LEAD LEVELS IN MILK AND ITS PRODUCTS FROM VARIOUS ENVIRONMENTS IN EGYPT

. Milk and its products are considered healthy due to their content of key nutritional elements. Despite their essential role in the human diet, they may be susceptible to contamination with many chemical pollutants from the surrounding environment, most notably heavy metals such as lead, which is considered toxic to consumers. This study evaluated lead concentrations in milk and dairy product samples collected from various Egyptian environments. The results showed significant differences (p < 0.05) in Pb concentrations in the samples collected from different environments in Egypt (industrial, traffic and rural). The average lead content in raw cow’s milk, sterilised milk, Domiati cheese, Ras cheese, processed cheese and yoghurt samples from industrial areas were 2.23, 0.33, 3.19, 5.10, 0.11 and 0.09 mg/kg, respectively. In traffic areas, lead concentrations were 1.83, 0.30, 2.72, 4.72, 0.13 and 0.09, and in rural areas, they were 0.61, 0.27, 1.52, 3.13, 0.13 and 0.09. The data showed that Pb levels in dairy product samples collected from industrial areas are very high compared with those collected from traffic areas. By contrast, the lowest Pb concentrations were recorded in samples from rural areas. It is recommended that lead levels in different foods, especially milk and its products, be evaluated at regular intervals.


INTRODUCTION
Milk and its products are considered healthy foods because they contain important sources of essential nutrients for the body that cannot be easily obtained from plant foods.These elements include proteins, fats, carbohydrates, minerals and vitamins.The importance of these elements increases in vulnerable groups during the main stages of life, such as pregnancy and lactation, childhood, adolescence and older age (FAO, 2023).Milk protein is a complete protein vital for growth, cell restoration, immune system regulation and muscle mass augmentation throughout the body.Milk also contains various fatty acids, whether saturated or non-saturated, which benefit health.Milk carbohydrates include milk sugar (lactose) and other sugars such as galactose and fructose, along with many nitrogen-containing carbohydrates that fuel the body's activities and generate heat.Regarding minerals, among the most important are calcium, which is necessary to maintain healthy teeth and bones and reduce the risk of osteoporosis with age; potassium, which is essential for the functioning of organs and tissues, including the heart, brain, muscles and nerves; zinc, which helps the immune system to function and plays a role in metabolism and magnesium, which is essential for nerve and muscle functions.Among the vitamins in milk, the most important are those that are soluble in fat (A, D, E, K), as well as B12 and B1, which are necessary for the health of the nervous system, muscles and cells.
Despite the benefits of dairy products for humans, they may contain traces of some environmental pollutants present near milk production outlets, such as toxic heavy metals.According to the toxicity of some heavy metals and potential human exposure to them, ATSDR (2022) has listed lead as the second priority contaminant in food.In addition, IARC (2023) indicated that inorganic lead is considered a group 2A carcinogen.Accordingly, the presence of toxic metals in milk indicates the cleanliness of the production environment (Kwon et al., 2017).Therefore, evaluating the levels of toxic metals in dairy products is necessary to control the quality of the product and is an important public health measure.
Environmental pollution with lead is related to different sources such as lead alkyl additives in petrol (Filella and Bonet, 2017), as well as manufacturing processes, waste incineration and burning of coal; therefore, the presence of high levels of lead in places where factories and manufacturing processes abound is not surprising (Shammi et al., 2021).Also, contamination of milk and its products with lead may occur from other sources, including contamination of the original milk, the water used in production, or the equipment and utensils used in packaging and storage (Abdelkhalek et al., 2015).
The contamination of milk and its products with lead represents a major risk to consumer health due to its long-term toxic effects.Once lead enters the body, it is distributed to body organs such as the brain, liver, kidneys and bones and is stored in the teeth, where it accumulates over time (WHO, 2023).The magnitude of lead toxicity is highly related to the developmental stage, sex, means and timing of exposure, amount of intake, biodegradation, degree of absorption and disposal mechanisms (Sharaf et al., 2008).Therefore, continued exposure to lead, even in small doses, may cause serious human diseases, such as irregular blood functions, by reducing haemoglobin formation (Rahimi, 2013).In addition, lead can exert strong carcinogenic activity against vital tissues in the human body and influence various body systems, particularly the cardiovascular, nervous, renal and skeletal systems (Geraldes et al., 2016).Lead in milk is of particular concern to newborns and young children as they depend to a great extent on milk and dairy products for their diets (Aggarwal et al., 2022).A high level of lead may cause headaches, memory disturbances and immobility; loss of consciousness and death can occur due to neurological damage (Rehman et al., 2018).
The level of environmental pollution with lead depends on the type and intensity of activity.Industrial areas are those designated and planned for industrial development and may contain heavy industry companies such as iron steel, cement, forgings, chemicals etc.Despite the importance of industrial complexes to economies and for accelerating economic growth, the most important disadvantages are noise, traffic congestion, air pollution, sewage and health problems.Traffic areas are those crowded with traffic on the roads, whether pedestrians, vehicles, cars or other means of transportation.One of the most important negative effects of traffic congestion is the increased environmental pollution due to the increased use of cars and the resulting health problems.Rural areas are communities with very low population, traffic and industrial densities and are characterised by calm and clean air.
Recently, with the increasing interest in food safety, researchers have paid attention to research on pollution and contaminants and their danger to public health.In Egypt, due to the spread of industrial areas, which are famous for various industries, traffic areas characterised by traffic congestion and agricultural activities on a large scale, all with the daily use of dairy and its products, there has been an urgent need to evaluate the risks of lead in such products in these areas.From this standpoint, this study was conducted to determine lead levels in raw milk and some of its products from various Egyptian locations, including some industrial and traffic areas, and compare them to lead levels in rural areas during the summer and winter periods, as well as to detect unacceptably contaminated milk samples by comparing their lead levels to the maximum permissible limits.

Description of the study areas
This study estimated the Pb concentrations in samples collected in three different environments in Greater Cairo, Egypt.
The first comprised the industrial areas of Helwan and Shoubra El-Kheima, which are among the most important industrial areas established in Egypt: the North Helwan Industrial area contains 16 factories, the Central Helwan Industrial area contains 10 heavy factories and the South Helwan Industrial area contains 6 factories in the field of iron and steel, as well as cement.The city of Shubra El Kheima includes many factories, most notably in textiles, ready-made clothing, light and consumer industries and factories of electrical cables, ceramics and concrete drainage pipes.
The second area is one of heavy traffic, represented by the Faysal area, one of those most crowded with cars and one of the largest and most densely populated residential neighbourhoods.It is a commercial area, and due to traffic congestion and population increase, this may lead to increased contamination of food exposed to the atmosphere.
The third area is rural, represented by some parts of El-Qaliubiya; these are rural areas far from factories and traffic, and samples were collected from farms and animal breeding facilities.Figure 1 shows a map of the sampling sites in Greater Cairo, Egypt, including three areas (industrial, traffic and rural).

Standard solution and acids
Standard lead element solution with a concentration of 1000 mg litre, as well as high-quality concentrated nitric acid, were purchased from Merck (Merck, Darmstadt, Germany).Re-distilled water was used to dilute the samples and wash the tools used to prepare and estimate Pb in the various samples.

Milk and dairy products
A total of 480 samples of raw cow's milk and its products (160 of each area) belonging to six different products (raw cow's milk, sterilised milk, Domiati cheese, Ras cheese, processed cheese and yoghurt) were collected from industrial (Shoubra El-Kheima and Helwan), heavy traffic (Faysal) and rural areas (El-Qaliubiya) throughout the summer and winter seasons in Greater Cairo (Egypt) as shown in (Table 1), and the collected samples were stored at −20°C until analysis.

Methods and procedures
Milk and dairy product samples were extracted according to the AOAC (2000), and Pb contents were estimated with an atomic absorption spectrophotometer (PG-990 Instruments Ltd) with a flame atomiser (airacetylene), equipped with a 10-cm burner and a deuterium lamp for background correction.
Maximum absorbance was obtained by adjusting the cathode lamp to the proper wavelength (217.0 nm).The results obtained were statistically analysed according to the method of Snedecor and Cochran (1980).The least significant difference (L.S.D) test was used to determine significant differences between means and to separate means at (p ≤ 0.05) using the SPSS package version 15.0.

Method validity
Quality assurance rules and precautions were implemented to ensure the reliability of the results, including thorough calibration of instruments, validation of methods and maintenance of cleanliness through washing and protocols to avoid contamination.To evaluate the efficiency of the method used to extract the samples, known lead concentrations (0.1, 0.2 and 0.4 mg/kg) were added to milk and Domiati cheese samples.The data showed that lead recovery in fortified samples was between 96.0% and 97.0% in milk and between 94.0% and 95.0% in Domiati cheese.The detection limit for lead was calculated and reported as 0.012 mg/L for both sample types.

RESULTS
The concentrations of Pb in raw and sterilised milk, Domiati cheese, Ras cheese, processed cheese and yoghurt collected from the three different environments were determined, and the data are presented in Table 2 and Figure  2. The data revealed that Pb levels in the collected samples were differentiated according to the sample collection areas, and there were significant differences (p ≤ 0.05) in lead concentrations.The results obtained indicate that the highest mean Pb concentrations were found in raw milk, sterilised milk, Domiati cheese and Ras cheese from industrial areas, which were 2.23, 0.33, 3.18 and 5.10 mg/kg, respectively.
However, the highest level of Pb in processed cheese and yoghurt was found in traffic areas, which recorded 0.13 and 0.09 mg/kg, consecutively.In addition, it was noted that there were no significant differences (p ≥ 0.05) in lead concentrations between processed cheese samples collected from traffic areas and those collected from rural areas.Regarding the samples collected from rural areas, the mean concentrations of Pb were 0.61, 0.27, 1.52, 3.13 and 0.09 mg/kg in raw milk, sterilized milk, Domiati cheese, Ras cheese and yoghurt, respectively.These levels were lower than those detected in the other two areas.
Lead levels in raw milk and its products in the present study were determined during two periods, summer and winter.The data in Table 3 show that the highest Pb concentrations were found in samples collected from industrial and traffic areas during the winter season, which were recorded as 2.87 and 2.07 mg/kg, respectively, but no significant differences (p ≥ 0.05) were found between Pb concentrations in samples collected from rural areas in the summer and winter.
Statistical analysis confirmed that Pb concentrations in industrial and traffic areas differed significantly between the winter and summer (p ≤ 0.05).

DISCUSSION
Milk and dairy products are a key component of the human diet and are considered by diverse cultures as one of the main food groups, as they contain many essential minerals, vitamins, proteins, fats and carbohydrates (FAO, 2023).Despite these benefits, milk and dairy products may contain traces of some environmental pollutants generated near milk production outlets, such as toxic metal elements.The levels of toxic metals in the environment are increasing with increasing industrial or agricultural emissions (Ngo et al., 2021).Lead is considered one of the most important toxic metals transferred to dairy products and other foods and causes major health problems for the consumer.Many sources cause the transfer of lead to dairy products, including direct pollution through pastures and crops used as animal feed and soil pollution (Gakidou et al., 2017), water used in production, equipment and tools used in packaging and storage (Abdelkhalek et al., 2015) and environmental emissions resulting from industrial practices close to milk production outlets (Ngo et al., 2021).
The lead levels in milk and its products vary according to the degree of contamination of production environments and animal feed (Bousbia et al., 2019).In the current study, the lead content of milk (raw and sterilised) and dairy products (Domiati cheese, Ras cheese, processed cheese and yoghurt) was high in all three environments.The results revealed that the highest concentrations of Pb were recorded in samples collected from industrial areas, followed by traffic, and then rural areas.The data showed that Domiati and Ras cheese contained high levels of lead compared to other dairy products, and this may be due to the transfer of lead from milk to cheese during manufacturing processes.Also, heat evaporation during the manufacture of milk products concentrates the milk, thus concentrating pollutants in the final products.
In addition, some studies have proven that lead is linked to milk caseins, which are transferred to the curd during the coagulation step in cheese processing (Yabrir, et al., 2016).On the other hand, lead in raw milk may be due to the contamination of drinking water and animal feed with lead due to industrial waste, which is secreted in milk.
When the concentrations of Pb in samples collected from the three environments were compared with the permissible limits, the data indicated that the mean Pb levels were higher than the maximum permissible level of 0.02 mg/kg wet weight set by the commission regulation (EUR-Lex, 2015).
From the results obtained, there are differences in lead levels according to the locations where the samples were collected, as follows.

Industrial areas
Considering the levels of Pb in milk and dairy product samples collected in this study from industrial areas (Helwan and Shubra El Kheima), the average concentrations were 2.23, 0.33, 3.18, 5.10, 0.11 and 0.09 mg/kg in raw milk, sterilised milk, Domiati cheese, Ras cheese, processed cheese and yoghurt samples, respectively (Table 2 and  Figure 2).Comparing these levels of Pb with those of other levels in previous Egyptian research, Khalil (2018) reported a large discrepancy between the metal contents in the sites of Aswan, especially Edfu, Kom-Ombo and Toshka, where the concentrations of heavy metals were higher in the city of Edfu compared to other sites.
The variations were due to the intensity of industrial activity, which is higher in the city of Edfu than in the city of Toshka, which is considered a nonindustrial area.In the Egyptian Beni Suef Governorate, Abdou et al. (2017) reported that mean Pb levels in raw milk samples collected from traditional and industrial sites from four various locations in Beni Suef were 0.40, 0.18, 0.28 and 0.38 (mg/kg) in Somosta (famous for agriculture and fishing), Ehnasia (famous for urban and agricultural projects), Beni Suef (famous for cement factories and other agriculture-based industries such as cotton ginning and textile manufacturing) and Naser (famous for manufacturing and exporting handmade carpets), respectively (Table 4).In addition, El Sayed et al. (2011) reported that cow's milk samples collected from Helwan and Shubra recorded Pb levels 0.28 and 0.58 mg/kg, consecutively (Table 4).
A comparison of Pb levels in this study with those in studies conducted in Romania (Miclean et al., 2019), India (Kumar, 2019), Iran (Sobhanardakani, 2018) and Kazakhstan (Sarsembayeva et al., 2020) revealed lower Pb concentrations of 24.0, 0.21, 32.83 and 11.60 µg/kg, respectively (Table 4).In China, Su et al. (2021) reported that the highest Pb concentration (2.68 μg/kg) in raw milk (Table 4) was detected in Tianjin, which was in an industrial area compared to Pb levels (2.29 μg/kg) in raw milk samples from Hohhot (nonindustrial area).They reported that the levels of metals in raw milk vary in some areas due to the differences in contamination levels (Boudebbouz et al., 2021).In Poland, Sujka et al. (2019) reported that the Pb content in samples of dairy and milk products was related to the level of industrialisation in the sample collection area.Higher levels of Pb were found in products from Silesia (the most industrialised region of Poland) than in those from the other (non-industrialised) regions, which recorded a mean value of 0.23 mg/kg.
In 2022, Zyambo et al. (2022) studied the extent of Pb contamination of cow's milk from cattle raised near a Pb and zinc mine in Kabwe, Zambia.They reported that Pb levels in cow's milk had different concentration patterns depending on the season, distance and location of the farmer from the Pb and zinc mine, with average Pb levels generally ranging between 0.60 and 2.22 µg/kg and 0.50 and 4.24 µg/kg in the wet and dry seasons, consecutively.In Rajasthan, a state in northern India, Ram Bilas Meena, Meena and Dakshene (2022) reported that mean Pb levels were 0.15 mg/kg (Table 4).
Generally, Pb contamination levels in milk and dairy products in industrial areas depend on several factors related to geographical locations and the extent of industrial activity as well as the resulting exhausts and the Pb they carry in these sites, which contaminates animal feed, water and soil (Meshref et al., 2014).
Moreover, the food chain is subject to Pb contamination as a result of absorption by plants from the polluted environment (Nachiyunde et al., 2013); consequently, Pb accumulates in the body and tissues of farm animals and is excreted in milk (Younus et al., 2016).In addition, climatic conditions, the lactation period of farm animals and animal feed ingredients may have a significant impact on Pb levels in milk (Bousbia et al., 2019).

Traffic areas
This study reported the extent of contamination of milk and its products by Pb in traffic areas in Cairo that suffer from traffic and population congestion, such as the Faysal area specifically, the sample collection area.The results indicated that the Pb content in the samples of milk and its products under investigation was high.The mean levels of Pb in raw milk, sterilised milk, Domiati cheese, Ras cheese, processed cheese and yoghurt were 1.83, 0.30, 2.72, 4.72, 0.13 and 0.09 mg/kg, respectively (Table 2 and Figure 2).
In general, Pb levels in the samples of dairy products during this research were higher than those in some other Egyptian studies.This may be due to many causes, the most important of which is the heavy traffic in the sample collection area and the accompanying Pb exhaust that pollutes the environment.Therefore, the main source of Pb in this area is heavy traffic emissions, although many Egyptian national projects are using unleaded gasoline and natural gas cylinders.The level of Pb in the atmosphere varies from one country to another, depending on the density of motor vehicles.
Previous Egyptian studies (Table 4) confirmed high lead concentrations in raw milk and its products from local markets, some of which were higher than the permissible international levels.A comparison of lead levels in raw milk in the present study with those in studies conducted in Cairo (Abou-Arab et al., 2008), Alexandria (Amer et al., 2021), Beni-Suef (Meshref et al., 2014), Mansoura (Elafify et al., 2023), Damanhour (Saleh et al., 2019) and Assiut (Wafy, 2019) reported lower Pb concentrations of 0.03, 0.05, 0.21, 0.10, 0.11 and 0.08 mg/kg, respectively.By contrast, Malhat et al. (2012) detected higher levels of Pb in El-Qaliubiya in 2012.On the other hand, Khalil (2018) found that Pb concentrations in the types of buffalo milk, cow's milk, goat's milk and sheep milk samples collected from the markets of Aswan City varied, as these concentrations ranged between (0.16 and 0.73), (0.21 and 0.43), (0.14 and 0.25) and (0.15 and 0.74 mg/kg), respectively.
In general, these concentrations are lower than those detected in the current study.Regarding Pb levels in sterilised milk, the data proved that these levels were higher than those detected in Cairo (Abou-Arab et al., 2008) and Alexandria (Gomaa et al., 2008), which recorded 0.02 and 0.21 mg/kg, consecutively.

Concerning
Ras cheese, Pb concentrations were higher than those detected in the samples collected from El-Qaliubiya markets, which averaged 1.33 mg/kg (Elbarbary and Hamouda, 2015).Regarding Pb levels in processed cheese, they were lower than those detected in El-Qaliubiya (Elbarbary and Hamouda, 2015) and Menofia (Hussien and Nosir, 2017) samples, which were 0.57 and 0.43 mg/kg, respectively, and higher than those detected in Assiut samples (Abd El Rahim et al., 2012), which averaged 0.02 mg/kg.The results obtained show the variation in Pb levels in the collected samples.The levels of Pb in Domiati cheese and Ras cheese were much higher than the Pb percentage in milk and other products.This may be due to the type of milk used in manufacturing cheese, in which the level of Pb in cheese is concentrated, as well as the type of salt used (NaCl) in the cheese-making process (Abou-Arab and Abou Donia, 2002).
Similar results were obtained in France (Maas et al., 2011) and Romania (Năstăsescu et al., 2020); conversely, higher mean concentrations of Pb were detected in the Iğdır region in Turkey (Koyuncu and Alwazeer, 2019).

Rural areas
Regarding rural area samples in the present study, the data showed significantly lower Pb levels in raw milk, sterilised milk, Domiati cheese and Ras cheese than those detected in industrial and traffic areas, where the mean concentrations recorded were 0.61, 0.27, 1.52 and 3.13 mg/kg, respectively (Table 2 and Figure 2).As for Pb concentrations in the three areas from which samples of processed cheese and yoghurt were collected, their lead concentrations were very similar.
However, average Pb concentrations in rural area samples were higher than international permissible limits.Several studies in Egypt regarding the contamination of milk and its products collected from rural areas confirmed that Pb concentrations were higher than permissible international limits.In the present study, Pb levels were higher than those detected in Mansoura (Enb et al., 2009) as well as Sohag and Qena governorates (Ateteallah et al., 2015), which were 0.08 in raw buffalo's milk and 0.07 mg/kg in raw cow's milk and 0.09 mg/kg in raw buffalo's milk, respectively (Table 4).El Sayed et al. (2011) reported that the mean concentrations (mg/kg) of lead in raw milk samples collected from farms in Awseem, Menofia, Mansoura and Gharbia were 0.14, 0.61, 0.14 and 0.14, respectively, while Abdou et al. (2017) detected Pb levels in raw cow's milk samples from four dairy farms in different cities representing Beni Suef governorate, Egypt.
It was shown that the average Pb concentrations were 0.40 (Somosta City), 0.18 (Ehnasia City), 0.28 (Beni Suef City) and 0.38 mg/kg (Naser City).In addition, Mansour et al. (2019) reported that mean Pb levels in the samples of milk and dairy products collected from Sharkia governorate, Egypt were 2. 68, 2.04, 2.78, 1.85, 1.94 and 2.26 (mg/kg) in raw cow's milk, raw buffaloes' milk, raw sheep's milk, raw goat's milk, UHT milk and Kariesh cheese, respectively.The mean values of Pb in the examined samples reported herein were relatively higher than those obtained for raw milk (Table 4) by Bilandžić, (2016) in Croatia (11.40 μg/l), Ram Meena (Meena and Dakshene, 2022) in Rajasthan, India (0.04 mg/kg) and Sharifi (Sharifi et al., 2022) in Tehran province from Iran (0.003-0.19 mg/kg).Although Pb levels in rural areas were lower than those in industrial and traffic areas, Pb concentrations were higher than permissible international limits and higher than Pb concentrations in many local and international studies.
This may be due to many causes, such as raising pasture animals near industrial or traffic areas, which can be considered one of the possible causes of contamination of livestock feed and water, and as a result, high Pb contamination in raw milk.
Furthermore, the livestock farming environment and surrounding atmosphere may be contaminated with high concentrations of Pb, which in turn causes contamination of the milk at all stages of production.

Lead levels in the samples through the summer and winter seasons
The data revealed that Pb concentrations were variable among samples collected through two seasons (Table 3).There were significant differences (p ≤ 0.05) in Pb concentrations between samples collected from industrial and traffic areas through the summer and winter seasons.This variation depends on the production season and regional differences (Saleh et al., 2019).It is worth mentioning that the differences in Pb levels between samples confirm its variation and instability in the environment and thus the varying levels of pollution due to seasonal variation.Some studies have been conducted on the seasonal variation in Pb levels in the environment and atmosphere.In one of the studies on the city of Cairo during the period from the winter of 1998 to the winter of 1999, Meshref et al. (2014) reported that Pb levels were high in the winter compared to the summer, and these levels decreased during the period 2001 and 2002 in the atmosphere of the Faysal region, but Pb levels were also higher in winter than in summer (Rehman et al., 2018).

CONCLUSIONS AND RECOMMENDATIONS
Therefore, the main source of Pb in the area is likely to be heavy vehicle traffic, although it is also possible that historical pollution entering the food chain via contaminated soils plays a role.Further studies/references are needed to confirm these findings.

Figure 1 -
Figure 1 -A map of sampling sites in Greater Cairo, Egypt showing three districts (industrial, traffic and rural)

Figure 2 -
Figure 2 -Lead levels (mg/kg) in milk and some dairy products collected from industrial, traffic and rural areas in Greater Cairo, Egypt

Table 1 -
Milk and some dairy product samples collected from each area (industrial, traffic and rural) in Greater Cairo, Egypt

Table 2 -
Lead levels (mg/kg) in milk and some dairy products collected from industrial, traffic and rural areas in Greater Cairo,

Egypt Milk and its products Concentrations (mg/kg) LSD at 5% Industrial areas Traffic areas Rural areas Mean±SD Range Mean±SD Range Mean±SD Range
All values are means of sample number determinations in each area ± standard deviation (SD).Means within columns with different letters are significantly different (p ≤ 0.05).(%) samples outside the maximum permissible level of Pb (0.02 mg/kg wet weight) set by the commission regulation (EUR-Lex, 2015) were 100%

Table 3 -
Lead levels (mg/kg) in milk and some dairy products collected from industrial, traffic and rural areas in the summer and winter seasons

Table 4 -
Lead levels in raw milk and its products reported in the different countries studied (mg/kg)