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Short-term toxicity to fish

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Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
no guideline available
Principles of method if other than guideline:
The zebrafish 96-h embryo-larval bioassay according to Schulte & Nagel (1994) was used to assess and compare the developmental toxicities of nanoscale zinc oxide (nZnO), titanium dioxide (nTiO2) and alumina (nAl2O3) aqueous suspensions. Toxicological endpoints such as zebrafish embryos or larvae survival, hatching rate and malformation were noted and described within 96 h of exposure. A comparative experiment with their bulk counterparts (i.e., ZnO/bulk, TiO2/bulk and Al2O3/bulk) was conducted to understand the effect of particle size on their toxicities.
GLP compliance:
no
Specific details on test material used for the study:
Uncoated nanoscale ZnO (nZnO, purity >99.9%) was purchased from Nanjing High Technology NANO CO., LTD (Nanjing, Jiangshu province, China); the bulk counterpart ZnO/bulk (purity >99.0%) was purchased from The Third Chemical Regent Factory of Tianjin (Tianjin, China).
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: nZnO ws added to 100 mL of Milli-Q® water and dispersed by ultrasonication at room temperature for 0.5 h
- Particle size: size was determined using a dynamic light scattering device (B19000AT, Brookhaven Instrument Corporation, USA) for particles of < 1µm. Determined sizes and morphology were confirmed using TEM, Tecnai G2T20ST, Philips, Holand); size range: 50 - 360nm
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
TEST ORGANISM
- Common name: Zebrafish
- Source: Market in Tianjin, China
- Age at study initiation (mean and range, SD): eggs
- Method of breeding: adult fish kept in 250 L full glass aquaria; 26 ± 1°C; 14h/10h light/dark cycle; roughly 2:1 male/female sex ratio; food: frozen chironomids larvae, twice a day
- Spawning: Spawning was triggered once the light was turned on and completed within 30 min; Eggs collected were rinsed several times with cleaning and aerated water to remove the residue on the egg surface; Normally fertilised eggs were picked out under a stereomicroscope for the experiments
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Test temperature:
26+-1°C
Nominal and measured concentrations:
50, 10, 5, 1, 0.5, 0.1 mg/L and control (nominal)
Details on test conditions:
14h-10h light - dark cycle
Reference substance (positive control):
no
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
1.793 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
nano ZnO
Basis for effect:
mortality (fish)
Remarks on result:
other: 1.498-2.145 mg/L (95% CL)
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
1.55 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
bulk ZnO
Basis for effect:
mortality (fish)
Remarks on result:
other: 0.927-2.589 mg/l (95% CL)
Duration:
84 h
Dose descriptor:
EC50
Effect conc.:
2.065 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
nano ZnO
Basis for effect:
other: hatching rate
Remarks on result:
other: 1.687-2.529 mg/l (95% CL)
Duration:
84 h
Dose descriptor:
EC50
Effect conc.:
2.066 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
bulk ZnO
Basis for effect:
other: hatching rate
Remarks on result:
other: 1.472-2.897 mg/l (95% CL)
Details on results:
Metal oxide particle-mediated malformation (e.g., pericardial edema and tissue ulceration, body arcuation, etc.) in the embryos and larvae from both the control and treatments was observed. When pericardial edema and body arcuation were considered, there was not a significant difference between the results of the nZnO suspensions treatment and the control. However, tissue ulceration of post-hatch zebrafish larvae exposed to nZnO suspension was observed to occur at 72 hpf. For the nZnO suspension at 5 mg/L particle concentration treatment, tissue ulceration was observed in zebrafish larve at 72 hpf, and its proportion to the surviving zebrafish was 31.67%. The percentage increased sharply to 95.83% and l00% at 96 hpf and 108 hpf respectively, which demonstrated that tissue ulceration became more evident as the exposure time increased and eventually resulted in some fish death. Compared to 5mg/L treatment, nZnO suspension with lower particle concentration caused a lower tissue ulceration rate in zebrafish larvae, and the time when ulceration occurred was delayed.
Reported statistics and error estimates:
All experiments were repeated 3 times independently. Statistical analyses were carried out using standard ANOVA techniques, and data were illustrated as the mean with standard deviation (S.D.). A P value of <0.05 was considered statistically significant. The LC50 and EC50, and the associated 95% confidence intervals (95% CI) were calculated by the standard USEPA Probit Analysis Program (Version 1.5)
Sublethal observations / clinical signs:

Table:Toxicological effects of ZnO suspensions at different particle concentration on survival and hatching zebrafish embryos and larvae at 96/84 hpf (all values are presented as mean ± standard deviation, *p < 0.05, **p < 0.01 vs. control group)

96 hpf survival (%)

84 hpf Hatching rate (%)

Particle Concentration

Control (0 mg/L)

98.133 ± 3.233

98.333 ± 2.887

0.1 mg/L

96.467 ± 3.075

98.333 ± 2.887

0.5 mg/L

88.267 ±10.226

92.157 ± 6.585

1 mg/L

71.567 ± 9.241**

75.273 ± 3.199*

5 mg/L

28.133 ± 15.127**

29.803 ± 17.213**

10 mg/L

1.667 ± 2.887**

3.333 ± 5.774**

50 mg/L

0**

0**

Results and conclusions:

Of the substances tested ZnO was the most toxic material to zebrafish embryos and larvae, the 96-h LC50 of nZnO and ZnO/bulk aqueous suspensions on the zebrafish survival were 1.793 mg/L and 1.550 mg/L, respectively; and the 84-h EC50 on the zebrafish embryo hatching rate were 2.065 mg/L and 2.066 mg/L, respectively. The zebrafish developmental toxicity of either the nZnO or the ZnO/bulk suspensions had an obvious dose-depending property. Metal oxide nanoparticles with different chemical compositions have different zebrafish developmental toxicity. Stability of chemical compositions of nanomaterials itself should be considered as an important factor affecting their potential environmental impacts and biological effects.

Validity criteria fulfilled:
yes
Executive summary:

Similar LC50 values observed with nano-ZnO and bulk-ZnO treatment. No comparative data with Zn2+ ecotoxicity. Nominal dose concentrations reported, only.

Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
no guideline followed
Principles of method if other than guideline:
Briefly, 24 eggs (blastula stage) were transferred to the test wells of a 24-well multi-plate at 1 embryo/well, in which twenty wells contained 2 ml nZnO test solution and four wells contained 2 ml of culture medium per well. The experiment was performed in triplicate (i.e., a total of 12 embryos were used in the water control and 60 embryos in the exposure group) for each treatment. The development of the zebrafish embryos and larvae
was observed with an inverse microscope (Olympus, Japan) equipped with a digital camera and was documented photographically at specified time points (t = 6, 12, 24, 36, 48, 60, 72, 84 and 96 hours post-fertilization (hpf)). The endpoints used to assess developmental toxicity included
embryo/larvae survival and embryo hatching rate.
GLP compliance:
not specified
Remarks:
GLP compliance not specified in publication
Specific details on test material used for the study:
Uncoated nanoscale ZnO (nZnO) with a published particle size of 20 nm was purchased from Nanjing High Technology NANO CO., LTD (Nanjing, China). Supplied as a yellowish powder, it had a purity of >99.7% and a specific surface area >90 m2/g.
Analytical monitoring:
yes
Details on sampling:
In brief, water samples were taken from the nZnO test solutions immediately at the end of exposure and filtered through a 20 nm filter (Whatman International Ltd, Maidstone, UK). Given the size of nZnO aggregates ranging from a few hundred nanometers to several microns in diameter (see below), the 20 nm filter is expected to remove all the particles. After filtration, the released Zn2+ concentrations in the filtrates were then detected by GFAA.
Vehicle:
no
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
Zebrafish embryos were obtained from an AB strain of zebrafish (Danio rerio) reared in a fish room at The Biodesign Institute at Arizona State University (ASU). Adults were raised and maintained in a closed flow-through culture system (Aquatic Eco-Systems, Inc., FL) at 28 ± 0.5 °C with a photoperiod of 14 h light and 10 h dark. The zebrafish were fed twice a day with TetraMin dry flakes (Tetra, Melle, Germany).
Spawning is triggered once the light is turned on in themorning and is complete within 30 min. At 4–5 h post-fertilization (hpf), embryos were collected and rinsed several times with culture medium to remove residues on the egg surface. Healthy embryos at blastula stage were then selected for subsequent experiments
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Remarks on exposure duration:
h
Duration:
84 h
Dose descriptor:
LC50
Effect conc.:
23.06 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
nZnO
Basis for effect:
other: embryo hatching
Reported statistics and error estimates:
All exposure experiments were repeated three times independently, and data were recorded as the mean with standard deviation (SD). For the embryo/larvae bioassays, a one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons was used to detect significant differences between the control and treated groups. For gene expression experiments, quantitative real-time PCR analysis was performed using the BioRAD iQ5 software. For each gene, the fold change expressed as the mean ± SD (% control) was calculated using the CT approximation corrected for primer efficiency and normalized to 18S expression values. Statistical analyses were performed using the Student’s t test. For all of the data analysis, a p-value <0.05 was considered statistically significant.
Sublethal observations / clinical signs:

Many metal oxide nanoparticles are known to aggregate in water or culture medium. To characterize the physical behavior of nZnO in water, we performed quantitative analyses to determine the level and kinetics of the nZnO aggregation process. The addition of nZnO to the culture medium (pH = 8.0) resulted in visible milk-white aggregates that settled out of water column very quickly; more than 80% sank within the initial 3 h. After 48 h sedimentation, the nZnO aggregates almost entirely came out of water column, and a layer of these aggregates was accumulated at the bottom of the experimental container. Observed by SEM, these nZnO aggregates look like floccules with variable sizes from a few hundred nanometers to several microns in diameter, which is in line with the finding reported previously. Using DLS, the median values of particle size were detected to be 2196 (with range 1037–4650), 3144 (with range 1421–6823), 2540 (with range 1227–5158) and 2497 (with range 1150–4935) nm at 0, 0.5, 12 and 48 h, respectively.

The nZnO aggregates can potentially exert an adverse effect on aquatic organisms, given their rapid aggregation process. To address this issue, a zebrafish embryo model was employed to determine whether and how nZnO aggregates influence the embryonic development. In this study, zebrafish embryos were exposed to nZnO aggregates for 96 h, and a dose-dependent inhibition of embryo hatching was induced by nZnO aggregates. Calculated by the probit method, the 84 h EC50 of these nZnO aggregates on embryo hatching was 23.06 mg l−1 (95% CI, 12.714–59.486 mg l−1).

To determine whether our observed embryonic toxicity could be attributed to the released Zn2+, we analyzed the level of Zn2+ released from nZnO and tested the effect of this defined concentration of Zn2+ on zebrafish embryos. The released Zn2+ concentrations in the nZnO test solution at the end of exposure were measured, and the highest dissolution of 1.01 ± 0.43 mg l−1 released Zn2+ was found in 10 mg l−1 nZnO test solutions. On the other hand, the released Zn2+ concentrations in 5, 10, 50 and 100 mg l−1 nZnO test solutions were not significantly different (p > 0.05, unpublished observation). Thus, we examined the effect of 1.0 mg l−1 of soluble Zn2+ in comparison to the one with nZnO aggregates, and found that Zn2+ caused no apparent toxicity to zebrafish embryos. Although at higher concentrations, soluble Zn2+ exerted toxicity, their effect was less significant as compared to the similar concentrations of nZnO. In addition, the embryos treated with soluble Zn2+ did not show any significant (p > 0.05) malformation (such as pericardial edema) at any of the tested time points.

Validity criteria fulfilled:
yes
Executive summary:

Nominal LC50 observed very high. No comparison with bulk or Zn2+ ecotoxicity.

Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
no guideline followed
Principles of method if other than guideline:
Test concentrations for lethality were 1, 2, 5, 10, 30 and 50 mg L1 for nZnO, bulk ZnO respectively, with single distilled water as a control. Test solutions were prepared immediately prior to use, without the addition of salts or stabilizing agents. The suspensions were continuously dispersed for 20 min by the bath sonicator to aid mixing and to maintain their stability. Seven fish were randomly exposed to each replicate of every concentration for 96 h in 2 L glass beakers containing 1.5 L of suspensions. Each concentration was run in triplicate and under the same conditions with a natural light/dark cycle (12 h light/12 h dark). To ensure a constant concentration, all test solutions were changed every 24 h.
GLP compliance:
not specified
Remarks:
GLP compliance not specified in publication
Specific details on test material used for the study:
Nano-ZnO powder (30 nm advertised particle size) was purchased from Nano Applied Research Center of Nanjing University of Technology; bulk ZnO powder (500 nm advertised particle size) and ZnSO4.7H2O were purchased from Tianjin Guangcheng Chemical Reagent Co., Ltd. The surfaces of the nZnO particles were not modified and the advertised purities of nZnO and bulk ZnO particles were 99.0%, respectively.
Analytical monitoring:
yes
Details on sampling:
Since the concentrations of the nZnO and bulk ZnO suspensions changed over time, the sedimentation property of nZnO in the exposure solution was analyzed and compared with that of the bulk ZnO. According to the acute toxicity tests, four initial concentrations, i.e. 1, 10, 30, 50 mg L1 were prepared with distilled water for both the nZnO and bulk ZnO suspensions, respectively. Seven fish were exposed to 1.5 L of each suspension in a beaker and each concentration was run in triplicate. 10 mL of each water suspension was sampled from the middle of the beaker at 2 h, 6 h, 12 h, 24 h, 48 h, 72 h, 96 h and 120 h. The water samples were digested with 1 mL concentrated nitric acid and 3 mL mixed acid (HNO3 : HClO4, 2 : 1).
Vehicle:
not specified
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
Adult zebrafish, of mean age 120 d, mean length 3.02 0.33 cm, and mean weight 0.22 0.05 g were obtained from the Research Group for Molecular Pathology of Fishes (Institute of Hydrobiology, Chinese Academy of Sciences). Fish were acclimatized in dechlorinated tap water with a natural light/dark cycle for at least 7 d in laboratory before experimentation and fed twice daily with newly hatched brine shrimp (Artemia salina). The water temperature was maintained at 23 2 C and no fish died prior to experiments.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96
Remarks on exposure duration:
h
Test temperature:
23 +/-2°C
pH:
6.8–7.2
Dissolved oxygen:
5.10 mg/L
Nominal and measured concentrations:
1, 2, 5, 10, 30 and 50 mg/L for nZnO, bulk ZnO and Zn2+, respectively, with single distilled water as a control
Reference substance (positive control):
not specified
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
3.969 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
dissolved
Remarks:
Zinc
Basis for effect:
mortality (fish)
Remarks on result:
other: Nano ZnO
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
2.525 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
dissolved
Remarks:
Zinc
Basis for effect:
mortality (fish)
Remarks on result:
other: Bulk ZnO
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
7.48 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
dissolved
Remarks:
Zinc
Basis for effect:
mortality (fish)
Remarks on result:
other: Zinc sulphate
Reported statistics and error estimates:
Data were analyzed using the statistical package SPSS (Ver. 13.0, SPSS Inc., Chicago, IL, USA). The 96 h LC50 values were calculated by using a probit analysis method. Data for toxicity tests and bioaccumulation of zinc in zebrafish are expressed as mean SD from three independent experiments. A one-way analysis of variance with Tukey’s multiple comparisons was run to compare the differences between treatment and control groups. Differences were considered statistically significant when p < 0.05.
Sublethal observations / clinical signs:

The SEM image of nZnO powder shows that most nZnO particles were spherical and short-rod shaped, whereas bulk ZnO particles were of an irregular shape. The primary particle mean sizes and distributions of nZnO and bulk ZnO measured by SEM were 80+-49 nm (N=50) and 454 +-191 (N=40) nm, respectively, which corroborated the manufacturer’s information. However, TEM images of nZnO and bulk ZnO particles revealed that both nZnO and bulk ZnO particles in suspensions gathered into large aggregates of irregular shapes, which may be due to the aggregation of particles and a certain amount of uncertainty during the manufacturing process.

the average hydrodynamic diameter of nZnO aggregates increased dramatically with increasing concentrations, while bulk ZnO was relatively stable in particle size. At high concentrations (100–300 mg L1), the mean hydrodynamic diameter of nZnO was much larger than that of bulk ZnO. Previous studies had also reported this phenomenon in other nanomaterials, and concluded that smaller particles would readily form larger aggregates.In addition, the effects of pH on the zeta potential and the hydrodynamic diameter of nZnO in suspensions were investigated. The zeta potential of nZnO in suspensions had a strong dependence on the solution pH value. As the pH increased from 6.05 to 10.62, the zeta potential of nZnO ranged gradually from 10.7 to 16.6 mV, and the isoelectric point (IEP) of nZnO in suspensions was calculated to be approximately 8.7, which was a little lower than the reported value of 9.4 or 9.5.35–37 Thus in this study, in the suspensions with a pH range of 6.9–7.3 and low ionic strength, only some of the nZnO particles were liable to form aggregates. It was also found that there were mainly two different sized aggregates in the nZnO suspension, namely, small aggregates and large aggregates, and the solution pH had a great influence on the hydrodynamic diameter of the nZnO aggregates. When the pH value approached the IEP, the proportion of the small aggregates decreased while the large aggregates increased. This may be explained by the fact that as the pH moves further away from the IEP the absolute zeta potential increases therefore the particles become more stable and homogeneously dispersed.Particle agglomerates were expected to be the largest at the IEP. Therefore, it can be inferred that the aggregation behavior of nZnO in suspensions reduced its toxicity to zebrafish.

size distribution of nZnO and buk ZnO in suspensions:

 particle concentration (mg/L)  average diameter (nm)  range of diameters (nm) 
 nZnO 300  1381  489 -2456 
  100  1273  356 -1798 
  50  658.3  423 -1722 
   10  449.1 201 -1296 
bulk ZnO  300  742.1  486 -9720 
  100  744.4  428 -1076 
   50  731.3  445 -939
  10  810.7  708 -971 
Validity criteria fulfilled:
yes
Conclusions:
Tests not done according to standard protocol but good quality and considered useful for assessing acute aquatic ecotoxicity
Executive summary:

Based on the results of this study, it can be concluded that: (1) the acute toxicity of nZnO to zebrafish is not as high as that of bulk

ZnO at the same concentrations; (2) the aggregation and sedimentation of nZnO particles reduced the toxicity of nZnO considerably at high concentrations; (3) _OH radicals generated by nZnO under illumination played a small role in the toxicity of nZnO without causing acute mortality; (4) Zn2+ dissolved from nZnO and bulk ZnO contributed to the toxicity effects, but may not be the main mechanism of acute toxicity to zebrafish. In conclusion, the toxicity mechanisms of nZnO could not be adequately explained by its small size or nano-effect. The concentrations of nZnO, the solubility of nZnO in different mediums, as well as environmental conditions could have

a pronounced influence on the observed toxic effects of nZnO.

Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
no guideline followed
Principles of method if other than guideline:
Stock solutions (100 mg l−1) of nZnO were prepared by stirring nZnO vigorously in culture medium using a magnetic agitator at room temperature for 2 h. Experiments began as soon as the intact fertilized eggs were selected. The exposure protocol was similar to that established by Schulte and Nagel. Briefly, 24 eggs (blastula stage) were transferred to the test wells of a 24-well multi-plate (Costar® 24-Well Cell Culture Cluster, Corning Incorporated, USA) at 1 embryo/well, in which twenty wells contained 2 ml nZnO test solution and four wells contained 2 ml of culture medium per well. Test solutions were prepared immediately prior to use by diluting the stocks of nZnO with culture medium. During the preparation of diluted solution, the stock solution/mixture was continuously stirred with a magnetic stirrer to maintain the suspension at as stable of a concentration as possible. Since we have not encountered major releases of nZnO, little is
known about the ‘representative’ concentrations of the metal oxide NPs present in the environment. Thus, we selected a wide concentration gradient of nZnO, i.e., 100, 50, 10, 5, 1, 0.5, 0.1 mg l−1, in this study. The experiment was performed in triplicate (i.e., a total of 12 embryos were used in the water control and 60 embryos in the exposure group) for each treatment. The plates containing experimental embryos were placed in the fish room. At the end of the experiment,
water samples were collected immediately for water quality assessment and filtrate testing.
GLP compliance:
not specified
Remarks:
GLP compliance not specified in publication
Specific details on test material used for the study:
ZnO NPs were purchased from the Nano Applied Research Center of Nanjing University of Technology. The surface of ZnO NPs were not modified. Bulk ZnO and ZnSO4·7 H2O were purchased from Tianjin Guangcheng Chemical Reagent Co., Ltd.
Analytical monitoring:
no
Vehicle:
no
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
Adult zebrafish with a mean age of 120 d, mean length of 3.02± 0.33 cm, and mean weight of 0.22±0.05 g were obtained from the Research Group for Molecular Pathology of Fish (Institute of Hydrobiology, Chinese Academy of Sciences).
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96
Remarks on exposure duration:
h
Nominal and measured concentrations:
Test concentrations for lethality were 0, 2, 5, 10, 30 and 50 mg/L for ZnO NPs, bulk ZnO and Zn2+
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
4.92 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
nZnO
Basis for effect:
mortality (fish)
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
3.31 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
bulk ZnO
Basis for effect:
mortality (fish)
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
8.062 mg/L
Nominal / measured:
nominal
Conc. based on:
element
Remarks:
Zinc
Basis for effect:
mortality (fish)
Reported statistics and error estimates:
Data were analyzed using the statistical package SPSS (Ver. 16.0, SPSS Inc., Chicago, IL, USA). The 96-h LC50 values were calculated using a probit analysis method. Data for biochemical tests are expressed as the means±SD from three independent experiments, and a one-way analysis of variance with Tukey's multiple comparisons was run to compare the differences between treatment and control groups. Differences were considered statistically significant when p<0.05.
Sublethal observations / clinical signs:

In suspensions of ZnO NPs and of bulk ZnO, particles were collected into aggregates of irregular shape. The mean size of the NP aggregates increased with increasing concentrations. The initial diameter of individual NPs (about 30 nm as provided by the suppliers) was much smaller than the corresponding bulk particles. However, the measured diameter ranges for NPs in suspensions of different concentrations were 400–1400 nm for ZnO, which were similar to the size ranges of bulk particles in suspension. In fact, the Nano-Zetasizer has overestimated the actual particle size, as its measuring principle is based on hydrodynamic properties that are calculated by the Stokes–Einstein equation. This method yields a hydrodynamic diameter that is a calculated particle diameter of a sphere that has the same measured motion in the solute as the actual particle.

The acute toxicity of ZnO NPs, bulk ZnO and Zn2+ to zebrafish increased with particle concentration, demonstrating a dose dependency. When their concentrations exceeded 2 mg/ L, death of the zebrafish occurred with mortality increasing as particle concentration increased; 100% mortality was observed at 30 mg/L of ZnO NPs and bulk ZnO suspensions with calculated 96-h LC50 values of 4.92 mg/L and 3.31 mg/L, respectively.

size ranges in suspensions:

 particle concentration (mg/l)  average diameter (nm)  range of diameter (nm) 
 ZnO NP 300  1381  489 -2456 
  100  1273  358 -1798 
  50  658.3  423 -1722 
  10  449.1  201 -1269 
  616.5  504 -753 
 bulk ZnO 50  744.4  428 -1076 
Validity criteria fulfilled:
yes
Executive summary:

From the results of this study, it can be concluded that: Metal ions released by ZnO contributed to toxicities but were not the main lethal mechanism of the ZnO NPs and bulk ZnO suspensions tested

Endpoint:
fish embryo acute toxicity (FET)
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with national standard methods with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 236 (Fish embryo acute toxicity (FET) test)
Principles of method if other than guideline:
Two exposure scenarios have been chosen. First, embryos were exposed from 0 to 96 hour post fertilization (hpf). The embryos were protected by the chorion. Second, newly hatched eleuthero-embryos were exposed from 72 to 168 hpf. Embryos and eleuthero-embryos were exposed for the same period of time to 0.2, 1, 5 mg/L nZnO, as well as to the corresponding concentrations of 0.27, 1.30, 5.74 mg/L ZnCl2
GLP compliance:
not specified
Remarks:
GLP compliance not specified in publication
Specific details on test material used for the study:
Zinc oxide nanoparticles (nZnO) were supplied by Genes'Ink (Marseille, France).
Analytical monitoring:
yes
Vehicle:
no
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
Zebrafish eggs were supplied by Harland Laboratories (Itingen, Switzerland).
Test type:
static
Water media type:
other: Holtfreter's medium
Remarks:
(3.5 g NaCl, 0.2 g NaHCO3, 0.05 g KCl, 0.12 g CaCl2 and 0.1 g alginic acid per litre at pH 7).
Limit test:
no
Total exposure duration:
96 h
Remarks on exposure duration:
48 and 96h exposures at increasing hours post fertilisation
pH:
pH 7.0
Nominal and measured concentrations:
Measured. Control, 0.1, 0.5 and 2.2 mg/L ZnO nano and ZnCl2.
Reference substance (positive control):
no
Duration:
96 h
Dose descriptor:
other: effect concentration
Effect conc.:
0.2 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
element (dissolved fraction)
Remarks:
ZnO nano
Basis for effect:
other: Hatch success
Remarks on result:
other: 89%
Remarks:
not significantly different to control (100%)
Duration:
96 h
Dose descriptor:
other: effect concentration
Effect conc.:
0.27 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
element (dissolved fraction)
Remarks:
ZnCl2
Basis for effect:
other: Hatch success
Remarks on result:
other: 98%
Remarks:
not significantly different to control (100%)
Details on results:
At 72 hpf, hatching was significantly delayed in exposed embryos at all doses of nZnO and Zn(II) compared to controls. At 96 hpf, almost all individuals (89.1 ± 12.7% nZnO and 97.8 ± 2.6% Zn(II)) of the lowest dose groups (0.2 mg/L nZnO and 0.27 Zn(II)) hatched, whereas only a few embryos (3.1 ± 2.7% and 3.75 ± 3.1% nZnO; 7.1 ± 9.4% and 2 ± 2.7% Zn(II)) hatched in the mid and high dose groups (1 and 5 mg/L nZnO; 0.5, and 2.2 mg/L Zn(II)). No significant differences occurred between nZnO and corresponding Zn(II) concentrations.
Reported statistics and error estimates:
The data were graphically illustrated with GraphPad Prism 5 (GraphPad Software, San Diego, CA, U.S.). Significant differences between treatments were assessed by one way ANOVA followed by a Bonferroni post hoc test (p ≤ 0.05) to compare treatment means of nanoparticulate exposure with Zn(II) exposure as well as treatment means with respective controls. Results are given as mean ± standarddeviation of mean (SD). Differences were considered statistically significant at p ≤ 0.05.
Sublethal observations / clinical signs:

Other "non-classical" endpoints were also used in this study. These endpoints were results from a gene expression study, and as they showed some intersting results, they were included in the section "7. ENVIRONMENTAL HAZARD ASSESSMENT" (freshwater, chronic nano vs. ion toxicity comparison). Please note, this syudy was included in the freshwater, chronic nano vs. ion toxicity comparison as it is a test on the most sensitive life cycle stage, and therefore deemed conservatively representative of a chronic study. However, as these were not classical endpoints, they could not be encoded into IUCLID.

In brief:

Expressional changes of selected target genes were analysed after exposure to nZnO and Zn(II) for 48 h and 96 h in both embryos and eleuthero-embryos, respectively. In both stages, mRNA levels of genes related to oxidative stress including cat and Cu/Zn-sod, metallothionein (mt2), and immune responsive and pro-inflammatory genes (TNFα, IL-1β, c-jun, Stat1a, MxA) were determined by RT-qPCR. Expression of cat and Cu/Zn-sod showed a similar temporal pattern for both nZnO and Zn(II) with an up-regulation at 48 hpf in embryos and a downregulation at 96 hpf at the highest concentrations (Fig. 5). In eleuthero-embryo exposures cat and Cu/Zn-sod showed a downregulation after 48 h of exposure at 120 hpf, whereas no alterations occurred at 168 hpf after 96 h of exposure (Fig. 5). Transcripts of mt2 were significantly up-regulated in embryos at 48 hpf and 96 hpf at almost all nZnO and Zn(II) concentrations, and at 5 mg/L nZnO and 2.2 mg/L Zn(II) in eleuthero-embryos at 120 and 168 hpf (Fig. 5). Transcripts of the pro-inflammatory cytokines, tumor necrosis factor-α (TNFα) and interleukin-1β (IL-1β), showed a different pattern in theembryo than eleuthero-embryo-exposures. A tendency for downregulation of TNFα occurred in the embryo exposures (particularly at 96 hpf) after Zn(II) and nZnO treatment, while an induction occurred in eleuthero-embryo exposures at 120 hpf for both nZnO and Zn(II) (Fig. 6). The expression pattern of IL-1β was similar, showing an upregulation in eleuthero-embryo exposures, mainly after nZnO treatment. Additionally, the jun proto-oncogene (c-jun) was significantly up-regulated at 96 hpf in embryo exposures at mid and high doses of nZnO and Zn(II), but was not altered in eleuthero-embryo exposures (Fig. 6). Both nZnO and Zn(II) exposures resulted in a significant down-regulation of the anti-viral and immune-related gene Myxovirus resistance A (MxA), while the signal transduction and activation of transcription 1a (Stat1a) remained unaffected (Fig. 7).

Validity criteria fulfilled:
yes
Conclusions:
Tests not done according to standard protocol but considered useful as a weight of evidence approach.
Executive summary:

Fish embryo acute test considered useful as a proxy for a chronic study due to the senstive life cycle stage being tested. Hatching success and a suite of gene expression endpoints showed that, in general, nano and ion toxicity were comparable.

Description of key information

It is generally concluded from the data that the acute toxicity of the ZnO-NP is rather similar to the one of the zinc ion, as was also suggested by Adam et al. (2015) in their review.  Consequently, the existing classification for acute effects based on the Zn-ion is considered relevant for nano-ZnO, too.

Key value for chemical safety assessment

Additional information

Nano ZnO data set


The present short-term acute aquatic toxicity database for nano-ZnO, covers the main taxonomic groups (fish, invertebrates and algae): In total, 13 species are included, 1 freshwater fish, 5 invertebrates (2 freshwater and 3 marine) and 7 algae species (2 freshwater and 5 marine). In addition, 1 acute study on 1 sediment freshwater species is available. For reasons of comparison, in the tables attached, the data are presented as normalized values with the ecotoxicity value observed for the soluble zinc salts (Zn2+) as reference (ECx = 100%). The ratio ECx Zn2+/ECx nano ZnO is provided. The detail of the studies is in the IUCLID files. The full set of EC50 values are presented in the table of the attached word document.


It is generally concluded from the data that the acute toxicity of the ZnO-NP is rather similar to the one of the zinc ion, as was also suggested by Adam et al. (2015) in their review.  Consequently, the existing classification for acute effects based on the Zn-ion is considered relevant for nano-ZnO, too.