Testing ZnO nanoparticle ecotoxicity: linking time

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Nanotechnology industry represents a market in constant growth (Scown et al. ... Among metal oxide NPs, zinc oxide nanoparticles (ZnO. NPs), with an annual ...

Environmental Science and Pollution Research https://doi.org/10.1007/s11356-017-0815-3

RESEARCH ARTICLE

Testing ZnO nanoparticle ecotoxicity: linking time variable exposure to effects on different marine model organisms Simona Schiavo 1 & Maria Oliviero 1 & Jiji Li 1 & Sonia Manzo 1 Received: 18 July 2017 / Accepted: 20 November 2017 # Springer-Verlag GmbH Germany, part of Springer Nature 2017

Abstract Zinc oxide nanoparticles (ZnO NPs) are increasingly used in several personal care products, with high potential to be released directly into marine environment with consequent adverse impact on marine biota. This paper aimed to compare the ecotoxicological effect of ZnO NPs (< 100 nm) towards three marine organisms widely used in toxicity assessment: an algal species (Dunaliella tertiolecta), a bioluminescent bacterium (Vibrio fischeri), and a crustacean (Artemia salina). Bulk ZnO (ZnO bulk, 200 nm) and ionic zinc were also investigated for understanding the role of size and of ionic release in the ZnO toxic action. To this aim, different ecotoxicological tests were used: the inhibition of bioluminescence with V. fischeri at three exposure times (5, 15, and 30 min); the D. tertiolecta growth inhibition at 24, 48, and 72 h; the A. salina mortality at 24–96 h, and A. salina mortality and body growth each 3 days along chronic exposure (14 days). For all selected species, ZnO NPs toxicity was strictly dependent on the exposure time and different sensitivities were recorded: ZnO NPs were more toxic towards algae (EC50 2.2 mg Zn/L) but relatively less toxic towards bacteria (EC50 17 mg Zn/L) and crustaceans (EC50 96 h 58 mg Zn/L). During the 14-day chronic exposure of A. salina, ZnO NPs had a significant inhibition of vitality and body length (EC5014d 0.02 mg Zn/L), while the effect of ZnSO4 was not statistically different from the control. ZnO NP toxicity was related to zinc ions and to interactions of particle/ aggregates with target organisms and therefore to NP behavior in the testing matrix and to the different testing time exposures. Keywords Metal oxide nanoparticles . Acute/chronic exposure . Time-varying exposure . Artemia salina . Vibrio fischeri . Dunaliella tertiolecta

Introduction Nanotechnology industry represents a market in constant growth (Scown et al. 2010) that covers many sectors, from electronics to daily products (Marano and Guadagnini 2016). As consequence, the increasing use of nanoparticles (NPs) posed an urgent need to assess the risk linked to their release in the environment (Gottschalk et al. 2013). NP behavior, transport, and fate strongly depend on the environmental matrix and on physicochemical characteristics of the NPs. For example, the high salinity encourages the aggregation of NPs (Batley et al. 2013), while the presence of natural organic matter reduces the aggregation process,

Responsible editor: Cinta Porte * Sonia Manzo [email protected] 1

Enea, P.le E. Fermi 1 80055 Portici, Naples, Italy

stabilizing the NPs (Keller et al. 2010). Moreover, apart from aggregation and sedimentation processes, dissolution should also be considered in the case of metal oxide NPs. This implies NP transformation evolving with time in the matrix (Li et al. 2016), consequently, the toxic effects depend on NP physicochemical state and time exposure of the organisms. Among metal oxide NPs, zinc oxide nanoparticles (ZnO NPs), with an annual global production of about 550 t, are one of the most widely used, being commonly employed in manufacturing of cosmetics and sunscreens (Piccinno et al. 2012). For this reason, it is estimated that ZnO NPs could be easily discharged in marine environment also during bathing and swimming (Danovaro et al. 2008). Various studies investigated the aquatic toxicity of ZnO NPs (Bondarenko et al. 2013; Adam et al. 2014; Xiao et al. 2015), but toxicity data for marine species are still scarce though they are largely needed for the proper risk assessment in seawater. It was reported that ZnO NPs affected cell growth and viability in chronic test with microalgae (Manzo et al. 2013a; Suman et al. 2015) and exerted toxic effects in amphipods chronically

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exposed (Fabrega et al. 2012; Hanna et al. 2013). Acute effect (96 h) were also detected for the crustaceans Artemia salina (Ates et al. 2013) while no data are actually available about a longer exposure (i.e., 14 days). ZnO NPs damaged the larval development (Fairbairn et al. 2011; Manzo et al. 2013b; Wu et al. 2015) and impaired the immune system (Manzo et al. 2017) and the cytogenesis (Oliviero et al. 2017) in sub-chronic exposure of sea urchins. Moreover, a recent chronic toxicity study on marine copepod Tigriopus fulvus exposed to ZnO NPs was reported by Parlapiano et al. (2017). In this work, we tested the ZnO NPs toxic effects with test organisms largely employed as models in NP toxicity assessment (Heinlaan et al. 2008): Dunaliella tertiolecta, which represents a primary producer widely used in ecotoxicity studies (Aruoja et al. 2009), the bioluminescent bacterium Vibrio fischeri, a representative microorganism of the aquatic environment, mainly used for rapid determination of the toxicity of metal-containing NPs (Heinlaan et al. 2008), and the crustacean Artemia salina that, belonging to zooplankton, plays an important role in the energy flow of the food web in a marine environment (Parra et al. 2001). We evaluated, for each organism, the ZnO NPs effect evolution along different exposure phases with the aim to elucidate the relationship among exposure time, the relative ZnO NPs effect concentration (NOEC and EC50), and biological complexity. Bulk ZnO (ZnO bulk, 200 nm) and ionic zinc (ZnSO4) effects are investigated in order to assess, by comparison, the role of particle size and of ionic zinc release. In particular, we evaluated the following: & & & &

The growth inhibition (24, 48, and 72 h of exposure) on D. tertiolecta The inhibition of bioluminescence of V. fischeri at three different exposure time 5, 15, and 30 min The mortality with A. salina (24–96 h) The mortality and body growth with A. salina each 3 days along a chronic exposure (14 days)

Materials and methods

Particle dispersions Stock suspensions of ZnO NPs < 100 nm, ZnO bulk, and ZnSO4 were obtained by chemical dispersion in Artificial SeaWater (ASW) (ASTM 1998) (ASW, NaCl 0.4 M, MgCl 2 ∙6H 2 O 0.053 M, Na 2 SO 4 0.02 M, CaCl 2 ∗H 2 O 0.01 M, KCl 9 mM, NaHCO3 2 mM, KBr 0.8 mM, H3BO3 0.4 mM, SrCl2∗6H2O 0.09 mM, NaSiO3∗9H2O 0.07 mM) to the final concentration of 100 mg Zn/L (120 mg/L mass of ZnO NPs and ZnO bulk used to prepare the stock suspensions). Just before the tests, ZnO particles stock suspensions (ZnO NPs and ZnO bulk) were dispersed by bath-sonication for 30 min at 50 W. Test suspensions were then prepared in ASW. The physicochemical characterizations of ZnO materials in exposure medium (ASW) were previously thoroughly evaluated (Manzo et al. 2013a, b; Li et al. 2016) and randomly verified in the present work.

Test organisms The toxicity of ZnO particles and ZnSO4 was evaluated upon three different marine organisms: microalgae Dunaliella tertiolecta, bacteria Vibrio fischeri, and crustaceans Artemia salina. D. tertiolecta (Chlorophyceae: Chlamydomonadales) algae were cultured in sterilized standard medium (f/2 medium) made with artificial standard seawater (ASTM pH 8.0, 0.22 μm filtered). To provide inoculant for experiments, microalgae were incubated under cool continuous white fluorescent lights (about 58 μmol photons m−2 s−1) at 24 ± 1 °C with aeration for 5–7 days until log phase growth prevailed. V. fischeri bacteria were used in lyophilized form, and they were rehydrated with the reconstitution solution (Ecotox LDS S.r.l) just prior to perform the test. A. salina cysts were purchased from Ecotox LDS S.r.l and stored in the dark at 5 °C until use for experiments. To allow the hatching, 100 mg of cysts were transferred into Petri dishes with 120 mL of ASW and exposed to a light source (3000–4000 lx) for 1 h at 25 °C, then the cysts were incubated in the dark at the same temperature for 24 h. The hatched larvae were transferred in a new Petri dish with ASW (120 mL) for 24 h at 25 °C. Nauplii 48 h old were used for the tests.

Chemicals Algal growth inhibition test ZnO NPs (cod. 544906) and ZnSO4 (cod.7446-20-0) were purchased from Sigma-Aldrich S.r.l. According to the manufacturer, ZnO NPs had pristine size < 100 nm, surface area of 15–25 m2/g, and purity > 99%, while ZnSO4 had purity of 99%. ZnO bulk powder for pharmaceutical formulation was purchased from Galeno S.r.l. (Italy), and it had particle pristine size 200 nm, specific surface area of 4–7 m2/g, and purity > 99.9%.

Algal bioassay with D. tertiolecta was performed according to ISO 10253:2006 protocol (ISO 10253:2006) with some modifications. All glassware was acid-washed, rinsed with purified MilliQ water, and autoclaved before use. Starting from previous results (Manzo et al. 2013a) we design a 72-h algae growth inhibition test with a daily cell counting and selecting ZnSO4 as reference toxicant in order to allow the comparison

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Zn/L) in ASW for all chemicals (ZnO NPs, ZnO bulk, and ZnSO4) were sonicated for 30 min at 50 W before use. The plates were incubated at 25 °C in darkness for 96 h. No food was provided during the exposure. The deprivation from food did not induce any lethal effects on Artemia larvae even up to 96 h (Ates et al. 2013) (control surviving at 96 h > 90%). Larval mortality was determined after 24 and 96 h by counting dead larvae (i.e., those which exhibited no internal or external movement during 10-s observation). All treatments were performed in triplicate. A control group was also set up without the test chemicals. Test was performed at least three times.

among all test performed. Suspensions were prepared at the following concentrations: 0.1, 0.5, 1, 3, 5, and 10 mg Zn/L. After, all the suspensions were sonicated for 30 min at 50 W before the addition of micronutrients (f/2 medium) (Guillard 1975), deprived of metals, to avoid the addition of EDTA that would complex any free metal ions in the test media, and microalgae. Previously, microalgae (with a final density of 10 4 cells/mL) were rinsed three times with filtered (0.22 μm) autoclaved ASW. Test was performed at least three times. Test plates (transparent glass, 10 mL) were kept in a growth chamber constantly illuminated with a white fluorescent lamp (enhanced irradiation between 400 and 500 nm), at a temperature of 24 ± 1 °C for 72 h. A control group was also set up without the test chemicals. Algae growth was recorded daily (24, 48, 72 h) by cell counting with a Burker Chamber.

Artemia salina chronic toxicity test The chronic test with A. salina was performed according to Manfra et al. (2012, modified). Ten larvae of A. salina were transferred to the testing beakers containing 50 mL of the ZnO NPs, ZnO bulk, and ZnSO4 testing concentrations (0.03, 0.06, 0.12, 0.25, and 0.5 mg Zn/L) in ASW. Four replicates were prepared for each treatment, and a control group was also set up without the test chemicals. Unlike the original protocol, instead of media changing, that could modify the testing condition due to the behavior of NPs, the transfer of the organisms three times a week in new beakers with the same suspension was performed. Moreover, to guarantee the same NP physicochemical conditions in beakers and similuate a continuum in the exposure, all testing beakers (i.e., 24 beakers: 4 replicates for 6 changes for each NP concentration) were prepared at the beginning of the experiment and posed in the same experimental condition. The testing beakers were covered with parafilm (leaving a space for air passage) and maintained at 25 (± 2) °C for the whole test duration with illumination of 900 (± 100) lux (photoperiod ratio 14 to 10 h). After 3, 5, 7, 9, and 12 exposure days, the number of live organisms was counted in each test beaker to verify the survival rate then the alive organisms were transferred in the testing beaker prepared the first day of experiment and the food supplement was added. To feed the organisms’ aliquots of D. tertiolecta, microalgae suspension were added (105 cells/mL). After 7 and 14 days of treatment, the length of live crustaceans’ body was measured. Test was performed at least three times.

Vibrio fischeri bioluminescence test Lyophilized bacteria were rehydrated with the included reconstitution solution (ultrapure water) just prior to performing the test. Luminescent bacteria were incubated under constant temperature (15 °C) in glass vials (Azur Environmental) with 1 mL of ASW and ZnO NPs, ZnO bulk, and ZnSO4. Nine concentrations of each substance, ranging from 0.3 to 40 mg Zn/L, were prepared and sonicated. Test suspensions from 50 mg Zn/L onwards could not be measurable for the testing since the samples were turbid, and this could generate interferences. The luminescence of each individual vial was measured using Microtox Model 500 Analyzer at 5, 15, and 30 min. The sample toxicity was evaluated based on the decrease of the intensity of light produced by the luminescent bacteria. A control group was also set up without the test chemicals. Test was performed at least three times.

Artemia salina acute toxicity test The acute test with A. salina was performed according to the Organization for Economic Cooperation and Development, OECD 202 testing guidelines (OECD 2004). The bioassay was carried out in 50-ml glassware contaniners, with 5 nauplii per well. Three different concentrations (10, 50, and 100 mg

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Fig. 1 Growth inhibition (expressed as % of effect, see Materials and methods) for D. tertiolecta algae exposed to ZnO NPs (a), ZnO bulk (b), and ZnSO4 (c) for 24, 48, and 72 h. *Statistically different with respect to the control (p = 0.05)

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Data analysis All data presented in this study are reported as mean ± SD. One-way ANOVA was applied in order to test for significant differences between treatments and control (significance level was always set at p = 0.05). The EC50 were calculated using the linear interpolation method (inhibition concentration procedure (ICp)) (US EPA 1993). The bootstrap method is used to obtain the 95% confidence interval, because standard statistical methods for confidence interval calculations are not applicable. No observed effect concentration (NOEC) were determined by Dunnett’s test (US EPA 1989). Concentration–response functions were statistically determined by applying a best-fit procedure (Scholze et al. 2001). Statistical analysis was performed with SPSS13.0 software.

Results and discussion ZnO physicochemical behavior in ASW As we previously thoroughly assessed and verified in the present work, the instability of ZnO in seawater results in the formation of large aggregates (micrometric sized) that show a different sedimentation rate also in relation to the initial particle concentration and pristine size. Ionic zinc released into seawater reached the 65% of the initial solid loading at 10 mg Zn/L (Li et al. 2016) after few hours, while at 1 mg Zn/ L, about 50% after 24 h (Oliviero et al. 2017). After 48 and 72 h, the percentage of dissolution still remained the same. The initial mean size of ZnO NP aggregates was around 700 nm at 10 mg/L and around 1450 nm at 100 mg/L (Manzo et al. 2013b). The particle size increased up in the following 24 h to 1800 nm and 2500 nm for 10 and 100 mg/ L, respectively (Manzo et al. 2013a). The aggregation kinetics of ZnO bulk particles at 10 and 100 mg/L had a similar trend with an average size of 1350 and 2000 nm, respectively, that increased to 1800 and 3000 nm for 10 and 100 mg Zn/L. The 100 mg Zn/L aggregates showed a rapid sedimentation (30% after 2 h) differently from 10 mg Zn/L (30% for ZnO bulk and

Table 1 NOEC calculated for V. fischeri, D. tertiolecta, and A. salina exposed to each tested substance (ZnO NPs, ZnO bulk, and ZnSO4) ZnO NPs (mg Zn/L)

ZnO bulk (mg Zn/L)

ZnSO4 (mg Zn/L)

V. fischeri 5 min

41.95

81.9

0.3

15 min

41.95

81.9

0.3

30 min D. tertiolecta

1.28

81.9

0.3

24 h

0.1

> 10

0.5

48 h

1

> 10

0.1

72 h

1

0.5

0.1

A. salina 24 h

> 100

> 100

> 100

96 h 3 days

10 > 0.5

10 0.125

10 > 0.5

5 days 7 days

0.5 0.25

0.125 0.125

> 0.5 > 0.5

9 days

0.06

0.03

> 0.5

12 days 14 days

0.03 0.03

0.03 0.03

> 0.5 > 0.5

10% for ZnO NPs, after 4 h). In the main, the sedimentation rate of ZnO NPs has a slow, constant trend while ZnO NP aggregates show a steeper sedimentation curve. Dunaliella tertiolecta The effects of ZnO NPs, ZnO bulk, and ZnSO 4, upon microalgae growth in relation to the exposure time (24, 48, and 72 h), are shown in Fig. 1. After 24 h of ZnO NPs exposure (Fig. 1a), a 50% effect up to 3 mg Zn/L and an effect > 60% from 5 mg Zn/L was registered, while ZnO bulk (Fig. 1b) caused an effect not related to dose. Relative to ZnSO4 exposure (Fig. 1c), a doseindependent effect was observable with the maximum effect (80%) registered at 10 mg Zn/L. At 48 h, ZnO NPs showed a lower toxicity trend, with 40% effect up to 3 mg Zn/L (Fig. 1a), while ZnO bulk exerted the

Fig. 2 Comparison of EC50 values for V. fischeri, A. salina, and D. tertiolecta exposed to ZnO NPs (a), ZnO bulk (b), and ZnSO4 (c) at different times. Asterisks indicate that the EC50 values were not computable

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shading, limitations in nutrient uptake) (Wang et al. 2016). Accordingly, the microscope images showed a ZnO hetero aggregation with algae in the range of concentration 0.1– 3 mg Zn/L after 24 h (Fig. 3). The increasing toxic effects at 5 and 10 mg Zn/L could also be due to a cytotoxic effect induced by ZnO action around the algae, as previously reported (Schiavo et al. 2016). Moreover, in the first hours, ionic zinc released into seawater, reached the 50–65% of the initial solid loading (i.e., 1–10 mg Zn/L) (Li et al. 2016, Oliviero et al. 2017). However, by comparing ZnSO4 and ZnO NPs toxicity trend (Fig. 1a) with EC50 24 h values (1.8 and 2.2 mg Zn/L for ZnSO4 and ZnO NPs, respectively) (Fig. 2a, c), we can conclude that the effects of ZnO NPs may not be solely ascribed to ion release and that the pristine nanosize contributes to the final toxic effect. (Heinlaan et al. 2008; Wong et al. 2010; Ji et al. 2011).

Fig. 3 Optical microscopy images (×40 magnification) showing D. tertiolecta entrapped in ZnO NPs aggregates at 3 mg Zn/L (24 h)

same effect already at 1 mg Zn/L (Fig. 1b). Besides ZnSO4 showed a 40% growth inhibition starting from 1 mg Zn/L (Fig. 1c). The 72 h EC50 values ranged from 1.81 to 4.5 mg Zn/L (Fig. 2). The NOEC trend, for algae exposed to ZnO NPs (Table 1), evidenced a particular algae sensitivity after the first 24 h of exposure at very low Zn concentrations: 0.1 mg Zn/L was the lowest NOEC values and was obtained at 24 h for ZnO NPs (48–72 h ZnO NPs NOEC 1 mg Zn/L) and at 48 and 72 h for ZnSO4. Instead, the lowest ZnO bulk NOEC was registered at 72 h (0.5 mg Zn/L). Since the estimated environmental NP concentration ranges from nanograms to micrograms per liter (Keller and Lazareva 2013), algae should be considered potentially at risk. The ZnO NP toxicity was mainly attributed to the generation, in the aqueous matrix, of aggregates larger than ZnO bulk ones that act by entrapping the algal cells, inhibiting cell growth or the photosynthetic process and releasing zinc ions close to cells (Wong et al. 2010; Ji et al. 2011; Manzo et al. 2013a). Starting from the first hours, also at low concentration (0.1 mg Zn/L) (Fig. 1a), ZnO NP rapid aggregation in test medium induced adverse effects as mechanical damages (e.g., cell wall injuries) or indirect physical effects (e.g.,

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In Fig. 4, the inhibition of Vibrio fischeri bioluminescence after 5, 15, and 30 min of exposure to ZnO NPs (Fig. 4a), ZnO bulk (Fig. 4b), and ionic zinc (ZnSO4) was reported (Fig. 4c). After 5 and 15 min of exposure to ZnO particles, only a biostimulation of bacteria bioluminescence was observed. Instead, ionic zinc NOEC (Table 1) and EC50 values (Fig. 2c) corresponding to very low concentrations (NOEC 5– 15 min, 0.3 mg Zn/L; EC50 5 min, 2.5 mg Zn/L; EC50 15 min, 2.7 mg Zn/L) were calculated. After 30 min exposure time, ZnO NPs NOEC value of 1.28 mg Zn/L (Table 1) and EC50 of 17 mg Zn/L were registered (Fig. 2a); a Bbiostimulation^ was instead evident for ZnO bulk (Fig. 4b) while the NOEC value was similar to previous exposure times (81.9 mg Zn/L). ZnSO4 EC50 was 2.8 mg Zn/L (Fig. 2c), and NOEC value was 0.3 mg Zn/L (Table 1). The ZnO NP effects evidenced upon V. fischeri were in agreement with those previously reported in literature (Heinlaan et al. 2008; Mortimer et al. 2008; Sovova et al. 2009). Although the ZnO toxicity for V. fischeri was generally explained by ion dissolution (Heinlaan et al. 2008;

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Fig. 4 Percentage of effect (inhibition of bioluminescence) for Vibrio fischeri exposed to ZnO NPs (a), ZnO bulk (b), and ZnSO4 (c). *Statistically different with respect to the control (p = 0.05)

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Fig. 5 Acute toxicity (mortality) for A. salina exposed to ZnO NPs (a), ZnO bulk (b), and ZnSO4 (c) at different exposure time. *Statistically different with respect to the control (p = 0.05)

Bondarenko et al. 2013), in this work, we found that, below 1 mg Zn/L, both ZnO compounds were less toxic than ZnSO4. This could be explained by the immediate availability of Zinc ions resulted by ZnSO4 dissolution, while ZnO particles take some time to dissolve (Oliviero et al. 2017). Actually, as suggested by Heinlaan et al. (2008), ZnO particle dissolution could mainly occur after the intimate contact of the particles with bacterial cell wall that induces changes in microenvironment near the contact area. Artemia salina In Fig. 5a, the results of A. salina acute exposure to ZnO NPs are reported. After 24 h, only a slight effect (9.7%) at the highest tested concentration (100 mg Zn/L) was registered (NOEC, > 100 mg Zn/L; EC50 not computable) (Table 1; Fig. 2a). ZnO bulk (Fig. 5b) showed a similar trend (NOEC, > 100 mg Zn/L; EC50 not computable); while for ionic zinc (Fig. 5c) at 24 h, the highest registered effect was 22.5% (50 mg Zn/L) that also correspond to NOEC (> 100 mg Zn/ L) and EC50 (not computable). At 96 h, the ZnO NP toxicity reached 70% effect (NOEC 10 mg Zn/L; EC50 58.3 [40.00–71.43] mg Zn/L) (Table 1; Fig. 2a). For ZnO bulk, a similar trend was obtained with 80% effect at 100 mg Zn/L (Fig. 5b) (NOEC 10 mg Zn/L; EC50 38 [32.22–46.00] mg Zn/L) (Table 1; Fig. 2b); while for ionic zinc (Fig. 5c), the highest effect was 94.7% (50 mg Zn/L), and NOEC value was obtained at 10 mg Zn/L (Table 1).

ZnO NPs then affected A. salina only at the end (96 h) of the acute exposure and at high concentration (Fig. 5a). Similarly, Ates et al. (2013) highlighted the key role of exposure time in NP-mediated toxicity and reported comparable results. Moreover, for freshwater crustacean Daphnia magna, Zhu and co-authors (2010) evidenced toxic effects only after 72 h TiO2 NP exposure. In order to additionally explore the NP effects, we also tested a longer exposure of A. salina (14 days). ZnO NPs (Fig. 6a) exerted a lethal effect with a trend dependent on dose and exposure time. In the first week of exposure, at low ZnO NP concentrations (0.03, 0.06, and 0.12 mg Zn/L), no significant (p < 0.05) effects were detected while, during the second week, the number of dead individuals significantly increased with time (Fig. 6a). In fact, until 5 days of exposure, NOEC was always higher than 0.5 mg Zn/L (Table 1). After 7 days of exposure, a significant mortality in A. salina (2.75 and 0.75 mean number of live individuals, for 0.25 and 0.5 mg Zn/L, respectively) (Fig. 6a) was observed while, in the second week, the mortality increased and no live individuals at 12 days of exposure for 0.25 mg Zn/L and at 9 days for 0.5 mg Zn/L (Fig. 6a) were recorded. In addition, NOEC values decreased after 7–9 days of exposure down to 0.06 mg Zn/L (Table 1). The 14-day ZnO NP EC50 value was 0.02 [0.02–0.02] mg Zn/ L (Table 2, Fig. 2a). The individual growth was also evaluated after 7 and 14 days by body length measurement (Fig. 7). At all ZnO NPs concentrations, and at both observation times, a similar individual length mean decrement (about 60%) with respect to control was calculated (Fig. 7a).

Fig. 6 Mean number of live individuals for A. salina exposed to ZnO NPs (a), ZnO bulk (b), and ZnSO4 (c) for 14 days at different concentrations

Environ Sci Pollut Res Table 2 EC50 calculated for each tested substance (ZnO NPs, ZnO bulk, and ZnSO4) for Artemia salina (chronic toxicity test) ZnO NPs (mg Zn/L)

ZnO bulk (mg Zn/L)

ZnSO4 (mg Zn/L)

3 days

N.C.

0.1 [0.09–0.12]

N.C.

5 days 7 days

0.43 [−] 0.20 [0.17–0.29]

0.08 [0.07–0.09] 0.08 [0.07–0.09]

N.C. N.C.

9 days

0.05 [0.05–0.06]

0.03 [0.02–0.07]

N.C.

12 days 14 days

0.02 [0.02–0.02] 0.02 [0.02–0.02]

0.02 [0.02–0.02] 0.02 [0.02–0.02]

N.C. N.C.

spp., feed assimilation process (i.e., filtration, ingestion) is particularly susceptible to stressing action especially during the larval growth when the feeding efficiency increases with the number of thoracopods (Lavens and Sorgeloos 1991). Therefore, the observed chronic toxicity may be due to scarce food intake and then to the derived malnutrition possibly caused by a high accumulation of nanoparticles in the gut (Fig. 8). The consequent reduction of Artemia body length obtained during the chronic exposure (Fig. 7a) was previously observed only for crustaceans exposed to Ag NP (Zhao and Wang 2011). The comparison of A. salina dose response curves obtained for ZnO NPs and ZnSO4 (Fig. 5a, c) showed that the effect could not be solely ascribed to Zn ions. Actually, the hypothesis that the toxicity of ZnO nanoparticles is exclusively caused by dissolved ions is still somewhat debatable since several studies evidenced differences between responses to NPs and corresponding salts. Certainly, ZnO pristine size and the consequent behavior in testing media played a role in the toxic effect generation (Zhao et al. 2013; Yung et al. 2015). The ZnO bulk quick aggregation (particle size up to 1300 ± 200 nm) starting from the first exposure hours also could cause a feed hindrance for the organisms, making more rapid the overall toxic effect.

Values in brackets denote the upper and lower limits of 95% confidence interval N.C. Not computable

ZnO bulk concentrations of 0.03 and 0.06 mg Zn/L (Fig. 6b) induced a decrement of survival with increasing exposure time and the death of all individuals after 5 days of exposure starting from 0.25 mg Zn/L, and only after 3 days starting from 0.5 mg Zn/L (Fig. 6b). Up to 7 days exposure, NOEC was 0.125 mg Zn/L, instead, after 9 days, it decreased to 0.03 mg Zn/L (Table 1). EC50 values for ZnO bulk (Table 2) were 0.08 [0.07–0.09] mg Zn/L at 7 days and 0.02 [0.02–0.02] mg Zn/L at 14 days of treatment. After 7 days, we observed a significant (p < 0.05) decrease of mean length of live individuals at all ZnO bulk concentrations (Fig. 7b) and a similar situation could be noted after 14 days (Fig. 7b). The exposure of A. salina to ionic zinc (ZnSO4, Fig. 6c) induced a decrease of individual number; however, the observed mortality resulted not significantly (p < 0.05) different from control, the EC50s were not computable (Table 2) and NOEC values were always higher than 0.5 mg Zn/L (Table 1). The mean length of individuals exposed to ZnSO4 after 7 and 14 days resulted similar to not exposed ones (Fig. 7c). It was noteworthy that, at the end of the first exposure week, a phase of peculiar sensitivity occurred that lead to, at the 9th day, a 75% reduction for NOEC and EC50 values. This event could represent a Bcritical window^ (Mueller et al. 2016) in the regular development of Artemia: a period in which the emerging phenotype of the animal is particularly plastic or susceptible to an environmental stressor. In Artemia

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14 d

3.5

Most of the studies on the species sensitivity to ZnO NPs were performed with freshwater organisms (Heinlaan et al. 2008; Garcia-Gomez et al. 2014). These studies highlighted that the sensitivity mainly depends on the organism complexity and generally, microalgae and bacteria were more affected in comparison to crustaceans and fishes. Regarding marine environment, only Wong et al. (2010), to the best of our knowledge, reported the toxicity of ZnO towards different organisms such as microalgae, crustacean, and fish, reaching the same conclusion. Our results (Fig. 2) highlighted that besides to the single species sensitivity, it is important to identify the most sensitive phases for the organisms, along the time exposure. This is particularly important with those NPs, such as ZnO NPs, that give rise to different transformation processes (i.e., dissolution, aggregation) along time and in dependence to test media (Keller et al. 2010). 7d

c

14 d

2 1.5

* *

* *

1

* *

* *

*

2

*

1.5

*

1

* *

0.5

0.5

* * *

*

0.25

0.5

0

0 0

0.03

0.06

0.12

ZnO NPs mg (Zn/L)

0.25

0.5

14 d

3 *

2.5

length (mm)

* *

length (mm)

3

2.5

7d 3.5

3.5

3 length (mm)

Consideration upon marine organisms different sensitivity

2.5 2 1.5 1 0.5 0

0

0.03

0.06

0.12

ZnO Bulk (mg Zn/L)

0

0.03

0.06

0.12

0.25

0.5

ZnSO4 (mg Zn/L)

Fig. 7 Mean body length in A. salina larvae after 7 and 14 days of exposure to ZnO NPs (a), ZnO bulk (b), and ZnSO4 (c). *Statistically different with respect to the control (p = 0.05)

Environ Sci Pollut Res Fig. 8 a Microscopic observation (×10 magnification) of A. salina exposed to 0.5 mg Zn/L of ZnO NPs. b detail of the gut with ZnO NPs inside. Control in the inset

Among acute tests, V. fischeri resulted more sensitive to ZnO NPs then A. salina. In fact, many studies refer to Artemia as one of the most resistant specie in ecotoxicity studies (Guerra 2001; Minguez et al. 2014; Manzo et al. 2014), and this is particularly true for NP exposure (Falugi et al. 2012). Although a higher algae sensitivity to ZnO NPs with respect to other organisms was generally reported (Wong et al. 2010; Ji et al. 2011), A. salina chronically exposed resulted even more responsive. By the evaluation of ZnO NP effect evolution along different exposure phases, bacterium was more sensitive in the longer exposure (30 min), while algae showed the highest sensitivity after 24 h and Artemia resulted more sensitive to ZnO NPs only at 14-day exposures. These results should be taken into account in experimental design for ZnO toxicity evaluation.

Conclusions In this paper, the sensitivity of organisms exposed to ZnO NPs has been evaluated monitoring the effect along the test exposure phases. The comparison among acute and chronic exposure allowed to highlight the importance of a long exposure test in the assessing of ZnO NP toxicity and effects in long-term traits, such as organism growth. In particular, A. salina chronic test turned out to be very sensitive, more than algae chronic test. Actually, a Bcritical windows^ could be identified during the Artemia test, showing the most sensitive phase of the larval growth. In addition, this test seemed to be able to detect toxicity even at very low concentrations corresponding to the estimated environmental ones. From the evaluation of ZnO NP effects upon D. tertiolecta during the whole exposure, it could

A

B

be evidenced that algae were more sensitive after 24 h of exposure. In view of the actual NP environmental concentrations and of their peculiar physicochemical behavior in seawater, it is very important to select appropriate test battery, tailored for the specific NP class, able to detect toxicity at very low doses and with short exposure time. Funding sources This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

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