the Creative Commons Attribution 4.0 License.
the Creative Commons Attribution 4.0 License.
| 08 Aug 2025
Hydrothermal activity indirectly influences ice nuclei particles in seawater and nascent sea spray of the Subtropical Pacific Ocean
Abstract. Particles of marine origin may act as ice nuclei when clouds form and therefore influence cloud properties and lifetime. Here we investigate the abundance of Ice Nuclei Particles in bulk seawater (INPSW) collected in natural seawater of the Western Tropical South Pacific and in sea spray aerosol (INPSSA) artificially generated from the surface seawater. The study area was separated into two oligotrophic zones (the Melanesian Basin and the Western South Pacific Gyre), and a mesotrophic one (the Lau basin), characterized by high plankton biomass due iron fertilization by underwater hydrothermal activity of the Tonga volcanic arc. Our results show that INPSW were on average 80 % heat labile, strongly suggesting a biological origin. INPSW concentrations were two-fold higher in the Lau basin as compared to both oligotrophic areas at all freezing temperatures. This trend is consistent with a higher abundance of planktonic microorganisms, pigments and particulate organic carbon (POC) concentrations in the Lau basin. Over the whole cruise transect, medium to strong correlations were found between INPSW concentrations and pigments (notably with bacteriochlorophyll-a and carotene), bacterial abundance and POC. The heat stable fraction of INPSW exhibited correlations with Dissolved Organic Carbon (DOC) concentrations and were not as variable as the heat labile INPSW. In the nascent sea spray, INPSSA were also mostly heat labile in coherence with the INPSW. INPSSA were predominantly (60 %), submicron in size (presumed originating from film drops), but the supermicron INPSSA constituted 40 % of the INPSSA and were all heat labile (presumably originating from jet drops). Supermicron INPSSA were between 60 to 80 % heat stable with a high variability between samples, indicating different nature of the two fractions of INPs. Supermicron INPSSA were generally more abundant in the Lau basin, while submicron INPSSA did not exhibit any significant difference between the three regions. We report a transfer function of seawater INPs to SSA INPs of 1.70 m-2.LSW and 3.3 m-2.LSW for heat stable INPs, hinting that heat stable INPs were more efficiently transferred to the SSA. Our results suggest that hydrothermal activity indirectly enhances the INP concentration of surface waters, through boosting the biological activity, which results in increases of the ice forming ability of supermicron sea spray particle. Given the extent of hydrothermal activity throughout the global Ocean, its impact on cloud properties should be considered in future ocean-atmosphere interaction studies.
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Status: final response (author comments only)
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RC1: 'Comment on egusphere-2025-3580', Anonymous Referee #1, 02 Oct 2025
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AC1: 'Reply on RC1', Yannick Bras, 13 Nov 2025
REPLY TO SPECIFIC COMMENTS
P1L30: This sentence needs to be reformatted. Is it correct understood that the first number is the total INP transfer and the second is the heat stable INP transfer? If yes, please state more clearly.In the “hint” statement, please write what you are comparing to. “,hinting that heat stable INPs were more efficiently transferred to the SSA” compared to ...
Yes. New sentence : We report a transfer function of total seawater INPs to total SSA INPs of 1.70 m-2.LSW and 3.3 m-2.LSW for heat stable seawater INPs to heat stable SSA INPs, hinting that heat stable INPs were more efficiently transferred to the SSA compared to heat labile INPs.
P4L98: Description of the SSA experiments is lacking in detail. The authors reference papers of similar studies, however, the Sellegri et al. 2005 paper is possibly the same chamber (not clear) but run in that study without water or jets. I would remove this reference as it causes confusion. The tank described in Schwier et al. 2015 seems to be the same as described in the current study. I suggest adding a few more details to the current description of the sea spray tank. The authors write “jets” in plural – are there several or is it a single plunging jet? If there are several please describe this as not many tanks have several. What flow rate of water through the jet was used? What flow rate of particle filtered air was used? Was the tank temperature regulated?
We added a description of the tank used here and kept the reference Schwier et al. 2015, which first described it:
« SSA was generated using the technique first described in Schwier et al. (2015) for discrete samples, but in a continuous way as described in Trueblood et al. (2021). Briefly, the underway seawater (UWAY, depth ~5 m) is continuously injected in a 10 L tank through eight 1 mm- water jets located a few centimeters from the seawater surface at a rate of 1.25 LPM. Seawater jets hitting the seawater surface created bubbles that, uppon bursting at the surface, eject film and jet drops that evaporate to SSA. The set-up was designed to reproduce at best the conditions described in Fuentes et al. 2011, who measured a bubble size distribution with shape similar to ambiant bubble size distributions observed under open ocean conditions. Our system, under the operation conditions previously described, entrains air into the seawater at a rate that is equivalent to a 9 m s-1 wind speed (Sellegri et al. 2023). The flushing filtered air flowrate in the tank’s headspace was set to 20 LPM, and the bubble system regularly stopped to check that the flushing air contained no particles. The tank was not temperature controlled, but in the SST range observed during the TONGA campaign, it was previously shown that temperature induced changes in the SSA fluxes were negligeable (Sellegri et al. 2023). P6L151: Please check that ‘Sellegri et al 2005’ is the correct reference based on previous possible mix-up.
Yes, the tank used in Sellegri et al 2005 was different and we changed this reference to Schwier et al. 2015.
P6L156: Specify how blank samples were made. Were they also placed in the stage for ~24hours?
Blank samples were placed on the stage using the same procedure than the samples, however they were placed only for a few minutes as potential contaminations are expected to take place during filter manipulations rather than once in place.
P6L159: Samples were collected in November 2019. How long were they stored frozen before INP measurement?
They were stored for 1 to 1.5 year in the freezer.
P6L165: The volume of liquid added to the Eppendorf tubes is different for SW and SSA measurements (200µL and 400µL, respectively). The Vali equation takes into account the volume difference when calculating the concentration of INPs. This should be specified in the text, so that the reader understands that the to dataset can be compared regardless of the difference in volume.
This is true. New text proposition : "In order to reach INP concentrations above the limit of detection (LOD), the Eppendorf tubes were filled with 200 μL for seawater samples, and 400 μL for extracted filter samples. Even though the extraction volumes are different, the two datasets can be compared thanks to the Vali equation that takes into account the volume difference when calculating the concentration of INPs" P6L168: What does it mean the samples were “being agitated”?
The solution was placed on an agitator for 20 minutes in order to have a better extraction of the INPs into the solution.
P6L168: The process of filling half the tubes and heating is described twice in the same section. I suggest keeping the following text (start line 172) and rewriting the text starting in line 168.
“Half of the Eppendorf© tubes were then filled with the untreated sample. The seawater samples were then subjected to the same heat treatment as the filter samples, and the second half of the Eppendorf© tubes was filled using the heated samples (Fig. 2).”
Very good suggestion, we will use this sentence.
P6L170: Comment on the heat treatment study by Daily et al. 2022, where they find that wet INP heat tests at (> 90°C) have the potential to produce false positives. Why did the authors select 100°C? Daily, Martin I., et al. "An evaluation of the heat test for the ice-nucleating ability of minerals and biological material." Atmospheric Measurement Techniques 15.8 (2022): 2635-2665. Answer : We were not aware of this publication at the time of writing this paper, and the manipulations were performed in 2020-2021. We selected 100°C as was suggested by other colleagues, and because the only way we could heat the samples was using boiling water.
We will discuss this potential bias here and in the manuscript : P6L170 : Recent studies highlighted a potential bias introduced by wet heat treatment for detecting biogenic INPs (Daily et al., 2022 ; Alsante et al., 2023). By testing wet and dry heat treatment, Daily et al. (2022) concluded that dry heating at 250 °C was more effective at deactivating biogenic INPs than wet treatment, which could also alter the IN activity of mineral aerosols, thus inducing potential false-positives in studies that used it. Alsante et al. (2023) point that while heat treatment at -95 °C reduces the IN activity of the RuBisCO enzyme, it did not eliminate it completely, suggesting that heating might twist the protein molecules into new shapes that may also be effective INPs. However, in the case of RuBisCO, it’s nucleation temperature is shifted from -8 to -22°C, a temperature which is outside the range of detection limit. However, the bias highlighted by Daily et al. (2022) might be applicable in our study, and we acknowledge that the heat treatment gives an approximated evaluation of the contribution of proteins to the INP activity.
P20L446 : However, recent discussions discussed the potentiel artifacts caused by wet heating for discriminating between biogenic heat labile INPs and non biogenic heat stable INPs (Daily et al., 2022 ; Alsante et al., 2023). We believe that the results highlithted here are relevant despite such biases, nonetheless keeping in mind that the heat treatment here gives only an approximated insight of the contribution of proteinaceous INPs. P7L174: What is the limit of detection?
It would also be nice with an idea of the experimental uncertainty e.g. as a number of degrees Celsius. This would give the reader a better understanding of how significant differences between samples freezing temperatures are.
The limit of detection increases as the temperature decreases, but below -15°C it is in the order of 5 to 10 INP/mL. For temperature, the uncertainty is 0.2 °C.
P7L178: Show a figure of the blank sample INP results in the appendix. State what the “lower temperature that can be reached” is.
We now provide a figure of the blank sample INP results in the appendix. What we mean by “lower temperature that can be reached” is that since blank samples were completely frozen between -18 and -20°C, it was meaningless to go below -18°C for field samples.
P7L180: It is interesting that the heat treatment increased the IN activity. Add a comment on whether this has been seen in other studies (see comment above about paper by Daily et al. 2022).
Modified sentence : Heated blank samples showed increased IN activity at all temperatures compared to unheated blanks. This has not been seen in any other stydu, which hints that the heat treatment results in some contaminated samples. This could also be the result of false positives as highlighted by Daily et al. (2022), who showed that some biological material was barely affected by wet heat treatment at -95 °C.
Section 3.1: It is some really nice data on the seawater characteristics, and the text describes a comparison of the three waters as is viewed in Figure 2. However, I am missing some discussion/interpretation of the comparisons. E.g. What does it mean that the different planktonic microorganisms are in higher abundance in the LAU surface water? Is this expected?
It is expected that microorganisms are generally higher in the LAU waters because those waters are more nutrient-rich thanks to the underwater hydrothermal activity. These observations and discussions mostly took part in the other papers from this campaign and previous campaigns in the same region (e.g. Bonnet et al., 2018; Moutin et al., 2018; Bonnet et al., 2023).
We propose the following changes to the manuscript (P7L189) : ‘The sampled waters were classified into three distinct zones based on their biogeochemical characteristics, including nutrient and trace metal concentrations as well as chlorophyll levels (e.g., Bonnet et al., 2018; Moutin et al., 2018; Bonnet et al., 2023): the Melanesian waters (MEL), the Lau Basin waters (LAU), and the West Pacific Gyre (WGY) waters’.
P9L205: The difference in salinity and surface tension between the three waters seems very small (very close to the standard deviation stated). Why is this important to mention?
This is a good point. We will change the sentence to :
«Salinity and surface tension (Fig. 2c and 2d) were generally lower in the LAU […] than in the MEL […] and WGY [...]. However, those variations are very close to the aforementionned standard deviations, thus we can conclude that those differences are not significant and there can not explain the INP differences between waters. »
P9L223: Elaborate on this sentence “The MEL were oligotrophic waters, and WGY were ultraoligotrophic waters.”. How does this fit with the seawater characteristics in Figure 2.
We show in Figure 2 that the LAU is enriched, the MEL is poor in nutrients and thus biologic activity (eg oligotrophic) and WGY is extremely poor in nutrients/microorganisms (eg ultraoligotrophic)
P10L228: It is confusing for the reader to have just read a section where the water types are separated into categories: MEL, LAU, WGY, and now in this section the INP samples are separated into new categories: INP_sw, INP_tot, INP_HS, INP_HL. It needs to be clearly stated in the start of this section (3.2.1) what the new categories mean and from which waters these INP samples come from. We now describe all INP categories in the methods section, and these are recalled at the begnning of section 3.2.1 and 3.3 :
P7L187 : After treatment and blank correction, we define the different INP categories. INP measured in the bulk seawater are defined as INP_SW, INP measured in the SSA are defined as INP_SSA. For each INP_SW and INP_SSA, we define the subcategories of « total » INPs, which are the measured INPs without a heat treatment ; « heat stable » INPs, which are INPs which were not removed by the heat treatment ; and « heat labile » INPs, which are INPs that were removed by the heat treatment.
This should also be added to the methods section – a specification of where the INP water samples and air samples where taken (If at all stations, then state that). Also state earlier in the text of section 3.2.1 that the comparison of water types was not possible.
INP air and water samples were taken along the ship’s transect, and not specifically at the fixed stations.
What is the authors definition of heat stable INP (e.g. a specific decrease in IN activity or)?
We now recall them at the beginning of section 3.2.1. We define heat stable INPs as INPs that were not inactivated by the heat treatment. These generally correspond to non biological INPs, as the proteins of micro organisms are degraded by the heat treatment. However we canot exclude that biologically originating compounds are also heat stable and therfore we use the term heat-stable.
P15L313: Elaborate on the strong correlation with dissolved iron.
This is now included in the text (P15L313) :
Bonnet et al. (2023) showed that iron emitted by hydrothermal vents supply the surface waters, and thus stimulate the biological activity there. The strong correlation between HS INPs and iron hints that either HS INP abundance is indirectly influenced by iron (along the other nutrients) via its impact on biology, or that iron itself is an INP. Iron has been found to be an efficient INP in the atmosphere under various forms, e.g. as oxides (Chong et al., 2021) or associated with minerals (Ganguly et al., 2018). However, to our knowledge, the INA of dissolved iron has never been studies before.
P16L344: I suggest simply writing INP/aerosol instead of shortening to INP/aer as this is likely to confuse readers. I do not believe that “Aer.” is a commonly used abbreviation for units. Same for Figure A3.
Ok
Figure 7. In text above figure n_s = surface site density, but in figure caption n_s = nucleation site density. Select one name for the symbol. Several different labels are written in the caption referring to the same thing. Ns_super = Unheated SUPM?? Use the same label for both legend, caption and text describing the figures. What do the error bars represent?
I will keep n_s = surface site density and make the caption and text clearer. Error bars correspond to three standard deviation of sample blanks
P17L357: Split sentence after parenthesis. Elaborate on Córdoba2025 comment, if possible, give n_s values.
New text at P17L357 : Total values of nS were in the range of [...], i.e. 1 to 2 orders of magnitude lower than nS fitted for ambient marine aerosols sampled in the North Atlantic coastal research station Mace Head by McCluskey et al. (2018c) (MC18, shown in Fig. 7a). Our walues are lower by several orders of magnitude to the ones measured by Córdoba et al. (2025) in SSA artifically generated from various seawaters near the coast of Mexico, which were in the range of 105-1010 m-2 at -20 °C depending on the location. This difference is not explained by the aerosol concentrations, which were similar in both our study and Córdoba et al. (2025), with values of about 500 to 2000 cm-3 (Fig. A3). Most likely, the difference in nS is explained by the differing nature of seawaters.
P17L375: Elaborate on how this observation contrasts that of McCluskey2018b.
As the McCluskey et al. (2018b) study refered to ambiant aerosol sampled at a coastal site of the North Atlantic, therefore to very different aerosol than those studied here, it is likely that these differences between our results and those from the Cluskey et al. (2018b) are largely attribuable to these different natures. We now refer to another study that focused on southern hemisphere aerosols instead :
Page 17, line 370 : « In the submicron size range, all of nS,sub are heat labile, suggesting that small INP are dominated by heat labile organic material ejected as film drops. On the other hand, only 20% of INPs in the supermicron size range, mainly ejected to the atmosphere as jet drops, are heat labile on average (Figure 7b and 8b). Jet drops have a chemical composition that is close to the bulk seawater, where dissolved, aged and heat stable organic matter dominates over fresh particulate organic matter. In McCluskey et al. (2018c), the authors report on the heat lability of INP in ambiant aerosol particles sampled in the Southern Ocean, that show extremely variable contributions of heat-labile material to the INP population that could not be explained by the Chla content of the seawater over the ship pathway, nor by the organic contribution to the INP population. Therefore we expect that our results are also specific to a geographical area, season, and organic material quality. This observation contrasts with that made by McCluskey et al. (2018b), who categorized INP in two classes: INPs smaller than 0.2 µm that were mostly heat stable, and INPs larger than 0.2 µm that were mostly heat labile. However, we chose to separate the INPs at the size cutoff of 1 µm, where a larger difference in aerosol chemical properties is often observed as a result of differences in sea spray production mechanisms (film vs jet drops, De Leeuw et al.; 2011).
P19L404: It is not clear to me what this transfer function means. Please explain in further detail why the equation ns/INP_sw is used and explain the resulting number in words. In addition, it would be nice to add a reference to other work that has determined a transfer function in a similar way and compare numbers.
This function represents the ratio of INPs that were transfered from the seawater to the aerosols. We think that this ratio is important for models predicting cloud formation using INP data over the oceans, as it is generally easier to measure Inps in the seawater than in the air due to the experimental constraints. We are not aware of other work that calculated such transfer for INPs however.
The authors could also determine a flux of INPs from water to air using the sea spray tank experimental conditions.
Fluxes of INPs would be very specific to our sea spray generation system (that is equivalent to a 9 m s-1 wind speed flux). Ns should be independant of the sea spray flux that can be parameterized as a funciton of wind speed in many studies.
Citation: https://doi.org/10.5194/egusphere-2025-3580-AC1
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AC1: 'Reply on RC1', Yannick Bras, 13 Nov 2025
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RC2: 'Comment on egusphere-2025-3580', Anonymous Referee #2, 22 Oct 2025
GENERAL COMMENTS
Indirect effects (i.e. those related to cloud properties) remain a major source of uncertainty in the Earth’s radiative budget and climate models. Processes related to the formation of ice in clouds are a particular source of uncertainty. There is considerable interest in ice nucleating particles (INP) from the ocean in terms of sources, their abundance in the atmosphere, and how their properties affect the formation and properties of mixed phase clouds. Bars et al. focused their research on ice nucleating particles from the ocean, both in bulk seawater and in artificially generated sea spray aerosol (SSA). Their work took place in the oligotrophic Pacific Ocean, which was a good location as the subtropical gyres (large areas of oligotrophic ocean) cover about a third of the Earth’s surface. In addition, INP from marine sources are more likely to be important in these remote locations that are a great distance from continental sources of aerosol.
In line with previous work, Bras et al. concluded that the INP in sea spray and bulk seawater were biogenic in origin. Correlations between the concentrations of INP from seawater and indicators of biomass (pigments, bacteria concentrations, and particulate organic carbon) further indicate a connection between biological processes in the ocean and INP, and recent organic matter rather than the background pool of recalcitrant carbon. The authors try and make a connection between hydrothermal activity in the ocean to the formation and abundance of INP.
This work adds to measurements of INP associated with marine sources (which are limited), but I do not think it increases our conceptual or mechanistic understanding of INP from the ocean.
The authors try and link INP concentrations with hydrothermal activity. While the title indicates that this connection is ‘indirect’ it seems very tenuous. It may be that some of the relevant information is missing as it is presented in other papers from the TONGA project. However, I feel that if the authors want to make a case linking INP with hydrothermal activity, then this should be explicitly made in the manuscript. At present the only data making this connection is presented in an appendix rather than the main paper. Is it clear that the high biomass is supported by hydrothermal addition of trace elements, rather than other sources, such as upwelling of deep water or aeolian deposition?
SSA were generated using a technique described by Sellergi et al. (2005). While this is a published method, I would like to see some evidence that the size distribution of generated aerosol is representative of natural aerosol. Perhaps this could be added as supplementary material.
The freezing temperature of the blanks was relatively warm, which, as the authors acknowledge means that a proportion of INP cannot be detected by this method. Perhaps what can’t be detected is not as important as INP that freeze at relatively warm temperatures, but I think that the authors should discuss the implications of not being able to detect INP affecting freezing at temperatures below -18 degrees Centigrade.
SPECIFIC POINTS
Abstract
Line 25 – The word ‘coherence’ is probably the wrong word choice here.
Line 32 – 35: Is the connection to hydrothermal activity the most significant conclusion? I would argue that there is still debate over whether SSA and INP can be linked to recent production in the water or whether they are driven by the high concentration background of older, relatively recalcitrant carbon in the dissolved organic carbon (DOC) pool. The connection with recent biological activity, whatever the source of nutrients supporting it, is the more interesting result.
Introduction
Line 44: Replace ‘uplifted’ with ‘lofted’.
Line 50: warmer temperature than what? Inorganic INP? Not clear.
Line 66-67: I think the wording is misleading as it suggests that hydrothermal activity is the source of the INP. This is not the case, the INP are biogenic in origin, but hydrothermal inputs are providing nutrients to support that biological activity.
Methods
Figure 1 – The map is difficult to read.
Lines 121-122: Does freezing and thawing the samples change the surface tension measurements?
Line 125: Larger cells will be excluded from flow cytometry measurements. Perhaps this bias is not an issue as oligotrophic areas of the open ocean tend to be dominated by small phytoplankton. The authors should comment on this.
Line 129: Dissolved Organic Matter (DOM) does NOT include ‘dissolved oxygen’, though many organic molecules contain oxygen atoms as part of their structure.
Section 2.3: Should there be a diagram (e.g. in supplementary material) of the setup described in this section?
Lines 153-158: More methodological information needed. For example, what was the filtration rate?
Line 176: I think something is missing from this sentence. The word ‘either’ implies an alternative.
Line 186: Delete ‘and hypothesis’
Results and discussion
Line 191: Should this be changed to ‘Figure 2’?
Figure 2 – Salinity is a dimensionless unit as it is based on the ratios of conductivity measurements. Delete ‘PSU’ as it is not an accepted unit used by oceanographers.
Line 204: Explain ‘the second passing of LAU’. Not clear from the text that the sites or regions were visited more than once. Make sure that this is clear on Figure 1?
Lines 206-207: Delete ‘PSU” as salinity is a dimensionless unit.
Lines 214-215: change ‘nifHgene’ to ‘nifH gene’.
Lines 230-234: Heating the aerosol to denature protein INP is likely to underestimate the numbers and relative significance of proteins as INP. Some proteins are only partially denatured at temperatures around 100 degrees centigrade and some proteins will reform their structure on cooling (e.g. Alsante et al. 2023; Communications Earth and Environment 4:51 https://doi.org/10.1038/s43247-023-00707-7).
Line 248-249: ‘which are known to be active at warmer temperature….’ Add a reference to support this statement.
Figure 4 – Would it be helpful to add the locations of the hydrothermal inputs on this map?
Line 282: ‘Note that relationships to the surface seawater biogeochemistry……’ I found this sentence too vague.
Line 298: Lots of organisms, in addition to Prochlorococcus, contain alpha-carotenes.
Line 306: I don’t understand what a ‘heat sensitive organisms’ is. All organisms are heat-sensitive outside their temperature range.
Line 330: Change ‘refractory’ to ‘recalcitrant.’ Recalcitrant is the more applicable terms and it is widely used in the literature. Recalcitrant emphasizes resistance to biological degradation, which is more appropriate for DOM in the ocean. Refractory compounds are resistant to degradation from heat, chemical oxidation etc.
Figure 7a – Is there really a difference between heated and unheated aerosol?
Line 394-395: The fact that there was only a boost in SSA INP activity near one of the two undersea volcanoes could be used as an argument that hydrothermal activity was not a significant factor in determining INP activity.
Figure 9 - Could a parametric or even a non-parametric statistical test be used to test the hypothesis that there was a significant difference in ice nucleation between the different zones for different categories of aerosol?
Conclusion
Lines 410-446: I suggest shortening the conclusion. Much of it was redundant as it was repetitive of the previous text.
Citation: https://doi.org/10.5194/egusphere-2025-3580-RC2 -
AC2: 'Reply on RC2', Yannick Bras, 13 Nov 2025
- REPLY TO GENERAL COMMENTS
Indirect effects (i.e. those related to cloud properties) remain a major source of uncertainty in the Earth’s radiative budget and climate models. Processes related to the formation of ice in clouds are a particular source of uncertainty. There is considerable interest in ice nucleating particles (INP) from the ocean in terms of sources, their abundance in the atmosphere, and how their properties affect the formation and properties of mixed phase clouds. Bars et al. focused their research on ice nucleating particles from the ocean, both in bulk seawater and in artificially generated sea spray aerosol (SSA). Their work took place in the oligotrophic Pacific Ocean, which was a good location as the subtropical gyres (large areas of oligotrophic ocean) cover about a third of the Earth’s surface. In addition, INP from marine sources are more likely to be important in these remote locations that are a great distance from continental sources of aerosol.
In line with previous work, Bras et al. concluded that the INP in sea spray and bulk seawater were biogenic in origin. Correlations between the concentrations of INP from seawater and indicators of biomass (pigments, bacteria concentrations, and particulate organic carbon) further indicate a connection between biological processes in the ocean and INP, and recent organic matter rather than the background pool of recalcitrant carbon. The authors try and make a connection between hydrothermal activity in the ocean to the formation and abundance of INP.
This work adds to measurements of INP associated with marine sources (which are limited), but I do not think it increases our conceptual or mechanistic understanding of INP from the ocean.
Relationships between seawater INP concentrations and nascent SSA INP concentraitons (not « polluted » by other INP sources than the marine source) are not very commun in the litterature and we believe that our data set does bring new mechanistic understanding of INP from the oceans.
The authors try and link INP concentrations with hydrothermal activity. While the title indicates that this connection is ‘indirect’ it seems very tenuous. It may be that some of the relevant information is missing as it is presented in other papers from the TONGA project. However, I feel that if the authors want to make a case linking INP with hydrothermal activity, then this should be explicitly made in the manuscript. At present the only data making this connection is presented in an appendix rather than the main paper. Is it clear that the high biomass is supported by hydrothermal addition of trace elements, rather than other sources, such as upwelling of deep water or aeolian deposition?
We made it clearer in the introduction that the aim of the paper is to investigate INP activity in a context of elevated biological activity caused by the presence of hydrothermal sources :
P3L77 : « The WTSP is characterized by contrasted ocean provinces, with oligotrophic (i.e. nutrient poor) waters in the east, separated by an area of high biological activity caused by iron fertilization from hydrothermal activity and subsequent diazotrophic plankton blooms near the Tonga- Kermadec volcanic arc (Bonnet et al., 2018; Moutin et al., 2018; Bonnet et al., 2023). The link between hydrothermal activity, elevated iron concentrations, increased primary production, N2 fixation and elevated biomasses has already been established in the other publications of the TONGA project (e.g. Bonnet et al., 2023 ; Tilliette et al., 2022). Here we explore how the IN properties of seawater and artifically generated sea spray evolve in a context of elevated biological activity induced by the presence of hydrothermal sources, and when naturally mixed with particles of volcanic origin at the seafloor.
SSA were generated using a technique described by Sellergi et al. (2005). While this is a published method, I would like to see some evidence that the size distribution of generated aerosol is representative of natural aerosol. Perhaps this could be added as supplementary material.
The right reference is the Schwier et al. 2015 paper that describes the exact same set-up. This set-up was similar in jet configuration to the Fuentes 2011 set-up in which bubble size distributions (that is the key variable shaping the sea spray size distribution) is similar to natural bubble size distribution.
Producing bubble size distribution with stable conditions is the condition for making SSA INP variability study a function of biology and not other external factors such as wind speed and atmospheric transport.
The freezing temperature of the blanks was relatively warm, which, as the authors acknowledge means that a proportion of INP cannot be detected by this method. Perhaps what can’t be detected is not as important as INP that freeze at relatively warm temperatures, but I think that the authors should discuss the implications of not being able to detect INP affecting freezing at temperatures below -18 degrees Centigrade.
We now discuss this.
Page 7 line 187 : « Our method does not allow for the detection of INP at temperatures lower than -18°C. Therefore our study focuses on the role of biological material that is influencing initiation of ice formation at warmer temperatures. These warmer temperatures are relevant for low level clouds in the subtropical region. These low level clouds are directly in contact with biological marine sources, and when glaciation is initiated, the impact of INP active at temperatures lower than -18°C would have a limited impact as the cloud would precipitate before these very low temperatures are reached.
- REPLY TO SPECIFIC POINTS
Line 25 – The word ‘coherence’ is probably the wrong word choice here.
In line ?
Line 32 – 35: Is the connection to hydrothermal activity the most significant conclusion? I would argue that there is still debate over whether SSA and INP can be linked to recent production in the water or whether they are driven by the high concentration background of older, relatively recalcitrant carbon in the dissolved organic carbon (DOC) pool. The connection with recent biological activity, whatever the source of nutrients supporting it, is the more interesting result.
Line 44: Replace ‘uplifted’ with ‘lofted’.
ok
Line 50: warmer temperature than what? Inorganic INP? Not clear.
At temperatures above -20°C.
Line 66-67: I think the wording is misleading as it suggests that hydrothermal activity is the source of the INP. This is not the case, the INP are biogenic in origin, but hydrothermal inputs are providing nutrients to support that biological activity.
Agreed. Maybe « Even less is known on the ice nucleating properties of particles in regions influenced by hydrothermal activity », this way the interpretation is open (eg either biogenic INPs vs INPs generated by hydrothermal activity).
Figure 1 – The map is difficult to read.
We will increase the size of the map and fonts to make it more readible.
Lines 121-122: Does freezing and thawing the samples change the surface tension measurements?
We do not know if freezing and thawing the sample changes the surface tension measurements, we know that it changes the INP content. However, not freezing the samples before analysis would change them even more.
Line 125: Larger cells will be excluded from flow cytometry measurements. Perhaps this bias is not an issue as oligotrophic areas of the open ocean tend to be dominated by small phytoplankton. The authors should comment on this.
We acknowledge that flow cytometry primarily targets small cells (pico- and nanoplankton), which generally dominate in oligotrophic oceans. However, in this region of the subtropical South Pacific, larger diazotrophic organisms such as Trichodesmium can represent a substantial fraction of the plankton biomass during summer. These taxa were specifically considered in our study through quantitative PCR (qPCR) analyses. Finally, we also examined correlations with pigments, which integrate contributions from the entire plankton size spectrum. We therefore believe that our characterization of the plankton community composition is robust and representative of this region.
Line 129: Dissolved Organic Matter (DOM) does NOT include ‘dissolved oxygen’, though many organic molecules contain oxygen atoms as part of their structure.
I will rephrase this. Dissolved oxygen was one of the variables from this data set that I used however.
Section 2.3: Should there be a diagram (e.g. in supplementary material) of the setup described in this section?
RC1 also asked for a better desription of the set-up which we provided :
Page 4 line 97 :
« SSA was generated using the technique first described in Schwier et al. (2015) for discrete samples, but in a continuous way as descrbed in Trueblood et al. (2021). Briefly, the underway seawater (UWAY, depth ~5 m) is continuously injected in a 10 L tank through 8 1 mm- water jets located a few centimeters from the seawater surface at a rate of 1.25 LPM. Seawater jets hitting the seawater surface created bubbles that, uppon bursting a the surface, eject film and jet drops that evaporate to SSA. The set-up was designed to reproduce at best the conditions described in Fuentes et al. 2011, who measured a bubble size distribution with shape similar to ambiant bubble size distributions observed under open ocean conditions. Our system, under the operation conditions previously described, entrains air into the seawater at a rate that is equivalent to a 9 m s-1 wind speed (Sellegri et al. 2023). The flushing filtered air flowrate in the tank’s headspace was set to 20 LPM, and the bubble system regularly stopped to check that the flushing air contained no particles. The tank was not temperature controlled, but in the SST range observed during the TONGA campaign, it was previously shown that temperature induced changes in the SSA fluxes were negligeable (Sellegri et al. 2023).
Lines 153-158: More methodological information needed. For example, what was the filtration rate?
Line 176: I think something is missing from this sentence. The word ‘either’ implies an alternative.
Indeed : « or by the total number of aerosols »
Line 186: Delete ‘and hypothesis’
ok
Line 191: Should this be changed to ‘Figure 2’?
yes
Figure 2 – Salinity is a dimensionless unit as it is based on the ratios of conductivity measurements. Delete ‘PSU’ as it is not an accepted unit used by oceanographers.
We will delete PSU.
Line 204: Explain ‘the second passing of LAU’. Not clear from the text that the sites or regions were visited more than once. Make sure that this is clear on Figure 1?
The ship first went eastward, and so passed the zones in the order MEL-LAU-WGY, and then went back to Nouméa, passing the zones in the order WGY-LAU-MEL. On the map, this correspond to the increasing number of the stations (eg S1 was the first station, S2 the second one, etc)
Lines 206-207: Delete ‘PSU” as salinity is a dimensionless unit.
ok
Lines 214-215: change ‘nifHgene’ to ‘nifH gene’.
ok
Lines 230-234: Heating the aerosol to denature protein INP is likely to underestimate the numbers and relative significance of proteins as INP. Some proteins are only partially denatured at temperatures around 100 degrees centigrade and some proteins will reform their structure on cooling (e.g. Alsante et al. 2023)
We were not aware of this publication. As Reviewer 1 also suggested that 100°C would be too high a heating temperature that would produced false positives, we also mention this potential artefact and we stress that the heat treatment gives an approximated evaluation of the contribuiton of proteins to the INP activity.
We now mention this potential underestimation : P6L170 : Recent studies highlighted a potential bias introduced by wet heat treatment for detecting biogenic INPs (Daily et al., 2022 ; Alsante et al., 2023). By testing wet and dry heat treatment, Daily et al. (2022) concluded that dry heating at 250 °C was more effective at deactivating biogenic INPs than wet treatment, which could also alter the IN activity of mineral aerosols, thus inducing potential false-positives in studies that used it. Alsante et al. (2023) point that while heat treatment at -95 °C reduces the IN activity of the RuBisCO enzyme, it did not eliminate it completely, suggesting that heating might twist the protein molecules into new shapes that may also be effective INPs. However, in the case of RuBisCO, it’s nucleation temperature is shifted from -8 to -22°C, a temperature which is outside the range of detection limit. However, the bias highlighted by Daily et al. (2022) might be applicable in our study, and we acknowledge that the heat treatment gives an approximated evaluation of the contribution of proteins to the INP activity.
P20L446 : However, recent discussions discussed the potentiel artifacts caused by wet heating for discriminating between biogenic heat labile INPs and non biogenic heat stable INPs (Daily et al., 2022 ; Alsante et al., 2023). We believe that the results highlithted here are relevant despite such biases, nonetheless keeping in mind that the heat treatment here gives only an approximated insight of the contribution of proteinaceous INPs.
Line 248-249: ‘which are known to be active at warmer temperature….’ Add a reference to support this statement.
Refences added : Kanji et al., (2017) ; Huang et al., (2021)
Figure 4 – Would it be helpful to add the locations of the hydrothermal inputs on this map?
A previous version showed the LAU area but we decided to remove it because it added a lot of noise in the figure, but we can re-add it.
Line 282: ‘Note that relationships to the surface seawater biogeochemistry……’ I found this sentence too vague.
We want to point that there are not a lot of correlations between INPs at -13 °C and the other variables. Would changing this sentence to « Correlations to the surface seawater biogeochemistry [...] » be clearer ?
Line 298: Lots of organisms, in addition to Prochlorococcus, contain alpha-carotenes.
New sentence :
This is consistent with the correlation between INP concentrations and carotenes, as many organisms contain α-carotenes, such as the prokaryote Prochlorococcus (Ralf and Repeta, 1992)
Line 306: I don’t understand what a ‘heat sensitive organisms’ is. All organisms are heat-sensitive outside their temperature range.
Organisms that were destroyed by the heat treatment, so the aforementioned « heat labile INPs ». We now use the term « heat-labile organisms » :
P15L306 : « This suggests that a large part of the INP activity at the warmer temperatures can be explained by the concentration of heat-labile microorganisms »
Line 330: Change ‘refractory’ to ‘recalcitrant.’ Recalcitrant is the more applicable terms and it is widely used in the literature. Recalcitrant emphasizes resistance to biological degradation, which is more appropriate for DOM in the ocean. Refractory compounds are resistant to degradation from heat, chemical oxidation etc.
We will change the term in the whole text.
Figure 7a – Is there really a difference between heated and unheated aerosol?
Added P16 L349 : Performing a Wilcoxon-Mann-Whitney test on the data sets showed that the decrease in IN activity from heating was significant.
Line 394-395: The fact that there was only a boost in SSA INP activity near one of the two undersea volcanoes could be used as an argument that hydrothermal activity was not a significant factor in determining INP activity.
However, this increase is not observed near the Simone Volcano like for INP_SW. This might caused by the difference between both volcanoes : Panamax is closer to the ocean surface, while Simone is lower and more complex, hinting at less obvious or less direct biological response to hydrothermal emissions (Guieu et al. 2023). This nonetheless suggests that hydrothermal emissions may boost SSA INPs activity through an increase in biological activity and larger heat stable INPs.
Figure 9 - Could a parametric or even a non-parametric statistical test be used to test the hypothesis that there was a significant difference in ice nucleation between the different zones for different categories of aerosol?
We add this to the manuscript :
P18L389 :
We performed a Wilcoxon-Mann-Whitney test on the data sets in the different zones that showed that there is not enough evidence to reject the null hypothesis and conclude there is a signficant difference in ice nucleation between the different zones for different categories of aerosol.
Lines 410-446: I suggest shortening the conclusion. Much of it was redundant as it was repetitive of the previous text.
New version of the conclusion :
Daily INP concentrations were measured in both surface seawater and in artificially generated SSA from an underway seawater (5 m depth) in the region of the Tonga-Kermadec volcanic arc together with a range of other biogeochemical parameters, providing a unique opportunity to investigate the potential impact of ocean biological properties on the INP variability.
We show that INPs in the bulk surface seawater were generally active at temperatures <-12 °C, indicative of the presence of ice nucleating biological material, which was confirmed by the high fraction of heat labile INP. The INP concentrations in the surface seawater measured during TONGA were overall lower than reported from biologically richer seawaters (Wilson et al., 2015; Irish et al., 2017), but were comparable to other oligotrophic regions (McCluskey et al., 2018a; Trueblood et al., 2021). Surface seawater INP concentrations were about two-fold higher in the mesotrophic Lau basin compared to the oligotrophic Melanesian Basin waters and the ultra-oligotrophic West Pacific Gyre waters at all freezing temperatures, consistent with higher biological activity, and higher abundances of microorganisms such as Prochlorococcus, chlorophyl-a and carotenes, and POC in the Lau basin (e.g. Moutin et al., 2018; Bonnet et al., 2023; Mériguet et al., 2024). Over the whole campaign, medium to strong correlations were found between INP_SW and various biological markers, suggesting that the INP variability, and especially the heat labile fraction, was driven by the POC-type INPs as assumed by McCluskey et al. (2018b).
The nascent SSA generated from these seawaters exhibited generally low nS values compared to other marine regions. In agreement with the INPSW, SSA nS were mostly heat labile and therefore likely to be of biological/organic origin. Submicron SSA nS represented on average the majority of SSA INPs, although this was variable depending on the temperature. Submicron nS were all heat labile while supermicron nS were mainly heat stable, showing that submicron ns was likely the result of small heat labile POC via the ejection of film drops, while supermicron ns were more likely to be caused by heat stable DOC ejected in the form of jet drops.
We provide a transfer function linking SW and SSA INPs, evaluated at 1.70 m-2.LSW over the whole transect. This value was however doubled for heat stable INPs, hinting that heat stable INPs were more efficiently transferred from the seawater to the SSA in the form of jet drops. Our observations suggest that the hydrothermal emissions do not have any direct effect on the composition or properties of the INPs, but rather an indirect effect through the stimulation of biological activity, that, in turn, influences IN properties. This result highlights the importance of studying the marine IN activity along trophic gradients in contrasted waters, especially in regions influenced by hydrothermal activity. Such regions include the Tropical Ocean and the Southern Ocean where measurements are scarce. Combining INP measurements in the seawater and SSA with various biogeochemical measurements allows for a better understanding of the nature of marine INPs, and thus help improve atmospheric models that predict the formation of clouds over pristine marine environments.
Citation: https://doi.org/10.5194/egusphere-2025-3580-AC2
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AC2: 'Reply on RC2', Yannick Bras, 13 Nov 2025
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General comments
This is a very interesting and relevant manuscript investigating ice nucleating particles (INPs) in the Subtropical Pacific Ocean and the influence of hydrothermal activity on marine INPs. This study includes seawater samples collected during the TONGA campaign and sea spray aerosol samples generated from sea spray tank experiments during the campaign. They find that the hydrothermal activity indirectly influences INPs in seawater (as described by the title) through its stimulation of biological activity in the area. The studied regions are areas with little measurement data and the investigation of hydrothermal activity is a topic unexplored with respect to INPs. Therefore, the dataset is of high value to the scientific ice nucleation community.
The manuscript is well written and presented. However, the manuscript would benefit from added discussion/interpretation of the presented results and figures. In addition, the methods description is missing important details. See comments below.
Specific comments:
P1L30: This sentence needs to be reformatted. Is it correct understood that the first number is the total INP transfer and the second is the heat stable INP transfer? If yes, please state more clearly.
In the “hint” statement, please write what you are comparing to. “,hinting that heat stable INPs were more efficiently transferred to the SSA” compared to ...
P4L98: Description of the SSA experiments is lacking in detail. The authors reference papers of similar studies, however, the Sellegri et al. 2005 paper is possibly the same chamber (not clear) but run in that study without water or jets. I would remove this reference as it causes confusion. The tank described in Schwier et al. 2015 seems to be the same as described in the current study.
I suggest adding a few more details to the current description of the sea spray tank. The authors write “jets” in plural – are there several or is it a single plunging jet? If there are several please describe this as not many tanks have several. What flow rate of water through the jet was used? What flow rate of particle filtered air was used? Was the tank temperature regulated?
P6L151: Please check that ‘Sellegri et al 2005’ is the correct reference based on previous possible mix-up.
P6L156: Specify how blank samples were made. Were they also placed in the stage for ~24hours?
P6L159: Samples were collected in November 2019. How long were they stored frozen before INP measurement?
P6L165: The volume of liquid added to the Eppendorf tubes is different for SW and SSA measurements (200µL and 400µL, respectively). The Vali equation takes into account the volume difference when calculating the concentration of INPs. This should be specified in the text, so that the reader understands that the to dataset can be compared regardless of the difference in volume.
P6L168: What does it mean the samples were “being agitated”?
P6L168: The process of filling half the tubes and heating is described twice in the same section. I suggest keeping the following text (start line 172) and rewriting the text starting in line 168.
“Half of the Eppendorf© tubes were then filled with the untreated sample. The seawater samples were then subjected to the same heat treatment as the filter samples, and the second half of the Eppendorf© tubes was filled using the heated samples (Fig. 2).”
P6L170: Comment on the heat treatment study by Daily et al. 2022, where they find that wet INP heat tests at (> 90°C) have the potential to produce false positives. Why did the authors select 100°C?
Daily, Martin I., et al. "An evaluation of the heat test for the ice-nucleating ability of minerals and biological material." Atmospheric Measurement Techniques 15.8 (2022): 2635-2665.
P7L174: What is the limit of detection?
It would also be nice with an idea of the experimental uncertainty e.g. as a number of degrees Celsius. This would give the reader a better understanding of how significant differences between samples freezing temperatures are.
P7L178: Show a figure of the blank sample INP results in the appendix.
State what the “lower temperature that can be reached” is.
P7L180: It is interesting that the heat treatment increased the IN activity. Add a comment on whether this has been seen in other studies (see comment above about paper by Daily et al. 2022).
Section 3.1: It is some really nice data on the seawater characteristics, and the text describes a comparison of the three waters as is viewed in Figure 2. However, I am missing some discussion/interpretation of the comparisons. E.g. What does it mean that the different planktonic microorganisms are in higher abundance in the LAU surface water? Is this expected?
P9L205: The difference in salinity and surface tension between the three waters seems very small (very close to the standard deviation stated). Why is this important to mention?
P9L223: Elaborate on this sentence “The MEL were oligotrophic waters, and WGY were ultraoligotrophic waters.”. How does this fit with the seawater characteristics in Figure 2.
P10L228: It is confusing for the reader to have just read a section where the water types are separated into categories: MEL, LAU, WGY, and now in this section the INP samples are separated into new categories: INP_sw, INP_tot, INP_HS, INP_HL.
It needs to be clearly stated in the start of this section (3.2.1) what the new categories mean and from which waters these INP samples come from. This should also be added to the methods section – a specification of where the INP water samples and air samples where taken (If at all stations, then state that). Also state earlier in the text of section 3.2.1 that the comparison of water types was not possible.
What is the authors definition of heat stable INP (e.g. a specific decrease in IN activity or)?
P15L313: Elaborate on the strong correlation with dissolved iron.
P16L344: I suggest simply writing INP/aerosol instead of shortening to INP/aer as this is likely to confuse readers. I do not believe that “Aer.” is a commonly used abbreviation for units. Same for Figure A3.
Figure 7. In text above figure n_s = surface site density, but in figure caption n_s = nucleation site density. Select one name for the symbol.
Several different labels are written in the caption referring to the same thing. Ns_super = Unheated SUPM?? Use the same label for both legend, caption and text describing the figures.
What do the error bars represent?
P17L357: Split sentence after parenthesis. Elaborate on Córdoba2025 comment, if possible, give n_s values.
P17L375: Elaborate on how this observation contrasts that of McCluskey2018b.
P19L404: It is not clear to me what this transfer function means. Please explain in further detail why the equation ns/INP_sw is used and explain the resulting number in words. In addition, it would be nice to add a reference to other work that has determined a transfer function in a similar way and compare numbers.
The authors could also determine a flux of INPs from water to air using the sea spray tank experimental conditions.
Technical comments
P1L17: Change “study” to “studied”.
P1L18: add “to” after “due”.
P1L22: Consistently use capitol letters. Change to “Particulate Organic Carbon (POC)”.
P2L43: Remove s from “drops”.
P2L44: Consistently use capitol letters. Change to “Cloud Condensation Nuclei (CCNs)”.
P2L50: warmer temperatures compared to what?
P2L59: Split sentence between “coastal sites,” and “and heat labile”. It is confusing that the sentence starts by introducing a specific study and then later mentions other studies.
P3L66: missing parenthesis after references.
P3L73: Remove “the” in front of “bulk seawater” and “SSA”.
P3L91: I suggest replacing “West-East-West with” with “West-East-West reaching”.
Figure 1: I suggest increasing the font size in this figure, especially the legend is difficult to read.
Figure 1: Add to either figure or the caption which stations correspond to MEL, LAU and WGY. This is very helpful for the reader.
P4L100: Change “than” to “to”.
P5L119: Exchange link with details about instrument. The link is just to the campaign website.
P5L120: Add details on the dynotester instrument, e.g. company name.
P6L159: Remove “on land.”
P6L162: Specify the cooling rate for the experiments.
P7L176: Remove “either” or add another type of normalization. This sentence does not make sense.
Figure 2. What is the black square? I assume average/mean? Add this to caption along with a note that the y-axis’ are different and some are in log-scale. This is important when reader is comparing values from the three sites.
P9L220: Add to text how many sampling points for LAU?
P10L235: Add to caption what type of seawater SSW corresponds to.
P13L264: Should read Figure 5?
P13L275: The legend in figure 5. The mean datapoints are squares in the figure but not in the legend for red and blue.
P13L275: Use MEL and LAU as written in text not new abbreviations (Mel. B.).
P13L279: Pearsons’s test – missing an r.
P18L389: Should read Fig. B3 ?
P20L431: Specify what temperature? E.g. sea surface temperature, nucleation/freezing temperature.