June-Soo Park
email: jpark@dtsc.ca.gov

Olga Ioanna Kalantzi

Dianne Kopec

Myrto Petreas

Polychlorinated biphenyls (PCBs) and their hydroxylated metabolites (OH-PCBs) in harbor seal (Phoca vitulina) livers from San Francisco Bay, California and Gulf of Maine

Keywords: hydroxylated PCB metabolites, polychlorinated biphenyls, harbor seal liver, San Francisco Bay, Gulf of Maine

Bioaccumulation of endocrine disruptors in marine mammals positioned at the top of the food chain is of toxicological concern. Stranded four pup and ten adult harbor seal (Phoca vitulina) livers were collected from San Francisco Bay and the Gulf of Maine and analyzed for polychlorinated biphenyls (PCBs) and their hydroxylated metabolites (OH-PCBs). We used GC-ECD and GC-NCI/MS to investigate the presence of 28 PCBs and 8 OH-PCB metabolites, respectively. 28 PCB concentrations (di-to octa-CBs) ranged from 1.81 to 35.9 µg/g lipid with a median of 6.53 for the seal pups and 2.31 to 249 µg/g lipid with a median of 28.9 for the adult seals. 8 OH-PCB concentrations (penta-to hepta-OH-PCBs) ranged from 0.02 to 0.69 µg/g lipid with a median of 0.04 for the adult seals, i.e., at much lower concentrations than those for PCBs. Ratios of OH-PCBs to PCBs (0.24% on average) were comparable to those in beluga whale, but were lower than ratios in human livers. The OH-PCB profiles were slightly different between SFB and GOM seal livers although similar PCB congener patterns were observed. Generally, 4-OH-CB107 was found predominantly in seal livers and was the only OH-PCB detectable in most of seal pup livers. This study provides information on OH-PCBs in seals, adding to the scarce exposure data for these chemicals.

Introduction

Polychlorinated biphenyls (PCBs) are known endocrine disruptors that are persistent and lipophilic, and biomagnify through the food chain [START_REF] Wan | Characterization of trophic transfer for polychlorinated dibenzo-p-dioxins, dibenzofurans, non-and mono-ortho polychlorinated biphenyls in the marine food web of Bohai Bay, north China[END_REF]. Since marine mammals such as seals are positioned at the top of the marine food web and have a relatively long life span (25-30 years), they are exposed to organochlorines (Neale et al., 2005a;[START_REF] Shaw | Polychlorinated biphenyls and chlorinated pesticides in harbor seals (Phoca vitulina concolor) from the northwestern Atlantic coast[END_REF] and are consequently vulnerable to endocrine disruptive effects such as reproductive impairment [START_REF] Reijnders | Reproductive failure in common seals feeding on fish from polluted coastal waters[END_REF][START_REF] Reddy | Opportunities for using Navy marine mammals to explore associations between organochlorine contaminants and unfavorable effects on reproduction[END_REF], cancer [START_REF] Martineau | Cancer in wildlife, a case study: Beluga from the St. Lawrence estuary, Quebec, Canada[END_REF][START_REF] Ylitalo | The role of organochlorines in cancer-associated mortality in California sea lions (Zalophus californianus)[END_REF], thyroid alteration [START_REF] Sormo | Thyroid hormone status in gray seal (Halichoerus grypus) pups from the Baltic Sea and the Atlantic Ocean in relation to organochlorine pollutants[END_REF][START_REF] Tabuchi | PCB-related alteration of thyroid hormones and thyroid hormone receptor gene expression in free-ranging Harbor seals (Phoca vitulina)[END_REF], and immune suppression/infectious diseases [START_REF] Ross | The role of immunotoxic environmental contaminants in facilitating the emergence of infectious diseases in marine mammals[END_REF][START_REF] Beckmen | Organochlorine contaminant exposure and associations with hematological and humoral immune functional assays with dam age as a factor in free-ranging northern fur seal pups (Callorhinus ursinus)[END_REF]Hall et al., 2006;[START_REF] Hammond | Comparison of polychlorinated biphenyl (PCB) induced effects on innate immune functions in harbor and grey seals[END_REF]. PCBs are stored in the insulating blubber (lipid) and can be subsequently remobilized to the blood through lipid metabolism during the seasonal fasting [START_REF] Debier | Mobilization of PCBs from blubber to blood in northern elephant seals (Mirounga angustirostris) during the post-weaning fast[END_REF], posing a health risk.

PCB metabolites; hydroxyl and methylsulfone PCBs (OH-and MeSO 2 -PCBs) exert similar toxicological effects to PCBs in mammals, particularly thyroid alteration in laboratory rats [START_REF] Meerts | Placental transfer of a hydroxylated polychlorinated biphenyl and effects on fetal and maternal thyroid hormone homeostasis in the rat[END_REF], suggesting the need for their inclusion in risk assessment studies. Marine mammals, including seals, are capable of producing cytochrome P450 enzymes [START_REF] Teramitsu | Identification of novel cytochrome P450 1A genes from five marine mammal species[END_REF] that biotransform PCBs to more soluble/excretable forms, i.e., OH-PCBs. Accordingly, 837 mono OH-PCB congeners can be theoretically formed in the body via the mechanisms of arene oxidation/1,2 shift and/or direct oxygen insertion [START_REF] Letcher | Methylsulfon and hydroxylated metabolites of polychlorinated biphenyls[END_REF]. While most of the OH-PCBs tend to be excreted [START_REF] Letcher | Methylsulfon and hydroxylated metabolites of polychlorinated biphenyls[END_REF], some specific OH-PCBs have been retained predominantly in the blood of humans [START_REF] Fängström | Hydroxylated PCB metabolites and PCBs in serum from pregnant Faroese women[END_REF]Park et al., 2007a) and marine mammals [START_REF] Hoekstra | Hydroxylated and methylsulfone-containing metabolites of polychlorinated biphenyls in the plasma and blubber of bowhead whales (Balaena mysticetus)[END_REF][START_REF] Houde | Polychlorinated biphenyls and hydroxylated polychlorinated biphenyls in plasma of bottlenose dolphins (Tursiops truncatus) from the Western Atlantic and the Gulf of Mexico[END_REF] due to their preferential binding to blood proteins [START_REF] Letcher | Methylsulfon and hydroxylated metabolites of polychlorinated biphenyls[END_REF]. OH-PCBs are also retained in the livers of rats [START_REF] Bergman | Selective Retention of Hydroxylated Pcb Metabolites in Blood[END_REF], humans [START_REF] Guvenius | Metabolites of polychlorinated biphenyls in human liver and adipose tissue[END_REF], and beluga whales [START_REF] Mckinney | Organohalogen contaminants and metabolites in beluga whale (Delphinapterus leucas) liver from two Canadian populations[END_REF], presumably by mechanisms similar to pentachlorophenol conjugation to liver fatty acids and/or liver protein/enzymes [START_REF] Leighty | Conjugation of pentachlorophenol to palmitic acid by liver-microsomes[END_REF]. A set of structurally known OH-PCB metabolites preferentially retained in the body have an OH-group bound to a para carbon of a phenyl ring and chlorine atoms bound to carbon adjacent to the OH-group [START_REF] Lans | Structure-dependent, competitive interaction of hydroxy-polychlorobiphenyls, hydroxy-dibenzo-p-dioxins and hydroxy-dibenzofurans with human transthyretin[END_REF].

In this pilot research study we used liver samples collected from stranded, dead harbor seals from San Francisco Bay (SFB) and from an island off the Gulf of Maine (GOM). Although the causes of death of the harbor seals stranded on SFB and GOM shores were not known, it is possible that liver accumulation of PCBs and their metabolites may have contributed directly/indirectly to some causative effects, such as infectious disease and liver dysfunction. Thus, the circulation and bioaccumulation of these contaminants in the liver of seals is of concern.

Therefore, we investigated the distribution of possible PCB precursors and OH-PCB metabolites retained in liver tissues of harbor seals stranded in the SFB and the GOM coast where there are the scarce exposure data for OH PCBs in seals which often carry high PCB body burden (tens of ppm). The present study was also undertaken to provide data to compare contaminant exposure in other marine mammals and humans.

Materials and methods

Seal liver tissue sampling

Seal liver samples analyzed in this study were collected from SFB as part of a larger study that examined harbor seal population dynamics, health, contaminant residues and prey selection [START_REF] Kopec | Toxic pollutants, health indices and population dynamics of harbor seals in San Francisco Bay, 1989-1992[END_REF]. Liver samples were collected from stranded, dead harbor seals (Phoca vitulina) between 1989 and 1998 found along the shores SFB, an urban/industrialized estuary that hosts a year-round resident population of about 500 harbor seals. Liver tissues were also collected from stranded harbor seals between 2002 and 2003 from Mt. Desert Rock, an isolated island 20 miles offshore of the central Maine coast. Seals hauling at Mt. Desert Rock are part of the larger regional seal population in the GOM numbering over 74,000 harbor seals. As a pilot study, a subset of four pup and five adult seal livers from SFB and five male seal livers from GOM were included in the present study and analyzed for PCBs and OH-PCBs, as summarized in Table 1. All harbor seal tissue collections were authorized by the National Marine Fisheries Service (NMFS) in coordination with local stranding network facilities (College of the Atlantic, Bar Harbor, Maine and Museum of Vertebrate Zoology, University of California, Berkeley, California). SFB samples consisted of males and females while all GOM livers were from males. All seal liver samples were collected during necropsy and were immediately placed in clean vials (I-Chem) at -20 o C prior to analysis [START_REF] Kopec | Toxic pollutants, health indices and population dynamics of harbor seals in San Francisco Bay, 1989-1992[END_REF]. Seals were grouped by age class based on standard length [START_REF] Bigg | The harbour seal in British Columbia[END_REF].

Chemicals and standards

The following OH-PCB standards were purchased from Wellington Laboratory (TerraChem Inc., USA) and used as reference standards for the identification and quantification of the analytes: 2,3,3',4',5-pentachlorobiphenyl-4-ol (4-OH-CB107), 2,2',3,4',5,2,2',3',4,4 synthesized in hexane by using N-nitroso-N-methylurea (Sigma-Aldrich, USA) as described elsewhere [START_REF] Sandau | Analytical chemistry of hydroxylated metabolites of PCBs and other halogenated phenolic compounds in blood and their relationship to thyroid hormone and retinol homeostasis in humans and polar bears[END_REF]. Other chemicals and solvents used for the analysis include dichloromethane and hexane (trace analysis, Burdick & Jackson), methanol, methyl-tert butyl ether, and water (HPLC grade, Fisher Sci., USA), 2-propanol (99.9%, pesticide grade, Fisher Sci., USA), hydrochloric acid, sulfuric acid (98%), potassium hydroxide, potassium chloride, sodium hydroxide, and ethyl alcohol (94-96%, 200 proof) (Fisher Sci., USA), silica (200-400 mesh) (Sigma-Aldrich, USA). Organics in Cod liver Oil (SRM1588b, National Institute of Standards and Technology, Gaithersburg, MD) was used as a standard reference material for PCB analysis.

Analysis and clean up

The analyses were conducted at the ultra-clean laboratory of the Department of Toxic Substances Control, Berkeley, CA. The analytical method was modified from analyses of seal blubber [START_REF] She | PBDEs in the San Francisco Bay Area: measurements in harbor seal blubber and human breast adipose tissue[END_REF] and blood serum (Park et al., 2007a). In summary, liver samples (0.4-3 g) along with a method blank (sodium sulfate), and a standard reference material (NIST 1588b) were homogenized, spiked with PCB and OH-PCB surrogate standards (1-5 ng and 1 ng, respectively), and extracted with a mixture of methyl-tertbutyl ether (MTBE):hexane (1:1, v/v) (75 mL) with 2-propanol (10 mL). The samples went through two more extractions by successive shaking and centrifuging with MTBE:hexane mixture. Lipid content was determined gravimetrically using 7% (v/v) of the extracts. The extracts were treated with 1% KCl wash and KOH phase separation.

Only 7% (v/v) of the organic extracts were used for the PCB analysis to avoid saturation of the ECD. The phenolic compounds retained in the KOH solution were re-protonated by using 2M HCl (pH<2), extracted with MTBE:hexane (1:9, v/v) (4 mL), and derivatized overnight by using diazomethane. The organic extract was cleaned up by using concentrated sulfuric acid (98%). Further clean up and fractionation was conducted on the Pasteur pipette column packed with acidic silica gel (1:2 w/w, 0.6g) and activated silica (0.4g). The columns were eluted with 5 mL hexane and then 10 mL dichloromethane:hexane (1:1). The OH-PCB derivatives were cleaned up with concentrated sulfuric acid (98%), followed by passing through a Pasteur pipette column packed with sulfuric acidic silica gel (0.5g) and activated silica (0.1g). Injection standards were added before GC analysis.

Instruments

Twenty eight PCB congeners 49,52,56,66,70,74,,99,101,105,110,113,118,137,138,146,153,156,157,170,177,180,183,187,189,190,194,203) were -28, 49, 52,66, 70, 74, 101, 105, 110, 118, 138, 146, 153, 156, 157, 177, 180, 183, 187 and 194 were quantitated on the RTX-5MS column while 170,190,199,and 203 were quantitated on the DB-XLB column. We tested precision for the PCB and OH-PCB analytical methods as well as the gravimetric lipid determination by using duplicate liver samples within and between batches. We tested accuracy by using the SRM samples. Both precision and accuracy were within reasonable error ranges (±25%).

analyzed
To monitor the performance of the experimental procedures, 2.00 ng of 4'-OH-CB159, which, to our knowledge, has not been detected in any animal liver to date, were added to

Results and discussion

In Table 2 we report the ranges and medians of PCBs and OH-PCBs (on a lipid weight basis) measured in harbor seal liver tissues for four pups and five adults from SFB and five adult males from GOM.

PCBs

Concentrations of PCBs measured from SFB seal pup livers ranged from 1.81 to 35.9 µg/g fat while adult seal livers showed a wider range (2.31-249 µg/g fat). Because of the limited sample size we did not examine the impact of gender on SFB samples and geographical comparisons. In other study, females tend to have lower contaminant burdens than males since reproductively active females can transfer a significant portion of their PCB body burden to their offsprings through gestation or nursing (Neale et al., 2005b). During the winter, GOM seals may migrate southward along the coast to urban/industrialized areas (e.g., Massachusetts Bay) [START_REF] Waring | Short-term movements of radiotagged harbor seals in New England[END_REF] where they may be exposed to elevated PCB levels, while SFB seals reside in the Bay for most of the time.

CB-153 was the primary congener in both SFB and GOM adult seal livers, comprising 22 and 31% on average of 28 PCBs, respectively ( [START_REF] Mckinney | Organohalogen contaminants and metabolites in beluga whale (Delphinapterus leucas) liver from two Canadian populations[END_REF]. Although the number of congeners measured was different between the McKinney and our study, most PCB congeners predominantly found in the liver matrices, as well as the sample collection time, overlapped. In our study, 8 out of 14 seal livers (1 SFB male pup, 1 SFB female adult, 3 SFB male adults and 3 GOM male adults) showed PCB levels within or exceeding the estimated threshold level for adverse effects on immune/reproductive function (17-77 µg/g blubber fat) in aquatic animals as summarized elsewhere [START_REF] Shaw | Polychlorinated biphenyls and chlorinated pesticides in harbor seals (Phoca vitulina concolor) from the northwestern Atlantic coast[END_REF]. Seal liver (this study) and blubber PCB concentrations (Park et al., unpublished data) showed correlation (r=0.77), which is consistent with other studies [START_REF] Wolkers | Tissue-specific accumulation and lactational transfer of polychlorinated biphenyls, chlorinated pesticides, and brominated flame retardants in hooded seals (Cistophora cristata) from the Gulf of St. Lawrence: Applications for monitoring[END_REF]. Thus, it is possible that some of the stranded harbor seals, particularly one SFB seal pup with high PCB liver accumulation, might have suffered PCB-related adverse effects, such as infectious diseases and/or the conditions of the animals presented in Table 1.

OH-PCB metabolites

Although literature searches indicated that most of the OH-PCBs are retained in blood, we attempted to measure OH-PCBs in liver because major enzyme-mediated biotransformation of PCBs occurs in the liver. OH-PCBs were detected in almost all liver tissues of SFB and GOM harbor seals including SFB seal pups. 4-OH-CB107 was detected in almost all samples, followed by 4-OH-CB187 (67%), 4-OH-CB146 (60%), 3'-OH-CB138 (60%), and 3-OH-CB153 (47%). As shown in Figure 1 Several unidentified OH-PCB peaks were present in the SFB seal liver chromatogram.

Particularly, two peaks (a hexa and a hepta OH-PCB) observed between the peaks of 4'-OH-CB159 (internal standard) and 3'-OH-CB180 were fairly notable. When these unidentified OH-PCBs were quantified using response factors for 4-OH-CB107 and 4-OH-CB187 standards representing each hydroxyl homologue group, they comprised 16-27% (22% on average) of OH-PCBs (sum of identified and unidentified).

The harbor seal liver concentrations of OH-PCB metabolites (0.020~0.693 µg/g fat) in this study had a wider range than what has been reported for humans (0.007~0.175 µg/g fat) [START_REF] Guvenius | Metabolites of polychlorinated biphenyls in human liver and adipose tissue[END_REF] and beluga whales (<0.5-0.145 µg/g fat) [START_REF] Mckinney | Organohalogen contaminants and metabolites in beluga whale (Delphinapterus leucas) liver from two Canadian populations[END_REF] due to one extreme value. Excluding that value, the range narrowed (0.020~0.064 µg/g fat) to previously reported levels. The 8 OH-PCB levels identified in the harbor seals were much lower (~0.59 % of 28 PCBs, 0.24% on average) than those of parent PCBs. It should be noted that the sums of 28 PCBs includes congeners which are not precursors of 8 OH-PCBs and that, in addition, the presence of many OH-PCB congeners below the detection limits may bias their sum. These ratios were comparable to data from beluga whales in St. Lawrence River, but they were lower than data in human livers (usually between 1-10%). It is possible that these lower ratios in marine mammals relative to humans may be due to a reduced capacity in marine mammals to metabolize PCBs, or a higher Phase II conjugation process which rapidly depletes OH-PCBs [START_REF] Mckinney | Organohalogen contaminants and metabolites in beluga whale (Delphinapterus leucas) liver from two Canadian populations[END_REF]. This OH-PCB/PCB ratio was even higher in human blood (Park et al., 2007a,b) and marine mammal blood [START_REF] Hoekstra | Hydroxylated and methylsulfone-containing metabolites of polychlorinated biphenyls in the plasma and blubber of bowhead whales (Balaena mysticetus)[END_REF][START_REF] Houde | Polychlorinated biphenyls and hydroxylated polychlorinated biphenyls in plasma of bottlenose dolphins (Tursiops truncatus) from the Western Atlantic and the Gulf of Mexico[END_REF], indicating that OH-PCB metabolites preferentially bind to blood protein relative to liver fatty acids and/or hepatic proteins and enzymes. Since we did not measure lower chlorinated OH-PCBs (containing fewer than four chlorines) and also did not include the ones detected but not identified, we may have underestimated the total concentrations of OH-PCB metabolites in seal livers.

The OH-PCB profiles are presented in the bottom of Figure 2. This profile was slightly different between SFB and GOM seal livers although similar PCB congener patterns were observed in the two regions (Figure 2,top). This difference may result from biological (e.g., nutritional/reproductive status, age, gender, polymorphism) and environmental factors (e.g., temperature, migration) influencing enzyme activities and thereby metabolite formation in seals. For example, [START_REF] Wolkers | Biomarker responses and decreasing contaminant levels in ringed seals (Pusa hispida) from Svalbard, Norway[END_REF] reported that EROD activity negatively correlated to blubber content in seals, indicating contaminants get mobilized from blubber during fasting, and possibly induce the Phase I and II enzyme system. Seals also go through other seasonal lipid changes via molting and reproduction.

However, the contributions from other various OH-PCB exposure pathways including diet intake/food chain accumulation [START_REF] Campbell | Hydroxylated PCBs and other chlorinated phenolic compounds in lake trout (Salvelinus namaycush) blood plasma from the Great Lakes Region[END_REF], and even the abiotic aquatic environments [START_REF] Ueno | Detection of hydroxylated polychlorinated biphenyls (OH-PCBs) in the abiotic environment: Surface water and precipitation from Ontario, Canada[END_REF] are probably minor, as observed in a recent study [START_REF] Verreault | Comparative fate of organohalogen contaminants in two top carnivores in Greenland: Captive sledge dogs and wild polar bears[END_REF].

Although the profiles of OH-PCB congeners varied from sample-to-sample, 4-OH-CB107 was the dominant congener, similar to the results from the St. Lawrence River beluga whales [START_REF] Mckinney | Organohalogen contaminants and metabolites in beluga whale (Delphinapterus leucas) liver from two Canadian populations[END_REF]. This was the only congener detected in three SFB seal pup livers in this study, which raises concern due to its relationship to thyroid dysfunction reported for the experimental rat fetus [START_REF] Meerts | Placental transfer of a hydroxylated polychlorinated biphenyl and effects on fetal and maternal thyroid hormone homeostasis in the rat[END_REF]. The known possible PCB precursors (CB-118 and CB-105) of 4-OH-CB107 in animals [START_REF] Letcher | Methylsulfon and hydroxylated metabolites of polychlorinated biphenyls[END_REF] were also found in our samples, albeit they were not among the most dominant congeners. With 3-OH-CB153, 4-OH-CB146, 3'-OH-CB138, and 4-OH-CB187, they comprised 66 to 81% of OH-PCBs (identified and unidentified penta, hexa, and hepta OH-PCBs). They are the OH-PCB congeners primarily found in the blood of humans (Park et al., 2007a,b) and wildlife [START_REF] Park | Hydroxylated metabolites of polychlorinated biphenyls (PCBs) in California wild birds[END_REF] since their chemical structure preferentially binds to the blood transthyretin (TTR) receptor protein. This binding involves a hydroxyl group in either the para-or meta-position of a biphenyl ring, adjacent to chlorine atoms on both sides [START_REF] Lans | Structure-dependent, competitive interaction of hydroxy-polychlorobiphenyls, hydroxy-dibenzo-p-dioxins and hydroxy-dibenzofurans with human transthyretin[END_REF][START_REF] Letcher | Methylsulfon and hydroxylated metabolites of polychlorinated biphenyls[END_REF]. This also seems to apply to the seal liver matrix, indicating interaction of those OH-PCBs with liver enzyme/proteins as well as fatty acid conjugation. 4'-OH-CB130 was reported at considerable levels in human livers [START_REF] Guvenius | Metabolites of polychlorinated biphenyls in human liver and adipose tissue[END_REF] while it was not detected or detected at trace levels in our seal livers.

The pairs of PCB precursors and respective OH-PCB metabolites were correlated.

Since correlations could be driven by a single extreme value, that value was removed and the resulting unbiased correlations were: CB-153/3-OH-CB146 (r=0.67, p<0.05), CB-138/3-OH-CB138 (r=0.74, p<0.05), CB-187/4-OH-CB187 (r=0.66, p<0.05). However, the pairs of CB-118/4-OH-CB107 and CB-105/4-OH-CB107 showed weaker correlations (r=0.24 and 0.32, respectively), possibly because 4-OH-CB107 is labile [START_REF] Malmberg | Pharmacokinetics of two major hydroxylated polychlorinated biphenyl metabolites with specific retention in rat blood[END_REF]). In the body, PCBs are metabolized via the various transformation pathways mediated by the cytochrome P450 enzyme series (CYP1A, CYP2B, CYP3A).

Preferential CYP450 enzyme mediated-metabolic capacity of marine mammals towards OH-PCBs depends on the number and positioning of the chlorine atoms around the biphenyl ring [START_REF] Boon | Concentrationdependent changes of PCB patterns in fish-eating mammals: Structural evidence for induction of cytochrome P450[END_REF][START_REF] Li | Hepatic microsomal cytochrome P450 enzyme activity in relation to in vitro metabolism/inhibition of polychlorinated biphenyls and testosterone in Baltic grey seal (Halichoerus grypus)[END_REF][START_REF] Mckinney | Characterization and profiling of hepatic cytochromes P450 and phase II xenobiotic-metabolizing enzymes in beluga whales (Delphinapterus leucas) from the St. Lawrence River Estuary and the Canadian Arctic[END_REF]. For example, CB-118 and 105, having only ortho-and meta-unsubstituted sites and one or fewer ortho-Cl atoms, were subject to the CYP1A type enzyme-mediated metabolism, while CB-52, 49, 101 and 110, with meta-and para-vicinal H-atoms, were preferentially metabolized by CYP2B or CYP3A types of enzymes

We here report several OH-PCBs retained in harbor seal livers in quantifiable amounts. This is one of a few studies available on OH-PCB metabolites detected in liver tissues; rats [START_REF] Bergman | Selective Retention of Hydroxylated Pcb Metabolites in Blood[END_REF][START_REF] Haraguchi | Hydroxylation and methylthiolation of mono-ortho-substituted polychlorinated biphenyls in rats: Identification of metabolites with tissue affinity[END_REF], humans [START_REF] Guvenius | Metabolites of polychlorinated biphenyls in human liver and adipose tissue[END_REF], beluga whales [START_REF] Mckinney | Organohalogen contaminants and metabolites in beluga whale (Delphinapterus leucas) liver from two Canadian populations[END_REF], glaucous gulls [START_REF] Verreault | Organohalogen and metabolically-derived contaminants and associations with whole body constituents in Norwegian Arctic glaucous gulls[END_REF], polar bears [START_REF] Gebbink | Tissue-specific congener composition of organohalogen and metabolite contaminants in East Greenland polar bears (Ursus maritimus)[END_REF], and predatory birds [START_REF] Jaspers | Predatory bird species show different patterns of hydroxylated polychlorinated biphenyls (HO-PCBs) and polychlorinated biphenyls (PCBs)[END_REF]. Data on seal liver OH-PCB metabolites are especially valuable because of the difficulties in sampling, the rarity of the sample itself, and the complexity of liver analyses for OH-PCB metabolites.

There are no reports available to date on the toxicological index for hepatic OH-PCB metabolites. However, due to their endocrine disrupting properties, they should be included in the risk assessment of marine mammals' health.

Summary and conclusions

PCBs and OH-PCB metabolites were detected in livers of harbor seals from both the San Francisco Bay and the Gulf of Maine. OH-PCB metabolites were retained in seal liver, albeit at lower OH-PCBs to PCBs ratios compared to human liver, possibly due to a lower metabolic expression of CYP enzymes. The OH-PCB profiles were slightly different between SFB and GOM seal livers although similar PCB congener patterns were observed between the two regions. In general, 4-OH-CB107 was detected predominantly in seal livers and was the only OH-PCB detectable in most seal pup livers, raising toxicological concerns. These results should be treated with caution, as they are based on a small sample size. They do, however, highlight the need to include OH-PCB metabolites in the risk assessment of marine mammals, due to their potential endocrine disrupting properties. Further investigations are needed to better understand the relationship between exposure to these endocrine disruptors and possible health effects in seals.

Figure captions

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  ol (3'-OH-CB180), 2,2',3,3',4',5,5'-heptachlorobiphenyl-4-ol (4'-OH-CB172). 2,2',4,4',5,5'-hexachlorobiphenyl-3-ol (3-OH-CB153) was provided by Professor Åke Bergman (Stockholm University, Sweden). The numbering of PCBs and OH-PCBs is based on that specified by Ballschmitter and Zell (1980) and Letcher et al (2000), respectively. Internal surrogate standards for PCBs and OH-PCBs were CB-14, 65 and 166 for PCBs and 4'-OH-CB159 for OH-PCBs. Injection standards for PCBs and OH-PCB analyses were CB-30 and 204, and CB-209, respectively. Diazomethane was

  on a Varian 3800 gas chromatograph (GC) with 63 Ni electron capture detection (Varian Inc., Walnut Creek, CA) equipped with dual capillary columns; RTX-5MS (60m × 0.25 mm i.d., 0.25 µm thickness, Restek, Bellefonte, PA) and DB-XLB (60m × 0.25 mm i.d., 0.25 µm thickness, J&W Scientific, Folsom, CA). Carrier and make up gases were helium and nitrogen, respectively and set in constant flow mode. Injection (2 µL) was made in splitless mode with an injector temperature of 280 o C. The initial GC temperature was set to 80 o C and held for 1.6 min followed by a 15 o C/min increase to 135 o C, 1 o C/min to 261 o C, 3 o C/min to 295 o C and 1 o C/min to 300 o C held for 3.5 min. Post run was set to 320 o C for 1 min. Eight OH-PCBs were determined as methyl derivatives by using a Varian 3800 gas chromatograph equipped with a 1200L mass spectrometer (MS) (Varian Inc., Walnut Creek, CA. The MS was operated in negative chemical ionization (NCI) mode with electron energy of 70 eV and emission current of 300 µA using selected ion monitoring (SIM). The GC was equipped with a DB-5MS capillary column (60m × 0.25 mm i.d., 0.25 µm thickness, J&W Scientific, Folsom, CA, USA). Injection (2 µL) was made in split/splitless mode with an injector temperature of 250 o C. The initial GC temperature was set to 80 o C and held for 2 min followed by a 50 o C/min increase to 200 o C, 1 o C/min to 230 o C, and 30 o C/min to 300 o C and held for 4 min. Post run was set to 320 o C for 1 min. The temperatures for both ion source and quadrupole were set to 150 o C. We monitored the most intense ions, either molecular ion or fragmentation ion; [M -] for 4'-MeO-CB159, [(M+2-CH 3 ) -] for 4-MeO-CB187 and 4'-MeO-CB172, and [(M-HCl) -] or [(M+2-HCl) -] for the rest of the congeners. Carrier and reagent gases were helium and methane, respectively. 2.5. Quantification and QA/QC All glassware were washed, rinsed with acetone and hexane, and baked at 550 o C for 8 hours. Each batch consisted of one procedural blank, one SRM, and six samples. The five points of external calibration curves were used for the quantifications of PCBs and OH-PCBs. CB

  all samples. We derivatized OH-PCB standards simultaneously with the sample extracts for more accurate quantification. Any values lower than the LOQ (~0.48 ng/g fat) were replaced by LOQ/2 when calculating summary statistics. We conducted non-parametric tests (e.g., Spearman correlation) for PCB and OH-PCB data to assess their relationships by using Minitab statistical software.The average (± standard deviation) surrogate recoveries of CB-14, CB-65, and CB-166 for PCB analysis were 92±6%, 82±7%, and 82±19%, respectively. The recoveries from Standard Reference Materials (SRM 1588b Organics in Cod Liver Oil) ranged from 63% for CB-194 to 125% for CB-170. The average (± standard deviation) recovery of the OH-PCB surrogate (4'-OH-CB159) was 77±15%.

  , the two OH-PCB chromatograms analyzed as MeO-PCBs were presented for one of the calibration standards (top) and a SFB adult male seal liver (bottom). The standard chromatogram indicates that our GC temperature program resolved the possible co-elutions (e.g., 3-OH-CB153/4-OH-CB146, 3'-OH-CB138/4'-OH-CB130, and 3'-OH-CB180/4'-OH-CB172).

Fig. 1 .

 1 Fig. 1. GC chromatograms of calibration standard (top) and identified and unidentified

Fig. 2 .

 2 Fig. 2. Profiles of PCB precursors (top) and OH-PCB metabolites (bottom) observed in

Table 2

 2

	) and was a good

GOM, respectively. The nine PCB congeners presented in Table

2

accounted for more than 80% of 28 PCBs in SFB and GOM seal livers. The average concentrations of 28 PCBs measured in GOM harbor seal livers (28.3 ug/g fat) were comparable to 38 PCBs measured in beluga whale livers (31.9 ug/g fat) from the St. Lawrence River

Table 1

 1 Information of seal liver samples collected from San Francisco Bay (SFB) and Gulf of Maine (GOM)

	!	"	"

*length (cm)/weight (kg) to roughly quantify blubber thickness and general health.

Table 2

 2 Concentrations of PCBs and OH-PCBs measured from livers of dead and stranded harbor seals along SanFrancisco Bay (SFB) and Gulf of Maine (GOM)

			San Francisco Bay Seals			Gulf of Maine Seals
			Pups (N=4)		Adults (N=5)			Adults (N=5)
		min	max	median	min	max median	min	max	median
	PCBs (µg/g lipid)
	PCB118	0.02	0.48	0.07	0.03	0.32	0.17	0.03	0.19	0.08
	PCB146	0.03	1.52	0.24	0.05	8.88	1.36	0.24	2.84	0.89
	PCB153	0.18	8.58	1.32	0.24	67.3	9.26	1.45	22.2	7.27
	PCB105	0.001	0.15	0.03	0.01	0.12	0.06	0.003	0.07	0.04
	PCB138	0.16	6.13	0.97	0.23	38.9	5.39	1.09	13.5	4.23
	PCB187	0.09	3.45	0.68	0.62	38.6	4.53	0.37	6.73	2.25
	PCB183	0.04	1.37	0.22	0.06	9.77	1.23	0.18	1.99	0.69
	PCB180	0.11	4.00	0.60	0.15	42.3	3.86	0.39	4.58	2.39
	PCB170	0.04	1.72	0.39	0.07	10.0	1.40	0.29	2.22	0.83
	28 PCBs	1.81	35.9	6.53	2.31	249	35.6	5.60	63.7	22.4
	OH-PCBs (µg/g lipid)
	4-OH-CB107	<0.001 0.018	0.004 <0.001 0.123 0.026	0.008	0.033	0.017
	3-OH-CB153	<0.001 <0.001 <0.001 <0.001 0.036 <0.001 <0.001 0.006	0.002
	4-OH-CB146	<0.001 <0.001 <0.001 <0.001 0.218 0.002	0.003	0.018	0.008
	3'-OH-CB138	<0.001 0.003 <0.001 <0.001 0.104 <0.001 0.002	0.013	0.005
	4'-OH-CB130	<0.001 0.001 <0.001 <0.001 0.034 <0.001 <0.001 0.002 <0.001
	4-OH-CB187	<0.001 0.011 <0.001 0.013 0.149 0.019	0.004	0.019	0.008
	3'-OH-CB180	<0.001 <0.001 <0.001 <0.001 0.017 <0.001 <0.001 <0.001 <0.001
	4'-OH-CB172	<0.001 <0.001 <0.001 <0.001 0.011 <0.001 <0.001 <0.001 <0.001
	8 OH-PCBs	0.004	0.034	0.004	0.017 0.693 0.045	0.019	0.064	0.045

Acknowledgement and disclaimer

The authors would like to thank Joginder Dhaliwal and Dr. Reber Brown of the Department of Toxic Substances Control for their valuable analytical expertise. The ideas and opinions expressed herein are those of the authors and do not necessarily reflect the official position of the California Department of Toxic Substances Control.


Azra Bozcaarmutlu
email: bozcaarmutlu_a@ibu.edu.tr

Canan Sapmaz

Zuleyha Aygun

Emel Arinç

Assessment of pollution in the west black sea coast of turkey using biomarker responses in fish

Keywords: Black Sea, Biomonitoring, CYP1A, Catalase, Chemical pollution, EROD, Glutathione S-transferase, Mullet

Assessment of pollution in the west black sea coast of turkey using biomarker responses in fish

Azra Bozcaarmutlu, Canan Sapmaz, Zuleyha Aygun, Emel Arinç

INTRODUCTION

The Black Sea has been increasingly threatened by pollutants over the past decades as a result of accidental crude oil spills, dumping of toxic industrial wastes, discharge of domestic wastes from coastal settlements and industrial and domestic pollutants carried by rivers. Some industrial areas, rivers and coastal cities in the Black Sea have tendency to create local pollution [START_REF] Mee | The Black-Sea in crisis-A need for concerted international action[END_REF]. The Sakarya River, from its source in the West Anatolia, flows through many industrial and agricultural areas and drains into the Black Sea. It carries many pollutants to the Black Sea from inner regions of Turkey [START_REF] Tuncer | Land-based sources of pollution along the Black Sea coast of Turkey: concentrations and annual loads to the Black Sea[END_REF][START_REF] Barlas | A pilot study of heavy metal concentration in various environments and fishes in the upper Sakarya River basin, Turkey[END_REF]. Industrial areas found in Düzce and Adapazarı drain their discharges into Efteni Lake. The Melen Stream originates from this lake and flows through many industrial and agricultural areas. The Gülüç Stream is very close to the Ere li Harbour and Ere li Iron and Steel Factory. This small stream was highly polluted primarily by domestic waste discharges from Ere li and villages around the stream. Zonguldak and Ere li, with increasing population, shipping activities, uncontrolled discharges from industries and coastal settlements, are among the most polluted regions in the Black Sea. Zonguldak is also an important coal mining area in Turkey. It suffers from particulate discharges from the thermal power plant and coal processing wastes, resulting in permanently elevated turbidity levels along the coast of the city [START_REF] Tuncer | Land-based sources of pollution along the Black Sea coast of Turkey: concentrations and annual loads to the Black Sea[END_REF].

Chemical analyses of the sea water and sediment give valuable information about the levels of chemical pollutants. However, organisms are often exposed to complex mixtures of pollutants, including polychlorinated biphenyls (PCBs), polychlorinated dibenzodioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), polyaromatic hydrocarbons (PAHs), alkyltin compounds, and metals. Chemical analyses do not reveal the impact of these pollutants on organisms. The use of biochemical markers fulfills this purpose. Induction of CYP1A (P4501A) and associated EROD activity are the best characterized and used biochemical markers of exposure of fish to organic pollutants such as PCBs, PAHs and dioxins. CYP1A induction provides a useful "tool" for the assessment of exposure to these types of organic pollutants and acts as an early warning sign for potential harmful effects of many organic molecules [START_REF] Payne | Review and perspective on the use of mixed function oxygenase enzymes in biological monitoring[END_REF][START_REF] Goksøyr | The cytochrome P450 in fish, aquatic toxicology and environmental monitoring[END_REF][START_REF] Bucheli | Induction of cytochrome P450 as a biomarker for environmental contamination in aquatic ecosystem[END_REF][START_REF] Stegeman | Diversity and regulation of cytochrome P450 in aquatic species[END_REF][START_REF] Addison | The use of biological effects monitoring in studies of marine pollution[END_REF][START_REF] Arinç | Hepatic cytochrome P4501A and 7-ethoxyresorufin O-deethylase induction in mullet and common sole as an indicator of toxic organic pollution in zmir Bay, Turkey[END_REF][START_REF] Miller | Hepatic CYP1A levels and EROD activity in English sole: biomonitoring of marine contaminants in Vancouver Harbour[END_REF][START_REF] Arinç | Cytochrome P4501A and associated MFO induction in fish as a biomarker for toxic, carcinogenic pollutants in the aquatic environment[END_REF][START_REF] Arinç | Induced CYP1A mRNA, protein and catalytic activity in the liver of feral fish, leaping mullet, Liza saliens[END_REF]. Glutathione-S-transferases (GSTs) catalyse the conjugation of electrophilic compounds (or phase I metabolites) with reduced glutathione (GSH) and are one of the main enzymes involved in xenobiotic phase II metabolism. GSTs detoxify a number of environmental carcinogens and epoxide intermediates [START_REF] Gallagher | Channel catfish glutathione S-transferase isoenzyme activity toward (±)-anti-benzo[a]pyrene-trans-7,8-dihydrodiol-9,10-epoxide[END_REF]. Glutathione S-transferase activity measurement is also used as a marker of oxidative stress [START_REF] Rodriguez-Ariza | Biochemical indicators of oxidative stress in fish from polluted Littoral AreasRodriguez-Ariza[END_REF][START_REF] Martínez-Gómez | Monitoring biomarkers in fish (Lepidorhombus boscii and Callionymus lyra) from the northern Iberian shelf after the Prestige oil spill[END_REF]. The induction of antioxidant enzyme activities represents a cellular defense mechanism to neutralize toxic effects of reactive oxygen species. Benzo[a]pyrene as well as other PAHs and PCBs have been shown to cause significant alterations in the activities of antioxidant enzymes in laboratory experiments with molluscs and fish [START_REF] Otto | 3,3´,4,4´-tetrachlorobiphenyl effects on antioxidant enzymes and glutathione status in different tissues of rainbow trout[END_REF]Moon, 1995, Van der Oost et al., 2003). Changes in the levels of phase II enzymes, glutathione Stransferase and antioxidant enzymes have also been proposed as biomarkers of contaminants in a variety of marine organisms, including invertebrates, mussels, and fish [START_REF] Otto | 3,3´,4,4´-tetrachlorobiphenyl effects on antioxidant enzymes and glutathione status in different tissues of rainbow trout[END_REF]Moon, 1995, Livingstone, 2001;[START_REF] Orbea | Antioxidant enzymes and peroxisome proliferation in relation to contaminant body bordens of PAHs and PCBs in bivalve molluscs, crabs and fish from the Urdaibai and Plentzia estuarios (Bay of Biscay)[END_REF][START_REF] Sen | Biochemical characterization and distribution of glutathione S-transferases in leaping mullet (Liza saliens)[END_REF][START_REF] Ferreira | Oxidative stress biomarkers in two resident species, mullet (Mugil cephalus) and flounder (Platichthys flesus), from a polluted site in River Douro Estuary, Portugal[END_REF][START_REF] Martínez-Gómez | Monitoring biomarkers in fish (Lepidorhombus boscii and Callionymus lyra) from the northern Iberian shelf after the Prestige oil spill[END_REF].

The studies concerning the effects of pollutants on the Black Sea ecosystem are relatively few [START_REF] Telli Karakoç | Differences between blood and liver aromatic DNA adduct formation[END_REF][START_REF] Bozcaarmutlu | Biomonitoring of the toxic pollutants by using mullet liver enzymes in the West Black Sea Region of Turkey[END_REF]Bozcaarmutlu et al., , 2008)). Monitoring biological effects by using biomarkers and their implementation into environmental monitoring programmes in the Black Sea fall behind the advanced studies taking place in the USA, Canada and most of the Western and Southern Europe. In this study, our aim was to determine and monitor the extent of pollution in the West Black Sea Coast of Turkey by measuring CYP1A associated EROD activity, phase II enzyme, glutathione S-transferase and antioxidant enzymes, catalase and glutathione reductase activities and immunochemical detection of CYP1A protein level in mullet liver. Besides these biochemical parameters, total PAH concentrations were determined in fish liver tissues to show the presence of one of the inducer organic pollutants in fish samples. Mullet is chosen as a test organism. It is an economically important marine fish due to the marketing of their meats and eggs. It is a pelagic fish inhabiting usually inshore regions, enters lagoons and estuaries along the Atlantic coasts to the north of Bay of Biscay, also the whole of the Mediterranean, Black Sea and Sea of Azov. Since mullet is resistant to pollutants, it is widely used in biomonitoring programs of contaminated waters [START_REF] Arinç | Hepatic cytochrome P4501A and 7-ethoxyresorufin O-deethylase induction in mullet and common sole as an indicator of toxic organic pollution in zmir Bay, Turkey[END_REF][START_REF] Arinç | Cytochrome P4501A and associated MFO induction in fish as a biomarker for toxic, carcinogenic pollutants in the aquatic environment[END_REF][START_REF] Ferreira | Organochlorine contaminants in flounder (Platichthys flesus) and mullet (Mugil cephalus) from Douro estuary, and their use as sentinel species for environment monitoring[END_REF]. Biomarker enzyme activities such as components of cytochrome P450 system, glutathione S-transferase and antioxidant enzymes have been well characterized in mullet (Sen and Arinç, 1998a, b;[START_REF] Bozcaarmutlu | Inhibitory effects of divalent metal ions on liver microsomal 7-ethoxyresorufin O-deethylase (EROD) activity of leaping mullet[END_REF][START_REF] Bozcaarmutlu | Mechanism of inhibition of purified leaping mullet (Liza saliens) NADPH-cytochrome P450 reductase by toxic metals: aluminum and thallium[END_REF], 2008;[START_REF] Ferreira | Organochlorine contaminants in flounder (Platichthys flesus) and mullet (Mugil cephalus) from Douro estuary, and their use as sentinel species for environment monitoring[END_REF][START_REF] Sen | Biochemical characterization and distribution of glutathione S-transferases in leaping mullet (Liza saliens)[END_REF][START_REF] Bozcaarmutlu | Mechanism of inhibition of purified leaping mullet (Liza saliens) NADPH-cytochrome P450 reductase by toxic metals: aluminum and thallium[END_REF][START_REF] Sen | Effects of metals and detergents on biotransformation and detoxification enzymes of leaping mullet (Liza saliens)[END_REF].

MATERIALS AND METHODS

Chemicals

Acrylamide, anti-rabbit IgG alkaline phosphatase antibody, -amino caproic acid ( -ACA), 7-ethoxyresorufin, N-2-hydroxyethylpiperazine-N'-2-ethansulphonic acid (HEPES), -mercaptoethanol, N,N'-methylene bisacrylamide, phenylmethanesulfonyl fluoride (PMSF), resorufin sodium salt were purchased from Sigma-Aldrich, Saint Louis, Missouri, USA. Bromophenol blue, N,N-dimethylformamide and nitro blue tetrazolium chloride were purchased from Merck, Darmstadt, Germany. 5-bromo-4-chloro-3indolylphosphate disodium salt (BCIP), glucose-6-phosphate dehydrogenase, -D-glucose-6-phosphate monosodium salt, polyethylene sorbitan monolaurate (Tween 20), sodium dodecyl sulfate (SDS) were purchased from Calbiochem, California, USA. Ammonium persulfate, N, N, N', N' tetrametylethylene diamine (TEMED), trans blot tranfer medium (pure nitrocellulose membrane) were purchased from Bio-Rad Laboratories, California, USA. -Nicotinamide adenine dinucleotide phosphate disodium salt (NADP + ),nicotinamide adenine dinucleotide, reduced form (NADPH), phenazine methosulfate (PMS) were purchased from Applichem, Darmstadt, Germany. All other chemicals were of analytical grade and were obtained from commercial sources at the highest grade of purity available.

Sampling Sites and Fish

The fish samples were collected in August 2005, August 2006 and August 2007 from six different locations of the West Black Sea Region of Turkey. The fish sampling stations were shown in Figure 1. As indicated in this figure, Amasra was used as the reference site. Three different species of mullet, namely so-iuy mullet (Mugil soiuy), flathead mullet (Mugil cephalus) and golden grey mullet (Liza aurata), each weighing 600-750 g, were captured by fish net.

Figure 1

Preparation of Tissue Samples

Fish were killed by decapitation and livers were removed immediately by avoiding the gall bladders and flash frozen in liquid nitrogen. Tissues were taken from liquid nitrogen and cut into two pieces without thawing. One of the pieces was wrapped by freezing bags and covered by aluminum foil and then put back into liquid nitrogen. This piece was later used in PAH analysis. The other piece of the liver tissue was used in the preparation of microsomes and thawed on ice. Liver microsomes were prepared as described before [START_REF] Adali | Electrophoretic, spectral, catalytic and immunochemical properties of highly purified cytochrome-P450 from sheep lung[END_REF][START_REF] Arinç | Characterization of cytochrome P450 dependent mixed function oxidase system of gilthead seabream (Sparus aurata; Sparidae) liver[END_REF] except that homogenization solution contained 10 mM EDTA, 0.25 mM PMSF and 0.25 mM -ACA. The homogenate was centrifuged at 13300×g for 40 minutes. The microsomes were sedimented from the supernatant by centrifugation at 70000xg for 60 minutes. The supernatant fraction (cytosol) was removed and stored at -80 º C until use. The microsomal pellet was resuspended in 1.15% KCl solution containing 10 mM EDTA and resedimented by ultracentrifugation at 70000xg for 50 minutes. The washed microsomes were resuspended in 10% glycerol containing 10 mM EDTA. Aliquots of microsomes were gassed with nitrogen and stored in liquid nitrogen until used. All the procedures involved in the preparation of microsomes and cytosols were performed at 0-4°C.

Biochemical Methods

Protein concentrations of microsomes and cytosols were determined by the method of [START_REF] Lowry | Protein measurement with the Folin Phenol reagent[END_REF]. Bovine serum albumin was used as a standard.

EROD activity of the mullet liver microsomes was determined by the spectrofluorometric method of [START_REF] Burke | Ethoxyresorufin: Direct fluorometric assay of microsomal O-dealkylation which is preferentially induced by 3-methylcholanthrene[END_REF], with some modifications. Assay conditions optimized for gilthead seabream (Sparus aurata) liver microsomes by [START_REF] Arinç | Effects of in vivo benzo[a]pyrene treatment on liver microsomal mixed-function oxidase activities of gilthead seabream (Sparus aurata)[END_REF] were also used in this study. The reaction mixture contained 0.1 M potassium phosphate buffer, pH 7.8, 0.1 M NaCl, 2.4 mg bovine serum albumin (BSA), 1.5 M 7ethoxyresorufin, 50-100 g of fish liver microsomal protein, and 0.5 mM NADPH generating system in a final volume of 2.0 mL. The reaction was initiated by the addition of substrate and followed for 2 minutes in a spectrofluorometer. Finally, a known amount of resorufin was added as an internal standard to the reaction mixture and the increase in fluorescence was recorded. EROD activity was calculated using the fluorescence increase caused by the addition of resorufin.

Total glutathione S-transferase (GST) activity of mullet liver cytosols was determined according to the method of [START_REF] Habig | Glutathione S-transferases: the first enzymatic step in mercapturic acid formation[END_REF]. The reaction mixture contained 50 mM potassium phosphate buffer, pH 7.5, 1.3 mM GSH and fish liver cytosols. The reaction was initiated by the addition of 1 mM CDNB in a final volume of 3.0 mL. The thioether formation was followed at 340 nm for 2 minutes. Specific total glutathione Stransferase activity was calculated as nmol CDNB conjugate formed min -1 mg -1 protein at 25ºC, using 9.6 mM -1 cm -1 as an extinction coefficient ( 340 ).

Catalase (CAT) activity of mullet liver cytosols was determined according to the method of [START_REF] Aebi | Catalase in vitro[END_REF]. The reaction mixture contained 50 mM potassium phosphate buffer, pH 7.5, 10 mM H 2 O 2 and fish liver cytosols . The rate of reaction was followed by measuring the consumption of H 2 O 2 spectrophotometrically at 240 nm for 1 minute. Specific catalase activity was calculated as nmol H 2 O 2 consumed min -1 mg -1 protein at 25ºC, using 36.4 mM -1 cm -1 as an extinction coefficient ( 240 ).

Glutathione reductase (GR) activity of mullet liver cytosols was determined according to the method of Carlberg and Mannervick (1985). The reaction mixture contained 100 mM potassium phosphate buffer, pH 7.6, 0.5 mM EDTA, pH 7.6, 0.1 mM NADPH and fish liver cytosols in a final volume of 2.0 mL. The reaction was started by adding 1 mM oxidized glutathione (GSSG). Then the GSH formation was followed by measuring the NADPH utilization spectrophotometrically at 340 nm for 5 minutes. One unit of glutathione reductase activity was calculated as nmol NADPH oxidized min -1 mg -1 protein at 25ºC, using 6.22 mM -1 cm -1 as an extinction coefficient ( 340 ).

Western Blot Analysis

Cytochrome P4501A (CYP1A) protein levels were determined using the polyclonal antibodies produced against purified leaping mullet (Liza saliens) liver cytochrome P4501A [START_REF] Arinç | Hepatic cytochrome P4501A and 7-ethoxyresorufin O-deethylase induction in mullet and common sole as an indicator of toxic organic pollution in zmir Bay, Turkey[END_REF]. Western blot analysis of fish liver microsomes was carried out essentially as described by [START_REF] Towbin | Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications[END_REF]. Sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) was performed as described by [START_REF] Laemmli | Cleavage of structural proteins during the assembly of the head of bacteriophage T4[END_REF] and proteins were transferred from slab gels to nitrocellulose membrane by use of a Bio-Rad Trans-Blot electrophoretic transfer apparatus. The proteins on membranes were first reacted with 1:5000 diluted leaping mullet CYP1A antibodies and then with the enzyme-linked secondary antibody, and alkaline phosphatase activity was detected by the method of [START_REF] Ey | The use of alkaline phosphatase-conjugated antiimmunoglobulin with immunoblots for determining the specificity of monoclonal antibodies to protein mixture[END_REF]. The final images of the blots were photographed using a computer-based gel imaging instrument (Infinity 3000-CN-3000 darkroom, Vilber Lourmat, Marne-la-Vallee Cedex 1, France), and relative peak area (R.P.A.) of the blots was analyzed using the Scion Image software for Windows (Version 4.0.2, Scion Corporation, Maryland, USA) as a quantitative tool to determine CYP1A protein levels.

PAH Analysis

Spectrofluorometric measurements of the total PAH concentration in fish liver tissues were performed according to the method described by UNEP, 1992. The principle of the method is based on the extraction of PAHs by suitable pure solvent. Mullet livers were dried in an oven at 40 °C overnight. Dried tissue (0.2 g) was ground and refluxed with 20 mL of ethanol containing 0.75 g of KOH for 120 minutes by constant heating. Then 20 mL of PAH-free hexane was added to the flask and mixed very well and waited until the flask reached to room temperature. When the flask was attained to room temperature, it was poured into a separatory funnel and sufficient amount of distilled water was added. The lower aqueous phase was extracted further two times with 20 mL of PAHfree hexane. The fluorescence intensities of the pooled samples were measured at 310 nm excitation and 360 nm emission wavelengths. The fluorescence intensity of the pooled samples was compared with the fluorescence intensity of known concentrations of chrysene standard.

Statistical Analysis

Results were expressed as means ± standard error of the mean (S.E.M.). Data were analyzed by two-way ANOVA using years and sampling sites as independent variables. When significant differences were observed, Tukey HSD Post Hoc Test was applied for a pair-wise comparison. The analyses were carried out using the SPSS statistical package (SPSS 15.0 for Windows Evaluation Version). No significant sex dependent differences were observed in the fish samples. Therefore the data were pooled.

RESULTS

In this study 147 fish samples were collected from six different locations of the West Black Sea Coast of Turkey. In all samples, EROD, glutathione S-transferase, catalase and glutathione reductase activities were measured. Table 1 illustrates biomarker enzyme activity results. The lowest biomarker activities were measured in fish samples (so-iuy mullet, flathead mullet and golden grey mullet) caught from Amasra. The biomarker enzyme activities of different mullet species were compared with the corresponding mullet species caught from Amasra. This relatively clean site was considered as a reference site to demonstrate the extent of pollution.

Table 1 3

.1 7-Ethoxyresorufin O-deethylase (EROD) Activity and CYP1A Protein Level

The highest EROD activities were found in mullets (so-iuy mullets, flathead mullets, and golden grey mullets) caught from Zonguldak Harbour in all sampling years (Table 1). Mullets caught from Zonguldak Harbour had about 6-9 fold higher EROD activities than those obtained from Amasra. The highly elevated EROD activities were also measured in mullets captured from Ere li Harbour which were about 7-9 fold higher with respect to the values obtained from Amasra. Gülüç Stream's Mouth was the other highly polluted station in this study. EROD activities of mullets samples caught from this station were about 6 fold higher than those obtained from Amasra. In Figure 2, the data were presented separately for each species. Statistically significant differences were indicated in this figure with different letters (p<0.05). EROD activities of soiuy mullets caught from Zonguldak Harbour and Gülüç Stream's Mouth were significantly different from the souiy mullets caught from Amasra, Melen Stream's and Sakarya River's Mouths. EROD activities of flathead mullets caught from Zonguldak Harbour were significantly different from Amasra. EROD activities of golden grey mullets caught from Zonguldak and Ere li Harbours were significantly different from Amasra. EROD activities of mullets caught from Melen Stream's Mouth were 2 fold higher than Amasra, however these differences were not statistically significant. Significant differences between years were not found in EROD activities of fish caught from all sampling stations except Sakarya River's Mouth.

Figure 2 CYP1A protein level was also determined in mullet liver microsomes using the polyclonal antibodies produced against leaping mullet cytochrome P4501A. Figure 3 illustrates the western blot analysis results of mullets caught from six different stations in three sampling years. Polyclonal antibody anti-leaping mullet cytochrome P4501A showed strong cross-reactivity with liver microsomes of so-iuy mullets, flathead mullets and golden grey mullets caught from different stations of the West Black Sea. The CYP1A bands of mullets caught from Zonguldak and Ere li Harbours and Gülüç Stream's Mouth were stained more intensely than those obtained from Amasra. Liver microsomes of mullets caught from Melen Stream's Mouth and Sakarya River's Mouth showed moderate cross reactivity with polyclonal anti-mullet P4501A. Liver microsomes of mullets caught from Amasra showed low cross-reactivity with polyclonal antibody anti-mullet P4501A. The results clearly indicated that mullets caught from the polluted sites had both highly induced EROD activity and elevated CYP1A protein content. The relationship between CYP1A protein levels and EROD activities was also examined with correlation analysis (Figure 3). CYP1A protein levels were highly correlated with the EROD activity results in the mullet samples caught from different stations of the West Black Sea Coast of Turkey (R 2 ranged between 0.92-0.96).

Figure 3 When EROD activities of different mullet species were analyzed using ANOVA, species differences were not significant (p=0.714). When the species specific crossreactivity pattern of anti-leaping mullet CYP1A protein was compared, different mullet species having similar EROD activity showed similar degree of staining and gave almost the same EROD activity/CYP1A protein level (R.P.A.) ratio (Figure 4). The induction responses to organic pollutants, both with respect to EROD activities and CYP1A protein levels, were not different between these mullet species.

Figure 4 3.2 Glutathione S-transferase Activity

The highest total glutathione S-transferase activities were measured in mullets caught from Zonguldak Harbour in all sampling years which were about 4 fold higher than those obtained from Amasra (Table 1). Mullets caught from Gülüç and Melen Streams' Mouths also displayed highly elevated total glutathione S-transferase activities which were 3-4 fold higher than those obtained from Amasra. In Figure 5, the data were presented separately for each species. Glutathione S-transferase activities of soiuy mullets caught from Zonguldak Harbour, Melen and Gülüç Streams' Mouths were not significantly different from each other. However, they were significantly different from Amasra. Glutathione S-transferase activities of soiuy mullets caught from Sakarya River's Mouth (represented by "ab") were not statistically different from Zonguldak Harbour, Gülüç and Melen Stream's Mouths and Amasra. Glutathione S-transferase activities of golden grey mullets caught from Ere li Harbour (represented by "ab") were 2-3 fold higher than those obtained from Amasra, however these differences were not significantly different from Amasra and Zonguldak Harbour. Glutathione S-transferase activities of mullets caught from Ere li Harbour showed significant difference between years. The highest GST activities were measured in the fish samples caught in 2005.

Figure 5 3.3 Catalase Activity

The highest catalase activities were detected in mullets caught from Sakarya River's and Melen Stream's Mouths and Zonguldak Harbour in 2005, which were about 3 fold higher than those obtained from Amasra (Table 1). Fish sampled from Gülüç Stream's Mouth also displayed highly elevated catalase activities which were 2 fold higher than those obtained from Amasra. In Figure 6, the data were presented separately for each species. Catalase activities of soiuy mullets caught from Zonguldak Harbour, Melen Stream's and Sakarya River's Mouths were not statistically different from each other. However, they were significantly different from Amasra. Catalase activities of soiuy mullets caught from Gülüç Stream's Mouth (represented by "ab") were not statistically different from Zonguldak Harbour, Melen Stream's and Sakarya River's Mouths and Amasra. Catalase activities of flathead mullets caught from Zonguldak Harbour were significantly different from Amasra. Catalase activities of golden grey mullets caught from Zonguldak and Ere li Harbours were significantly different from Amasra. Although highly elevated catalase activities were measured in mullets caught in 2005, this enzyme activity significantly decreased in the mullets caught from Melen Stream's and Sakarya River's Mouths and Ere li Harbour in the following sampling years.

Figure 6 3.4 Glutathione Reductase Activity

Highly elevated glutathione reductase activities were measured in mullets caught from Zonguldak and Ere li Harbours, Gülüç and Melen Streams' Mouths (Table1). In 2005, mullets caught from Melen Stream's Mouth had the highest glutathione reductase activities among the other sampling sites which were 1.7 fold higher than those obtained from Amasra. Glutathione reductase activities of mullets caught from Gülüç Stream's Mouth were 1.6 fold higher than those obtained from Amasra in 2005 and 2006. Glutathione reductase activities of mullets captured from Ere li Harbour were 1.6 fold higher than those obtained from Amasra. In 2006, glutathione reductase activities of so-iuy mullets and golden grey mullets captured from Zonguldak Harbour were 1.5 and 1.4 fold higher than those obtained from Amasra, respectively. In Figure 7, the data were presented separately for each species. Glutathione reductase activities of soiuy mullets caught from Melen and Gülüç Streams' Mouths were significantly different from Amasra and Sakarya River's Mouth. Glutathione reductase activities of soiuy mullets caught from Zonguldak Harbour (represented by "ab") were not statistically different from Gülüç, Melen Streams', Sakarya River's Mouths and Amasra. Glutathione reductase activities of golden grey mullets caught from Zonguldak and Ere li Harbours were significantly different from Amasra. Significant differences between years were not found in glutathione reductase activities of fish caught from all sampling stations except Sakarya River's Mouth.

Figure 7 3.5 PAH Analysis

Besides biomarker enzyme activity measurements, total PAH concentrations were determined in mullets caught in 2006 to show the presence and extent of one of the organic pollutants in the sampling stations. Table 2 illustrates the total PAH concentrations in mullet liver tissues. The highest total PAH concentrations were measured in mullets caught from Zonguldak Harbour. Total PAH concentrations of fish caught from Zonguldak and Ere li Harbours and Gülüç Stream's Mouth were significantly different from Amasra. Total PAH concentrations of fish caught from Zonguldak Harbour were significantly different from fish caught from Ere li Harbour. However, total PAH concentrations of fish caught from Ere li Harbour and Gülüç Stream's Mouth were not significantly different from each other. The lowest total PAH concentrations were measured in mullets caught from Amasra. Total PAH concentrations of fish caught from Sakarya River's and Melen Stream's Mouths (represented as "bc") were not significantly different from Amasra, Ere li Harbour and Gülüç Stream's Mouth.

Table 2 The relationship between biomarker activities and total PAH concentrations was also examined with correlation analysis (Pearson). The highest positive correlation was found between EROD activities and total PAH concentrations (r=0.491, p<0.01). Glutathione reductase and glutathione S-transferase activities were slightly correlated with total PAH concentrations ((r=0.392, p<0.01) and (r=0.283, p<0.05), respectively). Correlation between total PAH concentrations and catalase activities was positive but not significant (r=0.210, p=0.083).

DISCUSSION

In this study, cytochrome P4501A (CYP1A) associated EROD activity, phase II enzyme, glutathione S-transferase and antioxidant enzymes, catalase and glutathione reductase activities were measured in mullet liver fractions to determine and monitor the extent of pollution in the West Black Sea Coast of Turkey. Highly elevated biomarker enzyme activities were found in the mullets caught from Zonguldak and Ere li Harbours, and Gülüç Stream's Mouth. Zonguldak is an important coal mining area in Turkey. The harbour of this city is polluted especially with coal processing wastes and discharges of industrial and domestic wastes. Cohen and coworkers (1994) have shown that coal byproducts such as coal-tar significantly induce EROD activity in medika liver. In addition, Vignier and coworkers (1994) have demonstrated that EROD activity significantly increases in winter flounder in coal-tar contaminated estuary. Creosote is a dark liquid made from coal tar. It is a complex mixture of organic compounds, containing about 85% PAHs. [START_REF] Hyötyläinen | Assessment of the bioactivity of creosote-contaminated sediment by liver biotransformation system of rainbow trout[END_REF] and Whyte and coworkers (2000) have demonstrated in different studies that the extracts of creosote-contaminated sediments highly induce EROD activities in rainbow trout. In this study, the lowest biomarker enzyme activities were measured in mullets caught from Amasra. The biomarker enzyme activities obtained from the other stations were compared with those obtained from Amasra. 6-9 fold increase in EROD activities and elevated CYP1A protein levels were found in mullets caught from Zonguldak Harbour with respect to Amasra. This result clearly indicates that coal mining activities create pollution in Zonguldak Harbour.

zmir Bay is one of the most polluted sites in Turkey. Highly elevated EROD activities have been reported in mullets (Liza saliens and Mugil cephalus) caught from the most polluted site of zmir Bay. In these studies, EROD activities of Liza saliens were reported as 1293±292 pmol min -1 mg -1 protein in 1995 and 1028±287 pmol min -1 mg -1 protein in 1999 [START_REF] Arinç | Hepatic cytochrome P4501A and 7-ethoxyresorufin O-deethylase induction in mullet and common sole as an indicator of toxic organic pollution in zmir Bay, Turkey[END_REF][START_REF] Arinç | Cytochrome P4501A and associated MFO induction in fish as a biomarker for toxic, carcinogenic pollutants in the aquatic environment[END_REF]. EROD activities of Mugil cephalus caught from the same site were 1398±410 pmol min -1 mg -1 protein in 1999 [START_REF] Arinç | Cytochrome P4501A and associated MFO induction in fish as a biomarker for toxic, carcinogenic pollutants in the aquatic environment[END_REF]. In our study, highly elevated EROD activities were found in mullets caught from Zonguldak and Ere li Harbours, and Gülüç Stream's Mouth, suggesting the presence of organic pollutants such as PAHs and PCBs in these sampling stations in the West Black Sea Coast of Turkey.

Glutathione S-transferases are involved in the detoxification reactions of xenobiotics. Induction of glutathione S-transferase have been reported in a number of field studies in fish exposed to organic contaminants (Lenartova et al., 1997;[START_REF] Pandey | Biomarkers of oxidative stress: a comparative study of river Yamuna fish Wallago attu (Bl. & Schn.)[END_REF][START_REF] Martínez-Gómez | Monitoring biomarkers in fish (Lepidorhombus boscii and Callionymus lyra) from the northern Iberian shelf after the Prestige oil spill[END_REF][START_REF] Sen | Biochemical characterization and distribution of glutathione S-transferases in leaping mullet (Liza saliens)[END_REF]. In our study, highly elevated total glutathione S-transferase activities measured in mullets caught from Zonguldak Harbour (950-1147 nmol min -1 mg -1 protein), Ere li Harbour (932±131 nmol min -1 mg -1 protein), Gülüç Stream's Mouth (1352±154 nmol min -1 mg -1 protein) and Melen Stream's Mouth (1192±140 nmol min -1 mg -1 protein) are at the same order of magnitude of that reported by [START_REF] Sen | Biochemical characterization and distribution of glutathione S-transferases in leaping mullet (Liza saliens)[END_REF] in mullets (Liza saliens) caught from highly polluted site of zmir Bay in Turkey (1183±35.9 nmol min -1 mg -1 protein).

Chronic exposure to organic pollutants increases reactive oxygen species (ROS) formation [START_REF] Livingstone | Contaminant reactive oxygen species production and oxidative damage in aquatic organisms[END_REF]. The antioxidant enzyme catalase is one of the most responsive enzymes to ROS [START_REF] Halliwell | Free Radicals in Biology and Medicine[END_REF]. Elevated catalase enzyme activities have been reported in several fish species in various laboratory and field studies [START_REF] Otto | 3,3´,4,4´-tetrachlorobiphenyl effects on antioxidant enzymes and glutathione status in different tissues of rainbow trout[END_REF]Moon, 1995, Orbea et al., 2002;[START_REF] Ferreira | Oxidative stress biomarkers in two resident species, mullet (Mugil cephalus) and flounder (Platichthys flesus), from a polluted site in River Douro Estuary, Portugal[END_REF]. Significantly high catalase activities were measured in mullets caught from Zonguldak and Ere li Harbours and Melen Stream's and Sakarya River's Mouths, indicating the presence of ROS generating pollutants in these regions. The other biomarker enzyme activity measured in this study was glutathione reductase. Glutathione reductase maintains GSH/GSSG homeostasis under oxidative stress conditions. Induction of glutathione reductase activity have been reported in various field studies in fish exposed to organic pollutants such as PAHs, PCBs and halogenated xenobiotics [START_REF] Rodriguez-Ariza | Biochemical indicators of oxidative stress in fish from polluted Littoral AreasRodriguez-Ariza[END_REF][START_REF] Van Der Oost | Fish bioaccumulation and biomarkers in environmental risk assessment: a review[END_REF][START_REF] Martínez-Gómez | Monitoring biomarkers in fish (Lepidorhombus boscii and Callionymus lyra) from the northern Iberian shelf after the Prestige oil spill[END_REF]. Significantly high glutathione reductase activities have been found in mullets caught from Zonguldak and Ere li Harbours, Gülüç and Melen Streams' Mouths, suggesting the presence of PAHs, PCBs and halogenated xenobiotics in these sampling sites.

Biomarker enzyme activities of fish caught from Sakarya River's Mouth displayed significant differences between sampling years. Sakarya River is the third longest river of Turkey (824 km). It rises from the center of Anatolia and joins with Porsuk, Göksu, Kirmir, Mudurnu and Çark Streams. The river runs through many industrial and agricultural areas before reaching to the Black Sea. The results of this study suggested the existence of qualitative and quantitative differences of contaminants in the sampling station between years. However, it is difficult to determine which chemicals specifically affected the biomarker activities in the environmental monitoring studies. Biomarkers reveal the interactive effects of chemical contaminants on organisms.

In the Black Sea, total PAH concentrations in fish samples have been determined by Telli-Karakoç and coworkers (2001) in mullet samples caught from Trabzon, Yomra. In our study, the highest total PAH concentrations were measured in so-iuy mullets and golden grey mullets caught from Zonguldak Harbour (54±5 g g -1 dried liver weight, n=13 and 47±6 g g -1 dried liver weight, n=9, respectively) are at the same order of magnitude of that reported by Telli-Karakoç and coworkers for mullets (Mugil saliens) (55.7±16.6 g g -1 dried liver weight) caught from Trabzon, Yomra. In our study, significantly high total PAH concentrations were also measured in mullets caught from Ere li Harbour and Gülüç Stream's Mouth. The sampling areas arranged in decreasing order of total PAH concentrations are Zonguldak Harbour>Gülüç Stream Mouth>Ere li Harbour>Melen Stream Mouth>Sakarya River Mouth>Amasra.

The relationship between biomarker activities and total PAH concentrations was also analyzed. Biomarker activities of fish were moderately related with total PAH concentrations, suggesting the presence of the other organic contaminants such as PCBs and/or dioxins in these regions besides polycyclic aromatic hydrocarbons.

CONCLUSION

In this study, the contribution of Turkish rivers and harbours present in the West Black Sea Region of Turkey to the pollution of the Black Sea was assessed using biomarker responses of fish. The elevated biomarker responses found in this study indicate that Zonguldak Harbour, Ere li Harbour and Gülüç Stream are highly polluted by polycyclic aromatic hydrocarbons and related contaminants. In addition, Melen Stream and Sakarya River also bring some PAHs, PCBs and other type of pollutants from the inner parts of Anatolia and Black Sea Region of Turkey. The results reported here also supported the conclusion of previous field studies that mullet is a suitable fish species for biomonitoring studies.

Figure 4

 4 Figure 4Comparison of anti-leaping mullet CYP1A cross reactivity pattern of different mullet species. The intensities of CYP1A protein bands of different mullet species present in the same western blot result (Figure3C) were compared together with corresponding EROD activities and EROD activity/CYP1A protein level (R.P.A.) ratios. Different mullet species having similar EROD activity showed similar degree of staining and gave almost the same EROD activity/CYP1A protein level (R.P.A.) ratio.

Figure 5

 5 Figure 5 Glutathione S-transferase (GST) activities of mullets caught from six sampling stations of the West Black Sea Coast of Turkey in 2005, 2006, and 2007. Homogenous subsets (groups do not differ significantly) were indicated by the same letters (p<0.05).

Figure 6

 6 Figure 6 Catalase activities of mullets caught from six sampling stations of the West Black Sea Coast of Turkey in 2005, 2006, and 2007. Homogenous subsets (groups do not differ significantly) were indicated by the same letters (p<0.05).

Figure 7

 7 Figure 7 Glutathione reductase activities of mullets caught from six sampling stations of the West Black Sea Coast of Turkey in 2005, 2006, and 2007. Homogenous subsets (groups do not differ significantly) were indicated by the same letters (p<0.05).

  Figure 1

Table 1 .

 1 Mean values of biomarker enzyme activities and Standard Error of Mean (SEM)

	Zone	Year	N	Fish Species	EROD a	GST b	CAT b	GR b
	Zonguldak Harbour	2005	7	Mugil soiuy	3237±539	1260±207	322±34	14.9±1.9
		2006	13	Mugil soiuy	2559±315	950±135	283±46	20.4±0.8
		2006	9	Liza aurata	2302±240	1067±208	163±16	16.2±1.6
		2007	5	Mugil cephalus	3017±565	1017±231	171±12	11.5±2.1
	Ere li Harbour	2005	16	Liza aurata	1871±391	932±131	195±18	17.4±0.7
		2006	12	Liza aurata	2037±280	585±64	138±12	18.1±0.8
		2007	5	Liza aurata	2233±464	406±29	121±12	17.9±1.3
	Gülüç Stream Mouth	2005	13	Mugil soiuy	2402±261	1352±154	281±32	20.8±0.9
		2006	11	Mugil soiuy	2415±426	830±154	224±30	20.7±1.3
	Melen Stream Mouth	2005	11	Mugil soiuy	1065±176	851±129	349±32	23.1±1.9
		2006	9	Mugil soiuy	818±217	1192±140	200±20	18.7±1.8
	Sakarya River Mouth	2005	10	Mugil soiuy	904±216	810±159	352±17	19.7±1.7
		2006	10	Mugil soiuy	438±62	735±134	257±32	9.6±0.7
		2007	4	Mugil soiuy	1642±470	1016±349	187±19	11.3±0.8
	Amasra	2006	3	Mugil soiuy	425±18	288±165	123±20	13.4±0.8
		2006	4	Mugil cephalus	541±22	263±62	117±21	11.3±0.5
		2007	5	Liza aurata	259±72	285±37	100±11	11.2±1.7
	a pmol min -1 mg -1 protein b nmol min -1 mg -1 protein

Table 2

 2 Total PAH concentrations in the livers of mullets collected from six sampling stations of the West Black Sea Coast of Turkey in 2006. Homogenous subsets (groups do not differ significantly) were indicated by the same letters (p<0.05). Fish sampling stations in the West Black Sea Coast of Turkey. * Reference site. Ethoxyresorufin O-deethylase (EROD) activities of liver microsomes of mullets caught from six sampling stations of the West Black Sea Coast of Turkey in 2005, 2006, and 2007. Homogenous subsets (groups do not differ significantly) were indicated by the same letters (p<0.05). Western blot analysis of CYP1A protein contents and correlation between microsomal EROD activities and CYP1A protein levels of mullets caught from different stations of the West Black Sea Coast of Turkey in 2005 (A), 2006 (B), and 2007 (C). 80 µg microsomal protein was applied to each slot.

	Zone	N	Fish Species	PAH Concentration *
	Zonguldak Harbour	13	Mugil soiuy	54 ± 5 (a)
		9	Liza aurata	47 ± 6 (a)
	Ere li Harbour	12	Liza aurata	28 ± 3 (b)
	Gülüç Stream Mouth	11	Mugil soiuy	35 ± 3 (ab)
	Melen Stream Mouth	9	Mugil soiuy	25 ± 3 (bc)
	Sakarya River Mouth	8	Mugil soiuy	21 ± 3 (bc)
	Amasra	3	Mugil soiuy	12 ± 2 (c)
		4	Mugil cephalus	11 ± 2 (c)
	* µg g -1 dry weight

(A) Line 1-9 (Mugil so-iuy) and Line 10-12 (Liza aurata); (B) Line 1-7 (Mugil so-iuy) and Line 8-12 (Liza aurata); (C) Line 1-4 (Liza aurata), Line 5-8 (Mugil so-iuy), Line 9-12 (Mugil cephalus). * Sampled in 2006 ** Sampled in 2007

polycyclic aromatic hydrocarbons (PAHs) in creosote contaminated microcosmes.

Polycyclic Aromatic Compounds 18,

ACKNOWLEDGMENTS

The authors thank all the members of the Ministry of Agriculture and Rural Affairs (Tarim ve Koy Isleri Bakanligi, Turkey) for their help in collecting the fish. They also would like to thank Munire Ozlem Cevik and Asiye Kumru for their technical support in the statistical analysis and Serif Akman for his help for providing some laboratory equipments. This research was supported by TUBITAK (The Scientific and Technological Research Council of Turkey) (Project No:104Y083).