international journal of green technology, 1-12 1 does the ...zinc oxide (zno) is a major...

International Journal of Green Technology, 2015, 1, 1-12 1

© 2015 Revotech Press

Does the Process for Production Nanoparticulate ZnO Play a Role in its Ecotoxicity?

Hudson C. Polonini1, Nádia R. B. Raposo1, Humberto M. Brandão2, Cauê Ribeiro3, Claude Yéprémian4, Alain Couté4, Yann Sivry5 and Roberta Brayner6,*

1Universidade Federal de Juiz de Fora, Núcleo de Pesquisa e Inovação em Ciências da Saúde (NUPICS),

Rua José Lourenço Kelmer, s/n, 36036-900, Juiz de Fora, Brasil; 2Empresa Brasileira de Pesquisa

Agropecuária (Embrapa Gado de Leite), 36038-330, Juiz de Fora, Brasil; 3Embrapa Instrumentação

Agropecuária, Rua XV de Novembro, 1452, 13560-970, São Carlos, Brasil; 4Muséum National d’Histoire

Naturelle, UMR 7245 CNRS-MNHN Molécules de Communication et Adaptation des Micro-organismes, 57 rue Cuvier, F-75005 Paris, France;

5Université Paris Diderot, Institute de Physique du Globe de Paris (IPGP),

Sorbonne Paris Cité, UMR 7154, CNRS, 1 rue Jussieu, F-75238 Paris, France and 6Université Paris Diderot,

Sorbonne Paris Cité, Interfaces, Traitements, Organisation et Dynamique des Systèmes (ITODYS), UMR 7086, CNRS, 15 rue Jean de Baïf, F-75205 Paris Cedex 13, France.

Abstract: The interaction between live organisms and nanosized particles has become a current focus in toxicology. The aims of the present work are: (i) to assess the zinc oxide (ZnO) toxicity and its mechanisms into the aquatic environment, using the green algae Chlorella vulgaris as biological indicator; (ii) to compare the ZnO behavior and toxic profile in synthetic (Bold’s Basal) and natural (Seine River Water, SRW) culture media; and (iii) to address whether the obtaining route is an issue in ZnO particles toxicity or not. Responses such as growth inhibition, cell viability, superoxide dismutase (SOD) activity, adenosine-5-triphosphate (ATP) content and photosynthetic efficiency were evaluated. The main conclusions are: (i) nanoparticulate ZnO have an statistically significant toxic effect on C. vulgaris growth since the lower concentration tested (1ppm), that seems to be mediated by a induced oxidative stress (probably due to extensive release of Zn2+ into the media); (ii) the ZnO behavior in synthetic and natural culture media were statistically similar, although the toxic effects were more pronounced in SRW; and (iii) the production process does not seem to be an issue in ZnO nanoparticles toxicity since all tested particles produced significant effects on microalgae growth

Keywords: Zinc oxide, Seine river water, Ecotoxicology, Characterization, Nanotechnology.

1. INTRODUCTION

Zinc oxide (ZnO) is a major nanomaterial that has unique optical, catalytic, semiconducting, piezoelectric, and magnetic properties [1,2]. Because it has been widely produced and applied in industry, concern about its ecotoxicity is raising, as evidenced by the increasing number of publications in this topic, specially the ones dealing with aquatic toxicology. To cite a few of these studies: Tang et al. [3] explored the toxicity of ZnO nanoparticles to Anabaena sp, a cyanobacteria (EC50 = 0.74 ± 0.01 mg L-1); Brayner et al. [4] reported biocidal effects of ZnO nanoparticles on E. coli bacteria (concentrations between 3.0 x 10-3 and 1.5 x 10-3 M inhibited bacterial growth by 85%, but concentrations between 1.5 x 10-3 and 10-3 M promoted an increase of E. coli colony forming units); Brayner et al. [5] also observed that ZnO killed A. flos-aquae and the euglenoid Euglena gracilis after 10 days of incubation in the presence of the nanoparticles; and Franklin et al.

*Address correspondence to this author at the Université Paris Diderot, Sorbonne Paris Cité, Interfaces, Traitements, Organisation et Dynamique des Systèmes (ITODYS), UMR 7086, CNRS, 15 rue Jean de Baïf, F-75205 Paris Cedex 13, France; Tel: + 33(0) 1 57 27 87 64; E-mail: [email protected]

[6] evaluated the toxicity using Pseudokirchneriella

subcapitata freshwater alga (72-h IC50 value near 60 g Zn L-1).

The aquatic toxicity assays are widely used because these ecosystems are the main enclosures of contaminants, whether they are coming from direct release into water bodies through discharge of effluents, released into the air or deposited in soils [7]. Yet, the use of primary producers as biological indicators is important because they are situated at the base of the food chain and any change in the dynamics of their communities can affect higher trophic levels of the ecosystem - they are also quite sensitive to changes in the environment and their life cycle is relatively short, what permits the observation of toxic effects in several generations [8].

In the present study, it was the aim of the authors to use another algae species not yet studied exposed to ZnO, the green algae Chlorella vulgaris (grown both in artificial and natural culture medium - Bold’s Basal, BB, or Seine River water, SRW, respectively), as a model organism to assess the aquatic toxicology of ZnO produced by different process. C. vulgaris are rounded or ellipsoid eukaryotic unicellular green algae, with an

2 International Journal of Green Technology, 2015, Vol. 1 Polonini et al.

average diameter of 5 μm. It has a parietal chloroplast containing chlorophyll a and b and carotenoids. Starch is the major carbohydrate reserve and its outer cell membrane is composed of three membrane sheets containing glucosamine [9].

Within this context, our objectives were: (i) to assess the toxicity of nanoparticulate ZnO obtained by different processes and its mechanisms into the aquatic environment, using C. vulgaris as biological indicator; (ii) to compare the ZnO behavior and toxic profile in synthetic and natural culture media; and (iii) to address whether the process for ZnO production is an issue in its particles toxicity or not.

2. EXPERIMENTAL SECTION

2.1. The Subjects of the Study: ZnO Nanoparticles and Chlorella Vulgaris Model-Organism

2.1.1. ZnO Samples

Five different samples of nanoparticulate ZnO were assayed. ZnO-lot1, ZnO-lot2 and ZnO-lot3 were obtained by evaporation of metallic zinc slags (also known as indirect or French process). The metallic zinc was evaporated with a higher heat treatment at 1000 °C, and then the cooled product were collected in the form of ZnO. Different “lot” numbers represent different days on which they were obtained (as the slags change and also the processing temperature, the particles can have different crystallization, size and shape). The other two samples, ZnO-NaOH and ZnO-KOH, were obtained by hydrothermal synthesis. For this, zinc acetate was dissolved in distilled water and then 15 mL of a 6 M solution of base was added. In this case, the bases were respectively NaOH and KOH. They were put in the hydrothermal reactor at 150 °C for 2 h, at high pressure. After that, the material was washed with distilled water.

2.1.2. Samples Characterization

The X-ray diffraction (XRD) patterns of the powders were recorded with a X’pert Pro diffractometer (PANalytical), equipped with a multichannel X’celerator detector, and using the Co K radiation (= 1.790307 Å), in the 2 range 5°-120°, with a scan step of 0.05° for 5s. The sample holder used was a Si monocrystal.

Morphological observation of powders by transmi- ssion electron microscopy (TEM) was obtained in a JEOL 100CX-II microscope operating with an acceler- ating voltage of 100 kV. Specimens’ aliquots in ethanol were prepared sonicated for 10 min at 200 W (VWR,

USA) prior to deposition on the carbon-coated TEM grids.

The degree of dissolution of the ZnO powders as a function of time (2, 4, 8, 24, 48, 72 and 96h) within the media (BB medium and SRW, chemical composition available as Supplementary Material) was evaluated by following a protocol proposed by Sivry et al. [10]. From a stock solution (100 μg mL–1) prepared at time 0h, aliquots (3.5 mL) were withdrawn at the specified time intervals and ultra-filtered using 3kDa filters (Microsep Advance Centrifugal Device, Pall Corporation), placed in a centrifuge (EBA 8, Hettich) at 20,000 g for 1h, and then 50μL of saturated nitric acid (HNO3) (with no trace of Zn2+) were added to the supernatant. All solutions were immediately frozen until elemental analysis (Zn2+) was performed by inductively coupled plasma optical emission spectrometry (ICP-OES) (iCAP 6200, Thermo Scientific). Detection limit was set as 1.0 ppb.

All reagents were analytical grade, and ultrapure water (18.2 M cm) was obtained with an Elga Pure-Lab UV.

2.1.3. C. vulgaris Cell Culture

C. vulgaris, a planktonic eukaryotic single-cell green algae, was grown in 275 mL (= 75 cm2) erlenmeyer flasks with air-permeable stoppers, in (i) sterile BB medium with pH adjusted to 7.0 using 1 M NaOH solution, or (ii) SRW (measured pH = 8.01). All cultures were kept at a controlled temperature of 20.0 ± 0.5 °C and a daily cycle of 16 h of luminosity (50-80 μmol m–2 s–1 photosynthetic photon flux, PPF), under ambient CO2 conditions.

SRW, representative of a highly anthrophized watershed, was collected near the Université Paris

Diderot, France (GPS: 48.831039°N, 2.381709°E). The sample was immediately filtered after collection through a 0.22 μm acetate membrane (Millipore) under vacuum to remove contaminants and microorganisms and stored in pre-cleaned, acid-washed polyethylene bottles, at 4 °C until analysis.

The composition of both media can be found as Supplementary Material.

2.2. Toxicological Assessment

Stock suspensions containing 1,000 μg mL–1 by weight of ZnO nanoparticles were obtained by sonicating aliquots of 10 mg in 10 mL of Seine water or

Does the Process for Production Nanoparticulate ZnO Play a Role in its Ecotoxicity? International Journal of Green Technology, 2015, Vol. 1, 3

SUPPLEMENTARY MATERIAL

1. Bold’s Basal (BB) Medium

The composition of the medium BB is found in Table S1. The final medium was obtained by mixing 10 mL of solution A, 10 mL of solution B, 1 mL of trace solution and water to 1000 mL. After homogenization, the pH was adjusted to 7.0 using 1M NaOH solution.

Table S1. Composition of Bold’s Basal Culture Medium

Component Mass (g/400 mL water)

NaNO3 10.0

(or NaCl) 6.1

CaCl2 2H2O 1.0

MgSO4 7H2O 3.0

Solution A

Fe-EDTA 0.8

Component Mass (g/400 mL water)

K2HPO4 3.0

KH2PO4 7.0 Solution B

NaCl 0.8

Component Mass (mg/100 mL water)

H3BO3 240.0

MnCl2 7H2O 180.0

MoO3 7H2O 10.0

ZnSO4 7H2O 22.0

CuSO4 7H2O 8.0

CoSO4 7H2O 9.0

Trace solution

VOSO4 2H2O 4.3

NaNO3: sodium nitrate; NaCl: sodium chloride; CaCl2: calcium chloride; MgSO4: magnesium sulphate; Fe-EDTA: erric edetate; K2HPO4: dibasic potassium phosphate; KH2PO4: monobasic potassium phosphate; H3BO3: boric acid; MnCl2: manganese chloride; MoO3: molybdenum trioxide; ZnSO4: zinc sulphate; CuSO4: copper sulphate; CoSO4: cobalt sulphate; VOSO4: vanadyl sulphate.

2. Seine River Water (SRW)

In this study, microorganisms grown in natural water samples from the Seine River were used. This river is 777 km long, with approximately 14 million cubic meters of water (annually circulating). Like all rivers, its composition varies throughout the year. Its pH is between 7.8 and 8.2, the average temperature varies depending on the season between 4 and 24 °C (mean = 11.6 °C) and its ionic strength is 6.5 mM. Table S2 summarizes the composition in terms of major chemical constituents, determined from 131 collections made between October 2007 and July 2009.

Table S2. Physico-Chemical Characterization of the Seine River

Component Quantity Component Quantity

pH 7.99 ± 0.11 Magnesium ( 10–4 M) 1.69 ± 0.24

Zeta potential (mV) – 67.95 ± 14.78 Calcium ( 10–3 M) 2.34 ± 0.22

Temperature (°C) 11.59 ± 4.88 Sodium ( 10–4 M) 4.63 ± 0.93

[O2] (%) 103.33 ± 3.61 Potassium ( 10–5 M) 8.04 ± 1.08

[O2] (mg L–1) 11.55 ± 1.35 Nitrates (μM) 394.62 ± 51.61

Conductivity (μS cm–1) 511.98 ± 34.21 Nitrites (μM) 1.99 ± 0.93

Total organic carbon (mg L–1) 2.69 ± 0.39 Ammonia (μM) 4.43 ± 2.81

Alkalinity (mM) 3.88 ± 0.72 Silicates (μM) 106.91 ± 31.98

Ionic strength (mM) 6.51 ± 0.42 Phosphates (μM) 2.12 ± 1.04

Source: Da Rocha A. Impact écotoxicologique de nanoparticules de ZnO et CdS préparées par la méthode polyol, sur la microalgue verte Chlorella vulgaris dans l’eau de Seine. 2014. Thesis (Doctorate in Chemistry)-Université Paris Diderot, Paris, 2014.


BB medium for 10 min at 200 W (VWR, USA). The sonication was used to break micrometric aggregates and stabilize the suspensions. Aliquots of these suspensions were then added to the batch cultures (exponential growth algae, prepared 3 days before the start of the test at a concentration of 5.0 105 cells mL–

1) to obtain the final ZnO nanoparticle concentrations of 1, 25, 50, 75 and 100 μg mL–1 (n=3 each). Therefore, this study uses higher concentrations than what is forecasted for contamination in natural water, aiming to assess the acute toxicity of the materials. The experiment was also conducted with Zn2+ at 25 μg mL–1 (concentration chosen based upon the results of the dissolution experiments).

The toxic response was evaluated as cell counting at 24, 48, 72 and 96h after the addition of ZnO nanoparticles, as a function of the exposure concentration in comparison with the average growth of replicate, un- exposed control cultures. Cell counting was performed with bright field microscopy using the Cellometer Auto X4 (Nexcelom, USA), which simultaneously calculates the percentage of cell viability (live/dead test, conducted with the trypan blue dye).

The average specific growth rate (μ) was calculated as the logarithmic increase in the cell counting from the following equation for each single flask of controls and treatments, from days 0 to 4 (calculated section-by-section):

μi j =In Xi In Xj

tj ti (1)

where Xi is the cell counting at time i and Xj is the same parameter at time j.

The percent inhibition of growth rate for each treatment replicate was then calculated as:

%Ir =μC μT

μC100 (2)

where %Ir is the percent inhibition in average specific growth rate, μC is the mean value for average specific growth rate (μ) in the control group, and μT is the average specific growth rate for the treatment replicate. Finally, the inhibition percentage for each BT powder was plotted against the logarithm of the BT concentrations, and then regression analysis was performed to obtain the values of the concentration of the BT powders suspended in test medium that results in a 50% reduction in the growth within the exposure period (ErC50).

2.3. Assessment of the Factors Linked to the Toxicity

2.3.1. Microscopic Observation

The interaction between the microalgae and ZnO nanoparticles (100 μg mL–1, after 72h of exposure) was observed using SEM and TEM. For SEM, control and microalgae after contact with ZnO nanoparticles were fixed with a mixture containing 2.5% of glutaraldehyde and 1.0% of picric acid in phosphate Sörengen buffer (0.1 M, pH = 7.4). Dehydration was achieved in a series of ethanol baths (from 50% to 100%), and then the samples were dried with a BAL-TEC CDP 030 supercritical point dryer. The images were obtained in a Zeiss Supra 40 microscope equipped with an in-lens detector (low excitation voltage = 2.5 kV and small working distance = 3 mm were used). For TEM, control and treatments were fixed with a mixture containing 2.5% of glutaraldehyde and 1.0% of picric acid in phosphate Sörengen buffer (0.1 M, pH = 7.4). Post-fixation using 4% osmium tetroxide (OsO4) was conducted and the dehydration was achieved in a series of ethanol baths (from 50% to 100%). The samples were processed for flat embedding in a Spurr resin, and then ultrathin sections were made using a Reicherd-Young Ultracut microtome (Leica). Sections were contrasted with a 4% aqueous uranyl acetate solution and Reynold’s lead citrate before visualization, which was performed in a Tecnai 12 operating at 80kV equipped with a 1Kx1K Keen View camera.

2.3.2. Effect of Particles on Algae Oxidative Stress

Superoxide dismutase (SOD), which catalyzes the dismutation of the superoxide anion (O2.-) into hydrogen peroxide and molecular oxygen, was quantified in the controls and treatments (1, 50 and 100 μg mL–1, at 24, 48, 72 and 96h of exposure) using a SOD assay kit-WST 19160 (Sigma-Aldrich, Germany). This allows very convenient SOD assaying by utilizing Dojindo’s highly water-soluble tetrazolium salt, WST-1 (2-(4-Iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl) -2H-tetrazolium, monosodium salt) that produces a water-soluble formazan dye upon reduction with a superoxide anion. The rate of the reduction with O2 is linearly related to the xanthine oxidase (XO) activity, and it is inhibited by SOD. Therefore, the IC50 (50% inhibition activity of SOD or SOD-like materials) can be colorimetrically determined. The controls and treatments were incubated at 37 °C for 20 min, and then read at 450 nm using an Envision multilabel plate reader (Perkin-Elmer, USA).


2.3.3. Effect of Particles on Algae Photosynthetic Activity

The photosynthetic activity of the controls and treatments (1, 50 and 100 μg mL–1, at 24, 48, 72 and 96h of exposure) was determined through the Pulsed Amplitude Modulation (PAM) method, using a Handy PEA (Hansatech, UK) fluorometer. This method uses the saturation pulse principle, in which a sample is subjected to a short pulse of light that saturates the photosystem II (PSII) reaction centers of the active chlorophyll molecules. This process suppresses photochemical quenching, which might otherwise reduce the maximum fluorescence yield. A ratio of variable over maximal fluorescence (Fv/Fm) can then be calculated, which approximates the potential quantum yield of PSII.

2.3.4. Effect of Particles on Algae Mitochondria

Intracellular levels of adenosine-5-triphosphate (ATP) in controls and treatments (1, 50 and 100 μg mL–

1, at 24, 48, 72 and 96h of exposure) were quantified using an ATP Bioluminescent assay (Sigma-Aldrich). The algae cell lyses was mechanically achieved in a vortex using glass beads followed by centrifugation (15 min at 2,000 g), in order to obtain the free ATP at the supernatant. The assay can quantitative bioluminescent determinate the ATP in samples containing 2 10–12 to 8 10–5 M. ATP is consumed and light is emitted when firefly luciferase catalyzes the oxidation of D-luciferin. When ATP is the limiting reagent, the light emitted is proportional to the ATP present. The relative luminescent units were detected with an En- vision multilabel plate reader equipped with a luminescent optical filter.

2.4. Statistical Analysis

Statistical analyses were performed using SPSS v.14.0. For comparisons among control and treatments, ANOVA test followed by Tukeys’ post-hoc test was conducted for the variables that meet the criteria of normality (Shapiro-Wilk test, p>0.05), homoscedasticity (variance homogeneity, Levene test, p>0.05) and independence (Durbin Watson test, p~2.0). For the variables that did violate the assumptions for ANOVA valitidy, a non-parametric Kruskal-Wallys test was conducted. Differences between groups were consi- dered statistically significant when p < 0.05.

3. RESULTS AND DISCUSSION

3.1. ZnO Characterization

The XRD pattern of the powders can be seen in Figure 1. By the exposed, ZnO-NaOH is hexagonal and

anisotropic [in the (00l) direction: 255 nm, with 0.1% of micro strain; in the (hk0) direction: 39 nm, with 0.06% of micro strain]. ZnO-KOH is hexagonal and anisotropic [in the (00l) direction: 103 nm, with 0.04% of micro strain; in the (hk0) direction: 46 nm, with 0.07% of micro strain]. ZnO-lot1, ZnO-lot2 and ZnO-lot3 have the (00l) direction larger than the (hk0), but any direction is above 100 nm [about 120 nm in (hk0) and 200 nm in (00l) direction].

Figure 1: XRD of (a) ZnO-lot1; (b) Zn-O lot2; (c) ZnO-lot3; (d) ZnO-NaOH; and (e) ZnO-KOH.

(a)

(b)

(c)

(d)

(e)


Morphologically, we can see that the particle grains are similar between ZnO-lot1, ZnO-lot2 and ZnO-lot3, and also between ZnO-NaOH and ZnO-KOH (Figure 2). As the difference among the first three is only the day of production, and given that the XRD patterns were very similar, we decided to use for our experiments only ZnO-lot1, as a representative of this process.

Figure 2: TEM micrographs of (a) ZnO-lot1; (b) Zn-O lot2; (c) ZnO-lot3; (d) ZnO-NaOH; and (e) ZnO-KOH.

A dissolution experiment was carried out (Figure 3) at pH 7.0 for BB and 8.01 for SRW. ZnO-NaOH and ZnO-KOH released more Zn2+ in the media than ZnO-

lot1 – approximately 22% after 96h in BB medium for both nanoparticles, against 12% for ZnO-lot1. The same happened in SRW, but the dissolution was even greater (almost up to 40% for ZnO-KOH). The higher porosity of these hydrothermal materials can have played a role in this greater Zn2+ leaching out process from the surface. Yet, the dissolution rates of the powders in BB medium or SRW were not greater than 40%, and this is the reason why this was the concentration of free Zn2+ ions chosen (40 μg mL–1, based upon the maximum concentration tested of 100 μg mL–1 for ZnO) to perform the toxicological assessment: to evaluate whether the released (and consequently penetrated) ions could be a potential reason for the toxicity.

Figure 3: Dissolution rate of ZnO powders in terms of Zn2+ leached out as a function of time.

Even with the high percentage of dissolution, it was still lower than a previous study by Rocha et al. [11], who found nearly 60% of dissolution after 96h (and almost 90% after 168 h), also in SRW. In this study, the authors used nanoparticles synthetized using zinc acetate in diethylene glycol medium, obtaining 50 nm length and 15 nm diameter. As these particles are smaller than the ones we used, we can hypothesize that the dissolution of ZnO is inversely proportional to its size, which explain both the differences (between ZnO-lot1 and ZnO-NaOH and ZnO-NaOH, and between our work and the one of Rocha et al.).

Although no zeta potential study was performed, which is a limitation of this study, previous works from our research group with ZnO from diverse synthesis routes showed that zero-point charge is generally expected to be around pH 9.0 [5,11]. According to these authors, this material tends to be slightly negative between pH 4 and 6, neutral between pH 8 and 10 and highly negative between pH 10 and 12.


3.2. Toxicological Assessment

The toxicological assessment was conducted using two culture media: the traditional BB culture medium and also the SRW, because artificial culture media may not be always fully representative of nanoparticles behavior into the environment. In Figure 4, we can see the growth of C. vulgaris as a function of time of exposure to ZnO nanoparticles. Reduction of growth in the exposed cells was observed; therefore we can infer that both ZnO nanoparticles were toxic for C. vulgaris

(p<0.05), in BB and SRW, and so the process for production did not interfere in the toxicity of ZnO. On the contrary, the effects seem to be inherent to the nature of ZnO.

Figure 4: Growth of C. vulgaris as a function of concentration and time of exposure to ZnO particles in (a) BB medium, and (b) Seine River Water.

In BB, the toxic effect occurred for all the concentrations tested, except for ZnO-lot1 and ZnO-KOH at 1 μg mL–1 (after 24h of exposure). In SRW, the non-visible effect in the fist day also occurred with ZnO-NaOH at 1 μg mL–1, but, after that, the inhibition of growth was more pronounced than in BB. Regarding the ErC50, results are shown in Table 1.

The effects were way more pronounced in SRW than in BB medium. This can be hypothesized to have occurred due to the presence of a contaminant from the SRW or a worse physiological/lower resistance caused by the paucity of nutrients of SRW (nitrate, phosphate, carbon sources such as glucose) [12]. This

trend was also observed when evaluating cell viability (Figure 5), i.e., C. vulgaris exposed to ZnO in both media had a decreased ability to exclude dye. These differences on behavior of the microalgae in the two media can be explained by the fact that the culture media can influence the expression of cell membrane proteins [13].

Table 1: ErC50 of ZnO Obtained by Different Processes, Accordingly to the Growth Media Used

ErC50 (μg mL–1

) Nanoparticulate ZnO

BB Medium Seine River Water

ZnO-lot 11.51 0.97

ZnO-NaOH 13.56 1.91

ZnO-KOH 16.32 1.15

Figure 5: C. vulgaris viability as a function of concentration and time of exposure to ZnO particles in (a) BB medium, and (b) Seine River Water.

The effect on growth of the free Zn2+, for their turn, suggest that the toxic events may have occurred in a great extend due to their release into the media. In a theoretical concentration similar to the released by the particles, the inhibition of algae growth was very similar to the higher concentrations of nano-ZnO. This Zn2+-mediated effect meets the findings from the works with ZnO previously cited here.

Besides the high probability of causal relation between the observed effects and the released Zn2+, some other hypothesis were tested, in order to try to explain the higher mortality caused by the nano-ZnO,


compared to the free ions. We evaluated the following the possible causes, already reported on literature: (i) a direct contact of the particles with the cell wall [14]; and/or (ii) an indirect effect through the generation of reactive oxygen species (ROS) [15]. Based on that, we performed some assays to assess which is the possible one involved in ZnO toxicity to C. vulgaris.

3.3. Assessment of the Factors Linked to the Toxicity

The possible adsorption and consequent internalization of ZnO through the cell membrane were assessed by SEM and TEM imaging (Figures 6 and 7). Although no conclusive report can be made based on

Figure 6: C. vulgaris in BB medium: SEM micrographs [(a) control, (c) exposed to ZnO-lot, (e) exposed to ZnO-NaOH, (g) exposed to ZnO-KOH] and TEM micrographs.in SRW [(b) control, (d) exposed to ZnO-lot, (f) exposed to ZnO-NaOH, (h) exposed to ZnO-KOH.


the imaging techniques used, we can have some indicatives of the phenomena involved. We can see by SEM that there probably is no ZnO nanoparticle adsorbed on microalgae membranes. In fact, it seems that they, as solid particles, damaged the cell by physical pressure into the membrane, deforming the microalgae. We can also see, by TEM, that there is no indicative of detectable particle inside the cells vesicles. What we can actually see is the formation of

particle aggregates within the algae culture, surrounding the cell. Yet, the TEM images showed that exposed cells produced a gum-like material. This is related to the cell ability of C. vulgaris to produce a sugar - residue composed by a high - molecular - weight polymer known as exopolysaccharide (EPS) as a form of adaptive protection [16]. This EPS could have prevented the direct contact of the nanoparticles with the cell, but not the contact of Zn2+, one of the possible

Figure 7: C. vulgaris in Seine River Water: SEM micrographs [(a) control, (c) exposed to ZnO-lot, (e) exposed to ZnO-NaOH, (g) exposed to ZnO-KOH] and TEM micrographs in SRW [(b) control, (d) exposed to ZnO-lot, (f) exposed to ZnO-NaOH, (h) exposed to ZnO-KOH. Larger aggregates are seen in this medium.


reasons the toxicity still occurred even with limited contact of the particles with the algae. The EPS could also help forming the particles aggregates surrounding the microalgae, preventing particle internalization. Zeta potential of C. vulgaris is negative in both media used (data not shown), then it coherent with an electrostatic repulsion the algae would exert upon the particles, and vice-versa, which hampers absorption. This would be helped by the production and release of EPS into the media. Yet, larger aggregates were seen in SRW, what can also account for the higher toxicity within this medium and that was previously reported by Rocha et al. [11].

The cell oxidative stress was also assessed, in terms of inhibition rate of the SOD activity (Figure 8). After 48h, there was a slight increase in activity in some concentrations points, but a more pronounced effect can be seen after 48h, for both nanoparticles in both media. This finding can be inferred to a higher exposure/production of ROS (that can be linked to the absorption of the Zn2+ free ions), and this is often related to the activation of cell apoptosis [17]. The aggregates found surrounding the microalgae could be causing a decrease in the availability of necessary nutrients for the microalgae growth and even of light, causing the microalgae stress [18,19]. In this context, the mortality observed can be linked to the exposure to ROS generated by ZnO nanoparticles.

Figure 8: Superoxide dismutase (SOD) activity of C. vulgaris as a function of concentration and time of exposure to ZnO particles in (a) BB medium, and (b) Seine River Water.

The increased ROS exposure can also be indirect seen in the inhibition of photosynthesis [20] (Figure 9). Indeed, the photosynthetic activity was found statistically decreased in the microalgae grown in both media since the first 24h of exposure to the ZnO nanoparticles, but there was a gradual negative trend along the days. This effect was already observed by Brayner et al. [5], for their synthesized nanoZnO.

Figure 9: Photosynthetic activity of C. vulgaris as a function of concentration and time of exposure to ZnO particles in (a) BB medium, and (b) Seine River Water.

The deregulation in the cells energetic metabolism can also be confirmed by the intracellular ATP content, a parameter related to the mitochondrial activity. The decrease in ATP content (Figure 10) therefore may reflect a decrease in the activity of the mitochondria, which is related to cell viability, since they are responsi- ble not only for the ATP production, but also for the ROS production and for the release of proteins that control the apoptosis [16]. This effect is more notice- able from the first 24h of exposure, in both media.

4. CONCLUSIONS

Given the exposed, the main conclusions from the present study are: (i) nanoparticulate ZnO have an statistically significant toxic effect on C. vulgaris growth (significant effects on growth and cell viability were observed after 24h of exposure, both in BB medium and SRW, although the effects were more pronounced in this last one) – the effect seems to be mediated by a induced oxidative stress (SOD activity was found increased in the algae), probably linked to the Zn2+ free


ions released into the media (up to 22% of dissolution); (ii) the ZnO behavior in synthetic and natural culture media were statistically similar, although the toxic effects were more pronounced in SRW (ErC50 was nearly 10 times lower in SRW than in BB medium); and (iii) the process for production does not seem to be an issue in ZnO nanoparticles toxicity since all tested particles produced significant effects on microalgae growth (ErC50 was 11.51 for Zn-lot, 13.56 for Zn-NaOH and 16.32 for Zn-KOH μg mL-1 in BB medium, and 0.97, 1.91 and 1.15, respectively, in SRW).

Figure 10: Adenosine-5-triphosphate (ATP) content of C. vulgaris as a function of concentration and time of exposure to ZnO particles in (a) BB medium, and (b) Seine River Water.

ACKNOWLEDGMENT

H. Polonini thanks CAPES (04/CII-2008-Project 7, Network Brazil Nanobiotec) and Programa Ciência sem Fronteiras/CNPq (245781/2012-9) for the scholarships granted. All authors thank Institut Jacques Monod (Université Paris Diderot, Paris, France); FAPEMIG; Ludovic Mouton (microscopy); and Sophie Nowak (XRD analysis).

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Received on 05-08-2015 Accepted on 21-08-2015 Published on 16-09-2015

© 2015 Polonini et al.; Licensee Revotech Press. This is an open access article licensed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/), which permits unrestricted, non-commercial use, distribution and reproduction in any medium, provided the work is properly cited.

international journal of green technology, 1-12 1 does the ...zinc oxide (zno) is a major...

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