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Biochemical of Anthracene in Milk Fish.
  Biochemical response of anthracene and benzo [a] pyrene inmilkfish  Chanos chanos L. Palanikumar n , A.K. Kumaraguru, C.M. Ramakritinan, M. Anand Department of Marine and Coastal Studies, School of Energy, Environment and Natural Resources, Madurai Kamaraj University, Madurai 625021, India a r t i c l e i n f o  Article history: Received 2 June 2011Received in revised form29 August 2011Accepted 30 August 2011 Keywords: AnthraceneBenzo [a] pyrene C. chanos Acute toxicityBioaccumulationBiomarkers a b s t r a c t Polycyclic aromatic hydrocarbons (PAHs) are common toxic pollutants found in the aquatic environ-ment, and the assessment of their impact on biota is of considerable concern. The aim of the presentresearch was to study the acute toxicity, bioaccumulation and biochemical response of milkfish  Chanoschanos  (Forsskal) to two selected PAHs: anthracene and benzo [a] pyrene. Acute toxicity test resultswere evaluated by the Probit analysis method and 96 h LC 50  values for  C. chanos  exposed to anthracenewas 0.030 mg l  1 and 0.014 mg l  1 for benzo [a] pyrene. Bioaccumulation concentration of anthracenewas high when compared to benzo [a] pyrene. Biomarkers indicative of neurotoxicity (acetylcholines-terase, AchE), oxidative stress (lipid peroxidation, LPO and catalase, CAT) and phase II biotransforma-tion of xenobiotics (glutathione S transferase, GST and reduced glutathione, GSH) were measured toassess effects of selected PAHs. Anthracene and benzo [a] pyrene increase LPO and CAT level of   C. chanos suggesting that these PAHs may induce oxidative stress. Both the PAHs inhibited AchE indicating thatthey have atleast one mechanism of neurotoxicity in common: the disruption of cholinergic transmis-sion by inhibition of AChE. An induction of   C. chanos  glutathione S-transferase (GST) activity was foundin fish exposed to benzo [a] pyrene, while an inhibition was observed after exposure to anthracene.These results suggest that GST is involved in the detoxification of benzo [a] pyrene, but not of anthracene. &  2011 Elsevier Inc. All rights reserved. 1. Introduction Polycyclic aromatic hydrocarbons (PAHs) are the most wide-spread organic pollutants. In addition to their presence in fossilfuels, they are also formed by incomplete combustion of fuelssuch as wood, coal, diesel, fat, tobacco, or incense. PAHs are foundwherever there is oil pollution and combustion wastes. Sedimentsof many marine and freshwater harbors and even remote oceanlocations are contaminated with PAHs (Oliva et al., 2010). PAHsand their halogenated forms are chemically stable, and due totheir lipophilic nature they can easily penetrate biological mem-branes and accumulate in organisms. PAHs are important envir-onmental pollutants because of their ubiquitous presence andcarcinogenicity (Tuvikene, 1995).Anthracene is a commercially important PAH produced inlarge quantities and extensively used as a reagent in organicsynthesis (Archer et al., 1979). Anthracene has also been usedfrequently as a model PAH for studies of environmental fate andtransport in aquatic systems (Ausmus et al., 1980) or physiologi-cal disposition in aquatic biota (Roubal et al., 1977). Benzo [a]pyrene (B [a] P) is classified as potent carcinogen and/or mutagen(Shaw and Connell, 1994). Sublethal amounts of B [a] P arecommonly found in marine environments especially after oil spillaccidents (Banni et al., 2010). Marine fish readily take uplipophilic organic contaminants such as B [a] P from the marineenvironment, with a variety of physiological effects (Walker andLivingstone, 1992), which is a drawback in the consumption of toxic marine fish as food.Aquatic animals have often been used in bioassays to monitorwater quality of effluents and surface waters (Brungs et al., 1978).Marine fish readily take up lipophilic organic contaminants suchas anthracene and B [a] P from the marine environment, with avariety of physiological effects (Walker and Livingstone, 1992),which is a drawback in the consumption of toxic marine fish asfood. PAHs have been found to induce adverse effects on fishgrowth (Hannah et al., 1982; Ostrander et al., 1990; Jifa et al., 2006; Kim et al., 2008), reproduction (Thomas, 1990; White et al., 1999; Monteverdi and DiGiulio, 2000) and survival (Collier and Varanasi, 1991; Hawkins et al., 1991). Furthermore, after biotransformation, these compounds may srcinate reactive pro-ducts that bind to DNA and may cause mutations or otheralterations on the genetic material (Hall and Glover, 1990;Marvin et al., 1995; Woodhead et al., 1999; Wahidulla and Rajamanickam, 2009). Contents lists available at SciVerse ScienceDirectjournal homepage: Ecotoxicology and Environmental Safety 0147-6513/$-see front matter  &  2011 Elsevier Inc. All rights reserved.doi:10.1016/j.ecoenv.2011.08.028 n Corresponding author. E-mail address: (L. Palanikumar). Please cite this article as: Palanikumar, L., et al., Biochemical response of anthracene and benzo [a] pyrene in milkfish  Chanos chanos .Ecotoxicol. Environ. Saf. (2011), doi:10.1016/j.ecoenv.2011.08.028 Ecotoxicology and Environmental Safety  ]  ( ]]]] )  ]]] – ]]]  Acute toxicity is the major subject of research for evaluatingthe impact of toxic effect of chemicals on fishes ( Johnson andFinley, 1980). The use of acute toxicity for testing the potentialhazards of chemical contaminants to aquatic animals is welldocumented (Henderson et al., 1960; Sanders and Cope, 1966; Hutchinson et al., 2006). Flow through toxicity tests werebelieved to provide a better estimate of toxicity than static orrenewal toxicity tests because they provide a greater control of toxicant concentrations, minimize changes in water quality,reduce accumulation of waste products in test exposure waters(Rand et al., 1995; ASTM, 1997; Welsh et al., 2008). Many PAH accumulation studies have been carried out todetermine the adverse effects of PAHs (Boleas et al., 1998; Wetzel and Van Vleet, 2004). Bioaccumulation results are integrated withbiochemical and toxicological data, providing more informationon the possible classes of contaminants, which cause adversebiological effects (Gaudy et al., 1991).In recent years, a relatively new concept in aquatic environ-mental study is the analysis of changes in various physiologicaland biochemical parameters in resident biota. The use of so-called‘biomarker’ has been adopted from ‘epidemiology’ or ‘moleculartoxicology’ (Verlecar et al., 2006). In fish, PAHs in general, aresubject to biotransformation in a first step by enzymes of thephase I enzymes. The first step in the xenobiotics metabolism isusually catalyzed by cytochrome P450-dependent monooxy-genases (phase I) and their products are subsequently coupledto endogenous metabolites (phase II) (Landis and Yu, 1995; Oliva et al., 2010). Therefore the role of PAHs detoxification deservesfurther research.The enzymatic activities of glutathione S-transferases (GST), afamily of multi-functional enzymes involved in phase II of biotransformation are related to cellular antioxidant defencesdue to the conjugation of electrophilic xenobiotics and oxidizedcomponents with glutathione (GSH) (Fitzpatrick et al., 1995). It isknown that oxidative damage is an important mechanism of toxicity induced by PAHs (Altenburger et al., 2003). The catalaseactivity (CAT) was selected as an oxidative stress biomarker, sinceit is an important enzymatic antioxidant defences (Livingstone,2001). Acetylcholinesterase activity is usually used in biomoni-toring programs as biomarker of exposure to organophosphoruspesticides and metals (Banni et al., 2005).Important aspect regarding the toxicity of petrochemical is thepotential that some of these compounds and mixtures seem tohave to inhibit the activity of acetylcholinesterase (AChE) and,thus, to disrupt cholinergic neurotransmission (Vieiria et al.,2008). In fact, several recent studies performed with invertebratesand fish reported inhibition of this enzyme after exposure to fueloil and/or to PAHs (Moreira et al., 2004; Zapata-Pe ´rez et al., 2004;Barsiene et al., 2006). However, no effects on AChE in fish exposedto PAHs have been reported ( Jifa et al., 2006). Therefore, this isalso a subject that needs further research, since this enzyme hasbeen used in biomonitoring studies (Lehtonen and Schiedek,2006; Monteiro et al., 2007). Fish play a major role for the flow of energy in aquaticecosystems. They are exposed continuously to contaminants inthe natural habitat and constitute an important part of humandiet, especially in the coastal region ( Jha, 2004). Fishes have beenthe most popular test organisms because they are presumed to bethe best understood organisms in the aquatic environment. Fishesexposed to PAHs in water column throughout their life cycle canserve as natural indicators of PAH contamination in surfacewaters (Logan, 2007).Milkfish  Chanos chanos  (Forsskal, 1775) is an important tropicalmarine fish and is cultured in the Philippines, Indonesia, India andTaiwan producing about 330,000 tonnes every year (Rabanal, 1988).The milkfish has been the subject of numerous studies of variedextent and depth by investigators in relation to biology, aquaculture,hatchery context and toxicity (Bagarinao, 1994; Magesh and Kumaraguru, 2006). In view of the above, the present investigationswere carried out to assess acute toxicity, bioaccumulation andbiomarker enzyme effects of anthracene and B [a] P on fingerlingsof the milkfish  C. chanos  (Forsskal) of the Gulf of Mannar, SoutheastCoast of India. 2. Materials and methods  2.1. Experimental animals Health and live specimens of a milkfish fingerlings  C. chanos  (Forsskal)(average size and weight 2.82 7 0.07 cm; 1.55 7 0.10 g) collected from Kundukaland Chinnapalam regions (Latitude 09 1 16.26 0 N and Longitude 079 1 12.88 0 E) of Pamban coast, Gulf of Mannar, Southeast coast of India. Initial disinfectiontreatment was carried out using benzyl konium chloride (1 mg l  1 ) for 1 h andKMnO 4  solution (1 mg l  1 ) for 1 h and then healthy individuals were separated,acclimatized for ten days in a large glass aquaria containing aged, filtered seawater(calcium hardness 385.4mg l  1 , magnesium hardness 1422.0 mg l  1 , dissolvedoxygen 6.44 mg l  1 , silicates 6.44 m g l  1 , in-organic phosphate 3.32 m g l  1 , nitrite–nitrogen 1.72 m mol l  1 , nitrate–nitrogen 4.40 mmoll  1 and ammonia 0.12 m g l  1 ).During this period, the fishes were fed on live brine shrimp (  Artemia sp ) nauplii(Cruz and Tamse, 1989) and starved 24 h prior to and during the experiment. Everyeffort as suggested by Bennett and Dooley (1982) was made to maintain optimalconditions during acclimatization. The study has been approved and partiallyfunded by Ministry of Earth Sciences — ICMAM, Chennai.  2.2. Determination of acute concentrations of anthracene and benzo [a] pyrene The acute toxicity bioassay procedure based on standard methods (Sprague,1973; OECD, 1993; APHA/AWWA/WEF, 1998) was conducted to determine the LC 50 values of anthracene (CAS no 120-12-1) and benzo [a] pyrene (CAS no 50-32-8)(Sigma-Aldrich Co, USA). Preliminary range finding tests was performed anddefinitive range concentrations were chosen i.e., 0.011, 0.023, 0.047, 0.094 and0.188 mg l  1 for anthracene and 0.002, 0.004, 0.007, 0.015 and 0.031 mg l  1 forbenzo [a] pyrene. The concentrations dissolved in test medium were estimated(UNESCO, 1984). The unfiltered experimental medium samples (1 L) were spikedwith the following internal standards: anthracene (1 m g) and benzo [a] pyrene(1 m g). The samples were consecutively extracted with 50 mL of hexane and 25 mL of dichloromethane. The organic extracts were combined, dried over anhydrous.Na 2 SO 4 , rotary-evaporated and fractionated in a glass column filled with neutralalumina (1 g) and silica gel (1 g), both five percent water deactivated. Two fractionswere eluted, the first one with 2 mL of hexane, which contained the aliphaticcompounds and the second with 10 mL of dichloromethane:hexane (30:70 v/v),which contained the PAHs.The second fraction was analyzed by Capillary Gas Chromatography — HighResolution Mass Spectrometry (GC/MS) system of Shimadzu GC/MS 2010 gaschromatograph. Separation took place in a DB-XLB column (60 m  0.25 mm  0.25 m m) (Agilent, Wilmington), temperature programmed from 50  1 C (4 min) to200  1 C at 6  1 C min  1 and finally to 325  1 C at 4  1 C min  1 , holding this temperaturefor 10 min (Gonzalez et al., 2006). Shimadzu data system was used for PAHanalysis. The recoveries of spiked PAHs standards ranged from 65to 102 percent.The range of concentrations present in test medium was 0.011, 0.022, 0.046, 0.094and 0.176 mg l  1 for anthracene and 0.001, 0.004, 0.007, 0.014 and 0.031 mg l  1 for benzo [a] pyrene. 0.05 percent acetone and seawater was maintained assolvent control and negative control. In control medium, concentrations of anthracene and benzo [a] pyrene was nil. The experiments were carried out inreplicate for a period of 96 h under flow-through test system and mortality of organisms were noted at an interval of 24 h.  2.3. Determination of PAHs bioaccumulation in fish tissues Dry and powdered whole body tissues of anthracene and benzo [a] pyreneexposed and control fish were used for estimation. All solvents used were HPLCgrade (Merck India Ltd., Mumbai). The extraction procedure was based on thestandard methods of  Al-Omair and Helaleh (2004). The Capillary GasChromatography — High Resolution Mass Spectrometry (GC/MS) system of Shi-madzu GC/MS 2010 gas chromatograph, equipped with auto-sampler, 30 m 0.25IDRTXs-5sil fused silica capillary column (Agilent, Wilmington) and Shimadzu datasystem was used for PAH analysis. Helium was used as the carrier gas and thecolumn head pressure was maintained at 10 psi to give an approximate flow rateof 1 ml min  1 . The injector and the transfer lines were maintained at 290  1 C and250  1 C, respectively. All injection volumes were 1 m l in splitless mode. The columntemperature was initially held at 70  1 C for 4 min, ramped to 300  1 C at a rate of  L. Palanikumar et al. / Ecotoxicology and Environmental Safety  ]  ( ]]]] )  ]]] – ]]] 2 Please cite this article as: Palanikumar, L., et al., Biochemical response of anthracene and benzo [a] pyrene in milkfish  Chanos chanos .Ecotoxicol. Environ. Saf. (2011), doi:10.1016/j.ecoenv.2011.08.028  10  1 C min  1 , then held at 300  1 C for 10 min (Anyakora et al., 2005). The massspectrometer was used in electron ionization mode and all spectra were acquiredusing a mass range of m/z 50–400 and automatic gain control (AGC).  2.4. Estimation of biomarker enzymes After the stipulated periods of treatment (96 h), the live fish were dissectedand tissues (head, gill and dorsal fin muscles) were isolated in ice-cold conditionfor further studies.  2.4.1. Protein The protein content in different fish tissues was determined by the methoddescribed by Lowry et al. (1951) using bovine serum albumin (BSA) as a standard.  2.4.2. Lipid peroxidation Head, gill and dorsal fin muscles were individually analyzed according toBuege and Aust (1978). Lipid peroxidation (LPO) was measured by the generationof thiobarbituric acid reactive species and quantified in terms of MDA equivalents.Its absorbance was measured at 532 nm with Systronics make double beam UVvisible spectrophotometer Model 2201 series. Each sample was run by triplicate.  2.4.3. Catalase Enzymatic activity was evaluated individually in head, gill and dorsal finmuscles of fish exposed to acute concentrations following the method describedby Bainy et al. (1996). Catalase activity (CAT) was measured by the rate of hydrogen peroxide (H 2 O 2 ) decomposition at 240 nm (Beutler, 1982) with Systro-nics make double beam UV visible spectrophotometer Model 2201 series. Eachsample was run by triplicate.  2.4.4. Acetyl choline esterase The activity of acetyl choline esterase (AChE) in head, gill and dorsal finmuscles of fish tissue was assayed according to the method of  Ellman et al. (1961).The reaction mixture (3 ml) contained sodium phosphate buffer (50 ml, pH 7.5),5,5,dithiobis-(nitrobenzoic acid), (DTNB, 0.5 mM prepared in 10 mM phosphatebuffer, pH 7.5 and 15 mg sodium bicarbonate added per 10 ml of solution), thesubstrate acetylthiocholine iodide (ATI, 0.5 mM) and enzyme protein (50–100 mg). For assays, the concentration of the substrate, DTNB and enzyme proteinin reaction mixture were chosen so as to give maximal reaction rate. The increasein absorbance was recorded at 412 nm and 28  1 C for 3 min in a Systronics makedouble beam UV visible spectrophotometer Model 2201 series. Measurement wasmade in triplicate for each tissue homogenate. Simultaneously two blanks werealso used. One containing phosphate buffer, DTNB and ATI but not enzyme proteinto determine hydrolysis of ATI and the second containing phosphate buffer, DTNBand enzyme protein but not substrate (ATI) to correct for any non-AChEdependent formation of thio nitro benzoic acid. The blank readings weresubtracted from the experimental absorbance increase per min. One unit of enzyme activity has been defined as the amount of enzyme required to catalyzethe hydrolysis of one micro mole of the ATI into product per minute underspecified experimental conditions. The specific activity of enzyme is expressed asunits of enzyme activity per mg protein. The extinction coefficient of the yellowanion (1.36  10 4 M  1 cm  1 ) was employed for calculating the enzyme activity(Ellman et al., 1961).  2.4.5. Glutathione S transferase Glutathione S transferase (GST) activity in head, gill and dorsal fin muscles of fish tissue was estimated according to the method of  Habig et al. (1974). Reactionmixtures contained 4.95 ml phosphate buffer (0.1 M, at pH 6.5):0.9 ml GSH(10 mM):0.15 ml CDNB (60 mM). One ml of reaction mixture was added to0.5 ml of the sample, with the final concentration of 1mMGSH and 1mMCDNBin the assay. The activity rate of GST was measured as the change in OD/minat 340 nm (ext. coefft. 9600 M  1 cm  1 ) in a Systronics make double beamUV visible spectrophotometer Model 2201 series and expressed as nmol min  1 mg protein  1 .  2.4.6. Reduced glutathione Reduced glutathione (GSH) content in head, gill and dorsal fin muscles wereindividually determined using a fluorometric assay (Jasco make fluorometerModel 6000 series) according to the method of  Hissin and Hilf (1976).  2.5. Statistical analysis Median lethal concentration (LC 50 ) values were calculated for 24, 48, 72 and96 h time points for each test series using the Probit analysis software (Finney,1971; USEPA, 1994). The differences in biomarkers in comparison to control for each PAHs were assessed by one way analysis of variance (ANOVA), Dunnett’s testwas employed to compare the significant difference between control and differentexposure concentrations (Zar, 1996). Analysis of variance was carried out usingGraph Pad prism software version 5.0. 3. Results  3.1. Influence of acetone Acetone (0.05 percent) was used as a solvent in the presentstudy. Control groups received equal volume of acetone. Theresults showed that the acute toxicity, bioaccumulation andbiomarker enzyme activity were not affected by acetone.  3.2. Acute toxicity The calculated 24, 48, 72 and 96 h acute LC 50  values and theirrespective 95 percent confidence limits for anthracene and benzo[a] pyrene exposed to fingerlings of   C. chanos  under flow-throughtest system was shown in Table 1. Non-locomotor and locomotorresponse in swimming behavior of fish was observed. Moreimmobilized response was observed in higher concentrations of anthracene and benzo [a] pyrene when compared to lowerconcentrations. Under acute effect, no activity was found in fishafter 80 h. At higher concentrations, fish showed erratic swim-ming movement. The death of the fish was confirmed by cessationof opercular movement. No mortality was observed in control andsolvent control group. Percentage mortality of   C. chanos  exposedto different acute concentrations of anthracene and benzo [a]pyrene is shown in Figs. 1 and 2. The mortality of fish increasedwith increase in concentrations of anthracene and benzo [a]pyrene as well as experimental duration.  3.3. Bioaccumulation Accumulation of anthracene and benzo [a] pyrene in thedifferent concentrations are summarized in Table 2. In controltissues, no accumulation was detected. Maximum increase inaccumulation was noticed with anthracene. This may be due toexposure of fish to higher concentration of anthracene i.e.,0.176 mg l  1 than that of benzo [a] pyrene i.e., 0.031 mg l  1 .  3.4. Biomarker  3.4.1. Lipid peroxidation Significant differences ( P  o 0.05) were detected among treat-ment and control fish. LPO level in  C. chanos  exposed to different  Table 1 Median lethal concentration (LC 50 ) values of anthracene and benzo [a] pyrene to milkfish  C. chanos  ( n ¼ 4) (Mean 7 SD).  Toxicants LC 50  (mg l  1 )  R  2  value Slope line equation 24 h 48 h 72 h 96 hAnthracene 0.433 7 0.054 0.241 7 0.029 0.098 7 0.008 0.030 7 0.004 0.959  Y  ¼ 0.538 þ 0.135  x Benzo [a] pyrene 0.088 7 0.017 0.071 7 0.018 0.049 7 0.015 0.014 7 0.001 0.971  Y  ¼ 0.116 þ 0.024  xL. Palanikumar et al. / Ecotoxicology and Environmental Safety  ]  ( ]]]] )  ]]] – ]]]  3 Please cite this article as: Palanikumar, L., et al., Biochemical response of anthracene and benzo [a] pyrene in milkfish  Chanos chanos .Ecotoxicol. Environ. Saf. (2011), doi:10.1016/j.ecoenv.2011.08.028  concentrations of PAHs is shown in Fig. 3a and b. Greater increasein LPO level was noticed in benzo [a] pyrene exposure. Of thethree tissues examined, maximum increase in LPO level wasnoticed in dorsal fin muscles of benzo [a] pyrene exposed animalsi.e., 73 percent in 0.031 mg l  1 .  3.4.2. Catalase CAT activity in  C. chanos  increased with increase in concentra-tions of anthracene and benzo [a] pyrene (Fig. 3c and d). Sig-nificant differences ( P  o 0.05) were observed between treatmentand control fish. Maximum increase in CAT level was noticed inbenzo [a] pyrene exposure. Of the three tissues tested, maximumincrease in CAT activity was noticed in dorsal fin muscles of benzo[a] pyrene exposed animals i.e., 65 percent in 0.031 mg l  1 .  3.4.3. Acetyl choline esterase The AchE levels of fish exposed to different acute concentra-tions of anthracene and benzo [a] pyrene decreased with increasein concentrations (Fig. 3e and f). Significant differences ( P  o 0.05)were detected among treatment and control fish. Maximumdecrease in AchE level was noticed in anthracene exposure. Of the three tissues tested, maximum reduction in AchE activity wasnoticed in gills of anthracene exposed animals i.e., 67 percent in0.176 mg l  1 . However, reduction in AchE activity was alsonoticed in the head and dorsal fin muscles of anthracene exposedanimals.  3.4.4. Glutathione S transferase Significant differences ( P  o 0.05) were detected among treat-ment and control fish. Increase in GST activity was observed inbenzo [a] pyrene exposed animals, while decrease in GST activitywas noticed in anthracene exposed fish (Fig. 3g and h). MaximumGST activity was noticed in the highest concentration of benzo [a]pyrene used i.e, 4.73 m g l  1 . Of the three tissues tested, maximumincrease in GST activity was noticed in gills of benzo [a] pyreneexposed animals i.e., 46 percent. However, increase in GSTactivity was also noticed in head and gill tissues of benzo [a]pyrene exposed animals. Maximum decrease in GST activity wasnoticed in the highest concentration of anthracene i.e.,11.17 m g l  1 . Of the three tissues tested, maximum decrease inGST activity was noticed in gills of anthracene exposed fish i.e., 66percent. This level was followed by dorsal fin muscles and headtissues. R  2  = 0.570 (24h)R  2 = 0.673 (48h)R  2  = 0.791 (72h)R  2  = 0.788 (96h)010203040506070Control    M  o  r   t  a   l   i   t  y   (   %   ) Concentration (mg.l -1 ) 24h48h72h96h24h48h72h96hSolvent control0.0010.0040.0070.0140.031 Fig. 2.  Percentage mortality of   C. chanos  exposed to different concentrations of benzo [a] pyrene.  Table 2 Bioaccumulation of anthracene and benzo [a] pyrene in whole body tissues of milkfish  C. chanos  ( n ¼ 3) (Mean 7 SD).  Anthracene Benzo [a] pyreneConcentration(mg l  1 ) Accumulation( l g g   1 dry weight)Concentration(mg l  1 ) Accumulation( l g g   1 dry weight) Control Not detected Control Not detected0.011 8.20 7 0.07 0.001 2.0 7 0.080.022 15.2 7 0.01 0.004 3.8 7 0.080.046 29.7 7 0.01 0.007 5.9 7 0.030.094 54.6 7 0.01 0.014 9.0 7 0.010.176 94.9 7 0.01 0.031 15.6 7 0.01 R  2  = 0.593 (24h)R  2  = 0.704 (48h)R  2  = 0.777 (72h)R  2  = 0.887 (96h)020406080100Control    M  o  r   t  a   l   i   t  y   (   %   ) Concentration (mg.l -1 ) 24h48h72h96h24h48h72h96hSolvent control0.0110.0220.0460.0940.176 Fig. 1.  Percentage mortality of   C. chanos  exposed to different concentrations of anthracene. L. Palanikumar et al. / Ecotoxicology and Environmental Safety  ]  ( ]]]] )  ]]] – ]]] 4 Please cite this article as: Palanikumar, L., et al., Biochemical response of anthracene and benzo [a] pyrene in milkfish  Chanos chanos .Ecotoxicol. Environ. Saf. (2011), doi:10.1016/j.ecoenv.2011.08.028

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Jul 23, 2017
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