Ocean Sci. J. (2012) 47(3):209-214 http://dx.doi.org/10.1007/s12601-0021-7
Available online at www.springerlink.com
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Spatial Variability of Biochemical Responses in Resident Fish after the M/V Hebei Spirit Oil Spill (Taean, Korea) Jee-Hyun Jung, Young Sun Chae, Ha Na Kim, Moonkoo Kim, Un Hyuk Yim, Sung Yong Ha, Gi Myung Han, Joon Geon An, Eunsic Kim, and Won Joon Shim* Oil & POPs Research Group, South Sea Research Institute, KIOST, Geoje 656-834, Korea Received 16 August 2012; Revised 30 August 2012; Accepted 11 September 2012 © KSO, KIOST and Springer 2012
Abstract − This study describes the spatial variation and the duration of the impacts from the Hebei Spirit oil spill using specific biochemical indices in resident benthic fish. Enzymatic activities and biliary PAHs metabolites were higher at the site closer to the spill area in four months after spill incident. Regarding our results of detoxification response, markers of Phase I followed a similar trend in accordance with levels of biliary metabolites, while markers of phase II and GST appeared relatively unchanged. Keywords − Hebei spirit, oil spill, petroleum hydrocarbons, biomarker, biliary metabolite
1. Introduction The Hebei Spirit oil spill (HSOS) on 7 December 2007 was one of largest tanker spills of recent years (approx. 10,900 tons, comparable to the Prestige oil spill in 2002 and Tasman Spirit in 2003) (ITOPF, 2008). High waves (up to four meters) and winds (10 to 14 m/s) for several days after spill led to widespread distribution and stranding of oil on the coastline of Taean (Kim et al. 2010). More than 150 km of coastline had been impacted by December 17. Suspended oyster culture and land-based fish aquaculture facilities were directly affected by the crude oil. There is continuing controversy about the impacts of submerged oil on marine biota. These observations prompted the implementation of a broad range of chemical and biological effects monitoring of the spill. Polycyclic aromatic hydrocarbons (PAHs) are considered the primary cause of toxicity in oil, mainly because of their *Corresponding author. E-mail:
[email protected]
carcinogenic and mutagenic properties (Aas et al. 2000). PAHs usually are metabolized to form epoxy- and hydroxyderivatives during phase I metabolism, and are subsequently converted into relatively water-soluble conjugates (Varanasi et al. 1989). Hepatic PAH metabolites are secreted into the bile and stored in the gall bladder before being released to the alimentary tract. Therefore, induction of the hepatic cytochrome P-450 system and its catalytic activity indicated by ethoxyresorufin O-de-ethylase (EROD) have been measured to evaluate the sub-lethal toxic effects of oil exposure. After the Prestige and the Sea Empress oil spill, the concentrations of total CYP1A followed a gradient depending on distance from the spill site, similar to the gradient seen in tissue PAH levels (Marigómez et al. 2006; Kirby et al. 1999). Even 10 years after Exxon Valdez oil spill (EVOS), liver CYP1A and EROD activity remained elevated in resident fish (Jewett et al. 2002). Additionally, there is a good correlation between biomarker responses and long-term effects on populations (Peterson et al. 2003). In this study, the possibility that other P450's were induced was investigated by measuring pentoxy-resorufin O-de-ethylase (PROD) activity which is widely assumed to be a catalysed by CYP2B, and is therefore a “marker” of CYP2B induction in mammals (Bozcaarmutlu and Arinc, 2008). In addition, cyanoethoxycoumarin O-deethylase (CN-ECOD) appears to be catalysed primarily by CYP1A, but may also be catalysed by other P450's (White 1988) and it has been shown to be sensitive to exposure of fish to hydrocarbons (Renton and Addison 1992; Addison et al. 1994). Marbled flounder (Pseudopleuronectes yokohamae) and flatfish (Paralichthys olivaceus) were selected as a target species
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for this study, to allow a comparison of the influences on hydrocarbon accumulation and effects. The species have limited migration patterns and are commercially important and are demersal species which feeds on benthic invertebrates (Shafieipour et al. 1999). The present work is the first account of the spatial monitoring of HSOS impacts on benthic fish and it summarizes the extent and duration of biochemical impacts using specific biochemical indices in resident fish.
2. Materials and Methods Site position and sampling The areas trawled are mapped in Fig. 1. In total five stations yield (St. A- St. E) fish for survey in the initial response cruise on 12th to 14th April 2008. The second survey from 17th to 22nd June 2009 was also conducted at six stations yield (St. F- St. K). Uihangri is a heavily oiled site and Oeyeondo considered as a “reference” site not directly affected by the spill. Fish were processed on the dockside in Shin-gin harbor. They were killed by a blow on the head, and length, weight, sex and gonad weights (in mature individuals) were recorded. The liver was dissected out and immediately frozen in liquid nitrogen. Liver by sampled in 2009 were pooled to two samples and stored at 80 °C until analysis. Time series oil contamination study has been conducted independently with fish sampling in sub-tidal regions in the spill site. Seven out of seventy four stations were selected near the fish sampling region in this study to evaluate temporal change of petroleum derived hydrocarbon concentrations (Fig. 1). One intertidal (St. 1) and six sub-tidal (Sts. 2-7) surface sediments were collected using a van Veen grab from 18th December 2007, 11 days post spill, to July 2009. Approximately, top 2 cm of sediment was collected with a stainless steel spatula. Samples were stored in pre-combusted amber bottles and kept at 20 °C until analysis. Petroleum hydrocarbon analysis in sediment PAHs residue analysis Twenty grams of the sediment sample fortified with surrogates (50 µL of 10 ppm O-terphenyl and 30 µL of mixture of deuterated acenaphthene, phenanthrene, chrysene, and perylene, 10 ppm each) was Soxhlet extracted using dichloromethane for 8 hours. The extracts were concentrated to 1 mL and solvent was exchanged with hexane. And they
Fig. 1. Map of the Taean, South Korea, showing fish sampling sites; Stations A,B,C,D and E were sampled at 1st survey in 2008 and stations F, G, H, I, J and K were sampled at 2nd survey in 2009. Uihangri (△ ) was heavily oiled and Oeyeondo (□ ) was a “reference” site not directly affected by the spill. The star indicates the oil spill site (36°52'00" N, 126°02'09"E)
were transferred to a 3 g activated silica gel column, which was topped with about 1 cm anhydrous granular sodium sulfate and had pre-conditioned using 20 mL of hexane, for sample clean-up and fractionation (Wang et al. 1994). Analyses for n-alkane distribution and TPH were performed on a Agilent 7890 gas chromatograph equipped with a flame-ionization detector (FID) and an HP 7673 auto-sampler. Analyses of PAH and biomarker compounds were performed on an HP model 5890 GC equipped with a model HP 5972 mass selective detector (MSD). System control and data acquisition were achieved with an HP G1034C MS ChemStation. Detailed chromatographic conditions, analysis quality control, and quantification methodology are referred to Wang et al. (1994) and Yim et al. (2005). Bile fluorescence analysis Synchronous scanning of fluorescent bile metabolites,
Spatial Variability of Biochemical Responses in Resident Fish after the M/V Hebei Spirit Oil Spill (Taean, Korea)
focusing on 1-hydroxypyrene (Ariese et al. 1993) was used as a rough indicator of fish exposure to PAH. The bile diluted as required in 50% ethanol, was analyzed as described by Jung et al. (2008, 2009). 1-Hydroxypyrene (Aldrich) was used as standard at concentrations (in 50% ethanol) in the range 50–500 nM. Enzyme activity Liver samples were homogenized and microsomes were prepared and suspended in 0.1 M phosphate buffer pH 7.6 essentially as described by Addison and Payne (1986). An aliquot of microsomes and supernatant were transferred and stored at -80 °C until analyzed. Microsomes were used in assays for ethoxyresorufin O-de-ethylase (EROD), pentoxyresorufin O-de-ethylase (PROD) and cyanoethoxy-resorufin O-deethylase (CN-ECOD) and 100,000×g supernatants were used for glutathione-S-transferase (GST) assay. EROD: EROD activity in microsomes was determined using reagent concentrations described by Addison and Payne (1986) in a fluorescence microplate reader with excitation and emission filter set at 544 nm and 590 nm respectively. CN-ECOD: CN-ECOD assays were carried out in a 96well microplate containing substrate at a concentration of 4.65 µM. The product was read in a fluorescence plate reader using excitation and emission wavelengths of 405 and 450 nm for 3-cyanoumbelliferone as described by Addison et al. (1994). PROD: PROD activity in microsomes was determined using reagent concentrations described by Bozcaarmutlu and Arinc (2008) in a fluorescence microplate reader with excitation and emission filter set at 544 nm and 590 nm respectively. GST: GST measurement was performed using modification of the method described in Habig et al. (1974). The enzyme activity was followed spectrophotometerically with a microplate reader at 340 nm. Protein concentrations in the sample were assayed by the bicinconchinic acid (BCA) method using a kit from Pierce Chemicals (Rockford, IL); the product was determined in a microplate reader at 550 nm, using bovine serum albumin as a standard.
3. Results and Discussion One-hundred fish were sampled at each survey. Most of
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fish was degenerated stage in reproductive cycle in this season. Total body length of marbled flounder and flatfish were 32.3 ± 4.78 cm and 32.4 ± 5.35 cm in the first survey and body weights were 332 ± 157. 6 and 311 ± 160 g, respectively. In the second survey, total body length of flatfish (39.2 ± 15.6 cm) and weight (938 ± 882 g) were larger than in fish from the first survey. Fig. 2(a) shows the variation of bile 1-OH-pyrene intensity in individual fish collected from the oil spill site. Bile fluorescence intensity was highest 4 months after the spill at Station A (76.5 ± 79.8) and B (112 ±121) and significantly elevated over that in fish (p
