Heavy Metal Residues in Milk and Milk Products and Their Detection Method

Abstract

Milk and milk products are an essential part of the human daily diet, and their consumption is steadily increasing. Milk is regarded as a complete food because it contains all of the macronutrients including protein, carbohydrates, fat and vitamins. Milk also has a high concentration of mineral elements (metals) such as sodium, potassium, iron, calcium, magnesium, selenium, copper and zinc. They are critical for proper body growth and maintenance but excess in these metals, particularly, heavy metals cause disturbances and pathological conditions. People nowadays are concerned about food safety issues involving microbial, chemical and physical hazards. Heavy metal residues such as cadmium (Cd), lead (Pb), arsenic (As) and mercury (Hg) pose a chemical hazard. These are the main contaminants. Heavy metals are any metallic chemical elements with a relatively high density (5 g/cc) whose levels must be monitored. Atomic absorption spectroscopy can be used to estimate the heavy metal contamination in milk and milk products.

1. Introduction

Milk is a whitish liquid containing protein, carbohydrates, fat, vitamin and trace mineral elements, which are produced by mammary gland of all mature female mammals. Milk production in India increased at a growth of 6.2% in 2020–2021 reaching 209.96 million tonnes [1]. Milk products include butter, ice cream, cheese, paneer etc. These are very important components of human diets because they contain good nutritive value and are thus widely consumed by children and adults, particularly elderly people all over the world. The advancement of industry and agriculture has resulted in the release of numerous heavy metals into the environment which is harmful to the health of both animals and humans. Animals ingest heavy metals from a variety of sources including soil, water, feed and fodder. Because the mammary gland is the most physiologically active component of an animal that resulted into heavy metals are reflected in milk (Figure 1). Central Pollution Control Board found that the presence of higher levels of mercury (above Environmental Protection Agency (EPA) permissible limits) in water from several Indian states and its highly toxic heavy metal & poison with a long retention time in the human body poses a threat to the body. Metals of various types of minerals can be divided into two categories based on their relative abundance in our bodies. i.e., macro minerals are those that the human body requires in relatively large quantities such as sodium, potassium, chlorine, calcium, phosphorus, magnesium and sulfur whereas micro/trace minerals such as selenium, iron, zinc, copper, cobalt, fluorine, iodine, manganese and molybdenum are required in little amounts. These are found in nearly all cells of the human body where they help to maintain general homeostasis and are necessary for our bodies to function normally. The excess mineral can have a negative impact on human health e.g., daily intake of high levels of sodium can lead to hypertension (Blood pressure).

Figure 1.

Source of heavy metal in milk.

2. Heavy metals

A heavy metal is any metallic element with a relatively high density (5 g/cm³) that is unsafe or poisonous even at low. Heavy metal is a broad term that refers to a class of metals and metalloids with atomic densities greater than 5 g/cm³ or five times that of water [2]. Heavy metals are cumulative toxins that can cause harm even at very low levels. The toxicity of these metals is divided into two categories

1. They have really no known metabolic function but when present in the body, they disrupt the normal cellular processes which causes toxic effects in a variety of organs.

2. Bioaccumulation or the ability to accumulate in biological tissues [3].

The growth in a chemical concentration in a biological organism over time in relation to the chemical concentration in the environment is referred to as bioaccumulation. The World Health Organization (WHO), CODEX and the Food Safety and Standard Authority of India (FSSAI) have determined metal maximum residual limit (MRL) values in food products. Heavy metals that exceed the MRL level in any food or food product harm human health.

3. Heavy metal problems in milk and milk products

Because it contains nutrients such as protein, fat, carbohydrate and minerals in which milk is considered nearly a complete food in our daily diet [4]. As industrial and agricultural processes expand, which result in the concentration of physical, chemical and biological hazards in the environment grows [5]. A significant quantity of heavy metals found in plants & animals eventually finds their way into food harming both the quality of final products and human health.

Metal levels in cow or buffalo milk are currently being examined intensively, particularly in industrialized & polluted areas of both developing and developed countries. According to reports, the basic ingredients in cow and buffalo milk are rather consistent and only alter slightly based on a variety of conditions such as lactation phase, nutrition quality and environmental conditions, all of which are primarily chemical contaminants.

Because milk and milk products are primarily consumed by infants and children, residues of lead, cadmium, arsenic and mercury are of great concern. As a result, their levels in food and food products must be monitored and controlled. Heavy metal level measurement is useful not only for determining risk to human health and assessing environmental quality but also for maintaining the high quality of final food and food products [6]. Many studies have been published on heavy metals in milk and their presence in milk and various milk products has been connected to lactating cows being exposed to pollution, consuming polluted feed and water and the manufacturing process of various milk products. Lead, mercury, arsenic and cadmium levels in milk from cows grazing in open fields in Kaduna were found to be higher than the WHO recommended limit daily intake (50 ppb) [7].

Heavy metal residues such as lead (Pb), arsenic (As), mercury (Hg) and cadmium (Cd) which pose a chemical hazard are described below.

3.1 Lead as heavy metal pollutant

Lead is one of the most dangerous metals for humans, plants and animals and it is among the most common metals in the environment due to human activities. Lead mines, coal combustion, wastewater applications, industrial waste & farmyard manure are the primary reason sources of lead in the environment [8]. Lead is a non-ferrous metal that is widely used in a variety of industries including the manufacture of plastics, storage battery alloys, ceramics, cable sheathing and even paints. The production of anti-knock compounds from petrol results in increased air pollution in the environment. Vehicle exhausts are a major source of lead contamination in the environment affecting the quality of food and food products as well as the health of animals [9]. Inhalation, ingestion and skin contact are the three main routes through which lead enters the human body system. Long-term doses of lead may cause thalassemia, pale skin, decreased muscle activity, stomach pain, vomiting, wrist joint paralysis and decreased fertility and birth abnormalities (Table 1). Prolonged exposure may also cause kidney damage, liver problems, nervous system damage and eventually death in humans [14]. The maximum acceptable concentration of lead for milk and milk products is 250 ppb according to FSSAI and WHO.

Heavy metals	Application	Human health consequences	Reference
Mercury	Metallurgy industries chemical manufacturing and metal finishing, use in thermometer	Memory issues increased heart rate, tremors, kidney, brain and liver damage	[10]
Arsenic	Metal plating electroplating leather, dye production	Ulcer, liver problems and kidney damage	[11]
Lead	Metal plating battery manufacturer automotive and petroleum industries	Abortion on the spur of the moment causes nervous system damage, kidney & brain damage and liver problems.	[12]
Cadim	Electroplating, fertilizers and battery manufacturing	Cancer, lung insufficiency disturbance in liver and kidney	[13]

Table 1.

Adverse effect of heavy metals on human health.

3.2 Arsenic as heavy metal pollutant

Arsenic can be found in water that has been contaminated with industrial or agrochemical waste. Ingestion of arsenic at low doses through food or water is the main route of this metalloid into the organism with absorption taking place in the human stomach and intestines and release into the bloodstream (Table 1). Arsenic can be found in both natural and man-made environments. Arsenic contamination has been documented in a variety of foods and food items including tap water, air, foods and beverages (Table 1). Drinking water contamination is increasing as a result of industrial operations and excessive groundwater withdrawal for irrigation [15].

3.3 Mercury as heavy metal pollutant

Almost all mercury compounds are extremely toxic and can harm human and animal health even at very low levels. Mercury is subject to bioaccumulation which is the process by which organisms (including humans) absorb toxins faster than their bodies resulting in mercury levels in their bodies building up over time and causing adverse health effects in humans [16].

Human activities such as the use of fossil fuels particularly coal for heat and energy, the production of metals, cement, caustic soda, the disposal of mercury-containing waste materials etc. are the main sources of mercury in food and the food chain. Human activities that contribute to contaminated air include increased industrialization and small-scale coal burning for heat and automobiles [17]. In many state areas, mercury concentrations have increased as a result of increased atmospheric deposition which harms both humans and animals (Table 1).

3.4 Cadmium as heavy metal pollutants pollutant

Cadmium is a poisonous element that can harm your health. Its existence in water, soil, beverages, herbal medications, milk products and other places has gained recent notice. Phosphate fertilizers, nonferrous smelters, sewage sludge application and fossil fuel combustion are all sources of cadmium in soil and plants. According to FSSAI, the MRL level of heavy metals in milk and various milk products should not exceed 1.5 ppm.

Cadmium is used in plating, alloying, pigments, polymers and batteries among other things which is poisonous to people and animals (Table 1) [18].

4. Heavy metal standard for various milk products

The Food Safety and Standard Authority of India (FSSAI), CODEX and WHO tolerable weekly intake of heavy metals established standards for heavy metals in milk and milk products such as lead, arsenic, mercury and cadmium (Table 2).

Heavy metals	Standard for milk and milk product
	FSSAI (mg/l)	CODEX (mg/l)	WHO weekly tolerance toxic heavy metal intake (mg/l)
Mercury	1000	500	5
Arsenic	110	20	15
Lead	250	140	25
Cadmium	150	200	25

Table 2.

Food Safety and Standards Authority of India (FSSAI), CODEX standard and WHO Weekly tolerance intake for milk and dairy products.

High Sn and Ni contents of some milk products samples from this Arak, iran might be potentially hazardous to consumers [19].

A total of 65 cow and 126 buffalo milk samples were collected from various Haryana districts, covering both industrial and non-industrial areas, and it was discovered that the milk samples collected from various Haryana districts contained Pb, Cd, As, and Hg levels below the maximum contamination level, making them safe for human consumption [20].

The amount of iron, copper, manganese, zinc, lead, cadmium and chromium in cow milk yoghurt had fallen by 0.40–15% and buffalo milk yoghurt had decreased by 0.50–15% according to a study. Nickel, cobalt and tin levels in cow milk yoghurt were down 50–100% while buffalo milk yoghurt was down 25–50%. The level of these metals in yoghurt is dramatically reduced as a result of the high acidity and bacterial activity in the production process [4]. Another study looked at heavy metals like cadmium, mercury, lead and arsenic in milk samples collected from the Livestock Production and Management Production Department at NDRI Karnal. They discovered that higher levels of lead, cadmium and mercury in various milk products could be due to high exposure to heavy metal sources in the soil and water near hazardous waste sites while higher levels of arsenic in various milk products were mainly due to use in veterinary medicine for the eradication of tapeworms in cattle (Table 3) [21].

Type of products	Type of heavy metal (ppb)
	Milk	Paneer	Dahi	Cheese	Khoa	Milk powder
Lead	4.55–8.16	3.97–6.28	4.12–9.79	3.16–10.93	11.69–13.89	3.99–5.01
Cadmium	9.96–11.89	3.38–9.53	7.84–11.50	9.16–10.90	16.91–26.41	7.73–10.2
Mercury	4.48–7.23	4.23–8.53	4.04–8.04	4.87–8.68	7.46–10.68	3.34–5.55
Arsenic	4.87–8.94	3.77–8.98	4.05–11.32	5.01–7.73	14.61–21.04	6.7–9.7

Table 3.

Concentration of heavy metal in milk and milk products.

Source: [21]

5. Reducing the contaminations

Many studies have looked at removing heavy metal pollution from many sources particularly water sources and this method can be used to disinfect a range of various dairy products mineral absorbents like smectite and Palygorsctite were used to absorb heavy metals in recent times [22]. Some other study was using sepiolite minerals and zeolites as heavy metal adsorbent materials and corrective agents [23]. Another study used a modified rice husk with different sodium bicarbonate concentrations to absorb low levels of cadmium in aquatic settings [24]. Penaud et al. [25] discovered that lactobacilli as probiotic agent could absorb heavy metals from products such as yoghurt.

Nurdin et al. [26] investigated the effect of medicinal herbs in the diet on the quantity of lead excreted in cow’s milk where researcher discovering that cumin, white turmeric and mango turmeric reduced the amount of lead in various milk products by 98.36, 99.33, and 99.37% respectively.

Heavy metals bind to lactobacilli-specific proteins (LAB) and are then biologically absorbed and removed [27].

6. Analytical methods for estimation of heavy metals in various milk products

6.1 Atomic absorption spectroscopy

Atomic Absorption Spectroscopy (AAS) became available for the first time in 1962. Since then, there have been several rapid developments such as a variety of fuels and oxidants in atomic absorption and emission spectroscopy such as flame atomic absorption spectroscopy (FAAS), graphite furnace atomic absorption spectroscopy (GFAAS), vapour atomic absorption spectroscopy (CVAAS) and hydride vapor atomic absorption spectroscopy (CVAAS).

AAS which measures ppb levels in various milk product samples which are exceedingly sensitive. The spectra formed when the sample is excited with radiation from a hollow cathode lamp. Transitions to higher energy levels occur as a result of absorbing ultraviolet or visible light and then measure the amount of energy in the form of photons of light absorbed by the sample and sends the signal to the detector. In this procedure, the wavelengths of light transmitted by the sample are measured and compared to the wavelengths that passed through it originally (Figure 2). Atomic absorption spectroscopy is a technique for identifying an element's concentration in a sample by measuring the intensity of external absorbed radiation by a sample atom at a wavelength characteristic of the element. The absorption of electromagnetic radiation by well-separated atoms or ions in the gaseous state is quantified using atomic absorption spectroscopy (AAS). The emission of radiation from atoms stimulated by heat or other methods is measured using atomic emission spectroscopy (AES).

Figure 2.

Atomic absorption spectroscopy.

6.2 Basic principle of AAS

In this system, the atoms of various elements absorb light at different wavelengths. When analyzing a sample containing a given element, light from excited atoms produces the proper wavelength combination to be absorbed by any elements present in a sample. A lamp containing atoms emits light from excited atoms resulting in a spectrum of wavelengths absorbed by any atoms in the sample is a way to find determining atom concentration in samples (Figure 2). Atomic Absorption Spectroscopy (AAS) atomizes the sample by converting ground state free atoms to a vapour state and passing a beam of electromagnetic radiation generated by excited atoms through the vaporized sample. The sample unique atoms absorb some of the radiation because more light is absorbed the maximum number of atoms are in the vapor which transfers the signal to the detector (Figure 3).

Figure 3.

Basic principle of atomic absorption spectroscopy.

6.3 Techniques of AAS

There are various techniques which can be used in AAS for the estimation of heavy metal residue in milk and milk products, which are described below:

Flame Atomic Absorption Spectroscopy is a widely used technique for estimating or detecting heavy metals, metalloids etc. in samples (ppb). A hollow cathode lamp emits radiation from a line source of the element of interest and samples are often delivered into the flame using a sprayer or nebulizer that creates minute sample droplets. The sample particles vaporize and break down into atoms, ions and electrons as the solvent in the droplets evaporates quickly. The power of the source is reduced because atoms in the sample absorb radiation released by the identical atom in the hollow cathode lamp. A monochromator is typically used to separate a spectral line of interest from any flame source background radiation. The monochromator for sodium is adjusted to pass radiation with a wavelength of 589 nm. A liquid sample is inhaled and combined as an aerosol with flammable gases in this approach (acetylene and air or acetylene and nitrous oxide). A flame with a temperature ranging from 2000°C to 3000 °C is used to ignite the mixture (depending on which fuel gas is used). An emitted light from a lamp with a cathode made of the elements to be evaluated is carried into a monochromator where it is converted to a signal and supplied to the detector via the flame (Figure 4).

Figure 4.

Flame atomic absorption spectroscopy (FAAS).

Graphite Furnace Atomic Absorption Spectroscopy (GF AAS) – Graphite furnace atomic absorption spectroscopy is a highly sensitive spectroscopic technique for measuring various metal concentrations in aqueous and liquid samples with outstanding detection limits (ppb). GFAAS has a number of advantages over conventional analytical processes including increased sensitivity and lower limit of detection, less spectrum interference and the ability to use very small sample quantities. These all contribute to enhanced heavy metal detection or estimation accuracy. The graphite furnace is a 3000°C capable high-temperature electro-thermal atomizer system. The thermal energy of the heated graphite furnace is used to break chemical bonds inside the sample which releases free ground-state atoms capable of absorbing light energy and sending a signal to the detector & displaying the results.

Metals in solution or samples can be easily recognized using graphite furnace atomic absorption spectrophotometry (GFAAS). The approach is simple, quick and may be used to estimate metals in a variety of environmental samples including groundwater, household & industrial wastes, extracts, soils, sludge, sediments and other wastes. Except for dissolved ingredient analyses, all samples in this system must be digested before being analyzed.

Graphite Furnace Atomic Absorption Spectroscopy is similar to flame AA in that the cloud of atoms is generated by heating a tiny electrically heated graphite tube or cuvette to a temperature of 3000–3200°C. The increased atom density and tube residence length improve furnace AAS detection limits allowing detection in the ppb or level range (Figure 5). A comparative study between the Flame Atomic Absorption Spectroscopy (FAAS) and Graphite Furnace Atomic Absorption Spectroscopy (GFAAS) is described in Table 4.

Figure 5.

Graphite furnace atomic absorption spectroscopy.

	Flame atomization	Graphite atomization
Amount of sample required	1 ml	5–20 μL
Atomization principle	Atomized through the heat of flame	Atomized by heat generated when a current is passed through a resistance bulb
Sample usage (Atomization efficiency)	Approx. 10%	Approx. 90%
Shape of adsorption signal	Stationary signal	Peak shaped signal
Reproducibility	<RSD 1%	Approx RSD 2–5%
Sensitivity	PPM Level	PPB Level
Measuring timing	Short (10–30 seconds)	Long (1–5 min)

Table 4.

Difference between flame atomic absorption spectroscopy (FAAS) and graphite furnace atomic absorption spectroscopy.

Vapour Generation Atomic Absorption Spectroscopy (VG AAS) – It consists of Hydride Generation Atomic Absorption Spectroscopy (HGAAS) or Cold Vapor Atomic Absorption Spectroscopy (CVAAS). This typical approach for analyzing or estimating different mineral elements, metals and some metalloids is atomic absorption spectroscopy (AAS). However, due to interferences, poor repeatability and detection limitations, hydride generation atomic absorption spectroscopy is frequently used to analyze metalloids such as antimony, arsenic and selenium. The hollow cathode lamp, air/acetylene flame, optical system and its detector utilized in AAS are all the same in HGAAS. Materials react with sodium borohydride and hydrochloric acid to produce a volatile hydride in this process for example Arsenic interacts with sodium borohydride to form H₃AS (Arsenic sodium hydride). The functions of the hydride generating system are as follows:

1. Aspiration of the liquid sample followed by mixing with sodium borohydride (NaBH4) and hydrochloric acid (HCL).

2. The reaction produces a volatile hydride of the analyte metalloid.

3. Fill the optical cell system with gaseous hydride.

Atomic Absorption Spectroscopy (AAS) is a widely used technique for determining mineral elements in samples. However, some elements mostly metalloids have been developed due to interferences, low repeatability and inadequate detection methods. However, it is more expensive than atomic absorption spectroscopy (Figure 6). In AAS, a nebulizer is required but not in HGAAS.

Figure 6.

Hydride generation atomic absorption spectroscopy.

7. Conclusion

Heavy metals are frequently thought to be extremely hazardous and harmful for the environment. People nowadays are concerned about food safety issues such as microbial, chemical, and physical risks. Heavy metal residues such as cadmium (Cd), lead (Pb), arsenic (As), mercury (Hg) and others are major pollutants in chemical hazards. Heavy metals do not naturally arise in milk as a consequence of human activities such as industrial and agricultural processes, but they can naturally occur in milk as a result of human activities such as industrial and agricultural processes. Polluted soils are a major source of Cd and Pb which can build up in milk through the food chain. Heavy metals have become pollutants in food for a variety of reasons resulting in a concern of health issues. Atomic Absorption Spectroscopy was used to determine the amount of heavy metal contamination in milk and milk products.

Acknowledgments

Sincere thanks are extended to the Institution of Eminence (IoE) scheme, Banaras Hindu University, Varanasi (U.P.) India for support under Incentive to Seed Grant under IoE Scheme (Devt Scheme No 6031 & PFMS Scheme No 3254).