Mapping Iodine Sources for Human Nutrition in Portugal Considering Geography, Seasonality, and Processing: Milk and Plant-Based Milk Alternatives

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

A topic of significant health and political importance is the issue of iodine deficiency and the changing sources of iodine supply, which is crucial in the context of public health.

Broad market sampling: collecting samples from major retail chains increases the representativeness of the study.

Use of a classic analytical method: the Sandell–Kolthoff method, following appropriate digestion, is the reference method, although it requires validation.

Consideration of differentiating factors: region, season, type of milk, and technological process.

Incomplete description of method validation:
Data on repeatability, standard deviation, limit of detection (LOD), and limit of quantification (LOQ) are missing.
The authors refer to previous work on this method, but in the original publication, such data should be provided directly for each series of measurements.
It is not clear whether reference materials (certified control samples) were used.

Inaccurate description of the sampling method:
The statement “one unit of each product” is insufficient—there is no information on the number of replicates, batch numbers, or expiration dates.
It is unclear whether products of the same brand but with different production dates were treated as independent samples.
This makes it impossible to assess inter-batch variability, a key aspect in the analysis of commercial foods.

The authors use parametric tests (t-tests) despite clearly skewed distributions, as evidenced by large differences in standard deviations and medians in the tables.
There is a lack of data on repeatability, standard deviation, limit of detection (LOD), and limit of quantification (LOQ). 
The authors refer to earlier work on this method, but in the original publication, such data should be provided directly for each series of measurements. 
It is not stated whether reference materials (certified control samples) were used. 
The description of the sampling method is inadequate: 
The statement ‘one unit of each product’ is insufficient, as there is no information on the number of replicates, batch numbers, or expiry dates. 
It is unclear whether products of the same brand but with different production dates were treated as independent samples. 
This omission makes it impossible to assess inter-batch variability, which is a key aspect in the analysis of commercial foods. 
Statistics and data analysis: 
The authors use parametric tests (t-tests) despite clearly skewed distributions, as indicated by large differences between standard deviations and medians in the tables. 
There is no regression analysis or multivariate modelling to identify independent predictors of iodine content (e.g. region, type of production, processing). 
Effect sizes are reported, but without interpretation of their biological significance. 
There is a lack of standardisation in comparisons between groups: 
Comparisons between seasons or regions did not account for differences in milk types (e.g. proportion of organic products).

This may lead to incorrect conclusions regarding the absence of seasonal differences. The authors state that the lack of differences between seasons is due to ‘maintaining stable cattle feeding practices’. This claim is unsupported, as no data on feed were collected.

An alternative explanation could be the lack of sensitivity of the method or an insufficient number of comparable samples between seasons.

Overly simplistic conclusions about the role of milk:
Estimates of milk’s contribution to iodine intake are based on data from nutrition surveys conducted ten years ago (IAN-AF 2015–2016), which raises questions about their current relevance. No analysis of the uncertainty of these estimates has been performed.

Lack of comparative analysis for plant-based drinks:
The authors do not statistically distinguish between fortified and non-fortified products in their analysis; they provide only global averages. This is a significant error, as this distinction is of greatest practical importance.

Overly superficial discussion of regional factors:
The observation of lower iodine content in milk from the Azores and southern Portugal is noteworthy, but a geochemical interpretation is missing. The authors do not refer to data on soil composition, water, or feed characteristics, so the conclusion remains speculative.

The abstract is verbose and does not clearly state the study's purpose. I recommend reducing its length by one third and explicitly stating the purpose, method, main results, and practical conclusion.

Introduction: The section on the WHO and supplementation in women can be shortened, as it digresses from the main aim of the study.

Methods: The calculation method for effect size (η², d) and the interpretation thresholds should be clearly specified.

Discussion: It is advisable to relate the results more directly to practical applications. For example, could the introduction of mandatory iodine fortification of plant-based beverages realistically improve iodine status?

The technical language should be improved in several places. For example, ‘milk presented considerable iodine’ should be revised to ‘cow’s milk contained moderate-to-high iodine levels’.

The authors declare some limitations, but not sufficiently. In this version, the following should be done:

1. Clearly indicate that the lack of data on feed, soil and water limits the interpretation of the sources of regional differences.
2. Point out that the data only refer to retail products — they do not include raw milk, fermented milk or processed products.
3. Emphasise that iodine content does not equal its bioavailability.
4. Make it clear that the data on milk consumption is almost a decade old, which significantly underestimates the relevance of the estimates.

The study addresses an important topic, but its analytical credibility and interpretative power are limited by its simplified methodology and insufficient reporting of results.

The article has potential, but requires significant refinement in order for the results to make a real contribution to the scientific literature and serve as a basis for health policy.

The article may be considered for publication after significant methodological, editorial and interpretative revisions.

Key recommendations for authors:

1. Add complete method validation data (LOD, LOQ, RSD, control materials).
2. Reorganise the methods section: clearly specify the number of samples, how they were selected and their repeatability.
3. Separate the analysis of fortified and non-fortified beverages.
4. Correct tables and units; introduce consistency in formatting.
5. Introduce multivariate analysis (e.g. ANOVA or regression) instead of only non-parametric tests.
6. Update references on milk consumption and iodine requirements.
7. Shorten the discussion and focus on the interpretation of results rather than repetitions of the literature.

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

The MS by Machado et al describe the iodine sources for human nutrition in Portugal: milk and
plant-based milk alternatives.

In the Materials and Methods section, the authors state that iodine concentration in milk samples was determined according to previously published methods, citing Sandell & Kolthoff (1937) and Machado et al. (2017). However, both of these references refer to the determination of iodine in urine samples, not in milk or food matrices.

The original Sandell–Kolthoff (1937) paper describes a catalytic colorimetric method based on the reduction of Ce(IV) by As(III), but it does not involve ammonium persulfate digestion. The later study by Machado et al. (2017) indeed incorporates ammonium persulfate oxidation, yet it is explicitly optimized for urine samples, addressing matrix-specific interferences relevant to biological fluids.

Therefore, the manuscript lacks a clear and adequate description or citation of a validated analytical procedure for iodine determination in milk samples.I recommend that the authors should either (a) provide a detailed description of the digestion and analytical steps actually applied to milk samples, or (b) cite a method specifically developed or validated for iodine determination in milk.

In the Abstract, the statement "Milk is potentially one of the major contributors for human iodine intake in most countries, including Portugal" is an overgeneralization.

The contribution of milk to dietary iodine intake varies markedly across regions and depends on multiple factors such as the use of iodized feed, the prevalence of iodized salt consumption, and dietary habits. While milk can be a major iodine source in some countries, this may not apply to others, where seafood and iodized salt play a more substantial role in total iodine intake.

It is suggested that the authors revise this statement to reflect regional variability.

  In Table 1 and 3,  the header "Iodine content (μg/100 mL)" is not clearly aligned,  making it ambiguous whether the unit refers to the column "N" or to "Mean, SD." This lack of alignment may confuse the reader. It is recommended that the tables be reformatted so that the unit is explicitly associated with the "Mean, SD" column, thereby improving clarity and accuracy in data presentation. 

 Τable 2  lacks a header indicating the unit of measurement for iodine content (e.g., "Iodine content (μg/100 mL)"), which is essential for data interpretation and consistency.  It is recommended to include the appropriate unit label in the column header to ensure clarity and uniformity across tables.  

 Table 5 needs to Table 5 appears to be split in the middle, and it is unclear why this is the case. Additionally, the column headers are truncated with hyphens (e.g., "vita-min"), which is not ideal. Finally, summing the numbers (N) in each row does not correspond to the values shown in the "Number of products" column.  These issues contribute to a serious problem in clarity and accuracy in data presentation.

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Reviewer 3 Report

Comments and Suggestions for Authors

Dear Authors

The paper “Mapping iodine sources for human nutrition in Portugal: milk and plant-based milk alternatives Interpretation depth” is extremely important for Public Health and provides a high impact directly relevant to nutrition policy and sustainable dietary transitions. Excellent balance and objectivity.

Some minor suggestions could improve the clarity and flow.

Find the detailed suggestions in the file

Comments for author File: Comments.pdf

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The article addresses an important public health issue: the assessment of iodine sources in the Portuguese diet in the context of dietary changes, specifically the increased consumption of plant-based drinks at the expense of milk. The topic is of social, health, and nutritional importance, particularly given the growing trend towards plant-based diets and the associated risk of iodine deficiency.

It would also be worthwhile to relate the results more broadly to European iodine deficiency prevention programmes and to emphasise their practical significance for health policy.
The authors have justified the importance of the topic well but have not fully developed the potential of the work into practical recommendations for public health.
The objective of the study is clearly stated: to determine the iodine content in milk and plant-based drinks in Portugal, taking into account seasonality and technological processing.
However, there is no clear research question or working hypothesis, which makes it difficult to assess whether the methodology fully corresponds to the objective.
In future studies, it would be useful to clarify the objectives in the form of measurable research questions and hypotheses (e.g. ‘Are plant-based drinks a significant source of iodine compared to cow's milk?’).
The title is correct, unambiguous, and appropriate to the scope of the work. It reflects the main topic of the study (comparison of milk and its plant-based alternatives in terms of iodine content).
However, it could be clarified further regarding seasonal variation or its significance for public health, which would increase its precision and citation potential.
The article follows the correct IMRAD structure (Introduction, Methods, Results, Discussion) and is logical and clear.
The results are presented reliably, but the discussion section needs to be expanded and should refer more strongly to European studies (e.g. comparisons with countries with similar dietary patterns).
The discussion lacks consideration of limitations regarding potential measurement errors or differences between commercial brands.
The final conclusions are too general and do not follow directly from the statistical data.

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors The authors do not clearly and in detail report the laboratory method they used.

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Round 3

Reviewer 2 Report

Comments and Suggestions for Authors

While the digestion conditions described by the authors are consistent with published Sandell–Kolthoff protocols for urine, the approach used for milk typically requires different—often stronger—digestion or ashing procedures. In the Machado et al. studies cited by the authors, ammonium persulfate digestion was validated specifically for urine matrices; these papers do not describe or validate the method for milk or dairy products. Because milk contains higher levels of fat, protein, and organically bound iodine, it presents greater analytical challenges compared to urine. For this reason, published Sandell–Kolthoff methods for milk commonly rely on acidic or more intensive digestion protocols, such as the preparation of an acidic digestion reagent (e.g., ammonium metavanadate dissolved in perchloric acid), or other matrix-appropriate pre-treatments.
Two relevant examples demonstrating the distinct digestion requirements for milk include:1. Hedayati M, Ordookhani A, Daneshpour MS, Azizi F. Rapid acid digestion and simple microplate method for milk iodine determination. J Clin Lab Anal. 2007;21(5):286-92. doi: 10.1002/jcla.20185. PMID: 17847102; PMCID: PMC6649143. 2. Vance KA, Makhmudov A, Shakirova G, Roenfanz H, Jones RL, Caldwell KL. Determination of iodine content in dairy products by inductively coupled plasma mass spectrometry. At Spectrosc. 2018 May 1;39(3):95-102.

Author Response

Please see the attachment.

Author Response File: Author Response.docx