Microsoft Word - Hornik et al NB 2008.doc Nova Biotechnologica 8-1 (2008) 55 BIOACCUMULATION OF 137Cs AND 60Co IN FRESHWATER PLANTS MIROSLAV HORNÍK, MARTIN PIPÍŠKA, JOZEF AUGUSTÍN Department of Biotechnology, University of SS. Cyril and Methodius, J. Herdu 2, Trnava, SK-917 01, Slovak Republic (hornikm@ucm.sk) Abstract: Contamination of the aquatic environment by the heavy metals and radionuclides has become a serious concern in the world. In our study, gamma-spectrometry of freshwater plants Bacopa monnieri and Egeria densa growing in cultivation media spiked with 137CsCl and 60CoCl2 was used for quantitative determination of bioaccumulation kinetic and distribution Cs+ and Co2+ ions in plant tissues. We found, that bioaccumulation of Cs and Co by fully immersed B. monnieri in Hoagland media (HM) was dependent on ion concentration in medium. Approx. 5-times lower Cs uptake 2.9 nmol/g (d.w.) was obtained in plants cultivated in 20% HM than from deionized water. The maximal Co uptake was 4-times higher than cesium uptake at the same conditions. Both Cs and Co were localized mainly in roots. The highest immobilization from roots to shoots was found in the case of Co uptake from deionized water with concentration ratio [Co]leaves : [Co]stem : [Co]root = 1.00 : 5.33 : 56.8. Cesium uptake by submerged plant E. densa was also strongly dependent on nutrients concentration in medium. However, in the case of cobalt uptake this dependence was less pronounced. Nutrients concentration also had a significant influence on distribution of Cs between stems and leaves of E. densa. Cesium was localized in leaves, however with increasing of nutrients concentration in cultivation media Cs was localized for account of stem. On the other hand, cobalt was immobilized mainly in leaves in whole range of nutrients concentration. Obtained data can serve as a models for understanding of phytoaccumulation of radionuclides from open water ponds and water channels in the vicinity of nuclear power plants and monovalent and bivalent metals from industrial sources of contamination. Keywords: cesium, cobalt, 137Cs, 60Co, bioaccumulation, freshwater plants 1. Introduction Heavy metals are toxic pollutants released into the surface and ground water as a result of different activities such as industries, mining, and agriculture. At present, a number of technologies can be used to remove of heavy metals from contaminated water such as filtration, reverse osmosis, solvent extraction, adsorption, chemical precipitation and ion-exchange (CHEREMISINOFF, 2002). However, these methods are not efficient in removing low heavy metals concentrations, can be relatively expensive and may fail to achieve legal limits. Contrary to this, phytoremediation, i.e. removal of pollutants by the use of plants offers a promising technology for heavy metal removal from waste water (STERNBERG, 2007; FIOL et al., 2005). Aquatic macrophytes have a great potential to accumulate heavy metals inside their plant body (PRASAD, 2007; OTTE and JACOB, 2006). Most studies on pollutant bioaccumulation in macrophytes are aimed at assessing removal efficiency or toxic effects without taking into account the metal bioaccumulation process by macrophytes, key knowledge not only to understand the behavior of macrophytes but also to optimize effluent depuration by means of artificial wetlands. Wetlands have significant merits of low capital 56 Horník, M. et al. and operating costs compare with conventional system as activated sludge, aerated lagoon system etc.. This technology has been used mainly in the case of mine waters (see e.g. LESLEY et al., 2008; LESLEY and YOUNGER, 2007; BATTY et al., 2005). The nuclear revolution (weapons testing or accidents at power production) has resulted in the large-scale release of cesium into the environment, in particular l34Cs (τ = 2.07 y) and the long-lived radionuclide 137Cs (τ = 30.2 y). Radiocesium 137Cs persists in aquatic systems, with environmental half-lives in monomitic lakes, meromitic lakes and flowing rivers 1.2, 6.7 and 1.4 years, respectively (AVERY, 1996). Its almost unlimited solubility in aquatic systems and chemical similarity to potassium means that it can be easily assimilated by terrestrial and aquatic organisms (MOULD, 2000; KERPEN, 1986). Large variations in the activity concentrations of 137Cs from Chernobyl nuclear accident in fish and lake water were still being observed in the 1990s and even in the 2000s (SAXÉN and ILUS, 2008; SAXÉN, 2007). Cobalt usually occurs in the environment in association with other metals such as copper, nickel, manganese and arsenic. Cobalt released by human activities comes mainly from: nickel, copper, silver, lead and iron mines and refineries; metal production facilities; industrial boilers that burn coal and oil; vehicles that burn gasoline; and incinerators that burn refuse and sewage sludge (PEREZ-ESPINOZA et al., 2005). Cobalt, as microelement for methanogenic bacteria, not for higher plants, can be also trapped in anaerobic bottom sediments. Cobalt 60Co (τ = 5.27 y) is also present in low-level radioactive wastes (CARON and MANKARIOS, 2004). Our previous papers were oriented to bioaccumulation of radionuclides by terrestrial vascular plants from defined water solutions (HORNÍK et al., 2007; BARÁTOVÁ et al., 2006). The objectives of this study are the investigation of 137Cs and 60Co uptake and their distribution in roots, stems and leaves of freshwater plants Bacopa monnieri and Egeria densa, in order to evaluate the role of nutrient concentration in bioaccumulation processes. B. monnieri is an emergent, wetland macrophyte. It grows very fast with its creeping stem in wetlands as a weed. It is well known from ancient times for its medicinal properties having active ingredients like bacosides and bacopasides. E. densa is an introduced invasive species, mainly via the aquarium trade. Also, this invasive submerged species is distributed in lakes and rivers of Europe. 2. Materials and methods 2.1 Plant material Freshwater plants Bacopa monnieri and Egeria densa were obtained from common aquarium shops. One week before experimentation, plants were pre-cultivated in 15 dm3 aquaria filled with 50% Hoagland medium (HM), (HOAGLAND, 1920) at artificial illumination with 12h/12h light/dark cycle (illumination with 2 tubes Brilliant daylight - 6000 K, 1 300 lm and Tropic sun – 4 700 K, 1 000 lm; Sera, D) and at 22±2°C. For experiments, healthy green plants of comparable weight (0.4-0.5 g Nova Biotechnologica 8-1 (2008) 57 fresh weight; f.w.) were used and three-times washed in deionized water before experiments. The following molar concentrations of salts were presented in full-strength HM (mM): MgSO4.7H2O – 1.5; KNO3 – 4.0; CaCl2 – 4.0; NaH2PO4.2H2O – 1.87; Na2HPO4.12H2O – 0.13; FeSO4.7H2O – 0.06; NaNO3 – 4.0; NH4Cl – 3.17; NH4NO3 – 2.0; H3BO3 – 0.14; Na2MoO4.2H2O – 0.0025; MnSO4.5H2O – 0.21; ZnSO4.7H2O – 0.023; CuSO4.5H2O – 0.033; pH 6.5. 2.2 Bioaccumulation experiments For bioaccumulation experiments, freshwater plants B. monnieri or E. densa (0.4- 0.5 g; f.w.) were placed on the bottom in 100 cm3 Erlenmayer flasks containing 50 cm3 deionized water or HM diluted with deionized water in the ratio 1:1, 1:3, 1:4, 1:7, 1:11 or 1:25, and spiked with both 137CsCl and 60CoCl2. Cultivation was carried out at artificial illumination with 12h/12h light/dark cycle (2 000 lx) and 22±2°C under occasional shaking. In time intervals, aliquots were taken and the remaining radioactivity in the cultivation media was estimated. At the end of the experiments, plants were washed in deionized water, dried at 60°C during 48 h and incorporated radioactivity of 137Cs or 60Co in leaves, stems or roots was measured by gamma- spectrometry. All the experiments were performed in triplicate series. 2.3 Radiometric analysis For radiometric determination 137Cs and 60Co in plants and cultivation media gamma-spectrometric scintillation detector 54BP54/2-X with well type crystal NaI(Tl) (Scionix, NL) with data processing software Scintivision32 (ORTEC, USA) were used. Counting time 600 s allowed obtaining data with measurement error <2 %, which do not reflect other source of errors. Standardized solutions of 137CsCl (5.723 MBq/cm3; 20 mg/dm3 CsCl + 3 g/dm3 HCl) and 60CoCl2 (5.571 MBq/cm 3; 20 mg/dm3 CoCl2 + 3 g/dm 3 HCl) were obtained from Czech metrology institute (CZ). 2.4 Speciation modeling Prediction of Cs and Co speciation in the nutrient solutions as a function of the total salt concentrations, solution pH and temperature was performed using the software Visual MINTEQ ver. 2.53. This speciation model allows the calculation of the composition of solution, in regard to formation of metal complexes. 3. Results and discussion Freshwater plants may take up nutrients from the sediments by roots or may absorb nutrients from the water column by foliar uptake. Determining the principal mode of toxic metals and radionuclides uptake by macrophytes is important because it affects the interpretation of the role of macrophytes in aquatic systems. If root 58 Horník, M. et al. uptake is the principal mode, then the metals and radionuclides content of the macrophytes represents remobilization from the sediments. If foliar uptake is the principal mode, then the metals and radionuclides content of the macrophytes represents a reduction in the water column concentration with the macrophytes serving as at least a temporary sink for these pollutants (KELLY and PINDER III, 1996). However, in these processes the speciation of metals and radionuclides in both water and sediment can play an important role. For example, MADRUGA and CARREIRO (1992) found in Tejo River water, that 60Co was almost 100% in cationic forms, however, in the presence of sediment there was a decrease in proportion of cationic forms (to 50%), with some anionic forms appearing. 0 1 2 3 4 5 6 7 8 0 4 8 12 16 20 B io ac cu m ul at io n of C s+ [n m ol /g ]; d. w . Time [d] 20% HM Deionized water 0 1 2 3 4 5 6 7 8 0 10 20 30 40 50 60 70 80 90 B io ac cu m ul at io n of C o2 + [n m ol /g ]; d. w . Time [d] 20% HM Deionized water Fig. 1. Bioaccumulation kinetics of Cs+ ions (A) and Co2+ (B) by freshwater plant B. monnieri (25 g/dm3; f.w.). Fully immersed plants were cultivated in deionized water or 20% Hoagland medium (HM), pH 6.5 containing 0.12 µmol/dm3 CsCl (17 kBq/dm3 137CsCl) and 0.11 µmol/dm3 CoCl2 (13 kBq/dm3 60CoCl2) at light/dark cycle 12h/12h (2 000 lx) and at 22±2°C. Total uptake of Cs: deionized water – 8.3%; 20% HM – 0.9%. Total uptake of Co: deionized water – 35%; 20% HM – 6.3%. Data represent average of three independent experiments. Bioaccumulation kinetics of Cs+ and Co2+ ions by emergent freshwater plant Bacopa monnieri are presented in Fig. 1A, B. Under given experimental condition, i.e. the initial concentration C0 = 0.12 µmol/dm 3 CsCl and biomass concentration 25 g/dm3 (f.w.) at 22°C the maximum cesium uptake was observed after 24 h and was stable within the next 7 days. As can be seen from Fig. 1A, bioaccumulation of Cs is dependent on ion concentration in medium. Cesium uptake 14.1 nmol/g (dry weight; d.w.) was obtained in deionized water after 8 days cultivation. Approximately 5-times lower 2.9 nmol/g (d.w.) was found at plants cultivation in 20% HM, where concentration of competing K+ and NH4 + ions was 0.8 and 1.2 mmol/dm3, respectively. Nevertheless, as can be calculated by Visual MINTEQ speciation program, cesium in 20% HM at pH 6.5 occurs practically as free cation (> 99% Cs+). Monovalent K+ and NH4 + ions act competitively on cesium uptake by plants. Vascular plants can accumulate Cs+ ions through both their leaves and through their roots. Substrate affinity i.e. Km values for Cs + uptake in shoots and roots of winter wheat is 25.5 and 16.7 μmol/dm3 respectively. According to SHAW et al. (1992) the measured transfer factor of roots is one order of magnitude greater than that of shoots. SMITH et al. (2003) in a whole-lake experiment at study to reduce the bioaccumulation of radiocesium 137Cs in fish in lakes contaminated A B Nova Biotechnologica 8-1 (2008) 59 by the Chernobyl accident found, that after the addition of 15 t of potassium chloride to Lake Svyatoe, Kostiukovichy resulted in a decrease in activity concentration of 137Cs to approximately 40% of pre-countermeasure values in a number of different fish species. However, the addition of 4 kg 133CsCl into an 11.4-ha, 157 000 m3 reservoir previously contaminated with 137Cs from past reactor operations at the US Department of Energy’s Savannah River Site near Aiken, South Carolina (USA) increased of 6.1 MBq of 137Cs (1.9 mg 137Cs) in the water column (PINDER III et al., 2006; 2005). These authors supposed, that possible sources for the increased 137Cs included release from the sediments (the principal source), release from the approx. 26 000 kg of aquatic macrophytes that occupied 80% of the reservoir, and wash-in from the pond’s watershed. 0 2000 4000 6000 8000 10000 Deionized water S pe ci fic r ad io ac tiv ity 1 37 C s [B q/ g] ; d .w . 20% Hoagland medium Root Stem Leaves 0 20000 40000 60000 80000 100000 120000 140000 Deionized water S pe ci fic r ad io ac tiv ity 6 0 C o [B q/ g] ; d .w . 20% Hoagland medium Root Stem Leaves Fig. 2. Specific radioactivities of 137Cs (A) and 60Co (B) in root, stem and leaves of B. monnieri after 8 days cultivation of fully immersed plants in deionized water or 20% HM, pH 6.5 containing 0.12 µmol/dm3 CsCl (17 kBq/dm3 137CsCl) and 0.11 µmol/dm3 CoCl2 (13 kBq/dm3 60CoCl2) at light/dark cycle 12h/12h (2 000 lx) and at 22±2°C. Data represent average of three independent experiments. Bioaccumulation kinetic of Co2+ ions by B. monnieri was identical with the process of Cs bioaccumulation at the same conditions, but only in the case of plants cultivation in 20% HM (Fig. 1B). Cobalt in 20% HM at pH 6.5 occurs practically as free cation (> 93% Co2+). When plants were cultivated in deionized water containing 0.11 µmol/dm3 CoCl2, the bioaccumulation of cobalt gradually increased to the maximum 62 nmol/g (d.w.) in 5 day of cultivation, and then slowly decreased to the value 54 nmol/g (d.w.) in 8 day of plants cultivation. The bioaccumulation of Co is also dependent on ion concentration in medium. It can be concluded, that cobalt was accumulated in more extent than cesium practically at the same conditions. ADAM and GARNIER-LAPLACE (2003) found similar results at accumulation of 137Cs and 60Co by freshwater alga Cyclotella meneghiana. From results of cesium distribution in plant organs of B. monnieri we found (Fig. 2A), that cesium is mainly localized in roots than in stems and leaves with concentration ratio [Cs]leaves : [Cs]stem : [Cs]root = 1.00 : 0.69 : 1.76 at plants cultivation in 20% HM and [Cs]leaves : [Cs]stem : [Cs]root = 1.00 : 1.22 : 2.14 at plants cultivation in deionized water. Similarly, cobalt was mainly immobilized in roots with concentration ratio [Co]leaves : [Co]stem : [Co]root = 1.00 : 0.39 : 1.49 at plants cultivation in 20% HM (Fig. 2B). In the case of plants cultivation in deionized water A B 60 Horník, M. et al. cobalt was much more immobilized in roots with concentration ratio [Co]leaves : [Co]stem : [Co]root = 1.00 : 5.33 : 56.8. Its generally known, that at emergent species of water plants root uptake is presumed to be the dominant mechanism of nutrients absorption. In this regard, mineral nutrients, mainly macroelements (Ca and Mg), from 20% HM are probably preferentially accumulated by roots than cobalt. 0 1 2 3 4 5 6 7 8 0 10 20 30 40 B io ac cu m ul at io n of C s+ [n m ol .g -1 ]; d. w . Time [d] Deionized water 8,3% HM 12,5% HM 25% HM 50% HM 0 1 2 3 4 5 6 7 8 0 20 40 60 80 100 120 B io ac cu m ul at io n of C o2 + [n m ol .g -1 ]; d. w . Time [d] Deionized water 8,3% HM 12,5% HM 25% HM 50% HM Fig. 3. Bioaccumulation kinetics of Cs+ (A) and Co2+ (B) ions by E. densa (20 g/dm3; f.w.). Fully immersed plants were cultivated in deionized water or diluted HM (8.3%, 12.5, 25% or 50%), pH 6.5 containing 0.12 µmol/dm3 CsCl (17 kBq/dm3 137CsCl) and 0.11 µmol/dm3 CoCl2 (13 kBq/dm3 60CoCl2) at light/dark cycle 12h/12h (2 000 lx) and 22±2°C. Total Cs uptake (%): deionized water – 46.4; 8.3% HM – 5.7; 12.5% HM – 6.4; 25% HM – 2.1; 50% HM – 3.3. Total Co uptake (%): deionized water – 70.3; 8.3% HM – 66.8; 12.5% HM – 72.0; 25% HM – 81.8; 50% HM – 70.6. Data represent average of three independent experiments. 0 10 20 30 40 50 0 20 40 60 80 100 B io ac cu m ul at io n of C s+ [n m ol /g ]; d. w . HM concentration [%] 0 20 40 60 80 100 B io ac cu m ul at io n of C o2 + [n m ol /g ]; d. w . 0 10 20 30 40 50 0 20 40 60 80 100 0 20 40 60 80 100 ra tio [C o] fo rm /[C o] to ta l HM concentration [%] Cs+ Co2+ CoSO 4 CoHPO 4 ra tio [C s] fo rm /[C s] to ta l Fig. 4. A. Dependence of bioaccumulation of Cs+ (-■-) and Co2+ (-○-) ions by E. densa (20 g/dm3; f.w.) on dilution of Hoagland medium at pH 6.5 after 8 d cultivation of fully immersed plants at light/dark cycle 12h/12h (2 000 lx) and 22±2°C. For details see Fig. 3. B. The percentage (ratio of concentration metal form to the total concentration of metal) of cesium in the form of Cs+ and cobalt in the forms Co2+ ions, CoSO4 or CoHPO4 in dependence on dilution of Hoagland medium at pH 6.5 and 22°C calculated by the speciation program Visual MINTEQ. As can be seen from Fig. 3A,B and Fig. 4A, Cs+ uptake by freshwater submerged plant Egeria densa is strongly dependent on ion concentration in medium. However in the case of cobalt uptake this dependence was less pronounced. Cesium uptake 46% (32.8 nmol/g; d.w.) in deionized water decreased to 6% (6.0 nmol/g; d.w.) in 8.3% A B A B Nova Biotechnologica 8-1 (2008) 61 HM, where concentration of competing K+ and NH4 + ions was 0.33 and 0.5 mmol/dm3, respectively. On the contrary, uptake of Co2+ ions increased with concentration of monovalent and bivalent cations in HM, however at highest concentration, i.e. 8 mmol/dm3 Me+ ions and 3 mmol/dm3 Me2+ ions in 50% HM cobalt uptake slightly decreased. The speciation of cesium and cobalt in individual cultivation media at pH 6.5 and 22°C can be seen in Fig. 4B. 0 10 20 30 40 50 0 1 2 60Co [6 0 C o] st em /[ 60 C o] le av es accum ulation in leaves HM concentration [%] accum ulation in stem 137Cs 0 1 2 [1 37 C s] st em /[ 13 7 C s] le av es Fig. 5. Influence of dilution of Hoagland medium on 137Cs (-■-) and 60Co (-○-) distribution in stems and leaves of E. densa (20 g/dm3; f.w.) after 8 d cultivation of fully immersed plants at light/dark cycle 12h/12h (2 000 lx) and 22±2°C. Data expressed as the ratio of concentration in stems to the concentration in leaves (Bq/g / Bq/g; d.w.). For details see Fig. 3 Concentration of mineral nutrients had also a significant influence on distribution of 137Cs between stems and leaves of submerged plant E. densa (Fig. 5). In the case of plants cultivation in deionized water containing 0.12 µmol/dm3 CsCl cesium was localized manly in leaves, however with increasing of nutrients concentration in cultivation media cesium was localized for account of stem. On the other hand, cobalt was immobilized mainly in leaves of E. densa in whole range of nutrients concentration. 4. 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