Seasonal baseline levels of physiological and biochemical parameters in polar cod (Boreogadus saida): Implications for environmental monitoring

Marine Pollution Bulletin 60 (2010) 1336–1345

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

Seasonal baseline levels of physiological and biochemical parameters in polar cod (Boreogadus saida): Implications for environmental monitoring Jasmine Nahrgang a,b,*, Lionel Camus a, Fredrik Broms b,c, Jørgen S. Christiansen b, Haakon Hop c a

Akvaplan-niva, Polar Environmental Centre, N-9296 Tromsø, Norway Department of Arctic and Marine Biology, University of Tromsø, N-9037 Tromsø, Norway c Norwegian Polar Institute, N-9296 Tromsø, Norway b

a r t i c l e

i n f o

a b s t r a c t

Keywords: Arctic pollutants Biomarker baseline Metabolism Oxidative stress Polar cod

Seasonality of biomarker baseline levels were studied in polar cod (Boreogadus saida), caught in Kongsfjorden, Svalbard, in April, July, September and December, 2006–2007. Physiological parameters (condition factor, gonado- and hepato-somatic indexes, energy reserves, potential metabolic activity and antifreeze activity) in polar cod were used to interpret the seasonality of potential biomarkers. The highest levels of ethoxyresorufin-O-deethylase (EROD) activity occurred concomitantly with the highest potential metabolic activity in July due to e.g. intense feeding. During pre-spawning, EROD showed significant inhibition and gender differences. Hence, its potential use in environmental monitoring should imply gender differentiation at least during this period. Glutathione S-transferase and catalase activities were stable from April to September, but changed in December suggesting a link to low biological activity. Knowledge of the biomarker baseline levels and their seasonal trends in polar cod is essential for a trustworthy interpretation of forthcoming toxicity data and environmental monitoring in the Arctic. Ó 2010 Elsevier Ltd. All rights reserved.

1. Introduction

Biomarkers are defined as biological responses to environmental chemicals at the sub-organismal level indicating a departure from the normal status (van Gestel and van Brummelen, 1996) and are developed to provide earlier and more sensitive signals than conventional endpoints such as growth, survival and reproduction. It is important to realise, however, that biomarkers are biochemical compounds and processes that play a primary role in the normal homeostasis of the organism and they are therefore influenced by natural biological and environmental cycles. Knowledge of the natural variability of biomarkers and their seasonal amplitude is critical for realistic interpretations of toxicity data and their application in environmental monitoring. For instance, an increase of a particular biomarker as part of a natural physiological cycle may be misinterpreted as an exposure effect (Sheehan and Power, 1999) since strong variation in biomarker levels occurs in relation to reproduction (Kopecka and Pempkowiak, 2008; Sanchez et al., 2008), food availability and water temperature (Viarengo et al., 1998). In the Arctic, the primary production is largely restricted to a short period of time, spring to summer, due to large seasonal fluctuations in ice cover and light (Wassmann et al., 2006). The large seasonal variation in food availability affects the physiology of marine ectotherms, including their energy reserves (Falk-Petersen et al., 1990) and metabolic activity (North, 1998). Furthermore, Arctic marine species experiencing sub-zero water temperatures produce seasonally antifreeze proteins (Desjardins et al., 2007).

Environmental monitoring programmes are designed to verify that environmental standards are being met and to detect sudden adverse changes in the environment (van der Oost et al., 2003). In the Arctic, the Arctic Monitoring and Assessment Program (AMAP, 2009) is the most comprehensive monitoring program that provides information on the status of the Arctic environment by compiling information from surveys and research studies. Existing data mainly report levels of chemicals in the marine environment and in tissues of Arctic organisms. However, there is a lack of basic knowledge on the potential adverse effects of pollutants on Arctic biological systems and of standardized procedures and assays for routine environmental monitoring. The Barents, Beaufort and Chukchi Seas are becoming major areas of concern in terms of petroleum related activities as some of the largest remaining oil and gas reserves are to be found in the Arctic (USGS, 2000). Therefore, there is a strong need to (1) document the environmental effects of oil related polycyclic aromatic hydrocarbons (PAHs) and (2) develop adapted monitoring tools, such as biomarkers for Arctic marine organisms.

* Corresponding author at: Akvaplan-niva, Polar Environmental Centre, N-9296 Tromsø, Norway. Tel.: +47 777 50 371; fax: +47 777 50 301. E-mail address: [email protected] (J. Nahrgang). 0025-326X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.marpolbul.2010.03.004

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Thus, a strong seasonality in physiological and biochemical parameters is expected in Arctic marine organisms. The polar cod (Boreogadus saida), a true Arctic gadoid fish with a circumpolar distribution, was chosen as a relevant indicator species for environmental monitoring in the Arctic (Nahrgang et al., 2010). Indeed, polar cod is a highly abundant fish, playing a key role in the Arctic ecosystem (Bradstreet and Cross, 1982; Tjelmeland, 2009) and which distribution overlaps with that of oil and gas activities in the Barents Sea. Furthermore, Stange and Klungsøyr (1997) considered its diet dominated by zooplankton, its short life-span and the low variation in contaminant levels among and within locations further advantages for its use as an indicator species. Although toxicity data are sparse for Arctic organisms in comparison to temperate species, some studies have shown the effects of oil related compounds on polar cod. For instance, crude oil exposed polar cod during sexual maturation showed reduced growth performance (Christiansen and George, 1995) and the induction of CYP1A activity in the liver (George et al., 1995). Furthermore, Ingebrigtsen et al. (2000) showed high total tissue levels of benzo(a)pyrene in the gills, olfactory organ, anterior kidney, liver, skin and intestinal wall while only traces where found in the muscle, brain and gonads of fish exposed via the water. Excretion in the polar cod occurs mainly via the bile due to its aglomerular kidneys (Christiansen et al., 1996). Recent studies of polar cod investigated biomarker responses to benzo(a)pyrene (Nahrgang et al., 2009b) and crude oil mixtures (Nahrgang et al., 2009a, 2010) and showed high and dose-dependent responsiveness of some biomarkers (e.g. CYP1A, GST mRNA expression and their activities, and DNA damage) in the liver. Finally, a study by Jonsson et al. (2010) reported low baseline levels of some biomarkers in polar cod in August–September and little variability within sampling locations around Svalbard. However, no information on the seasonality has been reported so far in polar cod. The goal of the present study was to determine baseline levels and seasonal variations of biochemical parameters in polar cod i.e. potential biomarkers for environmental monitoring. Hence, polar cod were caught in a pristine environment during different sea-

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sons. Physiological parameters (condition factor, gonadal status, energy reserves, potential metabolic activity and antifreeze activity) measured concomitantly provided valuable information on the biological cycle of polar cod to interpret the seasonality in the biochemical parameters. The investigated biochemical parameters are widely used biomarkers to assess the presence and effects of pollutants in organisms (reviewed by van der Oost et al., 2003) and are related to PAH metabolism (i.e. ethoxyresorufin-O-deethylase (EROD) and glutathione S-transferase (GST) activities and bile metabolites), and oxidative stress (i.e. catalase (CAT) activity).

2. Materials and methods 2.1. Fish sampling Polar cod were caught with Campelen Super 1800 bottom trawl in Kongsfjorden, Svalbard (Fig. 1), in April, September and December 2006 and July 2007 (Table 1). Temperature and depth profiles were measured with a Sea-Bird SBE9 CTD-rosette system. A fishlift, designed by the Institute of Marine Research, Norway, was used to prevent the fish from being injured during trawling (Holst and McDonald, 2000). Intact polar cod were kept 24 h on deck in a tank with running seawater to ensure bile accumulation. Blood was drawn from the caudal vein with syringes, gauge 30 (OmnicanÒ) rinsed with heparin. The serum was removed after centrifugation and stored at 80 °C prior to analysis of antifreeze activity. Fish were sacrificed by a sharp blow to the head. Total body weight (0.1 g wwt), fork length (0.1 cm) and gender were recorded (Table 2). However, due to time constrains during the April sampling, size and gender of polar cod were not recorded. Liver slices and bile were placed into separate cryovials, snap frozen in liquid nitrogen, and stored at 80 °C prior to analysis. In addition, a random subsample of polar cod was taken from each of the trawl hauls and fork length (0.1 cm), total body wet weight and weight of the gonads and liver (0.01 g) were recorded. The Fulton’s condition factor (K), the gonado-somatic index (GSI)

Fig. 1. The Svalbard Archipelago and a bathymetric map of Kongsfjorden (latitude 79° N), showing the stations where polar cod (Boreogadus saida) were sampled in April (A), September (S), December (D) 2006 and July (J) 2007.

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Table 1 Sampling site description (Kongsfjorden, Svalbard) in terms of temperature, salinity and depth, measured with a Sea-Bird SBE9 CTD-rosette system. Sampling date

Sampling name

30-04-06 28-08-06 02-12-06 25-07-07

April September December July

Location

Water temperature surface/bottom

Salinity surface/bottom

Trawling depth

N

E

(°C)

(psu)

(m)

78°57 79°01 78°33 79°02

11°58 11°26 12°06 11°24

0.3/0.3 6.0/1.9 3.0/4.0 7.0/2.5

34/35 32/35 35/35 34/35

300 300 300 240

Table 2 Summary of number (n), fork length (cm) and weight (g) of polar cod (Boreogadus saida) used for the biochemical analyses and the Fulton’s condition factor (K) and gonadosomatic index (GSI) of random males (#) and females ($), number given in parentheses, from the same trawl hauls, and at different sampling seasons. The hepato-somatic index (HSI) was determined on the same individuals as for K and GSI. In December, the HSI is however based on only 19 fish. Values are means ± SE. NA, not available. Asterisks indicate significant gender differences (p < 0.05). Letters (a, b, c, d) indicate significant difference (p < 0.05) between sampling months. Sampling

April July September December

n

14 29 22 27

Length (cm)

NA 12.5 ± 0.3a 14.8 ± 0.5b 14.5 ± 0.5b

Weight (g)

NA 11.7 ± 0.9a 19.6 ± 1.7b 17.3 ± 1.1b

(n)

(19) (26) (27) (39)

K

GSI (%)

HSI (%)

#

$

#

$

0.59 ± 0.01a,c 0.64 ± 0.01*,a,b 0.66 ± 0.01b 0.56 ± 0.02*,c

0.65 ± 0.03a,b 0.66 ± 0.01a 0.64 ± 0.01a,b 0.62 ± 0.01b

0.3 ± 0.04*,a 1.1 ± 0.1*,b 2.1 ± 0.2c 25.7 ± 4.4*,d

1.4 ± 0.2a 1.7 ± 0.1a 2.4 ± 0.1b 7.3 ± 0.2c

and the hepato-somatic index (HSI) were determined according to the equations:

K ¼ 100  sW=L3 ; GSI ¼ 100  ðtotal gonad wet weight=sWÞ; HSI ¼ 100  ðtotal liver weight=sWÞ; where sW is the somatic weight (whole body wet weight – gonad weight) (g) and L the fork length (cm). 2.2. Analyses 2.2.1. Physiological parameters The measure of energy reserves (lipids, proteins and carbohydrates) and electron transport system (ETS) activity were derived from the cellular energy allocation (CEA) assay (De Coen and Janssen, 1997). The biochemical measurements were performed on liver, the major metabolizing organ in fishes. Liver tissue were homogenized in a buffer pH 7.5 (0.1 M Trizma HCl/base buffer, 0.4 M MgSO4, 15% polyvinylpyrrolidone and 0.2% w/v Triton X100) and centrifuged for 10 min (3000g, 4 °C). Supernatants were split into 3 subsamples for determining energy reserves and ETS activity. For lipid extraction, the homogenates (200 ll) were mixed with 500 ll chloroform, 500 ll methanol and 250 ll dH2O before 5 min of centrifugation at 10000g. A 100 ll sample of the chloroform phase was then pipetted into glass reagent tubes, 500 ll of H2SO4 (95–97%) was added and the sample charred for 15 min at 200 °C. Samples were then diluted 1:6 in dH2O and read spectrophotometrically at 340 nm, as 4 replicates. Lipid concentrations (mg ml1) were determined against a glyceryl tripalmitate standard curve (0–0.5 mg ml1). For protein and carbohydrate concentrations, trichloroacetic acid (15%) was mixed with 300 ll of the homogenates and centrifuged at 10000g for 5 min. Pellets were dissolved in 500 ll NaOH (1 N) and incubated 30 min at 60 °C for dissolution of the pellets. Subsequently, 300 ll of HCl (1.67 N) was added to neutralize the solution. Protein concentration (mg ml1) was determined in 4 replicates, according to Bradford (1976) using Bovine Serum Albumin (BSA) as a standard. For carbohydrate determination, supernatants (50 ll) were mixed with H2SO4 (95–97%; 200 ll) and phenol (50 ll), distributed into microwells, as 4 replicates, and read spectrophotometrically at 490 nm. Carbohydrate concentration (mg ml1) was determined against a glycogen standard curve

4.3 ± 0.2a 5.3 ± 0.5b 7.1 ± 0.4c 7.0 ± 0.4c

(0–0.250 mg ml1). Lipids, proteins and carbohydrates were converted into Joule per unit liver wet weight (J g1 wwt) according to their specific enthalpy of combustion i.e. 39.5, 24.0 and 17.5 kJ g1, respectively (Gnaiger, 1983). The ETS activity reflects the potential metabolic activity that could be supported by the existing enzymatic ‘machinery’ (Cammen et al., 1990). For determination of the ETS activity (lmol O2 consumed min1 g1 wwt), a solution containing liver homogenates (50 ll) in buffered substrate solution (50 ll, 0.1 M Trizma HCl/base buffer pH 7.5, 0.3% Triton X-100) and NADH/NADPH (50 ll, 1.17 mM/250 lM) was pipetted as 4 replicates into microwells. The reaction was started by adding 100 ll 2-(p-iodophenyl)-3-(p-nitrophenyl)-5 phenyl tetrazolium chloride (4 mg ml1). Absorbance was measured kinetically at 490 nm at 20 °C for 10 min. The antifreeze activity in polar cod is related to the presence of antifreeze glycoproteins (AFGPs) and their capacity to depress the freezing point (FP) beyond the melting point (MP) of serum and other body fluids (Kao et al., 1986). The thermal hysteresis (TH) is the temperature difference between the FP and MP and is a direct measure of the antifreeze activity. A Clifton Nanoliter Osmometer (Clifton Technical Physics, Hartford, NY) was used to determine FP and MP of sera as described by Præbel and Ramløv (2005). Sera of six polar cod caught in December 06 and July 07 were analyzed repeatedly 6 times (subsamples) to ensure a precise reading and quality of the data. The data were read as the osmotic concentration (mOsm) and converted into temperature (°C) according to equation:

FP C ¼ ðmOsm 1:858Þ10001 :

2.2.2. Biochemical parameters Liver samples were homogenized at 4 °C with Potter–Elvehjem type homogenizer in a phosphate buffer pH 7.4 containing 50 mM Tris, 150 mM KCl, 1 mM EDTA, 1 mM dithiothreitol and 20% glycerol. Homogenates were centrifuged (10000g, 4 °C) for 30 min. Supernatants were subsequently centrifuged (50000g, 4 °C) during 2 h for extraction of the microsomal fraction. Pellets (microsomes) were dissolved in Tris buffer (pH 7.4) and stored at 80 °C. The cytosolic fraction was obtained by homogenizing the liver samples at 4 °C with Potter–Elvehjem type homogenizer in a phosphate buffer (pH 7.4) containing 1.5% NaCl and by successive centrifuga-

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tion of the supernatant (10000g for 30 min and 50000g for 2 h, 4 °C). The cytosolic fraction was stored at 80 °C. Ethoxyresorufin-O-deethylase activity was measured according to Eggens and Galgani (1992). The reaction mix consisted of 10 ll microsomal fraction in 100 mM of Tris–phosphate buffer (pH 8), with 2 lM ethoxyresorufin as substrate in a final volume of 230 ll. Reaction started by adding 0.25 mM NADPH in the microwells. The resorufin production was measured in four replicates during 20 min at 20 °C with a spectrometer plate reader PerkinElmer Victor at 544/584 nm excitation/emission wavelengths, respectively. A resorufin standard curve (0–2 lM) was used for determination of the reaction rates in pmol of resorufin produced min1 mg1 of total microsomal protein. Glutathione S-transferase (GST) activity was assayed in triplicate on the cytosolic fraction as described by Habig et al. (1974) and expressed as nmol min1 mg1 of total cytosolic protein. The reaction mixture contained 100 ll of cytosolic fraction, 100 ll reduced glutathione (20 mM), 100 ll 1-chloro-2,4-dinitrobenzene (20 mM), 1.7 ml phosphate buffer (100 mM, pH 7) in 2 ml cuvette. The appearance of the coupling compound dinitrophenyl thioether, formed by the reaction of reduced glutathione with 1-chloro-2,4dinitrobenzene, was followed at 340 nm (e = 9.6 mM1 cm1) during 1 min at 4 °C with a PerkinElmer UV/Vis spectrophotometer LAMBDA 35. Catalase (CAT) activity was measured in triplicate on the same cytosolic fraction and expressed in lmol min1 mg1 of total cytosolic protein. The decrease in absorbance was recorded at 4 °C at 240 nm (e = 40 M1 cm1) using 600 mM H2O2 as substrate (Clairborne, 1985) on the LAMBDA 35. Total protein concentrations in microsomal and cytosolic fractions of liver homogenates were determined colorimetrically (Bradford, 1976) for normalization of EROD, GST and CAT activities. The BSA was used as a standard reference. The fluorescent aromatic compounds (FACs) in the bile were measured with a PerkinElmer spectrofluorometer LS55 through synchronous fluorescence scan spectrometry (SFS). Bile extract was diluted 1:1600 in distilled water and relative fluorescence intensity from 250 to 500 nm excitation wavelengths was scanned with Dk 42 nm (Aas et al., 2000). The fluorescence spectrum of distilled water (triplicate) was subtracted from the fluorescence spectrum of each sample. Two groups of unidentified FACs were detected with fluorescence maxima at 282/324 and 355/397 nm excitation/emission wavelengths. 2.3. Statistics Statistical analyses were performed with SPSS Statistics™ 18. When requirements of normality and homogeneity of variances (Levene’s test) were met, a one-way ANOVA followed by Tukey HSD post hoc test, was used to test differences among the sampling months (i.e. for size, condition factor K, GSI and HSI). Requirements for normality and homogeneity of variances were not met for the other parameters measured. The t-test for equality of means (assuming heterogeneous variances) was used for the antifreeze activity to distinguish differences between July and December samples. The Robust Tests of Equality of Means (Welch ANOVA) was used to detect differences among sampling months for EROD, GST and CAT activities and bile metabolites and was followed by a multiple comparison test (Dunnett T3 test) to distinguish specific differences among months (tables inserted in figures; Welch, 1951). For all analyses, when gender differences were found, male and female data were treated separately (i.e. condition factor K, GSI, ETS and EROD activity). In this case, the April samples (gender unknown) were excluded from the statistical analyses but presented in the results as ‘‘mixed gender”. However, when no gender difference were observed for July, September and December data,

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the April data were included in the statistical analysis, assuming no gender differences in fish outside the reproductive season and pooled data were represented in the figures. The significance level applied for all analyses was p < 0.05. Results are shown as means ± SE.

3. Results 3.1. Environmental conditions The CTD profiles (data not shown) revealed the presence of Transformed Atlantic water masses (>1.0 °C, >34.7 psu) at each sampling time, except for April when local water prevailed (<1.0 °C, >34.4 psu) (Svendsen et al., 2002). Water temperature at the trawling depth was 0.3 °C in April, 2.5 °C in July, 1.9 °C in September, and 4 °C in December. A thermal stratification was observed in the surface layers with the presence of chlorophyll-a in July and September. The entire water column was homogenous in April and December in terms of temperature and no chlorophyll was detected. Water samples from Kongsfjorden, pumped from 80 m depth, were analyzed for PAH compounds in the autumn 2007 as part of a crude oil contamination experiment (Nahrgang et al., 2009a). The PAH concentration for most compounds was below the detection limit of the gas chromatograph (<0.001 lg L1). 3.2. Polar cod Polar cod caught in July were significantly smaller (i.e. length and weight) than in September and December (Table 2). No significant sex-specific differences were observed for either length or weight in any season. Fulton’s condition factor (K) showed higher levels in females compared to males in July and December and no gender differences were observed in April and September. Fish condition was significantly reduced in April and December in males while only in December compared to July for females. In April, K showed a large individual variability in females. The GSI increased continuously from April to December with a peak for both genders in December prior to the spawning season (December– March) (Hop et al., 1995). The GSI was significantly higher in females compared to males in April and July and significantly lower in December. No significant difference in GSI between genders was noted in September. Finally, the HSI showed a significant increase from April to September and no significant change between September and December. Furthermore no gender differences were found for any season. 3.3. Analyses 3.3.1. Physiological parameters Energy reserves (lipids, proteins and carbohydrates) showed no differences between males and females for any season. The major energy reserves in the liver were lipids, followed by proteins and carbohydrates (Table 3). The lipid reserves increased significantly from September to December, whereas proteins were lowest in July and maximal in September. Carbohydrates showed highest levels in April. The electron transport system (ETS) activity was significantly lower in females compared to males in September and December. Furthermore, the ETS activity was significantly reduced in December, compared to the other seasons, for both genders. In April, the ETS activity showed a high variance (Table 3). Antifreezes were active during both summer and winter seasons with a significant peak in December (0.94 ± 0.04 °C) compared to July (0.65 ± 0.04 °C), irrespective of gender.

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Table 3 Lipids, proteins, carbohydrates (J g1 wwt) and electron transport system (ETS) activity (lmol O2 min1 g1 wwt) in liver of polar cod (Boreogadus saida) caught in Kongsfjorden in April (n = 14), September (n = 22), December 2006 (n = 27) and July 2007 (n = 29). Values are represented as means ± SE. Gender differences (male #, female $) were observed for ETS activity, thus April was excluded from statistical analysis for this parameter. Asterisks indicate significant difference (p < 0.05) between males and females. Letters (a, b) indicate significant difference (p < 0.05) between sampling months. Sampling

Lipids (J g1 wwt)

Proteins (J g1 wwt)

Carbohydrates (J g1 wwt)

ETS activity (lmol O2 min1 g1 wwt) #

$

April July September December

17400 ± 230a,b 13800 ± 965b 15400 ± 400b 18800 ± 135a

515 ± 42a,b 425 ± 34a 574 ± 27b 494 ± 26a,b

189 ± 40a 25 ± 3b 34 ± 2b 34 ± 2b

345 ± 97 495 ± 63a 408 ± 38*,a 160 ± 21*,b

445 ± 54a 318 ± 21a 95 ± 9b

3.3.2. Biochemical parameters The EROD activity showed a marked seasonality with significantly higher levels in July (12.6 ± 3.1 and 11.6 ± 1.2 pmol min1 mg1 protein for males and females, respectively) compared to the other seasons (1.1 ± 0.4 pmol min1 mg1 protein) (Fig. 2). Differences between genders were found only in December with higher levels in males (p < 0.05). The GST, CAT activities and FACs in the bile did not reveal any differences between genders. The GST activity increased from July (139 ± 13 nmol min1 mg1) to December (486 ± 82 nmol min1 mg1) (Fig. 3). The CAT activity was significantly lower in December (79 ± 8 lmol min1 mg1) compared to the other seasons (139 ± 8– 205 ± 22 lmol min1 mg1) (Fig. 4). Two groups of unidentified FACs were detected with fluorescence maxima at 282/324 and 355/397 nm excitation/emission wavelengths, respectively (Fig. 5). The compounds at 282/324 nm showed a fluorescence maxima in April, while the compounds at 355/397 nm showed the highest fluorescence in July. 4. Discussion The sampling area, Kongsfjorden, is located on the western coast of Svalbard. Levels of pollutants in the specific sampling area are little known, except for some surveys (e.g. Hop et al., 2001).

Low levels of POPs (<240 ng g1 lipid weight) have been reported in polar cod from the Barents Sea (Stange and Klungsøyr, 1997; Hop et al., 2002) but no study has reported levels of PAHs in polar cod. A more significant presence of POPs and PAHs have been demonstrated in the sediments around Svalbard (Stange and Klungsøyr, 1997; Liping et al., 2009), including Kongsfjorden (Olsson et al., 1998). Exposure of polar cod to PAHs from the sediments is not relevant, however, considering its pelagic-sympagic life cycle (Lønne and Gulliksen, 1989). Levels of contaminants in Arctic marine fish are below threshold levels for biological effects (AMAP, 2009; Gabrielsen and Sydnes, 2009). Hence, seasonality in the biological parameters measured in the present study can be attributed to the natural biological cycle of polar cod. 4.1. Physiological parameters The characterization of condition factor (K), GSI, HSI, energy reserves, ETS activity and antifreeze activity provided valuable information on the fish physiological status. The lipids accounted for the major energy reserves in polar cod, supporting Hop et al. (1995). Lipids are playing an important role as energy reserve in Arctic organisms, for overwintering and reproduction when food availability becomes low (Falk-Petersen et al., 1990; Pörtner et al., 2005). The significant seasonal increase in lipid levels in

Fig. 2. Ethoxyresorufin-O-deethylase (EROD) activity (pmol min1 mg1 protein) in liver from polar cod (Boreogadus saida) caught in Kongsfjorden in April, September, December 2006 and July 2007. Plots represent the median (line), 25–75% percentiles (box), min–max (whisker) and sample size in parentheses. Asterisks in the figure indicate significant difference (*p < 0.05) between gender for the considered sampling month. The inserted tables indicate significant differences (ns = non significant, *p < 0.05, ** p < 0.01, ***p < 0.001) between sampling months. April data were not considered in the statistical analysis.

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Fig. 3. Glutathione S-transferase (GST) activity (nmol min1 mg1 protein) in liver from polar cod (Boreogadus saida) caught in Kongsfjorden in April, September, December 2006 and July 2007. There were no significant differences between genders, thus pooled data are presented. Plot and inserted table as in Fig. 2.

Fig. 4. Catalase (CAT) activity (lmol min1 mg1 proteins) in liver from polar cod (Boreogadus saida) caught in Kongsfjorden in April, September, December 2006 and July 2007. There were no significant differences between genders, thus pooled data are presented. Plot and inserted table as in Fig. 2.

polar cod from July to December (Table 3) supported Falk-Petersen et al. (1990) demonstrating a transfer of lipids through the Arctic food web in less than 6 month from the phytoplankton spring bloom starting in April–May, via the herbivorous zooplankton to zooplanktivorous fish in September. This is further supported by the significant increase in condition and HSI of polar cod from April to September. The reduction in condition towards December reflects the energy investment during gonadal maturation and is a known phenomenon in fish (e.g. Lambert and Dutil, 2000; Wuenschel et al., 2009). At the same time, the electron transport system (ETS) activity decreased three-folds from July to December, suggesting a decrease in the potential metabolic activity of the organism. To our

knowledge, no seasonal studies employing ETS activity measurements have been previously reported for fishes. Cammen et al. (1990) showed seasonal patterns of ETS activity in the polychaete (Nereis virens) and the amphipod (Corophium volutator) that could be related to changes in food availability and/or reproductive status of the organisms. In December during food shortage, adenosine triphosphate (ATP) requirements decrease together with locomotor activity (Clarke, 1993), which may explain partly the low ETS activity measured in polar cod. In April, the condition and HSI of polar cod were generally low compared to July and September levels, reflecting the poor condition of the fish following the winter period with low food availability. At the same time, the ETS activity and the carbohydrate levels

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Fig. 5. Fluorescent aromatic compounds (FACs) at an excitation/emission wavelength of 282/324 and 355/397 nm, respectively, measured through synchronous fluorescence scan spectrometry (SFS) of bile samples from polar cod (Boreogadus saida) caught in Kongsfjorden in April, September, December 2006 and July 2007. There were no significant differences between genders, thus pooled data are presented. Plot and inserted table as in Fig. 2.

showed large variances suggesting a transition period with physiological changes, such as increase in cellular respiration and glycogen synthesis. Indeed, our data suggest an energy mobilization and a physiological preparation to take advantage of the intense feeding period during spring. The liver metabolic organization varies seasonally, triggered by, for instance, changes in photoperiod. Levesque et al. (2005) showed experimentally that photoperiod is a prime driver of the physiology of Atlantic cod (Gadus morhua) from Newfoundland, where light conditions vary highly between summer and winter. Similarly, the reappearance of light and the rapid changes in photoperiod in Kongsfjorden between March and May, affects the physiology of polar cod, as suggested by the energetical changes occurring in April. The increased serum antifreeze activity from July to December supports the seasonality in antifreeze activity reported in temperate fish species that experience sub-zero conditions in winter. For instance, a strong seasonality TH was shown in Atlantic wolffish (Anarhichas lupus) (Desjardins et al., 2007), with almost complete loss in TH between July and September. The difference in polar cod TH between July and December was less pronounced, however, due to smaller seasonal temperature amplitude in Arctic waters. Our results support DeVries and Cheng (2005) who reported a substantial TH of 0.87 °C in polar cod caught in August in West Greenland (70° N, 53° W). The authors suggested either little loss in antifreeze activity in summer or an up-regulation of AFGPs in anticipation for winter. 4.2. Biochemical parameters The primary goal of this study was to characterize the natural range of baseline levels of potential biomarkers over the seasons in wild polar cod since these have not been reported for this species previously. In a recent study, Jonsson et al. (2010) reported levels of biomarkers (e.g. EROD activity and bile metabolites) in polar cod collected in September 2001 and August 2002 in Kongsfjorden, Wijdefjorden, Isfjorden and Hinlopen. Nevertheless, this study did not report seasonal variations. In the present study, EROD activity in polar cod was in the lower range of values reported for temperate fishes (Ronisz et al., 1999), but similar to activities

in Antarctic fishes ( 5 pmol min1 mg1), such as (Trematomus bernachii) (Benedetti et al., 2007) and (Pleuragramma antarcticum) (Regoli et al., 2005). The GST activity was in the range of values found in polar (Benedetti et al., 2007) and temperate (Sturve et al., 2006) fishes, while catalase (CAT) activity in polar cod (200 lmol min1 mg1) was higher than baseline values found in other polar fishes (1–70 lmol min1 mg1) (Regoli et al., 2005; Benedetti et al., 2007). In temperate fishes, CAT levels are extremely variable and reported up to 12000 lmol min1 mg1 in Atlantic cod (Sturve et al., 2006). The observed differences in biomarker baseline levels may be due to species specificities but also to difference in assay conditions. For instance, in the present study GST and CAT activities were measured at 4 °C to reflect in situ temperatures, while most studies have performed the assays at 18 °C (e.g. Regoli et al., 2005; Benedetti et al., 2007). As mentioned earlier, biochemical parameters, i.e. compounds and processes, play a primary role in the normal homeostasis of organisms and are therefore influenced by natural biological and environmental cycles. The effect of temperature has been extensively studied and is recognized to be involved in the seasonality of biomarkers in fishes from e.g. temperate regions where the annual thermal amplitude can be >20 °C (e.g. Kopecka and Pempkowiak, 2008). However, temperature is relatively stable and low in polar marine environments, as observed at the sampling location, where the largest temperature difference recorded was of 4 °C between December and April. In this respect, temperature likely has limited influence on the observed variation of baseline levels of biochemical parameters in polar cod in the present study. On the other hand, food availability in the Arctic represents a more remarkable stress for Arctic species, with important physiological consequences as observed in the present study (i.e. lipid reserves, ETS activity, condition factor and HSI). Laboratory studies have demonstrated that food availability and quality affect CYP1A enzyme activities (Wall and Crivello, 1999) and antioxidant parameters such as CAT and GST activities (Pascual et al., 2003; Rueda-Jasso et al., 2004). For instance, Pascual et al. (2003) showed that low food availability or starvation increases GST activity and inhibited CAT activity in gilthead seabream (Sparus aurata). Furthermore, food availability has an indirect effect on the biological

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activity of Arctic organisms, including changes in mobility, food consumption and respiration. This is supported in the present study by high ETS activity in polar cod during period of intense feeding (July) and low levels in December. Low food availability in April supported by the reduced condition and HSI of the fish may explain the very low EROD activities measured during this period of time. Conversely, the high biological activity and increased metabolism in July may explain the high EROD levels, as suggested by Dewaide and Henderson (1970), who reported highest activities of two drug-metabolizing enzymes in roach (Leuciscus rutilus) during the summer months. Furthermore, low biological activity in December may decrease ROS production (Cassini et al., 1993) and thus reduce CAT activity, as observed in the present study. Among the known factors affecting biochemical parameters in fish, the reproductive cycle plays a major role (Stegeman et al., 1982). Indeed, EROD activity shows a negative correlation with the GSI and circulating hormones in pre-spawning fishes, as observed in the present study. The suppression of CYP1A would be an adaptive response to maintain high tissue steroid hormone levels necessary for active reproductive processes (Arukwe et al., 2008). Differences between genders, as observed in polar cod in December, are well known in fishes particularly in the later stages of pre-spawning (Stegeman et al., 1982; George et al., 1995). Indeed, the 17b-estradiol, an estrogenic steroid hormone produced by female fish during the gonadal maturation process (Hop et al., 1995), inhibits EROD activity (Stegeman et al., 1982). Other factors, environmental or intrinsic to the fish’s physiology, may be involved in the observed biomarker variations. Among these factors, age and body size have been found to influence biomarkers (Martínez-Álvarez et al., 2005; Chandrasekara and Pathiratne, 2007). Hence, the difference in polar cod size among sampling months may have played a role in the seasonality observed in the present study, although the importance of this factor could not be determined. The fluorescent aromatic compounds (FACs) detected in the polar cod bile at 282/324 and 355/397 nm could not be identified as PAH metabolites based on their optimal excitation/emission wavelengths (Aas et al., 2000) and the lack of PAHs in the sea water. Numerous studies have reported PAH metabolites in the bile through fixed wavelength fluorescence (FF) or synchronous scan fluorescence (SFS), but, surprisingly, no studies have investigated the fluorescence baseline signal and the seasonality of endogenous aromatic metabolites in the bile of marine organisms. Endogenous metabolites may show high baseline levels in the bile of polar fishes compared to temperate fishes. Indeed, the lack of glomerular kidneys in polar cod and other polar fishes (Christiansen et al., 1996), in contrast to most temperate fishes, implies that a higher fraction of endogenous compounds are excreted via the bile rather than urine. Moreover, differences in the momentary feeding status of each specimen may explain the relatively strong variances observed (van den Hurk, 2006; Hanson and Larsson, 2008). Although wild polar cod are subjected to an important biliary excretion of endogenous compounds and strong changes in food availability over the year, the levels of FACs in wild fish were below levels of PAH metabolites in polar cod exposed to low bioavailable PAH concentrations (Nahrgang et al., 2009b, 2010). Hence, the baseline levels of FACs in polar cod bile should not pose any problem for recognizing PAHs in field-based toxicity assessments, but the compounds involved should be further identified. 4.3. Implication for environmental monitoring and future research As mentioned in the introduction, basic knowledge on the physiology and life cycles of Arctic marine organisms is relatively scarce in comparison to the extensive data existing for temperate species.

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Furthermore, standardized procedures for the assessment of adverse effects of anthropogenic pollutants to Arctic organisms are lacking to date and need to be implemented and calibrated. The present study, in combination with previous laboratory toxicity assessments by Nahrgang et al. (2009a,b, 2010), provides new and valuable information for the implementation of routine monitoring tools for polar cod, in terms of (i) calibration of analytical methodologies for polar cod, (ii) baseline levels and responsiveness of commonly used biomarkers and (ii) information on the physiology and life-cycle of the selected indicator species. Indeed, the present biomarkers have shown baseline values below induction levels by oil related compounds (Nahrgang et al., 2009a,b, 2010). Although, some biomarkers, such as EROD activity showed relatively high variations over the year, these variations may be dealt with by carefully designing sampling programs (ICES, 2007; Depledge, 2009). To help interpreting biomarker responses in the field, practical recommendations should be considered such as a thorough description of the sampled individuals e.g. gender, length, weight, age, gut contents and weights of gonads and liver for somatic index determination. Furthermore, gender differentiation for the biomarker analysis may be considered in the prespawning period, although the gender discrepancy found for EROD in December seemed minor as the baseline levels for both genders at this period were low and should not mask a PAH induced response. Indeed, in polar cod exposed to environmentally realistic crude oil concentrations from October to November, biomarkers showed inductions (e.g. EROD and GST activity) high above baseline values (e.g. Nahrgang et al., 2009a, 2010). By contrast, at periods of high baseline levels, such as in July for EROD activity, the biomarker behavior in response to oil related compounds is unknown as it may be masked or enhanced. This will need to be further investigated. Biomarkers are integrated in several guidelines and recommendation documents for environmental monitoring and risk assessments. For instance, the ICES Working Group on Biological Effects of Contaminants (ICES, 2006) and the workshop on Integrated Monitoring of Contaminants and their Effects in Coastal and Open-Sea Areas (ICES, 2007) attempted to develop a framework based on integrated indicators for assessing ecosystem health (Thain et al., 2008). To face challenges such as ecological relevance and responsiveness, involved in the use of biomarkers, an assessment criterion has been proposed including global baseline levels for all methods and levels deviating from the normal range (Thain et al., 2008). For instance, in most marine fish species EROD values above twice the upper limit of baseline values indicate an ecosystem influenced by planar organic contaminants (ICES, 2007). Likewise, the present study and the three experimental studies (Nahrgang et al., 2009a,b, 2010) allowed determining a range of baseline levels of several biomarkers and levels significantly higher than the baseline that were caused by environmentally relevant concentrations or doses of PAHs. The present dataset is thus important for the implementation of environmental programmes in the Arctic. PAHs and other anthropogenic pollutants have been shown to affect important physiological processes in several fish species, leading to reduced population fitness and survival. For instance, PAHs are well known to cause reproduction impairment through their endocrine disrupting effects, e.g. estrogenic and anti-estrogenic effects (Sol et al., 2000). Furthermore, some PAHs can suppress fish immune functions (Zelikoff, 1994) and thereby increase the susceptibility to diseases (Arkoosh et al., 1998). For future research, we therefore recommend the evaluation of a larger range of biomarkers of, for instance endocrine disruption and immunotoxicity that could inform on biological adverse effects on the population. Special focus should be given to critical times of the polar cod life cycle e.g. periods of low lipid reserves, during

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gonadal development and egg and larval developmental stages, implying studies during the Arctic winter and spring. These measurements would be particularly interesting for the prediction of population fitness, i.e. reproduction and population survival with regard to environmental risk assessment. 5. Conclusions Baseline studies of biomarkers in temperate organisms are often challenged by the concomitant effects of the natural biological cycle of the organism and anthropogenic contaminants (Hylland et al., 2009). Here, we studied polar cod from a pristine environment and determined the natural background levels of biochemical parameters that can be considered as values within the range of normal homeostasis for wild unexposed polar cod. Moreover, our results show that both physiological and biochemical parameters in polar cod vary with season in relation to major characteristics of the Arctic ecosystem. Finally, this study gives further insight to the general eco-physiological responses of polar cod to natural processes during periods of e.g. reproduction and feeding. In the present study, the GST and CAT activities showed relatively low seasonality and no gender differences. By contrast, EROD activity showed a stronger seasonal variability, which raises the questions whether the high baseline level of EROD activity (July) could mask a PAH exposure and, conversely, whether PAH exposed polar cod could induce EROD activity at a time (April) when it seems to be repressed. Further inter-annual studies would improve the characterization of the factors affecting the biomarkers and further explain the major trends observed in this study. Acknowledgements This study was funded by ConocoPhillips, Norge A.S. Travel expenses were partially supported by the PanAME grant sponsored by ConocoPhillips. Fredrik Broms was financed by the Research Council of Norway (Project MariClim 165112/S30). We thank the crew of R/V Jan Mayen for assisting with fish-lift and trawling. Iris Jæger is thanked for help with polar cod sampling and A.L. DeVries and C.-H.C. Cheng for their precious advices on the measurements of antifreeze activity. References Aas, E., Beyer, J., Goksøyr, A., 2000. Fixed wavelength fluorescence (FF) of bile as a monitoring tool for polyaromatic hydrocarbon exposure in fish: an evaluation of compound specificity, inner filter effect and signal interpretation. Biomarkers 5, 9–23. AMAP. 2009. Arctic Pollution 2009. Arctic Monitoring and Assessment Programme, Oslo, ISBN: 978-82-7971-050-9. Arkoosh, M.R., Casillas, E., Huffman, P., Clemons, E., Evered, J., Stein, J.E., Varanasi, U., 1998. Increased susceptibility of juvenile Chinook salmon from a contaminated estuary to Vibrio anguillarum. Trans. Am. Fish. Soc. 127, 360–374. Arukwe, A., Nordtug, T., Kortner, T.M., Mortensen, A.S., Brakstad, O.G., 2008. Modulation of steroidogenesis and xenobiotic biotransformation responses in zebrafish (Danio rerio) exposed to water-soluble fraction of crude oil. Environ. Res. 107, 362–370. Benedetti, M., Martuccio, G., Fattorini, D., Canapa, A., Barucca, M., Nigro, M., Regoli, F., 2007. Oxidative and modulatory effects of trace metals on metabolism of polycyclic aromatic hydrocarbons in the Antarctic fish Trematomus bernacchii. Aquat. Toxicol. 85, 167–175. Bradford, M.M.A., 1976. Rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein dye binding. Anal. Biochem. 72, 248–254. Bradstreet, M.S.W., Cross, W.E., 1982. Trophic relationships at High Arctic ice edges. Arctic 35, 1–12. Cammen, L.M., Corwin, S., Christensen, J.P., 1990. Electron-transport-system (ETS) activity as a measure of benthic macrofaunal metabolism. Mar. Ecol. Prog. Ser. 65, 171–182. Cassini, A., Favero, M., Albergoni, V., 1993. Comparative studies of antioxidant enzymes in red-blooded and white-blooded Antarctic teleost fish Pagothenia bernachii and Chionodraco hamatus. Comp. Biochem. Phys. C 106, 333–336. Chandrasekara, L.W.H.U., Pathiratne, A., 2007. Body size-related differences in the inhibition of brain acetylcholinesterase activity in juvenile Nile tilapia

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