Cytogenetic and Heamatological Profile of Sarotherodon melanotheron Exposed to Carica papaya L. In Fish Feed as Replacement for Rice Bran

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1 Americn-Eursin Journl of Toxicologicl Sciences 6 (1): 15-24, 2014 ISSN IDOSI Publictions, 2014 DOI: /idosi.ejts Cytogenetic nd Hemtologicl Profile of Srotherodon melnotheron Exposed to Cric ppy L. In Fish Feed s Replcement for Rice Brn S.O. Ayool, O.E. Akeredolu nd E.E. Ahiwe 1 Deprtment of Mrine Sciences, University of Lgos, Lgos Stte, Nigeri 2 Deprtment of Zoology, University of Lgos, Lgos Stte, Nigeri Abstrct: The qutic environment mkes up the mjor prt of our environment nd resources, its sfety is directly relted to the sfety of our helth nd food security. This study investigted the cytogenetic effect of blck jw Tilpi, Srotherodon melnotheron exposed to pwpw lef (Cric ppy L.) s replcement for rice brn for twelve weeks (0 dys). The hemtologicl effects of feeding Cric ppy lef mel rtions to Srotherodon melnotheron Juvenile were lso crried out. The study lso investigted the hemtologicl effects of feeding Cric ppy lef mel rtions to Srotherodon melnotheron Juvenile. The tretments of the pwpw leves for the experiment were T 0(0%), T 1(25%), T 2(50%), T 3(75%) nd T 4 (100%). The physico-chemicl prmeters showed significnt difference (p<0.05) nd positive strong correltion coefficient of temperture with dissolved oxygen nd ph (r = 0.8 nd r = 0. respectively). The men vlue for the physico chemicl prmeters mesured thorough out the experiment re s given thus: Temperture (26.47 ± 0.8), Dissolved oxygen (7.80 ± 0.64) nd ph (6.54 ± 0.43). The genotoxicity studies utilized micronucleus test of the exposed fish nd there ws no significnt difference (p > 0.05) in the micronuclei present in 30 dy in ll the tretment while, dy 60 nd 0 showed significnt difference (p < 0.05) in ll tretments. Anlysis of Vrince ( ANOVA) showed sttisticl significnt difference (p <0.05) in the blood prmeters: Men Hb (g/dl),pcv (%), WBC (10 /L), MCH (pg), Neutrophils (%) nd Lymphocytes (%) vlues t 0 %, 25%, 50%, 75% nd 100% level of inclusions. The trends observed in the hemoglobin, red blood cells nd pcked cell volumes' for the experimentl fish were indictions tht the dietry inclusion levels of 50%, 75% nd 100% fvoured n increse in hemtologicl prmeters. Cric ppy lef mel t 75% nd 100% inclusion in Clris griepinus Juvenile diet showed significnt weight gin nd feed utiliztion nd the rtion ws cost effective. It cn serve s promising replcement for mize when Physico-chemicl prmeters of culture wter were monitored within optimum rnge. It is evident from this finding tht exposure of test fish to pwpw (Cric ppy L.) replced for rice brn in fish feed does not led to cytogenetic dmge nd brings bout little brekge of nucleus to form micronucleus cells. Key words: Cytogenetic Hemtology Srotherodon melnotheron Cric ppy L Rice Brn INTRODUCTION consequently low yield in terms of qulity nd quntity, resulting in the scrcity of the commodity (fish) nd Feed is one of the mjor inputs in quculture eventully high cost of the few vilble ones to the production nd fish feed Technology is one of the lest disdvntge of the populce [2]. Fish feed ccount for t development sectors of quculture prticulrly in lest 60% of the totl cost of production [1]. Afric nd other developing countries of the World [1]. Fish is n importnt source of both food nd income High cost of fish feed ws observed s one of the to mny people in developing countries. problems hmpering quculture development [1]. Therefore, the need rises to serch for lterntive Expensive feeds will mrginlized or even nullify the feedstuffs tht re chep, vilble nd not in competitive profitbility of fish frming thereby incpcitting the demnd for humns, livestock nd industril uses. expnsion of frms to increse production nd In order to enhnce the commercil production of Corresponding Author: S.O. Ayool, Deprtment of Mrine Sciences, University of Lgos, Lgos Stte, Nigeri. 15

2 Am-Eurs. J. Toxicol. Sci., 6 (1): 15-24, 2014 fish, improve food security, reduce the level of poverty prticulrly fishes, hve not been exmined.c. ppy is in developing countries nd crete employment, rre exmple of species in which glucosinolte The feedstuff tht re chep, vilble nd not in (benzylglucosinolte) nd cynogenic glycoside competitive demnd for humns, livestock nd industril (prunsin) co-occur [12]; generlly plnts tht produce uses re referred to s un-conventionl ingredients [3]. glucosinoltes do not produce cynogenic glycoside Nonconventionl feed resources (NCFRs) re feeds tht nd vice vers. Excessive consumption of plnt prts tht re not usully common in the mrkets nd re not the contin cynogenic glycosides (leves nd roots, in the trditionl ingredients used for commercil fish feed cse of ppy) my induce dverse rections, due to the production [4,5]. Non-conventionl Feedstuffs re formtion of cynide in the digestive system of the ccredited for being non- competitive in terms of humn consumer, lthough none hve recorded for ppy. consumption, very chep to purchse, byproducts or Therefore, since there is pucity of informtion on wste products from griculture, frm mde feeds nd toxicity constituents of C. ppy L., this study is imed processing industries nd re ble to serve s form of t estimting the micronucleted cells of the Blck Jw wste mngement in enhncing good environmentl Tilpi exposed to inclusion of C. Ppy L in formulted impct. feed. The objectives of this study re to evlute the The qutic environment mkes up the mjor prt of potentil of pwpw s n lterntive feed stuff our environment nd resources; therefore, its sfety is in quculture nd s dietry protein source for directly relted to the sfety of our helth nd food S. melnotheron fingerling nd to exmine the security. Biomrkers nd bioindictors using fish micronucleus present in the blood smple of Blck jw micronucleus ssy in eco-genotoxicology offers severl Tilpi nd lso investigte the effect of different types of unique informtion not vilble from other concentrtion levels of Cric ppy lef-mel on the methods. These include: erly wrning on environmentl hemtology of S. melnotheron. dmge, the integrted effect of vriety of environmentl stresses on the helth of n orgnism nd the popultion, MATERIALS AND METHODS community nd ecosystem, reltionships between the individul responses of exposed orgnisms to pollution Experimentl Animl: Juvenile S. melnotheron used nd the effects t the popultion level, erly wrning of for the biossys (totl length 8.5 ± 0.72cm nd weight potentil hrm to humn helth bsed on the responses of.76 ± 0.85g) were purchsed from Deprtment of Mrine wildlife to popultion nd the effectiveness of remedition Sciences Aquculture unit, University of Lgos, Akok. efforts in decontminting wterwys [6]. The S. melnotheron ws kept in plstic tnk (34 27 Pwpw (Cric ppy) is common mn's fruits, 48.5 cm) which ws hlf filled with dechlorinted wter. Eno et l. nd Wilson et l. [7,8]. C. ppy is During cclimtiztion, the juvenile were fed with soft-wooded perennil plnt tht hs life spn of 5-10 commercil fish feed which contins 35% crude yers, lthough commercil plnttions re usully protein nd they were fed 5ml/L of their body weight. replnted [6]. It normlly grows s single-stemmed tree The Juvenile S. melnotheron were fed thrice dily with crown of lrge plmte leves emerging from the (morning, fternoon nd evening) nd the wter ws pex of the trunk, but plnt stnds my become chnged every dy to prevent the ccumultion of multi-stemmed when dmged []. The fleshy prt nd of wste metbolite nd food prticles. The Juvenile course the leves of Cric ppy re vilble S. melnotheron were mintined in the holding tnk for throughout the yer in the Tropics thus it is believed tht minimum of 14 dys to llow them cclimtize to the Cric ppy leves cn be possible substitute for lbortory conditions before commencement of the the expensive ingredient such s rice brn in tilpi fish experiment. rtion. Lohiy et l., [10] suggested tht ingestion of ppy Experiementl Feed: The feed fed to S. melnotheron seeds my dversely ffect the fertility of humn mles or in the course of the cclimtiztion ws commercil feed other mle mmmls. Eknem nd Okoronkwo [11] (Coppens) of 2mm. At the strt experiment, totl of reported high success in using pwpw seed powder in fifteen plstic tnks ech hving ten fishes were used for inducing sterility in mle tilpi when dministered the culturing of the fish. Ech of the vrying through feed. Despite their widespred use, their toxicity concentrtion of pwpw lef nd rice brn were nd effectiveness of C. ppy to qutic orgnisms, represented in triplicte with ten fishes in ech tnk. 16

3 Am-Eurs. J. Toxicol. Sci., 6 (1): 15-24, 2014 The fishes were constntly fed with the formulted feed of the experiment nd t the end (tht is, before chnge of (using person s squre method) with pwpw lef test medi). The prmeters mesured re dissolved used to replce rice brn t 0%, 25%, 50%, 75% nd 100%. oxygen, ph nd Temperture using pproprite digitl The control hd no inclusions of the pwpw lef instruments (Horiber U-10) nd were crried out t the (Cric ppy L.). Deprtment of Mrine Sciences lbortory, University of Lgos, Akok. Ingredients: The ingredient pwpw leves (Cric ppy) were dried nd grinded to powder form Determintion of Temperture ( C): The temperture of for the replcement with Rice brn. After cclimtiztion, the wter ws determined using mercury-in-glss 10 juvenile fishes were trnsferred to ech contined thermometer. The thermometer is clibrted in degree tretment of vrious percentges of replced feed. Celsius. It mesures the mximum temperture of the dy. Three replictes were followed for ech tretment. The feed ws pplied t the rte of 8% of body weight of Determintion of ph: The ph or Hydrogen ion the juvenile S. melnotheron throughout the experimentl concentrtions were determined using ph meter period of twelve weeks. (Model 405) with glss electrode. The electrodes were The diet ws fed twice dily (morning nd evening) stndrdized using buffer solution nd then wshed with in two equl portions. Dissolved oxygen nd ph of wter distilled wter. It ws therefter, wshed with the smple in ech contined tretment ws monitored by chnging wter to be tested, dipped into the smple wter nd the wter dily. The rnge of temperture remined C ph red on scle. during study period. The morphometric chrcteristics i.e. body weight nd totl lengths were recorded to observe Determintion of Dissolved Oxygen (DO) (Mg/l): their growth performnce. The ingredient used in The Dissolved oxygen (DO) in the wter throughout the formulting the fish feed were fish mel, soyben mel, experiment ws mesured using pproprite digitl groundnut cke, indomie, mize, minerl premix, vitmin instruments (Horiber U-10). The mesurement is crried premix, rice brn nd pwpw. The Person s squre by inserting the probe into the wter nd the redings method ws used in computing the feed with totl crude were tken. protein (C.P) of 35% (Tble 1). Cytogenetic Anlysis Physico-Chemicl Anlysis: Physico-chemicl Micronucleus Assy: Blood smples were collected from prmeters of the test medi were collected nd nlyzed ech tretment of the experiment. The peripherl blood during the 3months of exposure. Physicochemicl smers were obtined through the blood by mens of mesurements of test medi were mde t the beginning serum imprint following dissection s described by Tble 1: Percentge composition of feed ingredient used INGREDENTS T0 T1 T2 T3 T4 Fish mel CPLM Rice brn SBM GNC Tpioc Fish premix Tble Slt DCP TOTAL Key To: Tretment with 100% rice brn nd 0% pwpw lef T1: Tretment with 75% rice brn nd 25% pwpw lef T2: Tretment with 50% rice brn nd 50% pwpw lef T3: Tretment with 25% rice brn nd 75% pwpw lef T4: Tretment with 0% rice brn nd 100% pwpw lef 17

4 Am-Eurs. J. Toxicol. Sci., 6 (1): 15-24, 2014 Fgr et l. nd Plhres, Grisoli, [13, 14]. The slides were RESULTS then, ir-dried for 24h, fixed in methnol for 30min, followed by 10ml/L Giems (v/v) stining. Erythrocytes of Wter Qulity Prmeters: Wter qulity ech fish were exmined, from both peripherl blood. To prmeters in the bowls during the experimentl determine micronuclei in erythrocytes, the slides were period re presented in Tble 2. The vlues observed exmined using oil-immersion (x 1000). For the scoring of were within the tolernt rnge of S. melnotheron. micronuclei, the following criteri were dopted from [15]; The ph ws between , Dissolved oxygen the dimeter of the micronucleus (MN) should be less mg/l nd temperture C thn one- third of the min nucleus; MN should be (Tble 2). seprted from or mrginlly overlp with min nucleus s long s there is clers identifiction of the nucler Micronucleus Anlysis: The men frequencies of boundry; nd MN should hve similr stining s the micronucleus in Srotherodon melnotheron exposed min nucleus. to different tretments of Cric ppy L. replced with A drop of blood ws plced on ft-free slide. ricebrn in fish feed re presented in tble 3. The pltes A cover slip ws set on the slide t shrp ngle until it below show the photomicrogrph of micronucleus tking touched the blood drop. The blood spred long the side with the id of microscope (x 1000) showing the of the cover slip which ws then pulled rpidly nd presence of micronuclei in T 1, T 2, T 3 nd T4with none in evenly over the slide. Two slides per niml were the control (T 0)(Tbles 1-5). The bsence of micronuclei prepred. Micronuclei hve round or ovoid shpe nd in the control experiment ws due to the bsence of mximum dimeter of one third of tht of the cell pwpw lef in the diet. The lowest number of nucleus. These morphologicl chrcteristic ws used in micronucleus ws noticed in the blood smple of S. differentiting the megnucleus from the micronucleus melnotheron in tretment with 75% rice brn nd 25% present in the cytoplsm of the blood smple. pwpw lef t dy 30 while the highest number of micronucleus ws seen in the tretment with 100% Hemtologicl Exmintion of Fish: Some of the blood pwpw lef nd 0% rice brn t dy 0 of the collected ws used for hemtologicl nlysis. Blood experiment. smples were emptied into 5ml heprinized blood smpling The men frequencies of micronucleus in S. bottle treted with ethylene dimine tetr-cetic cid melnotheron exposed to different tretments of (EDTA) s n nticogulnt. Cric ppy L. replced with rice brn in fish feed showed tht there is significnt difference (p < 0.05) Sttisticl Anlysis: The sttisticl nlysis for the observed t Dy 0 nd Dy 60 while, Dy 30 showed no micronucleus test utilized nlysis of vrince (ANOVA) significnt difference (p > 0.05) in the micronuclei followed by Duncn s Multiple Rnge Test (DMRT). present (Figures 1, 2 &3). ANOVA (DMRT) showed tht Vlues were considered significnt when (p < 0.05). there ws significnt different (p <0.05) in the The dt from the blood were lso nlyzed using frequencies observed for micro nucleted cells in grphicl representtives. Anlysis ws performed using S. melnotheron exposed to ll tretments t dy 60 nd SPSS 18 for windows. dy 0. Tble 2: Wter qulity prmeters mesured during the experiment Prmeters Rnge Men ± S.E Temperture ( C) ± 0.8 Dissolved oxygen (mg/l) ± 0.64 ph ± 0.43 Tble 3: Men frequencies of micronucleus in blood of s. melnotheron exposed to different tretments of cric ppy l. replced with rice brn in fish feed Exposure period T0 T1 T2 T3 T4 DAY ± ± ± ± ±0.42 DAY 60 b 1.8±0.35 b 2.00±0.35 b 2.78±0.4 b 2.67± ±0.11 DAY ± ± ± ± ±

5 Am-Eurs. J. Toxicol. Sci., 6 (1): 15-24, 2014 Plte 1: Microgrph of Rice Brn 100% with 0% Pwpw Lef (Control T ) 0 Plte 3: Microgrph of Ricebrn 50% with Plte 2: Microgrph of Rice Brn 75% with 25% Pwpw Lef (T ) 1 Plte 4: Microgrph of Ricebrn 25%, Pwpw Lef 50% (T ) with Pwpw Lef 75% (T ) 2 3 Plte 5: Microgrph of Ricebrn 0% with Pwpw, Lef 100%.(T4) Fig 1: Micronucleus frequencies in the blood erythrocytes of Srotherodon melnotheron exposed to different tretments of Cric ppy L. replced with Rice brn in fish feed t DAY 30 [Mens ± SE brs with the sme lphbet in ech exposure period re not significntly different (p >0.05, DMRT)]. 1

6 Am-Eurs. J. Toxicol. Sci., 6 (1): 15-24, 2014 Fig 2: Micronucleus frequencies in the blood erythrocytes of Srotherodon melnotheron exposed to different tretments of Cric ppy L. replced with Rice brn in fish feed t DAY 60[Mens ± SE brs with the sme lphbet in ech exposure period re not significntly different (p >0.05, DMRT)]. Fig 3: Micronucleus frequencies in the blood erythrocytes of Srotherodon melnotheron exposed to different tretments of Cric ppy L. replced with Rice brn in fish feed t DAY 0 [Mens ± SE brs with the sme lphbet in ech exposure period re not significntly different (p >0.05, DMRT)]. Hemtologicl Prmeters: Results of Men Srotherodon melnotheron obtined from this study Hemtologicl prmeters of experiment fish smples were within cceptble rnge for different inclusion of were represented in Tble 4. The men hemoglobin Cric ppy lef mel t 50%, 75%, 100% dietry vlues of 5.±0.01 g/dl,6.4±0.06g/dl nd 8.33±0.01g/dl levels. The rnge of Men Corpusculr volume MCV for 50%, 75% nd 100% respectively were significntly (86.36±0.64, 86.25±0.36, 0.33± 0.67, 1.30±0.00 nd (p <0.05) higher thn the control (5.33±0.00) ±0.0 fl.) in (25%, 50%, 75% nd 100%) levels Result of Men red blood cell vlue slightly inclusions respectively were high. Result of the Men 12 incresed from 2.00±0.00 to 2.0±0.10(10 /l) in the Corpusculr Hemoglobin rnges (28.88± ±0.00 (50%, 75%, 100%) levels of inclusions. There ws pg) in this study. There ws no significnt difference significnt drop in the RBC count in the 25% level of in the men vlues of MCHC (33.33±0.00) between inclusion. Pcked cell volume vlues t 50%,75% nd inclusion levels. 100% were significntly higher thn the vlues in the Anlysis of Vrince ( ANOVA) showed sttisticl control nd 25% level of inclusions. significnt difference (p <0.05) in the men Hb (g/dl), The trends observed in the hemoglobin, red blood PCV (%), WBC (10 /L), MCH (pg), Neutrophils (%) nd cells nd pcked cell volumes' for the experimentl fish Lymphocytes (%) vlues t 0 %, 25%, 50%, 75% nd were indictions tht the dietry inclusion levels of 100% level of inclusions. No sttisticl significnt 50%,75% nd 100% fvoured n increse in difference(p >0.05) ws recorded between men MCHC hemtologicl prmeters. The white blood cell count (g/dl) vlues t 0%, 25%, 50%, 75% nd 100% level of in the 50% (4.18± /l) significntly higher thn inclusions. There ws sttisticl significnce (p <0.05) the control 25%(5.44± /l) nd 25% (1.60±0.10). 12 between men RBC (10 /L) counts between (0% nd The men vlues of pcked cell volume (PCV) in 25%), (0% nd 75% ) nd ( 0% nd 100%) levels of 20

7 Am-Eurs. J. Toxicol. Sci., 6 (1): 15-24, 2014 Tble 4: Men Hemtologicl Prmeters of Clris griepinus Fed with Different Levels of Cric ppy Leves Men±SD Hemtologicl Prmeters Control (0%) 25% 50% 75% 100% Hb (g/dl) 5.33± ± ± ± ±0.01 PCV (%) 16.33± ± ± ± ± RBC (10 / L) 2.00± ± ± ± ±0.10 WBC (10 /L) 5.44± ± ± ± ±0.02 MCV (fl) 80.36± ± ± ± ±0.0 MCH (pg) 26.66± ± ± ± ±0.05 MCHC (g/dl) 33.22± ± ± ± ±0.00 Neutrophils (%) 12.00± ± ± ± ±0.00 Lymphocytes(%) 87.67± ± ± ± ±0.00 Monocytes (%) Eiosinophils (%) Bsophils (%) Tble 5: Comprtive costs of different CPLM rtions INGREDENTS T0 T1 T2 T3 T4 Fish mel CPLM Rice brn SBM GNC Tpioc Fish premix Tble Slt DCP TOTAL COSTOF100Kg 17, , , , ,1.00 COST/TONNE 172, , , , ,10.00 inclusion. No significnt difference (p >0.05) between ech kg of experimentl rtion (0%, 25%, 50%, 75% nd (0% nd 50%) levels of inclusions. There ws significnt 100%) were (N17.3/kg, N16.2/kg, N16.1/kg, N15.6/kg, difference (p <0.05) in the men MCV (fl) vlues t 0%, N14./kg) respectively. 25%, 50%, 75% nd 100% levels of inclusions. There ws no difference (p >0.05) between the men vlue of MCV t DISCUSSION 25% nd 100% levels of inclusions. A significnt (p <0.05) positive correltion (r=0.527) occurred between men The cytogenetic effect of S. melnotheron exposed neurophils (%) nd MCV (pg). There ws significnt to different tretments of Pwpw (Cric ppy L.) (p <0.05) correltion between Neutrophils (%) nd MCH replced for rice brn in fish feed ws evluted for 0 (pg).a significnt negtive (p <0.05) correltion (r=-0.527) dys. Throughout the experiment vritions ws observed occurred between lymphocytes (%) nd MCV (fl). A in terms of growth rte of the fishes, micronucleus present significnt negtive (p <0.05) correltion (r=-0.537) in the smered blood smple nd the physico-chemicl occurred between lymphocytes nd MCH (pg). A positive prmeters of the wter. The presence of micronuclei in correltion between men vlues of neutrophils the blood smple of the fish ws due to the inclusion of (18.00±1.00%-35.00±0.00%) nd men MCV vlues pwpw lef in the diet which triggered the rpid cell (86.25±0.36%-1.30±0.00%) in juvenile fed with Cric division of the nucleus. These serves s biomrkers in ppy lef mel rtions. response to environmentl chemicls t the individul level or below demonstrting deprture from norml sttus Cost Benefit of Cric Ppy Lef Mel: Tble 5 [16]. The reltionship between the physico-chemicl showed the comprtive cost benefit of different inclusion levels of Cric ppy in prcticl diets of Srotherodon melnotheron. The cost of compounding prmeters lso ttribute to the growth of the fishes. The physico-chemicl prmeter for ph hs men vlue of 6.5 which flls within the FAO s limit rnge while 21

8 Am-Eurs. J. Toxicol. Sci., 6 (1): 15-24, 2014 the men vlue for wter temperture ws 26.5C which cell, white blood cell nd lymphocytes vlues with n lso flls below limit rnge. Dissolved oxygen hs men fterwrd progressive increse in men Hemoglobin, vlue of 7.80mg/L. pcked cell volume, Red blood cell, white blood cell nd The physico-chemicl prmeter showed significnt neurophils vlues. The men hemoglobin vlues of difference (p< 0.05) between Temperture, Dissolved 5.±0.01 g/dl,6.4±0.06g/dl nd 8.33±0.01g/dl for 50%, oxygen nd ph. Generlly, there ws strongly positive 75% nd 100% respectively were significntly (p <0.05) correltion mongst the prmeters (Temperture nd DO, higher thn the control (5.33±0.00).This is in consonnce r = 0.8*; Temperture nd ph, r = 0.*) which is n with the work of [23] for the Hemtologicl response of indiction tht there is n inter-reltionship tht the S. melnotheron fed dietry levels of Cric ppy lef physico-chemicl prmeter of the wter hs mjor mel. The red blood cell vlue slightly incresed from influence on the genetic mteril of the S. melnotheron ±0.00 to 2.0±0.10 (10 /l) in the (50%, 75%, 100%) According to Odeigh et l. [17], the impct of levels of inclusions. There ws significnt drop in the genotoxic wste wter on the environment nd the RBC count in the 25% level of inclusion. Pcked cell significnce to humn helth re difficult to predict, volume vlues t 50%, 75% nd 100% were significntly becuse wste wter re complex mixtures of chemicl higher thn the vlues in the control nd 25% level of substnces. Complete interprettion of their effect inclusions. The trends observed in the hemoglobin, red requires chemicl nlysis of the constituents tht my blood cells nd pcked cell volumes' for the experimentl indicte the components of the wste wter tht cn fish were indictions tht the dietry inclusion levels of persist nd ccumulte in exposed biot nd thus 50%,75% nd 100% fvoured n increse in potentilly pose hzrd to humn helth.fish re often hemtologicl prmeters. The white blood cell count in used s sentinel orgnism becuse they ply number of the 50% (4.18± /l) significntly higher thn the roles in the trophic web, ccumulte toxic substnces nd control.the vribility in WBC response of the fish might respond to low concentrtions of mutgens [18]. be due to the dietry inclusion levels of the lef mel in Therefore, the use of?sh biomrkers s indices of the the diets. The men vlues of pcked cell volume (PCV) in effects of pollution re of incresing importnce nd cn S. melnotheron obtined from this study were within permit erly detection of qutic environmentl problems cceptble rnge. It is higher thn vlues obtined by [1]. Gbriel et l. [1].This increse my be ssocited with the Fish serve s useful genetic models for the evlution inclusion of Cric ppy lef mel t 50%, 75%, 100% of pollution in qutic ecosystems [20]. The erythrocyte dietry levels. The rnge of Men Corpusculr micronucleus test hs been used with different fish hemoglobin MCV (86.36±0.64, 86.25±0.36, 0.33± 0.67, species to monitor qutic pollutnts displying 1.30±0.00 nd 86.21±0.0) in (25%, 50%, 75% nd 100%) mutgenic fetures [21]. Therefore, it is suggested tht levels inclusions respectively were higher but within the micronuclei tests in fish erythrocytes be crried out t rnge reported by Gbriel et l. [1]. The Men vrious times following tretments, thus mking it Corpusculr Hemoglobin rnges (28.88± ±0.00 possible to follow-up the chnging micronuclei pg) of this study were similr to findings of Nilz et l. frequencies. Studies of the micronuclei rtes of vrious [24].There were no significnt difference in the men fish species showed tht they generlly peked fter dy vlues of MCHC (33.33±0.00) between inclusion levels. forty of the tretment [22].The frequencies of This implies tht different levels of inclusions of Cric micronucleus in S. melnotheron exposed to different ppy hd no effect on this prmeter. A positive tretments of Pwpw (Cric ppy L.) replced with correltion between men vlues of neurophils Rice brn in fish feed showed tht there is significnt (18.00±1.00%-35.00±0.00%) nd men MCV vlues difference (p < 0.05) observed t Dy 60 nd 0 while Dy (86.25±0.36%-1.30±0.00%) is suggestive tht increse in 30 showed no significnt difference (p > 0.05). these prmeters ws due to feeding fish with Cric The hemtologicl response of the experimentl fish ppy lef mel rtions. A negtive correltion between fed vried dietry levels of Cric ppy lef mel s men MCH (pg) vlues (28.88±0.02 pg-30.43±0.00 pg) nd summrized on the tble showed significnt differences in lymphocytes vlues (82.00±0.00%-67.00±0.00) perhps the vrious hemtologicl indices for tretments (0%, could be n indiction tht the Cric ppy lef mel 25%, 50%, 75% nd 100%). However, it ws observed tht inclusion is immune-suppressive. Similr resons my be t 25% inclusion of Cric ppy there ws steep drop suggested for the reduction in men lymphocyte counts in the men Hemoglobin, pcked cell volume, red blood from 87.67±0.58 to 67.00±0.00%.A significnt increse 22

9 Am-Eurs. J. Toxicol. Sci., 6 (1): 15-24, 2014 in men weight of experimentl fish ws observed. 6. Villel, I.V., I.M. De Oliveir, J. D Silv nd There ws however progressive increse in men J.A. Henriques, DNA Dmge nd Repir weight of experimentl fish t 25%-75% inclusion from in Hemolymph Cells of Golden Mussel Exposed (10.13±7.3g-16.43±0.55).A drop in weight ws therefter to Environmentl Contminnts. Mutt. Res., observed t 100% level of inclusion. 605(1-2): Eno, A.E., O.I. Owo, E.H. Itm nd R.S. Kony, CONCLUSION Blood pressure depression by the fruit juice of Cric ppy (L.) in renl nd DOCA induced hypertension In conclusion, it is evident from this finding tht in the rt. Phytother. Res., 14: exposure of test fish to Pwpw (Cric ppy L.) 8. Wilson, R.K., T.K. Kwn, C.Y. Kwn nd G.J. Sorger, replced with Rice brn in fish feed does not led to Effects of ppy seed extrct nd benzyl cytogenetic dmge but brings bout little brekge of isothiocynte on vsculr contrction. Life Sci., nucleus to form micronucleus cells. The buildup of the 71: formtion regrding micronucleus indictes tht home. Villegs, V.N., 17. Ppy (Cric ppy). In: wste nd griculturl pollution cn increse the Verheji, E.W.M. nd R.E. Coronel (Eds.), Edible Fruits clstogenic effects on peripherl erythrocytes of qutic nd Nuts. Wgeningen University, Wgeningen, orgnisms nd could hve similr effects on humn Netherlnds, pp: popultion. The inclusion of Cric ppy in the feed of 10. Lohiy, N.K., B. Mnivnnn, P.K. Mishr, N. Pthk, the experimentl diets of (S. melnotheron) incresed S. Srirm, S.S. Bhnde nd S. Pnneerdoss, the growth nd hemtologicl prmeters of S. Chloroform extrct of Cric ppy seeds induces melnotheron t 50%, 75% nd 100% levels of inclusion long-term reversible zoospermi in lngur monkey. to replce rice brn in conventionl feed formultions. Asin J. Androl., 4: Eknem, S.B. nd T.E. Okoronkwo, Pwpw REFERENCES seed s fertility control gent on mle Nile tilpi. NAGA, World Fish Center Qurterly, 26(2): Gbriel, U.U., O.A. Akinrotimi, D.O. Bekibele, 12. Olfsdottir, E.S., L.B. Jorgensen nd D.N. Onunkwo nd P.E. Anynwu, Loclly J.W. Jroszewski, Cynogenesis in produced fish feed, potentils for cquculture glucosinolte-producing plnts: Cric ppy nd development in sub-shrn. Africn Journl of Cric quercifoli. Phytochemistry, 60: Agriculturl Reserch, 27: Fgr, A., A.M. El-shehwi nd M.A. Seehy, Adikwu, 12. Dietry crbonhydrte utiliztion in Micronucles Test in Fish Genome: A sensitive Tilpi (Oreochromis niloticus) J. Agric Sci Tech. Monitor for Aqutic Pollution. Africn Journl of 2(1): Biotechnology, 7(5): Ayool, S.O., Modern Fish Frming Techniques 14. Plhres, D. nd C.K. Grisoli, Comprison (Aquculture). Glmour books Ltd. Dugbe, Ibdn, between the Micronucleus Frequencies of Kidney Nigeri, pp: 180. nd Gill Erythrocytes in Tilpi fish, Following 4. Devendr, C., 185. Non-conventionl feed resources Mitomycin C Tretment. Genetic nd Moleculr in Asi nd the Pcific, 2nd edition FAO/APACA Biology, 25(3): Publiction No. 6 FAO Bngkok, pp: Fenech, M., W.P. Chng, M. Kirsch-Volders, 5. Mdu, C.T., O.A. Sogbesn nd L.M.O. Ibiyo, N. Hollnd, S. Bonssi nd E. Zeiger, Humn Some Non Conventionl Fish Feed Resources in Micronnucleus Project. "HUMN project: detiled Nigeri. In: Eyo, A.A., (Ed.), Proceeding of the Joint description of the scoring criteri for the cytokinesis- Fisheries Society of Nigeri/Ntionl Institute For block micronucleus ssy using isolted humn Freshwter Fisheries Reserch/FAO Ntionl Specil lymphocyte cultures." Mutt. Res., 534(1-2): Progrmme For Food Security Ntionl Workshop on 16. Wlker, C.H., S.P. Hopkin, R.M. Sibly nd Fish Feed Development nd Feeding Prctices in D.B. Pekll, Principles of Ecotoxicology, 2nd Aquculture Held t Ntionl Institute for Freshwter Edn. Tylor nd Frncis Group, Fetter Lne, London. Fisheries Reserch, 15th 1th Sept, New Buss, Principles of Ecotoxicology. CRC Press, 2006, pp: pp:

10 Am-Eurs. J. Toxicol. Sci., 6 (1): 15-24, Odeigh, P.G.C., O. Nurudeen nd O.O. Amund, 21. Sotome, K. nd M. Hyshi, Appliction of 17. Genotoxicity of oil field wstewter in Nigeri. se urchin micronucleus ssy to monitorin qutic Heredits, 126: polllution: influence of smple osmollity. 18. Cvs, T. nd S. Ergene-Gozukr, Genotoxicity Mutgenesis, 18(1): evlution of metronidzole using the piscine 22. Grisoli, C.K. nd C.M.T. Cordeiro, Vribility micronucleus test by cridine ornge fluorescent in micronucleus induction with different mutgens stining. Environ. Toxicol. Phr, 1: pplied to severl species of fish. Genet. Mol. Biol., 1. Vn Der Oost, R., J. Beyer nd N.P.E. Vermeulen, 23(1): Fish bioccumultion nd biomrkers in 23. Anynwu, D.C., A.B.I. Udedibie, D.I. Osuigwe nd environmentl risk ssessment: review. Environ. V.O. Ogwo, Hemtologicl responses of Toxicol. Phr, 13: hybrid of Heterobrnchus bidorslis nd Clris 20. Thir, M., hi-ud-din, C.S. Mll, Senthilkumr, griepinus fed dietry levels of Cric ppy lef A. Smeen nd N. Gnesh, Micronuclei s n mel. World Rurl Observtion, 3: 1. Evidence of DNA Dmge in Fresh Wter Ctfish 24. Nilz, L.R., M.M. Ligi, O.S. Dense, B.P. Rssi, Heteropneustes fossilis (Bloch) exposed to synthetic A.F. Celso nd P.F. Benedito, Hemthologicl sindoor. ARPN Journl of Agriculturl nd nd biochemicl vlues for Nile Tilpi cultured in Biologicl Science, 6(5): semi-intensive system. Act Scientirum Biologicl Sciences, Mring, 25(2):