Toxicity of PAMAM dendrimers to Chlamydomonas reinhardtii

Aquatic Toxicology 100 (2010) 187–193

Contents lists available at ScienceDirect

Aquatic Toxicology journal homepage: www.elsevier.com/locate/aquatox

Toxicity of PAMAM dendrimers to Chlamydomonas reinhardtii Anne-Noëlle Petit a,∗ , Philippe Eullaffroy b , Timothée Debenest a , Franc¸ois Gagné a a b

Environment Canada, 105 McGill Street, Montréal, Québec H2Y 2E7, Canada Laboratoire Plantes, Pesticides et Développement Durable, EA 2069, URVVC, BP 1039, Université de Reims Champagne-Ardenne, 51687 Reims Cedex 2, France

a r t i c l e

i n f o

Article history: Received 6 October 2009 Received in revised form 18 January 2010 Accepted 29 January 2010 Keywords: Aquatic toxicity Chlamydomonas reinhardtii Dendrimer Engineered nanoparticles Nanotoxicology Phytoplankton

a b s t r a c t In recent decades, a new class of polymeric materials, PAMAM dendrimers, has attracted marked interest owing to their unique nanoscopic architecture and their hopeful perspectives in nanomedicine and therapeutics. However, the potential release of dendrimers into the aquatic environment raises the issue about their toxicity on aquatic organisms. Our investigation sought to estimate the toxicity of cationic PAMAM dendrimers on the green alga, Chlamydomonas reinhardtii. Algal cultures were exposed to different concentrations (0.3–10 mg L−1 ) of low dendrimer generations (G2, G4 and G5) for 72 h. Potential adverse effects on Chlamydomonas were assessed using esterase activity (cell viability), photosynthetic O2 evolution, pigments content and chlorophyll a fluorescence transient. According to the median inhibitory concentration (IC50 ) appraised from esterase activity, toxicity on cell viability decreased with dendrimer generation number (2, 3 and 5 mg L−1 for G2, G4 and G5 dendrimers, respectively). Moreover, the three generations of dendrimers did not induce the same changes in the photosynthetic metabolism of the green alga. O2 evolution was stimulated in cultures exposed to the lowest generations tested (i.e. G2 and G4) whereas no significant effects were observed with G5. In addition, total chlorophyll content was increased after G2 treatment at 2.5 mg L−1 . Finally, G2 and G4 had positive effects on photosystem II (PSII): the amount of active PSII reaction centers, the primary charge separation and the electron transport between QA and QB were all increased inducing activation of the photosynthetic electron transport chain. These changes resulted in stimulation of full photosynthetic performance. Crown Copyright © 2010 Published by Elsevier B.V. All rights reserved.

1. Introduction In recent decades, engineered nanoparticles (ENPs) have found increasingly widespread use in many consumer products (Aitken et al., 2006). Among ENPs, dendrimers have attracted attention in a diverse array of biomedical applications because of their well-defined, mono-dispersive and stable molecular architecture (Duncan and Izzo, 2005; Yang and Kao, 2006; Bharali et al., 2009). Dendrimers are distinguished from the others branched polymers by their repeating patterns emanating from a central core and an outer surface of terminal functional units. Addition of successive layers, called generations (G), gradually increases the molecular weight of the dendrimer (Tomalia, 2005). Surface groups of dendrimers can be neutral, or positively/negatively charged. Poly(amidoamine) (PAMAM) dendrimers are the first and most extensively studied family of dendrimers for biomedical use (Tomalia, 2005). Their amphiphilic nature and easily modifiable surface makes them attractive vehicles for targeted drug

∗ Corresponding author at: Environment Canada, 105 McGill Street, 7 Floor, Montréal, Québec H2Y 2E7, Canada. Tel.: +1 514 283 0725. E-mail addresses: [email protected], [email protected] (A.-N. Petit).

or gene delivery and imaging of biological systems (Tomalia, 2005; Tomalia et al., 2007). Due to the exponential growth of biomedical applications of PAMAM dendrimers, there is an urgent need to investigate their possible toxic health effects on humans and the environment (Colvin, 2003; Nel et al., 2006). Recently, a series of studies have demonstrated that PAMAM dendrimers can cause toxic effects in bacteria and animal cells. Antibacterial properties have been observed in the Gram-negative bacteria Pseudomonas aeruginosa and Vibrio fischeri (Calabretta et al., 2007; Mortimer et al., 2008). Studies on mice and zebrafish embryos have revealed that dendrimer toxicity was dose- and generation-dependent (Roberts et al., 1996; Malik et al., 1999; King Heiden et al., 2007). Moreover, these studies have demonstrated that their toxicity was also closely related to the nature of terminal groups. Half-generation PAMAM dendrimers with anionic carboxylic acid terminal groups are less toxic compared to full generation PAMAM dendrimers with cationic amine terminal groups. Furthermore, positively charged dendrimers have been found to interact with blood components, destabilize cell mem´ branes, resulting in cell lysis (Rittner et al., 2002; Domanski et al., 2004; Hong et al., 2004). Nevertheless, no information is presently available on the toxicity of PAMAM dendrimers to plants, especially algae.

0166-445X/$ – see front matter. Crown Copyright © 2010 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.aquatox.2010.01.019

188

A.-N. Petit et al. / Aquatic Toxicology 100 (2010) 187–193

Green microalgae are the primary producers at the base of the aquatic food chain. Any disturbances may have consequences on the upper levels. In this context, microalgae are widely used in ecotoxicological tests (Morlon et al., 2005; Wang and Dei, 2006; Aruoja et al., 2009). The unicellular alga Chlamydomonas reinhardtii is a useful model organism to study responses to different environmental factors. Growth of this alga is rapid with cells attaining logarithmic phase in 2 or 3 days. This organism has also the sensitivity and ability to rapidly respond to a wide range of pollutants, especially with respect to water pollution (Reboud, 2002; Morlon et al., 2005; Wang and Dei, 2006; Rodríguez et al., 2007; Wang et al., 2007). Photosynthesis is the principal mode of energy metabolism of algae and alterations in algal photosynthesis could affect ecological balance. Hence, algal photosynthetic activity is widely studied for monitoring environmental degradation of aquatic ecosystems by direct measurement of O2 evolution and photosynthetic pigment content (Appenroth et al., 2001; Pan et al., 2008). To understand more precisely the target of pollutants on photosynthetic complex, chlorophyll (chl) a fluorescence techniques can be used. Measurement of chl a fluorescence induction curves provide powerful tools for the detection of toxic agents on photosynthesis of phytoplankton (Force et al., 2003; Papageorgiou et al., 2007). At high excitation irradiance, dark-adapted photosynthetic organisms show characteristic polyphasic fluorescence kinetics. The fluorescence intensity rises rapidly from the ‘origin’ (O) through two ‘inflections’ (J and I) to a ‘peak’ fluorescence level (P) (Strasser et al., 2000). Since there is general agreement that Photosystem II (PSII) is the primary and highly susceptible site to several types of stresses, the analysis of the fluorescence transients (O–J–I–P) according to the JIP-test (Strasser and Strasser, 1995) is widely used as a tool to detect and estimate the status of PSII under various environmental stresses (e.g., Appenroth et al., 2001; Pan et al., 2008; Eullaffroy et al., 2009). Photosynthesis as other population-based parameters, such as specific growth rate and biomass, does not provide information on the distribution of responses among the individual cells within the population. Flow cytometry, however, is a useful alternative to the standard algal population-based endpoints since it provides a fast and quantitative measurement of responses of individual microalgae to toxic stress (Franqueira et al., 2000; Hadjoudja et al., 2009; Rioboo et al., 2009). Taking into account all of these considerations, the present work sought to examine whether cationic PAMAM dendrimers are toxic to C. reinhardtii microalgae. Moreover, effects of these ENPs on viability and photosynthesis were evaluated, by measuring the pigment composition, photosynthetic oxygen evolution and the chl fluorescence emission of this green alga. 2. Materials and methods

Shaker, New Brunswick Scientifics, USA) under continuous illumination (40 ␮mol m−2 s−1 ) provided by white fluorescent lamps (Sylvania® Gro Lux F15W, Germany) at 25 ± 1 ◦ C and with constant rotary agitation (100 rpm). 2.2. Dendrimer exposure Exponentially growing cells were diluted with fresh medium to achieve test samples at around 1 × 106 cells mL−1 cell density. Algal culture cell densities were determined by using a coulter particle counter (ZI, Beckman Coulter Inc., USA). StarbustTM G2, G4 and G5 polyamidoamine (PAMAM) dendrimers solutions were purchased from Sigma–Aldrich (Canada). A short characterization of the dendrimers used is presented in Table 1. Dendrimer solutions were diluted in distilled water to make 500 mg L−1 stock solution. Aliquots of 25 mL of the algal culture in growing media were exposed to 0, 0.3, 1, 2.5 and 10 mg L−1 of dendrimer. For the control samples, the same media were used but in the absence of dendrimer. The algal samples treated with dendrimers were exposed to the same light intensity and temperature conditions as those used for growth culture. All measurements were done after 72 h of treatment. 2.3. Flow cytometric analysis Chl and esterase activities were evaluated using a three-color Guava EasyCyte Plus System cytometer (Guava Technologies Inc. Hayward, USA) with a laser emitting at 488 nm. The cytometer was calibrated before each experiment using the supplied Guava check kit (Guava Technologies Inc. Hayward, USA). Before each experiment, settings were adjusted. To respect the maximum limit of cell density recommended by the manufacturer (500 cells ␮L−1 ) to perform accurate analysis, algal solutions were diluted if necessary with cell growth medium. In a 96-well plate, 198 ␮L of algal solutions were exposed to 2 ␮L of fluorescein diacetate (FDA; CAS N. 596-09-8) stock solution in acetone (1.3 mg mL−1 ). Cells were incubated with FDA during 15 min in the dark according to Blaise and Ménard (1998). Non-algal particles were excluded from the analysis by setting an acquisition threshold value (100) for the forward-scatter (FSC) parameter. The flow rate was set at 0.59 ␮L s−1 and 1000 events were counted. Alterations of esterase activity resulting from exposure of algae to dendrimers were measure by changes in green fluorescence (fluorescein) to assess the cell viability. Two analysis markers were set up in flow cytometry histogram for green to separate cells with normal activity (high green fluorescence) from altered cells (low green fluorescence). Markers set in control samples were afterwards applied to others samples. Data processing was carried out using Cytosoft, Data acquisition and Analysis Software (Guava Technologies Inc. Hayward, USA). The data were displayed in Log mode.

2.1. Algal culture 2.4. Photosynthetic oxygen evolution C. reinhardtii (CC-125) culture was obtained from the Canadian Phycological Culture Centre (CPCC, Ontario, Canada), formerly known as the University of Toronto Culture Collection (UTCC). Cultures were grown in 250 mL Erlenmeyer flasks containing 100 mL of an autoclaved high salt growth medium (HSM) (Sueoka et al., 1967). Flasks were placed in an incubator (Innova 44 R Orbital

Photosynthetic oxygen production was determined using a Clark-type oxygen electrode (Oxygraph, Hansatech, UK) at 25 ◦ C with actinic light (about 50 ␮mol m−2 s−1 ) of photosynthetically active radiation. The measuring chamber was filled with 2 mL of sample taken from each flask and the cell suspension was continu-

Table 1 Characteristics of dendrimers investigated. Name, generation

Terminal groups

Number of terminal groups

Molecular formula

Molecular weight

Diameter (nm)

PAMAM 1,4-diaminobutane core, G2 PAMAM 1,4-diaminobutane core, G4 PAMAM 1,4-diaminobutane core, G5

–NH2 –NH2 –NH2

16 64 128

C144 H292 N58 O28 C624 H1252 N250 O124 C1264 H2528 N506 O252

3284 14,243 28,854

2.6 4.4 5.7

A.-N. Petit et al. / Aquatic Toxicology 100 (2010) 187–193

ously stirred during the measurements. Prior to measurement, the samples were adapted for 2 min, during which time, the oxygen evolution reached a stable level, and then the increase in the oxygen concentration was measured during the next 10 min. For each experimental measurement, the result was corrected for density of viable cells and expressed as a percentage of controls (100%). 2.5. Photosynthetic pigments content Photosynthetic pigment contents were extracted in 100% chilled methanol in dark. Absorbance was measured at 470, 653 and 666 nm. The content of chl pigments (chl a and b) and total carotenoids was calculated respectively using the Wellburn (1994) extinction coefficient equations. For each experimental measurement, the result was corrected for density of viable cells and expressed as a percentage of controls (100%). 2.6. Chlorophyll a fluorescence measurements A Plant Efficiency Analyser fluorometer (PEA, Hansatech Ltd., UK) was used to measure the rapid and polyphasic chl a fluorescence emission (JIP-test) according to Strasser et al. (1995). All samples were dark-adapted for 15 min before measurement. Approximately 3 × 106 algal cells were concentrated on 13 mm glass fiber filter (#AP20 013 00 Millipore). Filtration under low pressure did not induce additional physiological stress for algae, which may affect measurements. The PEA saturating flash was provided by an array of six light-emitting diodes with an excitation light of 3000 ␮mol photons m−2 s−1 at 650 nm. Diodes were focused on the sample surface to provide homogeneous illumination over an area of 4 mm in diameter. At room temperature, the fast fluorescence kinetics were measured in a time span from 50 ␮s to 1 s, with data acquisition every 10 ␮s. Each transient was analysed using the following data: (1) the minimal fluorescence yield F0 at 50 ␮s when all reaction centers (RCs) are open, (2) the maximal fluorescence yield FM (closed RC), (3) the fluorescence intensity F300 at 300 ␮s, (4) the fluorescence intensity FJ at the Jstep (2 ms), (5) the fluorescence intensity FI at the I-step (30 ms) and (6) area, total complementary area between fluorescence induction curve and FM . To visualize the effect of dendrimers on each step of the fluorescence transient, the curves were plotted as the relative variable fluorescence at any time (Vt) on a logarithmic time scale. From these data, the following fluorescence parameters were evaluated (Strasser et al., 2000; Appenroth et al., 2001; TsimilliMichael and Strasser, 2008): • the initial slope of the relative variable fluorescence corresponding to the rate of the primary quinone acceptor (QA ) reduction in the first ms, M0 = 4(F300 − F0 )/(FM − F0 ), • the relative variable fluorescence at the J-step, VJ = (FJ − F0 )/ (FM − F0 ), • the maximum quantum yield of PSII for primary photochemistry, P0 = FV /FM = (FM − F0 )/FM , • the quantum yield of electron transport, E0 = P0 ×  0 , • the efficiency/probability that a trapped exciton moves an electron into the transport chain further than QA − ,  0 = 1 − VJ , • the efficiency/probability that an exciton moves the reduced plastoquinone (PQ) to the photosystem I (PSI) end electron acceptors, RE0 /ET0 = (FM − FI )/(FM − FJ ), • the specific energy fluxes (per reaction center, RC): for absorption, ABS/RC = M0 (1/VJ )(1/P0 ), trapping, TR0 /RC = M0 /VJ , electron transport, ET0 /RC = (M0 /VJ ) ×  0 , and dissipation, DI0 /RC = ABS/RC − TR0 /RC, • the amount of active reaction centers per absorption (arbitrary units; proportional to the probability that a PSII

189

antenna chlorophyll a molecule is functioning as RC), RC/ABS = [(1 − F0 /FM )(FJ − F0 )]/[4(F300 − F0 )], • the performance indexes, PIABS = (RC/ABS) × (P0 /(1 − P0 )) × ( 0 /(1 −  0 )) and PITOT = PIABS [RE0 /(ET0 − RE0 )], expressing “potentials” for photosynthetic performance at the sequential energy bifurcations from exciton to PQ reduction and to the reduction of PSI end acceptors, respectively. Results were expressed as a percentage of controls (100%). 2.7. Statistical analyses Descriptive analyses were carried out to express the results and calculate the mean and the standard deviation. All experiments were carried out in triplicate and data were statistically analysed by a one-way analysis of variance (ANOVA). When significant differences were observed, means were compared using Tukey’s test. Significant differences at the significance P-level of 0.05 are represented by an asterisk (*). These analyses were carried out with Statistica® Software v7.0 (StatSoft). A plot of sample concentration versus the percentage of viability was used to perform a regression analysis in the linear portion of the graph to determine the median inhibitory concentration (IC50 ) and its 95% confidence intervals. These parameters were calculated using a logistic curve-fitting procedure according to the method described by Vindimian et al. (1999). These data were obtained using REGTOX, a Microsoft Excel® spreadsheet (http://eric.vindimian.9online.fr/). The Kendall tau rank correlation coefficient was calculated between the characteristics of the dendrimers (number of cations, molecular weight and diameter) and their physiological effects on C. reinhardtii (viability and fluorescence parameters). 3. Results 3.1. Effects of PAMAM dendrimers on viability of C. reinhardtii The effects of G2, G4 and G5 PAMAM dendrimers on the viability of C. reinhardtii were determined by flow cytometry (Fig. 1). Unexposed cells grown for 72 h in the HSM medium had viability ranging between 94 and 97%. A dose-dependent decline in cell viability was found after exposure to the dendrimers. At concentrations below 1 mg L−1 of PAMAM dendrimers, no significant increase of mortal-

Fig. 1. Cell viability (esterase activity) of Chlamydomonas reinhardtii cultures after 72 h of exposure to different concentrations of PAMAM dendrimer (G2, G4 and G5). All data were presented as the percent of viable cells with respect to the total amount of cells analysed by flow cytometry. Data are means ± S.D. (n = 3). Significant differences at P < 0.05 between control and treated cultures are marked by asterisks.

190

A.-N. Petit et al. / Aquatic Toxicology 100 (2010) 187–193

Table 2 IC50 values of PAMAM dendrimers for cell viability in Chlamydomonas reinhardtii, 72 h after treatment. Dendrimer

IC50 (mg L−1 )

Confidence intervals

G2 G4 G5

1.940 2.970 4.820

1.800 2.560 4.560

ity was observed in algal cells after 72 h of treatment. At 2.5 mg L−1 , loss of viability was close to 68% for G2, 38% for G4 and 7% for G5, in comparison with the control. PAMAM dendrimers applied at the highest concentration (10 mg L−1 ) induced algicidal effects characterized by a drastic viability loss of 99%, 98% and 95% for G2, G4 and G5 dendrimers, respectively. IC50 values obtained after exposure of C. reinhardtii for 72 h to these dendrimers are shown in Table 2. The IC50 was 1.940 (590 nM), 2.970 (208 nM) and 4.820 mg L−1 (167 nM) for PAMAM G2, G4 and G5, respectively. These endpoint values were significantly correlated with the dendrimer diameter (Kendall tau; P < 0.05). Cell mortality was most pronounced after treatment at the highest concentration (10 mg L−1 ). Therefore, at this concentration, photosynthetic parameters could not be reliably measured owing to a low viable population density. 3.2. Photosynthetic responses of C. reinhardtii to PAMAM dendrimers 3.2.1. Influence of PAMAM dendrimers to O2 evolution Algal exposure to the G5 dendrimer did not affect photosynthetic oxygen evolution while it increased with exposure to G2 and G4 dendrimers (Fig. 2.) Indeed, O2 evolution was significantly increased at concentrations below 1 mg L−1 of G4 dendrimer by 155% compared to the control. Oxygen evolution of algae exposed to 2.5 mg L−1 showed a further increase of 350% for G2 and 625% for G4.

2.070 3.370 5.060

IC50 (nM)

Confidence intervals

590 208 167

594 181 159

547 235 174

Total chl and carotenoids were not significantly affected by G4 and G5 dendrimers. However, chl content was significantly increased by 121% compared to the control at a G2 concentration of 2.5 mg L−1 . 3.2.3. Chlorophyll a fluorescence emission The polyphasic fast fluorescence induction test was performed in order to examine the effect of PAMAM dendrimers on the function of PSII. Fluorescence emission kinetics were affected in algae exposed to dendrimers as shown in Fig. 4. Chl a fluorescence transient of control samples exhibited a characteristic O–J–I–P shape composed of four clear transients while, following exposure to 1 mg L−1 of G2 and 2.5 mg L−1 of G4, visible changes in the fluorescence curves occurred (Fig. 4). The initial slope of the fluorescence curve (M0 ) and the fluorescence level at step J (VJ ) were decreased. Values of fluorescence parameters resulting from the shape changes of the O–J–I–P curves have been analysed and the results obtained are presented in Table 3. Only significant differences have been observed following exposure of G2 dendrimer at 1 mg L−1 and G4 at 2.5 mg L−1 . Therefore, only these data will be described. The initial slope (M0 ) at the origin of the fluorescence rise (measured between 50 and 300 ␮s), as well as the fluores-

3.2.2. Effects of PAMAM dendrimers on photosynthetic pigments content The pigment content of algal cells was determined by spectrophotometry after 72 h-exposure to PAMAM dendrimers (Fig. 3).

Fig. 2. Influence of different concentrations of PAMAM dendrimer (G2, G4 and G5) on photosynthetic O2 evolution of Chlamydomonas reinhardtii cultures after 72 h of exposure. All data were presented as the percent of corresponding control samples. Data are means ± S.D. (n = 3). Significant differences at P < 0.05 between control and treated cultures are marked by asterisks.

Fig. 3. Influence of different concentrations of PAMAM dendrimer (G2, G4 and G5) on photosynthetic pigment content (total chlorophyll and carotenoids) of Chlamydomonas reinhardtii cultures after 72 h of exposure. All data were presented as the percent of corresponding control samples. Data are means ± S.D. (n = 3). Significant differences at P < 0.05 between control and treated cultures are marked by asterisks.

A.-N. Petit et al. / Aquatic Toxicology 100 (2010) 187–193

191

Table 3 Parameters obtained from JIP-test for Chlamydomonas reinhardtii cells exposed for 72 h to PAMAM dendrimers. Treatment (mg L−1 )

PIABS

PITOT

M0

VJ

E0

P0

0

ABS/RC

ET0 /RC

DI0 /RC

TR0 /RC

Control

100

100

100

100

100

100

100

100

100

100

100

G2 0.30 G2 1.00 G2 2.50

158 383* 441

152 425* 206

83 61* 80

95 80* 88

111 147* 122

104 116* 106

107 126* 116

86 67* 89

95 97 108

96 86* 95

89 77* 94

G4 0.30 G4 1.00 G4 2.50

100 173 810*

107 206* 297*

101 93 86*

103 97 92*

97 118 133*

100 113 120*

96 104 111*

98 85 78*

94 99 104

100 88 83*

98 96 93*

G5 0.30 G5 1.00 G5 2.50

131 92 13

146 90 67

85 104 120

95 104 110

105 96 90

101 100 99

104 96 91

88 100 111

93 97 99

99 100 102

89 101 109

All parameters were normalized with controls. Numbers are percentages of the control values. Significant differences at P < 0.05 between control and treated cultures are marked by asterisks.

cence level at step J (2 ms) were significantly decreased by 39% and 20%, respectively, in algae exposed to 1 mg L−1 of G2 dendrimer, compared to the control. Similarly, the yield of electron transport (E0 ), which is the product of the yield of primary electron transport (P0 ) and the yield of electron transport per trapped exciton ( 0 ), was increased by 47%. Changes of E0 were due more to an increase of  0 (26%) rather than to an increase of P0 (16%). G2 dendrimer at 1 mg L−1 also induced an ABS/RC decrease of 33% compared to the control. In parallel, value of DI0 /RC parameter was 14% lower than in the control. These changes result in an increase in the performance indexes, PIABS and PITOT . These indexes are the overall expression of all the processes in the energy cascade from energy absorption to the reduction of the intersystem electron transport chain and the PSI end electron acceptors (Strasser et al., 2007; Tsimilli-Michael and Strasser, 2008). G2 at 1 mg L−1 increased strongly the values of both indexes by about 300%. Values of fluorescence parameters were also modified after exposure to 2.5 mg L−1 of G4. Firstly, M0 and VJ were significantly decreased by 14% and 8%, respectively. E0 was increased by 33% but contrary to G2, this rise was more related to an increase of  0 (20%) than P0 (11%). Similarly to G2, G4 at 2.5 mg L−1 induced an ABS/RC and DI0 /RC decreases of 22% and 17%, respectively. Finally, PIABS and PITOT were increased by about 700% and 200%, respectively, after 2.5 mg L−1 of G4. A significant correlation was observed between the photosynthetic performance index PIABS and the number of cations per dendrimer (Kendall tau; P < 0.05). No significant differences of the fluorescence parameters were shown in G5-treated algae.

Fig. 4. Representative curves of fluorescence transients in untreated and treated with PAMAM dendrimers cultures of Chlamydomonas reinhardtii, 72 h after treatment. Fluorescence values were normalized to initial fluorescence for ease of comparison.

4. Discussion Because of the versatile applications envisaged for dendrimerbased technologies, the use of these ENPs is of increasing importance in the pharmaceutical and medical fields. It will likely lead to the release of dendrimers into the aquatic environment. Although studies have been carried out on the toxicology and health implications of dendrimers, information is lacking concerning their behaviour in the aquatic environment and their ecotoxicity. To evaluate the effects of PAMAM dendrimers into aquatic systems, we studied their toxicity on both the viability and the photosynthesis of the green microalgae C. reinhardtii. In our study, we showed that PAMAM dendrimers have the potential to cause toxicity in C. reinhardtii. The median inhibitory concentration (IC50 ) for cell viability was 2, 3 and 5 mg L−1 for G2, G4 and G5 dendrimers, respectively. According to the EU-Directive 93/67/EEC classification scheme (CEC, 1996), PAMAM dendrimers of the tested generations are classified in the “toxic” group (range of 1–10 mg L−1 ) toward C. reinhardtii. Previous studies have reported impacts of ENPs on primary producers (e.g. green microalgae C. reinhardtii, Desmodesmus subspicatus, Pseudokirchneriella subcapitata, and the marine diatom Thalassiosira weissflogii) but they were mostly focused on metallic nanoparticles (Hund-Rinke and Simon, 2006; Franklin et al., 2007; Warheit et al., 2007; Blaise et al., 2008; Griffitt et al., 2008; Navarro et al., 2008; Wang et al., 2008; Aruoja et al., 2009; Miao et al., 2009). As our results suggest, most of these ENPs were found to be toxic to phytoplankton. For example, the algal 72 h-IC50 values based on growth inhibition for the most frequently examined ENP, titanium dioxide (TiO2 ), were 16–21 mg L−1 for P. subcapitata (Warheit et al., 2007) and 44 mg/L for D. subspicatus (Hund-Rinke and Simon, 2006). The toxicity of nanometals, like silver (Navarro et al., 2008; Miao et al., 2009) and zinc nanoparticles (Franklin et al., 2007; Aruoja et al., 2009), appear to result, at least in part, from particle dissolution. The single study which evaluated the impacts of organic nanoparticles on algae concerned carbon-based materials (Blaise et al., 2008). It revealed that fullerene-C60 was not toxic for P. subcapitata while singlewalled carbon nanotube (SWCNT) appeared in the “toxic” category (IC25 = 1.04 mg L−1 ). Numerous studies highlighting the toxicity of ENPs on algae agree with our results showing that these organisms appear particularly sensitive to these new emerging contaminants. Due to their ecological position at the base of the aquatic food web and their essential role in nutrient cycling and oxygen production, microalgae are critical components to many ecosystems. Functional investigation of the effects of PAMAM dendrimers on photosynthesis was performed to elucidate the mechanism of dendrimers interference with the photosynthetic apparatus of C. reinhardtii. To our knowledge, it is the first time that these dendrimers effects were evaluated on algae. Our results showed

192

A.-N. Petit et al. / Aquatic Toxicology 100 (2010) 187–193

that the three different generations of dendrimers studied did not induce the same changes in the photosynthetic metabolism of the green alga. While G2 dendrimer at 2.5 mg L−1 and G4 at 1 and 2.5 mg L−1 stimulated the photosynthetic O2 evolution of C. reinhardtii cells, G5 had no significant effects on the same process. In addition, G2 at 2.5 mg L−1 significantly increased total chlorophyll content of C. reinhardtii. To further assess the site of action of PAMAM dendrimers on the photosynthetic machinery, chl a fluorescence transient was also analysed. From the chl fluorescence rise of C. reinhardtii cells, we observed that G2 at 1 mg L−1 and G4 at 2.5 mg L−1 dendrimer resulted in decreased M0 and VJ levels. These results indicated that G2 and G4 activated the primary charge separation and the electron transport between the primary (QA ) and the secondary (QB ) quinone acceptors (Strasser et al., 1995; Strasser, 1997). Using the JIP-test analysis, we confirmed these results since the yield of electron transport (E0 ) was stimulated and mostly related to a rise of  0 for G2 and an increase of P0 for G4. It indicates that dendrimers stimulated the electron transport and it was more due to dark reactions after QA − for G2 and light-dependent reactions for G4. Furthermore, our results showed that dendrimers may lead to an increase of the amount of active reaction centers (ABS/RC) resulting in a decrease of the effective dissipation per active RC (DI0 /RC). The positive effects of dendrimers G2 and G4 on the PSII reaction center activity was confirmed by the performance indexes (PIABS and PITOT ). These parameters, which can be used as overall measures of the potential of the plants for energy conversion (Strasser et al., 2007; Tsimilli-Michael and Strasser, 2008), were increased after treatment at 1 mg L−1 of G2 and 1 and 2.5 mg L−1 of G4 dendrimers. In addition, a rise in photosynthetic O2 evolution was observed in G2 and G4 dendrimer-treated cells. From our results, it is clear that dendrimers caused a raise of the amount of active PSII reaction centers, the primary charge separation and the electron transport between QA and QB increasing the yield of electron transport and photosynthetic O2 evolution. These results demonstrate that there are different targets of dendrimers on PSII. These changes finally result in stimulation of full photosynthetic performance. Similarly, previous studies have documented an increase photosynthesis and chlorophyll production of algae exposed to a chemical stress (Tang et al., 1997; Zhou et al., 2006; Porsbring et al., 2009). At low concentrations of toxic agents, this phenomenon, called hormesis, has also been shown to stimulate the growth of aquatic plants (Calabrese, 2005; Cedergreen et al., 2007). Here, the high concentration tested combined to a decrease of the viability of C. reinhardtii suggests that the stimulation of the photosynthesis could not be explained by a hormetic effect of dendrimers on the green alga. Other nanoparticles, TiO2 , have also been found to stimulate chlorophyll synthesis, Rubisco activity and photosynthetic rate in spinach (Zheng et al., 2005, 2007). Nevertheless, contrary to our results for dendrimers, TiO2 improved the growth of plants (Zheng et al., 2005). The negative impact of G2 and G4 dendrimers on cell viability of C. reinhardtii associated with their positive effects on photosynthesis may be related to the cationic nature of terminal groups of these dendrimers (Roberts et al., 1996; Malik et al., 2000). Indeed, several reports have demonstrated that cationic macromolecules such as PAMAM dendrimers can destabilize and permeabilize cell membranes. This effect allows for the diffusion of molecules in and out (dendroporation) but leads to cell lysis once the concentrations inside the cell are too high (Kozlova et al., 2001; Rittner et al., 2002; Fischer et al., 2003). However, in this study, the size of the dendrimers could also influence the toxic potency (expressed as nM-IC50 ) since larger size dendrimers were more toxic than the smaller size dendrimer. This is confirmed by the Kendall tau showing a significant correlation between the dendrimer diameter and the IC50 values for the viability (P < 0.05). Dendrimers also have some unique properties which make it possible to encapsu-

late guest molecules due to the presence of internal cavities and open conformations (for low-generation dendrimers) (Naylor et al., 1989) and to chemically attach or physically adsorb agents onto the dendrimer surface (Roberts et al., 1990). Moreover, it has been reported that cationic PAMAM dendrimers internalize into cells and induce the formation of transient, nanoscale holes in both supported lipid bilayers and cellular membranes allowing a greatly enhanced exchange of materials across the cell membrane (Hong et al., 2004; Mecke et al., 2004, 2005). Since a significant correlation was noticed between the number of cations per dendrimer and the photosynthetic performance index PIABS (P < 0.05), we may suggest that the presence of PAMAM dendrimers (dendropores) could enhance nutrient assimilation and thus increase photosynthetic performance of C. reinhardtii. Indeed, nutrient availability is one of the principal environment factors that affect phytoplankton photosynthesis (Droop, 1974; MacIntyre et al., 2000). Many nutrients are crucial for photosynthesis such as nitrogen, a key constituent of photosynthetic enzymes, especially in the protein Rubisco (Lambers et al., 1998) or phosphorus which is involved in photosynthetic CO2 assimilation (Marschner, 1995). Nevertheless, further increases in the concentration of dendrimers may destroy algal cell membranes leading to loss of membrane permeability and cell lysis as confirmed by the high cell mortality in dendrimers-treated cells at the highest concentration. The lowest toxicity (expressed as mg L−1 ) and the non-significant effects on the photosynthesis of G5-dendrimer could be explained by the lowest efficacy to enter into algal cells for this high generation dendrimer. Indeed, it was already observed that high generation dendrimers (G5–G10) appear to be less efficient at mediating transfection in certain cell types than smaller generations (Kukowska-Latallo et al., 1996). In summary, the present work showed that the green alga C. reinhardtii is a good model to assess the toxicity of novel therapeutic nanodevices. To our knowledge, this is the first report on toxicity of dendrimers toward microalgae. As observed in bacteria and mammalian systems, cationic PAMAM dendrimers are toxic to C. reinhardtii. Nevertheless, a stimulation of the photosynthetic process was noticed in algal cells after exposure to G2 at 2.5 mg L−1 and G4 at 1 and 2.5 mg L−1 . Acknowledgements This work was funded by the nanotechnology research and the Genomics Research & Development Initiative of Environment Canada. References Aitken, R.J., Chaudhry, M.Q., Boxall, A.B.A., Hull, M., 2006. Manufacture and use of nano-materials: current status in the UK and global trends. Occup. Med. (Lond.) 56, 300–306. Appenroth, K.J., Stockel, J., Srivastava, A., Strasser, R.J., 2001. Multiple effects of chromate on the photosynthetic apparatus of Spirodela polyrhiza as probed by OJIP chlorophyll a fluorescence measurements. Environ. Pollut. 115, 49–64. Aruoja, V., Dubourguier, H.C., Kasemets, K., Kahru, A., 2009. Toxicity of nanoparticles of CuO, ZnO and TiO2 to microalgae Pseudokirchneriella subcapitata. Sci. Total Environ. 407, 1461–1468. Bharali, D.J., Khalil, M., Gurbuz, M., Simone, T.M., Mousa, S.A., 2009. Nanoparticles and cancer therapy: a concise review with emphasis on dendrimers. Int. J. Nanomedicine 4, 1–7. Blaise, C., Ménard, L., 1998. A micro-algal solid-phase test to assess the toxic potential of freshwater sediments. Water Qual. Res. J. Can. 33, 133–151. Blaise, C., Gagné, F., Férard, J.F., Eullaffroy, P., 2008. Ecotoxicity of selected nanomaterial to aquatic organisms. Environ. Toxicol. 23, 591–598. Calabrese, E.J., 2005. Paradigm lost, paradigm found: the re-emergence of hormesis as a fundamental dose response model in the toxicological sciences. Environ. Pollut. 138, 379–412. Calabretta, M.K., Kumar, A., McDermott, A.M., Cai, C., 2007. Antibacterial activities of poly(amidoamine) dendrimers terminated with amino and poly(ethylene glycol) groups. Biomacromolecules 8, 1807–1811. CEC (Commission of the European Communities), 1996. Technical Guidance Document in Support of Commission Directive 93/67/EEC on Risk Assessment for New

A.-N. Petit et al. / Aquatic Toxicology 100 (2010) 187–193 Notified Substances. Part II. Environmental Risk Assessment. Office for Official Publications of the European Communities, Luxembourg. Cedergreen, N., Streibig, J.C., Kudsk, P., Mathiassen, S.K., Duke, S.O., 2007. The occurrence of hormesis in plants and algae. Dose Response 5, 150–162. Colvin, V.L., 2003. The potential environmental impact of engineered nanomaterials. Nat. Biotechnol. 21, 1166–1170. ´ Domanski, D.M., Klajnert, B., Bryszewska, M., 2004. Influence of PAMAM dendrimers on human red blood cells. Bioelectrochemistry 63, 189–191. Droop, M.R., 1974. The nutrient status of algal cells in continuous culture. J. Mar. Biol. Assoc. U.K. 54, 824–855. Duncan, R., Izzo, L., 2005. Dendrimer biocompatibility and toxicity. Adv. Drug Deliv. Rev. 57, 2215–2237. Eullaffroy, P., Frankart, C., Aziz, A., Couderchet, M., Blaise, C., 2009. Energy fluxes and driving forces for photosynthesis in Lemna minor exposed to herbicides. Aquat. Bot. 90, 172–178. Fischer, D., Li, Y., Ahlemeyer, B., Krieglstein, J., Kissel, T., 2003. In vitro cytotoxicity testing of polycations: influence of polymer structure on cell viability and hemolysis. Biomaterials 24, 1121–1131. Force, L., Critchley, C., van Rensen, J.J.S., 2003. New fluorescence parameters for monitoring photosynthesis in plants. Photosynth. Res. 78, 17–33. Franklin, N.M., Rogers, N.J., Apte, S.C., Batley, G.E., Gadd, G.E., Casey, P.S., 2007. Comparative toxicity of nanoparticulate ZnO, bulk ZnO, and ZnCl2 to a freshwater microalga (Pseudokirchneriella subcapitata): the importance of particle solubility. Environ. Sci. Technol. 41, 8484–8490. Franqueira, D., Orosa, M., Torres, E., Herrero, C., Cid, A., 2000. Potential use of flow cytometry in toxicity studies with microalgae. Sci. Total Environ. 247, 119–126. Griffitt, R.J., Luo, J., Gao, J., Bonzongo, J.C., Barber, D.S., 2008. Effects of particle composition and species on toxicity of metallic nanomaterials in aquatic organisms. Environ. Toxicol. Chem. 27, 1972–1978. Hadjoudja, S., Vignoles, C., Deluchat, V., Lenain, J.F., Le Jeune, A.H., Baudu, M., 2009. Short term copper toxicity on Microcystis aeruginosa and Chlorella vulgaris using flow cytometry. Aquat. Toxicol. 94, 255–264. Hong, S.P., Bielinska, A.U., Mecke, A., Keszler, B., Beals, J.L., Shi, X.Y., Balogh, L., Orr, B.G., Baker, J.R., Holl, M.M.B., 2004. Interaction of poly(amidoamine) dendrimers with supported lipid bilayers and cells: hole formation and the relation to transport. Bioconjugate Chem. 15, 774–782. Hund-Rinke, K., Simon, M., 2006. Ecotoxic effect of photocatalytic active nanoparticles TiO2 on algae and daphnids. Environ. Sci. Pollut. Res. 13, 225–232. King Heiden, T.C., Dengler, E., Kao, W.J., Heideman, W., Peterson, R.E., 2007. Developmental toxicity of low generation PAMAM dendrimers in zebrafish. Toxicol. Appl. Pharmacol. 225, 70–79. Kozlova, N.O., Bruskovskaya, I.B., Okuneva, I.B., Melik-Nubarov, N.S., Yaroslavov, A.A., Kabanov, V.A., Menger, F.M., 2001. Interaction of a cationic polymer with negatively charged proteoliposomes. BBA-Biomembr. 1514, 139–151. Kukowska-Latallo, J.F., Bielinska, A.U., Johnson, J., Spindler, R., Tomalia, D.A., Baker, J.R., 1996. Efficient transfer of genetic material into mammalian cells using Starburst polyamidoamine dendrimers. Proc. Natl. Acad. Sci. (U.S.A.) 93, 4897–4902. Lambers, H., Chapin, F.S., Pons, T.L., 1998. Plant Physiological Ecology. Springer, New York, 540 pp. MacIntyre, H.L., Kana, T.M., Geider, R.J., 2000. The effect of water motion on shortterm rates of photosynthesis by marine phytoplankton. Trends Plant Sci. 5, 12–17. Malik, N., Evagorou, E.G., Duncan, R., 1999. Dendrimer-platinate: a novel approach to cancer chemotherapy. Anti-Cancer Drugs 10, 767–776. Malik, N., Wiwattanapatapee, R., Klopsch, R., Lorenz, K., Frey, H., Weener, J.W., Meijer, E.W., Paulus, W., Duncan, R., 2000. Dendrimers: relationship between structure and biocompatibility in vitro, and preliminary studies on the biodistribution of 125I-labelled polyamidoamine dendrimers in vivo. J. Control. Release 65, 133–148. Marschner, H., 1995. Mineral Nutrition of Higher Plants. Academic Press, San Diego, 889 pp. Mecke, A., Uppuluri, S., Sassanella, T.M., Lee, D.K., Ramamoorthy, A., Baker, J.R., Orr, B.G., Banaszak Holl, M.M., 2004. Direct observation of lipid bilayer disruption by polyamidoamine dendrimers. Chem. Phys. Lipids 132, 3–14. Mecke, A., Majoros, I.J., Patri, A.K., Baker, J.R., Banaszak Holl, M.M., Orr, B.G., 2005. Lipid bilayer disruption by polycationic polymers: the roles of size and chemical functional group. Langmuir 21, 10348–10354. Miao, A.J., Schwehr, K.A., Xu, C., Zhang, S.J., Luo, Z., Quigg, A., Santschi, P.H., 2009. The algal toxicity of silver engineered nanoparticles and detoxification by exopolymeric substances. Environ. Pollut. 157, 3034–3041. Morlon, H., Fortin, C., Floriani, M., Adam, C., Garnier-Laplace, J., Boudou, A., 2005. Toxicity of selenite in the unicellular green alga (Chlamydomonas reinhardtii): comparison between effects at the population and sub-cellular level. Aquat. Toxicol. 73, 65–78. Mortimer, M., Kasemets, K., Heinlaan, M., Kurvet, I., Kahru, A., 2008. High throughput kinetic Vibrio fischeri bioluminescence inhibition assay for study of toxic effects of nanoparticles. Toxicol. In Vitro 22, 1412–1417. Navarro, E., Baun, A., Behra, R., Hartmann, N.B., Filser, J., Miao, A.J., Quigg, A., Santschi, P.H., Sigg, L., 2008. Environmental behavior and ecotoxicity of engineered nanoparticles to algae, plants, and fungi. Ecotoxicology 17, 372–386. Naylor, A.M., Goddard III, W.A., Kiefer, G.E., Tomalia, D.A., 1989. Starburst dendrimers 5. Molecular shape control. J. Am. Chem. Soc. 111, 2339–2341. Nel, A., Xia, T., Mädler, L., Li, N., 2006. Toxic potential of materials at the nanolevel. Science 311, 622–627. Pan, X., Deng, C., Zhang, D., Wang, J., Mu, G., Chen, Y., 2008. Toxic effects of amoxicillin on the photosystem II of Synechocystis sp. characterized

193

by a variety of in vivo chlorophyll fluorescence tests. Aquat. Toxicol. 89, 207–213. Papageorgiou, G.C., Tsimilli-Michael, M., Stamatakis, K., 2007. The fast and slow kinetics of chlorophyll a fluorescence induction in plants, algae and cyanobacteria: a viewpoint. Photosynth. Res. 94, 275–290. Porsbring, T., Blanck, H., Tjellström, H., Backhaus, T., 2009. Toxicity of the pharmaceutical clotrimazole to marine microalgal communities. Aquat. Toxicol. 91, 203–211. Reboud, X., 2002. Response of Chlamydomonas reinhardtii to herbicides: negative relationship between toxicity and water solubility across several herbicide families. Bull. Environ. Contam. Toxicol. 69, 554–561. Rioboo, C., O˘ıConnor, E.J., Prado, R., Herrero, C., Cid, A., 2009. Cell proliferation alterations in Chlorella cells under stress conditions. Aquat. Toxicol. 94, 229–237. Rittner, K., Benavente, A., Bompard-Sorlet, A., Heitz, F., Divita, G., Brasseur, R., Jacobs, E., 2002. New basic membrane-destabilizing peptides for plasmid-based gene delivery in vitro and in vivo. Mol. Ther. 5, 104–114. Roberts, J.C., Adams, Y.E., Tomalia, D., Mercer-Smith, J.A., Lavallee, D.K., 1990. Using Starbust dendrimers as linker molecules to radiolabel antibodies. Bioconjugate Chem. 1, 305–308. Roberts, J.C., Bhalgat, M.K., Zera, R.T., 1996. Preliminary biological evaluation of polyamidoamine (PAMAM) Starburst (TM) dendrimers. J. Biomed. Mater. Res. A 30, 53–65. Rodríguez, M.C., Barsanti, L., Passarelli, V., Evangelista, V., Conforti, V., Gualtieri, P., 2007. Effects of chromium on photosynthetic and photoreceptive apparatus of the alga Chlamydomonas reinhardtii. Environ. Res. 105, 234–239. Strasser, B.J., Strasser, R.J., 1995. Measuring fast fluorescence transients to address environmental questions: the JIP-test. In: Mathis, P. (Ed.), Photosynthesis: From Light to Biosphere. Kluwer Academic Publishers, Dordrecht, pp. 977–980. Strasser, R.J., Srivastava, A., Govindjee, 1995. Polyphasis chlorophyll a fluorescence transient in plant and cyanobacteria. Photochem. Photobiol. 61, 32–42. Strasser, B.J., 1997. Donor side capacity of photosystem II probed by chlorophyll a fluorescence transients. Photosynth. Res. 52, 147–155. Strasser, R.J., Srivastava, A., Tsimilli-Michael, M., 2000. The fluorescence transient as a tool to characterize and screen photosynthetic samples. In: Yunus, M., Pathre, U., Mohanty, P. (Eds.), Probing Photosynthesis: Mechanism, Regulation and Adaptation. Taylor & Francis, London, pp. 443–480. Strasser, R.J., Tsimilli-Michael, M., Dangre, D., Rai, M., 2007. Biophysical phenomics reveals functional building blocks of plants systems biology: a case study for the evaluation of mycorrhization with Piriformospora indica. In: Varma, A., Oelmuller, R. (Eds.), Advanced Techniques in Soil Biology. Soil Biology Series. Springer, Germany, pp. 220–221. Sueoka, N., Chiang, K.S., Kates, J.R., 1967. Deoxyribonucleic acid replication in meiosis of Chlamydomonas reinhardtii. I. Isotopic transfer experiments with a strain producing eight zoospores. J. Mol. Biol. 25, 44–67. Tang, J.X., Hoagland, K.D., Siegfried, B.D., 1997. Differential toxicity of atrazine to selected freshwater algae. Bull. Environ. Contam. Toxicol. 59, 631–637. Tomalia, D.A., 2005. Birth of a new macromolecular architecture: dendrimers as quantized building blocks for nanoscale synthetic polymer chemistry. Prog. Polym. Sci. 30, 294–324. Tomalia, D.A., Reyna, L.A., Svenson, S., 2007. Dendrimers as multi-purpose nanodevices for oncology drug delivery and diagnostic imaging. Biochem. Soc. Trans. 35, 61–67. Tsimilli-Michael, M., Strasser, R.J., 2008. In vivo assessment of plants’ vitality: applications in detecting and evaluating impacts of mycorrhization on host plants’. In: Varma, A. (Ed.), Mycorrhiza: State of the Art, Genetics and Molecular Biology, Eco-function, Biotechnology, Eco-physiology, Structure and Systematics, 3rd ed. Springer, pp. 679–703. Vindimian, E., Garric, J., Flammarion, P., Thybaud, E., Babut, M., 1999. An index of effluent aquatic toxicity designed by partial least squares regression, using acute and chronic tests and expert judgements. Environ. Toxicol. Chem. 18, 2386–2391. Wang, J., Zhang, X., Chen, Y., Sommerfeld, M., Hu, Q., 2008. Toxicity assessment of manufactured nanomaterials using the unicellular green alga Chlamydomonas reinhardtii. Chemosphere 73, 1121–1128. Wang, W.X., Dei, R.C.H., 2006. Metal stoichiometry in predicting Cd and Cu toxicity to a freshwater green alga Chlamydomonas reinhardtii. Environ. Pollut. 142, 303–312. Wang, X.D., Liu, X.J., Yang, S., Li, A.L., Yang, Y.L., 2007. Removal and toxicological response of triazophos by Chlamydomonas reinhardtii. Bull. Environ. Contam. Toxicol. 78, 67–71. Warheit, D.B., Hoke, R.A., Finlay, C., Donner, E.M., Reed, K.L., Sayes, C.M., 2007. Development of a base set of toxicity tests using ultrafine TiO2 particles as a component of nanoparticle risk management. Toxicol. Lett. 171, 99–110. Wellburn, A.R., 1994. The spectral determination of chlorophylls a and b as well as total carotenoids, using various solvents with spectrophotometers of different resolution. J. Plant Physiol. 144, 307–313. Yang, H., Kao, W.J., 2006. Dendrimers for pharmaceutical and biomedical applications. J. Biomater. Sci., Polym. Ed. 17, 3–19. Zheng, L., Hong, F.S., Lu, S.P., Liu, C., 2005. Effect of nano-TiO2 on strength of naturally and growth aged seeds of spinach. Biol. Trace Elem. Res. 104, 83–91. Zheng, L., Su, M., Liu, C., Chen, L., Huang, H., Wu, X., Liu, X., Yang, F., Gao, F., Hong, F., 2007. Effects of nanoanatase TiO2 on photosynthesis of spinach chloroplasts under different light illumination. Biol. Trace Elem. Res. 119, 68–76. Zhou, W., Juneau, P., Qiu, B., 2006. Growth and photosynthetic responses of the bloom-forming cyanobacterium Microcystis aeruginosa to elevated levels of cadmium. Chemosphere 65, 1739–1746.