Evaluation of Cadmium and Lead Accumulation in Edible Horse Tissues: A Food Safety Perspective

Abstract

Horse meat is characterized by high nutritional value, but due to the specific physiology and long lifespan of horses, it represents a significant pathway for the bioaccumulation of toxic elements. The aim of this study was to examine the presence of cadmium (Cd) and lead (Pb) in muscle, liver and kidney samples of horses slaughtered in Serbia during 2023 and 2024. The toxic elements were determined by flame atomic absorption spectrometry (FAAS). The mean concentrations of cadmium and lead were 0.19 and 0.51 mg/kg in horse muscle; 2.31 and 0.74 mg/kg in horse liver; and 7.70 and 0.68 mg/kg in horse kidneys. Statistically significant differences in mean concentrations were observed between horse tissues, seasons and different age categories (p < 0.001), but there was no difference between sexes (male and female) (p > 0.05). Cadmium levels were above the maximum permitted limits in 93.2% of liver samples, 97.7% of kidney samples, and 31.1% of muscle samples tested. The data obtained indicate the need for continuous monitoring and strict control of animal traceability, especially those raised near ecological hotspots.

1. Introduction

Horse meat is recognized for its high nutritional value, characterized by a favorable fatty acid profile and high protein content and can be considered a good alternative for conventional meats [1]. However, its safety is often compromised by the accumulation of toxic elements, particularly cadmium (Cd) and lead (Pb), which are naturally present in the environment (soil, water and air) and can accumulate in plants and in animals grazing in polluted areas [2]. Cadmium is highly bioavailable and is primarily accumulated in the flowers, shoots, leaves and roots of many plant species [3]. It can be found in cereals and vegetables, followed by meat and edible offal (especially liver and kidney) of food-producing animals [4], where it tends to sequester in metabolically active organs [5]. Contrarily, lead, a potent neurotoxin, preferentially accumulates in soft tissues and bones [6]. Once absorbed, it binds to erythrocytes and travels via the blood to various tissues, eventually sequestering in bones and teeth [5,6]. According to the IARC (International Agency for Research on Cancer) [7], lead is often classified as Group 2B (possibly carcinogenic) or Group 2A (probably carcinogenic, specifically inorganic lead). Consequently, monitoring these toxic elements in horse edible tissues is vital for food safety and public health [8]. The accumulation of toxic elements, including cadmium, in meat and edible offal continues to present a major global food safety challenge due to their persistence in the environment and bioaccumulative properties in animal tissues [9,10]. Since horses have a longer lifespan than most livestock species and graze over large pasture areas, they are particularly susceptible to the bioaccumulation of toxic elements compared with other food-producing animals [11,12]. For instance, cadmium concentrations in horse muscle and edible offal have been reported to be approximately 25–50 times higher than in cattle, pigs and poultry, raising specific food safety concerns associated with horse-derived-food consumption [12,13]. Accordingly, assessing the concentrations of toxic elements in the meat and edible offal of all food-producing animals, including horses, is essential for ensuring food safety and protecting human health [11].

In Serbia and the surrounding Balkan countries, pollution deriving from toxic elements remains a critical environmental challenge [14]. Intensive industrial and mining activities, together with the long-term use of phosphate fertilizers and organic solids (manure and sewage sludge), have increased toxic element levels in surface soils, enabling their entry into the food chain through forage [14]. It is considered that more than 25,000 tons of cadmium are released into the environment every year [5].

The regulatory framework governing horse meat requires careful interpretation. In Serbia, national legislation on chemical contaminants in food is largely harmonized with European legislation. Specifically, Commission Regulation (EU) No. 2023/915 [8] (and its local equivalents [15]) establishes the maximum levels for cadmium in horse muscle (0.20 mg/kg), liver (0.50 mg/kg) and kidney (1.0 mg/kg). In contrast, the regulation of lead in horse tissues is not as specific, as maximum levels are established for muscle (0.10 mg/kg) and offal (0.20 mg/kg) in other livestock species, so horse edible tissues are often assessed by analogy based on these values. This lack of species-specific guidance highlights the need for country-based surveillance of the domestic horsemeat market. Therefore, the primary aim of this study was to determine the concentrations of cadmium and lead in horse muscle, liver and kidney samples to evaluate potential food safety and public health risks. Furthermore, the effects of slaughter age, sex and slaughter season on toxic element accumulation were examined to better identify risk factors associated with horsemeat and edible offal consumption.

2. Materials and Methods

2.1. Ethical Statement

The study was conducted in accordance with the Guide to Good Animal Welfare Practice for the Keeping, Care, Training and Use of Horses [16] and the European legislation on the protection of animals during transport [17] and at the time of slaughter [18]. Horses originated from two collection centers (Pećinci and Ruma, Srem District, Autonomous Province of Vojvodina, Serbia) and were slaughtered for human consumption in one accredited abattoir (Pećinci, Srem District, Serbia). No experimental or invasive procedures were performed in vivo. Muscle, kidney and liver samples were collected prior to carcass chilling and only after completion of the official post-mortem inspection by an official veterinary inspector. Data collection was deliberately carried out under standard commercial pre-slaughter conditions. Accordingly, this study did not fall within the scope of Directive 2010/63/EU [19] on the protection of animals used for scientific purposes and was, therefore, exempt from approval by a local animal welfare and ethical review body.

2.2. Experimental Animals, Pre-Slaughter Conditions and Slaughter Procedure

Farm 1, located in Pećinci, housed horses (Domestic Mountain Pony) for approximately 1–3 months. Farm 2, located in Ruma, housed horses of the same breed for periods ranging from 15 days to 6 months. At both farms, animals were purchased from local markets, fairs, households and auctions, raising concern about applied veterinary practices during animal breeding/husbandry as well as incoming controls in the abattoir. The horses’ diet on the farms consisted primarily of corn, livestock meal, and locally produced clover hay and silage, along with grass to meet the National Research Council’s dietary guidelines for horses [20]. At Farm 1, animals were kept in five pens (5 × 8 m, 6 × 8 m, 10 × 5 m, 5 × 5 m and 5 × 8 m) on concrete floors with straw bedding, while at Farm 2 they were kept in three pens (55 × 10 m, 30 × 4 m and 20 × 15 m) on concrete floors without bedding. Horses were tied during housing, except for foals at Farm 1. Both farms operated under a continuous-flow management system, with new horses introduced weekly and animals transported to the abattoir every two weeks. All horses were subjected to the same pre-slaughter handling (gentle), transport (same lorry and driver; transport time ≈ 1 h; loading density of 1.75 m²/horse), and lairage conditions (lairage time ≈ 3 h; lairage density of 3 m²/horse). The horses were slaughtered in the same accredited abattoir following standard industry practices, including stunning with a captive-bolt pistol, exsanguination by severing the neck blood vessels (a. carotis communis and v. jugularis) and dressing in accordance with the applicable European Union regulations [18]. Information regarding horse age, sex, food chain and origin was obtained from the farm and abattoir documentation.

2.3. Sample Collection

After evisceration and post-mortem inspection, muscle, liver and kidney samples were collected from slaughtered horses to determine lead and cadmium concentrations. Sampling occurred monthly over a one-year period (February 2023 to February 2024) from 132 horses (65 females and 67 males) aged between one and ten years. Muscle samples were taken from the longissimus lumborum muscle at the level of the 13th and 14th ribs, liver fragments were obtained from the caudate lobe, and kidney samples were collected from the renal cortex. Prior to analysis, tissues were trimmed to remove excess fat, connective tissue and major blood vessels. Muscle and liver samples were excised with a sterilized stainless-steel knife to obtain uncontaminated material, and no further homogenization was performed at that stage in order to avoid additional contamination. For the kidneys, a thin cross-sectional slice was taken close to, but not at, the center of the organ. This procedure was applied consistently to account for the inhomogeneous distribution of cadmium within kidney tissue. All samples were collected prior to carcass cooling, placed in individually labeled plastic bags, and transported to the Department of Food Hygiene and Technology, Faculty of Veterinary Medicine, University of Belgrade. Samples were stored frozen at −20 °C prior to the analysis of lead and cadmium concentrations.

2.4. Determination of Cadmium and Lead Concentrations

Before analysis, horse muscle, liver and kidney samples were thawed at 4 ± 1 °C and mechanically homogenized (Ultra-Turrax, IKA-Werke GmbH & Co. KG, Staufen, Germany) to obtain uniform homogenates suitable for toxic element determination. The concentration of cadmium and lead in the homogenized muscle, liver and kidney samples was determined according to EN 14082:2003 [21]. All chemicals used were of analytical purity. Deionized water (resistivity of 18.2 MΩ/cm at 25 °C), obtained from a Millipore Elix UV 10 system, was used for all dilutions. The reagents used for digestion—nitric acid (HNO₃, 65%) and hydrochloric acid (HCl, 38%)—were of trace metal grade. Calibration standards were prepared by diluting 1000 mg/L stock solutions.

For each determination, homogenate portions weighing approximately 10 ± 0.0001 g were utilized. Dry digestion of all homogenate portions was performed using an annealing furnace (Heraeus-Nigos 1011 P, Hanau, Germany). The homogenate portions were gradually heated by increasing the temperature at a rate of 50 °C/h from room temperature to 450 °C, at which they were maintained for 8 h. The resulting ash was dissolved in 5 mL of hydrochloric acid (6 M), and the solution was evaporated to dryness in a water bath. The residual precipitate was dissolved in approximately 10.0 mL of nitric acid (0.1 M), filtered and washed with deionized water to a final volume of 50 mL. Blank samples were prepared identically, excluding the tissue.

Following preparation, cadmium and lead concentrations were determined using a atomic absorption spectrometer equipped with an MHS system (AAnalyst 700, PerkinElmer Inc., Shelton, CT, USA). Elements were detected via flame atomic absorption spectrometry (FAAS) using an air–acetylene flame with hollow cathode lamps and a deuterium lamp for background correction. Measurements were performed in peak area mode against a calibration curve. The operating parameters for toxic element determination are summarized in

Table 1. Instrumental parameters for flame atomization (FAAS) using Analyst 700.

Toxic Element	Wavelength (nm)	Acetylene Flow (L/min)	Air Flow (L/min)	Slit (nm)
Cadmium (Cd)	228.8	2.0	17.0	0.7
Lead (Pb)	283.3	2.0	17.0	0.7

. Toxic element concentration was expressed in milligrams per kilogram (mg/kg) of the sampled tissue’s wet weight. All samples were analyzed in duplicate, and results are reported as mean ± standard deviation. Finally, the concentrations of cadmium and lead were compared with the maximum levels established by Commission Regulation (EU) 2023/915 [8]. The limit of detection (LoD) and limit of quantification (LoQ) for toxic elements in horse tissues for lead were (LoD) = 0.05 mg/kg, (LoQ) = 0.10 mg/kg and for cadmium were (LoD) = 0.007 mg/kg, (LoQ) = 0.010 mg/kg μg/L. The quality of the analyses was controlled using a certified reference material, BCR-185 (IRMM, Geel, Belgium) bovine liver. The concentrations determined in the reference material for cadmium and lead are shown in

Table 2. Certified concentration and measured values for the Certified Reference Material BCR-185 (bovine liver) (mg/kg).

Toxic Element	Assigned Values (mg/kg)	Measured Value ± Uncertainty (mg/kg)
Cadmium (Cd)	0.544 ± 0.017	0.535 ± 0.019
Lead (Pb)	0.172 ± 0.009	0.177 ± 0.021

and were within the permissible deviations specified in the delivered certificate.

The accuracy of the test method was also proven by the spike recoveries, with the addition of standard solutions in horse muscle samples. The mean recoveries were 104.46% for lead and 96.08% for cadmium. These results, derived from six replicates, are detailed in

Table 3. Concentration and recovery mean value of examined elements in horse muscle.

	Concentration of Element in Sample (mg/kg)	Concentration of Element in Sample After Spike (mg/kg)	Concentration of Spiked Element in Sample (mg/kg)	Added Concentration of Spiked Element in Sample (mg/kg)	Recovery Mean Value (%)
	S1	S2	S2 − S1	S3	S2 − S1/S3 × 100
Toxic Elements
Cadmium (Cd)	0.160	0.258	0.098	0.102	96.08
Lead (Pb)	0.440	0.651	0.211	0.202	104.46

.

2.5. Statistical Analysis

Statistical analysis was performed using IBM SPSS Statistics, version 23.0 [22]. The slaughtered horses were distributed into three age categories: under 2.5 years (Group A) (n = 44), from 2.5 to 4 years (Group B) (n = 46), and over 4 years (Group C) (n = 42). Furthermore, they were classified into two sex groups: mares, defined as fully developed females suitable for breeding (n = 65), and males, including both stallions (intact, ready for breeding) and geldings (castrated males) (n = 67). Finally, they were classified into four seasonal groups: (i) winter—individuals slaughtered from December to February (n = 33); (ii) spring—individuals slaughtered from March to May (n = 35); (iii) summer—individuals slaughtered from June to August (n = 33); and iv) autumn—individuals slaughtered from September to November (n = 31). Continuous variables were tested for normality using the Shapiro–Wilk test (p > 0.05), and outliers were excluded from further analysis if z-score > 3 (in total 12 samples across nine groups defined by sex, age and season). A general linear mixed model was used to estimate cadmium and lead concentrations in the muscle, liver and kidney of horses, with sex, slaughter age and season included as fixed factors. All two- and three-way interactions among the fixed effects were initially tested and subsequently excluded from the model when not statistically significant (p > 0.05). As no significant interactions were detected (p > 0.05), results were pooled across treatments and are presented in the tables and discussed in the text based on the main effects of each factor. The Bonferroni correction was applied for post hoc comparisons. The data are presented as means with standard errors of the means (SEM). Based on established cut-off values for cadmium concentration [8], univariate logistic regression was used to examine the individual associations of organ type, sex, slaughter age and season with elevated cadmium concentrations. All variables with p < 0.01 were included in the final multivariable model to determine their common effects, accounting for relative contributions. The Hosmer–Lemeshow test was used to assess the validity of the logistic models (p > 0.05). Results are presented as odds ratios (ORs) with 95% confidence intervals (CIs).

3. Results

The mean concentrations of cadmium and lead in the muscle, liver and kidney of slaughtered horses are shown in

Table 4. Cadmium and lead levels in muscle, liver and kidney of slaughtered horses (mg/kg, wet weight).

Toxic Elements	Mean	SEM	Minimum	Maximum
Cadmium (mg/kg)
Muscle	0.19	0.01	0.02	0.61
Liver	2.31	0.15	0.11	7.80
Kidney	7.70	0.26	0.01	17.60
Lead (mg/kg)
Muscle	0.51	0.02	0.21	0.82
Liver	0.74	0.02	0.20	1.54
Kidney	0.68	0.02	0.21	1.20

Abbreviations: SEM—standard error of the mean.

.

3.1. Cadmium Concentration in Horse Muscle, Liver and Kidney

Using a three-factorial general linear mixed model, individual effects of horse age and season were found for cadmium concentrations across the different organs, without interactive effects (p > 0.05) (

Table 5. Cadmium concentrations in horse tissues, according to sex, slaughter age and season.

Cadmium (mg/kg)	Sex		p-Value	Slaughter Age (Years)			p-Value	Sex				p-Value	SEM
	Male	Female		<2.5	2.5–4	>4		Winter	Spring	Summer	Autumn
Muscle	0.20 a	0.17 a	0.09	0.11 a	0.21 b	0.28 c	0.001	0.12 a	0.11 a	0.23 b	0.24 b	0.001	0.03
Liver	2.32 a	2.31 a	0.82	1.40 a	2.70 b	3.29 c	0.001	1.53 a	1.00 a	2.92 b	2.90 b	0.001	0.30
Kidney	14.44 a	14.61 a	0.88	9.00 a	16.94 b	20.29 c	0.001	9.81 a	6.81 a	18.10 b	18.08 b	0.001	1.27

Abbreviations: SEM—standard error of the mean. Different letters in the same row of the table denote statistical significance at the p < 0.05 level.

). The mean concentration of cadmium did not differ (p > 0.05) according to the sex of the slaughtered animal between the muscle, liver or kidney. Slaughter age significantly affected (p < 0.001) cadmium concentrations in the three tissue types. The highest mean concentrations of cadmium were recorded in the group of horses over 4 years old. Slaughter season significantly affected mean cadmium concentrations in all examined tissue types, with significantly higher levels observed in summer and autumn compared to winter and spring (p < 0.001). The highest concentrations of this toxic element were obtained during the summer season in the liver and kidneys, while the highest muscle concentration was recorded during autumn sampling.

3.2. Lead Concentration in Horse Muscle, Liver and Kidney

Using a three-factorial general linear mixed model, individual effects of horse age and season were found for lead concentrations across the different organs (

Table 6. Lead concentrations in horse tissues, according to sex, slaughter age and slaughter season.

Lead (mg/kg)	Sex		p-Value	Slaughter Age (Years)			p-Value	Season				p-Value	SEM
	Male	Female		<2.5	2.5–4	>4		Winter	Spring	Summer	Autumn
Muscle	0.53 a	0.49 a	0.10	0.32 a	0.44 b	0.67 c	0.001	0.48 a	0.41 a	0.49 a	0.62 b	0.04	0.04
Liver	0.72 a	0.74 a	0.85	0.43 a	0.74 b	1.14 c	0.001	0.74 a	0.60 a	0.72 a	0.81 a	0.17	0.07
Kidney	0.69 a	0.68 a	0.87	0.38 a	0.63 b	0.93 c	0.001	0.66 a	0.53 a	0.64 a	0.87 b	0.01	0.06

Abbreviations: SEM—standard error of the mean. Different letters in the same row of the table denote statistical significance at the p < 0.05 level.

), without interactive effects (p > 0.05). Sex did not have a significant influence on mean lead concentrations in any of the tested tissue types (p > 0.05). Slaughter age affected the lead concentrations in all tested horse tissues, whereby higher levels were detected in individuals older than 4 years than in the other two age categories (p < 0.001).

Statistical analysis showed differing results depending on the tissue type. Regarding the concentration of lead in the liver, no significant differences were observed among the tissues obtained during the four seasons (p > 0.001). In contrast, significantly higher mean lead concentrations were determined in muscle and kidney tissues during autumn rather than in other seasons (p < 0.001).

3.3. Factors Associated with Increased Cadmium Concentration in the Horse Tissues

Across all analyzed horse tissue samples (muscle, liver, and kidney), 74.0% exhibited cadmium concentrations exceeding the maximum permitted levels (95% confidence interval, CI: 69.4–78.2). Univariate logistic regression was used to determine individual associations between organ type and horse age with increased cadmium concentrations (

Table 7. Association between the individual investigated factors and increased cadmium concentration in horse tissues.

	n	Exceeded Concentration %	95% CI	OR	95% CI	p-Value
Organ						0.001
Liver	132	93.2	87.4–96.8	1.00
Muscle	132	31.1	23.3–39.7	0.04	0.01–0.07
Kidney	132	97.7	93.5–99.5	3.15	0.83–11.89
Sex						0.28
Male	195	76.4	69.8–82.2	1.00
Female	201	71.6	64.9–77.8	0.78	0.50–1.22
Slaughter age						0.003
<2.5 years	144	65.3	56.9–73.0	1.00
2.5–4 years	153	75.2	67.5–81.8	1.61	0.97–2.66
>4 years	99	84.8	76.2–91.3	2.98	1.56–5.69
Slaughter season						0.09
Summer	147	76.9	69.2–83.4	1.00
Autumn	99	79.8	70.5–87.2	1.19	0.64–2.21
Winter	90	70.0	59.4–79.2	0.70	0.39–1.27
Spring	60	63.3	49.9–75.4	0.52	0.27–0.99
Total	396	74.0	69.4–78.2

Abbreviations: CI—confidence interval; OR—odds ratio.

). All variables with p < 0.10 were tested for multicollinearity and included in the final multivariable logistic model to determine their common effects on cadmium accumulation (

Table 8. Final regression model: Adjusted odds ratios for exceeded cadmium concentration in horses.

	Adjusted OR	95% CI	p-Value
Organ
Liver	1.00
Muscle	0.02	0.01–0.04	0.02
Kidney	3.34	0.86–13.02	0.08
Slaughter age
<2.5 years	1.00
2.5–4 years	2.88	1.25–6.65	0.01
>4 years	7.96	2.94–21.51	0.0001
Slaughter season
Summer	1.00
Autumn	1.01	0.40–2.53	0.99
Winter	0.42	0.17–0.99	0.05
Spring	0.28	0.10–0.81	0.01

Abbreviations: CI—confidence interval; OR—odds ratio.

).

The probability of cadmium accumulation was 98% lower in muscle compared to the liver (p = 0.02). Compared to horses younger than 2.5 years, a 2.88-fold greater risk of exceeding cadmium limits was observed in horses aged 2.5–4.0 years (p = 0.01), while a 7.98-fold greater risk was found in horses older than 4 years (p = 0.001). Furthermore, the likelihood of exceeding cadmium limits was 58% lower in winter (p = 0.05) and 72% lower in spring (p = 0.01) compared to the summer season.

4. Discussion

4.1. Level of Cadmium and Lead Accumulation in Horse Tissues

According to Commission Regulation (EU) 2023/915 [8], the maximum level for cadmium in horse muscle is set at 0.20 mg/kg, reflecting the need to limit consumer exposure to this toxic metal. Although the mean cadmium concentration in muscle tissue recorded in the present study was slightly below the regulatory limit (0.19 mg/kg), the fact that 31.1% of individual samples exceeded the maximum level represents a substantial compliance concern for horse meat intended for the retail market and also a public health concern [11]. From a food safety and public health perspective, such exceedance rates suggest that a substantial proportion of horse meat entering the retail chain may contribute to dietary cadmium exposure beyond tolerable intake levels, particularly among high-consumption groups [23]. The situation is considerably more critical for edible offal, where cadmium accumulation is known to be markedly higher due to cadmium’s affinity for metallothioneins in detoxifying organs [24]. In the present study, exceedance rates of 93.94% in liver and 97.7% in kidney samples relative to European Union maximum cadmium limits (0.50 mg/kg and 1.0 mg/kg, respectively) suggest a systematic food safety risk associated with horse offal consumption. These findings are of particular concern given that horse liver and kidney are traditionally consumed in several European countries, including Serbia, and may significantly contribute to chronic cadmium exposure [25]. The mean cadmium concentrations observed in the present study (2.31 mg/kg in liver and 7.70 mg/kg in kidney) are consistent with accumulation patterns previously reported in horses from Poland [12], confirming that elevated cadmium levels in offal are widespread rather than region-specific. Although these values are lower than the extreme concentrations reported in horses from heavily industrialized regions of Turkey [26] and Kazakhstan [27], they remain comparable to levels observed in kidney samples from horses in Croatia, reinforcing the kidney as the primary target organ for cadmium accumulation [28]. Furthermore, Baldini et al. [29] reported significant differences in cadmium levels among horses from Poland, Lithuania and Hungary, with the highest concentrations in Polish horses, consistent with the region’s history of intensive industrial cadmium contamination [26]. More recently, Dimuccio et al. [11] demonstrated that horses originating from Italy and Spain exhibited significantly lower cadmium concentrations than horses imported from Poland, showing the strong influence of geographic origin on cadmium accumulation in edible tissues. It can be argued that geographic variability has important implications for food safety controls, as horses originating from highly industrialized regions may pose a greater toxicological risk if not subjected to targeted monitoring, underscoring the strong influence of regional environmental conditions on cadmium transfer into the food chain [30].

Analysis of lead accumulation in different horse tissues revealed the highest mean concentrations occurred in the liver (0.74 mg/kg), followed by the kidney (0.68 mg/kg) and muscle (0.51 mg/kg), reflecting the preferential accumulation of lead in metabolically active organs involved in detoxification and excretion [31,32]. From a food safety and public health perspective, these values are of concern due to the well-documented toxicological effects of lead and its cumulative nature in human consumers [33]. Although Commission Regulation (EU) 2023/915 [8] explicitly establishes maximum levels for lead in the meat and offal of cattle, sheep, pigs and poultry, it does not define a specific category for horse meat or edible offal, creating regulatory ambiguity for this species. In the absence of dedicated maximum limits for horses, the application of the strictest livestock standards represents a precautionary approach aimed at protecting consumer health and ensuring regulatory comparability across food-producing animals [13]. Furthermore, the lead concentrations observed in the present study exceeded those reported in Croatia (0.094 mg/kg) [28] and Germany (0.084 mg/kg) [34], indicating a potentially higher environmental or dietary exposure in the studied animal population. These differences may be attributed to regional environmental contamination, including soil and forage pollution, as well as horse age and management practices, all of which are recognized determinants of lead accumulation in livestock tissues [30].

Based on the results of this study, the elevated cadmium and lead concentrations in horse edible tissues are most likely linked to the geographic origin of the horses, as both collection centers were located in the Srem District of the Autonomous Province of Vojvodina, a region with a documented history of industrial and petrochemical pollution [35,36,37,38]. Environmental pollution resulting from the destruction of industrial facilities during the 1999 NATO bombardment, combined with atmospheric deposition, abnormal rainfall and subsequent flooding events, likely contributed to the long-term contamination of soils, surface waters, vegetables, crops and pastures [35,36]. Considering that the Autonomous Province of Vojvodina is one of the largest and most intensively cultivated agricultural regions in the Balkans, such legacy pollution provides a plausible pathway for toxic elements to transfer into grazing animals and, consequently, into the human food chain [37,38]. From a food safety and public health aspect, these findings highlight the importance of geographic origin and historical environmental burden as key risk factors and emphasize the need for region-based monitoring and traceability of horse meat and offal intended for human consumption. Moreover, the findings of this study underline the importance of traceability and origin-based risk assessment in managing toxic element contamination in horse meat and offal entering the food chain [13].

4.2. Association Between Slaughter Age and Cadmium and Lead Concentrations in Horse Tissues

In the present study, slaughter age had a significant effect on cadmium accumulation across all examined tissues, with horses older than four years exhibiting approximately 3-fold higher mean cadmium concentrations than younger animals, particularly in kidney and liver tissues. The findings of this study indicate that meat and edible offal obtained from younger rather than older horses pose comparatively lower food safety risks with respect to cadmium exposure. Comparable age-dependent accumulation trends were also documented in previous studies [28,29,34], with cadmium concentrations in some cases being up to 5-fold greater in older horses. This highlights the cumulative nature of cadmium exposure over the horse’s lifespan and confirms the consistency of this phenomenon across different production systems and geographic regions. In contrast, other authors [39] reported that cadmium levels in the kidney and liver increase with age up to a certain point and may subsequently decline in geriatric horses, a pattern also described in human populations and attributed to age-related physiological and metabolic changes (renal damage and the subsequent death of tubular cells) [9]. From a food safety and public health perspective, the progressive increase in cadmium concentration in horse tissues with advancing age is of particular concern, as this metal has an extremely long biological half-life and preferentially binds to metallothioneins, resulting in cumulative tissue retention over time and a high dietary exposure risk for consumers [24]. This phenomenon is particularly relevant in slaughter horses, which are often slaughtered at ages exceeding five years, in contrast to other food-producing animals that are typically slaughtered before two years of age [11]. As a consequence, slaughter horses experience prolonged exposure to contaminated soil, pasture and feed, resulting in a substantially greater cumulative toxic element burden [40].

In this study, slaughter age significantly affected lead concentrations in all examined horse tissues, with individuals older than four years exhibiting markedly higher mean levels than did younger age categories, confirming age as a critical determinant of lead accumulation. Considering the food safety and public health aspect, this finding is of great importance, because lead is a cumulative toxic element with slow elimination, resulting in progressive tissue accumulation over the animal’s lifespan [41]. Similar age-dependent increases in lead accumulations have been reported in horse meat and offal in Poland, where older individuals consistently showed higher lead burdens than younger horses [12]. The observed pattern is consistent with toxicokinetic studies demonstrating that prolonged exposure to contaminated feed, water, soil and atmospheric deposition leads to greater lead retention in edible tissues over time [30,32,42]. In addition, a concerning fact is that accumulation of lead was evident not only in detoxifying organs, i.e., liver and kidney, but also in muscle tissue, which is of direct relevance for consumer exposure due to its higher consumption rate [42]. This is of paramount concern because no safe exposure threshold for lead has been identified [8], and even low-level chronic intake has been associated with adverse neurological and cardiovascular effects in humans [33]. As previously mentioned, horses slaughtered for human consumption are often significantly older than other food-producing animals, which enhances the risk of elevated lead accumulation when compared to beef, pork or poultry products [11]. Consequently, meat, edible offal, and meat products derived from older horses may contribute disproportionately to dietary lead exposure in consumers, particularly when consumed regularly or in large quantities [33].

According to the findings of this study, the significant accumulation of toxic elements with slaughter age highlights the importance of age-based risk stratification and strongly supports the enhanced monitoring of older horses intended for human consumption to reduce consumer exposure to cadmium and lead through horse meat, edible organs and horse-derived meat products.

4.3. Association Between Sex and Cadmium and Lead Concentrations in Horse Tissues

In the present study, sex did not significantly influence mean cadmium or lead concentrations in horse muscle, liver or kidney tissues, indicating that sex-related physiological differences do not play a major role in the accumulation of these toxic elements. As a result, both male and female horses contribute equally to chronic dietary exposure to these toxic elements in consumers. Similar observations have been reported in horses [34,43] and other food-producing animals [44,45,46], where cadmium and lead tissue levels were largely independent of sex and were instead driven by environmental exposure intensity. From a food safety and public health perspective, these findings imply that both male and female horses are comparable potential sources of dietary exposure to the two toxic elements when raised under similar environmental conditions. Several studies [30,32,42] have demonstrated that the uptake and retention of cadmium and lead are primarily governed by external factors, such as contaminated soil, forage and feed, rather than intrinsic biological variables, including sex. In this study, males and female horses shared similar grazing habits, feeding regimes and management systems, which likely explains the absence of significant sex-related differences in toxic element burdens. Consequently, it can be argued that food safety risks associated with the aforementioned toxic elements in horse meat and offal cannot be mitigated through sex-based selection strategies. These findings further reinforce the need for food safety control measures to focus on environmental monitoring, age at slaughter and geographic origin rather than animal sex when assessing the risk of toxic element contamination in horse-derived food products.

4.4. Association Between Slaughter Season and Cadmium and Lead Concentrations in Horse Tissues

In the present study, slaughter season had a significant effect on cadmium concentrations in all examined tissues, with markedly higher mean levels observed in summer and autumn compared to in winter and spring. In addition, significantly higher mean lead concentrations were detected in muscle and kidney tissues of slaughter horses during autumn than in other seasons. This seasonal increase may be attributed to greater environmental exposure during late grazing periods (late summer and autumn), when farm animals, including horses, are often on pastures [47], which increases the risk of toxic element uptake in areas that are contaminated by atmospheric deposition, soil dust, feed sources or agricultural activities [40,48]. This seasonal pattern has also been linked to greater toxic element accumulation in forage plants during periods of intensive growth and to increased ingestion of soil with lower moisture and pH and greater organic matter content, which enhances uptake of cadmium and lead by grazing horses [40,49]. From a public health standpoint, the observed seasonal increase in cadmium and lead accumulation in edible horse tissues underscores the importance of considering slaughter season as a risk-modifying factor in the food safety assessment of horse meat and offal. These findings support the implementation of seasonally targeted monitoring and control strategies to reduce consumer exposure to these two toxic elements from horse-derived food products.

5. Conclusions

This study confirms that horse muscle, and to a greater extent, edible horse offal, are significant sources of cadmium and lead exposure, raising important food safety concerns. Although the mean cadmium level in muscle was close to the regulatory limit, the high proportion of non-compliant samples and the markedly elevated concentrations in liver and kidney indicate that horse offal consumption poses a substantial public health risk. Elevated mean lead concentrations across all tissues, together with the lack of horse-specific regulatory limits, further emphasize the need for clearer and more harmonized control measures. Slaughter age was identified as the primary factor influencing cadmium and lead accumulation, with horses older than four years showing substantially higher concentrations, supporting age-based risk stratification and enhanced monitoring of older animals. In contrast, the absence of sex-related differences highlights the dominant role of environmental exposure and lifespan in the accumulation of these toxic elements. Seasonal patterns, with higher toxic element levels in horse tissues detected during summer and autumn rather than during winter and spring, indicate that the season of slaughter should also be considered in food safety surveillance. Therefore, the findings obtained in the present study highlight the importance of monitoring slaughter horses throughout the entire production process and implementing the ‘Farm to Fork’ strategy. In parallel, this study confirms the need for the development of species-specific guidelines outlining maximum permitted limits of toxic elements. Future research should prioritize dietary exposure assessment and risk characterization in different consumer groups, longitudinal studies on accumulation dynamics in different edible tissues and investigations into environmental and management-related drivers of horse-derived food contamination. In addition, the establishment of horse-specific regulatory limits would significantly improve food safety throughout the horse meat chain continuum.