Edited by: Sylvia Gertrud Sander, University of Otago, New Zealand
Reviewed by: Randelle M. Bundy, Woods Hole Oceanographic Institution, USA; Luis Miguel Laglera, Universidad Islas Baleares, Spain
*Correspondence: Loes J. A. Gerringa
This article was submitted to Marine Biogeochemistry, a section of the journal Frontiers in Marine Science
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In the oxygen-rich layer of the Black Sea, above the permanent halocline, the Fe and nitrate concentrations are low where fluorescence is relatively high, indicating uptake by phytoplankton. In this study we used ligand exchange adsorptive cathodic stripping voltammetry (CLE-aCSV), using 2-(2-Thiazolylazo)-p-cresol (TAC) as measuring ligand, to investigate the role of Fe-binding dissolved organic ligands in keeping Fe in the dissolved phase and potentially biologically available. The conditional stability constant, logK′, was between 21 and 22 in most samples, which is on average lower than in ocean water. The Fe-binding dissolved organic ligand concentrations varied between 0.35 and 4.81 nEq of M Fe, which was higher than the dissolved concentration of Fe (DFe) as found in most samples. At two stations ligands were saturated in the surface. At one station ligands were saturated near the oxycline, where ligand concentrations seemed to increase, indicating that they play a role in keeping Fe in the dissolved phase across the redox gradient. At the fluorescence maximum (between 40 and 50 m), the dissolved organic ligand binding capacity (alphaFeL = K′*[L′]) of Fe was at its highest while the concentration DFe was at its lowest. Here, we find a statistically significant, positive relationship between fluorescence and the logarithm of alphaFeL, along with fluorescence and the ratio of the total ligand concentration over DFe. These relationships are best explained by phytoplankton utilizing Fe from Fe-binding organic ligands, resulting in an increase in free Fe-binding ligands.
The Black Sea is the largest permanently anoxic basin on Earth. It is vertically stratified, caused by dense Mediterranean seawater that sinks as it enters the basin via the Bosporus, flowing below the less dense surface waters that are strongly influenced by river input. A consequence of the strong vertical stratification is a permanent halocline between 50 and 120 m that separates the underlying anoxic layer, containing high sulfide concentrations, from the overlying oxygenated surface layer (OL); between them is a suboxic zone (Sorokin,
In the OL, the concentration of dissolved Fe (DFe) depends on external sources like dust and rivers, and on internal processes such as the dissolution of Fe-containing particles, and the presence of Fe-binding dissolved organic ligands (Rue and Bruland,
DOC concentrations in the OL range from ~125 μM near the OL-suboxic zone boundary to >180 μM at the surface (Ducklow et al.,
As far as we know, Fe-binding dissolved organic ligands have not yet been studied in the Black Sea, although Lewis and Landing (
Approximately 900 mL samples were taken from the ultra-clean CTD and filtered through a 0.2 μm filter using N2 overpressure in a clean-air laboratory unit (Rijkenberg et al.,
Samples were kept at 4°C in the dark. Fe-binding dissolved organic ligand characteristics were analyzed on board no more than 2 days after sampling. Immediately before the start of the analysis of the ligand characteristics, separate samples were taken from the un-acidified samples. These samples were acidified to pH 1.8 and measured according to the description in Section Flow Injection Analysis of DFe. Separate samples were not taken at station 11.
DOC data is from Margolin et al. (
Competing Ligand Exchange—adsorptive Cathodic Stripping Voltammetry (CLE-aCSV) was performed using two setups consisting of a μAutolab potentionstat (Metrohm Autolab B.V., formerly Ecochemie, the Netherlands), a 663 VA stand with a Hg drop electrode (Metrohm) and a 778 sample processor with ancillary pumps and Dosimats (Metrohm), all controlled using a laptop running Nova 1.9 (Metrohm Autolab B.V.). The VA stands were mounted on elastic-suspended wooden platforms in aluminum frames developed at the NIOZ to minimize motion-induced noise, while electrical noise and backup power was provided by Fortress 750 UPS systems for spike suppression and noise filtering (Best Power). Sample manipulations were performed in laminar flow cabinets.
Organic complexation of Fe was determined by CLE- aCSV using 2-(2-Thiazolylazo)-p-cresol (TAC) as a measuring ligand (Croot and Johanson,
1 | 40 | 0.17 | 0.004 | 22.03 | 0.32 | 0.18 | 1.25 | 0.19 | ||||||||||
2 | 9 | 0.56 | 0.000 | 22.19 | NA | 0.37 | 0.35 | 0.08 | ||||||||||
24 | 0.28 | 0.001 | 22.21 | 0.16 | 0.12 | 1.51 | 0.12 | |||||||||||
39 | 0.11 | 0.004 | 22.02 | 0.10 | 0.08 | 1.17 | 0.07 | |||||||||||
45 | 0.15 | 0.005 | 21.48 | 0.15 | 0.11 | 2.09 | 0.40 | |||||||||||
69 | 0.50 | 0.006 | 22.01 | 0.07 | 0.06 | 2.23 | 0.09 | |||||||||||
84 | 1.81 | 0.007 | 20.74 | 0.32 | 0.18 | 4.71 | 3.91 | |||||||||||
3 | 10 | 0.77 | 0.005 | 20.83 | 0.13 | 0.10 | 5.02 | 2.35 | ||||||||||
26 | 0.13 | 0.038 | 21.93 | 0.25 | 0.16 | 1.34 | 0.18 | |||||||||||
41 | 0.06 | 0.002 | 22.53 | 0.31 | 0.18 | 1.65 | 0.11 | |||||||||||
41 | 0.06 | 0.002 | 22.77 | NA | 0.52 | 21.25 | NA | 0.51 | 0.71 | 2.10 | 1.25 | 1.83 | ||||||
54 | 0.29 | 0.029 | 21.66 | 0.13 | 0.10 | 1.38 | 0.17 | |||||||||||
54 | 0.29 | 0.029 | 22.07 | 0.40 | 0.21 | 21.31 | NA | 0.68 | 0.81 | 0.58 | 3.05 | 13.74 | ||||||
70 | 0.26 | 0.008 | 21.94 | 0.15 | 0.11 | 1.57 | 0.15 | |||||||||||
85 | 4.76 | 0.078 | 21.26 | 0.08 | 0.07 | 4.81 | 0.51 | |||||||||||
4 | 9 | 0.92 | 0.016 | 21.28 | 0.09 | 0.07 | 2.65 | 0.41 | ||||||||||
25 | 0.37 | 0.010 | 21.95 | 0.06 | 0.06 | 1.48 | 0.06 | |||||||||||
40 | 0.36 | 0.007 | 21.65 | 0.12 | 0.10 | 2.16 | 0.26 | |||||||||||
40 | 0.36 | 0.007 | 24.38 | NA | 0.54 | 21.02 | 0.23 | 0.15 | 0.41 | 0.09 | 2.75 | 0.87 | ||||||
55 | 0.57 | 0.006 | 21.40 | 0.17 | 0.12 | 1.81 | 0.37 | |||||||||||
70 | 0.25 | 0.002 | 21.33 | 0.49 | 0.23 | 1.15 | 0.52 | |||||||||||
84 | 0.62 | 0.009 | 21.44 | 0.10 | 0.08 | 2.33 | 0.32 | |||||||||||
5 | 10 | 1.05 | 0.018 | 21.20 | 0.59 | 0.24 | 0.96 | 0.36 | ||||||||||
29 | 0.49 | 0.001 | 21.54 | 0.13 | 0.10 | 2.01 | 0.29 | |||||||||||
40 | 0.29 | 0.006 | 21.92 | 0.13 | 0.10 | 1.82 | 0.16 | |||||||||||
54 | 0.50 | 0.008 | 21.86 | 0.11 | 0.08 | 1.80 | 0.14 | |||||||||||
69 | 0.40 | 0.005 | 21.46 | 0.19 | 0.13 | 1.80 | 0.41 | |||||||||||
84 | 0.40 | 0.004 | 21.09 | 1.11 | 0.28 | 1.23 | 1.00 | |||||||||||
6 | 24 | 0.81 | 0.009 | 21.50 | 0.09 | 0.07 | 2.68 | 0.30 | ||||||||||
40 | 0.30 | 0.008 | 21.62 | 0.08 | 0.06 | 2.39 | 0.20 | |||||||||||
40 | 0.30 | 0.008 | 23.78 | NA | 0.44 | 20.90 | 0.15 | 0.11 | 0.32 | 0.09 | 2.83 | 0.60 | ||||||
70 | 0.65 | 0.005 | 21.24 | 0.11 | 0.09 | 2.55 | 0.48 | |||||||||||
84 | 0.53 | 0.004 | 21.40 | 0.15 | 0.11 | 2.36 | 0.48 | |||||||||||
11 | 10 | 1.09 | 0.009 | 22.02 | 0.17 | 0.12 | 1.73 | 0.13 | ||||||||||
25 | 1.98 | 0.011 | 21.69 | 0.25 | 0.16 | 2.76 | 0.32 | |||||||||||
55 | 0.96 | 0.020 | 21.91 | 0.10 | 0.08 | 2.67 | 0.15 | |||||||||||
70 | 0.70 | 0.015 | 21.77 | 0.14 | 0.11 | 2.31 | 0.21 | |||||||||||
85 | 0.46 | 0.006 | 21.84 | 0.13 | 0.10 | 1.94 | 0.17 | |||||||||||
85 | 0.46 | 0.006 | 23.01 | NA | 0.71 | 21.18 | NA | 0.35 | 0.61 | 0.74 | 2.41 | 0.77 | ||||||
12 | 10 | 4.18 | 0.030 | NA | NA | NA | 4.80 | NA | ||||||||||
35 | 4.79 | 0.028 | NA | NA | NA | 4.20 | NA |
Using [Lt] and K′, the concentrations of Fe bound to a natural Fe-binding ligand [FeL], the inorganic Fe [Fe′] and the natural unbound ligand [L′] were calculated using the assumption of chemical equilibrium and the mass balance DFe = [Fe3+](1+1010.1+K′[L′]) and [Lt] = [FeL]+[L′], respectively by repeated calculations using Newton's algorithm (Press et al.,
The ligand characteristics were calculated with two models, one assuming the presence of one ligand class and the other assuming the presence of two ligand classes (Table
The side reaction coefficient of the ligands (alphaFeL, given as log(alphaFeL)) was also calculated as the product of K′ and [L′]. In samples where two ligand classes could be discriminated, two values of alphaFeL were calculated: alphaFeL with the data from the one ligand model and the other with the data from the two ligand model (
The ratio [Lt]/DFe (Table
1 | 40 | 1.25 | 7.16 | 1.90E-13 | 1.06E-09 | 13.06 | ||
2 | 9 | 0.35 | 0.62 | 2.12E-10 | 1.32E-12 | 10.32 | ||
24 | 1.51 | 5.34 | 1.79E-13 | 1.24E-09 | 13.30 | |||
39 | 1.17 | 10.95 | 1.21E-13 | 1.06E-09 | 13.05 | |||
45 | 2.09 | 14.05 | 3.19E-13 | 1.95E-09 | 12.77 | |||
69 | 2.23 | 4.43 | 3.58E-13 | 1.72E-09 | 13.25 | |||
84 | 4.71 | 2.61 | 1.41E-11 | 2.92E-09 | 12.21 | |||
3 | 10 | 5.02 | 6.56 | 3.34E-12 | 4.30E-09 | 12.46 | ||
26 | 1.34 | 10.41 | 1.57E-13 | 1.22E-09 | 13.01 | |||
41 | 1.65 | 26.01 | 1.47E-14 | 1.60E-09 | 13.73 | |||
41 | 1.96 | 30.93 | 1.96E-14 | 6.50E-10 | 1.25E-09 | 13.61 | ||
54 | 1.38 | 4.72 | 7.34E-13 | 1.09E-09 | 12.70 | |||
54 | 3.86 | 13.24 | 2.70E-13 | 6.50E-10 | 2.92E-09 | 13.13 | ||
70 | 1.57 | 5.97 | 2.90E-13 | 1.32E-09 | 13.06 | |||
85 | 4.81 | 1.01 | 1.55E-10 | 2.06E-10 | 11.57 | |||
4 | 9 | 2.65 | 2.88 | 3.50E-12 | 1.74E-09 | 12.52 | ||
25 | 1.48 | 4.00 | 4.68E-13 | 1.11E-09 | 13.00 | |||
40 | 2.16 | 6.04 | 5.59E-13 | 1.78E-09 | 12.91 | |||
40 | 3.16 | 8.82 | 3.11E-14 | 5.91E-11 | 2.74E-09 | 14.16 | ||
55 | 1.81 | 3.18 | 2.28E-12 | 1.24E-09 | 12.49 | |||
70 | 1.15 | 4.51 | 1.66E-12 | 8.98E-10 | 12.28 | |||
84 | 2.33 | 3.77 | 1.64E-12 | 1.70E-09 | 12.67 | |||
5 | 10 | 0.96 | 0.91 | 1.41E-10 | 5.13E-11 | 10.91 | ||
29 | 2.01 | 4.12 | 1.16E-12 | 1.53E-09 | 12.72 | |||
40 | 1.82 | 6.34 | 2.83E-13 | 1.52E-09 | 13.11 | |||
54 | 1.80 | 3.59 | 6.69E-13 | 1.31E-09 | 12.97 | |||
69 | 1.80 | 4.53 | 1.23E-12 | 1.42E-09 | 12.61 | |||
84 | 1.23 | 3.03 | 4.93E-12 | 8.23E-10 | 12.01 | |||
6 | 24 | 2.68 | 3.29 | 1.73E-12 | 1.85E-09 | 12.77 | ||
40 | 2.39 | 8.05 | 4.28E-13 | 2.08E-09 | 12.94 | |||
40 | 3.15 | 10.62 | 1.26E-13 | 4.55E-11 | 2.81E-09 | 13.47 | ||
70 | 2.55 | 3.90 | 2.49E-12 | 1.90E-09 | 12.52 | |||
84 | 2.36 | 4.42 | 1.46E-12 | 1.81E-09 | 12.66 | |||
11 | 10 | 1.73 | 1.59 | 2.04E-12 | 6.44E-10 | 12.83 | ||
25 | 2.76 | 1.39 | 6.45E-12 | 7.88E-10 | 12.59 | |||
55 | 2.67 | 2.79 | 8.63E-13 | 1.70E-09 | 13.14 | |||
70 | 2.31 | 3.29 | 9.32E-13 | 1.60E-09 | 12.98 | |||
85 | 1.94 | 4.17 | 5.71E-13 | 1.49E-09 | 13.01 | |||
85 | 3.02 | 6.50 | 2.32E-13 | 2.12E-10 | 2.34E-09 | 13.40 | ||
12 | 10 | 4.80 | NA | NA | NA | |||
35 | 4.20 | NA | NA | NA |
The DFe concentrations required for data interpretation were measured at sea using an automated Flow Injection Analysis (FIA) (Klunder et al.,
Filtered (0.2 μm, Sartorius Sartobran 300) and acidified (pH 1.8, 2 ml/L 12M Baseline grade Seastar HCl) seawater was concentrated on a column containing iminodiacetic acid (IDA). IDA only binds with transition metals and not the interfering salts. The column was then washed with ultrapure water, and eluted with 0.4 M HCl (Suprapur, Merck). After mixing with 0.6 mM luminol (Aldrich), 0.6 M hydrogen peroxide (Suprapur, Merck) and 0.96 M ammonium (Suprapur, Merck) the reaction pH was ~ 10. The resulting oxidation of luminol with peroxide was catalyzed by Fe to produce a blue light that was detected with a photon counter. The Fe concentration was calculated using a standard calibration line, where a known amount of Fe was added to seawater containing low concentrations of Fe. Using this calibration line, a number of counts per nM Fe were obtained. Samples were analyzed in triplicate and average DFe concentrations and SEs are given (Table
Nitrate was determined on board colorimetrically (Grasshoff et al.,
The density structure of the Black Sea waters (as σΘ in kg m−3) is controlled by salinity resulting from the mixing between Mediterranean Sea and river waters. Temperature is less important for maintaining the basin's density structure. At the surface, temperature varies seasonally, resulting in a temperature minimum at ~50 m referred to as the cold intermediate layer (CIL). The CIL's maximum temperature boundaries are 8°C isotherms, with its core at σΘ≈14.6 kg m−3 (Konovalov and Murray,
Samples were taken at the same depths at each station, however, oxygen concentrations between stations were not consistent with respect to depth, while they were consistent with respect to σΘ (compare Figures
The boundaries of the OL and suboxic zone followed the contours of the σΘ isopycnal surfaces across the basin, which was also reflected in other parameters, like DOC, nitrate, DFe (Figures
At the surface (~10 m), oxygen concentrations were ~240 μM at all stations, increasing to a maximum of 320–340 μM at a depth of ~25 m, with the exception of station 12 near the Bosporus (Figures
At stations 2–6 DOC decreased from >180 μM near the surface to 125–130 μM at 85 m (Figure
Nitrate is low (<0.2 μM) at all stations in the upper ~40 m, increasing to a water-column maximum of 3.9–5.7 μM between ~50 m (at stations 3 and 12) and 150 m (station 6) (Figure
The fluorescence depth distribution is generally determined by nutrient availability from below (see also Figures
DFe concentrations generally ranged from 0 to ~2 nM in the upper 85 m at stations 1–11, with one sample at station 3 having a concentration of 4.67 nM at 85 m (Figure
Between 40 and 85 m, concentrations tended to increase slightly with depth at stations 1, 4, and 6 and increased considerably at stations 3 and 5. The high DFe concentrations coincided with samples taken from below the oxycline (Figure
[Lt] varied between 0.35 and 4.81 nEq of M Fe, with the highest concentration ranges found in the samples collected near the surface and at 85 m (Figure
The values of logK′ were between 20.74 and 22.55 for all samples, but most of them were between 21 and 22 (25 of 34 samples) (Table
In three samples (10 m samples at stations 2 and 5 and 85 m sample at station 3), the ligands were saturated resulting in a low ratio [Lt]/DFe (between 0.6 and 1). In all other samples [Lt]/DFe was between 1.5 and 26, with highest values typically at 40–54 m depth (Table
When the more complex two ligand model was applied, two ligand classes could be discriminated in 5 samples: station 3 at 40 and 54 m, station 4 at 40 m, station 6 at 40 m and station 11 at 85 m (Table
The low salinity of the OL in the Black Sea (17.17–20.71) would favor higher logK′ values, since ions have higher activities at lower ionic strength. To calculate the ligand characteristics we used a binding constant between our measuring ligand TAC and Fe of logβFe(TAC)2 = 22.4, as estimated by Croot and Johanson (
Samples were not kept at the ambient redox conditions prior to analysis, however, there wasn't a noticeable offset between DFe in our samples and those measured in samples that were immediately acidified. Even in the samples with ligands that were (nearly) saturated with Fe, including the only sample taken below the oxycline (from 85 m at station 3), Fe did not precipitate in the sample bottles prior to analysis. Thus, correct determinations of DFe were obtained. Apparently even after changing the redox conditions, the ligands kept Fe in the dissolved phase.
Compared to the Fe ligand characteristics measured in other marine environments, the logK′ of 21–22 obtained here is relatively low, while a [Lt] of 1–2.8 nEq of M Fe is similar to values found by others in a diverse range of oceans and seas (Rue and Bruland,
The Black Sea has a large river input, according to Margolin et al. (
At stations 1–6, DFe was slightly elevated at the surface and high near the redoxcline. In the upper 50 m of stations 11 and 12 near the Bosporus (excluding the surface sample at station 11), DFe was higher than in the rest of the basin. Rivers are the most probable sources of DFe for these coastal stations compared to the basin interior. At station 6, DFe is higher in the upper 40 m than at stations 1–5 and 11. According to the findings of Margolin et al. (
The anoxic water in the deep basin is known to be a source of Fe from below (Spencer and Brewer,
In the OL at stations 3 and 4, the Fe-binding dissolved organic ligands are higher near the surface than below, which is not observed in the other stations (Figure
[Lt] tend to be elevated in the suboxic zone below the oxycline (Figure
Complex redox cycling, such as the oxidation of reduced Fe(II) as it diffuses upwards, and the reduction of sinking Fe(III)(hydr)oxides occur near the redoxcline (Spencer and Brewer,
The ratio [Lt]/DFe had a maximum where ligands were relatively under saturated, corresponding to high fluorescence (Figures
Logalpha | Fluorescence | All | 34 | 0.19 | <0.01 | 0.20 | <0.01 | ||
-st11 | 29 | 0.28 | <0.001 | 0.31 | <0.001 | ||||
-st11 – 3 | 26 | 0.43 | <0.001 | 0.41 | <0.001 | ||||
[Lt]/DFe | Fluorescence | All | 34 | 0.35 | <0.001 | 0.39 | <0.001 | ||
-st11 | 29 | 0.53 | <0.001 | 0.35 | <0.001 | ||||
-st11 – 3 | 26 | 0.49 | <0.001 | 0.59 | <0.001 |
The increasing log(alphaFeL) results from an increase in [L′]. [L′] can increase due to an increase in [Lt], an accumulation of ligands by for example microbial production (Rue and Bruland,
Fe complexed by natural dissolved organic ligands is biologically available, as observed by Maldonado et al. (
Acknowledging the existence of two ligand classes in five samples improves the correlation between fluorescence with log(alphaFeL) and with [Lt]/DFe even more (Figure
Although a relationship between [Lt] and the fluorescence maximum has been observed before (Gerringa et al.,
Compared with ligand characteristics from open ocean environments the logK′ of 21–22 measured in the OL of the Black Sea is relatively low. This is probably due to the more coastal sources of DOC (i.e., terrigenous origin), and is likely not because of the Black Sea's lower salinity. [Lt] of 1–2.8 nEq of M Fe is similar to the average values found in other seas and oceans.
Sampling at different redox environments where different redox processes occur, did not affect the logK′ within the detection window of our method (Apte et al.,
A significant relationship existed between the alpha coefficient of the dissolved organic ligands and fluorescence, and also between the ratio [Lt]/DFe and fluorescence. These relationships are best explained by Fe bound by dissolved organic ligands being utilized by phytoplankton.
An interesting observation was that [Lt] increased near the suboxic zone at most stations. Transport of Fe over the oxic—anoxic boundary depends strongly on redox processes and the solubility of Fe. Organic complexation of Fe, affecting Fe solubility and redox processes, can therefore play an important role in the vertical transport of Fe in the Black Sea (Santana-Casiano et al.,
MR, LG, and HD were responsible for the design of this research. LG, JB, PL, and AM analyzed data. LG and MR did data interpretation. The first draft of the manuscript was made by LG and MR. All other co-authors contributed considerably to make the second and third drafts. All authors agreed on the final version of this manuscript.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
We thank Captain Pieter Kuijt and his crew of RV Pelagia for their hospitality and help during cruise 64PE373. We further thank everybody involved at Royal NIOZ who made this expedition possible. Jan van Ooijen measured nitrate. Sharyn Ossebaar measured hydrogen sulfide. Nikki Clargo and Lesley Salt measured oxygen for Sven Ober and Hendrik van Aken to calibrate the oxygen sensors. We thank Nikki Clargo for calculating pH values for us on a very short notice. We acknowledge the Dutch funding agency (project number: 822.01.015) of the national science foundation NWO for funding of this work as part of GEOTRACES. The data were collected within the GEOTRACES programme and can be requested at the British Ocean Data Centre (