lable at ScienceDirect

Plant Physiology and Biochemistry 71 (2013) 235e239

Contents lists avai

Plant Physiology and Biochemistry

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

Research article

Alleviation of silver toxicity by calcium chloride (CaCl2) inLemna gibba L.

Abdallah Oukarrouma,*, Marie-Hélène Gaudreault b, Laura Pirastru a, Radovan Popovic a,1

aDepartment of Chemistry and Biochemistry, University of Québec in Montréal, Case Postal 8888, Succursale Centre-Ville, Montréal, Québec H3C 3P8,Canadab Tête de Plume, Montréal, Québec H2H 2E6, Canada

a r t i c l e i n f o

Article history:Received 21 May 2013Accepted 29 July 2013Available online 7 August 2013

Keywords:Lemna gibbaSilver ionsCalciumFrond abscissionReactive oxygen species

* Corresponding author. Tel.: þ1 514 987 3000x141E-mail addresses: Oukarroum.abdallah@uqam

gmail.com (A. Oukarroum).1 In memory of Radovan Popovic.

0981-9428/$ e see front matter � 2013 Elsevier Mashttp://dx.doi.org/10.1016/j.plaphy.2013.07.019

a b s t r a c t

The toxicity effects of silver (Ag) and the protective role of calcium chloride (CaCl2) was studied in Lemnagibba L. (L. gibba) plants. Silver speciation showed that silver toxicity in L. gibba culture medium can beattributed to free ionic Agþ concentration. Frond abscission, intracellular reactive oxygen species (ROS)formation and intracellular uptake of Agþ were investigated when L. gibba plants were exposed to AgNO3

concentrations (0.5, 1, 5, and 10 mM) supplemented or not by 10 mM CaCl2. An increase in frondabscission, intracellular ROS and intracellular uptake of Agþ were detected in L. gibba plants for all testedconcentrations of AgNO3 after 24 h treatment. However, addition of 10 mM CaCl2 to the L. gibba culturemedium reduced the toxic effects of Ag by decreasing silver uptake into the plant and intracellular ROSformation. The results suggest that Ag-induced toxicity was attributed to Agþ accumulation and chloridewas able to protect L. gibba plants against Ag toxicity by formation of complexes with Ag and thenalleviation of the metal induced oxidative stress.

� 2013 Elsevier Masson SAS. All rights reserved.

1. Introduction

Silver ion is one of the most toxic metals in plants [1e3]. Silver isdischarged in the environment from its various applications in theimaging, electronic and electrical industries [4] and many toxicityreactions have been described in different organisms [2]. Thetoxicity of silver in the aqueous environment depends on theconcentration of active and free silver ions [5]. It was suggested thatsilver accumulation is due to adsorption to the cell surface ratherthan to active uptake into the cells [6]. Some studies reported that0.1 mg/L of silver nitrate (AgNO3) inhibits the growth of themicroalgae Chlorella vulgaris and Chlorella VT-1 [7] and reducedalgal photosynthetic yield [8].

It is well-known that metals, depending on their oxidationstates, can be highly reactive and, as a consequence, provoke anoxidative cellular damage [9,10]. Oxidative damage induced bymetals can be caused by an increasing cellular concentration ofreactive oxygen species (ROS) [11]. In most organisms, metal ionsinhibit photosynthetic electron transport due to ROS formation,which furthermore may affect biomass production [12e14]. At high

7; fax: þ1 514 987 4054..ca, abdallah.oukarroum@

son SAS. All rights reserved.

concentration of metals, cellular damage occurs because ROS levelsexceed the antioxidant capacity of the cell. On the other hand,reduction of toxic effects of various metals by calcium ions wasobserved [15,16].

In ponds and lakes, Lemna gibba plants (L. gibba) are known to beimportant contributors of biomass production serving as a sourceof food for different species [17]. This aquatic plant has also beenused as a suitable model for ecotoxicological studies due to its smallsize and fast growing rate [18,19]. In the present study, resultsobtained from frond abscission, bioaccumulation of silver andintracellular ROS formation were employed to assess the toxic ef-fects of AgNO3 on the L. gibba plants and the reverse effect of cal-cium chloride (CaCl2). The results from this study could facilitate abetter understanding of the potential toxicity risks of silver inaquatic environments and the potential detoxification by CaCl2.

2. Results and discussion

2.1. Distribution of the different ionic silver species in the culturemedium

Silver speciation was important to evaluate its toxicity onL. gibba plants and to calculate half-maximal response concentra-tions (EC50). As shown in Table 1, the calculated free ionic Agþ

concentration was increased when AgNO3 concentration increased

Table 1Distribution of the different ionic silver species in the culture medium I of Lemnagibba plants.

[AgNO3] (mM) Agþ (%) AgCl (aq)(%)

AgCl2�

(%)AgNO3 (aq)

(%)[Agþ](mM)

0.5 70.43 28.90 0.58 0.1 0.361 70.44 28.88 0.58 0.1 0.705 70.55 28.78 0.57 0.1 3.5310 70.69 28.65 0.57 0.1 7.070.5 þ 10 mM CaCl2 68.64 30.60 0.67 0.1 0.341 þ 10 mM CaCl2 68.67 30.58 0.66 0.1 0.695 þ 10 mM CaCl2 68.77 30.48 0.66 0.1 3.4410 þ 10 mM CaCl2 68.91 30.35 0.66 0.1 6.89

A. Oukarroum et al. / Plant Physiology and Biochemistry 71 (2013) 235e239236

in the culture medium and the distribution of the different silverspecies was not significantly changed between the different treat-ment concentrations. Therefore, the toxic effect of AgNO3 onL. gibba plants can be attributed to silver ions (Agþ). On the otherhand, addition of 10 mM of CaCl2 decreased the Agþ concentrationin the culture medium (Table 1) and we observed here that Cl-

formed complexes with Agþ (AgCl) in the culture medium.

2.2. Frond abscission

The effect of silver ions on the L. gibba fronds was evaluated 24 hafter adding AgNO3 to the nutrient medium. Release of daughterfronds from the mother frond and decreasing the size of colonieswas observed and it was the first visual symptom of Ag toxicity(Fig.1). Topp et al. [20] reported that silver caused an acceleration of

Fig. 1. Visual symptoms (frond abscission) on duckweed L. gibba colo

abscission stipes response and thus resulting in smaller colonies.Release of daughter fronds from the mother frond before maturityhas been reported also in Lemna aequinoctialiswild-type exposed tosix metals [21]. Henke et al. [22] showed that frond abscission inLemna minor was fast to a large metals. The mechanism of theabscission response has been analysed by Topp et al. [20] anddemonstrated that abscission of fronds is facilitated by secretingcells in the stipes that connect mother and daughter fronds. In ourstudy, under the influence of CaCl2, the response of frond abscissionwas reduced (Fig. 1) in plants exposed to AgNO3. Using the per-centage of frond abscission (FA %) as a quantitative measure, thedoseeresponse curve was presented in Fig. 2. It was assumed thatThe FA is 100% when all stipes have been broken, or when allpossible abscissions have been completed and only single frondsare present [22]. Treatment of plant for 24 h has a significantlyinhibitory effect on the FA (%) (p< 0.05). The FA (%) increased whenAgþ concentration increased and reached maximum at 10 mM ofAgNO3 (FA ¼ 100%). However addition of 10 mM CaCl2 to culturemedium reduced the effect of Agþ (p < 0.05). The higher concen-tration used in our experiment (10 mMAgNO3) diminished FA (%) by40% compared to the same concentration of AgNO3 treatment. In aprevious study, large differences between EFAC50 (Half-maximaleffective concentrations of frond abscission) of different clones of L.minor from different place of origin exposed to Agþ was observedby Topp et al. [20] and the calculated EFAC50 was ranged from0.47 to12 mM. Naumann et al. [23] reported by investigation of five growthparameters (frond number, fresh weight, dry weight, chlorophylland carotenoid contents) in L. minor L. clone St; that Agþ was themost toxic element compared to ten other metals and the

nies exposed 24 h to silver combined or not with 10 mM CaCl2.

A. Oukarroum et al. / Plant Physiology and Biochemistry 71 (2013) 235e239 237

calculated average of ErC50 (growth rate) was 0.4 mM. From thedoseeresponses curve presented in our investigation, a half-maximal response concentration of frond abscission (EFAC50) wascalculated and it was 0.96 and 1.94 mM respectively when L. gibbaplants were exposed for 24 h to Ag or supplemented with 10 mMCaCl2. Indeed, CaCl2 contributed to diminish Agþ toxicity.

2.3. Intracellular [Ag] determination

Accumulation of Ag inside the cell (and between cells) wasanalysed quantitatively. When L. gibba plants were exposed toAgNO3 in culture medium, an uptake of Agþ was found. Ag-inducedtoxicity expressed as a bioaccumulation of Agþ, increased pro-gressively with external nominal concentration of AgNO3.

However, Agþ uptake dropped significantly in plants supple-mented with 10 mM CaCl2. Total Agþ content in cells was0.14 � 1.5 � 10�3 mg/mg dry weight of L. gibba plants when plantswere treated with 10 mM of AgNO3 while it was0.090 � 1.1 � 10�3 mg/mg dry weight when plants were supple-mented with 10 mM CaCl2 (Table 2). These results indicated thatCaCl2 has an antagonistic effect against Agþ bioaccumulation andmight be resulted by competition for uptake. Such competition anduptake was observed in cells exposed to Cadmium (Cd2þ) [27].Indeed, some nonessential metal ions would enter plant cells viauptake systems for essential cations [28]. The accumulation ofintracellular Ag demonstrated an evident cellular toxicity by anincrease in ROS production. Lee et al. [25] have been demonstratedthat intracellular accumulation of silver in two green algae, Pseu-dokirchneriella subcapitata and Chlamydomonas reinhardtii wasresponsible of a direct toxicity rather than cell surface interactions.

2.4. Measurement of reactive oxygen species

In this study, we investigated ROS production upon Ag exposureor upon Ag and CaCl2 exposure. Formation of total ROS in cellsdetected by using the H2DCFDA fluorometric approach indicatesthat Ag stimulated intracellular ROS formation (Fig. 3A and B). ROS

Fig. 2. Doseeresponse curves of L. gibba exposed 24 h to silver effect combined or notwith 10 mM CaCl2. The free ionic Agþ concentration in the culture media used thisdoseeresponse curves were determined using the chemical equilibrium calculationsoftware Visual MINTEQ 2.61 and presented in Table 1.

formation is the well-known indicator for determining the degreeof oxidative stress. Fig. 3A showed that in plants growing in culturemedium, ROS formation was gradually increased and reaching250% compared to control (p < 0.05) for the highest AgNO3 con-centration (10 mM), which revealing a higher AgNO3 toxicity. In aprevious study Ag-induced ROS production was observed in C.reinhardtii and may be mostly due to indirect effect of the highaffinity of Agþ to thiol groups [24]. However, additionally of CaCl2reduced intracellular ROS formation in cells. It is known that thetoxic effects of metals on biological activity can be affected by thepresence of chelators which may reverse their toxicity. Indeed,addition of 10 mM CaCl2 to the L. gibba culture medium reduced thetoxic effects of Ag and the question that we could posed was whichCa2þ or Cl� was responsible for this antagonist effects? Then, whenL. gibba was exposed to different AgNO3 concentrations in growthmedium that was free of Cl�, we observed a higher ROS formation(660% at 10 mM) compared to L. gibba exposed to AgNO3 in culturemedium (with complete composition) (Fig. 3B). Addition of 10 mMCaCl2 to this culture medium free of Cl� provoked an increase by130% of ROS formation compared to control. We noted here thataddition of CaCl2 in culture medium has more antagonist effectcompared to culture medium free of Cl�, this was due to the exis-tence of Cl� already in the culture medium. We can assumed thatCl� plays an important role in this protection against Ag toxicity byformation of complexes with Ag (Table 1), in other words, thesecomplexes made Ag less bioavailable to plants and resulted indiminution of his toxic effect. Interestingly, Fortin and Campbell [5]and Lee et al. [26] expected that the toxicity of silver was lower inthe presence of chloride than in its absence in culture media ofthree algae species, due to chloride complexation and the conse-quent decrease in the free Agþ concentration.

Cl� was capable of reducing Agþ uptake by plants and then re-duces Ag toxicity by forming complexes with silver ions (AgCl) inthe medium (Table 1). Lee et al. [25] demonstrated that in Pseu-dokirchneriella subcapitata and C. reinhardtii, algae, silver toxicitywas attenuated in the presence of chloride due to chloridecomplexation and the consequent decrease in the free Agþ con-centration. Although Ca2þ must not to be neglected, it was reportedthat Ca2þ can stabilize cell membrane surfaces, influence the pH ofcells and prevent solute leakage from cytoplasm [29]. Addition Ca2þ

can significantly accelerated the growth and chlorophyll content ofChlorella vulgaris [30].

Aquatic ecosystems are directly or indirectly destinations ofdifferent substances as metals. Such metal pollution, may lead to anumber of metabolic transformations in living organisms. In thisstudy, chloride calcium treatment has demonstrated a protectiveeffect against silver toxicity. Thus, application of chloride calcium atdifferent level of waste water treatment could protect against thebioaccumulation of silver in L. gibba and moreover diminished thetrophic transfer of this metal.

In conclusion, frond abscission, intracellular ROS formation andAgþ intracellular accumulation were used to estimate the physio-logical alterations induced on L. gibba plants by Agþ or supple-mented with 10 mM CaCl2. We observed that Agþ was a very toxiccompound to L. gibba plants. However addition of 10 mM CaCl2 inthe culture medium resulted in formation of complexes betweenCl- and Ag; reduced the toxic effects of Agþ and showed a cellsprotection against Ag-induced oxidative stress.

3. Materials and methods

3.1. Biological material and AgNO3 treatment

L. gibba L. plants were obtained from the Canadian PhycologicalCulture Centre (formerly UTCC #310). Plants were grown in an

Table 2Total amount of intracellular Ag concentration in L. gibba plants (mg/mg dry weight) exposed 24 h to different concentrations of AgNO3 supplemented or not by 10 mM CaCl2.

Concentration 0.01 mg/L 0.1 mg/L 1 mg/L 10 mg/L

AgNO3 0.10 � 2.1 � 10�4 0.10 � 1.7 � 10�3 0.11 � 2.6 � 10�3 0.14 � 1.5 � 10�3

AgNO3 þ CaCl2 0.079 � 1.2 � 10�3 0.082 � 1.1 � 10�3 0.086 � 1.9 � 10�3 0.090 � 1.1 � 10�3

A. Oukarroum et al. / Plant Physiology and Biochemistry 71 (2013) 235e239238

inorganic culture medium as described by Frankart et al. [31]. Thismedium consisted of the following: KNO3, 202 mg/L; KH2PO4,50.3 mg/L; K2HPO4, 27.8 mg/L; K2SO4, 17.4 mg/L; MgSO4 � 7 H2O,49.6mg/L; CaCl2,11.1mg/L; FeSO4�7 H2O, 6mg/L; H3BO3, 5.72mg/L; MnCl2 � 4H2O, 2.82 mg/L; ZnSO4, 0.6 mg/L; (NH4)Mo7O24 � 4H2O, 0.043 mg/L; CuCl2 � 2 H2O, 0.078 mg/L; CoCl2 � 6 H2O,0.054mg/L. Before themediumwas autoclaved, its pHwas adjustedto 6.5 � 0.1 using 0.1 M HCl.

Experiments were done in a growing chamber CONVIRON(Controlled Environments Limited, Winnipeg, Manitoba, Canada)with a 16 h/8 h light/dark photoperiod. A light irradiance of100 mmol m�2 s�1 was provided by cool white fluorescent lamps(Sylvania GRO-LUX F40/GS/WS).

3.2. L. gibba L. plants exposure to AgNPs

Triple-fronded L. gibba plants, in the exponential growth phase,were used for experiments. Five triple-fronded L. gibba plants inthree replicates were treated during 24 h in plastic Petri dishescontaining growth medium having initial AgNPs concentrations of0, 0.5, 1, 5 and 10 mM supplemented or not by 10 mM CaCl2. The freeionic Agþ concentration in the culture media was determined usingthe chemical equilibrium calculation software Visual MINTEQ 2.61.

3.3. Frond abscission

The toxicity of AgNO3 was evaluated by the percentage of frondabscission (FA%). FA % was calculated according to Henke et al. [5]:

FAð%Þ ¼ ½ðCN24h � CN0Þ=FN24h � CN0� � 100

where CN is the number of colonies either at time zero 0 or 24 h andFN24h is the frond number at time 24 h.

Fig. 3. Change of intracellular reactive oxygen species (ROS) in L. gibba plants exposed 24 hdescribed by Frankart et al. [31]. (B) L. gibba plants were grown in a culture medium as descrand results are shown as the mean with standard deviations. Asterisk indicates statistical s

3.4. Intracellular [Agþ] determination

After 24 h of AgNPs treatments, L. gibba plants were recuperatedand washed in Ethylenediaminetetraacetic acid (EDTA) for 2 min toeliminate any free silver ions. Samples were dried at 105 �C for 24 h,weighted to calculate dry weight and then placed in an acid-washed glass tube in which 2 mL HNO3 and 500 ml H2O2 wasadded. The samples were allowed to digest 48 h at 120 �C beforebeing diluted in nanopure water for atomic absorption spectros-copy quantification of Ag using a Varian SpectrAA 220 FS system. Agconcentrations were normalised to the dry weight.

3.5. Measurement of reactive oxygen species (ROS)

The formation of reactive oxygen species (ROS) in L. gibba plantswas measured according to Ref. [32]. ROS formation was measuredby using the cell permeable indicator 20,70dichlorodihydro fluo-rescein diacetate (H2DCFDA) [33]). Cellular esterases hydrolyse theprobe to the non fluorescent 20,70dichlorodihydrofluorescein(H2DCF), which is better retained in the cells. In the presence of ROSand cellular peroxidases, H2DCF is transformed to the highly fluo-rescent 20,70dichlorofluorescein (DCF). All the fluorescence datawere collected using a fluorescence plate reader (SpectraMax M2eMulti-Mode Microplate Reader).

Change of intracellular reactive oxygen species (ROS) wasmeasured in L. gibba plants grown in a culturemedium as describedby Frankart et al. [31] and in a culture medium free of Cl�.

3.6. Data analysis and statistics

The experiments were done in triplicate for all treatments.Means and standard deviations were determined for each treat-ment. Significant differences between control and treated samples

to Agþ only or with 10 mM CaCl2. (A) L. gibba plants were grown in a culture medium asibed by Frankart et al. [31] but free of Cl�. The experiments were conducted in triplicateignificance between the control and AgNO3 treatments (p < 0.05).

A. Oukarroum et al. / Plant Physiology and Biochemistry 71 (2013) 235e239 239

were determined by using Student’s t-test where P values less than0.05 were considered to be significantly different.

Acknowledgements

This work was supported by the Natural Sciences and Engi-neering Research Council (NSERC, Canada) grants.

References

[1] G. Ouzounidou, H.A. Constantinidou, Changes in growth and physiology oftobacco and cotton under Ag exposure and recovery: are they of direct orindirect nature? Arch. Environ. Contam. Toxicol. 37 (1999) 480e487.

[2] H.T. Ratte, Bioaccumulation and toxicity of silver compounds: a review, En-viron. Toxicol. Chem. 18 (1999) 89e108.

[3] Q.S. Xu, J.Z. Hu, K.B. Xie, H.Y. Yang, K.H. Du, G.X. Shi, Accumulation and acutetoxicity of silver in Potamogeton crispus L, J. Hazard. Mater. 173 (2010) 186e193.

[4] T.W. Purcell, J.J. Peters, Sources of silver in the environment, Environ. Toxicol.Chem. 17 (1998) 539e546.

[5] C. Fortin, P.G.C. Campbell, Silver uptake by the green alga Chlamydomonasreinhardtii in relation to chemical speciation: influence of chloride, Environ.Toxicol. Chem. 19 (2000) 2769e2778.

[6] N.S. Fisher, J.R. Reinfelder, The trophic transfer of metals in marine systems,in: A. Tessier, D.R. Turner (Eds.), Metal Speciation and Bioavailability inAquatic Systems, John Wiley & Sons, London, 1995, pp. 363e406.

[7] A.H. Scragg, C. Bonnett, Inhibition of microalgal growth by silver nitrate,Biotechnol. Lett. 24 (2002) 169e172.

[8] E. Navarro, F. Piccapietra, B. Wagner, F. Marconi, R. Kaegi, N. Odzak, L. Sigg,R. Behra, Toxicity of silver nanoparticles to Chlamydomonas reinhardtii, Envi-ron. Sci. Technol. 42 (2008) 8959e8964.

[9] A. Mithöfer, B. Schulze, W. Boland, Biotic and heavy metal stress response inplants: evidence for common signals, FEBS Lett. 566 (2004) 1e5.

[10] M. Bertrand, I. Poirier, Photosynthetic organisms and excess of metals, Pho-tosynthetica 43 (2005) 345e353.

[11] G. Bartosz, Oxidative stress in plants, Acta Physiol. Plant 19 (1997) 47e64.[12] E. Pinto, T.C.S. Sigaud-Kutner, M.A.S. Leitão, O.K. Okamoto, D. Morse,

P. Colepicolo, Heavy metal-induced oxidative stress in algae, J. Phycol. 39(2003) 1008e1018.

[13] D.Dewez, L.Geoffroy,G.Vernet, R. Popovic,Determinationofphotosyntheticandenzymatic biomarkers sensitivity used to evaluate toxic effects of copper andfludioxonil in alga Scenedesmus obliquus, Aquat. Toxicol. 74 (2005) 150e159.

[14] S. Knauert, K. Knauer, The role of reactive oxygen species in copper toxicity totwo freshwater green algae, J. Phycol. 44 (2006) 311e319.

[15] Z. Rengel, Role of calcium in aluminum toxicity, New Phytol. 121 (1992)499e513.

[16] K. Lock, P. Criel, K.A.C. De Schamphelaere, H. Van Eeckhout, C.R. Janssen, In-fluence of calcium, magnesium, sodium, potassium and pH on copper toxicityto barley (Hordeum vulgare), Ecotoxicol. Environ. Saf. 68 (2007) 299e304.

[17] C.N. Fassett, A Manual of Aquatic Plants, sixth ed., Univ. Wisconsin Press,Madison, 1972.

[18] W. Wang, Literature review on duckweed toxicity testing, Environ. Res. 52(1990) 7e22.

[19] B.M. Greenberg, X.D. Huang, D.G. Dixon, Applications of the aquatic higherplant Lemna gibba for ecotoxicological assessment, J. Aquat. Ecosyst. Health 1(1992) 147e155.

[20] C. Topp, R. Henke, Á. Keresztes, W. Fischer, M. Eberius, K.J. Appenroth, A novelmechanism of abscission in fronds of Lemna minor L. and the effect of silverions, Plant Biol. 13 (2011) 517e523.

[21] T. Li, Z.T. Xiong, A novel response of wild-type duckweed (Lemna paucicostataHegelm.) to heavy metals, Environ. Toxicol. 19 (2004) 95e102.

[22] R. Henke, M. Eberius, K.J. Appenroth, Induction of frond abscission bymetals and other toxic compounds in Lemna minor, Aquat. Toxicol. 101(2011) 261e265.

[23] B. Naumann, M. Eberius, K.J. Appenroth, Growth rate based doseresponserelationships and EC-values of ten heavy metals using the duckweed growthinhibition test (ISO 20079) with Lemna minor L. clone St, J. Plant Physiol. 164(2007) 1656e1664.

[24] I. Szivák, R. Behra, L. Sigg, Metal-induced reactive oxygen species productionin Chlamydomonas reinhardtii (Chlorophyceae), J. Phycol. 45 (2009) 427e435.

[25] D.Y. Lee, C. Fortin, P.G.C. Campbell, Contrasting effects of chloride on thetoxicity of silver to two green algae, Pseudokirchneriella subcapitata andChlamydomonas reinhardtii, Aquat. Toxicol. 75 (2005) 127e135.

[26] D.Y. Lee, C. Fortin, P.G.C. Campbell, Influence of chloride on silver uptake bytwo green algae, Pseudokirchneriella subcapitata and Chlorella pyrenoidosa,Environ. Toxicol. Chem. 23 (2004) 1012e1018.

[27] P.M. Hinkle, E.D. Shanshala, E.J. Nelson, Measurement of intracellular cad-mium with fluorescent dyes. Further evidence for the role of calcium channelsin cadmium uptake, J. Biol. Chem. 267 (1992) 25553e25559.

[28] S. Clemens, Toxic metal accumulation, responses to exposure and mecha-nisms of tolerance in plants, Biochimie 88 (2006) 1707e1719.

[29] K.D. Hirschi, The Calcium Conundrum. Both versatile nutrient and specificsignal, Plant Physiol. 136 (2004) 2438e2442.

[30] A.H. EL-Naggar, M.M. EL-Sheekh, Abolishing cadmium toxicity in Chlorellavulgaris by ascorbic acid, calcium, glucose and reduced glutathione, Environ.Pollut. 101 (1998) 169e174.

[31] C. Frankart, P. Eullaffroy, G. Vernet, Photosynthetic responses of Lemna minorexposed to xenobiotics, copper, and their combinations, Ecotoxicol. Environ.Saf. 53 (2002) 439e445.

[32] T.S. Babu, T.A. Akhtar, M.A. Lampi, S. Tripuranthakam, D.G. Dixon,B.M. Greenberg, Similar stress responses are elicited by copper and ultravioletradiation in the aquatic plant Lemna gibba? Implication of reactive oxygenspecies as common signals, Plant Cell Physiol. 44 (2003) 1320e1329.

[33] I.B. Gerber, I.A. Dubery, Fluorescence microplate assay for the detection ofoxidative burst products in tobacco cell suspensions using 20 ,70-dichloro-fluorescein, Meth. Cell. Sci. 25 (2003) 115e122.