2 Key Lab of Active Material and Modern Analysis Technology, State Oceanic Administration (SOA), Qingdao 266061, China
Petroleum hydrocarbon is becoming one of the main pollutants in the marine environment with the development of offshore oil drilling and marine transportation. Aromatic hydrocarbon and its derivatives in petroleum hydrocarbon have toxic effects on marine organisms. High concentrations of pollutants can cause serious damage to the marine ecological system, and can even kill marine organisms (Tian et al., 2008). The population growth rates of common bivalves decreased by 2.33% from 1996 to 2005 in Bohai Bay, China(Xu et al., 2011). Thus, research on the toxic effects of petroleum hydrocarbons on marine organisms is important to assess ecological damage from marine oil spills.
Numerous pollutants consumed by organisms are usually metabolized by detoxification enzyme systems, which include 7-Ethoxyresorufin-Odeethylase(EROD), aryl hydrocarbon hydroxylase (AHH), and glutathione S-transferase(GST). The antioxidant defense system, which includes superoxide dismutase(SOD), catalase(CAT), and glutathione peroxidase(GPx), is important in eliminating reactive oxygen species produced in the metabolic processing of pollutants. Malondialdehyde (MDA)is a product of lipid peroxidation, its content often reflects the extent of lipid peroxidation and cell injury. Recent studies have focused on the effects of pollutants on the detoxification enzymes, antioxidant defense system, and MDA levels in fishes and bivalves (Akcha et al., 2000; Cheung et al., 2004; De LucaAbbott et al., 2005; Ren et al., 2006; Monari et al., 2007; Wang et al., 2007; Jung et al., 2008; Richardson et al., 2008; Faria et al., 2009; Nahrgang et al., 2010; González-Doncel et al., 2011). The detection of EROD, AHH, GST, GPx, SOD, CAT, MDA, and so on, and using them to reflect the effects of environmental pollutants on organisms are becoming an important aspect of molecular ecotoxicological research.
Bivalve molluscs are benthic organisms and are used extensively in biomonitoring programs. For example, they are deployed in caging experiments around platforms to monitor both chemical concentrations and biological responses (Hylland et al., 2008). Molluscs retain higher levels of pollutants than other organisms because of their sessile, filterfeeding lifestyle and low enzymatic degradation rate (Meador et al., 1995). Chlamys farreri is a popular and important marine scallop maricultured in China. In our previous work, we reported the effects of the water-soluble fraction(WSF)of crude oil on SOD and CAT activity and MDA content in C. farreri gills and digestive glands in laboratory experiments (Wang et al., 2013). In the present study, we analyzed petroleum hydrocarbon content in the scallops and examined the effects of WSF on C. farreri biomarkers in laboratory experiments. The biomarkers used included EROD, AHH, GST, and GPx. The relationship between biomarker activity and petroleum hydrocarbon content in the scallops was analyzed to evaluate the impacts of the toxicants. The results provide basic parameters for toxicological studies of oil pollution in C. farreri, and technical support for damage assessment of oil spills.2 MATERIAL AND METHOD 2.1 Chemicals
Crude oil was obtained from a National Offshore Oil Corporation platform(Tianjin, China). Dichloromethane and n-hexane were from Tedia Company Inc.(Ohio, USA); Resorufin(RF), Ethoxyresorufin(ERF), disodium salt of reduced form NADPH, and Bovine serum albumin(BSA) were purchased from Roche Co.(Basle, Switzerl and ); 2, 5-Diphenyloxazole(PPO), Kaumas brilliant blue G-250, and st and ard oil were purchased from Merck KGaA(Darmstadt, Germany), Sanl and Chemical Co. (California LA, USA), and National Marine Environmental Monitoring Center(Liaoning Province, China), respectively. GST and GPx reagent kits were obtained from the Nanjing Jiancheng Bioengineering Institute(Jiangsu Province, China). All other reagents used were domestic analytical reagents.2.2 Preparation of crude oil WSF
The crude oil WSF was prepared as follows. Crude oil and seawater were mixed at a ratio of 1:10(w/v), dispersed for 1 h with ultrasonic waves, and then deposited for 4 h. The water solution was separated as the stock solution, and sealed in a brown glass bottle at 4°C until further use. The WSF concentration of crude oil in the stock solution and all of the water samples was determined according to the specification of marine monitoring method( State Oceanic Administration, 2007).2.3 Animal treatment and experimental design
C. farreri were collected from aquaculture sites in Liuqing River Bay(Yellow Sea, Qingdao, China). After clearing their surfaces, the bivalves were maintained in seawater collected from Shilaoren Bay for an acclimation period of 1 week. The seawater was salinity 30.9 and pH 8.29. The scallops were fed with 100 mL of Isochrysis galbana(1×106–2×106 cells/mL) and dead scallops were removed daily. The seawater was renewed every morning and oxygen was provided by a continuous air-bubbler system. Scallops in good condition ranging in length from 4–5 cm were used in the exposure experiment.
The experiment was conducted in 60 L glass tanks (50 cm×40 cm×30 cm)with 30 L of seawater. According to the National Water Quality St and ards in China, the petroleum hydrocarbon concentration in the first and second class of seawater is below 0.05 mg/L, and that in the third and fourth class of seawater is below 0.30 and 0.50 mg/L, respectively. The petroleum hydrocarbon concentration in the blank seawater used in this study was 0.02 mg/L. The final petroleum hydrocarbon concentrations of WSF added seawater in the experiments were 0.18, 0.32, and 0.51 mg/L for the low-, middle-, and high-concentration groups, respectively. The scallops were divided into four groups: one in seawater as a control and three at different WSF concentrations. Each group contained 40 scallops, and one third of the water was renewed daily with seawater of corresponding WSF concentration to maintain the concentration in a relatively constant state. Other experimental conditions were similar to those used for acclimation. No dead animals were found during the experimental period.
Three scallops were r and omly selected and removed from each tank for dissection on days 1, 2, 4, 7, and 10. After the exposure experiment, the scallops were transferred to clean seawater to recover for 4 d. The gills and digestive glands of each scallop were quickly removed. The tissue samples were rinsed with cold distilled water, blotted, weighed, wrapped with aluminum foil paper, and then stored at -80°C until further biochemical analysis. The remaining scallop tissues were weighed and stored at -80°C for petroleum hydrocarbon analysis.2.4 Chemical analysis
Petroleum hydrocarbons were extracted from scallop tissues following the methods described by Jiang et al.(2012). The remaining soft tissue from each scallop was freeze-dried for 48 h and then homogenized to a powder using a tissue blender (IKA, Germany)for 2 min. The powder was then extracted in dichloromethane using ultrasonic waves. The extracted organic solution was purified by passing it through a silica gel column and then eluted by nhexane, until a constant volume of 10 mL was achieved. The solution was measured by a Hitachi F-4500 spectrofluorometer at an excitation wavelength of 310 nm and an emission wavelength of 360 nm, and then was quantified against an oil st and ard solution. The recovery rate of st and ard petroleum hydrocarbon with this method was between 76.5%–96.9%.2.5 Biochemical analysis
Tissue samples were thawed on ice and homogenized with cold phosphate buffer(0.014 mol/L NaCl, 0.003 mol/L KCl, 0.01 mol/L Na2HPO4·12 H2O, 0.002 mol/L KH2PO4, pH 7.4)(w/v=1:9)with a tissue grinder in an ice bath. The homogenate was centrifuged at 10 000×g for 30 min at 4°C(Hermle Z383K, Germany)to remove cell debris. The supernatant was collected and stored at -80°C until further biochemical analysis. All enzyme determinations were conducted in duplicate at room temperature.
EROD activity was determined through modified rapid termination of fluorescence spectrophotometry (Ren et al., 2006). The reaction mixture contained 100 μL supernatant, 10 μL 0.2 mmol/L ERF, 10 μL 6 mmo1/L NADPH, and 1.88 mL phosphate buffer (0.1 mol/L, pH 7.8, containing Na2EDTA 0.05 mol/L, 2–4°C). The reaction proceeded for 10 min at room temperature, and was then stopped by adding 0.5 mL ice-cold methanol. The fluorescence intensity was recorded at an excitation wavelength of 560 nm and an emission wavelength of 580 nm by a luminescence spectrofluorometer(Hitachi F4500, Japan). EROD activity was calculated according to an external st and ard curve using st and ard RF. Values were expressed as pmol per minute per mg of protein (pmol/(min∙mg protein)).
AHH activity was determined according to Wang et al.(2007). The reaction mixture contained 440 μL supernatant, 50 μL 2 mmol/L NADPH, 10 μL 3 mol/L MgCl2, and 500 μL Tris-HCl(pH 7.0). The mixture was pre-incubated at 30°C for 2 min and incubated at 37°C for 10 min after 20 μL 2.5 g/L PPO was added. The reaction was stopped by adding 1 mL ice-cold acetone. Approximately 3.25 mL n-hexane was added to the mixture prior to extraction. Two milliliter of the organic phase was collected and 5 mL 1 mol/L NaOH was added to re-extract. The water solution(3 mL) was measured at an excitation wavelength of 396 nm and an emission wavelength of 522 nm by a Hitachi F-4500 spectrofluorometer. The values were expressed as fluorescence intensity of protein(U/mg protein).
GST and GPx were assayed using GST assay kit (Colorimetric method) and GPx assay kit(Colorimetric method). One unit of GST activity was defined as the amount of glutathione conjugate formed by 1 μmol GSH and CDNB/min per mg protein at 37°C(U/mg protein). One unit of GPx activity was defined as 1 nmol GSH consumption during l min at 37°C(U/mg protein). The operations followed the instructions indicated in the kits.
All enzymes were normalized to total protein, which was determined using Coomassie Brilliant Blue G-250 following the method of Bradford(1976). BSA was used as the st and ard.2.6 Statistical analysis
Results are expressed as mean±SD. Data were analyzed using SPSS statistical software(Version 13.0; SPSS Inc., Chicago, IL, USA). Differences in petroleum hydrocarbon content and enzyme activity between exposure concentrations at the same exposure time were assessed by one-way ANOVA using Tukey’s tests. Differences were considered statistically significant at P<0.05 and highly significant at P<0.01.3 RESULT 3.1 Changes in petroleum hydrocarbon concentrations in scallops with exposure time
The petroleum hydrocarbon concentrations in the scallops in different treatments are shown in Fig. 1. The contaminant concentrations in the control group did not increase significantly, which varied from 102.8 to 136.31 mg/L dry weight(dw). While the petroleum hydrocarbon concentration in the lowconcentration group decreased to the lowest value of 63.70 mg/L dw on day 2, and then increased to 133.69 mg/L dw, which was higher than those of control group when prolong to day 7. In the middle and high-concentration treatment groups, petroleum hydrocarbon concentrations varied from 103.59 to 398.09 mg/L dw, and from 158.09 to 554.15 mg/L dw, respectively. The highest values were observed on day 10 in all treatment groups. The petroleum hydrocarbon concentrations in scallops in the lowconcentrations group were significant lower on day 1, and higher on day 10 when compared to that of control group. The significant higher values were observed on days 4, 7, and 10 in the middleconcentration groups, and the entire exposure period in the high-concentration group when compared to that of the control group. The regression curve showed that petroleum hydrocarbon concentrations in the three test groups increased linearly with time (R=0.965, 0.940, and 0.987, n=15, P<0.05, respectively). After recovery for 4 days, petroleum hydrocarbon concentrations fell in all three test groups, with those in the middle- and high concentration groups being significantly higher than that of the control group.3.2 Effects of WSF on EROD activity with exposure time
EROD responses to WSF in the scallops are shown in Fig. 2. EROD activity in the gills varied from 3.45 pmol/(min∙mg)to 5.87 pmol/(min∙mg)for the control group. EROD activities increased significantly at first then decreased with time for the three test groups. The highest values of 26.52, 16.52, and 24.58 pmol/(min∙mg)was observed on day 2, 4, and 2 in the low-, middle-, and high-concentration groups, respectively, which was more than 4 times of the control group. Moreover, the induction rates varied from 55.8% to 443% for the three test groups in the entire exposure period except on day 1 in the lowconcentration group(P<0.01). The induction degree of EROD activity in the gills in the three treatments exhibited a significantly positive correlation with WSF concentration(R=0.961, n=12, P<0.05)on day 1. After a recovery period of 4 days, the average values of EROD activity decreased in the four groups with increasing WSF concentration(R=0.948, n=12, P<0.05), with 4.74, 3.83, 2.26, and 1.45 pmol/(min∙mg) for the the control, low-, middle-, and highconcentration groups, respectively.
EROD activity in the digestive gl and varied from 9.33 to 11.90 pmol/(min∙mg)for the control group, which were higher than that in the gills. Significant induction of EROD activity in the digestive glands of the low-concentration group was observed within 4 days, induction degree was from 17.6% to 63.2%(as shown in Fig. 2b). Significant inhibition rate from 55.9% to 73.8% and from 33.4% to 61.9% was observed for digestive gl and EROD activity in the middle- and high-concentration groups on days 1, 4, and 10 compared to the control group. After a recovery period of 4 days, EROD activity in the lowconcentration group was significant higher(42.7%)than that in the control group, but those in both middle- and high-concentration groups were significant lower(35.6% and 42.3%)than that of the control group.3.3 Effects of WSF on AHH activity with exposure time
Figure 3 shows AHH responses to WSF in the scallops. AHH activity in the gills varied from 0.43 to 0.89 U/mg for the control group. On day 1, AHH activity in the gills in the treatment groups was varied from 5.56 to 12.98 U/mg and significantly induced by WSF, which was 10 times higher than that of the control group. Except on day 1, the highest value of AHH activity in the low-, middle-, and highconcentration groups was 2.33, 0.83, and 1.45 U/mg and observed on day 10, 7, and 2, respectively. Significant induction of AHH activity in the gills was observed on day 10 in the low-concentration group with induction rate of more than 400%, on days 4 and 7 in the middle-concentration group with 60.9% and 80.1% as induction rates, and on day 2 in the highconcentration group with 63.8% as induction rate. After recovery for 4 days, significant difference was existed for AHH activities in the gill tissues between the treatment groups and control group. AHH activity in the gill tissues in the low- and middle-concentration groups was 34.0% and 54.0% higher than that of the control group, while that in the high-concentration group was only 63.4% of that of control group.
AHH activity in the digestive gl and varied from 0.079 to 0.116 U/mg for the control group, which were much lower than that in the gills. For the three test groups, AHH activities increased at first then decreased with time. The highest values of 0.25, 0.32 and 0.47 U/ mg was observed on days 1, 2, and 1 in the low-, middle-, and high-concentration groups, respectively. AHH activity in the digestive glands was significantly induced on days 1 and 2 in the low-concentration group with the induction rate of 119% and 144%, and on days 2, 4, and 7 in the middle-concentration group with the induction rate ranged from 161% to 283%. As for the high-concentration group, AHH activity within 4 days varied from 1.84 to 4.07 times of that of the control group, and thus significant induction by WSF existed. After recovery for 4 days, AHH activity in the digestive glands in the low-concentration group was 2 times of that in the control group, while the middle-, high-concentration, and control groups had similar levels of AHH activity in the digestive glands, varying from 0.067 to 0.090 U/mg.3.4 Effects of WSF on GST activity with exposure time
GST responses to WSF in the scallops are shown in Fig. 4. GST activity in the gills ranged from 328.6 to 590.6 U/mg for the control group. The highest value of GST activity in the gills was 450.4, 340.0, and 468.1 U/mg for the low-, middle-, and highconcentration groups, respectively. And they was observed on day 2 and then decreased with time. Moreover, significant inhibition was observed during most of the exposure period compared to that of the control group, with the inhibition rates ranged from 13.3% to 95.9%. However, there were similar levels of the GST enzyme in the gills in all treatment groups on day 1, and also in the low-concentration and control groups on day 10. After recovery for 4 days, significant lower value of 73.5%, 67.3%, and 54.1% of GST activity of the control group in the gills was observed in the low-, middle-, and high-concentration group, respectively. GST activity and WSF concentration were negatively correlated on days 7 and 10, and on recovered for day 4(R=-0.711, -0.886, and -0.684, respectively, n=12, P<0.05).
GST activity in the digestive gl and varied from 90.30 to 148.90 U/mg for the control group, which were much lower than that in the gills. The highest GST activity value, i.e. 203.3, 194.0, and 200.1 U/mg, recorded in the digestive gl and was observed on day 4 in the low-, middle-, and high-concentration group, respectively. GST activity in the digestive glands was significantly induced on day 1 in the middleconcentration group with 28.2% as the induction rate, and after day 4 in all treatment groups with induction rate ranged from 33.3% to 90.2%. Similar values from 129.0 to 176.5 U/mg of GST activity were observed in the three treatment groups and control group on day 2, and there were no significant difference between them. After recovery for 4 days, there were no significant differences in GST activity in the digestive glands of the low- and middleconcentration groups(154.0 and 112.4 U/mg)when compared to that of the control group(126.5 U/mg), while significantly higher values of 207.6 U/mg were observed in the high-concentration group.3.5 Effects of WSF on GPx activity with exposure time
3.5 Effects of WSF on GPx activity with exposure time In the control group, GPx activity in the gills varied from 74.24 to 84.12 U/mg(Fig. 5). GPx activity in the gills increased at first then decreased with time in the three test groups. The highest GPx activity values of 67.57, 57.51, and 86.19 U/mg were observed on days 7, 2, and 2 in the gills in the low-, middle-, and highconcentration groups, respectively. The inhibition rate ranged from 18.01% to 64.24 for GPx activity in the gills throughout the exposure period in the three treatment groups(P<0.05 or P<0.01)except for day 2 in the high-concentration group, on which the induction rate of 13.3% was observed(P<0.01). After recovery for 4 days, significant lower value of 69.0%, 50.9% and 51.4% of that of control group GPx activity in the gills was observed in the low-, middle-, and high-concentration group, respectively.
GPx activity in the digestive gl and varied from 31.86 to 63.24 U/mg for the control group, which were much lower than that in the gills during the entire exposure period. GPx activity in the digestive glands increased with time in the low- and highconcentration groups, and the highest value, 74.81 and 89.38 U/mg, was recorded on days 7 and 10, respectively. However, GPx activity varied from 30.18 to 44.24 U/mg in the middle-concentration group, with the highest value being observed on day 4. GPx activity in the digestive glands was significantly inhibited on days 1 and 10 in the low-concentration group with 30.1% and 44.0% as the inhibition rate. And on days 7 and 10 in the middle-concentration group, the significant inhibition rate of 50.5% and 38.4% was observed. While in the high-concentration group, significant inhibition rate of 28.4% and induction rate of 41.3% were observed on days 4 and 10, respectively. GPx activity in all four groups ranged from 31.86 to 36.55 U/mg on day 2, and no significant difference existed between them. After recovery for 4 days, similar levels of GPx activity in the digestive glands were observed in the lowconcentration group, while significant lower values, only 56.7% and 52.9% of the control group, were observed in the digestive glands of the middle- and high-concentration groups.4 DISCUSSION
EROD is a family of cytochrome P450 isozymes, which are crucial in the metabolism of many xenobiotics. AHH is the most sensitive metabolic enzyme in mixed-functional oxidase(MFO)when exposed to PAHs(Sen and Semiz, 2007; Marohn et al., 2008; Zhang et al., 2010). Biological responses to these organic compounds can be evaluated by measuring the induction of EROD and AHH activity. The results of the present study revealed a substantial EROD activity dose-response in gill tissues on day 1 in WSF-exposed C. farreri. Previous study reported that the EROD activity in digestive gl and of C. farreri was significantly induced, and increased with the increasing BaP concentration(Pan et al., 2009). AHH activity in C. farreri tissues was significantly induced. This result is consistent with the findings of Wang et al.(2007), in which the AHH activities of gills and digestive gl and of C. farreri were significantly induced by PAHs. Negative relationships also existed between EROD activity in digestive glands and petroleum hydrocarbon concentrations in C. farreri tissues(R=-0.443, n=72, P=0.030 1). This result suggests that C. farreri is sensitive enough to be used as a petroleum hydrocarbon pollution biomonitor. Additionally, EROD and AHH activity in the different treatment groups exhibited induction, saturation, and inhibition phases in response to WSF pollution when compared to that of the control group.
GST is involved in the biotransformation of several pollutants. It may also participate in detoxifying strong electrophiles with toxic, mutagenic, and carcinogenic properties. GST activity has been extensively used as an environmental biomarker(Cheung et al., 2002, 2004; Damiens et al., 2007). In the present study, GST activity was higher in gills than in digestive glands, which is consistent with the result of a previously published study(Cheung et al., 2001). This may be attributed to the fact that the gills receive more exposure to environmental contaminants than the digestive glands do. The gills may have higher detoxification rates and consequently higher GST activity than the digestive gl and . The results also revealed that GST activity was induced in the gills and inhibited in the digestive glands. This could because the digestive gl and is the principal organ for detoxification, suggesting that the conjugation of glutathione with xenobiotics and their metabolites increased in organisms when exposed to WSF. Cheung et al.(2002)reported that. GST activity in the gill negatively correlated with petroleum hydrocarbon concentration in C. farreri tissues(R=-0.631, n=72, P=9.38×10-4). Richardson et al.(2008)observed a negative correlation between tissue B[a]P and GST activity in hepatopancreas of the mussel, Perna viridis.
The antioxidant defense system, which includes superoxide dismutase(SOD), catalase(CAT), and glutathione peroxidase(GPx), has an important function in the elimination of active oxygen free radicals. The inducibility of enzymes, such as GPx and SOD, is an important response to pollutantinduced stress, such as oxidative stress(Solé et al., 2000; Cheung et al., 2004). In this study, GPx activity in the gill tissues and digestive glands was mainly inhibited during the exposure period. The substrate competition between GPx and CAT might be the cause of the reduction in GPx, allowing for the induction of CAT during most of the exposure period (Wang et al., 2013). This result was consistent with previous findings(Guyton et al., 1996; Cheung et al., 2004). The results also revealed that GPx activity in gill tissues negatively correlated with petroleum hydrocarbon concentration in other tissues(R=-0.482, n=72, P=0.017). Similarly, Richardson et al.(2008)found negative correlations between gill GPx activity and contaminants(total PAH and OC pesticides). However, previous studies have revealed positive correlations between GPx activity and organochlorine(OC)body burden(i.e., PCBs, dichlorophenyltrichloroethane, and lindane)in mussels(Solé et al., 1994).5 CONCLUSION
Exposure to crude oil WSF had significant effects on detoxification enzymes in C. farreri. The highest enzyme activity values in the gills and digestive glands were observed at different times with varying WSF concentrations. EROD activity in the gills and digestive glands in the 0.18 mg/L group(lowconcentration)were significantly induced. AHH response to WSF pollution in the gills and digestive glands was induced in most cases. GST activity was significantly inhibited in the gills and induced in the digestive glands during most of exposure period. GPx activity was significantly inhibited in the gills throughout the exposure period in the three treatment groups, and no significant difference in digestive glands between the control and treatment groups was observed in most cases. EROD activity in digestive glands and GST and GPx activity in gill tissues were negatively correlated with petroleum hydrocarbon body burden. These are potentially effective biomarkers of petroleum hydrocarbon pollutants. Controlled laboratory experiments for simulating field exposure scenarios are particularly useful in ascertaining suitable biomarkers for monitoring complex contaminant mixtures in the marine environment.6 ACKNOWLEDGEMENT
We thank our colleagues Ms. QU Lingyun from the First Institute of Oceanography, SOA, for technical assistance.
|Akcha F, Izuel C, Venier P, Budzinski H, Burgeot T, Narbonne J F. 2000. Enzymatic biomarker measurement and study of DNA adduct formation in benzo[a]pyrene-contaminated mussels, Mytilus galloprovincialis. Aquat. Toxicol., 49 (4): 269-287.|
|Bradford M M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem., 72 (1-2): 248-254.|
|Cheung C C C, Zheng G J, Li A M Y, Richardson B J, Lam P K S. 2001. Relationships between tissue concentrations of polycyclic aromatic hydrocarbons and antioxidative responses of marine mussels, Perna viridis. Aquat. Toxicol., 52 (3-4): 189-203.|
|Cheung C C C, Zheng G J, Lam P K S, Richardson B J. 2002. Relationships between tissue concentrations of chlorinated hydrocarbons (polychlorinated biphenyls and chlorinated pesticides) and antioxidative responses of marine mussels, Perna viridis. Mar. Pollut. Bull., 45 (1-12): 181-191.|
|Cheung C C C, Siu W H L, Richardson B J, De Luca-Abbott S B, Lam P K S. 2004. Antioxidant responses to benzo[a] pyrene and Aroclor 1254 exposure in the green-lipped mussel, Perna viridis. Environ. Pollut., 128 (3): 393-403.|
|Damiens G, Gnassia-Barelli M, LoquèS F, Roméo M, Salbert V. 2007. Integrated biomarker response index as a useful tool for environmental assessment evaluated using transplanted mussels. Chemosphere, 66 (3): 574-583.|
|De Luca-Abbott S B, Richardson B J, McClellan K E, Zheng G J, Martin M, Lam P K S. 2005. Field validation of antioxidant enzyme biomarkers in mussels (Perna viridis) and clams (Ruditapes philippinarum) transplanted in Hong Kong coastal waters. Mar. Pollut. Bull., 51 (8-11): 694-707.|
|Faria M, Carrasco L, Diez S, Riva M C, Bayona J M, Barata C. 2009. Multi-biomarker responses in the freshwater mussel Dreissena polymorpha exposed to polychlorobiphenyls and metals. Comp. Biochem. Physiol. C Toxicol. Pharmachol., 149 (3): 281-288.|
|González-Doncel M, Segundo L S, Sastre S, Tarazona J V, Torija C F. 2011. Dynamics of BNF-induced in vivo ethoxyresorufin-O-deethylase (EROD) activity during embryonic development of medaka (Oryzias latipes). Aquat. Toxicol., 105 (3-4): 421-427.|
|Guyton K Z, Xu Q, Holbrook N. 1996. Induction of the mammalian stress response gene GADD153 by oxidative stress: role of AP-1 element. Biochem. J., 314 (Pt 2): 547- 554.|
|Hylland K, Tollefsen K E, Ruus A, Jonsson G, Sundt R C, Sanni S, Utvik T I R, Johnsen S, Nilssen I, Pinturier L, Balk L, Baršienė J, Marigòmez I, Feist S W, Børseth J F. 2008. Water column monitoring near oil installations in the North Sea 2001-2004. Mar. Pollut. Bull., 56 (3): 414- 429.|
|Jiang F H, Zhao M L, Wang X Y, Zheng L, Chen J H, Wang X R. 2012. Determination of petroleum hydrocarbon in mussels by fluorescence spectro-photometry with ultrasonic extraction. Mar. Environ. Sci., 31 (6): 906-909. (in Chinese with English abstract)|
|Jung J H, Kim S J, Lee T K, Shim W J, Woo S Kim D J, Han C H. 2008. Biomarker responses in caged rockfish (Sebastes schlegeli) from Masan Bay and Haegeumgang, South Korea. Mar. Pollut. Bull., 57 (6-12): 599-606.|
|Marohn L, Rehbein H, Kündiger R, Hanel R. 2008. The suitability of cytochrome-P4501A1 as a biomarker for PCB contamination in European eel (Anguilla anguilla). J. Biotechnol., 136 (3-4): 135-139.|
|Meador J P, Stein J E, Reichert W L, Varanasi U. 1995. Bioaccumulation of polycyclic aromatic hydrocarbons by marine organisms. Rev. Environ. Contam. Toxicol., 143 : 79-165.|
|Monari M, Cattani O, Serrazanetti G P, Selli A, Pagliuca G, Zironi E, O'Hara S C M, Livingstone D R. 2007. Effect of exposure to benzo[a]pyrene on SODs, CYP1A1/1A2- and CYP2E1 immunopositive proteins in the blood clam Scapharca inaequivalvis. Mar. Environ. Res., 63 (3): 200- 218.|
|Nahrgang J, Camus L, Carls M G, Gonzalez P, Jönsson M, Taban I C, Bechmann R K, Christiansen J S, Hop H. 2010. Biomarker responses in polar cod (Boreogadus saida) exposed to the water soluble fraction of crude oil. Aquat. Toxicol., 97 (3): 234-242.|
|Pan L Q, Ren J Y, Zheng D B. 2009. Effects of benzo(a)pyrene exposure on the antioxidant enzyme activity of scallop Chlamys farreri. Chin. J. Oceanol. Limnol., 27 (1): 43-53.|
|Ren J Y, Pan L Q, Miao J J. 2006. Effects of benzo(a)pyrene and benzo(k)fluoranthene mixture on the toxicology parameter of scallop Chlamys farreri. Acta Scien. Circum., 26 (7): 1 180-1 186. (in Chinese with English abstract)|
|Richardson B J, Mak E, De Luca-Abbott S B, Martin M, McClellan K, Lam P K S. 2008. Antioxidant responses to polycyclic aromatic hydrocarbons and organochlorine pesticides in green-lipped mussels (Perna viridis): do mussels "integrate” biomarker responses?. Mar. Pollut. Bull., 57 (6-12): 503-514.|
|Sen A, Semiz A. 2007. Effects of metals and detergents on biotransformation and detoxification enzymes of leaping mullet (Liza saliens). Ecotoxicol. Environ. Saf., 68 (3): 405-411.|
|Solé M, Porte C, Albaigés J. 1994. Mixed-function oxygenase system components and antioxidant enzymes in different marine bivalves: its relation with contaminant body burdens. Aquat. Toxicol., 30 (3): 271-283.|
|Solé M, Nasci C, Livingstone D R. 2000. Study of the biological impact of organic contaminants on mussels (Mytilus galloprovincialis L.) from the Venice Lagoon, Italy: responses of CYP1A-immunopositive protein and benzo(a)pyrene hydroxylase activity. Biomarkers, 5 (2): 129-140.|
|State Oceanic Administration. 2007. The Specification for Marine Monitoring, Part 4, Seawater Analysis. GB17378- 2007, Beijing, Maritime Press. p.42-44. (in Chinese)|
|Tian L F, Ren Z, Cui Y, Chen B J, Jiao G B, Wang X D, Zhang X G. 2008. Acute toxicity of Shengli crude oil and its impact on AKP activity of Paralichthys Olivaceus. Mar. Fish. Res., 29 (6): 95-100. (in Chinese with English abstract)|
|Wang J, Pan L Q, Miao J J. 2007. Effects of polycyclic aromatic hydrocarbons on aryl hydrocarbon hydroxylase activities of marine scallop Chlamys farreri. Mar. Sci., 31 (12): 19- 23. (in Chinese with English abstract)|
|Wang X Y, Feng L J, Jiang F H, Zheng L, Zhao M L, Xu Z H. 2013. Effects of water-soluble fraction of crude oil on the activity of antioxidant enzyme and malondialdehyde content of Chlamys farreri. J. Ocean Univ. Chin., 43 (7): 45-50.(in Chinese with English abstract)|
|Xu S S, Song J M, Yuan H M, Li X G, Li N, Duan L W, Yu Y. 2011. Petroleum hydrocarbons and their effects on fishery species in the Bohai Sea, North China. J. Environ. Sci., 23 (4): 553-559.|
|Zhang Y, Song J M, Yuan H M, Xu Y Y, He Z P, Duan L Q. 2010. Biomarker responses in the bivalve (Chlamys farreri) to exposure of the environmentally relevant concentrations of lead, mercury, copper. Environ. , 30 (1): 19-25.|