Resistance of Lophelia pertusa to coverage by sediment and petroleum drill cuttings

Marine Pollution Bulletin 74 (2013) 132–140

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Marine Pollution Bulletin journal homepage: www.elsevier.com/locate/marpolbul

Resistance of Lophelia pertusa to coverage by sediment and petroleum drill cuttings Elke Allers a,⇑, Raeid M.M. Abed b, Laura M. Wehrmann a,c,1, Tao Wang a, Ann I. Larsson d, Autun Purser e, Dirk de Beer a a

Max Planck Institute for Marine Microbiology, Celsiusstr. 1, 28359 Bremen, Germany Sultan Qaboos University, College of Science, Biology Department, P.O. Box 36, 123, Al Khoud, Oman Coral Reef Ecology Group (CORE), GeoBio-Center & Department of Earth and Environmental Science, Ludwig-Maximilians Universität, Richard Wagner Str. 10, 80333 München, Germany d Department of Marine Ecology, Tjärnö, University of Gothenburg, 452 96 Strömstad, Sweden e Jacobs University Bremen, Campus Ring 1, 28759 Bremen, Germany b c

a r t i c l e

i n f o

Keywords: Lophelia pertusa Cold-water coral reef Sedimentation Anoxia Cold-water coral-derived mucus Drill cuttings

a b s t r a c t In laboratory experiments, the cold-water coral Lophelia pertusa was exposed to settling particles. The effects of reef sediment, petroleum drill cuttings and a mix of both, on the development of anoxia at the coral surface were studied using O2, pH and H2S microsensors and by assessing coral polyp mortality. Due to the branching morphology of L. pertusa and the release of coral mucus, accumulation rates of settling material on coral branches were low. Microsensors detected H2S production in only a few samples, and sulfate reduction rates of natural reef sediment slurries were low (<0.3 nmol S cm3 d1). While the exposure to sediment clearly reduced the coral’s accessibility to oxygen, L. pertusa tolerated both partial low-oxygen and anoxic conditions without any visible detrimental short-term effect, such as tissue damage or death. However, complete burial of coral branches for >24 h in reef sediment resulted in suffocation. Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction Lophelia pertusa (L. 1758) is the most common reef-building scleractinian deep-water coral (Freiwald et al., 2004). It is reportedly found between 39 m (Rapp and Sneli, 1999) and 3380 m depth (Squires, 1959), between 4 °C and 12 °C (Teichert, 1958), and at densities between 27.35 kg m3 and 27.65 kg m3 (Dullo et al., 2008). In addition to building benthic reefs, L. pertusa was also observed to colonize structures of oil and gas platforms in the northern North Sea within the water depth range of 59– 132 m (Gass and Roberts, 2006). L. pertusa forms bush-like colonies that can reach a height of up to 2 m (Rogers, 1999). Cold-water corals, as sessile filter feeders, require nutrient delivery. Thus, they preferentially settle on elevated features where bottom current speeds are high, such as banks, seamounts and ridges. They are also found in areas where nutrient delivery is enhanced by other Abbreviations: S, sediment; DC, drill cuttings; S + DC, mix of both sediment and drill cuttings. ⇑ Corresponding author. Current address: University of Arizona, P.O. Box 210088, Tucson, AZ 85721, USA. Tel.: +1 520 626 6297; fax: +1 520 621 9903. E-mail address: [email protected] (E. Allers). 1 Current address: Department of Earth Sciences, University of California, Riverside, 900 University Ave., Riverside, CA 92521, USA. 0025-326X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.marpolbul.2013.07.016

hydrodynamic mechanisms, such as internal waves or downwelling events (Frederiksen et al., 1992; Dorschel et al., 2007; White, 2007; Mienis et al., 2009; Wagner et al., 2011). Coral thickets often grow into the prevailing current to optimally benefit from the nutrient supply (Wilson, 1979; Thiem et al., 2006; Buhl-Mortensen et al., 2010). High current flow generally prevents material from settling and burying corals, especially in the upper, current-exposed zone of living coral reefs (Dowdeswell et al., 1996; Dorschel et al., 2007; Mienis et al., 2007). Within the cold-water coral thickets, however, low energy microenvironments develop, which results in sedimentation of biogenic carbonate debris and allochthonous sediment particles between the coral branches of the thicket (Dorschel et al., 2007; De Haas et al., 2009; Mienis et al., 2009). Exposure of living corals to settling particle material is furthermore facilitated by anthropogenic activities, e.g. resuspension of sediments by fishing (Pilskaln et al., 1998), and following the release of drill cuttings produced during the drilling operations of the offshore oil and gas industry (Lepland and Mortensen, 2008; Trannum et al., 2010). Drill cuttings, drilling waste material from the offshore petroleum industry, are the broken up pieces of rock produced during drilling, combined with a thin layer of drilling fluid. This drilling fluid is used to cool the drill bit and push broken rock

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fragments from the drill hole. Although non-toxic, this fluid can contain high percentages of fine particulate material, the ‘weighting agent’ (commonly the mineral barite), which is added to ensure smooth drilling and to maintain a positive pressure within the well (Neff, 2005). Commonly during a drilling event, these drill cuttings are released to the ocean periodically over a period of 3–5 weeks (Purser and Thomsen, 2012). The effects of sedimentation on tropical stony corals have been studied for many years (reviewed in Rogers, 1990; Fabricius, 2005; Weber, 2009). Short-term sediment exposure affects these corals by reducing their zooxanthellate symbionts’ photosynthetic efficiency whilst increasing their respiration, resulting in bleaching and necrosis (Bak and Elgershuizen, 1976; Riegl and Branch, 1995; Philipp and Fabricius, 2003). Sediment rejection activities, through mucus production and polyp movement, further increase energy expenditure (Bak and Elgershuizen, 1976). In contrast, the effect of sediment coverage on cold-water corals is not very well understood. In a laboratory study on cold-water corals retrieved from the Gulf of Mexico, Brooke et al. (2009) tested the tolerance of two L. pertusa morphotypes to different loads of autoclaved sediment, as well as the response to complete short-term sediment coverage. The authors showed that L. pertusa was able to tolerate fairly high short-term sediment coverage of 2–4 days before coral mortality sets in and suggested that mortality was most likely due to oxygen deficiency. Sub-lethal effects on L. pertusa from exposure to both benthic sediment and drill-cuttings, on the other hand, include loss of tissues (Larsson and Purser, 2011) as well as reduced skeletal growth and reduced larval survival (Larsson et al., 2013). The present study aimed to assess in detail how anoxia develops at the coral surface following sediment coverage and to determine whether this had any impact on coral health. A particular focus of this study was to determine whether anaerobic microbial processes (such as sulfate reduction) on the coral surface would lead to coral damage. Laboratory experiments were carried out with cold-water corals and reef sediments retrieved from the Tisler Reef, Norwegian Skagerrak as well as with drill cuttings supplied by the company Statoil, Norway. Microsensors and 35S-tracer techniques were used to assess the development of anoxia at the coral surface following coverage. The hypothesis tested was that resuspended local material (reef sediments with the associated microbial community), or drill cuttings settling onto the coral would lead to oxygen depletion on the coral surface. Consequently, anaerobic microenvironments on the coral surface would form, with these providing suitable habitats for sulfate-reducing microbial assemblages. The production of H2S by these assemblages during sulfate reduction would represent a potential danger to the coral tissue as sulfide is a cell toxin (Bagarinao, 1992).

2. Material and methods 2.1. Site and Sampling Reef sediment and branches of white L. pertusa were collected from Tisler Reef (58°59 N, 10°57 E) located in the Norwegian Skagerrak at a depth of 70–160 m. The material was collected during ROV (Remotely Operated Vehicle) -assisted collection campaigns in April 2007, July 2007, and May 2008. Corals and sediment were kept at in situ temperature of approximately 8 °C and transported to the laboratory at the Marine Station of the Sven Lovén Centre for Marine Sciences, Tjärnö, Sweden, within hours of collection. Corals were transferred to aquaria supplied with flow-through of pre-filtered seawater from a depth of 40 m in the adjacent Koster Fjord. Two to seven days after collection, these coral fragments were used in experiments as described below. Drill cuttings for all presented experiments originated from the 17 1/200 section of

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a well drilled in the Smørbukk Sør drilling field during November 2006 and were supplied by Statoil. These cuttings came from a 1320 m long section of the drilling hole, which cut predominantly through claystone and occasionally layers of sand. 2.2. Sediment analyses and pre-treatment A subsample of the reef sediment from the May 2008 campaign was freeze-dried and analyzed for concentration of total carbon (TC), total nitrogen (TN) and total inorganic carbon (TIC). The concentration of total organic carbon (TOC) was calculated following the methods described in Wehrmann et al. (2009). Sediments for all experimental campaigns were sieved through 1 mm mesh size to remove any living macrofauna, the activity of which would bias results of microsensor deployments (bioturbation), and to remove larger fragments, which mostly comprised of biogenic carbonate debris that could break microsensors. Sieved sediments were stored refrigerated until used in experiments. Reef sediments and drill cuttings from the April and July 2007 collection campaigns were analysed for grain size using a Laser in situ Scattering and Transmissiometry device (LISST-100X, Sequoia Instruments). 2.3. Measurement of dry weight (dw)/wet weight (ww) ratios For uniform dosing of sediments and drill cuttings by dry weight, the dry weight/wet weight ratios were determined for each material type. The sediments were stirred and mixed thoroughly, with the wet weights of three replicate samples of each material type measured. Samples were dried at 60 °C until constant weight and the ratios were then calculated. 2.4. Sedimentation experiments In the first set of flow chamber experiments, the amount of settling sediment required for coverage of coral branches to be sufficiently deep to allow microsensor investigations was determined experimentally. Flow chambers of 10 cm  20 cm were set up with a water flow of 450 ml min1 and a water temperature of 8–9 °C. Twelve to eighteen fragments of L. pertusa, each 3–6 cm long and with at least 4 active polyps, were placed at the bottom of the flow chambers (Fig. 1) and left to recover from the stress of transfer for 24 h. Three sediment concentrations were tested: (i) 66 mg cm2 of sediment (dry weight, dw; designation 1), (ii) a 3 times higher load of 198 mg cm2 (dw; designation 3), and (iii) a 7 times higher load of 462 mg cm2 (dw; designation 7) (Fig. 2). These three concentrations were selected to be equivalent to that required to achieve a maximum depositional depth of 6.3 mm (an exposure threshold used by the offshore industry in risk assessment), i.e. 66 mg cm2 (Larsson and Purser, 2011) and exposures 3 and 7 times higher than this (3 and 7 exposures), which represent high levels of exposure, which may result from resuspension or continuous local drill cutting release. In all of the sedimentation load experiments, three different sediment types were studied: (a) Tisler Reef sediment (S), (b) drill cuttings (DC), and (c) a mixture of S and DC (S + DC, 1:1). In the field, these three treatment types would represent (1) typical reef sediments, as may be resuspended by bottom trawl activity in the vicinity of reefs or changes in the bottom water current regime, (2) settling waste drill cuttings released by a drilling rig and (3) waste drilling material mixed with resuspended material, as may be generated as a result of drill anchoring and/or the drilling process (Purser and Thomsen, 2012). This would represent exposure of the corals to drill cuttings amended with the in situ microbial community of the reef sediment. All sediment types were mixed thoroughly with seawater before addition to experimental flow chambers in order to obtain

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Fig. 1. Cross section of the flow chamber. Coral fragments lie at the bottom of the chamber. Water in- and outlets are on the left and right sides of the chamber, respectively. (a) Water flows into the chamber from the left and leaves the chamber on the right. (b) In order to allow settling of sediments, the water flow through the chambers was halted, then particles were added. (c) After approx. 12 h, clearance of the water indicated that the particles had settled and water flow was re-established.

homogenous suspensions. At the commencement of each experiment, the water flow through chambers was halted and the homogenized suspension was carefully resuspended into the seawater. After 12 h, the material had settled and the water cleared in each flow chamber, at which point flow through was re-established. Non-sediment exposed coral pieces served as control. 2.5. Microsensor measurements The flow chamber setups containing loads of 462 mg cm2 (7) of each of the sediment types (S, DC, or S + DC) were subsequently used for microsensor measurements. On eight of the following eleven days, steady-state O2, pH and H2S microprofiles were measured using a computer-controlled setup (software m-Profiler; www.microsen-wiki.net) at up to six different measurement points on two coral pieces (replicates). The measurement points were in the middle of sediment patches and represented the locations with thickest sediment cover on each coral piece. Vertical microsensor positioning with 1 lm precision was facilitated by a VT-80 linear positioner (Micos, Germany) equipped with a DC motor (Faulhaber, Germany). The microsensors were positioned at the coral surface with assistance of a dissecting microscope (SV6, Zeiss,

Germany). Measurements were carried out by moving the microsensor from the coral surface to the water column in 100 lm steps. The O2 and H2S microsensors were connected to a fast-responding picoammeter while the pH microsensor was connected to a millivoltmeter. For pH measurements, liquid ion-exchange (LIX) membrane potentiometric microsensors were prepared as described previously (Revsbech, 1989; Jeroschewski et al., 1996; Kühl et al., 1998). The signals were collected using a data acquisition card (DAQCard-AI-16XE-50, National Instruments). A more detailed description of the microsensor measurements is presented in the online supplementary material.  2 The total sulfide ([S2 ]) in the collected tot ] = [H2S] + [HS ] + [S calibration subsamples was determined colorimetrically (Cline, 1969; Fonselius, 1983). H2S concentrations for calibration were obtained as previously described (Millero et al., 1988; Jeroschewski et al., 1996). The total sulfide concentrations were calculated from the local H2S concentrations and pH values for selected measurements (Millero et al., 1988). 2.6. Burial experiment To determine the effect of complete burial of cold-water corals with natural (non-autoclaved) reef sediment, an additional

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Fig. 2. L. pertusa fragments covered by different amounts of sediment, (a) 1 (=66 mg cm2), (b) 3, (c) 7. (d) Mucus threads were observed after exposure of the corals to sedimentation.

experiment was conducted. In this burial experiment, sediment amended with lyophilized algal cells (Spirulina, Sigma–Aldrich, Steinheim, Germany) as a source of organic carbon was placed on six coral fragments in sufficient concentration to ensure total polyp coverage and burial. After 24 h, 48 h and 72 h of incubation in this anoxic sediment, two coral branches were retrieved from the flow chamber and returned to oxic 0.2 lm-filtered seawater to allow 24 h for recovery. Following this recovery period, each polyp was scored as ‘dead’ (no tentacles visible) or ‘alive’ (polyp’s tentacles partially or fully extended; after Sassaman and Mangum, 1972). 2.7. Sulfate reduction rates Two slurries were prepared from homogenized reef-associated surface sediment from the May 2008 collection campaign and 0.2 lm-filtered, anaerobic seawater in a 1:1 dilution. A 125 mg cm2 Spirulina solution (Spirulina powder, Sigma–Aldrich, Steinheim; in 0.2 lm-filtered seawater) was added to both slurries immediately after preparation. Slurries were kept at in situ temperature (8 °C) under N2 atmosphere in the dark throughout the course of the experiment. Slurries were pre-incubated for 54.5 h after preparation to allow for the degradation of added Spirulina in order to evoke elevated, detectable sulfate reduction rates (SRR), as it is known from cold-water reef sediments that anaerobic carbon mineralization usually proceeds at very low rates (Wehrmann et al., 2009). Subsequently, 50 ml of freshly-harvested coral mucus were added to one of the slurries (hereafter referred to as mucus-slurry) and the equal amount of anaerobic, 0.2 lm-filtered fjord water was added to the second slurry (control-slurry). Prior to the experiment, mucus was freshly collected from an additional 10 to 20 small (lengths: 3–10 cm) L. pertusa fragments. These fragments were originally sampled from Tisler Reef and kept in 40 l glass aquaria with flow-through water originating from 40 m in the Koster Fjord (see Site & Sampling) for 2–60 d prior to mucus sampling. Mucus was collected by exposing the coral fragments to air for 3–5 min. Mucus released during the first minute was discarded, with subsequent production collected in Petri dishes.

When necessary, 0.2 lm-filtered seawater was used to detach mucus from the coral fragments (final dilution: approximately 1:5, mucus:seawater). During the course of the experiment, three time points before, and nine time points after mucus or seawater addition (6–152 h after start of experiment) were selected for shortterm radiotracer incubations. During sampling, slurry jars were gassed with N2 to prevent O2-penetration. At every time point, triplicate slurry aliquots were transferred into 5 ml glass tubes sealed with butyl rubber stoppers at both ends. Five ll 35 SO2 4 -tracer solution (200 kBq, Hartmann Analytic) were injected into each vial and samples incubated for 6 h at 8 °C. Slurry aliquots for SO2 4 measurement were centrifuged and the supernatant fixed by adding ZnAc (2%, w/v). In a second experiment, a slurry of reef-associated sediment and anaerobic, filtered fjord water was prepared and divided into two jars. Large coral fragments were removed from the sediment prior to dilution. The slurries were amended with fresh L. pertusa mucus and seawater as control, respectively, giving an overall dilution of 1:1 between sediment and liquid (fjord water + additive). Sample handling was conducted in a glove bag purged with N2. Individual sub-samples of both slurries were distributed into 5 ml glass tubes sealed with butyl rubber stoppers. All samples were injected with 5 ll 35 SO2 4 -tracer solution (200 kBq, Hartmann Analytic) and incubated at 8 °C in the dark. Incubations were carried out in triplicates and stopped after 0 (blanks), 4, 8, 16, 32 and 48 h for the mucus slurry and 0, 4, 16 and 48 h for the control, respectively, in order to measure the cumulative sulfate reduction rates. All radiotracer incubations were terminated by transferring the samples into vials containing 10 ml ZnAc (20%, w/v). Reduced radiolabeled sulfur was separated by the cold distillation method described by Kallmeyer et al. (2004). Median sulfate reduction rates (SRR) per cm3 of undiluted sediment were calculated based on the pore-water sulfate concentration (in nmol cm-3sed corrected for porosity), the total amount of 35S–SO2 4 and the amount of total reduced inorganic sulfur species (TRI35S) produced during sulfate reduction as well as an estimated fractionation factor between 35 S and 32S of 1.06 after Jørgensen (1978). Sulfate concentrations

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were measured after a 100:1 dilution of the aliquots by non-suppressed anion exchange chromatography (Waters 510 HPLC Pump; Waters IC-Pak 50  4.6 mm anion exchange column; Waters 430 Conductivity detector) using IAPSO seawater (Canada) as reference standard. 3. Results 3.1. Sediment composition Sediment from Tisler Reef was composed of greenish-grey silt and clay particles (Table 1). The sediment, as measured from sediments collected during the May 2008 collection campaign, had a carbonate content of 17.8 wt% (excluding the large coral fragments that were removed prior to analyses), and a total organic carbon (TOC) content of 1.6 wt% dw. The drill cuttings consisted of claystone with some thin layers of sand (Table 1). They were composed of less clay and more silt than the reef sediment and were on average larger in grain size. The TOC content of the drill cuttings was 2.4 wt% dw. 3.2. Sediment coverage

3.3. Development of anoxia at the coral surface Uncovered corals had an oxygen concentration at the surface that was close to that in the surrounding seawater (approximately 280 lM, Fig. 3a). Sediment coverage led to a gradual decrease of oxygen concentration at the coral surfaces in all three sediment types (S, S + DC, DC), as shown in Fig. 3 by oxygen profiles from 2-3 selected days during the experimental period. At the surface of the coral branches covered by >3 mm of S or S + DC, the oxygen concentration decreased below 50 lM within three days. After six to eight days, complete anoxia had developed in these sediment types as well as in S + DC layers that were <3 mm in thickness. After eleven days, anoxia had also developed in the natural sediment layers that were <3 mm in thickness. Thus, the thicker the coral-covering sediment layer, the faster anoxia developed at the coral surface. Drill cuttings were less effective in inducing anoxia than natural sediment. On coral branches that were covered with DC, anoxia developed by day 11 in sediment layers >2 mm thick, whereas within the experimental timeframe it never fully developed in sediment layers <2 mm (Fig. 3d).

3.4. Effect of complete burial on Lophelia pertusa 2

The nominal sediment cover of 66 mg cm (1) and of the three times higher load of 198 mg cm2 (3) did not result in adequate sediment coverage of coral branches for microsensor measurements (Fig. 2a and b). The sediment-free surface area of the fragments in >75% of cases was greater than the area covered by sediment, independent of the sediment type used (Tables S1 and S2). The deposition of 462 mg cm2 (7) of sediment resulted in accumulation of sediments on top of the coral branches. However, even this high sediment load did not result in complete coverage, with most polyps still able to extend at least their tentacles above the sediment horizon (Fig. 2c). The thickness of sediment layers ranged from 1.5 to 15.3 mm (median 3 mm), revealing a large heterogeneity in sediment coverage across and even within branches (Fig. S1). As a result, oxygen measurements which were initially intended as replicate observations were then found to have been conducted in layers of, e.g., 15 mm and 1.5 mm sediment, respectively. In order to nonetheless interpret the results in a meaningful way, the data set was divided by its median into samples with coverage layers <3 mm or >3 mm. This division into two sub-datasets reduced the number of replicates per measurement, with on occasion a sole measurement being available. As a consequence, we decided not to test the microsensor data for significance, but rather report on trends based on replicated as well as non-replicated results. As the drill cuttings layers were generally thinner than the sediment layers (median 2 mm), this particular data set is referred to as <2 mm and >2 mm sediment layers. The investigated experimental sediment loads (i.e. 1, 3, and 7) did not lead to any visible damage of the coral fragments, as was indicated by the extended tentacles of the polyps following exposure.

After 24 h of complete sediment coverage, two coral fragments were retrieved from the sediment. During rinsing, the sediment was easily detached from the branches. After a 1 h recovery time in oxic seawater, all polyps’ tentacles were at least partially extended again. Coral branches that were exposed to the anoxic sediment for 48 h and 72 h were harder to clean as the sediment

Table 1 Sediment properties.

Grain size fractiona (% total) Clay (<3.9 lm) Silt (>3.9 lm, <62.5 lm) Carbonate (%wt.) TOC (%wt. %dw.) C/N ratio

Reef sediment (S)

Drill cuttings (DC)

47 51 17.8 1.6 9

25 70 n.a. 2.4 n.a.

n.a. – not analyzed. Size-classification after Wentworth (1922).

a

Fig. 3. Representative oxygen profiles from the coral surface (0 mm) through the sediment horizon (7 nominal sediment cover) and towards the free water: (a) Control without sediment, (b) thin and thick layers of reef sediment (S), (c) thin and thick layers of mixed reef sediment and drill cuttings (S + DC), (d) thin and thick layers of drill cuttings (DC).

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tended to stick to the coral surface. These corals did not appear to recover over the following 24 h, with their tentacles never extended. These polyps were considered dead. 3.5. Hydrogen sulfide (H2S) and total sulfide concentration at the coral surface Simultaneous with the depletion of oxygen, the H2S concentration increased at the coral surface. After 10 days of sediment coverage, H2S and total sulfide concentrations were observed to be heterogeneous across and within fragments (Fig. 4). Despite this heterogeneity, all locations showed a common trend: an increase in total sulfide towards the coral surface, with sulfide concentrations >0.20 mM at the coral surface (Fig. 4). Maximum H2S concentrations at the coral surface reached 1 mM. 3.6. Sulfate reduction Sulfate reduction rates (SRR) of reef sediments were low, as demonstrated by the results of the experimental run without Spirulina addition (Fig. 5; reaching a cumulative SRR of 0.6 nmol cm3 ± 0.4 nmol cm3 after 48 h). Within 16 h, the amount of sulfate reduced in the mucus-amended samples was significantly higher than in the control samples (0.31 ± 0.05 nmol cm3 and 0.12 ± 0.08 nmol cm3, respectively). Thus, the mucus did not inhibit sulfate reduction but rather led to an increase of sulfate reduction rates. In the slurry experiment with Spirulina addition, no significant difference between control and mucus-slurry was detected for 5 d (Fig. 6). However, at day 6 the rates in the mucus-slurry had strongly increased, with SRR twice as high as in the control (77.3 ± 8.1 nmol cm3 d1 and 30.4 ± 7.4 nmol cm3 d1, respectively).

Fig. 5. Cumulative sulfate reduction rates in reef sediment with (solid circles) and without (empty circles) coral mucus-amendment as monitored for 48 h. Error bars indicate standard deviation.

4. Discussion The sedimentation experiment showed that a very high sediment load is needed to achieve persistent sediment coverage of living L. pertusa branches. The gradual local decrease of oxygen at the coral surface where layers of sediments (S, DC, S + DC) built up did not observably affect L. pertusa for the duration of the sedimentation experiment of 11 days. The burial experiment, on the other hand, showed that the persistent absence of oxygen over the whole

Fig. 6. Sulfate reduction rates as measured in mucus- (solid circles) and controlslurry (open circles) incubations over >150 h (>6 d). Data points represent the median of triplicate measurements. Errors bars indicate the standard deviation. The period from 40 to 80 h is depicted enlarged in the center. The arrow indicates the time point of mucus addition.

polyp surface was detrimental for the coral. It can be concluded from these observations, that the conditions tested in the sedimentation experiment, mimicking resuspension and settling of benthic sediment and drill cuttings in the vicinity of oil and gas exploration and exploitation activities would not cause coral death within <12 days, even at concentrations 3 and 7 that required to achieve the 6.3 mm risk assessment guideline maximum depositional depth. 4.1. Development and effect of anoxia on L. pertusa branches

Fig. 4. Representative profiles of total sulfide concentrations in reef sediment covering 2 different fragments of L. pertusa (solid vs. empty) for 10 days. A distance of 0 mm corresponds to the coral surface. Two profiles were measured in a sediment layer <3 mm (designation: square), two were measured in a sediment layer >3 mm (designation: circles).

The sedimentation experiment demonstrated that a very high sediment load is needed to achieve persistent sediment coverage of living coral branches. The amount of sediment added was above natural sedimentation regimes in living cold-water coral ecosystems. Mass fluxes to cold-water coral ecosystems range from 0.5 to 3.7 g m2 d1 as reported by Duineveld et al. (2004) for Galicia Bank and Mienis et al. (2009) for the Southwest Rockall Trough margin. However, both studies also reported very high mass flux pulses, which they attributed to resuspension events resulting from strong currents. Increased internal wave activity at the Southwest Rockall Trough margin in winter and summer occurs annually and the consequent sediment resuspension may affect living corals

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during these periods (Mienis et al., 2009). Resuspension events can also be expected from reversals in the current flow direction, as fine-grained material deposited during slack water may then be exposed to higher current speeds causing its resuspension. Seasonal and periodic reversals of the current flow direction have been reported for Tisler Reef by Guihen et al. (2010) and Wagner et al. (2011). By simulating high-sedimentation events, this study mimics situations triggered by human activities such as hydrocarbon exploration and exploitation, or fishery activity. The threshold level for burial presently used by the oil companies on the Norwegian Continental Shelf in the risk assessment of drilling discharges is 6.3 mm (Smit et al. 2008).In the sedimentation experiment described here, exposures of concentrations to achieve this depth (6.3 mm) and 3 and 7 this depth were delivered to the corals during one depositional event. In an authentic drilling situation however, the discharges occur periodically throughout the drilling period and not all at once (Purser and Thomsen, 2012). In close vicinity of a drill well, this threshold depth of 6.3 mm is not enforced and thick layers of deposited material, often of coarse grain size may occur. Though all drilling campaigns are different, this area of thick deposition is measured in 10s of meters. An in depth survey of a 2006 drilling campaign showed that visible drill cutting accumulation seldom exceeded 100 m from the point of discharge (Gates and Jones, 2012). On the Norwegian margin drilling permission is only given to companies following an in depth survey of the intended drilling area, with potential coral reef structures mapped and possibly investigated by ROV video. Companies are also expected to carry out analysis of local current regimes. In the light of these surveys, computer models are employed to minimize the risk of coral reefs being exposed during drilling operations to concentrations of material sufficient to reach depositional depths of 6.3 mm (Rye et al., 2008). Although computer modeling techniques are improving rapidly, they are not 100% accurate and unusual current regimes may be experienced during a drilling event. Such unexpected occurrences are also factored into the planning of drilling campaigns. During a 2009–10 drilling campaign within the Morvin field on the Norwegian margin flow patterns were not as predicted, but drill cutting release point was sufficiently distant (>500 m) from the coral reefs ensuring that the 6.3 mm depth threshold was not reached despite the unexpected flow conditions (Tenningen et al., 2011). Lepland and Mortensen (2008) showed however that even at 4 km distance from a drill well traces of drill cuttings can be observed in seabed sediments in the vicinity of coral reefs years after a drilling event, as indicated by the measured Barium concentrations (Renaud et al., 2008). At the time of the drilling event described in Lepland and Mortensen (2008) the presence of the coral reefs at such close proximity to drilling was not known, and therefore no exposure risk assessments were carried out prior to drilling, nor settled material depth analyses conducted after drilling. Where branching morphology and mucus secretion of L. pertusa did not prevent settling of material onto the coral branches, layers of sediment (S, S + DC, DC) were observed. The present data describe the following trend: Anoxia developed gradually at the L. pertusa surface 1–2 weeks following sediment coverage. The decrease in oxygen concentration on the coral surface can be attributed to oxygen consumption by the coral itself and respiratory activity of the in situ sedimentary microbial community. With on-going uptake of oxygen, an oxygen gradient from the surrounding water to the coral surface developed. Microbial degradation targeted organic carbon in the sediment and with a high likelihood the labile organic carbon released by the coral in the form of mucus (Wild et al., 2009). Although little is known about the function of coral-derived mucus in cold-water coral reefs, it is suggested that cold-water corals, as

with warm-water corals, actively release mucus to enhance particle removal from their surface tissue (Wild et al., 2008). Anoxia on the coral-surface developed faster at coral branches covered by natural reef sediment than at those covered by drill cuttings, likely due to three factors. Firstly, the reef surface sediment hosts a microbial community that is adapted to suboxic to anoxic conditions and thus returns to normal activity soon after sediment settlement. Drill cuttings originate from the deep biosphere and have a lower microbial density (Jørgensen and Boetius, 2007). Secondly, the difference in organic matter concentration and composition affects the development of anoxia. Weber et al. (2012) observed a significant correlation between decreasing O2 concentrations in sediment layers covering tropical corals with increasing concentrations of organic carbon. Although the TOC content in the drill cuttings was higher than in the reef sediment, the organic matter in both sediment types probably differs greatly in degradation state. Therefore, the reef sediment collected in May 2008 was investigated for its degradation state using chlorophyll degradation products, i.e. the chlorin index. The chlorin index (CI), introduced by Schubert et al. (2005), ranges from 0.2 for fresh chlorophyll to 0.9 for strongly degraded material. The retrieved reef surface sediment had a CI of 0.7–0.8 (data not shown), which indicates subtly degraded organic material that is still available for degradation by the sedimentary microbial community on shorttime scales. The drill cuttings originated from sediment depth of up to 2000–3000 m and most probably had a more degraded character, given that the reactivity of buried organic matter generally decreases with sediment depth following the power law (Middelburg, 1989) and the low concentrations associated with the drilling fluids (celluloses). Thirdly, the larger average grain size of drill cuttings allows more oxygen to diffuse into the sediment covering the coral. Mixing of the reef sediment and the drill cuttings led to the inoculation of the drill cuttings with the reef sedimentary community, with anoxia developing on branches exposed to this treatment over a timescale between those observed for the natural sediment and drill cuttings treatments. Concomitant to the depletion of oxygen on the coral surface, H2S concentrations rose. The sulfide was likely either produced by sulfate reduction or originated from hydrolysis of organic matter (Weber, 2009; Weber et al. 2012). Further increase of the sulfide concentration would pose a threat to the coral as it is a toxin that leads to tissue damage and increased mortality. 4.2. The effect of coral-derived mucus on sulfate reduction in coral reef sediments The mucus released by L. pertusa did not show an inhibitory effect on sulfate reduction. The low sulfate reduction rates (SRRs) within the first 88 hours of the Spirulina-amended sediment-slurry incubations can be attributed to low concentrations of fermentative products available to the sulfate-reducing microbial community (Wehrmann et al., 2009). The increase within the following hours in both slurries (control and mucus-slurry) was most likely the result of the addition of Spirulina. The strong SRR increase in the mucus-slurry thereafter, however, indicates that the added mucus represented an additional carbon source for the sulfate-reducing microbial assemblage. 4.3. Survival strategy of L. pertusa during periods of increased sedimentation The gradual decrease of oxygen at the coral surface during the sedimentation experiment did not observably affect L. pertusa. In the natural environment, L. pertusa can occur at local oxygen minimum zones (e.g. 138–224 lM, Freiwald, 2002) and may therefore

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have a survival strategy for sub-oxic conditions. The burial experiment on the other hand showed that the persistent absence of oxygen over the whole polyp surface was detrimental for the coral. The complete coverage of coral fragments with anoxic sediment led to stress reactions in the coral (withdrawn tentacles, mucus release), but did not kill the coral unless exposure lasted >24 h. A similar finding was made by Brooke et al. (2009) with sediment tolerance experiments on two L. pertusa morphotypes from the Gulf of Mexico, showing that the corals were able to tolerate high sedimentation loads for several days before mortality was observed. Larsson and Purser (2011) showed that if L. pertusa is repeatedly exposed to low concentrations of fine sedimentary material (rather than large pulses of material as investigated here) the percentage of the surface, which remains covered by sediment increases slightly with each exposure, and will lead to polyp mortality if wholly smothered. The ability to cope with anoxic conditions was also observed previously in other Cnidaria, such as anemones (Sassaman and Mangum, 1972). The mechanism of how L. pertusa copes with oxygen deprivation is not yet understood (Beattie, 1971; Sassaman and Mangum, 1972, 1973; Mangum, 1980; Dodds et al., 2007 and references therein). That L. pertusa endured anoxia only for a comparatively short time (whereas anemones may survive for weeks; Mangum, 1980) and were slower to recover when returned to the oxic environment, suggests an anoxia-tolerance rather than an anaerobic metabolism, i.e. metabolic down-regulation rather than fermentation. Also, the ecophysiological study of L. pertusa by Dodds et al. (2007) demonstrated that the species is unable to maintain aerobic metabolic activity below 140 lM O2, which indicates a threshold in the oxic rather than the anoxic realm. The combined results of the presented study indicate that L. pertusa is resilient to sedimentation-induced O2 stress, but that this resilience is conditional. If coverage by sediment was complete and lasted long enough, the coral could not recover and died. Under natural conditions, situations in which this threshold is met or overstepped are considered rare. However, human activities, such as the above mentioned hydrocarbon exploration, discharge large amounts of material into the environment, particularly the drill cuttings and attached drilling fluids, and certainly have the potential to cover an entire coral reef. This scenario can be avoided, provided care is taken in assessing the benthic habitat and hydrodynamic conditions in the vicinity of a drilling site and in planning activities accordingly, as is common practice in European waters (Purser and Thomsen, 2012). The amount of sediment that is sufficient to bury a coral reef varies with the size and height of the reef. From the nominal sediment coverages tested in this study (1, 3, 7, and 20, the latter of which refers to complete burial), the L. pertusa fragments suffocated only under 20 cover. Thus, the critical parameter for short-term exposure is the fraction of the coral animal that is buried during sedimentation events – whether this be by natural and continuous or anthropogenic and ephemeral processes. In long-term scenarios, the coral – as a reef – will survive as long as the growth rate is higher than the sedimentation rate of resuspended material (Correa et al., 2012). L. pertusa is a low-oxygen resilient organism to which the most imminent threats are physical destruction caused by anchoring, bottom trawling and dredging (Fosså et al., 2002; Hall-Spencer et al., 2002; Althaus et al., 2009; Fosså and Skjoldal, 2010) and the possible long-term effects of sedimentation on growth rate and reproduction, as observed in shallow-water corals (Bak, 1978; Dallmeyer, 1982) and recently in L. pertusa (Larsson et al., 2013).

Disclosure statement The authors declare no conflict of interest. All authors have approved the final version of the manuscript. Each author’s

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contribution is listed in the following: RMMA, DdeB, EA, LMW, AIL designed the experiments. EA, RMMA, TW, LMW, AP, AIL performed research (TW, AIL, EA, LMW – collections of field samples; RMMA, TW, AIL, AP – sedimentation experiments; RMMA, TW, EA – microsensor measurements; LMW, EA – sulfate reduction rates; EA – Burial experiment). EA, DdeB, TW, AIL analysed data. EA, LMW, AP, DdeB, RMMA wrote the paper. Role of funding source This work was funded by the Max Planck Society and Statoil and is a CORAMM (Coral Risk Assessment, Monitoring and Modelling) group collaboration. The authors declare independence in experimental design and execution, as well as in data evaluation and interpretation and manuscript preparation and submission. Acknowledgements Katharina Kohls and Miriam Weber are gratefully acknowledged by E.A. for sharing their microsensor knowledge. Tomas Lundälv, Lisbeth Jonsson, Sandra I. Schöttner and Christian Wild were invaluable in the field and during experimental stages. Gabrielle Eickert and Ines Schroeder are acknowledged for providing excellent microsensors and support in handling them. For great assistance in laboratory analysis we thank Katsia Pabortsava. We also thank Timothy G. Ferdelman for helpful discussions on the radiotracer experiments. This paper is a CORAMM (Coral Risk Assessment, Monitoring and Modelling) group collaboration, funded by the Max Plank Society and Statoil. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.marpolbul.2013. 07.016. References Althaus, F., Williams, A., Schlacher, T.A., Kloser, R.J., Green, M.A., Barker, B.A., Bax, N.J., Brodie, P., Schlacher-Hoenlinger, M.A., 2009. Impacts of bottom trawling on deep-water ecosystems of seamounts are long-lasting. Mar. Ecol.-Prog. Ser. 379, 279–294. Bagarinao, T., 1992. Sulfide as an environmental factor and toxicant: tolerance and adaptations in aquatic organisms. Aquat. Toxicol. 24, 21–62. Bak, R.P.M., 1978. Lethal and sublethal effects of dredging on reef corals. Mar. Poll. Bull. 9, 14–16. Bak, R.P.M., Elgershuizen, J.H.B.W., 1976. Patterns of oil-sediment rejection in corals. Mar. Biol. 37, 105–113. Beattie, C.W., 1971. Respiratory adjustments of an estuarine coelenterate to abnormal levels of environmental phosphate and oxygen. Comp. Biochem. Physiol. 40B, 907–916. Brooke, S.D., Holmes, M.W., Young, C.M., 2009. Sediment tolerance of two different morphotyopes of the deep-sea coral Lophelia pertusa from the Gulf of Mexico. Mar. Ecol.-Prog. Ser. 390, 137–144. Buhl-Mortensen, L., Vanreusel, A., Gooday, A.J., Levin, L.A., Priede, I.G., BuhlMortensen, P., Gheerardyn, H., King, N.J., Raes, M., 2010. Biological structures as a source of habitat heterogeneity and biodiversity on the deep ocean margins. Mar. Ecol.-Evol. Persp. 31, 21–50. Cline, J.D., 1969. Spectrophotometric determination of hydrogen sulfide in natural waters. Limnol. Oceanogr. 14, 454–458. Correa, T.B.S., Grasmueck, M., Eberli, G.P., Reed, J.K., Verwer, K., Purkis, S.A.M., 2012. Variability of cold-water coral mounds in a high sediment input and tidal current regime, Straits of Florida. Sedimentology 59, 1278–1304. Dallmeyer, D.G., 1982. Effects of particulate peat on the behavior and physiology of the Jamaican reef-building coral Montastrea annularis. Mar. Biol. 68, 229–233. De Haas, H., Mienis, F., Frank, N., Richter, T., Steinacher, R., de Stitger, H.C., van der Land, C., van Weering, T.C.E., 2009. Morphology and sedimentology of (clustered) cold-water coral mounds at the south Rockall Trough margins, NE Atlantic Ocean. Facies 55, 1–26. Dodds, L.A., Roberts, J.M., Taylor, A.C., Marubini, F., 2007. Metabolic tolerance of the cold-water coral Lophelia pertusa (Scleractinia) to temperature and dissolved oxygen change. J. Exp. Mar. Biol. Ecol. 349, 205–214.

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