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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

ABSTRACTThe 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 TTnh 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 i1 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 pha167

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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 mL168

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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) so vi nghim thc 59,1 mg/L NO2-N (67 mgO2/kg/gi) nhng khc bit c ngha thng k (p