MicroPat, a 10-treatment microcosm mirroring the MesoPat mesocosm (treatment design as per MesoPat, but with six 20 L containers per treatment rather than a single HDPE tank) and two 16-treatment multistressor experiments (MultiPat/MultiArc) were conducted using artificial lighting in temperature-controlled rooms (Table 1). Coastal seawater, filtered through nylon mesh, was used to fill 20 L HDPE collapsible containers. The 20 L containers were arranged on custom-made racks with a light intensity of 80 µmol quanta m-2 s-1, approximating that at ∼3 m depth. Lamps (Phillips, MASTER TL-D 90 De Luxe 36W/965 tubes) were selected to match the solar spectrum as closely as possible. A diurnal light regime representing spring/summer light conditions at each field site was used (Table 2) and the tanks were agitated daily and after any additions (e.g. glucose, acid, or macronutrient solutions) in order to ensure a homogeneous distribution of dissolved components. In all 20 L scale experiments, macronutrients were added daily. One 20 L container from each treatment set was emptied for sampling each sample day.

The experimental matrix used for the MultiPat/MultiArc experiments duplicated the MesoPat/MesoArc design, with an additional pH manipulation: ambient and low pH. The pH of “low” pH treatments was adjusted by a single addition of HCl (trace metal grade) on day 0 only with pH measured prior to and after the addition (Table 2). Sample water from 20 L collapsible containers was extracted using a plastic syringe and silicon tubing which was mounted through the lid of each collapsible container. Throughout, where changes in Meso/Micro/Multi experiments are plotted against time, “day 0” is defined as the day the experimental gradient (zooplankton, DOC, pH, pCO2) was imposed. Time prior to day 0 was intentionally introduced during some experiments to allow water to equilibrate with ambient physical conditions after mesocosm filling. Fe(II) concentration varies on diurnal timescales, and thus during each experiment where a time series of Fe(II) or DFe concentration was measured, sample collection and analysis occurred at the same time each day.

Fe(II) concentrations and other key biogeochemical parameters were measured in ambient surface (∼10–20 cm depth) water at all three experiment locations: Comau fjord for Meso/Micro/MultiPat (Patagonia, November 2014), Kongsfjorden for Meso/MultiArc (Svalbard, June 2015), and Taliarte (Gran Canaria, March 2016). FIA apparatus was assembled in waterproof boxes on floating jetties. A 3 m PTFE sample line was then positioned to float approximately 1 m away from the jetty with seawater continuously pumped into the FIA using a peristaltic pump (MiniPuls 3, Gilson). The time delay between water inflow into the PTFE line and sample analysis was 60–120 s. Complementary chemical parameters (TdFe, DFe, DOC and pH) were determined on samples collected by hand using trace metal clean 1 L LDPE bottles. Salinity and temperature data were collected with a hand-held LF 325 conductivity meter (WTW) calibrated with KCl solution. To compare Fe(II)/H2O2 FIA data to discrete DFe/TdFe samples, the mean of seven FIA data points, corresponding to 14 min of sample intake and analysis time, was used.

A series of experiments was conducted during Meso/Micro/MultiPat, during Meso/MultiArc (n=79), and under laboratory conditions using filtered Atlantic seawater (n=46) to investigate the change in Fe(II) concentration when water was moved from ambient light into the dark. Fe(II) decay experiments were conducted inside the temperature-controlled rooms hosting the MultiPat/MultiArc experiments. As such, a constant temperature was maintained throughout these experiments. Sub-samples for Fe(II) analysis or decay experiments were always collected when the mesocosms had been untouched (i.e. no sampling or additions) for >12 h; thus, Fe(II) species could not plausibly have been directly perturbed by any external manipulation of the mesocosm/microcosm/multistressor experiments. After collection of unfiltered 1–2 L samples in transparent 2 L HDPE containers, the PTFE FIA sample line was placed into the sample bottle and continuous analysis for Fe(II) and H2O2 begun. After a stable chemiluminescence response was obtained (typically 2–4 min after first loading the sample), the sample bottle was moved to an Al foil-lined dark laminar flow hood and analysis continued for >1 h or until Fe(II) concentration fell below the detection limit (∼0.2 nM). The time at which the sample was moved into the dark was designated t=0. Subsamples for the determination of DFe and TdFe were retained from this time point.

Theoretical decay rate constants (k′) for these experiments were calculated using the formulation presented in Santana-Casiano et al. (2005) with measured pH, temperature, dissolved O2, and salinity as per Eq. (1), where T is temperature (K), pH is pHfree, and S is salinity (psu). O2 saturation was calculated as per Garcia and Gordon (1992) and then k′ was adjusted for measured O2 concentrations as per Eq. (2). Measured rate constants (kmeas) were derived from the gradient of ln⁡[Fe(II)] against time for each decay experiment from at least five sequential data points (Fe(II) concentration was obtained at 2 min intervals). log⁡k′=35.407-6.7109×pHfree+0.5342×pHfree2-5362.6T-0.04406×S0.51-(0.002847×S)2k=k′[O2] Dissolved O2 was measured using an Oxyminisensor (World Precision Instruments). Salinity and temperature for each experiment were measured using a hand-held LF 325 conductivity meter (WTW). Measured decay rates were determined, assuming pseudo-first-order kinetics, from linear regression of ln⁡[Fe(II)] for t 0–15 min. Fe(II) decay experiments under laboratory conditions used aged, filtered (0.2 µm) Atlantic water. This water was previously stored filtered in 1 m3 trace element clean HDPE containers for in excess of 1 year and maintained in the dark at experimental temperature for 3 d prior to commencing any experiment.

During MesoArc a “bookkeeping” exercise was conducted for the mesocosm experiment by the sub-sampling of all solutions added to the incubated seawater. Aqueous additions consisted of HCl solution (used to apply the pH gradient), macronutrient solution, glucose solution, and zooplankton. A short (1–2 h) 1 M HCl (trace metal grade) leach was applied to equipment placed within the mesocosm and also to the HDPE mesocosm containers prior to filling to provide a quantitative estimate of “leachable” Fe. Atmospheric deposition of Fe into the tanks when open was estimated by deploying open bottles of de-ionized water within the vicinity of the mesocosms for fixed time intervals of 1 h in triplicate on three occasions and recording the approximate extent of time when the mesocosm lids were removed. All additions to the MesoArc mesocosm experiment were volume weighted as per Eq. (3) using the mean (mid-experiment) mesocosm volume (Vmesocosm) and assuming that all additions were well mixed and TdFe behaved conservatively. Δ[TdFe]mesocosm=VadditionVaddition+Vmesocosm3×[TdFe]addition

In order to provide a rigorous assessment of Fe contamination during one experiment, Fe inputs were tracked in all additions to MesoArc and scaled to the mesocosm volume (initially 1200 L, declining by 15 % over the experiment duration). Volume weighting all additions (Table 3) to the MesoArc mesocosm experiment as per Eq. (3) produced a total mean concentration of 48 nM TdFe (Fig. 1). In addition to the uncertain variability arising as the mesocosms were filled, approximately 8 % (3.6 nM) of TdFe within the MesoArc experiment could be attributed to inadvertent addition (Fig. 1) over the experiment duration.

When MesoArc is compared to the two other mesocosms with a similar design (MesoPat and MesoMed), the TdFe inputs and the relative contribution of inadvertent TdFe addition were 66.9 nM TdFe with 4.8 % arising from inadvertent addition for MesoPat and 13.3 nM with 24 % TdFe arising from inadvertent addition for MesoMed (Fig. 1). Systematic contamination was in all cases a minor, yet measurable, source of TdFe for these inshore mesocosms. Strictly, the inadvertent input of TdFe varied between different treatments within each mesocosm experiment due to, for example, the variable volume of glucose solution used to create a DOC gradient (Table 1). However, these differences caused small or negligible changes in TdFe addition (Table 3).

Concentrations of both DFe and H2O2 (as per Hopwood et al., 2020) were measured at the highest resolution for the baseline treatments (no DOC addition, no zooplankton addition) during the mesocosm experiments. For MesoPat (Fig. 2), the initial concentration of DFe and H2O2 was estimated by using a Go-Flo bottle to sample at a depth of 10 m in the fjord (at which approximate depth the mesocosms were filled from). The apparent rise in H2O2 between day 0 and day 1 (Fig. 2) likely reflects the result of increased formation of H2O2 after pumping of water from ∼10 m depth into containers at the surface. NO3 was added daily (Table 2); hence, concentrations increased prior to the onset of a phytoplankton bloom. The decline in DFe likely reflects biological uptake and/or scavenging onto particle (>0.2 µm) or mesocosm container surfaces.

Less frequent temporal resolution was available for treatments other than the “baseline” (no DOC/zooplankton addition) treatment, but the decline in DFe during the MesoPat mesocosm was apparent across all measurements considered together. In addition to TdFe measurements from unfiltered water samples, particulate (>0.6 µm) Fe concentrations were also determined from wavelength dispersive X-ray fluorescence. WDXRF data were normalized to phosphorus (P) in order to discuss trends in the elemental composition of particles and are thus presented as the Fe:P (mol Fe mol-1 P) ratio. The initial Fe:P ratio in particles varied between the mesocosm field sites: MesoPat 0.34±0.09 and MesoArc 0.62±0.07. A similar trend however was observed during all experiments: a general decline in Fe:P across all treatments with time. Particulate Fe:P ratios on the final day of measurements were invariably lower than the initial ratio: MesoPat 0.09±0.04, MicroPat 0.05±0.01, MultiPat 0.07±0.03, and MesoArc 0.17±0.08. All of these ratios are high compared to literature values reported for offshore stations where the ratio for cellular material ranged from 0.005 to 0.03 mol Fe mol-1 P (Twining and Baines, 2013). However, this may simply reflect elasticity in Fe:P ratios which increase under high DFe conditions (Sunda et al., 1991; Sunda and Huntsman, 1995). Alternatively, it could reflect the inclusion of a large fraction of lithogenic material, which would be expected to have a higher Fe:P ratio than biogenic material (Twining and Baines, 2013).

Particles from ambient waters outside the mesocosms were collected and analysed at the Patagonia and Svalbard field sites in order to assist in interpreting the temporal trend in Fe:P. Suspended particles from Kongsfjorden (Svalbard) exhibited a Fe:P ratio of 3.01±0.06 mol Fe mol-1 P and suspended particles in Comau fjord (Patagonia) varied more widely, with a mean ratio of 0.54±0.41 mol Fe mol-1 P. Kongsfjorden surface waters are characterized by extremely high TdFe concentrations originating from particle-rich meltwater plumes (Hop et al., 2002) and thus the 3.0 Fe:P ratio can be considered to be a lithogenic signature. After ambient water was collected for the mesocosm experiments, the steady decline in particle Fe:P ratios throughout the experiments likely resulted partially from a settling or aggregation of lithogenic material after filling of the mesocosms. At the same time, a decline in the ratio of dissolved Fe:PO4 during each experiment, due to the daily addition of PO4 and minimal addition of new Fe, may also have led to reduced Fe uptake relative to P.

A key focus of this work was to determine the fraction of DFe present as Fe(II). During the Gran Canaria mesocosm, a detailed time series of Fe(II) concentrations was conducted. The timing of sample collection was the same daily (14:30 UTC) in order to minimize the effect of changing light intensity over diurnal cycles on measured Fe(II) concentrations. Over the duration of the Gran Canaria mesocosm, Fe(II) concentrations fell within the range 0.10–0.75 nM (Fig. 3a). On the first measured day (day -2) Fe(II) ranged from 0.13 nM (mesocosm 7, 700 µatm pCO2) to 0.63 nM (mesocosm 6, 1450 µatm pCO2), with an overall mean (± standard deviation) concentration of 0.41±0.12 nM. From days 9 to 20 strong variations were observed between treatments. Following nutrient addition on day 18, a phytoplankton bloom was evident in chlorophyll a data from day 19 or day 20, with chlorophyll a peaking on day 21 or later (Hopwood et al., 2018). An increase in Fe(II) was then evident from days 20 to 29 under bloom and post-bloom conditions (Fig. 3b).

Contrasting days 1 and 29, Fe(II) in all of the mesocosms except the 700 µatm pCO2 treatment experienced a measurable increase in Fe(II) concentration (+0.4, +0.4, +0.2, +0.2, +0.2, 0.0, and +0.3 nM). The 700 µatm pCO2 treatment was also anomalous with respect to slow post-bloom nitrate drawdown and elevated H2O2 concentration (100 nM H2O2 greater than other treatments under post-bloom conditions, Hopwood et al., 2018). Overall, despite the large gradient in pCO2 (400–1450 µatm and a corresponding measured pH range of 8.1–7.7), Fe(II) showed no significant correlation with pH (Pearson product moment correlation p0.32) (Fig. 3a).

In a companion text presenting H2O2 results from the same series of experiments (Hopwood et al., 2020), a series of experiments in the Mediterranean (MesoMed/MultiMed) is also included. During these Mediterranean experiments however the rapid oxidation rate of Fe(II) precluded the determination of Fe(II) concentrations. Fe(II) concentrations were universally <0.2 nM (i.e. below detection), and thus no Fe(II) results from the “Med” experiments are presented herein. During the MesoArc and MesoPat experiments, a series of decay experiments was conducted to investigate the stability of in situ Fe(II) concentrations. The 79 time points at the start of these experiments were made before water was moved from ambient lighting into the dark and can be considered in situ Fe(II) concentrations. Across the complete dataset, the properties known to affect the rate of Fe(II) oxidation in seawater varied over relatively large ranges for the various experiments: temperature 4.0–18 ∘C, salinity 22.7–33.8, pH 7.46–8.44, 315–449 µM O2, and 1–79 nM H2O2 (see Supplement). Initial Fe(II) concentrations ranged from 0.3 to 16 nM. Generally a decline in Fe(II) was observed immediately after transferring this sampled water to a dark box, yet this was not always the case. The Fe(II) concentration more often than not remained measurable (>0.2 nM) for the entire duration of the decay experiment. One hour after the transfer of water from ambient conditions into the dark, Fe(II) was below detection on only 2 out of 79 occasions, and on average 55 % of the initial Fe(II) concentration at t=0 remained.

In order to account for the many physio-chemical parameters that affect Fe(II) oxidation rates, theoretical pseudo-first-order rate constants (k′) were calculated for each decay experiment assuming pseudo-first-order kinetics (correlation coefficients are noted for each linear regression – Supplement). The rate constant, k (Eq. 1), thus accounts for the major effect of variations between experiments of salinity, temperature, pH, and O2 in a single constant (Fig. 4). Before comparing kmeas and k, an estimate of the uncertainty should also be made as differences between the two values may arise due to the relatively large combined error from propagating the uncertainty in S/T/pHfree/[O2], and in analytical error on Fe(II) measurements. The accuracy of Fe(II) measurements is challenging to quantify for a transient species with no appropriate reference material. In this case, the exact Fe(II) detection method used here was previously compared to another variation of the luminol chemiluminescence method (with pre-concentration, Bowie et al., 2002), and kmeas was determined with ±20 % difference between the two methods. The uncertainty in kmeas is therefore assumed to be ±20 % rather than the generally smaller uncertainty that can be calculated from linear regression of ln⁡[Fe(II)]. The uncertainty in calculated k was assessed by calculating the change resulting from the estimated uncertainty in measured salinity (±0.1), temperature (±0.5 ∘C), pHfree (±0.05), and O2 (±10 µM). The combined uncertainty is ±35 % for k. Reduced uncertainties are possible with closed thermostat systems where the uncertainty in all physical/chemical parameters (S/T/pH/O2) would be reduced; however, our objective here was to measure the decay rates of in situ Fe(II) concentrations, and thus the first priority was to commence measurements after sub-sampling rather than to stabilize physical/chemical conditions.

In order to further understand the cause of any systematic discrepancies in the dataset between measured kmeas and calculated k, an additional set of experiments was conducted using aged, filtered Atlantic seawater (Fig. 4). The background concentration of Fe(II) in this water was below detection (<0.2 nM) and the initial DFe concentration relatively low (0.98±0.39 nM). In a series of 46 decay experiments, Fe(II) spikes of 2–8 nM were added and then the decay in the dark monitored as per the Meso/Micro/Multi Arc/Pat in situ experiments.

Assembling and maintaining mesocosm-scale experiments under trace-element clean conditions is a logistically challenging exercise (e.g. Guieu et al., 2010) and thus it was desirable to conduct a thorough assessment of the extent to which Fe concentrations were subject to inadvertent increases during at least one experiment. All of the incubation experiments herein were conducted using coastal or near-shore waters. This is reflected in the low salinities of the MesoPat (27.5–28.0) and MesoArc (33.7–33.8) mesocosms. Both of these field sites were fjords with high freshwater input. Comau fjord (Patagonia, MesoPat) is situated in a region with high annual rainfall and receives discharge from rivers including the River Vodudahue. Kongsfjorden (Svalbard, MesoArc) receives freshwater discharge from numerous meltwater-fed streams and marine-terminating glaciers in addition to melting ice. Correspondingly high DFe and TdFe concentrations were thereby found in surface waters, universally >4 nM DFe. The Gran Canaria (initial S37.0) mesocosm cannot be considered to have had a coastal low-salinity signature from freshwater outflows, but was still conducted using near-shore waters which would generally be expected to contain higher Fe concentrations than offshore waters due to sedimentary sources of Fe (see, for example, Croot and Hunter, 2000). Despite the inshore basis of MesoArc, Fe contamination was a small, but significant, fraction of the TdFe added to the starting water (8 %, 3.6 nM, Fig. 1). It is not anticipated that this small TdFe addition will have had any adverse effect on the Fe redox chemistry results presented herein for the Meso/Micro/Multi Arc/Pat experiments.

Throughout all of the Meso/Micro/Multi Arc/Pat experiments, Fe(II) consistently constituted a large fraction of DFe (Table 4). The presence of 24 %–65 % of DFe in mesocosms as Fe(II) is not unexpected, as the photoreduction of Fe(III) species by sunlight is well characterized (Wells et al., 1991; Barbeau, 2006). Yet it also raises questions about how Fe speciation is modelled in these waters. DFe in the ocean is widely assumed to be characterized as “99 % complexed by organic species” (Gledhill and Buck, 2012) on the basis of extensive research using voltammetric titrations to determine the strength and concentration of Fe-binding ligands (Van Den Berg, 1995; Rue and Bruland, 1995). Yet these approaches exclusively measure Fe(III)-L species (Gledhill and Buck, 2012).

Here we should note that the method utilized during these incubation and diurnal experiments, flow injection analysis with a PTFE line inserted directly into the experiment water, is relatively well suited for establishing the in situ concentration of Fe(II) (O'Sullivan et al., 1991). Such an experimental setup ensures no unnecessary delay is introduced between the collection and analysis of a sample. When using an opaque sampler, such as a Go-Flo bottle typically deployed at sea for collection of trace element samples (Cutter and Bruland, 2012), the collection process inevitably displaces near-surface water from its ambient light conditions for a time period that constitutes >1 half-life of Fe(II) in warm, oxic seawater. Measured near-surface Fe(II) concentrations on samples from a rosette system would therefore always be expected to under-estimate in situ near-surface Fe(II) concentrations (O'Sullivan et al., 1991).

Fe(II) concentration was also quantified in ambient waters adjacent to the mesocosms and found to constitute a lower fraction of DFe (2 %–11 %). Most of the decay experiments, from which initial Fe(II) concentrations are reported (Table 4), were conducted at the end of Meso/Micro/Multi experiments, and thus it is not possible to assess the development of Fe(II) stability throughout a phytoplankton bloom. Nevertheless, the high fraction of DFe present as Fe(II) in these experiments (Table 4) relative to that observed in ambient waters is consistent with the increase in Fe(II) concentrations observed in Gran Canaria after the initiation of the phytoplankton bloom (day 19 onwards, Fig. 3b). The Meso/Micro/Multi Arc/Pat experiments had macronutrient additions daily, whereas the Gran Canaria experiment had macronutrient addition only on day 18. The conditions within the Meso/Micro/Multi Arc/Pat experiments during the time period in which decay experiments were conducted were therefore typical of those during, or shortly after, a phytoplankton bloom. Whilst chlorophyll a was not quantified for ambient waters, for which Fe(II) data are reported (Table 4), sampling in Svalbard (MesoArc, July 2015) and Patagonia (MesoPat, November 2014) occurred during relatively low-productivity phases of the annual cycle in primary production at these field sites (Hop et al., 2002; Iriarte et al., 2013). The ambient concentrations of Fe(II) measured at the mesocosm experiment field sites are therefore not necessarily directly comparable to Fe(II) concentrations measured after nutrient addition in the corresponding mesocosm experiments.

Fe(II) oxidation rates are relatively well constrained in seawater with varying temperature, salinity, pH, H2O2, and O2 concentrations from extensive series of experiments where the change in concentration of an Fe(II) spike was monitored with time and the rate constants for oxidation with O2 and H2O2 were then derived from first-order kinetics (Millero et al., 1987; King et al., 1995). Whilst dissolved O2 is the dominant oxidizing agent for Fe(II), H2O2 is also of importance as an Fe(II) oxidizing agent in surface seawater (Millero and Sotolongo, 1989; King and Farlow, 2000; González-Davila et al., 2005). The unusually low concentration of H2O2 within the Meso/Micro/Multi Arc/Pat experiments due to the enclosed HDPE mesocosm design and/or synthetic lighting (Hopwood et al., 2020) was therefore fortunate from a mechanistic perspective as it allows the simplification that O2 was the only major oxidizing agent. The much lower H2O2 concentrations (1–79 nM) present, compared to ambient surface waters, throughout the Meso/Micro/Multi Arc/Pat experiments should mean that Fe(II) decay rates during these experiments more closely match the oxidation rate constants used to derive Eq. (1) (which were derived for low-H2O2 conditions).

The decay experiments reported here still however differ in two critical respects from controlled oxidation rate experiments used to derive rate constants. First, the speciation of Fe(II) may differ. It is debatable to what extent Fe(II)-L species, analogous to Fe(III)-L species, exist in surface marine waters due to the absence of reliable techniques to probe Fe(II)-organic speciation (Statham et al., 2012). Yet there is consistent evidence that organic material affects Fe(II) oxidation rates (see below). Second, these decay experiments measure the change in Fe(II) concentration between light and dark conditions and not specifically the oxidation rate. If photochemical Fe(II) production was the sole Fe(II) source, and oxidation of Fe(II) via H2O2 and O2 were the only Fe(II) sink, then the decay rate measured here would approximate the oxidation rate determined under controlled laboratory conditions. However, there are possible biological sources of Fe(II) (Sato et al., 2007; Nuester et al., 2014), the possibility of biological uptake of Fe(II) (Shaked and Lis, 2012), and cross-reactivity with other reactive trace species (e.g. reactive oxygen species and Cu, Rijkenberg et al., 2006; Croot and Heller, 2012) to consider. These complexities make Fe(II) more challenging to model in natural waters compared to controlled conditions. This is especially the case at the low Fe(II) concentrations relevant to the surface ocean, where Fe(II) concentrations range from below detection up to ∼1 nM (Gledhill and Van Den Berg, 1995; Hansard et al., 2009; Sarthou et al., 2011).

Contrasting k with kmeas during Fe(II) decay experiments (Fig. 4), it is immediately apparent that the Fe(II) present within Meso/Micro/Multi Arc/Pat experiments was generally much more stable than would be predicted for an equivalent inorganic spike of Fe(II) added to water with the same physical/chemical properties; i.e. in most cases kmeas<k. Three plausible hypotheses can be conceived for the offset. i.

The measured rates here refer to relatively low initial Fe(II) concentrations (0.3–16 nM) compared to the concentrations at which rate constants have been derived (typically ∼20–200 nM), and the difference arises simply because the rate constants are not calibrated for low nanomolar starting concentrations.

Calculating the difference between calculated and measured k (Δk), it is evident that the largest differences were associated with the lowest initial Fe(II) concentrations (Fig. 4b). This is consistent with both hypothesis II and hypothesis III. Assuming that the dominant source of Fe(II) is photochemistry, the effects of both a secondary “dark” Fe(II) source and a limited fraction of Fe(II) existing in a more stable form with respect to oxidation would be most evident at the lowest initial Fe(II) concentration. Sources of Fe(II) other than photochemistry are plausible and may include, for example, zooplankton grazing due to the reduced pH and O2 within organisms (Tang et al., 2011; Nuester et al., 2014). Mesozooplankton addition was one of the three experimental variables manipulated during the Arctic/Patagonia experiments. However, no clear trend was evident with respect to Δk and the zooplankton addition status of the experiments. Mean Δk±SD(×10-2) for the high/low zooplankton treatments over all the experiments were 4.66±5.79 and 4.08±5.63, respectively. A dependency of Δk on the initial Fe(II) concentration (Fig. 4b), with [Fe(II)]t=0 likely very sensitive to multiple experimental factors such as the time of day that the sample was collected and the exact time delay between sample collection and the first time point for each Fe(II) decay experiment, would however make determining the relative importance of any other underlying causes challenging. In order to gain further insight into the potential role of zooplankton in Fe(II) release under dark conditions, a series of incubations was conducted with addition of the copepod Calanus finmarchichus to cultures of the diatom Skeletonema costatum (Hopwood et al., 2020). No changes in extracellular Fe(II) or H2O2 concentrations were evident across a gradient of copepods from 0 to 10 L-1. Whilst this suggests the role of high/low zooplankton treatments was minimal in short-term changes to ambient Fe(II) concentrations, the potential release of Fe(II) by zooplankton may of course be species-specific: different results may have been obtained with different zooplankton–prey combinations.

The high magnitude of Δk in some cases at low initial Fe(II) concentrations (Fig. 4) is consistent with the theory that Fe(II)-binding ligands are responsible for the observed stability of Fe(II) in some natural waters (Roy and Wells, 2011; Statham et al., 2012). The Fe(II)-binding capacity of any ligands present in a specific sample would be expected to become saturated as Fe(II) concentrations increased. The effect of Fe(II) ligands on the oxidation rate of an added Fe(II) spike would therefore become less evident as Fe(II) concentration increased because the fraction of Fe(II) present as Fe(II)-L species would decline; i.e. Δk would approach zero. This has an important methodological implication. The effect of cellular exudates, or natural organic material extracts, on Fe(II) oxidation rate is more often than not tested by adding reasonably high nanomolar Fe(II) spikes to solution and then following the Fe(II) decay with time (see, for example, Lee et al., 2017). By raising the initial Fe(II) concentration, such an approach may however systematically under-estimate the effect of organic material on Fe(II) stability at in situ Fe(II) concentrations.

The effect of organic material on Fe(II) is difficult to generalize as organic compounds can accelerate, retard, or have no apparent effect on Fe(II) oxidation rates via O2 (Santana-Casiano et al., 2000). However, there are now sufficient studies of Fe(II) behaviour to distinguish between the broad effects of allochthonous and autochthonous material. Extracts from the green algae Dunaliella tertiolecta (González et al., 2014), cyanobacteria Synechococcus (Samperio-Ramos et al., 2018b) and Microcystis aeruginosa (Lee et al., 2017), coccolithophore Emiliania huxleyi (Samperio-Ramos et al., 2018a), and diatoms Chaetoceros radicans (Lee et al., 2017) and Phaeodactylum tricornutum (Santana-Casiano et al., 2014) have all been found to retard Fe(II) oxidation rates. Furthermore, the effect of cellular exudates on the reaction constant appears to scale with increasing total organic carbon (Samperio-Ramos et al., 2018b). In contrast to the stabilization apparent in some cellular exudates, allochthonous material generally, although not universally, has the opposite effect, with an acceleration of Fe(II) oxidation rates reported both in coastal environments (Lee et al., 2017) and using terrestrially derived organic leachates (Rose and Waite, 2003). The generally positive effects of cellular exudates on Fe(II) stability with respect to oxidation determined in single-species studies is consistent with the stability of Fe(II) observed in almost all experiments here (Fig. 4), and this suggests that microbial cellular exudates are indeed a stabilizing influence on Fe(II) concentrations at a broad scale in coastal marine environments. Stabilization of Fe(II) by freshly produced exudates could explain the sustained increase in Fe(II) concentrations across all pCO2 treatments under post-bloom conditions in Gran Canaria (Fig. 3b) and the high fraction of DFe present as Fe(II) during all Meso/Micro/Multi Arc/Pat experiments (Table 4).

Apart from the influence of organic Fe(II) ligands on Fe(II) stability arising from the slower oxidation rates of some organically complexed Fe(II) species, Fe(II)-binding organics may also have a role in the generation of superoxide (O2-), which is speculated to be a dominant mechanism for the formation of Fe(II) in the dark (Rose, 2012). Experiments with 65–130 nM of protoporphyrin IX demonstrated increased formation of Fe(II) in the dark with both increasing porphyrin concentration and increasing irradiation of seawater prior to the onset of darkness (Rijkenberg et al., 2006). Whilst the rates of this process are challenging to investigate at the sub-nanomolar porphyrin and Fe(II) concentrations expected in the ocean's dark interior, the dark formation of Fe(II) mediated by reactive oxygen species' interactions with Fe(II)-organic complexes could potentially be important in both the diurnal cycling of Fe in the surface ocean and the non-photochemical formation of Fe(II) in the dark of the ocean's interior (Rose, 2012). From a mechanistic perspective, it is challenging to establish definitively from the experiments herein whether apparent Fe(II) stability arises from reduced oxidation rates due to Fe(II) complexation or dark Fe(II) formation via a mechanism, such as that proposed for superoxide, which involves Fe(II)-organic complexes. Both hypotheses are consistent with field observations, and it is also possible that both processes operate in parallel.