The mesocosm study, conducted in Taliarte Harbor, Gran Canaria in March 2016, used eight thermoplastic polyurethane bags with a 2 m diameter, a depth of ~3 m, a starting volume of ~8,000 L, and no lid or screen on top. The bags were mounted on a buoyant frame which was allowed to drift ~2–3 m away from a sampling jetty. After filling with ambient seawater (on 1 March, experiment day −4), pumped from outside Taliarte Harbor (depth 15 m), the mesocosms were allowed to function without nutrient addition for 21 days (Supplementary Figure 1). A pCO₂ gradient across the eight mesocosms was induced on day 0 by the addition of varying volumes of filtered, CO₂ saturated seawater using a custom made “spider” distribution device described by Riebesell et al. (2013). The pCO₂ gradient (400–1,450 μatm) was designed to be similar to that used during the offshore KOSMOS mesocosm experiment conducted in Gando Bay, Gran Canaria in September/October 2014 (Taucher, 2017, this Research Topic). A further top-up of pCO₂ saturated seawater was then made to the mesocosms as necessary to maintain the pCO₂ gradient (Supplementary Figure 1) following CO₂ outgassing. The precise volume of each mesocosm was determined (on day 18) by measuring salinity before, and after, the addition of 40 L freshwater to each mesocosm, similar to Czerny et al. (2013). A single macronutrient addition (3.1 μM nitrate, 1.5 μM silicate, and 0.2 μM phosphate) was then made (after day 18 sampling).

H₂O₂ samples were collected in opaque 125 mL high density polyethylene (HDPE) bottles (Nalgene) which were pre-cleaned (1 day soak in detergent, 1 week soak in 1 M HCl, three rinses with de-ionized water; 18.2 MΩ·cm, Milli-Q, Millipore) and dried in a laminar flow hood prior to use. Sample bottles were rinsed once with seawater and filled with no headspace by gently submerging the bottles within the mesocosms. Chlorophyll a, bacterial abundance, chromophoric dissolved organic matter (CDOM) and macronutrient concentrations were determined from depth integrated water samples collected using 2.5 m long custom-made samplers with an internal volume of ~10 L. These samplers were constructed from polypropylene tubing with valves at both ends. After filling, by submerging into the mesocosms and closing the valves, the samplers were removed and gently inverted to facilitate mixing. Samplers were then slowly drained through 1 cm diameter silicone tubing into pre-rinsed (de-ionized water and then mesocosm water) 25 L transparent HDPE containers, which were then transferred to shaded boxes and moved to a dark, refrigerated room for sub-sampling. Analysis of chlorophyll a and macronutrients then began immediately.

Chlorophyll a was measured by fluorometry as per Welschmeyer (1994) and macronutrient concentrations (nitrate + nitrite, phosphate, silicate) were determined by colorimetry as per Hansen and Koroleff (1999). CDOM absorption spectra were measured with a 100 cm, 250 μL capillary (LPC100CM) connected via an optical fiber to a light source (DH2000BAC) and a USB2000+UV-VIS ES detector (Ocean Optics). The system was controlled using Spectra-suite software (Ocean Optics). Samples were injected into the capillary with a peristaltic pump at a flow rate of 1 mL min⁻¹. Relative molecular weight was estimated from CDOM absorption by deriving the slope ratio (S_R) as the ratio of the slope of the shorter wavelength region (275–295 nm) to that of the longer wavelength region (350–400 nm; Helms et al., 2008). The spectral slopes were calculated from the linear regression of the log-transformed absorption spectra.

H₂O₂ was always analyzed within 1 h of collection via flow injection analysis (FIA) using the Co(II) catalyzed oxidation of luminol (Yuan and Shiller, 1999). A FIA system was assembled and operated exactly as per Hopwood et al. (2017) resulting in a detection limit of <1 nM. Calibrations were run daily, and with every new reagent batch, by at least six standard additions of diluted H₂O₂ (TraceSelect, Fluka) to aged (stored at room temperature for >48 h) seawater (unfiltered). The stability of H₂O₂ stock solution was checked by measuring absorbance at 240 nm using a quartz 10 cm cell and a USB4000 spectrometer (Ocean Optics). H₂O₂ (TraceSelect, Fluka) was sequentially diluted weekly to create stock solutions of 100 mM and 100 μM using de-ionized water. With respect to the measurement of chemical species other than H₂O₂, the H₂O₂ luminol based FIA method is expected to be robust for measurements in oxygenated surface seawater (Yuan and Shiller, 1999, 2004).

Bacterial counts were obtained on depth integrated water samples. Two mL water samples were fixed with 1% paraformaldehyde (final concentration) and stored at −80°C until analysis. Samples were analyzed by flow cytometry (FACSCalibur, Becton Dickinson), with a 15 mW laser set to excite at 488 nm (Gasol and del Giorgio, 2000). Subsamples (400 μL) for the determination of heterotrophic bacteria were stained with the fluorochrome SybrGreen-I (4 μL) at room temperature for 20 min and run at a flow rate of 16 μL min⁻¹. Cells were enumerated in a bivariate plot of 90° light scatter and green fluorescence. Molecular Probes latex beads (1 μm) were used as internal standards.

A CTD cast (using a CTD60M, Sea and Sun Technology) was conducted every sample collection day in all mesocosms. Additionally, a CTD cast was conducted on the sampling jetty adjacent to the mesocosms where a H₂O₂ sample in ambient seawater was also collected alongside every mesocosm sampling event. Photosynthetically active radiation (PAR) data was obtained from an ELDONET (Häder and Lebert, 2006) monitoring site located <200 m from the mesocosm jetty. Complete diurnal light profiles were available for experiment day 7 onwards, excluding days 12 and 13.

The diurnal change in H₂O₂ concentration in ambient seawater, and inside two mesocosms, was monitored by setting up FIA equipment on the mesocosm sampling jetty with a PTFE sampling line weighted to float ~10 cm below the water surface. Seawater was pumped continuously using a peristaltic pump (MiniPuls 3, Gilson) with a time delay between water inflow and analysis of ~60–120 s. The sample line was used without a filter and visually inspected regularly for blockage. Calibration was undertaken three times in every 24 h period by 6–8 standard additions of H₂O₂ into aged (>48 h in the dark) seawater. A coiled 3 m PTFE sample line, which could be extended to the bottom of the mesocosms, was used to determine whether H₂O₂ was vertically homogenous within the mesocosm bags. During the diurnal experiment monitoring ambient seawater, salinity was measured regularly (<3 h intervals) using a LF 325 conductivity meter (WTW) which was calibrated before use with a KCl solution.

During rain events, rainwater was collected by deploying open low density polyethylene (LDPE) bags adjacent to the mesocosms. Rainwater was diluted prior to analysis by spiking 200 μL unfiltered rainwater into 50 mL aged seawater with the H₂O₂ concentration in seawater measured before, and after, the rainwater spike.

Time series for the core parameters discussed herein are included in Supplementary Datasheet 1. Initial measurements in the mesocosms (2 March 2016, experiment day −3, after filling the mesocosms—but before any treatments were applied) verified that the enclosed waters were close to identical with respect to H₂O₂ concentration. Differences between the eight enclosures (range 38.3–40.9 nM, mean 39.3 ± 0.8 nM) were small compared to the detection limit of the analytical method (<1 nM; Croot et al., 2004; Hopwood et al., 2017) and the standard deviation of quadruple measurements of H₂O₂ in seawater collected on the sample jetty (mean 3.3 nM over the experiment duration). Depth profiles within a mesocosm bag (~3 m depth) were conducted on day 25 mid-afternoon, when any stratification was expected to be maximal, and verified that H₂O₂ concentration was relatively well-mixed with only a slight vertical gradient (increasing from 43 to 51 nM bottom-top, mean 47 ± 3.5 nM). On day 12, surface seawater collected using a small inflatable boat showed a range of H₂O₂ concentrations at different locations around Taliarte Harbor (39 ± 9.9 nM, n = 4), outside the harbor within 150 m of the coastline (63 ± 1.8 nM, n = 3) and outside the harbor >400 m offshore (38 ± 5.6 nM, n = 4). Ambient seawater concentrations within the harbor (used to determine a background concentration for comparison with the mesocosms) were therefore similar to those found in near-shore waters.

The imposition of a large pCO₂ gradient across the mesocosm bags after sampling on day 0 had no clear prolonged effect on observed H₂O₂ concentrations (Figure 1A). Temporal trends in H₂O₂ concentration were relatively similar in all mesocosms until day 9 of the experiment with the standard deviation ranging from 0.7 to 13 nM. Data for mesocosm 1 are shown only until day 3, after which exchange with surrounding seawater occurred following leakage, and monitoring was thus discontinued. After day 9 the enclosed waters diverged and some mesocosms experienced swings to very high (>300 nM) H₂O₂ concentrations compared to ambient water (Figure 1A). Over the duration of the mesocosm experiment, ambient surface seawater H₂O₂ concentrations ranged from only 20–92 nM.

The initial nitrate concentration present in ambient seawater was depleted rapidly after filling of the mesocosms (Figure 1C). Nitrate + nitrite fell to <0.1 μM in all mesocosms by day −1. Nitrate concentration then remained depleted until the macronutrient addition on day 18 (macronutrient addition occurred after sampling on day 18). Correspondingly, a small peak in chlorophyll a was observed in all mesocosms on day −1 (Figure 1B). Chlorophyll a then declined to low concentrations until a larger peak following the macronutrient addition on day 18. Maximum chlorophyll a was then observed in most mesocosms on days 21–22.

A notable exception to the general trend in mesocosm H₂O₂ under post-bloom conditions was mesocosm 7 (700 μatm pCO₂). On days 25–27, H₂O₂ concentrations in the majority of mesocosms dropped below those measured in ambient seawater, but H₂O₂ in mesocosm 7 always remained >100 nM (Figure 1A). Mesocosm 7 was also anomalous with respect to the bloom development. In mesocosm 7 only it appeared that grazing may have impeded bloom development after nutrient addition (this was consistent with higher meso-zooplankton abundances in mesocosm 7, data not shown), as evidenced by a noticeably slower decline in nitrate concentration (Figure 1C) and a late peak in chlorophyll a (Figure 1B).

Bacterial abundance was similar in all mesocosms until after nutrient addition (Figure 1D). Unlike chlorophyll a however, an increase in bacterial abundance was evident in all treatments prior to the nutrient addition on day 18. A small dip in bacterial abundance was then evident in most mesocosms between days 19 and 22. Under post-bloom conditions bacterial abundance was lowest in the anomalous treatment (700 μatm) and the 400 μatm pCO₂ mesocosm with notably elevated abundances in all pCO₂ enriched treatments. The observed trend in bacterial abundance can be interpreted as resulting from grazing pressure, and enhanced growth rates post-nutrient addition. Bacterial abundance pre-nutrient addition was inversely related to nanoeukaryotes' abundance due to grazing pressure. The increase in bacterial abundance from day 13 reflected a decline in the abundance of nanoeukaryotes. After nutrient addition (day 18), grazing on bacteria was probably considerable, but bacterial growth was enhanced sufficiently to overcome grazing pressure (except in the anomalous treatment). An in depth discussion of phytoplankton community structure over the experiment duration will be presented in a companion text.

The trend in CDOM over the experiment duration was similar across all pCO₂ treatments. The generally higher S_R at the start of the experiment (Figure 1E) corresponds to lower mean molecular weight and suggests an overall increase in CDOM molecular weight over the experiment duration. Photochemical bleaching would be expected to have had the opposite effect; to have produced low molecular weight CDOM from high molecular weight CDOM (i.e., to have increased S_R). Thus, the overall trend suggests that bacterial production of high molecular weight CDOM exceeded the rate of photochemical bleaching. Only between experiment days 7 and 13 was a sustained increase in S_R evident across most mesocosms and this corresponded to a temporary decline in bacterial abundance (Figure 1D).

Photosynthetically active radiation (PAR) data was available from day 7 until the end of the mesocosm experiment (Figure 1F) from a recording site close to the mesocosm jetty (<200 m displacement). Integrated daily PAR was subject to notable variations over the duration of the experiment ranging from a mean irradiance of 44 (day 25) to 102 (day 22) W m⁻².

The diurnal cycling of H₂O₂ was followed both in ambient seawater (Figure 2) and inside mesocosm numbers 6 (1,450 μatm pCO₂, Figure 3A) and 5 (400 μatm pCO₂, Figure 3B) on days 22 and 23, respectively. Gaps in the data series corresponded to periods when standard additions were analyzed, the FIA instrument was cleaned, or malfunctions occurred such as air bubbles which occasionally resulted in missed data collection points. Generally, a clear increase in H₂O₂ associated with daylight was evident in all three diurnal datasets (Figures 2, 3). Yet H₂O₂ concentrations were much higher throughout the diurnal cycle within the mesocosms compared to ambient waters. In all three diurnal cycles, peak H₂O₂ concentration occurred mid-late afternoon (times refer to UTC). In the two mesocosms where diurnal H₂O₂ concentration was followed, the range between peak and minimum H₂O₂ was similar (180–300 nM, mesocosm 5, day 23; 160–300 nM, mesocosm 6, day 22), despite the pronounced difference in pCO₂ (400 μatm [5] compared 1,450 μatm [6]) and the 2 h offset between the timing of peak daytime H₂O₂ concentration (Figure 3). In ambient seawater, the amplitude of diurnal variation in H₂O₂ (~20–30 nM, Figure 2) was very similar to that reported previously in the central Atlantic Ocean (25 nM, Yuan and Shiller, 2001).

Two curious features were notable in both mesocosm diurnal cycles (Figure 3). These features could only be observed by virtue of the very high data resolution and, to some extent, also the relatively high H₂O₂ concentrations within the mesocosms compared to ambient waters. First, peak H₂O₂ concentrations occurred >1 h after peak irradiance. PAR data from ELDONET was not available for the seawater diurnal study date (Figure 2), but noting the timing of peak H₂O₂, there was likely approximately a 1 h offset between peak irradiance and peak H₂O₂ here also. Second, the net decline in H₂O₂ concentration which occurred in the dark continued until sometime after sunrise in both mesocosm studies (Figure 3). An increase in H₂O₂ was not apparent until after PAR increased above ~100 W m⁻². This was not apparent in the ambient seawater study (Figure 2) where a relatively stable concentration of 18.1 ± 1.3 nM was maintained from 21:11 to 07:20 (sunrise) with a sustained rise in H₂O₂ thereafter. This time offset could simply therefore have related to the incident solar angle as the mesocosm walls may have reduced the rate of light induced H₂O₂ formation at low incident solar angles.

The open design of the mesocosms created the potential for significant atmospheric deposition of H₂O₂. Rain water H₂O₂ concentrations were quantified for the two rainfall events during the mesocosm experiment with sufficient amounts of deposition (>1 mm) to facilitate sampling (Table 1) and were similar to values reported elsewhere over the Atlantic (Zika et al., 1982; Kieber et al., 2001). H₂O₂ concentrations in rainwater are thought to be sufficiently high to offset the expected beneficial effect of rainwater derived nutrient and micro-nutrient addition on primary production when rainwater is mixed with seawater (Willey et al., 2004) unless the dilution factor is high. The calculated H₂O₂ addition to mesocosms resulting from rainfall in Gran Canaria was however modest relative to the mesocosm H₂O₂ concentrations due to the low ratio of rain:mesocosm volume (Table 1).

Photochemical processes are thought to be the dominant influence on ambient H₂O₂ concentrations in the surface ocean (O'Sullivan et al., 2005; Steigenberger and Croot, 2008), with large scale spatial variations typically explained in terms of latitudinal changes in light and ocean temperature (Yocis et al., 2000; Yuan and Shiller, 2001). In the high-resolution temporal experiments a clear diurnal cycle in H₂O₂ concentration was evident, both inside and outside the mesocosm bags (Figures 2, 3). The amplitude of H₂O₂ concentration over a diurnal cycle was however much greater inside the mesocosms (100 nM compared to 20–30 nM in ambient seawater). H₂O₂ concentrations over the 28 day duration of the mesocosm experiment (Figure 1A) were always measured at 14:00–15:00 (UTC) daily to ensure that the month long data series was not affected unduly by diurnal variation.

During overnight monitoring of ambient H₂O₂ it appeared that an equilibrium concentration of 18 nM was maintained in the dark (Figure 2). This was indicative of a dark production mechanism, such as that highlighted in prior work (Palenik and Morel, 1988; Moffett and Zafiriou, 1990), sufficient to offset H₂O₂ decay. Whilst photochemical processes are no doubt a major source of H₂O₂, inter-day changes in integrated PAR were only significantly correlated with the H₂O₂ trend in ambient seawater (Spearman rank correlation 0.73, p value 0.002, n = 14) and in the 400 μatm mesocosm (Table 2). In all other mesocosms there was no apparent correlation between integrated daily PAR and H₂O₂ concentration from days 7–29.

The two high-resolution diurnal cycles within mesocosms (Figure 3) revealed some interesting features that would not be apparent at lower H₂O₂ concentrations (due to the increased signal:noise ratio) or reduced sampling resolution. An offset between irradiance and H₂O₂ concentration was reflected in the later delayed peak in H₂O₂ compared to maximum irradiance. Partial shading of the mesocosms in the early morning and late afternoon could create a local PAR exposure that was reduced compared to that reported by a mounted sensor. However, as the displacement of the sensor was <200 m from the mesocosms, this could not explain the offset between peak H₂O₂ and peak irradiance in the mid-afternoon. Instead, we suggest that net biological production of H₂O₂ is slightly offset from irradiance. A critical factor, not measured herein, which likely affects the net rate of change in H₂O₂ concentration during daylight hours is the production rate of peroxidase or catalase enzymes. This process is known to be diurnally variable, with oxidative defenses more active during daylight hours (Morris et al., 2016). Whilst it is not clear if this response under natural conditions is triggered by increasing H₂O₂ induced stress or by a circadian rhythm, the rate and efficiency (which may also change with incident light) of enzymatic H₂O₂ removal are likely to be key influences on the shape of the diurnal H₂O₂ trend.

Between days −3 and 9 the range of H₂O₂ within the different mesocosms was small (<45 nM) and the behavior of chlorophyll a and nitrate very similar across all mesocosms (Figures 1A–C). During this early phase, H₂O₂ inside and outside the mesocosms was relatively well correlated across all pCO₂ conditions (positive Spearman Rank Coefficients of 0.79–0.93). Combined with the comparability of the diurnal H₂O₂ cycle in 400 and 1,450 μatm pCO₂ mesocosms (Figure 3), despite the extreme pCO₂ difference, this suggests that the direct effect of the pCO₂ gradient (applied after day 0) on H₂O₂ concentrations was minimal.

In contrast to the early stage of the experiment, H₂O₂ concentrations in the mesocosms diverged after day 9 and there was then no longer a statistically significant relationship between the paired H₂O₂ concentrations in and outside the mesocosm (Spearman Rank Correlation P > 0.05, Table 2) for any pCO₂ treatment. If irradiance was the dominant factor controlling extracellular H₂O₂ concentrations throughout the experiment, we would expect H₂O₂ concentrations to have remained similar across all mesocosms and to remain correlated with H₂O₂ in ambient water outside the mesocosms. Yet this was not the case. Variations in daily integrated PAR, available from day 7 onwards, could explain some of the variation in background seawater H₂O₂, but inter-day changes in PAR were only significantly correlated with H₂O₂ in the 400 μatm mesocosm (Table 2).

Formation of ROS generally increases with dissolved organic carbon concentration in estuarine waters and this is specifically linked to the presence of terrestrially derived humic material (Timko et al., 2014) which has a relatively large component of CDOM. However, CDOM properties are not clearly linked to increasing H₂O₂ concentrations in offshore seawater (O'Sullivan et al., 2005). The general decline in S_R throughout the mesocosms (from day 1 to day 27, Figure 1E) suggested that in situ production of higher molecular weight CDOM was sufficient to offset photo bleaching. However, this was not clearly related to any change in H₂O₂ concentration as no clear correlation was found between S_R and H₂O₂ either over the whole experiment or specifically for days 11+ (Table 2).

Similarly, the overall trend in H₂O₂ concentration could not be related directly to macronutrient depletion. The observed nitrate concentrations were similar across all mesocosms until after the addition of macronutrients on day 18 (Figure 3) and there was no consistent trend in H₂O₂ concentration in the days immediately following this addition. Most mesocosms obtained the highest measured H₂O₂ concentration between days 9–18. Yet mesocosm 6 was notable for obtaining a peak H₂O₂ concentration 3 days after the nutrient addition. A sustained drop in H₂O₂ concentration across all mesocosms (apart from the anomalous mesocosm 7) was only observed between days 23–25.The lowest H₂O₂ concentrations, both within the mesocosm experiment and relative to ambient seawater, were observed in this post-bloom phase when bacterial abundance peaked (Figure 1D) and when daily integrated PAR was persistently low for 3 days (Figure 1F).

H₂O₂ concentrations might be expected to be generally higher inside the mesocosms due to the nature of their design. Sunlight is attenuated with depth, so in a natural surface mixed layer H₂O₂ is formed predominantly at the surface and then physically mixed throughout the surface mixed layer (Johnson et al., 1989; Miller and Kester, 1994), although sub-surface H₂O₂ peaks can occasionally be observed at the chlorophyll a maxima (Yuan and Shiller, 2001; Croot et al., 2005; Steigenberger and Croot, 2008). The confinement of the surface 3 m within the mesocosm bags used here thereby encloses the water with the highest light exposure and, throughout most of this experiment, a high level of biological activity relative to ambient seawater, whilst removing the physical processes that would constantly act to mix H₂O₂ into deeper waters. The enclosure of seawater was thereby a likely factor contributing to the high H₂O₂ concentrations occurring during the middle (days 9–18) of the mesocosm experiment. Yet this alone does not explain the divergence in H₂O₂ levels between the various mesocosms after day 9 (Figure 1A). The timings of the initial mini-bloom after the mesocosms were filled, of the macronutrient addition and the subsequent bloom were (with the exception of mesocosm 7) relatively uniform (Figures 1B,C).

Two possible explanations for why H₂O₂ concentrations were so variable between mesocosms, which are not mutually exclusive, are that either the biological H₂O₂ source(s) or the biological H₂O₂ sink(s) varied because of different microbial communities across the pCO₂ gradient. An anti-correlation between bacterial abundance and extracellular H₂O₂ concentrations is therefore intriguing. Microorganisms are simultaneously both a H₂O₂ source and a H₂O₂ sink. ROS are generated as waste products from aerobic cellular metabolism (Kruk, 1998; Apel and Hirt, 2004), with H₂O₂ produced both directly and from the decay of other ROS (for example, the enzyme superoxide dismutase produces H₂O₂ from O2-; McCord and Fridovich, 1969). Conversely, biological H₂O₂ sinks arise from both synthesis of H₂O₂ destroying enzymes (catalase and peroxidase) and also, under some circumstances, serendipitously with increasing biomass. The quenching of extracellular H₂O₂ with increasing biomass is referred to as “self-shading” and has been described both in monocultures of Prochlorococcus, which lacks catalase-peroxidase genes (Morris et al., 2011) and at the community level (Barros and Colepicolo, 2003). It presumably arises from physical shading and/or non-enzymatic ROS sinks.

The anti-correlation between H₂O₂ concentrations in each mesocosm both with chlorophyll a and with total bacterial abundance over the duration of the experiment (Table 2) was statistically significant in most mesocosms. Overall there was a significant (P < 0.05) anti-correlation between chlorophyll a and H₂O₂ concentrations with negative correlation coefficients (Table 2) in mesocosms 3, 4, 8, and 2 (850–1,300 μatm pCO₂). The anti-correlation was slightly stronger when the initial phase of the experiment (Days −3 to 9, when the range of H₂O₂ concentrations across all mesocosms was relatively narrow) was excluded. A negative relationship between chlorophyll a and H₂O₂ concentrations could arise from an increasing biological H₂O₂ sink simply due to self-shading associated with increasing biomass, yet no correlation with chlorophyll a was found either at low (400, 700 μatm) or at the highest (1,450 μatm) pCO₂ treatments. For bacteria, the correlation followed the same pattern as chlorophyll a with respect to pCO₂, yet the effect was stronger and more significant. From day 11 onwards, the intermediate pCO₂ mesocosms (850–1,300 μatm) all exhibited a strong negative correlation (Spearman Rank Coefficients of −0.83 to −0.94) between bacteria cell counts and H₂O₂ concentration (P values < 0.001). This suggests that under post-bloom conditions, bacteria were an important net-sink for extracellular H₂O₂.

As both cellular H₂O₂ production and defense mechanism activity vary over diurnal cycles and at the species level, it is difficult to separate the source and sink terms within separate mesocosms and over the pCO₂ gradient. Yet in any case, the effects of a pCO₂ gradient on H₂O₂ concentration were apparently indirect, resulting from changes to bacterial abundance and community composition rather than directly arising as a consequence of perturbing the carbonate system alone.

Reported H₂O₂ concentrations in seawater incubated under laboratory conditions are typically 100–300 nM (e.g., Coe et al., 2016), but can be much higher depending on the buffer composition and irradiance exposure (Morris and Zinser, 2013). In a spot-check of baseline H₂O₂ concentrations in incubated seawater (with added nutrients) in our own laboratory, we found 42 ± 50 nM H₂O₂ in freshly made mixtures (made from seawater which was previously stored in the dark for >1 year) and 180 ± 130 nM in the same water after 72 h of incubation subject to a diel light cycle. In all these cases, the H₂O₂ concentrations in seawater incubated under laboratory conditions are thereby at the mid-high end of the range observed in the surface ocean (Price et al., 1998; Yuan and Shiller, 2001; Hopwood et al., 2017).

The unmodified concentrations of H₂O₂ in growth media are sufficiently high to prevent the cultivation of some microorganisms, including strains of Prochlorococcus, under laboratory conditions (Morris et al., 2011). However, uncertainties remain about what the effects of elevated H₂O₂ are at the community level. Whilst H₂O₂ is not widely investigated during mesocosm studies, field evidence suggests that increases in H₂O₂ to concentrations less than the peaks observed here (Figure 1A) can affect a broad range of biogeochemical processes. Additions of 10–100 nM H₂O₂ to water from 100 m in the North Atlantic were found to reduce the extracellular activity of β-glucosidase, alkaline phosphatase, and leucine aminopeptidase by 20–80% with the inhibition effect consistently strongest on β-glucosidase (Baltar et al., 2013). A similar small absolute increase of only 30 nM is reported to suppress rates of ammonia oxidation to below detection in surface (10 m depth) Antarctic seawater (Tolar et al., 2016) where the initial natural concentration of H₂O₂ was low (14 nM). Additional specific effects of increasing extracellular H₂O₂ concentrations in seawater can also be deduced from laboratory experiments. For example, an increase on the order of 100 nM H₂O₂ is sufficient to measurably decrease the half-life of Fe(II), thereby theoretically decreasing dissolved Fe bioavailability (González-Davila et al., 2005), and to double brominating activity in some diatoms (Hughes and Sun, 2016).

A particularly curious phenomenon observed here was the extremely high H₂O₂ concentrations observed during the pre-bloom phase of the experiment (Figure 1A). It is questionable to what extent this phenomenon would occur in a natural environment because small-scale mesocosms inevitably fail to mimic the mixing processes that occur within the natural water column. Nevertheless, the potential for oxidative stress from extracellular ROS to be increased under natural, or incubated, oligotrophic conditions should be investigated concurrently with other stressors to understand the interactive effect(s) on microbial functioning. As outlined above, the sensitivity of some enzymatic processes to increasing extracellular H₂O₂ concentrations and the involvement of H₂O₂ in the biogeochemical cycling of various chemicals including the micronutrient Fe, means that large perturbations to H₂O₂ during mesocosm experiments are undesirable.

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.