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

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript presents the potential of honey to be used as an indicator of pollution. Some more information should be provided to clarify the findings.

1. The study investigates the heavy metals in the honey from the polluted areas. The results show significant amounts of heavy metals in the honey. However, to draw a strong conclusion that honey can be an indicator of pollution, the heavy metals in the honey from the non-polluted areas should also be determined.
2. Introduction: The content in lines 51-89 can be shortened. The mechanisms or behaviors of bees that are related to the accumulation of heavy metals in honey should be given in detail in the “Introduction”.
3. Please include harvesting periods/ years and replications in the “Methodology”.
4. Are the units of heavy metal concentration shown in Table 1, Figure 2, and Figure 3 wet basis or dry basis?
5. From No. 4, if the wet basis is reported, total solid contents of each honey sample should also be reported.
6. The suggestion on how to reduce contamination or health risk in honey should also be mentioned.

Author Response

Author's comments: Dear Reviewer (R1),

Author's comments: I would like to express my sincere appreciation for the time and effort you have invested in reviewing my manuscript. Your thoughtful and constructive comments have been extremely valuable and have helped me to critically assess and improve the quality of the work. In the following sections, I have addressed each of your observations individually. Where applicable, I have implemented the recommended changes and provided clarifications or additional data to support the scientific accuracy and clarity of the manuscript. I believe these revisions have strengthened the overall content and presentation of the study. Thank you once again for your guidance and for contributing to the enhancement of this research. The recommendations received from Reviewer 1 have been implemented and are highlighted in red in the manuscript.

Reviewer's comments: Comments and Suggestions for Authors:

Reviewer's comments: The manuscript presents the potential of honey to be used as an indicator of pollution. Some more information should be provided to clarify the findings.

Reviewer's comments: The study investigates the heavy metals in the honey from the polluted areas. The results show significant amounts of heavy metals in the honey. However, to draw a strong conclusion that honey can be an indicator of pollution, the heavy metals in the honey from the non-polluted areas should also be determined.

Author's answer: The results obtained in this study clearly demonstrate that honey reflects the level of environmental contamination and support the conclusion that it can be used as a bioindicator of pollution. We analyzed honey samples from seven locations situated at varying distances from three major industrial sources (an industrial platform, a mining area, and a tailings dump). A consistent spatial pattern emerged: the closer the source of pollution, the higher the concentrations of heavy metals in honey. For example, Budeni, located 13.3 km from the industrial platform, recorded a cadmium level of 0.1371 mg/kg, which is considerably lower than Izvorul Ampoiului (2.2 km, Cd = 0.2618 mg/kg) and Presaca Ampoiului (7.3 km, Cd = 0.2481 mg/kg). This gradient supports a direct relationship between distance from the source and contamination levels. This study builds on the well-established scientific understanding that honey can serve as a bioindicator of heavy metal pollution. It is already documented that bees, through foraging, transfer environmental contaminants into honey, making it a reliable integrator of environmental quality. Our results confirm, reinforce, and add spatial and quantitative depth to these findings, highlighting honey’s practical value in environmental monitoring frameworks.

This finding is strongly supported by a large body of literature demonstrating similar results in various geographic regions and environmental contexts (see references 1–15):

1. Fadil, M.; Krasniqi, D.; Ahmet, M. Heavy metals in honey produced in some localities in Kosovo. Rasāyan J. Chem. 2020, 13(4), 2013–2036. doi:10.31788/RJC.2020.1345811
2. Rădulescu, H.; Ciobanu, O. Impact of air pollution on heavy metals content of honey. In Proceedings of the 18th International Multidisciplinary Scientific GeoConference SGEM2018, Sofia, Bulgaria,3-6 December, 2018, 18(4.3), 301–306. doi:10.5593/sgem2018V/4.3/S06.035
3. Mustapha, S.; Musa, A.K.; Vanhaelewyn, L.; Hung, Y.; Adeboye, A.A.; Orijemie, E.A.; Popoola, F.A. Honey as a sustainable indicator of heavy metals in tropical rainforest vegetation zone: An early warning monitoring approach. Int. J. Trop. Insect Sci. 2023, 43(4), 1263–1281. https://doi.org/10.1007/s42690-023-01038-y
4. Akbari, B.; Gharanfoli, F.; Khayyat, M.H.; Khashyarmanesh, Z.; Rezaee, R.; Karimi, G. Determination of heavy metals in different honey brands from Iranian markets. Food Addit. Contam. Part B 2012, 5(2), 105–111. https://doi.org/10.1080/19393210.2012.664173
5. Šerevičienė, V.; Zigmontienė, A.; Paliulis, D. Heavy metals in honey collected from contaminated locations: A case of Lithuania. Sustainability 2022, 14(15), 9196. https://doi.org/10.3390/su14159196
6. Mititelu, M.; Udeanu, D.I.; Docea, A.O.; Tsatsakis, A.; Calina, D.; Arsene, A.L.; Ghica, M. New method for risk assessment in environmental health: The paradigm of heavy metals in honey. Environ. Res. 2023, 236, 115194. https://doi.org/10.1016/j.envres.2022.115194
7. Erbilir, F.; Erdoğrul, Ö. Determination of heavy metals in honey in Kahramanmaraş City, Turkey. Environ. Monit. Assess. 2005, 109, 181–187. https://doi.org/10.1007/s10661-005-5848-2
8. Flamminii, F.; Consalvo, A.; Cichelli, A.; Chiaudani, A. Assessing mineral content and heavy metal exposure in Abruzzo honey and bee pollen from different anthropic areas. Foods 2024, 13(12), 1930. https://doi.org/10.3390/foods13121930
9. Bolelli, L.; Ferri, E.N.; Sangiorgi, S.; Porrini, C.; Ferrari, L.; Nenzioni, M.; Girotti, S. Honeybees as bioindicators in environmental monitoring: Practical applications and open online course. In Recent Advances in Environmental Science from the Euro-Mediterranean and Surrounding Regions; Springer: Cham, Switzerland, 2021; pp. 677–681. https://doi.org/10.1007/978-3-030-51210-1_107
10. Mehdi, Y.; Mutlaq, A.; Al-Balas, Q.; Azzi, E.; Bouadjela, L.; Taïbi, N.; Bachari, K. Physicochemical characterization and determination of chloramphenicol residues and heavy metals in Algerian honeys. Environ. Sci. Pollut. Res. 2018, 25, 33322–33333. https://doi.org/10.1007/s11356-018-3241-2
11. Bratu, I.; Georgescu, C. Chemical contamination of bee honey – identifying sensor of the environment pollution. J. Cent. Eur. Agric. 2005, 6(1), 467–470.
12. Karabagias, I.K.; Louppis, A.P.; Kontakos, S.; Papastephanou, C.; Kontominas, M.G. Characterization and Geographical Discrimination of Greek Pine and Thyme Honeys Based on Their Mineral Content, Using Chemometrics. Eur. Food Res. Technol. 2017, 243, 101–113. https://doi.org/10.1007/s00217-016-2727-8
13. He, J.Z.; Feng, Q.; Sun, P.L. Health risk assessment of six heavy metals in different sources of honey consumed in China. Adv. Mater. Res. 2013, 680, 86–93. https://doi.org/10.4028/www.scientific.net/AMR.680.86
14. Manouchehri, A.; Pirhadi, M.; Shokri, S.; Khaniki, G.J. The possible effects of heavy metals in honey as toxic and carcinogenic substances on human health: A systematic review. Uludag Bee J. 2021, 21(2), 237–246. doi:10.31467/uluaricilik.973053
15. Bora, F.D.; Babeș, A.C.; Călugăr, A.; Jitea, M.I.; Hoble, A.; Filimon, R.V.; Bunea, C.I. Unravelling heavy metal dynamics in soil and honey: A case study from Maramureș region, Romania. Foods 2023, 12(19), 3577.https://doi.org/10.3390/foods12193577

Reviewer's comments: Introduction: The content in lines 51-89 can be shortened. The mechanisms or behaviors of bees that are related to the accumulation of heavy metals in honey should be given in detail in the “Introduction”.

Author's answer: Thank you for your valuable feedback. We have addressed the comment by incorporating specific data, mechanisms, and cited research to explain how heavy metals are transferred into honey via environmental exposure, bee foraging behavior, and internal physiological processes. In particular, we added evidence showing how elements such as cadmium and lead accumulate in nectar-producing plants and how honey bees act as vectors, introducing contaminants into the hive during nectar and pollen collection. We also included recent findings that quantify metal concentrations in flowers, bee bodies, and hive products (e.g., Tomczyk et al., 2023; Di et al., 2020; Borsuk et al., 2021). To strengthen the scientific basis of the introduction, we further elaborated on the role of bee behavior—such as extended foraging ranges and lack of avoidance of contaminated resources—in facilitating contamination. Additionally, we provided physiological context by describing how metals are absorbed through the digestive tract, transported via the hemolymph, and either bioaccumulated in bee tissues or partially filtered during nectar processing (e.g., Hladun et al., 2016). These additions not only respond to the request for more data and interpretation but also establish a more coherent and evidence-based rationale for the study’s objectives. The revised introduction better frames honey’s role as a bioindicator and clarifies the environmental and methodological context of our investigation.

Reviewer's comments: Please include harvesting periods/ years and replications in the “Methodology”.

Author's answer: Thank you for your observation. The requested information has been added to the “Methodology - 2.1. Study Area and Environmental Background” section of the manuscript.

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.

Reviewer's comments: Are the units of heavy metal concentration shown in Table 1, Figure 2, and Figure 3 wet basis or dry basis?

Author's answer: Thank you for this valuable observation. The concentrations of heavy metals presented in Table 1, Figure 2, and Figure 3 are expressed on a dry weight basis. This approach was chosen to ensure consistency in data comparison and to eliminate the variability that can arise from differences in moisture content between honey samples. To enhance clarity and transparency, we have now explicitly stated the use of the dry basis in the legend of Table 1 as well as in the captions of Figures 2 and 3 in the revised manuscript. This clarification allows for a more accurate interpretation of the results and improves the scientific rigor of the presentation.

Reviewer's comments: From No. 4, if the wet basis is reported, total solid contents of each honey sample should also be reported.

Author's answer: As previously stated, the reported values are expressed on a dry weight basis; therefore, it is not necessary to include the percentage of total solids for each honey sample. This approach allows for direct comparability between samples, without the need for additional conversions, and ensures consistency in interpreting heavy metal concentrations across all data points.

Reviewer's comments: The suggestion on how to reduce contamination or health risk in honey should also be mentioned.

Author's answer: Thank you for this important observation. We agree that highlighting practical recommendations based on the study findings adds value to the manuscript, both scientifically and in terms of public health relevance. In response, we have added a concluding paragraph in the “Conclusion” section, emphasizing the importance of avoiding the placement of beehives near industrial, mining, or other environmentally contaminated areas. We also recommend the implementation of regular honey monitoring programs, particularly in regions with a known history of pollution. These measures are intended to reduce contamination risks and ensure the safety and quality of honey for consumers. The new paragraph is highlighted in the revised manuscript.

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 heavy metals, 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.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Dear Authors 

Reviewer 2 - Observation/comment/question

Ad. Abstract: The abstract contains no data. e.g. "while Cu and Zn, although essential trace elements, were present at elevated levels."

What is defined by "heavy metals," because IUPAC does not provide such a definition, and including Zn and Cu - nutrients with Cd and Pb seems to be rather related PTEs (potentially toxic elements). IUPAC in 2001 regulated the use of such an "ecological, not chemical, term" for incorrectly defining such elements - recommends PTEs. Interesting data on Cu content are given in the publication - Environmental Science and Pollution Research (2019) 26:371–380 https://doi.org/10.1007/s11356-018-3612-8. Can such contents in bees be related to the contents in honey?

Ad. INTRODUCTION: I did not connect people's health so "dramatically" with the content of Zn and Cu, Cd and Pb - firstly, fashion can be a source of microelements, e.g. Se, and the content should be connected with the amount consumed.

The knowledge introduction contains mainly generalities, without data and their interpretation.

Why were elements already so well recognized in honey, bees and other bee products chosen?

Figure 1 should be the basis of GA, but in itself it does not provide any significant information regarding the location and potential sources of contamination of plants or flowers. In my opinion it can be omitted.

Methodology: attention should be paid to chemometric aspects. The main errors in this type of studies are factors related to representativeness, correct determination of the mode mass and volume change after dissolution. The precision of volume determination is only at the level of +/- 1 mL (p. 8 l. 339). Then the results appear in such a record 1.4239 ± 0.39. Lack of understanding of the value and significance of the UNC measurement. This REQUIRES correction.

The limit of detection = LOD - not LoD.

Results and Discussion: Due to the large amount of information (Tab. 1), it is not easy to follow the results. Maybe a more advanced analysis than ANOVA, maybe PC graphs or others.

Ad. 3 - I wonder if it makes sense without the correlation of the distance from the source of contamination with the bee zeroing area? It is not always a straight line in bee flights, but rather it is correlated with the fields of melliferous plants.

Since Fig. 5 - it is that ! The interesting chemometric interpretation presented in this publication was used in research on bees - https://doi.org/10.1016/j.chemosphere.2021.130572.

Does this statistically significant change in pH (is the important the 2nd digit??? In pH? Of honey) have any significance for the health of bees or for consumers?

greetings 

Author Response

Author's comments: Dear Reviewer (R2),

Reviewer's comments: Reviewer 2 - Observation/comment/question

Author's comments: Thank you for your thorough and thoughtful review of our manuscript. We greatly appreciate the time and effort you have invested in evaluating our work and for the critical observations and suggestions that contribute meaningfully to the improvement of the manuscript. Your comments have raised important scientific, methodological, and terminological considerations, particularly regarding the classification of trace elements, the structure of the introduction, the robustness of the methodology, and the clarity of data presentation. We have carefully addressed each of your points and made corresponding modifications to the manuscript where appropriate. Specific changes have been highlighted in the revised version for clarity. Below, we provide detailed, point-by-point responses to each of your observations. The modifications suggested by this reviewer have been implemented in the manuscript and are highlighted in blue for clarity.

Reviewer's comments: Ad. Abstract: The abstract contains no data. e.g. "while Cu and Zn, although essential trace elements, were present at elevated levels."

Author's comments: Thank you for this helpful comment. In response, the abstract has been revised to include specific quantitative data for all four analyzed elements (Pb, Cd, Cu, Zn), along with the number of samples, sampling locations, and the main analytical techniques used. The updated version presents a concise summary of the key findings, including concentration ranges and spatial trends, in accordance with journal guidelines. These revisions improve the clarity, informativeness, and scientific value of the abstract. The revised abstract is now included in the manuscript and highlighted in blue for clarity.

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 (0.7211–1.6912 mg/kg) and Cd (0.0233–0.3765 mg/kg) levels consistently exceeded international safety thresholds, while Cu (0.6231–2.2256 mg/kg) and Zn (0.9157–1.9364 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.

Reviewer's comments: What is defined by "heavy metals," because IUPAC does not provide such a definition, and including Zn and Cu - nutrients with Cd and Pb seems to be rather related PTEs (potentially toxic elements).

Author's comments: Thank you for this valuable observation. In response, we have removed the term “heavy metals” and replaced it throughout the manuscript with “potentially toxic elements (PTEs)”, which better reflects the dual role of elements such as Cu and Zn—essential at trace levels, but potentially harmful when accumulated in excess. This change has been made in line with IUPAC recommendations and is highlighted in blue in the revised version of the manuscript.

Reviewer's comments: IUPAC in 2001 regulated the use of such an "ecological, not chemical, term" for incorrectly defining such elements - recommend PTEs.

Author's comments: Thank you for your insightful comment regarding the appropriate use of terminology in line with IUPAC recommendations (2001). We have thoroughly revised the entire manuscript and replaced all ecologically vague or chemically inaccurate terms such as “heavy metals” or “toxic metals” with the scientifically appropriate term “PTEs” (Potentially Toxic Elements), as recommended by IUPAC. We believe this change enhances the clarity and scientific rigor of the manuscript.

Reviewer's comments: Interesting data on Cu content are given in the publication - Environmental Science and Pollution Research (2019) 26:371–380 https://doi.org/10.1007/s11356-018-3612-8. Can such contents in bees be related to the contents in honey?

Author's comments: Thank you for your valuable comment and for referring us to the study by Sadowska et al. (2019). We agree that the accumulation mechanisms of Cu in bees and honey may differ, and we acknowledge that Cu, as an essential micronutrient, is not typically used as a direct marker of pollution. Nonetheless, we consider that investigating Cu content in both bees and honey can offer meaningful insights into the environmental exposure in the vicinity of an apiary. Several recent studies support the premise that the levels of potentially toxic elements (such as Cu, Zn, Cd, and Pb) in bees and honey are influenced by the location of hives relative to pollution sources:

• Capuano et al. (2022) demonstrated that honey bees are effective bioindicators of heavy metal pollution and clearly reflect spatial and temporal variability based on the proximity of hives to urban or industrial zones (Atmosphere, https://doi.org/10.3390/atmos13040624).
• Giacomelli et al. (2024) emphasized that honey can reflect environmental metal contamination, particularly in areas under industrial or agricultural influence, confirming the utility of honey as a matrix for environmental monitoring (Environmental Pollution, https://doi.org/10.1016/j.envpol.2024.125221).
• Porrini et al. (2021) highlighted that elemental composition in bees varies significantly depending on geographical and environmental context, with hive location being a major factor influencing the levels of trace metals (Environmental Research, https://doi.org/10.1016/j.envres.2021.112237).

These studies support our assumption that the simultaneous evaluation of trace metals in bees and honey is scientifically justified, and that potential correlations may reflect the degree of environmental contamination in the foraging area. Although our current study did not statistically test the correlation between these two matrices, the observed trends support further investigation. A corresponding note has now been included in the Discussion section of the revised manuscript to reflect this relevance and to highlight it as a future research direction.

 

Reviewer's comments: Ad. INTRODUCTION: I did not connect people's health so "dramatically" with the content of Zn and Cu, Cd and Pb - firstly, fashion can be a source of microelements, e.g. Se, and the content should be connected with the amount consumed.

Author's comments: Thank you for your insightful comment. We fully agree that the presence of trace elements such as Zn and Cu in honey should not be automatically associated with negative health outcomes, as these are essential micronutrients that play important physiological roles. In the revised manuscript, we have modified the relevant sentence in the Introduction to adopt a more balanced tone and to clarify that the potential health effects of trace elements depend on both their concentration and the amount of honey consumed. The updated text also highlights that risks may arise primarily from excessive intake of toxic elements such as Cd and Pb, and not from the presence of essential elements within normal dietary ranges. The revised sentence now reads:

Although honey is widely regarded as a safe and natural food, its trace element content warrants monitoring, particularly in regions exposed to environmental pollution [30–32]. 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 [30–32]. 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 [33,34]. 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.

Reviewer's comments: The knowledge introduction contains mainly generalities, without data and their interpretation.

Author's comments: Thank you for your valuable comment. In response, we have thoroughly revised the introduction to address the lack of specificity. We integrated recent scientific literature that includes quantitative data and their interpretation regarding the presence of heavy metals (e.g., Cd, Pb, Cu, Zn) in nectar, pollen, flowers, and hive products. We also added relevant examples showing how these contaminants reach honey, including concentrations reported in polluted areas and the differences observed after nectar processing. Furthermore, we included new explanatory paragraphs on the physiological pathways of metal uptake and distribution in honey bees, as well as the influence of bee behavior (e.g., foraging range, floral preferences) on contamination. These updates clarify the scientific rationale behind the study and more clearly connect the introduction to the research aims.

Reviewer's comments: Why were elements already so well recognized in honey, bees and other bee products chosen?

Author's comments: Thank you for your thoughtful comment. The choice of Cu, Zn, Cd, and Pb was guided by their environmental relevance and toxicological importance, particularly in the specific context of the Zlatna region in Romania. This area has a long history of mining and metallurgical activities, which are well known to have contributed to persistent heavy metal contamination in the local environment. Although these elements are indeed frequently studied in bee products, their concentrations are highly site-specific and can vary significantly depending on local pollution sources. In the case of Zlatna, previous reports have documented elevated levels of heavy metals in soil, vegetation, and water, but comparative data on honey and bees from this area remain scarce. By including both essential elements (Cu, Zn) and toxic ones (Cd, Pb), we aimed to evaluate not only potential contamination but also deviations from natural micronutrient balances due to environmental stress. This dual perspective enhances our ability to assess ecological risk and potential implications for food safety. Furthermore, analyzing these elements simultaneously in honey and bees, in a known contaminated region like Zlatna, provides valuable insights into how hive placement in such areas influences trace element accumulation. Therefore, despite being widely recognized, these elements retain high scientific and practical relevance in the specific environmental context of our study.

Reviewer's comments: Figure 1 should be the basis of GA, but in itself it does not provide any significant information regarding the location and potential sources of contamination of plants or flowers. In my opinion it can be omitted.

Author's comments: We thank the reviewer for the observation regarding Figure 1. We respectfully argue for its retention, as the figure serves a critical role in contextualizing the geographic location of the sampling sites (apiaries) relative to legacy industrial pollution sources, such as the Zlatna Industrial Platform, Larga de Sus Mine, and Haneș Tailings Dump. This spatial information forms the geographic foundation for the geospatial analysis (GA) presented in the manuscript, including the assessment of contamination gradients and the correlation with environmental variables (e.g., altitude, distance to source).

To clarify its purpose, we have revised the figure legend and corresponding text to emphasize how the positioning of the apiaries influences the likelihood of PTE transfer via contaminated floral resources (nectar/pollen). We hope this clarification justifies the inclusion of the figure, which we consider essential for interpreting the spatial distribution of contamination and exposure risk.

Reviewer's comments: Methodology: attention should be paid to chemometric aspects. The main errors in this type of studies are factors related to representativeness, correct determination of the mode mass and volume change after dissolution.

Author's comments: Reviewer's comments: ~studies are factors related to representativeness ~ Author's comments: To ensure analytical robustness and statistical validity in assessing contamination with potentially toxic elements (PTEs), a total of 48 individual multifloral honey samples were collected and analyzed. This sample size was established to capture the spatial, ecological, and biological variability of the study area. The samples were evenly distributed across multiple locations in the Zlatna region, characterized by varying degrees of exposure to historical and current pollution sources (e.g., mining industry, traffic, forested areas). Each honey sample was composed of material from three independent beehives within the same apiary, following established ecotoxicological sampling protocols. This approach ensured intra-site variability was accounted for and minimized biological uncertainty. The experimental design was calibrated to allow:

• reliable estimation of concentration means with acceptable confidence intervals (α = 0.05),
• application of robust statistical tests (e.g., ANOVA, PCA),
• detection of significant differences based on hive placement and environmental exposure.

Through both the number and distribution of samples, the dataset provides adequate spatial and ecological coverage, aligning with current requirements for environmental biomonitoring using apicultural products. This approach reinforces the validity of the study’s conclusions regarding regional contamination with PTEs and supports the role of honey as an integrative bioindicator.

Author's comments: Reviewer's comments: ~ correct determination of the mode mass ~ Author's comments: Thank you for emphasizing the importance of accurate mass determination. In this study, each honey sample was weighed using a calibrated analytical balance with a precision of ±0.001 g, which is annually certified by the National Institute of Metrology in accordance with national regulations and metrological standards. Exactly 1.000 ± 0.001 g of homogenized honey was used for each analysis. Prior to weighing, all samples were thoroughly homogenized to ensure compositional uniformity and to minimize subsampling errors. This standardized approach, aligned with internationally accepted protocols for trace element analysis, minimizes analytical uncertainty and reinforces the reliability of the reported results. This clarification has been added to the revised Materials and Methods section of the manuscript.

Author's comments: Reviewer's comments: ~ volume change after dissolution ~ Author's comments: Thank you for highlighting the importance of accurate volume adjustment following digestion. In our study, after mineralization, all samples were quantitatively transferred and the final volume was brought to exactly 25.00 mL using Class A volumetric flasks and ultrapure deionized water (18.2 MΩ·cm). All volumetric glassware used for dilution and preparation steps is annually verified and certified by the National Institute of Metrology, in accordance with national metrological standards. After volume adjustment, each solution was thoroughly mixed to ensure homogeneity prior to instrumental analysis. This approach minimizes volumetric errors and ensures the accuracy and comparability of measured concentrations.

Reviewer's comments: The precision of volume determination is only at the level of +/- 1 mL (p. 8 l. 339).

Author's comments: Thank you for your observation. We acknowledge that the original version of the manuscript did not explicitly clarify the volumetric accuracy used during sample preparation. In the revised version, we have specified that, after digestion, all samples were filtered, quantitatively transferred to Class A volumetric flasks, and the final volume was adjusted to exactly 25.00 mL using ultrapure deionized water. Furthermore, all volumetric glassware used was annually verified and certified by the National Institute of Metrology, ensuring traceability and compliance with standard analytical requirements. This clarification has been added to Section 2.3.2 (The Microwave-Assisted Digestion Methodology) to address your concern regarding volume precision and to reinforce the methodological rigor of the study.

Reviewer's comments: Then the results appear in such a record 1.4239 ± 0.39. Lack of understanding of the value and significance of the UNC measurement. This REQUIRES correction.

Author's comments: Thank you for your observation regarding the expression of results and the interpretation of the uncertainty (UNC). We have thoroughly revised the manuscript to ensure that all numerical results are reported with a number of significant figures consistent with their associated uncertainties. For example, the value previously reported as 1.4239 ± 0.39 has been corrected to 1.42 ± 0.39. Additionally, we have clarified in the text that the uncertainty represents the standard deviation derived from replicate measurements. We appreciate your insightful comment, which has helped improve the accuracy and clarity of the manuscript.

Reviewer's comments: The limit of detection = LOD - not LoD.

Author's comments: Thank you for the observation. The abbreviations LoD and LoQ have been replaced with the standardized forms LOD (Limit of Detection) and LOQ (Limit of Quantification) throughout the manuscript, in accordance with common scientific conventions.

Reviewer's comments: Results and Discussion: Due to the large amount of information (Tab. 1), it is not easy to follow the results. Maybe a more advanced analysis than ANOVA, maybe PC graphs or others.

Author's comments: Thank you for your insightful comment regarding the presentation of the results in Table 1. We acknowledge that the table contains a large volume of data; however, this format was intentionally chosen to ensure clarity and transparency in reporting the mean values, standard deviations, and statistical significance of the analyzed variants. The data are presented in one of the simplest and most commonly used forms in environmental and food sciences: mean ± standard deviation, accompanied by superscript letters to indicate statistically significant differences between variants (e.g., 1.5210 ± 0.23a). This format allows for a straightforward understanding of group-wise comparisons. A statistical analysis was performed (ANOVA followed by post-hoc tests) to identify significant differences between honey samples collected from distinct geographical areas (Area I – Zlatna Industrial Platform, Area II – Larga de Sus Mine, Area III – Haneș Tailings Dump). The results clearly demonstrate that metal levels in honey are influenced by the proximity to former industrial sources, despite the cessation of mining activities in 2004. Spatial distribution patterns of elements like Pb, Cd, Cu, and Zn highlight both local contamination and broader environmental transport processes (wind, water, topography), reinforcing the importance of honey as a bioindicator for long-term monitoring. We have also included information on international and national Maximum Permissible Limits (MPLs) for contextual reference and used standard significance notation (*** for p < 0.001, ** for p < 0.01, * for p < 0.05, ns for not significant) for quick interpretation of statistical relevance. While ANOVA provided a solid statistical basis for evaluating differences between groups, we appreciate the suggestion to include a more advanced multivariate approach. In our assessment, the current dataset—focused on a limited number of metals across clearly defined geographic areas—is well suited for the applied statistical tests and may not fully benefit from PCA at this stage. However, we remain open to incorporating such analysis if the editors consider it necessary for enhancing the clarity or depth of interpretation.

Reviewer's comments: Ad. 3 - I wonder if it makes sense without the correlation of the distance from the source of contamination with the bee zeroing area?

Author's comments: Thank you for this valuable observation. We agree that the correlation between the contamination sources and the bee foraging range is a relevant factor in interpreting the spatial distribution of metals in honey. In our study, sampling sites were selected at varying distances from known contamination sources (Zlatna Industrial Platform, Larga de Sus Mine, and Haneș Tailings Dump). While we did not perform a formal correlation analysis between contamination levels and distance from the sources, we did consider the typical foraging range of honey bees—approximately 1.5 to 3 km, with some studies reporting up to 5 km—when selecting the apiary locations. This range supports the assumption that the metal concentrations in honey reflect the environmental conditions within that radius. We acknowledge that a direct correlation analysis could provide additional insights. However, factors such as topography, wind direction, and water flow introduce environmental variability that may influence contaminant dispersion independently of linear distance. For this reason, such a correlation was beyond the scope of the current study, but we agree it represents a valuable direction for future work incorporating geospatial and ecological modeling.

Reviewer's comments: It is not always a straight line in bee flights, but rather it is correlated with the fields of melliferous plants.

Author's comments: Thank you for this insightful remark. We fully agree that bee foraging behavior is not determined solely by linear distance but is significantly influenced by the availability and distribution of melliferous plants. Bees tend to forage in areas with abundant nectar and pollen sources, and their flight paths may follow resource-rich zones rather than the shortest route. In our study, the apiaries were in regions with naturally occurring or cultivated melliferous vegetation, typical for the Apuseni region. While we did not map the precise floral composition or density surrounding each site, the general landscape provided sufficient foraging resources within the expected range (1.5–3 km). We acknowledge that this introduces variability in exposure to potential contaminants, and we consider this a valuable point for future research. Integrating vegetation mapping and floral resource analysis would enhance the ecological interpretation of honey contamination levels and bee foraging dynamics.

Reviewer's comments: Since Fig. 5 - it is that ! The interesting chemometric interpretation presented in this publication was used in research on bees - https://doi.org/10.1016/j.chemosphere.2021.130572.

Author's comments: Thank you for your comment. We are aware of the publication in Chemosphere (DOI: 10.1016/j.chemosphere.2021.130572), which applies chemometric methods in bee-related environmental research. However, the chemometric tools used in our study are standard and widely accepted in environmental and food sciences, and were applied here independently, based on the structure of our dataset. Our interpretation focuses specifically on honey contamination in a historically industrial region and integrates site-specific data and regulatory benchmarks, offering a distinct context and objective compared to the work. Therefore, while methodologically similar, the cited article did not influence our analysis directly, and we believe formal citation is not necessary in this case. We appreciate the reviewer’s suggestion, which helped us reflect further on the positioning of our study within the broader literature.

Reviewer's comments: Does this statistically significant change in pH (is the important the 2nd digit??? In pH? Of honey) have any significance for the health of bees or for consumers?

Author's comments: Thank you for this thoughtful and relevant question. We agree that while statistical significance can indicate measurable differences between samples, it is also important to assess whether such differences have practical or biological relevance. In the case of pH, although statistically significant variations were observed (sometimes affecting the second decimal), the biological impact of such minor changes is likely negligible in terms of honey’s safety for both bees and human consumers. The pH of honey typically ranges between 3.2 and 4.5, and all our measured values fall well within this natural range. Small fluctuations within this interval do not appear to influence honey's antimicrobial properties, quality, or acceptability. From the perspective of bee health, current literature suggests that bees are not directly affected by the pH of the stored honey, but rather by the overall availability and quality of nectar sources. Similarly, for human consumers, regulatory and quality standards do not impose strict limits on pH, and small variations at the second decimal place are not considered to pose health concerns. Therefore, while the observed pH differences are statistically valid, we recognize that their biological or toxicological relevance is limited, and we have clarified this point in the revised manuscript.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript entitled “Comprehensive Assessment of Heavy Metal Contamination in Honey from a Historically Polluted Agro-Industrial Landscape: Implications for Agricultural Sustainability and Food Safety” presents results on the concentration of various metals in a set of Romanian honeys (n=48).

However, the introduction is excessively long and, throughout the reading, resembles a bibliographic review more than an introductory text that provides context for the scientific work conducted. Many paragraphs, particularly those between lines 164 and 198, would be more appropriately placed in the discussion of results.

Similarly, the materials and methods section contains lengthy descriptions regarding the data obtained from other works, which may confuse the reader. This section should focus strictly on describing the experimental procedures and the samples used in the study.

Additionally, it would be beneficial to include graphs or more tables to present the results. The current text is quite long and combines the results obtained with comparisons to other data sets from different sources, making it difficult to read.

Minor Comments:

• Figure 3: Each element should be separated, as the Maximum Permissible Limits for Honey limits are not clearly visible, making the figure confusing.

• Figure 4: The purpose for comparing the mentioned countries is unclear, especially since similar studies have been conducted in several places of the world such as Asia, the Americas, and Africa. While this comparison may be relevant, it complicates the interpretation when the sites are not described and the data are sourced from other research. From this perspective, this figure could be moved to the supplemental files, as it does not directly represent the results of the current research. Such a graph would be more appropriate in a review article.

• Figure 7: This figure is confusing, and it is unclear where the data was obtained to create this graph. There is no further mention about this in the materials and methods section.

• Table 1: What does "MLA" means?

Author Response

Author's comments: Dear Reviewer (R3),

Reviewer's comments:  The manuscript entitled “Comprehensive Assessment of Heavy Metal Contamination in Honey from a Historically Polluted Agro-Industrial Landscape: Implications for Agricultural Sustainability and Food Safety” presents results on the concentration of various metals in a set of Romanian honeys (n=48).

Author's comments: We sincerely thank Reviewer 3 for the time dedicated to evaluating our manuscript and for the constructive comments provided. We appreciate your acknowledgement of the study's focus on heavy metal contamination in Romanian honey samples collected from historically polluted agro-industrial areas. Your observations have helped us to critically reflect on the clarity, relevance, and scientific rigor of our work. Below, we provide detailed responses to each of your comments, along with the corresponding revisions made to the manuscript. All suggested modifications and clarifications in the manuscript have been made using brown-colored text, to ensure transparency and to facilitate the identification of the revised sections.

Reviewer's comments: However, the introduction is excessively long and, throughout the reading, resembles a bibliographic review more than an introductory text that provides context for the scientific work conducted. Many paragraphs, particularly those between lines 164 and 198, would be more appropriately placed in the discussion of results.

Author's comments: Thank you for your observation regarding the structure and focus of the introduction. In response to your comment, we have revised and streamlined the first part of the Introduction by reducing general background content and avoiding repetition regarding honey’s nutritional and therapeutic attributes. This helped to shorten the section and focus it more directly on the environmental and analytical context of the study. Regarding the paragraphs between lines 164 and 198, we respectfully maintain them in the Introduction because they establish the scientific rationale for our study. These sections synthesize critical knowledge gaps such as the lack of harmonized methods, insufficient spatial assessments, and underrepresentation of Eastern European regions in the literature that directly justify our research questions. They also introduce relevant methodological frameworks (e.g., spatial modeling, use of honey as a bioindicator), which are essential for understanding the novelty and scope of our investigation. While we acknowledge that this section includes some references and data that resemble a literature review, we believe it is necessary to provide sufficient context for the scientific problem we address. The inclusion of regional data, regulatory thresholds, and comparisons with previous findings helps to position our study within the broader European research landscape and emphasizes the relevance of the Zlatna region as a case study. Therefore, we consider this content essential for orienting the reader and framing the objectives presented at the end of the Introduction. We trust that these improvements bring greater clarity, coherence, and scientific grounding to the manuscript.

Reviewer's comments:  Similarly, the materials and methods section contains lengthy descriptions regarding the data obtained from other works, which may confuse the reader.

Author's comments: Thank you for this valuable observation. We understand the concern regarding the inclusion of content from previous works in the Materials and Methods section. However, we would like to clarify that the current research builds upon a well-established methodology developed and applied by the corresponding author in earlier studies on heavy metal contamination in honey. The references to these prior publications are not meant to present external data or results, but rather to support and document the analytical procedures employed in this study—procedures that have been validated, published, and widely accepted in similar scientific contexts. Including these references ensures transparency, reproducibility, and methodological consistency. We have carefully structured the descriptions to focus strictly on the analytical workflow (e.g., sample preparation, instrumentation, detection techniques), without introducing interpretations or findings from earlier studies. Nevertheless, we will revise the section to make this distinction clearer and avoid any potential confusion for the reader.

Reviewer's comments: This section should focus strictly on describing the experimental procedures and the samples used in the study.

Author's comments: Thank you for this comment. We agree that the Materials and Methods section should focus strictly on describing the experimental procedures and the samples used in the current study. We would like to clarify that this has already been our approach: the section exclusively describes the sampling strategy, analytical methods, instrumentation, and statistical procedures applied in this research. Any references to previous works were included solely to document the origin and validation of the methods we used, not to introduce external data or discussion. Nonetheless, we have reviewed the section once more and made minor adjustments to ensure that the focus remains entirely on the methodology specific to this study and to avoid any potential confusion for the reader.

Reviewer's comments: Additionally, it would be beneficial to include graphs or more tables to present the results.

Author's comments: Thank you for this suggestion. From our perspective, we have already included key tables and figures that we believe effectively illustrate and support the main findings of the study. However, we understand the importance of data visualization and are open to further improving the clarity of our results. If the reviewer has specific recommendations regarding the type of graphs or data representations that would enhance the manuscript, we would be glad to implement them accordingly in the revised version.

Reviewer's comments: The current text is quite long and combines the results obtained with comparisons to other data sets from different sources, making it difficult to read.

Reviewer's comments: Thank you for this helpful observation. We understand that combining our results with extensive comparisons to literature may affect the clarity and readability of the manuscript. Our intention was to contextualize the findings and highlight their relevance by comparing them to previously published data. However, we acknowledge the reviewer’s concern and have revised the Results and Discussion section to better separate the presentation of our findings from comparisons with other studies. This restructuring aims to improve the flow of information and make it easier for the reader to distinguish between our data and literature-based references. We appreciate this suggestion, which has allowed us to enhance the clarity and structure of this section.

Reviewer's comments: Minor Comments:

Author's comments:  -

Reviewer's comments: Figure 3: Each element should be separated, as the Maximum Permissible Limits for Honey limits are not clearly visible, making the figure confusing.

Author's comments: Thank you for this observation. We acknowledge that including multiple elements in a single figure may reduce the visibility of the Maximum Permissible Limits (MPLs). However, we chose to retain the integrated format of Figure 3 to provide a comparative overview of all elements across the sampling sites. To improve clarity, we have added a detailed explanation in the manuscript that describes the figure structure, highlights the MPL positions, and guides the interpretation of each element. We believe this clarification enhances readability while preserving the comparative value of the figure.

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.

Reviewer's comments: Figure 4: The purpose for comparing the mentioned countries is unclear, especially since similar studies have been conducted in several places of the world such as Asia, the Americas, and Africa. While this comparison may be relevant, it complicates the interpretation when the sites are not described and the data are sourced from other research. From this perspective, this figure could be moved to the supplemental files, as it does not directly represent the results of the current research. Such a graph would be more appropriate in a review article.

Author's comments: Thank you for this important observation. We acknowledge the reviewer's concern regarding the clarity and relevance of Figure 4. Our intention in including this figure was not to directly compare site-specific conditions, but rather to provide a broader international context for interpreting the concentrations of heavy metals detected in our honey samples. The countries included in Figure 4 were selected based on the availability of comparable data in peer-reviewed studies that applied similar analytical methods and reported results using compatible units. This allows for a general-level comparison that helps position our findings within a global perspective—highlighting whether the contamination levels observed in our study area are unusually high, within expected limits, or relatively low compared to other regions of the world. We agree that this figure does not represent original data and that the included sites are not described in detail. To address this, we have revised the figure caption and manuscript text to clearly state that the data are from published studies, and that the purpose of the figure is strictly contextual and not intended for direct spatial or methodological comparison. We believe that this global frame of reference provides additional value for readers, especially in terms of understanding potential environmental and regulatory implications. Therefore, we have opted to retain Figure 4 in the main text, with improved clarification of its scope and limitations.

Reviewer's comments: Figure 7: This figure is confusing, and it is unclear where the data was obtained to create this graph. There is no further mention about this in the materials and methods section.

Author's comments: Thank you for this observation. We confirm that the data presented in Figure 7 were obtained entirely from the current study. We acknowledge that the methodology related to this figure was not clearly described in the original version of the manuscript, which may have led to confusion. In response to the reviewer’s comment, we have revised the Materials and Methods section to include a clear description of the data acquisition, processing, and graphical representation used for Figure 7. We have also updated the figure caption and the corresponding explanation in the Results and Discussion section to better integrate and contextualize the figure within the study. We appreciate this suggestion, which has helped us improve the transparency and clarity of the manuscript.

Materials and Methods:

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 (e.g., Figure 7). 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, 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.

Results and Discussion:

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.

Reviewer's comments: Table 1: What does "MLA" means?

Author's comments: Thank you for this observation. The abbreviation “MLA” stands for Maximum Legal Allowance. This term was defined in the Abbreviations section of the manuscript. However, to improve clarity and accessibility, we have now also included its definition in the caption of Table 1 to ensure that readers can easily understand its meaning without referring to other sections.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

No more comment.

Author Response

We would like to thank the reviewer for the valuable comments and constructive suggestions provided. Your feedback has helped us to significantly improve the clarity and scientific quality of our manuscript. We appreciate the time and effort you dedicated to the review process.

Reviewer 2 Report

Comments and Suggestions for Authors

I support those that were not taken into account, but I understand the responses to these comments.

Author Response

We would like to thank the reviewer for the valuable comments and constructive suggestions provided. Your feedback has helped us to significantly improve the clarity and scientific quality of our manuscript. We appreciate the time and effort you dedicated to the review process.