Determining the Authenticity of Ghanaian Honeys Using Stable Isotope Ratio Analysis (SIRA)

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

After careful consideration, I find that this manuscript demonstrates competent analytical work using EA‑IRMS. It applies recognized methodologies correctly. However, it suffers from essential conceptual weakness (representative sampling, authentication and inferential scope) that places this paper at the borderline of rejection. Principal concern is the absence of independent verification of samples designated as “authentic” or “farm derived”.It seriously undermines several focal claims of the paper.

Specific remarcs:

1. No independent proof of verification oh honey origin is provided for reference samples. Farm or beekeeper sourcing is treated as authentication. Yet no important proofs for thet are given. No analytical verification is provided, no documentation of production practices, supplemental feeding, chain‑of‑custody control. In honey fraud research, location alone is very inadequate to establish genuineness.
2. Study relies on honey protein δ¹³C as an internal benchmark. Than it uses consistency with this benchmark to declare samples as being authentic. This creates a problem of circular reasoning. In this case, protein isotopes function both as assumption and as evidence. It may be valid for detecting C4 sugar addition, but this approach doesn’t give proof of overall honey authenticity and cannot exclude C3 adulteration or indirect adulteration pathways.
3. Authors put claims about regional origin, supply chain structure, and post harvest as opposed to adulteration at production level. Thse claims are drawn from samples whose authenticity and origin were not independently verified. As a result, these interpretations are at best tentative, but are presented as confirmatory.
4. Manuscript over states its scope,referring to “authentic honey” and national reference baselines. However, the data supports only detection of C4 sugar adulteration which is relative to internal isotopic composition.

Taken altogether,all these issues mean that the conclusions presented in this paper exceed what the presented study design can reliably support. While the analytical measurements themselves are not put in question,it remains the question of the representative sampling. Lack of authenticated reference material represents a major methodological gap that cannot be corrected through minor revision. This manuscript may eventualy be suitable for publication but only if it is substantially reframed, taken seriously into consideration:

1. Significant verification of sample origin
2. Explicit limitation of “authenticity” to absence of detectable C4 sugar adulteration
3. Removal of implied certification of farm samples as authentic controls
4. Clear acknowledgment of the inability to assess C3 adulteration, feeding practices, or absolute provenance
5. Significant reduction and softening of claims regarding regional origin and policy implications

Short of such fundamental revision, thiss study risks misleading readers about the evidentiary strength of isotope based honey authentication.

Author Response

See-attached file.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript titled “Authenticity of Honey from Ghana: A Stable Isotope Ratio Analysis (SIRA) Approach” investigates the authenticity of honey samples from different regions of Ghana using stable isotope ratio analysis, with particular emphasis on the detection of C4 sugar adulteration through EA-IRMS methodology. In addition to carbon isotopes, the study also explores the potential application of nitrogen and sulphur isotopes for assessing geographical and environmental origin of honey samples. The topic is scientifically relevant and addresses an important issue related to food authenticity and honey fraud detection.

After careful evaluation of the manuscript, several aspects were identified that require further clarification and improvement in order to strengthen the scientific quality, methodological transparency, and overall presentation of the work. Detailed comments and suggestions are provided below with the aim of helping the authors improve the manuscript.

Abstract:
Overall, the abstract summarizes the study adequately. However, some parts are unnecessarily descriptive and resemble discussion rather than a concise overview of the work. Inclusion of the total number of analyzed samples would improve clarity. Better standardization of terminology related to “industrial syrups” and “C4 sugars” throughout the manuscript is also recommended. More emphasis on the analytical methodology and the principal findings would further strengthen this section.

Introduction

Comment 1:
Greater focus should be placed on the scientific background of honey adulteration and isotope-based authentication. Several paragraphs discussing biodiversity, climate, and the cultural importance of beekeeping are excessively detailed and only marginally connected to the main objective of the manuscript. Shortening these descriptive sections and focusing more on analytical approaches and current challenges in honey authenticity assessment would improve readability and scientific clarity.

Comment 2:
Current literature is mostly summarized descriptively without sufficient critical evaluation. Expanding the discussion regarding the limitations of AOAC 998.12, especially concerning the detection of C3 sugar adulteration, would significantly strengthen the manuscript. Additional comparison with recent LC-IRMS and LC-HRMS studies would also improve the scientific context of the work.

Comment 3:
Greater clarity is needed regarding the primary objective of the study. At present, it remains uncertain whether the manuscript mainly focuses on authenticity assessment, geographical origin determination, or market surveillance of adulterated honey. A clearer statement of the aims and hypotheses at the end of the introduction would help guide the reader through the study.

Results and Discussion

Comment 4:
Interpretation of the obtained results remains largely descriptive throughout this section. More detailed discussion regarding possible socio-economic or supply-chain-related causes of adulteration would improve the scientific impact of the manuscript. Incorporation of statistical analysis is strongly encouraged, as it would provide better support for the presented conclusions.

Comment 5:
Presentation of Tables 1 and 2 could be improved further. Grouping samples according to adulteration severity or adding statistical indicators would make interpretation easier for readers. Missing standard deviation values for certain δ¹⁵N measurements should also be checked carefully and corrected where necessary.

Comment 6:
Discussion of δ¹⁵N and δ³⁴S values is interesting; however, several conclusions appear somewhat speculative. Statements linking elevated δ¹⁵N values to intensive agricultural activity would benefit from additional references or environmental evidence. A more cautious interpretation of the “sea-spray effect” influencing sulphur isotope composition is also advisable.

Comment 7:
Scientific robustness of the study could be considerably improved through additional statistical analysis. Application of multivariate approaches such as PCA or discriminant analysis would provide stronger support for regional differentiation and authenticity assessment. Current interpretation relies mainly on descriptive comparison of isotope values.

Materials and Methods

Comment 8:
Methodological details are extensive, but the section would benefit from improved organization and conciseness. Climatic descriptions of the investigated regions are unnecessarily long and could be shortened substantially or transferred to supplementary material. Additional information regarding analytical replication, validation parameters, recovery studies, and quality assurance procedures should also be provided.

Comment 9:
Sampling strategy requires additional clarification. More information regarding the criteria used for selecting farms and retail outlets would improve transparency. It would also be useful to indicate whether seasonal or botanical variability was considered during sample collection. Given the broad conclusions presented in the manuscript, the relatively limited number of samples should be acknowledged as a study limitation.

Comment 10:
Description of the EA-IRMS system is generally appropriate, although several important analytical details remain missing. Inclusion of detection limits, calibration performance, repeatability data, and quality control procedures would improve the methodological quality of the manuscript. Clarification regarding the number of technical and biological replicates is also needed.

Conclusions

Comment 11:
Most conclusions simply restate previously presented results without providing deeper scientific interpretation. Statements regarding the establishment of a “national multi-isotope reference database” appear somewhat overstated considering the relatively limited dataset. A more balanced conclusion acknowledging the limitations of the study would strengthen the manuscript.

Comment 12:
Figure 3 is useful for visualizing the sampling regions, but graphical quality and resolution should be improved. Clearer labeling of sampling locations and inclusion of geographical coordinates would enhance clarity and reproducibility.

Author Response

See-attachement!

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

Thank you for the opportunity to review the manuscript entitled “Authenticity of Honey from Ghana: A Stable Isotope Ratio Analysis (SIRA) Approach”. The manuscript addresses a relevant and timely issue in food authenticity and fraud detection, particularly within the context of emerging African honey markets. The use of stable isotope ratio analysis (SIRA) to investigate honey adulteration and geographical differentiation is scientifically valuable and aligns well with the scope of the journal.

The study presents interesting regional comparisons and demonstrates the applicability of EA-IRMS for detecting C4 sugar adulteration. The inclusion of nitrogen and sulphur isotopes as complementary tracers is a strength. However, several aspects of the manuscript require substantial improvement in terms of scientific rigor, methodological clarity, interpretation depth, language quality, and manuscript structure.

 

1. The manuscript demonstrates the application of AOAC 998.12 methodology to Ghanaian honey samples. However, the scientific novelty remains insufficiently articulated. The authors should clarify: What is genuinely novel in this study beyond regional application? How does this work advance current knowledge in isotope-based honey authentication? Is the novelty related to the Ghanaian database, the multi-isotope approach, or the supply-chain interpretation? At present, much of the study appears confirmatory rather than innovative.
2. The study includes only 28 honey samples across multiple regions. While this may be acceptable for a preliminary study, the manuscript sometimes makes broad conclusions regarding “Ghana’s honey sector”. For example: “This indicates broader supply chain vulnerabilities within Ghana’s honey sector.”

Such statements appear overstated considering: the small sample size; limited geographical coverage; absence of seasonal variability; lack of floral characterization. The conclusions should be moderated accordingly.

3. The manuscript presents isotopic values descriptively, but no inferential statistical analysis was performed. This is a major limitation because the manuscript repeatedly discusses: regional differences; distinctions between farm and retail samples; isotopic separation between regions. The authors should consider: t-tests or Mann–Whitney tests; ANOVA/Kruskal-Wallis; multivariate analyses such as PCA or discriminant analysis.

A PCA combining δ¹³C, δ¹⁵N and δ³⁴S could substantially strengthen the geographical discrimination argument.

4. The discussion linking δ¹⁵N values to fertilizer use and agricultural intensification is speculative. For example: “These elevated values are likely associated with more intensive agricultural activity…”. However: no soil data were collected; no fertilizer analyses were performed; no botanical confirmation exists. Similarly, the “sea-spray effect” explanation for sulphur isotopes is plausible but insufficiently supported.
5. The manuscript repeatedly refers to floral and environmental signatures, yet no melissopalynological analysis or floral source confirmation was conducted.

This is problematic because: isotopic variation may depend on floral composition; different nectar sources can influence isotopic composition; botanical origin remains assumed rather than demonstrated.

6. Several analytical procedures lack sufficient detail for reproducibility. Examples include: how many analytical replicates were performed per sample; whether isotope measurements were conducted in duplicate/triplicate; quality assurance procedures; recovery efficiency during protein extraction; blank correction procedures. Additionally, Equation 2 should be better formatted and explained. The current presentation is visually confusing and interrupts readability.
7. The Introduction contains extensive general information about Ghanaian ecology, culture, and beekeeping, some of which is only loosely connected to the research question. Examples: long descriptions of honeybee behavior; general medicinal uses of honey; broad climate descriptions. The Introduction would benefit from substantial condensation and stronger focus on: honey adulteration; isotope authentication methods; analytical gaps in African honey studies.
8. The Discussion primarily repeats results rather than critically interpreting them within the broader literature. For example, several paragraphs simply restate: Δδ¹³C values; adulteration percentages; regional observations.

The discussion should instead: compare findings quantitatively with previous studies; discuss implications for food authentication systems; address analytical limitations of AOAC 998.12; discuss inability to detect C3 adulterants using this approach.

This last point is particularly important because the manuscript itself acknowledges this limitation in the Introduction, but it is not critically revisited later.

9. The manuscript contains numerous grammatical issues, awkward constructions, and inconsistent scientific writing. Examples: “The Honey production in Ghana is a rich and diverse industry”; “The area always receives hot temperatures”; “Bees do so much more than just produce honey”; “This unique combination contributes to its traditional use in Ghana for healing wounds”. Several sections sound informal and narrative rather than scientific.

Professional English language editing is strongly recommended.

10. Table 1 and Table 2 are informative but visually dense. The manuscript would benefit from: graphical visualization of isotope distributions; boxplots by region; scatterplots of δ¹³Cbulk vs Δδ¹³C; PCA or clustering analysis. Currently, interpretation relies heavily on large tables, which reduces readability.

 

Other Comments

- The manuscript alternates between: SIRA; SCIRA; stable isotope ratio analysis. Use one terminology consistently throughout.

- Several isotope symbols and units are inconsistently formatted: δ13C vs δ¹³C; spacing around ‰; superscript formatting.

- Several references are inconsistently formatted and require revision. Examples: missing italics; inconsistent journal abbreviations; spacing errors; inconsistent DOI formatting.

- Figure 3 is informative but visually basic. The map resolution and design could be improved for publication quality.

- Some information is repeated multiple times. Examples: authentic C3 range explanations; adulteration thresholds; descriptions of AOAC standards.

- The conclusion section is too long and partially repeats earlier results. It should focus on: key findings; analytical significance; future directions.

- The study focuses on C4 sugar detection. However, modern honey fraud increasingly involves C3 syrups (rice, beet). The manuscript should more explicitly discuss: analytical limitations of AOAC 998.12; why LC-IRMS or HRMS may be necessary; implications for regulatory monitoring.

 

The manuscript addresses an important topic and presents potentially valuable data regarding honey authenticity in Ghana. However, substantial revisions are needed to improve methodological rigor, statistical support, scientific interpretation, and language quality.

Author Response

See attachement!

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

I have red the new version of the paper. I see that the authors have taken my criticisms seriously and addressed most of the major conceptual objections. However, they have not completely solved the underlying methodological weakness since that weakness is largely impossible to solve retrospectively. Not without new samples and without independent authentication.

The revised paper explicitly acknowledges the lack of independent sample authentication and limits the interpretation of AOAC 998.12 to detection of C4 sugar adulteration. It also recognizes the inability to exclude C3 adulteration, and substantially softens claims regarding supply-chain attribution and provenance. These revisions address the principal concerns raised in the initial review. 

However, I would still prefer replacing several remaining uses of the word "authentic" with "AOAC-compliant" or "without detectable C4 adulteration". My original objection that insufficiently authenticated samples cannot be used to establish robust regional provenance claims resulted in softening of these claims. However statements like "contributed to the differentiation of honey-producing regions" and "demonstrates the usefulness of multi-isotope analysis for regional differentiation" remain prety much stronger than the data justify. Given that only 28 samples and unauthenticted controls, I need to see further softening.

What still bothers me is that the authors still continue to call farm samples "authentic". And yet, the manuscript simultaneously admits that farm samples are not independently authenticated. That is big inconsistency.  Authors should remove any implication that farm samples constitute authenticated controls. Moreover, the national baseline claims remain ambitious. Given 28 samples, 3 regions, no authentication, no seasonal replication and they still call this a Ghanaian baseline????

 The PCA interpretation remains overconfident. PCA separation is being discussed as regional differentiation. But still, the sample numbers are low, adulteration itself drives much of the separation and no validation set exists. The PCA section is better than before but still too enthusiastic.

In my belief authors tackled most of the points however there is still to be done. 

To conclude, in this form the paper is definitely not for accptance. However, I will give it the recomandation to the MAJOR REVISION NEEDED!.

Author Response

see attachement

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have made a considerable effort to address the comments raised during the first round of review, and the manuscript has improved significantly. The Introduction is now better focused on honey authenticity and isotope-based authentication, the objectives of the study are clearly stated, and the discussion has been strengthened with additional literature and a more balanced interpretation of the results. The inclusion of PCA analysis is also a valuable addition and provides further support for the interpretation of the isotope data.

A few minor issues should still be addressed before the manuscript is accepted:

1. Several editorial and formatting issues remain in the revised version. For example, the manuscript still contains an unresolved citation ("Adadi & Obeng [Error! Reference source not found.]") as well as a placeholder citation ("[reference]"). The authors should carefully review the manuscript to ensure that all references, citations, and cross-references are correctly formatted.
2. The interpretation of regional differentiation should remain somewhat cautious. Although the PCA analysis provides useful exploratory information and strengthens the discussion, the number of analyzed samples is relatively limited. Therefore, some statements related to regional characterization and geographical differentiation could be moderated to avoid overinterpretation of the results.

Overall, the authors have satisfactorily addressed the major concerns raised during the previous review round. Following correction of the remaining minor issues, the manuscript can be considered suitable for publication.

 

Report 3

After carefully reviewing the marked-up manuscript, I realized that I had overlooked the fact that the previously noted reference issues were shown as deleted text in the tracked-changes version. I apologize for this oversight.

Having reviewed the revised manuscript, I can confirm that my previous comments have been adequately addressed, and I have no further comments.

In my opinion, the manuscript is suitable for publication in its current form.

Author Response

see attachement

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

I have carefully evaluated the revised manuscript and would like to commend the authors for the substantial improvements made in response to the previous review comments. The manuscript has been significantly strengthened through the clearer articulation of its novelty, the inclusion of Principal Component Analysis (PCA), the improved discussion of methodological limitations, the moderation of conclusions regarding supply-chain interpretation, and the addition of important methodological details that enhance reproducibility.

The revised version now provides a more balanced interpretation of the findings and appropriately acknowledges the limitations associated with sample size, geographical coverage, botanical origin, traceability, and the inability of AOAC 998.12 to detect C3 sugar adulteration.

The study presents valuable baseline isotopic data for Ghanaian honey and demonstrates the usefulness of a multi-isotope approach for honey authenticity assessment and regional characterization.

At this stage, I consider that the scientific concerns raised during the previous review round have been satisfactorily addressed. Only a few minor editorial issues remain, including the correction of residual reference formatting errors (e.g., “Error! Reference source not found”), final language polishing, and verification of formatting consistency throughout the manuscript.

Therefore, I recommend acceptance of the manuscript after minor editorial corrections.

I congratulate the authors on their work and believe that the manuscript will make a useful contribution to the literature on food authenticity and isotope-based authentication methods.

Report 3

I have checked the latest version of the manuscript and the revisions made to the reference section. The formatting issue has been appropriately resolved, and the additional modifications appear satisfactory.

At this stage, I have no further comments regarding the references.

 

Author Response

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Author Response File: Author Response.pdf