Selenium Requirement of a Bloom-Forming Planktonic Algafrom Softwater and Acidified Lakes

John D. Wehr and Lewis M. BrownDepartment of Plant Sciences, The lJniversity of Westem Ontario, London, Ont. N6A SB7

Wehr, J. D., and L. M. Brown.1985. Selenium requirement of a bloom-forming planktonic alga from soft-water and acidified lakes. Can. J. Fish. Aquat. Sci. 42: 1783-17ffi.

Lakewide odor. episo_des have been associated with blooms of the planktonic alga Chrysochromulinabreviturrita Nicholls (Prymnes-ioph.yceae) in lakes influenced by acidic precipitationl We demonstrate anabsolute requirement in this flagellate for selenium (Se) in axeiic culture. The alga is capable of utilizingseveral forms of Se: selenite, selenate, dimethylselenide (DMSe), and selenometfiionine. This is believejto be the first demonstration of the utilization of DMSe as a Se source by any organism. As the microflora ofaquatic sediments recycle Se as volatile DMSe, this may have significahce'in iis biogeochemistry in acidi-figd Aa1qr.s.. Bioassays of lake waters from the Muskolia-Halibirton region of Oniario show thiat in onelake (Dickie) a simulated 59 lpike resulted.in a significant increase (70%lp < 0.05) in algal growth. CrowthinCinderLakewater,which.hadsupportedapop-ulation of C.brevituniti,wasmarginaiiysiimulated(24%,p < 0.05). Distant coal-fired powerplants, which have been implicated in the epiiodic'deposition of Se,may account in part for the unp.redictable blooms of the alga.'Chrysochromulina brevituirita cannot bemaintained in culture without this micronutrient, and sevelal Se iources may be important triggeringfactors for the initiation of blooms.

Dans les lacs sous l'influence de pr6cipitations acides, des p6riodes de pr6sence d,odeurs ont 6t6 associ6es) des prolif6rations pl6thoriques de l'algue planctonique Chrysochromulina breviturrita Nicholls (prym-nesiophyTae)..Les auteurs d6montrent qu'en culture ixeniqJe, le s6l6nium (Se) esi iUiolurnent n6ces-saire.i ce flagFl16 qui peut l'utiliser sous_piusieurs formes : s6i6nite, s6l6niate, dim6thyls6l6niure (DMSe)et s6l6nom6thionine. On croit que ceci'repr6sente la premidre preuve de I'utilisatioh du DMSe-par unorganisme c9.Tme source de Se. Le fait que la microflore des s6diments lacustres recycle le Se ious laforme de DMSe volatile- peut avoir une importance pour sa biog6ochimie dans les 6aux acides. Desanalyses.biologiq_ues d'6chantillons d'eau iecueillis dans des lac"s de la r6gion Muskoka-Haliburton(Ontario) ont r6vdl€. que dans le lac Dickie, un important apport simu16 de Se ientrain6 une prolif6rationphytoplanctonique importante (70Yo,p < 0,05.). Lians le lai'Cinder, habitat d,une population de C. brevi_turrita, la croissance n'a 6t6 qu'i peine stimul6e 94 %, p < 0,05). Des centrales ihermiques aliment6esau charbon situ6es A distance, qui ont joud un rdle dani les d6p6ts 6pisodiques de Se, peuvent ritre enPartie- responsables des pouss6es phytoplanctoniques impr6viiibles.' Chrysochromutini breviturrita nepeut 6tre.gat96 g! culture sans cet oligodl6ment dt plusiiurs sources de Se peuvent Ctre d,importantsfacteurs dans le d6clenchement des efflorescences.

Received April 4,1985Accepted July 23, 1985(JEr78)

eposition of acids from SO2-emitting sources havebeen identified as a primary cause of the acidificationof softwater lakes in the eastern United States andCanada (Dillon et al. 1978; Loucks and Glass 1984).

Anthropogenic emissions are estimated to account for more thanX)Vo of the total SO, emissions in eastern North America(Galloway and Whelpdale 1980), and principal sources are fromcoal combustion and metal smelting industries (Bubenick et al.1983). In southern Ontario a number of lakes within a broadregion of the Precambrian Shield now have reduced pH, whichhas been shown to be directly linked to anthropogenic SOzsources (La7*te and Dillon 1984). In addition to depressed pHand alkalinities in affected lakes, metal mobilization (Schindleret al. 1980b; Wright et al. 1980) and deposition (Jeffries andSnyder l98l) have been observed. Observational and experi-mental studies of biological responses have documented declinesin fish populations, simplification of plankton communities, and

Can. J. Fish. Aquat. Sci., Vol.42, 1985

reductions in microbial numbers and rates of organic matterdecomposition (Hendrey et al. 1976; Schindler and Turner1982; Bubenick et al. 1983).

Another biological effect, which is believed to be linked withacidification, has recently been observed. Since 1978 softrraterlakes in eastern North American have been affected by obnox-ious ("rotten cabbage") lakewide odors. Odorepisodes have allbeen associated with nearly unialgal blooms of the plankton algaChrysochromulina breviturrira Nicholls (Nicholls et al. 1982).In Ontario, blooms have developed in moderately acidic (pH5.5-6.2), poorly buffered (< 160 pEq HCO3-/L) lakes in theMuskoka-Haliburton region (Nicholls et al. 1982), which isinfluenced by acid precipitation (Dillon et al. 1978; Scheider etal.1979;LaZerte and Dillon 1984). In addition, following 2yrof artificial acidification of Lake 302 (south basin) with H2SO4,a bloom of Chrysochromulina spp. (containing both C. brevi-turrita Nicholls and C. laurentiana Kling (Kling l98l)), with

1783

ReEu le 4 avril 1985Acceptd le 23 iuillet 1985

odor, was observed (M. e. Turner, Freshwater Institute, Win-nipeg, Man., pers. comm.). Recent experiments in our labora-tory with an axenic culture of C. breviturrira isolated from abloom in one of the affected lakes in Central Ontario haveverified these field observations. A pH tolerance range of4.0-6.9 was established and experiments have also demon-strated inhibition of growth as a result of HCO3- additionsexceeding approximately 100 pEq/L (Wehr et al. 1985).

During the development of a chemically defined growthmedium for C. breviturrita we were aware that earlier workers(Pintner and Provasoli 1968) were unable to maintain consistentgrowth of marine Chrysochromulina spp. without the additionof selenium (Se) as a trace element. Subsequently we alsodiscovered that trace amounts of Se markedly improved thegrowth and long-term maintenance of C. breviturrita, Ourinterest in the role of Se was heightened, as it has been shownthat the principal sources of SO2 emission (coal combustion,metal smelting) also emit substantial quantities of Se (Andrenet al. 1975; National Acadamy of Sciences Committee 1976;Nriagu and Wong 1983). In one instance, deposition fromcoal-fired power plants resulted in marked increases in Se inthe water and biota of contaminated ponds (Gutenmann et al.1976). Acidified lakes within a 30-km radius of Cu-Ni smeltersin Sudbury, Ontario, were also Se enriched (Nriagu and Wong1983).

Our objectives in this sfudy were to (l) determine the degreeof Se requirement by C. breviturrita in culture, (2) establishwhether a dose-response relationship exists for Se, and (3)determine the potential for Se as a possible triggering factor inblooms of the alga in acidified lakes using bioassays.

Methods and Materials

Details of isolation and development of a chemically definedmedium have been given previously (Wehr et al. 1985) and onlya brief summary is given here. Chrysochromulina breviturritawas collected in July 1982 using surface water grab samplesfrom Cinder Lake in Ontario (78o56'W, 45"04'N) during anodiferous bloom and subsequently isolated into unialgal, axenicculture. Due to the possibility of confusion of the identity of C.breviturrita with a recently described species, C. laurentianaKling (Kling l98l), an exhaustive study of the scales of ourisolate was carried out using transmission electron microscopy(TEM). Using shadow cast, negative staining, and freeze-etchmethods with TEM on many occasions, it was determined thatonly C. breviturrita Nicholls emend Kling was present in thestrain (No. BTl30) used in this study (L. M. Brown, unpubl.).

The medium used was designed to approximate the low-alkalinity waters of acid-sensitive lakes in Ontario. The basalmedium without added Se (Muskoka No. 20) contained (M :mol/L) 103 p,M CaSOa' 2HzO, 44 pM MgSOa. 7H2O, I 00 pMNHaH2POa, 3 lt M CaCl2, 14 p,M KCl, l8 pM FeSOa'7H2O,8.l1tM H3BO3, and l.2p.M Na2EDTA.2H2O. Trace metalsincluded 4.5p,M MnCl2'4H2O, 84nM CoCl2.6H2, 78nMCuSOa, 76nM ZnCl2, 50nM NaMoOa, and 19nM VOSOa.2H2O. The "f12" vitamin mix (Nichols 1973), thiamine (0.3pM), biotin (2.5nM), and B12 Q.37nM) was also added.Media were adjusted to pH 6.0 with NaOH (approximate toral30 St"M), and a "Ross" semimicro combination electrode (Orionmodel 810300) was used to measure pH. Cultures were axenicand routinely tested for axenicity.

All glassware was acid washed (1070 HNO3, l2 h) and rinsedin deionized and distilled water prior to use. The water used for

1784

media and stock solutions was deionized and distilled. Mediawere routinely sterilized by autoclaving, except in treatmentsusing different oxidation states of Se (H2SeO3, H2SeOa) andorganic forms of Se. These latter treatments were filter sterilized(0.2-pm pore size). With one exception, experiments were runusing 125-mL Ehrlenmeyer flasks with 50 mL of medium.

An experiment with volatile selenides (dimethyselenide(DMSe), dimethyldiselenide (DMSe2)) and their controls usedsealed 7-mL "funnel top" ampoules (Wheaton Glass) containing3 mL of medium as follows. A conrainer with I 00 mL of Se-freemedium was inoculated with C. breviturrira and then bubbledfor 15 min with filtered (0.2-pm pore size) 5Vo CO2 in air, whichwas mixed using a gas proportioner (Matheson Ltd.). prelimi-nary experiments indicated that CO2 bubbling was necessary forgrowth in sealed containers. Aliquots (3 mL) of the inocula wereadded aseptically to sterile ampoules and sealed with sterileserum stoppers. Se was added using a lO-pI- syringe and theampoules were flame sealed. DMSe and DMSe2 stocks wereprepared in redistilled ethanol; hence, controls with inorganic Se(as HzSeOs) or no Se also had ethanol added. Supplien ofcompounds were Baker Chemical (H2SeO3), Alfa products(H2SeOa, DMSe, DMSe), and Sigma Chemical Company(seleno-or-methionine, or-methionine, selenocystamine di-hydrochloride).

Growth assays were carried out using lake water samplescollected l0 September 1984 from Cinder Lake (pH 6.40), theoriginal source of the isolate of C. breviturrita used in theexperiments, and from Dickie Lake (pH 6.36) (79'05'W,45'05'N), another softwater lake in the Muskoka region ofOntario. Waters were prefiltered (0.45-p.m pore size) and sup-plemented with either the f /2 vitamin mix ("control"), 100 pMNH4H2PO4 plus vitamins, 0.63 pM H2SeO3 plus vitamins, orall three additions. These enriched lake waters were adjusted topH 6.0, autoclaved, and inoculated as with the chemicallydefined medium.

Unfiltered samples of Dickie and Cinder lake waters werealso examined for the presence of C. breviturrilc using phase-confast microscopy. Counts of the dominant algal taxa werecarried out using Sedgwick-Rafter cells after treatnient withLugol's iodine. The total alkalinities (by titration with standard-ized 0.010 M H2SO4) of fresh samples of'Cinder and Dickielake waters were determined to be 36 and 14 p.fulL, respec-tively. During 1983, concentrations of total N and total Paverage approximately 480 pg N/L and l5 pg PiL, respectively,in these lakes (K. H. Nicholls, Ontario Ministry of the Environ-ment, Rexdale, Ont., pers. comm.). An inspection of theplankton from the unfiltered water samples revealed the presenceof a moderately large (approximately 250 cells/ml) populationof C. breviturrita in Cinder Lake only. Otherwise, the phyto-plankton communities were structurally similar at the time ofsampling.

To maintain C. breviturrita for more than two or threetransfers it was necessary for stock cultures to be grown in amedium supplemented with Se (0.63 p.M). Therefore, prior toexperiments cells were starved of Se in the following manner. Inlate exponential phase (between days 8 and l2), healthy cellsremained in a film at the water surface. The medium wasaspirated from the bottom and the majority of liquid (90-95Vo)removed aseptically from the cells. Cells were then inoculatedinto a large volume of Se-free medium (e.g. 800mL) for aperiod of at least 5 but no more than l0d. Starvation periodsexceeding this generally yielded unhealthy inocula or even celllysis. After the starvation period enough medium was aspirated

TAsr.e 1 . Comparison of growth of C . breviturrira in various mediarelative to enriched Cinder Lake water. Lake water was collected July1982 during a bloom (enrichment = 100 pM NH4H2PO4 and f/2vitamins; M20 = Muskoka No. 20 (see text); yeast extract concentra-tion : 0.5 g/L; added Al concentration : 100 pglL; added Se con-cenration : 50 p.g/L).

1.0

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j

3

Eosl-

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-r 0.3E

UI

o

! o-zo

=E0.1!,

o.;

Medium

Maximumgrowth rate Yield

(divisions/day) (l0scells/mL)

Enriched Cinder LakeM20 (basal medium without Se)M20 + yeast extractM20 + AlM20 * SeIv

0.70+0.140.65+0.070.68+0.170.59+0.100.78-+0. l0

3.09+0.320"30+0.053.04-f 0.250.23-|0.102.65{-0.30

0 0.0r 01

|---1_

r0 r00 1000 10000

tttttIt!ttI

JE

ul

o(,

-tv+)g

-Se

104

time (doys)Rc. l. Comparisons of growth of C. breviturrita in a definedmedium with and without Se added as H2SeO3 (+Se : 50pg Se/L(0.63p"M), n = 3, X t sp).

away to-produce an inoculum whose density was approximatelyI x lOs to 2 x lOt cells/ml-. Experimental regimes (flasks oiampoules) were inoculated to produce an initial density of 2 xl0'to 5 x l0'cells/ml.

Experiments were run at20"C with a subsurface irradiance of60-70pE.m-2.s-r ("cool white" fluorescent lights) on a l2hlight : l2h dark cycle. Cells were harvested (3 mL) asepricallyfor growth estimates. Growth was measured by increases in cellnumber. Counting was carried out using a Bio/physics (model6300 A) Cytograf laser-based particle counter (Ortho Instru-ments Division, Westwood, MA). Counts were calibrated usingcell suspensions of known densities from hemacytometercounts.Experiments were run in triplicate and usually repeated twice.

Can. J. Fish. Aquat. Sci., Vol.42, 1985

lSerYl (yg/LlFtc. 2. Effect of SeIv (as H2SeO3) concenfiations on maximumdivision rate and cell yield of C. breviturrita in a defined medium.Solid line on yield plot fitted by linear regression (n : 3 for 0-0.1,500 - l0000pg Se/L; n = 6 for 1.0, lO-l00pg Se/L; * * sp).

Results

The development of a chemically defined growth medium forC. breviturrita led to some comparisons between $owth insynthetic media, partially defined media, and enriched lakewater (Table l). One chemically defined medium, Muskoka No.20, would only support vigorous $owth (>105 cells/ml atstationary phase) for one or two subcultures after maintenancein enriched Cinder Lake water; yeast extract or selenite yieldedvigorous, reproducible growth comparable with enrichedCinderLake water (Table I ). Because yeast extract rendered the mediumundefined, its use was not continued. Typical growth curvesdemonstrated that the cell yield was significantly (ANOVA,p < 0.05) increased in a defined medium with selenite (Fig. l).This enhancement ranged from 5- to l0-fold depending on thedegree of Se starvation or carryover. Maximum division rates inthe two treatments (days 0-3) were not significantly different(p < 0.05). Further subcultures in the absence of Se resulted invirtually no growth and, ultimately, cell lysis.

A dose-response of C. breviturrira growth over a broadrange (greater than six orders of magnitude) of Se (as selenite)concentrations (Fig. 2) indicated a minimum'stimulation of cellyield in culture at0.1 pg/L (doubled yield). The growth responsebetween 0.05 and 100 pg/L had a significant relarionsiip tologle Se concentration (p < 0.01). The apparent optimum Seconcentration range in this medium was 50-500 pg/L. Above

t785

302010

r2= 0.994p<0.01

F---

0.3

0.2

0.r

Flc. 3. Growth responses of C. breviturrita to Se and/ornutrient additions in assay watercollected(10 September 1984) from Cinder and Dickie lakes of the Muskoka-Haliburton region of Ontario(control : f/2 vitamins only; N + P: l00pMNHaH2PO.1; SeIv: 0.6 1tM H2SeO3 (50p,g Se/L);N + P + SeIv = both additions; n : 3, .t I so).

E

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E

Eo.>

,o- 3 .\ -\S+q'Lr'Orte

$

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these concentrations, growth was increasingly inhibited. Maxi-mum division rates (Fig. 2) indicate no significant differencesover nearly the entire range, except a reduction at >5 mg Se/L,where marked inhibition of cell yield was also observed.

As growth in enriched lake water that had been collectedduring a bloom (July 1982) proved to be successful, assays wereconducted using water from two softwater lakes in which C.breviturrita has been observed previously, but during a "non-bloom" period: Cinder Lake and Dickie Lake. Growth of C.breviturrita was significantly stimulated relative to controls(p < 0.05) in both lake waters by the addition of N * P (Fig. 3).In Cinder Lake the addition of Se alone had a small but signi-ficant effect Q, < 0.05), but yield in Dickie Lake was increasedby 70Vo (p << 0.05). Growth in Dickie Lake water was alsogreater in the treatment with Se and N * P (2.42 x I 05 cells/ml)than N * P alone (1.99 x lOs cells/ml).

Both selenite (SeIv, as HzSeOl) or selenate (SevI, as HzSeO+)added to the chemically defined medium resulted in a similarstimulation relative to -Se controls (Table 2). Because anapparent stimulation may be due to inorganic Se alleviatingphosphate toxicity (Broyer et al. 1972), an experiment wasionducted over decreasing POa3- concentrations (Table 3).There was no Se-POa3- interaction, as a lO-fold decrease inPOo3- lfrom 100 to 10 pM) had no significant effect (p > 0.05)on growth of C. breviturrita in the presence or absence of Se.

Sulfur-selenium antagonism was also considered, using excessconcentrations (I0-fold increase) of SOa2- in the presence and

I 786

TAnls 2. Effects of Se oxidation state on growthof C. breviturrita, as maximum division rate andcell yield, in a defined medium (Selv = selenite as

HrSeOr; Sevl : selenate as H2SeOa; both +Se treat-ments 50 pg Se/L; n = 6;* i sp; *value significantlydifferent from -Se control at p < 0.05).

Maximumgrowth rate Yield

Se treatment (divisions/day) (105 cells/ml)

-SeSeIvSevI

0.65-f 0.100.70-f 0.l l0.78 -f 0.05

0.40-+0.142.26!0.30*2.32t0.24*

absence of Se (Table 3). No significant increase was observedwhen S replaced Se, nor did excess SOa2- affect yield in thepresence of Se.

The possible stimulatory effects of organic Se compounds(equivalent Se concentrations: 50 pg/L) were also examinedGig. a). Significant positive effects were found for chemicallyunrelated compounds; selenite (H2SeO3), DMSe, and or-seleno-methionine. The S-containing analogue of the latter compounddid not stimulate growth of C . breviturrita (p > 0.05) comparedwith the -Se control. Results were ambiguous with the additionof selenocystamine, and a significant inhibition of growth (p <0.05) was observed with DMSe2.

Dickie

Tlsr.E 3. Tests for interactions of SeIv (as H2SeO3) with S(as CaSO+) and with P (as NaH2POa), using two concentra-tions of SO+2- and three concentrations oi pOa3-, on celltt"tdr ot t. *uaar * ro).

Potential aon..noffiinterfering ion (pM) Sery (ld cells/ml)

SOa2- 3.03-'-0.130.25-f 0.05

2.89-f 0.380.26-+0.01

2.20-rO.220.30+0.06

2.36'+0.170.28-f 0.05

2.38+O.200.26+0.03

Fon3-

0.3

0.2

0. 1

flagellate Peridinium cinctum and the diatom Stephanodiscushantzschii, from circumneutral to slightly alkaline lakes (Lind-shdm and Rodhe 1978; Lindstnim 1983). However, unlike C.breviturrita, neither species responded positively to Sevl. Thedose-response was suggested as evidence ofa poisible bioassayof "available" Se (Lindstrom 1980).

One of the definitive methods for determining if Se is anessential micronutrient is that of establishing whether gowth isinhibited if the element is omined (Anderson anC Scaif teel;.This is satisfied for C. breviturnra (Tables l-3; Fig. l, 2). Wehave ruled out Se antagonism (Table 3) of phosphate toxicity, as

th_oryn in some higher plants (e.g. Broyer it al. 1972),'and,SOa'- interaction or antagonism (Schrift 1954; Wheeler et al.1982). Stimulation of C. brevirurrita growth by several chemi_cally unrelated sources of Se, SeIv, SevI, seienomethionine,and DMSe further suggests that there is a true requirement for Sewhich may perform some specific enzymatic function, such asin glutathione peroxidase in mammalian systems and formatedehydrogenase in bacterial systems (Stadtman 1979).

Assays of Dickie lake water suggest that when tested, Se maybe limiting growth of C. brevitutita and, could be a possibletriggering factor for blooms. This is in addition to the itimula-tlon by N and P, as would be expected in an oligotrophic lake.Growth in Cinder Lake, which had supported a iizabie popula_tion of C. brevitunita in-situ, was marginally but signiiica,nttystimulated. (24Vo, p < 0.05) by the Se addition.-Althougirconcentrations of total Se in freshwaters rarely exceed 0. I pgll.(l.3nIuO (Lindstnim 1983; Nriagu and Wong l9g3), ceil deisi_ties of blooms in affected lakes are roughly two orders ofmagnitude less than in culture (approximately 3 x 103 vs. 3 x105 cells/ml). Concentrations of SeIv or Seut necessary tostimulate growth in situ or in continuous culture may be lowerthan observed in the laboratory thus far.

Dissolved organic Se fractions (e.g. amino acids), which arealso utilized by the alga, are important in the biogeochemistry ofthe element in marine and fresh waters (Cutter and Bruland1984; Takayanagi and Wong 1984). Although similar growthstimulation by selenomethionine has been dimonstrated withother algae (Lindstr<im 1983), we believe this may be the fintdemonstration of utilization of DMSe as a Se source. As amajority of Se in acidified waters may be deposited in sediments(Schindler et al. 1980a; Nriagu and Wong tggl), it is interestingto note that the microflora and aquatic sediments are capable o1recycling Se as DMSe and other odor-producing forms (-Chau etal.1976).

One additional characteristic of the nearly unialgal bloomsof C. breviturrita is its abrupt and unpredictable 6ccurrence(Nicholls et al. 1982). Although atmospheric SO2 and Se emis-sions are fairly ubiquitous in eastern Nortn emerita (Andren etal.1975; Furr et al. 1977), a systematic, daily study of aerosolsidentified a _distinctly episodic nature to the transport of Se(along with SOo'- and As), which was linked to diitant coal_fired power plants (Parekh and Husain lggl). Such short_termpulses of Se, while potentially stimulator], may not be detectedin routine surveys of aqueous trace elementj. In addition torecycled organic Se from sediments, these atmospheric pulsesmay also prove to stimulate populations in lakes influenied byacid precipitation.

Acknowledgments

Financial support, provided by the Ontario Ministry of the Environ_ment, the Academic Development Fund of the University of Western

1781

100

1000

l0

50

100

1

lll+

E

tn

(l,U

c,o

=E

!to

i,i Y.*"i *lJFrc. 4. Growth responses of C. breviturrita (as cell yield) to organicSe compounds relative to controls and inorganic SeIv addition (all Seconcentrations 50 pg Se/L; SeIv : selenite as HzSeOi; DMSe =dimethylselenide; DMSez : dimethyldiselenide; Se-Met : seleno-oL-methionine; Met = pr-methionine without Se equimolar to Se-Met;Se-Cym = selenocystamine; rr : 3, X -r so).

Discussion

Physiological studies on marine Chrysochromulina spp.demonstrated an apparent requirement for Se, although dose-response relationships were erratic (Pintner and Provasoli 1968).Experiments with C. brevirurrita clearly demonstrated a signifi-cant growth response relative to SeIV concentrations, spanningmore than three orders of m_agnitude (0.05-l00pg Se/L). Asimilar dose-response to SeIv has been reported for the dino-

Can. J. Fish. Aquat. Sci., Vol.42, l98S

Ontario, and the Natural Sciences and Engineering Research Council ofCanada, is gratefully acknowledged' Technical assistance ofD. J. Rose

was most appreciated. Discussions with K. H' Nicholls (LimnologySection, Ontario Ministry of the Environment, Rexdale, Ont.) and J.

O. Nriagu (National Water Research Institute, Burlington, Ont.) during

the study were most helpful. The lake water samples for assays were

collected by staff of the Ontario Ministry of the Environment, Dorset,

Ont.

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