30 4 Vol. 30,No. 4 2 0 0 6 7 ACTA HYDROBIOLOGICA SINICA July, 2 0 0 6 (, 524025) :, ;, : 31,24hLC 50 48hLC 50 72hLC 50 96hLC 50 31419mg/ L 17513mg/ L 10012mg/ L 8910mg/ L ; 17, 13213mg/ L 6516mg/ L 5113mg/ L 3915mg/ L 31 ( P < 0105) 17 410mg/ L 810mg/ L, (SOD) (PO) (U a ) (U L ) ( P < 0105) ; ( P < 0105),, : ; ; ; :S966112 :A :100023207 (2006) 0420466206,, [1 ] ; [2 ] ;, [3 ] [4 ] ( Litopenaeus vannamei) [5,6 ],, 1 111 L. vannamei ( 112, 113 31 17, [7 ] 5, 3312 5910 10510 18617 33210mg/ L 2 011m 3, 10cm 20, 24h,100 %,,,24h 48h 27h 96h 29 1,pH 719 114 (NO 2 2N) 24h 48h 72h 96h (LC 50 ) [7 ], [8 Cs :Cs = 011 96hLC ] 50 115 015m 3, ),,1 :2005204206 ;:2006203227 :, (:C20823) : (1962 ),,,;Tel :075922362166,E2mail :ybcl @tom. com
4 : 467 2,2,, 1 410mg/ L, 2 810mg/ L, 100 %,,2,2,1 3d 1, 2 10cm 20 31,pH 719, 29 1, 615mg/ L, 3 18,, - [12 ] (PO) Ashida [13 ] L2dopa,2 48h,,;011mol/ L (ph = 614) A 570nm = 013,,;, (U L ) = (A 0 - A) / A 0 [14 ] [14], 011mol/ L (ph = 614) A 570nm = 013,, (U a ) = [ (A 0 - A) / A 0 ] 1/ 2 2 [9 ] 211 116 [ 10 ],,,7,,, 10 % ( = 1 1), 5910mg/ L, Eppendorf, 31, 24h 48h ; 117 17, 24h 48h,Eppendorf, 4, 2510 % 4510 %, 31, - 20 [10 ] 24h 48h 72h 96h Comassic [11 ], 17,, F 45100, (mg/ ml) = ( OD/ 20111 34195 30101, P < 0105,, OD) (SOD) ( 1,2) 1 Tab11 The result of the acute toxicity experiment of NO 22N on Litopenaeus. vannamei at different salinity NO 2 2N Mortality( %) 24h 48h 72h 96h 31 17 31 17 31 17 31 17 3312 0 2010 0 3010 715 3510 1215 4010 5910 0 2510 0 4510 715 5510 1215 8010 10510 715 3510 20 8010 5510 8510 7010 8510 18617 1215 8010 5510 9510 6010 9510 10010 9510 33210 5510 10010 6010 10010 10010 10010 10010 10010 2 Tab12 The median lethal and safe concentration of L. vannamei at different salinity Salinity 24hLC 50 48hLC 50 72hLC 50 96hLC 50 Cs 31 31419 17513 10012 8910 819 17 13213 6516 5113 3915 410 212 21211 24h, 24h 24h,,,, 1 410mg/ L
468 30 3 24h Tab13 Variation of NO 22N concentration during 24 hours under water2renew experimental condition NO 2 2N Test time (d) Control group 1 Experiment group 1 2 Experiment group 2 8 :00 16 :00 18 :00 8 :00 16 :00 18 :00 8 :00 16 :00 18 :00 5 01174 01015 01000 41228 31697 41000 71948 71577 81000 10 01024 01031 01000 41020 31982 41000 71110 81022 81000 15 01096 01098 01000 41666 31697 41000 71494 71556 81000, 2 810mg/ L, 011mg/ L, 2 1 1 ( 3) 21212,,, 1 3d 1, 2 ( 4), 2d, F 13146, P > 0105, ; 6 10 13 16 18d, F 61186 30116 55198 33110 4 Tab14 Variation of NO 22N concentration during the experiment under Group the experiment condition without renewing water NO 2 2N 2d 6d 10d 13d 16d 18d 1 01081 01117 01355 11506 31145 41707 2 01157 01779 11345 31470 61716 81374 29157, P < 0105, 213, 1 2 6613 % 6813 %, 615 % 1116 %, 4819 % 5619 %,2418 % 5916 %,2119 % 4319 %, 1819 % 2319 %( 5),, F 11107 10116 10163 12148 23195 20123, P < 0105,, 2 1 6212 %,1117 %, 4819 %,6211 %, 1213 %, 1519 % ( 6),, F 21103 25171 27192 25160 19153 28165, P < 0105,, 5 Tab15 Analyzing results on factors relating to anti2disease ability of L. vannamei under water2renew experimental condition Group SOD activity PO activity U a U L Blood serum protein (mg/ ml) Hemocyte number ( 10 6 cell/ ml) 501504 11708 01302 11353 151500 21740 1 401986 11333 01227 01692 141500 01923 2 381434 01958 01122 01583 131700 01869
4 : 469 6 Tab16 Analyzing results on factors relating to anti2disease ability of L. vannamei under the experiment condition without renewing water Group SOD activity PO activity U a U L Blood serum protein (mg/ ml) Hemocyte number ( 10 6 cell/ ml) 1 561143 41071 01972 11353 41339 21150 2 471244 31571 01368 01692 31831 01813 3 311,,,, 17 410mg/ L, 31 819mg/ L, 2,,, ( 1,2), 312,,,,,, [15 ], ( Pe2 naeus chinesis ) PO SOD [14 ] ;( Penaeus japoni2 cus) ( Macrobrachium rosenbergii) [16 19 ],,, 4819 % 5619 %,2418 % 5916 %,2119 % 4319 %, 1819 % 2319 % ; 6145 % 1116 % ; 6613 % 6813 % ( 3,4) 313,,, [1 ], Lactococcus garvieae, [19 ],( Penaeus monodom) MBV [20 ] [21 ],,,,,, 314,,,,,,,, ph,,,,, ph, : [ 1 ] Ding ML, Lin L, Li G Y, et al. Effects of organic pollution on Pe2 naeus chinesis bodyπs intraenvironment and externalenvironment [J ]. Oceanol et Limnol Sin, 1997,28 (1) :7 12[,.
470 30,..,1997,28 (1) :7 12 ] [ 2 ] Sun J J, Ding ML. Effect of ammonia2n on anti2disease ability of Pe2 naeus chinesis[j ]. Oceanol et Limnol Sin, 1999,30 (3) :267 272[,.., 1999,30 (3) :267 272] [ 3 ] Wu W N, Guo L J, Lin H S, et al. The analysis of shrimp maricultural water by factor analysis method2[j ]. Environ Monitoring in China, 1998,14 (2) :43 44[,,,..,1998,14 (2) :43 44] [ 4 ] Wu Z H, Liu C B, Liu C R, et al. Histopathological research on chronic poisoning of Penaeus chinensis by nitrite and ammonia[j ]. J Central China Normal University ( Nat. Sci), 1999,33 (1) :119 121[,,,..,1999,33 (1) : 119 121 ] [ 5 ] Huang X H, Li C L, Liu C W, et al. Study on Oocysyis borgei and Nannochloris oculata improving environment of shrimp pond and strengthening anti2disease ability of Penaeus vannamei[j ]. Acta Hy2 drobio Sin, 2002,26 (4) : 342 347 [,,,..,2002,26 (4) :342 347 ] [ 6 ] Zhou Q C, Ding Y, Zheng S X, et al. Effect of dietary vitamin C supplementation on growth and anti2disease ability of shrimp, Penaeus vannamei[j ]. Acta Hydrobio Sin, 2004,28 (6) :529 598 [,,,. C.,2004,28 (6) :529 598 ] [ 7 ] Zhou Y X, Zhang Z S. Methods of toxicity test to aquatic organisms [M]. Beijing : Agriculture Press, 1989,11 27[,.. :,1989,11 27 ] [ 8 ] Zang W L. Toxic effects of Cn 2 + Cu 2 + Cd 2 + and NH 3 on Chinese prawn[j ]. Chin. J. Oceanol. Limol, 1993,11 (3) :95 98 [ 9 ] Chen J R. Guide book of hydrochemistry[m]. Beijing : China Agri2 culture Science and Technology Press, 2000,5[.. :,2000,5 ] [ 10 ] Guan S H. Immunity, cell culture and disease researches on seawater aquiculture animals [ M]. Jinan : Shandong Science and Technology Press. 1999,1 31[.. :. 1999,1 31 ] [ 11 ] Chen Q. The experimental method of anti2ageing study[m]. Beijing : The Scientific and Technological Publishing Company of China Medicine, 1996,606 607[.. :,1996. 606 607 ] [12 ] Deng B Y, Yuan Q S, Li W J. The improved pyorgallol autoxidation methods on determining Superoxide Dismutase Activity [J ]. Prog. Biochem. BioPhys, 1991,18 (2) :163[,,..,1991,18 (2) :163 ] [ 13 ] Ashida M. Purification and characterization of pro2phenoloxidase from hemolymph of the silkworm Bombyx morl [J ]. Arch. phy., 1971,144 :749 762 Biochem Bio2 [14 ] Wang L, Li G Y,Mao Y X. Studies on the activities and characteris2 tics of the antibacteria, bacteriolysis and phenoloxidase in the haemolymph of Penaeus chinesis [J ]. Oceanol et Limnol Sin, 1995, 26 (2) :179 185[,,..,1995,26 (2) :179 185 ] [15 ] Cheng S Y, Chen J C. Haemocyanin oxygen affinity and the fraction2 ation of oxyhemocyanin and deoxyhemocyanin for Penaeus monodom exposed to elevated nitrite[j ]. Aquat Toxicol, 1999,45 :35 46 [16 ] Chen J C, Lee Y. Effects of nitrite exposure on acid2base balance, respiratory protein, and ion concentrations of giant freshwater prawn Macrobrachium rosenbergii at low ph[j ]. Arch Environ Contam Toxi2 col, 1997,33 :290 297 [17 ] Cheng S Y, Chen J C. The time2course changes of nitrogenous excre2 tion in the kuruma shrimp Penaeus japonicus following nitrite exposure [J ]. Aquat Toxicol,2001,51 :443 454 [18 ] Cheng S Y, Chen J C. Effects of nitrite exposure on the haemolymph electrolyte respiratory protein and free amino acid levels and water content of Penaeus japonicus[j ]. Aquat Toxicol,1998,44 :129 139 [ 19 ] Cheng W, Liu C H, Chen J C. Effects of nitrite on interaction beteen the giant freshwater prawn Macrobrachium rosenbergii and its pathogen Lactococcus[J ]. Dis Aquat Org, 2002,50 :189 197 [20 ] Li G S, He J G, Li G F, et al. Relationships Between Penaeus monodon Baculovirus Infection and Physical and Chemical Elements of Prawn2Farming Pond Water [J ]. Supplement to the Journal of Sun Yatsen University (Suppl),1996,26 30[,,,.. (),1996,26 30 ] [21 ] Chen P, Huang H, Chi X C, et al. The composition and ultrastruc2 ture of haemocytes in four species of penaeid shrimps[j ]. Acta Hy2 drobio Sin, 1998,22 (2) :158 163[,,,..,1998,22 (2) : 158 163 ]
4 : 471 THE TOXICITY OF NO 2 2N ON LITOPENAEUS VANNAMEI AND EFFECTS OF NO 2 2N ON FACTORS RELATING TO THE ANTI2DISEASE ABILITY HUANG Xiang2Hu, LI Chang2Ling, ZHENGLian, LIU Chu2Wu and ZHOU Jie ( Fisheries College, Guangdong Ocean University, Zhanjiang 524025) Abstract : NO 2 2N is one of the main contaminations and a great stress factor in the intensive prawn culture system. Therefore it is very important to study the toxicity of NO 2 2N on Litopenaeus vannamei and effects of NO 2 2N on factors relating to the anti2dis2 ease ability for the health culture of L. vannamei. Acute toxicity test of NO 2 2N to L. vannamei was done by the usual method of biological toxicity test at different salinity. According to the result of acute toxicity test, NO 2 2N was introduced into the culture system of L. vannamei at tow levels(4mg/ L,8mg/ L). Those without introducing NO 2 2N were used as controls. The water in the culture system was changed with proportion of 100 % every day. N a NO 2 was introduced into the same system with the same con2 centration of NO 2 2N. Without renewing water, two experiment group were set. The filth of group 1 was cleared once every 4 days, while group 2 was not. The blood serum of prawn was drawn out on day 18 and the changes of the hemocyte number, the content of blood serum protein and the activity of phenoloxidase, superoxide dismutase, antibacterial enzyme, lysozyme of L. vannamei were tested. The result shows that the salinity had remarkable effect on the toxicity of NO 2 2N. When the salinity was 31 in the culture system, the 24hLC 50,48hLC 50,72hLC 50,96hLC 50 was 31419mg/ L,17513mg/ L,10012mg/ L and 8910mg/ L, re2 spectively ; when the salinity was 17, the corresponding concentration was 13213mg/ L, 6516mg/ L,5113mg/ L and 3915mg/ L, respectively. All LC 50 of the experiment group with salinity of 31 was significantly higher( P < 0105) than that of the experiment group with 17. The toxicity of NO 2 2N was greater under the condition of lower salinity. NO 2 2N showed obvious effect on the anti2 disease ability of L. vannamei. Under water2renew condition, and the NO 2 2N was 4mg/ L and 8mg/ L in the culture system, the hemocyte number, the content of blood serum protein and the activity of phenoloxidase, superoxide dismutase, antibacterial en2 zyme, lysozyme of L. vannamei were declined 6613 %, 615 %, 2119 %, 1819 %, 2418 %, 4819 % and 6813 %, 1116 %, 4319 %, 2319 %,5916 %,5619 %, respectively in contrast to the control group. The activity of factors relating to the anti2disease ability was significantly lower( P < 0105) than that of the control group. Under the experiment condition without renewing water, the activity of factors that relate to the anti2disease ability in group 2 was significantly lower( P < 0105) than that in group 1. The result applies that the anti2disease ability of L. vannamei declines in the range of lower concentration of NO 2 2N and the higher NO 2 2N concentration is, the weaker anti2disease ability will become. Key words : Nitrite nitrogen, Litopenaeus vannamei, Anti2disease ability, Acute toxicity test