Uranium accumulation by aquatic plants from uranium-contaminated water in Central Portugal

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URANIUM ACCUMULATION BY AQUATICPLANTS FROM URANIUM-CONTAMINATEDWATER IN CENTRAL PORTUGALJoão Pratas a b , Paulo J. C. Favas b c , Carlos Paulo b , NelsonRodrigues a b & M. N. V. Prasad da Department of Earth Sciences, Faculty of Sciences and Technology,University of Coimbra, Portugalb Geosciences Center, Faculty of Sciences and Technology, Universityof Coimbra, Portugalc School of Life Sciences and the Environment, University of Trás-os-Montes e Alto Douro, Vila Real, Portugald Department of Plant Sciences, University of Hyderabad,Hyderabad, India

Available online: 29 Jun 2011

To cite this article: João Pratas, Paulo J. C. Favas, Carlos Paulo, Nelson Rodrigues & M. N. V. Prasad(2012): URANIUM ACCUMULATION BY AQUATIC PLANTS FROM URANIUM-CONTAMINATED WATER INCENTRAL PORTUGAL, International Journal of Phytoremediation, 14:3, 221-234

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International Journal of Phytoremediation, 14:221–234, 2012Copyright C© Taylor & Francis Group, LLCISSN: 1522-6514 print / 1549-7879 onlineDOI: 10.1080/15226514.2011.587849

URANIUM ACCUMULATION BY AQUATIC PLANTSFROM URANIUM-CONTAMINATED WATERIN CENTRAL PORTUGAL

Joao Pratas,1,2 Paulo J. C. Favas,2,3 Carlos Paulo,2

Nelson Rodrigues,1,2 and M. N. V. Prasad4

1Department of Earth Sciences, Faculty of Sciences and Technology,University of Coimbra, Portugal2Geosciences Center, Faculty of Sciences and Technology, University of Coimbra,Portugal3School of Life Sciences and the Environment, University of Tras-os-Montese Alto Douro, Vila Real, Portugal4Department of Plant Sciences, University of Hyderabad, Hyderabad, India

Several species of plants have developed a tolerance to metal that enables them to survivein metal contaminated and polluted sites. Some of these aquatic plants have been reportedto accumulate significant amounts of specific trace elements and are, therefore, usefulfor phytofiltration. This work focuses the potential of aquatic plants for the phytofiltrationof uranium (U) from contaminated water. We observed that Callitriche stagnalis, Lemnaminor, and Fontinalis antipyretica, which grow in the uraniferous geochemical provinceof Central Portugal, have been able to accumulate significant amounts of U. The highestconcentration of U was found in Callitriche stagnalis (1948.41 mg/kg DW), Fontinalisantipyretica (234.79 mg/kg DW), and Lemna minor (52.98 mg/kg DW). These resultsindicate their potential for the phytofiltration of U through constructed treatment wetlandsor by introducing these plants into natural water bodies in the uraniferous province ofCentral Portugal.

KEY WORDS: phytofiltration, Callitriche stagnalis, Fontinalis antipyretica, Lemna minor,in-situ remediation, aquatic macrophyte

INTRODUCTION

The use of plant species for the environmental clean-up of trace elements is based ontheir ability to concentrate trace elements in their tissues (Adriano et al. 2004). Bioconcen-tration involves certain specific processes in the soil/sediment/water/plant system. Plantsthat are capable of growing in trace element-rich substrates usually reveal the mineral com-position of those substrates, for example, in the soil, sediment and water (Kabata-Pendiasand Pendias 2001). These plant species are able to accumulate or exclude toxic metals andcan, therefore, tolerate the imposed stress. This function is of considerable significance

Address correspondence to Paulo J. C. Favas, School of Life Sciences and the Environment, University ofTras-os-Montes e Alto Douro, Apartado 1013, 5000-801 Vila Real, Portugal. E-mail: [email protected]

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222 J. PRATAS ET AL.

in the emerging areas of wastewater treatment and in the establishment of constructedtreatment wetlands (Prasad 2004; Rai 2009; Uysal and Taner 2009; Stout and Nusslein2010). However, the specific process of bioconcentration depends not only on plant traitsbut also on certain physico-chemical and geochemical processes. In natural sites, metal re-moval depends on factors including microbial biofilms on abiotic substrates and the growthof periphyton (Gray et al. 2001; Bradac et al. 2010).

Trace element contamination in aquatic ecosystems can be both abiotic (water andsediments) and biotic (organisms living in the contaminated environment). Metal uptakeby aquatic plants depends on the type of plant, direct absorption from the water columnto the plant surface followed by passive or active transport across membranes and, to alesser extent, root uptake (Rai et al. 1995; Brooks 1998; Nyquist and Greger 2007). Theseprocesses have been observed not only in submersed species (with poorly developed rootsystems) but also in free-floating plants (Brooks 1998). Additionally, the bioaccumulationof metals in aquatic plants also occurs due to absorption, adsorption and other mechanismsof metal retention. Holistically, these mechanisms fall under the term “bioaccumulation.”A physico-chemical process, biosorption is a term that is used to describe the set ofmechanisms for the removal of substances from solution by biological materials, includingabsorption, adsorption, ion exchange, surface complexation, and precipitation (Tsezos andVolesky 1981; Volesky and Holan 1995; Raize et al. 2004; Gadd 2009).

Certain aquatic plants, such as algae, bryophytes, and angiosperms, are consideredto be indicators of trace element pollution and have been successfully used as diagnostictools for monitoring environmental pollution (Markert 1993; Cenci 2000). There is littleavailable information about uranium (U) accumulation by aquatic plants, including possiblephytoremediation applications.

Among aquatic plants, algae are of considerable interest to ecological engineers dueto their ability to sequester U as evidenced by the fact that many algae can survive inabundance under extreme environmental conditions. Algae grow in a wide spectrum ofcontaminated waters from alkaline environments (Chara, Nitella) to acidic mine drainagewastewaters (Mougeotia, Ulothrix). Therefore, Kalin et al. (2005) suggested these algaecould provide a simple and long-term solution for removing U in waste streams throughthe processes of adsorption, reduction, and transformation.

Pettersson et al. (1993) identified the water lily (Nymphaea violacea) as an accumula-tor of several radionuclides when they observed high levels of U and Th series radionuclidesin plant roots, rhizomes and foliage in the vicinity of the Ranger Uranium Mine (Australia).At the same mine, a recent attempt to phytoremediate mine runoff water was tested us-ing Eleocharis dulcis (Overall and Parry 2004). Another example of a macrophyte-basedconstructed wetland being used for mine water remediation occurred at Schlema-AlbedoraUranium Mine in Germany using Phragmites australis and Carex gracillis (Gerth et al.2005). Lemna gibba (612.36 ± 143.6 mg/kg DW) is an example of a U accumulator plant(Mkandawire and Dudel 2005). Members of Lemnacea are the most favored plants forphytoremediation and have been intensively described in literature as duck weeds includ-ing Lemna, Spirodella, Wolffia, and Wolffiella (Miretzky et al. 2004; Rahman et al. 2007).Other aquatic plants that have been suggested as U accumulators include Zostera japonica(5.2–8.8 Bq/kg DW), Zostera marina (11.3–12.4 Bq/kg DW) (Kondo et al. 2003) and Typhalatifolia (9.0 mg/kg DW, roots + rhizome) (Soudek et al. 2007).

This work is a part of an ongoing study on the uraniferous geochemical provinceof Central Portugal in which the use of aquatic plants as indicators of U contaminationis being probed using aquatic plants with an emphasis on their potential use in emerging

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URANIUM ACCUMULATION BY AQUATIC PLANTS 223

phytotechnologies. In the present study, we analysed the aquatic community and identifiedpotential phytotechnologically useful species capable of immobilizing U on a long-termbasis for the ecological restoration of the contaminated sites.

Location and Geology

The studied area is located in the counties of Tabua, Nelas, and Oliveira do Hospital ofthe Centre of Portugal (Figure 1). This area is in the south-west part of the uraniferous regionof Beiras (Centre of Portugal). The uraniferous area occupies approximately 10,000 km2

and is part of the geotectonical Central-Iberian Zone. In this region, there are large oc-currences of several phases of hercynian granites that intrude on the formations of theAnte-Ordovician Schist-Graywake Complex. Discordantly Ordovician formations are sit-uated above this Complex. There are also occurrences of Tertiary deposits (Ferreira 1971;Ponte and Pereira 1991).

The uraniferous deposits are located on the hercynian granites, on the metasedimentsenclaves and in the metamorphism contact haloes. The occurrences of U define an archthat is located on the north-east border of the Serra da Estrela horst (Ferreira 1971; Neiva2003).

The principal uranium minerals present in these deposits are pechblende, autonite,thorbernite, uranocircite, and sabugalite within quartziferous gang or argyles (JEN 1966;Ferreira 1971; IGM 1998; Neiva 2003). Several of these deposits have been exploited either

Figure 1 Location of the study area on the map of Portugal (a) and simplified geological map (b) (based on Ponteand Pereira 1991). (Color figure available online.)

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by underground or surface mining methods. The main mineral processing method used waslixiviation (JEN 1966), especially during the last working activity phase (the last mineclosed in 2001). Many of the places were left in different stages of degradation. However,several programs for the environmental restoration of some of these old mines have beendeveloped.

MATERIALS AND METHODS

Field Sampling

The samples for this work were collected from running and in standing waters (lenticand lotic) in places where aquatic species could be observed. Samples of the waters and thevegetable species were taken at these sites. The plants collected represented free-floatingand rooted emergent plants. In the ponds, only free-floating plants were found. The plantmaterial was thoroughly washed with fresh water to remove sediment and other foreignobjects and then briefly cleaned with distilled water. The plants were collected in May 2006and were identified with the help of regional/local floras (Franco 1971, 1984; Franco andAfonso 1994, 1998; Sampaio 1988; Bracamonte and Domingo 2002) and the herbariumof the Department of Life Sciences of the University of Coimbra, Portugal (Table 1). Thewater pH was determined using a pH meter in the field. HCO3 was also measured in situusing the titration method. The water was transported in clean and sterilized plastic bottles.The plants were transported in clean plastic bags to the laboratory.

Sampling Treatment

All of the samples were processed in the Chemistry Laboratory of the Earth ScienceDepartment of the University of Coimbra. To determine the presence of U, As, and metals,the water samples were filtered with 0.45 µm Millipore cellulose membrane filters, and thepH was adjusted to 2 with 65% HNO3 (V/V). The plants were dried in an oven at 60◦C.The dried plant samples were ground into a homogenous powder for further analysis.

Analytical Procedures

The uranium in natural water and plants was estimated using a “Fluorat-02–2Manalyzer” (Lumex, Russia). This device measures the concentration of U in solution bythe intensity of the fluorescence of the uranyl-ions (λ = 530 nm). The water samples wereanalysed by a standard fluorimetric analysis (Lumex 2000). The detection limit for U in thewater samples was 0.1 µg/L. Certified reference material from the National Water ResearchInstitute of Canada (reference TMDA-62) was used to validate the results. Fluorometry wasthe methodology that was adopted for the determination of the U content in the plants. Thesamples were dry ashed at 450◦C, which was followed by dissolving the ash in 2.5 M nitricacid that was saturated with aluminium nitrate. The uranium was extracted into ethyl acetate.The ethyl acetate was then destroyed by ignition, and the residue was redissolved in 0.005%nitric acid (Huffman and Riley 1970; Van Loon and Barefoot 1989). The detection limit forU in the plant samples was 0.02 mg/L. Certified Virginia tobacco leaves (reference CTA-VTL-2, Polish certified reference material) were also analysed to validate the experimentalprocedure.

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Table 1 Aquatic plants identified in the studied area

Family Plant species

Alismataceae Alisma plantago-aquatica L.Baldellia alpestris (Cosson) Vasc.

Azolaceae Azolla caroliniana Willd.Boraginaceae Myosotis baetica (Perez Lara) Rocha AfonsoCallitrichaceae Callitriche brutia Petagna

Callitriche hamulata KochCallitriche lusitanica SchotsmanCallitriche stagnalis Scop.Callitriche truncata Guss. subsp. occidantalis (Rouy) Schotsman

Caryophyllaceae Sagina procumbens L.Cruciferae Rorippa nasturtium-aquaticum (L.) HayekCyperaceae Cyperus longus

Eleocharis palustris (L.) Roemer & SchultesScirpus holoschoenus L.

Fontanalaceae Fontinalis antipyretica HewdHaloragaceae Myriophyllum spicatum L.

Myriophyllum aquaticum (Velloso) Verdc.Iridaceae Iris pseudacorus L.Juncaceae Juncus articulatus L.

Juncus bufonius L.Juncus effusus L.Juncus heterophyllus DufresneJuncus pygmaeus C.Rich

Lemnaceae Lemna minor L.Lemna trisulca L.Spirodela polyrrhiza L.

Lythraceae Lythrum portula (L.) D.A.WebbLythrum hyssopifolium L.Rotala indica (Willd.) Koehne

Portulacaceae Montia fontana L. subsp. amporitana SennenPotamogetonaceae Potamogeton crispus L.

Potamogeton natans L.Potamogeton pectinatus L.Potamogeton perfoliatus L.Potamogeton pusillus L.

Ranunculaceae Ranunculus hederaceus L.Ranunculus peltatus Schrank subsp. saniculifolius (Viv.) C.D.K.CookRanunculus trichophyllus Chaix subsp. trichophyllus

Sparganiaceae Sparganium erectum L.Typhaceae Typha dominguensis Pers.

Typha latifolia L.Umbelliferae Apium nodiflorum (L.) Lag.

Hydrocotyle vulgaris L.Oenanthe crocata L.

To define the chemical characteristics of the stream water and also the occurrenceof other heavy-metal contamination related to the mineralization, several parameters weremeasured using current analytical methods, including Atomic Absorption Spectrometry(AAS, SOLAAR M Series equipment from Thermo–Unicam) for Ca, Mg, Na and K;coupled graphite furnace AAS for Fe, Mn, Cu, Zn, Cd, Co, Ni, Bi, Cr, Pb, and As; and theHigh Performance Anion Exchange Chromatography method for Cl, NO3, and PO4. The

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detection limits for major and trace elements in the water samples were 0.025 mg/L NO3,PO4, Cl; 0.02 mg/L Ca; 0.003 mg/L Mg; 0.015 mg/L Na, K; 2 µg/L Fe; 0.05 µg/L Mn;0.1 µg/L Cu, Pb; 0.25 µg/L As, Bi; 0.01 µg/L Zn; 0.05 µg/L Cd, Cr; 0.15 µg/L Co; and0.2 µg/L Ni.

Statistical Testing

We used a principal components analysis (PCA) to interpret the data. A PCA is usedto reduce the data dimensionality while preserving the variance-covariance structure of thedata. The analysis begins with p random variables X1, X2, . . . , Xp where no assumption ofmultivariate normality is required. If the principal components are derived from multivariatenormal random variables, then they have a noteworthy geometric property. The axes of theconstant density ellipsoids correspond to the principal components (Pereira et al. 2003). Inour study, PCA was performed using the AnDad C© (v. 7.10) software (CVRM 2002). Thedata matrix used in the analysis consisted of 61 samples and 21 variables (pH, HCO3, NO3,PO4, Cl, Ca, K, Mg, Na, Fe, Mn, Zn, Cu, Pb, Cd, Bi, Co, Cr, Ni, As, and U).

RESULTS AND DISCUSSION

A summary of the chemical analysis of the stream water samples is presented inTable 2. The observations made in the 61 sampling sites showed that U was detectedin the superficial waters in a wide range of concentrations (0.4–1220 µg/L). From ageographical interpretation of the sampling locations, it was clear that mine effluentscontributed significantly to the U contamination of the water.

Table 2 Chemical composition of the stream waters (n = 61)

Parameters Units Min Max Mean SD

pH 4.5 7.8 6.4 0.69HCO3 mg/L 0.00 171.41 31.26 25.20NO3 mg/L 0.09 28.38 3.77 4.51PO4 mg/L 0.04 13.34 1.98 2.34Cl mg/L 1.00 92.97 23.08 14.58Ca mg/L 1.17 218.27 15.94 33.57K mg/L 0.34 13.66 3.30 2.98Mg mg/L 0.85 80.71 5.96 12.02Na mg/L 7.57 80.06 18.10 12.48Fe µg/L 4.15 4363.40 332.31 691.43Mn µg/L 0.54 1767.04 216.59 470.37Zn µg/L 1.20 617.10 39.63 94.36Cu µg/L 0.42 139.42 9.41 18.31Pb µg/L 0.38 13.91 2.24 2.49Cd µg/L n.d. 17.43 0.79 2.71Bi µg/L 0.27 15.05 4.91 3.15Co µg/L n.d. 9.36 1.00 1.73Cr µg/L 0.05 36.96 2.48 6.16Ni µg/L 0.89 186.98 17.33 33.75As µg/L n.d. 50.43 4.47 7.31U µg/L 0.40 1220.40 35.45 161.99

n.d – not detected

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Figure 2 Projection of the variables in the two first factorial plans of the principal components analysis (PCA).(Color figure available online.)

The water pH ranged between 4.5 and 7.8 (Table 2). According to Wang et al. (2010),in an acid environment (pH < 4.0), U(VI) occurs predominately in the form of UO2

2+,whereas at higher pH ranges (4.0 < pH < 7.0), composite hydrolysed ionic species yieldcompounds such as UO2OH+, (UO2)2(OH)2

2+, and (UO2)3(OH)5+. When the pH is above

7.0, U(VI) precipitates easily.The hydrochemic data were analyzed by the PCA. The result of three principal axes,

which explained more than 60% of the total variance, are shown in Figure 2. Consideringthe retained axes and given the coordinate values, the following variable associations wereverified: a group composed of Cd, Zn, Mg, Mn, Ca, Ni, Co, and U (axis 1); another groupcomposed of K, Cl, Na, PO4, HCO3, and, in some way, NO3 and As (axis 2), which revealsthe independent behavior that of the first group; and a third group composed of Cu, Fe, Pb,and, in an unclear way, As (axis 3).

The PCA also highlighted sample “populations”, which are mostly distinct from oneanother (Figure 3). “Population” A is essentially composed of samples belonging to the

Figure 3 Projection of the samples in the first factorial plan of the principal components analysis (PCA). (Colorfigure available online.)

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local hydrochemical background. “Population” B results from the association of the mostU-contaminated samples. This “population” is located in the Cd, Zn, Mg, Mn, Ca, Ni, Co,and U association influence area. “Population” C is composed of the samples situated underthe influence of the K, Cl, Na, PO4, HCO3, NO3, and As group.

Based on the distinction of the samples derived from results of the PCA, the empirical(local) background level for U in superficial waters in the studied area was 3.24 µg/L. Thisvalue corresponds to the arithmetic mean concentrations of U in the “Population” A samplesin the results of the PCA. In most of the sampled sites, the U concentration was belowthis value. This value is exceeded in 23 sampling points, and in 7 of those points, theU concentration exceeded the limit established by USEPA for drinking water (30 µg/L)(USEPA 2000). At these points, there was a direct relation to mining activities because thestreams were directly fed by mine drainage.

Although we observed very low concentrations of U in the fresh waters in the studiedsites, we found a set of vegetable species with the ability to accumulate U in concentrationsthat are orders of magnitude higher than the surrounding environment. For this preliminarywork, we selected Callitriche stagnalis Scop., Lemna minor L., Fontinalis antipyretica L.,Apium nodiflorum (L.) Lag., and Oenanthe crocata L. from all of the collected speciesbecause they were the most representative species. The first three mentioned species accu-mulated U significantly.

A summary of the results of the chemical analysis is presented in Table 3 and inFigure 4. We also present the values of the natural background for U in these species(this background was assumed to be the arithmetical mean of the nonanomalous samplesresulting from the PCA) as well as the values of the bioconcentration factor (BCF), which isdefined as the ratio of the total concentration of elements in the plant to that in the growingwater (Zhu et al. 1999; Ha et al. 2011). However, in this study, we did not differentiatebetween adsorption to the tissue surface and true absorption into the plant interior; therefore,the cited values for BCF must be interpreted cautiously. This is a limitation of this study, but,

Table 3 Uranium in plant species (mg/kg, values on dry weight basis, DW) and in fresh waters (µg/L). Biocon-centration factor is BCF = concentration in plant/concentration in water. T/t is the relation between the maximumand background levels

Plant / Water / BCF n Mean Min Max SD Background T/t

Callitriche stagnalis 32 121.26 0.53 1948.41 386.25 0.66 2952Water 50.47 0.5 1220.4 215.9BCF 3.78×103 65.5 31.3×103 5.8×102

Fontinalis antipyretica 24 33.89 2.42 234.79 51.07 3.83 61Water 58.51 0.4 1220.4 253.4BCF 9.89 × 103 28.5 59.4 × 103 13.2 × 103

Lemna minor 10 3.84 0.02 8.54 2.98 0.40 21Water 5.18 1.2 18.6 5.09BCF 7.97 × 102 7.2 1.4 × 103 415.9

Apium nodiflorum 29 4.38 0.20 31.61 7.29 0.31 102Water 9.6 0.47 179 32.78BCF 1.8 × 103 35.1 21.9 × 103 41.6 × 102

Oenanthe crocata 20 2.24 0.03 35.15 7.78 0.12 293Water 2.71 0.4 12.38 2.73BCF 8.55 × 102 7.24 11 × 103 2.4 × 103

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Figure 4 Uranium concentration in aquatic plants (mg/kg, values on dry weight basis, DW).

for the remediation of water systems, it may not be necessary to specifically differentiatebetween adsorption and absorption.

Based on the ratio of the maximum concentration of the element in the plant material(T) to the natural background (t), the types of “physiological barriers” present were deter-mined according to the criteria and concept of Kovalevskii (1979) as applied to the selectionof suitable plant organs (biosamples) for biogeochemical prospecting. These criteria werealso recognized by Brooks (1983, 1993) and other authors (Pratas et al. 2005). Given thetypes of physiological barriers present and the correlation coefficients between the concen-trations in the water and plant materials, we determined the bioindication capacity of eachspecies.

The results showed a strong accumulation of U in the plants compared with theU present in the water. The highest concentrations of U were found in the submergedspecies Callitriche stagnalis, which showed an average BCF of 3.78 × 103. From theseobservations, we conclude the chosen plants, such as Callitriche stagnalis, appear to bekeystone species for further phytoremediation research to remove U from mining effluentsor in the phytoremediation of U-contaminated sites. This species also showed a high averageratio T/t (2952), would be classified as “barrier-free” (T/t > 300), and showed a very highlysignificant (P < 0.001) positive correlation with the U present in the water (Table 4).Therefore, this species serves as an excellent indicator of U pollution.

Table 4 Correlation coefficients between water and plant U concentrations data and between water pH and Uconcentrations in plants. Correlations in bold are very highly significant (P < 0.001)

L. minor

C. stagnalis F. antipyretica running water standing water A. nodiflorum O. crocatan = 32 n = 24 running water n = 10 standing water n = 14 n = 29 n = 20

U water 0.5575 0.0148 0.8909 0.7952 0.1128 0.0352pH water –0.5914 –0.0780 –0.2434 –0.0332 –0.5330 0.0083

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Table 5 Uranium in Lemna minor (mg/kg DW) and in running and standing waters (µg/L). Bioconcentrationfactor is BCF = concentration in plant/concentration in water. T/t is the relation between the maximum andbackground levels

n Mean Min Max Background T/t BCF (mean)

Plant concentration in running waters 10 3.84 0.02 8.54 0.40 21 7.97 × 102

Water 5.18 1.2 18.6Plant concentration in standing waters 14 9.14 0.28 52.98 0.45 118 2.87 × 103

Water 2.79 0.6 4.16

The bryophyte Fontinalis antipyretica, which normally grows on top of rocks inthe water showed a strong ability to monitor the contamination present in running waterbecause it accumulated large quantities of U. It is known that, in general, bryophytes havea great potential for rapid accumulation and exhibit seasonal fluctuations depending uponthe environmental contaminants (Cenci 2000). Because this species does not have a rootsystem, uptake occurs through the rhizoids as an ionic exchange between the environmentand the basal portion of the plant. The BCF for this species is high; it is on the order of9.89 × 103.

Free-floating Lemna minor shows good ability to accumulate U in a similar way, asobserved in Lemna gibba (Mkandawire and Dudel 2005). Lemna minor also showed a verysignificant (P < 0.001) positive correlation with the U present in the waters (Table 4) with anaverage BCF of 7.97 × 102 in running water and 2.87 × 103 in standing water. Therefore,it has great potential for U accumulation in running and standing waters. However, weobserved a discrepancy when the results from running waters were compared with thosefrom standing waters (Table 5).

Table 5 shows that the values of the maximum accumulation, the ratio T/t and theBCF were much higher in the standing waters compared with those of the running waters.This indicates a positive correlation between the concentration in the plant and the residencetime for this accumulation. If this is true, then there is a good opportunity for its use inphytoremediation in closed tanks where it could be grown. This obviously depends onfurther studies regarding its biomass and bio-productivity.

Several studies have shown that many factors affect the bioaccumulation (biosorption)of metals in aquatic ecosystems. Among the physico-chemical factors, pH is possibly themost important (Ebbs et al. 1998; Li et al. 2004; Gadd 2009; Kumar and Gaur 2011). Thiscan attributed to the influence of pH on the solution chemistry of metals and the activityof the functional groups of the biomass (Wang et al. 2010). Many microbial organisms,including bacteria, lichens, fungi and algae, have been studied for their U-binding capacity,and the maximum U uptake was most frequently observed between pH 4–5 (Li et al.2004; Bhat et al. 2008; Wang et al. 2010). Similar results have been found in severalother plant materials, such as dried roots (Bhainsa and D’Souza 2001) or coir pith (Parabet al. 2005). Srivastava et al. (2010) also observed in the aquatic plant Hydrilla verticillatathat the maximum U uptake rates occurred at pH 5. In the present study, only the speciesCallitriche stagnalis had a very highly significant (P < 0.001) negative correlation with thepH of the water (Table 4).

Apium nodiflorum and Oenanthe crocata, in spite of their high biomass and bio-productivity, did not show a significant accumulation in their aerial organs, and the values

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were not correlated with the U found in the water. However, Oenanthe crocata showeda high average T/t ratio (293; “high-barrier”). Therefore, more studies are needed onthese emergent species. It will be important to investigate the mechanism of U uptakeand accumulation by Apium nodiflorum and Oenanthe crocata, taking care to distinguishbetween the stems and roots and considering the water column and sediment. Overalland Parry (2004) showed that the U present in the stems of the emergent macrophyteEleocharis dulcis represented a very small proportion of what was held in the sediment andaccumulated by the plants’ roots.

CONCLUSION

This work focused on U-contaminated waters (running and standing) and the use ofnative plants for phytofiltration. The study area was composed of old uranium mines inCentral Portugal, and the investigation indicated the substantial scope of proper radiophy-toremediation and phytosociological investigation using the native flora.

The highest concentrations of U were found in the submerged species of Callitrichestagnalis (1948.41 mg/kg DW), in Fontinalis antipyretica (234.79 mg/kg DW) and in free-floating Lemna minor in standing waters (52.98 mg/kg DW). In contrast, the measuredconcentrations in the aboveground parts of emergent plants such as Apium nodiflorum andOenanthe crocata were significantly lower when compared with the previously mentionedspecies. It is possible that these species have developed mechanisms for inhibiting theabsorption of U, excluding this element from their tissues, or minimizing U translocationto their upper organs.

Among several factors that can affect the bioaccumulation (biosorption) of metals inaquatic ecosystems, pH is one of the most important. In the present study, water pH variedbetween 4.5 and 7.8; this range favors the occurrence of U composite hydrolyzed ionicspecies yield. That variation in pH and the effect on U speciation may have affected theresults. When there are differences in pH, U may be a chemical species that is less availablefor uptake and transport. In fact, Callitriche stagnalis is the only species that exhibited avery highly significant (P < 0.001) negative correlation with the water pH. In the case ofemergent macrophytes (Apium nodiflorum and Oenanthe crocata), there could have beenprecipitation of U in the roots that precluded uptake and transport. Therefore, more studiesare needed on the influence of this pH range on U speciation and its effect on U precipitationand/or uptake and transport by plants.

The ability of Callitriche stagnalis to concentrate U and its BCF of 3.78 × 103

(average) confirmed its high potential for the phytofiltration of U-contaminated waters.The abundance of Callitriche stagnalis, its biomass and relatively high bio-productivity andits ability to accumulate U and several other toxic elements (Robinson et al. 2006; Paulo2006) at the same time made this plant our first choice for the development of biopolishingmethodologies. Other species with high U uptake, such as Fontinalis antipyretica and Lemnaminor, can also be used in biological polishing applications either in monoculture systemsor in a combined system with Callitriche stagnalis representing the natural systems.

ACKNOWLEDGMENTS

This work was funded by a grant from the Portuguese Foundation of Science andTechnology (Project PTDC/CTE-GEX/66710/2006).

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Uranium accumulation by aquatic plants from uranium-contaminated water in Central Portugal

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