Introduction

The rapidly increasing process of industrialization and urbanization in developing nations has resulted in notable environmental issues, specifically the pollution of soil and water resources. Untreated industrial effluents, sewage, and agricultural runoff are significant sources of environmental pollution in various regions, including Pakistan. This pollution results in the buildup of heavy metals in the ecosystem1. These contaminants present a substantial risk to the safety of food, particularly in regions where wastewater is commonly employed for irrigation. Wastewater irrigation is a prevalent method used in urban and peri-urban areas, particularly in locations with limited water resources. It has been shown that approximately 7% of the global agricultural land is irrigated with wastewater. However, this percentage is likely to be higher due to underreporting2. Utilizing wastewater in agricultural practices is widely acknowledged as a significant contributor to soil and food pollution. This, in turn, poses health hazards to consumers as harmful heavy metals accumulate in crops and animal-derived goods3.

In Pakistan, the practice of utilizing wastewater for irrigation is prevalent, with approximately 350,000 hectares of land being directly irrigated and an additional 550,000 hectares indirectly impacted by wastewater4. This practice has resulted in the pollution of soil and water resources, specifically in urban areas like Karachi, Lahore, and Faisalabad, where untreated wastewater is frequently utilized for agricultural activities5. Repeatedly applying wastewater that is contaminated with heavy metals results in the buildup of these toxic substances in the soil. Consequently, these heavy metals are subsequently absorbed by food crops, so presenting significant health hazards to both animals and people6. Buffalo in Pakistan are widely recognized as the primary source of milk and are sometimes referred to as "black gold." Their milk is highly regarded for its nutritional value across all age groups of the population7. Buffaloes are often fed fodder and silage grown on land irrigated with contaminated wastewater. This practice has been identified as a key factor in the contamination of milk with heavy metals, which can have severe health implications for consumers7. Consuming milk derived from buffaloes and animals that have been exposed to contamination and have been grazing in polluted locations can potentially have severe consequences for human well-being8. Research has indicated that agricultural goods cultivated in polluted soils are a main contributor of toxic metals found in animal-derived food items, such as milk9.

Environmental pollution is the primary cause of the presence of heavy metals including cadmium (Cd), arsenic (As), and chromium (Cr) in animal feed. The primary origins of these harmful metals are industrial operations, inadequate waste management, and the utilisation of polluted water and soil in agricultural practices10,11. Contaminated feed consumed by dairy animals leads to the accumulation of metals in their tissues, which are eventually excreted into the milk7. Not only does this diminish the milk’s nutritional content, but it also presents significant health hazards to users. Extended exposure to high levels of heavy metals from the intake of milk can result in many health problems, such as neurotoxicity, renal impairment, and an elevated susceptibility to chronic illnesses12. It is essential to comprehend the influence of various feed types on the amounts of harmful trace metals in milk, particularly in Pakistan and other regions where buffalo milk is a primary source of nutrition13. The fresh buffalo milk available in markets is often sourced from animals that are nourished with a variety of feeds, such as alfalfa fodder, maize silage, or a combination of both14. Given the potential health risks, there is an urgent need for comprehensive studies that quantify the levels of these metals in milk based on different feeding practices and assess the associated health risks15.

Therefore considering all the above facts and figures this study has objectives (i) to examine the fluctuations in the physicochemical characteristics (pH, electrical conductivity, density, total solids, moisture, and fat content) of buffalo milk when different feed types (alfalfa, maize silage, and a combination of both) are used (ii) to assess and contrast the levels of toxic trace metals (Lead, Cadmium, Mercury, Arsenic, Manganese, Iron) in buffalo milk. (iii) to evaluate health hazards for adults and children by computing hazard quotients (HQ) and hazard indices (HI). (iv) to employ Principal Component Analysis (PCA) in order to discover the primary elements that have an impact on milk quality and trace metal levels, with a focus on different feed types. The findings aim to provide insights that could inform strategies for monitoring and controlling contamination in milk, thereby ensuring its safety for consumers.

Materials and methods

Study area

The study was carried out in Tehsil Daska, District Sialkot, an area known for its significant industrial and agricultural operations. The region is renowned for its industrial output, encompassing the creation of sports goods, surgical instruments, and various other manufacturing industries, all of which contribute to environmental pollution16. As per the report of the local government which shows significant maize cultivation in this area17. After a thorough field survey of this area, this is revealed that along with the maize the farmers of that area also use alfalfa as fodder. Alfalfa has rich nutritional value and fast a growing crop18. Rapid growth and readily availability of alfalfa made it more suitable for fodder. Farmers in the region frequently growing both of these crops and utilize untreated wastewater for irrigation, which presents a potential hazard of soil and water contamination with heavy metals18,19.The study regarded these environmental factors as important, as they could affect the levels of contamination in buffalo milk produced in this region19. The study region was chosen to examine the influence of nearby industrial and agricultural activities on the quality of buffalo milk, with a specific emphasis on the detection of harmful trace metals.

Sample collection, storage and transportation

A field survey was conducted to identify the farmers who feed their buffalos with various categories of feed i.e. alfalfa fodder, maize silage and a combination of both. Fifteen sampling points were selected across the study area (Fig. 1). At each point, milk was sampled from two lactating buffaloes for three consecutive days. It facilitated an effective assessment of milk quality and levels of contamination of the milk of buffaloes given these feed categories (alfalfa fodder, maize silage and its combination). A total of 90 milk samples were collected. The milk samples were collected from the farms situated near the city line. The samples were collected in clean glass bottles (500 mL) directly from the farms to avoid the chances of after-milking contamination. To ensure the quality of samples, these were properly labelled, and stored in ice during transportation and were immediately sent to the laboratories of the Pakistan Council of Scientific and Industrial Research and were stored at −20˚C for 24hrs20,21.

Fig. 1
figure 1

Location map of study area tehsil Daska, Sialkot.

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Physico-chemical attributes

The physico-chemical attributes of milk samples such as pH, conductivity, moisture content, and fat content was determined using the standard procedures and calibrated equipment. The pH values of the samples were measured using a digital pH meter (Jenway, UK)22while the electrical conductivity was determined by conductivity meter23. Moisture content (MC) was determined by the difference between the known weight of the milk sample and the determined weight of the total solid after evaporating the liquid component of the milk sample on a hot plate24.

$${\text{Moisture Content }}\left( {{\text{MC}}} \right){\text{ in milk }} = \, \left( {{\text{W1}} - {\text{W2}}} \right)/{\text{W1 }} \times { 1}00$$

Here, W1 = original weight of the milk in raw form, and W2 represents weight after drying (evaporation).

For the determination of the fat content routine Method “Gerber” (1,5) was used. The method consists of introducing 10 mL of H2SO4 into the butyrometer followed by pipetting 10 ml of milk sample into the butyrometer. Later 1 ml of amyl alcohol was pipetted onto the milk. The butyrometer was closed with a rubber stopper and shake vigorously until the liquids are thoroughly mixed. Then the butyrometer was placed in a Gerber centrifuge (two tubes on two opposite sides). At the end of the proper time approximately 5 min the butyrometer was placed in the heated water bath at 65°C for 5 min. Finally, the scale reading was calculated. The gravimetric method (drying at 102 °C) was used for the determination of total solid contents of the milk samples. An accurate quantity of approximately 5 ml of milk is weighed into pre- weight round bottom glass dish provided with a lid (5 cm diameter). The uncovered dish was placed in a boiling water bath until most of the moisture was driven off. The dish was transferred to a well-ventilated oven at 102 °C. The dish with the lid apart was dried for 2 hours in the oven and then the dish was covered with the lid cooled for 30 minutes in a desiccator and weighed. The dish and the lid were heated again for 30 minutes periods in the oven, cooled, and weighed until the difference between the two successive weightings did not exceed 1 mg.

Sample digestion and metals analysis

For the digestion of milk samples, 10 ml of HNO3was added to 1 ml of milk in a 100 ml conical flask and heated at 80 ℃ for 20 minutes. After allowing the flask to cool to ambient temperature, 5 ml of perchloric acid was added and heated at 180 ℃ until the volume reduces to 2–3ml. Deionized water was used to dilute the digested milk samples up to 50 ml. For metal analysis, samples that were prepared were placed onto an Atomic Absorption Spectrophotometer (AAS) (Thermo Scientific 3000 Series, Waltham, MA, USA) using a flame produced by a mixture of air and acetylene. Six potentially toxic metals i.e., Cd, As, Pb, Hg, Fe, and Mn were quantified in the digested samples of buffalo milk using an AAS (Perkin Elmer-Waltham, MA, USA, S# 8015050702), following the procedure as explained earlier by25. Stock solutions (1000 mg/L) of each metal were prepared using double-distilled water and used for preparing the working standards of different concentrations. For quality assurance, certified/standard reference material (NIST SRM 1515) was also analyzed under similar conditions, and it depicted an excellent recovery (98–100%). In addition, about 10% of the samples were used for inter-laboratory analysis, and the variation in the results was less than ± 3.0%26.

Health risk assessment

To evaluate the risks associated with consuming buffalo milk in the study area, the United States Environmental Protection Agency (USEPA) statistical health risk assessment tool was used. By using this tool, various parameters such as estimated daily intake (EDI), Target Hazard quotients (THQ) and hazard indices (HI) were computed for various trace metals. This standard methodology has been adapted globally in many recent studies as mentioned by Giri and Singh (2020) and Rafiq et al. (2022). The Provincial Tolerable Daily Intake (PTDI) values as mentioned by FAO/ WHO (JECFA, 2003) are also considered for the trace metals for which reference dose is not available.

The Estimated Daily Intake (EDI) of metal was determined by the following equation;

$${\text{Estimated Daily Intake }}\left( {{\text{EDI}}} \right) \, = {\text{ CM }} \times {\text{ IR}}/{\text{ Bw}}$$

Here, the CM was the concentration of metals determined in the buffalo’s milk samples, IR was the average ingestion rate and Bw was the body weight as mentioned in Table 1.

Table 1 Variables for Health Risk Assessment of Buffalo milk.
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Target health quotient (THQ)

Target hazard quotient (THQ) proposed by US EPA (1989) determines the ratio of the estimated daily intake (EDI; mg kg−1 d−1) of a metal through oral reference dose of that particular metal (RfD = mg kg−1 d−1)31. This presents the maximum intake of a specific metal daily dose without causing any adverse health impacts. The equation for calculating THQ was presented below:

$${\text{THQ }} = {\text{ EDI }}/{\text{ RfD}}$$

The dosage of a particular metal; HRI value of metal <1.00 were regarded as safe whereas the HRI >1.00 was considered as unsafe for human consumption.

$$HI=\prod_{i=1}^{n}THQi$$

Here, THQi was the target hazard quotient of a single metal. HI was the total hazard index for all the metals studied in this study and n=6.

Statistical analysis

Data collected in this study was validated and analyzed statistically using various descriptive statistics and geo-spatial tools. The correlation of various variables was determined using Principal Component Analysis (PCA) using XLSTAT software (Version 2021), and biplots were generated to compare the correlations among the observed data. Spatial distribution maps of various measured elemental concentrations were developed using ArcGIS software (Version 10.3).

Results

Physicochemical attributes

pH, EC and milk density

The findings of the current study revealed that various feed categories showed variation in the physico-chemical attributes of buffalo milk from the study area. The range of pH (5.12–9.13), electrical conductivity (995–1799 µS/cm), density (17.2 to 30.29 g mL−1) of all the milk samples. About 50% of milk samples showed variation in pH and EC from the permissible range given by the WHO. The WHO range given for buffalo milk is 6.75–6.8732. Maximum pH (9.13), EC (1799 µS/cm) and density (30.29 g mL−1) was observed in those samples where the buffalos were fed by fodder (Alfalfa), followed by in those samples where the buffalos were fed on the mixture of alfalfa and maize silage. While minimum pH (5.12), EC (995 µS/cm) and density (17.2 g mL−1) of buffalo milk was observed when the buffalo were fed to maize silage for one month (Fig. 2, 3, 4).

Fig. 2
figure 2

Spatial distribution maps of pH in milk samples buffalo based on three different feed categories. (a) fodder (alfalfa), (b) silage (maize) and (c) mixture of fodder and silage. Variation in the size of bars indicates the level of pH of buffalo milk samples of different study points.

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Fig. 3
figure 3

Spatial distribution maps of electrical conductivity in milk samples buffalo based on three different feed categories. (a) fodder (alfalfa), (b) silage (maize) and (c) mixture of fodder and silage. Variation in the size of bars indicates the level of EC of buffalo milk samples of different study points.

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Fig. 4
figure 4

Spatial distribution maps of density of milk samples buffalo based on three different feed categories. (a) fodder (alfalfa), (b) silage (maize) and (c) mixture of fodder and silage. Variation in the size of bars indicates the level of density of buffalo milk samples of different study points.

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Total solids, moisture and fat contents

It was observed that various feed categories showed variation in the total solids, moisture and fats contents of buffalo milk from the study area. The range of total solids (10.12–15.98 mg L−1), moisture contents (84.5–90%) and fat contents (3.9–10.9%) was observed in all the milk samples of the study area. About 80, 50 and 85% of milk samples showed variation in total solids, moisture and fats contents respectively, from the permissible range given by WHO. Minimum total solids content (10.12 mg L−1), moisture content (89.77%) and fat contents (10.9%) were observed in those samples where the buffalos were fed by fodder (Alfalfa), followed by in those samples where the buffalos were fed on the mixture of alfalfa and maize silage. While minimum total solids (15.98 mg L−1), moisture content (84.5%) and fat content (3.9%) of milk samples were observed when the buffalo were fed to maize silage for one month (Fig. 5, 6, 7).

Fig. 5
figure 5

Spatial distribution maps of total solids in milk samples buffalo based on three different feed categories. (a) fodder (alfalfa), (b) silage (maize) and (c) mixture of fodder and silage. Variation in the size of bars indicates the level of total solids of buffalo milk samples of different study points.

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Fig. 6
figure 6

Spatial distribution maps of moisture content in milk samples buffalo based on three different feed categories. (a) fodder (alfalfa), (b) silage (maize) and (c) mixture of fodder and silage. Variation in the size of bars indicates the level of moisture content of buffalo milk samples of different study points.

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Fig. 7
figure 7

Spatial distribution maps of fat content in milk samples buffalo based on three different feed categories. (a) fodder (alfalfa), (b) silage (maize) and (c) mixture of fodder and silage. Variation in the size of bars indicates the level of fat content of buffalo milk samples of different study points.

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Metals and metalloids attributes

Lead contents (mg L −1 )

It was observed that Pb concentration in buffalo milk varied based upon the feed category. Overall, the Pb concentration ranged from (0.00229–0.029 mg L−1) in all feed categories. The maximum concentration of Pb was detected in fodder fed buffalo milk with 67% samples above the permissible limit of WHO (0.02 mg L−1). While in mix category fed buffalos 47% of the samples were above the permissible limit, whereas lowest concentration of Pb was detected in maize silage fed buffalos milk (7%) (Fig. 8).

Fig. 8
figure 8

Spatial distribution maps of lead contents in milk samples buffalo based on three different feed categories. (a) fodder (alfalfa), (b) silage (maize) and (c) mixture of fodder and silage. Variation in the size of bars indicates the level of lead of buffalo milk samples of different study points.

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Cadmium contents (mg L −1 )

The findings of the current study revealed that feed categories showed variation in the Cd concentration of buffalo milk from the study area. The Cd concentration of all the milk samples ranged from (0.00122–0.058 mg L−1). The fodder fed buffalos milk contained highest concentration of Cd with 47% samples, followed by mixed fed with 41% samples and maize silage fed 13% samples were above the permissible limit given by WHO (0.01 mg L−1) (Fig. 9).

Fig. 9
figure 9

Spatial distribution maps of cadmium contents in milk samples buffalo based on three different feed categories. (a) fodder (alfalfa), (b) silage (maize) and (c) mixture of fodder and silage. Variation in the size of bars indicates the level of cadmium of buffalo milk samples of different study points.

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Mercury contents (mg L −1 )

Results showed that that feed categories showed variation in the mercury of buffalo milk from the study area. The Hg concentration of all the milk samples ranged from (0.00101–0.03 mg L−1). Maximum mercury concentration (0.03 mg L−1) was observed in those samples where the buffalos were fed by a mixture of fodder (Alfalfa) and maize silage, followed by in those samples where the buffalos were fed on the fodder (Alfalfa). While minimum mercury concentration (0.00101 mg L−1) of milk was observed when the buffalo were fed to maize silage for one month (Fig. 10).

Fig. 10
figure 10

Spatial distribution maps of mercury contents in milk samples buffalo based on three different feed categories. (a) fodder (alfalfa), (b) silage (maize) and (c) mixture of fodder and silage. Variation in the size of bars indicates the level of Hg of buffalo milk samples of different study point.

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Arsenic contents (mg L −1 )

It was noticed that feed categories showed variation in the As concentration of buffalo milk from the study area. The As concentration of all the milk samples ranged from (0.0007–0.198 mg L−1). The fodder fed buffalos milk contained highest concentration of As with 44% samples, followed by mixed fed with 34% samples and silage fed 23% samples above the permissible limit given by WHO (0.1 mg L−1) (Fig. 11).

Fig. 11
figure 11

Spatial distribution maps of arsenic contents in milk samples buffalo based on three different feed categories. (a) fodder (alfalfa), (b) silage (maize) and (c) mixture of fodder and silage. Variation in the size of bars indicates the level of arsenic of buffalo milk samples of different study points.

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Manganese contents (mg L −1 )

It was noticed that feed categories showed variation in the manganese contents of buffalo milk from the study area. The manganese of all the milk samples ranged from (0.079–0.4894 mg L−1). About 50% of milk samples showed variation from the permissible range (0.2–0.4 mg L−1) given by WHO. Maximum manganese concentration (0.4894 mg L−1) was observed in those samples where the buffalos were fed by fodder (Fig. 12).

Fig. 12
figure 12

Spatial distribution maps of manganese contents in milk samples buffalo based on three different feed categories. (a) fodder (alfalfa), (b) silage (maize) and (c) mixture of fodder and silage. Variation in the size of bars indicates the level of manganese of buffalo milk samples of different study points.

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Iron contents (mg L −1 )

The current study revealed that feed categories showed variation in the iron contents of buffalo milk. The iron contents in all the milk samples ranged from (1.006–5.14 mg L−1). About 40% of milk samples showed variation from the permissible range (5 mg L−1) of iron contents given by WHO. Minimum iron concentration (1.006 mg L−1) of milk was observed when the buffalo were fed to maize silage for one month (Fig. 13).

Fig. 13
figure 13

Spatial distribution maps of iron contents in milk samples buffalo based on three different feed categories. (a) fodder (alfalfa), (b) silage (maize) and (c) mixture of fodder and silage. Variation in the size of bars indicates the level of iron of buffalo milk samples of different study points.

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Health risk assessment

Overall, the hazard quotient calculated for adults showed that overall, for HMs the HI was less than 1 for all three types of fields i.e., fodder, silage and mix. This indicate that it is safe for consumption (Tables 2 and 3). The HI of buffalo milk fed on fodder, silage and mix were ranged from 0.06–0.18 with an average of 0.09 for adults and 0.42–1.32 with average 0.62 for children. For silage the HI ranged from 0.00–0.41 with average 0.07 and mix feed ranging from 0.00–0.39 with an average of 0.09, while in children the HI was in order fodder (0.42–1.32), silage (0.01–3.01) and mix (0.02–2.85). moreover, it can be observed that for children the HI is unsafe indicating potential to cause carcinogenic and other chronic impacts (Table 4).

Table 2 EDI, THQ of heavy metals (Lead, Cadmium and Mercury) in adults and children in the study area.
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Table 3 EDI, THQ of heavy metals (Arsenic, Manganese and Iron) in adults and children in study area.
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Table 4 HI of Heavy metals in adults and children consuming buffalo milk of three different feeds.
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Principle component analysis

The biplot derived from the Principal Component Analysis (PCA) depicts the associations between the physico-chemical and metallic characteristics of buffalo milk samples across several feed categories, specifically fodder, silage, and a combination of both. The study reveals that the first two principal components, PC1 and PC2, explain 17.2% and 13.6% of the overall variation in the data, respectively. The components mentioned here are essential for comprehending the impact of different feed types on milk composition as they capture the most important variations among the evaluated properties.

The biplot graphically illustrates the distribution of milk samples in relation to these components, with each sample’s position indicating its link with the feed categories. A larger displacement from the center of the plot suggests a greater variability in the measured characteristics of the milk samples.

The milk samples obtained from buffaloes that were fed with fodder displayed the most pronounced variance in all the evaluated characteristics, indicating that this particular type of feed has a major impact on the variability observed in milk composition. In contrast, milk samples from the category of cows fed with silage showed very little variance, suggesting a more uniform attribute profile. This trend indicates that fodder may contribute to more variability in the physico-chemical and metallic properties of milk compared to silage, which seems to have a stabilizing influence (Fig. 14).

Fig. 14
figure 14

Principal component analysis (PCA) of scores (sampling sites) and loadings (variables) showing association among measured parameters.

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Discussion

The analyzed data of this study indicate the significant influence of various feed categories on the physicochemical attributes and heavy metal concentrations in buffalo milk. Livestock feed play a significant role in the milk yield and overall quality of milk33. Understanding these relationships is crucial for ensuring the quality and safety of dairy products, especially considering their importance in human nutrition34.

It is observed that changing the silage given to buffaloes affects the pH level of their milk accordingly. Buffaloes fed maize silage have lower milk pH levels or an acidic nature, while those fed alfalfa silage show higher pH levels or an alkaline nature. It is because of the action of lactic acid bacteria, which metabolize water-soluble sugars into organic acids, predominantly lactic acid, through solid-state lactic acid fermentation. Consequently, this enzymatic process reduces the pH level of the silage, thereby inhibiting the growth of spoilage microorganism. While those buffaloes fed alfalfa silage show higher pH levels or an alkaline nature, because this fodder mostly grow on marginal lands and have ability to store extra minerals. Buffaloes fed mixed fodder exhibit less variation in milk pH, as the acidic and alkaline nature of the fodder neutralizes each other. Certain forages have alkaline or acidic properties that affect the overall acidity of the milk20,35,36,37. The contaminants in the feed of livestock not only affect the milk quality but also very harmful for the livestock’s health38. The current study shows that changes in EC are associated with changes in milk components such as minerals, salts, and proteins. The superior nutritional profile of alfalfa silage, characterized by its abundance of minerals and high protein content, surpasses that of maize silage. Consequently, the electrical conductivity (EC) of milk from buffaloes consuming maize is lower, while those fed alfalfa exhibit higher EC levels. This divergence in EC values reflects the heightened mineral and protein concentrations absorbed by buffaloes grazing on alfalfa, elucidating the nutritional disparities between the two fodder sources. The diet of buffaloes, including silage and fodder, has strong impact on the mineral content of milk, potentially influencing EC39. Total solids in milk include components like fat, protein, lactose, minerals, and some vitamins. The composition of the diet significantly influences the levels of various components in milk40. Electrical conductivity (EC), density, total solids, moisture, and fat contents of buffalo milk samples show the complex interplay between feed types and milk composition. Notably, deviations from WHO standards in a substantial percentage of samples underscore the potential implications for milk quality assurance and regulatory compliance. It’s essential to recognize the multifactorial nature of milk composition, with feed quality emerging as a critical determinant41.

Measured levels of fats contents and moisture contents were higher in the fodder category of feed as compared to silage and mixture because traditional livestock feed includes a variety of grains, seeds, and other components (Oselu et al. 2022). Depending on the specific formulation, feeds contain higher levels of fats and moisture, especially if they include oilseeds or added fats42. Buffaloes fed high-fibre diets, such as grass or silage, yield milk with slightly lower total solids. This is because high-fibre diets often lead to an increase in water intake by the buffaloes. As a result, the milk might have a higher proportion of water content relative to other solids, thus lowering the total solids percentage. High-fibre diets often result in a lower energy intake, which impacts the fat synthesis in the mammary glands, consequently leading to a slight reduction in the fat content of the milk43. The higher solid content, including fats, proteins, and minerals, results in a lower proportion of water in the milk. Changes in milk composition due to feed categories might be subtle and vary based on numerous factors, including the specific diet composition, individual variations among buffaloes, environmental conditions, and overall herd management practices. This shows that, in contrast to fodder, the chemical makeup of silage feed considerably influences the quality of milk and provides the safe quality of milk rather than fodder and mixture categories of feed44,45.

Heavy metal concentrations in buffalo milk, particularly lead (Pb), cadmium (Cd), mercury (Hg), arsenic (As), manganese (Mn), and iron (Fe), reveal distinct patterns across different feed categories. Fodder-fed buffalos exhibited higher concentrations of these heavy metals compared to those fed maize silage, suggesting a direct correlation between feed composition and heavy metal accumulation in milk. That is because alfalfa is cultivated on marginal lands and it also has an extensive root system which gives a more suitable environment for heavy metal accumulation as compared to maize silage46. There are multiple reasons why alfalfa production on marginal lands results in higher accumulation of potentially toxic elements, such as poor soil composition, limited crop rotation, poor water quality sources, and lack of monitoring and regulation. The metal content in maize silage can be influenced by various factors like soil composition, farming practices, and environmental conditions. While maize itself is not a significant accumulator of heavy metals, certain agricultural practices or contamination during the silage production affect metal levels in the plant. Therefore, the metal content in the milk of buffaloes fed maize silage varies, with levels influenced by the specific environmental conditions and farming practices associated with maize cultivation. Unfortunately, in Pakistan and many underdeveloped countries, fodder has been planted on marginal lands without consideration of its health effects or the consequences thereafter. This highlights the need for stringent monitoring of feed ingredients and their potential contaminants to safeguard milk quality and consumer health.

Spatial distributions of the physicochemical attributes of buffalo milk collected from multiple farms of various locations of study area showed variation. Measured levels of fats contents and moisture contents were higher in the fodder category of feed as compared to silage and mixture that because traditional livestock feed may include a variety of grains, seeds, and other components (Oselu et al. 2022). Depending on the specific formulation, feeds contain higher levels of fats and moisture, especially if they include oilseeds or added fats42. This shows that, in contrast to fodder, the chemical makeup of silage feed also considerably influences the quality of milk. Buffaloes fed fodder or un-processed formulated feeds, show higher levels of certain metals in their milk. Fodder grown on marginal lands or produced using agricultural products potentially contains higher levels of metals due to soil contamination, fertilizers, or other agricultural practices. Additionally, certain additives or components in concentrate feeds contribute to higher concentrations of specific metals in the milk.

The highest concentrations of Cd, Pb, Hg, As, Mn and Fe metals were determined in samples collected from farms who feed the animals with fodder. Different plants have varying capacities to absorb and accumulate heavy metals from the soil47. If the types of plants used for fodder have a higher affinity for accumulating heavy metals, it could contribute to higher concentrations in the milk48. The use of certain fertilizers and pesticides may introduce heavy metals into the soil. If these chemicals are used in greater amounts for fodder production compared to silage production, it could result in higher heavy metal concentrations in the fodder49. Pb was maximum in fodder category sample, Pb concentration for all the samples of silage category was within the permissible range given by WHO. Comparatively greater Pb concentrations were observed in this investigation than had previously been reported in Italy50, Egypt51, Azerbaijan52, and Iran53. The main reason for the highest Pb contents in the fodder category is be due to the higher affinity for lead uptake54. Certain plants have a higher affinity for lead uptake55. Plants with high lead absorption rates can transfer more lead to the animals consuming them56. The bioavailability of lead can vary depending on soil and plant types. Some forms of lead are more easily absorbed by plants, making them more likely to enter the food chain57.

This shows that, as compared to silage, the contamination of all metals in fodder is slightly above the permissible range given by WHO and FAO, while the mixture category shows both the values i.e., few within the permissible range while few above than permissible range. Hence, it shows that the silage category of milk is safe for drinking and health and is better than both the other categories. So, silage feed considerably influences the quality of milk in a better way and provides a safe quality of milk rather than fodder and mixture categories of feed58,59. For simple fodder the land is used which is irrigated with wastewater so heavy metal contents were detected more than silage. More accumulation of heavy metals in the soil makes toxic the soil and cultivated crops.

The Hazard Quotient (HQ) analysis provides valuable insights into the potential health risks associated with buffalo milk consumption. While the overall HQ values for adults indicate that milk consumption is within safe limits, alarming findings emerge for children, especially when fed on fodder or mixed diets. These results underscore the vulnerability of certain demographic groups to the adverse effects of heavy metal exposure through milk consumption, necessitating targeted interventions and regulatory measures to mitigate risks and ensure public health protection.

Milk is an ideal food source of instant energy and is considered beneficial due to its ease of digestion and dietary significance60,61. It is a valuable source of vitamins, proteins, fatty acids, calcium, amino acids and different bioactive ingredients which are essential for different regulatory processes, growth and maintenance of human health62,63,64. However, in recent years milk contamination has emerged as one of the major public health risk factors especially for infants who consume milk on a daily basis65. The contaminated milk may contain different toxic organic and inorganic compounds, heavy metals, mycotoxins and other pollutants which cause many physiological dysfunctions in humans66,67,68. It is well noted that rapid urbanization, industrialization and intensive agricultural practices have contaminated surroundings with lethal potentially toxic elements that ultimately find their ways into animal milk through different entry routes including metal-enriched animal fodder69,70,71. Hence, certain study models must be developed which can precisely estimate the concentration of various toxic metals in animal fodder and their bio transfer in milk and associated dairy products72,73.

Conclusions

The changes in food intake in animals are thus aimed at improving the milk production of a particular species by increasing the availability and enhancing the efficiency of energy, protein, and other vital nutrients. To optimize good quality milk production in buffalos, it is essential to provide good quality feed. Overall, the average concentration of metallic attributes was relatively higher than most of the previously reported levels, and specifically, the milk samples collected from the study area in the fodder feed category as compared to silage and mixture of silage and fodder. Furthermore, silage supplementation in buffalo may improve milk quality and reduce the metallic concentration in the milk samples as compared to the feed categories that are mostly produced on marginal lands and by wastewater irrigation. This might suggest that fodder plays an important role in contaminating milk. However, detailed studies should be conducted, specifically focused on regular monitoring of livestock feed and drinking water quality, animals grazing around the rivers where intensive farming is practised as well as rearing dairy animals as well as milk processing and transportation should be strictly ensured.

Data availability statement

The datasets used and/or analysed during the current study available from the corresponding author on reasonable request.