research article catalase activity in brown mussels...

Research ArticleCatalase Activity in Brown Mussels (Perna perna) under AcuteCadmium Lead and Copper Exposure and Depuration Tests

Kamel Boudjema12 Sidali Kourdali3 Nabila Bounakous3

Abdellah Meknachi3 and Abdelmalek Badis12

1Department of Industrial Chemistry Faculty of Technology University of Saad Dahlab at Blida PO Box 270 09000 Blida Algeria2Laboratory of Natural Products Chemistry and Biomolecules (LNPCB) University of Saad Dahlab at BlidaPO Box 270 09000 Blida Algeria3National Centre for Research and Development of Fisheries and Aquaculture (CNRDPA) 11 Boulevard AmirouchePO Box 67 Bousmail Tipaza Algeria

Correspondence should be addressed to Abdelmalek Badis badisabdelmalekyahoofr

Received 8 June 2014 Revised 24 November 2014 Accepted 3 December 2014 Published 25 December 2014

Academic Editor Robert A Patzner

Copyright copy 2014 Kamel Boudjema et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Brown mussels (Perna perna) were exposed to cadmium (Cd) lead (Pb) and copper (Cu) concentrations under acute exposureand exposure-depuration tests for the estimation of biochemical biomarker catalase (CAT) The acute tests showed accumulatedCd Pb and Cu in Perna perna correlated linearly with the exposure concentrations (1198772 = 0794 1198772 = 0891 and1198772 = 0985 for CdPb and Cu resp) The results of CAT increased significantly in tissues of treatment mussels after 72 h exposure when comparedto control The values of total protein were disturbed in exposed groups when compared with control These results suggest thatmetabolites and catalase activity were affected by heavy metal exposures Analysis using the Spearman rank correlation coefficientshowed that the CAT activity appeared to have a significant positive correlativity (119877

119904= 0921119877

119904= 0949 and119877

119904= 0949 for Cd Pb

and Cu resp) with the accumulated Cd Pb and Cu concentrations respectively The result of exposure-depuration tests showedthat there is a general tendency for CAT to decrease in depuration phase suggesting that the induction of catalase is metal andormixture of metals dependent

1 Introduction

The coastal zone receives a large amount of metal pollutionfromagricultural and industrial activity [1] InAlgeriametalstraces in surface and seawaters and sediments harboursoriginate from industrial activities namely tanneries andpapermills (Cd Zn Cu Hg Cr andNi) wastewater (Zn CuCd Pb and Ni) and agriculture by improper use of mineralpesticides [2ndash9]

Moreover pollution by heavy metals is a serious problemdue to their toxicity and ability to accumulate in the biota [10]At the cellular level heavymetals are often involved in oxida-tive stress which results in the production of reactive oxygenspecies (ROS) [11] ROS includes the superoxide radical(O2

minus) hydrogen peroxide (H2O2) and the hydroxyl radical

all of which affect mainly lipids proteins carbohydrates andnucleic acids [11]

Furthermore the importance of antioxidant enzymes isgenerally emphasized in the prevention of oxidative stressesby scavenging of ROSThe antioxidant system comprises sev-eral enzymes such as superoxide dismutase (SOD) catalase(CAT) and guaiacol peroxidase (GPx) [11 12] The catalase(EC 11116) although not responding to a group of contami-nants has been shown to be a part of the antioxidant defenceCatalase removes hydrogen peroxide from cells during basalaerobic metabolism or after a pollution-enhanced oxyradicalgeneration that can be within the limit of tolerance of theorganism [13] Besides it has been demonstrated that theactivity of CAT is induced upon metal exposure to animalspecies by some environmental parameters and chemicalspollution [11 14]

Among all kinds of aquatic organisms brown mussels(Perna perna) have been widely used in the evaluation of

Hindawi Publishing CorporationJournal of Marine BiologyVolume 2014 Article ID 830657 9 pageshttpdxdoiorg1011552014830657

2 Journal of Marine Biology

the quality of aquatic environments [15ndash17] This study chosePerna perna as the test animal and aimed to investigate theinfluence of cadmium lead and copper on catalase activitytotal proteins and bioaccumulation and their correlationduring 72 h of exposure In the other part we investigate thereversibility of catalase in exposure-depuration test (72 h10days) and if this induction of catalase is metal andor mixtureof metals dependent

2 Materials and Methods

Mussels Perna perna (45ndash55 cm shell length) were col-lected from natural beds at Figuier Boumerdes Algeria(36∘46101584005381015840N 3∘28101584037921015840E) After collection mussels wereimmediately transported to the laboratory and acclimatizedin tanks with aerated natural filtered seawater for a periodof 15 days Stock solutions of cadmium lead and copperwere freshly prepared by dissolving the proper metal salts ofcadmium sulfate lead (II) nitrate and copper (II) chloridedihydrate in deionized (double distilled) water Fresh stocksolutions were prepared daily These solutions were seriallydiluted to get the experimental concentration for the toxi-city test The experimental method includes static renewal(24-hour renewal) test Moreover for all tests (exposureexposure-depuration) the mussels were divided into rectan-gular polystyrene trays 42 times 28 times 19 cm (15 mussels per tray)in a total volume of 20 L of seawatertray Besides in exposuretest the specimens mussels were exposed to cadmium (Cd)lead (Pb) and copper (Cu) at different concentrations (CdPb from 005 to 8mgsdotLminus1 Cu from 0005 to 0025mgsdotLminus1)for up to 72 h (exposure phase) A tray with pure seawaterhad served as control However in exposure-depuration testmussels were exposed to cadmium (119862

1= 2mgsdotLminus1) lead

(1198622= 2mgsdotLminus1) copper (119862

3= 0015mgsdotLminus1) and mixture

(1198621+ 1198622+ 1198623) for up to 72 h and were then transferred in a

clean water for 10 days (depuration phase) In parallel a traywith pure seawater had served as control

21 Some Physicochemical Properties and Nutrients SaltsDissolved of Aqueous Medium Physicochemical propertiesand nutrients were measured daily in medium before andafter eachwater renewal (24-hour renewal) Determination oftemperature (∘C) salinity (Psu) dissolved oxygen (mgsdotLminus1)and pH is done using a multiparametric YSI 556 Ammo-nia (NH

34) nitrogen nitrous (N-NO

2

minus) nitrate nitrogen(N-NO

3

minus) and orthophosphate (P-PO4

minus) were estimated byfollowing standard analytical methods [18]

22 Biochemical Determinations In order to determine pro-teins levels and catalase activity the tissue samples of 15survived test animals were collected from each tray at thefinal stages of the exposure test (72 h)Moreover in exposure-depuration test 3 to 4 mussels were collected from each trayafter 72 h of exposure phase In the second phase of exposure-depuration test (depuration phase) mussels were collected inselected days (1st 4th 7th and 10th) To ensure the stabilityof organelles and pH 5 g musselrsquos tissue initially underwent a

homogenization in chilled mortar by mixing at a rate of 110wv in Tris (hydroxymethyl) aminomethane 20mM bufferpH 78 and then was centrifuged at 10000 g for 30min at 4∘CTheobtained supernatant (S9 fraction)was used to determinethe proteins levels the CAT activity

221 Total Protein Proteins were assayed by the method ofLowry et al [19] that combines a biuret reaction and a reac-tionwith Folin-Ciocalteu reagent Samples of the supernatantobtained above (S9 fraction) were diluted to 15 18 and 110After addition of 5mL Lowry reagent the mixture washomogenized for 10min and then completed by 05mLFolin-Ciocalteu reagent freshly diluted to 12 After stirringand temporary resting in the dark for at least 30min theabsorbance was read on spectrophotometer at 660 nm Thecalibration rangewas established frombovine serumalbumin(BSA) solution stock standard 76 g

222 Catalase Activity The CAT activity was determinedby the method of Lartillot described by Atli et al [20] Thekinetic approach is to track the amount of active enzyme(appearing or disappearing) per unit time Fifty 120583L of theS9 fraction was added to 100120583L of 30 H

2O2solution and

24mL of 75mM phosphate buffer at pH 7 and then placedin a cuvette of the spectrophotometer in kinetic mode Thedecomposition of hydrogen peroxide was monitored inkinetic mode at 280 nm for 2min

23 Metal Analysis For metal analysis the soft tissues fromeach individual were dried in an oven at 70∘C for 48 h [21]and then digested with aqua regia or pseudototal digestion(HCl-HNO

3-H2O) at 95∘C for two hours until the solution

was clear [5]The solution is diluted to 100mLwith deionizedwater (double distilled) and used for the chemical analysisCadmium lead and copper were then analysed by atomicabsorption spectrophotometry type Perkin Elmer Analyst700 air-acetylene oxidizing flame source was used Thedetermination limits levels of the AAS machine for Cd (II)Pb (II) and Cu (II) ions were 003mgsdotLminus1 02mgsdotLminus1 and02mgsdotLminus1 respectively

The quality of the attacks was monitored by analysis ofinternational standards which are certified concentrationsprovided by the Japan International Cooperation Agency(JICA) in Algeria We used as standards cod fish tissue (CRM74026-a with certified concentration Cu 125plusmn007mgsdotKgminus1dry weight)

24 Statistical Analysis All data were processed using SPSS170 for Windows software All parameters detected were sta-tistically compared by one-way analysis of variance and LSDmultiple comparison test amongst the means The relation-ships among the biochemical and chemical parameters werecalculated by Spearmanrsquos rank correlation Statistical analysiswas expressed asmeansplusmn SD with119875 lt 005 being consideredsignificant and 119875 lt 001 being considered extremely signifi-cant

Journal of Marine Biology 3

Table 1 Means of some physicochemical properties of the aqueous medium before and after the renewal (+) augmentation (minus)diminishment

Parameters Control Cd Test Cd Control Pb Test Pb Control Pb Test CuΔ119879 (∘C) +0810 minus0116 +1350 minus0106 minus0260 minus0087ΔpH +0030 +0056 +0006 +0150 +0740 +0217ΔSalinity (Psu) minus0010 +0033 minus0016 minus0110 +0860 +0242ΔDissolved oxygen nd nd +0070 +0226 nd ndΔNH34 (mgsdotLminus1) +0003 minus0006 minus0003 minus0014 +0001 minus0017ΔN-NO

2

minus (mgsdotLminus1) minus0073 minus0169 +0030 minus0009 +0511 +0563ΔN-NO

3

minus (mgsdotLminus1) +0032 minus0068 +0048 +0009 +0236 +0193ΔP-PO

4

minus (mgsdotLminus1) minus0030 minus0110 +0070 minus0423 minus0020 minus0957nd not determined

Table 2 Means (plusmnSD) of bioaccumulation of different concentrations of cadmium lead and copper after 72 h exposure and Pearsoncorrelation coefficients between metals exposure and metals accumulation

Tests of cadmium and lead Test of copperConcentration(mgsdotLminus1)

Tissue Cd content(mgsdotKgminus1 dwminus1)

Tissue Pb content(mgsdotKgminus1 dwminus1)

Concentration(mgsdotLminus1)

Tissue Cu content(mgsdotKgminus1 dwminus1)

Control lt3 lt20 Control 3192 plusmn 00001005 lt3 lt20 0005 5148 plusmn 00002020 1068 plusmn 00013 8012 plusmn 00001 0015 7864 plusmn 000011 7246 plusmn 00007 32526 plusmn 00003 0025 9199 plusmn 000012 7345 plusmn 00012 48180 plusmn 00004 mdash mdash4 24901 plusmn 00016 98214 plusmn 00002 mdash mdash8 38157 plusmn 00016 92272 plusmn 00001 mdash mdashCorrelation 0794a 0891a 0985aaHighly significant correlation (119875 lt 001)

3 Results and Discussion

31 Acute Toxicity

311 Some Physicochemical Properties and Nutrient SaltsDissolved of Aqueous Medium Table 1 shows any significantdifferences of the average difference of physicochemical prop-erties and nutrient salts dissolved of seawater before and afterthe tests Besides the effects of the variation of the abioticparameters have been reported on CAT activity and bioaccu-mulation of heavy metals [12 22ndash26] Moreover temperatureand salinity affected the filtration rate of mussels and theamount of oxygen consumed As a result this influencesnutriments uptake and therefore xenobiotics uptake [7 13]Volodymyr (2011) [12] reported the variation of the environ-mental conditions (temperature pH oxygen level salinitytransition metal ions and iron) known to be inducers ofoxidative stress in aquatic animals In addition Khessiba et al[24] reported that CAT activity increased in Mytilus gallo-provincialis with the increase of the temperature and salinityIn the present study the abiotic factors may be less importanteffects when compared to the stress level of the heavy metalscontamination for our specimens In fact the measurementsobtained of biomarker (CAT) and bioaccumulations of heavymetals of mussels are the direct result of the effect of metalscontamination

312 TissueMetal Content Table 2 shows that the accumula-tion ofCd Pb andCu inP perna is concentration-dependentBesides themetal accumulation inmussels increased linearlywith increasing concentration of metals in the medium Thecoefficient of correlation 1198772 values between metal concentra-tion in solution and that accumulated by mussels for Cd Pband Cu were 0794 0891 and 0985 respectively

Moreover during the accumulation processes of heavymetals the accumulation mechanism is an overall combina-tion of uptake metabolism and excretion processes [22] Inthis paper an increase in the bioaccumulation in exposedspecimens suggested the ability of P perna to bioaccumulatethese metals in their tissues Further the bioaccumulationcapacity of bivalves exposed to metal contamination in thelaboratory has been demonstrated in other marine musselssuch as Mytilus galloprovincialis [22] Perna viridis [27]and Mytilus edulis [28] Moreover the apparent biologicalaccumulation indicated that partitioning of this compoundbetween seawater and mussels was an important accumu-lation mechanism and a steady state was reached after ashort treatment time Furthermore Kamaruzzaan et al [27]reported that the rate of bioaccumulation of essential heavymetals in Perna viridis is faster than nonessential metals Inthis paper based on accumulated levels in the tissue of Pernaperna the bioaccumulation of cadmium lead and copperfollowed this order Cu2+ gt Pb2+ gt Cd2+ These results are


01020304050

Con

trol

005 0

1

015 0

2

025 0

5 1 2 3 4 6 8

a

ca a a

ba

aa

ab

aa

Prot

ein

y = 09467x + 17998

R2 = 04186(m

gmiddotm

gminus1 of

tiss

ue)

Exposure concentration of cadmium (mgmiddotLminus1)

(a)

01020304050

Con

trol

005 0

1

015 0

2

025 0

5 1 2 3 4 6 8

a

b b b a a a bc c

b b b

Prot

ein

y = 08419x + 26582

R2 = 03732

(mgmiddot

mgminus

1 of ti

ssue

)

Exposure concentration of lead (mgmiddotLminus1)

(b)

01020304050

Control 0005 0015 0025

aa

aa

Prot

ein

y = minus2944x + 4579

R2 = 05215

(mgmiddot

mgminus

1 of ti

ssue

)

Exposure concentration of copper (mgmiddotLminus1)

(c)

Figure 1 Mean (plusmn SD) variation of total protein in Perna perna of control and Cd-exposed (a) Pb-exposed (b) and Cu-exposed mussels (c)after 72 h exposure Values followed by the same letter are not statistically different (119875 gt 005) but values followed by the different letter arestatistically significant (119875 lt 005)

comparable to the study where the rate of bioaccumulationfollowed this order Fe gt Zn gt Cu gt Co gt Pb gt Cd [27]

313 Total Protein Perna perna exposed to cadmium leadand copper showed changes in metabolites The values oftotal protein were not significantly higher (119875 lt 005) inthe exposed concentrations (025 and 6mgsdotLminus1) of cadmiumwhen compared with control (Figure 1(a)) Furthermore thetotal level of protein significantly decreased (119875 lt 005) tocadmium concentration (0005mgsdotLminus1) Besides concentra-tions of cadmium (01 015 020 050 2 3 4 6 and 8mg Lminus1)showed no significant difference after 72 h of exposure whencompared with control (Figure 1(a)) However the values oftotal protein were significantly higher (119875 lt 001) in all theexposed concentration of lead when compared with control(Figure 1(b)) Though the mean values of proteins in thetissues of P pernamussels at 0005mgsdotLminus1 of copper exposureshowed no significant difference after 72 h exposure More-over the values of total protein were not significantly lower(119875 gt 005) in exposed concentrations (0015 and 0025mgsdotLminus1of copper) when compared with control (Figure 1(c)) More-over a weak correlation between the levels of total proteinand the exposure concentrations (1198772 = 0418 1198772 = 0373and 1198772 = 0521 for Cd Pb and Cu resp) indicates that thetotal level of protein had variation with no linearity in the CdPb and Cu treated P perna

Protein reserves appear to be affected by the chemical pol-lutant [25 26] However Rajkumar (2013) [26] reported thatthe metabolites of mussels given by the level of total proteincan be affected by heavy metal exposures At low or height

cadmium lead and copper concentrations and physiologicalconcentrations of H

2O2 protein damage is restricted to

amino acid modification at specific metal binding sites [25]Moreover Rajkumar and John Milton [25] observed anincrease of levels of total proteins in Perna viridis exposed todeferent concentrations of lead cadmium and zinc but nosignificant changes in copper exposure Besides no signifi-cant difference in protein contents was reported by Borkovicet al [29] in mussels M galloprovincialis collected in a placewhich is known for intensive industrial pollution during bothwinter and spring seasons However Rajkumar (2013) [26]reported that the total level of protein in Perna indica signifi-cantly decreased with increase in concentration of a trivalentarsenic in short toxicity test Though according to Moslehet al [30] depletion of proteins is a response to early defenseagainst chemical stress Thus depletion of protein levels canbe attributed to protein catabolism in response to energydemand To overcome stress the organisms need a lot ofenergy and this demand can induce protein catabolism inaddition the decrease of protein levels may be due to thelipoproteins formation which will be used to repair damagein cells tissues and organs [31]

314 Catalase Activities Perna perna exposed to cadmiumlead and copper showed changes in CAT antioxidantenzyme Furthermore all treatment groups show the catalaseactivity significantly increased with increase in concentrationin the medium (Figures 2(a) 2(b) and 2(c)) Moreover after72-hour exposure a very significant increase (119875 lt 001)in catalase activity was observed at 8 8 and 0025mgsdotLminus1cadmium lead and copper groups respectively (7205 plusmn 45


01020304050607080

Con

trol

005 0

1

015 0

2

025 0

5 1 2 3 4 6 8

bc

c

b

aa a

a a

aa

a

b

Cata

lase

y = 31842x + 18456

R2 = 0876

(Umiddotm

gminus1

of p

rote

in)


(a)

01020304050607080

Con

trol

005 0

1

015 0

2

025 0

5 1 2 3 4 6 8

a a a a aa a a

a

c c

cc

Cata

lase

y = 37162x + 94008R2 = 09433

(Umiddotm

gminus1

of p

rote

in)


(b)

01020304050607080

Control 0005 0015 0025

a

cc

a

Cata

lase

y = 19429x minus 1607

R2 = 08425

(Umiddotm

gminus1

of p

rote

in)


(c)

Figure 2 Mean (plusmn SD) variation of catalase activity in Perna perna of control and Cd-exposed (a) Pb-exposed (b) and Cu-exposed mussels(c) after 72 h exposure Values followed by the same letter are not statistically different (119875 gt 005) but values followed by the different letterare statistically significant (119875 lt 005)

5841 plusmn 1127 and 5832 plusmn 227Usdotmgminus1 of protein resp)compared to the control group

Besides in bivalves increased CAT activity is associatedwith tissue burden accompanied by heavy metals [32] CATis the primary scavengers of H

2O2in the cell Increased

CAT activity presently seen exposed to trace metals in Pperna further indicates that pollution stress has elevatedthe formation rate of H

2O2 These results are in agreement

with those reported by Rajkumar and John Milton [25] whoobserved a significant induction of catalase in Perna viridis byCd Cu Pb and Zn under short toxicity test Moreover theseresults are comparable to those found in other studies whereCAT activity increases at sites contaminated with metals[33] Also elevated activity of CAT was reported in Mytilusgalloprovincialis in the Adriatic Sea [29] Seasonal alterationsin the CAT activities were measured in the digestive gland ofthe brown mussel P perna [16]

However some heavy metals are required at structuraland catalytic sites in protein and are regarded as essentialmet-als others do not have a known biological function and arethus considered as nonessentialmetals [34] Furthermore theCATmechanism could also be better understood by control-ling the speciation of cadmium lead and copper which playsa fundamental role in the interactions betweenmetal ions andliving organisms [35] Moreover the effects of trace metalson antioxidant defence enzymes are more evident when dif-ferent species are compared Trace metals seem to influencedirectly the antioxidant defence enzyme due to biologicalfactors which regulate fluctuations of defences [36]

Therefore metal ions are well known inducers of oxida-tive stress [12 25 26] They can stimulate ROS production

via two different mechanismsThe first one is related with theinterference of metal-related processes and the second onewith the generation of free radicals by ions with changeablevalence (transition metal ions) via Haber-Weiss reaction [12]Additionally cadmium and lead the typical nonessential andessential metals are often found together in the environmentbecause of similar properties but biologically they havediverse properties Both of them can be toxic when presentabove the critical level [37] In addition cadmium and leadare not transition metals on the contrary copper (redoxactive metals) indirectly induces ROS production and lipidperoxidation by affecting the antioxidant systems and affects aphysiology of mussels [12 25] Determinations of sulfhydrylsgroup levels that metals have high affinity towards and totalprotein levels could be beneficial in estimating the toxicity ofmetals [38]

Besides presence of contaminants is likely responsible forelevated CAT activity in tissues of P perna In this paper aninduction of CAT observed in the tissues exposed to cad-mium lead and copper suggests greater utilization of CAT inorder to combatmetals present in thesemarinemedia More-over lead alters filtration and assimilation rates [39] changesexpression of stress proteins [40] and decreases immunityin mussels [41] In addition lead binds with glutathione anddecreasesGSH level [42] Similar responses of decreasedGSHlevel have been shown in the mussel Perna perna as anindicator of oxidative stress [15] Lead induces oxidative stressin mussels suggesting that lead along with other metals caninduce LPO and production of ROS [43] Contrary to cad-miumand lead copper homeostasis in aquatic animals entailson regulated uptake transport and excretion However there


0102030405060708090

Control

Exposure phase Depuration phase

ca

c

a aaa aaaa

b

Cata

lase

0 3 1 4 7 10Exposure-depuration time (days)

(Umiddotm

gminus1

prot

ein)

C1 = 2 mgmiddotLminus1

(a)


0102030405060708090

Control

a aaa a

aa aac

aaCata

lase


(Umiddotm

gminus1

prot

ein)


(b)


0102030405060708090

Control

aa

a aa

c

aaaaa a

Cata

lase


(Umiddotm

gminus1

prot

ein)


(c)


0102030405060708090

0 3 1 4 7 10

Control

aaaa a a

ac

Cata

lase

Exposure-depuration time (days)

Mixture (C1 + C2 + C3)(U

middotmgminus

1pr

otei

n)

(d)

Figure 3 Mean (plusmn SD) variation of catalase activity in Perna perna of control and Cd- (a) Pb- (b) Cu- (c) and mixture-exposed mussels (d)during 3 days of exposure and 10 days of depuration Values followed by the same latter are not statistically different (119875 gt 005) but Valuesfollowed by the different latter are statistically significant (119875 lt 005)

is some specificity of these processes in hydrobionts relatedwith the possibility of brachial uptake along with intestinalBoth ways are efficient but depend on many factors and arehighly regulated processes [44]

In this study lower concentrations of copper at0035mgsdotLminus1 were lethal since no mussels survived during24 h of exposition On the contrary Cd and Pb do not causeacute physiological or biochemical damage to Perna pernacompared with copper It seems that the toxicity of thesetwo metals occurs only during chronic exposures Howevercadmium and lead different from copper are redox inactivemetals whose toxic effects are induced mainly by acting onthe sulfhydryls groups of the proteins [12 45] Consequentlyit is possible that the concentrations of metals required forCd2+ and Pb2+ to induct this response enzyme are wellbelow those that cause a crisis in the organism or a visibledegradation of the marine ecosystem Besides variationsof CAT activity are consistent with other findings showingdecreased and increased activities exposed to cadmiumcopper lead and zinc [46] In this study the degree ofinduction of CAT activity can be classified as follows CAT(Cu2+) gt CAT (Cd2+) gt CAT (Pb2+) However statisticalresults showed the CAT in tissue was significantly positivelycorrelated (119877

119904= 0921 119877

119904= 0949 and 119877

119904= 0949 for Cd Pb

and Cu resp) with the accumulated Cd Pb and Cu in thetissue of Perna perna after 72 h exposure (Table 3)

32 Exposure-Depuration Test Figures 3(a) 3(b) 3(c) and3(d) show the induction and the return of catalase activities

Table 3 Pearson correlation coefficients between catalase activityand metals accumulation

Parameters Pearson correlationCadmium accumulated 0921a

Lead accumulated 0949a

Copper accumulated 0949aaHighly significant correlation (119875 lt 001)

to their initial level after exposure-depuration test (72 h10days) Overall in all the exposure-depuration tests a highlysignificant (119875 lt 001) increase in CAT activity was observedduring the phase of exposure for all tested xenobiotics (CdPb Cu and mixture) The high significant induction (119875 lt001) was recorded after 72 h exposure (5904 plusmn 333Usdotmgminus1protein) inmixture of copper cadmium (119862


(1198622= 2mgsdotLminus1) and copper (119862

3= 0015mgsdotLminus1) when

compared to controls (2924 plusmn 28Usdotmgminus1 of protein)Moreover the lowest average difference of physicochem-

ical properties and nutrient salts dissolved of seawater beforeand after the exposure-depuration tests does not represent apossible source of physiological perturbation for mussels andcatalase activity (Table 4)

Furthermore these results support the reproducibilityof results in acute exposure test Besides it appears thatthe induction of CAT activity is higher in mixture than inmetal only Therefore the nonspecific nature of its responseas a biomarker is an advantage of a state of mixed metal


Table 4 Means of abiotic parameters in exposure-depuration tests (exp exposure dep depuration (+) augmentation and (minus)diminishment)

Parameters Cd-exp-dep Pb-exp-dep Cu-exp-dep Mixture-exp-depControl Treated Control Treated Control Treated Control Treated

Δ119879 (∘C) minus0530 minus0280 +0250 +0500 +0250 +0440 minus0530 +0070ΔpH +0010 minus0090 +0010 minus0010 minus0420 minus0640 +0010 +0050ΔSalinity (Psu) minus0550 +0030 minus0910 minus0170 minus0910 +0360 minus0550 +0010

pollution [15] Moreover in this paper the degree of induc-tion of CAT activity can be classified as follows CAT (Cu2++Cd2+ + Pb2+)mixture gt CAT (Cu2+)only gt CAT (Cd2+)only gtCAT (Pb2+)only

The return of catalase activities to their initial level afterdecontamination test illustrated the adaptability of molluscsresponses which allows us to qualify them as good bioindi-cators The catalase seems to adapt to environment changeswhichmake us think that this biomarkermay be used tomon-itor chemical pollution In this paper the enzymatic activitydecreased for all the groups after the transfer of musselsin a pollutant-free water (decontamination phase)

Moreover after 1 day 3 days 7 days and 10 days of depura-tion CAT activity decreased in mussels exposed to 2mg Lminus1of lead to values of 2439 plusmn 359 2174 plusmn 18 2083 plusmn 637and 163plusmn1599Usdotmgminus1 protein respectively Reductions are3882 4795 5013 and 6171 respectively SimilarlyCAT activity decreases in mussels exposed to 0015mg Lminus1 ofcopper to values of 3755 plusmn 723 2796 plusmn 1576 2781 plusmn 876and 1900 plusmn 827Usdotmgminus1 protein after 1 day 3 days 7 daysand 10 days respectively Reductions are 3882 47955013 and 4407 respectively Also after 1 day 3 days 7days and 10 days of depuration CAT activity decreased inmussels exposed to 2mg Lminus1 of cadmium to values of 3131 plusmn901 4788 plusmn 749 3411 plusmn 292 and 1383 plusmn 3571Usdotmgminus1protein respectively Reductions are 3338 5039 5066and 6629 respectively

However in case of mixture the results showed the per-centages of mortality which are 26 20 and 20 after 24 48and 72 h exposure respectively and 100 after one day ofdepuration phase It appears that a lethal effect occurs indecontamination phase in mussels exposed to mixture Sev-eral authors have noted that the lethal effect occurs in depu-ration phase after the exposure Sunila (1981) [47] reportedthat the mortality ofMytilus edulis occurs in 6 days of depu-ration after the exposure at 04mgsdotLminus1 of copper indicatingthat the biological damage is irreversible

Though CAT has proved capability of reflecting the stateof the surrounding environment in a short time (10 days indecontamination) This biomarker seems to be able to reflectthe curability of themussels health status and the reversibilityof the physiological mechanismThe same observations werereported by Company et al [48] after six days of depurationcycle with the mussels Bathymodiolus azoricus that had fol-lowed 24 days of exposure to 25120583gsdotLminus1 copper CAT activitiesrecorded in gills were found below those of contaminationphase Also Leonie et al [49] observed a return of catalase

activity to initial levels after 7 days of depuration in the gillsof oysters exposed to different concentrations of copper

4 Conclusion

In conclusion the metabolites and antioxidant enzymesactivity was affected by heavy metal exposures and theystrongly have the potential as indicators of heavy metalcontamination and cadmium lead and copper in specificBesides the induction of catalase in P perna is metal andormixture concentration-dependent Hence the determinationof oxidative stress biomarker (CAT) in Perna pernamay serveas a convenient approach during pollution biomonitoringprogramme

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This work belongs to NPR (National Project Research) andwas financially supported by the Centre for Research andDevelopment of Fisheries and Aquaculture (CNRDPA) ofAlgeria The authors would like to express their gratitude toMr Abdelkader Boudjema for his valuable proofreading andlanguage polishing services

References

[1] J Usero J Morillo and I Gracia ldquoHeavy metal concentrationsin molluscs from the Atlantic coast of southern Spainrdquo Chemo-sphere vol 59 no 8 pp 1175ndash1181 2005

[2] ldquoMarine pollution from land-based activitiesrdquo in Proceedingsof the Seminar Spouse Algeria-Japan p 108 JICA ONEDDMATE 2012

[3] S Megateli S Semsari and M Couderchet ldquoToxicity andremoval of heavymetals (cadmium copper and zinc) by LemnagibbardquoEcotoxicology andEnvironmental Safety vol 72 no 6 pp1774ndash1780 2009

[4] S Grimes T Ruellet J C Dauvin and Z Boutiba ldquoEcologicalQuality Status of the soft-bottom communities on the algeriancoast general patterns and diagnosisrdquoMarine Pollution Bulletinvol 60 no 11 pp 1969ndash1977 2010

[5] M Z Taleb and Z Boutiba ldquoLa mouleMytilus galloprovincialisbioindicatrice de pollutionmarine cas du port drsquoOranrdquo Sciencesamp Technologie C vol 1 no 25 pp 59ndash64 2007


[6] Z M Taleb I Benali H Gherras et al ldquoBiomonitoring ofenvironmental pollution on the Algerian west coast using cagedmusselsMytilus galloprovincialisrdquoOceanologia vol 51 no 1 pp63ndash84 2009

[7] N Benamar M Bouderbala and Z Boutiba ldquoEvaluation de laconcentration en cadmium drsquoun poisson pelagique communSardinella aurita dans la baie drsquoOranrdquo Journal des SciencesHalieutique et Aquatique vol 1 no 1 pp 16ndash20 2010

[8] B Belhaouari O RouaneHacene S Bouhadiba andZ BoutibaldquoUtilisation drsquoun Gasteropode marin Osilinus turbinatus enbiosurveillance marine application aux metaux lourds dulittoral algerien occidentalrdquo Science Halieutique et Aquaculturevol 1 no 3 pp 89ndash96 2010

[9] S Grimes Les peuplements macrobenthiques des substratsmleubles algeriens organisation et structure [PhD thesis] Uni-versite Es Senia Oran Algerie 2010

[10] M S Islam and M Tanaka ldquoImpacts of pollution on coastaland marine ecosystems including coastal and marine fisheriesand approach for management a review and synthesisrdquoMarinePollution Bulletin vol 48 no 7-8 pp 624ndash649 2004

[11] L R Vieira C Gravato A M V M Soares F Morgado andL Guilhermino ldquoAcute effects of copper and mercury on theestuarine fish Pomatoschistus microps linking biomarkers tobehaviourrdquo Chemosphere vol 76 no 10 pp 1416ndash1427 2009

[12] I I Volodymyr ldquoEnvironmentally induced oxidative stress inaquatic animalsrdquo Aquatic Toxicology vol 101 no 1 pp 13ndash302011

[13] G W Winston D R Livingstone and F Lips ldquoOxygen reduc-tion metabolism by the digestive gland of the common marinemusselMytilus edulis Lrdquo Journal of Experimental Zoology vol255 no 3 pp 296ndash308 1990

[14] T J Runnalls D N Hala and J P Sumpter ldquoPreliminarystudies into the effects of the human pharmaceutical Clofibricacid on sperm parameters in adult Fathead minnowrdquo AquaticToxicology vol 84 no 1 pp 111ndash118 2007

[15] A L Dafre I D Medeiros I C Muller E C Ventura and AC D Bainy ldquoAntioxidant enzymes and thioldisulfide status inthe digestive gland of the brown mussel Perna perna exposedto lead and paraquatrdquo Chemico-Biological Interactions vol 149no 2-3 pp 97ndash105 2004

[16] D W Filho T Tribess C Gaspari F D Claudio M A Torresand A R M Magalhaes ldquoSeasonal changes in antioxidantdefenses of the digestive gland of the brown mussel (Pernaperna)rdquo Aquaculture vol 203 no 1-2 pp 149ndash158 2001

[17] H G Chen X P Jia W G Cai Q Lin and S W MaldquoAntioxidant responses and bioaccumulation in green-lippedmussels (Perna Viridis) under acute tributyltin chloride expo-surerdquo Bulletin of Environmental Contamination and Toxicologyvol 87 no 5 pp 506ndash511 2011

[18] A Aminot andMChaussepiedManuel des Analyses Chimiquesen Milieu Marin Centre National pour lrsquoExploitation des Oceans(CNEXO) Centre National pour lrsquoExploitation des OceansParis France 1983

[19] O H Lowry N J Rosebrough A L Farr and R J RandallldquoProtein measurement with the Folin phenol reagentrdquo TheJournal of Biological Chemistry vol 193 no 1 pp 265ndash275 1951

[20] G Atli O Alptekin S Tukel and M Canli ldquoResponse ofcatalase activity to Ag+ Cd2+ Cr6+ Cu2+ and Zn2+ in fivetissues of freshwater fish Oreochromis niloticusrdquo ComparativeBiochemistry and PhysiologymdashC Toxicology and Pharmacologyvol 143 no 2 pp 218ndash224 2006

[21] MMerzouki N Talib and J Sif ldquoIndice de condition et teneursde quelques metaux (Cu Cd Zn et Hg) dans les organes de lamoule Mytilus galloprovincialis de la cote drsquoEl Jadida (Maroc)enmai et juin 2004rdquo Bulletin de lrsquoInstitut Scientifique vol 31 no1 pp 21ndash26 2009

[22] S Casas and C Bacher ldquoModelling trace metal (Hg and Pb)bioaccumulation in the Mediterranean mussel Mytilus gallo-provincialis applied to environmental monitoringrdquo Journal ofSea Research vol 56 no 2 pp 168ndash181 2006

[23] A Khessiba P Hoarau M Gnassia-Barelli P Aissa and MRomeo ldquoBiochemical response of the mussel Mytilus gallo-provincialis from Bizerta (Tunisia) to chemical pollutant expo-surerdquoArchives of Environmental Contamination and Toxicologyvol 40 no 2 pp 222ndash229 2001

[24] A Khessiba M Romeo and P Aıssa ldquoEffects of some environ-mental parameters on catalase activity measured in the mussel(Mytilus galloprovincialis) exposed to lindanerdquo EnvironmentalPollution vol 133 no 2 pp 275ndash281 2005

[25] J S I Rajkumar and M C John Milton ldquoBiochemical changesinduced by cadmium copper lead and zinc exposure to Pernaviridis under longterm toxicity testrdquo International Journal ofPharma and Bio Sciences vol 2 no 3 pp 50ndash59 2011

[26] J S I Rajkumar ldquoReduced glutathione and acetylcholinesteraseexpressions in Perna indica exposed to trivalent arsenicrdquo Inter-national Journal of Biological Research vol 1 no 1 pp 1ndash4 2013

[27] B Y Kamaruzzaan M S Mohd Zahir B Akbar John et alldquoBioaccumulation of somemetals byGreenmussel Perna viridis(Linnaeus 1758) from Pekan Pahang Malaysiardquo InternationalJournal of Biological Chemistry vol 5 no 1 pp 54ndash60 2011

[28] A T Marshall and V Talbot ldquoAccumulation of cadmium andlead in the gills of Mytilus edulis X-ray microanalysis andchemical analysisrdquo Chemico-Biological Interactions vol 27 no1 pp 111ndash123 1979

[29] S S Borkovic J S Saponjic S Z Pavlovic et al ldquoThe activityof antioxidant defence enzymes in the mussel Mytilus gallo-provincialis from the Adriatic Seardquo Comparative Biochemistryand PhysiologymdashC Toxicology and Pharmacology vol 141 no 4pp 366ndash374 2005

[30] Y Y Mosleh S Paris-Palacios M T Ahmed F M MahmoudM A Osman and S Biagianti-Risbourg ldquoEffects of chitosan onoxidative stress and metallothioneins in aquatic worm Tubifextubifex (Oligochaeta Tubificidae)rdquo Chemosphere vol 67 no 1pp 167ndash175 2007

[31] J C Amiard and C Amiard-Triquet Les biomarqueurs danslrsquoevaluation de lrsquo etat ecologique desmilieux aquatiques LavoisierParis France Technique et Documentation 2008

[32] C C C Cheung W H L Siu B J Richardson S B DeLuca-Abbott and P K S Lam ldquoAntioxidant responses tobenzo[a]pyrene and Aroclor 1254 exposure in the green-lippedmussel Perna viridisrdquo Environmental Pollution vol 128 no 3pp 393ndash403 2004

[33] I Lima S M Moreira J R-V Osten A M V M Soaresand L Guilhermino ldquoBiochemical responses of the marinemusselMytilus galloprovincialis to petrochemical environmen-tal contamination along the North-western coast of PortugalrdquoChemosphere vol 66 no 7 pp 1230ndash1242 2007

[34] T K S Janssens D Roelofs andNMVan Straalen ldquoMolecularmechanisms of heavy metal tolerance and evolution in inverte-bratesrdquo Insect Science vol 16 no 1 pp 3ndash18 2009

[35] K Pynnonen ldquoEffect of pH hardness and maternal pre-exposure on the toxicity of Cd Cu and Zn to the glochidial


larvae of a freshwater clam Anodonta cygneardquo Water Researchvol 29 no 1 pp 247ndash254 1995

[36] M J Bebianno R Company A Serafim L Camus R PCosson and A Fiala-Medoni ldquoAntioxidant systems and lipidperoxidation in Bathymodiolus azoricus from Mid-AtlanticRidge hydrothermal vent fieldsrdquo Aquatic Toxicology vol 75 no4 pp 354ndash373 2005

[37] BHenrikHansen S RoslashmmaOslashAGarmo S A Pedersen P AOlsvik and R A Andersen ldquoInduction and activity of oxidativestress-related proteins during waterborne CdZn-exposure inbrown trout (Salmo trutta)rdquo Chemosphere vol 67 no 11 pp2241ndash2249 2007

[38] C Gravato M Teles M Oliveira and M A Santos ldquoOxidativestress liver biotransformation and genotoxic effects induced bycopper inAnguilla anguilla Lmdashthe influence of pre-exposure to120573-naphthoflavonerdquo Chemosphere vol 65 no 10 pp 1821ndash18302006

[39] M L Pessatti C Resgalla Jr R W Reis Fo J Kuehn LC Salomao and J D Fontana ldquoVariability of filtration andfood assimilation rates respiratory activity andmultixenobioticresistance (MXR) mechanism in the mussel Perna perna underlead influencerdquo Brazilian Journal of Biology vol 62 no 4 pp651ndash656 2002

[40] M Radlowska and J Pempkowiak ldquoStress-70 as indicator ofheavy metals accumulation in blue mussel Mytilus edulisrdquoEnvironment International vol 27 no 8 pp 605ndash608 2002

[41] F E Nieto-Fernandez K Alcide and C Rialas ldquoHeavy metalsand neuroimmunomodulation inMytilus edulisrdquoActa BiologicaHungarica vol 51 no 2ndash4 pp 325ndash329 2000

[42] H Gurer-Orhan H U Sabir and H Ozgunes ldquoCorrelationbetween clinical indicators of lead poisoning and oxidativestress parameters in controls and lead-exposed workersrdquo Tox-icology vol 195 no 2-3 pp 147ndash154 2004

[43] N T Prakash and K S J Rao ldquoModulations in antioxidantenzymes in different tissues of marine bivalve Perna viridis dur-ing heavymetal exposurerdquoMolecular andCellular Biochemistryvol 146 no 2 pp 107ndash113 1995

[44] C Kamunde C Clayton and C M Wood ldquoWaterborne vsdietary copper uptake in rainbow trout and the effects ofprevious waterborne copper exposurerdquo American Journal ofPhysiology Regulatory Integrative and Comparative Physiologyvol 283 no 1 pp R69ndashR78 2002

[45] G Sudama J Zhang J Isbister and J D Willett ldquoMetabolicprofiling in Caenorhabditis elegans provides an unbiasedapproach to investigations of dosage dependent lead toxicityrdquoMetabolomics vol 9 no 1 pp 189ndash201 2013

[46] F Regoli ldquoTrace metals and antioxidant enzymes in gillsand digestive gland of the mediterranean mussel Mytilus gal-loprovincialisrdquo Archives of Environmental Contamination andToxicology vol 34 no 1 pp 48ndash63 1998

[47] I Sunila ldquoToxicity of copper and cadmium toMytilus edulis L(Bivalvia) in brackish waterrdquo Annales Zoologici Fennici vol 18no 4 pp 213ndash223 1981

[48] R Company A Serafim R P Cosson et al ldquoAntioxidantbiochemical responses to long-term copper exposure in Bathy-modiolus azoricus from Menez-Gwen hydrothermal ventrdquo Sci-ence of the Total Environment vol 389 no 2-3 pp 407ndash4172008

[49] A Leonie H L S William W K C Eric et al AntioxidantEnzymes As Biomarkers of Environmental Stress in Oysters inPort Curtis Cooperative Research Centre for Coastal ZoneEstuary and Waterway Management Hong Kong 2006

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


the quality of aquatic environments [15ndash17] This study chosePerna perna as the test animal and aimed to investigate theinfluence of cadmium lead and copper on catalase activitytotal proteins and bioaccumulation and their correlationduring 72 h of exposure In the other part we investigate thereversibility of catalase in exposure-depuration test (72 h10days) and if this induction of catalase is metal andor mixtureof metals dependent

2 Materials and Methods

Mussels Perna perna (45ndash55 cm shell length) were col-lected from natural beds at Figuier Boumerdes Algeria(36∘46101584005381015840N 3∘28101584037921015840E) After collection mussels wereimmediately transported to the laboratory and acclimatizedin tanks with aerated natural filtered seawater for a periodof 15 days Stock solutions of cadmium lead and copperwere freshly prepared by dissolving the proper metal salts ofcadmium sulfate lead (II) nitrate and copper (II) chloridedihydrate in deionized (double distilled) water Fresh stocksolutions were prepared daily These solutions were seriallydiluted to get the experimental concentration for the toxi-city test The experimental method includes static renewal(24-hour renewal) test Moreover for all tests (exposureexposure-depuration) the mussels were divided into rectan-gular polystyrene trays 42 times 28 times 19 cm (15 mussels per tray)in a total volume of 20 L of seawatertray Besides in exposuretest the specimens mussels were exposed to cadmium (Cd)lead (Pb) and copper (Cu) at different concentrations (CdPb from 005 to 8mgsdotLminus1 Cu from 0005 to 0025mgsdotLminus1)for up to 72 h (exposure phase) A tray with pure seawaterhad served as control However in exposure-depuration testmussels were exposed to cadmium (119862


(1198622= 2mgsdotLminus1) copper (119862

3= 0015mgsdotLminus1) and mixture

(1198621+ 1198622+ 1198623) for up to 72 h and were then transferred in a

clean water for 10 days (depuration phase) In parallel a traywith pure seawater had served as control

21 Some Physicochemical Properties and Nutrients SaltsDissolved of Aqueous Medium Physicochemical propertiesand nutrients were measured daily in medium before andafter eachwater renewal (24-hour renewal) Determination oftemperature (∘C) salinity (Psu) dissolved oxygen (mgsdotLminus1)and pH is done using a multiparametric YSI 556 Ammo-nia (NH

34) nitrogen nitrous (N-NO

2

minus) nitrate nitrogen(N-NO

3

minus) and orthophosphate (P-PO4

minus) were estimated byfollowing standard analytical methods [18]

22 Biochemical Determinations In order to determine pro-teins levels and catalase activity the tissue samples of 15survived test animals were collected from each tray at thefinal stages of the exposure test (72 h)Moreover in exposure-depuration test 3 to 4 mussels were collected from each trayafter 72 h of exposure phase In the second phase of exposure-depuration test (depuration phase) mussels were collected inselected days (1st 4th 7th and 10th) To ensure the stabilityof organelles and pH 5 g musselrsquos tissue initially underwent a

homogenization in chilled mortar by mixing at a rate of 110wv in Tris (hydroxymethyl) aminomethane 20mM bufferpH 78 and then was centrifuged at 10000 g for 30min at 4∘CTheobtained supernatant (S9 fraction)was used to determinethe proteins levels the CAT activity

221 Total Protein Proteins were assayed by the method ofLowry et al [19] that combines a biuret reaction and a reac-tionwith Folin-Ciocalteu reagent Samples of the supernatantobtained above (S9 fraction) were diluted to 15 18 and 110After addition of 5mL Lowry reagent the mixture washomogenized for 10min and then completed by 05mLFolin-Ciocalteu reagent freshly diluted to 12 After stirringand temporary resting in the dark for at least 30min theabsorbance was read on spectrophotometer at 660 nm Thecalibration rangewas established frombovine serumalbumin(BSA) solution stock standard 76 g

222 Catalase Activity The CAT activity was determinedby the method of Lartillot described by Atli et al [20] Thekinetic approach is to track the amount of active enzyme(appearing or disappearing) per unit time Fifty 120583L of theS9 fraction was added to 100120583L of 30 H

2O2solution and

24mL of 75mM phosphate buffer at pH 7 and then placedin a cuvette of the spectrophotometer in kinetic mode Thedecomposition of hydrogen peroxide was monitored inkinetic mode at 280 nm for 2min

23 Metal Analysis For metal analysis the soft tissues fromeach individual were dried in an oven at 70∘C for 48 h [21]and then digested with aqua regia or pseudototal digestion(HCl-HNO

3-H2O) at 95∘C for two hours until the solution

was clear [5]The solution is diluted to 100mLwith deionizedwater (double distilled) and used for the chemical analysisCadmium lead and copper were then analysed by atomicabsorption spectrophotometry type Perkin Elmer Analyst700 air-acetylene oxidizing flame source was used Thedetermination limits levels of the AAS machine for Cd (II)Pb (II) and Cu (II) ions were 003mgsdotLminus1 02mgsdotLminus1 and02mgsdotLminus1 respectively

The quality of the attacks was monitored by analysis ofinternational standards which are certified concentrationsprovided by the Japan International Cooperation Agency(JICA) in Algeria We used as standards cod fish tissue (CRM74026-a with certified concentration Cu 125plusmn007mgsdotKgminus1dry weight)

24 Statistical Analysis All data were processed using SPSS170 for Windows software All parameters detected were sta-tistically compared by one-way analysis of variance and LSDmultiple comparison test amongst the means The relation-ships among the biochemical and chemical parameters werecalculated by Spearmanrsquos rank correlation Statistical analysiswas expressed asmeansplusmn SD with119875 lt 005 being consideredsignificant and 119875 lt 001 being considered extremely signifi-cant




2


3


4










31 Acute Toxicity





01020304050

Con

trol

005 0

1

015 0

2

025 0

5 1 2 3 4 6 8

a

ca a a

ba

aa

ab

aa

Prot

ein

y = 09467x + 17998

R2 = 04186(m

gmiddotm

gminus1 of

tiss

ue)


(a)

01020304050

Con

trol

005 0

1

015 0

2

025 0

5 1 2 3 4 6 8

a

b b b a a a bc c

b b b

Prot

ein

y = 08419x + 26582

R2 = 03732

(mgmiddot

mgminus

1 of ti

ssue

)


(b)

01020304050

Control 0005 0015 0025

aa

aa

Prot

ein

y = minus2944x + 4579

R2 = 05215

(mgmiddot

mgminus

1 of ti

ssue

)


(c)










01020304050607080

Con

trol

005 0

1

015 0

2

025 0

5 1 2 3 4 6 8

bc

c

b

aa a

a a

aa

a

b

Cata

lase

y = 31842x + 18456

R2 = 0876

(Umiddotm

gminus1

of p

rote

in)


(a)

01020304050607080

Con

trol

005 0

1

015 0

2

025 0

5 1 2 3 4 6 8

a a a a aa a a

a

c c

cc

Cata

lase

y = 37162x + 94008R2 = 09433

(Umiddotm

gminus1

of p

rote

in)


(b)

01020304050607080

Control 0005 0015 0025

a

cc

a

Cata

lase

y = 19429x minus 1607

R2 = 08425

(Umiddotm

gminus1

of p

rote

in)


(c)













0102030405060708090

Control


ca

c

a aaa aaaa

b

Cata

lase


(Umiddotm

gminus1

prot

ein)


(a)


0102030405060708090

Control

a aaa a

aa aac

aaCata

lase


(Umiddotm

gminus1

prot

ein)


(b)


0102030405060708090

Control

aa

a aa

c

aaaaa a

Cata

lase


(Umiddotm

gminus1

prot

ein)


(c)


0102030405060708090

0 3 1 4 7 10

Control

aaaa a a

ac

Cata

lase



middotmgminus

1pr

otei

n)

(d)




119904= 0921 119877

119904= 0949 and 119877

119904= 0949 for Cd Pb
























4 Conclusion




Acknowledgments


References




























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




2


3


4










31 Acute Toxicity





01020304050

Con

trol

005 0

1

015 0

2

025 0

5 1 2 3 4 6 8

a

ca a a

ba

aa

ab

aa

Prot

ein

y = 09467x + 17998

R2 = 04186(m

gmiddotm

gminus1 of

tiss

ue)


(a)

01020304050

Con

trol

005 0

1

015 0

2

025 0

5 1 2 3 4 6 8

a

b b b a a a bc c

b b b

Prot

ein

y = 08419x + 26582

R2 = 03732

(mgmiddot

mgminus

1 of ti

ssue

)


(b)

01020304050

Control 0005 0015 0025

aa

aa

Prot

ein

y = minus2944x + 4579

R2 = 05215

(mgmiddot

mgminus

1 of ti

ssue

)


(c)










01020304050607080

Con

trol

005 0

1

015 0

2

025 0

5 1 2 3 4 6 8

bc

c

b

aa a

a a

aa

a

b

Cata

lase

y = 31842x + 18456

R2 = 0876

(Umiddotm

gminus1

of p

rote

in)


(a)

01020304050607080

Con

trol

005 0

1

015 0

2

025 0

5 1 2 3 4 6 8

a a a a aa a a

a

c c

cc

Cata

lase

y = 37162x + 94008R2 = 09433

(Umiddotm

gminus1

of p

rote

in)


(b)

01020304050607080

Control 0005 0015 0025

a

cc

a

Cata

lase

y = 19429x minus 1607

R2 = 08425

(Umiddotm

gminus1

of p

rote

in)


(c)













0102030405060708090

Control


ca

c

a aaa aaaa

b

Cata

lase


(Umiddotm

gminus1

prot

ein)


(a)


0102030405060708090

Control

a aaa a

aa aac

aaCata

lase


(Umiddotm

gminus1

prot

ein)


(b)


0102030405060708090

Control

aa

a aa

c

aaaaa a

Cata

lase


(Umiddotm

gminus1

prot

ein)


(c)


0102030405060708090

0 3 1 4 7 10

Control

aaaa a a

ac

Cata

lase



middotmgminus

1pr

otei

n)

(d)




119904= 0921 119877

119904= 0949 and 119877

119904= 0949 for Cd Pb
























4 Conclusion




Acknowledgments


References




























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


01020304050

Con

trol

005 0

1

015 0

2

025 0

5 1 2 3 4 6 8

a

ca a a

ba

aa

ab

aa

Prot

ein

y = 09467x + 17998

R2 = 04186(m

gmiddotm

gminus1 of

tiss

ue)


(a)

01020304050

Con

trol

005 0

1

015 0

2

025 0

5 1 2 3 4 6 8

a

b b b a a a bc c

b b b

Prot

ein

y = 08419x + 26582

R2 = 03732

(mgmiddot

mgminus

1 of ti

ssue

)


(b)

01020304050

Control 0005 0015 0025

aa

aa

Prot

ein

y = minus2944x + 4579

R2 = 05215

(mgmiddot

mgminus

1 of ti

ssue

)


(c)










01020304050607080

Con

trol

005 0

1

015 0

2

025 0

5 1 2 3 4 6 8

bc

c

b

aa a

a a

aa

a

b

Cata

lase

y = 31842x + 18456

R2 = 0876

(Umiddotm

gminus1

of p

rote

in)


(a)

01020304050607080

Con

trol

005 0

1

015 0

2

025 0

5 1 2 3 4 6 8

a a a a aa a a

a

c c

cc

Cata

lase

y = 37162x + 94008R2 = 09433

(Umiddotm

gminus1

of p

rote

in)


(b)

01020304050607080

Control 0005 0015 0025

a

cc

a

Cata

lase

y = 19429x minus 1607

R2 = 08425

(Umiddotm

gminus1

of p

rote

in)


(c)













0102030405060708090

Control


ca

c

a aaa aaaa

b

Cata

lase


(Umiddotm

gminus1

prot

ein)


(a)


0102030405060708090

Control

a aaa a

aa aac

aaCata

lase


(Umiddotm

gminus1

prot

ein)


(b)


0102030405060708090

Control

aa

a aa

c

aaaaa a

Cata

lase


(Umiddotm

gminus1

prot

ein)


(c)


0102030405060708090

0 3 1 4 7 10

Control

aaaa a a

ac

Cata

lase



middotmgminus

1pr

otei

n)

(d)




119904= 0921 119877

119904= 0949 and 119877

119904= 0949 for Cd Pb
























4 Conclusion




Acknowledgments


References




























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


01020304050607080

Con

trol

005 0

1

015 0

2

025 0

5 1 2 3 4 6 8

bc

c

b

aa a

a a

aa

a

b

Cata

lase

y = 31842x + 18456

R2 = 0876

(Umiddotm

gminus1

of p

rote

in)


(a)

01020304050607080

Con

trol

005 0

1

015 0

2

025 0

5 1 2 3 4 6 8

a a a a aa a a

a

c c

cc

Cata

lase

y = 37162x + 94008R2 = 09433

(Umiddotm

gminus1

of p

rote

in)


(b)

01020304050607080

Control 0005 0015 0025

a

cc

a

Cata

lase

y = 19429x minus 1607

R2 = 08425

(Umiddotm

gminus1

of p

rote

in)


(c)













0102030405060708090

Control


ca

c

a aaa aaaa

b

Cata

lase


(Umiddotm

gminus1

prot

ein)


(a)


0102030405060708090

Control

a aaa a

aa aac

aaCata

lase


(Umiddotm

gminus1

prot

ein)


(b)


0102030405060708090

Control

aa

a aa

c

aaaaa a

Cata

lase


(Umiddotm

gminus1

prot

ein)


(c)


0102030405060708090

0 3 1 4 7 10

Control

aaaa a a

ac

Cata

lase



middotmgminus

1pr

otei

n)

(d)




119904= 0921 119877

119904= 0949 and 119877

119904= 0949 for Cd Pb
























4 Conclusion




Acknowledgments


References




























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


0102030405060708090

Control


ca

c

a aaa aaaa

b

Cata

lase


(Umiddotm

gminus1

prot

ein)


(a)


0102030405060708090

Control

a aaa a

aa aac

aaCata

lase


(Umiddotm

gminus1

prot

ein)


(b)


0102030405060708090

Control

aa

a aa

c

aaaaa a

Cata

lase


(Umiddotm

gminus1

prot

ein)


(c)


0102030405060708090

0 3 1 4 7 10

Control

aaaa a a

ac

Cata

lase



middotmgminus

1pr

otei

n)

(d)




119904= 0921 119877

119904= 0949 and 119877

119904= 0949 for Cd Pb
























4 Conclusion




Acknowledgments


References




























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology











4 Conclusion




Acknowledgments


References




























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

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