PONE-D-24-58553Assessment of decadal changes in coastal nitrogen sources in NW Spain with stable isotopes in macroalgae and musselsPLOS ONE

Dear Dr. Bode,

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Academic Editor

PLOS ONE

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Additional Editor Comments:

Comments to the authors

Since as a handling editor I had difficult times in getting your manuscript reviewed I took the liberty to do it my self being from the similar field. In addition to the comments received I have some more observations which I mention below. The authors are encouraged to go through.

The authors try to understand the relationships between the nitrogen uptake through N15 isotope (during the upwelling period) on macroalgae and the mussel. The data is collected over the years during the upwelling period.

My first question is both the experimental species are located in near shore/intertidal. One is a filter feeder and other I guess is attached to the substrata in very shallow waters. Whether the sampling location is affected by the upwelled waters.? How does authors prove this? This is not reflected by the salinity or temperature although some figures are given? The location is right next to shore so whether the hypothesis that upwelled might influence nitrate uptake is flawed? The authors need to justify. Why not change the orientation of the paper to “N15 isotope assimilation by intertidal species”?. Upwelling could be discussed as a paragraph in the discussion. Probably that’s the reason there is no change observed! Mussel responds strongly to calcium carbonate saturation and temperature fluctuation! The authors are encouraged to make a plot of showing the spatial extent of upwelling for any particular year to substantiate their scientific hypothesis. Also encouraged to plot the actual nitrate values from the location during monsoon and non-monsoon to show if any influence? A table showing a comparison of N15 assimilation by the (Mytilus galloprovincialis) from different other geographical area and for the macro algae can be added along with some discussion.

With the above suggestion the authors are encouraged to revise the present manuscript….

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: I Don't Know

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Reviewer #1: No

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Reviewer #1: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1:  The article being reviewed presents solid and well-structured research, with effective use of environmental data and isotopic analysis to assess changes in coastal nitrogen sources. The analysis covers a long period (1989–2023), allowing for the identification of possible long-term trends. However, some critical issues were noted regarding the description and clarity of the materials and methods section. Although the key concepts and main tools are described, essential details are missing, which may hinder the study’s reproducibility.

Several key issues were identified, including:

1. Lack of details on sampling and sample processing protocols.

o Rows 138–142: It is not clearly specified whether previous studies have used Bifurcaria bifurcata as a biomonitor and whether it could replace Fucus in this role.

o Row 151 mentions sampling a portion of the mussels adductor muscle but does not specify the exact quantity. Does this refer to a specific weight (grams) or length (centimetres), or is the quantity irrelevant?

2. Insufficient description of sample preservation. What measures were taken to prevent biological degradation after sampling?

3. Incomplete documentation of laboratory instruments and procedures.

o Row 121: It is recommended to specify exactly what the CTD is, including its model and parameters.

o Rows 151–153: The rationale behind the choice of drying or freeze-drying some samples is unclear. Was an existing protocol followed? If so, please specify which one. Otherwise, all steps leading to the preparation of samples for isotopic analysis should be described.

o Rows 142–143: If nitrogen isotopic measurements were extracted from the Environmental Specimen Bank of Galicia, it would be appropriate to specify exactly what type of information was selected. Were filters or other selection criteria used? Additionally, providing a link to this database would enhance transparency.

o Rows 149–150: It is not specified whether previous studies have observed correlations between isotopic signatures and shell size.

It is recommended that the materials and methods section be rewritten in a more comprehensive manner, as the current version lacks essential information needed to ensure reproducibility. If including all details in the main text would be overly cumbersome, they could be provided in tables, appendices, or supplementary materials, but they must be made available.

The quality of the figures is generally good, but some aspects could be improved to enhance readability and accessibility:

• Figure 1: Although latitude and longitude references are provided, the figure lacks a metric scale, which would aid in understanding the depicted distances. It is also suggested to use a colour other than black to indicate algae sampling areas, as this is difficult to distinguish from the land outline. Additionally, using a thicker symbol for Mytilus would improve visibility.

• To enhance accessibility, it is recommended to use a colour-blind-friendly palette, such as the one proposed by Wong, B. (2011). Points of View: Colour Blindness. Nat Methods, 8(441). https://doi.org/10.1038/nmeth.1618

It is advised that the authors carefully check the formatting of the article. For example:

• Row 738: 15N should be formatted as a superscript.

• Citations: References [19], [20], and [21] appear before [16], while [17] and [18] are missing. It is possible that in line 48, the citation indicated as [15, 19] should actually be [15–19].

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Reviewer #1: No

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Assessment of decadal changes in coastal nitrogen sources in NW Spain with stable isotopes in macroalgae and mussels

PONE-D-24-58553R1

Dear Dr. Bode,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Kind regards,

Rajdeep Roy

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Dear Dr. Bode

I am happy to say that you have made necessary corrections. The response to the questions raised by both the reviewers have been answered in a logical and scientific way. The manuscript has improved greatly. This is good contribution in the filed of oceanography.

Kind Regards

Academic Editor

Reviewers' comments: