DEVELOPMENTAL EFFECTS OF BIOACCUMULATED SELENIUM IN EGGS AND LARVAE OF TWO SALMONID SPECIES

Environmentl Toxicology nd Chemistry, Vol. 24, No. 9, pp. 2373 2381, 2005 2005 SETAC Printed in the USA 0730-7268/05 $12.00.00 DEVELOPMENTAL EFFECTS OF BIOACCUMULATED SELENIUM IN EGGS AND LARVAE OF TWO SALMONID SPECIES JODI HOLM, VINCE PALACE,* PAULA SIWIK, GEORGE STERLING, ROBERT EVANS, CHRISTOPHER BARON, JULIETA WERNER, nd KERRY WAUTIER University of Mnitob, Deprtment of Zoology, Winnipeg, Mnitob R3T 2N6, Cnd Deprtment of Fisheries nd Ocens, Freshwter Institute, Winnipeg, Mnitob R3T 2N6, Cnd Albert Sustinble Resource Development, Fisheries Mngement Brnch, Edson, Albert T6B 2X3, Cnd (Received 11 August 2004; Accepted 18 Mrch 2005) Abstrct Elevted concentrtions of Se hve been detected in cold, flowing wter hbitts ner urnium nd col mines in Cnd. Fish from these systems hve concentrtions of Se in their tissues tht exceed toxic effect thresholds tht hve been estblished for wrm-wter fishes. However, the pplicbility of toxic effect thresholds nd guidelines to cold wter, lotic hbitts is mtter of contention in the literture since most cses of Se toxicosis hve been documented in stnding, wrm-wter systems. To exmine the possibility of impired reproduction in wild rinbow trout (Oncorhynchus mykiss) nd brook trout (Slvelinus fontinlis) ner col mining ctivity in the northestern slopes region of Albert, Cnd, spwn from both species were collected from exposure nd reference sites. Gmetes were fertilized in the lbortory, rered to the swim-up stge, nd exmined for deformities. A significnt reltionship ws observed for rinbow trout between the mount of Se in eggs nd the incidence of developmentl bnormlities, specificlly crniofcil defects, skeletl deformities, nd edem. These ssocitions pproximte exponentil functions with probbilities tht 15% of the popultion would be ffected occurring between 8.8 nd 10.5 g Se per grm of wet egg weight, bsed on probit nlysis. These reltionships re similr to those described for centrrchids inhbiting seleniferous wrmwter lke. No such reltionships were estblished for brook trout. Keywords Rinbow trout Brook trout Selenium Toxicity threshold Tertogenesis INTRODUCTION The potentil for selenium (Se) contmintion of qutic ecosystems due to nthropogenic ctivities hs been recognized for over two decdes [1]. However, to dte lmost ll reported cses of Se poisoning in nturl fish popultions hve been obtined from stnding, wrm-wter hbitts. Consequently, the pplicbility of toxic effect thresholds nd guidelines developed from these systems to cold, flowing wter hbitts is mtter of contention in the literture [2 6]. Recently, elevted Se levels hve been detected in cold, flowing wter hbitts downstrem from urnium [7] nd col [5,8,9] mining ctivity in Cnd. Fish from these systems hve concentrtions of Se in their tissues tht exceed toxic effect thresholds tht hve been estblished for wrm-wter fishes [1], but to dte, no effects hve been reported. A study by Kennedy et l. [5] exmined deformities in the lrve of cutthrot trout from cold-wter system with elevted Se. They did not observe ny differences in the rtes of deformities in the progeny of the cutthrot trout tht could be ttributed to Se nd concluded tht the bsence of toxic responses my be the result of n evolved Se tolernce in this popultion. Extrpolting the results from this one study for ssessing risk to other species is tenuous. Moreover, the results of this pper hve been criticized on the bsis of the methodologies employed nd interprettion of the dt [10]. The conclusion tht slmonid fishes re more tolernt to the effects of Se thn their wrm-wter counterprts is of concern becuse of reclmtion strtegy employed nd proposed for further use by col mining compnies in the northestern * To whom correspondence my be ddressed (plcev@dfo-mpo.gc.c). slopes region of Albert, Cnd. Specificlly, col mining compnies in the region hve proposed to develop end pit lkes following mine pit closure. These lkes re creted when surfce pits re llowed to fill with wter once mining is completed. A problem rises becuse the physicl nd geochemicl chrcteristics of pit lkes my potentite the mobility of Se nd its entry into qutic food webs [11]. Although Se concentrtions in some of these end pit lkes nd t other downstrem sites hve been found to exceed wter qulity guidelines in Albert, no ssessment hs been mde of the potentil dverse effects in fish popultions in these systems [12]. The objective of this study ws to determine if exposure to elevted levels of Se results in tertogenesis in the progeny of two slmonid species. s nd fish collection MATERIALS AND METHODS Spwning rinbow trout (Oncorhynchus mykiss) nd brook trout (Slvelinus fontinlis) were collected over three consecutive yers (2000 2002) from the northestern slopes region of Albert ner Jsper, Cnd. In June 2000, rinbow trout were collected from Luscr Creek (higher Se site) nd Deerlick Creek (reference site) nd brook trout in October 2000 from Luscr Creek nd Cold Creek (reference site). Two dditionl sites were dded in 2001 nd 2002: Gregg River (intermedite Se site) nd Wmpus Creek (dditionl reference site for rinbow trout only), both of which re lso in the immedite study re. Selected wter chemistry dt for the 2000 2002 study sites pper in Tble 1. For rinbow trout, collection of spwning dults begn in the spring (My/June) when dily mximum wter tempertures reched 6 C, while for brook trout, 2373

2374 Environ. Toxicol. Chem. 24, 2005 J. Holm et l. Tble 1. Selected wter chemistry prmeters of sites smpled during spring nd fll 2000 2002, Albert, Cnd Wter temperture ( C) % Dissolved Conductivity oxygen ph ( s/cm) Se concentrtion ( g/l) Field sites Luscr Creek Gregg River Deerlick Creek Cold Creek Wmpus Creek 2 6 2 6 2 6 2 7 2 8 93 77 91 97 90 8.3 8 7.5 8 7.3 0.538 0.506 0.116 0.393 0.257 6 32 b 1.4 5.4 b 0.71 c 0.66 c 0.5 c Lbortory Freshwter Institute d 8.0 0.8 e 98 7.8 0.018 1.6 0.1 f Dt obtined using Hydrolb multiprobe (Houston, TX, USA). b Dt obtined from Csey nd Swiik [9]. c Dt from 2002 (V. Plce, unpublished observtion) using inductively coupled mss spectroscopy s described in Feng et l. [13]. d Freshwter Institute Lbortories, Winnipeg, MB R3T 2N6, Cnd. e Except for rinbow trout rised in 2001 where wter temperture ws 5.0 0.6 C. f Dt obtined using Hydride genertion with tomic fluorescence detection (PSA Anlyticl, Kent, UK). collections begn in the fll (October) when minimum wter tempertures reched 3 C. Ripe nd running mle nd femle trout were collected using Smith-Root type VII bckpck electrofisher (Vncouver, WA, USA). Fish were netted, seprted by sex, nd held in cges t the site of cpture for 24 h before smpling. At smpling, fish were individully nesthetized with 0.15 g/l MS-222 (tricine methne sulfonte; Sigm Chemicl, St. Louis, MO, USA) until they developed loss of equilibrium nd slowed operculr movement ( 3 min). Ech fish ws then blotted dry, weighed, nd mesured. Condition fctor (K) ws clculted using the formul [body wt (g)]/[fork length 3 (cm)] 100. After thoroughly drying the re surrounding the urogenitl opening, eggs or milt were expressed by light pressure on the bdomen. Eggs or milt not redily expressed with light pressure (i.e., underripe) or where evidence of prior spwning ws detected (i.e., blood) were discrded. Gmetes from ech fish were trnsported on ice in seprte, seled plstic bgs filled with oxygen nd protected from light. In 2000, fish were scrificed, nd muscle smple ws tken posterior to the operculr bone nterior to the dorsl fin nd bove the lterl line. To limit potentil impcts to lredy declining popultions, fish were not scrificed fter gmetes were stripped in subsequent yers. Egg subsmples were tken for Se nlysis in ll three yers. All tissues were frozen until nlyzed. Tissue Se determintion Concentrtions of Se in egg nd muscle tissues were determined using n inductively coupled plsm/mss spectrometry (ICP-MS) method described by Feng et l. [13] nd n nlyticl detection limit of 0.4 g/g. Becuse severl other elements re known to ffect the vilbility nd toxicity of Se [10 nd references therein], concentrtions of Al, Sb, B, Be, Bi, B, Cd, C, Cr, Co, Cu, Fe, Pb, Li, Mg, Mn, Hg, Mo, Ni, Ag, Sr, Tl, Th, Sn, Ti, U, V, nd Zn were lso mesured (for rinbow trout collected in 2000, only As, C, Cd, Hg, Pb, Se, nd Zn were nlyzed). All metl nd metlloid nlyses were performed t the Albert Reserch Council s certified lbortory in Vegreville (AB, Cnd) using protocols with qulity ssurnce nd qulity controls incorported s described by Feng et l. [13]. Correltion nlysis ws used to evlute reltionships between muscle Se concentrtions nd egg Se concentrtions (SPSS, Chicgo, IL, USA). Becuse the fish popultions used for these studies were reltively smll nd potentilly sensitive to repeted lethl smpling, only subsmple of the fish tht were used for more detiled reproductive studies were scrificed. These subsmples of fish in which muscle nd egg concentrtions of Se were determined were used for the regressions shown in Figure 1. Selenium concentrtions in tissues re reported on wet-weight bsis. Selenium concentrtions in eggs expressed on wet-weight bsis cn be converted to pproximte dry-weight concentr- Fig. 1. Correltion between muscle nd egg selenium (Se) concentrtions in rinbow trout (open symbols) nd brook trout (closed symbols) collected from Se-exposed nd reference sites in southestern Albert, Cnd. Ech symbol represents dt from tissues obtined from one femle.

Developmentl effects of Se in two slmonid fishes Environ. Toxicol. Chem. 24, 2005 2375 Tble 2. Biologicl prmeters of femle rinbow trout (men stndrd error) cptured in Luscr Creek (LC), Gregg River (GR), Deerlick Creek (DC), nd Wmpus Creek (WC) during spring 2000 2002 in Albert, Cnd Prmeter LC GR DC WC n (2000, 2001, 2002) Length (cm) Weight (g) Condition fctor Egg Se concn. ( g/g wet wt) Muscle Se concn. ( g/g, wet wt) 3, 10, 9 22.1 0.87 137 18 1.17 0.02 9.9 1.4 1.5 0.3 0, 6, 1 18.3 0.59 71 6.8 1.14 0.04 6.5 1.6 NS 4, 10, 6 20.2 0.87 93 14 1.04 0.03A 3.5 0.4A 0.5 0.2 0, 2, 4 18.4 1.27 75 20 1.09 0.05A 3.5 0.4A NS Mens lbeled with letters re significntly different from the sme prmeter men for Luscr Creek bsed on nlysis of vrince of rnked dt (Kruskl Wllis) followed by Dunnett s t test on the rnked dt ( ); NS not smpled. tions bsed on previous reports tht rinbow trout eggs contin 61% moisture [14]. Where this clcultion is used in the discussion, concentrtions re reported s clculted wet weight. Egg nd lrvl rering After trnsporttion from Albert to the Freshwter Institute lbortory in Winnipeg (MB, Cnd) ( 24 h), eggs from ech femle were fertilized with consistent volume (10 l/50 ml) of composite milt derived from three to five mles cptured t the sme site. Eggs were combined with the milt in stinless-steel bowl by gentle mixing with the tip of sterilized goose fether. After stnding for 60 s, the eggs nd milt were covered with dechlorinted Winnipeg City tp wter ( 100 ml; see wter chemistry in Tble 1) nd gently swirled for 3 min. An dditionl 500 ml of wter were then dded, nd the eggs were llowed to wter hrden for further 5 min. The eggs were then plced in verticl Heth try-type incubtors (Mrisource, Milton, WA, USA). The eggs from ech femle were ssigned rndomly to one of four trys, ech with 12 comprtments. Weekly monitoring of wter in trys showed no significnt differences in temperture, dissolved oxygen sturtion, or Se concentrtion (dt not shown). Eggs were incubted t 8 0.8 C nd received 6 L wter per minute except in 2001, when rinbow trout eggs were incubted t 5 0.6 C to more closely pproximte the mbient temperture of the strems from which the dult rinbow trout were cptured. Ded eggs nd fry were removed dily nd preserved in Dvidson s solution. At swim-up, when fry hd bsorbed lmost ll the egg yolk nd hd begun exogenous feeding, fry were scrificed with n overdose of tricine methnesulfonte (MS-222, 0.25 g/l) nd preserved in Dvidson s solution. Percent fertiliztion ws clculted by dividing the number of fertilized eggs (embryos) by the totl number of eggs. Eggs tht died before htch were considered fertilized when visul inspection reveled signs of clevge. Mortlity ws clculted bsed on the number of ded embryos or fry removed until the swim-up stge compred to the totl number of fertilized eggs. Assessment of morphologicl deformities in lrve All preserved fry tht survived to the swim-up stge were exmined using dissection microscope for gross externl mlformtions. Abnormlities were recorded in four ctegories s previously described [15]: crniofcil, skeletl, finfold, nd edem. Skeletl deformities included kyphosis (convex curvture of the thorcic region of the spine), lordosis (concve curvture of the lumbr region of the spine), nd scoliosis (lterl curvture of the spine) s well s stunting of the trunk or til. Crniofcil defects included reduction or bsence of the jws nd oculr deformities such s noticebly reduced eye dimeter (microphthlmi) or pigment irregulrities s well s symmetry. Finfold defects included noticeble reduction in thickness or bsence of fin. Edem included ccumultion of body fluid in the region of the yolk sc, pericrdium, or hed. To ccount for differences in spwn sizes between individul femles, counts for ech ctegory were converted into percentges bsed on the totl number of preserved fry obtined from ech femle. Fry length ws quntified by tking the men lengths of 10 rndomly selected fry per femle with dissecting microscope. Fry weight ws clculted by finding the verge weight of 50 rndomly selected fry from ech femle, weighed 10 t time on four-plce nlyticl blnce. Sttisticl nlysis All sttisticl nlyses were conducted with SAS Version 8.0 (Cry, NC, USA). Becuse the dt were not normlly distributed, differences in dult femle, egg, nd lrvl endpoints mong sites were nlyzed by nlysis of vrince of rnked dt (Kruskl Wllis) followed by Dunnett s t test on the rnked dt. In ddition, the reltionships between Se ccumultion in eggs nd tertogenesis were nlyzed by probit nlyses. Specificlly, for given level of Se in eggs, the probbility of tertogenic response is modeled s p Pr(Response) C (1 C) F ( log 10 [Se]) where p is the probbility of response, is vector of prmeter estimtes, F is the cumultive distribution function, C is the nturl (threshold) response rte, nd [Se] is the concentrtion of selenium in the eggs. Estimtors of b 1 were sttisticlly significnt in ll cses. An lph vlue of 0.05 ws used for ll sttisticl nlyses. Adult chrcteristics RESULTS No significnt differences in men length or weight were found in femle rinbow trout from ny of the sites (Tble 2). Luscr Creek rinbow trout hd significntly higher condition fctors thn trout cptured t Deerlick Creek (p 0.003). Brook trout smpled from Luscr Creek were hevier nd hd higher condition thn fish cptured t Cold Creek (p 0.001; Tble 3). Significntly higher verge concentrtions of Se were found in the eggs of rinbow trout (9.9 g/g) nd brook trout

2376 Environ. Toxicol. Chem. 24, 2005 J. Holm et l. Tble 3. Biologicl prmeters of femle brook trout (men stndrd error) cptured in Luscr Creek (LC), Gregg River (GR), nd Cold Creek (CC) during fll 2000 2002, Albert, Cnd Prmeter LC GR CC n (2000, 2001, 2002) Length (cm) Weight (g) Condition fctor Egg Se concn. ( g/g, wet wt) Muscle Se concn. ( g/g, wet wt) 11, 11, 8 22.2 0.73 138 17 1.16 0.02 7.8 0.7 3.8 0.5 0, 8, 3 16.4 0.78 50 7 1.08 0.02 6.6 0.4A NS 7, 5, 10 21.5 0.65 105 9 1.01 0.02A 1.3 0.1B 0.6 0.1A Mens lbeled with letters re significntly different from the sme prmeter men for Luscr Creek bsed on nlysis of vrince of rnked dt (Kruskl Wllis) followed by Dunnett s t test on the rnked dt ( ); NS not smpled. (7.8 g/g) cptured t Luscr Creek thn t the reference sites ( 4 g/g; p 0.001; Tbles 2 nd 3, respectively). Correspondingly, the men xil muscle Se concentrtion of both species were higher in fish cptured t Luscr Creek (1.5 nd 3.8 g/g) compred to reference fish ( 1 g/g; p 0.001). Brook trout eggs from the intermedite exposure site, the Gregg River, were lso significntly elevted in Se compred to reference vlues (p 0.001). Selenium in eggs ws positively correlted to Se in muscle tissue for both rinbow trout (r 0.864; p 0.012) nd brook trout (r 0.954; p 0.001; Fig. 1) bsed on subsmple of fish where both tissues were nlyzed. Of interest, the slope of the brook trout correltion ws steeper thn tht for rinbow trout, indicting tht rinbow trout ccumulted higher concentrtions of Se in eggs t lower muscle burdens thn did brook trout. Regression nlysis showed no significnt reltionship between the weight of femles nd the concentrtion of Se in their eggs. The concentrtions of elements, other thn Se, in the eggs of rinbow trout nd brook trout re shown in Tbles 4 nd 5, respectively. Strontium (Sr) ws the only element tht ws significntly elevted in the eggs of both species collected from Tble 4. Men trce element concentrtion ( g/g, wet wt) for rinbow trout eggs from Luscr Creek (LC), Gregg River (GR), Deerlick Creek (DC), nd Wmpus Creek (WC), Albert, Cnd, during spring 2000 2002 Element LC GR DC WC n b Ag Al As B B Be Bi C Cd Co Cr Cu Fe Hg Li Mg Mn Mo Ni Pb Sb Se Sn Sr Th Ti Tl U V Zn 22 2.13 0.70 0.4 0.63 0.09 0.48 0.07 520 34 0.04 0.4 0.5 2.76 0.17 17.40 1.22 661 24 0.63 0.06 0.02 0.002 0.02 0.004 9.93 1.47 0.02 0.01 2.92 0.34 0.04 8.18 0.36 0.001 0.001 0.002 0.15 0.01 30.91 1.61 7 0.64 0.20 0.83 0.003 0.25 0.02 501 16 0.01 0.04 0.01 2.73 0.16 18.19 4.46 0.01 660 18 0.48 0.04A 0.02 0.003 0.06 0.01 0.004 6.53 1.55 0.02 0.01 2.27 0.20A 0.01 0.002 7.26 0.20 0.002 0.001 0.001 0.14 0.01 26.60 1.00 20 1.13 0.23 1.05 0.03 0.38 0.023A 0.3 531 28 2.90 0.17 19.16 1.19 626 29 0.84 0.07 0.02 0.001 0.05 0.02 3.56 0.38A 0.05 0.02 1.10 0.107B 0.02 0.004 7.42 0.33 0.006 0.002 0.15 0.01 30.22 1.30 6 1.48 0.49 0.3 2.0 0.42 0.04 561 68 0.01 0.5 2.84 0.46 22.37 3.80 0.3 687 58 0.86 0.082B 0.02 0.003 0.02 0.01 3.50 0.45A 0.05 0.02 0.98 0.129B 0.09 0.052A 8.22 0.88 0.004 0.004 0.17 0.03 33.48 3.06 s not smpled in 2000. b Smple size for elements other thn As, C, Cd, Hg, Pb, Se, nd Zn, the only elements nlyzed in 2000, re 16 nd 19 for Luscr nd Deerlick creeks, respectively. Men stndrd error. Different letters indicte significnt differences between groups bsed on nlysis of vrince of rnked dt (Kruskl Wllis) followed by Dunnett s t test on the rnked dt ( ).

Developmentl effects of Se in two slmonid fishes Environ. Toxicol. Chem. 24, 2005 2377 Tble 5. Men trce element concentrtion ( g/g, wet wt) for brook trout eggs from Luscr Creek (LC), Gregg River (GR), nd Cold Creek (CC) during fll 2000 2002 Element LC GR CC n Ag Al As B B Be Bi C Cd Co Cr Cu Fe Hg Li Mg Mn Mo Ni Pb Sb Se Sn Sr Th Ti Tl U V Zn 30 4.07 2.22 0.3 12.93 4.38 0.20 0.02 0.45 0.10 0.07 420 19 0.03 0.01 0.06 0.01 0.19 0.08 2.81 0.11 10.41 0.880 0.5 434 24 0.85 0.110 0.02 0.002 0.23 0.05 0.12 0.06 7.78 0.70 0.06 0.04 3.97 0.240 4.28 0.14 0.01 0.001 0.4 23.27 1.05 11 0.01 0.001 3.98 1.62 0.04 6.53 1.87 0.30 0.060 2.36 0.12 429 38 0.01 0.03 0.02A 0.07 0.02 3.11 0.16 13.26 1.46 0.04 0.7 533 23 0.67 0.120 0.02 0.003 0.12 0.06 0.011 0.003 0.003 6.59 0.42A 0.04 0.01 0.94 0.11A 0.009 4.33 0.37 0.003 0.001A 0.002 0.07 24.67 1.48 22 0.01 0.001 3.55 0.78 5.68 1.56 0.13 0.02A 058 0.13 430 32 0.02 0.002 0.04 0.01 0.09 0.01 4.42 1.03A 16.75 2.49A 0.5 385 32 1.74 0.16A 0.02 0.002 0.25 0.13 0.09 0.05 1.24 0.10B 0.06 0.03 0.66 0.06A 0.06 4.43 0.42 0.004 0.001A 24.43 1.83 Men stndrd error. Different letters indicte significnt differences between groups bsed on nlysis of vrince of rnked dt (Kruskl Wllis) followed by Dunnett s t test on the rnked dt ( 0.05). Luscr Creek compred to other sites. A few other trce elements hd significnt site-specific differences in rinbow or brook trout but were not consistently different in the two species t the sme sites. The potentil for other elements to ntgonize or potentite the toxicity of Se hs been discussed elsewhere [10]. Embryo-lrvl chrcteristics The verge rte of fertiliztion for both rinbow trout nd brook trout ws greter thn 75% t ech site (Tbles 6 nd 7). Fertiliztion ws not significntly different mong sites for both species. Mortlity rtes of embryos of both species were lso similr between ll sites. The verge mortlity rte of rinbow trout embryos t ech site (18 37%) ws higher thn tht of brook trout ( 7%). The occurrence of tert in rinbow trout lrve ws relted to the loction of cpture of prentl fish (Tble 6). A significntly higher proportion of crniofcil defects nd edem were found to occur in lrve from Luscr Creek compred to Deerlick Creek (p 0.008 nd 0.025, respectively). In most cses, edem ppered s fluid surrounding the yolk sc or hert nd ws often ssocited with hemorrhging nd spinl curvtures. Edemtous fry were often shorter nd hd used less of their yolk thn fry tht hd not developed edem t the time of smpling. In ddition, fluid ccumulted in the hed, often resulting in spreding of the crnil fetures. The men percentge of skeletl defects (25%) ws double in lrve from Luscr Creek thn from ny other site ( 10%), but becuse of vribility between femles, no sttisticlly significnt differences were detected. The most frequent type of skeletl bnormlity ws concve curvture of the spine. In contrst, significntly higher incidence of finfold deformities ws observed in lrve from Wmpus Creek thn the other reference site (p 0.015). Almost ll these deformities were ttributble to differences in fin thickness t the Wmpus site. Rinbow trout lrve were of similr length t ll sites, but the lrve of trout cptured t Luscr Creek were significntly hevier thn those from Deerlick Creek (p 0.021). specific differences in lrvl deformity rtes were observed for brook trout in only one ctegory of tert (Tble 7). The incidence of crniofcil defects ws pproximtely fourfold higher in lrve from Luscr Creek (7.9%) compred to the reference site (2.1%; p 0.004). Crniofcil defects consisted primrily of shortened or misshpen jws followed by eye defects. Brook trout lrve from the reference site were significntly longer nd hevier thn lrve from Luscr Creek (p 0.003 nd 0.013, respectively). A residue response reltionship ws observed for rinbow trout between the concentrtion of Se in eggs nd the prevlence of tert (Fig. 2). Sttisticlly significnt reltionships between egg Se nd edem, crniofcil defects, nd skeletl defects pproximted exponentil functions. In ddition to evluting the pooled dt from ll three yers of the study, the dt from ech of the yers of the studies were exmined using the sme probit nlysis procedures. Significnt rel- Tble 6. Effects on rinbow trout embryos nd lrve (men stndrd error) spwned from fish cptured t Luscr Creek (LC), Gregg River (GR), Deerlick Creek (DC), nd Wmpus Creek (WC), Albert, Cnd, during fll 2000 2002 Prmeter LC GR DC WC Egg percent fertiliztion Embryo percent mortlity Totl fry evluted Lrvl percent crniofcil defects Lrvl percent skeletl defects Lrvl percent finfold defects Lrvl percent edem Lrvl length (mm) Lrvl weight (mg) 77.8 4.3 35.0 6.3 3,700 33.3 7.9 25.0 5.9 15.0 5.8 34.5 8.6 18.5 0.4 53.3 3.5 81.2 4.8 34.2 12.3 825 10.6 2.7 9.9 2.4 13.6 6.2 12.2 5.0 19.4 0.7 44.6 4.2 77.5 4.7 18.1 3.3 2,466 7.1 1.4A 9.2 2.8 5.4 1.4 6.1 1.6A 19.0 0.3 41.2 2.1A 77.5 9.8 37.3 14.1 808 12.0 3.9 7.9 3.4 42.2 17.8 23.3 15.4 19.2 0.4 40.6 3.4 Mens lbeled with letters re significntly different thn the sme prmeter men for Luscr Creek bsed on nlysis of vrince of rnked dt (Kruskl Wllis) followed by Dunnett s t test on the rnked dt ( ).

2378 Environ. Toxicol. Chem. 24, 2005 J. Holm et l. Tble 7. Effects on brook trout embryos nd lrve (men stndrd error) spwned from fish cptured t Luscr Creek (LC), Gregg River (GR), nd Cold Creek (CC), Albert, Cnd, during fll 2000 2002 Prmeter LC GR CC Egg percent fertiliztion Embryo percent mortlity Totl fry evluted Lrvl percent crniofcil defects Lrvl percent skeletl defects Lrvl percent finfold defects Lrvl percent edem Lrvl length (mm) Lrvl weight (mg) 92.8 1.3 6.5 1.6 12,703 7.9 1.8 2.0 0.4 1.9 0.8 1.0 0.5 17.4 0.2 31.7 1.6 78.4 5.5 2.9 0.7 2,124 2.3 0.8 0.8 0.2 3.1 1.8 0.3 0.2 17.9 0.3 31.3 1.6 89.1 4.2 6.9 2.6 5,350 2.1 0.6A 1.0 0.2 0.9 0.3 0.7 0.3 18.5 0.3A 37.8 1.5A Mens lbeled with letters re significntly different thn the sme prmeter men for Luscr Creek bsed on nlysis of vrince of rnked dt (Kruskl Wllis) followed by Dunnett s t test on the rnked dt ( ). Fig. 2. Reltionships between selenium (Se) concentrtions in subsmples of eggs obtined from dult femle rinbow trout collected t the Seexposed nd reference sites (AB, Cnd) nd deformities in the offspring from ech femle in four ctegories: crniofcil (A), skeletl (B), finfold (C), edem (D). Ech symbol represents dt from one femle s offspring. Open symbols re dt from 2000, shded symbols re from 2001, nd filled symbols re from 2002. Circles represent fish from Luscr Creek, tringles those from Gregg River, squres those from Deerlick Creek, nd dimonds those from Wmpus Creek.

Developmentl effects of Se in two slmonid fishes Environ. Toxicol. Chem. 24, 2005 2379 Fig. 3. Reltionships between selenium (Se) concentrtions in subsmples of eggs obtined from dult femle brook trout collected t the Seexposed nd reference sites (AB, Cnd) nd deformities in the offspring from ech femle in four ctegories: crniofcil (A), skeletl (B), finfold (C), edem (D). Ech symbol represents dt from one femle s offspring. Open symbols re dt for 2000, shded symbols re for 2001, nd filled symbols re for 2002. Circles represent fish from Luscr Creek, tringles those from Gregg River, nd squres those from Cold Creek. tionships were observed between the concentrtions of Se in eggs nd crniofcil deformities, skeletl deformities, nd edem for 2001 (n 28) nd 2002 (n 20), respectively. However, with only seven fish to include in the regression in 2000, the probit nlysis model ws not significntly predictive. Between 8.8 nd 10.5 g Se per grm of wet egg weight, 15% probbility ws observed tht eggs would exhibit ech of these effects, bsed on probit nlysis. In contrst, n ssocition between the occurrence of deformities nd Se concentrtions in eggs ws not evident for brook trout (Fig. 3). It should be noted tht deformities could not be ssessed in the offspring of one rinbow trout from the Gregg River nd one brook trout from Cold Creek becuse of complete mortlity in the first cse nd lck of Se nlysis of eggs in the ltter. DISCUSSION Elevted levels of Se were found in the tissues of two slmonid species cptured downstrem from col mining ctivity in the northestern slopes region of Albert. The levels of Se in the eggs of fish cptured t the high-se-exposure site exceeded the toxic effect threshold of 10 g/g (dry wt) in eggs [1]. However, only Se concentrtions in the muscle of brook trout exceeded Lemly s recommended threshold of 8 g/g (dry wt) specific to the tissue to void toxic effects. Despite elevted concentrtions of Se in their tissues, dult rinbow nd brook trout did not show histopthologicl lesions typiclly ssocited with Se in the gill, liver, or kidney (R.E. Evns, unpublished observtion). Histopthologicl effects of Se cn include hemtologicl chnges s well s gill, liver, kidney, nd ovry dmge s described in reder sunfish with liver concentrtions of 7.5 g Se/g (wet-wt clcultion) [16] nd green sunfish with muscle concentrtions of 13 g Se/g (wet-wt clcultion) [17]. In light of these findings, exmintions of the differences in species susceptibility of dults to Se-induced tissue dmge in dult fish wrrnt further considertion. Rinbow trout nd brook trout consistently hd higher con-

2380 Environ. Toxicol. Chem. 24, 2005 J. Holm et l. centrtions of Se in their eggs thn in muscle tissue, similr to the pttern of ccumultion observed for cutthrot trout [5], lrgemouth bss, nd bluegill sunfish [18]. Selenium concentrtions in the eggs of both species lso showed strong reltionship with Se concentrtions in muscle, suggesting tht mternl trnsfer of Se is efficient in both brook trout nd rinbow trout. A similr reltionship between Se concentrtions in muscle nd eggs hs been reported for rzorbck suckers [19] nd cutthrot trout [5]. Selenium levels in the tissues of femle rinbow nd brook trout in the present study were highly vrible. This ws true even for femles collected from the sme site. This vribility is likely consequence of fish mobility within the study re. For exmple, fish re free to move into or out of Luscr Creek vi the McLeod River. This mens tht exposure to Se-lden wters or prey items my be intermittent. In fct, fish cptured in Luscr Creek my feed t sites other thn Luscr Creek tht differ widely in their Se concentrtions. Similrly, trout cptured t the reference sites my hve spent significnt portion of their feeding times t sites with elevted Se concentrtions. This is one of the most importnt resons tht ctul tissue Se concentrtions must be considered in exmining potentil tertogenesis rther thn comprisons bsed only on cpture site. No significnt differences in fertiliztion or mortlity were found between the Se-exposure sites nd the reference sites for either species. This is consistent with results from other studies with bluegills [20,21], fthed minnows [22], perch [23], nd cutthrot trout [5]. The pprent lck of response in endpoints ssocited with the erly egg stge of development to elevted concentrtions of Se could be relted to the rte of consumption of the yolk [reviewed in 24]. Selenium is incorported into the pltelet components, lipovitellin, nd phosvitin of the egg yolk [25] nd exerts toxic effects only fter it hs been ssimilted by the developing lrvl fish. Prior to htch, the yolk is bsorbed t slow but stedily incresing rte. The rte of bsorption increses rpidly right before htch nd continues t reltively high nd constnt rte. During this second phse the yolk pltelets re preferentilly consumed over the oil globule component of the yolk. The rte of yolk consumption then slows s the pltelets re depleted. Thus, lthough Se is present in the egg yolk throughout development, it my ffect lrvl rther thn egg development becuse it is mobilized to greter degree fter htch. Tertogenic effects observed in fish from this study were consistent with those reported for other fish species tht hd ccumulted similr tissue burdens of Se. Gillespie nd Bumnn [21] reported tht eggs from femle bluegill sunfish cptured from Se-contminted reservoir produced up to 100% edemtous lrve t ovrin concentrtions of 7.0 g/ g. Bluegills [26] nd fthed minnows [27] exposed to elevted levels of Se in outdoor experimentl strems hd higher incidence of edem, lordosis, nd hemorrhging. These effects were identified t Se concentrtions of 4.5 to 5.9 g/g in ovries. A residue response reltionship between the mount of Se in eggs nd the frequency of lrvl deformities ws identified for rinbow trout in this study. All regression reltionships indicte tht t egg burdens ner 8 to 10 g/g, 15% of skeletl deformities, crniofcil defects, nd edem could be ttributed to Se. Moreover, t only slightly more elevted concentrtion of 12 g/g, skeletl, crnil nd edem could be ttributed to 30, 40, nd 70% of the popultion, respectively. Lemly [28] reported similr reltionship between whole-body Se concentrtions nd the frequency of deformed centrrchid fish species inhbiting Se-lden reservoir. Similr to the results from the rinbow trout in this study, the reltionship for centrrchids lso fit n exponentil function (cubic model), with the point of rpid rise in deformities occurring between 10 nd 12.5 g/g (wet-wt clcultion). Lemly [29] found tht the sme mthemticl reltionship lso held for lrvl centrrchids but tht the criticl threshold ws lower, rnging from 7.5 to 10 g/g (wet-wt clcultion). However, it should be noted tht we hve reported reltionships bsed on concentrtions of Se in eggs, while Lemly s dt re bsed on concentrtions in lrve, juveniles, nd dult tissues. The regression in Figure 1 shows tht Se burden of 10 g/g in the eggs of rinbow trout corresponds to pproximtely 1.8 g/g in muscle, vlue considerbly lower thn those reported by Lemly. The lower thresholds obtined from these studies do not support the need for higher Se criteri in cold-wter species compred to wrm-wter fishes [5], reviewed in [6]. The lck of comprble dose response in brook trout to the one observed for rinbow trout my indicte difference in the sensitivity of these closely relted species to the effects of Se. It should lso be noted tht the efficiency of mternl trnsfer of Se to eggs from the muscle during vitellogenesis my be lower in brook trout. Although both species showed significnt correltion between Se concentrtion in muscle nd eggs, the slope for brook trout ws steeper. This mens tht t equl muscle burdens, brook trout ccumulted lower concentrtions of Se in their eggs. While rinbow trout showed sevenfold increse in the men egg Se concentrtion (9.9 g/g) over the men muscle concentrtion (1.5 g/g), the men Se burden in brook trout eggs (7.7 g/g) ws only twice s high s the men muscle burden (3.8 g/g). Resons for these species differences in susceptibility nd comprtmentliztion of Se re uncler. Rinbow trout nd brook trout inhbiting Se-contminted strems downstrem of col mining ctivity ccumulted elevted levels of Se in their eggs compred to fish from the reference sites. Significnt dose response reltionships existed between the egg Se burdens nd the incidence of tertogenesis in rinbow trout lrve. The threshold vlue, between 8 nd 10 g/g, ws similr to those reported in previous studies for wrm-wter species inhbiting lentic wters. No such reltionships were found for brook trout, indicting potentil difference in species-specific sensitivity of slmonids to Se. The significnce tht Se-induced tert will ultimtely hve on the nturl popultions of slmonids requires further exmintion. Acknowledgement The uthors wish to cknowledge the excellent technicl ssistnce of Rudy Hwryluk, Don Hildebrndt, Suznne Kollr, Jeff Werner, Dnielle Godrd, Cecili Doebel, nd Lyndon Brinkworth. 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