Effects of Imidazolium Ionic Liquids on Growth, Photosynthetic

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Effects of Imidazolium Ionic Liquids on Growth, Photosynthetic Efficiency, and Cellular Components of the Diatoms Skeletonema marinoi and Phaeodactylum tricornutum Chiara Samorì,*,† Giorgia Sciutto,‡ Laura Pezzolesi,† Paola Galletti,† Franca Guerrini,† Rocco Mazzeo,‡ Rossella Pistocchi,† Silvia Prati,‡ and Emilio Tagliavini† †

Interdepartmental Research Centre for Environmental Sciences (CIRSA), University of Bologna, via S. Alberto 163, 48123 Ravenna, Italy ‡ M2ADL-Microchemistry and Microscopy Art Diagnostic Laboratory, University of Bologna, via Tombesi dall'Ova 55, 48123 Ravenna, Italy ABSTRACT: This article describes the toxic effects of imidazolium ionic liquids bearing alkyl (BMIM), monoethoxy (MOEMIM), and diethoxy (M(OE)2MIM) side chains toward two marine diatoms, Skeletonema marinoi and Phaeodactylum tricornutum. MOEMIM and M(OE)2MIM cations showed a lower inhibition of growth and photosynthetic efficiency with respect to their alkyl counterpart, with both algal species. However, a large difference in sensitivity was found between S. marinoi and P. tricornutum, the first being much more sensitive to the action of ionic liquids than the second one. The effects of salinity on BMIM Cl toxicity toward S. marinoi revealed that a decrease from salinity 35 to salinity 15 does not influence the biological effects toward the alga. Finally, Fourier transform infrared (FT-IR) microscopy of algal cells after ionic liquids exposure allowed us to detect an alteration of the organic cellular components related to silica uptake and organization. On the basis of these results, the different behavior of the two diatom species can be tentatively ascribed to different silica uptake and organization in outer cell walls.

’ INTRODUCTION Ionic liquids (ILs) are the most studied class of alternative green solvents during the last two decades, from a technological point of view, as well as from sustainability and environmental concerns.1,2 Although the low volatility limits their release in the atmosphere and their environmental impact, the solubility in water of many ILs is very high, and in view of the growing industrial interest, concern is rising for their eventual release in the environment through industrial effluents and the consequent contamination of aquatic ecosystems. Moreover, as demonstrated in the large number of (eco)-toxicological studies of the last years,3,4 many ILs are very toxic substances at different levels of biological organization and against a variety of biological targets. The mechanism of IL toxicity is still not well understood, and probably more than a single physiological system can be affected. All the (eco)-toxicological studies have assessed that it is strictly correlated with the lipophilicity of the ion pair, affording a basic toxicity which causes a nonspecific disturbance of the functioning of biological membranes. Recent studies have also demonstrated that ILs exhibit adsorption behavior even through ionic interactions between the negative charged groups on cell surfaces and the IL cations.5 r 2011 American Chemical Society

Algae are very important organisms for (eco)-toxicological assessment because they are a key part of aquatic ecosystems, playing the role of primary producers and providing energy to sustain higher trophic levels; moreover, algal tests are generally sensitive, rapid, and cost-effective. For these reasons, algal doseresponse bioassays have become widely used as biological tools in environmental impact studies. Among marine algae, diatoms are the most used test organisms for their sensitivity to metals and organic compounds. Thus far, the toxicity of ILs toward microalgae has been performed by using mainly green freshwater algal species6-18 and more recently, seawater diatoms and cyanobacteria.5,11,19 These studies have revealed that the toxicity of ILs is correlated to the structure of algal cell walls, known to play a critical role in the transport of materials, including toxins, in and out of the cell. Diatoms, in particular, show many negatively charged silica-associated organic components, as extracellular polysaccharides, proteins (frustulins, pleuralins, and silaffins), and long chain polyamines, and thanks to these functional groups on the cell surface, these algae are very good Received: October 5, 2010 Published: February 25, 2011 392

dx.doi.org/10.1021/tx100343p | Chem. Res. Toxicol. 2011, 24, 392–401

Chemical Research in Toxicology

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chlorophyll fluorescence through the pulse amplitude modulation (PAM) fluorometric method, were measured as end points. Chlorophyll fluorescence analysis was never used before as response to investigate the toxic effects of ILs on algae, but it is a very useful tool to study in vivo the functional properties of photosynthetic apparatus, being a nondestructive, versatile, highly sensitive, and accurate technique widely applicable to a vast number of toxicants.36,32 This technique measures the portion of the adsorbed light energy which is not used for photochemical transformations by algae and plants and thus is dissipated as heat or as light re-emission (fluorescence), mainly emitted as chlorophyll a. In the past decades, there have been considerable progress in practical applications of chlorophyll fluorometry following the introduction of the PAM fluorometric method,37,33 enabling one to provide information about the quantum yield, a parameter proportional to the photosynthetic efficiency of the photosystem II and whose inhibition can be a measure for sublethal toxic responses. Moreover, exploiting the method developed by Cornmell et al.,23 we have evaluated IL interaction with algal cellular components, by using microscopy-coupled transmission fourier transform infrared spectroscopy (FT-IR) in combination with a multivariate analysis approach (Principal Components Analysis, PCA); in fact, any possible interaction between IL and cellular components should be recorded in the IR spectra as a change in intensity or frequency of IR bands of either IL or cellular constituents.

Chart 1. Structures and Names of the Tested ILs

biosorbents for positively charged chemicals and toxicants as heavy metals and ILs. Moreover, the composition of the abiotic environment and its variations are important parameters. Salinity, for example, could be one of the crucial factors in the mitigation of the biological effects toward microalgae; in fact, high concentrations of inorganic anions and cations from highly saline environments could potentially reduce the permeability of ionic liquid cations through algal cell walls.11 In the present study, we evaluated the biological effects of a set of imidazolium-based ILs toward two diatoms, Skeletonema marinoi and Phaeodactylum tricornutum, possessing differently featured cell walls. The ionic liquids were chosen on the basis of our previous works 20,21 and literature data4,16 in which it has been reported that the introduction of ether-oxygen atoms in the lateral chain of imidazolium-based ILs strongly reduced the toxicity toward different biological targets. In the present work, the test kit of compounds consisted of three imidazolium chlorides (Chart 1), one bearing an alkyl lateral chain (1-butyl3-methylimidazolium chloride, BMIM Cl) and two having an alkoxy lateral chain (1-methoxyethyl-3-methylimidazolium chloride, MOEMIM Cl, and 1-(2-(2-methoxy-ethoxy)-ethyl)3-methylimidazolium chloride, M(OE)2MIM Cl). Our aim was to check the influence of the presence of ethoxy units on algal toxicity, contributing to a further step into the elucidation of ILs' structure-activity relationship. Concerning the choice of the algae, S. marinoi was recommended as a standard test organism due to its ecological relevance in the ocean and high sensitivity to pollutants. We used a strain typical of the Adriatic Sea (salinity around 35) and also checked the effect of lower salinities (25 and 15), well tolerated by the alga, on the toxicity of BMIM Cl. P. tricornutum has never been used before in ILs toxicity assays; however, it is a good target for checking possible toxic effects on the algal cell wall. In contrast to other diatoms that typically have silicified cell walls, P. tricornutum possesses an organic cell wall and does not require silica for its growth. When silica is available, specific morphotypes (oval, fusiform and triradiate) with different silica content in their cell walls can exist; among them, only the oval type has the typical silica valve of diatoms.22 Algal growth inhibition, evaluated in a 72 h growth inhibition assay, and photosynthetic algal activity, evaluated by measuring

’ MATERIALS AND METHODS Chemicals. All the chemicals were purchased from Aldrich; 1-methylimidazole, 1-chlorobutane, and 2-chloroethyl methyl ether were redistilled before use to limit the formation of colored impurities. The purities of all the ionic liquids were established to be g98% through proton nuclear magnetic resonance (1H NMR) spectroscopy by integration of proton signals with respect to an internal standard. All spectra were recorded using a 5-mm probe on a Varian Inova 400 spectrometer (Varian, Palo Alto, CA, USA) at 400 MHz. All spectral data were acquired in acetone-d6, D2O or CDCl3 with a known amount of tetrakis(trimethylsilyl)silane as an internal standard and a delay time between successive scans of 20 s to ensure complete proton relaxation. 2-(2-Methoxy-ethoxy)-ethyl chloride, BMIM Cl, MOEMIM Cl, and M(OE)2MIM Cl were synthesized according to the procedures reported in the literature.24,25 The 1H NMR and 13C NMR spectra were in agreement with the literature data. Test Organisms. Skeletonema marinoi (CCMP2497) used in the present study was obtained from the Provasoli-Guillard National Centre for Culture of Marine Phytoplankton, Bigelow, ME, whereas Phaeodactylum tricornutum (PTN0301) was kindly donated by Dr. J.W. Rijstenbil, the Netherland Institute of Ecology (NIOO-KNAW). The original strains of S. marinoi and P. tricornutum were isolated from the northern Adriatic Sea and from the North Sea, respectively. Cultures were maintained in 500 cm3 Erlenmeyer flasks containing 200 cm3 of sterilized f/2 medium, prepared according to the literature from seawater26 with the addition of silica (final concentration of 0.1 mM). The major part of the experiments was conducted at salinity 35, similar to that of the northern Adriatic Sea. The culture flasks were illuminated at 100-110 μmol photons m-2 s-1 from daylight type cool white lamps (16:8 h light-dark period), with the temperature controlled at 20 °C. Growth Inhibition Tests. The IL toxicity tests were conducted according to the OECD guidelines (2006)27 with slight modifications regarding the use of f/2 medium and the choice of the test strain. 393

dx.doi.org/10.1021/tx100343p |Chem. Res. Toxicol. 2011, 24, 392–401


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Figure 2. Dose-response relationship plotted for Phaeodactylum tricornutum exposed to BMIM Cl. The response (absorbance) was expressed as percentage of the control (normalized response), considering the control value as 100%. Error bars correspond to standard deviations of the experimental data.

Modulated Fluorescence Measurements. Chlorophyll fluorescence of photosystem II of the microalgae treated with different concentrations of ILs was evaluated with a PAM fluorometer. The model used was a 101-PAM connected to a PDA-100 data acquisition system, high power LED Lamp Control unit HPL-C, and LED-Array-Cone HPL-470 to supply actinic light and saturating pulses, and US-L665 to supply measuring light (H. Walz, Effeltrich, Germany). Algal samples were analyzed in cuvettes (10 10 mm) mounted on an optical unit ED-101US/M. The minimum constant fluorescence level of darkadapted and light-adapted cells (F0 and F0 0, respectively) was measured by using a low light intensity (1 μmol photons m-2 s-1). Maximum fluorescence level (Fm) was induced by a short saturating pulse of >3000 μmol photons m-2 s-1 for 0.8 s. Maximum fluorescence level under actinic light (F0 m) was measured by giving a saturating pulse after 5 min of illumination with actinic light of intensity similar to the one used for the growth of the algae. This was done to avoid photoinhibitory effects and to provide optimal conditions for photosynthetic activity. The length of illumination was chosen after verifying that the steady-state of electron transport was reached. The maximum and the effective quantum yields, respectively, Y(II) and Y0 (II), were calculated as follows:28,29

Figure 1. Dose-response relationships plotted for Skeletonema marinoi exposed to BMIM Cl (a), MOEMIM Cl (b) and M(OE)2MIM Cl (c). The response (absorbance) was expressed as percentage of the control (normalized response), considering the control value as 100%. Error bars correspond to standard deviations of the experimental data. Experiments were conducted in 100 cm3 sterile Erlenmeyer flasks, sealed with cotton, containing 66.5 cm3 of sterilized f/2 medium enriched with silica to which 7.5 cm3 of algal suspension in the log growth phase and 1 cm3 of different concentrations of an aqueous IL solution or f/2 medium (control cultures) were added. The initial optical density (OD) at 750 nm, measure of the cell number was 0.02 for both species, in each experiment. Two replicates were set up for each treatment concentration and for the control. Test concentrations, identified through a preliminary range-finding test, were arranged in a geometric series ranging from 1.0 mM to 8.0 mM for BMIM Cl, from 8.0 mM to 20 mM for MOEMIM Cl and M(OE)2MIM Cl (P. tricornutum), from 0.021 mM to 0.350 mM for BMIM Cl, from 0.7 mM to 4.0 mM for MOEMIM Cl, and from 4.0 mM to 8.0 mM for M(OE)2MIM Cl (S. marinoi). Each flask was placed at 20 °C and illuminated at 100-110 μmol photons m-2 s-1 from daylight type cool white lamps (16:8 h light-dark period); after 72 h of incubation, the number of cells in the cultures was counted by OD measurement at 750 nm, using a spectrophotometer (UV/vis, JASCO 7800, Tokyo, Japan). Salinity Experiments. The S. marinoi strain used in the present study was maintained at salinity 35. To perform the experiments at lower salinities, we followed a pattern of decreasing salinity by 5 from 35 to15, diluting with deionized water to obtain each specific salinity and acclimatizing the algal cells for 15 days to each condition before changing the salinity again.

YðIIÞ ¼ ðF m - F 0 Þ=F m

Y 0 ðIIÞ ¼ ðF 0m - F 00 Þ=F0m

Data Analysis. The 50% effect concentration (EC50) of each substance for the growth inhibition tests, the salinity experiments, and the evaluation of chlorophyll fluorescence inhibition was estimated by fitting the experimental concentration-response curves to a logistic model: ðtop - botÞ y ¼ bot þ ð1 þ ðx=EC50 Þc Þ where y = end point value; x = substance concentration; bot = expected end point value when the concentration of the toxicant is infinite (bottom asymptote); top = expected end point value when the concentration of the toxicant is zero (top asymptote); and c = slope parameter. The parameters of the equation, including the EC50, were estimated using the nonlinear regression procedures implemented in Statistica (Statsoft, Tulsa, OK, USA). Figures 1-5 were fit to this equation. 394



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Figure 4. Maximum and effective quantum yield (Y(II) and Y 0 (II)) inhibition of Skeletonema marinoi after 72 h of exposure to BMIM Cl (a), MOEMIM Cl (b), and M(OE)2MIM Cl (c). The response (absorbance) was expressed as the percentage of the control (normalized response), considering the control value as 100%.

Figure 3. Dose-response relationships plotted for Skeletonema marinoi exposed to BMIM Cl at salinity 35 (a), 25 (b) and 15 (c). The response (absorbance) was expressed as percentage of the control (normalized response), considering the control value as 100%. Error bars correspond to standard deviations of the experimental data. The differences in the EC50 values were evaluated by using each experimental replicate by itself and then comparing these replicates by an ANOVA method. The assumption of homogeneity of variances was tested by Cochran’s C test. Whenever a significant difference for the main effect was observed (P < 0.05), a Newman-Keuls test was also performed. FT-IR Spectroscopy. For the FT-IR analysis, S. marinoi cells were cultured in the presence of BMIM Cl at 0.1 mM (around the EC50 value) and 0.3 mM (above the EC50 value, at which no algal growth was observed) concentrations. P. tricornutum cells were cultured in the presence of BMIM Cl at a concentration of 1.9 mM (around the EC50 value) and in the presence of MOEMIM Cl at a concentration of 20 mM (at which concentration no growth inhibition was observed). Control and sample cultures were prepared in duplicate according to the procedure described above, and after 72 h of exposure to ILs, cells were collected by centrifugation at 3000 rpm for 10 min, washed with a physiological saline solution, and centrifuged again 3 times.23 Aliquots (1 μL) of cell suspensions and of each IL were applied on a gold mirror, and μFTIR spectra were recorded in reflection-absorption (RAS) mode, by using a Nicolet iN10MX FTIR microscope fitted with a MCT detector, cooled with liquid nitrogen. Spectra were acquired at a resolution of 4 cm-1 (16 spectra were averaged to improve the signal-to-noise ratio) and elaborated with OMNIC Picta software. The chemometric package V-PARVUS was used for PCA.30 The matrix data set was composed with intensity values of peaks as variables (columns) and spectra as objects (rows).

Figure 5. Maximum and effective quantum yields (Y(II) and Y0 (II)) inhibition of Phaeodactylum tricornutum after 72 h of exposure to BMIM Cl. The response (absorbance) was expressed as the percentage of the control (normalized response), considering the control value as 100%. The second derivative has been applied as a row pretreatment on the matrix data set. Second derivatives were performed with third-order smoothing polynomials through 15 points. 395



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Table 2. Influence of Salinity on BMIM Cl Toxicity Expressed As Skeletonema marinoi Growth Inhibitiona

Table 1. Influence of ILs on the Growth of Skeletonema marinoi and Phaeodactylum tricornutuma EC50 ( SE (mM) IL BMIM Clb MOEMIM Clc M(OE)2MIM Cl

d

Skeletonema marinoi

Phaeodactylum tricornutum

0.12 ( 0.01

1.26 ( 0.06

2.98 ( 0.08

>20

4.64 ( 0.16

>20

salinity

EC50 ( SE (mM)

35

0.12 ( 0.01

25 15

0.10 ( 0.01 0.14 ( 0.02

The 50% effect concentrations EC50 are expressed in mM, as EC50 ( standard error (SE). a

a The 50% effect concentrations EC50 are expressed in mM, as EC50 ( standard error (SE). b1-Butyl-3-methylimidazolium chloride. c1-Methoxyethyl-3-methylimidazolium chloride. d1-(2-(2-Methoxyethoxy)ethyl)3-methylimidazolium chloride.

A suggestion for the strong difference in sensitivity among the two diatoms could rely on the fact that P. tricornutum presents some structural differences with respect to S. marinoi. First, P. tricornutum does not require silica for its growth, and it is generally observed in cultures and in natural waters as a fusiform morphotype, the most stable of the three. This morphotype, used in the present study, typically contains silica (0.4-0.5% dry weight) in the form of irregular particles, clearly organized in bands in the girdle region of the wall, and does not form the typical frustul valves of diatoms.22 Second, P. tricornutum presents some differences with respect to S. marinoi in the organic negatively charged components on the cell surface, involved in silicon uptake and formation of the cell wall, especially in protein content and typology.31 We hypothesized that ILs can interact with functional groups on the algal cell surface and give different responses according to different silica organization, uptake and polymerization of the two species. This interaction could affect the growth of diatoms, such as S. marinoi, which need silica for the formation of valves, but it could be much less relevant for other diatoms, such as P. tricornutum, that lack such biological structures and are even able to survive with a reduced amount of silica (Si/C ratio for P. tricornutum is 0.011332 with respect to a ratio of 0.46 for S. marinoi33). Moreover, it is known that silica is taken up by marine diatoms from seawater in the form of the anion SiO(OH)3-.34 We can hypothesize that IL cations could act as scavengers for this anion species preventing the uptake by cells, with a mechanism similar to what was proposed by Lataza et al.11 to explain the effects of salinity on IL toxicity. Since P. tricornutum does not need silica for growing, the effects of this scavenging could be less pronounced than for S. marinoi. Influence of Salinity Variations on IL Toxicity. The impact of three different salinities (35, 25, and 15) on the biological effects of BMIM Cl against the growth of S. marinoi was also investigated. Table 2 shows the influence of salinity on the growth inhibition of S. marinoi exposed to BMIM Cl with respect to the control sample. In Figure 3, the growth inhibition of S. marinoi after 72 h of exposure to different concentrations of BMIM Cl at salinity 35 (Figure 3a), 25 (Figure 3b), and 15 (Figure 3c) is shown. The EC50 values at salinity 35, 25, and 15 were very similar, and their differences were not statistically significant (ANOVA, P > 0.05), indicating that different salinities did not give rise to different biological effects of the ionic liquid. Since the alga was adapted to each salinity for two weeks prior to toxicological experiments, after that the cell yields in the three cultures were similar (absorbance values of 0.152 at salinity 35, 0.103 at salinity 25, and 0.104 at salinity 15), we could assume a good adaptation of S. marinoi to each salinity regime and reasonably exclude a stress in cell growth due to changes in the environmental conditions. Moreover, the EC50 value obtained for S. marinoi

’ RESULTS AND DISCUSSION Growth Inhibition Assay. The effects of ILs on the growth of S. marinoi and P. tricornutum were estimated with respect to control samples (normalized response). In the case of P. tricornutum, cultures were composed essentially by the fusiform morphotype with only less than 1% of the triradiate morphotype. In Figure 1, the plots of growth inhibition of S. marinoi after 72 h of exposure to different concentrations of BMIM Cl (Figure 1a), MOEMIM Cl (Figure 1b), and M(OE)2MIM Cl (Figure 1c) are shown; in Figure 2, the plot of growth inhibition of P. tricornutum after 72 h of exposure to different concentrations of BMIM Cl is shown. The dose-response curves performed with BMIM Cl gave a parallel pattern for S. marinoi and P. tricornutum, although with different concentration ranges (0.02-0.35 mM for S. marinoi and 1.0-8.0 mM for P. tricornutum). In the case of the two etherfunctionalized ILs, tested with S. marinoi, the pattern of algal growth inhibition was similar and followed a typical sigmoid curve; P. tricornutum had no growth inhibition with etherfunctionalized IL up to 20 mM, as shown by the EC50 values measured after 72 h of exposure and listed in Table 1. As shown in Table 1, the EC50 values obtained for S. marinoi ranged from 0.12 to 4.64 mM, indicating BMIM Cl as the most toxic among the tested ILs and M(OE)2MIM Cl as the least. Also for P. tricornutum, the most toxic compound was BMIM Cl (EC50 value 1.26 ( 0.06 mM), although the sensitivity of this alga was one order of magnitude lower than that of S. marinoi, and the differences in the two EC50 values were statistically significant (ANOVA, P < 0.001); the EC50 values for both ether-functionalized ILs were higher than 20 mM, the maximum tested concentration with no effects on algal growth. It was clear that the introduction of one ethoxy unit in the lateral chain of imidazolium cations strongly reduced the toxic effects for both species, although at different levels. This trend confirmed what was found with the bacterium Vibrio fischeri, the crustacean Daphnia magna, and the green microalga Scenedesmus vacuolatus:20,21,16 for all the test species, as well as for S. marinoi and P. tricornutum, BMIM Cl resulted to be almost 10 times more toxic than MOEMIM Cl. This effect could be ascribed to a lower lipophilicity of the cation MOEMIM with respect to the cation BMIM. The further addition of another ethoxy moiety reduced the toxicity of M(OE)2MIM Cl with respect to MOEMIM Cl, at least for S. marinoi. For this alga, the differences among the three ionic liquids were statistically significant (ANOVA, P < 0.001), and the posthoc Newman-Keuls test indicated BMIM CI as the most toxic compound, and M(OE)2MIM Cl as the least one. 396



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Table 3. Influence of ILs on the Photosynthetic Efficiency of Skeletonema marinoi and Phaeodactylum tricornutuma EC50 ( SE (mM) Skeletonema

Phaeodactylum

marinoi

tricornutum

Y(II)e

Y0 (II)f

Y(II)e

BMIM Clb

0.17 ( 0.02

0.13 ( 0.02

2.53 ( 0.22

1.14 ( 0.13

MOEMIM Clc M(OE)2MIM Cld

3.78 ( 0.14 4.90 ( 0.19

2.94 ( 0.03 4.63 ( 0.16

>20 >20

>20 >20

IL

Y0 (II)f

a The 50% effect concentrations EC50 are expressed in mM, as EC50 ( standard error (SE). b 1-Butyl-3-methylimidazolium chloride. c 1-Methoxyethyl-3-methylimidazolium chloride. d 1-(2-(2-Methoxy-ethoxy)ethyl)-3-methylimidazolium chloride. e Maximum quantum yield. f Effective quantum yield.

Figure 6. FT-IR spectra (reflection-absorption mode) of Skeletonema marinoi cells after 72 h of exposure to BMIM Cl concentration of 304 μM (a), BMIM Cl concentration of 100 μM (b), and Skeletonema marinoi unexposed cells (c).

adapted to salinity 35 (0.12 ( 0.01 mM) was almost 100 times higher than the EC50 reported by Lataza et al.5 at salinity 8(3.32 ( 0.17 μM). Our results are apparently in disagreement with Lataza et al.11 who reported for other algal species a trend of increasing toxicity by reducing salinity, explainable as an increase of the permeability of the algal cell walls to ionic liquids. We believe that the differences could be ascribed to different initial conditions, the different algal strains having adapted to opposed salinity regimes and different experimental media. The strain of S. marinoi used in the present work, largely known to be eurythermal and euryhaline,35 was originally isolated from quite high saline waters (Adriatic Sea) and subsequently acclimatized to lower salinities, while Lataza and colleagues used strains isolated from low saline waters (Baltic Sea), then adapted to higher salinities. Moreover, in our case the experimental media at salinity 25 and 15 were obtained by diluting high salinity seawater with deionized water, while Lataza et al. started from low saline waters and increased the salinity by evaporating the medium. These different procedures should afford different culture media and could explain the different outcomes of toxicity tests. Chlorophyll Fluorescence Analysis. The efficiency of light energy conversion into chemical energy and its subsequent use by plants and algae may give useful information on the physiological state of the organisms.36 The maximum and the effective quantum yields of algal photosystem II, respectively, Y(II) and Y0 (II), were evaluated for algal control cultures and for treatments with ILs, after dark adaptation or light exposure, respectively. In Figure 4, the maximum and the effective quantum yield inhibitions of S. marinoi after 72 h of exposure to different concentrations of BMIM Cl (Figure 4a), MOEMIM Cl (Figure 4b), and M(OE)2MIM Cl (Figure 4c) are shown. Figure 5 shows the maximum and the effective quantum yield inhibition of P. tricornutum after 72 h of exposure to different concentrations of BMIM Cl. The EC50 values for all ILs after 72 h of exposure for both algal species are listed in Table 3. In the absence of ILs, Y(II) and Y0 (II) for S. marinoi were 0.501 and 0.336, respectively, while P. tricornutum had values of 0.400 and 0.278, respectively. In the case of S. marinoi, the pattern of chlorophyll fluorescence inhibition was in good agreement with the pattern of growth inhibition (Figure 1) for all the ionic liquids and the EC50 values for Y(II) and Y0 (II) were quite similar to those previously obtained in growth inhibition tests. Even in the case of P.

tricornutum the EC50 values neither for the inhibition of Y(II) nor for that of Y0 (II) by BMIM Cl were in line with that for the growth inhibition of the same alga. Since the reduction of the growth and photosynthetic efficiency of both algae were proportional, apart from the ionic liquid, we could assume a mechanism of IL action which affected the two end points in a very similar way. FT-IR Spectroscopy of Diatoms Exposed to ILs. Microscopy-coupled FT-IR spectroscopy is a helpful, but rarely applied, technique in (eco)-toxicological studies to characterize the physiological state and chemical composition of the microorganisms after toxicant exposure.38 The principal constituents of the diatom cell walls are peptidoglycan, polysaccharides, lipids, proteins, and silica. The reflection IR spectra allowed the detection of the typical organic and inorganic groups belonging to each membrane component of S. marinoi and P. tricornutum (Figures 6c and 7c), in agreement with the literature data.39-41 As expected, the less silicified diatom P. tricornutum showed a weaker band related to Si-O-Si adsorption than S. marinoi, which, on the contrary, was characterized by a strong absorption band around 1072 cm-1. For S. marinoi, FT-IR microscopy has been employed to evaluate the effects of BMIM Cl, already identified as the most toxic IL among the three tested (Figure 6); the FT-IR spectra of S. marinoi cells after 72 h of exposure to two concentrations of BMIM Cl, one close to the EC50 value and the other at a level where no algal growth could be observed (0.3 mM), were recorded and compared with the spectra of BMIM Cl (Figure 8a) and with unexposed control cells. The dose levels of BMIM Cl chosen in FT-IR experiments were high enough to afford measurable effects but not too high in order to check an eventual dose-effect relationship. For P. tricornutum, the effects of BMIM Cl were investigated by recording FT-IR spectra after the exposure of cells to a concentration of BMIM Cl close to the EC50 value (1.9 mM), in parallel with the experiment on S. marinoi. For the poorly effective MOEMIM Cl, the maximum concentration tested in growth inhibition tests (20 mM) was chosen (Figure 7). The rationale for this last experiment was to verify if some interaction of IL with the cellular components can occur without damaging the algal vitality. Before recording the spectra, the collected algal cells were washed three times with physiological saline solution, 397



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Figure 7. FT-IR spectrum (reflection-absorption mode) of Phaeodactylum tricornutum cells after 72 h of exposure to BMIM Cl concentration of 1900 μM (a), MOEMIM Cl concentration of 20000 μM (b), and Phaeodactylum tricornutum unexposed cells (c).

Figure 8. FT-IR spectra (reflection-absorption mode) of BMIM Cl (a) and MOEMIM Cl (b).

in order to remove the excess of ILs, not accumulated by the cells, and each trace of the inorganic and organic compounds coming from of the algal culture medium. After this procedure, no signal related to ILs (Figure 8) was detected with both algae. This observation suggests that ionic liquids were not taken up and accumulated by cells within the test period or that they were lost from the cells after 72 h of incubation.23 The FT-IR spectra of S. marinoi cells exposed to both BMIM Cl concentrations (1.0 mM and 0.3 mM) apparently showed few weak changes in terms of band intensity and shape with respect to unexposed control cells. Thus, in order to better evaluate these differences and to visualize the spectral data, they were processed by means of principal component analysis (PCA). Multivariate statistical analysis plays a crucial role in the interpretation of complex instrumental data (e.g., spectra), extracting the relevant information embodied in the multivariate signal, which is often not directly accessible.42 In particular, PCA is one of the most popular explorative techniques applied in the interpretation of multidimensional data.43,44 This chemometric approach allows the magnification of significant spectral differences and the reduction of spectral heterogeneity and unhelpful information. All relevant information contained in the original data is concentrated into few new variables: the principal components (PCs). They are orthogonal vectors in a new multidimensional data space and can be expressed as a linear combination of the original variables. Each PC explains a percentage of the whole variability (i.e., of the whole exploitable information) of the data set. The visual inspection of data is performed by the elaboration of score plots, where each object (the starting spectrum) can be seen as a point of a given coordinate (score) on the selected PC. The contribution of the original variables to each PC can be assessed by the loadings values. In fact, as already mentioned, the PCs are linear combinations of the original variables, and the coefficients which multiply the variables are called loadings. They represent the cosine value (director cosines) of the angle between a given PC and the original variables. These values may vary between -1 and þ1, indicating which of the original variables is the most important in the definition of a given PC. Thus, in the present work, particular attention has been paid to the examination of loadings values, in order to better identify the spectral regions more involved in the discrimination among sample subsets. Initial observation of spectra allowed the determination of the spectral ranges 674-1850 cm-1 and 2770-4000 cm-1, as the

most representative regions for all the investigated S. marinoi samples, and they were employed for the creation of a matrix data set. The wavenumber range 1850-2770 cm-1 was excluded due to the lack of significant spectral information in such a region. Principal component 2 (PC2, 63% of explained variance) and principal component 3 (PC3, 7% of explained variance) showed the highest discriminative ability and revealed well clustered groups related to the two types of treated samples and control cells (Figure 9a). The loading values allowed the identification of the absorption bands (spectral variables) mostly involved in the definition of each PC and, consequently, in the differentiation of sample spectra. In particular, a crucial role was played by the spectral ranges between 1000 and 1100 and the one around 960 cm-1, which are related to the silicate peaks (Figure 9b). This could support our initial hypothesis of interaction of ILs with those functional groups on the S. marinoi cell surface related to siliceous valve synthesis or organization. In addition, the analysis of loading values reveals that also the variability of the region around 1630-1660 cm-1 (region attributed to the amide I absorption band) is important in the discrimination effect of PC2. This evidence could support the hypothesis of IL interaction with proteins (frustulins, pleuralins, and silaffins) on the diatom cell surface. In the analysis of FT-IR spectra of P. tricornutum, PC1 (69% of explained variance) and PC2 (17% of explained variance) were selected as the most representative principal components according to their discriminative power. PCA allowed the characterization of three different and well-defined groups of spectra related to the three sample typologies under investigation (control cells and cells exposed to BMIM Cl and MOEMIM Cl). This result indicates differences among samples classes, which can be associated with MOEMIM Cl interaction with some cellular components, producing changes in IR spectra with respect to unexposed cells, but without inhibiting algal growth (Figure 10a). The loading values of the selected PC showed that the separation of different sample typologies was mainly related to changes of the band around 1640 cm-1 and in particular to 1740 cm-1, while the spectral range around 1050-1080 cm-1 seemed to be not relevant in the discriminatory purpose (Figure 10b). This could support our initial hypothesis about the effects of ILs for P. tricornutum: the most significant interactions seemed to occur with proteins (IR 398



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Figure 9. Principal component analysis of Skeletonema marinoi. Score plot of PC2 (63% of explained variance) and PC3 (7% of explained variance) (a) and loading profile of PC2 (b). 304BMIM, samples exposed at a 304 μM concentration of BMIM Cl; 100BMIM, samples exposed at a 100 μM concentration of BMIM Cl; C, unexposed cells.

Figure 10. Principal component analysis of Phaeodactylum tricornutum. Score plot of PC1 (69% of explained variance) and PC2 (17% of explained variance) and loading profile of PC1 (b). 1900BMIM, samples exposed at a 1900 μM concentration of BMIM Cl; 20000MOEMIM, samples exposed at a 20000 μM concentration of MOEMIM Cl; C, unexposed cells.

In order to provide an estimation of the discriminative ability of each PC selected in terms of separation between two categories, Fisher weights (FW) were calculated according to Harper et al.42 (Table 4). In the case of S. marinoi, the analysis of the FWs highlighted the discriminative ability of PC3 for all the three categories of samples under investigation. The highest values of FW was related to the separation between the cluster of untreated control cells and the samples after exposure to the highest concentration of BMIM Cl, while a lower FW value of PC3 was present between the two groups of the treated samples. In the case of P. tricornutum, both PC1 and PC2 showed an important role in the resulting clustering. In particular, the highest value of FW was related to the discrimination between the untreated control cells and the samples of P. tricornutum after exposure to BMIM Cl (PC1), while the lower FW values of PC2 were mainly related to the separation between control samples and samples exposed to MOEMIM Cl. Conclusions. In the present work, we have investigated the toxic effects of alkyl- and ether-functionalized imidazolium-based ILs toward S. marinoi and P. tricornutum. Ionic liquids bearing

Table 4. Fisher Weights (FW) of the First Three Principal Components (PC) for the Pair of Classes PC1 FW

PC2 FW

PC3 FW

Skeletonema marinoi BMIM100a-BMIM304b

0.4

1.4

1.6

BMIM100a-controlc

0.09

0.8

4.5

BMIM304b-controlc

0.1

0.5

8.1

Phaeodactylum tricornutum BMIMd-MOEMIMe

0.5

6.4

0.12

BMIMd-controlc MOEMIMe-controlc

18.6 3.8

1.1 6.8

0.17 0.04

a Samples exposed at a 100 μM concentration of BMIM Cl. b Samples exposed at a 304 μM concentration of BMIM Cl. c Unexposed cells. d Samples exposed at a 1900 μM concentration of BMIM Cl. e Samples exposed at a 20000 μM concentration of MOEMIM Cl.

bands around 1540 and 1650 cm-1) and with lipids (IR band around 1740 cm-1, attributed to CdO stretching of esters). 399



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(poly)ethoxy chains showed lower toxicity with respect to those bearing alkyl chains, confirming the well-known role of lipophilicity in determining the toxicity of these compounds. Another important indication about the mechanism of action of ILs arose from the differences in sensitivity of the two algae. The toxicity could be associated with the silica uptake and organization typical of diatoms, and for this reason, P. tricornutum, which does not require silica for growing, is largely less sensitive than S. marinoi. FT-IR microscopy afforded a support to both the mechanisms, showing specific interactions of ILs with silica, protein, and lipid groups exposed on the cellular surface. We presume that ILs, thanks to their amphiphilc character, could interact with functional groups present on the cell surface either through ionic or lipophilic interaction with acidic aminoacidic residues or lipid components, respectively. This action could interfere with the formation of the siliceous cell wall crucial for the survival of many diatoms, such as S. marinoi. However, at present we cannot definitely exclude that the effects of IL on algal growth are due also to other reasons; further investigation is needed for controlling the validity of the silica mechanism, and this will be scheduled for the future. Different from the literature, we did not find any effect of salinity on the toxicity of tested ILs toward S. marinoi. This result could be ascribed to different experimental procedures for obtaining algal media which, therefore, could influence the total amount of ionic species able to interact with ILs. Moreover, these differences also suggest the importance of the original environment of the algal strain in eco-toxicological assessments. High-salinity adapted algal samples probably could have developed a series of biological adaptations against the effects of organic or inorganic salts, preventing the enhancement of IL toxic effects in less saline environments. Therefore, the investigation of the physiological responses of biological targets with different mechanisms of adaptation and physiology, and detailed studies of the physical-chemical alteration of biological membranes are extremely important to further understand the mechanism of toxic action of ILs toward environmentally important living organisms.

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’ AUTHOR INFORMATION Corresponding Author

*Tel: þ39.0544.937353. Fax: þ39.0544.937411. E-mail: chiara. [email protected]. Funding Sources

We acknowledge the ministry MiUR and the University of Bologna (Polo Scientifico Didattico di Ravenna and RFO) for funding.

’ ACKNOWLEDGMENT We thank Dr. Fabio Moretti and Dr. Andrea Pasteris of the University of Bologna for helping in the statistical analysis, and Dr. Paolo Oliveri of the University of Genova for support with the principal components analysis. We are grateful to MacKenzie L. Zippay for English revision of the manuscript. ’ REFERENCES (1) Earle, M. J., and Seddon, K. R. (2002) Ionic liquids: green solvents for the future. ACS Symp. Ser. 819, 10–25. (2) Wasserscheid, P., and Welton, T. (2007) Ionic Liquids in Synthesis, 2nd ed., Wiley-VCH, Weinheim, Germany 400



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Effects of Imidazolium Ionic Liquids on Growth, Photosynthetic

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