Interaction between dietary calcium supplementation and chronic waterborne zinc exposure in juvenile rainbow trout, Oncorhynchus mykiss

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1 Comprtive Biohemistry nd Physiology, Prt C 143 (26) Intertion etween dietry lium supplementtion nd hroni wterorne zin exposure in juvenile rinow trout, Onorhynhus mykiss S. Niyogi,, C.M. Wood Deprtment of Biology, University of Sskthewn, 112 Siene Ple, Ssktoon, Sskthewn, Cnd S7N 5E2 Deprtment of Biology, MMster University, Hmilton, ON, Cnd L8S 4K1 Reeived 28 July 25; reeived in revised form 21 Deemer 25; epted 21 Deemer 25 Aville online 3 Ferury 26 Astrt This study investigted the effets of dietry C 2+ on rnhil C 2+ nd Zn 2+ uptke, new nd totl zin umultion in trget tissues (gill, liver nd kidney), lium nd zin homeostsis, nd ute tolerne to wterorne zin in fish hronilly exposed to wterorne zin. Juvenile rinow trout (Onorhynhus mykiss) were mintined on lium-enrihed diet [41.2 mg vs mg (ontrol) lium/g dry wt. of food] nd hroni wterorne zin exposure (2.3 μmol/l), oth seprtely nd in omintion, for 28 dys. Clium-supplemented diet in the sene of wterorne zin signifintly redued rnhil C 2+ nd Zn 2+ influx rtes, nd new nd totl zin umultions in trget tissues reltive to ontrol. However it did not protet ginst the ute zin hllenge. In ontrst, wterorne zin exposure signifintly inresed rnhil C 2+ nd Zn 2+ influx rtes, new nd totl zin onentrtions in trget tissues, nd ute zin tolerne reltive to ontrol. Interestingly, no suh hnges in ny of these prmeters were reorded in fish treted simultneously with elevted dietry C 2+ nd wterorne zin, exept ute zin tolerne whih ws highest mong ll the tretments. Thus, we onlude tht the intertions etween elevted dietry C 2+ nd wterorne zin n protet freshwter fish ginst wterorne zin toxiity. 25 Elsevier In. All rights reserved. Keywords: Fish; Dietry C 2+ ; Wterorne zin; C 2+ nd Zn 2+ uptke; Aute zin tolerne 1. Introdution Zin is mironutrient for fish, ut it eomes toxi t inresed wterorne onentrtions. Wterorne zin is ioville s free divlent tion, Zn 2+, whih shres, t lest in prt, ommon uptke pthwy with C 2+ in the gills of freshwter fish (Spry nd Wood, 1989; Hogstrnd et l., 1994, 1995, 1996, 1998). Beuse of this phenomenon, inresed wterorne zin speifilly disrupts C 2+ uptke ross the gills (Spry nd Wood, 1985; Hogstrnd et l., 1995, 1996), leding to hypolemi, whih my ulminte in the deth of fish within few dys, depending on the onentrtion. However, fish hronilly exposed to wterorne zin re often le to limte physiologilly, nd this limtion involves progressive redution in the rnhil influx rte of Zn 2+ nd restortion of plsm lium onentrtions Corresponding uthor. Tel.: ; fx: E-mil ddress: som.niyogi@ussk. (S. Niyogi). (Hogstrnd et l., 1994, 1995). Interestingly, these hnges re rought out y ltering the ffinity (K m ) of the shred pil C 2+ /Zn 2+ trnsport pthwy while keeping the mximum rte of rnhil C 2+ uptke (J mx ) reltively unffeted (Hogstrnd et l., 1995, 1998). These oservtions strongly suggest tht the gills ply very importnt role in mintining lium homeostsis under hroni wterorne zin exposure in freshwter fish. Not surprisingly, wter hrdness hs n meliorting effet ginst zin umultion in trget orgns (e.g., gills) nd toxiity in freshwter fish (Zitko nd Crson, 1976; Everll et l., 1989). Brdley nd Sprgue (1985) reported tht ten-fold derese in wter hrdness inreses ute zin toxiity to juvenile rinow trout, Onorhynhus mykiss, y ten times. Similrly, Alsop et l. (1999) showed tht six-fold redution in hrdness inreses toxiity out six times in trout. Alsop nd Wood (1999) demonstrted tht wterorne C 2+ redues oth short-term (3 h) wterorne zin uptke nd toxiity wheres wterorne Mg 2+ does only the former. Brron nd Aleke (2) showed tht elevted wterorne C 2+ redues oth /$ - see front mtter 25 Elsevier In. All rights reserved. doi:1.116/j.p

2 S. Niyogi, C.M. Wood / Comprtive Biohemistry nd Physiology, Prt C 143 (26) whole ody uptke nd gill umultion of zin in rinow trout through oth physiologil limtion nd ompetitive intertion. Although the protetive effets of wterorne C 2+ on zin toxiity re well doumented, virtully nothing is known out how dietry C 2+ ffets the uptke, umultion nd toxiity of wterorne zin. Freshwter fish hve two primry uptke pthwys for essentil ions (e.g., C 2+, N + ), the gills (wterorne ions) nd the gstrointestinl trt (dietry ions), nd they n regulte the totl uptke y hnging the proportion of eh kind of uptke depending on the environmentl situtions. For exmple, goldfish (Crssius urtus) fed with lium-defiient diet inrese their rnhil C 2+ uptke nd tilpi (Oreohromis mossmius) up-regulte intestinl C 2+ uptke when living in wter with low C 2+ onentrtion (Flik et l., 1995). Therefore, if the fish n quire more ions vi the gstrointestinl route, they my derese rnhil ion uptke rtes, nd therey susequently redue the uptke of metl(s) shring the ommon rnhil pthwy. For exmple, Cd 2+, like Zn 2+, shres the sme trnsport pthwy with C 2+ (Verost et l., 1989; Niyogi nd Wood, 24), nd reent experiments hve shown tht dietry C 2+ supplementtion deresed wterorne C 2+ uptke, nd susequently wterorne Cd 2+ uptke nd umultion in trget orgns under wterorne dmium exposures (Zohouri et l., 21; Bldisserotto et l., 24,, 25). These findings suggest tht lium-enrihed diet my hve similr meliorting effet s wterorne C 2+ ginst wterorne Cd 2+ toxiity prtiulrly in soft wters (low C 2+ ), nd wild metl-impted fish n enefit y preferentilly forging on lium-rih prey speies (e.g., mollusks, rustens) in nture. There is no ville dt (to the est of our knowledge) of C 2+ onentrtions in the nturl diet of ferl fish, ut C 2+ is ville in undne in rusten exoskeleton nd mollusk shells. Interestingly though, Sherwood et l. (2) reported tht wild yellow perh (Per flvesens) in metl-impted (dmium, opper nd zin) lkes tend to et reltively more invertertes thn fish ompred to perh in referene lkes, strtegy whih would likely inrese the C 2+ ontent of their diet. In view of this kground, the present study ws designed to investigte the intertions etween elevted dietry C 2 + (supplemented with CCO 3 ) nd hroni wterorne zin exposure in juvenile rinow trout. The min ojetives were to find out the influene of dietry C 2+ supplementtion on the following spets: (i) wterorne C 2+ nd Zn 2+ influx rtes, (ii) new nd totl zin umultion in trget orgns, (iii) lium nd zin homeostsis, nd (iv) tolerne to ute wterorne zin exposure. 2. Mterils nd methods 2.1. Experimentl fish Juvenile (1 15 g, n = 25) rinow trout, Onorhynhus mykiss, were otined from Humer Springs Trout Hthery, Mono Mills, ON, Cnd, nd limted to lortory onditions in single 5-L plsti tnk for 2 weeks. Lortory onditions inluded flow-through of dehlorinted Hmilton Muniipl tp wter from Lke Ontrio (N +.6; C 2+ 1.; Cl.7; Mg 2+.2; HCO 3 1.9, ll in mmol/l; dissolved orgni ron 3. mg/l; hrdness 12 mg/l s CCO 3 ; ph nd temperture 12±1 C). The fish were mintined on 2% dily rtion (dry food/wet ody weight) of ommeril grnulted 1. grde dry trout pellet (Corey Feed Mills, Frederiton, New Brunswik, Cnd) during the 2- week period of lortory limtion. The ommeril trout diet ontined rude protein 54% (minimum), rude ft 19% (minimum), rude fier 2% (mximum), phosphorus 11 mg/g, sodium 6 mg/g (ll in dry weight sis). Mesured lium nd zin onentrtions in the food were 21.2±1.3 mg/g dry weight (.53 mmol/g dry weight) nd 174.4±11.3 μg/g dry weight (2.68 μmol/g dry weight), respetively, n = 1 in oth mesurements. Fish were mintined under 12-h light nd 12-h drk yle during limtion nd throughout the entire period of the experiment Diet preprtion All diets were prepred with ommerilly ville trout feed tht ws used to feed the fish during limtion. This fish food ws ground in lender, followed y hydrtion with pproximtely 4% v/w deionized wter. To prepre the lium-supplemented tretment diet, the ontrol diet (21.2 mg lium/g dry wt. of food) ws supplemented with CCO 3 to yield n experimentl diet with 41.2 ± 2.8 mg lium/g dry wt. of food, n=8 (1.3 mmol lium/g dry wt. of food). CCO 3 ws dissolved in the deionized wter nd dded to the food pste. The resulting pste ws mixed nd extruded through pst mker, ir-dried, nd roken into smll pellets y hnd. The ontrol diet ws prepred y the sme method ut with the ddition of deionized wter only Experimentl design The experiment desried here omprised four tretments, eh replited in seprte 2-L tnks for 28 dys. Tretments inluded: (i) norml diet nd wter (ontrol), (ii) liumenrihed diet nd norml wter, (iii) norml diet nd wterorne zin (2.3 μmol/l), nd (iv) lium-enrihed diet nd wterorne zin (2.3 μmol/l). Following 2 weeks of lortory limtion, fish were eqully nd rndomly distriuted into one of two replites in eh tretment (n=6 per tretment). An exposure onentrtion of 2.3 μmol/l wterorne zin ws hieved vi onstnt drip of stok solution [ZnSO 4 7H 2 O (Fisher Sientifi, Toronto, ON, Cnd) diluted in deionized wter] from Mrriott ottle into hed tnk reeiving dehlorinted Hmilton tp wter. The hed tnk supplied the four experimentl tnks [replites of tretments (iii) nd (iv)] t flow rte of 7 ml/min. The mesured zin onentrtion in the exposure wter ws 2.35 ±.32 μmol/l (n =28). The remining two tretments of this study [replites of tretments (i) nd (ii)] were mintined on regulr dehlorinted wter t flow rte of 7 ml/min. The kground zin onentrtion in

3 96 S. Niyogi, C.M. Wood / Comprtive Biohemistry nd Physiology, Prt C 143 (26) the ontrol wter ws.15±.4 μmol/l (n=28). Fish were su-smpled on dy 7, 14, 21 nd 28 during the exposure for nlysis of rnhil C 2+ nd Zn 2+ uptke, newly umulted nd totl zin onentrtions in trget tissues (gill, liver nd kidney), nd lium nd zin onentrtions in plsm. Prior to the rndom seprtion of fish to the experimentl tretment tnks, ten fish from omined ontrol pool were similrly smpled nd nlyzed to generte dy dt. Fish were fed twie dy t 12-h intervls with the ontrol or lium-enrihed diets t rtion of 2% wet wt. of ody mss per dy, with hlf of the rtion eing delivered t eh feeding. Fish in ll tretment tnks were ulk-weighed weekly to djust the quntity of rtion every week. Unonsumed food from ll tretment tnks ws siphoned out 1 h fter the food ws provided. Anlysis of wter smples olleted 1 h post-feeding (one week) from tretments with lium-enrihed diet reveled no signifint differene in C 2+ onentrtion reltive to dehlorinted Hmilton tp wter (ontrol wter), thus there ws no detetle lehing of C 2+ from the diet into the wter. No mortlity ws reorded in ny of the tretment tnks during the entire experimentl period. No differenes in growth ourred mong ny of the tretments Brnhil C 2+ nd Zn 2+ uptke nlyses On eh smpling dy, fish were smpled 1 h post feeding for mesuring oth rnhil C 2+ nd Zn 2+ uptke rtes, seprtely, using the methods of Hogstrnd et l. (1994). Brnhil C 2+ uptke ws determined y exposing fish for 4 h to rdiotive 45 C in pproprite exposure wters. Exposure wters were tken diretly from the respetive experimentl tretment tnks tht housed the fish for 28 dys nd therefore ontined elevted wterorne zin or not, s pproprite. Fish (4 5 per replite, 9 1 per tretment) were pled into 5-L ler polyethylene gs filled with 3 l of exposure wter. Eh g ws provided with ertion, nd housed in wter th to mintin onstnt temperture (12 C). Five minutes efore the fish were introdued to the gs, 2 μci/l of 45 C ws dded [s CCl 2 (Perkin Elmer, USA); speifi tivity=12.26 mci/mg]. Duplite wter smples of 5 ml were tken t nd 4 h. After 4 h of exposure, fish were srified with n overdose of nestheti (5 mg/l uffered MS-222), rinsed for 1 min in 1 mmol/l C(NO 3 ) 2 to disple surfe-ound 45 C, lotted dry, individully weighed, nd digested in five volumes of 1 N HNO 3 for 48 h t 6 C. Lter, three replites (2 μl eh) from eh digested fish were mixed with 2 ml of id/orgniomptile sintilltion oktil (Ultim Gold, Perkin Elmer, USA) nd ounted for 45 C on liquid sintilltion ounter (LKB Wll 1217 Rket, Phrmi-LKB, Helsinki, Finlnd). One set of wter smples (5 ml eh) were mixed with 1 ml of sintilltion fluor (ACS, Amershm, USA) nd ounted for 45 C s well. Counting effiienies for 45 C were determined y internl stndrdiztion, i.e. y ddition/reovery of known mounts of 45 C. The remining wter smples were nlyzed for totl lium using ertified stndrd for lium (Fisher Sientifi, Cnd) on flme tomi sorption spetrometer (22 FS, Vrin, Austrli). The rnhil influx rte (J in )ofc 2+ (in μmol/kg h) in fish ws lulted ording to Hogstrnd et l. (1994): J in ¼ CPM F = ðsa w CE tþ where CPM F is the verge 45 C ounts in fish (ounts per min/ kg wet mss), SA w is the mesured men speifi tivity of 45 C in the wter [(ounts per min/l wter)/totl lium in wter (μmol/l)], CE is the reltive ounting effiieny of the tissue fluor system reltive to wter fluor system nd t is time. The rnhil Zn 2+ uptke rtes were mesured in fish from the wterorne zin exposed s well s the zin unexposed tretments. All mesurements were mde t the zin onentrtion of the exposure tnks (2.3 μmol/l). The flux hmers used were 24-L lk polyethylene ukets, eh filled with 2 L of dehlorinted wter diretly tken from the zin exposure tnks. Eh hmer ws erted nd pled into flow-through wter th to keep the fish t the sme temperture s the holding tnks (12 C). Five minutes efore the fish were introdued to the flux hmers, 3 μci/l of 65 Zn [s ZnCl 2 (Ok Ridge Ntionl Lortory, USA); speifi tivity = 1.97 mci/mg] ws dded to eh hmer. Fish (4 5 per replite, 9 1 per tretment) were trnsferred to the flux hmers nd were held there for 24 h. Qudruplite wter smples (1 ml eh) were olleted from the hmers t, 12, 18 nd 24 h. At the end of the exposure, fish were nesthetized (2 mg/l uffered MS-222), rinsed with len wter, nd lood ws olleted from the udl vein with heprinized 1-mL syringes. Blood smples were entrifuged t 1, g for 5 min to seprte plsm, nd the seprted plsm smples were ounted for 65 Zn tivity in gmm ounter (MINAXI Gmm 5 Series, Cnerr Pkrd, USA). Wter smples were similrly ounted for 65 Zn tivity nd totl zin ws mesured s ove y flme tomi sorption spetrosopy using ertified stndrd for zin (Fisher Sientifi). Counting effiienies for 65 Zn were evluted y ddition/reovery of known mounts of 65 Zn to plsm nd wter, nd no quenhing ws reorded. The rnhil Zn 2+ uptke rtes were lulted y the method desried y Spry nd Wood (1989). The protool ws sed on the reltionship etween the stedy-stte onentrtion of 65 Zn in plsm fter 24 h of exposure (C ss expressed in units of nmol/l of exogenous zin per ml of plsm, s lulted from the externl speifi tivity of zin) nd the influx of Zn 2+ (in nmol/kg h) estlished in rinow trout y Spry nd Wood (1989): J in ¼ 4:215 C ss þ 2:47 The lultion is not ffeted y the endogenous onentrtion of zin in the plsm. The Zn 2+ uptke ws mesured t only one onentrtion of the sustrte (zin) Determintion of newly umulted zin in trget orgns Fish used in rnhil Zn 2+ uptke mesurements were killed with single low to the hed immeditely fter the olletion of lood smples. Fish were rinsed for 1 min in 1 mmol/l ZnSO 4 to disple surfe-ound 65 Zn, lotted dry, individully weighed, nd gill, liver nd kidney were disseted

4 S. Niyogi, C.M. Wood / Comprtive Biohemistry nd Physiology, Prt C 143 (26) out. All the tissues were weighed seprtely, nd then ounted for 65 Zn tivity s desried efore. Agin potentil quenhing of 65 Zn ws heked s desried efore nd no quenhing ws notied in ny tissues. Newly umulted zin ws lulted y the following eqution: M New ¼ =ð=þ where M New is the newly umulted zin (nmol/g wet wt.), is the pm/g of tissue, is the pm/l of wter, nd is the totl zin onentrtion/l of wter. It is importnt to note here tht dt for nd re derived from the wter smples olleted during rnhil Zn 2+ uptke mesurements Determintion of totl zin onentrtions in trget orgns nd whole ody, nd plsm lium nd zin onentrtions Following the ounting of 65 Zn tivity, ll tissues (gill, liver nd kidney) long with the remining rss were digested seprtely in five volumes of 1 N HNO 3 for 48 h t 6 C. Totl zin onentrtions were mesured in digested smples fter pproprite dilution with 1% HNO 3 y flme tomi sorption spetrosopy s desried efore using ertified zin stndrd (Fisher Sientifi). Similrly, totl lium nd zin levels in plsm were nlyzed fter pproprite dilution y flme tomi sorption spetrosopy using plsm smples olleted during rnhil Zn 2+ influx mesurements. Whole ody totl zin onentrtions were lulted y dividing the sum of zin ontents (onentrtions multiplied y weight) of ll the tissues smpled plus the rss y the sum of weights of ll the tissues plus the rss Determintion of tolerne to ute wterorne zin exposure Tolerne to ute wterorne zin exposure in ll four tretments ws determined t dy 28 y medin lethl time (LT 5, the time when 5% of the popultion is expeted to die). Ten fish from eh tretment (five per replite) were trnsferred to 24-L flow-through polyethylene uket fitted with n irline. An exposure onentrtion of 82.7±4.1 μmol/l (n=5) wterorne zin ws hieved vi onstnt drip of ZnSO 4 stok solution from Mrriott ottle into hed tnk reeiving dehlorinted Hmilton tp wter, whih susequently supplied ll the test hmers t flow rte of 25 ml/min. This system ws designed to ensure tht fish from ll the tretments were exposed to identil wter qulity nd onstnt temperture (12 C). No food ws provided to fish during the test. Mortlity ws monitored every 6 h intervl for 72 h, nd ded fish were removed whenever found. LT 5 vlues ±95% onfidene intervl (CI) were lulted from plots of proit mortlity ginst log time (Lithfield, 1949) Sttistil nlyses All dt (exept LT 5 vlues) were nlyzed y three-wy nlysis of vrine (ANOVA, Sttisti version 6.) with time, dietry C 2+ nd wterorne zin onentrtions s independent vriles (t α =.5). Tukey's honestly signifint differene (HSD) test ws used to delinete differenes in men vlues. The ssumptions of ANOVA, i.e., homogeneity of vrines nd normlity of distriution, were tested using Levene's nd Shpiro Wilk's tests (oth t α =.5). All dt met these ssumptions. Men vlues were onsidered different t P.5. For medin lethl time (LT 5 ) tests, the groups were onsidered signifintly different if the 95% CI for the LT 5 vlues did not overlp (Lithfield, 1949). 3. Results 3.1. Brnhil C 2+ uptke Brnhil C 2+ influx rte signifintly deresed (F [1,144] = 7.15, P.1) y up to 29% in fish fed the lium-enrihed diet nd not exposed to wterorne zin reltive to the ontrol tretment, nd this effet existed during the entire exposure (Fig. 1A). In ontrst, exposure to wterorne zin nd norml diet signifintly inresed C 2+ influx rte (F [1,144] =11.49, P.1) y up to 39% reltive to the ontrol tretment. However, this effet ws oserved only t the initil stges (dys 7 nd 14), nd it leveled off y the end of the exposure (Fig. 1A). Interestingly, fish exposed to wterorne zin nd fed with the lium-enrihed diet did not show ny hnges reltive to the ontrol group in rnhil C 2+ influx rte throughout the entire exposure period (Fig. 1A) Brnhil Zn 2+ uptke Altertions in rnhil Zn 2+ influx rte showed quite similr pttern to those in rnhil C 2+ influx. A 24% derese (F [1,144] =4.7, P.5) in Zn 2+ influx rte ws reorded in fish fed with lium-supplemented diet ut not exposed to wterorne zin during the exposure, lthough the influx rte did not differ from tht of the ontrol group t the end (dy 28) (Fig. 1B). In ontrst, rnhil Zn 2+ influx rte inresed signifintly y up to 64% (F [1,144] =11.73, P.1) following exposure to wterorne zin nd norml diet, rehing its pek during dys 7 to 14 followed y grdul derese to the rte in the ontrol group on dy 28 (Fig. 1B). Agin, fish exposed to wterorne zin nd fed with the lium-enrihed diet did not show ny ltertions in rnhil Zn 2+ influx rte throughout the entire exposure period (Fig. 1B) Newly umulted zin in trget orgns Dietry C 2+ supplementtion lone signifintly deresed newly umulted zin onentrtions in the gill (F [1,144] = 12.22, P.1), liver (F [1,144] =7.1, P.1) nd kidney (F [1,144] =3.91, P.5) (Fig. 2A,B,C). In ontrst, wterorne zin exposure lone signifintly inresed newly umulted zin onentrtions in the gill (F [1,144] =11.97, P.1), liver (F [1,144] =11.45, P.1) nd kidney (F [1,144] =3.93, P.5), lthough new zin onentrtions in the liver nd kidney, ut not in the gill, dropped k to the ontrol onentrtion y the

5 98 S. Niyogi, C.M. Wood / Comprtive Biohemistry nd Physiology, Prt C 143 (26) (A) J in C 2+ (µmol/kg.h) (B) J in Zn 2+ (nmol/kg.h) rnhil C 2+ influx Time (dys) rnhil Zn 2+ influx Time (dys) Fig. 1. Brnhil C 2+ (A) nd Zn 2+ (B) influx rtes in different tretments: (i) white rs represent norml diet (21.2 mg lium/g dry wt. of food) nd wter (ontrol), (ii) lk rs represent lium-enrihed diet (41.2 mg lium/g dry wt. of food) nd norml wter, (iii) grey rs represent norml diet (21.2 mg lium/g dry wt. of food) nd wterorne zin (2.3 μmol/l), nd (iv) rosshthed rs represent lium-enrihed diet (41.2 mg lium/g dry wt. of food) nd wterorne zin (2.3 μmol/l). Dt re presented s men±stndrd error of men (S.E.M.), n=9 1. Brs with different letters re signifintly different (Tukey's HSD, P.5). end of the exposure (Fig. 2A,B,C). Interestingly, the omined tretment of dietry C 2+ supplementtion nd wterorne zin exposure did not show ny inrese in newly umulted zin onentrtions in ny of the trget tissues reltive to ontrol (Fig. 2A,B,C) Totl zin umultion in trget orgns nd whole ody Dietry C 2+ supplementtion lone redued the totl zin onentrtion in the gill (Fig. 3A) reltive to ontrol (F [1,144] = 4.1, P.5), ut not in the liver, kidney nd whole ody (Fig. 3B,C,D). Totl zin onentrtions inresed signifintly oth in the gill (F [1,144] =7.23, P.1) nd the liver (F [1,144] =4.29, P.5) in fish exposed to wterorne zin nd fed with norml diet reltive to the ontrol tretment, ut this inrese ws oserved only on dy 7 (Fig. 3A,B). No hnges in totl zin onentrtion were reorded in the kidney nd whole ody mong ny tretment groups during the entire exposure period (Fig. 3C,D). Agin, no ltertions in totl zin onentrtions d d ws oserved on ny smpling dy either in ny trget tissues or in the whole ody in fish treted with the lium-enrihed diet under wterorne zin exposure reltive to the ontrol tretment (Fig. 3A,B,C,D) Plsm lium nd zin onentrtions No signifint differenes were oserved either in plsm lium or plsm zin onentrtions mong ny tretment groups during the entire exposure period (Tle 1) Aute tolerne to wterorne zin Aute wterorne zin tolerne did not differ signifintly etween the exlusive dietry C 2+ supplementtion nd the (A) nmol zin/g wet wt. nmol zin/g wet wt. (B) (C) nmol zin/g wet wt gill Time (dys) liver Time (dys) Kidney Time (dys) d d d d d Fig. 2. New zin umultion in trget tissues [(A) gill, (B) liver, nd (C) kidney] in different tretments: (i) white rs represent norml diet (21.2 mg lium/g dry wt. of food) nd wter (ontrol), (ii) lk rs represent liumenrihed diet (41.2 mg lium/g dry wt. of food) nd norml wter, (iii) grey rs represent norml diet (21.2 mg lium/g dry wt. of food) nd wterorne zin (2.3 μmol/l), nd (iv) ross-hthed rs represent lium-enrihed diet (41.2 mg lium/g dry wt. of food) nd wterorne zin (2.3 μmol/l). Dt re presented s men±stndrd error of men (S.E.M.), n=9 1. Brs with different letters re signifintly different (Tukey's HSD, P.5).

6 S. Niyogi, C.M. Wood / Comprtive Biohemistry nd Physiology, Prt C 143 (26) (A) 1 gill (B) 1 liver nmol zin/g wet wt nmol zin/g wet wt (C) Time (dys) kidney (D) Time (dys) whole ody nmol zin/g wet wt nmol zin/g wet wt Time (dys) Time (dys) Fig. 3. Totl zin umultion in trget tissues [(A) gill, (B) liver, nd (C) kidney] nd whole ody (D) in different tretments: (i) white rs represent norml diet (21.2 mg lium/g dry wt. of food) nd wter (ontrol), (ii) lk rs represent lium-enrihed diet (41.2 mg lium/g dry wt. of food) nd norml wter, (iii) grey rs represent norml diet (21.2 mg lium/g dry wt. of food) nd wterorne zin (2.3 μmol/l), nd (iv) ross-hthed rs represent lium-enrihed diet (41.2 mg lium/g dry wt. of food) nd wterorne zin (2.3 μmol/l). Dt re presented s men±stndrd error of men (S.E.M.), n=9 1. Brs with different letters re signifintly different (Tukey's HSD, P.5). ontrol tretment groups (Fig. 4). However, tolerne signifintly inresed in fish treted with wterorne zin nd the norml diet s well s wterorne zin nd the lium-enrihed diet, with the ltter group hving the highest medin lethl time (LT 5 ) vlue (Fig. 4). 4. Disussion The present study hs reveled three importnt phenomen in reltion to dietry C 2+ supplementtion in juvenile rinow trout unexposed s well s exposed to hroni wterorne zin. First, tretment with dietry C 2+ supplementtion lone redues rnhil C 2+ nd Zn 2+ influx rtes s well s newly umulted zin onentrtions in trget tissues. Seond, dietry C 2+ supplementtion under hroni wterorne zin exposure prevents the inrese in rnhil C 2+ nd Zn 2+ influx indued y wterorne zin, nd therey prevents the rise in new nd totl zin onentrtions in trget tissues. Third, dietry C 2+ supplementtion signifintly inreses the tolerne to ute wterorne zin, ut only under simultneous hroni wterorne zin exposure. Rodgers (1984) demonstrted, y treting rook trout (Slvelinus fontinlis) with omintion of low wterorne nd dietry C 2+, tht minimum C 2+ uptke either vi gills or intestine is required for norml fish growth. In our study, the norml diet (prepred from ommeril trout how) hd 21.2 mg lium/g of foosd whih is well ove the minimum dietry requirement of 3 7 mg lium/g of food for norml growth in freshwter fish (O'Connell nd Gtlin, 1994). Similrly, wterorne C 2+ level in our study ws round 1 mmol/l (4 mg/l) whih is lso ove the minimum level (.575 mmol/l) required for rinow trout without dietry C 2 + (Ogino nd Tked, 1978). Therefore it n e onluded tht down-regultion of the rnhil C 2+ influx in zin-unexposed fish is medited y the dietry C 2+ supplementtion, similr to the findings of Bldisserotto et l. (24,, 25). Sine the present fish were lredy living in n environment with undnt C 2+, they proly ompensted the inresed intestinl C 2+

7 1 S. Niyogi, C.M. Wood / Comprtive Biohemistry nd Physiology, Prt C 143 (26) Tle 1 Totl plsm lium nd zin levels in different tretments Dy Tretments Plsm lium (mmol/l) Plsm zin (mmol/l) Norml wter+norml food 2.3±.9.133±.1 7 Norml wter+norml food 1.96±.1.128±.8 Norml wter+clium-enrihed 2.13±.9.126±.6 Wterorne zin 1.98±.1.135±.13 (2.3 μmol/l) +Norml food Wterorne zin 2.2± ±.5 (2.3 μmol/l) +Clium-enrihed 14 Norml wter+norml food 2.8±.8.135±.5 Norml wter+clium-enrihed 2.18±.1.124±.8 Wterorne zin 1.92± ±.5 (2.3 μmol/l) +Norml food Wterorne zin 2.5±.1.126±.11 (2.3 μmol/l) +Clium-enrihed 21 Norml wter+norml food 2.1± ±.11 Norml wter+clium-enrihed 2.1±.9.12±.5 Wterorne zin 1.95± ±.5 (2.3 μmol/l) +Norml food Wterorne zin 2.1±.1.129±.1 (2.3 μmol/l) +Clium-enrihed 28 Norml wter+norml food 1.99±.8.129±.9 Norml wter+clium-enrihed 2.5±.8.128±.8 Wterorne zin 2.1±.1.133±.5 (2.3 μmol/l) +Norml food Wterorne zin (2.3 μmol/l) +Clium-enrihed 2.13±.7.132±.7 The dt re presented s men±stndrd error of men (S.E.M.), n=9-1. No signifint differenes were oserved mong ny tretments during the entire exposure period in either of two prmeters. C 2+ or Zn 2+ influx rte t the end of the exposure (dy 28) in our study. It is resonle to ssume tht the inrese in rnhil Zn 2+ influx rte ourred due to the inrese in the rte of rnhil C 2+ influx in our study sine oth divlent tions shre the sme rnhil uptke pthwy (Spry nd Wood, 1989; Hogstrnd et l., 1994, 1995, 1996, 1998). Freshwter fish limte to hroni wterorne zin exposure (studied t identil zin onentrtion nd wter hemistry onditions s in our study) primrily y o-regulting the kineti properties of C 2+ /Zn 2+ trnsporters in the gill. The K m (inverse of ffinity) of C 2+ uptke in the gill dereses sustntilly wheres the J mx (mximum uptke rte) lters only slightly (Hogstrnd et l., 1994, 1995, 1998). These hnges our in onjuntion with drmti inrese of oth K m nd J mx of rnhil Zn 2+ uptke whih persists throughout the period of the hroni exposure (Hogstrnd et l., 1998). This pprent o-regultion of the C 2+ /Zn 2+ trnsporters ours to ompenste the loss of C 2+ in fish indued y the hroni wterorne zin exposure (Hogstrnd et l., 1998). Therefore, the inrese in oth rnhil C 2+ nd Zn 2+ uptke rtes in fish treted with hroni wterorne zin nd norml diet in our study proly ourred due to the hnges in kineti properties of C 2+ /Zn 2+ trnsporters in the gill. Interestingly, fish exposed to hroni wterorne zin ut fed with lium-supplemented diet in our study did not show ny modultions in either rnhil C 2+ or Zn 2+ influx rte throughout the exposure. Wterorne zin ompromises rnhil C 2+ uptke y inresed ompetition t the pil uptke sites (voltge insensitive C 2+ hnnels) nd/or ompetitive inhiition of solterl C 2+ trnsporters (C 2+ - ATPses) (Hogstrnd et l., 1994, 1995, 1996). It is likely tht dietry C 2+ supplementtion resulted into inresed intestinl C 2+ uptke whih in turn rpidly replenished the pprent loss of internl C 2+ level indued y the hroni wterorne zin uptke y lowering the gill uptke in order to mintin lium lne in the whole ody. The down-regultion in rnhil C 2+ influx rte therey resulted into redued rnhil Zn 2+ influx nd newly umulted zin onentrtions in trget tissues sine C 2+ nd Zn 2+ re tken up y the ommon pthwy in fish gills, t lest in prt (Spry nd Wood, 1989; Hogstrnd et l., 1994, 1995, 1996, 1998). Hogstrnd et l. (1995) reported n erly inrese (during first two weeks) followed y susequent derese in oth unidiretionl rnhil C 2+ nd Zn 2+ influx rtes in juvenile rinow trout exposed to 2.3 μmol/l wterorne zin onentrtion reltive to ontrol fish over period of 3 dys t lmost identil wter hemistry to our study. It is to e noted here tht unlike in our study they mesured the rte of C 2+ nd Zn 2+ influx not on the sme dys of the exposure ut the former prmeter on ertin dys nd the ltter on other dys. Nonetheless, the erly inrese followed y grdul derese of rnhil C 2+ nd Zn 2+ uptke in fish exposed to wterorne zin (2.3 μmol/l) nd fed with norml diet in our study re in resonle greement with their findings, lthough we did not oserve the signifint derese in either rnhil LT 5 (hours) Fig. 4. Medin lethl time (LT 5 ) vlues ginst ute wterorne zin hllenge (82.7 μmol/l) in different tretments: (i) white rs represent norml diet (21.2 mg lium/g dry wt. of food) nd wter (ontrol), (ii) lk rs represent lium-enrihed diet (41.2 mg lium/g dry wt. of food) nd norml wter, (iii) grey rs represent norml diet (21.2 mg lium/g dry wt. of food) nd wterorne zin (2.3 μmol/l), nd (iv) ross-hthed rs represent lium-enrihed diet (41.2 mg lium/g dry wt. of food) nd wterorne zin (2.3 μmol/l). Dt re presented s LT 5 vlue±95% CI, n=1. Brs with different letters re signifintly different (Lithfield, 1949).

8 S. Niyogi, C.M. Wood / Comprtive Biohemistry nd Physiology, Prt C 143 (26) exposure in fish. As result, fish proly were not required to up-regulte the rnhil C 2+ uptke proess. Alsop et l. (1999) reported highly signifint inreses in newly umulted zin onentrtions in the gills following exposure to 2.3 μmol/l of wterorne zin for 3 dys t lmost identil wter hemistry. They lso reported signifint elevtion of totl zin onentrtions in the gills (dy 1) nd liver (dy 2), whih susided to ontrol onentrtions y the end of the exposure (dy 3). Both these findings re in very good greement with our results in fish treted exlusively with wterorne zin exposure. However, there re no previous reports of signifint elevtion in newly umulted zin onentrtions in liver nd kidney in fish hronilly exposed to wterorne zin. Agin, no signifint inreses were notied either in newly umulted or totl zin onentrtions in ny trget tissues during the entire exposure in fish hronilly exposed to wterorne zin nd fed with the lium-supplemented diet. This is not surprising oservtion in view of the ft tht these fish did not show ny up-regultion of wterorne Zn 2+ uptke during the entire exposure. Zohouri et l. (21) showed tht lium-enrihed diet (53 mg lium/g of food, dded s CCl 2 2H 2 O) lso prevented the inrese of totl dmium umultion in trget tissues (gill, liver, kidney) s well s in whole ody of rinow trout hronilly exposed to wterorne dmium (.2 μmol/l), metl known to enter the fish gill y C 2+ uptke pthwy in similr mnner to Zn 2+ (Verost et l., 1989; Niyogi nd Wood, 24). Totl plsm lium or zin onentrtions did not differ signifintly mong the tretments during the entire exposure, suggesting no pprent disruption of lium nd zin homeostsis. Zohouri et l. (21) nd lter Bldisserotto et l. (24,, 25) reported no hnges in totl plsm lium onentrtions in rinow trout treted following exlusive tretments with lium-enrihed diet (3 6 mg lium/g of food) for durtions rnging from 7 to 3 dys. Although previous studies hve reported very smll ut signifint ltertions in totl plsm lium (Hogstrnd et l., 1995) nd plsm zin (Spry et l., 1988) in fish exposed to 2.3 μmol/l of wterorne zin t similr wter hemistry onditions, no suh hnges were reorded in our study. Furthermore, no signifint modultions were oserved in whole ody zin onentrtions mong the tretments throughout the entire exposure. Both lium nd zin re essentil elements for norml growth in freshwter fish, nd it is likely tht they re under tight homeostti ontrol. The tolerne to ute wterorne zin exposure inresed signifintly in fish hronilly exposed to wterorne zin for 28 dys, inditing the ourrene of physiologil limtion. Hogstrnd et l. (1994) lso reported notle inrese in tolerne to ute wterorne zin exposure (52 μmol/l) in rinow trout following 27 dys of exposure to hroni wterorne zin (2.3 μmol/l) t identil wter hemistry onditions. Interestingly, the fish treted simultneously with hroni wterorne zin nd lium-enrihed diet showed the gretest inrese in tolerne to ute wterorne zin hllenge ( 83 μmol/l). To our knowledge, this is the first report tht demonstrtes the tul protetive effet of dietry C 2+ supplementtion ginst the lethlity of wterorne zin exposure. However, it is to e noted here tht fish treted exlusively with elevted C 2+ diet did not show suh n inrese in LT 5 vlues, inditing the importnt implitions of intertions etween dietry C 2+ nd hroni wterorne zin exposure in induing the ute zin tolerne. The dditionl protetive effet of simultneous exposure to elevted dietry C 2+ nd hroni wterorne zin ginst lethl zin hllenge in omprison to hroni wterorne zin exposure lone my e ttriuted to the enhned ility to regulte internl lium lne in the former group when exposed to ute wterorne zin hllenge. Overll, the results of the present study led to the onlusion tht elevted dietry C 2+ ould protet freshwter fish ginst the hroni toxiity of wterorne zin sine this tretment inhiits the inrese in wterorne Zn 2+ uptke nd umultion indued y hroni wterorne zin exposure. Moreover, elevted dietry C 2+ under hroni wterorne zin exposure genertes mximum tolerne to ute wterorne zin exposure in fish. Thus, in the rel world, freshwter fish ould potentilly enefit y preferentilly forging on liumenrihed prey speies (mollusks nd rustens) in zinontminted quti eosystems. Aknowledgements This work ws supported y the Metls in the Environment Reserh Network (MITE-RN), whih ws funded y the Nturl Sienes nd Engineering Reserh Counil (NSERC) of Cnd. CMW is supported y the Cnd Reserh Chir (CRC) progrm. We thnk Sunit R. Ndell for providing tehnil ssistne in onduting this study. Referenes Alsop, D.H., Wood, C.M., Influene of wterorne tions on zin uptke nd toxiity in rinow trout, Onorhynhus mykiss. Cn. J. Fish. Aqut. Si. 56, Alsop, D.H., MGeer, J.C., MDonld, D.G., Wood, C.M., The osts of hroni wterorne zin exposure nd the onsequenes of zin limtion on the gill/zin intertions of rinow trout in hrd nd soft wter. Environ. Toxiol. Chem. 18, Bldisserotto, B., Kmunde, C., Mtsuo, A., Wood, C.M., 24. A protetive effet of dietry lium ginst ute wterorne dmium uptke in rinow trout. Aqut. Toxiol. 67, Bldisserotto, B., Kmunde, C., Mtsuo, A., Wood, C.M., 24. Aute wterorne dmium uptke in rinow trout is redued y dietry lium ronte. Comp. Biohem. Physiol. 137C, Bldisserotto, B., Chowdhury, M.J., Wood, C.M., 25. Effets of dietry lium nd dmium on dmium umultion, lium nd dmium uptke from the wter, nd their intertions in juvenile rinow trout. Aqut. Toxiol. 72, Brron, M.G., Aleke, S., 2. Clium ontrol of zin uptke in rinow trout. Aqut. Toxiol. 5, Brdley, R.W., Sprgue, J.B., The influene of ph, wter hrdness, nd lklinity on the ute lethlity of zin to rinow trout (Slmo girdneri). Cn. J. Aqut. Si. 42, Everll, N.C., MFrlne, N.A.A., Sedgwik, R.W., The effets of wter hrdness upon the uptke, umultion nd exretion of zin in the rown trout, Slmo trutt L. J. Fish Biol. 35,

9 12 S. Niyogi, C.M. Wood / Comprtive Biohemistry nd Physiology, Prt C 143 (26) Flik, G., Verost, P.M., Wendelr Bong, S.E., Clium trnsport proesses in fishes. In: Wood, C.M., Shuttleworth, T.J. (Eds.), Fish Physiology. Cellulr nd Moleulr Approhes to Fish Ioni Regultion, vol. 14. Ademi Press, Sn Diego, pp Hogstrnd, C., Wilson, R.W., Polgr, D., Wood, C.M., Effets of zin on the kinetis of rnhil uptke in freshwter rinow trout during dpttion to wterorne zin. J. Exp. Biol. 186, Hogstrnd, C., Reid, S.D., Wood, C.M., C 2+ versus Zn 2+ trnsport in the gills of freshwter rinow trout nd the ost of dpttion to wterorne Zn 2+. J. Exp. Biol. 198, Hogstrnd, C., Verost, P.M., Wendelr Bong, S.E., Wood, C.M., Mehnisms of zin uptke in gills of freshwter rinow trout: interply with lium trnsport. Am. J. Physiol. 27, R1141 R1147. Hogstrnd, C., We, N., Wood, C.M., Covrition in regultion of ffinity for rnhil zin nd lium uptke in freshwter rinow trout. J. Exp. Biol. 21, Lithfield Jr., D.J., A method for rpid grphi solution of time perent effet urves. J. Phrmol. Exp. Ther. 97, Niyogi, S., Wood, C.M., 24. Kineti nlyses of wterorne C nd Cd trnsport nd their intertions in the gills of rinow trout (Onorhynhus mykiss) nd yellow perh (Per flvesens), two speies differing gretly in ute wterorne Cd sensitivity. J. Comp. Physiol. 174B, O'Connell, L.P., Gtlin, D.M., Effets of dietry lium nd vitmin D 3 on weight gin nd minerl omposition of the lue tilpi (Oreohromis ureus) in low-lium wter. Aquulture 125, Ogino, C., Tked, H., Requirements of rinow trout for dietry lium nd phosphorus. Bull. Jpn. So. Si. Fish. 42, Rodgers, D.W., Amient ph nd lium onentrtion s the modifiers of growth nd lium dynmis of rook trout, Slvelinus fontinlis. Cn. J. Fish. Aqut. Si. 41, Sherwood, G.D., Rsmussen, J.B., Rown, D.J., Brodeur, J., Montel, A., 2. Bioenergeti ost of hevy metl exposure in yellow perh (Per flvesens): in situ estimtes with rdiotrer tehnique. Cn. J. Fish. Aqut. Si. 57, Spry, D.J., Wood, C.M., Ion flux rtes, id se sttus, nd lood gses in rinow trout, Slmo girdneri, exposed to toxi zin in nturl soft wter. Cn. J. Fish. Aqut. Si. 42, Spry, D.J., Wood, C.M., A kineti method for the mesurement of zin influx in vivo in the rinow trout, nd the effets of wterorne lium on flux rtes. J. Exp. Biol. 142, Spry, D.J., Hodson, P.V., Wood, C.M., Reltive ontriutions of dietry nd wterorne zin in rinow trout, Slmo girdneri. Cn. J. Fish. Aqut. Si. 45, Verost, P.M., Vn Rooij, J., Flik, G., Lok, A.C., Wendelr Bong, S.E., The movement of dmium through freshwter trout rnhil epithelium nd its interferene with lium trnsport. J. Exp. Biol. 145, Zitko, V., Crson, W.G., A mehnism of the effets of wter hrdness on the lethlity of hevy metls to fish. Chemosphere 5, Zohouri, M.A., Pyle, G.G., Wood, C.M., 21. Dietry C inhiits wterorne Cd uptke in Cd-exposed rinow trout, Onorhynhus mykiss. Comp. Biohem. Physiol. 13C,