• General comment

Comment: In this manuscript, three CLE-ACSV methods (TAC, SA5, SA25) for analyzing the concentration and conditional stability constants of organic Fe-binding ligands using several model ligands (DTPA, phytic acid, desferrioxamine B, ferrichrome, ferrioxamine E, vibriobactin, FA and HA). It is notable that not only comparing the analysis results from the CLE-ACSV titrations, but also this manuscript investigates and discusses the differences in the characteristics of the competing ligands SA and TAC and differences in analytical instruments. Although these points have been pointed out as possibilities for some time, there are few studies that discuss their effects based on the actual measured value. In the Conclusion, it is suggested that future studies of organic Fe-binding ligands in seawater requires alternatives to the current CLE-ACSV method, and I agree. Although the model ligands used in this manuscript are just “model” and can be considered different from the natural ligands in the ocean, understanding the characteristics of each CLE-ACSV method was exactly what was needed for research in this area. In addition to this, I am interested in the influence of the results on the multiple analytical windows method. In recent years, a multiple analytical windows technique with SA has been applied to determine the multiple classes of Fe-binding ligands in seawater (e.g., Bundy et al., 2014, which has been refereed in this manuscript). How do you think about the influence of these results on the evaluation of multiple analytical window method?

Answer: We thank the reviewer for her/his comments

The question about the multiple window approach is very interesting. We think the multiple analytical window approach is a very good step forward to obtain more details on metal speciation, but since it estimates more parameters one has to consider carefully the degrees of freedom in the estimation.

First, this approach will only work if distinct groups of organic metal binding ligands do exist and are not shielded by other organic ligands which have a continuum of ligand sites (thinking of humics and the work of Buffle). Continuing in this line of thinking, this approach has more chance of success with Cu because the difference in alpha factor for Cu-binding organic ligands is larger and concentrations are larger, making distinguishing more than one ligand group easier.

Second, by applying the multiple window approach the added ligand concentration [AL] varies. From the work of Abualhaija and also from our experiments we know that probably the formed metal-added ligand (Me_x-AL_y) species varies with the [AL]. Thus, the range of formed species and whether they are yes of no electro active must be known, together with the conditional binding strengths per Me_x-AL_y species.  It surely complicates matters. For example with SA, with increasing [AL], the non-electro-active species will increase (according to Abualhaija), it might even be at cost of the electro-active species, resulting in a decrease instead of the expected increase of the signal.

Third, the kinetic problem we discuss in our manuscript in the 25µMSA application will interfere with the multiple window approach. When a short equilibration is used the slow kinetics of the exchange between iron and natural organic ligands will probably be influenced differently at different [AL]. We think that overnight equilibration will largely overcome this problem.

Fourth, we suggest the possibility that formation of FeSA₂ is irreversible. We cannot prove this with the experiments we did. However, suppose we are right then this would interfere with the multiple window approach.

Comment: Overall, this manuscript is organized and well-written. I believe that this manuscript is going to have a strong influence on the development of research in this area. After responding to the following minor comments, I think this manuscript can be accepted.

Answer: We thank the reviewer for the interesting comment above and the suggested improvements below.

Minor comments

Comment: Line 17. Fe3+ should be Fe³⁺

Answer: Thank you

Comment: Line 125~160. The "Langmuir isotherm assumption" is often mentioned in this manuscript and the outline of the assumption itself is explained. However, as shown in Line 163(…assumption 2 or 6), for example, it is not clear from this manuscript alone what each assumption number refers to, so please indicate it in the text after Line 132.

Answer: Indeed, this is awkward, we added 1-6 in lines 125-160. 

Comment: Line 185~. Please indicate the temperature conditions of the samples during equilibration period for each experiment in the main text.

Answer: We added at line 207: “Equilibration between the samples and AL was attained at room temperature.” 

Comment: Line 227. Please show the reference information of the UV irradiation for the past experiments related to Co and Cu ligand analyses.

Answer: Rapp, et al. (2017) and Wuttig, et al. (2019) assessed the influence of quartz and FEP vessels on UV-digestion efficiency and contamination. No difference between a FEP bottle and a quartz cuvette was observed with regards to the efficiency of UV-digestion using either vessel material.

We added in the text the references: Rapp et al., 2017; Wuttig et al., 2019.

Comment:  Line 331~. The explanations of the In-cell experiments and bottle experiments are complicated to understand for people outside the field, so I thought it would be nice to have a schematic diagram as a supplementary figure.

Answer: We understand that it is somewhat complicated, we added a scheme in the Supplementary information as Figure S4

Comment: Line 385. “Using Eqs. (1) and (3) give”

Answer: Indeed, thank you, we changed the text as suggested.

Comment:  Figure 1. Please add [A] – [H] in each figure.

In figure [H], the HA concentration is 1 mg and 2 mg in the legend, so please correct it.

Answer: Apologies, this was the wrong figure, we replaced it with the correct one. Thank you for spotting this. Moreover, the complete figure is new, since we changed the colors (see comment at figure 2).

Comment: Line 434. Since the standard deviation can be shown with 3 or more data. If the number of data is 2, strictly speaking, the difference from the average value is the correct notation.

Answer: Thank you, we changed the text as suggested.

Comment: Figure 2. I sometimes suffered from distinguishing the color patterns in these figures. Could you change the color so that it can be easily distinguished in each series?

Answer: We chose these colors to make it easier for persons suffering from color blindness, but we agree that the result is not very good, we made new figures 1 and 2 with other colors.

Comment: Line 449-450. 2A and C?

Answer: Thank you, this is a mistake, we changed it as suggested

Comment: K^cond can be written as K^cond

Answer: The reviewer is correct. 

Comment: Line 461. sweater:    Seawater?

Answer: Yes! Thank you.

Comment: Line 475. Please insert the unit for [L].

Answer: We added nM Eq

Comment: Line 481. There are 3 significant digits and 4 significant digits of log K, so it is better to unify them (only the 0 in the 4th digit disappears?). I think this can be said for the entire manuscript and tables. Or is it due to a difference in method (TAC or SA)?

Answer: Yes we agree and changed all logK^cond  values into values with one digit.

Comment: Line 489. Buck et al. (2007)?

Answer: 2010. Thank you for pointing out this mistake, the 2010 is in the reference list.

Comment: Figure 6. The resolution of the figures is rough.

Answer: We improved the resolution.

Dear Dario Omanović. Thank you for your comment, thank you for your earlier elaborate comments, which improved the manuscript considerably. 

Below the remarks of the reviewer (Reveiwer), followed by our reactions (Answer)

Reviewer: This is an interesting manuscript and a welcome addition to this field, however in reading through its conclusions regarding the underestimation of the model ligand concentrations it is apparent that some key aspects have been overlooked in the analysis to date.

Answer:Thank you Peter Croot.

Reviewer:

1. Independent determination of ligand concentration

For many of the siderophore ligands the purity of commercial sources is not 100% and for desferrioxamine B it is typically 90-95% depending on the manufacturer and lot/batch number. Earlier works also suggested that some siderophores were not stable in solution as they were easily degraded, though other studies have shown that solutions can be stable for days to weeks (Hayes et al., 1994). It is important then to obtain an independent determination of the model ligand concentration, rather than simply assume 100% and using the mass weighed out initially. This is frequently done using ASV in clean KCl or NaCl solutions and titrating with Cu, as then there should be no interferences. In the current work there is no information on how the ligand concentration was assayed prior to analysis.  

Answer: This is an interesting point raised by the reviewer. We did not estimate the purity of the siderophores. A deviation from 100% purity of the siderophores can explain the deviation of [L] from the added amount. However, it does not change the differences between the applications and the discussion on this point. We added in the text that we did not do any research on purity of the siderophores:

At the methods section second line of 3.1.4 lines 238-240: “No tests were undertaken to check the purity of the siderophores. The solutions were used within two weeks after preparation, and kept in the refrigerator in the dark at 4◦C, which should at least for DFOB be short enough to prevent degradation (Hayes et al., 1994)”.

In the discussion we added at lines 525-528 (accepted version): “It is possible that the siderophores used are not of 100 % purity, which would result in a systematic underestimation of L. However, whilst it is interesting to note absolute values, our research focusses on differences between the three applications, which is should not be impacted by any impurities.”

Reviewer

1. Composition of seawater used in this study:

The paper states that a range of leftover samples were used in this study, though there is no information on their salinity or potential for having other metals which may complex the model ligands under the experimental conditions. For example Cu and V may also be present in seawater at significant concentrations to chelate DTPA, siderophores and fulvic/humic acids thus resulting in lower than expected ligand concentrations when titrated with iron. The question then is, which metals could be present under these conditions to outcompete iron for the model ligands tested?  This also is a reminder that all measurements done in natural waters are conditional measurements and this applies to the ligand concentration as well as the stability constant.

Answer:

The reviewer is right, we should have added information on this point. All water used was from the Atlantic Ocean, surface waters were not used. The same water was used per experiment for the three applications, thus the competition between metals for the model ligand should have been equivalent across all three methods. However other metals could have influenced the results. This should have been discussed. As you will see below in our answer, we used part of your text in the addition to the manuscript.

For your information below some info on the samples taken for ligands at depths>100m. We do not give this info in the manuscript, since it might be misleading, we do not know which samples were used, above all they were mixed and for every treatment the three applications received sample from the same UV irradiated mixture:

Parameter      average          stdev   unit      N

pH                   7.93                 0.08                 370

DFe                 0.64                 0.41     nM       434

DMn                0.23                 0.18     nM       434

Salinity            35.09              0.61                 434

We added in the method section at fourth line of section 3 Methods (line 186), after:”Consequently, one batch differs from others with respect to DFe content”:

“, and also potentially in other constituents, such as other trace metals. Since surface samples were not used we do not expect large differences in salinity, the average salinity was 35.09 ±0.61 (N=434), obtained as average of all samples >100 m depth taken for the ligand analysis in Gerringa et al. (2015).”

We added in the discussion section at 4.2 Titrations Line 424: “Differences due to variations in sample materials are assumed to be small. However, a variance in the content of metals that could compete with Fe for ligand sites can have influenced the results and might have caused an underestimation of the model ligand concentration and indirectly also have influenced the value of . This could not have influenced the comparison between the applications, since always the same mixed sample was used per experiment for the three applications. We again emphasize that CLE-AdCSV titrations in natural waters result in the derivation of conditional parameters and this applies to the ligand concentration as well as the stability constant”

Reviewer:

1. Phytic acid is not a strong iron chelator under seawater conditions:

While the earlier study on Phytic acid by Witter et al. (2000) suggested that this ligand was capable of chelating Fe(III) in seawater, subsequent work suggests this isn’t the case. Indeed calculations based on thermodynamic data (Crea et al., 2008; Torres et al., 2005) suggest that no significant complexes would be formed under seawater conditions. Voltammetric studies (Marolt and Pihlar, 2015) do indicate that both Fe(III) and Fe(II) complexes are formed however though they are very weak.  Ultrafiltration studies (Schlosser and Croot, 2008) also indicate that the conditional binding constants in seawater for Phytic acid are significantly lower than that reported in the kinetic titrations of Witter et al. (2000). While Purawatt et al. (2007) using FFF found that Phytic acid reacts with Fe(III) to form colloidal material. These results suggest that Phytic acid is not a strong iron chelator (Luther et al., 2021) and the results reported in the current manuscript should be reinterpreted along those lines.

Answer:

Thank you for this useful information, we indeed should have elaborated on this, since our estimations of  by all methods (except one duplicate obtained with TAC and this value has a large error) are lower than the values of 22.3 (with respect to Fe³⁺) given by Witter et al., 2000.

We added at section 4.2, line 516:

Reviewer:

Lastly a recent paper (Sanvito and Monticelli, 2021) has suggested that pH buffering is not required for measurements such as this though despite earlier works indicating that it is a critical parameter. One aspect where all speciation work could be improved, and the current work should be no exception, is to include the relevant information on the pH scale (Dickson et al., 2016) being used (NBS, total, seawater, free) to describe the system, along with temperature and salinity (ionic strength)  to fully describe the experimental system.

Answer:

We are also convinced that pH is a critical parameter. This applies for the actual measurement as well as the natural conditions (Avendano et al., 2016; Gledhill et al., 2015; Ye et al., 2020; Zhu et al., 2021). We added the pH scale we used, thank you for pointing out this omission. We used the NBS scale added now at the third line of the section 3.2 AL calibration.

author Luis Laglera gave a comment on the paper of Sanvito and Monticelli:

What Sanvito and Monticelli do is leave the pH drift exclusively at the limit layer of the electrode (microns thick) exclusively during the potential scan. In this period H2O2 and OH- are formed at the electrode surface as a result of the oxygen reduction reaction (half wave potential about -0.1 to -0.2 V). Their solution bulk pH is controlled by the natural carbonate buffer since they do not purge, this buffer is not affected. What they claim is that this substantial increase of pH during the few seconds of the voltammetric scan increases the sensitivity. This is something Luis checked personally.

Since the amount of OH- formed is so small, the pH of the limit layer can go to 9 (which can be determined by the drift of the peak potential) but the pH of the bulk of the solution remains constant leaving the sample unaffected (checked with a pH electrode inserted in the cell) (Laglera et al 2016). So, the pH is stable up to the quiescence period and then only the tiny percentage of complexes adsorbed onto the electrode which complexed iron is going to be reduced experience a rise in pH for a few seconds.

Answer references:

Avendaño, L., Gledhill, M., Achterberg, E. P., Rérolle, V. M. C., and Schlosser, C. Influence of ocean acidification on the organic complexation of iron and copper in Northwest European shelf seas; a combined observational and model study. Front. Mar. Sci. 3, 58, 2016 doi:10.3389/fmars.2016.00058.

Gerringa, L.J.A., Rijkenberg, M.J.A., Schoemann, V., Laan, P., de Baar, H.J.W. Organic complexation of iron in the West Atlantic Ocean. Mar Chem. 177:434-446 .doi.org/10.1016/j.marchem.2015.04.007, 2015.

Gledhill, M., Achterberg, E. P., Li, K., Mohamed, K. N., and Rijkenberg, M. J. A. Influence of ocean acidification on the complexation of iron and copper by organic ligands in estuarine waters. Mar. Chem. 177, 421–433. doi:http://dx.doi.org/10.1016/j.marchem.2015.03.016, 2015.

Laglera, L.M, Caprara, S., Monticelli, D., 2016. Towards a zero-blank, preconcentration-free voltammetric method for iron analysis at picomolar concentrations in unbuffered seawater. Talanta 177, 421–433. doi:http://dx.doi.org/10.1016/j.marchem.2015.03.016.

Ye, Y.; Völker, C.; Gledhill, M. Exploring the Iron-Binding Potential of the Ocean Using a

691 Combined PH and DOC Parameterisation. Global Biogeochem. Cycles 2020.

692 GBC20978. https://doi.org/10.1029/2019GB006425.

Zhu, K. Hopwood, M.J., Groenenberg, J. E. Engel, A., Achterberg, E.P. and Gledhill, M., 2021.Influence of pH and dissolved organic matter on iron speciation and apparent iron solubility in the Peruvian upwelling region. Environ. Sci. Technol. 2021, 55, 13, 9372–9383

Reviewer:

References:

Crea, F., De Stefano, C., Milea, D., Sammartano, S., 2008. Formation and stability of phytate complexes in solution. Coordination Chemistry Reviews 252, 1108-1120.

Dickson, A.G., Cam?es, M.F., Spitzer, P., Fisicaro, P., Stoica, D., Pawlowicz, R., Feistel, R., 2016. Metrological challenges for measurements of key climatological observables. Part 3: seawater pH. Metrologia 53, R26.

Hayes, D.M., Reilly, R.M., Lee, M.M.C., 1994. The Pharmaceutical Stability of Deferoxamine Mesylate The Canadian Journal of Hospital Pharmacy 47, 9-14.

Luther, G.W., Mullaugh, K.M., Hauser, E.J., Rader, K.J., Di Toro, D.M., 2021. Determination of ambient dissolved metal ligand complexation parameters via kinetics and pseudo-voltammetry experiments. Marine Chemistry 234, 103998.

Marolt, G., Pihlar, B., 2015. Potentiometric Determination of Phytic Acid and Investigations of Phytate Interactions with Some Metal Ions. 2015 62, 9.

Purawatt, S., Siripinyanond, A., Shiowatana, J. Flow field-flow fractionation-inductively coupled optical emission spectrometric investigation of the size-based distribution of iron complexed to phytic and tannic acids in a food suspension: implications for iron availability. Analytical And Bioanalytical Chemistry 389, 733-742, 2007.

Sanvito, F., Monticelli, D., 2021. Exploring bufferless iron speciation in seawater by Competitive Ligand Equilibration-Cathodic Stripping Voltammetry: Does pH control really matter? Talanta 229, 122300.

Schlosser, C., Croot, P.L., 2008. Application of cross-flow filtration for determining the solubility of iron species in open ocean seawater. Limnology and Oceanography: Methods 6, 630-642.

Torres, J., Dominguez, S., Cerda, M.F., Obal, G., Mederos, A., Irvine, R.F., Diaz, A., Kremer, C., 2005. Solution behaviour of myo-inositol hexakisphosphate in the presence of multivalent cations. Prediction of a neutral pentamagnesium species under cytosolic/nuclear conditions. Journal of Inorganic Biochemistry 99, 828.

We replied in the attached file, because of the length of the comments and answers .