Marine Pollution Bulletin 62 (2011) 327–339

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Marine Pollution Bulletin

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Integrated biomarker assessment of the effects exerted by treated producedwater from an onshore natural gas processing plant in the North Seaon the mussel Mytilus edulis

Steven Brooks a,⇑, Christopher Harman a, Beñat Zaldibar b, Urtzi Izagirre b,Tormod Glette c, Ionan Marigómez b

a Norwegian Institute for Water Research (NIVA), Gaustadalléen 21, NO-0349 Oslo, Norwayb Cell Biology in Environmental Toxicology Research Group, Department of Zoology and Animal Cell Biology, School of Science and Technology, University of the Basque Country, P.O.Box 644, E-48080 Bilbo, Basque Country, Spainc Det Norske Veritas (DNV), Maries vei 20, 1363 Høvik, Norway

a r t i c l e i n f o a b s t r a c t

Keywords:BiomarkersMonitoringProduced waterMusselsPassive samplers

0025-326X/$ - see front matter � 2010 Elsevier Ltd. Adoi:10.1016/j.marpolbul.2010.10.007

⇑ Corresponding author. Tel.: +47 22185100; mo22185200.

E-mail address: sbr@niva.no (S. Brooks).

The biological impact of a treated produced water (PW) was investigated under controlled laboratoryconditions in the blue mussel, Mytilus edulis. Mussel health status was assessed using an integrated bio-marker approach in combination with chemical analysis of both water (with SPMDs), and mussel tissues.Acyl-CoA oxidase activity, neutral lipid accumulation, catalase activity, micronuclei formation, lysosomalmembrane stability in digestive cells and haemocytes, cell-type composition in digestive gland epithe-lium, and the integrity of the digestive gland tissue were measured after 5 week exposure to 0%, 0.01%,0.1%, 0.5% and 1% PW. The suite of biomarkers employed were sensitive to treated PW exposure with sig-nificant sublethal responses found at 0.01–0.5% PW, even though individual chemical compounds of PWwere at extremely low concentrations in both water and mussel tissues. The study highlights the benefitsof an integrated biomarker approach for determining the potential effects of exposure to complex mix-tures at low concentrations. Biomarkers were integrated in the Integrative Biological Response (IBR/n)index.

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1. Introduction

The chemical composition of produced water (PW) can be verycomplex and quantitatively variable among PWs, but usually theircompounds include trace metals, organic acids, phenols, alkylphe-nols, polycyclic aromatic, aliphatic hydrocarbons and residual pro-duction and treatment chemicals and their breakdown products(emulsifiers, corrosion inhibitors, antifoaming agents, corrodedmaterials, etc.) (Roe Utvik, 1999; Neff, 2002; Johnsen et al.,2004). Most of these compounds usually occur at extremely lowconcentrations (i.e. [PAHs] total in North Sea PW < 7 lg/L;Strømgren et al., 1995). Moreover, once discharged into the sea,PW is rapidly diluted and dispersed and further volatilizationand biodegradation reduces the levels of marine contamination(Flynn et al., 1996). As a result, the chemical identification andquantification of these compounds can be difficult and on mostoccasions present at concentrations below their detection limit(Smith et al., 1998). In contrast, both acute and sublethal toxic

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effects have been reported in fish and invertebrates at exposureconcentrations below 1% PW (Strømgren et al., 1995; Stephenset al., 1996, 2000; Zhu et al., 2008; Hannam et al., 2009). Thegroups of PW compounds most likely to be contributing to itstoxicity include volatile (BTEX) and semi-volatile (GRO) PAHs,phenols and dissolved ions (Smith et al., 1998; Fisher and Bidwell,2006). However, in most cases the toxicity can not be attributed toindividual components (usually at non-toxic extremely low con-centrations) but to the properties of the mixture.

The Ormen Lange gas processing plant is situated at Nyhamnaon the island of Gossa, on the West coast of Norway, where pro-duced water, gas and condensate received by pipeline from the Or-men Lange gas field 100 km offshore in the North Sea is processed.Produced water from the onshore processing plant is diluted withcooling water within the Ormen Lange system before it is dis-charged into the surrounding coastal water environment by a sin-gle outfall pipe. Macro porous polymer extraction (MPPE)technology in combination with biological treatment, can reducedissolved and dispersed hydrocarbons with up to 99% removal,and is used at the Ormen Lange gas processing plant (AkerKværner, 2006). The MPPE technology and biological treatmentcan remove most aliphatic hydrocarbons, BTEX, PAHs and NPDs

328 S. Brooks et al. / Marine Pollution Bulletin 62 (2011) 327–339

and some polar compounds including alkyl phenols to a limited ex-tent (Aker Kværner, 2006).

The main objective of the present study was to evaluate undercontrolled laboratory conditions the potential biological impact ex-erted by a treated PW effluent (Ormen Lange processing plant),which was expected to present extremely low levels (often belowdetection limits) of individual contaminants in a highly complexmixture. Blue mussels, Mytilus edulis, were selected as a sensitivetarget species representative of the biota inhabiting receivingwaters in the North Sea. The health status of the mussel can be di-rectly related to the amount of environmental stress imposedthrough a variety of factors including contaminant exposure andtherefore, it provides important information on their surroundingenvironment including waterborne toxicity.

In order to assess mussel health status, an integrated biomarkerapproach was applied in combination with chemical analysis ofboth water, through the utilisation of semipermeable membranedevices (SPMDs), and mussel tissues. Biomarkers have been previ-ously applied in fish for monitoring the impact of PW dischargesfrom offshore platforms in North Sea and Northern Shelf of Austra-lia (Codi King et al., 2005; Sturve et al., 2006; Zhu et al., 2008; Abra-hamson et al., 2008; Hylland et al., 2009; Brooks et al., 2010). Dueto the complexity of PW effluents and the variety of biological re-sponses they can induce, multiple effect biomarkers were deter-mined to provide a sensitive evaluation of mussel healthfollowing PW exposure (Cajaraville et al., 2000; Brooks et al.,2009; Hylland et al., 2009). These included: (a) acyl-CoA oxidase(AOX) activity enhancement, as a measure of peroxisome prolifer-ation (Fahimi and Cajaraville, 1995); (b) intracellular neutral lipidsaccumulation (INLA) resulting from exposure to organic com-pounds (Marigómez and Baybay-Villacorta, 2003); (c) catalase(CAT) activity increase, as indication of enhanced antioxidant de-fences (Eertman et al., 1995); (d) micronuclei (MN) formation, asa measure of DNA damage (Heddle et al., 1983); (e) reduction inlysosomal membrane stability in digestive cells (LMSDC), as bio-marker of general stress (Moore 1976; UNEP/RAMOGE, 1999); (f)changes in cell type composition (augmented relative proportionof basophilic cells; VvBAS) in digestive gland epithelium, as indica-tion of general stress (Soto et al., 2002); (g) disrupted membraneintegrity in haemocytes (LMSHC), indicative of both either generalstress or immunodeficiency (Borenfround and Puerner, 1985; Loweand Pipe, 1994); and (h) changes in the integrity of the digestivegland tissue (CTD), as an early symptom of pathological damage(Garmendia et al., submitted). The biomarkers were integrated inthe Integrative Biological Response (IBR/n) index (Beliaeff andBurgeot 2002; Broeg and Lehtonen 2006). Integrative biomarkerindices may be used to provide comprehensive assessment ofmussels� health status, which is currently regarded as the bestavailable approach for monitoring pollution effects in marineecosystems (e.g. Broeg and Lehtonen 2006; Dagnino et al., 2007;Brooks et al., 2009; Garmendia et al., submitted).

2. Material and methods

2.1. Flow-through exposure and sampling

A laboratory flow-through dosing system was designed to ex-pose mussels and semipermeable membrane devices (SPMDs) toknown concentrations of the PW over a 5 week period. The PWwas collected September 2008 from the Ormen Lange processingplant whilst operating at approximately 50% maximum produc-tion. Approximately 3000 L were collected from the Observationtank (post treatment) in 3 � 1000 L plastic airtight containersand transported overnight by road to NIVA�s marine researchstation at Solbergstrand, Norway. The PW stock was diluted with

filtered (10 lm) seawater (SW) within the flow through systemto deliver environmentally relevant exposure concentrations of0.01%, 0.1%, 0.5% and 1% of the original PW concentration. ThePW stock was measured for PAHs and metals at the start of theexposure. The flow-through system was allowed to dose for 1 weekprior to the addition of the mussels and SPMDs in an attempt toachieve steady state conditions.

The design of the flow-through system included separate 100 Lmixing tanks, which were used to combine the PW with the dilu-tion SW. The outflow water from the mixer tanks, after an approx-imate 1 h residency time, was transported to the 50 L exposuretanks holding the mussels and SPMDs. The seawater flow ratewas calculated at 2.3 L/min, which was based on a mussel clear-ance rate of 0.033 L/min with 70 mussels in each exposure tank.This was to ensure that each mussel was exposed to fresh exposuremedium. The mussels used in the study were roped mussels ob-tained from a mussel hatchery in Rissa, Norway (www.snadderogs-naskum.no). The mussels were transported on ice by overnightcourier and placed in the exposure tanks on the morning of arrival.Only mussels between 4 and 6 cm in length were selected for theexposure. Within the tanks, mussels were held within nylon meshbags and suspended within the water column. Mussels were fed aconcentrated mixed algal feed (Shellfish diet 1800). Physicochem-ical readings of the exposure media, including pH, temperature,salinity, and dissolved oxygen, as well as flow rates, were checkedon a daily basis (Table 1). Feeding and general health checks of themussels and the dosing system were made every 2 day during the5 week exposure. The exposure was conducted in the autumn.

The test was terminated after 5 week. Mussels were removedfrom the exposure tanks and processed within 2 h of removal fromthe exposure tanks. Haemolymph samples were taken for LMSHC

and MN determinations. Digestive gland and gonad tissue were re-moved from individual mussels and preserved by either snapfreezing in liquid nitrogen or by fixing in formalin. Frozen digestivegland samples were processed for biochemistry and cytochemistry(AOX, CAT, VvNL and LMSDG). Fixed digestive glands and gonadswere dehydrated in alcohol and embedded in paraffin for histolog-ical examination (gamete development, digestive gland and gonadhistopathology), and determinations of tissue-level biomarkers(VvBAS, and CTD). Whole mussel homogenates were used to mea-sure organic (PAHs, NPDs) and metal concentrations.

2.2. Semipermeable membrane devices (SPMDs)

SPMDs were wound around stainless steel deployment spiders(Environmental Sampling Technologies, Saint Joseph, USA) andplaced directly into the exposure tanks. Three replicates per tankwere used. SPMDs were spiked with a mixture of deuterated PAHas performance reference compounds (PRCs) to allow for the deter-mination of sampling rates (Booij et al., 1998; Huckins et al., 2002)and were obtained from ExposMeter (Tavelsjo, Sweden).

2.2.1. Sampler extraction and chemical analysisThe exterior of the SPMDs were wiped clean before extraction

by dialysis with 2 � 150 mL hexane (Huckins et al., 1990a). Cleanup by GPC and analysis for PAHs and NPDs by GC–MS, proceededas described below for mussel samples. Quantification of individ-ual components was performed by using the relative response ofinternal standards. In order to correct for any possible contamina-tion during study procedures, control or ‘blank� SPMDs were used.These included field controls (FCs) that were exposed to the airduring deployment and retrieval and laboratory controls (LCs) thatfollow exposure to solvents, glassware etc. during sample work up.At least one of each type of control was used per 10 exposed sam-plers. Initial (time zero) concentrations of PRCs were also estab-lished from LCs.

Table 1Physicochemical properties of the seawater in each of the treatment tanks during the 5 week exposure (mean ± SD, n = 24).

Treatment Tank Temp (�C) Dissolved oxygen (mg/L) Salinity (‰) pH

Control 10.83 ± 1.74 6.51 ± 0.72 33.13 ± 0.46 7.88 ± 0.100.01% 10.77 ± 1.74 6.79 ± 0.90 33.25 ± 0.45 7.95 ± 0.080.1% 10.78 ± 1.75 6.49 ± 0.77 33.14 ± 0.48 7.89 ± 0.090.5% 10.73 ± 1.74 6.58 ± 0.89 33.03 ± 0.47 7.92 ± 0.081% 10.89 ± 1.74 6.64 ± 0.88 32.95 ± 0.42 7.89 ± 0.11

S. Brooks et al. / Marine Pollution Bulletin 62 (2011) 327–339 329

2.2.2. Calculation of sampling rates and water concentrationsAn empirical model, described in detail by Huckins et al. (2006),

was used in the calculation of water concentrations from SPMDaccumulations. In this model compound specific effects on uptakeare adjusted based on the log Kow of the analyte and site-specificfactors arising from differences in environmental variables are ad-justed by using the PRC data. In this way the uptake for each indi-vidual compound at each sampling station was established,expressed as a sampling rate (L/d). Where individual analytes werenot detected in SPMDs then the analytical detection limit was usedin calculations to provide a maximum theoretical concentration inthe water.

2.3. Biomarkers

2.3.1. Haemolymph analysis2.3.1.1. Lysosomal membrane stability in haemocytes (LMSHC). Theintegrity of lysosomal membranes of mussel haemocytes wasdetermined using the Neutral Red Retention (NRR) procedureadapted from Lowe and Pipe (1994). Approximately 0.1 mL of hae-molymph was removed from the adductor muscle of the musselwith a syringe containing approximately 0.1 mL physiological sal-ine. The haemolymph/saline solution was placed in a microcentri-fuge tube, from which a 40 lL sample was removed and pipettedonto the centre of a microscope slide. The slide was left in a darkhumid chamber for 15 min to allow the cells to adhere to the slide.After this time, the excess liquid was removed from the slide and40 lL of neutral red solution added (Sigma). The neutral red solu-tion was taken up inside the haemocytes and stored within lyso-somes. The ability of the lysosomes to retain the neutral redsolution was checked every 15 min by light microscopy (�400).The test was terminated and the time recorded when greater than50% of the haemocytes leaked the neutral red dye into the cytosol.

2.3.1.2. Micronuclei formation in mussel haemocytes (MN). Approx-imately 0.1 mL of haemolymph was removed from the posterioradductor muscle of each mussel with a hypodermic syringe con-taining 0.1 mL PBS buffer (100 mM PBS, 10 mM EDTA). The haemo-lymph and PBS buffer were mixed briefly in the syringe and placedon a microscope slide. The slide was then placed in a humid cham-ber for 15 min to enable the haemocytes to adhere to the slides. Ex-cess fluid was drained and the adhered haemocytes were fixed in1% glutaraldehyde for 5 min. Following fixation, the slides weregently rinsed in PBS buffer and left to air-dry overnight. The driedslides were brought back to the laboratory for further processing.Slides were stained with 1 lg/mL bisbenzimide 33258 (Hoechst)solution for 5 min, rinsed with distilled water and mounted inglycerol McIlvaine buffer (1:1). The frequency of micronuclei for-mation was measured on coded slides without knowledge of theexposure status of the samples to eliminate bias. The frequencyof micronuclei in haemocytes was determined microscopically at100� objective (final magnification �1000�). A total of 2000 cellswere examined for each experimental group of mussels. Only cellswith intact cellular and nuclear membranes were scored. MN werescored when: (a) nucleus and MN have a common cytoplasm, (b)colour intensity and texture of MN is similar to the nucleus, (c)the size of the MN is equal or smaller than 1/3 of the nucleus,

and (d) MN are apparent as spherical structures with a sharpcontour.

2.3.2. Enzyme activities in digestive glandFrozen digestive glands were individually homogenised in a

Braun-Potter homogeniser using TVBE buffer (1 mM sodium bicar-bonate, 1 mM EDTA, 0.1% ethanol and 0.01% Triton X-100;pH = 7.6). After homogenisation, samples were centrifuged at500g for 15 min. Supernatants were removed and diluted appropri-ately to perform the enzyme assays. Total protein of all sampleswas measured according to the Lowry method using a commercialprotein as standard (BioRad, California).

2.3.2.1. Catalase (CAT) activity. The activity of the antioxidant en-zyme catalase was measured as described by Porte et al. (1991)in frozen digestive gland samples (n = 10 mussels). Briefly, aftercentrifugation at 500g for 15 min, a small amount of sample wasstored for further analysis of AOX activity and the remainder wasprocessed to obtain mitochondrial and cytosolic fractions by cen-trifugation at 12,000g for 45 min and 100,000g for 90 min, respec-tively. CAT activity was determined in the mitochondrial andcytosolic fractions by measuring the consumption of H2O2 at240 nm (ext. coeff. 40 M�1 cm�1) using H2O2 50 mM as substratein potassium phosphate buffer 80 mM (pH = 7). Total CAT activitywas calculated as the sum of the activity of the two fractions.

2.3.2.2. Palmitoyl-CoA Oxidase (AOX) activity. Peroxisomal palmi-toyl-CoA oxidase (AOX) activity was determined spectrophoto-metrically (k = 502 nm), in 5 pools of 2 digestive glands perexperimental group, measuring the H2O2 dependent oxidation ofdichlorofluorescein diacetate (Molecular Probes, Eugene, Oregon,USA) catalyzed by an exogenous peroxidase, using 30 lM palmi-toyl-CoA as substrate, according to Small et al. (1985).

2.3.3. Digestive gland cytochemistryTen serial sections (10 lm thick) of frozen digestive gland (5

mussels per treatment) were cut in a Leica CM 3000 cryotome ontosuccessive serial slides and stored at �40 �C until processing.

2.3.3.1. Intracellular neutral lipid accumulation (INLA). One set ofcryotome slides were stained using the method of Lillie andAshburn�s Oil Red O (ORO). Histochemical controls were carriedout in a second set of slides by clearing lipids with a mixture of chlo-roform/methanol (1:1) for 1 h at room temperature before staining.Cryotome sections were transferred to a cabinet at 4 �C and fixedin Baker�s solution (+2.5% NaCl) for 15 min. Then sections weredried at room temperature, washed in isopropanol (60%) and rinsedfor 20 min in ORO staining solution. The ORO stock solution is a sat-urated (approximately 0.3%) solution of ORO (BDH, 34061) in iso-propanol. The staining solution was freshly made (it is only stablefor 1–2 h) by dissolving 60 mL stock solution in 40 mL distilledwater and filtering after a 10 min gap to stabilise the solution.Stained sections were differentiated in 60% isopropanol, washedin water, counterstained with 1% Fast Green FCF (Sigma, F-7252)for 20 min and mounted in Kaiser�s glycerine. Slides were viewedusing the 40� objective (final magnification�400�). Five measure-ments were made by image analysis to calculate volume density of

330 S. Brooks et al. / Marine Pollution Bulletin 62 (2011) 327–339

intracellular neutral lipids in digestive cells (VvNL) as VvNL = VNL/VC,where VNL is the volume of neutral lipids and VC the volume ofdigestive cells (Marigómez and Baybay-Villacorta, 2003).

2.3.3.2. Lysosomal membrane stability(LMSDC). The determination oflysosomal membrane stability was based on the time of acid labil-isation treatment required to produce the maximum stainingintensity according to UNEP/RAMOGE (1999), after demonstrationof hexosaminidase (Hex) activity in digestive cell lysosomes. Eightserial cryotome sections (10 lm) were subjected to acid labilisa-tion in intervals of 0, 3, 5, 10, 15, 20, 30 and 40 min in 0.1 M citratebuffer (pH 4.5 containing 2.5 % NaCl) in a shaking water bath at37 �C, in order to find out the range of pre-treatment time neededto completely labilise the lysosomal membrane, denoted as thelabilisation period (LP). Following this treatment, sections weretransferred to the substrate incubation medium for the demonstra-tion of Hex activity.

The incubation medium consisted of 20 mg naphthol AS-BI-N-acetyl-b-D glucosaminide (Sigma, N 4006) dissolved in 2.5 mL 2-methoxyethanol (Merck, 859), and made up to 50 mL with 0.1 M cit-rate buffer (pH 4.5) containing 2.5 % NaCl and 3.5 g low viscositypolypeptide (Sigma, P5115) to act as a section stabiliser. Sectionswere incubated in this medium for 20 min at 37 �C, rinsed in a salinesolution (3.0 % NaCl) at 37 �C for 2 min and then transferred to 0.1 Mphosphate buffer (pH 7.4) containing 1 mg/mL diazonium dye FastViolet B salt (Sigma, F1631), at RT for 10 min. Slides were then rap-idly rinsed in running tap water for 5 min, fixed for 10 min in Baker�sformol calcium containing 2.5% NaCl at 4 �C and rinsed in distilledwater. Finally, slides were mounted in Kaiser�s glycerine gelatineand sealed with nail varnish. The time of acid labilisation treatmentrequired to produce the maximum staining intensity was assessedunder the light microscope as the maximal accumulation of reactionproduct associated with lysosomes (UNEP/RAMOGE 1999). Fourdeterminations were made for each animal by dividing each sectionin the acid labilisation sequence into 4 approximately equal seg-ments and assessing the LP in each of the corresponding set of seg-ments. The mean LP value was then derived for each section,corresponding to an individual digestive gland.

2.3.4. Digestive gland and gonad histologyHistological sections (7 lm) were cut using a rotary microtome

Leitz 1512 (Ernest Leitz Wetzlar GmbH, Austria) and stained withhaematoxylin-eosin (H/E). Prevalence of parasites, haemocyteinfiltration and general condition of the digestive epithelium, theinterstitial connective tissue and the gonad tissue were systemat-ically recorded.

2.3.4.1. Epithelial cell-type composition (VvBAS)and tissue integrity indigestive gland (CTD ratio). The volume density of basophilic cells(VvBAS) was quantified by means of stereology as an indication ofwhether changes in cell-type composition occurred or not. Like-wise, the integrity of the digestive gland tissue was simultaneouslydetermined as the extent of the interstitial connective tissue rela-tive to the space occupied by digestive diverticula (connective-to-diverticula (CTD) ratio). Counts were made in one randomlyselected field in one digestive gland slide per mussel (10 musselsper sample). Slides were viewed at 40x objective (final magnifica-tion �400x) using a drawing tube attached to a Nikon Optiphotmicroscope. A simplified version of the Weibel graticule multipur-pose test system M-168 (Weibel 1979) was used, and hits on baso-philic cells (b), digestive cells (d), diverticular lumens (l) andinterstitial connective tissue (c) were recorded. CTD ratio was cal-culated as CTD = c/(b + d + l). VvBAS was calculated according to theDelesse�s principle (Weibel 1979), as VvBAS = VBAS/VEP, where VBAS isthe volume of basophilic cells and VEP the volume of digestivegland epithelium.

2.3.5. Integrative Biological Response indexThe Integrative Biological Response (IBR) index was developed

by Beliaeff and Burgeot (2002) in order to integrate biochemical,genotoxicity and histochemical biomarkers. Presently, AOX, CAT,LP, VvBAS and NRRT were used to calculate the IBR/n index. SinceAOX, CAT and VvBAS values are expected to increase in responseto environmental insult whereas LP and NRRT decrease, the inversevalues of these latter were used for calculations. The calculationmethod is based on relative differences between the biomarkersin each given data set. Thus, the IBR index is computed by sum-ming-up triangular star plot areas (a simple multivariate graphicmethod) for each two neighbouring biomarkers in a given dataset, according to the following procedure: (1) calculation of themean and standard deviation for each sample; (2) standardizationof data for each sample: xi� = (xi � x)/s; where, xi� = standardized va-lue of the biomarker; xi = mean value of a biomarker from eachsample; x = general mean value of xi calculated from all comparedsamples (data set); s = standard deviation of xi calculated from allsamples; (3) addition of the standardized value obtained for eachsample to the absolute standardized value of the minimum valuein the data set (yi = xi� + |xmin�|); (4) calculation of the Star Plot tri-angular areas by multiplication of the obtained standardized valueof each biomarker (yi) with the value of the next standardized bio-marker value (yi+1), dividing each calculation by 2 (Ai=(yi � yi+1)/2);and (5) calculation of the IBR index which is the summing-up of allthe Star Plot triangular areas (IBR =

PAi) (Beliaeff and Burgeot,

2002). Since the IBR value is directly dependent on the numberof biomarkers in the data set, we divided the obtained IBR valueby the number of biomarkers used in each case (n = 5) to calculateIBR/n, according to Broeg and Lehtonen (2006).

2.4. Mussel tissue chemistry

For each treatment group, triplicate mussel samples were takenfor analysis of selected metals and PAHs, including alkylatedhomologues of naphthalene, phenanthrene and dibenzothiophene(NPD). Five whole mussels per sample were removed from theirshells and placed in pyrolysed (560 �C) glass containers. The mus-sels were frozen and transported to NIVA on dry ice. All sampleswere stored at �20 �C until analyses.

Samples were defrosted, homogenised and a sub sample takenof approximately 5 g. Internal standards were added before extrac-tion by saponification. Analytes were then extracted twice with40 mL cyclohexane and dried over sodium sulphate. The extractswere reduced by a gentle stream of nitrogen and cleaned by gelpermeation chromatography (GPC) using the system describedpreviously (Harman et al., 2008). Analysis proceeded by gas chro-matography with mass spectrometric detection (GC–MS) withthe MS detector operating in selected ion monitoring mode(SIM). The GC was equipped with a 30 m column with a stationaryphase of 5% phenyl polysiloxane (0.25 mm i.d. and 0.25 lm filmthickness), and the injector operated in splitless mode. The initialcolumn temperature was 60 �C, which after two minutes wasraised stepwise to 310 �C. The carrier gas was helium and the col-umn flow rate was 1.2 mL/min. Quantification of individual com-ponents was performed by using the internal standard method.The alkylated homologues were quantified by baseline integrationof the established chromatographic pattern and the response fac-tors were assumed equal within each group of homologues.

3. Results

The mussels used in this experiment appeared to be de visu ingood condition throughout the exposure period, with < 1% mortal-ity recorded both in control and PW exposure treatments.

S. Brooks et al. / Marine Pollution Bulletin 62 (2011) 327–339 331

However, at the microscope certain loss of histological integrity inthe digestive gland tissue and epithelial thinning in digestivealveoli were found in both control and PW exposed mussels, albeitmuch more marked in the latter. Thus, 0.01–0.5% PW exposedmussels, and those exposed to 1% PW to a lesser extent, showeda severe reduction in the numbers of digestive diverticula, whichappeared sparse throughout a highly disorganized and eventuallyfibrous interstitial connective tissue (see CTD ratio below) and anextreme thinning of the digestive gland epithelium. In contrast,no significant parasitic infestation or pathological lesion was foundin any case. The histological examination of the gonad revealed noclear difference among mussels from the different treatmentgroups. Mature gametes were observed in most mussels for bothmales and females. Although in some specimens spawning hadalready taken place, it appeared to be incidental and could not berelated to PW exposure.

The results obtained regarding the concentrations of pollutantsmeasured in water (through SPMDs) and mussel tissues, the indi-vidual biomarker recorded in mussels and the integration of bio-markers as IBR/n index are reported in the next three sections.

3.1. Concentrations of pollutants in water and mussel tissues

Based on the chemical analysis of SPMDs, it was observed thatPAH concentrations were either low or undetected in all experi-mental tanks (Table 2). The PAHs that were detected include fluo-rene, phenanthrene, fluoranthene and pyrene. These compoundswere detected at background concentrations (

PPAH 0.7–2.0 ng/

L) with acceptable variation between replicates (average RSD15%). NPD compounds were also only present at very low concen-trations (4.6–6.3 ng/L), but were more variable (average RSD 50%).There were no apparent differences in PAH/NPD concentration be-tween the exposure treatments, based on the SPMD results, withthe exception of the treatment with 0.01% PW. This treatmenthad noticeably lower concentrations than the other treatmentsincluding the control tank. The PRC results (data not presented)showed that the total volume of water extracted during the fiveweek laboratory exposure was between 28 and 161 L, dependingon the compound.

Likewise, low or undetected concentrations of PAHs were foundin mussel tissues in all exposure tanks with no noticeable differ-ences between the exposure concentrations (Table 3). The PAH

Table 2The PAH concentration calculated from SPMDs exposed for 5 weeks to different concentra

ng/L Control 0.01% PW

1 2 3 1 2 3

Naphthalene a a a a a aAcenaphthylene <0.11 <0.10 <0.10 <0.10 <0.09 <0.09Acenaphthene 0.10 <0.09 0.09 0.11 0.08 <0.07Fluorene 0.34 0.32 0.27 0.25 0.17 0.19Dibenzothiophene <0.08 <0.07 <0.07 <0.07 <0.06 <0.06Phenanthrene 0.71 0.67 0.56 0.43 0.26 0.30Anthracene <0.07 <0.06 <0.06 <0.06 <0.05 <0.05Fluoranthene 0.14 0.14 0.13 0.10 0.08 0.09Pyrene 0.11 0.10 0.09 0.08 0.06 0.07Benz[a]anthracene <0.06 <0.05 <0.05 <0.05 <0.04 <0.04Chrysene <0.05 <0.05 <0.05 <0.04 <0.03 <0.04Benzo[b.j]fluoranthene <0.05 <0.05 <0.05 <0.04 <0.04 <0.04Benzo[k]fluoranthene <0.06 <0.06 <0.05 <0.05 <0.04 <0.04Benzo[e]pyrene <0.07 <0.06 <0.06 <0.05 <0.04 <0.05Benzo[a]pyrene <0.07 <0.06 <0.06 <0.05 <0.04 <0.04Perylene <0.07 <0.06 <0.06 <0.05 <0.04 <0.04Indeno[1.2.3-cd]pyrene <0.08 <0.08 <0.07 <0.07 <0.05 <0.06Dibenzo[ac/ah]anthracene <0.07 <0.07 <0.06 <0.06 <0.05 <0.05Benzo[g.h.I]perylene 0.09 <0.08 <0.08 <0.07 <0.06 <0.06SUM PAH <2.32 <2.17 <1.94 <1.74 <1.29 <1.38PAH EPA16 <2.11 <1.97 <1.76 <1.56 <1.15 <1.23

concentrations that were detected include naphthalene, phenan-threne, fluorene, fluoranthene and pyrene, which were present invery low ng/g (wt w). Metal concentrations in mussel tissues werealso low and no obvious relationship between metal concentrationand nominal exposure concentration was found (Table 4).

3.2. Biomarkers

Although no significant differences were found in AOX activitybetween the different exposure groups, a trend to increase AOXactivity with PW exposure up to 0.5% PW was found (Fig. 1A).AOX activity in mussels exposed to 0.5% PW was almost doublethat of the control group. However, the lowest AOX activity was re-corded in 1% PW exposed mussels. Neutral lipids were revealed asbright reddish purple deposits easily identified at the light micro-scope in the digestive alveoli, digestive ducts and stomach. Thestaining pattern was heterogeneous and not all the alveoli pre-sented the same degree of reactivity but in all the reactive alveoli,the ORO reaction product appeared clearly localized within com-partments of the endo-lysosomal system. As a result, VvNL valueswere highly variable and similar in all the experimental groupsbut in the 1% PW exposure treatment where VvNL was significantlythe lowest (Fig. 1B). CAT activity was not significantly dissimilaramong experimental groups but, like in the case of AOX activity,this enzyme activity was slightly higher in 0.01–0.5% PW treat-ments than in the control one, and the lowest values were recordedon exposure to 1% PW (Fig. 1C). The highest mean frequency of MNwas found in the haemocytes of mussels exposed to 1% PW(Fig. 1D). However, no significant differences in MN frequencywere found between the different exposure groups. Overall, therewas a low prevalence of MN in all groups ranging from 0.5 to 1.4MN per 1000 cells. LP in control mussels was unexpectedly low(13–15 min) but, nevertheless, LP values (<5 min) were signifi-cantly lower at exposures to 0.01–0.5% PW than in the controlgroup (ANOVA, Turkey�s test, p < 0.05; Fig. 1E). In contrast, the LPrecorded in mussels exposed to 1% PW was not significantly differ-ent from that recorded in the control group (Fig. 1E). Only VvBAS

values recorded in the 0.1% PW exposure group were significantlydifferent from those obtained in the control group (one-way ANO-VA, Duncan�s test, p < 0.05) but, overall, the same trend than forAOX and CAT activities can be depicted (Fig. 1F). The VvBAS valuesrecorded in all groups were reasonably high (>0.12 lm3/lm3) with

tions of produced water. a = high blank concentrations data not reported.

0.1% PW 0.5% PW 1% PW

1 2 3 1 2 3 1 2 3

a a a a a a a a a<0.10 <0.11 <0.11 <0.15 <0.15 <0.13 <0.12 <0.10 <0.110.09 0.13 0.11 0.14 0.16 0.12 <0.10 0.09 0.110.27 0.39 0.33 0.57 0.51 0.45 0.35 0.29 0.40<0.07 0.10 <0.08 0.11 <0.11 <0.10 <0.08 <0.07 <0.080.51 0.86 0.65 1.27 0.94 0.91 0.75 0.63 0.81<0.06 0.07 <0.07 <0.1 <0.1 <0.09 <0.07 <0.06 <0.070.11 0.14 0.12 0.19 0.16 0.14 0.14 0.11 0.140.09 0.11 0.11 0.16 0.13 0.12 0.13 0.11 0.13<0.05 <0.06 <0.05 <0.09 <0.09 <0.08 <0.06 <0.05 <0.06<0.05 <0.05 <0.05 <0.08 <0.08 <0.07 <0.06 <0.05 <0.06<0.05 <0.05 <0.05 <0.08 <0.08 <0.07 <0.06 <0.05 <0.06<0.05 <0.06 <0.06 <0.10 <0.10 <0.08 <0.07 <0.05 <0.07<0.06 <0.07 <0.07 <0.11 <0.11 <0.09 <0.08 <0.06 <0.07<0.06 <0.07 <0.06 <0.10 <0.10 <0.09 <0.07 <0.06 <0.07<0.06 <0.07 <0.06 <0.10 <0.10 <0.09 <0.07 <0.06 <0.07<0.07 <0.08 <0.08 <0.13 <0.13 <0.11 <0.09 <0.07 <0.09<0.06 <0.07 <0.07 <0.11 <0.11 <0.10 <0.08 <0.06 <0.08<0.08 <0.09 <0.09 <0.14 <0.14 <0.12 <0.10 <0.08 <0.09<1.88 <2.58 <2.22 <3.73 <3.32 <2.97 <2.49 <2.04 <2.57<1.69 <2.35 <2.01 <3.41 <2.99 <2.69 <2.26 <1.86 <2.35

Table 3The PAH concentration of whole mussel homogenates exposed for 5 weeks to different concentrations of produced water.

lg/kg (wet weight) Control 0.01% PW 0.1% PW 0.5% PW 1% PW

1 2 3 1 2 3 1 2 3 1 2 3 1 2 3

Naphthalene <0.8 2.0 3.2 2.0 2.0 1.4 4.1 2.8 4.3 0.92 0.90 2.9 <0.8 2.9 1.3C1-Naphthalenes <2 <2 <2 <2 <2 <2 2.1 <2 <2 <2 <2 2.6 <2 <2 <2C2-Naphthalenes 4.8 8.9 6.0 5.6 7.5 5.6 9.5 5.8 3.0 3.8 4.7 11 12 8.4 6.4C3-Naphthalenes 7.4 12 10 14 14 12 18 12 7.9 7.7 9.6 23 7.9 13 12Phenanthrene 1.2 1.1 0.80 1.2 1.6 1.8 1.9 1.4 1.9 0.93 0.75 1.8 1.1 2.1 1.0C1-Phenanthrenes 5.1 <2 2.3 4.5 5.6 6.8 4.5 3.8 2.5 2.2 <2 4.8 2.4 4.1 3.4C2-Phenanthrenes 3.0 2.3 <2 3.8 4.0 3.8 5.7 4.0 4.5 <2 <2 3.6 2.7 3.1 2.5C3-Phenanthrenes 5.0 2.5 <2 5.2 5.0 12 3.4 3.1 2.2 2.2 <2 4.4 4.6 3.8 4.4Dibenzothiophene <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5C1-Dibenzothiophenes <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2C2-Dibenzothiophenes <2 <2 <2 <2 <2 <2 2.1 <2 2.5 <2 <2 <2 <2 <2 <2C3-Dibenzothiophenes <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2Sum NPD <35.8 <39.3 <34.8 <44.8 <48.2 <51.9 <55.8 <41.4 <35.3 <28.25 <30.45 <60.6 <40 <45.9 <39.5Acenaphthylene <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5Acenaphthene <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5Fluorene <0.5 <0.5 <0.5 0.65 0.64 0.61 0.91 0.72 0.56 <0.5 <0.5 0.72 0.61 0.61 <0.5Anthracene <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5Fluoranthene 0.97 0.76 <0.5 1.1 1.4 0.77 1.2 1.3 0.97 0.72 0.73 0.88 0.75 1.3 0.88Pyrene 1.2 0.85 0.84 1.4 1.7 0.94 1.4 1.6 1.3 0.66 0.89 1.5 1.0 1.3 1.0Benzo(a)anthracenes <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5Chrysene <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5Benzo(b)fluoranthene <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 0.53 <0.5 <0.5 <0.5Benzo(k)fluoranthene <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5Benzo(e)pyrene 0.71 0.52 <0.5 0.60 0.82 0.52 0.58 0.73 0.70 <0.5 <0.5 0.96 0.55 0.64 <0.5Benzo(a)pyrene <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5Perylene <0.5 0.70 0.57 <0.5 <0.5 0.57 <0.5 <0.5 <0.5 <0.5 <0.5 0.55 <0.5 <0.5 <0.5Indeno(1,2,3-cd)pyrene <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5Dibenz(a,h)anthracene <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5Benzo(g,h,i)perylene <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 0.61 <0.5 <0.5 <0.5Sum PAH <45.18 <48.13 <43.21 <54.55 <58.76 <60.81 <65.89 <51.75 <44.83 <36.63 <39.07 <70.85 <48.91 <55.75 <48.38Sum PAH16 <10.17 <10.71 <11.34 <11.85 <12.84 <11.02 <15.01 <13.32 <14.53 <9.23 <9.27 <13.44 <9.76 <13.71 <10.18Lipid (%) 1.8 2.0 1.9 1.8 0.3 1.6 1.7 1.7 1.3 1.9 1.8 1.6 1.5 1.8 1.8

Table 4Metal concentrations of whole mussel homogenates from mussels exposed for 5 weeks to different concentrations of produced water.

mg/kg (wet weight) Control 0.01% PW 0.1% PW 0.5% PW 1% PW

1 2 3 1 2 3 1 2 3 1 2 3 1 2 3

Ag <0.005 <0.005 <0.005 0.005 0.007 0.007 0.007 0.005 0.006 0.009 0.007 0.008 0.005 0.009 0.008Al 4.3 4.5 4.1 6.1 4.9 3.1 4.0 5.4 2.7 3.1 3.1 6.2 3.8 4.8 4.1As 2.06 1.84 1.75 1.75 1.64 1.96 1.86 1.73 1.88 1.92 2.22 1.94 1.81 1.95 2.09Cd 0.098 0.099 0.094 0.106 0.093 0.107 0.100 0.084 0.105 0.092 0.079 0.109 0.092 0.106 0.088Cr 1.2 1.0 0.71 1.1 1.7 0.97 1.2 1.2 0.65 0.55 0.81 1.0 0.51 0.49 0.42Cu 1.19 1.13 1.12 1.10 1.01 0.74 1.07 1.16 1.10 1.50 1.00 0.94 1.03 1.05 1.26Fe 18 18 14 17 16 14 14 16 14 14 16 20 13 14 12Hg 0.009 0.008 0.007 0.008 0.008 0.008 0.009 0.008 0.008 0.009 0.008 0.008 0.007 0.008 0.008Ni 0.48 0.38 0.33 0.30 0.35 0.29 0.40 0.41 0.42 0.38 0.54 0.71 0.26 0.26 0.24Pb 0.07 0.04 0.04 0.05 0.04 0.06 0.05 0.05 0.06 0.05 0.06 0.05 0.05 0.05 0.05Zn 11.3 10.0 11.9 11.4 10.0 11.6 10.9 10.8 10.3 13 10.4 13.8 12.4 10.0 13.7

332 S. Brooks et al. / Marine Pollution Bulletin 62 (2011) 327–339

a high variability between mussels from the same experimentalgroup. Significantly higher neutral red retention time (NRRT) wasfound in the control group compared to all other groups (ANOVA,Turkey�s test, p < 0.05; Fig. 1G). NRRT in control mussels was�60 min and approached 20 min in mussels exposed to 0.5% and1% PW, they were not found to be significantly different from thevalues recorded in mussels exposed to 0.01% PW (NRRT �40 min). CTD ratios were similar and over 0.5 in all the experimen-tal groups and exhibited a great variability in PW exposed mussels.CTD ratios were significantly lower in mussels exposed to 1% PWthan in the other experimental groups (one-way ANOVA, Duncan�stest, p < 0.05; Fig. 1H).

3.3. Integrative Biological Response (IBR/n)

Five biomarkers (AOX, CAT, LP, VvBAS, and NRRT) were selectedand represented in the five axes of star plots (Fig. 2). The integrative

biomarker response was almost zero in the seawater control (SW;0% PW). AOX, CAT and LP were sensitive biomarkers in 0.01–0.5%PW (Fig. 2B-D), with VvBAS also relevant after 0.1% and 0.5% PWexposures (Fig. 2C and D) and NRRT in 0.5% and 1% PW treatments(Fig. 2D and E). Despite the weak effects reported for individualbiomarkers, IBR/n values were markedly higher in 0.1–0.5% PWtreatments than in the control and the 1% PW treatment(Fig. 2F), indicating a trend to decrease in mussel health statuson exposure up to 0.5% PW. Unexpectedly, affection was appar-ently lower in the 1% PW exposure group (Fig. 2F).

4. Discussion

The main objective of the present study was to evaluate, undercontrolled laboratory conditions, the potential biological impactexerted by a treated PW effluent (Ormen Lange processing plant),which was expected to present low levels (often below detection

* * *

14

12

10

8

6

4

2

LPD

C(m

in)

0 0.01 0.1 0.5 1

VvN

L(µ

m3 /µ

m3 )

0 0.01 0.1 0.5 1

0.15

0.12

0.09

0.06

0.03

0

CA

T A

ctiv

ity (m

mol

/ml)

0.2

0.4

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1.0

1.2

0 0.01 0.1 0.5 1

10

30

50

70

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NR

RT H

C (m

in)

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* *

**

0.3

VvB

AS

(µm

3 /µm

3 )

*

CTD

Rat

io (µ

m3 /µ

m3 )

0,5

1,0

1,5

2,0

2,5

0

*

0.12

0.02

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0.06

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AO

X A

ctiv

it (m

U/m

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0 0.01 0.1 0.5 1

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/100

0 ce

lls

5

4

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1

0

-1

0 0.01 0.1 0.5 1

0.2

0.1

00 0.01 0.1 0.5 1

*0 0.01 0.1 0.5 1

PW (%)0 0.01 0.1 0.5 1

(A) (B)

(C) (D)

(E) (F)

(G) (H)

Fig. 1. Biomarkers recorded in mussels exposed to known concentrations of produced water (PW). (A) Acyl-CoA oxidase (AOX) activity in digestive gland; (B) Volume densityof intracellular neutral lipids in digestive gland epithelium (VvNL); (C) Catalase (CAT) activity in digestive gland; (D) Frequency of micronuclei (MN) in blood cells; (E)Labilisation period (LP) of digestive cell lysosomes; (F) Volume density of basophilic cells in digestive gland epithelium (VvBAS); (G) Neutral red retention time (NRRT) inhaemocytes; and (H) Connective tissue to digestive diverticula (CTD) ratio in digestive gland tissue. Data expressed as mean, standard error (box), standard deviation (outerline) and outliers (black dots). *denotes significant difference from all other groups for graph B and significant difference from control (0) for all other graphs (ANOVA, Tukey;Mann–Whitney; p<0.05).

S. Brooks et al. / Marine Pollution Bulletin 62 (2011) 327–339 333

limits) of individual contaminants in a highly complex mixture.The biological endpoints measured were responsive to PW expo-sure. Moreover, although the measured biomarkers corresponded

to different levels of biological complexity and were determinedby different technologies and by different research labs, dependingon their expertise, they were found to be highly coherent and

0

1

2

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4AOX

CAT

LPBAS

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0.01 % PW

0

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(A) (B)

(C) (D)

(E) (F)

0.51

1.52

2.53

3.54

0 0.01 0.1 0.5 1PW (%)

IBR

/n

Fig. 2. Star plots (A–E) representing the five biomarkers (AOX, CAT, LP, VvBAS, and NRRT) used to compute the IBR/n index (F) in mussels exposed to known concentrations ofproduced water (PW). Biomarkers are orderly represented in the five axes of star plots according to their biological complexity level; AOX (metabolic response); CAT(subcellular response); LP (cellular response); VvBAS (tissue response); NRRT (systemic response).

334 S. Brooks et al. / Marine Pollution Bulletin 62 (2011) 327–339

sensitive. The suite of biomarkers revealed that exposure to concen-trations as low as 0.01–0.5% of treated PW for 5 weeks appearedto provoke a significant stress response in mussels, whereas ananomalous response was found in mussels exposed to 1% PW.

Mussels in the control group were found to be exhibiting aslight stress response, according to the histopathological examina-tion of the digestive gland (scarce digestive diverticula sparsethroughout disorganized interstitial connective tissue and thinningof the digestive gland epithelium) and the values recorded forsome biomarkers investigated (i.e. Lp < 20 min, VvBAS > 0.12 lm3/lm3 and NRRT < 90 min). Confounding factors including foodavailability and water quality can be ruled out since animals werefed every second day and physicochemical measurements (i.e.temp, pH, and dissolved oxygen) were found to remain stable dur-ing the exposure duration. Mussel mortality was minimal (<1%),suggesting that these mussels were in reasonable health prior tothe test exposure. No significant parasitic infestation or pathologi-cal lesions were found in any case and the histological examinationof the gonad revealed the presence of mature gametes in mostmussels, for both males and females. However, the removal andplacement of mussels from their natural environment into the

controlled laboratory environment is likely to impose stress factorsresulting in the stress response in mussels. However, despite thisslight stress response, control and PW exposed mussels wereseemingly different and dose–response trends in several biomark-ers were evident.

4.1. Concentrations of pollutants in water and mussel tissues

The chemistry data for the mussel and SPMDs were not found todifferentiate between the exposure groups and could not accountfor the biological effects observed. In this case the biological re-sponses were most likely caused by other contaminants not mea-sured. Although PAHs and NPDs are a crucial component of PW,there are also many other chemicals that have not been measured.Some examples include; alkylphenols, organic acids (such as naph-thenic acids), and decalins, which may have contributed towardsthe biological effects observed. It is however, impractical to mea-sure the thousands of chemicals present and this highlights thebenefits of sensitive biological effects measurements for theassessment of environmental risk of complex discharges.

S. Brooks et al. / Marine Pollution Bulletin 62 (2011) 327–339 335

Levels of PAH/NPD in exposure treatments (ca. 2 and 5 ng/L,respectively), are comparable to those previously described usingsimilar methods at the same facility and also as those measuredat reference sites offshore (Harman et al., 2009a,b). Analysis ofthe PW used in the exposure (results not shown) revealed that lev-els of PAH were generally below the limits of detection, althoughelevated levels of NPD compounds were present (up to 400 ng/L,C3 naphthalenes). Once diluted and allowing for evaporative lossesthese levels were not able to measurably increase the concentra-tions of NPD compounds above that already present.

4.2. Single biomarkers

Histopathological examination of the digestive gland and gonadcan help in interpreting biomarkers (e.g. responses may be influ-enced by gender, gamete developmental cycle, presence of para-sites or lesions that imply cell loss/hypertrophy/migration/etc.)and provides sensitive, useful and potential indications for thescreening of the mussel health status (Kim et al., 2006; Marigómezet al., 2006; Bignell et al., 2008; Garmendia et al., submitted). Thehistological examination of the gonad, which was mature, revealedno clear differences between the mussel treatment groups. In con-trast, PW exposed mussels showed severe loss of histologicalintegrity in digestive gland tissue and extreme thinning of thedigestive gland epithelium, which were less marked in mussels ex-posed to 1% PW. Histopathological alterations in digestive epithe-lium cells (vacuolisation and loss of digestive cells, hypertrophyof basophilic cells, epithelial thinning and atrophy) and loss of his-tological integrity in digestive gland tissue (including systemichaemocytosis and interstitial connective tissue oedema; sensuCouch, 1985) are characteristic traits in stressed mussels (Loweet al., 1981; Cajaraville et al., 1990, 1992; Marigómez et al.,2006; Wedderburn et al., 2000; Usheva et al., 2006; Kim et al.,2008; Aarab et al., 2008;. Garmendia et al. submitted).

4.2.1. Peroxisome proliferationPeroxisomes are membrane-bound organelles involved in lipid

metabolism, oxyradical homeostasis and several other importantcell functions (Cancio and Cajaraville, 2000), which under exposureto certain organic chemical compounds proliferate and enhancetheir metabolic activity (Fahimi and Cajaraville, 1995). Amongother substances, PAHs, oil derivatives and alkylphenols are knownto provoke peroxisome proliferation in marine fish and bivalves(Cajaraville et al., 2000). Peroxisome proliferation is accompaniedby the induction of AOX and other peroxisomal enzyme activities(Fahimi and Cajaraville, 1995), which has been proposed as expo-sure biomarker for organic pollutants (Cajaraville et al., 2000).The present results suggest that treated PW up to 0.5% of itsoriginal concentration acts as a peroxisome proliferator. Althoughdifferences between groups lacked statistical significance, adose–response trend was depicted and AOX activity in 0.5% PWtreated mussels was found to be twice that of the control group.PWs have been considered a source of peroxisome proliferatingagents probably associated to their petroleum accommodatedfraction or to alkylphenols (Sturve et al., 2006; Zhu et al., 2008)but, in contrast, Gorbi et al. (2009) did not find any variation inAOX activity in PW exposed marine fish.

4.2.2. Intracellular neutral lipids accumulationINLA in digestive cells may be considered indicative of exposure

to organic chemicals of different physicochemical properties(phthalates, benzo[a]pyrene, phenanthrene, fluoranthene oilWAF), although it can be also the result of membrane turnoverimpairment after severe exposure (Lowe and Pipe, 1994; Krish-nakumar et al., 1995; Marigómez and Baybay-Villacorta, 2003).INLA constitutes a prompt all-or-nothing response useful for

screening purposes but usually dose/response curves cannot beconstructed (Marigómez and Baybay-Villacorta, 2003). However,this biomarker cannot be considered alone due to natural variabil-ity between geographical locations and seasons (Cancio et al.,1999). In the present experimental conditions it seems that INLAdoes not occur in response to PW exposure, which might be inagreement with mussel tissue chemistry. Quite the opposite, theonly change observed is a severe reduction in the intracellular lev-els of neutral lipids on exposure to 1% PW, whose possible causesare discussed below.

4.2.3. Induction of antioxidant enzymesInduction of antioxidant enzymes such as CAT in fish liver and

molluscan digestive gland may be indicative of oxidative stress ex-erted by pollutants acting through enhanced generation of oxygenfree radicals (Burgeot et al., 1996; Regoli et al., 2004; Vlahogianniet al., 2007). In contrast, CAT activity can be inhibited in responseto severe pollution (Pampanin et al., 2005; Vlahogianni et al.,2007). Thus, CAT activity is induced in M. edulis on exposure tolow concentrations of PAHs whereas at high concentrations it isinhibited most likely due to their narcotic effect (Eertman et al.,1995). Regarding our results, the weak induction of CAT activity re-corded in mussels exposed to 0.01–0.5% PW might be indicativethat a weak oxidative stress was exerted by treated PW to whichmussels were responding through the protecting action of catalase.

4.2.4. Micronuclei formation (genotoxicity)Micronuclei formation is an index of chromosomal damage

based on the quantification of downstream aberrations after DNAdamage and reveals a time-integrated response to complex mix-tures of pollutants (Heddle et al., 1983). The MN test has been eval-uated in isolated mussel haemocytes and gill cells (Burgeot et al.,1996; Bolognesi et al., 1996; Dailianis et al. 2003; Viarengo et al.,2007). There is no evidence of genotoxicity in treated PW exposedmussels under the present laboratory conditions, with a low prev-alence of MN in all groups (0.5–1.4 MN/1000 haemocytes). The rea-son for this may be that the exposure concentration was below thethreshold level required to illicit genotoxicity in mussels. A previ-ous monitoring program reported significant increases in MN fol-lowing 30 days exposure to oil compounds (Baršiene et al., 2008).However, the exposure was related to an oil spill scenario withhigher contaminant concentrations than that exhibited in the pres-ent study. Likewise, increased frequency of MN has been reportedin mussels when exposed for 6 week to PW in the North Sea (Hyl-land et al., 2009; Brooks et al., 2010). Although it cannot be ex-cluded that increasing the length of exposure might result insignificant differences between control and exposure groups, theOrmen Lange treated PW would likely cause at most very lowgenotoxicity.

4.2.5. Lysosomal membrane destabilization in digestive cellsLysosomes of mussel digestive cells, which under normal condi-

tions are involved in food intracellular digestion (Robledo et al.,2006; Izagirre et al., 2008) and autophagy (Moore et al., 2007), playan important role in responses to toxic compounds through thesequestration and accumulation of toxic metals and organic xeno-biotics (Viarengo et al., 1987; Marigómez et al., 2002). Environ-mental stressors cause reduction in the stability of the lysosomalmembranes of mussel digestive cells, which is usually measuredin terms of reduced labilisation period (LP), a widely accepted gen-eral stress biomarker, and evaluated using the LMSDC (UNEP/RAMOGE 1999; ICES 2004; Marigómez et al. 2005). The values ofLP around 15 recorded in the control mussels min (<20 min; criti-cal threshold value; Marigómez et al., 2006) are in agreement withthe general histological examination that revealed that controlmussels were not in optimal conditions. However, exposure to

336 S. Brooks et al. / Marine Pollution Bulletin 62 (2011) 327–339

treated PW (0.01–0.5%) caused a significant and non dose-depen-dent reduction in LP beyond 5 min. Treated PW at concentrationsso low as 0.01% seem therefore to cause a severe stress in mussels,at least in the present experimental conditions.

4.2.6. Changes in cell-type composition in digestive gland epitheliumIn mussels, the digestive gland epithelium is comprised by two

cell types: digestive and basophilic cells. Under stress situations,including exposure to pollutants, the relative occurrence of baso-philic cells is apparently augmented (Rasmussen et al., 1983). Ithas been recently concluded that this apparent alteration resultsfrom digestive cell loss and basophilic cell hypertrophy (Zaldibaret al., 2007). It is considered a common response in molluscs topollutant induced stress (Cajaraville et al., 1990; Syasina et al.,1997; Usheva et al., 2006; Zaldibar et al., 2007). In Mytilus gallopro-vincialis from clean localities and under experimental control con-ditions, VvBAS is usually below 0.1 lm3/lm3 (Marigómez et al.,2006) but after exposure to pollutants VvBAS may surpass0.12 lm3/lm3 (Cajaraville et al., 1990; Marigómez et al., 2006;Garmendia et al., submitted). Presently, all the VvBAS values re-corded (also in controls) were always above 0.12 lm3/lm3, whichseemingly suggests that all the mussels were exhibiting some de-gree of stress, which was in agreement with histopathologicalobservations, LP and NRRT results. However, it is worth noting thatthe critical threshold values aforementioned for VvBAS refer to M.galloprovincialis and therefore the values might be different in M.edulis, although according to the few existing data (Bilbao et al.,2006; Hylland et al., 2009) differences between the two specieswould most likely be minimal. Nevertheless, this parameter clearlyindicated that treated PW provokes general stress in mussels, inagreement with LP results. VvBAS values were significantly twicehigher at exposure to 0.1% PW than in the control group. Overall,it seems that PW exposure provokes changes in the cell type com-position in the digestive gland epithelium but the results are notfully conclusive as control mussels seemed to be subjected to cer-tain stress.

4.2.7. Cytotoxicity in haemocytesNeutral red has been used for the quantification of cytotoxicity

based on the ability of viable haemocytes to incorporate and accu-mulate the weakly cationic dye within lysosomes (Borenfroundand Puerner, 1985). Neutral red measures membrane functionalintegrity and lysosomal functioning (Ivanova and Uhlig, 2008),and reflects the capacity of cellular processes to adapt to stressconditions (Lowe and Pipe, 1994; Lowe et al., 1995). NRRT in mus-sel haemocytes has been found to be affected by a wide range ofenvironmental stressors including metals and organic compoundssuch as phenanthrene, anthracene, chlorpene, benzo[a]pyrene andother PAHs typically found in PW discharges (Lowe et al., 1995;Moore et al., 1996; Fang et al., 2010). It has been reported that oxi-dative stress exerted by PAHs provokes cytoskeleton damage,which results in reduced endocytosis, phagocytosis and, overall,cell motility and is associated with lowered NRRT (Cajaravilleet al., 1996; Gómez-Mendikute et al., 2002). Although changes inNRRT are often associated to reduced lysosomal membrane stabil-ity (Lowe and Pipe, 1994), they are the result of a more general re-sponse that involves loss of integrity in overall cell membranes(Ivanova and Uhlig 2008), not only lysosomal ones, unlike in thecytochemical procedure to determine LP in digestive cells that isstrictly targeted to lysosomes (Marigómez et al., 2005). The symp-toms indicative of cellular injury that are revealed by the neutralred assay in mussel haemocytes include a reduction in the numberof lysosomes, an increase in lysosomal volume and a reduction ofthe overall cell size with associated changes in cell morphology(Moore et al., 1996). Pryor and Facher (1997) concluded that themussel haemocytes respond to pollutants by rounding up and

becoming inactive, which leads to immune suppression. Thus, im-mune suppression (reduced haemocyte number and phagocyticcapacity) was shown to be severe after Sea Empress oil spill inassociation with PAH exposure (Dyrynda et al., 2000). The immunesystem of mussels not only can be significantly affected by organicpollutants at high concentrations but also is very sensitive to lowconcentrations, which enhances their susceptibility to infectiousdiseases (Liu et al., 2009). Significantly lower NRRT was found inmussels exposed to treated PW compared to the control (ANOVA,Turkey�s test, p < 0.05; Fig. 1G) and thus mussels exposed to trea-ted PW were seemingly subjected to immune suppression (haemo-cyte function impairment). Similarly, in mussels exposed toconcentrations of non-treated PW up to 0.5% PW for 21 day hae-mocytes presented reduced cell viability and phagocytic capacityand increased cytotoxicity (Hannam et al., 2009).

4.2.8. Loss of tissue integrity in digestive glandIn mussels subjected to pollution stress, digestive diverticula

are apparently reduced in numbers and appear sparse surroundedby ample areas of disrupted interstitial connective tissue, mostprofiles of digestive alveoli appearing elongated or branched (Gar-mendia et al., submitted). Several authors found smaller numbersof digestive tubules per unit area in scallops, and mussels from pol-luted sites (Syasina et al., 1997; Usheva et al., 2006). Presently, CTDratio was quantified for the first time as a tissue-level biomarkerindicative of the histopathological condition of mussels. CTD ratioswere similar and over 0.5 in all the experimental groups with onlya slight increase in mussels exposed to 0.5% PW over the controlones. Seemingly due to the poor histological integrity found in con-trol mussels this biomarker was not very sensitive in the range of0.01–0.5% PW exposure concentrations.

4.2.9. Anomalous biological responses on exposure to 1% PWThe highest exposure (1% PW) used was clearly not effective.

Both chemical data and biological responses did not follow thetrend expected from lower exposures. The strong reduction inAOX and CAT activities recorded after 1% PW exposure might beexplained by a toxic effect on enzyme activity, as reported after se-vere exposure to pollutants (Bilbao et al., 2010). However, it wasaccompanied by reduction in VvNL, VvBAS and CTD and increase inLP, in some cases beyond control group values, and NRRT andMN were not different from the 0.5% PW exposure, which cannotbe simply explained by toxic action on enzyme activities. Theflow-through system was maintained and checked on a daily basiswith no reported problems in delivery and therefore, the reasonsfor these anomalies are unclear. Interestingly, it has been reportedthat at concentrations >1% PW, inorganic particles are removeddue to enhanced settlement of organic matter and there is alsoan increase in buoyant particles that are aggregates of oil dropletswith inorganic particles (Azetsu-Scott et al., 2007). This would re-sult in reduced waterborne contaminants as well as bioaccumula-tion and toxicity lower than expected from a dose–response curve.In addition, PW from gas reservoirs is thought to produce non-po-lar narcosis and alterations in cell membrane permeability (John-sen et al., 2004). Chemicals present in a mixture at very lowconcentrations may contribute to the narcotic activity of the mix-ture (Smith et al., 1998). Only minor effects where found in turbotlarvae exposed for up to 12 h to 1% PW, whereas exposure to 10%PW depressed heart rate, gill damage and augmented body choles-terol have been associated to PW induced narcosis (Stephens et al.,1996). In 50 d-old turbots, exposure to 0.01% PW (North Sea oil)stimulated swimming activity (avoidance defence mechanism)whereas exposure to 0.1–1% PW provoked a stress response andexposure to 1% PW caused a significant reduction in swimmingactivity, suggesting a narcotic action of the externals hydrocarbonmixture or those accumulated internally or their metabolites

S. Brooks et al. / Marine Pollution Bulletin 62 (2011) 327–339 337

(EROD activity was induced) (Stephens et al., 2000). Finally, a weakacidification (i.e., caused by the presence of low amounts of polarcompounds) may provoke metabolic alterations in mussels (Mich-aelidis et al., 2005), although no differences in the pH values of thetreatment water were found. Therefore it seems that the observedanomalies could be the consequence of either altered exposureconditions at 1% PW concentration (Azetsu-Scott et al., 2007) orthe result of a narcotic effect exerted by the complex mixture ofconstituents of the PW at low concentrations (Stephens et al.,1996, 2000; Smith et al., 1998), or maybe both. This is an interest-ing issue that deserves further research in order to advance in theunderstanding of the potential biological effects of PW on musselhealth.

4.3. Integrative Biological Response (IBR/n)

IBR has been previously applied to fishes and mussels includingdifferent suites of biomarkers (Beliaeff and Burgeot, 2002; Broegand Lehtonen 2006; Damiens et al. 2007; Pytharopoulou et al.,2008; Marigómez et al., in prep.). In general terms, the results ob-tained in these studies and their interpretation were comparable tothose presently achieved. It is worth noting that IBR and IBR/n pro-duce satisfactory discrimination between sites with differenthealth status whatever the combination of biomarkers is. Accord-ingly, IBR/n has successfully discriminated between PW treatmentsin the present study. Five biochemical, histochemical and histolog-ical biomarkers (AOX, CAT, LP, VvBAS, and NRRT) were used to cal-culate the IBR index developed by Belaieff and Burgeot (2002).Aware that different biomarker arrangements on the star plots pro-duce different IBR/n values (Broeg and Lehtonen 2006), biomarkerswere orderly represented in the five axes of star plots according totheir biological complexity level (AOX, metabolic response; CAT,subcellular; LP, cellular response; VvBAS, tissue response; NRRT;systemic response). Overall, IBR/n values were moderately higherafter PW exposure than in the control group and increased with% PW from 0.01% to 0.5% PW but were low on exposure to 1%PW. Star plots revealed details about the biological responses elic-ited at each sampling time and locality. Affects at the simplest lev-els of biological complexity, such as AOX and LP, were moremarked at lowest PW exposure (0.01%) whereas alterations pro-gressed to more complex biological levels (VvBAS and NRRT) atincreasing % PW exposures up to 0.05%. Eventually, only NRRTwas apparently altered after 1% PW treatment, which was unex-pected, as above discussed for single biomarkers.

5. Conclusion

The present paper confirms the utility of the integrative bio-marker approach based on a battery of biological responses elicitedat diverse levels of biological complexity for the assessment of PWsin which single pollutants are at concentrations below their detec-tion limits. There was good agreement found between the biolog-ical effects measurements showing that exposure to PWconcentrations at 0.01% and above had a marked effect on musselhealth. Exposure up to 0.5% PW provoked peroxisome prolifera-tion, induction of antioxidant enzymes and changes in cell typecomposition in the digestive gland epithelium and marked lyso-somal membrane destabilisation in digestive cells and cytotoxicityin haemocytes, as well as adverse effects in the integrity of diges-tive gland tissue, although CTD ratio was less responsive due to thehigh values recorded in the control group. Slight but recognizabletendencies were also envisaged for genotoxicity and intracellularlipid accumulation. Overall, though these changes and trends wereoften small and individually not significant, they were coherentlyrelated to each other. Thus, integration into the IBR/n index

showed an evident alteration in mussel health status at PW con-centrations in the range of 0.01–0.5%, which was interpreted as apollutant induced sublethal stress response. However, no relation-ship was found between the biological effects and the contaminantconcentrations measured, which was extremely marked on expo-sure to 1% PW. It is possible that additive or synergistic toxic ef-fects or non-polar narcosis occurred (Smith et al., 1998; Hannamet al., 2009), or even that other PW contaminants not measuredwere responsible for the biological effects observed. Inconsisten-cies between biological effects and mussel tissue chemistry havebeen previously reported in field studies, being attributed to eithereffects of contaminants that were not measured, to the combinedeffects of mixture toxicity resulting in a threshold effect, or tothe consequences (i.e. reduced feeding activity, growth and bioac-cumulation) of adaptive mechanisms or toxic effects elicited inmussels by pollutants themselves (Brooks et al., 2009; Garmendiaet al., submitted).

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