Ecotoxicology and Environmental Safety 114 (2015) 109–116

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Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv

Effect of cultivation media on the toxicity of ZnO nanoparticles to freshwater and marine microalgae Andriana F. Aravantinou a, Vasiliki Tsarpali b, Stefanos Dailianis b, Ioannis D. Manariotis a,n a b

Environmental Engineering Laboratory, Department of Civil Engineering, University of Patras, 26504 Patras, Greece Section of Animal Biology, Department of Biology, Faculty of Sciences, University of Patras, 26504 Patras, Greece

art ic l e i nf o

a b s t r a c t

Article history: Received 6 June 2014 Received in revised form 16 January 2015 Accepted 17 January 2015

The aim of this work was to investigate the effect of zinc oxide nanoparticles (ZnO NPs) on freshwater and marine microalgae cultivated in different media. Freshwater species Chlorococcum sp. and Scenedesmus rubescens were cultivated in modified Blue–Green medium (BG-11) and Bold's Basal Medium (BBM), and marine species Dunaliella tertiolecta, and Tetraselmis suesica, cultured in salt modified BG-11 and f/2 medium. The microalgae species were exposed for 96 h with a daily reading of algal growth rate, to different ZnO NPs concentrations (0.081–810 mg/L). Significant differences were observed on microalgae growth rates, with the marine being more sensitive than the freshwater species, as revealed by their half inhibitory concentration values (IC50). The IC50 values in freshwater species were affected by the culture medium. The lowest IC50 values (o 2.57 mg/L) were observed in the marine species. S. rubescens showed the less toxic effect in cultures with modified BG-11, compared to BBM cultures, with IC50 values 4810 mg/L and 14.27 mg/L after 96 h exposure time, respectively. ZnO nanoparticles appeared to have toxic effects in all species tested, depended on the species type, the exposure time, the NPs concentration, and mainly the culture medium. & 2015 Elsevier Inc. All rights reserved.

Keywords: Microalgae Toxicity Nanoparticles Zinc oxide Culture medium

1. Introduction The use of engineered metal oxide nanoparticles (NPs) has increased in recent years in material science and nanotechnology industries for a great variety of applications, including sensors, catalysts, and incorporation into commercial products (Godwin et al., 2009; TRS, 2004; USEPA, 2005). Among them, zinc oxide nanoparticles (ZnO NPs) is of great importance, with their annual global production to be estimated in 550 ton, classifying them third in production order after SiO2 (5550 ton) and TiO2 (3000 ton) (Piccinno and Gottschalk, 2012). ZnO NPs are used as ultraviolent light absorbents additives in sunscreens, toothpastes and beauty products (Serpone et al., 2007), as well as in rubber manufacture, production of solar cells and LCD, pigments, chemical fibers, electronics, and textiles (Bondarenko et al., 2013; Klaine et al., 2008). Finally, ZnO NPs have been also employed as antimicrobial agents (Padmavathy and Vijayaraghavan, 2008). The properties of nanomaterials raise concerns about their adverse effects, while their environmental impact remains a relatively unexplored area of research (Farre et al., 2009; Melegari et al., 2013). For instance, the aquatic environment is likely to be n

Corresponding author. Fax: þ 30 2610 996573. E-mail address: [email protected] (I.D. Manariotis).

http://dx.doi.org/10.1016/j.ecoenv.2015.01.016 0147-6513/& 2015 Elsevier Inc. All rights reserved.

the ultimate recipient of discharged nanomaterials (Klaine et al., 2008) and different critical reviews have been focused on the environmental hazard assessment of engineered NPs (Fang et al., 2007) and their toxic effects in fish (Handy et al., 2008) and invertebrates (Baun et al., 2008). In addition, since their surface properties are of great importance for their aggregation behavior, data regarding their mobility and interactions with aquatic biota, such as algae, are still scarce. Microalgae are a major constituent of the aquatic food chain. Marine microalgae play an important role in coastal ecosystems (Behrenfeld et al., 2006; Buffet et al., 2013), while freshwater microalgae are employed in natural wastewater treatment systems (Aravantinou et al., 2013). Many ways that NPs cause toxic effects on algal cells (e.g. membrane damage, protein binding, loss of function, mutation, mitochondrial damage, lysis, adhesion etc.) have been reported (Manzo et al., 2012; Rodea-Palomares et al., 2012; Xia et al., 2011). Although zinc is classified as an essential nutrient for aquatic organisms, algae can be highly sensitive to zinc. The toxicity of ZnO NPs, bulk ZnO and ionic zinc on various microorganisms have been shown to exert inhibitive and lethal effects on microalgae (Aruoja et al., 2009; Chen et al., 2012b; Ji et al., 2011; Ma et al., 2013; Manzo et al., 2013). The main mechanisms of ZnO NPs toxicity include particle dissolution to ionic zinc, particle-induced generation of reactive oxygen species (ROS), and photo-induced toxicity under ultraviolet radiation (Ma et al.,

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2013; Miao et al., 2010; Peng et al., 2011). However, although, a lot of studies reported toxicological tests with the use of ZnO NPs to algal species, the behavior of ZnO NPs as well as their effects on algal species cultivated in different culture media, remains still unclear. According to the latter the main goal of the present study was to investigate the effect of ZnO NPs on both freshwater (Chlorococcum sp. and Scenedesmus rubescens), and marine microalgae (Dunaliella tetrtiolecta and Tetraselmis suesica) cultivated in different media with different zinc concentrations. Freshwater species were cultivated in modified Blue Green medium (BG-11) and Bold's Basal medium (BBM), while saline species in salt modified BG-11 and f/2 medium. In addition, the surface properties of ZnO NPs were examined because of their aggregation, and mobility behavior. NPs and algae interactions were also investigated, since the diverse culture media could impact the algal growth and the behavior of nanoparticles. To our knowledge, this is the first comparative toxicity study of ZnO NPs on microalgae with different culture media.

2. Materials and methods 2.1. Nanoparticles ZnO NPs was purchased from Sigma-Aldrich, USA (catalog number 544906). The ZnO NPs had particle size smaller than 100 nm and a specific surface area of 15–25 m2/g, as reported by the manufacturer. A stock solution of 810 mg ZnO NPs/L in Milli-Q water was prepared and used in further experiments. Nanoparticles solution was dispersed using an ultrasonic bath (TranssonicTI-H-5, Elma Hans Schmidbauer GmbH & Co KG, Germany) before each experiment. 2.2. Microalgae Four different algae strains, were selected on the basis of their presence in municipal wastewater and their potential use for biofuel due to their lipids content. The S. rubescens SAG 5.95 and the Chlorococcum spec. SAG 22.83 are freshwater species and were obtained from the bank SAG Culture Collection of the University of Göttingen. The marine strains Dunaliella tertiolecta CCAP 19/6B and the T. suesica were obtained from the Department of Biology, University of Patras. 2.3. Culture medium Both freshwater species (Chlorococcum spec. and S. rubescens), were cultivated in ⅓N BG-11 (BG-11 enriched with one third times the nitrates) and BBM medium recommended for freshwater microalgal species (Chen et al., 2012a; Karemore et al., 2013). The ⅓N BG-11 and BBM media have quite different zinc concentration. The composition of the media employed is shown in Table A.1 (see Supporting information – Appendix A). The marine species (D. tertiolecta and T. suesica) were cultured in f/2 and salt ⅓N BG-11 (⅓N BG-11 supplemented with NaCl) media, which are very common for marine species cultivation (Aravantinou et al., 2013; Chen et al., 2012a; Tsiaka et al., 2013). The initial pH of ⅓N BG-11, BBM, salt ⅓N BG-11 and f/2 culture media was 7.5, 6.2, 8.3 and 8.2, respectively. The salt ⅓N BG-11 medium was supplemented with 35.0 g/L NaCl for the marine strains. 2.4. Nanoparticle exposure and algal growth studies The toxic effects of ZnO NPs on the four selected microalgae strains, (freshwater and marine strains), were determined

according to ΟECD 201 protocols and guidelines (OECD, 2011). The experiments were carried out in two phases. In the first phase (phase 1) all algae strains were cultivated in the investigated medium to obtain stable characteristics. During the exponential growth phase, and when the microalgal was acclimated in the medium, an appropriate volume was withdrawn and transferred to a 250 mL sterile flask, which contained 100 mL sterile medium to start the second phase (phase 2). The initial algal concentration was 104 cells/mL. The flasks were housed in a constant temperature walk-in room at an average temperature of 21 72 °C. Illumination was continuously provided by 36 W fluorescents lamps (cool daylight). The light intensity at the surface of the conical flasks was 100 μmol m  2 s  1. All procedures were performed under aseptic conditions. The nutrients and all components were sterilized. All media and cultivation apparatus were sterilized with an autoclave sterilizer (121 °C, 20 min). All conical flasks were closed with sterilized hydrophobic cotton wool to prevent contamination. Considering that environmental relevant concentrations may range from nanograms to milligrams per liter (Gottschalk et al., 2009; Luo et al., 2011), microalgae strains were exposed for different periods of time (24, 48, 72, and 96 h) to ZnO NPs concentrations ranging from 0.081 to 810 mg/L (0.081, 0.81, 8.1, 81, and 810 mg/L). Each experiment was performed in duplicate (2 technical replicates per ZnO NPs concentration and experiment). Every 24 h, the cell number was counted in triplicate using a Neubauer hemocytometer. Every batch toxicity test was accompanied by a positive control (with the use of K2Cr2O7, according to OECD (2011)). 2.5. Nanoparticles and algae characteristics The interaction of ZnO NPs with algal surface was studied by scanning electron microscopy (SEM) analysis (microscope JEOL 6300, JEOL Ltd.). An elemental analysis on the samples surface was conducted after 96 h exposure for all ZnO NPs concentrations tested. A volume of 1–2 mL of algae was transferred on slides, dehydrated in an oven and glued to SEM stubs with colloidal silver and sputter-coated with gold–palladium using a gold ion sputter coater (JEOL, JFC1100 Fine Coat). The samples were examined with a SEM operating at 20 kV. For each sample, at least three fields were observed at different magnifications between 2500 to 14,000  . The size distribution of each ZnO NPs powder was measured by dynamic light scattering (DLS) after sonication for 30 min (25 °C, 130 kHz) with a particle size analyzer (Zetasizer NanoZS, Malvern Instruments, UK). The ZnO NPs suspensions (0.081–81 mg/L) were prepared in the corresponding medium of the algal toxicity test. ZnO NPs size distribution was monitored every 24 h for 4 d. All suspensions were vigorously shaken prior to each measurement to resuspend any settled ZnO. The samples were placed in clean disposable cuvettes, and at least three consecutive measurements were performed at 25 °C. Each measurement consisted of at least 10 runs. The zinc concentration was determined after the addition of 8.1 mg/L of ZnO NPs (which corresponds to 6.5 mg/L of Zn2 þ ) in all media used and in algal cultures after 96 h exposure. An atomic absorption spectrometer (AAnalyst 800, Perkin-Elmer), equipped with air–acetylene flame was employed to measure zinc contents. The zinc concentration was determined after applying nitric acid digestion (APHA et al., 1998). Specifically, in each case, 100 mL of sample, was filtered through a 0.2 μm pore size syringe filter, and transferred to a 125-mL conical flask, in which 5 mL HNO3 was added. The solution was gently boiled and evaporated on a hot plate to 20 mL volume. Afterwards, the solution was transferred in a 100-mL volumetric flask with two 5-mL portions of water,

A.F. Aravantinou et al. / Ecotoxicology and Environmental Safety 114 (2015) 109–116

μ = lnX L –lnX 0/tL –t0

(1)

where XL is the number of cells at time tL (days), and X0 the initial number of cells at time t0. All experiments were carried out in duplicate and the toxicity of NPs on microalgae was calculated by the half inhibitory concentration values (IC50); the IC50 is the percentage of the ZnO concentration that causes 50% of growth inhibition per day. The mean IC50 value and the standard deviation was determined from 6 replicates, using Probit Analysis (IBM SPSS software), the level of

A

4

3. Results and discussion 3.1. ZnO NPs-induced effects on microalgae cultivated in different media ⅓N BG-11 and BBM culture media are common for freshwater algae, especially for Chlorococcum sp., whereas for marine algae, salt ⅓N BG-11 and f/2 culture medium were used. These media were selected not only because they are commonly used but also

50

40

40

30

30

20

20

10

10

0

0

C

50

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30

20

20

10

10

0

0

E

F

60

50

50

40

40

30

30

20

20

10

10

0

0

G

60

D

60

50

60

B

60

50

60

Cell number x10 (cells/mL)

After checking for homogeneity of the variance (Levene's test of equality of error variances), the significant differences among the parameters measured in algae were tested, with the use of Mann– Whitney U-test (p o0.05). Significant alterations in the growth rate (μ) observed in each algae treated with each concentration of ZnO NPs for a period of 24, 48, 72 and 96 h were tested with the use of the Friedman test (p o0.05).

H

60

50

50

40

40

30

30

20

20

10

10

0

T. suesica

60

(2)

Chlorococcum sp.

The specific growth rate (μ) was determined from the growth phase by the following equation:

Inhibition (%)=[(Ccontrol − Ctoxicity )/Ccontrol ]x100

S. rubescens

2.6. Data analysis

significance was accepted at p o0.05. The growth inhibition rate (I%) was calculated according to the OECD 201 guideline (OECD, 2011):

D.tertiolecta

adding these rinsings to the volumetric flask. After the solution had reached room temperature, it was diluted to the mark, mixed thoroughly and used for metal determination. The metal analysis method was verified with known concentrations of each metal tested (Pure Atomic Spectroscopy standards, purchased by PerkinElmer Life and Analytical Sciences, USA). Whenever “Filtered zinc” is used in the present paper, it refers to zinc, which passes a 0.2 μm pore size filter.

111

0 0

20

40

60

80

Exposure time (h)

Control

0.081

100

0.81

0

8.1

20

40

60

80

Exposure time (h)

81

100

810 mg ZnO/L

Fig. 1. Effect of different ZnO NPs concentrations on microalgae cell numbers over a period of 96 h. Chlorococcum sp. cultivated in (A) ⅓N BG-11 and (B) in BBM. S. rubescens cultivated in (C) ⅓N BG-11 and (D) in BBM. D. tertiolecta cultivated in (E) salt ⅓N BG-11 and (F) in f/2. T. suesica cultivated in (G) salt ⅓N BG-11 and (H) in f/2.

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due to their different ZnSO4  7H2O concentration (Table A.1), and consequently the different Zn2 þ content. As it is seen, among different culture media the Zn2 þ content significantly varies, a fact that could play an important role both in ZnO NPs dose effect and algal responses. Specifically, ⅓N BG-11 and salt ⅓N BG-11 contain 0.222 mg/L ZnSO4  7H2O, while BBM and f/2 contain 8.82 mg/L and 0.022 mg/L ZnSO4  7H2O, respectively. In case of freshwater algal species, the results showed a differential algal species response in terms of growth rate in different culture media. More specifically, Chlorococcum sp. showed a time depended growth rate increase when cultivated in BBM culture media, compared to those occurred in ⅓N BG-11 (Fig. 1A and B). On the other hand, S. rubescens showed almost similar trend of growth rates when cultivated either in BBM or ⅓N BG-11 culture media (Fig. 1C and D). Based on the different Zn2 þ content between the two media, it could be suggested that those two freshwater species posses different requirements for Zn2 þ in their dietary. However, the presence of different concentration of ZnO NPs in both culture media seems to differentially affect the growth rate of both algal species. In case of Chlorococcum sp. incubated with different ZnO NPs concentrations in BBM culture media (high initial Zn2 þ content), a significant growth rate inhibition was observed over time, while in algae incubated in ⅓N BG-11 (low initial Zn2 þ content) the presence of ZnO NPs did not seem to induce further alteration in growth rates, compared to control values. In case of S. rubescens whose growth rate does not seem to be mediated by the different content of initial Zn2 þ concentration in two culture media, the presence of ZnO NPs led to significant growth arrest over time, especially at increased ZnO NPs concentrations. The latter is more evident in case of S. rubescens cultivated in BBM culture medium in the presence of 810 mg/L ZnO NPs, where the growth rate at 72 and 96 h in BBM was almost zero, whereas in ⅓N BG-11 was 0.92 70.04 and 0.767 0.02 d  1 for the 72 and 96 h, respectively (Table A.2). According to the results of the present study, it seems that the toxic effects of ZnO NPs on both freshwater microalgae species depends merely on species dietary specific demand for Zn2 þ concentration as well as ZnO NPs mediated Zn2 þ . BBM has the highest Zn2 þ concentration, so at a first glance it can be assumed that the presence of a high ZnO NPs concentration may be more toxic for the algae. This cannot be supported by the present study because the number of cells and the related growth rates were higher in BBM than in ⅓N BG-11, especially for Chlorococcum sp. (Fig. 1A and B). On the other hand S. rubescens seems to better adjust in ⅓N BG-11 than in BBM, with the presence of ZnO NPs. The growth rate was similar without the presence of ZnO NPs in the control, whereas in the presence of ZnO NPs were

Table 2 Determination of the size of each microalgae species, before and after treatment for 96 h with 8.1 mg/L of ZnO NPs. Microalgae species

Medium

⅓N BG-11 BBM ⅓N BG-11 BBM salt ⅓N BG-11 f/2 salt ⅓N BG-11 f/2

Chlorococcum sp. S. rubescens D. tertiolecta T. suesica

Algae size (μm) without NPs

with NPs

8.90 70.10 13.27 70.020 7.6770.68 5.82 71.46 9.02 70.32 8.82 72.32 12.31 70.10 13.16 71.40

3.767 1.78n 4.29 7 0.23n 8.46 7 0.72 8.977 0.72n 6.92 7 0.82n 6.157 1.75 9.497 0.58n 8.99 7 1.27n

Note. The results are mean 7 SDs from the analysis of 10 cells in each case. Asterisks denote significant difference between ZnO NPs-treated cells and ZnO NPs-free cells in any case (Mann–Whitney U-test, p o 0.05).

different, especially in the highest concentration of NPs (Fig. 1C and D). The experimental results revealed that ZnO NPs affected the growth rate of Chlorococcum sp. and S. rubescens cultured in both media, even at very low concentrations (0.081 mg/L) (Fig. A.1, Table A.2). Despite the short time of the exposure period in two different media (⅓N BG-11 and BBM), the toxic effect of the low ZnO NPs concentration (0.081 mg/L) on the two freshwater algae was demonstrated at the first 24 h of exposure. Similar results were reported by Aruoja et al. (2009) who studied the toxicity of ZnO NPs and bulk ZnO on Pseudokirchneriella subcapitata cultured in OECD TG 201 medium, and mentioned that ZnO in both forms were toxic even at concentrations below of 0.1 mg/L. On the other hand, Chen et al. (2012b) reported that ZnO NPs did not affect the freshwater Chlorella sp. viability, cultivated in Algo-Gro freshwater medium, up to a concentration of 0.81 mg/L after a 24 h exposure time. Consequently, it seems that the toxicity of ZnO NPs to freshwater microalgae is related with the algal species and the culture medium. The growth rates of the two marine microalgae, D. tertiolecta and T. suesica, cultivated in two different media (salt ⅓N BG-11 and f/2) are also presented in Fig. 1E–H. D. tertiolecta and T. suesica, are common marine microalgae which have been employed for toxicity tests (Manzo et al., 2012, 2013; Tsarpali et al., 2012; Tsiaka et al., 2013). D. tertiolecta showed a steady growth during cultivation in salt ⅓N BG-11, whereas in f/2 medium seemed to need an adjustment time. Even though in the first days of cultivation, the algae growth rates in f/2 medium (Fig. 1E and F) were lower than the rates in salt ⅓N BG-11, at 96 h a similar cell number in both media was observed. On the other hand, T. suesica exhibited

Table 1 ZnO NP inhibitory concentrations (IC50 values) of algal species at 24, 48, 72 and 96 h exposure period. Microalgal

Medium

IC50 (mg/L) Exposure time (h)

D. tertiolecta T. suesica Chlorococcum sp. S. rubescens

f/2 Salt ⅓N BG-11 f/2 Salt ⅓N BG-11 BBMs ⅓N BG-11 BBMs ⅓N BG-11

24

48

72

96

0.88nab (0.13–9.52) 1.22n (ND) 0.13nab (0.11–0.17) 0.52nab (0.41–0.66) 30.82nabc (4.24–734.51) 37.36nabc (ND) 6.97nabc (0.82–65.13) 38.10nabc (17.37–98.74)

1.01nc (0.81–1.26) 1.24n (0.17–51.46) 0.82cd (0.66–1.03) 0.97cd (0.22–3.40) 0.77nad (0.07–3.30) 7.99nade (0.73–102.45) 42.68nade (8.86–471.45) 300.59nade (110.29–1314.63)

1.44a (ND) 1.34 (1.11–1.77) 1.97ac (0.30–12813.32) 2.41ac (0.36–14.64) 1.78be (0.19–9.94) 2.03bdf (0.42–7.38) 22.56nbdf (2.77–336.65) 4810nbd ( 4810 –ND)

1.50bc (ND) 1.33 (ND) 2.10bd (1.01–4.85) 2.57bd (0.64–10.11) 893.55ncde (244.60–7590.10) 0.75ncef (0.04–4.63) 14.27ncef (0.88–674.04) 4810nce ( 4810 – ND)

IC50 values (in terms of mg/L) and confidence intervals (lower and upper bound values within parenthesis), as obtained by Probit analysis, p o0.05, N ¼ 4). Values in each row that share the same letter are significantly different from each other. Values with asterisks in each column for each species are significantly different from each other (Mann– Whitney U test, p o 0.05).

A.F. Aravantinou et al. / Ecotoxicology and Environmental Safety 114 (2015) 109–116

*

1.2

1.0

1.0

0.8

0.8

0.6

0.6

0.4

0.4

0.0

1.2

0.2 1/3N BG-11

BBM

C

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1.0

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0.8

0.6

0.6

0.4

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0.2

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0.0

0.0 salt 1/3N BG-11

f/2

1/3N BG-11

BBM

D

*

0.2

B

*

A

*

% Zn content per algal surface

1.2

113

salt 1/3N BG-11

f/2

Fig. 2. Scanning electron microscopy (SEM) analysis of Zn2 þ on (A) Chlorococcum sp., (B) S. rubescens, (C) D. tertiolecta and (D) T. suesica surface cultured for 96 h in different culture media without NPs (dark bars) and with 8.1 mg L  1 ZnO NPs (light bars). The results are the mean 7SDs from the analysis of 10 samples in each case. Asterisks denote significant difference among values observed in ZnO NPs-treated algae with those in NPs-free algae cultures in any case (Mann–Whitney U-test, p o 0.05).

similar growth rate trend with the two media. The cultivation matrix has significant contribution on the toxic effects of ZnO NPs on algae species (Tables 1 and A.2). It should be also noted that Manzo et al. (2013) studied the exposure of several limit concentrations of nanoparticles, including ZnO on the growth inhibition of three algal species (T. suesica, D. tertiolecta and Isochrysis galbana). They concluded that the most sensitive organism, D. tertiolecta, underwent a very toxic effect owing to NP exposure, a fact that could lead to the suggestion that the aforementioned species is a target organism more suitable to reveal the ecotoxicity effects of these new contaminants. The latter was further reinforced by the fact that among the four algae species employed in the present study D. tertiolecta was the most sensitive organism. In contrast to freshwater species, the two marine species (D. tertiolecta and T. suesica), were not affected by ZnO NPs at a concentration of 0.081 mg/L. As it shown in Fig. 1E–H, the cell number without the presence of ZnO NPs coincides with the cell number at an exposure concentration of 0.081 mg ZnO/L. Previous studies (Manzo et al., 2013) on D. tertiolecta cultivated in standard culture media (Guillard medium) showed that the toxic effects were not significant at ZnO concentrations below of 0.08 mg/L. However, in the present study, total inhibition of algal growth (100%), was observed for concentrations Z8.1 mg/L of ZnO NPs (Figs. 1 and A.1). The high inhibition of algal growth was demonstrated in both marine species over the entire exposure period (24–96 h). This suggests that the damage in the algae cells was permanent, as it was observed by SEM images of the algae cells (data not shown). On the other hand, inhibition even at the lowest exposure concentration of ZnO NPs (0.081 mg/L) was observed for the freshwater species. A complete suppression (100% inhibition) was observed only in the highest exposure concentration (810 mg/L). It should be noted that the inhibition was lower when Chlorococcum sp. were cultivated in BBM than in ⅓N BG-11, probably due to the significantly different initial zinc content of culture media, and algae-specific dietary requirements. In contrast, the cultivation of S. rubescens in BBM showed much greater inhibition compared to ⅓N BG-11 medium. Moreover, S. rubescens inhibition was up to

20% after 96 h of exposure, even at the highest exposure concentration (810 mg/L) of ZnO NPs. In this case it could be supposed that shading, due to the high NPs concentration, did not contribute to additional inhibition probably due to NPs sedimentation effects. A similar observation was reported by Aruoja et al. (2009), who stated that there was not shading effect on algal growth even in the case of higher TiO2 concentrations, due to the large TiO2 clumps settling within 24 h, allowing sufficient light to pass though thus attenuating NPs inhibitory effects on algal growth rate. The toxic effect of ZnO NPs on algal growth could be estimated more adequately by the IC50 values (Table 1). As it seen, the highest IC50 values (correspond to less toxic effects of ZnO NPs) were observed for S. rubescens cultured in ⅓N BG-11 medium. The IC50 was increased with the exposure time, from 38.10 mg/L (17.37 to 98.74 mg/L) at 24 h, to greater than 810 mg/L at 96 h. It appears that S. rubescens was adjusted in the presence of ZnO NPs, and even at the highest concentrations the algal growth was not affected. The most toxic effects of ZnO NPs appeared as the lowest inhibitory concentration values, which were observed in the marine species. The IC50 values for D. tertiolecta and T. suesica were not greater than 1.50 and 2.57 mg/L, respectively, indicating that ZnO NPs were more toxic for D. tertiolecta. 3.2. Effect of ZnO NPs size and zinc release on algal species According to SEM analysis of microalgae size before and after exposure to ZnO NPs, our results showed a significant decrease of algal cells size in almost all cases of ZnO NPs-treated algal species (Table 2, Fig A.2). This is evident since it is known that viable algal cells reduce their size in order to minimize the effect of zinc ions released in ZnO NPs (Chen et al., 2012b). The latter further reinforced by data obtained after SEM analysis, which showed increased levels of zinc ions onto algal surface (Figs. 2 and A.3). On the other hand, the size of S. rubescens was increased in the presence of ZnO NPs, in BBM culture media (Table 2), which is in accordance with the increased size of Chorella sp. cells following

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2500

A

Table 3 Determination of Zn content in used media and microalgae cultures after 96 h exposure at 8.1 mg/L of ZnO NPs.

2000 1500 1000

Medium

Microalgal

Estimated Zn (mg/L)

Filtered Zn (mg/L)

Filtered Zn reductiona (%)

BBM

– Scenedesmus rubescens Chlorococcum sp. – Scenedesmus rubescens Chlorococcum sp. –

8.5

9.617 3.53 8.42 7 4.21

– 12.38

9.23 7 3.98 4.707 0.06 0.617 0.02n

3.9 – 87.02

4.23 7 3.88 7.007 0.06

10 –

4.23 7 3.49

39.57

4.25 7 2.82

39.28

5.727 0.02 4.53 7 0.04n

– 20.9

0.96 7 0.05n

83.3

500 0 2500

⅓N BG-11

B

2000

Salt ⅓N BG-11

1500 1000 500

Size (nm)

0 2500

f/2

C

2000

500

D

2000 1500 1000 500 0

0.081

81

E

800 600 400 200 0

6.505

a The values represent the percentage the reduction (%) of Zn content, in relation to algal-free medium in any case. n Asterisks indicate significant difference from algal free medium in each case (Mann–Whitney U-test, p o 0.05) Note. The addition of 8.1 mg/L of ZnO NPs corresponds to 6.5 mg/L of Zn.

1000

1000

6.55

Arithmetic mean 7standard deviation for 3 independent measurements in each case.

1500

0 2500

Dunaliella tertiolecta Tetraselmis suesica – Dunaliella tertiolecta Tetraselmis suesica

6.55

0.081 0.81 8.1 81 ZnO NPs Concentration (mg/L)

Fig. 3. Size of ZnO NPs at 0 h (dark bars) and after 96 h (light bars) in different culture media without microalgae: (A) BBM; (B) ⅓N BG-11; (C) salt ⅓N BG-11; (D) f/ 2; (E) Distilled water. Error bars represent standard deviation of 30 runs.

exposure to ZnO NPs at a concentration of 0.81 mg/L (Chen et al., 2012b). In this context, viable cells currently tested, possess decreased surface area in order thus minimizing zinc ions uptake without affecting zinc ions absorption. The above does not exclude the enhancement of ZnO NPs toxic effects observed in all species, at least in case of high concentration of ZnO NPs tested in the present study. The particle size of ZnO NPs suspensions typically showed two distinct populations, one with a mean peak of several nanometers

(o100 nm), and the presence of larger aggregates with a mean peak of several hundred nanometers (4 200 nm) (data not shown). Similar results with two distinctive populations for the particle size of ZnO NPs was mentioned by Franklin et al. (2007) and Peng et al. (2011). At the highest ZnO NPs concentration (810 mg/L) visible flocs were observed, which settled within seconds. The determination of particle size distribution by DLS was challenging due to the extensive aggregation of ZnO NPs and the wide range of particles size in the culture medium. Nevertheless, DLS is a good choice when specifically examining whether nanoparticles have aggregated in solution according to Franklin et al. (2007). As it is seen in Fig. 3A–E, the particle size values were considerably higher than the nominal value of 100 nm. The particle size was affected by the initial ZnO NPs concentration, the solution composition, and the exposure time. After 96 h of exposure the pH in all cultures increased due to algal growth, and ranged from 8 to 10, indicating limited ZnO NPs dissolution (Miao et al., 2010). The aggregation of NPs was decreased with exposure time for BBM, ⅓N BG-11 and salt ⅓N BG-11, and increased for f/2 and distilled water. It should be mentioned that f/2 medium had similar behavior with distilled water. In freshwater media, ZnO NPs formed larger aggregations in BBM compared to ⅓N BG-11. The size of ZnO NPs in ⅓N BG-11 media seems to be considerably affected by NaCl concentration and it almost doubled in salt ⅓N BG-11 after 96 h compared to ⅓N BG-11. In the presence of NaCl pH values in the media were close to 8.2, which possibly blocks the dissolution of ZnO NPs in the medium (Miao et al., 2010) and as a result induces a rapid agglomeration of the particles (Buffet et al., 2013). The concentration of the filtered zinc in the culture media with and without the presence of ZnO NPs is given in Table 3. A high percentage of the NPs (471.7%) was found in the filtrate in various media without algae. The zinc content in the filtrate was reduced in the presence of algae indicating zinc uptake or sorption by algae. The highest reduction of filtered zinc was observed by S. rubescens cultured in ⅓N BG-11, which was expected since it was the

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algal with less toxic effect, and the higher number of cells. The measurement of the dissolved zinc is essential in these studies in order to investigate its influence of dissolved zinc toxicity on species, and attributed the effects solely to the nanoparticle suspensions. In fact, since metal nanoparticles play an important role in their toxicity (Miao et al., 2010; Peng et al., 2011), many studies attributed the toxicity of ZnO NPs to their released zinc ions (Bondarenko et al., 2013; Miao et al., 2010; Peng et al., 2011). Specifically, although there is current data regarding zinc ions exposure, Franklin et al. (2007) found comparable toxicity of ZnO NPs, bulk ZnO and ZnCl2 using the freshwater microalgae P. subcapitata and the toxicity was attributed to dissolved Zn. Moreover, Aruoja et al. (2009) investigated the effect CuO, ZnO and TiO2 nanoparticles to P. subcapitata, reported that the most toxic metal oxide to algae was ZnO NPs and the toxicity was attributed to the soluble metal ions derived from nanoparticles. Despite the fact that ZnO NPs’ toxicity seems to be related with the zinc ions, other factors may also contribute to toxicity, which are associated with the physicochemical properties of nanomaterials in the culture media, and NPs surface interactions with target organisms (Manzo et al., 2013; Miao et al., 2010; Miller et al., 2010).

4. Conclusions The experimental results of this work indicate that the sensitivity of algae to ZnO NPs strongly depends on the species type, the concentration of NPs, the medium composition, and exposure time. Our results showed that the response of each species against ZnO NPs, after incubation in culture media, containing different amount zinc content as nutrient was based on species-specific demands of zinc uptake as well as its ability to be adsorbed onto algal surface. In this context, the presence of zinc, released from ZnO NPs in each case, could be probably toxic, resulting in both algal growth rate arrest and algal size alterations. Freshwater algal species (Chlorococcum sp. and S. rubescens) were more resistive in the presence of high concentration ZnO NPs, than the marine species (D. tertiolecta and T. suesica). ZnO NPs at low concentration (0.081 mg/L) were not toxic to the marine species, whereas in freshwater algal species were toxic. The physicochemical state and changes of NPs in the culture media is so far uncertain and generally poor characterized, therefore, more studies should be done and great care should be adopted when dealing with nanomaterials.

Acknowledgments This research work has been partially supported by “K. Karatheodoris” Grant by the Research Committee of the University of Patras (D.169).

Appendix A. Supplementary information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.ecoenv.2015.01. 016.

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