatures. In this study cow's milk was collected from Butajirra and Meskan districts South Central Ethiopia to determine its heavy metal levels by using inductively coupled plasma-optical emission spectrometry and microwave plasma atomic emission spectroscopy.

Description of the Study Area
A cross sectional study was conducted from July to August 2017 with respect to sample collection to determine the level of heavy metals in cow's milk in Butajirra and Meskan districts.
Butajirra is a town and separate district in south central Ethiopia located at the base of the Zebider massif in the Gurage zone of the Southern Nations Nationalities and People's Region (SNNPR). The town of Butajira is surrounded by Meskane and located about 130 km south of Addis Ababa, Ethiopia as shown in Figure 1 (Sinaga et al., 2015). According to Butajirra's livestock and fish development office there are about 14,000 milking cows and daily milk production is estimated to be greater than 3500 litters. The district consists of 30 peasant associations, 65 private herders and two agro-industries. The total population of the district is estimated to be 78,000. Four out of five "kebeles", (lower level of village administrative district in Ethiopia) (named kebeles 01, 02, 03 and 04) were purposively selected from Butajirra district.
According to the district's livestock and fish development office there are about 34,198 milking cows and the total population of the Meskan district is estimated to be 196,000. Three kebeles were selected from Meskan district based on their climatic condition to accommodate the three agro climatic conditions of Ethiopia. The selected sites were: Enseno town (lowland), Mekicho (middle land) and Huletenya Wolensho (highland) (Berhane and Byass, 2002).
The societies in Butajirra and Meskan districts are supported by farming in agro-pastoral settings. They grow cereals, false banana ('enset') and Catha eduslis Forsk (Khat) Berg et al., (2009),Nana (2016. In the study area there is a dietary practice serving individuals with milk during any time while having their meal. The daily milk consumption rate for Ethiopia was reported as 53 g/day (Belete et al., 2014). So, the study areas were chosen in expectation of high milk consumption.

Sample collection
The polyethylene sampling bottles were soaked in HNO3 and were rinsed with deionized water before collection of fresh cow's milk in order to avoid possible contamination.
For this study, a total of one hundred ninety-three healthy and lactating cows were randomly selected from Butajirra and Meskan districts; Eighty-five and one hundred eight cows respectively. The health of the selected lactating cows was ascertained by veterinarians practicing in the area. Samples were labeled into 17 different codes 8 of which were unmixed (taken from single cow) and the remaining 9 were mixed (prepared by taking about 6 ml from 20 cows). The purpose of collecting mixed samples was to minimize cost for determination. Eight unmixed samples were prepared from randomly selected four cows at Butajirra and four cows at Meskan; 125 mL milk was taken from each cow, and put directly into eight sterile screw cupped bottles. Nine homogenized samples were also prepared from randomly selected eighty-one cows at Butajirra and one hundred four cows at Meskan; about 6 mL milk was taken from each cow directly into nine sterile screw cupped bottles. Twenty cows were included in the preparation of each homogenized sample. These samples were collected manually at any time of the day from both districts by the farmers and the quality of sample collection process was directly observed.
The udder of each cow was washed with tap water and then rinsed thoroughly with distilled water before milking. The samples were kept in an ice box and were maintained under refrigeration for nine days until collection was completed. Finally, seventeen samples were transported to Addis Ababa and immediately kept in a deep freeze (-20 o c) until microwave digestion was carried out.

Chemicals and reagents
All the chemicals used were of analytical reagent grade. Nitric acid (68%), hydrogen peroxide

Apparatus
All glass wares were washed before use with deionized water to avoid contamination and then soaked in nitric acid, and rinsed with deionized water and finally the glasses were dried in oven.
The glass wares were kept in desiccator, to avoid contamination. Milk samples were digested by using milestone start D microwave digester (START D, Milestone, Basel, Switzerland). ICP-OES (Agilent 700 Series, California, USA) and MP-AES (Agilent 4200 Series, California, USA) for the determination of the heavy metals level in milk samples.

Sample digestion and preparation of analyte solution for ICP-OES and MP-AES
The optimized microwave digestion procedure was selected depending upon the clarity of digests, minimal digestion time, and minimum reagent volume, absence of undigested milk samples, simplicity and low heating temperature. In this study 3 mL of cow's milk from each sample was treated with 9 mL 68% nitric acid and 2 mL 32% hydrogen peroxide and were transferred to dried digestion vessels. The digestion vessels were placed in a microwave digestion system at 180 0 C (30 min) and kept until brown fumes disappeared, indicating completion of oxidation of organic matter. After heating, the closed sample bottle was cooled to room temperature to avoid foaming and splashing and the digestion vessels were opened carefully in a fume hood. The cold clear solution was filtered in to 100 mL volumetric flask using Whatman filter paper (0.45µm pore diameter membrane) to remove any suspended residues. 14ml, 1% nitric acid was added to the solution and diluted up to the mark with deionized water. Digestion of blanks was also performed in parallel with the milk samples keeping all the digestion parameters the same.

Determination of metal contents of each digested sample by ICP-OES and MP-AES
The measurements of levels of Ni, Zn, Fe, Cu, Cr, Mn, Pb and Cd were carried out with ICP-OES. Before analysis of the sample, both MP-AES and ICP-OES were optimized using standard solutions of the metals to give maximum signal strength by adjusting the parameters such as wavelength, nebulizer flow, pump speed and lamp current for each element as shown in Table 1. The correlation coefficients of calibration curves for Zn, Pb, K, and Ca are given in Table 2

Method Validation Precision and accuracy
Analytical results must be evaluated to decide on the best values to report and to establish the probable limits of errors of these values (Kikuchi et al., 2002). In this study the precision of the results were evaluated by percentage relative standard deviation of the results of three samples (N=3) and triplicate readings for each sample giving a total of nine measurements for a given bulk sample. On the other hand, the accuracy and validity of the measurements were determined by analyzing spiked samples. In this study, milk samples were spiked by adding 1mL from 1000 ppm metal standard solutions to three mL cow's milk.
The resulting mixtures were then digested, in triplicate for each sampled cow's milk, and analyzed in a similar manner as the milk sample. Then, the percentage recovery (% R) was calculated for each of the selected element using the following equation: In this study the variability in the method between the runs on the same day (intra-day variation), and the variability between runs on different days (inter day variation) was examined by using samples from a standard reference. In addition, reproducibility of the method was also checked by using samples from a standard reference.

Method Detection and Quantification Limits
For determination of the detection limits of the method, blank sample was digested following the same procedure as the milk samples and each of the blank samples was analyzed for Ni, Zn, Fe, Cu, Cr, Mn, Pb, and Cd by using ICP-OES and the same done for K, Na, Mg, Ca by using MP-AES. The method detection limits and method quantification limits for MP-AES and ICP-OES are shown in Table 3. The standard deviation for each element was calculated from the blank measurements to determine method detection and quantification limits of the instrument.

Statistical analyses
Microsoft Office Excel 2016 was used to calculate the descriptive statistics (like mean, standard deviation) and draw calibration graphs. One-way analysis of variance (ANOVA) was performed to compare the differences among the samples using SPSS 20.0. Origin was used to draw graph for each metal in all samples (in mg/L). P < 0.05 was considered significant.

Results and Discussion
The average heavy metal concentrations of the seventeen sampled cows' milk collected from Butajirra and Meskan districts, south central Ethiopia are shown in Tables 4 and 5 Table 6. Cd, Cr, Cu, Mn, Pb, Zn and Mg were found to be over the permissible limits in 12%, 6%, 53%, 65%, 82%, 6 % and 100 % of the samples respectively. The values over permissible limits of heavy metals in the study areas could be originated due to the commonly used underground water and animals' feed.
It is known that heavy metal contents in milk can vary widely due to many factors such as its secretion from the mammary gland, feeding, use of different water sources and industrial pollutions (Moreno et al., 1993,Caggianoa et al., 2005, Younus et al., 2016. As shown in Figure   2, reported by Belete et al (2014) in Ethiopia, Alem et al (2015) in Ethiopia, Maheswara and Murthy (2017) in Tanzania respectively. Cu is an essential trace element that plays a vital role in the physiology of animals for fetal growth and early post-natal development. Excess Cu in the body leads to Wilson's disease which is characterized by abdominal pain, fluid buildup in the legs or abdomen and problems with speech (Hassan andMasood, 2004 andLawal et al., 2006).  Malhat et al (2012) in Egypt respectively, and higher than 0 -0.93 ppm and 0.0 -0.39 ppm the study made by Ahmad et al (2016) in Bangladeshand Abdulkhaliq et al (2012) in Palestine respectively. Like Cu, high concentration of Pb in milk may result from consumption of contaminated feeding stuffs and the commonly used underground water in the redistricts. Pb has no beneficial biological function and is known to accumulate in the body. Pb exposure can cause adverse health effects, especially in young children and pregnant women. Pb is a neurotoxin that permanently interrupts normal brain development (Duruibe et al., 2007). Table 4 and 5 show that, the average Cr concentration, in cow's milk analyzed ranged from 0.00 -0.4 ppm which is lower than 0.845 -0.895 and 0.468-0.828 ppm reported by Belete et al (2014) and Akele et al (2017) in Ethiopia respectively and higher than 0.055 -0.075 and 0.0 -0.11 ppm reported by Alem et al (2015) in Ethiopia and Ahmad et al (2016) in Bangladesh respectively. Only sample CM13 one exceeded the permissible limit. Like other microelements Cr is essential to maintain the metabolic systems of human body but at higher level causing stomach upsets and ulcers, convulsions, kidney and liver damage, and even death (Qin et al., 2009).

Results presented in
All the average values of Fe in milk samples were found to be below permissible limit which  Malhat et al(2012)in Egypt while lower results 1.96 ̶ 3 .640 ppm were reported by (Bano et al., 2015) in Pakistan.
Tables 4 and 5 shows that the Cd level ranged from 0.0 -0.03 ppm. The average concentrations of Cd in this study is observed to be in agreement with 0.016-0.04 ppm and 0.022-0.057 ppm the report made by Abou-Arab et al (2008)in Egypt and Abdulkhaliq et al (2012)in Palestine respectively, but it is lower than the 0.200-0.288 ppm reported by (Malhat et al., 2012) in Egypt.
The level of Mn in milk sample from all the sites ranged from 0.0-0.7 ppm and 65% of the milk samples have above the limit. Lower levels 0.411 -0.441 ppm and 0.37 -0.52 were reported by Belete et al(2014) in Ethiopia andBano et al (2015) in Pakistan respectively. Exposure to high concentration of Mn is associated with mental and emotional disturbances, impaired male fertility, birth defects, and impaired bone development (Santamaria, 2008).
Magnesium was detected in all samples as shown in Tables 4 and 5. The levels of Mg in cow's milk in this study ranged from 159.6 -397.9 ppm which is lower than the report made by Birghila et al (2008) 919.8 ppm in Romania. The mean concentration of Mg in this study is higher than the maximum standard indicated for food composition as shown in Table 6. Tables 4 and 5 indicated that the concentration of K in this study ranged from 1114.2-1685.8 ppm and this is higher than the value reported by Qin et al (2009) in China, which was 1000 ppm and agrees with 1440 -1780 ppm reported by Zamberlin et al (2012) in Croatia and 1510-1660 ppm by Pereira (2014) in Portugal. The mean concentration of K in this study is lower than the maximum standard indicated for food composition as shown in Table 6.
The mean values of Ca in this study ranged from 380.1 -532.4 ppm that was found to be below the limit set by Chinese food Guidelines (820-1130 ppm) and which is also much lower than the concentration reported by Qin et al (2009) in China 1000 ppm and agree with 195 -1528 ppm ,the result reported by Bano et al (2015) in Pakistan. The level of Na in samples from all the sites ranged from 495.9 -1298.3 ppm and this is higher than 361-574 ppm value reported byQin et al (2009) in China and 400 -580 ppm by Zamberlin et al (2012)in Croatia. The mean concentration of Na in this study is lower than the maximum standard indicated for food composition as shown in Table 6. The high level of heavy metals in these districts could be from the contaminated feeding hay, agricultural inputs, and agricultural left over such as "geleba" (Dried hay of maize plantations after harvest), "Chid" (Teff (Ergratois teff) straw after harvest) and the commonly used underground water.
Assuming a value of 53 g / day for milk consumption in Ethiopia per Belete et al (2014) study, the daily intake of these metals are determined and are presented in Table 7. The estimated daily intake for each metal was calculated to assess the health risks associated with trace metals due to milk consumption. The last column shows the RDA values as set by institute of medicine 2011, FAO and WHO. The density of whole, full-fat milk is very close to the density of water, which is 1.0002 g/mL. Therefore, it is possible to assume that 53 mL is equal to 53 g of milk.
The daily intake of the metals depends on both the concentration and the amount of food consumed (Farid, 2004). Since the EDI values of all the analyzed metals are below the RDA values, milk consumption at this level is safe in the study area even for children. This could be due to the less daily milk consumption rate in Ethiopia compared with 113 g/day in Mumbai reported byTripathi et al (1997), 124g/day in Spain reported by Schuhmacher et al (1993) and 224 g/day in USA reported by (Lopez et al., 1995).

Conclusion
In this

Availability of data and materials:
All data generated in the study are included in the manuscript.    water + 9mL 68% HNO3 + 2 mL 32% H202.  Map of Gurage zone of Ethiopia displaying districts selected for the study. Note: The designations employed and the presentation of the material on this map do not imply the expression of any opinion whatsoever on the part of Research Square concerning the legal status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. This map has been provided by the authors.

Figure 2
The average values for each metal in all samples (in mg/L)