Introduction

Isotopes of plutonium and americium are typical components of radioactive nuclear wastes discharged into aquatic ecosystems. These nuclides dominate the long term radiotoxicity. The Yenisei River is contaminated with transuranium elements due to the operation of the Mining-and-Chemical Combine (ROSATOM), which has been producing weapon-grade plutonium since late 1950th. Actinides 239Np, 239,240Pu, 241Am and 243,244Cm have been detected in sediments, aquatic weeds of the Yenisei River, flood plain soils and berry shrubs in the flood plain [1–3]. Aquatic plants are an important component in migration of artificial radionuclides in the aquatic environment. Freshwater plants can accumulate transuranium elements in their biomass under natural [4, 5] and experimental conditions [6–8]. 241Am and 242Pu taken up from water by aquatic plant Elodea canadensis can tightly bind to the organic fraction of biomass [6, 8]. It has been shown that artificial radionuclides (137Cs, 60Co, 51Cr) bind with different biochemical molecules of plant biomass [9]. The role of biochemical components of biomass in biosorption of 241Am was estimated for yeasts [10] and fungi [11]. There are experimental data indicating that assimilation of artificial radionuclides by grazers depends on radionuclide distribution in microalgae cell compartments [12]. Hence, the data on the distribution of americium and plutonium in fractions of plant biomass can be used to understand the role of certain cell structures in uptake of actinides from water and to estimate further migration of these actinides in the aquatic environment. The purpose of this study was to estimate and compare the distributions of americium and plutonium in the intra- and extracellular compartments and biochemical components constituting the biomass of common freshwater macrophytes.

Experimental

Materials

The apical shoots (3–4 cm long) of common submerged macrophytes: vascular aquatic plants Elodea canadensis Michx., Ceratophyllum demersum L. and Myriophyllum spicatum L. and aquatic moss Fontinalis antipyretica Hedw. (5 cm long) were used for laboratory experiments. The plants were sampled in the Yenisei River upstream of the contaminated zone and acclimated to laboratory conditions during a week. In experiments we used filtered (0.2 μm RC-membranes, Schleicher & Schuell) Yenisei River water. 241Am (T1/2 = 434 y) was added to water as a 2 M HNO3 solution, 242Pu (T1/2 = 373000 y) and 238Pu (T1/2 = 88 y)—as 1 M HNO3 solutions. Isotopes were obtained in Rimex Ltd. (St. Petersburg, Russia).

Biosorption experiments

Initial activity concentration of 241Am in water was 1.1–1.2 kBq/l, 238Pu—630 ± 11 Bq/l; 242Pu—4 ± 1 (exp. No. 1) and 128 ± 13 (exp. No. 2) Bq/l. Isotopes were added to water, pH was adjusted to 7 with 0.1 M NaOH, and plants were placed in experimental vessels. The vessels were illuminated from the top for 14 h per day, 2 klx on the surface. For dark incubation, vessels were wrapped in aluminium foil. Water temperature was 19 °C.

The shoots were taken out of water after being maintained in the presence of isotopes for 2–6 days and rinsed with distilled water at pH 7 (adjusted with 0.1 M NaOH) for about 30 s.

Fractionation of plant biomass

To determine the dissolved intracellular portion of 241Am, the washed fresh shoots were homogenized in a glass cylinder homogenizer with a glass pestle in the presence of a tiny amount of distilled water, pH 7. The homogenized biomass was separated by filtering through membranes (0.2 μm, RC, Schleicher & Schuell).

For subsequent extraction of lipids, proteins and carbohydrates, rinsed shoots were dried at 60 °C. The dry shoots were finely ground and lipids were extracted from the samples with a mixture of isopropyl alcohol and trichloromethane (1:1 v/v) for 24 h at room temperature. Extract of lipids was separated from particulate biomass by filtration through glass fibre filters (25 mm dia., Millipore, Ireland) [13]. The biomass remaining on the filters was treated with 1 M NaOH for 1 h in boiling water bath to extract protein. The alkali extracts were separated from the biomass residue by filtration as described above; the glass fibre filters of dia. 47 mm were used. The filters with the biomass residue were washed with distilled water at the end of filtration. The biomass residue was not separated from the filters prior to measurements of γ-activity.

Sample preparation and measurement of isotope concentration

For mass spectrometry and γ-counting the samples of biomass were digested in the mixture of H2O2 (30%) and HNO3 (conc.) under heating. Liquid extracts were evaporated and the dry residues were also mineralised. The mineralised samples were transferred into scintillation vials and the final volume of samples was adjusted to 10 ml with distilled water. To avoid loss of activity, all glassware was rinsed with 1 M HNO3 after contact with extracted fractions of the biomass, and the rinse fluids were added to the corresponding biomass fractions. The loss of isotopes during fractionation did not exceed a few percent except for 242Pu, which will be discussed further in this paper.

241Am and 238Pu activities were measured in samples using a “Wallac 1480 Wizard 3” Gamma-counter (Perken Elmer, Finland). The activity of 238Pu was recorded using the characteristic X-ray line. The statistic standard deviation of γ-counting did not exceed 10%. Concentrations of 242Pu in samples (exp. No. 1) were measured on an α-spectrometer (7184, Eurisys Mesures, France) coupled to a PLUS-300 Si-low-background semiconductor detector. Sample preparation for α-spectrometry and the measurement technique were described by Bolsunovsky et al. [6]. Concentrations of 242Pu in samples (exp. No. 2) were measured on an Agilent-7500a ICP mass-spectrometer (Agilent Technologies, USA). Detection limit for 242Pu was 3 ng/l (or 0.4 Bq/l).

Results and discussion

Concentration of actinides in plant biomass

Activity concentration of americium taken up by apical shoots of macrophytes from water ranged from 528 to 1990 Bq/g of dry mass and was the highest in aquatic moss F. antipyretica (Table 1). The differences in americium biosorption by aquatic macrophytes could be due to differences in the surfaces areas of the plant species, resulting in dissimilarities in the number of binding sites on cell wall surface. The biosorption capacity also depends upon the plant’s epiphytic activity and metabolic stress response. The ability of aquatic bryophytes (F. antipyretica in particular) to absorb high concentrations of heavy metals, radionuclides and organic pollutants is well known [14]. Well developed detoxication mechanisms allow survival of bryophytes at high concentrations of pollutants in water [14, 15]. The accumulation of organic matter (epiphytic microorganisms and their exometabolites) and mineral (mainly carbonate) incrustations, which occur on vegetative organs of submerged macrophytes naturally, can increase the absorption of metals by plant surface [16]. This was proved experimentally for absorption of 241Am by leaves of E. canadensis [13].

Table 1 Activity concentrations of 241Am, 242Pu and 238Pu in dry biomass of macrophyte species (Bq/g), taken up from water for 2–6 days
Full size table

Plutonium accumulation was estimated for E. canadensis only. Activity concentration of 238Pu in the biomass of E. canadensis was 322–406 Bq/g, 242Pu—13–19 and 388–466 Bq/g in exp. No. 1 and No. 2, respectively. Activity concentrations of americium and plutonium accumulated in the biomass of macrophytes from water for 2–6 days did not differ considerably, indicating the equilibrium state in the system. Activity concentration of americium in the biomass of Elodea in the light was similar to its activity concentration in the dark, indicating the energy-independent (biosorption) mechanism of americium uptake from water. It has been shown experimentally that the biosorption mechanism of 241Am by Saccharomyces from water involves ion exchange, complexation and nonspecific adsorption in cell wall owing to static electricity [10]. We can expect similar mechanisms for submerged macrophytes.

Actinides in intra- and extracellular compartments of E. canadensis

Microscopy showed that homogenization of Elodea shoots destroyed the cells and most of the chloroplasts. The major concentrations of americium and plutonium were registered in the fraction of particles of homogenized biomass larger than 0.2 μm (Table 2), i.e. cell walls, membranes and organelles. Up to 10% of americium and up to 18% of plutonium activities were registered in the filtrate (dissolved fraction), which contained particles smaller than 0.2 μm, i.e. cytosol. Similar results were reported for americium in marine microalgae [17, 18]: up to 7% of 241Am was registered in cytoplasm, and the major portion of the radionuclide was bound to structural components (cell walls and plasmalemmae). Major role of cell walls in biosorption of 241Am was reported also for yeasts [10] and fungi [11]. Reinfelder and Fisher [19] referred to higher affinity of 241Am, as well as plutonium, to oxygen than to nitrogen or sulfur [20] when explained the low penetration of 241Am into cytoplasm of microalgae.

Table 2 Activity percentages in the dissolved intracellular fraction (cytosol) and the particulate biomass of E. canadensis (mean ± SD, n = 3)
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It was shown experimentally [19] that the larger concentration of the metal is dissolved in cytoplasm of microalgae, the more effectively it is assimilated by crustaceans. Due to low penetration of 241Am in cytoplasm of microalgae, the assimilation of 241Am by copepods was low (0.9%). Marine bivalves, however, assimilated up to 40% of 241Am from labeled microalgae [12]. The retention of 241Am ingested by calanoid copepod with labeled algae (4.5%) was more efficient than the retention of 237Pu (0.8%) [18].

Actinides in biochemical fractions of plant biomass

The distribution of americium in biochemical fractions of biomass was estimated for four macrophyte species: vascular aquatic plants E. canadensis, M. spicatum and C. demersum and aquatic moss F. antipyretica (Fig. 1). The lipid fraction of the biomass contained 0.1–0.8% of americium. As we estimated before, the total concentration of lipids in the biomass of E.canadensis was 3.6% [13]. The contribution of lipids to the adsorption of 241Am by fungi and yeasts was not considerable [10, 11], that correspond with our data for submerged macrophytes (Fig. 1).

Fig. 1
figure 1

Distribution of 241Am (% of total activity) among fractions of the biomass of four macrophyte species: Elodea canadensis, Myriophyllum spicatum, Ceratophyllum demersum and Fontinalis antipyretica. Mean ± SD, n = 3. * Elodea was incubated in the dark

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The concentration of americium in alkali extract of the biomass of macrophytes was 8–24% (Fig. 1). Alkali extract of E. canadensis biomass contained the major concentration of cellular protein (95 ± 5%) and the major concentration of carbohydrates (89 ± 13%) [13]. Total concentration of protein in the biomass of E. canadensis was 30%, carbohydrates—6% [13]. The effect of protein and carboxyl functional groups on the adsorption of americium was demonstrated for yeasts and fungi [10, 11]. The extraction of carboxyl functional groups from biomass of yeasts and fungi reduced the adsorption capacity of these microorganisms most effectively [10, 11], implying that the carboxyl groups may play more important role in adsorption of americium than protein. The contribution of protein to the biosorption of americium was most essential for yeasts [10] than for fungi [11].

The percent of 241Am in the biomass residue of macrophytes was 76–92% (Fig. 1). The biomass residue was considered to be mainly cellulose-like polysaccharides of cell walls. The biomass residue of Elodea in our experiments also contained 5% of cellular protein and 11% of carbohydrates [13]. The biomass residue comprised 19% of Elodea biomass [13] and 20% of F. antipyretica biomass. Other authors reported the content of cellulose in the tissues of Elodea and Fontinalis about 15%, lower concentration of cellulose was detected in Myriophyllum (5–10%) [21]. This proves our assumption of the cellulose basis of the biomass residue in our experiments. The results for Elodea are consistent with our results obtained previously [13]. The biochemical fractionations of americium absorbed by Elodea under dark and light conditions were similar (Fig. 1), supporting the mechanism of biosorption.

The distribution of 238Pu in the biochemical fractions of the biomass of Elodea (Fig. 2) was similar to the distribution of 241Am: the major portion of plutonium (88–96%) was detected in the biomass residue, i.e. bound to cellulose-like polysaccharides of cell walls. From 7 to 11% of 238Pu was recorded in the fraction of proteins and carbohydrates and 0.5–0.8% in the fraction of lipids. Hence, of the molecules constituting Elodea biomass, cellulose-like polysaccharides are principal concentrators of 241Am and 238Pu. The distributions of americium in the biomass of three vascular plant species and aquatic moss do not differ essentially (Fig. 1). The distribution of 241Am and 238Pu in the biochemical fractions of the biomass of Elodea did not change essentially during plant exposure in water containing actinides (Fig. 2). The results of biochemical fractionation of 242Pu are not presented here, because of essential loss (up to 60%) of 242Pu concentration during the preparation of the samples for mass spectrometry.

Fig. 2
figure 2

Distributions of 241Am and 238Pu (% of total activity) among biochemical fractions of the biomass of Elodea canadensis after 2–6 days of incubation in water containing radionuclides

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Other authors reported an important role of extractable polysaccharides and lipids in retention of such artificial isotopes as 137Cs, 60Co and 51Cr in the biomass of Elodea canadensis [9]. Compared to macrophytes, the biosorption of radionuclides in the biomass of cyanobacteria and microalgae was much higher due to higher concentration of these isotopes in the fraction of sugars [9]. But the concentration of isotopes in cellulose of cell walls was not taken into consideration.

Conclusions

Transuranium elements americium and plutonium taken up from water by E.canadensis apical shoots were mainly (90% for 241Am; 89% for 238Pu and 82–87% for 242Pu) absorbed by structural components of plant cells (mostly cell walls). About 10–18% of the activity was recorded in the dissolved fraction including cytosol.

The major portion of 241Am (76–92%) was bound to cellulose-like polysaccharides of cell walls of E. canadensis, M. spicatum, C. demersum and F. antipyretica, 8–24% of americium activity was registered in the fraction of proteins and carbohydrates, and just a small portion (<1%) in the lipid fraction. The distribution of 238Pu in the biochemical biomass fractions of Elodea was similar to that of 241Am. Hence, americium and plutonium had the highest affinity to cellulose-like polysaccharides of cell walls of freshwater submerged macrophytes.