Contrasting seasonal patterns in particle aggregation and DOM transformation in a sub-Arctic fjord

Digernes, Maria G.; Bodur, Yasemin V.; Amargant-Arumí, Martí; Müller, Oliver; Hawkes, Jeffrey A.; Kohler, Stephen G.; Dietrich, Ulrike; Reigstad, Marit; Paulsen, Maria Lund

doi:https://doi.org/10.5194/egusphere-2024-1314

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https://doi.org/10.5194/egusphere-2024-1314

Preprints

05 Jun 2024

| 05 Jun 2024

Contrasting seasonal patterns in particle aggregation and DOM transformation in a sub-Arctic fjord

Maria G. Digernes, Yasemin V. Bodur, Martí Amargant-Arumí, Oliver Müller, Jeffrey A. Hawkes, Stephen G. Kohler, Ulrike Dietrich, Marit Reigstad, and Maria Lund Paulsen

Abstract. Particulate (POM) and dissolved (DOM) organic matter in the ocean are important components of the Earth’s biogeochemical cycle and in constant dynamic change through physical and biochemical processes. However, they are mostly treated as two distinct entities, separated operationally by a filter. We studied the transition between the DOM and POM pools and its drivers in different seasons in a sub-Arctic fjord by monthly environmental sampling and performing aggregation-dissolution experiments. For the experiments, surface water (5 m) was either pre-filtered through a GF/F filter (0.7 µm), or left unfiltered, followed by 36 h incubations. Before and after the incubation, samples were collected for dissolved and particulate organic carbon concentrations (DOC, POC), microbial community (flow cytometry) and in-depth analysis of the molecular composition of DOM (HPLC-HRMS). During the biologically productive period, when environmental POC concentrations were high (April, June, September), the filtered water showed a rapid increase of POC concentrations by up to 88 % within 36 h, indicating net-aggregation processes. During this process in September, DOM lability decreased based on changes in average hydrogen saturation and aromaticity of DOM molecules. In contrast, during the winter period (December, February), when environmental POC concentrations were low, the experiments indicated a dissolution of POC with a net-loss up to 58 %. Simultaneously, the DOM pool became more labile during the incubation period indicated by changes in average hydrogen saturation, aromaticity, and oxygen saturation. In both periods, bacterial activity increased throughout the incubation, showing that bacterial degradation likely plays a role in the transformation of POM and DOM. Our data highlights the importance of both physically driven DOM aggregation and biologically driven POM dissolution during different periods of the year, together determining the fate of the OM pool in high latitude marine ecosystems.

Received: 06 May 2024 – Discussion started: 05 Jun 2024

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Maria G. Digernes, Yasemin V. Bodur, Martí Amargant-Arumí, Oliver Müller, Jeffrey A. Hawkes, Stephen G. Kohler, Ulrike Dietrich, Marit Reigstad, and Maria Lund Paulsen

Status: final response (author comments only)

RC1:
'Comment on egusphere-2024-1314', Anonymous Referee #1, 23 Jul 2024
The authors collected samples in a sub-Arctic fjord in different seasons and conducted incubation experiments to investigate changes in DOM and POM concentrations, DOM compositions and other metrics before and after incubations. While the study is interesting, there are two major concerns that need to be addressed before it can be considered for publication.
The authors attempted to interpret their experimental results in the context of POM and DOM transition, arguing that DOM aggregation and POM dissolution determine the fate of these organic matters. However, the results of the incubation experiments are not strong enough to support this interpretation because: 1) the trends of changes in POC and DOC concentrations during the incubation seem to be complex and they are not always having opposite trend. The authors also mentioned this in the section 5.3.2, and 2) the magnitudes of changes in POC and DOC concentrations are significantly different (for example,~ 2 μM changes in POC vs >200 μM changes in DOC in Sep incubation), making it difficult to compare these changes to draw conclusions about DOM aggregation and POM dissolution.

The authors also tried to compare the changes in DOM compositions before and after incubation. However, the analysis is questionable because the changes in metrics such as H/C, O/C, MW, and AI in Table 2 are small compared to the standard deviations. Most of the discussions are based on these changes, which are smaller than the errors. While the authors argue that these small changes are significant, they do not provide any statistical test reports to support this argument. The authors should conduct statistical analysis to support those small changes are still significant.

Major comments:
Lines 32-35: The sentence seems to imply that DOC cannot contribute to carbon sequestration, which is misleading. The authors should acknowledge that DOC can also contribute to carbon sequestration.
Line 44: The sources listed for DOM are incomplete. For example, zooplankton grazing can also release DOM from phytoplankton. I recommend referencing “Carlson, C. A., & Hansell, D. A. (2015). DOM sources, sinks, reactivity, and budgets. Biogeochemistry of marine dissolved organic matter, 65-126.”
Lines 50-55: The authors mention labile and semi-labile DOM but omit recalcitrant DOM, which has a turnover time of millennia. To be thorough and consistent with previous categorization of DOM based on lability, recalcitrant DOM should be included .
Line 145: I do not fully understand why using headlight with red light can reduce the possibility of biological production. Chlorophyll a absorbs red light.
Lines 253-254: Why pre-soaking SPE column sorbent with Methanol is needed ?
Line 259: The DOC recovery is highly variable. Could you explain why?
Line 280: Did the authors try other criteria for formula assignment, such as allowing nitrogen between 0 and 4 ?
Line 281: Why remove formulas which contained both nitrogen and sulfur, ¹³C and nitrogen or sulfur and ¹³C? Could the authors justify it?
Lines 395-400 and Figure 5: The changes in DOM parameters like H/C, ALmod, MW, and O/C are much smaller than the standard deviation. Although the authors state these small differences are statistically significant, statistical test results should be provided to justify this claim .

Minor comments:
Line 108: should be “characteristics” instead of “character”?
Line 131: (TPM and PIM)
Line 141: should be “every two months”?
Citation: https://doi.org/10.5194/egusphere-2024-1314-RC1
- AC2:
  'Reply on RC1', Maria Digernes, 06 Sep 2024
  We would like to thank the reviewer for their time and interest in our study.
  Please note: the responses to the reviewer comments are unfortunately swapped and we were not able to edit after submission. As a result, here are the responses to Reviewer #2 marked in bold and we refer Reviewer # 1 to the author response to Reviewer #2.
  Anonymous Referee #2
  This manuscript focuses on DOM-POM transformation in a sub-Arctic fjord in Norway. The authors use filtered and unfiltered incubations in different seasons to address changes in DOC and POC to understand aggregation and dissolution dynamics. The manuscript reads well, however below comments that need addressing.
  Comments:
  This is an interesting study and relevant research. However, the manuscript is lacking in statistical testing to support the presented results. Also the presented hypothesis should be clearly addressed at the end of the manuscript.
  
  We would also like to thank Reviewer #2 for their interest in our study and thorough comments. The response to point 2 in Reviewer #1 comments applies here as well: Regarding the changes in DOM metrics between sample treatment, we would like to clarify that the standard deviation calculations are done on the numerous compounds per sample. We have provided the standard error of the mean instead. The small standard error of mean (for each sample) and small standard error of difference between treatment means (for treatments t0 and t1) ensures that even small differences between sample averages are statistically significant.
  Following on the comment about statistical evidence, we performed a t-test to test our initial hypothesis; “biologically active periods with higher POM concentrations have a higher potential for aggregation of DOM via adsorption in comparison to the winter period”. We performed a t-test on the difference (t1-t0) in POC concentrations in winter vs productive period (the two groups “winter” and “productive” period had earlier been delineated by the SIMPROF test as shown in the manuscript). For the F treatments, this resulted in a significant (p= 0.04) difference in change of concentration between winter and the productive period. This leads us to accept our hypothesis, because we were interested in the seasonal aggregation potential of DOM, represented by the F treatments. For the UF treatments, however, this test was not significant (p= 0.34) which is likely due to aggregation, dissolution and biological processes taking place at the same time, which complicate the UF treatment results and its interpretation.
  Accordingly, we will add the following sentences to the manuscript:
  L294: “To test our main hypothesis (“biologically active periods with higher POM concentrations have a higher potential for aggregation of DOM via adsorption in comparison to the winter period”), we then performed a t-test on the difference (t1-t0) in experimental POC concentrations between the two biogeochemical periods delineated by the SIMPROF test (“winter” and “productive” period)”
  L362: “For the F treatment, a t-test revealed significant differences in the change of POC concentrations between winter and the productive period in the F treatment (p = 0.04); whereas this difference was not significant for the UF treatment (p>0.05).”
  L687: change of sentence to “our experiment demonstrates that transitions in the DOM–POM continuum are subject to contrasting seasonal conditions at high latitudes”
  L703: “Our results confirm our initial hypothesis where we postulated that the aggregation potential of DOM to POM is higher in the productive period compared to the winter period. Beyond that, we found that while DOM aggregation is dominant in the productive period, during winter POM seems to undergo dissolution.”
  I would also encourage the authors to add all the data to the supplement or to a data repository to benefit future research.
  
  We fully support making the data open access and have included DOIs for all datasets, which are now publicly available in a data repository. The code will also be made available with the publication along with mzXML files for running the DOM data analysis.
  Lines 101-102: There are other DOM-POM studies albeit from different environments, see e.g., Attermeyer et al. 2018 (Swedish rivers), Shakil et al 2020 (permafrost thaw streams in Canada), Keskitalo et al. 2022 (Kolyma River).
  
  We would like to thank the reviewer for bringing these studies to our attention and we plan to incorporate them into our revised manuscript.
  Lines 321-328: Scientific names of
  
  species should be in italics here and elsewhere.
  We agree and this has been fixed.
  Line 356-357: Could you clarify how there can be a decrease in POC in filtered waters where POC has been removed by filtering?
  
  Even after continuous refiltrations, POC can be measured in filtered water (Figure S1 and Valdes-Villaverde et al., 2020). This is due to the immediate self-assembly of EPS molecules in filtered water. Therefore, POC cannot be fully “removed” from filtered water and we explain the lower concentrations at t1 with a decrease of remnant POC in the sample. For clarification, we changed the sentence to the following: “In winter, (December and February), after 36 h of incubation, we measured lower POC concentrations in F water at t1 compared to t0 (a “decrease” of the mean POC concentration by −2.55 µM ± 0.8 (around -50% relative to t0)”.
  Lines 396-401: So was the statistical significance tested as the authors mention that the results are significant (after explaining about interpretation of signal intensity weighted averages)? It should be made easier for the reader to judge the significance of these results as the changes look so minor. I’ve read the explanation, but statistical testing could help to understand how meaningful the results are.
  
  We would like to refer the reviewer to answer in point 1.
  Line 441: Typo -> the end of the incubation should be T1 instead of T0.
  
  This typo is fixed.
  Line 522: Some of the increases in POM could be due to DOM adsorption to particles, or could you clarify if adsorption is included here under aggregation processes?
  
  Yes, DOM adsorption to POM could be occurring. For clarification, we added “adsorption of DOM to particles”
  Lines 565-565: Any thoughts why your results were different from Maie et al 2008?
  
  Maie et al. demonstrated that tannin aggregation increases with salinity, progressing from freshwater to seawater levels. In contrast, our experiments from September do not show a decrease of these highly oxygenated DOM compounds, likely due to a limited tannin source in this region as shown by the low seasonality differences in the tannins region of the van Krevelen diagrams. However, we did observe a reduction in more labile DOM components, characterized by a lower oxygen-to-carbon ratio, during the aggregation period. As discussed earlier, changes in DOM are not directly linked to POC aggregation, particularly in September and October (due to decreased SPEDOC recovery in this period), and it is likely that EPS continues to contribute to POC aggregation as this period is characterised by post-bloom conditions; however, EPS cannot be detected with our DOM extraction method.
  Line 573: I assume the abbreviation LMW refers to light molecular weight, but it should be defined here.
  
  LMW refers to low molecular weight compounds and is now added in the text.
  Line 617: So the difference between experimental and in situ POC concentrations is the sieving step? Could these differences be more clearly mentioned and also presented in a supplementary table for example (I see that the information exists in separate figures in the supplement, but it is rather difficult to compare).
  
  A parentheses was added to the sentence “Throughout the whole sampling period (September-August), experimental POC concentrations at t0 in UF water largely followed a similar seasonal pattern as field POC concentrations, although at lower levels (note that the water collected for the UF treatment was sieved through a 90µm mesh, while the water collected for field measurements was not).” Additionally, a clarifying table was added to the supplementary (S9), and referred to after the sentence “Experimental POC concentrations at the start of incubation (4.7 – 5.3 µM in F, and 5.6 – 6.0 in UF water; Fig. S4) were similar to field POC concentrations in winter (around 4 µM, Fig. S2y), which suggests that particles were of extremely low abundance and size during the winter period (Table S1).”
  Lines 633-636: The t-peaks should be mentioned already in the methods. Could you also clarify to what the statistics refer to on line 635 (e.g. which test and which samples exactly). Were the t-peaks not observed during other seasons than winter?
  
  T-peaks have been added to the methods section of the manuscript. These terrestrial peaks were found in the other months (see updated figure S7 attached) but did not change significantly during the incubations based on unpaired t-test results on each month's replicates for time 0 versus time 1 (ran as four independent tests at the 5% significance level).
  Lines 682-716 (conclusion and outlook): I suggest focusing on results and conclusion from your study while leaving out comparisons with other studies. Furthermore, this section is lacking a clear answer to your original hypothesis and the subsequent questions that were laid out in the introduction (on lines 104-107).
  
  Following this suggestion, the following sentence was deleted from the conclusion: “This contrasts with other observations that show aggregation during winter in a temperate region (Riley, 1963) and is possibly because temperate systems are not light-limited and primary production (and with that, EPS exudation) can take place throughout the year, even if it is reduced. “ Additionally, the used references were removed, as these were referred to earlier in the discussion and introduction to avoid repetition.
  To address the hypothesis more clearly in the conclusion, the changes mentioned for the 1st comment of Reviewer #2 apply here as well.
  Figures:
  Figure 2. Nice and useful figure summarising the sampling and incubation set-up.
  Figure 4. Add statistical testing on the changes in POC and DOC and different parameters before and after incubations. Were the changes significant? Also adding a panel of total organic carbon (DOC+POC) would be interesting.
  A panel for total DOC+POC was not added, because it follows the same patterns as DOC as the changes in POC are minimal compared to DOC. The changes in POC or the variation will not be reflected in this panel.
  We performed t-test on DOC and POC concentrations at t0 vs. t1 for each month. For DOC, the differences between t0 and t1 were significant in September (p<0.05) and December (p<0.05) incubations. However, for October (p=0.07) and February (p= 0.7), the differences were not statistically significant. Regarding POC, the changes between t0 and t1 were also not significant (p>0.05). The standard deviations in Figure 4a) demonstrate that the range of change in POC concentrations is wide, except during the winter months. DOC and POC quantification can have challenges due to its complex nature (Chow et al., 2022) and recommend increasing the sampling size in future studies. However, this was not feasible within the seasonal scale of our experiments. Therefore, in a revised manuscript, we would focus on the seasonal contrasts of DOM characteristics and POC. To highlight this, as mentioned in point #1, we conducted a t-test on the difference (t1-t0) in POC concentrations in winter vs productive period. This resulted in a significant (p= 0.04) difference in change of concentration between winter and the productive period.
  Moreover, increasing the incubation time could possibly have resulted in more distinct differences between t0 and t1; however, we wanted to explore the immediate changes in the OM pool, and many studies have shown before that aggregation occurs within shortest time scales (hours). Longer timescales of water body incubations would also be less reflective of ambient conditions and might lead to bottle effects.
  Figure 5. Why has standard deviation/error not been included in this figure?
  We thank the reviewer for bringing this up and have now added the standard error of the difference of means to the figure (see figure 5 attached). The standard error of the difference of means is particularly useful here for comparing means.
  Figure 6. This figure is difficult to read. The font sizes should be larger, especially the axis titles. The green color is explained in the caption, but I’d suggest adding that to the legend as well. Why are some bars separated from others with black lines but some aren’t? Why do some bars have black horizontal line at the top of the bar while others don’t? Some of the panels have lines next to the axis while others don’t, I suggest re-making this figure or using a different figure type.
  We appreciate the reviewers feedback on this figure and have revised figure 6 (attached) to a side-by-side histogram with two distinct colors for clearer comparison.
  Figure 7a. Could you add to the legend the meaning of the different colors (green, blue).
  Done
  Table 3. Wouldn’t it be more meaningful to compare the changes (in %) in POC during incubations to the experimental POC t0 concentrations and then after calculate how much that % is from in situ concentrations?
  We would like to ask for clarification on this comment.
  References mentioned in responses from authors:
  Chen, C.-S.; Shiu, R.-F.; Hsieh, Y.-Y.; Xu, C.; Vazquez, C. I.; Cui, Y.; Hsu, I. C.; Quigg, A.; Santschi, P. H.; Chin, W.-C. 2021. Stickiness of Extracellular Polymeric Substances on Different Surfaces via Magnetic Tweezers. Science of The Total Environment , 757, 143766.
  Chow, A. T.-S.; Ulus, Y.; Huang, G.; Kline, M. A.; Cheah, W.-Y. 2022. Challenges in Quantifying and Characterizing Dissolved Organic Carbon: Sampling, Isolation, Storage, and Analysis. Journal of Environmental Quality, 51 (5), 837–871.
  Goldberg SJ, Carlson CA, Hansell DA, Nelson NB, Siegel DA. 2009. Temporal dynamics of dissolved combined neutral sugars and the quality of dissolved organic matter in the northwestern Sargasso Sea. Deep-Sea Res. I 56:672–85
  Grasset, C.; Groeneveld, M.; Tranvik, L. J.; Robertson, L. P.; Hawkes, J. A. 2023. Hydrophilic Species Are the Most Biodegradable Components of Freshwater Dissolved Organic Matter. Environmental science & technology, 57 (36), 13463–13472.
  Kirchman DL, Meon B, Ducklow HW, Carlson CA, Hansell DA, Steward GF. 2001. Glucose fluxes and concentrations of dissolved combined neutral sugars (polysaccharides) in the Ross Sea and polar front zone, Antarctica. Deep-Sea Res. II 48:4179–97
  Valdes Villaverde, P., Almeda Jauregui, C., and Maske, H. 2020. Rapid abiotic transformation of marine dissolved organic material to particulate organic material in surface and deep waters, Biogeosciences Discuss. [preprint].
  Wangersky, P. J. 1993. Dissolved Organic Carbon Methods: A Critical Review. Marine Chemistry, 41 (1), 61–74.
  
  Citation: https://doi.org/10.5194/egusphere-2024-1314-AC2
RC2:
'Comment on egusphere-2024-1314', Anonymous Referee #2, 29 Jul 2024

This manuscript focuses on DOM-POM transformation in a sub-Arctic fjord in Norway. The authors use filtered and unfiltered incubations in different season to address changes in DOC and POC to understand aggregation and dissolution dynamics. The manuscript reads well, however below comments that need addressing.
Comments:
This is an interesting study and relevant research. However, the manuscript is lacking in statistical testing to support the presented results. Also the presented hypothesis should be clearly addressed at the end of the manuscript. I would also encourage the authors to add all the data to the supplement or to a data repository to benefit future research.
Lines 101-102: There are other DOM-POM studies albeit from different environments, see e.g., Attermeyer et al. 2018 (Swedish rivers), Shakil et al 2020 (permafrost thaw streams in Canada), Keskitalo et al. 2022 (Kolyma River).
Lines 321-328: Scientific names of species should be in italics here and elsewhere.
Line 356-357: Could you clarify how there can be a decrease in POC in filtered waters where POC has been removed by filtering?
Lines 396-401: So was the statistical significance tested as the authors mention that the results are significant (after explaining about interpretation of signal intensity weighed averages)? It should be made easier for the reader to judge the significance of these results as the changes look so minor. I’ve read the explanation, but statistical testing could help to understand how meaningful the results are.
Line 441: Typo -> the end of the incubation should be T1 instead of T0.
Line 522: Some of the increases in POM could be due to DOM adsorption to particles, or could you clarify if adsorption is included here under aggregation processes?
Lines 565-565: Any thoughts why your results were different from Maie et al 2008?
Line 573: I assume the abbreviation LMW refers to light molecular weight, but it should be defined here.
Line 617: So the difference between experimental and in situ POC concentrations is the sieving step? Could these differences be more clearly mentioned and also presented in a supplementary table for example (I see that the information exists in separate figures in the supplement, but it is rather difficult to compare).
Lines 633-636: The t-peaks should be mentioned already in the methods. Could you also clarify to what the statistics refer to on line 635 (e.g. which test and which samples exactly). Were the t-peaks not observed during other seasons than winter?
Lines 682-716 (conclusion and outlook): I suggest focusing on results and conclusion from your study while leaving out comparisons with other studies. Furthermore, this section is lacking a clear answer to your original hypothesis and the subsequent questions that were laid out in the introduction (on lines 104-107).
Figures:
Figure 2. Nice and useful figure summarising the sampling and incubation set-up.
Figure 4. Add statistical testing on the changes in POC and DOC and different parameters before and after incubations. Were the changes significant? Also adding a panel of total organic carbon (DOC+POC) would be interesting.
Figure 5. Why standard deviation/error has not been included in this figure?
Figure 6. This figure is difficult to read. The font sizes should be larger, especially the axis titles. The green color is explained in the caption, but I’d suggest adding that to the legend as well. Why some bars are separated from others with black lines but some aren’t? Why some bars have black horizontal line at the top of the bar while others don’t? Some of the panels have lines next to the axis while others don’t, I suggest re-making this figure or using a different figure type.
Figure 7a. Could you add to the legend the meaning of the different colors (green, blue).
Table 3. Wouldn’t it be more meaningful to compare the changes (in %) in POC during incubations to the experimental POC t0 concentrations and then after calculate how much that % is from in situ concentrations?
References:
Attermeyer, K., Catalán, N., Einarsdottir, K., Freixa, A., Groeneveld, M., Hawkes, J. A., Bergquist, J. and Tranvik, L. J.: Organic Carbon Processing During Transport Through Boreal Inland Waters: Particles as Important Sites, J. Geophys. Res. Biogeosciences, 123(8), 2412–2428, doi:10.1029/2018JG004500, 2018.
Keskitalo, K. H., Bröder, L., Jong, D., Zimov, N., Davydova, A., Davydov, S., Tesi, T., Mann, P. J., Haghipour, N., Eglinton, T. I. and Vonk, J. E.: Seasonal variability in particulate organic carbon degradation in the Kolyma River, Siberia, Environ. Res. Lett., 17, 034007, doi:https://doi.org/10.1088/1748-9326/ac4f8d, 2022
Shakil, S., Tank, S., Vonk, J. and Zolkos, S.: Low biodegradability of particulate organic carbon mobilized from thaw slumps on the Peel Plateau, NT, and possible chemosynthesis and sorption effects, Biogeosciences Discuss., (July), 1–25, 2021.

Citation: https://doi.org/10.5194/egusphere-2024-1314-RC2
- AC1:
  'Reply on RC2', Maria Digernes, 06 Sep 2024
  We would like to thank the reviewer for their time and interest in our study. Please see our responses to the comments in bold.
  Anonymous Referee #1
  The authors collected samples in a sub-Arctic fjord in different seasons and conducted incubation experiments to investigate changes in DOM and POM concentrations, DOM compositions and other metrics before and after incubations. While the study is interesting, there are two major concerns that need to be addressed before it can be considered for publication.
  The authors attempted to interpret their experimental results in the context of POM and DOM transition, arguing that DOM aggregation and POM dissolution determine the fate of these organic matters. However, the results of the incubation experiments are not strong enough to support this interpretation because: The trends of changes in POC and DOC concentrations during the incubation seem to be complex and they are not always having opposite trend. The authors also mentioned this in the section 5.3.2. The magnitudes of changes in POC and DOC concentrations are significantly different (for example,~ 2 μM changes in POC vs >200 μM changes in DOC in Sep incubation), making it difficult to compare these changes to draw conclusions about DOM aggregation and POM dissolution.
  
  We appreciate Reviewer #1’s interest in our study and their valuable comments. We fully acknowledge the mentioned limitations of our study and have addressed them openly, as they reflect inherent technical challenges faced in all studies on the DOM-POM transition. Our study was meticulously designed to be ultra-clean, free from organic matter contamination and to give as many insights into organic matter as possible using a wide range of techniques. These include DOM characterization (HPLC-HRMS), DOC concentration, POC/PN concentration, colorimetric determination of exopolymeric substances as well as biological parameters. To our knowledge, no other studies have examined all these parameters at a seasonal scale.
  We are not able to quantitatively determine the flux between DOM and POM likely due to the sticky nature of extracellular polymeric substances (Chen et al., 2021), which can cause DOC and POC to be adsorbed on the experimental containers leading to complex changes in the dissolved and particulate carbon budget (Vades-Villaverde et al., 2020). The increase in EPS is supported by the high non-recoverable SPE-DOC in September and October t1 indicating an increase in hydrophilic material (see point 8 below). Additionally, the magnitude differences between POC and DOC, as mentioned by the reviewer, meant that we could not infer that the decrease in POC is directly related to the increase in DOC, however we could infer that an increase in POC may be due to the aggregation of DOC. Further, we highlight the importance of high precision in DOC measurements, which is often a challenge (Chow et al., 2022; Wangersky, P., 1993), and we recommend a high number of replicates (5-10). We would suggest highlighting these limitations in a revised manuscript.
  Due to the mentioned limitation, our focus is on the seasonal variations in POC observations and the character of DOM, rather than on the quantitative differences between DOC and POC. Namely, we highlight key findings on seasonal contrasts, such as the aggregation of POC during biologically productive months and its dissolution in winter (see answer to Reviewer #2 point 1 for more details). Additionally, we explore the contrasting trends in DOM lability, which increases during the winter and decreases during the productive period. These individual findings have implications on the DOM-POM continuum on a seasonal scale.
  The authors also tried to compare the changes in DOM compositions before and after incubation. However, the analysis is questionable because the changes in metrics such as H/C, O/C, MW, and AI in Table 2 are small compared to the standard deviations. Most of the discussions are based on these changes, which are smaller than the errors. While the authors argue that these small changes are significant, they do not provide any statistical test reports to support this argument. The authors should conduct statistical analysis to support that those small changes are still significant.
  
  We appreciate the reviewer’s comment and would like to clarify that the high range of weighted average standard deviations compared to the weighted average differences is expected, given that the standard deviation provided is computed on the variability of all of the compounds with varying ratios in samples per treatment. The benefit of having hundreds of compounds is that the certainty of the weighted average is very high, leading to a low standard error of the mean. This ensures that even small differences between sample averages are statistically significant. So we have changed the standard deviation (done on all compounds) to the standard error of the mean. We apologize for the confusion this caused.
  To further illustrate this, we have updated the table 2 (attached) and Figure 5 (attached) in the manuscript to include the standard error of the mean per treatment and will revise the manuscript text for clarity.
  Major comments:
  Lines 32-35: The sentence seems to imply that DOC cannot contribute to carbon sequestration, which is misleading. The authors should acknowledge that DOC can also contribute to carbon sequestration.
  
  We agree with your comment and would rephrase this sentence to state that DOC plays an important role in carbon sequestration. We are revising it as: Marine DOM is one of the largest stocks of organic carbon on Earth, contributing to long term carbon storage in the ocean. Particulate organic matter (POM) also aids in carbon sequestration by potentially sinking to the seafloor and transporting carbon from the ocean surface.
  Line 44: The sources listed for DOM are incomplete. For example, zooplankton grazing can also release DOM from phytoplankton. I recommend referencing “Carlson, C. A., & Hansell, D. A. (2015). DOM sources, sinks, reactivity, and budgets. Biogeochemistry of marine dissolved organic matter, 65-126.”
  
  We think this is a good point to include the other DOM sources and have revised this sentence accordingly: DOM is generated and secreted by phytoplankton during their growth and can also be produced by zooplankton during grazing and excretion, as well as by bacterial and viral processes such as lysis and excretory release and through the dissolution of particles (Carlson & Hansell, 2015; Riley, 1963; Wagner et al., 2020).
  Lines 50-55: The authors mention labile and semi-labile DOM but omit recalcitrant DOM, which has a turnover time of millennia. To be thorough and consistent with previous categorization of DOM based on lability, recalcitrant DOM should be included.
  
  We have now modified the text based on your comment to incorporate recalcitrant DOM and its long residence time. Labile DOM, which constitutes less than 1% of the overall DOM reservoir, displays relatively short turnover times, typically ranging from hours to days (Hansell, 2013). Conversely, semi-labile and recalcitrant DOM persists in the ocean over more extended time scales, ranging from months to millennia (Fleurs et al., 2012; Hertkorn et al., 2006).
  Line 145: I do not fully understand why using a headlight with red light can reduce the possibility of biological production. Chlorophyll a absorbs red light.
  
  Chl a absorbs mainly in the blue part of the light spectrum, but indeed also absorbs red light. The reason we do not use green light is that it does not provide as good visibility for the human eye at low intensities. The light exposure (in terms of intensity and time) were minimal in the beginning and the end of the incubation, and therefore we are confident that this light exposure virtually did not impact the incubations. This can be confirmed by almost no increase of phytoplankton cells during winter (change of -101.5 ± 23.4 cells ml-1).
  Lines 253-254: Why pre-soaking SPE column sorbent with Methanol is needed ?
  
  A pre-soak time can potentially improve the activation of the sorbent and ensure that the solvent fully permeates into the sorbent.
  Line 259: The DOC recovery is highly variable. Could you explain why?
  
  The greatest variability in DOC recovery was attributed to seasonal differences in the recoverable DOC. This is illustrated in the boxplot figure attached. The highest SPE-DOC recovery, ranging from 74% to 85%, occurred in December t0. This indicates a higher proportion of hydrophobic DOM and likely CRAM material, which SPE-DOC strongly retains (Hertkorn et al., 2013). Interestingly, SPE-DOC recovery decreases during the December incubations, likely due to a reduction in these compounds. By late winter (February), SPE-DOC recovery declines further, reflecting a shift towards more labile material as observed in December incubations. Lower recoveries were also observed in September and October, months marked by high biological productivity. During this period, there is a higher proportion of hydrophilic material such as dissolved proteins and carbohydrates (Kirchman et. al 2001; Goldberg et. al 2009)—compounds that are not well retained by our SPE sorbent (Grasset et al 2023). Finally, the decrease in SPE-DOC recovery during September and October incubations also indicate components with low affinity for the SPE sorbent.
  Line 280: Did the authors try other criteria for formula assignment, such as allowing nitrogen between 0 and 4 ?
  
  The authors would like to emphasize that allowing too many nitrogens leads to false assignments to these formulas. Formulas with a higher number of nitrogens are not very commonly assigned (even in 21T FT-ICR data), and when they are, they are usually very minor peaks. These peaks wouldn’t make a large difference to the assigned peak intensities or mass values (in our data, in which they would coalesce with another peak), and even if they were resolved and assigned, would make very tiny differences to the overall metrics.
  Line 281: Why remove formulas which contain both nitrogen and sulfur, 13C and nitrogen or sulfur and 13C? Could the authors justify it?
  
  Same reason as point 9, to avoid false assignments, these peaks are uncommon and tiny.
  Lines 395-400 and Figure 5: The changes in DOM parameters like H/C, AImod, MW, and O/C are much smaller than the standard deviation. Although the authors state these small differences are statistically significant, statistical test results should be provided to justify this claim.
  
  We would like to refer the reader to our response in point 2.
  Minor comments:
  Line 108: should be “characteristics” instead of “character”? Done
  
  Line 131: (TPM and PIM). Done
  
  Line 141: should be “every two months”? Done
  
  References mentioned in responses from authors:
  Chen, C.-S.; Shiu, R.-F.; Hsieh, Y.-Y.; Xu, C.; Vazquez, C. I.; Cui, Y.; Hsu, I. C.; Quigg, A.; Santschi, P. H.; Chin, W.-C. 2021. Stickiness of Extracellular Polymeric Substances on Different Surfaces via Magnetic Tweezers. Science of The Total Environment , 757, 143766.
  Chow, A. T.-S.; Ulus, Y.; Huang, G.; Kline, M. A.; Cheah, W.-Y. 2022. Challenges in Quantifying and Characterizing Dissolved Organic Carbon: Sampling, Isolation, Storage, and Analysis. Journal of Environmental Quality, 51 (5), 837–871.
  Goldberg SJ, Carlson CA, Hansell DA, Nelson NB, Siegel DA. 2009. Temporal dynamics of dissolved combined neutral sugars and the quality of dissolved organic matter in the northwestern Sargasso Sea. Deep-Sea Res. I 56:672–85
  Grasset, C.; Groeneveld, M.; Tranvik, L. J.; Robertson, L. P.; Hawkes, J. A. 2023. Hydrophilic Species Are the Most Biodegradable Components of Freshwater Dissolved Organic Matter. Environmental science & technology, 57 (36), 13463–13472.
  Kirchman DL, Meon B, Ducklow HW, Carlson CA, Hansell DA, Steward GF. 2001. Glucose fluxes and concentrations of dissolved combined neutral sugars (polysaccharides) in the Ross Sea and polar front zone, Antarctica. Deep-Sea Res. II 48:4179–97
  Valdes Villaverde, P., Almeda Jauregui, C., and Maske, H. 2020. Rapid abiotic transformation of marine dissolved organic material to particulate organic material in surface and deep waters, Biogeosciences Discuss. [preprint], https://doi.org/10.5194/bg-2020-291.
  Wangersky, P. J. 1993. Dissolved Organic Carbon Methods: A Critical Review. Marine Chemistry, 41 (1), 61–74.
  
  Citation: https://doi.org/10.5194/egusphere-2024-1314-AC1
  - AC3: 'Reply on AC1', Maria Digernes, 06 Sep 2024
    
    Please note that the responses were unfortunately swapped so we refer Reviewer #2 to the response located at Reviewer #1 and vice versa.
    
    Citation: https://doi.org/10.5194/egusphere-2024-1314-AC3

Maria G. Digernes, Yasemin V. Bodur, Martí Amargant-Arumí, Oliver Müller, Jeffrey A. Hawkes, Stephen G. Kohler, Ulrike Dietrich, Marit Reigstad, and Maria Lund Paulsen

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https://doi.org/10.5194/egusphere-2024-1314-supplement

Maria G. Digernes, Yasemin V. Bodur, Martí Amargant-Arumí, Oliver Müller, Jeffrey A. Hawkes, Stephen G. Kohler, Ulrike Dietrich, Marit Reigstad, and Maria Lund Paulsen

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Short summary

Dissolved (DOM) and particulate organic matter (POM) are in constant exchange, but usually studied as distinct entities. We investigated the dynamics between POM and DOM in a sub-Arctic fjord across different seasons by conducting bi-monthly aggregation-dissolution experiments. During the productive period, POM concentrations increased in the experiment while DOM molecules became more recalcitrant. During the winter period, POM concentrations decreased whereas DOM molecules became more labile.


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