Integrated coastal monitoring of a gas processing plant using native and caged mussels

Science of the Total Environment 426 (2012) 375–386

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Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv

Integrated coastal monitoring of a gas processing plant using native and caged mussels Steven Brooks a,⁎, Christopher Harman a, Manu Soto b, Ibon Cancio b, Tormod Glette c, Ionan Marigómez b a b c

Norwegian Institute for Water Research (NIVA), Gaustadalléen 21, NO-0349 Oslo, Norway CBET Res Grp, R&D Centre for Experimental Marine Biology and Biotechnology (PIE), Univ Basque Country, Areatza Z/G, Plentzia-Bizkaia, E-48620 Basque Country, Spain Det Norske Veritas (DNV), Veritasveien 1, 1363 Høvik, Norway

a r t i c l e

i n f o

Article history: Received 30 November 2011 Received in revised form 15 March 2012 Accepted 20 March 2012 Available online 18 April 2012 Keywords: Biomarkers Process water Integrated monitoring Integrative Biological Response index Passive samplers

a b s t r a c t The biological effects of a coastal process water (PW) discharge on native and caged mussels (Mytilus edulis) were assessed. Chemical analyses of mussel tissues and semi permeable membrane devices, along with a suite of biomarkers of different levels of biological complexity were measured. These were lysosomal membrane stability in haemocytes and digestive cells; micronuclei formation in haemocytes; changes in celltype composition in the digestive gland epithelium; integrity of digestive gland tissue; peroxisome proliferation; and oxidative stress. Additionally the Integrative Biological Response (IBR/n) index was calculated. This integrative biomarker approach distinguished mussels, both native and caged, exhibiting different stress conditions not identified from the contaminant exposure. Mussels exhibiting higher stress responses were found with increased proximity to the PW discharge outlet. However, the biological effects reported could not be entirely attributed to the PW discharge based on the chemicals measured, but were likely due to either other chemicals in the discharge that were not measured, the general impact of the processing plant and or other activities in the local vicinity. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Process water (PW) is a mixture of produced water extracted along with natural gas and condensate from geological formations with additional water and process chemicals. It is a complex mixture containing thousands of different compounds mostly including trace metals, organic acids, phenols, polycyclic aromatic, aliphatic hydrocarbons (PAH) and residual production and treatment chemicals and their breakdown products (Johnsen et al., 2004; Neff, 2002; Roe Utvik, 1999). Due to improved PW treatment systems installed on both offshore and coastal oil and gas facilities, the concentrations of contaminants in PW released into the environment are often small. This is particularly the case for onshore plants where space limitations for treatment systems are not a factor, enabling discharge limits to be achieved around 10 fold lower than that for offshore platforms. Once discharged, the PW is then further diluted and dispersed in to the receiving waters, followed by volatilisation and biodegradation (Flynn et al., 1996). Consequently, the measurement of these compounds is difficult, often resulting in concentrations below detection limits. However, biological effects have frequently been reported following exposure to PW mixtures, with individual chemicals below their detection limits (Brooks et al., 2011b; Harman et al., 2009a, 2009b; Stephens et al., 1996, 2000; Zhao

⁎ Corresponding author. Tel.: +47 22185100, +47 92696421 (Mob); fax: +47 22185200. E-mail address: [email protected] (S. Brooks). 0048-9697/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.scitotenv.2012.03.059

et al., 2008). In these cases, the toxicity of the mixture and not the individual compounds was believed to be responsible for the biological effects observed. The Ormen Lange gas processing plant is situated on the island of Gossa, on the West coast of Norway. The plant processes water, gas and condensate received by pipeline from a gas field approximately 120 km offshore in the North Sea. The processing plant contains both a biological treatment system and macro porous polymer extract technology, which together can remove up to 99% of the dissolved and dispersed hydrocarbons (Aker Kværner, 2006). This includes most aliphatic hydrocarbons, volatiles such as benzene, toluene, ethlbenze and xylenes (BTEX), PAHs and their alkylated homologues, as well as some polar compounds such as alkyl phenols to a lesser extent (Aker Kværner, 2006). Previously, biological effect monitoring of the Ormen Lange PW has confirmed that it is comprised of extremely low concentrations of a variety of compounds that are difficult to detect chemically but appear to cause biological effects in exposed mussels (Brooks et al., 2011b). The main objective of the present study was to evaluate the potential biological effects of the Ormen Lange PW using native and caged mussels, Mytilus edulis. The combined use of native and caged mussels is useful to assess chronic pollution restraining the extent to which genetic variability and impending adaptive mechanisms against chronic environmental insult may affect biological response (Gomiero et al., 2011; Gorbi et al., 2008; Nigro et al., 2006). In addition, semi-permeable membrane devices (SPMDs) have also

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been used to support the biological effect measurements (Brooks et al., 2011b; Huckins et al., 1990, 2006). Using a combination of mussels and SPMDs is a valuable tool for estimating the fate and impact of chemical contaminants in PW that are discharged to the ocean. The health status of the mussels was evaluated using an integrated suite of biomarkers. The selected biomarker measurements can be directly related to the amount of environmental stress imposed through a variety of factors including organic compounds typically found in produced water. This approach will provide information on the potential toxicity of the receiving waters within the vicinity of the PW discharge. Due to the complexity of chemicals present in PW, which can induce a wide range of biological effects, the multiple biomarker approach has been found to be most effective (Brooks et al., 2009; Cajaraville et al., 2000; Garmendia et al., 2011a, 2011b, 2011c; Hylland et al., 2008). In the present study, the biomarkers measured included: 1) peroxisome proliferation measured as palmitoyl-CoA oxidase (AOX) activity (Fahimi and Cajaraville, 1995); 2) oxidative stress through catalase (CAT) activity measurements in the digestive gland (Eertman et al., 1995); 3) micronuclei (MN) formation as a measure of genetic damage (Baršienė and Andreikėnaitė, 2007); 4) the stability of the lysosomal membrane in both haemocytes (Neutral red retention, NRR; ICES, 2004; Lowe and Pipe, 1994) and digestive cells (LMS(d); Marigómez et al., 2005; UNEP/RAMOGE, 1999); 5) changes in the cell composition in the digestive gland epithelium in terms of volume density of basophilic cells (VvBAS; Marigómez et al., 2002; Soto et al., 2002); and 6) integrity of the digestive gland tissue through the connective

tissue to diverticula ratio (CTD; Brooks et al., 2011b; Garmendia et al., 2011b). The biomarkers were integrated using the Integrative Biological Response (IBR/n) Index (Beliaeff and Burgeot, 2002; Broeg and Lehtonen, 2006). IBR has been previously applied to fish and mussels including different suites of biomarkers (Baussant et al., 2009; Beliaeff and Burgeot, 2002; Broeg and Lehtonen, 2006; Brooks et al., 2011b; Damiens et al., 2007; Pytharopoulou et al., 2008; Raftopoulou and Dimitriadis, 2010). 2. Material and methods 2.1. Sample collection and processing 2.1.1. Native mussels With the aid of a small boat, M. edulis were collected in the first week of September from three sites in and around the processing plant (Fig. 1). The sites included a reference location positioned upstream from the PW discharge point (S3) and two exposure sites within the bay area, approximately 100–200 m from the PW discharge (S2 & S3). Mussels (3–5 cm length) were collected from below the water line at each site and stored in pre-chilled cooler boxes containing seawaterdampened paper towels. 2.1.2. Caged mussels Mussels were collected from a local shellfish supplier in Rissa, Norway and transported on ice to the field stations within 12 h. Mussels between 3.5 and 4.5 cm length were selected. A total of 80

500 m

6 5

100 m

4 3

Discharge outlet

1 2

S2 S3 S1 Ormen Lange

Fig. 1. The positions of the mussel cages (1–6 yellow dot) and shore mussels (S1-S3 red dot) with respect to the discharge outlet of the gas processing plant. Distances of the cages from the discharge point were as follows 50, 100, 110, 200, 1900 and 2000 m for Cages 1–6 respectively.

S. Brooks et al. / Science of the Total Environment 426 (2012) 375–386

mussels were placed in elasticated nylon mesh bags. The mesh bags were divided into 8 compartments holding 10 mussels per compartment to ensure that all mussels could attach freely to the nylon mesh and were equally exposed to the surrounding waters. The mussels were held at a depth of 25–30 m at all sites. A total of six sites were used (50 m, two at 100 m, and 200 m away from discharge point as well as two reference sites, Ref 1 and Ref 2; Fig. 1), and cages maintained for 6 weeks before sample collection. SPMDs were wound around stainless steel deployment spiders and held within a stainless steel mesh cage (Environmental Sampling Technologies, Saint Joseph, USA), which was secured to the rope directly below the mussels. Three replicates per cage were used. SPMDs were spiked with a mixture of deuterated PAH as performance reference compounds (PRCs) to allow for the determination of sampling rates in situ (Booij et al., 1998; Huckins et al., 2002) and were obtained from ExposMeter (Tavelsjo, Sweden). 2.1.3. Sample processing All mussels, both native and caged, were taken to the field laboratory and processed within 4 h of collection from the shore. Haemolymph samples were taken from the posterior adductor muscle of individual mussels and assessed for NRR and MN formation. Digestive gland and gonad tissue were removed from individual mussels and preserved by either snap freezing in liquid nitrogen or submersion in formalin. Digestive gland samples were used to measure lysosomal stability (LMS(d)), enzyme activity (i.e. AOX, CAT) and histological alterations (VvBAS, CTD). In addition, whole soft tissue samples were collected for the analysis of organic and metal contaminant concentrations. 2.2. Chemical analyses 2.2.1. Pollutant tissue burdens in mussels For each treatment group, triplicate mussel samples were taken for analysis of selected metals and PAHs, including alkylated homologues of naphthalene, phenanthrene and dibenzothiophene (NPD). Five whole mussels per sample were removed from their shells and placed in pyrolysed (560 °C) glass containers. The mussels were frozen and transported to NIVA on dry ice. All samples were stored at −20 °C until analyses. Samples were defrosted, homogenised and a sub sample taken of approximately 5 g. Internal standards were added before extraction by saponification. Analytes were then extracted twice with 40 mL cyclohexane and dried over sodium sulphate. The extracts were reduced by a gentle stream of nitrogen and cleaned by gel permeation chromatography (GPC) using the system described previously (Harman et al., 2008). Analysis proceeded by gas chromatography with mass spectrometric detection (GC–MS) with the MS detector operating in selected ion monitoring mode (SIM). The GC was equipped with a 30 m column with a stationary phase of 5% phenyl polysiloxane (0.25 mm i.d. and 0.25 μm film thickness), and the injector operated in splitless mode. The initial column temperature was 60 °C, which after 2 min was raised stepwise to 310 °C. The carrier gas was helium and the column flow rate was 1.2 mL/min. Quantification of individual components was performed by using the internal standard method. The alkylated homologues were quantified by baseline integration of the established chromatographic pattern and the response factors were assumed equal within each group of homologues. Recoveries of PAH from mussel tissue ranged between 70 and 120%. 2.2.2. Semipermeable membrane devices (SPMDs) The exterior of the SMPDs was wiped clean before extraction by dialysis with 2 × 150 mL hexane (Huckins et al., 1990). Clean up by GPC and analysis for PAHs and NPDs by GC–MS, proceeded as described above for mussel samples. Quantification of individual components was performed by using the relative response of internal standards. In order to correct for any possible contamination during

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study procedures, control or ‘blank’ SPMDs were used. These included field controls (FCs) that were exposed to the air during deployment and retrieval and laboratory controls (LCs) that follow exposure to solvents, glassware etc. during sample work up. At least one of each type of control was used per 10 exposed samplers. Initial (time zero) concentrations of PRCs were also established from LCs. An empirical model, described in detail by Huckins et al. (2006), was used in the calculation of water concentrations from SPMD accumulations. In this model compound specific effects on uptake are adjusted based on the log Kow of the analyte and site-specific factors arising from differences in environmental variables are adjusted by using the PRC data. In this way the uptake for each individual compound at each sampling station was established, expressed as a sampling rate (L/d). Where individual analytes were not detected in SPMDs then the analytical detection limit was used in calculations to provide a maximum theoretical concentration in the water, see Harman et al. (2009b) for more details. 2.3. Biomarkers 2.3.1. Haemolymph analysis 2.3.1.1. Lysosomal membrane stability in haemocytes. The integrity of lysosomal membranes of mussel haemocytes was determined using the Neutral Red Retention (NRR) procedure adapted from Lowe and Pipe (1994). Approximately 0.1 mL of haemolymph was removed from the adductor muscle of the mussel with a syringe containing approximately 0.1 mL physiological saline. The haemolymph/saline solution was placed in a microcentrifuge tube, from which a 40 μL sample was removed and pipetted onto the centre of a microscope slide. The slide was left in a dark 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 40 μL of neutral red solution added (Sigma). The neutral red solution was taken up inside the haemocytes and stored within lysosomes. The ability of the lysosomes to retain the neutral red solution was checked every 15 min by light microscopy (×40). The test was terminated and the time recorded when greater than 50% of the haemocytes leaked the neutral red dye into the cytosol. 2.3.1.2. Micronuclei formation in mussel haemocytes (MN). Approximately 0.1 mL of haemolymph was removed from the posterior adductor muscle of each mussel with a hypodermic syringe containing 0.1 mL PBS buffer (100 mM PBS, 10 mM EDTA). The haemolymph and PBS buffer were mixed briefly in the syringe and placed on a microscope slide. The slide was then placed in a humid chamber for 15 min to enable the haemocytes to adhere to the slides. Excess fluid was drained and the adhered haemocytes were fixed in 1% glutaraldehyde for 5 min. Following fixation, the slides were gently rinsed in PBS buffer and left to air-dry overnight. The dried slides were brought back to the laboratory for further processing. Slides were stained with 1 μg/mL bisbenzimide 33258 (Hoechst) solution for 5 min, rinsed with distilled water and mounted in glycerol McIlvaine buffer (1:1). The frequency of micronuclei formation was measured on coded slides without knowledge of the exposure status of the samples to eliminate bias (Baršienė and Andreikėnaitė, 2007). The frequency of micronuclei in haemocytes was determined microscopically at 100× objective (final magnification ~1000×). A total of 2000 cells (200 cells× 10 mussels) were examined for each experimental group of mussels. Only cells with intact cellular and nuclear membranes were scored. 2.3.2. Enzyme activities in digestive gland Frozen digestive glands were individually homogenised in a Braun-Potter homogeniser using TVBE buffer (1 mM sodium bicarbonate, 1 mM EDTA, 0.1% ethanol and 0.01% Triton X-100; pH = 7.6). After homogenisation, samples were centrifuged at 500 g for 15 min.

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Supernatants were removed and diluted appropriately to perform the enzyme assays. Total protein of all samples was measured using the DC protein assay (BioRad, California) based on the Lowry method and using a γ-globulin as standard. 2.3.2.1. Catalase (CAT) activity. The activity of the antioxidant enzyme catalase was measured basically as described by Porte et al. (1991) in frozen digestive gland samples (n = 10 mussels). CAT activity was determined in 5 pools (2 digestive glands per pool) per experimental group, by measuring the consumption of H2O2 at 240 nm (ext. coeff. 40 M − 1 cm − 1) using H2O2 50 mM as substrate in potassium phosphate buffer 80 mM (pH = 7). 2.3.2.2. Palmitoyl-CoA oxidase (AOX) activity. Peroxisomal palmitoylCoA oxidase (AOX) activity was determined spectrophotometrically (λ = 502 nm), in the same 5 pools per experimental group as used to measure CAT activity, measuring the H2O2 dependent oxidation of dichlorofluorescein diacetate (Molecular Probes, Eugene, Oregon, USA) catalysed by an exogenous peroxidase, using 30 μM palmitoylCoA as substrate, according to Small et al. (1985). 2.3.3. Digestive gland cytochemistry Ten serial sections (10 μm thick) of frozen digestive gland (5 mussels per treatment) were cut in a Leica CM 3000 cryotome onto successive serial slides and stored at −40 °C until processing. 2.3.3.1. Lysosomal membrane stability (LMS(d)). The determination of LMS was based on the time of acid labilisation treatment required to produce the maximum staining intensity according to UNEP/ RAMOGE (1999), after demonstration of hexosaminidase (Hex) activity in digestive cell lysosomes. Eight serial cryotome sections (10 μm) were subjected to acid labilisation in intervals of 0, 3, 5, 10, 15, 20, 30 and 40 min in 0.1 M citrate buffer (pH 4.5 containing 2.5% NaCl) in a shaking water bath at 37 °C, in order to find out the range of pre-treatment time needed to completely labilise the lysosomal membrane. Following this treatment, sections were transferred to the substrate incubation medium for the demonstration of Hex activity. The incubation medium consisted of 20 mg naphthol AS-BI-N-acetylβ-D glucosaminide (Sigma, N 4006) dissolved in 2.5 mL 2methoxyethanol (Merck, 859), and made up to 50 mL with 0.1 M citrate buffer (pH 4.5) containing 2.5% NaCl and 3.5 g low viscosity polypeptide (Sigma, P5115) to act as a section stabiliser. Sections were incubated in this medium for 20 min at 37 °C, rinsed in a saline solution (3.0% NaCl) at 37 °C for 2 min and then transferred to 0.1 M phosphate buffer (pH 7.4) containing 1 mg/mL diazonium dye Fast Violet B salt (Sigma, F1631), at RT for 10 min. Slides were then rapidly rinsed in running tap water for 5 min, fixed for 10 min in Baker's formol calcium containing 2.5% NaCl at 4 °C and rinsed in distilled water. Finally, slides were mounted in Kaiser's glycerine gelatine and sealed with nail varnish. The time of acid labilisation treatment required to produce the maximum staining intensity was assessed under the light microscope as the maximal accumulation of reaction product associated with lysosomes (UNEP/RAMOGE, 1999). Four determinations were made for each animal by dividing each section in the acid labilisation sequence into 4 approximately equal segments and assessing the labilisation period in each of the corresponding set of segments. The mean value was then derived for each section, corresponding to an individual digestive gland. 2.3.4. Digestive gland histology Histological sections (7 μm) were cut using a rotary microtome and stained with haematoxylin-eosin (H/E). Prevalence of parasites, haemocyte infiltration and general condition of the digestive epithelium, and the interstitial connective tissue were systematically recorded.

2.3.4.1. Epithelial cell-type composition (VvBAS) and tissue integrity in digestive gland (CTD ratio). The volume density of basophilic cells (VvBAS) was quantified by means of stereology as an indication of whether changes in cell-type composition occurred or not (Soto et al., 2002). Likewise, the integrity of the digestive gland tissue was simultaneously determined as the extent of the interstitial connective tissue relative to the space occupied by digestive diverticula (connective-to-diverticula (CTD) ratio) (Brooks et al., 2011b; Garmendia et al., 2011b). Counts were made in one randomly selected field in one digestive gland slide per mussel (10 mussels per sample). Slides were viewed at 40× objective (final magnification ~400×) using a drawing tube attached to a light microscope. A simplified version of the Weibel graticule multipurpose 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 calculated as CTD = c / (b + d + l). VvBAS was calculated according to the Delesse's principle (Weibel, 1979), as VvBAS = VBAS/VEP, where VBAS is the volume of basophilic cells and VEP the volume of digestive gland epithelium. 2.3.5. Integrative Biological Response index The Integrative Biological Response (IBR) index was developed by Beliaeff and Burgeot (2002) in order to integrate biochemical, genotoxicity and histochemical biomarkers. The IBR was modified to IBR/n, which took into account the number of biomarkers (Broeg and Lehtonen, 2006). Presently, CAT, MN, LP, CTD and NRR were used to calculate the IBR/n. The inverse values of LP and NRR were used since a decrease was reflective of adverse impact. For CAT, previous research has identified a reduction in CAT activity in mussels following long term exposure to chronically polluted areas as a result of adaptation (Regoli and Principato, 1995). Since a similar adaptation was considered to have occurred in the present study, the inverse of the values for CAT were used in the IBR/n calculation. The calculation method is based on relative differences between the biomarkers in each given data set. For a more detailed explanation see Beliaeff and Burgeot (2002) and Broeg and Lehtonen (2006). 2.4. Statistical analysis Homogeneity of variance was checked using the Levene's test, and where necessary, variables were log transformed to obtain homogeneity. The data were then subjected to analysis of variance (ANOVA) with a Tukey post-hoc test, p b 0.05. If homogeneity was not possible a Kruskal–Wallis non-parametric test was used to compare means (Sokal and Rohlf, 1981). 3. Results Low or undetected concentrations of PAHs and metals were measured in the whole tissue homogenates of native mussels collected from the three sampling sites (Table 1). However, a small PAH signature was found at Site 1 compared to the other two sites, suggesting exposure to low levels of PAHs such as phenanthrene and fluoranthene. Likewise, the tissue concentration of PAHs and metals in caged mussels was low or near the detection limits (Table 2). Based on the chemical analysis of SPMDs, it was observed that PAH concentrations were either low or undetected in all the samples (Table 3). 3.1. Native mussels The results of the biomarker analysis performed in native mussels are shown in Fig. 2. Despite an apparent reduction in AOX activity at Site 2 in comparison with Site 1, no significant difference between mean values was found (Fig. 2A), which can be attributed to the large intravariability observed. Elevated levels of CAT activity were measured in mussels from Site 1 compared to that measured in mussels from Site 2

S. Brooks et al. / Science of the Total Environment 426 (2012) 375–386 Table 1 PAH and metal concentrations in whole mussel homogenates of native shore mussels collected from the three sampling sites. Ten mussels were pooled per sample, data presented in μg/kg w.w. for PAH and mg/kg w.w for metals (mean ± SD, n = 3). Compound

Site 1

Site 2

Site 3

Naphthalene Acenaphthylene Acenaphthene Fluorene Phenanthrene Anthracene Fluoranthene Pyrene Benzo(a)anthracene Chrysene Benzo(b)fluoranthene Benzo(k)fluoranthene Benzo(a)pyrene Dibenz(a,h)anthracene Benzo(g,h,i)perylene Indeno(1,2,3-cd)pyrene Sum PAH16 Cadmium Copper Mercury Lead Zinc Lipid %

b4.5 b1.0 b1.0 1.23 ± 0.25 3.67 ± 0.46 1.67 ± 0.23 4.37 ± 0.58 2.07 ± 0.25 b1.0 1.1 b1.0 b1.1 b1.2 b1.3 b1.4 b1.5 13.3 ± 2.0 0.11 ± 0.02 0.93 ± 0.07 0.009 ± 0.001 0.057 ± 0.006 18.1 ± 2.14 1.6 ± 0.17

b 4.5 b 1.0 b 1.0 b 1.0 1.33 ± 0.21 b 1.0 1.15 ± 0.21 b 1.0 b 1.0 b 1.0 b 1.0 b 1.0 b 1.0 b 1.0 b 1.0 b 1.0 2.1 ± 0.87 0.12 ± 0.02 0.89 ± 0.09 0.010 ± 0.001 0.06 14.2 ± 1.16 1.43 ± 0.15

b4.5 b1.0 b1.0 b1.0 1.35 ± 0.35 b1.0 1.1 b1.0 b1.0 b1.0 b1.0 b1.0 b1.0 b1.0 b1.0 b1.0 1.9 ± 1.13 0.14 ± 0.01 1.09 ± 0.09 0.009 ± 0.001 0.06 ± 0.006 18.8 ± 1.57 1.6 ± 0.15

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(ANOVA, Tukey, p > 0.05). Lower CAT levels were also observed in mussels from Site 3, although due to higher variability no significant difference to Site 1 was found (Fig. 2B). Although slightly higher incidence of MN was formed in mussels from Site 3 compared to the other two sites, no statistically significant differences were found (Fig. 2C, ANOVA, Tukey, p > 0.05). The labilisation period (LP) of lysosomes from the digestive cells was significantly lower in mussels from the two exposure sites compared to Site 1 (Fig. 2D, ANOVA, Tukey, p > 0.05). VvBAS values recorded in mussels from Site 3 were significantly higher than those recorded in mussels from Site 1 (Fig. 2E). VvBAS values in Site 2 were higher but very variable and thus were not significantly different from those recorded in Site 1. Significantly elevated CTD ratio was found in mussels from Sites 2 (0.29 ± 0.095) and 3 (0.31 ± 0.09) compared to the reference Site 1 (0.158 ± 0.058) (ANOVA, Tukey, p >0.05; Fig. 2F). Significantly shorter NRR of lysosomes of haemocytes were observed in mussels collected from Sites 2 and 3 compared to Site 1 (ANOVA, Tukey, p b 0.05, Fig. 2G). The digestive gland tissue presented a normal histological integrity in the 3 sites with a well organised interstitial connective tissue, apparently unaltered epithelia in stomach and digestive gland and food material being processed in the mid-gut lumen (Fig. 3A–C). Overall, no significant parasitic infestation or pathological lesions were found. However, moderate thinning and vacuolisation of the digestive gland epithelium as well as an apparent reduction in the

Table 2 PAH and metal concentrations in whole mussel homogenates of caged mussels positioned for 6 weeks at varying distances from the PW discharge outfall. Ten mussels were pooled per sample, data presented in μg/kg w.w. for PAH and mg/kg w.w for metals (mean ± SD, n = 3). Compound

50 m

100 m

110 m

200 m

1900 m (Ref 2)

2000 m (Ref 1)

Naphthalene C1-Naphthalenes C2-Naphthalenes C3-Naphthalenes Phenanthrene C1-Phenanthrenes C2-Phenanthrenes C3-Phenanthrenes Dibenzothiophene C1-Dibenzothiophenes C2-Dibenzothiophenes C3-Dibenzothiophenes Sum NPD Acenaphthylene Acenaphthene Fluorene Anthracene Fluoranthene Pyrene Benzo(a)anthracenes Chrysene Benzo(b)fluoranthene Benzo(k)fluoranthene Benzo(e)pyrene Benzo(a)pyrene Perylene Indeno(1,2,3-cd)pyrene Dibenz(a,h)anthracene Benzo(g,h,i)perylene Sum PAH Sum PAH16 Ag Al As Cd Cr Cu Fe Hg Ni Pb Zn

b0.8 b2 b2 b2 0.81 ± 0.12 b2 3.0 ± 0.85 3.5 b0.5 b2 b2 b2 b21.17 ± 1.86 b0.5 b0.5 b0.5 b0.5 0.77 ± 0.2 b0.5 b0.5 b0.5 b0.5 b0.5 b0.5 b0.5 0.65 b0.5 b0.5 b0.5 b29.57 ± 1.86 b8.68 ± 0.4 0.02 ± 0.01 3.67 ± 1.01 2.7 ± 0.16 0.16 ± 0.02 0.17 ± 0.06 1.41 ± 0.11 11.33 ± 1.15 0.007 0.14 ± 0.02 0.04 ± 0.01 13.27 ± 1.31

b0.8 b2 2.2 2.7 ± 0.85 0.90 ± 0.02 2.5 3.1 ± 0.53 3.63 ± 1.23 b0.5 b2 b2 b2 b23.63 ± 2.15 b0.5 b0.5 b0.5 b0.5 0.64 ± 0.13 b0.5 b0.5 b0.5 b0.5 b0.5 b0.5 b0.5 b0.5 b0.5 b0.5 b0.5 b31.77 ± 2.26 b8.84 ± 0.15 0.011 3.77 ± 0.76 2.71 ± 0.07 0.15 ± 0.01 0.2 ± 0.10 1.29 ± 0.22 11.67 ± 1.53 0.007 0.14 ± 0.01 0.04 ± 0.01 13.9 ± 2.84

b0.8 b2 2.5 ± 0.42 3.2 ± 0.57 0.89 ± 0.08 b2 3.3 ± 0.42 3 b0.5 b2 b2 b2 b22.37 ± 2.31 b0.5 b0.5 b0.5 b0.5 0.78 ± 0.15 b0.5 b0.5 b0.5 b0.5 b0.5 b0.5 b0.5 b0.5 b0.5 b0.5 b0.5 b30.56 ± 2.5 b8.72 ± 0.37 0.014 3.97 ± 1.01 2.91 ± 0.09 0.15 ± 0.01 0.13 ± 0.06 1.42 ± 0.14 11.67 ± 0.58 0.007 0.12 ± 0.02 0.04 15.23 ± 3.95

b 0.8 b2 b2 2.45 ± 0.35 0.84 ± 0.04 b2 3.0 ± 0.36 b2 b 0.5 b2 b2 b2 b 21.44 ± 0.73 b 0.5 b 0.5 b 0.5 b 0.5 0.87 ± 0.09 b 0.5 b 0.5 b 0.5 b 0.5 b 0.5 b 0.5 b 0.5 b 0.5 b 0.5 b 0.5 b 0.5 b 29.81 ± 0.81 b 9.01 ± 0.12 0.017666667 3.27 ± 0.12 2.75 ± 0.14 0.14 ± 0.01 0.17 ± 0.06 1.26 ± 0.35 11.33 ± 0.58 0.007 0.12 ± 0.02 0.04 0.01 13.73 ± 0.85

b 0.8 b2 2.4 2.93 ± 0.64 0.97 ± 0.11 b2 3.5 ± 0.4 3.1 b 0.5 b2 b2 3.3 b 23.67 ± 1.59 b 0.5 b 0.5 b 0.5 b 0.5 0.96 ± 0.12 b 0.5 b 0.5 b 0.5 0.57 b 0.5 b 0.5 b 0.5 b 0.5 b 0.5 b 0.5 b 0.5 b 32.16 ± 1.54 b 9.26 ± 0.21 0.015 3.6 ± 0.66 2.91 ± 0.06 0.16 ± 0.01 0.1 1.79 ± 0.41 11.0 ± 1.0 0.008 0.12 ± 0.01 0.04 ± 0.01 13.27 ± 0.93

b 0.8 2.6 3.05 ± 1.34 3.0 ± 0.95 0.89 ± 0.07 b2 2.9 ± 0.36 b2 b 0.5 b2 b2 2.1 b 23.02 ± 1.31 b 0.5 b 0.5 b 0.5 b 0.5 0.7 ± 0.03 b 0.5 b 0.5 b 0.5 0.5 b 0.5 b 0.5 b 0.5 b 0.5 b 0.5 b 0.5 b 0.5 b 31.22 ± 1.32 b 8.89 ± 0.04 0.011333333 3.13 ± 0.32 2.88 ± 0.08 0.15 ± 0.02 0.17 ± 0.06 1.47 ± 0.26 11.33 ± 0.58 0.007 0.13 ± 0.02 0.04 ± 0.01 14.4 ± 1.64

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Table 3 PAH concentrations measured in SPMDs that were placed for 6 weeks in the water column at varying distances from the PW discharge outlet. Data presented as mean ± SD, n = 3 (ng/L), a = high blank values prevent reporting of this compound. Compound

50 m

100 m

110 m

200 m

1900 m (Ref 2)

2000 m (Ref 1)

Naphthalene Acenaphthylene Acenaphthene Fluorene Dibenzothiophene Phenanthrene Anthracene Fluoranthene Pyrene Benz[a]anthracene Chrysene Benzo[b.j]fluoranthene Benzo[k]fluoranthene Benzo[e]pyrene Benzo[a]pyrene Perylene Indeno[1,2,3-cd]pyrene Dibenzo[ac/ah]anthracene Benzo[g,h,I]perylene SUM PAH PAH EPA16

a b 0.07 b 0.19 0.26 ± 0.01 0.08 0.94 ± 0.04 b 0.03 0.15 ± 0.01 0.05 b 0.02 b 0.02 b 0.02 b 0.02 b 0.02 b 0.02 0.04 0.07 0.03 0.05 b 2.06 ± 0.08 b 1.93 ± 0.07

a b0.07 b0.19 0.23 ± 0.06 0.07 ± 0.02 0.89 ± 0.26 b0.03 0.14 ± 0.02 0.05 b0.02 b0.02 b0.02 b0.02 b0.02 b0.02 0.07 b0.02 b0.02 b0.02 b1.91 ± 0.36 b1.78 ± 0.35

a b0.07 b0.19 0.25 ± 0.08 0.07 ± 0.02 0.87 ± 0.25 0.04 0.14 ± 0.02 0.053 ± 0.01 b0.02 b0.02 b0.02 b0.02 b0.02 b0.02 b0.02 b0.02 b0.02 b0.02 b1.92 ± 0.43 b1.81 ± 0.40

a b 0.07 b 0.18 0.12 ± 0.02 0.05 ± 0.01 0.61 ± 0.16 b 0.03 0.14 ± 0.01 0.043 ± 0.01 b 0.02 b 0.02 b 0.02 b 0.02 b 0.02 b 0.02 b 0.02 b 0.02 b 0.02 b 0.02 b 1.43 ± 0.22 b 1.35 ± 0.21

a b 0.07 b 0.19 0.15 0.06 0.82 ± 0.1 b 0.03 0.28 ± 0.02 0.093 ± 0.01 b 0.02 0.033 ± 0.01 0.03 b 0.02 b 0.02 b 0.02 b 0.02 0.04 b 0.02 0.03 b 1.92 ± 0.16 b 1.82 ± 0.15

a b 0.07 b 0.18 0.12 ± 0.03 0.06 ± 0.01 0.75 ± 0.11 b 0.03 0.25 ± 0.03 0.08 ± 0.01 b 0.02 0.025 ± 0.01 0.023 ± 0.01 b 0.02 b 0.02 b 0.02 0.04 0.03 b 0.02 b 0.03 b 1.77 ± 0.22 b 1.67 ± 0.21

size of the digestive alveoli were recorded in all the mussels from Sites 2 and 3 (Fig. 3B and C). The IBR/n index was calculated from star plots of normalised biomarker data from, CAT, MN, LP, CTD and NRR (Fig. 4). The IBR/n was close to zero at site 1, with only MN showing any response. For mussels at Site 2, CAT, LP, CTD and NRR showed almost equal contribution to the IBR/n (0.95). The highest IBR/n value was obtained at site 3 (1.43) with all five biomarkers contributing almost equally.

digestive alveoli beyond that found in native mussels from Sites 2 and 3 (Fig. 3B and C). The IBR/n index was calculated from star plots of normalised biomarker data from, CAT, MN, LP, CTD and NRR (Fig. 6). The IBR/n was close to zero at the reference stations increasing with increased proximity to the discharge outlet. Highest IBR/n of approximately 3.5 was found in mussels 50 m from the discharge with almost equal contributions shown by CAT, MN, LP, CTD and NRR. 4. Discussion

3.2. Caged mussels It should be noted that 20% mortality was recorded in the caged mussels after their 6 weeks exposure. A significant reduction in NRR was found in mussels caged 50 m and 100 m from the discharge compared to the reference sites at 1900 m and 2000 m (ANOVA, Tukey p b 0.05; Fig. 5A). The frequency of MN was higher at the closest site to the PW discharge (50 m), although no significant differences between the groups were found (Fig. 5B). LP values were significantly higher in mussels from the station furthest from the discharge (2000 m) compared to all other groups (ANOVA, Tukey p b 0.05; Fig. 5C), although significantly lower LP values (b6 min) were recorded in mussels caged 50 m and 200 m from the discharge. VvBAS values were above 0.10 μm 3/μm 3 in all caged mussels, with no significant difference between the groups (Fig. 5D). Significant differences were found in the CTD ratio with a maximum value in mussels caged at 50 m from the PW discharge point and lowest in those caged at 200 m (ANOVA, Tukey p b 0.05; Fig. 5E). AOX activities at one of the reference stations, 1900 m from the discharge, were significantly higher than all other stations (ANOVA, Tukey p b 0.05). The other stations had very low AOX activities (b0.5; Fig. 5F). CAT activity was lowest in mussels 100 m from the discharge outlet, which was significantly lower than CAT activity in mussels 1900 m away (ANOVA, Tukey p b 0.05; Fig. 5B). In general terms, the digestive gland tissue of mussels caged 1900 m from discharge and 2000 m from discharge (Fig. 3D and E) exhibited a histological integrity similar to that observed in the native shore mussels from Site 1 (Fig. 3A). In contrast, mussels held in cages at 50 m (Fig. 3F), had alterations in digestive gland tissue that included a certain reduction in the extent of the diverticular mass, disorganisation of the ICT and thinning of the epithelium in the

The overall aim of the monitoring study was to determine the biological effects of the Ormen Lange PW discharge on the local marine environment using native (long-term response) and caged (short-term response) mussels in combination. Caged mussels have been successfully used in exposure studies to offshore PW discharges where they have been shown to bioaccumulate PAH compounds and show sensitive biological responses, which have led to their continued use in the Norwegian offshore water column monitoring programme (Hylland et al., 2008; Brooks et al., 2011a). However, despite the clear differences in many of the biological endpoints measured, discussed below, the mussel body burden data (PAH, metals) did not show any differences between the sites or between cages and were either low or undetected in all cases. Due to the treatment system installed at Ormen Lange (biological treatment and MPPE) the concentration of chemicals released to the environment is low and typical oil related compounds are almost undetected in the receiving waters using chemical methods (Brooks et al., 2011b). It is also conceivable that the changes in biological endpoints were not caused by either elevated concentrations of PAHs or metals, but rather other contaminants that were not measured in this study. Monitoring the environmental effects of a complex mixture at such low concentrations in the complexity of the field may not be effective using chemical methods. However, as shown in a previous study (Brooks et al., 2011b) the application of a suite of sensitive and integrative biomarker endpoints is able to clearly differentiate between groups of healthy and stressed mussels. 4.1. Biomarkers in native mussels The biological effects data have shown clear differences in the overall health status of the native mussels from the three sites, with

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Fig. 2. Measured biomarker responses in native mussels collected from the three coastal sites (mean ± SE (box), ± SD (outer line). A) Neutral red retention (NRR), n = 20; B) micronuclei (MN) formation, n = 10; C) Lysosomal membrane stability (LMS) measured as labilisation period (LP), n = 5; D) Volume of basophilic cells (VvBAS), n = 10; E) Connective to diverticula tissue (CTD) ratio, n = 5; F) Peroxisome proliferation measured as palmitoyl-CoA oxidase (AOX) activity, n = 5; G) Catalase (CAT) activity, n = 5. Groups labelled with a different letter are significantly different from each other (ANOVA, Tukey p b 0.05).

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Fig. 3. H/E stained sections of the digestive gland tissue of mussels from Site 1 (A), Site 2 (B) and Site 3 (C) and mussels caged at Ref 1 (Cage 5) (D), Ref 2 (Cage 6) (E) and 50 m (Cage 1) (F). S, stomach; D, digestive duct; ICT, interstitial connective tissue; arrowheads basophilic cells. Scale bars = 50 μm.

mussels from Sites 2 and 3 showing evidence of impaired health compared to the reference mussels from Site 1. The Sites 2 and 3 were the exposure sites that were located in the vicinity of the PW outlet. The direction of the main water currents within the bay has been previously identified as moving in a predominantly North-West direction out to sea (DNV report, 2008), although some circulatory current exists within the Ormen Lange bay. As a result exposure of the mussels from Sites 2 and 3 to the PW, and the chemicals within, would be expected. Although AOX activity induction and MN formation in mussels are well regarded as particularly sensitive endpoints to chronic pollution in environmental monitoring (Orbea and Cajaraville, 2006; Burgeot et al., 1996; Baršienė et al., 2008; Brooks et al., 2011a), these biomarkers were not able to differentiate between the 3 sites. In contrast, there was very good agreement between other biomarkers. Membrane stability measured in lysosomes of digestive cells revealed a significant reduction in the health status of mussels from Sites 2 and 3 compared to the reference group (Site 1). LP values over 20 min indicate a healthy condition; whereas LP values lower than 10 min indicate a severe stress situation (Viarengo et al., 2000). The native mussels from Sites 2 and 3 with LPs of around 5 min were characterised as severely stressed, whilst Site 1 mussels with an LP of around 15 min were exhibiting some stress. This conclusion was also supported by the pathological assessment of the digestive gland tissue, where increased thinning and vacuolisation of the digestive

gland epithelium as well as reduction in the size of the digestive alveoli were found to occur in mussels from Sites 2 and 3. Furthermore, VvBAS values higher than 0.12 μm 3/μm 3 were found in mussels from Sites 2 and 3, which were indicative of poor health whereas mussels from the reference site (Site 1) were considered healthy with a VvBAS b 0.10 μm 3/μm 3 (Marigómez et al., 2006). Overall, digestive cell loss, measured as the volume density of basophilic cells (VvBAS) in the digestive gland is considered a sensitive indicator of general stress (Zaldibar et al., 2007). VvBAS values below 0.10 μm 3/μm 3 indicate a healthy condition; whereas VvBAS values higher than 0.12 μm 3/μm 3 indicate a stress situation (Marigómez et al., 2006). The CTD ratio indicated an apparent reduction in the integrity of the digestive gland tissue in mussels from Sites 2 and 3 compared to the reference site (Site 1), in agreement with LP values. Moreover, membrane stability in haemocyte lysosomes exhibited the same trend. Threshold levels for NRR have been established, with mussels recording a NRR >120 min considered as healthy, between 120 and 50 min stressed but compensating, and severely stressed and probably exhibiting pathology if b50 min (OSPAR Commission, 2007). With this in mind, the exposed mussels at Sites 2 and 3 were considered severely stressed, whilst the reference mussels from Site 1 were experiencing some stress but compensating. However, the current view amongst experts (e.g. OSPAR and ICES), based on increasing field data, is that the NRR assessment criteria of 120 and 50 have been set too high and may require adjustment.

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Fig. 4. Integrative Biomarker response (IBR) in native mussels collected from the three coastal sites and histogram of the IBR/n calculated from areas connecting biomarker responsiveness in the star plots.

Although clear differences in health status were found between the reference mussels and those collected from the two sites within the Ormen Lange bay area, the mussels from the reference site were found to exhibit a low level stress response, identified from the LMS of both haemocytes and digestive cells. Nevertheless, the reference values for LMS in the digestive gland, which was used in this study, were established for M. galloprovincialis from the Bay of Biscay. These vary with season with baseline VvBAS below 0.10 μm3/μm3 in summer but below 0.05 in spring and autumn (Marigómez et al., 2006). Thus, it cannot be disregarded that baseline VvBAS values in North Sea M. edulis in late summer–autumn might be lower than 0.10 μm 3/μm 3 and therefore also mussels from Site 1 might be subjected to some source of environmental stress, although to a much lesser extent than Sites 2 and 3. The reason for the possible stress response in the reference mussels is not known, although it may suggest an additional source of exposure, such as from a nearby river, which has not yet been identified, or from other industrial and/or man-made activities. Fish farming activity using large cages of salmon and/or trout has been identified in the rough vicinity of the reference site. Potential impacts of fish farming practices on the local environment such as, organic loading, oxygen depletion, copper exposure from fish nets, and anti-parasitic drugs have been well documented (Wu, 1995) and may be a potential source of stressor to the reference mussels. Likewise, it is worth noting that a small PAH signature was found at Site 1 suggesting exposure to low levels of phenanthrene and fluoranthene. The present biomarker approach, including the calculation of the integrative IBR/n index, succeeded in distinguishing Site 1 from Site 2 and Site 3. In a previous study, IBR/n enabled mussel health status to be fully assessed after exposure to low concentrations of PW under laboratory conditions with individual chemicals at very low concentrations or below their detection limits (Brooks et al., 2011b). Aware that different biomarker arrangements on the star plots produce different IBR/n values (Broeg and Lehtonen, 2006), biomarkers were orderly represented in the five axes of start plots according to their biological complexity level. Star plots revealed details about the profile of the biological responses at each site, which were very similar in Site 2 and Site 3, with the exception of the lack of MN signal in Site 2 that accounts for a slightly lower IBR/n. However, due to the position of Sites 2 and 3 in relation to the PW outlet, and the lack of

The biomarker data for the caged mussels showed a good relationship between biological response and distance from the PW discharge outlet, with increasing signs of stress and poor health in mussels closest to the discharge compared to the reference groups (2000 m and 1900 m). However, 20% mortality in all caged mussels indicates that the mussels used were unduly stressed. The source of the caged mussels was a local mussel farm. Transplanting these mussels into their new environment combined with handling and transport pressures was thought to be the main factor in the higher than expected mortalities (Nigro et al., 2006; Orbea and Cajaraville, 2006; Gorbi et al., 2008). This is also likely to impact on some of the biomarkers measured including LP, VvBAS, and NRR as described below. LP values below 15 min, as found in both reference groups, suggest a certain degree of environmental stress, although LP was even lower in mussels closer to the PW discharge. Accordingly, VvBAS values were always over 0.12 μm 3/μm 3. As previously mentioned, baseline and critical values need to be properly fixed for North sea M. edulis. In any case, VvBAS seems to be a less sensitive parameter in caged mussels in this study. NRR in all groups, including reference groups, were below 60 min indicating stress. Although, significantly lower responses were found in the closest two stations (50 and 100 m). MN was only elevated in mussels closest to the outlet, whilst at all other groups had MN typical of North Sea background concentrations (Baršienė et al., 2008). Overall, differences in the biomarkers were observed amongst mussels caged at different distances from the PW outlet, with highest effects at 50 m and lowest effects at the reference sites, which was reflected in the IBR/n health index. Conversely, like in the case of native mussels, the concentration of PAHs in water (measured through the use of SPMDs) and PAHs and metals in mussel tissues were generally below or around detection limits and did not show a clear relationship with the biomarkers or the IBR/n index. The IBR/n index also provided clear signals of the biological effects of PW at exposure levels in the laboratory in which the concentration of individual chemicals in water and mussel tissues was low or below the detection limits (Brooks, et al., 2011b). Star plots revealed details about the profile of the biological responses elicited in mussels caged at different distances from the PW outlet with a clear increase in IBR/n for mussels at the closest station (50 m) and also at 100 m. IBR/n values in mussels at 110 and 200 m from the PW outlet were more similar to the mussels from the reference station. The difference between mussels at 100 and 110 m may be related to exposure to the plume with the former mussel group more in the path of the plume compared to the latter. However, the background concentrations of chemicals measured in the mussels and passive sampling devices does not provide supporting information for this. Comparison of the IBR/n of shore and caged mussels shows that the caged mussels at 50 m from the discharge outlet were the most affected with an IBR/n approximately 3 times that of all other mussel groups. The IBR/n for the exposed shore mussels was of similar value to caged mussels held 100 m from the discharge. 4.3. Concluding remarks There was good agreement found between the biological effect measurements showing that native shore mussels collected from Site 1 were significantly healthier than mussels from Sites 2 and 3. Likewise, mussels caged at longer distances from the PW outlet were healthier than those caged nearby the discharge outlet. However, no relationship was found between the biological effects and the

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contaminant concentrations measured. The Integrative Biological Response approach was able to differentiate between mussels exhibiting different stress conditions not identified from the contaminant exposure for both native and caged mussels. The biological effects observed in both caged and shore mussels were likely due either one or a combination of the following: 1) other chemicals in the discharge that were not measured; 2) the general impact of the processing plant and or 3) other activities in the local vicinity. Acknowledgements The authors wish to acknowledge the crew members of MS Amelie who assisted in the field work programme. This work was funded by Norske Shell. References Aker Kværner. BAT assessment report Ormen Lange Project Doc. 37-1A-AK-F15-00014; 2006. p. 124. Baršienė J, Andreikėnaitė L. Induction of micronuclei and other nuclear abnormalities in blue mussels exposed to crude oil from the North Sea. Ekologija 2007;53:9-15. Baršienė J, Rybakovas A, Förlin L, Šyvokienė J. Environmental genotoxicity studies in mussels and fish from the Göteborg area of the North Sea. Acta Zool Lituanica 2008;18:240–7. Baussant T, Bechmann RK, Taban IC, Larsen BK, Tandberg AH, Bjornstrand A, et al. Enzymatic and cellular responses in relation to body burden of PAHs in bivalve molluscs: a case study with chronic levels of North Sea and Barents Sea dispersed oil. Mar Pollut Bull 2009;58:1796–807. Beliaeff B, Burgeot T. Integrated biomarker response: a useful tool for ecological risk assessment. Environ Toxicol Chem 2002;21:1316–22. Booij K, Sleiderink H, Smedes F. Calibrating the uptake kinetics of semipermeable membrane devices using exposure standards. Environ Toxicol Chem 1998;17:1236–45. Broeg K, Lehtonen KK. Indices for the assessment of environmental pollution of the Baltic Sea coasts: integrated assessment of the multi-biomarker approach. Mar Pollut Bull 2006;53:508–22. Brooks S, Lyons B, Goodsir F, Bignell J, Thain J. Biomarker responses in mussels, an integrated approach to biological effects measurements. J Toxicol Environ Health A 2009;72:196–208. Brooks S, Harman C, Zaldibar B, Izagirre U, Glette T, Marigómez I. 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 2011a;62:327–39. Brooks S, Harman C, Grung M, Farmen E, Ruus A, Vingen S, et al. Water column monitoring of the biological effects of produced water from the Ekofisk offshore oil installation from 2006 to 2009. J Toxicol Environ Health A 2011b;74:582–604. Burgeot T, Woll S, Galgani F. Evaluation of the micronucleus test on Mytilus galloprovincialis for monitoring applications along French coasts. Mar Pollut Bull 1996;32:39–46. Cajaraville MP, Bebianno MJ, Blasco J, Porte C, Sarasquete C, Viarengo A. The use of biomarkers to assess the impact of pollution in coastal environments of the Iberian Peninsula: a practical approach. Sci Total Environ 2000;247:295–311. Damiens G, Gnassia-Barelli M, Loquès F, Roméo M, Salbert V. Integrated biomarker response index as a useful tool for environmental assessment evaluated using transplanted mussels. Chemosphere 2007;66:574–83. DNV report. Hydrographical measurements in Nyhamna. Report no:2009–2443; 2008. Eertman RHM, Groenink CLFMG, Sandee B, Hummel H, Smaal AC. Response of the blue mussel Mytilus edulis L. following exposure to PAHs or contaminated sediment. Mar Environ Res 1995;39:169–73. Fahimi HD, Cajaraville MP. Induction of peroxisome proliferation by some environmental pollutants and chemicals in animal tissues. Cell. Biology in environmental toxicology. Ed. Basque Country Press Service. 1995;221–255 Flynn SA, Butle E, Vance I. Produced water composition, toxicity and fate. In: Reed M, Johnsen S, editors. Environmental Science Research 52: Produced water 2. Environmental issues and mitigation techniques. NY: Plenum Press; 1996. p. 69–80. Garmendia L, Izagirre U, Cajaraville MP, Marigómez I. Application of a battery of biomarkers in mussel digestive gland to assess long-term effects of the Prestige oil spill in Galicia and Bay of Biscay: lysosomal responses. J Environ Monit 2011a;13:901–14. Garmendia L, Soto M, Vicario U, Kim Y, Cajaraville MP, Marigómez I. Application of a battery of biomarkers in mussel digestive gland to assess long-term effects of the Prestige oil spill in Galicia and Bay of Biscay: tissue-level biomarkers and histopathology. J Environ Monit 2011b;13:915–32. Garmendia L, Soto M, Ortiz-Zarragoitia M, Orbea A, Cajaraville MP, Marigómez I. Application of a battery of biomarkers in mussel digestive gland to assess longterm effects of the Prestige oil spill in Galicia and Bay of Biscay: correlation and multivariate analysis. J Environ Monit 2011c;13:933–42. Gomiero A, Da Ros L, Nasci C, Meneghetti F, Spagnolo A, Fabi G. Integrated use of biomarkers in the mussel Mytilus galloprovincialis for assessing off-shore gas platforms in the Adriatic Sea: results of a two-year biomonitoring program. Mar Pollut Bull 2011;62:2483–95. Gorbi S, Virno-Lamberdi C, Notti A, Benedetti M, Fattorini D, Moltedo G, et al. An ecotoxicological protocol with caged mussels, Mytilus galloprovincialis, for monitoring the impact of an offshore platform in the Adriatic sea. Mar Environ Res 2008;65:34–49. Harman C, Bøyum O, Tollefsen KE, Thomas KV, Grung M. Uptake of some selected aquatic pollutants in semipermeable membrane devices (SPMDs) and the polar organic chemical integrative sampler (POCIS). J Environ Monit 2008;10:239–47.

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