Integrated Biomarker Assessment of the Effects Exerted by Treated

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Integrated biomarker assessment of the effects exerted by treated produced water from an onshore natural gas processing plant in the North Sea on the mussel Mytilus edulis

Article in Marine Pollution Bulletin · November 2010 DOI: 10.1016/j.marpolbul.2010.10.007 · Source: PubMed

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Steven Brooks Christopher Harman Norwegian Institute for Water Research Norwegian Institute for Water Research

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Beñat Zaldibar Ionan Marigómez Universidad del País Vasco / Euskal Herriko… Universidad del País Vasco / Euskal Herriko…

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Marine Pollution Bulletin

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Integrated biomarker assessment of the effects exerted by treated produced water from an onshore natural gas processing plant in the North Sea on 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, Norway b 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, Spain c Det Norske Veritas (DNV), Maries vei 20, 1363 Høvik, Norway article info abstract

Keywords: The biological impact of a treated produced water (PW) was investigated under controlled laboratory Biomarkers conditions in the blue mussel, Mytilus edulis. Mussel health status was assessed using an integrated bio- Monitoring marker approach in combination with chemical analysis of both water (with SPMDs), and mussel tissues. Produced water Acyl-CoA oxidase activity, neutral lipid accumulation, catalase activity, micronuclei formation, lysosomal Mussels membrane stability in digestive cells and haemocytes, cell-type composition in digestive gland epithe- Passive samplers 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 PW were at extremely low concentrations in both water and mussel tissues. The study highlights the beneﬁts of 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. Ó 2010 Elsevier Ltd. All rights reserved.

1. Introduction effects have been reported in fish and invertebrates at exposure concentrations below 1% PW (Strømgren et al., 1995; Stephens The chemical composition of produced water (PW) can be very et al., 1996, 2000; Zhu et al., 2008; Hannam et al., 2009). The complex and quantitatively variable among PWs, but usually their groups of PW compounds most likely to be contributing to its compounds include trace metals, organic acids, phenols, alkylphe- toxicity include volatile (BTEX) and semi-volatile (GRO) PAHs, nols, polycyclic aromatic, aliphatic hydrocarbons and residual pro- phenols and dissolved ions (Smith et al., 1998; Fisher and Bidwell, duction and treatment chemicals and their breakdown products 2006). However, in most cases the toxicity can not be attributed to (emulsifiers, corrosion inhibitors, antifoaming agents, corroded individual components (usually at non-toxic extremely low con- materials, etc.) (Roe Utvik, 1999; Neff, 2002; Johnsen et al., centrations) but to the properties of the mixture. 2004). Most of these compounds usually occur at extremely low The Ormen Lange gas processing plant is situated at Nyhamna concentrations (i.e. [PAHs] total in North Sea PW < 7 lg/L; on the island of Gossa, on the West coast of Norway, where pro- Strømgren et al., 1995). Moreover, once discharged into the sea, duced water, gas and condensate received by pipeline from the Or- PW is rapidly diluted and dispersed and further volatilization men Lange gas field 100 km offshore in the North Sea is processed. and biodegradation reduces the levels of marine contamination Produced water from the onshore processing plant is diluted with (Flynn et al., 1996). As a result, the chemical identification and cooling water within the Ormen Lange system before it is dis- quantification of these compounds can be difficult and on most charged into the surrounding coastal water environment by a sin- occasions present at concentrations below their detection limit gle outfall pipe. Macro porous polymer extraction (MPPE) (Smith et al., 1998). In contrast, both acute and sublethal toxic technology in combination with biological treatment, can reduce dissolved and dispersed hydrocarbons with up to 99% removal, and is used at the Ormen Lange gas processing plant (Aker ⇑ Corresponding author. Tel.: +47 22185100; mob.: +47 92696421; fax: +47 22185200. Kværner, 2006). The MPPE technology and biological treatment E-mail address: [email protected] (S. Brooks). can remove most aliphatic hydrocarbons, BTEX, PAHs and NPDs

Please cite this article in press as: Brooks, S., et al. Integrated biomarker assessment of the effects exerted by treated produced water from an onshore natural gas processing plant in the North Sea on the mussel Mytilus edulis. Mar. Pollut. Bull. (2010), doi:10.1016/j.marpolbul.2010.10.007 2 S. Brooks et al. / Marine Pollution Bulletin xxx (2010) xxx–xxx and some polar compounds including alkyl phenols to a limited ex- filtered (10 lm) seawater (SW) within the flow through system tent (Aker Kværner, 2006). to deliver environmentally relevant exposure concentrations of The main objective of the present study was to evaluate under 0.01%, 0.1%, 0.5% and 1% of the original PW concentration. The controlled laboratory conditions the potential biological impact ex- PW stock was measured for PAHs and metals at the start of the erted by a treated PW effluent (Ormen Lange processing plant), exposure. The flow-through system was allowed to dose for 1 week which was expected to present extremely low levels (often below prior to the addition of the mussels and SPMDs in an attempt to detection limits) of individual contaminants in a highly complex achieve steady state conditions. mixture. Blue mussels, Mytilus edulis, were selected as a sensitive The design of the flow-through system included separate 100 L target species representative of the biota inhabiting receiving mixing tanks, which were used to combine the PW with the dilu- waters in the North Sea. The health status of the mussel can be di- tion SW. The outflow water from the mixer tanks, after an approx- rectly related to the amount of environmental stress imposed imate 1 h residency time, was transported to the 50 L exposure through a variety of factors including contaminant exposure and tanks holding the mussels and SPMDs. The seawater flow rate therefore, it provides important information on their surrounding was calculated at 2.3 L/min, which was based on a mussel clear- environment including waterborne toxicity. ance rate of 0.033 L/min with 70 mussels in each exposure tank. In order to assess mussel health status, an integrated biomarker This was to ensure that each mussel was exposed to fresh exposure approach was applied in combination with chemical analysis of medium. The mussels used in the study were roped mussels ob- both water, through the utilisation of semipermeable membrane tained from a mussel hatchery in Rissa, Norway (www.snadderogs- devices (SPMDs), and mussel tissues. Biomarkers have been previ- naskum.no). The mussels were transported on ice by overnight ously applied in fish for monitoring the impact of PW discharges courier and placed in the exposure tanks on the morning of arrival. from offshore platforms in North Sea and Northern Shelf of Austra- Only mussels between 4 and 6 cm in length were selected for the lia (Codi King et al., 2005; Sturve et al., 2006; Zhu et al., 2008; Abra- exposure. Within the tanks, mussels were held within nylon mesh hamson et al., 2008; Hylland et al., 2009; Brooks et al., 2010). Due bags and suspended within the water column. Mussels were fed a to the complexity of PW effluents and the variety of biological re- concentrated mixed algal feed (Shellfish diet 1800). Physicochem- sponses they can induce, multiple effect biomarkers were deter- ical readings of the exposure media, including pH, temperature, mined to provide a sensitive evaluation of mussel health salinity, and dissolved oxygen, as well as flow rates, were checked following PW exposure (Cajaraville et al., 2000; Brooks et al., on a daily basis (Table 1). Feeding and general health checks of the 2009; Hylland et al., 2009). These included: (a) acyl-CoA oxidase mussels and the dosing system were made every 2 day during the (AOX) activity enhancement, as a measure of peroxisome prolifer- 5 week exposure. The exposure was conducted in the autumn. ation (Fahimi and Cajaraville, 1995); (b) intracellular neutral lipids The test was terminated after 5 week. Mussels were removed accumulation (INLA) resulting from exposure to organic com- from the exposure tanks and processed within 2 h of removal from pounds (Marigómez and Baybay-Villacorta, 2003); (c) catalase the exposure tanks. Haemolymph samples were taken for LMSHC (CAT) activity increase, as indication of enhanced antioxidant de- and MN determinations. Digestive gland and gonad tissue were re- fences (Eertman et al., 1995); (d) micronuclei (MN) formation, as moved from individual mussels and preserved by either snap a measure of DNA damage (Heddle et al., 1983); (e) reduction in freezing in liquid nitrogen or by fixing in formalin. Frozen digestive lysosomal membrane stability in digestive cells (LMSDC), as bio- gland samples were processed for biochemistry and cytochemistry marker of general stress (Moore 1976; UNEP/RAMOGE, 1999); (f) (AOX, CAT, VvNL and LMSDG). Fixed digestive glands and gonads changes in cell type composition (augmented relative proportion were dehydrated in alcohol and embedded in paraffin for histolog- of basophilic cells; VvBAS) in digestive gland epithelium, as indica- ical examination (gamete development, digestive gland and gonad tion of general stress (Soto et al., 2002); (g) disrupted membrane histopathology), and determinations of tissue-level biomarkers integrity in haemocytes (LMSHC), indicative of both either general (VvBAS, and CTD). Whole mussel homogenates were used to mea- stress or immunodeficiency (Borenfround and Puerner, 1985; Lowe sure organic (PAHs, NPDs) and metal concentrations. and Pipe, 1994); and (h) changes in the integrity of the digestive gland tissue (CTD), as an early symptom of pathological damage 2.2. Semipermeable membrane devices (SPMDs) (Garmendia et al., submitted). The biomarkers were integrated in the Integrative Biological Response (IBR/n) index (Beliaeff and SPMDs were wound around stainless steel deployment spiders Burgeot 2002; Broeg and Lehtonen 2006). Integrative biomarker (Environmental Sampling Technologies, Saint Joseph, USA) and indices may be used to provide comprehensive assessment of placed directly into the exposure tanks. Three replicates per tank musselsÕ health status, which is currently regarded as the best were used. SPMDs were spiked with a mixture of deuterated PAH available approach for monitoring pollution effects in marine as performance reference compounds (PRCs) to allow for the deter- ecosystems (e.g. Broeg and Lehtonen 2006; Dagnino et al., 2007; mination of sampling rates (Booij et al., 1998; Huckins et al., 2002) Brooks et al., 2009; Garmendia et al., submitted). and were obtained from ExposMeter (Tavelsjo, Sweden).

2.2.1. Sampler extraction and chemical analysis 2. Material and methods The exterior of the SPMDs were wiped clean before extraction by dialysis with 2 Â 150 mL hexane (Huckins et al., 1990a). Clean 2.1. Flow-through exposure and sampling up by GPC and analysis for PAHs and NPDs by GC–MS, proceeded as described below for mussel samples. Quantification of individ- A laboratory flow-through dosing system was designed to ex- ual components was performed by using the relative response of pose mussels and semipermeable membrane devices (SPMDs) to internal standards. In order to correct for any possible contamina- known concentrations of the PW over a 5 week period. The PW tion during study procedures, control or ‘blankÕ SPMDs were used. was collected September 2008 from the Ormen Lange processing These included field controls (FCs) that were exposed to the air plant whilst operating at approximately 50% maximum produc- during deployment and retrieval and laboratory controls (LCs) that tion. Approximately 3000 L were collected from the Observation follow exposure to solvents, glassware etc. during sample work up. tank (post treatment) in 3 Â 1000 L plastic airtight containers At least one of each type of control was used per 10 exposed sam- and transported overnight by road to NIVAÕs marine research plers. Initial (time zero) concentrations of PRCs were also estab- station at Solbergstrand, Norway. The PW stock was diluted with lished from LCs.

Table 1 Physicochemical 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 ± 0.10 0.01% 10.77 ± 1.74 6.79 ± 0.90 33.25 ± 0.45 7.95 ± 0.08 0.1% 10.78 ± 1.75 6.49 ± 0.77 33.14 ± 0.48 7.89 ± 0.09 0.5% 10.73 ± 1.74 6.58 ± 0.89 33.03 ± 0.47 7.92 ± 0.08 1% 10.89 ± 1.74 6.64 ± 0.88 32.95 ± 0.42 7.89 ± 0.11

2.2.2. Calculation of sampling rates and water concentrations and (d) MN are apparent as spherical structures with a sharp An empirical model, described in detail by Huckins et al. (2006), contour. was used in the calculation of water concentrations from SPMD accumulations. In this model compound speciﬁc effects on uptake 2.3.2. Enzyme activities in digestive gland are adjusted based on the log Kow of the analyte and site-speciﬁc Frozen digestive glands were individually homogenised in a factors arising from differences in environmental variables are ad- Braun-Potter homogeniser using TVBE buffer (1 mM sodium bicar- justed by using the PRC data. In this way the uptake for each indi- bonate, 1 mM EDTA, 0.1% ethanol and 0.01% Triton X-100; vidual compound at each sampling station was established, pH = 7.6). After homogenisation, samples were centrifuged at expressed as a sampling rate (L/d). Where individual analytes were 500g for 15 min. Supernatants were removed and diluted appropri- not detected in SPMDs then the analytical detection limit was used ately to perform the enzyme assays. Total protein of all samples in calculations to provide a maximum theoretical concentration in was measured according to the Lowry method using a commercial the water. protein as standard (BioRad, California).

2.3. Biomarkers 2.3.2.1. Catalase (CAT) activity. The activity of the antioxidant enzyme catalase was measured as described by Porte et al. (1991) 2.3.1. Haemolymph analysis in frozen digestive gland samples (n = 10 mussels). Brieﬂy, after

2.3.1.1. Lysosomal membrane stability in haemocytes (LMSHC). The centrifugation at 500g for 15 min, a small amount of sample was integrity of lysosomal membranes of mussel haemocytes was stored for further analysis of AOX activity and the remainder was determined using the Neutral Red Retention (NRR) procedure processed to obtain mitochondrial and cytosolic fractions by cen- adapted from Lowe and Pipe (1994). Approximately 0.1 mL of hae- trifugation at 12,000g for 45 min and 100,000g for 90 min, respec- molymph was removed from the adductor muscle of the mussel tively. CAT activity was determined in the mitochondrial and with a syringe containing approximately 0.1 mL physiological sal- cytosolic fractions by measuring the consumption of H2O2 at À1 À1 ine. The haemolymph/saline solution was placed in a microcentri- 240 nm (ext. coeff. 40 M cm ) using H2O2 50 mM as substrate fuge tube, from which a 40 lL sample was removed and pipetted in potassium phosphate buffer 80 mM (pH = 7). Total CAT activity onto the centre of a microscope slide. The slide was left in a dark was calculated as the sum of the activity of the two fractions. humid chamber for 15 min to allow the cells to adhere to the slide. After this time, the excess liquid was removed from the slide and 2.3.2.2. Palmitoyl-CoA Oxidase (AOX) activity. Peroxisomal palmi- 40 lL of neutral red solution added (Sigma). The neutral red solu- toyl-CoA oxidase (AOX) activity was determined spectrophoto- tion was taken up inside the haemocytes and stored within lyso- metrically (k = 502 nm), in 5 pools of 2 digestive glands per somes. The ability of the lysosomes to retain the neutral red experimental group, measuring the H2O2 dependent oxidation of solution was checked every 15 min by light microscopy (Â400). dichloroﬂuorescein diacetate (Molecular Probes, Eugene, Oregon, The test was terminated and the time recorded when greater than USA) catalyzed by an exogenous peroxidase, using 30 lM palmi- 50% of the haemocytes leaked the neutral red dye into the cytosol. toyl-CoA as substrate, according to Small et al. (1985).

2.3.1.2. Micronuclei formation in mussel haemocytes (MN). Approx- 2.3.3. Digestive gland cytochemistry imately 0.1 mL of haemolymph was removed from the posterior Ten serial sections (10 lm thick) of frozen digestive gland (5 adductor muscle of each mussel with a hypodermic syringe con- mussels per treatment) were cut in a Leica CM 3000 cryotome onto taining 0.1 mL PBS buffer (100 mM PBS, 10 mM EDTA). The haemo- successive serial slides and stored at À40 °C until processing. lymph and PBS buffer were mixed briefly in the syringe and placed on a microscope slide. The slide was then placed in a humid cham- 2.3.3.1. Intracellular neutral lipid accumulation (INLA). One set of ber for 15 min to enable the haemocytes to adhere to the slides. Ex- cryotome slides were stained using the method of Lillie and cess fluid was drained and the adhered haemocytes were fixed in AshburnÕs Oil Red O (ORO). Histochemical controls were carried 1% glutaraldehyde for 5 min. Following fixation, the slides were out in a second set of slides by clearing lipids with a mixture of chlo- gently rinsed in PBS buffer and left to air-dry overnight. The dried roform/methanol (1:1) for 1 h at room temperature before staining. slides were brought back to the laboratory for further processing. Cryotome sections were transferred to a cabinet at 4 °C and fixed Slides were stained with 1 lg/mL bisbenzimide 33258 (Hoechst) in BakerÕs solution (+2.5% NaCl) for 15 min. Then sections were solution for 5 min, rinsed with distilled water and mounted in dried at room temperature, washed in isopropanol (60%) and rinsed glycerol McIlvaine buffer (1:1). The frequency of micronuclei for- for 20 min in ORO staining solution. The ORO stock solution is a sat- mation was measured on coded slides without knowledge of the urated (approximately 0.3%) solution of ORO (BDH, 34061) in iso- exposure status of the samples to eliminate bias. The frequency propanol. The staining solution was freshly made (it is only stable of micronuclei in haemocytes was determined microscopically at for 1–2 h) by dissolving 60 mL stock solution in 40 mL distilled 100Â objective (final magnification 1000Â). A total of 2000 cells water and filtering after a 10 min gap to stabilise the solution. were examined for each experimental group of mussels. Only cells Stained sections were differentiated in 60% isopropanol, washed with intact cellular and nuclear membranes were scored. MN were in water, counterstained with 1% Fast Green FCF (Sigma, F-7252) scored when: (a) nucleus and MN have a common cytoplasm, (b) for 20 min and mounted in KaiserÕs glycerine. Slides were viewed colour intensity and texture of MN is similar to the nucleus, (c) using the 40Â objective (final magnification 400Â). Five measure- the size of the MN is equal or smaller than 1/3 of the nucleus, ments were made by image analysis to calculate volume density of

intracellular neutral lipids in digestive cells (VvNL)asVvNL =VNL/VC, 2.3.5. Integrative Biological Response index where VNL is the volume of neutral lipids and VC the volume of The Integrative Biological Response (IBR) index was developed digestive cells (Marigómez and Baybay-Villacorta, 2003). by Beliaeff and Burgeot (2002) in order to integrate biochemical, genotoxicity and histochemical biomarkers. Presently, AOX, CAT,

2.3.3.2. Lysosomal membrane stability(LMSDC). The determination of LP, VvBAS and NRRT were used to calculate the IBR/n index. Since lysosomal membrane stability was based on the time of acid labil- AOX, CAT and VvBAS values are expected to increase in response isation treatment required to produce the maximum staining to environmental insult whereas LP and NRRT decrease, the inverse intensity according to UNEP/RAMOGE (1999), after demonstration values of these latter were used for calculations. The calculation of hexosaminidase (Hex) activity in digestive cell lysosomes. Eight method is based on relative differences between the biomarkers serial cryotome sections (10 lm) were subjected to acid labilisa- in each given data set. Thus, the IBR index is computed by sum- tion in intervals of 0, 3, 5, 10, 15, 20, 30 and 40 min in 0.1 M citrate ming-up triangular star plot areas (a simple multivariate graphic buffer (pH 4.5 containing 2.5 % NaCl) in a shaking water bath at method) for each two neighbouring biomarkers in a given data 37 °C, in order to find out the range of pre-treatment time needed set, according to the following procedure: (1) calculation of the to completely labilise the lysosomal membrane, denoted as the mean and standard deviation for each sample; (2) standardization labilisation period (LP). Following this treatment, sections were of data for each sample: xiÕ =(xi À x)/s; where, xiÕ = standardized va- transferred to the substrate incubation medium for the demonstra- lue of the biomarker; xi = mean value of a biomarker from each tion of Hex activity. sample; x = general mean value of xi calculated from all compared The incubation medium consisted of 20 mg naphthol AS-BI-N- samples (data set); s = standard deviation of xi calculated from all acetyl-b-D glucosaminide (Sigma, N 4006) dissolved in 2.5 mL 2- samples; (3) addition of the standardized value obtained for each methoxyethanol (Merck, 859), and made up to 50 mL with 0.1 M cit- sample to the absolute standardized value of the minimum value rate buffer (pH 4.5) containing 2.5 % NaCl and 3.5 g low viscosity in the data set (yi = xiÕ +|xminÕ|); (4) calculation of the Star Plot tri- polypeptide (Sigma, P5115) to act as a section stabiliser. Sections angular areas by multiplication of the obtained standardized value were incubated in this medium for 20 min at 37 °C, rinsed in a saline of each biomarker (yi) with the value of the next standardized bio- solution (3.0 % NaCl) at 37 °C for 2 min and then transferred to 0.1 M marker value (yi+1), dividing each calculation by 2 (Ai=(yi Â yi+1)/2); phosphate buffer (pH 7.4) containing 1 mg/mL diazonium dye Fast and (5) calculation of the IBR index whichP is the summing-up of all Violet B salt (Sigma, F1631), at RT for 10 min. Slides were then rap- the Star Plot triangular areas (IBR = Ai)(Beliaeff and Burgeot, idly rinsed in running tap water for 5 min, fixed for 10 min in BakerÕs 2002). Since the IBR value is directly dependent on the number formol calcium containing 2.5% NaCl at 4 °C and rinsed in distilled of biomarkers in the data set, we divided the obtained IBR value water. Finally, slides were mounted in KaiserÕs glycerine gelatine by the number of biomarkers used in each case (n = 5) to calculate and sealed with nail varnish. The time of acid labilisation treatment IBR/n, according to Broeg and Lehtonen (2006). required to produce the maximum staining intensity was assessed under the light microscope as the maximal accumulation of reaction 2.4. Mussel tissue chemistry product associated with lysosomes (UNEP/RAMOGE 1999). Four determinations were made for each animal by dividing each section For each treatment group, triplicate mussel samples were taken in the acid labilisation sequence into 4 approximately equal seg- for analysis of selected metals and PAHs, including alkylated ments and assessing the LP in each of the corresponding set of seg- homologues of naphthalene, phenanthrene and dibenzothiophene ments. The mean LP value was then derived for each section, (NPD). Five whole mussels per sample were removed from their corresponding to an individual digestive gland. shells and placed in pyrolysed (560 °C) glass containers. The mussels were frozen and transported to NIVA on dry ice. All samples 2.3.4. Digestive gland and gonad histology were stored at À20 °C until analyses. Histological sections (7 lm) were cut using a rotary microtome Samples were defrosted, homogenised and a sub sample taken Leitz 1512 (Ernest Leitz Wetzlar GmbH, Austria) and stained with of approximately 5 g. Internal standards were added before extrac- haematoxylin-eosin (H/E). Prevalence of parasites, haemocyte tion by saponification. Analytes were then extracted twice with infiltration and general condition of the digestive epithelium, the 40 mL cyclohexane and dried over sodium sulphate. The extracts interstitial connective tissue and the gonad tissue were systemat- were reduced by a gentle stream of nitrogen and cleaned by gel ically recorded. permeation chromatography (GPC) using the system described previously (Harman et al., 2008). Analysis proceeded by gas chro- 2.3.4.1. Epithelial cell-type composition (Vv )and tissue integrity in BAS matography with mass spectrometric detection (GC–MS) with digestive gland (CTD ratio). The volume density of basophilic cells the MS detector operating in selected ion monitoring mode (Vv ) was quantified by means of stereology as an indication of BAS (SIM). The GC was equipped with a 30 m column with a stationary whether changes in cell-type composition occurred or not. Like- phase of 5% phenyl polysiloxane (0.25 mm i.d. and 0.25 lm film wise, the integrity of the digestive gland tissue was simultaneously thickness), and the injector operated in splitless mode. The initial determined as the extent of the interstitial connective tissue rela- column temperature was 60 °C, which after two minutes was tive to the space occupied by digestive diverticula (connective- raised stepwise to 310 °C. The carrier gas was helium and the col- to-diverticula (CTD) ratio). Counts were made in one randomly umn flow rate was 1.2 mL/min. Quantification of individual com- selected field in one digestive gland slide per mussel (10 mussels ponents was performed by using the internal standard method. per sample). Slides were viewed at 40x objective (final magnifica- The alkylated homologues were quantified by baseline integration tion 400x) using a drawing tube attached to a Nikon Optiphot of the established chromatographic pattern and the response fac- microscope. A simplified version of the Weibel graticule multipur- tors were assumed equal within each group of homologues. pose test system M-168 (Weibel 1979) was used, and hits on basophilic cells (b), digestive cells (d), diverticular lumens (l) and interstitial connective tissue (c) were recorded. CTD ratio was cal- 3. Results culated as CTD = c/(b + d + l). VvBAS was calculated according to the DelesseÕs principle (Weibel 1979), as VvBAS =VBAS/VEP, where VBAS is The mussels used in this experiment appeared to be de visu in the volume of basophilic cells and VEP the volume of digestive good condition throughout the exposure period, with < 1% mortal- gland epithelium. ity recorded both in control and PW exposure treatments.

However, at the microscope certain loss of histological integrity in concentrations that were detected include naphthalene, phenan- the digestive gland tissue and epithelial thinning in digestive threne, fluorene, fluoranthene and pyrene, which were present in alveoli were found in both control and PW exposed mussels, albeit very low ng/g (wt w). Metal concentrations in mussel tissues were much more marked in the latter. Thus, 0.01–0.5% PW exposed also low and no obvious relationship between metal concentration mussels, and those exposed to 1% PW to a lesser extent, showed and nominal exposure concentration was found (Table 4). a severe reduction in the numbers of digestive diverticula, which appeared sparse throughout a highly disorganized and eventually fibrous interstitial connective tissue (see CTD ratio below) and an 3.2. Biomarkers extreme thinning of the digestive gland epithelium. In contrast, no significant parasitic infestation or pathological lesion was found Although no significant differences were found in AOX activity in any case. The histological examination of the gonad revealed no between the different exposure groups, a trend to increase AOX clear difference among mussels from the different treatment activity with PW exposure up to 0.5% PW was found (Fig. 1A). groups. Mature gametes were observed in most mussels for both AOX activity in mussels exposed to 0.5% PW was almost double males and females. Although in some specimens spawning had that of the control group. However, the lowest AOX activity was re- already taken place, it appeared to be incidental and could not be corded in 1% PW exposed mussels. Neutral lipids were revealed as related to PW exposure. bright reddish purple deposits easily identified at the light micro- The results obtained regarding the concentrations of pollutants scope in the digestive alveoli, digestive ducts and stomach. The measured in water (through SPMDs) and mussel tissues, the indi- staining pattern was heterogeneous and not all the alveoli pre- vidual biomarker recorded in mussels and the integration of bio- sented the same degree of reactivity but in all the reactive alveoli, markers as IBR/n index are reported in the next three sections. the ORO reaction product appeared clearly localized within com- partments of the endo-lysosomal system. As a result, VvNL values were highly variable and similar in all the experimental groups

3.1. Concentrations of pollutants in water and mussel tissues but in the 1% PW exposure treatment where VvNL was significantly the lowest (Fig. 1B). CAT activity was not significantly dissimilar Based on the chemical analysis of SPMDs, it was observed that among experimental groups but, like in the case of AOX activity, PAH concentrations were either low or undetected in all experi- this enzyme activity was slightly higher in 0.01–0.5% PW treat- mental tanks (Table 2). The PAHs that were detected include fluo- ments than in the control one, and the lowest values were recorded rene, phenanthrene, fluoranthene and pyrene.P These compounds on exposure to 1% PW (Fig. 1C). The highest mean frequency of MN were detected at background concentrations ( PAH 0.7–2.0 ng/ was found in the haemocytes of mussels exposed to 1% PW L) with acceptable variation between replicates (average RSD (Fig. 1D). However, no significant differences in MN frequency 15%). NPD compounds were also only present at very low concen- were found between the different exposure groups. Overall, there trations (4.6–6.3 ng/L), but were more variable (average RSD 50%). was a low prevalence of MN in all groups ranging from 0.5 to 1.4 There were no apparent differences in PAH/NPD concentration be- MN per 1000 cells. LP in control mussels was unexpectedly low tween the exposure treatments, based on the SPMD results, with (13–15 min) but, nevertheless, LP values (<5 min) were signifi- the exception of the treatment with 0.01% PW. This treatment cantly lower at exposures to 0.01–0.5% PW than in the control had noticeably lower concentrations than the other treatments group (ANOVA, TurkeyÕs test, p < 0.05; Fig. 1E). In contrast, the LP including the control tank. The PRC results (data not presented) recorded in mussels exposed to 1% PW was not significantly differ- showed that the total volume of water extracted during the five ent from that recorded in the control group (Fig. 1E). Only VvBAS week laboratory exposure was between 28 and 161 L, depending values recorded in the 0.1% PW exposure group were significantly on the compound. different from those obtained in the control group (one-way ANO- Likewise, low or undetected concentrations of PAHs were found VA, DuncanÕs test, p < 0.05) but, overall, the same trend than for in mussel tissues in all exposure tanks with no noticeable differ- AOX and CAT activities can be depicted (Fig. 1F). The VvBAS values ences between the exposure concentrations (Table 3). The PAH recorded in all groups were reasonably high (>0.12 lm3/lm3) with

Table 2 The PAH concentration calculated from SPMDs exposed for 5 weeks to different concentrations of produced water. a = high blank concentrations data not reported.

ng/L Control 0.01% PW 0.1% PW 0.5% PW 1% PW 123123123123123 Naphthalene aaaaaaaaaaaaaaa Acenaphthylene <0.11 <0.10 <0.10 <0.10 <0.09 <0.09 <0.10 <0.11 <0.11 <0.15 <0.15 <0.13 <0.12 <0.10 <0.11 Acenaphthene 0.10 <0.09 0.09 0.11 0.08 <0.07 0.09 0.13 0.11 0.14 0.16 0.12 <0.10 0.09 0.11 Fluorene 0.34 0.32 0.27 0.25 0.17 0.19 0.27 0.39 0.33 0.57 0.51 0.45 0.35 0.29 0.40 Dibenzothiophene <0.08 <0.07 <0.07 <0.07 <0.06 <0.06 <0.07 0.10 <0.08 0.11 <0.11 <0.10 <0.08 <0.07 <0.08 Phenanthrene 0.71 0.67 0.56 0.43 0.26 0.30 0.51 0.86 0.65 1.27 0.94 0.91 0.75 0.63 0.81 Anthracene <0.07 <0.06 <0.06 <0.06 <0.05 <0.05 <0.06 0.07 <0.07 <0.1 <0.1 <0.09 <0.07 <0.06 <0.07 Fluoranthene 0.14 0.14 0.13 0.10 0.08 0.09 0.11 0.14 0.12 0.19 0.16 0.14 0.14 0.11 0.14 Pyrene 0.11 0.10 0.09 0.08 0.06 0.07 0.09 0.11 0.11 0.16 0.13 0.12 0.13 0.11 0.13 Benz[a]anthracene <0.06 <0.05 <0.05 <0.05 <0.04 <0.04 <0.05 <0.06 <0.05 <0.09 <0.09 <0.08 <0.06 <0.05 <0.06 Chrysene <0.05 <0.05 <0.05 <0.04 <0.03 <0.04 <0.05 <0.05 <0.05 <0.08 <0.08 <0.07 <0.06 <0.05 <0.06 Benzo[b.j]ﬂuoranthene <0.05 <0.05 <0.05 <0.04 <0.04 <0.04 <0.05 <0.05 <0.05 <0.08 <0.08 <0.07 <0.06 <0.05 <0.06 Benzo[k]ﬂuoranthene <0.06 <0.06 <0.05 <0.05 <0.04 <0.04 <0.05 <0.06 <0.06 <0.10 <0.10 <0.08 <0.07 <0.05 <0.07 Benzo[e]pyrene <0.07 <0.06 <0.06 <0.05 <0.04 <0.05 <0.06 <0.07 <0.07 <0.11 <0.11 <0.09 <0.08 <0.06 <0.07 Benzo[a]pyrene <0.07 <0.06 <0.06 <0.05 <0.04 <0.04 <0.06 <0.07 <0.06 <0.10 <0.10 <0.09 <0.07 <0.06 <0.07 Perylene <0.07 <0.06 <0.06 <0.05 <0.04 <0.04 <0.06 <0.07 <0.06 <0.10 <0.10 <0.09 <0.07 <0.06 <0.07 Indeno[1.2.3-cd]pyrene <0.08 <0.08 <0.07 <0.07 <0.05 <0.06 <0.07 <0.08 <0.08 <0.13 <0.13 <0.11 <0.09 <0.07 <0.09 Dibenzo[ac/ah]anthracene <0.07 <0.07 <0.06 <0.06 <0.05 <0.05 <0.06 <0.07 <0.07 <0.11 <0.11 <0.10 <0.08 <0.06 <0.08 Benzo[g.h.I]perylene 0.09 <0.08 <0.08 <0.07 <0.06 <0.06 <0.08 <0.09 <0.09 <0.14 <0.14 <0.12 <0.10 <0.08 <0.09 SUM PAH <2.32 <2.17 <1.94 <1.74 <1.29 <1.38 <1.88 <2.58 <2.22 <3.73 <3.32 <2.97 <2.49 <2.04 <2.57 PAH EPA16 <2.11 <1.97 <1.76 <1.56 <1.15 <1.23 <1.69 <2.35 <2.01 <3.41 <2.99 <2.69 <2.26 <1.86 <2.35

Table 3 The 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 123123123123123 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 1.3 C1-Naphthalenes <2 <2 <2 <2 <2 <2 2.1 <2 <2 <2 <2 2.6 <2 <2 <2 C2-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 6.4 C3-Naphthalenes 7.4 12 10 14 14 12 18 12 7.9 7.7 9.6 23 7.9 13 12 Phenanthrene 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 1.0 C1-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 3.4 C2-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 2.5 C3-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 4.4 Dibenzothiophene <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 <0.5 C1-Dibenzothiophenes <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 C2-Dibenzothiophenes <2 <2 <2 <2 <2 <2 2.1 <2 2.5 <2 <2 <2 <2 <2 <2 C3-Dibenzothiophenes <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 Sum 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 <39.5 Acenaphthylene <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 <0.5 Acenaphthene <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 <0.5 Fluorene <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 <0.5 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 <0.5 Fluoranthene 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 0.88 Pyrene 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 1.0 Benzo(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 <0.5 Chrysene <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 <0.5 Benzo(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 <0.5 Benzo(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 <0.5 Benzo(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 <0.5 Benzo(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 <0.5 Perylene <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 <0.5 Indeno(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 <0.5 Dibenz(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 <0.5 Benzo(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 <0.5 Sum 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 <48.38 Sum 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 <10.18 Lipid (%) 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 4 Metal 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 123123123123 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 0.008 Al 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 4.1 As 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 2.09 Cd 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 0.088 Cr 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 0.42 Cu 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 1.26 Fe 18 18 14 17 16 14 14 16 14 14 16 20 13 14 12 Hg 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 0.008 Ni 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 0.24 Pb 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 0.05 Zn 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 a high variability between mussels from the same experimental biomarker response was almost zero in the seawater control (SW; group. Significantly higher neutral red retention time (NRRT) was 0% PW). AOX, CAT and LP were sensitive biomarkers in 0.01–0.5% found in the control group compared to all other groups (ANOVA, PW (Fig. 2B-D), with VvBAS also relevant after 0.1% and 0.5% PW TurkeyÕs test, p < 0.05; Fig. 1G). NRRT in control mussels was exposures (Fig. 2C and D) and NRRT in 0.5% and 1% PW treatments 60 min and approached 20 min in mussels exposed to 0.5% and (Fig. 2D and E). Despite the weak effects reported for individual 1% PW, they were not found to be significantly different from the biomarkers, IBR/n values were markedly higher in 0.1–0.5% PW values recorded in mussels exposed to 0.01% PW (NRRT treatments than in the control and the 1% PW treatment 40 min). CTD ratios were similar and over 0.5 in all the experimen- (Fig. 2F), indicating a trend to decrease in mussel health status tal groups and exhibited a great variability in PW exposed mussels. on exposure up to 0.5% PW. Unexpectedly, affection was appar- CTD ratios were significantly lower in mussels exposed to 1% PW ently lower in the 1% PW exposure group (Fig. 2F). than in the other experimental groups (one-way ANOVA, DuncanÕs test, p < 0.05; Fig. 1H). 4. Discussion

3.3. Integrative Biological Response (IBR/n) The main objective of the present study was to evaluate, under controlled laboratory conditions, the potential biological impact

Five biomarkers (AOX, CAT, LP, VvBAS, and NRRT) were selected exerted by a treated PW efﬂuent (Ormen Lange processing plant), and represented in the ﬁve axes of star plots (Fig. 2). The integrative which was expected to present low levels (often below detection

0.12 (A) 0.15 (B) 0.10

) 0.12 3 0.08 /µm

3 0.09

0.06 (µm 0.06 * 0.04 NL

Vv 0.03 0.02 AOX Activit (mU/mg) 0 0 0.01 0.1 0.5 1 0 0.01 0.1 0.5 1 1.2 5 (C) (D) 1.0 4 3 0.8 2 0.6 1

0.4 MN/1000 cells 0

0.2 -1 CAT Activity (mmol/ml)

0 0.01 0.1 0.5 1 0 0.01 0.1 0.5 1 14 (E) 0.3 * (F)

12 ) 3

10 /µm 0.2 * 3 (min) 8 * (µm

DC *

LP 6 BAS 0.1

4 Vv

2 0 00.010.10.51 0 0.01 0.1 0.5 1 90 (G) 2,5 (H) ) 3 70 2,0 /µm

* 3

(min) * 1,5 HC 50 *

* 1,0 NRRT 30 * 0,5

10 CTD Ratio (µm 0 0 0.01 0.1 0.5 1 0 0.01 0.1 0.5 1 PW (%) PW (%)

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 density of 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) in haemocytes; and (H) Connective tissue to digestive diverticula (CTD) ratio in digestive gland tissue. Data expressed as mean, standard error (box), standard deviation (outer line) 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). limits) of individual contaminants in a highly complex mixture. to different levels of biological complexity and were determined The biological endpoints measured were responsive to PW expo- by different technologies and by different research labs, depending sure. Moreover, although the measured biomarkers corresponded on their expertise, they were found to be highly coherent and

AOX AOX 4 (A) 4 (B) 3 3 2 2 NRRT 1 CAT NRRT 1 CAT 0 0

BAS LP BAS LP SW (0% PW) 0.01 % PW

AOX AOX 4 4 (C) (D) 3 3 2 2 NRRT CAT NRRT 1 CAT 1 0 0

BAS LP BAS LP 0.10 % PW 0.50 % PW

AOX 4 4 (F) (E) 3.5 3 3 2 2.5 NRRT 1 CAT 2

0 IBR/n 1.5 1 0.5

BAS LP 0 0.01 0.1 0.5 1 1 % PW PW (%)

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 of produced 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). sensitive. The suite of biomarkers revealed that exposure to concen- controlled laboratory environment is likely to impose stress factors trations as low as 0.01–0.5% of treated PW for 5 weeks appeared resulting in the stress response in mussels. However, despite this to provoke a significant stress response in mussels, whereas an slight stress response, control and PW exposed mussels were anomalous response was found in mussels exposed to 1% PW. seemingly different and dose–response trends in several biomark- Mussels in the control group were found to be exhibiting a ers were evident. slight stress response, according to the histopathological examination of the digestive gland (scarce digestive diverticula sparse throughout disorganized interstitial connective tissue and thinning 4.1. Concentrations of pollutants in water and mussel tissues of the digestive gland epithelium) and the values recorded for 3 some biomarkers investigated (i.e. Lp < 20 min, VvBAS > 0.12 lm / The chemistry data for the mussel and SPMDs were not found to lm3 and NRRT < 90 min). Confounding factors including food differentiate between the exposure groups and could not account availability and water quality can be ruled out since animals were for the biological effects observed. In this case the biological re- fed every second day and physicochemical measurements (i.e. sponses were most likely caused by other contaminants not mea- temp, pH, and dissolved oxygen) were found to remain stable dur- sured. Although PAHs and NPDs are a crucial component of PW, ing the exposure duration. Mussel mortality was minimal (<1%), there are also many other chemicals that have not been measured. suggesting that these mussels were in reasonable health prior to Some examples include; alkylphenols, organic acids (such as naph- the test exposure. No significant parasitic infestation or pathologi- thenic acids), and decalins, which may have contributed towards cal lesions were found in any case and the histological examination the biological effects observed. It is however, impractical to mea- of the gonad revealed the presence of mature gametes in most sure the thousands of chemicals present and this highlights the mussels, for both males and females. However, the removal and benefits of sensitive biological effects measurements for the placement of mussels from their natural environment into the assessment of environmental risk of complex discharges.

Levels of PAH/NPD in exposure treatments (ca. 2 and 5 ng/L, screening purposes but usually dose/response curves cannot be respectively), are comparable to those previously described using constructed (Marigómez and Baybay-Villacorta, 2003). However, similar methods at the same facility and also as those measured this biomarker cannot be considered alone due to natural variabil- at reference sites offshore (Harman et al., 2009a,b). Analysis of ity between geographical locations and seasons (Cancio et al., the PW used in the exposure (results not shown) revealed that lev- 1999). In the present experimental conditions it seems that INLA els of PAH were generally below the limits of detection, although does not occur in response to PW exposure, which might be in elevated levels of NPD compounds were present (up to 400 ng/L, agreement with mussel tissue chemistry. Quite the opposite, the C3 naphthalenes). Once diluted and allowing for evaporative losses only change observed is a severe reduction in the intracellular lev- these levels were not able to measurably increase the concentra- els of neutral lipids on exposure to 1% PW, whose possible causes tions of NPD compounds above that already present. are discussed below.

4.2. Single biomarkers 4.2.3. Induction of antioxidant enzymes Induction of antioxidant enzymes such as CAT in fish liver and Histopathological examination of the digestive gland and gonad molluscan digestive gland may be indicative of oxidative stress ex- can help in interpreting biomarkers (e.g. responses may be influ- erted by pollutants acting through enhanced generation of oxygen enced by gender, gamete developmental cycle, presence of para- free radicals (Burgeot et al., 1996; Regoli et al., 2004; Vlahogianni sites or lesions that imply cell loss/hypertrophy/migration/etc.) et al., 2007). In contrast, CAT activity can be inhibited in response and provides sensitive, useful and potential indications for the to severe pollution (Pampanin et al., 2005; Vlahogianni et al., screening of the mussel health status (Kim et al., 2006; Marigómez 2007). Thus, CAT activity is induced in M. edulis on exposure to et al., 2006; Bignell et al., 2008; Garmendia et al., submitted). The low concentrations of PAHs whereas at high concentrations it is histological examination of the gonad, which was mature, revealed inhibited most likely due to their narcotic effect (Eertman et al., no clear differences between the mussel treatment groups. In con- 1995). Regarding our results, the weak induction of CAT activity re- trast, PW exposed mussels showed severe loss of histological corded in mussels exposed to 0.01–0.5% PW might be indicative integrity in digestive gland tissue and extreme thinning of the that a weak oxidative stress was exerted by treated PW to which digestive gland epithelium, which were less marked in mussels ex- mussels were responding through the protecting action of catalase. posed to 1% PW. Histopathological alterations in digestive epithelium cells (vacuolisation and loss of digestive cells, hypertrophy 4.2.4. Micronuclei formation (genotoxicity) of basophilic cells, epithelial thinning and atrophy) and loss of his- Micronuclei formation is an index of chromosomal damage tological integrity in digestive gland tissue (including systemic based on the quantification of downstream aberrations after DNA haemocytosis and interstitial connective tissue oedema; sensu damage and reveals a time-integrated response to complex mix- Couch, 1985) are characteristic traits in stressed mussels (Lowe tures of pollutants (Heddle et al., 1983). The MN test has been eval- et al., 1981; Cajaraville et al., 1990, 1992; Marigómez et al., uated in isolated mussel haemocytes and gill cells (Burgeot et al., 2006; Wedderburn et al., 2000; Usheva et al., 2006; Kim et al., 1996; Bolognesi et al., 1996; Dailianis et al. 2003; Viarengo et al., 2008; Aarab et al., 2008;. Garmendia et al. submitted). 2007). There is no evidence of genotoxicity in treated PW exposed mussels under the present laboratory conditions, with a low prev- 4.2.1. Peroxisome proliferation alence of MN in all groups (0.5–1.4 MN/1000 haemocytes). The rea- Peroxisomes are membrane-bound organelles involved in lipid son for this may be that the exposure concentration was below the metabolism, oxyradical homeostasis and several other important threshold level required to illicit genotoxicity in mussels. A previ- cell functions (Cancio and Cajaraville, 2000), which under exposure ous monitoring program reported significant increases in MN fol- to certain organic chemical compounds proliferate and enhance lowing 30 days exposure to oil compounds (Baršiene˙ et al., 2008). their metabolic activity (Fahimi and Cajaraville, 1995). Among However, the exposure was related to an oil spill scenario with other substances, PAHs, oil derivatives and alkylphenols are known higher contaminant concentrations than that exhibited in the pres- to provoke peroxisome proliferation in marine fish and bivalves ent study. Likewise, increased frequency of MN has been reported (Cajaraville et al., 2000). Peroxisome proliferation is accompanied in mussels when exposed for 6 week to PW in the North Sea (Hyl- by the induction of AOX and other peroxisomal enzyme activities land et al., 2009; Brooks et al., 2010). Although it cannot be ex- (Fahimi and Cajaraville, 1995), which has been proposed as expo- cluded that increasing the length of exposure might result in sure biomarker for organic pollutants (Cajaraville et al., 2000). significant differences between control and exposure groups, the The present results suggest that treated PW up to 0.5% of its Ormen Lange treated PW would likely cause at most very low original concentration acts as a peroxisome proliferator. Although genotoxicity. differences between groups lacked statistical significance, a dose–response trend was depicted and AOX activity in 0.5% PW 4.2.5. Lysosomal membrane destabilization in digestive cells treated mussels was found to be twice that of the control group. Lysosomes of mussel digestive cells, which under normal condi- PWs have been considered a source of peroxisome proliferating tions are involved in food intracellular digestion (Robledo et al., agents probably associated to their petroleum accommodated 2006; Izagirre et al., 2008) and autophagy (Moore et al., 2007), play fraction or to alkylphenols (Sturve et al., 2006; Zhu et al., 2008) an important role in responses to toxic compounds through the but, in contrast, Gorbi et al. (2009) did not find any variation in sequestration and accumulation of toxic metals and organic xeno- AOX activity in PW exposed marine fish. biotics (Viarengo et al., 1987; Marigómez et al., 2002). Environ- mental stressors cause reduction in the stability of the lysosomal 4.2.2. Intracellular neutral lipids accumulation membranes of mussel digestive cells, which is usually measured INLA in digestive cells may be considered indicative of exposure in terms of reduced labilisation period (LP), a widely accepted gen- to organic chemicals of different physicochemical properties eral stress biomarker, and evaluated using the LMSDC (UNEP/ (phthalates, benzo[a]pyrene, phenanthrene, fluoranthene oil RAMOGE 1999; ICES 2004; Marigómez et al. 2005). The values of WAF), although it can be also the result of membrane turnover LP around 15 recorded in the control mussels min (<20 min; criti- impairment after severe exposure (Lowe and Pipe, 1994; Krish- cal threshold value; Marigómez et al., 2006) are in agreement with nakumar et al., 1995; Marigómez and Baybay-Villacorta, 2003). the general histological examination that revealed that control INLA constitutes a prompt all-or-nothing response useful for mussels were not in optimal conditions. However, exposure to

Garmendia et al., submitted). Presently, all the VvBAS values re- by ample areas of disrupted interstitial connective tissue, most corded (also in controls) were always above 0.12 lm3/lm3, which profiles of digestive alveoli appearing elongated or branched (Gar- seemingly suggests that all the mussels were exhibiting some de- mendia et al., submitted). Several authors found smaller numbers gree of stress, which was in agreement with histopathological of digestive tubules per unit area in scallops, and mussels from pol- observations, LP and NRRT results. However, it is worth noting that luted sites (Syasina et al., 1997; Usheva et al., 2006). Presently, CTD the critical threshold values aforementioned for VvBAS refer to M. ratio was quantified for the first time as a tissue-level biomarker galloprovincialis and therefore the values might be different in M. indicative of the histopathological condition of mussels. CTD ratios edulis, although according to the few existing data (Bilbao et al., were similar and over 0.5 in all the experimental groups with only 2006; Hylland et al., 2009) differences between the two species a slight increase in mussels exposed to 0.5% PW over the control would most likely be minimal. Nevertheless, this parameter clearly ones. Seemingly due to the poor histological integrity found in con- indicated that treated PW provokes general stress in mussels, in trol mussels this biomarker was not very sensitive in the range of agreement with LP results. VvBAS values were significantly twice 0.01–0.5% PW exposure concentrations. higher at exposure to 0.1% PW than in the control group. Overall, it seems that PW exposure provokes changes in the cell type com- 4.2.9. Anomalous biological responses on exposure to 1% PW position in the digestive gland epithelium but the results are not The highest exposure (1% PW) used was clearly not effective. fully conclusive as control mussels seemed to be subjected to cer- Both chemical data and biological responses did not follow the tain stress. trend expected from lower exposures. The strong reduction in AOX and CAT activities recorded after 1% PW exposure might be 4.2.7. Cytotoxicity in haemocytes explained by a toxic effect on enzyme activity, as reported after se- Neutral red has been used for the quantification of cytotoxicity vere exposure to pollutants (Bilbao et al., 2010). However, it was based on the ability of viable haemocytes to incorporate and accu- accompanied by reduction in VvNL, VvBAS and CTD and increase in mulate the weakly cationic dye within lysosomes (Borenfround LP, in some cases beyond control group values, and NRRT and and Puerner, 1985). Neutral red measures membrane functional MN were not different from the 0.5% PW exposure, which cannot integrity and lysosomal functioning (Ivanova and Uhlig, 2008), be simply explained by toxic action on enzyme activities. The and reflects the capacity of cellular processes to adapt to stress flow-through system was maintained and checked on a daily basis conditions (Lowe and Pipe, 1994; Lowe et al., 1995). NRRT in mus- with no reported problems in delivery and therefore, the reasons sel haemocytes has been found to be affected by a wide range of for these anomalies are unclear. Interestingly, it has been reported environmental stressors including metals and organic compounds that at concentrations >1% PW, inorganic particles are removed such as phenanthrene, anthracene, chlorpene, benzo[a]pyrene and due to enhanced settlement of organic matter and there is also other PAHs typically found in PW discharges (Lowe et al., 1995; an increase in buoyant particles that are aggregates of oil droplets Moore et al., 1996; Fang et al., 2010). It has been reported that oxi- with inorganic particles (Azetsu-Scott et al., 2007). This would re- dative stress exerted by PAHs provokes cytoskeleton damage, sult in reduced waterborne contaminants as well as bioaccumula- which results in reduced endocytosis, phagocytosis and, overall, tion and toxicity lower than expected from a dose–response curve. cell motility and is associated with lowered NRRT (Cajaraville In addition, PW from gas reservoirs is thought to produce non-po- et al., 1996; Gómez-Mendikute et al., 2002). Although changes in lar narcosis and alterations in cell membrane permeability (John- NRRT are often associated to reduced lysosomal membrane stabil- sen et al., 2004). Chemicals present in a mixture at very low ity (Lowe and Pipe, 1994), they are the result of a more general re- concentrations may contribute to the narcotic activity of the mix- sponse that involves loss of integrity in overall cell membranes ture (Smith et al., 1998). Only minor effects where found in turbot (Ivanova and Uhlig 2008), not only lysosomal ones, unlike in the larvae exposed for up to 12 h to 1% PW, whereas exposure to 10% cytochemical procedure to determine LP in digestive cells that is PW depressed heart rate, gill damage and augmented body choles- strictly targeted to lysosomes (Marigómez et al., 2005). The symp- terol have been associated to PW induced narcosis (Stephens et al., toms indicative of cellular injury that are revealed by the neutral 1996). In 50 d-old turbots, exposure to 0.01% PW (North Sea oil) red assay in mussel haemocytes include a reduction in the number stimulated swimming activity (avoidance defence mechanism) of lysosomes, an increase in lysosomal volume and a reduction of whereas exposure to 0.1–1% PW provoked a stress response and the overall cell size with associated changes in cell morphology exposure to 1% PW caused a significant reduction in swimming (Moore et al., 1996). Pryor and Facher (1997) concluded that the activity, suggesting a narcotic action of the externals hydrocarbon mussel haemocytes respond to pollutants by rounding up and mixture or those accumulated internally or their metabolites

(EROD activity was induced) (Stephens et al., 2000). Finally, a weak showed an evident alteration in mussel health status at PW con- acidification (i.e., caused by the presence of low amounts of polar centrations in the range of 0.01–0.5%, which was interpreted as a compounds) may provoke metabolic alterations in mussels (Mich- pollutant induced sublethal stress response. However, no relation- aelidis et al., 2005), although no differences in the pH values of the ship was found between the biological effects and the contaminant treatment water were found. Therefore it seems that the observed concentrations measured, which was extremely marked on expo- anomalies could be the consequence of either altered exposure sure to 1% PW. It is possible that additive or synergistic toxic ef- conditions at 1% PW concentration (Azetsu-Scott et al., 2007)or fects or non-polar narcosis occurred (Smith et al., 1998; Hannam the result of a narcotic effect exerted by the complex mixture of et al., 2009), or even that other PW contaminants not measured constituents of the PW at low concentrations (Stephens et al., were responsible for the biological effects observed. Inconsisten- 1996, 2000; Smith et al., 1998), or maybe both. This is an interest- cies between biological effects and mussel tissue chemistry have ing issue that deserves further research in order to advance in the been previously reported in field studies, being attributed to either understanding of the potential biological effects of PW on mussel effects of contaminants that were not measured, to the combined health. effects of mixture toxicity resulting in a threshold effect, or to the consequences (i.e. reduced feeding activity, growth and bioaccumulation) of adaptive mechanisms or toxic effects elicited in 4.3. Integrative Biological Response (IBR/n) mussels by pollutants themselves (Brooks et al., 2009; Garmendia et al., submitted). IBR has been previously applied to fishes and mussels including different suites of biomarkers (Beliaeff and Burgeot, 2002; Broeg and Lehtonen 2006; Damiens et al. 2007; Pytharopoulou et al., References 2008; Marigómez et al., in prep.). In general terms, the results obtained in these studies and their interpretation were comparable to Aarab, N., Pampanin, D.M., Naedal, A., Øysaed, K.B., Gastaldi, L., Bechmann, R.K., 2008. Histopathological alterations and histochemistry measurements in those presently achieved. It is worth noting that IBR and IBR/n pro- mussel, Mytilus edulis collected offshore from an aluminium smelter industry duce satisfactory discrimination between sites with different (Norway). Marine Pollution Bulletin 57, 569–574. health status whatever the combination of biomarkers is. Accord- Abrahamson, A., Brandta, I., Brunströma, B., Sundt, R.C., Jørgensen, E.H., 2008. Monitoring contaminants from oil production at sea by measuring gill EROD ingly, IBR/n has successfully discriminated between PW treatments activity in Atlantic cod (Gadus morhua). Environmental Pollution 153, 169–175. in the present study. Five biochemical, histochemical and histolog- Aker Kværner, 2006. BAT assessment report Ormen Lange Project Doc. 37-1A-AK- F15-00014. pp. 124. ical biomarkers (AOX, CAT, LP, VvBAS, and NRRT) were used to cal- Azetsu-Scott, K., Yeats, P., Wohlgeschaffen, G., Dalziel, J., Niven, S., Lee, K., 2007. culate the IBR index developed by Belaieff and Burgeot (2002). Precipitation of heavy metals in produced water: Influence on contaminant Aware that different biomarker arrangements on the star plots pro- transport and toxicity. Marine Environmental Research 63, 146–167. duce different IBR/n values (Broeg and Lehtonen 2006), biomarkers Baršiene˙ , J., Rybakovas, A., Förlin, L., Šyvokiene˙ , J., 2008. Environmental genotoxicity studies in mussels and fish from the Göteborg area of the North Sea. Acta were orderly represented in the five axes of star plots according to Zoologica Lituanica 18 (4), 240–247. their biological complexity level (AOX, metabolic response; CAT, Beliaeff, B., Burgeot, T., 2002. 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