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Keywords:

  • Toxicity;
  • Nanoparticles;
  • Ionic zinc;
  • Feeding inhibition;
  • Reproduction

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSIONS
  8. SUPPLEMENTAL DATA
  9. Acknowledgment
  10. REFERENCES
  11. Supporting Information

As the production of zinc oxide nanoparticles (ZnO-NPs) and other metal oxides is exponentially increasing, it is important to investigate potential environmental and health impacts of such nanoparticles. Nanoparticles' properties (e.g., size, dissolution rate) may change in different water media, and their characterization is essential to derive conclusions about toxicity results. Therefore, an aquatic model organism, Daphnia magna, was used to investigate the effect of ZnO-NPs with 2 different particle sizes (30 nm and 80–100 nm) and then compare these effects with ZnO microsized particles (>200 nm) and the ionic counterpart (in the form of ZnCl2) on immobilization, feeding inhibition, and reproduction endpoints. The 48-h median lethal concentration (LC50) for immobilization ranged between 0.76 mg Zn L−1 for the ionic zinc and 1.32 mg Zn L−1 for ZnO-NPs of 80 nm to 100 nm. For the chronic exposures, the reproduction output was impaired similarly among zinc exposures and possibly driven mainly by the zinc ionic form. The concentrations used showed a total dissolution after 48 h. On the other hand, feeding activity was more affected by the 30 nm ZnO-NPs than by the ionic zinc, showing that the particulate form was also playing an important role in the feeding inhibition of D. magna. Dissolution and particle size in the daphnia test media were found to be essential to derive conclusions on toxicity. Therefore, they can possibly be considered critical for evaluating nanoparticles' toxicity and fate. Environ Toxicol Chem 2014;33:190–198. © 2013 SETAC


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSIONS
  8. SUPPLEMENTAL DATA
  9. Acknowledgment
  10. REFERENCES
  11. Supporting Information

Metal oxide nanoparticles (NPs) (e.g., ZnO, TiO2) as well as fullerenes (e.g., C60) and carbon nanotubes (CNT) are among the most studied NPs in ecotoxicology as a consequence of their wide application in novel technologies [1-3], and their production rate is expected to continue to rise in the coming years [4]. Therefore, it is necessary to evaluate the hazard associated with engineered nanomaterials by evaluating their effects on organisms. For that, fate and distribution of nanomaterials in the environment should be assessed and studied to establish an accurate prediction on exposure and derive risk [5]. The development of nano-related detection techniques is of major importance to allow exposure estimations of nanomaterials that are being released to the environment.

As a consequence of the extensive production and use of engineered nanomaterials, they are likely to be released into the environment. These occurrences inevitably will lead to some degree of environmental and human exposure, instigating the need to catch up the fast growth of this multidisciplinary field [6].

As metal oxide NPs, zinc oxide NPs (ZnO-NPs) have received much attention as a result of their wide range of applications in nanotechnology. Because of their high ultraviolet radiation absorption [7], ZnO-NPs have been applied in a variety of personal care products such as sunscreens, toothpastes, and cosmetics [8, 9]. They can also be used in coatings [9], antifouling paints [10] environmental remediation processes [8], wastewater treatment [11], textiles [12], ceramics, rubber processing, food additives, and biosensors [13], or even used as catalysts in batteries [14] and antibacterial agents [15].

A considerable amount of literature already exists for ZnO-NPs, reporting the toxicity effects to several organisms, such as bacteria [16-18], algae [19-21], and fish [22, 23], as well as human cells [24]. A recent review published on this topic [25] lists the toxicity data available for ZnO NPs to aquatic organisms. But most of these studies are based on short-term or acute exposure tests, sometimes lacking ecological relevance, such as long-term effects or important ecological traits or endpoints. Additionally, NPs' size and solubility effects are often not fully considered and toxicity related.

It has been reported that when bulk counterparts are used to produce smaller particles, their physicochemical features change, increasing their surface reactivity [6], thus enabling them to interact or penetrate more efficiently with or into organisms, possibly triggering adverse responses [8, 13]. Therefore, size is an important NP characteristic to be considered when toxicity or fate studies are conducted.

Daphnia magna is a freshwater invertebrate that has been used extensively for the past 20 yr in regulatory testing and ecotoxicological research. Several features of this invertebrate make it suitable for laboratory testing [26]. In addition to their small size, high fecundity, short life cycle, reproduction by parthenogenesis, ubiquitous occurrence, and ease in laboratory handling [26], they can be used to evaluate functional traits (e.g., filtration, by feeding inhibition tests) or derive effects from the individual to the population scale (e.g., reproduction tests). Cladocerans have been considered a good toxicity model organism to predict the toxicity of pollutants to ecosystems due to their high sensitivity to environmental pollutants [27], including to the metal Zn [28], and representativeness in food-web chains as food and energy link between primary producers and secondary consumers [29].

To our knowledge, to date only acute or short-term effects of ZnO-NPs to D. magna have been studied, and the effect of particle size or solubility has not been taken into account [30-32]. Therefore, the present study aimed to evaluate the toxicity-related effects of size of ZnO particles to D. magna considering 2 different nanoparticulate sizes (30 nm and 80–100 nm) and a microsized (> 200 nm) particle, which were also related and compared with ionic counterparts. This was carried out by studying the effects on daphnids' immobilization, feeding activity, and reproduction on exposure to ZnO particles and ZnCl2 and by characterizing the exposure for all ZnO particles in American Standards for Testing and Materials (ASTM) hard water (transmission electron microscopy [TEM] and energy-dispersive X-ray spectroscopy), as well as their dissolution behavior in the same medium.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSIONS
  8. SUPPLEMENTAL DATA
  9. Acknowledgment
  10. REFERENCES
  11. Supporting Information

Chemicals

Zinc oxide Nanosun NPs (Zn10 with approximately 30 nm and Zn12 with approximately 80–100 nm) and the microsized form of ZnO (Zn13 with approximately 200 nm) were supplied by Nanotrade as powders. Zinc chloride (CAS number 7647-85-7; 98% purity) with a molar mass of 136.40 g mol−1 was purchased from Riedel-de Haën.

Preparation of suspensions

For the toxicity tests, ZnO stock dispersions (both nano and submicro) of 50 mg L−1 were prepared in ultrapure water (Milli-Q). All suspensions were prepared by sonication for approximately 30 min. Test suspensions were immediately prepared and stirred prior to test (immobilization and feeding inhibition) or for media renewal for the reproduction tests. No particle sedimentation was visualized during the tests' procedures. A 20-mg L−1 stock solution was prepared for zinc chloride in ASTM and shaken vigorously before use. For the reproduction tests, because it was necessary to renew the media every 2 d, the solution was stored at 4 °C during the test duration. The dispersions used for the toxicity tests were analyzed by inductively coupled plasma, using a Jobin Yvon Activa M, to confirm zinc concentration accuracy.

Nanoparticle characterization and dissolution

The specific surface area of ZnO powders was measured by the Brunauer-Emmett-Teller (BET) method (model Gemini 2360; Micromeritics AccuPyc). Density was determined using helium picnometry (Accupyc 1540; Micromeritics). Having the specific surface area as well as density, and assuming that all particles were spherical and identical, the average diameter of the particles was calculated. To confirm NPs' chemical identity as ZnO and calculate the crystallite size using the Scherrer's equation [33], X-ray diffraction with a Philips X'pert Pro Diffractometer (PANalytical) was performed. To prepare samples for TEM investigations, suspensions of approximately 100 mg L−1 of the ZnO were made in ASTM water by sonication in a low-power ultrasonic bath for 1 min. A drop of the dispersion was deposited on a copper TEM grid coated with holey carbon film right after dispersion. Then, dispersions were kept in the dark for 48 h and were quick-shaken and deposited on the grids. Experiments were carried out on a JEOL 2010 analytical TEM, which has a lanthanum hexaboride electron gun and can be operated between 80 kV and 200 kV. This instrument has a resolution of 0.19 nm, an electron probe size down to 0.5 nm, and a maximum specimen tilt of ± 10 degrees along both axes. The instrument is equipped with an Oxford Instruments LZ5 windowless energy dispersive X-ray spectrometer (EDS) controlled by INCA software. It has facilities for point analysis as well as mapping and line scanning through the SemiStem controller.

Conductivity and pH were measured using a Schott ProLab 2000 GLP equipped with pH electrode type A 161 1MDIN-ID, conductivity electrodes type LF 413 T-ID.

Hydrodynamic diameter size of particles was determined by a dynamic light scattering analyzer (Malvern Zetasizer Nano ZS), and the zeta potential was determined by a laser Doppler electrophoresis analyzer (Malvern Zetasizer Nano ZS).

A short-term dissolution study was performed to investigate the solubility and dissolution of ZnO in ASTM dispersions. First, a stock suspension of 1 wt% ZnO was prepared in distilled water. Then, from this stock suspension, an ASTM dispersion of ZnO 0.01% (mass concentration) was prepared. Zinc oxide suspensions were prepared in 100-mL sterile plastic containers (Margomed, PP), which were previously rinsed thoroughly with deionized water, and preparation and filtration of solutions were performed in a filtered-air laminar flow hood (SafeFlow 1.2; Euroclone-Bioair) to prevent contamination. All containers remained sealed during the experiment except when sampling was carried out (time 0, after 24 h, and after 48 h) or conductivity and pH were measured. Between subsequent time points, all containers were placed in a filtered-air laminar flow hood. Samples were stored at 23 °C. Before measuring the concentration of zinc, the collected suspensions were centrifuged at 19 064 g (Centrifuge MPW-251) for 20 min and passed through a 0.2-µm syringe filter (Art 6022N25001; Alchem) to separate the solution from ZnO-NPs.

Test organism and culture maintenance

The freshwater crustacean D. magna Strauss, clone K6, was used as the standard test organism. Daphnids were kept at a constant temperature of 20 ± 1 °C with a 16:8-h light:dark photoperiod and maintained in 3-L aquariums with reconstituted ASTM hard water [34]. Briefly, the ASTM hard water used for culturing and exposure was prepared by adding the following to Milli-Q water salt solutions: MgSO4, NaHCO3, KCl, and CaSO4. The final pH was 7.9 ± 0.3 (Supplemental Data, Table S1). Culture medium was renewed 3 times per week. Daphnids were fed with Pseudokirchneriella subcapitata at a concentration of 3 × 105 cell mL−1 and with 6 mL L−1 of seaweed extract (equivalent to a dissolved organic carbon of 5 mg L−1). Only the neonates from the third to fifth brood were used in toxicity tests to minimize variability. Neonates from the sixth brood were used to replace old cultures. Additionally, to verify organism sensitivity, acute tests with the reference compound potassium dichromate were performed at least twice a year.

Acute toxicity tests

Immobilization tests were performed in accordance with the Organisation for Economic Co-operation and Development (OECD) guideline 202 [35]. The tests were performed with 5 replicates for each concentration plus a control. Daphnids were not fed during the experiments. Tests were conducted at constant temperature of 20 ± 1 °C with a 16:8-h light:dark photoperiod.

Five neonates (<24 h old) randomly chosen were placed in 50-mL glass beakers per replicate exposed to a range of concentrations during the experimental setup. After 24 h and 48 h, immobilization (inability to swim after a gentle agitation of the glass beaker) and mortality were recorded, and the median lethal concentration (LC50) values were calculated. The nominal concentrations for ZnO-NPs (both NPs and submicro-sized) ranged from 0.25 mg Zn L−1 to 10 mg Zn L−1. For zinc (in the form of ZnCl2), the nominal concentrations ranged from 0.2 mg Zn L−1 to 6.4 mg Zn L−1.

Feeding inhibition tests

Neonates (<24 h old) were separated from the main culture to other aquarium until they were 4 d old to 5 d old, being equivalent to the fourth instars. This life stage allows the test to be carried out during a single moult cycle, avoiding molting interference in the feeding rates of daphnids as observed by McWilliam and Baird [36].

For each concentration tested, plus a control, 5 replicates with 5 individuals per treatment were used. The 5 neonates were randomly chosen and placed in 170-mL glass beakers with 100 mL of the test substance and fed for 24 h with the algae P. subcapitata at a concentration of 5 × 105 cell mL−1. To establish the initial algal concentrations, a blank set of 3 replicates per concentration and control (with no daphnids) was added to the experimental setup. In addition, another control containing ZnO-NP dispersions in ASTM hard water at the highest concentration tested was checked for the influence of NPs' spectral absorption on the measurements [37].

All of the glass beakers were kept in the dark to ensure uniform feeding rates during the 24-h exposure period by avoiding algae growth. After this exposure time, daphnids were transferred into 50-mL glass beakers containing clean ASTM hard water with food (5 × 105 cells mL−1) and allowed to feed for 4 h (postexposure) in the dark.

At the end of both periods (exposure and postexposure), each replicate was vigorously shaken to resuspend cells, and the absorbance values were determined at 440 nm by spectrophotometry (6505 Spectrophotometer UV-VIS; Jenway). Individual feeding rates for the 24-h exposure and the 4-h postexposure were determined according to Allen et al. [38].

Nominal test concentrations for ZnO 30 nm, ZnO 80 to 100 nm and ZnO > 200 nm ranged from 1.2 mg Zn L−1to 2.4 mg Zn L−1, from 2 mg Zn L−1 to 4 mg Zn L−1, and from 0.5 mg Zn L−1 to 3 mg Zn L−1, respectively. For the zinc (in the form of ZnCl2), the nominal test concentrations ranged from 0.4 mg Zn L−1 to 12.8 mg Zn L−1.

Chronic toxicity tests

The reproduction tests were conducted for 21 d according to OECD guideline 211 [39]. For each treatment and control, 10 replicates of 1 individual each (<24 h old) were used. The exposure was conducted in 50-mL glass beakers containing P. subcapitata at a concentration of 3 × 105 cells mL−1 and seaweed extract and kept at constant temperature of 20 ± 1 °C with a 16:8-h light:dark photoperiod. The renewal of the test medium for all treatments and control was carried out every 2 d. Additionally, organisms were fed daily during the test period.

Immobilization and mortality of the parent daphnids and offspring were assessed daily. The live neonates were counted and removed from the test beakers after each brood release. Test-medium parameters (pH, dissolved oxygen, and conductivity) were measured at the beginning, middle, and end of each test in both old and renewed medium. Daphnids were measured at the beginning of the test and also at the end to assess the differences of size between the control and the test concentrations.

Nominal concentrations used for ZnO 30 nm, ZnO 80 nm to 100 nm, and ZnO > 200 nm ranged between 0.125 mg Zn L−1 and 0.75 mg Zn L−1, between 0.125 mg Zn L−1and 0.75 mg Zn L−1, and between 0.038 mg Zn L−1 and 0.45 mg mg Zn L−1, respectively. For zinc (in the form of ZnCl2), the nominal concentrations ranged from 0.15 mg Zn L−1 to 0.4 mg Zn L−1.

Statistical analysis

All results were expressed as mg Zn L−1 to compare exposures to different zinc types. The 48-h LC50 values for the immobilisation of D. magna were calculated using the SigmaPlot software by a nonlinear regression with a sigmoidal function. The 24-h median effective concentration (EC50) and 4-h EC50 values for feeding activity and EC50 values for reproduction were calculated using a nonlinear regression by a logistic three-parameter equation or four-parameter equations.

To determine statistical differences between control and exposure treatments, data were analyzed by a one-way analysis of variance, followed by the Dunnett's test when appropriate. For data that failed the normality testing, a nonparametric Kruskal-Wallis test was performed followed by Dunn's method to access multiple comparisons between treatments and control. All significant differences were established at p < 0.05.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSIONS
  8. SUPPLEMENTAL DATA
  9. Acknowledgment
  10. REFERENCES
  11. Supporting Information

Characterization of ZnO particles and dissolution studies

Zinc oxide powder properties can be found in Supplemental Data, Table S2. The TEM images showed that 30-nm and 80-nm to 100-nm NPs were mainly equiaxial and rounded (Figure 1). At a concentration of approximately 100 mg Zn L−1, the appearance of the individual particles and agglomerates did not change significantly over a 48-h period. In some samples (mainly ZnO 80–100 nm), however, additional low contrast features appeared, which may have been contamination or reaction products of the ZnO with the salt ions in the ASTM water.

image

Figure 1. Transmission electron microscope images (TEM) of suspensions of approximately 100 mg Zn L−1 of ZnO particles of 30 nm (left), 80 nm to 100 nm (center), and >200 nm (right) at time 0 and after 48 h in ASTM hard water.

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In distilled water, NPs' agglomerate sizes (d/nm) were approximately 199 (for the 30 nm NPs), 209.5 (for the 80- to 100-nm NPs) and 769.2 for the >200-nm ZnO particles. In the ASTM media, suspensions showed larger agglomerate sizes: 1061 nm for the 30-nm NPs, 1353 nm for the 80-nm to 100-nm NPs and 1565 nm for the >200-nm ZnO particles (Table 1). Other characteristics of ZnO particles are described in Table 1.

Table 1. Concentration of dissolved Zn2+ at time 0, 24 h and 48 h of aqueous ZnO suspensions with different particle size in ASTM hard water with initial ZnO particle concentration of 100 mg Zn L–1
ZnO particleTime (h)Concentration (mg Zn L–1)pHConductivity (µS/cm)Size by DLS, Z-average d (nm)ZP by LDE [mV]
  1. DLS = dynamic light scattering; ZP = zeta potential; LDE = laser Doppler electrophoresis; ZnO-NP = zinc oxide nanoparticle.

ZnO-NP (30 nm)00.368.336391061–2.83
 240.418.286863373–1.9
 480.518.356854533–2.84
ZnO-NP (80–100 nm)00.348.326471353–2.63
 240.48.387342675–3.19
 480.438.327343560–2.02
>200 nm ZnO particle00.38.336251565–9.35
 240.4258.346342217–22.1
 480.88.246643365–19.8

Zinc analysis revealed a variation of less than 10% between nominal and measured concentrations. Therefore, nominal zinc exposures were used to calculate toxicity endpoints. At the concentration chosen for the dissolution experiment (100 mg Zn L−1), particle precipitation was observed. The results from the dissolution experiment can be found in Table 1. From the chemical data on the concentration of ionic zinc in solution, it can be observed that both NPs behave similarly after 48 h in suspension (0.51 mg Zn L−1 and 0.43 mg Zn L−1 for 30 nm and 80- to 100-nm NPs, respectively), with the submicrosized ZnO showing a higher dissolution than the nano-sized particles (0.8 mg Zn L−1). Results are presented for 24 h and 48 h, which are, respectively, the duration of the exposure period of a feeding inhibition test and acute test or the time interval between two media changes in a reproduction test. Time between media changes or test duration was less than or equal to 48 h; therefore, this experimental setup was sufficient to confirm the exposure to NPs and/or to the ionic form. The pH values measured over time for all dispersions were similar (Table 1), but the zeta potential showed a completely different pattern between both NPs (30-nm and 80- to 100-nm NPs) and the microsized particles (>200 nm). Zeta potential did not change during the 48-h period for the NPs, with values between –2.83 and –2.02 (Table 1). For the >200-nm particle, however, the zeta potential value at time 0 was –9.35, dropping to –19.8, after the 48 h (Table 1).

Acute toxicity of Zn forms to D. magna

The 48-h LC50 values for all Zn forms studied are shown in Table 2. Although ionic zinc showed a lower LC50 to D. magna when compared with both ZnO NPs (30 nm and 80–100 nm), the LC50 of microsized ZnO particles was similar to that obtained for the ionic form.

Table 2. Summary of the effects of all test compounds on immobilisation, feeding activities and reproduction of Daphnia magnaa
SubstanceAcute toxicity testsFeeding inhibition testsChronic toxicity tests
48 h LC50R224 h EC50R2NOECLOEC4 h EC50R2NOECLOEC21 d EC50R2NOECLOEC
  • a

    Results are expressed as mean ± standard error. Data are expressed as mg Zn L−1 for all ZnO forms.

  • LC50 = median lethal concentration; R2 = coefficient of determination; EC50 = median effective concentration; NOEC = no-observed-effect concentration; LOEC = lowest-observed-effect concentration; ZnO-NP = zinc oxide nanoparticle.

ZnCl20.761>3.2 <12.80.81.61.92 ± 0.250.8710.4>0.25 <0.400.15
ZnO 30 nm1.02 ± 0.240.9981.41 ± 0.030.97211.27 ± 0.030.97510.26 ± 0.030.8330.125
ZnO 80–100 nm1.10 ± 0.050.9982.00 ± 0.030.9841.61.91 ± 0.050.9511.60.36 ± 0.030.7990.1250.25
ZnO >200 nm0.89 ± 0.030.9981.89 ± 0.300.9300.81.21.71 ± 0.050.9550.81.20.30 ± 0.030.4930.300.45

Feeding behavior of D. magna exposed to different Zn forms

Feeding activities, measured as feeding rates (cell mL−1), generally showed a decrease during the exposure and postexposure period with increasing concentrations for all the Zn forms studied (Figure 2). No mortality was observed during the 24-h exposure during all tests. The smaller-sized ZnO-NPs (30 nm) appeared to be more toxic when compared with the other ZnO-NP and microsized particle, with an EC50 of 1.41 mg Zn L−1 (Table 2).

image

Figure 2. Feeding rates of Daphnia magna during 24-h exposure (black bars) and 4-h postexposure (white bars) at concentrations of (A) ionic zinc, (B) 30-nm ZnO nanoparticles, (C) 80- to 100-nm ZnO nanoparticles, and (D) ZnO microsized particles. Data is expressed as mg Zn L−1 mean values ± standard error. Asterisks indicate statistical differences (p < 0.05) between treatments and control.

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For the ionic zinc, mortality was observed in the 2 highest concentrations used (6.4 mg Zn L−1 and 12.8 mg Zn L−1). The EC50 for feeding rates for the 24-h period could not be calculated because the highest concentration used where no mortality occurred showed feeding inhibition less than 50% (EC50 > 3.2 mg Zn L−1).

For the postexposure period, it was observed that daphnids were not able to recover from the previous effects caused by the ionic zinc exposure, even at low concentrations (0.4 mg Zn L−1 and 0.8 mg Zn L−1), where effects were not detected during exposures. For the 2 NPs and the microsized particles, all concentrations tested in the exposure period were still inducing lower feeding rates than the control during the 4-h posterior period.

Chronic toxicity of different Zn forms to D. magna

The mortality of parent control animals at the end of all chronic tests was always ≤ 20%. Additionally, the mean number of live offspring produced per parent control daphnids in all toxicity tests ranged between 121 and 149 (data not shown), thus making all tests valid according to OECD guideline 211 [39].

Over the 21-d testing period pH ranged between 7.49 and 8.49, dissolved oxygen ranged between 8.47 mg L−1 and 12.63 mg L−1, and conductivity ranged between 451 µS/cm and 615 µS/cm. These values were obtained considering changes in media every 48 h and are in accordance with the validation criteria of the protocol adopted. The pH ranges for each chronic test are shown in Supplemental Data, Table S3. The highest concentration tested for ZnO-NPs 30 nm (0.75 mg Zn L−1) was not used in the data analysis because of to the high mortality rate that occurred during the last week of the test.

The number of neonates produced by each female was affected by zinc exposures (Figure 3). The lowest-observed-effect concentration (LOEC) values of 0.125 mg Zn L−1, 0.25 mg Zn L−1, and 0.45 mg Zn L−1were obtained for ZnO particles of 30 nm, 80 nm to 100 nm, and >200 nm, resulting in offspring reduction rates of 29%, 31%, and 62%, respectively. Table 2 provides EC50, showing similar results among exposures. For the ionic zinc, results showed a LOEC value of 0.15 mg Zn L−1, with a decrease of 24% on the reproduction output.

image

Figure 3. Effects of (A) ionic zinc, (B) 30-nm ZnO nanoparticles, (C) 80- to 100-nm ZnO nanoparticles, and (D) ZnO microsized particles on the number of neonates produced by Daphnia magna. Data is expressed as mg Zn L−1 mean values ± standard error. Asterisks indicate statistical differences (p < 0.05) between treatments and control.

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The females' length after 21 d of exposure showed LOEC values of 0.25 mg Zn L−1for the ionic zinc, 0.6 mg Zn L−1for ZnO-NPs 80 nm to 100 nm, and 0.12 mg Zn L−1 for ZnO microsized particles (Supplemental Data, Figure S1). On the other hand, no statistical differences were observed for the 21-d length of daphnids exposed to ZnO-NPs of 30 nm (Supplemental Data, Figure S1).

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSIONS
  8. SUPPLEMENTAL DATA
  9. Acknowledgment
  10. REFERENCES
  11. Supporting Information

It has been reported that at a water pH of 7.8, a 1 g L−1 dispersion of ZnO NPs will lead to a dissolution of 10 mg Zn L−1 (i.e., < 2%) [40]. In the study of Franklin et al. [19], the dissolution of 100 mg L−1 of 30-nm particles was carried out for 40 h, resulting in a dissolution of 15 mg L−1. This is not in accordance with the results of the present study, in which we observed a lower dissolution of 0.51 mg Zn L−1 for the 30-nm NPs. Although this was not expected, the discrepancies on the values found can be related to the quality of the NPs, such as the content of hydroxides (content of the amorphous and crystalline phases), but also on the media used for their dispersion.

The rate of dissolution of a particle is usually considered proportional to its surface area, considering also the same shape, which will lead to a faster dissolution for lower-scale particles when compared with larger-sized particles or bulk materials, for the same mass. In the present study, the >200-nm ZnO particles showed a higher dissolution in 48 h than the 2 NPs studied, which goes against this theory and also against results obtained by Franklin et al. [19], where similar dissolutions were observed between ZnO-NPs of 30 nm and bulk ZnO particles. Therefore, dissolution may be considered not just dependent on NP size but also on details of their nanostructure and most likely on the agglomerates' size and structure, which are also dependent on the methodology used for production and functionalization and the media in which they were dispersed.

The LC50 values for the acute toxicity tests to D. magna ranged between 0.76 mg Zn L−1 and 1.32 mg Zn L−1. For the same species and clone, Heijerick et al. [41] reported a 48-h EC50 value for the acute toxicity of Zn of 0.39 mg Zn L−1, which is lower than the LC50 values obtained in our results. The differences in toxicity may be due to the different concentrations of cations used to prepare both media (ASTM and M4 medium), because according to their results, the toxicity of Zn can be reduced by different concentrations of cations (e.g., Ca2+, Mg2+, Na+, K+) used in test media.

The acute toxicity of both NPs (30 nm and 80–100 nm) and the microsized ZnO particles were found to be similar and not dependent on the initial particle size. Differences in agglomerate sizes were observed after 48 h in ASTM hard water and were not directly related to their initial size. Previous studies have already reported similar toxicity between nanoscale ZnO particles and the respective bulk counterparts [22, 31, 42]. Although it is known that particle size plays an important role in the NPs' toxicity [14, 43], this was not observed in the present study, where there was no relationship between the toxicity and initial particle size, which can be justified by the fact that daphnids were not exposed to the individual NPs but instead to the agglomerates that may have modified its toxicity. This lack of relationship observed between the acute toxicity of nanomaterials and their size is in agreement with a previous study in which all Zn forms were very toxic, with similar LC50 values between them all [29].

From the EC50 values obtained for the feeding behavior of daphnids, based on zinc concentrations, it can be observed that test organisms were more sensitive to ZnO-NPs than to the ionic zinc, with lower values for the ZnO-NPs of smaller particle size (Table 2). Higher concentrations of ionic zinc (> 3.2 mg Zn L−1) will be needed to inhibit their feeding activity in 50% of the population. Therefore, considering that the EC50 values based on zinc for NPs were < 2 mg Zn L−1 and lower than the ionic zinc concentration inducing feeding inhibition, we assume that the concentrations of ZnO particles affecting D. magna were mainly constituted by the particulate form. In addition, ZnO agglomerates changed their size throughout the 48 h, with 30-nm ZnO-NPs showing larger agglomerates after this period.

Several studies have suggested that the toxicity of ZnO-NPs is mainly the result of the dissolution of zinc ions present in suspensions [19, 29]. This is not yet a consensual assumption, however, because other studies believe that their dissolution does not account for the total observed toxicity of ZnO-NPs [42], as the present study has also shown.

As mentioned above, particle size is one of the features that bring special attention to NPs studies. Their smaller size provides a larger surface area and reactivity, thus allowing them to penetrate into cells and organisms more efficiently. This will possibly induce higher toxicity effects where the same material in the bulk form may be inert [13, 14, 43]. This was partly observed in our results in the feeding behavior tests for the 2 NPs studied (30 nm and 80–100 nm), possibly because toxicity was mainly driven by the particulate form. For the acute toxicity, however, microsized ZnO particles presented a lower LC50 value when compared with those of ZnO-NPs (Table 2). In this last case, the acute toxicity was possibly driven by both the particulate and the ionic forms. When looking at the dissolution rates, the >200-nm particles released more ions during 48 h when compared with both NPs, which may then explain the similarity between LC50 values found between these and the ionic form.

It is also known that NPs are susceptible for aggregation in aqueous suspensions. This process can change their physicochemical properties, making them less available to cause toxicity [11, 44]. Therefore, regarding the zeta potential values obtained, we can consider that the particles of >200 nm with a highest negative value (–19.8) would be more stable than the NPs whose zeta potential values were closer to 0 due to electrostatic stabilization. Their dissolution was higher, however, which contradicts these findings to a certain extent.

For the postexposure period, it was observed that daphnids were not able to recover from the ionic zinc previous effects, as shown in Figure 2, even at low concentrations (0.4 mg Zn L−1 and 0.8 mg Zn L−1), where effects were not detected during the exposure periods. For the 2 NPs studied and the microsized particles, all concentrations tested in the exposure period were still inducing lower feeding rates than the control during the 4-h period. This highlights that even after short exposure periods (e.g., 24 h) there will be long-lasting effects in daphnids exposed to Zn forms.

The number of offspring produced was the most sensitive endpoint used in the present study. No published data about chronic toxicity of ZnO-NPs to the cladoceran D. magna was found in the literature for comparison. Taking into account the key water characteristics used in the ecotoxicological tests, however, Heijerick et al. [45] reported EC50 and no-observed-effect concentration values for Zn toxicity to D. magna of 0.0083 mg L−1 and 0.0055 mg L−1, respectively. These values are lower than those obtained in the present study, which again may be related to the cations composition of the test medium.Given the scarcity of studies concerning long-term effects of NPs, it becomes difficult to assess, compare, and understand their potential risk to aquatic environments.

Contrary to results of the immobilization tests and similarly on the feeding inhibition tests, the effect of ionic zinc on the reproduction endpoint did not allow us to observe a complete dose–response curve (EC50 > 0.25 mg Zn L−1). Concentrations inducing both sublethal and lethal effects on reproduction along the 21-d of exposure were very close, with a small interval between them (> 0.25 mg Zn L−1 and < 0.4 mg Zn L−1, respectively; Table 2). This also shows that acute effects on time are important to consider. In this case, results obtained in the reproduction test for the ionic zinc derived a lower LC50 value for 21 d than the value reported for the 48 h (0.76 mg Zn L−1and 0.21 mg Zn L−1for 48 h [without food] and 21 d [within the chronic assay], respectively).

Similar to our results, Sánchez-Ortíz et al. [46] also showed a decrease in population growth in 2 cladocerans species (Ceriodaphnia dubia and Daphnia pulex) with increasing concentrations of zinc in the medium. These authors observed no reproduction at 1 mg Zn L−1 and mortality after 1 wk at concentrations ≥ 0.25 mg Zn L−1. A similar pattern was observed in our results. From approximately the second week of exposure to ionic zinc, daphnids showed low reproduction rates and started to die in all the replicates of the highest 3 concentrations used (0.30 mg Zn L−1, 0.35 mg Zn L−1, and 0.40 mg Zn L−1), reaching adult mortality rates > 50%. Therefore, according to the standardized protocol followed, these data were not included in the statistical approach. Contrary to what was found in the present study, Sánchez-Ortíz et al. [46] showed that the population growth of D. pulex was higher at 0.125 mg Zn L−1 than in the control, resulting in an hormesis (stimulatory effect of sublethal concentrations).

When looking at the EC50 values obtained for reproduction performance (Table 2) and the dissolution results (Table 1), it can be concluded that the effects are driven mainly by the ionic form of Zn. At the end of the 48-h period of the dissolution experiments, the amount of zinc dissolved was similar to or higher than the EC50 causing reduction in the number of offspring.

During the reproduction tests in the present study, daphnids visibly showed difficulty changing their moults after 2 wk of exposure (except the control) and consequently died (data not shown). A recent study of D. magna suggested that exposures of waterborne zinc resulted in whole-body zinc burden and consequently leads to a decrease of Ca2+ body contents as a result of the competition between these 2 ions for ion regulatory surfaces [28]. The depletion of Ca2+ levels (hypocalcemia) inhibits the filtration rates of daphnids, leading inevitably to a decrease of food uptake [47]. As a consequence, less energy will be available to allow a normal growth rate and reproduction [28, 47]. These findings could then explain the results obtained in the present study for the low feeding rates and the reproduction output of daphnids at the highest concentrations tested. Therefore, we can hypothesize that surplus zinc levels can indirectly affect the moulting process of D. magna.

CONCLUSIONS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSIONS
  8. SUPPLEMENTAL DATA
  9. Acknowledgment
  10. REFERENCES
  11. Supporting Information

In the present study, differences in the initial size of NPs or their agglomerate size (nanostructure) during exposure did not play a direct role in toxicity. Dissolution experiments carried out provided essential information to derive conclusions on toxicity. Particles behave differently, considering their initial size and the media they are in, and this should be regarded by risk assessors. From our results it can be highlighted that NPs may impair daphnids' function in ecosystems, because their feeding rate was found to be more affected by the particulate Zn form than by the ionic form. In addition, NPs can act as an extra source of ionic zinc to the environment, inducing changes at low levels on the reproduction output of daphnids, and therefore on population dynamics.

Acknowledgment

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSIONS
  8. SUPPLEMENTAL DATA
  9. Acknowledgment
  10. REFERENCES
  11. Supporting Information

Work reported here was partly conducted in the NanoFATE, Collaborative Project CP-FP 247739 (2010–2014) under the 7th Framework Programme of the European Commission (FP7-NMP-ENV-2009, Theme 4), coordinated by C. Svendsen (Natural Environment Research Council Centre for Ecology and Hydrology, UK; www.nanofate.eu) and in the Central European Initiative project NANOFORCE, by the team members of IHPP.

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  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSIONS
  8. SUPPLEMENTAL DATA
  9. Acknowledgment
  10. REFERENCES
  11. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSIONS
  8. SUPPLEMENTAL DATA
  9. Acknowledgment
  10. REFERENCES
  11. Supporting Information

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