During the Antarctic expedition ANTXXVIII/3 on-board the R.V. Polarstern (January to March 2012; Wolf-Gladrow 2013), salps were sampled from the center of a stable diatom bloom at 51°12.38 S, 12°39.80 W. The chlorophyll-a concentrations within the diatom bloom reached up to 2 μg chlorophyll a L⁻¹ (Hoppe et al., 2015).

For experiments, salps were collected during the night at a sampling depth of 25 m using a bongo net with cod ends lined with plastic to minimize metal contamination. Seawater that was previously collected at a sampling depth of 25 m using a Teflon double diaphragm pump (Wilden A100 with Teflon fittings; Kelair Pumps Australia Pty Ltd) and polyethylene tubing was used to maintain the salps and rinse salps and FPs. The seawater, collected in using trace metal clean techniques, was then directly pumped into a shipboard laminar flow hood inside of a trace metal clean room container. The sampled seawater was either screened through a 200-μm plankton net (screened seawater, SSW) or 0.2-μm filter (filtered seawater, FSW) (Acropak 1000, PALL). The collected salps were gently rinsed three times with this FSW, placed in a 50 L tank filled with SSW and kept in the dark at the in situ seawater temperature (3°C). Every 6–8 h, approximately one third of the SSW was carefully replaced with fresh SSW. From the incubation tanks, FPs were collected with a clean plastic pipette followed by FSW rinse. The FPs were either placed in a 100 mL trace metal clean low density polyethylene (LDPE) bottle filled with FSW for the on-board experiments or stored at −80°C for laboratory experiments. The salp FPs were compact looking pellets packed with particles (described as “type 1” in Iversen et al., 2016).

These experiments aimed to measure short-term effects of freezing and light exposure on dFe and Fe-binding ligands release from salp FPs. Indeed, particulate organic matter may undergo photochemical transformations in the upper surface water that may lead to a production of dissolved organic matter (Pisani et al., 2011) likely to interact with Fe chemistry. In the same manner, freezing causes break-up of the particles within the FPs and will potentially result in release of dissolved organic compounds to the surrounding waters. Considering that salp FPs take about 4–24 h to travel the first 100 upper meters, all the incubations were performed over a period of 24 h (Iversen et al., 2016). The on-board experiments were done in FSW with and without FPs. Two 20 L carboys were filled with FSW and 40 FPs were added to one of them. For consistency, both carboys were rolled and shaken for 20 min in order to homogenize the FPs. The two homogenized seawater batches were then transferred into 4 L polycarbonate bottles that were exposed to three different experimental conditions. Two incubation bottles were (i) frozen at −20°C for 10 h followed by thawing in the dark at sea surface temperature (SST) for 24 h (“freezer”), (ii) incubated for 24 h in the dark (“dark”), or (iii) exposed for 24 h to natural visible sunlight at SST in an on-deck incubator with a “flow-through” seawater system (“VIS”). Finally, 2 L were sampled from each 20 L carboy into 2 L polycarbonate bottles for the analysis of initial parameters (“control”). Treatments “freezer” and “dark” were wrapped in several layers of black plastic to maintain dark conditions whilst treatment “VIS” was contained in clear plastic bags (double wrapped). At the end of the incubation period, unfiltered and filtered (0.2-μm, Nuclepore, Millipore) samples were collected from each incubation bottle for determination of Fe speciation, humic substance-like material (HS-like, weak Fe ligands), dFe and macronutrients. Samples for dFe and macronutrients were analyzed on-board whereas Fe speciation and HS-like samples were stored at −20°C until analysis at the University of Geneva.

We determined the amount Fe leaching from the FPs after a period of 48 h acidification at pH 2.0 (HCl, Ultrapur, Merck). Six FPs were placed in six 30 mL LDPE containers filled with 10 mL of filtered surface seawater (dFe = 0.15 nM) and kept at room temperature before analysis by voltammetry (Laglera et al., 2013) with internal calibration. FP suspensions were not filtered before analysis since voltammetry is not responsive to particulate Fe.

Laboratory experiments were performed in synthetic seawater [AQUIL media based on Price et al. (1989) using major salts only, pH = 8.13] to facilitate Fe binding ligands detection and to extend light exposure to ultraviolet light (“UV”). Four polytetrafluoroethylene-capped quartz sample tubes were filled with 10 mL of synthetic seawater and six FPs were added to two of them. One tube with FPs and one without FPs were placed under a sunlight simulator and exposed to UV and visible light (220–780 nm, 1940 μEinstein, ABET Technologies Sun 2000) for 30 min on ice prior to 24 h incubation in the dark at 4°C on an agitation plate (100 rpm; VWR STD 3500). The two other tubes were placed directly on the agitation plate without exposure to light (“dark”). After incubation, each sample was filtered on to a 0.2 μm polycarbonate filter for analysis of dFe, HS-like and Fe speciation. Samples for Fe speciation and HS-like were stored at −20°C and dFe samples were acidified [0.5% with quartz distilled hydrochloric acid (qHCl), VWR] and left at room temperature until analysis.

The total Fe content in FPs was determined after storing five FPs in a mixture of HCl/HNO3 (pH = 1.52) for 2 months (n = 2) prior being analyzed by Inductively Coupled Plasma Mass Spectrometry (Agilent 7700). Aluminum (Al), nickel (Ni), copper (Cu), zinc (Zn), and molybdenum (Mo) were also quantified. Standard solutions for ICP (Sigma) were used for the calibrations and the blank averages in counts per second were 343 (²⁷Al), 18076 (⁵⁶Fe), 437 (⁶⁴Zn), 2848 (⁶⁵Cu), 120 (⁶⁰Ni), and 247 (⁹⁸Mo).

DFe, HS-like and Fe speciation analyses were performed using a μAutolab potentiostat (Ecochemie, Netherlands) with a 663 Metrohm stand as in Norman et al. (2015) and Laglera et al. (2013). Fe speciation was measured by Competitive Ligand Exchange-Adsorptive Cathodic Stripping Voltammetry (CLE-AdCSV) as per Abualhaija and van den Berg (2014) in order to provide the concentration of Fe-binding ligands (L_T) and the conditional stability of their complexes with Fe (K'_Fe'L). Analysis of SAFe (Sampling and Analysis of Fe) reference sample S (0.101 ± 0.005 nM; n = 3), detection limit (0.05 nM) and side reaction coefficient for salycilaldoxime (SA) at a concentration of 5 μM (α_Fe'SA = 20.1) were in accordance with the literature (Abualhaija and van den Berg, 2014; Mahmood et al., 2015). Peak heights and the chemical speciation parameters were calculated with ECDSoft and proMCC software, respectively (Omanović et al., 2015). Titrations performed on AQUIL samples in the presence of 5 nM desferrioxamine B (DFB; Figure 1C) allowed us to validate our analytical method. Both L_T (4.61 ± 0.13 nM; n = 2) and stability constant (log K'_Fe'L of 12.25 ± 0.04) corresponded with reported values (Witter et al., 2000; Abualhaija and van den Berg, 2014). HS-like compounds were determined using the voltammetric method of Laglera et al. (2007) with standard addition on each sample of Suwannee River Fulvic Acid (SRFA, International Humic Substances Society, standard 1) for calibration. The limit of detection was 1.88 μg SRFA.L⁻¹. DFe was measured onboard by catalytic cathodic voltammetry (Laglera et al., 2013) and a summary of data can be found in (Hoppe et al., 2015). Macronutrients (nitrate (NO3−), nitrite (NO2−), orthophosphate (PO43−), and orthosilicate (Si (OH)₄) were determined colorimetrically on-board using a Technicon TRAACS 800 autoanalyzer (details in Hoppe et al., 2015).

All plastic-ware was trace metal cleaned as per the GEOTRACES guidelines (Cutter et al., 2010) to avoid contamination. Experimental samples were sealed in triple bags during equilibration/incubation periods and all the manipulations were carried out in an ISO Class 5 laminar flow hood.

Salp FP fluxes were collected with free-drifting sediment traps at 100 and 300 m and a detailed description is provided by Iversen et al. (2016). Salp FPs are very large and fairly easy to identify. They are very compact and much bigger than FPs from crustaceans like copepods, amphipods, and krill. They are further surrounded by mucus membrane which differentiate salp pellets from physically formed aggregates such as marine snow. The sediment traps were surface attached to a buoy that provide GPS positions during the deployment. Additionally, the sediment trap had two surface floats for buoyancy as well as 12 small air-filled balls (used as wave-breakers). The trap deployments lasted ~24 h. The collection cylinders at the two depths contained a viscous gel that preserved the size, shape, and structure of the collected aggregates, including salp FPs (Iversen et al., 2016). We measured the x, y, and z dimensions of each salp FP in order to determine its volume and used a carbon to volume ratio to calculate carbon fluxes from salp FPs at 100 and 300 m. The carbon to volume ratio was determined from pellets produced during the pellet production incubation (Iversen et al., 2016).

Statistical analysis was performed using Sigma Plot (Systat Software Inc.). Whether for the on-board or laboratory experiments, the various treatments (“freezer,” “dark,” and “VIS” for on-board incubations; “UV” and “dark” for laboratory experiments) did not have a significant impact on any parameter (one-way ANOVA, p = 0.167). A t-test was therefore performed to verify whether the FPs affected analytical parameters over all the treatments. Finally, the concentration differences related to the presence of FPs are presented as averages calculated over all the treatments.

Results from the determination of leachable Fe from salp FPs and total trace-metals content are summarized in (Table 2). Our analysis revealed a total Fe concentration of 0.33 ± 0.02 nmol Fe FP⁻¹ (n = 2). Fe was in a very refractory form since only 10% was released following acidification to pH 2.0 for 48 h (0.033 ± 0.040 nmol Fe.FP⁻¹, n = 6). Taking into account the total carbon content quantified in salp FPs collected during the same sampling period (17.6 ± 27 μg C FP⁻¹; Iversen et al., 2016), we estimated a Fe:C ratio of 225 μmol Fe mol C⁻¹. Finally, ICP-MS analysis of other trace elements showed that aluminum was the most abundant element found in the FPs (1.45 ± 0.13 nmol FP⁻¹) followed by Fe (0.33 ± 0.02 nmol FP⁻¹), zinc (0.177 ± 0.002 nmol FP⁻¹), copper (0.0.25 ± 0.006 nmol FP⁻¹), nickel (0.011 ± 0.001 nmol FP⁻¹), and molybdenum (0.003 ± 0.000 nmol FP⁻¹).

All measured parameters from the on-board incubation are summarized in the top part section of (Table 3). None of the experimental treatments (“freezer,” “dark,” and “VIS”) significantly increased PO42− or NO_x (NO3− + NO3−) concentrations (p = 0.49 and 0.90 respectively, n = 8). In contrast, an increase in Si(OH)₄ concentrations (p = 0.001, n = 8) by 0.53 ± 0.11 μmol L⁻¹ was observed. No variation in dFe concentration was detected for any of the experimental treatments (p = 0.26, n = 14) despite a potential release of 0.66 nmol Fe L⁻¹ estimated from the salp FPs total Fe content. Results from Fe-binding ligand titrations indicated that FPs did not significantly affect L_T or log K'_Fe'L of the ligand class detected in filtered samples (p = 0.14 and 0.65, n = 8; Figure 1A). Although for unfiltered samples, we observed a slight release of L_T from the FPs (between 0.07 and 0.22 nmol L_T FP⁻¹; Figure 1B), this effect was, however, not statistically significant (p = 0.088 and p = 0.46, n = 6). HS-like concentrations increased significantly in the presence of FPs (p = 0.031, n = 14) by 16.43 ± 9.45 and 30.86 ± 18.57 μg L⁻¹ for filtered and unfiltered samples, respectively, corresponding to a release of 8.22 ± 4.72 and 15.43 ± 9.28 μg HS-like.FP⁻¹ d⁻¹ (since we had a concentration of two FPs per liter).

All measured parameters from the laboratory incubation are summarized in the bottom section of the (Table 3). For both treatments (“UV” and “dark”), the presence of FPs did not have any impact on the dFe concentrations (p = 0.73, n = 4). Similarly to the on-board experiments, we observed a release of humic substances (p = 0.002, n = 4) of 8.74 ± 0.52 μg L⁻¹ corresponding to a daily release of 0.78 ± 0.05 μg HS-like FP⁻¹ d⁻¹. Based on the Fe organic complexation analyses, no ligands were detected in any of the samples (both treatments, with and without FPs) since the titration curves were virtually linear (Figure 1C).

FPs are composed predominantly of organic compounds directly related to the material ingested (Madin, 1982; Manno et al., 2015). Therefore, the increasing dSi(OH)₄ concentrations observed in presence of FPs in our on-board experiments can be linked to the high ingestion rates of the salps at the time of sampling. Accordingly, dSi(OH)₄ concentrations were depleted in the collected FSW (2.93 μmol L⁻¹, Table 3). Our incubations showed that salp FPs released 265 nmol dSi(OH)₄ FP⁻¹ d⁻¹. Using the daily salp FP production (Table 1) determined during the cruise, the integrated Si(OH)₄ release from salp FPs was 82.2 μmol dSi(OH)₄ m⁻² d⁻¹ in the upper 250 m (Table 4). Despite concentrations below 3 μmol L⁻¹ dSi(OH)₄ in the upper 40 m, the Si(OH)₄ standing stock in the upper water column was stable with values ranging between ~600 and 700 mmol dSi(OH)₄ m⁻² (Hoppe et al., 2015). Hence, the daily added dSi(OH)₄ concentrations from salp FPs were almost one order of magnitude below the standing stock, suggesting that the release observed of dSi(OH)₄ (corresponding to our three physico-chemical experimental treatments: “freezer,” “dark,” and “VIS”) would not have substantially contribute to the maintenance of the observed diatom bloom.

Satellite images showed that the diatom bloom maintained high chl a concentrations (> μg L⁻¹) for many weeks (weeks before and after our period of monitoring), implying sufficient Fe supply over all this period. DFe concentrations remained low at 0.1–0.2 in the upper 100 m during our 3 weeks sampling (Laglera et al., 2013; Hoppe et al., 2015). Due to the absence of major “external” Fe inputs at our sampling site, Fe recycling processes should have consequently played a major role in the maintaining of the diatom bloom. From our experimental results, the different treatments that we applied to the FPs did not lead to the release of dFe suggesting that salp FPs could have barely contributed to Fe recycling in the euphotic zone. However, we cannot discount biological processes such as zooplankton grazing on fecal products (coprophagy) and microbial degradation that were not evaluated in this work (Turner, 2002; Dagg et al., 2014; Morata and Seuthe, 2014). Acidification to pH 2.0, a treatment too mild to leach intracellular Fe from intact planktonic cells (Lannuzel et al., 2011), released only 10% of Fe associated with salp FPs (Table 2). In comparison, the same treatment was found to be strong enough to leach ~100% of the Fe in FPs egested by individuals of the SO copepod Calanus simillimus (Laglera et al., unpublished data). This difference might be explained by different ingestion procedures and gut passage efficiencies. Copepods crack and grind what they capture before ingestion, potentially allowing their FPs to be more easily degradable, contrary to the salp that are purely passive filterers (Kiørboe, 2011). In this sense, our results show that the Fe inside salp FPs is in extremely refractory form (possibly due to a low digestion efficiency), supporting the result that physico-chemical processes did not alter our salp FPs content. Accordingly, the zooplankton group dominant in a determined area of the ocean can have an important effect on the percentage of Fe that is recycled in the euphotic zone.

The Fe:C ratio estimated in our salp FPs (225 μmol Fe mol C⁻¹; Table 4) is relatively high in comparison with the commonly observed Fe:C ratios in plankton of HNLC waters, which are in the order of 10–50 μmol Fe mol⁻¹ C⁻¹ (Twining and Baines, 2013; Boyd et al., 2015). In the absence of other studies, it seems from our results that salps have neither significant Fe metabolic requirements nor do they accumulate Fe in their tissues. The enrichment of Fe in salp FPs determined during our experiments seems to demonstrate that most of the Fe ingested by the salps was packed into FPs, contrary to the copepods for whom half of ingested Fe was regenerated to dissolved forms (Schmidt et al., 1999) during the ingestion in a process denoted sloppy feeding. In the same way, using salp FP fluxes we determined an export at 100 and 300 m depth of ~30 nmol Fe m⁻² d⁻¹ and ~11.3 nmol Fe m⁻² d⁻¹ respectively (Table 4). In comparison to the total dFe stock in the upper 100 m at the sampling location (~10–15 μmol Fe m⁻²; Laglera et al., 2013; Hoppe et al., 2015), our export flux represents only ~0.1% of this dFe standing stock, which is in agreement with low Fe exports estimated in previous works where salp FPs represent ~0.3% of the estimated total Fe used by phytoplankton across the SO (Boyd et al., 2012). Taking into account previous estimates of new Fe supplies resulting from island wake (vertical flux of 4 nmol Fe m⁻² d⁻¹, Blain et al., 2007), atmospheric deposition (3.2–51.2 nmol Fe m⁻² d⁻¹, Wagener et al., 2008), and seasonal sea-ice melt (<10–800 nmol Fe m⁻² d⁻¹, Lancelot et al., 2009), our Fe sequestration flux (at 300 m) might significantly reduce new Fe supply in remote areas of limited inputs and high salp concentrations.

Regarding the other trace-metals, Fe was the most abundant essential element after Al, followed by Zn, Cu, Ni, and Mo, which is in accordance with the reported trace metal composition of marine phytoplankton (Twining and Baines, 2013). The high Al content determined in our salp FPs could be explained by the fact that the diatoms are able to take up significant amount of Al (both adsorption and incorporation into soft tissues) during bloom events (Moran and Moore, 1988; Ren et al., 2011) despite its recognized toxicity toward numerous organisms (Gillmore et al., 2016).

Fe-binding ligands are key compounds that control both Fe reactivity (Barbeau, 2006; Rijkenberg et al., 2006) and bioavailability to phytoplankton (Hassler et al., 2011a,b, 2015; Norman et al., 2014). In this study, we could not detect, after any treatment (“freezer,” “VIS,” “UV,” and “dark”), a release of ligands by salp FPs. Such an observation was not due to an absence of ligands in the pellets, but possibly to the reduction of the assimilation and digestion efficiency of the ingested food under blooming conditions referred to above (Von Harbou, 2009); i.e., salps could not break the hard frustule of a good percentage of the ingested diatoms.

Zooplankton grazing is known to be an important autochthonous source of colored and fluorescent organic matter, including marine humic substances (Ortega-Retuerta et al., 2009; Nakayama et al., 2011). In fact, Fe-binding HS-like compounds were released in the presence of salp FPs and we could calculate an HS-like potential flux from FPs of 2.5 and 0.24 mg HS-like m⁻² d⁻¹ for the field and laboratory work, respectively (Table 4). The difference could be explained by the fact that the field-work was carried out in SSW containing bacteria and viruses which could have enhanced the release of HS-like compounds. We therefore concluded that salp FPs could be a source of weak Fe-binding ligands with low log K'_Fe'L likely below our analytical detection limit (α_Fe'SA = 20.1), explaining why we did not detect them by CLE-AdCSV. In line with this, previous work on humic substances showed that the log K'_Fe'L values of their complexes with Fe were around 10.6 (Laglera and van den Berg, 2009), nearly 1 log unit below that of other major ligand groups. Hence, our results show that HS-like compounds contributed to the pool of photo-sensitive HS material likely to be autochtonously produced, such as exopolymeric substances or marine humic substances (Obernosterer and Herndl, 2000; Barbeau et al., 2003; Hassler et al., 2011a; Laglera et al., 2011). Unfortunately, to date, the nature of organic compounds contributing to the pool of HS material detected by CSV remains poorly characterized.

The SO plays a critical role in global ocean circulation (Lumpkin and Speer, 2007) and acts as a significant atmospheric CO₂ sink (Bopp et al., 2001; Sabine et al., 2004). As such, the ocean circulation dynamics of the SO globally affects biogeochemical cycles, biodiversity and climate regulation. The hypothesized increase in salp dominance in the SO due to a depletion of sea-ice (Atkinson et al., 2004) could enhance their annual contribution to carbon vertical flux. Compared to krill, salps consume more food more effectively (Perissinotto and Pakhomov, 1998), produce larger and denser FPs with high carbon content (Gleiber et al., 2012) and have higher sinking velocity (Phillips et al., 2009; Turner, 2015). Increasing abundances of salps over krill could imply important changes to the lower and higher trophic levels (Fraser and Hofmann, 2003) with strong implications for the biological pump activity and associated carbon sequestration.

Based on our incubation experimental settings, salps do not easily release strong Fe ligands as it is the case during grazing by other zooplankton groups such as copepods (Sato et al., 2007). However, the rapid release of weak Fe-binding ligands from salp FPs could affect Fe bioavailability by enhancing Fe solubility and accelerating Fe uptake (Hassler et al., 2011a,b). Therefore, Fe speciation in the presence of salps is difficult to predict as it will depend on food quality and egestion rate, both strongly dependent on the phytoplankton community and chlorophyll concentration present (Von Harbou, 2009).

Our study shows that salp FPs store high concentrations of Fe that are not released rapidly enough by physicochemical processes to enter the recycling loop before sinking below the euphotic zone. Differences in the decrease of organic carbon associated with the bulk of POC (which included particles such as single diatom cells, marine snow, and copepod and krill FPs) and with the salp FPs between the surface (e.g., site of production) and 100 m, indeed suggest that the export efficiencies of salp FPs are 2.5-fold higher (Figure 2). Nevertheless, the organic carbon associated with FPs decrease by a factor of two as they settle down to 100 m, suggesting that biological recycling of FPs occur. In our case, relatively low salp numbers, compared to other reports from the Southern Ocean (Pakhomov et al., 2002; Atkinson et al., 2004; Phillips et al., 2009), allowed that Fe export was kept to a minimum, resulting in less Fe export that what would be exported in situation of high salp abundances. Under such conditions, with a carbon export in the form of salp FPs of about 20 mg C m⁻² d⁻¹ at 300 m depth (Phillips et al., 2009), a Fe:C ratio of 225 μmol Fe mol C⁻¹ would generate an Fe export of ≈ 375 nmol Fe m⁻² d⁻¹ which would deplete dFe stocks in HNLC areas during a period of a few days with consequences for phytoplankton Fe limitation.

More studies on biological remineralization processes associated with salp FPs are needed to estimate whether salp FPs contribute to Fe export. Due to changes in the dominance of zooplankton predicted across the SO and its potential impact for Fe biogeochemistry, comparative studies on the impact of grazing activities and FP degradation rates between salps, krill and copepods are urgently needed.

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.