PONE-D-19-26525

Impact of zooplankton grazing on lipid biomarker distributions in the stratified Eastern Gotland Basin (Central Baltic Sea)

PLOS ONE

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Reviewers' comments:

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Comments to the Author

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Reviewer #1: Partly

Reviewer #2: Yes

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

Reviewer #1: N/A

Reviewer #2: N/A

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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5. Review Comments to the Author

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Reviewer #1: While the data quality is good and the manuscript well written, I found some major weak points in their arguments. The manuscript would require major revision before acceptance for publication.

Major comments

1. The author did not present their abundance data (both organisms and carbon content) relative to per unit volume of water.

- On Line 27, they wrote “very high cell counts…”. Cell counts are typically presented in the unit of individuals per unit volume of sample. I did not see any such data in the manuscript; the data presented were all relative proportions.

- Such data, including the abundance of phytoplankton and zooplankton per unit volume of water or the particulate organic carbon (POC) content per unit volume of water, are useful because these are the types of data commonly reported in oceanographic literature. Such information would allow the reader to place the results in a broader environmental context.

- While the discussion on the relationship between 14:0 n-alcohol and 14:0 FA is interesting (Line 358-380), I would like to see quantitative assessment that takes the zooplankton lipid export and sediment lipid influx into account. Ideally, such calculation should make use of water-column particle or POC flux data and sedimentation rate data. I do not think their comparison based on μg lipid/g Corg is correct, as most of the zooplankton biomass (Corg) is possibly degraded in the water column.

2. Their title “Impact of zooplankton grazing on lipid biomarker distributions in the stratified Eastern Gotland Basin” is misleading.

- When seeing such a title, most of the reader would expect a process-oriented study that examines which lipids are consumed or transformed by zooplankton digestion, which stay unaltered, and what might be the enrichment or depletion factors for different lipids.

- Discussion in the aforementioned direction was inadequate in the manuscript. Their data did show that labile cyanobacterial markers can be found in the shallow but not the deep zooplankton sample, indicating cycling of these markers (mainly polyunsaturated FA) in the upper water column. However, for the markers that are present both in the phytoplankton and surface sediment sample but not the zooplankton samples (i.e., 7-methylheptadecane), some further explanation is needed. This is crucial in that methylheptadecanes are “useful” biomarkers for paleoceanographic studies. I wonder how much (weight) zooplankton material was used for lipid extraction; perhaps a higher amount of material could improve the detection of methylheptadecanes in the samples.

- The same issue actually applies to those terrestrial biomarkers. These biomarkers are abundant in the surface sediment – is zooplankton grazing involved in the enrichment (active transport)? Or do these lipids settle together with other clastic material (passive transport), without passing the guts of the zooplankton? Again, I wonder if a higher amount of extraction material will help the authors to detect these compounds in zooplankton samples.

- I also hope that the authors can reorganize their discussion to make the manuscript more interesting. Instead of discussing different lipid groups, they can talk about different processes, such as “autochtonous lipids recycled in the water column”, “autochtonous lipids reaching seafloor via active transport”, “autochtonous lipids reaching seafloor via active transport”, etc.

- If the authors have no intention to add additional data to match their title, I suggest that they should use a different title.

Minor comments

L35: “time scale of decades”: I did not see any description/discussion related to time scale in the main text

L66: EGB: spell out the name

L77: Remove “;”

L113-119: Specify the mesh size of the Aptein-net

L130-134: Specify the filtered water volume. I wonder if an inadequate volume of filtered water resulted in the low lipid diversity on the filter

L145: Specify the weight of samples used for extraction

L185: The Chl a content in the upper mixed layer did not seem to reach 1.5 μg/L

L188: To me the salinity seems to reach 7 PSU

L214, L275 (and other places): “57 % PLFA”: percentage of what? “> 1 % of the total”: total of what?

L218: “the latter samples”: specify which samples are referred to

L231: replace “except” with “but not”

L238-239: What is the relationship between the statements “no separation between NL and PLFA was made” and “it can be expected that degradation processes and (proto-) kerogen formation would have influenced the biological distributions”?

L244-245: Change “… it was much less abundant (< 5 %) in the water column samples” to “… its proportion was much lower (< 5 %) in …”

L265: Which scientific name in the preceding sentence does “sitosterol” refer to?

L290-291: Report the concentration of 7-methylheptadecane in the topmost sediment

L314: Add “,” after “…(~15%)”

L320: Remove “still”

L322-327: The issue of 18:1ω9: Isotopic measurements of this lipid may help to quantify the relative contribution of different sources and reduce the level of speculation

L358: Change “amounts” to “concentrations”

L385: This does not seem to be a good header. Should we expect to see “surface water cyanobacteria” in sediment?

L421: Do you mean Fig. 8 (instead of Fig. 6)?

L427-432: The argument here seems somehow disconnected from the preceding text. The cited paper [49] is an arctic research; is the result applicable to other area? The sentence “phytoplankton and zooplankton are mainly degraded during summer by photodegradation in the upper water column” sounds counter-intuitive; didn’t the authors just show us rapid biological lipid cycling in the upper water column?

Fig. 5 (and supplementary tables): Specify the sampling depth of Filter sample

Fig. 7: Delimit C27, C28… sterols (in analogy to Fig. 8)

Fig. 7, 8: Provide common name of the sterols (if applicable) for easier cross-referencing in the text

Fig. 8: Sterol/tetrahymanol data of the depth 3-4 cm look strange. Why did the major lipid 24-ethylcholest-5-en-3β-ol disappear in this layer?

Table S1, S3, S5: Values of the Filter sample are unclear

Table S3, S4: Provide common name of the sterols (if applicable) for easier cross-referencing in the text

Reviewer #2: The authors present data of lipid distributions in the Eastern Gotland Basin and study how these biomarker signals are transformed during/after deposition in this stratified and partly anoxic environment. For this purpose, samples enriched in the main biomass producers phyto- and zooplankton were analyzed for the lipids fatty acids, n-alcohols, and sterols, and compared to samples from the underlying sediment. In contrast to the expectation that the lipids of primary producers are predominantly found in surface sediments deposited under (partly) anoxic conditions, the data instead show that transformed remnants of wax esters derived from zooplankton are the dominating biomarkers. Deeper sediment layers are mostly comprised of biomarkers from terrestrial plants, indicating that grazing of zooplankton strongly changes the composition of the primary producer-derived lipids during and after deposition, leading to unexpected results.

The manuscript is well-written and all experiments are described in sufficient detail. Figures and Tables follow a clear layout and all data is made available in the Supplementary Information. Previous literature is fairly treated and the experimental data support the conclusions that the authors draw. I do not have any major criticism of any kind, this was a pleasure to read – well done!

Detailed comments

L66: introduce EGB here, readers who have not read the abstract first will not know the abbreviation

L120: typo: “homogenous”

L148-154: Was the efficiency/recovery of the column chromatography tested? I suggest to rename the “phospholipid fraction” into “polar lipid fraction” to also account for other types of polar lipids (glyco, amino, etc.) which might be eluted (cf. Heinzelmann et al., 2014, AEM; “Critical Assessment of Glyco- and Phospholipid Separation by Using Silica Chromatography”).

L222: perhaps change to „virtually absent”

L265: check assignment of “sitosterol” to chemical formula

Table S4: use decimal point in stanol/stenol ratios

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Reviewer #1: No

Reviewer #2: No

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Zooplankton impact on lipid biomarkers in water column vs. surface sediments of the stratified eastern Gotland Bain (Central Baltic Sea)

PONE-D-19-26525R1

Dear Dr. Wittenborn,

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Academic Editor

PLOS ONE