Distribution of Se and its species in Myriophyllum spicatum and Ceratophyllum demersum growing in water containing se (VI)

Chemosphere 84 (2011) 1636–1641

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Distribution of Se and its species in Myriophyllum spicatum and Ceratophyllum demersum growing in water containing se (VI) Špela Mechora a, Petra Cuderman b, Vekoslava Stibilj b, Mateja Germ a,⇑ a b

Biotechnical Faculty, University of Ljubljana, Jamnikarjeva 101, SI-1000 Ljubljana, Slovenia Jozˇef Stefan Institute, Jamova Cesta 39, SI-1000 Ljubljana, Slovenia

a r t i c l e

i n f o

Article history: Received 1 April 2011 Received in revised form 5 May 2011 Accepted 6 May 2011 Available online 23 June 2011 Keywords: Myriophyllum spicatum Ceratophyllum demersum Se species Photochemical efficiency Chlorophyll

a b s t r a c t The uptake of Se (VI) by two aquatic plants, Myriophyllum spicatum L. and Ceratophyllum demersum L., and its effects on their physiological characteristics have been studied. Plants were cultivated outdoors under semi-controlled conditions and in two concentrations of Na selenate solution (20 lg Se L1 and 10 mg Se L1). The higher dose of Se reduced the photochemical efficiency of PSII in both species, while the lower dose had no effect on PSII. Addition of Se had no effect on the amounts of chlorophyll a and b. The concentration of Se in plants grown in 10 mg Se L1, averaged 212 ± 12 lg Se g1 DM in M. spicatum (grown from 8–13 d), and 492 ± 85 lg Se g1 DM in C. demersum (grown for 31 d). Both species could take up a large amount of Se. The amount of soluble Se compounds in enzyme extracts ranged from 16% to 26% in control, and in high Se solution from 48% to 36% in M. spicatum and C. demersum, respectively. Se-species were determined using HPLC-ICP–MS. The main soluble species in both plants was selenate (37%), while SeMet and SeMeSeCys were detected at trace levels. Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction Selenium (Se) is trace element that can function as both an essential nutrient and an environmental toxicant (Fan et al., 2002). It is found in the Earth’s crust, soils, minerals, in freshwater, seawater, and in sediments. There is widespread discharge of soluble Se from agricultural and industrial sources (Fan et al., 2002), including certain mining and petrochemical manufacturing operations (Lemly, 2004). The addition of Se to feed stuffs and soil fertilizers is common practice. Part of the Se added to feed is used by animals and part is spilled or secreted and passed to the environment in direct or indirect ways. In aquatic systems Se can occur in four oxidation states – elemental (Se0), selenite (Se+4), selenate (Se+6) and selenide (Se2) (Canton and Van Derveer, 1997). The form of Se present in a given system depends on the redox potential and pH. The oxidized forms of Se, selenite and selenate, are the most soluble and predominate in natural water systems (Environmental Protection Agency, 1987). Because of their high solubility these forms are more available for plants (Carvalho and Martin, 2001) and are potentially toxic to aquatic organisms. Elemental Se is stable, insoluble and poorly Abbreviations: F, steady state fluorescence; Fo, minimal chlorophyll a fluorescence yield in dark adapted samples; Fm, maximal chlorophyll a fluorescence yield in dark adapted samples; F 0m , maximal fluorescence of an illuminated sample; Fv, variable fluorescence; PSII, photosystem II; SeMet, selenomethionine; SeMeSeCys, selenomethylselenocysteine; SeCys2, selenocystine. ⇑ Corresponing author. Tel.: +386 1 320 33 00; fax: +386 1 257 33 90. E-mail address: [email protected] (M. Germ). 0045-6535/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.chemosphere.2011.05.024

assimilated by aquatic organisms, providing little toxic threat (Environmental Protection Agency, 1987). Once in the aquatic environment, Se can rapidly attain levels that are toxic to fish and wildlife because of its bioaccumulation in food chains and resulting dietary exposure (Lemly, 2004). Se contamination has led to devastation of wildlife populations such as the incidents at Belews Lake, NC and Kesterson Reservoir, CA (Browne and Lutz, 2010). The waterborne criterion for toxic chronic selenium exposure has been set at 5 lg L1 by the US EPA (US EPA, 1998). Concentrations in freshwaters of Slovenia are in the range of 0.07–0.15 ng Se g1. As reported by Fournier et al. (2010), typical concentrations of Se in freshwaters ranged from 0.01– 0.5 lg Se L1. In the Arkansas River basin concentrations ranged from 2 to 24 lg Se L1 (Canton and Van Derveer, 1997). Macrophytes are aquatic plants that can be used as indicators of low level environmental contamination that might otherwise be difficult to detect (Mazej and Germ, 2009). They have been used as indicators of trace element pollution since the early seventies (Phillips, 1977). Aquatic plants are suitable for wastewater treatment because they have a tremendous capacity for absorbing nutrients and other substances from the water (Boyd, 1970) and hence reduce the pollution. Aquatic plants can take up trace elements through their roots whereas, in submerged plants, leaves as well as roots take part in uptake. Macrophytes contribute to primary production in water and support both the detrital and herbivore food webs. Little is known about the ability of macrophytes to take up Se from the sediment

Š. Mechora et al. / Chemosphere 84 (2011) 1636–1641

and water, since this is a relatively recently recognized pollutant with an uncertain role (Carvalho and Martin, 2001). There is even less known about the effect of Se on the physiological characteristics of macrophytes. In the present study we have examined the uptake of Se, its distribution and speciation, as well as its effects on physiological and biochemical parameters in Myriophyllum spicatum L. and Ceratophyllum demersum L. These are cosmopolitan species, exhibiting similar life forms and colonizing mainly eutrophic stagnant and flowing waters (Martincˇicˇ et al., 1999). M. spicatum is rooted into the sediment while C. demersum is free floating in the water column. 2. Materials and methods 2.1. Plants and growth conditions Experiments were conducted under natural conditions at Ljubljana Botanical Garden, Slovenia. M. spicatum was obtained from Lake Bohinj (547 m asl, 46°170 N, 13°540 E, Slovenia) and planted on 16th of June, 2010 in plastic pots containing sediment from Lake Bohinj. Plastic pots were placed in three containers of size 120 cm  52 cm  54 cm, containing 160 L of tap water. C. demersum was obtained from a pond in the Botanical Garden (Ljubljana: 320 m asl, 46°350 N, 14°550 E, Slovenia) and placed in containers on 14th of June, 2010. It was grown in three containers containing 3 cm of sediment from Lake Bohinj. After two weeks of plant acclimatization, Na2SeO4 was added to the water. One container contained water with 20 lg Se L1, the second with 10 mg Se L1 and the third contained no detectable Se as a control for each species. The concentration of Se in water was measured every week and maintained at the desired levels. During the experiment air temperature, water temperature, humidity and rainfall were also monitored. High air temperatures increased water temperature in the containers to 32 °C. After 31 d of exposure, the plants were harvested, lyophilized and milled for analysis of Se. The exception was M. spicatum in 10 mg Se L1 solution. It was planted three times in the same period. When half of the plants died, we planted plants on freshly and a new group was created. Thus, the first group (I) was planted on June 29 and grew for 13 d, the second (II) was planted on July 12 and grew for 8 d, and the third group (III) was planted on July 20 and grew for 9 d. 2.2. Photochemical efficiency Chlorophyll fluorescence was measured in situ on ten vital plants from each container using a fluorometer (OS-500, Opti-Sciences, Tyngsboro, MA, USA). The potential quantum yield was evaluated in terms of the ratio Fv/Fm. Measurements of minimal (F0) and maximal (Fm) chlorophyll fluorescence were made after 15 min of darkness, provided by dark-adaptation clips. Fluorescence was excited with a saturating beam of ‘‘white light’’ (PPFD = 8 000 lmol m2 s1, 0.8 s). The effective quantum yield was determined with a saturating pulse of ‘‘white light’’ (PPFD = 9 000 lmol m2 s1, 0.8 s). The effective quantum yield of PSII (F 0m  FÞ=F 0m = DF/F 0m provided an estimate of the actual efficiency of energy conversion in PSII. F 0m is the maximum fluorescence of an illuminated leaf and F is the steady state fluorescence (Schreiber et al., 1995).

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Absorbance was measured with a UV/VIS Spectrometer System (Lambda 12, Perkin–Elmer, Norwalk, CT, USA). The amounts of pigment were calculated as described by Jeffrey and Humphrey (1975). 2.4. Determination of total Se concentration in plant Three plants from each container were analyzed for Se content. To 0.200 g of homogenized and lyophilized sample, 1.5 mL HNO3 and 0.5 mL H2SO4 were added and heated for 24 h at 80 °C. The temperature was then increased to 130 °C and maintained there for 60 min. H2O2 and 0.1 mL 40% HF were added to the cooled solution. After heating and cooling the samples again, 0.1 mL of V2O5 in H2SO4 was added. Se (VI) was reduced to Se (IV) by the addition of concentrated HCl and heating at 90 °C. The solution was diluted before determining the Se content, which was carried out by hydride generation atomic fluorescence spectrometry (Smrkolj and Stibilj, 2004). Working standard solutions of Se (IV) were prepared weekly by dilution of a standard stock solution with 0.5 M HCl (Cuderman et al., 2008). Each sample was analyzed at least in triplicate. The method is described in detail by Smrkolj and Stibilj (2004). The accuracy of the method was checked with the certified reference material Spinach Leaves, NIST 1570a and good agreement was found between the obtained, 115 ± 3 ng Se g1, and certified, 117 ± 9 ng Se g1, values. 2.5. Extraction and speciation Plants were extracted in duplicate as described Mazej et al. (2006). 8 mL water, containing 60 mg of protease XIV was added to 0.5 g of sample and incubated for 24 h at 37 °C. Extracts were centrifuged at 11 000 rpm for 60 min at 4 °C (5804R, Eppendorf). The supernatant was filtered through 0.45 and 0.22 lm Millex GV filters (Millipore Corporation) and used for Se speciation analysis by HPLC-ICP–MS. Supernatants and sediments were stored at 20 °C until analyzed for total Se by HG-AFS. Se species in supernatants were determined using an ion exchange HPLC system (Varian ProStar 210) coupled directly to a HPLC system (Agilent 1100, Waldbronn, Germany) coupled to an ICP–MS (Agilent 7500ce, Tokyo, Japan). For soluble Se species determination, a Hamilton PRP-X 100 anion-exchange column (4.1 mm  250 mm  10 lm) and a Zorbax 300-SCX cation-exchange column (4.6 mm  250 mm  5 lm) were used. 3 mM and 10 mM citrate buffer in 2% MeOH (pH 4.8) was used as the mobile phase for anion-exchange chromatography, and 3 mM pyridine solution in 2% MeOH (pH 2.1) was used as eluent for the cation-exchange chromatography. The flow rate was 0.5 mL min1 and the volume of the sample injected was 50 lL. The method and operating conditions were described in detail elsewhere (Cuderman et al., 2008). Limits of detection for SeMet and SeMeSeCys are 0.9 ng g1, for SeCys2 0.2 ng g1, for Se (IV) 1.1 ng g1 and for Se (VI) 0.1 ng g1 (Cuderman et al., 2008). There is no reference material with matrix similar to that of our samples. We therefore used NIST RM 8436, which has a certified value for SeMet. Good agreement was found between the obtained and certified value for SeMet (Wolf and Goldschmidt, 2004), the values being 1.2 ± 0.01 mg Se kg1 and 1.23 ± 0.09 mg Se gk1, respectively. Total Se in supernatants was determined using digestion in HNO3. Total Se in sediments was determined as described in Section 2.4.

2.3. Photochemical pigments 2.6. Statistical analysis For content of chlorophylls a and b, leaves of five plants from each container were measured. Chlorophyll was extracted with 90% acetone. Extracts were centrifuged in a refrigerated ultracentrifuge (2K15, Sigma, Osterode, Germany) at 4000 rpm for 3 min at 4 °C.

The significance of the difference between mean values was determined by the analysis of variance with LSD test. Differences at p < 0.05 were considered as statistically significant.

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germinates at 25 °C (Ke and Li, 2006), V. Americana L. at 22 °C (Jarvis and Moore, 2008) and M. spicatum at 22.5 °C (Xiao et al., 2010). In another study, the optimum temperature for growth of M. spicatum in Wisconsin was 32.3 °C (Titus and Adams, 1979). Although these studies measured temperature in the natural environment, we concluded that the influence of temperature on the growth of M. spicatum could be minimal.

3. Results and discussion 3.1. Photochemical efficiency Fluorescence measurements provide simple, rapid and sensitive methods for detecting the photoinhibitory effects of environmental stressors on higher plants and phytoplankton (Lichtenthaler, 1988). The effective and potential photochemical efficiencies of M. spicatum and C. demersum were high at the beginning of the experiment (Table 1). Potential photochemical efficiency was close to the theoretical maximum of 0.8 (Schreiber et al., 1995), which indicates an undamaged antenna complex (Germ et al., 2007). The lower concentration of Se did not affect the potential quantum yield of PSII in either of the species (Table 1). Plants of both species were exposed to low Se solution for 31 d. During this exposure M. spicatum formed new shoots. No formation of shoots was observed in control, therefore we concluded, that low concentration of Se was beneficial for the plant. Addition of 10 mg Se L1 reduced the potential and effective photochemical efficiencies of the two species (Table 1), but the effect was greater in M. spicatum. Thus, C. demersum is more tolerant to the high concentration of Se (10 mg Se L1). The lowest values of effective and of potential photochemical efficiency in M. spicatum in high Se solution were observed in group I, when some plants had already died. There were no differences between groups II and III. Towards the end of July the values again decreased (Table 1) while the plants were slowly dying. In C. demersum and group II of M. spicatum the photochemical efficiency was highest after 21 d of exposure (Table 1). This implies that some kind of recovery was occurring. Temperature influences the germination of water plants. Vallisneria natans (Lour.) H.

3.2. Photochemical pigments Addition of Se had no effect on the content of chlorophylls in either species. The amount of chlorophyll b in C. demersum was slightly increased by addition of 10 mg Se L1 on July 15 and 27 (Table 2), but the difference was not statistically significant. A reduction in chlorophyll a and b content was observed towards the end of the experiment in both species (Table 2). The same trend was seen in aquatic plants Eichhornia crassipes L., Pistia stratiotes L. and Spirodela polyrhiza (L.) Schleid. after exposure to Cu, Cd, Fe, Cr and Zn (Mishra and Tripathi, 2008). The reduction of chlorophyll content in macrophytes may be attributed to inhibition of chlorophyll synthesis which results in the loss of photosynthetic activity (Chandra and Kulshreshtha, 2004). 3.3. Determination of total Se concentration and identification of Se species The total Se content was found to be 0.5 and 1.2 lg Se g1 in controls of C. demersum and M. spicatum respectively (Table 3). In 20 lg Se L1 solutions, the content was similar to that in the control. Chara spp. accumulated 1 mg Se kg1 from water containing 20 lg Se L1 (Lin et al., 2002) which is higher than in our experiment. This plant plays an important role in the removal of Se from agricultural drainage water (Lin et al., 2002). In the study of Pollard and co-workers (2007), tissues of water plants growing in swamp containing 20 lg Se L1, contained from 1.16 lg Se g1 in Scirpus californicus (C.A. Mey) Palla to 4.70 lg Se g1 in Najas marina L. These concentrations are similar to those observed in our study. Entirely submerged species have been shown to accumulate relatively large amounts of trace elements (Fritioff and Greger, 2003), in accord with our findings. Similarly, in plants treated with 10 mg Se L1, the amount of Se was 200 times and 1000 times higher in M. spicatum and C. demersum than in controls. In the second group of M. spicatum tissues contained only 102 lg Se g1, in the first group 236 lg Se g1 and in the third group 297 lg Se g1 (Table 3). As Rai et al. (1995) reported, species like C. demersum, S. polyrhiza, Hydrodictyon reticulatum (L.) Lagerh., Bacopa monnieri (L.) Pennell, Hygrorrhiza aristata (Retz.) Nees. accumulate an appreciable amount of metals from pond water and are unspecific collectors of metals. M. spicatum is known to be capable of very effectively absorbing Pb and Cu from solution (Keshinkan et al., 2003). M. propinquum L. was pronounced to be an accumula-

Table 1 Photochemical efficiency of PSII in M. spicatum and C. demersum (n = 10). M. spicatum

C. demersum

Date

DF/Fm’

Fv/Fm

DF/Fm’

Fv/Fm

Control 17.6. 8.7. 20.7. 27.7.

0.71 ± 0.04f 0.55 ± 0.12bc 0.70 ± 0.04ef 0.62 ± 0.08cde

0.76 ± 0.03d 0.71 ± 0.05c 0.76 ± 0.03d 0.71 ± 0.04c

0.70 ± 0.04f 0.60 ± 0.14de 0.65 ± 0.07ef 0.49 ± 0.09

0.76 ± 0.04cd 0.72 ± 0.04bc 0.78 ± 0.01c 0.70 ± 0.06b

20 lg Se L1 8.7. 20.7. 27.7.

0.56 ± 0.11bc 0.70 ± 0.05c 0.67 ± 0.07ef 0.76 ± 0.04cd 0.65 ± 0.07def 0.76 ± 0.02d 0.61 ± 0.08de 0.76 ± 0.02cd 0.56 ± 0.06bc 0.72 ± 0.04cd 0.55 ± 0.09bcd 0.70 ± 0.04b

10 mg Se L1 8.7. 20.7. 27.7.

Group I 0.44 ± 0.10 0.50 ± 0.08 II 0.60 ± 0.15bcd 0.55 ± 0.07 b III 0.53 ± 0.11 0.64 ± 0.12b

0.42 ± 0.10 0.63 ± 0.09 0.56 ± 0.11bcd 0.63 ± 0.05 0.47 ± 0.09 0.61 ± 0.09

Values are means ± SD. Each column was tested separately. The values followed by the letters were significantly different at p < 0.05.

Table 2 Chlorophyll a and b in M. spicatum and C. demersum (n = 5). Parameter

Date

Chlorophyll a (mg g1 DM)

Chlorophyll b (mg g1 DM)

17.6. 7.7. 15.7. 27.7. 17.6. 7.7. 15.7. 27.7.

M. spicatum Control 5.17 ± 0.35 3.30 ± 1.84 5.86 ± 2.89 4.02 ± 0.95 4.40 ± 1.71 3.05 ± 1.95 5.65 ± 1.92 2.59 ± 0.92

C. demersum

20 lg Se L1

group

10 mg Se L1

7.42 ± 2.41 3.3 ± 2.36 2.58 ± 0.90

I II III

4.20 ± 1.19 3.78 ± 2.37 2.57 ± 1.70

1.39 ± 1.46 2.05 ± 1.50 1.38 ± 0.46

I II III

1.75 ± 0.5 3.94 ± 2.45 2.46 ± 1.64

Control 7.25b ± 2.56 6.47ab ± 0.68 4.20 ± 1.77 5.80 ± 1.39 4.18b ± 1.74 4.17 ± 2.87 2.98 ± 1.38 3.50 ± 0.93

Means ± SD. Each column was tested separately. The values followed by letters were significantly different at p < 0.05.

20 lg Se L1

10 mg Se L1

4.82 ± 1.43 5.17 ± 2.69 5.59 ± 1.64

5.00 ± 0.83 4.49 ± 1.20 5.02 ± 2.72

1.07 ± 0.47 4.53 ± 1.77 3.94 ± 1.20

1.27 ± 0.47 4.62 ± 1.42 4.22 ± 1.96

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tor plant for As, with over 1000 mg As kg1 (Robinson et al., 2006). Selected Myriophyllum species could therefore be used for remediation of contaminated waters. Tissues of C. demersum contained 492 lg Se g1 when grown in the higher concentration of Se (Table 3). Similarly, Robinson et al. (2006) found that C. demersum exposed to As took up 400 mg As kg1. C. demersum also took up large amounts of Mn, Cu, Pb and Fe (Rai et al., 1995). In macrophytes, the whole plant plays an important role in removing contaminants. Because of their high surface/biomass ratio, the uptake of metals by water plants is considerable (Baudo et al., 1981). C. demersum has a high surface/biomass ratio, thereby accounting for the high uptake of Se. M. spicatum in high Se solution lasted between 8 and 13 d. In all three groups of M. spicatum half of the plants in the container died. Thus, 10 mg Se L1 could prove a toxic concentration for M. spicatum. We also observed browning of shoots as a sign of toxicity. In contrast, C. demersum lasted for 31 d in high Se concentration. Se has been measured in a variety of aquatic plants (Bailey et al., 1995; Wu and Guo, 2002; Carvalho and Martin, 2001). In Ruppia

maritima L. exposed to concentrations around 6 mg Se L1 the content of Se was 200 lg Se g1 (Bailey et al., 1995), which is less than C. demersum and M. spicatum in our study. Cattails and swamp lilies grown in 200 mg Se L1 however contained only 80 and 30 lg Se g1 (Carvalho and Martin, 2001). This concentration in solution was very high and it is remarkable that plants were still growing. In our study, M. spicatum could not survive more than 13 d in 10 mg Se L1 solution. In the study of Wu and Guo (2002) Se concentrations in shoots of Potamogeton crispus L. and R. maritima, grown 10 d in 60 lg Se L1, were 363.7 lg Se kg1 and 384.2 lg Se kg1. In our experiment, M. spicatum took up more Se from a lower Se concentration and endured for longer. Similar amounts of Se were extracted from samples with water (Milli Q) or by protease XIV action. Little difference was obtained when using protease XIV instead of water (Table 3). A standard solution of the Se compounds prepared in water was stable, as well as in enzyme assisted extracts of control plants. We observed that Se species were stable in enzymatic extracts of Se enriched plants. In water extracts of M. spicatum we found 11% of soluble Se for control and 38% for 10 mg Se L1

Table 3 Se content in samples and supernatant, percent of solubility. Total Se lg g1 (DM)

Sample

M. spicatum 10 mg Se L1

C. demersum

Supernatant (lg g1) Extraction with water

Solubility (%)

Extraction with protease XIV

Solubility (%)

Control 20 lg Se L1 I II III

1.19 ± 0.2 0.77 ± 0.03 236 ± 7.2 102 ± 2.3 297 ± 25.9

0.13 ± 0.07 0.12 ± 0.01 100 ± 24 35 ± 3.8 113 ± 3.9

11 16 43 34 38

0.19 ± 0.02 0.15 ± 0.03 112 ± 2.2 42 ± 0.9 122 ± 0.4

16 19 48 42 41

Control 20 lg Se L1 10 mg Se L1 III

0.48 ± 0.01 0.57 ± 0.04 492 ± 84.7

0.07 ± 0.01 0.12 ± 0.01 176 ± 10.5

14 21 36

0.13 ± 0.02 0.14 ± 0.02 179 ± 3.5

26 24 36

Results are expressed on a dry matter basis and given as the average of three/four measurements ±SD.

Table 4 Content of soluble Se and its species in macrophytes. SeMetb

Se (VI) (%SeVI according to soluble Se)

sample

treatment/ group

soluble Se lg g1 a

SeMeSeCys

M. spicatum

20 lg Se L1 I II III 20 lg Se L1 10 mg Se L-1

0.12 ± 0.01(97)

0.002 ± 0.001

0.12 ± 0.01 (96)

101 ± 4(76) 35 ± .8(62) 113 ± 3.9(82) 0.12 ± 0.01(55)

0.3 ± 0.1 0.05 0.74 ± 0.3 0.004

77 ± 1 (76) 22 ± 1 (62) 92 ± 1 (82) 0.06 ± 0.01 (44)

176 ± 10 (64)

1.6

112 ± 1 (63)

20 lg Se L1 I II III 20 lg Se L1 10 mg Se L1

0.15 ± 0.03(32)

0.005 ± 0.001

112 ± 2 (80) 42 ± 1(95) 122 ± 1(82) 0.14 ± 0.02(32)

2.3 ± 0.4 1.3 2.5 ± 0.1 0.002 ± 0.001

179 ± 3(60)

4.7

Se (IV)

Wc

Xd

Ye

Zf

2.3 ± 0.1 0.5 ± 0.1

Se species (lg Se g-1) water extraction

10 mg Se L1 C. demersum

enzymatic extraction

M. spicatum

10 mg Se L1 C. demersum

0.004 ± 0.001

0.03 ± 0.01 (17)

0.02 ± 0.01

2.2 ± 0.09 1.3 ± 0.1 2.1 0.008 ± 0.003

0.5 ± 0.03 0.8 ± 0.02 0.003 ± 0.001

79 ± 9 (70) 36 ± 2 (85) 92 ± 1 (75) 0.03 ± 0.01 (23)

0.5 ± 0.1 0.2 ± 0.1 1.7 ± 0.1 0.005 ± 0.001

2.2 ± 0.5 0.9 ± 0.1 2.3 ± 0.7

0.8 ± 0.1 0.2 ± 0.1 0.8 ± 0.1

3.3 ± 1.1

1.8 ± 0.1

90 ± 1 (50)

0.8 ± 0.1

1.1 ± 0.1

6±1

Results are given as the average of three measurements ± SD. a in bracket is % of identified Se compounds according to soluble Se b Se as SeMet c Unknown Se species with the same retention time as SeCys2 (3.9 min), obtained on Hamilton PRP-X 100, estimated as Se in SeCys2. d Unknown Se species with retention time 11.6 min, obtained on Hamilton PRP-X 100, estimated as Se in SeMet. e Unknown Se species with retention time of 8.49 min, obtained on Zorbax 300-SCX, estimated as Se in SeMeSeCys. f Unknown Se species with retention time of 7.35 min, obtained on Zorbax 300-SCX, estimated as Se (IV).

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(a)

(b)

4.0E+04

4.0E+03

Intensitiy 78Se

Intensity 78Se

3.0E+04

2.0E+03

2.0E+04

1.0E+04

0.0E+00 0

1000

2000

3000

0.0E+00 0

Retention time (s)

500

1000

1500

Retention time (s)

Fig. 1. Chromatogram of enzyme extracts from group I of M. spicatum in 10 mg Se L1 obtained after separation on Hamilton PRP-X 100 (anion exchange column) (a) and Zorbax 300-SCX (cation exchange column) and (b) connected to ICP–MS.

treatment. In enzyme extracts the percentage of soluble Se in M. spicatum was almost the same. On the other hand, the percentage of soluble Se in control of C. demersum was slightly higher when using enzyme, but in high Se treatment there was no difference (Table 3). It means that protease XIV did not enhance the extraction efficiency. Se (VI) and traces of SeMeSeCys were found in water extracts of both species (Table 4). After enzyme extraction, we identified SeMet, SeMeSeCys, Se (IV), Se (VI) and, after high Se treatment, also three unidentified species X, Y and Z (Table 4; Fig. 1). On the anion exchange column an unknown species X occurred, with a retention time of 11.6 min (Fig. 1a). On the cation exchange column two unknown species Y and Z occurred with retention times of 8.49 min and 7.35 min (Fig. 1b). On the anion exchange column we observed a peak W with the same retention time as SeCys2 (Fig. 1), but on the cation exchange column we did not confirm SeCys2. Cubadda et al. (2010) suggested that this substance, with the same retention time as SeCys2, could be an oxide of SeMet, possibly an artifact of sample preparation. There is a lot of confusion about presence of SeCys2 in plants. Cubbada et al. (2010) and Ximénez-Embún et al. (2004) reported the presence of SeCys2 in wheat grain and lupine. In contrast, Mazej et al. (2006) instead of SeCys2 rather defined an unknown peak with the same retention time as SeCys2. On average, 75% of the soluble Se content in high Se treatment of M. spicatum was in the form of Se (VI) and, in C. demersum 50% (Table 4). The greater part was still in the form of Se (VI), with which the plants were treated. From that we conclude that metabolism in plants was incomplete. Published information on Se species in macrophytes is scarce, and comparison with our results is difficult. In Chara spp. 47% of Se was in organic form (Lin et al., 2002) in contrast to M. spicatum in which 75% was in inorganic form. In enzyme and water extracts of both species we detected SeMeSeCys (Table 4), which was confirmed with the standard addition method. In this case, no difference was found between enzyme and water extraction. The content of SeMeSeCys in M. spicatum ranged 1.3–2.5 lg Se g1 and in C. demersum up to 4.7 lg Se g1 (Table 4). In enzyme extracts we also detected SeMet, which was confirmed with the standard addition method. The content of SeMet in M. spicatum was 1.3–2.2 lg Se g1 and in C. demersum 3.3 lg Se g1 (Table 4). Wu and Guo (2002) reported that the content of SeMeSeCys was 0.216 ng Se g1 in P. crispus and 0.242 ng Se g1 in R. maritima, while the content of SeMet was 1.3 ng Se g1 all on the detection limit of gas chromatography. The same authors also reported that Se-cystine was not detected, while Se-cysteine was, which is confusing.

The reported common Se species in Se non-accumulator plants is SeMeSeMet, but we identified SeMeSeCys. Identification of SeMeSeCys in M. spicatum and C. demersum could suggest that these two species have an Se transformation pathway similar to that in accumulator plants. 4. Conclusions Macrophytes were grown in various solutions of Se. Se negatively affected the photochemical efficiency of PSII, but had no effect on the amounts of chlorophyll a and b. Both species took up a large amount of Se. C. demersum was exposed for 31 d in high Se solution and is more resistant than M. spicatum which survived for only a short period (8–13 d). High Se treatment led to the presence of 40% of soluble Se in both species. We identified Se (VI), which was dominant form of Se in the macrophytes (50–75%), SeMet, SeMeSeCys, Se (IV) and three unknown species. Acknowledgments The authors are grateful to Dr. R. Pain for critical reading of the manuscript. This research was financed by the Ministry of Higher Education, Science and Technology of the Republic of Slovenia through the program ‘‘Young researchers’’ (32059), ‘‘Biology of plants’’ (P1-0212) and the project (J4-2041). References Bailey, F.C., Knight, A.W., Ogle, R.S., Klaine, S.J., 1995. Effect of sulfate level on selenium uptake by Ruppia maritima. Chemosphere 30, 579–591. Baudo, R., Galanti, G., Guilizzoni, P., Varini, P., 1981. Relationship between heavy metals and aquatic organism in lake Mezzola hydrographic system (Northern Italy), 4. Metal concentration in six submerged aquatic macrophytes. Mem. Ist. Ital. Idrobiol. 39, 203–225. Boyd, C.E., 1970. Production, mineral nutrient accumulation and pigment concentration in Typha latifolia and Scripus americanus. Ecology 51, 285–290. Browne, R.A., Lutz, D., 2010. Lake ecosystem effects associated with top-predator removal due to selenium toxicity. Hydrobiologia 655, 137–148. Canton, S.P., Van Derveer, W.D., 1997. Selenium toxicity to aquatic life: An argument for sediment-based water quality criteria. Environ. Toxicol. Chem. 16, 1255–1259. Carvalho, K.M., Martin, D.F., 2001. Removal of aqueous selenium by four aquatic plants. J. Aquat. Plant Manage. 39, 33–36. Chandra, P., Kulshreshtha, K., 2004. Chromium accumulation and toxicity in aquatic vascular plants. Bot. Rev. 70, 313–327. Cubadda, F., Aureli, F., Ciardullo, S., D’Amato, M., Raggi, A., Acharya, R., Reddy, R.A.V., Prakash, N.T., 2010. Changes in selenium speciation associated with increasing tissue concentrations of selenium in wheat grain. J. Agric. Food Chem. 58, 2295– 2301. Cuderman, P., Kreft, I., Germ, M., Kovacˇevicˇ, M., Stibilj, V., 2008. Selenium species in selenium-enriched and drought exposed potato. J. Agric. Food Chem. 56, 9114– 9120.

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