Dynamics of hydrogen peroxide in a coral reef: Sources and sinks

Yeala Shaked1,2 and Rachel Armoza-Zvuloni 1,2

Received 15 August 2013; revised 31 October 2013; accepted 7 November 2013; published 18 December 2013.

[1] The dynamics of hydrogen peroxide (H2O2) was studied in the fringing coral reef offthe coast of Eilat, Red Sea. Diurnal changes in H2O2 concentrations in the reef lagoon weretypical of photochemically produced species. During the daytime H2O2 accumulated in thelagoon at low tide and exceeded open water concentrations by 100–250 nM. Elevated H2O2

decay kinetics (termed hereafter antioxidant activity) were also recorded in the lagoon at lowtide. The observed antioxidant activities were high enough to moderate H2O2 accumulationin the lagoon. In pursuit of the antioxidant source, the ability of corals to release antioxidantactivity to their surrounding water was examined in both natural and laboratory settings.Water collected in situ from surfaces of individual corals and next to a coral knoll containedhigh antioxidant activity. Incubation experiments revealed that many Red Sea corals releaseantioxidant activity to their externalmilieu. Besides serving a potential antioxidant source tothe reef system, the antioxidant activity detected on coral surfaces enabled corals to lowerH2O2 concentrations in their vicinity. The ability of corals to offset exogenous H2O2 wasvalidated in incubations with Stylophora pistillata in the absence of mixing. Conversely,corals subjected to mixing in a beaker were found to release H2O2, implying that corals mayact as both a sink and a source for H2O2 in the reef. This newly described ability of corals tochange H2O2 dynamics by releasing both H2O2 and antioxidants may bare importantimplications for coral physiology and interactions with the environment.

Citation: Shaked, Y., and R. Armoza-Zvuloni (2013), Dynamics of hydrogen peroxide in a coral reef: Sources and sinks,J. Geophys. Res. Biogeosci., 118, 1793–1801, doi:10.1002/2013JG002483.

1. Introduction

[2] Hydrogen peroxide (H2O2) is a commonly occurringreactive oxygen species (ROS) of great biological and chem-ical importance. In marine systems H2O2 was shown to play acentral role in the cycling of dissolved organic matter, tracemetals, pollutants, and a number of highly reactive free radi-cals [Avery et al., 2005;Moffett and Zika, 1987]. Open oceanH2O2 concentrations are typically below 100 nM and canreach ~300 nM in coastal water [Miller et al., 2005; Petasneand Zika, 1997]. It is primarily produced by photochemical re-actions involving colored dissolved organic matter (CDOM)and O2 [Yuan and Shiller, 2001; Zafiriou et al., 1984]. Lightabsorbed by CDOM induces an electron transfer to molecularoxygen, forming the superoxide anion radical which undergoesdisproportionation to form H2O2 [Zafiriou et al., 1984]. Otherpotential H2O2 sources include rain, redox reactions involv-ing metals, and biological production by phytoplankton[Hansard et al., 2010; Moffett and Zika, 1987; Palenik and

Morel, 1988; Shaked et al., 2010]. Biological enzymaticH2O2 production may proceed directly or through extracellu-lar superoxide which rapidly dismutate to H2O2 [Rose et al.,2010; Diaz et al., 2013; Shaked and Rose, 2013]. H2O2 isrelatively stable in seawater with a half-life ranging from afew hours in coastal environments to a several days in openocean oligotrophic waters [Petasne and Zika, 1997]. H2O2

decay can mostly be attributed to microbial enzymatic degra-dation and redox reactions with reduced metals [Petasne andZika, 1997; Wong et al., 2003].[3] On an intracellular level, H2O2 and other ROS are com-

mon by-products of normal aerobic metabolism and, at lowlevels, serve as important signaling molecules [Bartosz,2009]. Nonetheless, ROS may accumulate beyond the capac-ity of an organism to efficiently quench them or to repair theresulting damage. This state, known as oxidative stress,results in extensive damage to cellular components includingproteins, lipids, and DNA, all of which may eventually leadto cell demise [Apel and Hirt, 2004; Gechev and Hille,2005]. Because of the multifunctional roles of ROS, it isnecessary for the cells to tightly control the level of ROS inorder to avoid any oxidative damage, on the one hand, butnot eliminate ROS completely, on the other. Degradation ordetoxification of excess ROS is achieved by an array of enzy-matic and nonenzymatic antioxidants [Apel and Hirt, 2004;Bartosz, 2009].[4] At a first glance the intracellular levels of H2O2 and its

dynamics in seawater have little in common. Yet, since H2O2

is membrane permeable, its presence in seawater can cause

Additional supporting information may be found in the online version ofthis article.

1Interuniversity Institute for Marine Sciences, Eilat, Israel.2Institute of Earth Sciences, Hebrew University, Jerusalem, Israel.

Corresponding author: Y. Shaked, Institute of Earth Sciences, HebrewUniversity of Jerusalem and Interuniversity Institute for Marine Sciences,Eilat, 88103, Israel. (yeala.shaked@mail.huji.ac.il)

©2013. American Geophysical Union. All Rights Reserved.2169-8953/13/10.1002/2013JG002483

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JOURNAL OF GEOPHYSICAL RESEARCH: BIOGEOSCIENCES, VOL. 118, 1793–1801, doi:10.1002/2013JG002483, 2013

deleterious effects to marine organisms such as bacteria andphytoplankton [Baltar et al., 2013; Morris et al., 2011]. Onthe other hand, marine microbes can influence H2O2 dynam-ics in seawater by producing and degrading H2O2 [Hansardet al., 2010; Palenik and Morel, 1988; Rose et al., 2010;Wong et al., 2003]. In analogy, we hypothesize that coralscan influence H2O2 dynamics locally and are influenced byH2O2 concentrations in the reef. This research details our at-tempts to document H2O2 distribution and residence times ina natural coral reef and next to individual corals.[5] The interactions among corals and ROS in their sur-

roundings are of environmental significance since the wide-spread affliction of coral bleaching and disease outbreak areclosely linked to ROS production, accumulation, and oxida-tive stress [Lesser, 2006; Lesser, 2011]. Residing in shallow,warm, and strongly illuminated water, corals are likelysubjected to elevated concentrations of photochemically pro-duced H2O2 (and other ROS). In tropical regions, rain and run-off may further contribute H2O2 to the reef water, eitherdirectly or through input of trace metals that catalyze ROSphotoformation [Moffett and Zika, 1987]. In addition to abioticH2O2 sources, corals may contribute to the fluxes (or sinks) ofH2O2 in the reef ecosystem. ROS generated within corals bytheir photosynthetic symbionts [Suggett et al., 2008; Lesser,2011] may be released to the surrounding water via diffusionor active water exchange during feeding. Recently, we docu-mented in incubation experiments that the coral Stylophorapistillata releases superoxide and antioxidants to its externalmilieu [Saragosti et al., 2010]. It is possible that similar phe-nomena prevail in nature and result in measurable fluxes ofROS and antioxidants in the vicinity of corals.[6] Following up on our study of H2O2 photocycling in the

Gulf of Aqaba [Shaked et al., 2010], we chose to examine thedynamics of H2O2 in a shallow reef, where elevated fluxes ofphotochemically generated H2O2 may be harmful to corals.The proximity to shore, compact structure, and simple flowpattern make the fringing reef in the Eilat nature reserve aconvenient system for studying changes in biological, chem-ical, and physical parameters on a reef scale [Barnes andLazar, 1983; Silverman et al., 2004; Silverman et al., 2007;Yahel et al., 1998]. As part of a larger study, we areconducting on the dynamics of reactive oxygen species inthe external milieu of corals; in the current contribution, weseek to identify the potential influence of corals on H2O2

dynamics in their natural environment.[7] This field-based study combines sensitive techniques,

kinetic approaches, and statistical tools to follow H2O2 con-centrations and decay kinetics (antioxidant activity) on dif-ferent scales: the reef lagoon, surfaces of individual corals,and corals in a knoll. Laboratory experiments with coral col-onies were carried out to support the field observations.

2. Materials and Methods

2.1. H2O2 Measurements

[8] H2O2 concentration wasmeasured with a Varian spectro-fluorometer (Cary Eclipse) using the 4-hydroxyphenylaceticacid (POHPPA) technique [Miller et al., 2005]. Principally,POHPPA (4-hydroxyphenylacetic acid) in the presence ofhorseradish peroxidase reacts on a 1:1 basis with H2O2 toform a fluorescent dimer. The POHPPA reagent stock consistsof 0.25mM POHPPA (cat. H5004, Sigma-Aldrich Corp.),

70 units mL�1 of horseradish peroxidase (cat. P8125, Sigma-Aldrich Corp.), and 0.25M Tris at pH8.8. The reagent stockwas added to seawater samples at a 1:50 dilution and wasallowed to react in the dark for at least 10min prior to measure-ment. Excitation was set to 315±10 nm, and emission spectrawere collected from 360 to 440 nm with maximal emissionobtained at 406–410 nm. Calibration curves were run dailyusing filtered seawater spiked with dilute H2O2 standards.These standards were made fresh daily from 30% (w/w)H2O2 (Suprapur, Merck), which was checked for stability ina Varian spectrophotometer (Cary 50) based on its UVabsorbance at 240 nm (ε =38.1M�1 cm�1). The methodblank was determined in catalase-amended seawater (Sigma,25U mL�1). Detection limit, defined as 3 times the blank SD,was typically 10 nM, and the error on replicate analysis was10–15%. Laboratory light did not enhance POHPPA auto-fluorescence over the period of a few hours, and hence, thesample analysis was done in the light. Natural light inducedlow but measurable POHPPA autofluorescence over fewhours. Hence, POHPPA was preferably added to samples in-doors, and during dives, the POHPPA-containing vials werekept in dark containers. The florescent signals of dark keptPOHPPA-contained samples were stable for at least 6 h at roomtemperature and over 24 h when refrigerated. Since thePOHPPA method cannot distinguish between H2O2 and or-ganic peroxides, the lagoon water was repeatedly measuredalso with the H2O2-specific acridinium ester chemilumines-cent-based method [Miller et al., 2005]. The good agreementbetween the methods suggests that organic peroxides comprisean insignificant fraction of the total peroxide signal.

2.2. Antioxidant Activity (H2O2 Decay Kinetics)

[9] The rates of H2O2 decay in open seawater, lagoon, andcoral water were determined experimentally by spiking the sam-ple of interest with H2O2 and following its concentration overtime. The kinetic data were treated similarly to Hansard et al.[2010] and Rose et al. [2010] as explained below. Five to fortymilliliter samples were placed in acid-cleaned glass vials, spikedwith 1μM H2O2, mixed thoroughly, and then subsampled intocuvettes containing POHPPA reagent stock at time intervalsranging from a few minutes to a few hours. The experimentlength was modified according to the rate of H2O2 decay,changing from 0.5–2h in coral water to 24–36h in open sea-water. H2O2 decay rates in all our experiments followedpseudo-first-order kinetics as described by equation (1):

H2O2 loss rate ¼ �kantiox � H2O2½ � (1)

[10] The antioxidant activity of a sample is defined as thedecay constant �kantiox with a unit of h�1. Note that kantioxis always positive with higher values representing higherantioxidant activity. From kantiox, we can calculate H2O2

half-life (equation (2)), which is the time it takes half of itsconcentration to be degraded by antioxidant activity:

H2O2 half -life ¼ Ln 2ð Þkantiox

(2)

[11] The half-life provides an intuitive sense of the stabilityof H2O2 in a sample, where higher antioxidant activity resultsin a shorter half-life.

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[12] In a previous study, copper and iron (6 and 30 nM, re-spectively) had no effect on H2O2 decay rates in the Gulf ofAqaba open seawater [Shaked et al., 2010]. Nonetheless, carewas taken to prevent trace metal contamination during sam-pling, manipulation, and analysis. All vials, test tubes, tips,and cuvettes were precleaned with 10% HCl and washed withdouble distilled water (DDW, 18.2 MΩ, MQ Academic) priorto use, and reagents were analytical grade or higher.

2.3. Study Area and Sample Collection

2.3.1. Study Area[13] The study was conducted in the Gulf of Aqaba, the

northern extension of the Red Sea, surrounded by Israel,Jordan, Egypt, and Saudi Arabia. This region is characterizedby hot and dry climates, negligible precipitation (~3 cm yr�1)and runoff, and a high evaporation rate (~180 cm yr�1). Thelack of runoff and the water circulation pattern in the Gulfmake it a high-salinity (40.6‰), warm (20.5–27°C),oligotrophic ecosystem with relatively low phytoplanktonbiomass and low CDOM (colored dissolved organic matter)concentrations. Tidal fluctuations in sea level are typically70–90 cm, and midday solar flux varies from 1000–1200Wm�2 in summer to ~600–800Wm�2 in winter[Shaked and Genin, 2012].[14] Data were collected between 2011 and 2013 at the

Nature Reserve Reef (NRR), a high-latitude (~29.5N) fring-ing reef, located along the western shore of the Gulf of

Aqaba, approximately 10 km south of the city of Eilat. TheNature Reserve Reef flat is made up of a more or less contin-uous series of coral patches with an average width of about20m and extends for ~1 km parallel to the shoreline(Figures 1a and 1b) [Shaked et al., 2005]. The NRR is sepa-rated from the shore by a shallow lagoon about 30–40mwideand 1–2m deep. The lagoon is covered with coarse carbonatesand and gravel and has 10–20% live coral cover (Figure 1b)[Shaked and Genin, 2012]. Several small isolated knolls,such as the Moses Rock, are found at the northern end ofthe NRR (Figure 1a). Despite its modest dimensions, theNRR is highly diverse and it supports 97 scleractinian coralspecies of 40 genera and 13 families [Loya, 1972]. The flownext to the reef has on-shore and long-shore components thatare influenced by the wind and tide. The prevailing northerlywinds generate a long-shore southern current ranging from0 to 10 cm s�1, with an on-shore component of 0–2 cm s�1

[Silverman et al., 2007].2.3.2. Sampling Strategy and Sites[15] Previous studies in the NRR documented large varia-

tions in several physical and chemical parameters on adiurnal cycle in the reef lagoon [Silverman et al., 2004;Silverman et al., 2007]. Changes in temperature and salinitywere shown to reflect physical processes occurring in a smallwater body isolated from the sea during low tide. Changes inoxygen, pH, nutrients, and alkalinity were shown to reflectbiological processes in the reef that are accumulated in thelagoon. Adopting this approach, we investigated the dynam-ics of H2O2 in the reef lagoon in winter and in summer, asdetailed below.2.3.2.1. Lagoon Sampling[16] Winter—H2O2 concentrations were measured twice

over a diurnal cycle in the winter of 2011 in the southernNRR lagoon next to the Underwater Observatory (Figure 1a).Water was collected from the reef lagoon and seaward of thereef using a plastic bucket that was lowered from the observa-tory bridge. Twenty milliliter samples were immediately trans-ferred to opaque acid-cleaned glass vials containing 0.4mLPOHPPA reagent to prevent H2O2 loss via antioxidant activity.Antioxidant activities were not measured in January 2011.However, this same location was repeatedly sampled in differ-ent seasons at minimal tide and assayed for antioxidant activity.[17] Summer—H2O2 concentrations and antioxidant activi-

ties were measured in the northern NNR during an extremelow tide event in the summer of 2012 (31 August; Figures 1aand 1b). The lagoon (three discrete stations) and its source water(100m seaward from the reef flat) were sampled by swimmingin triplicate 40mL acid-cleaned glass vials, every 30minbetween 11:30 and 15:30. Samples were amended withPOHPPA reagent within 2–5min of sampling indoors and keptin the dark until analysis (~1 h). The assay for antioxidant activ-ity was initiated within 2–4 h from collection and run overnight.During minimal tide, some of the reef flat corals were exposedto air but were frequently washed by waves. Maximal irradia-tion at the time of sampling was ~700Wm2, and water temper-ature measured by a Hg-filled laboratory thermometer remainedconstant at 26± 1°C in all sites.2.3.2.2. Moses Rock Knoll Sampling[18] Three scuba diving campaigns were carried out in the

summer of 2012 to asses H2O2 concentrations (28 Augustand 11 September) and antioxidant activities (20 September)at a coral knoll site known as “Moses Rock”. This coral habitat

Moses Rock

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Figure 1. The study sites at the northern Gulf of Aqaba, RedSea, and underwater sampling techniques. (a) Aerial photo-graph of the Eilat Nature Reserve Reef (NRR) showing thefringing reef with its back lagoon and the sampling locations.(b) Close up on the lagoon, showing some of the samplingspots (red dots). (c) Close up on the “Moses Rock” coral knoll.Collection of water in scuba diving with syringes (d) next tocorals at the Moses Rock and (e) from individual Platygyrasp. coral. (f ) Injection of the sampled water to vials containingPOHPPA to avoid H2O2 loss due to antioxidant activity.

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is located on a sandy bottom at a depth of 9 m, 50 m off theshoreline and seaward from the fringing reef (Figures 1a).The ~7m in diameter, ~7m high knoll is densely populatedby diverse stony corals (such as Stylophora sp., Acroporasp., Favia sp., Platygyra sp., Porites sp., Pocillopora sp.,and Cypastrea sp.) and other reef inhabitants (such as seaanemones, soft corals, tunicates, and sponges; Figure 1c).Sampling was carried out at a depth of 4–5m by scuba diving.Water samples were collected 1–5 cm from various stonycorals (Figure 1d) using needle and syringe. A samplinglocation 5m upstream of the knoll was chosen as a referencepoint. Sampling for H2O2 was conducted with 2.5mL syringesthat were filled completely and injected underwater intoairtight acid-cleaned glass vials equipped with silicon lids(Figure 1f ). To avoid H2O2 degradation by antioxidants,50μL POHPPA was added to the vials prior to the dive.Vials were kept in the dark before and during the dive sincePOHPPA is light sensitive. At each location, 30–50 sampleswere collected to generate enough data for generatingfrequency distribution profiles.2.3.2.3. Coral Surface Sampling[19] Samples were collected next to the surface of 40 indi-

vidual massive Platygyra sp. corals and from the open waterabove the reef (as control) by scuba diving with syringes,avoiding physical contact with the corals (Figure 1d).Sampling for antioxidants was repeated twice (13 and 17March 2013) to increase sampling size and reaffirm findings,and was conducted at two depth (5 and 20m) to probe forlight effects. The 20mL samples were assayed for antioxi-dant activity within 2–4 h of sampling and again after 24 hof incubation at room temperature (25°C). In few samples,the antioxidant activity was assayed after heating to 80°Cfor 10min and in the presence of 0.1mM sodium azide(NaN3, Sigma). H2O2 samples collected in 2.5mL syringes(30 July 2013) were injected underwater to vials containingPOHPPA to avoid H2O2 loss due to antioxidants (Figures 1f ).

2.4. Experiments

[20] H2O2 and antioxidant release by corals were tested ex-perimentally in corals belonging to the most abundant coralgenera in the northern Red Sea reefs: Stylophora, Acropora,Favia, Platygyra, Porites, Pocillopora, Cyphastrea, Fungia,and Seriatopora. All corals were kept in water tables with run-ning natural seawater prior to and throughout the experimentperiod. Experiments were conducted under fluorescent labora-tory light, and water temperature was kept at 25 ± 1°C. Tominimize stress, coral fragments were gently transferred andsuspended in glass beakers and allowed to acclimate in run-ning seawater for a short period. Coral-containing beakerswere placed on a stirrer to ensure complete water mixing,and water samples were drawn regularly for H2O2 measure-ments and assayed for antioxidant activity. Water volumesvaried between experiments (100–500mL) to fit differentsizes of coral colonies and fragments (5–50 cm3). Hence, thesedata should not be regarded quantitatively, but rather as a dem-onstration of the release phenomena. In the no-flow experi-ment, six Stylophora pistillata fragments and seawater-onlycontrols (n=6) were treated with 500 nM H2O2 and sampledfor H2O2 every 10min for a total of 70min. At the end ofthe incubation, the control and coral waters were analyzedfor antioxidant activity.

2.5. Statistical Tools and Analyses

[21] Statistical analyses were carried out using Statistica8® software and R software version 2.12.2 (R Foundationfor Statistical Computing). Frequency distribution analysiswas applied for tracing the dynamics of H2O2 in the reef lagoonand at the “Moses Rock” knoll. To evaluate the differencesin frequency distribution profiles among sites, we used theKolmogorov-Smirnov (K-S) two-sample test. Differences inH2O2 concentrations and antioxidant activity between control(seawater) and the water collected on the surfaces of massivePlatygyra corals (Figure 5) were analysed using Student’s t test.In the no-flow laboratory experiments, differences in H2O2

concentrations over time between controls and coral treatments(Figure 7) were tested with a linear mixed-effects model (fit bymaximum likelihood) and computed with R software.

2.6. Additional Measurements

[22] Supporting data (sea level and solar irradiation), cour-tesy of the Israel National Monitoring Program at the Gulf ofEilat (NMP), were measured in a meteorological stationlocated 500m south of the NRR at the InteruniversityInstitute for Marine Sciences pier. Sea level was monitoredby a Campbell water pressure sensor (model CS408), globalradiation by a Kipp & Zonen pyranometer (model CM11B).Coastal current velocities were measured by an upwardlooking acoustic Doppler current profiler (RDI 600 kHz),located 1 km south of the Moses Rock at a depth of 30m[Shaked and Genin, 2012].

3. Results

3.1. Dynamics of H2O2 in the Reef Lagoon

3.1.1. Diurnal Cycle in Winter[23] In January 2011, H2O2 concentrations in the NRR la-

goon and in the open waters were examined over a diurnalcycle (Figure 2). Sampling was repeated twice, a week apart,on contrasting hydrographic conditions: when maximalradiation co-occurred with low tide (19 January; Figure 2a)and when maximal radiation co-occurred with high tide(26 January; Figure 2b). H2O2 concentrations in the open wa-ters were comparable between days, ranging from ~20 nM atnight and early morning to ~50 nM in the afternoon. At night,H2O2 concentrations in the lagoon remained low and similarto the water flushing the reef. On both days, the lagoon accu-mulated H2O2 during the daytime, and in the evening its con-centration dropped back to ~20 nM. When maximal radiationco-occurred with the low tide, significant H2O2 buildup(up to 140 nM ) was observed in the lagoon.3.1.2. Extreme Low Tide in Summer[24] On 31 August 2012, H2O2 concentrations and decay

rates in the NRR lagoon and the open water were examinedthroughout a strong low tide event. No distinct trend in timewas apparent, and hence, all lagoon data were pooledtogether and presented along with the open water data asfrequency distribution plots (Figure 3 and supporting infor-mation Table S1 and Figure S1). Such plots visualize the var-iability among values in a data set in addition to showing themost frequent values. H2O2 concentrations in the lagoon withan average of 174 ± 54 nM (mean ± SD, n = 74) werestatistically higher than in open water (105 ± 14 nM; n = 19,p< 0.001, K-S test). Moreover, H2O2 frequency distribution

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profiles varied significantly between locations, with homog-enous values in the open water as opposed to highly variableconcentrations in the lagoon ranging between 90 and 480 nM( p< 0.001, K-S test; Figure 3a).[25] H2O2 decay rates in the open water, termed hereafter

antioxidant activity and expressed by kantiox (equation 1),were relatively slow and homogenous, with an average kantioxof 0.042 ± 0.021 h�1 (mean ± SD, n = 14; Figure 3b). In thelagoon, antioxidant activities were much higher and morevariable, ranging from kantiox of 0.036 to 1.3 h�1 (n= 53;Figure 3b). The average lagoon antioxidant activity of0.21 ± 0.22 h�1 was statistically higher than the incomingwater ( p< 0.001, K-S test; Figure S1). Discrete measure-ments in the lagoon at low tide throughout the year reveal asimilar trend of elevated antioxidant activities (Figure S2).

3.2. Dynamics of H2O2 in a Coral Knoll

[26] H2O2 concentrations and antioxidant activity next todifferent corals residing in the “Moses Rock” knoll were ex-amined on several scuba diving campaigns (Figure 1). Onceagain, the frequency distribution profiles of the measured pa-rameters are presented, showing in all cases statistically dif-ferent distributions in the vicinity of corals in the knoll ascompared with the water upstream from the knoll (Figure 4;K-S test, Table S1). The antioxidant activity measured on20 September was high and heterogeneous next to the knollcorals (average kantiox = 0.18 ± 0.14 h

�1), while in the up-stream water it was low and homogenous (average kantiox =0.037 ± 0.024 h�1; Figure 4a). The H2O2 frequency distribu-tion profile measured on 11 September next to the knollcorals was shifted to lower values than in the upstream water( p< 0.001, K-S test; Figure 4b). The current was slow duringsampling (4.1 ± 3.2 cm s�1), and average H2O2 concentra-tions were 30 ± 11 nM (mean ± SD, n = 49) next to the corals

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Figure 2. Diurnal changes in H2O2 concentrations in the reef lagoon in winter. (a) Co-occurrence of lowtide and maximal solar irradiation (19 January 2011). (b) Co-occurrence of high tide and maximal solarirradiation (26 January 2011).

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Figure 3. (a) H2O2 concentrations (b) and antioxidantactivity in the reef lagoon measured during low tide in sum-mer (31 August 2012). In each panel, frequency distributionprofile of the lagoon data (red line) is compared with that ofwater seaward from the reef (blue line). Distribution profilesare statistically different among locations (Kolmogorov-Smirnov (K-S) two-sample test).

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as compared to 43 ± 11 nM (n = 46) in the upstreamwater. On28 August H2O2 concentrations upstream of the knoll werehigh and uniform (193 ± 34 nM, n = 32; Figure 4c), and cur-rent was fast during sampling (11 ± 7.7 cm s�1). The averageH2O2 concentration next to the knoll (213 ± 97 nM, n = 31)was not statistically different from the concentration in up-stream water. Nonetheless, its distribution was statisticallydifferent ( p< 0.05, K-S test), extending to both higher andlower values than the upstream water (Figure 4c).

3.3. Dynamics of H2O2 on the Surface of Massive Corals

[27] Water collected in situ from surfaces of 40 massivePlatygyra sp. corals were analyzed for antioxidant activityand H2O2. Antioxidant activity was found at the surface ofall corals with an average kantiox of 18 ± 14 h�1 (n= 63;

Figure 5a). No statistical differences were found between thedifferent days or the different depths (Figure S3). H2O2 decayin seawater collected further away from the corals was ordersof magnitude lower than at the coral surface averaging atkantiox of 0.014 ± 0.011 h�1 (n = 21, p< 0.001, Student’s ttest; Figure 5a). At the coral surface, H2O2 concentration isexpected to drop due to the high antioxidant activity (averagehalf-life ~2min). Indeed, lower H2O2 concentrations weremeasured on the surface of Platygyra sp. compared to theoverlying water ( p< 0.05, Student’s t test; Figure 5b). Theantioxidants collected from the coral surface maintained theiractivity over the tested period of 24 h (Figure S3). Theantioxidants appear to be enzymatic, since loss of activitywas observed upon heating to 80°C for 10min and uponthe use of sodium azide (Figure 6), an inhibitor of haemenzymes such as catalase and peroxidase [Sanchis-Seguraet al., 1999].

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Figure 4. H2O2 concentrations and antioxidant activitynext to corals at the Moses Rock knoll. In each panel, fre-quency distribution profile of the knoll data (red line) is com-pared with that of water upstream from the knoll (blue line).(a) Antioxidant activity sampled on 20 September showingelevated antioxidant activity next to the knoll corals. H2O2

concentrations sampled on (b) 11 September and (c) 28August showing coral-induced shifts in H2O2 distributions.

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2 (n

M)

k anti

ox

(hr-1

)

Mean Mean±SE Mean±SD

Mean Mean±SE Mean±SD

Figure 5. (a) Antioxidant activity and (b) H2O2 concentra-tions in water collected from the surfaces of individualPlatygyra sp. corals in the reef.

2

3

4

5

6

7

8

0 10 20 30 40 50 60

Ln

[H

2O2]

Time (min)

Coral water

Seawater

Sodium azide

Incubation at 80°C

Figure 6. Effect of heating to 80°C and 0.1mM sodiumazide on the antioxidant activity collected from the surfacesof individual Platygyra sp. corals in the reef.

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3.4. H2O2 and Antioxidant Release by Corals ina Beaker

[28] Incubation experiments with individual corals wereconducted to examine whether corals act as sinks or sourcesof H2O2, thereby altering H2O2 dynamics in situ. The abilityof corals to lower external H2O2 concentrations was exam-ined by adding 500 nM H2O2 to six beakers containing S.pistillata fragments. No mixing was applied to mimic theboundary layer conditions at the coral surface. H2O2 concen-trations remained unchanged in the seawater controls but de-clined rapidly and were stabilized at a low value of ~200 nMin the presence of S. pistillata fragments (Figure 7a). Thedifferences over time between H2O2 concentrations in thecontrols and coral treatments were tested with a linearmixed-effects model (fit by maximum likelihood) and werefound to be significantly different ( p <0.001). The rapidH2O2 decline in the presence of corals can be accounted forby the antioxidant activity that increased to 1.7 ± 0.7 h�1

(mean ± SE, n = 6) after 70min of incubation, correspondingto an H2O2 half-life of ~10min (Figure 7b). Another set ofincubation experiments tested the release of H2O2 andantioxidants by a variety local Red Sea corals subjected to

mild stirring. Both massive (Porites sp., Platygyra sp.,Cyphastrea sp., and Favia sp.) and branching corals(Acropora sp., Seriatopora sp., Pocillopora sp., andStylopora pistillata) were found to release H2O2 and anti-oxidant activity to the incubation water (Figures 8 and 9).No release was observed from the solitary coral Fungiasp. (Figures 8 and 9). No change in H2O2 concentrationand decay rates were seen in seawater-only controls.Release rates differed among corals, but no quantitativeconclusions should be drawn from these data due to vari-ability in coral sizes and water volumes.

4. Discussion

4.1. Characterization of H2O2 Dynamics—Methodsand Concepts

[29] In this study, a major emphasis was placed on the de-velopment of methodological and conceptual approaches forfollowing H2O2 production and decay in a coral reef systemon different spatial scales in both natural and experimentalsettings. Detection of small changes in natural H2O2 concen-trations over space and time was made possible using sensi-tive fluorometric-based methods and extensive samplingefforts. Fine-resolution detection of H2O2 (and antioxidants)next to corals was achieved by highly qualified scientific di-vers and by on-site underwater mixing between the reagentand sample to avoid H2O2 decay. A sensitive, accurate, andreproducible approach was adopted to assess H2O2 decay ki-netics in water samples, termed here antioxidant activity.This approach differs from standard antioxidant assays bythe use of a very low concentration H2O2 spike, longer decaycurves (up to 36 h), and the use of a decay rate constant(kantiox, equation 1) rather than initial H2O2 decay. This ap-proach enables H2O2 half-life calculation (equation 2) and al-lows studying the behavior of uncharacterized compoundsthat react with H2O2. In the field campaigns, frequency distri-bution analysis was applied in order to trace the dynamics ofH2O2 in the reef lagoon and next to Moses Rock knoll. Byhighlighting the values that diverge from the mean (mini-mum and maximum values), this approach allows detectingthe footprints of two opposing processes, in this case H2O2

0

100

200

300

400

500

600a b

H2O

2(n

M)

Time (min)

Corals Control

0 3

k antiox (hr-1)

BDL

0 10 20 30 40 50 60 70 1 2

Figure 7. Degradation of exogenous H2O2 by S. pistillataunder no-flow conditions. (a) Change with time in H2O2 con-centrations added at 500 nM to incubated corals (red line;n = 6) and to control beakers (blue line; n= 6; water only).(b) Antioxidant activity accumulated in the incubation watersat 70min. BDL= below detection limit.

00.20.40.60.8

11.21.41.61.8

22.22.4

k an

tio

x (h

r-1)

Time (min)

Platygyra sp.

Acropora sp.Stylophora sp.

Porites sp.

Seriatopora sp.

Pocylopora sp.

Fungia sp.

Figure 8. Release of antioxidant activity by a variety ofRed Sea corals to their surrounding water observed in incu-bation experiments with moderate stirring.

0

100

200

300

400

500

600

700

800

900

H2O

2(n

M)

Time (min)

Stylophora Acropora sp. Pocylopora sp.

Porites sp. Platygyra sp. Fungia sp.

Cyphastrea sp. Favites sp. Control

Figure 9. H2O2 release by a variety of Red Sea corals totheir surrounding water observed in incubation experimentswith moderate stirring.

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release and degradation. These methods and concepts may beapplicable to other studies which seek to characterize H2O2

fluxes rather than measure absolute concentrations and eval-uate the importance of antioxidant activity in regulatingH2O2 concentrations. For example, it is highly suitable formicrobial and phytoplankton studies where external ROSfluxes are thought to play a role in viral resistance, communi-cation, competition, mutualism, and programmed cell death[Baltar et al., 2013; Evans et al., 2006; Morris et al., 2011].

4.2. Dynamics of H2O2 in the Reef Lagoon

[30] H2O2 concentrations in the nearshore waters that enterthe reef change seasonally from ~20 nM in winter to ~100 nMin summer (Figures 2 and 3a). These concentrations and sea-sonal variations are in accord with values reported for theGulf of Aqaba offshore water, where photochemistry wasassigned as the primary source of H2O2 [Shaked et al., 2010].During both seasons, when low tide commenced at daytime,the reef lagoon accumulated additional 100–250 nM H2O2.Intense photochemical reactions in the water entrapped in theshallow lagoon probably account for this H2O2 buildup.Hence, corals residing on the reef flat or in the lagoon may besubjected at times to elevated H2O2 concentrations in theiroverlyingwater. The recordedH2O2 concentrations are not con-sidered toxic to corals [Higuchi et al., 2009]. However, theydemonstrate the changes in environmental conditions that corals(and other reef inhabitants) need to adjust to. Additionally, theseelevated H2O2 concentrations in combination with otherstressors (irritation and temperature) may enhance oxidativestress response [Lesser, 2011]. Compared to the open water,the lagoon summer H2O2 concentrations were not only higherbut far more variable (Figure 3a). This variability may stemfrom local sources and sinks of H2O2, as discussed below.[31] During low tide, high and variable antioxidant activity

accumulated in the reef lagoon (Figure 3b). These antioxi-dants lead to a drop in H2O2 half-life from 16–18 h in theopen water to 0.5–4 h in the lagoon. The average lagoonH2O2 half-life of ~3 h is shorter than the water residence timein the lagoon of 4–6 h [Silverman et al., 2007]. Hence, theseantioxidants can degrade some of the H2O2 before it is mixedback with the open water. As a result, the antioxidants mod-erate H2O2 accumulation in the lagoon and probably contrib-ute to the high variability in H2O2 concentrations. Thus, ineffect, the measured H2O2 concentrations in the lagoon reflecta balance between its production and degradation.[32] The antioxidant activity (H2O2 decay kinetics) in the

reef lagoon was 50 times greater than in the nearshore water.In search of a potential source for the antioxidants that accu-mulated in the reef lagoon we turned to corals. Naturallyoccurring corals, either solitary or clustered together in aknoll, all contained high antioxidant activity at their surface(Figures 4–6). Many Red Sea corals were found to release an-tioxidant activity to their surrounding water in incubation ex-periments (Figure 8). Assuming that antioxidants are washedoff coral surfaces and are diluted in seawater, we expecthigher activities next to corals than in the lagoon. Indeed, av-erage antioxidant activity on surfaces of Platygyra sp. coralswas ~100 times higher than in the lagoon water. We thus con-clude that the elevated antioxidant activity in the lagoon watermost likely originated from corals. In addition, the durabilityof the antioxidants from the Platygyra sp. surface (whichmaintained their activity over 24 h) is favorable in terms of

the potential effect on the reef system. Since corals are com-posed of a complex consortium of organisms, several potentialantioxidant contributors, such as the mucus-associated mi-crobes, should be considered in addition to the coral host.[33] Other possible antioxidant sources, such as phyto-

plankton, reduced metals, sediments, and various surface-at-tached macroalgae, are thought to be of minor importance tothe observed phenomenon. No sediment resuspension andmacroalgae detachment were seen during sampling (whenwave mixing was minimal due to the low tide). Based onour previous study on H2O2 interactions with iron and copper[Shaked et al., 2010], unrealistically high metal concentra-tions are required to generate such an effect. For the samereasons, microorganisms that are thought to govern H2O2 de-cay in the offshore water [Shaked et al., 2010] are discountedas a source of antioxidants in the lagoon. To account for the50 times higher H2O2 decay rates in the lagoon, massivebiomass is needed, while in fact, lower phytoplankton andbacteria densities were seen in the reef due to predation andfilter feeding [Yahel et al., 1998].

4.3. H2O2 Dynamics in Individual and Knoll Corals

[34] The antioxidants observed in the vicinity of massiveindividual corals and various corals in a knoll bear character-istics of enzymes (heat degradable and inhibited by sodiumazide, a haem enzyme inhibitor; Figure 6). Possessing antiox-idants at their surface, corals can offset external H2O2 con-centrations and maintain stable and low H2O2 in theirexternal milieu. Indeed, a shift to lower H2O2 concentrationsnext to Moses Rock and on surfaces of massive corals wereobserved in situ (Figures 4b, 4c, and 5b) and reaffirmed ina laboratory experiment with S. pistillata (Figure 7a).Common to all these observations is a restricted water flowor a slow current. We thus suggest that corals in situ can actas a sink for H2O2 under conditions of no or low flow.Such conditions likely prevail in the interior of large andintricate coral colonies in the reef, even in the presence ofcurrents. The ability of corals to offset exogenous H2O2

fluxes resulting mostly from photochemical reactions maybe beneficial for corals in maintaining a stable microenviron-ment and preventing oxidative damage. This may become par-ticularly important in the presence of high external H2O2

concentrations under conditions of metal contamination, hightemperature and irradiance, and when corals are covered by aminimal water layer or are exposed to air during low tide.[35] Corals subjected to mixing in a beaker were found to

release both H2O2 and antioxidants (Figures 8 and 9).However, a net accumulation of H2O2 was observed over atleast 30min in all tested corals, probably since the releasedantioxidant activity was still low. The experimentally ob-served net release of H2O2 from corals in the presence of flow(mixing) may also occur at sea. On a day with a relativelystrong current, elevated H2O2 concentrations were samplednext to the coral knoll, possibly originating from the corals(Figure 4c). Based on these data, we suggest that in the pres-ence of flow (or mixing) corals can serve as an H2O2 source.It is thus possible that aside from photochemistry, also coralscontribute to the accumulation of H2O2 in the lagoon duringlow tide.[36] Our data demonstrate that corals can influence H2O2

dynamics in their surrounding on different spatial scales.Individual massive corals were found to lower H2O2

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concentrations next to their surface (few centimeters) due toelevated antioxidant activity and possibly contribute antioxi-dants to the reef system. Knoll corals (and possibly otherknoll inhabitants) also possess external antioxidants andwere shown to alter H2O2 concentrations next to the knoll.This effect is restricted in space, estimated at a maximal dis-tance of ~0.5m based on a preliminary sampling campaign(data not shown). On the reef scale, antioxidants, probablyreleased from corals, accumulate in the lagoon when the wa-ter exchange with the open water is limited and may moder-ate the photochemically produced H2O2.[37] The phenomena of corals releasing both antioxidants

and H2O2 and changing H2O2 dynamics in their vicinity aredescribed here for the first time. Given the importance(and danger) of ROS to various biological processes, it maybe of great significance to the coral physiology and interac-tions with their symbiotic algae and microorganisms.Additionally, H2O2 may be involved in the chemical interac-tions between corals or other reef inhabitants. For example,the release of H2O2 by corals may serve as part of a chemicalwarfare against microorganisms and potential pathogens[Banin et al., 2003; Rosenberg and Falkovitz, 2004].Despite the moderate H2O2 concentrations detected in theexperimental waters, higher, possibly toxic concentrationsmay prevail at the diffusive boundary layer at the coral-waterinterface prior to dilution in the medium. Further research isrequired to explore the ecological significance and physio-logical role of H2O2 generation and degradation by coralsshown here to prevail in situ on different spatial scales.

5. Summary

[38] We show that H2O2 dynamics in the reef lagoon and inthe vicinity of corals may differ from that in nearshore water.At low tide, photochemistry accounts for H2O2 buildup in thelagoon, while high antioxidant activity, probably originatingfrom corals, moderates the lagoon H2O2 concentrations.High antioxidant activity is observed in water collected nextto corals, accompanied by lower H2O2 concentrations in thecoral vicinity.

[39] Acknowledgments. We wish to thank Ilan Shachar, AssafZvuloni, Yaron Halevy, and Avi Schneider for help with sampling, andHagar Lis and Roi Holzman for statistical analysis and writing assistance.This study was supported in part by the Israel Science Foundation grant248/11 (www.isf.org.il).

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