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NH HNG CA NITRITE LN MT S CH TIU SINH L C TRA (Pangasianodon hypophthalmus) GING


Th Thanh Hng1, Mai Diu Quyn2, Sjannie Lefevre3, Tobias Wang3, Mark Bayley3

ABSTRACT
The effect of nitrite on the physiology and hematology of stripped catfish (Pangasianodon hypophthalmus) fingerlings (15-20 g/fish) was studied at the College of Aquaculture and Fisheries, Can Tho University. The experiment to determine the acute toxicity of nitrite was randomly designed with 8 treatments (including a control of non-nitrite) and 3 replicates for each in 100-liter composite tanks. The result shows the medium lethal concentration (96h LC50) of nitrite to the stripped catfish was 75,6 mg/L NO2-N. The effects of nitrite on physiological, hematological changes of stripped catfish were conducted by exposing fish to various concentrations of nitrite including 59,1 mg/L (LC1096-hr); 66,4 mg/L (LC25-96-hr); 75,6 mg/L (LC50-96-hr) and control (0 mg/L). Three replicates for each treatment. The results showed that the ecrythrocyte count and percentage of Hct, Hb, MCV, MCH in fish blood were reduced in higher concentrations of nitrite if compared to those of control treatment. In contrast, the lymphocyte count, metHb and MCHC in fish blood increased in all nitrite contaminated treatments in comparasion with control treatment. Thus, the high concentration nitrite in the water leading to the increase of metHb concentration in the blood causing a phenomenon blood brown of stripped catfish. Keywords: Hematology, tripped catfish, nitrite, brown disease. Tilte: Study on the physiological and hematological changes of stripped catfish (Pangasianodon hypophthalmus) fingerling exposed to different nitrite concentrations

TM TT
nh hng ca nitrite ln cc ch tiu sinh l trn c tra (Pangasianodon hypophthalmus) giai on ging (1520 g) c thc hin ti Khoa Thy sn, i hc Cn th. Th nghim xc nh gi tr LC50-96 gi c b tr hon ton ngu nhin vi 8 nghim thc t 50 n 140 mg NO2-N/L v mt i
1 2

Khoa Thy sn, i hc Cn Th S Nng nghip v Pht trin nng thn tnh Kin Giang 3 B mn Sinh hc, i hc Aarhus, an Mch

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chng. Kt qu cho thy gi tr LC50-96 gi ca NO2-N l 75,6 mg/L. Th nghim nh gi nh hng ca nitrite ln cc ch tiu sinh l, huyt hc trn c tra c thc hin cc nng gm 59,1 mg/L (LC10-96 gi); 66,4 mg/L (LC25-96 gi); 75,6 mg/L (LC50-96 gi) v i chng. Mu c c thu vo cc thi im 1, 6, 24, 48, 72 v 96 gi sau khi th nghim bt u. Cc ch tiu huyt hc nh s lng hng cu, t l Hct, Hb, MCV, MCH u gim khi tip xc vi nng nitrite cng cao. Ngc li, s lng bch cu, metHb v MCHC tng khi tip xc vi nitrite. Nh vy, nitrite trong mi trng nc cao lm cho hm lng metHb trong mu c tra tng ln gy ra hin tng mu c c mu nu. T kha: Huyt hc, c tra, nitrite, bnh mu nu. 1 GII THIU Trong ao nui c tra cc ch tiu mi trng nh TAN, NO2- v NO3- c xu hng tng cao v cui v. Theo De Silva et al., (2010) th khi nui mt tn c tra bng thc n vin cng nghip s thi ra mi trng 46,0 kg nit v 14,0 kg phospho. Mi trng ao nui c tra nhim l mt trong nhng nguyn nhn lm cho t l c cht v hao ht nhiu (Thich, 2008). Trong nui thy sn ni chung khi ammonia thi ra th vi khun Nitrosomonas s chuyn ha ammonia thnh nitrite (Huey v Beitinger, 1980). Nitrite c xem l mt trong nhng kh c tn ti trong mi trng ao lm nh hng n sc khe ca c khi hm lng tng cao. c th nitrite c tch tr trong huyt tng v l nguyn nhn gy ra qu trnh oxy ha Hb (Hemoglobin) thnh metHb (Methemoglobin). metHb khng vn chuyn c oxy nn khi nitrite c th tr thnh c cht v ngay c khi mc thp th cht ny cng gy c cho nhiu loi c (Huertas et al., 2002). Trong ao nui c tra do th mt cao v lng thc n s dng nhiu (Lam et al., 2009) nn cht thi t phn c v thc n d tha chnh l ngun hnh thnh mt lng ln kh ammonia trong ao nui v tr thnh yu t bt li cho c. V th, nghin cu v nh hng ca nitrite ln sc khe ca c tra thng qua cc ch tiu sinh l v sinh ha l rt cn thit bit c gi mc nh hng n sc khe c nui. 2 PHNG PHP NGHIN CU Nghin cu ny c thc hin ti b mn Dinh dng v Ch bin Thy sn, Khoa Thy sn, Trng i hc Cn Th. Nghin cu gm 3 th nghm l: 2.1 Xc nh gi tr LC50-96 gi ca nitrite ln c tra Th nghim c tin hnh theo phng php nc tnh v khng thay nc trong 96 gi vi 8 nng nitrite gm 50, 65, 80, 95, 110, 125, 140 mg NO2N/L v 1 i chng (0 mg/L). Th nghim c b tr hon ton ngu nhin vi ba ln lp li trong b composit c th tch 100 lt. Nng nitrite c pha
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bng phng php ha tan NaNO2 vo mi trng nc theo phng trnh phn ly (NaNO2 Na + NO2-). Mi nghim thc c lp li vi 10 c tra khe mnh c gi trong b t 15-20 ngy trc khi tin hnh th nghim. Trong sut thi gian th nghim c khng c cho n. Theo di cc hot ng ca c v ghi nhn s c cht ti cc thi im gm 3, 6, 9, 12, 24, 48, 72 v 96 gi sau khi b tr. Gi tr LC50-96 gi s c tnh bng phng php Probit (Finney, 1971) s dng phn mm SPSS 16.0. 2.2 nh hng ca nitrite cc nng khc nhau ln tiu hao oxy v ngng oxy ca c tra Th nghim c tin hnh vi 4 nng nitrite gm 59,1 mg/L (LC10-96 gi); 66,4 mg/L (LC25-96 gi); 75,6 mg/L (LC50-96 gi) v i chng (0 mg/L NO2N/L). Mi nng th nghim c b tr trong su ln lp li. C th nghim khng cho n 2 ngy v nc th nghim c sc kh mt ngy trc khi s dng cho th nghim. Cho c vo bnh kn 2 L (1 con i vi th nghim tiu hao oxy v 2 con i vi th nghim ngng oxy). Nc c cho chy tun hon gia cc bnh trong 30 pht c quen vi mi trng mi. Sau 30 pht th nitrite c cho vo b v tip tc cho chy tun hon 30 pht v tin hnh thu mu xc nh hm lng oxy u th nghim v yn 15 pht ri thu mu xc nh oxy cui tnh ton tiu hao oxy ca c. Th nghim ngng oxy cng tin hnh tng t nhng n khi c trong bnh cht 50% (cht 1 con) th thu mu nc c thu phn tch hm lng oxy ha tan trong nc. 2.3 Th nghim xc nh nh hng ca nitrite ln ch tiu huyt hc ca c tra C t ao nui c chuyn v phng th nghim v thun ha t 1520 ngy trc khi th nghim. C chn th nghim c kch c ng u, khng b d tt hay xy xt. Th nghim gm 4 nghim thc gm cc nng nh th nghim xc nh ngng v tiu hao oxy (th nghim th 2) v mi nghim thc c lp li 3 ln. Mi nghim thc bao gm 24 b (100 L/b) v mi b c 5 c. Thi gian thu mu mu sau 1 g, 6 g, 24 g, 48 g, 72 g v 96 g sau khi c tip xc vi nitrite. C c thu bng vt li, mi gi thu ht tt c 5 con trong b. C sau khi thu s c cn khi lng v o chiu di. Mu c c thu ng mch ui bng ng tim 1 mL phn tch cc ch tiu huyt hc (hng cu, bch cu, hemoglobin, methemoglobin, hematorit). Cc yu t mi trng c o vo thi im lc thu cc ch tiu huyt hc. Hng cu v bch cu c m bng bung m Neurbar theo phng php thng thng bng cch nhum mu trong dung dch Natt Herrick v Wrights & Giemsa. Phng php o Hemoglobin bng thuc th Drabkin ( Th Thanh Hng v Nguyn vn T, 2010). Met-hemoglobin c o theo phng php dng ng tim ly 0,5 mL mu c, mu c tr trong ng eppenndorf, sau ly 10 L mu cho vo ng eppenndorf khc c cha 1 mL
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phosphate buffer (pH=7,3) v ly tm 18.000 vng trong 5 pht. Ly 1 mL sau ly tm cho vo cuvette v o cc bc sng t 480700 nm. S dng phn mm Sigma plot 10.0 tnh ton kt qu. Dung dch phosphate buffer (pH=7,3) c chun b bng cch pha dung dch A (ha tan 1,38 g NaH2PO4 vi nc ct thnh 500 mL) v dung dch B (ha tan 1,78 g Na2HPO4 vi nc ct thnh 500 mL) theo t l 30 mL v 70 mL sau iu chnh pH t n 7,3. Cc yu t mi trng nh NO2-N v hm lng oxy trong nc c phn tch theo phng php thng thng. S liu c phn tch gi tr trung bnh, lch chun (std) bng phn mm Excel; so snh s khc bit gia cc nghim thc da vo phn tch ANOVA v DUNCAN bng phn mm SPSS 11.5 (mc ngha p <0,05). 3 KT QU V THO LUN 3.1 Gi tr LC50 ca nitrite vi c tra Kt qu th nghim cho thy, t l cht ca c gia tng theo nng nitrite sau 6 gi tip xc, n thi im 24 gi th t l cht gn 100% hai nng 125 v 140 mg/L NO2-N (Hnh 1). Gi tr thp nht khng gy nh hng n hot ng ca c (t l sng 100%) qua 96 gi tip xc vi nitrite l 50 mg/L NO2N.

Hnh 1: T l cht ca c tra theo nng nitrite th nghim vo thi im 96 gi

Quan st hot ng ca c khi tip xc vi nitrite nhn thy hu ht cc nghim thc c nng cao c gim hot ng trong 24 gi u khi tip xc. Tuy nhin, thi gian c gim hot ng cng ngn khi tip xc vi nng nitrite cng cao. cc nng 110; 125 v 140 mg/L NO2-N th hot ng ca c tng cc gi u sau gim nhanh v n 9 gi sau tip xc (c cht gn 90%), c c hin tng bi l trn mt nc sau chm dn xung y b
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v cht. Mang c c mu nu, ton b cc c quan ni tng v mu ca c u chuyn sang mu nu sm (Hnh 2).

Hnh 2: Cc c quan ca c chuyn sang nu khi tip xc vi nitrite

84 82
Gi tr LC-50 (mg/L)

80 78 76 74 72 24 gi 48 gi 72 gi 96 gi Gi tr LC-50
Hnh 3: Cc gi tr LC50 t 24 gi n 96 gi th nghim

Gi tr LC50-96 gi ca nitrite vi c tra th nghim l 75,6 mg/L. Theo kt qu nghin cu ca Das et al., (2004) trn c Cirrhinus mrigala (kch thc 11,41,5 g v 11,20,3 cm) th LC50-96 gi l 10,4 mg/L NO2-N. Yanbo et al., (2006) nghin cu trn c r phi (Oreochromis niloticus) (kch thc 1,80,2 g v 4,90,2 cm) th thy gi tr LC5096 gi l 28,2 mg/L NO2-N (trong mi trng c Cl- 35,0 mg/L) v 44,7 mg/L NO2-N (trong mi trng c Cl- l 70,0
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mg/L). LC50-96 gi ca c tra trong th nghim ny thp hn nhiu so vi kt qu nghin cu ca Odrigues et al., (2007) trn c gi (0,880,06 g) th LC50 96 gi l 210 mg/L NO2-N. So vi cc loi gip xc th LC50-96 gi ca nittrite vi c tra cao hn nhiu; u trng tm cng xanh (Macrobrachium rosenbergii) 1014 ngy tui v khi lng t 80140 g/u trng th LC5096 gi l 8,60 mg/L NO2-N (David et al., 1976) hay trn tm nc ngt Macrobrachium malcolmsonii th LC5096 gi l 3,14 mg/L NO2-N (Chand v Sahoo, 2006). Nh vy, c tra l loi c kh nng chu c cao so vi mt s loi thy sn khc v iu ny c th l gii v c tra l loi c c quan h hp ph nn c th chu c mi trng c hm lng nitrite cao. 3.2 nh hng ca nitrite ln mt s ch tiu sinh l c tra Tiu hao oxy ca c tra trong iu kin khng c nitrite (nghim thc i chng) trung bnh l 74 mgO2/kg/gi khc bit khng c ngha thng k (p>0,05) so vi nghim thc 59,1 mg/L NO2-N (67 mgO2/kg/gi) nhng khc bit c ngha thng k (p<0,05) i vi nghim thc 66,4 mg/L NO2-N (56 mgO2/kg/gi) v nghim thc 75,6 mg/L NO2-N (42 mgO2/kg/gi). Nh vy, cng h hp ca c gim tt c cc nghim thc c hm lng nitrite so vi i chng (Hnh 3). Nh vy, kh nng tiu th oxy ca c gim khi nng nitrite cng tng, c th do c gim h hp gim s hp thu cht c vo c th thng qua mang. Cc nghim thc th nghim c nng nitrite cng cao th c gim h hp nhiu hn gim bt lng nitrite xm nhp vo c th do vy cng h hp hay tiu hao oxy ca c cng gim. Tuy nhin, d gim h hp nhng lng nitrite vn xm nhp vo c th c qua mang (thay i mu sc ca mang) v kt qu ny ph hp vi nhn nh ca Lewis v Morris (1986) cho rng khi hm lng nitrite trong nc cao, c tng hot ng ca mang lng nc qua mang nhiu hn nhng lng oxy hp thu vo c th vn thp bi v hemoglobin b chuyn thnh methemoglobin khng gn kt c vi oxy mang oxy vo c th c. Theo nghin cu ca Tilak et al., (2007) trn c chp (Cyprinus carpio) th thy khi c tip xc vi nitrite c nng cng cao th t l tiu th oxy gim dn. So vi cc ha cht khc nh kt qu nghin cu ca Lng Dim Trang (2009) trn c tra th tiu hao oxy ca c cng gim khi cho tip xc vi nng malachite green cng cao. Kt qu ca nghin cu ny v cc nghin cu trn cho thy c t trong mi trng nc c th lm gim cng h hp ca c.

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100 90
Tiu hao oxy (mg/kg/gi)

80 70 60 50 40 30 20 10 0 i chng 59,1 66,4 75,6 Nng nitrite (mg/L)

Hnh 3: Tiu hao ca c tra trong mi trng nc c nng NO2-N khc nhau

3.5 3.0
Ngng oxy (mg/L)

2.5 2.0 1.5 1.0 0.5 0.0 i chng 59,1 66,4 75,6

Nng nitrite (mg/L)

Hnh 4: Ngng oxy ca c tra trong mi trng nc c nng NO2-N khc nhau

Ngng oxy ca c tra cc mc nng nitrite khc nhau trong th nghim u cao v khc bit c ngha thng k (p<0,05) so vi i chng (1,85 mg/L). Tuy nhin, ngng oxy ca c gia cc nghim thc c nng nitrite 59,1 mg/L NO2-N (2,49 mg/L), nghim thc 66,4 mg/L NO2-N (2,69 mg/L) v
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nghim thc 75,6 mg/L NO2-N (2,77 mg/L) ca c khc bit khng c ngha thng k (p>0,05) (Hnh 4). Khi c b ng c nitrite th tn s h hp ca c s tng ln, biu hin ny th hin rt r khi quan st c cc nghim thc c nng nitrite cng cao. Khi mi trng nc c nhiu nitrite th c tm cch ngoi ln mt nc thc hin h hp kh tri nhng c cng phi thc hin qu trnh h hp bng mang nhm cung cp lng oxy cho c th, c h hp lin tc thng qua qu trnh trao i gia mang v mi trng nc nn nitrite cng vo c th nhiu v sau c t t chm xung y bnh bt ng v cht. Theo Murty (1988) (trch dn bi Th Thanh Hng, 1998) th khi b nhim thuc c th c cn tng cng nng lng thch ng vi mi trng bt li. Nhm c nng lng th c phi tng cng trao i cht ng thi tng tn s h hp cung cp oxy cho c th. Chnh v vy, nghim thc cng cao c cng tng tn s h hp nn c b ng c cng nng, c cht nhanh hn v vy ngng oxy cao hn so vi i chng. 3.3 nh hng ca nitrite ln cc ch tiu huyt hc ca c tra Kt qu th nghim cho thy t l huyt cu (hematocrit) ca c cc nghim thc c nng nitrite cao u gim theo thi gian v sai khc c ngha thng k (p<0,05) so vi i chng sau khi tip xc vi nitrite (Hnh 5). Sau 72 gi tip xc cc nghim thc 66,4 mg/L NO2-N v 75,6 mg/L NO2-N th t l huyt cu gim v khc bit c ngha thng k so vi nghim thc 59,1 mg/L NO2-N. thi im 96 gi th t l huyt cu gim t 38,2% (nghim thc i chng) xung 21,9% (nghim thc 75,6 mg/L NO2-N).
50 45
T l huyt cu (%)

40 35 30 25 20 15 10 5 0 1 6 24 48 72 96 i chng 59,1 66,4 75,6

Thi gian (gi)

Hnh 5: T l huyt cu ca c cc nghim thc c nng nitrite khc nhau

S lng hng cu gim c ngha thng k (p<0,05) so vi i chng sau khi c tip xc vi nitrite 24 gi nghim thc 66,4 mg/L v nghim thc 75,6 mg/L. nghim thc 75,6 mg/L NO2-N th s lng hng cu gim ng k
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(2,29 triu t bo/mm3) so vi i chng (2,72 triu t bo/ mm3) thi im 1 gi sau khi tip xc vi nitrite.
Bng 1: S lng hng cu (triu tb/mm3) ca c tra trong th nghim

Thi gian thu mu 1 gi 6 gi 24 gi 48 gi 72 gi 96 gi

i chng 2,720,40aA 2,810,28aA 2,720,23aA 2,850,20aA 2,710,20aA 2,710,24aA

Nghim thc 59,07 mg/L 66,39 mg/L 2,500,55aA 2,350,47aA 2,770,58aA 2,360,69aA 2,850,20aA 2,340,24bA 2,300,59bA 2,330,58bA 2,540,28abA 2,330,52bA 2,370,49abA 2,210,33bA

75,58 mg/L 2,290,23aA 2,350,41aA 2,320,45bA 2,300,35bA 2,290,22bA 2,260,44bA

S liu trnh by l trung bnh lch chun. Cc gi tr cng hng mang cng ch ci (a, b) th khc bit khng c ngha thng k (p>0,05). Cc gi tr cng ct mang cng ch ci A th khc bit khng c ngha thng k (p>0,05).

3.4 nh hng ca nitrite ln nng methemoglobin Kt qu th nghim th hin Hnh 6 cho thy cc nghim thc c hm lng nitrite cao trong nc th phn trm Methemoglobin (metHb) trong mu c tng c ngha thng k (p<0,05) so vi i chng, chim t 5368% so vi tng hm lng Hb. Hm lng metHb trong mu c tng cng cao nu thi gian tip xc ko di. cc nghim thc c nitrite cao th hm lng metHb trong mu c tng cao v khc bit c ngha thng k sau 48 gi tip xc. Trong cng nghim thc, metHb tng theo thi gian tip xc, hu ht cc nghim thc c cha nitrite trong nc th phn trm metHb u tng v khc bit c ngha thng k (p<0,05) cc thi im thu mu. nghim thc 75,6 mg/L NO2-N, hm lng methemoglobin tng cao nht (89,3%) thi im 96 gi v thp nht (15,8%) thi im 1 gi sau khi tip xc. Trong thi gian tip xc th c hot ng nhiu hn, c ln mt nc th kh tri lin tc nhng sau mt thi gian ngn th c chm xung y b v cht. Kt qu ca nghin cu ny tng t nh cc kt qu nghin cu v nh hng ca nitrite ln mt s loi c khc. Nghin cu ca Costa et al., (2004) trn c Colossoma macropomum cho thy hm lng metHb cng tng cao sau khi c tip xc vi nng nitrite cng cao (chim 6676% trong tng s Hb). Mt nghin cu c mng (319,5 g) th hm lng metHb tng c ngha thng k nghim thc 0,88 mg/L NO2-N v tng nh nghim thc 0,44 mg/L NO2-N nhng sai khc c ngha thng k so vi i chng. nghim thc NO2-N cao nht (14 mg/L) th sau tip xc 12 gi phn trm metHb l 68,7% (Almendras, 1987). Kt qu nghin cu trn c chp (Cyprinus carpio) cng cho kt qu tng t, khi cho c tip xc vi nng nitrite cao th nng metHb trong mu chim 90,5% v khc bit c ngha thng k so vi cc
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nghim thc cng nng nitrite nhng cha nhiu Cl- v nghim thc i chng (Svobodova, 2005). Huertas et al., (2002) c kt lun l phn trm metHb tng t 5687% cc nghim thc c nitrite so vi i chng khi th nghim trn c Acipenser baeri. Yanbo et al., (2006) th nghim nh hng ca nitrite trn c r phi (Oreochromis niloticus) th thy nng metHb tng c ngha thng k (p<0,05) so vi i chng (0 mg/L NO2-N) v tng cao nng nitrite cng cao (15 v 20 NO2-N mg/L) t 2,1689,6% so vi tng Hb.
100 90 80
MetHemoglobin (%)

70 60 50 40 30 20 10 0 1 6 24 48 72 96 Thi gian (gi) i chng 59,1 66,4 75,6

Hnh 6: Hm lng Methemoglobin trong mu c tra trong mi trng nc c nng NO2-N khc nhau.

Nh vy, khi cho c tip xc vi nitrite c nng cng cao th hm lng Hb cng gim nhnh hm lng metHb cng tng. Hm lng Hb gim thp l do s chuyn ha thnh metHb (Tilak et al.,2007, Th Thanh Hng v Nguyn Vn T, 2010); metHb khng c kh nng gn kt oxy v vy gy hin tng thiu oxy cho c th c v s gy ra tnh trng thiu oxy tng cao khi thi gian tip xc vi nitrite cng di. Khi hm lng metHb trong mu tng cao s gy du hiu mu nu. 4 KT LUN 4.1 Kt lun Gi tr LC50-96 gi ca nitrite ln c tra 1520 g/con l 75,6 mg/L. Nitrite gy c cho c thng qua vic lm gim hm lng hemoglobin nhng hm lng methemoglobin gia tng cao (chim 89,3% khi c tip xc vi nitrite nng LC50-96 gi). Nng nitrite cng cao trong nc th ngng oxy ca c tng v tiu hao oxy gim.
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4.2 xut Tip tc nghin cu nh hng ca nitrite ln cc c quan khc ca c nh c, gan, mang, no xc nh r thm mc nh hng ca nitrite. Th nghim nui tng trng trong thi gian di nhm xc nh tng trng ca c khi b nhim nitrite.

TI LIU THAM KHO


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