ELEMENTAL ANALYSIS AND HEALTH RISK ASSESSMENT OF COW AND BUFFALO MILK CHEESES

Abstract

This study quantified Cd, Cu, Fe, Mn, and Zn in ten commercial cow’s and buffalo milk cheese using flame atomic absorption spectrometry (FAAS) after microwave-assisted digestion with nitric acid and hydrogen peroxide. The analytical procedure was designed under the principles of green chemistry, employing reduced volumes of reagents, efficient microwave-assisted digestion, and minimal waste generation. Method accuracy was confirmed by recovery tests (80.1-120.0%), with limits of detection and quantification ranging from 0.2-3.0 and 0.6-9.0 mg kg^-1, respectively. In cow’s milk cheese, Cd (0.55 0.67 mg kg^-1), Cu (6.92 7.53 mg kg^-1), Mn (0.74 mg kg^-1), and Zn (12.71-66.63 mg kg^-1) were detected, while buffalo cheeses contained Cd (0.34 0.37 mg kg^-1), Cu (2.75 mg kg^-1), and Zn (8.14-49.33 mg kg^-1). Iron was below limit of detections in all samples. Notably, Cd concentrations in three cow’s milk cheese exceeded ANVISA’s (Brazilian Health Regulatory Agency) maximum permissible levels. However, risk assessment indicated that estimated daily intakes (EDI) for all metals were below provisional maximum tolerable daily intake (PMTDI), and hazard quotients (HQ) were < 1, indicating the absence of non-carcinogenic risks. These findings highlight the importance of monitoring Cd contamination in dairy products, while also demonstrating that environmentally friendly analytical strategies can ensure reliable results with reduced ecological impact.

Keywords:
cheese; FAAS; metals; health risk; green chemistry.

INTRODUCTION

Cheese plays an essential role in the human diet due to its richness in proteins, minerals, vitamins, and essential trace elements. Among the vast variety available, buffalo milk cheese stands out for containing the same nutrients as cow’s milk cheese, but in different amounts. It has more calcium, vitamin A, and protein, but it also has a higher fat content and, consequently, a higher caloric value. However, these characteristics can vary depending on the manufacturing process and the identity standards adopted.¹ This particular nutritional profile, combined with its characteristic flavor, makes buffalo milk cheese a highly valued product in national and international markets. Its production chain represents an important source of income, especially in the northern region of the country. Although buffalo farming has traditionally focused on meat production, dairy activity has proven to be a viable alternative for improving the socioeconomic sustainability of the agricultural sector in Brazil.²

Despite the nutritional benefits of buffalo milk and the expansion of buffalo farming in Brazil, a critical regulatory gap persists: there is no specific federal legislation for buffalo milk and its derivatives. This absence hinders the implementation of health control and inspection measures, while also creating restrictions on the commercialization of these products.³,⁴ Consequently, these products are often evaluated against standards developed for cow’s milk cheese, which is inadequate due to significant physicochemical differences.⁵ Although several studies in the literature⁶-¹³ address the composition of milk and its derivatives, focusing on the analysis of nutritional and bioactive components, much of this research has concentrated on cow’s milk cheese sources. Investigations into the inorganic composition of Brazilian cheeses, particularly those from non-bovine sources such as buffalo milk, remain scarce.

In this context, the presence of trace metals such as cadmium (Cd), copper (Cu), iron (Fe), manganese (Mn), and zinc (Zn) in cheese can represent both nutritional benefits and potential toxicological risks, depending on the concentrations present in the food. Among these metals, essential elements like Cu, Fe, and Zn play important biological roles in the human body, acting as enzyme cofactors, participating in cellular metabolism, and contributing to processes such as oxygen transport and the strengthening of the immune system.¹⁴,¹⁵ Mn is also considered an essential micronutrient, participating in the activity of antioxidant enzymes and in the metabolism of carbohydrates and lipids.¹⁶,¹⁷ However, at high levels, Mn becomes neurotoxic and is associated with the development of a parkinsonian syndrome (manganism) and other neurological dysfunctions.¹⁸ On the other hand, Cd is a potentially toxic metal with no known biological function, and it can cause adverse health effects in humans, such as kidney damage, bone damage, and bioaccumulation when ingested at high levels.¹⁹,²⁰ The presence of these metals in cheeses can be associated with environmental contamination, animal feed, or stages of industrial processing, making the monitoring of these elements essential to ensure the nutritional quality and food safety of the product.²¹ In light of this, advanced analytical techniques, such as atomic absorption spectrometry (AAS), have been widely used to determine essential and potentially toxic inorganic constituents in different types of cheese.⁶,¹⁰,²²-²⁵

The analysis of cheese using spectrometric techniques involves significant challenges due to the complex, high-fat, and protein-rich, which requires rigorous sample preparation to avoid interference and ensure accurate trace element determination. The main difficulties include complete organic matrix decomposition, managing high dissolved solids, spectral interferences, and avoiding contamination during preparation.²⁶

The combination of digestion with a diluted oxidizing mixture (HNO₃ and H₂O₂) and FAAS (flame atomic absorption spectrometry) offers significant advantages such as higher safety, reduced cost, lower blank values, improved efficiency for high-throughput laboratories, and compliance with green chemistry principles.²⁷-²⁹

The contamination of dairy products and their derivatives, particularly cheese by toxic metals has been documented in the literature,⁶,⁸,¹⁰ posing a significant risk to human health and raising concern due to chronic exposure to these contaminants. In this scenario, the United States Environmental Protection Agency (USEPA)³⁰ and the Joint FAO/WHO Expert Committee on Food Additives (JECFA)³¹ have established protocols, the use of which has been reported in the literature to assess the health risks associated with the consumption of cheese contaminated with metals, using indicators such as the estimated daily intake (EDI), the target hazard quotient (THQ), and the hazard index (HI).⁶,⁸,¹⁰,²²,²⁵,³⁰-³²

Given this context, the study of inorganic constituent levels in cow’s milk cheese and buffalo cheese becomes essential to assess the potential risk to human health from the possible presence of toxic components. Therefore, this study aimed to determine Cd, Cu, Fe, Mn, and Zn in cow’s milk cheese and buffalo cheeses sold in Belém PA using FAAS, and to conduct a health risk assessment through the EDI and hazard quotient (HQ) for Cd, Cu, Fe, Mn, and Zn.

EXPERIMENTAL

Samples

A total of ten cheese samples were collected, five of which were made from cow’s milk (COW 1-5) and the other five from buffalo milk (BUF 1-5), from local stores in the city of Belém (Pará State). Table 1 shows the types of cheeses studied.

Table 1
Types of cheeses

Sample	Cow’s milk cheese		Sample	Buffalo cheese
	Type	Code		Type	Code
1	mozzarella	COW 1	6	cream	BUF 1
2	prato	COW 2	7	mozzarella	BUF 2
3	coalho	COW 3	8	cream	BUF 3
4	ricotta	COW 4	9	mozzarella	BUF 4
5	minas	COW 5	10	ricotta	BUF 5

Reagents and analytical solutions

All solutions were prepared with analytical-grade reagents and ultrapure water (resistivity of 18.2 MΩ cm) from a Synergy-UV purification system (Millipore, Bedford, MA, USA). All materials were decontaminated in a 10% (v v^-1) HCl bath for 24 h and rinsed with ultrapure water before use. The nitric acid (Neon, Susano, SP, Brazil) used for preparing analytical solutions and for acid digestions was purified using a sub-distillation system (Berghof, BSB-939-IR, Germany). Hydrogen peroxide (35%, m m^-1) served as an auxiliary oxidant. Stock standard solutions of each metal (Cd, Cu, Fe, Mn, and Zn), with concentrations of 1000 mg L^-1 (SpecSol, Jacareí, SP, Brazil), were prepared by dilution in a nitric acid medium (5%, v v^-1).

Instrumentation

The samples were dried by lyophilization using a freeze dryer (model L101, Liotop, São Carlos, SP, Brazil) and subsequently ground in an analytical mill (model Q298A21, Quimis, Diadema, SP, Brazil). Acid digestion was carried out in a cavity microwave oven (START E, Milestone, Sorisole, Italy) equipped with a 12-position rotor and Teflon (TFM) vessels. All samples were weighed using an analytical balance with a readability of 0.0001 g (AP210 Analytical Plus, Ohaus, Switzerland).

The concentrations of Cd, Cu, Fe, Mn, and Zn in the cheese samples were determined by FAAS (iCE 3300 AA, Thermo Fisher Scientific, Cambridge, England). The air/acetylene flow rate (L min^-1) and the burner height (mm) was automatically optimized for each element studied before being determined by FAAS. Table 2 details the instrumental parameters employed.

Table 2
Instrumental parameters for the determination of Cd, Cu, Fe, Mn, and Zn by FAAS

Parameter	Instrumental conditions
	Cd	Cu	Fe	Mn	Zn
Wavelength / nm	228.8	324.8	248.3	279.5	213.9
Bandpass / nm	0.5	0.5	0.2	0.2	0.2
Lamp current / mA	3.0	4.0	6.0	5.0	5.0
Air/acetylene / (L min-1)	1.4	0.9	1.0	1.0	0.9
Measurement time / s	5.0	5.0	4.0	4.0	4.0
Nebuliser uptake / s	4	4	4	4	4
Burner height / mm	14.2	11.0	10.2	7.0	11.8

Analytical procedures

Microwave-assisted cheese sample digestion

All cheese samples were digested using the same heating program. The digestion procedure was adapted from the equipment manufacturer’s recommendations. Approximately 250 mg of each sample was digested with 4.0 mL of HNO₃ (14 mol L^-1), 2.0 mL of H₂O₂ (35%, m m^-1), and 2.0 mL of ultrapure water. The heating program consisted of four steps: (i) heating for 5 min to reach 120 °C; (ii) holding for 5 min at 120 °C; (iii) heating for 10 min to reach 180 °C; and (iv) holding for 10 min at 180 °C, followed by ventilation for 50 min. The applied power was set to 800 W for all steps. After cooling to room temperature, the digested solutions were transferred to volumetric flasks and diluted to 25.0 mL with ultrapure water. The analytical blank was prepared following the same procedure in the absence of the sample.

Elemental analysis of inorganic constituents by FAAS

The concentrations of Cd, Cu, Fe, Mn, and Zn were determined in the buffalo cheese and cow’s milk cheese samples by FAAS. An aliquot of the digest from each sample was diluted with ultra-pure water to obtain a final solution acidity of 5% (v v^-1).

Analytical curves were prepared in HNO₃ (5%, v v^-1) medium using stock solutions containing 1000 mg L ^-1 of each element in the following concentration ranges: 1 to 3 mg L^-1 for Cd, 2 to 10 mg L^-1 for Cu, 5 to 15 mg L^-1 for Fe, 1 to 3.5 mg L^-1 for Mn, and 0.5 to 1.5 mg L^-1 for Zn.

Evaluation of the analytical procedure

The FAAS analysis procedure was evaluated using the analyte spiking and recovery method. Thus, aliquots of 1.5, 2.5, and 3.5 mg L^-1 of Cd, Cu, Fe, Mn, and Zn were added to the digests samples from three different samples, respectively. Subsequently, the determination of Cd, Cu, Fe, Mn, and Zn was performed by FAAS.

On the other hand, the accuracy of the sample preparation procedure was evaluated by adding aliquots of Cd (1.25, 1.75, and 2.25 mg L^-1), Cu (3.0, 5.0, and 7.0 mg L^-1), Fe (6.0, 8.0, and 11.0 mg L^-1), Mn (1.25, 1.75, and 2.25 mg L^-1), and Zn (0.6, 0.8, and 1.15 mg L^-1) to three different samples. The samples were then subjected to microwave-assisted digestion. Subsequently, the elements under study were determined by FAAS to assess analyte recovery. This procedure aimed to evaluate whether there were losses of Cd, Cu, Fe, Mn, and Zn due to volatilization during sample preparation. The addition of different levels of these elements to different samples allowed for a more comprehensive assessment of the procedure’s robustness.

Evaluation of method sensitivity by FAAS

The limits of detection (LOD) and quantification (LOQ) were determined using the classical approach based on the blank signal.³³ The LOD was defined as the concentration that produces a signal equal to three times the standard deviation of the background noise, which was measured during the aspiration of a blank. Thus, the relationships LOD = 3s/m and LOQ = 10s/m were applied, where s is the standard deviation obtained from ten analytical blank replicates and m is the slope (sensitivity) of the calibration curve.

Indices used in the assessment of risks to human health

Estimated daily intake (EDI)

The estimated daily intake (EDI) assessment is calculated using the following Equation 1.²⁷

(1) $$\mathrm{EDI}=\frac{\mathrm{C}\times \mathrm{IR}}{\mathrm{BW}}$$

where C is the concentration of the element in the cheese (mg kg^-1), IR is the average daily intake of cheese (0.0015 kg day^-1), and BW is the average body weight (70 kg). The EDI is estimated in mg kg^-1 day^-1. For cadmium only, the IR is considered as the average monthly intake, and the EDI is estimated in mg kg^-1 month^-1.

Hazard quotient (HQ)

The non-carcinogenic risk assessment was performed using the Chemical Risk Calculator available on the Risk Assessment Information System (RAIS) website.³⁴ The parameters used are presented in Equation 2.

(2) $$\mathrm{HQ}=\frac{\mathrm{CDI}}{\mathrm{RfD}}$$

The hazard quotient (HQ) is the ratio of a single substance exposure level over a specified time period (e.g., subchronic) to a reference dose (RfD) for that substance derived from a similar exposure period.³⁰ For the elements in this study, the RfD values (mg kg^-1 day^-1) are as follows: 1.00 × 10^-4 (Cd), 2.00 × 10^-2 (Cu), 7.00 × 10^-1 (Fe), 1.40 × 10^-1 (Mn), and 3.00 × 10^-1 (Zn). The chronic daily intake (CDI), representing the long-term average daily dose (mg kg^-1 day^-1), was estimated via the following equation:

(3) $$\mathrm{CDI}=\frac{\mathrm{C}\times {10}^{-6}\times \mathrm{EF}\times \mathrm{ED}\times \mathrm{IR}}{\mathrm{AT}\times \mathrm{BW}}$$

where C is the concentration found in the cheese (mg kg^-1), EF is the exposure frequency (350 days year^-1), ED is the exposure duration (65 years), IR is the average daily intake of cheese (1500 mg day^-1), AT is the averaging time (365 days year^-1 × 65 years), and BW is the body weight (70 kg).

Statistical analysis

The Mann-Whitney U test (nonparametric) was used for the statistical analysis of the data obtained for Cd and Cu. On the other hand, the t-test was used for the Zn values found in the samples. No statistical tests were performed for Fe and Mn because their concentrations were below the limit of detection in all samples studied.

Green chemistry assessment

The proposed analytical procedure was evaluated using the modified index for green analytical procedures (MoGAPI), following the approach described in the literature.³⁵ This tool allows for a comprehensive assessment of the environmental impact of analytical methods, taking into account factors related to sample preparation, reagent consumption, instrumentation, energy consumption, and waste generation. The MoGAPI assessment was applied to estimate the compliance of the proposed method with the principles of green analytical chemistry.

RESULTS AND DISCUSSION

Analytical characteristics

Table 3 presents the analytical parameters for the content quantification of Cd, Cu, Fe, Mn, and Zn in cow’s milk cheese and buffalo cheese samples by FAAS, including LOD, LOQ, calibration equation, and the coefficient of determination (R²).

Table 3
Analytical performance parameters by FAAS

Analyte	LOD / (mg kg-1)	LOQ / (mg kg-1)	Calibration equation	R2
Cd	0.2	0.6	y = 0.2074x + 0.0053	0.9992
Cu	1.0	4.0	y = 0.0797x + 0.0161	0.9973
Fe	3.0	9.0	y = 0.0325x + 0.0069	0.9991
Mn	0.3	1.0	y = 0.1288x - 0.002	0.9999
Zn	1.0	3.0	y = 0.2647x + 0.0074	0.9958

The LODs ranged from 0.2 to 3.0 mg kg^-1, while the LOQs were between 0.6 and 9.0 mg kg^-1. The coefficients of determination were greater than 0.995 for all analytes, confirming adequate linearity of the calibration curves. All relative standard deviation (RSD) values were below 10%. These results demonstrate the good sensitivity and precision of the method.³⁶

Analytical procedure evaluation

The evaluation of the sample preparation procedure was performed using the analyte addition method in the cheese samples. Recovery rates for Cd, Cu, Fe, Mn, and Zn were 98.7-108.4%, 102.5-120.0%, 80.1-93.8%, 81.0-93.9%, and 91.5-97.1%, respectively. These results confirm that no significant losses of the analyzed elements occurred during sample preparation using microwave-assisted digestion.

The analyte spike recovery values obtained to evaluate the FAAS analysis procedure ranged from 81.3 to 111.7% for Cd, 100.5 to 119.1% for Cu, 90.8 to 104.3% for Fe, 81.3 to 91.7% for Mn, and 103.4 to 108.5% for Zn. According to ANVISA (Brazilian Health Regulatory Agency),³⁷ the confidence interval for precision and accuracy in the analysis of analyte levels must be between 80 and 120%. Therefore, the recovery values obtained for all elements in this study were satisfactory.

Determination of elements in cheese samples

Table 4 shows the average contents and respective standard deviations of Cd, Cu, Fe, Mn, and Zn in the cheese samples.

Table 4
Concentrations of Cd, Cu, Fe, Mn, and Zn in digested samples of cow’s milk cheese (COW) and buffalo cheese (BUF) (mean ± standard deviation, n = 3)

Sample	Concentration / (mg kg-1)
	Cd	Cu	Fe	Mn	Zn
COW 1	0.67 ± 0.07	< 1.0a	< 3.0a	< 0.3a	66.6 ± 0.2
COW 2	0.66 ± 0.06	< 1.0a	< 3.0a	0.74 ± 0.06	59.0 ± 6.2
COW 3	< 0.2a	6.9 ± 0.6	< 3.0a	< 0.3a	66.2 ± 0.9
COW 4	0.55 ± 0.04	7.5 ± 0.4	< 3.0a	< 0.3a	12.7 ± 1.1
COW 5	< 0.2a	< 1.0a	< 3.0a	< 0.3a	54.0 ± 0.9
BUF 1	0.37 ± 0.02	< 1.0a	< 3.0a	< 0.3a	25.8 ± 0.1
BUF 2	< 0.2a	< 1.0a	< 3.0a	< 0.3a	23.1 ± 0.7
BUF 3	< 0.2a	< 1.0a	< 3.0a	< 0.3a	28.5 ± 0.2
BUF 4	0.34 ± 0.03	2.75 ± 0.07	< 3.0a	< 0.3a	49.3 ± 1.9
BUF 5	< 0.2a	< 1.0a	< 3.0a	< 0.3a	8.1 ± 0.5

• ^a
  Limit of detection (LOD).

Zinc was the only element detected in all samples. Cadmium was quantified in half of the samples studied. Copper and manganese concentrations were below the limit of detections for most samples. Iron remained below the limit of detection in all cheese samples. The values found for Cd, Cu, Mn, and Zn were compared with those from other studies on cheese samples from different regions and using different techniques. These studies are presented in Table 1S of the Supplementary Material. The Cd and Cu values were higher than those reported in the literature (Table 1S) using other methodologies.⁶,⁸-¹⁰,¹³,²²,²⁵,³⁰,³⁸ The Mn value was lower than those observed by Al Sidawi et al.¹³ using GFAAS (graphite furnace atomic absorption spectrometry) and by Andrade et al.³⁸ using ICP OES (inductively coupled plasma optical emission spectrometry). On the other hand, the Zn values were similar to those reported by Kira and Maihara,⁹ who also employed a FAAS.

Cadmium concentrations in the cow’s milk cheese samples ranged from 0.55 ± 0.04 to 0.67 ± 0.07 mg kg^-1 (COW 1, COW 3, and COW 4), whereas the buffalo cheese samples contained levels between 0.34 ± 0.03 mg kg^-1 (BUF 1) and 0.37 ± 0.02 mg kg^-1 (BUF 4), respectively. Cadmium was not detected in the COW 3, COW 5, BUF 2, BUF 3, and BUF 5 samples. These values differ significantly from those reported by Al Sidawi et al.¹³ who found Cd concentrations between 0.002 ± 0.0015 and 0.007 ± 0.003 mg kg^-1 in similar cheeses. Similarly, the data presented here contrast with those of Elafify et al.,¹⁰ in which the recorded mean Cd levels ranged from 0.058 to 0.117 mg kg^-1, highlighting considerable variation between studies. The cadmium levels in the cow’s milk cheese samples (COW 1, COW 2, and COW 4) in this study raise concerns about food safety, since they exceed the maximum level permitted by ANVISA,³⁹ which is 0.5 mg kg^-1. While these levels do not pose an immediate acute risk, the bioaccumulative nature of this toxic metal indicates the necessity for ongoing monitoring of production chains. The presence of this contaminant in cheese is often linked to environmental contamination (e.g., soil, water or phosphate fertilizers used on pastures) rather than the physiology of the animal.

The copper levels determined in the cow’s milk cheeses ranged from 6.9 ± 0.6 to 7.5 ± 0.4 mg kg^-1 in samples COW 3 and COW 4. On the other hand, most buffalo cheeses had values below the limit of detection, with the exception of sample BUF 4 (2.75 ± 0.07 mg kg^-1). The Cu levels found in the samples in this study are below the maximum limit set by ANVISA,³⁹ which is 10 mg kg^-1. Sidawi et al.¹³ reported copper concentrations ranging from 1.26 ± 0.74 to 2.46 ± 2.32 mg kg^-1 in cheeses with similar characteristics, while Kira and Maihara⁹ found low copper levels ranging from 0.31 ± 0.03 to 0.45 ± 0.03 mg kg^-1. The observed difference suggests the influence of technological and environmental factors, such as the type of utensils used in production, processing conditions, and milk composition, which can significantly impact the trace metal content in dairy products. The results obtained in this study reinforce the need to expand research on the variability of copper concentration in different types of cheese, considering regional aspects and production practices, as well as to evaluate its implications for food quality and safety.

The iron contents found in the studied cow’s milk cheese and buffalo cheese samples were below the limit of detection. This may be because dairy products are not a good source of iron. Sidawi et al.¹³ found iron concentrations ranging from 69.09 ± 64.91 to 101.1 ± 91.16 mg kg^-1, while Kira and Maihara⁹ reported iron contents between 2.29 ± 0.01 and 7.74 ± 0.09 mg kg^-1. The discrepancy between the data obtained in this study and those reported by other authors suggests the influence of technological and environmental factors, which may directly affect iron absorption in dairy products.

In the present study, only sample COW 2 showed a detectable concentration of Mn, with a content of 0.74 ± 0.06 mg kg^-1. However, all buffalo cheese samples had levels below the limit of detection. These results differ from the mean values reported by Sidawi et al.,¹³ which ranged from 0.886 ± 0.595 to 2.348 ± 2.267 mg kg^-1, indicating higher concentrations. On the other hand, Capcarova et al.⁶ observed Mn levels between 0.08 ± 0.02 and 0.68 ± 0.37 mg kg^-1, while Kira and Maihara⁹ reported even lower values, ranging from 0.24 ± 0.02 to 0.30 ± 0.03 mg kg^-1. A comparison of the different studies reveals wide variability in manganese levels in cheeses, possibly attributed to factors such as the mineral composition of milk, regional differences in animal diets, environmental conditions, and technological practices employed in production.

Zinc concentrations in cow’s milk cheese samples ranged from 12.7 ± 1.1 to 66.6 ± 0.2 mg kg^-1, while values in buffalo milk cheese samples ranged from 8.1 ± 0.5 to 49.3 ± 1.9 mg kg^-1. These values were lower than those reported by Sidawi et al.¹³ (75.86 ± 52.528 to 124.8 ± 97.775 mg kg^-1), but higher than those recorded by Capcarova et al.⁶ (1.80 ± 0.08 to 2.33 ± 0.18 mg kg^-1). In contrast, the values reported by Kira and Maihara⁹ ranged from 6.90 ± 0.18 to 48.4 ± 1.2 mg kg^-1, demonstrating closer alignment with the outcomes observed in this study. The heterogeneity observed across these studies highlights the influence of various factors, including the mineral composition of the milk, the animals’ diet, the geographical origin and the technological procedures employed in cheese production. All of these factors directly impact the bioavailability and final accumulation of zinc. These differences emphasise the importance of considering production and environmental variables when evaluating the nutritional value and safety of these products.

Statistical analysis revealed significant differences in trace metal concentrations between cow’s milk cheese and buffalo cheeses. The Mann-Whitney test indicated significantly higher cadmium values in cow’s milk cheese than in buffalo cheeses (p = 0.03; r = 0.30), suggesting a moderate effect. Similarly, copper concentrations were higher in cow’s milk cheese (p = 0.02; r = 0.38), indicating a moderate to strong effect. Zinc showed a borderline difference according to the t-test (p = 0.06), but with a very high effect size (d = 1.38), which reinforced the trend of higher accumulation in cow’s milk cheese. Conversely, iron and manganese were below the limit of detections in both samples, precluding statistical comparison. In summary, the results show that cow’s milk cheese have significantly higher concentrations of Cd, Cu and Zn, while buffalo milk cheeses exhibit lower levels of these elements.

Health risk assessment

Health risk was assessed using the estimated daily intake (EDI) of the analyzed elements. Results are shown in Table 5.

Table 5
Estimated daily intake (EDI) of Cd, Cu, Fe, and Zn

Sample	Cd / (μg kg-1 month-1)	Cu / (mg kg-1 day-1)	Fe / (mg kg-1 day-1)	Zn / (mg kg-1 day-1)
COW 1	1.43 × 10-2	2.14 × 10-5	6.43 × 10-5	1.43 × 10-3
COW 2	1.41 × 10-2	2.14 × 10-5	6.43 × 10-5	1.27 × 10-3
COW 3	4.29 × 10-3	1.48 × 10-4	6.43 × 10-5	1.42 × 10-3
COW 4	1.18 × 10-2	1.61 × 10-4	6.43 × 10-5	2.72 × 10-4
COW 5	4.29 × 10-3	2.14 × 10-5	6.43 × 10-5	1.16 × 10-3
BUF 1	7.91 × 10-3	2.14 × 10-5	6.43 × 10-5	5.52 × 10-4
BUF 2	4.29 × 10-3	2.14 × 10-5	6.43 × 10-5	4.94 × 10-4
BUF 3	4.29 × 10-3	2.14 × 10-5	6.43 × 10-5	6.10 × 10-4
BUF 4	7.28 × 10-3	5.89 × 10-5	6.43 × 10-5	1.06 × 10-3
BUF 5	4.29 × 10-3	2.14 × 10-5	6.43 × 10-5	1.74 × 10-4
PMTDI	25	0.5	0.8	0.3
PMTDI: provisional maximum tolerable daily intake.

The EDI for all elements studied was below the provisional maximum tolerable daily intake (PMTDI) set by the FAO/WHO (JECFA).³¹ Furthermore, all hazard quotient (HQ) values were below 1. These results indicate that consumption of the cow’s milk cheese and buffalo cheese poses no appreciable non-carcinogenic health risk.

Table 6 presents the calculated HQ, which indicates the potential for non-carcinogenic risks from consumption of the cheeses in this study.

Table 6
Hazard quotient (HQ) values for Cd, Cu, Fe, Mn, and Zn

Sample	Cd	Cu	Fe	Mn	Zn	Total
COW 1	1.37 × 10-1	1.03 × 10-3	8.81 × 10-5	4.40 × 10-5	4.56 × 10-3	1.43 × 10-1
COW 2	1.35 × 10-1	1.03 × 10-3	8.81 × 10-5	1.08 × 10-4	4.05 × 10-3	1.41 × 10-1
COW 3	4.11 × 10-2	7.11 × 10-3	8.81 × 10-5	4.40 × 10-5	4.53 × 10-3	5.29 × 10-2
COW 4	1.13 × 10-1	7.74 × 10-3	8.81 × 10-5	4.40 × 10-5	8.71 × 10-4	1.22 × 10-1
COW 5	4.11 × 10-2	1.03 × 10-3	8.81 × 10-5	4.40 × 10-5	3.70 × 10-3	4.60 × 10-2
BUF 1	7.58 × 10-2	1.03 × 10-3	8.81 × 10-5	4.40 × 10-5	1.77 × 10-3	7.88 × 10-2
BUF 2	4.11 × 10-2	1.03 × 10-3	8.81 × 10-5	4.40 × 10-5	1.58 × 10-3	4.38 × 10-2
BUF 3	4.11 × 10-2	1.03 × 10-3	8.81 × 10-5	4.40 × 10-5	1.95 × 10-3	4.42 × 10-2
BUF 4	6.98 × 10-2	2.82 × 10-3	8.81 × 10-5	4.40 × 10-5	3.38 × 10-3	7.61 × 10-2
BUF 5	4.11 × 10-2	1.03 × 10-3	8.81 × 10-5	4.40 × 10-5	5.58 × 10-4	4.28 × 10-2

The EDI values for cadmium ranged from 4.29 × 10^-3 to 1.43 × 10^-2 μg kg^-1 month^-1. These values found were below the limit established by the PMTDI (25 μg kg^-1 month^-1) and suggests a low risk of chronic toxicity. The HQ values for Cd in the samples ranged from 4.11 × 10^-2 to 1.37 × 10^-1. All of these values are below 1, indicating that, according to the HQ assessment, exposure to cadmium in all samples does not pose an immediate health risk. However, considering that cadmium is a toxic metal with unknown biological function, cumulative exposures and possible additional sources of contamination should be monitored to avoid health risks. The establishment of the PMTDI for cadmium by JECFA is based on the long-term bioaccumulation of this metal in the kidneys, considering that the steady state of accumulation is not reached until 45 to 60 years of exposure. Although dietary exposure above the PMTDI for limited periods may pose a lower risk for younger age groups, there is reason for concern in regions where cadmium exposure during adulthood exceeds this limit.³¹

The copper concentrations showed low EDI values, ranging from 2.14 × 10^-5 to 1.61 × 10^-4 mg kg^-1 day^-1, remaining below the safe limit of 0.5 mg kg^-1 day^-1. The HQ values for Cu ranged from 1.03 × 10^-3 to 7.74 × 10^-3, all of which were below 1. These low HQ values indicate no significant risk of copper toxicity in the analyzed samples, reflecting a safe exposure level within tolerable limits.

The EDI of iron was only 6.43 × 10^-7 mg kg^-1 day^-1, below the PMTDI value of 0.8 mg kg^-1 day^-1. The HQ values for Fe were low in all samples. The HQ values indicate that exposure to iron does not pose a risk, as all values are below 1. Iron presents no risk of toxicity at these concentrations, and the values indicate that intake is within acceptable limits.

For manganese, the World Health Organization (WHO) does not establish PTMDI values that can be compared with the results of this study. The HQ values for Mn ranged from 4.40 × 10^-5 to 1.08 × 10^-4, with all values significantly below 1. The HQ values indicate that there is no risk of toxicity for manganese in the studied samples, reflecting safe exposure.

The estimated daily zinc intake ranged from 1.74 × 10^-4 to 1.43 × 10^-3 mg kg^-1 day^-1, remaining below the safe limit of 0.3 mg kg^-1 day^-1. The HQ values for Zn ranged between 5.58 × 10^-4 and 4.56 × 10^-3. All these HQ values below 1 indicate that there is no significant risk of toxicity, with exposures being controlled and within tolerable limits. Zinc is an essential element in human and animal nutrition. Studies with experimental animals have shown that high levels of zinc in the diet can cause anemia, as well as reduce copper and iron levels, in addition to decreasing the activity of several important enzymes in various tissues. These effects can also occur at lower dietary zinc levels, particularly when there is a copper deficiency.⁴⁰

The total HQ values, which consider the sum of the HQ for all elements, ranged from 4.28 × 10^-2 to 1.43 × 10^-1, indicating that the overall health risk is low. These values are below 1, meaning that, in general, the analyzed samples do not pose a significant toxicity risk, and exposure to the studied elements is safe. Despite the low risk indicated by all HQ values being below 1, the potential for chronic and cumulative exposure, particularly to elements like cadmium, must still be considered. Sustained monitoring is therefore crucial to ensure that exposure levels remain within safe limits.

Green chemistry assessment

The modified green analytical procedures index (MoGAPI) was used, as it provides a comprehensive assessment of the environmental impact at all stages of the analytical workflow to identify the green analytical chemistry parameters that are met by the methodology used in this study (Figure 1). The results indicate that the method exhibits several characteristics compatible with the principles of sustainable analytical chemistry, such as low reagent consumption (< 10 mL), minimal waste generation (1-10 mL), and the absence of additional treatment steps. Furthermore, the use of microwave-assisted digestion contributes to reduced analysis time and greater efficiency. However, some limitations were identified, mainly related to the use of concentrated nitric acid and hydrogen peroxide, which are classified as non-eco-friendly reagents, as well as the relatively high energy consumption of the microwave system (> 1.5 kWh per sample) and the FAAS (2.0 kWh). Nevertheless, MoGAPI suggests that the proposed method achieves a score considered satisfactory for analytical performance and environmental impact. Consequently, it can be considered a reasonably eco-friendly approach for elemental determination in complex food matrices.

Figure 1
MoGAPI assessment scores

CONCLUSIONS

This study confirmed the effectiveness of FAAS combined with microwave-assisted digestion for the determination of Cd, Cu, Fe, Mn, and Zn in cow’s milk cheese and buffalo cheese. The procedure proposed showed good sensitivity, precision, and accuracy, ensuring reliable quantification of trace metals in complex food matrices. The analytical parameters confirmed the robustness of the analytical procedure, while the results revealed significant differences between cow’s milk cheese and buffalo cheese. Cow’s milk cheese showed higher concentrations of cadmium, copper, and zinc, whereas iron and manganese were below limit of detections. Although the estimated daily intake (EDI) and hazard quotient (HQ) values for all elements were below internationally recognized safety thresholds, cadmium concentrations in some cow’s milk cheese samples exceeded the maximum limit established by ANVISA, raising concerns about long-term exposure. The data obtained highlight the influence of environmental and technological factors on metal accumulation in dairy products and reinforce the importance of continuous monitoring of production chains. The results found in this study emphasize the importance of robust analytical techniques and careful sample preparation for accurate trace metal determination in dairy products. Moreover, the use of microwave-assisted digestion with diluted oxidizing mixtures aligns with green chemistry principles, offering a safer, more efficient, and environmentally friendly approach. Sustained surveillance of production chains is essential to ensure nutritional quality and food safety, particularly regarding cadmium contamination in cow’s milk cheese.

SUPPLEMENTARY MATERIAL

Supplementary material for this work is available at http://quimicanova.sbq.org.br/, as a PDF file, with free access.

Supplementary PDF

Supplementary PDF

ACKNOWLEDGMENTS

The authors would like to thank CAPES, CNPq and the National Institute of Science and Technology on Molecular Sciences (CNPq Grant 406804/2022-2). They would also like to thank Google Gemini (Version 1.0.869192897, 16 February 2026) for colouring and styling the images in the graphical abstract figure.

DATA AVAILABILITY STATEMENT

The authors declare that all data from this research is available in the text.

REFERENCES

• ¹ Oliveira, R. L.; Ladeira, M. M.; Barbosa, M. A. A. F.; Matsushita, M.; Santos, G. T.; Bagaldo, A. R.; Oliveira, R. L.; Arq. Bras. Med. Vet. Zootec. 2009, 61, 736. [Crossref]
  » Crossref
• ² Marques, J. R. F.; Criação de Búfalos; Embrapa-SPI: Brasília, DF; Embrapa-CPATU: Belém, PA, 1998.
• ³ Simões, M. G.: Efeitos de Variações Sazonais e da Proporção Leite de Vaca:Leite de Búfala sobre Características Físico-Químicas e Microbiológicas de Queijos Artesanais do Marajó; PhD Thesis, Universidade Federal de Viçosa, Viçosa, Brasil, 2014. [Link] accessed in June 2026
  » Link
• ⁴ Pereira Junior, J. B.; Fernandes, K. G.; Müller, R. C. S.; Nóbrega, J. A.; Palheta, D. C.; Quim. Nova 2009, 32, 2333. [Crossref]
  » Crossref
• ⁵ Lourenço, L. F. H.; Sousa, C. L.; Simão Neto, M.; Lourenço Júnior, J. B.; Higiene Alimentar 2002, 16, 55. [Link] accessed in June 2026
  » Link
• ⁶ Capcarova, M.; Harangozo, L.; Arvay, J.; Toth, T.; Gabriny, L.; Binkowski, L. J.; Palsova, L.; Skalicka, M.; Pardo, M. L. G.; Stawarz, R.; Massanyi, P.; J. Environ. Sci. Health, Part B 2020, 55, 677. [Crossref]
  » Crossref
• ⁷ Herman-Lara, E.; Bolívar-Moreno, D.; Toledo-Lopez, V. M.; Cuevas Glory, L. F.; Lope-Navarrete, M. C.; Barron-Zambrano, J. A.; Díaz-Rivera, P.; Ramírez-Rivera, E. J.; Food Sci. Technol. 2019, 39, 517. [Crossref]
  » Crossref
• ⁸ Rocha, L. S.; Ramos, G. L. P. A.; Rocha, R. S.; Braz, B. F.; Santelli, R. E.; Esmerino, E. A.; Freitas, M. Q.; Mársico, E. T.; Bragotto, A. P. A.; Quitério, S. L.; Cruz, A. G.; Food Res. Int. 2023, 174, 113659. [Crossref]
  » Crossref
• ⁹ Kira, C. S.; Maihara, V. A.; Food Sci. Technol. 2007, 27, 446. [Crossref]
  » Crossref
• ¹⁰ Elafify, M.; EL-Toukhy, M.; Sallam, K. I.; Sadoma, N. M.; Abd-Elghany, S. M.; Abdelkhalek, A.; El-Baz, A. H.; Food Chem. Adv. 2023, 2, 100261. [Crossref]
  » Crossref
• ¹¹ Sousa, R. A.; Lisboa, T. P.; Revista do Instituto de Laticínios Cândido Tostes 2015, 70, 35. [Crossref]
  » Crossref
• ¹² de Barros, B. V.; Miranda, R. G.; Pereira, S. F. P.; Palheta, D. C.; Silva, C. S.; XII Safety, Health and Environment World Congress; São Paulo, Brazil, 2012. [Link] accessed in June 2026
  » Link
• ¹³ Al Sidawi, R.; Ghambashidze, G.; Urushadze, T.; Ploeger, A.; Foods 2021, 10, 2234. [Crossref]
  » Crossref
• ¹⁴ Grzeszczak, K.; Kwiatkowski, S.; Kosik-Bogacka, D.; Biomolecules 2020, 10, 1176. [Crossref]
  » Crossref
• ¹⁵ Chen, J.; Calderone, L. A.; Pan, L.; Quist, T.; Pandelia, M.-E.; Biochim. Biophys. Acta, Proteins Proteomics 2023, 1871, 140931. [Crossref]
  » Crossref
• ¹⁶ Li, L.; Yang, X.; Oxid. Med. Cell. Longevity 2018, 2018, 7580707. [Crossref]
  » Crossref
• ¹⁷ Horning, K. J.; Caito, S. W.; Tipps, K. G.; Bowman, A. B.; Aschner, M.; Annu. Rev. Nutr. 2015, 35, 71. [Crossref]
  » Crossref
• ¹⁸ Balachandran, R. C.; Mukhopadhyay, S.; McBride, D.; Veevers, J.; Harrison, F. E.; Aschner, M.; Haynes, E. N.; Bowman, A. B.; J. Biol. Chem. 2020, 295, 6312. [Crossref]
  » Crossref
• ¹⁹ Qu, F.; Zheng, W.; Toxics 2024, 12, 388. [Crossref]
  » Crossref
• ²⁰ Charkiewicz, A. E.; Omeljaniuk, W. J.; Nowak, K.; Garley, M.; Nikliński, J.; Molecules 2023, 28, 6620. [Crossref]
  » Crossref
• ²¹ Scutaraşu, E. C.; Trincă, L. C.; Foods 2023, 12, 3340. [Crossref]
  » Crossref
• ²² Barone, G.; Dambrosio, A.; Storelli, A.; Busco, A.; Ioanna, F.; Quaglia, N. C.; Giacominelli-Stuffler, R.; Storelli, M. M.; J. Food Compos. Anal. 2018, 66, 205. [Crossref]
  » Crossref
• ²³ Diniz, L. M. N.; Carrasco, T. S.; Medina, A. L.; Ribeiro, A. S.; Nunes, A. M.; Quim. Nova 2017, 40, 711. [Crossref]
  » Crossref
• ²⁴ Orak, H.; Altun, M.; Ercag, E.; Ital. J. Food Sci. 2005, 17, 95. [Link] accessed in June 2026
  » Link
• ²⁵ Christophoridis, C.; Kosma, A.; Evgenakis, E.; Bourliva, A.; Fytianos, K.; J. Food Compos. Anal. 2019, 82, 103238. [Crossref]
  » Crossref
• ²⁶ El Hosry, L.; El Hosry, R.; El Hosry, H.; El Hosry, G.; El Hosry, N.; Foods 2023, 12, 895. [Crossref]
  » Crossref
• ²⁷ Gałuszka, A.; Migaszewski, Z.; Namieśnik, J.; TrAC, Trends Anal. Chem. 2013, 50, 78. [Crossref]
  » Crossref
• ²⁸ Nóbrega, J. A.; Pirola, C.; Fialho, L. L.; Rota, G.; Jordão, C. E. K. M. A. C.; Pollo, F.; Talanta 2012, 98, 272. [Crossref] accessed in June 2026
  » Crossref
• ²⁹ Krug, F. J.; Rocha, F. R. P. In Métodos de Preparo de Amostras para Análise Elementar; Krug, F. J.; Rocha, F. R. P., eds.; EditSBQ: São Paulo, 2016, p. 224.
• ³⁰ United States Environmental Protection Agency (US EPA); Risk Assessment Guidance for Superfund Volume I: Human Health Evaluation Manual. Part A. Interim Report; US EPA, Washington, 1989. [Link] accessed in October 2025
  » Link
• ³¹ World Health Organization (WHO); Evaluations of the Joint FAO/WHO Expert Committee on Food Additives (JECFA); WHO: Geneva, Switzerland, 2021. [Link] accessed in June 2026
  » Link
• ³² Popovic, A. R.; Djinovic-Stojanovic, J. M.; Djordjevic, D. S.; Relic, D. J.; Vranic, D. V.; Milijasevic, M. P.; Pezo, L. L.; J. Food Compos. Anal. 2018, 67, 70. [Crossref]
  » Crossref
• ³³ Harris, D. C.; Quantitative Chemical Analysis, 9^th ed.; W. H. Freeman and Company: New York, 2015.
• ³⁴ Risk Assessment Information System. [Link] accessed in June 2026
  » Link
• ³⁵ Mansour, F. R.; Płotka-Wasylka, J.; Locatelli, M.; Analytica 2024, 5, 451. [Crossref]
  » Crossref
• ³⁶ Schlemmer, G.; Balcaen, L.; Todolí, J. L.; Hinds, M. W.; Elemental Analysis: An Introduction to Modern Spectrometric Techniques; Walter de Gruyter GmbH: Boston/Berlin, 2019.
• ³⁷ Agência Nacional de Vigilância Sanitária (ANVISA); Resolução da Diretoria Colegiada (RDC) No. 166, de 24 de julho de 2017, Dispõe sobre a Validação de Métodos Analíticos e dá outras Providências; Diário Oficial da União (DOU), Brasília, No. 141, de 25/07/2017, p. 87. [Link] accessed in June 2026
  » Link
• ³⁸ de Andrade, B. M.; Margalho, L. P.; Batista, D. B.; Lucena, I. O.; Kamimura, B. A.; Balthazar, C. F.; Brexó, R. P.; Pia, A. K. R.; Costa, R. A. S.; Cruz, A. G.; Granato, D.; Sant’Ana, A. S.; Luna, A. S.; de Gois, J. S.; J. Food Compos. Anal. 2022, 109, 104519. [Crossref]
  » Crossref
• ³⁹ Agência Nacional de Vigilância Sanitária (ANVISA); Resolução da Diretoria Colegiada (RDC) No. 160, de 1 de julho de 2022, Estabelece os Limites Máximos Tolerados (LMT) de Contaminantes em Alimentos; Diário Oficial da União (DOU), Brasília, No. 126, de 06/07/2022, p. 227. [Link] accessed in June 2026
  » Link
• ⁴⁰ World Health Organization (WHO); Evaluations of the Joint FAO/WHO Expert Committee on Food Additives (JECFA), Cadmium; Geneva, Switzerland, 2021. [Link] accessed in June 2026
  » Link

Edited by

• Associate Editor handled this article:
  Mario Henrique Gonzalez

Publication Dates

• Publication in this collection
  27 July 2026
• Date of issue
  2026

History

• Received
  12 Jan 2026
• Accepted
  29 May 2026
• Published
  25 June 2026