rXXXX American Chemical Society A dx.doi.org/10.1021/tx100343p | Chem. Res. Toxicol. XXXX, XXX, 000–000

ARTICLE

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Effects of Imidazolium Ionic Liquids on Growth, PhotosyntheticEfficiency, and Cellular Components of the Diatoms Skeletonemamarinoi and Phaeodactylum tricornutumChiara 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 imidazo-lium ionic liquids bearing alkyl (BMIM), monoethoxy (MO-EMIM), and diethoxy (M(OE)2MIM) side chains toward twomarine diatoms, Skeletonema marinoi and Phaeodactylum tricor-nutum. MOEMIM and M(OE)2MIM cations showed a lowerinhibition of growth and photosynthetic efficiency with respect totheir alkyl counterpart, with both algal species. However, a largedifference in sensitivity was found between S. marinoi and P.tricornutum, the first being much more sensitive to the action ofionic liquids than the second one. The effects of salinity onBMIMCl toxicity toward S. marinoi revealed that a decrease fromsalinity 35 to salinity 15 does not influence the biological effectstoward the alga. Finally, Fourier transform infrared (FT-IR)microscopy of algal cells after ionic liquids exposure allowed usto detect an alteration of the organic cellular components relatedto silica uptake and organization. On the basis of these results, the different behavior of the two diatom species can be tentativelyascribed to different silica uptake and organization in outer cell walls.

’ INTRODUCTION

Ionic liquids (ILs) are the most studied class of alternativegreen solvents during the last two decades, from a technologicalpoint of view, as well as from sustainability and environmentalconcerns.1,2 Although the low volatility limits their release in theatmosphere and their environmental impact, the solubility inwater of many ILs is very high, and in view of the growingindustrial interest, concern is rising for their eventual release inthe environment through industrial effluents and the consequentcontamination of aquatic ecosystems. Moreover, as demon-strated in the large number of (eco)-toxicological studies of thelast years,3,4 many ILs are very toxic substances at different levelsof biological organization and against a variety of biologicaltargets. The mechanism of IL toxicity is still not well understood,and probably more than a single physiological system can beaffected. All the (eco)-toxicological studies have assessed that it isstrictly correlated with the lipophilicity of the ion pair, affordinga basic toxicity which causes a nonspecific disturbance of thefunctioning of biological membranes. Recent studies have alsodemonstrated that ILs exhibit adsorption behavior even throughionic interactions between the negative charged groups on cellsurfaces and the IL cations.5

Algae are very important organisms for (eco)-toxicologicalassessment because they are a key part of aquatic ecosystems,playing the role of primary producers and providing energy tosustain higher trophic levels; moreover, algal tests are generallysensitive, rapid, and cost-effective. For these reasons, algal dose-response bioassays have becomewidely used as biological tools inenvironmental impact studies. Among marine algae, diatoms arethe most used test organisms for their sensitivity to metals andorganic compounds. Thus far, the toxicity of ILs toward micro-algae has been performed by using mainly green freshwateralgal species6-18 and more recently, seawater diatoms andcyanobacteria.5,11,19 These studies have revealed that the toxicityof ILs is correlated to the structure of algal cell walls, known toplay a critical role in the transport of materials, including toxins,in and out of the cell. Diatoms, in particular, show manynegatively charged silica-associated organic components, as ex-tracellular polysaccharides, proteins (frustulins, pleuralins, andsilaffins), and long chain polyamines, and thanks to thesefunctional groups on the cell surface, these algae are very good

Received: October 5, 2010

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biosorbents for positively charged chemicals and toxicants asheavy metals and ILs. Moreover, the composition of the abioticenvironment and its variations are important parameters. Sali-nity, for example, could be one of the crucial factors in themitigation of the biological effects toward microalgae; in fact,high concentrations of inorganic anions and cations from highlysaline environments could potentially reduce the permeability ofionic liquid cations through algal cell walls.11

In the present study, we evaluated the biological effects of aset of imidazolium-based ILs toward two diatoms, Skeletonemamarinoi and Phaeodactylum tricornutum, possessing differentlyfeatured cell walls. The ionic liquids were chosen on the basis ofour previous works 20,21 and literature data4,16 in which it hasbeen reported that the introduction of ether-oxygen atoms inthe lateral chain of imidazolium-based ILs strongly reduced thetoxicity toward different biological targets. In the present work,the test kit of compounds consisted of three imidazoliumchlorides (Chart 1), one bearing an alkyl lateral chain (1-butyl-3-methylimidazolium chloride, BMIM Cl) and two having analkoxy lateral chain (1-methoxyethyl-3-methylimidazoliumchloride, MOEMIM Cl, and 1-(2-(2-methoxy-ethoxy)-ethyl)-3-methylimidazolium chloride, M(OE)2MIM Cl). Our aim wasto check the influence of the presence of ethoxy units on algaltoxicity, contributing to a further step into the elucidation of ILs'structure-activity relationship.

Concerning the choice of the algae, S. marinoi was recom-mended as a standard test organism due to its ecologicalrelevance in the ocean and high sensitivity to pollutants. Weused a strain typical of the Adriatic Sea (salinity around 35) andalso checked the effect of lower salinities (25 and 15), welltolerated by the alga, on the toxicity of BMIM Cl. P. tricornutumhas never been used before in ILs toxicity assays; however, it is agood target for checking possible toxic effects on the algal cellwall. In contrast to other diatoms that typically have silicified cellwalls, P. tricornutum possesses an organic cell wall and does notrequire silica for its growth. When silica is available, specificmorphotypes (oval, fusiform and triradiate) with different silicacontent in their cell walls can exist; among them, only the ovaltype has the typical silica valve of diatoms.22

Algal growth inhibition, evaluated in a 72 h growth inhibitionassay, and photosynthetic algal activity, evaluated by measuring

chlorophyll fluorescence through the pulse amplitude modula-tion (PAM) fluorometric method, were measured as end points.Chlorophyll fluorescence analysis was never used before asresponse to investigate the toxic effects of ILs on algae, but it isa very useful tool to study in vivo the functional properties ofphotosynthetic apparatus, being a nondestructive, versatile,highly sensitive, and accurate technique widely applicable to avast number of toxicants.36,32 This technique measures theportion of the adsorbed light energy which is not used forphotochemical transformations by algae and plants and thus isdissipated as heat or as light re-emission (fluorescence), mainlyemitted as chlorophyll a. In the past decades, there have beenconsiderable progress in practical applications of chlorophyllfluorometry following the introduction of the PAM fluorometricmethod,37,33 enabling one to provide information about thequantum yield, a parameter proportional to the photosyntheticefficiency of the photosystem II and whose inhibition can be ameasure for sublethal toxic responses.

Moreover, exploiting the method developed by Cornmellet al.,23 we have evaluated IL interaction with algal cellularcomponents, by using microscopy-coupled transmission fouriertransform infrared spectroscopy (FT-IR) in combination with amultivariate analysis approach (Principal Components Analysis,PCA); in fact, any possible interaction between IL and cellularcomponents should be recorded in the IR spectra as a change inintensity or frequency of IR bands of either IL or cellularconstituents.

’MATERIALS AND METHODS

Chemicals. All the chemicals were purchased from Aldrich;1-methylimidazole, 1-chlorobutane, and 2-chloroethyl methyl etherwere redistilled before use to limit the formation of colored impurities.The purities of all the ionic liquids were established to beg98% throughproton nuclear magnetic resonance (1H NMR) spectroscopy by inte-gration of proton signals with respect to an internal standard. All spectrawere recorded using a 5-mm probe on a Varian Inova 400 spectrometer(Varian, Palo Alto, CA, USA) at 400 MHz. All spectral data wereacquired in acetone-d6, D2O or CDCl3 with a known amount of tetra-kis(trimethylsilyl)silane as an internal standard and a delay time betweensuccessive scans of 20 s to ensure complete proton relaxation.

2-(2-Methoxy-ethoxy)-ethyl chloride, BMIM Cl, MOEMIM Cl, andM(OE)2MIMCl were synthesized according to the procedures reportedin the literature.24,25 The 1H NMR and 13C NMR spectra were inagreement with the literature data.Test Organisms. Skeletonema marinoi (CCMP2497) used in the

present study was obtained from the Provasoli-Guillard National Centrefor Culture of Marine Phytoplankton, Bigelow, ME, whereas Phaeodac-tylum tricornutum (PTN0301) was kindly donated by Dr. J.W. Rijstenbil,the Netherland Institute of Ecology (NIOO-KNAW). The originalstrains of S. marinoi and P. tricornutum were isolated from the northernAdriatic Sea and from the North Sea, respectively. Cultures weremaintained in 500 cm3 Erlenmeyer flasks containing 200 cm3 ofsterilized f/2 medium, prepared according to the literature fromseawater26 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 wereilluminated at 100-110 μmol photons m-2 s-1 from daylight type coolwhite lamps (16:8 h light-dark period), with the temperature con-trolled at 20 �C.Growth Inhibition Tests. The IL toxicity tests were conducted

according to the OECD guidelines (2006)27 with slight modificationsregarding the use of f/2 medium and the choice of the test strain.

Chart 1. Structures and Names of the Tested ILs

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Experiments were conducted in 100 cm3 sterile Erlenmeyer flasks, sealedwith cotton, containing 66.5 cm3 of sterilized f/2 medium enriched withsilica to which 7.5 cm3 of algal suspension in the log growth phaseand 1 cm3 of different concentrations of an aqueous IL solution or f/2medium (control cultures) were added. The initial optical density (OD)at 750 nm, measure of the cell number was 0.02 for both species, in eachexperiment. Two replicates were set up for each treatment con-centration and for the control. Test concentrations, identified througha preliminary range-finding test, were arranged in a geometric seriesranging from 1.0 mM to 8.0 mM for BMIM Cl, from 8.0 mM to20 mM for MOEMIM Cl and M(OE)2MIM Cl (P. tricornutum), from0.021 mM to 0.350 mM for BMIM Cl, from 0.7 mM to 4.0 mM forMOEMIM 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 100-110μmol photons m-2 s-1 from daylight type cool white lamps (16:8 hlight-dark period); after 72 h of incubation, the number of cells in thecultures was counted by OD measurement at 750 nm, using a spectro-photometer (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 lowersalinities, we followed a pattern of decreasing salinity by 5 from 35 to15,diluting with deionized water to obtain each specific salinity andacclimatizing the algal cells for 15 days to each condition before changingthe salinity again.

Modulated Fluorescence Measurements. Chlorophyll fluor-escence of photosystem II of the microalgae treated with differentconcentrations of ILs was evaluated with a PAM fluorometer. Themodelused was a 101-PAM connected to a PDA-100 data acquisition system,high power LED Lamp Control unit HPL-C, and LED-Array-ConeHPL-470 to supply actinic light and saturating pulses, and US-L665 tosupply measuring light (H. Walz, Effeltrich, Germany). Algal sampleswere analyzed in cuvettes (10 � 10 mm) mounted on an optical unitED-101US/M. The minimum constant fluorescence level of dark-adapted and light-adapted cells (F0 and F00, respectively) was measuredby using a low light intensity (1 μmol photons m-2 s-1). Maximumfluorescence level (Fm) was induced by a short saturating pulse of >3000μmol photons m-2 s-1 for 0.8 s. Maximum fluorescence level underactinic light (F0m) was measured by giving a saturating pulse after 5 minof illumination with actinic light of intensity similar to the one used forthe growth of the algae. This was done to avoid photoinhibitory effectsand to provide optimal conditions for photosynthetic activity. Thelength of illumination was chosen after verifying that the steady-state ofelectron transport was reached. The maximum and the effective quantumyields, respectively, Y(II) and Y0(II), were calculated as follows:28,29

YðIIÞ ¼ ðFm - F0Þ=Fm Y 0ðIIÞ ¼ ðF0m - F00Þ=F0m

Data Analysis. The 50% effect concentration (EC50) of eachsubstance for the growth inhibition tests, the salinity experiments, andthe evaluation of chlorophyll fluorescence inhibition was estimated byfitting the experimental concentration-response curves to a logisticmodel:

y ¼ bot þ ðtop- botÞð1þ ðx=EC50ÞcÞ

where y = end point value; x = substance concentration; bot = expectedend point value when the concentration of the toxicant is infinite(bottom asymptote); top = expected end point value when the con-centration of the toxicant is zero (top asymptote); and c = slopeparameter. The parameters of the equation, including the EC50, wereestimated using the nonlinear regression procedures implemented inStatistica (Statsoft, Tulsa, OK, USA). Figures 1-5 were fit to thisequation.

Figure 1. Dose-response relationships plotted for Skeletonema marinoiexposed 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%. Errorbars correspond to standard deviations of the experimental data.

Figure 2. Dose-response relationship plotted for Phaeodactylum tri-cornutum exposed to BMIM Cl. The response (absorbance) wasexpressed as percentage of the control (normalized response), consider-ing the control value as 100%. Error bars correspond to standarddeviations of the experimental data.

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The differences in the EC50 values were evaluated by using eachexperimental replicate by itself and then comparing these replicates byan ANOVA method. The assumption of homogeneity of variances wastested by Cochran’s C test. Whenever a significant difference for themain effect was observed (P < 0.05), a Newman-Keuls test was alsoperformed.FT-IR Spectroscopy. For the FT-IR analysis, S. marinoi cells were

cultured in the presence of BMIMCl at 0.1mM(around theEC50 value) and0.3 mM (above the EC50 value, at which no algal growth was observed)concentrations.P. tricornutum cells were cultured in the presence of BMIMClat a concentration of 1.9mM(around the EC50 value) and in the presence ofMOEMIM Cl at a concentration of 20 mM (at which concentration nogrowth inhibition was observed). Control and sample cultures wereprepared in duplicate according to the procedure described above, andafter 72 h of exposure to ILs, cells were collected by centrifugation at3000 rpm for 10 min, washed with a physiological saline solution, andcentrifuged again 3 times.23 Aliquots (1 μL) of cell suspensions and ofeach IL were applied on a gold mirror, and μFTIR spectra were recordedin reflection-absorption (RAS) mode, by using a Nicolet iN10MX FT-IR microscope fitted with a MCT detector, cooled with liquid nitrogen.Spectra were acquired at a resolution of 4 cm-1 (16 spectra wereaveraged to improve the signal-to-noise ratio) and elaborated withOMNIC Picta software. The chemometric package V-PARVUS wasused for PCA.30 The matrix data set was composed with intensityvalues of peaks as variables (columns) and spectra as objects (rows).

The second derivative has been applied as a row pretreatment on thematrix data set. Second derivatives were performed with third-ordersmoothing polynomials through 15 points.

Figure 3. Dose-response relationships plotted for Skeletonema marinoiexposed to BMIM Cl at salinity 35 (a), 25 (b) and 15 (c). The response(absorbance) was expressed as percentage of the control (normalizedresponse), considering the control value as 100%. Error bars correspondto standard deviations of the experimental data.

Figure 4. Maximum and effective quantum yield (Y(II) and Y 0(II))inhibition of Skeletonema marinoi after 72 h of exposure to BMIMCl (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 5. Maximum and effective quantum yields (Y(II) and Y0(II))inhibition of Phaeodactylum tricornutum after 72 h of exposure to BMIMCl. The response (absorbance) was expressed as the percentage of thecontrol (normalized response), considering the control value as 100%.

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’RESULTS AND DISCUSSION

Growth InhibitionAssay. The effects of ILs on the growth ofS. marinoi and P. tricornutum were estimated with respect tocontrol samples (normalized response). In the case of P. tricor-nutum, cultures were composed essentially by the fusiformmorphotype 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 inhibitionof P. tricornutum after 72 h of exposure to different concentra-tions of BMIM Cl is shown.The dose-response curves performed with BMIM Cl gave a

parallel pattern for S. marinoi and P. tricornutum, although withdifferent concentration ranges (0.02-0.35 mM for S. marinoiand 1.0-8.0 mM for P. tricornutum). In the case of the two ether-functionalized ILs, tested with S. marinoi, the pattern of algalgrowth inhibition was similar and followed a typical sigmoidcurve; P. tricornutum had no growth inhibition with ether-functionalized IL up to 20 mM, as shown by the EC50 valuesmeasured 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 mosttoxic among the tested ILs andM(OE)2MIMCl as the least. Alsofor P. tricornutum, themost toxic compoundwas BMIMCl (EC50

value 1.26 ( 0.06 mM), although the sensitivity of this alga wasone order of magnitude lower than that of S. marinoi, and thedifferences in the two EC50 values were statistically significant(ANOVA, P < 0.001); the EC50 values for both ether-func-tionalized ILs were higher than 20 mM, the maximum testedconcentration 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 toxiceffects for both species, although at different levels. This trendconfirmed what was found with the bacterium Vibrio fischeri, thecrustaceanDaphnia magna, and the green microalga Scenedesmusvacuolatus:20,21,16 for all the test species, as well as for S. marinoiand P. tricornutum, BMIMCl resulted to be almost 10 timesmoretoxic than MOEMIM Cl. This effect could be ascribed to a lowerlipophilicity of the cation MOEMIM with respect to the cationBMIM.The further addition of another ethoxy moiety reduced the

toxicity of M(OE)2MIM Cl with respect to MOEMIM Cl, atleast for S. marinoi. For this alga, the differences among the threeionic liquids were statistically significant (ANOVA, P < 0.001),and the posthoc Newman-Keuls test indicated BMIM CI as themost toxic compound, and M(OE)2MIM Cl as the least one.

A suggestion for the strong difference in sensitivity among thetwo diatoms could rely on the fact that P. tricornutum presentssome structural differences with respect to S. marinoi. First, P.tricornutum does not require silica for its growth, and it isgenerally observed in cultures and in natural waters as a fusiformmorphotype, themost stable of the three. This morphotype, usedin the present study, typically contains silica (0.4-0.5% dryweight) in the form of irregular particles, clearly organized inbands in the girdle region of the wall, and does not form thetypical frustul valves of diatoms.22 Second, P. tricornutum pre-sents some differences with respect to S. marinoi in the organicnegatively charged components on the cell surface, involved insilicon uptake and formation of the cell wall, especially in proteincontent and typology.31

We hypothesized that ILs can interact with functional groupson the algal cell surface and give different responses according todifferent silica organization, uptake and polymerization of thetwo 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 asP. tricornutum, that lack such biological structures and are evenable to survive with a reduced amount of silica (Si/C ratio forP. 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 anionspecies preventing the uptake by cells, with a mechanism similarto what was proposed by Lataza et al.11 to explain the effects ofsalinity on IL toxicity. Since P. tricornutum does not need silica forgrowing, the effects of this scavenging could be less pronouncedthan for S. marinoi.Influence of Salinity Variations on IL Toxicity. The impact

of three different salinities (35, 25, and 15) on the biologicaleffects of BMIM Cl against the growth of S. marinoi was alsoinvestigated. Table 2 shows the influence of salinity on thegrowth inhibition of S. marinoi exposed to BMIMCl with respectto the control sample. In Figure 3, the growth inhibition ofS. marinoi after 72 h of exposure to different concentrationsof 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 todifferent biological effects of the ionic liquid. Since the alga wasadapted to each salinity for two weeks prior to toxicologicalexperiments, after that the cell yields in the three cultures weresimilar (absorbance values of 0.152 at salinity 35, 0.103 at salinity25, and 0.104 at salinity 15), we could assume a good adaptationof S. marinoi to each salinity regime and reasonably exclude astress in cell growth due to changes in the environmentalconditions. Moreover, the EC50 value obtained for S. marinoi

Table 1. Influence of ILs on the Growth of Skeletonemamarinoi and Phaeodactylum tricornutuma

EC50 ( SE (mM)

IL Skeletonema marinoi Phaeodactylum tricornutum

BMIM Clb 0.12( 0.01 1.26 ( 0.06

MOEMIM Clc 2.98( 0.08 >20

M(OE)2MIM Cld 4.64( 0.16 >20aThe 50% effect concentrations EC50 are expressed in mM, as EC50 (standard error (SE). b1-Butyl-3-methylimidazolium chloride. c1-Meth-oxyethyl-3-methylimidazolium chloride. d1-(2-(2-Methoxyethoxy)ethyl)-3-methylimidazolium chloride.

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

salinity EC50( SE (mM)

35 0.12( 0.01

25 0.10( 0.01

15 0.14( 0.02aThe 50% effect concentrations EC50 are expressed in mM, as EC50 (standard error (SE).

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adapted to salinity 35 (0.12 ( 0.01 mM) was almost 100 timeshigher than the EC50 reported by Lataza et al.5 at salinity8(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 toxicityby reducing salinity, explainable as an increase of the permeabilityof the algal cell walls to ionic liquids. We believe that the dif-ferences could be ascribed to different initial conditions, thedifferent algal strains having adapted to opposed salinity regimesand different experimental media. The strain of S. marinoi usedin the present work, largely known to be eurythermal and eury-haline,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 salinewaters (Baltic Sea), then adapted to higher salinities. Moreover,in our case the experimental media at salinity 25 and 15 wereobtained by diluting high salinity seawater with deionized water,while Lataza et al. started from low saline waters and increased thesalinity by evaporating the medium. These different proceduresshould afford different culture media and could explain thedifferent outcomes of toxicity tests.Chlorophyll Fluorescence Analysis. The efficiency of light

energy conversion into chemical energy and its subsequent useby plants and algae may give useful information on the physio-logical state of the organisms.36 The maximum and the effectivequantum yields of algal photosystem II, respectively, Y(II) andY0(II), were evaluated for algal control cultures and for treat-ments with ILs, after dark adaptation or light exposure, respec-tively. In Figure 4, the maximum and the effective quantum yieldinhibitions of S. marinoi after 72 h of exposure to differentconcentrations 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 yieldinhibition of P. tricornutum after 72 h of exposure to differentconcentrations of BMIMCl. The EC50 values for all ILs after 72 hof exposure for both algal species are listed in Table 3.In the absence of ILs, Y(II) and Y0(II) for S. marinoiwere 0.501

and 0.336, respectively, while P. tricornutum had values of 0.400and 0.278, respectively.In the case of S. marinoi, the pattern of chlorophyll fluores-

cence inhibition was in good agreement with the pattern ofgrowth inhibition (Figure 1) for all the ionic liquids and the EC50

values for Y(II) and Y0(II) were quite similar to those previouslyobtained 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 thegrowth inhibition of the same alga. Since the reduction of thegrowth and photosynthetic efficiency of both algae were propor-tional, apart from the ionic liquid, we could assume a mechanismof IL action which affected the two end points in a very similarway.FT-IR Spectroscopy of Diatoms Exposed to ILs. Micro-

scopy-coupled FT-IR spectroscopy is a helpful, but rarelyapplied, technique in (eco)-toxicological studies to characterizethe physiological state and chemical composition of the micro-organisms after toxicant exposure.38

The principal constituents of the diatom cell walls are pepti-doglycan, polysaccharides, lipids, proteins, and silica. The reflec-tion IR spectra allowed the detection of the typical organic andinorganic groups belonging to each membrane component ofS. marinoi and P. tricornutum (Figures 6c and 7c), in agreementwith the literature data.39-41 As expected, the less silicified diatomP. tricornutum showed a weaker band related to Si-O-Siadsorption than S. marinoi, which, on the contrary, was char-acterized 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 mosttoxic IL among the three tested (Figure 6); the FT-IR spectra ofS. marinoi cells after 72 h of exposure to two concentrations ofBMIM Cl, one close to the EC50 value and the otherat 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 ofBMIM Cl chosen in FT-IR experiments were high enough toafford measurable effects but not too high in order to check aneventual dose-effect relationship.For P. tricornutum, the effects of BMIM Cl were investigated

by recording FT-IR spectra after the exposure of cells to aconcentration of BMIM Cl close to the EC50 value (1.9 mM),in parallel with the experiment on S. marinoi. For the poorlyeffective MOEMIM Cl, the maximum concentration tested ingrowth inhibition tests (20 mM) was chosen (Figure 7). Therationale for this last experiment was to verify if some interactionof IL with the cellular components can occur without damagingthe algal vitality. Before recording the spectra, the collected algalcells were washed three times with physiological saline solution,

Table 3. Influence of ILs on the Photosynthetic Efficiency ofSkeletonema marinoi and Phaeodactylum tricornutuma

EC50 ( SE (mM)

Skeletonema

marinoi

Phaeodactylum

tricornutum

IL Y(II)e Y0(II)f Y(II)e Y0(II)f

BMIM Clb 0.17( 0.02 0.13( 0.02 2.53 ( 0.22 1.14 ( 0.13

MOEMIM Clc 3.78( 0.14 2.94( 0.03 >20 >20

M(OE)2MIM Cld 4.90( 0.19 4.63( 0.16 >20 >20aThe 50% effect concentrations EC50 are expressed in mM, as EC50 (standard error (SE). b 1-Butyl-3-methylimidazolium chloride. c 1-Meth-oxyethyl-3-methylimidazolium chloride. d 1-(2-(2-Methoxy-ethoxy)-ethyl)-3-methylimidazolium chloride. eMaximum quantum yield. fEffectivequantum yield. Figure 6. FT-IR spectra (reflection-absorption mode) of Skeletonema

marinoi cells after 72 h of exposure to BMIM Cl concentration of 304μM (a), BMIM Cl concentration of 100 μM (b), and Skeletonemamarinoi unexposed cells (c).

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in order to remove the excess of ILs, not accumulated by the cells,and each trace of the inorganic and organic compounds comingfrom of the algal culture medium. After this procedure, no signalrelated to ILs (Figure 8) was detected with both algae. Thisobservation suggests that ionic liquids were not taken up andaccumulated by cells within the test period or that they were lostfrom the cells after 72 h of incubation.23

The FT-IR spectra of S. marinoi cells exposed to both BMIMCl concentrations (1.0 mM and 0.3 mM) apparently showed fewweak changes in terms of band intensity and shape with respectto unexposed control cells. Thus, in order to better evaluate thesedifferences and to visualize the spectral data, they were processedby 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 multivariatesignal, which is often not directly accessible.42 In particular, PCAis one of the most popular explorative techniques applied in theinterpretation of multidimensional data.43,44 This chemometricapproach allows the magnification of significant spectral differ-ences and the reduction of spectral heterogeneity and unhelpfulinformation. All relevant information contained in the original datais concentrated into few new variables: the principal components(PCs). They are orthogonal vectors in a new multidimensionaldata space and can be expressed as a linear combination of theoriginal variables. Each PC explains a percentage of the wholevariability (i.e., of the whole exploitable information) of the dataset. The visual inspection of data is performed by the elaboration ofscore plots, where each object (the starting spectrum) can be seenas a point of a given coordinate (score) on the selected PC. Thecontribution of the original variables to each PC can be assessedby the loadings values. In fact, as already mentioned, the PCs arelinear combinations of the original variables, and the coefficientswhichmultiply the variables are called loadings. They represent thecosine value (director cosines) of the angle between a givenPC and the original variables. These values may vary between -1and þ1, indicating which of the original variables is the mostimportant in the definition of a given PC. Thus, in the present work,particular attention has been paid to the examination of loadingsvalues, in order to better identify the spectral regions more involvedin 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. marinoisamples, and they were employed for the creation of amatrix dataset. The wavenumber range 1850-2770 cm-1 was excluded dueto 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) showedthe highest discriminative ability and revealed well clusteredgroups related to the two types of treated samples and controlcells (Figure 9a).The loading values allowed the identification of the absorption

bands (spectral variables) mostly involved in the definition ofeach PC and, consequently, in the differentiation of samplespectra. In particular, a crucial role was played by the spectralranges between 1000 and 1100 and the one around 960 cm-1,which are related to the silicate peaks (Figure 9b). This couldsupport our initial hypothesis of interaction of ILs with thosefunctional groups on the S. marinoi cell surface related to siliceousvalve synthesis or organization. In addition, the analysis ofloading values reveals that also the variability of the regionaround 1630-1660 cm-1 (region attributed to the amide Iabsorption band) is important in the discrimination effect ofPC2. This evidence could support the hypothesis of IL interac-tion with proteins (frustulins, pleuralins, and silaffins) on thediatom cell surface.In the analysis of FT-IR spectra of P. tricornutum, PC1 (69% of

explained variance) and PC2 (17% of explained variance) wereselected as themost representative principal components accord-ing to their discriminative power. PCA allowed the characteriza-tion of three different and well-defined groups of spectra relatedto the three sample typologies under investigation (control cellsand cells exposed to BMIM Cl and MOEMIM Cl). This resultindicates differences among samples classes, which can be asso-ciated with MOEMIM Cl interaction with some cellular compo-nents, producing changes in IR spectra with respect to unexposedcells, but without inhibiting algal growth (Figure 10a). The loadingvalues of the selected PC showed that the separation of differentsample typologies was mainly related to changes of the bandaround 1640 cm-1 and in particular to 1740 cm-1, while thespectral range around 1050-1080 cm-1 seemed to be not relevantin the discriminatory purpose (Figure 10b). This could supportour initial hypothesis about the effects of ILs for P. tricornutum:themost significant interactions seemed to occurwith proteins (IR

Figure 7. FT-IR spectrum (reflection-absorption mode) of Phaeodac-tylum tricornutum cells after 72 h of exposure to BMIMCl concentrationof 1900 μM (a), MOEMIM Cl concentration of 20000 μM (b), andPhaeodactylum tricornutum unexposed cells (c). Figure 8. FT-IR spectra (reflection-absorption mode) of BMIM Cl

(a) and MOEMIM Cl (b).

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

In order to provide an estimation of the discriminative abilityof each PC selected in terms of separation between twocategories, Fisher weights (FW) were calculated according toHarper 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 ofsamples under investigation. The highest values of FW wasrelated to the separation between the cluster of untreated controlcells and the samples after exposure to the highest concentrationof BMIMCl, while a lower FW value of PC3was present betweenthe 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, thehighest value of FW was related to the discrimination betweenthe untreated control cells and the samples of P. tricornutum afterexposure to BMIM Cl (PC1), while the lower FW values of PC2were mainly related to the separation between control samplesand samples exposed to MOEMIM Cl.Conclusions. In the present work, we have investigated the

toxic effects of alkyl- and ether-functionalized imidazolium-basedILs toward S. marinoi and P. tricornutum. Ionic liquids bearing

Figure 10. Principal component analysis of Phaeodactylum tricornutum. Score plot of PC1 (69% of explained variance) and PC2 (17% of explainedvariance) and loading profile of PC1 (b). 1900BMIM, samples exposed at a 1900μMconcentration of BMIMCl; 20000MOEMIM, samples exposed at a20000 μM concentration of MOEMIM Cl; C, unexposed cells.

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 μMconcentration of BMIM Cl; C, unexposed cells.

Table 4. Fisher Weights (FW) of the First Three PrincipalComponents (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 18.6 1.1 0.17

MOEMIMe-controlc 3.8 6.8 0.04a Samples exposed at a 100 μM concentration of BMIM Cl. b Samplesexposed at a 304 μM concentration of BMIM Cl. cUnexposed cells.d Samples exposed at a 1900 μM concentration of BMIM Cl. e Samplesexposed at a 20000 μM concentration of MOEMIM Cl.

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(poly)ethoxy chains showed lower toxicity with respect to thosebearing alkyl chains, confirming the well-known role of lipophil-icity in determining the toxicity of these compounds. Anotherimportant indication about the mechanism of action of ILs arosefrom the differences in sensitivity of the two algae. The toxicitycould be associated with the silica uptake and organization typicalof diatoms, and for this reason, P. tricornutum, which does notrequire 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 lipidgroups exposed on the cellular surface. We presume that ILs,thanks to their amphiphilc character, could interact with func-tional groups present on the cell surface either through ionic orlipophilic interaction with acidic aminoacidic residues or lipidcomponents, respectively. This action could interfere with theformation of the siliceous cell wall crucial for the survival of manydiatoms, such as S. marinoi. However, at present we cannotdefinitely exclude that the effects of IL on algal growth are duealso to other reasons; further investigation is needed for control-ling the validity of the silica mechanism, and this will bescheduled 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 beascribed to different experimental procedures for obtaining algalmedia which, therefore, could influence the total amount of ionicspecies able to interact with ILs. Moreover, these differences alsosuggest the importance of the original environment of the algalstrain in eco-toxicological assessments. High-salinity adapted algalsamples probably could have developed a series of biologicaladaptations against the effects of organic or inorganic salts, prevent-ing 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 andphysiology, and detailed studies of the physical-chemical altera-tion of biological membranes are extremely important to furtherunderstand the mechanism of toxic action of ILs toward envir-onmentally important living organisms.

’AUTHOR INFORMATION

Corresponding Author*Tel: þ39.0544.937353. Fax: þ39.0544.937411. E-mail: chiara.samori3@unibo.it.

Funding SourcesWe acknowledge the ministry MiUR and the University ofBologna (Polo Scientifico Didattico di Ravenna and RFO) forfunding.

’ACKNOWLEDGMENT

We thank Dr. Fabio Moretti and Dr. Andrea Pasteris of theUniversity of Bologna for helping in the statistical analysis, andDr. Paolo Oliveri of the University of Genova for support withthe principal components analysis. We are grateful to MacKenzieL. Zippay for English revision of the manuscript.

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