Research Article Catalase Activity in Brown Mussels (Perna perna) under Acute Cadmium, Lead, and Copper Exposure and Depuration Tests

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1 Hindwi Pulishing Corportion Journl of Mrine Biology, Artile ID 83657, 9 pges Reserh Artile Ctlse Ativity in Brown Mussels (Pern pern) under Aute Cdmium, Led, nd Copper Exposure nd Depurtion Tests Kmel Boudjem, 1,2 Sidli Kourdli, 3 Nil Bounkous, 3 Adellh Meknhi, 3 nd Adelmlek Bdis 1,2 1 Deprtment of Industril Chemistry, Fulty of Tehnology, University of Sd Dhl t Blid, P.O. Box 27, 9 Blid, Algeri 2 Lortory of Nturl Produts Chemistry nd Biomoleules (LNPCB), University of Sd Dhl t Blid, P.O. Box 27, 9 Blid, Algeri 3 Ntionl Centre for Reserh nd Development of Fisheries nd Aquulture (CNRDPA) 11, Boulevrd Amirouhe, P.O. Box 67, Bousmil, Tipz, Algeri Correspondene should e ddressed to Adelmlek Bdis; disdelmlek@yhoo.fr Reeived 8 June 214; Revised 24 Novemer 214; Aepted 3 Deemer 214; Pulished 25 Deemer 214 Ademi Editor: Roert A. Ptzner Copyright 214 Kmel Boudjem et l. This is n open ess rtile distriuted under the Cretive Commons Attriution Liense, whih permits unrestrited use, distriution, nd reprodution in ny medium, provided the originl work is properly ited. Brown mussels (Pern pern) were exposed to dmium (Cd), led (P), nd opper (Cu) onentrtions under ute exposure nd exposure-depurtion tests for the estimtion of iohemil iomrker tlse (CAT). The ute tests showed umulted Cd, P, nd Cu in Pern pern orrelted linerly with the exposure onentrtions (R 2 =.794, R 2 =.891,ndR 2 =.985 for Cd, P, nd Cu, resp.). The results of CAT inresed signifintly in tissues of tretment mussels fter 72 h exposure when ompred to ontrol. The vlues of totl protein were distured in exposed groups when ompred with ontrol. These results suggest tht metolites nd tlse tivity were ffeted y hevy metl exposures. Anlysis using the Spermn rnk orreltion oeffiient showed tht the CAT tivity ppered to hve signifint positive orreltivity (R s =.921, R s =.949,ndR s =.949 for Cd, P, nd Cu, resp.) with the umulted Cd, P, nd Cu onentrtions, respetively. The result of exposure-depurtion tests showed tht there is generl tendeny for CAT to derese in depurtion phse, suggesting tht the indution of tlse is metl nd/or mixture of metls dependent. 1. Introdution The ostl zone reeives lrge mount of metl pollution from griulturlndindustriltivity [1]. In Algeri, metls tres, in surfe nd sewters nd sediments hrours, originte from industril tivities, nmely, tnneries nd ppermills(cd,zn,cu,hg,cr,ndni),wstewter(zn,cu, Cd, P, nd Ni), nd griulture y improper use of minerl pestiides [2 9]. Moreover, pollution y hevy metls is serious prolem due to their toxiity nd ility to umulte in the iot []. Attheellulrlevel,hevymetlsreofteninvolvedinoxidtive stress, whih results in the prodution of retive oxygen speies (ROS) [11]. ROS inludes the superoxide rdil (O 2 ), hydrogen peroxide (H 2 O 2 ), nd the hydroxyl rdil, ll of whih ffet minly lipids, proteins, rohydrtes, nd nulei ids [11]. Furthermore, the importne of ntioxidnt enzymes is generlly emphsized in the prevention of oxidtive stresses y svenging of ROS. The ntioxidnt system omprises severl enzymes suh s superoxide dismutse (SOD), tlse (CAT), nd guiol peroxidse (GPx) [11, 12]. The tlse (EC ), lthough not responding to group of ontminnts, hs een shown to e prt of the ntioxidnt defene. Ctlse removes hydrogen peroxide from ells during sl eroi metolism or fter pollution-enhned oxyrdil genertionthtnewithinthelimitoftolerneofthe orgnism [13]. Besides, it hs een demonstrted tht the tivity of CAT is indued upon metl exposure to niml speies y some environmentl prmeters nd hemils pollution [11, 14]. Among ll kinds of quti orgnisms, rown mussels (Pern pern) hveeenwidelyusedintheevlutionof

2 2 Journl of Mrine Biology thequlityof qutienvironments [15 17]. This study hose Pern pern s the test niml nd imed to investigte the influene of dmium, led nd opper on tlse tivity, totl proteins nd ioumultion, nd their orreltion during 72 h of exposure. In the other prt, we investigte the reversiility of tlse in exposure-depurtion test (72 h/ dys) nd if this indution of tlse is metl nd/or mixture of metls dependent. 2. Mterils nd Methods Mussels Pern pern ( m shell length) were olleted from nturl eds t Figuier, Boumerdes, Algeri ( N, E). After olletion, mussels were immeditely trnsported to the lortory nd limtized in tnks with erted nturl filtered sewter for period of 15 dys. Stok solutions of dmium, led, nd opper were freshly prepred y dissolving the proper metl slts of dmium sulfte, led (II) nitrte, nd opper (II) hloride dihydrte in deionized (doule distilled) wter. Fresh stok solutionswereprepreddily.thesesolutionswereserilly diluted to get the experimentl onentrtion for the toxiity test. The experimentl method inludes stti renewl (24-hour renewl) test. Moreover, for ll tests (exposure, exposure-depurtion), the mussels were divided into retngulr polystyrene trys m (15 mussels per try) in totl volume of 2 L of sewter/try. Besides, in exposure test, the speimens mussels were exposed to dmium (Cd), led (P), nd opper (Cu) t different onentrtions (Cd, P: from.5 to 8 mg L 1 ; Cu: from.5 to.25 mg L 1 ) forupto72h(exposurephse).atrywithpuresewter hd served s ontrol. However, in exposure-depurtion test, mussels were exposed to dmium (C 1 = 2mg L 1 ), led (C 2 =2mg L 1 ), opper (C 3 =.15 mg L 1 ), nd mixture (C 1 +C 2 +C 3 ) for up to 72 h nd were then trnsferred in len wter for dys (depurtion phse). In prllel, try with pure sewter hd served s ontrol Some Physiohemil Properties nd Nutrients Slts Dissolved of Aqueous Medium. Physiohemil properties nd nutrients were mesured dily in medium, efore nd fter eh wter renewl (24-hour renewl). Determintion of temperture ( C), slinity (Psu), dissolved oxygen (mg L 1 ), ndphisdoneusingmultiprmetriysi556.ammoni (NH 3,4 ), nitrogen nitrous (N-NO 2 ), nitrte nitrogen (N-NO 3 ), nd orthophosphte (P-PO 4 )wereestimtedy following stndrd nlytil methods [18] Biohemil Determintions. In order to determine proteinslevelsndtlsetivity,thetissuesmplesof15 survived test nimls were olleted from eh try t the finl stges of the exposure test (72 h). Moreover, in exposuredepurtion test 3 to 4 mussels were olleted from eh try fter 72 h of exposure phse. In the seond phse of exposuredepurtion test (depurtion phse), mussels were olleted in seleted dys (1st, 4th, 7th, nd th). To ensure the stility of orgnelles nd ph, 5 g mussel s tissue initilly underwent homogeniztion in hilled mortr y mixing t rte of 1/ w/v in Tris (hydroxymethyl) minomethne 2 mm uffer, ph 7.8, nd then ws entrifuged t g for 3 min t 4 C. The otined superntnt (S9 frtion) ws used to determine the proteins levels, the CAT tivity Totl Protein. Proteins were ssyed y the method of Lowry et l. [19] tht omines iuret retion nd retion with Folin-Ciolteu regent. Smples of the superntnt otined ove (S9 frtion) were diluted to 1/5, 1/8, nd 1/. After ddition of 5 ml Lowry regent, the mixture ws homogenized for min nd then ompleted y.5 ml Folin-Ciolteu regent freshly diluted to 1/2. After stirring nd temporry resting in the drk for t lest 3 min, the sorne ws red on spetrophotometer t 66 nm. The lirtion rnge ws estlished from ovine serum lumin (BSA) solution, stok stndrd 7.6 g% Ctlse Ativity. The CAT tivity ws determined y the method of Lrtillot desried y Atli et l. [2]. The kinetipprohistotrkthemountoftiveenzyme (ppering or disppering) per unit time. Fifty μl of the S9 frtion ws dded to μl of3%h 2 O 2 solution nd 2.4mLof75mMphosphteuffertpH7ndthenpled in uvette of the spetrophotometer in kineti mode. The deomposition of hydrogen peroxide ws monitored in kineti mode t 28 nm for 2 min Metl Anlysis. For metl nlysis, the soft tissues from eh individul were dried in n oven t 7 Cfor48h[21] nd then digested with qu regi or pseudototl digestion (HCl-HNO 3 -H 2 O) t 95 C for two hours until the solution ws ler [5]. The solution is diluted to ml with deionized wter (doule distilled) nd used for the hemil nlysis. Cdmium, led, nd opper were then nlysed y tomi sorption spetrophotometry type Perkin Elmer Anlyst 7; ir-etylene oxidizing flme soure ws used. The determintion limits levels of the AAS mhine for Cd (II), P (II), nd Cu (II) ions were.3 mg L 1,.2mg L 1,nd.2 mg L 1,respetively. The qulity of the ttks ws monitored y nlysis of interntionl stndrds whih re ertified onentrtions, provided y the Jpn Interntionl Coopertion Ageny (JICA) in Algeri. We used s stndrds od fish tissue (CRM 7426-, with ertified onentrtion, Cu: 1.25±.7 mg Kg 1 dry weight) Sttistil Anlysis. AlldtwereproessedusingSPSS 17. for Windows softwre. All prmeters deteted were sttistilly ompred y one-wy nlysis of vrine nd LSD multiple omprison test mongst the mens. The reltionships mong the iohemil nd hemil prmeters were lulted y Spermn s rnk orreltion. Sttistil nlysis ws expressed s mens ± SD, with P <.5 eing onsidered signifint nd P <.1 eing onsidered extremely signifint.

3 Journl of Mrine Biology 3 Tle 1: Mens of some physiohemil properties of the queous medium efore nd fter the renewl. (+): ugmenttion, ( ): diminishment. Prmeters Cd Test Cd P Test P P Test Cu ΔT ( C) ΔpH ΔSlinity (Psu) ΔDissolved oxygen nd nd nd nd ΔNH 3,4 (mg L 1 ) ΔN-NO 2 (mg L 1 ) ΔN-NO 3 (mg L 1 ) ΔP-PO 4 (mg L 1 ) nd: not determined. Tle 2: Mens (±SD) of ioumultion of different onentrtions of dmium, led, nd opper fter 72 h exposure nd Person orreltion oeffiients etween metls exposure nd metls umultion. Conentrtion (mg L 1 ) Tests of dmium nd led Tissue Cd ontent (mg Kg 1 dw 1 ) Tissue P ontent (mg Kg 1 dw 1 ) Conentrtion (mg L 1 ) Test of opper Tissue Cu ontent (mg Kg 1 dw 1 ) <3 < ±.1.5 <3 < ± ± ± ± ± ± ± ± ± ± ± ± ±.1 Correltion Highly signifint orreltion (P <.1). 3. Results nd Disussion 3.1. Aute Toxiity Some Physiohemil Properties nd Nutrient Slts Dissolved of Aqueous Medium. Tle 1 shows ny signifint differenes of the verge differene of physiohemil properties nd nutrient slts dissolved of sewter efore nd fter the tests. Besides, the effets of the vrition of the ioti prmeters hve een reported on CAT tivity nd ioumultion of hevy metls [12, 22 26]. Moreover, temperture nd slinity ffeted the filtrtion rte of mussels nd the mount of oxygen onsumed. As result, this influenes nutriments uptke nd therefore xenoiotis uptke [7, 13]. Volodymyr (211) [12] reported the vrition of the environmentl onditions (temperture, ph, oxygen level, slinity, trnsition metl, ions, nd iron) known to e induers of oxidtive stress in quti nimls. In ddition, Khessi et l. [24] reported tht CAT tivity inresed in Mytilus glloprovinilis withtheinreseofthetemperturendslinity. In the present study, the ioti ftors my e less importnt effets when ompred to the stress level of the hevy metls ontmintion for our speimens. In ft, the mesurements otined of iomrker (CAT) nd ioumultions of hevy metls of mussels re the diret result of the effet of metls ontmintion Tissue Metl Content. Tle 2 shows tht the umultion of Cd, P, nd Cu in P. pern is onentrtion-dependent. Besides, the metl umultion in mussels inresed linerly with inresing onentrtion of metls in the medium. The oeffiient of orreltion R 2 vlues etween metl onentrtion in solution nd tht umulted y mussels for Cd, P, nd Cu were.794,.891, nd.985, respetively. Moreover, during the umultion proesses of hevy metls, the umultion mehnism is n overll omintion of uptke, metolism, nd exretion proesses [22]. In this pper, n inrese in the ioumultion in exposed speimens suggested the ility of P. pern to ioumulte these metls in their tissues. Further, the ioumultion pity of ivlves exposed to metl ontmintion in the lortory hs een demonstrted in other mrine mussels suh s Mytilus glloprovinilis [22], Pern viridis [27], nd Mytilus edulis [28]. Moreover, the pprent iologil umultion indited tht prtitioning of this ompound etween sewter nd mussels ws n importnt umultion mehnism, nd stedy stte ws rehed fter short tretment time. Furthermore, Kmruzzn et l. [27] reported tht the rte of ioumultion of essentil hevy metls in Pern viridis is fster thn nonessentil metls. In this pper, sed on umulted levels in the tissue of Pern pern, the ioumultion of dmium, led, nd opper followed this order Cu 2+ > P 2+ > Cd 2+. These results re

4 4 Journl of Mrine Biology Protein (mg mg 1 of tissue) 5 y =.9467x R 2 = Protein (mg mg 1 of tissue) y =.8419x R 2 = Exposure onentrtion of dmium (mg L 1 ) Exposure onentrtion of led (mg L 1 ) () () Protein (mg mg 1 of tissue) y = 2.944x R 2 = Exposure onentrtion of opper (mg L 1 ) () Figure 1: Men (± SD) vrition of totl protein in Pern pern of ontrol nd Cd-exposed (), P-exposed (), nd Cu-exposed mussels () fter 72 h exposure. Vlues followed y the sme letter re not sttistilly different (P >.5) ut vlues followed y the different letter re sttistilly signifint (P <.5). omprletothestudywheretherteofioumultion followed this order Fe > Zn > Cu > Co > P > Cd [27] Totl Protein. Pern pern exposed to dmium, led, nd opper showed hnges in metolites. The vlues of totl protein were not signifintly higher (P <.5) in the exposed onentrtions (.25 nd 6 mg L 1 )ofdmium when ompred with ontrol (Figure 1()). Furthermore, the totl level of protein signifintly deresed (P <.5) to dmium onentrtion (.5 mg L 1 ). Besides, onentrtions of dmium (.1,.15,.2,.5, 2, 3, 4, 6, nd 8 mg L 1 ) showed no signifint differene fter 72 h of exposure when ompred with ontrol (Figure 1()). However, the vlues of totl protein were signifintly higher (P <.1) in ll the exposed onentrtion of led when ompred with ontrol (Figure 1()). Though, the men vlues of proteins in the tissues of P. pern mussels t.5 mg L 1 of opper exposure showed no signifint differene fter 72 h exposure. Moreover, the vlues of totl protein were not signifintly lower (P >.5) in exposed onentrtions (.15 nd.25 mg L 1 of opper) when ompred with ontrol (Figure1()). Moreover, wek orreltion etween the levels of totl protein nd the exposure onentrtions (R 2 =.418, R 2 =.373, nd R 2 =.521 for Cd, P, nd Cu, resp.) indites tht the totl level of protein hd vrition with no linerity in the Cd, P, nd Cu treted P. pern. Protein reserves pper to e ffeted y the hemil pollutnt [25, 26]. However, Rjkumr (213) [26]reportedtht the metolites of mussels given y the level of totl protein n e ffeted y hevy metl exposures. At low or height dmium, led nd opper onentrtions, nd physiologil onentrtions of H 2 O 2, protein dmge is restrited to mino id modifition t speifi metl inding sites [25]. Moreover, Rjkumr nd John Milton [25] oserved n inrese of levels of totl proteins in Pern viridis exposed to deferent onentrtions of led, dmium, nd zin, ut no signifint hnges in opper exposure. Besides, no signifint differene in protein ontents ws reported y Borković et l. [29] inmusselsm. glloprovinilis olleted in ple whih is known for intensive industril pollution during oth winter nd spring sesons. However, Rjkumr (213) [26] reported tht the totl level of protein in Pern indi signifintly deresed with inrese in onentrtion of trivlent rseni in short toxiity test. Though, ording to Mosleh et l. [3], depletion of proteins is response to erly defense ginst hemil stress. Thus, depletion of protein levels n e ttriuted to protein tolism in response to energy demnd. To overome stress, the orgnisms need lot of energy, nd this demnd n indue protein tolism; in ddition, the derese of protein levels my e due to the lipoproteins formtion whih will e used to repir dmge in ells, tissues, nd orgns [31] Ctlse Ativities. Pern pern exposed to dmium, led, nd opper showed hnges in CAT ntioxidnt enzyme. Furthermore, ll tretment groups show the tlse tivity signifintly inresed with inrese in onentrtion in the medium (Figures 2(), 2(),nd2()). Moreover, fter 72-hour exposure, very signifint inrese (P <.1) in tlse tivity ws oserved t 8, 8, nd.25 mg L 1 dmium, led, nd opper groups, respetively (72.5 ± 4.5,

5 Journl of Mrine Biology 5 Ctlse (U mg 1 of protein) y = x R 2 = Exposure onentrtion of dmium (mg L 1 ) () Ctlse (U mg 1 of protein) Ctlse (U mg 1 of protein) y = x 16.7 R 2 = Exposure onentrtion of opper (mg L 1 ) () y = x R 2 =.9433 Exposure onentrtion of led (mg L 1 ) () Figure 2: Men (± SD) vrition of tlse tivity in Pern pern of ontrol nd Cd-exposed (), P-exposed (), nd Cu-exposed mussels () fter 72 h exposure. Vlues followed y the sme letter re not sttistilly different (P >.5) ut vlues followed y the different letter re sttistilly signifint (P <.5) ± 11.27, nd58.32 ± 2.27 U mg 1 of protein, resp.), ompred to the ontrol group. Besides, in ivlves inresed CAT tivity is ssoited with tissue urden ompnied y hevy metls [32]. CAT is the primry svengers of H 2 O 2 in the ell. Inresed CAT tivity presently seen exposed to tre metls in P. pern further indites tht pollution stress hs elevted the formtion rte of H 2 O 2.Theseresultsreingreement with those reported y Rjkumr nd John Milton [25] who oserved signifint indution of tlse in Pern viridis y Cd, Cu, P, nd Zn under short toxiity test. Moreover, these results re omprle to those found in other studies, where CAT tivity inreses t sites ontminted with metls [33]. Also, elevted tivity of CAT ws reported in Mytilus glloprovinilis in the Adriti Se [29]. Sesonl ltertions in the CAT tivities were mesured in the digestive glnd of the rown mussel P. pern [16]. However, some hevy metls re required t struturl nd tlyti sites in protein nd re regrded s essentil metls; others do not hve known iologil funtion nd re thus onsidered s nonessentil metls [34]. Furthermore, the CAT mehnism ould lso e etter understood y ontrollingthespeitionofdmium,led,ndopperwhihplys fundmentl role in the intertions etween metl ions nd living orgnisms [35]. Moreover, the effets of tre metls on ntioxidnt defene enzymes re more evident when different speies re ompred. Tre metls seem to influene diretly the ntioxidnt defene enzyme due to iologil ftors, whih regulte flututions of defenes [36]. Therefore, metl ions re well known induers of oxidtive stress [12, 25, 26]. They n stimulte ROS prodution vi two different mehnisms. The first one is relted with the interferene of metl-relted proesses nd the seond one with the genertion of free rdils y ions with hngele vlene (trnsition metl ions) vi Her-Weiss retion [12]. Additionlly, dmium nd led, the typil nonessentil nd essentil metls, re often found together in the environment euse of similr properties, ut iologilly they hve diverse properties. Both of them n e toxi when present ove the ritil level [37]. In ddition, dmium nd led re not trnsition metls; on the ontrry, opper (redox tive metls) indiretly indues ROS prodution nd lipid peroxidtion y ffeting the ntioxidnt systems nd ffets physiology of mussels [12, 25]. Determintions of sulfhydryls group levels tht metls hve high ffinity towrds nd totl protein levels ould e enefiil in estimting the toxiity of metls [38]. Besides, presene of ontminnts is likely responsile for elevted CAT tivity in tissues of P. pern.inthispper,n indution of CAT oserved in the tissues exposed to dmium, led, nd opper suggests greter utiliztion of CAT in order to omt metls present in these mrine medi. Moreover, led lters filtrtion nd ssimiltion rtes [39], hnges expression of stress proteins [4], nd dereses immunity in mussels [41]. In ddition, led inds with glutthione nd dereses GSH level [42]. Similr responses of deresed GSH levelhveeenshowninthemusselpern pern, sn inditor of oxidtive stress[15]. Led indues oxidtive stress in mussels, suggesting tht led, long with other metls, n indue LPO nd prodution of ROS [43]. Contrry to dmium nd led, opper homeostsis in quti nimls entils on regulted uptke, trnsport, nd exretion. However, there

6 6 Journl of Mrine Biology Ctlse (U mg 1 protein) Exposure phse Depurtion phse Exposure-depurtion time (dys) Ctlse (U mg 1 protein) Exposure phse Depurtion phse Exposure-depurtion time (dys) C 1 =2mg L 1 C 2 =2mg L 1 () () Ctlse (U mg 1 protein) Exposure phse Depurtion phse Exposure-depurtion time (dys) Ctlse (U mg 1 protein) Exposure phse Depurtion phse Exposure-depurtion time (dys) C 3 =.15 mg L 1 Mixture (C 1 +C 2 +C 3 ) () (d) Figure 3: Men (± SD) vrition of tlse tivity in Pern pern of ontrol nd Cd- (), P- (), Cu- (), nd mixture-exposed mussels (d) during 3 dys of exposure nd dys of depurtion. Vlues followed y the sme ltter re not sttistilly different (P >.5) ut Vlues followed y the different ltter re sttistilly signifint (P <.5). is some speifiity of these proesses in hydroionts relted with the possiility of rhil uptke long with intestinl. Both wys re effiient ut depend on mny ftors nd re highly regulted proesses [44]. In this study, lower onentrtions of opper t.35 mg L 1 were lethl sine no mussels survived during 24 h of exposition. On the ontrry, Cd nd P do not use ute physiologil or iohemil dmge to Pern pern ompred with opper. It seems tht the toxiity of these two metls ours only during hroni exposures. However, dmium nd led, different from opper, re redox intive metls, whose toxi effets re indued minly y ting on the sulfhydryls groups of the proteins [12, 45].Consequently, it is possile tht the onentrtions of metls required for Cd 2+ nd P 2+ to indut this response enzyme re well elow those tht use risis in the orgnism or visile degrdtion of the mrine eosystem. Besides, vritions of CAT tivity re onsistent with other findings, showing deresed nd inresed tivities exposed to dmium, opper, led, nd zin [46]. In this study, the degree of indution of CAT tivity n e lssified s follows: CAT (Cu 2+ ) > CAT (Cd 2+ ) > CAT (P 2+ ). However, sttistil results showed the CAT in tissue ws signifintly positively orrelted (R s =.921, R s =.949,ndR s =.949 for Cd, P ndcu,resp.)withtheumultedcd,p,ndcuinthe tissue of Pern pern fter 72 h exposure (Tle 3) Exposure-Depurtion Test. Figures 3(), 3(), 3(), nd 3(d) show the indution nd the return of tlse tivities Tle 3: Person orreltion oeffiients etween tlse tivity nd metls umultion. Prmeters Cdmium umulted Led umulted Copper umulted Highly signifint orreltion (P <.1). Person orreltion to their initil level fter exposure-depurtion test (72 h/ dys). Overll, in ll the exposure-depurtion tests, highly signifint (P <.1) inrese in CAT tivity ws oserved during the phse of exposure for ll tested xenoiotis (Cd, P, Cu, nd mixture). The high signifint indution (P <.1) ws reorded fter 72 h exposure (59.4 ± 3.33 U mg 1 protein) in mixture of opper dmium (C 1 =2mg L 1 ), led (C 2 = 2mg L 1 ), nd opper (C 3 =.15 mg L 1 ), when ompred to ontrols (29.24 ±.28 U mg 1 of protein). Moreover, the lowest verge differene of physiohemil properties nd nutrient slts dissolved of sewter efore nd fter the exposure-depurtion tests does not represent possile soure of physiologil perturtion for mussels nd tlse tivity (Tle 4). Furthermore, these results support the reproduiility of results in ute exposure test. Besides, it ppers tht the indution of CAT tivity is higher in mixture thn in metl only. Therefore, the nonspeifi nture of its response s iomrker is n dvntge of stte of mixed metl

7 Journl of Mrine Biology 7 Tle 4: Mens of ioti prmeters in exposure-depurtion tests (exp: exposure, dep: depurtion, (+): ugmenttion, nd ( ): diminishment). Prmeters Cd-exp-dep P-exp-dep Cu-exp-dep Mixture-exp-dep Treted Treted Treted Treted ΔT ( C) ΔpH ΔSlinity (Psu) pollution [15]. Moreover, in this pper, the degree of indution of CAT tivity n e lssified s follows: CAT (Cu 2+ + Cd 2+ + P 2+ ) mixture > CAT (Cu 2+ ) only > CAT (Cd 2+ ) only > CAT (P 2+ ) only. The return of tlse tivities to their initil level fter deontmintion test illustrted the dptility of molluss responses whih llows us to qulify them s good ioinditors. The tlse seems to dpt to environment hnges, whih mke us think tht this iomrker my e used to monitor hemil pollution. In this pper, the enzymti tivity deresed for ll the groups fter the trnsfer of mussels in pollutnt-free wter (deontmintion phse). Moreover, fter 1 dy, 3 dys, 7 dys, nd dys of depurtion, CAT tivity deresed in mussels exposed to 2 mg L 1 of led to vlues of ± 3.59, ± 1.8, 2.83 ± 6.37, nd 1.63 ± U mg 1 protein, respetively. Redutions re 38.82%, 47.95%, 5.13%, nd 61.71%, respetively. Similrly, CAT tivity dereses in mussels exposed to.15 mg L 1 of opper to vlues of ± 7.23, ± 15.76, ± 8.76, nd 19. ± 8.27 U mg 1 protein fter 1 dy, 3 dys, 7 dys, nd dys, respetively. Redutions re 38.82%, 47.95%, 5.13%,nd44.7%,respetively.Also,fter1dy,3dys,7 dys, nd dys of depurtion, CAT tivity deresed in mussels exposed to 2 mg L 1 of dmium to vlues of ± 9.1, ± 7.49, ± 2.92, nd13.83 ± U mg 1 protein, respetively. Redutions re 33.38%, 5.39%, 5.66%, nd 66.29%, respetively. However, in se of mixture, the results showed the perentges of mortlity whih re 26, 2, nd 2% fter 24, 48, nd 72 h exposure, respetively, nd % fter one dy of depurtion phse. It ppers tht lethl effet ours in deontmintion phse in mussels exposed to mixture. Severl uthors hve noted tht the lethl effet ours in depurtion phse fter the exposure; Sunil (1981) [47] reported tht the mortlity of Mytilus edulis ours in 6 dys of depurtion fter the exposure t.4 mg L 1 of opper, inditing tht the iologil dmge is irreversile. Though, CAT hs proved pility of refleting the stte of the surrounding environment in short time ( dys in deontmintion). This iomrker seems to e le to reflet the urility of the mussels helth sttus nd the reversiility of the physiologil mehnism. The sme oservtions were reported y Compny et l. [48] fter six dys of depurtion yle with the mussels Bthymodiolus zorius tht hd followed24dysofexposureto25μg L 1 opper; CAT tivities reorded in gills were found elow those of ontmintion phse. Also, Leonie et l. [49] oservedreturnoftlse tivity to initil levels fter 7 dys of depurtion in the gills of oysters exposed to different onentrtions of opper. 4. Conlusion In onlusion, the metolites nd ntioxidnt enzymes tivity ws ffeted y hevy metl exposures nd they strongly hve the potentil s inditors of hevy metl ontmintion nd dmium, led, nd opper in speifi. Besides, the indution of tlse in P. pern is metl nd/or mixture onentrtion-dependent. Hene, the determintion of oxidtive stress iomrker (CAT) in Pern pern my serve s onvenient pproh during pollution iomonitoring progrmme. Conflit of Interests The uthors delre tht there is no onflit of interests regrding the pulition of this pper. Aknowledgments This work elongs to NPR (Ntionl Projet Reserh) nd ws finnilly supported y the Centre for Reserh nd Development of Fisheries nd Aquulture (CNRDPA) of Algeri. The uthors would like to express their grtitude to Mr. Adelkder Boudjem for his vlule proofreding nd lnguge polishing servies. Referenes [1] J. Usero, J. Morillo, nd I. Gri, Hevy metl onentrtions in molluss from the Atlnti ost of southern Spin, Chemosphere,vol.59,no.8,pp ,25. [2] Mrine pollution from lnd-sed tivities, in Proeedings of the Seminr Spouse Algeri-Jpn, p.8,jica,onedd, MATE, 212. [3] S. Megteli, S. Semsri, nd M. Couderhet, Toxiity nd removl of hevy metls (dmium, opper, nd zin) y Lemn gi, Eotoxiology nd Environmentl Sfety,vol.72,no.6,pp , 29. [4] S. Grimes, T. Ruellet, J. C. Duvin, nd Z. Bouti, Eologil Qulity Sttus of the soft-ottom ommunities on the lgerin ost: generl ptterns nd dignosis, Mrine Pollution Bulletin, vol.6,no.11,pp ,2. [5] M.Z.TlendZ.Bouti, LmouleMytilus glloprovinilis: ioinditrie de pollution mrine: s du port d Orn, Sienes &TehnologieC,vol.1,no.25,pp.59 64,27.

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9 Journl of Mrine Biology 9 lrve of freshwter lm Anodont ygne, Wter Reserh, vol.29,no.1,pp ,1995. [36]M.J.Beinno,R.Compny,A.Serfim,L.Cmus,R.P. Cosson, nd A. Fil-Médoni, Antioxidnt systems nd lipid peroxidtion in Bthymodiolus zorius from Mid-Atlnti Ridge hydrotherml vent fields, Aquti Toxiology, vol. 75, no. 4, pp , 25. [37] B.HenrikHnsen,S.Rømm,Ø.A.Grmo,S.A.Pedersen,P.A. Olsvik, nd R. A. Andersen, Indution nd tivity of oxidtive stress-relted proteins during wterorne Cd/Zn-exposure in rown trout (Slmo trutt), Chemosphere, vol. 67, no. 11, pp , 27. [38] C. Grvto, M. Teles, M. Oliveir, nd M. A. Sntos, Oxidtive stress, liver iotrnsformtion nd genotoxi effets indued y opper in Anguill nguill L. the influene of pre-exposure to β-nphthoflvone, Chemosphere,vol.65,no.,pp , 26. [39] M. L. Pesstti, C. Resgll Jr., R. W. Reis Fo, J. Kuehn, L. C.Slomo,ndJ.D.Fontn, Vriilityoffiltrtionnd food ssimiltion rtes, respirtory tivity nd multixenoioti resistne (MXR) mehnism in the mussel Pern pern under led influene, Brzilin Journl of Biology, vol. 62, no. 4, pp , 22. [4] M. Rdlowsk nd J. Pempkowik, Stress-7 s inditor of hevy metls umultion in lue mussel Mytilus edulis, Environment Interntionl,vol.27,no.8,pp.65 68,22. [41] F. E. Nieto-Fernndez, K. Alide, nd C. Rils, Hevy metls nd neuroimmunomodultion in Mytilus edulis, At Biologi Hungri,vol.51,no.2 4,pp ,2. [42] H. Gurer-Orhn, H. U. Sir, nd H. Özgüneş, Correltion etween linil inditors of led poisoning nd oxidtive stress prmeters in ontrols nd led-exposed workers, Toxiology,vol.195,no.2-3,pp ,24. [43] N. T. Prksh nd K. S. J. Ro, Modultions in ntioxidnt enzymes in different tissues of mrine ivlve Pern viridis during hevy metl exposure, Moleulr nd Cellulr Biohemistry, vol.146,no.2,pp.7 113,1995. [44] C. Kmunde, C. Clyton, nd C. M. Wood, Wterorne vs. dietry opper uptke in rinow trout nd the effets of previous wterorne opper exposure, Amerin Journl of Physiology: Regultory Integrtive nd Comprtive Physiology, vol. 283, no. 1, pp. R69 R78, 22. [45] G. Sudm, J. Zhng, J. Isister, nd J. D. Willett, Metoli profiling in Cenorhditis elegns provides n unised pproh to investigtions of dosge dependent led toxiity, Metolomis,vol.9,no.1,pp ,213. [46] F. Regoli, Tre metls nd ntioxidnt enzymes in gills nd digestive glnd of the mediterrnen mussel Mytilus glloprovinilis, Arhives of Environmentl Contmintion nd Toxiology, vol. 34, no. 1, pp , [47] I. Sunil, Toxiity of opper nd dmium to Mytilus edulis L. (Bivlvi) in rkish wter, Annles Zoologii Fennii, vol.18, no. 4, pp , [48] R. Compny, A. Serfim, R. P. Cosson et l., Antioxidnt iohemil responses to long-term opper exposure in Bthymodiolus zorius from Menez-Gwen hydrotherml vent, Siene of the Totl Environment, vol.389,no.2-3,pp , 28. [49] A. Leonie, H. L. S. Willim, W. K. C. Eri et l., Antioxidnt Enzymes As Biomrkers of Environmentl Stress in Oysters in Port Curtis, CoopertiveReserhCentreforCostlZone Estury nd Wterwy Mngement, Hong Kong, 26.

10 Interntionl Journl of Peptides BioMed Reserh Interntionl Hindwi Pulishing Corportion Advnes in Stem Cells Interntionl Hindwi Pulishing Corportion Hindwi Pulishing Corportion Virolog y Hindwi Pulishing Corportion Interntionl Journl of Genomis Hindwi Pulishing Corportion Journl of Nulei Aids Zoology Interntionl Journl of Hindwi Pulishing Corportion Hindwi Pulishing Corportion Sumit your mnusripts t The Sientifi World Journl Journl of Signl Trnsdution Hindwi Pulishing Corportion Genetis Reserh Interntionl Hindwi Pulishing Corportion Antomy Reserh Interntionl Hindwi Pulishing Corportion Enzyme Reserh Arhe Hindwi Pulishing Corportion Hindwi Pulishing Corportion Hindwi Pulishing Corportion Biohemistry Reserh Interntionl Interntionl Journl of Miroiology Hindwi Pulishing Corportion Interntionl Journl of Evolutionry Biology Hindwi Pulishing Corportion Hindwi Pulishing Corportion Moleulr Biology Interntionl Hindwi Pulishing Corportion Advnes in Bioinformtis Hindwi Pulishing Corportion Journl of Mrine Biology Hindwi Pulishing Corportion