Aquatic Toxicology (2012) Contents lists available at SciVerse ScienceDirect. Aquatic Toxicology

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1 Aqutic Toxicology (212) Contents lists ville t SciVerse ScienceDirect Aqutic Toxicology jou rn l h om ep ge: Accumultion of selenium in Ulv sp. nd effects on morphology, ultrstructure nd ntioxidnt enzymes nd metolites Michel Schivon,, Isell Moro, Elizeth A.H. Pilon-Smits c, Vlerio Mtozzo, Mrio Mlgoli, Frncesc Dll Vecchi DAFNAE, University of Pdov, vile dell Università 16, Legnro 352, Itly Biology Deprtment, University of Pdov, vi Ugo Bssi, Pdov 35131, Itly c Biology Deprtment, Colordo Stte University, Fort Collins, CO 8523, USA r t i c l e i n f o Article history: Received 15 My 212 Accepted 24 June 212 Keywords: Ulv Selenium Sulfur Antioxidnts Ultrstructure Photosynthetic ctivity s t r c t The impct of selenium (Se) on Ulv sp., green mcrolg nturlly growing in the Venice Lgoon, ws investigted. The lg ws provided for 1 dys with concentrtions of selente (N 2 SeO 4 ) rnging from to 1 M. Se ccumultion in the lgl iomss ws linerly relted to the selente dose nd this reltionship ws not ffected y the high sulfte concentrtion mesured in the sewter. The mount of Se mesured in the lg ws lwys reltively low nd not hzrdous to lgl consumers. However, Se induced the formtion of hydrogen peroxide (H 2 O 2 ) in Ulv sp. nd, s result, the ctivity of ntioxidnt enzymes (superoxide dismutse, SOD, nd ctlse, CAT) nd the mount of ntioxidnt metolites (phenols, flvonoids nd crotenoids) incresed, even when selente ws supplied to the mcrolg t low concentrtion ( M). This indicted tht different components of the ntioxidnt defence system plyed pivotl role in overcoming oxidtive dmge y Se in the mcrolg, nd explined the lck of morphologicl nd ultrstructurl ltertions in Ulv sp. exposed to selente. 212 Elsevier B.V. All rights reserved. 1. Introduction Selenium (Se) is very importnt element from n ecotoxicologicl point of view due to the nrrow concentrtion rnge existing etween its essentility nd toxic effect to humn nd niml helth (Pilon-Smits nd LeDuc, 29; Zhu et l., 29). In the qutic environments, Se occurs principlly in two oxidtion sttes, Se 3+ (selenite) nd Se 6+ (selente) (Plnt et l., 24). The rtio etween selenite nd selente depends on the wter ph nd on the presence of complexing gents nd orgnic mtter (Pyrzynsk, 1998). Generlly, selenite domintes under reducing conditions, while selente is minly found in oxidizing lkline wters. Furthermore, selente is highly solule nd thus more ioville thn selenite to qutic orgnisms (Chpmn et l., 21; Plnt et l., 24). Orgnic selenides cn lso exist in nturl wters, lthough t lower concentrtion thn inorgnic Se compounds (Fn et l., 22). Corresponding uthor. Tel.: ; fx: E-mil ddresses: michel.schivon@unipd.it (M. Schivon), isell.moro@unipd.it (I. Moro), epsmits@lmr.colostte.edu (E.A.H. Pilon-Smits), vlerio.mtozzo@unipd.it (V. Mtozzo), mrio.mlgoli@unipd.it (M. Mlgoli), frncesc.dllvecchi@unipd.it (F. Dll Vecchi). Uptke studies indicte tht selenite nd selente cn e incorported into lgl cells (De Alcntr et l., 1998; Wheeler et l., 1982) nd ffect growth in dose-dependent mnner (Umisová et l., 29). At low concentrtion, Se cts s eneficil element y promoting norml cell growth nd function, s oserved in plnts (Pilon-Smits et l., 29; Reunov et l., 27). For severl mrine unicellulr lge, including the green lg Chlmydomons reinhrdtii, Se hs even een recognized s n essentil nutrient, eing component of importnt seleno-enzymes similr to those identified in mmmls (Fu et l., 22; Hrrison et l., 1988; Novoselov et l., 22). However, t high dose Se is toxic to lge, leding to reduction of growth rte or ltertions in the levels of rective oxygen species (ROS) tht my cuse cellulr dmge (Fournier et l., 21; Pelh nd Cohen, 25; Umisová et l., 29; Wheeler et l., 1982). In the freshwter microlg Chlorell zofingiensis, the tretment with selenite cused n increse in ctivity of ntioxidnt enzymes, including superoxide dismutse (SOD) isoforms (Pelh nd Cohen, 25). In recent study, Chlorell vulgris ws shown to produce higher mount of phytocheltins nd glutthione (GSH) in response to toxic selente concentrtions (Simmons nd Emery, 211). The toxic effects of Se on mrine lge depend on the lg species (Adel-Hmid nd Skulerg, 26; Dzhi et l., 23; Wheeler et l., 1982), Se concentrtion, nd lso on the oxidtion stte of the element (Pstierov et l., 29; Umisová et l., 29). Indeed, X/$ see front mtter 212 Elsevier B.V. All rights reserved.

2 M. Schivon et l. / Aqutic Toxicology (212) lthough high cellulr concentrtion of either selenite or selente my cuse oxidtive stress or cell poptosis, selenite ws found to e less toxic thn selente in mny cses, t lest in microlge (Wheeler et l., 1982). The uptke of inorgnic Se species (selente nd selenite) is known to vry s function of ph over the rnge 5 9 (Riedel nd Snders, 1996; Tuzen nd Sri, 21). In C. reinhrdtii the mximum uptke of selente occurred t ph 8, wheres selenite uptke incresed significntly t the lower ph vlues (Riedel nd Snders, 1996). Selenium ccumultion in lge cn e lso ffected y the presence of certin mcronutrients, like phosphorus (P) nd sulfur (S) (Lee nd Wng, 21). Sulfte, in prticulr, is well-known ntgonist of selente (Fournier et l., 21; Simmons nd Emery, 211; Willims et l., 1994). In C. reinhrdtii the toxicity of selente ppered to e directly correlted to intrcellulr Se ccumultion, which ws directly dependent on the mient concentrtion of sulfte tht my compete with selente for the trnsport proteins (Fournier et l., 21). In the sme microlg nd in Selenstrum cpricornutum, incresing sulfte concentrtion in the growth sustrte resulted in sustntil decrese of selente (Riedel nd Snders, 1996; Willims et l., 1994) nd selenite (Morlon et l., 26) uptke, nd the green microlg Scenedesmus qudricud ws found to e more sensitive to selenite nd selente under S deficient conditions (Umisová et l., 29). Since S. cpricornutum cells supplied with different concentrtions of selente nd sulfte exhiited different cpcity to tke up selente even though the S:Se molr rtio ws mintined, the existence of different permese ffinities for sulfte nd selente nd/or of more permese systems for these ions in lge hs een hypothesized (Willims et l., 1994). Mcrolge my hve gret potentil s Se ioindictors, due to their wide distriution nd lrge sizes (Lee nd Wng, 21). Furthermore, numer of species, including Ulv sp., cn e introduced in the humn nd niml diet, especilly in the form of dietry supplements, eing considered rich source of nturl ntioxidnts (Dun et l., 26; Fleurence, 1999; Kud et l., 25; Zhng et l., 23). To our knowledge dt concerning the effects of Se in microlge re well documented, while no studies hve een performed to elucidte in detils the cellulr response to this element y mcrolge. On this ccount, the current reserch is imed t investigting the cpility to ccumulte nd tolerte Se y green lminr seweed Ulv sp., growing nturlly in the Venice Lgoon. Sulfur content in the lg nd in the sewter ws determined s potentil fctor ffecting Se ccumultion of Ulv. The effects of Se ccumultion in the lg were ssyed mesuring the ctivity of ntioxidnt enzymes nd quntifying ntioxidnt non-enzymtic metolites. Additionlly, nlyses of ultrstructure, morphology nd photosynthetic efficiency were performed. 2. Mterils nd methods 2.1. Algl mteril nd experimentl conditions Thlli of Ulv sp. were collected in Mrch 21 from the Venice Lgoon (Itly). Species elonging to this genus show very simple morphology nd certin degree of phenotypic plsticity, hevily influenced y environmentl conditions, mking difficult the delinetion of species, sed only on morphologicl fetures (Loughnne et l., 28). For this reson, we prefer to refer to Ulv sp., rther thn specific species. Once collected, thlli were thoroughly rinsed in sewter nd clened using soft rush to eliminte the epiphytes present on their surfce. Susequently, thlli were cut in 15 mm dimeter disks nd weighed. Disks of sme weight (±5% vrition) were plced in flsks contining 1 L of filtered sewter (Millipore GF/C, 1 2 m pore size), nd kept for 3 dys to cclimte inside climtic chmer with 14 h light/1 h drk cycle, t temperture of 16 C nd photon flux density of 8 mol m 2 s 1 ccording to Dll Vecchi et l. (27, 212). The initil ph of the sewter in the flsks ws 7.2. In ech flsk 1 disks were cultivted. After cclimtion, Se in the form of sodium selente (N 2 SeO 4, Sigm Aldrich, Steinheim, Germny) ws dded to the sewter t the following concentrtions: (control), 1, 5 or 1 M. The level of Se in the sewter efore selente ddition ws undetectle, eing elow the limit determined vi ICP-AES. The wide rnge of selente concentrtions ws useful to determine the reltionship etween physiologicl nd ultrstructurl chnges with incresing selente doses. For ech Se concentrtion five replictes were performed. Disks nd sewter were smpled t the eginning of the experiment nd t the 1th dy of tretment. Before nlyses, thlli were crefully wshed with distilled wter to remove ny Se ound to surfce. For dry weight mesurements, 2 disks from ech flsk were used Elementl nlysis of Se nd S Seweed thlli were dried for 48 h t 8 C, nd 1 mg of thlli dry weight per ech tretment were then digested in nitric cid (99%, v/v) s descried y Zrcins et l. (1987). Inductively coupled plsm tomic emission spectroscopy (ICP-AES, Spectrum CirosCCD, Kleve, Germny) ws used s descried y Fssel (1978) to determine ech digest s Se nd S concentrtions. The otined vlues were expressed in mg element kg 1 dry weight. The determintion of Se nd S ws performed in the sewter: (1) efore the ddition of selente; (2) immeditely fter the ddition of selente; (3) fter 1 dys from the ddition of selente either in the presence or sence of Ulv sp. thlli. Se nd S were directly quntified in 1 ml of filtered (.2 m) sewter smples using ICP-AES s descried y Fssel (1978). No preliminry digestion procedure ws performed efore the nlysis. Results were expressed in mg L Sulfte nd selente content Seweed thlli (5 mg) were ground in liquid nitrogen nd then 1 ml of distilled wter were dded. The smples were incuted for 2 h in heting lock t 85 C. The otined extrcts were filtered onto.45 m (Millipore) nd nlyzed for sulfte content y HPLC using Dionex IonPc AS11 4 mm column, coupled to gurd column AG 14 nd CD2 Conductivity Detector. The column ws eluted over period of 18 min with 3.5 mm N 2 CO 3 /1 mm NHCO 3 in H 2 O, t flow rte of.9 ml/min nd t 14 PSI pressure. For the mesurement of wter sulfte concentrtion, smples of sewter were first filtered onto.45. Then the smples were nlyzed vi HPLC using the sme procedure descried ove. To check the consistency of mient selente concentrtions during experiments, culture medium smples were nlyzed y HPLC s reported for sulfte. Sulfte contents in seweeds nd in sewters, s well s selente content in sewter, were expressed in mg kg 1 fresh weight nd mg L 1, respectively Quntifiction of pigments nd photosynthetic oxygen evolution Chlorophyll nd crotenoids were determined in thlli of Ulv sp. fter 3 nd 1 dys of tretment, using N,N-dimethylformmide (1:1) (Morn nd Porth, 198). The extrcts were kept in the drk for 1 dy t 4 C (Wellurn, 1993) nd then nlyzed

3 224 M. Schivon et l. / Aqutic Toxicology (212) spectrophotometriclly (124 Perkin-Elmer; Norwlk, CT, USA) t 664 nm for Chl, 647 nm for Chl, nd 48 nm for crotenoids. The concentrtions of chlorophylls nd crotenoids were clculted using the extinction coefficients ccording to Inskeep nd Bloom (1985) nd expressed in mg g 1 fresh weight. Photosynthesis ws mesured s oxygen relese in control nd Se-treted seweeds using n oxygen monitor (Ysi Model 53, Yellow Spring Instrument Co., OH, USA). For the nlysis, single disks were cut into 3 4 mm long segments to improve the efficiency of the method, ccording to Ishii et l. (1977) nd Rscio et l. (1991). Seweed disks were crefully rinsed with distilled wter nd the suspension medium used for the ssy ws the sewter used s the culture medium in the flsks. To verify the possile effects of Se on the integrity of photosynthetic pprtus, the oxygen evolution ws lso determined in control nd Se-treted Ulv disks t the end of the experimentl period (1 dys) using fresh filtered sewter (ph 7.2) s suspension medium. The mesurements were crried out under sturting light (14 mol photon m 2 s 1 PAR) using 15 W lmp (Philips, Achen, Germny) s light source. The tues were plced inside thermosttic th t 2 C nd kept stirred. The oxygen evolution rte ws expressed s mol O 2 mg 1 chlorophyll h 1.. Light nd electron microscopy Smples from control nd selente-treted thllus disks of Ulv sp. were fixed overnight t 4 C in 3% glutrldehyde in.1 M sodium ccodylte uffer (ph 6.9) nd post-fixed t 4 C for 2 h in 1% osmium tetroxide in the sme uffer. The specimens were dehydrted in grded series of ethyl lcohol nd propylene oxide nd emedded in rldite. Sections were cut using n ultrmicrotome (Ultrcut S, Reichert-Jung, Wien, Austri). For trnsmission electron microscopy, ultrthin sections (6 Å) stined with urnyl cette nd led citrte were oserved with trnsmission electron microscope (TEM 3, Hitchi, Tokyo, Jpn) operting t kv. For light microscopy, thin sections (1 m) stined with toluidine lue (1% sic toluidine nd 1% N tetrorte, 1:1, v/v) were oserved y DMR 5 Leic (Sweden) microscope, equipped with digitl imge cquisition system In situ determintion of hydrogen peroxide Intrcellulr production of hydrogen peroxide (H 2 O 2 ) in Ulv sp. ws mesured using dichlorofluorescein dicette (H 2 DCHF- DA, Moleculr Proes, Leiden, The Netherlnds) s fluorescent dye. Once inside cells, H 2 DCHF-DA cn e oxidized into highly fluorescent 2,7 -dichlorofluorescein (DCF) y intrcellulr H 2 O 2 nd other peroxides. Cells of control nd selente-treted lge were oserved using n epifluorescence microscope (LEICA DMR) t n excittion wvelength of 48 nm. A totl of 3 cells for ech smple were nlyzed for DFC positivity. Dt re expressed s percent Antioxidnt enzyme ctivity mesurement Superoxide dismutse (SOD) nd ctlse (CAT) ctivities were mesured in oth control nd selente-treted thllus disks of Ulv sp. Smples were homogenized in HCl 1 mm, KCl.15 M, sucrose.5 M ph 7.6 s extrction uffer, using n Ultrtturrx T8 (IKA). The homogentes were sonicted for 1 s t 4 C, centrifuged t 13,4 g for 3 min t 4 C, nd the superntnt (SN) ws collected for enzyme ctivity mesurement. Totl SOD ctivity ws mesured in SN with the xnthine oxidse/cytochrome c method ccording to Crpo et l. (1978). The rection mixture contined 1 L SN, 46.5 M K 2 PO 4 /K 2 HPO 4 (ph 8.6),.1 mm EDTA, 195 M hypoxnthine, 16 M cytochrome c, nd U xnthine oxidse. The cytochrome c reduction y superoxide nion generted y the xnthine oxidse/hypoxnthine rection ws detected t 55 nm t room temperture for 3 s. Enzyme ctivity ws expressed s U SOD mg 1 protein, one unit of SOD eing defined s the mount of smple tht cuses 5% inhiition in the ssy conditions. CAT ctivity ssy ws mesured ccording to the method of Aei (1984). Decreses in sornce of solution composed of 1 L of SN, 5 mm H 2 O 2 (ε =.436 mm 1 cm 1 ) in 5 mm phosphte uffer (KH 2 PO 4 /N 2 HPO 4, ph 7.8), were continuously recorded t 24 nm t 1 s intervls for 1 min. Results were expressed s U CAT mg 1 proteins, one unit of CAT eing defined s the mount of enzyme tht ctlyzes the dismuttion of 1 mol of H 2 O 2 in 1 min t 25 C. For oth SOD nd CAT ssys, the totl protein concentrtion in SN ws determined vi the Brdford method (1976) using ovine serum lumin (BSA) s stndrd Extrction nd mesurement of solule phenols nd flvonoids Solule phenolic cids were extrcted from control nd Setreted thlli disks of Ulv sp. y crushing them (1 g) in mortr in the presence of pure methnol (1:1, w/v). The extrcts were plced in n ice th for 1 h nd centrifuged t 3 g for 4 min t 4 C. The superntnts were stored t 2 C until use. Totl phenols were mesured ccording to Arnldos et l. (21). One ml of 2% N 2 CO 3 nd L of Folin Cioclteu regent (Sigm Aldrich) were dded to 1 L of phenolic extrct. After 15 min incution t 25 C in the drk, the sornce t 725 nm ws mesured. Gllic cid ws used s stndrd. Flvonoids were extrcted from thlli (1 g) in 5 ml of cidified methnol solution. The extrcts were kept t 4 C for 16 h efore mesuring the sornce t 3 nm. Phenols nd flvonoids were expressed s gllic cid equivlents g 1 fresh weight Sttisticl nlysis A one-wy nlysis of vrince (one-wy ANOVA) ws pplied to the dt. Sttisticl nlysis ws performed using SPSS 1. (Norusis, 1993). All proilities were two-tiled. Dt were checked for normlity nd homogeneity of vrince (Levene test) nd re presented s men ± SD of five replictes. Differences etween mens were evluted for significnce t P <.5 y using the Duncn s multiple rnge test (DMRT). Sttisticlly significnt differences t P <.5 were indicted y different letters reported in tles nd figures. Similr letters in tles nd figures indicte no significnt differences etween men vlues. 3. Results 3.1. Impct of Se on lgl growth nd culture medium ph The effect of selente on Ulv sp. growth ws evluted in terms of dry weight production reltive to the control tretment ( Se) t 1 dys (Fig. 1). The ppliction of selente to Ulv sp. incresed the dry weight of thlli (roughly 15% more thn the control). Interestingly, this increse ws the sme t ll externl selente concentrtions. The ph vlue of the sewter in the flsks efore the ddition of selente nd Ulv sp. thlli ws 7.2. After 1 dys, the ph of the sewter supplemented with selente without the thlli, ws slightly higher ( ) thn the ph of control sewter (Fig. 2). When Ulv thlli were cultivted in the flsks, the ph vlues were lower thn 8 in the first 6 dys, with no difference etween tretments (dt not shown). A remrkle increse of the sewter ph (from 8.46

4 M. Schivon et l. / Aqutic Toxicology (212) D.wt. % Se M Fig. 1. Effect of different selente concentrtions on dry weight (d.wt.) of Ulv sp. thlli. Vlues re reported s percent of control, which ws set t 1%. Different letters ove rs indicte significnt differences etween tretments (P <.5, ±SD). ph d c - Ulv + Ulv cd cd 1 Se cd 5 5 M cd 1 cd 1 Fig. 2. Vlues of ph in control sewter nd Se-dded sewter either used for Ulv sp. cultivtion (+Ulv) or let inside the flsks without lge ( Ulv). The vlues reported were mesured fter 1 dys since the eginning of the experiment. At time, the ph of sewter ws 7.2. Different letters ove rs indicte significnt differences etween tretments (P <.5, ±SD). t Se to 9.71 t 1 M Se) ws oserved t the end of the experiment (1 dys). It is noteworthy tht the ph mesured t 1 dys in sewter where Ulv sp. thlli were grown ws not significntly different etween control ( Se) nd, 1, 5 M Se Determintion of Se, S nd sulfte in Ulv sp. thlli, nd quntifiction of Se, S, sulfte nd selente in sewter The nlysis of nutrient concentrtions in the sewter ws used to ssess the potentil impct nd fte of Se in lge. The level of Se nd S in the sewter culture medium ws mesured t the eginning of the experiment nd fter 1 dys of Ulv sp. thllus cultivtion (Tle 1). The concentrtion of totl Se ws dditionlly mesured in the sewter contined in the flsks where Ulv thlli were not cultivted. This mesurement ws performed in order to evlute whether microorgnisms potentilly growing in the sewter contriuted to the removl of Se fter 1 dys. As reported in Tle 1, the concentrtion of totl Se did not chnge in sewter fter 1 dys exposure, nd results otined vi HPLC confirmed tht mient Se remined ll in the selente form during the experimentl period if Ulv sp. thlli were not cultivted in the flsks (dt not shown). Tle 1 Concentrtion of selenium (Se), sulfur (S) nd sulfte (SO4 2 ) in the sewter used either s the culture medium of Ulv sp. thlli (+Ulv) or let inside the flsks without thlli ( Ulv). The mesurements were performed t the eginning of the experiment (t = ), nd t the end (t = 1 dys). In ech column, vlues re the men of five replictes. Different letters indicte significnt differences mong tretments (P <.5, ±SD) nd mong conditions: t =, t = 1 dys ( Ulv) t = 1 dys (+Ulv). The sttisticl nlysis ws performed independently for Se, S nd SO4 2. Se ( M) Se (mg L 1 ) S (mg L 1 ) SO4 2 (mg L 1 ) t = t = 1 dys( Ulv) t = 1 dys(+ulv) t = t = 1 dys( Ulv) t = 1 dys(+ulv) t = t = 1 dys ( Ulv) t = 1 dys (+Ulv) <.5 <.5 < ± ± ± ± ± ± ±.12h.193 ±.17h.15 ±.1i ± ± ± ± ± ± ±.11f.767 ±.19f.635 ±.15g ± ± ± 22, ± ± ± ±.212d ±.35d ±.21e 924 ± ± ± ± ± ± ± ± ±.97c ± ± ± ± 69, ± ± ± ± ± ± ± ± ± ± ±

5 226 M. Schivon et l. / Aqutic Toxicology (212) Tle 2 Concentrtion of selenium (Se), sulfur (S) nd sulfte (SO 4 2 ) in thlli of Ulv sp. The mesurements were performed in thlli fter 1 dy-cultivtion period in the sence (control) or in the presence of vrying doses of selente. In ech column, vlues re the men of five replictes nd different letters indicte significnt differences etween tretments (P <.5, ±SD). Se ( M) Se (mg kg 1 d.wt.) S (g kg 1 d.wt.) SO 4 2 (mg kg 1 d.wt.) <.5f ± ± ±.26e ± ± ±.44d ± ± ±.243c ± ± ± ± ± ± ± ± Sulfur in sewter ws present lmost exclusively in the form of sulfte (Tle 1). Both totl S nd sulfte concentrtion were constnt throughout the experimentl period nd vlues were similr in the Se-tretment nd in the control, regrdless the presence of thlli (Tle 1). The ccumultion of Se in thlli of Ulv sp. linerly correlted with the culture medium concentrtion of selente (y =.293x, R 2 =.992) (Tle 2). Selente pprently did not ffect the content of totl S in the thlli, ut t doses s high s nd 1 M significntly incresed the level of sulfte (Tle 2). Tle 3 Photosynthetic oxygen evolution y Ulv sp. mesured using s suspension medium the sewter culture medium (ph > 8.5) or fresh sewter (ph = 7.2). O 2 mesurement ws performed in thlli fter 1 dy-cultivtion period either in the sence (control) or in the presence of vrying doses of selente. In ech column, vlues re the men of five replictes nd different letters indicte significnt differences etween tretments (P <.5, ±SD). Se ( M) mol O 2 mg 1 Chl h 1 Culture medium Fresh sewter 13.3 ± ± ±.93c 15.2 ± ± 1.2c ± ± ± ±.97c ± ± 1.98c ± 1.46 The content of chlorophyll (+) in thlli cultivted for 1 dys in the presence of selente did not chnge significntly compred to thlli of the minus Se condition, lthough t the 3rd dy n increse of these pigments ws oserved t the highest selente concentrtions (Fig. 3A). On the contrry, the level of crotenoids significntly incresed in the thlli exposed to selente oth t 3 nd 1 dys of exposure, especilly when Se ws supplied t high doses rnging from 5 to 1 M (Fig. 3B) Effect of Se on chlorophylls, crotenoids nd photosynthetic O 2 evolution The chlorophyll quntifiction ws performed in Ulv sp. thlli fter 3 dys nd 1 dys from the eginning of the experiment. A time 3 dys 1 dys mg Chl(+) g -1 f.wt B mg Cr g -1 f.wt time 3 dys 1 dys 1 Se M Se M 5 1 Fig. 3. Effect of different selente concentrtions on the level of chlorophyll (A) nd Crotenoids (B). The mesurements were performed fter 3 nd 1 dys of Ulv sp. cultivtion. Different letters indicte significnt differences etween tretments (P <.5, ±SD). Vlues re expressed s mg pigment g 1 fresh weight. Fig. 4. Light microscopy photogrphs of control (A) nd 1 dys-se-treted (B F) thlli of Ulv sp.

6 M. Schivon et l. / Aqutic Toxicology (212) The oxygrphic nlysis demonstrted lower photosynthetic O 2 evolution in thlli cultivted for 1 dys with Se thn in the control when the sewter contined in the flsks ws used s the suspension medium (Tle 3). However, if the photosynthetic O 2 emission ws mesured in Se-treted Ulv sp. thlli suspended in fresh sewter (ph = 7.2), the vlues of net photosynthesis were higher, lthough not significntly, thn those reported for the controls. A similr increse of photosynthetic O 2 evolution ws oserved during the first dys of Ulv sp. cultivtion in the presence of Se (dt not shown). Interestingly, the net photosynthesis did not chnge in thlli cultivted without Se when they were trnsferred to fresh sewter medium (Tle 3) Effects of Se on thllus morphology nd ultrstructure The thllus disks of Ulv sp. cultivted in the presence of selente concentrtions rnging from to 5 M shred similr thickness nd ilyered morphology with those of the control condition (Fig. 4A F). Only t the highest Se dose (1 M) ws the thickness of Ulv thlli reduced (Fig. 4F). With respect to cell ultrstructure, no significnt ltertion ws oserved etween control thllus disks nd the selente-treted ones (Fig. 5A F). Indeed, thllus cells of ll experimentl conditions showed norml orgniztion, with well-visile plstid chrcterized y the presence of undnt strch nd pyrenoid in the strom Cytochemicl nlysis nd effects of Se on ntioxidnt enzymes Ulv sp. thlli treted with selente ove 1 M showed the production of hydrogen peroxide, s evidenced y the presence of fluorescent signl in the cytoplsm of cells (Fig. 6). Interestingly, the ctivity of ntioxidnt enzymes SOD nd CAT incresed significntly for ll Se tretments (Fig. 7A nd B). The highest levels of SOD ctivity were oserved t M nd 1 M Se (Fig. 7A). The vrition pttern of CAT ctivity lso showed n increse reltive to the controls (Fig. 7B). Interestingly, the difference in SOD ctivity etween Se-treted nd control thlli ws higher (1 fold) thn tht reported for CAT (2 fold) Effects of Se on totl phenols nd flvonoids The exposure of Ulv sp. thlli to selente enhnced the level of phenolic compounds (Fig. 7C nd D). The totl phenol content incresed of 3.5 fold even t the lowest Se concentrtion pplied ( M) (Fig. 7C), while vlues of flvonoid content were pproximtely 1.5 fold higher in thlli cultivted with Se concentrtions rnging from 1 to 1 M thn in the control (Fig. 7D). Fig. 5. Trnsmission electron microscope detils of cells of control (A) nd 1 dys-se-treted (B F) thlli of Ulv sp. Note the chloroplsts with strch in the strom. No evident ltertions of ultrstructure re visile in cells.

7 228 M. Schivon et l. / Aqutic Toxicology (212) Fig. 6. Detection of H 2O 2 production with DCFH-DA of control (A) nd Se-treted (B F) thlli of Ulv sp. Note the green fluorescence signl in thlli of Ulv sp. cultivted with Se. (For interprettion of the references to color in this figure legend, the reder is referred to the we version of this rticle.) 4. Discussion Mcrolge my represent importnt ioindictors of metl pollution in freshwter nd mrine environments ecuse of their distriution, lrge size, longevity, presence t pollution sites, nd ility to ccumulte metls to stisfctory degree (Lee nd Wng, 21). The essentility of Se for mcrolge hs not een demonstrted yet, while for mny phytoplnkton species this element is needed t low doses (Arie nd Shiriw, 29). Toxic effects of Se on lge re generlly evluted using phytotoxicity tests sed on growth inhiition. In our study, the toxicity of selente to Ulv sp. ws ssyed y mesuring the effect of rod rnge of selente concentrtions on lgl growth during 1 dys-exposure period. Interestingly, selente t concentrtions up to 1 M did not inhiit Ulv sp. growth t ll tested doses; rther, this form of Se exerted positive effect on lgl iomss y incresing the dry weight of thlli y 15%. Despite the dvntges of low Se concentrtions on growth tht hve lso een reported for numer of lnd plnts (Hrtikinen, 25; Pilon-Smits et l., 29), inhiition of growth y selente is frequently oserved in microlge (Fournier et l., 21; Geoffroy et l., 27; Pstierov et l., 29; Reunov et l., 27). This inhiition is proly ecuse in most of these studies environmentlly relevnt Se concentrtions (up to 4 M) re used, which exceed the Se requirements for norml growth y microlge. Moreover, microlge generlly exhiit stronger cpcity of Se ioccumultion compred to mcrolge, so tht Se levels inside cells re often high enough to cuse toxicity (Fournier et l., 21; Geoffroy et l., 27; Reunov et l., 27; Umisová et l., 29). For exmple, S. qudricud cells ccumulted 373 mg Se kg 1 dry weight when exposed to 5 mg L 1 Se nd cultivted in the presence of 4 mm S (Umisová et l., 29). The cpcity of Ulv sp. to ccumulte Se ws likely ffected y the high sulfte concentrtion mesured in the culture medium. Indeed, owing to its chemicl similrity with selente, sulfte my

8 M. Schivon et l. / Aqutic Toxicology (212) A U SOD mg -1 protein C mg gllic cid equivlents g -1 f.wt d SOD c Phenols c c 1 5 Se M 1 5 Se M 1 1 B D mg gllic cid equivlents g -1 f.wt. U CAT mg -1 protein c CAT Flvonoids 1 5 Se M 1 5 Se M 1 1 Fig. 7. Effect of different selente concentrtions on superoxide dismutse (SOD, A) nd ctlse (CAT, B) ctivities, nd on the content of phenols (C) nd flvonoids (D). The mesurements were performed in thlli of Ulv sp. fter 1 dy-cultivtion period. Different letters indicte significnt differences etween tretments (P <.5, ±SD). The enzyme ctivity is expressed s U enzyme mg 1 protein, the content of phenols nd flvonoids s mg gllic cid equivlents g 1 fresh weight. reduce selente uptke y lge s consequence of the competition for trnsport into cells (Fournier et l., 21). In support of this hypothesis, there is rod evidence tht selente nd sulfte compete for uptke t the level of sulfte trnsporters in plnts nd microlge (Fournier et l., 21; Neumnn et l., 23; Riedel nd Snders, 1996; Schivon et l., 212; Terry et l., 2; Umisová et l., 29; Willims et l., 1994). In Chlorell vulgris nd Chlmidomons reinhrdtii, for instnce, the uptke of selente negtively correlted with sulfte concentrtions in the growth medium (Fournier et l., 21; Riedel nd Snders, 1996; Shrift, 1954). Interestingly, the liner reltionship etween Se content in Ulv sp. iomss nd mient selente concentrtion ws not ltered y the high sulfte concentrtion in sewter. A positive correltion etween Se nd selente ws lso oserved in the microlg S. qudricud (Umisová et l., 29). Selente nd sulfte re lso elieved to shre the sme ssimiltion pthwy in plnts nd microlge, which re usully more sensitive to selente when cultivted under S strvtion (Schivon et l., 212; Simmons nd Emery, 211; Umisová et l., 29). Vrition of sulfte content in lgl iomss my occur when lge sor selente, since the two nions re ntgonist sustrtes for the first enzyme of the S ssimiltory pthwy (Pilon-Smits et l., 1999; Schivon et l., 28, 212). In Ulv sp. sulfte ccumulted significntly more when thlli were exposed to high concentrtions of selente ( nd 1 M), while totl S level did not chnge in response to Se tretments. This my suggest tht the uptke of sulfte ws not ffected y selente, nd incresed sulfte ccumultion in thlli ws the result of the reduced delivery into the S metolic flux, due to Se interfering with S ssimiltion in this mcrolg. Selente pprently did not cuse significnt ltertions of Ulv sp. thllus morphology nd ultrstructure. Conversely, previous studies provided evidence of numer of ultrstructurl chnges induced y selente in microlge, nd highlighted chloroplsts s the first trget of Se cytotoxicity (Geoffroy et l., 27; Vítová et l., 211). The lck of chloroplst modifictions in Ulv sp. cells ws in greement with the lck of growth inhiition y Se. The Se tretments significntly up-regulted the levels of ntioxidnt compounds nd enzymes. These likely contriuted to the selente resistnce y Ulv sp. nd enhnced growth, due to their protective effects on memrne integrity. Crotenoids, in prticulr, re known to protect chloroplst memrnes from dmge cused y rective oxygen species (ROS) produced under stress (Young, 1991; Hvux, 1998). In our study, the formtion of hydroxide peroxide ws reveled in thlli of Ulv sp. exposed to selente, nd the concomitnt synthesis of crotenoids ws oserved. Despite the sence of chloroplst ltertions, the photosynthetic ctivity of Ulv sp. decresed fter 1 dys of selente exposure, s indicted y the oxygrphic nlysis. Previous studies hve shown in mny species of Ulvcee CO 2 -users the inhiition of the cpcity to sor CO 2 when ph increses (Axelsson et l., 2). At vlue s high s 9.5, ph ws proven to ffect lgl photosynthesis oth y decresing free CO 2 concentrtion in the medium nd y lowering the ffinity of the lge to free CO 2 (Azov, 1982). The progressive lkliniztion of the culture medium is often oserved when lge perform photosynthetic CO 2 fixtion in closed CO 2 systems (Grnum nd Myklestd, 22). Under such conditions, in fct, the inorgnic cron equilirium in the surrounding medium is shifted towrds the formtion of HCO 3, nd the ph increses s result (Riedel nd Snders, 1996). In our study, the high ph ppered not to e the only responsile for the net photosynthesis decrese oserved in ll Se-treted Ulv sp. thlli fter 1 dys. Indeed, the ph of the sewter used for the cultivtion of thlli nd enriched with, 1 or 5 M Se ws not significntly different from tht mesured in the control medium. Furthermore, the ph in the sewter without Se fter 1 dys of Ulv sp. cultivtion ws significntly higher thn tht reported for fresh

9 23 M. Schivon et l. / Aqutic Toxicology (212) sewter, ut no differences in photosynthetic oxygen evolution were evident in thlli. Selente tretment my hve ccounted for the reduction of net photosynthesis in Ulv sp. thlli. In the first dys of cultivtion, the net photosynthesis vlues mesured in Se-treted thlli were higher thn those determined in the controls, s well s fter 1 dys of Se exposure if thlli were trnsferred into fresh sewter. The higher photosynthesis rtes likely determined the stimultion of lgl iomss production during the first dys of Ulv sp. cultivtion, nd reduced the vilility of dissolved inorgnic cron in Se-enriched sewter t 1 dys with respect to the control medium. The recovery of the cpcity to produce photosynthetic O 2 y Se-treted thlli once trnsferred in fresh culture medium indicted tht selente did not compromise the photosynthetic pprtus of Ulv sp. Although Se did not injure Ulv sp. growth, morphology nd ultrstructure, there ws evidence of hydrogen peroxide (H 2 O 2 ) formtion in the lgl cells. H 2 O 2 represents the most stle form of ROS nd the enhncement of its synthesis suggests tht Se ws le to induce oxidtive stress in Ulv sp. to some extent, s reported for plnts (Freemn et l., 21; Gomes-Junior et l., 27; Grnt et l., 211; Tmoki et l., 28). Despite their role in oxidtive stress induction, ROS cn sometimes e correlted with Se resistnce in plnts depending on their tissue level, perhps cting s signl molecules in the induction of resistnce pthwys (Tmoki et l., 28). On this ccount, we cnnot exclude tht ROS production could e useful for cquisition of Se resistnce in Ulv sp., s previously reported for Aridopsis thlin (Tmoki et l., 28). Concomitnt to higher production of H 2 O 2, the ctivity of ntioxidnt enzymes, such s SOD nd CAT, ws significntly stimulted in Ulv sp. These enzymes re involved in the sequentil scvenging of ROS, s SOD ctlyzes the dismuttion of superoxide into oxygen nd H 2 O 2, nd CAT further converts H 2 O 2 into wter nd oxygen. In the present study, the two enzymes ppered to undergo differentil regultion y Se in Ulv sp., s the enhncement of SOD ctivity ws tightly dependent on Se ccumultion, while CAT ctivity ws induced to similr degree y most of tested selente doses. Increse of SOD ctivity ws lso oserved in Chlorell zofingiensis cells exposed to selenite (Pelh nd Cohen, 25), s well s in Ulv lctuc in response to ROS production cused y different Se compounds (Ross nd Vn Alstyne, 27). Unfortuntely, no informtion concerning Se effects on CAT ctivity in lge is ville in the literture. In ddition to the stimultion of ntioxidnt enzyme ctivity, the level of phenols nd flvonoids incresed in Ulv sp. thlli cultivted in the presence of selente. Phenolic compounds re known to ply severl importnt protective roles in plnts (Mzid et l., 211; Michlk, 26). Flvonoids, in prticulr, disply criticl function in plnt defence responses ccounting for secondry ROS-scvenging system in plnts exposed to severe/prolonged stress conditions (Fini et l., 211; Posmyk et l., 29). Our findings indicte tht, similrly to plnts, Ulv sp. possess highly efficient ntioxidnt mchinery to keep the level of ROS under tight control, which involves the ctivtion of oth enzymtic nd non-enzymtic ntioxidnts. 5. Conclusion The results otined in the present study show tht Ulv sp. ccumulted Se depending on selente dose, nd Se ccumultion in thlli interfered with S metolism, s indicted y the increse of sulfte:sulfur rtio. The resistnce of Ulv sp. to selente ppers to e due to its cpcity to keep cellulr Se t low levels nd to efficiently ctivte different metolic pthwys tht involve enzymtic nd non-enzymtic ntioxidnt compounds functioning in the scvenging of ROS produced upon Se ccumultion. References Adel-Hmid, M., Skulerg, O.M., 26. Effect of selenium on the growth of some selected green nd lue-green lge. Lkes nd Reservoir Mngement 1, Aei, H., Ctlse in vitro. Methods in Enzymology 15, Arnldos, T.L., Muñoz, R., Ferrer, M.A., Clderón, A.A., 21. Chnges in phenol content during strwerry (Frgri nnss, cv. Chndler) cllus culture. Physiologi Plntrum 113, Arie, H., Shiriw, Y., 29. Selenium utiliztion strtegy y microlge. Molecules 14 (12), Axelsson, L., Mercdo, J.M., Figuero, F.L., 2. Utiliztion of HCO 3 t high ph y the rown mcrolg Lminri scchrin. Europen Journl of Phycology 35, Azov, Y., Effect of ph on inorgnic cron uptke in lgl cultures. Applied nd Environmentl Microiology 43 (6), Brdford, M., Rpid nd sensitive method for the quntifiction of microgrm quntities of protein utilizing the principle of protein-dye inding. Anlyticl Biochemistry 72, Chpmn, P.M., Adms, W.J., Brooks, M.L., Delos, C.G., Luom, S.N., Mher, W.A., Ohlendorf, H.M., Presser, T.S., Shw, D.P., 21. Ecologicl Assessment of Selenium in the Aqutic Environment. CRC Press, Boc Rton, FL. Crpo, J.D., Mccord, J.M., Fridovich, I., Preprtion nd ssy of superoxide dismutses. Methods in Enzymology 53, Dll Vecchi, F., Moro, I., Mrzocchi, M., 27. Some effects of cdmium on Ulv letevirens Areschoug collected in Lgoon Venice. Algologicl Studies 125, Dll Vecchi, F., Mrzocchi, M., Mistro, S., Moro, I., 212. Morpho-physiologicl effects of cdmium on two Ulv species. Algologicl Studies 138, Dzhi, W., Zhodi, C., Shojing, L., Yhui, G., 23. Toxicity nd ccumultion of selenite in four microlge. Chinese Journl of Ocenology nd Limnology 21, De Alcntr, S., Lopes, C.C., Wgener, K., Controlled introduction of selenium into Chlorell cells. Indin Journl of Experimentl Biology 36, Dun, X.J., Zhng, W.W., Li, X.M., Wng, B.G., 26. Evlution of ntioxidnt property of extrct nd frctions otined from red lg. Polysiphoni urceolt. Food Chem. 95, Fn, T.W.-M., The, S.J., Hinton, D.E., Higshi, R.M., 22. Selenium iotrnsformtions into proteinceous forms y foodwe orgnisms of selenium-lden dringe wters in Cliforni. Aqutic Toxicology 57, Fssel, V.A., Quntittive elementl nlyses y plsm emission spectroscopy. Science 22, Fini, A., Brunetti, C., Di Ferdinndo, M., Ferrini, F., Tttini, M., 211. Stress-induced flvonoid iosynthesis nd the ntioxidnt mchinery of plnts. Plnt Signling nd Behvior 6 (5), Fleurence, J., Seweed proteins: iochemicl, nutritionl spects nd potentil uses. Trends in Food Science nd Technology 1, Fournier, E., Adm-Guillermin, C., Potin-Gutier, M., Pnnier, F., 21. Selente ioccumultion nd toxicity in Chlmydomons reinhrdtii: influence of mient sulphte ion concentrtion. Aqutic Toxicology 97, Freemn, J.L., Tmoki, M., Stushnoff, C., Quinn, C.F., Cpp, J.J., Devonshire, J., Fkr, S.C., Mrcus, M.A., McGrth, S.P., Vn Hoewyk, D., Pilon-Smits, E.A.H., 21. Moleculr mechnisms of selenium tolernce nd hyperccumultion in Stnley pinnt. Plnt Physiology 153 (4), Fu, L-H., Wng, X-F., Eyl, Y., She, Y-M., Donld, L.J., Stnding, K.G., Ben-Hyim, G., 22. A selenoprotein in the plnt kingdom. Journl of Biologicl Chemistry 227, Geoffroy, L., Gilin, R., Simon, O., Florini, M., Adm, C., Prdines, C., Cournc, L., Grnier-Lplce, J., 27. Effect of selente on growth nd photosynthesis of Chlmydomons reinhrdtii. Aqutic Toxicology 83 (2), Gomes-Junior, R.A., Grto, P.L., Gziol, S.A., Mzzfer, P., Le, P.J., Azevedo, R.A., 27. Selenium-induced oxidtive stress in coffee cell suspension cultures. Functionl Plnt Biology 34 (5), Grnt, K., Crey, N.M., Mendoz, M., Schulze, J., Pilon, M., Pilon-Smits, E.A.H., Vn Hoewyk, D., 211. Adenosine 5 -phosphosulfte reductse (APR2) muttion in Aridopsis implictes glutthione deficiency in selente toxicity. Biochemicl Journl 438, Grnum, E., Myklestd, S.M., 22. A photoiorector with ph control: demonstrtion y growth of the mrine ditom Skeletonem costtum. Journl of Plnkton Reserch 24 (6), Hrrison, P.J., Yu, P.W., Thompson, P.A., Price, N.M., Phillips, D.J., Survey of selenium requirements in mrine phytoplnkton. Mrine Ecology Progress Series 47, Hrtikinen, H., 25. Biogeochemistry of selenium nd its impct on food chin qulity nd humn helth. Journl of Trce Elements in Medicine nd Biology 18, Hvux, M., Crotenoids s memrne stilizers in chloroplsts. Trends in Plnt Science 3 (4), Inskeep, W.P., Bloom, P.R., Extinction coefficients of chlorophyll- nd chlorophyll- in N,N-dimethylformmide nd 9-percent cetone. Plnt Physiology 77 (2),

10 M. Schivon et l. / Aqutic Toxicology (212) Ishii, W.P., Ymgishi, T.Y., Murt, Y., One method for mesuring photosynthesis nd respirtion of lef slices with n oxygen electrode. Jpnese Journl of Crop Science 46, Kud, T., Tsunekw, M., Hishi, T., Arki, Y., 25. Antioxidnt properties of dried kymo-nori, rown lg Scytosiphon lomentri (Scytosiphonles, Pheophycee). Food Chem. 89, Lee, W.Y., Wng, W.X., 21. Metl ccumultion in the green mcrolg Ulv fscit: effects of nitrte, mmonium nd phosphte. Science of the Totl Environment 278 (1 3), Loughnne, C.J., McIvor, L.M., Rindi, F., Stengl, D.B., Guiry, M.D., 28. Morphology, rcl phylogeny nd distriution of distromtic Ulv (Ulvophycee, Chlorophyt) in Irelnd nd southern Britin. Phycologi 47, Mzid, M., Khn, T.A., Mohmmd, F., 211. Role of secondry metolites in defense mechnisms of plnts. Biology nd Medicine 3 (2), Michlk, A., 26. Phenolic compounds nd their ntioxidnt ctivity in plnts growing under hevy metl stress. Polish Journl of Environmentl Studies 15 (4), Morn, R., Porth, D., 198. Chlorophyll determintion in intct tissue using N,Ndimethylformmide. Plnt Physiology 77, Morlon, H., Fortin, C., Adm, C., Grnier-Lplce, J., 26. Selenite trnsport nd its inhiition in the unicellulr green lg Chlmydomons reinhrdtii. Environmentl Toxicology nd Chemistry 25, Neumnn, P.M., de Souz, M.P., Pickering, I.J., Terry, N., 23. Rpid microlgl metolism of selente to voltile dimethylselenite. Plnt, Cell nd Environment 26, Norusis, M.J., SPSS for Windows: se system user s guide, relese 6.. SPSS Inc., Chicgo. Novoselov, S., Ro, M., Onoshko, N., Zhi, H., Kryukov, G., Xing, Y., Weeks, D., Htfield, D., Gldyshev, V., 22. Selenoproteins nd selenocysteine insertion system in the model plnt cell system, Chlmydomons reinhrdtii. EMBO Journl 21, Pstierov, J., Krmrov, Z., Molnrov, M., Frgsov, A., 29. Comprison of the sensitivity of four freshwter mcrolge to selente nd selenite. Fresenius Environmentl Bulletin 18 (11), Pelh, D., Cohen, E., 25. Cellulr response of Chlorell zofingensis to exogenous selenium. Plnt Growth Regultion 45, Pilon-Smits, E.A.H., Hwng, S.B., Lytle, C.M., Zhu, Y.L., Ti, J.C., Brvo, R.C., Leustek, T., Terry, N., Overexpression of ATP sulfurylse in Brssic junce leds to incresed selente uptke, reduction nd tolernce. Plnt Physiology 119, Pilon-Smits, E.A.H., LeDuc, D.L., 29. Phytoremedition of selenium using trnsgenic plnts. Current Opinion in Biotechnology 2 (2), Pilon-Smits, E.A.H., Quinn, C.F., Tpken, W., Mlgoli, M., Schivon, M., 29. Physiologicl functions of eneficil elements. Current Opinion in Plnt Biology 12, Plnt, J.A., Kinniurgh, D.G., Smedley, P.L., Fordyce, F.M., Klinck, B.A., 24. Arsenic nd selenium. In: Hollnd, H.D., Turekin, K.K. (Eds.), Tretise on Geochemistry, vol. 9, Environ Geochem. Elsevier, Amsterdm, pp Posmyk, M.M., Kontek, R., Jns, K.M., 29. Antioxidnt enzymes ctivity nd phenolic compounds content in red cge seedlings exposed to copper stress. Ecotoxicology nd Environmentl Sfety 72 (2), Pyrzynsk, K., Specition of selenium compounds. Anlyticl Sciences 14, Rscio, N., Mrini, P., Tommsini, E., Bodner, M., Lrcher, W., Photosynthetic strtegies in leves nd stems of Egeri dens. Plnt 185, Reunov, Y.A., Aizdicher, N.A., Khristoforov, N.K., Reunov, A.A., 27. Effects of selenium on growth nd ultrstructure of the mrine unicellulr lg Dunliell slin (Chlorophyt). Russin Journl of Mrine Biology 33 (2), Riedel, G.F., Snders, G.J., The influence of ph nd medi composition on the uptke of inorgnic selenium y Chlmydomons reinhrdtii. Environmentl Toxicology nd Chemistry 15, Ross, C., Vn Alstyne, K.L., 27. Intrspecific vrition in stress-induced hydrogen peroxide scvenging y the ulvoid mcrolg Ulv lctuc. Journl of Phycology 43 (3), Schivon, M., Pilon-Smits, E.A.H., Wirtz, M., Hell, R., Mlgoli, M., 28. Interctions etween chromium nd sulfur metolism in Brssic junce. Journl of Environmentl Qulity 37, Schivon, M., Pittrello, M., Pilon-Smits, E.A.H., Wirtz, M., Hell, R., Mlgoli, M., 212. Selente nd molydte lter sulfte trnsport nd ssimiltion in Brssic junce L. Czern: implictions for phytoremedition. Environmentl nd Experimentl Botny, Simmons, D.B.D., Emery, R.J.N., 211. Phytocheltin induction y selente in Chlorell vulgris nd regultion of effect y sulfte levels. Environmentl Toxicology nd Chemistry 3 (2), Shrift, A., Sulfur selenium ntgonism. Antimetolite ction of selente on the growth of Chlorell vulgris. Americn Journl of Botny 41, Tmoki, M., Freemn, J.L., Pilon-Smits, E.A.H., 28. Coopertive ethylene nd jsmonic cid signling regultes selenite resistnce in Aridopsis. Plnt Physiology 146 (3), Terry, N., Zyed, A.M., de Souz, M.P., Trun, A.S., 2. Selenium in higher plnts. Annul Review of Plnt Physiology nd Plnt Moleculr Biology 51, Tuzen, M., Sri, A., 21. Biosorption of selenium from queous solution y green lge (Cldophor hutchinsie) iomss: equilirium, thermodynmic nd kinetic studies. Chemicl Engineering Journl 158, Umisová, D., Vìtovà, M., Douskovà, I., Bisovà, K., Hlvovà, M., Cizkovà, M., Mchàt, J., Douch, J., Zchleder, V., 29. Bioccumultion nd toxicity of selenium compounds in the green lg Scenedesmus qudricud. BMC Plnt Biology 9, Vítová, M., Bišová, K., Hlvová, M., Zchleder, V., Rucki, M., Čížková, M., 211. Glutthione peroxidse ctivity in the selenium-treted lg Scenedesmus qudricud. Aqutic Toxicology 12, Wellurn, A.R., The spectrl determintion of chlorophylls nd, s well s totl crotenoids, using vrious solvents with spectrophotometers of different resolution. Journl of Plnt Physiology 144, Wheeler, A.E., Zingro, R.A., Irgolic, K., The effects of selente, selenite nd sulphte on the growth of six unicellulr mrine lge. Journl of Experimentl Mrine Biology nd Ecology 57, Willims, M.J., Ogle, R.S., Knight, A.W., Buru, R.G., Effects of sulfte on selente uptke nd toxicity in the green lg Selenstrum cpricornutum. Archives of Environmentl Contmintion nd Toxicology 27 (4), Young, A.J., The photoprotective role of crotenoids in higher plnts. Physiologi Plntrum 83 (4), Zrcins, B.A., Crtwright, B., Spouncer, L.R., Nitric cid digestion nd multielement nlysis of plnt mteril y inductively coupled plsm spectrometry. Communictions in Soil Science nd Plnt Anlysis 18, Zhu, Y-G., Pilon-Smits, E.A.H., Zho, F-J., Willims, P.N., Mehrg, A.A., 29. Selenium in higher plnts: understnding mechnisms for iofortifiction nd phytoremedition. Trends in Plnt Science 14 (8), Zhng, Q., Yu, P., Li, Z., Zhng, H., Xu, Z., Li, P., 23. Antioxidnt ctivities of sulfted polyscchride frctions from Porphyr hitnesis. J. Appl. Phycol. 15,