Comprehensive Assessment of Potentially Toxic Element (PTE) Contamination in Honey from a Historically Polluted Agro-Industrial Landscape: Implications for Agricultural Sustainability and Food Safety

Abstract

Honey is increasingly recognized not only as a functional food but also as a potential bioindicator of environmental pollution. This study assessed the concentrations of four potentially toxic elements (PTEs)—lead (Pb), cadmium (Cd), copper (Cu), and zinc (Zn)—in 48 multifloral honey samples collected in 2023 from seven locations across a historically polluted agro-industrial region in Romania. Samples were analyzed using Flame Atomic Absorption Spectrometry (FAAS) and Graphite Furnace AAS (GFAAS), with quality control ensured through certified reference materials. Results revealed that Pb (0.72–1.69 mg/kg) and Cd (0.02–0.37 mg/kg) levels consistently exceeded international safety thresholds, while Cu (0.62–2.22 mg/kg) and Zn (0.91–1.93 mg/kg), although essential nutrients, were found in elevated concentrations. Spatial analysis indicated a general trend of higher contamination in sites located closer to former industrial facilities, influenced by factors such as altitude and atmospheric transport. These findings confirm the persistent environmental burden in post-industrial landscapes and support the use of honey as a cost-effective tool for pollution monitoring. The study underscores the need for targeted environmental policies, sustainable apicultural practices, and continued surveillance to protect ecosystem health and food safety.

1. Introduction

Honey is a natural product of ecological and nutritional relevance, valued for its rich composition of mainly fructose and glucose, as well as oligosaccharides, vitamins, minerals, amino acids, and bioactive compounds which confer antioxidant, antimicrobial, and anti-inflammatory properties [1,2,3,4,5,6,7]. It is also recognized for its ability to reflect environmental and landscape health. These characteristics support honey’s classification as a functional food relevant to sustainable agriculture and human health. Recently, however, attention has shifted toward its susceptibility to environmental pollutants, especially potentially toxic elements (PTEs) like cadmium, lead, copper, and zinc, which may affect its quality and safety. Honey’s water content (typically 17–20%) is crucial for its texture, microbial stability, and shelf-life, which are key factors in apicultural product quality and safety [3]. Honey is considered a functional food mainly due to its complex chemical composition. Beyond simple sugars like fructose and glucose, honey contains biologically active components such as proteins and amino acids (especially proline), which enhance its bioactivity [1,3,4]. It also contains essential micronutrients, including B-complex vitamins, vitamin C, and minerals like calcium, iron, magnesium, potassium, and zinc [3,5]. Together with phenolics, flavonoids, and enzymes, these compounds contribute to honey’s antioxidant, antimicrobial, and anti-inflammatory effects [4,6,7]. These properties support therapeutic effects such as wound healing, digestive regulation, immune modulation, and potential anticancer activity [8,9,10].

Honey’s multifunctionality stems from its bioactive compounds, enabling uses in food, pharmaceutical, and cosmetic sectors. As a natural sweetener and functional ingredient, it supports preservation and enhances nutritional quality through its antioxidant and antimicrobial properties [11,12,13]. In medicine, honey is valued for treating respiratory, gastrointestinal, and skin conditions, and is used in wound-healing materials and drug delivery systems [5,14,15]. In cosmetics, honey’s moisturizing and anti-aging effects make it a common ingredient in skincare and haircare products [16,17]. Honey is also used in advanced applications like cryopreservation and food preservation, supporting its role as a sustainable, bio-based resource in circular agriculture [18,19]. Honey’s link to environmental matrices via bee foraging highlights its growing use as a bioindicator of pollution, especially in industrially impacted agroecosystems. Heavy metals can enter the hive and accumulate in bee products through a complex transfer pathway that begins in the environment and is mediated by the foraging behavior and physiology of honey bees. The main sources of contamination include soil, air, and water in areas exposed to industrial, urban, or mining activities, where metals such as cadmium (Cd) and lead (Pb) can accumulate in the tissues of nectar-producing plants. Bees collect nectar and pollen from these plants, inadvertently introducing environmental contaminants into the hive. For instance, Tomczyk et al. (2023) [20] found that dandelion and goldenrod flowers growing in contaminated areas accumulated up to 0.94 mg/kg Cd and 0.89 mg/kg Pb, yet the honey produced from these sources contained only trace amounts (<0.015 mg/kg Cd and <0.043 mg/kg Pb), likely due to partial biological filtration during nectar processing. This transfer is heavily influenced by bee behavior: honey bees typically forage over large distances of up to several kilometers and do not avoid contaminated resources. Di et al. (2020) [21] demonstrated that bees actively consumed sugar syrup containing 0.3 mg/L Cd and 2 mg/L Cu, leading to Cu accumulation of up to 17.93 mg/kg in bee tissues, highlighting that behavioral avoidance is ineffective against contamination. Similarly, Sovrlić et al. (2022) [22] reported detectable arsenic (As) levels in honey collected up to 32 km from copper mining sites in Serbia, suggesting that bees’ extended foraging range allows them to transport pollutants over significant distances. Glevitzky et al. (2025) [23] further showed that honey samples from mining areas consistently exhibited elevated levels of various heavy metals, correlating with hive proximity to pollution sources. Once inside the bee’s body, contaminants are absorbed via the digestive tract and transported through the hemolymph to various tissues. Borsuk et al. (2021) [24] observed that during nectar processing, bees effectively reduced metal concentrations: iron decreased from 100.1 to 2.5 mg/kg, zinc from 54.3 to 2.1 mg/kg, copper from 5.8 to 0.7 mg/kg, and cadmium from 0.06 to 0.007 mg/kg, indicating an efficient filtration mechanism during food conversion. Nevertheless, metals can still bioaccumulate in beeswax and, to a lesser extent, in honey. Hladun et al. (2016) [25] demonstrated that cadmium and selenium accumulated differentially in bees depending on caste and tissue type: for example, queens exposed to 3.6 mg/kg Cd in diet accumulated 5.87 ± 0.24 µg/g in their tissues, compared to 4.08 ± 0.29 µg/g in workers. Similarly, selenium reached 11.8 ± 0.8 µg/g in queens and 8.5 ± 0.6 µg/g in workers, showing caste-based differences in metal processing. These findings underscore that honey bees, through both their ecological behavior and internal physiology, serve as vectors for environmental contaminants, and that hive products may retain measurable, albeit often reduced, levels of toxic metals.

In addition to its broad industrial and therapeutic uses, honey has emerged as a practical tool in environmental biomonitoring due to the foraging behavior of bees and their exposure to multiple environmental matrices including air, soil, and floral resources. This property enables honey to reflect the ecological quality of landscapes, especially in agro-industrial zones impacted by historical contamination [26,27,28,29]. Despite this potential, comparability between studies remains limited due to non-standardized methodologies and high variability linked to seasonality, anthropogenic activity, and site-specific factors [30,31,32]. Furthermore, the integration of geospatial variables—such as altitude, distance to pollution sources, and landscape configuration—remains underexplored in honey-based contamination studies [29,32]. These knowledge gaps hinder the development of effective environmental risk assessment models and limit the operational use of honey as a bioindicator. Addressing these limitations would enhance food safety governance and promote sustainable monitoring systems in agricultural ecosystems.

Heavy metal contamination in honey has become an increasing concern within agri-food systems, raising implications for both environmental integrity and public health. Owing to their close interaction with air, soil, water, and vegetation, honeybees and honey are now widely regarded as sensitive bioindicators of environmental pollution, particularly in detecting toxic metals such as lead and cadmium [26,27,28,29]. However, the interpretation and comparability of contamination data remain limited due to factors such as spatial variability, seasonal fluctuations, anthropogenic activities, and the lack of harmonized sampling and analytical methodologies [30,31,32]. The absence of internationally standardized maximum residue levels (MRLs) for potentially toxic elements (PTEs) in honey further complicates risk assessment and regulatory enforcement in the food sector [33,34,35].

Although honey is widely regarded as a safe and natural food, its trace element content warrants monitoring, particularly in regions exposed to environmental pollution [36,37,38]. While elements such as Zn and Cu are essential micronutrients important for human health, long-term excessive intake—especially of toxic elements like Cd and Pb—may present risks, depending on both concentration and consumption levels [36,37,38]. Therefore, assessing their presence in honey is relevant in the context of dietary exposure, especially for vulnerable population groups. Additionally, ecological factors, such as altered foraging behavior in contaminated landscapes and limited knowledge of detoxification mechanisms in bees (e.g., metallothionein expression), may lead to underestimation of true environmental loads [39,40]. Addressing these knowledge gaps through methodological standardization and expanded environmental monitoring could enhance the role of honey as a tool for agroecological surveillance and improve food safety governance.

In parallel with its increasing consumption and multifunctional use, honey has come under scrutiny due to its vulnerability to environmental contamination, particularly from potentially toxic elements (PTEs). As honeybees forage across large landscapes and interact with multiple environmental matrices, honey reflects both geogenic and anthropogenic sources of pollution, making it a valuable bioindicator of ecological health [26,27,28,29]. Naturally occurring elements such as cadmium, chromium, copper, iron, nickel, lead, and zinc originate from soil and can be absorbed by nectar-producing plants, subsequently entering the apicultural food chain [41,42]. However, anthropogenic activities, especially mining, petroleum refining, energy production, and industrial emissions, significantly elevate metal concentrations in surrounding agroecosystems [29,43,44]. Additional contamination may result from the use of fertilizers and pesticides, vehicular emissions, and airborne particulates in urban or peri-urban zones [45,46,47]. Studies have shown that honey collected near roads or industrial sites frequently exhibits elevated levels of Pb, Cd, Zn, Cr, and Mn, occasionally exceeding internationally recommended limits [25,26,30,31,32]. The spatial placement of apiaries plays a critical role in determining exposure risk, while factors such as soil properties, plant type, local climate, and bee behavior also modulate bioaccumulation patterns [29,48,49,50]. These findings emphasize the need for strategic apiary siting, environmentally conscious land use practices, and regular monitoring programs to ensure both honey safety and the ecological sustainability of apicultural systems [43,45].

Despite the increasing number of studies focused on potentially toxic elements (PTEs) in honey, significant gaps persist in terms of methodological standardization and regional harmonization in sampling design, analytical protocols, and data interpretation [29,30,32]. Very few investigations systematically consider spatial variability related to factors such as elevation, microclimate, or proximity to pollution sources, all of which are known to influence contaminant dispersion. Furthermore, regions in Eastern Europe with extensive mining histories remain underrepresented in global research efforts [48,49]. This gap is particularly relevant in post-industrial landscapes where beekeeping remains an active practice, yet contamination risks are poorly quantified and geographically underexplored. Accurate determination of trace-metal concentrations in honey is essential not only for assessing product quality and food safety but also for supporting regulatory frameworks in agricultural and environmental systems. To this end, advanced analytical tools have been adopted to meet the demands of high sensitivity, selectivity, and reproducibility. Atomic Absorption Spectroscopy (AAS) is widely used for trace metals such as cadmium, chromium, copper, lead, and zinc due to its precision and cost-effectiveness [38,46,51]. Inductively Coupled Plasma Mass Spectrometry (ICP-MS) offers superior sensitivity at parts-per-trillion levels and enables simultaneous multi-element detection, making it ideal for in-depth environmental risk assessments [52,53]. ICP-OES is particularly effective for high-throughput monitoring where speed and accuracy are essential [54,55]. These tools enable robust contamination assessments and contribute to evidence-based policy development while supporting the traceability and safety of apicultural products within sustainable agri-food chains.

The presence of potentially toxic elements (PTEs) in honey raises critical concerns for both public health and agro-environmental sustainability. Toxic elements such as cadmium (Cd), lead (Pb), and chromium (Cr) are known to bioaccumulate and pose significant health risks, including nephrotoxicity, skeletal damage, developmental disorders, and carcinogenic effects especially among vulnerable populations such as children [39,56,57]. From an agroecological perspective, honey functions as an effective bioindicator of cumulative pollution across air, soil, and vegetation, offering valuable insights into broader environmental degradation and threats to biodiversity [58,59,60]. To mitigate contamination, various remediation approaches have been explored, including bioremediation using sulfate-reducing bacteria for in situ stabilization of metals in soil [61,62], phytoremediation through the use of hyperaccumulator plants [54], and adsorption techniques involving nanomaterials such as graphene oxide [63]. Preventive strategies remain essential and include routine monitoring of honey and environmental matrices, enforcement of stricter emission controls in agriculture and industry, and targeted outreach programs for beekeepers and land managers [32,64]. Collectively, these interventions support the dual goals of food safety and environmental stewardship, contributing to the resilience of agricultural systems in polluted landscapes.

In Romania, a growing body of research has investigated heavy metal concentrations in honey, providing valuable insights into environmental contamination and food safety within diverse agricultural landscapes [32,47,61,65]. Commonly monitored elements include lead (Pb), cadmium (Cd), zinc (Zn), copper (Cu), chromium (Cr), iron (Fe), manganese (Mn), and nickel (Ni), with elevated concentrations often associated with industrial zones or peri-urban areas, as opposed to rural or protected ecosystems [28,66]. Reported lead concentrations typically range between 0.018 and 0.05 mg/kg, generally remaining below those observed in neighboring countries such as Poland [61]; however, site-specific exceedances for Pb and Cd have been documented [28,67]. While Zn and Cu levels were largely within regulatory limits, elevated values were occasionally observed near heavy industry [32]. Trace amounts of Cr, Ni, and Al were also detected, likely originating from atmospheric deposition and vehicular emissions [28]. Attempts to classify honey by botanical origin based on metal profiles showed moderate accuracy, whereas geographic origin proved less reliable [47]. Notably, apiary location emerged as a key determinant of contamination risk, with proximity to roads, agricultural fields, or industrial activities linked to higher trace-metal levels [65]. Although most samples complied with food safety thresholds, continued surveillance remains essential, particularly in vulnerable regions, to ensure the long-term integrity and marketability of Romanian honey.

Understanding how environmental and spatial factors influence the presence of potentially toxic elements (PTEs) in honey is crucial for managing food safety risks, informing evidence-based policies, and promoting sustainable apicultural practices. Romania, with its extensive history of mining and metallurgy, presents a particularly relevant context for such investigations, especially in regions where ecological degradation and legacy pollution remain persistent concerns [66,67]. A prime example is the Zlatna region, located in the Apuseni Mountains, which has experienced over 250 years of intensive copper extraction and smelting [68]. These activities, while economically significant, have resulted in long-term environmental damage, including elevated levels of potentially toxic elements (PTEs) such as Pb, Zn, Cu, Cd, and As, and the release of sulfur dioxide and nitrogen oxides into surrounding ecosystems [69,70]. Although industrial operations ceased officially in 2004, residual contamination from tailings, acid mine drainage, and untreated wastewater continues to affect the Ampoi Valley, particularly in areas like Larga and Haneș [71,72]. Soil samples consistently show concentrations of Pb, Cd, Zn, and Ni that exceed geochemical background levels, posing risks to land productivity and ecological recovery [73,74]. Additionally, forest ecosystems in the region have been severely impacted, with over 50,000 hectares exposed to air pollutants and more than 21,000 hectares affected by acid precipitation during the 1990s. Despite partial remediation efforts, the Zlatna region remains ecologically fragile, raising critical concerns for land use planning, beekeeping, and long-term agricultural resilience [69].

To what extent do spatial variables such as altitude and proximity to former mining and metallurgical sites influence the concentration of potentially toxic elements (PTEs) in honey collected from post-industrial agricultural landscapes in Romania? This study aims to qualitatively assess the presence of potentially toxic elements (PTEs), specifically lead (Pb), cadmium (Cd), copper (Cu), and zinc (Zn), in honey samples collected from a historically polluted agro-industrial region in Romania. The investigation evaluates how contamination levels are influenced by spatial variables such as altitude, elevation, and proximity to former mining and metallurgical sites. Measured concentrations are compared with international food safety thresholds to assess potential health risks, particularly for vulnerable consumer groups. Additionally, the study examines honey’s effectiveness as a cost-efficient biomonitoring tool for detecting environmental contamination across agroecosystems. The findings support site-specific risk assessment, sustainable apiculture, and the development of environmental monitoring strategies in post-industrial regions. Results are further contextualized within the broader European literature to highlight regional contamination patterns and the persistent challenges of ecological restoration and food safety in formerly industrialized zones. To address this research question, a systematic sampling and analytical protocol was implemented, as detailed in the following section.

This study contributes to bridging the gap between environmental diagnostics and food system safety in Eastern European agroecosystems, where industrial legacies continue to shape ecological and health outcomes. By evaluating how spatial factors such as altitude and proximity to former mining sites influence honey contamination, the findings support site-specific risk assessment and inform sustainable land use planning and environmental monitoring.

2. Materials and Methods

In order to investigate the presence and distribution of potentially toxic elements (PTEs) in honey from a historically industrialized region of Romania, this study employed a standardized and reproducible methodological framework. The section outlines the geographic and environmental characteristics of the study site, the procedures used for honey sampling and preservation, and the analytical workflow involving microwave-assisted digestion and determination of trace metals via Flame Atomic Absorption Spectrometry (FAAS) and Graphite Furnace AAS (GFAAS). Details regarding reagents, instrumentation, quality assurance protocols, and statistical analysis methods are provided to ensure analytical accuracy, traceability, and data robustness.

2.1. Study Area and Environmental Background

In 2023, a total of 24 honey samples (Apis mellifera) were collected from 4 stationary apiaries located in the villages of Trâmpoiele, Izvorul Ampoiului, Feneș, and Vâltori, all administratively affiliated with the town of Zlatna, Alba County, Romania. These areas are historically affected by heavy metal pollution due to legacy mining and smelting activities. All samples were multifloral, reflecting the polyfloral vegetation profile typical of the region. Sampling sites (SP1–SP4) were georeferenced and selected based on their spatial proximity to former pollution sources such as abandoned mines and smelters. Honey extraction followed traditional apicultural practices, and samples were stored in sterile glass containers at 4–5 °C under refrigeration until laboratory analysis. Additional details regarding site location, environmental background, and sampling rationale are presented in Figure 1.

The honey samples analyzed in this study were collected during the active harvesting season, specifically between May and June 2023, from seven geographically distinct locations situated at varying distances from identified industrial pollution sources. These timeframes correspond to the peak period of nectar flow in the region, ensuring that the collected honey represents current environmental conditions during active foraging. For each location, three independent honey samples were collected from different beehives to account for intra-site variability. Each sample was subsequently analyzed in triplicate under the same laboratory conditions. This approach was employed to increase the statistical robustness of the results, ensure measurement reproducibility, and minimize potential analytical errors. The sampling and replication strategy was designed in accordance with established protocols for environmental biomonitoring using honey, allowing for both spatial comparison and reliable assessment of heavy metal concentrations.

Figure 1 provides a geospatial representation of the location of the apiaries from which honey samples were collected, in relation to the main historical industrial pollution sources in the Zlatna region: the Zlatna Industrial Platform, Larga de Sus Mine, and Haneș Tailings Dump. This spatial positioning is essential for interpreting data on potentially toxic element (PTE) contamination, as both distance and relative location to pollution sources significantly influence bee exposure and the transfer of heavy metals into honey through floral resources (nectar and pollen). The map enables visualization of altitude differences, land use, and the spatial distribution of sampling sites, thus forming the foundation of the geospatial analysis presented in this study.

The Zlatna Industrial Platform, the Larga de Sus Mine, and the Haneș Tailings Dump are among the most significant legacy pollution sources in the Apuseni Mountains of western Romania, a region historically associated with intensive metallurgical and mining activity dating back over two centuries. These facilities were key contributors to the extraction and processing of non-ferrous metals, primarily copper, lead, and zinc, placing Zlatna among the most industrialized zones in Eastern Europe during the 20th century. While the economic relevance of these operations was considerable, the environmental costs have been substantial and long-lasting. Following the official cessation of large-scale industrial activities in the early 2000s, the area has remained ecologically vulnerable due to unremediated mining residues, abandoned infrastructure, and diffuse sources of contamination.

Residual pollution persists through various mechanisms, including windborne dispersal of metal-laden dust, surface runoff from tailings during precipitation events, and acid mine drainage, which lowers soil pH and enhances the solubility and mobility of potentially toxic elements (PTEs). These processes facilitate the long-range transport of contaminants such as cadmium, lead, arsenic, and zinc into adjacent agroecosystems, where they may accumulate in topsoil and be absorbed by crops and wild flora. This long-term, low-intensity exposure creates chronic stress conditions for terrestrial biodiversity, potentially altering microbial communities, inhibiting plant growth, and affecting pollinator health.

Notably, the proximity of former industrial sites to productive farmland and permanent apiaries amplifies the risk of contaminant bioaccumulation throughout the food chain. Nectar-producing plants, rooted in contaminated soils or exposed to atmospheric deposition, can serve as vectors for trace-metal uptake, ultimately transferring pollutants into honey via the foraging activity of bees. Over time, this mechanism may compromise not only the ecological function of pollinators but also the safety of honey as a food product.

Furthermore, the region’s complex topography and land use heterogeneity—ranging from steep forested slopes and regenerating pastures to intensively used agricultural fields—create spatially diverse exposure pathways that affect both the distribution and bioavailability of metals. These conditions offer an exceptional opportunity for studying environmental gradients and identifying landscape-level factors that mediate contaminant dynamics. Insights derived from this case study can inform evidence-based mitigation strategies, guide risk zoning and remediation planning, and support the implementation of high-resolution environmental monitoring systems tailored to post-industrial agricultural areas across Central and Eastern Europe.

2.2. Honey Sampling and Storage Protocol

The honey samples analyzed in this study were obtained through the local beekeepers’ association and certified for origin and authenticity, reflecting traditional apicultural practices characteristic of the study area. A total of 48 multifloral honey samples were collected during the active harvest season, representing dominant production trends in the region and capturing botanical diversity typical of polyfloral landscapes. Each sample was composed from three distinct hives within the same production lot to ensure representativeness. Extraction was carried out by centrifugation using sanitized stainless-steel equipment, and samples were collected in sterile glass containers without any thermal processing or pasteurization. Immediately after harvest, all samples were transported under controlled conditions and stored at 4–5 °C in darkness to preserve their chemical integrity prior to laboratory analysis. Handling procedures adhered to international food safety guidelines established by the Codex Alimentarius Commission [75], while sample preparation followed the Harmonized Methods of the International Honey Commission [75], ensuring methodological consistency and comparability. This approach supports high-quality traceability and minimizes potential variability introduced during processing. A complete description of each sample—including floral classification, geolocation, harvest year, extraction method, bee species, and relevant environmental or anthropogenic influences—is provided in Supplementary Table S1.

2.3. Sample Preparation and Microwave-Assisted Digestion

2.3.1. Analytical Approaches for Heavy Metal Analysis

To ensure analytical consistency and sample uniformity, all honey samples underwent a standardized preparation protocol prior to instrumental analysis. Crystallized samples were gently liquefied in a thermostated water bath at 65 °C for 30 min to restore fluidity without compromising bioactive compound integrity. Homogenization was achieved by manual stirring or vortex mixing to ensure representative subsampling. Precisely 1.00 g of each homogenized sample was then weighed using an analytical balance and transferred into sterile polypropylene tubes. Each portion was dissolved in 20 mL of ultrapure deionized water (resistivity 18.2 MΩ·cm) produced using a Milli-Q Integral Ultrapure Water-Type I system (Millipore, SAS, Molsheim, France), followed by brief reheating at 65 °C to facilitate complete solubilization of the organic matrix. This step optimized metal ion extraction and minimized matrix interferences during analysis. The entire sample handling procedure conformed to the Harmonized Methods of the International Honey Commission [75] and hygiene guidelines established by the Codex Alimentarius Commission [75]. All samples originated from traditionally extracted, unpasteurized multifloral honeys collected in the Zlatna region, and were stored in sterilized, sealed glass containers at 4–5 °C in the dark until analysis.

2.3.2. The Microwave-Assisted Digestion Methodology

For sample preparation and digestion, a standardized wet mineralization protocol was applied to enable reliable trace-metal determination. Exactly 1.00 g of homogenized, non-crystallized honey was weighed and dissolved in 100 mL of warm ultrapure deionized water (18.2 MΩ·cm resistivity) produced using a Direct-Q3UV Smart purification system (Millipore, SAS, Molsheim, France). Each solution was acidified with 0.5 mL of concentrated analytical-grade nitric acid (65% v/v; Merck, Darmstadt, Germany) to facilitate matrix breakdown and stabilize metal ions. Microwave-assisted digestion was conducted in triplicate using a TopWave Microwave Digestion System (Analytik Jena AG, Jena, Germany), equipped with 24 TFM-PTFE vessels. The digestion program included sequential heating steps at 145 °C, 170 °C, 190 °C, and a final step at 100 °C under pressure-controlled conditions. Procedural blanks were included to verify background levels and validate analytical integrity. To minimize cross-contamination, all digestion vessels were rigorously pre-cleaned by immersion in 50 mL of nitric acid, followed by soaking in 10% HNO₃ for 12 h and thorough rinsing with ultrapure water. After digestion, samples were filtered, quantitatively transferred to Class A volumetric flasks, and brought to exactly 25.00 mL using ultrapure deionized water. This procedure ensured complete organic matrix decomposition and optimal metal ion availability for quantification by FAAS and GFAAS. Supplementary Table S2 provides detailed operational parameters for the digestion protocol.

2.4. Instrumentation for FAAS and GFAAS

Quantitative determination of potentially toxic metals (Pb, Cd, Zn, Cu) in honey samples was carried out using Atomic Absorption Spectrometry (AAS), recognized for its specificity, sensitivity, and reliability in trace-metal analysis. All measurements were performed using a Shimadzu AA-6300 spectrometer (Shimadzu Corp., Kyoto, Japan), equipped with element-specific hollow cathode lamps, a Graphite Furnace (GFAAS) for electrothermal atomization, and a deuterium lamp (BGC-D2) for background correction. Flame AAS (FAAS) was used for zinc (Zn) and copper (Cu) quantification, operating with an air–acetylene flame under optimized conditions: burner gas flow of 2.0 L/min, lamp current of 3 mA, slit width of 0.7 nm, and analytical wavelengths of 213.9 nm for Zn and 324.8 nm for Cu. Air flow was maintained at 17 L/min to ensure stable combustion and atomization efficiency. Graphite Furnace AAS (GFAAS) was applied for the determination of lead (Pb) and cadmium (Cd), using wavelengths of 283.3 nm and 228.8 nm, respectively. A 20 µL aliquot of each sample was introduced into the graphite tube and subjected to a multi-step temperature program: drying at 100 °C (5 s) and 140 °C (15.5 s), pyrolysis at 700 °C (10 s), atomization at 1800 °C (Pb) or 1650 °C (Cd) for 0.5 s, followed by tube cleaning at 2600 °C (1.3 s). Calibration was performed using certified stock solutions (1000 mg/L; Merck) with at least three concentration levels per element. Blank samples were included to verify background signals and exclude contamination. All analyses were conducted in triplicate to ensure precision and statistical robustness. The methodology adhered to international quality control guidelines and harmonized protocols for food and environmental monitoring. A detailed overview of the instrumental parameters used for FAAS and GFAAS is provided in Supplementary Table S3.

2.5. Reagents and Calibration

All reagents used in the study were of analytical grade to ensure measurement accuracy and methodological reproducibility. Homogenized, uncrystallized honey samples were dissolved in warm ultrapure deionized water (resistivity 18.2 MΩ·cm) produced by a Direct Q3UV Smart system (Millipore, SAS, Molsheim, France). Wet digestion was initiated by the addition of 0.5 mL of concentrated trace-metal-grade nitric acid (65% v/v; Merck, Darmstadt, Germany) to each sample solution. Mineralization was carried out using a TopWave microwave digestion system (Analytik Jena AG, Germany), equipped with 24 TFM-PTFE digestion vessels. To prevent cross-contamination, vessels were pre-treated with 50 mL of concentrated HNO₃, soaked in 10% nitric acid for 12 h, and thoroughly rinsed with ultrapure water prior to use. The digestion protocol consisted of controlled heating steps at 145 °C, 170 °C, and 190 °C, followed by a cooling phase at 100 °C. All samples were digested in triplicate, and reagent blanks were included for quality control. Following digestion, the samples were filtered and diluted to the final volume with ultrapure water to prepare them for instrumental analysis. This protocol ensured complete decomposition of the organic matrix and minimized matrix interference during metal quantification.

External calibration for both Flame Atomic Absorption Spectrometry (FAAS) and Graphite Furnace AAS (GFAAS) was performed using a certified multi-element standard solution (Merck, Darmstadt, Germany). Calibration curves were generated through serial dilutions to cover the expected concentration range, ensuring linearity and analytical sensitivity. To correct for matrix effects and potential instrumental drift, internal standards—germanium (Ge), terbium (Tb), rhodium (Rh), and scandium (Sc) (50 µg/L in 1% HNO₃, Merck, Darmstadt, Germany)—were added at a concentration of 50 µg/L in 1% HNO₃ to all calibration standards, reagent blanks, and sample solutions. All dilutions and sample preparations were conducted under trace-metal clean laboratory conditions using high-purity reagents and acid-washed equipment. This procedure ensured the accuracy and consistency of trace- and ultra-trace-metal determinations across all honey samples analyzed in the study.

The pH of honey samples was determined using a calibrated Shimadzu (Shimadzu Corp., Kyoto, Japan) digital pH meter, following standardized procedures for food matrix analysis. Accurate pH measurement is essential for assessing honey freshness, microbial stability, and overall product quality, as acidity directly influences enzymatic activity, shelf-life, and resistance to fermentation. For each determination, approximately 10 g of honey was dissolved in 75 mL of ultrapure deionized water (18.2 MΩ·cm) and stirred gently until fully homogenized. Measurements were taken at room temperature after electrode stabilization to ensure consistency. This method offers reliable evaluation of the acidity level, a critical physicochemical parameter for honey classification and quality control within apicultural and agri-food systems.

2.6. Analytical Validation and Quality Assurance

The analytical performance of Flame AAS (FAAS) and Graphite Furnace AAS (GFAAS) was validated in accordance with Commission Regulation (EU) No. 2016/582, which amends Regulation (EC) No. 333/2007 on the determination of inorganic contaminants in food matrices. Validation focused on key performance indicators, including limits of detection (LOD), limits of quantification (LOQ), linearity, precision, and accuracy. LOD and LOQ values were calculated based on the standard deviation (σ) of repeated blank measurements, using 3σ and 10σ, respectively. Instrument calibration was performed with certified multi-element standard solutions (Merck, Darmstadt, Germany) across five concentration levels (2.5, 5, 10, 25, and 50 µg/L). Calibration curves showed excellent linearity, and blank samples were included to assess baseline signal levels. Analytical quality control was ensured through the inclusion of reagent blanks, duplicate samples, and recovery tests using honey spiked with 5 µg/L of each target analyte. Precision, expressed as Relative Standard Deviation (RSD%), remained below 5% for all elements. Accuracy, evaluated through recovery experiments, yielded recovery rates ranging from 90.32% to 113.12%, with associated uncertainties between 9% and 23%. The method’s repeatability was assessed using the Horwitz ratio (HorRat), with all values below the acceptability threshold of 2, confirming satisfactory intra-laboratory performance. Method validation was further supported using certified reference material BCR-151 (Honey) (European Commission, Joint Research Centre, Geel, Belgium). Recovery rates ranged from 92.0% to 104.4%, and RSD values remained under 5%, demonstrating the method’s robustness and compliance with regulatory standards for trace-metal analysis in honey. Detailed validation parameters, including LOD, LOQ, calibration data, and CRM-based recoveries, are presented in Supplementary Tables S4 and S5.

2.7. Statistical and Correlation Analysis

Statistical analyses were carried out using IBM SPSS Statistics software (Version 26, IBM Corp., Armonk, NY, USA). All measurements were performed in triplicate, and results are reported as mean values ± standard deviation (SD). To evaluate differences in heavy metal concentrations (Pb, Cd, Cu, Zn) across sampling locations, one-way analysis of variance (ANOVA) was applied, followed by Tukey’s Honest Significant Difference (HSD) post hoc test for pairwise comparisons. Statistical significance was set at p < 0.05. Prior to parametric testing, data were examined for normality and homogeneity of variance to ensure compliance with test assumptions. Pearson correlation coefficients were calculated to assess the relationship between metal concentrations and spatial variables, including altitude and distance from historical pollution sources. Repeatability and method performance were evaluated using the Horwitz ratio (HorRat), with values below 2 considered acceptable according to established analytical validation criteria. This statistical framework supports the robustness and interpretability of the quantitative results obtained in this study.

To enhance the spatial interpretation of heavy metal contamination patterns, selected data from the current study (Pb, Cd, Cu, Zn concentrations in honey samples) were synthesized and visualized using multivariate graphical formats, including composite charts and comparative spatial plots. All datasets used in these figures are original, obtained through the FAAS and GFAAS analyses described above. Figures were generated using IBM SPSS Statistics (Version 26) and Microsoft Excel 365 (Microsoft Corp., Redmond, WA, USA), following data normalization and standardization procedures where applicable. The goal of these visualizations was to illustrate distribution trends across sampling sites and to support interpretation of contamination dynamics in relation to geographical and environmental variables.

3. Results

3.1. Spatial and Environmental Factors Influencing Heavy Metal Accumulation in Honey Samples

Although copper (Cu) and zinc (Zn) are essential micronutrients involved in various metabolic and enzymatic processes in both humans and bees, they can become toxic when present in elevated concentrations. In the context of environmental contamination, especially in areas impacted by industrial and mining activities, these elements may accumulate in biological matrices such as honey to levels that raise toxicological concern. For this reason, and in line with current scientific recommendations, we have chosen to refer to cadmium (Cd), lead (Pb), copper (Cu), and zinc (Zn) as potentially toxic elements (PTEs). However, we acknowledge that the term “PTEs” lacks a precise definition and has been discouraged by IUPAC due to its ambiguity. We use it here solely for practical and comparative purposes, as it remains widely used in environmental literature. This terminology reflects both the possible environmental and health risks associated with these elements and the nuanced distinction between essential and non-essential metals when assessing ecological impact.

The assessment of lead (Pb) concentrations in honey samples provides clear evidence of the environmental legacy associated with historical mining and industrial operations in the Zlatna region (

Table 1. Heavy metal contamination in honey: variations in lead (Pb), copper (Cu), cadmium (Cd), and zinc (Zn) concentrations relative to altitude and proximity to pollution sources (dry wt.).

Location	Altitude of the Sampling Area Above Sea Level (m)	Distance from the Pollution Source (km)	Altitude of the Pollution Source Above Sea Level (m)	Pb mg/kg	Cu mg/kg	Cd mg/kg	Zn mg/kg	pH
				MLA	MLA	MLA	MLA
				0.10 mg/kg	5.00 mg/kg	0.01 mg/kg	5.00 mg/kg
Trâmpoiele	534	6.5 Area I	609	1.42 ± 0.39 a	1.73 ± 0.21 a	0.13 ± 0.01 b	1.92 ± 0.01 a	3.89 ± 0.01 ab
		1.5 Area II	706
		4.8 Area III	715
Izvorul Ampoiului	582	5.4 Area I	609	1.69 ± 0.19 a	2.22 ± 0.24 a	0.26 ± 0.01 a	1.93 ± 0.01 a	3.76 ± 0.02 d
		2.2 Area II	706
		4.7 Area III	715
Feneș	360	5.2 Area I	609	1.52 ± 0.23 a	1.95 ± 0.05 a	0.14 ± 0.01 b	1.92 ± 0.01 a	3.93 ± 0.01 a
		11.8 Area II	706
		13.3 Area III	715
Vîltori	502	3.6 Area I	609	0.72 ± 0.23 b	0.62 ± 0.03 b	0.02 ± 0.001 c	0.91 ± 0.01 b	3.86 ± 0.01 bc
		4.8 Area II	706
		7.3 Area III	715
Valea Mică	400	5.3 Area I	609	1.51 ± 0.32 a	1.71 ± 0.03 a	0.37 ± 0.01 a	1.54 ± 0.01 a	3.88 ± 0.02 bc
		11.3 Area II	706
		12.7 Area III	715
Presaca Ampoiului	373	7.3 Area I	609	1.57 ± 0.62 a	2.13 ± 0.03 a	0.24 ± 0.01 a	1.70 ± 0.01 a	3.81 ± 0.04 c
		13.6 Area II	706
		15.4 Area III	715
Budeni	692	13.3 Area I	609	1.51 ± 0.32 a	1.81 ± 0.03 a	0.13 ± 0.001 b	1.81 ± 0.01 a	3.9 ± 0.01 ab
		7.8 Area II	706
		8.1 Area III	715
F Value				9.87	8.23	7.45	5.66	71.00
p Value				<0.01	<0.01	<0.05	<0.05	<0.01
Significance				**	**	*	*	***

Different lowercase letters (a–d) following the mean ± standard deviation values indicate statistically significant differences between groups, as determined by Duncan’s multiple range test at a significance level of p < 0.05. Means sharing the same letter are not significantly different from each other. Asterisks denote significance levels based on one-way ANOVA: * p < 0.05, ** p < 0.01, *** p < 0.001.

). The primary pollution sources—Zlatna Industrial Platform (Area I), Larga de Sus Mine (Area II), and Haneș Tailings Dump (Area III)—have contributed to long-term metal dispersion through atmospheric deposition, surface runoff, and soil accumulation. Elevated Pb levels were detected in honey collected from Izvorul Ampoiului, Feneș, Trâmpoiele, and Presaca Ampoiului, all situated within 1.5 to 7.3 km of Areas I and II, indicating a strong local influence from industrial emissions and residual mining materials. Interestingly, samples from Budeni—located at a greater distance (7.8 to 13.3 km)—also exhibited high Pb concentrations, suggesting that lead contamination may be propagated via long-range atmospheric transport or hydrological pathways. These findings underscore the utility of honey as a sensitive bioindicator for assessing spatial patterns of environmental contamination in agroecosystems affected by industrial activity. The results hold important implications for food safety governance, agricultural sustainability, and land use planning in post-industrial rural regions (Table 1).

Interestingly, the lowest lead concentration was observed in samples from Vîltori (0.72 ± 0.23 mg/kg), despite its moderate proximity (3.6 to 7.3 km) to the identified pollution sources. This deviation from the expected spatial trend suggests that local environmental conditions—such as terrain morphology, vegetation cover, and prevailing wind patterns—may act as natural buffers, reducing airborne particulate deposition and limiting heavy metal transport. Such site-specific ecological features can significantly influence pollutant distribution in agroecosystems. Moreover, variation in floral composition may also contribute to lower Pb accumulation, as certain plant species demonstrate reduced uptake of trace metals, thereby minimizing their transfer into the nectar and, subsequently, into honey. These findings emphasize the heterogeneous impact of industrial and mining activities on honey contamination, shaped not only by distance from emission sources but also by biophysical and ecological variables. Among the pollution sources, the Zlatna Industrial Platform (Area I) appears to exert the most pronounced influence, with nearby sites consistently showing elevated Pb levels. Nevertheless, the effects of Larga de Sus Mine (Area II) and Haneș Tailings Dump (Area III) are also evident, particularly in locations with greater exposure to atmospheric fallout or hydrological runoff (Table 1).

To deepen the understanding of lead (Pb) contamination dynamics in agricultural landscapes, future research should incorporate isotopic analysis to accurately trace the origin of Pb in honey and its environmental reservoirs. Complementary investigations into meteorological patterns and topographical influences would enhance knowledge on pollutant transport mechanisms, especially in heterogeneous terrains. Comparative studies focusing on floral composition and plant-specific metal uptake are also recommended to evaluate how different nectar sources mediate Pb bioaccumulation in bee products. In addition, long-term monitoring across multiple seasons is essential to capture temporal variations in Pb deposition and to assess cumulative effects on soil quality, vegetation health, and apicultural productivity. These perspectives are critical for improving spatial risk assessment models and for informing food safety regulations in regions affected by legacy pollution. Considering honey’s widespread consumption and its role as a natural agricultural product, ongoing surveillance of heavy metal contamination is imperative to protect consumer health and ensure the sustainability of local food systems. The results of this study further highlight the urgency of implementing targeted pollution control strategies in former industrial and mining zones to reduce contaminant dispersion and mitigate its ecological and agricultural impacts (Table 1).

The evaluation of copper (Cu) concentrations in honey samples collected across varying distances from pollution sources offers valuable insight into the environmental footprint of historical industrial and mining operations in the Zlatna region. The main contamination hotspots—Zlatna Industrial Platform (Area I), Larga de Sus Mine (Area II), and Haneș Tailings Dump (Area III)—are recognized for their prolonged emission of potentially toxic elements (PTEs), which accumulate in soils, surface water, and vegetation. Given the foraging behavior of bees, the detection of Cu in honey serves as a proxy for the bioavailability of this metal in the surrounding ecosystem. The highest Cu levels were observed in samples from Izvorul Ampoiului (2.22 ± 0.24 mg/kg), Presaca Ampoiului (2.13 ± 0.03 mg/kg), and Feneș (1.95 ± 0.05 mg/kg), all located within 2.2 to 13.6 km from the principal pollution sources. These elevated values suggest the combined influence of airborne dispersion and soil-to-plant transfer mechanisms, emphasizing the capacity of both environmental media to contribute to the bioaccumulation of Cu in nectar-producing flora. The findings underscore honey’s effectiveness as a sensitive bioindicator for assessing spatial contamination patterns and the ecological consequences of metal exposure in agroecosystems affected by post-industrial land use (Table 1).

Moderate copper (Cu) concentrations were measured in honey samples from Trâmpoiele (1.73 ± 0.21 mg/kg), Valea Mică (1.71 ± 0.03 mg/kg), and Budeni (1.81 ± 0.03 mg/kg), despite their variable proximity to the main pollution sources. This spatial variability suggests that Cu dispersion and bioavailability are influenced by complex environmental factors, including soil composition, hydrological pathways, atmospheric dynamics, and vegetation cover. Additionally, floral diversity may modulate Cu accumulation in honey, as plant species differ in their capacity for metal uptake and translocation. Conversely, Vîltori exhibited the lowest Cu concentration (0.62 ± 0.03 mg/kg), despite being situated relatively close (3.6–7.3 km) to pollution sources. This anomaly may be explained by local mitigating conditions, such as dense vegetation that acts as a physical barrier against airborne particulates, or the prevalence of floral species with limited Cu absorption potential. Topographic shielding and meteorological factors—such as wind direction and rainfall patterns—may further limit the exposure of this site to contaminated dust and runoff. The results indicate that Cu contamination in honey is not solely a function of geographic distance from industrial sites, but also reflects the interplay of ecological, topographic, and atmospheric variables. Elevated Cu levels in areas near the Zlatna Industrial Platform (Area I) and Larga de Sus Mine (Area II) point to these sites as major sources of Cu emissions. However, the detection of substantial Cu levels in more remote locations underscores the potential for long-range environmental transport via windborne or hydrological vectors. Multi-seasonal monitoring is also essential to assess temporal fluctuations, while comparative studies involving other apicultural and horticultural products would help elucidate the broader agroecological impacts. These efforts are vital for supporting food safety policies and guiding sustainable land management in post-industrial rural regions (Table 1).

The levels in honey samples are strongly influenced by their geographic origin, particularly in relation to the Zlatna Industrial Platform (Area I), Larga de Sus Mine (Area II), and Haneș Tailings Dump (Area III). These sites, despite the cessation of industrial activities in 2004, continue to contribute to environmental pollution through residual soil contamination, tailings, and runoff. The observed spatial distribution of metals like Pb, Cd, Cu, and Zn reflects not only the proximity to these sources but also environmental transport mechanisms such as wind, water, and topography. Elevated metal levels even in distant locations indicate complex dispersion pathways, underscoring the need for long-term environmental monitoring using bioindicators like honey (Table 1).

The maximum permissible limits (MPLs) for these metals are based on international and national standards. For Pb, EU Regulation No. 1881/2006 (updated by EU 2023/915) sets a limit of 0.10 mg/kg, while Cd is recommended at 0.01 mg/kg by EFSA and WHO due to its high toxicity. Cu and Zn, although essential nutrients, are limited to 3–5 mg/kg in some national standards (e.g., Poland, India, Turkey) to prevent chronic exposure. These benchmarks are critical for ensuring honey safety and protecting public health.

In statistical analysis, significance levels are commonly indicated by asterisks: *** for p < 0.001 (highly significant), ** for p < 0.01 (significant), * for p < 0.05 (marginally significant), and “ns” for p ≥ 0.05 (not significant). This notation helps quickly assess the strength of statistical differences between groups.

All heavy metal concentrations are expressed on a dry weight basis (dry wt.) to ensure consistency across samples and to eliminate variability caused by moisture content. The analysis of cadmium (Cd) concentrations in honey samples from sites located at varying distances from major pollution sources reveals important patterns of environmental contamination and metal bioavailability in agroecosystems. As a highly toxic element with no biological role, Cd poses significant risks to food safety when mobilized through industrial emissions, mining residues, and soil degradation. In this study, the highest Cd level (0.37 ± 0.01 mg/kg) was detected in honey from Valea Mică, a site situated 5.3 to 12.7 km from key contamination sources such as the Zlatna Industrial Platform (Area I), Larga de Sus Mine (Area II), and Haneș Tailings Dump (Area III). This finding suggests that Cd dispersion is governed not only by spatial proximity but also by environmental variables such as topography, prevailing winds, and soil composition, which may facilitate long-range transport or localized accumulation. The elevated values in Valea Mică may also reflect legacy pollution from historical mining waste or contaminated hydrological systems influencing local flora. These results underscore the need for integrated environmental monitoring and highlight honey’s potential as a bioindicator for assessing heavy metal exposure in agricultural landscapes affected by industrial activity.

Moderately elevated cadmium (Cd) concentrations were identified in honey samples from Izvorul Ampoiului (0.26 ± 0.01 mg/kg) and Presaca Ampoiului (0.24 ± 0.01 mg/kg), despite their locations spanning 2.2 to 15.4 km from the primary pollution sources. These findings indicate that Cd bioavailability is strongly influenced by atmospheric deposition and soil leaching, which facilitate its uptake by nectar-producing plants. The detection of Cd at significant levels in more distant sites suggests that industrial and mining emissions exert a broader regional impact, underscoring the importance of long-range contaminant transport mechanisms in shaping heavy metal distribution within agricultural ecosystems.

Lower, yet still detectable, cadmium (Cd) concentrations were recorded in honey samples from Feneș (0.14 ± 0.01 mg/kg), Budeni (0.13 ± 0.001 mg/kg), and Trâmpoiele (0.13 ± 0.01 mg/kg), despite their varying distances from pollution sources. These relatively lower values may be attributed to local buffering factors such as dense vegetation cover, reduced soil Cd content, or limited atmospheric deposition. In regions like Feneș and Presaca Ampoiului, lower elevation may further influence contaminant retention through water runoff and sedimentation dynamics. Notably, Vîltori exhibited the lowest Cd level (0.02 ± 0.001 mg/kg), suggesting that site-specific environmental variables—such as soil pH, floral composition, and meteorological conditions—can significantly constrain Cd mobility and plant uptake, thereby reducing its transfer into honey. These findings reinforce the importance of microenvironmental factors in modulating contaminant bioaccumulation within agroecosystems.

Overall, the findings demonstrate that cadmium contamination in honey is governed by a complex interplay of environmental factors, including proximity to pollution sources, atmospheric dispersion, soil characteristics, and floral composition. Elevated Cd levels in certain locations underscore the metal’s environmental persistence and mobility, raising concerns about its potential impact on food safety and agroecological integrity. Given cadmium’s toxicity and the existence of strict regulatory thresholds in food products, sustained monitoring in historically industrialized regions is critical to evaluate long-term risks and support the development of resilient and safe agricultural systems.

To improve source attribution and environmental management, future research should employ cadmium isotope analysis to differentiate between geogenic and anthropogenic inputs. Complementary soil–plant bioaccumulation studies are recommended to clarify Cd transfer pathways into apicultural products. Incorporating seasonal monitoring could capture temporal dynamics in contamination levels, while comparative assessments with other locally produced foods would provide a broader perspective on heavy metal exposure risks within agricultural landscapes.

These results highlight the urgent need for enhanced environmental regulations and targeted remediation efforts to mitigate heavy metal contamination in impacted regions. Given honey’s role as both a dietary product and an ecological bioindicator, regular monitoring and pollution control measures are essential to safeguard public health and support sustainable agricultural practices.

Zinc (Zn) concentrations in the analyzed honey samples displayed clear spatial variability, with elevated levels detected in areas closer to major pollution sources, particularly near the Zlatna Industrial Platform (Area I) and Larga de Sus Mine (Area II). The highest Zn values were found in samples from Izvorul Ampoiului (1.93 ± 0.01 mg/kg), Trâmpoiele (1.92 ± 0.01 mg/kg), and Feneș (1.92 ± 0.01 mg/kg), suggesting enhanced exposure through atmospheric deposition and soil-mediated transfer. In contrast, lower Zn concentrations in Vîltori (0.91 ± 0.01 mg/kg) and Valea Mică (1.54 ± 0.01 mg/kg) may be attributed to site-specific differences in air quality, soil Zn content, and floral uptake capacity.

Although Zn is an essential micronutrient, excessive accumulation may have ecological and toxicological consequences. Its presence in honey reflects not only the level of environmental exposure, but also the bioavailability and uptake efficiency of local plant species. These findings support the use of honey as a bioindicator for Zn distribution in agroecosystems affected by industrial activity. Continued monitoring of Zn levels, along with studies on plant–soil–bee transfer dynamics, is essential to assess the long-term impact of industrial pollution on food safety and ecological resilience.

Figure 2 illustrates the spatial variation in heavy metal concentrations (Pb, Cu, Cd, Zn) in honey samples relative to distance from major pollution sources. While a general decline with increasing distance is typically anticipated, the observed patterns reveal a more complex dispersion dynamic. Lead (Pb) and copper (Cu) display irregular trends, suggesting the influence of secondary emission sources or soil-related retention effects. Zinc (Zn) concentrations remain relatively stable across distances, whereas cadmium (Cd) shows limited spatial variability, potentially reflecting distinct geochemical behavior influenced by factors such as soil pH and hydrology. These results emphasize that distance alone does not fully explain heavy metal distribution; instead, topographical features, atmospheric transport, and site-specific geochemical interactions appear to play a significant role. Further spatial modeling using GIS and geostatistical approaches is recommended to refine risk assessments and identify contamination pathways in agroecosystems.

These results enhance our understanding of zinc contamination in apicultural products and underscore the need to evaluate the environmental impact of industrial emissions in regions with ongoing or historical mining activity. Future studies should investigate the relationship between Zn concentrations in honey, soil, and vegetation to establish a more integrated framework for assessing human exposure to trace metals through honey consumption and for informing food safety strategies in agricultural landscapes.

Figure 3 presents the concentrations of lead (Pb), copper (Cu), cadmium (Cd), and zinc (Zn) in honey samples as a function of distance from pollution sources, overlaid with internationally recognized maximum permissible limits for honey. The results show that Pb levels exceed the regulatory threshold of 0.10 mg/kg across all sites, with no consistent decline by distance, indicating influence from persistent historical sources and atmospheric deposition. Cu concentrations also surpass the 0.40 mg/kg limit in all samples, with irregular spatial patterns likely driven by soil chemistry, pH, and local agricultural practices. Cd, although more mobile in acidic environments, shows highly variable concentrations (0.02–0.37 mg/kg), exceeding the 0.01 mg/kg limit and highlighting potential bioaccumulation risks. In contrast, Zn levels (0.91–1.93 mg/kg) remain well below the permissible limit of 5.00 mg/kg, suggesting lower risk, though continued monitoring is advised due to possible industrial inputs. The figure underscores the need for integrated risk assessment based on both environmental distribution and food safety standards.

Figure 3 illustrates the concentrations of individual heavy metals (Pb, Cd, Cu, Zn, etc.) detected in honey samples collected from the three studied areas. For improved clarity and visual interpretation, each element is presented separately. The horizontal dashed lines in each subplot represent the corresponding Maximum Permissible Limits (MPLs) established by international or national standards. This separation allows for a clearer comparison between measured concentrations and regulatory thresholds, highlighting cases where specific metals exceed safe limits. The visualization supports the assessment of potential health risks associated with environmental contamination in the studied region.

The results indicate that lead (Pb), copper (Cu), and cadmium (Cd) represent critical contamination risks, as their concentrations exceed permissible limits at all sampling sites. The absence of a consistent decline in metal levels with increasing distance from pollution sources suggests that dispersion is modulated by multiple environmental factors, including wind dynamics, soil retention capacity, pH variation, and hydrological flow. The elevated mobility of Cd, particularly in acidic soils, raises concerns regarding its bioavailability and potential for plant uptake. Persistently high levels of Pb and Cu, even in more distant locations, point to long-term contamination and the need for further investigation into their environmental persistence. To refine contamination risk assessments, future research should integrate soil pH profiling, geochemical characterization, and GIS-based spatial modeling to identify distribution patterns and contamination hotspots. In parallel, targeted remediation strategies—such as phytoremediation, soil stabilization, and the relocation of vulnerable apiaries—should be evaluated to mitigate exposure risks. Strengthening regulatory monitoring, particularly for Pb and Cd in apicultural products from post-industrial regions, is essential to ensure food safety and support sustainable agricultural practices.

Methodological differences (e.g., type of honey, seasonal variability, or analytical technique) are often not reported in comparative studies, limiting direct equivalence. Pb emerged as the most critical contaminant due to its neurotoxic potential, especially for vulnerable populations such as children and pregnant women. The highest Pb concentrations were recorded in Romanian samples (1.42–1.69 mg/kg), predominantly from areas with a legacy of mining and industrial emissions. These differences may be due to variations in analytical protocols (e.g., FAAS vs. ICP-MS), honey types (monofloral vs. polyfloral), or seasonal variability. In many cases, comparative studies lack full methodological details—including sample preparation, collection period, or floral origin—which limits direct comparability and requires cautious interpretation. Where methodological details were not reported in comparative studies, such differences should be interpreted with caution. In contrast, lower Pb levels were reported in honey from countries such as the Republic of Moldova, Turkey, Spain, and China (0.21–0.48 mg/kg), where stricter environmental regulations, lower pollution pressure, or variations in bee foraging behavior may mitigate exposure.

These observations are consistent with previous studies, including those by Bayir et al. [76] and Martins et al. [77], which attributed reduced Pb levels to more effective control of emission sources. Importantly, Pb concentrations in the Romanian honey samples exceeded the Codex Alimentarius and European Food Safety Authority (EFSA) limit of 0.10 mg/kg, underscoring potential risks to food safety and the urgent need for enhanced regulatory oversight and environmental remediation in high-risk regions. However, due to inconsistencies in reporting, these comparisons must be interpreted cautiously. In several referenced studies, methodological details such as the type of honey (monofloral vs. polyfloral), season of harvest, and analytical techniques (e.g., FAAS vs. ICP-MS) were not always specified. Where such information was missing, comparability was assumed with caution.

Figure 3 illustrates the variation in heavy metal concentrations (Pb, Cu, Cd, and Zn) in honey samples collected from different locations situated at varying distances from the pollution source. Each metal is represented by a distinct curve, and the values are compared against the maximum legal limits (MLA) established for honey: 0.10 mg/kg for lead (Pb), 5.00 mg/kg for copper (Cu), 0.01 mg/kg for cadmium (Cd), and 5.00 mg/kg for zinc (Zn). The colored dashed lines indicate these reference limits. In most cases, concentrations exceed the permissible values, especially for Pb and Cd, highlighting the negative influence of proximity to the contamination source on honey quality. This representation emphasizes the degree of contamination and enables a quick visual assessment of the potential risk to consumers.

Copper (Cu) concentrations exhibited marked regional variability, with the highest value reported in Finnish propolis (7.12 mg/kg), likely reflecting underlying geochemical differences or naturally Cu-rich soils. Romanian and Moldovan honey samples showed elevated Cu levels ranging from 1.73 to 2.22 mg/kg, exceeding the international threshold of 0.40 mg/kg [75,78], but remaining within certain national regulatory limits (e.g., 1.00 mg/kg in Romania). Where methodological details (e.g., honey type, harvest period, or analytical technique) were not specified in the comparative studies, results were interpreted with caution. Not all referenced studies provided this metadata (e.g., season, floral source, methodology), limiting the robustness of direct comparisons and requiring results to be interpreted as indicative rather than definitive. Where methodological details (e.g., honey type, harvest period, or analytical technique) were not specified in the comparative studies, results were interpreted with caution. However, it should be noted that some differences may arise from national sampling practices, agricultural inputs, or varying analytical methodologies (e.g., FAAS vs. ICP-MS). Where sampling conditions or analytical methods were not specified, assumptions regarding cross-national comparability must be considered tentative. Lower Cu concentrations reported in Turkey (0.67 mg/kg) and Spain (0.45 mg/kg) suggest that geographic, industrial, and agricultural variables influence Cu bioavailability. The widespread use of copper-based fungicides may also contribute to Cu accumulation in honey, as highlighted by Ru et al. [79] in China. Cadmium (Cd), a non-essential and highly toxic element, showed its highest concentration in Romanian agricultural honey samples (0.2618 mg/kg), significantly exceeding the Codex Alimentarius limit of 0.01 mg/kg [75]. This raises substantial food safety concerns, especially in regions historically affected by mining and industrial activity, and calls for stricter monitoring and mitigation strategies. It is important to note that not all comparative studies provided full methodological details. Differences in analytical instrumentation, sampling timeframes, or botanical origin of honey could influence the reported Cu values. Where this metadata was absent, conclusions should be treated as indicative rather than definitive.

Where these methodological parameters were unavailable, comparisons were interpreted as indicative rather than conclusive. Where such metadata were unavailable, cross-country comparisons should be interpreted as indicative rather than definitive. In cases where such information was not provided, comparisons are indicative rather than definitive. The findings presented in Figure 4 are consistent with previous studies, such as those by González-Paramás et al. [80], which reported elevated cadmium (Cd) levels in honey collected from areas with intensive agriculture and mining activity. In contrast, significantly lower Cd concentrations (0.03–0.05 mg/kg) were recorded in Spain, Turkey, and China, potentially reflecting lower industrial pressure or more effective environmental regulations in those regions. Unlike Pb and Cd, zinc (Zn) is an essential micronutrient with relatively low toxicity at moderate concentrations. In the present study, Zn levels ranged from 1.88 mg/kg (China) to 2.10 mg/kg (Spain), remaining well below the Codex Alimentarius limit of 5.00 mg/kg. Although Zn contamination appears less critical, elevated levels may still signal anthropogenic inputs such as industrial emissions, fertilizer application, or contamination from metal-processing industries, as previously noted by Ru et al. [81]. However, direct comparison may be limited by methodological differences such as the type of honey analyzed (e.g., mono vs. polyfloral), seasonal variations, and differences in sampling or analytical protocols. Where these details were not reported in the referenced studies, comparisons should be interpreted with caution. Although Zn levels were within permissible limits, the absence of consistent metadata across studies (e.g., floral composition, harvest season, and processing protocols) may affect cross-regional comparability. As such, observed differences may not be solely attributed to environmental contamination.

The pH of the analyzed honey samples ranged from 3.76 ± 0.02 to 3.93 ± 0.01, confirming their naturally acidic character, which is consistent with established honey quality parameters. The highest pH was recorded at SP3 (3.93 ± 0.01), while the lowest was found at SP2 (3.76 ± 0.02), with statistically significant differences among sampling sites (p < 0.001). These variations may be attributed to differences in floral source, soil chemistry, or local environmental conditions. All pH values fall within the recommended range for honey stability, supporting its microbiological safety and shelf-life.

3.2. Spatial and Environmental Drivers of Risk in Beekeeping Zones

The relationship between heavy metal concentrations in honey, distance from pollution sources, and altitude provides key insights into the spatial dynamics of environmental contamination in the study area.

Overall, higher levels of Pb, Cu, Cd, and Zn were observed in honey samples collected from sites located closer to industrial and mining facilities, while lower concentrations were generally associated with more distant or higher-altitude locations. Nevertheless, deviations from this trend suggest that metal deposition and bioaccumulation are influenced by a complex interplay of environmental variables, including topography, wind direction, vegetation cover, and soil properties. These findings highlight the need for spatially explicit monitoring strategies to accurately assess contamination risks in post-industrial agricultural landscapes.

In most cases, honey samples collected from locations in closer proximity to the Zlatna Industrial Platform (Area I), Larga de Sus Mine (Area II), and Haneș Tailings Dump (Area III) exhibited elevated concentrations of potentially toxic elements (PTEs). For instance, Izvorul Ampoiului, situated approximately 5.4 km from Area I and 2.2 km from Area II, recorded the highest levels of Pb (1.69 ± 0.19 mg/kg) and Cu (2.22 ± 0.24 mg/kg). Similarly, Presaca Ampoiului—located 7.3 km from Area I and 13.6 km from Area II—displayed increased Cu (2.13 ± 0.03 mg/kg) and Zn (1.70 ± 0.01 mg/kg) concentrations. These findings reinforce the role of spatial proximity to industrial and mining sources in influencing the bioaccumulation of potentially toxic elements (PTEs) in nectar-producing plants, ultimately affecting honey composition and quality.

However, certain anomalies challenge a purely distance-based interpretation of heavy metal dispersion. For example, Budeni—located 13.3 km from Area I—still exhibited elevated concentrations of Pb (1.51 ± 0.32 mg/kg) and Zn (1.81 ± 0.01 mg/kg). These findings suggest that additional environmental mechanisms, such as prevailing wind direction, watershed-mediated transport, and legacy deposition in soils, may contribute to contamination even at more distant sites. This underscores the need for multi-variable spatial assessments that consider not only proximity, but also geomorphology, hydrology, and long-term pollutant persistence.

Altitude emerges as an additional environmental factor influencing heavy metal accumulation in honey. The data indicate that lower-altitude sites, such as Feneș (360 m) and Presaca Ampoiului (373 m), recorded higher concentrations of Cd (0.14 ± 0.01 mg/kg and 0.24 ± 0.01 mg/kg, respectively) and Cu (1.95 ± 0.05 mg/kg and 2.13 ± 0.03 mg/kg, respectively). This pattern may be attributed to gravitational deposition of airborne contaminants, which tend to settle at lower elevations, especially in valley systems. Moreover, hydrological transport via rivers draining from polluted sites may contribute to localized soil and vegetation contamination, thereby affecting nectar quality and the subsequent heavy metal content in honey.

By contrast, honey samples from higher-altitude locations such as Budeni (692 m) generally showed slightly lower levels of heavy metal contamination, despite the site’s relative proximity to known pollution sources. This attenuation may be attributed to reduced atmospheric deposition at higher elevations, as airborne particulates tend to settle more readily in lower-lying areas. Additionally, variations in vegetation type and soil composition at higher altitudes may limit metal uptake by nectar-producing plants, thereby reducing the transfer of contaminants into honey.

The interaction between distance from pollution sources, altitude, and heavy metal accumulation in honey underscores the multifactorial nature of contamination dynamics in agroecological systems. While spatial proximity to industrial and mining sites remains a major driver of exposure, elevation significantly influences pollutant deposition patterns. Additional factors—such as prevailing wind direction, watershed-mediated transport, and soil geochemistry—further modulate the bioavailability and transfer of metals into apicultural products. These results highlight the importance of spatially integrated monitoring frameworks that account for both horizontal and vertical gradients of pollution. Future research should incorporate atmospheric dispersion modeling to refine predictions of deposition zones, as well as comparative studies on plant uptake mechanisms at varying altitudes. Longitudinal monitoring of honey and related environmental matrices is also essential to evaluate the long-term persistence of contamination in post-industrial landscapes.

3.3. Correlation Between Heavy Metal Concentrations in Honey, Distance from Pollution Sources, and Altitude

Statistical analysis of heavy metal concentrations (Pb, Cu, Cd, Zn) in honey samples revealed distinct correlations with both the distance from pollution sources and the altitude of the sampling locations. These associations offer valuable insight into the spatial dynamics of metal dispersion and their environmental bioavailability within the study area. As illustrated in Figure 5, the strength and direction of these correlations emphasize the influence of topographical and geographic variables on contamination patterns.

Correlation analysis revealed moderate positive associations between the concentrations of lead (Pb), copper (Cu), and zinc (Zn) in honey and the distance from pollution sources, with Pearson coefficients of 0.369, 0.329, and 0.395, respectively. These results challenge the conventional assumption of a linear decline in contamination with increasing distance and suggest that long-range atmospheric dispersion and hydrological transport may contribute to sustained metal deposition. Fine particulate matter containing potentially toxic elements (PTEs) can be carried over extended distances before settling on vegetation, while contaminated surface runoff and groundwater may facilitate metal uptake by plants, thus influencing honey composition beyond immediate proximity to pollution sources.

In contrast, cadmium (Cd) showed an almost negligible correlation with distance from pollution sources (r = −0.034), indicating that its presence in honey is not strongly influenced by spatial proximity alone. This pattern suggests that Cd bioavailability may be regulated by more complex site-specific factors, such as plant species composition, soil pH, and metal-binding dynamics. Unlike Pb and Cu, which are more prone to long-range atmospheric dispersion, Cd may remain localized in contaminated soils due to its lower mobility, resulting in inconsistent uptake by nectar-producing plants and variable accumulation in apicultural products.

Correlations between heavy metal concentrations and altitude were generally weak or negligible, indicating limited influence of elevation on metal accumulation in honey for most elements. However, cadmium (Cd) exhibited a moderate negative correlation with altitude (r = −0.312), suggesting that higher-altitude areas may be less affected by Cd deposition or bioavailability. This trend may reflect lower anthropogenic pressure, reduced soil contamination, or differences in plant uptake mechanisms at elevated sites. As Cd is commonly linked to industrial and mining sources, its reduced presence at higher altitudes underscores the potential buffering capacity of these landscapes in mitigating heavy metal transfer into the apicultural food chain.

Lead (Pb), copper (Cu), and zinc (Zn) exhibited minimal correlations with altitude (r = −0.019, −0.063, and 0.112, respectively), indicating that elevation does not exert a dominant influence on their distribution in honey. The weak negative trends observed for Pb and Cu may be attributed to gravitational settling and localized deposition processes, rather than altitude-driven climatic factors such as precipitation or temperature. Conversely, the slight positive correlation for Zn suggests that certain plant species at higher elevations may have an increased capacity for Zn uptake, potentially linked to edaphic conditions or adaptive physiological traits.

These results highlight the multifactorial nature of heavy metal dispersion and bioaccumulation in honey across heterogeneous landscapes. The moderate positive correlations observed for Pb, Cu, and Zn with distance from pollution sources suggest potential long-range atmospheric or hydrological transport mechanisms—particularly relevant in the case of Pb, which is known for its persistence in airborne particulates. In contrast, the generally weak associations with altitude indicate that local environmental variables, such as soil characteristics, vegetation type, and plant metal uptake dynamics, may exert a more substantial influence on metal bioavailability than elevation alone.

From an ecological standpoint, the detection of potentially toxic elements (PTEs) in honey collected even from sites distant from known pollution sources underscores the persistence and mobility of environmental contaminants. These findings reinforce the value of honey as a practical bioindicator for integrated air and soil pollution monitoring. Moreover, the spatial patterns observed suggest that while localized mitigation strategies may reduce immediate exposure near industrial sites, the broader implications of long-range atmospheric transport must be considered in environmental risk assessments and policy development.

3.4. Spatial Distribution of Heavy Metal Contamination in Relation to Distance from the Pollution Source

Assessing spatial patterns of heavy metal dispersion is essential for evaluating environmental contamination and its implications for ecosystem integrity and public health. Figure 5 presents scatter plots illustrating the relationship between concentrations of lead (Pb), copper (Cu), cadmium (Cd), and zinc (Zn) in honey and the distance from major pollution sources. The inclusion of regression lines and confidence intervals supports the identification of spatial trends and highlights the influence of both proximity and environmental variability on metal distribution across the landscape.

The observed weak positive correlations between lead (Pb, r = 0.37), copper (Cu, r = 0.33), and zinc (Zn, r = 0.40) concentrations and distance from pollution sources deviate from the expected decline typically associated with proximity-based contamination. These trends suggest that additional environmental and geospatial factors—such as secondary emission sources, landscape morphology, and hydrological pathways—may modulate metal dispersion patterns. For example, topographical variations can channel contaminated runoff toward more distant sites, while atmospheric deposition driven by wind and precipitation may result in diffuse distribution of metal particulates across the region. These findings point to the need for multifactorial environmental assessments when interpreting contamination gradients in post-industrial landscapes.

Among the potentially toxic elements (PTEs) analyzed, cadmium (Cd) exhibited the weakest correlation with distance from pollution sources (r = −0.03), as illustrated by the nearly flat regression trend in Figure 6. This pattern suggests that Cd dispersion is not predominantly influenced by spatial proximity, but rather by other site-specific factors such as soil chemistry, plant uptake variability, or legacy contamination.

Instead of being driven primarily by distance, cadmium (Cd) distribution appears to be modulated by local environmental conditions such as soil pH, adsorption–desorption dynamics, and potential point sources like agricultural inputs, including phosphate-based fertilizers that often contain Cd residues. The variability in Cd concentrations across sites may also reflect differences in soil retention and mobility, allowing greater dispersion in certain areas compared to more immobile elements like Pb or Cu. As shown in Figure 2, the widening confidence intervals—particularly for Pb and Zn—at greater distances emphasize the complexity of dispersion mechanisms. This suggests that multiple pathways, including groundwater transport, industrial leaching, or atmospheric inputs from vehicular emissions, contribute to heterogeneous contamination. Moreover, historical pollution events, land use variability, and vegetative cover may further shape the spatial patterns observed.

Several factors may account for the unexpected correlation patterns observed in heavy metal distribution. First, the persistence of lead (Pb) and copper (Cu) in soils—due to their strong affinity for organic matter and clay minerals—may result in relatively uniform concentrations across distances, reflecting their low mobility. Second, the presence of secondary contamination sources, such as legacy mining sites, industrial zones, and traffic-related emissions, likely contributes to localized metal inputs that disrupt distance-based trends. Third, hydrological processes and atmospheric deposition—including surface runoff, windborne transport, and rainfall—may redistribute metals far beyond their original emission points. Lastly, the unique behavior of cadmium (Cd) may be linked to its higher mobility in slightly acidic soils, leading to a more variable and less predictable spatial distribution.

3.5. Human Health Risk Assessment of Heavy Metal Exposure Through Honey Consumption

This analysis assesses the potential health risks associated with heavy metal exposure through honey consumption, stratified by age and sex categories. Using the measured concentrations of Pb, Cu, Cd, and Zn, the radar chart (Figure 7) illustrates relative risk levels on a standardized scale from 1 to 10. The evaluation incorporates biological vulnerability parameters, including differences in metabolic rate, organ development, and physiological sensitivity. These insights are particularly valuable for informing public health strategies, enhancing food safety regulations, and guiding targeted consumer protection initiatives, especially for vulnerable populations such as children and pregnant women.

Figure 7 illustrates the clustering of honey samples based on their heavy metal profiles. The grouping pattern suggests that samples collected from areas closer to former industrial sites (e.g., Zlatna Industrial Platform or Haneș Tailings Dump) tend to cluster separately from those collected at greater distances, reflecting higher concentrations of certain metals such as Pb and Cd. This pattern reinforces the spatial influence of historical contamination on the chemical composition of honey.

Among the analyzed elements, lead (Pb) presents the most significant health risk, particularly for sensitive groups such as infants (risk level = 9) and pregnant women (risk level = 10). This assessment is supported by the recorded Pb concentrations, which ranged from 0.7211 mg/kg to 1.6912 mg/kg—substantially exceeding the established regulatory limit of 0.10 mg/kg. In infants, Pb exposure is especially concerning due to its neurotoxic potential, which may result in cognitive deficits, behavioral disturbances, and impaired neurodevelopment. For pregnant women, the risk is amplified by the metal’s ability to cross the placental barrier, with documented associations to intrauterine growth restriction and adverse fetal outcomes.

Elderly individuals also exhibit elevated vulnerability to lead (Pb) exposure, with an estimated risk level of 7, primarily due to the cumulative nature of Pb toxicity. Chronic exposure is associated with increased risks of hypertension, renal dysfunction, and neurodegenerative conditions, including Alzheimer’s and Parkinson’s diseases. At Presaca Ampoiului (1.5745 mg/kg), Pb levels may aggravate oxidative stress and cardiovascular risks in the elderly. Although adults and adolescents demonstrated slightly lower risk levels (6–7), the highest recorded Pb concentration (1.6912 mg/kg at Izvorul Ampoiului) indicates that chronic low-level exposure remains a public health concern, even among individuals with fully developed detoxification mechanisms.

Cadmium (Cd) levels (0.0233–0.3765 mg/kg) exceeded the 0.01 mg/kg limit, posing high risks (level 9–10) for pregnant women and the elderly, due to its nephrotoxic and osteotoxic effects. At Valea Mică (0.3765 mg/kg), the value is nearly 38 times above the limit, suggesting severe risks. Infants and children (risk = 7–8) are also vulnerable due to Cd’s interference with calcium metabolism. The level at Izvorul Ampoiului (0.2618 mg/kg) indicates potential long-term effects in younger populations.

Copper (0.6231–2.2256 mg/kg) exceeded the safety threshold in all samples. The elderly individuals (risk = 7) and adults (risk = 6) face liver-related risks, especially at Izvorul Ampoiului. While risks are lower in children (risk = 4–5), vulnerable individuals (e.g., Wilson’s disease) remain at risk. Zinc (Zn), although an essential trace element, poses the lowest toxicological risk among the metals analyzed, with concentrations ranging from 0.9157 mg/kg to 1.9364 mg/kg—well below the Codex Alimentarius maximum limit of 5.00 mg/kg. Nonetheless, excessive Zn intake may disrupt physiological balance, potentially suppressing immune function, causing gastrointestinal disturbances, and impairing copper absorption.

The highest relative risk was observed in elderly individuals (risk level = 6), likely due to age-associated alterations in metal metabolism. For infants and children (risk level = 3–4), Zn toxicity remains minimal, given its critical role in growth, immune function, and enzymatic regulation. However, samples such as that from Trâmpoiele (1.9201 mg/kg) approach levels warranting attention, suggesting that long-term exposure—even within permissible limits—should be monitored to ensure nutritional safety in vulnerable populations. Lead (1.5745–1.6912 mg/kg) poses high risks, particularly for infants (risk = 9–10), due to neurotoxic effects and placental transfer. Chronic exposure remains a concern for all age groups.

The results of this health risk assessment underscore significant public health concerns related to heavy metal contamination in honey, particularly for vulnerable population groups. Among the elements examined, lead (Pb) and cadmium (Cd) present the highest toxicological risks, with concentrations such as 1.6912 mg/kg Pb in Izvorul Ampoiului and 0.3765 mg/kg Cd in Valea Mică substantially exceeding internationally accepted safety thresholds. These levels are especially alarming for infants, pregnant women, and elderly individuals due to their heightened physiological sensitivity. Lead exposure is well-documented for its neurodevelopmental toxicity in children and transplacental transfer potential during pregnancy, while cadmium is characterized by its cumulative nephrotoxic and osteotoxic effects, exacerbated by a prolonged biological half-life. These findings suggest that honey produced in historically contaminated areas may serve as a vector for chronic low-level exposure to toxic metals, reinforcing the need for targeted monitoring, stricter regulatory oversight, and risk communication strategies in affected agroecosystems.

An important finding of this study is that increased distance from pollution sources does not consistently correlate with reduced contamination levels in honey. Notably, elevated lead (Pb) concentrations—such as 1.5112 mg/kg detected at Budeni, located 13.3 km from the nearest industrial site—indicate that heavy metal dispersion extends beyond immediate proximity. This pattern suggests that secondary environmental pathways, including residual soil contamination, long-range atmospheric deposition, and hydrological transport, contribute to the widespread distribution of pollutants. These mechanisms challenge the assumption that distance alone is a reliable predictor of contamination intensity. Consequently, effective environmental risk assessments must incorporate a broader set of spatial and ecological variables to accurately capture the dynamics of metal mobility in agricultural landscapes affected by legacy industrial activity.

In light of the health risks identified, proactive regulatory measures are essential to safeguard vulnerable populations from heavy metal exposure through honey consumption. National food safety authorities and agricultural oversight bodies should intensify surveillance in regions affected by industrial emissions, mining legacies, and intensive agrochemical use. Routine screening for Pb, Cd, Cu, and Zn in apicultural products must be integrated into national monitoring programs to ensure compliance with international safety standards. Furthermore, contaminated honey sources should be promptly identified and excluded from commercial distribution to prevent long-term exposure risks. Strengthening regulatory frameworks and enhancing cross-sector collaboration can play a vital role in maintaining the integrity of apicultural supply chains and promoting consumer health.

Beyond regulatory measures, public awareness and community-level interventions are critical components of a comprehensive risk reduction strategy. Educational campaigns should inform consumers about the significance of honey provenance, encouraging the selection of certified, laboratory-tested products that comply with food safety standards. At the production level, promoting relocation of apiaries to low-contamination zones and supporting sustainable beekeeping practices can significantly reduce exposure risks. In parallel, the implementation of environmentally responsible agricultural and industrial policies—targeting the reduction in heavy metal emissions—will not only protect apicultural products but also advance broader goals of environmental health, sustainable land use, and resilient agri-food systems.

4. Discussion

This study reveals concerning levels of heavy metal contamination in honey from the Zlatna region, a historically industrialized area in Romania. The most critical findings include lead (Pb) and cadmium (Cd) concentrations that substantially exceed international food safety limits established by Codex Alimentarius and EU regulations. For instance, the highest values for Pb (1.6912 ± 0.19 mg/kg) and Cd (0.3765 ± 0.01 mg/kg) were recorded in Izvorul Ampoiului and Valea Mică, respectively—exceeding permissible thresholds by over 15 and 35 times. These results reflect the persistent environmental burden of mining and smelting activities, tailings mismanagement, and atmospheric emissions from past industrial operations.

Spatial variability in metal distribution underscores the complexity of environmental contamination dynamics. While one might expect a clear distance–decay gradient in pollution levels, the results show that remote locations like Budeni (13.3 km from emission sources) still exhibit elevated Pb concentrations (1.5112 ± 0.32 mg/kg). This suggests that atmospheric transport, runoff, and soil redistribution allow contaminants to travel and persist well beyond their original sources. Correlation analyses support this interpretation, with Cu and Zn displaying moderate positive relationships with distance (r = 0.33 and 0.40, respectively), indicating multifactorial dispersion influenced by local geography, wind patterns, and hydrology.

Altitude emerged as another key variable. Lower-elevation sites such as Feneș and Presaca Ampoiului, situated along valley corridors and downstream from major pollution sources, presented higher levels of Cd and Cu. This may result from gravitational deposition of airborne particles and accumulation via riverine systems. Conversely, higher-altitude locations like Budeni (692 m) generally showed lower contamination, likely due to limited pollutant deposition and distinctive soil–vegetation interactions that restrict metal uptake.

While zinc (Zn) and copper (Cu) are essential micronutrients, their concentrations in some samples approached or exceeded recommended dietary limits. Cu reached levels as high as 2.2256 ± 0.24 mg/kg—far above the safety threshold of 0.40 mg/kg—raising potential concerns about chronic exposure, particularly for sensitive individuals. Zn remained within acceptable limits but was consistently elevated (up to 1.9364 ± 0.01 mg/kg), suggesting ongoing environmental inputs, likely from fertilizers or industrial sources.

The toxicological implications of these findings are significant. Pb remains one of the most dangerous contaminants due to its neurotoxicity and ability to affect fetal development and child cognition. Cd, with its long biological half-life, poses serious risks of renal damage and bone demineralization. Even Cu and Zn, although physiologically necessary, can cause adverse effects at high levels, such as liver dysfunction or impaired immune function. These risks are particularly pronounced in vulnerable populations, including children, pregnant women, and the elderly.

Given this context, the integration of honey monitoring into environmental risk assessment frameworks is both justified and necessary. Honey’s ability to reflect the bioavailability of contaminants across landscapes makes it a practical, non-invasive tool for detecting and managing pollution in agroecosystems. In regions like Zlatna, where legacy contamination continues to affect land use and food production, targeted policy interventions—such as stricter air emission controls, soil remediation, and beekeeping relocation—are warranted. Moreover, incorporating advanced spatial tools like GIS, isotopic fingerprinting, and atmospheric dispersion models would enhance the precision and responsiveness of monitoring systems.

Ultimately, these results reinforce the dual value of honey: not only as a dietary product with high nutritional and economic importance, but also as a reliable bioindicator of environmental degradation in post-industrial rural contexts. A better understanding of its contaminant pathways supports both public health protection and the sustainable development of apiculture in ecologically fragile regions.

5. Conclusions

This study highlights a significant burden of environmental contamination in the Zlatna region, with honey functioning as both a nutritionally important product and a sensitive bioindicator of agroecosystem health. The findings reveal that lead (Pb) and cadmium (Cd) concentrations in numerous samples far exceed Codex Alimentarius and EU regulatory thresholds, with Pb levels reaching 1.6912 mg/kg and Cd levels 0.3765 mg/kg, up to 15–35 times above permissible limits. These results reflect the legacy of intense mining and metallurgical activity and the continued mobility of residual pollutants from tailings, soil, and air.

Importantly, contamination levels were not solely dependent on proximity to known pollution sources (e.g., Zlatna Industrial Platform, Larga de Sus Mine, Haneș Tailings Dump). Sites at considerable distances, such as Budeni (13.3 km), still showed Pb levels over 1.5 mg/kg, suggesting the influence of atmospheric dispersion, hydrological transport, and historical deposition patterns.

Altitude played a notable role, with lower-elevation sites (e.g., Feneș, Presaca Ampoiului) showing higher metal concentrations, likely due to gravitational settling and downstream accumulation, while higher-altitude locations exhibited lower contamination. This vertical gradient, combined with horizontal distance analysis, reinforces the complexity of metal transport mechanisms in heterogeneous terrain.

While Zn concentrations remained within the Codex limit (5.0 mg/kg), their elevated levels in some locations suggest continued environmental input, potentially from fertilizers or industrial residue. Copper (Cu) exceeded the safety threshold (0.40 mg/kg) in nearly all samples, with peak values surpassing 2.2 mg/kg, pointing to a need for further investigation into its sources and biological effects.

From a toxicological standpoint, Pb and Cd remain the most hazardous, especially for infants, children, pregnant women, and the elderly, due to their long biological half-lives and cumulative effects (neurotoxicity, nephrotoxicity, osteotoxicity). Cu and Zn, although essential, can also exert hepatotoxic and immunosuppressive effects at high concentrations.

Risk modeling by age and sex groups further confirmed the disproportionate impact of potentially toxic elements (PTEs) on vulnerable populations, with infants and pregnant women showing risk levels of 9–10 for Pb and Cd. This calls for targeted food safety regulations and enhanced consumer protection mechanisms.

The results clearly emphasize the need for integrated biomonitoring frameworks, combining honey analysis, environmental mapping, and advanced spatial tools (e.g., GIS, isotopic tracing, and atmospheric dispersion modeling). Furthermore, the study supports the use of honey as a low-cost, ecologically relevant bioindicator for ongoing surveillance in post-industrial rural landscapes.

To mitigate future risks, public health authorities should prioritize environmental remediation, stricter regulation of agro-industrial emissions, and strategic relocation of apiaries. Consumer awareness campaigns and clearer honey traceability standards could enhance both food safety and trust in local apicultural systems.

Based on the results obtained in this study, it is evident that honey collected from areas in close proximity to industrial and mining sites contains higher concentrations of potentially toxic elements (PTEs), particularly cadmium and lead. In light of these findings, a key preventive measure would be to avoid placing beehives near known sources of environmental contamination. Relocating apiaries to more remote or ecologically stable areas can significantly reduce the risk of contamination. Moreover, implementing regular monitoring programs for honey quality especially in high-risk or historically polluted regions can serve both as a protective strategy for public health and as a tool to ensure compliance with food safety standards. These actions would not only safeguard consumer health but also help maintain the ecological and commercial value of honey as a natural product.

In sum, this work underscores the dual role of honey as a functional food and a sentinel of environmental degradation. The Zlatna region exemplifies the broader challenges faced by post-industrial agricultural zones across Eastern Europe, and calls for coordinated policy responses at the interface of agricultural sustainability, environmental health, and food safety governance.