Effect of Different Concentrations of Waterborne Sodium on the Hatching Rate and Ions Content of Rainbow Trout (Oncorhynchus mykiss) Eggs

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1 Turkish Journl of Fisheries nd Aqutic Sciences 14: (2014) ISSN DOI: / v14_1_08 Effect of Different Concentrtions of Wterorne Sodium on the Htching Rte nd Ions Content of Rinow Trout (Oncorhynchus mykiss) Eggs Mehrdd Srkheil 1,*, Gholmrez Rfiee 1, Bgher Mojzi Amiri 1, Mehrdd Frhngi 1 1 University of Tehrn, Fculty of Nturl Resources, Deprtment of Fisheries nd Environmentl Sciences, P.O. Box: , Krj, Irn. * Corresponding Author: Tel.: ; Fx: ; E-mil: mehrddsrkheil@gmil.com Received 25 Septemer 2012 Accepted 24 Novemer 2013 Astrct With the im of finding the optimum concentrtion of wterorne sodium for use during egg incution period nd higher production of the rinow trout lrve in wter recircultion system, completely rndomize experiment ws conducted. The effect of different levels of wterorne sodium (2, 14, 50 nd 100 mg L -1 ) on eye pigmenttion, htching rtes, ions (N, K, P, Cu, nd Mn) nd wter content of eggs in rinow trout fish, Oncorhynchus mykiss, were determined. The newly fertilized eggs were introduced in wter recircultion incutors s experimentl units. The wter temperture ws etween C during the incution period. In tretments with N + concentrtion of 2 nd 14 mg L -1, htching rtes were significntly (P<0.05) higher thn other tretments. Increse of wterorne N + up to 100 mg L -1 significntly (P<0.05) decresed eye pigmenttion. In ll the tretments, wter content (%) of egg, compred to the newly fertilized egg significntly (P<0.05) decresed t the htching time. Egg ions content showed tht the elevtion of N + level to 14 mg L -1 motivte sodium uptke y eggs from wter nd highest sodium uptke occurred in tretment with [N + ] 100 mg L -1. In ll the tretments, potssium uptke occurred y eggs from environmentl wter. Egg potssium content in [N + ] 100 mg L -1 ws significntly (P<0.05) higher thn other tretments. In tretments with [N + ] 50 nd 100 mg L -1, prevention of Cu uptke y egg from mient wter occurred. Mn uptke y egg from wter ws only recorded in tretment with [N + ] 2 mg L -1. An increse in phosphorus content of the eggs ws lso recorded in tretments with [N + ] 50 nd 100 mg L -1. It ws concluded tht eye pigmenttion, htching rtes nd ions content of rinow trout eggs depend on the rte of wterorne sodium nd concentrtion of 14 mg L -1 cn e introduced s stndrd level, for htching the rinow trout eggs in wter recircultion system Keywords: Rinow trout, egg, htching rte, wterorne sodium, ion content. Introduction The emryogenic development is the min stge of the rering cycle of fish, especilly in cptivity (Depeche nd Billrd, 1994). Ionic composition of mient wter is known s the key fctor for the development of fertilized eggs (Vndervelden et l., 1991). Minerl requirements of fish emryo fulfill either from the existing storge during vitellogenesis or from the mient wter during incution periods (Lee nd Hu, 1983). Clcium nd sodium re the min undnt ctions in mient wter (Mekreth et l., 1978) nd cn e uptke from wter y the emryos of rinow trout (Brrett et l., 2001). However, Peterson et l., (1982) showed tht ions were not uptke y rinow trout eggs prior to htching. It is known tht, mitochondrion-rich cells (MRCs) re present in yolk sck memrne nd other ody surfces re the min ion-regultory sites in emryonic stge (Kneko et l., 2002). A numerous numer of MRCs in the yolk sc memrne of emryos nd lrve of Mozmique tilpi (Oreochromiss mossmicus) were identified (Kneko et l., 2002). It is known tht n increse in the N + content of emryonic rinow trout in the few dys prior to htching occurred, resulting in the presence of mitochondri rich cells in epitheli of oth yolk sck nd the developing gills (Shen nd Letherlnd, 1977). HA electriclly linking with the epithelil N chnnel (ENC), nd the electroneutrl NHE s two models were proposed for picl N + uptke/cid secretion functions in FW fish gills (Hwng, 2011). A specific NHE inhiitor, EIPA, ws found to decrese the N + uptke ctivity (y SIET) in medk (Oryzis ltipes) emryonic skin ionocytes (Wu et l., 2010). Horng nd collegues (2007) identified the vcuolr H + -ATPse (V-ATPse, H + pump) in the skin of zerfish (Dnio rerio) emryo Pulished y Centrl Fisheries Reserch Institute (CFRI) Trzon, Turkey in coopertion with Jpn Interntionl Coopertion Agency (JICA), Jpn

2 60 M. Srkheil et l. / Turk. J. Fish. Aqut. Sci. 14: (2014) tht distriuted minly in the picl memrne of H + - pump-rich cells nd knockdown of this pump reveled normlities, including suppression of cid-secretion from skin, growth retrdtion, trunk deformtion, nd loss of internl C 2+ nd N +. The physiologicl roles of clcium; influence on permeility of iologicl memrnes, uptke of certin metls y competitive inhiition which modultes their toxicity to fish (Mtsuo et l., 2004) nd preventing ion losses from the tissues to wter (Bijvelds et l., 1998) re well known. Ketol et l (1988) reported tht survivl of rinow trout eggs incuted in wterorne clcium concentrtion of mg L -1 increses nd higher C 2+ concentrtion reduces post-htch survivl of this species. Sodium is known s the key fctor regulting osmotic nd ionic grdient of egg plsm memrne nd lso sorption of some ions y egg is depended on sodium concentrtion in emryo tissues nd mient wter (Alderdic, 1988). Negtive correltion of copper ccumultion in rinow trout (Slomn et l., 2003) nd mngnese uptke y rook chrr (Slvelinus fontinlis) (Gonzlez et l., 1990) regrded with sodium uptke hs een reported. There is cler need to find whether ions ccumultion in emryonic tissues correltes with externl sodium concentrtion or not. Therefore, this reserch ws conducted to exmine whether or not externl N + concentrtion ffects the rinow trout eggs htching efficiency nd uptke of other ions (N, K, P, Cu nd Mn) y egg during the incution period. Mterils nd Methods Experimentl Tretments A completely rndomized experimentl design ws crried out t the Fculty of Nturl Resources, University of Tehrn, Krj, Irn. Fertilized rinow trout eggs were otined from Slmonids Reserch Institute, Kelrdsht, Irn. The 270 newly fertilized eggs were introduced in ech experimentl unit in which wterorne recircultion through incutors. The experimentl tretments were four wterorne sodium medi (2, 14, 50 nd 100 mg L -1 [N]) in triplictes. The incution medi (tretment) were prepred y dding nlyticl slt (Merck) [NCl, Tle 1. Ions concentrtion in different wterorne sodium tretments CCl 2, KCl, (MgCO 3 ) 4.Mg (OH) 2. 5H 2 O, FeCl 3.6H 2 O, ZnCl 2, MnCl 2.4H 2 O, CuSO 4.5H 2 O, N 2 HPO 4.12H 2 O] in to doule-deionized distilled wter. Wterorne ions concentrtion ws determined y ICP (GBC Integr XL) (Tle 1). Dissolved oxygen, temperture nd ph of wter were mesured using PC 300 instrument, totl mmoni of wter were mesured using photometer instrument (Plintest 8000 model). The ph, temperture, dissolved oxygen nd non ionized mmoni vried etween , 7.5-9ºC, (mg L -1 ) nd (mg L -1 ) respectively, mong tretments during incution period. Experimentl Procedure The newly fertilized eggs were introduced into the tretments, incution trys in recircultion wterorne sodium medi. Ech experimentl unit ws supplied with 45 L erted wter medi nd sterilized y UV lmp (246 nm, 20 w). Eye pigmenttion nd htching time of the eggs in ech tretment ws mesured during incution period. The htching rte (H) ws clculted y the method of Geertz Hnsennd nd Rmssen (1994): H = (numer of incuted eggs numer of ded eggs)/ (numer of incuted eggs) 100 Smpling Protocol Prior to llocting the fertilized eggs to trys nd over the time of htching, 100 eggs were smpled from the eggs tch nd their wet mss, wter nd ions content were mesured. For mesurement of the N, K, P, Cu nd Mn concentrtion of the wterorne, one liter of (1 L) wterorne ws lso smpled. Mesurement of Whole Egg Wter nd Ion Content Wet mss (W w, mg) of eggs ws determined fter lotting dry y sorent pper to remove the surfce moisture. Dry mss (W D, mg) ws determined fter plcing the eggs in 60ºC oven for 48 h,then cooling them in desicctor. Dried eggs were weighed nd whole ody wter content (%) ws clculted y the method of Brrett et l. (2001): Tretment s (mg L -1 ) (Men ± SD) Wterorne ions concentrtion (mg L -1 ) N C Mg K P Fe Cu Zn Mn 2.03± 54.55± 9.11± 1.94± 1.44± 0.055± 0.034± 0.026± 0.014± ± ± 9.12± 1.91± 1.45± 0.054± 0.033± 0.026± 0.015± ± ± 9.19± 1.92± 1.46± 0.053± 0.034± 0.024± 0.015± ± ± 9.23± 1.93± 1.45± 0.054± 0.032± 0.025± 0.014±

3 M. Srkheil et l. / Turk. J. Fish. Aqut. Sci. 14: (2014) 61 wter content = 100 (W w - W D ) W -1 w. The eggs were dried in 105ºC oven for 48 h nd 1g egg digested in 6 ml 1 N HNO 3 (Moopn, 1983), then concentrtions of N, K, P, Cu nd Mn were mesured y ICP (GBC Integr XL). Sttisticl Anlysis Dt were presented s Mens±SD. Percent vlues were trnsformed y rcsine method to e normlized, nd then nlyzed sttisticlly. Differences in wterorne N, K, P, Cu nd Mn concentrtions t the strt nd end of the experiment were recorded, egg mortlity rte prior nd fter eye pigmenttion were tested s well, using pired-smple T test. Differences etween the mens were nlyzed using one wy nlysis of vrince (ANOVA). Duncn s new multiple rnge test ws conducted to determine the significnt differences etween the mens using SPSS softwre (version 12.0). Sttisticl significnce ws ccepted t the level of P<0.05. Result Eye Pigmenttion nd Htching Rtes Eye pigmenttion ws pprent dys post fertiliztion nd htching occurred dys post fertiliztion. Eye pigmenttion rte of eggs which were exposed to the highest N + concentrtion (100 mg L -1 ) ws significntly (P<0.05) lower thn eggs exposed to other N + concentrtions (Tle 2). The htching rte of eggs ws significntly (P<0.05) different mong wterorne sodium tretments. The highest htching rte ws oserved in the [N + ] 2 mg L -1 tretment. The htching rtes of eggs were not significntly (P>0.05) different etween [N + ] 2 nd 14 mg L -1 tretments (Tle 2). The vrition of eggs mortlity rte prior nd fter eye pigmenttion were shown in the present study (Figure 1). In ll tretments, egg mortlity percent fter eye pigmenttion significntly incresed (P<0.05) compred to the prior of eye pigmenttion. Wet Mss nd Wter Content of Egg Wet mss of eggs which were incuted in different wterorne sodium concentrtions did not show ny significnt (P>0.05) chnge compred to the newly fertilized eggs. Different wterorne sodium concentrtions did not significntly (P>0.05) influence the wet mss of eggs (Tle 3). Incution of eggs in different wterorne sodium concentrtions significntly (P<0.05) Tle 2. Htching nd eye pigmenttion rtes of rinow trout eggs s function of different N + concentrtions utiliztion in different tretments Tretments (mg L -1 ) Eye pigmenttion (%) Htching rte (%) ± ± ± ± ± ± ± ±1.65 (Men ± SD) Mens with different letters in column re significntly different (P<0.05). n = Prior eye pigmenttion 20 After eye pigmenttion Egg mortlity (%) Sodium concentrtion (mg L -1 ) Figure 1. Comprison etween rinow trout eggs mortlity rte prior nd fter eye pigmenttion incuted in different sodium concentrtions. Brs represent Men ± SD. P<0.05. n=270

4 62 M. Srkheil et l. / Turk. J. Fish. Aqut. Sci. 14: (2014) decresed wter content (%) of eggs t htching time in reltion to the newly fertilized eggs. Egg wter content ws not significntly different mong tretments (Tle 3). Whole Egg ion Content The whole egg ions content re shown in Tle 4. The whole egg N + in tretment with [N + ] 14, 50 nd 100 mg L -1 significntly (P<0.05) incresed 40.89, nd 64.64% compred to the newly fertilized egg respectively. In ll the tretments, K content of whole egg significntly (P<0.05) incresed compred to the newly fertilized egg. In tretments with [N + ] 2 nd 14 mg L -1, no significnt (P>0.05) ltertion in whole egg P ws oserved reltive to the newly fertilized egg, ut incution of eggs in [N + ] 50 nd 100 mg L -1 incresed whole egg P content compred to the newly fertilized egg. Cu content of whole egg in [N + ] 2 nd 14 mg L -1 were ten nd six time higher thn the newly fertilized egg respectively. Incution of egg in tretment with [N + ] 2 mg L -1 incresed Mn content of whole egg y 48.8% compred to the newly fertilized egg. Incresing the wterorne sodium to 14, 50 nd 100 mg L -1 did not significntly (P>0.05) influenced Mn content of whole egg reltive to the newly fertilized egg. Wterorne Ions Content The vritions of wterorne ions content in different sodium tretments t the eginning of the experiment nd htching stge re shown in Tle 5. In ll tretments, wterorne N nd K significntly (P<0.05) decresed during eggs incution period. Altertion of wterorne P ws not significntly (P>0.05) different mong tretments during the incution period. Wterorne Cu significntly Tle 3. Whole egg wet mss nd wter content (%) in the newly fertilized egg nd incuted egg in different sodium tretments t htching stge Prmeter Newly fertilized Sodium Tretments (mg L -1 ) egg Wet mss (mg) 84.77± ± ± ± ±4.9 Wter content (%) 69.58± ± ± ± ±0.95 (Men ± SD). Mens with different letters in the sme row re significntly different (P<0.05). n = 20. Tle 4. Whole egg ions content (mg/g dry weight) in newly fertilized egg nd incuted egg in different tretments t htching stge Minerl Newly fertilized egg Sodium Tretments (mg L -1 N 0.74± ± ± ± ±0.08 c K 4.63± ± ± ± ±0.29 c P 29.61± ± ± ± ±0.29 Cu 0.01± ±0.001 c 0.065± ± ±0.001 Mn ± ± ± ± ± (Men ± SD). Mens with different letters in the sme row re significntly different (P<0.05). Tle 5. Vritions in wterorne ions concentrtion (mg L -1 ) in different tretments t the eginning of the experiment nd htching stge Minerl Time Sodium Tretments (mg L -1 ) N Beginning of Exp. 2.03± ± ± ±0.58 Htching stge 1.16± ± ± ±0.46 K Beginning of Exp. 1.94± ± ± ±0.054 Htching stge 0.56± ± ± ±0.011 P Beginning of Exp. 1.44± ± ± ±0.025 Htching stge 1.32± ± ± ±0.058 Cu Beginning of Exp ± ± ± ± Htching stge 0.017± ± ± ±0.006 Mn Beginning of Exp ± ± ± ± Htching stge 0.009± ± ± ± (Men ± SD). Mens with different letters in eginning of tril nd htching stge in ech sodium tretment re significntly different (P<0.05).

5 (P<0.05) decresed in tretments with [N + ] 2 nd 14 mg L -1, ut Cu reduction ws not significnt (P>0.05) in tretments with [N + ] 50 nd 100 mg L -1. Wterorne Mn significntly (P<0.05) decresed only in tretments with [N + ] 2 mg L -1 during the incution period. Discussion Eye Pigmenttion nd Htching Rtes Reduction of eye pigmenttion in tretment [N + ] 100 mg L -1 my e due to premture ursting of eggs from excessive wter sorption, so tht wter content (%) of eggs in this tretment ws higher thn other tretments (Tle 3). In ll tretments, higher eggs mortlity occurred fter eye pigmenttion (Figure 1), this phenomenon cn e ttriuted to N + uptke y egg fter eye pigmenttion. The findings of this study reveled tht different wterorne N + l evels ffect the eggs htchility. The eggs exposed to wterorne [N + ] of 2 nd 14 mg L -1 hd the highest htching rtes wheres reduction on htch rte in [N + ] 50 nd 100 mg L -1 ws recorded. Erly development of fish emryo involves the dorsl xis formtion nd the movement of epioly. It hs shown tht emryos of Misgurnus fossilis (Coitide) which were plced in 100 mm NCl solution cn not egin norml epioly nd xis formtion t erly lstul stge nd emryos hd irregulr structures nd different cell type. Influence of NCl on emryogenesis pper to e specific for N + nd is not osmotic (Minin nd Ivnov, 1996). Whole Egg Wter Content Whole egg wter content in different wterorne sodium decresed in reltion to the newly fertilized egg which indictes the wter loss during the incution period. In ll tretments, wterorne clcium concentrtion ws pproximtely constnt, 50 mg L -1, led to the prevention of wter sorption. Wter hrdness hs direct effect on the swelling of the newly fertilized egg (Spde nd Bristow, 1999). It hs shown tht higher Clcium concentrtion directly ffected the swelling of rinow shrk minnow (Eplzeorhynchus frentum) eggs due to n increse in the osmotic concentrtion of the incution wter (Aernthy, 2004). The result of the present study confirmed tht different wterorne sodium concentrtions hd not ny significnt (P>0.05) effect on wter uptke y egg. Egg wter content of % ws recorded mong tretments. Brrett et l (2001) reported the rte of 64.4% egg wter content for rinow trout. Ion Uptke y Emryos M. Srkheil et l. / Turk. J. Fish. Aqut. Sci. 14: (2014) 63 compred to the newly fertilized egg nd reduction of wterorne sodium concentrtion during the incution in these tretments indicte tht the trout eggs cn uptke sodium from wterorne. The liner reltionship etween [N + ] uptke rte y rinow Incresing the whole egg sodium content exposed to [N + ] tretment 14, 50 nd 100 mg L -1 trout egg nd externl [N + ] suggest tht N + cn enter into the egg (Brrett et l. 2001). Sodium uptke y eggs ws incresed long with n increse in wterorne [N + ], suggesting tht the rte of sodium uptke y egg relted to wterorne [N + ] levels. The results of whole egg K content nd wterorne K concentrtion showed tht potssium uptke y egg from wter in ll tretments nd the highest potssium uptke oserved in tretment with [N + ] 100 mg L -1. This phenomenon my e relted to the consequence of greter sodium uptke y egg (Tle 4). These findings suggest tht the prole mechnism of K uptke relte to the sodium uptke y egg from wter. Potssium uptke y river lmprey egg (Lmpetr fluvitilis) medited y two sic energy dependent trnsport mechnism N-K-pump nd N +, K +, 2Cl -1 co-trnsport (NKCC) (Sherstoitov et l. 2004). The NKCC is clss of integrl memrne proteins tht medite the movement of N +, K + nd Cl - ions cross the plsm memrne of niml cells in n electriclly neutrl mnner (Pyne nd Forush, 1995). Some studies reveled tht N +, K +, 2Cl -1 co-trnsport involved in sodium uptke function in qutic environments rich in sodium ions (Kirschner, 2004). Uptke of phosphorus y emryo nd lrve of fish from wter hs reported. For exmple, Win penny (1998) recorded phosphorus sorption y rown trout (Slmo trutt L.) from wter. The present dt conversely indicte lck of phosphorus uptke from environmentl wter nd different externl [N + ] do not ffect P uptke y eggs (Tle 4 nd 5). Lck of P uptke my e due to sufficient storges of P within the newly fertilized egg nd ctive P uptke my not e necessry. An increse in whole egg P content incuted in [N + ] 50 nd100 mg L -1 in reltion to the newly fertilized egg my e s consequence of incresing ctivity of ATP pumps for ions trnsporting, so tht uptke of N, K nd Mg (not shown) from wter were higher thn eggs incuted in [N + ] 2 nd 14 mg L -1. Cu uptke from wter environment occurred in eggs incuted in [N + ] 2 nd 14 mg L -1, s Cu content of the whole egg incresed in reltion to the newly fertilized egg nd wterorne Cu decresed during the incution period. Incution of eggs in [N + ] 50 nd 100 mg L -1 were prevented Cu uptke y eggs from wter. A theory suggests tht copper t lest in prt cross the gill through sodium chnnel on the picl memer nd some prt of trnsported copper cn e excluded y high externl sodium levels (Grosell nd Wood, 2002). Slomn et l. (2003) reported tht the differences in copper ccumultion in rinow trout my e due to differences in the sodium uptke rte. Sodium nd copper my shre

6 64 M. Srkheil et l. / Turk. J. Fish. Aqut. Sci. 14: (2014) common pthwy for up-tking through epithelil cells from externl medium. Copper sorption vi Cu-specific pthwy somehow ws modulted y the presence of externl N + (Hndy et l., 2002). The dt of this study suggest tht N + nd Cu sorption y egg from the externl wter proly tke plce vi the sme pthwy. Lower N + uptke rte y egg in [N + ] 2 nd 14 mg L -1 cn e ttriuted to N + nd Cu competition to entry through the sme pthwy nd higher Cu uptke rte. The results of whole egg Mn content nd wterorne Mn showed tht only eggs in tretment with [N + ] 2 mg L -1 sored Mn from the wterorne wheres incution of eggs in tretments with [N + ] 14, 50 nd 100 mg L -1 prevented Mn uptke from wter (Tle 4 nd 5). Mngnese is n cutely toxic t reltively high queous concentrtion nd its toxicity is ffected y wter hrdness (Stulefield et l. 1996). Gonzlez et l (1990) found tht mngnese uptke y gills of rook chrr (Slvelinus fontinlis) inversely correlted with ody sodium concentrtion. Mngnese uptke y eggs in [N + ] 2 mg L -1 my e due to the lck of sodium uptke from wter nd less sodium ccumultion in whole ody egg. Conclusion According to the results of this study, it is recommended tht the incution of rinow trout eggs in wterorne [N + ] from 14 to 100 mg L -1 nd [C 2+ ] concentrtion of 55 mg L -1 environmentlly re useful to reduce ccumultion nd toxicity of Cu nd Mn ions during the rinow trout emryogenesis. These results lso indicted the importnt role of wterorne N + levels in the improvement efficiency of rinow trout egg htching rte nd successful culture of this species in cptivity. Acknowledgements We re grteful to Deprtment of Fisheries nd Environmentl Sciences (Fculty of Nturl Resources, University of Tehrn) for finncil supporting. References Aernthy, M.A Effect of wter hrdness on the survivl of rinow shrkminnow (Eplzeorhynchos frentum) eggs nd lrve. Msc Thesis, Ginesville University of Florid. 63 pp. Alderdice, D.F Osmotic nd ionic regultion in teleost eggs nd lrve. In: WS, Hor Brrett, K.J., Mc Donld, D.G nd O, Donnell, M.J Chnges in ion content nd trnsport during development of emryonic rinow trout. Journl of Fish Biology, 59: doi: /j t00195.x Bijveld, M.J.C., Vn Der Velden, J.A., Kolr, Z nd Flik, G Mgnesium trnsport in freshwter teleost. Journl of Experimentl Biology, 201: Depeche, J. nd Billrd, R Emryonic in fish review. Society Frnce Ichthyology, Pris, 123 pp. DJ Rndll (Eds.) Fish Physiology. Acdemic Press, Sndiego, Geertz Hnsennd, P nd Rmssen, G Influence of ochre nd cidifiction on the survivl nd htching of rown trout eggs (Slmo trutt). In: R. Muller nd R. Lloyd (Eds.), Chronic Effects of Pollutnts on Freshwter Fish, FAO-Fishing New Books, Cmridge, 317 pp. Gonzlez, R.J., Grippo, R.S nd Dunson, W.A The disruption of sodium lnce in rook chrr, Slvelinus fontinlis (Mitchill), y mngnese nd iron. Journl of Fish Biology, 37 (5): doi: /j t02540.x Grosell, M nd Wood, C.M Copper uptke cross rinow trout gills: mechnisms of picl entry. Journl of Experimentl Biology, 205: Hndy, R.D., Eddy, F.B nd Bines, H Sodiumdependent copper uptke cross epitheli: review of rtionle with experimentl evidence from gill nd intestine. Biochimic et Biophysic Act, 1566: Horng, J.H., Lin, L.Y., Hung, C.J., Ktoh, F., Kneko, T nd Hwng, P.P Knockdown of V-ATPse suunit A (tp6v1) impirs cid secretion nd ion lnce in zerfish (Dnio rerio). The Americn Journl of Physiology - Regultory, Integrtive nd Comprtive Physiology, 292: doi: /jpregu Hwng, P.P., Lee, T.H. nd Lin, L.Y Ion regultion in fish gills: recent progress in the cellulr nd moleculr mechnisms. The Americn Journl of Physiology-Regultory, Integrtive nd Comprtive Physiology, 301: doi: /jpregu Kneko, T., Shirishi, K., Ktoh, F., Hsegw, S nd Hiroi, J Chloride cells during erly life stges of fish nd their functionl differentition. Fisheries Science, 68(1): 1-9. doi: /j x Ketol, G.H., Longcre, D., Greulich, A., Phetterplce, L nd Lshom, R High clcium concentrtion in wter increses mortlity of slmon nd trout eggs. The Progressive Fish-Culturist, 50: Kirschner, L.B The mechnism of sodium chloride uptke in hyper regulting qutic nimls. Journl of Experimentl Biology, 207: doi: /je Lee, C.S nd Hu, F Influence of C nd Mg ions on the egg survivl of grey mullet, Mugil cephlus L. Journl of Fish Biology, 22: dio: /j t04721.x Lin, Z.H., C.S nd Lin, L.Y Ammonium-dependent sodium uptke in mitochondrion-rich cells of medk (Oryzis ltipes) lrve. Americn Journl of Physiology-Cell Physiology, 298: doi: /jpcell Mckreth, F.J.H., Heron, J nd Tlling, J.F Wter Anlysis: Some Revised Methods for Limnologist. No.36, Freshwter Biologicl Assocition, U.K, 119 pp. Mtsuo, A.Y.O., Plyle, R.C., Vl, A.L nd Wood, C.M Physiologicl ction of dissolved orgnic mtter in rinow trout in the presence nd sence of copper: sodium uptke kinetics nd unidirectionl flux rtes in hrd nd soft wter. Aqutic Toxicology, 70:

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