Chemosphere 79 (2010) 1077–1083

Contents lists available at ScienceDirect

Chemosphere

journal homepage: www.elsevier .com/locate /chemosphere

Chromium(VI) bioremediation by aquatic macrophyte Callitriche cophocarpa Sendtn.

Joanna Augustynowicz a,*, Marek Grosicki a, Ewa Hanus-Fajerska b, Małgorzata Lekka c,Andrzej Waloszek d, Henryk Kołoczek a

a Department of Biochemistry, Faculty of Horticulture, University of Agriculture in Kraków, Al. 29 Listopada 54, 31-425 Kraków, Polandb Department of Botany, Faculty of Horticulture, University of Agriculture in Kraków, Al. 29 Listopada 54, 31-425 Kraków, Polandc Institute of Nuclear Physics, Radzikowskiego 152, 31-342 Kraków, Polandd Department of Plant Physiology and Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7, 30-387 Kraków, Poland

a r t i c l e i n f o

Article history:Received 9 December 2009Received in revised form 10 March 2010Accepted 11 March 2010Available online 10 April 2010

Keywords:AFMBioremediationCallitriche cophocarpaChromiumHeavy metals

0045-6535/$ - see front matter � 2010 Elsevier Ltd. Adoi:10.1016/j.chemosphere.2010.03.019

* Corresponding author. Tel.: +48 12 662 51 95; faxE-mail addresses: augustyn@ogr.ur.krakow.pl (J. A

ur.krakow.pl (E. Hanus-Fajerska), malgorzata.lekka@gwaloszek@uj.edu.pl (A. Waloszek), koloczek@ogr.ur.k

a b s t r a c t

Callitriche cophocarpa (water-starwort) – aquatic widespread macrophyte – was found to be an excellentchromium accumulator. The plants were exposed to various chromium(VI) concentration ranging from50 to 700 lM in a hydroponic culture up to ca. 3 weeks. Physiological conditions of shoots were moni-tored via measuring potential photosynthesis quantum efficiency (Fv/Fm) and photosynthetic pigmentcontents. Additionally, the structure of leaves was analyzed using optical and atomic force microscopy(AFM). It has been shown that plants grown in 50 lM Cr(VI) solution exhibited photosynthetic activityand shoot and leaf morphology similar to control plants. Moreover, at the same time the average Cr con-centration in their shoots reached about 470 mg kg�1 d.w. after 10 d and up to 1000 mg kg�1 d.w. after3 weeks of culture while in control plants did not exceed a few mg kg�1 d.w. Our results point to Callitri-che cophocarpa as a very promising species to be used in the investigation of chromium(VI) phytoreme-diation mechanisms as well as a good candidate for wastewaters remediation purpose.

� 2010 Elsevier Ltd. All rights reserved.

1. Introduction

There are two main sources of chromium in the environment.The first one is natural like ferrochromite (Fe2Cr2O4) and otherminerals in the Earth’s crust. The second one is connected withindustrial application of chromium, mainly in metallurgy, tanningand textile pigment production. This heavy metal (from transitiongroup VI-B) could be detected in several oxidations states, amongthem trivalent and hexavalent are the most stable. These twoforms exhibit distinct chemical and physical properties, as wellas different influence on living organisms (see Kotas and Stasicka(2000) for review). Cr(VI), mainly originated from anthropogenicsources, is found in waters as HCrO�4 ;Cr2O2�

7 and CrO2�4 which are

highly mobile anions. The dominant forms of Cr(III) are mainly cat-ionic CrOH2+�aq, CrðOHÞþ2 � aq or neutral Cr(OH)3�aq hydroxides. Incontrast to Cr(VI) trivalent chromium is poorly mobile because ofits ability to precipitate and to form complexes with organic li-gands such as humic acids (Nakayama et al., 1981). In oxygenatedaqueous systems like rivers the Cr(VI)/Cr(III) ratio depends also onpH of solution. Cr(VI) is found to be dominant form at pH P 7while Cr(III) at pH 6 6 (Campanella, 1996). Cr(III) is an essential

ll rights reserved.

: +48 12 413 38 74.ugustynowicz), ehanus@ogr.

mail.com (M. Lekka), andrzej.rakow.pl (H. Kołoczek).

element in a human diet. It is found to be crucial in sugar metab-olism as a component of glucose tolerance factor (GTF) (Schwartzand Mertz, 1959). On the other hand, due to the high redox poten-tial and facility to penetrate cell membranes, hexavalent chro-mium compounds are found to be extremely toxic carcinogens(Kotas and Stasicka, 2000).

From the ecological and economical point of view biologicalmethods of heavy metal remediation are of great interest. One ofthese methods, phytoextraction, is a kind of phytoremediation tech-nique that uses plants to extract a pollutant from the environmentand concentrate it in a plant body (Mulligan et al., 2001; Singh etal., 2003). Some of the plant species exhibit extraordinary abilityto accumulate metals in their tissues. A plant that can accumulatechromium to at least 1000 mg kg�1 d.w. when growing in its naturalhabitat is called, by definition, chromium hyperaccumulator (Bakerand Brooks, 1989; Reeves, 2006). In accordance with the above def-inition there are only few examples of chromium hyperaccumula-tors. They are locally distributed, mainly in hot-temperatureregions: Dicoma niccolifera and Sutera fodina in Zimbabwe (Wild,1974), the moss Aerobryopsis longissima from New Caledonia (Leeet al., 1977) and lastly, wetland Leersia hexandra native to southernpart of China (Zhang et al., 2007). Phytoremediation of pollutedwaters is chiefly based on the rhizofiltration – the accumulation ofan element in the root system of aquatic plants. In the group ofwater vegetation species able to efficiently remove Cr contaminantsare Eichornia crassipes (water hyacinth), Polygonum hydropiperoides

1078 J. Augustynowicz et al. / Chemosphere 79 (2010) 1077–1083

(smartweed) and Nymphaea spontanea (water lily). Likewise in ter-restrial plants, the Cr level in shoots of aquatic plants in most casesis considerably lower than in roots (Qian et al., 1999; review Zayedand Terry, 2003; Choo et al., 2006). Due to Cr(VI) higher toxicity theaccumulation potential of hexavalent Cr is about 10 or even 100times lower than that of a trivalent one (Zhang et al., 2007).

In the work reported here we studied the Cr accumulationcapacity of aquatic macrophyte Callitriche cophocarpa. The genusCallitriche (water-starwort), geographically cosmopolitan but cen-tered in temperate zones of both hemispheres, is classified to themonogeneric family Callitrichaceae. This family consists of numer-ous species, but it differs in number according to the stand underconsideration (Heigi, 1965; Heb et al., 1967; Erbar and Leins,2004). Five slender, obligatory submerged Callitriche perennial spe-cies are known to occur in Poland, among others C. cophocarpaSendtn. (syn. C. polymorpha Lönnr). C. cophocarpa is a cold-resistantspecies characteristic of Hottonion association. It mainly grows inrivers, but can also be encountered in stagnant or slow movingwater (Zajac and Zajac, 2001; Mirek et al., 2002; Baczkiewicz etal., 2007; Buczkowska et al., 2008).

Detoxification of Cr by macrophytes is strictly related to the oxi-dation state of the metal. One of the remediation mechanisms isreduction of the strong oxidizing Cr(VI) to less harmful and ofteninsoluble Cr(III) (Zazo et al., 2008). Due to the application of X-ray Fluorescence Spectroscopy (XRF) Espinoza-Quiñones and co-workers (2009) proved that reduction of Cr(VI) to Cr(III) occursduring metal biosorption in roots of Eichhornia crassipess, Salviniaauriculata and Pistia stratiotes. The authors did not indicate accu-mulation of other Cr intermediates like Cr(V) or Cr(IV) in the inves-tigated tissues. Moreover, by application of Electron ParamagneticResonance Spectroscopy (EPR), Cr(VI) detoxification in shoots androots of C. cophocarpa following its reduction to Cr(III) was also re-cently detected by Augustynowicz et al. (2009).

The performed experiments were focused on the accumulationpotential of chromium present in the medium in the hexavalentform which is highly toxic and mobile wastewater pollutant (seeabove). It was investigated the metal accumulation in shoots. C.cophocarpa may be particularly advantageous for that purpose.Firstly, considering bioremediation efficiency plants extracting apollutant in shoots are of greater interest. In this case the pollutioncould be effectively removed with plant biomass without any lossin root-sediment (soil) system. However, little information is avail-able about chromium remediation by shoots. Secondly, it is knownthat submerged macrophytes can reveal higher metal uptake thanfloating or partially submerged aquatic plants due to elevated sur-face/biomass ratio (Rai et al., 1995). Finally, C. cophocarpa is wide-spread and evergreen so it is able to metabolise all through theyear. Biochemical aspects of Cr influence on plant metabolism likephotosynthetic activity as well as chlorophyll a, chlorophyll b andcarotenoid content were analyzed. Additionally, microscopy obser-vation gave the information about chromium-induced morpholog-ical changes of leaves. Especially atomic force microscopy made itpossible to determine very fine changes of leaf epidermis structurecaused by Cr.

2. Materials and methods

2.1. Plant material

The identification of species was kindly provided by Prof. K.Szoszkiewicz (Poznan University of Life Sciences, Poland; Baczkie-wicz et al., 2007). Mature plants were collected from the canal ofthe Szreniawa river, southern Poland: 50� 140 280 0 N/20� 040 360 0 E,in July, September, October, November 2008 and April 2009. Thenthe material was exhaustively washed with tap and distilled water.

2.2. Chromium toxicity tests

Only healthy shoots were transferred into the sterile nutrientsolution for aquatic plants (Appenroth et al., 1996) containing dif-ferent Cr(VI) concentration. The composition of nutrient solutionwas: 8 mM KNO3, 1 mM Ca(NO3)2, 1 mM MgSO4, 60 lM KH2PO4,25 lM Fe(III)EDTA, 13 lM MnCl2, 5 lM H3BO3, 0.4 lM Na2MoO4

(POCh, Poland). According to pH in natural environment, pH ofmedium was set at 6.6. Chromium toxicity tests were performedup to 24 d. Cr(VI) was applied in the form of K2Cr2O7. Dichromatewas dissolved in nutrient media to obtain final concentration ofCr(VI): 50, 100, 400 or 700 lM. One and a half grams of freshweight of shoots were incubated in 300 ml medium under the lightregime of 16 h, 35 lmol photons m�2 s�1 (LF 36 W/54, Piła, Po-land): 8 h darkness, at 25 �C. The light intensity, measured directlyon a leaf surface, was comparable to the one detected in the naturalenvironment. In order to improve the exchange of nutrients thecultures were slightly rotary-shaken. After 10 d, new growingshoots of control and 50 as well as 100 lM Cr(VI)-treated plants,were cut off and transferred into fresh chromium solutions of theabove concentrations and culture for the next 2 weeks in the abovedescribed conditions. In oxygenated aqueous solutions, Cr(III)form, according to thermodynamic calculations, is stable atpH 6 6.0, while at pH P 7.0 the Cr(VI) oxidation form predomi-nates (Kotas and Stasicka, 2000). On the basis of the available datawe assumed that Cr(VI) would be the dominant form in solutionunder the above conditions during the toxicity tests.

2.3. Measurements of chromium content

The measurements of Cr content were performed after 5, 10 or24 d of experiment independently. Apex shoot (2–4 cm length)samples were washed three times with distilled water then dried(105 �C, 24 h). Dried tissue or aquatic sediment samples were min-eralized by gentle heating in nitric acid or in the mixture of nitricand perchloric acids (Fluka, purity p.a. plus) (1:3). Next, the sam-ples were diluted either to 10 ml (plants) or 25 ml (sediments)with distilled water and filtrated. Samples of the river water were50-fold concentrated. The total level of chromium was determinedat 357.9 nm by atomic absorption spectroscopy (AAS; Varian,Spectr AA-20B; Mulgrave, Victoria, Australia). Different oxidationstates of chromium were not distinguished.

2.4. Atomic force microscopy analysis

The topography of a surface of apex shoot leaves was detectedusing atomic force microscopy (AFM; Park Systems, model XE-120) working in a contact mode. Directly before AFM experimentsthe samples were washed with a culture medium, placed on a cov-erslip into a ‘‘liquid cell” and immobilized with the use of two me-tal clamps. Commercially available non-sharpened, gold coatedcantilevers (MLCT-AUHW, Veeco) were used with the nominalspring constant values of 0.01 N m�1 and radius of curvature of50 nm. The deflection of the cantilever was measured using the la-ser–photodiode technique. For a given condition, the topographyand error mode images were recorded at the scan rate of 1.5 Hzand the set point of 0.6 nN. The scan size was 80 lm � 80 lm(256 � 256 pixels). The details of the AFM technique are describedin the work of Lekka (2007).

2.5. Optical microscopy sections

The material was fixed in 2% glutaraldehyde in 0.1 M cacodylatebuffer (pH = 7.2). After dehydration in a graded ethanol series, thematerial was immersed in acetone and then embedded in Epon812 resin. One micrometer thick sections were cut on Tesla 490A

J. Augustynowicz et al. / Chemosphere 79 (2010) 1077–1083 1079

rotary ultramicrotome and stained with 0.1% methylene blue. Thesections were examined and photographed using Nicon EclipseC400 microscope.

2.6. Potential photosynthesis quantum yield

The ratio of Fv/Fm, where Fv – maximal variable fluorescence andFm – maximal fluorescence, was measured using PAM 210 chloro-phyll fluorometer (Heinz Waltz GmbH, Germany) on the basis ofthe standard procedure of this apparatus. Measuring light wasnot stronger than 0.1 lmol photons m�2 s�1, and saturating lightwas 3000 lmol photons m�2 s�1 for 0.8 s. Samples of apex shootleaves were preadapted in darkness for at least 15 min. The guideto the chlorophyll fluorescence method is given by Maxwell andJohnson (2000).

2.7. Photosynthetic pigments

Chlorophyll a, b and carotenoids were isolated using a methoddescribed by Swiderski (1998). Apex shoot samples of about 0.1 gwere homogenized in acetone (POCh, Poland) with a small amountof CaCO3 in order to neutralize organic acid and filtrated underpressure. The absorbance was measured (Jasco V-530 UV–Vis spec-trophotometer) at 470, 645 and 662 nm. Finally, the pigment con-tent was calculated in accordance with equations presented byLichtenthaler and Wellburn (1983).

2.8. Statistics

Up to five independent experiments were conducted and eachone in two independent series and several replicates. The resultswere analyzed by one-way analysis of variance (ANOVA;P = 0.05), and the means were compared using Tukey’s test(P = 0.01).

3. Results

3.1. Study of chromium accumulation

The level of chromium accumulation in shoot tissues was corre-lated with its concentration in medium (Fig. 1). In the case of 50and 100 lM Cr(VI) concentrations, the accumulation was saturatedafter the first 5 d of culture reaching the average level of 470 and

Fig. 1. Chromium accumulation (mg kg�1) in shoots of C. cophocarpa after 5- or 10-d incubation in different Cr(VI) concentration. Each value is an average of 8measurements. Error bars represent standard deviations.

560 mg kg�1 d.w., respectively. At the higher Cr concentration inculture media, 400 and 700 lM, a significant rise in the metal con-tent in shoots was observed. Moreover, the accumulation did notreach a plateau during the experiment. In the case of 700 lM chro-mium concentration, the metal level in shoots achieved almost4000 mg kg�1 d.w after 10 d of incubation. The observed decay ofCr content in nutrient solution accompanying its accumulationby shoots was ca. 7 lM in media including 50 and 100 lM Cr (atthe start of experiment) or ca. 70 lM in the case of 400 lM Cr solu-tion after 10 d of culture. No decrease in Cr content was observedin medium of its highest concentration.

The plants exposed to 50 and 100 lM Cr(VI) concentrationexhibited good physiological conditions (see Section 3.2). So inthe next part of the experiment the apex shoots of the plants aswell as of control ones were cut off and moved to the fresh solutionof respective Cr concentration for the next 2 weeks. The Cr accu-mulation in the shoots reached the average level of 995mg kg�1 d.w. for 50 lM Cr-treated tissues and 1140 mg kg�1 d.w.in the case of plants cultured in 100 lM Cr concentration. At thesame time 2-fold biomass growth for control and 50 lM Cr-treatedplants was to be found. No increase in biomass growth was de-tected in the case of 100 lM Cr concentration.

The bioaccumulation coefficient, calculated as a content of Cr infresh weight of shoots versus Cr content in fresh weight of nutrient

Fig. 2. Photographs of Callitriche cophocarpa apex shoots under binocular micros-copy (Nikon SMZ 1500) after 5-d incubation in Cr(VI). A – control, B – 50 lM Cr-treated plants, C – 400 lM Cr-treated plants.

1080 J. Augustynowicz et al. / Chemosphere 79 (2010) 1077–1083

solution, was the highest in the case of 50 lM Cr-treated plants, inthe second exposure test, and its average value was ca. 27.0.

The content of chromium in plants derived from a natural aqua-tic system did not exceed 3 mg kg�1 d.w. Sediments as well aswater from natural Callitriche environmental sites did not exhibitan elevated Cr content either. Detected average levels of the metalwere 14 mg kg�1 in the sediment and below 10 lg L�1 (0.2 lM) inwater.

3.2. Influence of Cr on physiological conditions of plants

3.2.1. Macro and microscopy analysisAs far as vegetative morphology is concerned, shoots of experi-

mental control were well-shaped, with regular internodes. Leaveswere comparable to those taken from the control sample in flowingwater of the river. Shoots continued to grow in experimental con-ditions in vitro, even if their vitality slightly decreased. The growthlimitation of plants treated with 50 lM Cr(VI) was not observedmacroscopically (compare Fig. 2A and B). Symptoms of Cr toxicity:shortening of internodes, chlorosis, necrosis, changes in the leafshape and reduction of the leaf area, appeared while increasingCr(VI) concentration in nutrient solutions (Fig. 2C).

Atomic force microscopy made it possible to visualize, after 1 dof culture, the Cr-induced very early changes of the leaf surface(Fig. 3D, E, G and H). These symptoms were not detected at thattime via light microscopy. The surface topography of the controlleaves showed irregular shape of the structures (Fig. 3A). Theheight of the structures varied from 1.0 lm to almost 2 lm whiletheir width (defined as a width taken at half height) was in therange of 8–10 lm. The calculated difference between the thinnest

Fig. 3. Typical topography and cross sections of C. cophocarpa leaves. Leaf surface (A, D,Scan size 80 lm � 80 lm, black arrows indicate stomata. Cross sections (C, F, I) were preH, I – 400 lM Cr-treated plants.

and the thickest structure was around 2 lm (Fig. 3B). The image ofsurface topography of leaves treated with 50 lM Cr(VI) is slightlydifferent, showing more regular, parallel structures (Fig. 3D). De-spite that, the observed height and width of the structures werein the same range (1–2 lm) (Fig. 3E). The calculated difference be-tween the thinnest and the thickest structure was around 3 lm,and it was slightly larger than in the case of non-treated leaves.After plant treatment with high concentration of Cr (400 lM),the structures present on the surface became irregular with awidth in the range of 6–12 lm (Fig. 3G). The difference betweenthe thinnest and the thickest structure was almost 3-fold largerthan in the case of non-treated leaves (Fig. 3H). In the cross sec-tions of Cr-treated leaves it was possible to perceive distortion ofanatomical features not earlier than on the fifth day of culture(Fig. 3I). However, the leaf samples of 50 lM Cr-treated plants(Fig. 3F) were usually comparable to the experimental control(Fig. 3C), even if sometimes symptoms of epidermis cell degenera-tion could be visible. In that case the shape of cells was also lessregular. Some other degeneration symptoms like: smaller andcurved blades, changes in chloroplast shape and finally plasmolysisoccurred while increasing Cr concentration in medium (Fig. 3I). Inthe case of 400- and 700 lM-Cr(VI)-treated plants some precipi-tates on the surface of the cranky cell wall were visualized. Theymight have been chromium aggregates. However, their oxidationstate is unknown.

3.2.2. Potential photosynthesis quantum efficiency (as measured bychlorophyll fluorescence-Fv/Fm)

During 10 d of the experiment a fast, significant decrease in po-tential fluorescence yield (Fv/Fm) of C. cophocarpa leaves exposed to

G) and respective height (lm) (B, E, H) were visualized by atomic force microscopy.pared with optical microscopy. A, B, C – control, D, E, F – 50 lM Cr-treated plants, G,

Fig. 4. Chlorophyll fluorescence as Fv/Fm of leaves exposed to different Cr(VI)concentration in relation to the level detected in plants growing in naturalenvironment (100% means Fv/Fm = 0.80). Each value is an average of 25 measure-ments. Error bars represent standard deviations.

Fig. 5. Photosynthetic pigments in shoots of C. cophocarpa after 5 or 10 d of culturein different Cr(VI) concentration. Pigment content was shown in relation to that ofcontrol plants from natural environment (100%) before exposing to Cr. A –chlorophyll a, 100% = 61.65 mg/100 g f.w. B – chlorophyll b, 100% = 18.48 mg/100 g f.w. C – carotenoids, 100% = 14.55 mg/100 g f.w. The letters inside barsindicate significant statistical differences between plants after 10 d of culture(P = 0.01). Each value is an average of 12 measurements. Error bars representstandard deviations.

J. Augustynowicz et al. / Chemosphere 79 (2010) 1077–1083 1081

400 and 700 lM Cr(VI) was observed. On the eighth day of exper-iment almost no photosynthetic activity was detected in plantstreated with the highest concentration of Cr. Contrary to these re-sults, Fv/Fm measured in control as well as in leaves growing in50 lM Cr was nearly stable and comparable to the average valueof 0.8 (100%) of plants growing in natural environment. The resultsobtained for plants cultured in 100 lM Cr solution are the mostinteresting. In this case the measuring parameter decreased, fromthe second to the fourth day of experiment, to the level detectedfor leaves growing in 700 lM of Cr. Then, after 6 d of incubationFv/Fm returned to the values statistically indifferent (P = 0.01) withcontrol (Fig. 4).

3.2.3. Photosynthetic pigmentsThe content of photosynthetic pigments in shoots of C. copho-

carpa decreased while increasing concentration of Cr in culturemedium. Significant differences were measured in the chlorophylla, b and carotenoid content between plants growing in the highestand lowest metal concentration (Fig. 5). However, pigment con-tents in shoots exposed to 50 lM of Cr were not statistically differ-ent in comparison to control plants. Chlorophyll a (Fig. 5A) andcarotenoids (Fig. 5C) were more sensitive to chromium. Chloro-phyll b (Fig. 5B) was the most stable in all cases. Carotenoid decaypattern was similar to the one observed for chlorophyll a, whereaschlorophyll b content changed in a different manner. According tothe analysis demonstrated in Fig. 5 chl b/chl a ratio in Cr-treatedshoots was inversely proportional to the metal concentration.

4. Discussion

The problem of heavy metal contamination consists in the factthat the metal cannot be degraded like other organic xenobioticsbut it must be extracted from a polluted area. For that reason phyt-orextraction takes advantage of biochemical pathways of livingplant organisms and represents promising cost-efficient technol-ogy. Research on the chromium phytoremediation is scarce. Sofar only few species able to highly accumulate or hyperaccumulatethis metal have been characterized, among them E. crassipes as themost pronounced Cr phytoremediator in water environment(Zayed and Terry, 2003; Paiva et al., 2009). In this work we inves-tigated C. cophocarpa, widespread perennial aquatic species, toexamine its potential of chromium accumulation. The negative

influence of Cr on living organisms is more pronounced with themetal in the hexavalent oxidation state. Cr(VI) is highly reactive,mobile and easily soluble in water. Additionally, Cr(VI) dominatesin slightly alkaline solutions which are characteristic of natural Cal-litriche states (Kotas and Stasicka, 2000; Zayed and Terry, 2003).The literature data point to rhizofiltration as a main mechanismof Cr remediation by aquatic as well as terrestrial plant species(Zayed and Terry, 2003). However, contrary to floating E. crassipesplants with a dense and long rooting system C. cophocarpa is a sub-merged macrophyte possessing delicate unbranched roots. There-fore, our work focused on shoots that being surrounded bysolution may remove the contaminant more effectively.

We tested Cr(VI) toxicity thresholds and the physiologicalresponses of the species to elevated Cr(VI) concentration. The

1082 J. Augustynowicz et al. / Chemosphere 79 (2010) 1077–1083

physiological condition of plants depended on Cr(VI) concentrationin nutrient solution. The atomic force microscopy method made itpossible to visualize tissue surface and to calculate topographyparameters connected with the very early influence of the metalions on a cell structure. Incubation of shoots in high Cr(VI) concen-trations manifested in a deep irregularity of the epidermis in com-parison to control and 50 lM of Cr-treated tissue with a regularparallel-like organization of the surface structure. The AFM analy-sis data sets reflecting the morphology of the leaf surface could berecommended as an early-warning biotest to recognize chromiumpollution in aquatic environment. It should be underlined thatdamage of cells induced by high Cr concentrations was confirmedvia optical microscopy. In the highest 400 and 700 lM Cr(VI) solu-tions Callitriche shoots exhibited also typical morphological symp-toms of Cr toxicity like: reduction of leaf area and inhibition of leafand stem growth, chlorosis and necrosis which are also typical ofother species exposed to Cr (see Shanker et al. (2005) for review).Leersia hexandra, lastly discovered Cr hyperaccumulator, showedobvious Cr(VI) toxicity symptoms in the metal concentration of570 lM (Zhang et al., 2007). The lethal influence of 400 and700 lM of Cr(VI) on Callitriche shoots was also shown by measure-ments of potential quantum photosynthesis efficiency. The resultsindicated no active photosynthesis in shoots exhibiting the highestCr concentrations on the eighth day of treatment.

Our results showed that Fv/Fm parameter was sensitive to chro-mium denying some literature communication describing it as notvery sensitive to heavy metal action (see Lichtenthaler et al. (2004)for review). Appenroth and co-workers (2001) found significant Cr-caused changes in photosynthesis parameters measured aschanges in the JIP (chlorophyll a fluorescent transient) test. Com-parison of the results presented in their work for Spirodela polyrh-iza with our data points to higher tolerance of C. cophocarpa to Cr.Additionally, our data showed that in shoots of C. cophocarpa incu-bated in 100 lM of studied metal anions, a mechanism responsiblefor a return of photosynthetic quantum yield to values comparableto control was induced. This tolerance mechanism is probably con-nected with a direct protection of the photosynthetic apparatus.However, based on the presented results we cannot explain its de-tail nature that will be the subject of further study. A decrease in aphotosynthetic pigment content is well known from many otherworks concerning Cr stress in plants (see Chandra and Kulshresh-tha (2004) for review) and it is attributed to the oxidative stress.The pigment content was statistically lower in plants incubatedin two highest concentrations of Cr, where very strong decliningof photosynthetic activity was observed, and it was more pro-nounced in plants after 10 d of incubation. Taking into consider-ation chl b/chl a ratios in plants incubated in various Crconcentrations (Fig. 5), it seems that Cr caused strong (photo)oxi-dative damage in the photosynthetic apparatus of those plants.Similar changes in a photosynthetic pigment content were ob-served during photooxidation of cucumber chloroplast (Waloszekand Wieckowski, 1993) or during other oxidative stresses (Guo etal., 2006).

Though the shoot tissues incubated in the highest metal con-centrations were not able to actively metabolise they accumulateda manifest amount of chromium (Fig. 1). However, it was observedthat in the case of 700 lM Cr concentration no decrease of Cr con-tent in medium following its bioaccumulation was detected. It maybe explained by small dry weight of plants which is ca. only 7% offresh weight in comparison to the very high amount of Cr in solu-tion. Due to lack of active metabolism it seems that the mechanismof Cr accumulation by shoots incubated in that lethal Cr concentra-tion is passive. Nevertheless, the process of adsorption by non-liv-ing plant biomass could be widely used by low-cost cleaning-uptechnology for the removal of metal ions. Gupta and co-workers(2001) as well as Mohanty et al. (2006) demonstrated high-adsorb-

ing capacity of Cr by Spirogyra and Eichhornia dried tissues, respec-tively. However, they provided no speciation analysis of the Cr-adsorbed form.

One hundred micromole of Cr was the border concentration atwhich some symptoms of the metal toxicity were found. At100 lM of Cr there were observed inhibition of growth and de-crease in photosynthetic pigment content. On the other hand, itwas proved in each kind of the conducted tests that the physiolog-ical state of plants growing in 50 lM of Cr(VI) was not significantlydifferent from the one characteristic for control plants. It must bepointed out that the level of Cr in polluted rivers reaches ca.1 lM (50 lg L�1) (Kabata-Pendias and Pendias, 1999). The shootcells incubated in 50 lM chromium concentration were effectivelyphotosynthesising with no significant changes in a cell structureconfirmed by macroscopic analysis as well as AFM and opticalmicroscopy measurements. At the same time they were able toaccumulate almost 500 mg kg�1 d.w. after 10-d incubation in chro-mium. Moreover, after the prolonged time of culture (up to ca.3 weeks) two-fold increase in Cr accumulation in these shootsaccompanying two-fold increase in their biomass was found. Inthis case, the concentration of Cr in fresh shoots was up to 27 timesgreater than in medium. The observed increase of Cr accumulationcould be a result of exchange of culture medium including freshnutrients as well as induction of new protection mechanisms. Thisphenomenon will be a subject of further investigation. Higherresistance of living organisms to Cr(III) than to Cr(VI) is well docu-mented. Leersia hexandra in the Cr(III) concentration of 200 lMaccumulated almost 4900 mg kg�1 d.w. in leaves, while in thesame Cr(VI) concentration ca. 600 mg kg�1 d.w. A bioaccumulationcoefficient concerning leaves of L. hexandra exposed to 100 lM ofCr(VI) concentration was about 55 (Zhang et al., 2007). The resultsare very interesting since chromium is known for being poorlytranslocated inside plant tissues and mainly accumulated andsequestrated by a root system (Lytle et al., 1998; Ganesh et al.,2008). Qian and co-workers conducted laboratory tests with 12wetland species accumulating 10 different trace elements, amongthem chromium in the form of dichromate. The highest chromiumaccumulation was found in the roots of smartweed –2980 mg kg�1 d.w., while in the same conditions the highest con-centration of Cr in shoots was only 44 mg kg�1 d.w. and was at-tained by Cyperus alternifolius (umbrella plant) (Qian et al., 1999).

We demonstrated that C. cophocarpa shoots are able to effec-tively accumulate Cr, still the exact mechanism of the accumula-tion demands further study. However, some of the resultsconcerning capacity of C. cophocarpa for reducing Cr(VI) were justpresented (Augustynowicz et al., 2009). The accumulation of Crmight be performed via Cr(III) adsorption following Cr(VI) reduc-tion. The uptake and further translocation of Cr ions should notbe neglected, either. Cr(III) uptake is known to be a passive process.Due to cationic nature, Cr (III) ions can be easily bound to the an-ionic functional groups of a plant/cell surface. They can also be eas-ily chelated by root exudates and then precipitated inside oroutside the rhizosphere (Suñe et al., 2007; Mishra and Tripathi,2009). On the other hand, Cr(VI) ions are hard-bound to the plantsurface. In contrast to Cr(III) transport, uptake of Cr(VI) is metabol-ically driven and carried out by sulphate transporters (Kaszycki etal., 2005; Appenroth et al., 2008; Kyzioł-Komosinska and Kukułka,2008). Further detailed analysis is required to elucidate the natureof hexavalent Cr detoxification by C. cophocrapa shoots.

The obtained results showed an extraordinary capacity ofshoots of submerged macrophyte C. cophocarpa Sendtn. to accumu-late Cr from the Cr(VI) solutions in the laboratory tests. ExploitingC. cophocarpa in order to reduce excessive chromium concentrationfrom both floating and stagnant waters seems to be a beneficial ap-proach. The next step of the investigations should be a study of thisaquatic species in contaminated aquatic environment for further

J. Augustynowicz et al. / Chemosphere 79 (2010) 1077–1083 1083

tests of its application as a natural filter. The species could also pro-vide a new molecular resource for studying Cr(VI) detoxificationmechanisms.

Acknowledgements

The authors are grateful to Prof. K. Szoszkiewcz (Poznan Univer-sity of Life Sciences, Poland) for botanical identification of the spe-cies. We are also indebted to Ms. Dorota Wójcik, Mr. ZbigniewZawodny for technical assistance during AAS measurements andDr. Michał Pniak, Dr. Anna Kostecka-Gugała for help while takingand preparing the photographs. This work was financially sup-ported by the Grant NN 304 326136 (H. Kołoczek in charge of) fromthe Polish Ministry of Science and Higher Education.

References

Appenroth, K.-J., Teller, S., Horn, M., 1996. Photophysiology of turion formation andgermination in Spirodela polyrhiza. Biol. Plant. 38, 95–106.

Appenroth, K.-J., Stöckel, J., Srivastava, A., Strasser, R.J., 2001. Multiple effect ofchromate on the photosynthetic apparatus of Spirodela polyrhiza as probed byOJIP chlorophyll a fluorescence measurements. Environ. Pollut. 115, 49–64.

Appenroth, K.-J., Luther, A., Jetschke, G., Gabrys, H., 2008. Modification of chromatetoxicity by sulphate in duckweeds (Lemnaceae). Aquat. Toxicol. 89, 167–171.

Augustynowicz, J., Kostecka-Gugała, A., Kołoczek, H., 2009. Analysis of kinetics ofCr(VI) reduction by aquatic phytoremediators. Environ. Prot. Nat. Resour. 41,210–219 (in Polish).

Baczkiewicz, A., Szoszkiewicz, K., Cichocka, J., Celinski, K., Drapikowska, M.,Buczkowska, K., 2007. Isozyme patterns of Callitriche cophocarpa, C. stagnalisand C. platycarpa from 13 Polish rivers. Biol. Lett 44, 103–114.

Baker, A.J.M., Brooks, R.R., 1989. Terrestrial higher plants which hyperaccumulatemetallic elements – a review of their distribution, ecology and phytochemistry.Biorecovery 1, 81–126.

Buczkowska, K., Szoszkiewicz, K., Baczkiewicz, A., 2008. Genetic variation in Polishpopulations of Callitriche cophocarpa. Acta Biol. Cracov. Bot. 50, 63–70.

Campanella, L., 1996. Problems of speciation of elements in natural waters: the caseof chromium and selenium. In: Caroli, S. (Ed.), Element Speciation in BioorganicChemistry. Wiley Interscience, New York, pp. 419–444.

Chandra, P., Kulshreshtha, K., 2004. Chromium accumulation and toxicity in aquaticvascular plants. Bot. Rev. 70, 313–327.

Choo, T.P., Lee, C.K., Low, K.S., Hishamuddin, O., 2006. Accumulation of chromium(VI) form aqueous solutions using water lilies (Nymphaea spontanea).Chemosphere 62, 961–967.

Erbar, C., Leins, P., 2004. Callitrichaceae. In: Kadereit, J. (Ed.), The Families andGenera of Vascular Plants. VII. Flowering Plants, Dicotyledons, Lamiales (exceptAcanthaceae including Avicenniaceae). Springer, Berlin, pp. 50–56.

Espinoza-Quiñones, F.R., Martin, N., Stutz, G., Tirao, G., Palácio, S.M., Rizzutto, M.A.,Módenes, A.N., Silva Jr., F.G., Szymansky, N., Kroumov, A.D., 2009. Root uptakeand reduction of hexavalent chromium by aquatic macrophytes as assessed byhigh-resolution X-ray emission. Water Res. 43, 4159–4166.

Ganesh, K.S., Baskaran, L., Rajasekaran, S., Sumathi, K., Chidambaram, A.L.A.,Sundaramoorthy, P., 2008. Chromium stress induced alterations inbiochemical and enzyme metabolism in aquatic and terrestrial plants.Colloids Surf. B 63, 159–163.

Guo, Y.-P., Zhou, H.-F., Zhang, L.-C., 2006. Photosynthetic characteristics andprotective mechanisms against photooxidation during high temperaturestress in two citrus species. Sci. Hortic. 108, 260–267.

Gupta, V.K., Shrivastava, A.K., Jain, N., 2001. Biosorption of chromium(VI) fromaqueous solutions by green algae Spirogyra species. Water Res. 35, 4079–4085.

Heigi, G., 1965. Illustriete Flora von Mittel-Europa Band V/1 Teil Dicotyledones,Linaceae–Violacea. Carl Hansen Verlag, München. pp. 190–202.

Heb, H.E., Landolt, E., Hirzel, R., 1967. Flora der Schweeitz und angrenzenderGebiete, Band 2: Nymphaeaceae bis Primulaceae. Birkhuäuser Verlag, Basel undStuttgart. pp. 670–674.

Kabata-Pendias, A., Pendias, H., 1999. Biogeochemistry of trace elements. PWN,Warszawa, p. 286 (in Polish).

Kaszycki, P., Gabrys, H., Appenroth, K.-J., Jaglarz, A., Sedziwy, S., Walczak, T.,Koloczek, H., 2005. Exogenously applied sulphate as a tool to investigatetransport and reduction of chromate in the duckweed Spirodela polyrhiza. PlantCell Environ. 28, 260–268.

Kotas, J., Stasicka, Z., 2000. Chromium occurrence in the environment and methodsof its speciation. Environ. Pollut. 107, 263–283.

Kyzioł-Komosinska, J., Kukułka, L., 2008. Application of minerals co-occurring inbrown coal deposits to removal of heavy metals from water and wastewater.Works and Studies 75, Polish Academy of Sciences, Zabrze (in Polish).

Lee, J., Brooks, R.R., Reeves, R.D., Jaffré, T., 1977. Plant–soil relationships in NewCaledonian serpentine flora. Plant Soil 46, 675–680.

Lekka, M., 2007. The Use of Atomic Force Microscopy as a Technique for theIdentification of Cancerous Cells. Habilitation Thesis, The HenrykNiewodniczanski Institute of Nuclear Physics Polish Academy of Sciences.<http://www.ifj.edu.pl/publ/reports/2007/2001.pdf?lang=pl>.

Lichtenthaler, H.K., Wellburn, A.R., 1983. Determination of total carotenoids andchlorophyll a and b of leaf extracts in different solvents. Biochem. Soc. Trans.603, 591–592.

Lichtenthaler, H., Buschmann, C., Knapp, M., 2004. Measurement of chlorophyllfluorescence kinetics (Kautsky effect) and the chlorophyll fluorescence decreaseratio (RFD-values) with the Pam-Fluorometer. In: Filek, M., Biesaga-Koscielniak,J., Marcinska, I. (Eds.), Analytical Methods in Plant Stress Biology. PolishAcademy of Sciences, Kraków.

Lytle, C.M., Lytle, F.W., Yang, N., Qian, J.-H., Hansen, D., Zayed, A., Terry, N., 1998.Reduction of Cr(VI) to Cr(III) by wetland plants: potential for in situ heavy metaldetoxification. Environ. Sci. Technol. 32, 3087–3093.

Maxwell, K., Johnson, G.N., 2000. Chlorophyll fluorescence – a practical guide. J. Exp.Bot. 51, 659–668.

Mirek, Z., Piekos-Mirkowa, H., Zajac, A., Zajac, M., 2002. Flowering Plants andPteridophytes of Poland. A Checklist. Szafer Institute of Botany. Polish Academyof Sciences, Kraków.

Mishra, V.K., Tripathi, B.D., 2009. Accumulation of chromium and zinc from aqueoussolutions using water hyacinth (Eichhornia crassipes). J. Hazard. Mater. 164,1059–1063.

Mohanty, K., Jha, M., Meikap, B.C., Biswas, M.N., 2006. Biosorption of Cr(VI) fromaqueous solutions by Eichhornia crassipes. Chem. Eng. J. 117, 71–77.

Mulligan, C.N., Yong, R.N., Gibbs, B.F., 2001. Remediation technologies for metal-contaminated soils and groundwater: an evaluation. Eng. Geol. 60, 193–207.

Nakayama, E., Kuwamoto, T., Tsurubo, S., Tokoro, H., Fujinaga, T., 1981. Effect ofnaturally occurring organic materials on the complex formation ofchromium(III). Anal. Chim. Acta 130, 289–294.

Paiva, L., de Oliveira, J., Azevedo, R., Ribeiro, D., da Silva, M., Vitória, A., 2009.Ecophysiological responses of water hyacinth exposed to Cr3+ and Cr6+. Environ.Exp. Bot. 65, 403–409.

Qian, J.-H., Zayed, A., Zhu, Y.-L., Yu, M., Terry, N., 1999. Phytoaccumulation of traceelements by wetland plants: III. Uptake and accumulation of ten trace elementsby twelve plant species. J. Environ. Qual. 28, 1448–1455.

Rai, U.N., Sinha, S., Tripathi, R.D., Chandra, P., 1995. Wastewater treatabilitypotential of some aquatic macrophytes: removal of heavy metals. Ecol. Eng. 5,5–12.

Reeves, R.D., 2006. Hyperaccumulation of trace elements by plants. In: Morel, J.-L.,Echevarria, G., Goncharova, N. (Eds.), Phytoremediation of Metal-ContaminatedSoils. Springer-Verlag, pp. 25–52.

Schwartz, K., Mertz, W., 1959. Chromium(III) and the glucose tolerant factor. Arch.Biochem. Biophys. 85, 292–295.

Shanker, A.K., Cervantes, C., Loza-Tavera, H., Avudainayagam, S., 2005. Chromiumtoxicity in plants. Environ. Int. 31, 739–753.

Singh, O.V., Labana, S., Pandey, G., Budhiraja, R., Jain, R.K., 2003. Phytoremediation:an overview of metallic ion decontamination from soil. Appl. Microbiol.Biotechnol. 61, 405–412.

Suñe, N.L., Sánchez, G., Caffaratti, S., Maine, M.A., 2007. Cadmium and chromiumremoval kinetics from solution by two aquatic macrophytes. Environ. Pollut.145, 467–473.

Swiderski, A., 1998. Analysis of pigments in plant material with traditional andmodified methods. PhD Thesis, University of Agriculture in Krakow (in Polish).

Waloszek, A., Wieckowski, S., 1993. Chloroplast pigment photobleaching and itseffect on low temperature fluorescence spectra of chlorophyll in greeningcucumber cotyledons. Photosynthetica 29, 509–520.

Wild, H., 1974. Indigenous plants and chromium in Rhodesia. Kiekia 9, 233–241.Zajac, A., Zajac, M., 2001. Distribution of Vascular Plants in Poland. Laboratory of

Computer Chronology, Institute of Botany. Jagiellonian University Press,Kraków. pp. 105–106.

Zayed, A.M., Terry, N., 2003. Chromium in the environment: factors affectingbiological remediation. Plant Soil 249, 139–156.

Zazo, J.A., Paull, J.S., Jaffe, P.R., 2008. Influence of plants on the reduction ofhexavalent chromium in wetland sediments. Environ. Pollut. 156, 29–35.

Zhang, X.-H., Liu, J., Huang, H.-T., Chen, J., Zhu, Y.-N., Wang, D.-Q., 2007. Chromiumaccumulation by the hyperaccumulator plant Leersia hexandra Swartz.Chemosphere 67, 1138–1143.