Dietary Pb Accumulation in Juvenile Freshwater Rainbow Trout (Oncorhynchus mykiss)

Transcription

1 rh. Environ. ontm. Toxiol. 51, (2006) DOI: /s Dietry P umultion in Juvenile Freshwter Rinow Trout (Onorhynhus mykiss) L.. lves, 1. N. Glover, 2. M. Woo 1 1 Deprtment of iology, MMster University, Hmilton, Ontrio, n, L8S 4K1 2 Ntionl Institute of Nutrition n Sefoo Reserh, Postoks 2029 Nornes, 5817 ergen, Norwy Reeive: 18 ugust 2005 /epte: 12 Mrh 2006 strt. Three ifferent iets mene with le (P) nitrte P(NO 3 ) 2 (7, 77, n 520 lg P/g ry weight) n P-free ontrol iet (0.06 lg P/g ry weight) were fe to juvenile freshwter rinow trout for 21 ys. ounting for mesure foo onsumption, the lulte oses per fish were 0.02, 3.7, 39.6, n lg/y, for the ontrol, low, intermeite, n high P tretments, respetively. The ptterns of P umultion over time were etermine in vrious tissues (gills, liver, kiney, intestine, rss), re loo ells (R), n plsm, s well s feeing, growth, hemtologil, n ionoregultory prmeters. P umultion ourre in ose-epenent mnner in ll tissues exept the plsm, where umultion ws miniml. Overll, when fe the highest P iet, the intestine exhiite the gretest P uren (17.8 lg P/g tissue wet weight), with high onentrtions lso foun in the kiney (2.4 lg P/g tissue wet weight) n liver (1.9 lg P/g) t the highest ietry P tretment y y 21. The Rs umulte sustntil mount of P (1.5 lg P/g) when ompre to the plsm (0.012 lg P/g) in the high tretment group. The perentge of P retine in the fish erese with inresing ietry P onentrtions. Growth, survivl, plsm protein, n hemtorit were not signifintly ffete y ietry P. Plsm 2+ levels erese t the eginning of the experiment, wheres Mg 2+ levels erese uring the mile of the experiment in oth the intermeite n high ietry tretments. oth the 2+ n Mg 2+ levels stilize y y 21. rnhil 2+ n N + influx rtes were not ffete y ietry P, exept on y 8 where N + influx rtes were signifintly elevte. The results of this stuy show tht P oes umulte internlly from the iet when present t levels within the rnge reporte in ontminte enthi invertertes in nture. We further ientify the intestine s potentil trget site of hroni toxiity of P vi the iet, n Rs s reservoir of ietry P. Le (P) is non-nutrient metl foun in the erthõs rust, n thus my enter the quti environment through nturl orresponene to: L.. lves; emil: lr.lves@e.g. proesses of geologil wethering n volni emissions (Demyo et l. 1982). The kgroun onentrtion of P in unontminte surfe wter hs een estimte to e 0.02 lg P/L (Flegl et l. 1987). P my lso enter the quti environment through nthropogeni prties suh s the mining, refining, n smelting of P (Sorensen 1991; Worl Helth Orgniztion 1995). In fish, the primry site of ute wterorne P toxiity is t the gills where inhiitory tions of P on 2+,N +, n l ) uptke hve een oumente (Vrnsi n Gmur 1978; Rogers et l. 2003, 2005; Rogers n Woo 2004). Nevertheless, there is eviene for speifi hroni toxi tion of wterorne P on erythroyte funtion in fish. Hoson et l. (1978) foun tht hroni wterorne P exposure (13 lg P/L; 32 weeks) to rinow trout resulte in signifint inreses in re loo ell (R) numers, ereses in R volumes, n -minolevulini i ehyrtse (LD) tivity, n enzyme tht tlyses the formtion of porphoilinogen from minolevulini i in the heme synthesis pthwy. This suggests tht fish expose hronilly to wterorne P my e t risk of nemi. Nevertheless, P toxiity my lso our vi the gstrointestinl trt (Sorensen 1991). respo et l. (1986) showe tht rinow trout tht were orlly ministere with P in the iet (10 lg P/g ry weight (w)/fish/y) h morphologil ltertions of the intestinl rush orer, whih resulte in n impirment of intestinl Nl sorption. Stuies hve emonstrte tht fish fe iets ontining metls (s,, u, P, n Zn) hve reue feeing tivity, growth, n survivl rtes (Frg et l. 1994; Woowr et l. 1994, 1995). Frg et l. (1994) reporte tht utthrot trout (Onorhynhus lrki) fe enthi mroinvertertes ontining high levels of metls (s, Zn,, n P) from ifferent ontminte sites long the oeur Õ lene River, Iho, h higher onentrtion of these metls in the stomh n pylori e when ompre to the gills n kiney. Norml P levels in unontminte enthi invertertes re usully less thn 1 lg/g w. However, oy urens up to 792 lg/g w hve een reporte in enthi invertertes in the South Fork of the oeur Õ lene River (Frg et l. 1994, 1999). While these stuies using nturlly ontminte iets hve isplye eviene of ietry P uptke n toxi effets to fish, the

2 616 L.. lves et l. lortory stuy of Hoson et l. (1978) reporte tht rinow trout fe iets ontminte with up to 118 lg P/g i not tke up ny P into internl tissue. lthough ute wterorne P intertions t the gills hve een well stuie (Dvies et l. 1976; Holome et l. 1976; Hoson et l. 1978, Rogers et l. 2003, 2005; Rogers n Woo 2004), the effets of ietry P on toxiologil n physiologil prmeters hve reeive very little ttention. The initil ojetive of this stuy ws to ssess whether three ifferent P-ontminte iets (7, 77, 520 lg P/g w) fe to juvenile rinow trout for 21 ys resulte in P umultion in speifi internl tissues: liver, kiney intestine, gills, rss, n loo. These onentrtions were speifilly selete to inlue the rnge of oses use y Hoson et l. (1978; lg P/g w) n the onentrtions foun in enthi invertertes (Frg et l. 1994, 1999; lg/g w). If gstrointestinl P uptke ourre, seon ojetive ws to evlute the pttern of tissue-speifi umultion over time, n to look for ny eviene of regultion or epurtion. In light of the known tions of wterorne P on R funtion, thir ojetive ws to test for possile hemtologil normlities in terms of plsm protein n hemtorit. finl ojetive ws to exmine if there were ny isruptions in growth n survivl rtes, plsm 2+ n Mg 2+ regultion, n N + n 2+ influx rtes from the wter, sine mny of these prmeters re ffete y wterorne P. Mterils n Methos Fish Juvenile rinow trout (Onorhynhus mykiss) (N = 368) weighing 3 6 g were otine from Humer Springs Trout Hthery (Orngeville, Ontrio). Upon rrivl t MMster University, Hmilton, Ontrio, fish were rnomly selete n ple in eight 200-L flow-through, erte holing tnks (46 fish per tnk). Eh tnk ws supplie with 0.8 L/min of ehlorinte Hmilton wter (in mm: N mm; l ) 0.7; ; Mg ; K , wter hrness in the form of O 3 = 140 ppm) n llowe to limte to mient onitions (11 13 n ph ) for t lest 7 weeks prior to the strt of the experiment. During the limtion perio no eths ourre. Fish were fe ommeril slmon fry pellets (NelsonÕs Silver up fish fee; Murry, Uth, US; 52% rue protein [min.]; 14% rue ft [min.]; 3% rue fier [mx.]; 12% sh [mx.]; n 1% soium [tul]) one ily to stition from the thir y of rrivl until the strt of the experiment. y this time, fish weighe out g. Eh tnk ws ssigne to one of four tretment groups, with eh tretment replite. The four tretment groups inlue nominlly P-free iet (ontrol), low P iet (nominlly 10 lg P/g w), n intermeite iet (nominlly 100 lg P/g w), n high P iet (nominlly 500 lg P/g w). The tul mesure men onentrtions were , , , n lg P/g w. Two weeks into the limtion perio, eight fish were rnomly selete from eh of the eight tnks n given pssive integrtive trnsponer (PIT) tg, ple into the peritonel vity uner MS-222 (3-minoenzoi i ethylester; 0.5 g/l) nesthesi. This ws one to estlish speifi growth rtes over the 21-y experiment. Diet Preprtion P-enrihe iets were me y ing le nitrte (P(NO 3 ) 2 ) into the sme ommeril slmon fry foo (Silver up Fee, Murry, Uth, US) s use uring the limtion perio. The ommeril foo ws pulverize into fine power using househol lener for pproximtely 2 minutes. Then, 500 g of the fine power ws hyrte with 40% v/w of oule istille wter (NNOpure II; Syron/rnste, oston, M, US) ontining ifferent proportions of issolve P(NO 3 ) 2 n lene in ommeril pst mker for 1.5 h in orer to hieve the intene oses of 10, 100, n 500 lg P/ g w foo. The pste ws then psse through utter where smll strns were roken into smll pellets. The ontrol iet ws prepre the sme wy exept P ws not e. The foo pellets were first irrie for 48 h, then rie in 60 oven to onstnt weight, n frozen until further use. The mesure onentrtion of P (Tle 1) ws etermine y het-igesting the foo pellets in five volumes of 1N HNO 3 t 60 for 48 h. The superntnt ws then ilute with 1% HNO 3 n 0.5% mmonium phosphte n mesure on grphite furne tomi sorption spetrophotometer (S; 220 Spetr; Vrin GT-110; Vrin, ustrli) with etetion limit of 0.06 lg P/L ginst ertifie multi-element P stnr (nhemi In., Quee), employing pproprite lnk smples n referene smples. Moisture ws mesure to e 6% y rying foo to onstnt weight in 60 oven. Feeing, Men Weight, Growth, n Foo onsumption totl of four replite tretments (0, 7, 77, n 520 lg P/g w, Tle 1) were teste in this stuy. On the thir y of the experiment, mehnil filure (low ertion) resulte in n ltertion in the exposure onitions in one of the replite 77-lg P/g w iet tretment tnks. This tnk ws eliminte from this experiment n thus the n vlue for this tretment ws hlf tht of ll others. Eh group ws fe one ily to stition, y pling pellets on the surfe of the wter every minute until the fish were no longer striking t the pellets. The pellets not eten on the surfe wter were remove n the ottom of eh tnk ws siphone pproximtely one hour post feeing to ontrol ny lehing of P from the foo n fees. The mount of ll foo onsume y the fish ws reore in orer to etermine the voluntry rtion. Unfiltere wter smples (10 ml) were tken ily efore n fter siphoning in orer to etermine the extent of ny wterorne P ontmintion. Wter ws iifie for metl nlysis. Men fish weights were lulte every week y ulk weighing ll fish from eh holing tnk. The men fish weight ws etermine y iviing the numer of fish in eh tnk y the totl iomss. Fish tht were tgge were iniviully weighe n their fork lengths were mesure. The onition ftor for oth tgge n ulkweighe fish ws etermine using the formul F ¼ ðw=l 3 Þ*100 ð1þ where W is the weight in grms n L is the fork length in entimeters. Speifi growth rtes (SGR) expresse on %/y sis (Riker 1979) were etermine using the lest-squres liner regression (SigmStt, version 3.0) through the nturl logrithm of the iniviul tgge fish weights versus time. The rtion of foo onsumption for ulk-weighe fish t stition (% oy wt/y) ws etermine using the formul r ¼ ðy/n*w)*100 ð2þ

3 Dietry P umultion in O. mykiss 617 Tle 1. Totl wterorne P onentrtions (lg/l) for ifferent tnk iets throughout the 3-week experiment Week ontrol (0 lg P/g) Low (7 lg P/g) Intermeite (77 lg P/g) High (520 lg P/g) Dt re represente s men 1 SEM, n = 28, for ll tretments, exept the 77-lg P/g ry weight tretment, n = 4. Vlues shring lowerse letters re not signifintly ifferent from other tretment vlues within the sme week (p < 0.05). where r is the rtion (% oy wt/y), y is the totl foo in grms fe to fish in eh tnk, n is the numer of fish in eh tnk, n w is the verge men fish wet weight in grms. Foo onversion effiieny (FE) (in %) for eh tretment ws expresse using the formul FE ¼ðSGR ve =rþ100 ð3þ where SGR ve is the men speifi growth rte (%/y), n r is the rtion (% oy wt/y). Tissue Smpling t y 0 two fish from eh tnk, n on ys 7, 14, n 21 six rnomly selete fish per tnk, were srifie with 1.0 g/l MS 222. Tgge fish were not smple. loo ws tken y ul punture using n ie-hille 250-lL gs tight Hmilton syringe pre-rinse with lithium heprin (50 i.u./ml). Some of the whole loo ws use for mesurement of hemtorit y pillry tue entrifugtion t 13,700 g for 2 min. The hemtorit ws re iretly from the tue, whih ws then roken in orer to extrt plsm for totl plsm protein (g/100 ml) y refrtometry (merin Optil, ufflo, NY) (lexner n Ingrm 1980). The rest of the whole loo ws entrifuge t 10,000 g for 2 min n plsm n Rs ollete, snpfrozen in liqui nitrogen, n store t )70 until further nlysis for plsm 2+,Mg 2+, totl plsm P, n totl R P onentrtions. The liver, kiney, intestine, n gill skets were issete, rinse, lotte, weighe, n kept t 4 until nlysis for P ontent. The gills were rinse with oule-istille wter n the intestinl trt ws flushe out with 0.9% Nl to remove ny soure of non-tissue P. The pylori ee were remove from the nterior intestine n ple with the rss, whih lso inlue the similrly flushe stomh. The presene of foo resiue ws note in the pylori ee. Plsm 2+ n Mg 2+ onentrtions were etermine y flme tomi sorption spetrophotometry (S; Vrin 220FS, ustrli). Smples were prepre y iluting the plsm with 0.5% Ll 3 n ssying it ginst similrly ilute known stnrs. P in plsm n wter smples were etermine in the sme mnner s outline for the nlysis of P in foo. Weighe liver, kiney, intestine, gill, rss, n R smples were igeste in 5 volumes of 1 N HNO 3 in 60 oven for 48 h. fter 48 h, these smples were entrifuge t 10,000 g for 20 min n the superntnt ws ppropritely ilute n mesure y grphite furne S for P umultion, s outline erlier for nlysis of P in foo. N + n 2+ Influx Rte On y 0 two rnomly selete fish from eh tnk, n on ys 8 n 22 six rnomly selete fish from eh tnk, were use to mesure the uniiretionl N + n 2+ influx rtes from the wter. t time 0, 0.1 li of 22 N n 10 li of 45 were e to 3-L flux hmers ontining 2 L of ehlorinte Hmilton tp wter, n llowe to mix for 10 min. For the entire flux perio (4 h), the flux hmers were prtilly sumerge on wet tle reeiving onstnt wter flow in orer to mintin temperture t fter 10-min isotope equilirtion perio, fish were e to the iniviul flux hmers. Two 5-mL wter smples were remove t 10 min, 2 h, n 4 h for etermintion of initil, mi, n finl ion onentrtion n riotivity mesurements. fter 4 h, fish were srifie y ing n overose of MS 222 to the flux hmers. Fish were immeitely remove n ple in plsti g ontining 200 ml of 10 mm Nl plus 10 mm l 2 Æ2H 2 O for 10 min, s ol isplement to remove ny loosely surfe-oun 22 N n 45. The fish were lotte ry, weighe, n ple in plsti vils to ount for 22 N on -ounter (nerr- Pkr 5000 Minxi). The -ounter i not etet 45 riotivity. Smples were prepre for sintilltion ounting of 45 using similr protool to Frnklin et l. (2005). In short, fish were iigeste in the sme mnner s for etermintion of the P tissue uren. The i-igeste smples were entrifuge t 5525g for 10 min (Sigm 4K15 Refrigerte entrifuge); 2.5 ml of the superntnt ws ilute with 10 ml of Ultim Gol oktil (Pkr iosiene, US). For wter smples, 5 ml of queous ounting sintilltion fluor (S, mershm, UK) ws e to 2 ml of wter. The proesse tissue n wter were ple in rk room overnight to reue hemiluminesene, then ounte on liqui sintilltion ounter (1217 Rket Liqui Sintilltion ounter, LK Wll, Finln). Sine 22 N is / emitter, the sintilltion ounter etete oth 45 n 22 N riotivity, so it ws neessry to istinguish the riotivity ue to 45 lone ( emitter) using the protool of Vn Ginneken n lust (1995). The 45 vlues from tissue igests were then quenh-orrete to the sme ounting effiieny s 45 foun in wter smples y the metho of externl stnr rtios (ESR), using 45 quenh urve tht ws generte from rss tissue of vrying weights, proesse the sme s ove n ounte in the sme oktil. The uniiretionl N + n 2+ influxes (lmol/kg/h) were lulte using the formul J in ¼ Fish PM/(MS.W.t.) ð4þ where MS is the men speifi tivity (PM/lmol) of wter in the ounter for 22 N or in the sintilltion ounter for 45, W is the weight of the fish (kg), t is the time in hours, n Fish PM is the totl ounts per minute of 22 N or 45 umulte y the fish uring the flux perio. Sttistis ll sttistil tests were performe using the omputer softwre Sigmstt (3.0.) or SYSTT 10. Dt were teste for homogeneity of vrines mong groups using the rtlett test. Those tht file were sujete to nturl logrithm or squre root trnsformtions to otin homogeneity mong groups. Dt tht oul not e normlize were sujete to non-prmetri nlysis, using the Kruskl-Wllis proeure, where ll rnks were orrete for ties. omprisons etween

4 618 L.. lves et l. Tle 2. Speifi growth rtes (SGR), voluntry rtions, foo onversion effiieny (FE), n onition ftors (F) of juvenile rinow trout fe to stition with iets ontining ifferent onentrtions of P Week 0 lg P/g 7 lg P/g 77 lg P/g 520 lg P/g SGR (%/y) Rtion (% oy wt/y) F FE (%) SGR n Rtions re men SEM. SGR ws lulte using the liner regression (SigmStt version 3) of the nturl logrithm of iniviul tgge fish weight versus time. The estimtes for eh fish were then verge. onition ftor (F) for ulk-weighe fish ws mesure using the formul F = (W/L 3 ) 100. FE ws lulte y the formul (SGR/verge rtion uring the week) multiplie y 100. Vlues shring lowerse letters re not signifintly ifferent from other tretment vlues within the sme week (p < 0.05). tretments n ys were one using two-wy nlysis of vrine (NOV), followe y Tukey (prmetri nlysis) or DunnÕs test (non-prmetri nlysis), s pproprite,. Neste t within the replite tnks within the sme tretments n etween ys were not signifintly ifferent, using the generl liner moel estimte in SYSTT 10. Results Wterorne P vlues (Tle 1) were not signifintly ove kgroun in the low (7 lg P/g) n intermeite (77 lg P/g) ietry P exposures, ut were signifintly elevte to out 10 lg/ L in the high (520 lg P/g) ietry P exposure, presumly ue to the lehing from the iet n/or fees. This suggests tht P umultion for the 520-lg P/g tretment my not e solely se on ietry P exposure, ut omintion of ietorne n wterorne P. No mortlity ws ssoite with the ietry P tretments throughout the ourse of the experiment. Speifi growth rtes, using tgge fish over 21 ys n voluntry rtions onsume eh week from ulk-weighe fish i not iffer mong tretment groups (Tle 2). Totl foo eten over 21 ys lso i not iffer etween tretments. onition ftors were extremely high n fish in ll tnks were onsiere overweight oth t the strt n en of the experiment (Tle 2). The foo onversion effiieny, lulte se on men SGR vlues of tgge fish, i not iffer onsistently mong tretments (Tle 2). t ll ietry P oses, P umultion ws seen in the gills (Figure 1), intestine (Figure 2), liver (Figure 3), kiney (Figure 4), n rss (Figure 5) uring the ourse of the 21-y experiment. t y 21, the orer of P onentrtion in speifi tissues ws intestine > rss > kiney > liver > gills. The intestine showe sustntil umultion with uren of 17.8 lg P/g tissue wet weight in the 520-lg P/g exposure, whih ws 445 greter thn the ontrol (0.04 lg P/g) on y 21. The intestine (17.8 lg P/g tissue wet weight), rss (2.7 lg P/g tissue wet weight), kiney (2.4 lg P/g tissue wet weight), n the liver (1.9 lg P/g tissue wet weight) ll exhiite their highest P urens on y 21 ompre to y 0, 7, n 14. In ontrst, the gills, t lest t the highest ose, h the gretest P umultion on y 7 (8.0 lg P/g tissue wet weight) n muh lower P uren (2.2 lg P/g tissue wet weight) y y 21. P uren in the intestine (Figure 2) inrese with time in ll ietry P tretments. When the perentge istriution of P uren in the whole fish is onsiere (Figure 8), the rss umulte out 80%, the intestine out 10%, while the gills, kiney, n liver me up the remining 10% of the P uren. These vlues re not surprising sine the rss mkes up 85 90% of the weight of the fish n it lso ontine the stomh n the pylori e; the ltter ontine some foo resiue. Notly, the iggest hnge from the % P istriution in the nonexpose fish ws muh lrger % ontriution from the intestine in ll ietry P tretments. The Rs umulte sustntil onentrtion of P y y 21 (Figure 6), with lmost no P present in the plsm (Tle 3). Inee, only t y 21 t the highest ose level ws the plsm P signifintly elevte. R P inrese from kgroun onentrtions of 0.05 lg P/g to out 1.5 lg P/g in the highest ose level y y 21, out 105 times more thn tht of the plsm. There were only few signifint effets of ietry P exposure on plsm 2+ n Mg 2+ regultion. Plsm Mg 2+ levels fell signifintly in the intermeite n high P tretments on y 14, ut h stilize y y 21 (Tle 3). similr pttern ws seen with the plsm 2+ of fish expose to the intermeite n high iets, where there ws signifint erese in levels on y 7 when ompre to the ontrol. The 2+ levels h reovere y y 14, n therefter remine stle. The hemtorit (30 40%) n the plsm protein ( g/100 ml) remine high throughout the experiment n were not ffete y the ietry P exposure. 2+ influx rtes from the wter were not signifintly ffete in juvenile rinow trout n remine stle throughout the experiment (Tle 4). However, on y 8, N + influx rtes were signifintly elevte in ll tretments, n this ourre to the gretest extent in the high P iet tretment (Tle 4). These effets h isppere y y 22.

5 Dietry P umultion in O. mykiss 619 Totl Gill P umultion(µg/g tissue wet weight) µg P/g 7 µg P/g 77 µg P/g 520 µg P/g Dy Fig. 1. Le umultion in the gills of juvenile rinow trout expose to ifferent levels of P in the iet over 21 ys. P tissue uren is expresse in lg g/g wet tissue weight. Dt represente s men 1 SEM, n = 12, exept the 77-lg P/g tretment, n = 6. Vlues shring lowerse letters re not signifintly ifferent (p < 0.05) etween tretment mens within y n vlues shring upperse letters re not signifintly ifferent etween ys within tretment mens using two-wy NOV with Tukey multiple omprison, or Kruskl-Wllis NOV on rnks with DunnÕs multiple omprison test Totl Intestine P umultion (µg/g tissue wet weight) µg P/g 7 µg P/g 77 µg P/g 520 µg P/g Dy D Fig. 2. Le umultion in the intestine of juvenile rinow trout expose to ifferent levels of P in the iet over 21 ys. The intestine oes not inlue the pylori e. Other etils s in legen of Figure 1 Disussion Dietry P onentrtions were hosen to mimi environmentlly relevnt onentrtions in terms of those foun in enthi invertertes t oth ontminte n unontminte sites in the environment (0 792 lg P/g w; Woowr et l. 1994; Frg et l. 1999), s well s to ut ross the sme rnge (4 118 lg P/g w) s use y Hoson et l. (1978) in n erlier ietry stuy. Dietry exposure to P t these mesure onentrtions ( 0.06, 7, 77, n 520 lg P/g) h few pprent verse effets on rinow trout uring the 21-y experiment. There were no mortlities n no signifint ifferenes were oserve in speifi growth rtes n rtions mong fish fe either the ontrol iet or the vrious P iets. These results re onsistent with Mount et l. (1994) who reporte no effets on survivl n growth of rinow trout fe ietry onentrtion s high s 170 lg P/g. In the present stuy, totl wterorne P ws signifintly elevte ove kgroun levels in the high ietry tretment tnks (from 1.5 to 10 lg P/L; Tle 2). Therefore, it is possile tht the P umultion in the tissues, espeilly in the gill, ws ttriutle to the omintion of oth ietry n wterorne P exposure in this tretment group only. The primry site of ute wterorne P uptke in fish is vi the gills (Vrnsi n Gmur 1978). Rinow trout expose to hroni wterorne P (13 lg P/L for 32 weeks) lso showe elevte gill P onentrtions (pproximtely 2.5 lg P/g) reltive to other tissues suh s the kiney n liver (Hoson et l. 1978). The gills in the present stuy i umulte P (to out 2.1 lg P/g on y 21), minly in the high ietry P tretment. Wterorne P levels (Tle 2) in the low n intermeite ietry tretments were similr to kgroun levels. This suggests ny umultion tht ws oserve in the gills n in the other tissues of fish in the low n

6 620 L.. lves et l. Totl Liver P umultion (µg/g tissue wet weight) µg P/g 7 µg P/g 77 µg P/g 520 µg P/g Dy Fig. 3. Le umultion in the liver of juvenile rinow trout expose to ifferent levels of P in the iet for 21 ys. Other etils s in legen of Figure 1 Totl Kiney P umultion (µg/g tissue wet weight) µg P/g 7 µg P/g 77 µg P/g 520 µg P/g D D Dy D Fig. 4. Le umultion in the kiney of juvenile rinow trout expose to ifferent levels of P in the iet for 21 ys. Other etils s in legen of Figure 1 intermeite tretments ws the result of ietry P, n not wterorne P. Notly, in the present stuy, the gill P uren sustntilly roppe from 8.0 lg P/g tissue wet on y 7 to 2.1 lg P/g tissue wet weight on y 21 (Figure 1), espite reltively onstnt wterorne P level (Tle 3). This suggests tht the gill P uren my hve een exrete, or reistriute to other tissues. P urens in the low ietry tretment in ll the tissues were lose to kgroun ontrol levels, whih suggests tht ietry onentrtion >7 lg P/g is neee to see n effet on P uren in internl tissues. Tissue umultion t (Figures 1 5) show tht the intestine h the gretest P uren mong ll the tissues nlyze. This is onsistent with Frg et l. (1994) who foun tht ult rinow trout fe metl-ontminte (s,, u, P, n Zn) enthi invertertes from the lrk Fork River, Montn, exhiite sustntil metl umultion in the gut tissues, with the highest onentrtion in the pylori e n stomh. In the present stuy, the stomh n pylori e were not nlyze iniviully, ut groupe with the rss. The presene of these tissues in the rss my explin the high proportion of le ssoite with this tissue, espeilly sine there ws some presene of foo resiue in the pylori e. We hypothesize tht just s the gills re the primry site of exposure n the site of wterorne P toxiity (Rogers et l., 2003, 2005, Rogers n Woo 2004), similr sitution my exist in terms of ietry P toxiity t the intestine. Longer-term ietry P stuies, ouple with physiologil n histologil mesurements of potentil sulethl impts, will e require to onfirm or refute this hypothesis. The high P uren in the intestine in this stuy my e ue to ining of P from the iet y muus whose seretion my e stimulte y ietry metls (Glover n Hogstrn 2002).

7 Dietry P umultion in O. mykiss 621 Totl rss P umultion (µg/g tissue wet weight) µg P/g 7 µg P/g 77 µg P/g 520 µg P/g Dy D Fig. 5. Le umultion in the rss of juvenile rinow trout expose to ifferent levels of P in the iet for 21 ys. Other etils s in legen of Figure 1 Totl P R umultion(µg/g) µg P/g 7 µg P/g 77 µg P/g 520 µg P/g Dy Fig. 6. Le umultion in the re loo ells of juvenile rinow trout expose to ifferent levels of P in the iet for 21 ys. Other etils s in legen of Figure 1 This muus lyer n t to sequester high levels of metls, n thus prevent the exposure of the unerlying epithelil tissue to potentilly toxi metl levels. Suh senrio hs previously een oserve in fish expose to intestinlly-perfuse zin (Glover n Hogstrn 2002). The trppe metl uren my susequently e sloughe off s result of movement of foo through the intestine. s suh, the high levels of P ssoite with the intestine, t lest in prt, my represent P tht is sore, ut not sore. The present results emonstrte tht ietry P n ross the intestinl epithelium n signifintly umulte in internl soft tissues suh s the liver (Figure 3), kiney (Figure 4), n Rs (Figure 6). When oho slmon (Onorhynhus kisuth) were expose to 150 lg P/L in se wter for 15 ys, twie s muh P ws foun in the posterior kiney (1.8 lg P/g) thn in the nterior kiney (0.5 lg P/g) (Reihert et l. 1979). These finings of signifint renl P umultion were onfirme y the present stuy, where kiney P uren t on y 21 in the high ietry P tretment ws 2.4 lg P/g tissue wet weight (Figure 4). Reihert et l. (1979) suggeste tht the high renl P onentrtion my e ssoite with the exretory/ionoregultory funtion of the posterior kiney in fish (Smith n ell 1976; Reihert et l. 1979). In prtiulr, sine 2+ n P re elieve to e ntgonists of one nother, these ions my e ompeting for trnsport sites t renl tuule ells. This oul result in P eoming trppe in the tuule ells n result in onsierle P tissue uren in this tissue. The lower P umultion of liver (Figure 3), when ompre to the intestine, kiney, n gills, suggests tht P is eposite preferentilly into internl soft tissues other thn the liver. The low levels of P in the liver my e expline y the ft tht P, in ontrst to mny other metls, is unle to inue the proution of the metl-ining protein metllothionein (MT) in the liver (Reihert et l. 1979; mpn et l. 2003). itionlly, s mentione ove, P my hieve uptke y pssge through 2+ trnsporters, n thus the presene n quntity of 2+ uptke pthwys my etermine tissue-speifi P istriution.

8 622 L.. lves et l. Tle 3. Plsm protein, hemtorit (%), n plsm 2+,Mg 2+, n P onentrtions over 21 ys in rinow trout fe ifferent onentrtions of P in the iet Dy Tretment (lg P/g ry wt.) Hemtorit (%) Plsm Protein (g/100 ml) Plsm 2+ (lm) Plsm Mg 2+ (lm) Plsm P (lg/l) l l Vlues shring lowerse letters re not signifintly ifferent from other tretment vlues within the sme week (p < 0.05). Vlues shring upperse letters re not signifintly ifferent from other tretment vlues etween ys. Tle 4. N + n 2+ influx rtes throughout the ourse of the experiment Dy Tretment (lg P/g) (lmol/kg h) N (lmol/kg/h) Vlues shring lowerse letters re not signifintly ifferent from other tretment vlues within the sme week (p < 0.05). When net P retention from the iet over 21 ys in the whole fish ws lulte, the low P iet h the highest net P retention (4.8%) followe y the high P exposure (1.1%) n the intermeite P exposure (0.8%). Therefore, the higher the ietry P onentrtion, the lower the proportion of ietry P retine, suggesting tht P levels my e regulte in terms of reue P sorption n/or n inrese P exretion. These umultion results ontrst with the stuy of Hoson et l. (1978), who foun tht ietry P (up to 118 lg P/g for 32 weeks) ws not tken up y juvenile rinow trout, n i not ffet the fish t ll. The ifferene my e tht the P(NO 3 ) 2 in the Hoson et l. (1978) stuy ws e to ommeril trout how omine with eef liver protein. It my e tht the eef liver, whih is high in iron (Fe), my hve reue P iovilility. Eviene in mmmls suggests tht Fe efiieny enhnes ietry P uptke, proly vi reue ompetition for uptke vi the ivlent metl ion trnsporter (ressler et l. 2004). This woul explin why Hoson et l. (1978) h signifint erese in loo iron in fish expose to ietry P n not wterorne P. In mmmls, uner stey stte onitions, out 96% of P in the whole loo is in the Rs (Worl Helth Orgniztion 1995). In this stuy, 99% of P ws in the Rs on y 21 in fish fe the 520-lg P/g iet. Given tht there ws n inrese in R P (Figure 6), portion of P umultion in the vrious tissues my e funtion of vsulriztion. Figure 7 plots the umultion of P in eh tissue (gill, intestine, liver, n kiney) tht my e expline y ounting for the mount of P present in trppe Rs in the tissue, using estimtes of 51 r R spes from Olson (1992). In ll three P iet tretments, out 9 14% of the P umulte in the kiney is estimte to e the result of trppe Rs in this tissue, wheres 86 91% of P umultion in the kiney is truly in the renl tissue. omprle figures for the liver n gill were only 1 3% in the trppe Rs. In the intestine, 0.3% of P umulte is the result of vsulriztion in terms of trppe R, while the rest hs een uilt up in the intestinl tissues. These t ouple with the emonstrte pity of the fish to tke up P suggests tht more stuies nee to e one in orer to etermine if exposure to ietry P over longer perio of time hs toxi tion in fish. 2+, tightly regulte ion in freshwter fish, is ontinuously sore from the wter vi the gills (Flik n Verost 1993). signifint erese in plsm 2+ levels in the intermeite n high P iets ourre on y 7 (Tle 3). However, 2+ levels reovere therefter. Notly, this reovery ourre without ny hnge in 2+ influx rtes from the wter (Tle 4). Stuies hve shown erese plsm 2+ levels in rinow trout expose to wterorne P, initing the presene of 2+ /P intertion t the gills (Rogers et l. 2003, 2005; Rogers n Woo 2004). The signifint erese in plsm 2+ levels in this stuy my e the result of 2+ /P intertion t the intestine (see isussion elow), whih in ition to the gill my t s n importnt 2+ uptke route (Flik n Verost 1993). In ition, the intermeite n high ietry P tretment groups exhiite signifint erese in Mg 2+ levels on y 14 with stiliztion therefter. This is not surprising sine most Mg 2+ normlly omes from the iet in fish (ijvels et l. 1998), n the presene of metls suh s P will potentilly erese oth Mg 2+ n 2+ sorption t the intestine. lthough there ws n inrese in P umultion in the liver, the orgn where the mjority of plsm proteins re forme, there were no signifint ifferenes in plsm protein onentrtions mong the tretments. The mjority of P ws oun to the Rs (Figure 6), ut there ws smll ut signifint inrese y y 21 in the plsm P levels in trout on the high P iet (Tle 4). Possily the Rs my hve

9 Dietry P umultion in O. mykiss % of umulte Tissue P within Rs µg P/g 77 µg P/g 520 µg P/g Kiney Gill Liver Intestine Fig. 7. Perentge of umulte P uren expline y P within the trppe Rs on y 21. The intestine oes not inlue the pylori e 100 % P Distriution Liver Kiney Gill Intestine rss Dietry P Level (µg/g ry weight) Fig. 8. Perent istriution of totl P uren on y 21 in ifferent tissues of juvenile rinow trout fe ifferent P iets eome sturte with P. Mnton n ook (1984) showe tht humn Rs hve the pity to fully in P, t loo P levels up to out 2.4 lmol P/L (0.5 lg P/g). However, when loo P onentrtions re ove this level there is n inrese in serum P levels. The t in the present stuy suggest tht the Rs in trout hve the pity to in out 4.8 lmol P/L (1.0 lgp/g) (Figure 6), s there ws signifint inrese in plsm P eyon this level (fter 21 ys in trout fe the high ietry P tretment). The ility of trout to potentilly in more P in the Rs, my e relte to the high oxygen rrying pity (higher hemtorit), n longer life spn of the erythroytes (150 ys), when ompre to humns (ushnell et l. 1985; Fnge 1992). Hemtorit ws not ffete in this stuy in ontrst to the wterorne P stuy of Hoson et l. (1978). This suggests tht the uner the onitions teste in the urrent experiment, nemi ws not inue. In future stuies, it will e of interest to iretly ssess possile ietry P effets on LD, sine wterorne P hs een shown to effet LD tivity (Hoson et l. 1978). The physiologil mehnism of ute wterorne P toxiity hs een hrterize s n inhiition of 2+ uptke n n inhiition of the N +,K + -TPse n roni nhyrse enzymes t the gill epithelium, whih result in isruptions in 2+ homeostsis n in N + n l ) regultion (Rogers et l. 2003, 2005; Rogers n Woo 2004). In ontrst, ietry P i not inhiit 2+ or N + influx rtes. Rogers et l. (2003) foun tht 2+ n N + influx rtes of juvenile rinow trout were inhiite y 65% n 50%, respetively, uring wterorne P exposures of 890 to 1200 lg/l. The gills umulte lose to 200 lg P/g t 96 h L50 wterorne P onentrtions of 1004 lg/l in the sme stuy. se on the ove stuies, it seems likely tht 2+ or N + influx rtes woul not e expete to hnge uring the present ietry exposure, sine the gill P uren ws only out 2.1 lg/g t y 21 in the highest ose tretment.

10 624 L.. lves et l. Nevertheless, wterorne P stuies showing mjor impts on 2+ n N + influx rtes t the gill suggest tht future experiments tht involve ietry P exposure in fish shoul onsier mesuring 2+ n N + influx rtes t the intestine, s well s intestinl N +, K + TPse tivity, sine the intestine in this stuy h the gretest P uren, whih oul potentilly interfere with 2+ n N + uptke t the intestine. In summry, the present stuy provies eviene tht ietry P, t levels representtive of those in nturlly ontminte iets, oes umulte in the internl tissues of rinow trout. Nevertheless, physiologil isturnes were miniml, n feeing, growth, n foo onversion effiieny were reltively unffete over the 21 ys of the experiment. Effets of this level of umultion over longer timefrme, n the possiility of neurologil n reproutive effets hve not een evlute. This present stuy hs provie sis to explore hroni ietry P effets on memrne trnsport in the intestine, n, given the high umultion in Rs, its potentil effet on the heme synthesis pthwy in future stuies. knowlegments. This work ws supporte y the Nturl Sienes n Engineering Reserh ounil of n RD Progrm, the Interntionl Le Zin Reserh Orgniztion, the Nikel Prouers Environmentl Reserh ssoition, the Interntionl opper ssoition, the opper Development ssoition, Tek-omino, Norn-Flonrige, n Ino. We thnk Dr. Peter hpmn (Goler ssoites) n Dr. Wlter Kuit (Tek-omino) for helpful omments on the mnusript, n two nonymous reviewers for onstrutive ritiisms..m.w. is supporte y the n Reserh hir Progrm. Referenes lexner J, Ingrm G (1980) omprison of five of the methos ommonly use to mesure protein onentrtion in fish ser. J Fish iol 16: ijvels MJ, Vn er Velen J, Kolr ZI, Flik G (1998) Mgnesium trnsport in freshwter teleosts. J Exp iol 201: ressler JP, Olivi L, heong JH, Kim Y, nnon D (2004) Divlent metl trnsporter 1 in le n mium trnsport. nn NY Si 1012: ushnell PG, Nikinm M, Oikri (1985) Metoli effets of ehyroieti i on rinow trout erythroytes. omp iohem Physiol 81: mpn O, Srsquete, lso J (2003) Effet of le on L-D tivity, metllothionein levels, n lipi peroxition in loo, kiney, n liver of the tofish Hlotrhus itylus. Eotox Environ Sfety 55: respo S, Nonnotte G, olin D, Lery L, Nonnotte L, uree (1986) Morphologil n funtionl ltertions inue in trout intestine y ietry mium n le. J Fish iol 28:69 80 Dvies PH, Goettl JR Jr, Sinley JR, Smith NF (1976) ute n hroni toxiity of le to rinow trout, Slmo girneri, in hr n soft wter. Wter Res 10: Demyo, Tylor M, Tylor KW, Hoson PV (1982) Toxi effets of le n le ompouns on humn helth, quti life, willife, plnts, n livestok. R rit Rev Environ ont 12: Fnge R (1992) Fish loo ells. In: Hor WS, Rnll DJ, Frrell P, (es) Fish Physiology, Vol. XII, prt : The riovsulr system. emi Press, Sn Diego, pp 1 54 Frg M, Woowr DF, rumugh W, Golstein JN, Monnell E, Hogstrn, rrows FT (1999) Dietry effets of metlsontminte invertertes from the oeur Õ lene River, Iho on utthrot trout. Trns m Fish So 128: Frg M, oese J, Woowr DF, ergmn HL (1994) Physiologil hnges n metl tissue umultion in rinow trout expose to fooorne n wterorne metls. Environ Toxiol hem 13: Flik G, Verost PM (1993) lium trnsport in fish gills n intestine. J Exp iol 184:17 29 Flegl R, Rosmn KJK, Stephenson MD (1987) Isotope systemtis of ontminnt les in Monterey y. Environ Si Tehnol 21: Frnklin NM, Glover N, Niol J, Woo M (2005) lium/ mium intertions t uptke surfes in rinow trout: wterorne versus ietry routes of exposure. Environ Toxiol hem 24: Glover N, Hogstrn (2002) In vivo hrteristion of intestinl zin uptke in freshwter rinow trout. J Exp iol 205: Hoson PV, lunt R, Spry DJ (1978) hroni toxiity of wterorne n ietry le to rinow trout (Slmo girneri) in Lke Ontrio wter. Wter Res 12: Holome GW, enoit D, Leonr EN, MKim JM (1976) Long term effets of le exposure on three genertions of rook trout (Slvelinus fontinlis). J Fish Res n 33: Mnton WI, ook JD (1984) High ury (stle isotope ilution) mesurements of le in serum n ererospinl flui. r J In Me 41: Mount DR, rth K, Grrison TD, rten K, Hokett JR (1994) Dietry n wterorne exposure of rinow trout (Onorhynhus mykiss) to opper, mium, le n zin using live iet. Environ Toxiol hem 13: Olson KR (1992) loo n extrellulr flui volume regultion: role of the renin-ngiotensin system, kllikrein-kinin system, n tril ntriureti pepties. In: Hor WS, Rnll DJ, Frrell P, (es) Fish Physiology, Vol. XII, prt : The riovsulr system. emi Press, Sn Diego, pp Reihert WL, Feerighi D, Mlins D (1979) Uptke n metolism of le n mium in oho slmon (Onorhynhus kisuth). omp iohem Physiol 63: Riker WE (1979) Growth rtes n moels. In: Hor WS, Rnll DJ, retts, (es) Fish Physiology, Vol. VIII: ioenergetis n growth. emi Press, Flori, pp Rogers JT, Woo M (2004) hrteriztion of rnhil le-lium intertions in freshwter rinow trout (Onorhynhus mykiss). J Fish iol 207: Rogers JT, Rihrs JG, Woo M (2003) Ionoregultory isruption s the ute toxi mehnism for le in rinow trout (Onorhynhus mykiss). qut Tox 64: Rogers JT, Ptel M, Gilmour KM, Woo M (2005) Mehnisms ehin P-inue isruption of N + n l ) lne in the rinow trout (Onorhynhus mykiss). m J Physiol R 289:R Smith LS, ell GR (1976) prtil guie to the ntomy n physiology of Pifi slmon. Fish Mr Serv Mis Spe Pul 27:14 Sorensen EM (1991) Le. In: Sorensen EM (e) Metl poisoning in fish. R Press, o Rton, pp Vn Ginneken L, lust R (1995) Sequentil etermintion of omine / n pure -emitter y gmm n liqui sintilltion ounting: pplition to the trnsport of metl ross fish gills. nl iohem 224:92 99 Vrnsi U, Gmur DJ (1978) Influene of wter-orne n ietry lium on uptke n retention of le y oho slmon (Onorhynhus kisuth). Tox ppl Phrm 46:65 75 Woowr DF, rumugh WG, DeLony J, Little EE, Smith E (1994) Effets on rinow trout fry of metls-ontminte iet

11 Dietry P umultion in O. mykiss 625 of enthi invertertes in the lrk Fork River, Montn. Trns m Fish So 123:51 62 Woowr DF, Frg M, ergmn HL, DeLony J, Little EE, Smith E, rrows FT (1995) Metls-ontminte enthi invertertes in the lrk Fork River, Montn: effets on ge-0 rown trout n rinow trout. n J Fish qut Si 52: Worl Helth Orgniztion (1995) Environmentl Helth riteri 165. Interntionl Progrmme on hemil Sfety. Genev