Phytoremediation of Water Polluted by Thallium ...

Phytoremediation of Water Polluted by Thallium, Cadmium, Zinc, and Lead with the Use of Macrophyte Callitriche cophocarpa Joanna Augustynowicz, Krzysztof Tokarz, Agnieszka Baran & Bartosz J. Płachno Archives of Environmental Contamination and Toxicology ISSN 0090-4341 Arch Environ Contam Toxicol DOI 10.1007/s00244-013-9995-0

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Author's personal copy Arch Environ Contam Toxicol DOI 10.1007/s00244-013-9995-0

Phytoremediation of Water Polluted by Thallium, Cadmium, Zinc, and Lead with the Use of Macrophyte Callitriche cophocarpa Joanna Augustynowicz • Krzysztof Tokarz Agnieszka Baran • Bartosz J. Płachno

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Received: 4 November 2013 / Accepted: 30 December 2013 Springer Science+Business Media New York 2014

Abstract The objective of the present work was to study the phytoremediation capacity of Callitriche cophocarpa concerning water contaminated with thallium (Tl), cadmium (Cd), zinc (Zn), and lead (Pb) derived from the natural environment. We found that after a 10-day incubation period, shoots of C. cophocarpa effectively biofiltrated the water so that it met (for Cd, Zn, and Pb) appropriate quality standards. The order of accumulation of the investigated elements by shoots (mg kg-1 dry weight) were as follows: Zn (1120) \ Tl (251) \ Cd (71) \ Pb (35). The order of bioconcentration factors were as follows: Cd (1177) \ Tl (1043) \ Zn (718) \ Pb (597). According to Microtox bioassay, C. cophocarpa significantly eradicated polluted water toxicity. During the experiment, the physiological status of plants was monitored by taking measurements of photosystem II activity (maximum efficiency of PSII, photochemical fluorescence quenching,

J. Augustynowicz (&) K. Tokarz Unit of Botany and Plant Physiology, Institute of Plant Biology and Biotechnology, Faculty of Horticulture, University of Agriculture in Krako´w, Al. 29 Listopada 54, 31-425 Krako´w, Poland e-mail: [email protected] K. Tokarz e-mail: [email protected] A. Baran Department of Agricultural and Environmental Chemistry, Faculty of Agriculture and Economics, University of Agriculture in Krako´w, Al. Mickiewicza 21, 31-120 Krako´w, Poland e-mail: [email protected] B. J. Płachno Department of Plant Cytology and Embryology, Jagiellonian University, Gronostajowa 9 St., 30-387 Krako´w, Poland e-mail: [email protected]

nonphotochemical fluorescence quenching, and quantum efficiency of PSII), photosynthetic pigment contents, and shoot morphology. Plants exposed to metallic pollution did not exhibit significant changes in their physiological status compared with the control. This work is potentially applicable to the future use of C. cophocarpa in the phytoremediation of polluted, natural watercourses.

The emission of heavy-metal compounds into water may originate from several sources, e.g., surface runoffs from soils where municipal sewage sludge was used or from landfill sites containing poorly protected industrial wastes. Several areas in Poland are at risk of severe heavy-metal contamination with the worst affected being located in the Upper Silesia region, Southern Poland. The dominant part of heavy-metal loads, when transported to the aquatic system is accumulated in sediments, which can easily be released when changing redox conditions or pH. Sources of monometallic contaminations are rare. Most pollution, however, is emitted as polymetallic similar to the ones found in the mining and smelting industries. The most mobile and well-described metal ions in water are as follows: Cd(II), Zn(II), Cr(III)/Cr(VI), and Ni(II) (KyziołKomosin´ska and Kukułka 2008). Although Cr(III) and Zn(II) are essential microelements found in low concentrations, their levels in an aquatic system are often significantly increased. Cd(II), Cr(VI), and also Pb(II), which is far less mobile, are widely known to be toxic to biota. In relation to the above-mentioned elements, relatively little information is available concerning thallium (Tl). Tl compounds are extremely dangerous, soluble environmental metallic pollutants. Tl an element from group 13 (former 3A) of the periodic table, is mainly recovered during Zn production. Zn ores usually contain high

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Author's personal copy Arch Environ Contam Toxicol

amounts of Tl, which is released into residues and dust by Zn industries. It is also produced as a byproduct in coal mining, lead smelting, and cement factories. In the environment, thallium exists in two, highly toxic oxidation states: more stable Tl(I) (e.g., TlCO3) and Tl(III) (e.g., Tl(OH)2þ ; Tl(OH)þ 2 , or Tl(OH)3) (Babula et al. 2008; Kabata-Pendias and Mukherjee 2007). Tl properties in the third oxidation state are similar to aluminum, whereas in the first oxidation state they bear a strong resemblance to alkali metals. Tl toxicity can be explained on the basis of its affinity to amino-, imino-, or sulfhydryl groups in active enzymatic centers. The most pronounced influence of Tl is related to its easy replacement with potassium (K), which causes disruption in K-controlled activity of enzymes and membrane processes, e.g., the mitochondrial respiratory chain (Leónard and Gerber 1997). The average concentration of Tl in river waters is *10 ng l-1. The concentration of Cd, Zn, and Pb is usually one or two orders of magnitude greater (Kabata-Pendias and Mukherjee 2007). In the present work, we investigated the phytoremediating potential of macrophyte (aquatic higher plant) Callitriche cophocarpa regarding water polluted by polymetallic contaminations (thallium [Tl], cadmium [Cd], zinc [Zn], and lead [Pb]). With global contamination on the increase, macrophytes provide an economically reasonable and ecologically sound approach to heavy metal/metalloid remediation in water systems (Malec et al. 2011). Callitriche cophocarpa belongs to the Callitrichaceae family and grows in stagnant or slow-moving water in the temperature zones of both hemispheres. Moreover, C. cophocarpa is one of the most common Callitriche species in Europe (Schotsman 1972). It must be also pointed out that C. cophocarpa is a submersed species. Submersed macrophytes are of greater interest than floating or emergent ones with respect to the phytoremediation of polluted waters because they are able to accumulate pollutants in their shoots. Thus, due to the high contact-area with surrounding water, metal-uptake occurs more efficiently (Chandra and Kulshreshtha 2004). The extraordinary phytoremediation capacity of C. cophocarpa toward chromium Cr(VI)/Cr(III) compounds was confirmed in our earlier works (Augustynowicz et al. 2010, 2013). Shoots of C. cophocarpa were incubated in the water derived from the Graniczna Woda stream located in the area of the industrial, highly contaminated mining region in Upper Silesia, Southern Poland. Rivers in this area are polluted by heavy-metal compounds emanating from chemical plants in Tarnowskie Go´ry and by zinc smelter plants in Miasteczko S´la˛skie (Reczyn´ska-Dutka 1986). The reason for our particular interest in this watercourse was the observation of the absence of any higher aquatic plants in the riverbed (Fig. 1a). In the area located in the

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neighborhood of the stream, the moss layer was quite well developed, but the level of vascular plant biodiversity was extremely low with the dominating species being Cardaminopsis halleri (Fig. 1b), which is a well-known facultative metallophyte (Zarzycki et al. 2004). The experimental flow of our study first covered chemical analysis of the water, measurements of elementaccumulation levels, and calculations of bioconcentration factors. The next step was to elaborate on a environmental toxicity test (Microtox) regarding Callitriche influence on water purification. This test relies on the use of luminescent marine bacteria Vibrio fischeri, which is a well-known tool in the environmental monitoring of polluted aquatic reservoirs (Kagalou et al. 2008). Vibrio bacteria use a major part of their metabolic energy for luminescence. Light emission results from the interaction of the enzyme luciferase, reduced flavin, and a long-chain aldehyde in the presence of oxygen and constitutes a part of the cell’s electron transport system (Ribo and Rogers 1990). Thus, any changes in the metabolism of V. fischeri under the influence of a toxic substance causes a decrease in the intensity of luminescence (Ribo and Kaiser 1987). Finally, we determined the physiological status of the plants after exposure to the contaminated water. Any type of abiotic stress factor that exceeded the optimum threshold implies that the rate of photosynthesis decreases. A decrease in photosynthetic carbon-fixation occurring under nutrient depletion and/or salt stresses may cause a photoinhibition of the photosystem II (PSII) reaction center. Under such circumstances, the over-reduction of electron transport chain leads to the production of reactive oxygen species (ROS). Hydroxyl radicals, superoxide anion, or hydrogen peroxide, which might be generated, affect the PSII reaction center and cause serious damage to the photosynthetic apparatus (Wilhelm and Selmar 2011). Therefore, measurements of PSII activity are good tools to assess the physiological status of plants. The above-stated goal was achieved by analysis of the following chlorophyll a fluorescence parameters: maximum efficiency of PSII (Fv/Fm), photochemical fluorescence quenching (qP), nonphotochemical fluorescence quenching (NPQ), and quantum efficiency of PSII (UPSII). In addition, photosynthetic pigment contents and plant morphology were evaluated.

Materials and Methods Plant Material and Incubation Tests Plants (Fig. 1d) were collected from the nonpolluted Dłubnia River in Southern Poland [50160 N/19560 E (Fig. 1c)] during spring and summer 2012. Then the


Fig. 1 Environmental conditions of the C. cophocarpa habitats and the source of contaminated water for experiments. a Highly polluted Graniczna Woda stream (Upper Silesia, Poland [50300 N/18490 E]). Note the absence of higher aquatic plants in the riverbed and the low number of marsh plants on the banks. Black and white arrows indicate the facultative metallophyte Cardaminopsis halleri growing in the

neighborhood of the Graniczna Woda stream. b Flowering C. halleri (indicated by white arrow on [b]). c Natural habitat of C. cophocarpa in the nonpolluted Dłubnia river (Małoposka, Poland [50160 N/ 19560 E]). Note the rich plant community of marsh plants on the river banks. d Collection of C. cophocarpa taken from natural stands (Dłubnia River) for the experiments

material was transported to the laboratory and exhaustively washed with tap and distilled water. Ten centimeter-length shoots were used for the experiments. Seven grams (14 g) of plant material was immersed in 300 ml (600 ml) of solution and incubated for 10 days in the phytotron under 16 h of light intensity at 80 lmol m-2 s-1 (LF 36W/54, Piła, Poland) and 8 h of darkness at 24 C. Two types of filtrated (pore size 60 lm; Millipore) media were used: the control water from the natural Callitriche stands and the polluted water derived from the Graniczna Woda stream situated in the area of the former reserve at De˛by Boruszowickie near Tarnowskie Go´ry, Upper Silesia

[50300 N/18490 E (Fig. 1a)]. The protocol for the water composition analysis is given later in the text. The average amount (mg l-1) of elements and ions, as well as other physicochemical parameters of the waters, are listed in Table 1. Chemical Analysis of Plant and Water Samples Before analysis of element contents, the plant material was thoroughly washed three times with distilled water and then dried for 24 h at 105 C. Digestion was performed in a mixture of H2O2 and HNO3 (6:1; v/v) (suprapure; Merck)

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Author's personal copy Arch Environ Contam Toxicol Table 1 Chemical composition as well as pH, Eh, and conductivity of polluted and control water used for experiments Element composition

Control water

Polluted water

9297 1994; PN-EN ISO 17294-1 2007), were applied to measure the chemical composition of the samples. The spectrometer was calibrated using the ICP multielement standard (Merck).

Cations Na

4.24

47.78

K

1.75

36.07

Mg

5.01

8.71

Ca

69.65

123.10

Sr

0.13

0.24

Ba Ti

0.03 \0.005

0.09 0.005

Zr

0.002

0.002

V

0.001

\0.005

Cr

\0.001

Mo

\0.0002

0.001

0.005

W

\0.002

\0.002 0.26

Mn

\0.002

Fe

0.02

0.86

Co

\0.0001

\0.001

Ni

\0.0001

0.002

Cu

\0.0005

0.002

Ag

\0.00002

\0.00002

Zn

\0.002

1.06

Cd

\0.0005

0.06

Hg Al

\0.0002 \0.005

\0.0002 0.13

Toxicity Test: Microtox Bioassay Toxicity of water samples was determined according to measurement of the luminescence intensity of V. fischeri bacteria. Samples were analyzed using Microtox M500 equipment based on Microtox Omni software (Strategic Diagnostics Inc., USA) according to the typical procedures (Microbics Corporation 1992). The system of risk assessment developed by Persoone et al. (2003) was used to estimate water toxicity according to the standard test procedure for water samples (81.9 % screening test). Inhibition of luminescence at 490 nm was measured before and after incubation of bacterial suspension (Mirotox Acute Toxicity Reagent; SDI, USA) with the analyzed sample, and then the toxicity effect I (%) was calculated according to Eq. 1: Ls I ¼ 1 100½%; ð1Þ Lc

Tl

\0.0001

0.24

Pb

\0.0005

0.05

where I is toxicity (%); Ls is the intensity of luminescence of the studied sample; and Lc is the intensity of luminescence of the reference sample (1,000 ll 2 % NaCl with 150 ll bacterial suspension). The results were classified as follows:

0.007

•

As

0.004 \0.01

\0.01

Cl

16.50

189.67

Br

\0.02

1.76

10.20

151.03

189.00

78.17

0.15

0.45

Se

•

Anions

SO4 HCO3 PO4 BO3

0.08

1.29

NO3

13.50

7.15

NO2

0.09

0.26

Other parameters pH Eh (mV) Conductivity (mS cm-1)

7.8 180

7.0 258

0.335

1.041 -1

Average value of element concentration in [mg l ]; n = 9 Concentration of four elements: Zn, Cd, Tl and Pb (typed in bold), surpassed the upper limits for surface waters

in the closed system of a microwave oven (Multiwave 3000; AntonPaar). Inductively coupled-plasma mass spectrometry (ICP-MS) (ELAN 6100; Perkin Elmer) (PN-EN ISO 9963-1 2001), as well as titration methods (PN-ISO

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• •

Class I: I \ 20 % (no significantly toxic effect, no acute hazard) Class II: 20 % B I \ 50 % (significantly toxic effect, low acute hazard) Class III: 50 % B I \ 100 % (significantly toxic effect, acute hazard) Class IV: I = 100 % (high acute hazard)

Determination of the Physiological Status of Plants Chlorophyll Fluorescence Measurements The impact of metallic contamination on photosynthetic apparatus was evaluated by analysis of chlorophyll a fluorescence. The induction kinetics as well as chlorophyllfluorescence parameters were measured using a chlorophyll fluorescence-monitoring system (FluorCam; Photon Systems Instruments, Czech Republic). The basic chlorophyll fluorescence yield (Fo) was recorded after dark adaptation for at least 20 min. The maximal chlorophyll fluorescence yield (Fm) was also measured by applying a 0.8-s saturating light pulse (2,000 lmol m-2 s-1). Variable fluorescence (Fv) was calculated as Fm - Fo, and Fv/Fm was


calculated as (Fm - Fo)/Fm. Maximum fluorescence yield during illumination (Fm0 ) was obtained with a saturating pulse during actinic light, whereas the level of minimum fluorescence yield during illumination (Fo0 ) was measured after turning off the actinic light. Ft represents the level of fluorescence during illumination. The photochemical fluorescence quenching (qP) was evaluated as qP = (Fm0 -Ft)/ (Fm0 - Fo), whereas nonphotochemical fluorescence quenching (NPQ) was evaluated as NPQ = (Fm - Fm0 )/ Fm0 , and the effective photochemical quantum yield of PSII (UPSII) was calculated as UPSII = (Fm0 - Ft)/Fm0 . Photosynthetic Pigment Contents Chlorophyll a, b, and carotenoids were isolated using a method described by S´widerski (1998). Shoots, 0.1 g, were homogenized in acetone (POCh, Poland) with a small amount of CaCO3 to neutralize the organic acid and then centrifuged for 15 min at 5,400 g at 4 C (Rotina 380-R; Hettich Zentrifugen, Germany). Absorbance was measured (UV–Vis spectrophotometer, HITACHI U-2900) at 470, 645, and 662 nm. Finally, the pigment contents were calculated in accordance with the equations presented by Lichtenthaler and Wellburn (1983). Microscopy Analysis Morphology of specimens was examined under a Olympus BX60 microscope equipped with a differential interference contrast and under a stereo microscope Zeiss Discover V12 using AxioVison 3.0 software. Statistics Three independent sets of experiments were performed with each set comprising a few replicates. The results were analyzed using one-way analysis of variance (ANOVA)/ Student t test to compare differences between objects based on Statistica 10 software. After rejection of the null hypothesis, least significant difference (LSD)-Fisher or Tukey’s tests were performed to determine the statistical significance of the results (a = 0.05).

Results Accumulation Test Analysis of water indicated the existence of pollution in the investigated stream (Table 1). According to the standards for surface waters set by both the Polish Ministry of the Environment(1) (Rozporza˛dzenie Ministra S´rodowiska z dn. 9 listopada 2011 r.) and the United States

Fig. 2 Accumulation levels (mg kg-1 dw) and BCFs of investigated elements in plants after 10-day incubation in contaminated water. Different letters indicate the statistically significant differences of the values of accumulation and BCFs separately according to ANOVA (a = 0.05; accumulation p \ 0.000; BCF p \ 0.000) and LSD-Fisher’s tests (a = 0.05; accumulation: Cd vs. Pb p \ 0.070, Cd vs. Tl p \ 0.000, Cd vs. Zn p \ 0.000, Pb vs. Tl p \ 0.000, Pb vs. Zn p \ 0.000, Tl vs. Zn p \ 0.000; BCF Cd vs. Pb p \ 0.000, Cd vs. Tl p \ 0.141, Cd vs. Zn p \ 0.000, Pb vs. Tl p \ 0.000, Pb vs. Zn p \ 0.181, and Tl vs. Zn p \ 0.001). Error bars represent SDs; n = 6

Environmental Protection Agency (2013)(2) (USEPA [Water Quality Standards]), concentrations of Zn, Cd, Tl, and Pb surpassed the upper limits (indicated in parentheses in mg l-1) in the following instances: Zn (B1.0000 (1)/ 0.1100 (2)), *10 times the limit(2); Cd (0.0015 (1)/0.0037 (2)), 40 (1) times the limit; Tl (B0.002 (1), (2), 120 (1), (2) times the limit; and Pb (B0.0072 (1)/0.0650 (2)), *7 (1) times the limit. Tl content significantly exceeded the water-quality standards. Therefore, in a subsequent analysis, we focused on these four toxic elements. A 10-day incubation of plants caused a significant decrease in the concentrations of investigated elements in the solution. The levels of the particular elements (mg l-1) in the contaminated water at the end of the experiment were as follows: Zn 0.0747 (±0.0030), Cd 0.0013 (±0.0009), Tl 0.0649 (±0.0000), and Pb 0.0007 ± 0.0000); n = 6. After a 10-day incubation period, the water reached the quality standards (compare previous text) except for Tl. The order of the decrease in the studied metallic elements in the solution was calculated as the difference between the element content at the beginning of the experiment (see Table 1) minus the element content at the end of the experiment. We measured the most significant decrease with Zn. The levels of the remaining elements decreased in the following order: Zn \ Tl \ Cd \ Pb. The amount of each particular element that disappeared from the solution was equivalent to the amount of this element accumulated by Callitriche shoots (Fig. 2). Callitriche cophocarpa exhibited significant accumulation levels of Zn and Tl, which reached average values of 1,120 and 251 mg kg-1 dry weight (dw), respectively. The Cd mean

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Author's personal copy Arch Environ Contam Toxicol Table 2 Toxicity of water before and after 10-day incubation of C. cophocarpa shoots measured according to luminescence intensity of V. fischeri (Microtox bioassay) against NaCl solution as a reference sample Group name

Specimen

Average toxicity (%)

Minimum toxicity (%)

X1

Control water start

-2.20a

-12.00

6.00

c

18.00

27.00

X2

Polluted water start

X3

Control water stop

X4

Polluted water stop

22.40

2.25ab 10.00b

Maximum toxicity (%)

0.00

5.00

6.00

14.00

Different letters indicate significant statistical differences between specimen according to ANOVA (a = 0.05; p \ 0.000) and Tukey’s tests (a = 0.05; X1 vs. X2 p \ 0.000, X1 vs. X3 p \ 0.530, X1 vs. X4 p \ 0.002, X2 vs. X3 p \ 0.000, X2 vs. X4 p \ 0.000, and X3 vs. X4 p \ 0.087); n [ \4; 6[

level was equal to 71 mg kg-1 dw, whereas the level of Pb reached 35 mg kg-1 dw. However, a bioconcentration factor (BCF), which is defined as the element concentration in shoots (mg g-1 dw) divided by its concentration in a solution (mg g-1), in plants exposed to contamination was highest for Cd (1177) and Tl (1043) (Cd and Tl had the same statistical significance) and then for Zn (718) and Pb (597) (Zn and Pb had the same statistical significance). Concentrations of Zn, Tl, and Cd in control samples were in the range between *1 and 5 mg kg-1 dw. However, we measured significant Zn content in the control plants, which reached an average value of 360 (±113) mg kg-1 dw. Microtox Bioassay

Fig. 3 Effects of heavy-metal pollution in water on the chlorophyll fluorescence parameters (mean ± SE) of Callitriche: Fv/Fm, qP, (NPQ, and UPSII. Student t test (a = 0.05) showed no statistically significant differences in pairs for Fv/Fm (p \ 0.260), qP (p \ 0.700), and UPSII (p \ 0.180) and a significant difference for NPQ (p \ 0.010), which is marked with a star. Error bars represent SDs; n=6

Table 3 Influence of contamination on chlorophyll fluorescence parameters (mean ± SE) of C. cophocarpa, Fo and Fv, after 10-day incubation Parameter

Control water

Polluted water

Significance

Fo Fv

114.7 ± 19.0 468.8 ± 82.0

156.5 ± 36.0 471.1 ± 85.4

** ns

** Represent significant differences in pairs (Student t test; a = 0.05; Fop \ 0.004, Fvp \ 0.900); n = 6

Callitriche cophocarpa showed the considerable influence on decreasing toxicity of contaminated water. At the start of the experiment, the mean toxic effect of the contaminated samples, measured as the means of luminescence intensity of V. fischeri, was 22.0 %, which represents the second class of acute toxicity. At the end of the experiment, the average toxic effect was equal to 10.0 % and was lower by 54.5 % in relation to the sample toxicity at the start of the incubation (Table 2). As a result of a 10-day incubation of C. cophocarpa, the water samples were classified as corresponding to class I, namely, with no toxicity. Control samples exhibited no toxic effects both at the start and at the end of the investigation. The control samples at the end of the experiment inhibited the luminescence of V. fischeri compared with the start of the experiment. Measurements of Photosynthetic Activity Plants exposed to the contaminated water in most cases did not show significant changes of fluorescence emission

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Fig. 4 Contents (mg 100 g-1 fresh weight) of chlorophyll a, chlorophyll b, and carotenoids in the control as well as plants exposed to polluted water after a 10-day incubation. No statistically significant differences were found according to Student t test (a = 0.05; chlorophyll a p \ 0.810, chlorophyll b p \ 0.892, carotenoids p \ 0.651). Error bars represent SDs; n = 6

Author's personal copy Arch Environ Contam Toxicol Fig. 5 Morphology of shoots (a, c) and epidermis (with epidermal trichomes [b, d]) of plants incubated in control (a, b) and contaminated water (c, d) after 10-day incubation. Bar indicates 5 mm (a, b) or 50 lm (b, d)

compared with the control plant samples (Fig. 3). In general, the growth of Callitriche in polluted water was characterized by an insignificant decrease in average values of Fv/Fm (6.2 %), qP (3.6 %), and UPSI (10.8 %). However, we observed only a slight decrease in Fv/Fm and qP in the case of plants subjected to contamination, whilst Fo was significantly affected and (Fv did not change significantly (Table 3). The only parameter that was statistically significantly different was a the NPQ, which increased by 32.2 % in plants subjected to contaminated water. No statistically significant differences were measured in the photosynthetic pigment content of C. cophocarpa shoots subjected to contamination compared with the control samples (Fig. 4). Microscopic Observations of Plant Morphology No significant differences in morphology were found between plants incubated in both the control and polluted water (Fig. 5a, c). Both groups of plants exhibited a similar size and shape of whole plant body and number of leaves as well as the ability to generate new shoots during the time of the experiment. Both types of plants also exhibited a similar epidermis morphology of leaves and stems. The epidermis formed typical epidermal trichomes (hairs) (Fig. 5b, d). The hairs had a simple structure and consisted of basal epidermal cells, stalk cells, and a multi-celled head (range 7–12 cells).

Discussion Heavy-metal compounds are among the most toxic and persistent pollutants in aquatic environments (Hesieh et al. 2004). In the group of Callitriche genus, Samecka-Cymerman and Kempers (2001) found C. verna to be a good indicator of water quality in relation to several heavy metals. Moreover, Favas et al. (2012) found high contents of arsenic (metalloid) in the aquatic species of the Callitrichaceae family: C. lustanica, C. brutia, and C. stagnalis. However, as far as we know, no studies have focused on use of C. cophocarpa for the purpose of Zn, Cd, and Pb phytoremediation. In particular, no data are available on the aquatic Callitriche species with respect to Tl phytoremediation. In general, research data regarding Tl compounds are also scarce. Heavy-metal ions can enter plant cells by way of the cation uptake system, which involves different metal channels/transporters. This phenomenon is a consequence of similar mechanisms of transport for various ions across cell membranes. Therefore, accumulation of metallic compounds is not only restricted to the one selected element (Babula et al. 2008). In this study, we proved the ability of C. cophocarpa to efficiently accumulate Tl, Cd, Zn, and Pb, elements, which are regarded as priority toxic pollutants by the USEPA. Our experiments were performed under laboratory conditions; however, the water (both

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contaminated and the control) originated from the natural environment. In the present work, we showed that C. cophocarpa accumulated the highest levels of Zn. However, we cannot explain the high level of Zn detected in the control samples, which were collected from unpolluted stands. To explain this result, a further exhaustive, molecular approach is needed; this was not within the remit of the current investigation. Compared with Cd and Pb, phytoextraction of Zn was also more pronounced in other aquatic plants, e.g., Salvinia auriculata, Pistia stratiotes, Eichhornia crassipes, Potamogeton natans, Ceratophyllum demersum, Polygonum amphibium, and Veronica beccabunga (Valitutto et al. 2006; Samecka Cymerman and Kempers 2007). In our research, high accumulation levels were also found in C. cophocarpa shoots with respect to Cd and Pb. The mean reported contents (mg kg-1 dw) of the aforementioned metallic elements in the cited aquatic vegetation were in the range [mg kg-1 dw] of 62–350, 0.2–5.0, and 1.3–29.0 for Zn, Cd and Pb, respectively (Valitutto et al. 2006; Samecka Cymerman and Kempers 2007). Regarding the reported macrophytes, C. cophocarpa exhibited significantly greater accumulation levels. After only a 10-day incubation period, the plants phytoextracted Cd, Zn, and Pb from the solution to the level that meets current waterquality standards. After 10 days, Tl was the only element that was not lower than the permissible surface-water standards. The above-mentioned result is a consequence of the extremely high toxicity of and high concentration of Tl in the studied solutions. The content of Tl was exceeded by [120 times with respect to the standard. Generally, Tl concentrations in plant leaves are \0.02 mg kg-1 dw; therefore, a tentative threshold value characterizing Tl hyperaccumulators is 100 mg kg-1 dw (van der Ent et al. 2013). Tl is reported to be considerably more toxic to biota than Hg, Cd, Pb, and Zn. Moreover, its aquatic toxicity is not affected by water hardness or concentration of organic (humic) substances (Peter and Viraraghavan 2005). It must be pointed out that C. cophocarpa has an ability to concentrate Tl very effectively; its BCFs were at the highest level ([1,000-fold) for both Tl and Cd. The level of Tl in macrophytes collected from the polluted water reservoir was reported to be in the range of 0.14–2.09 mg kg-1 dw (Valitutto et al. 2006) with a magnitude 2–3 times lower with respect to the results obtained in this study. Research on heavy-metal pollution often only determines total metal content. This goal is mainly achieved by using the spectroscopy techniques (e.g., atomic absorption spectroscopy) after acidification of the medium and mineralization of plant biomass. Such an approach is obviously important, although it does not provide enough information concerning the environmental impact of pollution. The bioavailability and toxicity of metallic elements to aquatic

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organisms is determined by their physical and chemical forms in which these elements appear in the water (Alonso et al. 2004; Deaver and Rodgers 1996). It was shown that metals found in the toxic forms in laboratory tests can be present in nontoxic forms in the field (Deaver and Rodgers 1996). Hesieh et al. (2004) explained that the difference in toxicity is related to free ion activity and not total metal concentration. Heavy metals in water can be present in an ionic form, which is the most mobile and toxic form for living organisms, or in the form of an ion associated with different ligands. Occurrence of the particular species of the metallic element depends mainly on physicochemical factors, such as pH, content of soluble oxygen, hardness, temperature, redox potential (Eh), and the presence of inorganic and organic ligands (Kabata-Pendias and Mukherjee 2007). Thus, instead of obtaining measurements of total element contents in solutions and in plant biomass, we performed a Microtox bioassay. At the beginning of the experiment, the water control sample showed negative mean value of toxicity. This means that the luminescence intensity of V. fischeri increased after exposure to the control water being a result of their lower toxicity compared with the reference medium (NaCl solution). Stimulation of the light emission by low concentrations of contaminants was also observed by other investigators with the observation classified as hormesis (Christofi et al. 2002; Grant and Briggs 2002; Łukawska-Matuszewska et al. 2009; Shen et al. 2009). Samples that exhibit hormesis are currently considered nontoxic. Although this is a widespread phenomenon, its elucidation requires further research. In contrast, at the end of the experiment we measured a slight increase in control-water toxicity. This was probably due to some degradation processes in plant tissue during the time of the experiment. In the natural environment (e.g., a river), plants are effectively aerated, but they are less so under laboratory conditions. Nevertheless, we found that after a 10-day incubation of C. cophocarpa shoots, the class II classification of acute water toxicity (of the contaminated samples) was no longer appropriate. This result confirmed the high phytoremediation potential of C. cophocarpa because this species significantly bioconcentrates Tl, Cd, Zn, and Pb, removes these elements from the solutions, and at the same time eradicates pre-existing acute toxicity. In addition to studies regarding the remediation of pollutants by Callitriche, we also performed measurements of the physiological status of plants after their exposure to polymetallic contamination. In our opinion, the analysis of bioremediation efficiency should always cover the metabolic status of an organism in addition to measurements of accumulation levels/BCFs. Photosynthesis is the fundamental metabolic process for plants, and it may be affected by heavy metals in many ways. The toxicity of metallic


elements may be manifested in the change in the chlorophyll content due to chlorophyll-biosynthesis enzyme inhibition (e.g., by Cd, Zn, Pb) (Geebelen et al. 2002; Omar 2002; Sharma and Dubey 2005). Furthermore, a serious PSII efficiency decrease, detected as a decrease in Fv/Fm and UPSII values, is caused by chlorophyll grana ultrastructure degradation (Sharma and Dubey 2005). It was shown that Pb can decrease chloroplast membrane permeability as a result of fatty acid dehydrogenation (Stefanov et al. 1995) and cause a decrease in energy transfer rate from the light-harvesting chl a/b protein complex (LHCII) to PSII, which is manifested by Fo decrease (Bilger and Schreiber 1986). Cations of heavy metals can substitute Mg in chlorophylls of LHCII or in the internal PSII antenna system as well as chl a dimmer in the reaction center, thus leading to their loss of function (Domıńguez et al. 2011; Ku¨pper et al. 1996, 2002; Mateos-Naranjo et al. 2008a, b). Moreover, substitution by Zn in active Rubisco CO2– Mg complex or irreversible bounding of Pb into the same complex results in a strongly decreased CO2 fixation rate according to the inhibition of Rubisco activity (Siborova 1988; Van Assche and Clijsters 1986). The PSII Fv/Fm value of the Callitriche control plants was slightly \0.83, which is a theoretical optimum (Maxwell and Johnson 2000). Exposure of plants to contaminated water caused a decrease in this value, albeit an insignificant one. A decrease in the Fv/Fm value might be caused either by an Fo increase or Fv decrease. The former is connected with antenna system damage, mainly in LHCII, whereas the latter may result from PSII reaction center damage (Domıńguez et al. 2011; Ku¨pper et al. 1996, 2002; MateosNaranjo et al. 2008a, b; Maxwell and Johnson 2000). In Callitriche plants, heavy metallic elements caused some disturbances in the antenna system, which was manifested by significantly greater Fo and NPQ values, whereas it did not significantly influence the PSII reaction center observed by other investigators (Domıńguez et al. 2011; Mateos-Naranjo et al. 2008a, b). Therefore, we can assume that C. cophocarpa has developed an efficient mechanism for avoiding serious photosynthetic apparatus damage. This conclusion is also confirmed by no significant changes in photosynthetic pigment contents. Moreover, as far as morphology is concerned, the plant shoots treated by heavy-metal compounds were well-shaped and had regular internodes. Epidermis, which is the first cell layer exposed to contamination, was undisturbed with properly formed trichomes. The results obtained in this study point to C. cophocarpa as a good candidate for the phytoremediation of water polluted by Tl as well as Cd, Zn, and Pb. This submersed macrophyte is able to efficiently bioconcentrate the aforementioned elements in shoot tissue while maintaining physiological status compared with the control. At the

same time, C. cophocarpa eradicates acute toxicity in the contaminated water. The next phase of our research will be focused on introducing this species into a polluted natural environment and on its application as a natural biofilter. Acknowledgments We are indebted to Mr. Wiesław Knap (AGH University of Science and Technology Krako´w, Poland) for the determination of water composition and Monika Bieniasz (University of Agriculture, Krako´w, Poland) for offering the stereo microscope Zeiss Discover V12. We are also grateful to Anna Kołton (University of Agriculture, Krako´w, Poland) for assistance with the statistical analysis. The critical reading of the manuscript and English corrections made by Arek Kulczyk (Harvard University, Boston, MA, USA) is also highly appreciated. The work was financed by Grant No. DEC-011/03/ B/NZ9/00952 given by the National Science Centre, Poland. B. J. Płachno gratefully acknowledges the scholarship awarded to Outstanding Young Scientists by the Minister of Science and Higher Education.

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