Review Reports

Round 1

Reviewer 1 Report

 

The authors have presented a very interesting research. However, some suggestions/ corrections are required before considering for publication.

 

All English language should be modified before next revision.

 

The authors only use the 16 Tufted Titmices, to divide into two groups. The number of samples are too less in scientific research, and the differences between them are also not marked, such as body length, injuries, gender, etc.

 

Importantly, the sections of result and discussion are required to remove redundant and useless content to clarity what the author’s want to express, and then to analyze what the reasons in your study. How to make sure your data is accurate in the case of feather replacement.

Author Response

Thank you for the review. I took advantage of one of the other reviewer's suggestions on my grammar, so I hope you will find my English better. I am a native English-speaker, so I was surprised to know that I needed significant improvement.

I appreciate your concern about the small sample size. Fortunately, the statistical tests take into account sample size, so I am confident in the results with regards to feather Mn. I tested the distributions to ensure they met the assumption of normality. See lines 234, 265-268. I also mentioned the small sample size as a weakness of our study (lines 293-296). The bird species is sexually monomorphic, so we were not able to assign sex (line 108). Estimating bird age is difficult and we did not have that skill so we did not do it. We agree this would have been useful and mention that at lines 348-355. We had already tested to see whether body mass index was different between our groups, but on your suggestion, we also compared the linear measurement of head+bill length (lines 238-239).

Upon suggestion of other reviewers, we removed selected passages that were redundant.

You mentioned a concern that our data might not be accurate due to feather replacement. Fortunately, that concern should be minimal because all the feathers were collected in early fall (lines 178-180), which means that all feathers were freshly grown from the latest molt. We inserted more information on feather molt in the Introduction to ensure this is understood by other readers (lines 82-91). If a feather had been lost, it takes about 30 days to grow back. We would have noticed and avoided any feather that was still in the growing process.

Reviewer 2 Report

General comments:

The manuscript entitled “Manganese concentrations in Tufted Titmouse feathers are highest near a metal processing plant” represents original, well structured, and informative study about the possibility of using bird feathers as bioindicators of Mn environmental contamination. The experimental design, sample size, methods used, and literature overview given are comprehensive. In my opinion, the manuscript can be published in the journal “Birds” after several specific issues are addressed and corrected.

Specific comments:

line 22: in bird chiks?

lines 22-23: I suggest rephrasing the sentence “We studied whether concentrations of Mn in feathers would correlate with the distance the sampling sites were from point-source emitters of Mn into the air”

to  “In this paper, we studied whether concentrations of Mn in birds feathers correlate with the distance from the point-source of Mn air emissions”

line 27: microwave plasma-atomic emission spectrometer (ICP-AES)

lines 27-29 correct this part “we found tail feathers collected  from the site closest to a steel plant had a significantly greater concentration of Mn than tail feathers collected at the site more distance to a plant”

to “we found  that tail feathers collected  from the closest distance to a steel plant had significantly higher concentration of Mn compared to the samples from the site with a longer distance to the emission source”

lines 30-32: I suggest dividing this sentence into two such as:

This is the first published study of Mn sequestration in Tufted Titmouse feathers. This study develops our general understanding in the potential for the use of birds feathers as non-invasive bioindicators of environmental metal exposure.

lines 33-34: I suggest modifying the set of key words. First, key words section and the title should not repeat each other, and there shouldn’t be repeated words in the “key words” section itself. For that reason, I suggest to remove from the list the common name of a studied species and leave only Latin name. For the same reason I suggest to remove manganese. Can the “key words” section be modified such as:

Keywords: Baeolophus bicolor,  bioindicators, feather analysis,  metal bioaccumulation, steel production, spectrometry.

Line 37: It is generally suggested to keep the formatting style such as the first section of the text is without an indent.

line 37: It would be more interesting if you will give one more sentence with the examples which birds people used? How? (in brief)

line 38: Mn itself isn’t an hazard, however it’s release or over production can be. Correct a logical flow here.

line 43: near mines and factories

lines 37-51: this paragraph is generally nicely written but you overuse the word “can”, check every sentence and delete it where it is unnecessary. In the scientific texts, it is better to be accurate with uncertain words “can”, “may”. So, state clearly where it is possible.

line 52: studies in human showed that…. Mn can’t alter something in studies but in humans. Then, you should say the studies showed that or say “ In humans, Mn alters…”

line 61: you don’t need to start a new paragraph here, combine with the previous one

line 70: our current study

lines 75-86: this information refers to the Material and Methods section, it says about sampling size. Please, transfer it to the next section

line 122: balances

For the materials and methods section could you please make sub-sections? This will improve readability of the text.

2.1. Birds trapping and feather sampling (lines 75-86; 93-107)

2.2. Birds morphometrics (lines 108-117)

2.3. Feather preprocessing (118-125)

2.4. Microwave plasma-atomic emission spectrometer (ICP-AES) analyses lines 125-135)

2.5. Statistical analyses (lines 135-146)

Did you mention somewhere the total number of your sample? How many birds were used, was it 16?

lines 189-190: is it generally better to collect tail feathers? Is this less destructive to the birds?

line 192: Correct the first sentence to “Our results demonstrate that tail feathers of Tufted Titmice accumulate and store Mn.”

Lines 193-195: specify your treatments, this needed to remind your readers what were those places.

Bridgeville (location close to Mn exposure site)  Latrobe (location at a longer distance from the exposure site)

lines 196-198: Were your documented values of Mn exposure about the same rates? Were the emission rates the same or comparable? Report this information somewhere? You stated in the abstract that the exposure is known. Try to search in the literature and speculate here about the possible exposure pathways. Via food? What previous studies about metal accumulation in birds say about the exposure pathways?

Check this paper https://www.sciencedirect.com/science/article/pii/S0013935122009021

It might be a very relevant reference and it provides an overview not only about aquatic bird. They also study Mn

line 226: correct to “ might contribute to these differences”.

line 227: which author? Specify here

lines 228-229: probably you can move this sentence to previous paragraph where you say that you don’t know the pathways of exposure (see my comment for the lines 196-198)- Logically, you can give your prediction and then set the paragraph describing the current knowledge of different bird species

lines 231-239: very interesting paragraph! Indeed, that can be. Do you know about average flying distances of this bird species? That might be essential information. You can also recommend to consider age and flying distances for future similar studies? If, of course, there is a reliable and non-invasive way to study the bird’s age.

line 241: here, I suggest substituting BMI for body mass, it can make a paper more reader-friendly

lines 247-262: I have a strong feeling that these two paragraphs fit much better to the introduction part. You can allocate this part somewhere around lines 52-59. Check your self, but it might fit better. In the discussion, I suggest to discuss more your interesting findings and the potential of developing non-invasive bioindicators.

Check this paper https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1566746/

It might sparkle good ideas for your conclusion.

Make your last sentence stronger and more convincing!

E.g. correct “Nevertheless, our study shows that Mn is in the environment, and there may be unforeseen consequences up and down the food chain”.

to

“Our results clearly demonstrate that Mn is accumulated in birds tail feathers, which can be used as non-invasive bioindicators of metal contamination. Presence of hazardous elements in the environment might possess unforeseen consequences for the food chains and should be constantly monitored”.

 

For the reference list it might be good either provide DOI for all references, or not provide them at all.

 

Your bird species is cute! If it is possible, can you add its picture somewhere? (This isn’t mandatory, but will give visible). The improvement of non-invasive methods is very needed.

Thank you for your interesting and nicely written paper!

 

Author Response

Dear reviewer, your comments were extraordinarily helpful; thank you. The first author on our paper was an undergraduate, and this is her thesis research. She and I continued to work together on the revisions, and it's been a good learning experience. I'm wondering if maybe you could tell we were inexperienced, and wanted to help in a constructive way. In any case, you get a gold star! Here are my comments on your suggestions:

old line 22: yes, these were bird chicks, but I changed this sentence and no longer reference chicks.

Lines 22-23: I changed this per your suggestion.

Lines 27-29: I changed this per your suggestion

Lines 30-32: I changed this per your suggestion

Lines 33-34: I took your advice, plus added one more keyword: biomonitoring.

Line 37: I checked the other articles in Birds and did not see paragraph indents different from ours, so I left this be.

Line 37: I took your suggestion and added reference to Silent Spring and to canaries in the mine. Maybe these are too literal and non-academic, but they are certainly well-known.

Old Line 38: I changed "hazard" to "byproduct." (see line 41)

Old line 43: corrected (now line 46)

Old lines 37-51: I cleaned this up so that I only use the words "can" or "may" three times in this paragraph, instead of the original six. As I made other textual modifications, I kept my word choices in mind.

Old Line 52: now deleted. Another reviewer wanted us to minimize reference to human studies.

Line 61: fixed

Old Line 70: fixed (now line 93).

Lines 75-86:  details on the Tufted Titmouse molt have now been moved to Methods (lines 113-118), and details on the study sites and their location in reference to steel plants was expanded on advice of the editor and placed in Methods (lines 119-164).

old line 122: We only used one balance, so we kept this word singular and not plural. (new line 201).

Methods: I made subsections as you and the editor recommended.

Methods: yes, the sample size was 16 birds. This was inferred in the earlier manuscript by adding eight and eight, but I made it more explicit now at line 178 and again at line 234.

Lines 189-190: I don't think tail or breast feather collection is destructive, as it is used in many studies and the feathers grow back. But since breast feathers are smaller and not used in flight at all, perhaps breast feathers are a better choice.

Old Line 192: I moved this to Line 298 but kept your suggested phrasing.

Old Lines 193-195: I made this change; see lines 300-301.

Old Lines 196-198: With your suggestion and that of another reviewer, we inserted this information at lines 138-164. You made a great point that we overlooked. In doing the extra research we found that not only were there two steel plants in Latrobe; there were three. The combined emissions of Mn were much higher in Latrobe than in Bridgeville, and this surprised us. This fact could have impacted our interpretation of results, but it turns out that prevailing wind direction matters a great deal when it comes to exposure to a point source. Fortunately for the design of our study, the Bridgeville bird feathers were collected directly down-wind from the factory there, and the Latrobe bird feathers were collected upwind of the three factories there. In this new revision, we emphasized wind direction just as much as distance between birds and the steel plants (see lines 145, 159-164, 344-347).

Also, you encouraged us to speculate on how the Mn got into the bird feathers based on previous studies. Our review of the literature shows that the exposure route is rarely known. We, like the previous authors, named ingestion of dust and  ingestion of contaminated food as the likely pathway. We also found a source that indicated that Mn does not accumulate in feathers at all [Borgheri et al., 2016], and that it is simply deposited on the feather surface from extrinsic environmental contact. This caused us to modify our word choice in places to allow for this possibility (compare old line 192 with new lines 298-299, and new lines 338-341); however, numerous sources published after 2016 have shown Mn does accumulate inside the feather structure (see lines 87-90, 315-317). We used the source you suggested; thank you.

Old line 226: fixed it. See line 337.

Old line 227: fixed it, see line 337.

Old lines 228-229: I moved this sentence to the start of the paragraph at line 315.

Old lines 231-239: I added the details you inquired about. See lines 349-355.

Old Line 241: fixed

Lines 247-262: Upon suggestion of the editor, this passage was deleted because it mentioned human health too much. 

Discussion: I made use of the reference you shared and used it as a starting point to write brand new concluding paragraphs (lines 356-378). You encouraged me to make the conclusion "stronger and more convincing," but the editor made the opposite suggestion, that I elaborate on our study's weaknesses more. I compromised and mixed in discussion of weaknesses throughout the Discussion (lines 293-296, 354-355, 373-374), but not in the last portion where you wanted the paper to be more convincing.

References: I gave the formatting job to my student and she really failed at it! I didn't realize how far off our references were from the journal's format. Fixed.

Thanks again for the review. I hope we've improved our work.

Reviewer 3 Report

This article is about manganese concentrations in feathers collected from tufted titmouse. I enjoyed reading the article, and I think the data included in it is essential for future research on this topic. The study is more of a preliminary study. I would like to read a more advanced study on this topic on a larger group of birds. 

Minor revision needed:

line 79 - 86 - this should be in the M&M section

line 252 - delete space bar before [9].

Author Response

Thank you for agreeing to review our paper. We made the two changes you suggested. The text in old lines 79-86 has now been moved to Methods where we added a whole subsection on the study sites.

Round 2

Reviewer 2 Report

The paper has been significantly improved! 

It can be published after several minor issues are addressed.

line 28: a spare space before "from the site"

line 29: set of birds, however

line 31: extra space

lines 34-35: key words should be in the alphabetic order 

lines 107-117: something had happen with the paragraph formatting

line 193: extra space 

line 214: extra space

line 296: nonnecessary space between paragraphs 

 

Please, do double check the layout and formatting of your manuscript. The work is important and interesting, and the presentation should be appropriate. 

Author Response

Dear reviewer, I made all the requested edits to the manuscript. Thank you very much for your time and effort to improve it.